# Patent

## Title
Diterpenoid compounds, compositions thereof and their use as anti-cancer or anti-fungal agents

## Abstract

The present invention relates to Diterpenoid Compounds, compositions comprising an effective amount of a Diterpenoid Compound, and methods useful for treating or preventing cancer or a neoplastic disorder comprising administering an effective amount of a Diterpenoid Compound. The compounds, compositions, and methods of the invention are also useful for inhibiting the growth of a cancer cell or neoplastic cell, or for inducing apoptosis in a cancer or neoplastic cell. The compounds, compositions, and methods of the invention are further useful for treating or preventing a fungal infection. The compounds, compositions, and methods of the invention are also useful for inhibiting the growth of a fungus.

## Background
This application claims the benefit of U.S. provisional application Ser. No. 60/431,096, filed Dec. 5, 2002, the entire disclosure of which is incorporated by reference herein in its entirety.


1. FIELD OF THE INVENTION
The present invention relates to Diterpenoid Compounds, compositions comprising an effective amount of a Diterpenoid Compound, and methods useful for treating or preventing cancer or a neoplastic disorder comprising administering an effective amount of a Diterpenoid Compound. The compounds, compositions, and methods of the invention are also useful for inhibiting the growth of a cancer cell or neoplastic cell, or for inducing apoptosis in a cancer or neoplastic cell. The compounds, compositions, and methods of the invention are further useful for treating or preventing a fungal infection. The compounds, compositions, and methods of the invention are also useful for inhibiting the growth of a fungus.
2. BACKGROUND OF THE INVENTION
2.1 Cancer and Neoplastic Disease 
Cancer affects approximately 20 million adults and children worldwide, and this year, more than 9 million new cases will be diagnosed (International Agency for Research on Cancer; www.irac.fr). According to the American Cancer Society, about 563,100 Americas are expected to die of cancer this year, more than 1500 people a day. Since 1990, in the United States alone, nearly five million lives have been lost to cancer, and approximately 12 million new cases have been diagnosed.
Currently, cancer therapy involves surgery, chemotherapy and/or radiation treatment or eradicate neoplastic cells in a patient (see, for example, Stockdale, 1998, “Principles of Cancer Patient Management”, in Scientific American: Medicine, vol. 3, Rubenstein and Federman, eds., Chapter 12, Section IV). All of these approaches pose significant drawbacks for the patient. Surgery, for example, can be contraindicated due to the health of the patient or can be unacceptable to the patient. Additionally, surgery might not completely remove the neoplastic tissue. Radiation therapy is effective only when the irradiated neoplastic tissue exhibits a higher sensitivity to radiation than normal tissue, and radiation therapy can also often elicit serious side effects. (Id.) With respect to chemotherapy, there are a variety of chemotherapeutic agents available for treatment of neoplastic disease. However, despite the availability of a variety of chemotherapeutic agents, traditional chemotherapy has many drawbacks (see, for example, Stockdale, 1998, “Principles Of Cancer Patient Management” in Scientific American Medicine, vol. 3, Rubenstein and Federman, eds., ch. 12, sect. 10). Almost all chemotherapeutic agents are toxic, and chemotherapy can cause significant, and often dangerous, side effects, including severe nausea, bone marrow depression, immunosuppression, etc. Additionally, many tumor cells are resistant or develop resistance to chemotherapeutic agents through multi-drug resistance.
Therefore, there is a significant need in the art for novel compounds and compositions, and methods that are useful for treating cancer or neoplastic disease with reduced or without the aforementioned side effects. Further, there is a need for cancer treatments that provide cancer-cell-specific therapies with increased specificity and decreased toxicity.
2.2 Fungal Infection and Related Health Issues 
Fungi are eukaryotic microorganisms and can occur as yeasts, molds, or as a combination of both forms. Some fungi are capable of causing superficial, cutaneous, subcutaneous, systemic or allergic diseases. Yeasts are microscopic fungi consisting of solitary cells that reproduce by budding. Molds, in contrast, occur in long filaments known as hyphae, which grow by apical extension.
Known fungal and mycotic pathogens include, but are not limited to, Absidia spp., Actinomadura madurae, Actinomyces spp., Allescheria boydii, Alternaria spp., Anthopsis deltoidea, Apophysomyces elegans, Arnium leoporinum, Aspergillus spp., Aureobasidium pullulans, Basidiobolus ranarum, Bipolaris spp., Blastomyces dermatitidis, Candida spp., Cephalosporium spp., Chaetoconidium spp., Chaetomium spp., Cladosporium spp., Coccidioides immitis, Conidiobolus spp., Corynebacterium tenuis, Cryptococcus spp., Cunninghamella bertholletiae, Curvularia spp., Dactylaria spp., Epidermophyton spp., Epidermophyton floccosum, Exserophilum spp., Exophiala spp., Fonsecaea spp., Fusarium spp., Geotrichum spp., Helminthosporium spp., Histoplasma spp., Lecythophora spp., Madurella spp., Malassezia furfur, Microsporum spp., Mucor spp., Mycocentrospora acerina, Nocardia spp., Paracoccidioides brasiliensis, Penicillium spp., Phaeosclera dematioides, Phaeoannellomyces spp., Phialemonium obovatum, Phialophora spp., Phoma spp., Piedraia hortai, Pneumocystis carinii, Pythium insidiosum, Rhinocladiella aquaspersa, Rhizomucor pusillus, Rhizopus spp., Saksenaea vasiformis, Sarcinomyces phaeomuriformis, Sporothrix schenckii, Syncephalastrum racemosum, Taeniolella boppii, Torulopsosis spp., Trichophyton spp., Trichosporon spp., Ulocladium chartarum, Wangiella dermatitidis, and Xylohypha spp. Other fungi that might have pathogenic potential include, but are not limited to, Thermomucor indicae-seudaticae, Radiomyces spp., and other species of known pathogenic genera. There are also reports implicating Saccharomyces as a human pathogen (e.g., Fungemia with Saccharomycetacea, H. Nielson, J. Stenderup, & B. Bruun, Scand. J. Infect. Dis. 22:581–584, 1990). In recent years, there has been a marked increase in the number of serious mycoses as a result of the growing number of immunosuppressed and immunocompromised individuals, such as transplant recipients, patients receiving chemotherapy, and HIV-infected individuals, and thus greater attention has been devoted to the need to develop safer and more effective antifungal agents.
Fungal infection is also a significant problem in veterinary medicine and in agriculture. Products that are susceptible to fungal infestation include wood products, textiles, plastics, paper, rubber, adhesives, emulsion polymers, leather, cosmetics, household disinfectants, deodorants, and paint (C. C. Yeager, Fungicides in Industry, in Antifungal Compounds, M. Siegel and H. Sisler, eds., Marcel Dekker Inc., NY, 1977).
2.2.1 Current Therapies 
The mechanism of action of four main classes of anti-fungal agents is summarized below:
Polyene Antifungal Drugs
Amphotericin, nystatin, and pimaricin interact with sterols in the cell membrane (ergosterol in fungi, cholesterol in humans) to form channels through which small molecules leak from the inside of the fungal cell to the outside.
Azole Antifungal Drugs
Fluconazole, itraconazole, and ketoconazole inhibit cytochrome P450-dependent enzymes (particularly C14-demethylase) involved in the biosynthesis of ergosterol, which is required for fungal cell membrane structure and function.
Allylamine and Morpholine Antifungal Drugs
Allylamines (naftifine, terbinafine) inhibit ergosterol biosynthesis at the level of squalene epoxidase. The morpholine drug amorolfine inhibits the same pathway at a later step.
Antimetabolite Antifungal Drugs
5-Fluorocytosine acts as an inhibitor of both DNA and RNA synthesis via the conversion of 5-fluorocytosine to 5-fluorouracil.
Many of the drugs currently available for treatment of mycoses have significant side effects or lack effectiveness against some important pathogens. For example, amphotericin B, an antifungal polyene macrolide antibiotic, has both short-term and long-term adverse effects, ranging from nausea and vomiting to kidney damage. Some evidence exists for the development of resistance to these drugs. There is therefore an ongoing need for novel antifungal drugs with few, if any, side effects and with effectiveness against pathogens for which current drugs are inadequate.
Citation of any reference in Section 2 of this application is not an admission that the reference is prior art to the application.
3. SUMMARY OF THE INVENTION
The present invention encompasses compounds having the Formula (I):




and pharmaceutically acceptable salts thereof, wherein:

Q1 and Q2 are independently ═O, ═S, ═NH or ═N—NHR, where R is —H, —C1–C10 alkyl, or -aryl;
Q3 is —O—, —S—, or —N(H)—;
R1 and R2 are independently —H, -halogen, -amino, —C1–C10 alkyl, —C1–C10 alkoxy, —C1–C10 (hydroxy)alkyl, —C1–C10 (amino)alkyl, —C1–C10 (halo)alkyl, —C2–C10 alkenyl, —C2–C10 alkynyl, (C3–C7)cycloalkyl, -aryl, C1–C10 (aryl)alkyl, or three- to seven-membered non-aromatic heterocycle, or R1, R2 and the carbon atom to which they are both attached are taken together to form a (C3–C7)cycloalkyl group or a three- to seven-membered non-aromatic heterocycle;
A is N or CR3; B is N or CR4; D is N or CR5; E is N or CR6, at least one of A, B, D and E being CR3, CR4, CR5 or CR6, respectively;
each R3, R4, R5 and R6 is independently —H, -halogen, —CN, —NH2, —NO2, —COOH, —C(O)NH2, —SH, —S(O)NH2, —S(O)2NH2, —C1–C10 (oxy)alkyl, —C1–C10 alkyl, —C1–C10 alkoxy, —C1–C10 (hydroxy)alkyl, —C1–C10 (amino)alkyl, —C1–C10 (halo)alkyl, —C2–C10 alkenyl, —C2–C10 alkynyl, —(C3–C7)cycloalkyl, -aryl, —C1–C10 (aryl)alkyl, three- to seven-membered non-aromatic heterocycle, five- to seven-membered aromatic heterocycle, —CH2OR11, —OCH2OR11, —OC(O)R11, —C(O)R11, —OC(O)OR11, —OC(O)NR11, —C(O)OR11, —C(O)NR11, —OP(O)(OR11)2, —SR11, —S(O)2NHR11, —SOR11, —S(O)2R11, —NHC(O)R11, —NHSOR11, or NHS(O)2R11; or
R3 and R4 and the carbon atoms to which they are attached are taken together to form a (C3–C7)cycloalkenyl group, a five- to seven-membered non-aromatic heterocycle, or a five- to seven-membered aromatic heterocycle; or
R5 and R6 and the carbon atoms to which they are attached are taken together to form a (C3–C7)cycloalkenyl group, a five- to seven-membered non-aromatic heterocycle, or a five- to seven-membered aromatic heterocycle; or
R4 and R5 and the carbon atoms to which they are attached are taken together to form a (C3–C7)cycloalkenyl group, a non-oxygen-containing five-membered non-aromatic heterocycle, a non-oxygen-containing five-membered aromatic heterocycle, a six- to seven-membered non-aromatic heterocycle or a six- to seven-membered aromatic heterocycle;
R7 is —H, —C1–C10 alkyl, or —C1–C10 alkoxy;
R8 and R9 are each independently —H, -halogen, —CN, —NH2, —NO2, —COOH, —C(O)NH2, —SH, —S(O)NH2, —S(O)2NH2, —C1–C10 (oxy)alkyl, —C1–C10 alkyl, —C1–C10 alkoxy, —C1–C10 (hydroxy)alkyl, —C1–C10 (amino)alkyl, —C1–C10 (halo)alkyl, —C2–C10 alkenyl, —C2–C10 alkynyl, —(C3–C7)cycloalkyl, -aryl, —C1–C10 (aryl)alkyl, three- to seven-membered non-aromatic heterocycle, five- to seven-membered aromatic heterocycle, —CH2OR11, —OCR11, —OC(O)R11, —C(O)R11, —OC(O)OR11, —OC(O)NR11, —C(O)OR11, —C(O)NR11, —OP(O)(OR11)2, —SR11, —SOR11, —S(O)2R11, —S(O)2NHR11, —NHSR11, —NHSOR11, or —NHS(O)2R11;
R10 is —H, —C1–C10 alkyl, —C3–C7 cycloalkyl, —C(O)C1–C10 alkyl, —C1–C10 (oxy)alkyl, —C(O)NH2, —C(O)NHR12, or -aryl;
R11 is —H, —C1–C10 alkyl, —(C3–C7)cycloalkyl, —C1–C10 (halo)alkyl, -aryl, —C2–C10 alkenyl, —C2–C10 alkynyl, —C1–C10 (aryl)alkyl, —C2–C10 (aryl)alkenyl, —C2–C10 (aryl)alkynyl, —C1–C10 (hydroxy)alkyl, —C1–C10 alkoxy, —C1–C10 (amino)alkyl,
a —(C3–C7)cycloalkyl unsubstituted or substituted with one or more —C1–C10 alkyl,
a three- to seven-membered non-aromatic heterocycle unsubstituted or substituted with one or more —C1–C10 alkyl, or
a three- to seven-membered aromatic heterocycle unsubstituted or substituted with one or more —C1–C10 alkyl, —C2–C10 alkenyl, or —C2–C10 alkynyl;
R12 is C1–C10 alkyl; and
each halogen is independently —F, —Cl, —Br or —I.
In one embodiment, the compound of Formula (I) is not:




or a pharmaceutically acceptable salt thereof.

In another embodiment, the compound or a pharmaceutically acceptable salt of the compound of Formula (I) is in isolated and purified form.
The present invention still further provides compounds having the Formula (II):




and pharmaceutically acceptable salts thereof, wherein:

Q1 and Q2 are independently ═O, ═S, ═NH or ═N—NHR, where R is —H, —C1–C10 alkyl, or -aryl;
Q3 is —O—, —S—, or —N(H)—;
R1 and R2 are independently —H, -halogen, -amino, —C1–C10 alkyl, —C1–C10 alkoxy, —C1–C10 (hydroxy)alkyl, —C1–C10 (amino)alkyl, —C1–C10 (halo)alkyl, —C2–C10 alkenyl, —C2–C10 alkynyl, (C3–C7)cycloalkyl, -aryl, C1–C10 (aryl)alkyl, or three- to seven-membered non-aromatic heterocycle, or R1, R2 and the carbon atom to which they are both attached are taken together to form a (C3–C7)cycloalkyl group or a three- to seven-membered non-aromatic heterocycle;
A is N or CR3; B is N or CR4; D is N or CR5; E is N or CR6, at least one of A, B, D and E being CR3, CR4, CR5 or CR6, respectively;
each R3, R4, R5 and R6 is independently —H, -halogen, —CN, —NH2, —NO2, —COOH, —C(O)NH2, —SH, —S(O)NH2, —S(O)2NH2, —C1–C10 (oxy)alkyl, —C1–C10 alkyl, —C1–C10 alkoxy, —C1–C10 (hydroxy)alkyl, —C1–C10 (amino)alkyl, —C1–C10 (halo)alkyl, —C2–C10 alkenyl, —C2–C10 alkynyl, —(C3–C7)cycloalkyl, -aryl, —C1–C10 (aryl)alkyl, three- to seven-membered non-aromatic heterocycle, five- to seven-membered aromatic heterocycle, —CH2OR11, —OCH2OR11, —OC(O)R11, —C(O)R11, —OC(O)OR11, —OC(O)NR11, —C(O)OR11, —C(O)NR11, —OP(O)(OR11)2, —SR11, —S(O)2NHR11, —SOR11, —S(O)2R11, —NHC(O)R11, —NHSOR11, or NHS(O)2R11; or
R3 and R4 and the carbon atoms to which they are attached are taken together to form a (C3–C7)cycloalkenyl group, a five- to seven-membered non-aromatic heterocycle, or a five- to seven-membered aromatic heterocycle; or
R5 and R6 and the carbon atoms to which they are attached are taken together to form a (C3–C7)cycloalkenyl group, a five- to seven-membered non-aromatic heterocycle, or a five- to seven-membered aromatic heterocycle; or
R4 and R5 and the carbon atoms to which they are attached are taken together to form a (C3–C7)cycloalkenyl group, a non-oxygen-containing five-membered non-aromatic heterocycle, a non-oxygen-containing five-membered aromatic heterocycle, a six- to seven-membered non-aromatic heterocycle or a six- to seven-membered aromatic heterocycle;
R7 is —H, —C1–C10 alkyl, or —C1–C10 alkoxy;
R8 and R9 are each independently —H, -halogen, —CN, —NH2, —NO2, —COOH, —C(O)NH2, —SH, —S(O)NH2, —S(O)2NH2, —C1–C10 (oxy)alkyl, —C1–C10 alkyl, —C1–C10 alkoxy, —C1–C10 (hydroxy)alkyl, —C1–C10 (amino)alkyl, —C1–C10 (halo)alkyl, —C2–C10 alkenyl, —C2–C10 alkynyl, —(C3–C7)cycloalkyl, -aryl, —C1–C10 (aryl)alkyl, three- to seven-membered non-aromatic heterocycle, five- to seven-membered aromatic heterocycle, —CH2OR11, —OCR11, —OC(O)R11, —C(O)R11, —OC(O)OR11, —OC(O)NR11, —C(O)OR11, —C(O)NR11, —OP(O)(OR11)2, —SR11, —SOR11, —S(O)2R11, —S(O)2NHR11, —NHSR11, —NHSOR11 or —NHS(O)2R11;
R10 is —H, —C1–C10 alkyl, —C3–C7 cycloalkyl, —C(O)C1–C10 alkyl, —C1–C10 (oxy)alkyl, —C(O)NH2, —C(O)NHR12, or -aryl;
R11 is —H, —C1–C10 alkyl, —(C3–C7)cycloalkyl, —C1–C10 (halo)alkyl, -aryl, —C2–C10 alkenyl, —C2–C10 alkynyl, —C1–C10 (aryl)alkyl, —C2–C10 (aryl)alkenyl, —C2–C10 (aryl)alkynyl, —C1–C10 (hydroxy)alkyl, —C1–C10 alkoxy, —C1–C10 (amino)alkyl,
a —(C3–C7)cycloalkyl unsubstituted or substituted with one or more —C1–C10 alkyl,
a three- to seven-membered non-aromatic heterocycle unsubstituted or substituted with one or more —C1–C10 alkyl, or
a three- to seven-membered aromatic heterocycle unsubstituted or substituted with one or more —C1–C10 alkyl, —C2–C10 alkenyl, or —C2–C10 alkynyl;
R12 is C1–C10 alkyl; and
each halogen is independently —F, —Cl, —Br or —I.
The present invention still further provides compounds having the Formula (III):




and pharmaceutically acceptable salts thereof, wherein:

Q1, Q2 and Q3 are independently ═O, ═S, ═NH or ═N—NHR, where R is —H, —C1–C10 alkyl, or -aryl;
R1 and R2 are independently —H, -halogen, -amino, —C1–C10 alkyl, —C1–C10 alkoxy, —C1–C10 (hydroxy)alkyl, —C1–C10 (amino)alkyl, —C1–C10(halo)alkyl, —C2–C10 alkenyl, —C2–C10 alkynyl, (C3–C7)cycloalkyl, -aryl, C1–C10 (aryl)alkyl, or three- to seven-membered non-aromatic heterocycle, or R1, R2 and the carbon atom to which they are both attached are taken together to form a (C3–C7)cycloalkyl group or a three- to seven-membered non-aromatic heterocycle;
A is N or CR3; B is N or CR4; D is N or CR5; E is N or CR6, at least one of A, B, D and E being CR3, CR4, CR5 or CR6, respectively;
each R3, R4, R5 and R6 is independently —H, -halogen, —CN, —NH2, —NO2, —COOH, —C(O)NH2, —SH, —S(O)NH2, —S(O)2NH2, —C1–C10 (oxy)alkyl, —C1–C10 alkyl, —C1–C10 alkoxy, —C1–C10 (hydroxy)alkyl, —C1–C10 (amino)alkyl, —C1–C10 (halo)alkyl, —C2–C10 alkenyl, —C2–C10 alkynyl, —(C3–C7)cycloalkyl, -aryl, —C1–C10 (aryl)alkyl, three- to seven-membered non-aromatic heterocycle, five- to seven-membered aromatic heterocycle, —CH2OR11, —OCH2OR11, —OC(O)R11, —C(O)R11, —OC(O)OR11, —OC(O)NR11, —C(O)OR11, —C(O)NR11, —OP(O)(OR11)2, —SR11, —S(O)2NHR11, —SOR11, —S(O)2R11, —NHC(O)R11, —NHSOR11, or NHS(O)2R11; or
R3 and R4 and the carbon atoms to which they are attached are taken together to form a (C3–C7)cycloalkenyl group, a five- to seven-membered non-aromatic heterocycle, or a five- to seven-membered aromatic heterocycle; or
R5 and R6 and the carbon atoms to which they are attached are taken together to form a (C3–C7)cycloalkenyl group, a five- to seven-membered non-aromatic heterocycle, or a five- to seven-membered aromatic heterocycle; or
R4 and R5 and the carbon atoms to which they are attached are taken together to form a (C3–C7)cycloalkenyl group, a non-oxygen-containing five-membered non-aromatic heterocycle, a non-oxygen-containing five-membered aromatic heterocycle, a six- to seven-membered non-aromatic heterocycle or a six- to seven-membered aromatic heterocycle;
R7 is —H, —C1–C10 alkyl, or —C1–C10 alkoxy;
R8 and R9 are each independently —H, -halogen, —CN, —NH2, —NO2, —COOH, —C(O)NH2, —SH, —S(O)NH2, —S(O)2NH2, —C1–C10 (oxy)alkyl, —C1–C10 alkyl, —C1–C10 alkoxy, —C1–C10 (hydroxy)alkyl, —C1–C10 (amino)alkyl, —C1–C10 (halo)alkyl, —C2–C10 alkenyl, —C2–C10 alkynyl, —(C3–C7)cycloalkyl, -aryl, —C1–C10 (aryl)alkyl, three- to seven-membered non-aromatic heterocycle, five- to seven-membered aromatic heterocycle, —CH2OR11, —OCR11, —OC(O)R11, —C(O)R11, —OC(O)OR11, —OC(O)NR11, —C(O)OR11, —C(O)NR11, —OP(O)(OR11)2, —SR11, —SOR11, —S(O)2R11, —S(O)2NHR11, —NHSR11, —NHSOR11, or —NHS(O)2R11;
R11 is —H, —C1–C10 alkyl, —(C3–C7)cycloalkyl, —C1–C10 (halo)alkyl, -aryl, —C2–C10 alkenyl, —C2–C10 alkynyl, —C1–C10 (aryl)alkyl, —C2–C10 (aryl)alkenyl, —C2–C10 (aryl)alkynyl, —C1–C10 (hydroxy)alkyl, —C1–C10 alkoxy, —C1–C10 (amino)alkyl,
a —(C3–C7)cycloalkyl unsubstituted or substituted with one or more —C1–C10 alkyl,
a three- to seven-membered non-aromatic heterocycle unsubstituted or substituted with one or more —C1–C10 alkyl, or
a three- to seven-membered aromatic heterocycle unsubstituted or substituted with one or more —C1–C10 alkyl, —C2–C10 alkenyl, or —C2–C10 alkynyl;
R12 is C1–C10 alkyl; and
each halogen is independently —F, —Cl, —Br or —I.
The present invention encompasses compounds having the Formula (IV):




and pharmaceutically acceptable salts thereof, wherein:

Q1, Q2 and Q3 are independently ═O, ═S, ═NH or ═N—NHR, where R is —H, —C1–C10 alkyl, or -aryl;
R1 and R2 are independently —H, -halogen, -amino, —C1–C10 alkyl, —C1–C10 alkoxy, —C1–C10 (hydroxy)alkyl, —C1–C10 (amino)alkyl, —C1–C10 (halo)alkyl, —C2–C10 alkenyl, —C2–C10 alkynyl, (C3–C7)cycloalkyl, -aryl, C1–C10 (aryl)alkyl, or three- to seven-membered non-aromatic heterocycle, or R1, R2 and the carbon atom to which they are both attached are taken together to form a (C3–C7)cycloalkyl group or a three- to seven-membered non-aromatic heterocycle;
A is N or CR3; B is N or CR4; D is N or CR5; E is N or CR6, at least one of A, B, D and E being CR3, CR4, CR5 or CR6, respectively;
each R3, R4, R5 and R6 is independently —H, -halogen, —CN, —NH2, —NO2, —COOH, —C(O)NH2, —SH, —S(O)NH2, —S(O)2NH2, —C1–C10 (oxy)alkyl, —C1–C10 alkyl, —C1–C10 alkoxy, —C1–C10 (hydroxy)alkyl, —C1–C10 (amino)alkyl, —C1–C10 (halo)alkyl, —C2–C10 alkenyl, —C2–C10 alkynyl, —(C3–C7)cycloalkyl, -aryl, —C1–C10 (aryl)alkyl, three- to seven-membered non-aromatic heterocycle, five- to seven-membered aromatic heterocycle, —CH2OR11, —OCH2OR11, —OC(O)R11, —C(O)R11, —OC(O)OR11, —OC(O)NR11, —C(O)OR11, —C(O)NR11, —OP(O)(OR11)2, —SR11, —S(O)2NHR11, —SOR11, —S(O)2R11, —NHC(O)R11, —NHSOR11, or NHS(O)2R11; or
R3 and R4 and the carbon atoms to which they are attached are taken together to form a (C3–C7)cycloalkenyl group, a five- to seven-membered non-aromatic heterocycle, or a five- to seven-membered aromatic heterocycle; or
R5 and R6 and the carbon atoms to which they are attached are taken together to form a (C3–C7)cycloalkenyl group, a five- to seven-membered non-aromatic heterocycle, or a five- to seven-membered aromatic heterocycle; or
R4 and R5 and the carbon atoms to which they are attached are taken together to form a (C3–C7)cycloalkenyl group, a non-oxygen-containing five-membered non-aromatic heterocycle, a non-oxygen-containing five-membered aromatic heterocycle, a six- to seven-membered non-aromatic heterocycle or a six- to seven-membered aromatic heterocycle;
R7 is —H, —C1–C10 alkyl, or —C1–C10 alkoxy;
R8 and R9 are each independently —H, -halogen, —CN, —NH2, —NO2, —COOH, —C(O)NH2, —SH, —S(O)NH2, —S(O)2NH2, —C1–C10 (oxy)alkyl, —C1–C10 alkyl, —C1–C10 alkoxy, —C1–C10 (hydroxy)alkyl, —C1–C10 (amino)alkyl, —C1–C10 (halo)alkyl, —C2–C10 alkenyl, —C2–C10 alkynyl, —(C3–C7)cycloalkyl, -aryl, —C1–C10 (aryl)alkyl, three- to seven-membered non-aromatic heterocycle, five- to seven-membered aromatic heterocycle, —CH2OR11, —OCR11, —OC(O)R11, —C(O)R11, —OC(O)OR11, —OC(O)NR11, —C(O)OR11, —C(O)NR11, —OP(O)(OR11)2, —SR11, —SOR11, —S(O)2R11, —S(O)2NHR11, —NHSR11, —NHSOR11, or —NHS(O)2R11;
R11 is —H, —C1–C10 alkyl, —(C3–C7)cycloalkyl, —C1–C10 (halo)alkyl, -aryl, —C2–C10 alkenyl, —C2–C10 alkynyl, —C1–C10 (aryl)alkyl, —C2–C10 (aryl)alkenyl, —C2–C10 (aryl)alkynyl, —C1–C10 (hydroxy)alkyl, —C1–C10 alkoxy, —C1–C10 (amino)alkyl,
a —(C3–C7)cycloalkyl unsubstituted or substituted with one or more —C1–C10 alkyl,
a three- to seven-membered non-aromatic heterocycle unsubstituted or substituted with one or more —C1–C10 alkyl, or
a three- to seven-membered aromatic heterocycle unsubstituted or substituted with one or more —C1–C10 alkyl, —C2–C10 alkenyl, or —C2–C10 alkynyl;
R12 is C1–C10 alkyl; and
each halogen is independently —F, —Cl, —Br or —I.
A compound of Formula (I), Formula (II), Formula (III), or Formula (IV), or a pharmaceutically acceptable salts thereof (a “Diterpenoid Compound”) is useful for treating or preventing cancer, a neoplastic disease or a fungal infection in a patient in need of such treatment or prevention. The Diterpenoid Compounds are also useful for inhibiting the growth of a cancer cell, neoplastic cell or fungus. The Diterpenoid Compounds are also useful for inducing cytotoxicity, e.g., through apoptosis, in a cancer cell or a neoplastic cell.
The present invention provides compositions comprising a pharmaceutically acceptable carrier and an effective amount of a Diterpenoid Compound. The compositions are useful for treating or preventing cancer, neoplastic disease or a fungal infection in a patient in need of such treatment or prevention. These compositions are also useful for inhibiting the growth of a cancer cell, neoplastic cell or fungus. These compositions are further useful for inducing cytotoxicity, e.g., through apoptosis, in a cancer cell or a neoplastic cell.
The invention further provides methods for treating or preventing cancer or a neoplastic disease, comprising administering to a patient in need of such treatment or prevention an effective amount of a Diterpenoid Compound.
The invention further provides methods for inhibiting the growth of a cancer cell or neoplastic cell, comprising contacting the cancer cell or neoplastic cell with an effective amount of a Diterpenoid Compound.
The invention further provides methods for inducing cytotoxicity, e.g., through apoptosis, in a cancer cell or neoplastic cell comprising contacting a cancer cell or neoplastic cell with an effective amount of a Diterpenoid Compound.
The invention further provides methods for inducing apoptosis in a cancer cell or neoplastic cell, comprising contacting a cancer cell or neoplastic cell capable of undergoing apoptosis with an effective amount of a Diterpenoid Compound.
In one embodiment, the Diterpenoid Compound is in isolated and purified form.
The invention further provides methods for treating or preventing a fungal infection, comprising administering to a patient in need of such treatment or prevention an effective amount of a Diterpenoid Compound.
The invention further provides methods for inhibiting the growth of a fungus, comprising contacting the fungus with an effective amount of a Diterpenoid Compound.



4. BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A is an isolation scheme for the isolation of Compounds 48 and 49 from dried biomass of Linum arboreum; FIG. 1B is a summary of the 1H-13C correlations observed for Compound 48.
FIG. 2 is a bar graph depicting Caspase activity in different cell lines following 16 hours of in vitro treatment with 1.6 μM of Compound 48 (10-Hydroxy-6-methoxy-1,1,4a,7-tetramethyl-1H,4aH-phenanthrene-2,9-dione).
FIG. 3 is a bar graph depicting cell survival of different cell types following 72 hours of in vitro treatment with Compound 48 (10-Hydroxy-6-methoxy-1,1,4a,7-tetramethyl-1H,4aH-phenanthrene-2,9-dione).
FIG. 4 is a graph depicting the weight of mice following treatment with Cisplatin or Compound 50. The time-points of administration of Cisplatin or Compound 50 are indicated by arrows on top of the graph.
FIG. 5 is a graph depicting the tumor size in mice following treatment with Cisplatin or Compound 50. The time-points of administration of Cisplatin or Compound 50 are indicated by arrows on top of the graph.
FIG. 6 is a graph depicting the net growth of Saccharomyces cerevisiae following treatment with Compound 50.
FIG. 7 is a graph depicting the net growth of Candida albicans following treatment with Compound 50.



5. DETAILED DESCRIPTION OF THE INVENTION
5.1 Definitions and Abbreviations
As used herein, the term “C1–C10 alkyl” means a saturated straight chain or branched non-cyclic hydrocarbon having from 1 to 10 carbon atoms. Representative saturated straight chain alkyls include -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl, -n-octyl, -n-nonyl and -n-decyl; while saturated branched alkyls include -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, -2-methylbutyl, 3-methylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylbutyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylpentyl, 2,2-dimethylhexyl, 3,3-dimtheylpentyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylpentyl, 3-ethylpentyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, 2-methyl-4-ethylpentyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2-methyl-4-ethylhexyl, 2,2-diethylpentyl, 3,3-diethylhexyl, 2,2-diethylhexyl, 3,3-diethylhexyl and the like.
As used herein, the term “C1–C10 alkoxy” means —O—(C1–C10 alkyl), wherein C1–C10 alkyl is defined above.
As used herein, the term “C1–C10 (hydroxy)alkyl” means C1–C10 alkyl, wherein C1–C10 alkyl is defined above, substituted with one or more —OH groups. Examples of C1–C10 (hydroxy)alkyl include, but are not limited to, hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 1-hydroxypropyl, 2-hydroxypropyl, 3-hydroxypropyl, 4-hydroxybutyl, 5-hydroxypentyl and the like.
As used herein, the term “C1–C10 (amino)alkyl” means C1–C10 alkyl, wherein C1–C10 alkyl is defined above, substituted with one or more —NH2 groups. Examples of C1–C10 (amino)alkyl include, but are not limited to, —CH2—NH2, —(CH2)2—NH2, —(CH2)3—NH2, —(CH2)4—NH2, —(CH2)5—NH2 and the like.
As used herein, the term “C1–C10 (halo)alkyl” means C1–C10 alkyl, wherein C1–C10 alkyl is defined above, substituted with one or more —F, —Cl, Br or —I groups. Examples of C1–C10 (halo)alkyl include, but are not limited to, trichloromethyl, trifluoromethyl, dichloromethyl, difluoromethyl, 1-fluoroethyl, 2-chloroethyl, 1-bromopropyl, 2-iodopropyl, 3-chloropropyl, 4-fluorobutyl, 5-chloropentyl and the like.
As used herein, the term “C2–C10 alkenyl” means a straight chain or branched non-cyclic hydrocarbon having from 2 to 10 carbon atoms and including at least one carbon-carbon double bond. Representative straight chain and branched C2–C10 alkenyls include -vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutylenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, -1-hexenyl, -2-hexenyl, -3-hexenyl, -1-heptenyl, -2-heptenyl, -3-heptenyl, -1-octenyl, -2-octenyl, -3-octenyl, -1-nonenyl, -2-nonenyl, -3-nonenyl, -1-decenyl, -2-decenyl, -3-decenyl and the like. In one embodiment, C2–C6 alkenyl is a subclass of C2–C10 alkenyl. The double bond of a C2–C10 alkenyl can be unconjugated or conjugated to another unsaturated group. A —C2–C10 alkenyl can be unsubstituted or substituted with, e.g., -amino, —C1–C10 (oxy)alkyl, -halogen, —COOH, —C(O)C1–C9 alkyl, —SH, ═S, —OH, and —C1–C10 alkoxy.
As used herein, unless otherwise specified the term “C2–C10 alkynyl” means a straight chain or branched non-cyclic hydrocarbon having from 2-10 carbon atoms and including at lease one carbon-carbon triple bond. Representative straight chain and branched C2–C10 alkynyls include -acetylenyl, -propynyl, -1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl, -3-methyl-1-butynyl, -4-pentynyl, -1-hexynyl, -2-hexynyl, -5-hexynyl, -1-heptynyl, -2-heptynyl, -6-heptynyl, -1-octynyl, -2-octynyl, -7-octynyl, -1-nonynyl, -2-nonynyl, -8-nonynyl, -1-decynyl, -2-decynyl, -9-decynyl, and the like. In one embodiment, C2–C6 alkynyl is a subclass of C2–C10 alkynyl. The triple bond of a C2–C10 alkynyl can be unconjugated or conjugated to another unsaturated group. A C2–C10 alkynyl can be unsubstituted or substituted with, e.g., -amino, —COOH, -halogen, C1–C10 (oxy)alkyl, —C(O)C1–C9 alkyl, —SH, ═S, —OH, C1–C10 alkoxy, and C1–C10 alkyl.
As used herein, the term “(C3–C7)cycloalkyl” means a monocyclic or bicyclic saturated ring consisting of carbon and hydrogen atoms and having 3-7 carbon atoms. A (C3–C7)cycloalkyl can be unsubstituted or substituted with, e.g., -amino, —COOH, -halogen, C1–C10 (oxy)alkyl, —C(O)C1–C9 alkyl, —SH, ═S, —OH, C1–C10 alkoxy, and C1–C10 alkyl. Examples of (C3–C7)cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl, and saturated cyclic and bicyclic terpenes.
As used herein, the term “(C3–C7)cycloalkenyl” means a monocyclic or bicyclic unsaturated ring consisting of carbon and hydrogen atoms and having 3-7 carbon atoms. A (C3–C7)cycloalkenyl can be unsubstituted or substituted with, e.g., -amino, —COOH, -halogen, C1–C10 (oxy)alkyl, —C(O)C1–C9 alkyl, —SH, ═S, —OH, C1–C10 alkoxy, and C1–C10 alkyl. Examples of (C3–C7)cycloalkyl include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, and cycloheptenyl, and unsaturated cyclic and bicyclic terpenes.
As used herein, the term “aryl” means a carbocyclic aromatic group. All of the ring atoms of an aryl group are carbon atoms. Aryl groups include compounds having one or more ring structures such as mono-, bi-, or tricyclic compounds as well as benzo-fused carbocyclic moieties such as 5,6,7,8-tetrahydronaphthyl and the like. In one embodiment, the aryl group is a monocyclic ring or bicyclic ring. Representative aryl groups include phenyl, tolyl, anthryl, fluorenyl, indenyl, azulenyl, phenanthryl and naphthyl. A carbocyclic aryl group can be unsubstituted or substituted with, e.g., -amino, —COOH, -halogen, C1–C10 (oxy)alkyl, —C(O)C1–C9 alkyl, —SH, ═S, —OH, C1–C10 alkoxy, and C1–C10 alkyl.
As used herein, the term “C1–C10 (aryl)alkyl” means C1–C10 alkyl, wherein C1–C10 alkyl is defined above, substituted with one or more aryl groups, wherein aryl is defined above. Examples of C1–C10 (aryl)alkyl include, but not limited to —(CH2)phenyl, —(CH2)2phenyl, —(CH2)3phenyl, —CH(phenyl)2, —CH(phenyl)3, —(CH2)tolyl, —(CH2)anthracenyl —(CH2)fluorenyl, —(CH2)indenyl, —(CH2)azulenyl, —(CH2)naphthyl, and the like.
As used herein, the term “C2–C10 (aryl)alkenyl” means C2–C10 alkenyl, wherein C2–C10 alkenyl is defined above, substituted with one or more aryl groups, wherein aryl is defined above.
As used herein, the term “C2–C10 (aryl)alkynyl” means C2–C10 alkynyl, wherein C2–C10 alkynyl is defined above, substituted with one or more aryl groups, wherein aryl is defined above.
As used herein, the term “three- to seven-membered aromatic heterocycle” means a heterocyclic ring that contains 3 to 7 ring atoms and that is aromatic. A three-membered heterocycle can contain up to 3 heteroatoms, and a 4- to 7-membered heterocycle can contain up to 4 heteroatoms, wherein the remaining atoms are carbon atoms. Each heteroatom is independently selected from nitrogen, which can be quaternized; oxygen; phosphorus and sulfur, including sulfoxide and sulfone. The heterocycle can be attached via any heteroatom or carbon atom. Representative three- to seven-membered aromatic heterocycles include, but are not limited to, pyridyl, furyl, thiophenyl, pyrrolyl, oxazolyl, imidazolyl, thiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, and triazinyl.
As used herein, the term “three- to seven-membered non-aromatic heterocycle” means a heterocyclic ring that contains 3 to 7 ring atoms and that is non-aromatic. A three-membered heterocycle can contain up to 3 heteroatoms, and a 4- to 7-membered heterocycle can contain up to 4 heteroatoms, wherein the remaining atoms are carbon atoms. Each heteroatom is independently selected from nitrogen, which can be quaternized; oxygen; phosphorus; and sulfur, including sulfoxide and sulfone. The heterocycle can be attached via any heteroatom or carbon atom. Representative three- to seven-membered non-aromatic heterocycles include, but are not limited to, morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperazinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, pyranyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, and tetrahydrothiopyranyl.
As used herein, the term “five- to seven-membered aromatic heterocycle” means a heterocyclic ring that contains 5 to 7 ring atoms and that is aromatic. A five- to seven-membered heterocycle can contain up to 4 heteroatoms, wherein the remaining atoms are carbon atoms. Each heteroatom is independently selected from nitrogen, which can be quaternized; oxygen; phosphorus; and sulfur, including sulfoxide and sulfone. The heterocycle can be attached via any heteroatom or carbon atom. Representative five- to seven-membered aromatic heterocycles include, but are not limited to, pyridyl, furyl, thiophenyl, pyrrolyl, furazanyl, oxazolyl, imidazolyl, thiazolyl, thiadiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, and triazinyl.
As used herein, the term “five- to seven-membered non-aromatic heterocycle” means a heterocyclic ring that contains 5 to 7 ring atoms and that is non-aromatic. A five- to seven-membered heterocycle can contain up to 4 heteroatoms, wherein the remaining atoms are carbon atoms. Each heteroatom is independently selected from nitrogen, which can be quaternized; oxygen; phosphorus; and sulfur, including sulfoxide and sulfone. The heterocycle can be attached via any heteroatom or carbon atom. Representative five- to seven-membered non-aromatic heterocycles include, but are not limited to, morpholinyl, pyranyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperazinyl, hydantoinyl, valerolactamyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, and tetrahydrothiopyranyl.
As used herein, the term “non-oxygen-containing five-membered non-aromatic heterocycle” means a heterocyclic ring that contains 5 ring atoms and that is non-aromatic. A five-membered heterocycle can contain up to 4 heteroatoms, wherein the remaining atoms are carbon atoms. Each heteroatom is independently selected from nitrogen, which can be quaternized; phosphorus; and sulfur, including sulfoxide and sulfone. The heterocycle can be attached via any heteroatom or carbon atom.
As used herein, the term “non-oxygen-containing five-membered aromatic heterocycle” means a heterocyclic ring that contains 5 ring atoms and that is aromatic. A five-membered heterocycle can contain up to 4 heteroatoms, wherein the remaining atoms are carbon atoms. Each heteroatom is independently selected from nitrogen, which can be quaternized; phosphorus; and sulfur, including sulfoxide and sulfone. The heterocycle can be attached via any heteroatom or carbon atom.
Examples of “halogen” are fluorine, chlorine, bromine, and iodine.
As used herein, the term “C1–C10 (oxy)alkyl” means C1–C10 alkyl, wherein C1–C10 alkyl is defined above, and wherein one of its carbon atoms is a C═O group. Examples of C1–C10 (oxy)alkyl include, but are not limited to, —C(O)CH3, —CH2CHO, —C(O)(CH2)2CH3, —CH2C(O)CH3, —(CH2)2CHO, —(CH2)3CHO, —(CH2)4CHO an the like.
As used herein, an “effective amount” when used in connection with a Diterpenoid Compound refers to that amount of the Diterpenoid Compound useful for treating or preventing cancer, a neoplastic disease or a fungal infection; for inhibiting the growth of a cancer cell, neoplastic cell or fungus; or for inducing cytotoxicity, e.g., through apoptosis, in a cancer cell or a neoplastic cell, alone or in combination with another active agent. As used herein, an “effective amount” when used in connection with another active agent refers to that amount of the other active agent that is useful for treating or preventing a particular disease or condition, alone or in combination with a Diterpenoid Compound.
As used herein, the term “treating cancer or a neoplastic disease” includes reducing the size of a tumor, ameliorating one or more symptoms associated with a cancer or a neoplastic disease, or inducing cytotoxicity, e.g., through apoptosis, selectively in cells of a cancer or neoplastic disease relative to a non-cancerous or non-neoplastic cell. The term “treating a cancer or a neoplastic disease” further includes arresting or retarding the progression of a cancer or a neoplastic disease.
As used herein, the term “pharmaceutically acceptable salt” refers to a salt prepared from an acid or a base including inorganic acids and bases and organic acids and bases. Suitable pharmaceutically acceptable salts of a Diterpenoid Compound having a —COOH group include, but are not limited to, metallic salts of aluminum, calcium, lithium, magnesium, potassium, sodium and zinc, or organic salts of lysine, N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. Acids useful for forming suitable salts with a Diterpenoid Compound having a nitrogen or sulfur atom include, but are not limited to, inorganic and organic acids such as acetic, alginic, anthranilic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethenesulfonic, formic, fumaric, furoic, galacturonic, gluconic, glucuronic, glutamic, glycolic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phenylacetic, phosphoric, propionic, salicylic, stearic, succinic, sulfanilic, sulfuric, tartaric acid, and p-toluenesulfonic acid. Other examples of salts are well known in the art, see, e.g., Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton Pa. (1990).
As used herein, the term “isolated and purified” means isolated from another component or from other components of a naturally occurring source (such as a plant or animal cell, including a hepatocyte; cell culture; tissue; in vivo fluid including intracellular and extracellular fluid, including blood and plasma; and ex vivo fluid including sputum, urine, sweat, semen, menstrual fluid, and milk) or from a synthetic organic chemical reaction mixture, and processed through one or more purifying steps that separate the compound of the invention from other molecules associated with it. When isolated and purified, the compound of the invention is at least about 95% pure. In one embodiment, the compound of the invention is at least about 98% pure. In another embodiment, the compound of the invention is at least about 99% pure.
When a first group is substituted with “one or more” second group(s), a hydrogen of the first group is replaced with the second group. In one embodiment, a first group is substituted with one, two or three second groups. In another embodiment, a first group is substituted with one or two second groups. In even another embodiment, a first group is substituted with one second group.
As used herein, the term “patient” refers preferably to an animal, including, but not limited, to a vertebrate such a chimpanzee, baboon, cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit, and guinea pig, and in one embodiment a mammal, and in a more specific embodiment a human.
A Diterpenoid Compound can have one or more chiral centers and, accordingly, can exist in the form of a diastereomer, a (+)- or (−)-enantiomer, a racemate, or a mixture thereof.










Abbreviations


















IBX
iodoxybenzoate



TBAF
tetra-n-butylammonium fluoride



BRDU
bromodeoxyuridine



i.v.
intravenous



Rpm
revolutions per minute



ATCC
American Type Culture Collection










The Diterpenoid Compounds are defined herein by their chemical structures and/or chemical names. Where a Diterpenoid Compound is referred to by both a chemical structure and a chemical name, and the chemical structure and chemical name conflict, the chemical structure is determinative of the compound's identity.
The Diterpenoid Compounds can exist in the form of a pharmaceutically acceptable salt, free base, solvate, hydrate, stereoisomer, clathrate, polymorph or prodrug thereof.
The invention can be understood more fully by reference to the following description, figures and illustrative examples, which are intended to exemplify non-limiting embodiments of the invention.
5.2 Formula I
As stated above, the present invention encompasses compounds having the Formula (I):




and pharmaceutically acceptable salts thereof, wherein Q1–Q3, R1–R10, A-E and halogen are defined above for Formula (I).

The Diterpenoid Compounds of Formula (I) are useful for treating or preventing cancer or neoplastic disease in a patient in need of such treatment or prevention. The Diterpenoid Compounds of Formula (I) are also useful for inhibiting the growth of a cancer cell or neoplastic cell. The Diterpenoid Compounds of Formula (I) are also useful for inducing cytotoxicity, e.g., through apoptosis, in a cancer cell or neoplastic cell. The Diterpenoid Compounds of Formula (I) are further useful for treating or preventing a fungal infection. The Diterpenoid Compounds of Formula (I) also useful for inhibiting the growth of a fungus.
In one embodiment, the Diterpenoid Compounds of Formula (I) are those wherein Q1 and Q2 are ═O and Q3 is —O—.
In another embodiment, the Diterpenoid Compounds of Formula (I) are those wherein A, B, D and E are CR3, CR4, CR5 and CR6, respectively.
In another embodiment, the Diterpenoid Compounds of Formula (I) are those wherein A is CR3, E is CR6, and R3 and R6 are hydrogen.
In another embodiment, the Diterpenoid Compounds of Formula (I) are those wherein D is CR5 and R5 is C1–C6 alkoxy, in another embodiment, —OCH3.
In another embodiment, the Diterpenoid Compounds of Formula (I) are those wherein B is CR4 and R4 is hydrogen.
In another embodiment, the Diterpenoid Compounds of Formula (I) are those wherein B is CR4, R4 is hydrogen, D is CR5 and R5 is C1–C6 alkoxy.
In another embodiment, the Diterpenoid Compounds of Formula (I) are those wherein B is CR4, R4 is hydrogen, D is CR5 and R5 is —OCH3.
In another embodiment, the Diterpenoid Compounds of Formula (I) are those wherein B is CR4 and R4 is C1–C10 alkyl, in another embodiment, —CH3.
In another embodiment, the Diterpenoid Compounds of Formula (I) are those wherein B is CR4 and R4 is C1–C10 (hydroxy)alkyl, in another embodiment, hydroxymethyl.
In another embodiment, R8 and R9 are hydrogen.
In another embodiment, R1 and R2 are independently C1–C10 alkyl, in another embodiment, —CH3.
In another embodiment, R10 is hydrogen.
In another embodiment, -Q3-R10 is —OH.
In another embodiment, R7 is C1–C10 alkyl, in another embodiment —CH3.
In one embodiment, Q1 and Q2 and Q3 are oxygen. In another embodiment, R1 and R2 are C1–C10 alkyl. In another embodiment, R8 and R9 are H. In another embodiment, R7 is C1–C10 alkyl. In another embodiment, R3 and R6 are H. In another embodiment, R4 and R5 are independently C1–C10 alkyl, C1–C10 alkoxy, or C1–C10 (hydroxy)alkyl.
In one embodiment, the Diterpenoid Compounds of Formula (I) are in isolated and purified form.
Illustrative Diterpenoid Compound of Formula (I) are:













and pharmaceutically acceptable salts thereof.
It is to be understood that Compound 50 is the racemate of its corresponding enantiomers Compound 50a and 50b; Compound 66 is the racemate of its corresponding enantiomers Compound 66a and 66b; Compound 67 is the racemate of its corresponding enantiomers Compound 67a and 67b; Compound 68 is the racemate of its corresponding enantiomers Compound 68a and 68b; and that Compound 69 is the racemate of its corresponding enantiomers Compound 69a and 69b.
5.3 Formula II
As stated above, the present invention encompasses compounds having the Formula (II):




and pharmaceutically acceptable salts thereof, wherein Q1–Q3, R1–R10, A-E and halogen are defined above for Formula (II).

The Diterpenoid Compounds of Formula (II) are useful for treating or preventing cancer or neoplastic disease in a patient in need of such treatment or prevention. The Diterpenoid Compounds of Formula (II) are also useful for inhibiting the growth of a cancer cell or neoplastic cell. The Diterpenoid Compounds of Formula (II) are also useful for inducing cytotoxicity, e.g., through apoptosis, in a cancer cell or neoplastic cell. The Diterpenoid Compounds of Formula (II) are further useful for treating or preventing a fungal infection. The Diterpenoid Compounds of Formula (II) also useful for inhibiting the growth of a fungus.
In one embodiment, the Diterpenoid Compounds of Formula (II) are those wherein Q1 and Q2 are ═O and Q3 is —O—.
In another embodiment, the Diterpenoid Compounds of Formula (II) are those wherein A, B, D and E are CR3, CR4, CR5 and CR6, respectively.
In another embodiment, the Diterpenoid Compounds of Formula (II) are those wherein A is CR3, E is CR6, and R3 and R6 are hydrogen.
In another embodiment, the Diterpenoid Compounds of Formula (II) are those wherein D is CR5 and R5 is C1–C6 alkoxy, in another embodiment, —OCH3.
In another embodiment, the Diterpenoid Compounds of Formula (II) are those wherein B is CR4 and R4 is hydrogen.
In another embodiment, the Diterpenoid Compounds of Formula (II) are those wherein B is CR4, R4 is hydrogen, D is CR5 and R5 is C1–C6 alkoxy.
In another embodiment, the Diterpenoid Compounds of Formula (II) are those wherein B is CR4, R4 is hydrogen, D is CR5 and R5 is —OCH3.
In another embodiment, the Diterpenoid Compounds of Formula (II) are those wherein B is CR4 and R4 is C1–C10 alkyl, in another embodiment, —CH3.
In another embodiment, the Diterpenoid Compounds of Formula (II) are those wherein B is CR4 and R4 is C1–C10 (hydroxy)alkyl, in another embodiment, hydroxymethyl.
In another embodiment, R8 and R9 are hydrogen.
In another embodiment, R1 and R2 are independently C1–C10 alkyl, in another embodiment, —CH3.
In another embodiment, R10 is hydrogen.
In another embodiment, -Q3-R10 is —OH.
In another embodiment, R7 is C1–C10 alkyl, in another embodiment —CH3.
In another embodiment, the Diterpenoid Compounds of Formula (II) are in isolated and purified form.
5.4 Formula III
As stated above, the present invention encompasses novel compounds having the Formula (III):




and pharmaceutically acceptable salts thereof, wherein Q1–Q3, R1–R9, A-E and halogen are defined above for Formula (III).

The Diterpenoid Compounds of Formula (III) are useful for treating or preventing cancer or neoplastic disease in a patient in need of such treatment or prevention. The Diterpenoid Compounds of Formula (III) are also useful for inhibiting the growth of a cancer cell or neoplastic cell. The Diterpenoid Compounds of Formula (III) are also useful for inducing cytotoxicity, e.g., through apoptosis, in a cancer cell or neoplastic cell. The Diterpenoid Compounds of Formula (III) are further useful for treating or preventing a fungal infection. The Diterpenoid Compounds of Formula (III) also useful for inhibiting the growth of a fungus.
In one embodiment, the Diterpenoid Compounds of Formula (III) are those wherein Q1, Q2 and Q3 are ═O.
In another embodiment, the Diterpenoid Compounds of Formula (III) are those wherein A, B, D and E are CR3, CR4, CR5 and CR6, respectively.
In another embodiment, the Diterpenoid Compounds of Formula (III) are those wherein A is CR3, E is CR6, and R3 and R6 are hydrogen.
In another embodiment, the Diterpenoid Compounds of Formula (III) are those wherein D is CR5 and R5 is C1–C6 alkoxy, in another embodiment, —OCH3.
In another embodiment, the Diterpenoid Compounds of Formula (III) are those wherein B is CR4 and R4 is hydrogen.
In another embodiment, the Diterpenoid Compounds of Formula (III) are those wherein B is CR4, R4 is hydrogen, D is CR5 and R5 is C1–C6 alkoxy.
In another embodiment, the Diterpenoid Compounds of Formula (III) are those wherein B is CR4, R4 is hydrogen, D is CR5 and R5 is —OCH3.
In another embodiment, the Diterpenoid Compounds of Formula (III) are those wherein B is CR4 and R4 is C1–C10 alkyl, in another embodiment, —CH3.
In another embodiment, the Diterpenoid Compounds of Formula (III) are those wherein B is CR4 and R4 is C1–C10 (hydroxy)alkyl, in another embodiment, hydroxymethyl.
In another embodiment, R8 and R9 are hydrogen.
In another embodiment, R1 and R2 are independently C1–C10 alkyl, in another embodiment, —CH3.
In another embodiment, R7 is C1–C10 alkyl, in another embodiment —CH3.
In another embodiment, the Diterpenoid Compounds of Formula (III) are in isolated and purified form.
An illustrative Diterpenoid Compound of Formula (III) is:




and pharmaceutically acceptable salts thereof.
It is to be understood that Compound 70 is the racemate of its corresponding enantiomers Compound 70a and 70b;
5.5 Formula IV
As stated above, the present invention encompasses novel compounds having the Formula (IV):




and pharmaceutically acceptable salts thereof, wherein Q1–Q3, R1–R9, A-E and halogen are defined above for Formula (IV).

The Diterpenoid Compounds of Formula (IV) are useful for treating or preventing cancer or neoplastic disease in a patient in need of such treatment or prevention. The Diterpenoid Compounds of Formula (IV) are also useful for inhibiting the growth of a cancer cell or neoplastic cell. The Diterpenoid Compounds of Formula (IV) are also useful for inducing cytotoxicity, e.g., through apoptosis, in a cancer cell or neoplastic cell. The Diterpenoid Compounds of Formula (IV) are further useful for treating or preventing a fungal infection. The Diterpenoid Compounds of Formula (IV) also useful for inhibiting the growth of a fungus.
In one embodiment, the Diterpenoid Compounds of Formula (IV) are those wherein Q1, Q2 and Q3 are ═O.
In another embodiment, the Diterpenoid Compounds of Formula (IV) are those wherein A, B, D and E are CR3, CR4, CR5 and CR6, respectively.
In another embodiment, the Diterpenoid Compounds of Formula (IV) are those wherein A is CR3, E is CR6, and R3 and R6 are hydrogen.
In another embodiment, the Diterpenoid Compounds of Formula (IV) are those wherein D is CR5 and R5 is C1–C6 alkoxy, in another embodiment, —OCH3.
In another embodiment, the Diterpenoid Compounds of Formula (IV) are those wherein B is CR4 and R4 is hydrogen.
In another embodiment, the Diterpenoid Compounds of Formula (IV) are those wherein B is CR4, R4 is hydrogen, D is CR5 and R5 is C1–C6 alkoxy.
In another embodiment, the Diterpenoid Compounds of Formula (IV) are those wherein B is CR4, R4 is hydrogen, D is CR5 and R5 is —OCH3.
In another embodiment, the Diterpenoid Compounds of Formula (IV) are those wherein B is CR4 and R4 is C1–C10 alkyl, in another embodiment, —CH3.
In another embodiment, the Diterpenoid Compounds of Formula (IV) are those wherein B is CR4 and R4 is C1–C10 (hydroxy)alkyl, in another embodiment, hydroxymethyl.
In another embodiment, R8 and R9 are hydrogen.
In another embodiment, R1 and R2 are independently C1–C10 alkyl, in another embodiment, —CH3.
In another embodiment, R7 is C1–C10 alkyl, in another embodiment —CH3.
In another embodiment, the Diterpenoid Compounds of Formula (IV) are in isolated and purified form.
5.6 Methods for Making  Diperpenoid Compounds 
The Diterpenoid Compounds can be obtained using conventional organic synthesis or by using the following illustrative methods shown in Schemes 1–7 below.
Diterpenoid Compounds of Formula (I) or (II) can be obtained from a tetralone-type precursor such as depicted by compounds 13 in Scheme 1. Nucleophilic addition of R7 using an appropriate organometallic reagent, such as a Grignard reagent (E. C. Ashby et al., J. Am. Chem. Soc., 89:1964 (1967)), followed by dehydration (C. Utermoehlen et al., J. Org. Chem., 52:5574 (1987)) provides compounds 14, which can undergo Diels-Alder cycloadditions with dienes such as compounds 15 (S. Danishefsky et al., J. Am. Chem. Soc., 101:7001 (1979)), with or without Lewis acid catalysis, to yield after desilylation tricyclic intermediates 16. The a, b-unsaturation of compounds 17 can be introduced by treating compounds 16 with a strong base such as lithium diisopropyl amide, followed by treatment with phenylselenium chloride, hydrogen peroxide and meta-chloroperoxybenzoic acid (M. Tius et al., J. Am. Chem. Soc., 114:5959 (1992)). Compounds 17 can then be oxidized with, for example, chromium trioxide/sulphuric acid or IBX in DMSO and oxygen with potassium t-butoxide in t-butanol to provide compounds 18 (Nicolaou et al., J. Am. Chem. Soc. 123:3183 (2001); Nicolaou et al., Angew. Chem. Int. Ed. 40:207 (2001)), which are in equilibrium with enols 19. Compounds 18a, 18b, 19a and 19b can be further modified to provide thioketones, imines, hydrazones, hydrazines, and arylhydrazones, as structurally described in Formulas (I), (II), (III) and (IV) by using simple organic transformations well recognised by chemists skilled in the art of organic synthesis.
Scheme 2 illustrates a synthesis of Diterpenoid Compounds of subgeneric formulas 5a, 5b, 6a, and 6b. This route uses compounds 21, which can be obtained (Scheme 3) in three steps from IBX-DMSO oxidation of aryl- or heteroaryl-substituted propanols 26 (Nicolaou et al., J. Am. Chem. Soc. 123:3183 (2001)), followed by Wittig reaction with aldehydes 27 (B. Maryanoff et al., J. Am. Chem. Soc., 107:217,(1985); A. Maercker, Organic Reactions, 14:270 (1965)), desilylation of resultant product 28 using a reagent such as, but not limited to, TBAF, and bromination using tribromophosphine in a solvent such as dichloromethane to provide bromide 21.
Carbonyl-containing compounds 20 can be coupled with intermediates 21 using reagents such as NaH or n-butyllithium in tetrahydrofuran to provide compounds 22 (S. Welch et al., J. Am. Chem. Soc, 101:6768 (1979)), which can then be cyclised to the tricyclic intermediates 23 using manganese acetate in acetic acid (B. Snider et al., J. Org. Chem., 50:3659 (1985)). The a, b-unsaturation of compounds 24 can be introduced by treating compounds 23 with lithium diisopropyl amide, phenylselenium chloride, hydrogen peroxide and meta-chloroperoxybenzoic acid (M. Tius et al., J. Am. Chem. Soc., 114:5959 (1992)). Oxidation of compounds 23 and 24 with, for example, chromium trioxide/acetic acid or oxygen and potassium t-butoxide in t-butanol, provides compounds 6a and 6b, respectively, which are in equilibrium with the enol forms of compounds 25a and 25b, respectively. Compounds 5a and 5b can be prepared from compounds 25a and 25b, respectively, by O-alkylating or O-acylating using well-known synthetic methods.
12-Methoxypodocarpa-8,11,13-trieneoic acid (29) is a useful starting material for Diterpenoid Compounds 11a, 11b, 12a, and 12b (Schemes 4 and 5). With reference to Scheme 4, compounds 30 can be obtained by treating compound 29 with a strong base such as n-butyllithium followed by alkylation using an alkyl halide, aldehyde, ketone, ester or epoxide. The resulting compounds 30 can then be treated with lead tetraacetate and monoperphtalic acid to provide epoxides 31 (R. Cambie and T. Fullerton, Aust. J. Chem., 24:2611 (1971)), which can then be treated with lithium diethylamide and n-lithioethelenediamine to yield the tricyclic compounds 32 (R. Cambie and T. Fullerton, Aust. J. Chem., 24:2611 (1971)). Compounds 33 can then be obtained by oxidizing compounds 32 with a reagent such as chromium trioxide and sulphuric acid, forming an enolate from the resultant ketone using a basic solution such as potassium t-butoxide in t-butanol and quenching the enolate with an alkylating agent, such as methyliodide (B. Snider et al., J. Org. Chem., 50:3659 (1985)). Reduction of compounds 33 with a metal such as palladium in a solvent/acid mixture such as ethanol and acetic acid provides the tricyclic ketones 34 (H. Thompson et al., J. Org. Chem., 41:2903 (1976)). Compounds such as 11a, where R5=OMe, can be obtained by treating compounds 34 with lithium diisopropyl amide, followed by phenylselenium chloride, hydrogen peroxide and meta-chloroperoxybenzoic acid, further followed by oxidation using, for example, chromium trioxide/acetic acid or oxygen with potassium t-butoxide in t-butanol (M. Tius et al., J. Am. Chem. Soc., 114:5959 (1992)). Oxidation of compounds 34 using chromium trioxide/acetic acid and oxygen with potassium t-butoxide in t-butanol provides 11b where R5=OCH3. Compounds 11a and 11b (R5=OMe) are in equilibrium with triketo compounds 12a and 12b, respectively.
Scheme 5 depicts an alternative method useful for the preparation of Diterpenoid Compounds 11a, 11b, 12a, and 12b. For example, compounds 35 having R5 functional groups other than —OCH3 can be obtained by demethylating compound 29 with a reagent such as boron trichloride, treating the resultant phenoxide with triflic anhydride or any other suitable triflating agent, and then displacing the triflate with an organometallic reagent such as a boronate or a cuprate. The conversion of compounds 35 to Diterpenoid Compounds 11a, 11b, 12a, and 12b can be achieved using the same or similar synthetic methods as described in Scheme 4 for converting compounds 29 to those of formula 11a, 11b (R5=OMe) and 12a, 12b (R5=OMe)(R. Cambie and T. Fullerton, Aust. J. Chem., 24:2611 (1971); M. Tius et al., J. Am. Chem. Soc., 114:5959 (1992)).




















Synthesis of Compound 50 
Compound 50 can be synthesized as illustrated in Scheme 6.







Compound 50a and Compound 50b can be synthesized as illustrated in Scheme 7.







5.7 Therapeutic/Prophylactic  Administration and Compositions 
Due to their activity, the Diterpenoid Compounds are advantageously useful in veterinary and human medicine. For example, the Diterpenoid Compounds are useful for treating or preventing cancer or neoplastic disease, inhibiting the growth of a cancer cell or neoplastic cell, inducing cytotoxicity, e.g., through apoptosis, in a cancer cell or neoplastic cell, treating or preventing a fungal infection, or inhibiting the growth of a fungus.
When administered to a patient, e.g., an animal for veterinary use or to a human for clinical use, or when made to contact a cell or tissue, the Diterpenoid Compounds can be in isolated and purified form.
The present compositions, which comprise a Diterpenoid Compound, can be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa) and can be administered together with another active agent. Administration can be systemic or local. Various delivery systems are known, e.g., encapsulation in liposomes, microparticles, microcapsules, capsules, etc., and can be used to administer a Diterpenoid Compound. In certain embodiments, more than one Diterpenoid Compound is administered to a patient. Methods of administration include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectally, by inhalation, or topically to the ears, nose, eyes, or skin. The mode of administration can be left to the discretion of the practitioner, and can depend in-part upon the site of the medical condition (such as the site of cancer or neoplastic disease or fungal infection).
In specific embodiments, it might be desirable to administer one or more Diterpenoid Compounds locally to the area in need of treatment. This can be achieved, for example, and not by way of limitation, by local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, by convection or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. In one embodiment, administration can be by direct injection at the site (or former site) of a cancer, tumor or neoplastic or pre-neoplastic tissue or fungal infection.
In certain embodiments, it might be desirable to administer one or more Diterpenoid Compounds by any suitable route, including intraventricular and intrathecal injection. Intraventricular injection can be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir.
Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent, or via perfusion in a fluorocarbon or synthetic pulmonary surfactant. In certain embodiments, the Diterpenoid Compounds can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
In another embodiment, the Diterpenoid Compounds can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527–1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353–365 (1989); Lopez-Berestein, ibid., pp. 317–327; see generally ibid.)
In yet another embodiment, the Diterpenoid Compounds can be delivered in a controlled-release system. In one embodiment, a pump can be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)). In yet another embodiment, a controlled-release system can be placed in proximity of the target of the Diterpenoid Compounds, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115–138 (1984)). Other controlled-release systems discussed in the review by Langer (Science 249:1527–1533 (1990)) can be used.
The present compositions comprise an effective amount of a Diterpenoid Compound, which can be in isolated and purified form, together with a suitable amount of a pharmaceutically acceptable carrier so as to provide a useful form for administration to the patient.
In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which a Diterpenoid Compound is administered. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents can be used. When administered to a patient, the Diterpenoid Compounds and pharmaceutically acceptable carriers can be sterile. Water is a useful carrier when the Diterpenoid Compound is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The present compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
The present compositions can take the form of solutions, suspensions, emulsion, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use. In one embodiment, the pharmaceutically acceptable carrier is a capsule (see e.g., U.S. Pat. No. 5,698,155). Other examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin.
In one embodiment, the Diterpenoid Compounds are formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, Diterpenoid Compounds intended for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the compositions can also include a solubilizing agent. Compositions for intravenous administration can optionally include a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the Diterpenoid Compound is to be administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the Diterpenoid Compound is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.
Compositions for oral delivery can be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs, for example. Orally administered compositions can contain one or more optionally agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of wintergreen, or cherry; coloring agents; and preserving agents, to provide a pharmaceutically palatable preparation. Moreover, where in tablet or pill form, the compositions can be coated to delay disintegration and absorption in the gastrointestinal tract thereby providing a sustained action over an extended period of time. Selectively permeable membranes surrounding an osmotically active driving compound are also suitable for orally administered Diterpenoid Compounds. In these later platforms, fluid from the environment surrounding the capsule is imbibed by the driving 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 delay material such as glycerol monostearate or glycerol stearate can also be used. Oral compositions can include standard carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, or magnesium carbonate. Such carriers can be of pharmaceutical grade.
The effective amount of the Diterpenoid Compound depends on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. However, suitable effective amounts for intravenous administration generally range from about 10 micrograms to about 1 gram per kilogram body weight, in one embodiment from about 20 micrograms to about 500 micrograms, about 400 micrograms to about 2 milligrams, about 1 milligram to about 5 milligram, about 2 milligram to about 20 milligram, about 10 milligram to about 60 milligram, about 50 milligram to about 200 milligram, about 100 milligram to about 500 milligram, or about 200 milligram to about 800 milligram of Diterpenoid Compound per kilogram body weight. In specific embodiments of the invention, the effective amount for an i.v. dose ranges from about 10 to about 40, about 40 to about 60, about 60 to about 100, or about 100 to about 200 micrograms per kilogram body weight. In other embodiments, the effective amount for an i.v. dose ranges from about 75 to about 150, about 150 to about 250, about 250 to about 375 or about 375 to about 500 or about 400 to about 800 micrograms per kilogram body weight. In specific embodiments of the invention, the effective amount for an i.v. dose ranges from about 0.5 to about 2, from about 1 to about 10, from about 10 to about 40, about 40 to about 60, about 60 to about 100, or about 100 to about 200 milligrams per kilogram body weight. In other embodiments, the effective amount for an i.v. dose ranges from about 75 to about 150, about 150 to about 250, about 250 to about 375 or about 375 to about 500 milligrams per kilogram body weight. Suitable effective amounts for intranasal administration generally range from about 0.01 pg/kg body weight to about 1 mg/kg, from about 0.5 mg/kg to about 800 mg/kg body weight. Suppositories generally contain an effective amount in the range of about 0.5% to about 10% by weight. Oral compositions can contain from about 10% to about 95% of Diterpenoid Compound. In specific embodiments of the invention, suitable effective amounts for oral administration generally range from about 0.1 micrograms to about 10 milligrams, from about 0.75 micrograms to about 1 milligram, from about 1 to about 500 micrograms, from about 200 micrograms to about 2 milligrams, from about 1 milligram to about 10 milligram, from about 5 milligram to about 50 milligram, from about 20 milligram to about 200 milligram, or from about 100 milligram to about 800 milligram of Diterpenoid Compound per kilogram body weight. In specific embodiments, the effective amount for an oral dose ranges from about 1 to about 10, about 10 to about 30, about 30 to about 90, or about 90 to about 150 micrograms per kilogram body weight. In other embodiments, the oral dose ranges from about 150 to about 250, about 250 to about 325, about 325 to about 450 or about 450 to about 1000 micrograms per kilogram body weight. In other embodiments, the oral dose ranges from about 150 to about 250, about 250 to about 325, about 325 to about 450 or about 450 to about 1000 milligrams per kilogram body weight. Effective amounts can be extrapolated from dose-response curves derived from in vitro or animal model test systems. Such animal models and systems are well known in the art. In one embodiment, for testing the effectiveness of a Diterpenoid Compound in an in vitro cell culture, concentrations from about 0.1 micromolar to about 10 micromolar, from about 0.2 micromolar to about 10 micromolar, from about 0.5 micromolar to about 5 micromolar, or from about 0.2 micromolar to about 5 micromolar can be used.
The invention also provides pharmaceutical packs or kits comprising one or more containers containing one or more Diterpenoid Compounds. Optionally associated with such container(s) can be instructions for use of one or more Diterpenoid Compounds or a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In one embodiment, e.g., when administered for the treatment or prevention of cancer, the kit can also contain one or more chemotherapeutic agents useful for treating cancer or a neoplastic disease to be administered prior to, subsequent to, or in combination with a Diterpenoid Compound. In another embodiment, e.g., when administered for the treatment or prevention of a fungal infection, the kit can also contain one or more other anti-fungal agents to be administered prior to, subsequent to or in combination with a Diterpenoid Compound. Such other anti-fungal agents include, but are not limited to, ketoconazole, itraconazole, amphotericin B, polyoxines, nikkomycines, carboxyamides, aromatic carbohydrates, carboxines, morpholines, inhibitors of sterol biosynthesis, and organophosphorus compounds.
The Diterpenoid Compounds can be assayed in vitro, and then in vivo, for the desired therapeutic or prophylactic activity, prior to use in humans. For example, in vitro assays can be used to determine whether administration of a specific Diterpenoid Compound or combination of Diterpenoid Compounds is preferred.
In one embodiment, a patient-tissue sample is grown in culture, and contacted or otherwise administered with a Diterpenoid Compound, and the effect of the Diterpenoid Compound upon the tissue sample is observed and compared with a non-contacted tissue. In other embodiments, a cell culture model is used in which the cells of the cell culture are contacted or otherwise administered with a Diterpenoid Compound, and the effect of the Diterpenoid Compound upon the tissue sample is observed and compared with a non-contacted cell culture. Generally, a lower level of proliferation or survival of the contacted cells compared to the non-contracted cells indicates that the Diterpenoid Compound is effective to treat or prevent cancer or a neoplastic disease. The Diterpenoid Compounds can also be demonstrated to be effective and safe using animal model systems.
In one embodiment, a fungus sample from an infected patient is grown in culture and contacted or otherwise administered with a Diterpenoid Compound, and the effect of the Diterpenoid Compound upon the growth of the fungus is observed and compared with a non-contacted tissue. Generally, a lower level of proliferation or survival of the contacted fungus compared to the non-contracted fungus indicates that the Diterpenoid Compound is effective to treat or prevent the fungal infection. The Diterpenoid Compounds can also be demonstrated to be effective and safe using animal model systems.
Other methods will be known to the skilled artisan and are within the scope of the invention.
5.8 Inhibition of Cancer and Neoplastic Cells and Disease 
The Diterpenoid Compounds can be shown to inhibit tumor cell proliferation, cell transformation or tumorigenesis in vitro and in vivo using a variety of assays known in the art, or described herein. Such assays may use cells of a cancer cell line, or cells from a patient. Many assays well-known in the art can be used to assess such survival and/or growth; for example, cell proliferation can be assayed by measuring (3H)-thymidine incorporation, by direct cell count, by detecting changes in transcription, translation or activity of known genes such as proto-oncogenes (e.g., fos, myc) or cell cycle markers (Rb, cdc2, cyclin A, D1, D2, D3, E, etc). The levels of such protein and mRNA and activity can be determined by any method well known in the art. For example, protein can be quantitated by known immunodiagnostic methods such as Western blotting or immunoprecipitation using commercially available antibodies (for example, many cell cycle marker antibodies are from Santa Cruz Inc.). mRNA can be quantitated using methods that are well known and routine in the art, for example, using northern analysis, RNase protection, the polymerase chain reaction in connection with the reverse transcription. Cell viability can be assessed by using trypan-blue staining or other cell death or viability markers known in the art. In a specific embodiment, the level of cellular ATP is measured to determined cell viability. Differentiation can be assessed, for example, visually based on changes in morphology.
The present invention provides for cell cycle and cell proliferation analysis using a variety of techniques known in the art, including but not limited to the following:
As one example, bromodeoxyuridine (BRDU) incorporation can be used as an assay to identify proliferating cells. The BRDU assay identifies a cell population undergoing DNA synthesis by incorporation of BRDU into newly synthesized DNA. Newly synthesized DNA can then be detected using an anti-BRDU antibody (see Hoshino et al., 1986, Int. J. Cancer 38, 369; Campana et al., 1988, J. Immunol. Meth. 107, 79).
Cell proliferation can also be examined using (3H)-thymidine incorporation (see e.g., Chen, J., 1996, Oncogene 13:1395–403; Jeoung, J., 1995, J. Biol. Chem. 270:18367–73). This assay allows for quantitative characterization of S-phase DNA synthesis. In this assay, cells synthesizing DNA will incorporate (3H)-thymidine into newly synthesized DNA. Incorporation can then be measured by standard techniques in the art such as by counting of radioisotope in a Scintillation counter (e.g. Beckman LS 3800 Liquid Scintillation Counter).
Detection of proliferating cell nuclear antigen (PCNA) can also be used to measure cell proliferation. PCNA is a 36 kilodalton protein whose expression is elevated in proliferating cells, particularly in early G1 and S phases of the cell cycle and therefore can serve as a marker for proliferating cells. Positive cells can be identified by immunostaining using an anti-PCNA antibody (see Li et al., 1996, Curr. Biol. 6:189–199; Vassilev et al., 1995, J. Cell Sci. 108:1205–15).
Cell proliferation can be measured by counting samples of a cell population over time (e.g. daily cell counts). Cells can be counted using a hemacytometer and light microscopy (e.g. HyLite hemacytometer, Hausser Scientific). Cell number can be plotted against time in order to obtain a growth curve for the population of interest. In one embodiment, cells counted by this method are first mixed with the dye Trypan-blue (Sigma), such that living cells exclude the dye, and are counted as viable members of the population.
DNA content and/or mitotic index of the cells can be measured, for example, based on the DNA ploidy value of the cell. For example, cells in the G1 phase of the cell cycle generally contain a 2N DNA ploidy value. Cells in which DNA has been replicated but have not progressed through mitosis (e.g. cells in S-phase) exhibit a ploidy value higher than 2N and up to 4N DNA content. Ploidy value and cell-cycle kinetics can be further measured using propidum iodide assay (see e.g. Turner, T., et al., 1998, Prostate 34:175–81). Alternatively, the DNA ploidy can be determined by quantitation of DNA Feulgen staining (which binds to DNA in a stoichiometric manner) on a computerized microdensitometrystaining system (see e.g., Bacus, S., 1989, Am. J. Pathol. 135:783–92). In an another embodiment, DNA content can be analyzed by preparation of a chromosomal spread (Zabalou, S., 1994, Hereditas. 120:127–40; Pardue, 1994, Meth. Cell Biol. 44:333–351).
The expression of cell-cycle proteins (e.g., CycA. CycB, CycE, CycD, cdc2, Cdk4/6, Rb, p21, and p27) provide information relating to the proliferative state of a cell or population of cells. For example, identification in an anti-proliferation signaling pathway can be indicated by the induction of p21cip1. Increased levels of p21 expression in cells result in delayed entry into G1 of the cell cycle (Harper et al., 1993, Cell 75:805–816; Li et al., 1996, Curr. Biol. 6:189–199). p21 induction can be identified by immunostaining using a specific anti-p21 antibody available commercially (e.g. Santa Cruz). Similarly, cell-cycle proteins can be examined by Western blot analysis using commercially available antibodies. In another embodiment, cell populations are synchronized prior to detection of a cell cycle protein. Cell cycle proteins can also be detected by FACS (fluorescence-activated cell sorter) analysis using antibodies against the protein of interest.
Detection of changes in length of the cell cycle or speed of cell cycle can also be used to measure inhibition of cell proliferation by the Diterpenoid Compounds. In one embodiment the length of the cell cycle is determined by the doubling time of a population of cells (e.g., using cells contacted or not contacted with one or more Diterpenoid Compounds). In another embodiment, FACS analysis is used to analyze the phase of cell cycle progression, or purify G1, S, and G2/M fractions (see e.g., Delia, D. et al., 1997, Oncogene 14:2137–47).
Lapse of cell cycle checkpoint(s), and/or induction of cell cycle checkpoint(s), can be examined using the methods described herein, or by any method known in the art. Without limitation, a cell cycle checkpoint is a mechanism that ensures that the different steps of cell division occur in a particular order. Checkpoint genes are defined by mutations that allow late events to occur without prior completion of an early event (Weinert, T., and Hartwell, L., 1993, Genetics, 134:63–80). Induction or inhibition of cell cycle checkpoint genes can be assayed, for example, by Western blot analysis, or by immunostaining, for example. Lapse of cell cycle checkpoints can be further assessed by the progression of a cell through the checkpoint without prior occurrence of specific events (e.g. progression into mitosis without complete replication of the genomic DNA).
In addition to the effects of expression of a particular cell cycle protein, activity and post-translational modifications of proteins involved in the cell cycle can play an integral role in the regulation and proliferative state of a cell. The invention provides for assays involved in detecting post-translational modifications (e.g. phosphorylation) by any method known in the art. For example, antibodies that detect phosphorylated tyrosine residues are commercially available, and can be used in Western blot analysis to detect proteins with such modifications. In another example, modifications such as myristylation, can be detected on thin layer chromatography or reverse phase h.p.l.c. (see e.g., Glover, C., 1988, Biochem. J. 250:485–91; Paige, L., 1988, Biochem J.; 250:485–91).
Activity of signaling and cell cycle proteins and/or protein complexes is often mediated by a kinase activity. The present invention provides for analysis of kinase activity by assays such as the histone Hi assay (see e.g., Delia, D. et al., 1997, Oncogene 14:2137–47).
The Diterpenoid Compounds can also be demonstrated to alter cell proliferation in cultured cells in vitro using methods which are well known in the art. Specific examples of cell culture models include, but are not limited to, for lung cancer, primary rat lung tumor cells (Swafford et al., 1997, Mol. Cell. Biol., 17:1366–1374) and large-cell undifferentiated cancer cell lines (Mabry et al., 1991, Cancer Cells, 3:53–58); colorectal cell lines for colon cancer (Park and Gazdar, 1996, J. Cell Biochem. Suppl. 24:131–141); multiple established cell lines for breast cancer (Hambly et al., 1997, Breast Cancer Res. Treat. 43:247–258; Gierthy et al., 1997, Chemosphere 34:1495–1505; Prasad and Church, 1997, Biochem. Biophys. Res. Commun. 232:14–19); a number of well-characterized cell models for prostate cancer (Webber et al., 1996, Prostate, Part 1, 29:386–394; Part 2, 30:58–64; and Part 3, 30:136–142; Boulikas, 1997, Anticancer Res. 17:1471–1505); for genitourinary cancers, continuous human bladder cancer cell lines (Ribeiro et al., 1997, Int. J. Radiat. Biol. 72:11–20); organ cultures of transitional cell carcinomas (Booth et al., 1997, Lab Invest. 76:843–857) and rat progression models (Vet et al., 1997, Biochim. Biophys Acta 1360:39–44); and established cell lines for leukemias and lymphomas (Drexler, 1994, Leuk. Res. 18:919–927, Tohyama, 1997, Int. J. Hematol. 65:309–317).
The Diterpenoid Compounds can also be demonstrated to inhibit cell transformation (or progression to malignant phenotype) in vitro. In this embodiment, cells with a transformed cell phenotype are contacted with one or more Diterpenoid Compounds, and examined for change in characteristics associated with a transformed phenotype (a set of in vitro characteristics associated with a tumorigenic ability in vivo), for example, but not limited to, colony formation in soft agar, a more rounded cell morphology, looser substratum attachment, loss of contact inhibition, loss of anchorage dependence, release of proteases such as plasminogen activator, increased sugar transport, decreased serum requirement, or expression of fetal antigens, etc. (see Luria et al., 1978, General Virology, 3d Ed., John Wiley & Sons, New York, pp. 436–446).
Loss of invasiveness or decreased adhesion can also be used to demonstrate the anti-cancer effects of the Diterpenoid Compounds. For example, an aspect of the formation of a metastatic cancer is the ability of a precancerous or cancerous cell to detach from primary site of disease and establish a novel colony of growth at a secondary site. The ability of a cell to invade peripheral sites reflects its potential for a cancerous state. Loss of invasiveness can be measured by a variety of techniques known in the art including, for example, induction of E-cadherin-mediated cell-cell adhesion. Such E-cadherin-mediated adhesion can result in phenotypic reversion and loss of invasiveness (Hordijk et al., 1997, Science 278:1464–66).
Loss of invasiveness can further be examined by inhibition of cell migration. A variety of 2-dimensional and 3-dimensional cellular matrices are commercially available (Calbiochem-Novabiochem Corp. San Diego, Calif.). Cell migration across or into a matrix can be examined using microscopy, time-lapsed photography or videography, or by any method in the art allowing measurement of cellular migration. In a related embodiment, loss of invasiveness is examined by response to hepatocyte growth factor (HGF). HGF-induced cell scattering is correlated with invasiveness of cells such as Madin-Darby canine kidney (MDCK) cells. This assay identifies a cell population that has lost cell scattering activity in response to HGF (Hordijk et al., 1997, Science 278:1464–66).
Alternatively, loss of invasiveness can be measured by cell migration through a chemotaxis chamber (Neuroprobe/Precision Biochemicals Inc. Vancouver, BC). In such assay, a chemo-attractant agent is incubated on one side of the chamber (e.g., the bottom chamber) and cells are plated on a filter separating the opposite side (e.g., the top chamber). In order for cells to pass from the top chamber to the bottom chamber, the cells must actively migrate through small pores in the filter. Checkerboard analysis of the number of cells that have migrated can then be correlated with invasiveness (see e.g., Ohnishi, T., 1993, Biochem. Biophys. Res. Commun. 193:518–25).
The Diterpenoid Compounds can also be demonstrated to inhibit tumor formation in vivo. A vast number of animal models of hyperproliferative disorders, including tumorigenesis and metastatic spread, are known in the art (see Table 317-1, Chapter 317, “Principals of Neoplasia,” in Harrison's Principals of Internal Medicine, 13th Edition, Isselbacher et al., eds., McGraw-Hill, New York, p. 1814, and Lovejoy et al., 1997, J. Pathol. 181:130–135). Specific examples include for lung cancer, transplantation of tumor nodules into rats (Wang et al., 1997, Ann. Thorac. Surg. 64:216–219) or establishment of lung cancer metastases in SCID mice depleted of NK cells (Yono and Sone, 1997, Gan To Kagaku Ryoho 24:489–494); for colon cancer, colon cancer transplantation of human colon cancer cells into nude mice (Gutman and Fidler, 1995, World J. Surg. 19:226–234), the cotton top tamarin model of human ulcerative colitis (Warren, 1996, Aliment. Pharmacol. Ther. 10 Supp 12:45–47) and mouse models with mutations of the adenomatous polyposis tumor suppressor (Polakis, 1997, Biochim. Biophys. Acta 1332:F127–F147); for breast cancer, transgenic models of breast cancer (Dankort and Muller, 1996, Cancer Treat. Res. 83:71–88; Amundadittir et al., 1996, Breast Cancer Res. Treat. 39:119–135) and chemical induction of tumors in rats (Russo and Russo, 1996, Breast Cancer Res. Treat. 39:7–20); for prostate cancer, chemically-induced and transgenic rodent models, and human xenograft models (Royai et al., 1996, Semin. Oncol. 23:35–40); for genitourinary cancers, induced bladder neoplasm in rats and mice (Oyasu, 1995, Food Chem. Toxicol 33:747–755) and xenografts of human transitional cell carcinomas into nude rats (Jarrett et al., 1995, J. Endourol. 9:1–7); and for hematopoietic cancers, transplanted allogeneic marrow in animals (Appelbaum, 1997, Leukemia 11 (Suppl. 4):S15–S17). Further, general animal models applicable to many types of cancer have been described, including, but not restricted to, the p53-deficient mouse model (Donehower, 1996, Semin. Cancer Biol. 7:269–278), the Min mouse (Shoemaker et al., 1997, Biochem. Biophys. Acta, 1332:F25–F48), and immune responses to tumors in rat (Frey, 1997, Methods, 12:173–188).
For example, a Diterpenoid Compound can be administered to a test animal, preferably a test animal predisposed to develop a type of tumor, and the test animal subsequently examined for a decreased incidence of tumor formation in comparison with controls not administered the Diterpenoid Compound. Alternatively, a Diterpenoid Compound can be administered to a test animal having a tumor (e.g., animals in which tumors have been induced by introduction of malignant, neoplastic, or transformed cells, or by administration of a carcinogen), and the tumors in the test animals can be subsequently examined for tumor regression and compared with controls that were not administered with the Diterpenoid Compound.
The Diterpenoid Compounds are useful for inhibiting the growth of a cancer cell or neoplastic cell and for inducing cytotoxicity, e.g., through apoptosis, of a cancer cell or neoplastic cell in vivo. Inhibiting the growth of a cancer cell or neoplastic cell and inducing cytotoxicity, e.g., through apoptosis, in a cancer cell or neoplastic cell in vivo is useful for treating, preventing and inhibiting the growth of a cancer. The Diterpenoid Compounds are useful for inhibiting the growth of a cancer cell or neoplastic cell and for inducing cytotoxicity, e.g., through apoptosis, in a cancer cell or neoplastic cell in vitro. Inhibiting the growth of a cancer cell or neoplastic cell and inducing cytotoxicity, e.g., through apoptosis, in a cancer cell or neoplastic cell in vitro is useful for assays to determine optimal concentration ranges of effectiveness of a Diterpenoid Compound.
5.8.1 Inducing Apoptosis in a Cancer Cell or a Neoplastic Cell 
Without being bound by theory, apoptosis is a morphologically and biochemically distinct form of cell death that occurs in response to a diverse range of stimuli, including irradiation and activation of death receptors such as Fas and the tumor necrosis factor receptor. Neoplastic transformation or cancerous growth of a cell can trigger apoptosis of that cell. Impaired apoptosis is therefore a significant factor in the aetiology of cancer and neoplastic diseases.
Morphologic criteria that can be used to describe apoptotic cells include condensation and margination of chromatin, cytoplasmic vacuolization, cellular shrinkage, increase in cellular density, nuclear fragmentation and apoptotic body formation.
Without being bound by theory, Applicants believe that the Diterpenoid Compounds induce apoptosis in a cancer cell or in a neoplastic cell. Moreover, without being bound by theory, Applicants believe that Diterpenoid Compounds induce apoptosis selectively in a cancer cell or in a neoplastic cell, relative to a non-cancer cell or non-neoplastic cell. In one embodiment, a Diterpenoid Compound induces apoptosis with at least 2-fold selectivity in a cancer cell or in a neoplastic cell, relative to a non-cancer cell or non-neoplastic cell. In certain embodiments, a Diterpenoid Compound induces apoptosis with at least 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold or 250-fold selectivity in a cancer cell or in a neoplastic cell, relative to a non-cancer cell or non-neoplastic cell. In certain embodiments, a Diterpenoid Compound induces apoptosis with at most 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold or 250-fold selectivity in a cancer cell and/or in a neoplastic cell, relative to a non-cancer cell or non-neoplastic cell. When selectivity in a cancer cell or neoplastic cell is n-fold, relative to a non-cancer or non-neoplastic cell, a Diterpenoid Compound induces apoptosis in n-times as many cancer cells or neoplastic cells than non-cancer cells or non-neoplastic cells.
Without being bound by theory, inducing apoptosis selectively in cancer cells or in neoplastic cells is useful for treating cancer or a neoplastic disease in a patient.
5.8.2 Treatment or Prevention of Cancer or a Neoplastic Disease in Combination with a Chemotherapy or Radiotherapy 
Cancer or a neoplastic disease, including, but not limited to, neoplasms, tumors, metastases, or any disease or disorder characterized by uncontrolled cell growth, can be treated or prevented by administration of an effective amount of a Diterpenoid Compound.
In certain embodiments, the present methods for treating or preventing cancer or a neoplastic disease comprise administering an effective amount of a Diterpenoid Compound and another active agent, such as a chemotherapeutic or anti-cancer agent, including, but not limited to, methotrexate, taxol, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, Cisplatin, carboplatin, mitomycin, dacarbazine, procarbizine, etoposides, campathecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, and docetaxel. In another embodiment, the other chemotherapeutic or anti-cancer agent includes, but is not limited to, those listed in Table 1.







TABLE 1





CHEMOTHERAPEUTICS AND OTHER ANTI-CANCER AGENTS
















Radiation:
γ-radiation







Alkylating agents








Nitrogen mustards:
cyclophosphamide



Ifosfamide



trofosfamide



Chlorambucil


Nitrosoureas:
carmustine (BCNU)



Lomustine (CCNU)


Alkylsulphonates
busulfan



Treosulfan


Triazenes:
Dacarbazine


Platinum containing compounds:
Cisplatin



carboplatin







Plant Alkaloids








Vinca alkaloids:
vincristine



Vinblastine



Vindesine



Vinorelbine


Taxoids:
paclitaxel



Docetaxol







DNA Topoisomerase Inhibitors








Epipodophyllins:
etoposide



Teniposide



Topotecan



9-aminocamptothecin



campto irinotecan



crisnatol







mytomycins:








mytomycin C
Mytomycin C







Anti-metabolites


Anti-folates:








DHFR inhibitors:
methotrexate



Trimetrexate


IMP dehydrogenase Inhibitors:
mycophenolic acid



Tiazofurin



Ribavirin



EICAR


Ribonuclotide reductase Inhibitors:
hydroxyurea



deferoxamine







Pyrimidine analogs:








Uracil analogs
5-Fluorouracil



Floxuridine



Doxifluridine



Ratitrexed


Cytosine analogs
cytarabine (ara C)



Cytosine arabinoside



fludarabine


Purine analogs:
mercaptopurine



Thioguanine







Hormonal therapies:


Receptor antagonists:








Anti-estrogens
Tamoxifen



Raloxifene



megestrol


LHRH agonists:
goscrclin



Leuprolide acetate


Anti-androgens:
flutamide



bicalutamide







Retinoids/Deltoids








Vitamin D3 analogs:
EB 1089



CB 1093



KH 1060


Photodyamic therapies:
vertoporfin (BPD-MA)



Phthalocyanine



photosensitizer Pc4



Demethoxy-hypocrellin A



(2BA-2-DMHA)


Cytokines:
Interferon-α



Interferon-γ



Tumor necrosis factor







Others:








Isoprenylation inhibitors:
Lovastatin


Dopaminergic neurotoxins:
1-methyl-4-phenylpyridinium ion


Cell cycle inhibitors:
staurosporine


Kinase inhibitors:
Imatinib


Actinomycins:
Actinomycin D



Dactinomycin


Bleomycins:
bleomycin A2



Bleomycin B2



Peplomycin


Anthracyclines:
daunorubicin



Doxorubicin (adriamycin)



Idarubicin



Epirubicin



Pirarubicin



Zorubicin



Mitoxantrone


MDR inhibitors:
verapamil


Ca2+ ATPase inhibitors:
thapsigargin









In other embodiments, the methods for treating or preventing cancer or a neoplastic disease comprise administering an effective amount of a Diterpenoid Compound and an effective amount of radiation therapy or another chemotherapeutic agent, in one embodiment, with a chemotherapeutic agent with which treatment of the cancer has not been found to be refractory. The Diterpenoid Compound can be administered to a patient that has also undergone surgery as treatment for the cancer.
In another specific embodiment, the invention provides methods for treating or preventing cancer that has shown to be refractory to treatment with a chemotherapy and/or radiation therapy.
In a specific embodiment, a Diterpenoid Compound is administered concurrently with chemotherapy or radiation therapy. In another specific embodiment, chemotherapy or radiation therapy is administered prior or subsequent to administration of a Diterpenoid Compound, preferably at least an hour, five hours, 12 hours, a day, a week, a month, more preferably several months (e.g., up to three months), subsequent to administration of the Diterpenoid Compound.
The chemotherapy or radiation therapy administered concurrently with, or prior or subsequent to, the administration of a Diterpenoid Compound can be accomplished using any method known in the art. The chemotherapeutic agents can be administered in a series of sessions, any one or a combination of the chemotherapeutic agents listed above can be administered. With respect to radiation therapy, any radiation therapy protocol can be used depending upon the type of cancer to be treated or prevented. For example, but not by way of limitation, x-ray radiation can be administered; in particular, high-energy megavoltage (radiation of greater that 1 MeV energy) can be used for deep tumors, and electron beam and orthovoltage x-ray radiation can be used for skin cancers. Gamma-ray emitting radioisotopes, such as radioactive isotopes of radium, cobalt and other elements, can also be administered to expose tissues to radiation.
Additionally, the invention provides methods for treating or preventing cancer or neoplastic disease with a Diterpenoid Compound as an alternative to chemotherapy or radiation therapy where the chemotherapy or the radiation therapy has proven or might prove too toxic, e.g., results in unacceptable or unbearable side effects, for the patient being treated. The patient being treated with the Diterpenoid Compound can, optionally, be treated with other cancer treatments such as surgery, radiation therapy or chemotherapy, depending on which treatment is found to be acceptable or bearable.
5.8.3 Cancer and Neoplastic Disease  Treatable or Preventable 
Cancers or neoplastic diseases and related disorders that can be treated or prevented by administration of an effective amount of a Diterpenoid Compound and cancer cells and neoplastic cells whose growth can be inhibited or in which cytotoxicity, e.g., through apoptosis, can be induced by contacting with an effective amount of a Diterpenoid Compound include but are not limited to those listed in Table 2 (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia):







TABLE 2





CANCERS AND NEOPLASTIC DISORDERS

















Leukemia









acute leukemia



acute lymphocytic leukemia



acute myelocytic leukemia









myeloblastic



promyelocytic



myelomonocytic



monocytic



erythroleukemia



chronic leukemia



chronic myelocytic (granulocytic) leukemia



chronic lymphocytic leukemia









Polycythemia vera



Lymphoma









Hodgkin's disease



non-Hodgkin's disease









Multiple myeloma



Waldenström's macroglobulinemia



Heavy chain disease



Solid tumors









sarcomas and carcinomas









fibrosarcoma



myxosarcoma



liposarcoma



chondrosarcoma



osteogenic sarcoma



chordoma



angiosarcoma



endotheliosarcoma



lymphangiosarcoma



lymphangioendotheliosarcoma



synovioma



mesothelioma



Ewing's tumor



leiomyosarcoma



rhabdomyosarcoma



colon carcinoma



pancreatic cancer



breast cancer



ovarian cancer



prostate cancer



squamous cell carcinoma



basal cell carcinoma



adenocarcinoma



sweat gland carcinoma



sebaceous gland carcinoma



papillary carcinoma



papillary adenocarcinomas



cystadenocarcinoma



medullary carcinoma



bronchogenic carcinoma



renal cell carcinoma



hepatoma



bile duct carcinoma



choriocarcinoma



seminoma



embryonal carcinoma



Wilms' tumor



cervical cancer



uterine cancer



testicular tumor



lung carcinoma



small cell lung carcinoma



bladder carcinoma



epithelial carcinoma



glioma



astrocytoma



medulloblastoma



craniopharyngioma



ependymoma



pinealoma



hemangioblastoma



acoustic neuroma



oligodendroglioma



meningioma



melanoma



neuroblastoma



retinoblastoma



NSCL-LC carcinoma



NSCL-adrenocarcinoma



Liver cancer



Breast epithelial cancer



Endothelial cancer



Bronchial epithelial cancer










In specific embodiments, cancer, malignancy or dysproliferative changes (such as metaplasias and dysplasias), or hyperproliferative disorders, are treated or prevented in the ovary, breast, colon, lung, skin, pancreas, prostate, bladder, cervix or uterus. In other specific embodiments, sarcoma, melanoma, or leukemia is treated or prevented.
In one embodiment, the Diterpenoid Compounds are useful for treating or preventing cancers including prostate cancer, such as hormone-insensitive prostate cancer, Neuroblastoma, Lymphoma (preferably follicular or Diffuse Large B-cell), Breast (for example Estrogen-receptor positive), Colorectal, Endometrial, Ovarian, Lymphoma (for example non-Hodgkin's), Lung (for example Small cell), or Testicular (for example germ cell).
In another embodiment, the Diterpenoid Compounds are useful for inhibiting the growth of a cell derived from a cancer or neoplasm such as prostate (in one embodiment, hormone-insensitive), Neuroblastoma, Lymphoma (in one embodiment, follicular or Diffuse Large B-cell), Breast (in one embodiment, Estrogen-receptor positive), Colorectal, Endometrial, Ovarian, Lymphoma (in one embodiment, non-Hodgkin's), Lung (in one embodiment, Small cell), or Testicular (in one embodiment, germ cell).
In other embodiments of the invention, the Diterpenoid Compounds are useful for inhibiting the growth of a cell, said cell being derived from a cancer or neoplasm in Table 2 or herein.
5.9 Inhibibition of Fungal Growth and  Treatment and Prevention of Fungal Infections 
The invention provides methods for treating or preventing a fungal infection, comprising administering to a patient in need of such treatment or prevention an effective amount of a Diterpenoid Compound. Fungal Infections that can be treated or prevented by administering an effective amount of a Diterpenoid Compound include, but are not limited to, Candida (including C. albicans, C. tropicalis, C.parapsilosis, C. stellatoidea, C. krusei, C. parakrusei, C. lusitanae, C. pseudotropicalis, C. guilliermondi, C. dubliniesis, C. famata or C. glabrata), Aspergillus (including A. fumigatus, A. flavus, A. niger, A. nidulans, A. terreus, A. sydowi, A. flavatus or A. glaucus), Cryptococcus, Histoplasma, Coccidioides, Paracoccidioides, Blastomyces, Basidiobolus, Conidiobolus, Rhizopus, Rhizomucor, Mucor, Asbidia, Mortierella, Cunninghamella, Saksenaea, Pseudallescheria, Paecilomyces, Fusarium, Trichophyton, Trichosporon Microsporum, Epidermophyton, Scytalidium, Malassezia, Actinomycetes, Sporothrix, Penicillium, Sacharomyces, Pneumocystis or Scopulariopsis infections.
In certain embodiments, such fungal infections in animals, including humans, can be a systemic, topical or mucosal infection.
In view of their antifungal activity, Diterpenoid Compounds are useful in the treatment of variety of fungal infections in animals, including humans. Such infections can be superficial, cutaneous, subcutaneous or systemic mycotic infections such as respiratory tract infections, gastrointestinal infections, cardiovascular infections, urinary tract infections, CNS infections, candidiasis and chronic muccocandidiasis and skin infections caused by fungi, cutaneous and mucocutaneous candidiasis, athletes foot, paronychia, fungal nappy rash, candida vulvitis, candida balanitis and otitis extema. They may also be used as prophylactic agents to prevent systemic and topical fungal infections. Use as prophylactic agents may be appropriate as part of a selective gut decontamination regimen in the prevention of infection in immunocomprised patients, e.g., AIDS patients and patients receiving transplant therapy.
The invention further provides a method for inhibiting the growth of a fungus comprising contacting the fungus with an effective amount of a Diterpenoid Compound. The fungi whose growth can be inhibited with a Diterpenoid Compound include Candida (including C. albicans, C. tropicalis, C.parapsilosis, C. stellatoidea, C. krusei, C. parakrusei, C. lusitanae, C. pseudotropicalis, C. guilliermondi, C. dubliniesis, C. famata or C. glabrata), Aspergillus (including A. fumigatus, A. flavus, A. niger, A. nidulans, A. terreus, A. sydowi, A. flavatus or A. glaucus), Cryptococcus, Histoplasma, Coccidioides, Paracoccidioides, Blastomyces, Basidiobolus, Conidiobolus, Rhizopus, Rhizomucor, Mucor, Asbidia, Mortierella, Cunninghamella, Saksenaea, Pseudallescheria, Paecilomyces, Fusarium, Trichophyton, Trichosporon Microsporum, Epidermophyton, Scytalidium, Malassezia, Actinomycetes, Sporothrix, Penicillium, Sacharomyces, Pneumocystis or Scopulariopsis. 
In certain embodiments, the Diterpenoid Compounds can be used as anti-fungal agents in vitro or in vivo. In a specific embodiment, the Diterpenoid Compounds can be used to prevent growth of a fungus wherever absence of fungal growth is desired, such as on or in food, medical instruments or devices, clothing, furniture and home appliances.
The following examples exemplify non-limiting aspects of the present invention.
6. EXAMPLES
6.1 Establishment and Manipulation of a Plant Cell  Suspension Culture of Linum arboreum  
A plant cell culture of Linum arboreum (Linaceae) was prepared using shoots of L. arboreum. The shoots were sterilized by immersion for 1 minute in 70% ethanol followed by immersion for 25 minutes in a solution of sodium hypochlorite (concentration 15%). The sterile shoots were chopped into small pieces of approximately 5 mm and placed upon solidified callus induction medium B5 (Gamborgs B5 recipe (Exp. Cell. Res. 50: 148 (1968)) containing 2,4-dichlorophenoxyacetic acid (2,4-D) (1 mg/L), kinetin (0.1 mg/L), sucrose (2%)). Callus initiations were incubated in continuous low light at 23_C. Upon establishment of callus, the material was used to initiate suspension cultures.
To establish suspension cultures, portions of established callus were placed in 100 mL conical flasks containing liquid medium B88, modified after Gamborgs B5 recipe to contain 2,4-D (1 mg/L), kinetin (0.1 mg/L), coconut water (10%)+3% sucrose. The liquid medium was replenished at 14 day intervals. After 4 months the established suspension culture was routinely maintained in a 250 mL conical flask, by transferring 40 mL of a 14 or 21 day old suspension culture into 100 mL fresh B88 medium. The culture was incubated at 25° C. in continuous low light and shaken at 140 rpm.
Accumulation of isolated compounds was induced in the Linum arboreum suspension culture according to either one of the following protocols.
(1) 2 L conical flasks each containing 750 mL of a secondary metabolite production medium B49 (Gamborgs B5, 5% sucrose, no hormones), were each inoculated with 260 mL of a 14-day old suspension culture grown on B88 medium. The cultures were incubated under low light conditions (approximately 30 lux) at 25° C. for 28 days.
(2) A 40 mL aliquot of a day 0 suspension growing on B88 medium was transferred to a 100 mL flask. On day 3, a sterile solution of 5-azacytidine (5-AC) in water was added for a final concentration of 3×10−5 M, and the resultant mixture was incubated for 11 days. At this point the 40 mL 5-AC-treated culture was subcultured, twice before inoculating 190 mL B49 production medium in a 500 mL flask with 70 mL of a 14 day-old suspension. The culture was incubated under low light conditions (approximately 30 lux) at 25° C. for 7 days following inoculation, filter-sterilized methyl jasmonate (250 uM final concentration) and an autoclaved Candida albicans preparation (50 mg/L final concentration) were added. The C. albicans preparation was obtained by growing a culture of strain ATCC28516 on YEPD media (yeast extract 1%, yeast peptone 2%, glucose 2%) to maximum cell density and twice autoclaving the total yeast culture prior to addition to plant cultures. The culture was incubated for a further 7 days before harvest of biomass.
6.2 Isolation of Coumpounds 48 and 49 
At harvest the plant cell culture of Linum arboreum, the culture was centrifuged and the supernatant liquid was decanted. The residual biomass was freeze-dried, and subsequently extracted. Biomass from the culture method (1) in Example 6.1 was used in the following isolation procedure.
81.85 g of dried biomass corresponding to a 6 L culture of Linum arboreum obtained above were placed in two 3 L glass beakers, 2 L of methanol was added to each beaker and stirred at room temperature for 20 hours. The mixture was then filtered under vacuum through a fritted funnel. After filtration the marc was washed twice with 250 mL portions of methanol and then the washings were combined with the main filtrate. The combined filtrate and washings were evaporated to dryness under vacuum using a rotary evaporator. The yield of the methanol extract (E11) was 22.8 g (see FIG. 1A).
The E11 extract was dissolved in 2 L of water and then partitioned with 2×1 L of butanol. The butanol phases were separated, combined and then concentrated to dryness. The yield of the butanol exact (E11B) was 7.65 g (33.5% from the methanol extract). 150 mL of HP-20 were placed in a 360 mL fritted glass column (3.8×30 cm) and equilibrated with 10% acetonitrile. Approximately 7.5 g of the E11B extract were re-dissolved in methanol and absorbed into 70 mL of HP-20. The absorbed HP-20 extract was placed on top of the HP-20 column and eluted with an aqueous acetonitrile gradient followed by methanol and acetone. The active fraction (A4) was eluted with methanol. It yielded approximately 420mg (1.8% from the methanol extract).
Approximately 1 mg of the A4 fraction was fractionated by HPLC using the Gem5.met method (see Experimental Methods below). Fractions were collected in a 96-well plates.
The remainder of the A4 fraction was triturated with 2×50 mL of hexane under mechanical stirring. After centrifugation, the soluble material was concentrated to dryness yielding 197 mg of a hexane fraction (B1). The hexane insoluble material was triturated with 2×50 mL of ethyl acetate. After centrifugation, the soluble material was concentrated to dryness yielding 40 mg of an ethyl acetate fraction (B2). The ethyl acetate insoluble material yielded 135 mg of a methanol soluble fraction (B3). All fractions, B1–B3 were analyzed by HPLC.
B1 and B2 fractions were subjected to preparative reverse phase HPLC using the Gem8p.met method (see Experimental Methods below). Fractions B1C6 and B2C5 (peaks at Rt ̃25.8 mm) were combined to yield 4.3 mg of Compound 48. Fraction B1C4 (peak at Rt ̃14.3 min) yielded 1.5 mg of Compound 49.
Experimental Methods:












HPLC: Gem5.met













Column:
Xterra TM RP18 (150 × 7.8 mm, 7μ)



Flow Rate:
3 mL/min



Detection:
UV @ 205, 220 and 280 nm











Solvent system:









Time (min.)
H2O (%)
ACN (%)





 0.0
80
20


 5.0
60
40


35.0
0
100


42.0
0
100


45.0
80
20


55.0
80
20










HPLC: Gem8n.met













Column:
Xterra TM RP18 (300 × 19 mm, 7μ)



Flow Rate: 
14 mL/min



Detection:
UV @ 205, 220 and 277 nm











Solvent system:









Time (min.)
H2O (%)
ACN (%)





 0.0
80
20


 5.0
55
45


50.0
0
100


65.0
0
100


70.0
80
20


85.0
80
20









Structure Elucidation:

Compound 48's 1H-NMR and 13C-NMR assignments were made based on the analysis of their 2D-NMR experiments. The mass spectra showed a peak at m/z 313 corresponding to M+1 ion. The UV spectra showed a maximum absorption at λ218, 238 (sh), 277 and 326 nm. The 1H-NMR spectra run in CDCl3 reveals the presence of four olefinic protons [δ 8.01 (s), δ 7.39 (d, J=10 Hz), δ6.94 (s), δ 6.29 (d, J=10 Hz)], four methyl groups [δ 2.29 (s), δ 1.69 (s), δ 1.60 (s), δ 1.57 (s)], one methoxy group at δ 3.99 (s) and one exchangeable proton at δ 7.22 (s). The 13C-NMR spectra run in CDCl3 showed signals for 19 carbons. Multiplicity information was obtained from the DEPT experiments. The olefinic proton at δ 7.39 COSY with the signal at δ 6.29. The magnitude of their coupling constant (J=10 Hz) is in agreement with the cis configuration. 2D-NMR experiments including COSY, HMQC and HMBC were performed in order to assign all proton and carbon signals (Table 3). 1H-13C correlations observed from the HMBC experiment are summarized in FIG. 1B.
Compound 49's mass spectra showed a peak at m/z 329 corresponding to M+1 ion. The UV spectra showed a maximum absorption at λ 216, 238 (sh), 277 and 327 nm. The 1H-NMR spectra run in CDCl3 reveals the presence of four olefinic protons [δ 8.21 (s), δ 7.38 (d, J=10 Hz), δ 7.02 (s), δ 6.30 (d, J=10 Hz)], three methyl groups [δ 1.70 (s), δ 1.60 (s), δ 1.58 (s)], one methoxy group at δ 4.04 (s), one methylene group at δ 4.77 and one exchangeable proton at δ 7.21(s). The 13C-NMR spectra run in CDCl3 showed signals for 19 carbons. Structure elucidation was performed by comparison of their 1H and 13C-NMR spectra with Compound 48. The difference appears to be in the aromatic ring where a methylene group is replacing a methyl group. NMR assignments are summarized in Table 4.







TABLE 3







NMR Assignments of Compound 48


 








 











13C-NMR
1H-NMR
1H-NMR


Atom #
(in CDCl3)
(in CDCl3)
(in MeOD)













 1
150.6
7.39 (1H, d, J=10 Hz)
7.65 (1H, d, J=10 Hz)


 2
128.4
6.29 (1H, d, J=10 Hz)
6.27 (1H, d, J=10 Hz)


 3
202.7
—
—


 4
48.1
—
—


 5
133.1
—
—


 6
41.7
—
—


 7
148.8
—
—


 8
120.8
—
—


 9
179.0
—
—


10
142.9
—
—


11
105.7
6.94 (1H, s)
7.24 (1H, s)


12
162.6
—
—


13
127.4
—
—


14
129.3
8.01 (1H, s)
7.92 (3H, s)


15
26.3
1.60 (3H, s)
1.58 (3H, s)


16
21.1
1.69 (3H, s)
1.67 (3H, s)


17
38.9
1.57 (3H, s)
1.56 (3H, s)


18
55.7
3.99 (3H, s)
4.02 (3H, s)


19
15.9
2.29 (3H, s)
2.26 (3H, s)


OH
—
7.22 (1H, s)
—
















TABLE 4







NMR Assignments of Compound 49


 








 











13C-NMR
1H-NMR
1H-NMR


Atom #
(in CDCl3)
(in CDCl3)
(in MeOD)













 1
150.1
7.38 (1H, d, J=10Hz)
7.67 (1H, d, J=10 Hz)


 2
128.6
6.30 (1H, d, J=10Hz)
6.30 (1H, d, J=10 Hz)


 3
202.5
—
—


 4
48.1
—
—


 5
133.3
—
—


 6
41.9
—
—


 7
150.5
—
—


 8
121.3
—
—


 9
178.8
—
—


10
142.9
—
—


11
106.3
7.02 (1H, s)
7.30 (1H, s)


12
161.8
—
—


13
129.8
—
—


14
127.7
8.21 (1H, s)
8.22 (1H, s)


15
26.3
1.60 (3H, s)
1.59 (3H, s)


16
21.1
1.70 (3H, s)
1.68 (3H, s)


17
38.9
1.58 (3H, s)
1.58 (3H, s)


18
55.9
4.04 (3H, s)
4.03 (3H, s)


19
61.0
4.77 (2H, s)
4.67 (2H, s)


10-OH
—
7.21 (1H, s)
—









6.3 Synthesis of Compound 50 
Compound 50 was synthesized as illustrated in Scheme 6 and summarized below.
Tetralone 51 was treated with methylmagnesium bromide, and the resulting alcohol was eliminated with hydrochloric acid to provide an alkene. The alkene was treated with m-chloroperbenzoic acid, and the resultant epoxide was treated with boron trifluoride to provide tetralone 52 (Murphy et al. J. Org. Chem., 1960, 25, 1386; Kuehne. J. Amer. Chem. Soc., 1961, 83, 1492; Taylor; Chiang. Tetrahedron Lett., 1977, 1827). A Robinson's annelation was performed on tetralone 52 using a modified method (Shishido, K. et al. J. Org. Chem., 1994, 59, 406–414), and the resulting enone 53 was reduced by a Birch reduction to provide ketone 54. Enone 55 was obtained using known methods (Grieco et al., J. Org. Chem., 1998, 63, 5929–5936) followed by the methylation of the enone to provide Compound 56. Chromium(VI)oxide was used for the benzylic oxidation according to Rutledge (Cambie et al., P. S. Aust. J. Chem., 1998, 51, 931–940) to give ketone 56. The potassium enolate of ketone 56 was oxidized with oxygen, and the resultant 1,2-diketone formed was enolized in-situ to provide Compound 50.
Synthesis of Compound 52
Methylmagnesium bromide (100 mL; 3M solution; 300 mmol) was added to a stirred solution of 7-methoxy-1-teralone 51 (25 g; 142 mmol) in THF (400 mL) at −78° C. The resultant mixture was heated to room temperature and stirred for three hours. The solution was cooled to 0° C. and water (60 mL) was added dropwise, followed by a solution of hydrochloric acid (60 mL conc. HCl and 30 mL water). The resultant solution was stirred at room temperature for one hour and then extracted with ethyl acetate (3×200 mL). The combined organic layers were washed with a saturated aqueous solution of sodium bicarbonate (100 mL), dried over sodium sulfate, decanted and concentrated in vacuo. The resultant residue was dissolved in dichloromethane (400 mL) and cooled to 0° C., and to it was added m-chloroperbenzoic acid (38 g; 77%; 170 mmol). The resultant mixture was stirred at 0° C. for 30 min and then quenched with a saturated aqueous solution of sodium bicarbonate (100 mL) and a solution of saturated aqueous sodium thiosulfate (50 mL). The organic and aqueous layers were separated, and the aqueous layer was extracted with dichloromethane (2×100 mL). The combined organic layers were dried over sodium sulfate, decanted and concentrated in vacuo to a final volume of 400 mL. The solution was cooled to 0° C., and to it was added boron trifluoride diethyletherate (0.9 mL; 7.1 mmol). After 15 min at 0° C. the reaction was quenched with a solution of saturated aqueous sodium bicarbonate (100 mL). The organic and aqueous layers were separated, and the aqueous layer was extracted with dichloromethane (2×100 mL). The combined organic layers were dried over sodium sulfate, decanted and concentrated in vacuo. The resultant residue was purified via column chromatography over silica gel (10% ethyl acetate in hexane eluent) to provide Compound 52 (20 g, 74%) as a colorless oil.
NMR 1H (300 MHz, CDCl3): δ (ppm) 1.46 (d, J=8 Hz, 3H), 2.53 (m, 2H), 3.0 (m, 2H), 3.48 (q, J=8 Hz, 1H), 3.82 (s, 3H), 6.77 (m, 2H), 7.13 (d, J=8 Hz, 1H).
Synthesis of Compound 53
To a stirred solution of potassium hydroxide (1.27 g; 22.7 mmol) in methanol (22 mL) and water (2.5 mL) at −15° C., was added, dropwise, Compound 52 (3.6 g; 18.9 mmol) in methanol (5 mL). After 30 min at −15° C., ethyl vinyl ketone (1.87 mL; 18.9 mmol) was added. The resultant solution was stirred 1 h at −15° C., heated at room temperature for 1 h and finally heated to 50° C. for 2 hrs. The methanol was removed in vacuo and the residue was dissolved in water (30 mL) and extracted with ethyl acetate (3×25 mL). The combined organic layers were dried over sodium sulfate, decanted and concentrated in vacuo. The resultant residue was purified via column chromatography over silica gel (10% ethyl acetate in hexane eluent) to provide Compound 53 (3.5 g, 72%) as a pale yellow solid.
NMR 1H (300 MHz, CDCl3): δ (ppm) 1.52 (s, 3H), 1.85 (s, 3H), 2.06 (m, 1H), 2.3–3.1 (m, 7H), 3.80 (s, 3H), 6.72 (dd, J=9 Hz and 2 Hz, 1H), 6.84 (d, J=2 Hz, 1H), 7.03 (d, J=9 Hz, 1H).
Synthesis of Compound 54
To a stirred solution of lithium (948 mg; 136 mmol) in liquid ammonia (75 mL) at −78° C., was added dropwise a solution of Compound 53 (3.5 g; 13.6 mmol) and t-butanol (1.3 mL; 13.6 mmol) in tetrahydrofuran (25 mL). After 1.5 hr at −78° C., isoprene (7 mL; 68 mmol) was added dropwise, and the reaction mixture was heated to room temperature to allow the ammonia to evaporate. A saturated aqueous solution of ammonium chloride (50 mL) was added to the residue, and the resultant mixture was extracted with ethyl acetate (3×50 mL). The combined organic layers were dried over sodium sulfate, decanted and concentrated in vacuo. The resultant residue was purified via column chromatography over silica gel (10% ethyl acetate in hexane eluent) to provide Compound 54 (1.64 g, 66%) as a white solid.
NMR 1H (300 MHz, CDCl3): δ (ppm) 1.12 (d, J=7 Hz, 3H), 1.38 (s, 3H), 1.63 (m, 2H), 1.86 (m, 1H), 1.97 (m, 1H), 2.40–2.69 (m, 4H), 2.86 (m, 2H), 3.79 (s, 3H), 6.71 (dd, J=9 Hz and 2 Hz, 1H), 6.85 (d, J=2 Hz, 1H), 7.00(d, J=9 Hz, 1H).
Synthesis of Compound 55
Compound 54 was converted to Compound 55 using known methods (Grieco, et al. J. Org. Chem., 1998, 63, 5929–5936).
NMR 1H (300 MHz, CDCl3): δ (ppm) 1.23 (d, J=7 Hz, 3H), 1.34 (s, 3H), 1.67 (m, 1H), 2.00 (m, 2H), 2.43 (m, 1H), 2.88 (m, 2H), 3.80 (s, 3H), 5.97 (d, J=10 Hz, 1H), 6.71 (dd, J=9 Hz and 2 Hz, 1H), 6.95 (d, J=2 Hz, 1H), 7.00 (d, J=9 Hz, 1H), 7.54 (d, J=10 Hz, 1H).
Synthesis of Compound 56
To a stirred suspension of potassium hydride (492 mg; 30% dispersion in mineral oil; 3.69 mmol) in tetrahydrofuran (8 mL) at 0° C., was added dropwise a solution of Compound 55 (315 mg; 1.23 mmol) in tetrahydrofuran (9 mL). After 30 min at 0° C., freshly distilled iodomethane (0.765 mL; 12.3 mmol) was added. The reaction mixture was stirred at 0° C. for 1 hr and then quenched with a solution of saturated aqueous ammonium chloride (10 mL). The organic and aqueous layers were separated, and the aqueous layer was extracted with ethyl acetate (3×20 mL). The combined organic layers were dried over sodium sulfate, decanted and concentrated in vacuo. The resultant residue was purified via column chromatography over silica gel (15% ethyl acetate in hexane eluent) to provide Compound 56 (240 mg, 54%).
NMR 1H (300 MHz, CDCl3): δ (ppm) 1.18 (s, 3H), 1.20 (s, 3H), 1.40 (s, 3H), 1.89 (m, 2H), 2.14 (dd, J=11 Hz and 4 Hz, 1H), 2.88 (m, 2H), 3.79 (s, 3H), 6.00 (d, J=10 Hz, 1H), 6.71 (dd, J=9 Hz and 2 Hz, 1H), 6.91 (d, J=2 Hz, 1H), 7.02 (d, J=9 Hz, 1H), 7.53 (d, J=10 Hz, 1H).
Synthesis of Compound 50
To a stirred solution of Compound 56 (240 mg; 0.89 mmol) in acetic acid (6 mL) at 0° C., was added, before freezing of the acetic acid, a solution of chromium(VI) oxide (222 mg; 2.23 mmol) in acetic acid/water (1.2 mL/0.3 mL) dropwise over 10 min. The reaction mixture was stirred at 0° C. for 15 min, and then water (10 mL) was added. The reaction mixture was extracted with ethyl acetate (3×10 mL), and the combined organic layers were washed with a solution of saturated aqueous sodium bicarbonate (10 mL), dried over sodium sulfate, filtered through a short pad of silica gel, washed with ethyl acetate (50 mL) and concentrated in vacuo. The resultant residue was dissolved in tetrahydrofuran (3 mL) and added dropwise to a stirred solution of potassium t-butoxide (300 mg; 2.67 mmol) in t-butanol (5.5 mL) and tetrahydrofuran (13 mL) at 0° C. The resultant solution was stirred at 0° C. under oxygen atmosphere (balloon) for 1 h and then quenched with a solution of saturated aqueous ammonium chloride (10 mL). The resultant reaction mixture was extracted with ethyl acetate (3×20 mL), and the combined organic layers were dried over sodium sulfate, filtered through a short pad of silica gel, washed with ethyl acetate (50 mL) and concentrated in vacuo. The resultant residue was triturated in cold ethanol (4 mL) and filtered to provide Compound 50 (130 mg, 49%) as a white solid.
NMR 1H (300 MHz, CDCl3): δ (ppm) 1.57 (s, 3H), 1.59 (s, 3H), 1.69 (s, 3H), 3.95 (s, 3H), 6.27 (d, J=10 Hz, 1H), 7.00 (dd, J=9 Hz and 2 Hz, 1H), 7.08 (d, J=2 Hz, 1H), 7.20 (s, 1H), 7.37 (d, J=10 Hz, 1H), 8.20 (d, J=9 Hz, 1H).
NMR 13C (75 MHz, CDCl3): δ (ppm) 202.6, 179.0, 164.2, 150.8, 150.6, 143.0, 133.6, 130.1, 128.6, 121.6, 113.8, 111.0, 56.0, 48.4, 42.1, 39.3, 26.7, 21.5.
MP: 167° C.–168° C.
Synthesis of Compound 50a and Compound 50b
Compound 50a and Compound 50b were synthesized as illustrated in Scheme 7.
Synthesis of Compound 61
Compound 61 was synthesized as described in Takahashi et al., Syn. Lett. 1999, 5, 6444–646; Corey, et al. J. Am. Chem Soc. 1997, 119, 9927–9928; and Rosales et al. J. Org. Chem. 2002, 67, 1167–1170.
To a solution of geraniol 57 (100 mol %; 5.0 mmole; 868 μL) in dry THF at −45° C. was added methanesulfonyl chloride (130 mol %; 6.5 mmole; 502 μL) and triethylamine (200 mol %; 10 mmole; 1.4 mL). The resultant solution was stirred at −45° C. for 45 min, then warmed to 0° C., at which point lithium chloride (400 mol %, 20 mmole, 848 mg as a solid) or lithium bromide (400 mol %, 20 mmole, 1.737 g as a solution in 8 mL of THF via cannula) was added. The resultant solution was stirred for 1 hr at 0° C. Water was added (50 mL), and the resultant mixture was extracted using hexane (2×30 mL). The combined organic solution was successively washed with saturated NaHCO3 and brine, dried over Na2SO4, filtered and evaporated to provide crude allylic halides 58 and 59, which were used directly, without further purification, in the next step.
To a solution of tetrakis(triphenylphosphine)palladium(0) (5 mol %; 50 μmol; 58 mg) in dry and freshly degassed THF was added allylic halide 58 or 59 (100 mol %; 1.0 mmole; 58: 217 mg; 59: 173 mg). The resulting solution was stirred at room temperature (20° C.) for 5 min then cooled to 0° C. p-Methoxybenzyl magnesium chloride 60 (130 mol %; 1.3 mmole; 5.2 mL of a 0.25 M solution in THF) was added via syringe and the reaction mixture was stirred at room temperature (20° C., 58: 5 hrs; 59: 3 hrs). Water was added (20 mL), and the mixture was extracted using Et2O (2×10 mL). The combined organic solution was dried over Na2SO4, filtered and evaporated. The crude reaction product was dissolved in pentane and filtered through a pad of silica gel, the pad was further rinsed with CHCl3. The CHCl3 filtrate was evaporated and the resultant residue was purified via column chromatography over silica gel using a gradient of 0–2% of EtOAc/Hexanes to obtain Compound 61 (162 mg; 63%). This reaction was also performed on larger scale using 3.0 mmole of allylic halide 58 to give 61 (517 mg; 65%).
MH+/Z calc. for C18H27O1=259.21; LRMS found=259.27
1H NMR (300 MHz, CDCl3): δ (ppm) 1.59 (s, 3H); 1.64 (s, 3H); 1.72 (s, 3H); 1.99–2.13 (m, 4H); 2.26–2.34 (m, 2H); 2.58–2.85 (m, 2H); 3.80 (s, 3H); 5.09–5.14 (m, 1H); 5.17–5.22 (m, 1H); 6.81–6.86 (m, 2H); 7.08–7.26 (m, 2H).
Synthesis of the Racemic Epoxide 63
The racemic epoxide 63 was synthesized as described in Ceruti, M. et al. J. Med. Chem. 1998, 41, 540–554.
To a solution of Compound 61 (100 mol %; 2.0 mmole; 517 mg) in THF (58 mL) at 0° C. was added water (50 mL) until the solution reached opalescence. The resultant mixture was stirred at 0° C., and N-bromosuccinimide (NBS; 110 mol %; 2.2 mmole; 392 mg) was added in portions over 10 min. Water was added during the addition to maintain opalescence of the solution. After the addition of NBS was completed the reaction mixture was stirred an additional 15 min at 0° C. and water was continuously added to maintain opalescence (total volume of water added: 20 mL). Saturated aqueous NaHCO3 was added (60 mL) and the resulting mixture was extracted using Et2O (3×60 mL). The organic layers were combined and washed using saturated aqueous NaHCO3 (60 mL) and brine (60 mL). The organic solution was dried over Na2SO4, filtered and evaporated. The resultant residue was purified via column chromatography over silica gel using a gradient of 0–6% EtOAc/Hexanes to provide a crude bromohydrin (487 mg; 69%).
1H NMR (300 MHz, CDCl3): δ (ppm) 1.34 (s, 3H); 1.34 (s, 3H); 1.54 (s, 3H); 1.76–1.84 (m, 1H); 1.93–1.99 (m, 1H); 2.12–2.16 (m, 2H); 2.27–2.34 (m, 2H); 2.59–2.65 (m, 2H); 3.80 (s, 3H); 3.92 (dd, J1=2.0 Hz, J2=11 Hz, 1H); 5.24–5.29 (m, 1H); 6.80–6.85 (m, 2H); 7.08–7.26 (m, 2H).
To a suspension of K2CO3 (300 mol %; 3.6 mmole; 498 mg) in methanol (20 mL) was added freshly prepared crude bromohydrin, above (100 mol %; 1.2 mmole; 426 mg). The reaction mixture was stirred at room temperature (20° C.) for 2.5 hrs (reaction was monitored by TLC analysis). Water was added and the resultant mixture was extracted with Et2O (3×50 mL). The organic layers were combined and washed using brine (2×50 mL). The organic solution was dried over Na2SO4, filtered and evaporated. The resultant residue was purified via column chromatography over silica gel using a gradient of 0–5% EtOAc/Hexanes to provide compound 63 (261 mg; 79%).
1H NMR (300 MHz, CDCl3): δ (ppm) 1.28 (s, 3H); 1.32 (s, 3H); 1.59 (s, 3H); 1.61–1.72 (m, 2H); 2.07–2.18 (m, 2H); 2.30 (dd, J1=7.2 Hz, J2=15 Hz, 2H); 2.60 (t, J=7.6 Hz, 2H); 2.70 (t, J=6.4 Hz, 1H); 3.79 (s, 3H); 5.21–5.26 (m, 1H); 6.80–6.84 (m, 2H); 7.08–7.13 (m, 2H).
Synthesis of Chiral Diol 62b
The chiral diol 62b was synthesized as described in Corey, et al. J. Am. Chem Soc. 1997, 119, 9927–9928; and Crispino, G. A. et al. Synthesis 1993, 777–779.
To a mixture of 61 (100 mol %; 1.9 mmole; 500 mg) and chiral catalyst (1,4-bis(dihydroquinidinephthalazine) ((DHQD)2PHAL) for the synthesis of the R enantiomer or 1,4-bis(dihydroquininephthalazine) ((DHQ)2PHAL) for the synthesis of the S enantiomer (5 mol %; 96 μmol; 75.4 mg), in a 1:1 solution of water:tert-butanol (20 mL) at 0° C. was added potassium carbonate (300 mol %; 5.8 mmole; 800 mg), potassium ferricyanide (300 mol %; 5.8 mmole; 1.90 g), methanesulfonamide (100 mol %; 1.9 mmole; 184 mg), and potassium osmate dihydrate (1 mol %, 19 μmole; 7.1 mg). An additional 15 mL of the 1:1 solution of water:tert-butanol was added, and the reaction mixture was stirred at 0° C. for 4.5 hrs. Saturated aqueous Na2S2O3 (10 mL) was added and the reaction was stirred at room temperature (20° C.) for an additional 45 min. Evaporation of the volatiles on rotary evaporator (no heat) was followed by extraction using EtOAc (4×30 mL). The organic layers were combined and washed with KOH (1M; 30 mL) and brine (30 mL). The organic solution was dried over Na2SO4, filtered and evaporated. Crude diol 62 was purified first by precipitation of tetrol (overoxydation product) in Et2O followed by column chromatography over silica gel using a gradient of 0–40% EtOAc/Hexanes to provide Compound 62a (184 mg; 33%) and Compound 62b (127 mg; 22%).
1H NMR (300 MHz, CDCl3): δ (ppm) 1.16 (s, 3H); 1.19 (s, 3H); 1.38–1.46 (m, 1H); 1.57 (s, 3H); 2.05–2.09 (m, 1H); 2.21–2.24 (m, 2H); 2.30 (dd, J1=7.6 Hz, J2=15 Hz, 2H); 2.56–2.63 (m, 2H); 3.29 (dd, J1=2.0 Hz, J2=11 Hz, 1H); 3.79 (s, 3H); 5.21–5.26 (m, 1H); 6.79–6.84 (m, 2H); 7.07–7.11 (m, 2H).
Synthesis of Chiral Epoxide 63b
The chiral epoxides 63a and 63b were synthesized as described in Corey, et al. J. Am. Chem Soc. 1997, 119, 9927–9928.
The following example is for a synthesis of 63a, but the same procedure was used for synthesis of 63b except that chiral diol 62b was used in place of chiral diol 62a. To a solution of chiral diol 62a (100 mol %; 0.60 mmole; 175 mg) in CH2Cl2 (4 mL) at 0° C. was added pyridine (800 mol %; 4.8 mmole; 388 μL) and methanesulfonyl chloride (480 mol %; 2.8 mmole; 223 μL). The reaction mixture was stirred at 0° C. for 5 min then for an additional 3 hrs at room temperature (20° C.). The solvent was evaporated by nitrogen gas circulation through the reaction flask. Methanol (12 mL) was added along with potassium carbonate (1667 mol %; 10 mmole; 1.44 g), and the resultant suspension was stirred at room temperature (20° C.) for 1–2 hrs (monitored by TLC analysis). Water (20 mL) was added, and the mixture was extracted using Et2O (2×20 mL). The organic layers were combined and washed using dilute Cu(II) nitrate (2×20 mL), water (20 mL), and brine (20 mL). The organic solution was dried over MgSO4, filtered and evaporated. Crude epoxide 63 was purified by column chromatography over silica gel using a gradient of 0–2% EtOAc/Hexanes to provide Compound 63a (105 mg; 64% over two steps) and Compound 63b (70 mg 64% over two steps starting from 0.40 mmole of 62b).
1H NMR (300 MHz, CDCl3): δ (ppm) 1.27 (s, 3H); 1.31 (s, 3H); 1.58 (s, 3H); 1.60–1.72 (m, 2H); 2.09–2.28 (m, 2H); 2.30–2.33 (m, 2H); 2.57–2.62 (m, 2H); 2.70 (t, J=6.4 Hz, 1H); 3.79 (s, 3H); 5.20–5.25 (m, 1H); 6.79–6.84 (m, 2H); 7.07–7.12 (m, 2H).
Synthesis of Compound 64b
Compounds 64a and 64b were synthesized as described in Corey, et al. J. Am. Chem Soc. 1997, 119, 9927–9928.
The following example is for synthesis of Compound 64a, but the same procedure was used for synthesis of Compound 64b except that chiral epoxide 63b was used in place of chiral epoxide 63a. To a solution of chiral epoxide 63a (100 mol %; 0.38 mmole; 105 mg) in dichloromethane (3 mL) at −78° C. was added a solution of methylaluminum dichloride (120 mol %; 0.46 mmole; 460 μL of a 1M solution in hexanes) in dichloromethane (3 mL) cooled at −78° C. via cannula. The reaction mixture was stirred at −78° C. for 4 hrs. Triethylamine (0.80 mL) and 1:1 water:methanol (0.80 mL) were added, the resultant mixture was poured onto half saturated aqueous ammonium chloride (20 mL) and extracted using dichloromethane (10 mL) and Et2O (2×20 mL). The organic layers were combined and washed using brine (30 mL). The organic solution was dried over Na2SO4, filtered and evaporated. The resultant residue was purified via column chromatography over silica gel using a gradient of 0–4% EtOAc/Hexanes to provide Compound 64a (52 mg; 50%) and 64b (38 mg, 56% starting from 0.25 mmole of 63b).
1H NMR (300 MHz, CDCl3): δ (ppm) 0.91 (s, 3H); 1.09 (s, 3H); 1.22 (s, 3H); 1.25–1.30 (m, 1H); 1.44–1.62 (m, 1H); 1.67–1.94 (m, 4H); 2.25–2.32 (m, 1H); 2.74–2.96 (m, 2H); 3.29–3.34 (m, 1H); 3.78 (s, 3H); 6.68–6.69 (m, 1H); 6.78–6.79 (m, 1H); 6.95–6.98 (m, 1H).
Synthesis of Compound 65b
Compounds 65a and 65b were synthesized as described in Ley, S. V. et al.; Marsden S. P. Synthesis 1994, 639–666.
The following example is for a synthesis of Compound 65a, but the same procedure was used for synthesis of Compound 65b except that Compound 64b was used in place of Compound 64a. To 4 Å dry powdered molecular sieves (121 mg) was added a solution of Compound 64a (100 mol %; 0.19 mmole; 52 mg) in dichloromethane (3 mL), 4-methylmorpholine N-oxide (200 mol %; 0.38 mmole; 45 mg), and tetrapropylammonium perruthenate (10 mol %; 19 μmole; 7.0 mg). The reaction mixture was stirred at room temperature (20° C.) for 1–2 hrs (monitored by TLC analysis). The reaction mixture was filtered through a pad of silica gel, which was rinsed using 5% EtOAc/Hexanes (40 mL). The filtrate was discarded, and the silica gel was washed with 100% EtOAc (40 mL). The filtrate was evaporated to provide Compound 65a (52 mg; 99%) and Compound 65b (36 mg; 96% starting from 0.14 mmole of 64b).
1H NMR (300 MHz, CDCl3): δ (ppm) 1.15 (s, 3H); 1.18 (s, 3H); 1.31 (s, 3H); 1.77–1.84 (m, 2H); 1.89–2.10 (m, 2H); 2.41–2.50 (m, 1H); 2.60–2.91 (m, 4H); 3.78 (s, 3H); 6.68–6.71 (m, 1H); 6.78–6.79 (m, 1H); 6.98–7.00 (m, 1H).
Enantiomeric excess of Compound 65a and Compound 65b was for Compound 65a (R enantiomer)=21.57 min 97% ee. Retention time (Rt) for Compound 65b (S enantiomer)=21.57 min 92% ee.
Synthesis Compound 56b
Compounds 56a and 56b were synthesized as described in Poigny, S. et al., J. Org. Chem. 1998, 63, 5890–5894; and Ito, Y. et al. J. Org. Chem. 1978, 43, 1011–1013.
The following example is for a synthesis of Compound 56a, but the same procedure was used for synthesis of Compound 56b except that Compound 65b was used in place of Compound 65a. To a solution of Compound 65a (100 mol %; 0.23 mmole; 64 mg) in dichloromethane (4 mL) at 0° C. was added trimethylsilyl trifluoromethanesulfonate (200 mol %; 0.46 mmole; 84 μL), and the resulting mixture was stirred at 0° C. for 5 min. Triethylamine (250 mol %; 0.58 mmole; 81 μL) was added, and the reaction mixture was stirred at 0° C. for 1 hr (monitored by TLC analysis). Saturated aqueous NaHCO3 was added (10 mL,) and the resultant mixture was extracted using Et2O (3×10 mL). The organic layers were combined and washed using brine (10 mL). The organic solution was dried over MgSO4, filtered and evaporated. Conversion of ketone 65a to its corresponding silyl enol ether appeared to be quantitative, and the silyl enol ether was used without further purification in the next step.
1H NMR (300 MHz, CDCl3): δ (ppm) 0.00 (s, 9H); 0.75 (s, 3H); 0.87 (s, 3H); 1.03 (s, 3H); 1.41–1.45 (m, 2H); 1.56–1.61 (m, 1H); 1.92–1.98 (m, 1H); 2.21–2.29 (m, 1H); 2.51–2.67 (m, 2H); 3.54 (s, 3H); 4.51–4.54 (m, 1H); 6.42–6.45 (m, 1H); 6.56–6.57 (m, 1H); 6.71–6.74 (m, 1H).
To a solution of the silyl enol ether above (100 mol % 0.23 mmole; 80 mg) in acetonitrile (4 mL) was added palladium (II) acetate (200 mol %; 0.47 mmole; 105 mg). The resultant reaction mixture was stirred at reflux for 1 hr. Palladium (II) acetate (100 mol %; 0.23 mmole; 105 mg) was added, and the reaction mixture was stirred at reflux for 30 min. The reaction mixture was cooled to room temperature (20° C.) and stirred for 11.5 hrs, then stirred at reflux for 1 hr. The reaction mixture was then cooled to room temperature (20° C.), filtered and evaporated. Crude enone 56a was purified via column chromatography over silica gel using a gradient of 0–4% EtOAc/Hexanes to provide Compound 56a (40 mg; 63%) and Compound 56b (40 mg; 65% starting from 0.23 mmole of 65b).
Synthesis of Compound 50a and Compound 50b
Compound 50a and Compound 50b were made from Compound 56a and 56b, respectively, according to the procedure, above, used to make Compound 50 from Compound 56. Enantiomeric excess of Compound 50a and Compound 50b was measured using HPLC analysis with a chiral column (Chiralcel OD-RH, 4.6×150 mm): mobile phase A=100 mM KPF6 in water (pH2), mobile phase B=100% acetonitrile, isocratic 40% acetonitrile 60% water at 0.5 mL/min over 60 min, column temperature 30° C., on a Waters Alliance HPLC System 2795 using a PDA detector (200 to 600 nm). Retention time (Rt) for Compound 50a (R enantiomer)=67.25 min 95% ee. Retention time (Rt) for Compound 50b (S enantiomer)=70.92 min 93% ee.
Synthesis of Compound 66, 67, 68 and 69
Compounds 66, 67, 68 and 69 were synthesized according to the method used to synthesize Compound 50, except:
for Compound 66, ethyl iodide was used instead of iodomethane;
for Compound 67, propenyl iodide was used instead of iodomethane;
for Compound 68, benzyl iodide was used instead of iodomethane; and
for Compound 69, CH3CH2C(O)C(═CH2)C(O)OCH3 was used instead of ethyl vinyl ketone.
Synthesis of Compound 70
Compound 70 was synthesized according to the method used to synthesize Compound 50, except that twice as much chromium(VI) oxide was used and that the corresponding oxidation was performed at 80° C.
6.4 Compound 48 Selectively Induces Apoptosis 
The ability of Compound 48 to selectively induce apoptosis in a variety of cancer cells is described below.
A wide range of anticancer agents, including chemotherapeutic agents, induces apoptosis in malignant cells in vitro. Without being bound by theory, apoptosis is a regulated process manifested by the activation of proteolytic cleavages resulting mostly from the action of a unique family of cysteine-proteases called caspases. To demonstrate the ability of Compound 48 to trigger caspase activation, lysates of cells treated with various concentrations of the Compound 48 were prepared. In particular, H1299 non-small cell lung carcinoma cells, C33A cervical carcinoma cells, Mrc-5 normal lung fibroblasts (American Type Culture Collection, Manassas, Va. USA) and HMEC normal mammary epithelial cells (Clonetics San Diego, Calif., USA) were maintained in RPMI 1640 media supplemented with 10% fetal calf serum (Sigma-Aldrich Inc., St. Louis, Mo. USA). Cells were harvested and suspended at 0.6×106 cells/mL in media. A 45 μL aliquot of cell-suspension was added to each well of a 96-well microtiter plate (PerkinElmer Life Sciences Inc, Boston, Mass. USA). Cells were incubated overnight in a 5% CO2, 95% humidity incubator at 37° C. and then, 5 μL of a 10% dimethyl sulfoxide (Sigma-Aldrich Inc., St. Louis, Mo. USA) solution containing various concentrations of Compound 48 or 5 μL of 10% dimethyl sulfoxide (solvent control) was added. The plates were further incubated for 16 hr. Cells were lysed in lysis buffer (50 mM Hepes pH 7.4; 0.1% Chaps; 10 mM EDTA; 10 mM DTT) and set aside for the caspase activity assay.
To demonstrate the caspase activity in the cell lysates, 0.35 μg of N-terminal biotinylated EGKRKGDEVDGVPDRRASV (SEQ ID NO: 1) peptide (Phoenix Pharmaceuticals Inc, Belmont, Calif. USA) was labeled with 1 mCi of 32P-γATP (PerkinElmer Life Sciences mc, Boston, Mass. USA) using 250 units of Protein Kinase A catalytic subunit from bovine heart (Sigma-Aldrich Inc., St. Louis, Mo. USA) in 500 μL of HMK buffer (20 mM pH 7.5 Tris-HCl; 0.1 M NaCl; 12 mM MgCl2; 1 mM DTT) at 37° C. for one hour. The reaction was then filtered using Sephadex G-10 Poly-Prep chromatography column (Amersham Biosciences, mc, Piscataway, N.J., USA). The labeled peptide was coupled to 1.25 mL of streptavidin sepharose beads (Amersham Biosciences, mc, Piscataway, N.J., USA) during 15 minutes at room temperature on a rotary mixer. The beads were washed seven times with 6 mL of 0.5M NaCl in PBS and resuspended in a total volume of 7.25 mL of 0.5 M NaCl PBS solution to which 9 mL of RPMI 1640 media was added. 96-well 0.45 μm MultiScreen-HV filter plates (Millipore, Bedford, Mass. USA) were then prewetted with 200 μl of 0.5M NaCl in PBS and 40 μL of beads suspension was added to each well. Each well was washed five times with 200 μL of 0.5 M NaCl in PBS. In each well, 50 μ1 of cell lysate was added together with 12.5 μl of 0.5 M NaCl in 30% glycerol solution to each well. The plates were incubated at 30C with shaking at 220 rpm overnight. On the next day, the filter plates containing the beads and the extract were placed on top of 96-well sample plates (PerkinElmer Life Sciences Inc, Boston, Mass. USA) containing 100 μL of Optiphase SuperMix liquid scintillant fluid (PerkinElmer Life Sciences Inc, Boston, Mass. USA) in each well and centrifuged at 1500 rpm for 10 minutes at room temperature. The number of radioactive counts per minute (cpm) in each well of the sample plate was measured using a liquid scintillation counter (PerkinElmer Life Sciences Inc, Boston, Mass. USA). The potency of caspase cascade activation was determined by the percentage increase in cpm in wells compared to cells treated with dimethyl sulfoxide only. Values two fold higher (200%) than control were considered positive and demonstrated that the compound triggered caspase activation in the cells.
As depicted in FIG. 2, results showed that caspases were activated in the cancer cell lines H1299 and C33A and not in the normal cell line following 16 hours incubation with 1.6 μM of Compound 48. These results demonstrate that Compound 48 induces apoptosis selectively in cancer cells and is useful for treating or preventing cancer, particularly lung or cervical cancer.
6.5 Compound 48 Selectively Affects Cancer-Cell Viability 
To demonstrate the effect of Compound 48 on cell viability, cellular ATP levels were measured following Compound 48 treatment. H1299 non-small cell lung carcinoma cells, C33A cervical carcinoma cells, Mrc-5 normal lung fibroblasts (American Type Culture Collection, Manassas, Va. USA) and HMEC normal mammary epithelial cells (Clonetics San Diego, Calif., USA) were cultured in RPMI 1640 media supplemented with 10% fetal calf serum (Sigma-Aldrich Inc., St. Louis, Mo. USA). The four cells lines were plated in 96-well microtiter plates (PerkinElmer Life Sciences Inc, Boston, Mass. USA) at a confluency that allowed them to reach confluence after 4 days of growth. One day after plating, the cells were treated either with 10 μM etoposide, 100 nM staurosporine or 1.6 μM of Compound 48. Stock solutions of each compound were prepared in dimethyl sulfoxide (Sigma-Aldrich Inc., St. Louis, Mo. USA), diluted in media then added to the cells. The total dimethyl sulfoxide on the cells was 1%. After 3 days of incubation, the ATP levels in the cells were quantified using the luminescent ViaLight detection system (Bio-Whittaker, MD, USA). The results were plotted relative to untreated control cells, which were set at 100.
As depicted in FIG. 3, Compound 48 has a significantly greater effect on the ATP levels in cancer cells than in normal cells. After 72 hours, treatment with 1.6 μM Compound 48 was significantly more effective at lowering ATP levels in cells of the cancer cell line H1299 and C33A compared with cells of the lines HMEC and MRC-5. Control compounds were tested to illustrate the significance of this effect. The anti-cancer drug etoposide as well as Compound 48, but not the protein kinase inhibitor staurosporine, are selectively cytotoxic towards cancer cells, particularly lung and cervical cancer. These results demonstrate that Compound 48 is selectively cytotoxic to cancer cells and is useful for treating or preventing cancer, particularly lung or cervical cancer.
6.6 Anti-Oncogenic Effects of Compound 50
6.6.1 Effects of Compound 50A and Compound  50B on Cancer Cell Viability In Vitro 
To demonstrate the effect of Compounds 50a and 50b, respectively, on cell viability, cellular ATP levels were measured before and after treating cell lines with each compound. The following cell lines were used: A549 human non-small cell lung carcinoma; C33A human cervical carcinoma; H1299 human non-small cell lung carcinoma; Hep-3B human hepatocellular carcinoma; MIA-Paca-2 human pancreatic carcinoma; PC-3 human prostatic adenocarcinoma (American Type Culture Collection, Manassas, Va., USA); HMEC normal human mammary epithelial cells; HUVEC normal human umbilical vein endothelial cells; and NHBE normal human bronchial epithelial cells (Cambrex Bio Science, Rockland, Me., USA). All tumor cell lines were cultured in RPMI 1640 media supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, Vt., USA), 100 U/mL penicillin, 100 μg/mL streptomycin and 2 mM L-glutamine (Invitrogen, Carlsbad, Calif., USA). HMEC, HUVEC and NHBE cell lines were cultured in MEGM, EGM-2 and BEGM media, respectively (Cambrex Bio Science, Rockland, Me., USA). The cell lines were plated in 96-well microtitre plate at a density of 2–4×103 cells per well. After 16 hours, the cells were treated with various concentrations of Compound 50a or Compound 50b. Stock solutions of Compound 50a and Compound 50b, respectively, were prepared in dimethyl sulfoxide at a concentration of 5 mM. Serial dilutions were prepared in RPMI, 10% FBS and 2% DMSO and then added to the cells. The total DMSO in the cell cultures was 1%. After 72 hours of incubation, the ATP levels in the cells were quantified using the Vialight HS kit (Cambrex Bio Science, Rockland, Me., USA). The results were plotted relative to untreated control cells, which were set at a value of 100. The IC50s were determined using a best-fit signioidal dose response curve with variable slope.
As depicted in Table 5, Compound 50a and Compound 50b showed greater efficacy in decreasing cellular ATP levels in the cancer cell lines than in normal cell lines. These results demonstrate that Compound 50a and Compound 50b are selectively cytotoxic to cancer cells and are useful for treating or preventing cancer, particularly NSCL-LC carcinoma, NSCL-adrenocarcinoma, liver cancer, pancreatic cancer, cervial cancer, prostate cancer or lung cancer.







TABLE 5







Anti-oncogenic effects of Compound 50a and Compound 50b
















IC50 of







Com-
IC50 of






pound
Compound






50a
50b



Cell line
Tissue type
Species
(μM)
(μM)





Tumor
H1299
NSCL-LC
Human
0.550
0.230




carcinoma



A549
NSCL-
Human
0.630
0.580




adrenocarinoma



Hep 3B
Liver
Human
0.970
0.770



MIA-
Pancreas
Human
0.660
0.250



Paca-2



C33A
Cervix
Human
0.500
0.450



PC-3
Prostate
Human
0.370
0.350


Normal
HMEC
Breast epithelial
Human
2.300
2.100



HUVEC
Endothelial
Human
1.700
1.300



NHBE
Bronchial
Human
1.200
1.200




epithelial





*Measurement of ATP levels were taken 72 h post-treatment and compared to untreated cells.






6.6.2 Effect of Compound 50 on Growth  of Cervical Tumor Cells In Vivo 
To demonstrate the anti-tumor activity of Compounds 50 in vivo, CB17 SCID/SCUD female mice (Charles River, Mass., USA) that were injected with C33A human cervical cancer cells were used. The resultant mice are a model for a human having cervical cancer.
The C33A human cervical cancer cells were maintained in RPMI supplemented with 10% inactivated fetal bovine serum and 1% penicillin-streptomycin-L-Glutamine, under 5% CO2 at 37° C., and passaged twice a week. The cells were grown at a confluency lower than 70% and then collected with Trypsin (Bio-Whittaker, MD, USA). The cells were then centrifuged and washed twice using phosphate buffered saline solution (PBS) and resuspended in PBS at 2×106 cells per 100 μl. Viability was examined by staining with Trypan Blue and only flasks with cell viability of greater than 95% were used for in vivo studies. 
C33A cells were transplanted subcutaneously into the flank of female CB17 SCID/SCID mice. Each mouse was inoculated with a suspension of 2×106 tumor cells per 100 μL on day zero. The following six treatment groups of ten mice each were used: (a) a negative control group treated with intravenous (i.v.) injection of vehicle, (b) a negative control group treated with intraperitoneal (i.p.) injection of vehicle, (c) a positive control group treated with i.p. injection of Cisplatin, (d) a group treated with i.v. injection of a Compound 50, (e) a group treated with i.p. injection of Compound 50, and (f) a group treated with subcutaneous (s.c.) injection of Compound 50.
Treatment started on day thirteen after C33A cells transplantation for Compound 50 treated groups and on day fourteen for Cisplatin treated group. Compound 50 was administered i.v., i.p., and s.c. once daily for five consecutive days at a dose of 10, 15 and 30 mg/kg, respectively. Compound 50 was prepared as a working solution of 1.5 mg/mL in a vehicle solution of 20% Cremaphor EL (Sigma, St. Louis, Mo., USA), 10% Ethanol and 5% Dextrose (Abbot Laboratories, QC, Canada). The negative control groups were treated with vehicle alone. The positive control group was treated once every 3 days for fifteen days at a dose of 3.5 mg/kg. Cisplatin was formulated in PBS on each day of the injection and was administered i.p.
The mice were weighed and the tumors measured on day 13 and every 2 to 3 days after treatment commenced. Observations continued for 36 days after initial tumor implantation. The changes in body weight and in the calculated tumor volume were plotted (FIGS. 4 and 5). Statistical analysis was performed using GraphPad Prism (GraphPad Software Inc., San Diego, Calif.). Two-way ANOVA was used to determine how the treatment affected tumor growth over time. Following the two-way ANOVA, post-tests were performed using the Benferroni method to determine the statistical difference between the mean tumor-size of the two groups being compared on every day that the tumors were measured. Four animals in the negative control injected i.v. with vehicle and two animals in the group treated i.v. with Compound 50 died during the observation period. Therefore, the data collected from these animals were excluded from all calculations and the total number of animals in the groups changed to six for the negative control injected i.v. with vehicle and eight for the group treated i.v. with Compound 50.
As shown in FIG. 4, mice treated with Compound 50 experienced a non-significant change in body weight, whereas Cisplatin treated positive control group experienced a weight loss of 19% on day 34.
As shown in FIG. 5, Compound 50 administered i.v. or i.p. at a dose of 10 or 15 mg/kg, respectively, once a day for five days resulted in a significant reduction (p<0.001) in tumor growth compared to mice treated i.v. with vehicle only. On day 36, animals treated i.v. with 10 mg/kg of Compound 50 had significantly (p<0.05) smaller mean tumor-size than animals treated with vehicle only. Similarly, animals treated i.p. with Compound 50 had a significantly (p<0.05) smaller mean tumor-size than animals treated with vehicle on day 34 and day 36. Furthermore, Compound 50 administered s.c. at a dose of 30 mg/kg once a day for five days resulted in a significant reduction (p<0.001) in tumor growth compared to mice treated s.c. with vehicle alone. Animals treated s.c. with Compound 50 had a significantly smaller mean tumor-size than animals treated with vehicle on day 34 (p<0.01) and day 36 (p<0.001).
As indicated in FIG. 5, Compound 50 significantly reduces the human cervical tumors implanted in SCID mice, an art-accepted model for human cervical cancer. Accordingly, Compound 50 is useful for inhibiting the growth of a cancer cell, particularly a cervical cancer cell, and for treating or or preventing cancer, particularly cervical cancer, in a patient.
6.7 Anti-Fungal Biological Activity of Compound 50
6.7.1 Effect of Compound 50 on the Growth  of the Yeast Saccharomyces cerevisiae  
To demonstrate that Compound 50 inhibits the growth of the yeast Saccharomyces cerevisiae, the strain W303a was cultured in YEP media, containing Bacto Yeast Extract and Bacto Peptone (Becton Dickinson Microbiology Systems, Sparks, Md., USA) and supplemented with one of three different carbon sources, 2% glucose (Sigma Chemical Co. St. Louis, Mo., USA), 2% galactose (BDH Laboratory Supplies, Poole, England), or 2% glycerol (EM Science, Gibbstown, N.J., USA). Yeast cells were first cultured overnight in 10 mL of YEP glucose-containing medium at 30° C. The following day, 5 μL of the overnight culture (5–6×103 cells) was added to 100 μL of YEP media supplemented with 2% of one of the three carbon sources mentioned above in a 96-well microtitre plate. To these cultures a dilution series of Compound 50 was added. Compound 50 was prepared as a stock solution in 100% dimethyl sulfoxide (DMSO) at a concentration of 10 mM. Serial dilutions were prepared in distilled water. The final DMSO concentration in each well was 0.5%. Yeast cells were grown at 30° C. and cell growth was monitored by reading the OD600 (optical density at 600 nm) with a Tecan ULTRA plate reader (TECAN U.S. Inc., Research Triangle Park, N.C., USA). As depicted in FIG. 6, Compound 50 significantly inhibited growth of the yeast Saccharomyces cerevisiae in dose-dependent manner. Accordingly, Compound 50, an illustrative Diterpenoid Compound, is useful for inhibiting the growth of a fungus or treating a fungal infection.
6.7.2 Effect of Compound 50 on Growth of the Fungus Candida  albicans  
To demonstrate that Compound 50 inhibits the growth of the fungus Candida albicans, the strain ATCC 10231 was cultured in YEP media, containing Bacto Yeast Extract and Bacto Peptone (Becton Dickinson Microbiology Systems, Sparks, Md., USA) and supplemented with 2% glucose (Sigma Chemical Co. St. Louis, Mo., USA). Fungus cells were first cultured overnight in 10 mL of YEP glucose-containing medium at 30° C. The following day, 5 μL of the overnight culture (5–6×103 cells) was added to 100 μL of YEP glucose-containing medium in a 96-well microtitre plate. To these cultures a dilution series of Compound 50 was added. Compound 50 was prepared as a stock solution in 100% dimethyl sulfoxide (DMSO) at a concentration of 10 mM. Serial dilutions were prepared in distilled water. The final DMSO concentration in each well was 0.5%. Fungus cells were grown at 30° C. and cell growth was monitored by reading the OD600 (optical density at 600 nm) with a Tecan Ultra plate reader (TECAN U.S. Inc. Research Triangle Park, N.C., USA). As depicted in FIG. 7, Compound 50 significantly inhibited growth of the fungus Candida albicans in a dose-dependent manner. Accordingly, Compound 50, an illustrative Diterpenoid Compound, is useful for inhibiting the growth of a fungus or treating a fungal infection.
6.8 Biological Activity of Compounds 66, 67, 68, 69 and 70
6.8.1 Effects of Compounds 66, 67, 68, 69 and 70 on Cancer Cell Viability In Vitro 
To demonstrate the effect of compounds 66, 67, 68, 69 and 70 on cell viability, cellular ATP levels were measured before and after treating cell lines with each compound. The selected tumor cell lines included C33A human cervical carcinoma and H1299 human non-small cell lung carcinoma (American Type Culture Collection, Manassas, Va., USA). The cell lines were cultured in RPMI 1640 media supplemented with 10% FBS (Hyclone, Logan, Vt., USA), 100 U/mL penicillin, 100 μg/mL streptomycin and 2 mM L-glutamine (Invitrogen, Carlsbad, Calif., USA). The cell lines were plated in 96-well microtitre plate at a density of 2–4×103 cells per well. After 16 hours, the cells were treated with various concentrations of compound 66, 67, 68, 69 and 70, respectively. Stock solutions of compounds 66, 67, 68, 69 and 70 were prepared in dimethyl sulfoxide at a concentration of 5 mM. Serial dilutions were prepared in RPMI, 10% FBS and 2% DMSO and then added to the cells. The total DMSO on the cells was 1%. After 72 hours of incubation the ATP levels in the cells were quantified using Vialight HS kit (Cambrex Bio Science, Rockland, Me., USA). The results were plotted relative to untreated control cells, which were set at a value of 100. The IC50s were determined using a best-fit sigmoidal dose response curve with variable slope.
As depicted in Table 6, treatment with Compound 66, 67, 68, 69 or 70 decreased the cellular ATP levels in the cancer cell lines. These results demonstrate that Compounds 66, 67, 68, 69 and 70 inhibit the growth of a cancer cell, particularly a C33A human cervical carcinoma cell and H1299 human non-small cell lung carcinoma cell, and are useful for treating or preventing cancer, particularly human cervical carcinoma and human non-small cell lung carcinoma, in a patient.







TABLE 6







The IC50s in μM of Compounds 66, 67, 68, 69


and 70 for anti-oncogenic effects*.












Compound
Formula
H1299
C33A
















66
I
1.440
1.550



67
I
0.950
1.190



68
I
2.070
2.530



69
I
1.360
0.870



70
III
16.810
15.890







*Measurement of ATP levels were taken 72 h post-treatment and compared to untreated cells.






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 that are functionally equivalent are within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art and are intended to fall within the scope of the appended claims.
A number of references have been cited, the entire disclosures of which have been incorporated herein in their entirety.

## Claims

1. A compound having the Formula (I):




or a pharmaceutically acceptable salt thereof, wherein:
Q1 and Q2 are independently ═O, ═S, ═NH or ═N—NHR, where R is —H, —C1–C10 alkyl, or -aryl;
Q3 is —O—, —S—, or —N(H)—;
R1 and R2 are independently —H, -halogen, -amino, —C1–C10 alkyl, —C1–C10 alkoxy, —C1–C10 (hydroxy)alkyl, —C1–C10 (amino)alkyl, —C1–C10 (halo)alkyl, —C2–C10 alkenyl, —C2–C10 alkynyl, (C3–C7)cycloalkyl, -aryl, C1–C10 (aryl)alkyl, or three- to seven-membered non-aromatic heterocycle, or R1, R2 and the carbon atom to which they are both attached are taken together to form a (C3–C7)cycloalkyl group or a three- to seven-membered non-aromatic heterocycle;
A is CR3; B is CR4; D is CR5; E is CR6;
each R3, R4, R5 and R6 is independently —H, -halogen, —CN, —NH2, —NO2, —COOH, —C(O)NH2, —SH, —S(O)NH2, —S(O)2NH2, —C1–C10 (oxy)alkyl, —C1—C10 alkyl, —C1–C10 alkoxy, —C1–C10 (hydroxy)alkyl, —C1–C10 (amino)alkyl, —C1–C10 (halo)alkyl, —C2–C10 alkenyl, —C2–C10 alkynyl, —(C3–C7)cycloalkyl, -aryl, —C1–C10 (aryl)alkyl, three- to seven-membered non-aromatic heterocycle, five- to seven-membered aromatic heterocycle, —CH2OR11, —OCH2OR11, —OC(O)R11, —C(O)R11, —OC(O)OR11, —OC(O)NR11, —C(O)OR11, —C(O)NR11, —OP(O)(OR11)2, —SR11, —S(O)2NHR11, —SOR11, —S(O)2R11, —NHC(O)R11, —NHSOR11, or NHS(O)2R11; or
R3 and R4 and the carbon atoms to which they are attached are taken together to form a (C3–C7)cycloalkenyl group, a five- to seven-membered non-aromatic heterocycle, or a five- to seven-membered aromatic heterocycle; or
R5 and R6 and the carbon atoms to which they are attached are taken together to form a (C3–C7)cycloalkenyl group, a five- to seven-membered non-aromatic heterocycle, or a five- to seven-membered aromatic heterocycle; or
R4 and R5 and the carbon atoms to which they are attached are taken together to form a (C3–C7)cycloalkenyl group, a non-oxygen-containing five-membered non-aromatic heterocycle, a non-oxygen-containing five-membered aromatic heterocycle, a six- to seven-membered non-aromatic heterocycle or a six- to seven-membered aromatic heterocycle;
R7 is —H, —C1–C10 alkyl, or —C1–C10 alkoxy;
R8 and R9 are each independently —H, -halogen, —CN, —NH2, —NO2, —COOH, —C(O)NH2, —SH, —S(O)NH2, —S(O)2NH2, —C1–C10 (oxy)alkyl, —C1–C10 alkyl, —C1–C10 alkoxy, —C1–C10 (hydroxy)alkyl, —C1–C10 (amino)alkyl, —C1–C10 (halo)alkyl, —C2–C10 alkenyl, —C2–C10 alkynyl, —(C3–C7)cycloalkyl, -aryl, —C1–C10 (aryl)alkyl, three- to seven-membered non-aromatic heterocycle, five- to seven-membered aromatic heterocycle, —CH2OR11, —OCR11, —OC(O)R11, —C(O)R11, —OC(O)OR11, —OC(O)NR11, —C(O)OR11, —C(O)NR11, —OP(O)(OR11)2, —SR11, —SOR11, —S(O)2R11, —S(O)2NHR11, —NHSR11, —NHSOR11, or —NHS(O)2R11;
R10 is —H, —C1–C10 alkyl, —C3–C7 cycloalkyl, —C(O)C1–C10 alkyl, —C1–C10 (oxy)alkyl, —C(O)NH2, —C(O)NHR12, or -aryl;
R11 is —H, —C1–C10 alkyl, —(C3–C7)cycloalkyl, —C1–C10 (halo)alkyl, -aryl, —C2–C10 alkenyl, —C2–C10 alkynyl, —C1–C10 (aryl)alkyl, —C2–C10 (aryl)alkenyl, —C2–C10 (aryl)alkynyl, —C1–C10 (hydroxy)alkyl, —C1–C10 alkoxy, —C1–C10 (amino)alkyl,
a —(C3–C7)cycloalkyl unsubstituted or substituted with one or more —C1–C10 alkyl,
a three- to seven-membered non-aromatic heterocycle unsubstituted or substituted with one or more —C1–C10 alkyl, or
a three- to seven-membered aromatic heterocycle unsubstituted or substituted with one or more —C1–C10 alkyl, —C2–C10 alkenyl, or —C2–C10 alkynyl;
R12 is C1–C10 alkyl; and
each halogen is independently —F, —Cl, —Br or —I;

with the proviso that the compound of Formula (I) is not:



10-Hydroxy-6-methoxy-1,1,4a,7-tetramethyl-1H,4aH-phenanthrene-2,9-dione or




10-Hydroxy-7-hydroxymethyl-6-methoxy-1,1,4a-trimethyl-1H,4aH-phenanthrene-2,9-dione, or a pharmaceutically acceptable salt thereof.

2. A compound of the formula:




or a pharmaceutically acceptable salt thereof, the compound or pharmaceutically acceptable salt thereof being in isolated and purified form.

3. A compound of the formula:




or a pharmaceutically acceptable salt thereof, the compound or pharmaceutically acceptable salt thereof being in isolated and purified form.

4. The compound or pharmaceutically acceptable salt of the compound of claim 1, wherein
Q1 and Q2 and Q3 are oxygen;
R1 and R2 are C1–C10 alkyl;
R8 and R9 are H;
R7 is C1–C10 alkyl;
R3 and R6 are H; and
R4 and R5 are independently C1–C10 alkyl, C1–C10 alkoxy, or C1–C10 (hydroxy)alkyl.

5. A compound having the structure:

6. The compound of claim 1, having the structure:







or a pharmaceutically acceptable salt thereof.

7. A compound having the Formula (II):




or a pharmaceutically acceptable salt thereof, wherein:
Q1 and Q2 are independently ═O, ═S, ═NH or ═N—NHR, where R is —H, —C1–C10 alkyl, or -aryl;
Q3 is —O—, —S—, or —N(H)—;
R1 and R2 are independently —H, -halogen, -amino, —C1–C10 alkyl, —C1–C10 alkoxy, —C1–C10 (hydroxy)alkyl, —C1–C10 (amino)alkyl, —C1–C10 (halo)alkyl, —C2—C10 alkenyl, —C2–C10 alkynyl, (C3–C7)cycloalkyl, -aryl, C1–C10 (aryl)alkyl, or three- to seven-membered non-aromatic heterocycle, or R1, R2 and the carbon atom to which they are both attached are taken together to form a (C3–C7)cycloalkyl group or a three- to seven-membered non-aromatic heterocycle;
A is CR3; B is CR4; D is CR5; E is CR6;
each R3, R4, R5 and R6 is independently —H, -halogen, —CN, —NH2, —NO2, —COOH, —C(O)NH2, —SH, —S(O)NH2, —S(O)2NH2, —C1–C10 (oxy)alkyl, —C1–C10 alkyl, —C1–C10 C1–C10 (hydroxy)alkyl, —C1–C10 (amino)alkyl, —C1–C10 (halo)alkyl, —C2–C10 alkenyl, —C2–C10 alkynyl, —(C3–C7)cycloalkyl, -aryl, —C1–C10 (aryl)alkyl, three- to seven-membered non-aromatic heterocycle, five- to seven-membered aromatic heterocycle, —CH2OR11, —OCH2OR11, —OC(O)R11, —C(O)R11, —OC(O)OR11, —OC(O)NR11, —C(O)OR11, —OP(O)(OR11)2, —SR11, —S(O)2NHR11, —SOR11, —S(O)2R11, —NHC(O)R11, —NHSOR11, or NHS(O)2R11; or
R3 and R4 and the carbon atoms to which they are attached are taken together to form a (C3–C7)cycloalkenyl group, a five- to seven-membered non-aromatic heterocycle, or a five- to seven-membered aromatic heterocycle; or
R5 and R6 and the carbon atoms to which they are attached are taken together to form a (C3–C7)cycloalkenyl group, a five- to seven-membered non-aromatic heterocycle, or a five- to seven-membered aromatic heterocycle; or
R4 and R5 and the carbon atoms to which they are attached are taken together to form a (C3–C7)cycloalkenyl group, a non-oxygen-containing five-membered non-aromatic heterocycle, a non-oxygen-containing five-membered aromatic heterocycle, a six- to seven-membered non-aromatic heterocycle or a six- to seven-membered aromatic heterocycle;
R7 is —H, —C1–C10 alkyl, or —C1–C10 alkoxy;
R8 and R9 are each independently —H, -halogen, —CN, —NH2, —NO2, —COOH, —C(O)NH2, —SH, —S(O)NH2, —S(O)2NH2, —C1–C10 (oxy)alkyl, —C1–C10 alkyl, —C1–C10 alkoxy, —C1–C10 (hydroxy)alkyl, —C1–C10 (amino)alkyl, —C1–C10 (halo)alkyl, —C2–C10 alkenyl, —C2–C10 alkynyl, —(C3–C7)cycloalkyl, -aryl, —C1–C10 (aryl)alkyl, three- to seven-membered non-aromatic heterocycle, five- to seven-membered aromatic heterocycle, —CH2OR11, —OCR11, —OC(O)R11, —C(O)R11, —OC(O)OR11, —OC(O)NR11, —C(O)OR11, —C(O)NR11, —OP(O)(OR11)2, —SR11, —SOR11, —S(O)2R11, —S(O)2NHR11, —NHSR11, —NHSOR11, or —NHS(O)2R11;
R10 is —H, —C1–C10 alkyl, —C3–C7, cycloalkyl, —C(O)C1–C10 alkyl, —C1–C10 (oxy)alkyl, —C(O)NH2, —C(O)NHR12, or -aryl;
R11 is —H, —C1–C10 alkyl, —(C3–C7)cycloalkyl, —C1–C10 (halo)alkyl, -aryl, —C2–C10 alkenyl, —C2–C10 alkynyl, —C1–C10 (aryl)alkyl, —C2–C10 (aryl)alkenyl, —C2–C10 (aryl)alkynyl, —C1–C10 (hydroxy)alkyl, —C1–C10 alkoxy, —C1–C10 (amino)alkyl,
a —(C3–C7)cycloalkyl unsubstituted or substituted with one or more —C1–C10 alkyl,
a three- to seven-membered non-aromatic heterocycle unsubstituted or substituted with one or more —C1–C10 alkyl, or
a three- to seven-membered aromatic heterocycle unsubstituted or substituted with one or more —C1–C10 alkyl, —C2–C10 alkenyl, or —C2–C10 alkynyl;
R12 is C1–C10 alkyl; and
each halogen is independently —F, —Cl, —Br or —I.

8. A compound having the Formula (III):




or a pharmaceutically acceptable salt thereof, wherein:
Q1, Q2 and Q3 are independently ═O, ═S, ═NH or ═N—NHR, where R is —H, —C1–C10 alkyl, or -aryl;
R1 and R2 are independently —H, -halogen, -amino, —C1–C10 alkyl, —C1–C10 alkoxy, —C1–C10 (hydroxy)alkyl, —C1–C10 (amino)alkyl, —C1–C10 (halo)alkyl, —C2–C10 alkenyl, —C2–C10 alkynyl, (C3–C7)cycloalkyl, -aryl, C1–C10 (aryl)alkyl, or three- to seven-membered non-aromatic heterocycle, or R1, R2 and the carbon atom to which they are both attached are taken together to form a (C3–C7)cycloalkyl group or a three- to seven-membered non-aromatic heterocycle;
A is CR3; B is CR4; D is CR5; E is CR6;
each R3, R4, R5 and R6 is independently —H, -halogen, —CN, —NH2, —NO2, —COOH, —C(O)NH2, —SH, —S(O)NH2, —S(O)2NH2, —C1–C10 (oxy)alkyl, —C1–C10 alkyl, —C1–C10 alkoxy, —C1–C10 (hydroxy)alkyl, —C1–C10 (amino)alkyl, —C1–C10 (halo)alkyl, —C2–C10 alkenyl, —C2–C10 alkynyl, —(C3–C7)cycloalkyl, -aryl, —C1–C10 (aryl)alkyl, three- to seven-membered non-aromatic heterocycle, five- to seven-membered aromatic heterocycle, —CH2OR11, —OCH2OR11, —OC(O)R11, —C(O)R11, —OC(O)OR11, —OC(O)NR11, —C(O)OR11, —C(O)NR11, —OP(O)(OR11)2, —SR11, —S(O)2NHR11, —SOR11, —S(O)2R11, —NHC(O)R11, —NHSOR11, or NHS(O)2R11; or
R3 and R4 and the carbon atoms to which they are attached are taken together to form a (C3–C7)cycloalkenyl group, a five- to seven-membered non-aromatic heterocycle, or a five- to seven-membered aromatic heterocycle; or
R5 and R6 and the carbon atoms to which they are attached are taken together to form a (C3–C7)cycloalkenyl group, a five- to seven-membered non-aromatic heterocycle, or a five- to seven-membered aromatic heterocycle; or
R4 and R5 and the carbon atoms to which they are attached are taken together to form a (C3–C7)cycloalkenyl group, a non-oxygen-containing five-membered non-aromatic heterocycle, a non-oxygen-containing five-membered aromatic heterocycle, a six- to seven-membered non-aromatic heterocycle or a six- to seven-membered aromatic heterocycle;
R7 is —H, —C1–C10 alkyl, or —C1–C10 alkoxy;
R8 and R9 are each independently —H, -halogen, —CN, —NH2, —NO2, —COOH, —C(O)NH2, —SH, —S(O)NH2, —S(O)2NH2, —C1–C10 (oxy)alkyl, —C1–C10 alkyl, —C1–C10 alkoxy, —C1–C10 (hydroxy)alkyl, —C1–C10 (amino)alkyl, —C1–C10 (halo)alkyl, —C2–C10 alkenyl, —C2–C10 alkynyl, —(C3–C7)cycloalkyl, -aryl, —C1–C10 (aryl)alkyl, three- to seven-membered non-aromatic heterocycle, five- to seven-membered aromatic heterocycle, —CH2OR11, —OCR11, —OC(O)R11, —C(O)R11, —OC(O)OR11, —OC(O)NR11, —C(O)OR11, —C(O)NR11, —OP(O)(OR11)2, —SR11, —SOR11, —S(O)2R11, —S(O)2NHR11, —NHSR11, —NHSOR11, or —NHS(O)2R11;
R11 is —H, —C1–C10 alkyl, —(C3–C7)cycloalkyl, —C1–C10 (halo)alkyl, -aryl, —C2–C10 alkenyl, —C2–C10 alkynyl, —C1–C10 (aryl)alkyl, —C2–C10 (aryl)alkenyl, —C2–C10 (aryl)alkynyl, —C1–C10 (hydroxy)alkyl, —C1–C10 alkoxy, —C1–C10 (amino)alkyl,
a —(C3–C7)cycloalkyl unsubstituted or substituted with one or more —C1–C10 alkyl,
a three- to seven-membered non-aromatic heterocycle unsubstituted or substituted with one or more —C1–C10 alkyl, or
a three- to seven-membered aromatic heterocycle unsubstituted or substituted with one or more —C1–C10 alkyl, —C2–C10 alkenyl, or —C2–C10 alkynyl;
R12 is C1–C10 alkyl; and
each halogen is independently —F, —Cl, —Br or —I.

9. The compound of claim 8 having the formula:



or a pharmaceutically acceptable salt thereof.

10. A compound having the Formula (IV):




or a pharmaceutically acceptable salt thereof, wherein:
Q1, Q2 and Q3 are independently ═O, ═S, ═NH or ═N—NHR, where R is —H, —C1–C10 alkyl, or -aryl;
R1 and R2 are independently —H, -halogen, -amino, —C1–C10 alkyl, —C1–C10 alkoxy, —C1–C10 (hydroxy)alkyl, —C1–C10 (amino)alkyl, —C1–C10 (halo)alkyl, —C2–C10 alkenyl, —C2–C10 alkynyl, (C3–C7)cycloalkyl, -aryl, C1–C10 (aryl)alkyl, or three- to seven-membered non-aromatic heterocycle, or R1, R2 and the carbon atom to which they are both attached are taken together to form a (C3–C7)cycloalkyl group or a three- to seven-membered non-aromatic heterocycle;
A is CR3; B is CR4; D is CR5; E is CR6;
each R3, R4, R5 and R6 is independently —H, -halogen, —CN, —NH2, —NO2, —COCH, —C(O)NH2, —SH, —S(O)NH2, —S(O)2NH2, —C1–C10 (oxy)alkyl, —C1–C10 alkyl, —C1–C10 alkoxy, —C1–C10 (hydroxy)alkyl, —C1–C10 (amino)alkyl, —C1–C10 (halo)alkyl, —C2–C10 alkenyl, —C2–C10 alkynyl, —(C3–C7)cycloalkyl, -aryl, —C1–C10 (aryl)alkyl, three- to seven-membered non-aromatic heterocycle, five- to seven-membered aromatic heterocycle, —CH2OR11, —OCH2OR11, —OC(O)R11, —C(O)R11, —OC(O)OR11, —OC(O)NR11, —C(O)OR11, —C(O)NR11, —OP(O)(OR11)2, —SR11, —S(O)2NHR11, —SOR11, —S(O)2R11, —NHC(O)R11, —NHSOR11, or NHS(O)2R11; or
R3 and R4 and the carbon atoms to which they are attached are taken together to form a (C3–C7)cycloalkenyl group, a five- to seven-membered non-aromatic heterocycle, or a five- to seven-membered aromatic heterocycle; or
R5 and R6 and the carbon atoms to which they are attached are taken together to form a (C3–C7)cycloalkenyl group, a five- to seven-membered non-aromatic heterocycle, or a five- to seven-membered aromatic heterocycle; or
R4 and R5 and the carbon atoms to which they are attached are taken together to form a (C3–C7)cycloalkenyl group, a non-oxygen-containing five-membered non-aromatic heterocycle, a non-oxygen-containing five-membered aromatic heterocycle, a six- to seven-membered non-aromatic heterocycle or a six- to seven-membered aromatic heterocycle;
R7 is —H, —C1–C10 alkyl, or —C1–C10 alkoxy;
R8 and R9 are each independently —H, -halogen, —CN, —NH2, —NO2, —COOH, —C(O)NH2, —SH, —S(O)NH2, —S(O)2NH2, —C1–C10 (oxy)alkyl, —C1–C10 alkyl, —C1–C10 alkoxy, —C1–C10 (hydroxy)alkyl, —C1–C10 (amino)alkyl, —C1–C10 (halo)alkyl, —C2–C10 alkenyl, —C2–C10 alkynyl, —(C3–C7)cycloalkyl, -aryl, —C1–C10 (aryl)alkyl, three- to seven-membered non-aromatic heterocycle, five- to seven-membered aromatic heterocycle, —CH2OR11, —OCR11, —OC(O)R11, —C(O)R11, —OC(O)OR11, —OC(O)NR11, —C(O)OR11, —C(O)NR11, —OP(O)(OR11)2, —SR11, —SOR11, —S(O)2R11, —S(O)2NHR11, —NHSR11, —NHSOR11, or —NHS(O)2R11;
R11 is —H, —C1–C10 alkyl, —(C3–C7)cycloalkyl, —C1–C10 (halo)alkyl, -aryl, —C2–C10 alkenyl, —C2–C10 alkynyl, —C1–C10 (aryl)alkyl, —C2–C10 (aryl)alkenyl, —C2–C10 (aryl)alkynyl, —C1–C10 (hydroxy)alkyl, —C1–C10 alkoxy, —C1–C10 (amino)alkyl,
a —(C3–C7)cycloalkyl unsubstituted or substituted with one or more —C1–C10 alkyl,
a three- to seven-membered non-aromatic heterocycle unsubstituted or substituted with one or more —C1—C10 alkyl, or
a three- to seven-membered aromatic heterocycle unsubstituted or substituted with one or more —C1–C10 alkyl, —C2–C10 alkenyl, or —C2–C10 alkynyl;
R12 is C1–C10 alkyl; and
each halogen is independently —F, —Cl, —Br or —I.

11. A composition comprising an effective amount of the compound or pharmaceutically acceptable salt of the compound of claim 1 and a pharmaceutically acceptable carrier.

12. A composition comprising an effective amount of the compound or pharmaceutically acceptable salt of the compound of claim 2 and a pharmaceutically acceptable carrier.

13. A composition comprising an effective amount of the compound or pharmaceutically acceptable salt of the compound of claim 3 and a pharmaceutically acceptable carrier.

14. A composition comprising an effective amount of the compound or pharmaceutically acceptable salt of the compound of claim 7 and a pharmaceutically acceptable carrier.

15. A composition comprising an effective amount of the compound or pharmaceutically acceptable salt of the compound of claim 8 and a pharmaceutically acceptable carrier.

16. A composition comprising an effective amount of the compound or pharmaceutically acceptable salt of the compound of claim 10 and a pharmaceutically acceptable carrier.