UREA DERIVATIVES OF POLYENE MACROLIDE ANTIBIOTICS

RELATED APPLICATION

This application claims the benefit of priority to U.S. Provisional Patent Application No. 62/049,592, filed September 12, 2014.

GOVERNMENT SUPPORT

This invention was made with government support under grant number

R01GM080436 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

For more than half a century amphotericin B (AmB) has served as the gold standard for treating systemic fungal infections. AmB has a broad spectrum of activity, is fungicidal, and is effective even against fungal strains that are resistant to multiple other agents. Surprisingly, clinically significant microbial resistance has remained exceptionally rare while resistance to next generation antifungals has appeared within just a few years of their clinical introduction. Unfortunately, AmB is also highly toxic. Thus, the effective treatment of systemic fungal infections is all too often precluded, not by a lack of efficacy, but by dose-limiting side effects. Some progress has been made using liposome delivery systems, but these treatments are prohibitively expensive and significant toxicities remain. Thus, less toxic, but equally effective, AmB derivatives stand to have a major impact on human health.

AmB is representative of a whole class of polyene macro lide antibiotics useful for the treatment of fungal infections. Similar to AmB, less toxic, but equally effective, derivatives of other polyene macrolide antibiotics stand to have a major impact on human health.

SUMMARY OF THE INVENTION An aspect of the invention is a urea derivative of a polyene macrolide antibiotic or a pharmaceutically acceptable salt thereof, comprising a urea-containing substructure represented by

wherein

R represents hydrogen, alkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, aminoalkyl, or -(CH ₂ ) _n -COOH;

n is 1, 2, 3, 4, 5, or 6; and

said urea derivative has antifungal activity;

provided that the urea derivative of the polyene macrolide antibiotic is not

An aspect of the invention is a pharmaceutical composition, comprising a urea derivative of the invention; and a pharmaceutically acceptable carrier.

An aspect of the invention is a method of inhibiting growth of a yeast or fungus, comprising contacting the yeast or fungus with an effective amount of a urea derivative of a polyene macrolide antibiotic or a pharmaceutically acceptable salt thereof; wherein said urea derivative comprises a urea-containing substructure represented by

wherein

R represents hydrogen, alkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, aminoalkyl, -(CH ₂ ) _n -COOH;

n is 1, 2, 3, 4, 5, or 6; and

said urea derivative has antifungal activity;

provided that the urea derivative of the polyene macrolide antibiotic is not

An aspect of the invention is a method of treating a fungal infection, comprising administering to a subject in need thereof a therapeutically effective amount of a urea derivative of a polyene macrolide antibiotic or a pharmaceutically acceptable salt thereof; wherein said urea derivative comprises a urea-containing substructure represented by

wherein

R represents hydrogen, alkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, aminoalkyl, -(CH ₂ ) _n -COOH;

n is 1, 2, 3, 4, 5, or 6; and

said urea derivative has antifungal activity;

provided that the urea derivative of the polyene macrolide antibiotic is not

An aspect of the invention is a method of making a urea derivative of a polyene m

Scheme 2

wherein 1 represents a substructure of a protected oxazolidinone derivative of a polyene macrolide antibiotic; and

each R is independently selected from the group consisting of hydrogen, halogen, straight- or branched-chain alkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, aryl, heteroaryl, aralkyl, heteroaralkyl, hydroxyl, sulfhydryl, carboxyl, amino, amido, azido, nitro, cyano, aminoalkyl, and alkoxyl;

provided that the polyene macrolide antibiotic is not amphotericin B.

In accordance with each of the foregoing aspects of the invention, in various embodiments, the polyene macrolide antibiotic is selected from the group consisting of amphotericin A, arenomycin B, candicidin D, candidin, candidoin, CE-108, etruscomycin, eurocidin D, eurocidin E, FR-008-VI, HA-2-91, hamycin A, levorin AO, levorin A3, mycoheptin, natamycin (pimaricin), nystatin Al, nystatin A2, nystatin A3, partricin A, polyfungin B, rimocidin, tetramycin A, tetramycin B, tetrin A, tetrin B, tetrin C, trichomycin A, trichomycin B, vacidin A, YS-822A, 3874 HI, 3874 H2, 3874 H3, and 67- 121-A.

DETAILED DESCRIPTION OF THE INVENTION

Amphotericin B (AmB), a widely known and used representative antifungal polyene macrolide antibiotic, comprises a polyhydroxylated, polyunsaturated macrolactone ring core, decorated with a mycosamine "appendage". Mycosamine is 3-amino-3,6-dideoxy-P- D-mannopyranose. The mycosamine portion of the molecule plays a key role in both the desired biological effects and the undesirable side-effects of Amphotericin B.

A lack of understanding of the mechanism(s) by which AmB is toxic to yeast and human cells has thus far hindered the rational development of a clinically successful derivative. The longstanding accepted mechanism of action of AmB has been ion channel formation within a cell's membrane leading to electrochemical gradient disruption and eventually cell death. This model suggests that development of a less toxic derivative requires selective ion channel formation in yeast versus human cells. Contrary to this longstanding model, our group recently discovered that the primary mechanism of action of AmB is not ion channel formation, but simple ergosterol binding. Gray, KC et al., Proc Natl Acad Sci USA 109:2234 (2012). Yeast and human cells possess different sterols, ergosterol and cholesterol, respectively. Therefore, the new model suggests a simpler and more actionable roadmap to an improved therapeutic index; i.e., a less toxic AmB derivative would retain potent ergosterol binding capability, but lack the ability to bind cholesterol.

Recently our group reported that removal of the C2' hydroxyl group from the mycosamine sugar produced a derivative, C2'deOAmB, which surprisingly retains ergosterol-binding ability, but shows no binding to cholesterol. Wilcock, BC et al, J Am Chem Soc 135:8488 (2013). Consistent with the preferential sterol binding hypothesis, in vitro studies demonstrated that C2'deOAmB is toxic to yeast, but not human cells.

To explain why removal of the C2' alcohol results in loss of cholesterol binding ability, while maintaining efficient ergosterol binding, we hypothesized that the AmB structure exists in a ground state conformation capable of binding both sterols. Removal of the C2' alcohol potentially results in a conformational change of the AmB structure which retains ergosterol binding ability but is incapable of binding cholesterol. A generic molecule is capable of binding two different ligands in a common binding site.

Modification at a site distal to the binding pocket alters the binding site conformation. This principle of allosteric modification causes preferential binding of one ligand over the other. To our knowledge, such ligand-selective allosteric effects have not been previously observed in small molecule-small molecule interactions. Encouragingly, ligand selective allosteric modifications have been observed in proteins which bind multiple ligands in a common binding site. We thus hypothesized that removal of the C2' alcohol allosterically modifies the sterol binding pocket, accounting for the decrease in cholesterol binding ability.

Interestingly, we noticed in a previously obtained X-ray crystal structure of N- iodoacyl AmB a prominent water bridged hydrogen bond joining the C2' alcohol to the C13 hemiketal. We recognized that if such a water bridged hydrogen bond helped rigidify the ground state conformation of AmB, it would follow that removal of the C2' alcohol abolishes this interaction and thereby potentially enables adoption of alternative ground state conformers having altered affinities for cholesterol and ergosterol. Intrigued by this capacity of the crystal structure to potentially rationalize our observations with

C2'deOAmB, we hypothesized that this crystal structure may represent the ground state conformation of AmB which is capable of binding both ergosterol and cholesterol.

Following this logic, we proposed that disruption or removal of any other rigidifying features observed in the crystal structure might similarly allow access to alternative ground state conformations and thereby alter the AmB sterol binding profile. Guided by this logic, careful inspection of the X-ray crystal structure revealed an additional intramolecular rigidifying feature with the potential of stabilizing the AmB ground state: a salt bridge between the C41 carboxylate and C3' ammonium.

Systematic modification of the group appended to the C16 carbon was targeted as the first series of derivatives to further probe this allosteric modification model. Multiple AmB derivatives modifying the C41 carboxylate have been reported including esters and amides among others. However, all previous AmB derivatives maintain a carbon atom appended to the C16 carbon. We hypothesized that appending a heteroatom to the C16 carbon would have a great impact on the salt bridge interaction. Therefore, we sought an efficient, chemoselective synthetic strategy to gain access to such a derivative.

We discovered that a short three-step sequence of Fmoc protection, methyl ketal formation, and Curtius rearrangement, promoted by diphenyl phosphoryl azide, provides an intermediate isocyanate which is trapped intramolecularly to generate oxazolidinone 1 (Scheme 1).

Scheme 1: Synthesis of C16 AmB derivatives

This facile sequence quickly generates gram quantities of versatile intermediate 1 in a chemoselective manner from AmB. Interception of 1 with a variety of amine

nucleophiles efficiently opens the oxazolidinone while concomitantly cleaving the Fmoc protecting group. For example, exposure of 1 to ethylene diamine, followed by methyl ketal hydrolysis in acidic water generates aminoethylurea (AmBAU) 2 in 42% yield.

Similarly, utilizing methylamine accesses methyl urea (AmBMU) 3 in 36% yield from 1. Exposure of 1 to β-alanine allylester followed by allyl removal with Pd(PPh ₃ ) ₄ and thiosalicylic acid yields ethylcarboxylateurea (AmBCU) 4. This versatile synthetic strategy allows efficient access to a diverse array of AmB urea derivatives and is capable of generating large quantities of urea derivatives due to its synthetic efficiency.

The strategy presented above can be used to access a wide variety of AmB derivatives with an amine appended to the C16 position. The opening of oxazolidinone 1 with a variety of nucleophiles (e.g., amines, alcohols, and phenols) could efficiently access a wide range of urea or carbamate derivatives. A small subset of the possible accessible derivatives is outlined in Scheme 2. Oxazolidinone 1 could be intercepted with primary amines to generate primary ureas, secondary amines to generate secondary ureas, and primary amines with alpha branching to create ureas with stereochemistry introduced at the alpha position. Additionally, oxazolidinone 1 could be opened with anilines to create aryl ureas, phenols to create aryl carbamates, or alcohols to generate alkyl carbamates.

Examples of amines include, without limitation, 1-(1-Naphthyl)ethylamine; l-(2- Naphthyl)ethylamine; l-(4-Bromophenyl)ethylamine; l,l-Diphenyl-2-aminopropane; 1,2,2- Triphenylethylamine; 1 ,2,3 ,4-Tetrahydro- 1 -naphthylamine; 1 ,2-Bis(2- hydroxyphenyl)ethylenediamine; l-Amino-2-benzyloxycyclopentane; 1-Aminoindane; 1- Benzyl-2,2-diphenylethylamine; 1-Cyclopropylethylamine; 1-Phenylbutylamine; 2-(3- Chloro-2,2-dimethyl-propionylamino)-3-methylbutanol; 2-

(Dibenzylamino)propionaldehyde; 2,2-Dimethyl-5-methylamino-4-phenyl-l ,3-dioxane; 2- Amino- 1 -fluoro-4-methyl- 1 , 1 -diphenylpentane; 2-Amino-3 ,3 -dimethyl- 1 , 1 -diphenylbutane; 2- Amino-3 -methyl- 1 , 1 -diphenylbutane; 2-Amino-3-methylbutane; 2-Amino-4-methyl- 1,1- diphenylpentane; 2-Aminoheptane; 2-Aminohexane; 2-Aminononane; 2-Aminooctane; 2- Chloro-6-fluorobenzylamine; 2-Methoxy-a-methylbenzylamine; 2-Methyl-l-butylamine; 2- Methylbutylamine; 3,3-Dimethyl-2-butylamine; 3,4-Dimethoxy-a-methylbenzylamine; 3- Amino-2-(hydroxymethyl)propionic acid; 3-Bromo-a-methylbenzylamine; 3-Chloro-a- methylbenzylamine; 4-Chloro-a-methylbenzylamine; 4-Cyclohexene-l,2-diamine; 4- Fluoro-a-methylbenzylamine; 4-Methoxy-a-methylbenzylamine; 7-Amino-5, 6,7,8- tetrahydro-2-naphthol; Bis [1-phenylethyl] amine; Bornylamine; cis-2-Aminocyclopentanol hydrochloride; cis-Myrtanylamine; cis-N-Boc-2-aminocyclopentanol;

Isopinocampheylamine; L-Allysine ethylene acetal; Methyl 3-aminobutyrate p- toluenesulfonate salt; N,N'-Dimethyl-l, -binaphthyldiamine; N,N-Dimethyl-1-(1- naphthyl)ethylamine; N,N-Dimethyl-l-phenylethylamine; Ν,α-Dimethylbenzylamine; N- allyl-a-methylbenzylamine; N-Benzyl-a-methylbenzylamine; sec-Butylamine; trans-2- (Aminomethyl)cyclohexanol; trans-2- Amino- 1,2-dihydro-l-naphthol hydrochloride; trans- 2-Benzyloxycyclohexylamine; a,4-Dimethylbenzylamine; a-Ethylbenzylamine; a- Methylbenzylamine; and β-Methylphenethylamine.

Taking a similar approach, the instant invention provides urea derivatives of any suitable mycosamine-containing polyene macrolide antibiotic. The present invention expressly excludes, however, the urea derivatives of AmB just described, namely, AmBAU, AmBMU, and AmBCU.

More than 200 mycosamine-containing polyene macrolides are known {Macrolide Antibiotics, Chemistry, Biology, and Practice; Omura, S., Ed.; Academic Press: New York, 1984); and 36 have been structurally characterized to a large extent: Beau, J.-M. et al. J. Am. Chem. Soc. 1990, 112, 4060-4061; Mechlinski, W. et al. Tetrahedron Lett. 1970, 44,

3873- 3876; Sowinski, P. et al. J. Antibiot. (Tokyo) 1996, 49, 1232-1235; Perez-Zuniga,

F.J. et al. J. Antibiot. (Tokyo) 2004, 57, 197-204; Gupte, T.E. et al. Indian J. Chem., Sect.

B. 2000, 39, 936-940; Lancelin, J.-M. et al. Tetrahedron Lett. 1989, 30, 4521-4524;

Pawlak, J. et al. Polish J. Chem. 2005, 79, 1673-1679; Zielinski, J. et al. J. Antibiot.

(Tokyo) 41, 1988, 1289-1291; Sowinski, P. et al. J. Antibiot. (Tokyo) 1995, 48, 1288-1291;

Ryu, G. et al. J. Nat. Prod. 1999, 62, 917-919; Hirota, H. et al. J. Antibiot. (Tokyo) 1991, 4,

181-186; Itoh, A. et al. J Antiobiot. (Tokyo) 1990, 43, 948-955; Nakagomi, K. et al. J.

Antibiot. (Tokyo) 1990, 43, 470-476; Vertesy, L. et al. J. Antibiot. (Tokyo) 1998, 51, 921- 928; Wright, J.J. et al. J. Chem. Soc. Chem. Comm. 1977, 710; Zielinski, J.; Borowy-

Borowski, H. et al. Tetrahedron Lett. 1979, 20, 1791-1794; Pawlak, J. et al. J. Antibiot.

(Tokyo) 1993, 46, 1598-1604; Zhou, Y. et al. Chem. Biol. 2008, 15, 629-638;

Kozhuharova, L. et al. World J. Microbiol. Biotechnol. 2008, 24, 1-5; Pawlak, J. et al.

Polish J. Chem. 2005, 79, 1667-1672; Borowski, E. et al. J. Antibiot. (Tokyo) 1978, 31, 117-123; Tweit, R.C. et al. J. Antibiot. (Tokyo) 1982, 35, 997-1012; Komori, T. J. Antibiot.

(Tokyo) 1990, 43, 778-782; Sowinski, P. et al. J. Antibiot. (Tokyo) 1989, 42, 1639-1642;

Macro lide Antibiotics, Chemistry, Biology, and Practice; Omura, S., Ed.; Academic Press:

New York, 1984; Macrolide Antibiotics, Chemistry, Biology, and Practice, Second Ed.;

Omura, S., Ed.; Academic Press: New York, 2002.

Structures of the 36 structurally characterized mycosamine-containing polyene macrolides are shown below.

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The compounds shown above can be generally characterized as comprising a C25-

C37 cyclic polyene macrolide aglycone moiety, linked via an oxygen atom to the anomeric carbon of a mycosaminyl moiety. The following motif is 100% conserved in these compounds:

All spectroscopic and structure-activity evidence collected thus far supports the conclusion that the above highly conserved motif is the sterol binding domain of amphotericin B (i.e., the portion of the molecule that directly binds ergosterol and cholesterol). The perfect conservation of this motif across the range of all known mycosamine-containing polyene macrolides supports the inventor's proposition that it is sterol binding domain in all (known and yet to be discovered) mycosamine-containing polyene macrolides.

In contrast, the compounds of the invention, i.e., urea derivatives of polyene macrolide antibiotics, comprise a corresponding motif or substructure represented by

wherein

R represents hydrogen, alkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, aminoalkyl, or -(CH ₂ )„-COOH; and

n is 1, 2, 3, 4, 5, or 6.

An aspect of the invention is a urea derivative of a polyene macrolide antibiotic or a pharmaceutically acceptable salt thereof, comprising a urea-containing substructure represented by

wherein

R represents hydrogen, alkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, aminoalkyl, or -(CH ₂ ) _n -COOH;

n is 1, 2, 3, 4, 5, or 6; and

said urea derivative has antifungal activity;

provided that the urea derivative of thepolyene macrolide antibiotic is not

In certain embodiments, the binding avidity for ergosterol of said urea derivative is at least 75 percent of the binding avidity for ergosterol of the counterpart polyene macrolide antibiotic. In various individual embodiments, the binding avidity for ergosterol of a compound of the invention is at least 80 percent, at least 85 percent, at least 90 percent, or at least 95 percent of the binding avidity for ergosterol of the counterpart polyene macrolide. In certain embodiments, the binding avidity for ergosterol of a compound of the invention is at least 100 percent of the binding avidity for ergosterol of the counterpart polyene macrolide.

In certain embodiments, the binding avidity for cholesterol of said urea derivative is less than or equal to 25 percent of the binding avidity for cholesterol of the counterpart polyene macrolide antibiotic. In various individual embodiments, the binding avidity for cholesterol of a compound of the invention is less than or equal to 20 percent, less than or equal to 15 percent, less than or equal to 10 percent, or less than or equal to 5 percent, of the binding avidity for cholesterol of the counterpart polyene macrolide. In an

embodiment, a compound of the invention has essentially no binding avidity for cholesterol.

In an embodiment, the binding avidity for ergosterol of a compound of the invention is at least 75 percent of the binding avidity for ergosterol of the counterpart polyene macrolide; and the binding avidity for cholesterol of the compound of the invention is less than or equal to 25 percent of the binding avidity for cholesterol of the counterpart polyene macrolide.

In certain embodiments, R represents alkyl, aminoalkyl, or -(CH ₂ ) _n -COOH.

In certain embodiments, R represents alkyl.

In certain embodiments, R represents aminoalkyl.

In certain embodiments, R represents -(CH ₂ ) _n -COOH.

In certain embodiments, R represents -(CH ₂ ) _n -COOH, and n is 1.

In certain embodiments, R represents -(CH ₂ ) _n -COOH, and n is 2.

In certain embodiments, R represents -(CH ₂ ) _n -COOH, and n is 3.

In certain embodiments, R represents -(CH ₂ ) _n -COOH, and n is 4.

In certain embodiments, R represents -(CH ₂ ) _n -COOH, and n is 5.

In certain embodiments, R represents -(CH ₂ ) _n -COOH, and n is 6.

In certain embodiments, the polyene macrolide antibiotic is selected from the group consisting of amphotericin A, arenomycin B, candicidin D, candidin, candidoin, CE-108, etruscomycin, eurocidin D, eurocidin E, FR-008-VI, HA-2-91, hamycin A, levorin AO, levorin A3, mycoheptin, natamycin (pimaricin), nystatin Al, nystatin A2, nystatin A3, partricin A, polyfungin B, rimocidin, tetramycin A, tetramycin B, tetrin A, tetrin B, tetrin C, trichomycin A, trichomycin B, vacidin A, YS-822A, 3874 HI, 3874 H2, 3874 H3, and 67- 121-A.

In certain embodiments, the polyene macrolide antibioti c is amphotericin A.

In certain embodiments, the polyene macrolide antibioti c is arenomycin B.

In certain embodiments, the polyene macrolide antibioti c is candicidin D.

In certain embodiments, the polyene macrolide antibioti c is candidin.

In certain embodiments, the polyene macrolide antibioti c is candidoin.

In certain embodiments, the polyene macrolide antibioti c is CE-108.

In certain embodiments, the polyene macrolide antibioti c is etruscomycin.

In certain embodiments, the polyene macrolide antibioti c is eurocidin D.

In certain embodiments, the polyene macrolide antibioti c is eurocidin E.

In certain embodiments, the polyene macrolide antibioti c is FR-008-VI.

In certain embodiments, the polyene macrolide antibioti c is HA-2-91.

In certain embodiments, the polyene macrolide antibioti c is hamycin A.

In certain embodiments, the polyene macrolide antibioti c is levorin AO.

In certain embodiments, the polyene macrolide antibioti c is levorin A3.

In certain embodiments, the polyene macrolide antibioti c is mycoheptin.

In certain embodiments, the polyene macrolide antibioti c is natamycin (pimaricin)

In certain embodiments, the polyene macrolide antibioti c is nystatin Al .

In certain embodiments, the polyene macrolide antibioti c is nystatin A2.

In certain embodiments, the polyene macrolide antibioti c is nystatin A3.

In certain embodiments, the polyene macrolide antibioti c is partricin A.

In certain embodiments, the polyene macrolide antibioti c is polyfungin B.

In certain embodiments, the polyene macrolide antibioti c is rimocidin.

In certain embodiments, the polyene macrolide antibioti c is tetramycin A.

In certain embodiments, the polyene macrolide antibioti c is tetramycin B.

In certain embodiments, the polyene macrolide antibioti c is tetrin A.

In certain embodiments, the polyene macrolide antibioti c is tetrin B.

In certain embodiments, the polyene macrolide antibioti c is tetrin C.

In certain embodiments, the polyene macrolide antibioti c is trichomycin A. In certain embodiments, the polyene macrolide antibiotic is trichomycin B.

In certain embodiments, the polyene macrolide antibiotic is vacidin A.

In certain embodiments, the polyene macrolide antibiotic is YS-822A.

In certain embodiments, the polyene macrolide antibiotic is 3874 HI .

In certain embodiments, the polyene macrolide antibiotic is 3874 H2.

In certain embodiments, the polyene macrolide antibiotic is 3874 H3.

In certain embodiments, the polyene macrolide antibiotic is 67-121 -A.

Compounds of the Invention

As used herein, a "compound of the invention" refers to a urea derivative of a polyene macrolide antibiotic or a pharmaceutically acceptable salt thereof, as set forth herein. Without meaning to be limiting, compounds of the invention include the following

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-36- P T/US2015/049647

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In certain embodiments, a compound of the invention is purified, i.e., isolated from other compounds and components including the corresponding (i.e., unmodified) polyene macrolide.

In certain other embodiments, a compound of the invention is present in a mixture together with the corresponding polyene macrolide. In an embodiment, a compound of the invention represents at least 50 percent of the polyene macrolide present in such mixture. In various individual embodiments, a compound of the invention represents at least 50 percent, at least 60 percent, at least 70 percent, at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, or at least 95 percent of the polyene macrolide present in such mixture.

Pharmaceutical Compositions of the Invention

An aspect of the invention is a pharmaceutical composition, comprising a compound of the invention; and a pharmaceutically acceptable carrier. As described in further detail below, the term "pharmaceutically acceptable carrier" means one or more compatible solid or liquid filler, diluent or encapsulating substances which are suitable for administration to a human or other subject.

In an embodiment, the pharmaceutical composition is formulated for systemic administration. For example, in an embodiment, the pharmaceutical composition is formulated for intravenous administration. As another example, in an embodiment, the pharmaceutical composition is formulated for oral administration.

In an embodiment, the pharmaceutical composition is formulated for intraperitoneal administration.

In an embodiment, the pharmaceutical composition is formulated for topical administration.

In an embodiment, the pharmaceutical composition is formulated for local administration.

In an embodiment, the pharmaceutical composition is formulated for intrathecal administration.

Methods of the Invention

An aspect of the invention is a method of inhibiting growth of a yeast or fungus. The method includes the step of contacting the yeast or fungus with an effective amount of a urea derivative of a polyene macrolide antibiotic or a pharmaceutically acceptable salt thereof; wherein said urea derivative comprises a urea-containing substructure represented by

wherein

R represents hydrogen, alkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, aminoalkyl, or -(CH ₂ )„-COOH;

n is 1, 2, 3, 4, 5, or 6; and

said urea derivative has antifungal activity;

provided that the urea derivative of the polyene macrolide antibiotic is not

AmBMU, AmBAU, or AmBCU.

Yeasts are eukaryotic organisms classified in the kingdom Fungi. Yeasts are typically described as budding forms of fungi. Of particular importance in connection with the invention are species of yeast that can cause infections in mammalian hosts. Such infections most commonly occur in immunocompromised hosts, including hosts with compromised barriers to infection (e.g., burn victims) and hosts with compromised immune systems (e.g., hosts receiving chemotherapy or immune suppressive therapy, and hosts infected with HIV). Pathogenic yeast include, without limitation, various species of the genus Candida, as well as of Cryptococcus . Of particular note among pathogenic yeasts of the genus Candida are C albicans, C tropicalis, C stellatoidea, C. glabrata, C. krusei, C. parapsilosis, C. guilliermondii, C. viswanathii, and C. lusitaniae. The genus

Cryptococcus specifically includes Cryptococcus neoformans. Yeast can cause infections of mucosal membranes, for example oral, esophageal, and vaginal infections in humans, as well as infections of bone, blood, urogenital tract, and central nervous system. This list is exemplary and is not limiting in any way.

In addition to yeasts, Fungi include other eukaryotic organisms including molds and mushrooms. A number of fungi (apart from yeast) can cause infections in mammalian hosts. Such infections most commonly occur in immunocompromised hosts, including hosts with compromised barriers to infection (e.g., burn victims) and hosts with

compromised immune systems (e.g., hosts receiving chemotherapy or immune suppressive therapy, and hosts infected with HIV). Pathogenic fungi (apart from yeast) include, without limitation, species of Aspergillus, Rhizopus, Mucor, Histoplasma, Coccidioides,

Blastomyces, Trichophyton, Microsporum, and Epidermophyton. Of particular note among the foregoing are A. fumigatus, A.flavus, A. niger, H. capsulatum, C. immitis, and B.

dermatitidis. Fungi can cause deep tissue infections in lung, bone, blood, urogenital tract, and central nervous system, to name a few. Some fungi are responsible for infections of the skin and nails.

As used herein, the phrase "effective amount" refers to any amount that is sufficient to achieve a desired biological effect.

As used herein, "inhibit" or "inhibiting" means reduce by an objectively

measureable amount or degree compared to control. In one embodiment, inhibit or inhibiting means reduce by at least a statistically significant amount compared to control. In one embodiment, inhibit or inhibiting means reduce by at least 5 percent compared to control. In various individual embodiments, inhibit or inhibiting means reduce by at least 10, 15, 20, 25, 30, 33, 40, 50, 60, 67, 70, 75, 80, 90, or 95 percent compared to control.

In certain embodiments, the binding avidity for ergosterol of said urea derivative is at least 75 percent of the binding avidity for ergosterol of the counterpart polyene macrolide antibiotic. In various individual embodiments, the binding avidity for ergosterol of a compound of the invention is at least 80 percent, at least 85 percent, at least 90 percent, or at least 95 percent of the binding avidity for ergosterol of the counterpart polyene macrolide. In certain embodiments, the binding avidity for ergosterol of a compound of the invention is at least 100 percent of the binding avidity for ergosterol of the counterpart polyene macrolide.

In certain embodiments, the binding avidity for cholesterol of said urea derivative is less than or equal to 25 percent of the binding avidity for cholesterol of the counterpart polyene macrolide antibiotic. In various individual embodiments, the binding avidity for cholesterol of a compound of the invention is less than or equal to 20 percent, less than or equal to 15 percent, less than or equal to 10 percent, or less than or equal to 5 percent, of the binding avidity for cholesterol of the counterpart polyene macrolide. In an

embodiment, a compound of the invention has essentially no binding avidity for

cholesterol.

In an embodiment, the binding avidity for ergosterol of a compound of the invention is at least 75 percent of the binding avidity for ergosterol of the counterpart polyene macrolide; and the binding avidity for cholesterol of the compound of the invention is less than or equal to 25 percent of the binding avidity for cholesterol of the counterpart polyene macrolide.

In certain embodiments, R represents alkyl, aminoalkyl, or -(CH ₂ ) _n -COOH.

In certain embodiments, R represents alkyl.

In certain embodiments, R represents aminoalkyl.

In certain embodiments, R represents -(CH ₂ ) _n -COOH.

In certain embodiments, R represents -(CH ₂ ) _n -COOH, and n is 1.

In certain embodiments, R represents -(CH ₂ ) _n -COOH, and n is 2.

In certain embodiments, R represents -(CH ₂ ) _n -COOH, and n is 3.

In certain embodiments, R represents -(CH ₂ ) _n -COOH, and n is 4.

In certain embodiments, R represents -(CH ₂ ) _n -COOH, and n is 5.

In certain embodiments, R represents -(CH ₂ ) _n -COOH, and n is 6.

In certain embodiments, the polyene macrolide antibiotic is selected from the group consisting of amphotericin A, arenomycin B, candicidin D, candidin, candidoin, CE-108, etruscomycin, eurocidin D, eurocidin E, FR-008-VI, HA-2-91, hamycin A, levorin AO, levorin A3, mycoheptin, natamycin (pimaricin), nystatin Al, nystatin A2, nystatin A3, partricin A, polyfungin B, rimocidin, tetramycin A, tetramycin B, tetrin A, tetrin B, tetrin C, trichomycin A, trichomycin B, vacidin A, -822A, 3874 HI, 3874 H2, 3874 H3, and 67- 121-A.

In certain embodiments, the polyene macro ide antibioti c is amphotericin A.

In certain embodiments, the polyene macro ide antibioti c is arenomycin B.

In certain embodiments, the polyene macro ide antibioti c is candicidin D.

In certain embodiments, the polyene macro ide antibioti c is candidin.

In certain embodiments, the polyene macro ide antibioti c is candidoin.

In certain embodiments, the polyene macro ide antibioti c is CE-108.

In certain embodiments, the polyene macro ide antibioti c is etruscomycin.

In certain embodiments, the polyene macro ide antibioti c is eurocidin D.

In certain embodiments, the polyene macro ide antibioti c is eurocidin E.

In certain embodiments, the polyene macro ide antibioti c is FR-008-VI.

In certain embodiments, the polyene macro ide antibioti c is HA-2-91.

In certain embodiments, the polyene macro ide antibioti c is hamycin A.

In certain embodiments, the polyene macro ide antibioti c is levorin AO.

In certain embodiments, the polyene macro ide antibioti c is levorin A3.

In certain embodiments, the polyene macro ide antibioti c is mycoheptin.

In certain embodiments, the polyene macro ide antibioti c is natamycin (pimaricin)

In certain embodiments, the polyene macro ide antibioti c is nystatin Al .

In certain embodiments, the polyene macro ide antibioti c is nystatin A2.

In certain embodiments, the polyene macro ide antibioti c is nystatin A3.

In certain embodiments, the polyene macro ide antibioti c is partricin A.

In certain embodiments, the polyene macro ide antibioti c is polyfungin B.

In certain embodiments, the polyene macro ide antibioti c is rimocidin.

In certain embodiments, the polyene macro ide antibioti c is tetramycin A.

In certain embodiments, the polyene macro ide antibioti c is tetramycin B.

In certain embodiments, the polyene macro ide antibioti c is tetrin A.

In certain embodiments, the polyene macro ide antibioti c is tetrin B.

In certain embodiments, the polyene macro ide antibioti c is tetrin C.

In certain embodiments, the polyene macro ide antibioti c is trichomycin A.

In certain embodiments, the polyene macro ide antibioti c is trichomycin B.

In certain embodiments, the polyene macro ide antibioti c is vacidin A.

In certain embodiments, the polyene macro ide antibioti c is YS-822A. In certain embodiments, the polyene macro lide antibiotic is 3874 HI .

In certain embodiments, the polyene macrolide antibiotic is 3874 H2.

In certain embodiments, the polyene macrolide antibiotic is 3874 H3.

In certain embodiments, the polyene macrolide antibiotic is 67-121 -A.

An aspect of the invention is a method of treating a fungal infection, comprising administering to a subject in need thereof a therapeutically effective amount of a urea derivative of a polyene macrolide antibiotic or a pharmaceutically acceptable salt thereof; wherein said urea derivative comprises a urea-containing substructure represented by

wherein

R represents hydrogen, alkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, aminoalkyl, or -(CH ₂ ) _n -COOH;

n is 1, 2, 3, 4, 5, or 6; and

said urea derivative has antifungal activity;

provided that the urea derivative of the polyene macrolide antibiotic is not

AmBMU, AmBAU, or AmBCU.

As used herein, the terms "treating" and "treat" refer to performing an intervention that results in (a) preventing a condition or disease from occurring in a subject that may be at risk of developing or predisposed to having the condition or disease but has not yet been diagnosed as having it; (b) inhibiting a condition or disease, e.g., slowing or arresting its development; or (c) relieving or ameliorating a condition or disease, e.g., causing regression of the condition or disease. In one embodiment the terms "treating" and "treat" refer to performing an intervention that results in (a) inhibiting a condition or disease, e.g., slowing or arresting its development; or (b) relieving or ameliorating a condition or disease, e.g., causing regression of the condition or disease.

A "yeast or fungal infection" as used herein refers to an infection with a yeast or fungus as defined herein. As used herein, a "subject" refers to a living mammal. In various embodiments a subject is a non-human mammal, including, without limitation, a mouse, rat, hamster, guinea pig, rabbit, sheep, goat, cat, dog, pig, horse, cow, or non-human primate. In one embodiment a subject is a human.

As used herein, a "subject having a yeast or fungal infection" refers to a subject that exhibits at least one objective manifestation of a yeast or fungal infection. In one embodiment a subject having a yeast or fungal infection is a subject that has been diagnosed as having a yeast or fungal infection and is in need of treatment thereof.

Methods of diagnosing a yeast or fungal infection are well known and need not be described here in any detail.

As used herein, "administering" has its usual meaning and encompasses

administering by any suitable route of administration, including, without limitation, intravenous, intramuscular, intraperitoneal, intrathecal, subcutaneous, direct injection (for example, into a tumor), mucosal, inhalation, oral, and topical.

As used herein, the phrase "therapeutically effective amount" refers to any amount that is sufficient to achieve a desired therapeutic effect, e.g., to treat a yeast or fungal infection.

In certain embodiments, the binding avidity for ergosterol of said urea derivative is at least 75 percent of the binding avidity for ergosterol of the counterpart polyene macrolide antibiotic. In various individual embodiments, the binding avidity for ergosterol of a compound of the invention is at least 80 percent, at least 85 percent, at least 90 percent, or at least 95 percent of the binding avidity for ergosterol of the counterpart polyene macrolide. In certain embodiments, the binding avidity for ergosterol of a compound of the invention is at least 100 percent of the binding avidity for ergosterol of the counterpart polyene macrolide.

In certain embodiments, the binding avidity for cholesterol of said urea derivative is less than or equal to 25 percent of the binding avidity for cholesterol of the counterpart polyene macrolide antibiotic. In various individual embodiments, the binding avidity for cholesterol of a compound of the invention is less than or equal to 20 percent, less than or equal to 15 percent, less than or equal to 10 percent, or less than or equal to 5 percent, of the binding avidity for cholesterol of the counterpart polyene macrolide. In an

embodiment, a compound of the invention has essentially no binding avidity for cholesterol. In an embodiment, the binding avidity for ergosterol of a compound of the invention is at least 75 percent of the binding avidity for ergosterol of the counterpart polyene macrolide; and the binding avidity for cholesterol of the compound of the invention is less than or equal to 25 percent of the binding avidity for cholesterol of the counterpart polyene macrolide.

In certain embodiments, R represents alkyl, aminoalkyl, or -(CH ₂ ) _n -COOH.

In certain embodiments, R represents alkyl.

In certain embodiments, R represents aminoalkyl.

In certain embodiments, R represents -(CH ₂ ) _n -COOH.

In certain embodiments, R represents -(CH ₂ ) _n -COOH, and n is 1.

In certain embodiments, R represents -(CH ₂ ) _n -COOH, and n is 2.

In certain embodiments, R represents -(CH ₂ ) _n -COOH, and n is 3.

In certain embodiments, R represents -(CH ₂ ) _n -COOH, and n is 4.

In certain embodiments, R represents -(CH ₂ ) _n -COOH, and n is 5.

In certain embodiments, R represents -(CH ₂ ) _n -COOH, and n is 6.

In certain embodiments, the polyene macrolide antibiotic is selected from the group consisting of amphotericin A, arenomycin B, candicidin D, candidin, candidoin, CE-108, etruscomycin, eurocidin D, eurocidin E, FR-008-VI, HA-2-91, hamycin A, levorin AO, levorin A3, mycoheptin, natamycin (pimaricin), nystatin Al, nystatin A2, nystatin A3, partricin A, polyfungin B, rimocidin, tetramycin A, tetramycin B, tetrin A, tetrin B, tetrin C, trichomycin A, trichomycin B, vacidin A, YS-822A, 3874 HI, 3874 H2, 3874 H3, and 67- 121-A.

In certain embodiments, the polyene macrolide antibiotic is amphotericin A.

In certain embodiments, the polyene macrolide antibiotic is arenomycin B.

In certain embodiments, the polyene macrolide antibiotic is candicidin D.

In certain embodiments, the polyene macrolide antibiotic is candidin.

In certain embodiments, the polyene macrolide antibiotic is candidoin.

In certain embodiments, the polyene macrolide antibiotic is CE-108.

In certain embodiments, the polyene macrolide antibiotic is etruscomycin.

In certain embodiments, the polyene macrolide antibiotic is eurocidin D.

In certain embodiments, the polyene macrolide antibiotic is eurocidin E.

In certain embodiments, the polyene macrolide antibiotic is FR-008-VI.

In certain embodiments, the polyene macrolide antibiotic is HA-2-91. In certain embodiments, the polyene macrolide antibiotic is hamycin A.

In certain embodiments, the polyene macrolide antibiotic is levorin AO.

In certain embodiments, the polyene macrolide antibiotic is levorin A3.

In certain embodiments, the polyene macrolide antibiotic is mycoheptin.

In certain embodiments, the polyene macrolide antibiotic is natamycin (pimaricin)

In certain embodiments, the polyene macrolide antibiotic is nystatin Al .

In certain embodiments, the polyene macrolide antibiotic is nystatin A2.

In certain embodiments, the polyene macrolide antibiotic is nystatin A3.

In certain embodiments, the polyene macrolide antibiotic is partricin A.

In certain embodiments, the polyene macrolide antibiotic is polyfungin B.

In certain embodiments, the polyene macrolide antibiotic is rimocidin.

In certain embodiments, the polyene macrolide antibiotic is tetramycin A.

In certain embodiments, the polyene macrolide antibiotic is tetramycin B.

In certain embodiments, the polyene macrolide antibiotic is tetrin A.

In certain embodiments, the polyene macrolide antibiotic is tetrin B.

In certain embodiments, the polyene macrolide antibiotic is tetrin C.

In certain embodiments, the polyene macrolide antibiotic is trichomycin A.

In certain embodiments, the polyene macrolide antibiotic is trichomycin B.

In certain embodiments, the polyene macrolide antibiotic is vacidin A.

In certain embodiments, the polyene macrolide antibiotic is YS-822A.

In certain embodiments, the polyene macrolide antibiotic is 3874 HI .

In certain embodiments, the polyene macrolide antibiotic is 3874 H2.

In certain embodiments, the polyene macrolide antibiotic is 3874 H3.

In certain embodiments, the polyene macrolide antibiotic is 67-121 -A.

In certain embodiments, the urea derivative is administered systemically. For example, in certain embodiments, the urea derivative is administered intravenously. As another example, in certain embodiments, the urea derivative is administered orally.

In certain embodiments, the urea derivative is administered intraperitoneally.

In certain embodiments, the urea derivative is administered intrathecally.

In an embodiment, the urea derivative is administered locally.

In certain embodiments, the urea derivative is administered topically.

As stated above, an "effective amount" refers to any amount that is sufficient to achieve a desired biological effect. Combined with the teachings provided herein, by choosing among the various active compounds and weighing factors such as potency, relative bioavailability, patient body weight, severity of adverse side-effects and preferred mode of administration, an effective prophylactic or therapeutic treatment regimen can be planned which does not cause substantial unwanted toxicity and yet is effective to treat the particular subject. The effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular compound of the invention being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular compound of the invention and/or other therapeutic agent without necessitating undue experimentation. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to some medical judgment. Multiple doses per day may be contemplated to achieve appropriate systemic levels of compounds.

Appropriate systemic levels can be determined by, for example, measurement of the patient's peak or sustained plasma level of the drug. "Dose" and "dosage" are used interchangeably herein.

Generally, daily intravenous doses of compounds of the invention will be, for human subjects, similar to or greater than usual daily intravenous doses of corresponding polyene macrolide comprising a mycosaminyl moiety. Similarly, daily other parenteral doses of compounds of the invention will be, for human subjects, similar to or greater than usual daily other parenteral doses of corresponding polyene macrolide comprising a mycosaminyl moiety.

In one embodiment, intravenous administration of a compound of the invention may typically be from 0.1 mg/kg/day to 20 mg/kg/day. In one embodiment, intravenous administration of a compound of the invention may typically be from 0.1 mg/kg/day to 2 mg/kg/day. In one embodiment, intravenous administration of a compound of the invention may typically be from 0.5 mg/kg/day to 5 mg/kg/day. In one embodiment, intravenous administration of a compound of the invention may typically be from 1 mg/kg/day to 20 mg/kg/day. In one embodiment, intravenous administration of a compound of the invention may typically be from 1 mg/kg/day to 10 mg/kg/day. Intravenous dosing thus may be similar to, or advantageously, may exceed maximal tolerated doses of a given

corresponding polyene macrolide comprising a mycosaminyl moiety.

Generally, daily oral doses of active compounds will be, for human subjects, from about 0.01 milligrams/kg per day to 1000 milligrams/kg per day. It is expected that oral doses in the range of 0.5 to 50 milligrams/kg, in one or more administrations per day, will yield therapeutic results. Dosage may be adjusted appropriately to achieve desired drug levels, local or systemic, depending upon the mode of administration. For example, it is expected that intravenous administration would be from one order to several orders of magnitude lower dose per day. In the event that the response in a subject is insufficient at such doses, even higher doses (or effective higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. Multiple doses per day are contemplated to achieve appropriate systemic levels of compounds.

For any compound described herein the therapeutically effective amount can be initially determined from animal models. A therapeutically effective dose can also be determined from human data for compounds of the invention which have been tested in humans and for compounds which are known to exhibit similar pharmacological activities, such as other related active agents. Higher doses may be required for parenteral administration. The applied dose can be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dose to achieve maximal efficacy based on the methods described above and other methods as are well-known in the art is well within the capabilities of the ordinarily skilled artisan.

The formulations of the invention are administered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients.

For use in therapy, an effective amount of the compound of the invention can be administered to a subject by any mode that delivers the compound of the invention to the desired surface. Administering the pharmaceutical composition of the present invention may be accomplished by any means known to the skilled artisan. Routes of administration include but are not limited to intravenous, intramuscular, intraperitoneal, intravesical (urinary bladder), oral, subcutaneous, direct injection (for example, into a tumor or abscess), mucosal (e.g., topical to eye), inhalation, and topical.

For intravenous and other parenteral routes of administration, a compound of the invention generally may be formulated similarly to the corresponding polyene macrolide comprising a mycosaminyl moiety. For example, C2'epiAmB can be formulated as a lyophilized preparation with desoxycholic acid, as a lyophilized preparation of liposome- intercalated or -encapsulated active compound, as a lipid complex in aqueous suspension, or as a cholesteryl sulfate complex. Lyophilized formulations are generally reconstituted in suitable aqueous solution, e.g., in sterile water or saline, shortly prior to administration.

For oral administration, the compounds of the invention can be formulated readily by combining the active compound(s) with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated. Pharmaceutical preparations for oral use can be obtained as solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or

polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Optionally the oral formulations may also be formulated in saline or buffers, e.g., EDTA for neutralizing internal acid conditions or may be administered without any carriers.

Also specifically contemplated are oral dosage forms of the above component or components. The component or components may be chemically modified so that oral delivery of the derivative is efficacious. Generally, the chemical modification

contemplated is the attachment of at least one moiety to the component molecule itself, where said moiety permits (a) inhibition of acid hydrolysis; and (b) uptake into the blood stream from the stomach or intestine. Also desired is the increase in overall stability of the component or components and increase in circulation time in the body. Examples of such moieties include: polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline. Abuchowski and Davis, "Soluble Polymer-Enzyme Adducts", In: Enzymes as Drugs, Hocenberg and Roberts, eds., Wiley-Interscience, New York, N.Y., pp. 367-383 (1981); Newmark et al, J Appl Biochem 4: 185-9 (1982). Other polymers that could be used are poly-l,3-dioxolane and poly-l,3,6-tioxocane. Preferred for pharmaceutical usage, as indicated above, are polyethylene glycol moieties. For the component (or derivative) the location of release may be the stomach, the small intestine (the duodenum, the jejunum, or the ileum), or the large intestine. One skilled in the art has available formulations which will not dissolve in the stomach, yet will release the material in the duodenum or elsewhere in the intestine. Preferably, the release will avoid the deleterious effects of the stomach environment, either by protection of the compound of the invention (or derivative) or by release of the biologically active material beyond the stomach environment, such as in the intestine.

To ensure full gastric resistance a coating impermeable to at least pH 5.0 is essential. Examples of the more common inert ingredients that are used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and shellac. These coatings may be used as mixed films.

A coating or mixture of coatings can also be used on tablets, which are not intended for protection against the stomach. This can include sugar coatings, or coatings which make the tablet easier to swallow. Capsules may consist of a hard shell (such as gelatin) for delivery of dry therapeutic (e.g., powder); for liquid forms, a soft gelatin shell may be used. The shell material of cachets could be thick starch or other edible paper. For pills, lozenges, molded tablets or tablet triturates, moist massing techniques can be used.

The therapeutic can be included in the formulation as fine multi-particulates in the form of granules or pellets of particle size about 1 mm. The formulation of the material for capsule administration could also be as a powder, lightly compressed plugs or even as tablets. The therapeutic could be prepared by compression.

Colorants and flavoring agents may all be included. For example, the compound of the invention (or derivative) may be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage containing colorants and flavoring agents.

One may dilute or increase the volume of the therapeutic with an inert material. These diluents could include carbohydrates, especially mannitol, a-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch. Certain inorganic salts may be also be used as fillers including calcium triphosphate, magnesium carbonate and sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell. Disintegrants may be included in the formulation of the therapeutic into a solid dosage form. Materials used as disintegrates include but are not limited to starch, including the commercial disintegrant based on starch, Explotab. Sodium starch glycolate,

Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite may all be used. Another form of the disintegrants are the insoluble cationic exchange resins. Powdered gums may be used as disintegrants and as binders and these can include powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants.

Binders may be used to hold the therapeutic agent together to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatin.

Others include methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both be used in alcoholic solutions to granulate the therapeutic.

An anti-frictional agent may be included in the formulation of the therapeutic to prevent sticking during the formulation process. Lubricants may be used as a layer between the therapeutic and the die wall, and these can include but are not limited to; stearic acid including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants may also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000.

Glidants that might improve the flow properties of the drug during formulation and to aid rearrangement during compression might be added. The glidants may include starch, talc, pyrogenic silica and hydrated silicoaluminate.

To aid dissolution of the therapeutic into the aqueous environment a surfactant might be added as a wetting agent. Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents which can be used and can include benzalkonium chloride and benzethonium chloride. Potential non-ionic detergents that could be included in the formulation as surfactants include lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the formulation of the compound of the invention or derivative either alone or as a mixture in different ratios.

Pharmaceutical preparations which can be used orally include push- fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. Microspheres formulated for oral administration may also be used. Such microspheres have been well defined in the art. All formulations for oral administration should be in dosages suitable for such

administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by inhalation, compounds of the invention for use according to the present invention may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

Also contemplated herein is pulmonary delivery of a compound of the invention (or salts thereof). The compound of the invention (or derivative) is delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream. Other reports of inhaled molecules include Adjei et al, Pharm Res 7:565-569 (1990); Adjei et al, Int J Pharmaceutics 63:135-144 (1990) (leuprolide acetate); Braquet et al, J

Cardiovasc Pharmacol 13(suppl. 5): 143-146 (1989) (endothelin-1); Hubbard et al, Annal Int Med 3:206-212 (1989) (a 1 -antitrypsin); Smith et al, 1989, J Clin Invest 84: 1145-1146 (a- 1 -proteinase); Oswein et al, 1990, "Aerosolization of Proteins", Proceedings of

Symposium on Respiratory Drug Delivery II, Keystone, Colorado, March, (recombinant human growth hormone); Debs et al., 1988, J Immunol 140:3482-3488 (interferon-gamma and tumor necrosis factor alpha) and Platz et al, U.S. Pat. No. 5,284,656 (granulocyte colony stimulating factor; incorporated by reference). A method and composition for pulmonary delivery of drugs for systemic effect is described in U.S. Pat. No. 5,451,569 (incorporated by reference), issued Sep. 19, 1995 to Wong et al.

Contemplated for use in the practice of this invention are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art.

Some specific examples of commercially available devices suitable for the practice of this invention are the Ultravent nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the Acorn II nebulizer, manufactured by Marquest Medical Products, Englewood, Colo.; the Ventolin metered dose inhaler, manufactured by Glaxo Inc., Research Triangle Park, North Carolina; and the Spinhaler powder inhaler, manufactured by Fisons Corp., Bedford, Mass.

All such devices require the use of formulations suitable for the dispensing of the compound of the invention (or salt thereof). Typically, each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to the usual diluents, adjuvants and/or carriers useful in therapy. Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated. Chemically modified compound of the invention may also be prepared in different formulations depending on the type of chemical modification or the type of device employed.

Formulations suitable for use with a nebulizer, either jet or ultrasonic, will typically comprise compound of the invention (or derivative) dissolved in water at a concentration of about 0.1 to 25 mg of biologically active compound of the invention per mL of solution. The formulation may also include a buffer and a simple sugar (e.g., for compound of the invention stabilization and regulation of osmotic pressure). The nebulizer formulation may also contain a surfactant, to reduce or prevent surface induced aggregation of the compound of the invention caused by atomization of the solution in forming the aerosol.

Formulations for use with a metered-dose inhaler device will generally comprise a finely divided powder containing the compound of the invention (or derivative) suspended in a propellant with the aid of a surfactant. The propellant may be any conventional material employed for this purpose, such as a chlorofluorocarbon, a

hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2- tetrafluoroethane, or combinations thereof. Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid may also be useful as a surfactant.

Formulations for dispensing from a powder inhaler device will comprise a finely divided dry powder containing compound of the invention (or derivative) and may also include a bulking agent, such as lactose, sorbitol, sucrose, or mannitol in amounts which facilitate dispersal of the powder from the device, e.g., 50 to 90% by weight of the formulation. The compound of the invention (or derivative) should advantageously be prepared in particulate form with an average particle size of less than 10 micrometers (μιη), most preferably 0.5 to 5 μιη, for most effective delivery to the deep lung.

Nasal delivery of a pharmaceutical composition of the present invention is also contemplated. Nasal delivery allows the passage of a pharmaceutical composition of the present invention to the blood stream directly after administering the therapeutic product to the nose, without the necessity for deposition of the product in the lung. Formulations for nasal delivery include those with dextran or cyclodextran.

For nasal administration, a useful device is a small, hard bottle to which a metered dose sprayer is attached. In one embodiment, the metered dose is delivered by drawing the pharmaceutical composition of the present invention solution into a chamber of defined volume, which chamber has an aperture dimensioned to aerosolize and aerosol formulation by forming a spray when a liquid in the chamber is compressed. The chamber is compressed to administer the pharmaceutical composition of the present invention. In a specific embodiment, the chamber is a piston arrangement. Such devices are commercially available.

Alternatively, a plastic squeeze bottle with an aperture or opening dimensioned to aerosolize an aerosol formulation by forming a spray when squeezed is used. The opening is usually found in the top of the bottle, and the top is generally tapered to partially fit in the nasal passages for efficient administration of the aerosol formulation. Preferably, the nasal inhaler will provide a metered amount of the aerosol formulation, for administration of a measured dose of the drug.

The compounds, when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium

carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active compounds may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen- free water, before use.

The compounds may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described above, compounds of the invention may also be formulated as a depot preparation. Such long acting formulations may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous or saline solutions for inhalation, microencapsulated, encochleated, coated onto

microscopic gold particles, contained in liposomes, nebulized, aerosols, pellets for implantation into the skin, or dried onto a sharp object to be scratched into the skin. The pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above. The pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of methods for drug delivery, see Langer R, Science 249: 1527- 33 (1990).

A compound of the invention and optionally other therapeutics may be administered per se (neat) or in the form of a pharmaceutically acceptable salt. When used in medicine the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof. Such salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic, tartaric, citric, methane sulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, and benzene sulphonic. Also, such salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.

Suitable buffering agents include: acetic acid and a salt (1-2% w/v); citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5%) w/v); and phosphoric acid and a salt (0.8- 2%> w/v). Suitable preservatives include benzalkonium chloride (0.003-0.03%) w/v);

chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).

Pharmaceutical compositions of the invention contain an effective amount of a compound of the invention and optionally other therapeutic agents included in a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" means one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration to a human or other vertebrate animal. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being commingled with the compounds of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficiency.

The therapeutic agent(s), including specifically but not limited to compounds of the invention, may be provided in particles. Particles as used herein means nanoparticles or microparticles (or in some instances larger particles) which can consist in whole or in part of the compound of the invention or the other therapeutic agent(s) as described herein. The particles may contain the therapeutic agent(s) in a core surrounded by a coating, including, but not limited to, an enteric coating. The therapeutic agent(s) also may be dispersed throughout the particles. The therapeutic agent(s) also may be adsorbed into the particles. The particles may be of any order release kinetics, including zero-order release, first-order release, second-order release, delayed release, sustained release, immediate release, and any combination thereof, etc. The particle may include, in addition to the therapeutic agent(s), any of those materials routinely used in the art of pharmacy and medicine, including, but not limited to, erodible, nonerodible, biodegradable, or nonbiodegradable material or combinations thereof. The particles may be microcapsules which contain the compound of the invention in a solution or in a semi-solid state. The particles may be of virtually any shape.

Both non-biodegradable and biodegradable polymeric materials can be used in the manufacture of particles for delivering the therapeutic agent(s). Such polymers may be natural or synthetic polymers. The polymer is selected based on the period of time over which release is desired. Bioadhesive polymers of particular interest include bioerodible hydrogels described in Sawhney H S et al. (1993) Macromolecules 26:581-7, the teachings of which are incorporated herein. These include polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate).

The therapeutic agent(s) may be contained in controlled release systems. The term "controlled release" is intended to refer to any drug-containing formulation in which the manner and profile of drug release from the formulation are controlled. This refers to immediate as well as non-immediate release formulations, with non-immediate release formulations including but not limited to sustained release and delayed release

formulations. The term "sustained release" (also referred to as "extended release") is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that preferably, although not necessarily, results in substantially constant blood levels of a drug over an extended time period. The term "delayed release" is used in its conventional sense to refer to a drug formulation in which there is a time delay between administration of the formulation and the release of the drug there from. "Delayed release" may or may not involve gradual release of drug over an extended period of time, and thus may or may not be "sustained release."

Use of a long-term sustained release implant may be particularly suitable for treatment of chronic conditions. "Long-term" release, as used herein, means that the implant is constructed and arranged to deliver therapeutic levels of the active ingredient for at least 7 days, and preferably 30-60 days. Long-term sustained release implants are well- known to those of ordinary skill in the art and include some of the release systems described above.

An aspect of the invention is a method of making a urea derivative of a polyene macr lide antibiotic according to any one of the six transformations shown in Scheme 2

Scheme 2

wherein

1 represents a substructure of a protected oxazolidinone derivative of a polyene macrolide antibiotic; and

each R is independently selected from the group consisting of hydrogen, halogen, straight- or branched-chain alkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, aryl, heteroaryl, aralkyl, heteroaralkyl, hydroxyl, sulfhydryl, carboxyl, amino, amido, azido, nitro, cyano, aminoalkyl, and alkoxyl;

provided that the polyene macro lide antibiotic is not amphotericin B.

In certain embodiments, the polyene macrolide antibiotic is selected from the group consisting of amphotericin A, arenomycin B, candicidin D, candidin, candidoin, CE-108, etruscomycin, eurocidin D, eurocidin E, FR-008-VI, HA-2-91, hamycin A, levorin AO, levorin A3, mycoheptin, natamycin (pimaricin), nystatin Al, nystatin A2, nystatin A3, partricin A, polyfungin B, rimocidin, tetramycin A, tetramycin B, tetrin A, tetrin B, tetrin C, trichomycin A, trichomycin B, vacidin A, YS-822A, 3874 HI, 3874 H2, 3874 H3, and 67- 121-A.

In certain embodiments, the polyene macrolide antibiotic is amphotericin A.

In certain embodiments, the polyene macrolide antibiotic is arenomycin B.

In certain embodiments, the polyene macrolide antibiotic is candicidin D.

In certain embodiments, the polyene macrolide antibiotic is candidin.

In certain embodiments, the polyene macrolide antibiotic is candidoin.

In certain embodiments, the polyene macrolide antibiotic is CE-108.

In certain embodiments, the polyene macrolide antibiotic is etruscomycin.

In certain embodiments, the polyene macrolide antibiotic is eurocidin D.

In certain embodiments, the polyene macrolide antibiotic is eurocidin E.

In certain embodiments, the polyene macrolide antibiotic is FR-008-VI.

In certain embodiments, the polyene macrolide antibiotic is HA-2-91.

In certain embodiments, the polyene macrolide antibiotic is hamycin A.

In certain embodiments, the polyene macrolide antibiotic is levorin AO.

In certain embodiments, the polyene macrolide antibiotic is levorin A3.

In certain embodiments, the polyene macrolide antibiotic is mycoheptin.

In certain embodiments, the polyene macrolide antibiotic is natamycin (pimaricin)

In certain embodiments, the polyene macrolide antibiotic is nystatin Al .

In certain embodiments, the polyene macrolide antibiotic is nystatin A2.

In certain embodiments, the polyene macrolide antibiotic is nystatin A3.

In certain embodiments, the polyene macrolide antibiotic is partricin A.

In certain embodiments, the polyene macrolide antibiotic is polyfungin B.

In certain embodiments, the polyene macrolide antibiotic is rimocidin.

In certain embodiments, the polyene macrolide antibiotic is tetramycin A. In certain embodiments, the polyene macro lide antibiotic is tetramycin B.

In certain embodiments, the polyene macrolide antibiotic is tetrin A.

In certain embodiments, the polyene macrolide antibiotic is tetrin B.

In certain embodiments, the polyene macrolide antibiotic is tetrin C.

In certain embodiments, the polyene macrolide antibiotic is trichomycin A.

In certain embodiments, the polyene macrolide antibiotic is trichomycin B.

In certain embodiments, the polyene macrolide antibiotic is vacidin A.

In certain embodiments, the polyene macrolide antibiotic is YS-822A.

In certain embodiments, the polyene macrolide antibiotic is 3874 HI .

In certain embodiments, the polyene macrolide antibiotic is 3874 H2.

In certain embodiments, the polyene macrolide antibiotic is 3874 H3.

In certain embodiments, the polyene macrolide antibiotic is 67-121 -A.

In certain embodiments, the method of making comprises treating a protected variant of a macrolide antibiotic with diphenyl phosphoryl azide (DPP A) to promote a stereospecific Curtius rearrangement in which the C16-C41 bond (numbering according to AmB structure) is cleaved and the resulting isocyanate is intramolecularly trapped by the neighboring C15 alcohol to form an oxazolidinone (1). See, for example, Scheme 1. This oxazolidinone is surprisingly reactive to ring-opening with primary amines under mild conditions to yield urea-containing derivatives having a C16-nitrogen bond. Interestingly, the parent heterocycle, 2-oxazolidinone, is unreactive under the same conditions.

In certain embodiments, the method entails directly converting the parent macrolide antibiotic to a corresponding urea derivative in a scalable one-pot operation involving serial addition of diphenyl phosphoryl azide (DPP A), an amine, and aqueous acid. Definitions

The term "alkyl" is art-recognized, and includes saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In certain embodiments, a straight-chain or branched-chain alkyl has about 30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straight chain, C3-C30 for branched chain), and alternatively, about 20 or fewer. Likewise, cycloalkyls have from about 3 to about 10 carbon atoms in their ring structure, and alternatively about 5, about 6, or about 7 carbons in the ring structure. The terms "alkenyl" and "alkynyl" are art-recognized and refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively.

Unless the number of carbons is otherwise specified, "lower alkyl" refers to an alkyl group, as defined above, but having from one to about ten carbons, alternatively from one to about six carbon atoms in its backbone structure. Likewise, "lower alkenyl" and "lower alkynyl" have similar chain lengths.

The term "aryl" is art-recognized and refers to 5-, 6- and 7-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. Those aryl groups having heteroatoms in the ring structure may also be referred to as "heteroaryls" or "aryl heterocycles" or

"heteroaromatics." The aromatic ring may be substituted at one or more ring positions with such substituents as, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF ₃ , -CN, or the like. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (the rings are "fused rings") wherein at least one of the rings is aromatic, e.g., the other cyclic rings may be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls. Nonlimiting examples of polycyclic aryls include naphthalene, anthracene, purines, and pyrene.

The term "heteroatom" is art-recognized and refers to an atom of any element other than carbon or hydrogen. Illustrative heteroatoms include boron, nitrogen, oxygen, phosphorus, sulfur and selenium.

The terms "alkoxyl" or "alkoxy" are art-recognized and refer to an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like.

The term "aralkyl" is art-recognized and refers to an alkyl group substituted with an aryl group (i.e., an aromatic or heteroaromatic group).

The term "azido" is art-recognized and refers to -N ₃ .

The term "carboxyl" is art-recognized and refers to -COOH.

The term "cyano" is art-recognized and refers to -CN. The term "halogen" is art-recognized and refers to -F, -CI, -Br or -I.

The term "hydroxyl" is art-recognized and refers -OH.

The term "nitro" is art-recognized and refers to -N0 ₂ .

The term "sulfhydryl" is art-recognized and refers to -SH.

The term "sulfonyl" is art-recognized and refers to -S0 ₂ .

The terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines, e.g., a moiety that may be represented by the general formulas:

R50

R50

/ +

N N R53

\

^R 51 R52

wherein R50, R51 and R52 each independently represent a hydrogen, an alkyl, an alkenyl, -(CH ₂ ) _m -R61 , or R50 and R51 , taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure; R61 represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and m is zero or an integer in the range of 1 to 8. In other embodiments, R50 and R51 (and optionally R52) each independently represent a hydrogen, an alkyl, an alkenyl, or -(CH ₂ ) _m -R61. Thus, the term "alkylamine" includes an amine group, as defined above, having a substituted or unsubstituted alkyl attached thereto, i.e., at least one of R50 and R51 is an alkyl group.

The term "amido" is art recognized as an amino-substituted carbonyl and includes a moiety that may be represented by the general formula:

wherein R50 and R51 are as defined above. Certain embodiments of the amide in the present invention will not include imides which may be unstable.

The term "aminoalkyl" is art recognized and refers to an alkyl group, as defined above, having an amino radical, as defined above, attached thereto. Representative aminoalkyl groups include aminomethyl, aminoethyl, and aminopropyl. EXAMPLES

Having now described the present invention in detail, the same will be more clearly understood by reference to the following examples, which are included herewith for purposes of illustration only and are not intended to be limiting of the invention.

Materials

Commercially available materials were purchased from Aldrich Chemical Co. (Milwaukee, WI), AKSci (Union City, CA), Fisher Scientific (Hampton, NH), Lipoid (Luwigshafen, Germany), and Silicycle (Quebec, Canada) and used without further purification unless noted otherwise. All solvents were dispensed from a solvent purification as described by Pangborn and coworkers (THF, Et ₂ 0: dry neutral alumina; DMSO, DMF, CH ₃ OH : activated molecular sieves). Pangborn AB et al, Organometallics 15: 1518-1520 (1996). Triethylamine and pyridine were freshly distilled under nitrogen from CaH ₂ .

Camphorsulfonic acid was recrystallized from ethanol. Water was doubly distilled or obtained from a Millipore (Billerica, MA) MilliQ water purification system.

Reactions

When necessary due to light and air sensitivity of particular compounds, manipulations were carried out under low light conditions and compounds were stored under an anaerobic atmosphere. All reactions were performed in oven- or flame-dried glassware under an atmosphere of argon unless otherwise indicated. Reactions were monitored by RP-HPLC using an Agilent 1200 Series HPLC system equipped with an Agilent Zorbax Eclipse C ₁₈ 3.5-um, 4.6 x 75 mm column with UV detection at 383 nm at 1.2 mL/min, or an Agilent 6230 ESI TOF LC/MS system equipped with an Agilent Zorbax Eclipse Ci8 1.8-μιη, 2.1 x 50 mm column with UV detection at 383 nm at 0.4 mL/min. Purification and Analysis

1H NMR spectra were recorded at room temperature on a Varian Unity Inova Narrow Bore spectrometer operating at a 1H frequency of 500 MHz with a Varian 5 mm ¹ H{ ¹³ C/ ¹⁵ N} pulsed-field gradient Z probe or an Agilent VNMRS 750 MHz spectrometer with a Varian 5mm indirect-detection probe ¹ H{ ¹³ C/ ¹⁵ N} probe equipped with Χ,Υ,Ζ-field gradient capability. Chemical shifts (δ) are reported in parts per million (ppm) downfield added tetramethylsilane (δ = 0.0). Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, dd = doublet of doublets, m = multiplet, b = broad, app = apparent), coupling constant (J) in Hertz (Hz) and integration. ¹³ C spectra were recorded at room temperature with a Varian Unity Inova spectrometer operating at a C frequency of 125 MHz with a 5 mm Nalorac gradient { ¹³ C/ ¹⁵ N} 1H quad probe or a Varian Unity Inova spectrometer operating at a ¹³ C frequency of 150 MHz and equipped with a Varian 5 mm 600 DB Auto X probe. Chemical shifts (δ) are reported downfield of added

tetramethylsilane (δ = 0.00) or are referenced to the carbon resonances in the NMR solvent ((CD ₃ ) ₂ SO, δ = 39.52, center line). MS analysis was performed with an Applied Biosystems Micromass Ultima system with ESI ionization. High resolution mass spectra (HRMS) were obtained at the University of Illinois mass spectrometry facility. All synthesized compounds gave HRMS within 5 ppm of the calculated values.

Large scale prep purification of macro lide antibiotics and their derivatives is carried out by preparative HPLC purification using either an Agilent 1100 or 1200 series instrument equipped with either a Waters SunFire Prep C ₁₈ OBD 5-μιη, 30 x 150 mm at 25 mL/min, or a Waters Sunfire Prep C ₁₈ OBD 5-μιη, 50 x 250 mm column at 75 mL/min with detection at 383 nm and an eluent of acetonitrile and 0.3% formic acid. The purity of compounds is determined by HPLC analysis using an Agilent Zorbax Eclipse C ₁₈ 1.8-um, 2.1 x 50 mm column with detection at 383 nm and an eluent of acetonitrile and 0.1% formic acid in water unless otherwise indicated.

Example 1

Synthesis of oxazolidinone intermediate (I)

A round bottom flask was charged with natamycin (5.0 g, 7.5 mmol, 1 equiv.) and Fmoc-succinimide (3.8 g, 11.2 mmol, 1.5 equiv.) which were dissolved in a 2: 1 mixture of DMF:MeOH (250 mL) at room temperature. Pyridine (0.98 mL, 43.1 mmol, 5.75 equiv.) was subsequently added and the reaction was stirred for 12 hours at room temperature. The reaction was then poured into diethyl ether (7.5 L). After stirring for 30 minutes, the resulting yellow precipitate was isolated via Buchner filtration using Whatman #50 filter paper to afford a yellow solid. The filter cake was dried on the filter for 10 minutes and then stored under vacuum for one hour.

The resulting powder was dissolved in 1 : 1 THF:MeOH (150 mL) and cooled to 0 °C. To this solution was added camphorsulfonic acid (964 mg, 4.15 mmol, 0.55 equiv.) and the resulting mixture was stirred for 1 hour at 0 °C. The reaction was then quenched at 0 °C with triethylamine (0.58 mL, 4.15 mmol, 0.55 equiv.). The reaction was concentrated in vacuo, removing approximately half of the solvent. The resulting saturated solution was poured into 1 :1 hexanes: diethyl ether (5.0 L). The white precipitate was collected via Buchner filtration using Whatman #50 filter paper and washed with diethyl ether (200 mL) to yield a white solid that was then stored under vacuum for one hour.

The resulting solid was dissolved in THF (375 mL). To this solution was added triethylamine (3.14 mL, 22.5 mmol, 2 equiv.) and then diphenyl phosphoryl azide (2.43 mL, 11.25 mmol, 1.5 equiv.). The reaction was warmed to 50 °C and stirred for 3 hours. After 3 hours, the solution was cooled to room temperature and concentrated to an orange foam that was purified by silica gel chromatography (1-10% MeOH-DCM) to give the intermediate oxazolidinone I as a white solid (1.78 g, 26% yield over three steps).

LC-MS: Calculated (C ₄ 9H ₅ 8N ₂ Oi4 + Na) ⁺ : 921.39. Observed: 921.30. Analytical HPLC (Phenomenex Kinetix C ₁₈ , 5 μιη, 4.6 x 100 mm, 0.5 mL/min, 10:90 to 60:40 H ₂ 0:MeCN (both containing 0.1% HC0 ₂ H) over 19 min) Retention Time = 12.988 min.

Example 2

Synthesis of NatMU (

Oxazolidinone intermediate I (200 mg, 0.22 mmol, 1 equiv.) was dissolved in THF (2.2 mL). To the resulting yellow solution was added methylamine (2 M in THF, 1.1 mL, 2.2 mmol, 10 equiv.), and the resulting mixture was left to stir at room temperature for 16 hours. After 16 hours, the reaction was diluted with methyl ter t-butyl ether (10 mL). The resulting precipitate was collected via Buchner filtration using Whatman #50 filter paper and washed with methyl tert-butyl ether. The collected solid was stored under vacuum for one hour before it was dissolved in a minimal amount of DMSO and purified by a single prep-HPLC purification (Interchim Strategy C ₁₈ , 5 μιη, 30 x 100 mm, 30 mL/min, 10:90 to 60:40 H ₂ 0:MeCN (both containing 0.3% HC0 ₂ H) over 60 min). Following prep-HPLC purification, the fractions containing the desired compound were stirred at 40 °C for 30 min before they were frozen and lyophilized to give NatMU 5 as a fluffy white solid (92.3 mg, 61%).

1H NMR (500 MHz, Methanol-^) δ 6.51 - 6.38 (m, 2H), 6.29 - 6.10 (m, 5H), 6.06 (d, J = 15.8 Hz, 1H), 6.00 - 5.91 (m, 1H), 5.66 - 5.57 (m, 1H), 4.80 - 4.72 (m, 1H), 4.56 (s, 1H), 4.49 _ 4.42 (m, 1H), 4.30 (t, J= 10.6 Hz, 1H), 4.07 (t, J= 9.2 Hz, 1H), 3.93 (s, 1H), 3.83 (td, J= 10.5, 4.6 Hz, 1H), 3.28 - 3.21 (m, 1H), 3.16 (d, J= 7.4, 2.0 Hz, 1H), 2.96 (d, J = 10.7 Hz, 1H), 2.85 - 2.78 (m, 1H), 2.72 (s, 3H), 2.40 (dd, J= 13.4, 6.1 , 2.5 Hz, 1H), 2.34 - 2.19 (m, 2H), 2.10 - 2.00 (m, 2H), 1.75 - 1.67 (m, 1H), 1.67 - 1.59 (m, 2H), 1.42 (t, J = 12.0 Hz, 1H), 1.36 - 1.21 (m, 8H).

LC-MS: Calculated (C ₃ 4H ₅ iN ₃ 0i ₂ + H) ⁺ : 694.35. Observed: 694.25

Analytical HPLC (Phenomenex Kinetix C ₁₈ , 5 μιη, 4.6 x 100 mm, 0.5 mL/min, 10:90 to 60:40 H ₂ 0:MeCN (both containing 0.1% HC0 ₂ H) over 12 min) Retention Time = 7.063 min. Example 3

Synthesis ofNatamycin Urea (6)

Compound 6 was synthesized in a manner similar to NatMU 5, except methylamine was replaced with piperidine.

1H NMR (500 MHz, Methanol-^) δ 8.55 (s, 1H), 6.52 - 6.37 (m, 2H), 6.30 - 6.10 (m, 5H), 6.07 (d, J= 15.8 Hz, 1H), 6.02 - 5.93 (m, 1H), 5.66 - 5.57 (m, 1H), 4.80 - 4.72 (m, 1H), 4.55 (s, 1H), 4.48 - 4.41 (m, 1H), 4.31 (t, J = 10.5 Hz, 1H), 4.18 (t, J = 9.2 Hz, 1H), 3.92 (td, J = 11.1, 4.8 Hz, 1H), 3.87 (d, J= 3.0 Hz, 1H), 3.43 - 3.35 (m, 5H), 3.24 - 3.18 (m, 1H), 3.18 - 3.14 (m, 1H), 2.86 - 2.77 (m, 2H), 2.44 - 2.37 (m, 1H), 2.29 - 2.20 (m, 2H), 2.11 - 2.01 (m, 2H), 1.76 - 1.68 (m, 1H), 1.68 - 1.60 (m, 3H), 1.60 - 1.52 (m, 4H), 1.43 (t, J = 12.0 Hz, 1H), 1.31 (d, J= 6.2 Hz, 3H), 1.29 - 1.20 (m, 4H). LC-MS: Calculated (C ₃ 8H ₅₇ N ₃ Oi2 + H) ⁺ : 748.39. Observed: 748.30

Analytical HPLC (Phenomenex Kinetix C ₁₈ , 5 μιη, 4.6 x 100 mm, 0.5 mL/min, 10:90 to 60:40 H ₂ 0:MeCN (both containing 0.1% HC0 ₂ H) over 12 min) Retention Time = 7.315 mm.

Example 4

Synthesis of Nat A

Oxazolidinone intermediate I (200 mg, 0.22 mmol, 1 equiv.) was dissolved in THF (2.2 mL). To the resulting yellow solution was added ethylenediamine (0.147 mL, 2.2 mmol, 10 equiv.), and the resulting mixture was warmed to 50 °C and stirred for two hours. After two hours, the reaction was cooled to room temperature and diluted with methyl tert- butyl ether (10 mL). The resulting precipitate was collected via Buchner filtration using Whatman #50 filter paper and washed with methyl tert-butyl ether. The collected solid was stored under vacuum for one hour before it was dissolved in a minimal amount of DMSO and purified by a single prep-HPLC purification (Interchim Strategy C ₁₈ , 5 μιη, 30 x 100 mm, 30 mL/min, 10:90 to 60:40 H ₂ 0:MeCN (both containing 0.3% HC0 ₂ H) over 60 min). Following prep-HPLC purification, the fractions containing the desired compound were stirred at 40 °C for 30 min before they were frozen and lyophilized to give NatAU 7 as an off-white solid (93.3 mg, 59%).

1H NMR (500 MHz, Methanol-^) δ 8.54 (br s, 2H), 6.53 - 6.38 (m, 2H), 6.29 - 6.10 (m, 5H), 6.07 (d, J= 15.8 Hz, 1H), 6.01 - 5.92 (m, 1H), 5.66 - 5.57 (m, 1H), 4.79 - 4.70 (m, lH), 4.56 (s, 1H), 4.48 - 4.40 (m, 1H), 4.31 (t, J = 10.5 Hz, 1H), 4.09 (t, J = 9.4 Hz, 1H), 3.97 (s, 1H), 3.87 (td, J= 10.7, 4.9 Hz, 1H), 3.39 (t, J = 9.7 Hz, 1H), 3.28 - 3.19 (m, 1H), 3.18 - 3.14 (m, 1H), 3.12 - 2.94 (m, 3H), 2.82 (d, J= 9.3 Hz, 1H), 2.44 - 2.36 (m, 1H), 2.32 - 2.19 (m, 2H), 2.13 - 2.01 (m, 2H), 1.76 - 1.67 (m, 1H), 1.66 - 1.56 (m, 2H), 1.44 (t, J = 12.0 Hz, 1H), 1.34 - 1.21 (m, 7H). LC-MS: Calculated + H) ⁺ : 723.37. Observed: 723.30 Analytical HPLC (Phenomenex Kinetix C ₁₈ , 5 μηι, 4.6 x 100 mm, 0.5 mL/min, 10:90 to 60:40 H ₂ 0:MeCN (both containing 0.1% HC0 ₂ H) over 12 min) Retention Time = 6.415 min.

Example 5

Synthesis ofNatamycin Urea (8)

Compound 8 was synthesized in a manner similar to NatAU 7, except

ethylenediamine was replaced with morpholine.

1H NMR (500 MHz, Methanol-^) δ 8.53 (s, 1H), 6.51 - 6.37 (m, 2H), 6.30 - 6.10 (m, 5H), 6.07 (d, J= 15.8 Hz, 1H), 6.01 - 5.92 (m, 1H), 5.66 - 5.57 (m, 1H), 4.81 - 4.72 (m, 1H), 4.57 (s, 1H), 4.47 - 4.40 (m, 1H), 4.30 (t, J = 10.7 Hz, 1H), 4.19 (dd, J = 10.5, 8.0 Hz, 1H), 3.98 - 3.87 (m, 2H), 3.67 (t, J = 4.9 Hz, 5H), 3.43 - 3.34 (m, 7H), 3.27 - 3.20 (m, 1H), 3.17 (dd, J= 7.4, 2.0 Hz, 1H), 2.98 (dd, J = 10.1 , 3.1 Hz, 1H), 2.83 (d, J= 8.8 Hz, 1H), 2.44 - 2.37 (m, 1H), 2.29 - 2.17 (m, 2H), 2.1 1 - 2.00 (m, 2H), 1.76 - 1.60 (m, 3H), 1.43 (t, J = 12.0 Hz, 1H), 1.31 (d, J = 6.2 Hz, 3H), 1.30 - 1.20 (m, 5H).

LC-MS: Calculated (C ₃ 7H ₅₅ N ₃ Oi3 + H) ⁺ : 750.37. Observed: 750.30

Analytical HPLC (Phenomenex Kinetix C ₁₈ , 5 μιη, 4.6 x 100 mm, 0.5 mL/min, 10:90 to 60:40 H ₂ 0:MeCN (both containing 0.1% HC0 ₂ H) over 12 min) Retention Time = 7.174 min. Example 6

Synthesis ofNatamyc rea (9)

Compound 9 was synthesized in a manner similar to NatAU 7, except

ethylenediamine was replaced with piperazine.

1H NMR (500 MHz, Methanol-^) δ 8.49 (s, 2H), 6.52 - 6.37 (m, 2H), 6.31 - 6.10 (m, 5H), 6.07 (d, J= 15.8 Hz, 1H), 6.01 - 5.92 (m, 1H), 5.67 - 5.57 (m, 1H), 4.80 - 4.72 (m, 1H), 4.57 (s, 1H), 4.45 - 4.37 (m, 1H), 4.30 (t, J= 10.5 Hz, 1H), 4.19 (dd, J= 10.5, 8.0 Hz, 1H), 3.98 - 3.88 (m, 2H), 3.66 - 3.52 (m, 4H), 3.37 (q, J= 9.8 Hz, 2H), 3.27 - 3.20 (m, 1H),

3.17 (dd, J= 7.3, 2.0 Hz, 1H), 3.09 (t, J= 5.2 Hz, 4H), 3.02 (dd, J= 10.1, 3.1 Hz, 1H), 2.82 (d, J= 9.0 Hz, 1H), 2.45 - 2.35 (m, 1H), 2.29 - 2.14 (m, 2H), 2.13 - 2.00 (m, 2H), 1.77 - 1.59 (m, 3H), 1.44 (t, J= 12.0 Hz, 1H), 1.31 (d, J= 6.1 Hz, 3H), 1.30 - 1.21 (m, 5H). LC-MS: Calculated (C ₃ 7H ₅₆ N ₄ Oi2 + H) ⁺ : 749.39. Observed: 749.30

Analytical HPLC (Phenomenex Kinetix C ₁₈ , 5 μιη, 4.6 x 100 mm, 0.5 mL/min, 10:90 to 60:40 H ₂ 0:MeCN (both containing 0.1% HC0 ₂ H) over 12 min) Retention Time = 6.386 min.

Example 7

Synthesis ofNatamycin Urea (10)

Compound 10 was synthesized in a manner similar to NatAU 7, except

ethylenediamine was replaced with 2-(2-methoxyethoxy)ethanamine.

1H NMR (500 MHz, Methanol-^) δ 8.54 (s, 1H), 6.52 - 6.37 (m, 2H), 6.31 - 6.10 (m, 5H), 6.06 (d, J= 15.8 Hz, 1H), 5.99 - 5.90 (m, 1H), 5.66 - 5.56 (m, 1H), 4.80 - 4.71 (m, 1H), 4.56 (s, 1H), 4.46 (dt, J= 7.9, 3.5 Hz, 1H), 4.30 (t, J = 10.6 Hz, 1H), 4.06 (dd, J = 10.4, 7.9 Hz, 1H), 3.91 (d, J= 3.1 Hz, 1H), 3.83 (td, J = 10.7, 4.9 Hz, 1H), 3.67 - 3.59 (m, 2H), 3.59 - 3.51 (m, 4H), 3.38 (s, 3H), 3.36 - 3.32 (m, 1H), 3.27 - 3.14 (m, 2H), 2.98 - 2.91 (m, 1H), 2.82 (d, J = 9.4 Hz, 1H), 2.45 - 2.36 (m, 1H), 2.33 - 2.19 (m, 2H), 2.10 - 1.99 (m, 2H), 1.76 - 1.67 (m, 1H), 1.67 - 1.59 (m, 2H), 1.42 (t, J= 12.0 Hz, 1H), 1.31 (d, J= 6.1 Hz, 3H), 1.30 - 1.19 (m, 4H).

LC-MS: Calculated (C ₃ 8H ₅ 9N ₃ Oi4 + H) ⁺ : 782.40. Observed: 782.30

Analytical HPLC (Phenomenex Kinetix C ₁₈ , 5 μιη, 4.6 x 100 mm, 0.5 mL/min, 10:90 to 60:40 H ₂ 0:MeCN (both containing 0.1% HC0 ₂ H) over 12 min) Retention Time = 7.083 mm. Example 8

Synthesis ofNatam cin Urea (11)

Compound 11 was synthesized in a manner similar to NatAU 7, except

ethylenediamine was replaced with (5)-l-aminopropan-2-ol.

1H NMR (500 MHz, Methanol-^) δ 8.53 (s, 1H), 6.52 - 6.37 (m, 2H), 6.31 - 6.10 (m, 5H), 6.06 (d, J= 15.8 Hz, 1H), 5.99 - 5.90 (m, 1H), 5.67 - 5.56 (m, 1H), 4.81 - 4.71 (m, 1H), 4.57 (s, 1H), 4.45 (dt, J= 8.2, 3.5 Hz, 1H), 4.30 (t, J= 10.6 Hz, 1H), 4.06 (dd, J= 10.4, 7.9 Hz, 1H), 3.93 (d, J= 3.1 Hz, 1H), 3.87 - 3.76 (m, 2H), 3.37 (t, J= 9.7 Hz, 1H), 3.28 - 3.13 (m, 3H), 3.06 - 2.94 (m, 2H), 2.82 (d, J= 9.0 Hz, 1H), 2.44 - 2.36 (m, 1H), 2.31 - 2.19 (m, 2H), 2.10 - 2.00 (m, 2H), 1.76 - 1.59 (m, 3H), 1.43 (t, J= 12.0 Hz, 1H), 1.31 (d, J= 6.1 Hz, 3H), 1.30 - 1.20 (m, 4H), 1.15 (d, J = 6.3 Hz, 4H). LC-MS: Calculated (C ₃ 6H ₅₅ N ₃ Oi3 + H) ⁺ : 738.37. Observed: 738.30

Analytical HPLC (Phenomenex Kinetix C ₁₈ , 5 μιη, 4.6 x 100 mm, 0.5 mL/min, 10:90 to 60:40 H ₂ 0:MeCN (both containing 0.1% HC0 ₂ H) over 12 min) Retention Time = 7.141 mm.

Example 9

Synthesis ofNatamyci rea (12)

Compound 12 was synthesized in a manner similar to NatAU 7, except

ethylenediamine was replaced with dimethylamine.

1H NMR (500 MHz, Methanol-^) δ 8.53 (s, 1H), 6.52 - 6.36 (m, 2H), 6.31 - 6.10 (m, 5H), 6.07 (d, J= 15.8 Hz, 1H), 6.01 - 5.93 (m, 1H), 5.67 - 5.56 (m, 1H), 4.81 - 4.72 (m, 1H), 4.56 (s, 1H), 4.48 - 4.40 (m, 1H), 4.30 (t, J = 10.8 Hz, 1H), 4.19 (dd, J = 10.5, 8.0 Hz, 1H), 3.98 - 3.89 (m, 2H), 3.40 - 3.31 (m, 2H), 3.22 (dq, J= 9.4, 6.3 Hz, 1H), 3.16 (dd, J = 7.5, 2.0 Hz, 1H), 2.97 (dd, J= 10.2, 3.1 Hz, 1H), 2.93 (s, 6H), 2.83 (d, J = 8.9 Hz, 1H), 2.44 - 2.36 (m, 1H), 2.30 - 2.18 (m, 2H), 2.12 - 2.00 (m, 2H), 1.76 - 1.60 (m, 3H), 1.43 (t, J = 12.0 Hz, 1H), 1.31 (d, J = 6.1 Hz, 3H), 1.30 - 1.20 (m, 4H).

LC-MS: Calculated (C ₃ 5H ₅ 3N ₃ Oi2 + H) ⁺ : 708.36. Observed: 708.25

Analytical HPLC (Phenomenex Kinetix C ₁₈ , 5 μιη, 4.6 x 100 mm, 0.5 mL/min, 10:90 to 60:40 H ₂ 0:MeCN (both containing 0.1% HC0 ₂ H) over 12 min) Retention Time = 7.041 min. Example 10

Synthesis ofNatamycin Urea (13)

Compound 13 was synthesized in a manner similar to NatAU 7, except

ethylenediamine was replaced with N,N-dimethylethylenediamine.

1H NMR (500 MHz, Methanol-^) δ 8.52 (s, 2H), 6.52 - 6.37 (m, 2H), 6.30 - 6.10 (m, 5H), 6.07 (d, J= 15.8 Hz, 1H), 6.00 - 5.92 (m, 1H), 5.66 - 5.56 (m, 1H), 4.80 - 4.71 (m, 1H), 4.56 (s, 1H), 4.48 - 4.40 (m, 1H), 4.31 (t, J= 10.6 Hz, 1H), 4.09 (dd, J= 10.4, 8.2 Hz, 1H), 3.95 (d, J= 3.1 Hz, 1H), 3.86 (td, J= 10.8, 4.8 Hz, 1H), 3.62 - 3.50 (m, 1H), 3.38 (t, J = 9.7 Hz, 1H), 3.28 - 3.19 (m, 1H), 3.16 (dd, J= 7.5, 2.0 Hz, 1H), 3.07 - 2.97 (m, 3H), 2.82 (d, J = 8.8 Hz, 1H), 2.75 (s, 6H), 2.44 - 2.36 (m, 1H), 2.31 - 2.19 (m, 2H), 2.12 - 2.00 (m, 2H), 1.76 - 1.68 (m, 1H), 1.67 - 1.58 (m, 2H), 1.43 (t, J = 12.0 Hz, 1H), 1.31 (d, J = 6.1 Hz, 3H), 1.29 (d, J= 6.1 Hz, 3H), 1.27 - 1.19 (m, 1H).

LC-MS: Calculated (C ₃ 7H ₅ 8N ₄ Oi2 + H) ⁺ : 751.41. Observed: 751.30

Analytical HPLC (Phenomenex Kinetix C ₁₈ , 5 μιη, 4.6 x 100 mm, 0.5 mL/min, 10:90 to 60:40 H ₂ 0:MeCN (both containing 0.1% HC0 ₂ H) over 12 min) Retention Time = 6.374 mm. Example 11

Synthesis ofNatamycin Urea (14)

Compound 14 was synthesized in a manner similar to NatAU 7, except

ethylenediamine was replaced with benzylamine.

1H NMR (500 MHz, Methanol-^) δ 8.52 (s, 1H), 7.36 - 7.29 (m, 4H), 7.27 - 7.21 (m, 1H), 6.50 - 6.37 (m, 2H), 6.30 - 6.10 (m, 5H), 6.06 (d, J= 15.8 Hz, 1H), 6.00 - 5.91 (m, 1H), 5.67 - 5.57 (m, 1H), 4.81 - 4.72 (m, 1H), 4.52 (s, 1H), 4.49 - 4.43 (m, 1H), 4.42 - 4.25 (m, 3H), 4.08 (dd, J= 10.4, 7.9 Hz, 1H), 3.90 (d, J = 3.2 Hz, 1H), 3.84 (td, J = 10.9, 4.8 Hz,

1H), 3.36 (t, J = 9.7 Hz, 1H), 3.26 - 3.18 (m, 1H), 3.16 (dd, J= 7.4, 2.0 Hz, 1H), 2.91 (d, J = 10.2 Hz, 1H), 2.82 (d, J= 9.0 Hz, 1H), 2.45 - 2.36 (m, 1H), 2.32 - 2.19 (m, 2H), 2.11 - 1.99 (m, 2H), 1.77 - 1.59 (m, 3H), 1.44 (t, J = 12.0 Hz, 1H), 1.31 (d, J = 6.1 Hz, 3H), 1.29 - 1.20 (m, 4H).

LC-MS: Calculated (C ₃ 7H ₅ 8N ₄ Oi2 + H) ⁺ : 770.38. Observed: 770.25

Analytical HPLC (Phenomenex Kinetix C ₁₈ , 5 μιη, 4.6 x 100 mm, 0.5 mL/min, 10:90 to 60:40 H ₂ 0:MeCN (both containing 0.1% HC0 ₂ H) over 12 min) Retention Time = 7.795 mm. Example 12

Synthesis ofNatamycin Urea (15)

Compound 15 was synthesized in a manner similar to NatAU 7, except

ethylenediamine was replaced with 1-phenylpiperazine.

1H NMR (500 MHz, Methanol-^) δ 8.54 (s, 1H), 7.25 (dd, J= 8.7, 7.3 Hz, 2H), 6.99 (d, J = 7.8 Hz, 2H), 6.86 (t, J= 7.3 Hz, 1H), 6.52 - 6.37 (m, 2H), 6.30 - 6.10 (m, 5H), 6.07 (d, J = 15.8 Hz, 1H), 6.02 - 5.94 (m, 1H), 5.67 - 5.56 (m, 1H), 4.81 - 4.72 (m, 1H), 4.57 (s, 1H), 4.49_ 4.42 (m, 1H),4.31 (t,J= 10.1 Hz, 1H), 4.21 (dd,J= 10.5,8.0 Hz, 1H), 3.95 (td,J = 11.0, 4.8 Hz, 1H), 3.90 (d,J=3.1 Hz, 1H), 3.59 (t, J= 5.1 Hz, 4H), 3.42 - 3.34 (m, 2H), 3.27-3.11 (m, 6H), 2.93 (dd,J= 10.0,3.1 Hz, 1H), 2.83 (d,J=8.8Hz, 1H), 2.45 - 2.36 (m, 1H), 2.30- 2.18 (m, 2H), 2.13 - 2.00 (m, 2H), 1.77-1.61 (m, 3H), 1.44 (t,J= 12.0 Hz, 1H), 1.31 (d,J=6.2 Hz, 3H), 1.30-1.21 (m, 4H).

LC-MS: Calculated (C ₄ 3H ₆ oN ₄ Oi2 + H) ⁺ : 825.42. Observed: 825.30

Analytical HPLC (Phenomenex Kinetix C ₁₈ , 5 μιη, 4.6 x 100 mm, 0.5 mL/min, 10:90 to 60:40 H ₂ 0:MeCN (both containing 0.1% HC0 ₂ H) over 12 min) Retention Time = 7.650 min. Example 13

Synthesis ofNatamycin Urea (16)

Compound 16 was synthesized in a manner similar to NatAU 7, except

ethylenediamine was replaced with histamine.

1H NMR (500 MHz, Methanol-^) δ 8.54 (br s, 1H), 7.67 (s, 1H), 6.91 (s, 1H), 6.51 - 6.37 (m, 2H), 6.31 - 6.10 (m, 5H), 6.06 (d, J = 15.8 Hz, 1H), 5.99 - 5.90 (m, 1H), 5.61 (d, J = 14.8, 9.7, 5.6 Hz, 1H), 4.80 - 4.71 (m, 1H), 4.56 (s, 1H), 4.48 - 4.39 (m, 1H), 4.29 (t, J = 10.6 Hz, 1H), 4.06 (dd, J = 10.4, 8.0 Hz, 1H), 3.95 (d, J= 3.1 Hz, 1H), 3.82 (td, J= 11.3, 10.5, 4.8 Hz, 1H), 3.44 - 3.34 (m, 3H), 3.29 - 3.22 (m, 1H), 3.16 (dd, J = 7.4, 1.9 Hz, 1H), 3.05 (dd, J = 10.1, 3.1 Hz, 1H), 2.86 - 2.72 (m, 3H), 2.45 - 2.34 (m, 1H), 2.31 - 2.18 (m, 2H), 2.10 - 1.98 (m, 2H), 1.76 - 1.67 (m, 1H), 1.67 - 1.58 (m, 2H), 1.42 (t, J = 12.0 Hz, 1H), 1.31 (d, J= 6.1 Hz, 3H), 1.30 - 1.19 (m, 4H).

LC-MS: Calculated (C ₃ 9H ₅₇ N ₅ Oi2 + H) ⁺ : 774.38. Observed: 774.20

Analytical HPLC (Phenomenex Kinetix C ₁₈ , 5 μιη, 4.6 x 100 mm, 0.5 mL/min, 10:90 to 60:40 H ₂ 0:MeCN (both containing 0.1% HC0 ₂ H) over 12 min) Retention Time = 6.369 mm. Example 14

Synthesis ofNatamycin Urea (17)

Compound 17 was synthesized in a manner similar to NatAU 7, except

ethylenediamine was replaced with [2-(lH-l,2,4-triazol-l-yl)ethyl]amine.

1H NMR (500 MHz, Methanol-^) δ 8.46 (s, 1H), 8.01 (s, 1H), 6.53 - 6.38 (m, 2H), 6.31 - 6.11 (m, 5 H), 6.07 (d, J= 15.8 Hz, 1H), 6.02 - 5.92 (m, 1H), 5.66 - 5.57 (m, 1H), 4.80

- 4.71 (m, 1H), 4.57 (s, 1H), 4.47 - 4.25 (m, 4H), 4.05 (t, 1H), 3.98 - 3.94 (m, 1H), 3.80 (td, J= 10.7, 4.8 Hz, 1H), 3.63 - 3.49 (m, 2H), 3.37 (t, J= 9.6 Hz, 1H), 3.29 - 3.21 (m,

1H), 3.17 (dd, J= 7.4, 2.0 Hz, 1H), 3.05 (d, J= 9.5 Hz, 1H), 2.83 (d, J= 8.6 Hz, 1H), 2.44

- 2.36 (m, 1H), 2.30 - 2.14 (m, 2H), 2.09 - 1.99 (m, 2H), 1.76 - 1.67 (m, 1H), 1.67 - 1.58 (m, 2H), 1.42 (t, J = 12.0 Hz, 1H), 1.35 - 1.17 (m, 8H). LC-MS: Calculated (C ₃ 7H ₅₄ N ₆ Oi2 + H) ⁺ : 775.38. Observed: 775.25

Analytical HPLC (Phenomenex Kinetix C ₁₈ , 5 μιη, 4.6 x 100 mm, 0.5 mL/min, 10:90 to 60:40 H ₂ 0:MeCN (both containing 0.1% HC0 ₂ H) over 12 min) Retention Time = 7.154 mm.

Example 15

Assessment of Biological Activity

Each derivative proposed herein is tested for biological activity against both yeast and human cells to determine its therapeutic index. A broth microdilution experiment determines the MIC (minimum inhibitory concentration) of each derivative against S. cerevisiae and the clinically relevant C. albicans, thereby establishing the antifungal activity of each novel derivative. To test for toxicity against human cells, each compound is exposed to a hemolysis assay against red blood cells which determines the concentration required to cause 90% lysis of human red blood cells (EH ₉₀ ). Additionally, each compound is exposed to human primary renal tubule cells to determine the toxicity of each compound against kidney cells. These assays when compared against the known values of

corresponding (i.e., unmodified) polyene macrolide antibiotic against the same cell lines determine the improvement in therapeutic index of each compound.

In Vivo Sterol Extraction Studies and Membrane Isolation

This assay is performed similar to that previously described. Anderson TM et al., Nat Chem Biol 10:400-406 (2014). Specifically, 75 mL overnight cultures of

Saccharomyces cerevisiae are grown to stationary phase (OD -1.7) in YPD media at 30 °C, shaking. 49.5 mL of this culture is transferred to a 50 mL Falcon centrifuge tube.

Cells are treated with 500 of DMSO, 500 μΜ test compound (final compound concentration of 5 μΜ). Falcon tubes are incubated in the shaking incubator at 30 °C for 2 hours. Tubes are inverted at the 1 hour timepoint to resuspend.

Yeast membranes are isolated using a modified version of Haas' spheroplasting and isosmotic cell lysis protocol and differential ultracentrifugation. After treatment time, tubes are centrifuged for 5 minutes at 3000 g at 23 °C. The supernatant is decanted and 5 mL of wash buffer (milliQ H ₂ 0 (89%), 1M aq. DTT (1%), and 1M aq. Tris buffer pH 9.4 (10%)) is added. Tubes are vortexed to resuspend and incubated in a 30 °C water bath for 10 minutes. Tubes are then centrifuged for 5 minutes at 3000 g at 23 °C and the supernatant decanted.

1 mL of spheroplasting buffer (1M aq. potassium phosphate buffer pH 7.5 (5%>), 4M aq. sorbitol (15%>), and YPD media (80%>)) and 100 of a 5 mg/mL aq. solution of lyticase from Arthrobacter luteus (L2524 Sigma- Aldrich) is added to each tube, vortexed to resuspend. Tubes are incubated in a 30 °C shaking incubator for 30 minutes. After incubation, tubes are centrifuged for 10 minutes at 1080 g at 4 °C and the supernatant decanted.

1 mL of PBS buffer and 20 μΐ _^ of a 0.4 mg/ml dextran in 8% Ficoll solution is added to each tube, mixed very gently to resuspend. This suspension is placed in an ice bath for 4 minutes and then transferred to a 30 °C water bath for 3 minutes.

The suspensions are transferred to 2 mL Eppendorf tubes, vortexed to ensure complete lysis, and centrifuged at 15,000 g at 4 °C to remove un-lysed cells and cell debris. The resulting supernatants are transferred to thick-wall polycarbonate ultracentrifuge tubes (3.5 mL, 13 x 51 mm, 349622 Beckman Coulter). PBS buffer is added to the tubes to bring the volume up to ~3 mL. The tubes are centrifuged for 1 hour at 100,000 g at 4 °C in a Beckman Coulter TLA- 100.3 fixed-angle rotor in a tabletop ultracentrifuge. The

supernatant is poured off. The remaining membrane pellet is resuspended in 1 mL PBS buffer. 750 of the suspension is transferred to a 7 mL vial and stored at -80 °C until further analysis.

Gas chromatography quantification of sterols

The suspension is allowed to warm to room temperature and 20 of internal standard (4 mg/mL cholesterol in chloroform) is added. They are dissolved in 3 mL 2.5% ethanolic KOH, which is vortexed gently, capped, and heated in a heat block on a hot plate at 90 °C for 1 hour. The vials are allowed to cool to room temperature. 1 mL of brine is added to the contents of each vial. Extraction is performed three times, each with 2 mL of hexane. Organic layers are combined, dried over MgS0 ₄ , filtered through Celite ^® 545, and transferred to another 7 mL vial. The contents of the vial are concentrated in vacuo. The lipid films are dried on high vac with P ₂ O ₅ for 30 minutes to remove residual water.

To the resulting lipid films, 100 pyridine and 100 μΐ, Ν,Ο- Bis(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane (T6381-10AMP Sigma-Aldrich) is added and vortexed gently. This solution is heated at 60 °C for 1 hour to produce TMS ethers. The vials are placed in an ice bath and the solvent is evaporated off by nitrogen stream. Vials are kept at low temperature to prevent evaporation of the sterol ethers along with the solvent. The resulting films are resuspended in 100 μΐ, of decane, filtered using a Supelco ISO-Disc PTFE Filter (4 mm x 0.2 μιη) and transferred to a GC vial insert for analysis.

Gas chromatography analysis is carried out on an Agilent 7890A gas chromatograph equipped with FID and Agilent GC 7693 Autosampler. Samples are separated on a 30 m, 0.320 mm ID, 0.25 μιη film HP-5 capillary column (19091 J-413 Agilent). Hydrogen is employed as a carrier gas with a flow rate of 4 mL/min. Nitrogen make-up gas, hydrogen gas, and compressed air are used for the FID. A split/splitless injector is used in a 20: 1 split. The injector volume is 2 The column temperature is initially held at 250 °C for 0.5 min, then ramped to 265 °C at a rate of 10 °C /min with a final hold time of 12.5 min. The injector and detector temperature are maintained at 270 °C and 290 °C, respectively. Growth Conditions and MIC Assay for C. albicans, C. tropicalis, C. parapsilosis, and C. glabrata

The organisms are maintained, grown, subcultured, and quantified on Sabouraud dextrose agar (SDA; Difco Laboratories, Detroit, MI). 24 hours prior to the study, the organisms are subcultured at 35 °C. MIC determinations are performed in duplicate on at least two occasions using the Clinical and Laboratory Standards Institute M27-A3 microbroth methodology.

C. albicans is generally grown and maintained as described previously. Stocks are stored in 15% glycerol at -80 °C; strains are generally grown in YPD media at 30 °C. Drugs are added directly to media from DMSO stocks.

Growth Conditions and MIC Assay for C. neoformans

C. neoformans MIC is determined as previously reported after 48 hours. Cruz MC et al, Antimicrob Agents Chemother 44: 143-149 (2000).

Growth Conditions and MIC Assay for C. fumigatus

The organisms are maintained, grown, subcultured, and quantified on potato dextrose agar (PDA; Difco Laboratories, Detroit, MI). MIC determinations are performed in duplicate on at least two occasions using the Clinical and Laboratory Standards Institute M28-A2 microbroth methodology at 48 hours.

Minimum Inhibitory Concentration (MIC) and Growth Assays

Susceptibility of wild-type strains to test compound, tert-butyl peroxide (Sigma-

Aldrich), geldanamycin and radicicol (A.G. Scientific) is determined in flat bottom, 96-well microtiter plates (Costar) using a broth microdilution protocol adapted from CLSI M27-A3. Overnight cultures (14-20 hr) are grown at 30 °C in YPD, and approximately 5xl0 ³ cells are seeded per well. For test compounds, MIC assays are performed at 37 °C in RPMI buffered with MOPS (0.165M) with 10% fetal bovine serum (Sigma- Aldrich) added; for tert-butyl peroxide, geldanamycin, and radicicol, MIC's are determined in YPD at 30 °C. MIC's are determined after 24h incubation as the concentration of compound resulting in no visible growth in wells. For quantitative display of growth at drug dilutions, OD ₆ oo is measured in a spectrophotometer (Tecan) and displayed as heat maps using Java TreeView 1.1.3.

Hemolysis Assays

Hemolysis experiments are performed following known procedures. Wilcock BC et al., J^m Chem Soc 135:8488-8491 (2013). WST-8 Cell Proliferation Assays

Primary Renal Proximal Tubule Epithelial Cells Preparation

Primary human renal proximal tubule epithelial cells (RPTECs) are prepared following known procedures. Wilcock BC et al, J Am Chem Soc 135 :8488-8491 (2013).

TERT1 Renal Proximal Tubule Epithelial Cells Preparation

TERT1 human renal proximal tubule epithelial cells (RPTECs) are purchased from ATCC (CRL-4031 , Manassas, VA) and immediately cultured upon receipt. Complete growth media is prepared using DMEM:F12 media (ATCC, 30-2006), triiodo-L-thyronine (Sigma, T6397), recombinant human EGF (Life Technologies, PHG031 1), ascorbic acid (Sigma, A4403), human transferrin (Sigma, T8158), insulin (Sigma 19278), prostaglandin El (Sigma, P7527), hydrocortisone (Sigma, H0888), sodium selenite (Sigma, S5261), and G418 (Sigma, A1720). Complete media is stored at 4 °C and used within 28 days. TERT1 RPTECs are grown in C0 ₂ incubator at 37 °C with an atmosphere of 95% air/5% C0 ₂ .

WST-8 Reagent Preparation

WST-8 reagent is prepared and stored following known procedures ⁹ . Wilcock BC et al, J Am Chem Soc 135 :8488-8491 (2013).

WST-8 Assay

A suspension of primary or TERT1 RPTECs in complete growth media is brought to a concentration of 1 x 10 ⁵ cells/mL. A 96-well plate is seeded with 99 of the cell suspension and incubated at 37 °C with an atmosphere of 95%> air/5%> C0 ₂ for 3 hours. Positive and negative controls are prepared by seeding with 100 of the cell suspension or 100 of the complete media. Compounds are prepared as 5 mM stock solutions in DMSO and serially diluted to the following concentrations with DMSO: 8000, 6000, 4000, 3000, 2000, 1500, 1000, 800, 600, 400, 300, 200, 100, 50, 25, 10, 5, 2.5, 1 , 0.5, 0.25, and 0.1 μΜ. 1 μΕ aliquots of each solution are added to the 96-well plate in triplicate, with each column representing a different concentration of the test compound. The 96-well plate is incubated at 37 °C with an atmosphere of 95% air/5% C0 ₂ for 24 hours. After incubation, the media is aspirated and 100 μΐ, of serum- free media is added and 10 μΐ, οΐ the WST-8 reagent solution is added to each well. The 96-well plate is mixed in a shaking incubator at 200 rpm for 1 minute and incubated at 37 °C with an atmosphere of 95% air/5% C0 ₂ for 2 hours. Following incubation, the 96-well plate is mixed in a shaking incubator at 200 rpm for 1 minute and absorbances are read at 450 nm using a Biotek HI Synergy Hybrid Reader (Wanooski, VT). Experiments are performed in triplicate and the reported cytotoxicity represents an average of three experiments.

Data Analysis

Percent hemolysis is determined according to the following equation:

% cell viability = _{x 10} o%

Concentration vs. percent hemolysis is plotted and fitted to 4-parameter logistic (4PL) dose response fit using OriginPro 8.6. The MTC is defined as the concentration to cause 90% loss of cell viability.

In Vivo Murine Efficacy Study

All studies are approved by the Animal Research Committee of the William S. Middleton Memorial VA Hospital (Madison, WI). Efficacy is assessed by CFU count in the kidneys of neutropenic mice with a disseminated fungal infection as described previously by Andes et al. A clinical isolate of Candida albicans (K-l) is grown and quantified on SDA. For 24 hours prior to infection, the organism is subcultured at 35 °C on SDA slants. A 10 ⁶ CFU/mL inoculum (CFU, colony forming units) is prepared by placing six fungal colonies into 5 mL of sterile, depyrogenated normal (0.9%) saline warmed to 35 °C. Six- week-old ICR/Swiss specific-pathogen- free female mice are obtained from Harlan Sprague Dawley (Madison, WI). The mice are weighed (23-27 g) and given intraperitoneal injections of cyclophosphamide to render neutropenia (defined as <100 polymorphonuclear leukocytes/mm ³ ). Each mouse is dosed with 150 mg/kg of cyclophosphamide 4 days prior to infection and 100 mg/kg 1 day before infection. Disseminated candidiasis is induced via tail vein injection of 100 of inoculum. Test compounds are reconstituted with 1.0 mL of 5% dextrose. Each animal in the treatment group is given a single 200 intraperitoneal (ip) injection of reconstituted test compound 2 hours post-infection. Doses are calculated in terms of mg of compound/kg of body weight. At each time point (6, 12, and 24 hours postinfection), three animals per experimental condition are sacrificed by C0 ₂ asphyxiation. The kidneys from each animal are removed and homogenized. The homogenate is diluted serially 10-fold with 9% saline and plated on SDA. The plates are incubated for 24 hours at 35 °C and inspected for CFU viable counts. The lower limit of detection for this technique is 100 CFU/mL. All results are expressed as the mean logio CFU per kidney for three animals. In Vivo Murine Toxicity Study

All studies are approved by the Animal Research Committee of the William S. Middleton Memorial VA Hospital (Madison, WI). Uninfected Swiss ICR mice are used for assessment of infusion toxicity. Groups of five mice are treated with single intravenous doses of test compound (reconstituted with 1.0 mL of 5% dextrose), or sterile pyrogen- free 0.85% NaCl administered via the lateral tail vein over 30 seconds. Dose levels studies included 0.5, 1, 2, 4, 8, 16, 32, and 64 mg/kg. Following administration mice are observed continuously for one hour and then every 6 hours up to 24 hours for signs of distress or death.

INCORPORATION BY REFERENCE

All patents and published patent applications mentioned in the description above are incorporated by reference herein in their entirety. EQUIVALENTS

Having now fully described the present invention in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious to one of ordinary skill in the art that the same can be performed by modifying or changing the invention within a wide and equivalent range of conditions, formulations and other parameters without affecting the scope of the invention or any specific embodiment thereof, and that such modifications or changes are intended to be encompassed within the scope of the appended claims.