WO2007130404A1 - Procédés d'utilisation de composés hétérocycliques [3.2.0] et d'analogues de ceux-ci pour le traitement du cancer du poumon - Google Patents

Procédés d'utilisation de composés hétérocycliques [3.2.0] et d'analogues de ceux-ci pour le traitement du cancer du poumon Download PDF

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WO2007130404A1
WO2007130404A1 PCT/US2007/010540 US2007010540W WO2007130404A1 WO 2007130404 A1 WO2007130404 A1 WO 2007130404A1 US 2007010540 W US2007010540 W US 2007010540W WO 2007130404 A1 WO2007130404 A1 WO 2007130404A1
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substituted
formula
compound
alkyl
alkoxy
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PCT/US2007/010540
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Michael A. Palladino
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Nereus Pharmaceuticals, Inc.
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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
    • A61K31/4015—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 having oxo groups directly attached to the heterocyclic ring, e.g. piracetam, ethosuximide
    • 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 invention disclosed herein relates to certain compounds and to methods for the preparation and the use of certain compounds in the fields of chemistry and medicine. Embodiments of the invention disclosed herein relate to methods of using heterocyclic compounds.
  • the compounds are used as proteasome inhibitors.
  • the compounds are used to treat lung cancer, such as non-small lung carcinoma, small lung carcinoma and mesothelioma.
  • 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.
  • Lung cancer is the leading cause of cancer-related deaths among men and women in the U.S., with approximately 160,000 estimated deaths in 2004.
  • lung cancer is cancer that starts in the lung compared to secondary lung cancer that starts in another location of the body and spreads to the lung.
  • primary lung cancer There are several different types of primary lung cancer. Examples include but are not limited to small cell lung cancer, non-small cell lung cancer and mesothelioma.
  • NSCLC non-small cell lung cancer
  • Small cell lung carcinoma is distinctive from other kinds of lung cancer (metastases are already present at the time of discovery) and accounts for approximately 110,000 cancer diagnoses annually.
  • Small-cell lung cancer (SCLC) tends to be more aggressive than NSCLC. Similar to NSCLC, the current first-line of standard of care is a platinum-based chemotherapy with individuals being treated having a median survival of approximately 9 months.
  • Mesothelioma is a type of cancer that affects the covering of the lung (i.e., the pleura), and is often caused by exposure to asbestos. Generally, only 1 out of 10 people who are diagnosed with mesothelioma survive 3 years and only 1 out of 20 survive 5 years. Mesothelioma can be difficult to treat with the current options being chemotherapy, radiation, or surgery.
  • 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.
  • Some embodiments disclosed herein relate to a method of treating a primary lung cancer cell comprising administering to an animal a compound described herein, or a pharmaceutically acceptable salt or pro-drug ester thereof.
  • Other embodiments disclosed herein related to a method of inhibiting the growth of a primary lung cancer cell comprising administering to an animal a compound described herein, or a pharmaceutically acceptable salt or pro-drug ester thereof.
  • the primary lung cancer cell can be a lung carcinoma cell such as a non-small lung carcinoma cell or a small lung carcinoma cell, or a mesothlioma cell.
  • non-small lung carcinoma cells include but are not limited to a squamous cell carcinoma cell, an adenocarcinoma cell, and a large cell carcinoma cell.
  • the primary cancer cell can be a chemoresistant cancer cell, a radioresistant cancer cell, or a cancer cell that is resistant to a biologic therapeutic.
  • 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 such as lung cancer.
  • the compounds can be administered or used in combination with treatments such as chemotherapy, radiation, and biologic therapies.
  • Some embodiments disclosed herein relates to a method of treating a lung cancer cell comprising contacting the cell with at least a first agent and a second agent, wherein the first agent can be a chemotherapeutic agent or a biologic, and the second agent can be a compound described herein, or a pharmaceutically acceptable salt or a pro-drug ester thereof.
  • Another embodiment disclosed herein relates to a method of treating a lung cancer cell comprising contacting the cell with at least a first agent and a second agent, wherein the first agent can be a chemotherapeutic agent or a biologic, and the second agent can be a compound having the structure of Formula 11-16, or a pharmaceutically acceptable salt or a pro-drug ester thereof.
  • chemotherapeutics include Alkaloids, alkylating agents, antibiotics, antimetabolites, enzymes, hormones, platinum compounds (carboplatin), 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)), topoisomerase I poison (SN-38), topoisomerase II poison, Camptothecin, nitrogen mustards (cyclophosphamide), Nitrosoureas, Pemetrexed (ALIMT A®), epidermal growth factor receptor-tyrosine kinase inhibitors, erlotinib (Tarceva), gefitinib (Iressa), bevasizumab (Avastin), epidermal growth factor receptor inhibitors, centuximab (Eributux), gemcitabine, histone deacetylase inhibitors, Carmustine, lomustine, dacarbazine, hydroxymethylmelarnine, thiotepa and mitocycin C, Dactino
  • 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-ASP ARAGINASE, 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 can be used in combination with the instant compounds, for example.
  • biologies can include agents such as TRAIL and antibodies to TRAIL receptors including 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.
  • the compounds can be conjugated to or delivered with an antibody.
  • the second agent e.g., a compound described herein
  • the second agent can be administered before the administration of the first agent (e.g., chemotherapeutic agent or biologic).
  • the second agent can be administered simultaneously with the administration of the first agent.
  • the second agent can be administered after the administration of the first agent (e.g., chemotherapeutic agent or biologic).
  • carcinoma cell lines include but are not limited to A549, CaLu-I, H157, H1299, H460, H358, a p53 null clone, H322, a p53 mutant, H520, H522, NSCLC-3, NSCLC-5, H138, Hl 155, H810, HBE135-E637, H1915, H650, H647, SHP-77, L5, L8, H23, H1770, H69, H44, CORL24, DMS 79, SCLC-21H, SCLC-22H, NCI-H889, -H1963, -H1417, -H187, -H510, -H209, - H526, -H146, -H1048, DMS 53, H69CIS200, H690X400, SBC-5, DMS70,
  • Some embodiments relate to uses of a compound having the structure of Formula I, and pharmaceutically acceptable salts and pro-drug esters thereof:
  • Rl can be separately selected from the group consisting of a hydrogen, a halogen, mono- substituted, poly-substituted or unsubstituted variants of the following residues: saturated C 1 -C24 alkyl, unsaturated C2-C24 alkenyl or C2-C24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino, aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, cycloalkylacyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, thio,
  • R 2 can be selected from the group consisting of hydrogen, a halogen, mono-substituted, poly-substituted or unsubstituted variants of the following residues: saturated C1-C24 alkyl, unsaturated C2-C24 alkenyl or C 2 -C 2 4 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl (including, for example, cyclohexylcarbinol), alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino, aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, cycloalkylacyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, and
  • R 3 can be selected from the group consisting of a halogen, mono-substituted, poly-substituted or unsubstituted variants of the following residues: saturated C 1 -C 24 alkyl, unsaturated C 2 -C 24 alkenyl or C2-C 24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino, aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, cycloalkylacyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl; and wherein each of
  • Ri is a substituted or unsubstituted Ci to C 5 alkyl.
  • methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, and pentyl are preferred.
  • Ri is not a substituted or unsubstituted, unbranched Ce alkyl.
  • 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- VI, and pharmaceutically acceptable salts and pro-drug esters thereof.
  • compositions which include a compound of a formula selected from Formulae I- VI.
  • the pharmaceutical compositions can further include an anti-microbial agent.
  • Still further embodiments relate to methods of inhibiting the growth of a cancer cell.
  • the methods can include, for example, contacting a cancer cell with a compound of a formula selected from Formulae I- VI, and pharmaceutically acceptable salts and pro-drug esters thereof.
  • Other embodiments relate to methods of inhibiting proteasome activity that include the step contacting a cell with a compound of a formula selected from Formulae I-VI, and pharmaceutically acceptable salts and pro-drug esters thereof.
  • NF- ⁇ B nuclear factor- kappa B
  • Other embodiments relate to methods of inhibiting nuclear factor- kappa B (NF- ⁇ B) activation including the step contacting a cell with a compound of a formula selected from Formulae I-VI, and pharmaceutically acceptable salts and pro-drug esters thereof.
  • Some embodiments relate to methods for treating an inflammatory condition, including administering an effective amount of a compound of a formula selected from Formulae I-VI to a patient in need thereof.
  • Further embodiments relate to methods for treating a microbial illness including administering an effective amount of a compound of a formula selected from Formulae I-VI to a patient in need thereof.
  • FIG. 1 shows the chemical structure of Salinosporamide A.
  • FIG. 2 shows the pan-tropical distribution of the Salinospora.
  • "X" denotes Salinospora collection sites.
  • FIG. 3 shows colonies of Salinospora.
  • FIG. 4 shows the typical 16S rDNA sequence of the Salinospora. Bars represent characteristic signature nucleotides of the Salinospora that separate them from their nearest relatives.
  • FIG. 5 shows Omuralide, a degradation product of the microbial metabolite Lactacystin. Also shown is a compound of Formula 11-16, also referred to as Salinosporamide A.
  • FIG. 6 illustrates lethal toxin-mediated macrophage cytotoxicity.
  • NPI- 0052 represents the compound of Formula 11-16.
  • FIG. 7 depicts the 1 H NMR spectrum of a compound having structure Formula 11-20.
  • FIG. 8 depicts the 1 H NMR spectrum of a compound having structure Formula II-24C.
  • FIG. 9 depicts the 1 H NMR spectrum of a compound having structure Formula 11-19.
  • FIG. 10 depicts the 1 H NMR spectrum of a compound having structure Formula II-2.
  • FIG. 11 depicts the mass spectrum of a compound having structure Formula II-2.
  • FIG. 12 depicts the 1 H NMR spectrum of a compound having structure Formula II-3.
  • FIG. 13 depicts the mass spectrum of a compound having structure Formula II-3.
  • FIG. 14 depicts the 1 H NMR spectrum of a compound having structure Formula II-4.
  • FIG. IS depicts the mass spectrum of a compound having structure Formula II-4.
  • FIG. 16 depicts the 1 H NMR spectrum of a compound having structure Formula II-5A.
  • FIG. 17 depicts the mass spectrum of a compound having structure Formula II-5A.
  • FIG. 18 depicts the 1 H NMR spectrum of a compound having structure Formula II-5B.
  • FIG. 19 depicts the mass spectrum of a compound having structure Formula II-5B.
  • FIG. 20 depicts the 1 H NMR spectrum of a compound having structure Formula IV-3C in DMSO-d 6 .
  • FIG. 21 depicts the 1 H NMR spectrum of a compound having structure Formula IV-3C in C 6 D 6 ZDMSO-Ci 6 .
  • FIG. 22 depicts the 1 H NMR spectrum of a compound having structure Formula II- 13C.
  • FIG. 23 depicts the 1 H NMR spectrum of a compound having structure Formula II-8C.
  • FIG. 24 depicts the 1 H NMR spectrum of a compound having structure Formula 11-25.
  • FIG. 25 depicts the 1 H NMR spectrum of a compound having structure Formula 11-21.
  • FIG. 26 depicts the 1 H NMR spectrum of a compound having structure Formula 11-22.
  • FIG. 27 shows inhibition of the chymotrypsin-like activity of rabbit muscle proteasomes.
  • FIG. 28 shows inhibition of the PGPH and Caspase-like activity of rabbit muscle proteasomes.
  • FIG. 29 shows inhibition of the chymotrypsin-like activity of human erythrocyte proteasomes.
  • FIG. 30 shows the effect of 11-16 treatment on chymotrypsin-mediated cleavage of LLVY-AMC substrate.
  • FIG. 31 shows nuclear factor-kappa B (NF- ⁇ B)/luciferase activity and cytotoxicity profiles of II- 16.
  • FIG. 32 shows reduction of I ⁇ B ⁇ degradation and retention of phosphorylated I ⁇ B ⁇ by 11-16 in HEK293 cells (A) and the HEK293 NF- ⁇ B/Luciferase reporter clone (B).
  • FIG. 33 shows accumulation of cell cycle regulatory proteins, p21 and p27, by ⁇ -16 treatment of HEK293 cells (A) and the HEK293 NF- ⁇ B/Luciferase reporter clone (B).
  • FIG. 34 shows activation of Caspase-3 by II- 16 in Jurkat cells.
  • FIG. 35 shows PARP cleavage by 11-16 in Jurkat cells.
  • FIG. 36 shows inhibition of LeTx-induced cytotoxicity by 11-16 in RAW264.7 cells.
  • FIG. 37 shows the effects of 11-16 treatment on PARP and Pro-Caspase 3 cleavage in RPMI 8226 and PC-3 cells.
  • FIG. 38 shows 11-16 treatment of RPMI 8226 results in a dose-dependent cleavage of PARP and Pro-Caspase 3.
  • FIG. 39 shows in vitro proteasome inhibition by 11-16, 11-17, and 11-18.
  • FIG. 40 shows proteasomal activity in PWBL prepared from II- 16 treated mice.
  • FIG. 41 shows epoxomicin treatment in the PWBL assay.
  • FIG. 42 shows intra-assay comparison.
  • FIG. 43 shows decreased plasma TNF levels in mice treated with LPS.
  • FIG. 44 depicts assay results showing the effect of Formula II-2, Formula II-3 and Formula II-4 on NF- ⁇ B mediated luciferase activity in HEK293 NF- ⁇ B/Luc Cells.
  • FIG. 45 depicts assay results showing the effect of Formula II-5A and Formula II-5B on NF- ⁇ B mediated luciferase activity in HEK293 NF- ⁇ B/Luc Cells
  • FIG. 46 depicts assay results showing the effect of Formula II-2, Formula II-3, and Formula II-4 on the chymotrypsin-like activity of rabbit 2OS proteasome.
  • FIG. 47 depicts the effect of Formula II-5 A and Formula II-5B on the chymotrypsin-like activity of rabbit 2OS proteasome.
  • FIG. 48 depicts the effect of Formulae II-2, II-3, and II-4 against LeTx- mediated cytotoxicity.
  • FIG. 49 depicts the 1 H NMR spectrum of a compound having structure Formula 11-17.
  • FIG. 50 depicts the 1 H NMR spectrum of a compound having structure Formula 11-18.
  • FIG. 51 depicts the 1 H NMR spectrum of the compound of Formula II- 26 in DMSO-d 6 .
  • FIG. 52 depicts the computer-generated ORTEP plot of the compound of Formula 11-26.
  • FIG. 53 depicts the 1 H NMR spectrum of the compound of Formula II-
  • FIG. 54 depicts the 1 H NMR spectrum of the compound of Formula II-
  • FIG. 55 depicts the 1 H NMR spectrum of the compound of Formula II-
  • FIG.56 depicts the 1 H NMR spectrum of the compound of Formula II-
  • FIG. 57 depicts the 1 H NMR spectrum of the compound of Formula II- 44 in DMSO-d6.
  • FIG. 58 depicts the 1 H NMR spectrum of the compound of Formula 1-7 in DMSO-d6.
  • FIG. 59 depicts the 1 H NMR spectrum of the compound of Formula II- 47 in DMSO-d6.
  • FIG. 60 depicts the 1 H NMR spectrum of the compound of Formula II- 38 in DMSO-d6.
  • FIG. 61 depicts the 1 H NMR spectrum of the compound of Formula II- 50 in DMSO-d6.
  • Embodiments of the invention include, but are not limited to, providing a method for the preparation of compounds, including compounds, for example, those described herein and analogs thereof, and to providing a method for producing pharmaceutically acceptable anti-microbial, anti-cancer, and anti-inflammatory compositions, for example.
  • the methods can include the compositions in relatively high yield, wherein the compounds and/or their derivatives are among the active ingredients in these compositions.
  • Other embodiments relate to providing novel compounds not obtainable by currently available methods.
  • embodiments relate to methods of treating cancer, inflammation, and infectious diseases, particularly those affecting humans.
  • one or more formulae, one or more compounds, or groups of compounds can be specifically excluded from use in any one or more of the methods of treating the conditions described herein.
  • compounds of Formula 11-16 can be excluded in some embodiments from the methods of treating cancer generally, for example, or a specific type of cancer.
  • the methods may include, for example, the step of administering an effective amount of a member of a class of new compounds.
  • Preferred embodiments relate to the compounds and methods of making and using such compounds disclosed herein, but not necessarily in all embodiments of the present invention, these objectives are met.
  • each stereogenic carbon can be of R or S configuration.
  • the specific compounds exemplified in this application can be depicted in a particular configuration, compounds having either the opposite stereochemistry at any given chiral center or mixtures thereof are also envisioned.
  • chiral centers are found in the derivatives of this invention, it is to be understood that the compounds encompasses all possible stereoisomers.
  • Some embodiments provide compounds, and methods of producing a class of compounds, pharmaceutically acceptable salts and pro-drug esters thereof, wherein the compounds are represented by Formula I:
  • the substituent(s) Ri, R 2 , and R 3 separately may include a hydrogen, a halogen, a mono-substituted, a poly-substituted or an unsubstituted variant of the following residues: saturated C1-C 2 4 alkyl, unsaturated C 2 -C 24 alkenyl or C2-C24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl (including for example, cyclohexylcarbinol), cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino, aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, cycloalkylacyl, hydroxy, alkylthio, arylthio, oxysulf
  • n can be equal to 1 or equal to 2.
  • the substituents can be the same or can be different.
  • R 3 is not a hydrogen.
  • Ri is a substituted or unsubstituted Ci to Cs alkyl.
  • Ci methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, and pentyl are preferred.
  • Ri is not a substituted or unsubstituted, unbranched C & alkyl.
  • R2 is not cyclohex-2-enyl carbinol when one of the Rj substituents is ethyl or chloroethyl and R 3 is methyl.
  • R ⁇ can be a formyl.
  • the compound may have the following structure 1-1:
  • Rg may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.
  • Rg may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.
  • the structure of Formula 1-1 may have the following stereochemistry :
  • Re may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.
  • R 2 can be a carbinol.
  • the compound may have the following structure 1-2:
  • Rg may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.
  • Rg may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.
  • the structure of Formula 1-2 may have the following stereochemistry:
  • R « may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.
  • exemplary compound of Formula I can be the compound having the following structure 1-3 :
  • R. 8 may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.
  • the compound of Formula 1-3 may have the following stereochemical structure:
  • Rg may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.
  • Another exemplary compound Formula I can be the compound having the following structure 1-4:
  • Rg may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.
  • the compound of Formula 1-4 may have the following stereochemical structure:
  • Rs may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.
  • Still a further exemplary compound of Formula I is the compound having the following structure 1-5:
  • Rg may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.
  • the compound of Formula 1-5 may have the following stereochemistry:
  • Ra may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.
  • Ra of Formula I can be, for example, a cyclohex-2-enyli denemethyl.
  • the compound may have the following structure of Formula 1-6:
  • Re may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.
  • the compound of Formula 1-6 may have the following stereochemistry:
  • Re may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.
  • R. 2 of Formula I can be, for example, a cyclohex-2-enylmethyl.
  • the compound may have the following structure of Formula 1-7:
  • Rg may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.
  • the compound of Formula 1-7 may have the following stereochemistry:
  • Re may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.
  • R 2 can be a cyclohexylalkylamine.
  • R2 can be a C-Cyclohexyl- methyleneamine.
  • R 2 can be a cyclohexanecarbaldehyde O-oxime.
  • R2 can be a cycloalkylacyl.
  • the substituent(s) Ri, R 3 , and R 4 separately may include a hydrogen, a halogen, a mono-substituted, a poly-substituted or an unsubstituted variant of the following residues: saturated C1-C 24 alkyl, unsaturated C 2 -C 24 alkenyl or C 2 -C24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxy carbonyl, alkoxy carbbnylacyl, amino, aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, cycloalkylacyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, thi
  • 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
  • R4 can be the same or different.
  • E 5 can be, for example, OH, O, ORi 0 , S, SRn, SO 2 Rn, NH, NH 2 , NOH, NHOH, NR12, and NHOR13, wherein R ⁇ o-13 may separately include, for example, hydrogen, a substituted or unsubstituted of any of the following: alkyl, an aryl, a heteroaryl, and the like.
  • Ri can be CH2CH 2 X, wherein X can be, for example, H, F, Cl, Br, and I.
  • R3 can be methyl.
  • R4 may include a cyclohexyl.
  • each of Ei, E3 and E4 can be O and E 2 can be NH.
  • Ri can be CH2CH2X, wherein X is selected from the group consisting of H, F, Cl Br, and I; wherein R4 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.
  • R2 is not cyclohex-2-enyl carbinol when one of the Ri substituents is ethyl or chloroethyl and R 3 is methyl.
  • Ri is a substituted or unsubstituted C
  • methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, and pentyl are preferred.
  • Ri is not a substituted or unsubstituted, unbranched C « alkyl.
  • an exemplary compound of Formula II has the following structure II- 1 :
  • R « may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.
  • the compound of Formula II has any of the following structures:
  • R4 may include a 7-oxa- bicyclo[4.1.0]hept-2-yl).
  • An exemplary compound of Formula II is the following structure II-5:
  • Rs may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.
  • At least one Rj may include a subsituted or an unsubstituted branched alkyl.
  • a compound of Formula II can be the following structure II-6:
  • Rs may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.
  • the compound of Formula II can be the following structure II-7:
  • Ri may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.
  • At least one R4 can be a cycloalkyl and E5 can be an oxygen.
  • An exemplary compound of Formula II can be the following structure II-8:
  • Rg may include, 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 II has the following structure II-9:
  • Re may include, for example, hydrogen, fluorine, chlorine, bromine and iodine; R can be hydrogen, and a substituted or unsubstituted alkyl, aryl, or heteroaryl, and the like.
  • a further exemplary compound of Formula II has the following structure H-IO:
  • Re may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.
  • E5 can be NH 2 .
  • An exemplary compound of Formula II has the following structure II- 11 :
  • R 8 may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.
  • At least one R 4 may include a cycloalkyl and Es can be NH 2 .
  • An exemplary compound of Formula II has the following structure 11-12:
  • R « may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.
  • a further exemplary compound of Formula II has the following structure 11-13:
  • Rg may include, for example, hydrogen (II- 13A), fluorine (II- 13B), chlorine (U- 13C), bromine (II- 13D) and iodine (II- 13E).
  • a still further exemplary compound of Formula II has the following structure 11-14:
  • Rs may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.
  • the compounds of Formula II may include as R4 at least one cycloalkene, for example. Furthermore, in some embodiments, the compounds may include a hydroxy at Es, for example.
  • a further exemplary compound of Formula II has the following structure 11-15:
  • 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 II may include a methyl group as Ri, for example.
  • a further exemplary compound, Formula 11-20 has the following structure and stereochemistry:
  • the compounds of Formula II may include hydroxyethyl as Rj, for example.
  • a further exemplary compound, Formula 11-21 has the following structure and stereochemistry:
  • the hydroxyl group of Formula 11-21 can be esterif ⁇ ed such that Ri may include ethylpropionate, for example.
  • An exemplary compound, Formula 11-22 has the following structure and stereochemistry:
  • the compounds of Formula II may include an ethyl group as R 3 , for example.
  • a further exemplary compound of Formula II has the following structure 11-23:
  • R & may include, for example, 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 Formula II-24C, where Rs is chlorine:
  • the compounds of Formula 11-15 may have the following stereochemistry, exemplified by the compound of Formula 11-25, where Rg is chlorine:
  • the compound of Formula 11-15 may have the following stereochemistry, exemplified by the compound of Formula 11-26, where R 8 is chlorine:
  • the compound of Formula II may have the following structure and stereochemistry, exemplified by Formula 11-27, where Rj is ethyl:
  • the compound of Formula II may have the following structure and stereochemistry, exemplified by Formula II-28, where Rj is methyl:
  • the compounds of Formula II may include azidoethyl as Ri, for example.
  • a further exemplary compound, Formula 11-29 has the following structure and stereochemistry:
  • the compounds of Formula II may include propyl as Rj, for example.
  • a further exemplary compound, Formula 11-30 has the following structure and stereochemistry:
  • the compound of Formula II may include cyanoethyl as Ri; for example, the compound of Formula 11-37 has the following structure and stereochemistry:
  • the compound of Formula II may include ethylthiocyanate as Ri; for example, the compound of Formula 11-38 has the following structure and stereochemistry:
  • the compounds of Formula II may include a thiol as Ri, for example.
  • the substituent Ri of the compound of Formula ⁇ may include a leaving group, for example, a halogen, as in compounds II- 18 or 11-19, or another leaving group, such as a sulfonate ester.
  • a leaving group for example, a halogen, as in compounds II- 18 or 11-19, or another leaving group, such as a sulfonate ester.
  • a sulfonate ester such as a sulfonate ester.
  • methane sulfonate (mesylate) of Formula 11-41 is the methane sulfonate (mesylate) of Formula 11-41 :
  • the substituent Rj of the compound of Formula II 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 II.
  • the compound of Formula 11-44 (a prodrug thioester of the compound of Formula 11-16) has the following structure and stereochemistry:
  • the compounds of Formula II may include an alkenyl group as R
  • alkenyl group
  • ethylenyl alkenyl group
  • the compounds can be prodrug esters or thioesters of the compounds of Formula II.
  • the compound of Formula 11-47 (a prodrug thioester of the compound of Formula 11-17) has the following structure and stereochemistry:
  • the compounds can be prodrug esters or thioesters of the compounds of Formula II.
  • 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 II.
  • the compound of Formula 11-50 has the following structure and stereochemistry:
  • separately may include, for example, a hydrogen, a halogen, a mono-substituted, a poly-substituted or an unsubstituted variant of the following residues: saturated C 1 -C 24 alkyl, unsaturated C 2 -C 24 alkenyl or C 2 -C 24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino, aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, thio, sulfoxide, sulf
  • R 4 can be, for example, a hydrogen, a halogen, a mono-substituted, a poly-substituted or an unsubstituted variants of the following residues: saturated C1-C24 alkyl, unsaturated C2-C24 alkenyl or C 2 -C 2 4 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino, aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl.
  • m can be equal to 1 or 2, and where m is equal to 2, the substituents can the same or different.
  • each of Ei, E 2 , E 3 , E4 and E 5 can be, for example, a substituted or unsubstituted heteroatom.
  • the heteroatom can be nitrogen, sulfur or oxygen.
  • R 2 is not cycIohex-2-enyl carbinol when one of the Ri substituents is ethyl or chloroethyl.
  • Ri is a substituted or unsubstituted Ci to C 5 alkyl.
  • methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, and pentyl are preferred.
  • Ri is not a substituted or unsubstituted, unbranched Ce alkyl.
  • the substituent(s) Ri R 3 , and R 5 may separately include a hydrogen, a halogen, a mono-substituted, a poly-substituted or an unsubstituted variants of the following residues: saturated C 1 -C 24 alkyl, unsaturated C 2 - C24 alkenyl or C2-C24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino, aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, thio, sulfoxide, sul
  • each of Ei, E 2 , E 3 , E4 and E 5 can be a heteroatom or substituted heteroatom, for example, nitrogen, sulfur or oxygen.
  • R3 is not a hydrogen
  • n is equal to 1 or 2.
  • n is equal to 2
  • the substituents can be the same or can be different.
  • m can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.
  • m is greater than 1 , the substituents can be the same or different.
  • Ri is a substituted or unsubstituted Ci to Cs alkyl.
  • Ci methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, and pentyl are preferred.
  • Ri is not a substituted or unsubstituted, unbranched Ce alkyl.
  • R 5 may give rise to a di-substituted cyclohexyl.
  • An exemplary compound of Formula IV is the following structure IV-I, with and without exemplary stereochemistry:
  • Re may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.
  • the substituent(s) R ⁇ and R 7 may separately include a hydrogen, a halogen, a mono-substituted, a poly-substituted or an unsubstituted variants of the following residues: saturated C1-C24 alkyl, unsaturated C2-C24 alkenyl or C2-C24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino, aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano,
  • an exemplary compound of Formula IV has the following structure IV-2:
  • Re may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.
  • an exemplary compound of Formula IV has the following structure IV-3:
  • R 8 may include, for example, hydrogen (1V-3A), fluorine (IV-3B), chlorine (IV-3C), bromine (IV-3D) and iodine (IV-3E).
  • an exemplary compound of Formula IV has the following structure IV-4:
  • Rg may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.
  • Some embodiments provide compounds, and methods of producing a class of compounds, pharmaceutically acceptable salts and pro-drug esters thereof, wherein the compounds are represented by Formula V:
  • the substituent(s) Ri and Rs may separately include a hydrogen, a halogen, a mono-substituted, a poly-substituted or unsubstituted variants of the following residues: saturated C1-C24 alkyl, unsaturated C2-C24 alkenyl or C 2 -C 24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino, aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, thio, sulfoxide, sulfone, sulf
  • each of Ei, E 2 , E 3 , E 4 and Es can be a heteroatom or substituted heteroatom, for example, nitrogen, sulfur or oxygen, n can be equal to 1 or 2, and when n is equal to 2, the substit ⁇ ents can be the same or different.
  • n can be equal to 1 or 2
  • m can be, for example, 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.
  • R 5 can be the same or different.
  • Ri is a substituted or unsubstituted Ci to Cs alkyl.
  • methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, and pentyl are preferred.
  • Ri is not a substituted or unsubstituted, unbranched Ce alkyl.
  • Some embodiments provide compounds, and methods of producing a class of compounds, pharmaceutically acceptable salts and pro-drug esters thereof, wherein the compounds are represented by Formula VI:
  • each Rj can be separately selected from the group consisting of a mono-substituted, poly-substituted or unsubstituted variants of the following residues: saturated C 1 -C 2 4 alkyl, unsaturated C2-C24 alkenyl, unsaturated C2-C24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino, aminocarbonyl, aminocarboyloxy, phenyl, cycloalkylacyl, alkylthio, arylthio, oxysulfonyl, carboxy, thio, sulfoxide, sulfone, boronic acid, boronic ester, and halogenated alkyl including
  • R 2 can be selected from the group consisting of hydrogen, a halogen, mono-substituted, poly-substituted or unsubstituted variants of the following residues: saturated C1-C24 alkyl, unsaturated C2-C24 alkenyl, unsaturated C2-C24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl (including, for example, cyclohexylcarbinol), cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino, aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, cycloalkylacyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano,
  • R 3 can be selected from the group consisting of a halogen, mono-substituted, poly-substituted or unsubstituted variants of the following residues: saturated C 1 -C 2 4 alkyl, unsaturated C 2 -C 2 4 alkenyl, unsaturated C2-C 24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino, aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, cycloalkylacyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, thio, sulfoxide, sulfone,
  • Formula VI-A wherein p can be 1 or 2.
  • Rj, R2, R 3 , RH, E I , E2, E3, and E4 are the same as previously defined in Formula VI.
  • R 2 is not cyclohex-2-enyl carbinol when one of the Ri substituents is ethyl or chloroethyl and R 3 is methyl.
  • Ri is a substituted or unsubstituted Ci to Cs alkyl.
  • Ci a substituted or unsubstituted Ci to Cs alkyl.
  • methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, and pentyl are preferred.
  • Ru can be selected from the group consisting of a halogen, mono-substituted, poly-substituted or unsubstituted variants of the following residues: saturated C1-C24 alkyl, unsaturated C2-C24 alkenyl or C 2 -C24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino, aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, cycloalkylacyl, hydroxy, heteroalkylthio, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, thio, thioester, sulfoxide,
  • R H is an alkylthiol or substituted alkylthiol
  • E 3 is an oxygen
  • some of the compounds of Formula VI can have the following structure referred to as Formula VI-I :
  • Ri can be separately selected from the group consisting of a mono-substituted, poly-substituted or unsubstituted variants of the following residues: saturated C 1 -C 24 alkyl, unsaturated C2-C24 alkenyl, unsaturated C2-C24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino, aminocarbonyl, aminocarboyloxy, phenyl, cycloalkylacyl, alkylthio, arylthio, oxysulfonyl, carboxy, thio, sulfoxide, sulfone, boronic acid, boronic ester, and halogenated alkyl including polyhalogen
  • R 3 can be selected from the group consisting of a halogen, mono-substituted, poly-substituted or unsubstituted variants of the following residues: saturated C 1 -C 24 alkyl, unsaturated C2-C24 alkenyl, unsaturated C2-C24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino, aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, cycloalkylacyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, thio, sulfoxide, sulfone, sulfon
  • R5 can be separately selected from the group consisting of a hydrogen, a halogen, mono-substituted, poly-substituted or unsubstituted variants of the following residues: saturated C1-C24 alkyl, unsaturated C2-C24 alkenyl, unsaturated C2-C 24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino, aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, oxy, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, thio, sulfoxide, sulfone, sulfonate este
  • R ⁇ is a substituted or unsubstituted Ci to C 5 alkyl.
  • methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, and pentyl are preferred.
  • Ri 4 can be selected from the group consisting of a halogen, mono-substituted, poly-substituted or unsubstituted variants of the following residues: saturated C 1 -C 24 alkyl, unsaturated C 2 -C24 alkenyl, unsaturated C 2 -C 2 4 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino, aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, cycloalkylacyl, hydroxy, heteroalkylthio, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, thio, thioester
  • an exemplary compound of Formula VI has the following structure and stereochemistry VI-IB:
  • Certain embodiments also provide pharmaceutically acceptable salts and pro-drug esters of the compound of Formulae I- VI, and provide methods of obtaining and purifying such compounds by the methods disclosed herein.
  • pro-drug ester 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-R-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.
  • pro-drug ester also refers to a chemical derivative of the compound that is rapidly transformed in vivo to yield the compound, for example, by hydrolysis in blood.
  • pharmaceutically acceptable salt refers to any pharmaceutically acceptable salts of a compound, and preferably refers to an acid addition salt of a compound.
  • Preferred examples of 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 Cj- 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-drug esters of the compound of Formulae I-VI 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.
  • the compounds can be used to treat microbial diseases, cancer, and inflammation.
  • Disease is meant to be construed broadly to cover infectious diseases, and also autoimmune diseases, non-infectious diseases and chronic conditions.
  • the disease is caused by a microbe, such as a bacterium, a fungi, and protozoa, for example.
  • the methods of use may also include the steps of administering a compound or composition comprising the compound to an individual with an infectious disease or cancer.
  • the compound or composition can be administered in an amount effective to treat the particular infectious disease, cancer or inflammatory condition.
  • the infectious disease can be, for example, one caused by Bacillus, such as B. anthracis and B. cereus.
  • the infectious disease can be one caused by a protozoa, for example, a Leishmania, a Plasmodium or a Trypanosoma.
  • the compound or composition can be administered with a pharmaceutically acceptable carrier, diluent, excipient, and the like.
  • the cancer can be, for example, a multiple myeloma, a colorectal carcinoma, a prostate carcinoma, a breast adenocarcinoma, a non-small cell lung carcinoma, small lung carconima, an ovarian carcinoma, a melanoma, and the like.
  • the inflammatory condition can be, for example, rheumatoid arthritis, asthma, multiple sclerosis, psoriasis, stroke, myocardial infarction, reperfusion injury, and the like.
  • 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, with bromine and chlorine being preferred.
  • alkyl means any unbranched or branched, substituted or unsubstituted, saturated hydrocarbon, with C1-C24 preferred, and Ci-Ce hydrocarbons being preferred, with methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and tert-butyl, and pentyl being most preferred.
  • C 1 -C 24 are preferred, with Ci-C ⁇ mono- and di- and per-halogen substituted saturated hydrocarbons and amino-substituted hydrocarbons more preferred.
  • 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, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl
  • cycloalkyl refers to any non-aromatic hydrocarbon ring, preferably having five to twelve atoms comprising the ring.
  • acyl refers to alkyl or aryl groups derived from an oxoacid, with an acetyl group being preferred.
  • alkenyl means any unbranched or branched, substituted or unsubstituted, unsaturated hydrocarbon including polyunsaturated hydrocarbons, with Cj-Ce 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.
  • aryl refers to aromatic hydrocarbon rings, preferably having five, six, or seven atoms, and most preferably having six atoms comprising the ring.
  • Heteroaryl and substituted heteroaryl refer to aromatic hydrocarbon rings in which at least one heteroatom, e.g., oxygen, sulfur, or nitrogen atom, is in the ring along with at least one carbon atom.
  • heterocycle refers to any cyclic compound containing one or more heteroatoms. The substituted aryls, heterocycles and 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 Ci-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 non-aromatic hydrocarbon ring, preferably having five to twelve atoms comprising the ring.
  • alkoxy carbonyl refers to any linear, branched, cyclic, saturated, unsaturated, aliphatic or aromatic alkoxy attached to a carbonyl group.
  • the examples include methoxycarbonyl group, ethoxycarbonyl group, propyloxycarbonyl group, isopropyloxycarbonyl group, butoxycarbonyl group, sec- butoxycarbonyl group, tert-butoxycarbonyl group, cyclopentyloxycarbonyl group, cyclohexyloxycarbonyl group, benzyloxycarbonyl group, allyloxycarbonyl group, phenyloxycarbonyl group, pyridyloxycarbonyl group, and the like.
  • 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.
  • Certain of the compounds of Formula I- VI 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, 11-17, 11-18 and 11-19, can be obtained synthetically or by fermentation. Exemplary fermentation procedures are provided below. Futher, the compounds of Formula 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.
  • FIG. 1 shows the chemical structure of 11-16. 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. Zaks, A., Curr Opin Chem Biol 5:130 (2001). 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).
  • the possible biosynthetic origins are acetyl-CoA, ethylmalonyl-CoA, phenylalanine and chlorine.
  • 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.
  • the production of compounds of Formulae 1-7, 11-16, 11-17, 11-18, II- 20,II-24C, 11-26, 11-27 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.
  • FIG. 2 shows some collection sites worldwide for the culture (CNB476), which is also refered to as Salinospora.
  • FIG. 3 shows colonies of Salinospora.
  • FIG. 4 shows the typical 16S rDNA sequence of the Salinospora. Bars represent characteristic signature nucleotides of the Salinospora that separate them from their nearest relatives.
  • 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 11-16, 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 degree C to 40 degree C, but it is preferable to conduct the fermentation at 22 degree C to 32 degree 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.
  • Some embodiments relate to methods of treating cancer, inflammation, and infectious diseases, particularly those affecting humans.
  • the methods may include, for example, the step of administering an effective amount of a member of a class of new compounds.
  • the compounds disclosed herein can be used to treat cancer, inflammation, and infectious disease.
  • the compounds have various biological activities.
  • the compounds have chemosensitizing activity, anti-microbial, anti-inflammation, radiosensitizing, and anti-cancer activity.
  • the compounds have proteasome inhibitory activity.
  • the proteasome inhibitory activity may, in whole or in part, contribute to the ability of the compounds to act as anti-cancer, anti-inflammatory, and anti-microbial agents.
  • the proteasome is a multisubunit protease that degrades intracellular proteins through its chymotrypsin-like, trypsin-like and peptidylglutamyl-peptide hydrolyzing (PGPH; and also know as the caspase-like activity) activities.
  • the 26S proteasome contains a proteolytic core called the 2OS proteasome and one or two 19S regulatory subunits.
  • the 2OS proteasome is responsible for the proteolytic activity against many substrates including damaged proteins, the transcription factor NF- ⁇ B and its inhibitor IKB, signaling molecules, tumor suppressors and cell cycle regulators.
  • compounds of Formula 11-16 were more potent (EC 50 2nM) at inhibiting the chymotrypsin-like activity of rabbit muscle proteasomes than Omuralide (EC 50 52 nM) and also inhibited the chymotrypsin-like activity of human erythrocyte derived proteasomes (EC 50 ⁇ 250pM).
  • FIG. 5 shows omuralide, which is a degradation product of Lactacystin, and it shows a compound of Formula 11-16.
  • Compounds of Formula 11-16 exhibit a significant preference for inhibiting chymotrypsin- like activity of the proteasome over inhibiting the catalytic activity of chymotrypsin.
  • Compounds of Formula 11-16 also exhibit low nM trypsin-like inhibitory activity ( ⁇ 10 nM), but are less potent at inhibiting the PGPH activity of the proteasome (EC 50 -350 nM).
  • HEK293 cells human embryonic kidney
  • Tumor Necrosis Factor-alpha TNF- ⁇
  • TNF- ⁇ Tumor Necrosis Factor-alpha
  • HEK293 cells were pre-treated for 1 hour with compounds of Formula 11-16 followed by TNF- ⁇ stimulation.
  • Treatment with compounds of Formula EI- 16 promoted the accumulation of phosphorylated I ⁇ B ⁇ suggesting that the proteasome-mediated I ⁇ B ⁇ degradation was inhibited.
  • a stable HEK293 clone (NF- ⁇ B/Luc 293) was generated carrying a luciferase reporter gene under the regulation of 5x NF- ⁇ B binding sites. Stimulation of NF- ⁇ B/Luc 293 cells with TNF- ⁇ increases luciferase activity as a result of NF- ⁇ B activation while pretreatment with compounds of Formula 11-16 decreases activity.
  • Western blot analyses demonstrated that compounds of Formula 11-16 promoted the accumulation of phosphorylated-I ⁇ B ⁇ and decreased the degradation of total I ⁇ B ⁇ in the NF- ⁇ B/Luc 293 cells.
  • Compounds of Formula 11-16 were also shown to increase the levels of the cell cycle regulatory proteins, p21 and p27.
  • Tumor cells can be more sensitive to proteasome inhibitors than normal cells. Moreover, proteasome inhibition increases the sensitivity of cancer cells to anticancer agents.
  • the cytotoxic activity of the compounds described herein, including Formula 11-16, were examined for cytotoxic activity against various cancer cell lines. Formula 11-16 was examined, for example, in the National Cancer Institute screen of 60 human tumor cell lines. Formula 11-16 exhibited selective cytotoxic activity with a mean GI 50 value (the concentration to achieve 50% growth inhibition) of less than 10 nM. The greatest potency was observed against SK-MEL-28 melanoma and MDA-MB-235 breast cancer cells [both with LCso(the concentration with 50% cell lethality) ⁇ 10 nM].
  • a panel of cell lines including human colorectal (HT-29 and LoVo), prostate (PC3), breast (MDA-MB-231), lung (NCI-H292), ovarian (OVC AR3), acute T- cell leukemia (Jurkat), murine melanoma (B16-F10) and normal human fibroblasts (CCD- 27sk) was treated with Salinosporamide A for 48h to assess cytotoxic activity.
  • HT-29, LoVo, PC3, MDA-MB-231, NCI-H292, OVC AR3, Jurkat, and B16-F10 cells were sensitive with EC 50 values of 47, 69, 78, 67, 97, 69, 10, and 33 nM, respectively.
  • the anti-anthrax activity of the described compounds was evaluated using an in vitro LeTx induced cytotoxicity assay.
  • the results indicate that Formula 11-16 is a potent inhibitor of LeTx -induced cytotoxicity of murine macrophage-like RAW264.7 cells.
  • Treatment of RAW264.7 cells with Formula 11-16 resulted in a 10-fold increase in the viability of LeTx-treated cells compared to LeTx treatment alone (average EC 50 of ⁇ 4 nM).
  • NF- ⁇ B transcription factor nuclear factor-kappa B
  • IKB inhibitor of NF- ⁇ B
  • Chemotherapy agents such as CPT-I l (Irinotecan) can activate NF- ⁇ B in human colon cancer cell lines including LoVo cells, resulting in a decreased ability of these cells to undergo apoptosis. Painter, R.B. Cancer Res 38:4445 (1978).
  • VelcadeTM is a dipeptidyl boronic acid that inhibits the chymotrypsin-like activity of the proteasome (Lightcap, et al, Clin Chem 46:673 (2000), Adams, et al, Cancer Res 59:2615 (1999), Adams, Curr Opin Oncol 14:628 (2002)) while enhancing the trypsin and PGPH activities.
  • VelcadeTM Proangiogenic chemokines/cytokines Growth Related Oncogene-alpha (GRO- ⁇ ) and Vascular Endothelial Growth Factor (VEGF) in squamous cell carcinoma, presumably through inhibition of the NF- ⁇ B pathway.
  • GRO- ⁇ proangiogenic chemokines/cytokines Growth Related Oncogene-alpha
  • VEGF Vascular Endothelial Growth Factor
  • anthracis anthracis
  • Anthrax spores are inhaled and lodge in the lungs where they are ingested by macrophages. Within the macrophage, spores germinate, the organism replicates, resulting ultimately in killing of the cell. Before killing occurs, however, infected macrophages migrate to the lymph nodes where, upon death, they release their contents allowing the organism to enter the bloodstream, further replicate, and secrete lethal toxins.
  • Hanna, et al Proc Natl Acad Sci U S A 90:10198 (1993). Anthrax toxins are responsible for the symptoms associated with anthrax.
  • LF lethal toxin
  • LeTx lethal toxin
  • LF has an enzymatic function, but requires PA to achieve its biological effect. Neither PA or LF cause death individually; however, when combined they cause death when injected intravenously in animals. Kalns, et al., Biochem Biophys Res Commun 297:506 (2002), Kalns, et al., Biochem Biophys Res Commun 292:41 (2002).
  • PA Protective antigen
  • LF the receptor-binding component of anthrax toxin
  • PA oligomerizes into a ring-shaped heptamer (see FIG. 6).
  • the complex formed between the PA heptamer and LF is taken into the cell by receptor-mediated endocytosis. Following endocytosis, LF is released into the cytosol where it attacks various cellular targets.
  • Lethal factor is a zinc dependent metal loprotease, which in the cytosol can cleave and inactivate signaling proteins of the mitogen-activated protein kinase kinase family (MAPKK). Duesbery, et al, Science 280:734 (1998), Bodart, et al, Cell Cycle 1 :10 (2002), Vitale, et al, J Appl Microbiol 87:288 (1999), Vitale, et al, Biochem J 352 Pt 3:739 (2000). Of the seven different known MAPK kinases, six have been shown to be cleaved by LF.
  • MAPK kinase pathways transduce various signals involved in cell death, proliferation, and differentiation making these proteins highly significant targets.
  • certain inhibitors that prevent LeTx-induced cell death do not prevent MAPBCK cleavage by LF suggesting that this activity is not sufficient for induction of cell death.
  • the receptor for PA has been identified and is expressed by many cell types. Escuyer, et al, Infect Immun 59:3381 (1991). Lethal toxin is active in a few cell culture lines of macrophages causing cell death within a few hours. Hanna, et al. , Proc Natl Acad Sci US A 90:10198 (1993), Kim, et al., JBiol Chem 278:7413 (2003), Lin, et al., Curr Microbiol 33:224 (1996). LeTx can induce both necrosis and apoptosis in mouse macrophage-like RAW264.7 and J774A.1 cells upon in vitro treatment.
  • 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.
  • sodium benzoate, ascorbic acid and esters of p-hydroxybenzoic acid can be added as preservatives.
  • antioxidants and suspending agents can be used.
  • 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. If desired, 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., Ophthalmologica, 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.
  • anti-inflammatory or anti-microbial compound for example, the compounds of Formulae I- VI or compositions including Formulae I-VI 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, anti-inflammatory or antimicrobial can be mixed with additional substances to enhance their effectiveness.
  • the anti-microbial is combined with an additional anti-microbial.
  • the anti-microbial is combined with a drug or medicament that is helpful to a patient that is taking anti-microbials.
  • the disclosed chemical compounds and the disclosed pharmaceutical compositions are administered by a particular method as an anti-microbial.
  • 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 rectal Iy, 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 are various methods for administration of the
  • compositions that include the described compounds, including those of Formulae I- VI, 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.
  • the products or compositions can be used alone or in combination with one another, or in combination with other therapeutic or diagnostic agents. These products can be utilized in vivo, ordinarily in a mammal, preferably in a human, or in vitro. In employing them in vivo, 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 microgram/kg and 100 mg/kg body weight, preferably between about 100 microgram/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's 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 al., 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.
  • 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.
  • such agents can be formulated and administered systemically or locally.
  • a variety of techniques for formulation and administration can be found in Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, PA(1990), which is incorporated herein by reference in its entirety.
  • 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. AU 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 compounds disclosed herein can be administered by either oral or a 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, intradermally, or the like. Controlled release formulations, depot formulations, and infusion pump delivery are similarly contemplated.
  • compositions disclosed herein in pharmaceutical compositions may also comprise a pharmaceutically acceptable carrier.
  • Such compositions can be prepared for storage and for subsequent administration.
  • 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).
  • such compositions can be formulated and used as tablets, capsules or solutions for oral administration; suppositories for rectal or vaginal administration; sterile solutions or suspensions for injectable administration.
  • 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 include, but are not limited to, 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.
  • the pharmaceutically effective amount of the composition required as a dose will depend on the route of administration, the type of animal 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.
  • compositions of the embodiment can be used alone or in combination with one another, or in combination with other therapeutic or diagnostic agents. These products can be utilized in vivo or in vitro.
  • the useful dosages and the most useful modes of administration will vary depending upon the age, weight and animal treated, the particular compounds employed, and the specific use for which these composition or compositions are employed.
  • the magnitude of a dose in the management or treatment for a particular disorder will vary with the severity of the condition to be treated and to the route of administration, and depending on the disease conditions and their severity, the compositions can be formulated and administered either systemically or locally.
  • a variety of techniques for formulation and administration can be found in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, PA (1990).
  • 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 as cellulose or sugar, or methylacetate.- methacrylate copolymer as a derivative of polyvinyl can be used as suspension agents; and plasticizers such as ester phthalates and the like can be used as suspension agents.
  • the compounds and compositions can be orally or non-orally administered to a human patient in the amount of about 0.001 mg/kg/day to about 10,000 mg/kg/day of the active ingredient, and more preferably about 0.1 mg/kg/day to about 100 mg/kg/day of the active ingredient at, preferably, one time per day or, less preferably, over two to about ten times per day.
  • the compound produced by the method of the embodiment may preferably be administered in the stated amounts continuously by, for example, an intravenous drip.
  • the preferred daily dose of the active or anti-infective ingredient would be about 0.07 mg/day to about 700 gm/day, and more preferable, 7 mg/day to about 7 grams/day. Nonetheless, as will be understood by those of skill in the art, in certain situations it can be necessary to administer the anti-cancer, anti-inflammatory or the anti- infective compound of the embodiment in amounts that excess, or even far exceed, the above-stated, preferred dosage range to effectively and aggressively treat particularly advanced cancerss or infections.
  • 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 anti-microbial, anticancer or anti-tumor 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.
  • the method of using a compound as an antimicrobial, anti-cancer or anti-inflammatory involves administering an effective amount of any of the compounds of Formulae I- VI or compositions of those compounds.
  • the method involves administering the compound represented by Formula II, to a patient in need of an anti-microbial, until the need is effectively reduced or more preferably removed.
  • needle is not an absolute term and merely implies that the patient can benefit from the treatment of the anti-microbial, the anti-cancer, or anti-inflammatory in use.
  • patient what is meant is an organism that can benefit by the use of an anti-microbial, anti-cancer or antiinflammatory agent.
  • any organism with B. anthracis, Plasmodium, Leishmania, Trypanosoma, and the like may benefit from the application of an antimicrobial that may in turn reduce the amount of microbes present in the patient.
  • any organism with cancer 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, may benefit from the application of an anti-cancer agent that may in turn reduce the amount of cancer present in the patient.
  • any organism with an inflammatory conditions such as, rheumatoid arthritis, asthma, multiple sclerosis, psoriasis, stroke, reperfusion injury, myocardial infarction, and the like, may benefit from the application of an antiinflammatory that may in turn reduce the amount of cells associated with the inflammatory response present in the patient.
  • the patient's health may not require that an anti-microbial, anti-cancer, or anti-inflammatory be administered, however, the patient may still obtain some benefit by the reduction of the level of microbes, cancer cells, or inflammatory cells present in the patient, and thus be in need.
  • the anti-microbial or anti-cancer agent is effective against one type of microbe or cancer, but not against other types; thus, allowing a high degree of selectivity in the treatment of the patient.
  • the anti-inflammatory can be effective against inflammatory conditions characterized by different cells associated with the inflammation. In choosing such an anti-microbial, anti-cancer or anti-inflammatory agent, the methods and results disclosed in the Examples can be useful.
  • the anti-microbial can be effective against a broad spectrum of microbes, preferably a broad spectrum of foreign, and, more preferably, harmful bacteria, to the host organism.
  • the anti-cancer and/or anti-inflammatory agent can be effective against a broad spectrum of cancers and inflammatory conditions/cells/substances.
  • the anti-microbial is effective against all microbes, even those native to the host. Examples of microbes that can be targets of anti-microbials, include, but are not limited to, B. anthracis, Plasmodium, Leishmania, Trypanosoma, and the like.
  • the anti-cancer agent is effective against a broad spectrum of cancers or all cancers.
  • cancers against which the compounds can be effective include 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.
  • exemplary inflammatory conditions against which the agents are effective include rheumatoid arthritis, asthma, multiple sclerosis, psoriasis, stroke, myocardial infarction, and the like.
  • Therapeutically effective amount 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.
  • the medical response is one sought by a researcher, veterinarian, medical doctor, or other clinician.
  • Anti-microbial refers to a compound that reduces the likelihood of survival of microbes, or blocks or alleviates the deleterious effects of a microbe.
  • the likelihood of survival is determined as a function of an individual microbe; thus, the anti -microbial will increase the chance that an individual microbe will die.
  • the likelihood of survival is determined as a function of a population of microbes; thus, the anti-microbial will increase the chances that there will be a decrease in the population of microbes.
  • anti-microbial means antibiotic or other similar term. Such anti-microbials are capable of blocking the harmful effects, destroying or suppressing the growth or reproduction of microorganisms, such as bacteria.
  • an anti-microbial will not change the likelihood of survival, but will change the chances that the microbes will be harmful to the host in some way. For instance, if the microbe secretes a substance that is harmful to the host, the anti-microbial may act upon the microbe to stop the secretion or may counteract or block the harmful effect.
  • an anti-microbial while, increasing the likelihood that the microbe(s) will die, is minimally harmful to the surrounding, non- microbial, cells.
  • 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 anti-cancer 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, anti-metabolites 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 onapri stone.
  • 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 anti-cancer 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.
  • Neoplasm refers to a cell or a population of cells, including a tumor or tissue (including cell suspensions such as bone marrow and fluids such as blood or serum), that exhibits abnormal growth by cellular proliferation greater than normal tissue. Neoplasms can be benign or malignant.
  • An "inflammatory condition” includes, for example, conditions such as ischemia, septic shock, autoimmune diseases, rheumatoid arthritis, inflammatory bowel disease, systemic lupus eythematosus, multiple sclerosis, asthma, osteoarthritis, osteoporosis, fibrotic diseases, dermatosis, including psoriasis, atopic dermatitis and ultraviolet radiation (UV)-induced skin damage, psoriatic arthritis, alkylosing spondylitis, tissue and organ rejection, Alzheimer's disease, stroke, atherosclerosis, restenosis, diabetes, glomerulonephritis, cancer, Hodgkins disease, cachexia, inflammation associated with infection and certain viral infections, including acquired immune deficiency syndrome (AIDS), adult respiratory distress syndrome and Ataxia Telangiestasia.
  • AIDS acquired immune deficiency syndrome
  • AIDS acquired immune deficiency syndrome
  • Ataxia Telangiestasia AIDS
  • a described compound preferably a compound having the Formulae I- VI, including those as described herein, is considered an effective anti-microbial, anti-cancer, or anti-inflammatory if the compound can influence 10% of the microbes, cancer cells, or inflammatory cells, for example, hi a more preferred embodiment, the compound is effective if it can influence 10 to 50% of the microbes, cancer cells, or inflammatory cells. In an even more preferred embodiment, the compound is effective if it can influence 50-80% of the microbes, cancer cells, or inflammatory cells. In an even more preferred embodiment, the compound is effective if it can influence 80-95% of the microbes, cancer cells, or inflammatory cells.
  • the compound is effective if it can influence 95-99% of the microbes, cancer cells, or inflammatory cells.
  • "Influence" is defined by the mechanism of action for each compound. Thus, for example, if a compound prevents the reproduction of microbes, then influence is a measure of prevention of reproduction. Likewise, if a compound destroys microbes, then influence is a measure of microbe death. Also, for example, if a compound prevents the division of cancer cells, then influence is a measure of prevention of cancer cell division. Further, for example, if a compound prevents the proliferation of inflammatory cells, then influence is a measure of prevention of inflammatory cell proliferation. 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 microbes, cancer cells or inflammatory cells, and need not be administered to a patient.
  • the compounds described herein can be administered directly to the products to reduce the risk of microbes in the products.
  • the compounds can be used to reduce the level of microbes present in the surrounding environment, such working surfaces.
  • 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.
  • the compounds can be left in the food products or on the work surfaces to allow for a more protection. 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.
  • proteasome inhibitors may make cells more sensitive to chemotherapy by inhibiting the activiation of the NF- ⁇ B pathway.
  • the inhibition of the NF- ⁇ B pathway decreases the Bcl-2 proteins which may help sensitize the cancer cells to chemotherapeutic agents and/or biologic therapies.
  • proteasome inhibitors may increase/upregulate the cyclin-dependent kinase inhibitors, p21 and p27kipl, which may help induce apoptosis of the cancer cells. See Scagliotti, G., "Proteasome Inhibitors in Lung Cancer," Critical Reviews in Oncology Hematolgy (2006) 58(3): 177-189.
  • a compound described herein is administered in combination with another chemotherapeutic or biologic agent.
  • combination therapy is used to treat lung cancer.
  • the two agents are administered simultaneously.
  • the compound described herein is administered prior to administration of the additional chemotherapeutic or biologic agent.
  • the additional chemotherapeutic or biologic agent is administered prior to administration of the compound described herein.
  • Non-limiting examples of additional chemotherapeutic or biologic agents include Alkaloids, alkylating agents, antibiotics, antimetabolites, enzymes, hormones, platinum compounds (carboplatin), 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)), topoisomerase I poison (SN-38), topoisomerase II poison, Camptothecin, nitrogen mustards (cyclophosphamide), Nitrosoureas, Pemetrexed (ALIMTA®), epidermal growth factor receptor-tyrosine kinase inhibitors, erlotinib (Tarceva), gefitinib (Iressa), bevasizumab (Avastin), epidermal growth factor receptor inhibitors, centuximab (Eributux), gemcitabine, histone deacetylase inhibitors, Carmustine, lomustine, dacarbazine, hydroxymethylmelamine, thiotepa and mitocycin C, Dactinomycin (Act
  • 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: Pre
  • proteasome inhibitors such as bortezomib can be used in combination with the instant compounds, for example.
  • biologies can include agents such as TRAIL and antibodies to TRAIL receptors including 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.
  • the compounds can be conjugated to or delivered with an antibody.
  • 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 degree 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 degree C and 250 rpm on a rotary shaker for 2 days.
  • the production cultures were incubated at 28 degree 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 degree 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.
  • 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 degree 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 degree C and 250 rpm on a rotary shaker for 2 days.
  • the production cultures were incubated at 28 degree 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 degree C and 250 rpm on rotary shaker for 4 days and achieved a titer of 350 - 400 mg/L for Compound 11-16.
  • 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 degree 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 degree C and 250 rpm on a rotary shaker for 2 days. Twenty ml each of the second seed culture was inoculated into 2.8L Fernbach flask containing of 400 ml of the vegetative medium.
  • the third seed cultures were incubated at 28 degree and 250 rpm on a rotary shaker for 2 days. 1.2L of the third seed culture was inoculated into a 42L fermentor containing 26L of Production Medium A. Production Medium B and Production Medium C, with the following composition, can also be used.
  • 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 degree C; agitation, 200 rpm; aeration, 13L/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 8L/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 4.5L 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, 11-16, 11-17, 11-20, II-24C, 11-26 and 11-28.
  • Sample containing II-24C and 13-26 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-Cl 8 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 0 C.
  • FIG. 7 depicts the IH NMR spectrum of a compound having the structure of Formula II- 20.
  • FIG. 51 depicts the 1 H NMR spectrum of a compound having the structure of Formula 11-26 in DMSO-d ⁇ .
  • FIG. 52 depicts the computer-generated ORTEP plot of the compound of Formula 11-26.
  • FIG. 54 depicts the 1 H NMR spectrum of a compound having the structure of Formula 11-28 in DMSO-d ⁇ .
  • 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 degree 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 0 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 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 11-18.
  • FIG. 49 depicts the 1 H NMR spectrum of a compound having the structure of Formula 11-17.
  • FIG. 50 depicts the 1 H NMR spectrum of a compound having the structure of Formula II-l 8.
  • HPLC conditions for the separation of compound formula 11-19 from unreacted 11-16 employed an isocratic HPLC method consisting of 24% ethyl acetate and 76% hexane, in which the majority of compound 11-19 eluted 2.5 minutes before compound 11-16. Equivalent fractions from each of 10 injections were pooled to yield 35 mg compound 11-19.
  • FIG. 12 depicts the NMR spectrum of the compound of Formula II-3 in DMSO-d6.
  • FIG. 13 depicts the low resolution mass spectrum of the compound of Formula D-3: m/z 282 (M+H), 304 (M+Na).
  • 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 ⁇ ; (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. Diol can also be synthesized by acid or base hydrolysis of epoxy compounds (Formula II-5 A 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, NaNa, NaOAc, HBr, HCl, etc. to creat various substituents on the cyclohexane ring, including a hydroxyl substituent.
  • HPLC conditions used for the purification were as follows: Phenomenex Luna 1Ou 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 ml/min. An ELSD was used to monitor the purification process. Compound of Formula IV-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 FV- 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/EtOAc 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 1Ou 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 ml/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).
  • Formula II-13C UV (Acetonitrile/H2 ⁇ ) ⁇ max 226 (sh) & 300 (sh) nm; ESMS, m/z 312 (M+H) + , 334 (M+Na) + ; HRMS (ESI), m/z 312.1017 [M+H] + , 4.5 ppm, C 15 H 19 NO 4 Cl; 1 H NMR inDMSO-d ⁇ (see FIG. 22).
  • the reaction mixture was acidified using 2 ml of 4% HCl solution in water and extracted with CH 2 CI 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 1Ou 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.
  • 11-31 UV (Acetonitrile/H 2 O) ⁇ m ⁇ 250 (sh) nm; ESMS m/z 328.1 (M+H) + & 350.0 (M+Na) + .
  • 11-32 UV (Acetonitrile/H 2 O) ⁇ max 250 (sh) nm; ESMS, m/z 328.1 (M+H) + & 350.0 (M+Na) + .
  • II- 49 UV (Acetonitrile/H 2 O) ⁇ max 250 (sh) and 320 nm; ESMS, m/z 326.0 (M+H) + , 343.1 (M+H 2 O) + & 348.0 (M+Na) + .
  • the ketone of the compounds of formula 11-31 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 Ch-
  • the organic layer can be evaporated to yield the mixtures of compounds of formulae 11-33, 11-34, 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 Cl 8 HPLC chromatography (ACE 5 ⁇ 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 II- 19 by cyano-de-halogenation or thiocyanato-de-halogenation, respectively.
  • Compound 11-19 can be treated with NaCN or KCN to obtain compound II- 37.
  • Compound 11-19 can be treated with NaSCN or KSCN to obtain compound 11-38.
  • Compound 11-38 was purified by normal phase HPLC using a Phenomenex Luna lO ⁇ Silica column (25cm x 21.2 mm ID) with a solvent gradient of 0 to 95% H 2 ⁇ /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 C18 HPLC purification in 2 injections (ACE 5 ⁇ C18-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.
  • any of the compounds of Formulae 11-16, H-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 (NaBHsCN) 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.
  • NaBHsCN 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 Nal ⁇ 4 in THF- H 2 O solution to yield dial derivatives which are reduced to the alcohol with NaBHt.
  • 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 H- 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_i, followed by NalO. ⁇ , 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).
  • the starting compound such as the compound of Formula 11-49
  • the cyclohexadiene derivative can be further treated, for example with DDQ, to aromatize the ring to a phenyl group.
  • the OTMS on the phenyl group can be removed, for example, with acid or base.
  • 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. NH 3
  • NaBRi sodium cyanoborohydride
  • Compound 11-48 UV (Acetonitrile/H 2 ⁇ ) A 1113x 235 (sh) nm; ESMS m/z 621.0 (M+H) + .
  • Compound II-48 was unstable in solution and converted to compound VI-IB which appeared as a mixture of 11-48 and VI-IB in the ratio of 7:3.
  • Compound VI-IB UV (Acetonitrile/H 2 O) X 1n ⁇ 235 (sh) nm; ESMS, m/z 585.2 (M+H) + .
  • each of the 60 human tumor cell lines were grown in RPMI 1640 medium, supplemented with 5% fetal bovine serum and 2 mM L-glutamine.
  • Cells were plated at their appropriate density in 96-well microtiter plates and incubated at 37°C, 5% CO 2 , 95% air and 100% relative humidity.
  • 100 ⁇ L of various 10-fold serial dilutions of Salinosporamide A were added to the appropriate wells containing 100 ⁇ L of cells, resulting in a final Salinosporamide A concentration ranging from 10 nM to 100 ⁇ M.
  • Cells were incubated for an additional 48 hours and a sulforhodamine B protein assay was used to estimate cell viability or growth.
  • GI 50 indicates the concentration that inhibits growth by 50%.
  • TGI indicates the concentration that completely inhibits growth.
  • LC 50 indicates the concentration that is lethal to 50% of the cells.
  • Salinosporamide A (1) is a potent compound with a mean GI 50 value of ⁇ 10 nM, and (2) displays good tumor selectivity of more than 1000-fold difference in both the mean TGI and mean LC 50 values between the most sensitive and resistant tumor cell lines.
  • B16-F10 (ATCC; CRL-6475), DU 145 (ATCC; HTB-81), HEK293 (ATCC; CRL-1573), HT-29 (ATCC; HTB-38), LoVo (ATCC; CCL-229), MDA-MB-231 (ATCC; HTB-26), MIA PaCa-2 (ATCC; CRL-1420), NCI-H292 (ATCC; CRL-1848), OVCAR-3 (ATCC, HTB-161), PANC-I (ATCC; CRL-1469), PC-3 (ATCC; CRL-1435), RPMI 8226 (ATCC; CCL-155) and U266 (ATCC; TIB-196) were maintained in appropriate culture media. The cells were cultured in an incubator at 37 0 C in 5% CO2 and 95% humidified air.
  • B16-F10, DU 145, HEK293, HT-29, LoVo, MDA-MB-231, MIA PaCa-2, NCI-H292, OVCAR-3, PANC-I, PC-3, RPMI 8226 and U266 cells were seeded at 1.25xlO 3 , 5xlO 3 , 1.5xlO 4 , 5xlO 3 , 5xlO 3 , IxIO 4 , 2xlO 3 , 4x10 3 , IxIO 4 , 7.5x10 3 , 5x10 3 , 2x10 4 , 2.5x10 4 cells/well respectively in 90 ⁇ l complete media into Corning 3904 black-walled, clear-bottom tissue culture plates.
  • 2OmM stock solutions of Formula 11-16 were prepared in 100% DMSO, aliquoted and stored at -80 0 C.
  • Formula 11-16 was serially diluted and added in triplicate to the test wells resulting in final concentrations ranging from of 20 ⁇ M to 0.2pM. The plates were returned to the incubator for 48 hours. The final concentration of DMSO was 0.25% in all samples.
  • the data were normalized to the average fluorescence of the cells treated with media + 0.25% DMSO (100% cell growth) and EC 50 values (the drug concentration at which 50% of the maximal observed growth inhibition is established) were determined using a standard sigmoidal dose response curve fitting algorithm (generated by XLfit 3.0, ID Business Solutions Ltd or Prism 3.0, GraphPad Software Inc).
  • the EC 50 values indicate that Formula II- 16 was cytotoxic against B 16- FlO, DU 145, HEK293, HT-29, LoVo, MDA-MB-231, MIA PaCa-2, NCI-H292, OVCAR-3, PANC-I, PC-3, RPMI 8226 and U266 cells.
  • II-4 ⁇ -5A, II-5B, II-8C, II-13C, 11-16, 11-17, 11-18, 11-19, 11-20, U-21, 11-22, II-24C, 11-25,
  • Assays were performed at a proteasome concentration of 1 ⁇ g/ml in a final volume of 200 ⁇ l in 96- well Costar microtiter plates.
  • Formulae II-2, II-4, 11-16, H-17, 11-18, 11-19, 11-21, 11-22 and 11-44 were tested as eight-point dose response curves with final concentrations ranging from 500 nM to 158 pM.
  • Formulae 1-7, II-5A, II-5B, 11-20, 11-29, 11-30 and 11-38 were tested at concentrations ranging from 1 ⁇ M to 0.32nM.
  • Formulae II-3 and VI-IA were tested as an eight-dose response curve with final concentrations ranging from 10 ⁇ M to 3.2 nM.
  • Formula 11-47 was tested at concentrations ranging from 5 ⁇ M to 1.6 nM, while Formulae II-8C, II-13C, II-24C, 11-25, 11-26, 11-27, 11-28, 11-31, 11-32 and IV-3C were tested with final concentrations ranging from 20 ⁇ M to 6.3nM.
  • the samples were incubated at 37°C for five minutes in a temperature controlled Fluoroskan Ascent 96-well microplate reader (Thermo Electron, Waltham, MA). During the preincubation step, the substrate was diluted 25-fold in SDS-containing assay buffer. After the preincubation period, the reactions were initiated by the addition of 10 ⁇ l of the diluted substrate and the plates were returned to the plate reader.
  • Formulae II-3, II-4, II-5A, II-5B, II-8C, II-13C, 11-17, 11-18, 11-20, 11-21 , 11-22, II-24C, 11-25, 11-26, 11-27, 11-28, 11-29, 11-30, IV-3C, 11-44 and VI-IA were tested at concentrations ranging from 20 ⁇ M to 6.3nM.
  • Formula II-2 was tested at concentrations ranging from lO ⁇ M to 3.2nM
  • Formula 11-16 and Formula 11-19 were tested at concentrations ranging from 5 ⁇ M to 1.58nM.
  • the SDS was omitted from the assay buffer and Boc-LRR-AMC was used as the peptide substrate.
  • Formula 11-20 was tested at concentrations ranging from 5 ⁇ M to 1.6 nM.
  • Formulae H-3, II-8C, II-13C, 11-17, 11-21, II- 22, II-24C, ⁇ -25, 11-26, 11-27, 11-28, 11-29, 11-30, IV-3C and VI-IA were tested at concentrations ranging from 20 ⁇ M to 6.3nM.
  • concentrations tested ranged from lO ⁇ M to 3.2nM
  • Formulae II-4, II-5A, 11-16, II- 18 and II- 19 were tested at concentrations ranging from l ⁇ M to 0.32nM.
  • 11-44 was tested at concentrations ranging from 2 ⁇ M to 632pM.
  • Results are shown in Table 3 and illustrate that among the tested compounds, Formulae II-5A, II-16, 11-18, 11-19, 11-20, 11-21, 11-22, 11-29, H-38 and 11-44 are the most potent inhibitors of the chymotrypsin-like activity of the 2OS proteasome with EC50 values ranging from 2 nM to 1 InM.
  • Formulae 1-7, II-2, II-4, II-5B, 11-17, 11-30 and 11-47 inhibit the proteasomal chymotrypsin-like activity with EC 50 values ranging from 13 nM to 88 nM, while the EC50 values of Formulae II-3, 11-26 and VI-IA ranged from 207 nM to 964 nM.
  • Formulae II-13C, II-24C, H-27, 11-28 and IV-3C inhibited the chymotrypsin-like activity with ECso values ranging from 1.4 ⁇ M to 10.6 ⁇ M.
  • EC 50 values for Formulae II-8C, 11-25, 11-31 and 11-32 were greater than 20 ⁇ M.
  • Formulae II-2, II-3, II-4, II-5A, II-5B, II-13C, 11-16, 11-17, II- 18, 11-19, II-20, 11-21, 11-22, ⁇ -24C, 11-26, II-29, ⁇ -30, II-44 and VI-IA were able to inhibit the trypsin-like activity of the 2OS proteasome.
  • Formulae II-4, II-5A, 11-16, 11-18, 11-19 and 11-29 inhibited the caspase-like activity with EC50 values ranging from 213nM to 85OnM, while Formulae II-2, II-5B, 11-17, ⁇ -20, 11-21, H-22, 11-30, 11-44 and VI-IA had EC 50 values ranging from 956 nM to 8.7 ⁇ M.
  • Results from a representative experiment evaluating Formula II-2, Formula II-3 and Formula II-4 are shown in FIG. 46 and illustrate that Formula II-2 and Formula II-4 inhibit the chymotrypsin-like activity of the proteasome with EC 50 values of 18.5 nM and 15nM respectively.
  • Formula II-3 is active in this assay with an EC 50 value of 890 nM. Similar results were obtained from an independent experiment.
  • Omuralide was prepared as a 10 mM stock in DMSO and stored in 5 ⁇ L aliquots at -8O 0 C.
  • Salinosporamide A was prepared as a 25.5 mM solution in DMSO and stored in aliquots at -8O 0 C.
  • the assay measures the hydrolysis of Suc-LLVY-AMC into Suc-LLVY and AMC.
  • the assays were performed in a microtiter plate (Corning 3904), and followed kinetically with measurements every five minutes.
  • the instrument used was a Thermo Lab Systems Fluoroskan, with the incubation chamber set to 37 0 C.
  • the assays were performed according to the manufacturer's protocol, with the following changes.
  • the proteasome was activated as described with SDS, and held on ice prior to the assay.
  • Salinosporamide A and Omuralide were serially diluted in assay buffer to make an 8-point dose-response curve.
  • Ten microliters of each dose were added in triplicate to the assay plate, and 190 ⁇ L of the activated proteasome was added and mixed.
  • the samples were pre-incubated in the Fluoroskan for 5 minutes at 37°C. Substrate was added and the kinetics of AMC were followed for one hour. All data were collected and plotted as the mean of triplicate data points.
  • the data were normalized to reactions performed in the absence of Salinosporamide A and modeled in Prism as a sigmoidal dose-response, variable slope
  • Salinosporamide A is a potent inhibitor of the chymotrypsin-like activity of the proteasome.
  • the EC 50 values for cytotoxicity were in the 10-200 nM range suggesting that the ability of Salinosporamide A to induce cell death was due, at least in large part, to proteasome inhibition.
  • the data suggest that Salinosporamide A is a potent small molecule inhibitor of the proteasome.
  • Omuralide can inhibit the PGPH activity (also known as the caspase- like) of the proteasome; therefore, the ability of Salinosporamide A to inhibit the PGPH activity of purified rabbit muscle 2OS proteasomes was assessed.
  • a commercially available fluorogenic substrate specific for the PGPH activity was used instead of the chymotrypsin substrate supplied in the proteasome assay kit described above.
  • Salinosporamide A (11-16) was prepared as a 20 mM solution in DMSO and stored in small aliquots at -8O 0 C.
  • the substrate Z-LLE-AMC was prepared as a 20 mM stock solution in DMSO, stored at -2O 0 C.
  • the proteasomes were activated with SDS and held on ice as per manufacturer's recommendation.
  • Salinosporamide A was diluted in DMSO to generate a 400-fold concentrated 8-point dilution series. The series was diluted 20-fold with assay buffer and preincubated with the proteasomes as described for the chymotrypsin-like activity. After addition of substrate, the samples were incubated at 37°C, and release of the fluorescent AMC was monitored in a fluorimeter. All data were collected and plotted as the mean of triplicate points. In these experiments, the EC 50 was modeled in Prism as normalized activity, where the amount of AMC released in the absence of Salinosporamide A represents 100% activity.
  • Salinosporamide A was prepared as a 20 mM solution in DMSO and stored in small aliquots at -8O 0 C.
  • the substrate, suc-LLVY-AMC was prepared as a 20 mM solution in DMSO and stored at -20 0 C.
  • the proteasomes were activated by SDS and stored on ice as with the experiments using rabbit muscle proteasomes.
  • Salinosporamide A was diluted in DMSO to generate a 400-fold concentrated 8-point dilution series. The series was then diluted 20-fold with assay buffer and pre-incubated with proteasomes at 37 0 C. The reaction was initiated with substrate, and the release of AMC was followed in a Fluoroskan microplate fluorimeter. Data were collected and plotted as the mean of triplicate points. Data were captured kinetically for 3 hours, and indicated that these reactions showed linear kinetics in this time regime. The data were normalized to reactions performed in the absence of Salinosporamide A and modeled in Prism as a sigmoidal dose-response, variable slope.
  • Salinosporamide A inhibits the proteasome is by the reaction of the ⁇ -lactone functionality of Salinosporamide A with the active site threonine of the proteasome. This covalent modification of the proteasome would block the active site, as this residue is essential for the catalytic activity of the proteasome. Fenteany, et al, J Biol Chem 273:8545 (1998).
  • Lactacystin has been shown to also inhibit cathepsin A (Ostrowska, et al, Int J Biochem Cell Biol 32:747 (2000), Kozlowski, et al., Tumour Biol 22:211 (2001), Ostrowska, et al., Biochem Biophys Res Commun 234:729 (1997)) and TPPII (Geier, et al., Science 283:978 (1999)) but not trypsin, chymotrypsin, papain, calpain (Fenteany, et al., Science 268:726 (1995)), thrombin, or plasminogen activator (Omura, et al, J Antibiot (Tokyo) 44:113 (1991)). Similar studies were initiated to explore the specificity of Salinosporamide A for the proteasome by evaluating its ability to inhibit the catalytic activity of a prototypical serine protease, chymotry
  • Salinosporamide A was prepared as a 20 mM solution in DMSO and stored in small aliquots at -8O 0 C.
  • Bovine pancreatic chymotrypsin was obtained from Sigma (Cat.
  • Salinosporamide A inhibits the chymotrypsin-like and PGPH activity of the proteasome. Preliminary studies indicate that Salinosporamide A also inhibits the trypsin-like activity of the proteasome with an EC50 value of -10 nM (data not shown).
  • the HEK293 NF- ⁇ B/luciferase reporter cell line is a derivative of the human embryonic kidney cell line (ATCC; CRL- 1573) and carries a luciferase reporter gene under the regulation of 5X NF- ⁇ B binding sites.
  • the reporter cell line was routinely maintained in complete DMEM medium (DMEM plus 10%(v/v) Fetal bovine serum, 2mM L-glutamine, 10 mM HEPES and Penicillin/Streptomycin at 100 IU/ml and 100 ⁇ g/ml, respectively) supplemented with 250 ⁇ g/ml G418.
  • the DMEM basal medium was replaced with phenol-red free DMEM basal medium and the G418 was omitted.
  • the cells were cultured in an incubator at 37 0 C in 5% CO 2 and 95% humidified air.
  • HEK293 NF- ⁇ B/luciferase cells were seeded at 1.5x10 4 cells/well in 90 ⁇ l phenol-red free DMEM complete medium into Corning 3917 white opaque-bottom tissue culture plates.
  • a 400 ⁇ M starting dilution was made in 100% DMSO and this dilution was used to generate an 8-point half log dilution series.
  • This dilution series was further diluted 4OX in appropriate culture medium and ten ⁇ l aliquots were added to the test wells in triplicate resulting in final test concentrations ranging from 1 ⁇ M to 320 pM.
  • a 2.5mM or 8mM starting dilution was made in 100% DMSO and the final test concentrations ranged from 6.3 ⁇ M to 2.OnM or 20 ⁇ M to 6.3nM respectively.
  • the plates were returned to the incubator for 1 hour.
  • 10 ⁇ l of a 50 ng/ml TNF- ⁇ solution, prepared in the phenol-red free DMEM medium was added, and the plates were incubated for an additional 6 hours.
  • the final concentration of DMSO was 0.25% in all samples.
  • NF-KLB regulates the expression of a large number of genes important in inflammation, apoptosis, tumorigenesis, and autoimmune diseases.
  • compounds capable of modulating or affecting NF- ⁇ B activity are useful in treating diseases related to inflammation, cancer, and autoimmune diseases, for example.
  • NF- KB complexes with IKB in the cytosol and upon stimulation, IKB is phosphorylated, ubiquitinated and subsequently degraded by the proteasome. The degradation of IKB leads to the activation of NF- ⁇ B and its translocation to the nucleus.
  • the EC 50 values to inhibit NF- ⁇ B-mediated luciferase activity are shown in Table 4 and demonstrate that compounds of Formulae II-2, II-4, II-5A, II-5B, 11-16, 11-17, 11-18, H-19, 11-20, ⁇ -21, 11-22, II-24C, ⁇ -26, 11-29, 11-30 and 11-44 inhibited NF- ⁇ B activity in this cell-based assay.
  • Results from a representative experiment evaluating Formula II-2, Formula II-3 and Formula II-4 revealed that pretreatment with Formula II-2 and Formula II-4 resulted in a dose-dependent decrease of luciferase activity in NF- ⁇ B/Luc 293 cells upon TNF- ⁇ stimulation.
  • the calculated EC50 to inhibit NF- ⁇ B inducible luciferase activity in this experiment was 73 nM for Formula II-2, while EC 50 value for Formula II-4 was 67 nM. Similar data were observed in a replicate experiment.
  • Results from a representative experiment evaluating Formula II-5 A and Formula II-5B are shown in FIG. 45 and illustrate that Formula II-5A and Formula II-5B inhibit NF- ⁇ B inducible luciferase activity with EC 50 values of 30 nM and 261 nM respectively. Similar data were observed in a replicate experiment.
  • NF- ⁇ B/Luc 293 cells were pre-treated with 8-point half-log serial dilutions of Salinosporamide A (ranging from 1 ⁇ M to 317 pM) for 1 hour followed by a 6 hour stimulation with TNF- ⁇ (10 ng/mL). NF- ⁇ B inducible luciferase activity was measured at 6 hours. Viability of NF- ⁇ B/Luc 293 cells, after treatment with Salinosporamide A for 24 hr, was assessed by the addition of resazurin dye, as previously described.
  • the ubiquitin-proteasome pathway is an essential proteolytic system involved in cell cycle control by regulating the degradation of cyclins and cyclin-dependent kinase (Cdk) inhibitors such as p21 and p27.
  • Cdk cyclin-dependent kinase
  • p21 and p27 protein levels are increased in the presence of proteasome inhibitors.
  • Jurkat cells were plated at 2 XlO cells / 3 mL per well in a 6-well plate and incubated at 37°C, 5% (v/v) CO 2 and 95% (v/v) humidity.
  • Salinosporamide A and Mitoxantrone (Sigma, St. Louis, MO. Cat # M6545), were prepared in DMSO at stock concentrations of 20 mM and 40 mM, respectively.
  • Mitoxantrone is a chemotherapeutic drug that induces apoptosis in dividing and non-dividing cells via inhibition of DNA synthesis and repair and was included as a positive control. Bhalla, et al., Blood 82:3133 (1993).
  • PARP poly (ADP-ribose) polymerase
  • Jurkat cells were maintained in RPMI supplemented with 10% Fetal Bovine Serum (FBS) at low density (2x10 s cells per mL) prior to the experiment. Cells were harvested by centrifugation, and resuspended in media to 1 X 10 6 cells per 3 mL. Twenty mL of the cell suspension were treated with 100 nM Salinosporamide A (20 mM DMSO stock stored at -8O 0 C), and a 3 mL aliquot removed and placed on ice for the To sample. Three mL aliquots of the cell suspension plus Salinosporamide A were placed in 6-well dishes and returned to the incubator.
  • FBS Fetal Bovine Serum
  • the western blot presented in FIG. 35 shows the cleavage of PARP within the Jurkat cells in a time-dependent fashion.
  • the cleaved form (denoted by the asterisk, ⁇ ) appears in the treated cells between 2 and 4 hrs after exposure to Salinosporamide A while the majority of the remaining PARP is cleaved by 24 hrs.
  • the Staurosporine treated cells (St) show rapid cleavage of PARP with most of this protein being cleaved within 4 hours.
  • RAW264.7 cells (ATCC # TIB-71) were adapted to and maintained in Advanced Dulbecco's Modified Eagle Medium (Invitrogen, Carlsbad, CA) supplemented with 5% fetal bovine serum (ADMEM, Mediatech, Hemdon, VA) at 37°C in a humidified 5% CO 2 incubator. Cells were plated overnight in ADMEM supplemented with 5% FBS at 37 0 C in a humidified 5% CO2 incubator at a concentration of 50,000 cells/well in a 96- well plate. Alternatively, cells cultured in DMEM supplemented with 10% fetal calf serum were also used and found to be amenable to this assay.
  • % viability 100* (observed OD -positive control)/ (negative control-positive control).
  • the data represented in FIG. 36 indicate that treatment with Salinosporamide A can prevent LeTx -induced cell death of macrophage-like RAW264.7 cells in vitro.
  • Treatment of RAW cells with either LF or PA alone or Salinosporamide A alone resulted in little reduction in cell viability, whereas treatment with LeTx resulted in approximately 0.27% cell viability as compared to controls.
  • Salinosporamide A may enhance macrophage survival by inhibiting the degradation of specific proteins and decreasing the synthesis of cytokines, which will ultimately lead to the inhibition of the lethal effects of anthrax toxins in vivo.
  • Salinosporamide A treatment alone produced very modest cytotoxicity at concentrations of 100 nM and above, treatment with lower, relatively nontoxic levels revealed a marked increase in RAW 264.7 cell viability in LeTx treated cells (FIG. 36).
  • the Salinosporamide A + LeTx treated group showed 82% cell- viability when pretreated with 12 nM Salinosporamide A, which was a concentration that showed 96% viability with Salinosporamide A alone.
  • the average EC 50 for Salinosporamide A in these studies was 3.6 nM.
  • Omuralide showed relatively little effect on cell viability until concentrations of 1 ⁇ M were reached.
  • Salinosporamide A promoted survival of RAW264.7 cells in the presence of LeTx indicating that this compound or it's derivatives can be a valuable clinical therapeutic for anthrax. In addition, it is worth noting that Salinosporamide A is much less cytotoxic on RAW 264.7 cells than for many tumor cells.
  • NF- ⁇ B can be to be critical to the growth and resistance to apoptosis in Multiple Myeloma and has also been reported to be constitutively active in various prostate cancer cell lines (Hideshima T et al. 2002, Shimada K et al. 2002 and Palayoor ST et al. 1999). NF- ⁇ B activity is regulated by the proteasomal degradation of its inhibitor IKBCX. Since Salinosporamide A has been shown to inhibit the proteasome in vitro and to interfere with the NF- ⁇ B signaling pathway, the activity of Salinosporamide A against the multiple myeloma cell line RPMI 8226 and the prostate cancer cell lines PC-3 and DU 145 was evaluated.
  • EC 50 values were determined in standard growth inhibition assays using Resazurin dye and 48 hour of drug exposure. Results from 2-5 independent experiments (Table 6) show that the EC50 values for Salinosporamide A against RPMI 8226 and the prostate cell lines range from 10-37nM.
  • RPMI 8226 cells were used to evaluate the effect of treating the cells for 8 hours with various concentrations of Salinosporamide A. Briefly, RPMI 8226 cells were treated with varying concentrations of Salinosporamide A (2345R01) for 8 hours and protein lysates were made. 25 ⁇ g of the lysates were then resolved under reducing/denaturing conditions and blotted onto nitrocellulose. The blots were then probed with anti-PARP or anti-caspase 3 antibodies followed by stripping and reprobing with an anti-actin antibody.
  • FIG. 38 demonstrates that Salinosporamide A induces a dose-dependent cleavage of both PARP and Pro-Caspase 3.
  • RPMI 8226 ATCC; CCL- 155) and U266 (ATCC; TIB- 196) were maintained in appropriate culture media. The cells were cultured in an incubator at 37 0 C in 5% CO2 and 95% humidified air.
  • RPMI 8226 cells and U266 were seeded at 2x10 4 and 2.5x10 4 cells/well respectively in 90 ⁇ l complete media into Corning 3904 black- walled, clear-bottom tissue culture plates. 2OmM stock solutions of the compounds were prepared in 100% DMSO, aliquoted and stored at -80 0 C. The compounds were serially diluted and added in triplicate to the test wells.
  • the final concentration range of Formulae 1-7, II-3, II-8C, II-5B, II-13C, 11-17, 11-20, 11-21, 11-22, II- 24C, 11-25, 11-26, 11-28, 11-29, 11-30, 11-31, 11-32, 11-38, IV-3C, VI-IA and 11-47 were from 20 ⁇ M to 6.32nM.
  • the final concentration of Formulae 11-16, 11-18, 11-19, 11-44 and 11-50 ranged from 632nM to 20OpM.
  • the final concentration range of Formulae II-2, II-4 and II-5A were from 2 ⁇ M to 632pM.
  • the final concentration of DMSO was 0.25% in all samples.
  • the data were normalized to the average fluorescence of the cells treated with media + 0.25% DMSO (100% cell growth) and EC 5 O values (the drug concentration at which 50% of the maximal observed growth inhibition is established) were determined using a standard sigmoidal dose response curve fitting algorithm (generated by XLfit 3.0 or XLfit 4.0, ID Business Solutions Ltd). The data are summarized in Tables 12 and 13.
  • Results from these growth inhibition assays show that, as expected, Paclitaxel did not retain its activity against MES-SA/Dx5 cells as reflected by the 408 fold increase in the EC50 values.
  • EC50 values for Salinosporamide A against MES-SA and MES-SA/Dx5 were similar. This illustrates that Salinosporamide A is able to inhibit the growth of the multi-drug resistant cell line MES-SA/Dx5 suggesting that Salinosporamide A does not seem to be a substrate for the P-glycoprotein efflux pump.
  • Salinosporamide A was evaluated against HL-60/MX2, the drug resistant derivative of the human leukemia cell line, HL-60, characterized by having a reduced Topoisomerase II activity and considered to have atypical multidrug resistance.
  • EC 50 values for growth inhibition were determined for Salinosporamide A against the HL- 60 and HL-60/MX2.
  • the DNA binding agent Mitoxantrone was included as a control, as HL-60/MX2 cells are reported to be resistant to this chemotherapeutic agent (Harker W.G. e/ ⁇ /. 1989).
  • Salinosporamide A was also shown to have activity against drug resistant multiple myeloma cell lines.
  • Salinosporamide A was shown to be active against MM. IR and Doxorubicin-resistant Dox-40 cell lines.
  • Salinosporamide A was shown to be active against cell lines obtained from human multiple myloma patients that had relapsed after multiple prior therapies with Dexamethasone, Bortezomib, and thalidomide.
  • Salinosporamide A is active against drug resistant multiple myeloma including multiple myeloma exhibiting resistance to doxorubicin, dexamethasone, bortezomib, and thalidomide.
  • the other compounds disclosed herein are active against drug resistant multiple myeloma including multiple myeloma exhibiting resistance to doxorubicin, dexamethasone, bortezomib, and thalidomide.
  • Salinosporamide A To establish an initial structure activity relationship (SAR) for Salinosporamide A, a series of Salinosporamide A analogs were evaluated against the multiple myeloma cell line RPMI 8226. EC 50 values were determined in standard growth inhibition assays using Resazurin dye and 48 hour of drug exposure.
  • mice were weighed and various Salinosporamide A concentrations (ranging from 0.01 mg/kg to 0.5 mg/kg) were administered intravenously as a single dose (qdxl) or daily for five consecutive days (qdx5). Animals were observed daily for clinical signs and were weighed individually twice weekly until the end of the experiment (maximum of 14 days after the last day of dosing). Results are shown in Table 10 and indicate that a single intravenous Salinosporamide A dose of up to 0.25 mg/kg was tolerated. When administered daily for five consecutive days, concentrations of Salinosporamide A up to 0.1 mg/kg were well tolerated. No behavioral changes were noted during the course of the experiment.
  • Results from the preliminary P450 inhibition screen showed that Salinosporamide A, when tested at lO ⁇ M, showed no or low inhibition of all P450 isoforms: CYP 1A2, CYP2C9 and CYP3A4 were inhibited by 3%, 6% and 6% respectively, while CYP2D6 and CYP2C19 were inhibited by 19% and 22% respectively.
  • Salinosporamide A was previously demonstrated to be a potent and specific inhibitor of the proteasome in vitro, with an IC 50 of 2 nM towards the chymotrypsin-like activity of purified 2OS proteasomes.
  • IC 50 2 nM towards the chymotrypsin-like activity of purified 2OS proteasomes.
  • a rapid and reproducible assay (adapted from Lightcap et al. 2000) was developed to assess the proteosome activity in whole blood.
  • Proteasome activity was determined by measuring the hydrolysis of a fluorogenic substrate specific for the chymotrypsin-like activity of proteasomes (suc- LLVY-AMC, Bachem Cat. 1-1395). Control experiments indicated that >98% of the hydrolysis of this peptide in these extracts is mediated by the proteasome. Assays were set up by mixing 5 ⁇ L of a PWBL from an animal with 185 ⁇ L of assay buffer (20 mM HEPES, 0.5 mM EDTA, 0.05% Triton X-100, 0.05% SDS, pH 7.3) in Costar 3904 plates.
  • Salinosporamide A To explore the in vivo activity of Salinosporamide A, male Swiss- Webster mice (5 per group, 20-25g in weight) were treated with various concentrations of Salinosporamide A. Salinosporamide A was administered intravenously and given its LogD 74 value of 2.4, suggestive of oral availability, Salinosporamide A was also administered orally. Salinosporamide A dosing solutions were generated immediately prior to administration by dilution of Salinosporamide A stock solutions (100% DMSO) using 10% solutol yielding a final concentration of 2% DMSO. The vehicle control consisted of 2% DMSO in 10% solutol.
  • Salinosporamide A was administered at 10 mL/kg and ninety minutes after administration the animals were anesthetized and blood withdrawn by cardiac puncture. Packed whole blood cells were collected by centrifugation, washed with PBS, and re- centrifuged. All samples were stored at -80 0 C prior to the evaluation of the proteasome activity.
  • FIG. 40 is a scatter plot displaying the normalized proteasome activity in PWBL's derived from the individual mice (5 mice per group), hi each group, the horizontal bar represents the mean normalized activity.
  • Epoxomicin is a peptide epoxide that has been shown to highly specific for the proteasome, with no inhibitory activity towards any other known protease (Meng et ⁇ /.,1999). Lysates from a vehicle control and also from animals treated intravenous (i.v.) with 0.1 mg/kg Salinosporamide A were incubated with varying concentration of Epoxomicin, and IC 50 values were determined. Palayoor et al., Oncogene 18:7389-94 (1999). As shown in FIG. 41, Epoxomicin caused a dose dependent inhibition in the hydrolysis of the proteasome substrate.
  • the IC 50 obtained in these experiments matches well with the 10 nM value observed using purified 2OS proteasomes in vitro (not shown). These data also indicate that the remaining activity towards this substrate in these lysates prepared from animals treated with 0.1 mg/kg Salinosporamide A is due to the proteasome, and not some other protease.
  • the residual activity seen in extracts treated with high doses of Epoxomicin is less than 2% of the total signal, indicating that over 98% of the activity observed with suc-LLVY-AMC as a substrate is due solely to the activity of the proteasomes present in the PWBL.
  • proteasome plays a role in the activation of many signaling molecules, including the transcription factor NF- ⁇ B via protealytic degradation of the inhibitor of NF- ⁇ B (IKB).
  • LPS signaling through the TLR4 receptor activates NF- ⁇ B and other transcriptional regulators resulting in the expression of a host of proinflammatory genes like TNF, IL-6, and IL- l ⁇ .
  • proinflammatory cytokines has been identified as a major factor in many diseases.
  • Inhibitors of TNF and IL- l ⁇ have shown efficacy in many inflammation models including the LPS murine model, as well as animal models of rheumatoid arthritis and inflammatory bowel disease.
  • mice Male Swiss Webster mice (12/group weighing 20-25g) were injected with LPS (2mg/kg) by the i.p. route. Thirty minutes later, mice were injected i.v. (tail vein) with Salinosporamide A at 2.5 mg/kg after approximately 5 minutes under a heat lamp. Ninety minutes after LPS injection, the mice were anesthetized with Isoflurane and bled by cardiac puncture to obtain plasma. Remaining blood pellet was then resuspended in 500 ⁇ L of PBS to wash away residual serum proteins and centrifuged again. Supernatant was removed and blood pellet frozen for analysis of proteasome inhibition in packed whole blood lysate.
  • Dosing solutions were prepared using a lOmg/mL Salinosporamide A stock solution in 100% DMSO.
  • a 10% solutol solution was prepared by diluting w/w with endotoxin-free water and a 1:160 dilution was made of the lOmg/ml Salinosporamide A stock. Animals were dosed i.v. with 4ml/kg.
  • a vehicle control solution was also prepared by making the same 1 :160 dilution with 100% DMSO into 10% solutol solution giving a final concentration of 9.375% solutol in water and 0.625% DMSO.
  • Measurements of plasma TNF were performed using the Biosource mTNF Cytoset kit (Biosource Intl., Camarillo, CA; catalog # CMC3014) according to manufacturer's instructions. Samples were diluted 1 :60 for the assay.
  • Chemotherapy agents such as CPT-I l (Irinotecan) can activate the transcription factor nuclear factor-kappa B (NF- ⁇ B) in human colon cancer cell lines including LoVo cells, resulting in a decreased ability of these cells to undergo apoptosis. Cusack, et al, Cancer Res 61 :3535 (2001). In unstimulated cells, NF- ⁇ B resides in the cytoplasm in an inactive complex with the inhibitory protein IKB (inhibitor of NF- ⁇ B).
  • VelcadeTM was found to inhibit the expression of proangiogenic chemokines/cytokines GRO- ⁇ and VEGF in squamous cell carcinoma, presumably through inhibition of the NF- ⁇ B pathway.
  • Sunwoo et al., CHn Cancer Res 7:1419 (2001). The data indicate that proteasome inhibition may not only decrease tumor cell survival and growth, but also angiogenesis.
  • Human colon adenocarcinoma (HT-29; HTB-38), prostate adenocarcinoma (PC-3; CRL-1435), breast adenocarcinoma (MDA-MB-231; HTB-26), non-small cell lung carcinoma (NCI-H292; CRL-1848), ovarian adenocarcinoma (OVCAR-3; HTB-161), multiple myeloma (RPMI 8226; CCL-155), multiple myeloma (U266; TIB-196) and mouse melanoma (B16 : F10; CRL-6475) cells were all purchased from ATCC and maintained in appropriate culture media. The cells were cultured in an incubator at 37 0 C in 5% CO 2 and 95% humidified air.
  • HT-29, PC-3, MDA-MB-231, NCI- H292, OVCAR-3 and B16-F10 cells were seeded at IxIO 4 , 4xlO 3 , IxIO 4 and 1.25x10 3 cells/ well respectively in 90 ⁇ l complete media into 96 well (Corning; 3904) black-walled, clear-bottom tissue culture plates and the plates were incubated overnight to allow cells to establish and enter log phase growth.
  • RPMI 8226 and U266 cells were seeded at 2x10 4 and 2.5x10 4 cells/well respectively in 90 ⁇ l complete media into 96 well plates on the day of the assay.
  • 2OmM stock solutions of the compounds were prepared in 100% DMSO and stored at -80 0 C. The compounds were serially diluted and added in triplicate to the test wells. Concentrations ranging from 6.32 ⁇ M to 632pM were tested for II-2 and II-4. II-3 and 11-17 were tested at concentrations ranging from 20 ⁇ M to 6.32nM. Formula 11-18 and 11-19 were tested at concentrations ranging from 2 ⁇ M to 20OpM. Formula II-5A and Formula II-5B were tested at final concentrations ranging from 2 ⁇ M to 632pM and 20 ⁇ M to 6.32nM respectively. The plates were returned to the incubator for 48 hours. The final concentration of DMSO was 0.25% in all samples. .
  • the data were normalized to the average fluorescence of the cells treated with media + 0.25% DMSO (100% cell growth) and EC 50 values (the drug concentration at which 50% of the maximal observed growth inhibition is established) were determined using a standard sigmoidal dose response curve fitting algorithm (XLfit 3.0, ID Business Solutions Ltd). Where the maximum inhibition of cell growth was less than 50%, an EC 50 value was not determined.
  • Table 12 summarize the growth inhibitory effects of Formulae II-2, II-3, II-4, II-5A, II-5B, 11-17, H-18 and 11-19 against the human colorectal carcinoma, HT-29, human prostate carcinoma, PC-3, human breast adenocarcinoma, MDA-MB-231, human non-small cell lung carcinoma, NCI-H292, human ovarian carcinoma, OVCAR-3, human multiple myelomas, RPMI 8226 and U266 and murine melanoma B16-F10 cell lines.
  • the EC 50 values indicate that the Formulae II-2, II-4, II-5A, II-5B, II- 18 and II- 19 were cytotoxic against the HT-29, PC-3, MDA-MB-231, NCI-H292, RPMI 8226, U266 and B16-F10 tumor cell lines.
  • II-2, II-5A, II-5B and 11-19 were also cytotoxic against the OVCAR-3 tumor cells.
  • Formula 11-17 was cytotoxic against MDA-MB-231, RPMI 8226, U266 and B16-F10 tumor cell lines.
  • MES-SA Human uterine sarcoma
  • MES-SA/Dx5 human acute promyelocytic leukemia cells
  • HL-60/MX2 human acute promyelocytic leukemia cells
  • HL-60/MX2 human acute promyelocytic leukemia cells
  • CRL-2257 human acute promyelocytic leukemia cells
  • the cells were cultured in an incubator at 37 0 C in 5% CO 2 and 95% humidified air.
  • MES-SA and MES-SA/Dx5 cells were both seeded at 3x10 3 cells/ well in 90 ⁇ l complete media into 96 well (Corning; 3904) black- walled, clear-bottom tissue culture plates and the plates were incubated overnight to allow cells to establish and enter log phase growth.
  • HL-60 and HL-60/MX2 cells were both seeded at 5x10 4 cells/ well in 90 ⁇ l complete media into 96 well plates on the day of compound addition. 2OmM stock solutions of the compounds were prepared in 100% DMSO and stored at -8O 0 C. The compounds were serially diluted and added in triplicate to the test wells.
  • Concentrations ranging from 6.32 ⁇ M to 2nM were tested for II- 2 and II-4. II-3 and 11-17 were tested at concentrations ranging from 20 ⁇ M to 6.32nM. Compound 11-18 was tested at concentrations ranging from 2 ⁇ M to 632pM. The plates were returned to the incubator for 48 hours. The final concentration of DMSO was 0.25% in all samples.
  • the data were normalized to the average fluorescence of the cells treated with media + 0.25% DMSO (100% cell growth) and EC 50 values (the drug concentration at which 50% of the maximal observed growth inhibition is established) were determined using a standard sigmoidal dose response curve fitting algorithm (XLfit 3.0, ID Business Solutions Ltd). Where the maximum inhibition of cell growth was less than 50%, an EC50 value was not determined.
  • the multidrug resistant MES-SA/Dx5 tumor cell line was derived from the human uterine sarcoma MES-SA tumor cell line and expresses elevated P- Glycoprotein (P -gp), an ATP dependent efflux pump.
  • P -gp P- Glycoprotein
  • Table 14 summarize the growth inhibitory effects of Formulae II-2, II-3, II-4, 11-17 and 11-18 against MES-SA and its multidrug resistant derivative MES-S A/Dx5.
  • Paclitaxel, a known substrate of the P-gp pump was included as a control.
  • HL-60/MX2 is a multidrug resistant tumor cell line derived from the human promyelocyte leukemia cell line, HL-60 and expresses reduced topoisomerase II activity.
  • Table 15 summarize the growth inhibitory effects of Formulae II-2, II-3, II-4, 11-17 and 11-18 against HL-60 and its multidrug resistant derivative HL-60/MX2. Mitoxantrone, the topoisomerase II targeting agent was included as a control.
  • RPMI 8226 (ATCC, CCL- 155), the human multiple myeloma cell line, was cultured in RPMI 1640 medium supplemented with 2 mM L-glutamine, 100 units/ml penicillin, 100 ⁇ g/ml streptomycin, 1 mM sodium pyruvate and 10% heat inactivated fetal bovine serum at 37°C, 5% CO 2 and 95% humidified air.
  • test compounds prepared in DMSO were appropriately diluted in culture medium and added to 2.5xl0 5 /ml RMPI 8226 cells.
  • the final test concentrations ranged from 1 nM to 100 nM.
  • Formula 11-17, Formula 11-20 and Omuralide (Calbiochem, San Diego, CA) the final test concentrations ranged from 1 nM to 10 ⁇ M.
  • DMSO was used as the vehicle control at a final concentration of 0.1%.
  • the cells were pelleted by centrifugation at 2,000 rpm for 10 sec at room temperature and washed 3X with ice-cold IX Dulbecco's Phosphate-Buffered Saline (DPBS, Mediatech, Herndon, VA).
  • the chymotrypsin-like activity of the 2OS proteasome was measured by using the Suc-LLVY-AMC fluorogenic peptide substrate (Boston Biochem, Cambridge, MA) in the proteasome assay buffer (20 mM HEPES, 0.5 mM EDTA, pH 8.0) containing a final concentration of 0.035% SDS.
  • the reactions were initiated by the addition of 10 ⁇ L of 0.4 mM Suc-LLVY-AMC (prepared by diluting a 10 mM solution of the peptide in DMSO 1 :25 with assay buffer) to 190 ⁇ L of the cell lysates and incubated in the Thermo Lab Systems Fluoroskan plate reader at 37°C.
  • the assay was performed in a microtiter plate (Corning 3904) and followed kinetically with measurements every five minutes for 2hr.
  • the total amount of protein used for each assay was 20 ⁇ g.
  • the final concentration of Suc-LLVY-AMC and DMSO was 20 ⁇ M and 0.2%, respectively. Results are presented as the percent inhibition of the 2OS proteasome chymotrypsin-like activity relative to the DMSO control.
  • Results in Table 16 show that exposure of RPMI 8226 cells to Formula 11-16, Formula 11-17, Formula 11-20 and Omuralide resulted in inhibition of the chymotrypsin-like activity of the 2OS proteasomes.
  • Formula 11-16 inhibits 85 ⁇ 7 % of the chymotrypsin-like activity of the 2OS proteasome at 5 nM.
  • Formula II- 16 is able to completely inhibit the chymotrypsin-like activity of the 2OS proteasome.
  • Formula 11-17, Formula 11-20 and Omuralide are only able to inhibit the chymotrypsin-like activity at 30 ⁇ 4 %, 66 ⁇ 3 % and 32 ⁇ 8 %, respectively.
  • PC-3 (ATCC, CRL-1435), the human prostate cancer cell line, was cultured in F12K medium supplemented with 2 mM L-glutamine, 100 units/ml penicillin, 100 ⁇ g/ml streptomycin and 10% heat inactivated fetal bovine serum at 37°C, 5% CO 2 and 95% humidified air.
  • test compounds prepared in DMSO were appropriately diluted in culture medium and added to 1.25xl0 5 /ml PC-3 cells.
  • the final test concentrations ranged from 1 nM to 50 nM.
  • Formula 11-17 Formula 11-20 and Omuralide (Calbiochem, San Diego, CA)
  • the final test concentrations ranged from 1 nM to 10 ⁇ M.
  • DMSO was used as the vehicle control at a final concentration of 0.1%.
  • the cells were washed 3X with ice-cold IX Dulbecco's Phosphate-Buffered Saline (DPBS, Mediatech, Herndon, VA).
  • the chymotrypsin-like activity of the 2OS proteasome was measured by using the Suc-LLVY-AMC fluorogenic peptide substrate (Boston Biochem, Cambridge, MA) in the proteasome assay buffer (20 mM HEPES, 0.5 mM EDTA, pH 8.0) containing a final concentration of 0.035% SDS.
  • the reactions were initiated by the addition of 10 ⁇ L of 0.4 mM Suc-LLVY-AMC (prepared by diluting a 10 mM solution of the peptide in DMSO 1 :25 with assay buffer) to 190 ⁇ L of the cell lysates and incubated in the Thermo Lab Systems Fluoroskan plate reader at 37°C.
  • the assay was performed in a microtiter plate (Corning 3904) and followed kinetically with measurements every five minutes for 2hr.
  • the total amount of protein used for each assay was 20 ⁇ g.
  • the final concentration of Suc-LLVY-AMC and DMSO was 20 ⁇ M and 0.2%, respectively. Results are presented as the percent inhibition of the 2OS proteasome chymotrypsin-like activity relative to the DMSO control.
  • Results in Table 17 show that exposure of PC-3 cells to Formula II- 16, Formula 11-17, Formula 13-20 and Omuralide resulted in inhibition of the chymotrypsin- like activity of the 2OS proteasomes similar to results obtained from RPMI 8226 cell- based experiments.
  • Formula 11-16 inhibits 69% of the chymotrypsin-like activity of the 2OS proteasome at 5 nM.
  • Formula II- 16 is able to completely inhibit the chymotrypsin-like activity of the 2OS proteasome.
  • Formula 11-17, Formula II- 20 and Omuralid ⁇ inhibit the chymotrypsin-like. activity at 26%, 57% and 36%, respectively.
  • RPMI 8226 CCL-155
  • HT-29 HTB-38
  • B16-F10 CTL-6475 cells
  • RPMI 8226 cells were maintained in RPMI 1640 media supplemented with 10%(v/v) FBS, 2mM L-glutamine, ImM sodium pyruvate and Penicillin/Streptomycin at 100 IU/ml and lOO ⁇ g/ml, respectively.
  • HT-29 cells were maintained in McCoys 5 A supplemented with 10%(v/v) FBS, 2mM L-glutamine, ImM sodium pyruvate, l%(v/v) non-essential amino acids, 1OmM HEPES and Penicillin/Streptomycin at 100 IU/ml and lOO ⁇ g/ml, respectively.
  • B16-F10 cells were maintained in DMEM supplemented with 10%(v/v) FBS, 2mM L-glutamine, 1OmM HEPES and Penicillin/Streptomycin at 100 IU/ml and lOO ⁇ g/ml, respectively. The cells were cultured in an incubator at 37 0 C in 5% CO 2 and 95% humidified air.
  • HT-29 and B16-F10 cells were seeded 5x10 3 , and 1.25x10 3 cells/ well respectively in 90 ⁇ l media containing 10%(v/v) FBS or l%(v/v) FBS into 96 well (Corning; 3904) black-walled, clear-bottom tissue culture plates. The plates were incubated overnight to allow cells to establish and enter log phase growth. RPMl 8226 cells were seeded at 2x10 4 cells/well in 90 ⁇ l RPMI media containing 10%(v/v) FBS or l%(v/v) FBS into 96 well black- walled, clear-bottom tissue culture plates.
  • 2OmM stock solutions of Formulae 11-16, 11-17 and Formula 11-18 were prepared in 100% DMSO, aliquoted and stored at -8O 0 C.
  • Formulae 11-16, 11-17 and Formula 11-18 were serially diluted in media containing 1% or 10% FBS and added in triplicate to the test wells.
  • the final concentration of Formula 11-16 ranged from 2 ⁇ M to 20OpM.
  • the final concentration range of Formula 11-17 was from 20 ⁇ M to 6.3nM.
  • the final concentration of Formula 11-18 ranged from 2 ⁇ M to 63OpM.
  • the plates were returned to the incubator for 48 hours.
  • the final concentration of DMSO was 0.25% in all samples.
  • the data were normalized to the average fluorescence of the cells treated with media + 0.25% DMSO (100% cell growth) and EC 50 values (the drug concentration at which 50% of the maximal observed growth inhibition is established) were determined using a standard sigmoidal dose response curve fitting algorithm (generated by XLfit 3.0, ID Business Solutions Ltd).

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Abstract

On décrit des procédés pour traiter le cancer du poumon comprenant l'administration à l'animal d'une quantité thérapeutiquement efficace d'un composé hétérocyclique, seul ou combiné à une autre substance thérapeutique.
PCT/US2007/010540 2006-05-03 2007-05-02 Procédés d'utilisation de composés hétérocycliques [3.2.0] et d'analogues de ceux-ci pour le traitement du cancer du poumon WO2007130404A1 (fr)

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WO2008124699A1 (fr) * 2007-04-06 2008-10-16 Nereus Pharmaceuticals, Inc. Procédé d'utilisation d'inhibiteurs du protéasome en combinaison avec des inhibiteurs d'histone désacétylase pour traiter un cancer
WO2009140287A1 (fr) * 2008-05-12 2009-11-19 Nereus Pharmaceuticals, Inc. Dérivés de salinosporamide à utiliser en tant qu'inhibiteurs des protéasomes
WO2010070365A1 (fr) 2008-12-18 2010-06-24 Bioblocks Magyarország Gyógyszerkémiai És Fejlesztö Kft. Hétérocycles-1,3 condensés avec un squelette monoterpénique, utilisation de ceux-ci et compositions pharmaceutiques comprenant de tels composés
US8637565B2 (en) 2002-06-24 2014-01-28 The Regents Of The University Of California Salinosporamides and methods for use thereof
US8722724B2 (en) 2004-12-03 2014-05-13 Triphase Research And Development I Corp. Compositions and methods for treating neoplastic diseases
US8986971B2 (en) 2006-09-22 2015-03-24 Triphase Research And Development I Corp. Salt formulations for the fermentation of marine microorganisms
US10703760B2 (en) 2016-08-19 2020-07-07 Celgene International Ii Sàrl Morphic forms of marizomib and uses thereof
US11980606B2 (en) 2016-06-01 2024-05-14 Celgene International Ii Sàrl Use of marizomib for the treatment of central nervous system (CNS) cancers

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006060809A2 (fr) * 2004-12-03 2006-06-08 Nereus Pharmaceuticals, Inc. Procedes d'utilisation de composes heterocycliques [3.2.0] et d'analogues de ces derniers
WO2007030662A1 (fr) * 2005-09-09 2007-03-15 Nereus Pharmaceuticals, Inc. Biosynthese et procedes de production de la salinosporamide a et de ses analogues

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Publication number Priority date Publication date Assignee Title
WO2006060809A2 (fr) * 2004-12-03 2006-06-08 Nereus Pharmaceuticals, Inc. Procedes d'utilisation de composes heterocycliques [3.2.0] et d'analogues de ces derniers
WO2007030662A1 (fr) * 2005-09-09 2007-03-15 Nereus Pharmaceuticals, Inc. Biosynthese et procedes de production de la salinosporamide a et de ses analogues

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* Cited by examiner, † Cited by third party
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US9713607B2 (en) 2002-06-24 2017-07-25 The Regents Of The University Of California Salinosporamides and methods of use thereof
US10314818B2 (en) 2002-06-24 2019-06-11 The Regents Of The University Of California Salinosporamides and methods of use thereof
US10912764B2 (en) 2002-06-24 2021-02-09 The Regents Of The University Of California Salinosporamides and methods of use thereof
US9078881B2 (en) 2002-06-24 2015-07-14 The Regents Of The University Of California Salinosporamides and methods of use thereof
US8637565B2 (en) 2002-06-24 2014-01-28 The Regents Of The University Of California Salinosporamides and methods for use thereof
US8722724B2 (en) 2004-12-03 2014-05-13 Triphase Research And Development I Corp. Compositions and methods for treating neoplastic diseases
US10610517B2 (en) 2004-12-03 2020-04-07 Celgene International Ii Sàrl Compositions and methods for treating neoplastic diseases
US8986971B2 (en) 2006-09-22 2015-03-24 Triphase Research And Development I Corp. Salt formulations for the fermentation of marine microorganisms
US10011814B2 (en) 2006-09-22 2018-07-03 Celgene International Ii Sàrl Salt formulations for the fermentation of marine microorganisms
US10793824B2 (en) 2006-09-22 2020-10-06 Celgene International II Sárl Salt formulations for the fermentation of marine microorganisms
WO2008124699A1 (fr) * 2007-04-06 2008-10-16 Nereus Pharmaceuticals, Inc. Procédé d'utilisation d'inhibiteurs du protéasome en combinaison avec des inhibiteurs d'histone désacétylase pour traiter un cancer
WO2009140287A1 (fr) * 2008-05-12 2009-11-19 Nereus Pharmaceuticals, Inc. Dérivés de salinosporamide à utiliser en tant qu'inhibiteurs des protéasomes
US7910616B2 (en) 2008-05-12 2011-03-22 Nereus Pharmaceuticals, Inc. Proteasome inhibitors
WO2010070365A1 (fr) 2008-12-18 2010-06-24 Bioblocks Magyarország Gyógyszerkémiai És Fejlesztö Kft. Hétérocycles-1,3 condensés avec un squelette monoterpénique, utilisation de ceux-ci et compositions pharmaceutiques comprenant de tels composés
US11980606B2 (en) 2016-06-01 2024-05-14 Celgene International Ii Sàrl Use of marizomib for the treatment of central nervous system (CNS) cancers
US10703760B2 (en) 2016-08-19 2020-07-07 Celgene International Ii Sàrl Morphic forms of marizomib and uses thereof
US11136332B2 (en) 2016-08-19 2021-10-05 Celgene International Ii Sàrl Morphic forms of marizomib and uses thereof

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