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WO2008124699A1 - Procédé d'utilisation d'inhibiteurs du protéasome en combinaison avec des inhibiteurs d'histone désacétylase pour traiter un cancer - Google Patents

Procédé d'utilisation d'inhibiteurs du protéasome en combinaison avec des inhibiteurs d'histone désacétylase pour traiter un cancer Download PDF

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Publication number
WO2008124699A1
WO2008124699A1 PCT/US2008/059592 US2008059592W WO2008124699A1 WO 2008124699 A1 WO2008124699 A1 WO 2008124699A1 US 2008059592 W US2008059592 W US 2008059592W WO 2008124699 A1 WO2008124699 A1 WO 2008124699A1
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Prior art keywords
compound
alkyl
group
salinosporamide
optionally substituted
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PCT/US2008/059592
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English (en)
Inventor
Michael Palladino
Kenneth C. Anderson
Dharminder Chauhan
Joya Chandra
David Mcconkey
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Nereus Pharmaceuticals, Inc.
Dana-Farber Cancer Institute
University Of Texas M.D. Anderson Cancer Center
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Application filed by Nereus Pharmaceuticals, Inc., Dana-Farber Cancer Institute, University Of Texas M.D. Anderson Cancer Center filed Critical Nereus Pharmaceuticals, Inc.
Publication of WO2008124699A1 publication Critical patent/WO2008124699A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/166—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the carbon of a carboxamide group directly attached to the aromatic ring, e.g. procainamide, procarbazine, metoclopramide, labetalol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/167—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the nitrogen of a carboxamide group directly attached to the aromatic ring, e.g. lidocaine, paracetamol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/336—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having three-membered rings, e.g. oxirane, fumagillin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/4045—Indole-alkylamines; Amides thereof, e.g. serotonin, melatonin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/427—Thiazoles not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4406—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 3, e.g. zimeldine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/473—Quinolines; Isoquinolines ortho- or peri-condensed with carbocyclic ring systems, e.g. acridines, phenanthridines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to certain compounds and to methods for the preparation and the use of certain compounds in the fields of chemistry and medicine.
  • Cancer is a leading cause of death in the United States. Despite significant efforts to find new approaches for treating cancer, the primary treatment options remain surgery, chemotherapy and radiation therapy, either alone or in combination. Surgery and radiation therapy, however, are generally useful only for fairly defined types of cancer, and are of limited use for treating patients with disseminated disease.
  • Chemotherapy is the method that is generally useful in treating patients with metastatic cancer or diffuse cancers such as leukemias. Although chemotherapy can provide a therapeutic benefit, it often fails to result in cure of the disease due to the patient's cancer cells becoming resistant to the chemotherapeutic agent. Due, in part, to the likelihood of cancer cells becoming resistant to a chemotherapeutic agent, such agents are commonly used in combination to treat patients.
  • infectious diseases caused, for example, by bacteria, fungi and protozoa are becoming increasingly difficult to treat and cure.
  • bacteria, fungi and protozoa are developing resistance to current antibiotics and chemotherapeutic agents.
  • examples of such microbes include Bacillus, Leishmania, Plasmodium and Trypanosoma.
  • Marine-derived natural products are a rich source of potential new anticancer agents and anti-microbial agents.
  • the oceans are massively complex and house a diverse assemblage of microbes that occur in environments of extreme variations in pressure, salinity, and temperature.
  • Marine microorganisms have therefore developed unique metabolic and physiological capabilities that not only ensure survival in extreme and varied habitats, but also offer the potential to produce metabolites that would not be observed from terrestrial microorganisms (Okami, Y. 1993 J Mar Biotechnol 1:59).
  • Representative structural classes of such metabolites include terpenes, peptides, polyketides, and compounds with mixed biosynthetic origins.
  • the embodiments disclosed herein generally relate to chemical compounds, including heterocyclic compounds and analogs thereof. Some embodiments are directed to the use of compounds as proteasome inhibitors.
  • the compounds are used to treat neoplastic diseases, for example, to inhibit the growth of tumors, cancers and other neoplastic tissues.
  • neoplastic diseases for example, to inhibit the growth of tumors, cancers and other neoplastic tissues.
  • the methods of treatment disclosed herein can be employed with any patient suspected of carrying tumorous growths, cancers, or other neoplastic growths, either benign or malignant ("tumor” or “tumors” as used herein encompasses tumors, cancers, disseminated neoplastic cells and localized neoplastic growths).
  • growths include but are not limited to breast cancers; hematologic malignancies including lymphomas, such as Hodgkin's lymphoma and non-hodgkin's lymphoma, leukemias, and multiple myelomas; osteosarcomas, angiosarcomas, fibrosarcomas and other sarcomas; leukemias; sinus tumors; ovarian, uretal, bladder, prostate and other genitourinary cancers; colon, esophageal and stomach cancers and other gastrointestinal cancers; rectal cancers; lung cancers (such as large cell carcinoma, small cell carcinoma, large cell carcinoma, squamous cell carcinoma, adenocarcinoma, and bronchioloalveolar carcinoma); lymphomas; myelomas; teratomas; pancreatic cancers; liver cancers; kidney cancers; endocrine cancers; skin cancers; eye cancers; cervical cancers; anal carcinomas; hepato
  • the tumor or growth to be treated can be any tumor or cancer, primary or secondary.
  • Certain embodiments relate to methods of treating neoplastic diseases in animals.
  • the method can include, for example, administering an effective amount of a compound to a patient in need thereof.
  • Other embodiments relate to the use of compounds in the manufacture of a pharmaceutical or medicament for the treatment of a neoplastic disease.
  • the compounds can be administered or used in combination with treatments such as chemotherapy, radiation, and biologic therapies.
  • the compounds can be administered or used with a chemotherapeutic agent.
  • chemotherapeutics include Alkaloids, alkylating agents, antibiotics, antimetabolites, enzymes, hormones, platinum compounds, immunotherapeutics (antibodies, T-cells, epitopes), BRMs, and the like.
  • examples include, Vincristine, Vinblastine, Vindesine, Paclitaxel (Taxol), Docetaxel, topoisomerase inhibibitors epipodophyllotoxins (Etoposide (VP- 16), Teniposide (VM-26)), Camptothecin, nitrogen mustards (cyclophosphamide), Nitrosoureas, Carmustine, lomustine, dacarbazine, hydroxymethylmelamine, thiotepa and mitocycin C, Dactinomycin (Actinomycin D), anthracycline antibiotics (Daunorubicin, Daunomycin, Cerubidine), Doxorubicin (Adriamycin), Idarubicin (Ida
  • the two major anticancer drugs in this category are 6-mercaptopurine and 6-thioguanine, Chlorodeoxyadenosine and Pentostatin, Pentostatin (2'-deoxycoformycin), pyrimidine antagonists, Avastin, Leucovorin, Oxaliplatin, fluoropyrimidines (5-fluorouracil(Adrucil), 5-fluorodeoxyuridine (FdUrd) (Floxuridine)), Cytosine Arabinoside (Cytosar, ara-C), Fludarabine, Hydroxyurea, glucocorticoids, antiestrogens, tamoxifen, nonsteroidal antiandrogens, flutamide, aromatase inhibitors Anastrozole(Arimidex), Cisplatin, 6-Mercaptopurine and Thioguanine, Methotrexate, Cytoxan, Cytarabine, L-Asparaginase, Steroids: Prednisone and Dex
  • proteasome inhibitors such as bortezomib can be used in combination with the instant compounds, for example.
  • biologies can include agents such as TRAIL antibodies to TRAIL, integrins such as alpha- V-beta-3 ( ⁇ V ⁇ 3) and / or other cytokine/growth factors that are involved in angiogenesis, VEGF, EGF, FGF and PDGF.
  • the compounds can be conjugated to or delivered with an antibody.
  • the above-described combination methods can be used to treat a variety of conditions, including cancer and neoplastic diseases, inflammation, and microbial infections.
  • the compounds are administered in combination with a histone deacetylase inhibitor (HDACi).
  • HDACi histone deacetylase inhibitor
  • the HDACi is selected from the group consisting of:
  • tubacin panobinostat
  • vorinostat suberoylanilide hydroxamic acid (SAHA)
  • the compounds are used to treat inflammatory conditions.
  • Certain embodiments relate to methods of treating inflammatory conditions in animals.
  • the method can include, for example, administering an effective amount of a compound to a patient in need thereof.
  • Other embodiments relate to the use of compounds in the manufacture of a pharmaceutical or medicament for the treatment of inflammation.
  • the compounds are used to treat infectious diseases.
  • the infectious agent can be a microbe, for example, bacteria, fungi, protozoans, and microscopic algae, or viruses. Further, the infectious agent can be B. anthracis (anthrax). In some embodiments the infectious agent is a parasite. For example, the infectious agent can be Plasmodium, Leishmania, and Trypanosoma. Certain embodiments relate to methods of treating infectious agents in animals. The method can include, for example, administering an effective amount of a compound to a patient in need thereof. Other embodiments relate to the use of compounds in the manufacture of a pharmaceutical or medicament for the treatment of infectious agents.
  • the present embodiments provide methods of treating cancer comprising administering to an animal a compound having the structure of any one of Formulas I and ⁇ , or a pharmaceutically acceptable salt or pro-drug thereof:
  • HDACi histone deacetylase inhibitor
  • the dashed lines represent a single or a double bond
  • each Ri is separately a hydrogen, a halogen, a cyano, a nitro, an azido, a hydroxy, or a thiocyano, or selected from the group consisting of optionally substituted: C 1 - C 24 alkyl, C 2 -C 24 alkenyl, C 2 -C 24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonyloxy, phenyl, cycloalkylacyl, alkylthio, arylthio, oxysulfonyl, carboxy, thio, sulfoxide, sulfone, sulfonate esters, boronic acids and
  • n is 1 or 2, where if n is 2, then each Ri can be the same or different;
  • m is 1 or 2, where if m is 2, then each R 4 can be the same or different;
  • R 2 is a hydrogen, a halogen, a cyano, a nitro, an azido, a hydroxy, or a thiocyano, or selected from the group consisting of optionally substituted: C 1 -C 24 alkyl, C 2 - C 24 alkenyl, C 2 -C 24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonyloxy, phenyl, cycloalkylacyl, alkylthio, arylthio, oxysulfonyl, carboxy, thio, sulfoxide, sulfone, sulfonate esters, boronic acids and est
  • R 3 is a halogen or selected from the group consisting of optionally substituted C 1 -C 24 alkyl, C 2 -C 24 alkenyl, C 2 -C 24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonyloxy, nitro, azido, phenyl, cycloalkylacyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl;
  • each of Ei, E 3 , E 4 and E 5 is an optionally substituted heteroatom
  • E 2 is an optionally substituted heteroatom or -CH 2 - group
  • each R 4 is separately a halogen, a cyano, a nitro, an azido, or a thiocyano, or selected from the group consisting of optionally substituted C 1 -C 24 alkyl, C 2 -C 24 alkenyl, C 2 -C 24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, hydroxy, aminocarbonyl, aminocarbonyloxy, phenyl, cycloalkylacyl, alkylthio, arylthio, oxysulfonyl, carboxy, thio, sulfoxide, sulfone, sulfonate esters, boronic acids and esters, and hal
  • compositions comprising a histone deacetylase inhibitor (HDACi) and a compound of any one of Formulas I and H:
  • HDACi histone deacetylase inhibitor
  • each Ri is separately a hydrogen, a halogen, a cyano, a nitro, an azido, a hydroxy, or a thiocyano, or selected from the group consisting of optionally substituted: C 1 - C 24 alkyl, C 2 -C 24 alkenyl, C 2 -C 24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonyloxy, phenyl, cycloalkylacyl, alkylthio, arylthio, oxysulfonyl, carboxy, thio,
  • R 3 is a halogen or selected from the group consisting of optionally substituted C 1 -C 24 alkyl, C 2 -C 24 alkenyl, C 2 -C 24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonyloxy, nitro, azido, phenyl, cycloalkylacyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl;
  • each of Ei, E 3 , E 4 and E 5 is an optionally substituted heteroatom
  • E 2 is an optionally substituted heteroatom or -CH 2 - group
  • each R 4 is separately a halogen, a cyano, a nitro, an azido, or a thiocyano, or selected from the group consisting of optionally substituted C 1 -C 24 alkyl, C 2 -C 24 alkenyl, C 2 -C 24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, hydroxy, aminocarbonyl, aminocarbonyloxy, phenyl, cycloalkylacyl, alkylthio, arylthio, oxysulfonyl, carboxy, thio, sulfoxide, sulfone, sulfonate esters, boronic acids and esters, and hal
  • kits for treating a neoplastic disease in an animal can include, for example, administering to the animal, a therapeutically effective amount of a compound of a formula selected from Formulae I and II, and pharmaceutically acceptable salts and pro-drug esters thereof.
  • the present embodiments provide methods of inhibiting the growth of a cancer cell comprising contacting the cell with a HDACi and a compound having the structure of any one of Formulas I and II:
  • the dashed lines represent a single or a double bond
  • each Ri is separately a hydrogen, a halogen, a cyano, a nitro, an azido, a hydroxy, or a thiocyano, or selected from the group consisting of optionally substituted: C 1 - C 24 alkyl, C 2 -C 24 alkenyl, C 2 -C 24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonyloxy, phenyl, cycloalkylacyl, alkylthio, arylthio, oxysulfonyl, carboxy, thio, sulfoxide, sulfone, sulfonate esters, boronic acids and
  • n is 1 or 2, where if n is 2, then each Ri can be the same or different;
  • m is 1 or 2, where if m is 2, then each R t can be the same or different;
  • R 2 is a hydrogen, a halogen, a cyano, a nitro, an azido, a hydroxy, or a thiocyano, or selected from the group consisting of optionally substituted: C 1 -C 24 alkyl, C 2 - C 24 alkenyl, C 2 -C 24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonyloxy, phenyl, cycloalkylacyl, alkylthio, arylthio, oxysulfonyl, carboxy, thio, sulfoxide, sulfone, sulfonate esters, boronic acids and est
  • R 3 is a halogen or selected from the group consisting of optionally substituted C 1 -C 24 alkyl, C 2 -C 24 alkenyl, C 2 -C 24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonyloxy, nitro, azido, phenyl, cycloalkylacyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl;
  • each of Ei, E 3 , E 4 and E 5 is an optionally substituted heteroatom
  • E 2 is an optionally substituted heteroatom or -CH 2 - group
  • each R 4 is separately a halogen, a cyano, a nitro, an azido, or a thiocyano, or selected from the group consisting of optionally substituted C 1 -C 24 alkyl, C 2 -C 24 alkenyl, C 2 -C 24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, hydroxy, aminocarbonyl, aminocarbonyloxy, phenyl, cycloalkylacyl, alkylthio, arylthio, oxysulfonyl, carboxy, thio, sulfoxide, sulfone, sulfonate esters, boronic acids and esters, and hal
  • the present embodiments provide methods of inducing apoptosis of a cancer cell comprising contacting the cell with a HDACi and a compound having the structure of any one of Formulas I and II:
  • each Ri is separately a hydrogen, a halogen, a cyano, a nitro, an azido, a hydroxy, or a thiocyano, or selected from the group consisting of optionally substituted: C 1 - C 24 alkyl, C 2 -C 24 alkenyl, C 2 -C 24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonyloxy, phenyl, cycloalkylacyl, alkylthio, arylthio, oxysulfonyl, carboxy, thio,
  • R 3 is a halogen or selected from the group consisting of optionally substituted C 1 -C 24 alkyl, C 2 -C 24 alkenyl, C 2 -C 24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonyloxy, nitro, azido, phenyl, cycloalkylacyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl;
  • each of Ei, E 3 , E 4 and E 5 is an optionally substituted heteroatom
  • E 2 is an optionally substituted heteroatom or -CH 2 - group
  • each R 4 is separately a halogen, a cyano, a nitro, an azido, or a thiocyano, or selected from the group consisting of optionally substituted C 1 -C 24 alkyl, C 2 -C 24 alkenyl, C 2 -C 24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, hydroxy, aminocarbonyl, aminocarbonyloxy, phenyl, cycloalkylacyl, alkylthio, arylthio, oxysulfonyl, carboxy, thio, sulfoxide, sulfone, sulfonate esters, boronic acids and esters, and hal
  • Fig. 1 shows Salinosporamide A (NPI-0052) inhibits the proteolytic activities of the 2OS proteasome in leukemia cell lines.
  • Fig. 2 shows in vitro and in vivo effects of Salinosporamide A (NPI-0052).
  • Fig. 3 shows Salinosporamide A (NPI-0052) induced apoptosis through caspase activation and mitochondrial perturbations.
  • Fig. 4 shows the role of caspase-8 and FADD in Salinosporamide A (NPI- 0052) induced apoptosis.
  • Fig. 5 shows the free radical scavenger, NAC, protects from Salinosporamide A (NPI-0052) induced apoptosis.
  • Fig. 6 shows low doses of Salinosporamide A (NPI-0052) and HDAC inhibitors induce synergistic apoptosis.
  • Fig. 7 shows schematic representation of the mechanism of action of Salinosporamide A (NPI-0052).
  • Fig. 8 shows the effect of the combination of MS-275 with bortezomib and the combination of MS-275 with Salinosporamide A to induce synergistic apoptosis.
  • Fig. 9 shows that Salinosporamide A (10 nM) enhances the activity of vorinostat (SAHA, 1 ⁇ M) in Hodgkin's HD-LM2 Lymphoma cell lines.
  • Fig. 10 shows that Salinosporamide A (50 nM) enhances the activity of vorinostat (SAHA, 5 ⁇ M) in Hodgkin's L428 cells lines and that Salinosporamide A (50 nM) enhances the activity of vorinostat (SAHA, 5 ⁇ M) in KM-H2 cell lines.
  • Fig. 11 shows the effect of combinations of Salinosporamide A (14 nm) with MS-275 (0.5 ⁇ M, 2 ⁇ M and 3 ⁇ M) in a human myeloma cell line (RPMI 8226 MM cells), the effect of combinations of Salinosporamide A (18 nM) with MS-275 (1 ⁇ M) and the effect of combinations of Salinosporamide A (16 nM) with MS-275 (1.5 ⁇ M).
  • Fig. 12 shows the effect of combinations of Salinosporamide A (12 nM, 14 nM and 18 nM) with MS-275 (1.5 ⁇ M) in a human myeloma cell line (OPM-I MM cells), and the effect of combinations of Salinosporamide A (18 nm and 20 nm) with MS-275 (2 ⁇ M) in a human myeloma cell line (OPM-I MM cells).
  • Fig. 13 shows the effect of combinations of Salinosporamide A (16 nM and 18 nM) with MS-275 (0.5 ⁇ M, 1 ⁇ M, 1.5 ⁇ M and 2 ⁇ M) in a human myeloma cell line (DHL-6 MM cells).
  • Fig. 14 shows the effect of combinations of Salinosporamide A (12 nM, 14 nM, and 20 nM) with MS-275 (0.5 ⁇ M, 1 ⁇ M, 1.5 ⁇ M and 2 ⁇ M) in a human myeloma cell line (Dox-6 MM cells).
  • Fig. 15 shows the effect of combinations of Salinosporamide A (14 nM, 16 nM, and 20 nM) with MS-275 (0.5 ⁇ M, 1 ⁇ M, 1.5 ⁇ M and 2 ⁇ M) in a human myeloma cell line (Dox-40 MM cells).
  • Fig. 16 shows the effect of combinations of Salinosporamide A (12 nM, 14 nM, 18 nM and 20 nM) with MS-275 (0.5 ⁇ M, 1 ⁇ M, and 2 ⁇ M) in a human myeloma cell line (LR-5 MM cells).
  • Fig. 17 shows the effect of combinations of Salinosporamide A (3 nM, 5 nM, and 7 nM) with MS-275 (0.25 ⁇ M, 0.5 ⁇ M, and 1 ⁇ M) in a human myeloma cell line (MM. IR cells.
  • Fig. 18A shows activity of Salinosporamide A (10 nM) and vorinostat (5 ⁇ M) alone and in combination on SB-2 line.
  • Fig. 18B shows activity of Salinosporamide A (10 nM) and vorinostat (5 ⁇ M) alone and in combination on WM-266-4 melanoma cell line.
  • Fig. 19 shows activity of Salinosporamide A (NPI-0052, 10 nM) and vorinostat (SAHA, 5 ⁇ M) alone and in combination on MeWo melanoma cell lines.
  • Fig. 20 shows growth inhibition effects of Salinosporamide A (NPI-0052) at doses of 5 nM to 500 nM as single agent or in combination with Vorinostat (SAHA, 2 ⁇ M) in various lung cancer cell lines.
  • Fig. 21 shows growth inhibition effects of Salinosporamide A (NPI-0052) at doses of 5 nM to 500 nM as single agent or in combination with Vorinostat (2 ⁇ M) in various lung cancer cell lines.
  • Fig. 22A-H shows isobologram analyses for eight lung carcinoma cell lines treated with Salinosporamide A (NPI-0052) and vorinostat (SAHA).
  • Fig. 23 shows that human pancreatic carcinoma cell lines are resistant to treatment with gemcitabine alone, when gemcitabine (1 ⁇ M or 10 ⁇ M) is used in combination with Salinosporamide A a dosage effect is observed.
  • Fig. 24 shows the effect of Salinosporamide A (NPI-0052) with vorinostat (SAHA) increases apoptosis compared to treatment with the individual agents alone.
  • Fig. 25 shows HDAC inhibitor MS-275 can decrease mRNA expression of 2OS proteasome ⁇ subunits in Jurkat cells.
  • Fig. 26 shows vorinostat can decrease mRNA expression of 2OS proteasomal ⁇ 5 subunit in Jurkat cells as measured at 12 hours and 18 hours. The expression of ⁇ 5 mRNA was analyzed by real time PCR.
  • Fig. 27 shows MS-275 in combination with Salinosporamide A or bortezomib causes Histone-3 to hyperacetylate in Jurkat T-cells.
  • Fig. 28 shows combination of Salinosporamide A with vorinostat causes hyperacetylation of Histone-3 in jurkat cells, the addition of N-acetyl cysteine (NAC) inhibits the hyperacetylation of Histone-3.
  • NAC N-acetyl cysteine
  • Fig. 29 shows histone-3 ubiquitination is not affected by Salinosporamide A (NPI-0052)or the combination of Salinosporamide A (NPI-0052) with MS-275.
  • Fig. 30 shows effects of vorinostat pretreatment of human Jurkat ALL cells followed by treatment with bortezomib.
  • Fig. 31 shows effects of simultaneous treatment of human Jurkat ALL cells with vorinostat and bortezomib.
  • Fig. 32 shows effects of vorinostat pretreatment of human Jurkat ALL cells followed by treatment with Salinosporamide A.
  • Fig. 33 shows effects of simultaneous treatment of human Jurkat ALL cells with vorinostat and Salinosporamide A.
  • Fig. 34 shows effect of MS-275 and Salinosporamide A in combination and alone on superoxide levels in Jurkat T-cells.
  • Fig. 35 shows effect of MS-275 and Salinosporamide A in combination and alone on superoxide levels in Caspase-8 deficient cells.
  • Fig. 36 shows vorinostat in combination with Salinosporamide A or bortezomib causes formation of reactive oxygen species (ROS), when N-acetylcysteine (NAC) is included in the combinations the amount of ROS decrease as measured by mean fluorescence.
  • ROS reactive oxygen species
  • NAC N-acetylcysteine
  • Fig. 37 shows N-acetylcysteine (NAC) decreases formation of ROS when combined with vorinostat (Zolinza) and Salinosporamide A but not when combined with vorinostat and bortezomib (Velcade).
  • Fig. 38 shows combination of vorinostat (SAHA) and Salinosporamide A (NPI) does not induce apoptosis in cells that are caspase-8 deficient as strongly as cells that are not caspase-8 deficient.
  • NAC N-acetylcysteine
  • Fig. 39 shows regulation of NF -KB Activity in vitro by vorinostat and Salinosporamide A alone and in combination.
  • Fig. 40 shows regulation of NF- ⁇ B Activity in vitro in human pancreatic carcinoma cells by vorinostat and Salinosporamide A alone and in combination.
  • Fig. 41 shows combination of Salinosporamide A and vorinostat exhibit enhanced activity in an orthotopic pancreatic tumor model.
  • co-administration or use “in combination,” it is meant that the two or more agents may be found in the patient's bloodstream at the same time, regardless of when or how they are actually administered.
  • the agents are administered simultaneously.
  • administration in combination is accomplished by combining the agents in a single dosage form.
  • the agents are administered sequentially.
  • the agents are administered through the same route, such as orally.
  • the agents are administered through different routes, such as one being administered orally and another being administered i.v.
  • the pharmacokinetics of the two or more agents are substantially the same.
  • HDACi histone deacetylase inhibitor
  • each Ri is separately a hydrogen, a halogen, a cyano, a nitro, an azido, a hydroxy, or a thiocyano, or selected from the group consisting of optionally substituted: C 1 - C 24 alkyl, C 2 -C 24 alkenyl, C 2 -C 24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonyloxy, phenyl, cycloalkylacyl, alkylthio, arylthio, oxysulfonyl, carboxy, thio, sulfoxide, sulfone, sulfonate esters, boronic acids and
  • n is 1 or 2, where if n is 2, then each Ri can be the same or different;
  • m is 1 or 2, where if m is 2, then each R t can be the same or different;
  • R 2 is a hydrogen, a halogen, a cyano, a nitro, an azido, a hydroxy, or a thiocyano, or selected from the group consisting of optionally substituted: C 1 -C 24 alkyl, C 2 - C 24 alkenyl, C 2 -C 24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonyloxy, phenyl, cycloalkylacyl, alkylthio, arylthio, oxysulfonyl, carboxy, thio, sulfoxide, sulfone, sulfonate esters, boronic acids and est
  • each of Ei, E 3 , E 4 and E 5 is an optionally substituted heteroatom
  • E 2 is an optionally substituted heteroatom or -CH 2 - group
  • each R 4 is separately a halogen, a cyano, a nitro, an azido, or a thiocyano, or selected from the group consisting of optionally substituted C 1 -C 24 alkyl, C 2 -C 24 alkenyl, C 2 -C 24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, hydroxy, aminocarbonyl, aminocarbonyloxy, phenyl, cycloalkylacyl, alkylthio, arylthio, oxysulfonyl, carboxy, thio, sulfoxide, sulfone, sulfonate esters, boronic acids and esters, and hal
  • E 5 can be, for example, OH, O, ORio, S, SR ⁇ , SO 2 R n , NH, NH 2 , NOH, NHOH, NR i2 , and NHORi 3 , wherein Ri 0 - I3 may separately include, for example, hydrogen, a substituted or unsubstituted of any of the following: alkyl, an aryl, a heteroaryl, and the like. R 3 can be methyl. Furthermore, R 4 may include a cyclohexyl. Also, each of Ei, E 3 and E 4 can be O and E 2 can be NH.
  • Ri can be CH 2 CH 2 X, wherein X is selected from the group consisting of hydrogen, fluorine, chlorine, bromine, and iodine; wherein R 4 may include a cyclohexyl; wherein R 3 can be methyl; and wherein each of Ei, E 3 and E 4 separately can be O and E 2 can be NH.
  • R 1 can be alkyl optionally substituted with a boranic ester or boranic ester.
  • the boronic ester can be B(OMethyl) 2 , B(OEthyl) 2 , B(OPropyl) 2 , B(OPhenyl) 2 , and the like.
  • the cancer can be selected from the group consisting of breast cancer, sarcoma, leukemia, uretal cancer, bladder cancer, colon cancer, rectal cancer, stomach cancer, lung cancer, lymphoma, liver cancer, kidney cancer, endocrine cancer, skin cancer, melanoma, angioma, brain cancer and central nervous system (CNS) cancer.
  • the cancer can be leukemia, lymphoma, and the like.
  • the cancer can comprise a tumor.
  • the tumor can be a refractory solid tumor.
  • the method can further comprise coadministering a chemotherapeutic agent.
  • the compound is Salinosporamide A
  • the HDACi and the compound having the structure of any one of Formulas I and II can work in a synergistic manner to treat cancer.
  • the HDACi can be selected from the group consisting of:
  • tubacin panobinostat
  • vorinostat suberoylanilide hydroxamic acid (SAHA)
  • the HDACi can be (pyridin-3-yl)methyl 4-(2- aminophenylcarbamoyl)benzylcarbamate (MS-275), valproic acid, vorinostat, and the like.
  • HDACi histone deacetylase inhibitor
  • each Ri is separately a hydrogen, a halogen, a cyano, a nitro, an azido, a hydroxy, or a thiocyano, or selected from the group consisting of optionally substituted: C 1 - C 24 alkyl, C 2 -C 24 alkenyl, C 2 -C 24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonyloxy, phenyl, cycloalkylacyl, alkylthio, arylthio, oxysulfonyl, carboxy, thio,
  • R 3 is a halogen or selected from the group consisting of optionally substituted C 1 -C 24 alkyl, C 2 -C 24 alkenyl, C 2 -C 24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonyloxy, nitro, azido, phenyl, cycloalkylacyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl;
  • each of Ei, E 3 , E 4 and E 5 is an optionally substituted heteroatom
  • E 2 is an optionally substituted heteroatom or -CH 2 - group
  • each R 4 is separately a halogen, a cyano, a nitro, an azido, or a thiocyano, or selected from the group consisting of optionally substituted C 1 -C 24 alkyl, C 2 -C 24 alkenyl, C 2 -C 24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, hydroxy, aminocarbonyl, aminocarbonyloxy, phenyl, cycloalkylacyl, alkylthio, arylthio, oxysulfonyl, carboxy, thio, sulfoxide, sulfone, sulfonate esters, boronic acids and esters, and hal
  • E 5 can be, for example, OH, O, ORio, S, SRn, SO 2 Rn, NH, NH 2 , NOH, NHOH, NRi 2 , and NHORi 3 , wherein Ri 0 -I 3 may separately include, for example, hydrogen, a substituted or unsubstituted of any of the following: alkyl, an aryl, a heteroaryl, and the like.
  • R 3 can be methyl.
  • R 4 may include a cyclohexyl.
  • each of Ei, E 3 and E 4 can be O and E 2 can be NH.
  • Ri can be CH 2 CH 2 X, wherein X is selected from the group consisting of hydrogen, fluorine, chlorine, bromine, and iodine; wherein R 4 may include a cyclohexyl; wherein R 3 can be methyl; and wherein each of Ei, E 3 and E 4 separately can be O and E 2 can be NH.
  • R 1 can be alkyl optionally substituted with a boranic ester or boranic ester.
  • the boronic ester can be B(OMethyl) 2 , B(OEthyl) 2 , B(OPropyl) 2 , B(OPhenyl) 2 , and the like.
  • the compound is Salinosporamide A.
  • the HDACi can be selected from the group consisting of (pyridin-3-yl)methyl 4-(2-aminophenylcarbamoyl)benzylcarbamate (MS-275), APHA compound 8, Apicidin, (-)- Depudecin, sodium butyrate, scriptaid, sirtinol, trichostatin A, valproic acid, vorinostat and the like.
  • the HDACi can be (pyridin-3-yl)m ethyl 4-(2- aminophenylcarbamoyl)benzylcarbamate (MS-275), HDACi is valproic acid, vorinostat, and the like.
  • Some embodiments provide a method of inhibiting the growth of a cancer cell comprising contacting the cell with a HDACi and a compound having the structure of any one of Formulas I and II:
  • each Ri is separately a hydrogen, a halogen, a cyano, a nitro, an azido, a hydroxy, or a thiocyano, or selected from the group consisting of optionally substituted: C 1 - C 24 alkyl, C 2 -C 24 alkenyl, C 2 -C 24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonyloxy, phenyl, cycloalkylacyl, alkylthio, arylthio, oxysulfonyl, carboxy, thio,
  • R 3 is a halogen or selected from the group consisting of optionally substituted C 1 -C 24 alkyl, C 2 -C 24 alkenyl, C 2 -C 24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonyloxy, nitro, azido, phenyl, cycloalkylacyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl;
  • each of Ei, E 3 , E 4 and E 5 is an optionally substituted heteroatom
  • E 2 is an optionally substituted heteroatom or -CH 2 - group
  • each R 4 is separately a halogen, a cyano, a nitro, an azido, or a thiocyano, or selected from the group consisting of optionally substituted C 1 -C 24 alkyl, C 2 -C 24 alkenyl, C 2 -C 24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, hydroxy, aminocarbonyl, aminocarbonyloxy, phenyl, cycloalkylacyl, alkylthio, arylthio, oxysulfonyl, carboxy, thio, sulfoxide, sulfone, sulfonate esters, boronic acids and esters, and hal
  • E 5 can be, for example, OH, O, ORio, S, SRn, SO 2 Rn, NH, NH 2 , NOH, NHOH, NRi 2 , and NHORi 3 , wherein Ri 0 -I 3 may separately include, for example, hydrogen, a substituted or unsubstituted of any of the following: alkyl, an aryl, a heteroaryl, and the like.
  • R 3 can be methyl.
  • R 4 may include a cyclohexyl.
  • each of Ei, E 3 and E 4 can be O and E 2 can be NH.
  • Ri can be CH 2 CH 2 X, wherein X is selected from the group consisting of hydrogen, fluorine, chlorine, bromine, and iodine; wherein R 4 may include a cyclohexyl; wherein R 3 can be methyl; and wherein each of Ei, E 3 and E 4 separately can be O and E 2 can be NH.
  • Ri can be alkyl optionally substituted with a boranic ester or boranic ester. Typical boranic ester groups include, but are in no way limited to, B(OMethyl) 2 , B(OEthyl) 2 , B(OPropyl) 2 , B(OPhenyl) 2 , and the like.
  • the compound is Salinosporamide A.
  • the HDACi can be selected from the group consisting of (pyridin-3-yl)methyl 4-(2-aminophenylcarbamoyl)benzylcarbamate (MS-275), APHA compound 8, Apicidin, (-)- Depudecin, sodium butyrate, scriptaid, sirtinol, trichostatin A, valproic acid, vorinostat and the like.
  • the HDACi can be (pyridin-3-yl)m ethyl 4-(2- aminophenylcarbamoyl)benzylcarbamate (MS-275), HDACi is valproic acid, vorinostat, and the like.
  • Some embodiments provide a method of inducing apoptosis of a cancer cell comprising contacting the cell with a HDACi and a compound having the structure of any one of Formulas I and II:
  • each Ri is separately a hydrogen, a halogen, a cyano, a nitro, an azido, a hydroxy, or a thiocyano, or selected from the group consisting of optionally substituted: C 1 - C 24 alkyl, C 2 -C 24 alkenyl, C 2 -C 24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonyloxy, phenyl, cycloalkylacyl, alkylthio, arylthio, oxysulfonyl, carboxy, thio,
  • R 3 is a halogen or selected from the group consisting of optionally substituted C 1 -C 24 alkyl, C 2 -C 24 alkenyl, C 2 -C 24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonyloxy, nitro, azido, phenyl, cycloalkylacyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl;
  • each of Ei, E 3 , E 4 and E 5 is an optionally substituted heteroatom
  • E 2 is an optionally substituted heteroatom or -CH 2 - group
  • each R 4 is separately a halogen, a cyano, a nitro, an azido, or a thiocyano, or selected from the group consisting of optionally substituted C 1 -C 24 alkyl, C 2 -C 24 alkenyl, C 2 -C 24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, hydroxy, aminocarbonyl, aminocarbonyloxy, phenyl, cycloalkylacyl, alkylthio, arylthio, oxysulfonyl, carboxy, thio, sulfoxide, sulfone, sulfonate esters, boronic acids and esters, and hal
  • E 5 can be, for example, OH, O, ORi 0 , S, SR ⁇ , SO 2 R n , NH, NH 2 , NOH, NHOH, NRi 2 , and NHORi 3 , wherein R 10- I 3 may separately include, for example, hydrogen, a substituted or unsubstituted of any of the following: alkyl, an aryl, a heteroaryl, and the like. R3 can be methyl. Furthermore, R4 may include a cyclohexyl. Also, each of Ei, E 3 and E 4 can be O and E 2 can be NH.
  • Ri can be CH 2 CH 2 X, wherein X is selected from the group consisting of hydrogen, fluorine, chlorine, bromine, and iodine; wherein R 4 may include a cyclohexyl; wherein R 3 can be methyl; and wherein each of Ei, E 3 and E 4 separately can be O and E 2 can be NH.
  • R 1 can be alkyl optionally substituted with a boranic ester or boranic ester.
  • the boronic ester can be B(OMethyl) 2 , B(OEthyl) 2 , B(OPropyl) 2 , B(OPhenyl) 2 , and the like.
  • the compound is Salinosporamide A.
  • the HDACi can be selected from the group consisting of (pyridin-3-yl)methyl 4-(2-aminophenylcarbamoyl)benzylcarbamate (MS-275), APHA compound 8, Apicidin, (-)- Depudecin, sodium butyrate, scriptaid, sirtinol, trichostatin A, valproic acid, vorinostat and the like.
  • the HDACi can be (pyridin-3-yl)m ethyl 4-(2- aminophenylcarbamoyl)benzylcarbamate (MS-275), HDACi is valproic acid, vorinostat, and the like.
  • Some embodiments provide a compound having the structure of Formula
  • the dashed lines represent a single or a double bond
  • each Ri is separately a hydrogen, a halogen, a cyano, a nitro, an azido, a hydroxy, or a thiocyano, or selected from the group consisting of optionally substituted: C 1 - C 24 alkyl, C 2 -C 24 alkenyl, C 2 -C 24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonyloxy, phenyl, cycloalkylacyl, alkylthio, arylthio, oxysulfonyl, carboxy, thio, sulfoxide, sulfone, sulfonate esters, boronic acids and
  • n is 1 or 2, where if n is 2, then each Ri can be the same or different;
  • R 2 is a hydrogen, a halogen, a cyano, a nitro, an azido, a hydroxy, or a thiocyano, or selected from the group consisting of optionally substituted: C 1 -C 24 alkyl, C 2 - C 24 alkenyl, C 2 -C 24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonyloxy, phenyl, cycloalkylacyl, alkylthio, arylthio, oxysulfonyl, carboxy, thio, sulfoxide, sulfone, sulfonate esters, boronic acids and est
  • R 3 is a halogen or selected from the group consisting of optionally substituted C 1 -C 24 alkyl, C 2 -C 24 alkenyl, C 2 -C 24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonyloxy, nitro, azido, phenyl, cycloalkylacyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl;
  • each of Ei, E 3 , and E 4 is an optionally substituted heteroatom
  • E 2 is an optionally substituted heteroatom or -CH 2 - group.
  • compounds having the structure of Formula I have the structure of Formula II:
  • each Ri is separately a hydrogen, a halogen, a cyano, a nitro, an azido, a hydroxy, or a thiocyano, or selected from the group consisting of optionally substituted: C 1 - C 24 alkyl, C 2 -C 24 alkenyl, C 2 -C 24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonyloxy, phenyl, cycloalkylacyl, alkylthio, arylthio, oxysulfonyl, carboxy, thio,
  • each of Ei, E 3 , E 4 and E 5 is an optionally substituted heteroatom
  • E 2 is an optionally substituted heteroatom or -CH 2 - group
  • each R 4 is separately a halogen, a cyano, a nitro, an azido, or a thiocyano, or selected from the group consisting of optionally substituted C 1 -C 24 alkyl, C 2 -C 24 alkenyl, C 2 -C 24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, hydroxy, aminocarbonyl, aminocarbonyloxy, phenyl, cycloalkylacyl, alkylthio, arylthio, oxysulfonyl, carboxy, thio, sulfoxide, sulfone, sulfonate esters, boronic acids and esters, and hal
  • n can be equal to 1, while in others it can be equal to 2.
  • the substituents can be the same or can be different.
  • R 3 is not a hydrogen.
  • m can be equal to 1 or 2, and when m is equal to 2, R 4 can be the same or different.
  • E 5 can be, for example, OH, O, ORio, S, SR ⁇ , SO 2 R n , NH, NH 2 , NOH, NHOH, NR i2 , and NHORi 3 , wherein Ri 0 - I3 may separately include, for example, hydrogen, a substituted or unsubstituted of any of the following: alkyl, an aryl, a heteroaryl, and the like. R 3 can be methyl. Furthermore, R 4 may include a cyclohexyl. Also, each of Ei, E 3 and E 4 can be O and E 2 can be NH.
  • Ri can be CH 2 CH 2 X, wherein X is selected from the group consisting of hydrogen, fluorine, chlorine, bromine, and iodine; wherein R 4 may include a cyclohexyl; wherein R 3 can be methyl; and wherein each of Ei, E 3 and E 4 separately can be O and E 2 can be NH.
  • R 1 can be alkyl optionally substituted with a boranic ester or boranic ester.
  • the boronic ester can be B(OMethyl) 2 , B(OEthyl) 2 , B(OPropyl) 2 , B(OPhenyl) 2 , and the like.
  • R 2 is not cyclohex-2-enyl carbinol when one of the Ri substituents is ethyl or chloroethyl and R3 is methyl.
  • Ri can be an optionally substituted C 1 to C 5 alkyl.
  • Ri can be methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl and the like.
  • R 1 is not a substituted or unsubstituted, unbranched C 6 alkyl.
  • E5 can be OH.
  • the compound may have the following Formula 1-1 :
  • R 8 can be selected from the group consisting of hydrogen, fluorine, chlorine, bromine and iodine.
  • Formula 1-1 may have the following stereochemistry:
  • Rg can be selected from the group consisting of hydrogen, fluorine, chlorine, bromine and iodine.
  • Rg can be selected from the group consisting of hydrogen, fluorine, chlorine, bromine and iodine.
  • Still a further exemplary compound of Formula II is a compound having the following Formula 1-2:
  • R 8 can be selected from the group consisting of hydrogen, fluorine, chlorine, bromine and iodine.
  • Formula 1-2 may have the following stereochemistry:
  • Rg can be selected from the group consisting of hydrogen, fluorine, chlorine, bromine and iodine.
  • An exemplary compound of Formula II can heve the following Formula II- 1 :
  • R 8 can be selected from the group consisting of hydrogen, fluorine, chlorine, bromine and iodine.
  • Exemplary stereochemistry can be as follows:
  • the compound of Formula I can have any of the following structures of Foumulae II-2, II-3, and II-4:
  • R 4 may include a 7-oxa-bicyclo[4.1.0]hept- 2-yl).
  • An exemplary compound of Formula I is the following Formula II-5:
  • Rg can be selected from the group consisting of hydrogen, fluorine, chlorine, bromine and iodine.
  • At least one R 4 may include an optionally substituted branched alkyl.
  • a compound of Formula I can be the following Formula ⁇ -6:
  • R 8 can be selected from the group consisting of hydrogen, fluorine, chlorine, bromine and iodine.
  • the compound of Formula I can be the following Formula ⁇ -7:
  • R 8 can be selected from the group consisting of hydrogen, fluorine, chlorine, bromine and iodine.
  • At least one R 4 can be an optionally substituted cycloalkyl and E5 can be an oxygen.
  • R 4 can be cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like.
  • An exemplary compound of Formula I can have the structure of Formula II-8:
  • Rg can be, for example, hydrogen (II-8A), fluorine (II-8B), chlorine (II-8C), bromine (II-8D) and iodine (II-8E).
  • E5 can be an amine oxide, giving rise to an oxime.
  • An exemplary compound of Formula I has the following structure of Formula II-9:
  • R 8 can be selected from the group consisting of hydrogen, fluorine, chlorine, bromine and iodine; R can be a hydrogen, or an optionally substituted substituent selected from the group consisting of alkyl, aryl, heteroaryl, and the like.
  • a further exemplary compound of Formula I has the following structure of Formula 11-10:
  • Rg can be selected from the group consisting of hydrogen, fluorine, chlorine, bromine and iodine.
  • E 5 can be NH 2 .
  • An exemplary compound of Formula I has the following structure of Formula II- 11 :
  • R 8 can be selected from the group consisting of hydrogen, fluorine, chlorine, bromine and iodine.
  • At least one R 4 can be an optionally substituted cycloalkyl and E5 can be NH 2 .
  • R 4 can be cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like.
  • An exemplary compound of Formula I has the following structure of Formula 11-12:
  • Rg can be selected from the group consisting of hydrogen, fluorine, chlorine, bromine and iodine.
  • a further exemplary compound of Formula I has the following structure of Formula 11-13:
  • R 8 may include, for example, hydrogen (II-13A), fluorine (II-13B), chlorine (II-13C), bromine (II-13D) and iodine (II-13E).
  • a compound of Formula I can have the following structure of F ormul a II- 14 :
  • Rg can be selected from the group consisting of hydrogen, fluorine, chlorine, bromine and iodine.
  • the radical R 4 of a compound of Formula II can be an optionally substituted cycloalkene.
  • the compounds of Formula II may include a hydroxy at E 5 , for example.
  • a further exemplary compound of Formula II has the following structure of Formula 11-15:
  • Rg can be selected from the group consisting of hydrogen, fluorine, chlorine, bromine and iodine
  • R 8 may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.
  • the compounds of Formulae 11-16, 11-17, 11-18 and 11-19 can be obtained by fermentation, synthesis, or semi-synthesis and isolated/purified as set forth below. Furthermore, the compounds of Formulae 11-16, 11-17, 11-18 and 11-19 can be used, and are referred to, as "starting materials" to make other compounds described herein.
  • the compounds of Formula I may include a methyl group as R 1 , for example.
  • a further exemplary compound, structure 11-20 has the following structure and stereochemistry:
  • the compounds of Formula I may include hydroxyethyl as Ri, for example.
  • a further exemplary compound, Formula 11-21 has the following structure and stereochemistry:
  • the hydroxyl group of Formula 11-21 can be esterified such that Ri may include ethylpropionate, for example.
  • An exemplary compound, structure 11-22, has the following structure and stereochemistry:
  • the compounds of Formula I may include an ethyl group as R3, for example.
  • a further exemplary compound of Formula I has the following structure of Formula 11-23:
  • Rg can be selected from the group consisting of hydrogen, fluorine, chlorine, bromine and iodine.
  • exemplary stereochemistry can be as follows:
  • the compounds of Formula 11-23 may have the following structure and stereochemistry, exemplified by structure of Formula II-24C, where R.8 is chlorine:
  • the compounds of Formula 11-15 may have the following stereochemistry, exemplified by the compound of Formula 11-25, where Rs is chlorine:
  • the compound of Formula 11-15 may have the following stereochemistry, exemplified by the compound of Formula 11-26, where Rs is chlorine:
  • the compound of Formula I may have the following structure and stereochemistry, exemplified by the structure of Formula 11-27, where Ri is ethyl: ⁇ -27
  • the compound of Formula I may have the following structure and stereochemistry, exemplified by the structure of Formula 11-28, where R 1 is methyl:
  • the compounds of Formula I may include azidoethyl as R 1 , for example.
  • a further exemplary compound, Formula 11-29 has the following structure and stereochemistry:
  • the compounds of Formula I may include propyl as Ri, for example.
  • a further exemplary compound, Formula 11-30 has the following structure and stereochemistry: ⁇ -30
  • the compound of Formula I may include cyanoethyl as Ri; for example, the compound of Formula 11-37 has the following structure and stereochemistry:
  • the compound of Formula I may include ethyl thiocyanate as Ri; for example, the compound of Formula 11-38 has the following structure and stereochemistry:
  • the compounds of Formula I may include a thiol as Ri, for example.
  • the substituent Ri of the compound of Formula I may include a leaving group, for example, a halogen, as in compounds of Formulae 11-18 or 11-19, or another leaving group, such as a sulfonate ester.
  • a leaving group for example, a halogen, as in compounds of Formulae 11-18 or 11-19, or another leaving group, such as a sulfonate ester.
  • a sulfonate ester is the methane sulfonate (mesylate) of Formula 11-41 :
  • the substituent Ri of the compound of Formula I may include electron acceptors.
  • the electron acceptor can be, for example, a Lewis acid, such as a boronic acid or ester.
  • the electron acceptor can be, for example, a Michael acceptor.
  • the compounds can be prodrug esters or thioesters of the compounds of Formula I.
  • the compound of Formula 11-44 (a prodrug thioester of the compound of structure 11-16) has the following structure and stereochemistry:
  • the compounds of Formula I may include an alkenyl group as Ri, for example, ethyl enyl.
  • a further exemplary compound, Formula 11-46 has the following structure and stereochemistry:
  • the compounds can be prodrug esters or thioesters of the compounds of Formula I.
  • the compound of Formula 11-47 (a prodrug thioester of the compound of structure 11-17) has the following structure and stereochemistry:
  • the compounds can be prodrug esters or thioesters of the compounds of Formula I.
  • the compound of Formula 11-48 has the following structure and stereochemistry:
  • the compound can be prodrug ester or thioester of the compounds of Formula I.
  • the compound of Formula 11-50 has the following structure and stereochemistry:
  • An exemplary compound of Formula I is the following Formula IH-I, with and without exemplary stereochemistry:
  • Rs can be selected from the group consisting of hydrogen, fluorine, chlorine, bromine and iodine.
  • the substituent(s) K 6 and R 7 may each separately be selected from a hydrogen, a halogen, a nitro, a cyano, or an optionally substituted substituent selected from the group consisting Of C 1 -C 24 alkyl, C 2 -C 24 alkenyl, C 2 - C 24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino, aminocarbonyl, aminocarbonyloxy, azido, phenyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, and
  • an exemplary compound of Formula I has the following Formula ⁇ i-2:
  • R 8 may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.
  • an exemplary compound of Formula I has the following
  • R 8 may include, for example, hydrogen (III-3A), fluorine (III-3B), chlorine (III-3C), bromine (III-3D) and iodine (III-3E).
  • an exemplary compound of Formula I has the following
  • Rg may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.
  • Certain embodiments also provide pharmaceutically acceptable salts and pro-drug esters or thioesters of the compound of Formulae I and II, and provide methods of obtaining and purifying such compounds by the methods disclosed herein.
  • pro-drug especially when referring to a pro-drug ester of the compound of Formula I synthesized by the methods disclosed herein, refers to a chemical derivative of the compound that is rapidly transformed in vivo to yield the compound, for example, by hydrolysis in blood or inside tissues.
  • pro-drug ester refers to derivatives of the compounds disclosed herein formed by the addition of any of several ester- or thioester-forming groups that are hydrolyzed under physiological conditions.
  • pro-drug ester groups examples include pivoyloxymethyl, acetoxymethyl, phthalidyl, indanyl and methoxymethyl, as well as other such groups known in the art, including a (5-i?-2-oxo-l,3- dioxolen-4-yl)methyl group.
  • Other prodrugs can be prepared by preparing a corresponding thioester of the compound, for example, by reacting with an appropriate thiol, such as thiophenol, Cysteine or derivatives thereof, or propanethiol, for example.
  • Other examples of pro-drug ester groups can be found in, for example, T. Higuchi and V. Stella, in "Pro-drugs as Novel Delivery Systems", Vol. 14, A.C.S.
  • pharmaceutically acceptable salt refers to any pharmaceutically acceptable salts of a compound, and preferably refers to an acid addition salt of a compound.
  • pharmaceutically acceptable salt are the alkali metal salts (sodium or potassium), the alkaline earth metal salts (calcium or magnesium), or ammonium salts derived from ammonia or from pharmaceutically acceptable organic amines, for example C 1 -C 7 alkylamine, cyclohexylamine, triethanolamine, ethylenediamine or tris-(hydroxymethyl)-aminomethane.
  • the preferred examples of pharmaceutically acceptable salts are acid addition salts of pharmaceutically acceptable inorganic or organic acids, for example, hydrohalic, sulfuric, phosphoric acid or aliphatic or aromatic carboxylic or sulfonic acid, for example acetic, succinic, lactic, malic, tartaric, citric, ascorbic, nicotinic, methanesulfonic, p-toluensulfonic or naphthalenesulfonic acid.
  • pharmaceutically acceptable inorganic or organic acids for example, hydrohalic, sulfuric, phosphoric acid or aliphatic or aromatic carboxylic or sulfonic acid, for example acetic, succinic, lactic, malic, tartaric, citric, ascorbic, nicotinic, methanesulfonic, p-toluensulfonic or naphthalenesulfonic acid.
  • Preferred pharmaceutical compositions disclosed herein include pharmaceutically acceptable salts and pro-drugs of the compound of Formula I obtained and purified by the methods disclosed herein. Accordingly, if the manufacture of pharmaceutical formulations involves intimate mixing of the pharmaceutical excipients and the active ingredient in its salt form, then it is preferred to use pharmaceutical excipients which are non- basic, that is, either acidic or neutral excipients.
  • halogen atom means any one of the radio- stable atoms of column 7 of the Periodic Table of the Elements, i.e., fluorine, chlorine, bromine, or iodine.
  • alkyl means any unbranched or branched, substituted or unsubstituted, fully saturated (no double or triple bonds) hydrocarbon group.
  • the alkyl group may have 1 to 24 carbon atoms (whenever it appears herein, a numerical range such as “1 to 24" refers to each integer in the given range; e.g., "1 to 24 carbon atoms” means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 24 carbon atoms, although the present definition also covers the occurrence of the term "alkyl” where no numerical range is designated).
  • the alkyl group may also be a medium size alkyl having 1 to 10 carbon atoms.
  • the alkyl group could also be a lower alkyl having 1 to 5 carbon atoms.
  • the alkyl group of the compounds may be designated as "C 1-6 alkyl” or similar designations.
  • “C 1-6 alkyl” indicates that there are one to six carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec- butyl, t-butyl, pentyl and hexyl.
  • Typical alkyl groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, hexyl, and the like.
  • substituted has its ordinary meaning, as found in numerous contemporary patents from the related art. See, for example, U.S. Patent Nos. 6,509,331; 6,506,787; 6,500,825; 5,922,683; 5,886,210; 5,874,443; and 6,350,759; all of which are incorporated herein in their entireties by reference. Specifically, the definition of substituted is as broad as that provided in U.S. Patent No.
  • substituted alkyl such that it refers to an alkyl group, preferably of from 1 to 10 carbon atoms, having from 1 to 5 substituents, and preferably 1 to 3 substituents, selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyacylamino, cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, keto, thioketo, thiol, thioalkoxy, substituted thioalkoxy, thiocyanate, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, azido
  • cycloalkyl refers to any non-aromatic hydrocarbon ring, preferably having three to twelve atoms comprising the ring.
  • acyl refers to alkyl or aryl groups derived from an oxoacid, with an acetyl group being preferred.
  • alkoxycarbonylacyl refers to an acyl group substituted with an alkoxycarbonyl group.
  • Typical alkoxycarbonylacyl groups include, but are in no way limited to, CH 3 OC(O)CH 2 C(O)-, CH 3 CH 2 CH 2 OC(O)CH 2 C(O)-, 4- ethoxycarbonylbenzoyl-, 4-methoxycarbonylbenzoyl-, 4-propoxycarbonylbenzoyl-, 3-tert- butoxycarbonylbenzoyl-, and the like.
  • amino refers to amine radicals, wherein one or both hydrogen atoms are optionally replaced by substituents such as alkyl, and aryl groups.
  • Typical amino groups include, but are in no way limited to, -NH 2 , -NHMe, -NHEt, -NHCH 2 phenyl, -N(Me)(phenyl), -N(Et)(Me), -N(Phenyl)(Et), -N(Et)(CH 2 phenyl), -N(CH 2 phenyl)(phenyl), and the like.
  • aminocarbonyl refers to a carbonyl substituted with an amino.
  • Typical aminocarbonyl groups include, but are in no way limited to, -C(O)NH 2 , -C(O)NHMe, -C(O)NHEt, -C(O)NHCH 2 phenyl, -C(O)N(Me)(phenyl), -C(O)N(Et)(Me), -C(O)N(Phenyl)(Et), -C(O)N(Et)(CH 2 phenyl),
  • acyloxy refers to an acyl group attached to an oxygen with the oxygen being the attachment point.
  • Typical acyloxy groups include, but are in no way limited to, MeC(O)O-, PhenylC(O)O-, and the like.
  • alkenyl as used herein, means any unbranched or branched, substituted or unsubstituted, unsaturated hydrocarbon including polyunsaturated hydrocarbons, with C 1 -C 6 unbranched, mono-unsaturated and di-unsaturated, unsubstituted hydrocarbons being preferred, and mono-unsaturated, di-halogen substituted hydrocarbons being most preferred.
  • cycloalkenyl refers to any non-aromatic hydrocarbon ring, preferably having five to twelve atoms comprising the ring and having at least one unsaturated bond.
  • heterocycle or “heterocyclic” refer to any non-aromatic cyclic compound containing one or more heteroatoms. In polycyclic ring systems, the one or more heteroatoms, may be present in only one of the rings.
  • a heterocycle or heterocyclic group may be substituted or unsubstituted. The substituted heterocycle or heterocyclic group can be substituted with any substituent, including those described above and those known in the art.
  • aryl refers to a carbocyclic (all carbon) ring or two or more fused rings (rings that share two adjacent carbon atoms) that have a fully delocalized pi-electron system.
  • Typical aryl groups include, but are in no way limited to, benzene, naphthalene, azulene and the like.
  • An aryl group may be substituted or unsubstituted.
  • the substituted aryls can be substituted with any substituent, including those described above and those known in the art.
  • heteroaryl refers to an aromatic heterocyclic group, whether one ring or multiple fused rings. In fused ring systems, the one or more heteroatoms, may be present in only one of the rings.
  • the hetero atom is an element other than carbon, including but not limited to, nitrogen, oxygen and sulfur.
  • Typical heteroaryl groups include, but are in no way limited to, indole, oxazole, benzoxazole, isoxazole, benzisoxazole, thiazole, benzothiazole, isothiazole, imidazole, benzimidazole, pyrazole, pyridazine, pyridine, pyrimidine, purine, pyrazine, pteridine, pyrrole, phenoxazole, oxazole, isoxazole, oxadiazole, benzopyrazole, indazole, quinolizine, cinnoline, phthalazine, quinazoline, quinoxaline, and the like.
  • a heteroaryl group of this invention may be substituted or unsubstituted.
  • the substituted heteroaryls can be substituted with any substituent, including those described above and those known in the art.
  • alkoxy refers to any unbranched, or branched, substituted or unsubstituted, saturated or unsaturated ether, with C 1 -C 6 unbranched, saturated, unsubstituted ethers being preferred, with methoxy being preferred, and also with dimethyl, diethyl, methyl-isobutyl, and methyl-tert-butyl ethers also being preferred.
  • cycloalkoxy refers to any cycloalkyl attached to an oxygen atom with the oxygen being the attachment point to the rest of the molecule.
  • arylalkoxy refers to an alkoxy group substituted with an aryl group.
  • arylalkoxy can be methoxy substituted with an aryl group, such as benzyloxy and the like.
  • arylalkoxycarbonyl refers to an arylalkoxy group attached to a carbonyl group with the carbonyl being the attachment point to the rest of the molecule.
  • Typical arylalkoxycarbonyl groups include, but are in no way limited to, benzyl oxycarbonyl (i.e., PhenylCH 2 OC(O)-) and the like.
  • cycloalkyl refers to any non-aromatic hydrocarbon ring.
  • alkoxycarbonyl refers to any linear, branched, cyclic, saturated, unsaturated, aliphatic or aryl alkoxy attached to a carbonyl group with the carbonyl group being the attachment point to the rest of the molecule.
  • Typical alkoxycarbonyl groups include, but are in no way limited to, ethoxycarbonyl group, propyl oxycarbonyl group, isopropyl oxycarbonyl group, butoxy carbonyl group, sec- butoxycarbonyl group, tert-butoxy carbonyl group, cyclopentyloxycarbonyl group, cyclohexyl oxycarbonyl group, benzyl oxycarbonyl group, allyloxycarbonyl group, phenyl oxycarbonyl group, pyridyl oxycarbonyl group, and the like.
  • alkoxycarbonyloxy refers to an alkoxycarbonyl group attached to an oxygen with the oxygen being the attachment point to the rest of the molecule.
  • Typical alkoxycarbonyloxy groups include, but are in no way limited to, MeOC(O)O-, methoxycarbonyloxy group, ethoxycarbonyl oxy group, propyl oxycarbonyl oxy group, isopropyloxycarbonyloxy group, butoxycarbonyloxy group, sec-butoxycarbonyloxy group, tert-butoxycarbonyloxy group, cyclopentyloxycarbonyloxy group, cyclohexyl oxycarbonyl oxy group, allyloxycarbonyloxy group, benzyl oxycarbonyl oxy group and the like.
  • alkoxycarbonyloxy groups refer to aryloxy and heteroaryloxy groups such as, phenyl oxycarbonyl oxy group, pyridyl oxycarbony
  • the terms “pure,” “purified,” “substantially purified,” and “isolated” as used herein refer to the compound of the embodiment being free of other, dissimilar compounds with which the compound, if found in its natural state, would be associated in its natural state.
  • the compound may comprise at least 0.5%, 1%, 5%, 10%, or 20%, and most preferably at least 50% or 75% of the mass, by weight, of a given sample.
  • the terms "derivative,” “variant,” or other similar term refers to a compound that is an analog of the other compound.
  • Certain of the compounds of Formula I can be obtained and purified or can be obtained via semi-synthesis from purified compounds as set forth herein.
  • the compounds of Formula 11-15 preferably, Formulae 11-16 (Salinosporamide A), 11-17, 11-18 and 11-19, can be obtained synthetically or by fermentation. Exemplary fermentation procedures are provided below.
  • the compounds of structure 11-15, preferably, Formulae 11-16, 11-17, 11-18 and 11-19 can be used as starting compounds in order to obtain/synthesize various of the other compounds described herein. Exemplary non- limiting syntheses are provided herein.
  • the compound of Formula 11-16 may be produced through a high-yield saline fermentation (-350 - 400 mg/L) and modifications of the conditions have yielded new analogs in the fermentation extracts. Additional analogs can be generated through directed biosynthesis. Directed biosynthesis is the modification of a natural product by adding biosynthetic precursor analogs to the fermentation of producing microorganisms (Lam, et al, J Antibiot (Tokyo) 44:934 (1991), Lam, et al, J Antibiot (Tokyo) 54: 1 (2001); which is hereby incorporated by reference in its entirety).
  • Biotransformation reactions are chemical reactions catalyzed by enzymes or whole cells containing these enzymes.
  • Microbial natural products are ideal substrates for biotransformation reactions as they are synthesized by a series of enzymatic reactions inside microbial cells. Riva, S., Curr Opin Chem Biol 5: 106 (2001).
  • Ethylmalonyl-CoA is derived from butyryl-CoA, which can be derived either from valine or crotonyl-CoA. Liu, et al, Metab Eng 3:40 (2001). Phenylalanine is derived from shikimic acid.
  • compounds such as structure 11-16 and its analogs may be produced synthetically, e.g., such as described in United States Application Serial No. 11/697,689, which is incorporated by reference in its entirety.
  • the production of compounds of Formulae 1-7, II- 16, 11-17, 11-18, 11-20,11- 24C, ⁇ -26, ⁇ -27 and 11-28 can be carried out by cultivating strain CNB476 and strain NPS21184, a natural variant of strain CNB476, in a suitable nutrient medium under conditions described herein, preferably under submerged aerobic conditions, until a substantial amount of compounds are detected in the fermentation; harvesting by extracting the active components from the fermentation broth with a suitable solvent; concentrating the solvent containing the desired components; then subjecting the concentrated material to chromatographic separation to isolate the compounds from other metabolites also present in the cultivation medium.
  • the culture (CNB476) was deposited on June 20, 2003 with the American Type Culture Collection (ATCC) in Rockville, MD and assigned the ATCC patent deposition number PTA-5275.
  • Strain NPS21184 a natural variant of strain CNB476, was derived from strain CNB476 as a single colony isolate. Strain NPS21184 has been deposited to ATCC on April 27, 2005. The ATCC deposit meets all of the requirements of the Budapest treaty.
  • the culture is also maintained at and available from Nereus Pharmaceutical Culture Collection at 10480 Wateridge Circle, San Diego, CA 92121.
  • mutants such as those produced by the use of chemical or physical mutagens including X-rays, etc. and organisms whose genetic makeup has been modified by molecular biology techniques, may also be cultivated to produce the starting compounds of Formulae U- 16, 11-17, and 11-18.
  • Production of compounds can be achieved at temperature conducive to satisfactory growth of the producing organism, e.g. from 16°C to 40 0 C, but it is preferable to conduct the fermentation at 22°C to 32°C.
  • the aqueous medium can be incubated for a period of time necessary to complete the production of compounds as monitored by high pressure liquid chromatography (HPLC), preferably for a period of about 2 to 10 days, on a rotary shaker operating at about 50 rpm to 400 rpm, preferably at 150 rpm to 250 rpm, for example.
  • HPLC high pressure liquid chromatography
  • the production of the compounds can also be achieved by cultivating the production strain in a bioreactor, such as a fermentor system that is suitable for the growth of the production strain.
  • the sources of carbon include glucose, fructose, mannose, maltose, galactose, mannitol and glycerol, other sugars and sugar alcohols, starches and other carbohydrates, or carbohydrate derivatives such as dextran, cerelose, as well as complex nutrients such as oat flour, corn meal, millet, corn, and the like.
  • the exact quantity of the carbon source that is utilized in the medium will depend in part, upon the other ingredients in the medium, but an amount of carbohydrate between 0.5 to 25 percent by weight of the medium can be satisfactorily used, for example.
  • These carbon sources can be used individually or several such carbon sources can be combined in the same medium, for example. Certain carbon sources are preferred as hereinafter set forth.
  • the sources of nitrogen include amino acids such as glycine, arginine, threonine, methionine and the like, ammonium salt, as well as complex sources such as yeast extracts, corn steep liquors, distiller solubles, soybean meal, cotttonseed meal, fish meal, peptone, and the like.
  • the various sources of nitrogen can be used alone or in combination in amounts ranging from 0.5 to 25 percent by weight of the medium, for example.
  • nutrient inorganic salts which can be incorporated in the culture media, are the customary salts capable of yielding sodium, potassium, magnesium, calcium, phosphate, sulfate, chloride, carbonate, and like ions. Also included are trace metals such as cobalt, manganese, iron, molybdenum, zinc, cadmium, and the like.
  • the compounds disclosed herein are used in pharmaceutical compositions.
  • the compounds preferably can be produced by the methods disclosed herein.
  • the compounds can be used, for example, in pharmaceutical compositions comprising a pharmaceutically acceptable carrier prepared for storage and subsequent administration.
  • embodiments relate to a pharmaceutically effective amount of the products and compounds disclosed above in a pharmaceutically acceptable carrier or diluent.
  • Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro edit. 1985), which is incorporated herein by reference in its entirety.
  • Preservatives, stabilizers, dyes and even flavoring agents can be provided in the pharmaceutical composition.
  • compositions can be formulated and used as tablets, capsules, or elixirs for oral administration; suppositories for rectal administration; sterile solutions, suspensions for injectable administration; patches for transdermal administration, and sub- dermal deposits and the like.
  • injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • Suitable excipients are, for example, water, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride, and the like.
  • the injectable pharmaceutical compositions may contain minor amounts of nontoxic auxiliary substances, such as wetting agents, pH buffering agents, and the like.
  • absorption enhancing preparations for example, liposomes, can be utilized.
  • compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or other organic oils such as soybean, grapefruit or almond oils, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • compositions for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).
  • PVP polyvinylpyrrolidone
  • disintegrating agents can be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings for this purpose, concentrated sugar solutions can be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments can be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • concentrated sugar solutions can be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments can be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • Such formulations can be made using methods known in the art (see, for example, U.S. Patent Nos.
  • compositions well known in the pharmaceutical art for uses that include topical, intraocular, intranasal, and intraauricular delivery.
  • Pharmaceutical formulations include aqueous ophthalmic solutions of the active compounds in water-soluble form, such as eyedrops, or in gellan gum (Shedden et al., Clin. Ther., 23 (3): 440-50 (2001)) or hydrogels (Mayer et al., Ophthalmologic ⁇ 210(2): 101-3 (1996)); ophthalmic ointments; ophthalmic suspensions, such as microparticulates, drug-containing small polymeric particles that are suspended in a liquid carrier medium (Joshi, A.
  • lipid-soluble formulations (Aim et al., Prog. Clin. Biol. Res., 312:447-58 (1989)), and microspheres (Mordenti, Toxicol. Sci., 52(1): 101-6 (1999)); and ocular inserts. All of the above-mentioned references, are incorporated herein by reference in their entireties.
  • suitable pharmaceutical formulations are most often and preferably formulated to be sterile, isotonic and buffered for stability and comfort.
  • Pharmaceutical compositions may also include drops and sprays often prepared to simulate in many respects nasal secretions to ensure maintenance of normal ciliary action.
  • suitable formulations are most often and preferably isotonic, slightly buffered to maintain a pH of 5.5 to 6.5, and most often and preferably include anti-microbial preservatives and appropriate drug stabilizers.
  • Pharmaceutical formulations for intraauricular delivery include suspensions and ointments for topical application in the ear. Common solvents for such aural formulations include glycerin and water.
  • ком ⁇ онентs to formulate the compounds of Formulae I and II as an anti-cancer agent, known surface active agents, excipients, smoothing agents, suspension agents and pharmaceutically acceptable film-forming substances and coating assistants, and the like can be used.
  • alcohols, esters, sulfated aliphatic alcohols, and the like can be used as surface active agents; sucrose, glucose, lactose, starch, crystallized cellulose, mannitol, light anhydrous silicate, magnesium aluminate, magnesium methasilicate aluminate, synthetic aluminum silicate, calcium carbonate, sodium acid carbonate, calcium hydrogen phosphate, calcium carboxymethyl cellulose, and the like can be used as excipients; magnesium stearate, talc, hardened oil and the like can be used as smoothing agents; coconut oil, olive oil, sesame oil, peanut oil, soya can be used as suspension agents or lubricants; cellulose acetate phthalate as a derivative of a carbohydrate such
  • the compounds of Formulae I and II or compositions including compounds of Formulae I and II can be administered by either oral or non-oral pathways.
  • it can be administered in capsule, tablet, granule, spray, syrup, or other such form.
  • it can be administered as an aqueous suspension, an oily preparation or the like or as a drip, suppository, salve, ointment or the like, when administered via injection, subcutaneously, intraperitoneally, intravenously, intramuscularly, or the like.
  • the anti-cancer agent can be mixed with additional substances to enhance their effectiveness.
  • the disclosed chemical compounds and the disclosed pharmaceutical compositions are administered by a particular method as an anticancer, anti-microbial or anti-inflammatory.
  • Such methods include, among others, (a) administration though oral pathways, which administration includes administration in capsule, tablet, granule, spray, syrup, or other such forms; (b) administration through non-oral pathways, which administration includes administration as an aqueous suspension, an oily preparation or the like or as a drip, suppository, salve, ointment or the like; administration via injection, subcutaneously, intraperitoneally, intravenously, intramuscularly, intradermally, or the like; as well as (c) administration topically, (d) administration rectally, or (e) administration vaginally, as deemed appropriate by those of skill in the art for bringing the compound of the present embodiment into contact with living tissue; and (f) administration via controlled released formulations, depot formulations, and infusion pump delivery.
  • modes of administration and as further disclosure of modes of administration, disclosed herein
  • compositions that include the described compounds required as a dose will depend on the route of administration, the type of animal, including human, being treated, and the physical characteristics of the specific animal under consideration.
  • the dose can be tailored to achieve a desired effect, but will depend on such factors as weight, diet, concurrent medication and other factors which those skilled in the medical arts will recognize.
  • a compound represented by Formulae I and II can be administered to a patient in need of an anti-cancer agent, until the need is effectively reduced or preferably removed.
  • the products or compositions can be used alone or in combination with one another, or in combination with other therapeutic or diagnostic agents.
  • products can be utilized in vivo, ordinarily in a mammal, preferably in a human, or in vitro.
  • the products or compositions can be administered to the mammal in a variety of ways, including parenterally, intravenously, subcutaneously, intramuscularly, colonically, rectally, vaginally, nasally or intraperitoneally, employing a variety of dosage forms. Such methods may also be applied to testing chemical activity in vivo.
  • the useful in vivo dosage to be administered and the particular mode of administration will vary depending upon the age, weight and mammalian species treated, the particular compounds employed, and the specific use for which these compounds are employed.
  • the determination of effective dosage levels can be accomplished by one skilled in the art using routine pharmacological methods. Typically, human clinical applications of products are commenced at lower dosage levels, with dosage level being increased until the desired effect is achieved. Alternatively, acceptable in vitro studies can be used to establish useful doses and routes of administration of the compositions identified by the present methods using established pharmacological methods.
  • dosages In non-human animal studies, applications of potential products are commenced at higher dosage levels, with dosage being decreased until the desired effect is no longer achieved or adverse side effects disappear.
  • the dosage may range broadly, depending upon the desired affects and the therapeutic indication. Typically, dosages can be between about 10 mg/kg and 100 mg/kg body weight, preferably between about 100 mg/kg and 10 mg/kg body weight. Alternatively dosages can be based and calculated upon the surface area of the patient, as understood by those of skill in the art. Administration is preferably oral on a daily or twice daily basis.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. See for example, Fingl et ah, in The Pharmacological Basis of Therapeutics, 1975, which is incorporated herein by reference in its entirety. It should be noted that the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity, or to organ dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity).
  • the magnitude of an administrated dose in the management of the disorder of interest will vary with the severity of the condition to be treated and to the route of administration. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency, will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above can be used in veterinary medicine.
  • Suitable administration routes may include oral, rectal, transdermal, vaginal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • the agents of the embodiment can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer.
  • physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art.
  • Use of pharmaceutically acceptable carriers to formulate the compounds herein disclosed for the practice of the embodiment into dosages suitable for systemic administration is within the scope of the embodiment. With proper choice of carrier and suitable manufacturing practice, the compositions disclosed herein, in particular, those formulated as solutions, can be administered parenterally, such as by intravenous injection.
  • the compounds can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration. Such carriers enable the compounds of the embodiment to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • Agents intended to be administered intracellular ⁇ can be administered using techniques well known to those of ordinary skill in the art. For example, such agents can be encapsulated into liposomes, then administered as described above. All molecules present in an aqueous solution at the time of liposome formation are incorporated into the aqueous interior. The liposomal contents are both protected from the external micro- environment and, because liposomes fuse with cell membranes, are efficiently delivered into the cell cytoplasm. Additionally, due to their hydrophobicity, small organic molecules can be directly administered intracellularly.
  • these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • the preparations formulated for oral administration can be in the form of tablets, dragees, capsules, or solutions.
  • the pharmaceutical compositions can be manufactured in a manner that is itself known, for example, by means of conventional mixing, dissolving, granulating, dragee-making, levitating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
  • Compounds disclosed herein can be evaluated for efficacy and toxicity using known methods.
  • the toxicology of a particular compound, or of a subset of the compounds, sharing certain chemical moieties can be established by determining in vitro toxicity towards a cell line, such as a mammalian, and preferably human, cell line. The results of such studies are often predictive of toxicity in animals, such as mammals, or more specifically, humans.
  • the toxicity of particular compounds in an animal model such as mice, rats, rabbits, dogs or monkeys, can be determined using known methods.
  • the efficacy of a particular compound can be established using several art recognized methods, such as in vitro methods, animal models, or human clinical trials.
  • the compound produced by methods of the embodiment inhibits the progression of the disease when it is dissolved in an organic solvent or hydrous organic solvent and it is directly applied to any of various cultured cell systems.
  • Usable organic solvents include, for example, methanol, methylsulfoxide, and the like.
  • the formulation can, for example, be a powder, granular or other solid inhibitor, or a liquid inhibitor prepared using an organic solvent or a hydrous organic solvent.
  • a preferred concentration of the compound produced by the method of the embodiment for use as an anticancer compound is generally in the range of about 1 to about 100 ⁇ g/mL, the most appropriate use amount varies depending on the type of cultured cell system and the purpose of use, as will be appreciated by persons of ordinary skill in the art. Also, in certain applications it can be necessary or preferred to persons of ordinary skill in the art to use an amount outside the foregoing range.
  • needle is not an absolute term and merely implies that the patient can benefit from the treatment of the anti-cancer agent in use.
  • patient what is meant is an organism that can benefit by the use of an anticancer agent.
  • any organism with cancer such as, pancreatic cancer.
  • the patient's health may not require that an anti-cancer agent be administered, however, the patient may still obtain some benefit by the reduction of the level of cancer cells present in the patient, and thus be in need.
  • the anti- anti-cancer agent is effective against one type of cancer, but not against other types; thus, allowing a high degree of selectivity in the treatment of the patient.
  • the anti-cancer agent is effective against a broad spectrum of cancers or all cancers.
  • cancers, against which the compounds can be effective include pancreatic cancer, a colorectal carcinoma, a prostate carcinoma, a breast adenocarcinoma, a non-small cell lung carcinoma, an ovarian carcinoma, multiple myelomas, a melanoma, and the like.
  • "Therapeutically effective amount,” “pharmaceutically effective amount,” or similar term means that amount of drug or pharmaceutical agent that will result in a biological or medical response of a cell, tissue, system, animal, or human that is being sought. In a preferred embodiment, the medical response is one sought by a researcher, veterinarian, medical doctor, or other clinician.
  • Anti-cancer agent refers to a compound or composition including the compound that reduces the likelihood of survival of a cancer cell.
  • the likelihood of survival is determined as a function of an individual cancer cell; thus, the anticancer agent will increase the chance that an individual cancer cell will die.
  • the likelihood of survival is determined as a function of a population of cancer cells; thus, the anti-cancer agent will increase the chances that there will be a decrease in the population of cancer cells.
  • anti-cancer agent means chemotherapeutic agent or other similar term.
  • a "chemotherapeutic agent” is a chemical compound useful in the treatment of a neoplastic disease, such as cancer.
  • chemotherapeutic agents include alkylating agents, such as a nitrogen mustard, an ethyleneimine and a methylmelamine, an alkyl sulfonate, a nitrosourea, and a triazene, folic acid antagonists, antimetabolites of nucleic acid metabolism, antibiotics, pyrimidine analogs, 5-fluorouracil, cisplatin, purine nucleosides, amines, amino acids, triazol nucleosides, corticosteroids, a natural product such as a vinca alkaloid, an epipodophyllotoxin, an antibiotic, an enzyme, a taxane, and a biological response modifier or antibodies to biological response modifiers or other agents; miscellaneous agents such as a platinum coordination complex, an anthracenedione, an anthracycline, a substituted
  • Adriamycin Doxorubicin, 5- Fluorouracil, Cytosine arabinoside ("Ara-C"), Cyclophosphamide, Thiotepa, Busulfan, Cytoxin, Taxol, Toxotere, Methotrexate, Cisplatin, Melphalan, Vinblastine, Bleomycin, Etoposide, Ifosfamide, Mitomycin C, Mitoxantrone, Vincreistine, Vinorelbine, Carboplatin, Teniposide, Daunomycin, Carminomycin, Aminopterin, Dactinomycin, Mitomycins, Esperamicins, Melphalan, and other related nitrogen mustards. Also included in this definition are hormonal agents that act to regulate or inhibit hormone action on tumors, such as tamoxifen and onapristone.
  • chemotherapeutics include alkaloids, alkylating agents, antibiotics, antimetabolites, enzymes, hormones, platinum compounds, immunotherapeutics (antibodies, T-cells, epitopes), BRMs, and the like.
  • Examples include, Vincristine, Vinblastine, Vindesine, Paclitaxel (Taxol), Docetaxel, topoisomerase inhibibitors epipodophyllotoxins (Etoposide (VP- 16), Camptothecin, nitrogen mustards (cyclophosphamide Cytoxan), Nitrosoureas, Carmustine, lomustine, dacarbazine, hydroxymethylmelamine, thiotepa and mitocycin C, Dactinomycin (Actinomycin D), anthracycline antibiotics (Daunorubicin, Daunomycin, Cerubidine), Doxorubicin (Adriamycin), Idarubicin (Idamycin), Anthracenediones (Mitoxantrone), Bleomycin (Blenoxane), Plicamycin (Mithramycin, Antifolates (Methotrexate (Folex, Mexate)), purine antimetabolites (6-mercap
  • the two major anticancer drugs in this category are 6-mercaptopurine and 6-thioguanine, Chlorodeoxyadenosine and Pentostatin, Pentostatin (2'-deoxycoformycin), pyrimidine antagonists, Avastin, Leucovorin, Oxaliplatin, fluoropyrimidines (5-fluorouracil(Adrucil), 5- fluorodeoxyuridine (FdUrd) (Floxuridine)), Cytosine Arabinoside (Cytosar, ara-C), Fludarabine, L-asparaginase, Hydroxyurea, glucocorticoids, antiestrogens, tamoxifen, nonsteroidal antiandrogens, flutamide, aromatase inhibitors Anastrozole(Arimidex), Cisplatin, 6-Mercaptopurine and Thioguanine, Methotrexate, Cytoxan, Cytarabine, L- Asparaginase, Steroids:
  • proteasome inhibitors such as bortezomib and carfilzomib (PR-171) can be used in combination with the instant compounds, for example.
  • biologies can include agents such as TRAIL, antibodies to TRAIL and agonistic antibodies TRAIL death receptors DR4 and DR5, integrins such as alpha- V-beta-3 ( ⁇ V ⁇ 3) and / or other cytokine/growth factors that are involved in angiogenesis, VEGF, EGF, FGF and PDGF and antibodies to these cytokines/growth factors such as Erbitux.
  • the compounds can be conjugated to or delivered with an antibody.
  • the additional chemotherapeutic is a histone deacetylase inhibitor (HDACi).
  • HDACi histone deacetylase inhibitor
  • panobinostat
  • SAHA suberoylanilide hydroxamic acid
  • the additional chemotherapeutic is a vascular disrupting agents (VDA).
  • VDAs include combratostatin CA4P and NPI- 2358.
  • NPI-2358 is represented by the following formula:
  • the anti-cancer agent may act directly upon a cancer cell to kill the cell, induce death of the cell, to prevent division of the cell, and the like.
  • the anticancer agent may indirectly act upon the cancer cell by limiting nutrient or blood supply to the cell, for example.
  • Such anti-cancer agents are capable of destroying or suppressing the growth or reproduction of cancer cells, such as a colorectal carcinoma, a prostate carcinoma, a breast adenocarcinoma, a non-small cell lung carcinoma, an ovarian carcinoma, multiple myelomas, a melanoma, and the like.
  • a described compound preferably a compound having the Formula I, including those as described herein, is considered an effective anti-cancer agent if the compound can influence 10% of the cancer cells, for example.
  • the compound is effective if it can influence 10 to 50% of the cancer cells.
  • the compound is effective if it can influence 50-80% of the cancer cells.
  • the compound is effective if it can influence 80-95% of the cancer cells.
  • the compound is effective if it can influence 95-99% of the cancer cells.
  • "Influence" is defined by the mechanism of action for each compound.
  • a compound prevents the division of cancer cells
  • influence is a measure of prevention of cancer cell division. Not all mechanisms of action need be at the same percentage of effectiveness.
  • a low percentage effectiveness can be desirable if the lower degree of effectiveness is offset by other factors, such as the specificity of the compound, for example.
  • a compound that is only 10% effective, for example, but displays little in the way of harmful side-effects to the host, or non-harmful microbes or cells can still be considered effective.
  • the compounds described herein are administered simply to remove cancer cells and need not be administered to a patient.
  • the compounds can be administered ex vivo to a cell sample, such as a bone marrow or stem cell transplant to ensure that only non-cancerous cells are introduced into the recipient. After the compounds are administered they may optionally be removed. Whether or not this is an option will depend upon the relative needs of the situation and the risks associated with the compound, which in part can be determined as described in the Examples below.
  • Strain CNB476 was grown in a 500-mL flask containing 100 mL of vegetative medium consisting of the following per liter of deionized water: glucose, 4 g; Bacto tryptone, 3 g; Bacto casitone, 5 g; and synthetic sea salt (Instant Ocean, Aquarium Systems), 30 g.
  • the first seed culture was incubated at 28°C for 3 days on a rotary shaker operating at 250 rpm.
  • Five mL each of the first seed culture was inoculated into three 500-ml flasks containing of 100 mL of the vegetative medium.
  • the second seed cultures were incubated at 28°C and 250 rpm on a rotary shaker for 2 days.
  • the production cultures were incubated at 28°C and 250 rpm on roatry shakers for 1 day. Approximately 2 to 3 grams of sterile Amberlite XAD-7 resin were added to the production cultures. The production cultures were further incubated at 28°C and 250 rpm on rotary shakers for 5 days and achieved a titer of Compound 11-16 of about 200 mg/L.
  • the culture broth was filtered through cheese cloth to recover the Amberlite XAD-7 resin.
  • the resin was extracted with 2 times 6 liters ethyl acetate followed by 1 time 1.5 liters ethyl acetate.
  • the combined extracts were dried in vacuo. The dried extract, containing 3.8 grams the compound of Formula 11-16 and lesser quantities of compounds of formulae 11-20 and II-24C, was then processed for the recovery of the compounds of Formula 1-7, 11-16, II- 20, II-24C, 11-26 and 11-28.
  • Strain NPS21184 was grown in a 500-mL flask containing 100 mL of vegetative medium consisting of the following per liter of deionized water: glucose, 8 g; yeast extract, 6 g; Hy-Soy, 6 g; and synthetic sea salt (Instant Ocean, Aquarium Systems), 30 g.
  • the first seed culture was incubated at 28°C for 3 days on a rotary shaker operating at 250 rpm.
  • Five mL of the first seed culture was inoculated into 500-mL flask containing of 100 mL of the vegetative medium.
  • the second seed cultures were incubated at 28°C and 250 rpm on a rotary shaker for 2 days.
  • the production cultures were incubated at 28°C and 250 rpm on rotary shakers for 1 day. Approximately 2 to 3 grams of sterile Amberlite XAD-7 resin were added to the production culture. The production culture was further incubated at 28°C and 250 rpm on rotary shaker for 4 days and achieved a titer of 350 - 400 mg/L for Compound II- 16. [0346] Alternatively, the production of the compounds can be achieved in a 42L fermentor system using strain NPS21184.
  • Strain NPS21184 was grown in a 500-mL flask containing 100 mL of vegetative medium consisting of the following per liter of deionized water: glucose, 8 g; yeast extract, 6 g; Hy-Soy, 6 g; and synthetic sea salt (Instant Ocean, Aquarium Systems), 30 g.
  • the first seed culture was incubated at 28°C for 3 days on a rotary shaker operating at 250 rpm.
  • Five mL of the first seed culture was inoculated into 500-mL flask containing of 100 mL of the vegetative medium.
  • the second seed cultures were incubated at 28°C and 250 rpm on a rotary shaker for 2 days.
  • Production Medium C consisting of the following per liter of deionized water: starch, 15 g; yeast extract 6 g; Hy-Soy, 6 g; ferric sulfate, 40 mg; potassium bromide, 100 mg; calcium carbonate, 1 g; and synthetic sea salt (Instant Ocean, Aquarium Systems), 30 g.
  • the fermentor cultures were operated at the following parameters: temperature, 28°C; agitation, 200 rpm; aeration, 13 L/min and back pressure, 4.5 psi.
  • approximately 600 grams of sterile Amberlite XAD-7 resin were added to the fermentor culture. The production culture was further incubated at the above operating parameters until day 4 of the production cycle.
  • the aeration rate was lowered to 8 L/min.
  • the fermentor culture achieved a titer of about 300 mg/L for Compound 11-16.
  • the culture broth was filtered through cheese cloth to recover the Amberlite XAD-7 resin.
  • the resin was extracted with 2 times 4.5 liters ethyl acetate followed by 1 time 1.5 liters ethyl acetate.
  • the combined extracts were dried in vacuo. The dried extract was then processed for the recovery of the Compounds of Formulae 1-7, II- 16, ⁇ -17, ⁇ -20, ⁇ -24C, ⁇ -26 and 11-28.
  • EXAMPLE 3 PURIFICATION OF COMPOUND OF FORMULAE 1-7, 11-16, 11-20, II-24C, 11-26 AND II-
  • Sample containing II-24C and 11-26 generated from the process described above were further separated using reversed-phase preparative HPLC as follows.
  • the sample containing II-24C 70 mg was dissolved in acetonitrile at a concentration of 10 mg/mL, and 500 ⁇ L was loaded on an HPLC column of dimensions 21 mm i.d. by 15 cm length containing Eclipse XDB-C 18 support.
  • the solvent gradient increased linearly from 15% acetonitrile /85% water to 100% acetonitrile over 23 minutes at a flow rate of 14.5 mL/min.
  • the solvent composition was held at 100% acetonitrile for 3 minutes before returning to the starting solvent mixture.
  • Compound 11-26 eluted at 17.5 minutes while compound II-24C eluted at 19 minutes under these conditions.
  • Crystalline 11-26 was obtained using a vapor diffusion method.
  • Compound 11-26 (15 mg) was dissolved in 100 ⁇ L of acetone in a 1.5 mL v-bottom HPLC vial. This vial was then placed inside a larger sealed vessel containing 1 mL of pentane. Crystals suitable for X-ray crystallography experiments were observed along the sides and bottom of the inner vial after 48 hours of incubation at 4°C.
  • a Biotage Flash 75Li system with a Flash 75L KP-SiI cartridge was used to process the filtered crude extract (10.0 g), enriched in Compound 11-16 and containing Compound of Formula 1-7.
  • the crude extract was dissolved to a concentration of 107 mg/mL in acetone and loaded directly onto the cartridge.
  • the following solvent step gradient was then run through the cartridge at a flow rate between 235 mL/min and 250 mL/min
  • Strain CNB476 was grown in a 500-mL flask containing 100 mL of the first vegetative medium consisting of the following per liter of deionized water: glucose, 4 g; Bacto tryptone, 3 g; Bacto casitone, 5 g; and synthetic sea salt (Instant Ocean, Aquarium Systems), 30 g.
  • the first seed culture was incubated at 28 C for 3 days on a rotary shaker operating at 250 rpm.
  • the first seed culture was inoculated into a 500-mL flask containing 100 mL of the second vegetative medium consisting of the following per liter of deionized water: starch, 1O g; yeast extract, 4 g; peptone, 2 g; ferric sulfate, 40 mg; potassium bromide, 100 mg; calcium carbonate, 1 g; and sodium bromide, 30 g.
  • the second seed cultures were incubated at 28°C for 7 days on a rotary shaker operating at 250 rpm. Approximately 2 to 3 gram of sterile Amberlite XAD-7 resin were added to the second seed culture.
  • the second seed culture was further incubated at 28°C for 2 days on a rotary shaker operating at 250 rpm.
  • Five ml of the second seed culture was inoculated into a 500-ml flask containing 100 mL of the second vegetative medium.
  • the third seed culture was incubated at 28°C for 1 day on a rotary shaker operating at 250 rpm. Approximately 2 to 3 gram of sterile Amberlite XAD-7 resin were added to the third seed culture.
  • the third seed culture was further incubated at 28°C for 2 days on a rotary shaker operating at 250 rpm. Five ml of the third culture was inoculated into a 500-ml flask containing 100 mL of the second vegetative medium.
  • the fourth seed culture was incubated at 28°C for 1 day on a rotary shaker operating at 250 rpm. Approximately 2 to 3 gram of sterile Amberlite XAD-7 resin were added to the fourth seed culture. The fourth seed culture was further incubated at 28°C for 1 day on a rotary shaker operating at 250 rpm. Five mL each of the fourth seed culture was inoculated into ten 500-mL flasks containing 100 mL of the second vegetative medium. The fifth seed cultures were incubated at 28°C for 1 day on a rotary shaker operating at 250 rpm. Approximately 2 to 3 grams of sterile Amberlite XAD-7 resin were added to the fifth seed cultures.
  • the fifth seed cultures were further incubated at 28°C for 3 days on a rotary shaker operating at 250 rpm.
  • Four mL each of the fifth seed culture was inoculated into one hundred and fifty 500-mL flasks containing 100 mL of the production medium having the same composition as the second vegetative medium.
  • Approximately 2 to 3 grams of sterile Amberlite XAD-7 resin were also added to the production culture.
  • the production cultures were incubated at 28°C for 6 day on a rotary shaker operating at 250 rpm.
  • the culture broth was filtered through cheese cloth to recover the Amberlite XAD-7 resin.
  • the resin was extracted with 2 times 3 liters ethyl acetate followed by 1 time 1 liter ethyl acetate.
  • the combined extracts were dried in vacuo.
  • the dried extract containing 0.42 g of the compound Formula 11-17 and 0.16 gram the compound of Formula 11-18, was then processed for the recovery of the compounds.
  • the resulting samples were dried in vacuo using no heat to remove the aqueous solvent mixture.
  • the spectroscopic data for these samples of compound 11-16 and compound 11-18 were found to be identical with those of samples prepared from earlier purification methods.
  • the sample of compound 11-18 was found to contain 8% of the lactone hydrolysis product and was further purified by washing through a normal phase silica plug (1 cm diameter by 2 cm height) and eluting using a solvent mixture of 20% EtOAc / 80% Hexanes (25 mL).
  • the resulting sample was found to contain pure compound II- 18.
  • Compounds of Formula II-5 A and Formula II-5B can be synthesized from compound of Formula 11-16 by epoxidation with mCPBA.
  • Diols can be synthesized by Sharpless dihydroxylation using AD mix- ⁇ and ⁇ : AD mix- ⁇ is a premix of four reagents, K 2 OsO 2 (OH) 4 ; K 2 CO 3 ; K 3 Fe(CN) 6 ; (DHQ) 2 - PHAL [l,4-bis(9-O-dihydroquinine)phthalazine] and AD mix- ⁇ is a premix of K 2 OsO 2 (OH) 4 ; K 2 CO 3 ; K 3 Fe(CN) 6 ; (DHQD) 2 -PHAL [l,4-bis(9-O- dihydroquinidine)phthalazine] which are commercially available from Aldrich.
  • the diol can also be synthesized by acid or base hydrolysis of epoxy compounds (Formula II-5A and II- 5B) which may be different to that of products obtained in Sharpless dihydroxylation in their stereochemistry at carbons bearing hydroxyl groups
  • any of the compounds of Formulae 11-16, 11-17 and 11-18 can be used as the starting compound.
  • compound of Formula 11-16 is used.
  • the starting compound is dissolved in t-butanol/water in a round bottom flask to which is added AD mix- ⁇ or ⁇ and a magnetic stir bar.
  • the reaction is monitored by silica TLC as well as mass spectrometer.
  • the pure diols are obtained by usual workup and purification by flash chromatography or HPLC.
  • the structures are confirmed by NMR spectroscopy and mass spectrometry. In this method both hydroxyl groups are on same side.
  • the epoxy ring is opened with various nucleophiles like NaCN, NaN 3 , NaOAc, HBr, HCl, etc. to creat various substituents on the cyclohexane ring, including a hydroxyl substituent. Examples:
  • HPLC conditions used for the purification were as follows: Phenomenex Luna 10 ⁇ m Silica column (25 cm x 21.2 mm ID) with a solvent gradient of 25% to 80% EtO Ac/Hex over 19 min, 80 to 100% EtOAc in 1 min, then 5 min at 100% EtOAc at a flow rate of 14.5 niL/min. An ELSD was used to monitor the purification process. Compound of Formula III-3C eluted at about 18 min (2.2 mg).
  • Reductive ring opening of epoxides (II-5): The compound of Formula is treated with metalhydrides like BH 3 -THF complex to make compound of Formula III-4.
  • the solvent volume was reduced to one third, absorbed on silica gel, poured on top of a 20 cc silica flash column and eluted in 20 mL fractions using a gradient of Hexane/EtO Ac from 10 to 100%.
  • the fraction eluted with 30% EtOAc in Hexane contained a mixture of rotamers of Formula II-13C in a ratio of 1.5:8.5.
  • the mixture was further purified by normal phase HPLC using the Phenomenex Luna 10 ⁇ m Silica column (25 cm x 21.2 mm ID) with a solvent gradient of 25% to 80% EtO Ac/Hex over 19 min, 80 to 100% EtOAc over 1 min, holding at 100% EtOAc for 5 min, at a flow rate of 14.5 niL/min.
  • An ELSD was used to monitor the purification process.
  • Compound of Formula II-13C eluted at 13.0 and 13.2 mins as a mixture of rotamers with in a ratio of 1.5:8.5 (7 mg).
  • the reaction mixture was acidified using 2 mL of 4% HCl solution in water and extracted with CH 2 Cl 2 .
  • the organic layer was evaporated to yield mixture of compound of formulae 11-25 and 11-16 in a 9.5:0.5 ratio as a white solid, which was further purified by normal phase HPLC using a Phenomenex Luna 10 ⁇ m Silica column (25 cm x 21.2 mm ID).
  • the mobile phase was 24% EtO Ac/76% Hexane, which was held isocratic for 19 min, followed by a linear gradient of 24% to 100% EtOAc over 1 min, and held at 100% EtOAc for 3 min; the flow rate was 25 mL/min.
  • An ELSD was used to monitor the purification process.
  • a rotamer mixture of the Compound of Formula II-13C (20 mg) was dissolved in acetone (4 mL) in a scintillation vial (20 mL) to which a catalytic amount (3 mg) of 10% (w/w) Pd/C and a magnetic stir bar were added.
  • the reaction mixture was stirred at room temperature for about 15 hours.
  • the reaction mixture was filtered through a 0.2 ⁇ m Gelman Acrodisc to remove the catalyst.
  • the ketone of the compounds of formula II-31 and 11-32 can be reduced by using sodium borohydride at 0 to -10 0 C in monoglyme solvent for about 14 minutes.
  • the reaction mixture can be acidified using 4% HCl solution in water and extracted with CH 2 Cl 2 .
  • the organic layer can be evaporated to yield the mixtures of compounds of formulae 11-33, II- 34, 11-35 and 11-36 which can be separated by chromatographic methods.
  • a 2.4 mg portion of compound 11-29 was further purified using additional C18 HPLC chromatography (ACE 5 ⁇ m C18-HL, 150 mm X 21 mm ID) using an isocratic solvent gradient consisting of 35% acetonitrile/65% H 2 O. Under these conditions compound 11-29 eluted after 20 minutes, while Compound 11-16 eluted after 21.5 minutes. The resulting sample consisted of 1.1 mg Compound 11-29 was used for characterization in biological assays.
  • the compounds of Formulae 11-37 and 11-38 can be prepared from the compound of Formula 11-19 by cyano-de-halogenation or thiocyanato-de-halogenation, respectively.
  • Compound 11-19 can be treated with NaCN or KCN to obtain compound 11-37.
  • Compound 11-19 can be treated with NaSCN or KSCN to obtain compound II- 38.
  • Compound 11-38 was purified by normal phase HPLC using a Phenomenex Luna 10 ⁇ m Silica column (25cm x 21.2 mm ID) with a solvent gradient of 0 to 95% H 2 O/acetonitrile over 21 min, at a flow rate of 14.5 mL/min. Diode array detector was used to monitor the purification process. Compound 11-38 (3.0 mg, 34% yield) eluted at 18.0 min as a pure compound. 11-38: UV Acetonitrile/H 2 O ⁇ max 203 (sh) nm; ESMS m/z 337.1 (M+H) + & 359.1 (M+Na) + .
  • Thiols and thioethers of the Formula 11-39 can be formed by dehalogenation of the compound of Formula 11-19.
  • the compound of the Formula 11-41 can be prepared by treatment of the compound of Formula 11-21 (or a protected derivative of 11-21, where the C-5 alcohol or lactam NH are protected, for example) with methyl sulfonyl chloride (mesyl chloride) in pyridine, for example, or by treatment with mesyl chloride in the presence of triethylaminde.
  • methyl sulfonyl chloride meyl chloride
  • Other sulfonate esters can be similarly prepared.
  • the alkene of the Formula 11-46 can be prepared by dehydroiodination of the compound of Formula 11-19, or by hydro-mesyloxy elimination of the compound of Formula 11-41, for example, by treatment with base.
  • the compound of the Formula 11-43 A can be prepared by treatment of the compound of Formula 11-19 with triphenyl phosphine to make a phosphorus ylide, which can be treated with various aldehydes, for example, glyoxylic acid methyl ester, to make Formula II-43A.
  • the reaction was quenched by washing the THF solution through a plug of silica gel (1 cm diameter by 2 cm length) along with further washing using a solution of 50% EtOAc / 50% hexanes (50 mL).
  • the combined silica plug washes were dried in vacuo and subjected to further Cl 8 HPLC purification in 2 injections (ACE 5 ⁇ m C 18-HL, 150 mm X 21 mm ID) using an isocratic solvent gradient consisting of 35% acetonitrile/65% H 2 O.
  • Compound 11-30 eluted under these conditions at 23.5 minutes and yielded 2.4 mg material (27% isolated yield) at 90.8% purity as measured by analytical HPLC.
  • An alternative normal phase purification method can be utilized using Phenomenex Luna 10 ⁇ m Silica column (25cm x 21.2 mm ID) with a solvent gradient consisting of 100% hexanes/ethyl acetate to 0% hexanes over 20 minutes. Compound 11-30 eluted under these conditions at 16.5 minutes and yielded 3.0 mg material (41% isolated yield) at 97.1% purity as measured by analytical HPLC.
  • Compound 11-30 can also be obtained by saline fermentation of strain CNB476.
  • CNB476 was transferred to 500-mL flasks containing 100 mL production medium consisting of the following per liter of deionized water: starch, 1O g; yeast extract, 4 g; Hy-Soy, 4 g; ferric sulfate, 40 mg; potassium bromide, 100 mg; calcium carbonate, 1 g; and synthetic sea salt, 30 g.
  • the production cultures were incubated at 28°C and 250 rpm for 1 day. Approximately 2 g of sterile Amberlite XAD-7 resin was added to the production cultures. The production cultures were further incubated for 5 days. The resin was recovered from the broth and extracted with ethyl acetate. The extract was dried in vacuo. The dried extract (8 g) was then processed for the recovery of Compound 11-30.
  • the crude extract was processed by flash chromatography using a Biotage Flash system.
  • the flash chromatography was developed by the following step gradient: i) Hexanes (IL); ii) 10% EtOAc in hexanes (IL); iii) 20% EtOAc in hexanes, first elution (IL); iv) 20% EtOAc in hexanes, second elution (IL); v) 20% EtOAc in hexanes, third elution (IL); vi) 25% EtOAc in hexanes (IL); vii) 50% EtOAc in hexanes (IL); viii) EtOAc (IL).
  • any of the compounds of Formulae 11-16, 11-17 and 11-18 can be used as the starting compound.
  • the secondary hydroxyl group in the starting compound is oxidized using either of the following reagents: pyridinium dichromate (PDC), pyridinium chlorochromate (PCC), Dess-Martin periodinane or oxalyl chloride (Swern oxidation) (Ref: Organic Syntheses, collective volumes I- VIII).
  • PDC pyridinium dichromate
  • PCC pyridinium chlorochromate
  • Dess-Martin periodinane or oxalyl chloride (Swern oxidation)
  • Dess-Martin periodinane can be used as a reagent for this reaction.
  • the resulting keto compound is treated with hydroxylamine or methoxy amine to generate oximes. Examples:
  • keto derivatives for example Formula II-8 and 11-13, are treated with sodium cyanoborohydride (NaBH 3 CN) in the presence of various bases to yield amine derivatives of the starting compounds which are subsequently hydrogenated with 10% Pd/C, H 2 to reduce the double bond in the cyclohexene ring.
  • NaBH 3 CN sodium cyanoborohydride
  • Any compound of Formulae 11-16, 11-17 and 11-18 can be used as a starting compound.
  • the Starting Compounds can be protected, for example, at the alcohol and/or at the lactam nitrogen positions, and treated with OsO 4 and NaIO 4 in THF-H 2 O solution to yield dial derivatives which are reduced to the alcohol with NaBH 4 .
  • the protecting groups can be removed at the appropriate stage of the reaction sequence to produce II-7 or II-6.
  • a starting compound of any of Formulae 11-16, 11-17 or 11-18 is dehydrated, for example, by treatment with mesylchloride in the presence of base, or, for example, by treatment with Burgess reagent or other dehydrating agents.
  • the resulting dehydrated compound is treated with OsO 4 , followed by NaIO 4 , or alternatively by ozonolysis, to yield an aldehyde group at the lactone-lactam ring junction.
  • a Starting Compound, such as the ketone of Formula II-13C, is treated with Pd/C to produce a cyclohexadiene derivative.
  • the new double bond can be at any position of the cyclohexene ring.
  • the ketone can be reduced, for example, with sodium borohydride, to obtain the corresponding secondary alcohol(s).
  • the cyclohexadiene derivative can be further treated, for example with DDQ, to aromatize the ring to a phenyl group.
  • the ketone can be reduced, for example, with sodium borohydride, to obtain the corresponding secondary alcohol(s).
  • Reductive amination is performed on the aldehyde group using various bases (eg. NH 3 ) and sodium cyanoborohydride to yield amine derivatives.
  • bases eg. NH 3
  • sodium cyanoborohydride e.g. NaBH 4
  • the aldehyde is reduced with NaBH 4 to form alcohols in the side chain.
  • a mixture obtained by thoroughly blending 1 g of a compound obtained and purified by the method of the embodiment, 98 g of lactose and 1 g of hydroxypropyl cellulose is formed into granules by any conventional method.
  • the granules are thoroughly dried and sifted to obtain a granule preparation suitable for packaging in bottles or by heat sealing.
  • the resultant granule preparations are orally administered at between approximately 100 mL/day to approximately 1000 mL/day, depending on the symptoms, as deemed appropriate by those of ordinary skill in the art of treating cancerous tumors in humans.
  • a mixture obtained by thoroughly blending 1 g of a compound obtained and purified by the method of the embodiment, 98 g of lactose and 1 g of hydroxypropyl cellulose is formed into granules by any conventional method.
  • the granules are thoroughly dried and sifted to obtain a granule preparation suitable for packaging in bottles or by heat sealing.
  • the resultant granule preparations are orally administered at between approximately 100 mL/day to approximately 1000 mL/day, depending on the symptoms, as deemed appropriate by those of ordinary skill in the art of treating cancerous tumors in humans.
  • Jurkat, K562, ML-I and 12.1 FADD deficient Jurkat human leukemia cell lines were purchased from American Type Culture Collection (Rockville, MD). Caspase- 8 deficient Jurkat cells, 19.2, were obtained from University of Texas, M. D. Anderson Cancer Center, Houston, TX. All cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS (Hyclone, Logan, UT), L-glutamate and penicillin/streptomycin (Sigma, St. Louis, MO). Cells were maintained at 37°C with 5% CO 2 . Peripheral blood were obtained from a Philadelphia positive (Ph+) ALL patient.
  • FBS heat-inactivated FBS
  • L-glutamate L-glutamate
  • penicillin/streptomycin Sigma, St. Louis, MO
  • Mononuclear cells were isolated using double density Ficoll-Hypaque gradients composed of Histopaque 1077 and 1119 (Sigma, St. Louis, MO) as previously described (see Chandra J, Ralphbarth J, Le S, et al. Involvement of reactive oxygen species in adaphostin-induced cytotoxicity in human leukemia cells. Blood. 2003; 1 02:4512-4519). Reagents
  • Salinosporamide A was obtained from Nereus Pharmaceuticals, bortezomib was obtained from the M.D. Anderson Cancer Center pharmacy.
  • Fluorogenic substrates, suc-LLVY-amc and z-LLE-amc were from AG Scientific, Inc. (San Diego, CA) and boc-LLR-amc from Bachem (King of Prussia, PA).
  • NAC and staurosporine were purchased from Sigma (St.
  • H2DCF-DA 6-carboxy-2',7' dichlorodihydrofluorescein
  • HEt dihydroethidium
  • TMRE tetramethylrhodamine ethyl ester
  • Antibodies were obtained from: Caspase-8, caspase-9, caspase-3, FADD, and Bid (Cell Signaling, Beverly, MA); CH-I l (MBL International, Woburn, MA); p27 (Transduction Laboratories, San Diego, CA); cytochrome c (BD PharMingen, San Diego, CA); and actin (Sigma, St. Louis, MO).
  • the caspase inhibitors zVAD-fmk, IETD-fmk and LEHD-fmk were purchased from Calbiochem (San Diego, CA).
  • Annexin V-FITC was purchased from BD Bioscience (Franklin Lakes, NJ). 2OS Proteasome Activity Assay
  • the chymotrypsin-like, trypsin-like and caspase-like activity of the 2OS proteasome of leukemia cells can be determined by measurement of fluorescence generated from the cleavage of the fluorogenic substrates suc-LLVY-amc, boc LRR-amc and z-LLE- amc respectively (see Lightcap ES, McCormack TA, Pi en CS, Chau V, Adams J, Elliott PJ. Proteasome inhibition measurements: clinical application. Clin Chem. 2000;46:673-683).
  • Apoptosis was assessed by propidium iodide (PI) staining followed by fluorescence-activated cell sorting (FACS) analysis as described previously (see Chandra J, Ralphbarth J, Le S, et al. Involvement of reactive oxygen species in adaphostin-induced cytotoxicity in human leukemia cells. Blood. 2003; 1 02:4512-4519). Following incubation with different doses of Salinosporamide A for 24 h, cells were pelleted by centrifugation and resuspended with PBS containing 50 ⁇ g/mL PI, 0.1 % Triton-X-1 00, and 0.1 % sodium citrate.
  • synergism was determined using isobologram analysis based on the Chou and Talalay method with Calcusyn (Biosoft, Ferguson, MO) software program 32.
  • a combination index (CI) value ⁇ 1.0 indicates synergism: from 0.1-0.3 indicates strong synergism, and from 0.3-0.7 synergism.
  • In vivo data were analyzed with linear regression to estimate the rate of change (i.e., the slope of the regression line) of the WBC count over time for the mice treated with diluent or Salinosporamide A.
  • Salinosporamide A inhibits the 2OS proteolytic activities in leukemia cells.
  • cell lines representative of CML, ALL and AML K562, Jurkat and ML-I respectively. Cells were incubated with 1 ⁇ M Salinosporamide A for 1 h and the chymotrypsin-like, caspase-like and trypsin-like activities were measured using distinct fluorogenic peptides.
  • Figures IA and IB show that 1 ⁇ M Salinosporamide A inhibited the chymotrypsin-like and caspase-like activity by greater than 90% compared to diluent in the three leukemia cells lines, whereas the trypsin-like activity was inhibited to a lesser extent (Figure 1C).
  • Figure ID The accumulation of p27 ( Figure ID) and p21 (data not shown), both known proteasome protein substrates, in cells treated with Salinosporamide A served as functional confirmation of proteasome inhibition.
  • Salinosporamide A was more effective than bortezomib at inhibiting the chymotrypsin-like, caspase-like and trypsin-like activities in Jurkat cells.
  • Salinosporamide A induces apoptotic cell death in vitro and in vivo. The cytotoxic effects of Salinosporamide A in K562, Jurkat and ML-I cells were examined next. These cell lines were exposed to 1 nM - 1 ⁇ M Salinosporamide A for 24 h. Salinosporamide A induced DNA fragmentation, which peaked at 200 nM, in a dose dependent manner as measured by PI staining in the three leukemia cell lines ( Figure 2A).
  • Salinosporamide A achieved significant proteasome inhibition but did not cause DNA fragmentation.
  • Bid is a pro-apoptotic BH-3 domain containing member of the Bcl-2 family and is a substrate for caspase-8 36.
  • Exposure to 1 ⁇ M Salinosporamide A for 8 h induced the cleavage of Bid ( Figure 3E), generating a 15 kDa fragment, tBid, which can translocate to mitochondria.
  • Figure 3E Exposure to 1 ⁇ M Salinosporamide A for 8 h induced the cleavage of Bid ( Figure 3E), generating a 15 kDa fragment, tBid, which can translocate to mitochondria.
  • tBid which can translocate to mitochondria.
  • FIG. 3G shows the presence of cytochrome c in cytosol after 4 h of Salinosporamide A treatment but not in diluent treated cells. Accordingly, levels of pro-caspase-9 were decreased in Salinosporamide A treated cells, indicating activation of the zymogen ( Figure 3G).
  • Caspase-3 activity was assessed after Salinosporamide A exposure in 12.1 and 19.2 cells.
  • Caspase-8 and FADD deficient cell lines moderately increased caspase-3 activity 3-fold, whereas wild type Jurkat cells exhibited a 6-fold increase as compared to control ( Figure 4B).
  • caspase-8 and FADD mediate Salinosporamide A-induced caspase-3 activity.
  • Salinosporamide A interacts with HDACi to induce synergistic apoptosis. It was examined whether the HDACi, MS-275 and VPA, can be combined with Salinosporamide A to enhance apoptosis in leukemia cells. Jurkat cells were treated with increasing doses of MS-275 (1 - 5 ⁇ M) or VPA (1 - 5 mM) and low doses of Salinosporamide A (10 nM or 5 nM) for 24 h. The combination of 2.5 ⁇ M or 5 ⁇ M MS-275 and 10 nM Salinosporamide A significantly increases DNA fragmentation (p ⁇ 0.05) when compared to cells exposed to a single agent (Figure 6A).
  • caspase-3 activation was potentiated in parental Jurkat cells treated with the combination of Salinosporamide A and MS-275 (Figure 6B; p ⁇ 0.001).
  • caspase-8 deficient cells did not display additive or synergistic effects.
  • Combined treatment of Salinosporamide A and MS-275 resulted in an increase of intracellular superoxide levels (Figure 6B inset).
  • Exposure of Jurkat cells to low doses of VPA (1 or 2.5 mM) and Salinosporamide A (5 nM) also displayed significantly enhanced apoptosis (Figure 6D, p ⁇ 0.05).
  • Salinosporamide A inhibits all three activities associated with the 2OS proteasome in leukemia cells (Figure 1) and induces apoptosis in a variety of leukemia cells.
  • Cells representative of ALL, CML, AML, as well as mononuclear cells from a Ph+ ALL patient are sensitive to Salinosporamide A ( Figure 2A and 5C).
  • In vivo administration of Salinosporamide A also decreases tumor burden in leukemia bearing mice ( Figure 2E).
  • Salinosporamide A inhibits the 2OS proteolytic activities in leukemic cells to different degrees, blocking the chymotrypsin-like and caspase-like activities more effectively than the trypsin-like activity ( Figure IA-C).
  • Salinosporamide A was found to be more potent than bortezomib in inhibiting the rate-limiting activity of the proteasome ( Figure IE). It has been reported that proteasome activities are allosterically regulated and that inhibition of multiple sites of the proteasome is necessary to block significant protein degradation, Salinosporamide A's pattern of inhibition may impact proteasome function.
  • the mode of apoptosis induction by Salinosporamide A may also be influenced by this unique differential inhibition of proteasome activities.
  • Caspase-8 inhibition appears to be critical for Salinosporamide A's cytotoxicity whereas caspase-9 inhibitors did not significantly protect against DNA fragmentation or caspase-3 activation ( Figure 3A, D). Nevertheless, pro-enzyme caspase-9 disappearance is detected by western blot ( Figure 3G) indicating activation of caspase-9 by Salinosporamide A. Since upstream caspases, caspase-8 in this case, can cause activation of other caspases, it is likely that the disappearance of pro- caspase-9 is via this mechanism.
  • caspase-8 is activating caspase-9 since experiments using caspase-8 inhibitors, or ALL cell lines lacking caspase-8m or FADD showed diminished mitochondrial perturbations, caspase-3 activity, and DNA fragmentation ( Figures 3 A, 3D and 4B, 4D). These results place caspase-8 at the apex of the apoptotic cascade triggered by Salinosporamide A ( Figure 7).
  • Use of lymphocyte models with total caspase-8 and FADD deficiency extends and confirms data from multiple myeloma cell lines transfected with dominant- negative caspase-8, caspase-9 or FADD constructs.
  • NAC The antioxidant, conferred protection against Salinosporamide A induced caspase-3 activity and apoptosis (Figure 5A, 5B). Furthermore, increased peroxide and superoxide levels are detectable after exposure to Salinosporamide A ( Figure 3H). Since caspase-8 activation proceeds in the presence of NAC ( Figure 5E), and caspase-8 inhibitors do not affect Salinosporamide A's ability to raise intracellular superoxide levels (Figure 5F), these data place ROS alterations in a parallel pathway. Consistent with this model, NAC does not alter proteasome activity (Figure 5D). Several structurally dissimilar proteasome inhibitors are reported to cause elevated levels of intracellular superoxide and intracellular peroxides. The source of this oxidant production is of interest since inhibition of ROS by NAC prevents cytotoxicity in numerous models.
  • HDACi have been shown to raise intracellular ROS levels and these results indicate that the combination of Salinosporamide A and MS-275 cause a further increase in superoxide than seen with either agent alone ( Figure 6B inset). Thus it is conceivable that this greater oxidative challenge may contribute to the synergistic effects. These results also implicate caspase-8 in the synergistic apoptosis induced by Salinosporamide A and MS-275 ( Figure 6B). This data reveals the potential of administering Salinosporamide A at low doses (nontoxic) with HDACi, such as vorinostat (suberoylanilide hydroxamic acid (SAHA) or Zolinza®), for clinical benefit.
  • SAHA suberoylanilide hydroxamic acid
  • Zolinza® Zolinza®
  • Salinosporamide A is a potent proteasome inhibitor with potential therapeutic value in several hematologic malignancies, such as refractory solid tumors and lymphoma.
  • Salinosporamide A, and histone deacetylase inhibitors synergistically induce apoptosis in leukemia cell lines in a caspase-8 and oxidant dependent manner. Furthermore, these lethal effects with Salinosporamide A and HDACi were more potent than those obtained when HDACi were combined with the reversible proteasome inhibitor, bortezomib (Velcade). To determine the mechanisms by which Salinosporamide A and HDACi may be synergizing, inhibition of proteasome activity and histone acetylation was examined, the proximal targets of these compounds.
  • SAHA suberoylanilide hydroxamic acid
  • Salinosporamide A interacts with HDACi (MS-275) to induce synergistic apoptosis. It was examined whether the HDACi, MS-275, can be combined with Salinosporamide A to enhance apoptosis in acute myeloid leukemia (AML) cells. The effect of Salinosporamide A in combination with MS-275 was compared to the effect of Velcade in combination with MS-275. The combination of Salinosporamide A with MS-275 had a greater synergistic effect compared to bortezomib (Velcade) with MS-275 ( Figure 8).
  • Salinosporamide A enhances the activity of vorinostat (SAHA) in Hodgkin's Lymphoma cell lines.
  • SAHA vorinostat
  • HD-LM2 Human Reed- Sternberg cell lymphoma
  • Salinosporamide A (10 nM) and vorinostat (0.5 ⁇ M) showed activity greater than the control. Additionally, when Salinosporamide A (10 nM) and vorinostat (0.5 ⁇ M) were used in combination synergistic activity was observed. Activity of Salinosporamide A and vorinostat on MeWo melanoma cell lines was observed ( Figure 19).
  • the IC50 for Salinosporamide A used alone ranges from about 10 nM (large cell carcinoma H1341 cell line) to about 300 nM (adenocarcinoma HCC4006 cell line).
  • the lung carcinoma cell lines were treated with Salinosporamide A at doses of 5 nM to 500 nM as single agent or in combination with vorinostat (SAHA) (2 ⁇ M).
  • SAHA vorinostat
  • the results indicate that the combination of vorinostat (SAHA) plus Salinosporamide A has additive effects on growth inhibition in the lung cancer cell lines tested ( Figures 20 and 21).
  • isobologram analysis show the effect of combinations of vorinostat (SAHA) plus Salinosporamide A in various lung carcinoma cell lines: H441 (Figure 22A); A549 (Figure 22B); H322C (Figure 22C); HCC4006 (Figure 22D); Hl 341 (Figure 22E); Hl 96 ( Figure 22F); Hl 57 ( Figure 22G); and H226 (Figure 22 H).
  • SAHA vorinostat
  • Salinosporamide A in various lung carcinoma cell lines: H441 (Figure 22A); A549 (Figure 22B); H322C (Figure 22C); HCC4006 (Figure 22D); Hl 341 (Figure 22E); Hl 96 ( Figure 22F); Hl 57 ( Figure 22G); and H226 (Figure 22 H).
  • the HDAC inhibitor MS-275 can decrease mRNA expression of 2OS proteasome ⁇ subunits in Jurkat cells ( Figure 25). The reletaive expression of 2OS proteasome ⁇ l, ⁇ 2, and ⁇ 5 subunits were all decreased when treated with MS-275 (5 ⁇ M) for 24 hours.
  • the HDAC inhibitor vorinostat can decrease mRNA expression of 2OS proteasomal ⁇ 5 subunit in Jurkat cells as measured at 12 hours and 18 hours ( Figure 26). The expression of ⁇ 5 mRNA was analyzed by reqal time PCR were all decreased when treated with MS-275 (5 ⁇ M) for 24 hours.
  • the HDAC inhibitor MS-275 in combination with Salinosporamide A or bortezomib causes Histone-3 to hyperacetylate (Figure 27).
  • Jurkat T-cells were treated for 6 hours with MS-275 (5 ⁇ M), Salinosporamide A (10 nM), bortezomib (10 nM) and combinations of MS-275 (5 ⁇ M) with Salinosporamide A (10 nM) or bortezomib (10 nM). It can be seen from the data that the combination treatments cause an increase in the acetylation of Ac-H3 and H3. Additionally, the data shows that Salinosporamide A (10 nM) causes an increase in the acetylation of H3 relative to control.
  • the HDAC inhibitor vorinostat in combination with Salinosporamide A or bortezomib causes formation of reactive oxygen species (ROS) ( Figure 36).
  • ROS reactive oxygen species
  • N- acetylcysteine (NAC) is included in the combinations the amount of ROS decrease as measured by mean fluorescence.
  • N-acetylcysteine (NAC) decreases formation of ROS when combined with vorinostat and Salinosporamide A but not vorinostat and bortezomib ( Figure 37).
  • mPANC-96 is a drug-resistant mesenchymal pancreatic tumor. Luciferase-transduced mPANC-96 cells were used to generate orthotopic tumors in nude mice. The tumor volumes were monitored by bioluminescence imaging after injection of luciferin. Salinosporamide A was administered IP at 0.15 mg/kg weekly. Vorinostat was administered IP 5 days/week at 50 mg/kg. Tumor measurements were made after 3 weeks of therapy. The combination of Salinosporamide A (0.15 mg/kg weekly) and Vorinostat (5 days/week at 50 mg/kg) showed enhanced reduction in tumor volume compared to control.

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Abstract

L'invention concerne des procédés de traitement du cancer comportant l'administration à un animal d'une quantité thérapeutique efficace d'inhibiteurs du protéasome et d'un ou de plusieurs inhibiteurs d'histone désacétylase. Ledit animal est un mammifère, de préférence un être humain ou un rongeur.
PCT/US2008/059592 2007-04-06 2008-04-07 Procédé d'utilisation d'inhibiteurs du protéasome en combinaison avec des inhibiteurs d'histone désacétylase pour traiter un cancer WO2008124699A1 (fr)

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US9078881B2 (en) 2002-06-24 2015-07-14 The Regents Of The University Of California Salinosporamides and methods of use thereof
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US11136332B2 (en) 2016-08-19 2021-10-05 Celgene International Ii Sàrl Morphic forms of marizomib and uses thereof

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