WO2006089406A1 - Diterpenoid compounds, compositions thereof and their use as anti-cancer or anti-fungal agents - Google Patents

Diterpenoid compounds, compositions thereof and their use as anti-cancer or anti-fungal agents Download PDF

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WO2006089406A1
WO2006089406A1 PCT/CA2006/000248 CA2006000248W WO2006089406A1 WO 2006089406 A1 WO2006089406 A1 WO 2006089406A1 CA 2006000248 W CA2006000248 W CA 2006000248W WO 2006089406 A1 WO2006089406 A1 WO 2006089406A1
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alkyl
aryl
alkynyl
alkenyl
cancer
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PCT/CA2006/000248
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WO2006089406A8 (en
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Pierre Beauparlant
Giorgio Attardo
Samuel Fortin
Laurent Belec
Sasmita Tripathy
Lionel Dumas
Gerson Gonzalez
Daniel Rabouin
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Gemin X Biotechnologies Inc.
Galileo Pharmaceuticals, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D303/00—Compounds containing three-membered rings having one oxygen atom as the only ring hetero atom
    • C07D303/02—Compounds containing oxirane rings
    • C07D303/12—Compounds containing oxirane rings with hydrocarbon radicals, substituted by singly or doubly bound oxygen atoms
    • C07D303/32—Compounds containing oxirane rings with hydrocarbon radicals, substituted by singly or doubly bound oxygen atoms by aldehydo- or ketonic radicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • DITERPENOID COMPOUNDS COMPOSITIONS THEREOF AND THEIR USE AS ANTI-CANCER OR ANTI-FUNGAL AGENTS
  • 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 cytotoxicity 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.
  • 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.
  • chemotherapeutic agents there are a variety of chemotherapeutic agents available for treatment of neoplastic disease.
  • 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).
  • chemotherapeutic agents are toxic, and chemotherapy can cause significant, and often dangerous, side effects, including severe nausea, bone marrow depression, immunosuppression, etc.
  • tumor cells are resistant or develop resistance to chemotherapeutic agents through multi-drug resistance.
  • 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., Epidermoph
  • Saccharomyces as a human pathogen (e.g., Fungemia with Saccharomycetacea, H. Nielson, J. Stenderup, & B. Bruun, Scand. J. Infect. Dis. 22:581-584, 1990).
  • 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 (CC. Yeager, Fungicides in Industry, in Antifungal Compounds, M. Siegel and H. Sisler, eds., Marcel Dekker Inc., NY, 1977).
  • 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.
  • Allylamines inhibit ergosterol biosynthesis at the level of squalene epoxidase.
  • the morpholine drug amorolfine inhibits the same pathway at a later step.
  • 5-Fluprocytosine acts as an inhibitor of both DNA and RNA synthesis via the conversion of 5-fluorocytosine to 5-fluorouracil.
  • amphotericin B an antifungal polyene macrolide antibiotic
  • amphotericin B an antifungal polyene macrolide antibiotic
  • the present invention encompasses compounds having the Formula (I):
  • the present invention encompasses compounds having the Formula (IA):
  • the present invention encompasses compounds having the Formula (IB):
  • the present invention encompasses compounds having the Formula (II):
  • R 1 is -H, -C(O)NH 2 , -S(O)NH 2 , -S(O) 2 NH 2 , -C 1 -C 10 (oxy)alkyl, -C 1 -Ci 0 alkyl, -C 1 -C 10 (hydroxy)alkyl, -C 1 -C 10 (amino)alkyl, -C 1 -C 1O (halo)alkyl, -C 2 -C 10 alkenyl, -C 2 -CiO alkynyl, - (C 3 -C 7 ) cycloalkyl, -aryl, -C 1 -C 1O (aryi)alkyl, three- to seven-membered non-aromatic heterocycle, five- to seven-membered aromatic heterocycle, -CH 2 OR 2 , -C(O)R 2 , -C(O)OR 2 , - C(O)NR 2 , -P
  • Ri is -CH 3 .
  • the present invention encompasses compounds having the Formula (IIA):
  • Ri is -H, -C(O)NH 2 , -S(O)NH 2 , -S(O) 2 NH 2 , -C 1 -C 10 (oxy)alkyl, -C 1 -Ci 0 alkyl, -C 1 -C 10 (hydroxy)alkyl, -C 1 -C 10 (amino)alkyl, -C 1 -C 10 (halo)alkyl, -C 2 -C 10 alkenyl, -C 2 -Ci 0 alkynyl, - (C 3 -C 7 ) cycloalkyl, -aryl, -Ci-Ci 0 (aryl)alkyl, three- to seven-membered non-aromatic heterocycle, five- to seven-membered aromatic heterocycle, -CH 2 OR 2 , -C(O)R 2 , -C(O)OR 2 , - C(O)NR 2 , -P(
  • Ri is -CH 3 .
  • the present invention encompasses compounds having the Formula (HB):
  • R 1 is -H, -C(O)NH 2 , -S(O)NH 2 , -S(O) 2 NH 2 , -Ci-C 10 (oxy)alkyl, -C 1 -C 10 alkyl, -C 1 -C 10 (hydroxy)alkyl, -C 1 -C 10 (amino)alkyl, -C 1 -C 10 (halo)alkyl, -C 2 -C 10 alkenyl, -C 2 -C 10 alkynyl, - (C 3 -C 7 ) cycloalkyl, -aryl, -C 1 -Ci 0 (aryl)alkyl, three- to seven-membered non-aromatic heterocycle, five- to seven-membered aromatic heterocycle, -CH 2 OR 2 , -C(O)R 2 , -C(O)OR 2 , - C(O)NR 2 , -P(O)(
  • R 1 is -CH 3 .
  • the invention provides 4-hydroxy-8-methoxy-3,3,9b- trimethylphenanthro[4,3-b]oxirene-2,5(laH,3H,9bH,9cH)-dione (Compound 1) and pharmaceutically acceptable salts thereof.
  • the invention provides (laR,9bR,9cR)-4-hydroxy- 8-methoxy-3,3,9b-trimethylphenanthro[4,3-b]oxirene-2,5(laH,3H,9bH,9cH)-dione (Compound Ia) and pharmaceutically acceptable salts thereof.
  • the invention provides (laS,9bS,9cS)-4-hydroxy- 8-methoxy-3,3,9b-trimethylphenanthro[4,3-b]oxirene-2,5(laH,3H,9bH,9cH)-dione (Compound Ib) and pharmaceutically acceptable salts thereof.
  • the compound or a pharmaceutically acceptable salt of the compound of Formula (I), Formula (IA), or Formula (IB), Formula (II), Formula (IIA), or Formula (HB) is in isolated and purified form. It is recognized that the compound or a pharmaceutically acceptable salt of the compound of Formula (I), Formula (IA), or Formula (IB), Formula (II), Formula (IIA), or Formula (HB) in a composition, such as a pharmaceutical composition, is preferably in isolated and purified form. In a preferred embodiment, the compound or a pharmaceutically acceptable salt of the compound of Formula (I), Formula (IA), or Formula (IB), Formula (II), Formula (IIA), or Formula (HB) is in solid form.
  • a compound of Formula (I), Formula (IA), or Formula (IB), Formula (II), Formula (IIA), or Formula (HB) or a pharmaceutically acceptable salt thereof is useful for treating or preventing cancer, a neoplastic disease or a fungal infection in a patient in need of such treatment or prevention.
  • a Diterpenoid Compound is also useful for inhibiting the growth of a cancer cell, neoplastic cell or fungus.
  • a Diterpenoid Compound is 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.
  • 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.
  • Fig. 1 illustrates the variation in body weight of SCID mice over time following treatment with Compound 1 at a dose of 2 mg/kg and 10 mg/kg, respectively.
  • Fig. 2 illustrates the changes in tumor volume in SCID mice which were implanted with C33 A human cervical cancer cells and treated with Compound 1 at a dose of 2 mg/kg and 10 mg/kg, respectively.
  • C 1 -C 1O 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-
  • C 1 -Ci O alkoxy means -0-(Ci-C 1 O alkyl), wherein C 1 -C 1 O alkyl is defined above.
  • C 1 -C 1O (hydroxy)alkyl means C 1 -C 1O alkyl, wherein C 1 -C 1 O alkyl is defined above, substituted with one or more -OH groups.
  • Examples of C 1 -Ci O (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.
  • amino acid means any naturally occurring amino acids and non-naturally occurring amino acids such as D-amino acids.
  • amino acids see, e.g., L. Stryer, Biochemistry, W.H. Freeman and Company, New York.
  • An amino acid can be substituted with a protecting group.
  • Suitable protecting groups for amino and amido groups include acetyl, tert-butoxy-C(O)-, benzyloxy-C(O)-, and the like.
  • Suitable protecting groups for hydroxy include benzyl and the like.
  • Suitable protecting groups for carboxy moieties include benzyl, tert-butyl, and the like.
  • Other suitable protecting groups are well known to those of ordinary skill in the art and include those found in T. W. Greene, Protecting Groups in Organic Synthesis, John Wiley & Sons, Inc. 1981.
  • C 1 -C 1O (amino)alkyl means C 1 -C 1O alkyl, wherein C 1 -C 1O alkyl is defined above, substituted with one or more -NH 2 groups.
  • Examples of Ci-C 1O (amino)alkyl include, but are not limited to, -CH 2 -NH 2 , -(CH 2 ) 2 -NH 2 , -(CH 2 ) 3 -NH 2 , -(CH 2 ) 4 - NH 2 , -(CH 2 ) 5 -NH 2 and the like.
  • C 1 -CiO (halo)alkyl means Ci-C 10 alkyl, wherein C 1 -Ci O alkyl is defined above, substituted with one or more -F, -Cl, Br or -I groups.
  • Examples of C 1 - Cio (halo)alkyl include, but are not limited to, trichloromethyl, trifluoromethyl, dichloromethyl, difluoromethyl, 1-fluoroethyl, 2-chloroethyl, 1-bromopropyl, 2- iodopropyl.S-chloropropyl, 4-fluorobutyl, 5-chloropentyl and the like.
  • C 2 -C 10 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 C 2 -C 1O alkenyls include - vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutylenyl, -1-pentenyl, -2-pentenyl, - 3-methyl-l-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
  • C 2 -C 6 alkenyl is a subclass of C 2 -Ci O alkenyl.
  • the double bond of a C 2 -C 1O alkenyl can be unconjugated or conjugated to another unsaturated group.
  • C 2 -C 1O 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 C 2 -C 1O alkynyls include -acetylenyl, -propynyl, -1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl, - 3-methyl-l-butynyl, -4-pentynyl, -1-hexynyl, -2-hexynyl, -5-hexynyl, -1-heptynyl, -2- heptynyl, -6-heptynyl, -1-octynyl, -2-octynyl, -7-octynyl, -1
  • C 2 -C 6 alkynyl is a subclass of C 2 -C 1O alkynyl.
  • the triple bond of a C 2 -C 1O alkynyl can be unconjugated or conjugated to another unsaturated group.
  • (C 3 -C- 7 ) cycloalkyl means a monocyclic or bicyclic saturated ring consisting of carbon and hydrogen atoms and having 3-7 carbon atoms.
  • Examples of (C 3 -C 7 ) cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl, and saturated cyclic and bicyclic terpenes.
  • (C 3 -C 7 ) cycloalkenyl means a monocyclic or bicyclic unsaturated ring consisting of carbon and hydrogen atoms and having 3-7 carbon atoms.
  • Examples of (C 3 -C 7 ) cycloalkyl include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, and cycloheptenyl, and unsaturated cyclic and bicyclic terpenes.
  • 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.
  • the aryl group is a monocyclic ring or bicyclic ring.
  • Representative aryl groups include phenyl, tolyl, anthryl, fluorenyl, indenyl, azulenyl, phenanthryl and naphthyl.
  • C 1 -C 10 (aryl)alkyl means C 1 -C 10 alkyl, wherein C 1 -C 10 alkyl is defined above, substituted with one or more aryl groups, wherein aryl is defined above.
  • C 1 -C 10 (aryl)alkyl examples include, but not limited to -(CH 2 )phenyl, - (CH 2 ) 2 ⁇ henyl, -(CH 2 ) 3 phenyl, -CH(phenyl) 2 , -CH(phenyl) 3 , -(CH 2 )tolyl, -(CH 2 )anthracenyl, -(CH 2 )fluorenyl, -(CH 2 )indenyl, -(CH 2 )azulenyl, -(CH 2 )naphthyl, and the like.
  • C 7 -C 12 (aryl)alkyl means C 7 -C 12 alkyl, wherein C 7 -Ci 2 alkyl is defined above, substituted with one or more aryl groups, wherein aryl is defined above.
  • C 2 -C 10 (aryl)alkenyl means C 2 -C 10 alkenyl, wherein C 2 -C 10 alkenyl is defined above, substituted with one or more aryl groups, wherein aryl is defined above.
  • C 2 -C 10 (aryl)alkynyl means C 2 -C 10 alkynyl, wherein C 2 -Ci 0 alkynyl is defined above, substituted with one or more aryl groups, wherein aryl is defined above.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • halogen examples are fluorine, chlorine, bromine, and iodine.
  • Examples of C 1 - C 10 (oxy)alkyl include, but are not limited to, -C(O)CH 3 , -CH 2 CHO, -C(O)(CH 2 ) 2 CH 3 , - CH 2 C(O)CH 3 , -(CH 2 ) 2 CHO, -(CH 2 ) 3 CHO, -(CH 2 ) 4 CHO an the like.
  • 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.
  • 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.
  • 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.
  • peptide is a sequence of two to six amino acids. In certain embodiments, a peptide is two, three, four, five, or six amino acids long.
  • 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.
  • Illustrative 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.
  • inorganic and organic acids such as acetic, alginic, an
  • a salt of a Diterpenoid Compound is mesylate or tartrate.
  • Other examples of salts are well known in the art, see, e.g., Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton PA (1990).
  • isolated means separated 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.
  • 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
  • a compound of the invention is at least about 90% pure. In certain embodiments, a 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.
  • a hydrogen of the first group is replaced with the second group.
  • a first group is substituted with one, two or three second groups.
  • a first group is substituted with one or two second groups.
  • a first group is substituted with one second group.
  • 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.
  • Diterpenoid Compounds of the invention can have one or more chiral centers and/or double bonds and, therefore, exist as stereoisomers, such as double-bond isomers (Le., geometric isomers), enantiomers, or diastereomers.
  • stereoisomers such as double-bond isomers (Le., geometric isomers), enantiomers, or diastereomers.
  • the compound if a chemical structure depicted herein does not indicate the stereochemistry at a chiral center, the compound encompasses all of the corresponding enantiomers and stereoisomers, that is, both the stereomerically pure form (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) and enantiomeric and stereoisomeric mixtures, e.g., racemates.
  • Diterpenoid Compound differs only by the placement of a proton and the corresponding location of a double-bond (tautomerism), the chemical structures depicted herein, and therefore the compounds of the invention or the compounds to be used with the methods of the invention, encompass all of the corresponding tautomers and the mixture of the tautomers.
  • stereomerically pure means a composition that comprises one stereoisomer of a compound and is substantially free of other stereoisomers of that compound.
  • a stereomerically pure composition of a compound having one chiral center will be substantially free of the opposite enantiomer of the compound.
  • a stereomerically pure composition of a compound having two chiral centers will be substantially free of other diasteroemers of the compound.
  • a typical stereomerically pure compound comprises greater than about 80% by weight of stereoisomer of the compound and less than about 20% by weight of other stereoisomers the compound, more preferably greater than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of the other stereoisomers of the compound, even more preferably greater than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of the other stereoisomers of the compound, and most preferably greater than about 97% by weight of one stereoisomer of the compound and less than about 3% by weight of the other stereoisomers of the compound.
  • Enantiomeric and stereoisomeric mixtures of compounds of the invention can be resolved into their component enantiomers or stereoisomers by well-known methods, such as chiral-phase gas chromatography, chiral-phase high performance liquid chromatography, crystallizing the compound as a chiral salt complex, or crystallizing the compound in a chiral solvent.
  • Enantiomers and stereoisomers can also be obtained from stereomerically or enantiomerically pure intermediates, reagents, and catalysts by well-known asymmetric synthetic methods.
  • the Diterpenoid Compounds can exist in the form of a pharmaceutically acceptable salt, free base, solvate, hydrate, stereoisomer, clathrate, polymorph or prodrug thereof.
  • the present invention encompasses compounds having the Formula (I):
  • the present invention encompasses compounds having the Formula (IA):
  • the present invention encompasses compounds having the Formula (IB):
  • R 15 is H, -Ci-Ci 0 alkyl, C 7 -Ci 2 arylalkyl, Cj-Cioaminoalkyl, Q-Ciohaloalkyl, or Ci-Cio hydroxyalkyl.
  • the present invention encompasses compounds having the Formula (ILA):
  • the present invention encompasses compounds having the Formula (IIB):
  • a Diterpenoid Compound of Formula (I), Formula (IA) or Formula (IIB), Formula (II), Formula (IIA) or Formula (IIB) is useful for treating or preventing cancer or neoplastic disease in a patient in need of such treatment or prevention.
  • a Diterpenoid Compound of Formula (I), Formula (IA) or Formula (IB), Formula (II), Formula (IIA) or Formula (IIB) is also useful for inhibiting the growth of a cancer cell or neoplastic cell.
  • a Diterpenoid Compound of Formula (I), Formula (IA) or Formula (IB), Formula (II), Formula (IIA) or Formula (IIB) is also useful for inducing cytotoxicity, e.g., through apoptosis, in a cancer cell or neoplastic cell.
  • a Diterpenoid Compound of Formula (I), Formula (IA) or Formula (IB), Formula (II), Formula (IIA) or Formula (IIB) is further useful for treating or preventing a fungal infection.
  • a Diterpenoid Compound of Formula (I), Formula (IA) or Formula (IB), Formula (II), Formula (IIA) or Formula (IIB) is also useful for inhibiting the growth of a fungus.
  • the invention provides 4-hydroxy-8-methoxy-3,3,9b- trimethylphenanthro[4,3-b]oxirene-2,5(laH,3H,9bH,9cH)-dione (Compound 1) and pharmaceutically acceptable salts thereof.
  • Compound 1 4-hydroxy-8-methoxy-3,3,9b- trimethylphenanthro[4,3-b]oxirene-2,5(laH,3H,9bH,9cH)-dione
  • the invention provides (laR,9bR,9cR)-4-hydroxy- 8-methoxy-3,3,9b-trimethylphenanthro[4,3-b]oxirene-2,5(laH,3H,9bH,9cH)-dione (Compound Ia) and pharmaceutically acceptable salts thereof.
  • the invention provides (laS,9bS,9cS)-4-hydroxy- 8-methoxy-3,3,9b-trimethylphenanthro[4,3-b]oxirene-2,5(laH,3H,9bH,9cH)-dione (Compound Ib) and pharmaceutically acceptable salts thereof.
  • the Diterpenoid Compounds can be obtained using conventional organic synthesis or by using the following illustrative methods shown in Schemes 1 to 8 below. Methods that can be used for the synthesis of the Diterpenoid Compounds are described, e.g., in Hijfte L.V., Little R.D., Petersen J.L., and Moeller K.D., J. Org. Chem. 1987, 52, 4647-4660; Numazawa M. and Tachibana M., Steroids, 1994, 59, 579-585; and Yang N.C. and Finnegan R.A. J. Am. Chem. Soc. 1958, 80, 5845-5848.
  • Diterpenoid Compounds of Formula (HB) can be obtained from a compound of Formula (IIIB) as depticted in Scheme If (R 1 is as defined above).
  • Diterpenoid Compounds of Formula (I) can also be obtained from a compound of Formula (IV) as depticted in Scheme 2a (A, B, D, and E are as defined above).
  • Scheme 2a A, B, D, and E are as defined above.
  • Diterpenoid Compounds of Formula (IA) can be obtained from a compound of Formula (IVA) as depticted in Scheme 2b (A, B, D, and E are as defined above).
  • Scheme 2b A, B, D, and E are as defined above.
  • Diterpenoid Compounds of Formula (II) can also be obtained from a compound of Formula (III) as depticted in Scheme 2d (R is as defined above).
  • Diterpenoid Compounds of Formula (IB) can also be obtained from a compound of Formula (HB) as depticted in Scheme 2f (R 1 is as defined above).
  • Compound 1 can be obtained from a Compound 2 as depticted in Scheme 2g.
  • Compound Ia can be obtained from Compound 2a as depticted in Scheme 2h.
  • Compound Ib can be obtained from Compound 2b as depticted in Scheme 2i.
  • Compounds of Formula (IV) can be obtained from a tetralone-type precursor such as depicted by compounds 13 in Scheme 3.
  • Nucleophilic addition of -CH 3 using an appropriate organometallic reagent such as, a Grignard reagent (E.G. Ashby et al., J. Am. Chem. Soc, 89:1964 (1967)), followed by dehydration (C. Utermoehlen et al., /. 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.
  • the a, b- unsaturation of compounds 17 can be introduced by treating compounds 16 with a strong base such as lithium diisopropyl amide (LDA), followed by treatment with phenylselenium chloride (PhSeCl), hydrogen peroxide or meta-chloroperoxybenzoic acid (mCPBA; M. Tius et al., J. Am. Chem. Soc, U4:5959 (1992)).
  • a strong base such as lithium diisopropyl amide (LDA)
  • PhSeCl phenylselenium chloride
  • mCPBA meta-chloroperoxybenzoic acid
  • 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, i.e., compounds of Formula (IV).
  • Compounds of Formula III can be obtained analogously to the synthesis scheme for compounds of Formula IV.
  • Compounds 21 can be used as a starting material for the synthesis of Diterpenoid Compounds.
  • Compounds 21 can be obtained (Scheme 4) in three steps from IBX-DMSO oxidation of aryl-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.
  • 12-Methoxypodocarpa-8,ll,13-trieneoic acid (29) is a useful starting material for Compound 2.
  • compounds 29 can 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.
  • Compound 2 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)). Compound 2 is in equilibrium with triketo Compound 3.
  • the present invention also provides prodrugs of the Diterpenoid Compounds of the invention.
  • Illustrative prodrugs of the Diterpenoid Compounds of the invention are:
  • the invention provides methods for treating cancer in a patient, comprising administering to the patient an effective amount of a prodrug of a Diterpenoid Compound of the invention, e.g., Compound 4, Compound 4a, Compound 4b, Compound 5, Compound 5a, Compound 5b, Compound 6, Compound 6a, or Compound 6b.
  • a prodrug of a Diterpenoid Compound of the invention e.g., Compound 4, Compound 4a, Compound 4b, Compound 5, Compound 5a, Compound 5b, Compound 6, Compound 6a, or Compound 6b.
  • a prodrug of a Diterpenoid Compound of the invention e.g.
  • Compound 4, Compound 4a, Compound 4b, Compound 5, Compound 5a, Compound 5b, Compound 6, Compound 6a, or Compound 6b is useful for treating or preventing cancer or neoplastic disease in a patient in need of such treatment or prevention.
  • a prodrug of a Diterpenoid Compound of the invention e.g., Compound 4, Compound 4a, Compound 4b, Compound 5, Compound 5a, Compound 5b, Compound 6, Compound 6a, or Compound 6b, is also useful for inhibiting the growth of a cancer cell or neoplastic cell.
  • a prodrug of a Diterpenoid Compound of the invention e.g., Compound 4, Compound 4a, Compound 4b, Compound 5, Compound 5a, Compound 5b, Compound 6, Compound 6a, or Compound 6b
  • a prodrug of a Diterpenoid Compound of the invention e.g., Compound 4, Compound 4a, Compound 4b, Compound 5, Compound 5a, Compound 5b, Compound 6, Compound 6a, or Compound 6b, is further useful for treating or preventing a fungal infection.
  • a prodrug of a Diterpenoid Compound of the invention e.g., Compound 4, Compound 4a, Compound 4b, Compound 5, Compound 5a, Compound 5b, Compound 6, Compound 6a, or Compound 6b, is also useful for inhibiting the growth of a fungus.
  • the present invention also provides additional prodrugs of the Diterpenoid Compounds of the invention.
  • Prodrugs include derivatives of Diterpenoid Compounds that can hydrolyze, oxidize, or otherwise react under biological conditions (in vitro or in vivo) to provide an active Diterpenoid Compound of the invention.
  • Examples of prodrugs include, but are not limited to, derivatives and metabolites of a compound of the invention that include biohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, and biohydrolyzable phosphate analogues.
  • prodrugs of Diterpenoid Compounds with carboxyl functional groups are the lower alkyl esters of the carboxylic acid.
  • the carboxylate esters are conveniently formed by esterifying any of the carboxylic acid moieties present on the molecule.
  • Prodrugs can typically be prepared using well-known methods, such as those described by Burger's Medicinal Chemistry and Drug Discovery 6 th ed. (Donald J. Abraham ed., 2001, Wiley) and Design and Application of Prodrugs (H. Bundgaard e d., 1985, Harwood Academic Publishers Gmfh).
  • Biohydrolyzable moieties of a Diterpenoid Compound do not interfere with the biological activity of the compound but can confer upon that compound advantageous properties in vivo, such as uptake, duration of action, or onset of action; or (ii) are biologically inactive but are converted in vivo to the biologically active compound.
  • biohydrolyzable esters include, but are not limited to, lower alkyl esters, alkoxyacyloxy esters, alkyl acylamino alkyl esters, and choline esters.
  • biohydrolyzable amides include, but are not limited to, lower alkyl amides, ⁇ -amino acid amides, alkoxyacyl amides, and alkylaminoalkylcarbonyl amides.
  • biohydrolyzable carbamates include, but are not limited to, lower alkylamines, substituted ethylenediamines, aminoacids, hydroxyalkylamines, heterocyclic and heteroaromatic amines, and polyether amines. 5.5.1 SYNTHESIS OF PRODRUGS: COMPOUND 4, 5, AND 6
  • the Diterpenoid Compounds are advantageously useful in veterinary and human medicine.
  • 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.
  • the Diterpenoid Compounds 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.
  • compositions which comprise a Diterpenoid Compound
  • Various delivery systems are known, e.g., encapsulation in liposomes, microparticles, microcapsules, capsules, etc., and can be used to administer a Diterpenoid Compound.
  • 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).
  • Diterpenoid Compounds 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.
  • 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.
  • 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.
  • the Diterpenoid Compounds can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • 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.)
  • the Diterpenoid Compounds can be delivered in a controlled-release system, hi 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)).
  • polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (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)).
  • 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.
  • 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.
  • 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.
  • 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, polymethylated castor oil (CREMAPHOR EL) and the like.
  • the pharmaceutical carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like.
  • auxiliary, stabilizing, thickening, lubricating and coloring agents can be used.
  • 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.
  • 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.
  • 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.
  • the pharmaceutically acceptable carrier is a capsule (see e.g., U.S. Patent No. 5,698,155).
  • suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.
  • the Diterpenoid Compounds are formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • Diterpenoid Compounds intended for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • 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.
  • 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.
  • 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.
  • sweetening agents such as fructose, aspartame or saccharin
  • flavoring agents such as peppermint, oil of wintergreen, or cherry
  • coloring agents such as peppermint, oil of wintergreen, or cherry
  • preserving agents to provide a pharmaceutically palatable preparation.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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, hi 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.
  • 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.
  • the kit when administered for the treatment or prevention of cancer, 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.
  • the kit when administered for the treatment or prevention of a fungal infection, can also contain one or more other anti-fungal agents to be administered prior to, subsequent to or in combination with a Diterpenoid Compound.
  • 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.
  • in vitro assays can be used to determine whether administration of a specific Diterpenoid Compound or combination of Diterpenoid Compounds is preferred.
  • 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.
  • a cell culture model is used in which the cells of the celi 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.
  • 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.
  • 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.
  • 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.
  • 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 ( 3 H)-thymidine incorporation, by direct cell count, by detecting changes in transcription, translation or activity of known genes such as proto-oncogenes (e.g.,fo$, myc) or cell cycle markers (Rb, cdc2, cyclin A, Dl, D2, D3, E, etc).
  • proto-oncogenes e.g.,fo$, myc
  • cell cycle markers Rb, cdc2, cyclin A, Dl, D2, D3, E, etc.
  • the levels of such protein and mRNA and activity can be determined by any method well known in the art.
  • protein can be quantitated by known immunodiagnostic methods such as Western blotting or immunoprecipitation using commercially availably 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.
  • 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:
  • 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 ( H)-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.
  • cells synthesizing DNA will incorporate ( 3 H)-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).
  • PCNA proliferating cell nuclear antigen
  • 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.
  • Sigma Trypan-blue
  • DNA content and/or mitotic index of the cells can be measured, for example, based on the DNA ploidy value of the cell.
  • cells in the Gl 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
  • 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).
  • 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.l35:783-92).
  • 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).
  • cell-cycle proteins e.g., CycA. CycB, CycE, CycD, cdc2, Cdk4/6, Rb, p21, and p27
  • cell-cycle proteins 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 p21 C ⁇ l . Increased levels of p21 expression in cells result in delayed entry into Gl 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).
  • cell-cycle proteins can be examined by Western blot analysis using commercially available antibodies.
  • 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.
  • 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).
  • FACS analysis is used to analyze the phase of cell cycle progression, or purify Gl, 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 checkpoints can be examined using the methods described herein, or by any method known in the art.
  • 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).
  • 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.
  • post-translational modifications e.g. phosphorylation
  • antibodies that detect phosphorylated tyrosine residues are commercially available, and can be used in Western blot analysis to detect proteins with such modifications.
  • 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).
  • kinase activity 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 Hl 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, MoI. 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.
  • the Diterpenoid Compounds can also be demonstrated to inhibit cell transformation (or progression to malignant phenotype) in vitro.
  • 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 anticancer effects of the Diterpenoid Compounds.
  • 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 (Calbiochern-Novabiochem Corp. San Diego, CA). 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.
  • 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.
  • HGF hepatocyte growth factor
  • This assay identifies a cell population that has lost cell scattering activity in response to HGF (Hordijk et al., 1997, Science 278:1464-66).
  • loss of invasiveness can be measured by cell migration through a chemotaxis chamber (Neuroprobe/ Precision Biochemicals Inc. Vancouver, BC).
  • 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).
  • the cells 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.l93:518-25).
  • the Diterpenoid Compounds can also be demonstrated to inhibit tumor formation in vivo.
  • a vast number of animal models of hyperpfoliferative 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • Diterpenoid Compounds induce apoptosis in a cancer cell or in a neoplastic cell.
  • Diterpenoid Compounds induce apoptosis selectively in a cancer cell or in a neoplastic cell, relative to a non-cancer cell or non-neoplastic cell.
  • 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 nonneoplastic cell.
  • 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.
  • a Diterpenoid Compound 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.
  • inducing apoptosis selectively in cancer cells or in neoplastic cells is useful for treating cancer or a neoplastic disease in a patient.
  • 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.
  • 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, if osf amide, nitrosoureas, Cisplatin, carboplatin, mitomycin, dacarbazine, procarbizine, etoposides, campathecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, and docetaxel.
  • Taxoids Paclitaxel
  • mitomycins mitomycin C
  • Anti-metabolites Anti-folates: DHFR inhibitors: methotrexate Trimetrexate
  • 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- ⁇ Interferon- ⁇ Tumor necrosis factor
  • Isoprenylation inhibitors Lovastatin
  • Dopaminergic neurotoxins l-methyl-4-phenylpyridinium ion Kinase inhibitors: Staurosporine
  • MDR inhibitors verapamil Ca consult2+ ATPase inhibitors: Thapsigargin
  • 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.
  • the invention provides methods for treating or preventing cancer that has shown to be refractory to treatment with a chemotherapy and/or radiation therapy.
  • a Diterpenoid Compound is administered concurrently with chemotherapy or radiation therapy.
  • 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.
  • any radiation therapy protocol can be used depending upon the type of cancer to be treated or prevented.
  • x-ray radiation can be administered; in particular, high-energy megavoltage (radiation of greater than 1 MeV energy) can be used for deep tumors, and electron beam and ortho voltage 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.
  • 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.
  • 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):
  • cancer, malignancy or dysproliferative changes are treated or prevented in the ovary, breast, colon, lung, skin, pancreas, prostate, bladder, cervix or uterus.
  • sarcoma, melanoma, or leukemia is treated or prevented.
  • 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).
  • 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).
  • the cancer to be treated is Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), Acute Myeloid Leukemia/Other Myeloid Malignancies, Adrenocortical Carcinoma, ABDS-related Lymphoma, AIDS-related Malignancies, Alveolar Soft Part Sarcoma, Anal Cancer, Anaplastic Astrocytoma, Anaplastic Carcinoma, Thyroid, Angiosarcoma, Astrocytomas/Gliomas, Atypical Teratoid Rhabdoid Tumor, Basal Cell Carcinoma, Bile Duct Cancer, Bladder Cancer, Brain Stem Glioma (low grade and high grade), Burkitt's Lymphoma, Cancer of Unknown Primary (CUP), Carcinoid Tumor (gastrointestinal - usually appendix), Cervical Cancer, Childhood Leukemia, Childhood Hodgkin's Disease, Childhood Liver Cancer, Childhood Non-Hodgkin's Lymphoma, Childhood
  • ALL Acute
  • 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).
  • 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 Testi
  • 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.
  • 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. glabratd), Aspergillus (including A.
  • Cryptococcus 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.
  • such fungal infections in animals can be a systemic, topical or mucosal infection.
  • 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 externa.
  • 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 immunoconiprised 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.
  • the Diterpenoid Compounds can be used as anti-fungal agents in vitro or in vivo.
  • 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.
  • mice that were injected with C33 A human cervical cancer cells (American Type Culture Collection, Manassas, VA USA) 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% CO 2 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 X 10 6 cells per 100 ⁇ L. Viability was examined by staining with Trypan Blue and only flasks with cell viability of greater than 95% were used for in vivo studies.
  • PBS phosphate buffered saline solution
  • C33A cells were transplanted subcutaneously into the flank of female CB 17 SCID/SCID mice. Each mouse was inoculated with a suspension of 2 X 10 6 tumor cells per 100 ⁇ L of PBS on day zero.
  • Compound 1 was administered intravenously (i.v.) once daily for five consecutive days at a dose of 2 mg/kg and 10 mg/kg, respectively.
  • Compound 1 was prepared as a working solution of 1.5 mg/mL in vehicle solution.
  • the mice were weighed and the tumors measured on day thirty six and every 2 to 3 days after treatment commenced. Observations continued for 57 days after initial tumor implantation. The changes in body weight and in the calculated tumor volume were plotted ( Figures 1 and 2).
  • Statistical analysis was performed using GraphPad Prism (GraphPad Software Inc., San Diego, CA). 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.
  • Compound 1 significantly reduces the human cervical tumors implanted in SCID mice, an art-accepted model for human cervical cancer. Accordingly, Compound 1 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.

Abstract

The present invention relates to diterpenoid compounds of formula (I) and compositions comprising an effective amount of a diterpenoid compound according to formula (I), compounds of formula (I) wherein A, B, D or E can either represent a nitrogen atom or a carbon atom which is optionally subsituted. There are also disclosed methods useful for treating or preventing cancer or neoplastic disorders comprising administering an effective amount of a diterpenoid compound. The present compounds, compositions, and methods thereof are further useful for treating or preventing fungal infections. They are also useful for inhibiting the growth of a cancer cell or neoplastic cell, for inducing apoptosis in a cancer or neoplastic cell or useful for inhibiting the growth of a fungus.

Description

DITERPENOID COMPOUNDS, COMPOSITIONS THEREOF AND THEIR USE AS ANTI-CANCER OR ANTI-FUNGAL AGENTS
Related Applications
This application claims the benefit of U.S. Provisional Application Serial No. 60/655,613, filed February 22, 2005, 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 cytotoxicity 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). AU 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., Epidermophytonfloccosum, 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, Tliermomucor 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). hi 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 (CC. 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-Fluprocytosine 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):
Figure imgf000005_0001
(I) and pharmaceutically acceptable salts thereof, wherein:
A is N or CR4; B is N or CR5; D is N or CR6; E is N or CR7, at least one of A, B, D and E being CR4, CR5, CR6 or CR7, respectively; each R4, R5, R6 and R7 is independently -H, -OH, -halogen, -CN, -NH2, -NO2, - COOH, -C(O)NH2, -SH, -S(O)NH2, -S(O)2NH2, -C1-C10 (oxy)alkyl, -Ci-C10 alkyl, -C1-C]0 alkoxy, -Ci-C10 (hydroxy)alkyl, -C1-C10 (amino)alkyl, -C1-C10 (halo)alkyl, -C2-C10 alkenyl, - C2-C10 alkynyl, -(C3-C7) cycloalkyl, -aryl, -C1-Ci0 (aryl)alkyl, three- to seven-membered non- aromatic heterocycle, five- to seven-membered aromatic heterocycle, -CH2ORn, -OCH2ORn, -OC(O)Rn, -C(O)Rn, -OC(O)ORn, -OC(O)NRn, -C(O)ORn, -C(O)NRn, -OP(O)(ORn)2, -SR11, -S(O)2NHRn, -SORn, -S(O)2Rn, -NHC(O)Rn, -NHSORn, NHS(O)2R11, -OPO(ORi5)2, -O-arylPO(ORi5)2, or -O-alkylarylPO(ORi5)2 ; wherein Rn is -H, -Ci-Ci0 alkyl, -(C3-C7) cycloalkyl, -Ci-Ci0 (halo)alkyl, -aryl, -C2- Cio alkenyl, -C2-Ci0 alkynyl, -Ci-Ci0 (aryl)alkyl, -C2-Ci0 (aryl)alkenyl, -C2-Ci0 (aryl)alkynyl, -Ci-Ci0 (hydroxy)alkyl, -Ci-Ci0 alkoxy, -Ci-Ci0 (amino)alkyl, a -(C3-C7) cycloalkyl unsubstituted or substituted with one or more -Ci-Ci0 alkyl, a three- to seven-membered non- aromatic heterocycle unsubstituted or substituted with one or more -Ci-C10 alkyl, or a three- to seven-membered aromatic heterocycle unsubstituted or substituted with one or more -C1- C10 alkyl, -C2-Ci0 alkenyl, or -C2-Ci0 alkynyl; and wherein Rj5 is H, -Ci-Ci0 alkyl, C7-Ci2arylalkyl, Ci-Ci0aminoalkyl, Ci-Ci0haloalkyl, or Ci-Cio hydroxyalkyl.
In certain, more specific embodiments, A is CR4; B is CR5; D is CR6; and E is CR7, wherein R4, R5, R6 and R7 are as defined above.
The present invention encompasses compounds having the Formula (IA):
Figure imgf000006_0001
(IA)
and pharmaceutically acceptable salts thereof, wherein:
A is N or CR4; B is N or CR5; D is N or CR6; E is N or CR7, at least one of A, B, D and E being CR4, CR5, CR6 or CR7, respectively; each R4, R5, R6 and R7 is independently -H, -OH, -halogen, -CN, -NH2, -NO2, - COOH, -C(O)NH2, -SH, -S(O)NH2, -S(O)2NH2, -C1-C10 (oxy)alkyl, -C1-Ci0 alkyl, -C1-C10 alkoxy, -C1-C10 (hydroxy)alkyl, -C1-Ci0 (amino)alkyl, -C1-Ci0 (halo)alkyl, -C2-Ci0 alkenyl, - C2-Ci0 alkynyl, -(C3-C7) cycloalkyl, -aryl, -Ci-Ci0 (aryl)alkyl, three- to seven-membered non- aromatic heterocycle, five- to seven-membered aromatic heterocycle, -CH2ORn, -OCH2ORn, -OC(O)Rn, -C(O)Rn, -OC(O)ORn, -OC(O)NRn, -C(O)OR11, -C(O)NRn, -OP(O)(ORn)2, -SRn, -S(O)2NHR11, -SOR11, -S(O)2R11, -NHC(O)R11, -NHSOR11, NHS(O)2R11, -OPO(OR15)2, -O-arylPO(OR15)2, or -O-alkylarylPO(OR15)2; wherein R11 is -H, -Ci-Ci0 alkyl, -(C3-C7) cycloalkyl, -Ci-Ci0 (halo)alkyl, -aryl, -C2- Cio alkenyl, -C2-Ci0 alkynyl, -Ci-Ci0 (aryl)alkyl, -C2-Ci0 (aryl)alkenyl, -C2-Ci0 (aryl)alkynyl, -Ci-Ci0 (hydroxy)alkyl, -Ci-C10 alkoxy, -C1-C10 (amino)alkyl, a -(C3-C7) cycloalkyl unsubstituted or substituted with one or more -Ci-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; and wherein Ri5 is H, -C1-C10 alkyl, C7-C12arylalkyl, d-Qoaminoalkyl, Q-Qohaloalkyl, or C i -C 10 hydroxyalkyl.
In certain, more specific embodiments, A is CR4; B is CR5; D is CR6; and E is CR7, wherein R4, R5, R6 and R7 are as defined above.
The present invention encompasses compounds having the Formula (IB):
Figure imgf000008_0001
(IB)
and pharmaceutically acceptable salts thereof, wherein:
A is N or CR4; B is N or CR5; D is N or CR6; E is N or CR7, at least one of A, B, D and E being CR4, CR5, CR6 or CR7, respectively; each R4, R5, R6 and R7 is independently -H, -OH, -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-Ci0 (hydroxy)alkyl, -C1-C10 (amino)alkyl, -C1-CiO (halo)alkyl, -C2-C10 alkenyl, - C2-Ci0 alkynyl, -(C3-C7) cycloalkyl, -aryl, -C1-Ci0 (aryl)alkyl, three- to seven-membered non- aromatic heterocycle, five- to seven-membered aromatic heterocycle, -CH2ORn, -OCH2OR11, -OC(O)R11, -C(O)R11, -OC(O)OR11, -OC(O)NR11, -C(O)ORn, -C(O)NRn, -OP(O)(ORn)2, -SR11, -S(O)2NHR11, -SORn, -S(O)2R11, -NHC(O)R11, -NHSOR11, NHS(O)2R11, -OPO(OR15)2, -O-arylPO(ORi5)2, or -O-alkylarylPO(ORi5)2 wherein 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-CiO (aryl)alkynyl, -C1-C1O (hydroxy)alkyl, -C1-CiO alkoxy, -Ci-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 -Ci-C10 alkyl, or a three- to seven-membered aromatic heterocycle unsubstituted or substituted with one or more -C1- Cio alkyl, -C2-CiO alkenyl, or -C2-CiO alkynyl; and wherein R15 is H, -C1-C10 alkyl, C7-Ci2arylalkyl, Q-Cioaminoalkyl, Q-Qohaloalkyl, or Ci-Ciohydroxyalkyl.
In certain, more specific embodiments, A is CR4; B is CR5; D is CR6; and E is CR7, wherein R4, R5, R6 and R7 are as defined above.
The present invention encompasses compounds having the Formula (II):
Figure imgf000009_0001
(H)
and pharmaceutically acceptable salts thereof, wherein:
R1 is -H, -C(O)NH2, -S(O)NH2, -S(O)2NH2, -C1-C10 (oxy)alkyl, -C1-Ci0 alkyl, -C1-C10 (hydroxy)alkyl, -C1-C10 (amino)alkyl, -C1-C1O (halo)alkyl, -C2-C10 alkenyl, -C2-CiO alkynyl, - (C3-C7) cycloalkyl, -aryl, -C1-C1O (aryi)alkyl, three- to seven-membered non-aromatic heterocycle, five- to seven-membered aromatic heterocycle, -CH2OR2, -C(O)R2, -C(O)OR2, - C(O)NR2, -P(O)(OR2)2, -S(O)2NHR2, -SOR2, -S(O)2R2, -NHC(O)R2, -NHSOR2, or NHS(O)2R2 ; and wherein R2 is -H, -C1-Ci0 alkyl, -(C3-C7) cycloalkyl, -Ci-C10 (halo)alkyl, -aryl, -C2-Ci0 alkenyl, -C2-Ci0 alkynyl, -Ci-Ci0 (aryl)alkyl, -C2-Ci0 (aryl)alkenyl, -C2-Ci0 (aryl)alkynyl, - Ci-Cio (hydroxy)alkyl, -Ci-Ci0 alkoxy, -Ci-Ci0 (amino)alkyl, a -(C3-C7) cycloalkyl unsubstituted or substituted with one or more -Ci-Ci0 alkyl, a three- to seven-membered non- aromatic heterocycle unsubstituted or substituted with one or more -Ci-Cio alkyl, or a three- to seven-membered aromatic heterocycle unsubstituted or substituted with one or more -Ci- Cio alkyl, -C2-Ci0 alkenyl, or -C2-Ci0 alkynyl.
In a specific embodiment, Ri is -CH3.
The present invention encompasses compounds having the Formula (IIA):
Figure imgf000010_0001
(HA)
and pharmaceutically acceptable salts thereof, wherein:
Ri is -H, -C(O)NH2, -S(O)NH2, -S(O)2NH2, -C1-C10 (oxy)alkyl, -C1-Ci0 alkyl, -C1-C10 (hydroxy)alkyl, -C1-C10 (amino)alkyl, -C1-C10 (halo)alkyl, -C2-C10 alkenyl, -C2-Ci0 alkynyl, - (C3-C7) cycloalkyl, -aryl, -Ci-Ci0 (aryl)alkyl, three- to seven-membered non-aromatic heterocycle, five- to seven-membered aromatic heterocycle, -CH2OR2, -C(O)R2, -C(O)OR2, - C(O)NR2, -P(O)(OR2)2, -S(O)2NHR2, -SOR2, -S(O)2R2, -NHC(O)R2, -NHSOR2, or NHS(O)2R2; and wherein R2 is -H, -Ci-Ci0 alkyl, -(C3-C7) cycloalkyl, -Ci-Ci0 (halo)alkyl, -aryl, -C2-C10 alkenyl, -C2-Ci0 alkynyl, -Ci-Ci0 (aryl)alkyl, -C2-Ci0 (aryl)alkenyl, -C2-Ci0 (aryl)alkynyl, - Ci-Ci0 (hydroxy)alkyl, -Ci-Ci0 alkoxy, -Ci-Ci0 (amino)alkyl, a -(C3-C7) cycloalkyl unsubstituted or substituted with one or more -Ci-Ci0 alkyl, a three- to seven-membered non- aromatic heterocycle unsubstituted or substituted with one or more -Ci-Ci0 alkyl, or a three- to seven-membered aromatic heterocycle unsubstituted or substituted with one or more -Ci- Cio alkyl, -C2-Ci0 alkenyl, or -C2-Ci0 alkynyl.
In a specific embodiment, Ri is -CH3.
The present invention encompasses compounds having the Formula (HB):
Figure imgf000011_0001
(HB)
and pharmaceutically acceptable salts thereof, wherein:
R1 is -H, -C(O)NH2, -S(O)NH2, -S(O)2NH2, -Ci-C10 (oxy)alkyl, -C1-C10 alkyl, -C1-C10 (hydroxy)alkyl, -C1-C10 (amino)alkyl, -C1-C10 (halo)alkyl, -C2-C10 alkenyl, -C2-C10 alkynyl, - (C3-C7) cycloalkyl, -aryl, -C1-Ci0 (aryl)alkyl, three- to seven-membered non-aromatic heterocycle, five- to seven-membered aromatic heterocycle, -CH2OR2, -C(O)R2, -C(O)OR2, - C(O)NR2, -P(O)(OR2)2, -S(O)2NHR2, -SOR2, -S(O)2R2, -NHC(O)R2, -NHSOR2, or NHS(O)2R2; and wherein R2 is -H, -Ci-Ci0 alkyl, -(C3-C7) cycloalkyl, -Ci-Ci0 (halo)alkyl, -aryl, -C2-Ci0 alkenyl, -C2-Ci0 alkynyl, -Ci-Ci0 (aryl)alkyl, -C2-Ci0 (aryl)alkenyl, -C2-Ci0 (aryl)alkynyl, - C1-Ci0 (hydroxy)alkyl, -C1-Ci0 alkoxy, -C1-C10 (amino)alkyl, a -(C3-C7) cycloalkyl unsubstituted or substituted with one or more -C1-Ci0 alkyl, a three- to seven-membered non- aromatic heterocycle unsubstituted or substituted with one or more -Ci -Ci0 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.
In a specific embodiment, R1 is -CH3.
hi an illustrative embodiment, the invention provides 4-hydroxy-8-methoxy-3,3,9b- trimethylphenanthro[4,3-b]oxirene-2,5(laH,3H,9bH,9cH)-dione (Compound 1) and pharmaceutically acceptable salts thereof.
Figure imgf000012_0001
Compound 1
In another illustrative embodiment, the invention provides (laR,9bR,9cR)-4-hydroxy- 8-methoxy-3,3,9b-trimethylphenanthro[4,3-b]oxirene-2,5(laH,3H,9bH,9cH)-dione (Compound Ia) and pharmaceutically acceptable salts thereof.
Figure imgf000012_0002
Compound Ia
In a further illustrative embodiment, the invention provides (laS,9bS,9cS)-4-hydroxy- 8-methoxy-3,3,9b-trimethylphenanthro[4,3-b]oxirene-2,5(laH,3H,9bH,9cH)-dione (Compound Ib) and pharmaceutically acceptable salts thereof.
Figure imgf000013_0001
Compound Ib
In certain embodiments, the compound or a pharmaceutically acceptable salt of the compound of Formula (I), Formula (IA), or Formula (IB), Formula (II), Formula (IIA), or Formula (HB) is in isolated and purified form. It is recognized that the compound or a pharmaceutically acceptable salt of the compound of Formula (I), Formula (IA), or Formula (IB), Formula (II), Formula (IIA), or Formula (HB) in a composition, such as a pharmaceutical composition, is preferably in isolated and purified form. In a preferred embodiment, the compound or a pharmaceutically acceptable salt of the compound of Formula (I), Formula (IA), or Formula (IB), Formula (II), Formula (IIA), or Formula (HB) is in solid form.
A compound of Formula (I), Formula (IA), or Formula (IB), Formula (II), Formula (IIA), or Formula (HB) or a pharmaceutically acceptable salt 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. A Diterpenoid Compound is also useful for inhibiting the growth of a cancer cell, neoplastic cell or fungus. A Diterpenoid Compound is 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. 1 illustrates the variation in body weight of SCID mice over time following treatment with Compound 1 at a dose of 2 mg/kg and 10 mg/kg, respectively.
Fig. 2 illustrates the changes in tumor volume in SCID mice which were implanted with C33 A human cervical cancer cells and treated with Compound 1 at a dose of 2 mg/kg and 10 mg/kg, respectively.
5. DETAILED DESCRIPTION OF THE INVENTION
5.1 DEFINITIONS AND ABBREVIATIONS
As used herein, the term "C1-C1O 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-CiO alkoxy" means -0-(Ci-C1O alkyl), wherein C1-C1O alkyl is defined above.
As used herein, the term "C1-C1O (hydroxy)alkyl" means C1-C1O alkyl, wherein C1-C1O alkyl is defined above, substituted with one or more -OH groups. Examples of C1-CiO (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 "amino acid" means any naturally occurring amino acids and non-naturally occurring amino acids such as D-amino acids. For structures of amino acids, see, e.g., L. Stryer, Biochemistry, W.H. Freeman and Company, New York. An amino acid can be substituted with a protecting group. Suitable protecting groups for amino and amido groups include acetyl, tert-butoxy-C(O)-, benzyloxy-C(O)-, and the like. Suitable protecting groups for hydroxy include benzyl and the like. Suitable protecting groups for carboxy moieties include benzyl, tert-butyl, and the like. Other suitable protecting groups are well known to those of ordinary skill in the art and include those found in T. W. Greene, Protecting Groups in Organic Synthesis, John Wiley & Sons, Inc. 1981.
As used herein, the term "C1-C1O (amino)alkyl" means C1-C1O alkyl, wherein C1-C1O alkyl is defined above, substituted with one or more -NH2 groups. Examples of Ci-C1O (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-CiO (halo)alkyl" means Ci-C10 alkyl, wherein C1-CiO alkyl is defined above, substituted with one or more -F, -Cl, Br or -I groups. Examples of C1- Cio (halo)alkyl include, but are not limited to, trichloromethyl, trifluoromethyl, dichloromethyl, difluoromethyl, 1-fluoroethyl, 2-chloroethyl, 1-bromopropyl, 2- iodopropyl.S-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-C1O alkenyls include - vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutylenyl, -1-pentenyl, -2-pentenyl, - 3-methyl-l-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-CiO alkenyl. The double bond of a C2-C1O alkenyl can be unconjugated or conjugated to another unsaturated group. A -C2-C10 alkenyl can be unsubstituted or substituted with, e.g., -amino, -C1-C1O (oxy)alkyl, -halogen, -COOH, - C(O)C1-C9 alkyl, -SH, =S, -OH, and -C1-Ci0 alkoxy.
As used herein, unless otherwise specified the term "C2-C1O 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-C1O alkynyls include -acetylenyl, -propynyl, -1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl, - 3-methyl-l-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-C1O alkynyl. The triple bond of a C2-C1O alkynyl can be unconjugated or conjugated to another unsaturated group. A C2-C1O alkynyl can be unsubstituted or substituted with, e.g., -amino, -COOH, -halogen, Ci-C10 (oxy)alkyl, -C(O)C1-C9 alkyl, -SH, =S, -OH, C1-C10 alkoxy, and Ci-Ci0 alkyl.
As used herein, the term "(C3-C-7) 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- Cio(oxy)alkyl, -C(O)Ci-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-C1O (oxy)alkyl, -C(O)C1-C9 alkyl, -SH, =S, -OH, C1-Ci0 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)2ρhenyl, -(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 "C7-C12 (aryl)alkyl" means C7-C12 alkyl, wherein C7-Ci2 alkyl is defined above, substituted with one or more aryl groups, wherein aryl is defined above.
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-Ci0 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-C1O (oxy)alkyl" means C1-C1O alkyl, wherein C1-CiO 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 "peptide" is a sequence of two to six amino acids. In certain embodiments, a peptide is two, three, four, five, or six amino acids long.
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. Illustrative 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. In a preferred embodiment, a salt of a Diterpenoid Compound is mesylate or tartrate. 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" means separated 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.
As used herein the term "purified" means that the compound is free of other components such that the compound is pure. In certain embodiments, a compound of the invention is at least about 90% pure. In certain embodiments, a 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. It is recognized that Diterpenoid Compounds of the invention can have one or more chiral centers and/or double bonds and, therefore, exist as stereoisomers, such as double-bond isomers (Le., geometric isomers), enantiomers, or diastereomers. According to the invention, if a chemical structure depicted herein does not indicate the stereochemistry at a chiral center, the compound encompasses all of the corresponding enantiomers and stereoisomers, that is, both the stereomerically pure form (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) and enantiomeric and stereoisomeric mixtures, e.g., racemates.
Similarly, where a Diterpenoid Compound differs only by the placement of a proton and the corresponding location of a double-bond (tautomerism), the chemical structures depicted herein, and therefore the compounds of the invention or the compounds to be used with the methods of the invention, encompass all of the corresponding tautomers and the mixture of the tautomers.
As used herein and unless otherwise indicated, the term "stereomerically pure" means a composition that comprises one stereoisomer of a compound and is substantially free of other stereoisomers of that compound. For example, a stereomerically pure composition of a compound having one chiral center will be substantially free of the opposite enantiomer of the compound. A stereomerically pure composition of a compound having two chiral centers will be substantially free of other diasteroemers of the compound. A typical stereomerically pure compound comprises greater than about 80% by weight of stereoisomer of the compound and less than about 20% by weight of other stereoisomers the compound, more preferably greater than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of the other stereoisomers of the compound, even more preferably greater than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of the other stereoisomers of the compound, and most preferably greater than about 97% by weight of one stereoisomer of the compound and less than about 3% by weight of the other stereoisomers of the compound.
Enantiomeric and stereoisomeric mixtures of compounds of the invention can be resolved into their component enantiomers or stereoisomers by well-known methods, such as chiral-phase gas chromatography, chiral-phase high performance liquid chromatography, crystallizing the compound as a chiral salt complex, or crystallizing the compound in a chiral solvent. Enantiomers and stereoisomers can also be obtained from stereomerically or enantiomerically pure intermediates, reagents, and catalysts by well-known asymmetric synthetic methods.
It should be noted that if there is a discrepancy between a depicted structure and a name given that structure, the depicted structure controls. In addition, if the stereochemistry of a structure or a portion of a structure is not indicated with, for example, bold or dashed lines, the structure or portion of the structure is to be interpreted as encompassing all stereoisomers of it and racemic mixtures thereof.
Abbreviations
IBX iodoxybenzoate
TBAF tetra- n -butylammonium fluoride
BRDU bromodeoxyuridine i.v. intravenous
Rpm revolutions per minute
ATCC American Type Culture Collection
LC/MS Liquid Chromatography / Mass Spectrometry
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
The present invention encompasses compounds having the Formula (I):
Figure imgf000023_0001
(D
and pharmaceutically acceptable salts thereof, wherein:
A is N or CR4; B is N or CR5; D is N or CR6; E is N or CR7, at least one of A, B, D and E being CR4, CR5, CR6 or CR7, respectively; each R4, R5, R6 and R7 is independently -H, -OH, -halogen, -CN, -NH2, -NO2, -COOH, -C(O)NH2, -SH, -S(O)NH2, -S(O)2NH2, -C1-C10 (oxy)alkyl, -C1-C10 alkyl, -C1-CJ0 alkoxy, -Ci-Ci0 (hydroxy)alkyl, -C1-CiO (amino)alkyl, -C1-Ci0 (halo)alkyl, -C2-CiO 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, -OCH2ORn, -OC(O)Rn, -C(O)R11, -OC(O)ORn, -OC(O)NRn, -C(O)ORn, -C(O)NRn, -OP(O)(ORn)2, -SRn, -S(O)2NHRn, -SORn, -S(O)2Rn, -NHC(O)Rn, -NHSORn, NHS(O)2Ru, -OPO(ORi5)2, -O-arylPO(OR15)2, or -O-alkylarylPO(OR15)2; wherein R11 is -H, -C1-C1O alkyl, -(C3-C7) cycloalkyl, -C1-C1O (halo)alkyl, -aryl, -C2- C10 alkenyl, -C2-C1O alkynyl, -C1-C1O (aryl)alkyl, -C2-C10 (aryl)alkenyl, -C2-C10 (aryl)alkynyl, -C1-C10 (hydroxy)alkyl, -C1-C10 alkoxy, -C1-C1O (amino)alkyl, a -(C3-C7) cycloalkyl unsubstituted or substituted with one or more -C1-CiO 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; and wherein Ri5 is H, -Ci-Ci0 alkyl, C7-C12arylalkyl, Q-Qoaminoalkyl, Ci-C10haloalkyl, or Ci-Ci0 hydroxyalkyl. In certain, more specific embodiments, A is CR4; B is CR5; D is CR6; and E is CR7, wherein R4, R5, R6 and R7 are as defined above.
The present invention encompasses compounds having the Formula (IA):
Figure imgf000024_0001
(IA)
and pharmaceutically acceptable salts thereof, wherein:
A is N or CR4; B is N or CR5; D is N or CR6; E is N or CR7, at least one of A, B, D and E being CR4, CR5, CR6 or CR7, respectively; each R4, R5, R6 and R7 is independently -H, -OH, -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-CiO (hydroxy)alkyl, -C1-C1O (amino)alkyl, -Ci-C1O (halo)alkyl, -C2-C1O alkenyl, - C2-C1O alkynyl, -(C3-C7) cycloalkyl, -aryl, -C1-C1O (aryl)alkyl, three- to seven-membered" non- aromatic heterocycle, five- to seven-membered aromatic heterocycle, -CH2OR11, -OCH2ORn, -OC(O)R11, -C(O)R11, -OC(O)ORn, -OC(O)NR11, -C(O)ORn, -C(O)NRn, -OP(O)(ORn)2, -SRn, -S(O)2NHR11, -SOR11, -S(O)2R11, -NHC(O)R11, -NHSORn, NHS(O)2R11, -OPO(OR15)2, -O-arylPO(OR15)2, or -O-alkylarylPO(OR15)2 ; wherein Rn is -H, -C1-C10 alkyl, -(C3-C7) cycloalkyl, -C1-C10 (halo)alkyl, -aryl, -C2- C10 alkenyl, -C2-C10- alkynyl, -C1-C1O (aryl)alkyl, -C2-C10 (aryl)alkenyl, -C2-CiO (aryl)alkynyl, -C1-C10 (hydroxy)alkyl, -C1-Ci0 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 -Ci- C10 alkyl, -C2-Ci0 alkenyl, or -C2-Ci0 alkynyl; and wherein R1S is H, -C1-C1O alkyl, C7-C12arylalkyl, Q-Cioaminoalkyl, Q-Ciohaloalkyl, or C1-C1O hydroxyalkyl.
In certain, more specific embodiments, A is CR4; B is CR5; D is CR6; and E is CR7, wherein R4, R5, R6 and R7 are as defined above.
The present invention encompasses compounds having the Formula (IB):
Figure imgf000025_0001
(IB)
and pharmaceutically acceptable salts thereof, wherein:
A is N or CR4; B is N or CR5; D is N or CR6; E is N or CR7, at least one of A, B, D and E being CR4, CR5, CR6 or CR7, respectively; each R4, R5, R6 and R7 is independently -H, -OH, -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, -Ci-C10 (hydroxy)alkyl, -C1-C10 (amino)alkyl, -C1-C10 (halo)alkyl, -C2-Ci0 alkenyl, - C2-C10 alkynyl, -(C3-C7) cycloalkyl, -aryl, -Ci-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)ORn, -OC(O)NR11, -C(O)OR11, -C(O)NR11, -OP(O)(ORn)2, -SR11, -S(O)2NHR11, -SOR11, -S(O)2R11, -NHC(O)R11, -NHSOR11, NHS(O)2Rn, -OPO(ORiS)2, -O-arylPO(OR15)2, or -O-alkylarylPO(OR15)2 ; wherein Rn is -H, -C1-C10 alkyl, -(C3-C7) cycloalkyl, -C1-C10 (halo)alkyl, -aryl, -C2- C10 alkenyl, -C2-Ci0 alkynyl, -C1-C10 (aryl)alkyl, -C2-C10 (aryl)alkenyl, -C2-C10 (aryl)alkynyl, -C1-Ci0 (hydroxy)alkyl, -C1-C10 alkoxy, -C1-C10 (amino)alkyl, a -(C3-C7) cycloalkyl unsubstituted or substituted with one or more -Cj-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 -Q- C1O alkyl, -C2-CiO alkenyl, or -C2-CiO alkynyl; and wherein R15 is H, -Ci-Ci0 alkyl, C7-Ci2arylalkyl, Cj-Cioaminoalkyl, Q-Ciohaloalkyl, or Ci-Cio hydroxyalkyl.
In certain, more specific embodiments, A is CR4; B is CR5; D is CR6; and E is CR7, wherein R4, R5, R6 and R7 are as defined above.
5.3 FORMULA II
As stated above, the present invention encompasses compounds having the Formula (II):
Figure imgf000026_0001
(H) and pharmaceutically acceptable salts thereof, wherein Ri is as defined above for Formula (II). '
The present invention encompasses compounds having the Formula (ILA):
Figure imgf000026_0002
(HA) and pharmaceutically acceptable salts thereof, wherein Ri is as defined above for Formula (IIA).
The present invention encompasses compounds having the Formula (IIB):
Figure imgf000027_0001
(HB) and pharmaceutically acceptable salts thereof, wherein R1 is as defined above for Formula (IB).
A Diterpenoid Compound of Formula (I), Formula (IA) or Formula (IIB), Formula (II), Formula (IIA) or Formula (IIB) is useful for treating or preventing cancer or neoplastic disease in a patient in need of such treatment or prevention. A Diterpenoid Compound of Formula (I), Formula (IA) or Formula (IB), Formula (II), Formula (IIA) or Formula (IIB) is also useful for inhibiting the growth of a cancer cell or neoplastic cell. A Diterpenoid Compound of Formula (I), Formula (IA) or Formula (IB), Formula (II), Formula (IIA) or Formula (IIB) is also useful for inducing cytotoxicity, e.g., through apoptosis, in a cancer cell or neoplastic cell. A Diterpenoid Compound of Formula (I), Formula (IA) or Formula (IB), Formula (II), Formula (IIA) or Formula (IIB) is further useful for treating or preventing a fungal infection. A Diterpenoid Compound of Formula (I), Formula (IA) or Formula (IB), Formula (II), Formula (IIA) or Formula (IIB) is also useful for inhibiting the growth of a fungus.
In an illustrative embodiment, the invention provides 4-hydroxy-8-methoxy-3,3,9b- trimethylphenanthro[4,3-b]oxirene-2,5(laH,3H,9bH,9cH)-dione (Compound 1) and pharmaceutically acceptable salts thereof. >
Figure imgf000028_0001
Compound 1
In another illustrative embodiment, the invention provides (laR,9bR,9cR)-4-hydroxy- 8-methoxy-3,3,9b-trimethylphenanthro[4,3-b]oxirene-2,5(laH,3H,9bH,9cH)-dione (Compound Ia) and pharmaceutically acceptable salts thereof.
Figure imgf000028_0002
Compound Ia
In a further illustrative embodiment, the invention provides (laS,9bS,9cS)-4-hydroxy- 8-methoxy-3,3,9b-trimethylphenanthro[4,3-b]oxirene-2,5(laH,3H,9bH,9cH)-dione (Compound Ib) and pharmaceutically acceptable salts thereof.
Figure imgf000029_0001
Compound Ib
TAUTOMERIC REACTION
It is believed that compounds of Formula (I), Formula (IA), and Formula (IB) can be converted into their respective tautomers of Formula (I), Formula (IA), and Formula (IB).
Figure imgf000029_0002
Formula (I)
Figure imgf000029_0003
Formula (IA)
Figure imgf000030_0001
Formula (IB)
It is believed that compounds of Formula (II), Formula (IIA), and Formula (HB) can be converted into their respective tautomers of Formula (II), Formula (IIA), and Formula (DB).
Figure imgf000030_0002
Formula (II)
Figure imgf000030_0003
Formula (IIA)
Figure imgf000031_0001
Formula (IIB)
It is further believed that Compound 1, Compound Ia, and Compound Ib can be converted into their respective tautomers.
Figure imgf000031_0002
Compound 1
Figure imgf000031_0003
Compound Ia
Figure imgf000032_0001
Compound Ib
5.4 METHODS FOR MAKING DITERPENOID COMPOUNDS
The Diterpenoid Compounds can be obtained using conventional organic synthesis or by using the following illustrative methods shown in Schemes 1 to 8 below. Methods that can be used for the synthesis of the Diterpenoid Compounds are described, e.g., in Hijfte L.V., Little R.D., Petersen J.L., and Moeller K.D., J. Org. Chem. 1987, 52, 4647-4660; Numazawa M. and Tachibana M., Steroids, 1994, 59, 579-585; and Yang N.C. and Finnegan R.A. J. Am. Chem. Soc. 1958, 80, 5845-5848.
Diterpenoid Compounds of Formula (I) can be obtained from a compound of Formula (IV) as depticted in Scheme Ia (A, B, D, and E are as defined above). Scheme Ia
Figure imgf000032_0002
Formula (IV) Formula (I) Diterpenoid Compounds of Formula (IA) can be obtained from a compound of Formula (IVA) as depticted in Scheme Ib (A, B, D, and E are as defined above). Scheme Ib
Figure imgf000033_0001
Formula (IVA) Formula (IA)
Diterpenoid Compounds of Formula (IB) can be obtained from a compound of Formula (IVB) as depticted in Scheme Ic (A, B, D, and E are as defined above). Scheme Ic
Figure imgf000033_0002
Formula (IVB) Formula (IB)
Diterpenoid Compounds of Formula (II) can be obtained from a compound of Formula (III) as depticted in Scheme Id (R1 is as defined above). Scheme Id
Figure imgf000034_0001
Formula (III) Formula (II)
Diterpenoid Compounds of Formula (IIA) can be obtained from a compound of Formula (IIIA) as depticted in Scheme Ie (R1 is as defined above). Scheme Ie
Figure imgf000034_0002
Formula (IIIA) Formula (IIA)
Diterpenoid Compounds of Formula (HB) can be obtained from a compound of Formula (IIIB) as depticted in Scheme If (R1 is as defined above). Scheme If
Figure imgf000035_0001
Formula (IIIB) Formula (IIB)
Compound 1 can be obtained from Compound 2 as depticted in Scheme Ig. Scheme Ig
Figure imgf000035_0002
Compound 2 Compound 1
Compound Ia can be obtained from Compound 2a as depticted in Scheme Ih. Scheme Ih
Figure imgf000036_0001
Compound 2a Compound Ia
Compound Ib can be obtained from Compound 2b as depticted in Scheme Ii. Scheme Ii
Figure imgf000036_0002
Compound 2b Compound Ib
Diterpenoid Compounds of Formula (I) can also be obtained from a compound of Formula (IV) as depticted in Scheme 2a (A, B, D, and E are as defined above). Scheme 2a
Figure imgf000037_0001
Formula (IV) Formula (I)
Diterpenoid Compounds of Formula (IA) can be obtained from a compound of Formula (IVA) as depticted in Scheme 2b (A, B, D, and E are as defined above). Scheme 2b
Figure imgf000037_0002
Formula (IVA) Formula (IA)
Diterpenoid Compounds of Formula (IB) can be obtained from a compound of Formula (IVB) as depticted in Scheme 2c (A, B, D, and E are as defined above). Scheme 2c
Figure imgf000038_0001
Formula (IVB) Formula (IB)
Diterpenoid Compounds of Formula (II) can also be obtained from a compound of Formula (III) as depticted in Scheme 2d (R is as defined above).
Scheme 2d
Figure imgf000038_0002
Formula (III) Formula (II)
Diterpenoid Compounds of Formula (IIA) can also be obtained from a compound of Formula (IIIA) as depticted in Scheme 2e (R1 is as defined above). Scheme 2e
Figure imgf000039_0001
Formula (IIIA) Formula (HA)
Diterpenoid Compounds of Formula (IB) can also be obtained from a compound of Formula (HB) as depticted in Scheme 2f (R1 is as defined above).
Scheme 2f
Figure imgf000039_0002
Formula (HIB) Formula (IIB)
Compound 1 can be obtained from a Compound 2 as depticted in Scheme 2g.
Scheme 2g
Figure imgf000040_0001
Compound 2 Compound 1
Compound Ia can be obtained from Compound 2a as depticted in Scheme 2h.
Scheme 2h
Figure imgf000040_0002
Compound 2a Compound Ia
Compound Ib can be obtained from Compound 2b as depticted in Scheme 2i.
Scheme 2i
Figure imgf000041_0001
Compound 2b Compound Ib
Compounds of Formula (IV) can be obtained from a tetralone-type precursor such as depicted by compounds 13 in Scheme 3. Nucleophilic addition of -CH3 using an appropriate organometallic reagent, such as, a Grignard reagent (E.G. Ashby et al., J. Am. Chem. Soc, 89:1964 (1967)), followed by dehydration (C. Utermoehlen et al., /. 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 (LDA), followed by treatment with phenylselenium chloride (PhSeCl), hydrogen peroxide or meta-chloroperoxybenzoic acid (mCPBA; M. Tius et al., J. Am. Chem. Soc, U4:5959 (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, i.e., compounds of Formula (IV). Compounds of Formula III can be obtained analogously to the synthesis scheme for compounds of Formula IV.
Compounds 21 can be used as a starting material for the synthesis of Diterpenoid Compounds. Compounds 21 can be obtained (Scheme 4) in three steps from IBX-DMSO oxidation of aryl-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, 1Φ-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.
12-Methoxypodocarpa-8,ll,13-trieneoic acid (29) is a useful starting material for Compound 2. With reference to Scheme 5, compounds 29 can 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)). Compound 2 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)). Compound 2 is in equilibrium with triketo Compound 3.
Scheme 3
Figure imgf000043_0001
13 14
Figure imgf000043_0002
(Formula IV)
Scheme 4
Figure imgf000044_0001
28 21
Scheme 5
Figure imgf000045_0001
Compound 2 can be synthesized as illustrated in Scheme 6.
SCHEME 6
Figure imgf000046_0001
51
I) LiHMDS1 TMSCI PhSeCI
2) H2O2, CH2CI2/Pyridine
Figure imgf000046_0002
Figure imgf000046_0003
Compound 2a and Compound 2b can be synthesized as illustrated in Scheme 7.
SCHEME 7
Figure imgf000047_0001
Figure imgf000047_0002
Figure imgf000048_0001
5.5 PRODRUGS
The present invention also provides prodrugs of the Diterpenoid Compounds of the invention. Illustrative prodrugs of the Diterpenoid Compounds of the invention are:
Figure imgf000048_0002
Figure imgf000049_0001
Figure imgf000049_0002
Figure imgf000049_0003
Figure imgf000050_0001
Figure imgf000051_0001
a pharmaceutically acceptable salt thereof (Bn is benzyl). hi certain embodiments, the invention provides methods for treating cancer in a patient, comprising administering to the patient an effective amount of a prodrug of a Diterpenoid Compound of the invention, e.g., Compound 4, Compound 4a, Compound 4b, Compound 5, Compound 5a, Compound 5b, Compound 6, Compound 6a, or Compound 6b. A prodrug of a Diterpenoid Compound of the invention, e.g. , Compound 4, Compound 4a, Compound 4b, Compound 5, Compound 5a, Compound 5b, Compound 6, Compound 6a, or Compound 6b, is useful for treating or preventing cancer or neoplastic disease in a patient in need of such treatment or prevention. A prodrug of a Diterpenoid Compound of the invention, e.g., Compound 4, Compound 4a, Compound 4b, Compound 5, Compound 5a, Compound 5b, Compound 6, Compound 6a, or Compound 6b, is also useful for inhibiting the growth of a cancer cell or neoplastic cell. A prodrug of a Diterpenoid Compound of the invention, e.g., Compound 4, Compound 4a, Compound 4b, Compound 5, Compound 5a, Compound 5b, Compound 6, Compound 6a, or Compound 6b, is also useful for inducing cytotoxicity, e.g., through apoptosis, in a cancer cell or neoplastic cell. A prodrug of a Diterpenoid Compound of the invention, e.g., Compound 4, Compound 4a, Compound 4b, Compound 5, Compound 5a, Compound 5b, Compound 6, Compound 6a, or Compound 6b, is further useful for treating or preventing a fungal infection. A prodrug of a Diterpenoid Compound of the invention, e.g., Compound 4, Compound 4a, Compound 4b, Compound 5, Compound 5a, Compound 5b, Compound 6, Compound 6a, or Compound 6b, is also useful for inhibiting the growth of a fungus.
The present invention also provides additional prodrugs of the Diterpenoid Compounds of the invention. Prodrugs include derivatives of Diterpenoid Compounds that can hydrolyze, oxidize, or otherwise react under biological conditions (in vitro or in vivo) to provide an active Diterpenoid Compound of the invention. Examples of prodrugs include, but are not limited to, derivatives and metabolites of a compound of the invention that include biohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, and biohydrolyzable phosphate analogues. In certain embodiments, prodrugs of Diterpenoid Compounds with carboxyl functional groups are the lower alkyl esters of the carboxylic acid. The carboxylate esters are conveniently formed by esterifying any of the carboxylic acid moieties present on the molecule. Prodrugs can typically be prepared using well-known methods, such as those described by Burger's Medicinal Chemistry and Drug Discovery 6th ed. (Donald J. Abraham ed., 2001, Wiley) and Design and Application of Prodrugs (H. Bundgaard e d., 1985, Harwood Academic Publishers Gmfh). Biohydrolyzable moieties of a Diterpenoid Compound (i) do not interfere with the biological activity of the compound but can confer upon that compound advantageous properties in vivo, such as uptake, duration of action, or onset of action; or (ii) are biologically inactive but are converted in vivo to the biologically active compound. Examples of biohydrolyzable esters include, but are not limited to, lower alkyl esters, alkoxyacyloxy esters, alkyl acylamino alkyl esters, and choline esters. Examples of biohydrolyzable amides include, but are not limited to, lower alkyl amides, α-amino acid amides, alkoxyacyl amides, and alkylaminoalkylcarbonyl amides. Examples of biohydrolyzable carbamates include, but are not limited to, lower alkylamines, substituted ethylenediamines, aminoacids, hydroxyalkylamines, heterocyclic and heteroaromatic amines, and polyether amines. 5.5.1 SYNTHESIS OF PRODRUGS: COMPOUND 4, 5, AND 6
Illustrative prodrugs of Compound 1, Compounds 4, 5, and 6, can be synthesized as described below in Scheme 8. Prodrugs of other Diterpenoid Compounds can be synthesized analogously.
Scheme 8
Figure imgf000053_0001
5.6 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, hi 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, Florida (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, polymethylated castor oil (CREMAPHOR EL) 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. Patent 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, hi 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 celi 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.
5.7 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.,fo$, myc) or cell cycle markers (Rb, cdc2, cyclin A, Dl, 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 availably 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 ( H)-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 Gl 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 Gl 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.l35:783-92). 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 p21Cφl. Increased levels of p21 expression in cells result in delayed entry into Gl 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 Gl, 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 checkpoints), 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 Hl 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, MoI. 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 anticancer 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 (Calbiochern-Novabiochem Corp. San Diego, CA). 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.l93:518-25).
The Diterpenoid Compounds can also be demonstrated to inhibit tumor formation in vivo. A vast number of animal models of hyperpfoliferative 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.
5.7.1 INDUCING APOPTOSIS IN A CANCER CELL QR 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 nonneoplastic 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.
5.7.2 TREATMENT OR PREVENTION OF CANCER
OR A NEOPLASTIC DISEASE IN COMBINATION
WITH 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, if osf amide, 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 l
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
Oxaliplatin
Plant Alkaloids Vinca alkaloids: Vincristine
Vinblastine
Vindesine
Vinorelbine
Taxoids: Paclitaxel
Docetaxol
DNA Topoisomerase Inhibitors Epipodophyllins: Etoposide
Teniposide
Topotecan
9-aminocamptothecin campto irinotecan crisnatol
mitomycins: mitomycin C Mitomycin 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: Goserelin
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: l-methyl-4-phenylpyridinium ion Kinase inhibitors: Staurosporine
Imatinib mesylate
Actinomycins: Actinomycin D
Dactinomycin Bleomycins: Bleomycin A2
Bleomycin B2
Peplomycin
Anthracyclines: Daunorubicin
Doxorubicin (adriamycin)
Idarubicin
Epirubicin
Pirarubicin
Zorubicin
Mitoxantrone
MDR inhibitors verapamil Ca „2+ 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 than 1 MeV energy) can be used for deep tumors, and electron beam and ortho voltage 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.
5.7.3 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 t-cell leukemia acute lymphocytic leukemia acute myelocytic leukemia myeloblastic promyelocytic myelomonocytic Monocytic erythroleukemia chronic leukemia chronic myelocytic (granulocytic) leukemia chronic lymphocytic leukemia Hairy cell leukemia Polycythemia vera Lymphoma
Hodgkin's disease non-Hodgkin's disease Multiple myeloma Waldenstrom's macroglobulinemia Heavy chain disease Myelodysplastic syndrome 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
Anal carcinoma
Rectal carcinoma
Cancer of unknown primary
Thyroid carcinoma
Gastric carcinoma
Head and Neck carcinomas
Non-small cell lung carcinoma
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 certain specific embodiments, the cancer to be treated is Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), Acute Myeloid Leukemia/Other Myeloid Malignancies, Adrenocortical Carcinoma, ABDS-related Lymphoma, AIDS-related Malignancies, Alveolar Soft Part Sarcoma, Anal Cancer, Anaplastic Astrocytoma, Anaplastic Carcinoma, Thyroid, Angiosarcoma, Astrocytomas/Gliomas, Atypical Teratoid Rhabdoid Tumor, Basal Cell Carcinoma, Bile Duct Cancer, Bladder Cancer, Brain Stem Glioma (low grade and high grade), Burkitt's Lymphoma, Cancer of Unknown Primary (CUP), Carcinoid Tumor (gastrointestinal - usually appendix), Cervical Cancer, Childhood Leukemia, Childhood Hodgkin's Disease, Childhood Liver Cancer, Childhood Non-Hodgkin's Lymphoma, Childhood Rhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Cholangiocarcinoma (cancer of the bile ducts), Chondromsarcoma, Chordoma, Choroid Plexus Tumors, includes choroid plexus carcinoma & papilloma, Chronic Myelogenous Leukemia (CML), Clear Cell Sarcoma, CNS Lymphoma, Colon Cancer, Craniopharyngiomas, Cutaneous T-CeIl Lymphoma, Dermatofibrosarcoma Protuberans, Ductal Carcinoma - Invasive, Ductal Carcinoma in Situ (DCIS) (Non-invasive), Endometrial Cancer, Ependymoma, Epithelioid Sarcoma, Esophageal, Ewings Tumors and Primitive Neuroectodermal Tumors, Extraskeletal Chondrosarcoma, Extraskeletal Osteosarcoma, Fibrilary Astrocytoma, Fibrosarcoma, Follicular Carcinoma of Thyroid, Gallbladder Cancer, Gastric (stomach) Cancer, Gastrointestinal Stromal Tumor (GIST), Germ Cell Tumor, Germinoma, Germ Cell Tumor, Mixed Germ Cell Tumor, Gestational Trophoblastic Tumor (GTD) (placenta), Glioblastoma Multiformae (Also known as Astrocytoma Grade IVJ, Gliomas/Astrocytoma, Granular Cell Myoblastoma, Hairy Cell Leukemia, Hemangiosarcoma, Hepatobiliary, Hepatocellular (primary liver cancer), Hodgkin's Disease, Hurthle Cell Carcinoma of the Thyroid, Hypopharyngeal Cancer, Inflammatory Breast, Islet Cell Carcinoma (endocrine pancreas), Kaposi's Sarcoma, Kidney (Renal Cell) Cancer, Laryngeal Cancer, Leiomyosarcoma, Leukemia, Lip and Oral Cavity Cancer, Liposarcoma, Liver Cancer, Adult Primary (hepatocellular carcinoma), Liver cancer, Metastatic Lobular Carcinoma - Invasive, Lobular Carcinoma in Situ (LCIS) (Non-invasive), Lung Cancer, Lymphangiosaroma, Lymphoma, Male Breast Cancer, Malignant Fibrous Histiocytoma (MFH), Malignant Hemangiopericytoma, Malignant Mesenchymoma, Malignant Mesothelioma, Malignant Peripheral Nerve Sheath Tumor, Malignant Schwannoma, Malignant Thymoma, Medullary Carcinoma of the Thyroid, Medulloblastoma, Melanoma, Meningiomas, Mesenchymoma, Mesothelioma, Merkel Cell Carcinoma, Metastatic Cancer (may include lung, brain, spine, bone, lymph nodes, other), Multiple Myeloma/Plasma Cell Neoplasm, Mycosis Fungoides, Myelodysplastic Syndrome, Myeloproliferative Disorders, Nasopharyngeal Cancer, Neuroblastoma, Neurofibrosarcoma, Nipple (Paget's Disease of the Breast), Non-Hodgkin's Lymphoma (NHL), Non-Small Cell Lung, Oligodendroglioma, Oropharyngeal Cancer, Osteosarcoma, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor, Pancreatic Cancer, Papillary Carcinoma of the Thyroid, Paranasal Sinus and Nasal Cavity Cancer, Parathyroid Cancer, Penile Cancer, Peripheral Neuroectodermal Tumors, Pheochromocytoma (adrenal cancer), Pilocytic Astrocytoma, Pineal Parenchymal Tumor, Pineal Tumors, includes Pineoblastoma, Pituitary Tumor, includes Pituitary Adenoma, Primitive Neuroectodermal Tumors (Ewing's family of tumors), Primitive Neuroectodermal Tumors, Supratentorial, Primary Central Nervous System Lymphoma (CNS Lymphoma), Prostate Cancer, Rectal Cancer, Renal Pelvis and Ureter Cancer, Transitional Cell, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Schwannomas, Sezary Syndrome, Small Cell Lung, Small Intestine Cancer, Squamous Cell Neck Cancer, Stomach (Gastric) Cancer, Synovial sarcoma, T-CeIl Lymphoma, Cutaneous, Testicular Cancer, Thyroid Cancer, Urethral Cancer, Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma, Vulvar Cancer, Waldenstrom's Macroglobulinemia, Wilms' Tumor and Other Childhood Kidney Tumors.
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.
5.8 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. glabratd), Aspergillus (including A. fumigatus, A. flavus, A. niger, A. nidulans, A. terreus, A. sydowi, Aflavatus 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 externa. 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 immunoconiprised 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.
I
The following examples exemplify non-limiting aspects of the present invention. 6. EXAMPLES 6.1 ANTI-ONCOGENIC EFFECTS OF COMPOUND 1
6.1.1 SYNTHESIS OF COMPOUND 1 (METHOD A)
Sodium bicarbonate (725 mol%; 20.2 mmole; 1.70 g) is added to a stirred solution of Compound 2 (100 mol%; 2.79 mmole; 834 mg) in tetrahydrofuran (8.0 mL, dry), followed by addition of water (4.0 mL) and hydrogen peroxide (30% wt in water, 948 mol%; 26.5 mmole; 3.0 mL). The reaction mixture is stirred at room temperature for 40 h. Water (50 mL) is added, the resulting solution is extracted using ethyl acetate (2 x 50 mL). The organic layers are combined and washed successively using a solution of sodium thiosulfate (saturated aqueous, 50 mL) and brine (50 mL). The organic layer is dried over magnesium sulfate, filtered, and concentrated in vacuo to give Compound 1 (805 mg, 92% yield, >99% purity). Alternatively, the final product can be purified by column chromatography over silica gel using a gradient of 0-9 % of ethyl acetate in hexanes.
1H NMR (300 MHz, CDCl3): δ (ppm) 1.43 (s, 3H); 1.53 (s, 3H); 1.67 (s, 3H); 3.70 (d, J = 4.5 Hz, IH); 3.94 (s, 3H); 4.16 (d, J = 4.5 Hz, IH); 7.02 (m, 2H); 7.18 (s, IH); 8.20 (d, / = 7.9 Hz, IH).
13C NMR (300 MHz, CDCl3): δ (ppm) 21.53; 24.10; 28.99; 42.85; 46.78; 55.57; 56.00; 64.23; 111.48; 113.69; 121.75; 130.19; 132.44; 143.96; 149.28; 164.06; 179.18; 208.95
6.1.2 SYNTHESIS OF COMPOUND 1 (METHOD B)
To a stirred solution of Compound 2 (100 mol%, 6.70 mmole, 2.00 g), in dichloromethane (40 mL) at O0C, is added benzyltrimethylammonium hydroxide (Triton B, 40 % wt solution in methanol, 150 mol%;10.0 mmole; 4.0 mL) followed by tert-butyl hydroperoxide (5.0-6.0 M in decane, 300 mol%; 20.1 mmole; 4.0 mL). The mixture is warmed to room temperature and stirred overnight (18 h). Distilled water (100 mL) is added along with ethyl acetate (100 mL). The organic layer is washed with brine (2 x 100 mL). The combined aqueous layers are re-extracted using ethyl acetate (50 mL). The combined organic layers are dried over sodium sulfate, filtered and concentrated in vacuo to give practically pure compound. The white solid is recrystallized in 75 mL of hot ethyl acetate to give pure Compound 1 (1.52 g, 72 %).
6.1.3 EFFECT OF COMPOUND 1 ON GROWTH OF CERVICAL TUMOR CELLS
/N VIVO
To demonstrate the anti-tumor activity of Compound 1 in vivo, CB 17 SCID/SCID female mice (Charles River, MA, USA) that were injected with C33 A human cervical cancer cells (American Type Culture Collection, Manassas, VA USA) 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 X 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 CB 17 SCID/SCID mice. Each mouse was inoculated with a suspension of 2 X 106 tumor cells per 100 μL of PBS on day zero. The following treatment groups of eight mice each were used: (a) a negative control group treated with vehicle solution of 20% Cremaphor EL (Sigma, St. Louis, MS, USA), 10% Ethanol and 5% Dextrose (Abbot Laboratories, QC, Canada), (b) a group treated with 2 mg/kg of Compound 1, and (c) a group treated with 10 mg/kg of Compound 1.
Treatment started on day thirty six after C33A cells transplantation. Compound 1 was administered intravenously (i.v.) once daily for five consecutive days at a dose of 2 mg/kg and 10 mg/kg, respectively. Compound 1 was prepared as a working solution of 1.5 mg/mL in vehicle solution. The mice were weighed and the tumors measured on day thirty six and every 2 to 3 days after treatment commenced. Observations continued for 57 days after initial tumor implantation. The changes in body weight and in the calculated tumor volume were plotted (Figures 1 and 2). Statistical analysis was performed using GraphPad Prism (GraphPad Software Inc., San Diego, CA). 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.
As shown in Figure 1, mice treated with Compound 1 at 2 mg/kg or 10 mg/kg, respectively, experienced a non-significant change in body weight as compared to mice treated with vehicle only.
As shown in Figure 2, Compound 1 administered i.v. at a dose of 2 mg/kg once a day for five days resulted in a significant reduction (p=0.011) in tumor growth compared to mice treated i.v. with vehicle only. On Day 57, after initial tumor implantation, the mice treated with 2 mg/kg of Compound 1 had on average smaller tumors (p<0.05) compared to mice treated with vehicle only in the control group. The T/C values on Day 47 and Day 57, for mice treated with Compound 1 at a dose of 2 mg/kg, were 43% and 66%, respectively.
As shown in Figure 2, Compound 1 administered i.v. at a dose of 10 mg/kg once a day for five days resulted in a significant reduction (p<0.0001) in tumor growth compared to mice treated i.v. with vehicle only. On Day 57, after initial tumor implantation, the mice treated with 10 mg/kg of Compound 1 had on average smaller tumors (p<0.001) compared to mice treated with vehicle only in the control group. The T/C values on Day 47 and Day 57, for mice treated with Compound 1 at a dose of 10 mg/kg, were 21% and 48%, respectively.
As indicated in Figure 2, Compound 1 significantly reduces the human cervical tumors implanted in SCID mice, an art-accepted model for human cervical cancer. Accordingly, Compound 1 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.
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

What is claimed is:
1. A compound having the Formula (I):
Figure imgf000080_0001
(I)
or a pharmaceutically acceptable salt thereof, wherein:
A is N or CR4; B is N or CR5; D is N or CR6; E is N or CR7, at least one of A, B, D and E being CR4, CR5, CR6 or CR7, respectively; each R4, R5, R6 and R7 is independently -H, -OH, -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-Ci0 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)(ORn)2, -SR11, -S(O)2NHR11, -SOR11, -S(O)2R11, -NHC(O)R11, -NHSOR11, NHS(O)2R1L -OPO(ORIS)2, -O-arylPO(OR15)2, or -O-alkylarylPO(OR15)2 ; wherein R11 is -H, -C1-Ci0 alkyl, -(C3-C7) cycloalkyl, -C1-Ci0 (halo)alkyl, -aryl, -C2- C10 alkenyl, -C2-C10 alkynyl, -C1-Ci0 (aryl)alkyl, -C2-C10 (aryl)alkenyl, -C2-Ci0 (aryl)alkynyl, -Ci-Ci0 (hydroxy)alkyl, -C1-C10 alkoxy, -C1-Cj0 (amino)alkyl, a -(C3-C7) cycloalkyl unsubstituted or substituted with one or more -C1-Ci0 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-Ci0 alkenyl, or -C2-C10 alkynyl; and wherein R15 is H, -C1-Ci0 alkyl, C7-C12arylalkyl, Q-doaminoalkyl, Q-Ciohaloalkyl, or Ci-Cjohydroxyalkyl.
2. A compound having the Formula (IA):
Figure imgf000081_0001
(IA)
or a pharmaceutically acceptable salt thereof, wherein:
A is N or CR4; B is N or CR5; D is N or CR6; E is N or CR7, at least one of A, B, D and E being CR4, CR5, CR6 or CR7, respectively; each R4, R5, R6 and R7 is independently -H, -OH, -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-C1O (hydroxy)alkyl, -C1-C1O (amino)alkyl, -C1-C1O (halo)alkyl, -C2-C1O alkenyl, - C2-C1O alkynyl, -(C3-C7) cycloalkyl, -aryl, -C1-C1O (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)(ORn)2, -SR11, -S(O)2NHR11, -SOR11, -S(O)2R11, -NHC(O)R11, -NHSOR11, NHS(O)2R11, -OPO(OR15)2, -O-arylPO(OR15)2, or -O-alkylarylPO(ORi5)2; wherein R11 is -H, -C1-Ci0 alkyl, -(C3-C7) cycloalkyl, -C1-C10 (halo)alkyl, -aryl, -C2- Cio alkenyl, -C2-C1O alkynyl, -Ci-C1O (aryl)alkyl, -C2-CiO (aryl)alkenyl, -C2-C10 (aryl)alkynyl, -Ci-Cio (hydroxy)alkyl, -C1-Ci0 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-Ci0 alkyl, or a three- to seven-membered aromatic heterocycle unsubstituted or substituted with one or more -Ci- Cio alkyl, -C2-Ci0 alkenyl, or -C2-Cj0 alkynyl; and wherein R15 is H, -C1-C10 alkyl, C7-C12arylalkyl, Q-Qoaminoalkyl, Q-Qohaloalkyl, or Ci-Cio hydroxyalkyl.
3. A compound having the Formula (IB):
Figure imgf000082_0001
(IB)
or a pharmaceutically acceptable salt thereof, wherein:
A is N or CR4; B is N or CR5; D is N or CR6; E is N or CR7, at least one of A, B, D and E being CR4, CR5, CR6 or CR7, respectively; each R4, R5, R6 and R7 is independently -H, -OH, -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-Ci0 alkenyl, - C2-Ci0 alkynyl, -(C3-C7) cycloalkyl, -aryl, -Ci-C10 (aryl)alkyl, three- to seven-membered non- aromatic heterocycle, five- to seven-membered aromatic heterocycle, -CH2ORj1, -OCH2ORn, -OC(O)R11, -C(O)R11, -OC(O)ORn, -OC(O)NRn, -C(O)ORn, -C(O)NRn, -OP(O)(ORn)2, -SR11, -S(O)2NHR11, -SORn, -S(O)2R11, -NHC(O)R11, -NHSOR11, NHS(O)2Rn, -OPO(ORi5)2, -O-arylPO(OR15)2, or -O-alkylarylPO(OR15)2 wherein Rn is -H, -Ci-Ci0 alkyl, -(C3-C7) cycloalkyl, -Ci-Ci0 (halo)alkyl, -aryl, -C2- Ci0 alkenyl, -C2-Ci0 alkynyl, -Ci-Ci0 (aryl)alkyl, -C2-Ci0 (aryl)alkenyl, -C2-Ci0 (aryl)alkynyl, -Ci-Cio (hydroxy)alkyl, -Ci-Ci0 alkoxy, -Ci-Ci0 (amino)alkyl, a -(C3-C7) cycloalkyl unsubstituted or substituted with one or more -Ci-Ci0 alkyl, a three- to seven-membered non- aromatic heterocycle unsubstituted or substituted with one or more -Ci-Ci0 alkyl, or a three- to seven-membered aromatic heterocycle unsubstituted or substituted with one or more -C1- Ci0 alkyl, -C2-C10 alkenyl, or -C2-Ci0 alkynyl; and wherein R15 is H, -Ci-C10 alkyl, C7-C12arylalkyl, Ci-C10aminoalkyl, C)-C10haloalkyl, or C1-C10 hydroxyalkyl.
4. The compound of claim 1, 2, or 3, wherein A is CR4; B is CR5; D is CR6; and E is CR7.
5. A compound having the Formula (II):
Figure imgf000083_0001
(H) or a pharmaceutically acceptable salt thereof, wherein:
R1 is -H, -C(O)NH2, -S(O)NH2, -S(O)2NH2, -C1-C10 (oxy)alkyl, -C1-C10 alkyl, -C1-Ci0 (hydroxy)alkyl, -Ci-C10 (amino)alkyl, -Ci-Ci0 (halo)alkyl, -C2-C10 alkenyl, -C2-Ci0 alkynyl, - (C3-C7) cycloalkyl, -aryl, -Ci-Ci0 (aryl)alkyl, three- to seven-membered non-aromatic heterocycle, five- to seven-membered aromatic heterocycle, -CH2OR2, -C(O)R2, -C(O)OR2, - C(O)NR2, -P(O)(ORz)2, -S(O)2NHR2, -SOR2, -S(O)2R2, -NHC(O)R2, -NHSOR2, NHS(O)2R2; and wherein R2 is -H, -Ci-Ci0 alkyl, -(C3-C7) cycloalkyl, -Ci-Qo (halo)alkyl, -aryl, -C2-Ci0 alkenyl, -C2-Ci0 alkynyl, -Ci-Ci0 (aryl)alkyl, -C2-Ci0 (aryl)alkenyl, -C2-Ci0 (aryl)alkynyl, - Ci-Ci0 (hydroxy)alkyl, -C1-Ci0 alkoxy, -Ci-Ci0 (amino)alkyl, a -(C3-C7) cycloalkyl unsubstituted or substituted with one or more -Ci-Ci0 alkyl, a three- to seven-membered non- aromatic heterocycle unsubstituted or substituted with one or more -Ci-Ci0 alkyl, or a three- to seven-membered aromatic heterocycle unsubstituted or substituted with one or more -Q- Ci0 alkyl, -C2-Ci0 alkenyl, or -C2-CiO alkynyl.
6. A compound having the Formula (IIA):
Figure imgf000084_0001
(HA) or a pharmaceutically acceptable salt thereof, wherein:
R1 is -H, -C(O)NH2, -S(O)NH2, -S(O)2NH2, -C1-C10 (oxy)alkyl, -C1-C10 alkyl, -C1-C10 (hydroxy)alkyl, -C1-C10 (amino)alkyl, -C1-C10 (halo)alkyl, -C2-Ci0 alkenyl, -C2-C10 alkynyl, - (C3-C7) cycloalkyl, -aryl, -C1-Ci0 (aryl)alkyl, three- to seven-membered non-aromatic heterocycle, five- to seven-membered aromatic heterocycle, -CH2OR2, -C(O)R2, -C(O)OR2, - C(O)NR2, -P(O)(OR2)2, -S(O)2NHR2, -SOR2, -S(O)2R2, -NHC(O)R2, -NHSOR2, or NHS(O)2R2; and wherein R2 is -H, -C1-C10 alkyl, -(C3-C7) cycloalkyl, -C1-C10 (halo)alkyl, -aryl, -C2-Ci0 alkenyl, -C2-C10 alkynyl, -C1-C10 (aryl)alkyl, -C2-C10 (aryl)alkenyl, -C2-C10 (aryl)alkynyl, - Ci-Ci0 (hydroxy)alkyl, -Ci-C10 alkoxy, -C1-C10 (amino)alkyl, a -(C3-C7) cycloalkyl unsubstituted or substituted with one or more -Ci-C10 alkyl, a three- to seven-membered non- aromatic heterocycle unsubstituted or substituted with one or more -C1-Ci0 alkyl, or a three- to seven-membered aromatic heterocycle unsubstituted or substituted with one or more -Q- Ci0 alkyl, -C2-C10 alkenyl, or -C2-Ci0 alkynyl.
7. A compound having the Formula (IIB):
Figure imgf000085_0001
(HB) or a pharmaceutically acceptable salt thereof, wherein:
R1 is -H, -C(O)NH2, -S(O)NH2, -S(O)2NH2, -C1-C10 (oxy)alkyl, -C1-C10 alkyl, -C1-CJ0 (hydroxy)alkyl, -Ci-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, -CH2OR2, -C(O)R2, -C(O)OR2, - C(O)NR2, -P(O)(OR2)2, -S(O)2NHR2, -SOR2, -S(O)2R2, -NHC(O)R2, -NHSOR2, or NHS(O)2R2; and wherein R2 is -H, -C1-Ci0 alkyl, -(C3-C7) cycloalkyl, -C1-C10 (halo)alkyl, -aryl, -C2-Ci0 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-Ci0 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-Ci0 alkenyl, or -C2-C10 alkynyl.
8. A compound having the formula:
Figure imgf000086_0001
Compound 1;
Figure imgf000086_0002
Compound Ia;
Figure imgf000086_0003
Compound Ib; or a pharmaceutically acceptable salt thereof.
9. A pharmaceutical composition comprising (i) a compound having the Formula (I):
Figure imgf000087_0001
(D
or a pharmaceutically acceptable salt thereof, wherein:
A is N or CR4; B is N or CR5; D is N or CR6; E is N or CR7, at least one of A, B, D and E being CR4, CR5, CR6 or CR7, respectively; each R4, R5, R6 and R7 is independently -H, -OH, -halogen, -CN, -NH2, -NO2, - COOH, -C(O)NH2, -SH, -S(O)NH2, -S(O)2NH2, -Ci-C10 (oxy)alkyl, -C1-C10 alkyl, -C1-C10 alkoxy, -C1-C10 (hydroxy)alkyl, -C1-C10 (amino)alkyl, -C1-C10 (halo)alkyl, -C2-C1O alkenyl, - C2-CiO 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)Rn, -C(O)Rn, -OC(O)ORn, -OC(O)NR11, -C(O)OR11, -C(O)NRn, -OP(O)(OR1O2, -SRn, -S(O)2NHR11, -SORn, -S(O)2R11, -NHC(O)R11, -NHSOR11, NHS(O)2R11, -OPO(OR15)2, -O-arylPO(OR15)2, or -O-alkylarylPO(OR15)2 ; wherein Rn is -H, -C1-C10 alkyl, -(C3-C7) cycloalkyl, -C1-C1O (halo)alkyl, -aryl, -C2- Cio alkenyl, -C2-C10 alkynyl, -C1-C10 (aryl)alkyl, -C2-Ci0 (aryl)alkenyl, -C2-Ci0 (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-CiO alkyl, or a three- to seven-membered aromatic heterocycle unsubstituted or substituted with one or more -Ci- C1O alkyl, -C2-C1O alkenyl, or -C2-Ci0 alkynyl; wherein Ri5 is H, -C1-C10 alkyl, C7-C12arylalkyl, CrCioaminoalkyl, Q-Qohaloalkyl, or C1-C10 hydroxyalkyl; and (ii) a pharmaceutically acceptable carrier.
10. A pharmaceutical composition comprising (i) a compound having the Formula (IA): '
Figure imgf000088_0001
(IA)
or a pharmaceutically acceptable salt thereof, wherein:
A is N or CR4; B is N or CR5; D is N or CR6; E is N or CR7, at least one of A, B, D and E being CR4, CR5, CR6 or CR7, respectively; each R4, R5, R6 and R7 is independently -H, -OH, -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-Ci0 alkynyl, -(C3-C7) cycloalkyl, -aryl, -Ci-Ci0 (aryl)alkyl, three- to seven-membered non- aromatic heterocycle, five- to seven-membered aromatic heterocycle, -CH2ORi1, -OCH2OR11, -OC(O)R11, -C(O)R11, -OC(O)ORn, -OC(O)NR11, -C(O)OR11, -C(O)NR11, -OP(O)(ORn)2, -SR11, -S(O)2NHR11, -SOR11, -S(O)2R11, -NHC(O)Rn, -NHSORn, NHS(O)2Ri l, -OPO(ORiS)2, -O-arylPO(OR15)2, or -O-alkylarylPO(OR15)2; l wherein Rn 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-Ci0 (hydroxy)alkyl, -C1-Ci0 alkoxy, -Ci-Ci0 (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-Ci0 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; wherein R15 is H, -C1-C10 alkyl, C7-Ci2arylalkyl, Ci-C10aminoalkyl, C1-C10 haloalkyl, or C1-C10 hydroxyalkyl; and (ii) a pharmaceutically acceptable carrier.
11. A pharmaceutical composition comprising (i) a compound having the Formula (IB):
Figure imgf000089_0001
(IB)
or a pharmaceutically acceptable salt thereof, wherein:
A is N or CR4; B is N or CR5; D is N or CR6; E is N or CR7, at least one of A, B, D and E being CR4, CR5, CR6 or CR7, respectively; each R4, R5, R6 and R7 is independently -H, -OH, -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)ORn, -OC(O)NR11, -C(O)OR11, -C(O)NR11, -OP(O)(ORn)2, -SR11, -S(O)2NHR11, -SOR11, -S(O)2R11, -NHC(O)R11, -NHSOR11, NHS(O)2R11, -OPO(OR15)2, -O-arylPO(OR15)2, or -O-alkylarylPO(OR15)2 wherein Rn is -H, -C1-Ci0 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- Cio alkyl, -C2-Ci0 alkenyl, or -C2-Ci0 alkynyl; wherein Ri5 is H, -Ci-Ci0 alkyl, C7-Ci2arylalkyl, Ci-Ci0aminoalkyl, Ci-Ci0haloalkyl, or C1-C10 hydroxyalkyl; and (ii) a pharmaceutically acceptable carrier.
12. The pharmaceutical composition of claim 9, 10, or 11, wherein A is CR4; B is CR5; D is CR6; and E is CR7.
13. A pharmaceutical composition comprising (i) a compound having the Formula (H):
Figure imgf000090_0001
(H) or a pharmaceutically acceptable salt thereof, wherein:
R1 is -H, -C(O)NH2, -S(O)NH2, -S(O)2NH2, -C1-C10 (oxy)alkyl, -C1-C10 alkyl, -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, -CH2OR2, -C(O)R2, -C(O)OR2, - C(O)NR2, -P(O)(OR2)2, -S(O)2NHR2, -SOR2, -S(O)2R2, -NHC(O)R2, -NHSOR2, or NHS(O)2R2; wherein R2 is -H, -C1-Ci0 alkyl, -(C3-C7) cycloalkyl, -C1-Ci0 (halo)alkyl, -aryl, -C2-C10 alkenyl, -C2-C10 alkynyl, -C1-Ci0 (aryl)alkyl, -C2-C10 (aryl)alkenyl, -C2-Ci0 (aryl)alkynyl, - C1-Ci0 (hydroxy)alkyl, -Ci-Ci0 alkoxy, -Ci-Ci0 (amino)alkyl, a -(Ca-C7) cycloalkyl unsubstituted or substituted with one or more -Ci-Ci0 alkyl, a three- to seven-membered non- aromatic heterocycle unsubstituted or substituted with one or more -Ci-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; and (ii) a pharmaceutically acceptable carrier.
14. A pharmaceutical composition comprising (i) a compound having the Formula (IIA):
Figure imgf000091_0001
(HA) or a pharmaceutically acceptable salt thereof, wherein:
R1 is -H, -C(O)NH2, -S(O)NH2, -S(O)2NH2, -C1-Ci0 (oxy)alkyl, -C1-C10 alkyl, -C1-C10 (hydroxy)alkyl, -C1-C10 (amino)alkyl, -C1-C10 (halo)alkyl, -C2-CiO alkenyl, -C2-C1O alkynyl, - (C3-C7) cycloalkyl, -aryl, -C1-C10 (aryl)alkyl, three- to seven-membered non-aromatic heterocycle, five- to seven-membered aromatic heterocycle, -CH2OR2, -C(O)R2, -C(O)OR2, - C(O)NR2, -P(O)(OR2)2, -S(O)2NHR2, -SOR2, -S(O)2R2, -NHC(O)R2, -NHSOR2, or NHS(O)2R2; wherein R2 is -H, -C1-C10 alkyl, -(C3-C7) cycloalkyl, -Ci-C10 (halo)alkyl, -aryl, -C2-C10 alkenyl, -C2-C10 alkynyl, -C1-C10 (aryl)alkyl, -C2-C10 (aryl)alkenyl, -C2-C10 (aryl)alkynyl, - C1-CjO (hydroxy)alkyl, -C1-C1O alkoxy, -Ci-Ci0 (amino)alkyl, a -(C3-C7) cycloalkyl unsubstituted or substituted with one or more -Ci-Ci0 alkyl, a three- to seven-membered non- aromatic heterocycle unsubstituted or substituted with one or more -Ci-Ci0 alkyl, or a three- to seven-membered aromatic heterocycle unsubstituted or substituted with one or more -C1- Ci0 alkyl, -C2-Ci0 alkenyl, or -C2-CI0 alkynyl; and (ii) a pharmaceutically acceptable carrier.
15. A pharmaceutical composition comprising (i) a compound having the Formula (IIB):
Figure imgf000092_0001
(HB) or a pharmaceutically acceptable salt thereof, wherein:
R1 is -H, -C(O)NH2, -S(O)NH2, -S(O)2NH2, -C1-Ci0 (oxy)alkyl, -C1-C10 alkyl, -C1-C10 (hydroxy)alkyl, -C1-C10 (amino)alkyl, -C1-Ci0 (halo)alkyl, -C2-Ci0 alkenyl, -C2-C10 alkynyl, - (C3-C7) cycloalkyl, -aryl, -C1-Ci0 (aryl)alkyl, three- to seven-membered non-aromatic heterocycle, five- to seven-membered aromatic heterocycle, -CH2OR2, -C(O)R2, -C(O)OR2, - C(O)NR2, -P(O)(OR2)2, -S(O)2NHR2, -SOR2, -S(O)2R2, -NHC(O)R2, -NHSOR2, or NHS(O)2R2; wherein R2 is -H, -Ci-Ci0 alkyl, -(C3-C7) cycloalkyl, -C1-Ci0 (halo)alkyl, -aryl, -C2-Ci0 alkenyl, -C2-Ci0 alkynyl, -Ci-C10 (aryl)alkyl, -C2-C10 (aryl)alkenyl, -C2-C10 (aryl)alkynyl, - C1-Ci0 (hydroxy)alkyl, -C1-C10 alkoxy, -Ci-C10 (amino)alkyl, a -(C3-C7) cycloalkyl unsubstituted or substituted with one or more -Ci-Ci0 alkyl, a three- to seven-membered non- aromatic heterocycle unsubstituted or substituted with one or more -Ci-Ci0 alkyl, or a three- to seven-membered aromatic heterocycle unsubstituted or substituted with one or more -Ci- Cio alkyl, -C2-CiO alkenyl, or -C2-Ci0 alkynyl; and (ii) a pharmaceutically acceptable carrier.
16. A pharmaceutical composition comprising (i)
Figure imgf000092_0002
Compound 1;
Figure imgf000093_0001
Compound Ia;
Figure imgf000093_0002
Compound Ib; or a pharmaceutically acceptable salt thereof; and (ii) a pharmaceutically acceptable carrier.
17. A method 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 compound having the Formula (I):
Figure imgf000093_0003
(D or a pharmaceutically acceptable salt thereof, wherein:
A is N or CR4; B is N or CR5; D is N or CR6; E is N or CR7, at least one of A, B, D and E being CR4, CR5, CR6 or CR7, respectively; each R4, R5, R6 and R7 is independently -H, -OH, -halogen, -CN, -NH2, -NO2, - COOH, -C(O)NH2, -SH, -S(O)NH2, -S(O)2NH2, -C1-C10 (oxy)alkyl, -C1-C10 alkyl, -Ci-C10 alkoxy, -C1-Ci0 (hydroxy)alkyl, -Ci-C10 (amino)alkyl, -C1-C10 (halo)alkyl, -C2-C10 alkenyl, - C2-Ci0 alkynyl, -(C3-C7) cycloalkyl, -aryl, -C1-C10 (aryl)alkyl, three- to seven-membered non- aromatic heterocycle, five- to seven-membered aromatic heterocycle, -CH2ORn, -OCH2OR11, -OC(O)R11, -C(O)R11, -OC(O)ORn, -OC(O)NRn, -C(O)ORn, -C(O)NR11, -OP(O)(OR1O2, -SRn, -S(O)2NHRn, -SORn, -S(O)2Rn, -NHC(O)Rn, -NHSORn, NHS(O)2R11, -OPO(OR15)2, -O-arylPO(OR15)2, or -O-alkylarylPO(OR15)2 ; wherein Rn is -H, -C1-Ci0 alkyl, -(C3-C7) cycloalkyl, -Ci-C10 (halo)alkyl, -aryl, -C2- C10 alkenyl, -C2-C10 alkynyl, -C1-C10 (aryl)alkyl, -C2-C10 (aryl)alkenyl, -C2-C10 (aryl)alkynyl, -Ci-Ci0 (hydroxy)alkyl, -C1-C10 alkoxy, -C1-Ci0 (amino)alkyl, a -(C3-C7) cycloalkyl unsubstituted or substituted with one or more -Ci-Ci0 alkyl, a three- to seven-membered non- aromatic heterocycle unsubstituted or substituted with one or more -Ci-C10 alkyl, or a three- to seven-membered aromatic heterocycle unsubstituted or substituted with one or more -Ci-, Cio alkyl, -C2-Ci0 alkenyl, or -C2-Ci0 alkynyl; and wherein Ri5 is H, -Ci-Ci0 alkyl, C7-Ci2arylalkyl, Ci-Ci0aminoalkyl, Ci-Ci0 haloalkyl, or C1-C10 hydroxyalkyl.
18. A method 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 compound having the Formula (IA):
Figure imgf000094_0001
(IA) or a pharmaceutically acceptable salt thereof, wherein:
A is N or CR4; B is N or CR5; D is N or CR6; E is N or CR7, at least one of A, B, D and E being CR4, CR5, CR6 or CR7, respectively; each R4, R5, R6 and R7 is independently -H, -OH, -halogen, -CN, -NH2, -NO2, - COOH, -C(O)NH2, -SH, -S(O)NH2, -S(O)2NH2, -C1-C10 (oxy)alkyl, -C1-C10 alkyl, -CrC10 alkoxy, -C1-Ci0 (hydroxy)alkyl, -Ci-Ci0 (aminό)alkyl, -C1-C10 (halo)alkyl, -C2-C10 alkenyl, - C2-Ci0 alkynyl, -(C3-C7) cycloalkyl, -aryl, -Ci-Ci0 (aryl)alkyl, three- to seven-membered non- aromatic heterocycle, five- to seven-membered aromatic heterocycle, -CH2OR11, -OCH2ORn, -OC(O)Rn, -C(O)Rn, -OC(O)ORn, -OC(O)NRn, -C(O)ORn, -C(O)NRn, -OP(O)(ORn)2, -SRn, -S(O)2NHRn, -SORn, -S(O)2Rn, -NHC(O)Rn, -NHSORn, NHS(O)2R11, -OPO(OR15)2, -O-arylPO(OR15)2, or -O-alkylarylPO(OR15)2; wherein R11 is -H, -Ci-Ci0 alkyl, -(C3-C7) cycloalkyl, -C1-C10 (halo)alkyl, -aryl, -C2- Cio alkenyl, -C2-C10 alkynyl, -C1-C10 (aryl)alkyl, -C2-C10 (aryl)alkenyl, -C2-Ci0 (aryl)alkynyl, -C1-C10 (hydroxy)alkyl, -Ci-Ci0 alkoxy, -Ci-Ci0 (amino)alkyl, a -(C3-C7) cycloalkyl unsubstituted or substituted with one or more -Ci-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- Ci0 alkyl, -C2-C10 alkenyl, or -C2-C10 alkynyl; and wherein R15 is H, -C1-C10 alkyl, C7-C12arylalkyl, Ci-Ci0aminoalkyl, Ci-Ci0haloalkyl, or Ci-Cio hydroxyalkyl.
19. A method 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 compound having the Formula (IB):
Figure imgf000096_0001
(IB)
or a pharmaceutically acceptable salt thereof, wherein:
A is N or CR4; B is N or CR5; D is N or CR6; E is N or CR7, at least one of A, B, D and E being CR4, CR5, CR6 or CR7, respectively; each R4, R5, R6 and R7 is independently -H, -OH, -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-C1O (hydroxy)alkyl, -C1-CiO (amino)alkyl, -C1-C10 (halo)alkyl, -C2-C1O alkenyl, - C2-C1O alkynyl, -(C3-C7) cycloalkyl, -aryl, -C1-C1O (aryl)alkyl, three- to seven-membered non- aromatic heterocycle, five- to seven-membered aromatic heterocycle, -CH2OR11, -OCH2OR11, -OC(O)R11, -C(O)Rn, -OC(O)OR11, -OC(O)NRn, -C(O)OR11, -C(O)NRn, -OP(O)(ORn)2, -SR11, -S(O)2NHR11, -SOR11, -S(O)2R11, -NHC(O)R11, -NHSORn, NHS(O)2Rn, -OPO(OR15)2, -O-arylPO(ORi5)2, or -O-alkylarylPO(OR15)2 wherein R11 is -H, -C1-C10 alkyl, -(C3-C7) cycloalkyl, -C1-C10 (halo)alkyl, -aryl, -C2- Cio alkenyl, -C2-C10 alkynyl, -C1-C1O (aryl)alkyl, -C2-C1O (aryl)alkenyl, -C2-C10 (aryl)alkynyl, -C1-C10 (hydroxy)alkyl, -Ci-Cio alkoxy, -C1-C1O (amino)alkyl, a -(C3-C7) cycloalkyl unsubstituted or substituted with one or more -C1-C1O alkyl, a three- to seven-membered non- aromatic heterocycle unsubstituted or substituted with one or more -C1-Ci0 alkyl, or a three- to seven-membered aromatic heterocycle unsubstituted or substituted with one or more -C1- Cio alkyl, -C2-C1O alkenyl, or -C2-C1O alkynyl; and wherein R15 is H, -C1-CiO alkyl, C7-C12arylalkyl, d-Qoaminoalkyl, C1-C1O haloalkyl, or C1-C1O hydroxyalkyl.
20. The method of claim 17, 18, or 19, wherein A is CR4; B is CR5; D is CR6; and E is CR7.
21. A method 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 compound having the Formula (II):
Figure imgf000097_0001
(H) or a pharmaceutically acceptable salt thereof, wherein:
R1 is -H, -C(O)NH2, -S(O)NH2, -S(O)2NH2, -C1-C10 (oxy)alkyl, -C1-C10 alkyl, -C1-C10 (hydroxy)alkyl, -C1-C10 (amino)alkyl, -C1-C1O (halo)alkyl, -C2-C10 alkenyl, -C2-C1O alkynyl, - (C3-C7) cycloalkyl, -aryl, -Cj-C1O (aryl)alkyl, three- to seven-membered non-aromatic heterocycle, five- to seven-membered aromatic heterocycle, -CH2OR2, -C(O)R2, -C(O)OR2, - C(O)NR2, -P(O)(OR2)2, -S(O)2NHR2, -SOR2, -S(O)2R2, -NHC(O)R2, -NHSOR2, or NHS(O)2R2 ; and wherein R2 is -H, -C1-C10 alkyl, -(C3-C7) cycloalkyl, -C1-C10 (halo)alkyl, -aryl, -C2-C10 alkenyl, -C2-C10 alkynyl, -Ci-C1O (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.
22. A method 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 compound having the Formula (IIA):
Figure imgf000098_0001
(IIA) or a pharmaceutically acceptable salt thereof, wherein:
R1 is -H, -C(O)NH2, -S(O)NH2, -S(O)2NH2, -C1-C10 (oxy)alkyl, -C1-C10 alkyl, -Ci-C10 (hydroxy)alkyl, -C1-C10 (amino)alkyl, -C1-C10 (halo)alkyl, -C2-Ci0 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, -CH2OR2, -C(O)R2, -C(O)OR2, - C(O)NR2, -P(O)(OR2)2, -S(O)2NHR2, -SOR2, -S(O)2R2, -NHC(O)R2, -NHSOR2, or NHS(O)2R2 ; and wherein R2 is -H, -Ci-Ci0 alkyl, -(C3-C7) cycloalkyl, -Ci-Ci0 (halo)alkyl, -aryl, -C2-Ci0 alkenyl, -C2-Ci0 alkynyl, -Ci-Ci0 (aryl)alkyl, -C2-Ci0 (aryl)alkenyl, -C2-Ci0 (aryl)alkynyl, - Ci-Ci0 (hydroxy)alkyl, -Ci-C)0 alkoxy, -Ci-Ci0 (amino)alkyl, a -(C3-C7) cycloalkyl unsubstituted or substituted with one or more -Ci-Ci0 alkyl, a three- to seven-membered non- aromatic heterocycle unsubstituted or substituted with one or more -Ci-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-Ci0 alkynyl.
23. A method 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 compound having the Formula (IIB):
Figure imgf000099_0001
(HB) or a pharmaceutically acceptable salt thereof, wherein:
R1 is -H, -C(O)NH2, -S(O)NH2, -S(O)2NH2, -C1-C10 (oxy)alkyl, -C1-C10 alkyl, -C1-C10 (hydroxy)alkyl, -Ci-Ci0 (amino)alkyl, -C1-C10 (halo)alkyl, -C2-CiO alkenyl, -C2-C10 alkynyl, - (C3-C7) cycloalkyl, -aryl, -Ci-C10 (aryl)alkyl, three- to seven-membered non-aromatic heterocycle, five- to seven-membered aromatic heterocycle, -CH2OR2, -C(O)R2, -C(O)OR2, - C(O)NR2, -P(O)(OR2)2, -S(O)2NHR2, -SOR2, -S(O)2R2, -NHC(O)R2, -NHSOR2, or NHS(O)2R2 ; and wherein R2 is -H, -C1-C10 alkyl, -(C3-C7) cycloalkyl, -C1-Ci0 (halo)alkyl, -aryl, -C2-Ci0 alkenyl, -C2-Ci0 alkynyl, -Ci-Ci0 (aryl)alkyl, -C2-Ci0 (aryl)alkenyl, -C2-Ci0 (aryl)alkynyl, - Ci-Cio (hydroxy)alkyl, -Ci-C)0 alkoxy, -Ci-Ci0 (amino)alkyl, a -(C3-C7) cycloalkyl unsubstituted or substituted with one or more -Ci-C10 alkyl, a three- to seven-membered non- aromatic heterocycle unsubstituted or substituted with one or more -Ci-Ci0 alkyl, or a three- to seven-membered aromatic heterocycle unsubstituted or substituted with one or more -C1- Cio alkyl, -C2-Ci0 alkenyl, or -C2-C10 alkynyl.
24. A method 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 compound having the formula:
Figure imgf000100_0001
Compound 1;
Figure imgf000100_0002
Compound Ia;
Figure imgf000100_0003
Compound Ib; or a pharmaceutically acceptable salt thereof.
25. A method for inhibiting the growth of a cancer cell or neoplastic cell comprising contacting the cancer cell or neoplastic cell with an effective amount of the compound of claim 1, 2, 3, 5, 6, 7, or 8.
26. A method for inhibiting the growth of a cancer cell or neoplastic cell comprising contacting the cancer cell or neoplastic cell with an effective amount of the compound of claim 4.
27. A method for inducing cytotoxicity in a cancer cell or neoplastic cell comprising contacting the cancer cell or neoplastic cell with an effective amount of the compound of claim 1, 2, 3, 5, 6, 7, or 8.
28. A method for inducing cytotoxicity in a cancer cell or neoplastic cell comprising contacting the cancer cell or neoplastic cell with an effective amount of the compound of claim 4.
29. The method of claim 17, 18, 19, 21, 22, 23, or 24, wherein the cancer or neoplastic disease is Leukemia, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, myeloblastic leukemia, Promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, chronic leukemia, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, Polycythemia vera, Lymphoma, Hodgkin's disease, non-Hodgkin's disease, Multiple myeloma, Waldenstrom's macroglobulinemia, Heavy chain disease, 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 or Bronchial epithelial cancer.
30. The method of claim 20, wherein the cancer or neoplastic disease is Leukemia, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, myeloblasts leukemia, Promyelocyte leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, chronic leukemia, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, Polycythemia vera, Lymphoma, Hodgkin's disease, non-Hodgkin's disease, Multiple myeloma, Waldenstrom's macroglobulinemia, Heavy chain disease, 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 or Bronchial epithelial cancer.
31. The method of claim 26 or 28, wherein the cancer cell or neoplastic cell is a Leukemia, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, myeloblastic leukemia, Promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, chronic leukemia, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, Polycythemia vera, Lymphoma, Hodgkin's disease, non- Hodgkin's disease, Multiple myeloma, Waldenstrom's macroglobulinemia, Heavy chain disease, 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 or Bronchial epithelial cancer cell.
32. The method of claim 25, wherein the cancer cell or neoplastic cell is a Leukemia, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, myeloblastic leukemia, Promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, chronic leukemia, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, Polycythemia vera, Lymphoma, Hodgkin's disease, non- Hodgkin's disease, Multiple myeloma, Waldenstrom's macroglobulinemia, Heavy chain disease, 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 or Bronchial epithelial cancer cell.
33. The method of claim 27, wherein the cancer cell or neoplastic cell is a Leukemia, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, myeloblastic leukemia, Promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, chronic leukemia, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, Polycythemia vera, Lymphoma, Hodgkin's disease, non- Hodgkin's disease, Multiple myeloma, Waldenstrom's macroglobulinemia, Heavy chain disease, 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 or Bronchial epithelial cancer cell.
34. The method of claim 27, wherein the cytotoxicity is apoptosis.
35. The method of claim 28, wherein the cytotoxicity is apoptosis.
36. A method for treating a fungal infection comprising administering to a patient in need of such treatment an effective amount of the compound of claim 1, 2, 3, 5, 6, 7, or 8.
37. A method for treating a fungal infection comprising administering to a patient in need of such treatment an effective amount of the compound of claim 4.
38. A method for inhibiting the growth of a fungus comprising contacting the fungus with an effective amount of the compound of claim 1, 2, 3, 5, 6, 7, or 8.
39. A method for inhibiting the growth of a fungus comprising contacting the fungus with an effective amount of the compound of claim 4.
40. The method of claim 37 or 39, wherein the fungus is Candida, Aspergillus, 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.
41. The method of claim 36, wherein the fungus is Candida, Aspergillus, 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.
42. The method of claim 38, wherein the fungus is Candida, Aspergillus, 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.
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US20120196880A1 (en) * 2010-12-17 2012-08-02 Eric Anderson Pyrazolyl and pyrimidinyl tricyclic enones as antioxidant inflammation modulators
US8513436B2 (en) * 2010-12-17 2013-08-20 Reata Pharmaceuticals, Inc. Pyrazolyl and pyrimidinyl tricyclic enones as antioxidant inflammation modulators
US9884809B2 (en) 2010-12-17 2018-02-06 Reata Pharmaceuticals, Inc. Pyrazolyl and pyrimidinyl tricyclic enones as antioxidant inflammation modulators
US11192852B2 (en) 2010-12-17 2021-12-07 Reata Pharmaceuticals, Inc. Pyrazolyl and pyrimidinyl tricyclic enones as antioxidant inflammation modulators
US11814338B2 (en) 2010-12-17 2023-11-14 Reata Pharmaceuticals, Inc. Pyrazolyl and pyrimidinyl tricyclic enones as antioxidant inflammation modulators
US11059792B2 (en) 2015-02-12 2021-07-13 Reata Pharmaceuticals, Inc. Imidazolyl tricyclic enones as antioxidant inflammation modulators
US11292781B2 (en) 2016-12-16 2022-04-05 Reata Pharmaceuticals, Inc. Pyrimidine tricyclic enone derivatives for inhibition of ROR-gamma and other uses

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