AU712015B2 - Triterpene derivatives with immunosuppressant activity - Google Patents

Description

WO 97/16068 PCT/US96/17211 -1- 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 I

'A

WO 97/16068 PCT/US96/17211 -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 H Formula 1(a) b is a single H bond and R is OAc b CH O Formula 1(b) b is a double H H bond and R is OAc OH 'COOCH3 O CH CH 3 OAc Formula 1(c) b is a single 0 A bond and R is OH OAc CH3 OAcOAc

S

OR 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(d) and which have the relative stereochemistry depicted above.

11l WO 97/16068 PCT/US96/17211 -3- SUMMARY OF THE INVENTION This invention relates to a class of triterpene derivatives of the general structural Formula I

R

2 9 a R 29 b C 12 i 19 21 11 17 2 1 H 9 H 1OH 6 2

O

2

CH

3 3 b 10 2 5 1 15 OAc O 2 4 6 OAC ,a OAc 23 a 4

I

are useful as immunosuppressives.

As an immunosuppressive, 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 immunosuppressive agent 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 A1 WO97/16068 PCT/US96/17211 -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 comea, dystrophia epithelialis coreae, coreal 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 bums 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 97/16068 PCT/US96/17211 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 drug, 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:

R 9a R 29 b 2 1 H9 14 H1 28 0 2

CH

3 O~c E4 a R or a pharmaceutically acceptable salt, crystal form or hydrate, wherein: X is: 0, S, NEor Hand R1; a is: a single bond, or a double bond when R 4 isabsent; b andecare .independently: a single bond or a double bond; n is: 1 to4; n is: I to 4; .:96 s or 1; 10 s is: 0Oorl1;

R

1 is: H, or

(CI-C

6 )-alkyl, wherein alkyl. is unsubstituted or substituted with one, two 0* **or three sub stituents selected from the group consisting of: Br, Cl, F, 1,

(CI-C

6 )-alkoxy, vinyl, cyano, oxo, nitro, hydroxy, CHO, CO 2 H, COCI-C 6 alkyl, C0 2

CI-C

6 -alkyI, CONRaR 2 NRIaR 2 NR laCOCI -C 6 -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, (Ci-C 6 )-alkoxy, phenyl., phenoxy, cyano, nitro, hydroxy, CHO, CO 2 H, COCI-C 6 -alkyl, CO 2

CI-C

6 -alkyl, CONRlaR 2 NR~aR 2 NIR ICOC I-C 6 -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 0, S, N, unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, 1, (CI-C 6 )-alkoxy, cyano, nitro, hydroxy, CHO, CO 2 H, COCI-C 6 -alkyl,

CO

2

CI-C

6 -alkyl, CONR aR, NRa R, NRlaCoCl-C 6 alkyl, any two [R:\LIBAA]07569.doc TAB 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

1 and R 2 are independently; a) (Ci-C 6 )-alkyl, or b) aryl, wherein aryl is phenyl or naphthyl.

R

3 is: a) -(CI-C 6 )-alkyl, alkyl as defined above; b) -(CI-C6)-alkenyl, wherein alkenyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (Ci-C 6 )-alkoxy, cyano, oxo, nitro, hydroxy, CHO, CO 2 H, COC 1

-C

6 alkyl, CO 2 C1-C 6 -alkyl, CONRIaR 2 NRlaR 2 NRlaCOCi-C6-alkyl, aryl as ~defined above, and heteroaryl as defined above; 15 c) -(Ci-C 6 )-alkynyl, wherein alkynyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (Ci-C 6 )-alkoxy, cyano, oxo, nitro, hydroxy, CHO, CO 2 H, COC 1

-C

6 i alkyl, CO 2 C1-C 6 -alkyl, CONRaR 2 NRlaR 2 NRlaCOCi-Co-alkyl, aryl as defined above, and heteroaryl as defined above, i4: 20 d) -aryl, aryl as defined above, or e) -heteroaryl, heteroaryl as defined above; wherein R 1 and R 2 are as defined above.

R

4 is: a) absent and a is a double bond; 25 b) -H, c) -OH, d)

=O,

e) -O[(C=O)Or]sCi-Clo-alkyl, alkyl as defined above, f) -O[(C=0)Or]sC 2 -Clo-alkenyl, as defined above, g) -O[(C=O)Or]sC 2 -C6-alkynyl, alkynyl as defined above, h) -O[(C=0)O]s(C3-C 7 )-cycloalkyl, i) -O[(C=0)Or]saryl, aryl as defined above, j) -O[(C=O)Or]sheteroaryl, heteroaryl as defined above, k) -O(CH2)nO(CH 2 )mheteroaryl, heteroaryl as defined above, 1) -O(CH2)nO(CH 2 )maryl, aryl as defined above, m) -OC(=O)NRlaR 2 [R:\LIBAA]07569.doc:TAB 8 n1) -0S0 2 or o) -NR aR2; wherein Ria and R 2 are as defined above.

R

29 a and R 2 Ob are independently: a) -H, c) -(CH 2

),NR

1

R

2 d) -(CH2)s0-[(C=0)r]sCClio-alkyl, alkyl as defined above, e) -(CH2)sO[(C=0)Or]C 2 -Clo.alkenyl, alkenyl as defined above, f) -(CH2)s0-[(C=0)r],aryl, aryl as defined above, g) -(CH2),-0[(C=0)c0r],heteroaryl, heteroaryl as defined above, h) -(CH2)s;-0(CH2)nO0(CH 2 )mheteroaryl, heteroaryl as defined above, i) -(CH2)s0(CH 2 )nO(CH 2 ),aryl, aryl as defined above, see*:j)

-(CH

2 )r-0C(=o)NR'p 2 k) -(CH 2 ),-0S0 2

R',

-(CI-C6)-alkyl, alkyl as defined above, or M) -(C2-C 6 )-alkenyl, alkenyl as defined above; *0or R21 and R29 can be taken together to be =0 or =C(Ci-Cjoalkyl) 2 alkyl being as defined above.

20 An embodiment of the invention are the compounds of Formula I c 1 128 0 2

CH

3 0 24560~ 4 OAc a R or a pharmaceutically acceptable salt, crystal form or hydrate, wherein: X is: 0, S, NH; a is: a single bond; b and care independently: a single bond or a double bond; n is: Ilto 4; In is: 1lto 4; [R:\LIBAA]07569 doc:TAB ris: 0 or l; sis: 0 or 1; R' is: a) H, or s b) (C 1

I-C

6 )-alkyl, wherein alkyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (Ci-C 6 )-alkoxy, vinyl, cyano, oxo, nitro, hydroxy, CHO, CO 2 H, COCI-C 6 alkyl, CO 2 C1-C 6 -alkyl, CONRaR2, NRlaR 2 NRlaCOC-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, (Ci-C6)-alkoxy, phenyl, phenoxy, cyano, nitro, hydroxy, CHO, CO 2 H, COCi-C 6 -alkyl, CO 2 C1-C 6 -alkyl, CONRIaR 2 NRIlaR 2 NR IaCOCi-C 6 -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 1 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 0, S, N, unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (CI-C 6 )-alkoxy, cyano, nitro, hydroxy, CHO, CO 2 H, COC 1

-C

6 -alkyl,

CO

2 C1-C 6 -alkyl, CONRIaR 2 NRlaR 2 laCOCi-C 6 -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 Sbenzo-fused ring; 25 wherein R' and R 2 are as defined above.

R

3 is: a) -(CI-C 6 )-alkyl, alkyl as defined above; b) -(CI-C 6 )-alkenyl, wherein alkenyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (CI-C 6 )-alkoxy, cyano, oxo, nitro, hydroxy, CHO, CO 2 H, COCI 1

-C

6 alkyl, C0 2

C

1

-C

6 -alkyl, CONRIaR 2 NRlaR 2 NRlaCOC-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 the group consisting of: Br, Cl, F, I, (C1-C 6 )-alkoxy, cyano, oxo, nitro, hydroxy, CHO, C0 2 H, COCI-C 6 [R:\LIBAA]07569.doc TAB a.

a a..

a.

a a..

a a a a a a a.

alkyl, CO 2

CI-C

6 -alkyl, CONRaR 2 NRIaR 2 NRlaCOCi

-C

6 .alkyl, aryl as defined above, and heteroaryl as defined above, d) -aryl, aryl as defined above, or e) -heteroaryl, heteroaryl as defined above; wherein R la and R 2 are as defined above.

R' is: a) absent and a is a double bond, b) -H, c) -OH, d) =0O, e) O0[(C=0)rjsCj-Coalkyl, alkyl as defined above, 0) O[(C=O)OrjsC 2 -Cio-alkenyl, as defined above, g) -O[(C=O)OrJsC 2

-C

6 .alkynyl, alkynyl as defined above, h) O0[(C=O)Or1,s(C 3

-C

7 )..cycloalkyl, i) O0[(C=O)Orlsaryl, aryl as defined above, j) -O[(C=O)Or]sheteroaryl, heteroaryl as defined above, k) -O(CH2)nO(CH 2 heteroaryl, heteroaryl as defined above, 1) -O(CH2)nO(CH 2 )maryl, aryl as defined above, Mn) -OC(=O)NR 1 R2, n) -0S0 2 R or o) -NRlIaR 2 wherein R la and R 2 are as defined above.

R

29 a and R 2 9 b are independently: a) -H, b) -(CH2)s-O[(C=O)OrjsCi-Cio-alkyl, alkyl as defined above, c) -(CH2)s0[I(CzrO)Or]sC 2 -C o-alkenyl, alkenyl as defined above, d) -(CH2)s-O[(C=O)Or],C 2

-C

6 -alkynyl, alkynyl as defined above, e) -(CH2)s-O[(C=0)0Q1,(C 3

-C

7 )-cycloalkyl, f) -(CH2)s0O[(C0)OrsarYl, aryl as defined above, g) -(CH2),s-O[(C=oyOr]sheteroaryl, heteroaryl as defined above, h) -(CH2)s-O(CH2)nO(CH 2 )mheteroaryl, heteroaryl as defined above, i) -(CH2)s-O(CH 2 )nO(CH 2 ),aryl, aryl as defined above, j) -(CH2),;-OC(=O)NR 1

R

2 k) -(CH 2

)S-OSO

2

R

3 1) I-C 6 )-alkyl, alkyl as defined above, or M) -(C 2

-C

6 )-alkenyl, alkenyl as defined above; 25 [R:\4IBAA]07569.docTAB or R 29a and R 29 b can be taken together to be =0 or =C(Ci-Co1alkyl) 2 alkyl as defined above.

An embodiment of this embodiment of the invention are the compounds of Formula I 9 2 R29a 2 9 b 12 H18 172J 2 1 H OH 28 02CH 3 9 1OH b- 3 1025 6 15 OAc 0, 24 6A OAc OAc -4 OAc E4 23 4 a R

I

or a pharmaceutically acceptable salt, crystal form or hydrate, wherein: Xis:

O;

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: Oorl; R is: a) H, or b) (Ci-C 6 )-alkyl, wherein alkyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (Ci-C 6 )-alkoxy, vinyl, cyano, oxo, nitro, hydroxy, CHO, CO 2 H, COCI-C 6 alkyl, C0 2

C

1

-C

6 -alkyl, CONRlaR 2 NRlaR 2 NRlaCOC

-C

6 -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, (Ci-C 6 )-alkoxy, phenyl, phenoxy, cyano, nitro, hydroxy, CHO, CO 2 H, COCi-C 6 -alkyl, CO 2 C1-C 6 -alkyl, CONRIaR 2 NRlaR 2 NR aCOCi-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, [R:\LIBAA]07569.doc:TAB

M

two or three substituents selected from the group consisting of: Br, Cl, F, 1, (CI-C 6 )-alkoxy, cyano, nitro, hydroxy, CHO, CO 2 H, COCI-C 6 -alkyl, C0 2

CI-C

6 -alkyl, CONR~aR 2 NRIaR 2 IPRIaCoCI -C 6 -alkyl, any two adjacent substituents can be joined to form a 6- or 7-membered fused s 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-ftised ring; wherein R laand R 2 are as defined above.

R 3 is: a) I-C 6 )-alkyl, alkyl as defined above, b) -aryl, aryl as defined above, or c) -heteroaryl, heteroaryl as defined above.

R

4 is: a) -0[(C=0)r]sC-Clo-alkyl, alkyl as defined above, 15 b) O0[(C0)Orl,(C 3

-C

7 )-cycloalkyl, c) -O[(C=O)Or.]saryl, aryl as defined above, d) -O[(C=O)Or]sheteroaryl, heteroaryl as defined above, e *O C 2 n H h t r a y h t r a y s d f n d a o e e) 0O(CH 2 )nO(CH 2 )mhraryl, traryl as defined above, g) -OC(=O)NR R or -0S0 2 R :::wherein Rlaand R 2 are as defined above.

R29 and R 2 9 b are independently:

-H,

25 b) H C O Ol C l -lk l y sd fn d a o e c 8 Cl-Clo-alkeyl, alkyl as defined above, c) -(CH2)s-O[(C=O)Or],C 2 -CL-alkenyl, alkenyl as defined above, d) -(CH2)s0O[(CzrO)Or1,C3-C7alkynlalkyl a eiedaoe f) -(CH2)s0O[(C=0),ryC 3 Ca)ylakyiedabve -(CH2)s0O[(C0)Or1,eraryl, traryl as defined above, h0 1 ]H2heteroary, heteroaryl as defined above, i) -(CH 2

),-O(CH

2

),O(CH

2 aryl, aryl as defined above, j) -(CH 2

),-OC(=O)NR

1

R

2 k) -(CH 2

),-OSO

2

R

3 1) -(C 1

-C

6 )-alkyl, alkyl as defined above, or M) -(C 2

-C

6 )-alkenyl, alkenyl as defined above; [R:\LIBAA]O7569.doc:TAB 13 or R 29a and R 29 b can be taken together to be =0 or =C(Ci-Co1alkyl) 2 alkyl as defined above.

An embodiment of this embodiment are the compounds of structural Formula I or a pharmaceutically acceptable salt, crystal form or hydrate, wherein:

R

4 is: a) -O[(C=0)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, CO 2 H, COC 1

-C

6 -alkyl, C0 2

C

1

-C

6 alkyl, CONRaR 2 NRlaR 2 NRlaCOCI-C 6 -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=0)Or]sheteroaryl, wherein heteroaryl is defined as a 5 or 6- '*io 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-C 6 )-alkoxy, cyano, nitro, hydroxy, CHO, CO 2 H, COC 1

-C

6 -alkyl, CO 2 Ci-C 6 -alkyl, CONR'aR 2 SNRaR2 N R2 NRaCOC-C 6 -alkyl any two of adjacent substituents can be joined to form a 6- or 7-membered fused ring said ring containing 1 or 20 2 oxygen atoms and the remainder carbon atoms, or any two adjacent substituents can be joined together to form a benzo-fused ring; wherein Ria and R 2 are as defined above.

An embodiment of the invention are the compounds of Formula I R29a R29b *9 2 12 HI18 7 2 C 17 OAc SR 4

I

or a pharmaceutically acceptable salt, crystal form or hydrate, wherein: X is: Hand R; a is: a single bond; b and c are [R:\LIBAA]07569.doc:TAB independently: a single bond or a double bond; n is: 1 to 4; m is: 1 to 4; ris: 0 or 1; s sis: 0 or 1; R' is: a) H, or b) (CI-C 6 )-alkyl, wherein alkyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, C1, F, I, (Ci-C 6 )-alkoxy, vinyl, cyano, oxo, nitro, hydroxy, CHO, CO 2 H, COCI-C 6 alkyl, CO 2 C1-C 6 -alkyl, CONRIaR2, NRlaR 2 NRlaCOCI-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, (CI-C 6 )-alkoxy, phenyl, phenoxy, cyano, nitro, hydroxy, 9.

S* 15 CHO, CO 2 H, COCI-C 6 -alkyl, CO2C1-C 6 -alkyl, CONRaR 2 NRlaR 2 ~NRlaCOCi-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 20 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-C 6 )-alkoxy, cyano, nitro, hydroxy, CHO, CO 2 H, COCI-C 6 -alkyl,

CO

2

CI-C

6 -alkyl, CONRIaR 2 NRlaR 2 NRlaCOCi-C6-alkyl, any two adjacent substituents can be joined to form a 6- or 7-membered fused 25 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; wherein Rla and R 2 are as defined above.

R

3 is: a) -(CI-C 6 )-alkyl, alkyl as defined above; b) -(Ci-C6)-alkenyl, wherein alkenyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (Ci-C 6 )-alkoxy, cyano, oxo, nitro, hydroxy, CHO, CO 2 H, COCI-C 6 alkyl, CO2C1-C 6 -alkyl, CONRiaR2, NlaR2, NRlaCOCi-C6-alkyl, aryl as defined above, and heteroaryl as defined above; [R:\LIBAA]07569.doc:TAB C) -(CI-C 6 )-alkynyl, wherein alkynyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, 1, (CI-C 6 )-alkoxy, cyano, oxo, nitro, hydroxy, CHO, CO 2 H, COCI-C 6 alkyl, CO 2

CI-C

6 -alkyl, CON7R~aR 2 NRlaR 2 NRlaCOCi -C 6 -alky1, aryl as defined above, and heteroaryl as defined above, d) -aryl, aryl as defined above, or e) -heteroaryl, heteroaryl as defined above; wherein R la and R 2 are as defined above.

R' is: a) absent and a is a double bond, b) -H, c) -OH, d) =0, -O[(C=O)Or],Ci-Clo-alkyl, alkyl as defined above, 15 f) -O[(C=O)Or]sC 2 Cio-alkenyl, as defined above, g) -O[(C=O)Or]sC 2

-C

6 -alkynyl, alkynyl as defined above, h1) -O[(C=O)Or1s(C3-C 7 )-cycloalkyl, i) -O[(C0)Orlsaryl, aryl as defined above, j) -O[(C=O)O,],heteroaryl, heteroaryl as defined above, k) -O(CH2)nO(CH 2 )m..heteroaryl, heteroaryl as defined above, 91) 0O(CH2)nO(CH 2 )maryl, aryl as defined above, *l 29 -OC(=O)NRlaR n) -0S0 2

R

3 or -NR laR2; 25 wherein R la and R 2 are as defined above.

R 2aand R 2 9 b are independently: a) -H, b) -(CH2)-O[(C=0)rlsCClLo-alkyl, alkyl as defined above, C) -(CH 2 2 -C io-alkenyl, alkenyl as defined above, d) -(CH2)s-O[(C=O)Or]sC 2

-C

6 -alkynyl, alkynyl as defined above, e) -(CH2)s,-O[(C=O)Or1,(C 3

-C

7 -cycloalky, f) -(CH2)s,-O[(C=O)Orlsaryl, aryl as defined above, g) -(CH2),-O[(C=O)Orlsheteroaryl, heteroaryl as defined above, h) -(CH2)s-O(CH2)nO(CH 2 )mIheteroaryl, heteroaryl as defined above, i) -(CH2)s-O(CH 2

).O(CH

2 ),maryl, aryl as defined above, j) -(CH 2 )s-OC(=O)NR'R 2 [R:\LIBAA]07569.doc:TAB k) -(CH 2 )s-OS0 2

R

3 1) -(Ci-C 6 )-alkyl, alkyl as defined above, or m) -(C 2

-C

6 )-alkenyl, alkenyl as defined above; or R29a and R29b can be taken together to be =0O or =C(CI-Cloalkyl) 2 alkyl being as s defined above.

An embodiment of this embodiment of the invention are the compounds of Formula I R29a R 2 9 b 9 2 12 1 7 22 1 1 28 02CH3 2 H 9 H 14OH 16 3 10 25 6 15 OAc X~ 57 O 24 6 OAc OAc 4 Ac 23 4 a R aR or a pharmaceutically acceptable salt, crystal form or hydrate, wherein: .X is: H and R'; 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; 15 r is: 0 or 1; sis: 0 or 1; R' is: a) H, or b) (CI-C 6 )-alkyl, wherein alkyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (Ci-C 6 )-alkoxy, vinyl, cyano, oxo, nitro, hydroxy, CHO, CO 2 H, COC 1

-C

6 alkyl, CO 2 C1-C 6 -alkyl, CONRIaR 2 NRlaR 2 NRlaCOCi-C 6 -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, C1, F, I, (Ci-C 6 )-alkoxy, phenyl, phenoxy, cyano, nitro, hydroxy, CHO, CO 2 H, COC 1

-C

6 -alkyl, C0 2

C

1

-C

6 -alkyl, CONRaR 2 NRlaR 2 NRIaCOCi-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 [R:\LIBAAJ07569.doc:TAB 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-C 6 )-alkoxy, cyano, nitro, hydroxy, CHO, CO 2 H, COC 1

-C

6 -alkyl, C0 2

C

1

-C

6 -alkyl, CONR'aR 2 NRlaR 2 NRlaCOCC 6 -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; wherein Rla and R2 are as defined above.

R

3 is: a) -(Ci-C 6 )-alkyl, alkyl as defined above; Soo**: b) -(C 1

-C

6 )-alkenyl, wherein alkenyl is unsubstituted or substituted with one, 9.

s15 two or three substituents selected from the group consisting of: Br, Cl, F, I, (CI-C 6 )-alkoxy, cyano, oxo, nitro, hydroxy, CHO, CO 2 H, COCI-C 6 alkyl, C0 2

C

1

-C

6 -alkyl, CONR1aR 2 NRlaR 2 NRlaCOCi-C 6 -alkyl, aryl as defined above, and heteroaryl as defined above; c) -(CI-C 6 )-alkynyl, wherein alkynyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, SI, (CI-C 6 )-alkoxy, cyano, oxo, nitro, hydroxy, CHO, CO 2 H, COC 1

-C

6 alkyl, C0 2

C

1

-C

6 -alkyl, CONRIaR 2 NRlaR 2 laCOC-C6-alkyl, aryl as defined above, and heteroaryl as defined above, d) -aryl, aryl as defined above, or 25 e) -heteroaryl, heteroaryl as defined above; wherein Rla and R 2 are as defined above.

R

4 is: a) -OH, b) -O[(C=O)Or]sCI-CIo-alkyl, alkyl as defined above, c) -O[(C=0)Or]s(C 3

-C

7 )-cycloalkyl, d) -O[(C=O)Or]saryl, aryl as defined above, e) -O[(C=0)Or]sheteroaryl, heteroaryl as defined above, f) -O(CH 2 )nO(CH 2 )mheteroaryl, heteroaryl as defined above, g) -O(CH 2 )nO(CH 2 )maryl, aryl as defined above, h) -OC(=O)NR'aR2, or i) -OSO 2

R

3 [R:\LIBAA]07569.doc:TAB

R

2 9 a and R 29 b are independently: a) -H, b) -(CH2)-0[(C=0)0rl sCl-Clo-alkyl, alkyl as defined above, C) -(CH2)-0[(C=0)0Q],C 2 -Cio-alkenyl, alkenyl as defined above, d) -(CH2)s;-0[(C=0)Or]C 2

-C

6 -alkynyl, alkynyl as defined above, e) -(CH2)s-O[(C=0)0rls(C 3

-C

7 -cycloalkyl, f) -(CH2)-[(C0O)Orjsaryl, aryl as defined above, g) -(CH2),-0[(C=0)0r],heteroaryl, heteroaryl as defined above, h) -(CH2)s-0(CH2)n0(CH 2 )m..heteroary1, heteroaryl as defined above, i) -(CH2)s-0(CH 2 )n0(CH 2 )maryl, aryl as defined above, j) -(CH 2 ),-OC(=0)NR'R 2 k) -(CH 2 )s-0S0 2

R',

1) -(CI-C 6 )-alkyl, alkyl as defined above, or SM) -(C2-C 6 )-alkenyl, alkenyl as defined above, n) =C(Cj-CjO-alkYl) 2 alkyl as defined above; or R 29 and R 29 can be taken together to be =0 or =C(Cj-CjOalkyl) 2 alkyl being as defined above.

S..An embodiment of the invention is a compound selected from the group consisting of: 4,6,7,15,1 6 -pentakis(acetyloxy)-2 2 2-epoxy- 18 -hydroxy-22-methoxycarbonyl[6(x,7(x 15 P, 16 6f,20oc,21 lf, 2 2f]D:A-Fiedo-A-homo-27,3 O-dinor-24-oxaoleana- 1 -en-3 -one; 4,6,7,15,1 6 -pentakis(acetyloxy)- 18 -hydroxy-22-methoxycarbonyl[6a, 7oc 15P, 16 D:A-Friedo-A-homo27,3O-dinor-24.oxaoleana- 1,21 -dien-3-one; 4,6,7,15,1 6 -pentakis(acetyloxy)-2 ,22-epoxy- 1 8 -hydroxy-22-methoxycarbonyl[6u.7cc 15P3, 16f,20x,2 1 1,22f3]D :A-Friedo-Ahomo27,3O-dinor24-oxaoleana- 1 -ene; 4,6,7,15,1 6 -pentakis(acetyloxy)- 1 8 -hydroxy-22-methoxycarbonyl[6ca, 15f3, 1 63,20cX] D:A-Friedo-A-homo-27,3 O-dinor-24-oxaoleana. 1,21 -diene; [RALIBAAIO7569.docTAB EDITORIAL NOTE NUMBER 74781/96 THIS SPECIFICATION DOES NOT CONTAIN PAGES 19-22.

19 to 22 *0* to.

1.

be#

G**

4..A]769dc:A WO 97/16068 PCTIUS96/1 7211 23 4,6,7,15,1 6 -pentakis (acetyloxy)- Il 8 -hydroxy-22-methoxycarbony..

[6Qx,7cx, 15f3, l 6 f]D:A-FriedoAhomo273dinor24oxaolea 1 ,20(29),2 I -trien-3-one; 4,6,7,15,1 6-pentakis (acetyloxy)- IlX-hydroxy-22-methoxycarbony..

[6u,7ux, 1 Sf3, l 6 f]D:A-Friedo-A-.homo-.27,30-dinor-24-.oxaolea- 1,21 dien-3 2 0-dione; 4 -(2-bromobenzoyl)oxy-6,7, 15,1 6-tetrakis (acetyloxy)- I 8-hydroxy-22methoxycarbonyl[6ux7Qx I Sf3.16f3,20x] :A-Friedo-A-homo..27,30-dinor- 24 -oxaoleana-. 1,21 -dien-3 -one; 4,6,7,15,1 6 -pentakis(acetyloxy).2 2 2 -epoxy- I 8 -hydroxy-22-methoxy-.

carbonyl[6cx,7Qx, 1 5p, 1 6f3,20ux,21lf, 2 2f3]D:A-Friedo-.A-.homo-.27,30 dinor- 2 4-oxaoena3-one; 4,6,7,15,1 6 -pentakis (acetyloxy)- IX-hydroxy-22-methoxycarbonyl- 6 u.,7Q, I 1 6 f3.

2 Ox]D:AFriedoAhomo273 dinor 2 4 ale 21 -en-3-one; 4,6,7,15.1 6 -pentakis(acetyloxy)-2 1 22 -epoxy- I 8-hydroxy-22methoxycarbonyl [6a,7c, I f3.1 6f,2Ocx.21 lf.

2 23]D:A-Friedo-A-homno.

2 7 3 O-dinor-24-oxaolean; 4,6,7,15,1 6 -pentakis(acetyloxy)-

I

8 -hydroxy-22-methoxycarbonyI.

[6u,7u., 1Sf3. I 6 f3 2 ix]D:AFri iedA-omo o730dinor 2 4 oala 21 -ene; 4,6,7,15.1 6 -pentakis(acetyloxy)- IX-hydroxy-22-methoxycarbony..

[6ux,7u, I 5f.1 6 f]D:AFriedoAhomo.27,3dinor24oxaola 2 2 9 21 -dien-3-one 4,6,7,15,1 6 -pentakisacetyjoxy)-

I

8 -hydroxy-22-methoxycarbonyl- [6oc,7cx, 15f3. I 6 f]D:A-Friedo-A-homo-27,30 dinor-24..oxaolean- 21 -en- 3 WO 97/16068 PCT/US96/17211 -24- 4 -(2-bromobenzoyl)oxy-6,7,15,1 6 -tetrakis(acetyloxy)-18-hydroxy-22methoxycarbonyl[6a,7a,15p,160,20a]D:A-Friedo-A-homo-27,30dinor-24-oxaoleana-21 -en-3-one; 4 -(2-bromobenzoyl)oxy-6,7,15,1 6 -tetrakis(acetyloxy)-21,22-epoxy-18hydroxy-22-methoxycarbonyl 153, 16p,20ac,21 P,223]D:A- Friedo-A-homo-27, 3 0-dinor-24-oxaoleana-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, norbomyl, 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, 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, (Cl-C6)-alkoxy, phenyl, phenoxy, cyano, oxo, nitro, hydroxy, WO 97/16068 PCT/US96/17211 CHO, CO2H, COC -C6-alkyl, CO2C -C6-alkyl, CONR1R 2

NR

1

R

2 NR ICOCI-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 -C6-alkyl, CO2C -C6-alkyl, CONR1R 2

NR

1

R

2 NR ICOCl-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 oxygens 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 WO 97/16068 PCT/US96/17211 26 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 97/16068 PCT/US96/17211 -27- REACTION SCHEME

A

12 H 1 8 'OAc OAc OAc

I

Li,

NH

3

THF

'OAc OAc As seen in Scheme A, compound I, 4 ,5,6,15,16-pentakis (acetyloxy)-21, 2 2 -epoxy- 18-hydroxy-22-methoxycarbonyl[6a, 7X, 153, 163, 210, 2 2 P]D:A-Freido-A-homo27,30-dinor 24 -oxaleana 1,20( 2 9 )-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 reference. The resultant lactone can then be converted to the oxepin analog by procedures described in Reaction Scheme

B.

WO 97/16068 PCTJUS96/1 7211 28 It should also be noted that compounds of Formula I having the 1 l,l2-double bond can be prepared using the starting material, 4,6,7,15, l 6 -pentakisacetoxy-.21I, 2 2 -epoxy- IlX-hydroxy-22-methoxy.

carbonyl [6Qx,7(, 1 S3, 16f3,21 f, 2 2 f]D:A-FreidoA-.homo2730dinor- 2 4 oxaleana-l,l1 2

O(

2 9 )-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.

REACTION4 SCH-EME B3 'OAc tAc 28\ OAc OI (b =double bond) 11 (b =single bond) 1. LiAIH(OtBU) 3 2. Et 3 SiH,'

BF

3 OEt 2 11 29 '"OAc OAc WO 97/16068 PCT/US96/17211 -29- As seen in Scheme B, compound I 4 ,6,7,15,16-pentakis(acetyloxy)- 21, 2 2 -epoxy-18-hydroxy-22-methyoxycarbonyl- [6a,7a, 153,16,21, 2 2 1]D:A-Freido-A-homo-27,30-dinor- 2 4 oxaoleana-1, 2 0( 2 9 )-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 on 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.

I WO 97/16068 PCT/US96/17211 REACTION SCHEME C 2 P02CH 3 QAc OAc 1. LiAIH(OtBu) 3 2. Et 3

AI

'OAc OAc 2 4 OAc SOAc In a variation of Scheme B, 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 97/16068 PCT/US96/17211 -31 REACTION SCHEME D

WCI

6 nBuLi THF, 2PO 2

CH

3 OAc 2 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 97/16068 PCT/US96/17211 -32- REACTION SCHEME

E

'OAc

HCI/THF

or

CH

3 'C0 2

CH

3

CH

3 4 OH 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(OCH 3

)CH

3 (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 97/16068 PCT/US96/17211 -33 REACTION SCHEME

F

M H H r Y r CO2CH3 'OAc C0 2

CH

3 S[0] '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 97/16068 PCT/US96/1 7211 34 REACTION SCHEME

G

0 0 2 0H 3 (PhO) 3 MePI HMPT, 75 0

C

'OAc The C4 hydroxy group can be dehydrated to give the olefin. Reaction of the alcohol with tris-phenoxymethylphosphonium iodide in hexamethyiphosphorous triamide (HMPT) at 75 0 C will achieve this conversion.

WO 97/16068 PCTIUS96/1 7211 35 REACTION SCHEAMEL

H

R 4 00CI CO 2 CH3

CH

3

-CO

2

CH

3 'OAc CDI, R 1

R

2

NH

i; WO 97/16068 PCT/US96/17211 -36- REACTION SCHEME H (CONT'D) CDI, R4'OH lI 'C0 2

CH

3 'OAc

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. R4 can be an alkyl carbonate which is depicted in the scheme as OC(=0)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 1

I

WO 97/16068 PCT/US96/17211 -37alcohol 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 97/16068 PCT/US96/1 7211 38 REACTION SCHEME

I

MeO0 H HYnW niPJCO 2

CH

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

WO 97/16068 PCT/US96/17211 -39- REACTION SCHEME

J

'OAc OAc

[C

6

H

5 3

P]

3 RhCI THF,

H

2 12 H18 'OAc OAc OAc 23 OAc 23 R WC1 6 nBuLi THF, 50 0

C

29 '3n "C0 2

CH

3 OAc Lactone or ether (oxepin) derivatives which have or have not been derivatized at C4 can be selectively hydrogenated at the position to give the methyl analog. This conversion can be achieved by i WO 97/16068 PCT/US96/17211 hydrogenation with tris(triphenylphosphine)rhodium(I)chloride (Wilkinson's catalyst) in THF at 15 to 80psi for several days.

The C21-C22 epoxide of lactone or ether derivatives can then be converted to the olefin by use of a WCI6/BuLi complex in tetrahydrofuran (THF) by procedures described in reaction scheme

D.

REACTION SCHEME

K

29 19 21 12 H8 191j

S

1 HC17

H

9 H OH ,PO 2

CH

3 OAc

[C

6 eH) 3

P]

3 RhCI THF,

H,

'OAc OAc OAc 23 R WO 97/16068 PCT/US96/17211 -41 In an alternative to Reaction Scheme J, diene lactone or ether (oxepin) derivatives (Reaction Scheme D) which have or have not been derivatized at C4 can be selectively hydrogenated at the position to give the methyl analog. This conversion can be achieved by hydrogenation with Tris(triphenylphosphine)rhodium(I)chloride (Wilkinson's catalyst) in THF at 15 to 80 psi for several days.

WO 97/16068 PCT/US96/17211 -42- REACTION SCHEME

L

OAc [0 6

H

5 3

P]

3 RhCI THF,

H

2 1 29 'OAc QAc

WCI

6 nBuLi THFE GAc The C20-29 olefin can be selectively converted to the corresponding ketone by a variety of oxidative cleavage procedures.

WO 97/16068 PCT/US96/17211 -43- Ozonization (03) at reduced temperatures, preferrably at -78 0 C in dichloromethane and methanol gives the ketone in good yield.

Alternatively, the C20 ketone can be prepared by sequential reaction with osmium tetroxide or ruthenium tetroxide and sodium periodate.

The epoxide can then be removed by procedures described in reaction scheme

D.

WO 97/16068 PCTIUS96/1 7211 44 REACTION SCHEME

M

QAc QAc IMe 4 N BH (OAc) flH 2 9O 2

CH

3 DAc 3 2

PO

2

CH

3 )Ac 2

PO

2

CH

3 WCi 6 nBuLi OAc 2.j4A The C20 ketone can be reduced to its corresponding alcohol by using a of reducing agents. In particular, tetramethylarnmonium WO 97/16068 PCT/US96/17211 triacetoxyborohydride in THF is effective. The epoxide can then be removed by procedures described in reaction scheme

D.

WO 97/16068 PCTIUS96/1 7211 46 REACTION SCHEME

N

1 2

H.

11

HQ

OAc OAc

BH

3 .THF, Me 3

NO

'OAc OAc WC1 6 nBuLi 'OAc OAc WO 97/16068 PCT/US96/17211 -47- The C20-29 olefin can be converted to the hydroxymethyl derivative. One procedure involves reaction with diborane in THF followed by oxidative workup with trimethylamine-Noxide (Me3NO).

The epoxide is then removed by procedures described in reaction scheme D.

WO 97/1 6068 PCT/US96/1 7211 48- REACTION SCHEME 0 R'

R"

0 2 0H 3 12 l "OAc ~'OAc I R 2 9 am OAc The C20 ketone derivative can be reacted to produce olefins with a variety of well known olefination agents. A particularly useful WO 97/16068 PCT/US96/17211 -49reagent is the Horer-Emmons-type of olefination reagent This produces a mixture of geometric isomers.

Alternatively, the C20 ketone can be reacted with nucleophiles

(R

2 9 to give C20 substituted hydroxy derivatives.

In general, Grignard reagents

(R

2 9 a'MgBr) or alkyllithium reagents

(R

2 9 a'Li) are utilized in aprotic solvents such as diethyl ether or THF.

It should be understood that

R

2 9 a' and R29b' are used to represent a portion of the R 2 9a and R29b definitions, e.g. R 2 9 a can be a substituted olefin which is depicted in the scheme as

R'

and R" representing alkyl substituents.

REACTION SCHEME

P

O 0 19 21 12

H

18 221 1 H 13 OH ,O02CH 3 OAc OAc OAc

R'R

2

NH

NaCNBH 3 1 H 2

PO

2

CH

3 2, 6~ L OAc Ac OAc 2, 4 3Rc WO 97/16068 PCT/US96/17211 Amines at C20 can be prepared from the C20 ketone by reaction with an amine R 1

R

2 NH in a variety of solvents with a reducing agent such as sodium cyanoborohydride.

REACTION SCHEME 0 OAc OAc WO 97/16068 PCTIUS96/1 7211 51 REACTION SCHEME 0 (CONT) OAc 2

PO

2

CH

3 'OAc

NR

1

R

2 'C 020 H 3 28 OAc The C20 bydroxy derivative (Reaction Scheme M) or the C20 hydroxymethyl derivative (Reaction Scheme N) can be converted to WO 97/16068 PCT/US96/17211 -52ether, ester, carbonate, carbamate, sulfonate and other related derivatives by procedures commonly practiced in the art and as described in Reaction Scheme H.

The C20 hydroxymethyl derivative may also be derivatized as a methanesulfinate ester or triflate ester by standard procedures. The methansulfinate or triflate can then be reacted with an amine NR1R2H to give amine derivatives.

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, corea, lung, pancreas, intestinum tenue, limb, muscle, 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 coreae, corneal i WO 97/16068 PCT/US96/17211 53 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 bums 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 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 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 infarction): 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 disease such as cataracta, siderosis, retinitis, pigmentosa, senile macular degeneration, vitreal scarring and corneal alkali bur; dermatitis such as erythema multiforme, linear IgA ballous dermatitis and cement dermatitis; and others such as gingivitis, periodontitis, sepsis, pancreatitis, diseases caused by environmental pollution (for example, air pollution), aging, carcinogenis, metastasis of carcinoma and hypobaropathy; 15 disease caused by histamine or leukotriene-C 4 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 20 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-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 [R:\LIBAA]07569.doc:TAB WO 97/16068 PCT/US96/17211 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 Kv1.3 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, muscle, 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 WO 97/16068 PCT/US96/17211 -56keratitis, conical cornea, dystrophia epithelialis comeae, corneal leukoma, ocular pemphigus, Mooren's ulcer, Scieritis, 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 bums 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 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; 57 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 infarction): 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 lungoxygen 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 bur; dermatitis such as erythema multiforme, linear IgA ballous dermatitis and cement dermatitis; and others such as gingivitis, periodontitis, sepsis, pancreatitis, diseases caused by environmental pollution (for 15 example, air pollution), aging, carcinogenis, metastasis of carcinoma and hypobaropathy; disease caused by histamine or leukotriene-C 4 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 20 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-on-chronic" liver failure (acute liver failure or chronic liver diseases), and moreover are useful for various diseases because of their useful activity such as augmention of chemotherapeutic effect, preventing or treating [R:\LIBAA]07569.doc:TAB WO 97/16068 PCT/US96/17211 -58activity 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, 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, Kv 1.3, that is found on human T-lymphocytes.

WO 97/16068 PCT/US96/17211 -59- 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 dissimilar 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 Kv 1.3 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 Kv 1.3 in human T cells, charybdotoxin was subsequently shown to inhibit four different K+ channels (Kv1.3 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, WO 97/16068 PCT/US96/17211 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 Kv 1.3, 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 Kv 1.3.

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® 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), leflunomide (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 itM in any of the assays thereby demonstrating and confirming the utility of the compounds of the invention as Kvl.3 inhibitors and immunosuppressants.

TCELLIL-2

ASSAY

Peripheral blood mononuclear (MNC) cells from healthy donors were separated by density centrifugation with ficoll-hypaque

M

WO 97/16068 PCT/US96/17211 -61 (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 tl/well. The various dilutions of test compound were then added in triplicate wells at 25 gl/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 supernatants 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 WO 97/16068 PCT/US96/17211 -62- 6 cells/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% C02 95% air, 20 tl/well of anti-CD3 (Ortho Diagnostic, NJ) at final conc. of 0.3 ng/ml was added, followed by 50 tl 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 measurement of tritiated thymidine incorporation. During the last 18- 24 hrs. of culturing, the cells were pulse-labeled with 2 tCi/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 KVI.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 pCi/ml, Dupont-NEN) in the glutamine supplemented IMDM. After aspiration of the media, 100 ptl of Low K Buffer (in mM, 6.5 KC1, 125 NaCI, 1 CaC12, 2 MgCl2, 10 HEPES, pH WO 97/16068 PCT/US96/17211 -63 adjusted to 7.2 with NaOH) is added to each well followed by 100 .1 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 Kv1.3 channels are opened by depolarization of the cells with High K Buffer (final concentrations, in mM, 63.25 KCI, 68.25 NaCI, 1 CaC12, 2 MgC1 2 10 HEPES, pH adjusted to 7.2 with NaOH) also containing test compounds. To measure 86Rb+ efflux through the channels, aliquots of 100 p1 are taken from each well after a given time and added to plates containing 100 pl MicroScint-40 (Packard) for counting by liquid scintillation techniques. MicroScint-40 (100 ptl) is then added to each well of the cell plate to determine the remaining 86 Rb+ activity. The efflux counts are normalized for the total amount of 8 6 Rb+ 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 Kvl.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 3( 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, intracisteral and parenteral. The term WO 97/16068 PCT/US96/17211 -64parenteral" as used herein refers to modes of administration which include subcutaneous, intravenous, intramuscular, intraarticular injection or infusion, intrastemal 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 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.

-M

WO 97/16068 PCT/US96/17211 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 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 insuffiation 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.

WO 97/16068 PCT/US96/17211 -66- 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.

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.

I

I

WO 97/16068 PCT/US96/17211 67-

INJECTABLE

A parenteral composition suitable for administration by injection is prepared by stirring 1.5% by weight of active ingredient in 10% 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 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

I

A Method Of Extracting The Compounds Of Formula 1(a) and 1(b) From Spachea correa WO 97/16068 PCT/US96/17211 68 H H 1. H b4 CH0 Formula 1(a) b is a single H bond and R is OAc OH

COOCH,

0 CH3 C

H

3 OAc Formula 1(b) b is a double A Ac OAc bond and R is OAc CH,\ OAc 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 and eluted with a 50 minute gradient of acetonitrile:water (1:1, v/v) to 100% acetonitrile, delivered at 1 ml/min, to afford 4 mg of compound 1(a) and 1 mg of I(b).

Homogeneity of the preparations was ascertained in several TLC systems, such as E. Merck silica gel 60F254, methylene chlorideethyl acetate 1:1, Rf l(a) 0.4, Rf 1(b) 0.3; Whatman KCI8, 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

I

WO 97/16068 PCT/US96/17211 -69resolution (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 (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 C40H520 16 M+ 728.3040 C38H48014 M-acetic acid 668.2834 C36H440 12 M-2 x acetic acid 334.1417 C18H220 6 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, 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 C40H520 1 6 derived by scanning HR EI-MS.

The IH NMR spectra of compound of Formula(a) is provided as Figure 1. The spectra was recorded at 400 MHz in CD2C12 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 65.32 as the internal standard.

WO 97/16068 PCT/US96/17211 The mass spectra of the compound of Formula 1(b) was obtained as above. The following results were obtained.

Observed m/z 786.3075 726.2886 666.2651 606.2451 489.2099 471.1992 Formula C40H50016 C38H46014 C36H42012 C34H38010 C26H3309 C26H3108 Assignment

M+

M-acetic acid M-2 x acetic acid M-3 x acetic acid base peak 13 C 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.

EXAMPLE 2 A Method Of Extracting The Compounds Of Formula l(c) And 1(d) From Spachea Correa H ,H

'COOCH

3 Formula 1(c) b is a single bond and R is OH Formula 1(d) b is a double bond and R is OH OAc WO 97/16068 PCT/US96/17211 -71 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 1(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 2( UV spectra and color reactions on TLC plates. Material from the methylene 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 2.1x 50mm C8 column at 0.2ml/min. with a mobile phase of 45% acetonitrile/0.0lM 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 1(c) has a .1 WO 97/16068 PCT/US96/17211 72retention 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 C38H500 16 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 0 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.

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 S 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 j_ WO 97/16068 PCTIUS96/17211 -73 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 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(a) ranges from about 2 to about 3.5 column volumes of solvent; that for the compound of Formula I(b) 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 l(a) and 1(b) can be precipitated and or crystallized from concentrated methanol solutions.

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 l(a) and l(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 1(a) and S(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(a) ranges from about 2 to about 3.5 column volumes of solvent; that for the compound of Formula l(b) is about 3 to WO 97/16068 PCT/US96/17211 -74about 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(a) and 1(b) can be precipitated and or crystallized from concentrated methanol solutions.

EXAMPLE 4,6,7,15,1 6 -Pentakis(acetyloxy)-21, 2 2 -epoxy-18-hydroxy-22methoxycarbonyl[6a,7a, 15P,16p,20a,210, 2 2 p]D:A-Friedo-A-homo- 2 7 .30-dinor-24-oxaoleana-1 -en-3-one

O

H OH C 0 2CH3 O OAc OAc OAc AcO.OAc A solution of 50mg (63.7 tmole) of 4,6.7,15,16- Pentakis(acetyloxy)-21,2 2 -epoxy- 18-hydroxy-22-methoxycarbonyl- [6a,7(a,150,160,210, 2 2 ]D:A-Friedo-A-homo-27,30-dinor- 2 4 oxaoleana-1,20( 2 9 )-dien-3-one in 50ml of dry THF was degassed under reduced pressure and saturated with nitrogen, the procedure being repeated several times. 25mg of Wilkinson's catalyst (PPh3)3RhCI were added and the solution was degassed and saturated with hydrogen in the previously described manner. The reaction vessel was then pressurized with H2 to 15 psi (latm) and shaken for 65h at 25°C. After that time the solvent was removed under reduced pressure. The residue was dissolved in a small amount of ethyl acetate/hexanes (ca. I mL) and filtered through 30g of silica gel eluting with 500ml of ethyl acetate hexanes The first fractions, containing the Wilkinsoncatalyst (approx. 50mL) were discarded. The fractions containing the WO 97/16068 PCT/US96/l 7211 75 product were combined. After removing the solvent under reduced pressure the crude product was dried in vacuo and purified by HPLC to afford 20.8 mg of the title compound as a white solid. I H NMR

(CDCI

3 5 1.5 3H, J 8.6 Hz, C29-H), 2.4 (in, IlH, C20-H); Mass Spectrum (APCI): m/e 808.

EXAMPLE 6 4,6,7,15,1 6 -Pentakis(acetyloxy)-

I

8 -hydroxy-22-methoxycarbonyl- [6Qx,7cx, 15f3, l 6 f, 2 0a]D:AFriedoAhomo273dinor.

2 4 ale 1,21 -dien-3-one OO~c AcO~c A solution of 48.2 mg 104 inmole) of tungsten hexachloride in 8 mL of dry tetrahydrofuran was cooled to -78'C under nitrogen. Then 0. 152 mL (0.208 mmole) of 1.6M butyllithium was added and the soltuion was allowed to warm to room temperature over mm. Then a solution of 20.2 mng (0.026 inmole) of 4,6,7,15,16- Pentakis(acetyloxy)-21I 2 2 -epoxy- I 8-hydroxy-22-methoxycarbonyJ- 6 (x,7u. 15f,16f3.2 lP 2 2 f3]D:A-Friedo-.A-.homo.27,3O.dinor- 2 4 oxaoleana- 1 -en-3-one in 2 mL of dry tetrahydrofuran was added and the solution was heated under nitrogen at 50*C for 18 h. The mixture was applied to a 10 cm colun 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 15.9 mng of the title compound as a white solid; Mass Spectrum (APCI) 792

(M+NH-

4 WO 97/16068 PCT/US96/17211 76 EXAMPLE 7 4,6,7,15,16-Pentakis(acetyloxy)-21, 2 2-epoxy-18-hydroxy-22methoxycarbonyl[6a,7a,15,16,20a,210,22p]D:A-Friedo-A-homo- 2 7 .30-dinor-24-oxaoleana- I-ene

O

H OH CO2CH3 OAc O A c O OAc S OAc AcO Ac A solution of 213 mg (0.27 mmole) of 4,6,7,15,16pentakis(acetyloxy)-21, 2 2 -epoxy-1 8 -hydroxy-22-methoxycarbonyl- [6a,7a, 15,16p,20a,21

P,

2 2 p]D:A-Friedo-A-homo-27,30-dinor-24oxaoleana- I -en-3-one in 2 mL of dichloromethane was cooled to 0°C.

Then 0.68 mL of a 1.02M solution of lithium tri-tertbutoxyaluminum hydride was added and the solution was stirred at 0OC for 18h. The reaction was quenched with 10 mL of 2.5M aqueous H2S04 and then was diluted with 50 mL of dichloromethane. After the layers were separated, the organic phase was dried over Na2SO 4 and concentrated.

The residue was dissolved in a mixture of 5 mL of dichloromethane and 1 mL of triethylsilane. Then 0.8 mL of boron trifluoride-etherate was added and the solution was stirred at room temperature. After 2 h, the reaction was quenched by addition of saturated aqueous NaHCO3 solution and dichloromethane. The layers were separated and the organic layer was washed with and brine, saturated aqueous NaHCO3, dried over MgSO4, and concentrated. The residue was first filtered through a plug of silica gel and then purified by HPLC (Waters RCM, Prep Nova-Pak HR Silica, 25mm x 100 mm) WO 97/1 6068 PCT/US96/1 7211 77 using 8:4:1 hexane:t-butylmethylether:acetonitrile to afford 60.5 mg of the title compound as a white solid; I H NMR (CDCI3) 8 2.41 (in, I1H, C20-H), 3.3 8 I H, C21 3.69 (dd, 2H,. AB, J 12 Hz, C24-H), 5.5 (in, IlH, ClI-H), 5.57 (in, I1H, C2-H); Mass Spectrum (APCI): m/e 794 (M+NH 4 EXAMPLE 8 4,6,7,15,1 6 -Pentakis(acetyloxy)- IX-hydroxy-22-methoxycarbonyv.

[6Qx,7cx, 1 5P3, 1l 6

P,

2 Oul]D:A-Friedo-Ahomo-273.d'mor-2.4-oxaoleana- 1,21 -diene

O~OA

AcO A solution of 233 mg (0.56 inmole) of tungsten hexachioride in 8 mL of dry tetrahydrofuran was cooled to -78*C under nitrogen. Then 0.70 mL (1.12 mmole) of 1.6M butyllithiuin was added and the solution was allowed to warm to room temperature over mmn. Then a solution of Ill mg 141 minole) of 4,6,7,15,16- Pentakis(acetyloxy-2 1,22-epoxy- Il 8 -hydroxy-22-methoxycarbonyl.

[6ux,7u. 150,1 60,20a,21 1, 2 2 f]D:A-Friedo-A-homo.2730.dinor-24oxaoleana- I -ene in 2 mL of dry tetrahydrofuran was added and the solution was heated under nitrogen at 50 0 C for 18 h. The mixture was applied to a 10 cm colun of silica gel, which was washed w,ith 2:1 ethyl acetate-hexane. The eluate was concentrated and purified by silica gel chromatography with 2:1 ethyl acetate-hexane to afford 95 mng WO 97/16068 PCTIUS96/1 7211 78 of the title compound as a white solid; I H NMR (CDCl 3 6 2.66 (in, I1H, C20-H), 3.64 and 3.67 (dd, AB, 2H-, J 12.2 Hz, C24-H), 6.52 I H, C21 Mass Spectrum (APCI) 77 8 (M+NH4).

EXAMPLE 9 4,6,7,15,1 6 -pentakis(acetyloxy)- 1 8 -hydroxy-22-methoxycarbonyl.

[6ux,7cx, 1 5P, 1 6 f 3 ]D:A-Friedo-A-.homo.273O.dinor24-oxaoleana- 1 .20(29).2 I -trien-3-one DO~c AcQ A solution of 233 mng (0.56 mmole) of tungsten hexachioride in 8 mL of dry tetrahydrofuran was cooled to -78'C under nitrogen. Then 0.70 mL 12 mmole) of 1.6M butyllithium was added and the solution was allowed to warm to room temperature over 30 min.

Then a solution of 11 I g 141 minole) of 4.6,7,15,16pentakis(acetyloxy-2 1 2 2-epoxy- I 8-hydroxy-22methoxycarbony][6a,7ux, 15 5j,1 0 j,2lf 3 2 2 f 3 A-Fri edo..-A -homo..27,3 0 dinor-24-oxaoleana I 2

O(

2 9 )-dien-3-one in 2 ml of dry tetrahydrofuran was added and the solution was heated under nitrogen at 55 0 C for 14 h.

The mixture was applied to a 10 cm colun 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 95 mng of the title compound as a white solid; I H NMR (CDCI3) 67.10 lH, C21-H); Mass Spectrum (APCI) 790 (M+NI-4).

WO 97/1 6068 PCTJUS96/1721

I

79 EXAMPLE 4,6,7,15,1 6 -pentakis(acetYloxy)- I 8 -hydroxy-22-methoxycarbony..

[6ux,7cx, 1 5P3, 1 6 f 3 ]D:A-Friedo-A-homo-.27,30 dinor-24-oxaolea 1,21 dien-3,20-dione AcO~KAc A solution of 120.5 mg of 4 6 7 l5,16-pentakis(acetyloxy)-

I

8 -hydroxy-22-methoxycarbonyl [6u,7ux, 1 50,16f3]D :A-Friedo-A-homo.

2 7 3 O-dinor24oxaoleana1,2(29)222trien3-one in 40 ml of (1:1, CH2CI2/CH3OH) was cooled to -78*C and 03 was bubbled into the solution until it contained a blue color. The solution was then purged with nitrogen for 3 minutes and 0.3 ml] of Me2S was added. The solution was allowed to warmed to 25'C for 14 hours. Volitiles were removed by vaccum and the residue was purified by chromatography on silica gel using 25 ethyl acetate-hexane to afford 100.2 mg of the title compound as a white solid; I H NMR (CDC1 3 6 6.63 I 2.84 (d, I H, J 17 Hz), 2.74 I1H, J 17 Hz); Mass Spectrum (APCI) m/e 792 (M+NH4).

WO 97/1 6068 PCT/US96/1 7211 80 EXAMPLE I I 4 -(2-Bromobenzoyl)oxy..6,7, 15,1 6 -Tetrakis(acetyloxy)- 1 8 -hydroxy-22methoxycarbonyl[6cx,7(x, 1$5, 1 6 f,200a]D:A-Friedo-A-.homo-.27,30dinor-24-oxaoleana- 1.21 -dien-3-one

CO

2

CH

3 Step A: 6,7,15,1 6 -Tetrakis(acetyloxy)-21I, 2 2-epoxy-4, 18dihydroxy-22-methoxycarbonyI [6Q,7Qx, 1 Sf3,- 1 6f,21lf, 2 2 N]D:A-Friedo-A-.homo..27,3O..dinor-24oxaoleana- 1 2

O(

2 9 )-dien-3-one 0 nHu C 0 2

CH

3 A solution of 102.1 mg (0.130 mimole) of 4,6,7,15,16pentaki s(acetyloxyy.2 1 2 2 -epoxy- Il 8 -hydroxy-22-methoxycarbonyl.

WO 97/16068 PCTIUS96/1 7211 81- [6ix,7cc, 1 5f3,l6t3,21lJ, 2 2 f0]D:A-Friedo-A-homo..27,3O-dinor.24oxaoleana-1I,20(29)-dien-3-one in 4 mL of tetrahydrofuran and 2 mL of 3M aqueous HCI was heated at 40'C for 24h. The solution was diluted with dichloromethane 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 (CDCI3) 8 4.20 IH, J 4.3 Hz, C4-H); Mass Spectrum (APCI): m/e 764 (M+NH4) Step B: 4 2 -Bromobenzoyloxy)-6,7, 15,1 6 -Tetrakis(acetyloxy).

21 2 2-epoxy- I 8 -hydroxy-22-methoxycarbony[6x7(x 16j3,210f, 2 2 01]D: A-Fri edo-A-homo-.2730.dinor-24oxaoleana- I 2 0( 2 9 )-dien-3-one 00 0 02H To sluio o 1.5mg(2.5p~ol f ,,1,6 terki~ceyox)2 22eox-, ~dhdOAy"~ehxcroy.

[6~7u 1Sf.16f.2lf.

2 f3D:-FieO-Achm.7Oio2 purified by HPL l(Wters RoM 17. Poosg 103. gme c) usin WO 97/1 6068 PCTIUS96/1 7211 82 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; I H NMR 5 5.67 (1 H, 7.40-7.43 (in, 2H), 7.72 (dd, I H, J 2.2, 6.9 Hz), 7.78 (dd, I1H, J 2.3, 6.9 Hz); Mass Spectrum (APCI): rn/c 946, 948 7 9 Br-M+NH4, 8 1 Br-M+NH4) Step2 C: 4 -(2-Bromobenzoyl)oxy-6,7, 15,1 6 -Tetrakis(acetyloxy)- 2 l, 2 2-epoxy-I 8-hydroxy-22-methoxycarbonyl.

[6ux,7cx, 150f,16f3,2ocx,2 1 22 1]D:A-Friedo-A-homo..27,30..

dinor-24-oxaoleana-I -en-3-one Br0 A~ sltoof000g(10 H.moe of-2brmoenoyoy),715

I

6 teraisactloy -2I 2 -eox-A8 hyroy-2.mthxyaronl6~~ 5f,163, I1 3 223D:-Fieo-.

ho* 2 .0io.2oalaai0(9Ade~n in2mco r shaken A fortio 72 at0. fte tha time the solvent was remveude TH a easdudrreduced pressure. The resdutwsrisoled int asllmontoehy aedcete/esune (1:1 (ca.du wa L)adfisleed thrug a salmon siic gethl

I

WO 97/16068 PCT/US96/1 7211 83 eluting with 5O0mI of ethyl acetate hexanes The first fractions, containing the Wilkinson-catalyst (approx. 5OmL) were discarded. The fractions containing the product were combined. After removing the solvent under reduced pressure the crude product was dried in vacuo and purified by HPLC (Waters RCM, Prep Nova-Pak HIR Silica, x 100 mm) using 8:4:1 heaetbtlehlehraeoirl to afford 71.2 mg (71 of the title compound as a white solid; I NMR (CDCl 3 8 1.5 3H, J 8.6 Hz, C29-H), 2.4 (in, IH, C20-H); 5.67 (III, C4-H), 7.40-7.43 (in, 2H), 7.72 (dd, IlH, J 2.2, 6.9 Hz), 7.78 (dd, I1H, J 2.3, 6.9 Hz); Mass Spectrum (APCI) m/e 948, 950 7 9 Br-M+NH4, 8 1 Br- M+NH-4).

Step D: 4 2 -Bromobenzoyl)oxy-6,7, 15,1 6 -tetrakis(acetyloxy). 18hydroxy-22-methoxycarbonyl [6cx,7Qx, I $,1I603,20a]D :A Friedo-A-Ihomo.27 3 O-dinor-24 -aleana. 1.21 -dien-3 -one OAc Br0 0 This compound is prepared from 4 2 -bromobenzoyl)oxy.

6,7,15,1 6 -tetrakis(acetyloxy)-21I, 2 2 -epoxy-1I8-hydroxy-22.

methoxycarbonyl[6Qx,7u, 1Sf,16f3,20(x,2 I f 3 22f]D:A-Friedo-.A-.homo.

7 3 O-dinor-24-.oxaoleana-I -en-3-one using the procedures described in Example 8.

WO 97/16068 PCTIUS96/1 7211 -84 EXAMPLE 12 4,6,7,15,1 6-pentakis (acetyloxy)-2 1 ,22-epoxy- I 8-hydroxy-22methoxycarbonyl[6,7cx, 1$P, 1 6P3,21 P,22P13D:A-Friedo-A-homo-.27,30dinor- 2 4 -oxaoleana-20(29)en3-one 29 u 19 21 12 '-18 22 0 11 1 21 H; 9 1' 3

O

4 H 16 0 2

CH

3 3 1025 15 QAc 0 245 6 2 0 4 Oc D~c As described in Scheme I, 4,5,6,15,1 6-pentakis(acetyloxy)- 1 o 2 1,22-epoxy- I 8 -hydroxy-22-methoxycarbonyl[6ax7(X 1 5P, 1 6P3,2 1 P,- 2 2 f 3 ]D:A-Freido-A-homo-.27,30-dinor-.24-.oxaoleanal 1.20(29)-dien-3one, 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.

M

WO 97/16068 PCT/US96/17211 EXAMPLE 13 4,6,7,15,1 6 -Pentakis(acetyloxy)-21, 2 2 -epoxy-18-hydroxy-22-methoxycarbonyl[6a,7a, 153,16,20a,21

P,

2 2 ]D:A-Friedo-A-homo-27,30dinor- 24 -oxaoleana-3-one

O

H f OH COCH3 O O A c OAc j: I OAc OAc AcO

A

A solution of 50mg (63.7 gmole) of 4,6,7,15,16- Pentakis(acetyloxy)-21, 2 2-epoxy- 18-hydroxy-22-methoxycarbonyl- [6a,7a, 15p3,161,21, 2 2 03]D:A-Friedo-A-homo-27,30-dinor-24oxaoleana-20(29)-en-3-one in 50ml of dry THF is degassed under reduced pressure and saturated with nitrogen, the procedure being repeated several times. 25mg of Wilkinson's catalyst (PPh3)3RhCl are added and the solution is degassed and saturated with hydrogen in the previously described manner. The reaction vessel is then pressurized with H2 to 15 psi (latm) and shaken for 65h at 25 0 C. After that time the solvent is removed under reduced pressure. The residue is dissolved in a small amount of ethyl acetate/hexanes (ca. 1 mL) and filtered through 30g of silica gel eluting with 500ml of ethyl acetate hexanes The first fractions, containing the Wilkinson-catalyst (approx. 50mL) is discarded. The fractions containing the product is combined. After removing the solvent under reduced pressure the crude product is dried in vacuo and purified by HPLC to produce the title compound.

WO 97/16068 PCT/US96'1721

I

86 EXAMPLE 14 4,6,7,15,1 6 -Pentakis(acetyloxy)- IX-hydroxy-22-methoxycarbonyl- [6Q,7cx, 1 Sf3, l 6 f, 2 0x]D:A-Friedo.Ahomo273dor 2 4 xolea 2 l1-en-3-one

C~

A solution of 48.2 mg (0.104 mmole) of tungsten hexachioride in 8 m-L of dry tetrahydrofuran is cooled to -78'C under nitrogen. Then 0. 152 mL (0.208 mmole) of 1 .6M butyllithium is added and the soltujon is allowed to warm to room temperature over 30 min.

Then a solution of 20.2 mg (0.026 mmole) of 4, 6 ,7 ,15 ,16- Pentakis(acetyloxy)-2 1 2 2 -epoxy- Il 8 -hydroxy-22.methoxycarbonyl.

[6Q..7u 1, 1 60.20(x,21 f 3 2 2 f]D:A-Friedo-A-.homo.273Odinor- 2 4 oxaoleana-3-one in 2 mL of dry tetrahydrofuiran is added and the solution is heated under nitrogen at 50*C for 18 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.

WO 97/16068 PCTUS96/1 7211 87 EXAMPLE 4,6,7,15,1 6 -Pentakis(acetyloxy)-21I, 2 2 -epoxy.. I 8-hydroxy-22methoxycarbonyl[6a,7(x 1 53, 1 61,20x,2 I0, 2 2

I

3 ]D:A-Friedo-A..homo.

2 7 3 O)-dinor-24-oxaolean E6ACc AcO A solution of 213 mg (0.27 mmole) of 4,6,7,15,16pentaki s acetyloxy)-21 2 2 -epoxy- Il 8 -hydroxy-22-methoxycarbonyI- [6(x,7cx, I 5j3,16f3,2Ocx,21lf 3 2 2 1]D:A-Friedo..A-.homo-.273O.dinor- 2 4 oxaoleana-3-.one in 2 mL of dichloro-methane is cooled to 0 0 C. Then 0.68 mL of a 1 .02M solution of lithium tri-tertbutoxyaluminum hydride is added and the solution is stirred at 0 0 C for 18h. The reaction is quenched with 10 mL of 2.5M aqueous H2S04 and then is diluted with 50 mL of dichioromethane. After the layers are separated, the organic phase is dried over Na2SO 4 and concentrated.

The residue is dissolved in a mixture of 5 mL of dichloromethane and I mL of triethylsilane. Then 0.8 mL of boron trifluorideetherate is added and the solution is stirred at room temperature. After 2 h, the reaction is quenched by addition of saturated aqueous NaHCO3 solution and dichloromethane. The layers are separated and the organic layer is washed with and brine, saturated aqueous NaHCO3, dried over MgSO4, and concentrated. The residue is purified by silica gel chromatography using S (hxae -uyIehleh :aeoiti 8:4:1) to produce the title compound.

WO 97/16068 PCT/US96'1 7211 EXAMPLE 16 4,6,7,15,1 6 -Pentakis(acetyloxy>-

I

8 -hydroxy-22-methoxycarbonyl.

[6ax,7cx, I 5j3,16f3,20QX]D :A-Friedo-A-homo.27 3 O-dinor-24-oxaoleana- 21 -ene AcO~' A solution of 233 mg (0.56 mmnole) of tungsten hexachloride in 8 ml. of dry tetrahydrofuran is cooled to -78 0 C under nitrogen. Then 0.70 ML 12 mmole) of 1.6M butyllithium is added and the soltuion is allowed to warm to room temperature over 30 min.

Then a solution of Ill mg 141 mmole) of 4,6,7,15,16pentaki s(acetyl oxy)-2 l, 2 2-epoxy- I 8-hydroxy-22-methoxycarbonyj [6a,7(x, 15f3,1 6 f,20a,21lf, 2 2 f]D:A-FriedoAhomo273Odinor-24oxaolean in 2 mL of dry tetrahydrofuran is added and the solution is heated under nitrogen at 50*C for 18 h. The mixture is applied to a cm column of silica gel, which was washed with 2:1 ethyl acetatehexane. The eluate is concentrated and purified by silica gel chromatography with 2:1 ethyl acetate-hexane to produce the title compound.

WO 97/16068 PCTIUS96/1 7211 89 EXAMPLE 17 4,6,7,15,1 6 -pentakis(acetyloxy> lS-hydroxy-22-methoxycarbonyl.

[6Qx,7u, I 5f3, l 6 f]D:A-Friedo-Ahomo27,30Odinor-24-oxaolea.

2 0( 2 9 21l-dien-3 -one :OACc 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 is added and the solution is allowed to warm to room temperature over 30 min.

Then a solution of Ill mg 141 mmole) of 4 6 7 ,l5,16-pentakis- (acetyloxy)-2 I 22-epoxy- I 8 -hydroxy-22-methoxycarbonyl.

[6cc,7cx. 15 f.16 Pf,210f, 2 2 0 1 D: A-Fri edo-A -homo..-273 0.dinor-24oxao leana-20(29)-en.3.-one in 2 mil of dry tetrahydrofuran is added and the solution is heated under nitrogen at 55 0 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.

M

WO 97/16068 PCT/US96/1 7211 90 EXAMPLE 18 4,6,7,15,1 6 -pentakis(acetyloxy). 1 8 -hydroxy-22-methoxycarbonyl.

[6Qx,7cx, 1Sf,16]:-reoAhm-7,0dnr2-xolaa 21-en- 3,20-dione OAc OAc A solution of 120.5 mg of 4, 6 ,7 ,15 ,16-pentakis (acetyloxy)- l 8 -hydroxy-22-methoxycarbony[6a,7(x15Sf, 16f3,2 1 ,223] D:A-Friedo-A-homo-.27,3dinor24-.oxaoleana 2 0( 2 9 2 1 -dien-3 -one in ml] of CH2CI2/CH 3 OI-) is cooled to -78'C and 03 is bubbled into the solution until it contained a blue color. The solution is then purged with nitrogen for 3 minutes and 0.3 ml of Me2S is added. The solution is allowed to warmed to 25'C for 14 hours. Volitiles are removed by vaccum and the residue is purified by chromatography on silica gel using 25 ethyl acetate-hexane to produce the title compound.

WO 97/16068 PCTIUS96/1 7211 91 EXAMPLE 19 4 -(2-Bromobenzoyloxy-6 ,7,15,1 6 -tetrakis(acetyloxy)- I 18-hydroxy-22methoxycarbonyl [6Qx,7Q, 1 5 f, 1 6f,20ax]D:A-Friedo-A..homo-27,30..

dinor-24-oxaoleana-21 -en.3-one 0C0 2

CH

3 OAc Step A: 6,7,15,1 6 -Tetrakis(acetyloxy)-21 ,22-epoxy-4, 18dihydroxy-22-methoxycarbonyl[6Ux7c I f1 6f3,21 j,22p3]- D:A-Friedo-A-homo..27,30-dinor-94-oxaoleana 20(99)-en- 3-one OAc OAc A solution of of 4 6 7 l,16-pentakis'acetyloxy)-21,22epoxy-I 8 -hydroxy-22-methoxycarbonyl[6Uc7u I Sf3.1 6J,2 1 ,22J3]D:A- WO 97/16068 PCT/US96,I 7211 92 Friedo-Ahomo.27,3dinor24oxaoleana2(2 9 )n 3 -one in 4 mL of tetrahydrofuran and 2 mEL 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. 1 M phosphate buffer (pH then is dried over MgSO4 and concentrated. The residue was purified by silica gel chromatography with 2:1 ethyl acetate-hexane to produce the title compound.

Step B: 4 -(2-Bromobenzoyloxy-6 15,1 6 -tetrakis(acetyloxy).

2 l, 2 2-epoxy- I 8 -hydroxy-22-methoxycarbonyl I1f3,16f3,2 I f,22f]D:A-Friedo-.A..homo.27,30dinor- 2 4 -ox aoleana-.20f29)-.en3-one Br 0 -K9c To a solution of 6 7 ,lS,16tetrakis(acetyloxy)21,22-epoxy- 4, 19dhyrx-l-etoyaf3yl6.7u101 6f.2 I1,22f3]D:A- Friedo-Ahomo273dinor4oxaoleana2(2 9 )n 3 -oein 0.5 mL pyridine is added 27.5 mL (237 ptmole) 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 I-PLC (Waters RCM, gt 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.

I

WO 97/16068 PCT/US96/17211 -93- Step.C: 4-(2-Bromobenzoyloxy)-6,7,15,1 6 -tetrakis(acetyloxy).

21,22-epoxy-18-hydroxy-22-methoxycarbonyl- [6a,7a, 150,16,20a,21 ,223]D:A-Friedo-A-homo-27,30dinor- 24 -oxaoleana-3-one H 0 O2CH3

O

OAc OAOAc Br 0 Ac 1 0 A solution of of 4 -(2-bromobenzoyloxy)-6,7,15,16tetrakis(acetyloxy)-21, 2 2 -epoxy- 18-hydroxy-22-methoxycarbonyl- 151, 160,20x,21

P.

2 2 P]D:A-Friedo-A-homo-27,30-dinor-24oxaoleana-20(29)-en-3-one in 20ml of dry THF is degassed under reduced pressure and saturated with nitrogen, the procedure being repeated several times. Then 75 mg of Wilkinson's catalyst [(PPh3)3RhCl are added and the solution is degassed and saturated with hydrogen in the previously described manner. The reaction vessel is then pressurized with H2 to 50 psi (3.5 atm) and shaken for 72h at 25 0

C.

After that time the solvent is removed under reduced pressure. The residue is dissolved in a small amount of ethyl acetate/hexanes (ca.

1 mL) and filtered through 30g of silica gel eluting with 500ml of ethyl acetate hexanes The first fractions, containing the Wilkinsoncatalyst (approx. 50mL) are discarded. The fractions containing the product are combined. After removing the solvent under reduced pressure the crude product is dried in vacuo and purified by HPLC to produce the title compound.

WO 97/16068 PCT/US96'1 7211 94 Step D: 4-( 2 -lBromobenzoyloxy)-6,7,15,1 6 -tetrakis(acetyloxy)- 18hydroxy-22-methoxycarbonyl..[6c,7ux, 1513,1603,20ax]D

:A-

Friedo-A-homo-2730dinor-24..oxaoleana-2 1I -en-3 -one HC 0 2 0H 3 OAc OAc This compound is prepared from 4 2 -bromobenzoyloxy)- 6, 7,15,1 6-tetrakis(acetyloxy)-21 ,22-epoxy- 1 -hydroxy-22-methoxycarbonyl [6a.,7Q, 1 5P3,1 6j3,20Qx,21 j,221]D:A-Ffiedo-A-homo-.27,30dinor- 24 -oxaoleana- I -en-.3-one using the procedures described in Example 16.

Claims (3)

1. A compound of structural Formula I: R29a R29b 9 2 12 H18 17 2J 11 2 1 H OH 16 28 02CH3 9 14 3 25 6 15 OAc 0 24 -6 OAc OAc -4 OAc

23- 4 aR I or a pharmaceutically acceptable salt, crystal form or hydrate, wherein: 5 Xis: O, S, NHorH andR 1 a is: a single bond, or a double bond when R 4 is absent; and c are independently: a single bond or a double bond; nis: lto4; m is: 1 to 4; Sr is: 0or l; sis: 0 orl; R is: a) H, or 15 b) (Ci-C 6 )-alkyl, wherein alkyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (Ci-C 6 )-alkoxy, vinyl, cyano, oxo, nitro, hydroxy, CHO, CO 2 H, COCi-C 6 alkyl, C0 2 C 1 -C 6 -alkyl, CONRlaR 2 NRlaR 2 NRlaCOC-C 6 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, (Ci-C 6 )-alkoxy, phenyl, phenoxy, cyano, nitro, hydroxy, CHO, CO 2 H, COCi-C 6 -alkyl, CO 2 Ci-C 6 -alkyl, CONRaR 2 NRlaR 2 NRaCOC-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, [R.\LIBAA]07569.doc:TAB S S *5* S *5* 5* S S S S S S a. S S. 55 I, (Ci-C 6 )-alkoxy, cyano, nitro, hydroxy, CHO, CO 2 H, COCI-C 6 -alkyl, CO 2 CI-C 6 -alkyl, CONRlaR 2 N7RlaR 2 NklaCOCI.C 6 -all 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; *Rla and R 2 are independently; a) (Ci-C 6 )-alkyl, or b) aryl, wherein aryl is phenyl or naphthyl. *R 3 is: a) I-C 6 )-alkyl, alkyl as defined above; b) i-C6)-alkenyl, wherein alkenyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, 1, (CI-C 6 )-alkoxy, cyano, oxo, nitro, hydroxy, CHO, CO 2 H, COCI-C 6 alkyl, CO2CI-C 6 -alkyl, CONIlaR 2 NRlaR 2 NRlaCOCl -C 6 -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 the group consisting of: Br, Cl, F, I, (CI-C 6 )-alkoxy, cyano, oxo, nitro, hydroxy, CHO, CO 2 H, COCI-C 6 alkyl, C02CI-C 6 -alkyl, CONRIaR 2 NRIaR 2 NRlaC0C C 6 alyl aryl as defined above, and heteroaryl as defined above, d) -aryl, aryl as defined above, or e) -heteroaryl, heteroaryl as defined above; wherein R la and R 2 are as defined above. R' is: a) absent and a is a double bond; b) -H, c) -OH, d) =0, e) -O[(C=O)Or1sCi-CIo-alkyl, alkyl as defined above, f) O0[(CO)Or]sC2-Clo-alkenyl, as defined above, g) -O[(C=0)Or],C 2 -C 6 -alkynyl, alkynyl as defined above, h) O0[(C0)Orls(C 3 -C 7 )-cycloalkyl, i) -O[(C=zO)0,]saryl, aryl as defined above, j) O0[(C0)Orlsheteroaryl, heteroaryl as defined above, k) -O(CH2)nO(CH 2 )mheteroaryl, heteroaryl as defined above, [RALIBAA]07569.doc:TAB 1) 0O(CH2)nO(CH 2 )m..aryl, aryl as defined above, mn) -OC(=0)NR R a, n) -0S0 2 R or o) -NRaR2; wherein and R 2 are as defined above. R 29 a and R 2 9 b are independently: a) -H, c) -(CH 2 ),NR 1 R, d) -(CH2)s0-[(C=0)r],CI-Clo.alkyl, alkyl as defined above, e) -(CH 2 )s-0[(C=0)OrlC 2 -C io-alkenyl, alkenyl as defined above, f) -(CH2)s0[(C=0)rjsaiyl, aryl as defined above, -(CH2),-0[(C=O)Orlsheteroaryl, heteroaryl as defined above, h) -(CH2)s0(CH2)nO(CH 2 )mheteroaryl, heteroaryl as defined above, -~(CH2),sO(CH 2 ).O(CH 2 )maryl, aryl asdefined above, j) -(CH 2 ),-0C(=O)NR 1 R, -(CH 2 8 -OSO 2 R', 1) -(CI-C 6 )-alkyl, alkyl as defined above, or -(C 2 -C 6 )-alkenyl, alkenyl as defined above; or R29 and R 2bcan be taken together to be =0 or =C(Ci-Cloalkyl) 2 alkyl being as defined above. 2. The compound of structural Formula 1, as recited in claim 1, *9 2 C a7 1 IH1 28 0 2 CH 3 94 1 Xis 25,6 I or inphraedently acsingle bond, oryal oubl bonhd; ae heen [R \L1BAA107569.doc:TAB 98 n is: 1 to 4; m is: 1 to 4; ris: 0 or 1; sis: 0 or 1; s R' is: a) H, or b) (C 1 I-C 6 )-alkyl, wherein alkyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (CI-C 6 )-alkoxy, vinyl, cyano, oxo, nitro, hydroxy, CHO, CO 2 H, COCI-C 6 alkyl, CO 2 C1-C 6 -alkyl, CONRIaR2, NlaR2, NRlaCOCi-C 6 -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-C 6 )-alkoxy, phenyl, phenoxy, cyano, nitro, hydroxy, CHO, CO 2 H, COCi-C 6 -alkyl, C0 2 C 1 -C 6 -alkyl, CONRaR 2 laR 15 NR IaCOCI-C 6 -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, (Ci-C 6 )-alkoxy, cyano, nitro, hydroxy, CHO, CO 2 H, COCi-C 6 -alkyl, CO 2 CI-C 6 -alkyl, CONRIaR 2 NRlaR 2 NRlaCOC -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; wherein Ria and R 2 are as defined above. R 3 is: a) -(Ci-C 6 )-alkyl, alkyl as defined above; b) -(C 1 -C 6 )-alkenyl, wherein alkenyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (Ci-C 6 )-alkoxy, cyano, oxo, nitro, hydroxy, CHO, C0 2 H, COCI 1 -C 6 alkyl, CO 2 CI-C 6 -alkyl, CONRIaR 2 NRlaR 2 NRlaCOCi-C 6 -alkyl, aryl as defined above, and heteroaryl as defined above; C) -(C1-C 6 )-alkynyl, wherein alkynyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, [R:\LIBAA]07569 doc:TAB 1, (CI-C 6 )-alkoxy, cyano, oxo, nitro, hydroxy, CHO, CO 2 H, COCI-C 6 alkyl, C0 2 CI-C 6 -alkyl, CONRIaR 2 NRlaR 2 NRlaC0C I-C 6 -alkyl, aryl as defined above, and heteroaryl as defined above, d) -aryl, aryl as defined above, or e) -heteroaryl, heteroaryl as defined above; wherein Ria and R 2 are as defined above. R 4 is: a) absent and a is a double bond, b) -H, c) -OH, d) =O, e) -0[(C=0)r],C-Co-alkyl, alkyl as defined above, f) -0[(C=0)r]sC 2 -Coalkenyl, as defined above, -O[(C=O)Orl,;C 2 -C 6 -alkynyl, alkynyl as defined above, h) -0[(C=0)rs(C 3 -C 7 )-cycloalkyl, i) -0[(C=0)rsaryl, aryl as defined above, 0[(C=0)r]heteroaryl, heteroaryl as defined above, k) 0O(CH 2 )nO(CH 2 ),mheteroaryl, heteroaryl as defined above, 0O(CH 2 )nO(CH 2 )maryl, aryl as defined above, 20 mn) -O(ON -0S0 2 R 3 or o) -NRRa; wherein Rila and R 2 are as defined above. RW 9 and R 2 9b are independently: a) -H, b) -(CH2)s0O[(C0)OrlCp-Clo-alkyl, alkyl as defined above, c) -(CH 2 2 -Clo-alkenyl, alkenyl as defined above, d) -(CH 2 )s-0[(C0)OrlsC 2 -C 6 alkynyl, alkynyl as defined above, e) -(CH 2 )s0[(C0)Orls(C 3 -C 7 )-cycloalkyl, f) -(CH 2 )s0O[(C0)Orlsaryl, aryl as defined above, g) -(CH 2 )s0[(C0)Orlsheteroaryl, heteroaryl as defined above, h) -(CH 2 ),s0(CH 2 )nO(CH 2 )mheteroaryl, heteroaryl as defined above, i) -(CH 2 )s0(CH 2 )nO(CH 2 )maryl, aryl as defined above, j) -(CH 2 )s-OC(=O)NR'R 2 k) -(CH 2 ),-OSO 2 R, 1) -(CI-C 6 )-alkyl, alkyl as defined above, or [R:\LIBAA]07569.doc:TAB 100 m) -(C 2 -C 6 )-alkenyl, alkenyl as defined above; or R29a and R 29 b can be taken together to be =0 or =C(Ci-Cloalkyl) 2 alkyl as defined above. 3. The compound of structural Formula I, as recited in claim 2, R29a R9b 9 2 12 H18 2J 1 17 2 1 I H 1 16 28 02CH3 9 14 3b 10 25 6 15 OAc X 57 O 24 6 OAc OAc -4 OAc 23j4 aR 5 I 4 I or a pharmaceutically acceptable salt, crystal form or hydrate, wherein: Xis: O; 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; s is: 0 or 1; R' is: a) H, or b) (Ci-C 6 )-alkyl, wherein alkyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (Ci-C 6 )-alkoxy, vinyl, cyano, oxo, nitro, hydroxy, CHO, CO 2 H, COCI-C 6 alkyl, C0 2 C 1 -C 6 -alkyl, CONRIaR 2 NRlaR 2 NRlaCOCI -C 6 -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, (CI-C 6 )-alkoxy, phenyl, phenoxy, cyano, nitro, hydroxy, CHO, CO 2 H, COC1-C 6 -alkyl, CO 2 C1-C 6 -alkyl, CONRaR 2 NRlaR2 NRlaCOCi-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 [R:\LIBAA]07569.doc:TAB 101 heteroatoms selected from 0, S, N, unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, 1, (CI-C 6 )-alkoxy, cyano, nitro, hydroxy, CHO, CO 2 H, COCI-C 6 -alkyl, C0 2 CI-C 6 -alkyl, CONR~aR 2 NRlaR 2 NIaC0CI C 6 alky1 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 formn a benzo-fused ring; wherein R laand R 2 are as defined above. R 3 is: -(CI-C 6 )-alkyl, alkyl as defined above, -aryl, aryl as defined above, or -heteroaryl, heteroaryl as defined above. S *5 S S S. S. *55 S.. S S .5.5 S S S. S5 S S S5S555 S S. 55 S S S R 4 is: a) -O[(C=0)Or]sCI-Cio-alkyl, alkyl as defined above, b) O0[(C=O)Ors(C 3 -C 7 )-cycloalkyl, c) -O[(C=0)Orlsaryl, aryl as defined above, d) -0[(C=0)Orjsheteroaiyl, heteroaryl as defined above, e) -0(CH2)nO(CH 2 )mheteroaryl, heteroaryl as defined above, f) 0O(CH2)nO(CH 2 )maryl, aryl as defined above, g) -OC(=0)NR' R or h) -0S0 2 R wherein R la and R 2 are as defined above. R 2aand R 2 9 b are independently: a) -H, b) -(CH 2 )s0-[(C=0)0r],Ci -Cio-alkyl, alkyl as defined above, c) -(CH 2 )s-0[(C=0)OrlC 2 -C io-alkenyl, alkenyl as defined above, d) -(CH2)-O[(C0)OrlsC 2 -C 6 -alkynyl, alkynyl as defined above, e) -(CH2)s-O[(C=0)Orls(C 3 -C 7 )-cycloalkyl, f) -(CH2)s-0[(C=0)Orlsaiyl, aryl as defined above, g) -(CH2)s;-0[(C=O)Orlsheteroaryl, heteroaryl as defined above, h) -(CH2)s-0(CH 2 )n0(CH 2 )n.heteroaryl, heteroaryl as defined above, i) -(CH2)s-O(CH 2 )nO(CH 2 )maryl, aryl as defined above, j) -(CH 2 -OC(=0)NfR 1 R k) -(CH 2 ),-0S0 2 R 3 1) I-C 6 )-alkyl, alkyl as defined above, or [R:\LIBAAJ07569.doc:TAB 102 m) -(C 2 -C 6 )-alkenyl, alkenyl as defined above; or R 2 9a and R 29b can be taken together to be =0 or =C(Ci-Cloalkyl) 2 alkyl as defined above. 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) -0[(C=0)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, CO 2 H, COCi-C 6 -alkyl, CO 2 C 1 -C 6 alkyl, CONRaR2, NRaR2, NRlaCOC1-C 6 -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=0)Or]sheteroaryl, wherein heteroaryl is defined as a 5 or 6- '5 membered ring substituted with one and two heteroatoms selected from O, S, N, unsubstituted or substituted with one, two or three substituents i selected from the group consisting of: Br, Cl, F, I, (Ci-C 6 )-alkoxy, cyano, nitro, hydroxy, CHO, CO 2 H, COCi-C 6 -alkyl, CO 2 C1-C 6 -alkyl, CONRIaR 2 NRIaR 2 NRlaCOCi-C6-alkyl any two of adjacent substituents can be 20 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; wherein Ria and R 2 are as defined above. 5. The compound of structural Formula I, as recited in claim 1, R29a R29b 1 H I O 28 0 2 CH 3 3 1 25 6 15 OAc O 24 6 OAc OAc _4 OAc 23 4 a R I or a pharmaceutically acceptable salt, crystal form or hydrate, wherein: X is: H and R'; i a is: a single bond; [R:\LIBAA]07569.doc:TAB 103 b and c are independently: a single bond or a double bond; n is: 1 to 4; m is: 1 to 4; ris: 0orl; s is: Oorl; R' is: a) H, or b) (Ci-C 6 )-alkyl, wherein alkyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (Ci-C 6 )-alkoxy, vinyl, cyano, oxo, nitro, hydroxy, CHO, CO 2 H, COC 1 -C 6 alkyl, CO 2 Ci-C 6 -alkyl, CONRaR 2 NRlaR 2 NRaCOCi-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: 15 iBr, Cl, F, I, (Ci-C 6 )-alkoxy, phenyl, phenoxy, cyano, nitro, hydroxy, CHO, C02H, COC 1 -C 6 -alkyl, CO 2 C1-C6-alkyl, CONRaR2, NRlaR2, NR aCOCi-C 6 -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 20 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-C 6 )-alkoxy, cyano, nitro, hydroxy, CHO, CO 2 H, COCi-C 6 -alkyl, o: CO 2 C1-C 6 -alkyl, CONRaR 2 NRlaR 2 NRlaCOC-C 6 -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; wherein Ria and R 2 are as defined above. R 3 is: a) -(CI-C 6 )-alkyl, alkyl as defined above; b) -(C 1 -C 6 )-alkenyl, wherein alkenyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, I, (C 1 -C 6 )-alkoxy, cyano, oxo, nitro, hydroxy, CHO, CO 2 H, COC 1 -C 6 alkyl, C0 2 C 1 -C 6 -alkyl, CONRlaR 2 NRlaR 2 NRlaCOCi-C6-alkyl, aryl as defined above, and heteroaryl as defined above; [R:\LIBAA]07569.doc:TAB c) 1 -C 6 )-alkynyl, wherein alkynyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of: Br, Cl, F, 1, (Cl-C 6 )-alkoxy, cyano, oxo, nitro, hydroxy, CHO, CO 2 H, COCI-C 6 alkyl, CO 2 CI-C 6 -alkyl, CONR la R 2 NRlaR' .TRlaCOCl-C 6 -aflkyl, aryl. as defined above, and heteroaryl as defined above, d) -aryl, aryl as defined above, or e) -heteroaryl, heteroaryl as defined above; wherein Rila and R 2 are as defined above. R 4 is: a) absent and a is a double bond, b) -H, c) -OH, d) =0, -0[(C=0)r],C-Cio-alkyl, alkyl as defined above, 15 f) 0O[(C0)Or1sC 2 -Cjoalkenyl, as defined above, *Vog) -O[(C=O)Or]sC 2 -C 6 -alkynyl, alkynyl as defined above, h1) -0[(C=0)r1s(C 3 -C 7 )-cycloalkyl, i) -0[(C=0)r],aryl, aryl as defined above, -0[(C=0)r]sheteroaryl, heteroaryl as defined above, k) 4J((3H2)nO(CH 2 )mnheteroaryl, tieteroaryl as defined aoe 0O(CH 2 )nO(CH 2 )mnaryl, aryl as defined above, mn) -OC(=O)NR 1 Ra, n) -0S0 2 R or 0) -NRIaR 2 wherein R la and R 2 are as defined above. R29 and R 29 b are independently: a) -H, b) -(CH2)s-O[(C'=O)Or]sCp-Cioalkyl, alkyl as defined above, c) -(CH2)s0O[(C0)OrlsC 2 -Clo-alkenyl, alkenyl as defined above, d) -(CH 2 )s0-[(C0)Or]sC 2 -C 6 -alkynyl, alkynyl as defined above, e) -(CH2)s0-[(C0)Orls(C 3 -C 7 -cycloalky, f) -(CH 2 )s0O[(C=0)r]saryl, aryl as defined above, g) -(CH 2 ),s-O[(C=O)Orlsheteroaryl, heteroaryl as defined above, h) -(CH 2 )s0(CH 2 )nO(CH 2 )mheteroaryl, heteroaryl as defined above, i) -(CH 2 ),s0(CH 2 )nO(CH 2 )marYl, aryl as defined above, j) -(CH 2 1 R 2 [R:\LIBAA]07569.doc:TAB 105 k) -(CH 2 )-OS0 2 R 3 1) -(Ci-C 6 )-alkyl, alkyl as defined above, or m) -(C 2 -C 6 )-alkenyl, alkenyl as defined above; or R2a and R2b can be taken together to be =0 or =C(Ci-Cloalkyl) 2 alkyl being as defined above. 6. The compound of structural Formula I, as recited in claim R29a R29b 9 2 12 H18 J C 17 11 1 1 28 0 2 CH 3 -1 g I 14OH 16 3 b 125 6 15 OAc X 57 O 24 5 6 OAc OAc 4 ()Ac 23* 23 4 V. aR a R J or a pharmaceutically acceptable salt, crystal form or hydrate, wherein: S., *X is: H and R'; 10 a is: a single bond; b and c are independently: a single bond or a double bond; n is: 1 to 4; Sm is: 1 to 4; 15 ris: 0 or 1; sis: 0 or 1; R' is: a) H, or b) (C 1 -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-C 6 )-alkoxy, vinyl, cyano, oxo, nitro, hydroxy, CHO, CO 2 H, COC 1 -C 6 alkyl, CO 2 C1-C 6 -alkyl, CONRIaR 2 NRlaR 2 NRlaCOC-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, (CI-C 6 )-alkoxy, phenyl, phenoxy, cyano, nitro, hydroxy, CHO, CO 2 H, COCI-C6-alkyl, C0 2 C 1 -C 6 -alkyl, CONRIaR 2 NRlaR 2 NR IaCOC 1 -C 6 -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 [R:\LIBAA]07569.doc:TAB C 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, (C 1 -C 6 )-alkoxy, cyano, nitro, hydroxy, CHO, CO 2 H, COC 1 -C 6 -alkyl, C0 2 C 1 -C 6 -alkyl, CONRaR2, NlaR2, NRlaCOCi-C 6 -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; wherein Rla and R 2 are as defined above. R 3 is: -(Ci-C 6 )-alkyl, alkyl as defined above; b) -(Ci-C 6 )-alkenyl, wherein alkenyl is unsubstituted or substituted with one, S* 15 two or three substituents selected from the group consisting of: Br, Cl, F, I, (Ci-C 6 )-alkoxy, cyano, oxo, nitro, hydroxy, CHO, CO 2 H, COCI-C 6 yCalkyl, C0 2 C 1 -C 6 -alkyl, CONRaR 2 RlaR 2 RlaCOC-C 6 -alkyl, aryl as defined above, and heteroaryl as defined above; c) -(C 1 I-C 6 )-alkynyl, wherein alkynyl is unsubstituted or substituted with one, S' 20 two or three substituents selected from the group consisting of: Br, Cl, F, *a I, (Ci-C 6 )-alkoxy, cyano, oxo, nitro, hydroxy, CHO, CO 2 H, COC 1 -C 6 alkyl, C0 2 C 1 -C 6 -alkyl, CONRiaR2, NRaR2, NRlaCOC 1 -C 6 -alkyl, aryl as defined above, and heteroaryl as defined above, d) -aryl, aryl as defined above, or e) -heteroaryl, heteroaryl as defined above; wherein RIa and R 2 are as defined above. R 4 is: a) -OH, b) -O[(C=0)r]sCi-Clo-alkyl, alkyl as defined above, c) -O[(C=O)O]s(C 3 -C 7 )-cycloalkyl, d) aryl as defined above, e) -O[(C=O)O,],heteroaryl, heteroaryl as defined above, f) -O(CH 2 )nO(CH 2 )mheteroaryl, heteroaryl as defined above, g) -O(CH 2 )nO(CH 2 )maryl, aryl as defined above, h) -OC(=O)NRaR2, or i) -OS 2 R; [R:\LIBAA]07569.doc:TAB R29 and R 2 9 b are independently: a) -H, b) -(CH 2 )-0[(C=0)r]Ci-Co-alkyl, alkyl as defined above, c) 2 -Clo-alkenyl, alkenyl as defined above, d) -(CH 2 2 -C-alkyny1, alkynyl as defined above, e) -(CH2)-0[(C=0)r1s(C 3 -C 7 -cycloalkyl, f) -(CH2)s-0[(C=0)Orbsaryl, aryl as defined above, g) -(CH2)s-0[(C=0)Or1sheteroaryl, heteroaryl as defined above, h) -(CH2)s,-0(CH 2 )nO(CH 2 )m..heteroaryl, heteroaryl as defined above, i) -(CH 2 )s-0(CHA)O(CH 2 aryl, aryl as defined above, j) -(CH 2 )s-OC(=0)NRR 2 k) -(CH 2 )s-0 2 R 3 1) I-C 6 )-alkyl, alkyl as defined above, or M) -(C 2 -C 6 )-alkenyl, alkenyl as defined above, n) =C(CI-Clo-alkyl) 2 alkyl as defined above; or R 29 a an 2 bcan be taken together to be =0 or =C(Ci-Cloalkyl) 2 alkyl being as defined above. 7. A compound selected from the group consisting of: 4,6,7,15,1 6-pentakis(acetyloxy)-2 1,22-epoxy-i 18-hydroxy-22-methoxycarbony [6,7c, 15 P3, *0 20 16 6j,20a,2 1 1,22f3]D :A-Friedo-A-homo-27,3 O-dinor-24-oxaoleana- 1 -en-3 -one; 4,6,7,15,1 6-pentakis(acetyloxy)- 18-hydroxy-22-methoxycarbonyl[6(,7a, 15f3,l61,20c.] D :A-Friedo-A-homo-27,30-dinor-24-oxaoleana- 1,21 -dien-3-one; 4,6,7,15,1 6-pentakis(acetyloxy)-2 1,22-epoxy- I 8-hydroxy-22-methoxycarbony[6o,7a, 15 P3, 16 Pf,2ocx,21 P3,22f3]D :A-Friedo-A-homo-27,3 O-dinor-24-oxaoleana- 1 -ene; 25 4,6,7,1 5,1 6-pentakis(acetyloxy)- 1 8-hydroxy-22-methoxycarbonyl [&C,70C, 15P, 16 6P,20C~] D:A-Friedo-A-homo-27,30-dinor-24-oxaoleana- 1,21 -diene; 4,6,7,15,1 6-pentakis(acetyloxy)- 1 8-hydroxy-22-methoxycarbonyl-[6a,7c, 15 P3, 1 63]D :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-[6(,7L, 15 P3, 1 63]D :A- Friedo-A-homo-27,3 O-dinor-24-oxaoleana- 1,21 -dien-3 4-(2-bromobenzoyl)oxy-6,7, 15,1 6-tetrakis(acetyloxy)- 1 8-hydroxy-22-methoxycarbonyl [6cc,7cc, 15 P3, 1 6J,2Ocg :A-Friedo-A-homo-27,30-dinor-24-oxaoleana- 1,21 -dien-3-one; 4,6,7,15,1 6-pentakis(acetyloxy)-2 1,22-epoxy-i1 8-hydroxy-22-methoxycarbonyl[6cc,7a., 15 f0, 1 6P,2Oa,2 1 P,22 P]D :A-Friedo-A-homo-27,3 O-dinor-24-oxaoleana-3 -one; [R:\LIBAAj07569.doc:TAB 108 4,6,7,15,16-pentakis(acetyloxy)- 1 8 -hydroxy-22-methoxycarbonyl-[6a,7a,15 ,1 61,20a] D:A-Friedo-A-homo-27,30-dinor-24-oxaoleana-2 1 -en-3-one; 4,6,7,15,16-pentakis(acetyloxy)-21,22-epoxy-1 8 -hydroxy-22-methoxycarbonyl[6c,7c,15 1 6 P,20ac,21 1,22P]D:A-Friedo-A-homo-27,30-dinor-24-oxaolean; 4,6,7,15,16-pentakis(acetyloxy)- 18-hydroxy-22-methoxycarbonyl-[6 15P,16P,20a] D:A-Friedo-A-homo-27,3 O-dinor-24-oxaoleana-21-ene; 4,6,7,15,16-pentakis(acetyloxy)- 1 8 -hydroxy-22-methoxycarbonyl-[6a,7a, 15P,160]D:A- Friedo-A-homo-27,30-dinor-24-oxaoleana-20(29),2 1 -dien-3-one; 4,6,7,15,16-pentakis(acetyloxy)- 1 8 -hydroxy-22-methoxycarbonyl-[6a,7a, 15P,16P]D:A- Friedo-A-homo-27,30-dinor-24-oxaoleana-21-en-3,20-dione; 4-(2-bromobenzoyl)oxy-6,7,15,16-tetrakis(acetyloxy)- 1 8 -hydroxy-22-methoxycarbonyl [6a,7ca,15P,1 6P,20]D:A-Friedo-A-homo-27,3 0-dinor-24-oxaoleana-21-en-3-one; 4-(2-bromobenzoyl)oxy-6,7,15,16-tetrakis(acetyloxy)-21,22-epoxy- 8-hydroxy-22- 0 0 methoxycarbonyl[6a,7u, 15p,16P,20a,21 ,22P]D:A-Friedo-A-homo-27,30-dinor-24- .0 15 oxaoleana-3-one. 8. An immunosuppressive 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 0 of claims 1 to 8 or a pharmaceutically acceptable crystal form or hydrate thereof. 10. The pharmaceutical formulation of claim 9, comprising in addition, a second immunosuppressive agent selected from the group consisting of azathioprine, brequinar sodium, deoxyspergualin, mizaribine, mycophenolic acid morpholino ester, cyclosporin, FK-506 and rapamycin. 25 11. A method treating a condition in a mammal, the treatment of which is effected or facilitated by K,1.3 inhibition, the method comprising the administration to the mammal, in an amount that is effective at inhibiting Kv1.3, of a compound of formula 1 as recited in any one of claims 1 to 8 or of a formulation as recited in claim 9 or claim 12.A compound of formula 1 as recited in any one of claims 1 to 8 or the formulation as recited in claim 9 or claim 10, when used for treating a condition in a mammal, the treatment of which is effected or facilitated by K,1.3 inhibition. 13. Use of a compound of formula 1 as recited in any one of claims 1 to 8 for the manufacture of a medicament for treating a condition in a mammal, the treatment of which is effected or facilitated by Kv1.3 inhibition. 14. The method as recited in claim 11, the compound or formulation as recited in claim 12 or the use as recited in claim 13, wherein the condition is selected from the group [R:\LIBAA]07569.doc:TAB 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, seborrhoea dermatitis, Lichen planus, Pemphigus, bullous pemphigoid, Epidermolysis bullosa, urticaria, angioedemas, vasculitides, erythemas, cutaneous eosinophilias, Lupus erhtyematosus, acne, Alopecia areata, keratoconjunctivitis, vernal conjunctivitis, uveitis associated with Behcet's disease, keratitis, herpetic keratitis, conical cornea, dystrophia epithelialis comeae, corneal cataracta, siderosis, retinitis, pigmentosa, senile macular degeneration, vitreal scarring, corneal alkali bum; dermatitis erythema multiform, linear IgA ballous dermatitis and cement dermatitis, gingivitis, periodontitis, sepsis, pancreatitis, diseases caused by environmental pollution, 15 aging, carcinogenis, metastasis of carcinoma and hypobaropathy; disease caused by histamine or leukotriene-C 4 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 20 failure, "acute-on-chronic" liver failure, augmentation of chemotherapeutic effect, preventing or treating activity of cytomegalovirus infection, HCMV infection, and anti- inflammatory activity; and treatment of immunodepression or a disorder involving immunodepression, including AIDS, cancer, senile dementia, trauma, chronic bacterial Sinfection, and certain central nervous system disorders. 25 15. The method, compound or use as recited in claim 14, wherein the condition is an autoimmune disease. 16. A method of preventing or treating the resistance to transplantation or transplantation rejection of an organ or tissue in a patient, which method comprises the administration of a compound as recited in any one of claims 1 to 8 or of a formulation of claim 9 or claim 17. A compound as recited in any one of claims 1 to 8 or a formulation as recited in claim 9 or claim 10 when used for preventing or treating the resistance to transplantation rejection of an organ or tissue in a patient. 18. The use of a compound as recited in any one of claims 1 to 8 for the manufacture of a medicament for preventing or treating the resistance to transplantation rejection of an organ or tissue in a patient. [R:\LIBAA]07569.doc:TAB 110 19. A method of suppressing the immune system in a subject, which method comprises the administration to the subject of an immune suppressing amount of a compound of formula I as recited in any one of claims 1 to 8 or of a formulation as recited in claim 9 or claim 20. A compound as recited in any one of claims 1 to 8 or a formulation as recited in claim 9 or claim 10 when used for suppressing the immune system in a subject. 21. The use of a compound as recited in any one of claims 1 to 8 for the manufacture of a medicament for suppressing the immune system in a subject. 22. The method as recited in claim 19, comprising the coadministration of a second 0o immunosuppressive agent. 23. A method of preventing or treating resistance by transplantation of an organ or tissue, graft-versus-host disease 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 15 mellitus, posterior uveitis, allergic encephalomyelitis, glomerulonephritis, infectious 0 diseases caused by pathogenic microorganisms, inflammatory and hyperproliferative skin diseases, psoriasis, atopical dermatitis, contact dermatitis, eczematous dermatitises, seborrhoea dermatitis, Lichen planus, Pemphigus, bullous pemphigoid, Epidermolysis bullosa, urticaria, angioedemas, vasculitides, erythemas, cutaneous eosinophilias, Lupus 20 erythematosus, acne, Alopecia areata, keratoconjunctivitis, vemal 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, S reversible obstructive airway disease, bronchial asthma, allergic asthma, intrinsic asthma, 25 extrinsic asthma and dust asthma, chronic or inveterate asthma, late asthma and airway hyper-responsiveness, bronchitis, gastric ulcers, vascular damage caused by ischaemic diseases and thrombosis, ischaemic bowel diseases, inflammatory bowel diseases, necrotising enterocolitis, intestinal lesions associated with thermal burs 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, polyneuritis, multiple neuritis, mononeuritis, radiculopathy, hyperthyroidism, Basedow's disease, pure red cell aplasia, aplastic anaemia, hypoplastic anaemia, idiopathic thrombocytopenic purpura, autoimmune hemolytic anaemia, agranulocytosis, pernicious anaemia, megaloblastic anaemia, anerythroplasia, osteoporosis, sarcoidosis, fibroid lung, idiopathic interstitial pneumonia, dermatomyositis, [R:\L1BAA]07569.doc.TAB 111 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 and/or promoting hair generation and hair growth; muscular dystrophy; Pyoderma and Sezary's syndrome, Addison's disease, ischaemia-reperfusion injury of organs which occurs upon preservation, transplantation or ischaemic disease, for example, thrombosis and cardiac infraction, endotoxin-shock, pseudomembranous colitis, colitis caused by drug or radiation, ischaemic 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 bur; dermatitis erythema multiform, linear IgA ballous dermatitis and cement dermatitis, gingivitis, periodontitis, o* 15 sepsis, pancreatitis, diseases caused by environmental pollution, aging, carcinogenis, metastasis of carcinoma and hypobaropathy; disease caused by histamine or leukotriene-C 4 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 20 cirrhosis, hepatic failure, fulminant hepatic failure, late-onset hepatic failure, "acute-on- chromic" liver failure, augmentation of chemotherapeutic effect, preventing or treating activity of cytomegalovirus infection, HCMV infection, and anti-inflammatory activity; and treatment of immunodepression or a disorder involving immunodepression, including AIDS, cancer, senile dementia, trauma, chronic bacterial infection, and certain central I 25 nervous system disorders, which method comprises the administration of a compound as recited in any one of claims 1 to 8 or of a formulation as recited in claim 9 or claim

24. A compound as recited in any one of claims 1 to 8 or a formulation as recited in claim 9 or claim 10 when used for preventing or treating resistance by transplantation of an organ or tissue, graft-versus-host disease 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, seborrhoea dermatitis, Lichen planus, Pemphigus, bullous pemphigoid, Epidermolysis bullosa, urticaria, angioedemas, vasculitides, erythemas, cutaneous eosinophilias, Lupus [R:\LIBAA]07569.doc:TAB i 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 hyper-responsiveness, bronchitis, gastric ulcers, vascular damage caused by ischaemic diseases and thrombosis, ischaemic bowel diseases, inflammatory bowel diseases, necrotising enterocolitis, intestinal lesions associated with thermal bums 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, polyneuritis, multiple neuritis, mononeuritis, radiculopathy, hyperthyroidism, Basedow's disease, pure red cell aplasia, aplastic anaemia, 15 hypoplastic anaemia, idiopathic thrombocytopenic purpura, autoimmune hemolytic I: anaemia, agranulocytosis, pernicious anaemia, megaloblastic anaemia, 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, 20 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 *9 dystrophy; Pyoderma and Sezary's syndrome, Addison's disease, ischaemia-reperfusion 25 injury of organs which occurs upon preservation, transplantation or ischaemic disease, for example, thrombosis and cardiac infraction, endotoxin-shock, pseudomembranous colitis, colitis caused by drug or radiation, ischaemic 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 multiform, 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-C 4 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 [R:\LIBAA]07569.doc:TAB I IC cirrhosis, hepatic failure, fulminant hepatic failure, late-onset hepatic failure, "acute-on- chromic" liver failure, augmentation of chemotherapeutic effect, preventing or treating activity of cytomegalovirus infection, HCMV infection, and anti-inflammatory 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. The use of a compound as recited in any one of claim 1 to 8 or a formulation as recited in claim 9 or claim 10 in the manufacture of a medicament for preventing or treating resistance by transplantation of an organ or tissue, graft-versus-host disease 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 15 dermatitis, contact dermatitis, eczematous dermatitises, seborrhoea 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 coreae, corneal leukoma, 20 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, 9°9 gastric ulcers, vascular damage caused by ischaemic diseases and thrombosis, ischaemic 25 bowel diseases, inflammatory bowel diseases, necrotising enterocolitis, intestinal lesions associated with thermal bums 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, polyneuritis, multiple neuritis, mononeuritis, radiculopathy, hyperthyroidism, Basedow's disease, pure red cell aplasia, aplastic anaemia, hypoplastic anaemia, idiopathic thrombocytopenic purpura, autoimmune hemolytic anaemia, agranulocytosis, pernicious anaemia, megaloblastic anaemia, 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, [R;\LIBAA]07569.doc:TAB L I 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, ischaemia-reperfusion injury of organs which occurs upon preservation, transplantation or ischaemic disease, for example, thrombosis and cardiac infraction, endotoxin-shock, pseudomembranous colitis, colitis caused by drug or radiation, ischaemic 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 bur; dermatitis erythema multiform, 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-C 4 release; Behcet's disease, autoimmune 15 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- top. A/non-B hepatitis, cirrhosis, alcoholic cirrhosis, hepatic failure, fulminant hepatic failure, a. late-onset hepatic failure, "acute-on-chromic" liver failure, augmentation of chemotherapeutic effect, preventing or treating activity of cytomegalovirus infection, 20 HCMV infection, and anti-inflammatory 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. Dated 30 August, 1999 Merck Co., Inc. Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON [R:\LIBAA]07569.doc:TAB