WO2000054760A2 - Bicyclo (3.3.1) nonenes useful for the treatment of diabetes and hypertriglyceridemia - Google Patents

Abstract

Bicyclo[3.3.1]nonenes, pharmaceutical compositions containing the nonenes and methods of using the compounds and compositions as hypoglycemic or hypotriglyceridemic agents are described. The bicyclo[3.3.1]nonenes and corresponding compositions are useful for lowering blood glucose levels, treating hyperglycemia and treating diabetes. The nonenes and corresponding compositions are also useful for lowering serum triglyceride levels and treating hypertriglyceridemia.

Description

BICYCLO [3.3.1] NONENES USEFUL FOR THE TREATMENT OF DIABETES

RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No. 09/270,489 (attorney docket no. SHBOT003) filed March 15, 1999, which is herein incorporated by reference.

1. FIELD OF INVENTION

The present invention relates to bicyclo [3.3.1] nonenes use in treating hypertriglyceridemia, treating diabetes mellitus, lowering serum triglyceride levels and lowering blood glucose, particularly from Hypericum spp.

2. BACKGROUND OF THE INVENTION 2.1 USES OF HYPERICUM SPP

Hypericum spp, and Clusea spp. are described as belonging to the family Clusiaceae, Guttiferae or Hypericaceae. Hypericum perforatum (Clusiaceae), is a perennial plant which grows widely throughout Europe, Asia, North America and North Africa. [Bombardelli, E.,et al, Fitoterapia 66(1}: 43 (1995)]. Members of the neotropical genus Clusia are found in North and South America, [de Oliveira, CM. A., et al, Tetrahedron Letts. 37(36^:6427 (1996). Plant extracts of these genera have been used in traditional medicine to treat a variety of maladies, including as a promoter of wound healing [Milliken, W., et al Economic Botany 50d):10 (1996), and Gurevich, et al, Antibiotix 16ι 510-513 (1971)]; as an antibacterial [Trifunovic, S, et al, Phytochemistry 49(5}: 1305 (1998)]; and as an antibiotic [Sakar, M.K., et al, Fitoterapia 59:49, (1998)]. Hypericum spp. members are currently being investigated for use in the treatment of depression [G. Loakmann et al, Pharmacopsychiat 31 (Supplement): 54-59], Bhattacharya, S. K., et al, Pharmacopsychiat 31 (Supplement): 22-29 (1998)]. 2.2 COMPOUNDS ISOLATED FROM CLUSIA AND HYPERICUM SPP 2.2.1 BICYCLO 13.3.11 NONENES

Bicyclo [3.3.1] nonenes have been characterized as being acvlphloroglucinol derivatives [Erdelmeier. et al, Pharmacopsychiac 31 (Supp): 2 (1998)]: benzophenones Cerrini, S.. et al, Phytochemistry 32(4}: 1023 (1992) or bicyclic tetraketones in the enol form [Bystrov, N.S.. et al, Tetrahedron Lett. 32: 2791 (1975)]. generally of the formula (I)

(I) (with the R groups as defined in the references)

Examples of naturally occuring bicyclo-nonenes of formula I include, for example, hyperforin (Compound 1) and adhyperforin (Compound 2). Hyperforin is also known as bicyclo[3.3.1]non-3-ene-2,9-dione, 4-hydroxy-6-methyl-l,3,7-tris(3- methyl-2-butenyl)-5-(2-methyl-l-oxopropyl)-6-(4-methyl-3-pentenyl)-, (lR,5S,6R,7S); 4-hydroxy-l-isobutyryl-8-methyl-3,5,7-tris(3-methyl-2-butenyl)-8-(4- methyl-3-pentenyl)-e o-bicyclo[3.3.1]non-3-ene-2,9-dione, 4-hydroxy- l-isobutyryl-8- methyl-3,5,7-tris(3-methylbuten-2-yl)-8-(4-methylpenten-3-yl)-exo- bicyclo[3.3.1]non-3-ene-2,9-dione, 4-hydroxy-l-oxobutyl-8-methyl-3,5,7-tris(3- methyl-2-butenyl)-8-(4-methyl-3-pentenyl)-exo-bicyclo[3.3.1]non-3-ene-2,9-dione and 4-hydroxy- 1 -(2 -methyl- 1 -oxopropyl)-8-methyl-3 ,5 ,7-tris(3 -methyl-2-butenyl)-8- (4-methyl-3-pentenyl)-e o-bicyclo[3.3.1]non-3-ene-2,9-dione. (Compound 1) [Gurevich, et al, Antibiotix 16: 510-513 (1971)]. Adhyperforin is also known as bicyclo[3.3.1 ]non-3-ene-2,9-dione, 4-hydroxy-6-methyl-l,3,7-tris(3-methyl-2- butenyl)-5-(2-methyl-l-oxobutyl)-6-(4-methyl-3-pentenyl)-, (1R,5S,6R,7S); 4- hydroxy-l-(2-methylbutyryl)-8-e.γo-methyl-3,5,7-tris(3-methylbuten-2-yl)-8-(4- methylpenten-3-yl)-eλ'o-bicyclo[3.3.1 ]non-3-ene-2,9-dione, 4-hydroxy- 1 -(2- methylbutyryl)-8-exo-methyl-3,5.7-tris(3-methyl-2-butenyl)-8-(4-methyl-3-pentenyl)- e o-bicyclo[3.3.1 ] non-3-ene-2.9-dione (Compound 2) and 4-hydroxy- l-(2-methyl-l - oxobutyl)-8-cΛO-methyl-3.5.7-tris(3-methyl-2-butenyl)-8-(4-methyl-3-pentenyl)-exo- bicyclo[3.3.1 ] non-3-ene-2,9-dione. (Compound 2) [Maisenbacher, et al. Planta Med. 58(4): 291-293 (1992)].

3. SUMMARY OF THE INVENTION

The present invention provides methods of using isolated or purified bicyclo[3.3.1 ]nonenes of the formulae (II)a, (IΙI)a or mixtures thereof, as well as pharmaceutically acceptable salts thereof. Particularly, the present invention provides methods for blood glucose levels, treating hyperglycemia, and treating NTDDM comprising administering to a mammal a therapeutically effective amount of an isolated or a purified bicyclo[3.3.1]nonene of formula (II)a:

wherein

R, is selected from the group consisting of hydroxy and oxygen;

R₂ is selected from the group consisting of hydroxy, oxygen, and benzoyl; wherein both R, and R₂ are not simultaneously oxygen R₃ is selected from the group consisting of hydrogen, benzoyl, methyl, methylhalide. 3-methyl-2-butenyl, and -(CH₂)_xCOOR₄; wherein X is 0 to 2; wherein

R₂ and R₃ can form a ring, said ring selected from the group consisting of a furan ring and a pyran ring; wherein R₄ selected from the group consisting of hydrogen and C,-

C₃ alkyl; R₅ is selected from the group consisting of hydrogen and C,-C₆ alkyl;

R₆ is selected from the group consisting of 3-methyl-2-butenyl, isobutyryl, 2-methylbutyryl, and benzoyl;

R₇ is selected from the group consisting of hydrogen, methyl, methylhalide, 3-methyl-2-butenyl, and -(CH₂)_xCOOR₄, wherein X is 0 to 2; wherein R₄ selected from the group consisting of hydrogen and C,-C₃ alkyl; and R_s is selected from the group consisting of hvdrogen. methyl, methvlhahde, 4-meth\ l-3-penten> l. and -(CH_;)_λCOOR₄, w herein X is 0 to 2, and wherein R₄ selected from the group consisting of hydrogen and C,-C alkyl

Such methods optionally contain a pharmaceutically acceptable earner and optionally another hypotriglyceridemic or hypoglycemic agent useful for loweπng serum tπglyceπde levels, blood glucose or treatment of diabetes mel tus

Preferred compounds for use in loweπng glucose levels include

3-benzovl-4-hydroxy-8,8-dιmethyl-l,5,7-tπs(3-methyl-2- butenyl)bιcyclo[3 3 l]non-3-ene-2.9-dιone, 5-benzovl-4-hvdroxy-8.8-dιmethvl-l,3,7-tπs(3-methyl-2-butenyl)-exo- bιcyclo[3 3 l]non-3-ene-2.9-dιone,

4-hydroλ\-5-(3'-hydroxybenzoyl)-8,8-dιmethyl-l,3,7-tπs(3-methyl-2- butenyl)-eγσ-bιcyclo[3 3 l]non-3-ene-2,9-dιone,

4-hydroxy-8-e o-methyl-5,7-e o-bιs(3-methylbut-2-enyl)-l-(2-methyl- l-oxopropyl)-8-e«Jo(4-methylpent-3-enyl)bιcyclo[3 3 l]non-3-ene-2,9-dιone,

4-hydroxv-8-ero-methyl-5,7-e o-bιs(3-methyl-2-butenyl)-l-(2-methyl- l-oxobutyl)-8-e«Jo(4-methyl-3-pentenyl)bιcyclo [3 3 l]non-3-ene-2,9-dιone,

1 -(2 -methyl- 1 -oxopropyl)-2, 12-dιoxo-3 , 10β-bιs(3-methyl-2-butenyl)- 6β-(l -methyl- l-hydroxyethyl)-l 1 β-methyl-1 lα(4-methyl-3-pentenyl)-5-oxatncyclo- [6 3 1 0^{4 8}]-3-dodecene, and l-(2-methyl-l -oxopropyl)-2,12-dιoxo-3,10β-bιs(3-methyl-2-butenyl)- 6-hydroxy-l l β-methyl-l l α-4(methyl-3-pentenyl)-5-oxatπcyclo[6 3 1 0^{4 8}]-3- dodecene

Especially preferred compounds include 4-hydroxy-l-ιsobutyryl-8-methyl-3,5,7-tπs-(3-methyl-2-butenyl)-8-(4- methyl-3-pentenyl)-e o-bιcyclo[3 3 l]non-3-ene-2,9-dιone (Compound 1), and

4-hydroxy- 1 -(2-methylbutyryl)-8-(4-methyl-3-pentenyl)-8-methyl- 3,5,7-tns-(3-methyl-2-butenyl)-exo-bicyclo[3 3 l]non-3-ene-2,9-dιone (Compound 2)

In another embodiment of the present invention, the present invention provides methods for loweπng blood glucose levels, treating hyperglycemia and treating NIDDM compπsing administeπng to a mammal a therapeutically effective amount of an isolated or a puπfied bιcyclo[3 3 l]nonene of the formula (IΙI)a

Formula (IΙI)a

wherein

R', is selected from the group consisting of O and OH;

R'₂ is selected from the group consisting of O and OH, wherein R', and R'_: cannot simultaneously be O; and

R'₃ is H or CH₃; and a pharmaceutically acceptable carrier.

Such methods optionally contain optionally another hypoglycemic agent useful for lowering blood glucose or treatment of diabetes mellitus.

Especially preferred bicyclo[3.3.1]nonene compounds of Formula (IΙI)a include: 4-hydroxy-l-isobutyryl-8-methyl-3,5,7-tris-(3-methyl-2-butenyl)-8-(4- methyl-3-pentenyl)-e,vo-bicyclo[3.3.1]non-3-ene-2,9-dione (Compound 1); and

4-hydroxy- 1 -(2-methylbutyryl)-8-(4-methyl-3-pentenyl)-8-methyl- 3,5,7-tris-(3-methyl-2-butenyl)-e o-bicyclo[3.3.1]non-3-ene-2,9-dione (Compound 2).

The present invention also provides methods of using isolated or purified bicyclo nonenes of the formula (II)b or mixtures thereof, as well as pharmaceutically acceptable salts thereof. Particularly, the present invention provides methods for lowering serum triglyceride levels, and treating hypertriglyceridemia comprising administering to a mammal a therapeutically effective amount of an isolated or a purified bicyclo[3.3.1 ]nonene of formula (II)b:

(II)b wherein

R", is selected from the group consisting of hydroxy, C,-C, alkoxy. and oxygen,

R"_: is selected from the group consisting of hydroxy, C,-C₃ alkoxy, oxygen, and benzoyl, wherein both R", and R"₂ are not simultaneously oxygen

R"₃ is selected from the group consisting of hydrogen, methyl, methylhalide, 3-methyl-2-butenyl, and -(CH,\COOR"₄, wherein X is 0 to 2. wherein R"₂ and R'\ can form a πng, said πng selected from the group consisting of a furan πng and a pyran πng, wherein R"₄ selected from the group consisting of hydrogen and C,-C₃ alkyl,

R"₅ is selected from the group consisting of hydrogen and C_rC₆ alkyl,

R"₆ is selected from the group consisting of 3-methyl-2-butenyl, isobutyryl, 2-methylbutyryl, and benzoyl; said benzoyl optionally substituted with hydroxy;

R"₇ is selected from the group consisting of hydrogen, methyl, methylhalide. 3-methyl-2-butenyl. and -(CH,)_xCOOR"₄, and

R"₈ is selected from the group consisting of hydrogen, methyl, methylhalide, 4-methyl-3-pentenyl, and -(CH₂)_xCOOR"₄; and a pharmaceutically acceptable earner.

Such methods optionally contain another hypotπglyceπdemic or hypoglycemic agent useful for loweπng serum tnglycende levels, blood glucose or treatment of diabetes melhtus Preferred compounds include

3-benzoyl-4-hydroxy-8,8-dιmethyl-l,5,7-tπs(3-methyl-2- butenyl)bιcyclo[3.3 1 ]non-3-ene-2,9-dιone,

3-benzoyl-4-methoxy-8,8-dιmethyl-l,5,7-tπs(3-methyl-2- butenyl)bιcyclo[3 3 l]non-3-ene-2,9-dιone, 5-benzoyl-4-hydroxy-S.8-dimethyl-1.3.7-tris(3-methyl-2-butenyl)-e.ro- bicyclo[3.3.1 ]non-3-ene-2,9-dione;

5-benzoyl-4-methoxy-8.8-dimethyl-l ,3,7-tris(3-methyl-2-butenyl)-e o- bicyclo[3.3.1 ]non-3-ene-2,9-dione; 4-hydroxy-5-(3'-hydroxybenzoyl)-8.8-dimethyl-l,3,7-tris(3-methyl-2- butenyl)-e.γo-bicyclo[3.3.1 ]non-3-ene-2,9-dione;

5-(3'-hydroxybenzoyl)-4-methoxy-8.8-dimethyl-l,3,7-tris(3-methyl-2- butenyl)-e.vo-bicyclo[3.3.1]non-3-ene-2,9-dione;

4-hydroxy-8-e o-methyl-5,7-exo-bis(3-methyl-2-butenyl)-l-(2-methyl- l-oxopropyπ-8-eHcfo(4-methyl-3-pentenyl)bicyclo [3.3.1]non-3-ene-2.9-dione; and

4-hydroxy-8-exo-methyl-5,7-exo-bis(3-methyl-2-butenyl)-l-(2-methyl- l-oxobutyl)-8-e/; o(4-methyl-3-pentenyl)bicyclo [3.3.1]non-3-ene-2,9-dione.

Additional preferred compounds include the oxidation derivatives for example, 1 -(2-methyl- 1 -oxopropyl)-2, 12-dioxo-3 , 10β-bis(3-methyl-2-butenyl)-

6β-( 1 -methyl- 1 -hydroxyethyl)- 1 1 β-methyl- 1 1 α(4-methyl-3-pentenyl)-5-oxatricyclo- [6.3.1.0^{4 8}]-3-dodecene; and

1 -(2-methyl- 1 -oxopropyl)-2, 12-dioxo-3 , 10β-bis(3-methyl-2-butenyl)- 6-hydroxy-l l β-methyl- 1 1 α-4(methyl-3-pentenyl)-5-oxatricyclo[6.3.1.0^4,8]-3- dodecene.

In another embodiment of the present invention, the present invention provides methods for lowering serum triglyceride levels and treating hypertriglyceridemia comprising the steps of administering to a mammal a therapeutically effective amount of an isolated or a purified bicyclo[3.3.1 ]nonene of the formula (IΙI)b:

Formula (IΙI)b wherein

R', is selected from the group consisting of O and OH; R'₂ is selected from the group consisting of O and OH. wherein R', and R'₂ cannot simultaneously be 0; and R'₃ is H or CH,; and a pharmaceutically acceptable carrier.

Such methods optionally contain another hypotriglyceridemic agent useful for lowering serum triglyceride levels or treatment of hypertriglyceridemia. An especially preferred compound for lowering serum triglyceride levels is 4-hydroxy- 1 -isobutyryl-8-methyl-3,5,7-tris-(3-methyl-2-butenyl)-8-(4- methyl-3-pentenyl)-exo-bicyclo[3.3.1 ]non-3-ene-2,9-dione (Compound 1).

The present invention may be understood more fully by reference to the following figures, detailed description and illustrative examples which are intended to exemplify non-limiting embodiments of the invention.

4. DESCRIPTION OF THE FIGURES

Fig. 1 is a bar graph showing the plasma glucose levels (mg/dL) of diabetic mice treated with varying doses of hyperforin (Compound 1) and adhyperforin (Compound 2). Animals were dosed at 0, 24 and 48 hours. All data points N = 8. *p<0.05; **p<0M ; ***p<0.000\ (analysis of vanance (ANOVA), one factor).

Fig. 2 is a line graph showing the reduction of post-prandial plasma glucose levels (mg.dL) of db/db mice treated with vehicle only; and 40 and 80 mg/kg q.d. of Compound 1. -•- represents vehicle; -D- represents Compound 1 administered at 40 mg/kg q.d.; -Δ- represents Compound 1 administered at 80 mg/kg q.d.; and -O- represents metformin administered at 250 mg/kg q.d.. Compound 1 was administered to the animals at 0, 8, 24, and 48 hours, followed by a glucose bolus (2g/kg) at 51 hours, and plasma glucose levels were measured at 0, 15, 30, 60 and 120 minutes after the glucose load. All data points N = 8. *p < 0.05; **p < 0.01, ***p < 0.001 (analysis of variance (ANOVA), one factor). Fig. 3 is a bar graph showing the reduction of the area under the curves of Fig. 2 for db/db mice treated with vehicle only; 250 mg/kg q.d. of metformin and 40 and 80 mg/kg q.d. of Compound 1. Compound 1 was administered to the animals at 0, 8, 24. and 48 hours, followed by a glucose bolus (2g/kg) at 51 hours. All data points N = 8.

Figure 4 is a line graph showing triglyceride levels in diabetic rats treated with bicyclo[3.3.1]nonenes at 40, 80 and 160 mg/kg q.d.^' of Compound 1 ; metformin (250 mg/kg b.i.d) in citrate buffer vehicle; and vehicle alone. -O- represents vehicle; — ■— represents Compound 1 in citrate buffer administered at 40 mg per kg of body weight, q.d.; —•- represents Compound 1 in citrate buffer administered at 80 mg per kg of body weight, q.d.; --♦-- represents Compound 1 in citrate buffer administered at 160 mg per kg of body weight, q.d.; —A— represents metformin in citrate buffer administered at 250 mg per kg of body weight, b.i.d. N in all cases is 8. * <0.05 (analysis of variance (ANOVA), one factor). Figure 5 is a line graph showing glucose levels in diabetic rats treated with bicyclo[3.3.1]nonenes at 40, 80 and 160 mg/kg q.d. of Compound 1 ; metformin (250 mg/kg b.i.d) in citrate buffer vehicle; and vehicle alone. -O- represents vehicle; --■— represents Compound 1 in citrate buffer administered at 40 mg per kg of body weight, q.d.; --•-- represents Compound 1 in citrate buffer administered at 80 mg per kg of body weight, q.d.; --♦-- represents Compound 1 in citrate buffer administered at 160 mg per kg of body weight, q.d.; --A-- represents metformin in citrate buffer administered at 250 mg per kg of body weight, b.i.d. N in all cases is 8. **p<0.0l; ***/?<0.0001 (analysis of variance (ANOVA), one factor).

Figure 6 is a line graph showing glucose levels in C57B1 6J ob/ob mice treated with a St. John's wort ("SJW") extract as compared to mice treated with vehicle. As shown, mice treated with SJW extract exhibited significantly reduced plasma glucose concentrations at all time points.

5. DETAILED DESCRIPTION OF THE INVENTION 5.1 BICYCLO 13.3.11 NONENES OF FORMULAE flDa. (IDb. QIDa and flinb

In accordance with the present invention, the methods for lowering blood glucose or treating diabetes mellitus comprise the steps of administering to a mammal m need of such therapeutic use. a therapeutic amount of the bicyclo [3 3 1 ] nonenes represented by formula (II)a

Formula (II)a

wherein

R, is selected from the group consisting of hydroxy and oxygen R is selected from the group consisting of hydroxy, oxygen, and benzoyl The benzoyl can be optionally substituted with a substituent selected from the group consisting of 0-2 hydroxyls, a halogen and 0-2 nitros Those skilled in the art will recognize that if R, is oxygen, then double bonds exist between the R, and C- 2; and C-3 and C-4 Likewise for the tautomer thereof, if R, is hydroxy, R₂ is oxygen, then a double bond exists between the R₂ and C-4 and between C-2 and C-3. Both R, and R₂ are not simultaneously oxygen if a double bond is to be present between C- 2/C-4 and C-3.

R₃ is selected from the group consisting of hydrogen, methyl, methylhalide, 3-methyl-2-butenyl, and -(CH₂)_xCOOR₄ X is 0 to 2. R₄ is optionally selected from the group consisting of hydrogen and C,-C₃ alkyl R₂ and R₃ can form a nng. Examples of the πng formed include furan and pyran nngs. The πng can be substituted with a hydrogen, hydroxy, and a C,-C₆ alkyl group, the alkyl group can be optionally substituted with a hydrogen or a hydroxy group, e.g , isobutoxy. R₄ can be selected from the group of substituents consisting of hydrogen and C,-C₃ alkyl. See Bystrov, N.S., et al, 1975 A 3-methyl-2-butenyl group is also known as an isoprenyl group. The isoprenyl units can also be optionally substituted with additional isoprenyl units

R_< is selected from the group consisting of hydrogen, C,-C₆ alkyl, and -(CH₂)_xCOOR₄ X and R₄.are the same as defined above. R₆ is selected from the group consisting of 3-methyl-2-butenyl, isobutyryl. 2-methylbutyryl, and benzoyl. Isobutyryl is also known as a 2-methyl- 1- oxoproyl group. A 2-methylbutyryl group is also known as a 2-methyl- 1 -oxobutyl group. R-, is selected from the group consisting of hydrogen, methyl, methylhalide. 3-methyl-2-butenyl, -(CH,)_xCOOR X and R₄ are the same as defined above. A methylhalide can include, but is not limited to a methyl chloride or a methyl bromide group.

R₈ is selected from the group consisting of hydrogen, methyl, methylhalide. 4-methyl-3-pentenyl, -(CH,)_xCOOR₄ and pharmaceutically acceptable salts thereof. X and R₄ are the same as defined above. A 4-methyl-3-pentenyl group is also known as a homo-isoprenyl group.

Such methods optionally administer the bicyclo[3.3.1] nonenes of formula (IΙ)a with a pharmaceutically acceptable carrier or vehicle and optionally with another hypoglycemic agent useful for lowering blood glucose levels.

Prefeπed embodiments of the compounds of formula (II)a are the compounds:

3-benzoyl-4-hydroxy-8,8-dimethyl-l,5,7-tris(3-methyl-2- butenyl)bicyclo[3.3.1]non-3-ene-2,9-dione; 5-benzoyl-4-hydroxy-8,8-dimethyl-l,3,7-tris(3-methyl-2-butenyl)-exo- bicyclo[3.3.1]non-3-ene-2,9-dione;

4-hydroxy-5-(3'-hydroxybenzoyl)-8,8-dimethyl-l,3,7-tris(3-methyl-2- butenyl)-exo-bicyclo[3.3.1]non-2-ene-2,9-dione;

4-hydroxy-8-exo-methyl-5,7-exo-bis(3-methyl-2-butenyl)-l-(2-methyl- l-oxopropyl)-8-e«t o(4-methyl-3-pentenyl)bicyclo [3.3.1]non-3-ene-2,9-dione; and

4-hydroxy-8-exo-methyl-5,7-exo-bis(3-methyl-2-butenyl)-l-(2-methyl- l-oxobutyl)-8-ercJo(4-methyl-3-pentenyl)bicyclo [3.3.1]non-3-ene-2,9-dione.

Additional preferred compounds include the oxidation derivatives of the bicyclo[3.3.1 ]nonenes, e.g., l-(2-methyl-l-oxopropyl)-2,12-dioxo-3,10β-bis(3- methyl-2-butenyl)-6β-( 1 -methyl- 1 -hydroxyethyl)- 1 1 β-methyl- 1 lα(4-methyl-3- pentenyl)-5-oxatricyclo-[6.3.1.0^4,8]-3-dodecene; and 1 -(2-methyl- l -oxopropyl)-2, 12- dioxo-3,10β-bis(3-methyl-2-butenyl)-6-hydroxy-l l β-methyl-l lα-4(methyl-3- pentenyl)-5-oxatricyclo[6.3.1.0^4,8]-3-dodecene. Especially preferred compounds include 4-hydroxy- 1 -ιsobutyryl-8-

l-2-butenyl)-8-(4-methyl-3-pentenyl)-exo- bιcyclo[3.3.1 ]non-3-ene-2.9-dιone (Compound 1). and 4-hydroxy- 1 -(2- methylbutyryl)-8-(4-methyl-3-pentenyl)-8-methyl-3,5,7-tπs-(3-methyl-2-butenyl)- exo-bιcyclo[3.3.1]non-3-ene-2,9-dιone (Compound 2).

In another embodiment of the present invention, the present .m ention provides methods for low eπng blood glucose levels, treating hyperglycemia and treating NIDDM compnsmg administeπng to a mammal a therapeutically effective amount of an isolated or a puπfied bicyclo nonene or pharmaceutical salt of the formula (IΙI)a.

Formula (IΙI)a wherein

R', is selected from the group consisting of O and OH;

R'₂ is selected from the group consisting of O and OH, wherein R', and R'₂ cannot simultaneously be O;

R'₃ is H or CH₃; and a pharmaceutically acceptable carrier.

Such methods optionally contain another hypoglycemic agent useful for loweπng blood glucose or treatment of diabetes mel tus.

Compound 1 Compound 2

Compound 1 may also be known as hyperforin, 4-hydroxy- 1 - isobutyryl-8-methyl-3,5,7-tris(3-methyl-2-butenyl)-8-(4-methyl-3-pentyryl)-exo- bicyclo[3.3.1]non-3-ene-2,9-dione , 4-hydroxy- 1 -(2-methyl- 1 -oxobutyl)-8-methyl- 3,5,7-tris(3-methyl-2-butenyl)-8-(4-methyl-3-pentyryl)-exo-bicyclo[3.3.1]non-3-ene- 2,9-dione. See also those listed in Section 2.2.1. Compound 2 may also be known as adhyperforin, 4-hydroxy-l -(r-methylbutyryl)-8-methyl-3,5,7-tris(3-methyl-2- butenyl)-8-(4-methyl-3-pentyl)-exo-bicyclo[3.3.1]non-3-ene-2,9-dione, or 4-hydroxy- l-(2-methyl-l-oxobutyl)-8-methyl-3,5,7-tris(3-methyl-2-butenyl)-8-(4-methyl-3- pentyl)-exo-bicyclo[3.3.1]non-3-ene-2,9-dione. See also those listed in Section 2.2.1. In accordance with the present invention, the methods for lowering serum triglyceride levels or treating hypertriglyceridemia comprise the steps of administering to a mammal in need of such therapeutic use, a therapeutic amount of isolated and purified bicyclo[3.3.1]nonenes represented by formula (II)b:

Formula (II)b

wherein R", is selected from the group consisting of hydroxy, C,-C-, alkoxy and oxygen;

R"₂ is selected from the group consisting of hydroxy, oxygen, benzoyl and C,-C₃ alkoxy. The benzoyl can be optionally substituted with a substituent selected from the group consisting of 0-2 hydroxyls, a halogen and 0-2 nitros, If R", is oxygen, then double bonds exist between the R", and C-2: and C-3 and C-4. If R", is a hydroxyl group, then R"₂ is oxygen, and a double bond exist between R"₂ and C-4 and between C-2 and C-3. Again, both R", and R"₂ are not simultaneously oxygen.

R"₃ is selected from the group consisting of hydrogen, benzoyl, methyl, methylhalide. 3-methyl-2-butenyl; and -(CH₂) COOR"₄. X can be from 0 to 2. R"₂ and R"₃ can form a ring as described earlier. R"₄ can selected from the group consisting of hydrogen and C,-C₃ alkyl. The methylhalide can include those groups as earlier described.

R"₅ is selected from the group consisting of hydrogen and C,-C₆ alkyl. R"₆ is selected from the group consisting of 3-methyl-2-butenyl, isobutyryl, 2-methylbutyryl, and benzoyl. The benzoyl group can be optionally substituted with a hydroxy group.

R"₇ is selected from the group consisting of hydrogen, methyl, methylhalide, 3-methyl-buten-2-yl, and -(CH₂)_xCOOR"₄, wherein X and R"₄ are defined as above.

R"₈ is selected from the group consisting of hydrogen, methyl, methylhalide, 4-methyl-3-pentenyl, -(CH₂)_xCOOR"₄ and pharmaceutically acceptable salts thereof, wherein X and R"₄ are defined as above

Such methods optionally administer the bicyclo[3.3.1] nonenes of formula (II)b with a pharmaceutically acceptable carrier or vehicle and optionally with another hypotriglyceridemic agent or hypolipodemic agent useful for lowering serum triglyceride levels or treatment of hypertriglyceridemia.

Preferred embodiments of the compounds of formula (II)b are the

Compounds: 3-benzoyl-4-hydroxy-8,8-dimethyl-l,5,7-tris(3-methyl-2- butenyl)bicyclo[3.3.1]non-3-ene-2,9-dione;

3-benzoyl-4-methoxy-8,8-dimethyl-l,5,7-tris(3-methyl-2- butenyl)bicyclo[3.3.1]non-3-ene-2,9-dione; [clusianone methyl derivative]

5-benzoyl-4-hydroxy-8,8-dimethyl-1.3,7-tris(3-methyl-2-butenyl)-exo- bicyclo[3.3.1]non-3-ene-2,9-dione; 5-benzoyl-4-methoxy-8.8-dimethyl-1.3.7-tris(3-methyl-2-butenyl)-exo- bicyclo[3.3.1 Jnon-3-ene-2,9-dione;

4-hydroxy-5-(3'-hydroxybenzoyl)-8,8-dimethyl-l,3,7-tris(3-methyl-2- butenyl)- γo-bicyclo[3.3.1 ]non-3-ene-2,9-dione; 5-(3 ^'-hydroxybenzoyl)-4-methoxy-8,8-dimethyl-l,3,7-tris(3-methyl-2- butenyl)- γo-bicyclo[3.3.1]non-3-ene-2,9-dione;

4-hydroxy-8-exo-methyl-5,7-exo-bis(3-methylbut-2-enyl)-l-(2-methyl- l-oxoprop\ )-8-e«<io(4-methylpent-3-enyl)bicyclo[3.3.1]non-3-ene-2,9-dione; and

4-hydroxy-8-exo-methyl-5,7-exo-bis(3-methylbut-2-enyl)-l-(2-methyl- 1-2 oxobutyl)-8-e/2(io(4-methylpent-3-enyl)bicyclo[3.3.1 ]non-3-ene-2,9-dione;

Additional compounds include the following oxidation derivatives:

1 -(2-methyl- 1 -oxopropyl)-2, 12-dioxo-3, 10β-bis(3-methyl-2-butenyl)- 6β-( 1 -methyl- 1 -hydroxyethyl)- 11 β-methyl- 1 lα(4-mefhyl-3-pentenyl)-5-oxatricyclo- [6.3.1.0⁴'⁸]-3-dodecene; and 1 -(2-methyl- 1 -oxopropyl)-2, 12-dioxo-3, 10β-bis(3-methyl-2-butenyl)-

6-hydroxy- 1 1 β-methyl- 1 1 -4(methyl-3-pentenyl)-5-oxatricyclo[6.3.1.0⁴'^s]-3- dodecene.

Especially preferred compound is 4-hydroxy- 1 -isobutyryl-8-methyl- 3,5,7-tris-(3-methyl-2-butenyl)-8-(4-methyl-3-pentenyl)-exo-bicyclo[3.3.1]non-3-ene- 2, 9-dione (Compound 1).

The present invention may be understood more fully by reference to the figures, detailed description and illustrative examples which are intended to exemplify non-limiting embodiments of the invention.

The bicyclo [3.3.1] nonenes of formulae (II)a, (II)b, (IΙI)a, and (IΙI)b can be isolated directly from Hypericum spp. or Clusia spp., preferably from H. perforatum, or semi-synthesized and isolated from a reaction mixture. Either way, the isolated bicyclo [3.3.1 ] nonenes of formulae (II)a, (II)b, (IΙI)a, and (IΙI)b can be obtained in purified form, preferably in substantially purified form, via extraction, solid phase extraction, column chromatography, recrystallization or other means known to those skilled in the art.

As used in reference to the present invention, a glucose lowering agent is the same as a hypoglycemic agent or an antihyperglycemic agent. Also as used in reference to the present invention, a serum tnglycende loweπng agent is the same as a hypolipidemic agent or an antihyperhpidemic agent.

The term "hyperhpidemia" refers to the presence of an abnormally elevated level of lipids m the blood Hyperhpidemia can appear in at least three forms: (1) hypercholesterolemia, i e . an elevated cholesterol level; (2) hypertπglyceπdemia. i c , an elevated tnglycende level, and (3) combined hyperhpidemia. e , a combination of hyperchlosterolemia and hypertnglyceπdemia.

5.2 PROCESSES FOR ISOLATING HYPERFORINS FROM HYPERICUM AND CLUSEA SPP.

Compounds 1 and 2 can be isolated from Hvpertcum spp., preferably H perforatum, using the illustrative methods descπbed below or other standard extraction and purification techniques known to those of ordinary skill in the art. For example, the isolation of hyperifonn from Hypericum perforatum (Bystrov, N.S., et al, 1977)); of adhyperfonn from Hypericum perforatum (Maisenbacher, P., et al, (1992)); of hyperevolutm A (also known as bιcyclo[3.3.1]non-3-ene-2,9-dione, 4- hydroxy-8-methyl-5,7-bis(3-methyl-2-butenyl)-l-(2-methyl-l-oxopropyl)-8-(4- methyl-3-pentenyl)-, (1R,5R,7S,8R)) and B (also known as bicyclo[3.3.1]non-3-ene- 2,9-dione, 4-hydroxy-8-methyl-5,7-bis(3-methyl-2-butenyl)-l-(2-methyl-l-oxobutyl)- 8-(4-methyl-3-pentenyl)) from Hypericum revolutum (Decosterd, L.A., et al, Helvetica Chimica Ada 72. 464 (1989)); of clusianone from C spiritu-santensis (male) (de Ohveira, C.M.A., et al, Tetrahedron letts 37:(36) 6427 (1996)); of nemorosone from C rosea, C grandiflora, C. tnsignis, and C. nemorosa (de Oliveira, et al, 1996) have all been reported. In addition, the compounds so obtained can be purified by chromatography, recrystallization or other purification methods known to those skilled in the art.

5.3 DERIVATIVES OF BICYCLO [3.3.11 NONENES

Semi-synthetic derivatives of bιcyclo[3.3.1]nonenes have been prepared. De Oliveira synthesized the methyl (methoxy) deπvatives of clusianone, nemorosone, hydroxy-nemorosone (de Oliveira, et al, 1996). Bystrov synthesized methyl, tetracarboxvhc acid, tetramethyl ester. den\ atιves of hyperfonn In addition, dilactone, diester diacid, dibromide den\ atιves of the tetracarboxvhc acid hyperfonn deπvatives were also synthesized (Bystrov, N S , et al, Tetrahedron Lett 32 2791 (1975)) Bystrov also synthesized crystalline 3.5-dmιtro benzoate. methyl, acid acetyl, tetrahydro, and octahydro denvatives of hyperfonn. [Bystrov, N S., et al, Bioorgamcheskava Khttnna 4 (6) 791 (1978)] Brondz, I, et al. synthesized &p- bromobenzoate denvative Brondz, I, et al, Ada Chem Scand 37 263 (1983)

5.3.1 ISOLATION AND PURIFICATION OF BICYCLO [3.3.11 NONENES

Plant matenal from H\perιcum spp , preferably P perforatum (Clusiaceae) is initially extracted with a solvent to provide a crude extract containing the identified bicyclo [3 3 1] nonenes By "plant matenal" is meant any part of the Hypericum plant, such as bark, leaves, flowers, roots and stems The plant matenal may optionally be shredded, ground, macerated, or otherwise treated pnor to extraction. Alternatively, the plant matenal may already be m a powdered, shredded, ground, macerated, or comminuted state when used herein. Suitable extraction solvents include polar solvents, non-polar solvents, or mixtures thereof. Useful polar solvents include, but are not limited to, acetonitπle, methanol, ethanol, isopropanol, acetone, butanol, ethyl acetate, tetrahydro furan, dimethylformamide, n- methylpyrrohdinone, dimethyl sulfoxide, water and mixtures thereof. Useful non- polar solvents include, benzene, toluene, pentane, hexane, heptane, higher alkane and other hydrocarbon solvents, such as petroleum ether.

Extracting the plant matenal with solvent can be performed at a temperature of about room temperature to about the reflux temperature of the chosen solvent or solvent system, preferably at room temperature, for between about 1 and 24 hours, preferably between about 2 to 12 hours, most preferable for about 6 hours, m order to maximize the amount of bicyclo [3.3.1] nonenes that can be isolated from the plant matenal The plant matenal may also be agitated, soaked, passed through or otherwise exposed to the solvent to facilitate the extraction process For example, the plant matenal can be mechanically mixed, sonicated, or otherwise agitated in the solvent bv methods known bv those skilled m the art The plant material may be washed with a polar solvent prior to the extractions of the bicyclo nonenes. The purpose of the wash is to remove very polar compounds from the plant material. The solvent is selected so that the bicyclo[3.3.1 ]nonenes are left in the plant material whereas the very polar compounds are removed. Subsequent extraction of the nonenes will result in an enriched extract devoid of the very polar compounds. Preferably the plant material is washed with a mixture of a C1-C3 alcohol, more preferably a methanol/water mixture, the meixture containing from about 30% methanol to about 80% methanol (e.g., methanol water mixtures from about 3: 10 to about 8:2). Most preferably the plant material is washed with a mixture of 1 : 1 methanol/water.

The resulting crude extract can then be filtered to remove undesired impurities therefrom and to afford a filtrate containing the bicyclo [3.3.1] nonenes. Suitable filtering methods include passing the crude extract through a synthetic hydrophobic sorbent such as HP-20C, diatomaceous earth, silica gel; or a fritted funnel. Various types of chromatographic gels or resins discussed infra, can also be used to filter the crude extract. Centrifugation of solutions or diluted solutions of the crude extract can also be employed to remove undesired impurities therefrom.

The crude filtrate can be concentrated, preferably in vacuo, and the resulting residue further purified by being partitioned between two partitioning solvents, so as to enhance the yield and overall purity of the isolated bicyclo [3.3.1] nonenes. It is important that the partitioning solvents are immiscible in each other. Preferably, one of the partitioning solvents is a non-aqueous solvent such as benzene, toluene, diethyl ether, ethyl methyl acetate, chloroform, carbon tetrachloride, pentane, hexane, heptane, higher alkane (C < 7) solvents, dichloromethane (DCM) and other hydrocarbon solvents, such as petroleum ether, known by those skilled in the art to be immiscible in water or capable of dissolvating bicyclo [3.3.1] nonenes. The aqueous solvent should preferably be capable of dissolving impurities found in the plant material.

The organic phase, containing the bicyclo [3.3.1] nonenes, is separated, optionally combined, and then concentrated to dryness to afford a crude concentrate which is enriched in bicyclo [3.3.1] nonenes. The previously described extraction and filtering steps can be repeated to increase the yield and overall purity of the isolated bicyclo [3.3.1] nonenes. The crude concentrate can be further purified by standard techniques known to those skilled m the art to ultimately afford isolated bιcyclo[3 3 l ]nonenes Exemplary puπfication techniques include recrystalhzation and chromatographv Preferably, the crude concentrate is puπfied using liquid chromatography, for example high performance liquid chromatography, \ acuum flash chromatography and adsorption chromatography

Ion suppressing agents can be used if the process uses chromatography Exemplary ion-suppression agents include weak acids, e g , acetic acid, and buffers, e g ammonium acetate, ammonium carbonate, and sodium phosphate

V aπous types of chromatography techniques include solid phase extraction (SPE), open column or gravity chromatography, flash chromatography, vacuum liquid chromatography, low pressure and medium pressure chromatography and high performance chromatography Suitable resins or sorbents include absorbents such as silica gel, silica-based bonded phases such as CI S, C8, phenyl, amino diol Synthetic resins made from polymenc acrylate or polystyremc divinyl benzene such as HP-20 or CHP-20 to selectively retain or pass the bicyclo [3 3 1] nonenes according to polaπty can also be used Size, molecular weight, or cellulosic charactenstics of the desired resm matenal may be used to separate the bicyclo [3 3 1] nonene by selective use of molecular exclusion or silica based resms.

An appropnate gradient solution is used to wash and separate the bιcyclo[3 3 l]nonenes from the crude concentrate on the column filled with the desired resm A suitable gradient may include an initial wash of a solvent followed by an elution solvent Suitable elution solvents contain a high percentage of acetonitπle (ACN), methanol, acetone, dichloromethane, ether/hexane or any other organic solvent or mixtures thereof that can release hyperfonn from the resm matenal, and into an ennched fraction The enriched fraction will contain the bιcyclo[3 3 l]nonenes, or mixtures thereof The elution solvent can contain up to 50%) water, so as to adjust or optimize the polanty thereof For example, for FfP-20 or C-18 resin equilibrated in 4 1 methanol/water (v/v), the elution solvent can be a gradient of increasing methanol or for silica gel resin equilibrated m hexane, the elution solvent can be a gradient of increasing ether m a ether/hexane concentration solution

High performance liquid chromatography (HPLC), thm layer chromatography (TLC) and nuclear magnetic resonance (NMR) analysis can be used to determine which ot the eluting fractions is an enπched fraction, and which ennched fractions contain the desired bicyclo [3 3 1] nonenes HPLC can also be used to determine the precise amount of [3 3 1 ] nonenes present in an enπched fraction Optionally , different elutmg fractions can be combined and subjected to the TLC and NMR analyses descπbed above The enπched fractions can optionally be repuπfied using either the same or a different eluent system

The resulting fractions containing the bicyclo [3 3 1] nonenes are concentrated, optionally in vacuo The fractions containing the bicyclo [3 3 1] nonenes from the chromatography methods descnbed above can be combined and further punfied by successive iterations of the above, or by recrystalhzation or other types of chromatography Optionally, successive recrystalhzation or chromatography puπfications may be performed to obtain punfied bicyclo [3 3 1] nonenes.

The resulting fractions containing the bicyclo [3 3 1] nonenes can be air sensitive and degrade pnor to chemical reduction, when exposed to light and air. (Maisenbacher, P , et al, 1992) To maximize yield and maintain activity dunng extraction and storage, the solvents and solutions can be nnsed, bubbled or flushed with inert gases, e.g , nitrogen or helium before use, and the resulting extracted compounds, for example, hyperfonn and adhyperfonn, can be stored under the same inert gases Alternatively or optionally, the extraction and/or storage can be earned out in the absence of light or under an inert gas. See Dr Willmar Schwabe GMBH & Co [Erdelmeier, C, et al], Republic of South Africa Patent Application 968114.

Using the above punfication techniques, the isolated bicyclo [3.3.1] nonenes can be punfied or substantially punfied. By "substantially punfied" is meant that the bicyclo [3.3 1] nonenes of formula (II) have a degree of punty of at least 90% By "punfied" is meant that the bicyclo [3.3.1] nonenes of formula (II) have a degree of punty of at least 95% Alternatively, extraction methods which result in an ennched extract charactenzed by bιcyclo[3 3 l]nonenes of formulae (II) or (III) having a degree of punty of less than 95%o, e.g., at least about 2.5% by weight, greater than about 3% by weight, greater than about 5%, greater than about 10%, greater than about 30%), or greater than about 60%o by weight are also contemplated by the inventors. 5.4 DERIVATIVES OF BICYCLO [3.3.11 NONENES

Also included within the scope of the present invention are methylated, carboxylic acid ether and acetate derivatives of bicyclo [3.3.1] nonenes which are useful for lowering serum triglycerides. blood glucose and treating diabetes mellitus, hyperglycemia, hyperhpidemia and hypertriglyceridemia. Bicyclo [3.3.1 ] nonenes analogues can be made semi synthetically according to those of skill in the art. For example, isolation and preparation of the 3.5-dinitrobenzoate, C-methylhyperforin, hyperforin methyl ether, hyperforin acetate, and partially and fully hydrogenated. reduced, oxidized and acylated analogues thereof has been reported (see de Oliveira, et al, 1996; Trifunovic, S., et al, 1998); Bystrov, (1975); Bystrov, (1978). Once the bicyclo [3.3.1 ] nonenes of formulae (II)a, (II)b, (IΙI a and (IΙI)b have been synthesized, they can be purified or substantially purified, using conventional chromatography, recrystalhzation or other purification techniques known to those skilled in the art. In addition, the hydroxyl groups of these bicyclo nonenes can be acetylated by methods well known to those skilled in the art, for example, using acetyl chloride (Greene, Protective Groups in Organic Synthesis 101. (1981).

It is to be pointed out that any hydroxyl groups not so methylated or acetylated can participate in the formation of those pharmaceutically acceptable salts of hyperforin described above.

5.5 METHODS FOR USE OF THE HYPERFORIN OF FORMULAE (ID AND

⁽III⁾

Due to the activity of the bicyclo [3.3.1] nonenes of the present invention, the bicyclo [3.3.1] nonenes of formulae (II)b and (IΙI)b or pharmaceutically acceptable salts thereof are advantageously useful in veterinary and human medicine for therapeutic treatment of elevated serum triglyceride levels and hypertriglyceridemia.

Due to the activity of the bicyclo [3.3.1] nonenes of the present invention, the bicyclo [3.3.1] nonenes of formulae (II)a and (IΙI)a or pharmaceutically acceptable salts thereof are advantageously useful in veterinary and human medicine for therapeutic treatment of diabetes melhtus Additionally, the bicyclo [3 3 1] nonenes of iormulae (II)a and (IΙI)a can be ad\ antageously used as hyφoglycemic agents to reduce blood glucose m situations of acute stress such as expeπenced by animals or patients with hyperthermia. trauma sepsis, burns or those undergoing general anesthesia Hyperglycemia sometimes associated with severe head injury, cerebral-thrombosis, encephalitis and heat stroke can also be therapeutically treated with the bicyclo [3 3 1] nonenes of formulae (II)a and (IΙI)a Additionally, the bicyclo [3 3 1 ] nonenes of forumulae (II)a and (IΙI a are useful as hypoglycemic agents for rare congenital metabolic glycogen storage disease associated with hyperglycemia The bιc\clo [3 3 1] nonenes of formulae (II)a and (IΙI)a used in the methods descπbed herein are particularly suited to control hyperglycemia in patients whose blood glucose cannot be controlled by diet alone Furthermore, the bicyclo [3 3 1] nonenes of formulae (II)a and (IΙI)a are capable of loweπng blood glucose levels without an accompanying increase in uπne glucose levels When administered to a mammal for vetennary use or to a human for clinical use, the bicyclo [3 3 1] nonenes of formulae (II)a, (II)b, (IΙI)a and (IΙI)b are administered in isolated form By "isolated" is meant that the bicyclo [3 3 1] nonenes are separated from other components of either (a) a natural source such as a plant or cell culture, or (b) a synthetic organic chemical reaction mixture Preferably, via conventional techniques, the bicyclo [3 3 1] nonenes of formulae (II)a, (II)b, (IΙI)a and (IΙI)b are substantially punfied, preferably punfied

When administered to a mammal for vetennary use or to a human for clinical use, the bicyclo [3 3 1] nonenes of formulae (II)a, (II)b, (IΙI)a and (IΙI)b can be used alone or in combination with any physiologically acceptable earner or excipient suitable for enteral or parenteral administration Where used for parenteral administration, the physiologically acceptable earner must be stenle and suitable for in vivo use m a human, or for use in a vetennary clinical situation

The bicyclo [3 3 1] nonenes of formulae (II)a, (II)b, (IΙI)a and (IΙI)b can be used in the form of a pharmaceutical preparation, for example, in solid, semisohd or liquid form, which contains at least one of the bicyclo [3 3 1] nonenes of formulaw (II)a, (II)b, (IΙI)a or (IΙI)b, preferably hyperfonn or adhyperfoπn, including their therapeutically active salts, as a bioactive component, alone or in combination with another antitnglycendemic, antidiabetic. antihyperglycemic. or blood glucose lowenng compound, in a mixture ith a earner or an excipient suitable for enteral or parental administration The bicyclo [3 3 1] nonenes of formulae (II)a, (II)b, (IΙI)a and (IIDb may be compounded, for example, with a pharmaceutically acceptable earner for solid compositions such as tablets, pellets or capsules, capsules containing liquids, suppositones. solutions, emulsions, suspensions or any other form suitable for use Suitable earners include, for example, steπle water, stenle physiological saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea and the like In addition, auxiliary, stabilizing, thickening, lubncatmg and colonng agents may be used The bιcvclo[3 3 l]nonenes of formulae (II)a and (IΙI)a are present in the compositions m an amount sufficient to produce a desired effect upon diabetes, blood glucose levels, or hyperglycemia The bιcyclo[3 3 l]nonenes of formulae (II)b and (IIDb are present in the compositions in an amount sufficient to produce a desired effect upon serum tnglycende levels, or hypertnglyceπdemia

The compositions of this invention may be administered by a vanety of methods including orally, intramuscularly, intravenously, subcutaneously, transdermally, rectally or by inhalation While the preferred mode of administration is through the oral mode, the precise mode of administration is left to the discretion of the practitioner They are advantageously effective when administered orally.

Compositions for oral administration may be m the form of tablets, troches, lozenges, aqueous or oily suspensions, granules or powders, emulsions, capsules, syrups or elixirs Orally administered compositions may contain one or more agents, such, as sweetening agents such as fructose, aspartame or sacchann; fiavonng agents such as peppermint, oil of wmtergreen, or cherry, colonng agents and preserving agents to provide a pharmaceutically palatable preparation Moreover, compositions m tablet form may be coated to delay disintegration and absorption in the gastrointestinal tract thereby providing a sustained action over an extended penod of time Selectively permeable membranes surrounding an osmotically active dnvmg compound are also suitable orally administered compositions In these later platforms, fluid from the environment surrounding the capsule is imbibed by the dnvmg compound, which swells to displace the agent or agent composition through an aperture These delivery platforms can provide an essentially zero order delivery profile as opposed to the spiked profiles of immediate release formulations A time delav matenal such as glycerol monosterate or glycerol sterate may also be used Aqueous suspensions containing the bicyclo [3.3.1] nonenes of formulae (II)a, (II)b, (IIDa and (IΙI)b may also contain one or more preservatives, such as, for example, ethyl or «-propyl-/ hydroxy-benzoate, one or more coloring agents, flavoring agents or sweetening agents. In a further embodiment, this invention comprises the use of a bicyclo

[3.3.1] nonenes of formulae (II)a, (II)b, (IΙI)a and (IΙI)b, preferably in isolated or purified form, when administered at a dose of less than or about equal to 1,000 mg per kg of body weight per day, preferably from about 0.25 to about 500 mg per kg of body weight per day. In still a further embodiment, the invention comprises the use of a bicvclo [3.3.1] nonenes of formulae (II) and (III) at a dose of about 2 to about 350 mg/kg body weight/day of compound to be utilized in an amount which results in the compositions exhibiting a therapeutically effective hypoglycemic, antihyperglycemic or antidiabetic activity. The dosage of the present compositions for treatment or prevention of hyperglycemia or diabetes or for reducing blood glucose levels, depends on the route and frequency of administration was well as the age, weight and physical condition of the patient. Generally the daily dosage is in the range of about 1 to about 1000 mg/kg body weight/day, preferably about 10 to about 350 mg/kg body weight/day. Treatment can be repeated as needed, depending upon the dosage and need, for example, a dosage of about 40 or 80 mg/kg body weight/day of patient/animal can be administered in dividing doses to prevent or treat diabetes or hyperglycemia or to lower blood glucose. Treatment can be continued, for example; reduced to the desired until the blood glucose level is level or to be maintained at a desired level. The appropriate dosage of the compositions can be readily determined by the skilled medical practitioner. Treatment can be repeated as needed, depending upon the dosage and need, for example, a dosage of about 80 or 160 mg/kg body weight/day of patient/animal can be administered in dividing doses to prevent or treat hypertriglyceridemia or to lower serum triglyceride levels. Treatment can be continued, for example; reduced to the desired until the serum triglyceride level is level or to be maintained at a desired level. The appropriate dosage of the compositions can be readily determined by the skilled medical practitioner.

For the treatment of diabetes, hyperglycemia effecting a lowering of blood glucose, a composition of present invention may be administered which contains a bicyclo [3.3.1 ] nonene of formulae (II)a and (IΙI)a or a pharmaceutically or the acceptable salt thereof as described above, together with another antidiabetic. antihyperglycemic or blood glucose lowering agent including, but not .limited to insulin: a biguanide such as metformin or buformin; a sulfonylurea such as acetohexamide, chlorpropamide, tolazamide, tolbutamide, glyburide, glypizide or glyclazide; a non-sulfonylurea insulin secretagogue such as nateglinide; a thiazolidinedione such as troglitazone. pioglitazone, rosiglitozone or ciglitazone; an α-glucosidase inhibitor such as acarbose or miglitol; a β-adrenoceptor agonist such as PL-316, 243, etc. or any other known antidiabetic or hypolipidemic agent. Alternatively, the compositions comprising a bicyclo [3.3.1] nonenes of forumulae (II)a and (IΙI)a or a pharmaceutically acceptable salt thereof can be administered in combination with, prior to, concurrent with or subsequent to the administration of another antidiabetic, antihyperglycemic, blood glucose lowering agent, as described above.

Although the present inventors do not wish to be limited to any particular mechanism of action to explain the hypoglycemic activity of the bicyclo [3.3.1] nonenes of formulae (II)a and (IΙI)a, it is envisaged that they may advantageously be useful for treatment of both insulin-dependent or type I diabetes (formerly termed juvenile-onset or ketosis prone diabetes) and non-insulin-dependent or Type 2 diabetes (formerly termed adult-onset, maturity onset or nonketotic diabetes). The bicyclo [3.3.1] nonenes of formulae (II)a, (II)b, (IΙI)a and (IΙI)b can optionally be administered in an effective amount as pharmaceutically acceptable carboxylate or phenolate salts using counter ions such as sodium, potassium, lithium, calcium, magnesium, zinc and iron.

Furthermore, the bicyclo [3.3.1] nonenes of formula (II)a, (II)b, (IΙI)a and (IΙI)b or pharmaceutically acceptable salts thereof can be used for research purposes, for example; to investigate the mechanism and activity of antihyperglycemic or antihypertriglyceridemic agents.

The compositions can be formulated and administered in the same manner as detailed below. "Formulation" is defined as a pharmaceutical preparation that contains a mixture of various excipients and key ingredients that provide a relatively stable, desirable and useful form of a compound or drug. For the present invention, "formulation" is included within the meaning of the term "composition." The nonenes of the present invention can be used effectively alone or in combination with one or more additional active agents depending on the desired target therapy (see, e.g., Turner, N. et al Prog. Drug Res. ( 1998) 51 : 33-94; Haffher, S. Diabetes Care (1998) 21 : 160-178; and DeFronzo. R. et al (eds.). Diabetes Reviews (1997) Vol. 5 No. 4). A number of studies have investigated the benefits of combination therapies with oral agents (see, e.g., Mahler, R., J. Chn. Endocrinol. Metab. (1999) 84: 1 165-71 : United Kingdom Prospective Diabetes Study Group: UKPDS 28, Diabetes Care (1998) 21 : 87-92; Bardin. C. W.,(ed.). CURRENT THERAPY IN ENDOCRINOLOGY AND METABOLISM. 6^th Edition (Mosby - Year Book, Inc., St. Louis, MO 1997); Chiasson, J. et al, Ann. Intern. Med. (1994) 121 : 928-935; Coniff, R. et al, Clin. Ther. (1997) 19: 16-26; Coniff, R. et al., Am. J. Med. (1995) 98: 443-451 ; and Iwamoto. Y. et al. Diabet. Med. (1996) 13 365-370; Kwiterovich, P. Am. J. Cardwl (1998) 82(12A): 3U-17U). These studies indicate that diabetes and hyperhpidemia modulation can be further improved by the addition of a second agent to the therapeutic regimen. Combination therapy includes administration of a single pharmaceutical dosage formulation which contains a nonene of the invention and one or more additional active agents.

Another example of combination therapy can be seen in modulating diabetes (or treating diabetes and its related symptoms, complications, and disorders), wherein the nonenes of this invention can be effectively used in combination with, for example, sulfonylureas (such as chlorpropamide, tolbutamide, acetohexamide, tolazamide, glyburide, gliclazide, glynase, glimepiride, and glipizide), biguanides (such as metformin), thiazolidinediones (such as ciglitazone, pioglitazone, troglitazone, and rosiglitazone); dehydroepiandrosterone (also referred to as DHEA or its conjugated sulphate ester, DHEA-SO4); antiglucocorticoids; TNFα inhibitors; α- glucosidase inhibitors (such as acarbose, miglitol, and voglibose), pramlintide (a synthetic analog of the human hormone amylin), other insulin secretogogues (such as repaglinide, gliquidone, and nateglinide), and insulin.

A further example of combination therapy can be seen in modulating hyperhpidemia (treating hyperhpidemia and its related complications), wherein the nonenes of this invention can be effectively used in combination with, for example, statins (such as fluvastatin, lovastatin, pravastatin or simvastatin). bile acid-binding resins (such as colestipol or cholestyramine), nicotinic acid, probucol, betacarotene, vitamin E, or vitamin C. In accordance with the present invention, a therapeutically effectrv e amount of a nonene of this invention can be used for the preparation of a pharmaceutical composition useful for treating diabetes, treating hyperhpidemia, treating obesity, loweπng tnglycende levels, lowenng cholesterol levels, and raising the plasma le\ el of high density hpoprotein

The following examples are set forth to assist m understanding the invention and should not. of course, be construed as specifically limiting the invention descnbed and claimed herein Such vanations of the inventions which would be withm the purview of those in the art, including the substitution of all equivalents now known or later developed, including changes in formulation or minor changes in expenmental design, are to be considered to fall withm the scope of the invention incorporated herein

6. EXAMPLE: ISOLATION AND CHARACTERIZATION OF BICYCLO [3.3.11 NONENES COMPOUNDS

6.1 MATERIALS AND METHODS

Analytical high performance liquid chromatography (HPLC) was performed on a Hitachi Model D-6500 Chromatography Data Station equipped with a L-6200A pump, AS-2000 autosampler, a Pπmesphere C18 HC 4 x 50 mm (5 μm) HPLC column, an L-4500 A diode arrary detector, and a Sedex 55 light scattenng detector connected m senes. All chromatographic runs were performed at ambient temperature HPLC grade solvents were used without further puπfication. Standards (Compounds 1 and 2) were made from punfied samples.

Nuclear magnetic resonance (NMR) spectra were recorded on a Vaπan Unity Plus 400 or a Vanan Unity 400 spectrometer. NMR spectra of compounds were recorded m either deuterated DMSO or CDC1₃. One and two-dimensional NMR expeπments, including Distortionless Enhancement Polanzation Transfer (DEPT), H- H Correlation Spectroscopy (COSY), Heteronuclear Multiple Quantum Correlation (HMQC), Heteronuclear Mutiple Bond Correlation (HMBC) MS spectra were recorded on a Kratos MS-50 in high resolution power electron impact scanning mode, 70 ev Resolution was set to 2000, scanning rate 10 sec/decay, temperature gradient from 50° to 300° C increased at a rate of 50°/mιn Infrared (IR) spectra were recorded on a Perkin-Elmer 1600 Seπes FTIR. Ultraviolet (UN) was taken directly from the Hitachi diode-array UV detector on the HPLC system. Optical rotations were measured using a Jasco Digital polarimeter.

6.2 ISOLATION AND PREPARATION OF BICYCLO [3.3.11 NONENES

USING SOLVENT EXTRACTION

6.2.1 ISOLATION OF COMPOUND 1 AND COMPOUND 2 FROM HYPERICUM PERFORA TUM (CLUSIACEAE)

Powdered aerial parts of (6.6 kg) Hypericum perforatum were extracted with 37 L of 4: 1 ethanol water at room temperature for 24 hours. The resulting crude extract was filtered through a CELITE® bed (200 g) and the resulting crude filtrate evaporated to dryness to provide 1.7 kg of a dark green extract (25.8% yield from H. perforatum). 1.28 kg of the dark green extract was partitioned between 35 L of 1 :1 (v/v) dichloromethane (DCM):water. The aqueous phase was separated and concentrated to dryness to afford 248 g of a crude concentrate (5.0% yield from H perforatum). 53 g of the crude concentrate were applied onto a 1 L glass column (25 cm x 9 cm i.d.) containing 700 ml of HP-20 resin in 4:1 methanol/water. The column was eluted at a flow rate of 80 mL/min. with 2 column volumes (2 L) in 4: 1 methanol/water, 2 column volumes (2 L) of in 9: 1 methanol/water, 2 column volumes (2 L) of 100%) methanol, and 2 column volumes (2 L) DCM. Two L fractions were collected and concentrated at reduced pressure in a rotary evaporator. The third fraction collected (eluted with 100%) methanol) yielded 34.9 g of a dark green material (3.3%o yield from H. perforatum). The dark green material was chromatographed using a semi-preparative HPLC system (column Primesphere 5, C18 HC, 250 x 21.2 mm with a pre-column Primesphere 5, C18 HC, 60 x 21.2 mm). An isocratic mobile phase containing 80: 15:5 acetonitrile/methanol/water, was used with a flow 10 mL/min, UV detection at 270 nm, 55 minute run. This yielded 26.4 mg of hyperforin per injection with a retention time of 36 minutes (1.3% from H. perforatum) and 6.5 mg of adhyperforin per injection with a retention time of 43.5 minutes (0.3% from H. perforatum). 6.2.2 ISOLATION OF COMPOUND 1 FROM HYPERICUM PERFORA TUM

Aerial parts of Hypericum perforatum (2.87 kg) were extracted with 20 L of 100% methanol for 24 hours at room temperature using a Silverson multi- purpose immersion type mixer - emulsifier for overhead stirring. The resulting crude extract was filtered through a fritted funnel, and the resulting marc was washed with an additional 2 L of 100% methanol. The crude extract supernatents were combined and the resulting 22 L was mixed at room temperature with 6 L of water. The resulting methanolic/aqueous suspension was loaded onto a 12 L HP-20 column, the column being pre-equilibrated with 4: 1 methanol/water. The column was washed with an additional 10 L of 4: 1 methanol/water, followed by 42 L of 9: 1 methanol/water. The column was then eluted with 97:3 methanol/water and ten 5 L fractions were collected. Fractions 3 - 10 were the hyperforin (Compound 1 ) enriched fractions. Fraction 3 was diluted with 1.13 L of water and loaded onto a 1.7 L C18 (J.T. Baker, Inc., Phillipsburg, New Jersey) chromatography column (4.8 x 94 cm.), pre-equilibrated with 4: 1 methanol/water (containing 0.1% HOAc). The column was eluted with 9:1 methanol/water (containing 0.1% HOAc). Thirty-three 400 mL fractions were collected and assayed by HPLC. The enriched fractions, containing Compound 1, fractions 21 - 25, were combined, evaporated, and concentrated at a reduced pressure to a reddish oil using a rotary evaporator. The material was placed in a vacuum oven overnight. This afforded 2.8 g of a reddish oil which was identified as hyperforin (Compound 1) having >95%o purity. The overall yield from the plant was 1.0%. The isolation of hyperforin (Compound 1) is illustrated schematically below.

Hypericum perforatum 2.8 kg

Primary Extraction

marc MeOH Extract (585 g)

HP20 column

LE Fl-Fll F12 F13-F14 F15-F17 F18-F19 452 g 29 g 7 g 35 g 19 g 6 g

C18 column

F1-F19 F20 F26-F27 F28-F33 1.7 g C.5 g 0.46 g 1.5 g

Compound 1

F21-F25

2.8 g

6.2.3 ISOLATION OF COMPOUNDS 1 AND 2 FROM HYPERICUM

PERFORATUM

Ground aerial portions of Hypericum perforatum (SI g) were stirred at room temperature with 890 ml of 100%o methanol containing 0.1% acetic acid for 3 hours. The acetic acid being added as a ion suppression agent. The resulting extract was filtered through 100 g of HP-20 which was washed with an additional 425 ml of methanol. The filtrate and wash were combined to give 1.25 L of filtered Hypericum extract. To this was added 313 ml of water. The resulting red suspension was pumped onto a pre-equilibrated (4:1 methanol/water containing 0.1% acetic acid) C18 column (2.5 x 90 cm). After loading, the column was washed with an additional 500 ml of 4: 1 methanol/water (containing 0.1% acetic acid). The column was eluted with 3.5 L 9: 1 methanol/water (containing 0.1% acetic acid). Thirteen 225 ml fractions were collected. Fractions 9-1 1 were combined and evaporated under reduced pressure (rotary evaporation) then dried in a vacuum oven at 40°C overnight. This gave 1.77 g of Compound 1 (> 95%) as a reddish sticky solid. F14-F15 were combined, evaporated and dried in a similar fashion to give 122 mg of Compound 2 (> 95%) as an amber oil. The isolation of Compounds 1 and 2 is illustrated schematically below.

Hypericum perforatum aerial parts

87 g

Primary Extraction

eOH Extract Spent Plant

Solid Phase Extraction (HP-20C)

HP20E

Wash

17.5 g 1.4 g

C18 Chromatography

LE F1 -F8 F12 -F13 14 . 9 g 2 . 26 g 0 .40 g

Compound 1 Compound 2 F9 -F11 F14 -F15 1 . 77 g 0 . 12 g

6.2.3 PREPARATION OF AN EXTRACT OF HYPERICUM PERFORATUM CONTAINING 20% COMPOUND 1

Ten grams of plant ground material was placed into a sintered glass funnel. To the funnel was added 130 ml of 1 :1 methanol/water. The funnel was allowed to slowly drain over a one hour period at which time 114 ml of wash had been collected. The wash was assayed and discarded. To the funnel was added 100 ml of methanol. The funnel was again allowed to drain over a one hour period at the end of which 100 ml of extract enriched in Compound 1 had been collected. The extract was dried on a rotary evaporator yielding 1.1 g of a dark residue containing 20%) Compound 1 by weight. Hypericum perforatum aerial parts

10 g

Plant Wash (1:1 methanol/water)

Washed Plant 1:1 methanol/water

6.2.4 PREPARATION OF AN EXTRACT OF HYPERICUM PERFORATUM CONTAINING 60% COMPOUND 1

Ten grams of plant ground material was placed into a sintered glass funnel. To the funnel was added 130 ml of methanol. The funnel was allowed to slowly drain over a one hour period at which time 102 ml extract containing 2.2 g of solids (12%) Compound 1 by weight). To the extract was added 30 ml of water and the resulting milky suspension was poured into a sintered glass funnel containing 15 g of pre-equilibrated HP-20C. The filtrate (124 ml) was assayed and discarded. The HP-20C resin was washed with 170 ml of 4:1 methanol/water, followed by 110 ml of 9:1 methanol/water and finally 220 ml of methanol. The column was cleaned with 150 ml of acetone. The 100% methanol extract contained 330 mg solids (60%> Compound 1). Hypericum perforatum aerial parts

10 g

Primary Extraction

MeOH Extract Spent Plant

7

Extract 2 330 mg 60% Compound

6.2.5 Preparation of Methyl Enol Ethers

6.2.5.1 Compound la

Compound 1 (836 mg), as isolated as described in Sections 6.2.1-6.2.3, was dissolved into 5 ml of a 1 :1 mixture of methanol and dichloromethane. To this solution was added trimethylsilydiazomethane reagent until color persisted. The reaction was allowed to stand for 1 hour after which it was evaporated and the resulting residue was chromatographed on a Bakerbond 40 μm C18 column (1 X 50 cm). Column elution was isocratic using 92:8 methanol/water. Thirty-six 20 ml fractions were collected. Fractions 11-18 were combined and evaporated to dryness, giving 282 mg of a colorless oil identified Compound la.

6.2.5.2 Compound 2a

Compound 2 (100 mg), as isolated as described in Sections 6.2.1-6.2.3, was dissolved into 2 ml of a 1 : 1 mixture of methanol and dichloromethane. To this solution was added trimethylsilydiazomethane reagent until color persisted. The reaction w as allowed to stand for 1 hour after w hich it w as blown down w ith nitrogen and the resulting residue w as chromatographed on a Bakerbond 40 μm C18 column (1 X 50 cm) Column elution w as isocratic using 95 5 methanol/water. Thirty-seven 20 ml fractions w ere collected Fractions 36-37 were combined and evaporated to dryness. giving 7 mg of a colorless oil identified Compound 2a

6.3 STRUCTURE ELUCIDATION OF THE HYPERFORIN COMPOUNDS

6.3.1 Compound 1

Compound 1 R=H Compound la R=Me

The isolation and structural elucidation of Compound 1 were reported in 1978 (Brondz, et al, (1978)). The absolute configuration reported in 1983 (Brondz, et al, (1983)) Charactenzation was earned out using the methyl denvative. HREIMS gave an NT of 550 4042 observed vs. 550.4022 calculated (Δ 3.6 ppm) thus giving the molecular formula of C₃₆H₅₄O₄. IR absorbances were observed at 3446, 2924, 1719, 1645, 1602, 1445, 1337, 1235, 1058 cm '. UV λ_maλ was observed at 273 nm Optical rotation was [a]_D ²¹=+39 (c 3 2. methanol)

Compound 1 (836 mg) was dissolved into 5 ml of a 1 : 1 mixture of methanol and dichloromethane To this solution was added trimethylsilydiazomethane reagent until color persisted. The reaction was allowed to stand for 1 hour after which it was evaporated and the resulting residue was chromatographed on a Bakerbond 40 μm C18 column (1 X 50 cm). Column elution was isocratic using 92:8 methanol/water. Thirty-six 20 ml fractions were collected. Fractions 11-18 were combined and evaporated to dryness, giving 282 mg of a colorless oil identified Compound la.

Table 1 shown below lists the assigned '^"C and ^!H chemical shifts for Compound 1 a. Assignments were based on one and two-dimensional NMR experiments known to those skilled in the art of structure elucidation and include Distortionless Enhancement Polarization Transfer (DEPT), H-H Correlation Spectroscopy (COSY), Heteronuclear Multiple Quantum Correlation (HMQC), Heteronuclear Mutiple Bond Correlation (HMBC).

TABLE 1

NMR Data for Compound 1 a

Spectra obtained in CDC1₃

¹³C @ 100 MHz: d; 'H @ 400 MHz: d, integral, multiplicity,

J(Hz)

o overlapped

6.3.2 Compound 2

Compound 2 R=H Compound 2a R=Me

The isolation and characterization of Compound 2 was reported in 1992 (Maisenbacher, et al, (1992)). Compound 2 gave a UV_^ at 273.3 nm. IR absorbances were recorded at 2925. 1723, 1602, 1450, 1376, 1233, 1076. 842 cm^"1 EIMS data of Compound 2 showed a M⁺ peak at 550 nm suggesting a molecular formula of C₃₆H₅₄O₄.

Compound 2 (100 mg) was dissolved into 2 ml of a 1 :1 mixture of methanol and dichloromethane. To this solution was added trimethylsilydiazomethane reagent until color persisted. The reaction was allowed to stand for 1 hour after which it was blown down with nitrogen and the resulting residue was chromatographed on a Bakerbond 40 μm C18 column (1 X 50 cm). Column elution was isocratic using 95:5 methanol/water. Thirty-seven 20 ml fractions were collected. Fractions 36-37 were combined and evaporated to dryness, giving 7 mg of a colorless oil identified Compound 2a.

Complete NMR assigments for 2a can be found in the table below (Table 2). Assignments were based on one and two-dimensional NMR experiments known to those skilled the art of structure elucidation and include Distortionless Enhancement Polanzation Transfer (DEPT), H-H Correlation Spectroscopy (COSY), Heteronuclear Multiple Quantum Conelation (HMQC). Heteronuclear Mutiple Bond Conelation (HMBC).

Compaπsion of la and 2a showed that the ¹ C-NMR and DEPT spectra of 2a contained one additional methvlene signal. Closer examination of the ¹³C-NMR data showed that 2a contained all of the signals of la except for those corresponding to the isobutyryl group at C-l . Instead 2a contained the 5 signals expected for a 2- methylbutyryl group. The chemical shift value for C-l assured placement of the 2- methylbutyryl group at that position. Complete NMR assigments for 2a can be found in the table below. Assignments were based on one and two-dimensional NMR expeπments known to those skilled in the art of structure elucidation and include Distortionless Enhancement Polanzation Transfer (DEPT), H-H Correlation Spectroscopy (COSY), Heteronuclear Multiple Quantum Correlation (HMQC), Heteronuclear Mutiple Bond Correlation (HMBC).

TABLE 2

NMR Data for 2a

Spectra obtained in CDC1₃

¹³C @ 100 MHz: d; Η @ 400 MHz: d, integral, multiplicity,

J(Hz)

This example illustrates the effectiveness of the bicyclo[3.3.1.]nonenes of formulae (II)a and (IΙI)a in reducing plasma glucose levels in C57BL/ks diabetic (db/db) mice, i.e., an art-recognized model of non-insulin dependent diabetes mellitus (NIDDM).

7.1.1 In vivo Experiments, General

The following experiments are set forth to assist in understanding the invention and should not, of course, be construed as specifically limiting the invention described and claimed herein. Such variations of the inventions which would be within the purview of those in the art, including the substitution of all equivalents now known or later developed, including changes in formulation or minor changes in experimental design, are to be considered to fall within the scope of the invention as hereinafter claimed.

This example illustrates the effectiveness of the bicyclo[3.3.1]nonenes of formulae (II)a and (IΙI)a, e.g. 4-hydroxy- l-isobutyryl-3, 5, 7-tris(3-methyl-2- butenyl)-8-(4-methyl-3-pentenyl)-eΛ'o-biocyclo[3.3.1 ]non-3-ene-2.9-dione (Compound 1 ); and 4-hydroxy- l-(2-methylbutyryl)-3, 5, 7-tris(3-methyl-2-butenyl)-8- (4-methyl-3-pentenyl)-eλO-bicyclo[3.3.1]non-3-ene-2,9-dione (Compound 2), in reducing plasma glucose levels in C57BL/ks diabetic (db/db) mice, i.e., an art recognized model of non-insulin dependent diabetes mellitus (NIDDM).

7.1.2 Materials and Methods

Genetically altered obese diabetic mice (designated C57BL/ks diabetic or db/db) were purchased from the Jackson Laboratory (Bar Harbor, ME, USA), and served as experimental animals. Male animals between the ages of 8-9 weeks were employed in the studies described here. Animals were housed (4 mice/cage) under standard laboratory conditions at approximately 22 °C and 50% relative humidity, and were maintained on a diet of Purina rodent chow and water ad libitum. Prior to treatment, blood was collected from the tail vein of each animal. Mice that had plasma glucose levels between 350 and 600 mg/dL were used. Each treatment group consisted of eight mice that were distributed so that the mean glucose levels were equivalent in each group at the start of the study. Db/db mice received, orally by gavage, once daily: the experimental compound administered at 40 or 80 mg kg/day (unless otherwise noted), or metforin admimstered at 250 mg/kg for 1-3 days. Test compounds were delivered in a 0.5 m citrate buffer liquid vehicle containing 1% (v/v) Tween 60® (polyoxyethylene sorbitan monosterate), and up to 10% (v/v) dimethyl sulfoxide (DMSO) in a volume of 10 ml/kg. Blood was sampled from the tail vein at three hours, twenty-seven and fifty-one hours after initial dosing (the first administration) of the particular compound in non-fasted conditions. Blood samples were analyzed for plasma glucose levels. Individual body weights and mean food consumption (each cage) were also measured daily. The synthetic test substances were prepared as descπbed previously (vide supra) Metformin (1.1-dιmethylbιguanιde) w as obtained from Groupe Lipha (Lyon, France), carboxymethyl cellulose and Tween 60 were purchased from Sigma Chemical Co. (St. Louis, MO, USA, catalog #'s C-4888, and p-1629, respectively). Plasma glucose levels were determined colonmetncally using the glucose oxidase method (Sigma Chemical Co., Sigma catalog #315) Significant differences between groups (companng compound-treated to vehicle-treated) were evaluated using analysis of vanance and Fisher's post-hoc test.

7.1.3 Results

7.1.3.1 Compounds 1 & 2

As shown in Fig. 1 and in Table 4. below, oral administration of Compound 1 (4-hydroxy- l-ιsobutyryl-3, 5, 7-tns(3-methyl-2-butyl)-8-(4-methyl-3- pentenyl)-(non-3-ene-2,9-dιone) and Compound 2 (4-hydroxy- 1-(1 -methylbutyryl)-8- methyl-3,5,7-tns(3-methyl-2-butenyl)-8-(4-methyl-3-pentenyl)-e o- bιcyclo[3.3.1]non-3-ene-2,9-dιone) at a dose level of 40 and 80 mg/kg to db/db mice produced statistically significant reduction in plasma glucose, relative to vehicle (control). The test substances were evaluated in a seπes of experiments which are summanzed in Table 4 below.

The db/db mice received doses of Compound 1 or Compound 2 at 0, 24, and 48 hours. Blood glucose levels were measured at 0, 3, 27, and 51 hours as read left to right in Figure 1.

Single doses of Compound 1 (40 and 80 mg/kg) were given to db/db mice at twenty-four and forty-eight hours after the initial oral administration resulted in statistically significant reductions in plasma glucose relative to vehicle controls at either three, twenty-seven or fifty-one hours or at all timepoints after oral administration. Three hours after the initial dosing of 40 mg/kg and 80 mg/kg, mean glucose levels for the active expeπmental Compound 1 declined 91.5 mg dL (p = 0.0059) and 164.6 mg/dL (p < 0.0001), respectively, from the baseline value.

Twenty-seven hours after the initial dosing, three hours after the second dosmg, mean glucose levels for the active expenmental Compound 1 (80 mg/kg) declined 172.4 mg/dL (p < 0.0001) from the baseline values. Fifty-one hours after the initial dosmg, three hours after the third dosmg mean glucose le\ els for the active expenmental Compound 1 (40 mg/kg and 80 mg/kg declined 56 9 mg/dL (p = 0 0198) and 128 5 mg/dL (p ^ 0 0001), respectr e , from the baseline values

Single doses of Compound 2 (40 and 80 mg/kg) w ere given to db/db mice at forty-eight hours after the initial oral administration resulted in statistically significant reductions m plasma glucose relative to vehicle controls at either three or fifty-one h after initial oral administration Three hours after the initial dosmg, mean glucose levels for the actι\ e expenmental Compound 2 (40 mg/kg and 80 mg/kg) declined 98 0 mg/dL (p = 0 0023) and 150 6 mg/dL (p < 0 0001), respectively, from the baseline value Fifty-one hours after the initial dos g, three hours after the second dosing, mean glucose le\ els for the active expenmental Compound 2 (40 mg/kg and 80 mg/kg) declined 192 8 mg/dL (p < 0 0001) and 186 4 mg,dL (p < 0 0001), respectively, from the baseline values

Compound 1 (40 mg/kg) administered to mice also showed a trend in reducing plasma glucose relative to vehicle controls at 3 hours after the second dosmg, twenty-seven hours after the initial oral administration Twenty-seven hours after the initial dosmg, mean glucose levels for the active expenmental Compound 1 /citrate buffer declined 66 8 mg/dL (p = 0 1072) from the baseline value

Compound 2 (40 mg/kg) administered also showed a trend in reducing plasma glucose relative to vehicle controls at 3 hours after the second dosmg, fifty- one hours after the initial oral administration Fifty-one hours after the initial dosmg, mean glucose levels for the active expenmental Compound 2 suspended m citrate buffer declined 48 1 mg/dL (p = 0 0574) from the baseline value

By companson, the known hypoglycemic agent metformin, given at 250 mg/kg. lowered plasma glucose levels by approximately 153 2 mg/dL

(p<0 0001), three hours after the initial dose, 158 0 mg/dL (p<0 0001), twenty-seven hours after the initial dose, 151 6 mg/dL (p<0 0001), three hours after the third dose and fifty-one hours after the initial dose, three hours after the third dose

As shown in Table 5. below, the antihyperglycemic effect of Compounds 1 and 2 at dosage regimes of 40 and 80 mg/kg occurred in the absence of any significantly adverse effect on food intake or body weight Body weights were not affected in animals treated duπng the test peπod (Table 5) The data in Tables 4 and 5 indicate that the aforementioned bicyclo [3.3.1] nonenes are effective hypoglycemic agents in a rodent model of insulin resistance, obesity, and NIDDM.

Table 4. Effects of test substances on glucose-lowering in diabetic db/db mice.

* Statistical significance evaluated using unpaired t-test and Fisher's post-hoc test. NS- not significant at p=0.05 level

Table 5. Effects of test substances on body weights and food consumption in diabetic db/db mice.

7.1.3.2 St. John's Wort Extract Enriched With Compound 1

Male, 8-9 weeks old, C57BL'6J ob/ob mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). Animals were housed (4-5 mice/cage) under standard laboratory conditions at 22 °C and 50% relative humidity, and were maintained on a diet of Purina rodent chow and water ad libitum. Prior to treatment, blood was collected from the tail vein of each animal. Mice that had non-fasting plasma glucose levels between 250 and 400 mg/dL were used. Each treatment group consisted of 10 mice that were distributed so that the mean glucose levels were equivalent in each group as the start of the study. Mice were dosed orally by gavage once a day for 7 days with either vehicle or St. John's Wort ("SJW") extract. The amount of SJW extract dosed equaled to 20 mg/kg of hyperforin in pure form. The extract was delivered in a liquid formulation contained 5%o (v/v) dimethyl sulfoxide (DMSO), 1%) (v/v) tween 80 and 0.9 % (w/v) methylcellulose. The gavage volume was 10 ml/kg. Blood samples were taken at 6, 30 and 150 hours after the first dose and analyzed for plasma glucose. Plasma glucose concentrations were determined colorimetrically using glucose oxidase method (Sigma Chemical Co, St. Louis, MO, USA). Significance difference between groups (comparing drug-treated to vehicle- treated) was evaluated using Student unpaired t-test. As illustrated in Fig.6, SJW extract significantly reduced plasma glucose concentrations at all time points as compared to the vehicle control group.

8. EXAMPLE: EFFECTS ON GLUCOSE DISPOSAL

This example illustrates the effectiveness of the bicyclo [3.3.1] nonenes of formulae (II)a and (IΙI)a , e.g., (4-hydroxy- 1 -isobutyryl-8-methyl-3, 5,7- tris(3-methyl-2-butenyl)-8-(4-methyl-3-pentenyl-exo-bicyclo[3.3.1]non-3-ene-2,9- dione) in glucose disposal in obese diabetic db/db mice, i.e., an art-recognized model of non-insulin dependent diabetes mellitus (NIDDM). 8.1 MATERIALS AND METHODS

Animals and experimental conditions were the same as those described in Section 7.1.2, above, except as follows: Db/db mice received, orally by gavage, at 0, 8, 24 and 48 hours, either vehicle, Compound 1 administered at 40 and 80 mg/kg. Food was withdrawn at 61 hours. The animals were administered with a dose at 72 hours, and sampled at the 75th hour, followed by an oral glucose load (2g/kg). Blood was obtained from the tail vein at 15, 30, 60 and 120 minutes following the glucose load; and analyzed for plasma glucose levels.

8.2 RESULTS

Figs. 2 and 3 show the effect of Compound 1 in an oral glucose tolerance test (OGTT) in db/db mice. At dosage levels of 40 and 80 mg/kg,

Compound 1 significantly suppressed the postprandial glucose levels compared to vehicle treated animals at all timepoints after the glucose load, relative to the vehicle. The oral glucose tolerance, i.e., the transfer or "disposal" of glucose from the bloodstream to the tissues was significantly improved at 40 mg/kg and 80 mg/kg, relative to vehicle, as indicated in Fig. 4 by the reduction of the area under the curves of Fig. 2. These data indicate that Compound 1 enhances glucose utilization and improves the rate of glucose disposal in a rodent model of insulin resistance, obesity, and NIDDM.

9. EXAMPLE: EFFECTS OF COMPOUND 1 TREATMENT ON SERUM

TRIGLYCERIDE LEVELS IN A RODENT MODEL OF NON INSULIN DEPENDENT DIABETES MELLITUS

The following experiments demonstrate that hyperforin is a safe and effective treatment for reducing serum triglyceride (TG) in diabetic animals. 9.1. MATERIALS AND METHODS

Male Sprague Dawley rats, 250 g body weight, obtained from Charles River Laboratories, Hollister. CA, were fed a 20%> high fat diet obtained from Harlan Teklad. Madison, Wl, for two weeks and then injected with 45 mg/kg streptozotocin, i.e., 2-deoxy-2[([methyl-nitrosoamino]-carbonyl)-amino]D-glucopyranose ("STZ"), obtained from ICN Pharmaceuticals, Inc., Costa Mesa CA (catalogue # 100557) by intravenous injection three days prior to the start of the study.

Compound 1 isolated as described in Sections 5.3.1 and 6, was kept under nitrogen until just before preparation and use of the Compound 1 /Citrate buffer suspension ("Compound 1/ Citrate Buffer"). Compound 1 /Citrate Buffer was prepared by vortexing Compound 1 in DMSO in one-tenth of the final volume. An amount of Citrate Buffer (0.05 M, pH 4.5) sufficient to achieve the final volume (e.g., 10 ml/kg) was added to the Compound 1 DMSO suspension and then vortexed prior to use, and mixed intermittently during the administration procedure.

Blood samples from tail snip bleeds were collected and analyzed as described above. Data are expressed as the mean ± SEM. Data were analyzed by analysis of variance with a Fisher's Protected Least Significant Difference post-hoc test. Ap value of less than 0.05 is considered significant.

9.2. EXPERIMENTAL PROTOCOL

Male Sprague Dawley rats were fed a high fat diet and injected with streptotocin to make them diabetic and insulin resistant. Animals were pre-screened by blood sampling and analysis for serum glucose levels. Animals with hyperglycemia (>300 mg/dl) were randomly sorted into five treatment groups (n = 8). Animals were orally gavaged with an citrate buffer vehicle (10%> DMSO, 1 % Tween in 0.05 M Citrate Buffer, pH 4.5 (10 ml kg)) or Compound 1 (hyperforin) formulated in Citrate buffer at 40, 80 or 160 mg/kg once a day for four days. Blood samples were taken before dosing and at two hours post dosing on days 2-4. Blood samples were also taken at four hours post dosing on day 2. Blood samples were analyzed for serum glucose and triglycerides (TG) levels. Body w eight and food consumption ere measured daily as gross indicators of animal health and appetite

9.3 RESULTS AND DISCUSSION

Tnglycende data for the expenment are shown in Figure 4 Hyperfonn significantly lowered TG concentrations (p<0 05) at doses of 40, 80 and 160 mg/kg when administered in the citrate buffer \ ehicle (Citrate Buffer)

Glucose data for the expenment is shown in Figure 5 Hyperfonn when administered in the q d 80 and 160 mg/kg dosmg regimens employed in the present study significantly lowered (p< 0001) glucose levels In addition, hyperfonn when administered in the q d 40 mg/kg dosmg regimen employed m the present study exhibited a trend in lowenng glucose levels (p - 0 14)

Compound 1 (hyperfonn) significantly lowered food consumption across the study at dosage regimens of 80 (23%.) and 160 (41%) mg/kg (p < 0 05)

Hyperfonn at a dosage regimen of 40 mg/kg did not have a significant effect on food consumption in this expenment Hyperfonn did not significantly affect body weight across all dosage levels In addition, all the animals appeared healthy and there were no deaths m either expenment

The present invention is not to be limited in scope by the specific embodiments disclosed in the examples which are intended as illustrations of a few aspects of the invention and any embodiments which are functionally equivalent are withm the scope of this invention. Indeed, vaπous modifications of the invention in addition to those descπbed herein will become apparent to those skilled m the art and are intended to fall withm the appended claims

A number of references have been cited and the entire disclosures of which are incorporated herein by reference.

Claims

What is claimed is:

1. A method for lowering blood glucose levels comprising administering to a mammal a therapeutically effective amount of an isolated or a purified compound of the formula (II)a:

or a pharmaceutically acceptable salt thereof, wherein

R, is selected from the group consisting of hydroxy and oxygen; R₂ is selected from the group consisting of hydroxy, oxygen and benzoyl; wherein both R, and R₂ are not simultaneously oxygen R₃ is selected from the group consisting of hydrogen, methyl, methylhalide, 3-methyl-2-butenyl; and -(CH₂)_xCOOR₄; wherein X is 0 to 2; wherein R₂ and R₃ can form a ring, said ring selected from the group consisting of a furan ring and a pyran ring; wherein R₄ is selected from the group consisting of hydrogen and C, - C₃ alkyl; R₅ is selected from the group consisting of hydrogen and C,-C₆ alkyl;

R₆ is selected from the group consisting of 3-methyl-2-butenyl, isobutyryl, 2-methylbutyryl, and benzoyl;

R₇ is 3-methyl-2-butenyl; and

R₈ is selected from the group consisting of hydrogen, methyl, methylhalide, 4-methyl-3-pentenyl, and -(CH₂)_xCOOR₄.

2. The according to claim 1 wherein the compound is selected from the group consisting of

4-hydroxy- l-isobutyryl-8-methyl-3, 5, 7-tris(3-methyl-2-butenyl)-8-(4- methyl-3-pentenyl)-exo-bicyclo [3.3.1] nonene-2,9-dione; 4-hydroxy- l-(2-methylbut>τyl)-8-exo-methyl-3.5, 7-tris(3-methyl-2- butenyl)-8-evo-bicyclo[3.3.1]non-3-ene-2,9-dione;

3-benzoyl-4-hydroxy-8.8-dimethyl-l,5,7-tris(3-methyl-2-butenyl)-exo- bicyclo[3.3.1 ]non-3-ene-2.9-dione; 5-benzoyl-4-hydroxy-8,8-dimethyl-l,3,7-tris(3-methyl-2-butenyl)-e o- bicyclo[3.3.1 ]non-3-ene-2.9-dione;

4-hydroxy-5-(3'-hydroxybenzoyl)-8,8-dimethyl-l,3,7-tris(3-methyl-2- butenyl)-e.TO-bicyclo[3.3.1]non-3-ene-2,9-dione;

4-hydroxy-8-e o-methyl-5,7-e o-bis(3-methyl-2-butenyl)-l-(2-methyl- 1 -oxopropyl)-8-e» Jo(4-methyl-3-pentenyl)bicyclo[3.3.1 ]non-3-ene-2,9-dione; and

4-hydroxy-8-exo-methyl-5,7-e o-bis(3-methyl-2-butenyl)-l-(2-methyl- 1 -oxobutyl)-8-e«<io(4-methyl-3-pentenyl)bicyclo[3.3.1 ]non-3-ene-2,9-dione.

3. The method according to claim 1 wherein the compound is a oxidation derivative selected from the group consisting of

1 -(2-methyl- 1 -oxopropyl)-2, 12-dioxo-3, 10β-bis(3-methyl-2-butenyl)- 6β-(l -methyl- 1 -hydroxyethyl)- 11 β-methyl- 11 α(4-methyl-3-pentenyl)-5-oxatricyclo- [6.3.1.0⁴'⁸]-3-dodecene; and l-(2-methyl-l-oxopropyl)-2,12-dioxo-3,10β-bis(3-methyl-2-butenyl)- 6-hydroxy- 11 β-methyl- 1 1 α-4(methyl-3-ρentenyl)-5-oxatricyclo[6.3.1.0⁴'⁸]-3- dodecene.

4. The method according to claim 1 wherein the mammal is hyperglycemic.

5. The method according to claim 1 wherein the mammal is suffering from NIDDM.

6. The method according to claim 1, wherein the compound is administered at a dose of about 0.5 to about 1000 mg per kg per day.

7. The method according to claim 1, wherein the compound is a pharmaceutically acceptable salt of sodium, potassium, lithium, calcium, magnesium, zinc or iron.

8. The method according to claim 1, wherein the compound is administered in conjunction with another antihyperglycemic agent selected from the group consisting of a sulfonylurea, a non-sulfonylurea insulin secretagogue, a biguanide, a thiazolidinedione. a β- adrenoceptor agonist, an α-glycosidase inhibitor and insulin.

9. The method according to claim 8, wherein the sulfonylurea is selected from the group consisting of acetohexamide, chlorpropamide, tolazamide, tolbutamide, glyburide, glypizide and glycazide.

10. The method according to claim 8 wherein the biguanide is metformin or buformin.

11. The method according to claim 8, wherein the α- glucosidase inhibitor is acarbose or miglatol.

12. The method according to claim 8, wherein the thiazolidinedione is selected from the group consisting of troglitazone, pioglitazone, rosiglitozone and ciglitazone.

13. A method for lowering blood glucose comprising administering to a mammal a therapeutically effective amount of a composition comprising an isolated or a purified compound of the formula (IΙI)a:

Formula (IΙI)a

or a pharmaceutically acceptable salt thereof, wherein R", is selected from the group consisting of O and OH;

R'_: is selected from the group consisting of O and OH, wherein R', and R'₂ are not simultaneously O; and

R^* ₃ is H or CH₃.

14. The method according to claim 13, wherein the compound is selected from the group consisting of:

4-hydroxy-l-isobutyryl-8-methyl-3,5,7-tris(3-methyl-2-butenyl)-8-(4- methyl-3-pentenyl)-e o-bicyclo [3.3.1] nonene-2,9-dione; and

4-hydroxy- l-(2-methylbutyryl)-8-exo-m ethyl 3,5,7-tris(3-methyl-2- butenyl)-8-e o-bicyclo[3.3.1]non-3-ene-2,9-dione.

15. The method according to claim 13, wherein the composition consists essentially of the compound of formula (IΙI)a.

16. The method according to claim 13, wherein the compound is administered at a dose of about 0.5 to about 1000 mg per kg per day.

17. The method according to claim 13, wherein the compound is a pharmaceutically acceptable salt of sodium, potassium, lithium, calcium, magnesium, zinc or iron.

18. The method according to claim 13, wherein the compound is administered in conjunction with another antihyperglycemic agent selected from the group consisting of a sulfonylurea, a non-sulfonylurea insulin secretagogue, a biguanide. a thiazolidinedione, a β-adrenoceptor agonist, an α-glycosidase inhibitor and insulin.

19. The method according to claim 18, wherein the sulfonylurea is selected from the group consisting of acetohexamide. chlorpropamide, tolazamide, tolbutamide. glyburide, glypizide and glycazide.

20. The method according to claim 18, wherein the biguanide is selected from the group consisting of metformin and buformin.

21. The method according to claim 18, wherein the α-glucosidase inhibitor is selected from the group consisting of acarbose and miglatol.

22. The method according to claim 18, wherein the thiazolidinedione is selected from the group consisting of troglitazone, pioglitazone, rosiglitozone and ciglitazone.

23. A method for lowering serum triglyceride levels comprising administering to a mammal a therapeutically effective amount of an isolated or a purified compound of the formula (II)b:

(II)b

or a pharmaceutically acceptable salt thereof, wherein R", is selected from the group consisting of hydroxy, oxygen and C,-C₆ alkoxy;

R"_: is selected from the group consisting of hydroxy, oxygen, benzoyl and C,-C₆ alkoxy; wherein both R", and R"_: are not simultaneously oxygen; R"₃ is selected from the group consisting of hydrogen, methyl, methylhalide, 3-methyl-2-butenyl; and -(CH₂) COOR"₄; wherein X is 0 to 2; wherein R"₂ and R"₃ can form a ring, said ring selected from the group consisting of a furan ring and a pyran ring; wherein R"₄ is selected from the group consisting of hydrogen and C] - C₃ alkyl; R"₅ is selected from the group consisting of hydrogen and C,-C₆ alkyl;

R"₆ is selected from the group consisting of 3-methyl-2-butenyl, isobutyryl, 2-methylbutyryl, and benzoyl;

R'\ is 3-methyl-2-butenyl; and

R"₈ is selected from the group consisting of hydrogen, methyl, methylhalide, 4-methyl-3-pentenyl, and -(CH₂)_xCOOR"₄.

24. The method according to claim 23 wherein the compound is selected from the group consisting of

4-hydroxy-l-isobutyryl-8-methyl-3,5,7-tris(3-methyl-2-butenyl)-8-(4- methyl-3-pentenyl)-exo-bicyclo [3.3.1] nonene-2,9-dione;

4-hydroxy-l-(2-methylbutyryl)-8-e o-methyl-3,5,7-tris(3-methyl-2- butenyl)-8-exo-bicyclo[3.3.1]non-3-ene-2,9-dione;

3-benzoyl-4-hydroxy-8,8-dimethyl-l,5,7-tris(3-methyl-2-butenyl)-e o- bicyclo[3.3.1]non-3-ene-2,9-dione; 5-benzoyl-4-hydroxy-8,8-dimethyl-l,3,7-tris(3-methyl-2-butenyl)-exo- bicyclo[3.3.1]non-3-ene-2,9-dione;

4-hydroxy-5-(3'-hydroxybenzoyl)-8,8-dimethyl-l,3,7-tris(3-methyl-2- butenyl)-exo-bicyclo[3.3.1]non-3-ene-2,9-dione;

4-hydroxy-8-e o-methyl-5,7-exo-bis(3-methyl-2-butenyl)-l-(2-methyl- l-oxopropyl)-8-endo(4-methyl-3-pentenyl)bicyclo[3.3.1]non-3-ene-2,9-dione; and

4-hydroxy-8-e o-methyl-5,7-exo-bis(3-methylbut-2-enyl)-l-(2-methyl- 1-2 oxobutyl)-8-e«<io(4-methylpent-3-enyl)bicyclo[3.3.1]non-3-ene-2,9-dione.

25. The method according to claim 23 wherein the compound is an oxidation derivative selected from the group consisting of l -(2-methyl-l -oxopropyl)-2.12-dioxo-3,10β-bis(3-methyl-2-butenyl)- 6β-( 1 -methyl- 1 -hydroxyethyl)- 1 1 β-methyl- 1 1 α(4-methyl-3-pentenyl)-5-oxatricyclo- [6.3.1.0⁴'⁸]-3-dodecene; and

1 -(2-methyl- 1 -oxopropyl)-2.12-dioxo-3 , 10β-bis(3-methyl-2-butenyl)- 6-hydroxy- 1 1 β-methyl- 1 1 α-4(methyl-3-pentenyl)-5-oxatricyclo[6.3.1.0⁴'⁸]-3- dodecene.

26. The method according to claim 23, wherein the compound is administered at a dose of about 0.5 to about 1000 mg per kg per day.

27. The method according to claim 23, wherein the compound is a pharmaceutically acceptable salt of sodium, potassium, lithium, calcium, magnesium, zinc or iron.

28. The method according to claim 23, wherein the compound is administered in conjunction with another antihyperlipidemic agent.

29. A method for lowering serum triglyceride levels comprising administering to a mammal a therapeutically effective amount of a composition comprising an isolated or a purified compound of the formula (IΙI)b:

Formula (IΙI)b

or a pharmaceutically acceptable salt thereof, wherein

R', is selected from the group consisting of O and OH; R', is selected from the group consisting of O and OH. wherein R', and R', are not simultaneously O; and R'₃ is H or CH₃.

30. The method according to claim 29, wherein the compound is selected from the group consisting of:

4-hydroxy- l-isobutyryl-8-methyl-3, 5, 7-tris(3-methyl-2-butenyl)-8-(4- methyl-3-pentenyl)-exo-bicyclo [3.3.1] nonene-2,9-dione; and

4-hydroxy- l-(2-methylbutyτyl)-8-e o-methyl 3,5,7-tris(3-methyl-2- butenyl)-8-e o-bicyclo[3.3.1 ]non-3-ene-2,9-dione.

31. The method according to claim 29, wherein the composition consists essentially of the compound of formula (IΙI)b.

32. The method according to claim 29, wherein the compound is administered at a dose of about 0.5 to about 1000 mg per day.

33. The method according to claim 29, wherein the compound is a pharmaceutically acceptable salt of sodium, potassium, lithium, calcium, magnesium, zinc or iron.

34. The method according to claim 29, wherein the compound is administered in conjunction with another antihyperlipidemic agent.

35. A method for lowering blood glucose levels comprising administering to a mammal a therapeutically effective amount of a Hypericum spp. extract enriched in bicyclo[3.3.1]nonenes of formula (II)a:

(II)a or a pharmaceutically acceptable salt thereof, wherein

R, is selected from the group consisting of hydroxy and oxygen;

R, is selected from the group consisting of hydroxy, oxygen and benzoyl; wherein both R, and R_: are not simultaneously oxygen

R₃ is selected from the group consisting of hydrogen, methyl, methylhalide, 3-methyl-2-butenyl; and -(CH₂) COOR₄; wherein X is 0 to 2; wherein R₂ and R, can form a ring, said ring selected from the group consisting of a furan ring and a pyran ring; wherein R₄ is selected from the group consisting of hydrogen and C - C₃ alkyl;

R₅ is selected from the group consisting of hydrogen and C,-C₆ alkyl;

R₆ is selected from the group consisting of 3-methyl-2-butenyl, isobutyryl, 2-methylbutyryl, and benzoyl;

R₇ is 3-methyl-2-butenyl; and R₈ is selected from the group consisting of hydrogen, methyl, methylhalide, 4-methyl-3-pentenyl, and -(CH₂)_xCOOR₄.

36. A method according to claim 35, wherein the bicyclo[3.3.1]nonenes have a degree of purity at least about 2.5%> by weight.

37. A method according to claim 35, wherein the bicyclo[3.3.1]nonenes have a degree of purity greater than about 5%o by weight.

38. A method for lowering blood glucose levels comprising administering to a mammal a therapeutically effective amount of a Hypericum spp. extract enriched in bicyclo[3.3.1]nonenes of formula (IΙI)b:

Formula (IΙI)b

or a pharmaceutically acceptable salt thereof, wherein R', is selected from the group consisting of O and OH;

R'₂ is selected from the group consisting of O and OH, wherein R', and R', are not simultaneously O; and

R'₃ is H or CH₃.

39. A method according to claim 38, wherein the bicyclo[3.3.1]nonenes have a degree of purity at least about 2.5%> by weight.

40. A method according to claim 38, wherein the bicyclo[3.3.1]nonenes have a degree of purity greater than about 5%> by weight.