AU700036B2 - Triterpene derivatives with immunosuppressant activity - Google Patents

Description

WO 97/16438 PCT/US96/17481 TITLE OF THE INVENTION TRITERPENE DERIVATIVES WITH IMMUNOSUPPRESSANT

ACTIVITY

BACKGROUND OF THE INVENTION Immunoregulatory abnormalities have been shown to exist in a wide variety of "autoimmune" and chronic inflammatory diseases including systemic lupus erythematosis, chronic rheumatoid arthritis, type I and II diabetes mellitus, inflammatory bowel disease, biliary cirrhosis, uveitis, multiple sclerosis and other disorders such as Crohn's disease, ulcerative colitis, bullous pemphigoid, sarcoidosis, psoriasis, ichthyosis, Graves ophthalmopathy and asthma.

Although the underlying pathogenesis of each of these conditions may be quite different, they have in common the appearance of a variety of autoantibodies and self-reactive lymphocytes. Such selfreactivity may be due, in part, to a loss of the homeostatic controls under which the normal immune system operates.

Similarly, following a bone-marrow or an organ transplantation, the host lymphocytes recognize the foreign tissue antigens and begin to produce antibodies which lead to graft rejection.

One end result of an autoimmune or a rejection process is tissue destruction caused by inflammatory cells and the mediators they release. Anti-inflammatory agents such as NSAID's act principally by blocking the effect or secretion of these mediators but do nothing to modify the immunologic basis of the disease. On the other hand, cytotoxic agents, such as cyclophosphamide, act in such a nonspecific fashion that both the normal and autoimmune responses are shut off.

Indeed, patients treated with such nonspecific immunosuppressive agents are as likely to succumb from infection as they are from their autoimmune disease.

Cyclosporin A (CsA), which was approved by the US FDA in 1983 is currently the leading drug used to prevent rejection of transplanted organs. In 1993, FK-506 (Prograf) was approved by the US FDA for the prevention of rejection in liver transplantation. CsA WO 97/16438 PCT/US96/17481 -2and FK-506 act by inhibiting the body's immune system from mobilizing its vast arsenal of natural protecting agents to reject the transplant's foreign protein. In 1994, CsA was approved by the US FDA for the treatment of severe psoriasis and has been approved by European regulatory agencies for the treatment of atopic dermatitis.

Though they are effective in fighting transplant rejection, CsA and FK- 506 are known to cause several undesirable side effects including nephrotoxicity, neurotoxicity, and gastrointestinal discomfort.

Newer, safer drugs exhibiting less side effects are constantly being searched for in the field.

Four active components of Spachea correa were recently identified which inhibit thymidine uptake of T cells and are useful as immunosuppressive agents in animals, including man.

H yH Formula 1(a) b is a single H bond and R is OAc b I CH O Formula 1(b) b is a double H bond and R is OAc 4 OH "COOCH3 O CH CH OAc Formula 1(c) b is a single O bond and R is OH I -OAc OAc CH OcOA

C

CH, ORA Formula 1(d) b is a double bond and R is OH These compounds are useful as immunosuppressive agents in animals, including man. The present invention describes newly developed immunosuppressive compounds derived from the compounds described in Formulae 1(a) through 1 and which have the relative stereochemistry depicted above.

WO 97/16438 PCT/US96/17481 -3- SUMMARY OF THE INVENTION A class of triterpene derivatives of the general structural Formula I 29 C 12 H 18 19 22 17 2 1 H 9 H13 OH 2

O

2

CH

3 2 14 16 3 b 10 25 15 OAc X 24 6 26 OAc OAc 23a R

I

are useful as immunosuppressants.

As immunosuppressants, the compounds of this invention are useful in the treatment of autoimmune diseases, the prevention of rejection of foreign organ transplants and/or related afflictions, diseases and illnesses. Also within the scope of this invention are pharmaceutical formulations comprising a compound of Formula I and a pharmaceutical carrier, as well as, pharmaceutical formulations comprising a compound of Formula I, a second immunosuppressant compound and a pharmaceutical carrier.

DETAILED DESCRIPTION OF THE INVENTION A. Scope of the Invention The present invention is related to compounds of formula I, including but not limited to those specified in the examples, which are useful in a mammalian subject for the treatment and prevention of the resistance by transplantation of organs or tissue, graft-versus-host diseases brought about by medulla ossium transplantation; rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type I diabetes uveitis, juvenileonset or recent-onset diabetes mellitus, posterior uveitis, allergic WO 97/16438 PCT/US96/17481- -4encephalomyelitis, glomerulonephritis, infectious diseases caused by pathogenic microorganisms, inflammatory and hyperproliferative skin diseases, psoriasis, atopical dermatitis, contact dermatitis, eczematous dermatitises, seborrhoeis dermatitis, Lichen planus, Pemphigus, bullous pemphigoid, Epidermolysis bullosa, urticaria, angioedemas, vasculitides, erythemas, cutaneous eosinophilias, Lupus erythematosus, acne, Alopecia areata, keratoconjunctivitis, vernal conjunctivitis, uveitis associated with Behcet's disease, keratitis, herpetic keratitis, conical cornea, dystrophia epithelialis comeae, corneal leukoma, ocular pemphigus, Mooren's ulcer, Scleritis, Graves' opthalmopathy, Vogt- Koyanagi-Harada syndrome, sarcoidosis, etc.; pollen allergies, reversible obstructive airway disease, bronchial asthma, allergic asthma, intrinsic asthma, extrinsic asthma and dust asthma, chronic or inveterate asthma, late asthma and airway hyper-responsiveness, bronchitis, gastric ulcers, vascular damage caused by ischemic diseases and thrombosis, ischemic bowel diseases, inflammatory bowel diseases, necrotizing enterocolitis, intestinal lesions associated with thermal burs and leukotriene B4-mediated diseases, Coeliac diseases, proctitis, eosinophilic gastroenteritis, mastocytosis, Crohn's disease, ulcerative colitis, migraine, rhinitis, eczema, interstitial nephritis, Good-pasture's syndrome, hemolytic-uremic syndrome, diabetic nephropathy, multiple myositis, Guillain-Barre syndrome, Meniere's disease, polyneuritis, multiple neuritis, mononeuritis, radiculopathy, hyperthyroidism, Basedow's disease, pure red cell aplasia, aplastic anemia, hypoplastic anemia, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, agranulocytosis, pernicious anemia, megaloblastic anemia, anerythroplasia, osteoporosis, sarcoidosis, fibroid lung, idiopathic interstitial pneumonia, dermatomyositis, leukoderma vulgaris, ichthyosis vulgaris, photoallergic sensitivity, cutaneous T cell lymphoma, arteriosclerosis, atherosclerosis, aortitis syndrome, polyarteritis nodosa, myocardosis, scleroderma, Wegener's granuloma, Sjogren's syndrome, adiposis, eosinophilic fascitis, lesions of gingiva, periodontium, alveolar bone, substantia ossea dentis, glomerulonephritis, male pattern alopecia or alopecia senilis by preventing epilation or providing hair germination WO 97/16438 PCT/US96/17481 and/or promoting hair generation and hair growth; muscular dystrophy; Pyoderma and Sezary's syndrome, Addison's disease, ischemiareperfusion injury of organs which occurs upon preservation, transplantation or ischemic disease, for example, thrombosis and cardiac infraction, endotoxin-shock, pseudomembranous colitis, colitis caused by drug or radiation, ischemic acute renal insufficiency, chronic renal insufficiency. toxinosis caused by lung-oxygen or dnrg. for example, paracort and bleomycins), lung cancer, pulmonary emphysema, cataracta, siderosis, retinitis, pigmentosa, senile macular degeneration, vitreal scarring, corneal alkali bum; dermatitis erythema multiforme, linear IgA ballous dermatitis and cement dermatitis, gingivitis, periodontitis, sepsis, pancreatitis, diseases caused by environmental pollution, aging, carcinogenis, metastasis of carcinoma and hypobaropathy; disease caused by histamine or leukotriene-C4 release; Behcet's disease, autoimmune hepatitis, primary biliary cirrhosis sclerosing cholangitis), partial liver resection, acute liver necrosis, necrosis caused by toxin, viral hepatitis, shock, or anoxia, B-virus hepatitis, non-A/non-B hepatitis, cirrhosis, alcoholic cirrhosis, hepatic failure, fulminant hepatic failure, late-onset hepatic failure, "acute-onchronic" liver failure, augmention of chemotherapeutic effect, preventing or treating activity of cytomegalovirus infection, HCMV infection, and antiinflammatory activity; and treatment of immunodepression or a disorder involving immunodepression, including AIDS, cancer, senile dementia, trauma, chronic bacterial infection, and certain central nervous system disorders.

More particularly this invention relates to compounds of the general structural formula I: WO 97/16438 PCT/US96/17481 -6- 2 P02CH3 or a pharmaceutically acceptable salt, crystal form or hydrate, wherein: Xis: a is: O; S, NH, H and R 1 a single bond, or a double bond when R 4 is absent; b and c are independently: a single bond, or a double bond; n is: m is: r is: s is: 1 to 4; 1 to 4; 0or 1; 0or 1;

R

1 and R 2 a) b) are independently: H, or (C1-C6)-alkyl, wherein alkyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (C1-C6)-alkoxy, vinyl, cyano, oxo, nitro, hydroxy, CHO, CO2H, COC -C6-alkyl, CO2C 1-C6-alkyl, CONR 1R 2 NR 1R 2 NR 1COC I-C6-alkyl, aryl, wherein aryl is defined as phenyl or naphthyl, WO 97/16438 PCT/US96/17481 -7unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (Cl-C6)-alkoxy, phenyl, phenoxy, cyano, nitro, hydroxy, CHO, CO2H, COCI-C6-alkyl, CO2C1-C6-alkyl,

CONR

1

R

2

NR

1

R

2

NR

1 COC1-C6-alkyl and any two of adjacent substituents can be joined to form a 6- or 7membered fused ring said ring containing I or 2 oxygen atoms and the remainder carbon atoms, heteroaryl, wherein heteroaryl is defined as a 5 or 6-membered ring substituted with one and two heteroatoms selected from O, S, N, unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (CI-C6)-alkoxy, cyano, nitro, hydroxy, CHO, CO2H, COC 1-C6-alkyl, CO2C -C6-alkyl, CONR 1R 2 NR 1R 2 NR 1COC 1-C6-alkyl, any two adjacent substituents can be joined to form a 6- or 7-membered fused ring said ring containing 1 or 2 oxygen atoms and the remainder carbon atoms, or any two adjacent substituents can be joined together to form a benzo-fused ring;

R

3 is: a) -(Cl-C6)-alkyl, alkyl as defined above; b) -(C1-C6)-alkenyl, wherein alkenyl is unsubstituted or substituted with one, two or three substituents selected from from the group consisting of: Br, Cl, F, I, (Cl-C6)-alkoxy, cyano, oxo, nitro, hydroxy, CHO, CO2H, COCI-C6-alkyl, CO2C 1-C6-alkyl, CONR R 2 NR 1R 2 NR COC 1-C6-alkyl, aryl as defined above, and heteroaryl as defined above; c) -(C1-C6)-alkynyl, wherein alkynyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (C1-C6)-alkoxy, cyano, oxo, nitro, hydroxy, CHO, CO2H, COCl-C6-alkyl, CO2C -C6-alkyl, CONR 1R 2 NR 1R 2 NR 1COC -C6-alkyl, aryl as defined above, and heteroaryl as defined above, WO 97/16438 PCT/US96/17481- -8d) -aryl, aryl as defined above, or e) -heteroaryl, heteroaryl as defined above;

R

4 is: a) absent and a is a double bond; b) -H, c) -OH.

d) =O, e) -O[(C=O)Or]sCl-C10-alkyl, alkyl as defined above, f) -O[(C=O)Or]sC2-Co1-alkenyl, as defined above, g) -O[(C=O)Or]sC2-C6-alkynyl, alkynyl as defined above, h) -O[(C=O)Or]s(C3-C7)-cycloalkyl, i) -O[(C=O)Or]saryl, aryl as defined above, j) -O[(C=O)Orlsheteroaryl, heteroaryl as defined above, k) -O(CH2)nO(CH2)mheteroaryl, heteroaryl as defined above, 1) -O(CH2)nO(CH2)maryl, aryl as defined above, m) -OC(=O)NRl R 2 n) -OS02R 3 or o) -NR 1

R

2 An embodiment of the invention are the compounds of structural Formula I, OAc OAc or a pharmaceutically acceptable salt, crystal form or hydrate, wherein: WO 97/16438 PCT/US96/17481 -9- X is: a is: O, S, or NH; a single bond; b and c are independently: a single bond or a double bond; n is: m is: r is: s is: 1 to 4; 1 to 4; 0or 1; 0or 1;

R

1 and R 2 a) b) are independently: H, or (C1-C6)-alkyl, wherein alkyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (Cl-C6)-alkoxy, vinyl, cyano, oxo, nitro, hydroxy, CHO, CO2H, COCl-C6-alkyl, CO2C -C6-alkyl, CONR 1R 2 NR 1R 2 NR 1 COC -C6-alkyl, aryl, wherein aryl is defined as phenyl or naphthyl, unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (C1-C6)-alkoxy, phenyl, phenoxy, cyano, nitro, hydroxy, CHO, CO2H, COC1-C6-alkyl, CO2C1-C6-alkyl,

CONR

1

R

2

NRIR

2 NR COC1-C6-alkyl and any two of adjacent substituents can be joined to form a 6- or 7membered fused ring said ring containing 1 or 2 oxygen atoms and the remainder carbon atoms, heteroaryl, wherein heteroaryl is defined as a 5 or 6-membered ring substituted with one and two heteroatoms selected from O, S, N, unsubstituted or substituted with one, two or three WO 97/16438 PCT/US96/17481 substituents selected from the group consisting of: Br, Cl, F, I, (C1-C6)-alkoxy, cyano, nitro, hydroxy, CHO, CO2H, COC1-C6-alkyl, CO2C1-C6-alkyl, CONR 1

R

2

NR

1

R

2 NR ICOC 1-C6-alkyl, any two adjacent substituents can be joined to form a 6- or 7-membered fused ring said ring containing 1 or 2 oxygen atoms and the remainder carbon atoms. or any two adjacent substituents can he joined together to form a benzo-fused ring;

R

3 is: a) -C6)-alkyl, alkyl as defined above; b) -(C1-C6)-alkenyl, wherein alkenyl is unsubstituted or substituted with one, two or three substituents selected from from the group consisting of: Br, Cl, F, I, (C1-C6)-alkoxy, cyano, oxo, nitro, hydroxy, CHO, CO2H, COCi-C6-alkyl, CO2C -C6-alkyl, CONR 1R 2

NR

1

R

2 NR 1COC 1-C6-alkyl, aryl as defined above, and heteroaryl as defined above; c) 1-C6)-alkynyl, wherein alkynyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (C1-C6)-alkoxy, cyano, oxo, nitro, hydroxy, CHO, CO2H, COC 1-C6-alkyl, CO2C -C6-alkyl, CONR R 2 NR 1R 2 NR 1COC 1-C6-alkyl, aryl as defined above, and heteroaryl as defined above, d) -aryl, aryl as defined above, or e) -heteroaryl, heteroaryl as defined above;

R

4 is: a) absent and a is a double bond; b) -H, c) -OH, d) =O, e) -O[(C=0)Or]sCI-C10-alkyl, alkyl as defined above, f) -O[(C=O)Or]sC2-C 10-alkenyl, as defined above, g) -O[(C=O)OrlsC2-C6-alkynyl, alkynyl as defined above, WO 97/16438 PCT/US96/17481 11 -O[(C=O)Or]s(C3-C7)-cycloalkyl, -O[(C=O)Or]saryl, aryl as defined above, -O[(C=O)Orlsheteroaryl, heteroaryl as defined above, -O(CH2)nO(CH2)mheteroaryl, heteroaryl as defined above, -O(CH2)nO(CH2)maryl, aryl as defined above, -OC(=O)NR1 R 2 -OSO9R 3 or

-NR

1

R

2 An embodiment of this embodiment of the invention are the of structural Formula I, compounds 'OAc OAc OAc or a pharmaceutically acceptable salt, crystal form or hydrate, wherein: X is: a is: a single bond; b and c are independently: a single bond or a double bond; n is: 1 to 4; m is: 1 to 4; WO 97/16438 PCT/US96/17481 12r is: s is: RI and R 2 a) b) 0or 1; 0or 1; are independently: H, or (C -C6)-alkyl, wherein alkyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (CI-C6)-alkoxy, vinyl, cyano, oxo, nitro, hydroxy, CHO, CO2H, COCi-C6-alkyl, CO2C 1-C6-alkyl, CONR 1R 2 NR 1R 2 NR 1COC l-C6-alkyl, aryl, wherein aryl is defined as phenyl or naphthyl, unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (C1-C6)-alkoxy, phenyl, phenoxy, cyano, nitro, hydroxy, CHO, CO2H, COC1-C6-alkyl, CO2C1-C6-alkyl,

CONR

1

R

2

NR

1

R

2 NR COC1-C6-alkyl and any two of adjacent substituents can be joined to form a 6- or 7membered fused ring said ring containing 1 or 2 oxygen atoms and the remainder carbon atoms, heteroaryl, wherein heteroaryl is defined as a 5 or 6-membered ring substituted with one and two heteroatoms selected from O, S, N, unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (CI-C6)-alkoxy, cyano, nitro, hydroxy, CHO, CO2H, COC -C6-alkyl, CO2C -C6-alkyl, CONR 1

R

2

NR

1

R

2 NR 1COC -C6-alkyl, any two adjacent substituents can be joined to form a 6- or 7-membered fused ring said ring containing 1 or 2 oxygen atoms and the remainder carbon atoms, or any two adjacent substituents can be joined together to form a benzo-fused ring; WO 97/16438 PCT/US96/17481 13

R

3 is: a) 1-C6)-alkyl, alkyl as defined above, b) -aryl, aryl as defined above, or c) -heteroaryl, heteroaryl as defined above;

R

4 is: a) -O[(C=O)Or]sCl-C0I-alkyl, alkyl as defined above, b) -O[(C=O)Or]s(C3-C7)-cycloalkyl, c) -O[(C=O)Or]saryl, aryl as defined above, d) -O[(C=O)Orlsheteroaryl, heteroaryl as defined above, e) -O(CH2)nO(CH2)mheteroaryl, heteroaryl as defined above, f) -O(CH2)nO(CH2)maryl, aryl as defined above, g) -OC(=O)NRI R 2 or h) -OS02R 3 A preferred embodiment of this embodiment are the compounds of structural Formula I, as recited in above,or a pharmaceutically acceptable salt, crystal form or hydrate, wherein:

R

4 is: a) -O[(C=O)Or]saryl, wherein aryl is defined as phenyl or naphthyl, unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (Ci-C6)-alkoxy, phenyl, phenoxy, cyano, nitro, hydroxy, CHO, CO2H, COCI-C6-alkyl, CO2CI-C6-alkyl,

CONR

1

R

2

NR

1

R

2

NR

1 COC1-C6-alkyl and any two of adjacent substituents can be joined to form a 6- or 7membered fused ring said ring containing 1 or 2 oxygen atoms and the remainder carbon atoms, or b) -O[(C=O)Or]sheteroaryl, wherein heteroaryl is defined as a or 6-membered ring substituted with one and two heteroatoms selected from O, S, N, unsubstituted or substituted with one, two or three substituents selected from WO 97/16438 PCT/US96/17481 14the group consisting of: Br, Cl, F, I, (C1-C6)-alkoxy, cyano, nitro, hydroxy, CHO, CO2H, COC1-C6-alkyl, CO2C 1-C6-alkyl, CONR 1

R

2 NR 1R 2 NR 1COC 1-C6-alkyl, any two adjacent substituents can be joined to form a 6or 7-membered fused ring said ring containing 1 or 2 oxygen atoms and the remainder carbon atoms, or any two adjacent substituents can be joined together to form a benzo-fused ring.

Another embodiment of this invention are the compounds of structural Formula I, 'OAc OAc 4 Ac 23 a 4 or a pharmaceutically acceptable salt, crystal form or hydrate, wherein: X is: a is: H and RI; a single bond; b and c are independently: a single bond or a double bond; n is: m is: 1 to 4; 1 to 4; WO 97/16438 PCT/US96/17481 r is: s is:

R

1 and R 2 a) b) 0or 1; 0or 1; are independently: H, or (C1 -C6)-alkyl, wherein alkyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (C1-C6)-alkoxy, vinyl, cyano, oxo, nitro, hydroxy, CHO, CO2H, COCI-C6-alkyl, CO2C -C6-alkyl, CONR 1R 2 NR R 2 NR 1COC -C6-alkyl, aryl, wherein aryl is defined as phenyl or naphthyl, unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (Cl-C6)-alkoxy, phenyl, phenoxy, cyano, nitro, hydroxy, CHO, CO2H, COCl-C6-alkyl, CO2C1-C6-alkyl, CONR R 2 NR R 2

NR

1 COC -C6-alkyl and any two of adjacent substituents can be joined to form a 6- or 7membered fused ring said ring containing 1 or 2 oxygen atoms and the remainder carbon atoms, heteroaryl, wherein heteroaryl is defined as a 5 or 6-membered ring substituted with one and two heteroatoms selected from O, S, N, unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (C1-C6)-alkoxy, cyano, nitro, hydroxy, CHO, CO2H, COC I-C6-alkyl, CO2C -C6-alkyl, CONR 1

R

2 NR R 2 NR 1COC1-C6-alkyl, any two adjacent substituents can be joined to form a 6- or 7-membered fused ring said ring containing 1 or 2 oxygen atoms and the remainder carbon atoms, or any two adjacent substituents can be joined together to form a benzo-fused ring;

R

3 is: a) -(Cl-C6)-alkyl, alkyl as defined above; WO 97/1 6438 PCTIUS96/17481 16 b) I -C6)-alkenyl, wherein alkenyl is unsubstituted or substituted with one, two or three substituents selected from from the group consisting of: Br, Cl, F, 1, (C1I-C6)-alkoxy, cyano, oxo, nitro, hydroxy, CHO, CO2H, COC I -C6-alkyl, C02C 1 -C6-alkyl, CONR I R 2 NR I R 2 NR I COC I -C6-alkyl, aryl as defined above, and heteroaryl as defined above; c) I -C6,)-alkynyl, wherein alkvnvl is unsiihstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, 1, (C I -C6)-alkoxy, cyano, oxo, nitro, hydroxy, CHO, CO2H, COC I -C6-alkyl, C02C I -C6-alkyl, CONR I R 2 NR I R 2 NR 1 COC I -C6-alkyl, aryl as defined above, and heteroaryl as defined above, d) -aryl, aryl as defined above, or e) -heteroaryl, heteroaryl as defined above;

R

4 is: a) absent and a is a double bond; b) -H, c) -OH, d) =0, e) (C=O)Orl sClI-C1IO-alkyl, alkyl as defined above, f) -O(C0)OrsC2-C10-alkenyl, as defined above, g) -O[(C=O)Or]sC2-C6-alkynyl, alkynyl as defined above, h) -O[(C0)Ors(C3-C7)-cycloalkyl, 1) -OI(C=O)Orlsaryl, aryl as defined above, j) (C=O)Orl sheteroaryl, heteroaryl as defined above, k) -O (CH2)nO (CH2)mheteroaryl, heteroaryl as defined above, 1) -O(CH2)nO(CH2)maryl, aryl as defined above, m) -OC(=O)NR I R 2 n) -OSO2R 3 or o) -NR IR 2 An embodiment of this embodiment are the compounds of structural Formula 1, WO 97/16438 PCT/US96/17481 17- 'OAc OAc or a pharmaceutically acceptable salt, crystal form or hydrate, wherein: Xis: a is: H and R1; a single bond; b and c are independently: a single bond or a double bond; n is: m is: r is: s is: 1 to 4; 1 to 4; 0or 1; 0or 1; R1 and R 2 a) b) are independently: H, or (CI-C6)-alkyl, wherein alkyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (Cl-C6)-alkoxy, vinyl, cyano, oxo, nitro, hydroxy, CHO, CO2H, COC -C6-alkyl, CO2C I-C6-alkyl, CONR R 2 NR 1R 2 NR 1COC 1-C6-alkyl, aryl, wherein aryl is defined as phenyl or naphthyl, WO 97/16438 PCT/US96/17481 18 unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (C1-C6)-alkoxy, phenyl, phenoxy, cyano, nitro, hydroxy, CHO, CO2H, COC1-C6-alkyl, CO2CI-C6-alkyl,

CONR

1

R

2

NR

1

R

2

NR

1 COC1-C6-alkyl and any two of adjacent substituents can be joined to form a 6- or 7membered fused ring said ring containing 1 or 2 oxygen atoms and the remainder carbon atoms, heteroaryl, wherein heteroaryl is defined as a 5 or 6-membered ring substituted with one and two heteroatoms selected from O, S, N, unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (C1-C6)-alkoxy, cyano, nitro, hydroxy, CHO, CO2H, COC -C6-alkyl, CO2C 1-C6-alkyl, CONR IR 2 NR 1R 2 NR 1 COC -C6-alkyl, any two adjacent substituents can be joined to form a 6- or 7-membered fused ring said ring containing 1 or 2 oxygen atoms and the remainder carbon atoms, or any two adjacent substituents can be joined together to form a benzo-fused ring;

R

3 is: a) -(C1-C6)-alkyl, alkyl as defined above; b) -(Cl-C6)-alkenyl, wherein alkenyl is unsubstituted or substituted with one, two or three substituents selected from from the group consisting of: Br, Cl, F, I, (C1-C6)-alkoxy, cyano, oxo, nitro, hydroxy, CHO, CO2H, COC1-C6-alkyl, CO2C 1-C6-alkyl, CONR 1R 2 NR 1R 2 NR 1COC 1-C6-alkyl, aryl as defined above, and heteroaryl as defined above; c) I-C6)-alkynyl, wherein alkynyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (Cl-C6)-alkoxy, cyano, oxo, nitro, hydroxy, CHO, CO2H, COC1-C6-alkyl, CO2C 1-C6-alkyl, CONR 1

R

2 NR 1R 2 NR 1COC -C6-alkyl, aryl as defined above, and heteroaryl as defined above, WO 97/16438 WO 9716438PCTIUS96/17481 19 d) -aryl, aryl as defined above, or e) -heteroaryl, heteroaryl as defined above;

R

4 is: a) -OH, b) -O[(C0)Or~sCi-CO-alkyl, alkyl as defined above, C) -Or(C=O)Orjs(C3-C7)-cycloalkyl, d) -O[(C=O)Orlsaryl, aryl as defined above, e) -01 (C=O)Orl sheteroaryl, heteroaryl as defined above, f) -O(CH2)nO(CH2)mheteroaryl, heteroaryl as defined above, g) -O(CH2)nO(CH2)maryl, aryl as defined above, h) -OC(=O)NR I R 2 or i) -0S02R 3 An embodiment of the invention is a compound selected from the group consisting of: 4,6,7,15,1 6-pentakis(acetyloxy)- 1 8-hydroxy-22-methoxycarbonyl- [6cx,7Qx, 1513,163] D :A-Friedo-A-homo-27 ,30-dinor-24-oxaoleana- 1 ,20(29),2 1 -trien-3-one; 4-(2-bromobenzoyl)oxy-6 15,1 6-tetrakis (acetyloxy)- 1 8-hydroxy-22methoxycarbonyl [6(x,7Qx, 153,1 61]D :A-Friedo-A-homo-27 ,30-dinor-24oxaoleana- 1 ,20(29),2 1 -trien-3-one; 4,6,7,15,1 6-pentakis(acetyloxy)- I 8-hydroxy-22-methoxycarbonyl- [6cx,7a, 15P3,1613]D :A-Friedo-A-homo-27 ,30-dinor-24-oxaoleana- 1 ,20(29),2 I -triene; 4,6,7,15,1 6-pentakis (acetyloxy)-2 1,22-epoxy- I 8-hydroxy-22methoxycarbonyl[6,7ix, 15P3,16P3,21 P,2213]D:A-Friedo-A-homo-27,30dinor-24-oxao leana-20(29)-en- 3-one; WO 97/16438 PCTIUS96/17481 20 4,6,7,15,16-pentakis(acetyloxy)- 18-hydroxy-22-methoxycarbonyl- [6ac,7, 150, 16P]D:A-Friedo-A-homo-27,30-dinor-24-oxaoleana- 20(29),21-dien-3-one; 4-(2-bromobenzoyl)oxy-6,7,15,16-tetrakis(acetyloxy)- I 8-hydroxy-22methoxycarbonyl[6a,7, 150,160]D:A-Friedo-A-homo-27,30-dinor-24oxaoleana-20(29),21-dien-3-one; 6,7,15,1 6-tetrakis(acetyloxy)-2 1 ,22-epoxy-4,18 -dihydroxy-22methoxycarbonyl[6ax,7ax, 15P,160,21 P,22P]D:A-Friedo-A-homo-27,30dinor-24-oxaoleana-20(29)-en-3-one; and 4-(2-bromobenzoyl)oxy-6,7,15,16-tetrakis(acetyloxy)-21,22-epoxy- 18hydroxy-22-methoxycarbonyl[6a,7, 15P,161,21 ,220]D:A-Friedo-Ahomo-27,30-dinor-24-oxaoleana-20(29)-en-3 -one.

The compounds of the present invention have asymmetric centers and this invention includes all of the optical isomers and mixtures thereof.

In addition compounds with carbon-carbon double bonds may occur in Z- and E- forms with all isomeric forms of the compounds being included in the present invention.

As used herein, the term "alkyl" includes those alkyl groups of a designated number of carbon atoms of either a straight, branched, or cyclic configuration. Examples of "alkyl" include methyl, ethyl, propyl, isopropyl, butyl, sec-and tert-butyl, pentyl, hexyl, heptyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, norbomrnyl, and the like. "Alkoxy" represents an alkyl group of indicated number of carbon atoms attached through an oxygen bridge, such as methoxy, ethoxy, propoxy, butoxy and pentoxy.

"Alkenyl" is intended to include hydrocarbon chains of a specified number of carbon atoms of either a straight- or branchedconfiguration and at least one unsaturation, which may occur at any point along the chain, such as ethenyl, propenyl, butenyl, pentenyl, WO 97/16438 PCT/US96/17481 -21 dimethyl pentenyl, and the like, and includes E and Z forms, where applicable. "Halogen", as used herein, means fluoro, chloro, bromo and iodo.

The term "aryl" is defined as a phenyl or naphthyl ring which is optionally substituted at any available carbon atoms with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (C1-C6)-alkoxy, phenyl, phenoxy, cyano, oxo, nitro, hydroxy, CHO, CO2H, COCI-C6-alkyl, CO2C1-C6-alkyl,

CONR

1

R

2

NRIR

2 NR COCl-C6-alkyl. The aryl may also be substituted with a fused or 7-membered ring containing one or two oxygens and the remaining ring atoms being carbon, the fused or 7-ring being selected from the group consisting of: dioxolanyl, dihydrofuranyl, dihydropyranyl, and dioxanyl.

The term "heteroaryl" as utilized herein is intended to include the following a 5 or 6-membered ring substituted with one or two heteroatoms selected from O, S, N, and is unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (C1-C6)-alkoxy, cyano, nitro, hydroxy, CHO CO2H, COC 1-C6-alkyl, CO2C 1-C6-alkyl, CONR 1

R

2 NR R 2 NR 1COC -C6-alkyl, any two adjacent substituents can be joined to form a 6- or 7-membered fused ring said ring containing 1 or 2 oxygen atoms and the remainder carbon atoms, or any two adjacent substituents can be joined together to form a benzo-fused ring. Heteroaryl groups within the scope of this definition include but are not limited to: acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl, benzotriazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, and pyrrolyl which are substituted or unsubstituted as defined above.

In the compounds of Formula I, the heteroaryl group may be optionally substituted with the substituents listed above at any available carbon atom or nitrogen atom (if present), but compounds bearing certain substitutents, directly substituted to a nitrogen may be relatively unstable and are not preferred. The heteroaryl may also be fused to a second or 7-membered ring containing one or two WO 97/16438 PCT/US96/17481 -22oxygens selected from the the remaining ring atoms being carbon, selected from the group consisting of: dioxolanyl, dihydrofuranyl, dihydropyranyl, and dioxanyl.

Pharmaceutically acceptable salts include both the metallic (inorganic) salts and organic salts; a list of which is given in Remington's Pharmaceutical Sciences, 17th Edition, pg. 1418 (1985). It is well known to one skilled in the art that an appropriate salt form is chosen based on physical and chemical stability, flowability, hydroscopicity and solubility. As will be understood by those skilled in the art, pharmaceutically acceptable salts include, but are not limited to salts of inorganic acids such as hydrochloride, sulfate, phosphate, diphosphate, hydrobromide, and nitrate or salts of an organic acid such as malate, maleate, fumarate, tartrate, succinate, citrate, acetate, lactate, methanesulfonate, p-toluenesulfonate or palmoate, salicylate and stearate. Similarly pharmaceutically acceptable cations include, but are not limited to sodium, potassium, calcium, aluminum, lithium and ammonium (especially ammonium salts with secondary amines).

Preferred salts of this invention for the reasons cited above include potassium, sodium, calcium and ammonium salts. Also included within the scope of this invention are crystal forms, hydrates and solvates of the compounds of Formula I.

WO 97/16438 PCT/US96/17481 -23- REACTION SCHEME A OAc OAc OAc Li, NH 3

THF

OAc OAc As seen in Scheme A, compound I, 4 ,5,6,15,16-pentakis (acetyloxy)-21,22-epoxy-18-hydroxy-22-methoxycarbonyl[6a, 7a, 16P, 211, 22p]D:A-Freido-A-homo-27,30-dinor-24-oxaoleana- 1,20(29)-diene-3-one, isolated from Spachea correa in liquid ammonia with lithium metal will result in the reduction of the Cl olefin group to produce the saturated lactone. Alternative methods for reducing the Cl olefin group and/or the C20(29) olefin that are known in the art may also be employed. US Serial Number 08/476,806 filed on June 7, 1995 describes the isolation of compound I and is hereby incorporated by WO 97/16438 PCT/US96/17481 -24reference. The resultant lactone can then be converted to the oxepin analog by procedures described in Reaction Scheme B.

It should also be noted that compounds of Formula I having the 11,12-double bond can be prepared using the starting material, 4,6,7,15,16-pentakisacetoxy-21,22-epoxy-I 8-hydroxy-22methoxycarbonyl[6a,7a,15p,16p,21 3,22p]D:A-Freido-A-homo-27,30dinor-24-oxaleana- 11 1,20(29)-trien-3-one, isolated from Spachea correa and following the procedures described herein. However, there may be reactions where it will not be possible to selectively operate on one of the double bonds, for example, ozonolysis.

REACTION SCHEME B 29 19 21 12 1-18 22 0 11 17 2 H 9 HL OH 2 pO 2

CH

3 14 16 b 10 25 8 15 OAc O 5 7 26OA S24 6 A OAc OAc 2 OAc OAc I (b is a double bond) II (b is a single bond) 1. LiAIH(OtBu) 2. Et 3 SiH, BF 3 OEt 2 'OAc OAc WO 97/16438 PCT/US96/17481 As seen in Scheme B, compound I [(4,6,7,15,16pentakis(acetyloxy)-21,22-epoxy-I 8 -hydroxy-22-methyoxycarbonyl- [6a,7a, 15 163,21 P,22p]D:A-Freido-A-homo-27,30-dinor-24oxaoleana-1, 20(29)-dien-3-one] isolated from Spachea correa can be converted to its oxepin analog in a two step process. US Serial Number 08/476,806 filed June 7, 1995, describing the isolation of compound I and is hereby incorporated by reference. Lactone I is first reduced to the lactol. This can be accomplished by using a variety of reducing agents including di-isobutylaluminum hydride (DIBAL-H) and sodium bis(2-methoxyethoxy)aluminum hydride (Red-Al). A more optimal reducing agent is the use of lithium tri-t-butoxyaluminum hydride in an inert solvent such as dichloromethane at reduced temperatures, preferably 0°C. The purified lactol intermediate is then reacted with triethylsilane and a Lewis acid such as borontrifluoride diethyl etherate to give the ether (oxepin) analog of I.

WO 97/16438 PCT/US96/17481 26 REACTION SCHEME C 1 H 'OAc OAc 1. LiAIH(OtBu) 3 2. Et 3

AI

12 Hia OAc 2" OAc SOAc In a variation of Scheme B, ether (oxepin) derivatives substituted at C3 can also be prepared. Thus in Reaction Scheme C, lactone I is first reduced to the lactol as described in Reaction Scheme B.

The purified lactol intermediate is then reacted with a trialkylaluminum reagent, as exemplified in this scheme by triethylaluminum (Et3Al) to give the ethyl derivative. The allyl derivative can be prepared with allyltrimethylsilane and a Lewis acid, such as borontrifluoride diethyl etherate.

WO 97/16438 PCT/US96/17481 -27- REACTION SCHEME D 1 H

WCI

6 nBuLi THF, 50 °C SWIG ui

'CO

2

CH

3 OAc -4 OAc SOAc The C21-C22 epoxide of lactone or ether derivatives can be converted to the olefin by use of a WCl6/BuLi complex in tetrahydrofuran (THF) by procedures developed by Sharpless et al. (J.

Am. Chem. Soc., 94, 6538-6540, 1972). This conversion can be achieved before or after any of the reaction schemes described.

WO 97/16438 PCT/US96/17481 -28 REACTION SCHEME E

CO

2

CH

3 OAc

HCI/THF

or

CO

2

CH

3 Lactone or ether derivatives can be selectively de-acetylated at C4 to give the corresponding alcohol by reacting it with an aqueous solution of HCI (preferably 2M to 3M concentration) in THF. It can also be prepared by reacting I with CH3(C1)AI[N(OCH3)CH3] (Weinreb reagent) in inert solvents such as THF, toluene or methylene chloride.

If a product from this reaction contains the epoxide, it can be removed by the method described in Reaction Scheme D.

WO 97/16438 PCT/US96/17481 -29- REACTION SCHEME F

CO

2

CH

3 OAc

CO

2

CH

3 OAc 0-

CH

3 The C4 hydroxy group can be oxidized to the corresponding ketone by a variety of oxidizing agents. The Jones reagent (chromic acid and sulfuric acid in H20), pyridinium chlorochromate, and oxalyl chloride plus DMSO all will achieve this conversion.

WO 97/16438 PCT/US96/17481 REACTION SCHEME G (PhO) 3 MePI HMPT, 75 °C i P0)MP The C4 hydroxy group can be dehydrated to give the olefin. Reaction of the alcohol with tris-phenoxymethylphosphonium iodide in hexamethylphosphorous triamide (HMPT) at 75 0 C will achieve this conversion.

WO 97/16438 WO 9716438PCTIUS96/17481 31 REACTION SCHEME H R 4 COCl Mel H H O H CO 2

CH

3 b Me me OAc O OAc OO~c MQ rIc OAO Oc el 0-5J, M 4 CD1 R0 RH CO 2

CH

3 'GAc

NR

1 R 2 WO 97/16438 PCT/US96/17481 -32- REACTION SCHEME H (CONT'D) CDI, R4'OH

CO

2

CH

3 'OAc 'OAc

OH

3 CH3 4

R

4 'OH, Tf20, base 'OAc As depicted in Reaction Scheme H, esters at C4 can be prepared by reaction of a preformed carboxylic acid chloride with the C4 alcohol derivative (Reaction Scheme E) in a basic solvent such as pyridine. It should be understood that R 4 is used to represent a portion of the R 4 definition, e.g. R 4 can be an alkyl carbonate which is depicted in the scheme as OC(=O)OR 4

R

4 representing the alkyl substituent.

The acid chlorides, when not purchased, are prepared by stirring the carboxylic acids in reagents such as oxalyl chloride or thionyl chloride.

Esters may also be prepared by reaction of the acid chloride and C4 WO 97/16438 PCT/US96/17481 -33 alcohol with silver cyanide (AgCN) in an aprotic solvent such as HMPA.

C4 sulfonate derivatives are prepared in a similar manner by reaction with sulfonyl chlorides.

C4 carbonate and carbamate derivatives are prepared by first reacting the C4 alcohol derivative with carbonyldiimidazole (CDI) to obtain the imidazolecarbonyl intermediate which is then reacted with an alcohol or amine (R 1

R

2 NH) to give the corresponding carbonate or carbamate derivatives.

C4 ether derivatives can also be prepared. The best procedure involves reacting an alcohol with trifluoromethanesulfonic anhydride (Tf20, triflic anhydride) to obtain the preformed triflate in dichloromethane at reduced temperature, preferably -78 C. To this solution is added the triterpene alcohol, the reaction mixture is warmed to room temperature and stirring is continued until reaction is complete.

Ethers may also be prepared by heating a mixture of triterpene C4 alcohol, the appropriate alkylhalide and an excess of silver oxide in an aprotic invert solvent such as THF.

WO 97/16438 PCT/US96/17481 -34- REACTION SCHEME I

CO

2

CH

3

CH

3

NHR

1

R

2 NaCNBH 3 C02CH 3 'OAc Amines at C4 can be prepared from the C4 ketone described in Reaction Scheme F by reaction with an amine NHRIR 2 in a variety of solvents with a reducing agent such as sodium cyanoborohydride.

UTILITY

The present invention is related to compounds of formula I, including but not limited to those specified in the examples, which are useful in a mammalian subject for the treatment and prevention of immunemediated diseases such as the resistance by transplantation of organs or tissue such as heart, kidney, liver, medulla ossium, skin, cornea, lung, pancreas, intestinum tenue, limb, muscle, WO 97/16438 PCTIUS96/17481 35 nervus, duodenum, small-bowel, pancreatic-islet-cell, including xeno transplants, etc.; graft-versus-host diseases brought about by medulla ossium transplantation; autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type I diabetes uveitis, juvenileonset or recent-onset diabetes mellitus, posterior uveitis, allergic encephalomyelitis, glomerulonephritis, and the like: and further infectious diseases caused by pathogenic microorganisms. Further uses may include the treatment and prophylaxis of inflammatory and hyperproliferative skin diseases and cutaneous manifestations of immunologically mediated illnesses, such as psoriasis, atopical dermatitis, contact dermatitis and further eczematous dermatitises and further eczematous dermatitises, seborrhoeis dermatitis, Lichen planus, Pemphigus, bullous pemphigoid, Epidermolysis bullosa, urticaria, angioedemas, vasculitides, erythemas, cutaneous eosinophilias, Lupus erythematosus, acne and Alopecia areata; various eye diseases (autoimmune and otherwise) such as keratoconjunctivitis, vernal conjunctivitis, uveitis associated with Behcet's disease, keratitis, herpetic keratitis, conical cornea, dystrophia epithelialis corneae, comeal leukoma, ocular pemphigus, Mooren's ulcer, Scleritis, Graves' opthalmopathy, Vogt-Koyanagi-Harada syndrome, sarcoidosis, etc.; pollen allergies, reversible obstructive airway disease, which includes condition such as asthma (for example, bronchial asthma, allergic asthma, intrinsic asthma, extrinsic asthma and dust asthma), particularly chronic or inveterate asthma (for example, late asthma and airway hyper-responsiveness), bronchitis and the like; inflammation of mucous and blood vessels such as gastric ulcers, vascular damage caused by ischemic diseases and thrombosis, ischemic bowel diseases, inflammatory bowel diseases, necrotizing enterocolitis, intestinal lesions associated with thermal burs and leukotriene B4-mediated diseases; intestinal inflammations/allergies such as Coeliac diseases, proctitis, eosinophilic gastroenteritis, mastocytosis, Crohn's disease and ulcerative colitis; food-related allergic diseases which have symptomatic manifestation remote from the gastrointestinal tract migraine, WO 97/16438 PCT/US96/17481 -36rhinitis and eczema); renal diseases such as interstitial nephritis, Goodpasture's syndrome, hemolytic-uremic syndrome and diabetic nephropathy; nervous diseases such as multiple myositis, Guillain-Barre syndrome, Meniere's disease, polyneuritis, multiple neuritis, mononeuritis and radiculopathy; endocrine diseases such as hyperthyroidism and Basedow's disease; hematic diseases such as pure red cell aplasia. aplastic anemia, hypoplastic anemia. idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, agranulocytosis, pernicious anemia, megaloblastic anemia and anerythroplasia; bone diseases such as osteoporosis; respiratory diseases such as sarcoidosis, fibroid lung and idiopathic interstitial pneumonia; skin disease such as dermatomyositis, leukoderma vulgaris, ichthyosis vulgaris, photoallergic sensitivity and cutaneous T cell lymphoma; circulatory diseases such as arteriosclerosis, atherosclerosis, aortitis syndrome, polyarteritis nodosa and myocardosis; collagen diseases such as scleroderma, Wegener's granuloma and Sjogren's syndrome; adiposis; eosinophilic fascitis; periodontal disease such as lesions of gingiva, periodontium, alveolar bone and substantia ossea dentis; nephrotic syndrome such as glomerulonephritis; male pattern alopecia or alopecia senilis by preventing epilation or providing hair germination and/or promoting hair generation and hair growth; muscular dystrophy; Pyoderma and Sezary's syndrome; Addison's disease; active oxygenmediated diseases, as for example organ injury such as ischemiareperfusion injury of organs (such as heart, liver, kidney and digestive tract) which occurs upon preservation, transplantation or ischemic disease (for example, thrombosis and cardiac infraction): intestinal diseases such as endotoxin-shock, pseudomembranous colitis and colitis caused by drug or radiation; renal diseases such as ischemic acute renal insufficiency and chronic renal insufficiency; pulmonary diseases such as toxinosis caused by lung-oxygen or drug (for example, paracort and bleomycins), lung cancer and pulmonary emphysema; ocular diseases such as cataracta, siderosis, retinitis, pigmentosa, senile macular degeneration, vitreal scarring and comeal alkali bum; dermatitis such as erythema multiforme, linear IgA ballous dermatitis and cement WO 97/16438 PCT/US96/17481 -37dermatitis; and others such as gingivitis, periodontitis, sepsis, pancreatitis, diseases caused by environmental pollution (for example, air pollution), aging, carcinogenis, metastasis of carcinoma and hypobaropathy; disease caused by histamine or leukotriene-C4 release; Behcet's disease such as intestinal-, vasculo- or neuro-Behcet's disease, and also Behcet's which affects the oral cavity, skin, eye, vulva, articulation, epididymis, lung, kidney and so on. Furthermore, the compounds of the invention are useful for the treatment and prevention of hepatic disease such as immunogenic diseases (for example, chronic autoimmune liver diseases such as the group consisting of autoimmune hepatitis, primary biliary cirrhosis and sclerosing cholangitis), partial liver resection, acute liver necrosis necrosis caused by toxin, viral hepatitis, shock, or anoxia), B-virus hepatitis, non-A/non-B hepatitis, cirrhosis (such as alcoholic cirrhosis) and hepatic failure such as fulminant hepatic failure, late-onset hepatic failure and "acute-onchronic" liver failure (acute liver failure on chronic liver diseases), and moreover are useful for various diseases because of their useful activity such as augmention of chemotherapeutic effect, preventing or treating activity of cytomegalovirus infection, particularly HCMV infection, and antiinflammatory activity; and The compounds of the present invention may also be used in the treatment of immunodepression or a disorder involving immunodepression, such as AIDS, cancer, senile dementia, trauma (including wound healing, surgery and shock) chronic bacterial infection, and certain central nervous system disorders.

A method of treating a condition in a mammal, the treatment of which is effected or facilitated by Ky 1.3 inhibition, comprising the administration, in an amount that is effective at inhibiting Kv 1.3, of a compound of Formula I. The method of treating a condition in a mammal, the treatment of which is effected or facilitated by Kv1.3 inhibition, wherein the condition is selected from the group consisting of: immunemediated diseases such as the resistance by transplantation of organs or tissue such as heart, kidney, liver, medulla ossium, skin, cornea, lung, pancreas, intestinum tenue, limb, WO 97/16438 PCT/US96/17481 -38muscle, nervus, duodenum, small-bowel, pancreatic-islet-cell, including xeno transplants, etc.; graft-versus-host diseases brought about by medulla ossium transplantation; autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type I diabetes uveitis, juvenileonset or recent-onset diabetes mellitus, posterior uveitis, allergic encephalomyelitis, glomerulonephritis, and the like: and further infectious diseases caused by pathogenic microorganisms. Further uses may include the treatment and prophylaxis of inflammatory and hyperproliferative skin diseases and cutaneous manifestations of immunologically mediated illnesses, such as psoriasis, atopical dermatitis, contact dermatitis and further eczematous dermatitises and further eczematous dermatitises, seborrhoeis dermatitis, Lichen planus, Pemphigus, bullous pemphigoid, Epidermolysis bullosa, urticaria, angioedemas, vasculitides, erythemas, cutaneous eosinophilias, Lupus erythematosus, acne and Alopecia areata; various eye diseases (autoimmune and otherwise) such as keratoconjunctivitis, vernal conjunctivitis, uveitis associated with Behcet's disease, keratitis, herpetic keratitis, conical cornea, dystrophia epithelialis comeae, coreal leukoma, ocular pemphigus, Mooren's ulcer, Scleritis, Graves' opthalmopathy, Vogt-Koyanagi-Harada syndrome, sarcoidosis, etc.; reversible obstructive airway disease, which includes condition such as asthma (for example, bronchial asthma, allergic asthma, intrinsic asthma, extrinsic asthma and dust asthma), particularly chronic or inveterate asthma (for example, late asthma and airway hyperresponsiveness), bronchitis and the like; inflammation of mucous and blood vessels such as gastric ulcers, vascular damage caused by ischemic diseases and thrombosis, ischemic bowel diseases, inflammatory bowel diseases, necrotizing enterocolitis, intestinal lesions associated with thermal burns and leukotriene B4-mediated diseases; intestinal inflammations/allergies such as Coeliac diseases, proctitis, eosinophilic gastroenteritis, mastocytosis, Crohn's disease and ulcerative colitis; food-related allergic diseases which have symptomatic manifestation remote from the gastrointestinal tract migraine, rhinitis and WO 97/16438 PCT/US96/17481 -39eczema); renal diseases such as interstitial nephritis, Good-pasture's syndrome, hemolytic-uremic syndrome and diabetic nephropathy; nervous diseases such as multiple myositis, Guillain-Barre syndrome, Meniere's disease, polyneuritis, multiple neuritis, mononeuritis and radiculopathy; endocrine diseases such as hyperthyroidism and Basedow's disease; hematic diseases such as pure red cell aplasia, aplastic anemia, hypoplastic anemia, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, agranulocytosis, pernicious anemia, megaloblastic anemia and anerythroplasia; bone diseases such as osteoporosis; respiratory diseases such as sarcoidosis, fibroid lung and idiopathic interstitial pneumonia; skin disease such as dermatomyositis, leukoderma vulgaris, ichthyosis vulgaris, photoallergic sensitivity and cutaneous T cell lymphoma; circulatory diseases such as arteriosclerosis, atherosclerosis, aortitis syndrome, polyarteritis nodosa and myocardosis; collagen diseases such as scleroderma, Wegener's granuloma and Sjogren's syndrome; adiposis; eosinophilic fascitis; periodontal disease such as lesions of gingiva, periodontium, alveolar bone and substantia ossea dentis; nephrotic syndrome such as glomerulonephritis; male pattern aleopreia or alopecia senilis by preventing epilation or providing hair germination and/or promoting hair generation and hair growth; muscular dystrophy; Pyoderma and Sezary's syndrome; Addison's disease; active oxygen-mediated diseases, as for example organ injury such as ischemia-reperfusion injury of organs (such as heart, liver, kidney and digestive tract) which occurs upon preservation, transplantation or ischemic disease (for example, thrombosis and cardiac infraction): intestinal diseases such as endotoxinshock, pseudomembranous colitis and colitis caused by drug or radiation; renal diseases such as ischemic acute renal insufficiency and chronic renal insufficiency; pulmonary diseases such as toxinosis caused by lung-oxygen or drug (for example, paracort and bleomycins), lung cancer and pulmonary emphysema; ocular diseases such as cataracta, siderosis, retinitis, pigmentosa, senile macular degeneration, vitreal scarring and corneal alkali bum; dermatitis such as erythema multiforme, linear IgA ballous dermatitis and cement dermatitis; and WO 97/16438 PCT/US96/17481 others such as gingivitis, periodontitis, sepsis, pancreatitis, diseases caused by environmental pollution (for example, air pollution), aging, carcinogenis, metastasis of carcinoma and hypobaropathy; disease caused by histamine or leukotriene-C4 release; Behcet's disease such as intestinal-, vasculo- or neuro-Behcet's disease, and also Behcet's which affects the oral cavity, skin, eye, vulva, articulation, epididymis, lung, kidney and so on. Furthermore, the compounds of the invention are useful for the treatment and prevention of hepatic disease such as immunogenic diseases (for example, chronic autoimmune liver diseases such as the group consisting of autoimmune hepatitis, primary biliary cirrhosis and sclerosing cholangitis), partial liver resection, acute liver necrosis necrosis caused by toxin, viral hepatitis, shock, or anoxia), B-virus hepatitis, non-A/non-B hepatitis, cirrhosis (such as alcoholic cirrhosis) and hepatic failure such as fulminant hepatic failure, lateonset hepatic failure and "acute-on-chronic" liver failure (acute liver failure on chronic liver diseases), and moreover are useful for various diseases because of their useful activity such as augmention of chemotherapeutic effect, preventing or treating activity of cytomegalovirus infection, particularly HCMV infection, and antiinflammatory activity; and immunodepression or a disorder involving immunodepression, such as AIDS, cancer, senile dementia, trauma (including wound healing, surgery and shock), chronic bacterial infection, and certain central nervous system disorders.

An embodiment of the invention is a method for the treatment of autoimmune diseases. Another embodiment of the invention is a method for the prevention of rejection of foreign organ transplants comprising administering to a patient in need of such treatment a therapeutically effective amount of a compound of formula I.

One end result of an autoimmune or a rejection process is tissue destruction caused by inflammatory cells and the mediators they release. Anti-inflammatory agents such as NSAID's and corticosteroids act principally by blocking the effect or secretion of these mediators, WO 97/16438 PCT/US96/17481 -41 but do nothing to modify the immunologic basis of the disease. On the other hand, cytotoxic agents, such as cyclophosphamide, act in such a nonspecific fashion that both the normal and autoimmune responses are shut off. Indeed, patients treated with such nonspecific immunosuppressive agents are as likely to succumb from infection as they are from their autoimmune disease.

Cyclosporin A, which was approved by the US FDA in 1983, is currently the leading drug used to prevent rejection of transplanted organs. The drug acts by inhibiting the body's immune system from mobilizing its vast arsenal of natural protecting agents to reject the transplant's foreign protein. Though cyclosporin A is effective in fighting transplant rejection, it is nephrotoxic and is known to cause several undesirable side effects including kidney failure, abnormal liver function and gastrointestinal discomfort.

Newer, safer drugs exhibiting fewer side effects are constantly being searched for in the field. The present invention provides for immunosuppressant agents which are inhibitors of a voltage dependent potassium channel, Kv1.3, that is found on human T-lymphocytes.

Potassium channels modulate a number of cellular events such as muscle contraction, neuro-endocrine secretion, frequency and duration of action potentials, electrolyte homeostasis, and resting membrane potential. These channels comprise a family of proteins that have been classified according to their biophysical and pharmacological characteristics. Inhibition of K+ channels, in their role as modulators of the plasma membrane potential in human T -lymphocytes, has been postulated to play a role in eliciting immunosuppressive responses. In regulating membrane potential, K+ channels play a role in the regulation of intracellular Ca++ homeostasis, which has been found to be important in T-cell activation. The biochemical characterization of K+ channels is underdeveloped, due to the paucity of selective high affinity probes.

Functional voltage-gated K+ channels can exist as multimeric structures formed by the association of either identical or WO 97/16438 PCT/US96/17481 -42dissimilar subunits. This phenomena is thought to account for the wide diversity of K+ channels. However, subunit compositions of native K+ channels and the physiologic role that particular channels play are, in most cases, still unclear.

The Kv1.3 channel is a voltage-gated potassium channel that is found in neurons, blood cells, osteoclasts and T-lymphocytes.

The Chandy and Cahalan laboratories proposed a hypothesis that blocking the Kv1.3 channel would elicit an immunosuppressant response. (Chandy et al., J. Exp. Med. 160, 369, 1984; Decoursey et al., Nature, 307, 465, 1984). However, the K+ channel blockers employed in their studies were non-selective. Until research with the peptide margatoxin, a peptide found in scorpion venom, no specific inhibitor of the Kv1.3 channel existed to test this hypothesis. Although a laboratory (Price et al., Proc. Natl. Acad. Sci. USA, 86, 10171, 1989) showed that charybdotoxin would block Ky 1.3 in human T cells, charybdotoxin was subsequently shown to inhibit four different K+ channels (Kv 1.3 and three distinct small conductance Ca++ activated K+ channels) in human T-lymphocytes, limiting the use of this toxin as a probe for the physiological role of Kv 1.3 (Leonard et al., Proc. Natl. Acad. Sci. USA, 89, 10094, 1992). Margatoxin, on the other hand, blocks only Kv1.3 in T-cells, and has immunosuppressant activity in both in vitro and in vivo models. (Lin et al., J. Exp. Med, 177, 637, 1993). Since the compounds of the embodiments of this invention produce blockade of Kv1.3, they will also inhibit T-cell activation.

Also within the scope of this invention is a method of treating a condition in a mammal, the treatment of which is effected or facilitated by K 1.3 inhibition, comprising the administration of a pharmaceutical composition comprising a suitable pharmaceutical carrier and a compound of Formula in an amount that is effective at inhibiting Kv1.3.

Also within the scope of this invention is a combination therapy comprising a compound of formula I and one or more immunosuppressant agents. These immunosuppressant agents within the scope of this invention include, but are not limited to, IMUREK® WO 97/16438 PCT/US96/17481 -43 azathioprine sodium, brequinar sodium, SPANIDIN® gusperimus trihydrochloride (also known as deoxyspergualin), mizoribine (also known as bredinin), CELLCEPT® mycophenolate mofetil, NEORAL® Cyclosporin A (also marketed as different formulation of Cyclosporin A under the trademark SANDIMMUNE®), PROGRAF® tacrolimus (also known as FK-506) and RAPIMMUNE® sirolimus (also known as rapamycin), lefhinomide (also known as HWA-486), glucocortcoids, such as prednisolone and its derivatives, antibody therapies such as orthoclone (OKT3) and Zenapax and antithymyocyte globulins, such as thymoglobulins.

Using the methodologies described below, representative compounds of the invention were evaluated and found to exhibit values of at least <10 gM in any of the assays thereby demonstrating and confirming the utility of the compounds of the invention as Kv 1.3 inhibitors and immunosuppressants.

T CELL IL-2 ASSAY Peripheral blood mononuclear (MNC) cells from healthy donors were separated by density centrifugation with ficoll-hypaque (LSM, Organon Teknika, Durham, NC), followed by rosetted with neuraminidase treated sheep red blood cells (SRBC). After another centrifugation with leucocyte separation medium (LSM), the SRBC of the rosetted T cells were then lysed with ammonium chloride lysing buffer (GIBCO, Grand Island, NY). Such purified T cells were resuspended at 3 X 106/ ml in RPMI 1640 culture medium (GIBCO) supplemented with 10% fetal calf serum (Sigma, St. Louis, MO), 100 mM glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, and I penn-strep (GIBCO). The cell suspension was immediately distributed into 96 well round-bottom microculture plates (Costar) at 200 Wl/well. The various dilutions of test compound were then added in triplicate wells at 25 ktl/well, incubated for 30 min at 37°C. lonomycin (125 ng/ml), and PMA (1 or 5 ng/ml), were added to the appropriate wells. The culture plates were then incubated at 37 0 C in a humidified atmosphere of 5% C02 95% air for 18-24 hours. The WO 97/16438 PCT/US96/17481 -44supernatants were removed, and assayed for IL-2 with an IL-2 capture ELISA, using monoclonal anti-IL-2, and biotinylated goat anti-IL-2 antibodies (unconjugated antibodies purchased from R&D System, Minneapolis, MN). The ELISA was developed with streptavidin conjugated peroxidase (Zymed, San Francisco, CA) and substrate for peroxidase (Sigma). Mean OD and units of IL-2 of the replicate wells were calculated from standard curve, created with recombinant IL-2 (Collaborative Biomedical Products, Bedford, MA) and the results were expressed as concentration of compound required to inhibit IL-2 production of T cells by T CELL PROLIFERATION ASSAY Peripheral blood mononuclear cells (MNC) from healthy donors were separated by density centrifugation with ficoll-hypaque (LSM, Organon Teknika, Durham, NC). After washing the MNC with complete media (RPMI 1640 medium with 5% fetal calf serum, 100 mM glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acid, and 1% penn-strep, obtained from GIBCO, Grand Island, NY), they were then irradiated at 7500 RADS, and resuspended at 4-4.5 x 106cells/ml in complete media. Another aliquot of MNC were rosetted with neuraminidase treated SRBC. After another centrifugation with LSM, the sheep red blood cells (SRBC) of these rosetted T cells were then lysed with ammonium chloride lysing buffer (GIBCO, Grand Island, NY). After washing 2X with complete media, these purified T cells were also resuspended at 2-2.5 x 10 6 cells/ml in complete media.

The various dilutions of the compound were added in triplicates at ul/well of a 96 well flat-bottom microculture plate (Costar, Cambridge, MA). T cell suspension was then immediately distributed into the wells at 100 ul/well. After incubating the cells with compound for 30 min. at 37 0 C in a humidified atmosphere of 5% CO2 95% air, 20 pl/well of anti-CD3 (Ortho Diagnostic, NJ) at final conc. of 0.3 ng/ml was added, followed by 50 ptl of the irradiated MNC. The culture plates were then incubated at 37 0 C in a humidified atmosphere of 5% C02 95% air for 72 hours. The proliferation of T lymphocytes was assessed by WO 97/16438 PCT/US96/17481 measurement of tritiated thymidine incorporation. During the last 18- 24 hrs. of culturing, the cells were pulse-labeled with 2 gCi/well of tritiated thymidine (NEN, Cambridge, MA). The cultures were harvested on glass fiber filters using a multiple sample harvester (MACH-II, Wallac,Gaithersburg, MD). Radioactivity of filter discs corresponding to individual wells was measured by standard liquid scintillation counting methods (Betaplate Scint Counter. Wallac). Mean counts per minute of replicate wells were calculated and the results were expressed as concentration of compound required to inhibit tritiated thymidine uptake of T cells by KV1.3-RUBIDIUM EFFLUX ASSAY CHO cells transfected with Kv1.3 channels at site densities of approximately 40,000 sites/cell are plated into 96 well culture plates and maintained in Iscove's Modified Dulbecco's Medium (IMDM, with L-Glutamine and HEPES, JRH Biosciences). Cells are incubated overnight with 86 Rb+ (3 gCi/ml, Dupont-NEN) in the glutamine supplemented IMDM. After aspiration of the media, 100 gl of Low K Buffer (in mM, 6.5 KCI, 125 NaCI, 1 CaCl2, 2 MgCl2, 10 HEPES, pH adjusted to 7.2 with NaOH) is added to each well followed by 100 [l test samples in Low K Buffer also containing 0.2% BSA and 2 mM ouabain.

Samples are tested at either 1 gg/ml for routine screening or at a variety of concentrations encompassing at least 1/10 to 10 times the putative

IC

5 0 of test compound to determine potency. After a fixed preincubation time, which is usually 10 min, the samples are aspirated.

The Ky 1.3 channels are opened by depolarization of the cells with High K Buffer (final concentrations, in mM, 63.25 KCI, 68.25 NaCI, 1 CaCl 2 2 MgCl2, 10 HEPES, pH adjusted to 7.2 with NaOH) also containing test compounds. To measure 86Rb+ efflux through the channels, aliquots of 100 pl are taken from each well after a given time and added to plates containing 100 jil MicroScint-40 (Packard) for counting by liquid scintillation techniques. MicroScint-40 (100 pl) is then added to each well of the cell plate to determine the remaining 86Rb+ activity. The efflux counts are normalized for the total amount of WO 97/16438 PCT/US96/17481 -46- 86Rb+ that was in the cells by adding the efflux counts to the cell plate counts. Activity is determined by inhibition of the efflux window that is established using a saturating concentration of margatoxin (MgTX), a 39 amino acid peptide that is a potent blocker of Ky 1.3 channels (IC 5 0 100 pM).

DOSAGE FORMS As an immunosuppressive, these compounds are useful in the treatment of autoimmune diseases, the prevention of rejection of foreign organ transplants and/or related afflictions, diseases and illnesses.

The compounds of this invention can be administered for the treatment of autoimmune diseases, the prevention of rejection of foreign organ transplants and/or related afflictions, diseases and illnesses according to the invention by any means that effects contact of the active ingredient compound with the site of action in the body of a warmblooded animal. For example, administration, can be oral, topical, including transdermal, ocular, buccal, intranasal, inhalation, intravaginal, rectal, intracisternal and parenteral. The term "parenteral" as used herein refers to modes of administration which include subcutaneous, intravenous, intramuscular, intraarticular injection or infusion, intrasternal and intraperitoneal.

The compounds can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic agents or in a combination of therapeutic agents.

They can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.

For the purpose of this disclosure, a warm-blooded animal is a member of the animal kingdom possessed of a homeostatic mechanism and includes mammals and birds.

The dosage administered will be dependent on the age, health and weight of the recipient, the extent of disease, kind of concurrent treatment, if any, frequency of treatment and the nature of WO 97/16438 PCT/US96/17481 47 the effect desired. Usually, a daily dosage of active ingredient compound will be from about 1-500 milligrams per day. Ordinarily, from 10 to 100 milligrams per day in one or more applications is effective to obtain desired results. These dosages are the effective amounts for the treatment of autoimmune diseases, the prevention of rejection of foreign organ transplants and/or related afflictions, diseases and illnesses.

The active ingredient can be administered orally in solid dosage forms, such as capsules, tablets, troches, drag6es, granules and powders, or in liquid dosage forms, such as elixirs, syrups, emulsions, dispersions, and suspensions. The active ingredient can also be administered parenterally, in sterile liquid dosage forms, such as dispersions, suspensions or solutions. Other dosages forms that can also be used to administer the active ingredient as an ointment, cream, drops, transdermal patch or powder for topical administration, as an ophthalmic solution or suspension formation, eye drops, for ocular administration, as an aerosol spray or powder composition for inhalation or intranasal administration, or as a cream, ointment, spray or suppository for rectal or vaginal administration.

Gelatin capsules contain the active ingredient and powdered carriers, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract.

Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.

In general, water, a suitable oil, saline, aqueous dextrose (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene gycols are suitable carriers for parenteral solutions. Solutions for parenteral administration preferably contain a WO 97/16438 PCT/US96/17481 48 water soluble salt of the active ingredient, suitable stabilizing agents, and if necessary, buffer substances. Antioxidizing agents such as sodium bisulfite, sodium sulfite, or ascorbic acid, either alone or combined, are suitable stabilizing agents. Also used are citric acid and its salts and sodium EDTA. In addition, parenteral solutions can contain preservatives, such as benzalkonium chloride, methyl- or propylparaben. and chlorobutanol.

Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, A. Osol, a standard reference text in this field.

For administration by inhalation, the compounds of the present invention may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or nebulisers. The compounds may also be delivered as powders which may be formulated and the powder composition may be inhaled with the aid of an insufflation powder inhaler device. The preferred delivery system for inhalation is a metered dose inhalation (MDI) aerosol, which may be formulated as a suspension or solution of a compound of Formula I in suitable propellants, such as fluorocarbons or hydrocarbons.

For ocular administration, an ophthalmic preparation may be formulated with an appropriate weight percent solution or suspension of the compounds of Formula I in an appropriate ophthalmic vehicle, such that the compound is maintained in contact with the ocular surface for a sufficient time period to allow the compound to penetrate the corneal and internal regions of the eye.

Useful pharmaceutical dosage-forms for administration of the compounds of this invention can be illustrated as follows:

CAPSULES

A large number of unit capsules are prepared by filling standard two-piece hard gelatin capsules each with 100 milligrams of powdered active ingredient, 150 milligrams of lactose, 50 milligrams of cellulose, and 6 milligrams magnesium stearate.

WO 97/16438 PCT/US96/17481 -49- SOFT GELATIN CAPSULES A mixture of active ingredient in a digestible oil such as soybean oil, cottonseed oil or olive oil is prepared and injected by means of a positive displacement pump into gelatin to form soft gelatin capsules containing 100 milligrams of the active ingredient. The capsules are washed and dried.

TABLETS

A large number of tablets are prepared by conventional procedures so that the dosage unit is 100 milligrams of active ingredient, 0.2 milligrams of colloidal silicon dioxide, 5 milligrams of magnesium stearate, 275 milligrams of microcrystalline cellulose, 11 milligrams of starch and 98.8 milligrams of lactose. Appropriate coatings may be applied to increase palatability or delay absorption.

INJECTABLE

A parenteral composition suitable for administration by injection is prepared by stirring 1.5% by weight of active ingredient in by volume propylene glycol. The solution is made to volume with water for injection and sterilized.

SUSPENSION

An aqueous suspension is prepared for oral administration so that each 5 milliliters contain 100 milligrams of finely divided active ingredient, 100 milligrams of sodium carboxymethyl cellulose, milligrams of sodium benzoate, 1.0 grams of sorbitol solution, U.S.P., and 0.025 milliliters of vanillin.

The same dosage forms can generally be used when the compounds of this invention are administered stepwise or in conjunction with another therapeutic agent. When drugs are administered in physical combination, the dosage form and administration route should be selected depending on the compatibility of the combined drugs. Thus WO 97/16438 PCT/US96/17481 the term coadministration is understood to include the administration of the two agents concomitantly or sequentially, or alternatively as a fixed dose combination of the two active components.

The following examples illustrate the preparation of the compounds of Formula I and as such are not to be considered as limiting the invention set forth in the claims appended hereto.

EXAMPLE 1 A Method Of Extracting The Compounds Of Formula l(a) and 1(b) From Spachea correa H

H

H

b 0CH O Formula 1(a) b is a single H H bond and R is OAc SOH COOCH3 O CH 3CH, OAc Formula 1(b) b is a double 0-CH3 A OAc bond and R is OAc C OAc CH OAc

OR

One gram of an ethanol extract of the roots of Spachea correa was partitioned between 100 ml of hexane (twice) and 100 ml of 90% aqueous methanol. After separation of the phases, the defatted methanol was concentrated down under vacuum to give an aqueous suspension. This was diluted out to 100 ml with water and extracted, with 100 ml of methylene chloride.

The bioactive methylene chloride extract was dried down to give 12 mg of residue. This was first fractionated by preparative thin layer chromatography (TLC) on a 20 cm by 20 cm E. Merck silica gel 60F254 plate of Imm thickness using methylene chloride-ethyl acetate 1:1 as solvent, then by high performance liquid chromatography (HPLC) using a Zorbax RxC8 4.6 mm x 25 cm column, operated at 0 C and eluted with a 50 minute gradient of acetonitrile:water (1:1, WO 97/16438 PCT/US96/17481 -51 v/v) to 100% acetonitrile, delivered at 1 ml/min, to afford 4 mg of compound 1(a) and 1 mg of 1(b).

Homogeneity of the preparations was ascertained in several TLC systems, such as E. Merck silica gel 60F254, methylene chlorideethyl acetate 1:1, Rf 1(a) 0.4, Rf l(b) 0.3; Whatman KC18, methanolwater 9:1, Rf 1(a) 0.65, Rf 1(b) 0.75 and by HPLC using a Zorbax RxC8 column, acetonitrile-water 3:2, k' 1(a) 4.15, k' 1(b) 3.30; and by

NMR.

Mass spectra were recorded on JEOL SX-102A (electron impact, EI,903V) and JEOL HX110 (Fast Atom Bombardment, FAB) mass spectrometers. Exact mass measurements were performed at high resolution (HR-EI) using perfluorokerosene (PFK) as the internal standard. Trimethylsilyl derivatives were prepared with a 1:1 mixture of BSTFA-pyridine at room temperature The FAB spectrum was run in a matrix of dithiothreitol dithioerthritol (20/80).

The compound of Formula 1(a) runs underivatized by El.

The molecular ion is observed a m/z 788 and three successive loses of acetic acid are observed. The base peak is observed a m/z 334. The compound does not silylate. Scanning HR-EI indicated a molecular formula of C40H52016. A table of the critical HR-EI data is given below.

Observed m/z Formula Assignment 788.3220 C40H52016 M+ 728.3040 C38H48014 M-acetic acid 668.2834 C36H44012 M-2 x acetic acid 334.1417 C18H2206 base peak 13 C NMR spectra were recorded for the compound of Formula 1(a) in CD2C12 at 100 MHz on a Varian Unity 400 NMR spectrometer at 20 0 C. Chemical shifts are given in ppm relative to tetramethylsilane (TMS) at zero ppm using the solvent peak at 53.8 ppm as internal standard. The following data were observed: 15.0, 15..2, 16.8, 17.1, 20.7*, 20.9, 21.1, 21.6, 21.8, 22.2, 35.6, 40.8*, 42.1, 43.6, WO 97/16438 PCT/US96/17481 -52- 45.1, 47.5, 49.3*, 53.5, 59.1, 62.6, 63.5, 66.1, 66.7*, 68.4*, 69.9, 73.9, 75.0, 75.6, 77.1*, 119.4, 123.7, 138.9, 143.0, 167.7, 169.2, 169.3*, 170.25, 170.31, 170.8, 171.3 ppm (where the signifies the observation as broad resonances). The carbon count of 40 is in agreement with the molecular formula C40H52016 derived by scanning HR EI-MS.

The 1H NMR spectra of compound of Formula(a) is provided as Figure 1. The spectra was recorded at 400 MHz in CD2Cl? on a Varian Unity 400 NMR spectrometer at 25 0 C. Chemical shifts are in ppm relative to TMS at zero ppm using the solvent peak at 55.32 as the internal standard.

The mass spectra of the compound of Formula 1(b) was obtained as above. The following results were obtained.

Observed m/z Formula Assignment 786.3075 C40H50016 M+ 726.2886 C38H46014 M-acetic acid 666.2651 C36H42012 M-2 x acetic acid 606.2451 C34H38010 M-3 x acetic acid 489.2099 C26H3309 base peak 471.1992 C26H3108 13C NMR spectra were recorded for the compound of Formula l(b) using the procedure described above. The following results were observed: 14.8, 14.9, 17.3, 20.8, 20.9, 21.3, 21.7, 21.8, 21.9, 27.1, 35.1, 40.6, 42.3, 45.4, 48.1, 50.4, 53.5, 54.1, 57.8, 63.7, 66.2, 67.8, 68.6, 71.4, 73.3, 73.8, 74.4, 119.5, 121.1, 124.3, 137.1, 138.9, 143.3, 167.6, 168.6, 169.3, 169.5, 169.9, 171.0, 171.7 ppm.

The carbon count of 40 is in agreement with the molecular formula C40H50016 derived by scanning HR EI-MS.

WO 97/16438 PCT/US96/17481 -53 EXAMPLE 2 A Method Of Extracting The Compounds Of Formula 1(c) And 1(d) From Spachea Correa H

H

H IN b CH O Formula 1(c) b is a single H Oc bond and R is OH OH

COOCH

3 O CH3 CH3 OAc Formula 1(d) b is a double O A bond and R is OH OAc CH OAc

OR

Analogs of the compounds of Formula 1(a) and 1(b) could be detected in the crude extract and fractions thereof when the process of Example 1 was carried out on a larger scale. Thus, 50 g of ethanol extract were partitioned as described in Example 1 using 900 ml of each solvent at each step.

Partial purification of the methylene chloride extract was achieved by column chromatography on E. Merck silica gel 60 (120 ml), eluting with a step gradient of ethyl acetate in methylene chloride.

The step gradient was designed so that the column was washed first with 100% methylene chloride and then with methylene chloride- ethyl acetate mixtures of 9:1, 8:2, 3:2, 2:1, 1:1, 1:2, 2:8 and 1:9. Ultimately the column was washed with 100% ethyl acetate. Fractions eluted with methylene chloride-ethyl acetate 3:2 were enriched in compound of Formula 1(a) and These were resolved by HPLC using a Zorbax RxC8 9 mm x 25 cm column, maintained at 50 0 C and eluted at 4 ml/min with acetonitrile-water 1:1 v/v. Three identical runs finally afforded 100 mg and 20 mg respectively of l(a) and 1(b) after crystallization from methanol. Later-eluting fractions from the silica gel column above were found to contain at least two related compounds based on UV spectra and color reactions on TLC plates. Material from the WO 97/16438 PCT/US96/17481 -54methylene chloride-ethyl actate 1:1 and 1:2 washings were combined and evaporated down. Separation was achieved on the same HPLC column as above, eluting with a 50 minute gradient of 30% to acetonitrile in water. Two identical runs gave 6 mg of purified compound Fractions containing the compound of Formula 1(d) were again processed by HPLC (same column) using acetonitrile-water 3:7 delivered isocratically, to yield 2 mg of purified Formula 1 (d) The mass spectra of these compounds were recorded on a Finnigan TSQ700 mass spectrometer (electrospray ionization, ESI).

The samples were analyzed by LC/MS using a 2.1xl50mm CS column at 0.2ml/min. with a mobile phase of 45% acetonitrile/0.01M aqueous ammonium acetate at 50 0 C. Component 1(d) had a retention time of 10.5 min. and a molecular weight of 744 which is observed a m/z: 745 762 (M+NH3), 786 (M H MeCN). Component l(c) has a retention time of 11.8 and a molecular weight of 746 which is observed at m/z: 747 764 (M+NH3) and 788 (M H MeCN).

The 13 C NMR spectra obtained for the compound of Formula 1(c) using the conditions previously described is as follows: 15.1 16.9, 19.8, 20.8, 20.91, 20.94, 21.9, 22.3, 35.6, 40.6, 42.2, 43.9, 45.0, 47.7, 50.8, 53.5, 55.6, 61.8, 63.5, 66.0, 67.6 69.8, 70.0, 73.9, 75.0, 75.6, 119.3, 123.7, 139.0, 144.4, 167.8, 169.2, 169.5, 170.1, 170.4, 171.4 ppm.

The carbon count of 38 is in agreement with the molecular formula C38H50016 derived by scanning HR EI-MS.

EXAMPLE 3 Separation By HPLC Compounds of this invention were characterized by the following behavior during HPLC separation on a Zorbax RxC8 4.6 mm x 25 cm column, maintained at 50°C and eluted at 1 ml/min with acetonitrile-water 3:2 v/v): Compound k' 4.15; k'=3.30; k'=2.30; k'=2.10.

WO 97/16438 PCT/US96/17481 Analyses using this HPLC system can be used to quantify the compounds in the crude extract or other mixtures, by comparing the absorbance of HPLC peaks at a wavelength of 220 nm with that produced by injections of known (weighed) amounts of pure standards.

EXAMPLE 4 Additional Purification Procedure A simplified purification process allows for rapid fractionation of even larger amounts of crude extract and the preparation of gram amounts of the compounds of Formula 1(a) and 1(b).

The ethanol extract is first dissolved at 20 grams per 150 ml in methanol. This solution is diluted with 150 ml of water and then extracted three times with methylene chloride using 150 ml of methylene chloride each time. The pooled methylene chloride extracts are evaporated down and fractionation proceeds by repeated column chromatography on silica gel. One employs methylene chloridemethanol 97:3 in a first step; the mixed compounds of Formula and 1(b) thus obtained are resolved by chromatographing on fresh silica gel eluted with methylene chloride-ethyl acetate 3:1. Volume of elution for the compound of Formula 1 ranges from about 2 to about 3.5 column volumes of solvent; that for the compound of Formula 1 is about 3 to about 4.5 column volumes. Finally, advantage is taken of the low solubility of these compounds, and, after total resolution by chromatography, the compounds of Formula 1 and 1 can be precipitated and or crystallized from concentrated methanol solutions.

WO 97/16438 PCT/US96/17481 -56- EXAMPLE 4,6,7,15,16-Pentakis(acetyloxy)- 18-hydroxy-22-methoxycarbonyl- [6,7a, 15 3,16 3]D:A-Friedo-A-homo-27,30-dinor-24-oxaoleana- 1, 20(29).21 -trien-3-one H OH CO2CH3 Ol OAc O OAc OAc AcO

A

A solution of 233 mg (0.56 mmole) of tungsten hexachloride in 8 mL of dry tetrahydrofuran was cooled to -78°C under nitrogen. Then 0.70 mL (1.12 mmole) of 1.6 M butyllithium was added and the solution was allowed to warm to room temperature over min. Then a solution of 111 mg (0.141 mmole) of 4,6,7,15,16pentakis(acetyloxy)-21,22-epoxy-18-hydroxy-22-methoxycarbonyl- [6a,7a,153,16p,210,22p]D:A-Friedo-A-homo-27,30-dinor-24oxaoleana-l,20(29)-dien-3-one in 2 mL of dry tetrahydrofuran was added and the solution was heated under nitrogen at 55°C for 14 h. The mixture was applied to a 10 cm column of silica gel, which was washed with 2:1 ethyl acetate-hexane. The eluate was concentrated and purified by silica gel chromatography with 2:1 ethyl acetate-hexane to afford mg of the title compound as a white solid; 1H NMR (CDC13) 6 7.10 1H, C21-H); Mass Spectrum (APCI) 790 (M+NH4).

WO 97/16438 WO 9716438PCTIUS96/1 7481 57 EXAMPLE 6 4-(2-Bromobenzoyl)oxy-6,7, 15,1 6-tetrakis(acetyloxy)- 1 8-hydroxy-22methoxycarbonyl [6(x,7cc, 1 50,1 6f]D :A-Friedo-A-homo-27 ,30-dinor-24oxaoleana- 1 ,20(29),21 -trien-3-one QAc GAc Step A: 6,7,15,1 6-Tetrakis(acetyloxy)-2 1,22-epoxy-4, 18dihydroxy-22-methoxycarbonyl- [6(x,7ux, 1 503,1 6P,21 1,2213]D:A-Friedo- A-homo-27 .30-dinor-24-oxaoleana- 1 .20(29)-dien-3 -one 0

CO

2

CH

3 I II GAc Ao Ac A solution of 102.1 mg 130 mmole) of 4,6,7,15,16pentakis(acetyloxy)-2 1,22-epoxy- I 8 -hydroxy-22-methoxycarbonyl.

WO 97/16438 WO 9716438PCTIUS96/1 7481 58 [6(x,7a, 1513,1613,21 1,221]D:A-Friedo-A-homo-27,30-dinor-24oxaoleana- 1,20(29)-dien-3-one in 4 mL of tetrahydrofuran and 2 mL of 3M aqueous HI was heated at 40*C for 24h. The solution was diluted with dichioromethane and the layers were separated. The organic layer was washed with 0. 1 M phosphate buffer (pH then was dried over MgSO4 and concentrated. The residue was purified by silica gel chromatography with 2:1 ethyl acetate-hexane to afford 44,9 mg of the title compound as a white solid IH NMR (CDCl3) 8 4.20 1H, J 4.3 Hz, C4-H); Mass Spectrum (APCI): m/e 764 (M+NI-4).

Step B: 4-(2-Bromobenzoyl)oxy-6 15,1 6-tetrakis(acetyloxy)- 21,22-epoxy- 1 8-hydroxy-22-methoxycarbonyl- [6ax,7Q, 1 51,1613,21 J,2213D:A-Friedo-A-homo-27,30dinor-24-oxaoleana- 1 .20(29)-dien-3-one 0 H O H C0 2 0H 3 0 ~OA~c SOAc Ac Br0 0 To a solution of 17.5 mg (23.5 g~mole) of 6,7,15,16tetrakis(acetyloxy)-2 1,22-epoxy-4, 1 9-dihydroxy-22-methoxycarbonyl- [6cx,7ax, 15 P, 1613,21 1,22f3]D:A-Friedo-A-homo-27,30-dinor-24-.

oxaoleana- 1,20(29)-dien-3 -one in 0.5 mL pyridine was added 27.5 mL (237 pgmole) of benzoyl chloride. The solution was stirred at room temperature for 4 h, then was concentrated under reduced pressure.

WO 97/16438 WO 97/ 6438PCTIUS96/1 7481 59 The residue was first filtered through a plug of silica gel and then purified by HPLC (Waters RCM, [t Porosil, 10 mm X 10 cm) using a mixture of 9.6:6 (5:4:1 hexane-methyl tert-butyl etheracetonitrile:hexane) to afford 17.3 mg of the title compound as a white solid; 11H NMR 8 5.67 (1 H, C4-H), 7.40-7.43 (in, 2H), 7.72 (dd, I1H, J 6.9 Hz), 7.78 (dd, I1H, J 6.9 Hz); Mass Spectrum (APCD': m/e 946, 948 7 9 Br-M+NH-4, 8 1 Br-M+NH,4).

Step C: 4-(2-Bromobenzoyl)oxy-6,7, 15,1 6-tetrakis(acetyloxy)- 18hydroxy-22-methoxycarbonyl- [6ac,7(, 1 5P ~1 61]D :A- Friedo-A-homo-27 ,30-dinor-24-oxaoleana- 1 ,20(29),2 1trien-3-one H O H 00 2

CH

3 0 OA~c O, Ac bAc

OAC

Br 1

A

0 A solution of 218 mg (0.55 mmole) of tungsten hexachioride in 8 ruL of dry tetrahydrofuran was cooled to -78'C under nitrogen. Then 0.69 mL 10 inmole) of 1 .6M butyllithium was added and the solution was allowed to warm to room temperature over min. Then a solution of 50.2 mg (0.141 mmole) of 4-(2bromobenzoyloxy)-6,7,15,l 6-tetrakis(acetyloxy)-2 1,22-epoxy- 18hydroxy-22-methoxycarbonyl[6x7, 1 5P, 1 60,2 10f,223] D :A-Friedo-Ahomo-27,3 0-dinor-24-oxaoleana- 1 ,20(29)-dien-3 -one in 2 mL of dry tetrahydrofuran was added and the solution was heated under nitrogen at WO 97/16438 WO 9716438PCT/US96/17481 60 for 14 h. The mixture was washed with 2.5 MNaOH and brine, then was dried over MgSO4. The filtrate was purified by HPLC (Waters RCM, p. Porosil, 25mm x 10cm) using a 8.9:4 mixture of (5:4:1 hexane:methyl tert-butyl ether: acetonitrile):hexane to afford 31.3 mg of the title compound as a white solid; 1H NMR (CDC13) 8 5.67 (1 H, C4-H), 7. 10 I1H, C21 7.40-7.43 (in, 2H), 7.72 (dd, I1H, J 2.2, 6.9 Hz). 7.78 (dd. 1H. J 2.3. 6.9 Hz); Mass Spectnim (APCI) m/e 930, 932 7 9 Br-M+NH4, 8 1 Br-M+NH4).

EXAMPLE 7 4,6,7,15,1 6-Pentaki s(acetylox 21,22 -epoxy- I 8-hydroxy-22methoxycarbonyl [6a,7x, 153,1 6fPD :A-Friedo-A-homo-27 ,30-dinor-24oxaoleana- 1,20(29),21 -triene H OH 0 2

CH

3 QAc 0Q OAc AcO' Ip" Step A: 4,6,7,15,1 6-Pentakis(acetyloxy)-2 1,22-epoxy- I 8-hydroxy- 22-methoxycarbonyl [6uc,7cc, 1 503,16j3,21 P,22f3]D:A-Friedo- A-homo-27,30-dinor-24-oxaoleana- 1 .20(29)-dien-3 -ol WO 97/16438 PCT/US96/17481 -61 0 H OH C0 2

CH

3 HO OAc S',n

OAC

OOAc AcO A solution of 3.0 g (3.8 mmole) of 4,6,7,15,16pentakis(acetyloxy)-21,22-epoxy-18-hydroxy-22-methoxycarbonyl- [6a,7a, 15p3,160,21 ,22p]D:A-Friedo-A-homo-27,30-dinor-24oxaoleana-l,20(29)-dien-3-one in 20 mL of dry dichloromethane was cooled to 0°C under nitrogen. Then 9 mL of a 1M solution of lithium tri-(tert-butoxy)aluminum hydride was added dropwise and the solution was stirred at 0'C. After 18 h, the reaction was quenched by dropwise addition of 20 mL of 2M aqueous H2S04 and the mixture was diluted with 200 mL of ether. The layers were separated and the aqueous layer was washed with two 100 mL portions of ether. The organic layers were sequentially washed with 20 mL of 2M aqueous H2S04 and brine, then were combined, dried over MgSO4, and concentrated to afford 2.9 g of the title compound, which was used directly in the next step.

WO 97/16438 PCT/US96/17481- -62- Step B: 4,6,7,15,16-Pentakis(acetyloxy)-21,22-epoxy-18-hydroxy- 22-methoxycarbonyl [6a,7a, 153,163,21 P,22p]D:A-Friedo- A-homo-27,30-dinor-24-oxaoleana-1.20(29)-diene H OH C0 2

CH

OAc OAc OAc AcO A sample of 2.9 g of crude 4,6,7,15,16-pentakis(acetyloxy)- 21,22-epoxy-1 8-hydroxy-22-methoxycarbonyl- [6a,7a, 15P, 16p,210,22p]D:A-Friedo-A-homo-27,30-dinor-24oxaoleana-l,20(29)-dien-3-ol was dissolved in 10 mL of dry dichloromethane under nitrogen. To this was added 10 mL of triethylsilane, and the solution was stirred at room temperature for min. Then 2 mL (20 mmole) of boron trifluoride etherate was added and the mixture was stirred at room temperature for 15 min. The reaction was quenched by addition of 10 mL of saturated aqueous KHCO3 solution and the resulting mixture was partitioned between ether and water. The water layer was washed with ether and the organic extracts were washed with brine, then were combined, dried over MgSO4, and concentrated. The residue was purified by chromatography on silica gel using 30% ethyl acetate-hexane to afford 2.13 g of the title compound as a white solid; 1 H NMR (CDC13) 4.14, 4.34 (dd, AB, 2H, J 12 Hz, C3-H); Mass Spectrum (APCI) m/e 792 (M+NH4).

WO 97/16438 PCT/US96/17481 -63 Step C: 4,6,7,15,16-Pentakis(acetyloxy)- 18-hydroxy-22-methoxycarbonyl[6a,7a, 15P, 16p]D:A-Friedo-A-homo-27,30-dinor- 24-oxaoleana- 1.20(29),21 -triene SOH CO 2

CH

3 OAc OAc OAc AcO Ac A solution of 119 mg (0.300 mmole) of tungsten hexachloride in 4 mL of dry tetrahydrofuran was cooled to -78°C under nitrogen. Then 0.38 mL (0.61 mmole) of 1.6M butyllithium was added and the solution was allowed to warm to room temperature over min. A solution of 34.6 mg (0.045 mmole) of 4,6,7,15,16pentakis(acetyloxy)-21,22-epoxy-I 8-hydroxy-22-methoxycarbonyl- [6a,7a, 15p,16p,21 ,22p]D:A-Friedo-A-homo-27,30-dinor-24oxaoleana-1,20(29)-diene in 2 mL of dry tetrahydrofuran was added and the solution was heated under nitrogen at 50'C for 14 h. The mixture was diluted with 20 mL ether, and the mixture was washed with M NaOH and brine, dried over MgSO4, and concentrated. The residue was purified by HPLC (Waters RCM, p. Porosil, 25mm x using a 6.75:7.0 mixture of (5:4:1 hexane:methyl tert-butyl ether:acetonitrile):hexane to to afford 27 mg of the title compound as a white solid; 1H NMR (CDC13) 5 1H, C21-H); Mass Spectrum (APCI) 776 (M+NH4).

WO 97/16438 WO 9/1 438PCTIUS96/17481 64 EXAMPLE 8 4,6,7,15,1 6-Pentakis(acetyloxy)-2 1,22-epoxy- I 8-hydroxy-22methoxycarbonyl [6&x,7Qx, 1 5P~,1 6P,21 f,22P] D: A-Friedo-A-homo-27,30dinor-24-oxaoleana-20(29)-en-3 -one

IH

24 OAc OAc OAc As described in Scheme 1, 4,5,6,15,16-pentakis(acetyloxy)- 21,22-epoxy- I 8-hydroxy-22-methoxycarbony 7cc, 1513, 16P3, 21J3, 2213] D:A-Freido-A-homo-27 ,30-dinor-24-oxaoleana- 1 ,20(29)-diene-3one, isolated from Spachea correa in liquid ammonia with lithium metal will result in the reduction of the ClI olefin group to produce the saturated lactone.

EXAMPLE 9 4,6,7,15,1 6-Pentakis(acetyloxy)- 1 8-hydroxy-22-methoxycarbonyl- [6ux,7u, 1513,1 61]D:A-Friedo-A-homo-27,30-dinor-24ox aoleana-20(29) .21I -dien-3 -one WO 97/16438 PCT/US96/17481 H OH

CO

2

C

H

3 0 OAc O ",OAqAc OAc AcO OAc OAc AcO A solution of 233 mg (0.56 mmole) of tungsten hexachloride in 8 mL of dry tetrahydrofuran is cooled to -78 C under nitrogen. Then 0.70 mL (1.12 mmole) of 1.6M butyllithium was added and the solution is allowed to warm to room temperature over 30 min.

Then a solution of 111 mg (0.141 mmole) of 4,6,7,15,16pentakis(acetyloxy)-21,22-epoxy-18-hydroxy-22methoxycarbonyl [6a,7, 1503,163,21 P,223]D:A-Friedo-A-homo-27,30dinor-24-oxaoleana-20(29)-en-3-one in 2 mL of dry tetrahydrofuran is added and the solution is heated under nitrogen at 55C for 14 h. The mixture is applied to a 10 cm column of silica gel, which is washed with 2:1 ethyl acetate-hexane. The eluate is concentrated and purified by silica gel chromatography with 2:1 ethyl acetate-hexane to produce the title compound.

EXAMPLE 4-(2-Bromobenzoyl)oxy-6,7,15,16-tetrakis(acetyloxy)- 18-hydroxy-22methoxycarbonyl[6a,7a, 153P,16p]D:A-Friedo-A-homo-27,30-dinor-24oxaoleana-20(29).21-dien-3-one WO 97/16438 WO 9716438PCT/US96/17481 66 OAc Step A: 6,7,15,1 6-Tetrakis(acetyloxy)-2 1,22-epoxy-4, 18dihydroxy-22-methoxycarbonyl[6x,7cx, 15$,1 6f,21 j3,22p] D :A-Friedo-A-homo-27 ,30-dinor-24-oxaoleana-20(29)-en- 3-one I _OAc OAc H O,

A

A solution of 102.1 mg (0.130 mmnole) of 4,6,7,15,16- 1 0 pentakis(acetyloxy)-2 1,22-epoxy- I 8-hydroxy-22-methoxycarbonyl- [6x,7c, 1 5P, 1 6f,21 J,22J3ID:A-Friedo-A-homo-27,3O-dinor-24.

oxaoleana-20(29)-en-3 -one in 4 mL of tetrahydrofuran and 2 mL of 3M aqueous HCI is heated at 40 0 C for 24h. The solution is diluted with dichloromethane and the layers are separated. The organic layer is washed with 0.I1M phosphate buffer (pH then is dried over MgSO4 WO 97/16438 WO 9716438PCT/US96/1 7481 67 and concentrated. The residue is purified by silica gel chromatography with 2:1 ethyl acetate-hexane to produce the title compound.

Step B: 4-(2-Bromobenzoyl)oxy-6,7, 15,1 6-tetrakis(acetyloxy)- 21,22-epoxy- I 8-hydroxy-22-methoxycarbonyl- [6ci,7o 153,16P,21 I,22fP]D:A-Friedo-A-homo-27,3Odinor-24-oxaoieana -20(29 -en- 3 -orie 0 00 2

CH

3 To a solution of 17.5 mg (23.5 tmole) of 6, 7, 15, 16tetrakis(acetyloxy)-2 1,22-epoxy-4, 1 8-dihydroxy-22-methoxycarbonyl- [6a,7ux, 1 50,1 60,21 1,22j3]D:A-Friedo-A-homo-27,30-dinor-24oxaoleana-20(29)-en-3 -one in 0.5 mL pyridine is added 27.5 mL (237 jimole) of benzoyl chloride, The solution is stirred at room temperature for 4 h, then is concentrated under reduced pressure. The residue is first filtered through a plug of silica gel and then purified by HPLC (Waters RCM, ji Porosil, 10 mm X 10 cm) using a mixture of 9.6:6 (5:4:1 hexane-methyl tert-butyl ether-acetonitrile :hexane) to produce the title compound.

WO 97/16438 WO 9716438PCTIUS96/17481 68 Step C: 4-(2-Bromobenzoyl)oxy-6 15,1 6-tetrakis(acetyloxy)- 18hydroxy-22-methoxycarbonyl [6ux,7u, 1 5P3, 1 6P]D:A-Friedo- A-homo-27.30-dinor-24-oxaoleana-20(29),2 1 -dien-3-one H O H 00 2 0H 3 0 O A c OAc

'OAC

Br >9__IAc K 0 A solution of 218 mg (0.55 mmole) of tungsten hexachioride in 8 mL of dry tetrahydrofuran is cooled to -78'C under nitrogen. Then 0.69 mL 10 mmole) of 1.6M butyllithium is added and the solution is allowed to warm to room temperature over 30 min.

Then a solution of 50.2 mg (0.141 mmole) of 4-(2-bromobenzoyl)oxy- 6,7,15,1 6-pentakis(acetyloxy)-21 ,22-epoxy- 1 8-hydroxy-22methoxycarbonyl-[6(x,7uc, 1 5P3, 16f3,21 f,22P]D:A-Friedo-A-homo-27,30dinor-24-oxaoleana-20(29)-en- 3-one in 2 mL of dry tetrahydrofuran is added and the solution is heated under nitrogen at 55*C for 14 h. The mixture is applied to a 10 cm column of silica gel, which is washed with 2:1 ethyl acetate-hexane. The eluate is concentrated and purified by silica gel chromatography with 2:1 ethyl acetate-hexane to produce the title compound.

Claims (7)

1. A compound of structural Formula I: 29 C 12 H18 2 2 1 H 9 H3 OH 2 pO 2 CH 3 2 14 16 3 b 10 25 8 15 OAc X 6 2 6 OAC O 24 6 OAc E OAc OAc 23 a R I or a pharmaceutically acceptable salt, crystal form or hydrate, wherein: Xis: O,S,NHor H andR 1 a is: a single bond, or a double bond when R 4 is absent; b and c are independently: a single bond or a double bond; n is: 1 to 4; m is: 1 to 4; r is: 0 or 1; sis: 0or 1; R1 and R 2 are independently: a) H, or b) (Cl-C6)-alkyl, wherein alkyl is unsubstituted or substituted with one, two or three substituents selected from the group WO 97/16438 PCT/US96/17481 consisting of: Br, Cl, F, I, (C1-C6)-alkoxy, vinyl, cyano, oxo, nitro, hydroxy, CHO, CO2H, COCI-C6-alkyl, CO2C 1-C6-alkyl, CONR 1R 2 NR 1R 2 NR COC -C6-alkyl, aryl, wherein aryl is defined as phenyl or naphthyl, unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F. 1. (C -C6)-alkoxy. phenyl, phenoxy. cyano. nitro, hydroxy, CHO, CO2H, COCl-C6-alkyl, CO2C1-C6-alkyl, CONR 1 R 2 NR R 2 NR 1 COC1-C6-alkyl and any two of adjacent substituents can be joined to form a 6- or 7- membered fused ring said ring containing 1 or 2 oxygen atoms and the remainder carbon atoms, heteroaryl, wherein heteroaryl is defined as a 5 or 6-membered ring substituted with one and two heteroatoms selected from O, S, N, unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (C1-C6)-alkoxy, cyano, nitro, hydroxy, CHO, CO2H, COC -C6-alkyl, CO2C 1-C6-alkyl, CONR 1R 2 NR 1R 2 NR 1 COC -C6-alkyl, any two adjacent substituents can be joined to form a 6- or 7-membered fused ring said ring containing 1 or 2 oxygen atoms and the remainder carbon atoms, or any two adjacent substituents can be joined together to form a benzo-fused ring; R 3 is: a) -(C1-C6)-alkyl, alkyl as defined above; b) -(CI-C6)-alkenyl, wherein alkenyl is unsubstituted or substituted with one, two or three substituents selected from from the group consisting of: Br, Cl, F, I, (C1-C6)-alkoxy, cyano, oxo, nitro, hydroxy, CHO, CO2H, COC1-C6-alkyl, CO2C 1-C6-alkyl, CONR 1R 2 NR 1 R 2 NR 1COC 1-C6-alkyl, aryl as defined above, and heteroaryl as defined above; c) -(CI-C6)-alkynyl, wherein alkynyl is unsubstituted or substituted with one, two or three substituents selected from WO 97/16438 WO 97/ 6438PCTIUS96/1 7481 71 the group consisting of: Br, Cl, F, 1, (CI-C6)-alkoxy, cyano, oxo, nitro, hydroxy, CHO, CO2H, COC I-C6-alkyl, C02C 1 -C6-alkyl, CONR I R 2 NR I R 2 NR 1 COC I -C6-alkyl, aryl as defined above, and heteroaryl as defined above, d) -aryl, aryl as defined above, or e) -heteroaryl, heteroaryl as defined above; R 4 is: a) absent and a is a double bond; b) -H, c) -OH, d) =0, e) -O[(C=O)Or]sCi-Cio-alkyl, alkyl as defined above, f) -O[(C=O)Or]sC2-Cl1 -alkenyl, as defined above, g) -O[(C=O)OrsC2-C6-alkynyl, alkynyl as defined above, h) -OI(C0)Orls(C3-C7)-cycloalkyl, i) -O[(C=O)Orlsaryl, aryl as defined above, j) -O[(C0)Orlsheteroaryl, heteroaryl as defined above, k) -O(CH2)nO(CH2)mheteroaryl, heteroaryl as defined above, 1) -O(CH2)nO(CH2)maryl, aryl as defined above, m) -OC(=O)NR I R 2 n) -OSO2R 3 or o) -NR 1 R 2 WO 97/16438 PCT/US96/17481 -72-

2. The compound of structural Formula I, as recited in Claim 1, 29 1 H 19 211 c 12 118 22 1 11 17 1 H 9 H13 OH 2 pO 2 CH, 3 b 10 25 8 15 OAc X 24 6 26 OAc OAc OAc 23 a R I or a pharmaceutically acceptable salt, crystal form or hydrate, wherein: X is: O, S, or NH; a is: a single bond; b and c are independently: a single bond or a double bond; n is: 1 to 4; m is: 1 to 4; r is: 0 or 1; sis: 0 or 1; R 1 and R 2 are independently: a) H, or b) (Cl-C6)-alkyl, wherein alkyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (Cl-C6)-alkoxy, vinyl, cyano, I WO 97/16438 PCT/US96/17481 -73 oxo, nitro, hydroxy, CHO, CO2H, COC1-C6-alkyl, CO2C -C6-alkyl, CONR 1R 2 NR R 2 NRI COC 1-C6-alkyl, aryl, wherein aryl is defined as phenyl or naphthyl, unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (Cl-C6)-alkoxy, phenyl, phenoxy, cyano, nitro, hydroxy,HHO. CO2H COCI-C6-alkyl, CO2C1-C6-alkyl, CONR 1 R 2 NR1R 2 NR 1 COC1-C6-alkyl and any two of adjacent substituents can be joined to form a 6- or 7- membered fused ring said ring containing 1 or 2 oxygen atoms and the remainder carbon atoms, heteroaryl, wherein heteroaryl is defined as a 5 or 6-membered ring substituted with one and two heteroatoms selected from O, S, N, unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (C1-C6)-alkoxy, cyano, nitro, hydroxy, CHO, CO2H, COC -C6-alkyl, CO2C I-C6-alkyl, CONR IR 2 NR 1 R 2 NR 1 COC I-C6-alkyl, any two adjacent substituents can be joined to form a 6- or 7-membered fused ring said ring containing 1 or 2 oxygen atoms and the remainder carbon atoms, or any two adjacent substituents can be joined together to form a benzo-fused ring; R 3 is: a) 1-C6)-alkyl, alkyl as defined above; b) -(Ci-C6)-alkenyl, wherein alkenyl is unsubstituted or substituted with one, two or three substituents selected from from the group consisting of: Br, Cl, F, I, (C1-C6)-alkoxy, cyano, oxo, nitro, hydroxy, CHO, CO2H, COC -C6-alkyl, CO2C -C6-alkyl, CONR 1R 2 NR 1R 2 NR 1 COC -C6-alkyl, aryl as defined above, and heteroaryl as defined above; c) -C6)-alkynyl, wherein alkynyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (C1-C6)-alkoxy, WO 97/16438 WO 9716438PCT[US96/17481 74 cyano, oxo, nitro, hydroxy, CHO, CO2H, COC I -C6-alkyl, C02C I -C6-alkyl, CONR I R 2 NR I R 2 NR 1 COC I -C6-alkyl, aryl as defined above, and heteroaryl as defined above, d) -aryl, aryl as defined above, or e) -heteroaryl, heteroaryl as defined above; R 4 is: a) absent and a is a double bond; b) -H, c) -OH, d) =0, e) -OI(C0O)Orl sC I-C I -alkyl, alkyl as defined above, f) -O[(C0)Or]sC2-CO-alkenyl, as defined above, g) -Ojj(C=O)Or]sC2-C6-alkynyl, alkynyl as defined above, h) -O(C0)Orls(C3-C7)-cycloalkyl, i) -O(C0O)Orlsaryl, aryl as defined above, j) -O[(C0)Orsheteroaryl, heteroaryl as defined above, k) -O (CH2)nO(CH2)mheteroaryl, heteroaryl as defined above, 1) -O(CH2)nO(CH2)maryl, aryl as defined above, m) -OC(=O)NRI R 2 n) -OSO2R 3 or o) -NR 1 R 2 WO 97/16438 PCT/US96/17481

3. The compound of structural Formula I, as recited in Claim 2, 11 1 I U OAc OAc or a pharmaceutically acceptable salt, crystal form or hydrate, wherein: Xis: a is: 0; a single bond; b and c are independently: a single bond or a double bond; n is: m is: r is: s is: R 1 and R 2 a) b) 1 to 4; 1 to 4; 0or 1; 0or 1; are independently: H, or (C1-C6)-alkyl, wherein alkyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (Cl-C6)-alkoxy, vinyl, cyano, WO 97/16438 PCT/US96/17481 -76- oxo, nitro, hydroxy, CHO, CO2H, COC1-C6-alkyl, CO2C 1-C6-alkyl, CONR1R2, NRR 2 NRR 2 N 1 COC -C6-alkyl, aryl, wherein aryl is defined as phenyl or naphthyl, unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (C1-C6)-alkoxy, phenyl, phenoxy, cyano, nitro, hydroxy. CHO. COH. COC -C6-alkyl. CO2Cl-C6-alkvl. CONR 1 R 2 NR 1 R 2 NR 1 COC1-C6-alkyl and any two of adjacent substituents can be joined to form a 6- or 7- membered fused ring said ring containing 1 or 2 oxygen atoms and the remainder carbon atoms, heteroaryl, wherein heteroaryl is defined as a 5 or 6-membered ring substituted with one and two heteroatoms selected from O, S, N, unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (C1-C6)-alkoxy, cyano, nitro, hydroxy, CHO, CO2H, COC -C6-alkyl, CO2C -C6-alkyl, CONR 1R 2 NR 1R 2 NR 1 COC I-C6-alkyl, any two adjacent substituents can be joined to form a 6- or 7-membered fused ring said ring containing 1 or 2 oxygen atoms and the remainder carbon atoms, or any two adjacent substituents can be joined together to form a benzo-fused ring; R 3 is: a) -(C1-C6)-alkyl, alkyl as defined above, b) -aryl, aryl as defined above, or c) -heteroaryl, heteroaryl as defined above; R 4 is: a) -O[(C=O)Or]sC1-Co1-alkyl, alkyl as defined above, b) -O[(C=O)Or]s(C3-C7)-cycloalkyl, c) -O[(C=O)Or]saryl, aryl as defined above, d) -O[(C=O)Or]sheteroaryl, heteroaryl as defined above, e) -O(CH2)nO(CH2)mheteroaryl, heteroaryl as defined above, C WO 97/16438 PCT/US96/17481 -77- f) -O(CH2)nO(CH2)maryl, aryl as defined above, g) -OC(=O)NRl R 2 or h) -OS02R 3

4. The compound of structural Formula I, as recited in Claim 3, or a pharmaceutically acceptable salt, crystal form or hydrate, wherein: R 4 is: a) -O[(C=O)Or]saryl, wherein aryl is defined as phenyl or naphthyl, unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (C1-C6)-alkoxy, phenyl, phenoxy, cyano, nitro, hydroxy, CHO, CO2H, COC1-C6-alkyl, CO2C1-C6-alkyl, CONR 1 R 2 NR 1 R 2 NR 1 COCl-C6-alkyl and any two of adjacent substituents can be joined to form a 6- or 7- membered fused ring said ring containing 1 or 2 oxygen atoms and the remainder carbon atoms, or b) -O[(C=O)Or]sheteroaryl, wherein heteroaryl is defined as a or 6-membered ring substituted with one and two heteroatoms selected from O, S, N, unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (Cl-C6)-alkoxy, cyano, nitro, hydroxy, CHO, CO2H, COCl-C6-alkyl, CO2C 1-C6-alkyl, CONR IR 2 NR IR 2 NR 1COC I-C6-alkyl, any two adjacent substituents can be joined to form a 6- or 7-membered fused ring said ring containing 1 or 2 oxygen atoms and the remainder carbon atoms, or any two adjacent substituents can be joined together to form a benzo-fused ring. WO 97/16438 PCT/US96/17481 78 The compound of structural Formula I, as recited in Claim 1, OAc SOAc 23 a R 4 OAc or a pharmaceutically acceptable salt, crystal form or hydrate, wherein: X is: a is: H and R1; a single bond; b and c are independently: a single bond or a double bond; n is: m is: r is: s is: R 1 and R 2 a) b) 1 to 4; 1 to 4; 0or 1; 0or 1; are independently: H, or (Cl-C6)-alkyl, wherein alkyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (Cl-C6)-alkoxy, vinyl, cyano, WO 97/16438 PCT/US96/17481 -79- oxo, nitro, hydroxy, CHO, CO2H, COC -C6-alkyl, CO2C -C6-alkyl, CONR IR 2 NR 1R 2 NR 1 COCI -C6-alkyl, aryl, wherein aryl is defined as phenyl or naphthyl, unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (C1-C6)-alkoxy, phenyl, phenoxy, cyano, nitro, hydroxy, CHO, CO2H, COC!-C6-alkyl. CO2Cl-C6-alkyl, CONR 1 R 2 NR 1 R 2 NRICOC1-C6-alkyl and any two of adjacent substituents can be joined to form a 6- or 7- membered fused ring said ring containing 1 or 2 oxygen atoms and the remainder carbon atoms, heteroaryl, wherein heteroaryl is defined as a 5 or 6-membered ring substituted with one and two heteroatoms selected from O, S, N, unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (C1-C6)-alkoxy, cyano, nitro, hydroxy, CHO, CO2H, COC -C6-alkyl, CO2C -C6-alkyl, CONR 1R 2 NR 1R 2 NR ICOC1-C6-alkyl, any two adjacent substituents can be joined to form a 6- or 7-membered fused ring said ring containing 1 or 2 oxygen atoms and the remainder carbon atoms, or any two adjacent substituents can be joined together to form a benzo-fused ring; R 3 is: a) -(Cl-C6)-alkyl, alkyl as defined above; b) -(Cl-C6)-alkenyl, wherein alkenyl is unsubstituted or substituted with one, two or three substituents selected from from the group consisting of: Br, Cl, F, I, (CI-C6)-alkoxy, cyano, oxo, nitro, hydroxy, CHO, CO2H, COC -C6-alkyl, CO2C I-C6-alkyl, CONR R 2 NR R 2 NR 1 COC1 -C6-alkyl, aryl as defined above, and heteroaryl as defined above; c) -(C1-C6)-alkynyl, wherein alkynyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (CI-C6)-alkoxy, WO 97/16438 WO 97/ 6438PCT/US96/1 7481 cyano, oxo, nitro, hydroxy, CHO, CO2H, COC I -C6-alkyl, C02C I -C6-alkyl, CONR I R 2 NR I R 2 NR 1 COC 1 -C6-alkyl, aryl as defined above, and heteroaryl as defined above, d) -aryl, aryl as defined above, or e) -heteroaryl, heteroaryl as defined above; R 4 is: a) absent and a is a double bond; b) -H, c) -OH, d) =0, e) -OL(C=O)Or]sCi -Clo-alkyl, alkyl as defined above, f) -O[(C0)Or]sC2-CO-alkenyl, as defined above, g) -OI(C=O)Or]sC2-C6-alkynyl, alkynyl as defined above, h) -O(C0O)Or] s(C3-C7)-cycloalkyl, i) -O[(C=O)Orlsaryl, aryl as defined above, j) -O sheteroaryl, heteroaryl as defined above, k) -O(CH2)nO(CH2)mheteroaryl, heteroaryl as defined above, 1) -O(CH2)nO(CH2)maryl, aryl as defined above, m) -OC(=O)NR I R 2 n) -OSO2R 3 or o) -NR 1 R 2 WO 97/16438 PCT/US96/17481 -81

6. The compound of structural Formula I, as recited in Claim 2 O 2 H 3 OAc I or a pharmaceutically acceptable salt, crystal form or hydrate, wherein: X is: a is: H and R 1 a single bond; b and c are independently: a single bond or a double bond; n is: m is: r is: s is: R 1 and R 2 a) b) 1 to 4; 1 to 4; 0or 1; 0or 1; are independently: H, or (Cl-C6)-alkyl, wherein alkyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (C1-C6)-alkoxy, vinyl, cyano, WO 97/16438 PCT/US96/17481

82- oxo, nitro, hydroxy, CHO, CO2H, COC I-C6-alkyl, CO2C -C6-alkyl, CONR R 2 NR 1R 2 NR 1COC -C6-alkyl, aryl, wherein aryl is defined as phenyl or naphthyl, unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (C1-C6)-alkoxy, phenyl, phenoxy, cyano, nitro, hydroxy. CHO, CO2H. COCI-C6-alkyl, C02C1-C6-alkyl CONR 1 R 2 NR 1 R 2 NR 1 COC1-C6-alkyl and any two of adjacent substituents can be joined to form a 6- or 7- membered fused ring said ring containing 1 or 2 oxygen atoms and the remainder carbon atoms, heteroaryl, wherein heteroaryl is defined as a 5 or 6-membered ring substituted with one and two heteroatoms selected from O, S, N, unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (C1-C6)-alkoxy, cyano, nitro, hydroxy, CHO, CO2H, COC 1 -C6-alkyl, CO2C -C6-alkyl, CONR 1R 2 NR 1 R 2 NR 1COC 1-C6-alkyl, any two adjacent substituents can be joined to form a 6- or 7-membered fused ring said ring containing 1 or 2 oxygen atoms and the remainder carbon atoms, or any two adjacent substituents can be joined together to form a benzo-fused ring; R 3 is: a) -(Cl-C6)-alkyl, alkyl as defined above; b) -(CI-C6)-alkenyl, wherein alkenyl is unsubstituted or substituted with one, two or three substituents selected from from the group consisting of: Br, Cl, F, I, (C1-C6)-alkoxy, cyano, oxo, nitro, hydroxy, CHO, CO2H, COCl-C6-alkyl, CO2C1 -C6-alkyl, CONR 1 R 2 NR 1R 2 NR 1COC 1-C6-alkyl, aryl as defined above, and heteroaryl as defined above; c) -(Cl-C6)-alkynyl, wherein alkynyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (Cl-C6)-alkoxy, WO 97/16438 WO 97/ 6438PCTIUS96/17481 83 cyano, oxo, nitro, hydroxy, CHO, CO2H, COC1-C6-alkyl, C02C I -C6-alkyl, CONk 1 I R 2 NR I R 2 NR I COC I -C6-alkyl, aryl as defined above, and heteroaryl as defined above, d) -aryl, aryl as defined above, or e) -heteroaryl, heteroaryl as defined above; p 4 a) -OH, b) -O[(C=O)OrsC1-CO-alkyl, alkyl as defined above, c) -OII(C=O)Orls(C3-C7)-cycloalkyl, d) -Or(C=o)orlsaryl, aryl as defined above, e) -O[(C=O)Orlsheteroaryl, heteroaryl as defined above, f) -O(CH2)nO(CH2)mheteroaryl, heteroaryl as defined above, g) -O(CH2)nO(CH2)maryl, aryl as defined above, h) -OC(=O)NRI R 2 or i) -0S02R 3 7. A compound selected from the group consisting of: 4,6,7,15,1 6-pentakis(acetyloxy)- 1 8-hydroxy-22-methoxycarbonyl. [6cc,7ax, 1i5P, 1 6f3]D :A-Friedo-A-homo-27 ,30-dinor-24-oxaoleana- 1 ,20(29),21 -trien-3-one; 4 -(2-bromobenzoyl)oxy-6 ,7,15, 16-tetrakis(acetyloxy)- 1 8-hydroxy-22- methoxycarbonyl [6cx,7cz, 15Sf, 1 6f3D:A-Friedo-A-homo-27 ,30-dinor-24- oxaoleana- 1 ,20(29),2 1 -trien-3 -one; 4,6,7,15,1 6-pentakis(acetyloxy)- 1 8-hydroxy-22-methoxycarbonyl- [6ux,7u, 15Pf, 1 6f3]D :A-Fiedo-A-homo-27 30 -dinor-24-oxaoleana- 1 ,20(29),2 1 -triene; 4,6,7,15,1 6-pentakis(acetyloxy)-2 1,22-epoxy- I 8-hydroxy-22- methoxycarbonyl [6Q,7ux, 1 3,1 6f,21 f,22f3D:A-Friedo-A-homo-27,30- dinor-24-oxaoleana-20(29)-en-3 -one; 4,6,7,15,16-pentakis(acetyloxy)-18-hydroxy-22-methoxycarbonyl-[6a,7a,15,16p]D:A-Friedo-A- homo-27,30-dinor-24-oxaoleana-20(29),21-dien-3-one; 4-(2-bromobenzoyl)oxy-6,7,15,16-tetrakis (acetyloxy)-18-hydroxy-22-methoxycarbonyl-[6a,7a, 1 5,1 61]D:A-Friedo-A-homo-27,30-dinor-24- oxanleana-20(29),21-dien-3-one: 6,7,15,16-tetrakis(acetyloxy)-21,22-epoxy-4,18-dihydroxy-22- methoxycarbonyl[6a,7a,15p,16p,21fp,22p]D:A-Friedo-A-homo-27,30-dinor-24-oxaoleana-20(29)-en- 3-one; and 4-(2-bromobenzoyl)oxy-6,7,15,16-tetrakis(acetyloxy)-21,22-epoxy-18-hydroxy-22-methoxy carbonyl[6a,7a,1 5p,16p,21p1,22p]D:A-Friedo-A-homo-27,30-dinor-24-oxaoleana-20(29)-en-3-one. 8. An immunosuppressant triterpene derivative, substantially as hereinbefore described with reference to any one of the examples. 9. A pharmaceutical formulation comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of the compound of formula I, as recited in any one of claims 1 to 8 or a pharmaceutically acceptable crystal form or hydrate thereof. The pharmaceutical formulation of claim 9, comprising in addition, a second S. immunosuppressive agent comprising azathioprine, brequinar sodium, deoxyspergualin, nizaribine, S. 15 mycophenolic acid morpholino ester, cyclosporin, FK-506 and rapamycin. 11. A method treating a condition in a mammal, the treatment of which is effected or facilitated by Kv1.3 inhibition, comprising the administration, in an amount that is effective at inhibiting Kv1.3, of a compound of Formula I. 12. The method of treating a condition in a mammal the treatment of which is effected or 20 facilitated by Kv1.3 inhibition, as recited in claim 11, wherein the condition is selected from the group consisting of: resistance by transplantation of organs or tissue, graft-versus-host diseases brought about by medulla ossium transplantation; rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type I diabetes uveitis, juvenile-onset or recent-onset diabetes mellitus, posterior uveitis, allergic encephalomyelitis, glomerulonephritis, infectious diseases caused by pathogenic microorganisms, inflammatory and hyperproliferative skin diseases, psoriasis, atopical dermatitis, contact dermatitis, eczematous dermatitises, seborrhoeis dermatitis, Lichen planus, Pemphigus, bullous pemphigoid, Epidermolysis bullosa, urticaria, angioedemas, vasculitides, erythemas, cutaneous eosinophilias, Lupus erythematosus, acne, Alopecia areata, keratoconjunctivitis, vernal conjunctivitis, uveitis associated with Behcet's disease, keratitis, herpetic keratitis, conical cornea, dystrophia epithelialis corneae, corneal leukoma, ocular pemphigus, Mooren's ulcer, Scleritis, Graves' opthalmopathy, Vogt-Koyanagi-Harada syndrome, sarcoidosis, etc.; pollen allergies, reversible obstructive airway disease, bronchial asthma, allergic asthma, intrinsic asthma, extrinsic asthma and dust asthma, chronic or inveterate asthma, late asthma and airway hyperresponsiveness, bronchitis, gastric ulcers, vascular damage caused by ischemic diseases and thrombosis, ischemic bowel diseases, inflammatory bowel diseases, necrotising enterocolitis, intestinal lesions associated with thermal burns and leukotriene B 4 -mediated diseases, Coeliac diseases, proctitis, eosinophilic gastroenteritis, mastocytosis, Crohn's disease, ulcerative colitis, migraine, rhinitis, eczema, interstitial nephritis, Good-pasture's syndrome, hemolytic-uremic syndrome, diabetic nephropathy, multiple myositis, Guillain-Barre syndrome, Meniere's disease, 4o;pplyneuritis, multiple neuritis, mononeuritis, radiculopathy, hyperthyroidism, Basedow's disease, pure LIBC/03635 86 diabetes uveitis, juvenile-onset or recent-onset diabetes mellitus, posterior uveitis, allergic encephalomyelitis, glomerulonephritis, infectious diseases caused by pathogenic microorganisms, inflammatory and hyperproliferative skin diseases, psoriasis, atopical dermatitis, contact dermatitis, eczematous dermatitises, seborrhoeis dermatitis, Lichen planus, Pemphigus, bullous pemphigoid, Epidermolysis bullosa, urticaria, angioedemas, vasculitides, erythemas, cutaneous eosinophilias, Lupus erythematosus, acne, Alopecia areata, keratoconjunctivitis, vernal conjunctivitis, uveitis associated with Behcet's disease, keratitis, herpetic keratitis, conical cornea, dystrophia epithelialis corneae, corneal leukoma, ocular pemphigus, Mooren's ulcer, Scleritis, Graves' opthalmopathy, Vogt- Koyanagi-Harada syndrome, sarcoidosis, etc.; pollen allergies, reversible obstructive airway disease, bronchial asthma, allergic asthma, intrinsic asthma, extrinsic asthma and dust asthma, chronic or inveterate asthma, late asthma and airway hyperresponsiveness, bronchitis, gastric ulcers, vascular damage caused by ischemic diseases and thrombosis, ischemic bowel diseases, inflammatory bowel diseases, necrotising enterocolitis, intestinal lesions associated with thermal burns and leukotriene B 4 mediated diseases, Coeliac diseases, proctitis, eosinophilic gastroenteritis, mastocytosis, Crohn's 15 disease, ulcerative colitis, migraine, rhinitis, eczema, interstitial nephritis, Good-pasture's syndrome, hemolytic-uremic syndrome, diabetic nephropathy, multiple myositis, Guillain-Barre syndrome, Meniere's disease, polyneuritis, multiple neuritis, mononeuritis, radiculopathy, hyperthyroidism, Basedow's disease, pure red cell aplasia, aplastic anemia, hypoplastic anemia, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, agranulocytosis, pernicious anemia, 20 megaloblastic anemia, anerythroplasia, osteoporosis, sarcoidosis, fibroid lung, idiopathic interstitial *pneumonia, dermatomyositis, leukoderma vulgaris, ichthyosis vulgaris, photo allergic sensitivity, cutaneous T cell lymphoma, arteriosclerosis, atherosclerosis, aortitis syndrome, polyarteritis nodosa, myocardosis, scleroderma, Wegener's granuloma, Sjogren's syndrome, adiposis, eosinophilic fascitis, lesions of gingiva, periodontium, alveolar bone, substantia ossea dentis, glomerulonephritis, male pattern alopecia or alopecia senilis by preventing epilation or providing hair germination and/or promoting hair generation and hair growth; muscular dystrophy; Pyoderma and Sezary's syndrome, Addison's disease, ischemia-reperfusion injury of organs which occurs upon preservation, transplantation or ischemic disease, for example, thrombosis and cardiac infraction, endotoxin-shock, pseudomembranous colitis, colitis caused by drug or radiation, ischemic acute renal insufficiency, chronic renal insufficiency, toxinosis caused by lung-oxygen or drug, for example, paracort and bleomycins, lung cancer, pulmonary emphysema, cataracta, siderosis, retinitis, pigmentosa, senile macular degeneration, vitreal scarring, corneal alkali burn; dermatitis erythema multiforme, linear IgA ballous dermatitis and cement dermatitis, gingivitis, periodontitis, sepsis, pancreatitis, diseases caused by environmental pollution, aging, carcinogenis, metastasis of carcinoma and hypobaropathy; disease caused by histamine or leukotriene-C4 release; Behcet's disease, autoimmune hepatitis, primary biliary cirrhosis sclerosing cholangitis, partial liver resection, acute liver necrosis, necrosis caused by toxin, viral hepatitis, shock, or anoxia, B-virus hepatitis, non-A/non-B hepatitis, cirrhosis, alcoholic cirrhosis, hepatic failure, fulminant hepatic failure, late-onset hepatic failure, "acute-on- chronic" liver failure, augmention of chemotherapeutic effect, preventing or treating activity of .ytomegalovirus infection, HCMV infection, and antiinflammatory activity; and treatment of /LIBC/13635 immunodepression or a disorder involving immunodepression, including AIDS, cancer, senile dementia, trauma, chronic bacterial infection, and certain central nervous system disorders which comprises the administration of a compound of any one of claims 1 to 8 or of a formulation of claim 9 or claim 19. A method of treating a condition in a mammal, the treatment of which is effected or facilitated by Kv1.3 inhibition, comprising the administration of a pharmaceutical formulation comprising a pharmaceutical carrier and a compound of formula I, in an amount that is effective at inhibiting Kv1.3. A method of treating a condition in a mammal, the treatment of which is effected or facilitated by Kv1.3 inhibition, comprising the coadministration of a therapeutically effective amount of a compound of formula I, as recited in any one of claims 1 to 8, with a second immunosuppressive agent. Dated 28 May, 1998 Merck Co., Inc. 15 Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON LIBC/03635 q '4 j ITERNATIONAL SEARCH REPORT International application No. PCTIUS96/17481 A. CLASSIFICATION OF SUBJECT MATTER IPC(6) :CO7D 313/06; A61K 31/365 US CL :514/183; 549/266, 268 According to International Patent Classification (IPC) or to both national classification and IPC B. FIELDS SEARCHED Minimum documentation searched (classification system followed by classification symbols) U.S. 514/183; 549/266, 268 Documentation searched other than muuimumn documentation to the extent that such documents are included in the fields searched Electronic data base consulted during the international search (name of data base and, where practicable, search terms used) CAS C. DOCUMENTS CONSIDERED TO DE RELEVANT Category's Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No. A US, A, 4,453,967 (MORI) 12 June 1984, see entire 1-19 document. Y Phytochemistry, Vol. 29, No. 7, issued 1990, ABREU et al.; 1-19 "A Nor-triterpenoid From Lophanthera Lactescens," pages

2257-2261, see pages 2257-2261. E Further documents are listed in the continuation of Box C. See patent family annex. Special categories of cited documnenta: *T lae document published after the itemiational filing dat o Opriority daoe aed no" i conflict with the application hut cited to undertand the 'A documemidefining the smerai state of the an which ia not considered priciple or theory underlying the invmntion to be of particular relevance earie doumet ublshe o oraftr he ntenaionl flig dte X. document of pailicular relevance; the claimed invention cannot be E erlir dcuentpubishd n o afertheintrnaioal ~*-considered noelo cannot be considered to involve an inventive step -L document which may throw doubts on priority claim(s) or which is when the documenti taken alone cited to establish the publication date of another citation or other Y special reaso (as specified) Y doctiment of paticular relevance; the claimed invention cannot be considered to involve an inventive step whe th docmn is 0. document referring to an oral diaclosuare. uae. exhibition or other combined with one or more other such documents, such combination being obvious to a petson skilled in the art document published prior to the international filing date but later than document meber of the same patent family the priority date claimed Date of the actual completion of the international search Date of mailing of the international search report 17 JANUARY 1997 0 5 MAR 197 Name and mailing address of the ISA/US Authorized officer Commnissioner of Patents and Trademaurks Y Box PCT P.K.WPt'X aco Washington, D.C. 20231 Facsimile No. (703) 305-3230 Telephone No. (703) 308-1235 Form PCT/15AJ210 (second sheet)(July 1992)* .4 INTERNATIONAL SEARCH REPORT International application No. S PCTIUS96/17481 Box I Observations where certain claims were found unsearchable (Continuation of item 1 of first sheet) This international report has not been established in respect of certain claims under Article 17(2Xa) for the following reasons: 1. O Claims Nos.: because they relate to subject matter not required to be searched by this Authority, namely: 2. O[ Claims Nos.: because they relate to parts of the international application that do not comply with the prescribed requirements to such an extent that no meaningful international search can be carried out, specifically: 3. Claims Nos.: because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a). Box II Observations where unity of invention is lacking (Continuation of item 2 of first sheet) This International Searching Authority found multiple inventions in this international application, as follows: Please See Extra Sheet. 1. E As all required additional search fees were timely paid by the applicant, this international search report covers all searchable claims. 2. D As all searchable claims could be searched without effort justifying an additional fee, this Authority did not invite payment of any additional fee. 3. L As only some of the required additional search fees were timely paid by the applicant, this international search report covers only those claims for which fees were paid, specifically claims Nos.: 4. No required additional search fees were timely paid by the applicant. Consequently, this international search report is restricted to the invention first mentioned in the claims; it is covered by claims Nos.: Remark on Protest D The additional search fees were accompanied by the applicant's protest. D No protest accompanied the payment of additional search fees. Form PCT/ISA/210 (continuation of first sheet(l))(July 1992)* INTERNATIONAL SEARCH REPORT International application No. PCT/US96/17481 BOX II. OBSERVATIONS WHERE UNITY OF INVENTION WAS LACKING This ISA found multiple inventions as follows: I. Claims 1-10, 13, 14, 18 and 19; drawn to compounds, pharmaceutical formulations and method of treating a condition in a mammal facilitated by K, 13 inhibitor. II. Claims 11, 16, 17 drawn to method of preventing or treating resistance of transplantation, classified in Class 514, subclass 183. III. Claims 12, 15 drawn to method of suppressing immune system; with coadministration of second agent, classified in Class 514, subclass 183. The Groupings of the invention lack unity of invention because Groups I, II and III are drawn to different methods of use. If one of the methods is found in prior art, it would not make a reference to other methods, 37 CFR 1.475(d). The different methods of use have different etiologies, involving different cascade systems. The claims, iterfntio, arconcit. so lined by a specl lnic featu wh ti m g f PCT Rui 13.2u As foZm a il invention concept. Form PCT/ISA/210 (extra shect)(July 1992)*