BRPI0517134A2 - methods of using heterocyclic compounds [3.2.0] and their analogues - Google Patents

Abstract

METHODS FOR THE USE OF HETEROCYCLICAL COMPOUNDS [3.2.0) AND THEIR ANALOGS. Methods for treating cancer, inflammatory conditions, and / or infectious diseases in an animal are described, comprising: administering to the animal a therapeutically effective amount of a heterocyclic compound. The animal is a mammal, preferably a human or a rodent.

Description

"METHODS OF USE OF HETEROCYCLIC COMPOUNDS [3.2.0] AND ANALOGS"

Patent Related Information

This document claims priority under 35 U.S.C. § 119 (e) for U.S. Provisional Applications No. 60 / 633,379, filed December 3, 2004; 60 / 643,922, filed January 13, 2005; 60/658,884, filed March 4, 2005; and 60 / 676,533, filed April 29, 2005 which are entitled METHODS OF USE OF HETEROCYCLIC COMPOUNDS [3.2.0] AND ANALOGS OF THEM; each of which is incorporated herein by reference in its entirety.

Field of the Invention

The present invention relates to certain compounds and methods for the preparation and use of certain compounds in the fields of chemistry and medicine. Embodiments of the invention described herein relate to methods of using heterocyclic compounds. In some embodiments, the compounds are used as proteasome inhibitors. In other embodiments, the compounds are used to treat inflammation, cancer, and infectious diseases.

Description of Related Art

Cancer is the leading cause of death in the United States. Despite significant efforts to find new approaches to treating cancer, primary treatment options remain, such as surgery, chemotherapy, and radiation therapy, both alone and in combination. However, surgery and radiation therapy are generally only useful for very defined cancers, and are of limited use to treat patients with disseminated disease. Chemotherapy is the method that is generally useful for treating patients with metastatic cancer or diffuse cancers such as leukemias. Although chemotherapy may provide a therapeutic benefit, it often does not result in cure of the disease due to the patient's cancer cells that become 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. Similarly, infectious diseases caused by, for example, bacteria, fungi and protozoa are becoming increasingly difficult to treat and cure. For example, more and more bacteria, fungi and protozoans are developing resistance to current antibiotics and chemotherapeutic agents. Examples of such microbes include Bacilus, Leishmania, Plasmodium and Trypanosoma.

In addition, an increasing number of diseases and medical conditions are classified as inflammatory diseases. Such diseases include conditions ranging from asthma to cardiovascular disease. These diseases continue to affect more people worldwide despite new therapies and medical advances.

Thus, there is a need for chemotherapeutic antimicrobial agents, and additional anti-inflammatory agents, to treat cancer, inflammatory diseases and infectious diseases. Continued efforts are being made by individual researchers, gyms and companies to identify new and potentially useful chemotherapies and antimicrobial agents.

Natural marine derived products are a rich source of new anti-cancer agents and potential anti-microbial agents. Oceans are massively complex and host a diverse array of microbes that occur in environments with extreme variations in pressure, salinity, and temperature. Marine microorganisms have developed unique metabolic and physiological capacities that not only guarantee survival in extreme and varied habitats, but also offer the potential to produce metabolites that would not be observed in terrestrial microorganisms (Okami, Y. 1993 J. Mar. Biotechnol. 1:59). Representative structural classes of such metabolites include terpenes, peptides, polychacides, and compounds with mixed biosynthetic origins. Many of these molecules have been shown to have anti-tumor, anti-bacterial, anti-fungal, anti-inflammatory or immunosuppressive activities (Bull, AT et al., 2000 35 Microbiol. Mol. Biol. Rev. 64: 573; Cragg, GM & DJ Newman, 2002 Trends Pharmacol Sci 23: 404; Kerr, RG & SS Kerr, 1999 Exp. Opinion Ther. Patents 9: 1207; Moore, BS, 1999 Nat. Prod. Rep. 16: 653; Faulkner , DJ, 2001 Nat. Prod. Rep. 18: 1; Mayer, AM & VK Lehmann, 2001 Anticancer Res. 21: 2489), validating the usefulness of this source for isolating invaluable therapeutic agents. Further isolation of new anti-cancer and antimicrobial agents that represent alternative mechanistic classes from those currently on the market will help with resistance, including any mechanism-based resistance that may have been created in pathogens for bio purposes. -terrorism.

Summary of the Invention

Embodiments described herein generally relate to chemical compounds, including heterocyclic compounds and analogs thereof. Some forms of incorporation are directed to the use of compounds as proteasome inhibitors.

In other embodiments, the compounds are used to treat neoplastic diseases, for example to inhibit the growth of tumors, cancers and other neoplastic tissues. The treatment methods described herein may be employed with any patient suspected of having tumoral, cancerous, or other neoplastic, benign or malignant growth ("tumor" or "tumors" as used herein encompasses tumors, cancers, disseminated neoplastic cells and localized neoplastic growths). Examples of such growths include but are not limited to breast cancers; osteosarcomas, angiosarcomas, fibrosarcomas and other sarcomas; leukemias; sinus tumors; ovarian, uretal, bladder, prostate and other genitourinary cancers; colon, esophageal and stomach cancers, and other gastrointestinal cancers; lung cancers; lymphomas; myelomas; pancreatic cancers; liver cancers; kidney cancers; endocrine cancers; skin cancers; melanomas; angiomas; and cancers of the brain or central nervous system (CNS; glioma). In general, the tumor or growth to be treated can be any primary or secondary tumor or cancer. Certain forms of incorporation relate to methods for treating neoplastic diseases in animals. The method may include, for example, administering an effective amount of a compound to a patient in need. Other embodiments relate to the use of compounds in the manufacture of a pharmaceutical or medicament for treating a neoplastic disease.

The compounds may be administered or used in combination with treatments such as chemotherapy and biological and radiation therapies. In some embodiments the compounds may be administered or used with a chemotherapeutic agent. Examples of such chemotherapeutic agents include alkaloids, alkylating agents, antibiotics, anti-metabolites, enzymes, hormones, platinum compounds, immunotherapeutics (antibodies, T cells, epitopes), BRMs, and the like. Examples include Vincristine, Vinblastine, Vindesine, Paclitaxel (Taxol), Docetaxel, Epipodophyllotoxins, Topisomerase Inhibitors (Etoposide (VP - 16), Teniposide (VM-26)), Camptothecin, Nitrogen Mustards (Cyclophosphamide), Nitrosoureas, , lomustine, dacarbazine, hydroxymethylmelamine, thiotepa and mitocycin C, Dactinomycin (Actinomycin D), anthracycline antibiotics (Daunorubicin, Daunomycin, Cerubidine), Doxorubicin

(Adriamycin), Idarubicin (Idamycin), Anthracenediones

(Mitoxantrone), Bleomycin (Blenoxane), Plicamycin (Mitramycin), Antifolates (Methotrexate (Folex, Mexato)), Purine anti-metabolites (6-mercaptopurine (6-MP, Purinetol) and 6-thioguanine (6-TG). The two main anti-cancer drugs in this category are 6-mercaptopurine and 6-thioguanine, Chloro-deoxyadenosine and Pentostatin, Pentostatin (2'-deoxy-coformycin), pyrimidine antagonists, fluoropyrimidines (5-fluorouracil (Adrucil ), 5-Fluoro-deoxyuridine (FdUrd) (Floxuridine), Cytosine Arabinoside (Cytosar, ara-C), Fludarabine, L-Asparaginase, Hydroxyurea, Glucocorticoids, Antiestrogens, Tamoxifen, Non-Steroidal Anti-Androgens , flutamide, aromatase inhibitors Anastrozole (Arimidex), Cisplatin, 6-Mercaptopurine and Thioguanine, Methotrexate, Cytoxane, Cytarabine, L-Asparaginase, Steroids: Prednisone and Dexamethasone. examples, for example. may include agents such as screening antibodies, integrins such as alpha-V-beta-3 (ανβ3) and / or other cytokine / growth factors that are involved in angiogenesis, VEGF, VEGF, FCF and PDGF. In some aspects, the compounds may be conjugated to, or may be distributed with, an antibody. The combination methods described above may be used to treat a variety of conditions, including cancer and neoplastic disease, inflammation, and microbial infections.

In still other embodiments, the compounds are used to treat inflammatory conditions. Certain forms of incorporation relate to. methods for treating inflammatory conditions in animals. The method may include, for example, administering an effective amount of a compound to a patient in need. Other embodiments relate to the use of compounds in the manufacture of a pharmaceutical or medicament for treating inflammation.

In certain embodiments, the compounds are used to treat infectious diseases. The infectious agent may be a microbe, for example bacteria, fungi, protozoa, and microscopic algae, or viruses. Further, the infectious agent may be B. Anthracis (anthrax). In some embodiments the infectious agent is a parasite. For example, the infectious agent may be Plasmodium, Leishmania, and Trypanosoma. Certain forms of incorporation relate to methods for treating infectious agents in animals. The method may include, for example, administering an effective amount of a compound to a patient in need. Other embodiments relate to the use of compounds in the manufacture of a pharmaceutical or medicament for treating infectious agents.

Some embodiments relate to uses of a compound having the structure of formula I, and related pharmaceutically acceptable prodrug salts and esters:

<formula> formula see original document page 6 </formula>

Formula I

where the dotted lines represent a single or double bond, where R1 may be selected separately 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 C 2 -C 24 alkenyl or C 2 -C 24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonyl acyl, amino, aminocarbonyl, aminocarbonoxy, nitro, azido, phenyl, cycloalkylacyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, thio, sulfoxide, sulfone, esters sulfonate, thio cyano, boronic acids and esters, and halogenated alkyl including polyhalogenated alkyl, where η is 1 or 2, and if η is 2, then R 1 may be the same or different;

where R2 may be selected from the group consisting of hydrogen, a halogen, mono-substituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, C2-C24 alkenyl or unsaturated C2-C24 alkynyl , acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl (including, for example, cyclohexylcarbinol), alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonyl -acyl, amino, amino-carbonyl, amino-carboxy, nitro, azido, phenyl, cycloalkyl-acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl;

where R3 may be selected from the group consisting of a halogen, mono-substituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, C2-C24 alkenyl or unsaturated C2-C24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonoxy, nitro azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl; and wherein each E1, E2, E3 and E4 is a substituted or unsubstituted heteroatom; in the treatment of cancer, inflammation, and infectious diseases.

Preferably, in some embodiments, R1 is a substituted or unsubstituted C1-C5 alkyl. For example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, and pentyl are preferred. In some embodiments, R1 is not an unsubstituted, substituted or unsubstituted C6 alkyl.

Other forms of incorporation relate to methods for treating a neoplastic disease in an animal. For example, the methods may include administering to the animal a therapeutically effective amount of a compound of a formula selected from Formulas I-VI7 and related pharmaceutically acceptable prodrug salts and esters.

Further embodiments relate to pharmaceutical compositions which include a compound of a formula selected from Formulas I-VI. The pharmaceutical compositions may additionally include an antimicrobial agent.

Further embodiments further relate to methods for inhibiting the growth of a cancer cell. For example, the methods may include contacting a cancer cell with a compound of a formula selected from Formulas I-VI, and related pharmaceutically acceptable prodrug salts and esters.

Other embodiments relate to methods for inhibiting proteasome activity including the step of contacting a cell with a compound of a formula selected from Formulas I-VI, and related pharmaceutically acceptable prodrug salts and esters.

Other forms of incorporation relate to methods for inhibiting activation of nuclear kappa factor B (FN-kB) including the step of contacting a cell with a compound of a formula selected from Formulas I-VI, and salts and esters pro related pharmaceutically acceptable drugs.

Some embodiments relate to methods for treating an inflammatory condition, including administering an effective amount of a compound of a formula I selected from Formula I-VI to a patient in need.

Further embodiments relate to methods for treating a microbial disease including administering an effective amount of a compound of a formula selected from Formulas I-VI to a patient in need.

Brief Description of the Drawings

The accompanying drawings, which are incorporated herein and form part of this specification, merely illustrate certain preferred embodiments of the present invention. Along with the rest of the description, they serve to explain to those skilled in the art preferred methods for making certain compounds of the invention. In the drawings:

Fig. 1 shows the chemical structure of Salinosporamide A.

Fig. 2 shows the pan-tropical distribution of Salinospora. "X" denotes Salinospora collection locations.

Fig. 3 shows Salinospora colonies.

Fig. 4 shows the typical Salinospora 16S rDNA sequence. The bars represent Salinospora's signature nucleotides that separate them from their closest relatives.

Fig. 5 shows Omuralide, a breakdown product of the Lactacystine microbial metabolite. Also shown is a compound of Formula 11-16, also called Salinosporamide A.

Fig. 6 illustrates macrophage cytotoxicity mediated by a lethal toxin. NPI-0052 represents the compound of Formula 11-16.

Fig. 7 depicts the 1 H NMR spectrum of a structure having the combination of Formula 11-20.

Fig. 8 describes the 1H NMR spectrum of a compound having the structure of Formula II-24C.

Fig. 9 depicts the 1 H NMR spectrum of a compound having the structure of Formula 11-19.

Fig. 10 depicts the 1 H NMR spectrum of a compound having the structure of Formula II-2. Fig. 11 describes the mass spectrum of a compound having the structure of Formula II-2.

Fig. 12 describes the 1 H NMR spectrum of a compound having the structure of Formula II-3.

Fig. 13 describes the mass spectrum of a compound having the structure of Formula II-3.

Fig. 14 describes 1 H NMR spectrum of a compound having the structure of Formula II-4.

Fig. 15 describes the mass spectrum of a compound having the structure of Formula II-4.

Fig. 16 describes 1H NMR spectrum of a compound having the structure of Formula II-5A.

Fig. 17 describes the mass spectrum of a compound having the structure of Formula II-5A.

Fig. 18 describes the 1 H NMR spectrum of a compound having the structure of Formula II-5B.

Fig. 19 describes the mass spectrum of a compound having the structure of Formula II-5B.

Fig. 20 describes the 1H NMR spectrum of a compound having the structure of Formula IV-3C in DMSO-d6.

Fig. 21 describes the 1H NMR spectrum of a compound having the structure of Formula IV-3C in C6D6 / DMSO-d6.

Fig. 22 describes the 1 H NMR spectrum of a compound having the structure of Formula II-13C.

Fig. 23 describes the 1H NMR spectrum of a compound having the structure of Formula II-8C.

Fig. 24 describes the 1 H NMR spectrum of a compound having the structure of Formula 11-25.

Fig. 25 describes the 1H NMR spectrum of a compound having the structure of Formula 11-21.

Fig. 26 describes the 1 H NMR spectrum of a compound having the structure of Formula 11-22.

Fig. 27 shows inhibition of chymotrypsin-like activity of rabbit muscle proteasomes.

Fig. 28 shows inhibition of caspase-like and HPPG-like activity of rabbit muscle proteasomes. Fig. 29 shows inhibition of chymotrypsin-like activity of human erythrocyte proteasomes.

Fig. 30 shows the effect of 11-16 treatment on chymotrypsin-mediated division of LLVY-AMC substrate.

Fig. 31 shows luciferase / nuclear kappa factor B (FN-κΒ) activity and cytotoxicity profiles of 11-16.

Fig. 32 shows a reduction in ΙκΒα degradation and retention of 11-16 phosphorylated ΙκΒα in HEK293 (A) cells and in the FN-KB / Luciferase reporting clone in HEK293 (B).

Fig. 33 shows the accumulation of cell cycle regulatory proteins, p21 and p27, by treatment with II-16 in HEK293 (A) cells and in the FN-KB / Luciferase reporting clone in HEK293 (B).

Fig. 34 shows the activation of Caspase-3 by II-16 in Jurkat cells.

Fig. 35 shows the division of PARP by 11-16 into Jurkat cells.

Fig. 36 shows TxLe-induced inhibition of cytotoxicity by 11-16 in RAW264.7 cells.

Fig. 37 shows the effects of II-16 treatment on PARP and Pro-Caspase 3 division in RPMI8226 and PC-3 cells.

Fig. 38 shows the results of treatment with RPMI 8226 11-16 in a dose dependent division 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 11-16 treated rats.

Fig. 41 shows epoxomycin treatment in the PWBL assay.

Fig. 42 shows the comparison of intra-assays.

Fig. 43 shows decreased plasma TNF levels in LPS-treated rats.

Fig. 44 describes the results of an assay showing the effect of Formula II-2, Formula II-3 and Formula ΙΙ-4 on FN-κΒ-mediated luciferase activity in FN-κΒ / Luc HEK293 cells.

Fig. 45 describes the results of an assay showing the effect of Formula II-5A and Formula II-5B on FN-κΒ-mediated luciferase activity mediated in FN-κΒ / Luc HEK293 cells.

Fig. 46 describes the results of an assay showing the effect of Formula II-2, Formula II-3, and Formula II-4 on rabbit 20S proteasome chymotrypsin-like activity.

Fig. 47 describes the effect of Formula II-5A and Formula II-5B on rabbit 20S proteasome chymotrypsin-like activity.

Fig. 48 describes the effect of Formulas II-2, II-3, and II-4 against TxLe mediated cytotoxicity.

Fig. 49 describes the 1 H NMR spectrum of a compound having the structure of Formula 11-17.

Fig. 50 describes the 1 H NMR spectrum of a compound having the structure of Formula 11-18.

Fig. 51 describes the 1H NMR spectrum of a compound having the structure of Formula 11-26 in DMSO-d6.

Fig. 52 describes the computer generated ORTEP curve of the compound of Formula 11-26.

Fig. 53 describes the 1 H NMR spectrum of a compound having the structure of Formula 11-27 in DMS0-d6.

Fig. 54 describes the 1H NMR spectrum of a compound having the structure of Formula 11-28 in DMS0-d6.

Fig. 55 describes the 1H NMR spectrum of a compound having the structure of Formula 11-29 in DMS0-d6.

FIG. 56 describes the 1H NMR spectrum of a compound having the structure of Formula 11-30

Fig. 57 describes the 1H NMR spectrum of a compound having the structure of Formula 11-44 in DMS0-d6.

Fig. 58 describes the 1H NMR spectrum of a compound having the structure of Formula 1-7 in DMS0-d6.

Fig. 59 describes the 1H NMR spectrum of a compound having the structure of Formula 11-47 in DMS0-d6. Fig. 60 describes the 1H NMR spectrum of a compound having the structure of Formula 11-38 in DMSO-d6.

Fig. 61 describes the 1H NMR spectrum of a compound having the structure of Formula 11-50 in DMSO-d6.

Detailed Description of Incorporation Form

Preferred

Numerous references are cited here. Cited references, including U.S. patents cited herein, are to be incorporated by reference in their entirety in this specification.

Embodiments of the invention include, but are not limited to, providing a method for the preparation of compounds, including compounds, for example, described herein and analogs thereof, and providing a method for producing antimicrobial, anticancer, and pharmaceutically acceptable antiinflammatory drugs, for example. The methods may include compositions in relatively high yield, where the compounds and / or derivatives thereof are among the active ingredients in these compositions. Other embodiments relate to providing new compounds not obtained by the currently available methods. In addition, forms of incorporation relate to methods for treating cancer, inflammation, and infectious diseases, particularly those affecting humans. In some embodiments, one or more Formulas, one or more compounds, or groups of compounds may be specifically excluded from use in any one or more of the methods for treating the conditions described herein. As an illustrative example, the compounds of Formula 11-16 may be excluded in some embodiments of methods for 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 for making and using such compounds described herein, but not necessarily in all embodiments of the present invention, these objects are achieved.

For the compounds described herein, each stereogenic carbon may be of R or S configuration. While specific compounds exemplified in this application may be described in a particular configuration, compounds having stereo-chemistry opposite any given chiral center or related mixtures are also targeted. . When chiral centers are found in the derivatives of this invention, it should be understood that the compounds encompass all possible stereoisomers.

Formula I Compounds

Some embodiments provide compounds and methods for producing a class of related pharmaceutically acceptable prodrug compounds, salts and esters, wherein the compounds are represented by Formula I:

<formula> formula see original document page 14 </formula>

In certain embodiments the substituent (s) R1, R2, and R3 may separately include a hydrogen, a halogen, a monosubstituted, a polysubstituted or unsubstituted variant of the following residues: C1-6 alkyl Unsaturated C24-C24 alkenyl or unsaturated C2-C24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl (including for example cyclohexylcarbinol), cycloalkenyl, alkoxy, cycloalkyl alkoxy, aryl, heteroaryl, aryl-alkoxycarbonyl, alkoxycarbonyl-acyl, amino, aminocarbonyl, amino-carboxy, nitro, azido, phenyl, cycloalkyl-acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, thio, sulfoxide, sulfone, sulfonate esters, thio cyano, boronic acids and esters, and halogenated alkyl including polyhalogenated alkyl. Further, in certain embodiments, each E1, E2, E3 and E4 may be a substituted or unsubstituted heteroatom, for example, a heteroatom selected separately from the group consisting of nitrogen, sulfur and oxygen.

In some embodiments η may be 1 or 2. When η is 2, the substituents may be the same or different. Also, in some embodiments R3 is not a hydrogen.

Preferably, in some embodiments, R1 is a substituted or unsubstituted C1-C5 alkyl. For example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, and pentyl are preferred. In some embodiments, R1 is not a substituted or unsubstituted unbranched C6 alkyl.

In some embodiments, R 2 is not carbinol cyclohex-2-enyl when one of the substituents on R 1 is ethyl or chloroethyl and R 3 is methyl.

Preferably R2 may be a formyl. For example, the compound may have the following structure 1-1:

<formula> formula see original document page 15 </formula>

Formula I-I

R 8 may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.

Preferably, the structure of Formula I-I may have the following stereo-chemical:

<formula> formula see original document page 15 </formula>

R 8 may include, for example, hydrogen, fluorine, chlorine, bromine and iodine. Preferably R2 may be a carbinol. For example, the compound may have the following structure 1-2:

<formula> formula see original document page 16 </formula>

Formula 1-2

R 8 may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.

As an example, the structure of Formula 1-2 may have the following stereo chemical:

<formula> formula see original document page 16 </formula>

R 8 may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.

An exemplary compound of Formula I may be the compound having the following structure 1-3:

<formula> formula see original document page 16 </formula>

Formula 1-3 R8 may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.

The compound of Formula 1-3 may have the following stereo-chemical structure:

<formula> formula see original document page 17 </formula>

R 8 may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.

Another exemplary compound of Formula I may be the compound having the following structure 1-4:

<formula> formula see original document page 17 </formula>

Formula 1-4

<formula> formula see original document page 17 </formula>

R8 may include, for example, hydrogen,

fluorine, chlorine, bromine and iodine.

Preferably, the compound of Formula 1-4 may be

have the following stereo-chemical structure: <formula> formula see original document page 18 </formula>

R 8 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:

<formula> formula see original document page 18 </formula>

Formula 1-5

R8 may include, for example, hydrogen,

fluorine, chlorine, bromine and iodine.

For example, the compound of Formula 1-5 may have the following stereo chemical:

<formula> formula see original document page 18 </formula> R8 may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.

In some embodiments, R 2 of Formula I may be, for example, a cyclohex-2-enyl denemethyl. For example, the compound may have the following structure of Formula 1-6:

<formula> formula see original document page 19 </formula>

Formula 1-6

R 8 may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.

Preferably, the compound of Formula 1-6 may have the following stereo chemical:

<formula> formula see original document page 19 </formula>

R 8 may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.

In further embodiments, R 2 of Formula I may be, for example, a cyclohex-2-enyl methyl. For example, the compound may have the following structure of Formula 1-7: <formula> formula see original document page 20 </formula>

Formula 1-7

R 8 may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.

Preferably, the compound of Formula 1-7 may have the following stereo chemical:

<formula> formula see original document page 20 </formula>

R 8 may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.

In other embodiments, R 2 may be a cyclohexylalkylamine.

Also, in other embodiments, R 2 may be a C-Cyclohexyl methylene amine. In others, R 2 may be a cyclohexanecarbaldehyde O-oxime.

Further, in some embodiments, R 2 may be a cycloalkyl acyl.

Compounds of Formula II Other embodiments provide compounds, and methods for producing a class of related pharmaceutically acceptable prodrug compounds, salts and esters, wherein the compounds are represented by Formula II:

<formula> formula see original document page 21 </formula>

Formula II

In certain embodiments the substituent (s) R1, R3, and R4 may separately include a hydrogen, a halogen, a monosubstituted, a polysubstituted or unsubstituted variant of the following residues: C1-6 alkyl Unsaturated -C24 saturated, C2 -C24 alkenyl or C2 -C24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, aryl -alkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonoxy, nitro, azido, phenyl, cycloalkylacyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, thio, sulfoxide sulfone, sulfonate esters, thio cyano, boronic acids and esters, and halogenated alkyl including polyhalogenated alkyl. Further, in certain embodiments, each E1, E2, E3 and E4 may be a substituted or unsubstituted heteroatom, for example a substituted heteroatom or atom selected from the group consisting of nitrogen, sulfur and oxygen.

In some embodiments η may be 1 while in others it may be 2. When η is 2, the substituents may be the same or may be different. Also, in some embodiments R 3 is not a hydrogen, m may be 1 or 2, and when m is 2, R 4 may be the same or different.

E5 may be, for example, OH, O, OR10, S, SRn, SO2 Rn, NH, NH2, NOH, NHOH, NR12, and NHOR13, where R10-13 may separately include, for example, hydrogen, a substituted or unsubstituted. substituted from any of the following: alkyl, aryl, heteroaryl, and the like. Also, R 1 may be CH 2 CH 2 X, where X may be, for example, H, F, Cl, Br, and I. R 3 may be methyl. In addition, R4 may include a cyclohexyl. Also, each E1, E3 and E4 may be 0 and E2 may be NH. Preferably, R 1 may be CH 2 CH 2 X, where X is selected from the group consisting of H, F, Cl Br, and I; where R4 may include a cyclohexyl; where R3 may be methyl; and where each E1, E3 and E4 may be separately 0 and E2 may be NH.

In some embodiments, R 2 is not cyclohex-2-enyl carbinol when one of the substituents R 1 is ethyl or chloroethyl and R 3 is methyl.

Preferably, in some embodiments R1 is a substituted or unsubstituted C1 for C5 alkyl. For example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, and pentyl are preferred. In some embodiments, R1 is not an unsubstituted, substituted or unsubstituted C6 alkyl.

For example, an exemplary compound of Formula II has the following structure II-I:

<formula> formula see original document page 22 </formula>

Formula II-I

R 8 may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.

An exemplary stereo-chemical may be as <formula> formula see original document page 23 </formula>

In preferred embodiments, the compound of Formula II has any of the following structures:

<formula> formula see original document page 23 </formula>

The following exemplary stereo-chemistry for compounds having structures II-2, II-3, and II-4, respectively, is shown below: <formula> formula see original document page 24 </formula>

In other embodiments where R 4 may include a 7-oxa-bicyclo [4.1.0] hepto-2-yl. An exemplary compound of Formula II is the following structure II-5:

<formula> formula see original document page 24 </formula>

Formula II-5

R 8 may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.

The following examples of compounds having the structure of Formula II-5 are presented below: <formula> formula see original document page 25 </formula>

In other additional embodiments, at least one R 4 may include a substituted or unsubstituted branched alkyl. For example, a compound of Formula II may be of the following structure II-6:

<formula> formula see original document page 25 </formula>

Formula II-6

R8 may include, for example, hydrogen,

fluorine, chlorine, bromine and iodine.

The following example is a stereo chemistry for a compound having the structure of Formula II-6: <formula> formula see original document page 26 </formula>

As another example, the compound of Formula II may be of the following structure II-7:

<formula> formula see original document page 26 </formula>

Formula II-7

R 8 may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.

The following exemplary stereo-chemistry for a compound having the structure of Formula II-7 is shown: <formula> formula see original document page 27 </formula>

In other embodiments, at least one R4 may be a cycloalkyl and E5 may be an oxygen. An exemplary compound of Formula II may be the following structure II-8:

Formula II-8

<formula> formula see original document page 27 </formula>

R 8 may include, for example, hydrogen (II-8A), fluorine (II-8B), chlorine (II-8C), bromine (II-8D) and iodine (II-8E).

The following exemplary stereo-chemistry is given for a compound having the structure of Formula II-8: <formula> formula see original document page 28 </formula>

In some embodiments E5 may be an amine oxide, giving rise to an oxime. An exemplary compound of Formula II has the following structure II-9:

<formula> formula see original document page 28 </formula>

Formula II-9

R 8 may include, for example, hydrogen, fluorine, chlorine, bromine and iodine; R may be hydrogen, and a substituted or unsubstituted alkyl, aryl, or heteroaryl, and the like.

The following exemplary stereo-chemistry is given for a compound having the structure of Formula II-9: <formula> formula see original document page 29 </formula>

An additional exemplary compound of Formula II has the following structure 11-10:

<formula> formula see original document page 29 </formula>

R 8 may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.

The following exemplary stereo-chemistry is given for a compound having the structure of Formula 11-10:

<formula> formula see original document page 29 </formula> In some embodiments, E5 may be NH2. An exemplary compound of Formula II has the following structure 11-11:

<formula> formula see original document page 30 </formula>

Formula II-Il

R 8 may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.

The following exemplary stereo-chemistry is given for a compound having the structure of Formula 11-11:

<formula> formula see original document page 30 </formula>

In some embodiments, at least one R 4 may include a cycloalkyl and E5 may be NH 2. An exemplary compound of Formula II has the following structure 11-12: <formula> formula see original document page 31 </formula>

R 8 may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.

The following exemplary stereo-chemistry is given for a compound having the structure of Formula 11-12:

<formula> formula see original document page 31 </formula>

An additional exemplary compound of Formula II has the following structure 11-13: <formula> formula see original document page 32 </formula>

Formula 11-13

R 8 may include, for example, hydrogen (II-13A), fluorine (II-13B), chlorine (II-13C), bromine (II-13D) and iodine (II-13E).

The following exemplary stereo chemistry is given for a compound having the structure of Formula 11-13:

<formula> formula see original document page 32 </formula>

Yet another exemplary compound of Formula II has the following structure 11-14: <formula> formula see original document page 33 </formula>

R 8 may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.

The following exemplary stereo-chemistry is given for a compound having the structure of Formula 11-14:

<formula> formula see original document page 33 </formula>

In some embodiments, compounds of Formula II may include as R 4 at least one cycloalkene, for example. In addition, in some embodiments, the compounds may include a E5 hydroxy, for example. An additional exemplary compound of Formula II has the following structure: 11 <formula> formula see original document page 34 </formula>

Formula 11-15

R 8 may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.

Exemplary stereo chemistry may be as follows:

<formula> formula see original document page 34 </formula>

The following exemplary stereo-chemistry is given for compounds having structures 11-16, 11-17, II-18, and 11-19, respectively: <formula> formula see original document page 35 </formula>

The compounds of Formulas 11-16, 11-17, 11-18 and 11-19 may be obtained below by fermentation, synthesis, or semi-synthesis and isolated / purified as set forth below. In addition, the compounds of Formulas 11-16, 11-17, 11-18 and 11-19 may be used, and are referred to as "starting materials" to make other compounds described herein.

In some embodiments, compounds of Formula II may include a methyl group such as RI, for example. An additional exemplary compound of Formula 11-20 has the following stereo-chemical structure: <formula> formula see original document page 36 </formula>

In some embodiments, compounds of Formula II may include hydroxyethyl such as RI, for example. An additional exemplary compound of Formula 11-21 has the following stereo-chemical structure:

<formula> formula see original document page 36 </formula>

In some embodiments, the hydroxyl group of Formula 11-21 may be esterified such that R 1 may include ethyl propionate, for example. An exemplary compound of Formula 11-22 has the following stereo-chemical structure: <formula> formula see original document page 37 </formula>

In some embodiments, the compounds of Formula II may include an ethyl group such as R 3, for example. An additional exemplary compound of Formula II has the following structure 11-23:

<formula> formula see original document page 37 </formula>

R 8 may include, for example, hydrogen, fluorine, chlorine, bromine and iodine. The exemplary stereo-chemistry can be as follows: <formula> formula see original document page 38 </formula>

In some embodiments, the compounds of Formula 11-23 may have the following stereochemical structure, exemplified by Formula II-24C where R8 is chloro:

<formula> formula see original document page 38 </formula>

In some embodiments, compounds 5 of Formula 11-15 may have the following stereo-chemical, exemplified by the compound of Formula 11-25, where R8 is chlorine: <formula> formula see original document page 39 </formula>

In some embodiments, the compound of Formula 11-15 may have the following stereo chemistry, exemplified by the compound of Formula 11-26, where R 8 is chlorine:

<formula> formula see original document page 39 </formula>

In some embodiments, the compound of Formula II may have the following stereo-chemical structure, exemplified by Formula 11-27, where R 1 is ethyl: In some embodiments, the compound of Formula II may have the following structure and chemical formula, exemplified by Formula 11-28, where R1 is methyl:

<formula> formula see original document page 40 </formula>

In some embodiments, the compounds of Formula II may include azidoethyl such as RI, for example. An additional exemplary compound of Formula 11-29 has the following stereochemical structure:

<formula> formula see original document page 40 </formula>

In some embodiments, compounds of Formula II may include propyl as RI, for example. An additional exemplary compound of Formula 11-30 has the following stereo-chemical structure: <formula> formula see original document page 41 </formula>

Still further exemplary compounds of Formulas 11-31 and 11-32 have the following stereo-chemical structure:

<formula> formula see original document page 41 </formula>

Formulas 11-31 and 11-32

Other exemplary compounds of Formulas 11-33, 11-34, 11-35 and 11-36 have the following stereo-chemical structure: <formula> formula see original document page 42 </formula>

Formulas 11-33 - 11-36

In some embodiments, the compound of Formula II may include cyanoethyl as RI, for example. The compound of Formula 11-37 has the following structure and is stereo-chemical:

<formula> formula see original document page 42 </formula>

In another embodiment, the compound of Formula II may include ethyl thio cyanate such as RI, for example. The compound of Formula 11-38 has the following structure and stereo-chemistry: <formula> formula see original document page 43 </formula>

In some embodiments, compounds of Formula II may include a thiol such as RI, for example. A further exemplary compound of Formula 11-39 has the following stereochemical structure where R = H, alkyl, aryl, 5 or substituted alkyl or aryl:

<formula> formula see original document page 43 </formula>

In a further exemplary compound, the sulfur of the compound of Formula 11-39 may be oxidized to a sulfoxide (n = 1) or a sulfone (n = 2), for example as in the compound of Formula 11-40: <formula> formula see original document page 44 </formula>

In some embodiments, the R 1 substituent of the compound of Formula II may include a residual group, for example a halogen, as in compounds 11-18 or 11-19, or another residual group, such as a sulfonate ester. An example is methane sulfonate (mesylate) of Formula 11-41:

<formula> formula see original document page 44 </formula>

In some embodiments, the R 1 substituent of the compound of Formula II may include electron acceptors. For example, the electron acceptor may be a Lewis acid, such as an ester or boronic acid. An exemplary compound of Formula 11-42 has the following stereo-chemical structure where η = 0, 1, 2, 3, 4, 5, or 6, for example, and where R = H or alkyl, for example: <formula> formula see original document page 45 </formula>

More exemplary compounds of Formula 11-42 are the compounds of Formula II-42A where n = 2 and R = H, and the compound of Formula II-42B where n = 1 and R = H:

<formula> formula see original document page 45 </formula>

In some embodiments where the substituent R 1 of the compound of Formula I includes an electron acceptor, the electron acceptor may be, for example, a Michael acceptor. An exemplary compound of Formula II-43 has the following structure where η = O, 1, 2, 3, 4, 5, 6, and where Z is an electron withdrawing group, for example, CHO, COLOR, COLOR, CONH2 , CN, NO2, SOR, SO2R, etc .: <formula> formula see original document page 46 </formula>

An additional exemplary compound of Formula 11-43 is the compound of Formula II-43A, where n = 1 and Z = CO 2 CH 3:

<formula> formula see original document page 46 </formula>

In some embodiments, the compounds may be prodrug esters or thioesters of the compounds of Formula II. For example, the compound of Formula 11-44 (a prodrug thioester of the compound of Formula 11-16) has the following stereo-chemical structure: <formula> formula see original document page 47 </formula>

In some embodiments, the compounds of Formula II may include an alkenyl group such as R1, for example ethylenyl. An additional exemplary compound of Formula 11-46 has the following structure and stereo-chemical:

<formula> formula see original document page 47 </formula>

In some embodiments, the compounds may be prodrug esters or thioesters of the compounds of Formula II. For example, the compound of Formula 11-47 (a prodrug thioester of the compound of Formula 11-17) has the following structure and stereo-chemical:

<formula> formula see original document page 47 </formula> In some embodiments, the compounds may be prodrug esters or thioesters of the compounds of Formula II. For example, the compound of Formula 11-48 has the following structure and stereo-chemical:

<formula> formula see original document page 48 </formula>

Another exemplary compound of Formula 11-49 has the following stereochemical structure:

<formula> formula see original document page 48 </formula>

In some embodiments, the compound may be a prodrug ester or thioester of the compounds of Formula II. For example, the compound of Formula 11-50 (prodrug ester of the compound of Formula 11-16) has the following structure and stereo-chemical:

<formula> formula see original document page 48 </formula> Formula III compounds

Other embodiments provide compounds and methods for producing a class of related pharmaceutically acceptable prodrug compounds, salts and esters, wherein the compounds are represented by Formula III:

<formula> formula see original document page 49 </formula>

Formula III

In certain embodiments, the substituent (s) for R1 may separately include, for example, a hydrogen, a halogen, a monosubstituted, a polysubstituted or unsubstituted variant of the following residues: alkyl Saturated C 1 -C 24, C 2 -C 24 alkenyl or unsaturated C 2 -C 24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, aryl-alkoxycarbonyl, alkoxycarbonyl-acyl, amino, aminocarbonyl, aminocarboxy, nitro, azido, phenyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, thio, sulfoxide, sulfone, esters of sulfonate, thio cyano, boronic acids and esters, and halogenated alkyl including polyhalogenated alkyl. For example, η can be 1 or 2.

In certain embodiments, R4 may be, for example, a hydrogen, a halogen, a monosubstituted, poly-substituted or unsubstituted variant of the following residues: saturated C1-C24 alkyl, C2-C24 alkenyl or C2-alkynyl Unsaturated C24, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, amino -carbonyl, aminocarboxy, nitro, azido, phenyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl. In some embodiments m may be 1 or 2, and where m is 2, the substituents may be the same or different. Also, each E1, E2, E3, E4 and E5 may be, for example, a substituted or unsubstituted heteroatom. For example, the hetero atom may be nitrogen, sulfur or oxygen.

In some embodiments, R 2 is not cyclohexa-2-enylcarbinol when one of the substituents on R 1 is ethyl or chloroethyl.

Preferably, in some embodiments, R1 is a substituted or unsubstituted C1-C5 alkyl. For example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, and pentyl are preferred. In some embodiments, R1 is not an unsubstituted, substituted or unsubstituted C6 alkyl.

Formula IV Compounds

Other embodiments provide compounds and methods for producing a class of related pharmaceutically acceptable prodrug compounds, salts and esters, wherein the compounds are represented by Formula IV:

<formula> formula see original document page 50 </formula>

In certain embodiments, the substituent (s) R1, R3, and R5 may separately include a hydrogen, a halogen, a mono-substituted, poly-substituted or unsubstituted variant of the following residues: alkyl Unsaturated C1 -C24, unsaturated C2 -C24 alkenyl or C2 -C24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, aryl -alkoxycarbonyl, alkoxycarbonyl-acyl, amino, aminocarbonyl, aminocarbonoxy, nitro, azido, phenyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, thio, sulfoxide, sulfone, sulfonate esters thio-cyano, boronic acids and esters, and halogenated alkyl including polyhalogenated alkyl. Also, each E1, E2, E3, E4 and E5 may be a heteroatom or a substituted heteroatom, for example nitrogen, sulfur or oxygen. In some embodiments, R3 is not a hydrogen, and η is 1 or 2. When η is 2, the substituents may be the same or different. Also, m may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. When m is greater than 1, the substituents may be the same or different.

Preferably, in some embodiments, R1 is a substituted or unsubstituted C1-C5 alkyl. For example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, and pentyl are preferred. In some embodiments, R1 is not an unsubstituted, substituted or unsubstituted C6 alkyl.

In some embodiments R 5 may give rise to a disubstituted cyclohexyl. An exemplary compound of Formula IV is the following structure IV-I, with and without exemplary stereo chemistry:

<formula> formula see original document page 51 </formula>

R 8 may include, for example, hydrogen, fluorine, chlorine, bromine and iodine. The substituents R6 and R7 may separately include a hydrogen, a halogen, a mono-substituted, poly-substituted or unsubstituted variant of the following residues: unsaturated C1-C24 alkyl, C2-C24 alkenyl or unsaturated C2-C24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, amino carboxyoxy, nitro, azido, phenyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl. Further, R6 and R7 may both be the same or different.

For example, An exemplary compound of Formula IV has the following structure IV-2:

<formula> formula see original document page 52 </formula>

Formula IV-2

R 8 may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.

The exemplary stereo-chemistry may be as follows:

<formula> formula see original document page 52 </formula>

For example, an exemplary compound of Formula IV has the following structure IV-3: <formula> formula see original document page 53 </formula>

R 8 may include, for example, hydrogen (IV-3A), fluorine (IV-3B), chlorine (IV-3C), bromine (IV-3D) and iodine (IV-3E).

Exemplary structure and stereo-chemistry may be as follows:

<formula> formula see original document page 53 </formula>

An additional exemplary stereo-chemical structure may be as follows: <formula> formula see original document page 54 </formula>

Formula IV-3C

For example, An exemplary compound of Formula IV has the following structure IV-4:

<formula> formula see original document page 54 </formula>

Formula IV-4

R 8 may include, for example, hydrogen, fluorine, chlorine, bromine and iodine.

Exemplary stereo-chemistry may be as follows: <formula> formula see original document page 55 </formula>

Formula V Compounds

Some embodiments provide compounds, and methods for producing a class of related pharmaceutically acceptable prodrug compounds, salts and esters, wherein the compounds are represented by Formula V:

<formula> formula see original document page 55 </formula>

Formula V

In certain embodiments, the substituent (s) R1 and R5 may separately include a hydrogen, a halogen, a mono-substituted, poly-substituted or unsubstituted variant of the following residues: C1 -C24 alkyl saturated, unsaturated C2 -C24 alkenyl or C2 -C24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl alkoxycarbonyl acyl, amino, aminocarbonyl, aminocarbonoxy, nitro, azido, phenyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, thio, sulfoxide, sulfone, sulfonate esters, thio cyano, boronic acids and esters, and halogenated alkyl including polyhalogenated alkyl. In certain embodiments, each E1, E2, E3, E4 and Ε5 may be a hetero atom or a substituted hetero atom, for example nitrogen, sulfur or oxygen, η may be 1 or 2, and when η equals 2, the substituents may be the same or different. Preferably, m may be, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. When m is greater than 1, R 5 may be the same or different.

Preferably, in some embodiments, R1 is a substituted or unsubstituted C1-C5 alkyl. For example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, and pentyl are preferred. In some embodiments, R1 is not an unsubstituted, substituted or unsubstituted C6 alkyl.

Formula VI Compounds

Some embodiments provide compounds, and methods for producing a class of related pharmaceutically acceptable prodrug compounds, salts and esters, wherein the compounds are represented by Formula VI:

<formula> formula see original document page 56 </formula>

Formula VI

Where R1 may be selected separately from the group consisting of mono-substituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, unsaturated C2-C24 alkenyl or C2-C24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonoxy, phenyl, cycloalkyl acyl, thioalkyl, thioaryl, oxysulfonyl, carboxy, thio, sulfoxide, sulfone, esters and boronic acids, and halogenated alkyl including polyhalogenated alkyl. η is equal to 1 or 2, and if η is equal to 2, then R1 may be the same or different;

where R2 may 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, C2-C24 alkenyl or unsaturated C2-C24 alkynyl , acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl (including, for example, cyclohexylcarbinol), cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonyl -acyl, amino, amino-carbonyl, amino-carboxy, nitro, azido, phenyl, cycloalkyl-acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, thio, sulfoxide, sulfone, sulfonate esters thiocyano, boronic acids and esters, and halogenated alkyl including polyhalogenated alkyl;

where R3 may 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 C2 -C24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonyl acyl, amino, amino carbonyl, aminocarboxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, thio, sulfoxide, sulfone, esters sulfonate, thio cyano, boronic acids and esters, and halogenated alkyl including polyhalogenated alkyl; wherein each E1, E2, E3 and E4 may be a substituted or unsubstituted heteroatom;

In some embodiments, R 2 is not cyclohexa-2-enylcarbinol when one of the substituents on R 1 is ethyl or chloroethyl and R 3 is methyl;

In some embodiments, preferably R1 is a substituted or unsubstituted C1-C5 alkyl. For example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, and pentyl are preferred;

where R14 may be selected from the group consisting of halogen, mono-substituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, unsaturated C2-C24 alkenyl or C2-C24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonyl acyl, amino, aminocarbonyl, aminocarbonoxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, thio, thioesters, sulfoxide, sulfone, sulfonate esters, thiocyano, and halogenated alkyl including polyhalogenated alkyl.

Preferably, in some embodiments, R 14 is a substituted alkylthiol or alkylthiol, and E 3 is an oxygen.

For example, in some embodiments some of the compounds of Formula VI may have the following structure, called Formula VI-I:

<formula> formula see original document page 58 </formula>

where R1 may be selected separately from the group consisting of mono-substituted, poly-substituted or 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, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonoxy, phenyl, cycloalkyl acyl, thioalkyl, thioaryl, oxysulfonyl, carboxy, thio, sulfoxide, sulfone, esters and boronic acids, and halogenated alkyl including polyhalogenated alkyl. η is equal to 1 or 2, and if η is equal to 2, then R1 may be the same or different;

where R3 may be selected from the group consisting of halogen, mono-substituted, poly-substituted or 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, arylalkoxycarbonyl, alkoxycarbonyl acyl, amino, aminocarbonyl, aminocarbonoxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, thio, sulfoxide, sulfone, sulfonate esters, thio cyano, boronic acids and esters, and halogenated alkyl including polyhalogenated alkyl; wherein each E1, E2, E3 and E4 may be a substituted or unsubstituted heteroatom;

where R5 may be selected separately from the group consisting of hydrogen, halogen, monosubstituted, poly-substituted or unsubstituted variants of the following residues: saturated C1-C24 alkyl, C2-C24 alkenyl or non-substituted C2-C24 alkynyl saturated, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, amino-carboxy, nitro, azido, phenyl, oxide, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, thio, sulfoxide, sulfone, sulfonate esters, thio cyano, boronic acids and esters, and halogenated alkyl including polyhalogenated alkyl, where m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 and if m is more than 1, then R5 may be the same or different; and wherein the substituent R 5 may form a ring; and wherein each E1, E2, E3, E4 and E5 is a substituted or unsubstituted heteroatom;

Preferably in some embodiments, R1 is a substituted or unsubstituted C1-C5 alkyl. For example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, and pentyl are preferred;

where R14 may be selected from the group consisting of halogen, mono-substituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, unsaturated C2-C24 alkenyl or C2-C24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonyl acyl, amino, aminocarbonyl, aminocarbonoxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, thio, thioesters, sulfoxide, sulfone, sulfonate esters, thiocyano, and halogenated alkyl including polyhalogenated alkyl.

For example, the compound has the following structure

VI-1A:

<formula> formula see original document page 60 </formula>

Formula VI-IA

The exemplary stereo-chemistry may be as follows:

<formula> formula see original document page 60 </formula>

For example, an exemplary compound of Formula VI has the following structure and stereo-chemical VI-IB:

<formula> formula see original document page 60 </formula>

Formula VI-IB

Another example, the compound of Formula VI has the following structure and stereo-chemical VI-IC: <formula> formula see original document page 61 </formula>

Formula VI-IC

Certain embodiments also provide pharmaceutically acceptable prodrug salts and esters of the compound of Formulas I-VI, and provide methods for obtaining and purifying such compounds by the methods described herein.

The term "prodrug ester", especially when referring to a prodrug ester of the compound of Formula I synthesized by the methods described herein, refers to a chemical derivative of the compound that is rapidly transformed in vivo to obtain the compound. for example by hydrolysis in blood or within tissues. The term "prodrug ester" refers to derivatives of the compounds described herein formed by the addition of any of several ester or thioester-forming groups which are hydrolyzed under physiological conditions. Examples of prodrug ester groups include pivoyloxymethyl, acetoxymethyl, phthalidyl, indanyl and methoxymethyl, as well as other groups known in the art, including a group (5-R-2-oxo-1,3-dioxolen-4-yl ) methyl. Other prodrugs may 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 prodrug 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. Symposium Series, American Chemical Society (1975); and "Bioreversible Carriers in Drug Design: Theory and Application", edited by E. B. Roche, Pergamon Press: New York, 14-21 (1987) (providing examples of esters useful as prodrugs for carboxyl-containing compounds). Each of the above references is incorporated herein by reference in its entirety.

The term "prodrug ester" as used herein also refers to a chemical derivative of the compound that is rapidly transformed in vivo to obtain the compound, for example by hydrolysis in blood.

The term "pharmaceutically acceptable salt" as used herein, and particularly when referring to a pharmaceutically acceptable salt of a compound, including Formulas I-VI, and Formula I-VI as produced and synthesized by the methods described herein, refers to to any pharmaceutically acceptable salts of a compound, and preferably refers to an acid addition salt of a compound. Preferred examples of pharmaceutically acceptable salts are alkali metal salts (sodium or potassium), alkaline earth metal salts (calcium or magnesium), or ammonium salts derived from ammonia or pharmaceutically acceptable organic amines, for example C1-C7 alkylamine cyclohexylamine, triethanolamine, ethylenediamine or tris- (hydroxymethyl) aminomethane. With respect to compounds synthesized by the method of this embodiment which are basic amines, preferred examples of pharmaceutically acceptable salts are acid addition salts of pharmaceutically acceptable inorganic or organic acids, for example hydrohalic, sulfuric, phosphoric or aliphatic acid. or aromatic or sulfonic carboxylic acid, for example acetic, succinic, lactic, malic, tartaric, citric, ascorbic, nicotinic, methanesulfonic, p-toluenesulfonic or naphthalenesulfonic acid.

Preferred pharmaceutical compositions described herein include pharmaceutically acceptable prodrug salts and esters of the compound of Formulas I-VI obtained and purified by the methods described herein. Suitably, if the manufacture of pharmaceutical formulations involves intimate mixing of the pharmaceutical excipients and the active salt form, then it is preferred to use non-basic pharmaceutical excipients, ie acidic or neutral excipients.

It will also be appreciated that the phrase "compounds and compositions comprising the compound", or any similar phrase, means encompassing compounds in any form suitable for pharmaceutical distribution, as discussed further in detail herein. For example, in certain embodiments, compounds or compositions comprising the same may include a pharmaceutically acceptable salt of the compound.

In one embodiment the compounds may be used to treat microbial diseases, cancer, and inflammation.

"Disease" should be interpreted as broadly covering infectious diseases, as well as autoimmune diseases, noninfectious diseases, and chronic conditions. In a preferred embodiment, the disease is caused by a microbe, such as bacteria, fungi, and protozoa, for example. Methods of use may also include the steps of administering a compound or composition including the compound to an individual with an infectious disease or cancer. The compound or composition may be administered in an amount effective to treat the particular infectious disease, cancer or inflammatory condition.

Infectious disease can be, for example, caused by Bacilli, such as B. Anthracis and B. Cereus. Infectious disease may be caused by protozoa, for example, Leishmanial Plasmodium or Trypanosoma. The compound or composition may be administered with a pharmaceutically acceptable carrier, diluent or excipient, and the like.

The cancer may be, for example, a multiple myeloma, a colorectal carcinoma, a prostate carcinoma, a breast adenocarcinoma, a non-small cell lung carcinoma, an ovarian carcinoma, a melanoma, and the like.

The inflammatory condition may be, for example, rheumatic arthritis, asthma, multiple sclerosis, psoriasis, stroke, myocardial infarction, reperfusion damage, and the like.

The term "halogen atom" as used herein means any of the radio-stable atoms in column 7 of the Periodic Table of Elements, i.e. fluorine, chlorine, bromine, or iodine, with bromine and chlorine being preferred.

The term "alkyl" as used herein means any unsubstituted or branched, substituted or unsubstituted saturated hydrocarbon with C1-C24 being preferred, and C1-C6 hydrocarbons being also preferred with methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl and pentyl being more preferred. Among substituted saturated hydrocarbons, C1-C24 are preferred, with C1-C6 saturated hydrocarbons and mono- and disubstituted and halogen-substituted amino-substituted hydrocarbons being more preferred.

The term "substituted" has its common meaning, as found in numerous contemporary patents of the related state of art. For example, see 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 being incorporated herein in their entirety by reference. Specifically, the definition of "substituted" is as broad as that provided in US Patent No. 6,509,331 which defines the term "substituted alkyl" such that it refers to an alkyl group, preferably of 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, amine, substituted amine, amino acyl, amino acyloxy, oxy acylamine, cyano, halogen, hydroxyl, carboxyl, carboxyalkyl, queto, thioqueto, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, hetero aryl, heteroaryloxy, heterocyclic, hetero-cyclooxy, hydroxy-amine, alkoxy-amine, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-hetero aryl, -SO 2 -alkyl, -SO 2 -substituted alkyl, -SO 2 -aryl and -SO 2 -heteroaryl. The other patents listed above also provide standard definitions for the term "substituted" which are well understood by those skilled in the art.

The term "cycloalkyl" refers to any non-aromatic hydrocarbon ring, preferably having five to twelve atoms comprising the ring. The term "acyl" refers to alkyl or aryl groups derived from an oxo acid, with an acetyl group being preferred.

The term "alkenyl" as used herein means any unsubstituted or branched, unsubstituted or unsubstituted hydrocarbon, including polyunsaturated hydrocarbons, with unsubstituted, mono-unsaturated and di-unsubstituted C1-6 hydrocarbons. unsaturated, being preferred, and mono-unsaturated dihalogen substituted hydrocarbons, being more preferred. The term "cycloalkenyl" refers to any non-aromatic hydrocarbon ring, preferably having five to twelve atoms comprising the ring.

The terms "aryl", "substituted aryl", "heteroaryl", and "substituted heteroaryl" as used herein refer to aromatic hydrocarbon rings preferably having five, six, or seven atoms, and preferably having six atoms, comprising the ring. "Heteroaryl" and "substituted heteroaryl" refer to aromatic hydrocarbon rings in which at least one heteroatom, for example an oxygen, sulfur, or nitrogen atom is in the ring together with at least one atom of carbon. The term "heterocycle" or "heterocyclic" refers to any cyclic compound containing one or more hetero atoms. Substituted aryls, heteroaryls and heterocycles may be substituted with any substituent, including those described above and those known in the state of the art.

The term "alkoxy" refers to any unsaturated or saturated, unbranched or branched, substituted or unsubstituted ether, with unbranched, saturated, unsubstituted C1-C6 ethers being preferred, with methoxy being preferred, and also with dimethyl, diethyl, methyl isobutyl, and methyl tert-butyl ethers also being preferred. The term "cycloalkoxy" refers to any non-aromatic hydrocarbon ring, preferably having five to twelve atoms comprising the ring. The term "alkoxycarbonyl" refers to any linear, branched, cyclic, saturated or unsaturated aromatic or aliphatic alkoxy attached to a carbonyl group. Examples include the methoxycarbonyl group, the ethoxycarbonyl group, the propyloxycarbonyl group, the isopropyloxycarbonyl group, the butoxycarbonyl group, the sec-butoxycarbonyl group, the tert-butoxycarbonyl group, the cyclohexyloxycarbonyl group, the benzyloxycarbonyl group. , the allyloxycarbonyl group, the phenyloxycarbonyl group, the pyridyloxycarbonyl group, and the like.

The terms "pure", "purified", "substantially purified", and "isolated" as used herein refer to the compound of the form of incorporation being free of other dissimilar compounds with which the compound is found in its natural state. , would be associated in its natural state. In certain embodiments described herein as "pure", "purified", "substantially purified", or "isolated", the compound may comprise at least 0.5%, 1%, 5%, 10%, or 20%, and most preferably at least 50% or 75% by weight of a given sample.

The terms "derivative", "variant", or other similar terms refer to a compound that is an analog of another compound.

Certain compounds of Formula I-VI may be obtained and purified or may be obtained by semi-synthesis from purified compounds as described herein. Generally, but not limited to, the compounds of Formula 11-15, and preferably Formulas 11-16, 11-17, 11-18 and 11-19 may be obtained synthetically or by fermentation. Exemplary fermentation procedures are provided below. In addition, the compounds of Formula 11-15, and preferably of Formulas 11-16, 11-17, 11-18 and 11-19 may be used as starting compounds to obtain / synthesize various other compounds described herein. Non-limiting exemplary syntheses are provided herein.

Formula 11-16 is currently produced by a high yield saline fermentation (~ 350-400 mg / L) and changing conditions have yielded new analogs in the fermentation extracts. Fig. 1 shows chemical structure 11-16. Additional analogs may be generated by directed biosynthesis. Targeted biosynthesis is the modification of a natural product by adding biosynthetic precursor analogs to the fermentation of producing microorganisms (Lam, et al., J. Antlbiot. (Tokyo), 44: 934 (1991); Lam, et al., J. Antibiot. (Tokyo) 54: 1 (2001), which are incorporated herein by reference in their entirety).

Exposing the producing culture to analogs of acetic acid, phenylalanine, valine, butyric acid, shikimic acid, and halogens, preferably other than chlorine, may lead to the formation of new analogs. The new analogues produced can be easily detected in crude extracts by CLAP and LC-MS. For example, after manipulating the medium with different concentrations of sodium bromide, a bromine analog, Formula 11-18, was successfully produced in a shake flask culture at a 14 mg / L titer.

A second approach to generating new analogs is through bio-transformation. Bio-transformation reactions are chemical reactions catalyzed by enzymes or whole cells containing these enzymes. Zaks, A., Curr. Opin. Cheni Biol. 5: 130 (2001). Microbial natural products are ideal substrates for bio-transformation reactions as they are synthesized by a series of enzymatic reactions within microbial cells. Riva, S. Curr. Opin. Chem. Biol. 5: 106 (2001).

Given the structure of the compounds described, including those of Formula 11-15, for example, possible biosynthetic origins are acetyl-CoA, ethylmalonyl-CoA, phenylalanine and chlorine. Ethylmalonyl-CoA is derived from butyryl-CoA, which can either be derived from either valine or crotonyl-CoA. Liu, et al., Metab. Eng. 3:40 (2001). Phenylalanine is derived from shikimic acid.

Production of Compounds of Formulas 1-7, 11-16, 11-17, 11-18, 11-20, II-24C, 11-26, 11-27 and 11-28

Production of compounds of Formulas 1-7, 11-16, 11-17, 11-18, 11-20, II-24C, 11-26, 11-27 and 11-28 can be performed by cultivating a CNB47 strain. 6 and an NPS21184 strain, a natural variant of the CNB476 strain, in a satisfactory nutrient medium under the conditions described herein, preferably under submerged aerobic conditions, until a significant amount of compounds are detected in fermentation; harvesting by extraction the active components of the fermentation broth with a satisfactory solvent; concentrating the solvent containing the desired components; The concentrated material is then subjected to chromatographic separation to isolate compounds from other metabolites also present in the culture medium.

Fig. 2 shows some worldwide collection sites for the crop (CNB476) which is also referred to as Salinospora. Fig. 3 shows Salinospora colonies. Fig. 4 shows the typical Salinospora 16S rDNA sequence. The bars represent Salinospora's signature signature nucleotides, which separate it from its closest relatives.

The culture (CNB476) was deposited on June 20, 2003 with the American Type Culture Collection (ATCC) in Rockville, MD, USA, and is designated under ATCC patent application number PTA-5275. . The NPS21184 strain, a natural variant of the CNB476 strain, was derived from the CNB476 strain as a single isolated colony. The NPS21184 lineage was deposited with the ATCC on April 27, 2005. The ATCC deposit meets all the requirements of the Budapest treaty. The culture is also maintained and available from the Nereus Pharmaceutical Culture Collection at 10480 Wateridge Circle, San Diego, CA 92121. In addition to the specific microorganism described herein, it should be understood that mutants, such as those produced by the use of mutations. physical or chemical including X-rays, etc. and organisms whose genetic makeup has been modified by molecular biology techniques can also be cultured to produce the starting compounds of Formulas 11-16, 11-17, and 11-18.

Fermentation of CNB476 strain and NPS21184 strain

Compound production may be achieved at a temperature consistent with satisfactory growth of the producing organism, for example 16 degrees C to 40 degrees C, but it is preferable to conduct fermentation at 22 degrees C to 32 degrees C. The aqueous medium may be incubated. for a period of time required to complete the production of compounds as monitored by high pressure liquid chromatography (HPLC), preferably for a period of about 2 to 10 days, in a rotary mixer operating at approximately 50 rpm to 400 rpm, preferably at 150 rpm to 250 rpm, for example. Compound production can also be achieved by cultivating the production lineage in a bioreactor, such as a fermenter system that is suitable for growth of the production lineage.

The growth of microorganisms can be achieved by those of ordinary skill in the art by the use of an appropriate medium. Broadly, carbon sources include glucose, fructose, mannose, maltose, galactose, mannitol and glycerol, other sugars and sugar alcohols, gums and other carbohydrates, or carbohydrate derivatives such as dextran, cerellose, as well as complexes. of nutrients such as oatmeal, corn bran, millet, corn, and the like. The exact amount of carbon source that is used in the medium will depend in part on the other ingredients in the medium, but an amount of carbohydrate between 0.5 to 25 percent by weight of the medium can be used satisfactorily, for example. These carbon sources may be used individually or several such carbon sources may be combined in the same medium, for example. Certain carbon sources are preferred, as explained below.

Nitrogen sources include amino acids such as glycine, arginine, threonine, methionine and the like, ammonium salt, as well as complex sources such as yeast extracts, corn-derived liquors, soluble distillates, soybean meal, soybean meal. cottonseed, fish meal, peptone, and the like. The various nitrogen sources may be used alone or in combination in amounts ranging from 0.5 to 25 weight percent of the medium, for example.

Among the inorganic nutrient salts that may be incorporated into the culture medium are customary salts capable of obtaining sodium, potassium, magnesium, calcium, phosphate, sulfate, chloride, carbonate, and similar ions. Trace metals such as cobalt, manganese, iron, molybdenum, zinc, cadmium, and the like are also included.

Biological Activity and Uses of Compounds

Embodiment forms relate to methods for 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. Thus, the compounds described herein may be used to treat cancer, inflammation, and infectious diseases.

The compounds have various biological activities. For example, the compounds have chemosensitizing, antimicrobial, anti-inflammation, radiosensitizing, and anti-cancer activity.

The compounds have proteasome inhibitory activity. 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 multi-sub-unit of protease that degrades intracellular proteins through its activities as chymotrypsin, trypsin and peptidylglutamyl peptide hydrolysis (HPPG, also known as caspase-like activity). The 26S proteasome contains a proteolytic nucleus called the 20S proteasome and one or two 19S regulatory subunits. The 20S proteasome is responsible for proteolytic activity against many substrates including damaged proteins, FN-κΒ transcription factor and its IkB inhibitor, signaling molecules, tumor suppressors and cell cycle regulators. There are three distinct protease activities within the proteasome: 1) chemotrypsin type; 2) trypsin type; and 3) peptidylglutamyl peptide hydrolysis (HPPG) activity.

As an example, the compounds of Formula 11-16 were more potent (2nM EC50) to inhibit rabbit muscle proteasome chemotrypsin-like activity than Omuralide (52 nM EC50) and also inhibited chymotrypsin-like activity. of human erythrocyte derived proteasomes (EC50 ~ 250 pM). Fig. 5 shows omuralide, which is a degradation product of Lactacystine, and shows a compound of Formula II-16. The compounds of Formula 11-16 show a significant preference for inhibiting proteasome chymotrypsin-like activity over inhibiting catalytic activity. of chymotrypsin. The compounds of Formula 11-16 also exhibit low inhibition of trypsin-like activity (~ 10 nM), but are less potent to inhibit proteasome HPPG activity (-350 nM EC50).

Additional studies characterized the effects of the compounds described herein, including studies of Formula 11-16 on

The signaling path of FN-κΒ / ΙκΒ. HEK293 (human embryonic kidney) cell treatment with Tumor Necrosis Alpha-Factor

(TNF-α) induces proteasome-mediated phosphorylation and degradation of ΙκΒα followed by activation of FN-κΒ. To confirm proteasome inhibition, HEK293 cells were pretreated for

1 hour with compounds of Formula 11-16 followed by TNF-α stimulation. Treatment with the compounds of Formula 11-16 promoted

accumulation of phosphorylated ΙκΒα suggesting that proteasome-mediated degradation of ΙκΒα was inhibited.

In addition, a stable HEK293 clone (FN-kB / Luc 293) was generated carrying a luciferase reporter gene under the regulation of 5x FN-κΒ binding sites. Stimulation of FN-kB / Luc 293 cells with TNF-α enhances luciferase activity as a result of FN-κ pré activation while pretreatment with compounds of Formula 11-16 decreases activity. Western blot analyzes showed that compounds of Formula 11-16 promoted accumulation of phosphorylated ΙκΒα and decreased total degradation of ΙκΒα in FN-kB / Luc 293 cells. Compounds of formula 11-16 also demonstrated to increase protein levels. cell cycle regulators, p21 and p27.

Tumor cells may be more sensitive to proteasome inhibitors than normal cells. In addition, proteasome inhibition increases cancer cell sensitivity to anti-cancer agents. Cytotoxic activity of compounds described herein, including Formula 11-16, was examined for cytotoxic activity against various cancer cell lines. For example, formula 11-16 was examined on the National Cancer Institute screen for 60 human tumor cell lines. Formula 11-16 exhibited selective cytotoxic activity with an average IC50 value (the concentration to achieve 50% growth inhibition) of less than 10 nM. The greatest potential was observed against SK-MEL-28 melanoma and MDA-MB-235 breast cancer cells [both with LC50 (50% concentration of cell lethality) <10 nM].

A panel of human (HT-29 and LoVo), prostate (PC3), breast (MDA-MB-231), lung (NC1-H292), ovarian (OVCAR3), human colorectal cell lines T-cell (Jurkat), murine melanoma (B16-F10) and normal human fibroblasts (CCD-27sk) therapy was treated with Salinosporamide A for 48 h to evaluate cytotoxic activity. HT-29, LoVo, PC3, MDA-MB-231, NC1-H292, OVCAR3, Jurkat, and B16-F10 cells were sensitive with EC50 values of 47, 69, 78, 67, 97, 69, 10, and 33 nM, respectively. In contrast, the EC 50 values for CCD-27sk cells was 196 nM. Treatment of Jurkat cells with Salinosporamide A on the approximate EC50 resulted in activation of Caspase-3 and PARP splitting confirming apoptosis induction.

The anti-anthrax activity of the described compounds was evaluated using a TxLe induced in vitro cytotoxicity assay. As an example, the results indicate that Formula II-16 is a potent inhibitor of TxLe induced cytotoxicity of RAW2 64.7 cells as murine macrophages. Treatment of RAW2 64.7 cells of formula 11-16 resulted in a 10-fold increase in viability of TxLe treated cells compared to TxLe treatment alone (mean EC50 <4 nM).

Potential Chemosensitizing Effects of Formula 11-16

Further studies characterized the effects of the compounds described herein on the FN-kB / IkB signaling pathway (see Examples). In unstimulated cells, nuclear transcription factor factor-kappa B (FN-κΒ) resides in the cytoplasm in an inactive complex with the inhibitor protein IkB (inhibitor of FN-kB). Various stimuli may cause IkB phosphorylation by IkB kinase, followed by ubiquitination and proteaome degradation. Following ΙκΙ degradation, FN-κΒ translocates to the nucleus and regulates gene expression, affecting many cellular processes including inhibition of apoptosis. Chemotherapy agents such as CPT-Il (Irinotecan) may activate FN-κΒ 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). Velcade® is a dipeptidyl boronic acid that inhibits proteasome chemotrypsin-like activity (Lightcap, et al., Clin. Chem. 46: 673 (2000), Adams, et al., Cancer Res. 59: 2615 (1999) Adams Curr Opin Opinion Oncol 14: 628 (2002)) while increasing trypsin and HPPG activities. Recently approved as a proteasome inhibitor, Velcade® (PS-341; Millennium Pharmaceuticals, Inc.) has been shown to be directly toxic to cancer cells and also to increase CPT-Il cytotoxic activity in LoVo cells in vitro and in a model. xenographic LoVo inhibiting proteasome degradation of IkB. Blum, et al. Ann. Intern Med. 80: 249 (1974). In addition, Velcade® has been found to inhibit the expression of Oncogene-alpha (CRO-α) -related growth of proangiogenic chemokines / cytokines and Vascular Endothelial Growth Factor (VEGF) in squamous cell carcinomas, presumably by FN-κΒ pathway inhibition. Dick, et al., J. Biol. Chem. 271: 7273 (1996). These data suggest that proteasome inhibition may not only decrease tumor cell survival and growth, but also angiogenesis.

Anti Anthrax Activity

Another potential application for proteasome inhibitors comes from recent studies in Category A bio-defense against agent B. Anthracis (anthrax). Anthrax spores are inhaled and lodged in the lungs where they are ingested by macrophages. Within macrophages, spores germinate, the organism replicates, ultimately resulting in cell death. Before death, however, infected macrophage cells migrate to the lymph nodes where, at death, they release their contents allowing the body to enter the bloodstream, and later reproduce, and secrete lethal toxins. Hanna, et al., Proc. Natl. Acad. Know. 90: 10198 (1993). Anthrax toxins are responsible for the symptoms associated with anthrax. Two proteins that play a key role in anthrax pathogenesis are the protective antigen (AP, 83 kDa) and the lethal factor (FL, 90 kDa) which are collectively known as the lethal toxin (TxLe). FL has an enzymatic function, but requires AP to achieve its biological effect. Neither AP or FL causes death individually; however, when combined they cause death when injected intravenously into animals. Kalns, et al., Biochem, Biophys. Res. Commun. 297: 506 (2002), Kalns, et al. Biochem. Biophys. Res. Commun. 292: 41 (2002).

The protective antigen (AP), the anthrax toxin binding / receptor component, is responsible for carrying the lethal factor in the host cell. The AP oligomerizes in a ring as a heptamer (see Fig. 6). Each heptamer, bound to its receptor on the surface of a cell, has the ability to bind to three FL molecules. The complex formed between the AP heptamer and FL is taken up in the cell by receptor mediated endocytosis. After endocytosis, FL is released into the cytosol where it attacks various cellular targets. Mogridge, et al., Biochemistry 41: 1079 (2002), Lacy, et al., J. Biol. Chem. 277: 3006 (2002), Bradley, et al., Nature 414: 225 (2001).

Lethal factor (FL) is a zinc-dependent metalloprotease, which in cytosol can split and deactivate the kinase family mitogen-activated protein kinase (QPAMQ) signaling proteins. Duesbery, et al., Science 280: 734 (1998), Bodart, et al., Cell Cyele 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 PAMQ kinases, six have been shown to be divided by FL. Within the cell, PAMQ kinase pathways translate various signals involved in cell death, proliferation, and differentiation making these proteins highly significant targets. However, certain inhibitors that prevent TxLe-induced cell death do not prevent the division of QPAMQ by FL suggesting that this activity is not sufficient to induce cell death. Kim, et al., J. Biol. Chem. 278: 7413 (2003), Lin, et al., Curr. Microbiol. 33: 224 (1996).

Studies have suggested that proteasome inhibition may prevent TxLe-induced cell death. Tang, et al., Infect. Immun. 67: 3055 (1999). The data showed that proteasome activity is required to kill cells such as TxLe-mediated RAW264.7 macrophages and that proteasome inhibitors protect TxLe RAW264.7 cells. Proteasome inhibition did not block MEK1 division, suggesting that the TxLe pathway is not blocked downstream of MEK1 division in these studies. Additionally, there is no increase in proteasome activity in TxLe treated cells. These data suggested that a new and potent proteasome inhibitor such as the compounds described herein may also prevent TxLe-induced cell death as illustrated in fig. 6

The receptor for AP has been identified expressed by many cell types. Escuyer, et al., Infect. Immun. 59: 3381 (1991). Lethal toxin is active in some macrophage cell culture lines causing cell death within a few hours. Hanna, et al., Proc. Natl. Acad. Know. U.S.A. 90: 10198 (1993), Kim, et al., J. Biol. Chem. 278: 7413 (2003), Lin, et al., Curr. Microbiol. 33: 224 (1996). TxLe can induce necrosis and apoptosis in rat cells such as RAW264.7 and J774A.1 macrophages in an in vitro treatment.

The results indicate that the compounds described herein act as a potent inhibitor of TxLe-induced cytotoxicity of RAW264.7 cells as murine macrophages. Treatment of RAW264.7 cells with, for example, compounds of Formula 11-16, resulted in a 10-fold increase in viability of TxLe treated cells compared to TxLe treatment alone (mean EC50 <4 nM), and then provides valuable therapy for anthrax infections. For example, formula 11-16 promoted the survival of RAW264.7 cells as macrophages in the presence of TxLe indicating that this compound and its derivatives provide valuable clinical therapy for anthrax infection.

Pharmaceutical Compositions

In one embodiment, the compounds described herein are used in pharmaceutical compositions. The compounds may preferably be produced by the methods described herein.

For example, the compounds may be used in pharmaceutical compositions comprising a pharmaceutically acceptable carrier prepared for storage and subsequent administration. Also, embodiments relate to a pharmaceutically effective amount of the products and compounds described 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 editors, 1985) which is incorporated herein by reference in its entirety. Preservatives, stabilizers, dyes and flavoring agents may be provided in the pharmaceutical composition. For example, sodium benzoate, ascorbic acid and p-hydroxybenzoic acid esters may be added as preservatives. In addition, antioxidants and suspending agents may be used.

The compositions, particularly those of Formulas I-VI, may be formulated and used as tablets, capsules, or elixirs for oral administration; suppositories for rectal administration; sterile solutions, suspensions for injectable administration; adhesives for transdermal administration, subdermal deposition, and the like. Injectables may be prepared in conventional forms, either as liquid solutions or suspensions, satisfactory solid forms for solution or suspension in liquid prior to injection, or as emulsions. For example, suitable excipients are water, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, hydrochloride cysteine, and the like. In addition, if desired, injectable pharmaceutical compositions may contain secondary amounts of non-toxic auxiliary substances, such as wetting agents, pH buffering agents, and the like. If desired, absorption enhancing preparations (e.g., liposomes) may be used.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in a water soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Satisfactory lipophilic solvents or carriers 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. Optionally, the suspension may also contain satisfactory stabilizers or solubility enhancing agents of the compounds to allow the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use may be obtained by combining the active compounds with a solid excipient, optionally milling the resulting mixture, and processing the granule mixture, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Satisfactory excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as corn gum, wheat gum, rice gum, potato gum, gelatin, tragacanth gum, methyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, and / or polyvinyl pyrrolidone (PVP) . If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a related salt such as sodium alginate. Dragee cores are provided with adequate covers. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol and / or titanium dioxide, lacquer solutions, and satisfactory organic solvents or solvent mixtures. . Dyes or pigments may be added to tablets or dragee layers for identification or characterization of different combinations of active compound doses. Such formulations may be made using state-of-the-art methods (see, for example, U.S. Patent Nos. 5, 773.888 (injectable compositions); 5,726,181 (poorly water-soluble compounds); 5,707,641 (therapeutically active proteins or peptides) ); 5,667,809 (lipophilic agents); 5,576,012 (solubilizing polymeric agents); 5,707,615 (antiviral formulations); 5,683,676 (particulate medicines); 5,654,286 (topical formulations); 5,688,529 (oral suspensions); 5,445,829 (extended release formulations); 5,653,987 (liquid formulations), 5,641,515 (controlled release formulations) and 5,601,845 (spheroid formulations), all of which are incorporated herein by reference in their entirety.

Further described herein are various pharmaceutical compositions well known in the pharmaceutical art including for topical, intraocular, intranasal, and intraocular release. Pharmaceutical formulations include aqueous ophthalmic solutions of the active compounds in water-soluble form, such as eye drops, or in gel 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 microparticles, which contain small polymeric drug particles that are suspended in a liquid carrier medium (Joshi, A. 1994 J. Ocul. Pharmacol. 10: 29-45), lipid soluble formulations (Alm et al., Prog. Clin Biol. Res., 312: 447-58 (1989)), and microspheres (Mordenti, Toxicol. Sci., 52 (1): 101-6 (1999)); and eye inserts. All aforementioned references are incorporated herein by reference in their entirety. Such satisfactory pharmaceutical formulations are often and preferably formulated to be sterile, isotonic and buffered for stability and comfort. Pharmaceutical compositions may also include drops and aerosols often prepared to simulate nasal secretions in many respects to ensure maintenance of normal ciliary action. As described in Remington's Pharmaceutical Sciences (Mack Publishing, 18th Edition) which is incorporated herein by reference in its entirety, and well known to those skilled in the art, satisfactory formulations are often and preferably isotonic, slightly buffered to maintain a pH of 5. , 5 to 6.5, and often and preferably include appropriate antimicrobial condoms and drug stabilizers. Pharmaceutical formulations for in-ear release include suspensions and ointments for topical application to the ear. Common solvents for such ear formulations include glierin and water.

When used as an anti-cancer, anti-inflammatory or anti-microbial compound, for example, the compounds of Formulas I-VI or compositions including Formulas I-VI may be administered by oral or non-oral routes. When administered orally, they may be administered in capsules, tablets, granules, spray, syrups, or otherwise. When administered non-orally, they may be administered as an aqueous suspension, oily preparation or the like or as a drip, suppository, ointment, ointment or the like, or administered by injection, subcutaneously, intraperitoneally, intravenously, intramuscularly, or the like.

In one embodiment, the anti-cancer, anti-inflammatory or anti-microbial may be mixed with additional substances to increase their effectiveness. In one embodiment, the antimicrobial is combined with an additional antimicrobial. In another embodiment, the antimicrobial is combined with a drug or medication that is useful for a patient taking antimicrobials.

Administration Methods

In an alternative embodiment, the disclosed chemical compounds and pharmaceutical compositions are administered by a particular method as an antimicrobial. Such methods include, among others, (a) administration by oral routes, the administration of which includes administration in capsules, tablets, granules, spray, syrups, or other forms; (b) administration by non-oral routes, the administration of which includes administration as an aqueous suspension, an oily or similar preparation or as a drip, suppository, ointment, ointment or the like; administration by injection, subcutaneously, intraperitoneally, intravenously, intramuscularly,

intradermally, or the like; as well as (c) topical administration, (d) rectal administration, or (e) vaginal administration, as deemed appropriate by those skilled in the art to bring the compound of the present embodiment into contact with living tissue; and (f) administration by controlled release formulations, deposition formulations, and infusion pump release. As additional examples of such modes of administration and as a further disclosure of modes of administration, various methods for administering the described chemical compounds and pharmaceutical compositions including modes of administration by intraocular, intra nasal, and intra atrial routes are described herein. .

The pharmaceutically effective amount of the compositions including the described compounds, including those of Formulas I-VI, required as a dose, will depend upon the route of administration, the type of animal being treated, including human, and the physical characteristics of the specific animal in question. consideration. The dose may be adjusted to achieve a desired effect, but will depend on factors such as weight, diet, concurrent medications, and other factors that those skilled in the medical arts will recognize.

By practicing the methods of the present embodiment, the products or compositions may be used alone or in combination with each other, or in combination with other therapeutic or diagnostic agents. These products may be used in vivo, commonly in a mammal, preferably in a human, or in vitro. Employing them in vivo, the products or compositions may 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 test chemical activity in vivo.

As will be readily apparent to one of ordinary skill in the art, 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 they are administered. These compounds are employed. Determination of effective dosage levels, which are the dosage levels required to achieve the desired result, can be performed by those skilled in the art using routine pharmacological methods.

Typically, human clinical applications of products begin at lower dosage levels, with the dosage level being increased until the desired effect is achieved. Alternatively, acceptable in vitro studies may be used to establish useful doses and routes of administration of the compositions identified by the present methods using established pharmacological methods.

In non-human animal studies, potential product applications start at higher dosage levels, with dosing decreasing until the desired effect is no longer achieved or adverse side effects disappear. The dosage may vary widely, depending on the desired effects and the therapeutic indication. Typically, the dosages may be from about 10 micrograms / kg to 100 mg / kg body weight, preferably from about 100 micrograms / kg to 10 mg / kg body weight. Alternatively dosages may be based and calculated on the patient's surface area as understood by those skilled in the art. Administration is preferably oral on a daily basis, or twice a day.

The exact formulation, route of administration and dosage may be chosen by the individual physician in view of the patient's condition. See for example, Fingi, 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 and when to terminate, discontinue, or adjust administration due to toxicity or organ dysfunction. Conversely, the attending physician would also be able to adjust treatment to higher levels if the clinical response is not adequate (preventing toxicity). The magnitude of a dose administered in the management of the disease of interest will vary with the severity of the condition being treated and the route of administration. The severity of the condition may, for example, be assessed in part by standardized prognostic assessment methods. Further, the dose and perhaps the frequency of the dose will also vary according to the age, body weight, and response of the individual patient. A program comparable to the one discussed above can be used in veterinary medicines.

Depending on the specific conditions treated, such agents may be formulated and administered systemically or locally. A variety of formulation and administration techniques 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. Satisfactory routes of administration may include oral, rectal, transdermal, vaginal, transmucosal, or intestinal administration; parenteral distribution, including intramuscular, subcutaneous, intramedullary as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal injections, or intraocular injections.

For injection, the agents of the present embodiment may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. For such transmucosal administration, appropriate penetrants to the barrier to be penetrated are used in the formulation. Such penetrants are generally known from the state of the art. The use of pharmaceutically acceptable carriers to formulate the compounds described herein for practicing the incorporation forms at dosages satisfactory for systemic administration is within the scope of the invention. With the appropriate choice of carrier and satisfactory manufacturing practice, the compositions described herein, in particular those formulated as solutions, may be administered parenterally by intravenous injection. The compounds may be readily formulated using pharmaceutically acceptable carriers well known in the art at dosages suitable for oral administration. Such carriers enable the compounds of the invention to be formulated as tablets, pills, capsules, liquids, gels, syrups, dregs, suspensions and the like for oral ingestion by a patient to be treated.

Agents intended to be administered intracellularly may be administered using techniques well known to those of ordinary skill in the art. For example, such agents may be encapsulated in liposomes, and then administered as described above. All molecules present in an aqueous solution at the time of liposome formation are incorporated into the aqueous interior. Liposome contents are protected from the external microenvironment and, because liposomes fuse with cell membranes, they are efficiently distributed in the cytoplasm of cells. Additionally, due to their hydrophobicity, small organic molecules can be directly administered intracellularly.

Determination of effective amounts is well within the capability of those skilled in the art, especially in view of the detailed description provided herein. In addition to the active ingredients, these pharmaceutical compositions may contain satisfactory pharmaceutically acceptable carriers including excipients and auxiliaries which facilitate the processing of the active compounds into pharmaceutically usable preparations. Preparations formulated for oral administration may be in the form of tablets, pills, capsules, or solutions. Pharmaceutical compositions may be manufactured in a known manner, for example by conventional processes such as mixing, dissolving, granulating, dredging, levitating, emulsifying, encapsulating, packaging, or lyophilizing.

The compounds described herein may be evaluated for efficiency and toxicity using known methods. For example, the toxicology of a particular compound or subset of compounds sharing certain chemical media may be established by determining in vitro toxicity for a cell line, such as a mammalian cell line, and preferably, of human. The results of such studies are often predictive of toxicity in animals, such as mammals, or more specifically, humans. Alternatively, the toxicity of particular compounds may be determined in an animal model, such as rats, mice, rabbits, dogs or monkeys, using known methods. The effectiveness of a particular compound may be established using various state of the art methods, such as in vitro methods, animal models, or human clinical experiments.

State-of-the-art in vitro models exist for almost every kind of condition, including conditions weakened by the compounds described herein, including cancer, cardiovascular disease, and various immune deficiencies, and infectious diseases. Similarly, acceptable animal models can be used to establish the effectiveness of chemicals in treating such conditions. In selecting a model to determine efficacy, the skilled artisan may be guided by the state of the art to choose an appropriate model, dose, route of administration, and regimen. Of course, human clinical experiments can also be used to determine the efficacy of a compound in humans.

When used as an anti-microbial, anti-cancer, or anti-inflammatory agent, the compounds described herein may be administered by oral or non-oral routes. When administered orally, they may be administered as a capsule, tablet, granule, spray, syrup, or the like. When administered non-orally, they may be administered as an aqueous suspension, an oily preparation or the like, or as a drip, suppository, ointment, ointment or the like, when administered by injection, subcutaneously, intraperitoneally, intravenously, intramuscularly, intradermally, or similar. Controlled release formulations, deposition formulations, and infusion pump delivery are similarly contemplated.

Compositions described herein in pharmaceutical compositions may also comprise a pharmaceutically acceptable carrier. Such compositions may 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). For example, such compositions may 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 may be prepared in conventional forms, either as liquid solutions or suspensions, satisfactory solid forms for solution or suspension in liquid prior to injection, or as emulsions.

Satisfactory excipients include, but are not limited to, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, hydrochloride cysteine, and the like. In addition, if desired, injectable pharmaceutical compositions may contain minor amounts of non-toxic auxiliary substances, such as wetting agents, pH protecting agents, and the like.

If desired, absorption enhancing preparations (e.g., liposomes) may be used.

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 may be adjusted to achieve a desired effect, but will depend on factors such as weight, diet, competing medications, and other factors that those skilled in the medical arts will recognize.

The products or compositions of the incorporation forms as described above may be used alone or in combination with each other or in combination with other therapeutic or diagnostic agents. These products may be used in vivo or in vitro. Useful dosages and most useful modes of administration will vary, depending on age, weight and animal treated, the particular compounds employed, and the specific use for which this composition or compositions are employed. The magnitude of a dose in the management or treatment for a particular disease will vary with the severity of the condition being treated and the route of administration, and depending on the condition of the disease and its severity, the compositions may be formulated and administered systemically or locally. A variety of formulation and administration techniques can be found in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, PA (1990).

To formulate the compounds of Formulas I-VI as an antimicrobial, anti-cancer, or anti-inflammatory agent, known surface active agents, excipients, softening agents, suspending agents and pharmaceutically acceptable film-forming substances and coverage aids. , and the like, may be used. Preferably alcohols, esters, sulfated aliphatic alcohols, and the like may be used as surface active agents; sucrose, glucose, lactose, starch, crystallized cellulose, mannitol, light anhydrous silicate, magnesium aluminate, magnesium metasilicate aluminate, synthetic aluminum silicate, calcium carbonate, sodium acid carbonate, calcium hydrogen phosphate, carboxymethyl cellulose calcium, and the like may be used as excipients; Magnesium stearate, talc, hardened oil and the like may be used as softening agents; coconut oil, olive oil, sesame oil, peanut oil, soybean oil may be used as suspending or lubricating agents; cellulose acetate phthalate as a derivative of a carbohydrate such as cellulose or sugar, or a methylacetate methacrylate copolymer, may be used as a polyvinyl derivative as suspending agents; and plasticizers such as phthalate ester and the like may be used as suspending agents. In addition to the above preferred ingredients, sweeteners, fragrances, colorants, preservatives and the like may be added to the administered formulation of the compound produced by the method of the invention, particularly when "the compound will be administered orally.

The compounds and compositions may be administered orally or non-orally to a human patient in the amount of from 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. daily to about 100 mg / kg / day of the active ingredient, preferably once daily or, less preferably, more than two to approximately ten times daily. Alternatively and also preferably, the compound produced by the method of incorporation forms may preferably be administered continuously in the amounts specified, for example, by an intravenous drip. Thus, for the example of a patient weighing 70 kilograms, the preferred daily dose of the active or anti-infective ingredient would be from about 0.07 mg / day to about 700 mg / day, and more preferably 7 mg / day to about 7 kg. grams / day. However, as will be appreciated by those skilled in the art, in certain situations it may be necessary to administer the anti-cancer, anti-inflammatory or anti-infective compound of the incorporation forms in amounts that exceed, or even far exceed, the range. dosage form described above to effectively and aggressively treat particularly advanced infections or cancers.

In case the antimicrobial produced by the incorporation form methods is used as a biochemical test reagent, the compound produced by the incorporation form methods inhibits disease progression when it is dissolved in an organic solvent or organic water solvent and is applied directly to any of several cultured cell systems.

Usable organic solvents include, for example, methanol, methyl sulfoxide, and the like. For example, the formulation may be a powder, granular, or other solid inhibitor, or a liquid inhibitor prepared using an organic solvent or an organic water solvent. While a preferred concentration of the compound produced by the incorporation method for use as an anti-microbial, anti-cancer or anti-tumor compound is generally in the range of about 1 to 100 μg / ml, the most appropriate amount of use will vary depending on type of cultured cell system and purpose of use as will be appreciated by those of ordinary skill in the art. Also, in certain applications it may be necessary or preferred that those of ordinary skill in the art use an amount outside the aforementioned range.

In one embodiment, the method for using a compound as an anti-microbial, anti-cancer or anti-inflammatory involves administering an effective amount of any of the compounds of Formulas I-VI or compositions of such compounds. In a preferred embodiment, the method involves administering the compound represented by Formula II to a patient in need of an antimicrobial, until the need is effectively reduced or preferably removed.

As should be understood by those skilled in the art, "need" is not an absolute term and only implies that the patient can benefit from the use of antimicrobial treatment. By "patient" is meant an organism that can benefit from the use of an antimicrobial. For example, any organism with B. Anthracis, Plasmodium Leishmania, Trypanosoma, and the like can benefit from the application of an antimicrobial that can in turn reduce the amount of microbes present in the patient. As another example, any cancerous organism, such as colorectal carcinoma, prostate carcinoma, breast adenocarcinoma, non-small cell lung carcinoma, ovarian carcinoma, melanoma, multiple myelomas, and the like may benefit from application. of an anti-cancer agent that can in turn reduce the amount of cancer present in the patient. In addition, any organism with inflammatory conditions such as rheumatic arthritis, asthma, multiple sclerosis, psoriasis, stroke, reperfusion damage, myocardial infarction, and the like may benefit from the application of an anti-inflammatory which may in turn reduce the amount of cells associated with the inflammatory response present in the patient. In one embodiment, the patient's health may not require an anti-microbial, anti-cancer, or anti-inflammatory drug to be administered, but the patient may still gain some benefit from reducing the level of microbes, cancer, or inflammatory cells present in the patient, and is thus in need. In one embodiment, the antimicrobial or anticancer agent is effective against one type of microbe or cancer, but not against other types; thus, it allows a high degree of selectivity in the treatment of the patient. In other embodiments, the anti-inflammatory may be effective against inflammatory conditions characterized by different cells associated with inflammation. Choosing such anti-microbial, anti-cancer or anti-inflammatory agent, the methods and results described in the Examples may be useful. In an alternative embodiment, the antimicrobial may be effective against a broad spectrum of microbes, preferably a broad spectrum of foreign bacteria, and more preferably harmful to the host organism. In embodiments, the anti-cancer and / or anti-inflammatory agent may be effective against a broad spectrum of cancers and inflammatory conditions / cells / substances. In yet another embodiment, the antimicrobial is effective against all microbes, even those native to the host. Examples of antimicrobial microbes include, but are not limited to, B. Antrhacis, Plasmodium, Leishmania, Trypanosoma, and the like. Still in additional embodiments, the anti-cancer agent is effective against a broad spectrum of cancers or all cancers. Examples of cancers against which the compounds may be effective include colorectal carcinoma, prostate carcinoma, breast adenocarcinoma, non-small cell lung carcinoma, ovarian carcinoma, multiple myelomas, melanoma, and the like. Exemplary inflammatory conditions against which the agents are effective include rheumatic arthritis, asthma, multiple sclerosis, psoriasis, stroke, myocardial infarction, and the like.

"Therapeutically effective amount", "pharmaceutically effective amount", or similar terms means the amount of drug or pharmaceutical agent that will result in a biological or medical response being sought in a cell, tissue, system, animal, or human. In a preferred embodiment, the medical response is sought by a researcher, veterinarian, medical doctor, or other clinician.

"Anti-microbial" refers to a compound that reduces the likelihood of survival of microbes. In one embodiment, the probability of survival is determined as a function of an individual microbe; thus the antimicrobial will increase the chance that an individual microbe will die. In one embodiment, the probability of survival is determined as a function of a population of microbes; thus, the antimicrobial will increase the chances of a decrease in the microbial population. In one embodiment, antimicrobial means antibiotic or other similar term. Such antimicrobials are capable of destroying or suppressing the growth or reproduction of microorganisms such as bacteria. For example, such antibacterials and other antimicrobials are described in Antibioties, Chemotherapeutics and Antibacterial Agents for Disease Control (M. Grayson, editor, 1982), and E. Gale et al. , The Molecular Basis of Antibiotic Aetion 2nd edition (1981). In another embodiment, an antimicrobial will not change the likelihood of survival, but will change the chances that microbes are somehow harmful to the host. For example, if the microbe secretes a substance that is harmful to the host, the antimicrobial may act on the microbe to stop secretion. In one embodiment, an antimicrobial, while increasing the likelihood that the microbe (s) will die, is minimally detrimental to neighboring, non-microbial cells. In an alternative embodiment, it is not important how harmful the antimicrobial is to neighboring, non-microbial cells as long as it reduces the probability of survival of the microbe.

"Anti-cancer agent" refers to a compound or composition including the compound that reduces the likelihood of survival of a cancer cell. In one embodiment, the probability 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. In one embodiment, survival probability is determined as a function of a cancer cell population; Thus, the anti-cancer agent will increase the chances that there will be a decrease in the cancer cell population. In one embodiment, anti-cancer agent means chemotherapeutic agent or other similar term.

A "chemotherapeutic agent" is a chemical compound useful in treating a neoplastic disease, such as cancer. Examples of chemotherapeutic agents include alkylating agents such as nitrogen mustard, ethylene imine and methylmelamine, alkyl sulfonate, nitrosourea, triazene, folic acid antagonists, nucleic acid metabolism anti-metabolites, antibiotics, pyrimidine analogs, fluorouracil, cisplatin, purine nucleosides, amines, amino acids, triazole nucleosides, corticosteroids, natural products such as vinca alkaloid, epipodophyllotoxin, antibiotics, enzymes, taxanes, biological response modifiers or antibodies to biological response modifiers, or others. agents; mixed agents such as platinum coordination complex, anthracenedione, anthracycline, substituted urea, methyl hydrazine derivatives, or adrenocortical suppressors; or a hormone or antagonist such as an adrenocorticosteroid, a progestin, an estrogen, an antiestrogen, an androgen, an anti-androgen, or an analogue of a gouadotropin releasing hormone. Specific examples include Adriamycin, Doxorubicin, 5-Fluorouracil, Cytosine arabinoside ("Ara-C"), Cyclophosphamide, Thio-tepa, Busulfan, Cytoxine, Taxol, Taxotero, Methotrexate, Cisplatin, Melfalane, Vinblastine, Bleomycin, Ephosamide, Etoposide C, Mitoxantrone, Vincristine, Vinorelbine, Carboplatin, Teniposide, Daunomycin, Carminomycin, Aminopterin, Dactinomycin, Mitomycin,

Speramycin, Melfalane, and other related nitrogen mustards. Also included in this definition are hormonal agents that act to regulate or inhibit the action of hormones in tumors such as tamoxifen and onapristone.

The anti-cancer agent can act directly on a cancer cell to kill the cell, induce cell death, prevent cell division, and similar cases. Alternatively, the anti-cancer agent may act indirectly on the cancer cell by limiting the cell's nutrients or blood supply, for example. Such anti-cancer agents are capable of destroying or suppressing cancer cell growth or reproduction, such as a colorectal carcinoma, a prostate carcinoma, a breast adenocarcinoma, a non-small cell lung carcinoma, an ovarian carcinoma, multiple myelomas, a melanoma, and the like.

A "neoplastic disease" or "neoplasm" refers to a cell or cell population, including a tumor or tissue (including cell suspensions such as bone marrow and fluids such as blood or serum), which exhibits abnormal growth through a greater cell proliferation than normal tissue. Neoplasms can be benign or malignant.

An "inflammatory condition" includes, for example, conditions such as ischemia, septic shock, autoimmune diseases, rheumatic arthritis, inflammatory bowel disease, systemic lupus erythematosus, multiple sclerosis, asthma, osteoarthritis, osteoporosis, fibrotic diseases, dermatoses, including psoriasis, atopic dermatitis and ultraviolet (UV) induced skin damage, psoriatic arthritis, alkylating spondylitis, organ and tissue rejection, Alzheimer's disease, stroke, arterosclerosis, restenosis, diabetes, glomerulonephritis, cancer, Hodgkins disease , cachexia, inflammation associated with infections and certain viral infections, including acquired immune deficiency syndrome (AIDS), adult respiratory distress syndrome and Ataxia Telangiestasia.

In one embodiment, a compound described, preferably a compound having Formulas I-VI, including those described herein, is considered effective as anti-microbial, anti-cancer, or anti-inflammatory if the compound can influence 10% of the microbes. , cancer cells, or inflammatory cells, for example. In a more preferred embodiment, the compound is effective if it can influence 10 to 50% of microbes, cancer cells, or inflammatory cells. In a more preferred embodiment, the compound is effective if it can influence 50-80% of microbes, cancer cells, or inflammatory cells. In a more preferred embodiment, the compound is effective if it can influence 80-95% of microbes, cancer cells, or inflammatory cells. In a more preferred embodiment, the compound is effective if it can influence 95-99% of microbes, cancer cells, or inflammatory cells. "Influence" is defined by the mechanism of action of 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 microbial death. Also, for example, if a compound prevents cancer cell division, then influence is a measure of cancer cell division prevention. For example, additionally, if a compound prevents the proliferation of inflammatory cells, then influence is a measure of the prevention of inflammatory cell proliferation. Not all mechanisms of action need to have the same percentage of effectiveness. In an alternative embodiment, a low percentage of effectiveness may be desirable if the lower degree of effectiveness is offset by other factors, such as compound specificity, for example. Thus a compound that is only 10% effective, for example, but exhibits few harmful side effects to the host, or to non-harmful microbes or cells, can still be considered effective.

In one embodiment, the compounds described herein are administered simply to remove microbes, cancer cells or inflammatory cells, and need not be administered to a patient. For example, in situations where microbes may present a problem, such as in food products, the compounds described herein may be administered directly to the products to reduce the risk of microbes in said products. Alternatively, the compounds may be used to reduce the level of microbes present in the surrounding environment, such as on working surfaces. As another example, the compounds may be administered ex vivo to a cell sample, such as a bone marrow or stem cell transplant to ensure that only non-cancerous cells will be introduced into the recipient. Once the compounds are administered they may optionally be removed. This may be particularly desirable in situations where operating surfaces or food products may come into contact with other surfaces or organisms that could present a risk of being damaged by the compounds. In an alternative embodiment, the compounds may be left on food products or on working surfaces to allow for greater protection. Whether or not this is an option will depend on the situation-related needs and risks associated with the compound, which can in part be determined as described in the examples below.

The following non-limiting examples describe preferred embodiments of the methods. Variations in the details of the particular methods employed and the precise chemical compositions obtained will undoubtedly be appreciated by those skilled in the art.

EXAMPLES

Example 1

FERMENTATION OF THE COMPOUNDS OF FORMULAS 1-7, II- 16, 11-17, 11-20, II-24C, 11-26, AND 11-28 USING CNB476 LINE

The CNB476 strain was grown in a 500 ml flask containing 100 ml of a vegetative medium consisting of the following per liter of deionized water: glucose, 4 g; Tripton bacto, 3 g; Casitone Bacto, 5 g; and synthetic sea salt (Instant Ocean, Aquarium Systems), 30 g. The first seed culture was incubated at 28 degrees C for 3 days in a rotary mixer operating at 250 rpm. Three five ml portions of the first seed culture were each inoculated into a 500 ml vial containing 100 ml of the vegetative medium. The second seed culture was incubated at 28 degrees C and 250 rpm in a rotary mixer for 2 days. Thirty-five five ml portions of the second seed culture were each inoculated into a 500 ml vial containing 100 ml of the vegetative medium. The third seed culture was incubated at 28 degrees C and 250 rpm in a rotary mixer for 2 days. Four hundred five ml portions of the third seed culture were each inoculated into a 500 ml vial containing 100 ml Production Medium A consisting of the following per liter of deionized water: starch, 10 g; yeast extract, 4 g; Hi-soy, 4 g; ferric sulfate, 40 mg; potassium bromide, 100 mg; calcium carbonate, 1 g; and synthetic sea salt (Instant Ocean, Aquarium Systems), 30 g. Production cultures were incubated at 28 degrees C and 250 rpm in rotary shakers for 1 day. Approximately 2 to 3 grams of sterile Amberlite XAD-7 resin was added to the production cultures. Production cultures were further incubated at 28 degrees C and 250 rpm in rotary shakers for 5 days and achieved a Compound 11-16 titer of approximately 200 mg / L. The culture broth was filtered through a perforated tissue to recover Amberlite XAD-7 resin. The resin was extracted twice with 6 liters of ethyl acetate followed by 1 time with 1.5 liters of ethyl acetate. The combined extracts were vacuum dried. The dried extract containing 3.8 grams of the compound of formula 11-16 and smaller amounts of the compounds of formula 11-20 and II-24C was then processed for recovery of the compounds of Formulas I-7, 11-16, 11. -20, II-24C, 11-26 and 11-28.

Example 2

FERMENTATION OF COMPOUNDS 1-7, 11-16, 11-17, II-20, II-24C, 11-26 AND 11-28, USING NPS21184 LINE

The 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; Hi-soy, 6 g; and synthetic sea salt (Instant Ocean, Aquarium Systems), 30 g. The first seed culture was incubated at 28 degrees C for 3 days in a rotary mixer operating at 250 rpm. Five ml of the first seed culture were inoculated into a 500 ml vial containing 100 ml of the vegetative medium. The second seed culture was incubated at 28 degrees C and 250 rpm in a rotary mixer for 2 days. Five ml of the second seed culture were inoculated into a 500 ml vial containing 100 ml of the vegetative medium. The third seed culture was incubated at 28 degrees C and 250 rpm in a rotary mixer for 2 days.

Five ml of the third seed culture was inoculated into a 500 ml vial containing 100 ml of Production Medium B consisting of the following per liter of deionized water: starch, 20 g; yeast extract, 4 g; Hi-soy, 8 g; ferric sulfate, 40 mg; potassium bromide, 100 mg; calcium carbonate, 1 g; and synthetic sea salt (Instant Ocean, Aquarium Systems), 30 g. Production cultures were incubated at 28 degrees C and 250 rpm in rotary shakers for 1 day. Approximately 2 to 3 grams of sterile Amberlite XAD-7 resin was added to the production culture. The production culture was further incubated at 28 degrees C and 250 rpm in a rotary mixer for 4 days and reached a titer of 350 - 400 mg / L for Compound 11-16. Alternatively, compound production can be achieved in a 42 L fermenter system using the NPS21184 strain. The 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; Hi-soy, 6 g; and synthetic sea salt (Instant Ocean, Aquarium Systems), 30 g. The first seed culture was incubated at 28 degrees C for 3 days in a rotary mixer operating at 250 rpm. Five ml of the first seed culture was inoculated into a 500 ml vial containing 100 ml of the vegetative medium. The second seed culture was incubated at 28 degrees C and 250 rpm in a rotary mixer for 2 days. Twenty ml of the second seed culture was inoculated into a 2.8 L Fernbach flask containing 400 ml of the vegetative medium. The third seed culture was incubated at 28 degrees C and 250 rpm in a rotary mixer for 2 days. 1.2 L of the third seed culture was inoculated into a 42 L fermentor containing 26 L of Production Medium A. Beo Production Medium Production Medium C with the following composition can also be used. Production Medium C consists of the following per liter of deionized water: starch, 15 g; yeast extract 6 g; Hi-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. Fermenter cultures were operated on the following parameters: temperature, 28 degrees C; stirring, 200 rpm; aeration, 13 L / min and back pressure, 4.5 psi. At 36 to 44 hours of the production cycle, approximately 600 grams of sterile Amberlite XAD-7 resin was added to the fermenter culture. The production culture was further incubated at the above operating parameters until the 4th day of the production cycle. The aeration rate has been decreased to 8 L / min. On day 5 of the production cycle, the fermenter culture reached a titer of about 300 mg / L for Compound 11-16. The culture broth was filtered through a perforated tissue to recover Amberlite XAD-7 resin. The resin was extracted twice with 4.5 liters of ethyl acetate followed by 1 time with 1.5 liters of ethyl acetate. The combined extracts were vacuum dried. The dried extract was then processed for recovery of the compounds of Formulas 1-7, 11-16 11-17, 11-20, II-24C, 11-26 and 11-28.

Example 3

PURIFICATION OF COMPOUNDS OF FORMULAS 1-7, II- 16, 11-20, II-24C, 11-26 AND 11-28

3A: Purification of Compounds of Formulas II-16, 11-20, II-24C, 11-26 and 11-28

The pure compounds of Formulas 11-16, 11-20, II-24C, 11-26 and 11-28 were obtained by flash chromatography followed by HPLC. Eight grams of crude extract containing 3.8 grams of Compound 11-16 and smaller amounts of 11-20, II-24C, 11-26 and 11-28 were processed by flash chromatography using a Biotage Flash 40i system and a Flash cartridge. 40M (KP-Sil Silica, 32-63 μπι, 90 grams). Flash chromatography was performed by the following step gradient:

<table> table see original document page 97 </column> </row> <table>

Fractions containing Compound 11-16 in an amount greater than or equal to 70% UV purity by HPLC were pooled and subjected to HPLC purification as described below to give Compound 11-16, together with 11-20 and II-24C as pure compounds. <table> table see original document page 98 </column> </row> <table>

The enriched fraction in Compound 11-16 (described above; ~ 70% purity with respect to Compound 11-16) was dissolved in acetone (60 mg / ml). Portions (950 μΐ) of this solution were injected into a normal phase HPLC column using the conditions described above. Compound 11-16 typically eluted after 14 minutes and compounds II-24C and 11-26 co-eluted with a single peak at 11 min. When source samples containing compounds 11-17, 11-20 and 11-28 were processed, Compound 11-17 eluted within 22 minutes, while 11-20 and 11-28 co-eluted within 23 minutes during washing. with 100% ethyl acetate. Fractions containing Compound 11-16 and smaller analogs were pooled based on the composition of the present compounds and evaporated under reduced pressure on a rotary evaporator. This process yielded pure Compound A as well as separate fractions containing minor Compounds 11-20, II-24C, 11-26 and 11-28, which were further purified as described below.

Samples containing the II-24C and 11-26 generated by the procedure described above were separated using reverse phase preparative HPLC as follows. The sample (70 mg) was dissolved in acetonitrile at a concentration of 10 mg / ml, and 500 μl were loaded onto a d.i-sized HPLC column. (inner diameter) 21 mm by 15 cm long containing an Eclipse XDB-C18 bracket. The solvent gradient increased linearly from 15% acetonitrile / 85% water to 100% acetonitrile in 23 minutes at a flow rate of 14.5 ml / min. The solvent composition was maintained at 100% acetonitrile for 3 minutes before returning to the initial 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 μl acetone in a 1.5 mL ν-bottom CLAP vessel.

This vial was then placed into a tightly sealed larger container containing 1 ml of pentane. Satisfactory crystals for X-ray crystallography experiments were observed along the sides and bottom of the inner flask after 48 hours incubation at 4 ° C. Crystallographic data were collected on a Bruker SMART APEX CCD X-ray diffractometer (F (000) = 2656, Μοκα radiation, λ = 0.71073 Â, μ = 0.264 mm "1, T = 100 ° K) at UCSD Crystallography Facility and the refinement method used was that of least squares in full matrix at F2.PNI-2065 crystals data: C15H2OCINO4, MW = 313.77, tetragonal, space group P4 (l) 2 (l) 2, a = b = 11,4901 (3) Â, c = 46,444 (2) Â, α = β = γ = 90 °, vol = 6131.6 (3) A3, Z = 16, Pcaic = 1, 360 g cm "3, crystal size, 0.30 χ 0.15 χ 0.07 mm3, range 1,7, 1.75-26.00 °, 35367 collected reflections, 6025 independent reflections (Rint = O, 0480), Final R (Ι> 2σ (Ι)): R1 = 0.0369, WR2 = 0.0794, GOF = 1.060.

To separate 11-28 from 11-20, an isocratic counter phase method was employed. The sample (69.2 mg) containing both compounds was dissolved in acetonitrile at a concentration of 10 mg / ml, and 500 μl were loaded onto a counter phase CLAP column (ACE 5 μ C18-HL, 15 cm X 21 mm di) by injection. A 27% acetonitrile / 63% water isocratic solvent system at a flow rate of 14.5 ml / min was used to separate Compounds 11-28 and 11-20, which eluted after 14 and 16 minutes, respectively. Fractions containing the compounds of interest were immediately evaporated under reduced pressure at room temperature on a rotary evaporator. The samples were then loaded onto a short silica column and eluted with 10 ml of 70% hexane / 30% acetone to remove additional impurities.

Samples generated from the normal-phase CLAP preparation method described above that contained 11-20 but were free of 11-28 could also be triturated with 100% EtOAc to remove minor lipophilic impurities.

Compound of Formula 11-16: UV (Aeetonitrile / H2 O) λ max 225 (sh) nm. Low Res Mass: m / z 314 (M + H), 336 (M + Na).

Compound of Formula 11-20: (Aeetonitrile / H2 O) λmaχ 225 (sh) nm. Low Res Mass: m / z 266 (M + H); HRMS (ESI), m / z 266.1396 (M + H), Acalc = 1.2 ppm. Fig. 16 describes the 1H NMR spectrum of a compound having the structure of Formula 11-20.

Compound of Formula II-24C: (Acetonitrile / H2 O) λmax 225 (sh) nm. Low Res Mass: m / z 328 (M + H), 350 (M + Na); HRMS (ESI), m / z 328, 1309 (M + H), Acalc = -2.0 ppm, C 16 H 23 NO 4 Cl. Fig. 19 describes the 1H NMR spectrum of a compound having the structure of Formula II-24C.

Compound of Formula 11-26: (Aeetonitrile / H2 O) λmax 225 (sh) nm; HRMS (ESI), m / z 314.1158 (M + H), Acalc = -0.4 ppm, C15 H21 NO4 Cl. Fig. 23 describes the 1H NMR spectrum of a compound having the structure of Formula 11-26 in DMS0-d6.

Compound of Formula 11-28: (Acetonitrile / H2 O) λmaχ 225 (sh) nm; HRMS (ESI), m / z 266, 1388 (M + H), Acalc = -1.8 ppm, C 14 H 20 NO 4. Fig. 26 describes the 1 H NMR spectrum of a compound having the structure of Formula 11-28 in DMS0-d6.

3B: Purification of Formula 1-7 Compound

A Biotage Flash 75Li system with a KP-Sil 75L Flash cartridge was used to process the filtered crude extract (10.0 g), enriched in compound 11-16 and containing the compound of Formula 1-7. The crude extract was dissolved at a concentration of 107 mg / ml in acetone and loaded directly into the cartridge. The following solvent step gradient was then processed through the cartridge at a flow rate between 235 ml / min and 250 ml / min.

1. 10% EtOAc in n-Heptane (3.2 L)

2. 25% EtOAc in n-Heptane (16 L)

3. 30% EtOAc in n-Heptane (5.4 L)

The enriched fractions of compound 11-16 were pooled and concentrated by rotavap until -5% of the total pooled volume of solvent remained. The solvent was removed, leaving behind the white solid.

A crystallization was then performed on the solid by dissolving the sample (4.56 g) in 1: 1 acetone: n-heptane (910 ml). The solvent was slowly evaporated using a rotary evaporator until the solvent was reduced to approximately 43% of its original volume. The (supernatant) solution was removed and concentrated (598 mg).

The supernatant was dissolved in acetone (80 mg / ml). Portions (500 µl) of this solution were injected into a normal phase HPLC column using the conditions described above for normal phase purification of Compounds 11-16, II-24C, 11-26 and 11-28. The compound of Formula 1-7 eluted at 7.5 minutes as a pure compound.

Compound of Formula 1-7 (Fig. 58): UV (Acetonitrile / H2 O) λmax 225 (sh) nm. Low Res Mass: m / z 298 (M + H), 320 (M + Na).

Example 4

FERMENTATION OF COMPOUNDS OF FORMULAS 11-17, II-

18, and 11-27

Fermentation of Starting Compounds 11-17 and 11-18 and Formula 11-27 Compound

The CNB476 strain 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; Tryptone Bacto, 3 g; Casitone Bacto, 5 g; and synthetic sea salt (Instant Ocean, Aquarium Systems), 30 g. The first seed culture was incubated at 28 degrees C for 3 days in a rotary mixer operating at 250 rpm. Five ml of 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, 10 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 culture was incubated at 28 degrees C for 7 days in a rotary mixer operating at 250 rpm. Approximately 2 to 3 grams of sterile Amberlite XAD-7 resin was added to the second seed culture. The second seed culture was further incubated at 28 degrees C for 2 days in a rotary mixer operating at 250 rpm. Five ml of the second seed culture was inoculated into a 500 ml vial containing 100 ml of the second vegetative medium. The third seed culture was incubated at 28 degrees C for 1 day in a rotary mixer operating at 250 rpm. Approximately 2 to 3 grams of sterile Amberlite XAD-7 resin was added to the third seed culture. The third seed culture was further incubated at 28 degrees C for 2 days in a rotary mixer operating at 250 rpm. Five ml of the third culture was inoculated into a 500 ml vial containing 100 ml of the second vegetative medium. The fourth seed culture was incubated at 28 degrees C for 1 day in a rotary mixer operating at 250 rpm. Approximately 2 to 3 grams of sterile Amberlite XAD-7 resin was added to the fourth seed culture. The fourth seed culture was incubated at 28 degrees C for 1 day in a rotary mixer operating at 250 rpm. Ten five ml portions of the fourth seed culture were each inoculated into 500 ml vials containing 100 ml of the second vegetative medium. The fifth seed culture was incubated at 28 degrees C for 1 day in a rotary mixer operating at 250 rpm. Approximately 2 to 3 grams of sterile Amberlite XAD-7 resin was added to the fifth seed culture. The fifth seed culture was further incubated at 28 degrees C for 3 days in a rotary mixer operating at 250 rpm. One hundred and fifty four ml portions of the fifth seed culture were each inoculated into a 500 ml flask containing 100 ml of production medium having the same composition as the second vegetative medium. Approximately 2 to 3 grams of sterile Amberlite XAD-7 resin was also added to the production culture. Production cultures were incubated at 28 degrees C for 6 days in a rotary mixer operating at 250 rpm. The culture broth was filtered through a perforated tissue to recover Amberlite XAD-7 resin. The resin was extracted 2 times with 3 liters of ethyl acetate followed by 1 time with 1 liter of ethyl acetate. The combined extracts were dried in vacuo. The dried extract containing 0.42 g of Compound 11-17 and 0.16 grams of Compound 11-18 was then processed for recovery of the compounds.

Example 5

PURIFICATION OF COMPOUNDS OF FORMULAS 11-17, II-18 AND 11-27

Pure compounds 11-17 and 11-18 were obtained by reverse phase HPLC as described below:

<table> table see original document page 103 </column> </row> <table>

The crude extract (100 mg) was dissolved in 15 ml of acetonitrile. Portions (900 μΐ) of this solution were injected into a reverse phase HPLC column using the conditions described above. Compounds 11-17 and 11-18 eluted at 7.5 and 9 minutes, respectively. Fractions containing pure compounds were first concentrated using nitrogen to remove organic solvent. The remaining solution was then frozen and lyophilized to dry.

An alternative purification method for Compounds 11-17 and 11-18 was developed for large scale purification and the involved fractionation of the crude extract on a normal phase VLC column. Under these conditions, sufficient amounts of several minor metabolites were identified, including Compound 11-27. The crude extract (2.4 g) was dissolved in acetone (10 ml) and this solution adsorbed onto silica gel (10 cc) by vacuum drying. The adsorbed crude extract was loaded onto a normal phase silica VLC column (250 silica gel, column dimensions diameter = 2.5 cm by 15 cm long) and washed with a hexane / EtOAc step gradient, increasing the percentage of hexane in the 5% steps (100 ml solvent per step). Most of Compound 11-16 eluted in the 60% hexane / 40% EtOAc wash while most of Compound 11-17 eluted in the 50% hexane / 50% ethyl acetate wash. Final separation of the compounds was achieved using HPLC C18 chromatography (ACE 5 μ C18-HL, 150 mm X 21 mm d.i.) using an isocratic solvent system consisting of 35% ACN / 65% H 2 O. Under these conditions Compound 11-27 eluted in 11 minutes, Compound 11-17 eluted in 12.00 minutes, traces of Compound 11-16 eluded in 23.5 minutes, and Compound 11-18 eluded in 25.5 minutes. minutes The resulting samples were vacuum dried without any heat to remove the aqueous solvent mixture. The spectroscopic data for these Compound 11-16 and Compound 11-18 samples were identical to those prepared by the above purification methods. The Compound 11-18 sample contained 8% of the lactone hydrolysis product and was further purified by washing through a normal phase silica plug (1 cm in diameter by 2 cm in height) and eluting using a mixture of 20% EtOAc / 80% Hexane solvent (25 mL). The resulting sample contained pure Compound 11-18.

The fractions containing Compound 11-27 described above were further purified using semi-normal preparative phase HPLC (Phenomenex Luna Si 10 μ, 100 Å; 250 χ 10 mm di) using an increasing solvent gradient of 100% hexane to 100 % EtOAc for 20 minutes with a flow rate of 4 ml / min. Compound 11-27 eluted as a pure compound after 11.5 minutes (0.8 mg, 0.03% yield isolated from dry weight weight).

Compound of formula 11-17: UV (Acetonitrile / H2 O) λ max 225 (sh) nm. High Res. Mass (APCI): m / z 280, 156 (M + H), Acalc = 2.2 ppm, C 15 H 22 NO 4. Fig. 49 shows the 1 H NMR spectrum of a compound having the structure of formula 11-17.

Compound of formula 11-18: UV (Acetonitrile / H2 O) λ max 225 (sh) nm. High Res. Mass (APCI): m / z 358.065 (M + H), Acalc = -1.9 ppm, C15 H21 NO4 Br. 1H NMR in DMS0-d6. Fig. 50 shows the 1 H NMR spectrum of a compound having the structure of formula II-18.

Compound 11-27: UV (Acetonitrile / H2 O) λ max 225 (sh) nm. MS (HR-ESI), m / z 280, 1556 (M + H), Acalc = 2.7 ppm (C15H22NO4); 1H NMR DMSO-d6, see fig. 53. Example 6

PREPARATION OF THE COMPOUND OF FORMULA 11-19 FROM COMPOUND 11-16

A sample of Compound 11-16 (250 mg) was added to a solution of sodium iodide acetone (1.5 g in 10 mL) and the resulting mixture was stirred for 6 days. The solution was then filtered through a 0.45 micron syringe filter and injected directly into a normal phase silica CLAP column (Phenomenex Luna 10 μ, Silica, 25 cm χ 21.2 mm) in 0.95 portions. ml. HPLC conditions for the separation of the compound of Formula 11-19 from unreacted Compound 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 of each of the 10 injections were pooled to obtain 35 mg of Compound 11-19.

Compound 11-19: UV (Acetonitrile / H2 O) λπ λ 225 (sh) nm. ESMS, m / z 406.0 (M + H); HRMS (ESI), m / z 406.0513 (M + H) +, Acalc = -0.5 ppm, C 15 H 2INO 4 I; 1H NMR DMS0-d6, see fig. 9).

<formula> formula see original document page 105 </formula>

Example 7

SUMMARY OF COMPOUNDS OF FORMULAS II-2, II-3, AND II-4

The compounds of Formulas II-2, II-3 and II-4 may be synthesized from Compounds 11-16, 11-17, and 11-18, respectively, by catalytic hydrogenation.

Exemplary Representation of Synthesis <formula> formula see original document page 106 </formula>

Example 7Α: Catalytic Hydrogenation of Starting Compound 11-16

Compound of formula 11-16 (10 mg) was dissolved in acetone (5 mL) in a scintillation vial (20 mL) to which was added 10% (w / w) Pd / C (1-2 mg) and a magnetic stir bar. The reaction mixture was stirred in a hydrogen atmosphere at room temperature for approximately 15 hours. The reaction mixture was filtered through a 3 cc silica column and washed with acetone. The filtrate was filtered again through a 0.2 µm Gelman Acrodisc to remove any traces of catalyst. The solvent was evaporated from the filtrate under reduced pressure to obtain the compound of Formula II-2 as a pure white powder. UV (acetonitrile / H2 O): λ max 225 (sh) nm. Fig. 10 depicts the NMR spectrum of the compound of Formula II-2 in DMSO-d6. Fig. 11 describes the low resolution mass spectrum of the compound of Formula II-2: m / z 316 (M + H), 338 (M + Na).

Example 7B: Catalytic Hydrogenation of the Compound of Formula 11-17

Compound of formula 11-17 (5 mg) was dissolved in acetone (3 mL) in a scintillation vial (20 mL) to which was added 10% (w / w) Pd / C (about 1 mg). and a magnetic stir bar. The reaction mixture was stirred in a hydrogen atmosphere at room temperature for approximately 15 hours. The reaction mixture was filtered through a 0.2 μπι Gelman Acrodisc to remove the catalyst. The solvent was evaporated from the filtrate to obtain the compound of Formula II-3 as a white powder which was purified by normal phase HPLC using the following conditions:

<table> table see original document page 107 </column> </row> <table>

The compound of Formula II-3 eluted in 22.5 min as a pure compound. UV (acetonitrile / H2 O): λ max 225 (sh) nm. Fig. 12 describes the NMR spectrum of the compound of Formula 11-3 in DMSO-d6. Fig. 13 describes the low resolution mass spectrum of the compound of Formula II-3: m / z 282 (M + H), 304 (M + Na).

Example 7C: Catalytic Hydrogenation of the Compound of Formula 11-18

3.2 mg of Compound 11-18 was dissolved in acetone (3 mL) in a scintillation vial (20 mL) to which was added 10% (w / w) Pd / C (approximately 1 mg) and a magnetic stir bar. The reaction mixture was stirred under a hydrogen atmosphere at room temperature for approximately 15 hours. The reaction mixture was filtered through a 0.2 μια Gelman Acrodisc to remove the catalyst. The solvent was evaporated from the filtrate to obtain the compound of Formula II-4 as a white powder which was further purified by normal phase HPLC using the following conditions:

<table> table see original document page 107 </column> </row> <table>

The Compound of Formula II-4 eluted in 16.5 min as a pure compound. UV (acetonitrile / H2 O): λ max 225 (sh) nm. Fig. 14 describes the NMR spectrum of the compound of Formula II-4 in DMSO-d6. Fig. 15 describes the low resolution mass spectrum of the compound of Formula II-3: m / z 360 (M + H), 382 (M + Na).

Additionally, high resolution mass spectrometry data were obtained for compounds II-2, II-3, and II-4. Compound II-2: HRMS (ESI), m / z 316.1305 [M + H] +, Acalc = -3.5 ppm, C15 H23 NO4 Cl. Compound II-3: HRMS (ESI), m / z 282, 1706 [M + H] Acalc = 0.3 ppm, C 15 H 24 NO 4. Compound II-4: HRMS (ESI), m / z 360, 0798 [M + H] +, Acalc = -3.4 ppm, C15 H23 NO4 Br.

Example 8

SUMMARY OF THE COMPOUNDS OF FORMULA II-5A AND II-5B The compounds of Formula II-5A and Formula II-5B may be synthesized from the compound of Formula 11-16 by epoxidation with AmCPB.

Compound of formula 11-16 (101 mg, 0.32 mmol) was dissolved in methylene chloride (30 mL) in a 100 mL round bottom flask to which 79 mg (0.46 mmol) of meta-chloroperbenzoic acid was added. (AmCPB) and a magnetic stir bar. The reaction mixture was stirred at room temperature for approximately 18 hours. The reaction mixture was poured into a 20 cc silica flash column and eluted with 120 mL of CH 2 Cl 2, 75 mL of 1: 1 ethyl acetate / hexane and finally with 40 mL of 100% ethyl acetate. The 1: 1 ethyl acetate / hexane fractions obtained a mixture of epoxy derivative diastereomers of Formulas II-5A and II-5B, which were separated by normal phase HPLC using the following conditions:

<table> table see original document page 108 </column> </row> <table>

The compounds of Formulas II-5A (major product) and II-5B (minor product) eluted at 21.5 and 19 min, respectively, as pure compounds. Compound II-5B was further chromatographed on a 3 cc silica flash column to remove traces of the chlorobenzoic acid reagent. Chemical Structures:

<formula> formula see original document page 109 </formula>

Structural characterization

Formula II-5A: UV (Acetonitrile / H2 O) λ max 225 (sh) nm. Low Res Mass: m / z 330 (M + H), 352 (M + Na). HRMS (ESI), m / z 330.1099 [M + H] +, Acalc = -2.9 PPM, C15 H21 NO5 Cl. Figs. 16 and 17, respectively, describe the 1H NMR spectrum of Formula II-5A and the mass spectrum of Formula II-5A.

Formula II-5B: UV (Acetonitrile / H2 O) λmax 225 sh) nm. Low Res Mass: m / z 330 (M + H), 352 (M + Na). HRMS (ESI), m / z 330, 1105 [M + H] +, Acalc = -0.9 PPM, C15 H21 NO5 Cl. Figs. 18 and 19, respectively, describe the 1H NMR spectrum of II-5B and the mass spectrum of II-5B.

Example 9

SUMMARY OF COMPOUNDS OF FORMULAS IV-I, IV-2, IV-3 AND IV-4

Synthesis of diol derivatives (Formula IV-2)

Diols can be synthesized by non-acute dihydroxylation using AD mix-α and β; AD mix-a is a premix of four reagents, K2OsO2 (OH) 4; K 2 CO 3; K3 Fe (CN) 6; (DHQ) 2 -PHAL [1,4-bis (9-0-dihydroquinine) phthalazine] and AD mix-β is a premix of K2OsO2 (OH) 4; K 2 CO 3; K3 Fe (CN) 6; (DHQD) 2-PHA1 [1,4-bis (9-0-dihydroquinidine) phthalazine] which are commercially available from the company Aldrich. Diol can also be synthesized by acid or base hydrolysis of epoxy compounds (Formulas II-5A and II-5B) which may be different from products obtained by non-acute dihydroxylation at their stereochemistry with carbons bearing groups. hydroxyl.

Non-acute dihydroxylation of Compounds 11-16, 11-17 and 11-18

Any of the compounds of formulas 11-16, II- 17 and 11-18 may be used as a starting compound. In the example below, the compound of formula 11-16 is used. The starting compound is dissolved in t-butanol / water in a round bottom flask to which AD mix-α or β is added and a magnetic stir bar. The reaction is monitored by silica TLC as well as by a mass spectrometer. Pure diols are obtained by common work 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 the same side.

<formula> formula see original document page 110 </formula>

Nucleophilic ring opening of (II-5) compounds

The epoxy ring is opened with various nucleophiles such as NaCN, NaN3, NaOAc, HBr, etc. to replace multiple groups on the cyclohexane ring with one hydroxyl group on one side. Examples: <formula> formula see original document page 111 </formula>

The epoxy is opened with HCl to make Formula IV-

<formula> formula see original document page 111 </formula>

The compound of Formula II-5A (3.3 mg) was dissolved in acetonitrile (0.5 ml) in a 1 dram flask to which 5% HCl (500 μm) and a magnetic stir bar were added. The reaction mixture was stirred at room temperature for about one hour. The reaction was monitored by mass spectrometry. The reaction mixture was injected directly into a normal phase HPLC to obtain the compound of Formula IV-3C as a pure compound without any processing. The HPLC conditions used for purification were as follows: Phenomenex Luna 10μ silica column (25 cm χ 21.2 mm di) with a 25% to 80% EtOAc / Hex solvent gradient for 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. The compound of Formula IV-3C eluted at approximately 18 min (2.2 mg). Compound of Formula IV-3C: UV (Acetonitrile / H2 O) λmaχ 225 (sh) nm; ESMS, m / z 366 (M + H), 388 (M + Na); HRMS (ESI), m / z 366.0875 [M + H] +, Acalc = 0.0 ppm, C 15 H 22 NO 5 Cl 2; 1H NMR in DMS0-d6 (Fig. 20). Stereochemistry of the compound of Formula IV-3C was determined based on coupling constants observed in the 1: 1 cyclohexane ring of C6D6 / DMS0-d6 (Fig. 21).

<formula> formula see original document page 112 </formula>

Reducing epoxide ring opening (II-5): The compound of the Formula treated with metal hydroxides as a BH3-THF complex to make compound of Formula IV-4. <formula> formula see original document page 113 </formula>

Example 10

SUMMARY OF THE COMPOUNDS OF FORMULAS II-13C AND II-8C The compound of formula 11-16 (30 mg) was

It was dissolved in CH 2 Cl 2 (6 mL) in a scintillation vial (20 mL) to which was added Dess-Martin Periodinane (122 mg) and a magnetic stir bar. The reaction mixture was stirred at room temperature for approximately 2 hours. Reaction progress was monitored by TLC (Hex: EtOAc, 6: 4) and analytical HPLC. From the reaction mixture, the volume of solvent was reduced to one third, absorbed on silica gel, poured onto a 20 cc silica flash column and eluted into 20 ml fractions using a 10 to 100 Hexane / EtOAc gradient. %. The fraction eluted with 30% EtOAc in Hexane contained a mixture of Formula II-13C rotamers in a ratio of 1.5: 8.5. The mixture was further purified by normal phase HPLC using the Phenomenex Luna 10 μ (25 cm χ 21.2 mm di) silica column with a solvent gradient of 25% to 80% EtOAc / Hex for 19 min, 80 ° C. 100% EtOAc for 1 min, maintained at 100% EtOAc for 5 min, at a flow rate of 14.5 ml / min. An ELSD was used to monitor the purification process. The compound of Formula II-13C eluted at 13.0 and 13.2 min as a mixture of rotamers with a ratio of 1.5: 8.5 (7 mg). Formula II-13C: UV (Acetonitrile / H2 O) λ max 226 (sh) and 300 (sh) nm; ESMS, m / z 312 (M + H) +, 334 (M + Na) +; HRMS (ESI), m / z 312.1017 [Μ + Η] Acalc = 4.5 ppm, C15 H19 NO4 Cl; 1H NMR in DMS0-d6 (see Fig. 22).

The mixture of Formula II-13C rotamers (4 mg) was dissolved in acetone (1 ml) in a scintillation vial (20 ml) to which a catalytic amount (0.5 mg) of 10% (wt / wt) was added. ) of Pd / C and a magnetic stir bar. The reaction mixture was stirred in a hydrogen atmosphere at room temperature for approximately 15 hours. The reaction mixture was filtered through a 0.2 μιη Gelman Acrodisc to remove the catalyst. The solvent was evaporated from the filtrate to obtain the compound of Formula II-8C as a colorless gum which was further purified by normal phase HPLC using a Phenomenex Luna 10 μ (25 cm χ 21.2 mm di) silica column with a solvent gradient 25% to 80% EtOAc / Hex for 19 min, 80 to 100% EtOAc for 1 min, maintained at 100% EtOAc for 5 min at a flow rate of 14.5 ml / min. An ELSD was used to monitor the purification process. The compound of Formula II-8C (1 mg) eluted at 13.5 min as a pure compound. Formula II-8C: UV (Acetonitrile / H2 O) λπιβχ 225 (sh) nm; ESMS, m / z 314 (M + H) +, 336 (M + Na) +; HRMS (ESI), m / z 314.1149 [M + H] +, Acalc = 3.3 ppm, C 15 H 2INO 4 Cl; 1H NMR in DMS0-d6 (see Fig. 23).

<formula> formula see original document page 114 </formula>

Example 11

SYNTHESIS OF COMPOUND 11-25 FROM COMPOUND II-13C The mixture of Formula II-13C rotamers (5 mg) was dissolved in dimethoxy ethane (monoglime; 1.5 ml) in a scintillation vial (20 ml). ) to which water (15 μΐ (1% of final solution concentration)) and a magnetic stir bar were added. The above solution was cooled to -78 ° C in a dry ice-acetone bath, and a solution of sodium borohydride (3.7 mg NaBH4 in 0.5 ml monoglime (created to allow a slow addition)) was added by dripping. The reaction mixture was stirred at -78 ° C for approximately 14 minutes. The reaction mixture was acidified using 2 ml of a 4% HCl solution in water and extracted with CH 2 Cl 2. The organic layer was evaporated to give the compound of Formula 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 silica column. Phenomenex Luna 10 μ (25 cm χ 21.2 mm di). The mobile phase was 24% EtOAc / 76% Hexane, which was kept isocratic for 19 min, followed by a linear gradient of 24% to 100% EtOAc for 1 min, and maintained at 100% EtOAc for 3 min; The flow rate was 25 ml / min. An ELSD was used to monitor the purification process. The compound of Formula 11-25 (1.5 mg) eluted at 11.64 min as a pure compound. Compound of Formula 11-25: UV (Acetonitrile / H 2 O) λ max 225 (sh) nm; ESMS, m / z 314 (M + H) +, 336 (M + Na) HRMS (ESI), m / z 314.1154 [M + H] +, Acalc = -0.6 ppm, C 15 H 21 NO 4 Cl; 1H NMR in DMS0-d5 (see Fig. 24).

<formula> formula see original document page 115 </formula> Example 12

SUMMARY OF THE COMPOUNDS OF FORMULAS 11-31, 11-32 AND 11-49 FROM II-13C; AND COMPOUNDS OF FORMS 11-33, 11-34, 11-35 AND 11-36 FROM 11-31 AND 11-32

A mixture of rotamers 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) was added. ) of Pd / C and a magnetic stir bar. The reaction mixture was stirred at room temperature for approximately 15 hours. The reaction mixture was filtered through a 0.2 μπι Gelman Acrodisc to remove the catalyst. The solvent was evaporated from the filtrate to obtain a mixture of hydroxy derivative diastereomers of Formulas II-31 and ΊΙ-32 (1: 1), and a smaller compound 11-49 which was separated by reverse phase HPLC using an ACE column. 5 μ C18 (150 mm χ 22 mm di) with a solvent gradient of 90% to 30% H20 / Acetonitrile for 15 min, 70 to 100% Acetonitrile for 5 min, maintained at 100% Acetonitrile for 4 min, at a flow rate of 14.5 ml / min. A diode array detector was used to monitor the purification process. Compounds 11-31 (2 mg), 11-32 (2 mg) and 11-49 (0.2 mg) eluted at 10.6, 10.8 and 11.54 min, respectively, as pure compounds. Compound 11-31: UV (Acetonitrile / H 2 O) λ max 250 (sh) nm; ESMS m / z 328.1 (M + H) + and 350.0 (M + Na) +. Compound 11-32: UV (Acetonitrile / H2 O) λ max 250 (sh) nm; ESMS, m / z 328.1 (M + H) + and 350.0 (M + Na) +. Compound 11-49: UV (Acetonitrile / H 2 O) λ max 250 (sh) and 320 nm; ESMS, m / z 326.0 (M + H) +, 343.1 (M + H 2 O) + and 348.0 (M + Na) +.

In an alternative method, compounds 11-31, II- 32 and 11-49 were separated by normal phase HPLC using a Phenomenex Luna 10 μ (25 cm χ 21.2 mm di) silica column with a solvent gradient. 10% to 100% Hexane / EtOAc for 24 min, maintained at 100% EtOAc for 3 min at a flow rate of 14.5 ml / min. ELSD was used to monitor the purification process.

The ketone of the compounds of Formulas 11-31 and II-32 may be reduced by using sodium borohydride at 0 to -10 ° C in monoglime solvent for approximately 14 minutes. The reaction mixture may be acidified using a 4% HCl solution in water and extracted with CH 2 Cl 2. The organic layer may be evaporated to obtain mixtures of compounds of Formulas 11-33, 11-34, 11-35 and 11-36 which may be separated by chromatographic methods.

<formula> formula see original document page 117 </formula>

Example 13

SUMMARY OF COMPOUND FORMULA 11-21 FROM COMPOSITE 11-19

Acetone (7.5 mL) was vigorously mixed with 5 N NaOH (3 mL) and the resulting mixture evaporated in vacuo to a minimum volume. A 100 μΐ sample of this solution was mixed with the compound of Formula 11-19 (6.2 mg) in acetone (1 ml) and the resulting biphasic mixture was vortexed for 2 minutes. The reaction solution was immediately subjected to a preparative C18 HPLC. Purification conditions involved a linear gradient of 10% acetonitrile / 90% water to 90% acetonitrile / 10% water for 17 minutes using a 5 μ C18 ACE CLAP column with dimensions 22 mm (di) per 150 mm in length.

The compound of Formula 11-21 eluted at 9.1 minutes under these conditions to obtain 0.55 mg of compound. Compound of Formula II-21: UV (Acetonitrile / H2 O) λmax 225 (sh), ESMS, m / z 296.1 (M + H); 1H NMR in DMSOd6 (see fig. 25).

<formula> formula see original document page 118 </formula>

Example 14

SUMMARY OF COMPOUND FORMULA 11-22 FROM COMPOSITE 11-19

A 60 mg sample of sodium propionate was added to a solution of the compound of Formula 11-19 (5.3 mg) in DMSO (1 ml) and the mixture was sonicated for 5 minutes, however sodium propionate did not dissolve completely. .

After 45 minutes, the solution was filtered through a 0.45 μ syringe filter and purified directly using CLAP. Purification conditions involved a linear gradient of 10% acetonitrile / 90% water to 90% acetonitrile / 10% water for 17 minutes using a 5 μ C18 ACAP 5 µ C18 CLAP column per 150 mm in length. Under these conditions, the compound of Formula 11-22 eluted at 12.3 minutes to obtain 0.7 mg of compound (15% isolated yield). UV (Acetonitrile / H2 O) λιι χ 225 (sh), ESMS, m / z 352.2 (M + H); HRMS (ESI), m / z 352.1762 [M + H] +, Acalc = 0.6 ppm, C18H26NO6; 1H NMR in DMS0-d6 (see Fig. 26).

<formula> formula see original document page 119 </formula>

Example 15

Synthesis of the compound of Formula 11-29 from 11-19

A sample of NaN3 (80 mg) was dissolved in DMSO (1 ml) and transferred to a flask containing Compound 11-19 (6.2 mg) which was contaminated with approximately 10% of the contaminating Compound 11-16 by 12.5. minutes (4.2 mg, 85% yield). A 2.4 mg portion of Compound 11-29 was further purified using HPLC C18 chromatography (ACE 5 μ C18 - HL, 150 mm X 21 mm di)) using an isocratic solvent gradient consisting of 35% acetonitrile / 65 ° C. % H2O. Under these conditions Compound 11-29 eluted after 20 minutes, while Compound 11-16 eluted after 21.5 minutes. The resulting sample consisting of 1.1 mg of Compound 11-29 was used for characterization in biological assays.

Compound 11-29: UV (Acetonitrile / H2 O) λmax 225 (sh), ESMS, m / z 321.1 (M + H); 1H NMR in DMS0-d6 (see Fig. 55). <formula> formula see original document page 120 </formula>

Example 16

SUMMARY OF COMPOUNDS OF FORMULAS 11-37 AND 11-38 FROM Compound 11-19

The compounds of Formula 11-37 and 11-38 may be prepared from the compound of Formula 11-19 by cyano dehalogenation or thio cyanate dehalogenation, respectively. Compound 11-19 may be treated with NaCN or KCN to obtain Compound 11-37. Alternatively, Compound 11-19 may be treated with NaSCN or KSCN to obtain Compound 11-38.

Synthesis of the compound of Formula 11-38 from mmol) was dissolved in 1.5 ml acetone in a scintillation vial (20 ml) to which sodium thio cyanate (10.0 mg, 0.1234 mmol) was added. ), triethylamine (5 μΐ, 0.03597 mmol) and a magnetic stir bar., The reaction mixture was stirred at room temperature for 72 hours. The reaction mixture was concentrated in vacuo to obtain Compound 11-38, which was purified by normal phase HPLC using a Phenomenex Luna 10 μ (25cm χ 21.2 mm di) column with a solvent gradient of 0 ° C. 95% H2 O / Acetonitrile for 21 min at a flow rate of 14.5 ml / min. A 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.

Compound 11-38: UV (Acetonitrile / H2 O) λ max 203 (sh) nm; ESMS m / z 337.1 (M + H) + and 359.1 (M + Na) +. <formula> formula see original document page 121 </formula>

Example 17

SUMMARY OF THE COMPOSITE OF FORMULA 11-39 FROM

11-19

The thiols and thioethers of Formula 11-39 may be formed by dehalogenation of the compound of Formula 11-19. Thiols (R = H) may be formed by treating Compound 11-19 with NaSH, for example, while thioethers (R = alkyl) may be formed by treating Compound 11-19 with thiol salts, or alternatively by treatment with the thiols themselves performing the reaction in benzene in the presence of DBU.

<formula> formula see original document page 121 </formula>

Example 18

SUMMARY OF THE COMPOSITE OF FORMULA 11-40 FROM

11-39

Sulfoxides (n = 1) and sulfones (n = 2) of Formula 11-40 may be formed by oxidizing thioethers of Formula II- 39, for example with hydrogen peroxide or other oxidizing agents. <formula> formula see original document page 122 </formula>

Example 19

SUMMARY OF COMPOUND FORMULA 11-41 FROM COMPOSITE 11-21

The compound of Formula 11-41 may be prepared by treating the compound of Formula 11-21 (or a protected derivative of 11-21, where 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 triethylamine. Other sulfonate esters may be similarly prepared.

<formula> formula see original document page 122 </formula>

Example 20

Synthesis of Compound 11-46 FROM Compound 11-19 or 11-41 Alkene of Formula 11-46 may be prepared by dehydro-iodination of the compound of Formula 11-19, or by elimination of hydro-mesyloxy of the compound of Formula 11-41, for example by treatment with base.

<formula> formula see original document page 123 </formula>

Example 21

SUMMARY OF COMPOUND FORMULA II-42A

Synthesis of boronic acids or esters, for example the compound of Formula II-42A, can be achieved as outlined in the retro-synthetic scheme below. Hydroboration of the alkene of Formula 11-46 results in the corresponding alkyl borane, which may be converted to the corresponding boronic acid or ester, for example the compound of Formula II-42A.

<formula> formula see original document page 123 </formula>

Example 22

SUMMARY OF COMPOUND FORMULA II-43A

The compound of Formula II-43A may be prepared by treating the compound of Formula 11-19 with triphenyl phosphine to make a phosphorus ilide, which may be treated with various aldehydes, for example glyoxylic acid methyl ester, to make the Formula II-43A. <formula> formula see original document page 124 </formula>

Example 23

SUMMARY OF COMPOUND FORMULA 11-30 FROM COMPOSITE 11-19

A portion of CuI (100 mg) was placed in a 25 ml vial with pear-shaped bottom and air-blended for 30 minutes; air flow was maintained by the vial throughout the course of the reaction. The vessel was cooled to -78 ° C prior to the addition of dry THF (5 ml) followed by the immediate dripping of a solution of dry lithium methyl lithium (5.0 ml, 1.6 M) by stirring. vigorously. A solution of Compound 11-19 in dry THF (12 mg Compound 11-19, 1 mL THF) was slowly added to the colorless dialkylcuprate solution and the resulting mixture stirred at -78 ° C for 1 hour. The reaction was quenched by washing the THF solution by a silica gel plug (1 cm diameter by 2 cm length) along with an additional wash using a 50% EtOAc / 50% hexane solution (50 mL). The combined wash with the silica plug was vacuum dried and subjected to additional 2-injection CLAP C18 purification (ACE 5 μ C18-HL, 150 mm X 21 mm di) using an isocratic solvent gradient consisting of 35% ACN / 65% H 2 O. Compound 11-30 eluted under these conditions in 23.5 minutes and obtained 2.4 mg of material (27% isolated yield) at 90.8% purity as measured by analytical HPLC. An alternative normal phase purification method may be used using a Phenomenex Luna 10 μ (25cm χ 21.2 mm di) column with a solvent gradient consisting of 100% hexane / ethyl acetate to 0% hexane for 20 minutes. Compound 11-30 eluted under these conditions within 16.5 minutes and obtained 3.0 mg of material (41% isolated yield) at 97.1% purity measured by analytical HPLC.

Compound 11-30: UV (Acetonitrile / H2 O) λmax 225 (sh), ESMS, m / z 294.1 (M + H); HRMS (ESI), m / z 294.1696 [M + H] +, Acalc = -3.2 ppm, C16H24NO4; 1H NMR in DMSOd6 (see fig. 56).

<formula> formula see original document page 125 </formula>

Example 24

SUMMARY OF COMPOUND FORMULAS 11-44. AND VI-IA FROM COMPOSITE 11-16

The compound of Formula 11-16 (30 mg, 0.096 mmol) was dissolved in CH 2 Cl 2 (9 mL) in a scintillation vial (20 mL) to which triethylamine (40 μΐ, 0.29 mmol), methyl-3 mercapto propionate (thiol, 250 μΐ) and a magnetic stir bar. The reaction mixture was stirred at room temperature for approximately 4 hours. The solvent was evaporated from the reaction mixture to obtain a mixture of compounds of Formulas 11-44 and VI-IA (19: 1), which were separated by reverse phase HPLC using a 5 μ C18 ACE (150 mm χ 22) column. mm di) with a solvent gradient of 35% to 90% H2 O / Acetonitrile for 17 min, 90 to 100% Acetonitrile for 1 min, maintained at 100% Acetonitrile for 1 min at a flow rate of 14, 5 ml / mm. A diode array detector was used to monitor the purification process. Compounds 11-44 (20 mg) and VI-IA (1 mg) eluted at 11.68 and 10.88 min, respectively, as pure compounds. Compound 11-44: UV (Aeetonitrile / H2 O) λ max 240 (sh) nm; ESMS m / z 434.0 (M + H) + and 456.0 (M + Na) +. Compound VI-1A: UV (Aeetonitrile / H2 O) λ max 220 (sh) nm; ESMS, m / z 398.0 (M + H) + and 420.0 (M + Na) +. <formula> formula see original document page 126 </formula>

Example 25

OXIDATION OF A SECONDARY HYDROXYL GROUP IN INITIAL COMPOUNDS AND REACTION WITH HYDROXY OR METOXY-AMINES

Any of the compounds of formulas 11-16, II- 17 and 11-18 may be used as a starting compound. The secondary hydroxyl group in the starting compound is oxidized using any of the following reagents: pyridinium dichromate (DCP), pyridinium chlorochromate (CCP), Dess-Martin periodinane or oxalyl chloride (Swern oxidation) (Ref: Organic Sintheses, Vol. I-VII). Preferably, Dess-Martin periodinane may be used as a reagent for this reaction. (Ref: Fenteany G. et al., Science, 1995, 268, 726-73). The resulting queto compound is treated with hydroxylamine or methoxy amine to generate oximes.

Examples:

<formula> formula see original document page 126 </formula>

Example 26

REDUCING Amination of Queto derivatives

Queto derivatives, for example of formulas II-8 and 11-13, are treated with sodium cyano-borohydride (NaBH3CN) in the presence of various bases to obtain amine derivatives of the starting compounds, which are subsequently hydrogenated with 10%. % Pd / C / H2 O to reduce double bond in the cyclohexene ring.

Example:

<formula> formula see original document page 127 </formula>

Example 27

CYCLE-HEXENE RING OPENING

Any of the compounds of formulas 11-16, II- 17 and 11-18 may be used as a starting compound. The starting compound may be protected, for example, at the lactam alcohol and / or nitrogen positions, and treated with OsO4 and NaIO4 in a THF-H2O solution to obtain dial derivatives that are reduced to alcohol with NaBH4. Protecting groups may be removed at the appropriate phase of the reaction sequence to produce II-7 or II-6. Example:

<formula> formula see original document page 127 </formula>

Starting Compound Example 28

ALCOHOL DEHYDRATION FOLLOWED BY ALDEIDE FORMATION AT LACTAMO-LACTONA RING JOINT

A starting compound of any of formulas 11-16, 11-17 or 11-18 is dehydrated, for example by treatment with mesylchloride in the presence of a base, or for example by treatment with a Burgess reagent or other agents. dehydrating agents. The resulting dehydrated compound is treated with OsO4, followed by NaIO4, or alternatively ozonolysis, to obtain an aldehyde group at the lactone-lactam ring junction.

<formula> formula see original document page 128 </formula>

Example 29

CYCLE-HEXENO RING OXIDATION TO PRODUCE CYC-HEXADIENES OR A PHENYL RING

An starting compound, such as the ketone of Formula 11-13 C, is treated with Pd / C to produce a cyclohexadiene derivative. The new double bond may be in any position of the cyclohexene ring. Ketone may be reduced, for example, with sodium borohydride to obtain the corresponding secondary alcohol (alcohols). Alternatively, the cyclohexadiene derivative may be further treated, for example, with DDQ, to aromatize the ring to a phenyl group.

Similarly, ketone may be reduced, for example, with sodium borohydride to obtain the corresponding secondary alcohol (s). <formula> formula see original document page 129 </formula>

As an alternative method, the starting compound, such as the compound of Formula 11-49, may be treated, for example, with TMSCl to yield a cyclohexadiene derivative. The cyclohexadiene derivative may be further treated, for example, with DDQ, to aromatize the ring to a phenyl group. OTMS on the phenyl group may be removed, for example, with an acid or base. Similarly, ketone may be reduced, for example, with sodium borohydride to obtain the corresponding secondary alcohol (s). <formula> formula see original document page 130 </formula>

Example 30

VARIOUS REACTIONS ON ALDEIDE DERIVATIVES

Wittig reactions are performed on the aldehyde group of formula I-I using various phosphorus ilides [e.g., (triphenylphosphoranylidene) ethane] to obtain an olefin. Side chain double bonding is reduced by catalytic hydrogenation.

Example: <formula> formula see original document page 131 </formula>

Reductive amination is performed on the aldehyde group using various bases (e.g. NH 3) and sodium cyano borohydride to obtain amine derivatives. Alternatively, the aldehyde is reduced with NaBH4 to form side chain alcohols.

Example:

<formula> formula see original document page 131 </formula>

Organometallic addition reactions may be performed on the carbonyl aldehyde to obtain various substituted secondary alcohols. Examples: <formula> formula see original document page 132 </formula>

Example 31

SUMMARY OF COMPOUND FORMULA 11-47 FROM COMPOSITE 11-17

The compound of Formula 11-17 (25 mg, 0.0896 mmol) was dissolved in CH 2 Cl 2 (9 mL) in a scintillation vial (20 mL) to which were added triethylamine (38 µl, 0.27 mmol), methyl 3-mercapto propionate (thiol, 250 μl) and a magnetic stir bar. The reaction mixture was stirred at room temperature for approximately 4 hours. The solvent was evaporated from the reaction mixture to obtain the compound of Formula 11-47 which was further purified by normal phase HPLC using a Phenomenex Luna 10 μ (25 cm χ 21.2 mm di) silica column with a gradient. 10% to 100% Hexane / EtOAc solvent for 24 min, maintained at 100% EtOAc for 3 min, at a flow rate of 14.5 ml / min. ELSD was used to monitor the purification process. Compound 11-47 (15 mg) eluted at 10.98 min as a pure compound.

Compound 11-47: UV (Acetonitrile / H2 O) λιη 3χ 240 (sh) nm; ESMS m / z 400.1 (M + H) + and 422.1 (M + Na) +.

<formula> formula see original document page 133 </formula>

Example 32

SUMMARY OF COMPOUND FORMULAS 11-48 AND VI-IB FROM COMPOUND 11-16

The compound of Formula 11-16 (15 mg, 0.048 mmol) was dissolved in a 1: 1 ratio of ACN / DMSO (8 mL) in a scintillation vial (20 mL) to which triethylamine (40 µl, 0.29 mmol), Glutathione (44.2 mg, 0.144 mmol) and a magnetic stir bar. The reaction mixture was stirred at room temperature for approximately 3 hours. The solvent was evaporated from the reaction mixture to obtain the compound of Formula 11-48 which was purified by reverse phase HPLC using a 5 μ C18 ACE column (150 mm χ 22 mm di) with a 10% solvent gradient at 70% H2 O / Acetonitrile for 15 min, 70 to 100% Acetonitrile for 5 min, maintained at 100% Acetonitrile for 4 min at a flow rate of 14.5 ml / min. A diode array detector was used to monitor the purification process. Compound 11-48 (10 mg) eluted as a pure compound at 8.255 min. Compound 11-48: UV (Aeetonitrile / H2 O) λmax 235 (sh) nm; ESMS m / z 621.0 (M + H) +. Compound 11-48 became unstable in solution and converted to compound VI-IB which appeared as a mixture of 11-48 and VI-IB in a 7: 3 ratio. Compound VI-1B: UV (Acetonitrile / H2 O) λ max 235 (sh) nm; ESMS, m / z 585.2 (M + H) +. <formula> formula see original document page 134 </formula>

Example 33

SUMMARY OF COMPOUND FORMULA 11-50 AND VI-IC FROM 11-16

The compound of Formula 11-16 (10 mg, 0.032 mmol) was dissolved in CH 2 Cl 2 (9 mL) in a scintillation vial (20 mL) to which triethylamine (26.5 μl, 0.192 mmol), N-Acetyl-L- Cysteine methyl ester (17 mg, 0.096 mmol) and a magnetic stir bar were added. The reaction mixture was stirred at room temperature for approximately 4 hours. The solvent was evaporated from the reaction mixture to obtain the mixture of compounds of Formulas 11-50 and VI-IC which were further purified by normal phase HPLC using a Phenomenex Luna 10 μ (25 cm χ 21.2) silica column. mm di) with a 10% to 100% Hexane / EtOAc solvent gradient for 24 min, maintained at 100% EtOAc for 3 min, at a flow rate of 14.5 ml / min. ELSD was used to monitor the purification process. Compounds 11-50 (2 mg) and VI-IC (0.2 mg) eluted at 10.39 and 10.57 min, respectively, as pure compounds. Compound 11-50: UV (Acetonitrile / H2 O) λ max 230 (sh) nm; ESMS m / z 491.1 (M + H) + and 513.0 (M + Na) +. Compound VI-1C: UV (Acetonitrile / H2 O) λ max 215 (sh) nm; ESMS m / z 455.1 (M + H) + and 577.0 (M + Na) <formula> formula see original document page 135 </formula>

Example 34 IN VITRO BIOLOGY

Initial studies of a compound of Formula 11-16 which is also called Salmosporamide A employed the National Cancer Institute (NCI) visualization panel, which consists of 60 human tumor cell lines representing leukemia, melanoma and lung, colon, brain, ovarian, breast, prostate and kidney cancers. A detailed description of the viewing procedure can be found in the hypertext transfer protocol (http: //) "dtp.nci.nih.gov/branches/btb/ivclsp.html".

In summary, each of the 60 human tumor cell lines grew in RPMI 1640 medium supplemented with 5% fetal bovine serum and 2 mM L-glutamine. Cells were plated at their appropriate density in 96-partition microtiter plates and incubated at 37 ° C, 5% CO2, 95% air and 100% relative humidity. After 24 hours, 100 μΙ; From several series of 10-fold diluted Salinosporamide A dilutions were added to the appropriate partitions containing 100 μΐ cells, resulting in a final Salinosporamide A concentration ranging from 10 nM to 100 μΜ. Cells were incubated for an additional 48 hours and a sulforhodamine B protein assay was used to estimate cell viability or growth.

Three dose response parameters were calculated as follows:

GI50 indicates concentration that inhibits growth by 50%

TGI indicates the concentration that completely inhibits growth. LC50 indicates the concentration that is lethal to 50% of cells.

An example of a study evaluating Salinosporamide A on the NCI screen is shown in Table 1 below.

Data indicate that the mean GI50 value of Salinosporamide A was less than 10 nM. The extensive range (> 1000-fold difference) observed in mean TGI and mean LC50 values for the most sensitive and most resistant tumor cell lines illustrate that Salinosporamide A exhibits good selectivity and does not appear to be a general toxin. In addition, mean TGI data suggest that Salinosporamide A shows preferred specificity for melanoma and breast cancer cell lines. The assay was repeated and showed similar results.

NCI tumor screen results showed that Salinosporamide A: (1) is a potent compound with an average GI50 value <10 nM, and (2) has good tumor selectivity of more than 1,000 times the difference in both. mean TGI and mean LC50 values among the most sensitive and resistant tumor cell lines.

Table 1: Relative Sensitivity of NCI 60 Human Tumor Cell Lines to Salinosporamide A <table> table see original document page 137 </column> </row> <table>

Example 35

INHIBITION OF TUMOR CELL LINES GROWTH

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. Cells were cultured in an incubator at 37 ° C in 5% CO2 and 95% air humidity.

For cell growth inhibition assays, 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 were seeded at 1.25χ103, 5x103, 1.5x105, 5x103, 5x103, Ix104, 2x103, 4x103, 7.5x103, 5x103, 2x104, 2.5x104 cells / partition respectively in 90 μΐ. a complete medium of Corning 3904 black-walled, light-bottomed tissue culture plates. 20 mM standard solutions of Formula 11-16 were prepared in 100% DMSO, portioned and stored at -80 ° C. Formula 11-16 was serially diluted and added triplicate to the test partitions resulting in final concentrations in the range of 20 μΜ to 0.2 pM. The plates were returned to the incubator for 48 hours. The final DMSO concentration was 0.25% in all samples.

After 48 hours of drug exposure, 10 μl of Resazurin 0.2 mg / ml (obtained from Sigma-Aldrich Chemical Co.) in Mg2 + free phosphate buffered saline, Ca2 + was added to each partition and the plates were returned to the incubator. for 3-6 hours. Since living cells metabolize Resazurin, the fluorescence of the Resazurin reduction product was measured using a Fusion Microplate Fluorometer (Packard Bioscience) with filters of λεχ = 535 nm and λβπι = 590 nm. Resazurin dye in a cell-free medium was used to determine background, which was subtracted from the data for all experimental partitions. Data were normalized to mean fluorescence of cells treated with a DMSO medium + 0.25% (100% cell growth) EC50 values (the drug concentration at which 50% of the observed maximum 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 Incorporation.).

The data in Table 2 summarize the growth inhibitory effects of Formula 11-16 against 13 diverse human and rat tumor cell lines.

Table 2

Average EC50 values of Formula 11-16 against various tumor cell lines

<table> table see original document page 138 </column> </row> <table> <table> table see original document page 139 </column> </row> <table>

* where η (number of independent experiments) = 2, the mean value is displayed

EC50 values indicate that Formula 11-16 was cytotoxic against B16-F10, DU 145, HEK293, HT-29, LoVo, MDA-MB-231, MIA PaCa-2, NCI-H292, OVCAR-3 cells. PANC-I, PC-3, RPMI 8226 and U266.

Example 36

IN VITRO INHIBITION OF PROTEASOM ACTIVITY BY FORMULAS I-7, I-1-2, II-3, II--4, II-5A, II-5B, II-8C, II- 13C, 11-16, 11-17, II-18, 11-19, 11-20, H-21, 11-22, II-24C, II-25, 11-26, 11-27, II-28, 11-29, 11-30, 11 -31, II-32, II -38, IV-3C, II-44, VI-AIA and II -47

All compounds were prepared as standard 20 mM solutions in DMSO and stored in small portions at -80 ° C. Purified rabbit muscle 20S proteasome was obtained from CalBiochem. To increase proteasome chymotrypsin-like activity, the assay buffer (20 mM HEPES, pH 7.3, 0.5 mM EDTA, and 0.05% Triton X100) was supplemented with SDS resulting in a final SDS concentration of 0.035%. The substrate used was suc-LLVI-AMC, a fluorogenic peptide substrate specifically divided by proteasome chemotrypsin-like activity. Assays were performed at a proteasome concentration of 1 μς / πιΐ in a final volume of 200 μΐ on 96 partition Costar microtiter plates. Formulas II-2, II-4, 11-16, 11-17, 11-18, 11-19, 11-21, 11-22 and 11-44 were tested with eight-point dose response curves with concentrations ranging from 500 nM to 158 pM. Formulas II-5A, II-5B, 11-20, II- 29, 11-30 and 11-38 were tested at concentrations ranging from 1 μΜ to 0.32 nM. Formulas II-3 and VI-IA were tested with an eight-point dose response curve with final concentrations ranging from 10 μΜ to 3.2 nM. Formula 11-47 was tested at concentrations ranging from 5 μΜ to 1.6 nM, while Formulas 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 μΜ to 6.3 nM. Samples were incubated at 37 ° C for five minutes in a 96-well Fluoroskan Ascent temperature controlled microplate reader (Thermo Electron, Waltham, MA). During the preincubation step, the substrate was diluted 25-fold in an SDS-containing assay buffer. After the preincubation period, reactions were initiated by the addition of 10 μΐ of the diluted substrate and the plates returned to the plate reader. The final substrate concentration in the reactions was 20 μΜ. The fluorescence of the split peptide substrate was measured at Xex = 390 nm and λβπι = 460 nm. All data were collected every five minutes for more than 1.5 hours and plotted as the average of triplicate data points. EC50 values (the drug concentration at which 50% of the maximum relative fluorescence unit is inhibited) were calculated by Prisma (GraphPad Software) using a sigmoidal dose responsive variable slope model. To evaluate the activity of compounds against caspase-like activity of 20S proteasomes, reactions were performed as described above except that Z-LLE-AMC was used as a peptide substrate. Formulas 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, 11-27, 11-28, 11-29, 11-30, IV-3C, 11-44 and VI-IA were tested at concentrations ranging from 20 μΜ to 6.3 nM. Formula II-2 was tested at concentrations ranging from 10 μΜ to 3.2 nM, while Formula 11-16 and Formula 11-19 were tested at concentrations ranging from 5 μΜ to 1.58 nM. For the evaluation of compounds against proteasome trypsin-like activity, 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 μΜ to 1.6 nM. Formulas II-3, II-8C, II-13C, 11-17, 11-21, 11-22, II-24C, 11-25, 11-26, 11-27, 11-28, 11-29, 11-30, IV-3C and VI-IA were tested at concentrations ranging from 20 μΜ to 6.3 nM. For Formulas II-2 and II-5B the concentrations tested ranged from 10 μΜ to 3.2 nM, while Formulas II-4, II-5A, 11-16, 11-18 and 11-19 were tested at concentrations ranging from from 1 μΜ to 0.32 nM. Formula 11-44 was tested at concentrations ranging from 2 μΜ to 632 pM.

Results (mean EC50 values) are shown in Table 3 and illustrate that among the compounds tested, Formulas II-5A, 11-16, 11-18, 11-19, 11-20, 11-21, 11-22, 11-29, 11-38 and 11-44 are the most potent inhibitors of 20S proteasome chymotrypsin-like activity with EC50 values ranging from 2.2 nM to 11 nM. Formulas 1-7, II-2, II-4, II-5B, 11-17, 11-30 and II- 47 inhibit chymotrypsin-like proteasomal activity with EC50 values ranging from 13 nM to 88 nM, while the value EC50 of Formulas II-3, 11-26 and VI-IA ranged from 207 nM to 964 nM. Formulas II-13C, II-24C, 11-27, 11-28 and IV-3C inhibited chymotrypsin-like activity with EC50 values ranging from 1.4 μΜ to 10.6 μΜ. EC50 values of Formulas II-8C, 11-25, 11-31 and 11-32 were greater than 20 μΜ. Under the conditions tested, Formulas II-2, II-3, 11-4, II-5A, II-5B, II-13C, 11-16, 11-17, 11-18, II-19, 11-20 , 11-21, 11-22, II-24C, 11-26, 11-29, 11-30, 11-44 and VI-IA could inhibit 20S proteasome trypsin-like activity.

Formulas II-4, II-5A, 11-16, 11-18, 11-19 and 11-29 inhibited caspase-like activity with EC50 values ranging from 213 nM to 850 nM, while Formulas II-2, II-5B, 11-17, 11-20, 11-21, II- 22, 11-30, 11-44 and VI-IA had EC50 values ranging from 8.7 μΜ to 956 nM. Table 3

Effects of Formulas 1-7, II-2, II-3, II-4, II-5A, II-5B, II-8C, II-13C, 11-16, 11-17, 11-18, 11-19 , 11-20, II-21, 11-22, II-24C, 11-25, 11-26, 11-27, 11-28, 11-29, 11-30, II-31, 11-32, 11 -38, IV-3C, 11-44, VI-IA and 11-47 on the varied enzymatic activities of purified rabbit 20S proteasomes

<table> table see original document page 142 </column> </row> <table> <table> table see original document page 143 </column> </row> <table>

EC50 values from one or two independent experiments are shown. Where η> 3, the mean value ± standard deviation of EC50 is presented; ** η = 3, standard deviation not applicable; ND = not determined. Results of 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 proteasome chymotrypsin-like activity with EC50 values of 18.5 nM and 15 nM respectively. Formula II-3 is active in this assay with an EC50 value of 890 nM. Similar results were obtained from an independent experiment.

Results of a representative experiment evaluating Formula II-5A and Formula II-5B are shown in fig. 47 and illustrate that Formula II-5A and Formula II-5B inhibit proteasome chymotrypsin-like activity with EC50 values of 6 nM and 88 nM respectively. Similar results were obtained in an independent experiment.

Example 37

SALINOSPORAMIDE A (11-16) INHIBITING THE ACTIVITY OF THE RIO MUSCLE 20S PROTEASOMY CHEMYTRIPSIN

The effect of Salinosporamide A (11-16) on proteasomes was examined using commercially available equipment from Calbiochem (# 539158 in the catalog), which uses a fluorogenic peptide substrate to measure rabbit muscle 20S proteasome activity (Kit Proteasoma Calbiochem 20S). This peptide substrate is specific for the activity of proteasome chymotrypsin-like enzymes.

Omuralide was prepared as a 10 mM standard in DMSO and stored in 5 μl portions at -80 ° C. Salinosporamide A was prepared as a 25.5 mM solution in DMSO and stored in portions at -80 ° C. The assay measured the hydrolysis of Suc-LLVI-AMC to Suc-LLVI and AMC. The released coumarin (AMC) was measured fluorometrically using λβχ = 390 nm and λέπι = 460 nm. Assays were performed on 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 at 37 ° C. The tests were performed according to the manufacturer's protocol, with the following changes. The proteasome was activated as described with SDS, and kept on ice before the assay. Salinosporamide A and Omuralide were serially diluted in a test buffer to make an 8-point dose response curve. Ten microliters of each dose were added in triplicate to the assay plate, and 190 μΐί of activated proteasome were added and mixed. Samples were preincubated in Fluoroskan for 5 minutes at 37 ° C. Substrate was added and AMC kinetics was followed for one hour. All data were collected and plotted as the triplicate mean of the data points. Data were normalized for reactions performed in the absence of Salinosporamide A and modeled by Prisma as a variable slope responsive to sigmoidal dose.

Similar to the results obtained for in vitro cytotoxicity (Table 2), Feling et al., Angew. Chem. Int. Ed. Engl. 42: 355 (2003), the EC50 values in the 20S proteasome assay showed that Salinosporamide A was approximately 40 times more potent than Omuralide, with an average value of 1.3 nM versus 49 nM, respectively (Fig. 27). This experiment was repeated and the mean EC50 in both trials was determined to be 2 nM for Salinosporamide A and 52 nM for Omuralide.

Salinosporamide A is a potent chymotrypsin-like activity inhibitor of. proteasome. EC50 values for cytotoxicity are in the range of 10-200 nM suggesting that Salinosporamide A's ability to induce cell death was due, at least in large part, to proteasome inhibition. The data suggest that Salinosporamide A is a potent inhibitor of small proteasome molecules.

Example 38

SALINOSPORAMIDE A (11-16) INHIBITING HPGP ACTIVITY OF RABBIT MUSCLE PROTEASOMAS 2TH

Omuralide may inhibit HPGP (also known as caspase-like) activity of the proteasome; then, the ability of Salinosporamide A to inhibit HPGP activity of purified rabbit muscle 20S proteasomes was evaluated. A commercially available fluorogenic substrate specific for HPGP activity was used instead of the chymotrypsin substrate provided in the proteasome assay equipment described above. Salinosporamide A (11-16) was prepared as a 20 mM solution in DMSO and stored at -80 ° C in small portions. Z-LLE-AMC substrate was prepared as a 20 mM standard solution in DMS0 stored at -20 ° C. The source of proteasomes was the equipment commercially available from Calbiochem (# 539158 in the catalog). As with the chymotrypsin substrate, the proteasome can divide Z-LLE-AMC into free Z-LLE and AMC. Activity can then be determined by measuring the fluorescence of the released AMC (λεχ = 390 nm and λβπι = 460 nm). The proteasomes were activated with SDS and kept on ice as recommended by the manufacturer. Salinosporamide A was diluted in DMSO to give an 8-point dilution series concentrated 400 times. The series was diluted 20-fold with a assay buffer and preincubated with proteasomes as described for chymotrypsin-like activity. After substrate addition, samples were incubated at 37 ° C, and fluorescent AMC release was monitored on a fluorimeter. All data were collected and plotted as the mean of triplicate points. In these experiments, the EC50 was modeled on Prisma as normalized activity, where the amount of AMC released in the absence of Salinosporamide A represents 100% activity. As before, the chosen model was a sigmoidal dose response with a variable slope.

The data revealed that Salinosporamide A inhibited HPGP activity in rabbit muscle 20S proteasomes with an EC50 of 350 nM (Fig. 28). A replicated experiment was performed which gave a predicted EC50 of 610 nM. These results indicate that Salinosporamide A blocks the in vitro HPGP activity of purified rabbit muscle 20S proteasomes, although with lower potency than that seen for chemotrypsin-like activity.

Example 39

INHIBITION OF CHEMIOTRIPSIN TYPE ACTIVITY OF HUMAN ERYTHROCYTE 20S PROTEASOMAS

The ability of Salinosporamide A (11-16) to inhibit chemotrypsin-like activity of human erythrocyte 20S proteasomes was evaluated in vitro. The calculated EC50 value was approximately 3 nM (Fig. 29). These data indicate that the inhibitory effect of Salinosporamide A is not limited to rabbit skeletal muscle proteasomes.

Salinosporamide A was prepared as a 20 mM solution in DMSO and stored in small portions at -80 ° C. The substrate, suc-LLVI-AMC, was prepared as a 20 mM solution in DMSO and stored at -20 ° C. Human Erythrocyte 20S proteasomes were obtained from BIOMOL (Cat. No. SE-221). The proteasome can divide suc-LLVI-AMC into suc-LLVI and free AMC and the activity can then be determined by measuring the released AMC fluorescence (λβχ = 390 nm and λθΐη = 460 nm). The proteasomes were activated by SDS and stored on ice as for experiments using rabbit muscle proteasomes. Salinosporamide A was diluted in DMSO to give an 8-point dilution series concentrated 400 times. The series was then diluted 20-fold with assay buffer and preincubated with proteasomes at 37 ° C. The reaction was initiated with substrate, and AMC release was followed on a Fluoroskan micro-plate 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. Data were normalized for reactions performed in the absence of Salinosporamide A and modeled on Prisma as a response to sigmoidal dose with variable inclination.

Replicated experiments were performed using separate batch human erythrocyte proteasomes resulting in an EC50 value range of approximately 4 nM. These results indicate that in vitro chymotrypsin-like activity of human erythrocyte 20S proteasomes is sensitive to Salinosporamide A.

Formula 11-16 also showed inhibition of trypsin-like and caspase-like activities of human erythrocyte proteasomes. For the trypsin type, the studies showed an EC50 value of about 9 nM, and for the caspase type an EC50 value of about 390 nM. Further studies of chymotrypsin-like activity in human erythrocytes resulted in an EC50 of about 250 pM. In addition, studies have shown that Formula 11-16 is specific for the proteasome, showing little or no effect on other proteolytic enzymes. For example, Formula II-16 when tested for inhibition of Chymotrypsin, Cathepsin B and Thrombin, respectively, had EC50 values of 18,000 nM,> 200,000 nm, and> 200,000 nM, respectively.

Example 40

SALINOSPORAMIDE SPECIFICITY (11-16)

One possible mechanism by which Salinosporamide A inhibits proteasome is that by the reaction of functionality of Salinosporamide A β-lactone 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 proteasome catalytic activity. Fenteany, et al., J. Biol. Chem. 273: 8545 (1998). A structurally related compound, Lactacystin, has also been shown to inhibit cathepsin A (Ostrowska, et al., Int. J. Biochem. Cell. Biol. 32: 747 (2000), Kozlowski, et al., Tumor Biol. 22: 211 ( 2001), Ostrowska, et al., Biochem, Biophis, 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 proteasome by assessing its ability to inhibit the catalytic activity of a prototypic serine protease, chymotrypsin.

Salinosporamide A was prepared as a 20 mM solution in DMSO and stored in small portions at -80 ° C. The substrate, suc-LLVI-AMC, was prepared as a 20 mM solution in DMSO and stored at -20 ° C. The proteolytic division of this substrate by both proteasomes and chymotrypsin releases the AMC fluorescent product, which can be monitored on a fluorimeter (λεχ = 390 nm and λέπι = 460 nm). Bovine pancreatic chymotrypsin was obtained from Sigma (Cat. No. C-4129), and prepared as a 5 mg / ml solution in a assay buffer (10 mM HEPES, 0.5 mM EDTA, 0.05% Triton X- 100, pH 7.5) daily. Just prior to the assay, chymotrypsin was diluted to 1 µg / mL (0.2 µg / partition) in a assay buffer and kept on ice. Salinosporamide A was diluted in DMSO to generate an 8-point dose response curve. The high final Salinosporamide A concentrations required to achieve complete chymotrypsin inhibition required that the diluted enzyme be added directly to the compound dilution series. The inclusion of 1% DMSO (the final solvent concentration in the test partitions) in the reaction had no significant effect on chymotrypsin activity on this substrate. Reactions were preincubated for 5 minutes at 37 ° C and reactions were initiated by the addition of substrate. Data were kinetically collected for one hour at 37 ° C in Fluoroskan and plotted as the mean of triplicate data points. Data were normalized for reactions performed in the absence of Salinosporamide A, and modeled on Prisma as a response to sigmoidal dose with variable inclination. Normalized data for Salinosporamide A inhibition of rabbit 20S proteasome chemotrypsin-like activity were included in the same graph.

The average inhibition observed in two experiments using salinosporamide A pretreatment of chymotrypsin was 17.5 μΜ (Fig. 30 shows a representative experiment). The data indicate that there is a preference for Salinosporamide A-mediated inhibition of proteasome chymotrypsin-like activity in inhibiting chymotrypsin catalytic activity.

Thus, Salinosporamide A inhibits chymotrypsin and HPGP proteasome activity. Preliminary studies indicate that Salinosporamide A also inhibits trypsin-like proteasome activity with an EC50 value of ~ 10 nM (data not shown).

Example 41

Inhibition of FN-KB-mediated LUCIFERASE ACTIVITY BY FORMULAS II-2, II-3, II-4, II-5A, II-5C, II-13C, 11-16, II-17, 11- 18, 11-19, 11-20, 11-21, II-22, II-24C, II-25, 11-26, II -27, 11-28, 11-29, 11-30, 11-44, VI-IA and IV-3C; HEK293 CELL LINING FN-kB LUCIFERASE REPORTER

The HEK293 luciferase reporter cell line FN-κΒ is a derivative of the human kidney embryonic cell line (ATCC; CRL-1573) and carries a luciferase reporter gene under 5X regulation of the FN-κ ligação binding sites. The reporter cell line was routinely maintained in a complete DMEM medium (DMEM plus 10% (v / v) fetal bovine serum, 2 mM L-glutamine, 10 mM HEPES and Penicillin / Streptomycin at 100 IU / ml and 100 μg / ml / respectively) supplemented with 250 μg / ml G418. When performing luciferase analysis, basal DMEM medium was replaced with red phenol free basal DMEM medium and G418 was omitted. Cells were cultured in an incubator at 37 ° C in 5% CO2 and 95% air humidity.

For FN-kB-mediated luciferase assays, HEK293 FN-KB / luciferase cells were seeded at 1.5 x 10 4 cells / partition in 90 μΐ of a complete phenol-free DMEM complete medium in Corning 3917 opaque white background tissue culture plates For Formulas II-2, II-4, II-5A, and 11-18, an initial dilution of 400 μΜ was made in 100% DMSO and this dilution was used to generate an 8-point half-dilution series. . This dilution series was further diluted 40X in an appropriate culture medium and ten μΐ portions were added to the triplicate test partitions resulting in final test concentrations ranging from 1 μΜ to 320 pM. For Formulas II-3, II-5B, II-8C, II-13C, 11-17, 11-20, 11-21, 11-22, II-24C, 11-25, II-26, 11-27 , 11-28, 11-29, 11-30, VI-IA and IV-3C, an initial 8 mM dilution was made in 100% DMSO and the same procedure was followed as described above resulting in varying final test concentrations. from 20 μΜ to 6.3 nM. For Formulas 11-16, 11-19 and 11-44, an initial dilution of 127 μΜ was made in 100% DMSO and final test concentrations ranged from 317 nM to 0.1 nM. For formula 11-20, an initial dilution of 8 mM was made in 100% DMSO and the final test concentrations ranged from 6.3 μΜ to 2.0 nM or 20 μΜ to 6.3 nM respectively. The plates were returned to the incubator for 1 hour. After 1 hour of pretreatment, 10 μΐ of a 50 ng / ml TNF-α solution prepared in red phenol-free DMEM medium was added, and the plates were incubated for an additional 6 h. The final DMSO concentration was 0.25% in all samples. At the end of TNF-α excitation, 100 μl of a Steady Lite HTS luciferase reagent (Packard Bioscience) was added to each partition and the plates were allowed to sit for 10 min at room temperature before measuring luciferase activity. Relative luciferase units (ULR) were measured using a Fusion microplate fluorometer (Packard Bioscience). EC50 values (the drug concentration at which 50% of the maximum relative luciferase unit inhibition is established) were calculated on the Prisma (GraphPad Software) using a sigmoidal dose response model with variable slope.

FN-κΒ Activation Inhibition by Formulas II-2, II-3, II-4, II-5A, II-5B, II-8C, II-13C, 11-16, 11-17, 11-18, 11 -19, 11-20, 11-21, 11-22, II-24C, 11-25, 11-26, 11-27, 11-28, 11-29, 11-30, 11-44, VI-IA E IV-3C

FN-κΒ regulates the expression of a large number of important genes in inflammation, apoptosis, tumor genesis, and autoimmune diseases. In its inactive form, FN-κΒ is complexed with IkB in the cytosol, and in excitation, IkB is phosphorylated, ubiquitinated and subsequently degraded by the proteasoma. IkB degradation leads to FN-κΒ activation and its translocation to the nucleus. The effects of Formulas II-2, II-3, II-4, II-5A, II-5B, II-8C, II-13C, 11-16, 11-17, 11-18, 11-19, 11- 20, 11-21, 11-22, II-24C, II-25, 11-26, 11-27, 11-28, 11-29, 11-30, 11-44, VI-IA and IV-3C in the FN-κΒ activation were. evaluated by evaluating FN-κΒ-mediated luciferase activity in HEK293 FN-kB / Luc cells on TNF-α excitation.

Pretreatment of FN-kB / Luc 293 cells with Formulas II-2, II-4, II-5A, II-5B, 11-16, 11-17, 11-18, 11-19, II-20 , 11-21, 11-22, II-24C, 11-26, 11-29, 11-30 and 11-44 resulted in a dose-dependent decrease of luciferase activity on TNF-α excitation. Mean EC50 values for inhibiting FN-κΒ-mediated luciferase activity are shown in Table 4 and demonstrate that the compounds of Formulas II-2, II-4, II-5A, II-5B, 11-16, 11- 17, 11-18, 11-19, 11-20, 11-21, 11-22, II-24C, 11-26, 11-29, 11-30 and 11-44 inhibited FN-κΒ activity in this assay. cell based.

Table 4

EC 50 values of Formulas II-2, II-3, II-4, II-5A, II-5B, II-8C, II-13C, 11-16, 11-17, 11-18, 11-19, 11-20, II-21, 11-22, II-24C, 11-25, 11-26, 11-27, 11-28, 11-29, 11-30, II-44, VI-IA and IV- 3C from FN-KB mediated luciferase reporter gene assays

<table> table see original document page 152 </column> </row> <table> <table> table see original document page 153 </column> </row> <table>

* EC50 values from two independent experiments are shown. Where η> 3, the mean value of EC50 ± standard deviation is displayed. ** The assay was also performed with compound II-20, and resulted in an EC50 value of 154 nM, which was not included in the calculation of the average EC50 value.

Results from a representative experiment evaluating Formula II-2, Formula II-3 and Formula II-4 (Fig. 44) revealed that pretreatment with Formula II-2 and Formula II-4 resulted in a dose-dependent decrease of luciferase activity in FN-kB / Luc293 cells in TNF-α stimulation. The EC50 calculated to inhibit FN-κΒ-inducible luciferase activity in this experiment was 73 nM for Formula ΙΙ-2, while the EC50 value for Formula II-4 was 67 nM. Similar data were observed in a replicated experiment.

Results of a representative experiment evaluating Formula II-5A and Formula II-5B are shown in fig.

45 and illustrate that Formula II-5A and Formula II-5B inhibit FN-κΒ-inducible luciferase activity with EC50 values of 30 nM and 261 nM respectively. Similar data were observed in a replicated experiment.

Example 42

EFFECT OF SALES INOS PORAMIDA A ON FN-KB SIGNALING WAY

Experiments were performed to study the role of Salinosporamide A in the FN-κΒ signaling pathway. A stable clone HEK293 (FN-kB / Luc 293) was generated carrying a luciferase reporter gene under regulation of 5X FN-κΒ binding sites. Excitation of this cell line with TNF-α leads to increased luciferase activity as a result of FN-κΒ activation.

FN-kB / Luc 293 cells were pretreated with 8-point serial half-dilutions of Salinosporamide A (ranging from 1 μΜ to 317 pM) for 1 hour followed by a 6-hour excitation with TNF-a (10 ng / ml). FN-κΒ-induced luciferase activity was measured at 6 hours. The viability of FN-kB / Luc 293 cells after treatment with Salinosporamide A for 24 hours was evaluated by the addition of resazurin tincture as previously described.

Pre-treatment of FN-kB / Luc 293 cells with Salinosporamide A resulted in a dose-dependent decrease in luciferase activity on TNF-α excitation (Fig. 31, right y-axis). The EC50 calculated for inhibition of FN-κΒ / luciferase activity was ~ 7 nM. A cytotoxicity assay was performed simultaneously and showed that this concentration of Salinosporamide A did not affect cell viability (Fig. 31, left y-axis). These representative data suggested that the observed decrease in luciferase activity by treatment with Salinosporamide A was mainly due to a signaling-mediated FN-KB event rather than cell death.

Example 43

In addition to the FN- κΒ luciferase reporter gene assay, the effect of Salinosporamide A on phosphorylated ΙκΒα levels and total ΙκΒα was assessed by Western blot. Endogenous protein levels were evaluated in both HEK293 cells and the FN-kB / Luc 293 reporter clone.

Cells were pretreated for 1 hour with Salinosporamide A at the indicated concentrations followed by excitation with 10 ng / ml TNF-α for 30 minutes. Antibodies against the total and phosphorylated forms of ΙκΒα were used to determine the endogenous level of each protein, and anti-tubulin antibodies were used to confirm equal protein loading.

As shown in fig. 32, treatment of both Salinosporamide A cell lines at 50 and 500 nM not only reduced total ΙκΒα degradation but also retained phospho-ΙκΒα level when stimulated with TNF-α. These results strongly support the mechanism of action of Salinosporamide A as a proteasome inhibitor, which prevents the degradation of phosphorylated ΙκΒα on TNF-α excitation.

Example 44

EFFECT OF SALINOSPORAMIDE A ON CELL CYCLE REGULATORY PROTEINS

The ubiquitin proteasome pathway is an essential proteolytic system involved in cell cycle control through regulation of cyclin degradation and cyclin-dependent kinase (QDC) inhibitors such as p21 and p27. Pagano, et al., Science 269: 682 (1995), Kisselev, et al., Chem. Biol. 8: 739 (2001), King, et al., Science 274: 1652 (1996). In addition, p21 and p27 protein levels are increased in the presence of proteasome inhibitors. Fukuchi, et al., Biochim. Biophys. Minutes 1451: 206 (1999), Takeuchi, et al. Jpn. J. Cancer Res. 93: 774 (2002). Then, Western blot analysis was performed to evaluate the effect of Salinosporamide A treatment on endogenous p21 and p27 levels using HEK293 cells and the FN-KB / Luciferase reporter clone HEK293.

The Western blots shown in fig. 33 were retested using antibodies against p21 and p27 to determine the endogenous level of each protein, and anti-tubulin antibodies were used to confirm equal protein loading.

As shown in figs. 33A and 33B, preliminary results indicated that p21 and p27 protein levels were elevated when both cell lines were treated with Salinosporamide A at various concentrations. The data showed that Salinosporamide A acts by inhibiting proteasome activity thus preventing TNF-α induced FN-κΒ activation. Additionally, this proteasome inhibition results in the accumulation of QDC inhibitors, p21 and p27, which have been reported to sensitize cells to apoptosis. Pagano, et al., Science 269: 682 (1995), King, et al., Science 274: 1652 (1996).

Example 4 5

CASPASE-3 ACTIVATION BY SALINOSPORAMIDE A (II-16)

To determine whether Salinosporamide A induces apoptosis, its effect on induction of Caspase-3 activity was evaluated using Jurkat cells (American Type Culture Collection) TIB-152, acute human T-cell leukemia. ).

Jurkat cells were plated at 2x10 6 cells / 3 ml per partition in a 6-partition plate and incubated at 37 ° C, 5% (v / v) CO2 and 95% (v / v) humidity . Salinosporamide A and Mitoxantrone (Sigma, St. Louis, MO. Cat. No. M6545) were prepared in DMSO at standard concentrations of 20 mM and 40 mM, respectively. Mitoxantrone is a chemotherapeutic drug that induces apoptosis by dividing and non-dividing cells by inhibiting and repairing DNA synthesis and has been included as a positive control. Bhalla, et al., Blood 82: 3133 (1993). Cells were treated with EC50 concentrations (Table 5) and incubated for 19 hours prior to evaluation of Caspase-3 activity. Cells treated with 0.25% DMSO served as negative control. The cells were collected by centrifugation and the medium removed. Cell pellets were processed for the Caspase-3 activity assay as described in the manufacturer's protocol (Molecular Probes EnzChek Caspase-3 Assay Kit (E-13183; see Appendix G, which forms part of this description and also is available as a hypertext transfer protocol at www.probes.com/media/pis/mpl3183.pdf In summary, the cell pellets were lysed on ice, mixed with the components of the EnzChek Caspase-3 kit in 96-partition plate, and then incubated in the dark for 30 minutes prior to the split-benziloxycarbonyl-DEVD-AMC fluorescence reading using a Packard Fusion instrument with λβχ = 485 nm and λθίτι = 530 nm filters. Lysates were determined using the BCA Protein Assay Kit (Pierce) and these values were used for normalization.

Data from representative experiments indicated that Salinosporamide A treatment of Jurkat cells results in cytotoxicity and activation of Caspase-3 (Table 5, Fig. 34).

Table 5: EC50 Values of Salinosporamide A and Mitoxantrone Cytotoxicity against Jurkat Cells

<table> table see original document page 157 </column> </row> <table>

Example 4 6

PARP DIVISION BY SALINOSPORAMIDE A IN JURKAT CELLS

To assess Salinosporamide A's ability to induce apoptosis in Jurkat cells, the division of poly (ADP-ribose) polymerase (PARP) was monitored. PARP is a 116 kDa nuclear protein that is one of the major intracellular targets of Caspase-3. Decker, et al. , J. Biol. Chem. 275: 9043 (2000), Nicholson, D.W., Nat. Biotechnol. 14: 297 (1996). The PARP division generates a stable 89 kDa product, and this process can be monitored using Western Spot. The division of PARP through caspase is a hallmark of apoptosis, and as such serves as an excellent marker for this process.

Jurkat cells were maintained in RPMI supplemented with 10% Low Density Fetal Bovine Serum (FBS) (2x10 5 cells per ml) prior to the experiment. Cells were harvested by centrifugation, and resuspended in a medium to 1x10 4 cells for every 3 mL. Twenty mL of the cell suspension was treated with 100 nM Salinosporamide A (20 mM DMSO stored at -80 ° C), and a 3 mL portion was removed and placed on ice as sample T0. Three mL portions of the cell suspension plus Salinosporamide A were placed in 6-partition plates and returned to the incubator. As a positive control for PARP division, an identical cell suspension was treated with 350 nM Staurosporine, a known apoptosis inducer (Sigma S5921, 700 μΜ DMSO stored at -20 ° C). Samples were removed at 2, 4, 6, 8, and 24 hours for Salinosporamide A-treated cells, and at 4 hours for Staurosporine control. For each time point, the cells were recovered by brief centrifugation, the cells were washed with 400 μL of PBS, and the cells were backwashed. After removal of PBS, the pellets were stored at -20 ° C before. SDS Each cell pellet was resuspended in 100 µL of a NuPAGE Sample Buffer (Invitrogen 4 6-5030) and 10 µL of each sample was separated into 10% NuPAGE Bis-Tris Gels (Invitrogen NB302). Following electro-transfer to nitrocellulose, the membrane was PARP-tested with a rabbit polyclonal antibody (Cell Signaling 9542), followed by a goat anti-rabbit alkaline phosphatase-conjugated secondary antibody (Jackson 11-055-045). Bound antibodies were detected colorimetrically using BCIP / NBT (Roche 1681451).

The Western Blot shown in fig. 35 shows the division of PARP within Jurkat cells in a time dependent manner. The split form (denoted by the asterisk, *) appears in treated cells 2 to 4 hours after exposure to Salinosporamide A while most of the remaining PARP is divided into 24 hrs. Cells treated with Staurosporine (St) show rapid division of PARP with most of this protein being split within 4 hours. These data strongly suggest that Salinosporamide A may induce apoptosis in Jurkat cells.

Example 47

ΆΝΤI ANTRAZ ACTIVITY

To analyze the ability of Salinosporamide A or other compounds to prevent cell death resulting from exposure to LeTx, RAW2 64.7 macrophage cell type and recombinant LF and PA lethal toxin components were used as an in vitro model system for cytotoxicity analysis. , as described below.

RAW264.7 cells (ATCC # TIB-71) were adapted and maintained in Advanced Dulbecco's Modified Eagle Medium (Invitrogen, Carlsbad, CA) supplemented with 5% fetal bovine serum (ADMEM, Mediatech, Herndon, VA) at 37 ° C in a 5% CO2 moistened incubator. Cells were plated overnight in ADMEM supplemented with 5% FBS at 37 ° C in a 5% CO 2 wetted incubator at a concentration of 50,000 cells / partition in a 96 partition plate. Alternatively, DMEM-grown cells supplemented with 10% fetal calf serum were also used and judged mild for this assay. The medium was removed the next morning and replaced with serum free ADMEM with or without Salinosporamide A or Omuralide at doses ranging from 1 μΜ to 0.5 nM for an 8-point dose response. Compounds were prepared from a standard 1 mg / mL DMSO solution and diluted in ADMEM to final concentration. After a 15 minute preincubation, 200 ng / ml LF or 400 ng / ml PA alone or in combination (LeTx) was added to the cells. Recombinants LF and PA were obtained from List Biological Laboratories and stored as 1 mg / ml standard solutions in sterile water containing 1 mg / ml BSA at -80 ° C as described by the manufacturer. Cells were incubated for 6 hours at 37 ° C, followed by the addition of Resazurin as previously described. The plates were incubated for an additional 6 hours before assessing cell viability by measuring fluorescence. Data are a summary of three experiments with three to six replicates per experiment and are expressed as percent viability using the DMSO (negative) and LeTx (positive) controls to normalize the data using the following equation:

Feasibility% = 100 χ (Positive Control Observed Data) / (Negative Control - Positive Control).

The data shown in fig. 36 indicate that Salinosporamide A treatment can prevent LeTx-induced cell death of RAW2 64.7 macrophage-like cells in vitro. Treatment of RAW cells with either LF, PA alone or Salinosporamide A alone resulted in a small reduction in cell viability, whereas treatment with LeTx resulted in approximately 0.27% cell viability compared to controls. Salinosporamide A may increase macrophage cell survival by inhibiting specific protein degradation and decreasing cytokine synthesis, which will ultimately lead to inhibition of the lethal effects of anthrax toxins in vivo.

Although treatment with Salinosporamide A alone produced very modest cytotoxicity at concentrations of 100 nM and above, treatment with lower, relatively non-toxic levels revealed a remarkable increase in the viability of RAW264.7 cells in LeTx-treated cells (Fig. 36). For example, 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 EC50 for Salinosporamide A in these studies was 3.6 nM. In contrast, Omuralide showed a relatively small effect on cell viability until concentrations of 1 μΜ were reached. Even at this high concentration of Omuralide, only 37% viability was observed indicating that Salinosporamide A is a more potent inhibitor of LeTx-induced RAW264.7 cell death. Consistent with these data, Tang et al., Infect. Immun. 67: 3055 (1999) found that EC50 concentrations for MG132 and Lactacystin (the precursor of Omuralide) in the LeTx assay were 3 μΜ. Taken together, these data illustrate that Salinosporamide A is a more potent inhibitor of LeTx-induced cytotoxicity than any other compound described to date. Salinosporamide A promoted the survival of RAW264.7 cells in the presence of LeTx indicating that this compound or its derivatives may represent valuable clinical therapy for anthrax. In addition, it is worth noting that Salinosporamide A is much less cytotoxic in RAW264.7 cells than for many tumor cells.

Example 48

SALINOSPORAMIDE ACTIVITY AGAINST MULTIPLE MYELOMA AND PROSTATE CANCER CELL LINES FN-κΒ appears to be critical for growth and

resistance to apoptosis in Multiple Myeloma and has also been reported to be constitutively active in several prostate cancer cell lines (Hideshima T. et al. 2002, Shimada K. et al. 2002 and Palayoor S.T. et al. 1999). FN-κΒ activity is regulated by the proteasomal degradation of its inhibitor ΙκΒα. Since Salinosporamide A has been shown to inhibit proteasome in vitro and interfere with the FN-κΒ signaling pathway, Salinosporamide A activity against RPMI 8226 multiple myeloma cell line and PC-3 prostate cancer cell lines and DU 145 was evaluated.

EC50 values were determined in standard growth inhibition assays using Resazurin dye and 48 h of drug exposure. Results from independent experiments 2-5 (Table 6) show that EC50 values for Salinosporamide A against RPMI 8226 cell lines and prostate cell lines ranges from 10-37 nM.

Table 6: Salinosporamide EC50 Values Ά

(11-16) against Prostate Tvimor and Multiple Myeloma Cell Lines

<table> table see original document page 161 </column> </row> <table> 161 Salinosporamide A's ability to induce apoptosis in RPMI 8226 and PC-3 cells was evaluated by monitoring the division of PARP and Pro-Caspase 3 using Western Spot analysis. Briefly, PC-3 and RPMI 8226 cells were treated with 100 nM Salinosporamide A (2345R01) for 0, 8 or 24 hours. Total protein lysates were made and 20 μς of the lysates were then resolved under reducing / denaturing conditions and stained with nitrocellulose. The spots were then tested with anti-PARP or anti-caspase-3 antibodies followed by removal and retesting with an anti-actin antibody.

The results of these experiments illustrate that Salinosporamide A treatment of RPMI 8226 cells leads to division of PARP and pro-caspase 3 in a time dependent manner (Fig. 37). RPMI 8226 cells appear to be more sensitive to Salinosporamide A than PC-3 cells since PARP division induction is already noticeable by 8 hours and complete by 24 hours. In contrast, in PC-3 cells PARP splitting is remarkable within 24 hours, whereas pro-caspase 3 splitting is not detected in this experiment (Fig. 37).

RPMI 8226 cells were used to evaluate the effect of 8 hour cell treatment 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 μg of the lysates were then resolved under reducing / denaturing conditions and stained with nitrocellulose. The spots were then tested with anti-PARP or anti-caspase-3 antibodies followed by removal and re-testing with an anti-actin antibody. Fig. 38 demonstrates that Salinosporamide A induces a dose dependent division of both PARP and Pro - Caspase 3.

Example 4 9

MULTIPLE MYELOMA GROWTH INHIBITION

HUMAN BY FORMULAS 1-7, II-2, II-3, II-4, II-5A, II-5B, II-8C, II-13C, 11-16, 11-17, 11-18, 11-19 , 11-20, 11-21, 11-22, II-24C, 11-25, 11-26, 11-28, 11-29, 11-30, 11-31, II-32, II- 38, IV-3C, II-44, VI-1A, 11-47 and 11-50; RPMI 8226 AND U266 CELLS Human multiple myeloma cell lines, RPMI 8226 (ATCC; CCL-155) and U266 (ATCC; TIB-196) were maintained in appropriate culture media. Cells were cultured in an incubator at 37 ° C in 5% CO2 and 95% air humidity.

For cell growth inhibition assays, RPMI 8226 and U266 cells were seeded at 2x104 and 2.5x104 cells / partition respectively in 90 μΐ of a complete medium on Corning 3904 black-walled, light-bottomed culture plates. Standard 20 mM solutions of the compounds were prepared in 100% DMSO, portioned and stored at -80 ° C. Compounds were serially diluted and added in triplicate to the test partitions. The final concentration range of Formulas I-7, I-1-3, 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, II- 44, VI-IA and 11-47 were from 20 μΜ to 6 32 nM. The final concentration of Formulas 11-16, 11-18 and 11-19 ranged from 632 nM to 200 pM. The final concentration range of Formulas II-2, II-4 and II-5A was 2 μΜ to 632 pM. The final DMSO concentration was 0.25% in all samples.

After 48 hours of drug exposure, 10 μΐ of 0.2 mg / ml resazurin (obtained from Sigma-Aldrich Chemical Co.) in Mg2 +, Ca2 + phosphate-free buffered saline, was added to each partition and plates were returned. to the incubator for 3-6 hours. Since living cells metabolize Resazurin, the fluorescence of the Resazurin reduction product was measured using a Fusion Microplate Fluorometer (Packard Bioscience) with filters λβχ = 535 nm and λέπι = 590 nm. Resazurin dye in a cell-free medium was used to determine background, which was subtracted from the data for all experimental partitions. Data were normalized to mean fluorescence of cells treated with medium + 0.25% DMSO (100% cell growth) and EC50 values (the drug concentration at which 50% of the maximum 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.). Data are summarized in Tables 12 and 13. Example 50

SALINOSPORAMIDE Δ (11-16) HOLD DRUG-RESISTANT CELL LINES ACTIVITY

Salinosporamide A EC50 values against the human uterine sarcoma MES-SA cell line and its multi-drug resistant MES-SA / Dx5 derivative were determined to assess whether Salinosporamide A retains activity against a superimposed cell line. expressed by an efflux pumping of P-glycoprotein. Paclitaxel, a known substrate for p-glycoprotein pumping, was included as a control.

Table 7: Salinosporamide EC EC50 values against MES-SA and drug resistant MES-SA / Dx5 derivative

<table> table see original document page 164 </column> </row> <table>

The results of these growth inhibition assays (Table 7) show that, as expected, Paclitaxel does not retain its activity against MES-SA / Dx5 cells as reflected by the 408-fold increase in EC50 values. EC50 values for Salinosporamide A against MES-SA and MES-SA / Dx5 were similar. This illustrates that Salinosporamide A may inhibit the growth of multi-drug resistant MES-SA / Dx5 cell lines suggesting that Salinosporamide A does not appear to be a substrate for p-glycoprotein efflux pumping.

In addition, Salinosporamide A has been evaluated against HL-60 / MX2, the drug-resistant derivative of the human leukemia cell line, HL-60, which has reduced Topo-isomerase II activity and is considered to have atypical resistance to multi-drug. EC 50 values for growth inhibition were determined for Salinosporamide A against HL-60 and HL-60 / MX2. Mitoxantrone DNA binding agent was included as a control, just as HL-60 / MX2 cells are reported to be resistant to this chemotherapeutic agent (Harker W. G. et al. 1989).

Table 8: Salinosporamide A EC50 values against HL-60 and drug resistant HL-60 / MX2 derivative

<table> table see original document page 165 </column> </row> <table>

The data in Table 8 reveal that Salinosporamide A could retain its activity against HL-60 / MX2 cells relative to HL-60 cells, indicating that Salinosporamide A is active in cells expressing reduced topoisomerase II activity. In contrast, Mitoxantrone was approximately 29 times less active against HL-60 / MX2 cells.

Salinosporamide A has also been shown to have activity against drug resistant multiple myeloma cell lines. For example, Salinosporamide A has been shown to be active against doxorubicin resistant MM.IR and Dox-40 cell lines. In addition, Salinosporamide A has been shown to be active against cell lines obtained from multiple myeloma human patients who had relapsed after previous multiple therapies with Dexamethasone, Bortezomiba, and thalidomide. Thus, Salinosporamide A is active against drug resistant multiple myeloma, including multiple myeloma exhibiting resistance to doxorubicin, dexamethasone, bortezomiba, and thalidomide. Similarly, the other compounds described herein are active against drug resistant multiple myeloma including multiple myeloma exhibiting resistance to doxorubicin, dexamethasone, bortezomib, and thalidomide.

Example 51

SALINOSPORAMIDE A AND MISCELLANEOUS ANALOGUE: RELATION OF STRUCTURE ACTIVITY

To establish an initial structure activity relationship (RAE) for Salinosporamide A, a series of Salinosporamide A analogues were evaluated against the multiple myeloma RPMI 8226 cell line. EC50 values were determined in standard growth inhibition assays using Resazurin dye and 48 hours of drug exposure.

The results of this initial RAE series (Table 9) indicate that the addition of a halogen group to the ethyl group appears to increase cytotoxic activity.

Table 9: Salinosporamide A Initial RAE Series

<table> table see original document page 166 </column> </row> <table>

Example 52 IN VIVO BIOLOGY DETERMINATION OF MAXIMUM TOLERATED DOSE (DMT) In vivo studies were designed to determine Salinosporamide A DMT when administered intravenously to female BALB / c mice.

BALB / c 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). The animals were observed daily for clinical signs and were weighed individually twice a week until the end of the experiment (maximum 14 days after the last day of dosing). Results are shown in Table 10 and indicate that a single intravenous dose of Salinosporamide A up to 0.25 mg / kg was tolerated. When administered daily for five consecutive days, Salinosporamide A concentrations up to 0.1 mg / kg were well tolerated. No behavioral changes were noted during the course of the experiment.

Table 10: Determination of Salinosporamide DMT

A in BALB / c female mice

<table> table see original document page 167 </column> </row> <table>

Example 53

PRELIMINARY EVALUATION OF SALINOSPORAMIDE A ABSORPTION, DISTRIBUTION, METABOLISM AND ELIMINATION (ADME) CHARACTERISTICS Studies to begin the evaluation of Salinosporamide A ADME properties were performed. These studies consisted of the evaluation of solubility by determination of LogD74 and a preliminary screen to detect inhibition of cytochrome P450 enzyme. The results of these studies showed an estimated solubility of Salinosporamide A in PBS (pH 7.4) of 9.6 μΜ (3g / ml) and a LogD74 value of 2.4. This value of LogD74 is within the accepted limits compatible with drug development (LogD74 <5.0) and suggests oral availability. Results from the preliminary P450 inhibition screen showed that Salinosporamide A, when tested at 10 μΜ, showed no or low inhibition of all P450 isoforms: CYP1A2, CYP2C9 and CYP3A4 were inhibited by 3%, 6% and 6% respectively. while CYP2D6 and CYP2C19 were inhibited by 19% and 22%, respectively.

Example 54

SALT INO S PORAMIDE A AND ITS EFFECTS IN VIVO ON FULL BLOOD PROTEASOMA ACTIVITY

Salinosporamide A has previously been shown to be a potent and specific proteasome inhibitor in vitro, with an IC50 of 2 nM for chymotrypsin-like activity of purified 20S proteasomes. To monitor Salinosporamide A activity in vivo, a rapid and reproducible assay (adapted from Lightcap et al. 2000) was developed to evaluate proteosome activity in whole blood.

In summary, frozen whole blood samples were thawed on ice for one hour and resuspended in 700 μΕ of 5 ii frozen EDTA, pH 8.0, to lyse cells through hypotonic shock. This represents approximately 2-3 times the volume of accumulated whole blood cells. The lysis was allowed to proceed for one hour, and the cellular debris was removed by centrifugation at 14,000 χ g for 10 minutes. The supernatant (Accumulated Complete Blood Lysate, LSCA) was transferred to a fresh tube, and the pellet discarded. LSCA protein concentration was determined by BCA analysis (Pierce) using BSA as a standard. Approximately 80% of the samples had a total protein concentration between 800 and 1,200 μg / mL. Proteasoma activity was determined by measuring the hydrolysis of a fluorogenic substrate specific for proteasome chemotrypsin-like activity (suc-LLVI-AMC, Bachem Cat. No. 1-1395). Control experiments indicated that> 98% of the hydrolysis of this peptide in these extracts is proteasome mediated. Assays were set up by mixing 5 μ] ^ of an animal LSCA with 185 μΙ <of a assay buffer (20 mM HEPES, 0.5 mM EDTA, 0.05% Triton X-100, 0.05% SDS, pH 7.3) in Costar 3904 plates. Titration experiments revealed that there is a linear relationship between protein concentration and hydrolysis rate if the protein concentration in the assay is between 200 and 1,000 μg. Reactions were initiated by the addition of 10 μΕ 0.4 mM suc-LLVI-AMC (prepared by diluting a 10 mM solution of the peptide in 1:25 DMSO with assay buffer), and incubated in a fluorometer (Fluoroskan Labsystems) at 37 ° C. Substrate hydrolysis results in free AMC release, which was fluorometrically measured using λβχ = 390 nm and λβιη = 460 nm. The hydrolysis rate in this system is linear for at least one hour. The hydrolysis rate of each sample is then normalized to fluorescent relative units per milligram protein (URF / mg).

To explore the in vivo activity of Salinosporamide A, Swiss male Webster rats (5 per group, 20-25g by weight) were treated with various concentrations of Salinosporamide A. Salinosporamide A was administered intravenously and given its LogD74 value of 2.4. suggesting oral availability, Salinosporamide A was also administered orally. Salinosporamide A dosing solutions were generated immediately prior to administration by diluting standard Salinosporamide A solutions (100% DMS0) using 10% solutol to obtain a final concentration of 2% DMSO. The control vehicle consisted of 2% DMS0 in 10% solutol. One group of animals was not dosed with either vehicle or Salinosporamide A to establish a baseline for proteasoma activity. Salinosporamide A or vehicle was administered at 10 mL / kg and ninety minutes after administration the animals were anesthetized and blood was removed by cardiac puncture. Accumulated whole blood cells were collected by centrifugation, washed with PBS, and re-centrifuged. All samples were stored at -80 ° C prior to evaluation of proteasome activity.

To certify that the substrate hydrolysis observed in these experiments was only due to proteasome activity, dose response experiments were performed on the extracts using the highly specific proteasome inhibitor Epoxomycin. LSCA lysates were diluted 1:40 in a assay buffer, and 180 μL was added to Costar 3904 plates. Epoxomycin (Calbochem Cat. No. 324800) was serially diluted in DMSO to generate a dose response curve of. eight points, diluted 1:50 in assay buffer, and 10 μL added to the triplicate diluted LSCA. Samples were preincubated for 5 minutes at 37 ° C, and substrate-initiated reactions as seen above. Dose response curves were analyzed using Prisma using a variable slope sigmoidal dose response as a model.

Fig. 40 is a scattered plot showing normalized proteasome activity in LSCA derivatives of individual mice (5 mice per group). In each group, the horizontal bar represents the average of normalized activity. These data show that Salinosporamide A causes a profound decrease in proteasome activity in LSCA, and that this inhibition is dose dependent. In addition, these data indicate that Salinosporamide A is active in oral administration.

The specificity of the assay was shown by examining the effect of a known proteasome inhibitor, Epoxomycin, on peptide substrate hydrolysis. Epoxomycin is a peptide epoxide that has been shown to be highly proteasome specific, with no inhibitory activity for any other known protease (Meng et al., 1999). Lysates from a control vehicle and also from intravenously (i.v.) treated animals with 0.1 mg / kg Salinosporamide A were incubated with varying concentrations of Epoxomycin, and IC 50 values were determined. Palayoor et al., Oncogene 18: 7389-94 (1999). As shown in fig. 41, Epoxomycin caused a dose-dependent inhibition in proteasome substrate hydrolysis. The IC50 obtained in these experiments well coincides with the 10 nM value observed using in vitro purified 20S proteasomes (not shown). These data also indicate that the remaining activity for this substrate in these lysates prepared from animals treated with 0.1 mg / kg Salinosporamide A is due to proteasome, not some other protease. Residual activity seen in extracts treated with high doses of Epoxomycin is less than 2% of the total signal, indicating that more than 98% of the activity observed with suc-LLVI-AMC as a substrate is due solely to the activity of LSCA proteasomes.

Comparison of intra-run variation in baseline activity and Salinosporamide A's ability to inhibit proteasomal activity was also evaluated. In fig. 42, the results of separate assays performed separately several weeks apart are shown. Qureshi, et al., J. Immunol. 171 (3): 1515-25 (2003). For clarity, only the control vehicle and matching dose results are shown. While there was some variation in proteasome activity in individual animal-derived LSCA in the control groups, the overall mean was very similar between the two groups. Animals treated with Salinosporamide A (0.1 mg / kg i.v.) also showed very similar residual activity and mean inhibition. This suggests that the results between trials can be compared reliably.

Example 55

LPS-INNUCTED FNT INHIBITION BY

SALINOSPORAMIDE TO

Studies suggest that proteasomes play a role in activating many signaling molecules, including FN-κΒ transcription factor via proteolytic degradation of the FN-κΒ inhibitor (IkB). LPS signaling by the TLR4 receptor activates FN-κΒ and other transcriptional regulators resulting in the expression of a host of proinflammatory genes such as TNF, IL-6, and IL-β. Continued expression of proinflammatory cytokines has been identified as a major factor in many diseases. TNF and IL-β inhibitors have been shown to be effective in many models of inflammation including the murine LPS model as well as animal models of rheumatic arthritis and inflammatory bowel disease. Recent studies have suggested that proteasome inhibition may prevent LPS-induced TNF secretion (Qureshi et al., 2003). These data suggest that Salinosporamide A, a new and potent proteasome inhibitor, may prevent TNF secretion in vivo in the high dose murine LPS model.

To assess Salinosporamide A's ability to inhibit rat plasma LPS-induced TNF levels in vivo, in vivo studies were initiated at BolderBioPATH, Inc. in Boulder, CO. The following methods outline the draft protocol for these studies.

Swiss Webster male rats (12 / group weighing 20-25 g) were injected with LPS (2 mg / kg) by route i.p .; Thirty minutes later, rats were injected i.v. (tail vein) with Salinosporamide Aa 2.5 mg / kg after approximately 5 minutes under a heat lamp. Ninety minutes after LPS injection, rats were anesthetized with Isoflurane and bled through cardiac puncture to obtain plasma. The remaining blood pellet was then resuspended in 500 µl PBS to wash away the residual serum proteins and centrifuged again. The supernatant was removed and the blood pellet frozen for analysis of proteasome inhibition in accumulated whole blood lysate.

Table 11

<table> table see original document page 172 </column> </row> <table> <table> table see original document page 173 </column> </row> <table>

Dosing solutions were prepared using a standard 10 mg / mL solution of Salinosporamide A in 100% DMSO. A 10% solutol solution was prepared diluted wt / wt with endotoxin free water, and a 1: 160 dilution was made with the standard 10 mg / ml Salinosporamide A. Animals were dosed i.v. at 4 ml / kg. A control vehicle solution was also prepared by making the same 1: 160 dilution with 100% DMSO in 10% solutol solution giving a final concentration of 9.375% solutol in water and 0.625% DMSO. TNF plasma measurements were made using the Biosource mFNT Cytoset equipment (Biosource Intl., Camarillo, CA; No. CMC3014 in the catalog) according to the manufacturer's instructions. Samples were diluted 1:60 for the assay.

Data from two independent experiments with at least ten replicated animals per group indicated that treatment with 0.125 or 0.25 mg / kg Salinosporamide A decreased LPS-induced TNF secretion in vivo. A representative experiment is shown in fig. 43. These data show that treatment of animals with 0.25 mg / kg Salinosporamide Thirty minutes after the injection of 2 mg / kg LPS resulted in a significant reduction in TNF serum levels. Accumulated whole blood samples were also analyzed for ex vivo proteasome inhibition revealing 70 ± 3% inhibition in 0.125 mg / kg treated animals, and 94 ± 3% in 0.25 mg / kg treated animals. No significant differences were seen in proteasome inhibition in animals treated with or without LPS. Salinosporamide A reduces LPS-induced TNF plasma levels by approximately 65% when administered at 0.125 or 0.25 mg / kg i.v. in rats 30 minutes after LPS treatment.

Example 56

IN VITRO POTENTIAL CHEMICAL SENSITIVE EFFECTS OF SALINOSPORAMIDE A

Chemotherapy agents such as CPT-Il (Irinotecan) may activate nuclear factor-kappa B (FN-κΒ) transcription 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, FN-κΒ resides in the cytoplasm in an inactive complex with the inhibitor protein IkB, (FN-κΒ inhibitor). Various stimuli may cause IkB phosphorylation by IkB kinase, followed by ubiquitination and IkB degradation by the proteasome. Following ΙκΒ degradation, FN-κΒ translocates to the nucleus and regulates gene expression, affecting many cellular processes, including over-regulation of survival genes thereby inhibiting apoptosis.

The recently approved proteasome inhibitor, Velcade® (PS-341; Millennium Pharmaceuticals, Inc.), is directly toxic to cancer cells and may also increase the cytotoxic activity of CPT-Il against LoVo cells in vitro and in a LoVo xenographic model. by inhibiting proteasome-induced IkB degradation. Adams J., Eur. J. Haematol. 70: 265 (2003). In addition, VelcadeTM has been found to inhibit expression of pro-angiogenic chemokines / cytokines GRO-α and VEGF in squamous cell carcinomas, presumably by inhibition of the FN-κΒ pathway. Sunwoo, et al., Clin. Cancer Res. 7: 1419 (2001). The data indicate that proteasome inhibition can not only decrease tumor cell survival and growth, but also angiogenesis.

Example 57

INHIBITION OF GROWTH OF MYELOMA AND MULTIPLE MELANOMA, COLON, PROSTATE, BREAST, LUNG AND 0VARIAN0

Human colon adenocarcinoma cells (HT-29; HTB-38), prostate adenocarcinoma (PC-3; CRL-1435), breast adenocarcinoma (MDA-MB-231; HTB-26), non-cell lung carcinoma -small (NCI-H292; CRL-1848), adenocarcinoma

(OVCAR-3; HTB-161), multiple myeloma (RPMI 8226; CCL-155), multiple myeloma (U266; TIB-196) and rat melanoma (B16-FlO; CRL-6475) were all purchased from ATCC and maintained in appropriate culture media. Cells were cultured in a 37 ° C incubator at 5% CO2 and 95% air humidity. For cell growth inhibition assays, HT-29, PC-3, MDA-MB-231, NCI-H292, OVCAR-3, and B16-F10 cells were seeded at 5x103, 5x103, Ix104, 4x104, and 1x104. 25x103 cells / partition respectively in 90 μΐ of a complete medium in light-walled, black-walled, 96-partition tissue culture plates (Corning; 3904), and the plates were incubated overnight to allow the cells would settle and enter the growth phase. RPMI 8226 and U266 cells were seeded at 2x104 and 2.5x104 cells / partition respectively in 90 μl of a complete medium in 96 partition plates on the day of the assay. Standard 20 mM solutions of the compounds were prepared in 100% DMSO and stored at -80 ° C. Compounds were serially diluted and added in triplicate to the test partitions. Concentrations ranging from 6.32 μΜ to 632 pM were tested for II-2 and II-4. II-3 and 11-17 were tested at concentrations ranging from 20 μΜ to 6.32 nM. Formulas 11-18 and II-19 were tested at concentrations ranging from 2 μΜ to 200 pM. Formula II-5A and Formula II-5B were tested at final concentrations ranging from 2 μΜ to 632 pM and 20 μΜ to 6.32 nM respectively. The plates were returned to the incubator for 48 hours. The final DMSO concentration was 0.25% in all samples.

After 48 hours of drug exposure, 10 μΐ of 0.2 mg / ml resazurin (obtained from Sigma-Aldrich Chemical Co.) in Mg2 +, Ca2 + phosphate-free buffered saline, was added to each partition and plates were returned. to the incubator for 3-6 hours. Since living cells metabolize Resazurin, the fluorescence of the Resazurin reduction product was measured using a Fusion Microplate Fluorometer (Packard Bioscience) with filters λβχ = 535 nm and Àem = 590 nm. Resazurin dye in a cell-free medium was used to determine background, which was subtracted from the data for all experimental partitions. Data were normalized to mean fluorescence of cells treated with medium + 0.25% DMSO (100% cell growth) and EC50 values (the drug concentration at which 50% of the maximum 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%, the EC50 value was not determined.

The data in Table 12 summarize the growth inhibitory effects of Formulas II-2, II-3, II-4, II-5A, II-5B, 11-17, 11-18 and 11-19 against carcinoma cell lines. human colorectal, HT-29, human prostate carcinoma, PC-3, human breast adenocarcinoma, MDA-MB-231, non-small cell human lung carcinoma, NCI-H292, human ovarian carcinoma, OVCAR-3, human multiple myeloma, RPMI 8226 and U266, and murine melanoma B16-F10.

Table 12

EC50 values of Formulas II-2, II-3, II-4, II-5A, II-5B, 11-17, 11-18 and 11-19 against various tumor cell lines

<table> table see original document page 176 </column> </row> <table>

* Where η = 3, the mean ± standard deviation is presented; NT = not tested

EC 50 values indicate that Formulas II-2, II-4, II-5A, II-5B, 11-16, 11-17, 11-18 and 11-19 were cytotoxic against HT-Tumor cell lines. 29, PC-3, MDA-MB-231, NCI-H292, RPMI 8226, U266 and B16-F10. II-2, II-5A, II-5B and 11-19 were also cytotoxic against OVCAR-3 tumor cells. Formula 11-17 was cytotoxic against tumor cell lines MDA-MB-231, RPMI 8226, U266 and B16-F10.

The data in table 13 summarize the growth inhibitory effects of Formulas 1-7, II-2, II-3, II-4, II-5A, II-5B, II-8C, II-13C, 11-16, 11. -17, 11-18, 11-19, 11-20, II-21, 11-22, II-24C, 11-25, 11-26, 11-28, 11-29, 11-30, 11-31 , II- 32, 11-38, IV-3C, 11-44, VI-1A, 11-47 and 11-50 against human multiple myeloma cell lines RPMI 8226 and U266.

Table 13

EC50 mean values of Formulas 1-7, II-2, II-3, II-4, II-5A, II-5B, II-8C, II-13C, 11-16, 11-17, 11-18, 11-19, 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, 11-44, VI-1A, 11-47 and 11-50 against RPMI 8226 and U266 cells

<table> table see original document page 177 </column> </row> <table> <table> table see original document page 178 </column> </row> <table> <table> table see original document page 179 < / column> </row> <table>

Where η> 3, the mean value of EC50 ± standard deviation is displayed; * η = 3, standard deviation not applicable; ND not determined.

EC 50 values indicate that Formulas II-2, II-4, II-5A, II-5B, 11-16, 11-17, 11-18, 11-19, 11-20, 11-22, II- 24C, 11-26, 11-29 and 11-30 were cytotoxic against RPMI 8226 and U266 cells. Formulas 1-7, 11-38, 11-44, VI-1A, 11-47 and 11-50 were cytotoxic against RPMI 8226 cells. Formulas 11-21 and IV-3C were cytotoxic against U266 cells.

Example 58

MES-SA, MES-SA / DX5, HL-60, and HL-60 / MX2 TUMOR CELL LINES GROWTH INHIBITION

Human uterine sarcoma cells (MES-SA; CRL-1976), its multi-drug resistant derivative (MES-SA / Dx5; CRL-1977), human acute promyelocytic leukemia (HL-60; CCL-240) and its Multi-drug resistant derivative (HL-60 / MX2; CRL-2257) were purchased from ATCC and maintained in appropriate culture media. The cells were cultured in a 37 ° C incubator at 5% CO2 and 95% air humidity.

For cell growth inhibition assays, MES-AS and MES-AS / Dx5 cells were both seeded at 3x10 ^ 3 cells / partition in 90 μl of a complete medium in light-walled, light-bottomed tissue culture plates. 96 partitions (Corning; 3904), and the plates were incubated overnight to allow cells to settle and grow. HL-60 and HL-60 / MX2 cells were both seeded at 5x10 4 cells / partition in 90 µl of complete medium in 96-partition plates on the day of the assay. Standard 20 mM solutions of the compounds were prepared in 100% DMSO and stored at -80 ° C. Compounds were serially diluted and added in triplicate to the test partitions. Concentrations ranging from 6.32 μΜ to 2 nM were tested for II-2 and II-4. II-3 and 11-17 were tested at concentrations ranging from 20 μΜ to 6.32 nM. Compound 11-18 was tested at concentrations ranging from 2 μΜ to 632 pM. The plates were returned to the incubator for 48 hours. The final DMSO concentration was 0.25% in all samples.

After 48 hours of drug exposure, 10 μm 0.2 mg / ml resazurin (obtained from Sigma-Aldrich Chemical Co.) in Mg2 +, Ca2 + phosphate buffered saline, was added to each partition and plates were returned. to the incubator for 3-6 hours. Since living cells metabolize Resazurin, the fluorescence of the Resazurin reduction product was measured using a Fusion microplate fluorometer (Packard Bioscience) with filters λβχ = 535 nm and Xem = 590 nm. Resazurin dye in a cell-free medium was used to determine background, which was subtracted from the data for all experimental partitions. Data were normalized to mean fluorescence of cells treated with medium + 0.25% DMSO (100% cell growth) and EC50 values (the drug concentration at which 50% of the maximum 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%, the EC50 value was not determined.

The multi-drug resistant MES-SA / Dx5 tumor cell line was derived from the MES-SA human uterine sarcoma tumor cell line and expressed high P-Glycoprotein (P-gp), an ATP-dependent efflux pumping . The data in Table 14 summarize the growth inhibitory effect of Formulas II-2, II-3, II-4, 11-17 and 11-18 against MES-SA and its multi-drug resistant MES-SA / Dx5 derivative. Paclitaxel, a known substrate for P-gp pumping, was included as a control. Table 14

EC50 values of Formulas II-2, II-3, II-4, II- 17 and 11-18 against MES-SA and MES-SA / Dx5 tumor cell lines

<table> table see original document page 181 </column> </row> <table>

* Amount of change times = EC50 rate values (MES-SA / Dx5: MES-SA)

EC50 values indicate that II-2, II-4, 11-17 and 11-18 have cytotoxic activity against both MES-AS and MES-SA / Dx5 tumor cell lines. The multi-drug resistant phenotype was confirmed by the observation that Paclitaxel was ~ 800-fold less active against resistant MES-SA / Dx5 cells.

HL-60 / MX2 is a multi-drug resistant tumor cell line derived from the human promyelocytic leukemia cell line, HL-60, and expresses reduced topoisomerase II activity. The data presented in Table 15 summarize the growth inhibitory effect of Formulas II-2, II-3, II-4, 11-17 and 11-18 against HL-60 and its multi-drug resistant derivative HL-60 / MX2. . Mitoxantrone, the topoisomerase II targeting agent, was included as a control. Table 15

<table> table see original document page 182 </column> </row> <table>

* Amount of change times = EC50 rate values (MES-SA / Dx5: MES-SA)

EC50 values indicate that II-2, H-4 and 11-18 retained cytotoxic activity against HL-60 and HL-60 / MX2 tumor cell lines. The multidrug resistant phenotype was confirmed by the observation that Mitoxantrone was ~ 30 times less active against resistant HL-60 / MX2 cells.

Example 59

THE EFFECTS OF FORMULA 11-16, FORMULA 11-17, FORMULA 11-20 AND OMURALIDE ON CHEMYTRYPSIN TYPE 20S ACTIVITY IN RPMI 8226 CELLS

RPMI 8226 (ATCC, CCL-155), the human multiple myeloma cell line, was grown in an RPMI medium supplemented with 2 mM L-glutamine, 100 units / ml penicillin, 100 μg / ml streptomycin, 1 mM of sodium pyruvate, 10% heat-inactivated fetal bovine serum at 37 ° C, 5% CO2, and 95% air humidity. To assess inhibitory effects on 20S proteasome chymotrypsin-like activity, test compounds prepared in DMSO were appropriately diluted in culture medium and added to 2.5 χ 105 / ml RMPI 8226 cells. For Formula 11-16, Final test concentrations ranged from 1 nM to 100 nM. For Formula 11-17, Formula 11-20 and Omuralida (Calbiochem, San Diego, CA), the final test concentrations ranged from 1 nM to 10 μΜ. DMSO was used as the vehicle control at a final concentration of 0.1%. After 1 h incubation of the RMPI 8226 cells with the compounds, the cells were pelleted by centrifugation at 2,000 rpm for 10 seconds at room temperature and washed 3 times with ice cold Phosphate Buffered Dulbecco Saline (SDTF, Mediatech, Herndon, VA). SDTF-washed cells were lysed on ice for 15 min in lysis buffer (20 mM HEPES, 0.5 mM EDTA, 0.05% Triton X-100, pH = 7.3) supplemented with an inhibitor cocktail. protease (Roche Diagnostics, Indianapolis, IN). Cell debris was pelleted by centrifugation at 14,000 rpm for 10 min, 4 ° C, and supernatants (cell lysates) were transferred to a new tube. Protein concentration was determined by the BCA protein assay kit (Pierce Biotechnology, Rockford, IL). The 20S proteasome chemotrypsin-like activity was measured 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. Reactions were initiated by the addition of 10 μΙ; of 0.4 mM Suc-LLVY-AMC (prepared by diluting a 10 mM solution of the peptide in 1:25 DMSO with assay buffer) to 190 μL of cell lysates and incubated in Thermo Lab Systems Fluoroskan plate reader at 37 ° C. The released coumarin (AMC) was measured fluorometrically using Xex = 390 nm and λέπι = 460 nm. The assay was performed on a micro-titer plate (Corning 3904) and followed kinetically with measurements every five minutes for 2 h. The total amount of protein used for each assay was 20 μg. The final concentration of Suc-LLVY-AMC and DMSO was 20 μΜ and 0.2%, respectively. The results are presented as the percentage inhibition of 20S proteasome chemotrypsin type activity in relation to DMSO control.

The 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 20S proteasome chemotrypsin-like activity. Among them, Formula II-16 inhibits 85 + 7% of 20S proteasome chymotrypsin-like activity at 5 nM. At 100 nM, Formula 11-16 can completely inhibit 20S proteasome chymotrypsin-like activity. At 100 nM, Formula 11-17, Formula 11-20 and Omuralide can only inhibit chymotrypsin-like activity by 30 ± 4%, 66 ± 3% and 32 ± 8%, respectively.

Table 16

Determination of chymotrypsin-like activity of 20S proteasomes derived from BMPI 8226 cells treated with formula 11-16, formula 11-17, formula 11-20 and Omuralida

<table> table see original document page 184 </column> </row> <table>

ND: not determined

Example 60

THE EFFECTS OF FORMULA 11-16, FORMULA 11-17, FORMULA 11-20 AND OMURALIDE ON PROTEASOMA 2K CHYTOTRIPSIN TYPE ACTIVITY IN PC-3 CELLS

PC-3 (ATCC, CRL-1435), the human prostate cancer cell line, was grown in an F12K medium supplemented with 2 mM L-glutamine, 100 units / ml penicillin, 100 μg / ml streptomycin and 10% heat-inactivated fetal bovine serum at 37 ° C, 5% CO2 and 95% air humidity.

To assess inhibitory effects on 20S proteasome chymotrypsin activity, test compounds prepared in DMSO were adequately diluted in a culture medium and added at 1.25 * 105 / ml PC-3 cells. For Formula 11-16, the final test concentrations ranged from 1 nM to 50 nM. For Formula 11-17, Formula 11-20 and Omuralida (CalBiochem, San Diego, CA, USA), the final test concentrations ranged from 1 nM to 10 μΜ. DMSO was used as vehicle control at a final concentration of 0.1%. After 1h incubation of the PC-3 cells with the compounds, the cells were washed 3x with 1x Dulbecco frozen phosphate buffered saline (DPBS, Mediatech, Herndon, VA, USA). DPBS-washed cells were lysed on ice for 15 min in lysis buffer (20 mM HEPES, 0.5 mM EDTA, 0.05% Triton X-100, pH 7.3) supplemented with an inhibitor cocktail. protease (Roche Diagnostics, Indianapolis, EM, USA). Cell debris was pelleted by centrifugation at 14,000 rpm for 10 min, 4 ° C and supernatants (cell lysates) were transferred to a new tube. Protein concentration was determined by BCA protein assay equipment (Pierce Biotechnology, Rockford, IL, USA). The 20S proteasome chymotrypsin activity was measured using the Suc-LLVY-AMC fluorogenic peptide substrate (Boston Biochem, Cambridge, MA, USA) in the proteasome assay buffer (20 mM HEPES, 0.5 mM EDTA, pH 8.0) containing a final concentration of 0.035% SDS. Reactions were initiated by the addition of 10 μL of 0.4 mM Suc-LLVY-AMC (prepared by diluting a 10 mM solution of peptide in 1:25 DMSO with assay buffer) to 190 μL of cell lysates and incubated. Thermo Lab Systems Fluoroskan plate reader at 37 ° C. Released coumarin (AMC) was fluorometrically measured using λθχ = 390 nm and λem = 460 nm. The assay was performed on a micro-titer plate (Corning 3904) and followed kinetically with measurements every five minutes for 2 hours. The total amount of protein used for each assay was 20 μg. The final Suc-LLVY-AMC and DMSO concentrations were 20 μΜ and 0.2%, respectively. Results are presented as the percent inhibition of 20S proteasome chymotrypsin activity relative to DMSO control.

The results in Table 17 show that exposure of PC-3 cells to Formula 11-16, Formula 11-17, Formula 11-20 and Omuralide resulted in inhibition of 20Ss proteasome chymotrypsin activity similar to results obtained from cell-based experiments. RPMI 8226. Formula 11-16 inhibited 69% of the 5 nM proteasome 20S chymotrypsin activity. At 50 nM, Formula 11-16 could completely inhibit the 20S proteasome chymotrypsin activity. At 100 nM, Formula 11-17, Formula 11-20 and Omuralide inhibited chymotrypsin activity by 26%, 57% and 36%, respectively.

Table 17

Determination of 20Ss proteasome chymotrypsin activity derived from PC-3 cells treated with Formula 11-16, Formula 11-17, Formula 11-20 and Omuralide

<table> table see original document page 186 </column> </row> <table>

ND: not determined

Example 61

INHIBITION OF GROWTH OF HUMAN MULTIPLE MYELOMA CELLS, RPMI 8226, HUMAN COLON ADENOCARCINOMA, HT-29 AND MURINA MELANOMA, B16-F10, IN ME MEDIUM CONTAINING 1% OR 10% SERUM

The growth inhibiting activity of Formulas 11-16, 11-17 and Formula 11-18 against human multiple myeloma cells, RPMI 8226, human colon adenocarcinoma, HT-29 and rat melanoma, B16-F10 in the presence of 1% or 10% fetal bovine serum (FBS) was determined.

RPMI 8226 (CCL-155), HT-29 (HTB-38), and B16-F10 (CRL-6475) cells were purchased from ATCC. RPMI 8226 cells were maintained in RPMI 1640 medium supplemented with 10% (vol / vol) SBF, 2 mM L-glutamine, 1 mM sodium pyruvate and 100 IU / ml Penicillin / Streptomycin and 100 μg / ml respectively. HT-29 cells were maintained in McCoys 5A medium supplemented with 10% (vol / vol) SBF, 2 mM L-glutamine, 1 mM sodium pyruvate, 1% (vol / vol) non-amino acids. 10 mM HEPES and Penicillin / Streptomycin at 100 IU / ml and 100 μg / ml respectively. B16-F10 cells were maintained in DMEM medium supplemented with 10% (vol / vol) SBF, 2 mM L-glutamine, 10 mM HEPES and Penicillin / Streptomycin at 100 IU / ml and 100 μg / ml, respectively. . Cells were cultured in a 37 ° C incubator at 5% CO2 and 95% air humidity.

For cell growth inhibition assays, HT-29 and B16-F10 cells were seeded at 5 * 103 and 1.25 * 103 cells / well respectively, in 90 μl of a medium containing 10% (vol / vol) of FBS or 1% (vol / vol) of FBS in 96-well, black, transparent bottom wall tissue culture plates (Corning 3904). The plates were incubated to allow the cells to settle and grow overnight. RPMI 8226 cells were seeded at 2 * 10 4 cells / well in 90 µl RPMI medium containing 10% (vol / vol) SBF or 1% (vol / vol) SBF in black wall tissue culture plates, Transparent bottom with 96 containers. Standard 20 mM solutions of Formulas 11-16, 11-17 and Formula 11-18 were prepared in 100% DMSO, partitioned and stored at -80 ° C.

Formulas 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 vessels. The final concentration of Formula 11-16 ranged from 2 μΜ to 200 pM. The final concentration range of Formula 11-17 was 20 μΜ to 6.3 nM. The final concentration of Formula 11-18 ranged from 2 μΜ to 630 pM. The plates were returned to the incubator for 48 hours. The final DMSO concentration was 0.25% in all samples.

After 48 hours of drug exposure, 10 μl of 0.2 mg / ml resazurin (obtained from Sigma-Aldrich Chemical Co.) in Mg2 +, Ca2 + phosphate-free buffered saline was added to each well and plates were returned to incubator for 3-6 hours. Since living cells metabolize Resazurin, the fluorescence of the Resazurin reduction product was measured using a Fusion microplate fluorometer (Packard Bioscience) with filters of λβχ = 535 nm and λβπι = 590 nm. Resazurin dyeing in a cell-free medium was used to determine background, which was subtracted from the data for all experimental recipients. Data were normalized to the mean fluorescence of cells treated with medium + 0.25% DMSO (100% cell growth) and EC50 values (the drug concentration at which 50% of the observed maximum growth inhibition is established) were determined using a standard sigmoidal dose response curve fitting algorithm (generated by XLfit 3.0, ID Business Solutions Ltd.).

The data in Table 18 summarize the growth inhibitory effect of Formulas 11-16, 11-17 and Formula 11-18 against human multiple myeloma cell line, RPMI 8226, in medium containing 1% or 10% FBS. .

Table 18

EC50 values of Formulas 11-16, 11-17 and Formula 11-18 against RPMI 8226 cells in medium containing 1% or 10% FBS

<table> table see original document page 188 </column> </row> <table>

EC50 values indicate that Formulas 11-16, 11-17 and Formula 11-18 were cytotoxic against RPMI 8226 cells in a medium containing 1% or 10% FBS. There was a less than three-fold decrease in the average EC50 of Formulas 11-16, 11-17, and Formula 11-18 when tested on medium containing 10% FBS relative to medium containing 1% FBS.

The data in Table 19 summarize the growth inhibitory effect of Formula 11-16 against human colon adenocarcinoma, HT-29, and murine melanoma, B16-F10 cell lines, in a medium containing 1% or 10% of SBF. Table 19

EC50 values of Formula 11-16 against HT-29 and B16-F10 cells in medium containing 1% or 10% FBS

<table> table see original document page 189 </column> </row> <table>

EC50 values indicate that Formula 11-16 was cytotoxic against HT-29 and B16-F10 cells in a medium containing 1% or 10% FBS. There was less than a two-fold decrease in the average EC50 of Formula 11-16 when tested on medium containing 10% FBS relative to medium containing 1% FBS. Taken together, these data show that with respect to in vitro cytotoxic activity against tumor cell lines, Formulas 11-16, 11-17 and Formula 11-18 maintain similar biological activity in the presence of 1% or 10% FBS. .

Example 62

ANTIZE LETHAL TOXIN INHIBIT

Anthrax toxin is responsible for the symptoms associated with anthrax. In this disease, B. Anthracis spores are inhaled and lodge in the lungs where they are ingested by macrophages. Within macrophages, spores germinate, reproduce, resulting in cell death. Before this death occurs, however, infected macrophages migrate to lymph nodes where, at their death, they release their contents, allowing organisms to enter the bloodstream, reproduce later, and secrete lethal toxins.

Two proteins called protective antigen (AP; 83 kDa) and lethal factor (FL; 90 kDa) play a key role in anthrax pathogenesis. These proteins are collectively known as lethal toxin (TxLe). When combined, AP and FL cause death when injected intravenously into animals. Lethal toxin is also active in some macrophage cell culture lines causing cell death within hours. TxLe can induce necrosis and apoptosis in RAW264.7 cells as rat macrophages in in vitro treatment. In Vitro Cell-Based Assay for Lethal Toxin-Mediated Cytotoxicity Inhibitors

RAW264.7 cells (obtained from the American Type Culture Collection, ATCC) were adapted and maintained in an RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 1% Penicillin / Streptomycin (medium 37 ° C in a 5% CO2 incubator with 95% air humidity. For

In the assay, cells were plated overnight in complete medium at a concentration of 50,000 cells / well in a 96 well plate. The medium was removed the next day and replaced with a complete serum free medium with or without varying concentrations of Formulas II-2, II-3, 11-4, II-5A, II-5B, II-13C, 11-17 11-18 and IV-3C starting at 330 nM and diluting at half-intervals for an 8-point dose response. After a 45 minute preincubation, 1 μg / ml FL and 1 μg / ml AP alone or in combination (FL: AP, also called lethal toxin (TxLe)) were added to the cells. Recombinant FL and AP were obtained from the List Biological Laboratories laboratory. Additional cards without TxLe were included as a control. The cells were then incubated for six hours followed by the addition of 0.02 mg / ml resazurin stain (Molecular Probes, Eugene, OR, USA) prepared in Mg ++, Ca ++ phosphate buffered saline (Mediatech, Herndon, VA, USA). . Plates were then incubated for an additional 1.5 hours prior to cell viability assessment. Since resazurin is metabolised by living cells, cytotoxicity or viability of cells can be assessed by measuring fluorescence using 530 nm excitation and 590 nm emission filters.

Data are expressed as percent viability when compared to a DMSO alone (high) control and TxLe alone (low) control using the following equation: Viability percentage = 100 * ((measured OD - lowest control) / (higher control - lower control)).

Inhibition of Lethal Anthrax Mediated Cytotoxicity in RAW264.7 cells

The data in fig. 48 summarize the effects of Formula II-2, Formula II-3 and Formula II-4 against cell line TxLe-mediated cytotoxicity as murine macrophage cell RAW264.7. Treatment of RAW264.7 cells with Formula II-2 and Formula II-4 resulted in an increase in viability of treated TxLe cells with EC50 values of 14 nM (Fig. 48). EC50 values for Formula II-3 for TxLe protection were not determined at the concentrations tested (EC50> 330 nM, the maximum concentration assessed). The data in Table 20 show the effects of Formulas II-5A, II-5B, II-13C, 11-17, 11-18 and IV-3C against TxLe-mediated cytotoxicity of the RAW264.7 macrophage murine cell line . Treatment of RAW264.7 cells with Formulas II-5A and 11-18 showed an increase in viability of treated TxLe RAW264.7 cells, with EC50 values of 3 nM and 4 nM, respectively. Treatment with Formula II-5B and Formula 11-17 resulted in an increase in viability of treated TxLe cells with an EC50 value of 45 nM. EC50 values for Formulas II-13C and IV-3C for TxLe protection could not be determined at the concentrations tested (EC50> 330 nM, the maximum concentration assessed).

Table 20

EC50 values for inhibition of lethal anthrax toxin mediated RAW264.7 cell cytotoxicity

<table> table see original document page 191 </column> </row> <table>

Example 63

ACTIVITY-STRUCTURE RELATIONSHIP OF THE RL SIDE CHAIN

A structure-activity relationship for the R1 side chain of the described compounds and specifically of the compounds of Formulas (I), (II), (III), (IV) and (V) was deduced by analyzing the relative activity of various compounds having the Formula: <formula> formula see original document page 192 </formula>

These compounds were analyzed for cytotoxicity of RPMI cells and for inhibition of NF-κΒ activities and as chymotrypsin, as trypsin, and as 2OS proteasome caspase. Results of 6 representative compounds are presented in Table 21.

Table 21

EC50 values for compounds having varied R1 groups

<table> table see original document page 192 </column> </row> <table>

The results of the above assays can be interpreted to suggest that compounds having R1 groups of chloroethyl, bromoethyl, or iodoethyl are potent proteasome inhibitors and exhibit very potent cytotoxicity. In contrast, compounds having R1 groups of methyl, ethyl, or hydroxyethyl exhibited relatively lower cytotoxicity (3-fold power decrease), lower NF-kB inhibition (3-fold power decrease), and proteasome as caspase (2 to 10 times less potent) and as trypsin (20 to 50 times less potent). Without being bound by any particular theory, this application realizes that the above results support the hypothesis that the increased activity of compounds containing Cl, Br, or I in the R1 group may be due to the halogen property of being a good residual group. . This hypothesis is supported by the fact that the opening of the lactone ring of Compound 11-16 is observed to form a cyclic ether through nucleophilic substitution where chlorine is displaced according to the following reaction:

<formula> formula see original document page 193 </formula>

It is hypothesized that in compounds having a good residual group on the IR side chain, such as Compounds 11-16, 11-18, and 11-19, the nucleophilic addition of the proteasome to the β-lactone ring forms a cyclic ether of similar to the above reaction. It is hypothesized that the cyclic ether, or its formation, interacts favorably with the proteasome.

Without being bound by any particular theory, this application realizes that the above results also support the alternative hypothesis that a second nucleophile in the proteasome displaces the residual group, thus forming a 2-point covalent attaché between the compound and the enzyme. In this case, the residual group functionality in the R1 collateral chain promotes increased interaction between compound and enzyme and thus promotes increased activity. Thus, it is expected that compounds having other residual groups in the R1 side chain may exhibit high activity.

Without being bound by any particular theory, this application realizes that the above results also support the hypothesis of a single point residual group. As an example, the presence of a halogen or other residual group on the R1 side chain promotes the distribution of the compound to its target, such as an intracellular target or other biological target, thereby enhancing its therapeutic effect. An example of a single point residual group is illustrated in the diagram shown below.

"Residual group" as used herein refers to any atom or medium that is capable of being displaced by another atom or medium in a chemical reaction. More specifically, in some embodiments, "residual group" refers to the atom or medium that is displaced in a nucleophilic substitution reaction. In some embodiments, "residual groups" are any atoms or means which are conjugated bases of a strong acid. Non-limiting features and examples of residual groups can be found, for example, in Organic Chemistry, 2a. ed., Francis Carey (1992), pages 328-331; Introduction to Organic Chemistry, 2a. ed., Andrew Streitwieser and Clayton Heathcock (1981), pages 169-171; and Organic Chemistry, 5a. ed., John McMurry (2000), pages 398 and 408; all incorporated herein by reference in their entirety.

Example 64

ACTIVITY-STRUCTURE RELATIONS

The data listed in the above tables illustrate various preferred embodiments. With respect to Formula II, compounds having an R 1 halogenated substituent are preferred and such compounds were generally equipotent in the assays described above. Most preferred is R1 n-halogenated ethyl. Also most preferred are compounds with a E5 hydroxy group and the attached carbon is in an S configuration (compounds having the stereochemistry of compound II-18, for example). Oxidation of a hydroxy group to a ketone is less preferred.

In one embodiment, the preferred substituent on R4 is cyclohexene. In another preferred embodiment, cyclohexene is oxidized to an epoxide. Less preferred are the double bond hydrogenated compounds of the cyclohexene substituent.

In addition in some embodiments, preferably R 3 is methyl, with ethyl being less preferred.

Example 65

ANGIOGENESIS INHIBITION

Angiogenesis is an important physiological process, without which embryonic development and wound healing would not occur. However, excessive or improper angiogenesis is associated with various diseases, conditions, and adverse treatment outcomes. Examples of disease types and conditions associated with excessive angiogenesis include inflammatory diseases such as immune and nonimmune inflammation, rheumatic arthritis, chronic joint rheumatism, and psoriasis; diseases associated with improper or untimely vessel invasion, such as diabetic retinopathy, neovascular glaucoma, prematurity retinopathy, macular degeneration, corneal graft rejection, retrolental fibroplasia, rubella, capillary proliferation in atherosclerotic plaques and osteoporosis; and cancer-associated diseases, including for example, solid tumors, tumor metastases, blood-borne tumors such as leukemia, angiofibromas, Kaposi's sarcoma, benign tumors such as hemangiomas, acoustic neuromas, neurofibromas, tracomas, and pyogenic granulomas, as well as other cancers that require neovascularization to support tumor growth. Additional examples of angiogenesis dependent diseases include, for example, Osier-Webber syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia; haemophilia joints and granulation of wounds.

In addition, excessive angiogenesis is also associated with clinical problems as part of biological and mechanical implants (tissue / organ implantation, stenting, etc.). The compositions may be used immediately to inhibit angiogenesis, and thus in the treatment of such conditions. . Other diseases in which angiogenesis plays a role, and to which compounds and compositions can be used immediately, are known to those skilled in the art.

The particular discussion of angiogenesis in various pathophysiological conditions such as cancer, rheumatic arthritis, diabetic retinopathy, age-related macular degeneration, endometriosis and obesity is found in Folkman, J. (1985) Tumor angiogenesis Adv. Cancer Res. 1985; 43: 175-203; Folkman, J. (2001), Angiogenesis-dependent diseases, Semin. Oncol. 28, 536-42; Grosios, K., Wood, J., Esser, R., Raychaudhuri, A. & Dawson, J. (2004), Angiogenesis inhibition by the novel VEGF receptor tyrosine kinase inhibitor, PTK787 / ZK222584, causing significant anti-arthritic effects on models of rheumatic arthritis. Ignite Res. 53, 133-42; Hull, M.L., Charnock-Jones, D.S., Chan, C.L., Bruner-Tran, K.L., Osteen, K.G., Tom, B.D., Fan, T.P. & Smith, S.K. (2003). Anti-angiogenic agents are effective endometriosis inhibitors, J. Clin. Endocrinol. Metab. 88, 2889-99; Liu, L. & Meydani, M. (2003). Angiogenesis inhibitors may regulate adiposity, Nutr. Rev. 61, 384-7; Mousa, S.A. & Mousa, A.S. (2004). Angiogenesis inhibitors: current & future directions, Curr. Pharm. Off 10, 1-9. Each of the references described above being incorporated herein by reference in their entirety.

The compounds described herein inhibit angiogenesis.

This is evidenced, for example, by the compound of formula 11-16 which blocks vascular endothelial growth factor (VEGF) induced migration of multiple myeloma cells in a trans-recipient migration assay. The other compounds described herein are tested in a transhipper migration assay and inhibit migration.

The compounds described herein show angiogenesis inhibitory activity in any other miscellaneous angiogenesis assays and tests, including one or more of the following. The compounds described herein show anti-angiogenic activity in various other in vitro and in vivo assays. Examples include: In vitro assays for the evaluation of anti-angiogenesis compounds include: 1) Boyden's modified chamber assay that assesses endothelial cell migration in response to proangiogenic factors (Alessandri G., Raju K., Gullino PM, (1983) "Mobilization of capillary endothelium in vitro induced by effectors of angiogenesis in vivo", Cancer Res., 43 (4): 1790-7.); 2) Differentiation analysis such as the Matrigel assay in which tubular endothelial cell attachment, migration and differentiation are analyzed (Lawley TJ, Kubota Y., 1989), "Induction of morphologic differentiation of endothelial cells in culture". ", J. Invest. Dermatol., August 93 (2 Supplement.): 59S-61S); and 3) Organ culture assays in which external endothelial (and other cell) growth is monitored (Nicosia RF, Ottinetti A., (1990). "Growth of micro vessels in serum-free matrix of rat aorta. quantitative assay of angiogenesis in vitro ", Lab. Invest., Jul; 63 (1): 115-22.). Some in vivo assays for the evaluation of angiogenesis inhibitors are: 1) Sponge Implantation Assays, during which sponges containing cells and / or angiogenic factors and the test substance are implanted subcutaneously in animals for in vivo angiogenesis study (Plunkett). ML, Hailey JA, (1990), "An in vivo quantitative angiogenesis model using tumor cells entrapped in alginate", Lab. Invest., April 1990; 62 (4): 510-7), 2) Chick chorioallantoic membrane assay , in which test compounds are inserted through a window cut into the eggshell. The lack of a mature immune system in the 7-8 day old chick embryo allows the study of tumor-induced angiogenesis (Folkman J., (1985). Tumor angiogenesis, Adv. Cancer Res., 1985; 43: 175- 203.) and 3) Various tumor models in which specific histological analyzes can be used to examine the effect on blood vessels, such as vascular density (CD31 / CD34 lineage), blood flow, and concomitant tumor necrosis / apoptosis. (TUNNEL lineage). Examples of in vitro assays include human umbilical vein endothelial cell (HUVEC) tests, aortic, capillary endothelial cells (HUVEC); endothelial cell proliferation analyzes; DNA synthesis analysis of endothelial cells; external growth analysis of endothelial cells (aortic ring); endothelial cell migration analysis (aforementioned; chemokinesis (colloidal gold), chemotaxis (Boyden's chamber)); endothelial cell tube formation assays; endothelial apoptosis assays; endothelial cell viability assays (trypan blue); endothelial cell lines transfected by angiogenesis factors; and magnetized micro beads in endothelial cells. Each of the references in this paragraph being incorporated herein by reference in their entirety.

Examples of in vivo assays include transparent chamber testing (e.g., rabbit ear, hamster cheek, cranial window, and dorsal skin); matrix implants (e.g., subcutaneous injection using sodium alginate, a subcutaneous disc (polyvinyl foam implant), rat dorsal air bladder, sponge implants); micro-pocket corneal analyzes, for example in rabbits and other rodents; anterior eye / iris chamber implant analysis, rat and elimination analyzes; ameroid constriction (heart) in pigs and dogs; rabbit hind limb ischemia tests; tissue vascularization (intradermal inoculation, peritoneal cavity / omentuin with implant; tumor implants, for example in rabbits, rats or mice.

Also, ex vivo assays are performed using the described compounds. Examples include MCP (chick chorioallantoic membrane assay) and polymer gel vertical MCP. Immunological assays such as serum analysis, urine analysis, cerebrospinal fluid assays and immunohistochemical tissue assays.

Some of the above assays are described in the following documents, each of which is incorporated herein by reference in its entirety. Grant et al., In Vitro Cell. Dev. Biol. 27A: 327-336 (1991); Min et al., Cancer Res. 56: 2428-2433 (1996); Schnaper et al., J. Cell. Physiol. 165: 107-118 (1995); Schnaper et al. , J. Cell. Physiol. 165: 107-118 (1995); Oikawa et al., Cancer Lett. 59: 57-66 (1991). Embodiments relate to methods of using the compounds and compositions described herein, alone or in combination with other agents, to inhibit angiogenesis and treat or alleviate diseases and conditions associated with excessive or improper angiogenesis. Preferably, inhibition occurs with respect to vascularization in connection with a disease associated with angiogenesis, such as cancer or any of the other diseases described above, and those known to those skilled in the art. The compounds and compositions may be distributed in an appropriate inhibitory amount. Inhibitory amount is intended to mean an amount of a compound or composition required to effect a decrease in the extent, amount or rate of neovascularization when administered to a tissue, animal or individual. The dosage of compound or composition required to be therapeutically effective will depend, for example, on the angiogenesis-dependent disease to be treated, the route and form of administration, the potency and active half-life of the molecule being administered, the weight and condition of the drug. animal or individual, and prior or simultaneous therapies. Application of the appropriate amount of the method may be determined by those skilled in the art using the guidance provided herein.

For example, the amount may be extrapolated in vitro or in the in vivo angiogenesis assays described above. One skilled in the art will recognize that the patient's condition needs to be monitored throughout the course of therapy and that the amount of composition administered may be adjusted accordingly.

The present compounds and compositions may be, and are, well used in conjunction with other angiogenesis inhibitors.

Angiogenic inhibitors are already known in the art and may be prepared by known methods. For example, angiogenic inhibitors include integrin inhibiting compounds such as alpha-V-beta-3 (ανβ3) integrin inhibiting antibodies, cell adhesion proteins or their functional fragments containing a cell adhesion binding sequence.

Additional angiogenic inhibitors include, for example, angiostatin, functional angiostatin fragments, endostatin, fibroblast growth factor (FCF) inhibitors, FCF receptor inhibitors, VEGF inhibitors, VEGF receptor inhibitors, vascular permeability factor inhibitors. (FPV), FPV receptor inhibitors, thrombospondin, platelet factor 4, interferon-alpha, interferon-gamma, interferon-inducible protein 10, interleukin 12, gro-beta, and the prolactin N-terminal 16 KDa fragment, thalidomide, and other mechanisms for inhibiting angiogenesis.

Thus, the methods may include the step of administering a compound or composition to an animal suffering from a condition associated with excessive angiogenesis. The methods may further include administering the immediate compound or composition together with other anti-angiogenesis drugs or together with other therapies to treat the condition (for example, with a chemotherapeutic or immunotherapeutic to treat cancer).

The compounds or compositions may be distributed to any patient and / or disease accordingly. Examples include intravenous, oral, intramuscular, intraocular, intranasal, intraperitoneal, and the like.

The following references provide additional teachings regarding methods of use, administration and analysis for inhibiting angiogenesis: Angiogenesis Protocols (Methods in Molecular Medicine), by J. Clifford Murray, Human Press (March 15, 2001), ISBN: 0896036987; Angiogenesis Tumor by R.J. Bicknell, Claire E. Lewis, Napoleone Fe., Oxford University Press (September 1, 1997), ISBN: 0198549377; and Angiogenesis in Health and Disease: Basic Mechanisms and Clinical Applications, by Gabor M. Rubanyi, Mareei Dekker (November 1, 1999), ISBN: 0824781023. Each book is incorporated herein by reference in its entirety, in particular the protocols and methods. incorporated herein.

Example 66

FORMULATION TO BE ORALLY OR SIMILAR

A mixture obtained by thoroughly mixing 1 g of a compound obtained and purified by the incorporation method, 98 g of lactose and 1 g of hydroxypropyl cellulose is formed into granules by any conventional method. The granules are thoroughly dried and sieved to obtain a suitable granule preparation for bottle wrapping or heat sealing. The resulting granule preparations are orally administered from about 100 ml / day to about 1,000 ml / day, depending on the symptoms, as deemed appropriate by those of ordinary skill in the art of treating cancerous tumors in humans.

Example 64

TREATMENT OF GLEEVEC RESISTANT AND RESISTANT TUMORS

Chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL) cells in most cases share a chromosome abnormality not found in normal cells. The abnormality, designated as t (9; 22), is a reciprocal translocation between chromosomes 9 and 22. This translocation results in chromosome 9 being longer than normal and chromosome 22 being shorter than normal. Fracture on chromosome 22 happens in the middle of a gene called BCR. The resulting chromosome 22, that is, Philadelphia chromosome (F1), has a BCR section fused to most of the proto-oncogene designated c-ABL. This fused fragment forms an abnormal "bcr-abl" oncogene that encodes an abnormally tyrosine kinase that is constantly activated and stimulates cell division and resistance to apoptosis.

Gleevec® (IST-571, 571 transduction signal inhibitor, or imatinib mesylate) is a selective inhibitor of tyrosine kinase BCR-ABL, the causative mutation of CML. Gleevec® is also a Stem Cell Factor (FCT) tyrosine receptor kinase inhibitor for c-KIT, and inhibits Platelet Derived Growth Factor (PDGF) receptor and FCT-mediated cell events. In clinical studies, Gleevec® has been shown to shrink gastrointestinal stromal tumors (TEGI) in 60 percent of those treated. Similar dramatic responses were observed with CML. However, resistance to Gleevec® develops in all patients through multiple proposed mechanisms. Without being bound by any particular theory, the possible mechanisms of resistance to Gleevec® may be the result of bcr-abl overexpression, mutations in the abl kinase domain where Gleevec® is attached, or activation of other oncogenic pathways. .

The compounds described herein are used to treat sensitive and resistant Gleevec® tumors. This is proven, for example, by the compound of Formula 11-16 which has been shown to induce apoptosis in CML cell lines, positive Philadelphia chromosome acute lymphoblastic leukemia (F + ALL; including recently isolated patient + F + ALL cells) and acute myeloid leukemia (AML). The compound of Formula 11-16 may be used as a sole agent or in combination with Gleevec®. In particular, the compound of Formula 11-16 may be used as a sole agent or in combination with Gleevec® in the treatment of positive or resistant Philadelphia chromosome positive leukemia.

The compound of Formula 11-16 or any other compound including the compounds of Formulas I, II, III, IV, V, and VI may be used to treat Gleevec® sensitive and resistant tumors.

The above examples are provided solely to assist in understanding embodiments. Thus, those skilled in the art will appreciate that the methods may provide derivatives of the compounds.

One skilled in the art would readily appreciate that the present invention is well adapted to accomplish its purposes and to achieve the purposes and advantages mentioned, as well as those inherent herein. The methods and procedures described herein are representative of preferred embodiments and are exemplary, and are not intended as limitations on the scope of the invention. Modifications and other uses that will eventually occur to those skilled in the art are encompassed within the spirit of the invention.

It will be readily apparent to one skilled in the art that various substitutions and modifications may be made to the embodiments described herein without departing from the scope and spirit of the invention. All patents and publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention belongs. All patents and publications are incorporated herein by reference for the same scope as each individual publication has been specifically and individually indicated to be incorporated by reference.

The invention described herein is illustrative and suitably practicable in the absence of any element or elements, limitation or limitations, not specifically described herein. The terms and expressions that have been used are used as terms of description rather than limitation, and it is not intended that the use of such terms and expressions shall indicate the exclusion of equivalences of or any of the features shown and described herein. It is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that while the present invention has been described herein specifically for preferred embodiments and optional features, modification and variation of the concepts described herein may be resorted to by those skilled in the art, and such modifications and variations are deemed to fall. within the scope of embodiments of the invention.

Claims (85)

1. "METHODS FOR USING HETEROCYCLIC COMPOUNDS [3.2.0] AND ANALOGS", characterized by the use of a compound in the manufacture of a medicament for the treatment of a drug-resistant cell where the compound has the structure and related pharmaceutically acceptable prodrug salts and esters: where the dashed lines represent a single or double bond, where R1 is selected separately from the group consisting of a hydrogen, a halogen, and mono- substituted, poly-substituted variants. substituted or unsubstituted residues of the following residues: unsaturated C1-C24 alkyl, unsaturated C2-C24 alkenyl or C2-C24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarboxy, nitro, azido, phenyl, cycloalkylacyl, hydroxy, thioalkyl, thioalkyl arila, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl, where η is 1 or 2, and if η is 2, then R 1 may be the same or different; where R2 is 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 C2-C24 alkynyl, acyl acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonoxy nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl; where R3 is 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 C2-C24 alkynyl, acyl, acyloxy alkylalkylcarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonoxy, nitro azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl; wherein each E1, E2, E3 and E4 is a substituted or unsubstituted heteroatom; and where the drug resistant cell is a cancer cell selected from the group consisting of leukemia, ovarian cancer, urethral cancer, bladder cancer, prostate cancer, rectal cancer, stomach cancer, lymphoma, multiple myeloma, pancreatic cancer, cancer Liver disease, kidney cancer, endocrine cancer, a sarcoma, skin cancer, angioma, and brain cancer. "METHODS FOR USE" according to claim 1, characterized in that the compound has the structure of Formula II, and related pharmaceutically acceptable prodrug salts and esters: <formula> formula see original document page 205 < where dashed lines represent a single or double bond, where R1 is selected separately from the group consisting of a hydrogen, a halogen, monosubstituted, poly-substituted or unsubstituted variants of the following residues: C1-C24 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, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonoxy, nitro, azido, phenyl, cycloalkylacyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including alkyl poly halo gened, where η is 1 or 2, and if η is 2, then R1 may be the same or different; where R3 is selected separately from the group consisting of a hydrogen, a halogen, mono-substituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, C2-C24 alkenyl or unsaturated C2-C24 alkynyl acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, amino -carbonyloxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl; where R4 is selected separately from the group consisting of a hydrogen, a halogen, mono-substituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, C2-C24 alkenyl or unsaturated C2-C24 alkynyl acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, amino -carbonyloxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl, where m is 1 or 2, and if m is 2, then R4 may be the same or different; and wherein each E1, E2, E3, E4 and E5 is a substituted or unsubstituted heteroatom. "METHODS FOR USE" according to claim 1, characterized in that the compound has the structure of Formula III, and related pharmaceutically acceptable prodrug salts and esters: <formula> formula see original document page 207 < where dashed lines represent a single or double bond, where R1 is selected separately from the group consisting of a hydrogen, a halogen, monosubstituted, poly-substituted or unsubstituted variants of the following residues: C1-C24 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, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonoxy, nitro, azido, phenyl, cycloalkylacyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including alkyl polyhalogen ada, where η is 1 or 2, and if η is 2, then R1 may be the same or different; where R4 is selected separately from the group consisting of a hydrogen, a halogen, monosubstituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, C2-C24 alkenyl or unsaturated C2-C24 alkynyl acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, amino -carboyloxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl, where m is 1 or 2, and if m equals 2, then R4 may be the same or different; and wherein each E1, E2, E3, E4 and E5 is a substituted or unsubstituted heteroatom. "METHODS FOR USE" according to claim 1, characterized in that the compound has the structure of Formula IV, and related pharmaceutically acceptable prodrug salts and esters: <formula> formula see original document page 208 < where dashed lines represent a single or double bond, where R1 is selected separately from the group consisting of a hydrogen, a halogen, monosubstituted, poly-substituted or unsubstituted variants of the following residues: C1-C24 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, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonoxy, nitro, azido, phenyl, cycloalkylacyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including alkyl polyhalogen ada, where η is 1 or 2, and if η is 2, then R1 may be the same or different; consists of a hydrogen, a halogen, mono-substituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, unsaturated C2-C24 alkenyl or C2-C24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy cycloalkoxy, aryl, heteroaryl, aryl alkoxycarbonyl, alkoxycarbonyl acyl, amino, aminocarbonyl, aminocarboxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thio aryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl; where R5 is selected separately from the group consisting of a hydrogen, a halogen, monosubstituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, C2-C24 alkenyl or unsaturated C2-C24 alkynyl , acyl, acyloxy, alkyloxycarbonyloxy, where R3 is selected separately from the group aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, aryl alkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonoxy, nitro, azido, phenyl, cycloalkylacyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl, where m is 0, 1, 2, 3, 4, -5, 6, 7, 8, 9, 10, or 11, and if m is greater than 1, then R5 may be the same or different; and wherein each E1, E2, E3, E4 and E5 is a substituted or unsubstituted heteroatom. "METHODS FOR USE" according to claim 1, characterized in that the compound has the structure of Formula V, and related pharmaceutically acceptable prodrug salts and esters: where the dotted lines represent a single or double bond. where R1 is selected separately from the group consisting of hydrogen, halogen, monosubstituted, poly-substituted or unsubstituted variants of the following residues: saturated C1-C24 alkyl, C2-C24 alkenyl or non-substituted C2-C24 alkynyl saturated, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, amino-carboxy, nitro, azido, phenyl, cycloalkyl-acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl, where η is equal to 1 or 2, and if η equals 2, then R1 may be the same or different; where R5 is selected separately from the group consisting of hydrogen, halogen, monosubstituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, C2-C24 alkenyl or unsaturated C2-C24 alkynyl acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, amino -carbonyloxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl, where m is 0, 1, 2 , 3, 4, - 5, 6, 7, 8, 9, 10, or 11, and if m is greater than 1, then R5 may be the same or different; and wherein each E1, E2, E3, E4 and E5 is a substituted or unsubstituted heteroatom. "METHODS FOR USE" according to claim 1, characterized in that the compound has the structure of Formula VI, and related pharmaceutically acceptable prodrug salts and esters: Formula VI <formula> formula see original document page Where R1 may be selected separately from the group consisting of mono-substituted, poly-substituted or unsubstituted variants of the following residues: saturated C1-C24 alkyl, C2-C24 alkenyl or unsaturated C2-C24 alkynyl acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, amino -carbonyloxy, phenyl, cycloalkyl acyl, thioalkyl, thioaryl, oxysulfonyl, carboxy, thio, sulfoxide, sulfone, esters and boronic acids, and halogenated alkyl including polyhalogenated alkyl; η is equal to 1 or 2, and if η is equal to 2, then R1 may be the same or different; where R2 may 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, C2-C24 alkenyl or unsaturated C2-C24 alkynyl acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl (including, for example, cyclohexylcarbinol), cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonyl -acyl, amino, amino-carbonyl, amino-carboxy, nitro, azido, phenyl, cycloalkyl-acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, thio, sulfoxide, sulfone, sulfonate esters thio-cyano, boronic acids and esters, and halogenated alkyl including polyhalogenated alkyl; where R3 may be selected from the group consisting of halogen, mono-substituted, poly-substituted or 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, aryl-alkoxycarbonyl, alkoxycarbonyl-acyl, amino, aminocarbonyl, aminocarbonoxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, thio, sulfoxide, sulfone, sulfonate esters, thio cyano, boronic acids and esters, and halogenated alkyl including polyhalogenated alkyl; wherein each E1, E2, E3 and E4 may be a substituted or unsubstituted heteroatom. "METHODS FOR USE" according to any one of claims 1, 2, 3, 4, 5 or 6, characterized in that the compound is: <formula> formula see original document page 211 </formula> R8 is selected from the group consisting of HF, Cl, Br and I. "METHODS FOR USE" according to claim 7, characterized in that the compound has the structure of Formula 11-16, and related pharmaceutically acceptable prodrug salts and esters of claims 1, 2, 3, 4, 5, 6, 7, or 8, characterized by the fact that the drug-resistant cell is chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), sarcoma, or myeloma. multiple. claim 1, 2, 3, 4, 5, 6, 7, 8 or 9, characterized in that the drug resistant cell is MES-SA / Dx5, HL-60 / MX2, MM.IR multiple myeloma, or Doxorubicin resistant Dox-40 multiple myeloma. claim 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, characterized in that the drug to which the cell is resistant is bortezomiba, Gleevec®, mitoxantrone, doxorubicin, daunorubicin, dactinomycin, vincristine, taxol, colchicine, mitomycin C, rituximab, dexamethasone, thalidomide, paclitaxel or melphalan. [3.2.0] AND ANALOGS ", characterized in that they comprise a method for inhibiting cell growth. <formula> formula see original document page 212 </formula> 9. "METHODS FOR USE" according to any of 10. "METHODS FOR USE" according to any 11. "METHODS FOR USE" according to any 12. "METHODS FOR USE OF DRUG-RESISTANT HETEROCYCLIC COMPOUNDS comprising contacting the cell with a compound having the structure of Formula I, and related pharmaceutically acceptable prodrug salts and esters: <formula> formula see original document page 213 </formula> Formula I where the dotted lines represent a single or double bond, where R 1 is selected separately from the group consisting of a hydrogen, a halogen, monosubstituted, polysubstituted or 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, arylalkoxycarbonyl alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonoxy, nitro, azido, phenyl, cycloalkylacyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and alkyl haloge nothing including polyhalogenated alkyl, where η is 1 or 2, and if η is 2, then R 1 may be the same or different; where R2 is selected from the group consisting of hydrogen, a halogen, mono-substituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, unsaturated C2-C24 alkenyl or C2-C24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, amino carboxyoxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl; where R3 is selected from the group consisting of a halogen, monosubstituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, unsaturated C2-C24 alkenyl or C2-C24 alkynyl, acyl, acyloxy alkylalkylcarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonoxy, nitro azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl; wherein each E1, E2, E3 and E4 is a substituted or unsubstituted heteroatom; and where cancer is leukemia, ovarian cancer, urethral cancer, bladder cancer, prostate cancer, rectal cancer, stomach cancer, lymphoma, multiple myeloma, pancreatic cancer, liver cancer, kidney cancer, endocrine cancer> a sarcoma, skin cancer, angioma, or brain cancer. "METHODS FOR USE" according to claim 12, characterized in that the compound has the structure of Formula II, and related pharmaceutically acceptable salts and esters: single or double, where R 1 is selected separately from the compound. A group consisting of hydrogen, halogen, monosubstituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, unsaturated C2-C24 alkenyl or C2-C24 alkynyl, acyl, acyloxy, alkyloxy -carbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonoxy, nitro, azido , phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl, where η is 1 or 2, and if η is 2 , so R1 may be the same or different; where R3 is selected separately from the group consisting of a hydrogen, a halogen, mono-substituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, C2-C24 alkenyl or unsaturated C2-C24 alkynyl acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, amino -carbonyloxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl; where R4 is selected separately from the group consisting of hydrogen, halogen, monosubstituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, C2-C24 alkenyl or unsaturated C2-C24 alkynyl acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, amino -carboyloxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl, where m is 1 or 2, and if m equals 2, then R4 may be the same or different; and wherein each E1, E2, E3, E4 and E5 is a substituted or unsubstituted heteroatom. "METHODS FOR USE" according to claim 12, characterized in that the compound has the structure of Formula III, and related pharmaceutically acceptable prodrug salts and esters: <formula> formula see original document page 215 < where dashed lines represent a single or double bond, where R1 is selected separately from the group consisting of a hydrogen, a halogen, monosubstituted, poly-substituted or unsubstituted variants of the following residues: C1-C24 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, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonoxy, nitro, azido, phenyl, cycloalkylacyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including alkyl poly- halogenated, where η is 1 or 2, and if η is 2, then R1 may be the same or different; where R4 is selected separately from the group consisting of a hydrogen, a halogen, mono-substituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, C2-C24 alkenyl or unsaturated C2-C24 alkynyl acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, amino -carboyloxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl, where m is 1 or 2, and if m equals 2, then R4 may be the same or different; and wherein each E1, E2, E3, E4 and E5 is a substituted or unsubstituted heteroatom. "METHODS FOR USE" according to claim 12, characterized in that the compound has the structure of Formula IV, and related pharmaceutically acceptable prodrug salts and esters: where the dotted lines represent a single or double bond. where R1 is selected separately from the group consisting of hydrogen, halogen, monosubstituted, poly-substituted or unsubstituted variants of the following residues: saturated C1-C24 alkyl, C2-C24 alkenyl or non-substituted C2-C24 alkynyl saturated, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, amino-carboxy, nitro, azido, phenyl, cycloalkyl-acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl, where η is equal to 1 or 2, and if η equals 2, then R1 may be the same or different; where R3 is selected separately from the group consisting of a hydrogen, a halogen, mono-substituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, C2-C24 alkenyl or unsaturated C2-C24 alkynyl acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, amino -carbonyloxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl; where R5 is selected separately from the group consisting of hydrogen, halogen, monosubstituted, poly-substituted or unsubstituted variants of the following residues: saturated C1-C24 alkyl, C2-C24 alkenyl or unsaturated C2-C24 alkynyl acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, amino -carbonyloxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl, where m is 0, 1, 2 , 3, 4, -5, 6, 7, 8, 9, 10, or 11, and if m is greater than 1, then R5 may be the same or different; and wherein each E1, E2, E3, E4 and E5 is a substituted or unsubstituted heteroatom. "METHODS FOR USE" according to claim 12, characterized in that the compound has the structure of Formula V, and related pharmaceutically acceptable salts and esters: single or double, where R 1 is selected separately from the compound. A group consisting of hydrogen, halogen, monosubstituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, unsaturated C2-C24 alkenyl or C2-C24 alkynyl, acyl, acyloxy, alkyloxy -carbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonoxy, nitro, azido , phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl, where η is 1 or 2, and if η is 2 , so R1 may be the same or different; where R5 is selected separately from the group consisting of hydrogen, halogen, monosubstituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, C2-C24 alkenyl or unsaturated C2-C24 alkynyl acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, amino -carbonyloxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl where the dotted lines represent a bond including polyhalogenated alkyl, where m is equals 0, 1, 2, 3, 4, -5, 6, 7, 8, 9, 10, or 11, and if m is greater than 1, then R5 may be the same or different; and wherein each E1, E2, E3, E4 and E5 is a substituted or unsubstituted heteroatom. 17. "METHODS FOR USE" according to claim 12, characterized in that the compound has the structure of Formula VI, and related pharmaceutically acceptable prodrug salts and esters: <formula> formula see original document page 219 < Formula VI wherein R1- may be selected separately from the group consisting of a mono-substituted, poly-substituted or unsubstituted variant of the following residues: saturated C1-C24 alkyl, C2-C24 alkenyl or unsubstituted C2-C24 alkynyl - saturated, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl aminocarbonoxy, phenyl, cycloalkyl acyl, thioalkyl, thioaryl, oxysulfonyl, carboxy, thio, sulfoxide, sulfone, esters and boronic acids, and halogenated alkyl including polyhalogenated alkyl; η is equal to 1 or 2, and if η is equal to 2, then R1 may be the same or different; where R2 may 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, C2-C24 alkenyl or unsaturated C2-C24 alkynyl acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl (including, for example, cyclohexylcarbinol), cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonyl -acyl, amino, amino-carbonyl, amino-carboxy, nitro, azido, phenyl, cycloalkyl-acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, thio, sulfoxide, sulfone, sulfonate esters thio-cyano, boronic acids and esters, and halogenated alkyl including polyhalogenated alkyl; where R3 may be selected from the group consisting of halogen, mono-substituted, poly-substituted or 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, aryl-alkoxycarbonyl, alkoxycarbonyl-acyl, amino, aminocarbonyl, aminocarbonoxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, thio, sulfoxide, sulfone, sulfonate esters, thio cyano, boronic acids and esters, and halogenated alkyl including polyhalogenated alkyl; wherein each E1, E2, E3 and E4 may be a substituted or unsubstituted heteroatom. "METHODS FOR USE" according to claims 12, 13, 14, 15, 16 or 17, characterized in that the compound is: wherein R8 and selected from the group consisting of H, F, Cl, Br and I. "METHODS FOR USE" according to claim 18, characterized in that the compound has the structure of Formula 11-16, and related pharmaceutically acceptable prodrug salts and esters: <formula> formula see original document page 221 </formula> "METHODS FOR USE" according to claims 12, 13, 14, 15, 16, 17, 18 or 19, characterized in that the drug-resistant cell is a chronic myeloid leukemia (CML), a leukemia Acute lymphoblastic (ALL), acute myeloid leukemia (AML), sarcoma, or multiple myeloma. "METHODS FOR USE" according to claims 12, 13, 14, 15, 16, 17, 18, 19 or 20, characterized in that the drug-resistant cell is MES-SA / Dx5, HL- 60 / MX2, MM.IR multiple myeloma, or doxorubicin-resistant multiple myeloma Dox-40. "METHODS FOR USE" according to claims 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21, characterized in that the drug to which the cell is resistant is bortezomiba, Gleevec. ®, mitoxantrone, doxorubicin, daunorubicin, dactinomycin, vincristine, taxol, colchicine, mitomycin C, rituximab, dexamethasone, thalidomide, paclitaxel or melphalan. 23. "METHODS FOR USING HETEROCYCLIC COMPOUNDS [3.2.0] AND ANALOGS", characterized by the use of a compound in the manufacture of a medicament for use in the treatment of a disease having characteristics of excessive or improper angiogenesis, where the compound has the structure of Formula I, and related pharmaceutically acceptable prodrug salts and esters: <formula> formula see original document page 222 </formula> where dashed lines represent a single or double bond, where R1 is selected separately from the group consisting of hydrogen, halogen, monosubstituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, unsaturated C2-C24 alkenyl or C2-C24 alkynyl, acyl, acyloxy , alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, amino-carbonyl, amino-carboxy, nitro, azido, phenyl, cycloalkyl-acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl, where η is equals 1 or 2, and if η equals 2, then R1 may be the same or different; where R2 is selected from the group consisting of hydrogen, a halogen, mono-substituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, unsaturated C2-C24 alkenyl or C2-C24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, amino carboxyoxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl; where R3 is selected from the group consisting of a halogen, mono-substituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, unsaturated C2-C24 alkenyl or C2-C24 alkynyl, acyl, acyloxy alkylalkylcarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonoxy, nitro azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl; wherein each E1, E2, E3 and E4 is a substituted or unsubstituted heteroatom; and where cancer is leukemia, ovarian cancer, urethral cancer, bladder cancer, prostate cancer, rectal cancer, stomach cancer, lymphoma, multiple myeloma, pancreatic cancer, liver cancer, kidney cancer, endocrine cancer, a sarcoma, skin cancer, angioma, or brain cancer. 24. "METHODS FOR USE" according to claim 23, characterized in that the compound has the structure of Formula II, and related pharmaceutically acceptable salts and esters: single or double, where R 1 is selected separately from the compound. A group consisting of hydrogen, halogen, monosubstituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, unsaturated C2-C24 alkenyl or C2-C24 alkynyl, acyl, acyloxy, alkyloxy -carbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonoxy, nitro, azido, phenyl , cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl, where η is 1 or 2, and if η is 2, then R1 may be the same or different; where R3 is selected separately from the group consisting of a hydrogen, a halogen, mono-substituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, C2-C24 alkenyl or unsaturated C2-C24 alkynyl acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, amino -carbonyloxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl; where R4 is selected separately from the group consisting of a hydrogen, a halogen, monosubstituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, C2-C24 alkenyl or unsaturated C2-C24 alkynyl acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, amino -carboyloxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl, where m is 1 or 2, and if m equals 2, then R4 may be the same or different; and wherein each E1, E2, E3, E4 and E5 is a substituted or unsubstituted heteroatom. "METHODS FOR USE" according to claim 23, characterized in that the compound has the structure of Formula III, and related pharmaceutically acceptable prodrug salts and esters: <formula> formula see original document page 224 < where dashed lines represent a single or double bond, where R1 is selected separately from the group consisting of a hydrogen, a halogen, monosubstituted, poly-substituted or unsubstituted variants of the following residues: C1-C24 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, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonoxy, nitro, azido, phenyl, cycloalkylacyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including alkyl poly -halogenated, where η is 1 or 2, and if η is 2, then R1 may be the same or different; where R4 is selected separately from the group consisting of a hydrogen, a halogen, monosubstituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, C2-C24 alkenyl or unsaturated C2-C24 alkynyl acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, amino -carboyloxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl, where m is 1 or 2, and if m equals 2, then R4 may be the same or different; and wherein each E1, E2, E3, E4 and E5 is a substituted or unsubstituted heteroatom. "METHODS FOR USE" according to claim 23, characterized in that the compound has the structure of Formula IV, and related pharmaceutically acceptable prodrug salts and esters: where the dotted lines represent a single or double bond. where R1 is selected separately from the group consisting of a hydrogen, a halogen, monosubstituted, poly-substituted or unsubstituted variants of the following residues: saturated C1-C24 alkyl, C2-C24 alkenyl or non-substituted C2-C24 alkynyl saturated, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, amino-carboxy, nitro, azido, phenyl, cycloalkyl-acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl, where η is equal to 1 or 2, and if η is equal to 2, then R1 may be the same or different; where R3 is selected separately from the group consisting of a hydrogen, a halogen, mono-substituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, C2-C24 alkenyl or unsaturated C2-C24 alkynyl acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, amino -carbonyloxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl; where R5 is selected separately from the group consisting of hydrogen, halogen, monosubstituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, C2-C24 alkenyl or unsaturated C2-C24 alkynyl acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonyloxy , nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl, where m is 0, 1, 2, 3 , 4, - 5, 6, 7, 8, 9, 10, or 11, and if m is greater than 1, then R5 may be the same or different; and wherein each E1, E2, E3, E4 and E5 is a substituted or unsubstituted heteroatom. "METHODS FOR USE" according to claim 23, characterized in that the compound has the structure of Formula V, and related pharmaceutically acceptable prodrug salts and esters: <formula> formula see original document page 227 < where dashed lines represent a single or double bond, where R1 is selected separately from the group consisting of a hydrogen, a halogen, monosubstituted, poly-substituted or unsubstituted variants of the following residues: C1-C24 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, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonoxy, nitro, azido, phenyl, cycloalkylacyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including alkyl polyhalogenated a, where η is 1 or 2, and if η is 2, then R1 may be the same or different; where R5 is selected separately from the group consisting of a hydrogen, a halogen, monosubstituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, C2-C24 alkenyl or unsaturated C2-C24 alkynyl acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, amino -carbonyloxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl, where m is 0, 1, 2 , 3, 4, - 5, 6, 7, 8, 9, 10, or 11, and if m is greater than 1, then R5 may be the same or different; and wherein each E1, E2, E3, E4 and E5 is a substituted or unsubstituted heteroatom. 28. "METHODS FOR USE" according to claim 23, characterized in that the compound has the structure of Formula VI, and related pharmaceutically acceptable prodrug salts and esters: Formula VI <formula> formula see original document page Where R1 may be selected separately from the group consisting of a mono-substituted, poly-substituted or unsubstituted variants of the following residues: saturated C1-C24 alkyl, C2-C24 alkenyl or non-substituted C2-C24 alkynyl saturated, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarboxy, phenyl, cycloalkyl acyl, thioalkyl, thioaryl, oxysulfonyl, carboxy, thio, sulfoxide, sulfone, esters and boronic acids, and halogenated alkyl including polyhalogenated alkyl, η is equal to 1 or 2, and if η is equal to 2, then R1 may be the same or different; where R2 may 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, C2-C24 alkenyl or unsaturated C2-C24 alkynyl acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl (including, for example, cyclohexylcarbinol), cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonyl -acyl, amino, amino-carbonyl, amino-carboxy, nitro, azido, phenyl, cycloalkyl-acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, thio, sulfoxide, sulfone, sulfonate esters thio-cyano, boronic acids and esters, and halogenated alkyl including polyhalogenated alkyl; where R3 may be selected from the group consisting of halogen, mono-substituted, poly-substituted or 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, aryl-alkoxycarbonyl, alkoxycarbonyl-acyl, amino, aminocarbonyl, aminocarbonoxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, thio, sulfoxide, sulfone, sulfonate esters, thio cyano, boronic acids and esters, and halogenated alkyl including polyhalogenated alkyl; wherein each E1, E2, E3 and E4 may be a substituted or unsubstituted heteroatom. 29. "METHODS FOR USE" according to claims 23, 24, 25, 26, 27 or 28, characterized in that the compound is: wherein R8 and selected from the group consisting of H, F, Cl, Br and I. 30. "METHODS FOR USE" according to claim 29, characterized in that the compound has the structure of Formula 11-16, and related pharmaceutically acceptable prodrug salts and esters: <formula> formula see original document page 230 </formula> "METHODS FOR USE" according to claims 23, 24, 25, 26, 27, 28, 29 or 30, characterized in that the disease is an inflammatory condition. 32. "METHODS FOR USE" according to claim 31, characterized in that the inflammatory condition is immune and non-immune inflammation, rheumatic arthritis, chronic joint rheumatism, and psoriasis. 33. "METHODS FOR USE" according to claims 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32, characterized in that the disease has characteristics of improper or inappropriate invasions of blood vessels. . 34. "METHODS FOR USE" according to claim 33, characterized in that the disease is diabetic retinopathy, neovascular glaucoma, prematurity retinopathy, macular degeneration, corneal graft rejection, retrolental fibroplasia, rubella, plaque capillary proliferation. atherosclerosis, osteoporosis, leukemia, angiofibroma, Kaposi's sarcoma, hemangioma, acoustic neuroma, neurofibroma, trachoma and pyogenic granuloma. 35. "METHODS FOR USE" according to claims 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34, characterized in that they comprise the use of an anti-angiogenic agent. . 36. "METHODS FOR USE" according to claim 35, characterized in that the anti-angiogenic agent is an alpha-V-beta-3 (ανβ3), an integrin inhibiting antibody, a protein for adhesion to cell, a functional cell adhesion protein fragment containing a cell adhesion binding sequence, angiostatin, a functional fragment of angiostatin, endostatin, a fibroblast growth factor (FCF) inhibitor, an FCF receptor inhibitor , a VEGF inhibitor, a VEGF receptor inhibitor, a vascular permeability factor (FPV) inhibitor, a VFP receptor inhibitor, thrombospondin, a platelet factor 4, an interferon-alpha, an interferon-gamma, a interferon inducible protein 10, an interleukin 12, gro-beta, a 16kDa N-terminal fragment of prolactin, and thalidomide. 37. "METHODS FOR USE" according to claims 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36, characterized in that the animal is a mammal. 38. "METHODS FOR USE" according to claims 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36, characterized in that the animal is a human. 39. "METHODS FOR USE" according to claims 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36, characterized in that the animal is a rodent. 40. "METHODS FOR USE" according to claims 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36, comprising the steps of: identify an individual who would benefit from the administration of an angiogenesis inhibiting agent; and perform the method on the individual. 41. "METHODS FOR USING HETEROCYCLIC COMPOUNDS [3.2.0] AND ANALOGS", characterized in that they comprise a method for inhibiting angiogenesis comprising administering to an animal a compound having the structure of Formula I, and salts and esters pro Related pharmaceutically acceptable drugs: <formula> formula see original document page 231 </formula> where dashed lines represent a single or double bond, where R1 is selected separately from the group consisting of a hydrogen, a halogen, mono variants - substituted, polysubstituted or unsubstituted residues of the following residues: unsaturated C1-C24 alkyl, unsaturated C2-C24 alkenyl or C2-C24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkyl -alkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, aryl-alkoxycarbonyl, alkoxycarbonyl-acyl, amino, aminocarbonyl, aminocarboxy, nitro, azido, phenyl, cycloalkyl α-acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl, where η is 1 or 2, and if η is 2, then R 1 may be same or different; where R2 is selected from the group consisting of hydrogen, a halogen, mono-substituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, unsaturated C2-C24 alkenyl or C2-C24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, amino carboxyoxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl; where R3 is 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 C2-C24 alkynyl, acyl, acyloxy alkylalkylcarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonoxy, nitro azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl; wherein each E1, E2, E3 and E4 is a substituted or unsubstituted heteroatom; and where cancer is leukemia, ovarian cancer, urethral cancer, bladder cancer, prostate cancer, rectal cancer, stomach cancer, lymphoma, multiple myeloma, pancreatic cancer, liver cancer, kidney cancer, endocrine cancer, a sarcoma, skin cancer, angioma, or brain cancer. 42. "METHODS FOR USE" according to claim 41, characterized in that the compound has the structure of Formula II, and related pharmaceutically acceptable salts and esters: single or double, where R 1 is selected separately from the compound. A group consisting of hydrogen, halogen, monosubstituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, unsaturated C2-C24 alkenyl or C2-C24 alkynyl, acyl, acyloxy, alkyloxy -carbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonoxy, nitro, azido , phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl, where η is 1 or 2, and if η is 2 , so R1 may be the same or different; where R3 is selected separately from the group consisting of a hydrogen, a halogen, mono-substituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, C2-C24 alkenyl or unsaturated C2-C24 alkynyl acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, amino -carbonyloxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl; where R4 is selected separately from the group consisting of a hydrogen, a halogen, mono-substituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, C2-C24 alkenyl or unsaturated C2-C24 alkynyl acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, amino -carboyloxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl, where m is 1 or 2, and if m equals 2, then R4 may be the same or different; and wherein each E1, E2, E3, E4 and E5 is a substituted or unsubstituted heteroatom. 43. "METHODS FOR USE" according to claim 41, characterized in that the compound has the structure of Formula III, and related pharmaceutically acceptable prodrug salts and esters: where the dotted lines represent a single or double bond. where R1 is selected separately from the group consisting of hydrogen, halogen, monosubstituted, poly-substituted or unsubstituted variants of the following residues: saturated C1-C24 alkyl, C2-C24 alkenyl or non-substituted C2-C24 alkynyl saturated, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, amino-carboxy, nitro, azido, phenyl, cycloalkyl-acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl, where η is equal to 1 or 2, and if η equals 2 , so R1 may be the same or different; where R4 is selected separately from the group consisting of a hydrogen, a halogen, monosubstituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, C2-C24 alkenyl or unsaturated C2-C24 alkynyl acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, amino -carboyloxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl, where m is 1 or 2, and if m equals 2, then R4 may be the same or different; and wherein each E1, E2, E3, E4 and E5 is a substituted or unsubstituted heteroatom. 44. "METHODS FOR USE" according to claim 41, characterized in that the compound has the structure of Formula IV, and related pharmaceutically acceptable prodrug salts and esters: where the dotted lines represent a single or double bond. where R1 is selected separately from the group consisting of hydrogen, halogen, monosubstituted, polysubstituted or unsubstituted variants of the following residues: saturated C1-C24 alkyl, C2-C24 alkenyl or non-substituted C2-C24 alkynyl saturated, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, amino carboxy, nitro. azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl, where η is 1 or 2, and if η is equal to 2, then R1 may be the same or different; where R3 is selected separately from the group consisting of a hydrogen, a halogen, mono-substituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, C2-C24 alkenyl or unsaturated C2-C24 alkynyl acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, amino -carbonyloxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl; where R5 is selected separately from the group consisting of a hydrogen, a halogen, monosubstituted, polysubstituted or unsubstituted variants of the following residues: saturated C1-C24 alkyl, C2-C24 alkenyl or unsaturated C2-C24 alkynyl acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, amino -carbonyloxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl, where m is 0, 1, 2 , 3, 4, - 5, 6, 7, 8, 9, 10, or 11, and if m is greater than 1, then R5 may be the same or different; and wherein each E1, E2, E3, E4 and E5 is a substituted or unsubstituted heteroatom. 45. "METHODS FOR USE" according to claim 41, characterized in that the compound has the structure of Formula V, and related pharmaceutically acceptable prodrug salts and esters: <formula> formula see original document page 237 < where dashed lines represent a single or double bond, where R1 is selected separately from the group consisting of a hydrogen, a halogen, monosubstituted, poly-substituted or unsubstituted variants of the following residues: C1-C24 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, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonoxy, nitro, azido, phenyl, cycloalkylacyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including alkyl polyhalogen ada, where η is 1 or 2, and if η is 2, then R1 may be the same or different; where R5 is selected separately from the group consisting of a hydrogen, a halogen, monosubstituted, polysubstituted or unsubstituted variants of the following residues: saturated C1-C24 alkyl, C2-C24 alkenyl or unsaturated C2-C24 alkynyl acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, amino -carbonyloxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl, where m is 0, 1, 2 , 3, 4, - 5, 6, 7, 8, 9, 10, or 11, and if m is greater than 1, then R5 may be the same or different; and wherein each E1, E2, E3, E4 and E5 is a substituted or unsubstituted heteroatom. 46. "METHODS FOR USE" according to claim 41, characterized in that the compound has the structure of Formula VI, and related pharmaceutically acceptable prodrug salts and esters: <formula> formula see original document page 238 < Formula VI wherein R1 may be selected separately from the group consisting of a monosubstituted, poly-substituted or unsubstituted variants of the following residues: saturated C1-C24 alkyl, C2-C24 alkenyl or non-substituted C2-C24 alkynyl saturated, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarboxy, phenyl, cycloalkyl acyl, thioalkyl, thioaryl, oxysulfonyl, carboxy, thio, sulfoxide, sulfone, esters and boronic acids, and halogenated alkyl including polyhalogenated alkyl. η is 1 or 2, and if η is 2, then R1 may be the same or different; where R2 may 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, C2-C24 alkenyl or non-C2-C24 alkynyl saturated, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl (including, for example, cyclohexylcarbinol), cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxy carbonyl acyl, amino, amino carbonyl, amino carboxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, thio, sulfoxide, sulfone, esters of sulfonate, thio cyano, boronic acids and esters, and halogenated alkyl including polyhalogenated alkyl, where R3 may be selected from the group consisting of a halogen, mono-substituted, poly-substituted or unsubstituted variants of the following residues: alkyl Saturated C1-C24 alkenyl Unsaturated C2 -C24 or C2 -C24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxy carbonyl acyl, amino, amino carbonyl, amino carboxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, thio, sulfoxide, sulfone, esters of sulfonate, thio cyano, boronic acids and esters, and halogenated alkyl including polyhalogenated alkyl; wherein each E1, E2, E3 and E4 may be a substituted or unsubstituted heteroatom. 47. "METHODS FOR USE" according to claims 41, 42, 43, 44, 45 or 46, characterized in that the compound is: <formula> formula see original document page 239 </formula> selected from the group consisting of H, F, Cl, Br and I. 48. "METHODS FOR USE" according to claim 47, characterized in that the compound has the structure of Formula 11-16, and related pharmaceutically acceptable prodrug salts and esters: <formula> formula see original document page 240 </formula> 49. "METHODS FOR USE" according to claims 41, 42, 43, 44, 45, 46, 47 or 48, characterized in that the animal is suffering from a disease, where the disease is an inflammatory condition. "METHODS FOR USE" according to claim 49, characterized in that the inflammatory condition is immune and non-immune inflammation, rheumatic arthritis, chronic joint rheumatism, and psoriasis. 51. "METHODS FOR USE" according to claim 49 or 50, characterized in that the disease has characteristics of improper or untimely invasions of blood vessels. 52. "METHODS FOR USE" according to claim 51, characterized in that the disease is diabetic retinopathy, neovascular glaucoma, retinopathy of prematurity, macular degeneration, corneal graft rejection, retrolental fibroplasia, rabeosis, plaque capillary proliferation. atherosclerosis, osteoporosis, leukemia, angiofibroma, Kaposi's sarcoma, hemangioma, acoustic neuroma, neurofibroma, trachoma and pyogenic granuloma. 53. "METHODS FOR USE" according to claims 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 or 52, characterized in that the use of an anti-angiogenic agent . 54. "METHODS FOR USE" according to claim 53, characterized in that the anti-angiogenic agent is an alpha-V-beta-3 (ανβ3), an integrin inhibiting antibody, a protein for adhesion to cell, a functional cell adhesion protein fragment containing a cell adhesion binding sequence, angiostatin, a functional fragment of angiostatin, endostatin, a fibroblast growth factor (FCF) inhibitor, an FCF receptor inhibitor , a VEGF inhibitor, a VEGF receptor inhibitor, a vascular permeability factor (FPV) inhibitor, a VFP receptor inhibitor, thrombospondin, a platelet factor 4, an interferon-alpha, an interferon-gamma, a interferon inducible protein 10, an interleukin 12, gro-beta, a 16kDa N-terminal fragment of prolactin, and thalidomide. 55. "METHODS FOR USE" according to claims 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 or 54, characterized in that the animal is a mammal. 56. "METHODS FOR USE" according to claims 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55, characterized in that the animal It is a human. 57. "METHODS FOR USE" according to claims 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 or 56, characterized in that The animal is a rodent. 58. "METHODS FOR USE" according to claims 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56 or 57, characterized in that to understand the steps of: identifying an individual who would benefit from the administration of an agent that inhibits angiogenesis; and perform the method on the individual. 59. "Methods for the use of heterocyclic compounds [3.2.0] and their analogues", characterized in that they comprise a method for treating cancer that is sensitive or resistant to Gleevec® comprising administering to a animal a compound having the structure of Formula I , and related pharmaceutically acceptable prodrug salts and esters: <formula> formula see original document page 242 </formula> where dashed lines represent a single or double bond, where R1 is selected separately from the group consisting of a hydrogen, a halogen, monosubstituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, unsaturated C2-C24 alkenyl or C2-C24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cyclo -alkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, aryl-alkoxycarbonyl, alkoxycarbonyl-acyl, amino, aminocarbonyl, aminocarbonoxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl, where η is 1 or 2, and if η is 2, then R1 may be the same or different; where R2 is selected from the group consisting of hydrogen, a halogen, mono-substituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, unsaturated C2-C24 alkenyl or C2-C24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, amino carboxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl ,. thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl; where R3 is selected from the group consisting of a halogen, mono-substituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, unsaturated C2-C24 alkenyl or C2-C24 alkynyl, acyl, acyloxy alkylalkylcarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonoxy, nitro azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl; wherein each E1, E2, E3 and E4 is a substituted or unsubstituted heteroatom; and where cancer is leukemia, ovarian cancer, urethral cancer, bladder cancer, prostate cancer, rectal cancer, stomach cancer, lymphoma, multiple myeloma, pancreatic cancer, liver cancer, kidney cancer, endocrine cancer, a sarcoma, skin cancer, angioma, or brain cancer. 60. "METHODS FOR USE" according to claim 59, characterized in that the compound has the structure of Formula II, and related pharmaceutically acceptable prodrug salts and esters: where the dotted lines represent a single or double bond. where R1 is selected separately from the group consisting of hydrogen, halogen, monosubstituted, poly-substituted or unsubstituted variants of the following residues: saturated C1-C24 alkyl, C2-C24 alkenyl or non-substituted C2-C24 alkynyl saturated, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, amino-carboxy, nitro, azido, phenyl, cycloalkyl-acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl, where η is equal to 1 or 2, and if η is equal to 2, en so R1 may be the same or different; where R3 is selected separately from the group consisting of a hydrogen, a halogen, mono-substituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, C2-C24 alkenyl or unsaturated C2-C24 alkynyl acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, amino -carbonyloxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl; where R4 is selected separately from the group consisting of a hydrogen, a halogen, monosubstituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, C2-C24 alkenyl or unsaturated C2-C24 alkynyl acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, amino -carboyloxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl, where m is 1 or 2, and if m equals 2, then R4 may be the same or different; and wherein each E1, E2, E3, E4 and E5 is a substituted or unsubstituted heteroatom. 61. "METHODS FOR USE" according to claim 59, characterized in that the compound has the structure of Formula III, and related pharmaceutically acceptable prodrug salts and esters: <formula> formula see original document page 244 < where dashed lines represent a single or double bond, where R1 is selected separately from the group consisting of a hydrogen, a halogen, monosubstituted, poly-substituted or unsubstituted variants of the following residues: C1-C24 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, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonoxy, nitro, azido, phenyl, cycloalkylacyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including alkyl poly- halogenated, where η is 1 or 2, and if η is 2, then R1 may be the same or different; where R4 is selected separately from the group consisting of a hydrogen, a halogen, monosubstituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, C2-C24 alkenyl or unsaturated C2-C24 alkynyl acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, amino -carboyloxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl, where m is 1 or 2, and if m equals 2, then R4 may be the same or different; and wherein each E1, E2, E3, E4 and E5 is a substituted or unsubstituted heteroatom. 62. "METHODS FOR USE" according to claim 59, characterized in that the compound has the structure of Formula IV, and related pharmaceutically acceptable prodrug salts and esters: <formula> formula see original document page 245 < where dashed lines represent a single or double bond, where R1 is selected separately from the group consisting of a hydrogen, a halogen, monosubstituted, poly-substituted or unsubstituted variants of the following residues: C1-C24 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, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonoxy, nitro, azido, phenyl, cycloalkylacyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including alkyl poly- halogenated, where η is 1 or 2, and if η is 2, then R1 may be the same or different; where R3 is selected separately from the group consisting of hydrogen, halogen, monosubstituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, C2-C24 alkenyl or unsaturated C2-C24 alkynyl acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, amino -carbonyloxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl; where R5 is selected separately from the group consisting of hydrogen, halogen, monosubstituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, C2-C24 alkenyl or unsaturated C2-C24 alkynyl acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, amino -carbonyloxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl, where m is 0, 1, 2 , 3, 4, -5, 6, 7, 8, 9, 10, or 11, and if m is greater than 1, then R5 may be the same or different; and wherein each E1, E2, E3, E4 and E5 is a substituted or unsubstituted heteroatom. 63. "METHODS FOR USE" according to claim 59, characterized in that the compound has the structure of Formula V, and related pharmaceutically acceptable prodrug salts and esters: <formula> formula see original document page 247 < where dashed lines represent a single or double bond, where R1 is selected separately from the group consisting of a hydrogen, a halogen, monosubstituted, poly-substituted or unsubstituted variants of the following residues: C1-C24 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, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonoxy, nitro, azido, phenyl, cycloalkylacyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including alkyl polyhalogen da, where η is 1 or 2, and if η is 2, then R1 may be the same or different; where R5 is selected separately from the group consisting of a hydrogen, a halogen, monosubstituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, C2-C24 alkenyl or unsaturated C2-C24 alkynyl acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, amino -carbonyloxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl, where m is 0, 1, 2 , 3, 4, - 5, 6, 7, 8, 9, 10, or 11, and if m is greater than 1, then R5 may be the same or different; and wherein each E1, E2, E3, E4 and E5 is a substituted or unsubstituted heteroatom. 64. "METHODS FOR USE" according to claim 59, characterized in that the compound has the structure of Formula VI, and related pharmaceutically acceptable prodrug salts and esters: Formula VI <formula> formula see original document page Where R1 may be selected separately from the group consisting of hydrogen, halogen, monosubstituted, poly-substituted or unsubstituted variants of the following residues: saturated C1-C24 alkyl, C2-C24 alkenyl or alkynyl Unsaturated C2 -C24, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino aminocarbonyl, aminocarbonoxy, phenyl, cycloalkyl acyl, thioalkyl, thioaryl, oxysulfonyl, carboxy, thio, sulfoxide, sulfone, boronic acids and esters, and halogenated alkyl including polyhalogenated alkyl; η is equal to 1 or 2, and if η is equal to 2, then R1 may be the same or different; where R2 may 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, C2-C24 alkenyl or unsaturated C2-C24 alkynyl acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl (including, for example, cyclohexylcarbinol), cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonyl -acyl, amino, amino-carbonyl, amino-carboxy, nitro, azido, phenyl, cycloalkyl-acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, thio, sulfoxide, sulfone, sulfonate esters thio-cyano, boronic acids and esters, and halogenated alkyl including polyhalogenated alkyl; where R3 may 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 C2-C24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, aryl-alkoxycarbonyl, alkoxycarbonyl-acyl, amino, aminocarbonyl, aminocarbonoxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, thio, sulfoxide, sulfone, sulfonate esters, thio cyano, boronic acids and esters, and halogenated alkyl including polyhalogenated alkyl; wherein each E1, E2, E3 and E4 may be a substituted or unsubstituted heteroatom. 65. "METHODS FOR USE" according to claims 59, 60, 61, 62, 63 or 64, characterized in that the compound is: F, Cl, Br and I. 66. "METHODS FOR USE" according to claim 65, characterized in that the compound has the structure of Formula 11-16, and related pharmaceutically acceptable prodrug salts and esters: <formula> formula see original document page 250 </formula> 67. "METHODS FOR USE" according to claims 59, 60, 61, 62, 63, 64, 65 or 66, characterized in that the cancer is chronic myeloid leukemia, Philadelphia chromosome positive acute lymphoblastic leukemia, and acute myeloid leukemia. 68. "METHODS FOR USE" according to claims 59, 60, 61, 62, 63, 64, 65 or 66, characterized in that they comprise the use of Gleevec®. 69. "METHODS FOR USE" according to claims 59, 60, 61, 62, 63, 64, 65 or 66, characterized in that the animal is a mammal. 70. "METHODS FOR USE" according to claims 59, 60, 61, 62, 63, 64, 65 or 66, characterized in that the animal is a human. 71. "METHODS FOR USE" according to claims 59, 60, 61, 62, 63, 64, 65 or 66, characterized in that the animal is a rodent. 72. "Methods for the use of heterocyclic compounds [3.2.0] and their analogues", characterized in that they comprise a method for treating cancer comprising administering to an animal a compound having the structure of Formula VI, and salts and esters pro. Related pharmaceutically acceptable drugs: <formula> formula see original document page 251 </formula> Formula VI where R1 may be selected separately from the group consisting of mono-substituted, poly-substituted or unsubstituted variants of the following residues: C1-alkyl Unsaturated -C24 saturated, C2 -C24 alkenyl or C2 -C24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, aryl -alkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonoxy, phenyl, cycloalkylacyl, thioalkyl, thioaryl, oxysulfonyl, carboxy, thio, sulfoxide, sulfone, esters and boric acids nicos, and halogenated alkyl including halogenated poly-alkyl; η is equal to 1 or 2, and if η is equal to 2, then R1 may be the same or different; where R2 may 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, C2-C24 alkenyl or unsaturated C2-C24 alkynyl acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl (including, for example, cyclohexylcarbinol), cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonyl -acyl, amino, amino-carbonyl, amino-carboxy, nitro, azido, phenyl, cycloalkyl-acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, thio, sulfoxide, sulfone, sulfonate esters thio-cyano, boronic acids and esters, and halogenated alkyl including polyhalogenated alkyl; where R3 may be selected from the group consisting of halogen, mono-substituted, poly-substituted or unsubstituted variants of the following residues: unsaturated C1-C24 alkyl, unsaturated C2-C24 alkenyl or C2-C24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, aryl-alkoxycarbonyl, alkoxycarbonyl-acyl, amino, aminocarbonyl, aminocarbonoxy, nitro, azido, phenyl, cycloalkyl acyl, hydroxy, thioalkyl, thioaryl, oxysulfonyl, carboxy, cyano, thio, sulfoxide, sulfone, sulfonate esters, thio cyano, boronic acids and esters, and halogenated alkyl including polyhalogenated alkyl; wherein each E1, E2, E3 and E4 may be a substituted or unsubstituted heteroatom. 73. "METHODS FOR USE" according to claim 72, characterized in that the cancer is a multiple myeloma. 74. "METHODS FOR USE" according to claim 72, characterized in that the cancer is a prostate cancer. 75. "METHODS FOR USE" according to claim 72, characterized in that the cancer is an ovarian cancer. 76. "METHODS FOR USE" according to claim 72, characterized in that the cancer is a pancreatic cancer. 77. "METHODS FOR USE" according to claim 72, characterized in that they comprise the use of a chemotherapeutic agent. 78. "METHODS FOR USE" according to claims 72, 73, 74, 75, 76 or 77, characterized in that the chemotherapeutic agent is Adriamycin, Doxorubicin, 5-Fluorouracil, Cytosine arabinoside ("Ara-C" ), Cyclophosphamide, Thiotepa, Busulfan, Cytoxine, Taxol, Toxotero, Methotrexate, Cisplatin, Melfalane, Vinblastine, Bleomycin, Etoposide, Ifosfamide, Mitomycin C, Mitoxantrone, Vincristine, Vinorelbine, Carboplatinin, Mycotinin, Carboplatinin, Mycotoxin , Speramycin, Melfalane, Tamoxifen or Onapristone. 79. "METHODS FOR USE" according to claim 72, characterized in that the cancer is a drug resistant cancer. 80. "METHODS FOR USE" according to claim 79, characterized in that the drug-resistant cancer is sarcoma, multiple myeloma, or leukemia. 81. "METHODS FOR USE" according to claim 80, characterized in that drug-resistant cancer exhibits at least one of the following: elevated levels of P-glycoprotein efflux pump, increased expression of multidrug resistance. -associated with MRP1-encoded protein 1, reduced drug absorption, altered drug target or increased repair of drug-induced DNA damage, altered apoptotic pathway or activation of cytochrome P450 enzymes. 82. "METHODS FOR USE" according to claim 72, characterized in that the animal is a mammal. 83. "METHODS FOR USE" according to claim 72, characterized in that the animal is a human. 84. "METHODS FOR USE" according to claim 72, characterized in that the animal is a rodent. 85. "METHODS FOR USE" according to claim 72, comprising the steps of: identifying an individual who would benefit from administration of an anti-cancer agent; and perform the method on the individual.