COMPOSITIONS AND METHODS FOR THE TREATMENT OF FUNGAL INFECTIONS

Background

The need for novel antifungal treatments is significant and is especially critical in the medical field. Immunocompromised patients provide perhaps the greatest challenge to modern health care delivery. The most common pathogens associated with invasive fungal infections are the opportunistic yeast, Candida albicans, and the filamentous fungus, Aspergillus fumigatus.

The development of antifungal treatment regimens has been a continuing challenge. The FDA has approved several antifungal agents of different chemical classes, e.g., polyenes, pyrimidines, azoles, and echinocandins. Among these, polyene antifungal agents exhibit potent antifungal activities by inserting into fungal membranes to disrupt membrane integrity. These polyene antifungal agents form complexes with ergosterol and structurally related sterols within the lipid bilayer, thus altering membrane structure and promoting leakage of cellular components. Polyene antifungal agents typically include large lactone rings with three to eight conjugated carbon-carbon double bonds and may also contain a sugar moiety and an aromatic moiety.

Even though many potent polyene macrolide antifungal agents have been isolated, the use of this class of antibiotics has been limited by the lack of absorption, high toxicity, and low tolerability.

Amphotericin B remains the preferred polyene antifungal agent for treatment of life-threatening mycotic infections.

Because of the shortcomings of existing antifungal treatments, there is a need in the art for improved antifungal therapies having greater efficacy, bioavailability, and reduced toxicity.

Summary

The disclosure relates to compositions, methods for inhibiting fungal growth, and methods for the treatment of fungal infections. In particular, such compositions include bifunctional molecules comprising a polyene antifungal agent moiety that binds to ergosterol and structurally related sterols in the fungal membrane and a chemotaxis receptor ligand moiety that interacts with a chemotaxis receptor. Such compositions are useful in methods for the inhibition of fungal growth and in methods for the treatment of fungal infections, such as those caused by a fungus of the genus Aspergillus or Candida.

Accordingly, in one aspect, the disclosure features a compound (e.g., a synthetic bifunctional non-antibody compound), or a pharmaceutically acceptable salt thereof, comprises, consists essentially of, or consists of a chemotaxis receptor ligand moiety, E, covalently conjugated to a polyene antifungal agent moiety through linker, L. In some embodiments, the linker is a polypeptide. In some embodiments, the chemotaxis receptor ligand moiety, E, is attached to the linker, L, through an amide bond and/or the polyene antifungal agent moiety is attached to the linker, L, through an amide bond. I n some embodiments, the linker is attached to a carbonyl group of the chemotaxis receptor ligand moiety, E. In some embodiments, the compound is formed from a linker that is a diamine. In some embodiments, the diamine linker is conjugated to a carbonyl group of the polyene antifungal agent moiety to form an amide linkage. In some embodiments, the diamine linker is conjugated to a carbonyl of the chemotaxis receptor ligand moiety, E, to form an amide linkage.

In some embodiments, the polyene antifungal agent is a 67-121 -A, 67-121 -C, amphotericin B, arenomvcin B, aurenin, aureofungin A, aureotuscin, candidin, chinin, demethoxyrapamycin, dermostatin A, dermostatin B, DJ-400-B^ DJ-400-B ₂ , elizabethin, eurocidin A, eurocidin b, tilipin I, tilipin I I, tilipin I I I, filipin IV, fungichromin, gannibamycin, hamycin, levorin A ₂ , lienomycin, lucensomycin, mycoheptin, mycoticin A, mycoticin B, natamycin, nystatin A, nystatin A ₃ , partricin A, partricin B, perimycin A, pimaricin, polifungin B, rapamycin, rectilavendomvcin, rimocidin, roflamycoin, tetramycin A, tetramycin B, tetrin A, or tetrin B. In some embodiments, the polyene antifungal agent is selected from amphotericin B, natamycin, and nystatin. In some embodiments, the polyene antifungal agent is amphotericin B. In some embodiments, the polyene antifungal agent is natamycin.

In some embodiments, the chemotaxis receptor ligand is a ligand to a formyl peptide receptor family. In some embodiments, the chemotaxis receptor ligand is a ligand to FPR1 , FPR2, FPR3, FPRL1 , and/or FPRL2. In some embodiments, the chemotaxis receptor ligand is a ligand to neuropilin 1 , CXCR1 , and/or CXCR2.

In some embodiments, the chemotaxis receptor ligand is selected from a chemotactic peptide, a tuftsin peptide, and an acetyl-proline-glycine-proline (PGP) peptide.

In some embodiments, the chemotaxis receptor ligand moiety, E, is a chemotactic peptide and the chemotactic peptide includes an amino acid having the formula:

R ¹⁴ -X1 -X2-X3-X4-X5-X6-X7-X8-X9- Formula V

wherein X1 is any amino acid residue; X2-X9 are any amino acid residue or is absent; R ¹⁴ is hydrogen or

^{R1 Λ} Aιο / ; wherein X ₁₀ is a bond, NH, or O; and R is hydrogen, optionally substituted C1 -C6 alkyl, optionally substituted C1 -C6 heteroalkyi, optionally substituted C2-C6 alkenyl, optionally substituted C2- C6 heteroalkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted C3-C1 0 cycloalkyl, optionally substituted C4-C10 cycloalkenyl, optionally substituted C4-C10 cycloalkynyl, optionally substituted oxime, optionally substituted hydrazone, optionally substituted C6-C12 aryl, or optionally substituted C2-C6 heterocyclic. In some embodiments, if R ¹⁴ is hydrogen, X2 is not absent.

In some embodiments, the chemotactic peptide includes an amino acid having the formula:

R ¹⁴ -X1 -X2-X9- Formula VI

wherein X1 is any amino acid residue; X2 is a leucine residue, isoleucine residue, or absent; and X9 is any amino acid residue.

In some embodiments, the chemotactic peptide includes an amino acid having the formula:

R ¹⁴ -X1 -X2-X9- Formula VI

wherein X1 and X9 are each independently hydrophobic amino acid residues and X2 is a hydrophobic amino acid residue, or is absent. In some embodiments, the X1 is a methionine residue, an

oxymethionine residue, or a norleucine residue. I n some embodiments, X2 is a leucine residue, a isoleucine residue, or is absent. In some embodiments, X9 is a phenylalanine residue, a 1 -amino-2- phenylcyclopropane-1 -carboxylic acid residue, a methionine residue, an (O-benzyl) serine residue, a 2- pyridiylalanine residue, or a 4-pyridylalanine residue. In some embodiments, R ¹⁴ is -C(0) H. I n some embodiments, H ^" is -(J(U)(JH ₃ . In some embodiments, R ¹⁴ is -C(0)OCH ₂ CH(CH ₃ ) ₂ . I n some embodiments, R ¹⁴ is -C(0)N H-(4-chlorophenyl).

In some embodiments, the chemotaxis receptor ligand moiety, E, is selected from the group consisting of

Formula E-l

wherein R ²⁴ is H or C1 -C3 alkyi; wherein q ¹ is 1 or 2; and wherein -NH-A ⁴ -C(0)- and -NH-A ⁵ -C(0)- are each independently a basic amino acid residue;

Formula E-l la Formula E-l lb wherein R ²⁵ is H, C1 -C6 alkyi or phenyl; wherein t ¹ is 1 or 2; wherein u ¹ is 1 , 2, 3, 4, 5, or 6; and wherein v ¹ is 1 or 2; and

Formula E-l l l

wherein -NH-A ¹ -C(0)- and -NH-A ² -C(0)- are each independently a hydrophobic amino acid residue; wherein -NH-A ³ -C(0)- is a hydrophobic amino acid residue or is absent; wherein R ²⁶ is H, C1 -C6 alkyi, OR ²⁸ , or NR ²⁹ ; wherein R ²⁷ is heterocyclyl, heteroaryl, or heteroalkenyl; wherein R ²⁸ and R ²⁹ are each independently C1 -C1 0 alkyi or phenyl optionally substituted with C1 -C3 alkyi or halo; and wherein w ¹ is 0, 1 , 2, or 3.

In some embodiments, the chemotaxis receptor ligand moiety, E, is selected from:

a)

Formula Ε-Γ

wherein R ²⁴ is H or C1 -C3 alkyi; wherein q ¹ is 1 or 2; and wherein -NH-A ⁴ -C(0)- and -NH-A ⁵ -C(0)- are each independently a basic amino acid residue;

Formula E-lla' Formula E-llb'

wherein R ²⁵ is H, C1 -C6 alkyi or phenyl; wherein t ¹ is 1 or 2; wherein u ¹ is 1 , 2, 3, 4, 5, or 6; and wherein v ¹ is 1 or 2; and

Formula E- I I I'

wherein -NH-A ¹ -C(0)- and -NH-A ² -C(0)- are each independently a hydrophobic amino acid residue; wherein -NH-A ³ -C(0)- is a hydrophobic amino acid residue or is absent; wherein R ²⁶ is H, C1 -C6 alkyi,

28 29 27 28 29

OR , or NR ; wherein R is heterocyclyl, heteroaryl, or heteroalkenyl; wherein R and R are each independently C1 -C1 0 alkyi or phenyl optionally substituted with C1 -C3 alkyi or halo; and wherein w ¹ is 0, 1 , 2, or 3.

In some embodiments, the chemotaxis receptor ligand moiety, E, is

Formula E-l "

wherein R ²⁴ is H or methyl; wherein r ¹ and s ¹ are each individually 1 , 2, 3, or 4; wherein q ¹ is 1 or 2; and wherein the basic amino acid residue is selected from the group consisting of a lysine residue, an ornithine residue, an arginine residue, a diaminobutyric acid residue, and a diaminopropanoic acid residue. In some embodiments, R ²⁴ is methyl; and the basic amino acid residue is the lysine or arginine residue.

In some embodiments, the chemotaxis receptor ligand moiety, E, is

Formula I I I"

wherein R ²⁷ is a C3-C1 0 heteroalkyl, heteroaryl, or heteroalkenyl having 1 -5 O, S, or N heteroatoms.

In some embodiments, R ²⁷ is piperidyl, tetrahydrofuranyl, pyridyl, pyrazinyl, pyrimidinyl triazinyl, thiophenyl, or furanyl. In some embodiments, R ²⁷ is 2-pyridyl, 4-pyridyl, or -NHC(NH)NH _2; w ¹ is 3;

-NH-A ¹ -C(0)- is a methionine residue; and -NH-A ² -C(0)- is a leucine residue. In some embodiments, the chemotaxis receptor ligand moiety, b, is

 In some embodiments, the chemotaxis receptor ligand moiety, b, has the structure selected trom:

In some embodiments, the chemotaxis receptor ligand moiety, E, has the structure selected from:

In some embodiments, the chemotaxis receptor ligand moiety, E, has the structure selected from:

In some embodiments, the linker in the compound or the pharmaceutically acceptable salt thereof includes a non-reactive linking moiety of 1 -1 00 atoms in length. In some embodiments, the linker has the structure:

G ¹ -(Z ¹ ) _b -(Y ¹ ) _c -(Z ² ) _d -(R ¹⁶ )-(Z ³ ) _e -(Y ² ) _f -(Z ⁴ ) _g -G ²

Formula VII

wherein G ¹ is a bond between the linker and the polyene antifungal agent moiety; G ² is a bond between the chemotaxis receptor ligand moiety, E, and the linker; each of Z ¹ , Z ² , Z ³ , and Z ⁴ is independently selected from optionally substituted C1 -C4 alkylene, optionally substituted C1 -C3 heteroalkylene, optionally substituted C3-C9 cycloalkylene, optionally substituted C6-C12 arylene, optionally substituted C2-C6 heterocyclylene, O, S, and NR ¹⁷ ; each R ¹⁷ is independently hydrogen, optionally substituted C1 - C4 alkyl, optionally substituted C2-C4 alkenyl, optionally substituted C2-C4 alkynyl, optionally substituted C2-C6 heterocyclyl, optionally substituted C6-C12 aryl, or optionally substituted C1 -C7 heteroalkyl; Y ¹ and Y ² are independently carbonyl, thiocarbonyl, sulphonyl, or phosphoryl; b, c, d, e, f, and g are, independently, 0 or 1 ; and R ¹⁶ is optionally substituted C1 -C1 0 alkylene, optionally substituted C2-C1 0 alkenylene, optionally substituted C2-C1 0 alkynylene, optionally substituted C3-C9 cycloalkylene, optionally substituted C2-C6 heterocyclylene, optionally substituted C6-C12 arylene, optionally substituted C2-C100 polyethylene glycolene, or optionally substituted C1 -C1 0 heteroalkylene, or a chemical bond, provided that R ¹⁶ is not a chemical bond when b, c, d, e, f, and g are 0; or G ¹ -(Z ¹ ) _b -(Y ¹ ) _c - (Z ² ) _d -(R ¹⁶ )-(Z ³ ) _e -(Y ² ) _f -(Z ⁴ ) _g -G ² is a bond linking the polyene antifungal agent moiety to the chemotaxis receptor ligand moiety.

Formula VIII Formula IX Formula X

Formula XII

Formula XIV Formula XV

wherein i, k, I, and m are independently 0 to 12; h is 1 to 6; j is 1 to 7; t is 1 to 5; u is 0 to 4; v is 1 to 4; w is 0 to 3; A is optionally substituted C3-C8 cycloalkylene, optionally substituted C2-C6 heterocyclylene, or optionally substituted C6-C12 arylene; R ¹⁸ and R ¹⁹ are independently hydrogen, amino, fluoro, hydroxyl, optionally substituted C1 -C6 alkyl, optionally substituted C1 -C6 heteroalkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted C3-C1 0 cycloalkyl, optionally substituted C4-C1 0 cycloalkenyl, optionally substituted C4-C1 0 cycloalkynyl, optionally substituted (J6-(J12aryl, optionally substituted C2-C6 heterocyclyl, optionally substituted C3-C1 0 cycloalkyloxy, optionally substituted C4- C10 cycloalkenyloxy, optionally substituted C4-C10 cycloalkynyloxy, optionally substituted C6- C12aryloxy, or optionally substituted C2-C6 heterocyclyloxy, or R ¹⁸ and R ¹⁹ combine together with the carbon atom(s) to which they are bound to form optionally substituted C3-C1 0 cycloalkyi, optionally substituted C4-C10 cycloalkenyl, optionally substituted C4-C10 cycloalkynyl, optionally substituted C6- C12aryl, or optionally substituted C2-C6 heterocyclyl; and each of Z ¹ and Z ⁴ is independently selected from optionally substituted C1 -C4 alkylene, optionally substituted C1 -C3 heteroalkylene, optionally substituted C3-C9 cycloalkylene, optionally substituted C6-C12 arylene, optionally substituted C2-C6 heterocyclylene, O, S, and NR ¹⁷ ; each R ¹⁷ is independently hydrogen, optionally substituted C1 -C4 alkyl, optionally substituted C2-C4 alkenyl, optionally substituted C2-C4 alkynyl, optionally substituted C2-C6 heterocyclyl, optionally substituted C6-C12 aryl, or optionally substituted C1 -C7 heteroalkyi.

In some embodiments, each of Z ¹ and Z ⁴ is independently carbonyl or N H.

the linker is

Formula XII

wherein m is 0 and wherein each of Z ¹ and Z ⁴ is indepdently NH.

Formula XVI Formula XVII Formula XVIII Formula XIX Formula XX

Formula XXI

wherein n ¹ , o, and p ¹ are 1 to 4; and R ¹⁸ and R ¹⁹ are independently hydrogen, amino, fluoro, hydroxyl, optionally substituted C1 -C6 alkyl, optionally substituted C1 -C6 heteroalkyi, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted C3-C1 0 cycloalkyi, optionally substituted C4-C1 0 cycloalkenyl, optionally substituted C4-C1 0 cycloalkynyl, optionally substituted C6-C12aryl, optionally substituted C2-C6 heterocyclyl, optionally substituted C3-C1 0 cycloalkyloxy, optionally substituted C4- C10 cycloalkenyloxy, optionally substituted C4-C10 cycloalkynyloxy, optionally substituted C6- C12aryloxy, or optionally substituted C2-C6 heterocyclyloxy, or R ¹⁸ and R ¹⁹ combine together with the carbon atom to which they are bound to form optionally substituted C3-C1 0 cycloalkyi, optionally substituted C4-C10 cycloalkenyl, optionally substituted C4-C10 cycloalkynyl, optionally substituted C6- C12aryl, or optionally substituted C2-C6 heterocyclyl.

In some embodiments, the the linker is:

Formula XXII

wherein x is an integer from 0 to 12; wherein Z ¹ is optionally substituted C1 -C2 alkylene, optionally substituted C1 -C3 heteroalkylene, optionally substituted C3-C9 cycloalkylene, optionally substituted C6- C12 arylene, optionally substituted C2-C6 heterocyclylene, O, S, or NR ¹⁷ ; and each R ¹⁷ is independently hydrogen, optionally substituted C1 -C4 alkyl, optionally substituted C2-C4 alkenyl, optionally substituted C2-C4 alkynyl, optionally substituted C2-C6 heterocyclyl, optionally substituted C6-C12 aryl, or optionally substituted C1 -C7 heteroalkyl. In some embodiments, Z ¹ is NH and x is 0.

In another aspect, a compound is selected from:



or a pharmaceutically acceptable salt thereof.

In some embodiments, the comp lly acceptable thereof, has the structure:

Formula I

wherein each R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , and R ⁷ is independently hydrogen, hydroxyl, -SH, amino, nitro, cyano, halo, optionally substituted C1 -C6 alkyl, optionally substituted C6-C10 aryl C1 -C6 alkyl, optionally substituted C1 -C6 alkoxy, optionally substituted C1 -C6 alkanoyi, amide, carboxy, ester, or includes -LE ; wherein at least one of R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , and R ⁷ includes -LE; R ⁸ is hydrogen or optionally substituted C2-C6 heterocyclyl; m is an integer from 5 to 13; n is an integer from 3 to 8; p is 0, 1 , or 2; A' is ; each of B' and C' is independently hydrogen, hydroxyl, C1 -C6 alkyl, oxo, or B' and C join to form -0-; wherein the dashed bond is a single bond or double bond, provided that the compound of Formula I includes at least two double bonds; or a pharmaceutically acceptable salt thereof. In some embodiments, R ⁷ is -LE.

In some embodiments, the compound, or pharmaceutically acceptable thereof, has the structure:

Formula II

wherein each of R ¹ , R ² , and R ³ is independently hydrogen, hydroxyl, optionally substituted C1 -C6 alkyl, or optionally substituted C6-C1 0 aryl C1 -C6 alkyl; each R ⁴ is independently hydrogen, hydroxyl, optionally substituted C1 -C6 alkyl, or oxo; each of R ⁵ and R ⁶ is independently hydrogen, hydroxyl, or C1 -C6 alkoxy;

R ⁹ is -LE; R ¹⁰ is hydrogen or hydroxyl; R ¹¹ is hydroxyl or amine; R ¹² is hydrogen ; m is an integer from 5 to 13; n is an integer from 3 to 8; p is 0, 1 , or 2; q is 1 , 2 or 3 ; A' is or each of B' and C is independently hydrogen, hydroxyl, C1 -C6 alkyl, oxo, or B' and C join to form -0-; and wherein the dashed bond is a single bond or double bond, provided that the compound of Formula I I includes at least two double bonds. In some embodiments, R ⁹ is -NR ¹³ (CH ₂ ) _a NR ¹³ E or - NR ¹³ (CH ₂ ) _a O(CH ₂ ) _a NR ¹³ E; each R ¹³ is independently hydrogen or C1 -C3 alkyl; and each a independently is 2, 3, or 4.

In some embodiments, the compound, or pharmaceutically acceptable thereof, has the structure:

_ wherein R is -LE. In some embodiments, R is -NR'(CH ₂ ) _a NR'E or -NR'(CH ₂ ) _a O(CH ₂ ) _a NR'E; each R' is independently hydrogen or C1 -C3 alkyl; and each a independently is 2, 3, or 4.

In some embodiments, the compound, or pharmaceutically acceptable thereof, has the structure: , wherein R" is -LE. In some embodiments, R" is -N HCH ₂ CH ₂ NH E or -NHCH ₂ CH ₂ OCH ₂ CH ₂ NHE.

In some embodiments, the compound, or pharmaceutically acceptable thereof, has the structure:

, wherein R is -LE. In some embodiments, R is -NR'(CH ₂ ) _a NR'E or -NR'(CH ₂ ) _a O(CH ₂ ) _a NR'E; each R' is independently hydrogen or C1 -C3 alkyl; and each a independently is 2, 3, or 4.

In some embodiments, the compound, or pharmaceutically acceptable thereof, has the structure:

, wherein R" is -LE. In some embodiments, R" is -N HCH ₂ CH ₂ NH E or -NHCH ₂ CH ₂ OCH ₂ CH ₂ NHE.

In some embodiments, the compound, or pharmaceutically acceptable thereof, has the structure:

, wherein R is -LE. In some embodiments, R is -NR'(CH ₂ ) _a NR'E or -NR'(CH ₂ ) _a O(CH ₂ ) _a NR'E; each R' is independently hydrogen or C1 -C3 alkyl; and each a independently is 2, 3, or 4.

In some embodiments, the compound, or pharmaceutically acceptable thereof, has the structure:

, wherein R" is -LE. In some embodiments, R" is -N HCH ₂ CH ₂ NH E or -NHCH ₂ CH ₂ OCH ₂ CH ₂ NHE.

In some embodiments, the compound, or pharmaceutically acceptable thereof, has the structure:

, wherein L is the linker.

In some embodiments, L is a bond covalently linking the polyene antifungal agent moiety to the chemotaxis receptor ligand moiety, E.

In some embodiments, the compound, or pharmaceutically acceptable thereof, has the structure:

Formula III

wherein each of R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , and R ⁷ is independently hydrogen, hydroxyl, -SH, amino, nitro, cyano, halo, optionally substituted C1 -C6 alkyl, optionally substituted C6-C1 0 aryl C1 -C6 alkyl, optionally substituted C1 -C6 alkoxy, optionally substituted C1 -C6 alkanoyi, amide, carboxy, ester, or -LE; each of R ⁹ and R is independently hydrogen, hydroxyl, -SH, amino, nitro, cyano, halo, optionally substituted (J1 -U6 alkyl, optionally substituted C6-C1 0 aryl C1 -C6 alkyl, optionally substituted C1 -C6 alkoxy, optionally substituted C1 -C6 alkanoyl, amide, carboxy, ester, -LE, or R ⁹ and R ¹⁰ together form -0-; wherein at least one of R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁹ and R ¹⁰ is -LE; R ⁸ is hydrogen or optionally substituted C2-C6

0 s an integer from 3 to 1 3; n is an _\ A ₀ A heterocyclyl; m i integer from 3 to 8; p is 0, 1 , or 2; A' is ^¾ or O

/Λ ^ ; and each of B' and C is independently hydrogen, hydroxyl, C1 -C6 alkyl, oxo, or B' and C join to form -0-; or a pharmaceutically acceptable salt thereof. In some embodiments, R ⁷ includes -LE.

In some embodiments, the compound, or pharmaceutically acceptable thereof, has the structure:

Formula IV

wherein each of R ¹ , R ² , and R ³ is independently hydrogen, hydroxyl, optionally substituted C1 -C6 alkyl, or optionally substituted C9-C10 aryl C1 -C6 alkyl; each R ⁴ is independently hydrogen, hydroxyl, optionally substituted C1 -C6 alkyl, or oxo; each of R ⁵ and R ⁶ is independently hydrogen, hydroxyl, or C1 -C6 alkoxy; each of R ⁹ and R ¹⁰ is independently hydrogen, hydroxyl, optionally substituted C1 -C6 alkyl, oxo, or R ⁹ and R ¹⁰ join to form -0-; R ¹¹ is -LE; R ¹² is hydrogen or hydroxyl; R ¹³ is hydroxyl or amine; R ¹⁴ is hydrogen

integer from 3 to 13; n is an integer from 3 to 8; p is 0, 1 , or 2; A' is or and each of B' and C is independently hydrogen, hydroxyl, C1 -C6 alkyl, oxo, or B' and C join to form -0-. In some embodiments, R ¹¹ is -NR ¹⁵ (CH ₂ ) _a NR ¹⁵ E or

-NR ¹⁵ (CH ₂ ) _a O(CH ₂ ) _a NR ¹⁵ E; each R ¹⁵ is independently hydrogen or C1 -C3 alkyl; and each a

independently is an integer from 2 to 4.

In some embodiments, the compound, or pharmaceutically acceptable thereof, has the structure: , wherein R is -LE. I n some embodiments, R is -

NR'(CH ₂ ) _a N R'E or -NR'(CH ₂ ) _a O(CH ₂ ) _a NR'E; each R' is independently hydrogen or C1 -C3 alkyl; and each a independently is an integer from 2 to 4.

In some embodiments, the compound, or pharmaceutically acceptable thereof, has the structure:

NHCH ₂ CH ₂ N HE or -NHCH ₂ CH ₂ OCH ₂ CH ₂ NHE.

In some embodiments, the compound, or pharmaceutically acceptable thereof, has the structure:

, wherein L is the linker. In some embodiments, L is a bond covalently linking the polyene antifungal agent moiety to the chemotaxis receptor ligand moiety, E.

In some embodiments, the compound, or pharmaceutically acceptable thereof, has a concentration of the compound or pharmaceutically acceptable salt that induces maximal neutrophil migration is less than or equal to 1 0,000 nM. In some embodiments, the concentration of the compound or pharmaceutically acceptable salt that induces maximal neutrophil migration is less than or equal to 1 ,000 nM. In some embodiments, the concentration of the compound or pharmaceutically acceptable salt that induces maximal neutrophil migration is less than or equal to 1 00 nM.

In some embodiments, the compound or pharmaceutically acceptable salt inhibits fungal growth. In some embodiments, the IC ₅₀ of the compound or pharmaceutically acceptable salt is less than or equal to 1 ,000 nM. In some embodiments, the IC ₅₀ of the compound or pharmaceutically acceptable salt is less than or equal to 100 nM. In some embodiments, the IC ₅₀ of the compound or pharmaceutically acceptable salt is less than or equal to 1 0 nM. In another aspect, the disclosure includes a pharmaceutical composition including any one or more of the compounds, or the pharmaceutically acceptable salts thereof, described herein and a pharmaceutically acceptable excipient.

In another aspect, the disclosure includes a method for the treatment of a subject having a fungal infection or presumed to have a fungal infection. The method includes administering to the subject an effective amount of a compound, or a pharmaceutically acceptable salt thereof, described herein.

In another aspect, the disclosure includes a method for the prophylactic treatment of a fungal infection in a subject in need thereof. The method includes administering to the subject an effective amount of a compound, or a pharmaceutically acceptable salt thereof, described herein.

In some embodiments, the fungal infection is caused by a fungus of the genus Aspergillus or

Candida. In some embodiments, the fungal infection is aspergillosis. In some embodiments, the aspergillosis is invasive aspergillosis. In some embodiments, the aspergillosis is pulmonary aspergillosis. In some embodiments, the fungal infection is caused by Aspergillus fumigatus. In some embodiments, the fungal infection is candidiasis. In some embodiments, the candidiasis is an intra-abdominal abscess, peritonitis, a pleural cavity infection, esophagitis, candidemia, or invasive candidiasis. In some embodiments, the fungal infection is caused by Candida albicans.

In some embodiments, the subject is immunocompromised. In some embodiments, the subject has been diagnosed with humoral immune deficiency, T cell deficiency, neutropenia, asplenia, or complement deficiency. In some embodiments, the subject is being treated or is about to be treated with immunosuppresive drugs.

In some embodiments, the subject has been diagnosed with a disease which causes immunosuppression. In some embodiments, the disease is cancer or acquired immunodeficiency syndrome. In some embodiments, the cancer is leukemia, lymphoma, or multiple myeloma. In some embodiments, the subject has undergone or is about to undergo hematopoietic stem cell transplantation. In some embodiments, the subject has undergone or is about to undergo an organ transplant.

In some embodiments, the administering includes administering intramuscularly, intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly,

intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, locally, by inhalation, by injection, or by infusion.

As used herein, the term "alkyl," "alkenyl," and "alkynyl" include straight-chain, branched-chain and cyclic monovalent substituents, as well as combinations of these, containing only C and H when unsubstituted. Examples include methyl, ethyl, isobutyl, cyclohexyl, cyclopentylethyl, 2-propenyl, 3-butynyl, and the like. The term "cycloalkyi," as used herein, represents a monovalent saturated or unsaturated non-aromatic cyclic alkyl group having between three to nine carbons (e.g., a C3-C9 cycloalkyi), unless otherwise specified, and is exemplified by cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicyclo[2.2.1 .jheptyl, and the like. When the cycloalkyi group includes at least one carbon-carbon double bond, the cycloalkyi group can be referred to as a "cycloalkenyl" group.

Exemplary cycloalkenyl groups include cyclopentenyl, cyclohexenyl, and the like. When the cycloalkyi group includes at least one carbon-carbon triple bond, the cycloalkyi group can be referred to as a

"cycloalkynyl" group. For the purposes herein, cycloalkenyl excludes aryl. When a list refers to what is otherwise the general term, e.g., alkyl, alkenyl, or alkynyl, and the cyclic form, e.g., cycloalkyi, cycloalkenyl, and cycloalkynyl, it will be understood that what is otherwise the general term refers only to acyclic radicals.

Typically, the alkyl, alkenyl, and alkynyl groups contain 1 -12 carbons (e.g., C1 -C12 alkyl) or 2-12 carbons (e.g. , C2-C12 alkenyl or C2-C12 alkynyl). In some embodiments, the groups are C1 -C8, C1 -C6, C1 -C4, C1 -C3, or C1 -C2 alkyl groups; or C2-C8, C2-C6, C2-C4, or C2-C3 alkenyl or alkynyl groups. Further, any hydrogen atom on one of these groups can be replaced with a substituent as described herein. For example, the term "aminoalkyl" refers to an alkyl group, as defined herein, comprising an optionally substituted amino group (e.g., NH ₂ ).

Heteroalkyi, heteroalkenyl, and heteroalkynyl are similarly defined and contain at least one carbon atom but also contain one or more O, S, or N heteroatoms or combinations thereof within the backbone residue whereby each heteroatom in the heteroalkyi, heteroalkenyl, or heteroalkynyl group replaces one carbon atom of the alkyl, alkenyl, or alkynyl group to which the heteroform corresponds. In some embodiments, the heteroalkyi, heteroalkenyl, and heteroalkynyl groups have C at each terminus to which the group is attached to other groups, and the heteroatom(s) present are not located at a terminal position. As is understood in the art, these heteroforms do not contain more than three contiguous heteroatoms. In some embodiments, the heteroatom is O or N. The term "heterocyclyl," as used herein represents cyclic heteroalkyi or heteroalkenyl that is, e.g., a 3-, 4-, 5-, 6-, or 7-membered ring, unless otherwise specified, containing one, two, three, or four heteroatoms independently selected from the group consisting of N, O, and S. The 5-membered ring has zero to two double bonds, and the 6- and 7- membered rings have zero to three double bonds. The term "heterocyclyl" also represents a heterocyclic compound having a bridged multicyclic structure in which one or more carbons and/or heteroatoms bridges two non-adjacent members of a monocyclic ring, e.g., a quinuclidinyl group. The term

"heterocyclyl" includes bicyclic, tricyclic, and tetracyclic groups in which any of the above heterocyclic rings is fused to one, two, or three carbocyclic rings, e.g., an aryl ring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, a cyclopentene ring, or another monocyclic heterocyclic ring, such as indolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, benzofuryl, benzothienyl and the like. When a list refers to what is otherwise the general term, e.g., heteroalkyi, heteroalkenyl, or heteroalkynyl, and the cyclic form, e.g., heterocyclyl, it will be understood that what is otherwise the general term refers only to acyclic radicals.

Heteroalkyi, heteroalkenyl, or heteroalkynyl substituents may also contain one or more carbonyl groups. Examples of heteroalkyi, heteroalkenyl and heteroalkynyl groups include CH ₂ OCH ₃ , CH ₂ N(CH ₃ ) ₂ , CH ₂ OH, (CH ₂ ) _n NR ₂ , OR, COOR, CONR ₂ , (CH ₂ ) _n OR,(CH ₂ ) _n COR, (CH ₂ ) _n COOR, (CH ₂ ) _n SR, (CH ₂ ) _n SOR, (CH ₂ ) _n S0 ₂ R, (CH ₂ ) _n CON R ₂ , NRCOR, N RCOOR, OCONR ₂ , OCOR, and the like where the R group in these examples of heteroalkyi, heteroalkenyl and heteroalkynyl groups contains at least one C and the size of the substituent is consistent with the definition of e.g., alkyl, alkenyl, and alkynyl, as described herein (e.g., n is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , or 12).

As used herein, the terms "alkylene," "alkenylene," "alkynylene," and the prefix "alk" refer to divalent or trivalent groups having a specified size, typically C1 -C2, C1 -C3, C1 -C4, C1 -C6, or C1 -C8 for the saturated groups (e.g. , alkylene or alk) and C2-C3, C2-C4, C2-C6, or C2-C8 for the unsaturated groups (e.g., alkenylene or alkynylene). They include straight-chain, branched-chain, and cyclic forms as well as combinations of these, containing only C and H when unsubstituted. Because they are divalent, they can link together two parts of a molecule. Examples are methylene, ethylene, propylene, cyclopropan-1 , 1 -diyl, ethylidene, 2-butene-1 ,4-diyl, and the like. These groups can be substituted by the groups typically suitable as substituents for alkyi, alkenyl and alkynyl groups as set forth herein. I hus C=0 is a C1 alkylene that is substituted by =0, for example. For example, the term "alkaryl," as used herein, represents an aryl group, as defined herein, attached to the parent molecular group through an alkylene group, as defined herein, and the term "alkheteroaryl" refers to a heteroaryl group, as defined herein, attached to the parent molecular group through an alkylene group, as defined herein. The alkylene and the aryl or heteroaryl group are each optionally substituted as described herein.

Heteroalkylene, heteroalkenylene, and heteroalkynylene are similarly defined as divalent groups having a specified size, typically C1 -C3, C1 -C4, C1 -C6, or C1 -C8 for the saturated groups and C2-C3, C2-C4, C2-C6, or C2-C8 for the unsaturated groups. They include straight chain, branched chain and cyclic groups as well as combinations of these, and they further contain at least one carbon atom but also contain one or more O, S, or N heteroatoms or combinations thereof within the backbone residue, whereby each heteroatom in the heteroalkylene, heteroalkenylene or heteroalkynylene group replaces one carbon atom of the alkylene, alkenylene, or alkynylene group to which the heteroform corresponds. As is understood in the art, these heteroforms do not contain more than three contiguous heteroatoms.

The term "alkoxy" represents a chemical substituent of formula -OR, where the R in -OR is an optionally substituted alkyi group (e.g., C1 -C6 alkyi group), unless otherwise specified. In some embodiments, the alkyi group can be substituted, e.g., the alkoxy group can have 1 , 2, 3, 4, 5, or 6 substituent groups as defined herein.

The term "alkoxyalkyl" represents a heteroalkyl group, as defined herein, that is described as an alkyi group that is substituted with an alkoxy group. Exemplary unsubstituted alkoxyalkyl groups include between 2 to 12 carbons. In some embodiments, the alkyi and the alkoxy each can be further substituted with 1 , 2, 3, or 4 substituent groups as defined herein for the respective group.

The term "amino," as used herein, represents -N(R ^N1 ) ₂ , where each R ^N1 is, independently, H, OH, N0 ₂ , N(R ^N2 ) ₂ , S0 ₂ OR ^N2 , S0 ₂ R ^N2 , SOR ^N2 , an /V-protecting group, alkyi, alkenyl, alkynyl, alkoxy, aryl, alkaryl, cycloalkyl, alkcycloalkyl, heterocyclyl (e.g., heteroaryl), alkheterocyclyl (e.g., alkheteroaryl), or two R ^N1 combine to form a heterocyclyl or an /V-protecting group, and where each R ^N2 is, independently, H, alkyi, or aryl. I n a preferred embodiment, amino is -N H ₂ , or -NR ^N1 , where R ^N1 is, independently, OH, N0 ₂ , NH ₂ , NR ^N2 ₂ , S0 ₂ OR ^N2 , S0 ₂ R ^N2 , SOR ^N2 , alkyi, or aryl, and each R ^N2 can be H, alkyi, or aryl. The term "aminoalkyl," as used herein, represents a heteroalkyl group, as defined herein, that is described as an alkyi group, as defined herein, substituted by an amino group, as defined herein. The alkyi and amino each can be further substituted with 1 , 2, 3, or 4 substituent groups as described herein for the respective group. For example, the alkyi moiety may comprise an oxo (=0) substituent.

"Aromatic" moiety or "aryl" moiety refers to any monocyclic or fused ring bicyclic system which has the characteristics of aromaticity in terms of electron distribution throughout the ring system and includes a monocyclic or fused bicyclic moiety such as phenyl or naphthyl; "heteroaromatic" or

"heteroaryl" also refers to such monocyclic or fused bicyclic ring systems containing one or more heteroatoms selected from O, S and N. The inclusion of a heteroatom permits inclusion of 5-membered rings to be considered aromatic as well as 6-membered rings. Thus, typical aromatic/heteroaromatic systems include pyridyl, pyrimidyl, indolyl, benzimidazolyl, benzotriazolyl, isoquinolyl, quinolyl, benzothiazolyl, benzofuranyl, thienyl, furyl, pyrrolyl, thiazolyl, oxazolyl, isoxazolyl, benzoxazolyl, benzoisoxazolyl, imidazolyl and the like. Because tautomers are theoretically possible, phthalimido is also considered aromatic. Typically, the ring systems contain 5-12 ring member atoms or 6-1 0 ring member atoms. I n some embodiments, the aromatic or heteroaromatic moiety is a 6-membered aromatic rings system optionally containing 1 -2 nitrogen atoms. More particularly, the moiety is an optionally substituted phenyl, pyridyl, indolyl, pyrimidyl, pyridazinyl, benzothiazolyl, benzimidazolyl, pyrazolyl, imidazolyl, isoxazolyl, thiazolyl, benzothiazolyl, indolyl, or imidazopyridinyl. Even more particularly, such moiety is phenyl, pyridyl, thiazolyl, imidazopyridinyl, or pyrimidyl and even more particularly, it is phenyl.

"O-aryl" or "O-heteroaryl" refers to aromatic or heteroaromatic systems which are coupled to another residue through an oxygen atom. A typical example of an O-aryl is phenoxy. Similarly, "arylalkyl" refers to aromatic and heteroaromatic systems which are coupled to another residue through a carbon chain, saturated or unsaturated, typically of C1 -C8, C1 -C6, or more particularly C1 -C4 or C1 -C3 when saturated or C2-C8, C2-C6, C2-C4, or C2-C3 when unsaturated, including the heteroforms thereof. For greater certainty, arylalkyl thus includes an aryl or heteroaryl group as defined above connected to an alkyl, heteroalkyl, alkenyl, heteroalkenyl, alkynyl, or heteroalkynyl moiety also as defined above. Typical arylalkyls would be an aryl(C6-C12)alkyl(C1 -C8), aryl(C6-C12)alkenyl(C2-C8), or aryl(C6-C12)alkynyl(C2- C8), plus the heteroforms. A typical example is phenylmethyl, commonly referred to as benzyl.

The term "hydrophobic amino acid residue" refers to an amino acid residue having relatively low- water solubility. Hydrophobic amino acid residues include, but are not limited to, leucine, isoleucine, alanine, phenylalanine, valine, proline, tyrosine, cysteine, methionine, tryptophan, and unnatural amino acid residues of similar hydrophobicity.

The term "chemotaxis receptor ligand," as used herein refers to a molecule, such as a peptide, that can be recognized by and interact with a chemotaxis receptor. The chemotaxis receptor ligand may be derived from prokaryotic or eukaryotic organisms, such as fungi, bacteria, or mammals. In some embodiments, the chemotaxis receptor ligand, or a chemotactic recognition receptor ligand, includes host immune stimulating factors. For example, once the chemotaxis receptor of a host organism interacts with the the chemotaxis receptor ligand, the chemotaxis receptor ligand may stimulate immune responses in the host organism.

The term "chemotactic peptide," as used herein refers to peptides that interact with chemotaxis receptors and induce chemotaxis.

The term "chemotaxis receptor," as used herein refers to receptors that are used by eukaryotic cells to sense the presence of chemotactic stimuli and include formyl peptide receptors (FRP), chemokine receptors (e.g., CCR, CXCR), and leukotriene receptors (BLT, such as BLT1 , BLT2). For example, the chemokine receptor family may include CCR1 -CCR1 0, CXCR1 -CXCR7, CX3CR1 , and XCR1 . The ligands for these receptors include those naturally occurring, framents derived from those ligands, and synthetically generated ligands.

The term "diamine linker" refers to a linker, e.g., a polypeptide linker, that includes an amine group (i.e., -NH ₂ ) at each of its two termini. For example, the amine at one terminus of the diamine linker can conjugate to the carbonyl group of the chemotaxis receptor ligand moiety, E; the amine at the second terminus of the diamines linker can conjugate to the carbonyl group of the polyene antifungal agent moiety.

The term "chemotaxic activity," as used herein refers to the concentration of a compound that induces maximal neutrophil migration at a concentration that is less than or equal to 10,000 nM as measured in accordance with the Transwell Migration Assay in Example 20 herein. In some aspects, a concentration of a compound that induces maximal neutrophil migration of less than or equal to 1 000 nM or less than or equal to 1 00 nM in accordance with the I ranswell Migration Assay, is indicative ot a compound having chemotactic activity. The concentration of a compound assumes the presence of a compound, and therefore, these amounts would be greater than 0 nM.

By "fungal infection" is meant the pathogenic growth of fungus in a host organism (e.g., a human subject). For example, the infection may include the excessive growth of fungi that are normally present in or on the body of a subject or growth of fungi that are not normally present in or on a subject. More generally, a fungal infection can be any situation in which the presence of a fungal population(s) is damaging to a host body. Thus, a subject is "suffering" from a fungal infection when an excessive amount of a fungal population is present in or on the subject's body, or when the presence of a fungal population(s) is damaging the cells or other tissue of the subject.

The term "inhibiting fungal growth" refers to to any slowing, stabilizing, interruption, suppression, delay, killing, or inhibition of growth of fungi. The compounds described herein that inhibit fungal growth display a minimal inhibitory concentration (MIC), e.g., less than 32 pg/mL (e.g., less than 30 pg/mL, 20 pg/mL, 1 0 pg/mL, 5 pg/mL, 1 pg/mL). Examples 1 8 and 1 9 describe M ICs of compounds described herein. Inhibiting fungal growth includes, for example, inhibiting the growth of resting fungal cells, which can include spore germination, mycelia development, and/or the formation of fruiting structures on the fungus (e.g., sporangia/sporophores). Fungal growth can be produced by a fungus of the genus Aspergillus or Candida, such as Aspergillus fumigatus or Candida albicans.

"Halo" or "halogen" may be any halogen atom, especially F, CI, Br, or I, and more particularly it is fluoro or chloro.

The term "haloalkyl," as used herein, represents an alkyl group, as defined herein, substituted by a halogen group (i.e., F, CI, Br, or I). A haloalkyl may be substituted with one, two, three, or, in the case of alkyl groups of two carbons or more, four halogens. Haloalkyl groups include perfluoroalkyls. In some embodiments, the haloalkyl group can be further substituted with 1 , 2, 3, or 4 substituent groups as described herein for alkyl groups.

The term "hydrazone" as used herein, represents the group having the structure: where each R in the hydrazone is independently hydrogen, alkyl, or aryl.

The term "hydroxyl," as used herein, represents an -OH group.

The term "hydroxyalkyl," as used herein, represents an alkyl group, as defined herein, substituted by one to three hydroxy groups, with the proviso that no more than one hydroxy group may be attached to a single carbon atom of the alkyl group, and is exemplified by hydroxymethyl, dihydroxypropyl, and the like.

The term "immunocompromised," as used herein refers to an immune response that has been weakened by a condition or an immunosuppressive agent.

The term "/V-protecting group," as used herein, represents those groups intended to protect an amino group against undesirable reactions during synthetic procedures. Commonly used /V-protecting groups are disclosed in Greene, "Protective Groups in Organic Synthesis," 3 ^rd Edition (John Wiley & Sons, New York, 1999), which is incorporated herein by reference. /V-protecting groups include acyl, aryloyl, or carbamyl groups such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2- bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, a-chlorobutyryl, benzoyl, 4- chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and chiral auxiliaries such as protected or unprotected D, L or D, L-amino acid residues such as alanine, leucine, phenylalanine, and the like; sulfonyl-containing groups such as benzenesulfonyl, p-toluenesulfonyl, and the like; carbamate forming groups such as benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl,

3,5-dimethoxybenzyl oxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2- nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, 1 -(p-biphenylyl)-l - methylethoxycarbonyl, a,a-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxy carbonyl, t- butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2,2,2,-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxy carbonyl, fluorenyl-9- methoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl,

phenylthiocarbonyl, and the like, alkaryl groups such as benzyl, triphenylmethyl, benzyloxymethyl, and the like and silyl groups such as trimethylsilyl, and the like. Preferred /V-protecting groups are formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, alanyl, phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc), and benzyloxycarbonyl (Cbz).

An "oxo" group is a substituent having the structure =0, where there is a double bond between a carbon and an oxygen atom.

O

i

|-o-p-o-|

The term "phosphoryl" as used herein, represents the group having the structure: OH The term "polyene antifungal agent," as used herein refers to compounds that bind to ergosterol and structurally related sterols in the fungal membrane and disrupt membrane structure integrity, thus causing leakage of cellular components from a fungus that causes infection at less than 5 μΜ (e.g., less than 4μΜ, 3μΜ, 2μΜ, 1 μΜ, 500ηΜ, or 1 0ΟηΜ) or displays an MIC less than 32 μg/mL (e.g., less than 30 μg/mL, 20 μg/mL, 1 0 μg/mL, 5 μg/mL, 1 μg/mL). The term "sulfonyl" as used herein, represents the group having the structure:

Typical optional substituents on aromatic or heteroaromatic groups include independently halo, such as chloro, fluoro, CN, N0 ₂ , CF ₃ , OCF ₃ , COOR', CONR' ₂ , OR', SR', SOR', S0 ₂ R', N R' ₂ ,

NR'(CO)R',NR'C(0)OR', N R'C(0)NR' ₂ , NR'S0 ₂ NR' ₂ , or NR'S0 ₂ R', where each R' is independently H or an optionally substituted group selected from alkyl, alkenyl, alkynyl, cycloalkyi, cycloalkenyl, cycloalkynyl, heterocyclyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, and aryl (all as defined above); or the substituent may be an optionally substituted group selected from alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, aryl, heteroaryl, O-aryl, O-heteroaryl and arylalkyl. I

Optional substituents on a non-aromatic group (e.g., alkyl, alkenyl, and alkynyl groups), are typically selected from the same list of substituents suitable for aromatic or heteroaromatic groups, except as noted otherwise herein. A non-aromatic group may also include an optional substituent selected from =0 and =NOR' where R' is H or an optionally substituted group selected from alkyl, alkenyl, alkynyl, cycloalkyi, cycloalkenyl, cycloalkynyl, heterocyclyl, heteroalkyl, heteroalkenyl, heteralkynyl, heteroaryl, and aryl (all as defined above). In general, a substituent group (e.g., alkyl, alkenyl, alkynyl, or aryl, including all heterotorms defined above) may itself optionally be substituted by additional substituents. The nature of these substituents is similar to those recited with regard to the substituents on the basic structures above. Thus, where an embodiment of a substituent is alkyl, this alkyl may optionally be substituted by the remaining substituents listed as substituents where this makes chemical sense, and where this does not undermine the size limit of alkyl per se; e.g., alkyl substituted by alkyl or by alkenyl would simply extend the upper limit of carbon atoms for these embodiments, and is not included. However, alkyl substituted by aryl, amino, halo (preferably chloro or fluoro) and the like would be included. For example, where a group is substituted, the group may be substituted with 1 , 2, 3, 4, 5, or 6 substituents. Optional substituents include, but are not limited to: C1 -C6 alkyl or heteroaryl, C2-C6 alkenyl, or heteroalkenyl, C2- C6 alkynyl or heteroalkynyl, halogen; aryl, heteroaryl, azido(-N ₃ ), nitro (-N0 ₂ ), cyano (-CN),

acyloxy(-OC(=0)R'), acyl (-C(=0) R'), alkoxy (-OR'), amido (-NR'C(=0)R", or -C(=0)NRR'), amino (-N RR'), carboxylic acid (-C0 ₂ H), carboxylic ester (-C0 ₂ R'), carbamoyl (-OC(=0)NR'R" or -NRC(=0)OR'), hydroxy (-OH), isocyano (-NC), sulfonate (-S(=0) ₂ OR), sulfonamide (-S(=0) ₂ NRR' or -N RS(=0) ₂ R'), or sulfonyl (-S(=0) ₂ R), where each R or R' in the optional substituents is selected, independently, from H, C1 -C6 alkyl, or heteroaryl, C2-C6 alkenyl, or heteroalkenyl, C2-C6 alkynyl ,or heteroalkynyl, aryl, or heteroaryl. A substituted group may have, for example, 1 , 2, 3, 4, 5, 6, 7, 8, or 9 substituents. Acyclic alkyl groups may not be substituted with aryl or heterocyclyc unless specifically stated.

In some embodiments, chemotaxis receptor ligands include amino acid residues. The amino acid residue may be of a naturally occurring amino acid residue (e.g., Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val), or the amino acid residue may be of a non- naturally occurring amino acid residue. A "non-naturally occurring amino acid residue" is an amino acid residue which is not naturally produced or found in a mammal. Examples of non-naturally occurring amino acid residues include D-amino acid residues; an amino acid residue having an acetylaminomethyl group attached to a sulfur atom of a cysteine; a pegylated amino acid residue; the omega amino acid residues of the formula NH ₂ (CH ₂ ) _n COOH where n is 2-6, neutral nonpolar amino acid residues, such as sarcosine, t-butyl alanine, t-butyl glycine, N-methyl isoleucine, and norleucine; oxymethionine;

phenylglycine; citrulline; methionine sulfoxide; cysteic acid; ornithine; diaminobutyric acid; and hydroxyproline.

The term an "effective amount" of an agent, as used herein, is that amount sufficient to effect beneficial or desired results, such as clinical results, and, as such, an "effective amount" depends upon the context in which it is being applied. For example, in the context of administering an agent that is used for the treatment of a fungal infection, an effective amount of an agent is, for example, an amount sufficient to slow down or reverse the progression of the infection as compared to the response obtained without administration of the agent.

The term "pharmaceutical composition," as used herein, represents a composition containing a compound described herein formulated with a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is manufactured or sold with the approval of a governmental regulatory agency as part of a therapeutic regimen for the treatment of disease in a mammal. Pharmaceutical compositions may be formulated to be administered intramuscularly, intravenously (e.g., as a sterile solution free of particulate emboli and in a solvent system suitable for intravenous use), intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravagmally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally (e.g., a tablet, capsule, caplet, gelcap, or syrup), topically (e.g., as a cream, gel, lotion, or ointment), locally, by inhalation, injection, infusion, continuous infusion, localized perfusion bathing target cells directly, catheter, lavage, in cremes, or lipid compositions.

A "pharmaceutically acceptable excipient," as used herein, refers any ingredient other than the compounds described herein (for example, a vehicle capable of suspending or dissolving the active compound) and having the properties of being nontoxic and non-inflammatory in a patient. Excipients may include, for example: antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspensing or dispersing agents, sweeteners, or waters of hydration. Exemplary excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

The term "pharmaceutically acceptable salt," as used herein, represents those salts of the compounds described that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response and the like and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in: Berge et al., J. Pharmaceutical Sciences 66:1 -19, 1 977 and in Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P. H. Stahl and C.G. Wermuth), Wiley- VCH, 2008. The salts can be prepared in situ during the final isolation and purification of the compounds described herein or separately by reacting the free base group with a suitable organic acid.

The compounds may have ionizable groups so as to be capable of preparation as

pharmaceutically acceptable salts. These salts may be acid addition salts involving inorganic or organic acids or the salts may, in the case of acidic forms of the compounds, may be prepared from inorganic or organic bases. Frequently, the compounds are prepared or used as pharmaceutically acceptable salts prepared as addition products of pharmaceutically acceptable acids or bases. Suitable pharmaceutically acceptable acids and bases are well-known in the art, such as hydrochloric, sulfuric, hydrobromic, acetic, lactic, citric, or tartaric acids for forming acid addition salts, and potassium hydroxide, sodium hydroxide, ammonium hydroxide, caffeine, various amines, and the like for forming basic salts. Methods for preparation of the appropriate salts are well-established in the art.

Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2- hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine and the like.

The term "prophylactic treatment," as used herein, refers to treatment initiated, for example, prior to ("pre-exposure prophylaxis") or following ("post-exposure prophylaxis") an event that precedes the onset of the disease, disorder, or conditions (e.g., when a subject is being treated or is about to be treated with immunosuppresive drugs, a subject has been diagnosed with a disease which causes immunosuppression (e.g., cancer such as, leukemia, lymphoma, multiple myeloma, or acquired immunodeficiency syndrome), a subject has undergone or is about to undergo hematopoietic stem cell transplantation, or a subject has undergone or is about to undergo an organ transplant). Prophylactic treatment that includes administration of a compound described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, can be acute, short-term, or chronic. The doses administered may be varied during the course of prophylactic treatment.

As used herein, the term "subject" can be a human, non-human primate, or other mammal, such as but not limited to dog, cat, horse, cow, pig, turkey, goat, fish, monkey, chicken, rat, mouse, and sheep.

As used herein, and as well understood in the art, "to treat" a condition or "treatment" of a fungal infection is an approach for obtaining beneficial or desired results, such as clinical results. Beneficial or desired results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions; diminishment of extent of disease, disorder, or condition; stabilized (i.e., not worsening) state of disease, disorder, or condition; preventing spread of disease, disorder, or condition; delay or slowing the progress of the disease, disorder, or condition; amelioration or palliation of the disease, disorder, or condition; and remission (whether partial or total), whether detectable or undetectable. "Palliating" a disease, disorder, or condition means that the extent and/or undesirable clinical manifestations of the disease, disorder, or condition are lessened and/or time course of the progression is slowed or lengthened, as compared to the extent or time course in the absence of treatment.

The term "unit dosage form" refers to a physically discrete unit suitable as a unitary dosage for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with any suitable pharmaceutical excipient or excipients. Exemplary, non-limiting unit dosage forms include a tablet (e.g., a chewable tablet), caplet, capsule (e.g., a hard capsule or a soft capsule), lozenge, film, strip, gelcap, and syrup.

In some cases, the compounds may contain one or more chiral centers. The compounds include each of the isolated stereoisomer^ forms as well as mixtures of stereoisomers in varying degrees of chiral purity, including racemic mixtures. It also encompasses the various diastereomers, enantiomers, and tautomers that can be formed.

Compounds useful may also be isotopically labeled compounds. Useful isotopes include hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, and chlorine, (e.g., ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁸ 0, ¹⁷ 0, ³¹ P, ³² P, ³⁵ S, ¹⁸ F, and ³⁶ CI). Isotopically labeled compounds can be prepared by synthesizing a compound using a readily available isotopically labeled reagent in place ot a non-isotopically labeled reagent. In some embodiments, the compound, or a composition that includes the compound, has the natural abundance of each element present in the compound.

The compounds described herein are also useful for the manufacture of a medicament useful to treat fungal infections.

Other features and advantages will be apparent from the following detailed description and the claims.

Detailed Description

Synthetic bifunctional compounds (e.g., non-antibody compounds) are useful in the treatment of fungal infections including a chemotaxis receptor ligand moiety, E, and a polyene antifungal agent moiety. The inventors have found that compounds of this type can have increased antifungal activity due to their ability to bind to the fungal cell wall through forming complexes with ergosterol and structurally related sterols within the lipid bilayer, thereby driving a concentration gradient near the locus of infection, whereby the chemotaxis receptor ligand then serves as a gradient against which neutrophils chemotax to the site. Compounds include, without limitation, compounds 1 -1 6.

Polyene antifugal agents

Polyene antifungal agents belong to a class of antifungal antibiotics that contains a macrocylic lactone ring with a series of three to eight conjugated carbon-carbon double bonds and may also contain a sugar moiety and an aromatic moiety. Polyene antifungal agents can insert into fungal membranes by binding to ergosterol or structurally similar sterols in the lipid bilayer, therein forming aggregates and ergosterol-antibiotic complexes. The antifungal effects of these agents are generally attributed to the disruption of fungal cell membrane, causing leakage of K ⁺ and Na ⁺ ions and other cellular components. Several classes of polyene antifungal agents include but are not limited to the 67-121 -A, 67-121 -C, amphotericin B, arenomvcin B, aurenin, aureofungin A, aureotuscin, candidin, chinin,

demethoxyrapamycin, dermostatin A, dermostatin B, DJ-400-B _! , DJ-400-B ₂ , elizabethin, eurocidin A, eurocidin B, filipin I , filipin I I, filipin I I I, filipin IV, fungichromin, gannibamycin, hamycin, levorin A ₂ , lienomycin, lucensomycin, mycoheptin, mycoticin A, mycoticin B, natamycin, nystatin A, nystatin A ₃ , partricin A, partricin B, perimycin A, pimaricin, polifungin B, rapamycin, rectilavendomvcin, rimocidin, roflamycoin, tetramycin A, tetramycin B, tetrin A, or tetrin B class of antifungal drugs.

Exemplary polyene antifungal agents are described herein and include, without limitation, compounds having the structure:

Formula ll

where each of R ¹ , R ² , and R ³ is independently hydrogen, hydroxyl, optionally substituted C1 -C6 alkyl, or optionally substituted aryl(C6-C12)alkyl(C1 -C8); each R ⁴ is independently hydrogen, hydroxyl, optionally substituted C1 -C6 alkyl, or oxo; each of R ⁵ and R ⁶ is independently hydrogen, hydroxyl, or C1 -C6 alkoxy; R ⁹ is hydroxyl, amino, or a linker; R ¹⁰ is hydrogen or hydroxyl; R ¹¹ is hydroxyl or amine; R ¹² is hydrogen or

; m is an integer from 5 to 13; n is an integer from 3 to 8; p is 0, 1 , or 2; q is 1 , 2 or 3; is or Λ ₀ Λ/ each of B' and C is independently hydrogen, hydroxyl, C1 -C6 alkyl, oxo, or B' and C join to form -0-; where R ⁹ is -NR ¹³ (CH ₂ ) _a NR ¹³ or -NR ¹³ (CH ₂ ) _a O(CH ₂ ) _a NR ¹³ ; each R ¹³ is independently hydrogen or C1 -C3 alkyl; and each a independently is an integer from 2 to 4 wherein the dashed bond is a single bond or double bond, provided that the compound of Formula II includes at least two double bonds.

Another exemplary formula is:

Formula IV

where each of R ¹ , R ² , and R ³ is independently hydrogen, hydroxyl, optionally substituted C1 -C6 alkyl, or optionally substituted C6-C12 aryl- C1 -C6 alkyl; each R ⁴ is independently hydrogen, hydroxyl, optionally substituted C1 -C6 alkyl, or oxo; each of R ⁵ and R ⁶ is independently hydrogen, hydroxyl, or C1 -C6 alkoxy; each of R ⁹ and R ¹⁰ is independently hydrogen, hydroxyl, optionally substituted C1 -C6 alkyl, oxo, or R ⁹ and R ¹⁰ join to form -0-; R ¹¹ is hydroxyl, amino, or a linker; R ¹² is hydrogen or hydroxyl; R ¹³ is hydroxyl or

amine; R is hydro from 3 to 13; n is an integer from 3 to 8; p is 0, 1 , or 2; q is 1 , 2 or 3; A' is or and each of B' and C is independently hydrogen, hydroxyl, C1 -C6 alkyl, oxo, or B' and C join to form -0-. Specifically, R ¹¹ is -NR ¹⁵ (CH ₂ ) _a NR ¹⁵ or - NR ¹⁵ (CH ₂ ) _a O(CH ₂ ) _a NR ¹⁵ ; each R ¹⁵ is independently hydrogen or C1 -C3 alkyl; and each a independently is an integer from 2 to 4.

Other polyene antifungal agents are known in the art, such as amphotericin B, natamycin, and nystatin, which have the structures shown below: CH^

HO ' NH ₂ amphotericin B

natamycin

nystatin

Other exemplary polyene antifungal agents include, without limitation:

C2'-deoxyamphotericin B

rimocidin

In some embodiments, any of the above compounds could be utilized as a polyene antifungal agent.

Chemotaxis Receptors

Chemotaxis receptors are receptors of the innate immune system. I he innate immune system represents a defense mechanism that a host uses immediately or within several hours after exposure to an antigen which is non-specific to such antigen. Unlike adaptive immunity, innate immunity does not recognize every possible antigen. Instead it is designed to recognize a few highly conserved structures present in many different pathogens. In some aspects, the structures recognized are pathogen- associated molecular patterns and include LPS from the gram-negative cell wall, peptidoglycan, lipotechoic acids from the gram-positive cell wall, the sugar mannose, fucose, N-acetyl glucosamine, bacterial DNA, N-formylmethionine found in bacterial proteins, double stranded RNA from viruses, and glucans from fungal cell walls.

Chemotaxis includes migration of immune cells and activation of immune cells. Generally, activating an immune cell includes, for example, directing their migration to sites of inflammation and or infection, stimulating phagocytosis as well as the release of toxic mediators, both internally and externally, which kill infectious agents and or infected cells, the secretion of an array of cytokines and vasoactive medaitors, the presentation of antigens to foster both humoral (antibodies from B-cells) and cellular responses (T lymphocytes), and the adherence of platelets to affected surfaces promoting coagulation together with the release of immune active mediators. .

Most body defense cells have pattern recognition receptors for these common pathogen associated molecular patterns. Consequently, there is an immediate response against the invading pathogen. Pathogen-associated molecular patterns can also be recognized by a series of soluble pattern recognition receptors (PRRs) in the blood that function as opsonins and initiate the complement pathways. Taken together, the innate immune system is thought to recognize approximately 1 0 ³ molecular patterns.

As used herein "PRRs," refers to either surface PRRs or soluble PRRs. The cell surface PRRs can be subdivided into two functionally different classes: endocytic pattern recognition receptors and signaling pattern recognition receptors. Endocytic pattern recognition receptors are found on the surface of immune cells and promote the attachment of pathogens to phagocytes and their subsequent engulfment and destruction.

The exemplary PRRs include mannose receptors (MR), formyl peptide receptors (FPRs), toll-like receptors (TLRs), CD14, and nucleotide binding oligomerization domain proteins (NOD). Binding of ligands to these receptors also promotes the synthesis and secretion of intracellular regulatory molecules

(immune modulating signals) such as cytokines that are crucial to initiating innate immunity and adaptive immunity.

Any PRR known in the art or described herein can be used in the compounds described herein. Formyl Peptide Receptors

The formyl peptide receptor family belongs to the class of chemotaxis receptors. FPRs are G- protein coupled receptors expressed primarily in neutrophils and some cells of macrophage or phagocyte lineage. The best characterized ligands for these receptors are peptides or protein fragments containing N-formyl methionine residues, a hallmark of proteins of prokaryotic origin. As such, these peptides serve as potent immunological homing signals for sites of bacterial infection, signaling several phases of neutrophil response and activation, including chemoattraction, stimulation of production and release of immunosignaling molecules (e.g., interleukins, cytokines), as well as degranulation, a cellular process that includes the production and release of both chemical (e.g. , hydrogen peroxide and other reactive oxygen species) and enzymatic agents (e.g., elastase and other digestive enzymes) capable of mediating destruction of the foreign agent or pathogen.

In humans, five related FPR family members have been identified: formyl peptide receptor 1 (FPR1 ), FPR2, FPR3, formyl peptide receptor-like 1 (FPRL1 ), and FPRL2. A naturally occurring ligand for FPR is formyl-methionine-leucine-phenylalanine (fMLF) while an unnatural ligand to FPR is formyl- methionine-leucine-(2-pyridyl)alanine.

The cellular response mediated by the formyl peptide receptor includes cellular polarization and transmigration, generation of superoxide 02 radicals through respiratory burst oxidase, degranulation and release of a variety of various degenerative enzymes, as well as phagocytosis. In some embodiments, compounds herein interact with FPR and induce at least one of the above cellular responses.

Formyl Peptide Receptor Ligands

Formyl peptide receptor ligands are known in the art. Some are known to be agonists such as a naturally occurring ligand for FPR is formyl-methionine-leucine-phenylalanine (fMLF), as noted above. Some are known to be antagonists such as the non-naturally occurring isobutyloxycarbonyl-methione- leucine-phenylalanine (ibMLF) that bind to the FPR and interfere with the activity of agonists. Several synthetic mimetics of fMLF have been shown to induce an immune response. These formyl peptides as well as the antagonists can be used as the chemotaxis receptor ligands in the compounds described herein, such as compounds of Formulas l-IV.

Chemotaxis receptor ligand moieties include, but are not limited to peptides comprising the formula:

R ¹⁴ -X1 -X2-X3-X4-X5-X6-X7-X8-X9- wherein X1 is any amino acid residue; each of X2-X9 isany amino acid residue or is absent; R ¹⁴ is hydrogen or ^{R1 Λ} A° */ ; wherein X ₁₀ is a bond, NH, or O; and R is hydrogen, optionally substituted C1 - C6 alkyl, optionally substituted C1 -C6 heteroalkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted C3-C10 cycloalkyl, optionally substituted C4-C10 cycloalkenyl, optionally substituted C4-C1 0 cycloalkynyl, optionally substituted oxime, optionally substituted hydrazone, optionally substituted C6-C12 aryl, or optionally substituted C2-C6 heterocyclic.

In some aspects, the chemotaxis receptor ligands include two amino acid residues, three amino acid residues, four amino acid residues, or five amino acid residues, in particular, formyl-methionine- leucine (fML), fMLF, fML(2-pyridyl-ala), fML(4-pyridyl-ala), ibMLF, fMLF-beta-ala, N-acetyl-MLF-beta-ala, ibMLF-beta-ala, fM IFL, fMIVIL, N-(p-chlorophenyl-aminocarbonyl)-MLF, fMLF(N-epsilon-guanidinyl)K, fMLF(N-alpha-guanidinyl)K, fMLM, fML(0-benzyl)serine, N-acetyl-PGP, and TKPR.

In particular embodiments, the chemotaxis receptor ligand moiety is a chemotactic peptide, which can comprise, consist essentially of, or consist of an amino acid having the formula:

R ¹⁴ -X1 -X2-X9- Formula VI where X1 is any amino acid residue; X2 is leucine or isoleucine, or is absent; X9 is any ammo acid residue; R ¹⁴ is hydrogen or ; where X ₁₀ is a bond, NH, or O; and R ¹⁵ is hydrogen, optionally substituted C1 -C6 alkyl, optionally substituted C1 -C6 heteroalkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted C3-C10 cycloalkyl, optionally substituted C4-C1 0 cycloalkenyl, optionally substituted C4-C1 0 cycloalkynyl, optionally substituted oxime, optionally substituted hydrazone, optionally substituted aryl, or optionally substituted heterocyclic.

In certain embodiments, the chemotactic peptide can comprise, consist essentially of, or consist of an amino acid residue having the formula:

R14-X1 -X2-X9-

Formula VI

where X1 and X9 are hydrophobic amino acid residues and X2 is a hydrophobic amino acid residue or is absent.

Amino acid residues that can be included in the chemotactic peptide can be selected from naturally occurring amino acid residues (e.g., Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val), or non-naturally occurring amino acid residues. A "non- naturally occurring amino acid residue" is an amino acid residue which is not naturally produced or found in a mammal. Examples of non-naturally occurring amino acid residues include D-amino acid residues; an amino acid residue having an acetylaminomethyl group attached to a sulfur atom of a cysteine; a pegylated amino acid residue; the omega amino acid residues of the formula N H ₂ (CH ₂ ) _n COOH where n is 2-6, neutral nonpolar amino acid residues, such as sarcosine, t-butyl alanine, t-butyl glycine, N-methyl isoleucine, and norleucine; oxymethionine; phenylglycine; citrulline; methionine sulfoxide; cysteic acid; ornithine; diaminobutyric acid; diaminopropionic acid; and hydroxyproline.. Other amino acid residues are a-aminobutyric acid, α-amino-a-methylbutyrate, aminocyclopropane-carboxylate, aminoisobutyric acid, aminonorbornyl-carboxylate, L-cyclohexylalanine, cyclopentylalanine, L-N-methylleucine, L-N- methylmethionine, L-N-methylnorvaline, L-N-methylphenylalanine, L-N-methylproline, L-N-methylserine, L-N-methyltryptophan, D-ornithine, L-N-methylethylglycine, L-norleucine, a-methyl-aminoisobutyrate, a- methylcyclohexylalanine, D-a-methylalanine, D-a-methylarginine, D-a-methylasparagine, D-a- methylaspartate, D-a-methylcysteine, D-a-methylglutamine, D-a-methylhistidine, D-a-methylisoleucine, D- a-methylleucine, D-a-methyllysine, D-a-methylmethionine, D-a-methylornithine, D-a-methylphenylalanine, D-a-methylproline, D-a-methylserine, D-N-methylserine, D-a-methylthreonine, D-a-methyltryptophan, D- a-methyltyrosine, D-a-methylvaline, D-N-methylalanine, D-N-methylarginine, D-N-methylasparagine, D-N- methylaspartate, D-N-methylcysteine, D-N-methylglutamine, D-N-methylglutamate, D-N-methylhistidine, D-N-methylisoleucine, D-N-methylleucine, D-N-methyllysine, N-methylcyclohexylalanine, D-N- methylornithine, N-methylglycine, N-methylaminoisobutyrate, N-(1 -methylpropyl)glycine, N-(2- methylpropyl)glycine, D-N-methyltryptophan, D-N-methyltyrosine, D-N-methylvaline, γ-aminobutyric acid, L-t-butylglycine, L-ethylglycine, L-homophenylalanine, L-a-methylarginine, L-a-methylaspartate, L-a- methylcysteine, L-a-methylglutamine, L-a-methylhistidine, L-a-methylisoleucine, L-a-methylleucine, L-a- methylmethionine, L-a-methylnorvaline, L-a-methylphenylalanine, L-a-methylserine, L-a- methyltryptophan, L-a-methylvaline, N-(N-(2,2-diphenylethyl) carbamylmethylglycine, 1 -carboxy-1 -(2,2- diphenyl-ethylamino) cyclopropane, 4-hydroxyproline, ornithine, 2-aminobenzoyl (anthraniloyl), U- cyclohexylalanine, 4-phenyl-phenylalanine, L-citrulline, a-cyclohexylglycine, L-1 ,2,3,4- tetrahydroisoquinoline-3-carboxylic acid, L-thiazolidine-4-carboxylic acid, L-homotyrosine, L-2- furylalanine, L-histidine (3-methyl), N-(3-guanidinopropyl)glycine, O-methyl-L-tyrosine, O-glycan-serine, meta-tyrosine, nor-tyrosine, L-N,N',N"-trimethyllysine, homolysine, norlysine, N-glycan asparagine, 7- hydroxy-1 ,2,3,4-tetrahydro-4-fluorophenylalanine, 4-methylphenylalanine, bis-(2-picolyl)amine, pentafluorophenylalanine, indoline-2-carboxylic acid, 2-aminobenzoic acid, 3-amino-2-naphthoic acid, asymmetric dimethylarginine, L-tetrahydroisoquinoline-1 -carboxylic acid, D-tetrahydroisoquinoline-1 - carboxylic acid, 1 -amino-cyclohexane acetic acid, D/L-allylglycine, 4-aminobenzoic acid, 1 -amino- cyclobutane carboxylic acid, 2 or 3 or 4-aminocyclohexane carboxylic acid, 1 -amino-1 -cyclopentane carboxylic acid, 1 -aminoindane-1 -carboxylic acid, 4-amino-pyrrolidine-2-carboxylic acid, 2-aminotetraline- 2-carboxylic acid, azetidine-3-carboxylic acid, 4-benzyl-pyrolidine-2-carboxylic acid, tert-butylglycine, b- (benzothiazolyl-2-yl)-alanine, b-cyclopropyl alanine, 5,5-dimethyl-1 ,3-thiazolidine-4-carboxylic acid, (2R,4S)4-hydroxypiperidine-2-carboxylic acid, (2S,4S) and (2S,4R)-4-(2-naphthylmethoxy)-pyrolidine-2- carboxylic acid, (2S,4S) and (2S,4R)4-phenoxy-pyrrolidine-2-carboxylic acid, (2R,5S)and(2S,5R)-5- phenyl-pyrrolidine-2-carboxylic acid, (2S,4S)-4-amino-1 -benzoyl-pyrrolidine-2-carboxylic acid, t- butylalanine, (2S,5R)-5-phenyl-pyrrolidine-2-carboxylic acid, 1 -aminomethyl-cyclohexane-acetic acid, 3,5- bis-(2-amino)ethoxy-benzoic acid, 3,5-diamino-benzoic acid, 2-methylamino-benzoic acid, N- methylanthranylic acid, L-N-methylalanine, L-N-methylarginine, L-N-methylasparagine, L-N- methylaspartic acid, L-N-methylcysteine, L-N-methylglutamine, L-N-methylglutamic acid, L-N- methylhistidine, L-N-methylisoleucine, L-N-methyllysine, L-N-methylnorleucine, L-N-methylornithine, L-N- methylthreonine, L-N-methyltyrosine, L-N-methylvaline, L-N-methyl-t-butylglycine, L-norvaline, a-methyl- γ-aminobutyrate, 4,4'-biphenylalanine, a-methylcylcopentylalanine, α-methyl-a-napthylalanine, a- methylpenicillamine, N-(4-aminobutyl)glycine, N-(2-aminoethyl)glycine, N-(3-aminopropyl)glycine, N- amino-a-methylbutyrate, a-napthylalanine, N-benzylglycine, N-(2-carbamylethyl)glycine, N-

(carbamylmethyl)glycine, N-(2-carboxyethyl)glycine, N-(carboxymethyl)glycine, N-cyclobutylglycine, N- cyclodecylglycine, N-cycloheptylglycine, N-cyclohexylglycine, N-cyclodecylglycine, N- cylcododecylglycine, N-cyclooctylglycine, N-cyclopropylglycine, N-cycloundecylglycine, N-(2,2- diphenylethyl)glycine, N-(3,3-diphenylpropyl)glycine, N-(3-guanidinopropyl)glycine, N-(1 - hydroxyethyl)glycine, N-(hydroxyethyl))glycine, N-(imidazolylethyl))glycine, N-(3-indolylyethyl)glycine, N- methyl-y-aminobutyrate, D-N-methylmethionine, N-methylcyclopentylalanine, D-N-methylphenylalanine, D-N-methylproline, D-N-methylthreonine, N-(1 -methylethyl)glycine, N-methyl-napthylalanine, N- methylpenicillamine, N-(p-hydroxyphenyl)glycine, N-(thiomethyl)glycine, penicillamine, L-a-methylalanine, L-a-methylasparagine, L-a-methyl-t-butylglycine, L-methylethylglycine, L-a-methylglutamate, L-a- methylhomophenylalanine, N-(2-methylthioethyl)glycine, L-a-methyllysine, L-a-methylnorleucine, L-a- methylornithine, L-a-methylproline, L-a-methylthreonine, L-a-methyltyrosine, L-N-methyl- homophenylalanine, N-(N-(3,3-diphenylpropyl) carbamylmethylglycine, L-pyroglutamic acid, D- pyroglutamic acid, O-methyl-L-serine, O-methyl-L-homoserine, 5-hydroxylysine, a-carboxyglutamate, phenylglycine, L-pipecolic acid (homoproline), L-homoleucine, L-lysine (dimethyl), L-2-naphthylalanine, L- dimethyldopa or L-dimethoxy-phenylalanine, L-3-pyridylalanine, L-histidine (benzoyloxymethyl), N- cycloheptylglycine, L-diphenylalanine, O-methyl-L-homotyrosine, L- -homolysine, O-glycan-threoine, Ortho-tyrosine, L-N,N'-dimethyllysine, L-homoarginine, neotryptophan, 3-benzothienylalanine, isoquinoline-3-carboxylic acid, diaminopropionic acid, homocysteine, 3,4-dimethoxyphenylalanine, 4- chlorophenylalanine, L-1 ,2,3,4-tetrahydronorharman-3-carboxylic acid, adamantylalanine, symmetrical dimethylarginine, 3-carboxythiomorpholine, D-1 ,2,3,4-tetrahydronorharman-3-carboxylic acid, 3- aminobenzoic acid, 3-amino-1 -carboxymethyl-pyridin-2-one, 1 -amino-1 -cyclohexane carboxylic acid, 2- aminocyclopentane carboxylic acid, 1 -amino-1 -cyclopropane carboxylic acid, 2-aminoindane-2-carboxylic acid, 4-amino-tetrahydrothiopyran-4-carboxylic acid, azetidine-2-carboxylic acid, b-(benzothiazol-2-yl)- alanine, neopentylglycine, 2-carboxymethyl piperidine, b-cyclobutyl alanine, allylglycine, diaminopropionic acid, homo-cyclohexyl alanine, (2S,4R)- 4-hydroxypiperidine-2-carboxylic acid, octahydroindole-2- carboxylic acid, (2S,4R) and (2S,4R)-4-(2-naphthyl), pyrrolidine-2-carboxylic acid, nipecotic acid, (2S,4R)and (2S,4S)-4-(4-phenylbenzyl) pyrrolidine-2-carboxylic acid, (3S)-1 -pyrrolidine-3-carboxylic acid, (2S,4S)-4-tritylmercapto-pyrrolidine-2-carboxylic acid, (2S,4S)-4-mercaptoproline, t-butylglycine, N,N- bis(3-aminopropyl)glycine, 1 -amino-cyclohexane-1 -carboxylic acid, N-mercaptoethylglycine, and selenocysteine.

In certain embodiments, X1 is selected from a methionine, oxymethionine, or norleucine residue, or is absent. In certain embodiments, X2 is selected from a leucine and isoleucine, or is absent residue. In certain embodiments, X9 is selected from a phenylalanine, 1 -amino-2-phenylcyclopropane-1 -carboxylic acid, methionine, (O-benzyl)serine, 2-pyridiylalanine, 4-pyridylalanine residue. In certain embodiments, R ¹⁴ is -H. I n certain embodiments, R ¹⁴ is -C(0)H. In certain embodiments, R ¹⁴ is -C(0)CH ₃ . In other embodiments, R ¹⁴ is -C(0)OCH ₂ CH(CH ₃ ) ₂ . . In still other embodiments, R ¹⁴ is -C(0)N-4-chlorophenyl. In various embodiments, each of the aforementioned selections for X1 , X2, X9, and R ¹⁴ can each be considered independently and in various combinations with each other.

In some embodiments, the chemotaxis receptor ligand moiety, E, is

In some embodiments, the chemotaxis receptor ligand moiety, E, has the structure selected from:

In some embodiments, the chemotaxis receptor ligand moiety, E, has the structure selected from:

In some embodiments, the chemotaxis receptor ligand moiety, E, has the structure selected from:

In some embodiments, the chemotaxis receptor ligand that may be used to make the compounds described herein including a chemotaxis receptor ligand comprises, consists essentially of, or consists of the structure:

Methionylnorleucyl-leucyl-phenylalanyl-phenylalanine (MNIeLFF) as an FPR ligand as disclosed in J Med 1 80, 21 91 -7 (1 994) herein incorporated by reference. fMLX-OMe as FPR ligands are disclosed Bioorg Med Chem, 21 , 668-675 (2013), herein incorporated by reference, wherein X-OMe has the structure:

N-terminal oximic or formyl hydrazonic containing peptides as FPR ligands are shown in J. Peptide Res., 2000, 55, 1 02-109, herein incorporated by reference, have the structure:

n = 2-5 n = 2-5

B38 B39 β-Peptido sulfonamides as FPR ligands are disclosed in IL AHMACU 59 (2004) 953-963, herein incorporated by reference, including Boc-Met-Tau-Phe-OMe; HCO-Met-Tau-Phe-OMe; Met-Tau-Phe- OMe, Boc-Met- ³ -HLeu-Phe-OMe; HCO-Met- ³ -HLeu-Phe-OMe; Boc-Met-Leu-i|j[CH ₂ S0 ₂ ]-Phe-OMe; HCO-Met-Leu^[CH ₂ S0 ₂ ]-Phe-OMe; Boc-Met-Tau-Phe-Phe-OMe; HCO-Met-Tau-Phe-Phe-OMe.

3V,5V-dimethylphenyl-ureido containing peptides as FPR ligands are shown in European Journal of Pharmacology 436 (2002) 187- 1 96, herein incorporated by reference, including 3V,5V-dimethylphenyl- ureido-Phe-D-Leu-Phe-D-Leu-Phe-olo; 3V,5V-dimethylphenyl-ureido-Phe-D-Leu-Phe-D-Leu-Glu; 3V,5V- dimethylphenyl-ureido-Phe-D-Leu-Phe-D-Leu-Tyr. Proline containing peptides as FPR ligands are shown in Bioorganic & Medicinal Chemistry 1 4 (2006) 2253-2265, herein incorporated by reference, having the structure:

Peptides as FPR ligands are also disclosed in Amino Acids (2008) 35 329-338, herein incorporated by reference, having the structure:

R = Boc or CHO R = Boc or CHO

R = Boc or CHO

Other peptides as FPR ligands are shown in Pept Res. 6, 298-307 (1 993), herein incorporated by reference, having the structure:

Fluoromodified peptides as FPR ligands are shown in J. Peptide Sci. 1 0, 67-81 (2004), herein incorporated by reference, HCO-Met-(S)-DfeGly-Phe-N H ₂ ; HCO-Met-(R)-DfeGly-Phe-NH ₂ ; HCO-Met-(S)- (aTfm)Ala-Phe-N H ₂ ; HCO-Met-(R)-(aTfm)Ala-Phe-NH ₂ ; HCO-Met-Aib-Phe-NH ₂ ; HCO-Met-(R)-(aDfm)Ala- Phe-N H ₂ Further peptides as FPR ligands are disclosed in Biochemistry 1 9, 2404-241 0 (1980), herein incorporated by reference, including HCONIe-Leu-Phe-OH ; HCO-Nva-Leu-Phe-OH; HCO-Hep-Leu-Phe- OH; HCO-lle-Leu-Phe-OH ; HCO-Met-Ala-Leu-Phe-OH; HCO-Met-Leu-Phe-Lys-OH. More peptides as FPR ligands are shown in Eur. J. Immunol. 35, 2486-2495 (2005), herein incorporated by reference. Hybrid α/β peptides as FPR ligands are shown in Amino Acids 30, 453^59 (2006), herein incorporated by reference, including those having the structure:

R = H or OtBu

Hybrid ap3-Peptides as FPR ligands are shown in J. Peptide Sci. 1 0, 51 0-523 (2004), herein incorporated by reference, including those having the structure:

R = Boc or HCO

X = CH ₂ or bond

Y = CH ₂ or bond

Z = CH ₂ or bond

Isopeptide bond containing peptides as FPR ligands are shown in J. Peptide Res., 59, 283-291 (2002), herein incorporated by reference. Peptides containing constrained mimics of phenylalanine as FPR ligands are shown in Arch. Pharm. Pharm. Med. Chem. 331 , 170-1 76 (1 998), herein incorporated by reference, including those having the structure:

R ₁ = -CH ₂ Ph, R ₂ , R ₃ = H

R-i , R ₂ = -CH ₂ Ph, R ₃ = H or

R-i = H, R ₂ , R ₃ = -CH ₂ Ph

Other proline containing peptides as FPR ligands are disclosed in Bioorganic & Medicinal Chemistry 1 7, 251 -259 (2009), herein incorporated by reference. More peptides as FPR ligands are disclosed in J. Peptide Sci. 7, 56-65 (2002), herein incorporated by reference, including those having the structure:

Ac _n c where n = cyclic ring size

3 = cyclopropane, 5 = cyclopentane, etc

Pseudopeptide as FPR ligands are disclosed in J. Peptide Res. 58, 56-66 (2001 ), herein incorporated by reference, including those with the structure: R = CI; n = 2,3, or 5 or R = -(CH ₂ ) ₃ CH ₃ or

R = Me; n = 2,3, or 5 R— -CH2CH2SCH3

Further peptides as FPR ligands are shown in Bioorganic Chemistry 34, 298-318 (2006), herein incorporated by reference. Other peptides as FPR ligands are shown in II Farmaco 58, 1 121 (2003), herein incorporated by reference, including HCO-Met- -Ala-Phe-OMe, Boc-Met-Tau-Phe-OMe, and HCO- Met-Tau-Phe-OMe. Peptides with alkyl spacers as FPR ligands are shown in II Farmaco 56, 851 -858 (2001 ), herein incorporated by reference, including those having the structure:

R = 0-tBu or n = 3, 4, or 5

R = H

Tetrapeptides as FPR ligands are shown in Amino Acids 37, 285-295 (2009), herein incorporated by reference, including those having the structure:

Peptides containing a substituted glycine as FPR ligands are shown in Eur. J. Med. Chem. 27, 19-26 (1 992), herein incorporated by reference, including those having the structure:

= CH ₃ or allyl

R ₂ = Boc or HCO

X = CH ₂ , S, or NH

Peptides with a para-substituted phenylalanine as FPR ligands are shown in J. Pept. Sci. 18, 418-426 (2012), herein incorporated by reference. Other peptides as FPR ligands are shown in Pept Sci, 269-272 (2002), herein incorporated by reference. Centrally modified pseudopeptides as FPR ligands are shown in Amino Acids 33, 477^87 (2007), herein incorporated by reference. Further peptides as FPR ligands are shown in TREN DS in Immunology Vol.23 No.1 1 November 2002, herein incorporated by reference. More peptides as FPR ligands are shown in Cytokine & Growth Factor Reviews 1 7 (2006) 501 -519, herein incorporated by reference.

An additional example of a chemotaxis receptor ligand is tuftsin (also called a tuftsin peptide herein), a tetrapeptide (Thr-Lys-Pro-Arg) produced by enzymatic cleavage of the Fc-domain of the heavy chain of imm unoglobulin G , such as

Tuftsin

Tuftsin binds to specific receptors on the surface of macrophages and polymorphonuclear leukocytes, stim ulating their migration and phagocytic activity. It also influences antibody formation.

A further embodiment of a chemotaxis receptor ligand is N-acetyl-proline-glycine-proline (PGP) peptide, a chemotactic peptide, which is a collagen-breakdown product that attracts neutrophils. In certain aspects the acetyl PGP ligand is N-acetyl-L-prolyl-glycyl-L-proline having the following structure:

A tuftsin or PGP peptide moiety may be conjugated to a polyene antifungal agent moiety through the terminal carboxylic acid group of an amino acid residue. A tuftsin moiety that may be conjugated to a polyene antifungal agent moiety through the terminal carboxylic acid group of the tuftsin moiety includes the following structure:

An N-acetyl-L-prolyl-glycyl-L-proline (PGP) peptide moiety that may be conjugated to a polyene antifungal agent moiety through the terminal carboxylic acid group of the PGP peptide moiety includes the structure:

Linkers

In some embodiments, the compounds include a linker. The linker component is, at its simplest, a bond, but typically provides a linear, cyclic, or branched molecular skeleton having pendant groups covalently linking two moieties (e.g., moieties of the chemotaxic receptor ligand and the polyene antifungal agent).

Thus, linking of the moieties (e.g., moieties of the chemotaxic receptor ligand and the polyene antifungal agent) is achieved by covalent means, involving bond formation with one or more functional groups. Examples of chemically reactive functional groups which may be employed for this purpose include, without limitation, amino, hydroxyl, sulfhydryl, carboxyl, carbonyl, carbohydrate groups, vicinal diols, thioethers, 2-aminoalcohols, 2-aminothiols, guanidinyl, imidazolyl, and phenolic groups.

The covalent linking of the moieties may be effected using a linker that contains reactive moieties capable of reaction with such functional groups present in either moiety. For example, an amine group of a moiety may react with a carboxyl group of the linker, or an activated derivative thereof, resulting in the formation of an amide linking the two.

Examples of moieties capable of reaction with sulfhydryl groups include a-haloacetyl compounds of the type XCH ₂ CO- (where X=Br, CI, or I), which show particular reactivity for sulfhydryl groups, but which can also be used to modify imidazolyl, thioether, phenol, and amino groups as described by Gurd, Methods Enzymol. 1 1 :532 (1967). N-Maleimide derivatives are also considered selective towards sulfhydryl groups, but may additionally be useful in coupling to amino groups under certain conditions. Reagents such as 2-iminothiolane (Traut et al., Biochemistry 12:3266 (1973)), which introduce a thiol group through conversion of an amino group, may be considered as sulfhydryl reagents if linking occurs through the formation of disulfide bridges.

Examples of reactive moieties capable of reaction with amino groups include, for example, alkylating and acylating agents. Representative alkylating agents include:

(i) a-haloacetyl compounds, which show specificity towards amino groups in the absence of reactive thiol groups and are of the type XCH ₂ CO- (where X=Br, CI, or I), for example, as described by Wong Biochemistry 24:5337 (1979);

(ii) N-maleimide derivatives, which may react with amino groups either through a Michael type reaction or through acylation by addition to the ring carbonyl group, for example, as described by Smyth et al., J. Am. Chem. Soc. 82:4600 (1 960) and Biochem. J. 91 :589 (1964);

(iii) aryl halides such as reactive nitrohaloaromatic compounds;

(iv) alkyl halides, as described, for example, by McKenzie et al. , J. Protein Chem. 7:581 (1988);

(v) aldehydes and ketones capable of Schiff's base formation with amino groups, the adducts formed usually being stabilized through reduction to give a stable amine;

(vi) epoxide derivatives such as epichlorohydrin and bisoxiranes, which may react with amino, sulfhydryl, or phenolic hydroxyl groups;

(vii) chlorine-containing derivatives of s-triazines, which are very reactive towards nucleophiles such as amino, sufhydryl, and hydroxyl groups;

(viii) aziridines based on s-triazine compounds detailed above, e.g., as described by Ross, J.

Adv. Cancer Res. 2:1 (1 954), which react with nucleophiles such as amino groups by ring opening;

(ix) squaric acid diethyl esters as described by Tietze, Chem. Ber. 124:1215 (1 991 ); and (x) α-haloalkyl ethers, which are more reactive alkylating agents than normal alkyl halides because of the activation caused by the ether oxygen atom, as described by Benneche et al., Eur. J. Med. Chem. 28:463 (1 993).

Representative amino-reactive acylating agents include:

(i) isocyanates and isothiocyanates, particularly aromatic derivatives, which form stable urea and thiourea derivatives respectively;

(ii) sulfonyl chlorides, which have been described by Herzig et al., Biopolymers 2:349 (1 964);

(iii) acid halides;

(iv) active esters such as nitrophenylesters or N-hydroxysuccinimidyl esters;

(v) acid anhydrides such as mixed, symmetrical, or N-carboxyanhydrides;

(vi) other useful reagents for amide bond formation, for example, as described by M. Bodansky, Principles of Peptide Synthesis, Springer- Verlag, 1984;

(vii) acylazides, e.g., wherein the azide group is generated from a preformed hydrazide derivative using sodium nitrite, as described by Wetz et al., Anal. Biochem. 58:347 (1 974); and

(viii) imidoesters, which form stable amidines on reaction with amino groups, for example, as described by Hunter and Ludwig, J. Am. Chem. Soc. 84:3491 (1962).

Aldehydes and ketones may be reacted with amines to form Schiff's bases, which may advantageously be stabilized through reductive amination. Alkoxylamino moieties readily react with ketones and aldehydes to produce stable alkoxamines, for example, as described by Webb et al., in Bioconjugate Chem. 1 :96 (1 990).

Examples of reactive moieties capable of reaction with carboxyl groups include diazo compounds such as diazoacetate esters and diazoacetamides, which react with high specificity to generate ester groups, for example, as described by Herriot, Adv. Protein Chem. 3:169 (1 947). Carboxyl modifying reagents such as carbodiimides, which react through O-acylurea formation followed by amide bond formation, may also be employed.

It will be appreciated that functional groups in either moiety (e.g., the chemotaxic receptor ligand moiety or the polyene antifungal agent moiety) may, if desired, be converted to other functional groups prior to reaction, for example, to confer additional reactivity or selectivity. Examples of methods useful for this purpose include conversion of amines to carboxyls using reagents such as dicarboxylic anhydrides; conversion of amines to thiols using reagents such as N-acetylhomocysteine thiolactone, S- acetylmercaptosuccinic anhydride, 2-iminothiolane, or thiol-containing succinimidyl derivatives;

conversion of thiols to carboxyls using reagents such as a -haloacetates; conversion of thiols to amines using reagents such as ethylenimine or 2-bromoethylamine; conversion of carboxyls to amines using reagents such as carbodiimides followed by diamines; and conversion of alcohols to thiols using reagents such as tosyl chloride followed by transesterification with thioacetate and hydrolysis to the thiol with sodium acetate.

So-called zero-length linkers, involving direct covalent joining of a reactive chemical group of one moiety with a reactive chemical group of the other without introducing additional linking material may, if desired, be used with the compounds herein.

More commonly, however, the linker will include two or more reactive moieties, as described above, connected by a spacer element. The presence of such a spacer permits bifunctional linkers to react with specific functional groups within either moiety (e.g., the chemotaxic receptor ligand moiety or the polyene antifungal agent moiety), resulting in a covalent linkage between the two. I he reactive moieties in a linker may be the same (homobifunctional linker) or different (heterobifunctional linker, or, where several dissimilar reactive moieties are present, heteromultifunctional linker), providing a diversity of potential reagents that may bring about covalent attachment between the two moieties.

Spacer elements in the linker typically consist of linear or branched chains and may include a C1 -

C10 alkylene, C2-C1 0 alkenylene, C2-C1 0 alkynylene, C2-C6 heterocyclylene, C6-C12 arylene, C7-C14 ary alkylene, C3-C1 0 heterocyclyl alkylene, C2-C100 polyethylene glycolene, or C1 -C1 0 heteroalkylene.

In some instances, the linker is described by any one of Formulas VI I-XXI I.

Examples of homobifunctional linkers useful in the preparation of the compounds include, without limitation, diamines and diols selected from ethylenediamine, propylenediamine and

hexamethylenediamine, ethylene glycol, diethylene glycol, propylene glycol, 1 ,4-butanediol, 1 ,6- hexanediol, cyclohexanediol, and polycaprolactone diol.

In certain embodiments, the chemotaxis receptor ligand or (b) is attached to said linker via an amide bond (e.g., at the C-terminus of a chemotactic peptide) and said polyene antifungal agent or (a) is attached to said linker via an amide bond (e.g., at a terminal primary or secondary amine such as, at R ⁹ of Formula I I or R ^{1 1} of Formula IV).

Treatment of Fungal Infections

The disclosure includes compositions and methods for treating or preventing a disease or condition associated with a fungal infection by administering a compound described herein. Compounds may be administered by any appropriate route for treatment or prophylactic treatment of a disease or condition associated with a fungal infection. These may be administered to humans, domestic pets, livestock, or other animals with a pharmaceutically acceptable diluent, carrier, or excipient.

Administration may be topical, parenteral, intravenous, intra-arterial, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal,

intraperitoneal, intranasal, aerosol, by suppositories, or oral administration.

The compounds described herein can be used to treat, for example, invasive aspergillosis, pulmonary aspergillosis, intra-abdominal abscess, peritonitis, a pleural cavity infection, esophagitis, candidemia, and invasive candidiasis.

Methods of treating a fungal infection in a subject include administering to the subject a compound or pharmaceutical composition in an amount sufficient to treat the infection. In particular embodiments, the pharmaceutical composition is administered intravenously or topically. The pharmaceutical composition can be administered to treat a blood stream infection, tissue infection (e.g., lung, kidney, or liver infection) in the subject, or any other type of infection described herein. The fungal infection being treated can be an infection selected from, invasive aspergillosis, pulmonary aspergillosis, intra-abdominal abscess, peritonitis, a pleural cavity infection, esophagitis, candidemia, and invasive candidiasis. In certain embodiments, the infection being treated is an infection by Candida albicans, C. parapsilosis, C. glabrata, C. guilliermondii, C. krusei, C. lusitaniae, C. tropicalis, Aspergillus fumigatus, A. flavus, A. terreus. A. niger, A. candidus, A. clavatus, or A. ochraceus.

Methods for the prophylactic treatment of a fungal infection in a subject include administering to the subject a compound or pharmaceutical composition. In particular embodiments, the pharmaceutical composition is administered at least once over a period of 1 -30 days (e.g., 1 , 2, 3, 4, or 5 times over a period of 1 -30 days). For example, the methods can be used tor prophylatic treatment in subjects being prepared for an invasive medical procedure (e.g., preparing for surgery, such as receiving a transplant, stem cell therapy, a graft, a prosthesis, receiving long-term or frequent intravenous catheterization, or receiving treatment in an intensive care unit), in immunocompromised subjects (e.g., subjects with cancer, with HIV/AI DS, or taking immunosuppressive agents), or in subjects undergoing long term antibiotic therapy.

Method of stabilizing, or inhibiting the growth of fungi, or killing fungi include contacting the fungi or a site susceptible to fungal growth with a compound or pharmaceutical composition, or a

pharmaceutically acceptable salt thereof.

Pharmaceutical Compositions

For use as treatment of human and animal subjects, the compounds can be formulated as pharmaceutical or veterinary compositions. Depending on the subject to be treated, the mode of administration, and the type of treatment desired - e.g., prevention, prophylaxis, or therapy - the compounds are formulated in ways consonant with these parameters. A summary of such techniques is found in Remington: The Science and Practice of Pharmacy, 21 ^s ' Edition, Lippincott Williams & Wilkins, (2005); and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1 988-1 999, Marcel Dekker, New York, each of which is incorporated herein by reference.

The compounds described herein may be present in amounts totaling 1 -95% by weight of the total weight of the composition. The composition may be provided in a dosage form that is suitable for intraarticular, oral, parenteral (e.g., intravenous, intramuscular), rectal, cutaneous, subcutaneous, topical, transdermal, sublingual, nasal, vaginal, intravesicular, intraurethral, intrathecal, epidural, aural, or ocular administration, or by injection, inhalation, or direct contact with the nasal, genitourinary, gastrointesitnal, reproductive or oral mucosa. Thus, the pharmaceutical composition may be in the form of, e.g., tablets, capsules, pills, powders, granulates, suspensions, emulsions, solutions, gels including hydrogels, pastes, ointments, creams, plasters, drenches, osmotic delivery devices, suppositories, enemas, injectables, implants, sprays, preparations suitable for iontophoretic delivery, or aerosols. The compositions may be formulated according to conventional pharmaceutical practice.

In general, for use in treatment, the compounds described herein may be used alone, as mixtures of two or more compounds or in combination with other pharmaceuticals. An example of other pharmaceuticals to combine with the compounds described herein would include pharmaceuticals for the treatment of the same indication. Another example of a potential pharmaceutical to combine with the compounds described herein would include pharmaceuticals for the treatment of different yet associated or related symptoms or indications. Depending on the mode of administration, the compounds will be formulated into suitable compositions to permit facile delivery. Each compound of a combination therapy may be formulated in a variety of ways that are known in the art. For example, the first and second agents of the combination therapy may be formulated together or separately. Desirably, the first and second agents are formulated together for the simultaneous or near simultaneous administration of the agents.

The compounds may be prepared and used as pharmaceutical compositions comprising an effective amount of a compound described herein and a pharmaceutically acceptable carrier or excipient, as is well known in the art. In some embodiments, the composition includes at least two ditterent pharmaceutically acceptable excipients or carriers.

Formulations may be prepared in a manner suitable for systemic administration or topical or local administration. Systemic formulations include those designed for injection (e.g., intramuscular, intravenous or subcutaneous injection) or may be prepared for transdermal, transmucosal, or oral administration. The formulation will generally include a diluent as well as, in some cases, adjuvants, buffers, preservatives and the like. The compounds can be administered also in liposomal compositions or as microemulsions.

For injection, formulations can be prepared in conventional forms as liquid solutions or suspensions or as solid forms suitable for solution or suspension in liquid prior to injection or as emulsions. Suitable excipients include, for example, water, saline, dextrose, glycerol and the like. Such compositions may also contain amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, such as, for example, sodium acetate, sorbitan monolaurate, and so forth.

Various sustained release systems for drugs have also been devised. See, for example, U.S. patent No. 5,624,677, which is herein incorporated by reference.

Systemic administration may also include relatively noninvasive methods such as the use of suppositories, transdermal patches, transmucosal delivery and intranasal administration. Oral administration is also suitable for compounds. Suitable forms include syrups, capsules, and tablets, as is understood in the art.

Each compound of a combination therapy, as described herein, may be formulated in a variety of ways that are known in the art. For example, the first and second agents of the combination therapy may be formulated together or separately.

The individually or separately formulated agents can be packaged together as a kit. Non-limiting examples include, but are not limited to, kits that contain, e.g., two pills, a pill and a powder, a suppository and a liquid in a vial, two topical creams, etc. The kit can include optional components that aid in the administration of the unit dose to patients, such as vials for reconstituting powder forms, syringes for injection, customized IV delivery systems, inhalers, etc. Additionally, the unit dose kit can contain instructions for preparation and administration of the compositions. The kit may be manufactured as a single use unit dose for one patient, multiple uses for a particular patient (at a constant dose or in which the individual compounds may vary in potency as therapy progresses); or the kit may contain multiple doses suitable for administration to multiple patients ("bulk packaging"). The kit components may be assembled in cartons, blister packs, bottles, tubes, and the like.

Formulations for oral use include tablets containing the active ingredient(s) in a mixture with non- toxic pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers (e.g. , sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates, or alginic acid); binding agents (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, carboxymethylcellulose sodium,

methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents, glidants, and antiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc). Other pharmaceutically acceptable excipients can be colorants, flavoring agents, plasticizers, humectants, buffering agents, and the like.

Two or more compounds may be mixed together in a tablet, capsule, or other vehicle, or may be partitioned. In one example, the first compound is contained on the inside of the tablet, and the second compound is on the outside, such that a substantial portion of the second compound is released prior to the release of the first compound.

Formulations for oral use may also be provided as chewable tablets, or as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin), or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil. Powders, granulates, and pellets may be prepared using the ingredients mentioned above under tablets and capsules in a conventional manner using, e.g., a mixer, a fluid bed apparatus or a spray drying equipment.

Dissolution or diffusion controlled release can be achieved by appropriate coating of a tablet, capsule, pellet, or granulate formulation of compounds, or by incorporating the compound into an appropriate matrix. A controlled release coating may include one or more of the coating substances mentioned above and/or, e.g., shellac, beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl distearate, glycerol palmitostearate, ethylcellulose, acrylic resins, dl- polylactic acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone, polyethylene, polymethacrylate, methylmethacrylate, 2-hydroxymethacrylate, methacrylate hydrogels, 1 ,3 butylene glycol, ethylene glycol methacrylate, and/or polyethylene glycols. In a controlled release matrix formulation, the matrix material may also include, e.g., hydrated methylcellulose, carnauba wax and stearyl alcohol, carbopol 934, silicone, glyceryl tristearate, methyl acrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and/or halogenated fluorocarbon.

The liquid forms in which the compounds and compositions can be incorporated for

administration orally include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

Generally, when administered to a human, the oral dosage of any of the compounds of the combination will depend on the nature of the compound, and can readily be determined by one skilled in the art. Typically, such dosage is normally about 0.001 mg to 2000 mg per day, desirably about 1 mg to 1000 mg per day, and more desirably about 5 mg to 500 mg per day. Dosages up to 200 mg per day may be necessary.

Administration of each drug in a combination therapy, as described herein, can, independently, be one to four times daily for one day to one year, and may even be for the life of the patient. Chronic, long-term administration may be indicated.

The following Examples are intended to illustrate the synthesis of a representative number of compounds and the use of these compounds for the induction of chemotaxis and antifungal activity. Accordingly, the Examples are intended to illustrate but not to limit the disclosure herein. Additional compounds not specifically exemplified may be synthesized using conventional methods in combination with the methods described herein. EXAMPLES

General Methods

Analytical H PLC was performed using the following column and conditions: Atlantis T3, 3 micron, 3.0 x 75 mm; 50°C, water/CH ₃ CN + 0.1 % formic acid, 5 to 95% CH ₃ CN over 1 1 min + 2 min hold.

Preparative HPLC was performed using the following column: Agilent ZORBAX SB-CN, 7 μιη, 21 .2 x 250 mm, CH ₃ CN/H ₂ O/0.1 % Acetic Acid various linear gradients as necessary at 20 mL/min.

Rapid LC: A Waters BEH C18, 3.0 x 30 mm, 1 .7 μιη, was used at a temperature of 50°C and at a flow rate of 1 .5 mL/min, 2 L injection, mobile phase: (A) water with 0.1 % formic acid and 1 % acetonitrile, mobile phase (B) methanol with 0.1 % formic acid; retention time given in minutes. Method details: (I) runs on a Binary Pump G1312B with UV/Vis diode array detector G1315C and Agilent 6130 mass spectrometer in positive and negative ion electrospray mode with UV PDA detection with a gradient of 15- 95% (B) in a 2.2 min linear gradient (I I) hold for 0.8 min at 95% (B) (I I I) decrease from 95-15% (B) in a 0.1 min linear gradient (IV) hold for 0.29 min at 1 5% (B).

Polar Stop-Gap: An Agilent Zorbax Bonus RP, 2.1 x 50mm, 3.5 μιη, was used at a temperature of 50°C and at a flow rate of 0.8 mL/min, 2 L injection, mobile phase: (A) water with 0.1 % formic acid and 1 % acetonitrile, mobile phase (B) methanol with 0.1 % formic acid; retention time given in minutes. Method details: (I) runs on a Binary Pump G1312Bwith UV/Vis diode array detector G1315C and Agilent 6130 mass spectrometer in positive and negative ion electrospray mode with UV-detection at 220 and 254 nm with a gradient of 5-95% (B) in a 2.5 min linear gradient (I I) hold for 0.5 min at 95% (B) (I I I) decrease from 95-5% (B) in a 0.1 min linear gradient (IV) hold for 0.29 min at 5% (B).

NMR Spectra were acquired on either of two instruments: (1 ) Agilent (formerly Varian) Unitylnova 400 M Hz NMR spectrometer equipped with a 5mm Automation Triple Broadband (ATB) probe. The ATB probe was simultaneously tuned to 1 H, 1 9F and 13C. (2) Agilent (formerly Varian) Unitylnova 500 M Hz NMR spectrometer. Several NM R probes are available for use with the 500 M Hz NMR spectrometer, including both 3 mm and 5 mm 1 H 13C15N probes and a 3 mm X1 H 19F NM R probe (usually X is tuned to 13C). For typical 1 H NM R spectra, the pulse angle was 45 degrees, 8 scans were summed and the spectral width was 1 6 ppm (-2 ppm to 1 4 ppm). A total of 32768 complex points were collected during the 5.1 second acquisition time, and the recycle delay was set to 1 second. Spectra were collected at 25 ^S C. 1 H NMR Spectra are typically processed with 0.3 Hz line broadening and zero-filling to 131 072 points prior to Fourier transformation.

Synthesis of peptide fragment coupling partners: In some cases, discreet coupling partners were isolated and purified. In others, the couplings partners were prepared and used directly without isolation.

Compounds may be made using synthetic methods known in the art, including procedures analogous to those disclosed below.

For some of the preparations the following general conditions were utilized.

Preparative HPLC was performed using the following:Teledyne Isco H P C1 8, 50g column. Eluent: CH ₃ CN/H ₂ 07 0.1 % formic Acid or 0.1 % trifluoro acetic acid; various linear gradients as necessary at 40 mL/min on the Isco Combiflash Rf LC unit. UV Detection at 220 and 254 nm.

OR Luna 5 micron C18, 1 00 A°, AXIA 1 00 x 30 mm. Eluent: CH ₃ CN/H ₂ 07 0.1 % formic Acid or 0.1 % trifluoro acetic acid; various linear gradients as necessary at 25 mL/min on the Gilson System; 215 Liquid Handler, Gilson UV-VIS 155, Gilson 305 pump and Detector. UV detection at 220 and 254 nm. Analytical LC/MS: High resolution liquid chromatography mass spectrometry (HHbS-LC/M!.;) was performed using a Waters Q-TOF Premier mass spectrometer with an electrospray probe coupled with an Agilent 1 1 00 HPLC system with a diode array detector set to collect from 1 90nm to 400 nm. A gradient of 0.1 % formic acid in water (A) and 0.1 % formic acid in acetonitrile (B) was run from 15%B to 95% over 15min using a 1 00x3.0 2.6u Phenomenex Kinetex C1 8 column at 30°C.

Liquid chromatography mass spectrometry was performed using an Agilent 6120 mass spectrometer an electrospray probe coupled coupled with an Agilent 1 100 H PLC system with a variable wavelength detector set to either 220nm or 254nm. A gradient of 0.1 % formic acid in water (A) and 0.1 % formic acid in acetonitrile (B) was run from 15%B to 99% over 3.5min using a 50x3.0 2.6u Phenomenex Kinetex C18 column at 30 °C.

1 H-NM R spectra were acquired on a Bruker 300 MHz system using a 5mm QNP probe.

Synthesis of peptide fragment coupling partners: I n some cases, discreet coupling partners were isolated and purified. In others, the couplings partners were prepared and used directly without isolation. The description of the isolated compounds is described in this section, while the coupling partners that were used directly are embedded within the examples of final compounds.

Some peptides or derivatized peptides were obtained from a Contract Research Organization or other commercial source. In general, peptides are considered readily available from such sources by standard peptide synthesis procedures.

Compounds herein may be made using synthetic methods known in the art, including procedures analogous to those disclosed below.

Example 1 : Synthesis of Intermediates

Synthesis of fMLF-OSu (lnt-1)

lnt-1

Procedure 1

(N-formyl)-MLF-OH (1 00 mg, 0.23 mmol, 1 eq) was dissolved in 4mL dry DMF. DCC (50 mg, 0.24 mmol, 1 .05 eq) and N-hydroxysuccinimide (27 mg, 0.23 mol, 1 eq) were added and the reaction was stirred overnight at RT. The product was used directly in subsequent reactions without purification.

Procedure 2

A solution of (N-formyl)-MLF-OH (0.866 g, 1 .49 mmol) in DMF (65 mL) was charged with HOSu (0.171 g, 1 .49 mmol) and DCC (0.322 g, 1 .56 mmol), and allowed to stir 48 h at rt, during which additional HOSu (0.034 g, 0.30 mmol) and DCC (0.064 g, 0.31 mmol) were added at t = 24 h. The reaction was used in subsequent steps directly without prior isolation of fMLF-OSu. MS (M+H) 535.0. Alternatively, the crude fMLF-OSu may be isolated by removal of the solvent under reduced pressure.

Synthesis of (N-iso-BOC)-MLF-OSu (lnt-2)

lnt-2

(N-iso-BOC)-MLF-OH (50 mg, 0.1 mmol, 1 eq) was dissolved in 1 ml_ dry DMF. EDCI (28 mg, 0.15 mmol, 1 .5 eq) and N-hydroxysuccinimide (12 mg, 0.1 mmol, 1 .05 eq) were added. When H PLC analysis indicated no (N-iso-BOC)-MLF-OH the reaction was used directly in subsequent reactions without purification.

Step a) Preparation of N ^a -F OC-N ^E -Z-Lysine tert-Butyl Ester

N ^E -Z-Lysine tert-butyl ester (300 mg, 0.81 mmoles) and FMOC-OSu (299 mg, 0.89 mmoles, 1 .1 eq) were dissolved in 3ml_ dry DM F and TEA (1 12 μΙ_, 0.81 mmoles, 1 eq) was added. The reaction was stirred at room temperature for 1 hour. The reaction was diluted with 1 0% aqueous LiCI and extracted 3 times with 10% heptanes in ethyl acetate. The organic extractions were combined and back extracted with 10% aqueous LiCI. The organic solution was dried over sodium sulfate, filtered, and evaporated. Yield 51 9 mg. Target Yield 452 mg. LC/MS, 559.0 [M+H] ⁺ . ¹ H NM R d ₆ -DMSO is consistent with the desired product and indicated trace amounts of DMF and ethyl acetate. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 1 .38 (s, 10 H) 1 .50 - 1 .72 (m, 1 H) 2.92 - 3.05 (m, 1 H) 3.79 - 3.90 (m, 1 H) 4.16 - 4.25 (m, 1 H) 4.26 - 4.33 (m, 1 H) 5.00 (s, 1 H) 7.20 - 7.26 (m, 1 H) 7.29 - 7.37 (m, 7 H) 7.41 (s, 2 H) 7.59 - 7.66 (m, 1 H) 7.72 (d, J=7A2 Hz, 2 H) 7.89 (d, J=7.42 Hz, 2 H) Step b) Removal of Z-group

The product from Step a) was dissolved in 20 mL ethanol and 2 mL acetic acid. 150 mg of 5% Pd/C was added and the reaction was placed under a hydrogen atmosphere (balloon pressure). After stirring overnight, the reaction was filtered and the solvent was removed under reduced pressure.

LC/MS, 425.2 [M+H] ⁺ . The crude product was used without purification.

Step c) Preparation of N ^E -fMLF-N ^a -FMOC-K tert-Butyl Ester

The product from Step b) above (344 mg, 0.81 mmol, 1 .7 eq) was dissolved in 7 mL dry DMF and fMLF-OSu (lnt-1 ) (246 mg, 0.46 mmol) dissolved in 2 mL dry DMF was added dropwise. After 1 0 minutes the product was purified by prep-HPLC using a 60% to 1 00% methanol in water gradient over 5 minutes. Yield 120 mg. LC/MS, 844.2 [M+H] ⁺ . ¹ H NMR (400 M Hz, DMSO-d ₆ ) δ ppm 0.80 (d, J=6.64 Hz, 3 H) 0.85 (d, J=6.64 Hz, 3 H) 1 .09 - 1 .34 (m, 5 H) 1 .34 - 1 .44 (m, 12 H) 1 .44 - 1 .64 (m, 3 H) 1 .67 - 1 .81 (m, 1 H) 1 .81 - 1 .92 (m, 1 H) 2.01 (s, 3 H) 2.34 - 2.44 (m, 2 H) 2.76 - 2.86 (m, 1 H) 2.87 - 2.98 (m, 2 H) 2.99 - 3.14 (m, 1 H) 3.77 - 3.88 (m, 1 H) 4.1 6 - 4.36 (m, 4 H) 4.36 - 4.48 (m, 2 H) 6.28 (s, 1 H) 7.17 (d, J=7.22 Hz, 3 H) 7.22 (d, J=6.25 Hz, 3 H) 7.33 (dd, J=15.23, 7.61 Hz, 3 H) 7.42 (td, J=7.42, 0.98 Hz, 3 H) 7.61 (d, J=7.81 Hz, 1 H) 7.72 (d, J=7.22 Hz, 2 H) 7.84 (d, J=7.42 Hz, 2 H) 7.89 (dd, J=7.52, 2.44 Hz, 3 H) 7.93 (d, J=8.20 Hz, 1 H) 7.99 - 8.03 (m, 1 H) 8.08 (d, J=8.00 Hz, 1 H) 8.30 (d, J=8.00 Hz, 1 H)

Step d) Removal of the tert-Butyl Ester

The product from Step c) above (120 mg, 0.14 mmol) was dissolved in 20 mL formic acid and stirred at room temperature for two hours. The formic acid was removed under reduced pressure. The residue was diluted twice with toluene and evaporated to remove residual formic acid. The product was used without further purification. Yield 134 mg. LC/MS, 788.2 [M+H] ⁺ .

Synthesis of fMLF-Diaminoethylamide (lnt-4)

lnt-4

fMLF-OSu (lnt-1 ) (246 mg, 0.46 mmol) was dissolved in 5 mL dry DM F and cooled to 0 °C.

FMOC-2-(2-aminoethoxy)-ethylamine HCI (1 84 mg, 0.51 mmol, 1 .1 eq) was added then DI PEA (88 uL, 65 mg, 0.51 mmol, 1 .1 eq) was added. The reaction was stirred until the fMLF-OSu was consumed.

Piperidine (250 uL, 5% V/V) was added and the reaction was stirred at 0 °C for 20 minutes. The reaction was filtered and the product was purified by prep-H PLC using a 5% to 1 00% methanol in water (0.1 % TEA) gradient. Yield 150 mg. LC/MS, 524.2 [M+H] ⁺ .

Synthesis of fMLF-N ^e -FMOC-Lysine (lnt-5)

Step a) Preparation of fMLF-(N ^E -Z-Lysine) ferf-Butyl Ester

A solution of N ^E -Z-Lysine ferf-butyl ester 0.852 g, 2.29 mmol) and fMLF (1 .00 g, 2.29 mmol) in DMF (20 mL) was charged with HATU (0.869 g, 2.29 mmol) and 2,4,6-collidine (0.554 g, 4.57 mmol). After stirring at rt for 40 min, the reaction was diluted with EtOAc (200 mL) and washed sequentially with saturated LiCI (3 x 50 mL), saturated NaHC0 ₃ (2 x 50 mL), and saturated NaCI (2 x 50 mL), then dried over Na ₂ S0 ₄ , filtered, and concentrated under reduced pressure. The crude product was then chromatographed on a 40 g Isco RediSep silica cartridge eluting with a gradient from 1 00% heptane to 100% EtOAc, and then to 1 00% MeOH Yield: 1 .49 g as a white solid. ¹ H NM R (400 MHz, Chloroform-d) δ ppm 0.84 (d, J=5.22 Hz, 3 H) 0.87 (d, J=6.20 Hz, 3 H) 1 .20 - 2.06 (m, 9 H) 1 .44 (s, 9 H) 2.08 (s, 3 H) 2.50 (t, J=7.35 Hz, 2 H) 2.81 (s, 1 H) 2.92 - 3.37 (m, 4 H) 4.30 - 4.75 (m, 4 H) 5.04 - 5.19 (m, 2 H) 5.22 - 5.29 (m, 1 H) 6.32 - 6.52 (m, 1 H) 6.74 (d, J=7.13 Hz, 1 H) 6.83 - 7.00 (m, 2 H) 7.1 0 - 7.47 (m, 10 H) 7.69

- 7.85 (m, 1 H) 8.04 - 8.20 (m, 1 H). MS (M+H) 756.2.

Step b) Preparation of fMLFK-O-ferf-Butyl Ester

A solution of fMLF-(N ^E -Z-lysine) ferf-butyl ester (1 .96 g, 2.60 mmol) in EtOH (300 mL) was charged with 5% Pd on carbon (5.52 g), then evacuated and blanketed under H ₂ via balloon 3 times. After stirring at rt for 19 h, the reaction was filtered through celite, and the filter cake was eluted with 9:1 EtOAc:MeOH, collecting 4 x 500 mL fractions. Fractions containing product (1 and 2) were pooled and concentrated under reduced pressure, and the isolated product was not purified further. Yield: 1 .38 g as a white solid. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 0.79 (d, J=6.54 Hz, 3 H) 0.85 (d, J=6.54 Hz, 3 H) 1 .21

- 1 .90 (m, 9 H) 1 .40 (s, 9 H) 2.01 (s, 3 H) 2.31 - 2.44 (m, 2 H) 2.58 (t, J=6.76 Hz, 2 H) 2.75 - 3.1 0 (m, 2 H) 3.19 - 3.50 (m, 2 H) 3.99 - 4.70 (m, 6 H) 7.1 1 - 7.26 (m, 5 H) 7.93 (d, J=8.25 Hz, 1 H) 8.00 (s, 1 H) 8.04 (d, J=8.25 Hz, 1 H) 8.21 (d, J=7.52 Hz, 1 H) 8.29 (d, J=8.00 Hz, 1 H). MS (M+H) 622.2.

Step c) Preparation of fMLF-(N ^E -FMOC-Lysine) tert-Butyl Ester

A solution of fMLFK-O- ferf-butyl ester (1 .37 g, 2.20 mmol) in DMF (20 mL) was charged with Fmoc-OSu (0.818 g, 2.42 mmol). After stirring at rt for 1 0 min, the reaction was diluted with EtOAc (250 mL) and washed with saturated LiCI (3 x 50 mL, with MeOH added to break an emulsion when needed), then dried over Na ₂ S0 ₄ , filtered, and concentrated under reduced pressure. The crude product was then chromatographed on a 40 g Isco RediSep silica cartridge elutmg with a gradient trom 1 00% heptane to 100% EtOAc, and then to 1 00% MeOH. Yield: 1 .62 g as a white solid. ¹ H NM R (400 MHz, DMSO-d ₆ ) δ ppm 0.77 (d, J=6.54 Hz, 3 H) 0.82 (d, J=6.54 Hz, 3 H) 1 .15 - 1 .90 (m, 9 H) 1 .38 (s, 9 H) 1 .99 (s, 3 H) 2.28 - 2.44 (m, 2 H) 2.76 - 3.1 0 (m, 2 H) 3.94 - 4.70 (m, 1 1 H) 7.09 - 7.27 (m, 5 H) 7.28 - 7.37 (m, 3 H) 7.40 (t, J=7.49 Hz, 2 H) 7.68 (d, J=7.52 Hz, 2 H) 7.88 (d, J=7.47 Hz, 2 H) 8.00 (s, 1 H) 8.04 (d, J=8.30 Hz, 1 H) 8.15 (d, J=8.39 Hz, 1 H) 8.28 (d, J=7.1 7 Hz, 1 H) 8.33 - 8.46 (m, 1 H). MS (M+H) 844.2.

Step d) Removal of the tert-Butyl Ester to Provide lnt-5

A solution of fMLF-(N ^E -FMOC-Lysine) tert-butyl ester (1 .61 g, 1 .91 mmol) in formic acid (20 mL) was stirred at rt for 3 h. The reaction was then concentrated under reduced pressure, chromatographed on a 50 g Isco Gold RediSep C-18 reversed phase silica cartridge, eluting with a gradient from 99.9:0.1 H ₂ 0:HOAc to 99.9:0.1 MeOH:HOAc. The isolated product was then lyophilized from 1 :1 H ₂ 0:CH ₃ CN (8 mL). Yield: 0.304 g. ¹ H NM R (400 MHz, DMSO-d ₆ ) δ ppm 0.79 (d, J=6.39 Hz, 3 H) 0.84 (d, J=6.49 Hz, 3 H) 1 .12 - 1 .92 (m, 9 H) 2.00 (s, 3 H) 2.38 (t, J=6.66 Hz, 2 H) 2.73 - 3.10 (m, 4 H) 3.31 (br. s., 2 H) 4.07 - 4.63 (m, 7 H) 7.09 - 7.36 (m, 8 H) 7.37 - 7.46 (m, 2 H) 7.68 (d, J=7.27 Hz, 2 H) 7.83 - 7.97 (m, 3 H) 7.98 - 8.06 (m, 2 H) 8.07 - 8.14 (m, 1 H) 8.27 (d, J=7.71 Hz, 1 H) 12.20 - 12.95 (m, 1 H). MS (M+H) 788.2.

Synthesis of Amphotericin-B-2-(amino)-ethyl-amide (lnt-6)

Mycosamine-N-FMOC-Amphotericin B (prepared as in Example 1 , step a) ) (256 mg, 0.224 mmoles) and ethylenediamine-mono-FMOC-HCI (1 07 mg, 0.335 mmoles, 1 .5 eq) were dissolved in 5 mL dry DMF and cooled to 0 °C. DIPEA (97 μΐ, 72 mg, 0.559 mmoles, 2.5 eq) and COM U (96 mg, 0.224 mmoles, 1 eq) were added. When no starting material remained, piperidine (66 uL, 57 mg, 0.671 mmoles, 3 eq) was added, and the reaction was allowed to warm to room temperature. When the intermediate was consumed, the reaction was quenched with acetic acid (1 02 uL, 107 mg, 1 .79 mmoles, 8 eq), and the product was purified by prep-H PLC using a 20% to 1 00% methanol/0.1 % acetic acid in water/0.1 % acetic acid gradient. Yield 48.6 mg. LC/MS, 988 [M+23] ⁺ . ¹ H NMR (400 M Hz, DMSO-d ₆ ) δ ppm 0.91 (d, J=7.03 Hz, 3 H) 1 .04 (d, J=6.44 Hz, 3 H) 1 .1 1 (d, J=6.2b Hz, 4 H) 1 .14 (d, J=6.0b Hz, 4 H) 1 .86 (s, 9 H) 2.54 (s, 4 H) 8.1 1 (t, J=5.08 Hz, 1 H).

Synthesis of C2'-Deoxyamphotericin B Diaminoethyl Ether Amide (lnt-7)

Step a) Synthesis of C2'-Deoxymycosamine-N-FMOC-Amphotericin B

C2'-Deoxyamphotericin B is prepared as described in the following reference (and references cited therein): B. C. Wilcock, et ai. "C2'-OH of Amphotericin B Plays an Important Role in Binding the Primary Sterol of Human Cells but Not Yeast Cells" J. Am. Chem. Soc. 2013, 135, 8488-849.

C2'-Deoxyamphotericin B (0.242 g, 0.266 mmol, 1 eq) is dissolved in 6 ml_ dry DM F. FMOC-OSu (0.132 g, 0.31 9 mmol, 1 .2 eq) is added, and the reaction is stirred at RT for about 2 h. The reaction is poured into ~60 ml_ ethyl ether. The solids are filtered and washed with additional ether. The solids are dried under vacuum to yield the product with a molecular mass of 1 129.56.

Step b) Synthesis of C2'-Deoxymycosamine-N-FMOC-Amphotericin-FMOC-2-(2-aminoethox y)-ethyl- amide

C2'-Deoxymycosamine-N-FMOC-amphotericin B (0.287 g, 0.20 mmol, 1 eq) and FMOC-2-(2- aminoethoxy)-ethylamine HCI (0.1 09 g,0.30 mmol, 1 .5 eq) are dissolved in 6 mL dry DMF and cooled to 0°C. COM U (0.128 g, 0.30 mmol, 1 .5 eq) and DI PEA (0.091 g, 0.70 mmol, 3.5 eq) are added. The reaction is stirred at OC for about 1 hr. The product is precipitated with the addition of ~60 mL ethyl ether and MTBE. The solids are filtered and washed with additional ether. The solids are dried under vacuum to yield impure product. The solids are washed with methanol to yield product with a molecular mass of 1437.71 . Step c) Removal of FMOC Groups

The product of step b above (0.2 g, 0.139 mmol, 1 eq) is dissolved in 1 mL DMF and piperidin (41 uL, 36 mg, 0.42 mmol, 3 eq) is added. The reaction is stirred for about 30 minutes. The product i precipitated from the reaction and is filtered. The product is washed with methanol and dried under vacuum to give the product with a molecular mass of 993.58.

Synthesis of Amphotericin B Diaminoethyl Ether Amide (lnt-8)

Procedure 1

Step a) Synthesis of Mycosamine-N-FMOC-Amphotericin B

Amphotericin B (1 g, 1 .082 mmol, 1 eq) was dissolved in 20 ml_ dry DM F. FMOC-OSu (0.445 g, 1 .32 mmol, 1 .22 eq) was added and the reaction was stirred at RT for 2 h. The reaction was poured into ~200 ml_ ethyl ether. The solids were filtered and washed with additional ether. The solids were dried under vacuum to yield 1 .09 g product. LC/MS, 1 168.2 [M+23] ⁺ . H PLC (ELSD) T _R 3.395 min (1 00%). ¹ H NMR is consistent with the desired product indicating four methyl groups from 0.90 to 1 .2 ppm and the FMOC aryl protons from 7.3 to 7.925 ppm. ¹ H NM R (400 M Hz, DMSO-d6) δ ppm 0.91 (d, J=7.03 Hz, 3 H) 1 .04 (d, J=6.05 Hz, 3 H) 1 .1 1 (d, J=6.25 Hz, 4 H) 1 .17 (d, J=5.47 Hz, 3 H) 7.33 (dq, J=7.61 , 3.71 Hz, 1 H) 7.42 (t, J=7.42 Hz, 2 H) 7.73 - 7.79 (m, 2 H) 7.89 (d, J=7.42 Hz, 2 H) Step b) Synthesis of Mycosamine-N-FMOC-Amphotericin-FMOC-2-(2-aminoethoxy)-ethyl- amide

Mycosamine-N-FMOC-Amphotericin (2.27 g, 1 .98 mmol, 1 eq) and FMOC-2-(2-aminoethoxy)- ethylamine HCI (1 .079 g, 2.97 mmol, 1 .5 eq) were dissolved in 60ml_ dry DM F and cooled to 0 °C. COMU (0.932 g, 2.1 8 mmol, 1 .5 eq) and DI PEA (0.894 g, 6.9 mmol, 3.5 eq) were added. The reaction was stirred at 0C for 1 hr. The product was precipitated with the addition of ~600 mL ethyl ether and MTBE. The solids were filtered and washed with additional ether. The solids were dried under vacuum to yield 2.48 g of impure product, H PLC [ELSD] 98.6%. The solids were washed with methanol to yield 1 .9 g of product. HPLC T _R 4.125 min (99%). TY 2.8 g, 68%. Step c) Removal of FMOC Groups

The product of step b (1 .9 g, 1455 mmol, 1 eq) was dissolved in 8mL DM F and piperidine (387 uL, 334 mg, 3.92 mmol, 3 eq) was added. The reaction was stirred for 30 minutes. The product precipitated from the reaction and was filtered. The product was washed with methanol and dried under vacuum to give 0.602 g of product. LC/MS, 1 01 0 [M+23] ⁺ . H PLC [ELSD] T _R 2.601 min (1 00%).

Procedure 2

Step a) Synthesis of Mycosamine-N-FMOC-Amphotericin B

A solution of amphotericin B (5.00 g, 5.41 mmol) and Fmoc-OSu (2.01 g, 5.95 mmol) in DMF (1 00 mL) stirred at rt for 1 9 h. The reaction was then diluted with f-butyl methyl ether (1 .0 L), and the resulting mixture was filtered through a fritted funnel. The filter cake was then washed with additional f-butyl methyl ether (2 x 1 00 mL), dried under vacuum, and not purified further. Yield: b. // g as a yellow solid. ¹ H NM R (400 MHz, DMSO-d ₆ ) δ ppm 0.91 (d, J=7.08 Hz, 3 H) 1 .04 (d, J=6.35 Hz, 3 H) 1 .09 - 1 .13 (m, 4 H) 1 .17 (d, J=5.27 Hz, 3 H) 6.92 (d, J=8.44 Hz, 1 H) 7.28 - 7.38 (m, 2 H) 7.38 - 7.48 (m, 2 H) 7.76 (dd, J=7.47, 3.76 Hz, 2 H) 7.89 (d, J=7.52 Hz, 2 H). MS (M+Na) 1 169.4.

Step b) Synthesis of Mycosamine-N-FMOC-Amphotericin-FMOC-2-(2-aminoethoxy)-ethyl- amide

A solution of mycosamine-N-FMOC-amphotericin B (4.56 g, 3.98 mmol) in DMF (90 mL) was chilled to ~0°C in an ice/water bath, then charged with H ₂ NCH ₂ CH ₂ OCH ₂ CH ₂ NHFmoc-HCI (1 .59 g, 4.38 mmol), COM U (1 .87 g, 4.38 mmol), and DI PEA (1 .54 g, 1 1 .9 mmol). After warming to rt and stirring for 2 h, the reaction was diluted with f-butyl methyl ether (900 mL), and the resulting mixture was filtered through a fritted funnel. The filter cake was then washed with additional f-butyl methyl ether (2 x 100 mL), dried under vacuum, and not purified further. Yield: 6.05 g as a yellow solid. ¹ H NMR (400 MHz, DMSO- d ₆ ) 5 ppm 0.92 (d, J=6.98 Hz, 3 H) 1 .04 (d, J=6.35 Hz, 3 H) 1 .09 - 1 .13 (m, 4 H) 1 .1 6 (d, J=4.49 Hz, 3 H) 6.86 - 6.99 (m, 1 H) 7.20 - 7.26 (m, 1 H) 7.28 - 7.36 (m, 4 H) 7.37 - 7.46 (m, 4 H) 7.61 (d, J=7.52 Hz, 1 H) 7.71 - 7.79 (m, 4 H) 7.81 - 7.92 (m, 4 H). No distinct molecular ion observed by MS.

Step c) Removal of FMOC Groups to Yield lnt-8

A solution of the mycosamine-N-FMOC-amphotericin-FMOC-2-(2-aminoethoxy)-ethyl- amide from step b) above (6.05 g, th. 5.79 g, 3.98 mmol) in DM F (30 mL) was charged with piperidine (1 .02 g, 1 1 .9 mmol). After stirring at rt for 1 .25 h, the reaction was diluted with f-butyl methyl ether (300 mL), and the resulting mixture was filtered through a fritted funnel. The filter cake was then washed with additional f- butyl methyl ether (2 x 1 00 mL), dried under vacuum, and not purified further. Yield of lnt-1 : 4.12 g (>1 00% (1 02%)) as a yellow solid. ¹ H NM R (400 M Hz, DMSO-d ₆ ) δ ppm 0.91 (d, J=7.03 Hz, 3 H) 1 .04 (d, J=6.30 Hz, 3 H) 1 .08 - 1 .13 (m, 4 H) 1 .14 (d, J=5.91 Hz, 3 H), 8.00 - 8.07 (m, 1 H). MS (M+H) 101 0.4.Ξ Synthesis of Nystatin Diaminoethyl Ether Amide (lnt-9)

Step a) Synthesis of N-FMOC-Nystatin

Nystatin (0.246 g, 0.266 mmol, 1 .0 eq) is dissolved in 6 mL dry DMF. FMOC-OSu (0.132 g, 0.31 9 mmol, 1 .2 eq) is added, and the reaction is stirred at RT for about 2 h. The reaction is poured into ~60 mL ethyl ether. The solids are filtered and washed with additional ether. The solids are dried under vacuum to yield the product with a molecular mass of 1 147.57.

Step b) Synthesis of N-FMOC-Nystatin-FMOC-2-(2-aminoethoxy)-ethyl-amide

N-FMOC-Nystatin (0.230 g, 0.20 mmol, 1 eq) and FMOC-2-(2-aminoethoxy)-ethylamine HCI (0.109 g,0.30 mmol, 1 .5 eq) are dissolved in 6 mL dry DM F and cooled to 0 °C. COM U (0.1 28 g, 0.30 mmol, 1 .5 eq) and DI PEA (0.091 g, 0.70 mmol, 3.5 eq) are added. The reaction is stirred at 0 ^S C for about 1 hr. The product is precipitated with the addition of ~60 mL ethyl ether and MTBE. The solids are filtered and washed with additional ether. The solids are dried under vacuum to yield impure product. The solids are washed with methanol to yield product with a molecular mass of 1455.72.

Step c) Removal of FMOC Groups

The product of step b above (0.202 g, 0.139 mmol, 1 eq) is dissolved in 1 mL DM F and piperidine (41 uL, 36 mg, 0.42 mmol, 3 eq) is added. The reaction is stirred for about 30 minutes. The product is precipitated from the reaction and is filtered. The product is washed with methanol and dried under vacuum to give the product with a molecular mass of 101 1 .59.

Preparation of N-formyl-D-methionyl-D-leucyl-D-phenylalanine (lnt-10)

lnt-10

Step a) Preparation of N-Formyl-D-Methionine

D-Methionine (3g, 200mmol) was added into a solution of 1 1 ml_ of acetic anhydride and 4.5 ml_ of formic acid cooled in an ice-water bath. The mixture was stirred at room temperature for 4 hours, then the reaction solution was quenched with methanol and concentrated to provide the product which was used in the next step without purification.

Step b) Preparation of N-Formyl-D-Methionyl-D-Leucine Methyl Ester

(D)-N-Formyl-Methionine (1 .8g, 1 00 mmol) and D-leucine methyl ester (1 .5 g, 1 00 mmol) were dissolved in 20 ml_ THF, then HATU (3.8 g, 10 mmol) was added to the mixture and the reaction was stirred overnight. Subsequently, the mixture was diluted with ethyl acetate, washed with NaHC0 ₃ and NH ₄ CI solution and then brine. The organic phase was dried, concentrated and purified to provide 2.1 g of the title compound.

Step c) Preparation of (D, D, D)-fMLF methyl ester

The dipeptide from Step b (1 .5g, 50 mmol) was dissolved into 15 ml_ of a 1 :1 :1 mixture of (THF/H20/MeOH), then LiOH(140 mg, 1 .1 eq) was added and the solution was stirred at RT for 4 hours. The mixture was concentrated to dryness and the residue was dissolved in 20 ml_ DMF. D-Phenylalanine methyl ester (1 .0 g, 55 mmol, 1 .1 equi.) was added, followed by HATU (2 g, 52 mmol). The solution was stirred overnight and diluted with ethyl acetate, washed with NaHC0 ₃ and then NH ₄ CI solution and then brine. The resultant organic solution was dried and purified to provide 1 .5 g of (D, D,D)-fMLF methyl ester.

Step d) Hydrolysis of the methyl ester to give (D, D, D)-fMLF

(D,D,D)-fMLF methyl ester (1 .5 g, 34 mmol) was dissolved in 15 ml_ of a 1 :1 :1 mixture of (THF/H20/MeOH) and LiOH (120 mg, 1 .5 eq) was added. The solution was stirred at RT for 4 hours, the solvent was removed and the residue purified by reversed phase LC on an ISCO instrument to provide 960 mg of the product. ¹ H-NM R (300 M Hz, DMSO d6) : 12.68(1 H, S), 8.28(1 H, d, J=9 Hz), 8.12 (1 H, d, J=6Hz), 8.04(1 H, d, J=9Hz), 8.01 (1 H, s), 7.25(5H, m), 4.42 (1 H, m), 4.31 (1 H, m), 3.07 (1 H, dd, J1 =3Hz, J2=12Hz), 2.92 (1 H, dd, J1 =9Hz, J2=15Hz), 2.39 (1 H, t, J=6Hz), 1 .742H, m), 1 .55 (1 H, m), 1 .43 (2H, m), 0.88 (3H, d=6Hz), 0.83 (3H, d, J=6Hz) Step e) Formation of the N-Hydroxysuccinimide ester (lnt-1 0)

(D,D,D)-(N-formyl)-MLF (1 00 mg, 0.23 mmol, 1 .0 eq) was dissolved in 4 mL of dry DM F. DCC (50 mg, 0.24 mmol, 1 .05 eq) and N-hydroxysuccinimide (27 mg, 0.23 mol, 1 .0 eq) were added and the reaction was stirred overnight at ambient temperature. The product was used directly in subsequent reactions without purification

Synthesis of -Acetyl-L-Prolyl-Glycyl-L-Proline (lnt-11)

Step a) Synthesis of Z-Gly-Pro tert-Butyl Ester

A solution of Z-Gly (1 .087 g, 5.2 mmol), and N-methylmorpholine (0.571 mL, 5.2 mmol) in THF

(15mL), was cooled to -15°C, and treated with isobutyl chloroformate (0.679 mL, 5.2 mmol). After stirring for 10 min at -15°C, the mixture was treated with L-proline t-butyl ester-HCI (0.831 g, 4.0 mmol), and N- methylmorpholine (0.571 mL, 5.2 mmol). Work-up: the reaction mixture was diluted with ethyl acetate (50 mL), washed with 1 N HCL, 1 N NaHC0 ₃ , and brine, then dried with anhydrous Na ₂ S0 ₄ . This solution was filtered and concentrated to give 1 .45 g of a white solid. This material was used in the next step without further purification.

Step b) Synthesis of Gly-Pro tert-Butyl Ester

A mixture of Z-Gly-Pro tert-butyl ester (1 .45 g, 5.2 mmol), 5% Pd/C (0.700 g), and methanol (20 mL) were stirred under a H ₂ atmosphere for 1 h, at which time LCMS showed complete removal of the Z- group. The reaction was filtered to remove palladium on carbon and concentrated to give 0.913 g of a solid. This material was used in the next reaction without further purification.

Step c) Synthesis of N-Acetyl-L-Pro-Gly-L-Pro tert-Butyl Ester

A solution of N-acetyl-L-proline (0.754 g, 4.80 mmol), N-methylmorpholine (0.528 mL, 4.80 mmol), and TH F (15mL) was cooled to -15° C then treated with isobutyl chloroformate (0.627 mL, 4.80 mmol). The mixture was stirred at this temperature for 15 min then treated with Gly-Pro-OtBu ester prepared in Step b (0.913 g, 4.00 mmol). After stirring for 1 h, the mixture was concentrated and purified by reversed phase liquid chromatography (RPLC) using an Isco CombiFlash liquid chromatograph eluted with 10% to 1 00% acetonitrile and water, using 0.1 % formic acid as the modifier to give 0.439 g of product.

Step d) Synthesis of N-Ac-L-Prolyl-Glycyl-L-Proline (lnt-1 1 )

N-Ac-L-Pro-Gly-L-Pro-OtBu (0.439g, 1 .95 mmol), was dissolved in DCM (3.0 mL), and treated with TFA (3.0 mL) at RT. After 1 h, the mixture was concentrated and purified by reverse phase liquid chromatography (RPLC) using an Isco CombiFlash liquid chromatograph eluted with 1 0% to 1 00% acetonitrile and water, using 0.1 % formic acid as the modifier. The product containing fractions were combined, concentrated and dried to yield 0.240 g of product. ¹ H-NMR (300 M Hz, CDCI ₃ ) δ 7.54 (s, 1 4.64-4.51 (m,2H), 3.77-3.48 (m, 6H), 2.42-1 .92 (m, 1 1 H); LC/MS, 312.2 [M/2+H] ⁺ calculated 312.15.

Preparation of N-formyl-L-Methionyl-L-Leucyl-L-Phenylalanyl-L-Arginine Diaminoethylether Monoamide (l

Step a)

N-Boc-Arg (300mg, 1 .1 mmol) was added to 1 0ml THF, followed by FMOC-2-(2-aminoethoxy)- ethylamine HCI (400mg, 1 .1 mmol, 1 .0 eq), EDCI(250mg, 1 .2 eq) and HOBt (150mg, 1 .1 eq). The resultant mixture was cooled in an ice-water bath, then 0.3 ml NMM (2.5mmol, 2.5eq) was added and the reaction mixture was stirred overnight. The mixture was partitioned between 50 ml of ethyl acetate and 50 ml of brine. The organic layer was separated and washed with 1 0 ml_ of saturated NaHC03, ammonium chloride and brine respectively, then dried over anhydrous sodium sulfate. The organic layer was filtered and stripped to dryness. The resulting residue was treated with 5 ml TFA to remove the Boc protection group. The TFA solution was stirred for 0.5 hour and then concentrated and applied onto a C1 8 reversed phase ISCO chromatography column and eluted to provide 240 mg of the desired compound. ¹ H NMR (300 M Hz, CDCI3) δ ppm 8.61 (s, 1 H), 8.46(s, HO, 7.75(d, J=7.5Hz, 2H), 7.61 (d, J=7.2Hz, 2H), 7.40(t, J=7.5Hz, 2H), 7.30(t, J=7.5Hz, 2H), 4.40(m, 2H) 4.20~4.00(m, 3H) 3.35(m, 6H), 2.02(m, 1 H), 1 .60(m, 3H).

Step b)

N-Formyl-L-methionyl-L-leucyl-L-phenylalanine (200 mg, 0.46 mmol) and the product from Step a) above (200 mg, 0.41 mmol) were dissolved in 6 ml of dry DM F and cooled to 0 °C. COM U (250 mg, 0.5 mmol, 1 .1 eq) and NMM (0.25 ml, 2 mmol, 4 eq) were added. The reaction was stirred overnight. The DMF solution was applied to a reversed phase C1 8 ISCO chromatography column and eluted to provide 170 mg of the desired product. ¹ H NMR (300 M Hz, CDCI3) 5 ppm 8.37(s, 1 HO, 8.14(s, 1 H) 7.81 (d, J=6Hz, 2H) 7.67(d, J=9Hz, 2H) 7.43(t, J=6Hz, 2H) 7.35(d, J=9Hz, 2H) 7.23(m, 5H) 4.54~4.22(m, 4H), 3.52(m, 2H) 3.1 9~2.88(m, 6H) 2.52(m, 2H) 2.03(s, 3H) 1 .60~1 .2(m, 1 2H), 0.89(m, 6H).

Step c)

The product from Step b) above (90 mg, 0.1 0 mmol) and 0.05ml piperidine were added to 2 ml DMF. The resulant mixture was stirred for an additional 1 hour, and the reaction mixture was purified by C18 reversed phase chromatography (ISCO) to provide 60 mg of lnt-12. ¹ H NMR (300 MHz, CDCI3) δ ppm 8.20(s, 1 H), 7.27(m, 5H) 6.30d, J=10.4Hz, 1 H) 5.95(m, 1 H) 5.49-5.1 7(m, 2H) 4.56~4.28(m, 4H), 3.67~3.01 (m, 8H), 2.59~2.34(m, 6H), 2.1 0(s, 3H), 1 .81 -1 .70(m, 2H), 1 .35(m, 3H), 0.91 (d, J=6.3Hz, 3H), 0.85(d, J=6.3Hz, 3H) Preparation of N-Succinyl-L-Prolyl-Glycyl-L-Proline (lnt-13)

Step a)

DCC (430 mg, 2.1 mmol) was added into a solution of Boc-Gly-L-Pro dipeptide (540mg, 2.0 mmol) in 20 mis of THF at room temperature followed by 9-fluorenylmethanol (400mg, 2mmol) and 15 mg of DMAP. The reaction mixture was stirred overnight and then 20g of silica gel was added and the mixture was stripped to dryness. The residue was loaded into an empty sample-cartridge (ISCO) and the product was washed out gradually by increasing the ethyl acetate gradient in hexanes to provide 520 mg product . ¹ H NM R (300 MHz, CDCI ₃ ) δ ppm 7.78 (d, J7.5Hz, 2H), 7.55(d, J=7.2Hz, 2H), 7.40(t, J=7.5Hz, 2H), 7.31 (t, J=5.7Hz, 2H), 4.71 (m, 2H) 4.55 (ddd, J1 =3Hz, J2=6Hz, J3=20Hz, 1 H) 4.23 (t, J=5.4Hz, 1 H) 4.48 (dd, J=3Hz, 1 H), 3.90 (dd, J 1 =5.4Hz, J2=17Hz, 1 H), 3.60(dd, J1 =4.0Hz, J2=1 7Hz), 2.02 (2H, m), 3.26 (1 H, m), 1 .72 (m, 1 OH), 1 .47(1 H, b)

Step b)

The product from Step a) above (450mg, 1 .0 mmol) was added into 5 ml TFA, then the solution was stirred for 0.5 hour and was stripped to dryness. The resulting residue was redissolved in 15 ml THF, and then Boc-Pro-OH (230 mg, 1 .1 eq), EDCI (200 mg, 1 .05 eq) and HOBt (150mg, 1 .1 eq) were added. The resulting solution was cooled in an ice-water bath, then 0.4 ml NMM (3mmol, 3 eq) was added and the reaction mixture was stirred overnight. The mixture was partitioned into 50 ml ethyl acetate and 50 ml brine. The organic layer was separated and washed with 20 ml saturated NaHC0 ₃ , ammonium chloride and brine respectively. Then the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to dryness. The resultant residue was treated with 5 ml TFA again to remove the Boc protecting group. The TFA solution was stirred for 0.5 hour and then stripped of the TFA and applied to a reversed phase C1 8 column (ISCO) and chromatographed to provide 230 mg of the desired product. ¹ H NMR (300 M Hz, CDCI ₃ ) δ ppm 7.75 (d, J=7.2Hz, 2H), 7.56 (d, J=7.2Hz, 2H), 7.36 (m, 4H), 4.64 (m, 2H) 4.21 (m, 1 H), 3.93 (1 H, m), 3.34 (2H, m), 2.00-1 .65 (6H, m), 1 .30 (m, 2H)

lnt-13

Step c) Preparation of l nt-13

The tripeptide from Step b) above (200mg, 0.45 mmol) was dissolved in 1 0 ml DCM containing 1 ml pyridine. The solution was cooled in an ice-water bath, then succinic anhydride (200 mg, 2 mmol) was added to the cooled solution and stirred overnight. The reaction was stripped to dryness and purified by C18 reversed phase chromatography to provide 120mg of product. ¹ H NM R (300 MHz, CDCI3) δ ppm 7.83(d, J=6Hz, 2H, 7.63(d, J=6Hz, 2H), 7.43-7.41 (m, 4H), 4.64(m, 2H), 4.46(m, 2H), 4.36~4.22(3H, m) 4.0(1 H, m), 3.6(m, 3H), 3.36(m, 8H), 2.63(m, 3H), 2.33(m, 1 H), 2.1 8-1 .3(m, 4H).

Preparation of N-Fmoc-L-Threoninyl-N-Fmoc-L-Lysinyl-L-Prolyl Arginine Diaminoethyl ether Monoamide (lnt-14)

Step a)

N-Boc-L-Arginine (1 .4 g, 5 mmol), azido-PEG1 -amine, (700 mg, 0.55 mmol, 1 .1 eq ) and COMU (2.30g, 5mmol, 1 .0 eq) in 1 0 ml DMF was cooled in an ice-water bath, then 0.3 ml of NMM (2.5mmol, 2.5eq) was added. The reaction mixture was stirred overnight. The reaction was partitioned between 100 ml ethyl acetate and 1 00 ml brine. The organic layer was separated and washed with 30 ml saturated NaHC0 ₃ , ammonium chloride and brine respectively, then dried and concentrated by rotary evaporation. The resultant residue was treated with 5 ml TFA to remove the Boc protecting group. The TFA solution was stirred for 0.5 hour and then concentrated and the residue was applied to a C1 8 reversed phase ISCO column to provide 1 .2 g. of desired product. MS (M+H) ⁺ 387.

Step b)

2'Azidoethyloxaethyl argininylamide from Step a) above (300 mg, 1 mmol), Boc-Pro-OH (220 mg, 1 mmol), and COM U (430 mg, 1 .0 eq) in 10 ml DMF was cooled in an ice-water bath, then 0.3ml NMM (2.5mmol, 2.5eq) was added and the reaction mixture was stirred overnight. The mixture was partitioned between 50 ml ethyl acetate and 50 ml brine. The organic layer was separated and washed with 10 ml saturated NaHC0 ₃ , ammonium chloride and brine respectively, then dried and concentrated on a rotary evaporator. The resulting residue was purified by C1 8 reversed phase chromatography (ISCO) to provide 350 mg of the product. ¹ H NMR (300 M Hz, CDCI3) δ ppm 4.44 (t, J 1 =6.9Hz, J2=15Hz, 2H, 4.27 (m, 1 H), 3.68 (m, 1 H), 3.44(t, J=6.9Hz, 4H), 3.30 (m, 4H), 2.12-1 .89 (m, 4H) 1 .45(s, 9H), 1 .40 (t, j=7.2Hz, 4H)

Step c)

The product from Step b) above (300mg, 0.6mmol) was treated with 5 ml of TFA for 1 hour, then the solution was stripped to dryness. The residue was redissolved into 1 0 ml THF, followed by Boc- Lys(Fmoc)-OH (350mg, 0.6mmol) ), EDCI (1 60mg, 0.8mmol, 1 .3 eq) and HOBt (1 OOmg, 0.74mmol,

1 .2eq). The resulting mixture was cooled down by an ice-water bath, then 0.3ml NMM (2.5mmol, 4eq) was added and the reaction mixture was stirred overnight. I he reaction was partitioned between bOml ethyl acetate and 50 ml brine. The organic layer was separated and washed with 1 0 ml saturated NaHC0 ₃ , ammonium chloride and brine respectively, then dried over anhydrous sodium sulfate. The organic solution was filtered out and stripped to dryness. The resulting residue was treated with 5 ml TFA to remove the Boc protection group. The TFA solution was stirred for 0.5 hour and then concentrated and subjected to reversed phase C1 8 chromatography (ISCO) to provide 260 mg of the desired tripeptide. ¹ H NMR (300 M Hz, CDCI3) δ ppm 8.33 (s, 2H), 7.83(d, J=7.5Hz, 2H) 7.65(d, J=7.5Hz, 2H), 7.41 (t, J=7.2Hz, 2H) 7.32(t, J=7.5Hz, 2H) 4.50(m, 1 H) 4.36(m, 2H) 4.23(m, 2H), 3.73-3.1 8(8H, m), 2.25(m, 2H),2.09(m, 4H), 1 .86(m, 2H), 1 .54(m, 2H), 1 .40(m, 2H).

Step d)

The product from Step c) above (200mg, 0.27mmol), FMoc-Thr-OH (1 OOmg, 0.3mmol) ), EDCI (1 00 mg, 0.4 mmol, 1 .3 eq) and HOBt (70 mg, 0.5 mmol, 1 .3eq) in DM F. The resultant mixture was cooled in an ice-water bath, then 0.12 ml NMM (1 mol, 3eq) was added and the reaction mixture was stirred overnight. The mixture was partitioned between 50 ml of ethyl acetate and 50 ml of brine. The organic layer was separated and washed with 1 0 ml saturated NaHC0 ₃ , ammonium chloride and brine respectively and then dried over sodium sulfate. The organic layer was filtered and stripped to dryness and then applied to reversed phase C1 8 chromatography (ISCO) to provide the desired product. ¹ H NMR (300 M Hz, CDCI3) δ ppm 7.73(m, 4H), 7.55(m, 4H), 7.36(m, 4H), 7.26(m, 4H), 7.15(m, 2H), 4.33~4.17(m, 4H), 4.02(m, 1 H), 3.90(m, 1 H), 3.78(m, 1 H), 3.52~3.04(m, 1 0H), 2.37(m, 1 H), 2.05(2H, m), 1 .90(2H, m), 1 .64(4H, m), 1 .41 (m, 4H), 1 .27(m, 3H), 1 .21 (d, J=6Hz, 3H), 1 .13(m, 2H), 0.8(m, 2H).

Step e) Preparation of lnt-14

The azide (0.100 mmol) prepared as in Step d) above is treated with triphenylphosphine (0.1 05 mmol) in 5% aqueous THF (10 ml_s) and stirred until there is complete disappearance of starting azide. The mixture is concentrated and purified by reversed phase HPLC. The fractions that contain the compound with an exact mass of 1 030.5276 are pooled and lyophilized to provide the product.

Preparation of Mycosamine-N-FMOC-Amphotericin B (lnt-15)

A solution of amphotericin B (5.00 g, 5.41 mmol) and Fmoc-OSu (2.01 g, 5.95 mmol) in DMF (1 00 mL) stirred at rt for 1 9 h. The reaction was then diluted with f-butyl methyl ether (1 .0 L), and the resulting mixture was filtered through a fritted funnel. The filter cake was then washed with additional f-butyl methyl ether (2 x 1 00 mL), dried under vacuum, and not purified further. Yield: 5.77 g as a yellow solid. ¹ H NM R (400 MHz, DMSO-d ₆ ) δ ppm 0.91 (d, J=7.08 Hz, 3 H) 1 .04 (d, J=6.35 Hz, 3 H) 1 .09 - 1 .13 (m, 4 H) 1 .17 (d, J=5.27 Hz, 3 H) 6.92 (d, J=8.44 Hz, 1 H) 7.28 - 7.38 (m, 2 H) 7.38 - 7.48 (m, 2 H) 7.76 (dd, J=7.47, 3.76 Hz, 2 H) 7.89 (d, J=7.52 Hz, 2 H). MS (M+Na) 1 169.4.

Preparation of N-FMOC-2'-Deoxyamphotericin B (lnt-16)

A solution of 2'-deoxyamphotericin B (5.0 g, 5.5 mmol) and Fmoc-OSu (2.0 g, 6.0 mmol) in DM F (1 00 mL) is stirred at room temperature until a significant amount of the polyene starting material is consumed. The reaction is diluted with f-butyl methyl ether (1 .0 L), and the mixture is filtered through a fritted funnel. The filter cake is then washed with additional f-butyl methyl ether (2 x 1 00 mL), dried under vacuum to provide lnt-1 6 with an exact mass of 1 129.561 0.

Example 2: Synthesis of Compound 1

Compound 1

Amphotericin B diaminoethyl ether amide (lnt-8) (121 mg, 0.120 mmol, 1 .00 eq) was dissolved in 4mL dry DMF and cooled to 0 °C. (N-iso-BOC)-MLF-OSu (lnt-2) in DM F (0.1 mmol, 0.8 eq) was added dropwise. The reaction was stirred at 0 °C for 30 minutes. I he product was purified by semi-prep HPLC using a methanol/0.1 % acetic acid and water 0.1 % acetic acid gradient. The desired fractions were combined and evaporated under vacuum at 30 ^ to yield 45 mg of product as the acetate salt. LC/MS, 1500 [M-H] ^" . HPLC [ELSD] T _R 7.751 min (1 00%). ¹ H NMR (400 MHz, DMSO-d6) δ ppm 0.80 (d, J=6.44 Hz, 3 H) 0.86 (t, J=7.03 Hz, 8 H) 0.91 (d, J=7.22 Hz, 3 H) 1 .04 (d, J=6.44 Hz, 3 H) 1 .1 1 (d, J=6.44 Hz, 3 H) 1 .14 (d, J=5.86 Hz, 3 H) 1 .88 (s, 5 H) 2.02 (s, 3 H) 2.73 (s, 2 H) 2.89 (s, 2 H)

Example 3: Synthesis of Compound 2

Compound 2

Procedure!

Amphotericin B diaminoethyl ether amide (lnt-8) (1 00 mg, 0.0990 mmol, 1 .00 eq) was dissolved in 4 mL dry DMF and cooled to 0 °C. 1 mL of the fMLF-OSu (lnt-1 ) solution in DM F from Procedure 1 in lnt-1 was added dropwise. After stirring for 1 hr, HPLC indicated that compound had been consumed. The DM F was removed under vacuum at RT and the residue was dissolved in 1 0mL DMSO. The product was purified by semi-prep H PLC using a methanol/0.1 % acetic acid and water/0.1 % acetic acid gradient. The desired fractions were combined and evaporated under vacuum to yield 47 mg of product as the acetate salt. LC/MS, 1430 [M+H] ⁺ . HPLC [ELSD] T _R 6.727 min (1 00%). ¹ H NM R (400 MHz, DMSO-d6) δ ppm 0.80 (d, J=7.03 Hz, 3 H) 0.85 (d, J=6.64 Hz, 3 H) 0.91 (d, J=7.22 Hz, 3 H) 1 .04 (d, J=6.44 Hz, 4 H) 1 .1 1 (d, J=6.64 Hz, 4 H) 1 .15 (d, J=5.86 Hz, 4 H) 1 .84 - 1 .91 (m, 1 H) 2.01 (s, 3 H) 2.54 (s, 1 H) 5.1 5 - 5.28.

Procedure 2

fMLF-OSu (lnt-1 ) from Procedure 2 was charged with lnt-8 (2.00 g, 1 .98 mmol). After stirring at room temperature for 1 .75 h, the reaction was diluted with f-butyl methyl ether (650 mL), and the resulting mixture was filtered through a fritted funnel. The filter cake was then washed with additional f-butyl methyl ether (2 x 1 00 mL) and dried under vacuum. The crude product was then mixed with celite (10.8 g) and chromatographed on a 50 g Isco Gold RediSep C-18 reverse phase silica cartridge, eluting with a gradient from 99.9:0.1 H ₂ 0:HOAc to 99.9:0.1 MeOH:HOAc. The isolated product was then lyophilized from H ₂ 0 (~8 mL). Yield: 0.532 g as a yellow solid. ¹ H NM R (400 M Hz, DMSO-d ₆ ) δ ppm 0.80 (d, J=6.44 Hz, 3 H) 0.85 (d, J=6.54 Hz, 3 H) 0.91 (d, J=7.03 Hz, 3 H) 1 .04 (d, J=6.49 Hz, 3 H) 1 .07 - 1 .13 (m, 4 H) 1 .15 (d, J=5.86 Hz, 3 H) 2.01 (s, 3 H) 7.08 - 7.28 (m, 5 H) 7.89 (d, J=7.91 Hz, 1 H) 7.95 - 8.00 (m, 2 H) 8.01 (s, 1 H) 8.08 (d, J=8.00 Hz, 1 H) 8.27 - 8.35 (m, 1 H). MS (M+H) 1430.2. Analytical H PLC t _R = 7.265 min; purity = 1 00% by ELSD detection. Example 4: Synthesis of Compound 3

lnt-8 (26 mg, 0.03 mmol) and N ^E -fMLF-N ^a -FMOC-K (lnt-3) (20.3 mg, 0.03 mmol) were dissolved in 4 mL dry DM F and cooled to 0 °C. HATU (10.3 mg, 0.03 mmol, 1 .1 eq) was added followed by 2,4,6- lutidine (6.8 μί, 6.24 mg, 0.05 mmol, 2eq). After 30 minutes, piperidine (15 μί, 12.8 mg, 0.15 mmol, 5 eq) was added and the reaction was warmed to room temperature. After stirring for two hours, the product was purified by prep-H PLC using a 40% to 1 00% methanol in water (0.1 % acetic acid) gradient over 5 minutes. Yield 1 8.3 mg. LC/MS, 779.4 [(M+2H)/2] ⁺ . H PLC (UV at 220 nm), T _R 6.136 min (70.7%) ; 21 .6% different isomer. ¹ H NM R (400 M Hz, DMSO-d ₆ ) δ ppm 0.79 (d, J=6.44 Hz, 2 H) 0.84 (d, J=6.64 Hz, 2 H) 0.91 (d, J=7.03 Hz, 3 H) 1 .04 (d, J=6.25 Hz, 3 H) 1 .12 (dd, J=1 0.93, 6.25 Hz, 7 H) 2.02 (s, 2 H) 7.12 - 7.29 (m, 5 H) 7.80 - 7.87 (m, 1 H) 7.87 - 7.93 (m, 1 H) 8.15 - 8.21 (m, 1 H) 8.32 - 8.38 (m, 1 H).

Example 5: Synthesis of Compound 4

Step a) Preparation of N-FMOC Natamycin

Natamycin (266 mg, 0.4 mmol) was slurried in 15 mL dry DMF and FMOC-OSu (150 mg, 0.44 mmol, 1 .1 1 eq) was added. The reaction was stirred at room temperature until complete. The reaction was poured into 500 mL MTBE and filtered, then washed with additional MTBE. The product was dried under vacuum in the dark overnight. Yield 31 1 mg. LC/MS, 91 0.0 [M+Na] ⁺ . ¹ H NMR (400 M Hz, DMSO- d ₆ ) 5 ppm 1 .16 (d, J=4.64 Hz, 3 H) 1 .25 (d, J=5.98 Hz, 3 H) 1 .44 - 1 .66 (m, 2 H) 1 .84 (dd, J=1 1 .1 0, 3.90 Hz, 1 H) 1 .89 - 2.04 (m, 3 H) 2.1 9 (dd, J=23.67, 1 0.49 Hz, 1 H) 2.32 - 2.44 (m, 1 H) 2.74 - 2.78 (m, 1 H) 3.16 (d, J=5.74 Hz, 2 H) 3.23 (d, J=7.08 Hz, 1 H) 3.42 (t, J=8.54 Hz, 1 H) 3.56 - 3.65 (m, 1 H) 3.94 - 4.07 (m, 1 H) 4.08 - 4.1 8 (m, 1 H) 4.22 (br. s., 3 H) 4.32 - 4.4b (m, 2 H) 4.59 - 4. /5 (m, 2 H) 5.24 - 5.45 (m, 1 H) 5.54 - 5.69 (m, 1 H) 5.87 (dd, J=15.13, 8.91 Hz, 1 H) 6.14 (s, 8 H) 6.51 (dd, J=14.15, 1 1 .1 0 Hz, 1 H) 6.91 (d, J=8.79 Hz, 1 H) 7.28 - 7.38 (m, 2 H) 7.42 (t, J=7.32 Hz, 2 H) 7.76 (dd, J=7.14, 3.97 Hz, 2 H) 7.89 (d, J=7.57 Hz, 2 H)

Step b) Coupling and Deprotection

N-FMOC Natamycin prepared in Step a) (100 mg, 0.1 1 mmol) was dissolved in 5 ml_ dry DMF and cooled to 0 °C. fMLF-Diaminoethylamide (lnt-5) (59 mg, 0.1 1 mmol, 1 eq) was added. COM U (48 mg, 0.1 1 mmol, 1 eq) was added, then DI PEA (41 uL, 03.5 mg, 0.24 mmol, 2.1 eq) was added. The reaction was stirred at 0 °C for two hours until N-FMOC Natamycin was consumed. Piperidine (325 uL, 6.5% V/V) was added and the reaction was stirred at 0°C for 20 minutes. The reaction was quenched with 500 uL acetic acid and the product was purified by prep-H PLC using a 30% to 1 00% methanol in water (0.1 % acetic acid) gradient over 5 minutes. Yield 14 mg. LC/MS, 1 1 71 .2 [M+H] ⁺ . HPLC (UV at 220 nm), T _R 6.01 0 min (93%). HPLC (ELSD), T _R 6.156 min (1 00%). ¹ H NM R (400 MHz, DMSO-d ₆ ) δ ppm 0.79 (d, J=6.48 Hz, 3 H) 0.85 (d, J=6.59 Hz, 3 H) 1 .14 (d, J=6.04 Hz, 4 H) 1 .21 - 1 .28 (m, 3 H) 1 .33 - 1 .44 (m, 3 H) 1 .44 - 1 .63 (m, 4 H) 1 .67 - 1 .79 (m, 1 H) 1 .93 (s, 3 H) 2.19 (dd, J=21 .1 9, 10.54 Hz, 1 H) 2.33 (dd, J=9.44, 2.53 Hz, 1 H) 2.39 (d, J=2.42 Hz, 3 H) 2.74 (d, J=8.02 Hz, 1 H) 2.79 - 3.05 (m, 5 H) 3.08 - 3.20 (m, 2 H) 3.22 (dd, J=7.63, 1 .70 Hz, 2 H) 3.25 - 3.36 (m, 4 H) 3.39 (dd, J=1 1 .42, 5.71 Hz, 3 H) 3.59 (d, J=2A2 Hz, 1 H) 4.05 (td, J=10.57, 4.56 Hz, 2 H) 4.13 (t, J=1 0.27 Hz, 2 H) 4.20 (d, J=10.65 Hz, 1 H) 4.24 (s, 2 H) 4.25 - 4.33 (m, 2 H) 4.36 - 4.53 (m, 3 H) 4.60 - 4.72 (m, 2 H) 5.54 - 5.67 (m, 1 H) 5.85 (dd, J=15.26, 8.68 Hz, 1 H) 5.97 - 6.33 (m, 9 H) 6.50 (dd, J=14.39, 1 0.65 Hz, 1 H) 7.1 1 - 7.29 (m, 5 H) 7.97 (d, J=8 3 Hz, 1 H) 7.99 - 8.07 (m, 3 H) 8.15 (d, J=8 3 Hz, 1 H) 8.33 (d, J=8.35 Hz, 1 H).

Example 6: Synthesis of an Acetate Salt of Compound 5

Compound 5

Step a) Coupling of lnt-8 and lnt-5

A solution of lnt-8 (0.1 00 g, 0.0990 mmol) in DMF (3.0 mL) was charged with lnt-5 (0.078 g, 0.099 mmol), HATU (0.040 g, 0.1 0 mmol), and 2,4,6-collidine (0.024 g, 0.20 mmol). After stirring at rt for 1 .3 h, the reaction was diluted with f-butyl methyl ether (30 mL), and the resulting mixture was filtered through a fritted funnel. The filter cake was then washed with additional f-butyl methyl ether (2 x 20 mL), dried under vacuum, and not purified further. Yield: 0.208 g (>100% (1 1 8%)) as a yellow solid. MS

(M+H+Na)/2 901 .4. Step b) Removal of FMOC Protecting Group to Give Compound 5

A solution of the coupling product from Step a) above (theoretical 0.1 76 g, 0.989 mmol) in DM F (3.0 mL) was charged with piperidine (0.025 g, 0.30 mmol). After stirring at rt for 2 h, the crude reaction was purified directly by semi-prep HPLC, eluting with a gradient from 30:70 A:B to 100% B (A = 99.9:0.1 H ₂ 0:HOAc; B = 99.9:0.1 MeOH:HOAc). The isolated product was then lyophilized from 1 :1 H ₂ 0:CH ₃ CN (8 mL). Yield: 0.037 g as a yellow solid. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 0.80 (d, J=6.49 Hz, 3 H) 0.85 (d, J=6.54 Hz, 3 H) 0.91 (d, J=6.88 Hz, 3 H) 1 .04 (d, J=6.39 Hz, 3 H) 1 .09 - 1 .12 (m, 4 H) 1 .14 (d, J=6 0 Hz, 3 H) 2.01 (s, 3 H) 7.1 1 - 7.33 (m, 5 H) 7.91 - 7.97 (m, 1 H) 7.99 - 8.04 (m, 2 H) 8.01 (s, 1 H) 8.06 - 8.1 1 (m, 1 H) 8.1 1 - 8.1 7 (m, 1 H) 8.32 - 8.38 (m, 1 H). MS (M+H+H)/2 779.4. Analytical H PLC t _R = 5.931 min; purity = 97% by ELSD detection.

Example 7: Synthesis of Compound 6

Amphotericin-B-2-(amino)-ethyl-amide (lnt-6) (15 mg, 0.0138 mmoles) was dissolved in 10 mL dry DMF and cooled to 0 °C. A solution of (N-isobutyloxycarbonyl)MLF-OSu in DMF (lnt-2) (0.0138 mmoles) was added dropwise until LC/MS indicated that no starting material remained. The DMF was removed under vacuum and the product was purified by prep-H PLC using a 30% to 1 00% methanol in water/0.1 % acetic acid gradient. Yield 1 1 mg, 54%. LC/MS, 1458.4 LM+HJ ^' . Ή NM H (400 M Hz, UMSU- d ₆ ) 5ppm 0.80 (d, J=6.44 Hz, 4 H) 0.86 (t, J=6.64 Hz, 1 0 H) 0.91 (d, J=7.03 Hz, 3 H) 1 .04 (d, J=6.25 Hz, 3 H) 1 .12 (dd, J=12.10, 6.25 Hz, 6 H) 2.02 (s, 2 H) 2.54 (s, 20 H) 7.09 (t, J=6.35 Hz, 1 H) 7.14 (d, J=6.64 Hz, 1 H) 7.1 9 (d, J=7.42 Hz, 3 H) 7.24 (d, J=6.44 Hz, 2 H) 7.32 (d, J=8.00 Hz, 1 H) 7.90 (d, J=8.79 Hz, 2 H) 8.00 (d, J=8.79 Hz, 1 H) 8.24 (t, J=7.20 Hz, 1 H).

Example 8: Preparation of Compound 7

Compound 7

fMLF-OSu (lnt-1 ) (0.053 g, 0.1 mmol, 1 .0 eq) in 1 ml_ of dry DM F is charged with lnt-7 (0.099 g,

0.10 mmol, 1 .0 eq). The mixture is stirred at room temperature for about 2 h and the reaction is diluted with f-butyl methyl ether (about 60 ml_) and the resulting mixture is filtered through a fritted funnel. The filter cake is washed with additional f-butyl methyl ether (about 2 x 10 ml_) and dried under vacuum. The crude product is then purified by reverse phase H PLC. The fractions containing the desired product are pooled and lyophilized to give a product with a molecular mass of 1412.77.

Example 9: Preparation of Co

Compound 8

fMLF-OSu (lnt-1 ) (0.053 g, 0.1 mmol, 1 .0 eq) in 1 mL of dry DM F is charged with lnt-9 (0.1 01 g, 0.10 mmol, 1 .0 eq). The mixture is stirred at room temperature for about 2 h and the reaction is diluted with f-butyl methyl ether (about 60 mL) and the resulting mixture is filtered through a fritted funnel. The filter cake is washed with additional f-butyl methyl ether (about 2 x 10 mL) and dried under vacuum. The crude product is then purified by reverse phase H PLC. The fractions containing the desired product are pooled and lyophilized to give a product with a molecular mass of 1503.83.

Example 10: Preparation of Compound 9

Step a) Preparation of N-Succinyl-L-prolyl-glycinyl-L-prolyl N-hydroxysuccinyl Ester

N-Succinyl-L-prolyl-glycinyl-L-prolyl (lnt-13) (63 mg, 0.1 0 mmol, 1 eq) was dissolved in 2 ml_ of dry DMF. DCC (22 mg, 0.1 1 mmol, 1 .05 eq) and N-hydroxysuccinimide (1 2 mg, 0.1 mol, 1 eq) were added and the reaction mixture was stirred overnight at RT. The product was used directly in subsequent reactions without purification.

Step b)

The solution containing N-succinyl-L-prolyl-glycinyl-L-prolyl N-hydroxysuccinyl ester

from Step a) above was charged with amphotericin-aminoethoxy-ethyl amide (lnt-6) (100 mg, 0.1 mmol) in 2 ml DM F. After stirring at room temperature for 4 hours, 0.05 ml piperidine was added and the resulting mixture was stirred for another 1 hour, and then purified by Gilson H PLC and the fractions concentrated by lyophilization to provide 8 mg of the product as a yellow solid. ¹ H NMR (300 M Hz, DMSO-d ₆ ) δ ppm 0.90 (d, J=6.9 Hz, 3 H) 1 .01 (d, J=7.2 Hz, 3 H) 1 .07 (d, J=8 Hz, 3 H), 1 .15(d, J=4.5Hz, 3H) 7.69(s, 1 H) 7.91 (s, 1 H) 8.17 (s, 1 H), 8.36 (s, 1 H). MS 681 [M+H]72.

Example 11. Preparation of Compound 10

Step a) Preparation of NAc-L-prolyl-glycyl-L-proline N-hydroxysuccinimide ester

NAc-L-prolyl-glycyl-L-proline (63 mg, 0.20 mmol, 1 eq) was dissolved in 4 ml_ of dry DM F. DCC (42 mg, 0.21 mmol, 1 .05 eq) and N-hydroxysuccinimide (24 mg, 0.21 mol, 1 eq) were added and the reaction was stirred overnight at RT. The product was used directly in subsequent reactions without purification

Step b)

A solution of NAc-L-prolyl-glycyl-L-proline N-hydroxysuccinimide ester prepared in Step a) above was charged with amphotericin-aminoethoxy-ethyl amide (lnt-8) (200 mg, 0.2 mmol) in 4 ml of DM F. After stirring at room temperature for 4 hour, the mixture was purified by Gilson H PLC and the product - containing pooled fractions lyophilized to provide 18 mg ot the desired compound as a yellow solid. Ή NMR (300 M Hz, DMSO-d ₆ ) δ ppm 0.91 (d, J=6. Hz, 3 H) 1 .04 (d, J=5A Hz, 3 H) 1 .1 1 (d, J=6.3 Hz, 3 H), 1 .15(d, J=6Hz, 3H) 1 .97(s,3H) 7.87(s, 1 H), 8.20(s, 1 H), 7.98(s, 1 H). MS 652 [M+H]72.

Example 12. Preparation of Compound 11

Example 13. Preparation of Compound 12

D,D, D-fMLF-OSu (lnt-10) (0.23 mmol) in 4 ml_s of dry DMF, prepared according to the procedure described in Example 1 for lnt-1 0, was charged with amphotericin B aminoethoxyethyl amide (lnt-8) (200 mg, 0.2 mmol). After stirring at room temperature for 4 hours, the reaction was purified by Gilson H PLC and lyophilized to provide 32 mg of the product as a yellow solid. ¹ H NMR (300 M Hz, DMSO-d ₆ ) δ ppm 8.34 (m, 3H) 8.05 (m, 1 H) 8.01 (s, 1 H) 7.22 (m, 5 H) 2.01 (s, 3 H) 1 .15(d, J=5.1 Hz, 3H1 .1 1 (d, J=6 Hz, 3 H), ) 1 .04 (d, J=6.0 9 Hz, 3 H) 0.90 (d, J=6.9 Hz, 3 H) 0.74 (d, J=6.3 Hz, 3 H) 0.70 (d, J=7.5 Hz, 3 H). MS 715 [M+H]72.

In a procedure analogous to Step b) in the preparation of Compound 4 in Example 5 but starting with lnt-14 (0.1 00 mmol) and lnt-15 (0.1 00 mmol), amphotericin B-diamidoethylether-RPKT conjugate with a molecular weight of 1492.82 may be prepared.

Example 15. Preparation of Compound 14

In a procedure analogous to the preparation of Compound 13 in the Example above but starting with lnt-14 and lnt-1 6, 2'-deoxyamphotericin B-diamidoethylether-RPKT conjugate with a molecular weight of 1476.82 may be prepared.

In a procedure analogous to the preparation of Compound 13 (Example 14) but starting with I nt- 12 and l nt-15, amphotericin B-diamidoethylether-RFLfM conjugate with a molecular weight of 1585.96 may be prepared.

Example 17. Preparation of Compound 16

Compound 16

N-Formyl-L-methionyl-L-leucinyl-L-4-pyridinylalanine (lnt-1 7) (50 mg, 0.1 1 mmol), COMU (50 mg, 0.12 mmol, 1 .1 eq) and NMM (0.12ml, 1 mmol, 2 eq) were added to 4 ml DM F cooled in an ice-water bath, followed by amphotericin-aminoethoxy-ethyl amide (lnt-8) (200 mg, 0.2 mmol) in 4 ml DM F. After stirring at room temperature overnight, the reaction was purified by Gilson H PLC and lyophilized to provide 1 8 mgs of the desired product as a yellow solid. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ ppm 8.42 (d, J=4.2Hz, 2H) 8.30 (s, 2H) 8.01 (s, 1 H) 7.22 (d, J=4.2Hz, 2 H) 2.02 (s, 3 H) 1 .15(d, J=6Hz, 3H) 1 .1 1 (d, J=6 Hz, 3 H), ) 1 .04 (d, J=5A, 3H) 0.92 (d, J=6.3 Hz, 3 H) 0.86 (d, J=6.3 Hz, 3 H) 0.80 (d, J=6.3 Hz, 3 H). MS 715 [M+H]72.

Example 18: Antifungal Activity of Compounds

Test Organisms

The test organisms consisted of strains from the Micromyx collection. Reference isolates were originally received from the American Type Culture Collection (ATCC; Manassas, VA). Organisms received at Micromyx were initially streaked for isolation on Sabouraud dextrose or potato dextrose agar. Colonies were picked by swab from the medium and resuspended in the appropriate broth containing cryoprotectant. The suspensions were aliquoted into cryogenic vials and maintained at -80 ^S C.

Prior to testing, Candida isolates were streaked from the frozen vials on Sabouraud dextrose agar. The yeast isolates were incubated at overnight at 35 ^S C before use. The fungal isolates were incubated at least 7 days on Sabouraud dextrose agar slants at 35 ^S C before harvesting.

Test Media

Isolates were tested in RPM I medium (Catalog No. SH3001 1 .04; Lot No. AWA92121 B; HyClone

Labs, Logan, UT) which was prepared according to CLSI guidelines. The pH of the medium was adjusted to 7.0 with 1 N NaOH. The medium was sterile filtered using a 0.2 μιη PES filter and stored at 4°C until used. Minimal Inhibitory Concentration (MIC) Assay Procedure

The M IC assay method employed automated liquid handlers to conduct serial dilutions and liquid transfers. Automated liquid handlers included the Multidrop 384 (Labsystems, Helsinki, Finland), Biomek 2000 and Biomek FX (Beckman Coulter, Fullerton CA). The wells in columns 2-12 in standard 96-well microdilution plates (Costar 3795) were filled with 150 μΙ of the correct diluent (50% DMSO for investigational compounds, 1 00% DMSO for comparator compounds). These would become the 'mother plates' from which 'daughter' or test plates would be prepared. Stocks were diluted to 40X the desired top concentration in the test plates in the indicated solvent, and 300 μΙ_ ot the 40X stock was dispensed into the appropriate well in Column 1 of the mother plates. The Biomek 2000 was used to make serial serial 2-fold dilutions through Column 1 1 in the "mother plate". The wells of Column 12 contained no drug and served as the organism growth control wells.

The daughter plates were loaded with 1 85 μΙ_ per well of RPM I described above using the

Multidrop 384. The daughter plates were prepared using the Biomek FX which transferred 5 μΙ_ of drug solution from each well of a mother plate to the corresponding well of the daughter plate in a single step.

A standardized inoculum of each organism was prepared. For Candida, colonies were picked from the streak plate and a suspension was prepared in RPMI medium equal to a 0.5 McFarland standard, then diluted 1 :1 00 in RPM I and transferred to compartments of sterile reservoirs divided by length (Beckman Coulter). For the Aspergillus isolates, previously prepared and quantitated suspensions were used to make dilutions in RPMI to reach 20X the final concentration. These dilutions were also transferred to compartments of sterile reservoirs divided by length (Beckman Coulter). The final concentration of the Aspergillus isolates was approximately 0.2-2.5 x 10 ⁴ CFU/mL.

The Biomek 2000 was used to inoculate the plates. Daughter plates were placed on the Biomek

2000 work surface reversed so that inoculation took place from low to high drug concentration. The Biomek 2000 delivered 1 0 μΙ_ of standardized inoculum into each well. Thus, the wells of the daughter plates ultimately contained 1 85 μΙ_ of RPMI, 5 μΙ_ of drug solution, and 10 μΙ_ of inoculum. The final concentration of DMSO in the test well was 2.5% for the evaluated comparators and 1 .25% for the investigational agents.

Plates were stacked 3 high, covered with a lid on the top plate, placed into plastic bags, and incubated at 35 ^S C for approximately 24-48 hr prior to reading. Plates were read when inoculum was confluent in growth wells. Plates were viewed from the bottom using a plate viewer. An un-inoculated solubility control plate was observed for evidence of drug precipitation. MICs were read where visible growth of the organism was inhibited (Table 1 ).

Table 1 : Antifungal Activity for Select Compounds

MIC=minimum inhibitory concentration; M ECs shown against Aspergillus spp. for caspofungin acetate Example 19. Antifungal Activity of Compounds

MIC and MEC assays were performed according to CLSI broth microdilution guidelines (M27-A3, M27- S4, and M38-A2) with the exception of using a 1 00 μΙ_ assay volume and preparing stock compounds at 50X final concentration. Briefly, starting solutions of all antifungal agents were prepared in 1 00% DMSO. Stock concentrations were made at 50X the highest final assay concentration and serially diluted 2-fold, 12 times in a 96-well PCR plate (VWR 83007-374). Candida and Aspergillus suspensions from

Sabouraud dextrose agar plate cultures were prepared in 0.85% saline at 0.5 McFarland standard (~0.1 OD ₅₃₀ nm). Candida suspensions were diluted 1 :500 in RPMI (M P Biomedicals, cat no. 1 060124;

buffered with MOPS and adjusted with NaOH to pH 7.0) to a concentration of -0.5-2.5 x 1 0 ³ CFU/mL and Aspergillus suspensions were diluted 1 :50 in RPMI to -0.4-5 x 10 ⁴ CFU/mL final concentration. 98 μΙ_ of each cell suspension in RPMI were added to test wells in a 96-well assay plate (Costar cat. no. 3370). A Beckman Multimek 96 liquid handling robot was used to dispense 2 μΙ_ of each 50X stock compound into the plate containing 98 μΙ_ of each strain in RPMI (2% final solvent concentration). Plates were shaken then incubated at 35 °C for 24-48 h prior to reading. M IC values were read visually at 50% growth inhibition for echinocandins (24 h) and at 1 00% growth inhibition for amphotericin B (48 h). MEC values (24 h) were read with the aid of a microscope at the lowest echinocandin concentration where rounded/compact hyphal morphology was observed. The data are shown in Tables 2a and 2b.

Table 2a: Antifungal Activity for Select Compounds

Table 2b: Antifungal Activity for Select Compounds

Example 20: Chemotactic Activity of Compounds

Human Neutrophil Transwell Migration Assay

Method 1 .

Neutrophils were purified from human peripheral blood utilizing PolymorphPrep. Red blood cells were lysed with a lysis buffer and the cells were washed, counted and resuspended in assay buffer. Neutrophils (approximately 50,000 to 100,000 in number) were placed over 5-micon pore sized transwell plates in 24-well format with test compounds in the bottom chamber of each well. A negative control of media alone in the bottom chamber was set up in triplicate. A positive control where the neutrophils were added directly to the bottom well was set up in triplicate.

The plates were incubated at 37 ^s C for 45 minutes to allow for migration. After this time, migration was confirmed visually and then an aliquot (1 /6 ^th of total cells) from each well was taken for ATP detection via luminescence with ATPIite (PerkinElmer) to quantify the number of migrated cells. The averaged reading for random migration detected in the negative control was subtracted from all readings to obtain the corrected migration number. These numbers were then divided by the averaged reading from the positive control to get the % Neutrophil Migration of the maximum migration possible. Method 2.

Neutrophils were purified from human peripheral blood utilizing PolymorphPrep. Red blood cells were lysed with a lysis buffer and the cells were washed, counted and resuspended in assay buffer. Neutrophils (approximately 50,000 to 100,000 in number) were placed over 5-micon pore sized transwell plates in 96-well format with test compounds in the bottom chamber of each well. Each test compound was set up in duplicate. A negative control of media alone in the bottom chamber was set up in replicates of six. A positive control where the neutrophils were added directly to the bottom well was set up in replicates of six.

The plates were incubated at 37 ^s C for 45 minutes to allow for migration. After this time, migration was confirmed visually and the ATP levels were detected via luminescence with ATPIite (PerkinElmer) to quantify the number of migrated cells. The averaged (average) reading for random migration detected in the negative control was subtracted from all readings to obtain the corrected migration number. These numbers were then divided by the averaged reading from the positive control to get the % Neutrophil Migration of the maximum migration possible at the indicated nanamolar (nM) concentrations of Compound (Table 3).

Table 3: Neutrophil Migration for Select Compounds

Other Embodiments

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations following, in general, the principles described herein and including such departures from the present disclosure come within known or customary practice within the art to which the disclosure pertains and may be applied to the essential features hereinbefore set forth.

All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. The disclosure herein incorporates by reference U.S. Provisional Application No. 61 /982, 134 in its entirety.