AMPHOTERICIN B DERIVATIVE WITH REDUCED TOXICITY

RELATED APPLICATION

This application claims benefit of U.S. Provisional Patent Application No.

61/807,871, filed April 3, 2013.

GOVERNMENT SUPPORT

This invention was made with government support under Grant No. R01 GM080436 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

The polyene macrolide natural product, amphotericin B (AmB, 1), is the archetype for both small molecules that form ion channels in living cells ¹ and antibiotics that are inherently refractory to microbial resistance. ² AmB is also, unfortunately, highly toxic, ³ which often limits its effective utilization as the last line of defense against life-threatening systemic fungal infections. Because both the incidence of such fungal infections and resistance to all other classes of antifungals are on the rise, ² finding a way to improve the therapeutic index of AmB has become an increasingly important problem. Some progress has been made with liposomal formulations, but they are often prohibitively expensive, ⁴ and substantial toxicity still remains. ⁵ Despite 50 years of extensive efforts worldwide, a clinically viable derivative of AmB with an improved therapeutic index has yet to emerge. ⁶

SUMMARY OF THE INVENTION An aspect of the invention is C2'deOAmB, represented by

or a pharmaceutically acceptable salt thereof.

An aspect of the invention is a pharmaceutical composition, comprising C2'deOAmB, represented by

or a pharmaceutically acceptable salt thereof; 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 C2'deOAmB, represented by

or a pharmaceutically acceptable salt thereof.

An aspect of the invention is a method of treating a yeast or fungal infection, comprising administering to a subject in need thereof a therapeutically effective amount of C2'deOAmB, represented by

or a pharmaceutically acceptable salt thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts structures of amphotericin B (AmB) and synthetic derivatives (AmdeB and C2'deOAmB) thereof. Also depicted are structures of mycosamine, ergosterol, and cholesterol.

FIG. 2 depicts a scheme (Scheme 1) for synthesis of C2'deOAmB.

FIG. 3 depicts a scheme (Scheme 2) for synthesis of C2'deOAmB.

FIG. 4A is a graph depicting binding of AmB, AmdeB, and C2'deOAmB to ergosterol. FIG. 4B is a graph depicting binding of AmB, AmdeB, and C2'deOAmB to cholesterol. NS: not statistically significant.

FIG. 5 is a series of four photomicrographs depicting human renal epithelial cells treated with DMSO (negative control) or 2 μΜ AmB, AmdeB, or C2'deOAmB. DETAILED DESCRIPTION OF THE INVENTION

Amphotericin B (AmB) is a clinically vital antimycotic but its use is limited by its toxicity. Binding ergosterol, independent of channel formation, is the primary mechanism by which AmB kills yeast, and binding cholesterol may primarily account for toxicity to human cells. A leading structural model predicts that the C2' hydroxyl group on the mycosamine appendage is critical for binding both sterols. To test this, this functional group was synthetically deleted and the sterol binding capacity of the resulting derivative, C2'deOAmB, was directly characterized via isothermal titration calorimetry. Surprisingly, C2'deOAmB retains the capacity to bind ergosterol but shows no evidence of binding cholesterol. Moreover, C2'deOAmB is nearly equipotent to AmB against yeast, but demonstrates essentially no toxicity to human cells. C2'deOAmB thus represents a powerful probe of AmB function and a promising new antifungal agent with an

unprecedented increase in therapeutic index relative to AmB. AmB is generally obtained from a strain of Streptomyces nodosus. It is currently approved for clinical use in the United States for the treatment of progressive, potentially life -threatening fungal infections, including such infections as systemic candidiasis, aspergillosis, cryptococcosis, blastomycosis, coccidioidomycosis, histoplasmosis, and mucormycosis, among others. It is generally formulated for intravenous injection.

Amphotericin B is commercially available, for example, as Fungizone® (Squibb),

Amphocin® (Pfizer), Abelcet® (Enzon), and Ambisome® (Astellas). Due to its unwanted toxic side effects, dosing is generally limited to a maximum of about 1.0 mg/kg/day and total cumulative doses not to exceed about 3 g in humans.

It has for many decades been widely accepted that AmB primarily kills both yeast and human cells via membrane permeabilization. ⁷ Guided by this model, extensive efforts have focused on the development of derivatives that selectively form ion channels in yeast vs. human cells. ^7b_e

In contrast to this classic model, we recently discovered that AmB primarily kills yeast by simply binding ergosterol (2); i.e., channel formation is not required. ⁸ This suggests that simply binding cholesterol (3) may alternatively account for the toxicity of

AmB to human cells, and that efforts to improve the therapeutic index of this clinically vital antimycotic can focus on the much simpler problem of maximizing the relative binding affinity for ergosterol vs. cholesterol. To enable the rational pursuit of this objective, we aimed to understand at the atomistic level the physical underpinnings of these very rare and interesting examples of biologically relevant small molecule-small molecule interactions. In this vein, we have previously found that deletion of the mycosamine appendage from AmB eliminates its capacity to bind both ergosterol and cholesterol . ⁸ The resulting derivative,

amphoteronolide B (AmdeB, 4), was also found to be non-toxic to yeast. ⁸ ' ⁹ The roles played by each heteroatom contained in the mycosamine appendage, however, have remained unclear.

In the leading structural model, AmB binds both ergosterol and cholesterol via a similar complex in which the C2' hydroxyl group of AmB forms a critical hydrogen bond to the 3-β hydroxyl group on each sterol (FIG. I). ¹⁰ However, strong evidence for or against this hypothesis is lacking. For example, computer simulations ¹¹ have suggested that such a hydrogen bond plays an important role in binding ergosterol, but not cholesterol.

Alternatively, previous studies comparing the membrane permeabilizing activities of conformationally restricted derivatives of AmB ^10c concluded that such a hydrogen bond plays a key role with both sterols. A series of C41 methyl ester derivatives of AmB further modified at C2' yielded conflicting results: epimerization at C2' led to retention of both membrane permeabilizing and antifungal activities whereas epimerization and methyl etherification of the C2' hydroxyl group resulted in substantial reductions in both activities. ^10a Most importantly, none of these prior studies directly measured sterol binding.

In accordance with the invention, the C2' hydroxyl group was deleted from AmB and the impact of this deletion on binding ergosterol and cholesterol was determined.

An aspect of the invention is C2'deOAmB, represented by

or a pharmaceutically acceptable salt thereof. Methods for making C2'deOAmB are disclosed herein below. In accordance with this and other aspects of the invention, the invention specifically embraces stereoisomers of the compound C2'deOAmB. In particular, the invention specifically embraces mixtures of individual stereoisomers of the compound C2'deOAmB, as well as isolated individual stereoisomers of the compound C2'deOAmB.

A "compound of the invention" as used herein refers to C2'deOAmB and any of the foregoing pharmaceutically acceptable salts and stereoisomers thereof.

An aspect of the invention is a pharmaceutical composition, comprising

C2'deOAmB, represented by

or a pharmaceutically acceptable salt thereof; 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, diluents or encapsulating substances which are suitable for administration to a human or other subject.

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 C2'deOAmB, represented by

or a pharmaceutically acceptable salt thereof.

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. Fungi include, in addition to yeasts, 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, central nervous system, to name a few. Some fungi are responsible for infections of the skin and nails.

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.

An aspect of the invention is a method of treating a yeast or fungal infection, comprising administering to a subject in need thereof a therapeutically effective amount of C2'deOAmB, represented by

or a pharmaceutically acceptable salt thereof.

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, subcutaneous, direct injection (for example, into a tumor), mucosal, inhalation, oral, and topical.

In one embodiment, the administration is systemically administering.

In one embodiment, the administration is topically administering. As used herein, the phrase "effective amount" refers to any amount that is sufficient to achieve a desired biological effect. A therapeutically effective amount is an amount that is sufficient to achieve a desired therapeutic effect, e.g., to treat a yeast or fungal infection.

Compounds of the invention can be combined with other therapeutic agents. The compound of the invention and other therapeutic agent may be administered simultaneously or sequentially. When the other therapeutic agents are administered simultaneously, they can be administered in the same or separate formulations, but they are administered substantially at the same time. The other therapeutic agents are administered sequentially with one another and with compound of the invention, when the administration of the other therapeutic agents and the compound of the invention is temporally separated. The separation in time between the administration of these compounds may be a matter of minutes or it may be longer.

Examples of other therapeutic agents include other antifungal agents, including AmB, as well as other antibiotics, anti-viral agents, anti-inflammatory agents,

immunosuppressive agents, and anti-cancer agents. 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 active compounds of the invention, e.g., C2'deOAmB, will be, for human subjects, similar to or greater than usual daily intravenous doses of AmB. Similarly, daily other parenteral doses of active compounds of the invention, e.g., C2'deOAmB, will be, for human subjects, similar to or greater than usual daily other parenteral doses of AmB.

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 AmB. 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 (e.g., AmB). 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, the compounds of the invention, e.g., C2'deOAmB, generally may be formulated similarly to AmB. For example, C2'deOAmB 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 (i.e., compounds of the invention, and other therapeutic agents) 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, the compounds 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 the compounds of the invention

(or derivatives 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 C/m 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). 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 compound of the invention (or derivative). 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, the compounds 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).

The compounds 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 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 the compound 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.

It will be understood by one of ordinary skill in the relevant arts that other suitable modifications and adaptations to the compositions and methods described herein are readily apparent from the description of the invention contained herein in view of information known to the ordinarily skilled artisan, and may be made without departing from the scope of the invention or any embodiment thereof. 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.

EXAMPLES

General Methods

Materials

Commercially available materials were purchased from Sigma-Aldrich, Alfa Aesar, Strem, Avanti Polar Lipids, Fisher Scientific or Julich, and were used without further purification unless stated otherwise. Amphotericin B was a generous gift from Bristol- Myers Squibb Company. Camphorsulfonic acid was recrystallized from the ethyl acetate prior to use. All solvents were dispensed from a solvent purification system that passes solvents through packed columns according to the method of Pangborn and coworkers (THF, Et ₂ 0, CH ₂ C1 ₂ , toluene, dioxane, hexanes : dry neutral alumina; DMSO, DMF, CH ₃ OH : activated molecular sieves). Ermishkin, LN et al. (1976) Nature 262:698-699. 2,6-Lutidine and pyridine were freshly distilled under nitrogen from CaH ₂ . Vinyl acetate was freshly distilled under nitrogen from CaCl ₂ . EtOAc and EtOH were freshly distilled under nitrogen from activated molecular sieves. Water was doubly distilled or obtained from a Millipore MilliQ water purification system.

Reactions Due to the light and air sensitivity of polyenes, all manipulations of polyenes were carried out under low light conditions and compounds were stored under an argon atmosphere. All reactions were performed in oven- or flame-dried glassware under an atmosphere of argon unless otherwise indicated. Reactions were monitored by analytical thin layer chromatography performed using the indicated solvent on E. Merck silica gel 60 F ₂₅₄ plates (0.25 mm). Compounds were visualized using a UV (λ ₂₅₄ ) lamp or stained by a solution of /?-anisaldehyde, KMn0 ₄ , or eerie ammonium molybdate (CAM) stain.

Alternatively, reactions were monitored by RP-HPLC using an Agilent 1100 series HPLC system equipped with a SYMMETRY® C ₁₈ 5 micron 4.6 x 150 mm column (Waters Corp. Milford, MA) with UV detection at 383 nm and the indicated eluent and flow rate of 1 mL/min.

Purification and Analysis

Flash chromatography was performed as described by Still and coworkers ¹¹ using the indicated solvent on E. Merck silica gel 60 230-400 mesh. Still, WC et al. (1978) J. Org. Chem. 43:2923. 1H NMR spectra were recorded at 23 °C on one of the following instruments: Varian Unity 400, Varian Unity 500, Varian Unity Inova 500NB. Chemical shifts (δ) are reported in parts per million (ppm) downfield from tetramethylsilane and referenced internally to the residual protium in the NMR solvent (CHCI ₃ , δ = 7.26, centerline; CD ₃ C(0)CHD ₂ , δ = 2.04, center line; CD ₃ S(0)CHD ₂ , δ = 2.50, center line) or to added tetramethylsilane. Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, dd = doublet of doublets, ddd = doublet of doublet of doublets, td = triplet of doublets, m = multiplet, b = broad, app. = apparent), coupling constant (J) in Hertz (Hz) and integration. ¹³ C spectra were recorded at 23 °C with a Varian Unity 500. Chemical shifts (δ) are reported downfield of

tetramethylsilane and are referenced to the carbon resonances in the NMR solvent (CDC1 ₃ , δ = 77.0, center line; CD ₃ C(0)CD ₃ , δ = 29.8, center line; CD ₃ S(0)CD ₃ , δ = 39.5, center line) or to added tetramethylsilane. High resolution mass spectra (HRMS) were obtained at the University of Illinois mass spectrometry facility. All synthesized compounds gave HRMS within 5 ppm of calculated values.

Extinction Coefficient Determination

General procedure

A sample of dried compound was massed in a tared vial using a Mettler Toledo MT5 microbalance. This sample was then dissolved in DMSO to create a concentrated stock solution. A portion of this concentrated stock solution was diluted by a factor of five with DMSO to create a dilute stock solution. To achieve the final concentration for UV/Vis experiments, a volume of the dilute stock solution was diluted to 0.5 mL with MeOH. For each compound, UV/vis experiments were performed using five different final

concentrations, and each concentration was prepared three times to obtain an average absorbance. The average absorbance was plotted against the concentration. The data was fitted with a linear least squares fit using Excel, and the slope of the fitted line was used as the extinction coefficient. The extinction coefficients were as follows: AmB (ε ₄ ο ₆ = 164,000), AmdeB (ε ₄₀₆ = 102,000), C2'deOAmB (ε ₄₀₆ = 73,000).

Example 1. Synthesis of C2'deOAmB

In pursuit of a definitive experiment, we aimed to delete the C2' hydroxyl group from AmB and determine the impact on binding ergosterol and cholesterol. Synthesis of the targeted C2'deOAmB derivative, however, represented a major challenge. This is because, in addition to all of the other problems associated with chemically manipulating this very complex and sensitive natural product, ^8"9 2-deoxy sugars are notoriously less compatible with many reagents than their oxygenated counterparts. ¹² To maximize the chances of success, we thus pursued two different synthetic strategies in parallel (Schemes 1 and 2; depicted in FIG. 2 and FIG. 3, respectively) . In the first approach, we targeted the synthesis of C2'deOAmB via site-selective deoxygenation of the decahydroxylated natural product (Scheme 1). Pursuit of this very challenging problem led to the discovery that site-selective and site-divergent functionalizations can be achieved simply by modifying the electronic properties of achiral reagents. ¹³ Harnessing this phenomenon, we achieved the highly site-selective acylation of the C2' hydroxyl group to generate intermediate 6, ¹³ and subsequent persilylation, deacylation, and deoxygenation of the (2' hydroxyl group generated protected C2'deOAmB 7. ¹³ We initiated deprotection of this intermediate by first removing all of the silyl groups with HF-pyridine. We then employed potassium hydroxide to deprotect the methyl ester and CSA to concomitantly remove the /?-methoxybenzylidenc acetals and methyl ketal. Consistent with the sensitivity of 2-deoxysugars, these studies revealed that C2'deOAmB derivatives are substantially more prone to chemical decomposition than their

mycosaminoylated counterparts, and such decomposition manifested itself in low yields for these transformations. This problem was particularly evident in the final step. Specifically, removal of the phenylacyl group from the C3' amine using penicillin G amidase (PGA), a reaction that was previously successful with both AmB and C35deOAmB, ^8a resulted in a low yield of C2'deOAmB 5 as an inseparable mixture containing a variety of

deglycosylated byproducts.

Importantly, extensive knowledge gained during these studies bolstered an alternative semisynthetic approach ^10a ' ¹⁴ that ultimately proved to be much more productive (Scheme 2). In this route, we first generated a C2'-deoxygenated mycosamine (acosamine) donor from known intermediate 8. ^10a ' ¹⁵ After much exploration, it was determined that the TBS-protected derivative of this 2,3 epoxy alcohol can be regioselectively opened at C2' using lithium triethylborohydride to give intermediate 9. The resulting alcohol was mesylated and displaced by sodium azide to produce intermediate 10. Subsequent removal of the PMB group on the hydroxyl of the anomeric carbon generated the deoxysugar donor 11. Importantly, 11 is protected in such a way that the functional groups at C3' and C4' are inert to all of the subsequently required transformations yet readily unmasked at the end of the synthesis using very mild conditions.

To efficiently prepare a similarly protected macrolide acceptor, we first generated intermediate 13 having suitably stable yet readily cleavable silyl ethers protecting all of the hydroxyl groups and the carboxylic acid at C41. The mycosamine appendage was then oxidatively cleaved, and subsequent diastereoselective reduction at ci9 ^8b ' ⁹ ' ¹⁶ resulted in protected AmdeB derivative 14. Glycosidation 14 with 11 proceeded smoothly to yield the alternatively protected C2'deOAmB derivative 15 as a 1 : 1 mixture of a and β anomers. Derivative 15 proved to be much more amenable to deprotection than 7.

Specifically, concomitant deprotection of all nine of the silyl protecting groups proceeded smoothly in 78% overall yield, and the resulting a and β anomers were readily separated by HPLC. Finally, very efficient deprotections of the C3' azide with trimethylphosphine (57%) and the C13 hemiketal with aqueous acid (80%>) completed the synthesis of

C2 ^* deOAmB 5.

Details of the synthesis are as follows.

TIPSOTf

2,6-Lutidine

Hexane 0°C

TIPS ester 13 Intermediate 12 (15.8 g, 7.20 mmol, 1 eq) was azeotropically dried with toluene and placed under vacuum overnight. Hexane (240 mL) and 2,6-lutidine (2.9 mL, 25.2 mmol, 3.5 eq) were added. The resulting solution was cooled to 0 °C and triisopropylsilyl triflate (2.9 mL, 10.8 mmol, 1.5 eq) was added slowly over 15 min. The reaction was quenched after 1 hr with saturated aqueous sodium bicarbonate and extracted with ether. The organic layer was washed with copper sulfate, water, and finally saturated sodium chloride. The organic layer was dried with sodium sulfate and filtered. The solvent was removed under reduced pressure, and column chromatography (Si0 ₂ ; EthenHexane 5:95→ 1 :4) purification yielded the 13 as a yellow-orange solid (15.2 g, 6.5 mmol, 90 %).

TLC (Ether:Hexane 0.1% Et ₃ N 3 :7) R _f = 0.72, stained by CAM

1H NMR (500 MHz, CD ₃ C(0)CD ₃ )

δ 7.86 (d, J = 7.5 Hz, 2H), 7.69 (d, J = 7.5 Hz, 2H), 7.41 (t, J = 7.5 Hz, 2H), 7.33 (t, J = 7.5 Hz, 2H), 6.53-6.05 (m, 12H), 5.51 (m, 1H), 5.34 (m, 1H), 4.65 (m, 2H), 4.47 (m, 3H), 4.34 (m, 2H), 4.24 (m, 2H), 4.13 (m, 1H), 3.98 (m, 2H), 3.90 (m, 1H), 3.83 (m, 1H), 3.66 (m, 2H), 3.45 (m, 1H), 3.27 (m, 1H), 3.15 (s, 3H), 2.56 (m, 1H), 2.42 (m, 2H), 2.10-2.01 (m, 3H), 1.94-1.59 (m, 12H), 1.50 (m, 1H), 1.38-1.30 (m, 4H), 1.23 (m, 4H), 1.16 (m, 20H), 1.07-0.89 (m, 85H), 0.78-0.55 (m, 56H).

¹³ C NMR (125 MHz, CD ₃ C(0)CD ₃ )

δ 172.3, 170.5, 156.2, 145.0, 142.1, 139.0, 135.7, 135.3, 135.2, 134.7, 133.8, 132.9, 132.8, 132.7, 132.5, 131.4, 130.7, 130.6, 128.4, 127.8, 125.8, 125.7, 120.7, 101.2, 99.5,

76.7, 74.6, 74.0, 73.9, 73.2, 71.1, 69.4, 68.0, 67.5, 67.3, 67.2, 59.0, 58.2, 48.2, 48.0,

47.8, 44.3, 43.4, 42.1, 41.2, 37.2, 35.6, 27.4, 19.9, 19.2, 19.0, 18.4, 18.2, 12.9, 11.3, 7.6, 7.5, 7.4, 7.3, 6.4, 6.2, 6.1, 6.0, 5.9, 5.8.

MS (ESI)

Calculated for Ci26H ₂₃ iNOi ₉ Siio (M + Na) ⁺ : 2365.5

Found: 2365.1

Allylic alcohol 14

Intermediate 13 (12.5 g, 5.35 mmol, 1 eq) was azeotropically dried with toluene and placed under vacuum overnight. THF (100 mL) was added. The resulting solution was cooled to 0 °C, and DDQ (1.82 g, 8.03 mmol, 1.5 eq) and CaC0 ₃ (5.3 g, 53.5 mmol, 10 eq) were added. The reaction was warmed to room temperature and quenched after 30 min with saturated aqueous sodium bicarbonate and extracted with ether. The organic layer was washed with water and then saturated sodium chloride. The organic layer was dried with sodium sulfate and filtered. The solvent was removed under reduced pressure, and flash column chromatography (Si0 ₂ ; Ether:Hexane 1 :4) purification yielded the enone as a dark red solid. This intermediate is sensitive to silica gel and was immediately subjected to the next reaction conditions.

TLC (Ether :Hexane 3: 17)

R _f = 0.35, stained by CAM

MS (ESI)

Calculated for C ₉ Hi8oNOi ₄ Si ₈ (M + Na) ⁺ : 1768.1

Found: 1768.0

The enone intermediate was azeotropically dried with toluene. THF (10 mL) and MeOH (20 mL) was added. The resulting solution was cooled to 0 °C, and NaBH ₄ (1.08 g, 28.6 mmol, 5.3 eq) was added. The reaction was quenched after 30 min with 1 M aqueous ammonium chloride and extracted with ether. The organic layer was washed with water and then saturated sodium chloride. The organic layer was dried with sodium sulfate and filtered. The solvent was removed under reduced pressure, and flash column

chromatography (Si0 ₂ ; Ether:Hexane 1 :9→ 1 :4) purification yielded 14 as a yellow-orange solid. This intermediate is not stable to long term storage and extended periods on silica gel. (4.5 g, 2.57 mmol, 48 % 2 steps).

TLC (Ether:Hexane 1 :4)

R _f = 0.44, stained by CAM

1H NMR (500 MHz, CD ₃ C(0)CD ₃ )

δ 6.49-6.10 (m, 13H), 5.53 (m, 1H), 4.68 (m, 1H), 4.50 (m, 2H), 4.22 (m, 1H), 4.15 (m,

1H), 4.06 (m, 1H), 4.00 (m, 1H), 3.91 (d, J = 4 Hz, 1H), 3.83 (m, 1H), 3.68 (m, 1H), 3.63 (m, 1H), 3.15 (s, 3H), 2.55 (m, 2H), 2.42 (m, 1H), 2.36 (m, 1H), 2.13 (m, 1H), 2.01 (m, 2H), 1.95-1.70 (m, 8H), 1.63 (m, 3H), 1.49 (m, 1H), 1.31 (m, 3H), 1.18-1.14 (m, 20H), 1.07-0.96 (m, 69H), 0.77-0.61 (m, 43H).

¹³ C NMR (125 MHz, CD ₃ C(0)CD ₃ )

δ 172.2, 170.5, 139.6, 138.6, 134.8, 134.7, 134.0, 133.3, 133.1, 133.0, 132.8, 132.7, 131.7, 131.6, 130.8, 127.9, 101.1, 76.7, 74.0, 73.2, 71.0, 69.3, 69.1, 67.4, 67.3, 59.2, 48.0, 47.8, 44.4, 43.5, 41.8, 41.3, 40.6, 35.5, 27.4, 19.8, 19.2, 18.4, 18.3, 12.8, 11.2, 7.7, 7.6, 7.5, 7.4, 7.3, 6.4, 6.2, 6.1, 5.9, 5.8.

MS (ESI)

Calculated for (M + Na) ⁺ : 1770.2

Found: 1770.2

Octaol SI1

Intermediate 14 (2.5 g, 1.29 mmol, 1 eq) was azeotropically dried with toluene and placed under vacuum overnight. Hexane (80 mL) was added followed by activated 4 angstrom molecular sieves. The resulting solution was allowed to stir at room temperature while the sugar donor was prepared. The sugar donor 11 (739 mg, 2.57 mmol, 2.0 eq) was dissolved in DCM (26 mL). Diphenyl sulfoxide (911 mg, 4.50 mmol, 3.5 eq) and activated 4 angstrom molecular sieves were added. The reaction was stirred for 4 hours at room temperature. 2,6-lutidine (675 μΐ,, 5.79 mmol, 4.5 eq) was added, and the reaction was cooled to -60 °C. Triflic anhydride (1 M in DCM) (2.57 mL, 2.57 mmol, 2 eq) was added slowly. The reaction was warmed to -20 °C and stirred for 1.5 hrs. 2,6-lutidine (600 μί, 5.15 mmol, 4.0 eq) was added to the solution of 14, and it was cooled to -30 °C. The sugar donor reaction was cannulated over to the solution of 14. The reaction was warmed to 0 °C for lhr. The reaction was quenched with saturated aqueous sodium bicarbonate and extracted with ether. The organic layer was washed with copper sulfate, water, and saturated sodium chloride. The organic layer was dried with sodium sulfate and filtered. The solvent was removed under reduced pressure, and column chromatography (Si0 ₂ ; EthenHexane 3:47) purification yielded the glycosidated intermediate 15 as a mixture of isomers ranging from 1 : 1 to 2: 1 α:β (2.12 g, 1.06 mmol, 82 %). The isomers were inseparable at this stage and were taken directly on the next reaction.

15

TLC (Ether :Hexane 1 : 19)

R _f = 0.25, stained by CAM

MS (ESI)

Calculated for C105H205N3O16S19 (M + Na) ⁺ : 2039.3

Found: 2039.9

The glycosidated intermediate 15 (710 mg, 352 μιηοΐ, 1 eq) was azeotropically dried with toluene in a teflon vial. THF (3 mL) was added, and the solution was cooled to 0 °C. Pyridine (3 mL) in a teflon vial was cooled to 0 °C, and MeOH (0.5 mL) was added. 70% HF-pyridine was added slowly to the pyridine-MeOH solution at 0 °C. This solution was transferred slowly to the THF solution of glycosylated intermediate. The reaction was allowed to stir for 12 hours at room temperature. The reaction was quenched at 0 °C with excess MeOTMS and diluted with toluene. The solution was concentrated under reduced pressure and diluted again with toluene. This process was repeated 3 times to remove all of the pyridine. The product is base sensitive, especially if water is present, - care must be taken not to concentrate directly to solid with pyridine present. Reversed phase HPLC purification (C18 Si0 ₂ ; MeCN:5 mM NH ₄ OAc in H ₂ 0 1 : 19→ 19: 1 over 30 minutes) allowed the a and β isomers to be separated and yielded 260 mg, 275 μιηοΐ, 78 %.

HPLC (C18 Si0 ₂ ; MeCN:5 niM NH ₄ OAc in H ₂ 0 1 : 19→ 19: 1 over 30 minutes)

1H NMR (500 MHz, CD ₃ S(0)CD ₃ )

δ 6.32-6.05 (m, 12H), 5.81 (m, 1H), 5.60 (m, 1H), 4.97 (m, 1H), 4.58 (m, 1H), 4.43 (m, 1H), 3.99 (m, 1H), 3.84 (m, 1H), 3.73 (m, 2H), 3.52 (m, 2H), 3.32 (m, 1H), 3.21 (m, 1H), 3.10 (m, 1H), 3.00 (s, 3H), 2.93 (m, 2H), 2.29 (m, 1H), 2.16 (m, 2H), 2.01 (m, 2H), 1.76 (m, 1H), 1.68 (m, 1H), 1.52-1.23 (m, 14H), 1.15 (d, J = 5.5 Hz, 3H), 1.11 (d, J= 5.5 Hz, 3H), 1.03 (d, J = 6 Hz, 3H), 0.89 (d, J = 7 Hz, 3H).

HRMS (ESI)

Calculated for C ₄₈ H ₇₃ N ₃ Oi ₆ (M + Na) ⁺ : 970.4889

Found: 970.4897

Amine SI2

Intermediate SI1 (19 mg, 20 μιηοΐ, 1 eq) was dissolved in DMSO (657 μί). Added water (36 μί, 200 μιηοΐ, 100 eq) and trimethyl phosphine (1 M) (60 μΐ,, 60 μιηοΐ, 3 eq). The reaction was heated to 55 °C for 3 hrs. Reversed phase HPLC purification (CI 8 Si0 ₂ ; MeCN:5 mM NH ₄ OAc in H ₂ 0 1 : 19→ 19: 1 over 30 minutes) yielded SI2 (10.5 mg, 11.4 μιηοΐ, 57 %).

HPLC (C18 Si0 ₂ ; MeCN:5 mM NH ₄ OAc in H ₂ 0 1 : 19→ 19: 1 over 30 minutes)

R, = 14.3 min

1H NMR (500 MHz, CD ₃ S(0)CD ₃ )

δ 6.34-6.06 (m, 12H), 5.90 (m, 1H), 5.62 (m, 1H), 4.94 (m, 1H), 4.63 (m, 1H), 4.52 (m, 1H), 3.97 (m, 1H), 3.90 (m, 1H), 3.73 (m, 2H), 3.56 (m, 1H), 3.38 (m, 1H), 3.30 (m, 1H), 3.25 (m, 1H), 3.15 (m, 1H), 2.95 (m, 5H), 2.25 (m, 4H), 2.03 (m, 1H), 1.77 (m, 3H), 1.53-1.24 (m, 13H), 1.17 (d, J = 5 Hz, 3H), 1.11 (d, J = 6 Hz, 3H), 1.03 (d, J = 6 Hz, 3H), 0.89 (d, J= 6.5 Hz, 3H).

HRMS (ESI)

Calculated for C ₄₈ H ₇₅ NOi ₆ (M + H) ⁺ : 922.5164

Found: 922.5169

C2 deOAmB

Intermediate SI2 (5 mg, 5.42 μιηοΐ, 1 eq) was placed in a vial. 180 of a 180 mM solution of CSA in 2: 1 THF:H ₂ 0 was added. The reaction was stirred for 30 min. Reversed phase HPLC purification (C18 Si0 ₂ ; MeCN:5 niM NH ₄ OAc in H ₂ 0 1 : 19→ 19: 1 over 30 minutes) yielded C2*deOAmB (3.9 mg, 4.34 μιηοΐ, 80 %).

HPLC (C18 Si0 ₂ ; MeCN:5 mM NH ₄ OAc in H ₂ 0 1 : 19→ 19: 1 over 30 minutes)

R _t = 15.1 min

1H NMR (500 MHz, CD ₃ S(0)CD ₃ )

δ 6.47-5.94 (m, 1 1H), 5.73 (m, 1H), 5.42 (m, 2H), 5.23 (m, 1H), 4.77 (m, 1H), 4.61 (m, 1H), 4.38 (m, 1H), 4.26 (m, 1H), 4.15 (m, 1H), 4.06 (m, 1H), 3.99 (m, 1H), 3.70-3.20 (m, 4H), 3.09 (m, 1H), 2.92 (m, 1H), 2.36-2.16 (m, 5H), 1.99 (m, 1H), 1.83-1.72 (m, 4H), 1.56-1.51 (m, 4H), 1.39-1.23 (m, 7H), 1.15 (d, J = 5.5 Hz, 3H), 1.1 1 (d, J = 6 Hz, 3H), 1.03 (d, J= 6 Hz, 3H), 0.91 (d, J = 6.5 Hz, 3H).

HRMS (ESI)

Calculated for C ₄₇ H ₇₃ NOi ₆ (M + H) ⁺ : 908.5008

Found: 908.5007

Epoxide SI3

To a 1 L round bottom flask containing 8 and stir bar under argon was added DCM (325 mL) and DMF (65 mL) and the resulting mixture was stirred to homogeneity. To the mixture was then added sequentially imidazole (31.0 g, 491 mmol) and TBSCl (51.4 g, 341 mmol). The flask was fitted with a reflux condenser and, under argon maintenance, was stirred at 40 °C for 16 h. The reaction was cooled to room temperature and diluted with Et ₂ 0 (1 L) and sat'd aq NaHC0 ₃ (1 L). The aqueous layer was separated and extracted with Et ₂ 0 (500 mL). The combined organic fractions were washed with brine, dried over Na ₂ S0 ₄ , filtered and concentrated in vacuo to give a white solid. The product was purified by dry column vacuum chromatography (DCVC) (Hex:EtOAc, 20: 1→ 3: 1) to afford SI3 as a white solid (40.0 g 92%). Pedersen, DS et al. (2001) Synthesis 16:2431-2434.

SI3

TLC (Ether:Hexane 4: l)

R _f = 0.35, stained by CAM

1H NMR (500 MHz, CDC1 ₃ )

δ 7.30 (d, J= 8.5 Hz, 2H), 6.88 (d, J= 9 Hz, 2H), 4.92 (d, J= 3 Hz, 1H), 4.70 (d, J= 12

Hz, 1H), 4.54 (d, J = 12 Hz, 1H), 3.85 (m, 1H), 3.81 (s, 3H), 3.61 (dd, J = 8.5, 1.5 Hz,

1H), 3.41 (app t, J = 4 Hz, U) 3.25 (dd, J = 4, 1 Hz, 1H), 1.15 (d, J = 6 Hz, 3H), 0.911

(s, 9H), 0.14 (s, 3H), 0.110 (s, 3H).

1 ³ C NMR (500 MHz, CDC1 ₃ )

δ 159.4, 129.9, 114.0, 92.0, 72.4, 68.9, 64.9, 55.4, 54.1, 25.9, 18.1, 17.7, -3.9, -4.6. HRMS (ESI)

Calculated for C ₂ oH ₃₂ 0 ₅ Si (M + Na) ⁺ : 403.1917

Found: 403.1918

SI3 9

Alcohol 9

Epoxide intermediate SI3 (8 g, 21 mmol, 1 eq) was dissolved in THF (263 mL). The resulting solution was cooled to 0 °C, and LiHBEt ₃ (1 M in THF) (105 mL, 105 mmol, 5eq) was added slowly. The reaction heated to 60 °C for 2.5 hrs. The reaction was cooled to 0 °C and quenched with 1 M ammonium chloride. The mixture was extracted with ether. The organic layer was washed with water and saturated sodium chloride. The organic layer was dried with sodium sulfate and filtered. The solvent was removed under reduced pressure, and column chromatography (Si0 ₂ ; Ether:Hexane 1 :4→ 1 :3) purification yielded 9 as an oil (5.47 g, 14.3 mmol, 68 %).

9

TLC (Ether:Hexane 3:7)

R _f = 0.38, stained by CAM

1H NMR (500 MHz, CDC1 ₃ )

δ 7.27 (d, J= 8.5 Hz, 2H), 6.88 (d, J= 9 Hz, 2H), 4.87 (d, J= 4 Hz, 1H), 4.66 (d, J= 12 Hz, 1H), 4.44 (d, J= 11.5 Hz, 1H), 4.04 (m, 1H), 3.92 (m, 1H), 3.81 (s, 3H), 3.32 (dd, J = 3 Hz, J = 9.5 Hz, 1H), 3.19 (m, 1H), 2.14 (dd, J = 3.5 Hz, J = 15 Hz, 1H), 1.89 (td, J = 3.5 Hz, J = 14.5 Hz, 1H), 1.26 (d, J= 6.5 Hz, 3H), 0.93 (s, 9H), 0.12 (s, 6H).

¹³ C NMR (125 MHz, CDC1 ₃ )

δ 159.5, 129.9, 114.0, 95.7, 75.1, 68.9, 68.0, 63.7, 55.5, 35.7, 26.1, 18.4, -4.0, -4.4.

HRMS (ESI)

Calculated for C ₂ oH ₃₄ 0 ₅ Si (M + Na) ⁺ : 405.2073

Found: 405.2078

9 SI4

Mesylate SI4

Intermediate 9 (4.83 g, 12.6 mmol, 1 eq) was dissolved in THF (15 mL). Pyridine (10.2 mL, 126 mmol, 10 eq) and MsCl (3.17 mL, 41 mmol, 3.25 eq) were added. The reaction was stirred overnight. The reaction was then quenched with saturated aqueous sodium bicarbonate and extracted with ether. The organic layer was washed with 1 M ammonium chloride, water, and saturated sodium chloride. The organic layer was dried with sodium sulfate and filtered. The solvent was removed under reduced pressure, and column chromatography (Si0 ₂ ; EthenHexane 2:3) purification yielded SI4 as a solid (4.24 g, 9.2 mmol, 73 %).

SI4 TLC (Ether:Hexane 2:3)

R _f = 0.27, stained by CAM

1H NMR (500 MHz, CDC1 ₃ )

δ 7.27 (d, J = 9 Hz, 2H), 6.88 (d, J = 8.5 Hz, 2H), 4.90 (dd, J = 3 Hz, J = 8 Hz, 2H),

4.66 (d, J = 11.5 Hz, 1H), 4.35 (d, J= 11 Hz, 1H), 4.13 (m, 1H), 3.80 (s, 3H), 3.43 (dd,

J = 3 Hz, J = 9 Hz, 1H), 2.91 (s, 3H), 2.41 (dd, J = 3 Hz, J= 15 Hz, 1H), 1.96 (m, 1H),

1.23 (d, J= 6.5 Hz, 3H), 0.93 (s, 9H), 0.14 (s, 3H), 0.11 (s, 3H).

1 ³ C NMR (125 MHz, CDC1 ₃ )

δ 159.4, 130.2, 129.6, 113.9, 94.8, 77.5, 72.7, 69.1, 64.3, 55.5, 39.9, 34.5, 26.0, 18.3, -

3.9, -4.6.

HRMS (ESI)

Calculated for C ₂ iH ₃₆ 0 ₅ SiS (M + Na) ⁺ : 483.1849

Found: 483.1848

SI4 10

Azide 10

Intermediate SI4 (1.6 g, 3.47 mmol, 1 eq) was dissolved in DMF (15 mL). Sodium azide (1.6 g, 24.3 mmol, 7 eq) was added. The reaction heated to 160 °C for 1.5 hrs. The reaction was cooled to room temperature. The reaction was quenched with saturated aqueous sodium bicarbonate and extracted with ether. The organic layer was washed with water, and saturated sodium chloride. The organic layer was dried with sodium sulfate and filtered. The solvent was removed under reduced pressure, and column chromatography (Si0 ₂ ; Ether:Hexane 1 : 19) purification yielded 10 as a solid (1.13 g, 2.78 mmol, 80 %).

10

TLC (Ether :Hexane 1 : 19)

R _f = 0.30, stained by CAM

1H NMR (500 MHz, CDC1 ₃ ) δ 7.29 (d, J = 9 Hz, 2H), 6.91 (d, J = 8.5 Hz, 2H), 4.91 (d, J = 3 Hz, 2H), 4.61 (d, J =

11.5 Hz, 1H), 4.39 (d, J = 11 Hz, 1H), 3.82 (s, 3H), 3.71 (m, 2H), 3.10 (t, J = 9 Hz, 1H),

2.19 (dd, J= 5 Hz, J = 13.5 Hz, 1H), 1.73 (td, J= 4 Hz, J = 12.5 Hz, 1H), 1.27 (d, J= 6

Hz, 3H), 0.94 (s, 9H), 0.22 (s, 3H), 0.13 (s, 3H).

1 ³ C NMR (125 MHz, CDC1 ₃ )

δ 159.6, 129.9, 129.8, 114.1, 95.4, 76.7, 68.9, 68.8, 61.8, 55.5, 35.9, 26.2, 18.7, 18.4, -

3.9, -4.0.

HRMS (ESI)

Calculated for C ₂₀ H ₃₃ N ₃ O ₄ Si (M + Na) ⁺ : 430.2138

Found: 430.2156

10 11

Alcohol 11

Intermediate 10 (6.5 g, 15.9 mmol, 1 eq) was dissolved in DCM:H ₂ 0 9: 1 (160 mL). The solution was cooled to 0 °C, and DDQ (4.3 g, 19.1 mmol, 1.2 eq) was added. The reaction was warmed to room temperature and stirred for 2 hrs. The reaction was quenched with saturated aqueous sodium bicarbonate and extracted with ether. The organic layer was washed with water, and saturated sodium chloride. The organic layer was dried with sodium sulfate and filtered. The solvent was removed under reduced pressure, and column chromatography (Si0 ₂ ; Ether :Hexane 1 : 19) purification followed by (C 18 Si0 ₂ ,

water:MeCN 1 :4) yielded 11 as a solid consisting of a 2: 1 mixture of anomers (3.47 g, 12.1 mmol, 76 %).

11

TLC (EtOAc:Hexane 1 :4)

R _f = 0.31, stained by CAM

(H ₂ 0:MeCN 1 :4)

R _f = 0.50, stained by CAM

1H NMR (500 MHz, CD ₃ C(0)CD ₃ ) δ 5.62 (d, J = 6.5 Hz, 1H), 5.33 (m, 2H), 5.22 (m, 2H), 4.84 (m, 1H), 3.84 (m, 2H), 3.67 (m, 2H), 3.47 (m, 1H), 3.29 (m, 1H), 3.07 (m, 3H), 2.25 (dd, J = 12.5 Hz, J= 2 Hz, 1H), 2.1 1 (dd, J = 13 Hz, J = 1 Hz, 2H), 1.65 (td, J = 3 Hz, J = 12.5 Hz, 2H), 1.50 (m, 1H), 1.19 (d, J = 6.5 Hz, 3H), 1.14 (d, J = 6.5 Hz, 6H), 0.90 (m, 28H), 0.19 (m, 9H), 0.12 (s, 9H).

¹³ C NMR (125 MHz, CD ₃ C(0)CD ₃ )

δ 94.31 , 90.96, 77.559, 76.90, 73.53, 68.49, 64.64, 62.38, 38.86, 36.80, 26.27, 18.99, 18.96, 18.64, -4.04, -4.09.

HRMS (ESI)

Calculated for Ci ₂ H ₂₅ N ₃ 0 ₃ Si (M + Na) ⁺ : 310.1563

Found: 310.1566

Example 2. C2'deOAmB Binds Ergosterol but Not Cholesterol

With multiple milligrams of this key probe in hand, we initially tested whether deletion of the C2' hydroxyl impacts the capacity of AmB to bind ergosterol via an optimized isothermal titration calorimetry (ITC)-based assay (FIG. 4A). Specifically, we first titrated an aqueous solution of AmB with a suspension of large unilamellar vesicles (LUVs) comprised of only l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and the net exotherm was recorded. We then repeated this titration using POPC LUVs containing 10% ergosterol. Consistent with our previous results, ⁸ we observed a significant increase in net exotherm when switching to ergosterol-containing LUVs, indicating a direct AmB-sterol binding interaction. No such binding was observed when the same pair of titrations was repeated with AmdeB (FIG. 4 A). ⁸ Surprisingly, when C2'deOAmB was subjected to the same experiments, a significant increase in net exotherm was observed demonstrating a retained capacity for this functional group-deficient derivative to bind ergosterol. Thus, contrary to the leading model, the C2' hydroxyl group on AmB is not required for ergosterol binding.

Even more surprisingly, when we repeated these same binding studies with cholesterol, we observed a very different result. Specifically, after confirming binding and no binding of cholesterol for AmB and AmdeB, respectively, ⁸ we tested the cholesterol binding capacity of C2'deOAmB (FIG. 4B). In contrast to the results with ergosterol, C2 'deOAmB showed no evidence of binding cholesterol. Thus, the C2' hydroxyl group is required to bind cholesterol, but not ergosterol. Details of this experiment are as follows: Isothermal Titration Calorimetry General Information

Experiments were performed using a NanoITC isothermal titration calorimeter (TA Instruments, Wilmington, DE). Solutions of the compounds to be tested were prepared by diluting a 60.0 mM stock solution of the compound in DMSO to 600 μΜ with K buffer (5.0 mM HEPES/KHEPES, pH = 7.4). The final DMSO concentration in the solution was 1% v/v. POPC LUVs were prepared and phosphorus and ergosterol content was quantified as described below. The LUV solutions were diluted with buffer and DMSO to give a final phospholipid concentration of 12.0 mM in a 1% DMSO/K buffer solution. Immediately prior to use, all solutions were incubated at 37 °C for 30 minutes and degassed under vacuum at 37 °C for 10 minutes. The reference cell of the instrument (volume = 0.190 mL) was filled with a solution of 1% v/v DMSO/K buffer.

L UV Preparation Palmitoyl oleoyl phosphatidylcholine (POPC) was obtained as a 20 mg/mL solution in CHCI ₃ from Avanti Polar Lipids (Alabaster, AL) and was stored at -20 °C under an atmosphere of dry argon and used within 1 month. A 4 mg/mL solution of ergosterol in CHCI ₃ was prepared monthly and stored at 4 °C under an atmosphere of dry argon. Prior to preparing a lipid film, the solutions were warmed to ambient temperature to prevent condensation from contaminating the solutions. A 13 x 100 mm test tube was charged with 800 POPC and 230 of the ergosterol solution. For cholesterol-containing liposomes, a 13 x 100 mm test tube was charged with 800 μΐ, POPC and 224 μΕ of the ergosterol solution. For sterol-free liposomes, a 13 x 100 mm test tube was charged with 800 μΐ, POPC. The solvent was removed with a gentle stream of nitrogen and the resulting lipid film was stored under high vacuum for a minimum of eight hours prior to use. The film was then hydrated with 1 mL of K buffer and vortexed vigorously for approximately 3 minutes to form a suspension of multilamellar vesicles (MLVs). The resulting lipid suspension was pulled into a Hamilton (Reno, NV) 1 mL gastight syringe and the syringe was placed in an Avanti Polar Lipids Mini-Extruder. The lipid solution was then passed through a 0.20 μιη Millipore (Billerica, MA) polycarbonate filter 21 times, the newly formed large unilamellar vesicle (LUV) suspension being collected in the syringe that did not contain the original suspension of MLVs to prevent the carryover of MLVs into the LUV solution.

Determination of Phosphorus Content

Determination of total phosphorus was adapted from the report of Chen and coworkers. Chen, PS et al. (1956) Anal. Chem. 28: 1756. The LUV solution was diluted tenfold with K buffer and three 10 μί samples of the diluted LUV suspension were added to three separate 7 mL vials. Subsequently, the solvent was removed with a stream of N ₂ . To each dried LUV film, and a fourth vial containing no lipids that was used as a blank, was added 450 of 8.9 M H ₂ SO ₄ . The four samples were incubated open to ambient atmosphere in a 225 °C aluminum heating block for 25 min and then removed to 23 °C and cooled for 5 minutes. After cooling, 150 of 30% w/v aqueous hydrogen peroxide was added to each sample, and the vials were returned to the 225 °C heating block for 30 minutes. The samples were then removed to 23 °C and cooled for 5 minutes before the addition of 3.9 mL water. Then 500 of 2.5% w/v ammonium molybdate was added to each vial and the resulting mixtures were then vortexed briefly and vigorously five times. Subsequently, 500 μΐ, of 10%> w/v ascorbic acid was added to each vial and the resulting mixtures were then vortexed briefly and vigorously five times. The vials were enclosed with a PTFE lined cap and then placed in a 100 °C aluminum heating block for 7 minutes. The samples were removed to 23 °C and cooled for approximately 15 minutes prior to analysis by UV/Vis spectroscopy. Total phosphorus was determined by observing the absorbance at 820 nm and comparing this value to a standard curve obtained through this method and a standard phosphorus solution of known concentration.

Determination of Ergosterol Content

Ergosterol content was determined spectrophotometrically. A 50 portion of the LUV suspension was added to 450 μΐ, 2: 18:9 hexane:isopropanol:water (v/v/v). Three independent samples were prepared and then vortexed vigorously for approximately one minute. The solutions were then analyzed by UV/Vis spectroscopy and the concentration of ergosterol in solution was determined by the extinction coefficient of 10400 L mol ^"1 cm ^"1 at the UV _max of 282 nm and was compared to the concentration of phosphorus to determine the percent sterol content. The extinction coefficient was determined independently in the above ternary solvent system. LUVs prepared by this method contained between 7 and 14%) ergosterol. Titration Experiment

Titrations were performed by injecting the LUV suspension at ambient temperature into the sample cell (volume = 0.191 mL) which contained the 600 μΜ solution of the compound in question at 25 °C. The volume of the first injection was 0.23 μί. Consistent with standard procedure (Heerklotz, H et al. (2000) Biochim. Biophys. Acta 1508:69), due to the large error commonly associated with the first injection of ITC experiments, the heat of this injection was not included in the analysis of the data. Next, six 7.49 injections of the LUV suspension were performed. The spacing between each injection was 720 seconds to ensure that the instrument would return to a stable baseline before the next injection was made. The rate of stirring for each experiment was 300 rpm.

Data Analysis

NanoAnalyze software (TA Instruments) was used for baseline determination and integration of the injection heats, and Microsoft Excel was used for subtraction of dilution heats and the calculation of overall heat evolved. To correct for dilution and mixing heats, the heat of the final injection from each run was subtracted from all the injection heats for that particular experiment. See, for example, te Welscher, YM et al. (2008) J. Biol. Chem. 283:6393. By this method, the overall heat evolved during the experiment was calculated using the following formula:

n

overall ^ > ^-^^ injection ^^injection

i=\ where i = injection number, n = total number of injections, ^ti _injection ⁼ heat of the i ^th injection, h" _njection = the heat of the final injection of the experiment.

Example 3. C2'deOAmB Exerts Antifungal Activity In Vitro

We next tested the activity of AmB, AmdeB, and C2'deOAmB against two ergosterol-containing strains of yeast, S. cerevisiae and C. albicans, with the latter representing the most common cause of life -threatening systemic fungal infections in humans.

Consistent with our previous results ⁸ and the ergosterol binding data described above, we observed potent antifungal activity for AmB against both cell lines and no antifungal activity for AmdeB. Importantly, and consistent with the observation of retained ergosterol binding, when we tested C2'deOAmB in these same assays, we observed retention of potent antifungal activity against both of these yeast cell lines (Table 1).

Table 1. In Vitro Characteristics of AmB, AmdeB, and C2'deOAmB

Details of this experiment are as follows: Growth Conditions for S. cerevisiae

S. cerevisiae was maintained with yeast peptone dextrose (YPD) growth media consisting of 10 g/L yeast extract, 20 g/L peptone, 20 g/L dextrose, and 20 g/L agar for solid media. The media was sterilized by autoclaving at 250 °F for 30 min. Dextrose was subsequently added as a sterile 40% w/v solution in water (dextrose solutions were filter sterilized). Solid media was prepared by pouring sterile media containing agar (20 g/L) onto Corning (Corning, NY) 100 x 20 mm polystyrene plates. Liquid cultures were incubated at 30 °C on a rotary shaker and solid cultures were maintained at 30 °C in an incubator.

Growth Conditions for C. albicans

C albicans was cultured in a similar manner to S. cerevisiae except both liquid and solid cultures were incubated at 37 °C.

Broth Microdilution Minimum Inhibitory Concentration (MIC) Assay The protocol for the broth microdilution assay was adapted from the Clinical and

Laboratory Standards Institute document M27-A2. Clinical and Laboratory Standards Institute. Reference Method for Broth Dilution Antifungal Susceptibility Testing, M27-A2, Approved Standard 2 ^nd Ed. Vol. 22, Number 15, 2002. 50 mL of YPD media was inoculated and incubated overnight at either 30 °C (S. cerevisiae) or 37 °C (C. albicans) in a shaker incubator. The cell suspension was then diluted with YPD to an OD ₆ oo of 0.10 (~5 x 10 ⁵ cfu/mL) as measured by a Shimadzu (Kyoto, Japan) PharmaSpec UV-1700 UV/Vis spectrophotometer. The solution was diluted 10-fold with YPD, and 195 μΙ_, aliquots of the dilute cell suspension were added to sterile Falcon (Franklin Lakes, NJ) Microtest 96-well plates in triplicate. Compounds were prepared either as 400 μΜ (AmB, C2'deOAmB) or 2 mM (AmdeB) stock solutions in DMSO and serially diluted to the following concentrations with DMSO: 1600, 1200, 800, 400, 320, 240, 200, 160, 120, 80, 40, 20, 10 and 5 μΜ. 5 μΐ aliquots of each solution were added to the 96-well plate in triplicate, with each column representing a different concentration of the test compound. The concentration of DMSO in each well was 2.5% and a control well to confirm viability using only 2.5% DMSO was also performed in triplicate. This 40-fold dilution gave the following final concentrations: 50, 40, 30, 20, 10, 8, 6, 4, 1, 0.5, 0.25 and 0.125 μΜ. The plates were covered and incubated at 30 °C (S. cerevisiae) or 37 °C (C. albicans) for 24 hours prior to analysis. The MIC was determined to be the concentration of compound that resulted in no visible growth of the yeast. The experiments were performed in duplicate and the reported MIC represents an average of two experiments.

Example 4. C2'deOAmB Is Not Toxic to Human Cells In Vitro

Finally, we probed the activity of these same three compounds against human cells. Two of the most important toxic side effects associated with AmB are anemia and nephrotoxicity caused by damage to red blood cells and renal proximal tubule cells, respectively. ⁵ ^ ¹⁵ Consistent with literature precedent, AmB causes 90%> hemolysis of human red blood cells at a concentration of 8.5 μΜ. This is defined as the minimum hemolytic concentration (MHC). In stark contrast, we found that the corresponding MHCs for AmdeB and C2'deOAmB, both of which do not bind cholesterol, to be >500 μΜ (Table 1). Similarly, AmB causes 90% loss of cell viability of primary human renal proximal tubule epithelial cells at a concentration of 2.4 μΜ [the minimum toxic concentration (MTC)]. Again, in stark contrast to AmB, both AmdeB and, most importantly,

C2'deOAmB showed no evidence of toxicity up to their limits of solubility. ¹⁶ As shown in FIG. 5, microscopy further revealed that human primary renal cells treated with AmB showed severe abnormalities compared to DMSO treated controls. In contrast, cells treated AmdeB and C2'deOAmB showed no visual evidence of toxicity. Details of this experiment are as follows:

Hemolysis Assays

Erythrocyte Preparation

The protocol for the hemolysis assay was adapted from the report of Paquet and coworkers. Paquet, V et al. (2008) Chem. Eur. J. 14:2465-2481. Whole human blood

(sodium heparin) was purchased from Bioreclamation LLC (Westbury, NY) and stored at 4 °C and used within two days of receipt. To a 2.0 mL Eppendorf tube, 1 mL of whole human blood was added and centrifuged at 10,000 g for 2 minutes. The supernatant was removed and the erythrocyte pellet was washed with 1 mL of sterile saline and centrifuged at 10,000 g for 2 minutes. The saline wash was repeated for a total of three washes. The erythrocyte pellet was suspended in 1 mL of RBC buffer (10 mM NaH ₂ P0 ₄ , 150 mM NaCl, 1 mM MgCl ₂ , pH 7.4) to form the erythrocyte stock suspension.

Minimum Hemolysis Concentration (MHC) Assay

Compounds were prepared as 1.03 mM (AmB) or 12.8 mM (C2'deOAmB and AmdeB) stock solutions in DMSO and serially diluted to the following concentrations with DMSO: 7689, 5126, 2563, 2050, 1538, 1025, 769, 513, 384, 256, 205, 154, 103, 77, 51, 26 μΜ. To a 0.2 mL PCR tube, 24 of RBC buffer and 1 of compound stock solution were added, which gave final concentrations of 500, 300, 200, 100, 80, 60, 40, 30, 20, 15, 10, 8, 6, 4, 3, 2, 1 μΜ. Positive and negative controls were prepared by adding 1 μΐ _^ of DMSO to MilliQ water or RBC buffer, respectively to 0.2 mL PCR tube. To each PCR tube, 0.63 μΐ, of the erythrocyte stock suspension was added and mixed by inversion. The samples were incubated at 37 °C for 2 hours. The samples were mixed by inversion and centrifuged at 10,000 g for 2 minutes. 15 μΐ _^ of the supernatant from each sample was added to a 384-well place. Absorbances were read at 540 nm using a Biotek HI Synergy Hybrid Reader (Wanooski, VT). Experiments were performed in triplicate and the reported MHC represents an average of three experiments.

Data Analysis

Percent hemolysis was determined according to the following equation:

% hemolysis = ^A . _x

nuo.p _OS nuj. _ne g

- Al - Concentration vs. percent hemolysis was plotted and fitted to 4-parameter logistic (4PL) dose response fit using OriginPro 8.6. Sebaugh, JL (201 1) Pharmaceut. Statist. 10: 128- 134. The MHC was defined as the concentration to cause 90% hemolysis. WST-8 Cell Proliferation Assays

Primary Renal Proximal Tubule Epithelial Cells Preparation

Primary human renal proximal tubule epithelial cells (RPTECs) were purchased from ATCC (Manassas, VA) and immediately cultured upon receipt. Complete growth media was prepared using renal epithelial cell basal medium (ATCC, PCS-400-030), renal epithelial cell growth kit (ATCC, PCS-400-040), and penicillin-streptomycin (10 units/mL and 10 μg/mL). Complete media was stored at 4 °C in the dark and used within 28 days. Primary RPTECs were grown in C0 ₂ incubator at 37 °C with an atmosphere of 95% air/5% C0 ₂ .

WST-8 Reagent Preparation WST-8 cell proliferation assay kit (10010199) was purchased from Cayman

Chemical Company (Ann Arbor, MI) and stored at -20 °C and used within 6 months of receipt. WST-8 reagent and electron mediator solution were thawed and mixed to prepare the WST-8 reagent solution. The solution was stored at -20 °C and used within one week.

WST-8 Assay A suspension of primary RPTECs in complete growth media was brought to a concentration of 1 x 10 ⁵ cells/mL. A 96-well plate was seeded with 99 xL 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 were prepared by seeding with 100 of the cell suspension or 100 of the complete media. Compounds were prepared as 5 mM (AmB), 20 mM (C2'deOAmB), and 50 mM (AmdeB) stock solutions in DMSO and serially diluted to the following concentrations with DMSO: 50000, 40000, 30000, 20000, 10000, 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 were added to the 96-well plate in triplicate, with each column representing a different concentration of the test compound. The 96-well plate was incubated at 37 °C with an atmosphere of 95% air/5% C0 ₂ for 24 hours. After incubation, the media was aspirated and 100 μΐ, of serum- free media was added and 10 μΐ, of the WST-8 reagent solution was added to each well. The 96-well plate was 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 was mixed in a shaking incubator at 200 rpm for 1 minute and absorbances were read at 450 nm using a Biotek Hl Synergy Hybrid Reader (Wanooski, VT). Experiments were performed in triplicate and the reported cytotoxicity represents an average of three experiments.

Data Analysis

Percent hemolysis was determined according to the following equation: % hemolysis = ^{Abs'samPle ~ M} s. _neg . _χ

Concentration vs. percent hemolysis was plotted and fitted to 4-parameter logistic (4PL) ⁸ dose response fit using OriginPro 8.6. The MTC was defined as the concentration to cause 90%) loss of cell viability.

Microscopy

Cells were imaged using an AMG (Bothell, WA) EVOS fl Microscope. Images were taken using transmitted light at lOx objective.

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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.

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