WO2009085360A2

WO2009085360A2 - Citreamicin antibiotic with a sugar residue

Info

Publication number: WO2009085360A2
Application number: PCT/US2008/078732
Authority: WO
Inventors: Aaron Peoples; Qibo Zhang; Andrew Staley; Lucy Ling; Charles Moore; Kim Lewis
Original assignee: Novobiotic Pharmaceuticals Llc
Priority date: 2007-10-04
Filing date: 2008-10-03
Publication date: 2009-07-09
Also published as: WO2009085360A8; WO2009085360A3

Abstract

This application relates to novel neocitreamicins and their analogs; processes for their preparation; pharmaceutical compositions comprising these neocitreamicins; and methods of using such compounds.

Description

CITREAMICIN ANTIBIOTIC WITH A SUGAR RESIDUE

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. §119(e) to United States Patent Application Serial Nos. 60/997,674 filed October 4, 2007, and 61/076,429 filed June 27, 2008, each of which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention is in the field of microbial chemistry. More specifically, the invention relates generally to novel citreamicin antibiotics.

BACKGROUND OF THE INVENTION

The success of antimicrobials against disease-causing microbes is one of medicine's great achievements. Antibiotics have saved numerous lives and blunted the complications of many diseases and infections. However, the currently available antibiotics are not as effective as they once were.

Over time, some bacteria have developed ways to circumvent the effects of these known antibiotics, and in recent years there has been a worldwide increase in infections caused by microbes resistant to multiple microbial agents. Other microbes such as viruses, fungi, and parasites have also developed resistance to known antimicrobials. Antimicrobial resistance provides a survival advantage to microbes and has made it harder to eliminate microbial infections from the body. This increased difficulty in fighting microbial infections has led to an increased risk of developing infections in hospitals or other settings.

With the increased availability and ease of global travel, rapid spread of resistant microbes around the world is becoming a serious problem. In the community, microbial resistance can result from nosocomial acquisition of drug-resistant pathogens (e.g., methicillin resistant Staphylococcus aureus, vancomycin resistant Enterococci), emergence of resistance due to use of antibiotics within the community (e.g., pencillin resistant Neisseria gonorrheae), acquisition of resistant pathogens as a result of travel (e.g., antibiotic-resistant Shigella), or as a result of using antimicrobial agents in animals with subsequent transmission of resistant pathogens to humans (e.g., antibiotic resistant Salmonella). Antibiotic resistance in hospitals has usually resulted from overuse of antibiotics and has been a serious problem with MRSA, MDR-GNB, and VRE.

Diseases such as tuberculosis, malaria, gonorrhea, and childhood ear infections are now more difficult to treat than they were just a few decades ago. Drug resistance is a significant problem for hospitals harboring critically ill patients who are less able to fight off infections without the help of antibiotics. Unfortunately, heavy use of antibiotics in these patients selects for changes in microbes that bring about drug resistance. These drug resistant bacteria are resistant to our strongest antibiotics and continue to prey on vulnerable hospital patients.

In view of these problems, there is an increasing need for novel antibiotics to deal with microbial infections and the problem of increasing drug resistance.

SUMMARY OF THE INVENTION

This application relates to novel neocitreamicins and their analogs; to processes for their preparation; to pharmaceutical compositions comprising these neocetreamicins; and to methods of using such compounds.

In a first aspect, the disclosure is directed to a compound having formula (I)

or an enantiomer, diastereomer, tautomer, or pharmaceutically acceptable salt or solvate thereof, wherein: Ri is alkyl or substituted alkyl, alkenyl or substituted alkenyl, alkynyl or substituted alkynyl, cycloalkyl or substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted heterocycle, or aryl or substituted aryl; R-₂ is hydrogen, halogen, cyano, nitro, alkyl or substituted alkyl, cycloalkyl or substituted cycloalkyl, aryl or substituted aryl, or C(=O)R_c, wherein R₀ is alkyl or substituted alkyl, cycloalkyl or substituted cycloalkyl, or aryl or substituted aryl; R₃ is hydrogen or C(=O)alkyl; and R₄ is hydrogen, alkyl or substituted alkyl.

In some embodiments of this aspect, the compound has the structure of formula (Ia)

wherein R₁ is alkyl or substituted alkyl, alkenyl or substituted alkenyl, alkynyl or substituted alkynyl, cycloalkyl or substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted heterocycle, or aryl or substituted aryl.

In other embodiments, the compound has the structure of formula (II)

or an enantiomer, diastereomer, tautomer, or pharmaceutically acceptable salt or solvate thereof, wherein the compound is isolated and purified.

hi some embodiments, the compound has the structure of formula (III)

or an enantiomer, diastereomer, tautomer, or pharmaceutically acceptable salt or solvate thereof.

In certain embodiments, the compound is isolated from an aerobically grown strain of Nocardia G0655, wherein the compound characterized by at least two of the following:(a) a molecular weight of 825.81 (Q-TOF LC-MS); (b) an ultraviolet absorption spectra substantially the same to that shown in FIG. 2; a proton nuclear magnetic resonance substantially the same to that shown in FIG. 3; a carbon- 13 nuclear magnetic resonance substantially the same to that shown in FIG. 4; a COSY nuclear magnetic resonance substantially the same to that shown in FIG. 5; a HSQC nuclear magnetic resonance substantially the same to that shown in FIG. 6; a HMBC nuclear magnetic resonance substantially the same to that shown in FIG. 7; and a ROESY spectrum substantially the same to that shown in FIG. 8.

In other embodiments, the invention provides pharmaceutical composition comprising the above compounds and a pharmaceutically-acceptable excipient, carrier or diluent. In some embodiments, the pharmaceutical composition further comprises an agent selected from the group consisting of an antineoplastic agent, an antibiotic, an antifungal agent, an antiviral agent, and combinations thereof. In specific embodiments, the antineoplastic agent is selected from the group consisting of an antimetabolite, a purine or pyrimidine analogue, an alkylating agent, a crosslinking agent, and an intercalating agent.

In another aspect, the disclosure provides a method of producing a compound of formula (II)

the method comprising cultivating a strain of Nocardia G0655 in a culture medium comprising assimilable sources of carbon, nitrogen, and inorganic salts under aerobic conditions to produce an assayable amount of the compound of formula (II). In certain embodiments, the method further comprises isolating and purifying the compound of formula (II) from the culture medium.

In another aspect, the disclosure is directed to a method of treating a disorder in a mammal in need thereof by administering to the mammal a therapeutically effective amount of a compound of formula (I),

or an enantiomer, diastereomer, tautomer, or pharmaceutically acceptable salt or solvate thereof, wherein: R₁ is alkyl or substituted alkyl, alkenyl or substituted alkenyl, alkynyl or substituted alkynyl, cycloalkyl or substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted heterocycle, or aryl or substituted aryl; R₂ is hydrogen, halogen, cyano, nitro, alkyl or substituted alkyl, cycloalkyl or substituted cycloalkyl, aryl or substituted aryl, or C(=O)R_c, wherein R₀ is alkyl or substituted alkyl, cycloalkyl or substituted cycloalkyl, or aryl or substituted aryl; R₃ is hydrogen or C(=O)alkyl; and R₄ is hydrogen, alkyl or substituted alkyl. In a specific embodiment, the compound administered has the formula (II)

In some embodiments, the disorder is caused by an agent selected from the group consisting of a bacterium, a fungus, a virus, a protozoan, a helminth, a parasite, and combinations thereof.

In a embodiment, the disorder is caused by a bacterium. In specific embodiments, the bacterium is selected from the group consisting of Helicobacter pylori, Borelia burgdorferi, Legionella pneumophilia, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellular, Mycobacterium kansaii, Mycobacterium gordonae, Mycobacteria sporozoites, Staphylococcus aureus, Staphylococcus epidermidis, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus dysgalactia, Streptococcus faecalis, Streptococcus bovis, Streptococcus pneumoniae, pathogenic Campylobacter sporozoites, Enterococcus sporozoites, Haemophilus influenzae, Pseudomonas aeruginosa, Bacillus anthracis, Corynebacterium diphtheriae, Corynebacterium jeikeium, Corynebacterium sporozoites, Erysipelothrix rhusiopathiae, Clostridium perfringens, Clostridium tetani, Clostridium difficile, Enter obacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sporozoites, Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, and Actinomyces israelii. In certain embodiments, the bacterium is a Gram-positive bacterium, hi specific embodiments, the Gram-positive bacterium is selected from the group consisting of Streptococcus, Staphylococcus, Methicillin Resistant Staphylococcus aureus (MRSA), Enterococcus, Corynebacteria, Listeria, Bacillus, Erysipelothrix, and Actinomycetes. In certain embodiments, the bacterium is a Gram-negative bacterium.

In another embodiment, the disorder is caused by a virus. In certain embodiments, the virus is selected from the group consisting of Retroviridae, Picornaviridae, Calciviridae, Togaviridae, Flaviridae, Coronaviridae, Rhabdoviridae, Filoviridae, Paramyxoviridae, Orthomyxoviridae, Bungaviridae, Arenaviridae, Reoviridae, Birnaviridae, Hepadnaviridae, Parvoviridae, Papovaviridae, Adenoviridae, Herpesviridae, Poxviridae, and Iridoviridae. In other embodiments, virus is selected from the group consisting of influenza virus, human immunodeficiency virus, and herpes simplex virus.

In other embodiments, the disorder is caused by a protozoan, hi certain embodiments, the protozoan is selected from the group consisting of Trichomonas vaginalis, Giardia lamblia, Entamoeba histolytica, Balantidium coli, Cryptosporidium parvum and Isospora belli, Trypansoma cruzi, Trypanosoma gambiense, Leishmania donovani, and Naegleria fowleri.

In yet other embodiments, the disorder is caused by a helminth. In certain embodiments, the helminth is selected from the group consisting of Schistosoma mansoni, Schistosoma cercariae, Schistosoma japonicum, , Schistosoma mekongi, Schistosoma hematobium, Ascaris lumbricoides, Strongyloides stercoralis, Echinococcus granulosus, Echinococcus multilocularis, Angiostrongylus cantonensis, Angiostrongylus constaricensis, Fasciolopis buski, Capillaria philippinensis, Paragonimus westermani, Ancylostoma diidodenale, Necator americanus,. Trichinella spiralis, Wuchereria bancrofti, Brugia malayi, and Brugia timori, Toxocara canis, Toxocara cati, Toxocara vitulorum, Caenorhabiditis elegans, and Anisakis species. In other embodiments, the disorder is caused by a parasite. In certain embodiments, the parasite is selected from the group consisting of Plasmodium falciparum, Plasmodium yoelli, Hymenolepis nana, Clonorchis sinensis, Loa loa, Paragonimus westermani, Fasciola hepatica, and Toxoplasma gondii.

In yet other embodiments, the disorder is caused by a fungus. In certain embodiments, the fungus is selected from the group consisting of Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans, Candida tropicalis, Candida glabrata, Candida krusei, Candida par apsilosis, Candida dubliniensis, Candida lusitaniae, Epidermophyton floccosum, Microsporum audouinii, Microsporum canis, Microsporum canis var. distortum Microsporum cookei, Microsporum equinum, Microsporum ferrugineum, Microsporum fulvum, Microsporum gallinae, Microsporum gypseum, Microsporum nanum, Microsporum persicolor, Trichophyton ajelloi, Trichophyton concentricum, Trichophyton equinum, Trichophyton flavescens, Trichophyton gloriae, Trichophyton megnini, Trichophyton mentagrophytes var. erinacei, Trichophyton mentagrophytes var. interdigitale, Trichophyton phaseoliforme, Trichophyton rubrum, Trichophyton rubrum downy strain, Trichophyton rubrum granular strain, Trichophyton schoenleinii, Trichophyton simii, Trichophyton soudanense, Trichophyton terrestre, Trichophyton tonsurans, Trichophyton vanbreuseghemii, Trichophyton verrucosum, Trichophyton violaceum, Trichophyton yaoundei, Aspergillus fumigatus, Aspergillus flavus, and Aspergillus clavatus.

The disclosure also provides, in another aspect, a method of inhibiting the growth of a tumor cell, comprising contacting the tumor cell with an effective amount of compound of formula (I)

or an enantiomer, diastereomer, tautomer, or pharmaceutically acceptable salt or solvate thereof, wherein: R₁ is alkyl or substituted alkyl, alkenyl or substituted alkenyl, alkynyl or substituted alkynyl, cycloalkyl or substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted heterocycle, or aryl or substituted aryl; R₂ is hydrogen, halogen, cyano, nitro, alkyl or substituted alkyl, cycloalkyl or substituted cycloalkyl, aryl or substituted aryl, or C(=O)R_c, wherein R₀ is alkyl or substituted alkyl, cycloalkyl or substituted cycloalkyl, or aryl or substituted aryl; R₃ is hydrogen or C(=O)alkyl; and R₄ is hydrogen, alkyl or substituted alkyl. In a specific embodiment, the administered compound has formula (II)

or an enantiomer, diastereomer, tautomer, or pharmaceutically acceptable salt or solvate thereof. In some embodiments of this aspect, the tumor cell is selected from the group consisting of a breast cancer cell, an ovarian cancer cell, a colon cancer cell, a prostate cancer cell, a liver cancer cell, a lung cancer cell, a gastric cancer cell, an esophageal cancer cell, a urinary bladder cancer cell, a melanoma cell, a leukemia cell, and a lymphoma cell. In specific embodiments, the tumor cell is a drug-resistant tumor cell.

In certain embodiments, the method further comprises administering a chemotherapeutic agent selected from the group consisting of an antimetabolite, a purine or pyrimidine analogue, an alkylating agent, a crosslinking agent, and an intercalating agent, wherein the chemotherapeutic agent is administered before, after or substantially simultaneously with the compound.

In another aspect, the disclosure is directed to a method of treating cancer in a mammal in need thereof, the method comprising administering to the mammal a therapeutically effective amount of compound of formula (I)

or an enantiomer, diastereomer, tautomer, or pharmaceutically acceptable salt or solvate thereof, wherein: Ri is alkyl or substituted alkyl, alkenyl or substituted alkenyl, alkynyl or substituted alkynyl, cycloalkyl or substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted heterocycle, or aryl or substituted aryl; R₂ is hydrogen, halogen, cyano, nitro, alkyl or substituted alkyl, cycloalkyl or substituted cycloalkyl, aryl or substituted aryl, or Q=O)R_c, wherein R₀ is alkyl or substituted alkyl, cycloalkyl or substituted cycloalkyl, or aryl or substituted aryl; R₃ is hydrogen or C(=O)alkyl; and R₄ is hydrogen, alkyl or substituted alkyl. In a specific embodiment, the administered compound has formula (II)

In certain embodiments, the cancer is selected from the group consisting of breast cancer, ovarian cancer, colon cancer, prostate cancer, liver cancer, lung cancer, gastric cancer, esophageal cancer, urinary bladder cancer, melanoma, leukemia, and lymphoma.

In other embodiments, the method further comprises administering a chemotherapeutic agent selected from the group consisting of an antimetabolite, a purine or pyrimidine analogue, an alkylating agent, a crosslinking agent, and an intercalating agent, wherein the chemotherapeutic agent is administered before, after or substantially simultaneously with the compound.

In another aspect, the disclosure provides an isolated G0655 strain deposited with the Agricultural Research Service (ARS) Culture Collection, U.S. Department of

Agriculture, 1815 North University Street, Peoria, Illinois 61604, U.S.A. and having the designation NRRL 50106.

In yet another aspect, the disclosure provides a culture consisting essentially of a Nocardia species, having the identifying characteristics of strain G0665 (NRRL 50106). The disclosure also provides, in another aspect, a method of preparing a compound of formula (I),

or an enantiomer, diastereomer, tautomer, or pharmaceutically acceptable salt or solvate thereof, wherein: Rj is alkyl or substituted alkyl, alkenyl or substituted alkenyl, alkynyl or substituted alkynyl, cycloalkyl or substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted heterocycle, or aryl or substituted aryl; R₂ is hydrogen, halogen, cyano, nitro, alkyl or substituted alkyl, cycloalkyl or substituted cycloalkyl, aryl or substituted aryl, or C(=O)R_C, wherein R₀ is alkyl or substituted alkyl, cycloalkyl or substituted cycloalkyl, or aryl or substituted aryl; R₃ is hydrogen or C(=O)alkyl; and R₄ is hydrogen, alkyl or substituted alkyl,

comprising converting a compound of formula (III)

(HI), or an enantiomer, diastereomer, tautomer, or pharmaceutically acceptable salt or solvate thereof, for a time and under conditions effective to form the compound of formula (I). In another aspect, the disclosure provides a method of preparing a compound of formula (Ia)

or an enantiomer, diastereomer, tautomer, or pharmaceutically acceptable salt or solvate thereof, wherein: Rj is alkyl or substituted alkyl, alkenyl or substituted alkenyl, alkynyl or substituted alkynyl, cycloalkyl or substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted heterocycle, or aryl or substituted aryl, comprising reacting a compound of formula (III)

or an enantiomer, diastereomer, tautomer, or pharmaceutically acceptable salt or solvate thereof, with a compound of formula RiC(=O)OH,

or RiC(O)-O-(O)Ri, for a time and under conditions effective to form the compound of formula (Ia). DESCRIPTION OF THE FIGURES

The foregoing and other objects of the present disclosure, the various features thereof, as well as the disclosure itself may be more fully understood from the following description, when read together with the accompanying drawings in which:

Fig. 1 is a schematic representation of the structure of Neocitreamicin II.

Fig. 2 is a graphic representation of the HPLC analysis of a solid phase extract of the Nocardia strain G0655 monitored at 254 nm. The Neocitreamicin II peak is shown as (2).

Fig. 3 is a schematic representation of the proton nuclear magnetic resonance spectra of neocitreamicin II.

Fig. 4 is a schematic representation of the carbon- 13 nuclear magnetic resonance spectra of neocitreamicin II.

Fig. 5 is a schematic representation of the COSY nuclear magnetic resonance spectra of neocitreamicin II.

Fig. 6 is a schematic representation of the HSQC nuclear magnetic resonance spectra of neocitreamicin II.

Fig. 7 is a schematic representation of the a HMBC nuclear magnetic resonance spectra of neocitreamicin II.

Fig. 8 is a graphic representation of selected ROESY correlations for Neocitreamicin II.

This disclosure relates to novel neocitreamicins and their analogs; to processes for their preparation; to pharmaceutical compositions comprising such neocitreamicins; and to methods of using such compounds. Throughout this application, various patents, patent applications, and publications are referenced. The inventions of these patents, patent applications, and publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the disclosure described and claimed herein. The instant disclosure will govern in the instance that there is inconsistency between the patents, patent applications, and publications and this disclosure.

DETAILED DESCRIPTION

Definitions

For convenience, certain terms employed in the specification, examples, and appended claims are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The initial definition provided for a group or term herein applies to that group or term throughout the present specification individually or as part of another group, unless otherwise indicated.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

The term "or" is used herein to mean, and is used interchangeably with, the term "and/or," unless context clearly indicates otherwise.

The term "about" is used herein to mean a value - or + 20% of a given numerical value. Thus, about 60% means a value of between 60% - 20% of 60 and 60% + 20% of 60 (i.e., between 48% and 72%).

The term "substantially the same" is used herein to mean that two comparing subjects share at least 90% of common feature. In certain embodiments, the common feature is at least 95%. In certain other embodiments, the common feature at least 99%. The term "isolated" is used herein to mean purified to a state beyond that in which it exists in nature. For example an isolated compound can be substantially free of cellular material or other contaminating materials from the cell from which the compound is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. In some embodiments, the preparation of a compound having less than about 50% (by dry weight) of contaminating materials from the cell, or of chemical precursors is considered to be substantially pure. In other embodiments, the preparation of a compound having less than about 40%, about 30%, about 20%, about 10%, about 5%, about 1% (by dry weight) of contaminating materials from the cell, or of chemical precursors is considered to be substantially pure.

The terms "alkyl" and "alk" refers to a straight or branched chain alkane (hydrocarbon) radical containing from 1 to 12 carbon atoms, e.g., 1 to 6 carbon atoms. Exemplary "alkyl" groups include methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, isobutyl pentyl, hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl, dodecyl, and the like. The term "CpC₄ alkyl" refers to a straight or branched chain alkane (hydrocarbon) radical containing from 1 to 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, and isobutyl. "Substituted alkyl" refers to an alkyl group substituted with one or more substituents, e.g. 1 to 4 substituents, at any available point of attachment. Exemplary substituents include but are not limited to one or more of the following groups: hydrogen, halogen (e.g., a single halogen substituent or multiple halo substituents forming, in the latter case, groups such as CF₃ or an alkyl group bearing CCl₃, cyano, nitro, CF₃, OCF3, cycloalkenyl, alkynyl, heterocycle, aryl, OR_a, SR_a, S(=O)Rc S(=O)₂R_e, P(=O)₂R_e, S(=O)₂OR_e, P(=O)₂OR_e, NRbRc, NR_bS(=O)₂R_e, NR_bP(=O)₂Re,

C(=O)NR_bRc, OC(=O)R_a, OC(=O)NR_bRc, NR_bC(=O)OR_e, NRdCeO)NRbR₀, NR_dS(=O)₂NR_bRc,

NR_dP^O)₂NRbR₀, NR_bC(=O)R_a, or NR_bP(=O)₂Rs, wherein each R_a is hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, heterocycle, or aryl; R_b, R₀ and R_d are independently hydrogen, alkyl, cycloalkyl, heterocycle, aryl, or said R_b and R₀ together with the N to which they are bonded optionally form a heterocycle or substituted heterocycle; and each R_e is alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, heterocycle, or aryl. In the aforementioned exemplary substituents, groups such as alkyl, cycloalkyl, alkenyl, alkynyl, cycloalkenyl, heterocycle and aryl can themselves be optionally substituted.

The term "alkenyl" refers to a straight or branched chain hydrocarbon radical containing from 2 to 12 carbon atoms and at least one carbon-carbon double bond. Exemplary such groups include ethenyl or allyl. "Substituted alkenyl" refers to an alkenyl group substituted with one or more substituents, e.g., 1 to 4 substituents, at any available point of attachment. Exemplary substituents include, but are not limited to, alkyl or substituted alkyl, as well as those groups recited above as exemplary alkyl substituents. The exemplary substituents can themselves be optionally substituted.

The term "alkynyl" refers to a straight or branched chain hydrocarbon radical containing from 2 to 12 carbon atoms and at least one carbon to carbon triple bond. Exemplary such groups include ethynyl. "Substituted alkynyl" refers to an alkynyl group substituted with one or more substituents, e.g., 1 to 4 substituents, at any available point of attachment. Exemplary substituents include, but are not limited to, alkyl or substituted alkyl, as well as those groups recited above as exemplary alkyl substituents. The exemplary substituents can themselves be optionally substituted.

The term "cycloalkyl" refers to a fully saturated cyclic hydrocarbon group containing from 1 to 4 rings and 3 to 8 carbons per ring. Exemplary such groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, etc. "Substituted cycloalkyl" refers to a cycloalkyl group substituted with one or more substituents, e.g., 1 to 4 substituents, at any available point of attachment. Exemplary substituents include, but are not limited to, nitro, cyano, alkyl or substituted alkyl, as well as those groups recited above as exemplary alkyl substituents. The exemplary substituents can themselves be optionally substituted. Exemplary substituents also include spiro-attached or fused cyclic substituents, especially spiro-attached cycloalkyl, spiro-attached cycloalkenyl, spiro- attached heterocycle (excluding heteroaryl), fused cycloalkyl, fused cycloalkenyl, fused heterocycle, or fused aryl, where the aforementioned cycloalkyl, cycloalkenyl, heterocycle and aryl substituents can themselves be optionally substituted.

The term "cycloalkenyl" refers to a partially unsaturated cyclic hydrocarbon group containing 1 to 4 rings and 3 to 8 carbons per ring. Exemplary such groups include cyclobutenyl, cyclopentenyl, cyclohexenyl, etc. "Substituted cycloalkenyl" refers to a cycloalkenyl group substituted with one more substituents, e.g., 1 to 4 substituents, at any available point of attachment. Exemplary substituents include but are not limited to nitro, cyano, alkyl or substituted alkyl, as well as those groups recited above as exemplary alkyl substituents. The exemplary substituents can themselves be optionally substituted. Exemplary substituents also include spiro-attached or fused cyclic substituents, especially spiro-attached cycloalkyl, spiro-attached cycloalkenyl, spiro-attached heterocycle (excluding heteroaryl), fused cycloalkyl, fused cycloalkenyl, fused heterocycle, or fused aryl, where the aforementioned cycloalkyl, cycloalkenyl, heterocycle and aryl substituents can themselves be optionally substituted.

The term "aryl" refers to cyclic, aromatic hydrocarbon groups that have 1 to 5 aromatic rings, especially monocyclic or bicyclic groups such as phenyl, biphenyl or naphthyl. Where containing two or more aromatic rings (bicyclic, etc), the aromatic rings of the aryl group may be joined at a single point {e.g., biphenyl), or fused {e.g., naphthyl, phenanthrenyl and the like). "Substituted aryl" refers to an aryl group substituted by one or more substituents, e.g., 1 to 3 substituents, at any point of attachment. Exemplary substituents include, but are not limited to, nitro, cycloalkyl or substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl, cyano, alkyl or substituted alkyl, as well as those groups recited above as exemplary alkyl substituents. The exemplary substituents can themselves be optionally substituted. Exemplary substituents also include fused cyclic groups, especially fused cycloalkyl, fused cycloalkenyl, fused heterocycle, or fused aryl, where the aforementioned cycloalkyl, cycloalkenyl, heterocycle and aryl substituents can themselves be optionally substituted. The terms "heterocycle" and "heterocyclic" refer to fully saturated, or partially or fully unsaturated, including aromatic (i.e., "heteroaryl") cyclic groups (for example, 4 to 7 membered monocyclic, 7 to 11 membered bicyclic, or 8 to 16 membered tricyclic ring systems) which have at least one heteroatom in at least one carbon atom-containing ring. Each ring of the heterocyclic group containing a heteroatom may have 1 , 2, 3, or 4 heteroatoms selected from nitrogen atoms, oxygen atoms and/or sulfur atoms, where the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quaternized. (The term "heteroarylium" refers to a heteroaryl group bearing a quaternary nitrogen atom and thus a positive charge.) The heterocyclic group may be attached to the remainder of the molecule at any heteroatom or carbon atom of the ring or ring system. Exemplary monocyclic heterocyclic groups include azetidinyl, pyrrolidinyl, pyrrolyl, pyrazolyl, oxetanyl, pyrazolinyl, imidazolyl, imidazolinyl, imidazolidinyl, oxazolyl, oxazolidinyl, isoxazolinyl, isoxazolyl, thiazolyl, thiadiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, furyl, tetrahydrofuryl, thienyl, oxadiazolyl, piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolodinyl, 2- oxoazepinyl, azepinyl, hexahydrodiazepinyl, 4-piperidonyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, triazolyl, tetrazolyl, tetrahydropyranyl, morpholinyl, thiamorpholinyl, thiamorpholinyl sulfoxide, thiamorpholinyl sulfone, 1,3-dioxolane and tetrahydro-l,l-dioxo thienyl, and the like. Exemplary bicyclic heterocyclic groups include indolyl, isoindolyl, benzothiazolyl, benzoxazolyl, benzoxadiazolyl, benzothienyl, benzo[d][l,3]dioxolyl, 2,3-dihydrobenzo[b][l,4]dioxinyl, quinuclidinyl, quinolinyl, tetrahydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuryl, benzofurazanyl, chromonyl, coumarinyl, benzopyranyl, cinnolinyl, quinoxalinyl, indazolyl, pyrrolopyridyl, furopyridinyl (such as furo[2,3-c]pyridinyl, furo[3,2-b]pyridinyl] or furo[2,3-b]pyridinyl), dihydroisoindolyl, dihydroquinazolinyl (such as 3,4-dihydro-4-oxo-quinazolinyl), triazinylazepinyl, tetrahydroquinolinyl and the like. Exemplary tricyclic heterocyclic groups include carbazolyl, benzidolyl, phenanthrolinyl, acridinyl, phenanthridinyl, xanthenyl and the like. "Substituted heterocycle" and "substituted heterocyclic" (such as "substituted heteroaryl") refer to heterocycle or heterocyclic groups substituted with one or more substituents, e.g., 1 to 4 substituents, at any available point of attachment. Exemplary substituents include, but are not limited to, cycloalkyl or substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl, nitro, oxo (i.e., = O), cyano, alkyl or substituted alkyl, as well as those groups recited above as exemplary alkyl substituents. The exemplary substituents can themselves be optionally substituted. Exemplary substituents also include spiro-attached or fused cyclic substituents at any available point or points of attachment, especially spiro-attached cycloalkyl, spiro-attached cycloalkenyl, spiro- attached heterocycle (excluding heteroaryl), fused cycloalkyl, fused cycloalkenyl, fused heterocycle, or fused aryl, where the aforementioned cycloalkyl, cycloalkenyl, heterocycle and aryl substituents can themselves be optionally substituted.

The terms "halogen" and "halo" refer to chlorine, bromine, fluorine, or iodine.

The term "carbocyclic" refers to aromatic or non-aromatic 3 to 7 membered monocyclic and 7 to 11 membered bicyclic groups, in which all atoms of the ring or rings are carbon atoms. "Substituted carbocyclic" refers to a carbocyclic group substituted with one or more substituents, e.g., 1 to 4 substituents, at any available point of attachment. Exemplary substituents include, but are not limited to, nitro, cyano, OR_a, wherein R_a is as defined hereinabove, as well as those groups recited above as exemplary cycloalkyl substituents. The exemplary substituents can themselves be optionally substituted.

Unless otherwise indicated, any heteroatom with unsatisfied valences is assumed to have hydrogen atoms sufficient to satisfy the valences.

The term "heating" includes, but not limited to, warming by conventional heating (e.g., electric heating, steam heating, gas heating, etc.) as well as microwave heating.

The term "pharmaceutically-acceptable excipient, carrier, or diluent" as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject pharmaceutical agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier is "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.

The term "Neocitreamicin II" is used herein to mean the compound of formula II

The term "treating" with regard to a subject, refers to improving at least one symptom of the subject's disorder. Treating can be curing the disorder or condition, or improving it.

The term "disorder" is used herein to mean, and is used interchangeably with, the terms disease, condition, or illness, unless context clearly indicates otherwise.

The term "microbe" is used herein to mean an organism such as a bacterium, a virus, a protozoan, or a fungus, especially one that transmits disease.

The phrase "effective amount" as used herein means that amount of one or more agent, material, or composition comprising one or more agents of the present disclosure that is effective for producing some desired effect in an animal. It is recognized that when an agent is being used to achieve a therapeutic effect, the actual dose which comprises the "effective amount" will vary depending on a number of conditions including, but not limited to, the particular condition being treated, the severity of the disease, the size and health of the patient, the route of administration. A skilled medical practitioner can readily determine the appropriate dose using methods well known in the medical arts.

The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings, animals and plants without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

Throughout the specifications, groups and substituents thereof may be chosen to provide stable moieties and compounds.

Compounds

The present disclosure provides, in part, a compound of formula (I)

or an enantiomer, diastereomer, tautomer, or pharmaceutically acceptable salt or solvate thereof, wherein the symbols have the following meanings and are, for each occurrence, independently selected:

Ri is alkyl or substituted alkyl, alkenyl or substituted alkenyl, alkynyl or substituted alkynyl, cycloalkyl or substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted heterocycle, or aryl or substituted aryl; R₃ is hydrogen, halogen, cyano, nitro, Q=O)R₀, alkyl or substituted alkyl, cycloalkyl or substituted cycloalkyl, aryl or substituted aryl;

R₆ is hydrogen or C(=O)alkyl; and

R₀ is alkyl or substituted alkyl, cycloalkyl or substituted cycloalkyl, or aryl or substituted aryl.

In the compound of formula (I), R₁ may be alkyl or substituted alkyl. Ri may also be Cj-₆ alkyl, aryl, or substituted aryl.

In the compound of formula (I), R₃ may be hydrogen, aryl, or substituted aryl, or C(=O)R₀.

In the compound of formula (I), R₆ may be hydrogen or C(=O)alkyl.

The present disclosure also provides, in part, a compound of formula (I) having the structure of formula (Ia)

wherein Ri is alkyl or substituted alkyl, alkenyl or substituted alkenyl, alkynyl or substituted alkynyl, cycloalkyl or substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted heterocycle, or aryl or substituted aryl.

In the compound of formula (Ia), R₁ may be alkyl or substituted alkyl, C]-₆ alkyl, or aryl or substituted aryl. The present disclosure further provides, in part, an isolated compound of formula

(II)

or an enantiomer, diastereomer, tautomer, or pharmaceutically acceptable salt or solvate thereof. The compound of formula (II) may be substantially pure, for example, more than 90% pure, more than 95% pure, or more than 99% pure, and/or may be isolated from the aerobic growth of a strain of Nocardia G0655 (NRRL 50106).

The present disclosure also provides a compound of formula (III)

The present disclosure provides, in part, a compound isolated from the aerobic growth of a strain of Nocardia G0655 (NRRL 50106) characterized by: (a) a molecular weight of about 825.81 (Q-TOF LC-MS); (b) an ultraviolet absorption spectra substantially similar to that shown in FIG. 2; (c) a proton nuclear magnetic resonance substantially similar to that shown in FIG. 3; (d) a carbon- 13 nuclear magnetic resonance substantially similar to that shown in FIG. 4; (e) a COSY nuclear magnetic resonance substantially similar to that shown in FIG. 5; (f) a HSQC nuclear magnetic resonance substantially similar to that shown in FIG. 6; (g) a HMBC nuclear magnetic resonance substantially similar to that shown in FIG. 7;and/or Qx) a ROESY spectrum substantially similar to that shown in FIG. 8.

The compounds of the present disclosure may form salts which are also within the scope of this disclosure. Reference to a compound of the present disclosure herein is understood to include reference to salts thereof, unless otherwise indicated. The term "salt(s)", as employed herein, denotes acidic and/or basic salts formed with inorganic and/or organic acids and bases. In addition, when a compound of the present disclosure contains both a basic moiety, such as but not limited to a pyridine or imidazole, and an acidic moiety such as but not limited to a carboxylic acid, zwitterions ("inner salts") may be formed and are included within the term "salt(s)" as used herein. Pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts are useful, other salts are also useful, e.g., in isolation or purification steps which may be employed during preparation. Salts of the compounds of the present disclosure may be formed, for example, by reacting a compound I, II or III with an amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt precipitates or in an aqueous medium followed by lyophilization.

The compounds of the present disclosure which contain a basic moiety, such as but not limited to an amine or a pyridine or imidazole ring, may form salts with a variety of organic and inorganic acids. Exemplary acid addition salts include acetates (such as those formed with acetic acid or trihaloacetic acid, for example, trifluoroacetic acid), adipates, alginates, ascorbates, aspartates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, cyclopentanepropionates, digluconates, dodecylsulfates, ethanesulfonates, fumarates, glucoheptanoates, glycerophosphates, hemisulfates, heptanoates, hexanoates, hydrochlorides, hydrobromides, hydroiodides, hydroxy-ethanesulfonates (e.g., 2-hydroxy- ethanesulfonates), lactates, maleates, methanesulfonates, naphthalenesulfonates (e.g., 2- naphthalenesulfonates), nicotinates, nitrates, oxalates, pectinates, persulfates, phenylpropionates (e.g., 3-phenylpropionates), phosphates, picrates, pivalates, propionates, salicylates, succinates, sulfates (such as those formed with sulfuric acid), sulfonates, tartrates, thiocyanates, toluenesulfonates such as tosylates, undecanoates, and the like.

The compounds of the present disclosure which contain an acidic moiety, such but not limited to a carboxylic acid, may form salts with a variety of organic and inorganic bases. Exemplary basic salts include ammonium salts, alkali metal salts such as sodium, lithium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases (for example, organic amines) such as benzathines, dicyclohexylamines, hydrabamines (formed with N,N-bis(dehydroabietyl) ethylenediamine), N-methyl-D-glucamines, N-methyl-D-glycamides, t-butyl amines, and salts with amino acids such as arginine, lysine and the like. Basic nitrogen-containing groups may be quaternized with agents such as lower alkyl halides (e.g. methyl, ethyl, propyl, and butyl chlorides, bromides and iodides), dialkyl sulfates (e.g. dimethyl, diethyl, dibutyl, and diamyl sulfates), long chain halides (e.g. decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides), aralkyl halides (e.g. benzyl and phenethyl bromides), and others.

Prodrugs and solvates of the compounds of the disclosure are also contemplated herein. The term "prodrug" as employed herein denotes a compound that, upon administration to a subject, undergoes chemical conversion by metabolic or chemical processes to yield the compounds of the present disclosure. Solvates of the compounds of the present disclosure include, for example, hydrates.

Compounds of the present disclosure, and salts thereof, may exist in their tautomeric form (for example, as an amide or imino ether). All such tautomeric forms are contemplated herein as part of the present disclosure. All stereoisomers of the compounds of the present disclosure (for example, those which may exist due to asymmetric carbons on various substituents), including enantiomeric forms and diastereomeric forms, are contemplated within the scope of this disclosure. Individual stereoisomers of the compounds of the disclosure may, for example, be substantially free of other isomers {e.g., as a pure or substantially pure optical isomer having a specified activity), or may be admixed, for example, as racemates or with all other, or other selected, stereoisomers. The chiral centers of the present disclosure may have the S or R configuration as defined by the IUPAC 1974 Recommendations. The racemic forms can be resolved by physical methods, such as, for example, fractional crystallization, separation or crystallization of diastereomeric derivatives or separation by chiral column chromatography. The individual optical isomers can be obtained from the racemates by any suitable method, including without limitation, conventional methods, such as, for example, salt formation with an optically active acid followed by crystallization.

Compounds of the present disclosure, subsequent to their preparation, may be isolated and purified to obtain a composition containing an amount by weight equal to or greater than 99% ("substantially pure" compound), which is then used or formulated as described herein.

All configurational isomers of the compounds of the present disclosure are contemplated, either in admixture or in pure or substantially pure form. The definition of compounds of the present disclosure embraces both cis (Z) and trans (E) alkene isomers, as well as cis and trans isomers of cyclic hydrocarbon or heterocyclic rings.

Throughout the specifications, groups and substituents thereof may be chosen to provide stable moieties and compounds. Methods of Preparation

The compound of formula (II) as shown below,

can be prepared through aerobically growing a strain of Nocardia G0655 under conditions conducive to provide the compound of formula (II). The growing of the strain of Nocardia G0655 is conducted in a liquid media consisting of assimilable sources of carbon, nitrogen, and inorganic salts. The substantially pure form of the compound of formula (II) can be obtained by further isolating and purifying the compound of formula (II).

Neocitreamicin II is produced by the Nocardia G0655 isolate that is deposited with the Agricultural Research Service (ARS) Culture Collection, U.S. Department of Agriculture, 1815 North University Street, Peoria, Illinois 61604, U.S.A. as NRRL 50106 under the provisions of the Budapest Treaty.

The G0655 isolate was obtained from a sandy soil sample collected in Falmouth using the technology for isolating "unculturable" microorganisms described in U.S. Patent No. 7,011,957. This technology makes use of a growth chamber that is sealed with a semi-permeable membrane, and thus is permeable to diffusion of components from the environment but not to cells of microorganisms. The growth chamber is designed to allow for the growth, isolation into pure culture, and characterization of microorganisms that are "uncultivable" at the present time. This desired result can be achieved because the conditions inside the chamber closely resemble, if they are not identical to, the natural environment of the microorganisms. One version of such a chamber is formed from a solid substrate, e.g., a glass or silicon slide or stainless steel washer, having an orifice which is sandwiched by two robust membranes, e.g., polycarbonate or other inert material, glued onto the substrate. The membranes have pore sizes, e.g., 0.025 μm - 0.03 μm, that are sufficiently small to retain all microorganisms inside the chamber but which are sufficiently large to permit components from the environment to diffuse into the chamber and waste products to diffuse out of the chamber. After one membrane is sealed onto the bottom of the substrate, the chamber is partially filled with a suspension of cells in an appropriate growth medium.

The specific procedures used to isolate the G0655 isolate are described in detail in Example 1. Of course, any other method known to one of skill in the art may also be used to isolate the microorganism from an environment of interest.

The isolation of Neocitreamicin II from the fermentation broth can be achieved by any means known in the art, such as the methods described in Example 1. Neocitreamicin II can be purified by any method known in the art including, but not limited to, normal phase chromatography, reverse phase chromatography, countercurrent chromatography, ion exchange chromatography, supercritical fluid chromatography or size exclusion chromatography.

The isolated Neocitreamicin II can then be purified by any method known in the art including, but not limited to, high performance liquid chromatography, normal phase chromatography, reverse phase chromatrgraphy, countercurrent chromatography, ion exchange chromatography, supercritical fluid chromatography or size exclusion chromatography.

The isolated Neocitreamicin II can be used as is or modified chemically. The compound of formula (III) as shown below,

can be prepared from the compound of formula (II) via hydrolysis. The hydrolysis can be achieved by, for example, contacting the compound of formula (II) with a base, such as NaOH, KOH, LiOH, K₂CO₃, Na₂CO₃, Cs₂CO₃, KHCO₃, and NaHCO₃.

The compound of formula (I) having the structure of formula (Ia)

wherein Ri is alkyl or substituted alkyl, alkenyl or substituted alkenyl, alkynyl or substituted alkynyl, cycloalkyl or substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted heterocycle, or aryl or substituted aryl, can be prepared by reacting the compound of formula (III) with an acid of formula Rj COOH, or an acid chloride of formula RjCOCl, to provide the compound of (Ia). The compound of formula (I)

Ri is alkyl or substituted alkyl, alkenyl or substituted alkenyl, alkynyl or substituted alkynyl, cycloalkyl or substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted heterocycle, or aryl or substituted aryl; R₃ is hydrogen, halogen, cyano, nitro,

alkyl or substituted alkyl, cycloalkyl or substituted cycloalkyl, aryl or substituted aryl; R₆ is hydrogen or C(=O)alkyl; and R₀ is alkyl or substituted alkyl, cycloalkyl or substituted cycloalkyl, or aryl or substituted aryl, can be prepared from the compound of formula (III) according to Scheme 1 (below). Compound (III-l) can be obtained from Compound III via hydrolysis. The hydrolysis can be achieved by, for example, contacting the compound of formula (III) with an acid such as TFA. Protection of the phenol group of Compound (III-l) affords Compound (III-2), wherein P can be an ester or ether. See, e.g., Greene et al., Protective Groups in Organic Synthesis, (3^rd Ed.), pp. 246-292: "Protection For Phenols and Catechols." Compound (III-2) can further react with an acid of formula Rj COOH, or an acid chloride of formula R₁COCl, to provide Compound (III-3a), wherein Y is OP (e.g., an ester or ether). Compound (III-3b) can be prepared from Compound (III-3a) by removing the protecting group P. Compound (III-3a) can undergo Friedel-Crafts acylation to afford Compound (III- 4a), wherein R₃ is C(=O)R_c in which R₀ is alkyl, cycloalkyl or aryl. Compound (III-3a) can also undergo bromination or nitration to provide Compound (III-4b) wherein R₃ is Br and Compound (III-4c) wherein R₃ is nitro. The bromide in Compound (III-4b) (when R₃ is Br) may be converted to alkyl, cycloalkyl, or aryl under Suzuki coupling conditions (palladium-catalyzed cross coupling between appropriate organoboronic acid and halides). See, e.g., Kirchhoff, et al, J. Am. Chem. Soc. (2002), 124:13662-13663; Cui, et al, Synthesis (2007) 393-399; Li, et al, J. Org. Chem. (2007) 72:4067-4072; Liu, et al, Synthesis (2006) 860-864; Kingston, et al, J. Org. Chem. (2007) 72:2816-2822; Marion, et al, J. Am. Chem. Soc. (2006) 128:4101-4111 ; Billingsley, et al, Chem. Int. Ed (2006) 45:3484-3488. In addition, the bromide in Compound (III-4b) (when R₃ is Br and OP is ester) may be converted to cyano through a nucleophilic substitution.

Compound (III-5) can be prepared from Compound (III-4a, III-4b or III-4c) by removing the protecting group P. Compound (1-1) wherein R₆ is H can be prepared from Compound (III-5) and 6-methyltetrahydro-2H-pyran-2,4,5-triol under Mitsunobu reaction conditions. See, e.g., Lepore, et al, J. Org. Chem. (2003) 68:8261-8263; Dandapani, et al, Tetrahedron (2002) 58:3855-3864; But, et al, J. Am. Chem. Soc. (2006) 128:9636- 9637; Keith, et al, J. Org. Chem. (2006) 71 :7113-7116. Alternatively, the phenol group in Compound (III-5) can be replaced with a leaving group X to provide Compound (III-6). X can be, for example, -O-SO₂-CH3, -0-SO₂-CF₃, or a moiety of the structure:

in which Z is -NO₂, halogen, -CH₃ or -CF₃. Replacement of X with 6- methyltetrahydro-2H-pyran-2,4,5-triol can afford Compound (1-1). Compound (1-2) can be prepared from Compound (1-1) by selectively acylating the OH group.

lll-3a, Y = OP, P is a protecting groi HWb₁Y = OH.

Alternatively, Scheme 2 describes that Compound (III- 5 a) can be prepared from Compound (III-3b) through a Friedel-Crafts rearrangement via intermediate (III-4a), wherein Rc can be alkyl, cycloalkyl, or aryl.

Scheme 2

Methods of Treatment

In some aspects, the disclosure relates to methods of inhibiting the growth of a pathogen. The method involves contacting the pathogen with an effective amount of one or more neocitreamicin compounds of the disclosure thereby inhibiting the growth of the pathogen compared with the growth of the pathogen in the absence of treatment with a compound of the disclosure. In certain embodiments, the method reduces the growth of the pathogen compared with the growth of the pathogen in the absence of treatment with a compound of the disclosure. In other instances, the treatment results in the killing of the pathogen. Non-limiting examples of a pathogen include, but are not limited to, a bacterium, a fungus, a virus, a protozoan, a helminth, a parasite, and combinations thereof. These methods may be practiced in vivo, ex vivo, or in vitro.

The anti-bacterial activity of the neocitreamicin compounds of the disclosure with respect to a specific bacterium can be assessed by in vitro assays such as monitoring the zone of inhibition and the minimal inhibitory concentration (MIC) assays described in detail in Example 3.

The anti-fungal activity of the neocitreamicin compounds of the disclosure can be determined, for example, by following the viability of the desired fungal pathogens (such as Candida albicans, and Aspergillus species) for example as described in Sanati et al, A new triazole, voriconazole (UK-109,496), blocks sterol biosynthesis in Candida albicans and Candida krusei, Antimicrob. Agents Chemother., 1997 Nov.; 41(11): 2492-2496. Anti-viral properties of the neocitreamicin compounds of the disclosure can be determined, for example, by monitoring the inhibition of influenzae neuraminidase or by assaying viral viability as described in Tisdale (2000), Rev. Med. Virol, 10(l):45-55. Anti-protozoan activity of the neocitreamicin compounds of the disclosure can be determined by following the viability of protozoan parasites such as Trichomonas vaginalis and Giardia lamblia as described in Katiyar et al., (1994) Antimicrob. Agents Chemother., 38(9): 2086-2090. Anthelminthic activity of the neocitreamicin compounds of the disclosure can be determined, for example, by following the effect of the compounds on the viability of nematodes such as Schistosoma mansoni, Schistosoma cercariae and Caenorhabditis elegans as described in Mølgaard P. et al. (1994), J. Ethnopharmacol, 42(2): 125-32.

In other aspects, the disclosure is directed to methods of treating a disorder in a subject in need thereof, comprising administering to the subject an effective amount of one or more neocitreamicin compounds described herein. In certain embodiments, the disorder is caused by a pathogen such as, but not limited to, a bacterium, a fungus, a virus, a protozoan, a helminth, a parasite, or a combination thereof.

In some embodiments, the disorder is caused by a bacterium. The neocitreamicin compounds described herein can be useful against both Gram-positive and Gram-negative bacteria. Non-limiting examples of Gram-positive bacteria include Streptococcus, Staphylococcus, Enterococcus, Corynebacteria, Listeria, Bacillus, Erysipelothrix, and Actinomycetes. In some embodiments, the methods of the disclosure are used to treat an infection by one or more of: Helicobacter pylori, Legionella pneumophilia, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansaii, Mycobacterium gordonae, Mycobacteria sporozoites,

Staphylococcus aureus, Staphylococcus epidermidis, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae pyogenes (Group B Streptococcus), Streptococcus dysgalactia, Streptococcus faecalis, Streptococcus bovis, Streptococcus pneumoniae, pathogenic Campylobacter sporozoites, Enterococcus sporozoites, Haemophilus influenzae, Pseudomonas aeruginosa, Bacillus anthracis, Bacillus subtilis, Escherichia coli, Corynebacterium diphtheriae, Corynebacterium jeikeium, Corynebacterium sporozoites, Erysipelothrix rhusiopathiae, Clostridium perfringens, Clostridium tetani, Clostridium difficile, Enter obacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides thetaiotamicron, Bacteroides uniformis, Bacteroides vulgatus, Fusobacterium nucleatum, Streptobacillus moniliformis, Leptospira, and Actinomyces israelii, hi specific embodiments, the compounds described herein are useful in treating an infection by Methicillin Resistant Staphylococcus aureus (MRSA) or by Vancomycin Resistant Entercocci (VRE). MRSA contributes to approximately 19,000 deaths annually in the United States and although most of these deaths are due to hospital-acquired MRSA (HA- MRSA), it is the community-acquired MRSA (CA-MRSA) that is actually more virulent, and known to kill previously healthy individuals. The virulence of the CA-MRSA is in part due to the expression of phenol soluble modulins or PSM peptides. Accordingly, in treating CA-MRSA, a neocitreamicin compound of the disclosure can be used in combination with an agent that modulates the expression and/or activity of virulence factors, such as, but not limited to, PSM peptides. The neocitreamicin compounds of the disclosure may be used to treat spirochetes such as Borelia burgdorferi, Treponema pallidium, and Treponema per tenue.

The neocitreamicin compounds described herein may also be useful in treating viral disorders. Non-limiting examples of infectious viruses that may be treated by the methods of the disclosure include: Retroviridae {e.g., human immunodeficiency viruses, such as HIV-I (also referred to as HTLV-III, LAV or HTLV-III/LAV), or HIV-III; and other isolates, such as HIV-LP; Picornaviridae {e.g., polio viruses, hepatitis A virus; enteroviruses, human coxsackie viruses, rhinoviruses, echoviruses); Calciviridae {e.g., strains that cause gastroenteritis); Togaviridae {e.g., equine encephalitis viruses, rubella viruses); Flaviridae {e.g., dengue viruses, encephalitis viruses, yellow fever viruses); Coronaviridae {e.g., coronaviruses, severe acute respiratory syndrome (SARS) virus); Rhabdoviridae {e.g., vesicular stomatitis viruses, rabies viruses); Filoviridae {e.g., ebola viruses); Paramyxoviridae {e.g., parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae {e.g., influenza viruses); Bungaviridae {e.g., Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arenaviridae (hemorrhagic fever viruses); Reoviridae {e.g., reoviruses, orbiviurses and rotaviruses); Birnaviridae; Hepadnaviridae {e.g, Hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae {e.g., herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes viruses); Poxviridae {e.g., variola viruses, vaccinia viruses, pox viruses); and Iridoviridae {e.g., African swine fever virus); and unclassified viruses {e.g., the etiological agents of Spongiform encephalopathies, the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), the agents of non-A, non- B hepatitis (class l=internally transmitted; class 2=parenterally transmitted {i.e., Hepatitis C); Norwalk and related viruses, and astro viruses). For example, the compounds of the disclosure can be used to treat a influenza virus, human immunodeficiency virus, and herpes simplex virus. In some embodiments, the neocitreamicin compounds of the disclosure may be useful to treat disorders caused by fungi. Non-limiting examples of fungi that may be inhibited by the compounds of the disclosure include, but are not limited to, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans, Candida tropicalis, Candida glabrata, Candida krusei, Candida parapsilosis, Candida dubliniensis, Candida lusitaniae, Epidermophyton floccosum, Microsporum audouinii, Microsporum canis, Microsporum canis var. distortum Microsporum cookei, Microsporum equinum, Microsporum ferrugineum, Microsporum fulvum, Microsporum gallinae, Microsporum gypseum, Microsporum nanum, Microsporum per sicolor, Trichophyton ajelloi, Trichophyton concentricum, Trichophyton equinum, Trichophyton flavescens, Trichophyton gloriae, Trichophyton megnini, Trichophyton mentagrophytes var. erinacei, Trichophyton mentagrophytes var. inter digitale, Trichophyton phaseoliforme, Trichophyton rubrum, Trichophyton rubrum downy strain, Trichophyton rubrum granular strain, Trichophyton schoenleinii, Trichophyton simii, Trichophyton soudanense, Trichophyton terrestre, Trichophyton tonsurans, Trichophyton vanbreuseghemii, Trichophyton verrucosum, Trichophyton violaceum, Trichophyton yaoundei, Aspergillus fumigatus, Aspergillus flavus, and Aspergillus clavatus.

In yet other embodiments, the neocitreamicin compounds described herein may be useful in treating disorders caused by protozoans. Non-limiting examples of protozoa that can be inhibited by the neocitreamicin compounds of the disclosure include, but are not limited to, Trichomonas vaginalis, Giardia lamblia, Entamoeba histolytica, Balantidium coli, Cryptosporidium parvum and Isospora belli, Trypansoma cruzi, Trypanosoma gambiense, Leishmania donovani, and Naegleria fowler i.

The neocitreamicin compounds described herein may also be useful in treating disorders caused by helminths. Non-limiting examples of helminths that can be inhibited by the compounds of the disclosure include: Schistosoma mansoni, Schistosoma cercariae, , Schistosoma japonicum, , Schistosoma mekongi, Schistosoma hematobium, Ascaris lumbricoides, Strongyloides stercoralis, Echinococcus granulosus, Echinococcus miiltilocularis, Angiostrongylus cantonensis, Angiostrongylus constaricensis, Fasciolopis buski, Capillaria philippinensis, Paragonimus westermani, Ancylostoma dudodenale, Necator americanus,. Tήchinella spiralis, Wuchereria bancrofti, Brugia malayi, and Brugia timori, Toxocara canis, Toxocara cati, Toxocara vitulorum, Caenorhabiditis elegans, and Anisakis species.

The neocitreamicin compounds described herein may be useful in treating disorders caused by parasites. Non-limiting examples of parasites that can be inhibited by the neocitreamicin compounds of the disclosure include Plasmodium falciparum, Plasmodium yoelli, Hymenolepis nana, Clonorchis sinensis, Loa loa, Paragonimus westermani, Fasciola hepatica, and Toxoplasma gondii. For example, the parasite may be a malarial parasite.

The neocitreamicin compounds of the disclosure are also envisioned for use in treating other disorders such as, but not limited to: cardiovascular disease, endocarditis, atherosclerosis, stroke, infections of the skin including burn wounds and skin infections in diabetics (e.g., diabetic foot ulcers), ear infections, upper respiratory tract infections, ulcers, nosocomial pneumonia, community-acquired pneumonia, sexually transmitted diseases, urinary tract infections, septicemia, toxic shock syndrome, tetanus, infections of the bones and joints, Lyme disease, treatment of subjects exposed to anthrax spores, hypercholesterolemia, inflammatory disorders, aging-related diseases, channelopathies, autoimmune diseases, graft-versus-host diseases and cancer.

For example, the neocitreamicin compounds of the disclosure can be used to treat an inflammatory disease. Examples of inflammatory diseases include, but are not limited to: arthritis, osteoarthritis, rheumatoid arthritis, asthma, inflammatory bowel disease, inflammatory skin disorders, multiple sclerosis, osteoporosis, tendonitis, allergic disorders, inflammation in response to an insult to the host, sepsis, and systematic lupus erythematosus. Anti-inflammatory activity of the compounds of the disclosure can be assessed, for example, by measuring the ligand binding ability of the compounds to the formylpeptide receptor (FPR) family of G protein-coupled receptors (see, Young S. M.et al. (2005), J. Biomol. Screen. 10(4):374-82) or by measuring the effect of such compounds on the secretion of pro-inflammatory cytokines in THP-I cells after lipopolysaccharide stimulation (Singh et al. (2005), Clin. Chem., 51(12):2252-6.). The neocitreamicin compounds of the disclosure inhibit metalloenzymes such as collagenases that destroy connective tissue and joint cartilage causing inflamed joints. For example, the neocitreamicin compounds of the disclosure can be used to treat rheumatoid arthritis. The neocitreamicin compounds may be administered in combination (either prior to, at the same time as, or after) with minocycline.

The neocitreamicin compounds of the disclosure may also be used to treat a channelopathy. Channelopathies are diseases caused by disturbed function of ion channel subunits or the proteins that regulate them. Non-limiting examples of channelopathies includeAlternating hemiplegia of childhood, Bartter syndrome, Brugada syndrome, Congenital hyperinsulinism, Cystic fibrosis, Episodic Ataxia, Erythromelalgia, Generalized epilepsy with febrile seizures plus, Hyperkalemic periodic paralysis, Hypokalemic periodic paralysis, Long QT syndrome, Malignant hyperthermia, Migraine, Myasthenia Gravis, Myotonia congenita, Neuromyotonia, Nonsyndromic deafness, Paramyotonia congenita, Periodic paralysis, Retinitis pigmentosa, Romano-Ward syndrome, Short QT syndrome, and Timothy syndrome. The effect of the compounds of the disclosure on channelopathies can be assayed, for example, via in vitro assays that utilize the desired ion channel, e.g. , cystic fibrosis (CF) transmembrane conductance regulator (see, Fulmer S.B. et al. (1995), Proc. Natl. Acad. ScL USA., 92(15):6832-6).

The neocitreamicin compounds of the disclosure are also useful for treating an aging-related disease. Non-limiting examples of aging-related diseases include Alzheimer's disease, and Parkinson's disease. The ability of the compounds of the disclosure to treat aging-related diseases can be tested, for example, by assays that monitor the compounds' activity on sirtuins, the NAD(+)-dependent histone/protein deacetylases (see, Borra (2004), Biochem. 43(30):9877-87). In addition, the neocitreamicin compounds of the disclosure can be used to treat an autoimmune disease. Non-limiting examples of autoimmune diseases include Acute disseminated encephalomyelitis, Addison's disease, Ankylosing spondylitis, Antiphospholipid antibody syndrome, aplastic anemia, Autoimmune hepatitis, Autoimmune Oophoritis, Celiac disease, Crohn's disease, Diabetes mellitus type 1, Gestational pemphigoid, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, Idiopathic thrombocytopenic purpura, Kawasaki's Disease, Lupus erythematosus, Multiple sclerosis, Myasthenia gravis, Opsoclonus myoclonus syndrome (OMS), Optic neuritis, Ord's thyroiditis, Pemphigus, Pernicious anaemia, Primary biliary cirrhosis, Rheumatoid arthritis, Reiter's syndrome, Sjogren's syndrome, Takayasu's arteritis, Temporal arteritis, Warm autoimmune hemolytic anemia, and Wegener's granulomatosis. The immunosuppressive properties of the neocitreamicin compounds of the disclosure can be measured, for example, by utilizing the mixed lymphocyte reaction assay (see, Itoh et al. (1993), Antϊbiot. (Tokyo) 46(10):1575-81).

Additionally, the neocitreamicin compounds of the disclosure can be used to treat a cancer. For example, the neocitreamicin compounds are used to inhibit the growth of a cancer or tumor cell, or to kill the cancer or tumor cell. Examples of cancers include, but are not limited to, breast cancer, ovarian cancer, colon cancer, prostate cancer, liver cancer, lung cancer, gastric cancer, esophageal cancer, urinary bladder cancer, melanoma, leukemia, and lymphoma. The compounds of the disclosure may be administered with a chemotherapeutic agent. Non-limiting examples of chemotherapeutic agents include antimetabolites, purine or pyrrolidine analogs, alkylating agents, crosslinking agents, and intercalating agent. The chemotherapeutic agent can be administered before, after, or substantially simultaneously with a compound of the disclosure. Anti-cancer activity of the compounds of the disclosure can be determined using, for example, cytotoxicity assays comparing the cytotoxicity of the compound of interest against cancer cells and normal (non-cancerous) mammalian cells (see, Roomi et al. (2006), Med. Oncol. 23(1): 105-11) or by measuring angiogenic properties (see, Ivanov o al. (2005), Oncol. Rep. 14(6): 1399- 404). The neocitreamicin compounds of the disclosure may be administered to treat hypercholesterolemia. For example, the neocitreamicin compounds of the disclosure can be administered to a subject to reduce the levels of low density lipoprotein (LDL) compared with the levels of LDL prior to administration of the compound to the subject. The compounds of the disclosure are administered to a subject to increase the levels of high density lipoprotein (HDL) compared with the levels of HDL prior to administration of the compound to the subject. Cholesterol lowering activities of the compounds of the disclosure can be assayed, for example, by determining the ability of the compound of interest to inhibit 3-hydroxy-3methylglutaryl-coenzyme A reductase (HMGCR), and/or on other enzymes involved in the mevalonate pathway downstream of HMGCR (see, Gerber et al. (2004), Anal. Biochem., 329(l):28-34). Neocitreamicin compounds of the disclosure can also be assessed for their potential to increase high density lipoprotein ("good" cholesterol) by measuring their ability to up-regulate scavenger receptor class B type I (SR-BI), the high-affinity high-density lipoprotein (HDL) receptor (see, Yang et al. (2007), Biomol. Screen., 12(2):211 -9).

The neocitreamicin compounds of the disclosure may be used to treat a cardiovascular disease, and/or Chlamydia pneumoniae infection that results in complications of atherosclerosis, cardiovascular disease, and stroke. They may also be used to treat endocarditis.

The neocitreamicin compounds of the disclosure may also be used as adjunct therapy for the treatment of the disorders described above, or to inhibit the growth of an infective agent compared with the growth of the infective agent in the absence of being treated by a compound of the disclosure. Non-limiting examples of infective agents include bacteria, fungi, viruses, protozoa, helminths, parasites, and combinations thereof. The neocitreamicin compounds may be used to inhibit the agent in vivo or in vitro. Formulation

The disclosure also provides a pharmaceutical composition comprising at least one of the neocitreamicin compounds of the disclosure (or an enantiomer, diastereomer, tautomer, or pharmaceutically-acceptable salt or solvate thereof), and a pharmaceutically- acceptable carrier. These neocitreamicin compositions are suitable for administration to a subject (e.g., a mammal such as a human). The pharmaceutical composition can be used for treating a disorder. Non-limiting examples of disorders are provided above.

The neocitreamicin compounds can be administered in a pharmaceutically- acceptable carrier. Any suitable carrier known in the art may be used. Carriers that efficiently solubilize the agents are preferred. Carriers include, but are not limited to, a solid, liquid, or a mixture of a solid and a liquid. The carriers may take the form of capsules, tablets, pills, powders, lozenges, suspensions, emulsions, or syrups. The carriers may include substances that act as flavoring agents, lubricants, solubilizers, suspending agents, binders, stabilizers, tablet disintegrating agents, and encapsulating materials. The phrase "pharmaceutically-acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

Non-limiting examples of materials which can serve as pharmaceutically- acceptable carriers include: sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution, ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations.

The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single-dosage form will vary depending upon the subject being treated, the particular mode of administration, the particular condition being treated, among others. The amount of active ingredient that can be combined with a carrier material to produce a single-dosage form will generally be that amount of the compound that produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, from about 5 percent to about 70 percent or from about 10 percent to about 30 percent.

Methods of preparing these formulations or compositions include the step of bringing into association a compound of the disclosure with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a neocitreamicin compound of the present disclosure with liquid carriers, or timely divided solid carriers, or both, and then, if desired, shaping the product.

In solid dosage forms of the disclosure for oral administration (e.g., capsules, tablets, pills, dragees, powders, granules, and the like), the active ingredient is mixed with one or more additional ingredients, such as sodium citrate or dicalcium phosphate, and/or any of the following: fillers or extenders, such as, but not limited to, starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; binders, such as, but not limited to, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; humectants, such as, but not limited to, glycerol; disintegrating agents, such as, but not limited to, agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; solution retarding agents, such as, but not limited to, paraffin; absorption accelerators, such as, but not limited to, quaternary ammonium compounds; wetting agents, such as, but not limited to, cetyl alcohol and glycerol monostearate; absorbents, such as, but not limited to, kaolin and bentonite clay; lubricants, such as, but not limited to, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and coloring agents. In the case of capsules, tablets, and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols, and the like.

In powders, the carrier is a finely-divided solid, which is mixed with an effective amount of a finely-divided agent. Powders and sprays can contain, in addition to a compound of this disclosure, excipients, such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

Tablets for systemic oral administration may include one or more excipients as known in the art, such as, for example, calcium carbonate, sodium carbonate, sugars {e.g., lactose, sucrose, mannitol, sorbitol), celluloses {e.g., methyl cellulose, sodium carboxymethyl cellulose), gums {e.g., arabic, tragacanth), together with one or more disintegrating agents {e.g., maize, starch, or alginic acid, binding agents, such as, for example, gelatin, collagen, or acacia), lubricating agents {e.g., magnesium stearate, stearic acid, or talc), inert diluents, preservatives, disintegrants {e.g., sodium starch glycolate), surface-active and/or dispersing agent. A tablet may be made by compression or molding, optionally with one or more accessory ingredients.

In solutions, suspensions, emulsions or syrups, an effective amount of the neocitreamicin compound is dissolved or suspended in a carrier, such as sterile water or an organic solvent, such as aqueous propylene glycol. Other compositions can be made by dispersing the agent in an aqueous starch or sodium carboxymethyl cellulose solution or a suitable oil known to the art. The liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as, but not limited to, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols, and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming, and preservative agents.

Suspensions, in addition to the active compound, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth, and mixtures thereof.

Formulations of the pharmaceutical compositions for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compounds of the disclosure with one or more suitable non-irritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature but liquid at body temperature and, thus, will melt in the rectum or vaginal cavity and release the agents. Formulations suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams, or spray formulations containing such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration of a compound of this disclosure include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active neocitreamicin compound may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any preservatives, buffers, or propellants that may be desired. Ointments, pastes, creams, and gels may contain, in addition to an active compound, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Transdermal patches have the added advantage of providing controlled delivery of a compound of the present disclosure to the body. Such dosage forms can be made by dissolving or dispersing the agents in the proper medium. Absorption enhancers can also be used to increase the flux of the agents across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the neocitreamicin compound in a polymer matrix or gel.

The neocitreamicin compounds are administered in a therapeutic amount to a patient in need of such treatment. Such an amount is effective in treating a disorder of the patient. This amount may vary, depending on the activity of the agent utilized, the nature of the disorder, and the health of the patient. The term "therapeutically-effective amount" is used to denote treatments at dosages effective to achieve the therapeutic result sought. Furthermore, a skilled practitioner will appreciate that the therapeutically-effective amount of the neocitreamicin compound may be lowered or increased by fine-tuning and/or by administering more than one neocitreamicin compound, or by administering a neocitreamicin compound together with a second agent (e.g., antibiotics, antifungals, antivirals, NSAIDS, DMARDS, steroids, etc.). Therapeutically-effective amounts may be easily determined, for example, empirically by starting at relatively low amounts and by step-wise increments with concurrent evaluation of beneficial effect (e.g., reduction in symptoms). The actual effective amount will be established by dose/response assays using methods standard in the art (see, e.g., Johnson et al. (1993), Diabetes. 42:1179). As is known to those in the art, the effective amount will depend on bioavailability, bioactivity, and biodegradability of the neocitreamicin compound.

A therapeutically-effective amount is an amount that is capable of reducing the symptoms of the disorder in a subject. Accordingly, the amount will vary with the subject being treated. Administration of the neocitreamicin compound may be hourly, daily, weekly, monthly, yearly, or a single event. For example, the effective amount of the neocitreamicin compound may comprise from about 1 μg/kg body weight to about 100 mg/kg body weight. In one embodiment, the effective amount of the compound comprises from about 1 μg/kg body weight to about 50 mg/kg body weight. In a further embodiment, the effective amount of the compound comprises from about 10 μg/kg body weight to about 10 mg/kg body weight. When one or more neocitreamicin compounds or agents are combined with a carrier, they may be present in an amount of about 1 weight percent to about 99 weight percent, the remainder being composed of the pharmaceutically-acceptable carrier.

The disclosure also provides for kits that comprise at least one neocitreamicin compound of the disclosure. The kits may contain at least one container and may also include instructions directing the use of these materials. In another embodiment, a kit may include an agent used to treat the disorder in question with or without such above- mentioned materials that may be present to determine if a subject has an inflammatory disease.

Administration of the Formulation

Methods of administration of the formulations of the disclosure comprising the neocitreamicin compounds of the disclosure described herein can be by any of a number of methods well known in the art. These methods include local or systemic administration. Exemplary routes of administration include oral, parenteral, transdermal, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal (e.g., nebulizer, inhaler, aerosol dispenser), colorectal, rectal, intravaginal, and any combinations thereof. In addition, it may be desirable to introduce the pharmaceutical compositions of the disclosure into the central nervous system by any suitable route, including intraventricular and intrathecal injection. Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Methods of introduction may also be provided by rechargeable or biodegradable devices, e.g., depots. Furthermore, it is contemplated that administration may occur by coating a device, implant, stent, or prosthetic. The compounds of the disclosure can also be used to coat catheters in any situation where catheters are inserted in the body.

The subject neocitreamicin compounds can be administered as part of a combinatorial therapy with other agents. Combination therapy refers to any form of administration combining two or more different therapeutic compounds such that the second compound is administered while the previously administered therapeutic compound is still effective in the body (e.g., the two compounds are simultaneously effective in the patient, which may include synergistic effects of the two compounds). For example, the different therapeutic compounds can be administered either in the same formulation or in a separate formulation, either simultaneously or sequentially. Thus, an individual who receives such treatment can have a combined (conjoint) effect of different therapeutic compounds.

For example, neocitreamicin compounds of the disclosure may be used in combination with other known antibiotics. The neocitreamicin compounds of the disclosure may either be administered sequentially or substantially at the same time. Varying the antibiotic can be helpful in reducing the ability of the pathogen to develop resistance to the drug. Non-limiting examples of antibiotics include penicillins (e.g., natural penicillins, penicillinase-resistant penicillins, antipseudomonal penicillins, aminopenicillins), tetracyclines, macrolides (e.g., erythromycin), lincosamides (e.g., clindamycin), streptogramins (e.g., Synercid), aminoglycosides, and sulfonamides. In some embodiments, the neocitreamicin compounds of the disclosure are used in combination with compounds that target virulence factors such as, but not limited to, phenol-soluble modulins. In some embodiments, the neocitreamicin compounds of the disclosure are used in combination with compounds that target the efflux pumps of the pathogens. In the case of inflammatory conditions, the subject neocitreamicin compounds can be administered in combination with one or more other agents useful in the treatment of inflammatory diseases or conditions. Agents useful in the treatment of inflammatory diseases or conditions include, but are not limited to, anti-inflammatory agents, or antiphlogistics. Antiphlogistics include, for example, glucocorticoids, such as cortisone, hydrocortisone, prednisone, prednisolone, fluorcortolone, triamcinolone, methylprednisolone, prednylidene, paramethasone, dexamethasone, betamethasone, beclomethasone, fluprednylidene, desoxymethasone, fluocinolone, flunethasone, diflucortolone, clocortolone, clobetasol and fluocortin butyl ester; immunosuppressive agents such as anti-TNF agents (e.g., etanercept, infliximab) and IL-I inhibitors; penicillamine; non-steroidal anti-inflammatory drugs (NSAIDs) which encompass antiinflammatory, analgesic, and antipyretic drugs such as salicyclic acid, celecoxib, difunisal and from substituted phenylacetic acid salts or 2-phenylpropionic acid salts, such as alclofenac, ibutenac, ibuprofen, clindanac, fenclorac, ketoprofen, fenoprofen, indoprofen, fenclofenac, diclofenac, flurbiprofen, piprofen, naproxen, benoxaprofen, carprofen and cicloprofen; oxican derivatives, such as piroxican; anthranilic acid derivatives, such as mefenamic acid, flufenamic acid, tolfenamic acid and meclofenamic acid, anilino- substituted nicotinic acid derivatives, such as the fenamates miflumic acid, clonixin and flunixin; heteroarylacetic acids wherein heteroaryl is a 2-indol-3-yl or pyrrol-2-yl group, such as indomethacin, oxmetacin, intrazol, acemetazin, cinmetacin, zomepirac, tolmetin, colpirac and tiaprofenic acid; idenylacetic acid of the sulindac type; analgesically active heteroaryloxyacetic acids, such as benzadac; phenylbutazone; etodolac; nabunetone; and disease modifying antirheumatic drugs (DMARDs) such as methotrexate, gold salts, hydroxychloroquine, sulfasalazine, ciclosporin, azathioprine, and leflunomide. Other therapeutics useful in the treatment of inflammatory diseases or conditions include antioxidants. Antioxidants may be natural or synthetic. Antioxidants are, for example, superoxide dismutase (SOD), 21-aminosteroids/aminochromans, vitamin C or E, etc. Many other antioxidants are well known to those of skill in the art. The subject compounds may serve as part of a treatment regimen for an inflammatory condition, which may combine many different anti-inflammatory agents. For example, the neocitreamicin compounds may be administered in combination with one or more of an NSAID, DMARD, or immunosuppressant. In one embodiment of the application, the subject compounds may be administered in combination with methotrexate. In another embodiment, the subject antibodies may be administered in combination with a TNF-α inhibitor.

In the case of cardiovascular disease conditions, and particularly those arising from atherosclerotic plaques, which are thought to have a substantial inflammatory component, the subject compounds can be administered in combination with one or more other agents useful in the treatment of cardiovascular diseases. Agents useful in the treatment of cardiovascular diseases include, but are not limited to, β-blockers such as carvedilol, metoprolol, bucindolol, bisoprolol, atenolol, propranolol, nadolol, timolol, pindolol, and labetalol; antiplatelet agents such as aspirin and ticlopidine; inhibitors of angiotensin- converting enzyme (ACE) such as captopril, enalapril, lisinopril, benazopril, fosinopril, quinapril, ramipril, spirapril, and moexipril; and lipid-lowering agents such as mevastatin, lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, and rosuvastatin.

In the case of cancer, the subject neocitreamicin compounds can be administered in combination with one or more anti-angiogenic factors, chemotherapeutics, or as an adjuvant to radiotherapy. It is further envisioned that the administration of the subject compounds will serve as part of a cancer treatment regimen, which may combine many different cancer therapeutic agents.

Reference will now be made to specific examples illustrating the invention. It is to be understood that the examples are provided to illustrate selected embodiments and that no limitation to the scope of the disclosure is intended thereby. EXAMPLES

EXAMPLE 1

Isolation of Neocitreamicin II An aliquot of soil collected from a sandy soil sample collected in Falmouth, MA, was the starting point for the isolation of the bacterial strain G0655 that produces Neocitreamicin II.

The sandy soil sample was ground using a mortar and pestle. The soil (1 gm) was added to 10 ml of deionized water. After gentle shaking for 30 minutes, the supernatant was diluted in Ringer's solution (Sodium chloride, 2.15g; potassium chloride, 0.075g, calcium chloride, 0.083g; distilled water, 1 liter). A 100 μl volume aliquot of a 1:10,000 dilution was inoculated into a diffusion chamber by mixing with 3 mL of HMA (3- (Nmorpholino)propanesulfonic acid, 0.25 g; Bacto-agar, 1O g; ferrous sulphate, 0.25 mg; manganese chloride, 0.25 mg; nickel sulphate, 0.25 mg; zinc sulphate, 0.25 mg; calcium chloride, 0.083 g; humic acid, 0.063g; distilled water, 1 liter), and the mixture poured into a sterile diffusion chamber. The diffusion chamber consisted of a steel washer sealed on one side with a 0.03 micron pore-sized polycarbonate membrane (see, U.S. Patent No. 7,011,957). Once the agar solidified, the open face of the chamber was sealed with another 0.03 micron pore-sized polycarbonate membrane, and the chamber placed on top of moist soil so that there was good contact between the chamber contents and the same soil the inoculum came from. After 28 days incubation the surface membrane (facing away from the soil) was peeled off, and the chamber contents were transferred to a sterile Petri dish. The chamber contents were transferred to a sterile Petri dish, and visible colonies picked by stabbing colonies with a sterile 28 Gauge wire and streaked onto the surface of 2% SMS agar (10 ml of 2% SMS agar in sterile 10 cm Petri dish). Several colonies were picked in this way. After 1 to 3 weeks growth on the agar surface, colonies were further purified by streaking onto sterile 2% SMS agar.

G0655, the producer of Neocitreamicin II, was one of these colonies directly picked from the diffusion chamber. 16S rDNA analysis of the strain G0655 was performed to identify its taxonomic identity at the genus level. Once the colonies of G0655 were shown to be pure by visual examination under a dissecting microscope, chromosomal DNA was isolated from approximately 10⁶ cells after a 5 min vigorous agitation in the presence of 50 mg of glass beads and 100 μl of H₂O in a 0.5 ml Eppendorf tube. PCR amplification of the gene encoding the 16S rRNA was carried out using the chromosomal DNA, GoTaq Green Master Mix (Promega M7122), and universal primers Bac8F (5'-AGR GTT TGA TCC TGG CTC AG-3' (SEQ ID NO:1)) and 1492R (5'-TAC GGY TAC CTT GTT ACG ACT T-3' (SEQ ID NO:2)). (Baker et al. (2003), J Microbiol. Meth. 55:541-555). PCR thermocycler parameters included 30 cycles of 95 ⁰C for 30 s, 45 ⁰C for 30 s, and 72 °C for 105 s. The amplified DNA fragment was sequenced at Macrogene (Rockville, MD) using primer 782R (5'-ACC AGG GTA TCT AAT CCT GT-3' (SEQ ID NO:3)) and compared by BLAST alignment to the nucleotide collection in GenBank. The 16S rRNA gene sequence similarity studies (first half of the gene, from base pair 20 to base pair 710) using nucleotide sequence comparison (Blast) to GenBank database placed strain G0655 as a member of the genus Nocardia. The 3 most closely related Nocardia species are Nocardia acidovorans (GenBank accession no. AM402972; 98.7%), Nocardia nova (GenBank accession no. AB 162790; 97.8%), and Nocardia pseudobrasiliensis (GenBank accession no. AF430042; 97.8%). G0655 was deposited with the Agricultural Research Service (ARS) Culture Collection and assigned designation NRRL 50106.

The fermentation procedure for G0655 was conducted as described below. A colony of isolate G0655 was homogenized using a sterile pestle in a 1.5 ml Eppendorf tube containing 200 μl sterile water. The bacterial homogenate was transferred to a 250 ml Erlenmeyer flask containing 40 ml of seed broth (1.5% glucose, 1.5% glycerol, 1.5% malt extract, 2.5% yeast extract, 0.5% casamino acids, and 0.1% calcium carbonate). The seed broth was incubated for 4 days at 28 °C on a rotary shaker (1 inch throw, 200 rpm) prior to production media inoculation at 2.5% (v/v). Production was performed in 500 ml aliquots of medium R4 (1.0% glucose, 0.1% yeast extract, 0.01% casamino acids, 0.3% L- proline, 1.0% MgCl₂-OH₂O, 0.02% K₂SO₄, 0.56% N-Tris(hydroxymethyl)methyl-2- aminoethanesulfonic acid) in 2 liter tri-baffled flasks at 28 ⁰C on a rotary shaker (2.5 inch throw, 200 rpm). After 2 days of incubation, 2 g of sterile Diaion HP-20 resin was added to each flask. Fermentations were continued for an additional 7 days prior to harvest.

The isolation procedure for Neocitreamicin II is as follows. The bacterial cell mass and HP-20 resin from 5 liters of fermentation broth were collected by filtration. The mixture was washed with 500 ml of 20% acetone in water and then with 500 ml 100% acetone. The wash was discarded and the acetone portion was evaporated to dryness, leaving a brown solid which was extracted with hexanes (2 x 40 ml). The suspension was centrifuged at 1660 g at 4⁰C for 10 minutes. The hexanes extract was discarded and the remaining reddish brown pellet was dried overnight. The solid was dissolved in 30 ml of methanol and then 70 ml of water was added, resulting in some precipitation. The suspension was adsorbed onto an Alltech Extract-Clean SPE C18-HC (10 g) cartridge using a vacuum pump. The cartridge was then sequentially washed with 40 mL portions of 20%, 30%, 50%, 70%, 90% and 100% of methanol in water. The final portion contained the majority of the active components and appeared deep red in color. This solution was then injected in 500 μL portions into semi-preparative HPLC for purification. Neocitreamicin II (2) eluted at 25.3 minutes. The corresponding HPLC fractions were pooled and lyophilized after removal of acetonitrile under reduced pressure to afford 5 mg as yellow powder.

EXAMPLE 2

Elucidation of the Structure of Neocitreamicin II

Neocitreamicins II (2) was isolated from the production fermentation broth by solid phase extraction and reversed phase chromatography (Fig. 2). The molecular formula of 2 was determined as C₄₄H₄₃NOj₅ on the basis of positive ion HR ESI-MS ([M+H]⁺ m/z 826.2714, calcd. for 826.2711 ) and NMR spectra data, indicating the presence of 24 double bond equivalents. All NMR spectra were taken on a Bruker-DRX- 500 spectrometer equipped with a 5 mm QNP probe. High resolution ESI-LC-MS data were recorded on a MicroMass Q-To f-2 spectrometer equipped with an Agilent 1100 solvent delivery system and a DAD using a Phenomenex Gemini-C18 reversed phase column (50 x 2.0 mm, 3 μm particle size).

The UV spectra of 2 is virtually identical to those reported for citreamicins. (Carter et al. (1990), J. Antibiot. 43:504-512). The structure of 2 was elucidated by interpretation of 2D NMR spectra (COSY, HSQC, and HMBC) and comparison with reported citreamicin data. The ¹H and ¹³C NMR signals of 2 are in good accordance to those of citreamicins for the common moieties of the structure. The 2,4-dimethylvalerate side chain was determined using data from COSY and HMBC experiments {see, Table 1).

Table 1 'H and '³C NMR data of 2 (5Q0 and 125 MHz, δm ppm, J in Hz)

2 (DMSO-tf₆)

1 62 1

Ia 19 1 1 81 (3H, s) 1, Ib, 2

Ib 649 4 66 (IH, d, 11 6) 1, 2

440 (IH, d, 1 1 6)

2 171 3

4 93 8

4a 25 4 1 71 (3H, s) 4, 5

5 39 8 3 74 (IH, d, 14 8) 4, 6, 7, 27

3 66 (IH, d, 14 8) 4, 6

6 135 4

7 118 0 7 51 (lH, brs) 5, 25, 27

8 136 7

9 132 5 8 19 (IH, AB) 11, 12, 24

10 1243 8 21 (IH, AB)

11 130 3

12 177 6 13 1₅51

I₅ 1₅46

16 1209 781 (IH, d, 90) 15, 18,20

17 1253 758 (IH, dd, 90,30) 18

18 1495

18-OH exchanged

19 1095 765 (IH, d, 3 O) 18,21 0 1260

2 (DMSO-(Z₆) o Sc" HMBC 1 1731 2 1193 3 1805 4 1402 5 1186 6 1611 6-OH 1341 (IH, s) 25,26,27 7 1069 8 1661

1' 1754 ' 367 251 (under DMSO) l',2a',3' a' 169 105 (3H, d, 68) r.2',3' ' 427 146(lH,m) r,2',2a\4',4a'

118-125(lH,m) 2', 2a', 4a' ' 253 152(lH,m) 4a'a' 223 081(3H,d, 63) 3', 4', 4a', 5' ' 222 O 85 (3H, d, 63) 3', 4', 4a', 5'

1" 968 549 (IH, dd, 98, 2 O) 2" 345 202-207 (IH, m)

181 (IH, m)

3" 656 396 (IH, m) 4", 5"

4" 713 494 (IH, d, 29) 3", 1-Ac

5" 691 399 (IH, m) 6"

6" 165 107 (3H, d, 64) 4", 5"

1-Ac 1703

2-Ac 208 2 ll(3H,s) 1-Ac

Assignments confirmed by HSQC correlations

An HMBC correlation was also observed between H- 19 and the C-21 carbonyl carbon, indicating the G ring substitution is at C-18 instead of C-17. The remaining ¹H and ¹³C NMR signals of 2 were easily attributed to a deoxysugar moiety through COSY, HSQC, and HMBC correlations. A clear HMBC correlation between H-4" and the carbonyl carbon of the acetyl group indicates that the deoxysugar is acetylated at 4" position, which is also consistent with the downfield chemical shift of H-4" (4.94 ppm). Although no HMBC correlation was observed between the anomeric proton and C-18, a ROESY experiment showed correlations from the anomeric proton to H-17 and H- 19, supporting the deoxysugar is attached to position 18 (Fig. 3). The anomeric proton occupies an axial position as indicated by the large coupling constant (J= 9.7 Hz). ROESY correlations were also observed between H-I" and H-3" and between H-I" and H- 5", indicating the hydroxyl and the methyl groups are in equatorial positions. Furthermore, because only a small coupling constant (2.9 Hz) was observed for H-4", the acetoxy group occupies an axial position. Thus, the deoxysugar moiety was determined to be an acetylated oliose derivative.

EXAMPLE 3

Properties of Strain G0655

When grown on several standard laboratory media used for isolation and identification of environmental microorganisms, strain G0655 was found to share many morphological characteristics with members of the actinobacteria, including the ability to sporulate, the production of diffusible pigments, and the formation of firm mycellial colonies (see, Table 2).

Table 2 Culture characteristics of strain G0655 Medium Description^ab

Tryptone-yeast extract agar G: Poor, flat, white

(ISP-I) A: Poor, white

R: Light yellow (241, 236, 126)

S: None

Yeast extract-malt extract agar G: Abundant, raised, beige (231, 217, 135)

(ISP-2) A: Good, velvety, yellowish

R: Orange brown (174, 132, 32)

S: None

Oatmeal agar G: Moderate, flat, white

(ISP-3) A: Poor, white

R: White

S: None

Inorganic salts-starch agar G: Poor, flat, white

(ISP-4) A: Poor

R: White

S: None

Commeal agar G: Abundant, raised, white

A: Good, velvety, white

R: Orange brown (204, 123, 64)

S: None

Potato Dextrose agar G: Moderate, flat, white A: Poor, white

R: Light brown (240, 204, 128)

S: None

R4 agar ^c G: Abundant, raised, light beige (253, 251, 170) A: Good, velvety, yellowish

R: Dark brown (129, 109, 41)

S: None

SMS agar^d G: Abundant, raised, white

A: Good, velvety, white R: Orange brown (177, 150, 56)

S: None

^a G: growth, A: aerial mycelium, R: reverse, S: soluble pigment. b For accurate color depiction use Microsoft Windows color palette and enter the 3 listed values in the order given for red, green and blue. c R4 agar: 10 g/1 glucose, 1 g/1 yeast extract, 0.1 g/1 casamino acids, 3 g/1 L-proline, 10 g/1 MgCl₂-OH₂O, 4 g/1 CaCl₂-2H₂O, 0.2 g/1 K₂SO₄, 5.6 g/1 TES, 1 ml Trace Metal Solution, 15 g/1 agar. d SMS agar: 0.125 g/1 casein, 0.1 g/1 potato starch, 1 g/1 casamino acids, 15 g/1 agar.

EXAMPLE 4

Determination of the Minimal Inhibitory Concentration of Neocitreamicin II

Minimal inhibitory concentrations of neocitreamicins against a set of target bacterial strains were determined in liquid growth medium. An 80 μg/ml stock solution of a test compound in DMSO was two-fold serially diluted with DMSO. In a 96-well microtiter plate, 10 μl of each compound solution was mixed with 90 μl of a log-phase target cells dilution at OD_6OOmTi = 0.001 in Mueller-Hinton Broth or Brain Hearth infusion Broth (Enterococcus strains only). This achieved final concentrations of the test compounds from 8 μg/ml to 0.06 μg/ml. After 18 hours of incubation at 37 ⁰C, minimal inhibitory concentrations were determined by visual inspection

Neocitreamicin II showed good antibacterial activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis .

Table 3 Antimicrobial activities of 2

Microbroth MIC (μg/ml)

Test Organism 2

Bacillus sub t His IAl 0.12 Staphylococcus aureus (MRSA NRS 1 ) 1.0

Sl aureus (MRSA NRS2) 0.50

S. aureus (MRSA NRS71) 0.50

Enterococcus faecalis (VRE 51299) 0.06

E. faecalis (VRE 51575) 0.25 Escherichia coli K- 12 >8.0

EQUIVALENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

CLAIMSWe claim:

1. A compound of formula (I)

or an enantiomer, diastereomer, tautomer, or pharmaceutically acceptable salt or solvate thereof, wherein:

Ri is alkyl or substituted alkyl, alkenyl or substituted alkenyl, alkynyl or substituted alkynyl, cycloalkyl or substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted heterocycle, or aryl or substituted aryl;

R₂ is hydrogen, halogen, cyano, nitro, alkyl or substituted alkyl, cycloalkyl or substituted cycloalkyl, aryl or substituted aryl, or C(=O)R_c, wherein R₀ is alkyl or substituted alkyl, cycloalkyl or substituted cycloalkyl, or aryl or substituted aryl;

R₃ is hydrogen or C(=O)alkyl; and

R₄ is hydrogen, alkyl or substituted alkyl.

2. The compound of claim 1 having the structure of formula (Ia)

3. The compound of claim 1 having the structure of formula (II)

4. A compound of formula (III)

5. A pharmaceutical composition comprising the compound of claim 1 and a pharmaceutically-acceptable excipient, carrier or diluent.

6. The pharmaceutical composition of claim 5, further comprising an agent selected from the group consisting of an antineoplastic agent, an antibiotic, an antifungal agent, an antiviral agent, and combinations thereof.

7. A pharmaceutical composition comprising the compound of claim 2 and a pharmaceutically-acceptable excipient, carrier or diluent.

8. The pharmaceutical composition of claim 7, further comprising an agent selected from the group consisting of an antineoplastic agent, an antibiotic, an antifungal agent, an antiviral agent, and combinations thereof.

9. A pharmaceutical composition comprising the compound of claim 3 and a pharmaceutically-acceptable excipient, carrier or diluent.

10. The pharmaceutical composition of claim 9, further comprising an agent selected from the group consisting of an antineoplastic agent, an antibiotic, an antifungal agent, an antiviral agent, and combinations thereof.

11. A pharmaceutical composition comprising the compound of claim 4 and a pharmaceutically-acceptable excipient, carrier or diluent.

12. The pharmaceutical composition of claim 11 , further comprising an agent selected from the group consisting of an antineoplastic agent, an antibiotic, an antifungal agent, an antiviral agent, and combinations thereof.

13. A compound isolated from an aerobically grown strain of Nocardia G0655, the compound characterized by:

(a) a molecular weight of 825.81 (Q-TOF LC-MS);

(b) an ultraviolet absorption spectra substantially the same to that shown in FIG. 2;

(c) a proton nuclear magnetic resonance substantially the same to that shown in FIG. 3;

(d) a carbon- 13 nuclear magnetic resonance substantially the same to that shown in FIG. 4;

(e) a COSY nuclear magnetic resonance substantially the same to that shown in FIG. 5;

(f) a HSQC nuclear magnetic resonance substantially the same to that shown in FIG. 6; and

(g) a HMBC nuclear magnetic resonance substantially the same to that shown in FIG. 7.

14. A pharmaceutical composition comprising the compound of claim 13 and a pharmaceutically-acceptable excipient, carrier or diluent.

15. The pharmaceutical composition of claim 14, further comprising an agent selected from the group consisting of an antineoplastic agent, an antibiotic, an antifungal agent, an antiviral agent, and combinations thereof.

16. The pharmaceutical composition of claim 15, wherein the agent is an antineoplastic agent.

17. The pharmaceutical composition of claim 16, wherein the antineoplastic agent is selected from the group consisting of an antimetabolite, a purine or pyrimidine analogue, an alkylating agent, a crosslinking agent, and an intercalating agent.

18. A method of producing a compound of formula (II)

the method comprising cultivating a strain of Nocardia G0655 in a culture medium comprising assimilable sources of carbon, nitrogen, and inorganic salts under aerobic conditions to produce an assayable amount of the compound of formula (II).

19. The method of claim 18, further comprising isolating and purifying the compound of formula (II) from the culture medium.

20. A compound of formula (II) prepared by the method of claim 18.

21. A method of treating a disorder in a mammal in need thereof, the method comprising administering to the mammal a therapeutically effective amount of a compound of formula (I)

R₃ is hydrogen or C(=O)alkyl; and

R₄ is hydrogen, alkyl or substituted alkyl.

22. The method of claim 21 , wherein the disorder is caused by an agent selected from the group consisting of a bacterium, a fungus, a virus, a protozoan, a helminth, a parasite, and combinations thereof.

23. The method of claim 22, wherein the agent is a bacterium.

24. The method of claim 23, wherein the bacterium is a Gram-positive bacterium.

25. The method of claim 24, wherein the Gram-positive bacterium is selected from the group consisting of Streptococcus, Staphylococcus, Enter ococcus, Corynebacteria, Listeria, Bacillus, Erysipelothrix, and Actinomycetes.

26. The method of claim 24, wherein the Gram-positive bacterium is Methicillin Resistant Staphylococcus aureus (MRSA).

27. The method of claim 23, wherein the bacterium is a Gram-negative bacterium.

28. The method of claim 23, wherein the bacterium is selected from the group consisting of Helicobacter pylori, Borelia burgdorferi, Legionella pneumophilia, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansaii, Mycobacterium gordonae, Mycobacteria sporozoites, Staphylococcus aureus, Staphylococcus epidermidis, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus dysgalactia, Streptococcus faecalis, Streptococcus bovis, Streptococcus pneumoniae, pathogenic Campylobacter sporozoites, Enterococcus sporozoites, Haemophilus influenzae, Pseudomonas aeruginosa, Bacillus anthracis, Corynebacterium diphtheriae, Corynebacterium jeikeium, Corynebacterium sporozoites, Erysipelothrix rhusiopathiae, Clostridium perfringens, Clostridium tetani, Clostridium difficile, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sporozoites, Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, and Actinomyces israelii.

29. The method of claim 22, wherein the agent is a virus.

30. The method of claim 29, wherein the virus is selected from the group consisting of Retroviridae, Picornaviridae, Calciviridae, Togaviridae, Flaviridae, Coronaviridae, Rhabdoviridae, Filoviridae, Paramyxoviridae, Orthomyxoviridae, Bungaviridae, Arenaviridae, Reoviήdae, Birnaviridae, Hepadnaviridae, Parvoviridae, Papovaviridae, Adenoviridae, Herpesviridae, Poxviridae, and Iridoviridae.

31. The method of claim 29, wherein the virus is selected from the group consisting of influenza virus, human immunodeficiency virus, and herpes simplex virus.

32. The method of claim 22, wherein the agent is a protozoan.

33. The method of claim 32, wherein the protozoan is selected from the group consisting of Trichomonas vaginalis, Giardia lamblia, Entamoeba histolytica, Balantidium coli, Cryptosporidium parvum and Isospora belli, Trypansoma cruzi, Trypanosoma gambiense, Leishmania donovani, and Naegleriafowleri.

34. The method of claim 22, wherein the agent is a helminth.

35. The method of claim 32, wherein the helminth is selected from the group consisting of Schistosoma mansoni, Schistosoma cercariae, , Schistosoma japonicum, , Schistosoma mekongi, Schistosoma hematobium, Ascaris lumbricoides, Strongyloides stercoralis, Echinococcus granulosus, Echinococcus multilocularis, Angiostrongylus cantonensis, Angiostrongylus constaricensis, Fasciolopis buski, Capillar ia philippinensis, Paragonimus westermani, Ancylostoma dudodenale, Necator americanus,. Trichinella spiralis, Wuchereria bancrofti, Brugia malayi, and Brugia timori, Toxocara canis, Toxocara cati, Toxocara vitulorum, Caenorhabiditis elegans, and Anisakis species

36. The method of claim 22, wherein the agent is a parasite.

37. The method of claim 36, wherein the parasite is selected from the group consisting of Plasmodium falciparum, Plasmodium yoelli, Hymenolepis nana, Clonorchis sinensis, Loa loa, Paragonimus westermani, Fasciola hepatica, and Toxoplasma gondii.

38. The method of claim 31, wherein the parasite is a malarial parasite.

39. The method of claim 22, wherein the agent is a fungus.

40. The method of claim 39, wherein the fungus is selected from the group consisting of Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans, Candida tropicalis, Candida glabrata, Candida krusei, Candida parapsilosis, Candida dubliniensis, Candida lusitaniae, Epidermophyton floccosum, Microsporum audouinii, Microsporum canis, Microsporum canis var. distortum Microsporum cookei, Microsporum equinum, Microsporum ferrugineum, Microsporum fulvum, Microsporum gallinae, Microsporum gypseum, Microsporum nanum, Microsporum persicolor, Trichophyton ajelloi, Trichophyton concentricum, Trichophyton equinum, Trichophyton flavescens, Trichophyton gloriae, Trichophyton megnini, Trichophyton mentagrophytes var. erinacei, Trichophyton mentagrophytes var. interdigitale, Trichophyton phaseoliforme, Trichophyton rubrum, Trichophyton rubrum downy strain, Trichophyton rubrum granular strain, Trichophyton schoenleinii, Trichophyton simii, Trichophyton soudanense, Trichophyton terrestre, Trichophyton tonsurans, Trichophyton vanbreuseghemii, Trichophyton verrucosum, Trichophyton violaceum, Trichophyton yaoundei, Aspergillus fumigatus, Aspergillus flavus, and Aspergillus clavatus.

41. The method of claim 21, wherein the compound administered has the formula (II)

42. A method of inhibiting the growth of a tumor cell, comprising contacting the tumor cell with an effective amount of compound of formula (I)

Ri is alkyl or substituted alkyl, alkenyl or substituted alkenyl, alkynyl or substituted alkynyl, cycloalkyl or substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted heterocycle, or aryl or substituted aryl; R₂ is hydrogen, halogen, cyano, nitro, alkyl or substituted alkyl, cycloalkyl or substituted cycloalkyl, aryl or substituted aryl, or C(=O)R_c, wherein R₀ is alkyl or substituted alkyl, cycloalkyl or substituted cycloalkyl, or aryl or substituted aryl;

R₃ is hydrogen or C(=O)alkyl; and

R₄ is hydrogen, alkyl or substituted alkyl.

43. The method of claim 42, wherein the tumor cell is selected from the group consisting of a breast cancer cell, an ovarian cancer cell, a colon cancer cell, a prostate cancer cell, a liver cancer cell, a lung cancer cell, a gastric cancer cell, an esophageal cancer cell, a urinary bladder cancer cell, a melanoma cell, a leukemia cell, and a lymphoma cell.

44. The method of claim 42, further comprising administering a chemotherapeutic agent selected from the group consisting of an antimetabolite, a purine or pyrimidine analogue, an alkylating agent, a crosslinking agent, and an intercalating agent, wherein the chemotherapeutic agent is administered before, after or substantially simultaneously with the compound.

45. The method of claim 42, wherein the tumor cell is a drug-resistant tumor cell.

46. The method of claim 42, wherein the tumor cell is killed by the compound.

47. The method of claim 42, wherein the administered compound has formula (II)

48. A method of treating cancer in a mammal in need thereof, the method comprising administering to the mammal a therapeutically effective amount of compound of formula (I)

R₃ is hydrogen or C(=O)alkyl; and

R₄ is hydrogen, alkyl or substituted alkyl.

49. The method of claim 48, wherein the cancer is selected from the group consisting of breast cancer, ovarian cancer, colon cancer, prostate cancer, liver cancer, lung cancer, gastric cancer, esophageal cancer, urinary bladder cancer, melanoma, leukemia, and lymphoma.

50. The method of claim 48, further comprising administering a chemotherapeutic agent selected from the group consisting of an antimetabolite, a purine or pyrimidine analogue, an alkylating agent, a crosslinking agent, and an intercalating agent, wherein the chemotherapeutic agent is administered before, after or substantially simultaneously with the compound.

51. The method of claim 48, comprising administering a compound of formula (II)

52. An isolated G0655 strain deposited with the Agricultural Research Service (ARS) Culture Collection and assigned designation NRRL 50106.

53. A culture consisting essentially of a Nocardia species, having the identifying characteristics of strain G0665 (NRRL 50106).

54. A method of preparing a compound of formula (I)

R₃ is hydrogen or C(=O)alkyl; and

R₄ is hydrogen, alkyl or substituted alkyl,

comprising converting a compound of formula (III)

or an enantiomer, diastereomer, tautomer, or pharmaceutically acceptable salt or solvate thereof, for a time and under conditions effective to form the compound of formula (I).

55. A method of preparing a compound of formula (Ia)

R₁ is alkyl or substituted alkyl, alkenyl or substituted alkenyl, alkynyl or substituted alkynyl, cycloalkyl or substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted heterocycle, or aryl or substituted aryl, comprising reacting a compound of formula (III)

or an enantiomer, diastereomer, tautomer, or pharmaceutically acceptable salt or solvate thereof,

with a compound of formula R₁CC=O)OH, R]C(=O)C1, or RiC(=O)-O-(=O)Ri, for a time and under conditions effective to form the compound of formula (Ia).