Thiohydantoin derivatives and uses thereof

Abstract

The present invention provides novel compounds (e.g., compounds of Formula (I)), and pharmaceutically acceptable salts, solvates, hydrate, polymorphs, co-crystals, tautomers, stereoisomers, isotopically labeled derivatives, prodrugs, and compositions thereof. Also provided are methods and kits comprising the inventive Compounds, or compositions thereof, for treating and/or preventing a fungal or protozoan infection, inhibiting the activity of a fungal or protozoan enzyme, killing a fungus or protozoan, or inhibiting the growth of a fungus or protozoan. The fungus may be a Candida species, Sacchawmyces species, or other pathogenic fungal species. The compounds of the invention may inhibit the activity of fungal or protozoan mitochondrial phosphate carrier protein.

Claims

1. An inhibitor of fungal or protozoan mitochondrial phosphate carrier protein Formula (I): ##STR00146## or a pharmaceutically acceptable salt thereof, wherein: ##STR00147## R.sup.A is halogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, OR.sup.A1, N(R.sup.A1).sub.2, SR.sup.A1,CN, SCN, C(NR.sup.A1)R.sup.A1, C(NR.sup.A1)OR.sup.A1, C(NR.sup.A1)N(R.sup.A1).sub.2, C(O)R.sup.A1, C(O)OR.sup.A1, C(O)N(R.sup.A1).sub.2, NO.sub.2, NR.sup.A1C(O)R.sup.A1, NR.sup.A1C(O)OR.sup.A1, NR.sup.A1C(O)N(R.sup.A1).sub.2, OC(O)R.sup.A1, OC(O)OR.sup.A1, OC(O)N(R.sup.A1).sub.2, or a nitrogen protecting group when attached to a nitrogen atom; each instance of R.sup.A1 is independently hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom; m is 0; n is 1 or 2; R.sup.C is C(O)R.sup.C1, C(O)OR.sup.C1, C(O)N(R.sup.C1).sub.2, S(O).sub.2R.sup.C1, S(O).sub.2OR.sup.C1, or S(O).sub.2N(R.sup.C1).sub.2; and each instance of R.sup.C1 is independently hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, or an oxygen protecting group when attached to an oxygen atom; provided that the Compound of Formula (I) is not ##STR00148##

2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R.sup.A is halogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, OR.sup.A1, or CN.

3. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein n is 1.

4. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein n is 2.

5. The compound of claim 1, wherein the compound is of the formula: ##STR00149## or a pharmaceutically acceptable salt thereof.

6. The compound of claim 1, wherein the compound is of the formula: ##STR00150## or a pharmaceutically acceptable salt thereof.

7. The compound of claim 1, wherein the compound is of the formula: ##STR00151## ##STR00152## ##STR00153## ##STR00154## or a pharmaceutically acceptable salt thereof.

8. A pharmaceutical composition comprising a compound of claim 1, or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable excipient.

9. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R.sup.A is F, Cl, Br, CH.sub.2F, CHF.sub.2, CF.sub.3, Me, Et, Pr, Bu, Ph, OMe, OEt, OPr, OBu, or CN.

10. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R.sup.A is F, Cl, or CN.

11. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R.sup.A is F.

12. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R.sup.A1 is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, or an oxygen protecting group.

13. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R.sup.C is C(O)Me, C(O)Et, C(O)Pr, C(O)Bu, C(O)OMe, C(O)OEt, C(O)OPr, C(O)OBu, C(O)OBn, C(O)OPh, C(O)NH.sub.2, C(O)NHMe, C(O)N(Me).sub.2, S(O).sub.2Me, S(O).sub.2CF.sub.3, S(O).sub.2OMe, S(O).sub.2OEt, S(O).sub.2NH.sub.2, S(O).sub.2NHMe, or S(O).sub.2N(Me).sub.2.

14. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R.sup.C is C(O)OMe, C(O)OEt, or C(O)OPr.

15. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R.sup.C is C(O)OEt.

16. The compound of claim 1, wherein the compound is of the formula: ##STR00155## or a pharmaceutically acceptable salt thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows some known inhibitors of fungal mitochondria oxidative phosphorylation. Ilicicolin H (1) has demonstrated 100-fold selectivity for fungal mitochondria in biochemical assays but displays moderate toxicity against HeLa cells. Antimycin (2) and funiculosin (3) are potent, but non-selective, inhibitors of mitochondrial function.

(2) FIG. 2 shows the stability of the probe compound I-B-4 (ML316) and compound I-A-10 (CID56604835) in PBS Buffer (pH 7.4, 23 C.).

(3) FIGS. 3A-B show the dose response curves for initial hit compound I-A-1 (CID3889161). Compound I-A-1 was used over a range of concentrations up to 26 M in the primary assay and secondary assays. Dose curves were generated with Genedata Condeseo and show normalized percent activity for the individual doses. C. albicans CaCi-2 growth inhibition (AID 623899), IC.sub.50=0.3 M (FIG. 3A); Murine 3T3 fibroblasts growth inhibition (AID 602394), IC.sub.50>26 M (FIG. 3B).

(4) FIG. 4 shows the critical path for the probe development.

(5) FIGS. 5A-C show the C. albicans and fibroblast dose response curves for probe compound I-B-4 (ML316). C. albicans CaCi-2 growth inhibition (AID 623899), IC.sub.50=0.04 M (FIG. 5A); C. albicans CaCi-17 growth inhibition (AID 623897), IC.sub.50=1.0 M (FIG. 5B); Murine 3T3 fibroblasts growth inhibition (AID 602394), IC50>26 M (FIG. 5C); Dose curves were generated with Genedata Condeseo and show normalized percent activity for the individual doses.

(6) FIGS. 6A-D show the S. cerevisiae and C. glabrata dose response curves for probe compound I-B-4 (ML316). S. cerevisiae on glycerol growth inhibition (AID 623969), IC.sub.50=0.004 M (FIG. 6A); S. cerevisiae on glucose growth inhibition (AID 623971), IC50>3.0 M (FIG. 6B); C. glabrata on glycerol growth inhibition (AID 623965), IC.sub.50=0.011 M (FIG. 6C); C. glabrata on glucose growth inhibition (AID 623967), IC.sub.50>3.0 M (FIG. 6D). Dose curves were generated with Genedata Condeseo and show normalized percent activity for the individual doses.

(7) FIG. 7 depicts the SAR (structure-activity relationship) strategy based on the initial hit compound I-A-1 (CID3889161). Key SAR findings for each site of diversification are provided in italics.

(8) FIG. 8 shows the inhibitory activities of compound I-B-4 (ML316) against C. albicans CaCi-2 growth in absence and presence of fluconazole (FCZ). IC.sub.50=120 nM for I-B-4 (ML316) alone. IC50=10 nM for I-B-4 (ML316) with FCZ.

(9) FIG. 9 shows the interaction of I-B-4 (ML316) with fluconazole against C. albicans CaCi-2 (FCZ-R). Strain I-B-4-R is a derivative of the CaCi-2 strain selected for resistance to I-B-4 (ML316). These data show that Candida from a fluconazole-resistant background that evolves resistance to I-B-4 (ML316) loses the ability to grow on glycerol media (indicative of a loss of respiration) and becomes hypersensitive to fluconazole (32 g/mL).

(10) FIG. 10 shows results of genome sequencing of three independent selections in C. albicans ATCC 200950 for resistance to I-B-4 (ML316); in all three selections, the same mutation in the MIR1 protein (N184T) emerged.

(11) FIG. 11 shows an alignment of the MIR1 protein sequences from different yeast species and in human (H. sapiens). The resistant mutation is N184T in the listed yeast species and corresponds to a particular amino acid in the human protein. The mutation confers resistance to I-B-4 (ML316).

(12) FIG. 12 shows the inhibitory activities of compound I-B-4 (ML316) against S. cerevisiae strain BY4743 and several derived strains. Data is provided for cells containing MIR1/MIR1, MIR1/mir1, MIR1/MIR1+CENMIR1, and the MIR1 resistant mutation N184T.

(13) FIG. 13 shows the growth of untransformed and transformed S. cerevisiae mir1+ cells in both glycerol and glucose media. The mutant cells were transformed with either the MIR1 (under the control of a TEF promoter) or the human SLC25A3 gene (under either the control of a TEF, GPD, or ADH promoter).

(14) FIG. 14 shows that compound I-B-4 (ML316) and a combination of I-B-4 (ML316) and fluconazole (FCZ) were cytotoxic to fluconazole-sensitive Candida albicans strain SC5314 (WT: wild type).

(15) FIG. 15 shows that compound I-B-4 (ML316) and a combination of I-B-4 (ML316) and fluconazole (FCZ) were cytotoxic to fluconazole-resistant Candida albicans strain CaCi-2.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

(16) Provided herein are novel thiohydantoin derivatives and uses thereof. In one aspect, the present invention provides compounds described herein. In certain embodiments, the compounds are of Formula (I). These compounds have been found to be antifungal agents, antiprotozoan agents, and/or chemosensitizers capable of reversing the resistance of a fungus to an antifungal agent (e.g., fluconazole) and/or resistance of a protozoon to an antiprotozoan agent. Without wishing to be bound by any particular theory, the provided compounds may inhibit the activity of fungal mitochondrial phosphate carrier protein. The invention also provides pharmaceutical compositions and kits comprising the compounds described herein. Also provided are methods of using the compounds described herein (e.g., compounds of Formula (I)), to treat and/or prevent a fungal or protozoan infection, kill a fungus or protozoon, and/or inhibit the growth of a fungus or protozoon. In certain embodiments, the fungus described herein is a Candida species. In certain embodiments, the fungus described herein is a Saccharomyces species. In certain embodiments, the compounds described herein are used in the inventive methods in combination with one or more additional pharmaceutical agents (e.g., an antifungal agent (such as an azole antifungal agent such as fluconazole and/or antiprotozoan agent). In certain embodiments, the fungus or protozoon described herein is resistant to the additional antifungal agent.

(17) Compounds

(18) In one aspect of the present invention, the present invention provides compounds of Formula (I):

(19) ##STR00008##
and pharmaceutically acceptable salts, solvates, hydrates, polymorphs, co-crystals, tautomers, stereoisomers, isotopically labeled derivatives, and prodrugs thereof;
wherein:

(20) Ring A is a substituted or unsubstituted aryl ring, or substituted or unsubstituted heteroaryl ring;

(21) each instance of R.sup.A is independently hydrogen, halogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, OR.sup.A1, N(R.sup.A1).sub.2, SR.sup.A1, CN, SCN, C(NR.sup.A1)R.sup.A1, C(NR.sup.A1)OR.sup.A1, C(NR.sup.A1)N(R.sup.A1).sub.2, C(O)R.sup.A1, C(O)OR.sup.A1, C(O)N(R.sup.A1).sub.2, NO.sub.2, NR.sup.A1C(O)R.sup.A1, NR.sup.A1C(O)OR.sup.A1, NR.sup.A1C(O)N(R.sup.A1).sub.2, OC(O)R.sup.A1, OC(O)OR.sup.A1, OC(O)N(R.sup.A1).sub.2, or a nitrogen protecting group when attached to a nitrogen atom, or optionally two R.sup.A groups are joined to form a substituted or unsubstituted carbocyclic, substituted or unsubstituted heterocyclic, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl ring;

(22) each instance of R.sup.A1 is independently hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom, or optionally two R.sup.A1 groups are joined to form a substituted or unsubstituted heterocyclic ring;

(23) k is 0, 1, 2, 3, 4, or 5;

(24) each instance of R.sup.B is independently hydrogen, halogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, OR.sup.B1, N(R.sup.B1).sub.2, SR.sup.B1, CN, SCN, C(NR.sup.B1)R.sup.B1, C(NR.sup.B1)OR.sup.B1, C(NR.sup.B1)N(R.sup.B1).sub.2, C(O)R.sup.B1, C(O)OR.sup.B1, C(O)N(R.sup.B1).sub.2, NO.sub.2, NR.sup.B1C(O)R.sup.B1, NR.sup.B1C(O)OR.sup.B1, NR.sup.B1C(O)N(R.sup.B1).sub.2, OC(O)R.sup.B1, OC(O)OR.sup.B1, or OC(O)N(R.sup.B1).sub.2, or optionally two R.sup.B groups are joined to form a substituted or unsubstituted carbocyclic, substituted or unsubstituted heterocyclic, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl ring;

(25) each instance of R.sup.B1 is independently hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom, or optionally two R.sup.B1 groups are joined to form a substituted or unsubstituted heterocyclic ring;

(26) m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

(27) n is 1 or 2;

(28) R.sup.C is hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, C(NR.sup.C1)R.sup.C1, C(NR.sup.C1)OR.sup.C1, C(NR.sup.C1)N(R.sup.C1).sub.2, C(O)R.sup.C1, C(O)OR.sup.C1, C(O)N(R.sup.C1).sub.2, S(O)R.sup.C1, S(O)OR.sup.C1, S(O)N(R.sup.C1).sub.2, S(O).sub.2R.sup.C1, S(O).sub.2OR.sup.C1, S(O).sub.2N(R.sup.C1).sub.2, C(CN)NOR.sup.C1, CF.sub.2R.sup.C1,

(29) ##STR00009##
or a nitrogen protecting group; and

(30) each instance of R.sup.C1 is independently hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, or an oxygen protecting group when attached to an oxygen atom, or optionally two R.sup.C1 groups are joined to form a substituted or unsubstituted heterocyclic ring.

(31) In certain embodiments, the present invention provides compounds of Formula (I), and pharmaceutically acceptable salts thereof.

(32) Compounds of Formula (I) include a substituted or unsubstituted aryl ring, or substituted or unsubstituted heteroaryl ring as Ring A. In certain embodiments, Ring A is a substituted aryl ring. In certain embodiments, Ring A is an unsubstituted aryl ring. In certain embodiments, Ring A is a monocyclic aryl ring. In certain embodiments, Ring A is substituted phenyl. In certain embodiments, Ring A is of the formula:

(33) ##STR00010##
In certain embodiments, Ring A is of the formula:

(34) ##STR00011##
In certain embodiments, Ring A is of the formula:

(35) ##STR00012##
In certain embodiments, Ring A is of the formula

(36) ##STR00013##
In certain embodiments, Ring A is of the formula:

(37) ##STR00014##
In certain embodiments, Ring A is of the formula:

(38) ##STR00015##
In certain embodiments, Ring A is the formula:

(39) ##STR00016##
In certain embodiments, Ring A is of the formula:

(40) ##STR00017##
In certain embodiments, Ring A is of the formula:

(41) ##STR00018##
In certain embodiments, Ring A is of the formula:

(42) ##STR00019##
In certain embodiments, Ring A is of the formula:

(43) ##STR00020##
In certain embodiments, Ring A is unsubstituted phenyl. In certain embodiments, Ring A is a bicyclic aryl ring. In certain embodiments, Ring A is substituted naphthyl. In certain embodiments, Ring A is unsubstituted naphthyl. In certain embodiments, Ring A is a tricyclic aryl ring. In certain embodiments, Ring A is substituted anthracenyl. In certain embodiments, Ring A is unsubstituted anthracenyl. In certain embodiments, Ring A is an optionally substituted aryl ring fused with one or more optionally substituted carbocyclic, optionally substituted heterocyclic, optionally substituted aryl, or optionally substituted heteroaryl groups wherein the point of attachment is on the aryl ring.

(44) Ring A of Formula (I) may also be an optionally substituted heteroaryl ring. In certain embodiments, Ring A is a substituted heteroaryl ring. In certain embodiments, Ring A is an unsubstituted heteroaryl ring. In certain embodiments, Ring A is a monocyclic heteroaryl ring. In certain embodiments, Ring A is a 6-membered monocyclic heteroaryl ring. In certain embodiments, Ring A is a 6-membered monocyclic heteroaryl ring, wherein only one of the six atoms in the ring of the heteroaryl is nitrogen. In certain embodiments, Ring A is of the formula:

(45) ##STR00021##

(46) In certain embodiments, Ring A is a 6-membered monocyclic heteroaryl ring, wherein only two of the six atoms in the ring of the heteroaryl are nitrogen. In certain embodiments, Ring A is of the formula:

(47) ##STR00022##
In certain embodiments, Ring A is of the formula:

(48) ##STR00023##
In certain embodiments, Ring A is of the formula:

(49) ##STR00024##

(50) In certain embodiments, Ring A is a 6-membered monocyclic heteroaryl ring, wherein only three of the six atoms in the ring of the heteroaryl are nitrogen. In certain embodiments, Ring A is of the formula:

(51) ##STR00025##
In certain embodiments, Ring A is of the formula:

(52) ##STR00026##
In certain embodiments, Ring A is of the formula:

(53) ##STR00027##

(54) In certain embodiments, Ring A is a 5-membered monocyclic heteroaryl ring. In certain embodiments, Ring A is a 5-membered monocyclic heteroaryl ring, wherein only one of the five atoms in the ring of the heteroaryl is nitrogen, oxygen, or sulfur. In certain embodiments, Ring A is of the formula:

(55) ##STR00028##
In certain embodiments, Ring A is of the formula:

(56) ##STR00029##
In certain embodiments, Ring A is of the formula:

(57) ##STR00030##

(58) In certain embodiments, Ring A is a 5-membered monocyclic heteroaryl ring, wherein only two of the five atoms in the ring of the heteroaryl are independently nitrogen, oxygen, or sulfur. In certain embodiments, Ring A is of the formula:

(59) ##STR00031##
In certain embodiments, Ring A is of the formula:

(60) ##STR00032##
In certain embodiments, Ring A is of the formula:

(61) ##STR00033##
In certain embodiments, Ring A is of the formula:

(62) ##STR00034##
In certain embodiments, Ring A is of the formula:

(63) ##STR00035##
In certain embodiments, Ring A is of the formula:

(64) ##STR00036##

(65) In certain embodiments, Ring A is a 5-membered monocyclic heteroaryl ring, wherein only three of the five atoms in the ring of the heteroaryl are independently nitrogen, oxygen, or sulfur. In certain embodiments, Ring A is of the formula:

(66) ##STR00037##
In certain embodiments, Ring A is of the formula:

(67) ##STR00038##
In certain embodiments, Ring A is of the formula:

(68) ##STR00039##

(69) In certain embodiments, Ring A is a 5-membered monocyclic heteroaryl ring, wherein only four of the five atoms in the ring of the heteroaryl are nitrogen, oxygen, or sulfur. In certain embodiments, Ring A is of the formula:

(70) ##STR00040##

(71) In certain embodiments, Ring A is bicyclic heteroaryl, wherein the point of attachment may be on any atom of the bicyclic heteroaryl ring system, as valency permits. In certain embodiments, Ring A is a monocyclic heteroaryl ring fused with phenyl. In certain embodiments, Ring A is a 5-membered monocyclic heteroaryl ring fused with phenyl. In certain embodiments, Ring A is a 6-membered monocyclic heteroaryl ring fused with phenyl. In certain embodiments, Ring A is a monocyclic heteroaryl ring fused with another monocyclic heteroaryl. In certain embodiments, Ring A is a 5-membered monocyclic heteroaryl ring fused with another 5-membered monocyclic heteroaryl. In certain embodiments, Ring A is a 5-membered monocyclic heteroaryl ring fused with a 6-membered monocyclic heteroaryl. In certain embodiments, Ring A is a 6-membered monocyclic heteroaryl ring fused with another 6-membered monocyclic heteroaryl.

(72) Ring A of compounds of Formula (I) may include one or more substituents R.sup.A. In certain embodiments, at least one R.sup.A is H. In certain embodiments, at least one R.sup.A is halogen. In certain embodiments, at least one R.sup.A is F. In certain embodiments, at least one R.sup.A is Cl. In certain embodiments, at least one R.sup.A is Br. In certain embodiments, at least one R.sup.A is I (iodine). In certain embodiments, at least one R.sup.A is substituted acyl. In certain embodiments, at least one R.sup.A is unsubstituted acyl. In certain embodiments, at least one R.sup.A is substituted alkyl. In certain embodiments, at least one R.sup.A is unsubstituted alkyl. In certain embodiments, at least one R.sup.A is C.sub.1-6 alkyl. In certain embodiments, at least one R.sup.A is methyl. In certain embodiments, at least one R.sup.A is substituted methyl. In certain embodiments, at least one R.sup.A is CH.sub.2F. In certain embodiments, at least one R.sup.A is CHF.sub.2. In certain embodiments, at least one R.sup.A is CF.sub.3. In certain embodiments, at least one R.sup.A is Bn. In certain embodiments, at least one R.sup.A is ethyl. In certain embodiments, at least one R.sup.A is substituted ethyl. In certain embodiments, at least one R.sup.A is (CH.sub.2).sub.2Ph. In certain embodiments, at least one R.sup.A is propyl. In certain embodiments, at least one R.sup.A is butyl. In certain embodiments, at least one R.sup.A is pentyl. In certain embodiments, at least one R.sup.A is hexyl. In certain embodiments, at least one R.sup.A is substituted alkenyl. In certain embodiments, at least one R.sup.A is unsubstituted alkenyl. In certain embodiments, at least one R.sup.A is vinyl. In certain embodiments, at least one R.sup.A is substituted alkynyl. In certain embodiments, at least one R.sup.A is unsubstituted alkynyl. In certain embodiments, at least one R.sup.A is ethynyl. In certain embodiments, at least one R.sup.A is substituted carbocyclyl. In certain embodiments, at least one R.sup.A is unsubstituted carbocyclyl. In certain embodiments, at least one R.sup.A is cylcopropyl. In certain embodiments, at least one R.sup.A is cylcobutyl. In certain embodiments, at least one R.sup.A is cyclopentyl. In certain embodiments, at least one R.sup.A is cyclohexyl. In certain embodiments, at least one R.sup.A is cycloheptyl. In certain embodiments, at least one R.sup.A is substituted heterocyclyl. In certain embodiments, at least one R.sup.A is unsubstituted heterocyclyl. In certain embodiments, at least one R.sup.A is substituted aryl. In certain embodiments, at least one R.sup.A is unsubstituted aryl. In certain embodiments, at least one R.sup.A is substituted phenyl. In certain embodiments, at least one R.sup.A is unsubstituted phenyl. In certain embodiments, at least one R.sup.A is substituted naphthyl. In certain embodiments, at least one R.sup.A is unsubstituted naphthyl. In certain embodiments, at least one R.sup.A is substituted heteroaryl. In certain embodiments, at least one R.sup.A is unsubstituted heteroaryl. In certain embodiments, at least one R.sup.A is monocyclic heteroaryl. In certain embodiments, at least one R.sup.A is 5-membered monocyclic heteroaryl. In certain embodiments, at least one R.sup.A is 5-membered monocyclic heteroaryl, wherein only one of the five atoms in the ring of the heteroaryl is nitrogen, oxygen, or sulfur. In certain embodiments, at least one R.sup.A is 5-membered monocyclic heteroaryl, wherein only two of the five atoms in the ring of the heteroaryl are independently nitrogen, oxygen, or sulfur. In certain embodiments, at least one R.sup.A is 5-membered monocyclic heteroaryl, wherein only three of the five atoms in the ring of the heteroaryl are independently nitrogen, oxygen, or sulfur. In certain embodiments, at least one R.sup.A is tetrazolyl. In certain embodiments, at least one R.sup.A is 6-membered monocyclic heteroaryl. In certain embodiments, at least one R.sup.A is 6-membered monocyclic heteroaryl, wherein only one of the six atoms in the ring of the heteroaryl is nitrogen. In certain embodiments, at least one R.sup.A is 6-membered monocyclic heteroaryl, wherein only two of the six atoms in the ring of the heteroaryl are nitrogen. In certain embodiments, at least one R.sup.A is triazinyl. In certain embodiments, at least one R.sup.A is tetrazinyl. In certain embodiments, at least one R.sup.A is bicyclic heteroaryl, wherein the point of attachment may be on any atom of the bicyclic heteroaryl ring system, as valency permits. In certain embodiments, at least one R.sup.A is a monocyclic heteroaryl ring fused with phenyl. In certain embodiments, at least one R.sup.A is a 5-membered monocyclic heteroaryl ring fused with phenyl. In certain embodiments, at least one R.sup.A is a 6-membered monocyclic heteroaryl ring fused with phenyl. In certain embodiments, at least one R.sup.A is a monocyclic heteroaryl ring fused with another monocyclic heteroaryl. In certain embodiments, at least one R.sup.A is a 5-membered monocyclic heteroaryl ring fused with another 5-membered monocyclic heteroaryl. In certain embodiments, at least one R.sup.A is a 5-membered monocyclic heteroaryl ring fused with a 6-membered monocyclic heteroaryl. In certain embodiments, at least one R.sup.A is a 6-membered monocyclic heteroaryl fused with another 6-membered monocyclic heteroaryl. In certain embodiments, at least one R.sup.A is OR.sup.A1. In certain embodiments, at least one R.sup.A is -OMe. In certain embodiments, at least one R.sup.A is -OEt. In certain embodiments, at least one R.sup.A is -OPr. In certain embodiments, at least one R.sup.A is -OBu. In certain embodiments, at least one R.sup.A is O(pentyl). In certain embodiments, at least one R.sup.A is O(hexyl). In certain embodiments, at least one R.sup.A is -OPh. In certain embodiments, at least one R.sup.A is -OBn. In certain embodiments, at least one R.sup.A is O(CH.sub.2).sub.2Ph. In certain embodiments, at least one R.sup.A is OH. In certain embodiments, at least one R.sup.A is SR.sup.A1. In certain embodiments, at least one R.sup.A is SH. In certain embodiments, at least one R.sup.A is N(R.sup.A).sub.2. In certain embodiments, at least one R.sup.A is NH.sub.2. In certain embodiments, at least one R.sup.A is CN. In certain embodiments, at least one R.sup.A is SCN. In certain embodiments, at least one R.sup.A is C(NR.sup.A1)R.sup.A1, C(NR.sup.A1)OR.sup.A1, or C(NR.sup.A1)N(R.sup.A1).sub.2. In certain embodiments, at least one R.sup.A is C(O)R.sup.A1, C(O)OR.sup.A1, or C(O)N(R.sup.A1).sub.2. In certain embodiments, at least one R.sup.A is NO.sub.2. In certain embodiments, at least one R.sup.A is NR.sup.A1C(O)R.sup.A1, NR.sup.A1C(O)OR.sup.A1, or NR.sup.A1C(O)N(R.sup.A1).sub.2. In certain embodiments, at least one R.sup.A is OC(O)R.sup.A1, OC(O)OR.sup.A1, or OC(O)N(R.sup.A).sub.2. In certain embodiments, at least one R.sup.A is a nitrogen protecting group when attached to a nitrogen atom. In certain embodiments, at least one R.sup.A is Bn, BOC, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, or Ts when attached to a nitrogen atom.

(73) In compounds of Formula (I), two R.sup.A groups may be joined to form a substituted or unsubstituted carbocyclic ring. In certain embodiments, two R.sup.A groups are joined to form a substituted or unsubstituted cyclopropyl ring. In certain embodiments, two R.sup.A groups are joined to form a substituted or unsubstituted cyclobutyl ring. In certain embodiments, two R.sup.A groups are joined to form a substituted or unsubstituted cyclopentyl ring. In certain embodiments, two R.sup.A groups are joined to form a substituted or unsubstituted cyclohexyl ring. In certain embodiments, two R.sup.A groups are joined to form a substituted or unsubstituted cycloheptyl ring. In certain embodiments, two R.sup.A groups are joined to form a substituted or unsubstituted cyclooctyl ring. In certain embodiments, two R.sup.A groups are joined to form a substituted or unsubstituted cyclononyl ring. In certain embodiments, two R.sup.A groups are joined to form a substituted or unsubstituted cyclodecyl ring.

(74) In certain embodiments, two R.sup.A groups are joined to form a substituted or unsubstituted heterocyclic ring. In certain embodiments, two R.sup.A groups are joined to form a substituted or unsubstituted 4-membered heterocyclic ring. In certain embodiments, two R.sup.A groups are joined to form a substituted or unsubstituted 5-membered heterocyclic ring. In certain embodiments, two R.sup.A groups are joined to form a substituted or unsubstituted 6-membered heterocyclic ring. In certain embodiments, two R.sup.A groups are joined to form a substituted or unsubstituted 7-membered heterocyclic ring. In certain embodiments, two R.sup.A groups are joined to form a substituted or unsubstituted 8-membered heterocyclic ring. In certain embodiments, two R.sup.A groups are joined to form a substituted or unsubstituted 9-membered heterocyclic ring. In certain embodiments, two R.sup.A groups are joined to form a substituted or unsubstituted 10-membered heterocyclic ring.

(75) In certain embodiments, two R.sup.A groups are joined to form a substituted or unsubstituted aryl ring. In certain embodiments, two R.sup.A groups are joined to form a substituted or unsubstituted monocyclic aryl ring. In certain embodiments, two R.sup.A groups are joined to form a substituted or unsubstituted phenyl ring. In certain embodiments, two R.sup.A groups are joined to form a substituted or unsubstituted bicyclic aryl ring. In certain embodiments, two R.sup.A groups are joined to form a substituted or unsubstituted naphthyl ring.

(76) In certain embodiments, two R.sup.A groups are joined to form a substituted or unsubstituted heteroaryl ring. In certain embodiments, two R.sup.A groups are joined to form a substituted or unsubstituted monocyclic heteroaryl ring. In certain embodiments, two R.sup.A groups are joined to form a substituted or unsubstituted 5-membered monocyclic heteroaryl ring. In certain embodiments, two R.sup.A groups are joined to form a substituted or unsubstituted 6-membered monocyclic heteroaryl ring. In certain embodiments, two R.sup.A groups are joined to form a substituted or unsubstituted, bicyclic heteroaryl ring. In certain embodiments, two R.sup.A groups are joined to form a substituted or unsubstituted, 5,6-membered bicyclic heteroaryl ring. In certain embodiments, two R.sup.A groups are joined to form a substituted or unsubstituted, 6,5-membered bicyclic heteroaryl ring. In certain embodiments, two R.sup.A groups are joined to form a substituted or unsubstituted, 6,6-membered bicyclic heteroaryl ring.

(77) In certain embodiments, at least one R.sup.A1 is H. In certain embodiments, at least one R.sup.A1 is substituted acyl. In certain embodiments, at least one R.sup.A1 is unsubstituted acyl. In certain embodiments, at least one R.sup.A1 is acetyl. In certain embodiments, at least one R.sup.A1 is substituted alkyl. In certain embodiments, at least one R.sup.A1 is unsubstituted alkyl. In certain embodiments, at least one R.sup.A1 is C.sub.1-6 alkyl. In certain embodiments, at least one R.sup.A1 is methyl. In certain embodiments, at least one R.sup.A1 is ethyl. In certain embodiments, at least one R.sup.A1 is propyl. In certain embodiments, at least one R.sup.A1 is butyl. In certain embodiments, at least one R.sup.A1 is pentyl. In certain embodiments, at least one R.sup.A1 is hexyl. In certain embodiments, at least one R.sup.A1 is substituted alkenyl. In certain embodiments, at least one R.sup.A1 is unsubstituted alkenyl. In certain embodiments, at least one R.sup.A1 is vinyl. In certain embodiments, at least one R.sup.A1 is substituted alkynyl. In certain embodiments, at least one R.sup.A1 is unsubstituted alkynyl. In certain embodiments, at least one R.sup.A1 is ethynyl. In certain embodiments, at least one R.sup.A1 is substituted carbocyclyl. In certain embodiments, at least one R.sup.A1 is unsubstituted carbocyclyl. In certain embodiments, at least one R.sup.A1 is cylcopropyl. In certain embodiments, at least one R.sup.A1 is cylcobutyl. In certain embodiments, at least one R.sup.A1 is cyclopentyl. In certain embodiments, at least one R.sup.A1 is cyclohexyl. In certain embodiments, at least one R.sup.A1 is cycloheptyl. In certain embodiments, at least one R.sup.A1 is substituted heterocyclyl. In certain embodiments, at least one R.sup.A1 is unsubstituted heterocyclyl. In certain embodiments, at least one R.sup.A1 is substituted aryl. In certain embodiments, at least one R.sup.A1 is unsubstituted aryl. In certain embodiments, at least one R.sup.A1 is substituted phenyl. In certain embodiments, at least one R.sup.A1 is unsubstituted phenyl. In certain embodiments, at least one R.sup.A1 is substituted heteroaryl. In certain embodiments, at least one R.sup.A1 is unsubstituted heteroaryl. In certain embodiments, at least one R.sup.A1 is substituted pyridyl. In certain embodiments, at least one R.sup.A1 is unsubstituted pyridyl. In certain embodiments, at least one R.sup.A1 is a nitrogen protecting group when attached to a nitrogen atom. In certain embodiments, at least one R.sup.A1 is Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, or Ts when attached to a nitrogen atom. In certain embodiments, R.sup.A1 is an oxygen protecting group when attached to an oxygen atom. In certain embodiments, R.sup.A1 is silyl, TBDPS, TBDMS, TIPS, TES, TMS, MOM, THP, t-Bu, Bn, allyl, acetyl, pivaloyl, or benzoyl when attached to an oxygen atom. In certain embodiments, R.sup.A1 is a sulfur protecting group when attached to a sulfur atom. In certain embodiments, R.sup.A1 is acetamidomethyl, t-Bu, 3-nitro-2-pyridine sulfenyl, 2-pyridine-sulfenyl, or triphenylmethyl when attached to a sulfur atom. In certain embodiments, two R.sup.A1 groups are joined to form a substituted heterocyclic ring. In certain embodiments, two R.sup.A1 groups are joined to form an unsubstituted heterocyclic ring. In certain embodiments, two R.sup.A1 groups are joined to form a substituted heteroaryl ring. In certain embodiments, two R.sup.A1 groups are joined to form an unsubstituted heteroaryl ring.

(78) In certain embodiments, k is 0. In certain embodiments, k is 1. In certain embodiments, k is 2. In certain embodiments, k is 3. In certain embodiments, k is 4. In certain embodiments, k is 5.

(79) Compounds of Formula (I) may include zero, one, two, or more substituents R.sup.B. In certain embodiments, at least one R.sup.B is H. In certain embodiments, at least one R.sup.B is halogen. In certain embodiments, at least one R.sup.B is F. In certain embodiments, at least one R.sup.B is Cl. In certain embodiments, at least one R.sup.B is Br. In certain embodiments, at least one R.sup.B is I (iodine). In certain embodiments, at least one R.sup.B is substituted acyl. In certain embodiments, at least one R.sup.B is unsubstituted acyl. In certain embodiments, at least one R.sup.B is substituted alkyl. In certain embodiments, at least one R.sup.B is unsubstituted alkyl. In certain embodiments, at least one R.sup.B is C.sub.1-6 alkyl. In certain embodiments, at least one R.sup.B is methyl. In certain embodiments, at least one R.sup.B is substituted methyl. In certain embodiments, at least one R.sup.B is CH.sub.2F. In certain embodiments, at least one R.sup.B is CHF.sub.2. In certain embodiments, at least one R.sup.B is CF.sub.3. In certain embodiments, at least one R.sup.B is benzyl (Bn). In certain embodiments, at least one R.sup.B is ethyl. In certain embodiments, at least one R.sup.B is substituted ethyl. In certain embodiments, at least one R.sup.B is (CH.sub.2).sub.2Ph. In certain embodiments, at least one R.sup.B is propyl. In certain embodiments, at least one R.sup.B is butyl. In certain embodiments, at least one R.sup.B is pentyl. In certain embodiments, at least one R.sup.B is hexyl. In certain embodiments, at least one R.sup.B is substituted alkenyl. In certain embodiments, at least one R.sup.B is unsubstituted alkenyl. In certain embodiments, at least one R.sup.B is vinyl. In certain embodiments, at least one R.sup.B is substituted alkynyl. In certain embodiments, at least one R.sup.B is unsubstituted alkynyl. In certain embodiments, at least one R.sup.B is ethynyl. In certain embodiments, at least one R.sup.B is substituted carbocyclyl. In certain embodiments, at least one R.sup.B is unsubstituted carbocyclyl. In certain embodiments, at least one R.sup.B is cylcopropyl. In certain embodiments, at least one R.sup.B is cylcobutyl. In certain embodiments, at least one R.sup.B is cyclopentyl. In certain embodiments, at least one R.sup.B is cyclohexyl. In certain embodiments, at least one R.sup.B is cycloheptyl. In certain embodiments, at least one R.sup.B is substituted heterocyclyl. In certain embodiments, at least one R.sup.B is unsubstituted heterocyclyl. In certain embodiments, at least one R.sup.B is substituted aryl. In certain embodiments, at least one R.sup.B is unsubstituted aryl. In certain embodiments, at least one R.sup.B is substituted phenyl. In certain embodiments, at least one R.sup.B is unsubstituted phenyl. In certain embodiments, at least one R.sup.B is substituted naphthyl. In certain embodiments, at least one R.sup.B is unsubstituted naphthyl. In certain embodiments, at least one R.sup.B is substituted heteroaryl. In certain embodiments, at least one R.sup.B is unsubstituted heteroaryl. In certain embodiments, at least one R.sup.B is monocyclic heteroaryl. In certain embodiments, at least one R.sup.B is 5-membered monocyclic heteroaryl. In certain embodiments, at least one R.sup.B is 5-membered monocyclic heteroaryl, wherein only one of the five atoms in the ring of the heteroaryl is nitrogen, oxygen, or sulfur. In certain embodiments, at least one R.sup.B is 5-membered monocyclic heteroaryl, wherein only two of the five atoms in the ring of the heteroaryl are independently nitrogen, oxygen, or sulfur. In certain embodiments, at least one R.sup.B is 5-membered monocyclic heteroaryl, wherein only three of the five atoms in the ring of the heteroaryl are independently nitrogen, oxygen, or sulfur. In certain embodiments, at least one R.sup.B is tetrazolyl. In certain embodiments, at least one R.sup.B is 6-membered monocyclic heteroaryl. In certain embodiments, at least one R.sup.B is 6-membered monocyclic heteroaryl, wherein only one of the six atoms in the ring of the heteroaryl is nitrogen. In certain embodiments, at least one R.sup.B is 6-membered monocyclic heteroaryl, wherein only two of the six atoms in the ring of the heteroaryl are nitrogen. In certain embodiments, at least one R.sup.B is triazinyl. In certain embodiments, at least one R.sup.B is tetrazinyl. In certain embodiments, at least one R.sup.B is bicyclic heteroaryl, wherein the point of attachment may be on any atom of the bicyclic heteroaryl, as valency permits. In certain embodiments, at least one R.sup.B is a monocyclic heteroaryl fused with phenyl. In certain embodiments, at least one R.sup.B is a 5-membered monocyclic heteroaryl fused with phenyl. In certain embodiments, at least one R.sup.B is a 6-membered monocyclic heteroaryl fused with phenyl. In certain embodiments, at least one R.sup.B is a monocyclic heteroaryl fused with another monocyclic heteroaryl. In certain embodiments, at least one R.sup.B is a 5-membered monocyclic heteroaryl fused with another 5-membered monocyclic heteroaryl. In certain embodiments, at least one R.sup.B is a 5-membered monocyclic heteroaryl fused with a 6-membered monocyclic heteroaryl. In certain embodiments, at least one R.sup.B is a 6-membered monocyclic heteroaryl fused with another 6-membered monocyclic heteroaryl. In certain embodiments, at least one R.sup.B is OR.sup.B1. In certain embodiments, at least one R.sup.B is -OMe. In certain embodiments, at least one R.sup.B is -OEt. In certain embodiments, at least one R.sup.B is -OPr. In certain embodiments, at least one R.sup.B is -OBu. In certain embodiments, at least one R.sup.B is O(pentyl). In certain embodiments, at least one R.sup.B is O(hexyl). In certain embodiments, at least one R.sup.B is -OPh. In certain embodiments, at least one R.sup.B is -OBn. In certain embodiments, at least one R.sup.B is O(CH.sub.2).sub.2Ph. In certain embodiments, at least one R.sup.B is OH. In certain embodiments, at least one R.sup.B is SR.sup.B1. In certain embodiments, at least one R.sup.B is -SMe. In certain embodiments, at least one R.sup.B is SH. In certain embodiments, at least one R.sup.B is N(R.sup.B1).sub.2. In certain embodiments, at least one R.sup.B is N(Me).sub.2. In certain embodiments, at least one R.sup.B is NHMe. In certain embodiments, at least one R.sup.B is NHAc. In certain embodiments, at least one R.sup.B is NH.sub.2. In certain embodiments, at least one R.sup.B is CN. In certain embodiments, at least one R.sup.B is SCN. In certain embodiments, at least one R.sup.B is C(NR.sup.B1)R.sup.B1, C(NR.sup.B1)OR.sup.B1, or C(NR.sup.B1)N(R.sup.B1).sub.2. In certain embodiments, at least one R.sup.B is C(O)R.sup.B1. In certain embodiments, at least one R.sup.B is C(O)OR.sup.B1. In certain embodiments, at least one R.sup.B is C(O)OMe. In certain embodiments, at least one R.sup.B is C(O)N(R.sup.B1).sub.2. In certain embodiments, at least one R.sup.B is C(O)N(Me).sub.2. In certain embodiments, at least one R.sup.B is C(O)NHMe. In certain embodiments, at least one R.sup.B is C(O)NH.sub.2. In certain embodiments, at least one R.sup.B is NO.sub.2. In certain embodiments, at least one R.sup.B is NR.sup.B1C(O)R.sup.B1, NR.sup.B1C(O)OR.sup.B1, or NR.sup.B1C(O)N(R.sup.B1).sub.2. In certain embodiments, at least one R.sup.B is OC(O)R.sup.B1, OC(O)OR.sup.B1, or OC(O)N(R.sup.B1).sub.2.

(80) In compounds of Formula (I), two R.sup.B groups may be joined to form a substituted or unsubstituted carbocyclic ring. In certain embodiments, two R.sup.B groups are joined to form a substituted or unsubstituted cyclopropyl ring. In certain embodiments, two R.sup.B groups are joined to form a substituted or unsubstituted cyclobutyl ring. In certain embodiments, two R.sup.B groups are joined to form a substituted or unsubstituted cyclopentyl ring. In certain embodiments, two R.sup.B groups are joined to form a substituted or unsubstituted cyclohexyl ring. In certain embodiments, two R.sup.B groups are joined to form a substituted or unsubstituted cycloheptyl ring. In certain embodiments, two R.sup.B groups are joined to form a substituted or unsubstituted cyclooctyl ring. In certain embodiments, two R.sup.B groups are joined to form a substituted or unsubstituted cyclononyl ring. In certain embodiments, two R.sup.B groups are joined to form a substituted or unsubstituted cyclodecyl ring.

(81) In certain embodiments, two R.sup.B groups are joined to form a substituted or unsubstituted heterocyclic ring. In certain embodiments, two R.sup.B groups are joined to form a substituted or unsubstituted 4-membered heterocyclic ring. In certain embodiments, two R.sup.B groups are joined to form a substituted or unsubstituted 5-membered heterocyclic ring. In certain embodiments, two R.sup.B groups are joined to form a substituted or unsubstituted 6-membered heterocyclic ring. In certain embodiments, two R.sup.B groups are joined to form a substituted or unsubstituted 7-membered heterocyclic ring. In certain embodiments, two R.sup.B groups are joined to form a substituted or unsubstituted 8-membered heterocyclic ring. In certain embodiments, two R.sup.B groups are joined to form a substituted or unsubstituted 9-membered heterocyclic ring. In certain embodiments, two R.sup.B groups are joined to form a substituted or unsubstituted 10-membered heterocyclic ring.

(82) In certain embodiments, two R.sup.B groups are joined to form a substituted or unsubstituted aryl ring. In certain embodiments, two R.sup.B groups are joined to form a substituted or unsubstituted monocyclic aryl ring. In certain embodiments, two R.sup.B groups are joined to form a substituted or unsubstituted phenyl ring. In certain embodiments, two R.sup.B groups are joined to form a substituted or unsubstituted bicyclic aryl ring. In certain embodiments, two R.sup.B groups are joined to form a substituted or unsubstituted naphthyl ring.

(83) In certain embodiments, two R.sup.B groups are joined to form a substituted or unsubstituted heteroaryl ring. In certain embodiments, two R.sup.B groups are joined to form a substituted or unsubstituted monocyclic heteroaryl ring. In certain embodiments, two R.sup.B groups are joined to form a substituted or unsubstituted, 5-membered monocyclic heteroaryl ring. In certain embodiments, two R.sup.B groups are joined to form a substituted or unsubstituted, 6-membered monocyclic heteroaryl ring. In certain embodiments, two R.sup.B groups are joined to form a substituted or unsubstituted, bicyclic heteroaryl ring. In certain embodiments, two R.sup.B groups are joined to form a substituted or unsubstituted, 5,6-membered bicyclic heteroaryl ring. In certain embodiments, two R.sup.B groups are joined to form a substituted or unsubstituted, 6,5-membered bicyclic heteroaryl ring. In certain embodiments, two R.sup.B groups are joined to form a substituted or unsubstituted, 6,6-membered bicyclic heteroaryl ring.

(84) In certain embodiments, at least one R.sup.B1 is H. In certain embodiments, at least one R.sup.B1 is substituted acyl. In certain embodiments, at least one R.sup.B1 is unsubstituted acyl. In certain embodiments, at least one R.sup.B1 is acetyl. In certain embodiments, at least one R.sup.B1 is substituted alkyl. In certain embodiments, at least one R.sup.B1 is unsubstituted alkyl. In certain embodiments, at least one R.sup.B1 is C.sub.1-6 alkyl. In certain embodiments, at least one R.sup.B1 is methyl. In certain embodiments, at least one R.sup.B1 is ethyl. In certain embodiments, at least one R.sup.B1 is propyl. In certain embodiments, at least one R.sup.B1 is butyl. In certain embodiments, at least one R.sup.B1 is pentyl. In certain embodiments, at least one R.sup.B1 is hexyl. In certain embodiments, at least one R.sup.B1 is substituted alkenyl. In certain embodiments, at least one R.sup.B1 is unsubstituted alkenyl. In certain embodiments, at least one R.sup.B1 is vinyl. In certain embodiments, at least one R.sup.B1 is substituted alkynyl. In certain embodiments, at least one R.sup.B1 is unsubstituted alkynyl. In certain embodiments, at least one R.sup.B1 is ethynyl. In certain embodiments, at least one R.sup.B1 is substituted carbocyclyl. In certain embodiments, at least one R.sup.B1 is unsubstituted carbocyclyl. In certain embodiments, at least one R.sup.B1 is cylcopropyl. In certain embodiments, at least one R.sup.B1 is cylcobutyl. In certain embodiments, at least one R.sup.B1 is cyclopentyl. In certain embodiments, at least one R.sup.B1 is cyclohexyl. In certain embodiments, at least one R.sup.B1 is cycloheptyl. In certain embodiments, at least one R.sup.B1 is substituted heterocyclyl. In certain embodiments, at least one R.sup.B1 is unsubstituted heterocyclyl. In certain embodiments, at least one R.sup.B1 is substituted aryl. In certain embodiments, at least one R.sup.B1 is unsubstituted aryl. In certain embodiments, at least one R.sup.B1 is substituted phenyl. In certain embodiments, at least one R.sup.B1 is unsubstituted phenyl. In certain embodiments, at least one R.sup.B1 is substituted heteroaryl. In certain embodiments, at least one R.sup.B1 is unsubstituted heteroaryl. In certain embodiments, at least one R.sup.B1 is substituted pyridyl. In certain embodiments, at least one R.sup.B1 is unsubstituted pyridyl. In certain embodiments, at least one R.sup.B1 is a nitrogen protecting group when attached to a nitrogen atom. In certain embodiments, at least one R.sup.B1 is Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, or Ts when attached to a nitrogen atom. In certain embodiments, R.sup.B1 is an oxygen protecting group when attached to an oxygen atom. In certain embodiments, R.sup.B1 is silyl, TBDPS, TBDMS, TIPS, TES, TMS, MOM, THP, t-Bu, Bn, allyl, acetyl, pivaloyl, or benzoyl when attached to an oxygen atom. In certain embodiments, R.sup.B1 is a sulfur protecting group when attached to a sulfur atom. In certain embodiments, R.sup.B1 is acetamidomethyl, t-Bu, 3-nitro-2-pyridine sulfenyl, 2-pyridine-sulfenyl, or triphenylmethyl when attached to a sulfur atom. In certain embodiments, two R.sup.B1 groups are joined to form a substituted heterocyclic ring. In certain embodiments, two R.sup.B1 groups are joined to form an unsubstituted heterocyclic ring. In certain embodiments, two R.sup.B1 groups are joined to form a substituted heteroaryl ring. In certain embodiments, two R.sup.B1 groups are joined to form an unsubstituted heteroaryl ring.

(85) In certain embodiments, m is 0. In certain embodiments, m is 1. In certain embodiments, m is 2. In certain embodiments, m is 3. In certain embodiments, m is 4. In certain embodiments, m is 5. In certain embodiments, m is 6. In certain embodiments, m is 7. In certain embodiments, m is 8. In certain embodiments, m is 9. In certain embodiments, m is 10.

(86) In certain embodiments, n is 1. In certain embodiments, n is 2.

(87) In certain embodiments, n is 1, and m is 0. In certain embodiments, n is 2, and m is 0.

(88) In certain embodiments, n is 1, m is 0, and Ring A is monosubstituted phenyl. In certain embodiments, n is 2, m is 0, and Ring A is monosubstituted phenyl.

(89) Compounds of Formula (I) include a substituent R.sup.C attached to the 3-nitrogen atom of the thiohydantoin moiety. In certain embodiments, R.sup.C is H. In certain embodiments, R.sup.C is substituted acyl. In certain embodiments, R.sup.C is unsubstituted acyl. In certain embodiments, R.sup.C is substituted alkyl. In certain embodiments, R.sup.C is unsubstituted alkyl. In certain embodiments, R.sup.C is C.sub.1-6 alkyl. In certain embodiments, R.sup.C is methyl. In certain embodiments, R.sup.C is substituted methyl. In certain embodiments, R.sup.C is CH.sub.2F. In certain embodiments, R.sup.C is CHF.sub.2. In certain embodiments, R.sup.C is CF.sub.3. In certain embodiments, R.sup.C is Bn. In certain embodiments, R.sup.C is ethyl. In certain embodiments, R.sup.C is substituted ethyl. In certain embodiments, R.sup.C is (CH.sub.2).sub.2Ph. In certain embodiments, R.sup.C is propyl. In certain embodiments, R.sup.C is butyl. In certain embodiments, R.sup.C is pentyl. In certain embodiments, R.sup.C is hexyl. In certain embodiments, R.sup.C is substituted alkenyl. In certain embodiments, R.sup.C is unsubstituted alkenyl. In certain embodiments, R.sup.C is vinyl. In certain embodiments, R.sup.C is substituted alkynyl. In certain embodiments, R.sup.C is unsubstituted alkynyl. In certain embodiments, R.sup.C is ethynyl. In certain embodiments, R.sup.C is substituted carbocyclyl. In certain embodiments, R.sup.C is unsubstituted carbocyclyl. In certain embodiments, R.sup.C is cylcopropyl. In certain embodiments, R.sup.C is cylcobutyl. In certain embodiments, R.sup.C is cyclopentyl. In certain embodiments, R.sup.C is cyclohexyl. In certain embodiments, R.sup.C is cycloheptyl. In certain embodiments, R.sup.C is substituted heterocyclyl. In certain embodiments, R.sup.C is unsubstituted heterocyclyl. In certain embodiments, R.sup.C is substituted aryl. In certain embodiments, R.sup.C is unsubstituted aryl. In certain embodiments, R.sup.C is substituted phenyl. In certain embodiments, R.sup.C is unsubstituted phenyl. In certain embodiments, R.sup.C is substituted naphthyl. In certain embodiments, R.sup.C is unsubstituted naphthyl. In certain embodiments, R.sup.C is C(NR.sup.C1)R.sup.C1, C(NR.sup.C1)OR.sup.C1, or C(NR.sup.C1)N(R.sup.C1).sub.2. In certain embodiments, R.sup.C is C(O)R.sup.C1. In certain embodiments, R.sup.C is C(O)Me, C(O)Et, C(O)Pr, or C(O)Bu. In certain embodiments, R.sup.C is C(O)OR.sup.C1. In certain embodiments, R.sup.C is C(O)OMe. In certain embodiments, R.sup.C is C(O)OEt. In certain embodiments, R.sup.C is C(O)OPr. In certain embodiments, R.sup.C is C(O)OBu. In certain embodiments, R.sup.C is C(O)N(R.sup.C1).sub.2. In certain embodiments, R.sup.C is C(O)N(Me).sub.2. In certain embodiments, R.sup.C is C(O)NHMe. In certain embodiments, R.sup.C is C(O)NH.sub.2. In certain embodiments, R.sup.C is S(O)R.sup.C1, S(O)OR.sup.C1, or S(O)N(R.sup.C1).sub.2. In certain embodiments, R.sup.C is S(O).sub.2R.sup.C1. In certain embodiments, R.sup.C is S(O).sub.2Me, S(O).sub.2CF.sub.3, S(O).sub.2Et, S(O).sub.2Pr, or S(O).sub.2Bu. In certain embodiments, R.sup.C is S(O).sub.2OR.sup.C1. In certain embodiments, R.sup.C is S(O).sub.2OMe, S(O).sub.2OEt, S(O).sub.2OPr, or S(O).sub.2OBu. In certain embodiments, R.sup.C is S(O).sub.2N(R.sup.C1).sub.2. In certain embodiments, R.sup.C is S(O).sub.2N(Me).sub.2, S(O).sub.2NHMe, or S(O).sub.2NH.sub.2. In certain embodiments, R.sup.C is C(CN)NOR.sup.C1. In certain embodiments, R.sup.C is C(CN)NOMe, C(CN)NOEt, C(CN)NOPr, or C(CN)NOBu. In certain embodiments, R.sup.C is CF.sub.2R.sup.C1. In certain embodiments, R.sup.C is CHFR.sup.C. In certain embodiments, R.sup.C is

(90) ##STR00041##

(91) In certain embodiments, R.sup.C is unsubstituted heteroaryl. In certain embodiments, R.sup.C is substituted heteroaryl. In certain embodiments, R.sup.C is heteroaryl substituted with (R.sup.D).sub.p,

(92) wherein:

(93) each instance of R.sup.D is independently hydrogen, halogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, OR.sup.D1, N(R.sup.D1).sub.2, SR.sup.D1, CN, SCN, C(NR.sup.D1)R.sup.D1, C(NR.sup.D1)OR.sup.D1, C(NR.sup.D1)N(R.sup.D1).sub.2, C(O)R.sup.D1, C(O)OR.sup.D1, C(O)N(R.sup.D1).sub.2, NO.sub.2, NR.sup.D1C(O)R.sup.D1, NR.sup.D1C(O)OR.sup.D1, NR.sup.D1C(O)N(R.sup.D1).sub.2, OC(O)R.sup.D1, OC(O)OR.sup.D1, OC(O)N(R.sup.D1).sub.2, or a nitrogen protecting group when attached to a nitrogen atom, or optionally two R.sup.D groups are joined to form a substituted or unsubstituted carbocyclic, substituted or unsubstituted heterocyclic, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl ring;

(94) each instance of R.sup.D1 is independently hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom, or optionally two R.sup.D1 groups are joined to form a substituted or unsubstituted heterocyclic ring; and

(95) p is 0, 1, 2, 3, or 4.

(96) In certain embodiments, R.sup.C is monocyclic heteroaryl. In certain embodiments, R.sup.C is 6-membered monocyclic heteroaryl. In certain embodiments, R.sup.C is 6-membered monocyclic heteroaryl, wherein only one of the six atoms of the heteroaryl ring is nitrogen. In certain embodiments, R.sup.C is of the formula:

(97) ##STR00042##

(98) In certain embodiments, R.sup.C is 6-membered monocyclic heteroaryl, wherein only two of the six atoms in the ring of the heteroaryl are nitrogen. In certain embodiments, R.sup.C is of the formula:

(99) ##STR00043##

(100) In certain embodiments, R.sup.C is 6-membered monocyclic heteroaryl, wherein only three of the six atoms of the heteroaryl ring are nitrogen. In certain embodiments, R.sup.C is of the formula:

(101) ##STR00044##

(102) In certain embodiments, R.sup.C is 5-membered monocyclic heteroaryl. In certain embodiments, R.sup.C is 5-membered monocyclic heteroaryl, wherein one of the five atoms of the heteroaryl ring is nitrogen, oxygen, or sulfur. In certain embodiments, R.sup.C is of the formula:

(103) ##STR00045##

(104) In certain embodiments, R.sup.C is 5-membered monocyclic heteroaryl, wherein two of the five atoms of the heteroaryl ring are independently selected from the group consisting of nitrogen, oxygen, and sulfur. In certain embodiments, R.sup.C is of the formula:

(105) ##STR00046##

(106) In certain embodiments, R.sup.C is 5-membered monocyclic heteroaryl, wherein three of the five atoms of the heteroaryl ring are independently selected from the group consisting of nitrogen, oxygen, and sulfur. In certain embodiments, R.sup.C is of the formula:

(107) ##STR00047##

(108) In certain embodiments R.sup.C is a 5-membered monocyclic heteroaryl, wherein four of the five atoms of the heteroaryl ring are independently selected from the group consisting of nitrogen, oxygen, and sulfur. In certain embodiments, R.sup.C is of the formula:

(109) ##STR00048##

(110) In certain embodiments, R.sup.C is of the formula:

(111) ##STR00049##

(112) In certain embodiments, R.sup.C is bicyclic heteroaryl, wherein the point of attachment may be on any atom of the bicyclic heteroaryl ring system, as valency permits. In certain embodiments, R.sup.C is monocyclic heteroaryl fused with phenyl. In certain embodiments, R.sup.C is 5-membered monocyclic heteroaryl fused with phenyl. In certain embodiments, R.sup.C is 6-membered monocyclic heteroaryl fused with phenyl. In certain embodiments, R.sup.C is monocyclic heteroaryl fused with another monocyclic heteroaryl. In certain embodiments, R.sup.C is 5-membered monocyclic heteroaryl fused with another 5-membered monocyclic heteroaryl. In certain embodiments, R.sup.C is 5-membered monocyclic heteroaryl fused with a 6-membered monocyclic heteroaryl. In certain embodiments, R.sup.C is 6-membered monocyclic heteroaryl fused with another 6-membered monocyclic heteroaryl.

(113) In certain embodiments, R.sup.C1 is H. In certain embodiments, R.sup.C1 is substituted acyl. In certain embodiments, R.sup.C1 is unsubstituted acyl. In certain embodiments, R.sup.C1 is acetyl. In certain embodiments, R.sup.C1 is substituted alkyl. In certain embodiments, R.sup.C1 is unsubstituted alkyl. In certain embodiments, R.sup.C1 is C.sub.1-6 alkyl. In certain embodiments, R.sup.C1 is methyl. In certain embodiments, R.sup.C1 is ethyl. In certain embodiments, R.sup.C1 is propyl. In certain embodiments, R.sup.C1 is butyl. In certain embodiments, R.sup.C1 is pentyl. In certain embodiments, R.sup.C1 is hexyl. In certain embodiments, R.sup.C1 is substituted alkenyl. In certain embodiments, R.sup.C1 is unsubstituted alkenyl. In certain embodiments, R.sup.C1 is vinyl. In certain embodiments, R.sup.C1 is substituted alkynyl. In certain embodiments, R.sup.C1 is unsubstituted alkynyl. In certain embodiments, R.sup.C1 is ethynyl. In certain embodiments, R.sup.C1 is substituted carbocyclyl. In certain embodiments, R.sup.C1 is unsubstituted carbocyclyl. In certain embodiments, R.sup.C1 is cylcopropyl. In certain embodiments, R.sup.C1 is cylcobutyl. In certain embodiments, R.sup.C1 is cyclopentyl. In certain embodiments, R.sup.C1 is cyclohexyl. In certain embodiments, R.sup.C1 is cycloheptyl. In certain embodiments, R.sup.C1 is substituted heterocyclyl. In certain embodiments, R.sup.C1 is unsubstituted heterocyclyl. In certain embodiments, R.sup.C1 is substituted aryl. In certain embodiments, R.sup.C1 is unsubstituted aryl. In certain embodiments, R.sup.C1 is substituted phenyl. In certain embodiments, R.sup.C1 is unsubstituted phenyl. In certain embodiments, R.sup.C1 is substituted heteroaryl. In certain embodiments, R.sup.C1 is unsubstituted heteroaryl. In certain embodiments, R.sup.C1 is substituted pyridyl. In certain embodiments, R.sup.C1 is unsubstituted pyridyl. In certain embodiments, R.sup.C1 is a nitrogen protecting group when attached to a nitrogen atom. In certain embodiments, R.sup.C1 is Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, or Ts when attached to a nitrogen atom. In certain embodiments, R.sup.C1 is an oxygen protecting group when attached to an oxygen atom. In certain embodiments, R.sup.C1 is silyl, TBDPS, TBDMS, TIPS, TES, TMS, MOM, THP, t-Bu, Bn, allyl, acetyl, pivaloyl, or benzoyl when attached to an oxygen atom. In certain embodiments, two R.sup.C1 groups are joined to form a substituted heterocyclic ring. In certain embodiments, two R.sup.C1 groups are joined to form an unsubstituted heterocyclic ring. In certain embodiments, two R.sup.C1 groups are joined to form a substituted heteroaryl ring. In certain embodiments, two R.sup.C1 groups are joined to form an unsubstituted heteroaryl ring.

(114) When R.sup.C of compounds of Formula (I) is heteroaryl, R.sup.C may include one or more substituents R.sup.D. In certain embodiments, at least one R.sup.D is H. In certain embodiments, at least one R.sup.D is halogen. In certain embodiments, at least one R.sup.D is F. In certain embodiments, at least one R.sup.D is Cl. In certain embodiments, at least one R.sup.D is Br. In certain embodiments, at least one R.sup.D is I (iodine). In certain embodiments, at least one R.sup.D is substituted acyl. In certain embodiments, at least one R.sup.D is unsubstituted acyl. In certain embodiments, at least one R.sup.D is substituted alkyl. In certain embodiments, at least one R.sup.D is unsubstituted alkyl. In certain embodiments, at least one R.sup.D is C.sub.1-6 alkyl. In certain embodiments, at least one R.sup.D is methyl. In certain embodiments, at least one R.sup.D is substituted methyl. In certain embodiments, at least one R.sup.D is CH.sub.2F. In certain embodiments, at least one R.sup.D is CHF.sub.2. In certain embodiments, at least one R.sup.D is CF.sub.3. In certain embodiments, at least one R.sup.D is benzyl (Bn). In certain embodiments, at least one R.sup.D is ethyl. In certain embodiments, at least one R.sup.D is substituted ethyl. In certain embodiments, at least one R.sup.D is (CH.sub.2).sub.2Ph. In certain embodiments, at least one R.sup.D is propyl. In certain embodiments, at least one R.sup.D is butyl. In certain embodiments, at least one R.sup.D is pentyl. In certain embodiments, at least one R.sup.D is hexyl. In certain embodiments, at least one R.sup.D is substituted alkenyl. In certain embodiments, at least one R.sup.D is unsubstituted alkenyl. In certain embodiments, at least one R.sup.D is vinyl. In certain embodiments, at least one R.sup.D is substituted alkynyl. In certain embodiments, at least one R.sup.D is unsubstituted alkynyl. In certain embodiments, at least one R.sup.D is ethynyl. In certain embodiments, at least one R.sup.D is substituted carbocyclyl. In certain embodiments, at least one R.sup.D is unsubstituted carbocyclyl. In certain embodiments, at least one R.sup.D is cylcopropyl. In certain embodiments, at least one R.sup.D is cylcobutyl. In certain embodiments, at least one R.sup.D is cyclopentyl. In certain embodiments, at least one R.sup.D is cyclohexyl. In certain embodiments, at least one R.sup.D is cycloheptyl. In certain embodiments, at least one R.sup.D is substituted heterocyclyl. In certain embodiments, at least one R.sup.D is unsubstituted heterocyclyl. In certain embodiments, at least one R.sup.D is substituted aryl. In certain embodiments, at least one R.sup.D is unsubstituted aryl. In certain embodiments, at least one R.sup.D is substituted phenyl. In certain embodiments, at least one R.sup.D is unsubstituted phenyl. In certain embodiments, at least one R.sup.D is substituted naphthyl. In certain embodiments, at least one R.sup.D is unsubstituted naphthyl. In certain embodiments, at least one R.sup.D is substituted heteroaryl. In certain embodiments, at least one R.sup.D is unsubstituted heteroaryl. In certain embodiments, at least one R.sup.D is monocyclic heteroaryl. In certain embodiments, at least one R.sup.D is 5-membered monocyclic heteroaryl. In certain embodiments, at least one R.sup.D is 5-membered monocyclic heteroaryl, wherein only one of the five atoms of the heteroaryl ring is nitrogen, oxygen, or sulfur. In certain embodiments, at least one R.sup.D is 5-membered monocyclic heteroaryl, wherein only two of the five atoms of the heteroaryl ring are independently nitrogen, oxygen, or sulfur. In certain embodiments, at least one R.sup.D is 5-membered monocyclic heteroaryl, wherein only three of the five atoms of the heteroaryl ring are independently nitrogen, oxygen, or sulfur. In certain embodiments, at least one R.sup.D is tetrazolyl. In certain embodiments, at least one R.sup.D is 6-membered monocyclic heteroaryl. In certain embodiments, at least one R.sup.D is 6-membered monocyclic heteroaryl, wherein only one of the six atoms of the heteroaryl ring is nitrogen. In certain embodiments, at least one R.sup.D is 6-membered monocyclic heteroaryl, wherein only two of the six atoms of the heteroaryl ring are nitrogen. In certain embodiments, at least one R.sup.D is triazinyl. In certain embodiments, at least one R.sup.D is tetrazinyl. In certain embodiments, at least one R.sup.D is bicyclic heteroaryl, wherein the point of attachment may be on any atom of the bicyclic heteroaryl ring system, as valency permits. In certain embodiments, at least one R.sup.D is a monocyclic heteroaryl ring fused with phenyl. In certain embodiments, at least one R.sup.D is a 5-membered monocyclic heteroaryl ring fused with phenyl. In certain embodiments, at least one R.sup.D is a 6-membered monocyclic heteroaryl ring fused with phenyl. In certain embodiments, at least one R.sup.D is a monocyclic heteroaryl ring fused with another monocyclic heteroaryl. In certain embodiments, at least one R.sup.D is a 5-membered monocyclic heteroaryl ring fused with another 5-membered monocyclic heteroaryl. In certain embodiments, at least one R.sup.D is a 5-membered monocyclic heteroaryl ring fused with a 6-membered monocyclic heteroaryl. In certain embodiments, at least one R.sup.D is a 6-membered monocyclic heteroaryl fused with another 6-membered monocyclic heteroaryl. In certain embodiments, at least one R.sup.D is OR.sup.D1. In certain embodiments, at least one R.sup.D is -OMe. In certain embodiments, at least one R.sup.D is -OEt. In certain embodiments, at least one R.sup.D is -OPr. In certain embodiments, at least one R.sup.Dis -OBu. In certain embodiments, at least one R.sup.D is O(pentyl). In certain embodiments, at least one R.sup.D is O(hexyl). In certain embodiments, at least one R.sup.D is -OPh. In certain embodiments, at least one R.sup.D is -OBn. In certain embodiments, at least one R.sup.D is O(CH.sub.2).sub.2Ph. In certain embodiments, at least one R.sup.D is OH. In certain embodiments, at least one R.sup.D is SR.sup.D1. In certain embodiments, at least one R.sup.D is SH. In certain embodiments, at least one R.sup.D is N(R.sup.D1).sub.2. In certain embodiments, at least one R.sup.D is NH.sub.2. In certain embodiments, at least one R.sup.D is CN. In certain embodiments, at least one R.sup.D is SCN. In certain embodiments, at least one R.sup.D is C(NR.sup.D1)R.sup.D1, C(NR.sup.D1)OR.sup.D1, or C(NR.sup.D1)N(R.sup.D1).sub.2. In certain embodiments, at least one R.sup.D is C(O)R.sup.D1, C(O)OR.sup.D1, or C(O)N(R.sup.D1).sub.2. In certain embodiments, at least one R.sup.D is NO.sub.2. In certain embodiments, at least one R.sup.D is NR.sup.D1C(O)R.sup.D1, NR.sup.D1C(O)OR.sup.D1, or NR.sup.D1C(O)N(R.sup.D1).sub.2. In certain embodiments, at least one R.sup.D is OC(O)R.sup.D1, OC(O)OR.sup.D1, or OC(O)N(R.sup.D1).sub.2. In certain embodiments, at least one R.sup.D is a nitrogen protecting group when attached to a nitrogen atom. In certain embodiments, at least one R.sup.D is Bn, BOC, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, or Ts when attached to a nitrogen atom.

(115) When R.sup.C of compounds of Formula (I) is heteroaryl substituted with two or more R.sup.D groups, two R.sup.D groups may be joined to form a substituted or unsubstituted carbocyclic ring. In certain embodiments, two R.sup.D groups are joined to form a substituted or unsubstituted cyclopropyl ring. In certain embodiments, two R.sup.D groups are joined to form a substituted or unsubstituted cyclobutyl ring. In certain embodiments, two R.sup.D groups are joined to form a substituted or unsubstituted cyclopentyl ring. In certain embodiments, two R.sup.D groups are joined to form a substituted or unsubstituted cyclohexyl ring. In certain embodiments, two R.sup.D groups are joined to form a substituted or unsubstituted cycloheptyl ring. In certain embodiments, two R.sup.D groups are joined to form a substituted or unsubstituted cyclooctyl ring. In certain embodiments, two R.sup.D groups are joined to form a substituted or unsubstituted cyclononyl ring. In certain embodiments, two R.sup.D groups are joined to form a substituted or unsubstituted cyclodecyl ring.

(116) When R.sup.C of compounds of Formula (I) is heteroaryl substituted with two or more R.sup.D groups, two R.sup.D groups are joined to form a substituted or unsubstituted heterocyclic ring. In certain embodiments, two R.sup.D groups are joined to form a substituted or unsubstituted 4-membered heterocyclic ring. In certain embodiments, two R.sup.D groups are joined to form a substituted or unsubstituted 5-membered heterocyclic ring. In certain embodiments, two R.sup.D groups are joined to form a substituted or unsubstituted 6-membered heterocyclic ring. In certain embodiments, two R.sup.D groups are joined to form a substituted or unsubstituted 7-membered heterocyclic ring. In certain embodiments, two R.sup.D groups are joined to form a substituted or unsubstituted 8-membered heterocyclic ring. In certain embodiments, two R.sup.D groups are joined to form a substituted or unsubstituted 9-membered heterocyclic ring. In certain embodiments, two R.sup.D groups are joined to form a substituted or unsubstituted 10-membered heterocyclic ring.

(117) When R.sup.C of compounds of Formula (I) is heteroaryl substituted with two or more R.sup.D groups, two R.sup.D groups are joined to form a substituted or unsubstituted aryl ring. In certain embodiments, two R.sup.D groups are joined to form a substituted or unsubstituted monocyclic aryl ring. In certain embodiments, two R.sup.D groups are joined to form a substituted or unsubstituted phenyl ring. In certain embodiments, two R.sup.D groups are joined to form a substituted or unsubstituted bicyclic aryl ring. In certain embodiments, two R.sup.D groups are joined to form a substituted or unsubstituted naphthyl ring.

(118) When R.sup.C of compounds of Formula (I) is heteroaryl substituted with two or more R.sup.D groups, two R.sup.D groups are joined to form a substituted or unsubstituted heteroaryl ring. In certain embodiments, two R.sup.D groups are joined to form a substituted or unsubstituted monocyclic heteroaryl ring. In certain embodiments, two R.sup.D groups are joined to form a substituted or unsubstituted, 5-membered monocyclic heteroaryl ring. In certain embodiments, two R.sup.D groups are joined to form a substituted or unsubstituted, 6-membered monocyclic heteroaryl ring. In certain embodiments, two R.sup.D groups are joined to form a substituted or unsubstituted, bicyclic heteroaryl ring. In certain embodiments, two R.sup.D groups are joined to form a substituted or unsubstituted, 5,6-membered bicyclic heteroaryl ring. In certain embodiments, two R.sup.D groups are joined to form a substituted or unsubstituted, 6,5-membered bicyclic heteroaryl ring. In certain embodiments, two R.sup.D groups are joined to form a substituted or unsubstituted, 6,6-membered bicyclic heteroaryl ring.

(119) In certain embodiments, R.sup.D1 is H. In certain embodiments, R.sup.D1 is substituted acyl. In certain embodiments, R.sup.D1 is unsubstituted acyl. In certain embodiments, R.sup.D1 is acetyl. In certain embodiments, R.sup.D1 is substituted alkyl. In certain embodiments, R.sup.D1 is unsubstituted alkyl. In certain embodiments, R.sup.D1 is C.sub.1-6 alkyl. In certain embodiments, R.sup.D1 is methyl. In certain embodiments, R.sup.D1 is ethyl. In certain embodiments, R.sup.D1 is propyl. In certain embodiments, R.sup.D1 is butyl. In certain embodiments, R.sup.D1 is pentyl. In certain embodiments, R.sup.D1 is hexyl. In certain embodiments, R.sup.D1 is substituted alkenyl. In certain embodiments, R.sup.D1 is unsubstituted alkenyl. In certain embodiments, R.sup.D1 is vinyl. In certain embodiments, R.sup.D1 is substituted alkynyl. In certain embodiments, R.sup.D1 is unsubstituted alkynyl. In certain embodiments, R.sup.D1 is ethynyl. In certain embodiments, R.sup.D1 is substituted carbocyclyl. In certain embodiments, R.sup.D1 is unsubstituted carbocyclyl. In certain embodiments, R.sup.D1 is cylcopropyl. In certain embodiments, R.sup.D1 is cylcobutyl. In certain embodiments, R.sup.D1 is cyclopentyl. In certain embodiments, R.sup.D1 is cyclohexyl. In certain embodiments, R.sup.D1 is cycloheptyl. In certain embodiments, R.sup.D1 is substituted heterocyclyl. In certain embodiments, R.sup.D1 is unsubstituted heterocyclyl. In certain embodiments, R.sup.D1 is substituted aryl. In certain embodiments, R.sup.D1 is unsubstituted aryl. In certain embodiments, R.sup.D1 is substituted phenyl. In certain embodiments, R.sup.D1 is unsubstituted phenyl. In certain embodiments, R.sup.D1 is substituted heteroaryl. In certain embodiments, R.sup.D1 is unsubstituted heteroaryl. In certain embodiments, R.sup.D1 is substituted pyridyl. In certain embodiments, R.sup.D1 is unsubstituted pyridyl. In certain embodiments, R.sup.D1 is a nitrogen protecting group when attached to a nitrogen atom. In certain embodiments, R.sup.D1 is Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, or Ts when attached to a nitrogen atom. In certain embodiments, R.sup.D1 is an oxygen protecting group when attached to an oxygen atom. In certain embodiments, R.sup.D1 is silyl, TBDPS, TBDMS, TIPS, TES, TMS, MOM, THP, t-Bu, Bn, allyl, acetyl, pivaloyl, or benzoyl when attached to an oxygen atom. In certain embodiments, R.sup.D1 is a sulfur protecting group when attached to a sulfur atom. In certain embodiments, R.sup.D1 is acetamidomethyl, t-Bu, 3-nitro-2-pyridine sulfenyl, 2-pyridine-sulfenyl, or triphenylmethyl when attached to a sulfur atom. In certain embodiments, two R.sup.D1 groups are joined to form a substituted heterocyclic ring. In certain embodiments, two R.sup.D1 groups are joined to form an unsubstituted heterocyclic ring. In certain embodiments, two R.sup.D1 groups are joined to form a substituted heteroaryl ring. In certain embodiments, two R.sup.D1 groups are joined to form an unsubstituted heteroaryl ring.

(120) In certain embodiments, p is 0. In certain embodiments, p is 1. In certain embodiments, p is 2. In certain embodiments, p is 3. In certain embodiments, p is 4.

(121) In certain embodiments, the compound of Formula (I) is of the formula:

(122) ##STR00050##
or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.

(123) In certain embodiments, the compound of Formula (I) is of the formula:

(124) ##STR00051##
or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.

(125) In certain embodiments, the compound of Formula (I) is of the formula:

(126) ##STR00052##
or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.

(127) In certain embodiments, the compound of Formula (I) is of the formula:

(128) ##STR00053##
or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.

(129) In certain embodiments, the compound of Formula (I) is of the formula:

(130) ##STR00054##
or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.

(131) In certain embodiments, the compound of Formula (I) is of the formula:

(132) ##STR00055##
or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.

(133) In certain embodiments, the compound of Formula (I) is of the formula:

(134) ##STR00056##
or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.

(135) In certain embodiments, the compound of Formula (I) is of the formula:

(136) ##STR00057##
or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.

(137) In certain embodiments, the compound of Formula (I) is of the formula:

(138) ##STR00058##
or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.

(139) In certain embodiments, the compound of Formula (I) is of the formula:

(140) ##STR00059##
or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.

(141) In certain embodiments, the compound of Formula (I) is of the formula:

(142) ##STR00060##
or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.

(143) In certain embodiments, the compound of Formula (I) is of the formula:

(144) ##STR00061##
or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.

(145) In certain embodiments, the compound of Formula (I) is of the formula:

(146) ##STR00062##
or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.

(147) In certain embodiments, the compound of Formula (I) is of the formula:

(148) ##STR00063##
or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.

(149) In certain embodiments, the compound of Formula (I) is of the formula:

(150) ##STR00064##
or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.

(151) In certain embodiments, the compound of Formula (I) is of the formula:

(152) ##STR00065##
or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.

(153) In certain embodiments, the compound of Formula (I) is of the formula:

(154) ##STR00066##
or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.

(155) In certain embodiments, the compound of Formula (I) is of the formula:

(156) ##STR00067## ##STR00068## ##STR00069## ##STR00070##
or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.

(157) In certain embodiments, the compound of Formula (I) is not of Formula (I-A-1), or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.

(158) The compounds described herein (e.g., compounds of Formula (I)) are shown to be antifungal agents, antiprotozoan, and/or chemosensitizers. In certain embodiments, the provided compounds are useful for reversing drug (e.g., fluconazole) resistance in fungi. In certain embodiments, the provided compounds are useful for reversing drug resistance in protozoa. Without wishing to be bound by a particular theory, these compounds may act by inhibiting the activity of fungal or protozoan mitochondrial phosphate carrier protein. In certain embodiments, the compound inhibits the activity of fungal or protozoan mitochondrial phosphate carrier protein without inhibiting, or at least not substantially, inhibiting the activity of the host's mitochondrial phosphate carrier protein (e.g., human phosphate carrier protein). In certain embodiments, the compounds described herein are specific antifungal agents, that is, for example, the compounds described herein do not inhibit normal enzymatic activity in a subject (e.g., a human), biological sample, or plant as much as the compounds inhibit the analogous activity in a fungus or protozoon. Thus, in some embodiments, it is desired that the compounds have high specificity for the fungal or protozoan target. The specificity of the inhibitor may be evaluated by comparing the IC.sub.50 value with respect to the fungal or protozoan enzyme (i.e., target IC.sub.50) as compared to that of the analogous host (e.g., human) enzyme (i.e., anti-target IC.sub.50). An IC.sub.50 value is defined as the concentration of the compound required to inhibit 50% of the enzymatic activity (e.g., mitochondrial phosphate carrier protein activity). In certain embodiments, the compound described herein exhibits an IC.sub.50 value of <100 M. In certain other embodiments, the compound exhibits an IC.sub.50 value of <50 M. In certain other embodiments, the compound exhibits an IC.sub.50 value of <40 M. In certain other embodiments, the compound exhibits an IC.sub.50 value of <30 M. In certain other embodiments, the compound exhibit an IC.sub.50 value of <20 M. In certain other embodiments, the compound exhibits an IC.sub.50 value of <10 M. In certain other embodiments, the compounds exhibit IC.sub.50 values<7.5 M. In certain embodiments, the compound exhibits an IC.sub.50 value of <5 M. In certain other embodiments, the compound exhibits an IC.sub.50 value of <2.5 M. In certain embodiments, the compound exhibits an IC.sub.50 value of <1 M. In certain embodiments, the compound exhibits an IC.sub.50 value of <0.75 M. In certain embodiments, the compound exhibits an IC.sub.50 value of <0.5 M. In certain embodiments, the compound exhibits an IC.sub.50 value of <0.25 M. In certain embodiments, the compound exhibits an IC.sub.50 value of <0.15 M. In certain embodiments, the compound exhibits an IC.sub.50 value of <0.1 M. In certain other embodiments, the compound exhibit an IC.sub.50 value of <75 nM. In certain other embodiments, the compound exhibits IC.sub.50 value of <50 nM. In certain other embodiments, the compound exhibits an IC.sub.50 value of <5 nM. In certain other embodiments, the compound exhibits IC.sub.50 value of <10 nM. In other embodiments, the compound exhibits an IC.sub.50 values of <7.5 nM. In other embodiments, the compound exhibits an IC.sub.50 value of <5 nM.

(159) In certain embodiments, the compound inhibits the fungal and/or protozoan enzyme at a concentration at least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, or 1000-fold lower than the concentration needed to inhibit the host's analogous enzyme to the same extent. In certain embodiments, the enzyme being inhibited is mitochondrial phosphate carrier protein.

(160) In certain embodiments, the compound exhibits an IC.sub.50 value of <100 nM when combined with an additional pharmaceutical agent. In certain embodiments, the compound exhibits an IC.sub.50 value of <80 nM when combined with an additional pharmaceutical agent. In certain embodiments, the compound exhibits an IC.sub.50 value of <60 nM when combined with an additional pharmaceutical agent. In certain embodiments, the compound exhibits an IC.sub.50 value of <40 nM when combined with an additional pharmaceutical agent. In certain embodiments, the compound exhibits an IC.sub.50 value of <20 nM when combined with an additional pharmaceutical agent. In certain embodiments, the compound exhibits an IC.sub.50 value of <10 nM when combined with an additional pharmaceutical agent. In certain embodiments, the additional pharmaceutical agent is another antifungal agent (e.g., an azole, e.g., fluconazole) or another antiprotozoan agent.

(161) Provided herein are methods for the identification of fungal or protozoan-selective inhibitors of the MIR1 mitochondrial phosphate transporter by chemical screening. For example, in one embodiment, the method uses a S. cerevisiae BY4741 mir1 deletion strain, which cannot grow on media containing glycerol as a sole carbon source. To restore mitochondrial phosphate transport and growth on glycerol media through Mir1 activity, a gene encoding for a mitochondrial phosphate transporter is transformed into this strain. This gene may be either fungal or protozoan phosphate transporter genes (including, but not limited to, Mir1 homologs from Saccharomyces cerevisiae (NCBI gene ID 85340) or Candida albicans (NCBI gene Id 3635000) or human SLC25A3 (NCBI gene ID 5250). Specific mutations of the yeast or human MIR1 or SLC25A3 gene (including S. cerevisiae N184T mutant) may be included to optimize expression or function of the protein in yeast. In this method, strains expressing one of these genes are assayed for growth in the presence of screening compounds or thiohydantoin analogs to identify compounds that inhibit growth of strains expressing MIR1 of pathogens but not inhibit growth of strains expressing human SLC25A3.

(162) In addition or alternatively, mitochondria are purified from these or other strains expressing different mitochondrial phosphate transporters and specific inhibition of mitochondrial phosphate transport activity is assayed. Mitochondrial phosphate uptake inhibition is measured by using an in vitro 32P method (see, e.g., Zara V, Dietmeier K, Palmisano A, et al. Yeast mitochondria lacking the phosphate carrier/p32 are blocked in phosphate transport but can import preproteins after regeneration of a membrane potential. Mol Cell Biol. 1996; 16(11):6524-31), in which 32P phosphoric acid is added to the purified mitochondria in the presence of the screening compounds to be tested, and phosphate accumulation is measured by .sup.32P radioactivity counts in mitochondrial pellets after centrifugation.

(163) The exogenously introduced gene (e.g., human SLC25A3) may be codon optimized for expression in the fungal or protozoal cell in which it is to be expressed. Additionally or alternately, the gene encoding a human mitochondrial phosphate transporter may be modified to omit at least a portion of the human mitochondrial targeting sequence or to encode a fungal or protozoal mitochondrial targeting sequence (MTS) instead of or in addition to at least a portion of the human mitochondrial targeting sequence. Without wishing to be bound by any theory, doing so may enhance proper mitochondrial membrane localization of the protein when expressed in fungal or protozoal cells. The MIR1 protein is normally localized to the inner mitochondrial membrane (IMM). In one embodiment, a targeting sequence from any fungal (or protozoal) protein that is localized to the IMM for purposes of achieving appropriate localization of the human homolog is used. The predicted mitochondrial targeting sequence of SLC25A3 is within the N-terminal 49 amino acids. In some embodiments, an N-terminal region of varying lengths, e.g., about the first 20, 25, 30, 35, 40, 45, 50 amino acids. In another embodiment, a chimeric MIR1 protein can be used.

(164) Pharmaceutical Compositions, Kits, and Administration

(165) The present invention also provides pharmaceutical compositions comprising a compound described herein, or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystals, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, and optionally a pharmaceutically acceptable excipient.

(166) In certain embodiments, the compound described herein, or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, is provided in an effective amount in the pharmaceutical composition. In certain embodiments, the effective amount is a therapeutically effective amount. In certain embodiments, the effective amount is a prophylactically effective amount. In certain embodiments, the effective amount is an amount useful for the treatment and/or prevention of a fungal or protozoan infection. The effective amount of the compound in the composition may be useful for the treatment and/or prevention of a fungal or protozoan infection as a single agent or in combination with another agent such as another antifungal agent (e.g., fluconazole) or another antiprotozoan agent. In certain embodiments, the effective amount is an amount useful for inhibiting the activity of a fungal or protozoan enzyme. In certain embodiments, the effective amount is an amount useful for killing a fungus or inhibiting the growth of a fungus. An effective amount of a compound may vary from about 0.001 mg/kg to about 1000 mg/kg in one or more dose administrations for one or several days (depending on the mode of administration). In certain embodiments, the effective amount per dose varies from about 0.001 mg/kg to about 1000 mg/kg, from about 0.01 mg/kg to about 750 mg/kg, from about 0.1 mg/kg to about 500 mg/kg, from about 1.0 mg/kg to about 250 mg/kg, and from about 10.0 mg/kg to about 150 mg/kg.

(167) Pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacology. In general, such preparatory methods include the steps of bringing the compound described herein (i.e., the active ingredient) into association with a carrier or excipient, and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping, and/or packaging the product into a desired single- or multi-dose unit.

(168) Pharmaceutical compositions can be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a unit dose is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

(169) Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. The composition may comprise between 0.1% and 100% (w/w) active ingredient.

(170) Pharmaceutically acceptable excipients used in the manufacture of provided pharmaceutical compositions include inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and perfuming agents may also be present in the composition.

(171) Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, and mixtures thereof.

(172) Exemplary granulating and/or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose, and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and mixtures thereof.

(173) Exemplary surface active agents and/or emulsifiers include natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite (aluminum silicate) and Veegum (magnesium aluminum silicate)), long chain amino acid derivatives, high molecular weight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylene sorbitan monolaurate (Tween 20), polyoxyethylene sorbitan (Tween 60), polyoxyethylene sorbitan monooleate (Tween 80), sorbitan monopalmitate (Span 40), sorbitan monostearate (Span 60), sorbitan tristearate (Span 65), glyceryl monooleate, sorbitan monooleate (Span 80)), polyoxyethylene esters (e.g. polyoxyethylene monostearate (Myrj 45), polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. Cremophor), polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether (Brij 30)), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F-68, Poloxamer P-188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and/or mixtures thereof.

(174) Exemplary binding agents include starch (e.g. cornstarch and starch paste), gelatin, sugars (e.g. sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums (e.g. acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum), and larch arabogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohol, and/or mixtures thereof.

(175) Exemplary preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, antiprotozoan preservatives, alcohol preservatives, acidic preservatives, and other preservatives. In certain embodiments, the preservative is an antioxidant. In other embodiments, the preservative is a chelating agent.

(176) Exemplary antioxidants include alpha tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.

(177) Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, and the like), citric acid and salts and hydrates thereof (e.g., citric acid monohydrate), fumaric acid and salts and hydrates thereof, malic acid and salts and hydrates thereof, phosphoric acid and salts and hydrates thereof, and tartaric acid and salts and hydrates thereof. Exemplary antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.

(178) Exemplary antifungal preservatives include butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.

(179) Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol.

(180) Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.

(181) Other preservatives include tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Glydant Plus, Phenonip, methylparaben, Germall 115, Germaben II, Neolone, Kathon, and Euxyl.

(182) Exemplary buffering agents include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and mixtures thereof.

(183) Exemplary lubricating agents include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and mixtures thereof.

(184) Exemplary natural oils include almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils. Exemplary synthetic oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and mixtures thereof.

(185) Liquid dosage forms for oral and parenteral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredients, the liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (e.g., cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. In certain embodiments for parenteral administration, the conjugates of the invention are mixed with solubilizing agents such as Cremophor, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and mixtures thereof.

(186) Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can be a sterile injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

(187) The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

(188) In order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This can be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form may be accomplished by dissolving or suspending the drug in an oil vehicle.

(189) Compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing the conjugates of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol, or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.

(190) Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, (c) humectants such as glycerol, (d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, (e) solution retarding agents such as paraffin, (f) absorption accelerators such as quaternary ammonium compounds, (g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, (h) absorbents such as kaolin and bentonite clay, and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may include a buffering agent.

(191) Solid compositions of a similar type can be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the art of pharmacology. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of encapsulating compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type can be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polethylene glycols and the like.

(192) The active ingredient can be in a micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings, and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active ingredient can be admixed with at least one inert diluent such as sucrose, lactose, or starch. Such dosage forms may comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may comprise buffering agents. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of encapsulating compositions which can be used include polymeric substances and waxes.

(193) Dosage forms for topical and/or transdermal administration of a compound of this invention may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants and/or patches. Generally, the active ingredient is admixed under sterile conditions with a pharmaceutically acceptable carrier or excipient and/or any needed preservatives and/or buffers as can be required. Additionally, the present invention contemplates the use of transdermal patches, which often have the added advantage of providing controlled delivery of an active ingredient to the body. Such dosage forms can be prepared, for example, by dissolving and/or dispensing the active ingredient in the proper medium. Alternatively or additionally, the rate can be controlled by either providing a rate controlling membrane and/or by dispersing the active ingredient in a polymer matrix and/or gel.

(194) Suitable devices for use in delivering intradermal pharmaceutical compositions described herein include short needle devices such as those described in U.S. Pat. Nos. 4,886,499; 5,190,521; 5,328,483; 5,527,288; 4,270,537; 5,015,235; 5,141,496; and 5,417,662. Intradermal compositions can be administered by devices which limit the effective penetration length of a needle into the skin, such as those described in PCT publication WO 99/34850 and functional equivalents thereof. Alternatively or additionally, conventional syringes can be used in the classical mantoux method of intradermal administration. Jet injection devices which deliver liquid vaccines to the dermis via a liquid jet injector and/or via a needle which pierces the stratum corneum and produces a jet which reaches the dermis are suitable. Jet injection devices are described, for example, in U.S. Pat. Nos. 5,480,381; 5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569,189; 5,704,911; 5,383,851; 5,893,397; 5,466,220; 5,339,163; 5,312,335; 5,503,627; 5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941,880; 4,940,460; and PCT publications WO 97/37705 and WO 97/13537. Ballistic powder/particle delivery devices which use compressed gas to accelerate the compound in powder form through the outer layers of the skin to the dermis are suitable.

(195) Formulations suitable for topical administration include, but are not limited to, liquid and/or semi-liquid preparations such as liniments, lotions, oil-in-water and/or water-in-oil emulsions such as creams, ointments, and/or pastes, and/or solutions and/or suspensions. Topically administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of the active ingredient can be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients described herein.

(196) A pharmaceutical composition of the invention can be prepared, packaged, and/or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers, or from about 1 to about 6 nanometers. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant can be directed to disperse the powder and/or using a self-propelling solvent/powder dispensing container such as a device comprising the active ingredient dissolved and/or suspended in a low-boiling propellant in a sealed container. Such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. Alternatively, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. Dry powder compositions may include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.

(197) Low boiling propellants generally include liquid propellants having a boiling point of below 65 F. at atmospheric pressure. Generally the propellant may constitute 50 to 99.9% (w/w) of the composition, and the active ingredient may constitute 0.1 to 20% (w/w) of the composition. The propellant may further comprise additional ingredients such as a liquid non-ionic and/or solid anionic surfactant and/or a solid diluent (which may have a particle size of the same order as particles comprising the active ingredient).

(198) Pharmaceutical compositions of the invention formulated for pulmonary delivery may provide the active ingredient in the form of droplets of a solution and/or suspension. Such formulations can be prepared, packaged, and/or sold as aqueous and/or dilute alcoholic solutions and/or suspensions, optionally sterile, comprising the active ingredient, and may conveniently be administered using any nebulization and/or atomization device. Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, and/or a preservative such as methylhydroxybenzoate. The droplets provided by this route of administration may have an average diameter in the range from about 0.1 to about 200 nanometers.

(199) Formulations described herein as being useful for pulmonary delivery are useful for intranasal delivery of a pharmaceutical composition of the invention. Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 to 500 micrometers. Such a formulation is administered by rapid inhalation through the nasal passage from a container of the powder held close to the nares.

(200) Formulations for nasal administration may, for example, comprise from about as little as 0.1% (w/w) to as much as 100% (w/w) of the active ingredient, and may comprise one or more of the additional ingredients described herein. A pharmaceutical composition of the invention can be prepared, packaged, and/or sold in a formulation for buccal administration. Such formulations may, for example, be in the form of tablets and/or lozenges made using conventional methods, and may contain, for example, 0.1 to 20% (w/w) active ingredient, the balance comprising an orally dissolvable and/or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension comprising the active ingredient. Such powdered, aerosolized, and/or aerosolized formulations, when dispersed, may have an average particle and/or droplet size in the range from about 0.1 to about 200 nanometers, and may further comprise one or more of the additional ingredients described herein.

(201) A pharmaceutical composition of the invention can be prepared, packaged, and/or sold in a formulation for ophthalmic administration. Such formulations may, for example, be in the form of eye drops including, for example, a 0.1/1.0% (w/w) solution and/or suspension of the active ingredient in an aqueous or oily liquid carrier or excipient. Such drops may further comprise buffering agents, salts, and/or one or more other of the additional ingredients described herein. Other opthalmically-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form and/or in a liposomal preparation. Ear drops and/or eye drops are contemplated as being within the scope of this invention.

(202) Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with ordinary experimentation.

(203) Compounds provided herein are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject or organism will depend upon a variety of factors including the disease being treated and the severity of the disorder; the activity of the specific active ingredient employed; the specific composition employed; the age, body weight, general health, sex, and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific active ingredient employed; the duration of the treatment; drugs used in combination or coincidental with the specific active ingredient employed; and like factors well known in the medical arts.

(204) The compounds and compositions provided herein can be administered by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and/or drops), mucosal, nasal, bucal, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol. Specifically contemplated routes are oral administration, intravenous administration (e.g., systemic intravenous injection), regional administration via blood and/or lymph supply, and/or direct administration to an affected site. In general, the most appropriate route of administration will depend upon a variety of factors including the nature of the agent (e.g., its stability in the environment of the gastrointestinal tract), and/or the condition of the subject (e.g., whether the subject is able to tolerate oral administration).

(205) The exact amount of a compound required to achieve an effective amount will vary from subject to subject, depending, for example, on species, age, and general condition of a subject, severity of the side effects or disorder, identity of the particular compound, mode of administration, and the like. The desired dosage can be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage can be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations).

(206) In certain embodiments, an effective amount of a compound for administration one or more times a day to a 70 kg adult human may comprise about 0.0001 mg to about 3000 mg, about 0.0001 mg to about 2000 mg, about 0.0001 mg to about 1000 mg, about 0.001 mg to about 1000 mg, about 0.01 mg to about 1000 mg, about 0.1 mg to about 1000 mg, about 1 mg to about 1000 mg, about 1 mg to about 100 mg, about 10 mg to about 1000 mg, or about 100 mg to about 1000 mg, of a compound per unit dosage form.

(207) In certain embodiments, the compounds described herein may be at dosage levels sufficient to deliver from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, preferably from about 0.1 mg/kg to about 40 mg/kg, preferably from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, and more preferably from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic and/or prophylactic effect.

(208) It will be appreciated that dose ranges as described herein provide guidance for the administration of provided pharmaceutical compositions to an adult. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.

(209) It will be also appreciated that a compound or composition, as described herein, can be administered in combination with one or more additional pharmaceutical agents (e.g., therapeutically and/or prophylactically active agents). The compounds or compositions can be administered in combination with additional pharmaceutical agents that improve their activity (e.g., potency and/or efficacy in inhibiting one or more fungal or protozoan enzymes), bioavailability, reduce and/or modify their metabolism, inhibit their excretion, and/or modify their distribution within the body of a subject. It will also be appreciated that the therapy employed may achieve a desired effect for the same disorder, and/or it may achieve different effects.

(210) The compound or composition can be administered concurrently with, prior to, or subsequent to, one or more additional pharmaceutical agents, which may be useful as, e.g., combination therapies. Pharmaceutical agents include therapeutically active agents. Pharmaceutical agents also include prophylactically active agents. Each additional pharmaceutical agent may be administered at a dose and/or on a time schedule determined for that pharmaceutical agent. The additional pharmaceutical agents may also be administered together with each other and/or with the compound or composition described herein in a single dose or administered separately in different doses. The particular combination to employ in a regimen will take into account compatibility of the inventive compound with the additional pharmaceutical agent(s) and/or the desired therapeutic and/or prophylactic effect to be achieved. In general, it is expected that the additional pharmaceutical agent(s) utilized in combination be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually.

(211) Exemplary additional pharmaceutical agents include, but are not limited to, antifungal agents, antiprotozoan agents, anti-bacterial agents, anti-viral agents, anti-inflammatory agents, and pain-relieving agents. The antifungal agents that may be used in combination with the compounds described herein include, but are not limited to, polyene antifungal agents (e.g., amphotericin B, candicidin, filipin, hamycin, natamycin, nystatin, rimocidin) and azole antifungal agents (e.g., imidazole antifungal agents (e.g., bifonazole, butoconazole, clotrimazole, econazole, fenticonazole, isoconazole, ketoconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, tioconazole), triazole antifungal agents (e.g., albaconazole, fluconazole, isavuconazole, itraconazole, posaconazole, ravuconazole, terconazole, voriconazole), and thiazole antifungal agents (e.g., abafungin). In certain embodiments, the antifungal agents are antifungal agents against which resistance is developed by the target fungus. Exemplary antiprotozoan agents include, but are not limited to, eflornithine, furazolidone, melarsoprol, metronidazole, ornidazole, paromomycin sulfate, pentamidine, pyrimethamine, tinidazole, artemisinin, artesunate, atovaquone, chloroquine, amodiaquine, lumefantrine, mefloquine, sulfadoxine, dihydroartemisinin, piperaquine, quinine, primaquine, suramin, fexinidazole, nifurtimox, and nitazoxanide. In some embodiments, an antiprotozoan agent is a lincosamide antibiotic, such as clindamycin. In some embodiments, an antiprotozoan agent is a macrolide antibiotic, such as azithromycin. In certain embodiments, the antiprotozoan agents are antiprotozoan agents against which resistance is developed by the target protozoon. Pharmaceutical agents include small organic molecules such as drug compounds (e.g., compounds approved for human or veterinary use by the U.S. Food and Drug Administration as provided in the Code of Federal Regulations (CFR)), peptides, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNAs, RNAs, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins, and cells.

(212) Also encompassed by the invention are kits (e.g., pharmaceutical packs). The kits provided may comprise an inventive pharmaceutical composition or compound and a container (e.g., a vial, ampule, bottle, syringe, and/or dispenser package, or other suitable container). In some embodiments, provided kits may optionally further include a second container comprising a pharmaceutical excipient for dilution or suspension of an inventive pharmaceutical composition or compound. In some embodiments, the inventive pharmaceutical composition or compound provided in the first container and the second container are combined to form one unit dosage form.

(213) Thus, in one aspect, provided are kits including a first container comprising a compound described herein, or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, or a pharmaceutical composition thereof. In certain embodiments, the kits described herein are useful in preventing and/or treating a fungal or protozoan infection in a subject. In certain embodiments, the kits are useful in inhibiting the activity of a fungal or protozoan enzyme (e.g., mitochondrial phosphate carrier protein) in a subject or biological sample. In certain embodiments, the kits are useful in killing a fungus or inhibiting the growth of a fungus. In certain embodiments, the kits are useful in killing a protozoon or inhibiting the growth of a protozoon. In certain embodiments, the kits further include instructions for administering the compound, or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, or a pharmaceutical composition thereof. In certain embodiments, the kits and instructions provide for treating and/or preventing a fungal or protozoan infection. In certain embodiments, the kits and instructions provide for inhibiting the activity of a fungal or protozoan enzyme. In certain embodiments, the kits and instructions provide for killing a fungus or inhibiting the growth of a fungus. In certain embodiments, the kits and instructions provide for killing a protozoon or inhibiting the growth of a protozoon. The kit may include one or more additional pharmaceutical agents described herein (such as antifungal agents (e.g., azole antifungal agents (e.g., fluconazole) and polyene antifungal agents (e.g., amphotericin B)) and/or antiprotozoan agents as a separate composition.

(214) Methods of Treatment and Uses

(215) In one aspect, the present invention provides methods for the treatment and/or prevention of a fungal or protozoan infection in a subject. In certain embodiments, the infection in the subject is caused by a fungus. In certain embodiments, the fungus is yeast. In certain embodiments, the fungus is a mold. In certain embodiments, the fungus that causes the fungal infection is a Candida species. In certain embodiments, the fungus is a Candida albicans strain. In certain embodiments, the fungus is Candida albicans CaCi-2. In certain embodiments, the fungus is Candida albicans CaCi-17. In certain embodiments, the fungus is Candida albicans strain 90028. In certain embodiments, the fungus is Candida albicans strain Gu5. In certain embodiments, the fungus is Candida albicans strain CTBT. In certain embodiments, the fungus is a Candida glabrata strain. Additional Candida species include, but are not limited to, C. krusii, C. rugosa, C. parapsilosis, C. tropicalis, C. dubliniensis, C. lusitaniae, C. guilliermondii, C. famata, C. kefyr, C. pelliculosa, C. lipolytica, C. inconspicua, C. sake, C. lambica, C. norvegensis, and C. zeylanoides. In certain embodiments, the fungus is a Saccharomyces species. In certain embodiments, the fungus is a Saccharomyces cerevisiae strain. In certain embodiments, the fungus is Saccharomyces cerevisiae W303 reporter strain (ATCC 208352). Additional Saccharomyces species include, but are not limited to, Saccharomyces bayanus, Saccharomyces boulardii, Saccharomyces bulderi, Saccharomyces cariocanus, Saccharomyces cariocus, Saccharomyces chevalieri, Saccharomyces dairenensis, Saccharomyces ellipsoideus, Saccharomyces eubayanus, Saccharomyces exiguus, Saccharomycesflorentinus, Saccharomyces kluyveri, Saccharomyces martiniae, Saccharomyces monacensis, Saccharomyces norbensis, Saccharomyces paradoxus, Saccharomyces pastorianus, Saccharomyces spencerorum, Saccharomyces turicensis, Saccharomyces unisporus, Saccharomyces uvarum, and Saccharomyces zonatus. In certain embodiments, the fungus is a Aspergillus species. In certain embodiments, the fungus is a Aspergillus terreus strain. Additional Aspergillus species include, but are not limited to, A. clavatus, A. fumigatus, A. niger, and A. flavus. In certain embodiments, the fungus is a Cryptococcus species. In certain embodiments, the fungus is a Histoplasma species. In certain embodiments, the fungus is a Rhizopus species. In certain embodiments, the fungus is a Mucor species. In some embodiments, the fungus is a member of the genus Coccidioides. In some embodiments, the fungus is a member of the phylum Ascomycota. In some embodiments, the fungus is a member of the phylum Basidiomycota. In some embodiments, the fungus is a member of the phylum Chytridiomycota. In some embodiments, the fungus is a member of the phylum Glomeromycota. In some embodiments, the fungus is a member of the phylum Zygomycota. The fungus may be any fungus including, but not limited to, a member of a genus selected from the group consisting of Aspergillus, Blastomyces, Candida, Coccidioides, Cryptococcus, Epidermophytum, Fusarium, Histoplasma, Malassezia, Microsporum, Mucor, Paracoccidioides, Pneumocystis, Pseudallescheria, Rhizopus, Scedosporium, Sporothrix, Stachybotrys, Saccharomyces, Trichophyton, Trichosporon, Bipolaris, Exserohilum, Curvularia, Alternaria, and Cladophialophora.

(216) In some embodiments, the fungus of the genus Blastomyces is Blastomyces dermatitidis. In some embodiments, the fungus of the genus Coccidioides is Coccidioides immitis or Coccidioides posadasii. In some embodiments, the fungus of the genus Cryptococcus is Cryptococcus neoformans, C. gattii, C. albidus, C. laurentii, or C. uniguttulas. In some embodiments, the fungus of the genus Epidermophyton is E. floccosum. In some embodiments, the fungus of the genus Fusarium is Fusarium graminearum Fusarium oxysporum fsp. cubense, a member of the Fusarium solani complex, Fusarium oxysporum, Fusarium verticillioides, or Fusarium proliferatum. In some embodiments, the fungus of the genus Histoplasma is Histoplasma capsulatum. In some embodiments, the fungus of the genus Malassezia is Malassezia furfur. In some embodiments, the fungus of the genus Mucor is M. circinelloides. In some embodiments, the fungus of the genus Paracoccidioides is Paracoccidioides brasiliensis. In some embodiments, the fungus of the genus Penicillium is Penicillium marneffei. In some embodiments, the fungus of the genus Pichia is Pichia anomala, Pichia guilliermondi. In some embodiments, the fungus of the genus Pneumocystis is Pneumocystis carinii or Pneumocystis jirovecii. In some embodiments, the fungus of the genus Pseudallescheria is Pseudallescheria boydii. In some embodiments, a parasite of the genus Rhizopus is Rhizopus oryzae. In some embodiments, the fungus of the genus Rhodotorula is Rhodotorula rubra. In some embodiments, the fungus of the genus Scedosporium is Scedosporium apiospermum. In some embodiments, the fungus of the genus Schizophyllum is Schizophyllum commune. In some embodiments, the fungus of the genus Sporothrix is Sporothrix schenckii. In some embodiments, the fungus of the genus Trichophyton is Trichophyton mentagrophytes, Trichophyton rubrum, Trichophyton verrucosum, Trichophyton tonsurans, or Trichophyton violaceum. In some embodiments, the fungus of the genus Trichosporon is Trichosporon asahii, Trichosporon cutaneum, Trichosporon inkin, or Trichosporon mucoides. In some embodiments, a member of the genus Exserohilum is Exserohilum rostratum, E. meginnisii, or E. longirostratum.

(217) In certain embodiments, the fungus is a fungus resistant to one or more azole antifungal agents. In certain embodiments, the fungus is a fungus resistant to fluconazole. In certain embodiments, the fungus is a fungus resistant to one or more polyene antifungal agents. In certain embodiments, the fungus is a fungus resistant to amphotericin B.

(218) In some embodiments, the fungus is a pathogen that affects one or more cultivated plants. In some embodiments, the fungus is a member of the genus Magnaporthe, Ophiostoma, Cryphonectria, Thielaviopsis, Verticillium, Fusarium, Ustilago, Alternaria, Rhizoctonia, Phakospora, Puccinia, or Cochliobolus. In some embodiments, a member of the genus Plasmodium is a causative agent of malaria, e.g., Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale curtisi, Plasmodium ovale wallikeri, Plasmodium malariae, or Plasmodium knowlesi. In some embodiments, a member of the genus Cryptosporidium is C. parvum, C. hominis, C. canis, C. felis, C. meleagridis, or C. muris.

(219) The protozoan infection in the subject may be caused by a protozoon. In some embodiments, the protozoon is an Apicomplexan, e.g., a member of the genus Babesia, Plasmodium, Cryptosporidium, Isospora, or Toxoplasma. In some embodiments, the member of the genus Isospora is Isospora belli. In some embodiments, the member of the genus Babesia is Babesia microti or Babesia divergens. In some embodiments, the protozoon is Toxoplasma gondii. In some embodiments, the protozoon is a member of a genus of amoebae. In some embodiments, the protozoon is a member of the genus Entamoeba. In some embodiments, a member of the genus Entamoeba is Entamoeba histolytica, Entamoeba dispar, or Entamoeba moshkovskii. In some embodiments, the protozoon is a member of the genus Naegleria, e.g., Naegleria fowleri. In some embodiments, the protozoon is a member of the genus Balamuthia, e.g., Balamuthia mandriallaris. In some embodiments, the protozoon is a member of the genus Acanthameba. In some embodiments, a member of the genus Giardia is Giardia lamblia (sometimes referred to as Giardia duodenalis or Giardia intestinalis). In certain embodiments, the protozoon is a member of genus Sarcocystis, e.g., Sarcocystis bovohominis, Sarcocystis suihominis, or Sarcocystis bovicanis. In certain embodiments, a protozoon is a member of genus Cyclospora, e.g., Cyclospora cayetanensis. In certain embodiments, the protozoon is a member of genus Neospora, e.g., Neospora caninum. In certain embodiments, the protozoon is a member of genus Theileria, e.g., Theileria parva. In certain embodiments, the protozoon is a member of genus Trichomonas, e.g., Trichomonas vaginalis. In some embodiments, the protozoon is a kinetoplastid. In some embodiments, a kinetoplastid is a trypanosomatid, e.g., a member of the genus Leishmania (e.g., L. donovani, L. major, L, tropica, or L. braziliensis) or a member of the genus Trypanosoma (e.g., T. brucii, T. cruzii, T. congolense, or T. equiperdum).

(220) In certain embodiments, the compounds described herein, or a composition thereof, inhibits a eukaryotic parasite.

(221) In certain embodiments, the compounds described herein, or a composition thereof, inhibits an enzyme of a microbial organism that requires mitochondrial respiratory function for virulence in a host of the microbial organism.

(222) The compounds of the present invention, or pharmaceutical compositions thereof, may inhibit the fungal or protozoan mitochondrial phosphate carrier protein. In certain embodiments, the compounds described herein (e.g., compounds of Formula (I)), or pharmaceutical compositions thereof, inhibit the fungal or protozoan mitochondrial phosphate carrier protein with selectivity over the analogous human enzyme. This activity results in single-agent killing of pathogenic Saccharomyces molds. In vitro this activity sensitizes fungal or protozoan pathogens (e.g., Candida albicans) to the commonly used antifungal agents (e.g., azole antifungal agents (e.g., fluconazole) and polyene antifungal agents (e.g., amphotericin B)) and/or antiprotozoan agents, and it is hypothesized that these compounds would have enhanced activity in a whole animal model of infection. In addition, these compounds may have use in the control of agricultural fungal or protozoan pathogens.

(223) In certain embodiments, the subject described herein is a human. In certain embodiments, the subject is an animal. The animal may be of either sex and may be at any stage of development. In certain embodiments, the subject is a fish. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a domesticated animal, such as a dog, cat, cow, pig, horse, sheep, or goat. In certain embodiments, the subject is a companion animal such as a dog or cat. In certain embodiments, the subject is a livestock animal such as a cow, pig, horse, sheep, or goat. In certain embodiments, the subject is a zoo animal. In another embodiment, the subject is a research animal such as a rodent (e.g., mouse, rat), dog, pig, or non-human primate. In certain embodiments, the animal is a genetically engineered animal. In certain embodiments, the animal is a transgenic animal. In certain embodiments, the subject is immunocompromised. For example, the subject may have reduced immune system function as a result of a disease such as HIV infection, acquired immunodeficiency syndrome (AIDS), cancer (e.g., solid tumor or leukemia), bone marrow disorder, or a genetic immunodeficiency. In some embodiments, the subject is immunocompromised as a result of a medication, e.g., immunosuppressive therapy or chemotherapy, bone marrow transplant, stem cell transplant, or exposure to radiation. In some embodiments, reduced immune system function comprises neutropenia. In some embodiments, the subject suffers from a nosocomial fungal infection.

(224) Another aspect of the present invention involves methods of preventing a fungal infection in a subject who was or may be exposed to a fungus. In certain embodiments, the subject has been exposed to a fungus. In certain embodiments, the subject may have been exposed to a fungus. In these circumstances, the subject may not have developed the signs or symptoms of a fungal infection.

(225) Another aspect of the present invention involves methods of preventing a protozoan infection in a subject who was or may be exposed to a protozoon. In certain embodiments, the subject has been exposed to a protozoon. In certain embodiments, the subject may have been exposed to a protozoon. In these circumstances, the subject may not have developed the signs or symptoms of a protozoan infection.

(226) In another aspect, the present invention provides methods of inhibiting the activity of a fungal enzyme in a subject or biological sample. All types of biological samples described herein or known in the art are contemplated as being within the scope of the invention. In certain embodiments, the activity inhibited by the inventive methods is the activity of fungal mitochondrial phosphate carrier protein.

(227) In another aspect, the present invention provides methods of inhibiting the activity of a protozoan enzyme in a subject or biological sample. In certain embodiments, the activity inhibited by the inventive methods is the activity of protozoan mitochondrial phosphate carrier protein.

(228) Another aspect of the present invention relates to methods of killing a fungus or inhibiting the growth of a fungus. In certain embodiments, the fungus is killed. In certain embodiments, the growth of the fungus is inhibited.

(229) Another aspect of the present invention relates to methods of killing a protozoon or inhibiting the growth of a protozoon. In certain embodiments, the protozoon is killed. In certain embodiments, the growth of the protozoon is inhibited.

(230) In certain embodiments, the methods described herein include administering to a subject, contacting a biological sample, or contacting a fungus and/or protozoon with an effective amount of a compound described herein, or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, or a composition thereof. In certain embodiments, the methods described herein include administering to a subject, contacting a biological sample, or contacting a fungus and/or protozoon with an effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof, or a composition thereof. In certain embodiments, the compound, or a composition thereof, is administered to a subject. In certain embodiments, the compound, or a composition thereof, is contacted with a biological sample. In certain embodiments, the compound, or a composition thereof, is contacted with a fungus and/or protozoon. In certain embodiments, the compound, or a composition thereof, is contacted with a fungus and/or protozoon in an agricultural setting. In certain embodiments, the compound, or a composition thereof, is contacted with a fungus and/or protozoon in an environmental setting. In certain embodiments, the compound, or a composition thereof, is contacted with a fungus and/or protozoon in a healthcare setting. In certain embodiments, the compound, or a composition thereof, is contacted with a fungus and/or protozoon in a clinical setting. In certain embodiments, the compound, or a composition thereof, is contacted with a fungus and/or protozoon in a research setting. In certain embodiments, the compound, or a composition thereof, is contacted with a fungus and/or protozoon in or on a subject.

(231) In certain embodiments, the compound, or a composition thereof, is contacted with a fungus and/or protozoon in or on a plant. In certain embodiments, the plant is a land plant. In certain embodiments, the plant is a non-vascular land plant. In certain embodiments, the plant is a vascular land plant. In certain embodiments, the plant is a seed plant. In certain embodiments, the plant is a cultivated plant. In certain embodiments, the plant is a dicot. In certain embodiments, the plant is a monocot. In certain embodiments, the plant is a flowering plant. In some embodiments, the plant is a cereal plant, e.g., maize, corn, wheat, rice, oat, barley, rye, or millet. In some embodiments, the plant is a legume, e.g., a bean plant, e.g., soybean plant. In some embodiments, the plant is a tree or shrub. In certain embodiments, the compound is contacted with a leaf, branch, trunk, root, or seed. In some embodiments, the compound is contacted with a plant by spraying, dusting, introducing the compound into soil into which a seed is to be planted or has been planted or in which a plant is growing. A compound of the invention may be formulated with one or more vehicles appropriate for use in agricultural setting and/or may be present in a composition together with one or more other compounds useful for agricultural purposes, such as other antifungal and/or antiprotozoan agent(s). In some embodiments, a compound of the invention is used to inhibit fungal and/or protozoan growth on a harvested plant material, e.g., crops or seeds, which may be stored for future use. In certain embodiments, the compound, or a composition thereof, is contacted with a fungus and/or protozoon in or on the soil. In certain embodiments, the compound, or a composition thereof, is contacted with a fungus and/or protozoon in water. The compounds described herein may be capable of killing or controlling various fungi and/or protozoa without adversely affecting plants, such as cultivated plants. The compounds described herein may also be readily decomposable and/or may present no substantial acute and chronic toxicity to mammals, such as humans.

(232) The methods of the present invention may further comprise administering to a subject, contacting a biological sample, or contacting a fungus and/or protozoon with one or more additional pharmaceutical agents in combination with a compound described herein, or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, or composition thereof. In certain embodiments, the additional pharmaceutical agents are as described herein. The additional pharmaceutical agent may be an antifungal agent. In certain embodiments, the additional pharmaceutical agent is an azole antifungal agent. In certain embodiments, the additional pharmaceutical agent is fluconazole. In certain embodiments, the additional pharmaceutical agent is a polyene antifungal agent. In certain embodiments, the additional pharmaceutical agent is amphotericin B. The additional pharmaceutical agent may also be an antiprotozoal agent. In certain embodiments, the additional pharmaceutical agent is an inhibitor of fungal and/or protozoan mitochondrial phosphate carrier protein.

(233) In certain embodiments, the additional pharmaceutical agent inhibits synthesis of a component of the fungal plasma membrane, such as ergosterol. In certain embodiments, the additional pharmaceutical agent inhibits a fungal enzyme. In certain embodiments, the fungal enzyme is involved in synthesis of a component of the fungal membrane. In certain embodiments, the enzyme is fungal lanosterol 14-demethylase. In certain embodiments, the additional pharmaceutical agent is an allylamine. Allylamines inhibit squalene epoxidase, an enzyme required for ergosterol synthesis. In some embodiments, the allylamine is amorolfin, butenafine, naftifine, or terbinafine.

(234) In certain embodiments, the additional pharmaceutical agent inhibits synthesis of a component of the fungal cell wall, such as fungal glucan. In some embodiments, the additional pharmaceutical agent inhibits fungal enzyme 1,3- glucan synthase. In some embodiments, the additional pharmaceutical agent is an echinocandin, such as caspofungin, micafungin, or anidulafungin. In some embodiments, the additional pharmaceutical agent is a polyene antifungal agent, such as amphotericin B, amphotericin A, nystatin, candicidin, filipin, hamycin, natamycin, timocidin, filipin, pimaricin, rimocidin, eurocidin, candidin, perimycin, levorin, or trichomycin.

(235) The inventive compounds or compositions may synergistically augment the fungal- and/or protozoan-inhibitory activity of the additional pharmaceutical agent(s). Therefore, the combination of the inventive compound and the additional pharmaceutical agent(s) may be useful in inhibiting the activity of a fungal and/or protozoan enzyme that is resistant to the additional pharmaceutical agent(s) in the absence the inventive compounds. The combination of the inventive compound and the additional pharmaceutical agent(s) may also be useful in treating and/or preventing a fungal and/or protozoan infection caused by a fungus and/or protozoon resistant to the additional pharmaceutical agent(s) in the absence of the inventive compound. The combination of the inventive compound and the additional pharmaceutical agent(s) may further be useful in killing a fungus and/or protozoon or inhibiting the growth of a fungus and/or protozoon resistant to the additional pharmaceutical agent(s) in the absence of the inventive compound. In some embodiments, resistance is innate resistance. In some embodiments, resistance is acquired resistance. Resistance may be acquired as a result of a mutation. In some embodiments, a mutation is in a gene that encodes a target of an antifungal and/or antiprotozoan agent or a gene in the same biosynthetic pathway. In some embodiments, a mutation is in a gene that encodes a transcription factor that results in overexpression of a target of an antifungal and/or antiprotozoan agent or a gene in the same biosynthetic pathway.

(236) In certain embodiments, a compound of the invention or composition comprising such compound may be used to inhibit fungal and/or protozoan growth on or in an inanimate object or in an environment such as the interior of a building that may contain one or more fungal and/or protozoan cells or spores.

(237) In some embodiments, the methods of the invention comprise detecting a fungus or protozoan. In some embodiments, the methods of the invention comprise diagnosing a subject as having a fungal infection or a protozoal infection. Methods for detecting fungi and protozoa, and methods for diagnosis of fungal or protozoal infections, are known in the art. In some embodiments, the methods of the invention comprise obtaining a sample and testing the sample for presence of a fungus, fungal product, protozoan, or protozoal product. In some embodiments, the methods of the invention comprise obtaining a biological sample from a subject and testing the sample for presence of a fungus, fungal product, protozoan, or protozoal product. In some embodiments, the methods of the invention comprise obtaining a biological sample from a subject and testing the sample for presence of an antibody to a fungus, protozoan, fungal antigen, or protozoal antigen. In some embodiments, a fungus produces a mycotoxin. In some embodiments, a sample is tested for presence of a mycotoxin. In some embodiments, a sample contains a mycotoxin.

(238) In some embodiments, a sample comprises soil, plant material, dust, air, and/or water. In some embodiments, a sample is obtained from a food, beverage, or medication, In some embodiments, a sample is obtained by collecting a material from an inanimate object, e.g., an inanimate object found in a healthcare setting or environmental setting. In some embodiments, an inanimate object is a medical device, wall, floor, heating, air conditioning, or ventilating system.

(239) Another aspect of the invention relates to methods of screening a library of compounds to identify one or more compounds that are useful in the methods of the invention. In certain embodiments, the one or more compounds identified are useful for treating and/or preventing a fungal and/or protozoan infection in a subject. In certain embodiments, the one or more compounds identified are useful for inhibiting the activity of a fungal and/or protozoan enzyme in a subject or biological sample. In certain embodiments, the one or more compounds identified are useful for killing a fungus or inhibiting the growth of a fungus. In certain embodiments, the one or more compounds identified are useful for killing a protozoon or inhibiting the growth of a protozoon. In certain embodiments, the library of compounds is a library of compounds described herein. In certain embodiments, the methods of screening a library include providing at least two different compounds described herein, or pharmaceutically acceptable salts, solvates, hydrates, polymorphs, co-crystals, tautomers, stereoisomers, isotopically labeled derivatives, or prodrugs thereof; and performing at least one assay using the different compounds described herein, to identify one or more compounds that potentiates the effect of an antifungal agent, reverses the resistance of a fungus to a particular antifungal agent, and/or that kills a fungus or inhibits the growth of a fungus. In certain embodiments, the methods of screening a library include providing at least two different compounds described herein, or pharmaceutically acceptable salts, solvates, hydrates, polymorphs, co-crystals, tautomers, stereoisomers, isotopically labeled derivatives, or prodrugs thereof; and performing at least one assay using the different compounds described herein, to identify one or more compounds that potentiates the effect of an antiprotozoan agent, reverses the resistance of a protozoon to a particular antiprotozoan agent, and/or that kills a protozoan or inhibits the growth of a protozoan.

(240) In some aspects, the disclosure provides a method of identifying an antifungal agent or antiprotozoal agent, the method comprising identifying an agent that inhibits expression or activity of a fungal or protozoal mitochondrial phosphate carrier protein. In some embodiments the agent is identified in a high throughput screen. In some embodiments the agent does not substantially inhibit human SLC25A3. In some embodiments the agent does not substantially inhibit proliferation or survival of human cells. In some embodiments the agent has an IC.sub.50 for SLC25A3 at least 5, 10, 20, 50, 100, 250, 500, or 1,000 times as great (or more) as its IC.sub.50 for a fungal or protozoal mitochondrial carrier protein (e.g., MIR1).

(241) In some embodiments the agent is a small molecule. In some aspects, a small molecule as used herein, is an organic molecule that is less than about 2 kilodaltons (kDa) in mass. In some embodiments, a small molecule is less than about 1.5 kDa, or less than about 1 kDa. In some embodiments, a small molecule is less than about 800 daltons (Da), 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, or 100 Da. Often, a small molecule has a mass of at least 50 Da. In some embodiments, a small molecule contains multiple carbon-carbon bonds and can comprise one or more heteroatoms and/or one or more functional groups important for structural interaction with proteins (e.g., hydrogen bonding), e.g., an amine, carbonyl, hydroxyl, or carboxyl group, and in some embodiments at least two functional groups. Small molecules often comprise one or more cyclic carbon or heterocyclic structures and/or aromatic or polyaromatic structures, optionally substituted with one or more of the above functional groups.

(242) In some embodiments the method of identifying comprises contacting isolated fungal or protozoal mitochondria with a test agent and assaying the ability of the test agent to inhibit phosphate transport into the mitochondria. In some embodiments isolated fungal or protozoal mitochondria are contacted with a test agent and labeled phosphate (optionally .sup.32P-labeled phosphate), and the ability of the test agent to inhibit phosphate transport into the mitochondria is measured.

(243) In some embodiments, the method of identifying comprises (a) contacting fungal or protozoal cells that have reduced or absent expression or activity of endogenous mitochondrial phosphate transporter polypeptide (e.g., that have a deletion or disruption of the endogenous MIR1 gene), wherein the cells are rescued by an introduced nucleic acid encoding a fungal or protozoal mitochondrial phosphate transporter polypeptide, with a test agent; (b) contacting fungal or protozoal cells that have reduced or absent expression or activity of endogenous mitochondrial phosphate transporter polypeptide (e.g., that have a deletion or disruption of the endogenous MIR1 gene), wherein the cells are rescued by an introduced nucleic acid encoding at least a portion of a mammalian, e.g., human, mitochondrial phosphate transporter polypeptide, with said test agent; and (c) identifying the test agent as an inhibitor of expression or activity of the fungal or protozoal mitochondrial phosphate transporter polypeptide if the test agent inhibits growth of fungal or protozoal cells of (a) but does not substantially inhibit growth of fungal or protozoal cells of (b). In some embodiments the cells are cultured in or on media that contains glycerol as the carbon source. In some embodiments the yeast cells have a deleted, disrupted, or otherwise disabled PIC2 gene (S. cerevesiae Gene ID: 856779; RefSeq accession numbers NM_001178944.3.fwdarw.NP_010973.3) or homolog thereof in a different fungal species or protozoal species. PIC2 encodes a second mitochondrial phosphate transporter that may provide a very slight rescue of the growth defect of MIR1 deletion strains. It may normally be expressed at a lower level than MIR1.

(244) In some aspects, an agent identified using a method described herein may be considered a candidate antifungal or antiprotozoal agent. In some embodiments a method of identifying an antifungal agent or antiprotozoal agent further comprises testing the ability of a candidate antifungal agent identified as described herein to inhibit growth of a fungal or protozoal cells in vitro in vivo, or both. In some embodiments tests are performed using a test animal, e.g., a test mammal such as a rodent, e.g., a mouse, rat, hamster, or guinea pig, or a rabbit, or a non-human primate. In some embodiments test may be performed using an avian, bovine, ovine, porcine, or other mammalian species.

(245) The different compounds described herein may be generated by synthetic methods such as combinatorial chemistry (see, e.g., Ecker et al., Bio/Technology, (1995) 13:351-360 and U.S. Pat. No. 5,571,902). In certain embodiments, the different compounds are provided by liquid-phase or solution synthesis. In certain embodiments, the different compounds are provided by solid-phase synthesis. In certain embodiments, the different compounds are provided by a high-throughput, parallel, or combinatorial synthesis. In certain embodiments, the different compounds are provided by a low-throughput synthesis. In certain embodiments, the different compounds are provided by a one-pot synthesis. The different compounds may be provided robotically or manually. In certain embodiments, the step of providing at least two different compounds of the present invention include arraying into at least two vessels at least two different compounds of the present invention wherein the compounds are bound to solid supports, cleaving the compounds from the solid supports, and dissolving the cleaved compounds in a solvent. The solid supports include, but do not limit to, beads (e.g., resin beads and magnetic beads), hollow fibers, solid fibers, plates, dishes, flasks, meshes, screens, and membranes. In certain embodiments, the solid supports are beads. In certain embodiments, one solid support is capable of supporting at least 50 nmol of a compound. In certain embodiments, one solid support is capable of supporting at least 100 nmol of a compound. In certain embodiments, one solid support is capable of supporting at least 200 nmol of a compound. Each vessel may contain one or more support-bound compounds of the present invention. In certain embodiments, each vessel contains one support-bound compounds of the present invention. The solid supports and/or the compounds may be labeled with one or more labeling agents for the identification or detection of the compounds. The vessels may be wells of a microtiter plate. The solvent may be an inorganic solvent, organic solvent, or a mixture thereof. The steps of arraying, cleaving, and dissolving may be performed robotically or manually.

(246) Typically, the methods of screening a library of compounds involve at least one assay. In certain embodiments, the assay is performed to detect one or more characteristics associated with the treatment of a fungal or protozoan infection. The assay may be an immunoassay, such as a sandwich-type assay, competitive binding assay, one-step direct test, two-step test, or blot assay. The step of performing at least one assay may be performed robotically or manually.

(247) In yet another aspect, the present invention provides the compounds described herein, and pharmaceutically acceptable salts, solvates, hydrates, polymorphs, co-crystals, tautomers, stereoisomers, isotopically labeled derivatives, prodrugs, and compositions thereof, for use in the treatment and/or prevention of a fungal or protozoan infection in a subject.

(248) Another aspect of the present invention relates to the compounds described herein, and pharmaceutically acceptable salts, solvates, hydrates, polymorphs, co-crystals, tautomers, stereoisomers, isotopically labeled derivatives, prodrugs, and compositions thereof, for use in inhibiting the activity of a fungal or protozoan enzyme in a subject or biological sample.

(249) In still another aspect, the present invention provides the compounds described herein, and pharmaceutically acceptable salts, solvates, hydrates, polymorphs, co-crystals, tautomers, stereoisomers, isotopically labeled derivatives, prodrugs, and compositions thereof, for use in killing a fungus or inhibiting the growth of a fungus.

(250) In yet another aspect, the present invention provides the compounds described herein, and pharmaceutically acceptable salts, solvates, hydrates, polymorphs, co-crystals, tautomers, stereoisomers, isotopically labeled derivatives, prodrugs, and compositions thereof, for use in killing a protozoon or inhibiting the growth of a protozoan.

(251) In certain embodiments, the provided compounds, or compositions thereof, are useful as a pesticide (e.g., a fungicide).

(252) In certain embodiments, the provided compounds, or compositions thereof, are useful as a disinfectant.

(253) In some embodiments, a compound described herein (e.g., a compound of Formula (I)) is tested in an animal model of a fungal or protozoan infection. For example, in some embodiments, a non-human animal such as a rodent (e.g., a mouse) or non-human primate is experimentally infected with a fungus or protozoan. A compound described herein is administered one or more times to the non-human animal. In some embodiments, an additional antifungal or antiprotozoan agent is also administered. The ability of the compound, or compound combination (i.e., the compound and the additional antifungal or antiprotozoan agent), to reduce mortality or morbidity due to the fungus or protozoan or to reduce fungal or protozoan burden in one or more organs, tissues, or the blood is assessed at one or more time points.

EXAMPLES

(254) In order that the invention described herein may be more fully understood, the following examples are set forth. The synthetic and biological examples described in this application are offered to illustrate the compounds, pharmaceutical compositions, and methods provided herein and are not to be construed in any way as limiting their scope.

Example 1

Compounds Screening Strategy

(255) The various metabolic pathways of medically relevant fungi have recently come under investigation for their roles in fungal pathogenicity and the acquisition of drug resistance (Shingu-Vazquez et al., Eukaryot. Cell 2011, 10(11):1376-83; Martins et al., J. Bioenerg. Biomembr. 2011, 43(1):81-8). Specifically, the mitochondrion and its numerous components and functions are emerging as factors in determining the effectiveness of current antimycotic therapies in controlling human fungal infections. In vitro experiments have shown that loss of mitochondrial DNA (mtDNA) in C. glabrata correlates to increased resistance to azole antifungals (Brun et al., J. Antimicrob. Chemother. 2005, 56:307-314). Paradoxically, clinical isolates of azole-resistant C. glabrata rarely show impairment of mtDNA function. The significance of the seemingly contradictory in vitro assays remains to be seen (Bouchara et al., J. Med. Microbiol. 2000, 49(11):977-84; Ferrari et al., PLoS Pathog. 2009, 5(1):e1000268).

(256) The importance of mitochondrial function to fungal virulence is no better understood. C. glabrata with dysfunctional or missing mtDNA, also known as petite strains, are documented as being less pathogenic than non-petite strains (Brun et al., J. Antimicrob. Chemother. 2005, 56:307-314). However, this observation is only reproducible when the genetic damage is artificially induced with chemical reagents such as ethidium bromide. The work of Ferrari et al. showed the C. glabrata petite mutant clinical isolate BPY41 was actually more virulent than its non-petite parent BPY40 in murine infection models (Ferrari et al., Antimicrob. Agents Chemother. 2011, 55(5):1852-60). The 15-day survival rate of mice infected with BPY41 was 25% compared to 65% when infected with BPY40. Similarly, murine fungal loads of BPY41 after 7-day infection were 10- to 100-fold greater on average. Interestingly, when BPY40 was treated with ethidium bromide to generate a petite mutant, this newly generated petite strain was noticeably less pathogenic than BPY41, with a 15-day survival rate of 85% but similar fungal loads to its parent strain BPY40 (Ferrari et al., Antimicrob. Agents Chemother. 2011, 55(5):1852-60). These conflicting observations underscore the need to better understand the role mitochondria play in pathogenic fungi.

(257) Similar work with Candida albicans has also proven challenging because C. albicans petite mutants are more difficult to obtain (Abuhatab et al., Biochem. Soc. Trans. 1992, 20(1):63S; Aoki et al., J. Med. Vet. Mycol. 1987, 25(4):269-77). In one case, Cheng, et al. were able to produce a viable petite mutant by passing C. albicans SC5314 through murine spleens by intravenous inoculation (Cheng et al., Cell Microbiol. 2007, 9(2):492-501; Cheng et al., Antimicrob. Agents Chemother. 2007, 51(5):1855-8). After five serial passages, a mutant strain named P5 was isolated and characterized. P5 exhibited several phenotypes characteristic of respiratory mutants, including the inability to proliferate on glucose-deficient media. Subsequent oxygen consumption measurements suggested that while the electron transport chain was intact and functional, it was no longer coupled to ATP synthesis (Cheng et al., Antimicrob. Agents Chemother. 2007, 51(5): 1855-8). With regards to clinical relevance, P5 was determined to be 10-fold less susceptible to fluconazole and voriconazole, more resistant to phagocytosis and neutrophils, and less sensitive to superoxide generators. In addition, P5 sustained a completely non-lethal infection in mice for up to 60 days. In contrast, SC5314 infection resulted in 100% mortality within 9 days (Cheng et al., Cell Microbiol. 2007, 9(2):492-501).

(258) The reported work with C. glabrata and C. albicans petite mutants suggests that modifying fungal mtDNA in laboratory settings with chemical agents cannot be relied upon to investigate the clinical behavior of these invasive fungi. Similarly, clinically isolated Candida petite mutants may not be amenable to further experimentation. The isolation of petite mutants from patients rarely occurs, and for the few strains harvested thus far, the occurrence of nuclear DNA mutations cannot be ruled out. Identifying specific mitochondrial contributions to Candida clinical behavior is thus complicated because the selective pressures of the host environment cannot be reasonably and reliably controlled to allow modification of only fungal mtDNA.

(259) An alternative, untested approach would be to modulate fungal mitochondria function during an active infection. Unfortunately, the current toolbox of mitochondria modulators (FIG. 1) target the oxidative phosphorylation pathway and do not discriminate between fungal or mammalian mitochondria (von Jagow et al., Methods Enzymol. 1986, 126:253-71; Ueki et al., Curr. Opin. Anti-Infective Invest. Drugs 2000, 2(4):387-398; Sridhara et al., J. Pharmaceutical. Res. 2011, 4(2):496-500; Mathre, Pest Biochem. Physiol. 1971, 1(2):216-224). Disruption of this process is typically a fatal proposition for both the invading fungi and its mammalian host. A possible fungal-selective mitochondria inhibitor is the antibacterial agent ilicicolin H (FIG. 1, compound 1) (Hayakawa et al., J. Antibiot. 1971, 24(9):653-4). Studies by Trumpower et al. have shown that ilicicolin H binds to purified S. cerevisiae cytochrome bc.sub.1 complex with 100-fold selectivity over isolated bovine bc.sub.1 complex (IC.sub.50=0.003 M and 0.200 M, respectively) (Gutierrez-Cirlos et al., J. Biol. Chem. 2004, 279(10):8708-14; Rotsaert et al., Biochim. Biophys. Acta 2008, 1777(2):211-9). However, compound 1 displays considerable toxicity against HeLa cells (MIC=2 g/mL or 4.6 M) (Hayakawa et al., J. Antibiot. 1971, 24(9):653-4). Follow-up studies with ilicicolin H have been slow to emerge, possibly because there are no longer any commercial sources of this natural product.

(260) Here compounds identified are capable of modulating cellular respiration in fungi, preferably by affecting mitochondrial function, without triggering a similar response in mammalian cells. It has been reported that fungal mitochondria possess several proteins lacking human orthologs (Shingu-Vazquez et al., Eukaryot. Cell 2011, 10(11):1376-83; Okamoto et al., Annu. Rev. Genet. 2005, 39:503-36). In this Example, a phenotypic assay tree is adopted to identify modulators of these unique targets. Such compounds are of great value for interrogating the metabolic requirements of fungal virulence and may be useful as new antifungal drugs that operate in a completely unexploited target space.

Example 2

Preparation of the Compounds

(261) Compounds of Formula (I) (e.g., probe compound I-B-4 (ML316)) may be prepared by the synthetic sequence outlined below in Scheme 1. Cyclohexanone was subjected to a Strecker reaction with 4-fluoroaniline and trimethylsilyl cyanide. The resulting adduct 4 was cyclized to the hydantoin 5 with sodium cyanate, converted to the thiohydanatoin 6, and acylated with ethyl chloroformate to provide compound I-B-4.

(262) ##STR00071##

2.1. General Methods

(263) All reagents and solvents were purchased from commercial vendors and used as received. NMR spectra were recorded on a Bruker 300 MHz or Varian UNITY INOVA 500 MHz spectrometer as indicated. Proton, fluorine, and carbon chemical shifts are reported in parts per million (ppm; ) relative to tetramethylsilane, CFCl.sub.3, or CDCl.sub.3 solvent (.sup.1H 0, .sup.19F 0, .sup.13C 77.16, respectively). NMR data are reported as follows: chemical shifts, multiplicity (obs=obscured, app=apparent, br=broad, s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet); coupling constant(s) in Hz; integration. Unless otherwise indicated, NMR data were collected at 25 C. Flash chromatography was performed using 40-60 um Silica Gel (60 mesh) on a Teledyne Isco Combiflash Rf system. Tandem liquid chromatography/mass spectrometry (LCMS) was performed on a Waters 2795 separations module and Waters 3100 mass detector. Analytical thin layer chromatography (TLC) was performed on EM Reagent 0.25 mm silica gel 60-F plates. Visualization was accomplished with UV light and aqueous potassium permanganate (KMnO.sub.4) stain followed by heating. High-resolution mass spectra were obtained at the MIT Mass Spectrometry Facility with a Bruker Daltonics APEXIV 4.7 Tesla Fourier Transform Ion Cyclotron Resonance mass spectrometer. Compound purity and identity were determined by UPLC-MS (Waters, Milford, Mass.). Purity was measured by UV absorbance at 210 nm. Identity was determined on a SQ mass spectrometer by positive electrospray ionization. Mobile Phase A consisted of either 0.1% ammonium hydroxide or 0.1% trifluoroacetic acid in water, while mobile Phase B consisted of the same additives in acetonitrile. The gradient ran from 5% to 95% mobile Phase B over 0.8 minutes at 0.45 mL/min. An Acquity BEH C18, 1.7 m, 1.050 mm column was used with column temperature maintained at 65 C. Compounds were dissolved in DMSO at a nominal concentration of 1 mg/mL, and 0.25 L of this solution was injected.

2.2. 1-((4-fluorophenyl)amino)cyclohexanecarbonitrile (4)

(264) ##STR00072##

(265) Cyclohexanone (1.0 g, 10.2 mmol) was dissolved in glacial acetic acid (15 mL) and cooled to 10 C. 4-Fluoroaniline (1.4 g, 12.2 mmol, 1.2 equiv.) was added in portions to the reaction, and the resulting mixture was stirred at 10 C. for 15 minutes. Trimethylsilyl cyanide (1.4 mL, 11.2 mmol, 1.1 equiv.) was then added. The reaction was warmed to room temperature and stirred for 15 hours. The reaction was poured into ice-cold ammonium hydroxide solution (30 mL, 20% v/v in water) to give a basic solution (pH>10). This mixture was then extracted with dichloromethane (260 mL). The combined organic extracts were washed with brine (80 mL), shaken over magnesium sulfate, filtered, and concentrated under reduced pressure. The crude residue was diluted with hexanes, and the precipitated solids were collected by filtration. After washing with more hexanes, the solids were air-dried and used without further purification (1.7 g, 76%). .sup.1H NMR (300 MHz, CDCl.sub.3): 7.02-6.88 (m, 4H), 3.46 (br. s, 1H), 2.29-2.19 (m, 2H), 1.85-1.54 (m, 7H), 1.41-1.22 (m, 1H); MS m/z (ESI+): 219 (M+H).

2.3. 1-(4-fluorophenyl)-1,3-diazaspiro[4.5]decane-2,4-dione (5)

(266) ##STR00073##

(267) Compound 4 (1.7 g, 7.8 mmol) was suspended in glacial acetic acid (20 mL) and treated with sodium cyanate (0.81 g, 12.5 mmol, 1.6 equiv.). The resulting mixture was heated to 60 C. and stirred at this temperature for 3 hours. A solution of concentrated hydrochloric acid (3 mL) in water (2 mL) was added to the reaction. The resulting mixture was then heated to 90 C. and stirred at this temperature for 30 minutes. The reaction was cooled to room temperature then poured into water (50 mL). The precipitated solids were collected by filtration, washed with water, and air-dried (1.13 g, 55%). .sup.1H NMR (300 MHz, CDCl.sub.3): 8.24 (s, 1H), 7.22-7.10 (m, 4H), 2.15-1.92 (m, 4H), 1.73-1.45 (m, 5H), 1.07-0.90 (m, 1H); MS m/z (ESI+): 263 (M+H).

2.4. 1-(4-fluorophenyl)-2-thioxo-1,3-diazaspiro[4.5]decan-4-one (6)

(268) ##STR00074##

(269) A solution of compound 5 (0.54 g, 2.1 mmol) in toluene (17 mL) was treated with Lawesson's reagent (0.46 g, 1.1 mmol, 0.55 equiv.). This mixture was heated to reflux and stirred for 5 hours. The reaction was cooled to room temperature and concentrated under reduced pressure. The crude material was purified by column chromatography over silica gel (hexanes/ethyl acetate: 100/0 to 90/10) to give the title compound as a solid (0.50 g, 87%). .sup.1H NMR (300 MHz, CDCl.sub.3): 9.13 (s, 1H), 7.23-7.15 (m, 4H), 2.17-1.96 (m, 4H), 1.75-1.48 (m, 5H), 1.07-0.91 (m, 1H); MS m/z (ESI.sup.+): 279 (M+H).

2.5. Ethyl 1-(4-fluorophenyl)-4-oxo-2-thioxo-1,3-diazaspiro[4.5]decane-3-carboxylate (I-B-4)

(270) ##STR00075##

(271) Compound 6 (0.26 g, 0.93 mmol) was dissolved in dichloromethane (9.3 mL) and cooled to 0 C. Triethylamine (0.18 mL, 1.9 mmol, 2.0 equiv.) was added, followed by ethyl chloroformate (0.26 mL, 1.9 mmol, 2.0 equiv.). The reaction was stirred at 0 C. for 2 hours before quenching with water (10 mL). The layers were separated and the aqueous phase was extracted with dichloromethane (310 mL). The combined organic layers were dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The crude material was purified by column chromatography over silica gel (hexanes/ethyl acetate: 100/0 to 80/20) to give the title compound as a white solid (87.1 mg, 27%). .sup.1H NMR (500 MHz, CDCl.sub.3): 7.25-7.17 (m, 4H), 4.53 (q, J=7.1 Hz, 2H), 2.13-2.02 (m, 4H), 1.77-1.65 (m, 3H), 1.57 (td, J=13.5, 4.2 Hz, 2H), 1.47 (t, J=7.1 Hz, 3H), 1.06-0.95 (m, 1H); .sup.19F NMR (282 MHz, CDCl.sub.3): 110.88 (tt, J=7.6, 5.3 Hz); .sup.13C NMR (125 MHz, CDCl.sub.3): 177.3, 171.7, 162.9 (d, J.sub.C-F=250.6 Hz), 148.5, 132.4 (d, J.sub.C-CF=8.9 Hz), 130.4, 116.9 (d, J.sub.C-CF=22.9 Hz), 67.3, 65.8, 32.4, 23.9, 20.6, 13.9; HRMS m/z (ESI.sup.+): calculated for C.sub.17H.sub.19FN.sub.2O.sub.3 SNa [M+Na]373.0993. found 373.1100.

Example 3

Analytical Assays of the Compounds

(272) Compounds of Formula (I) (e.g., compound I-B-4 (ML316)) were analyzed by UPLC, .sup.1H, .sup.19F, and .sup.13C NMR spectroscopy, and high-resolution mass spectrometry. The data obtained from NMR and mass spectroscopy are consistent with the structure of the compounds, and UPLC (Ultra Performance Liquid Chromatography) indicates an isolated purity of greater than 95%.

(273) 3.1. Solubility

(274) Solubility was determined in phosphate buffered saline (PBS) pH 7.4 with 1% DMSO. Each compound was prepared in duplicate at 100 M in both 100% DMSO and PBS with 1% DMSO. Compounds were allowed to equilibrate at room temperature with a 250 rpm orbital shake for 24 hours. After equilibration, samples were analyzed by UPLC-MS (Waters, Milford, Mass.) with compounds detected by SIR detection on a single quadrupole mass spectrometer. The DMSO samples were used to create a two-point calibration curve to which the response in PBS was fit.

(275) 3.2. PBS Stability

(276) Stability was determined in the presence of PBS pH 7.4 with 0.1% DMSO. Each compound was prepared in duplicate on six separate plates and allowed to equilibrate at room temperature with a 250-rpm orbital shake for 48 hours. One plate was removed at each time point (0, 2, 4, 8, 24, and 48 hours). An aliquot was removed from each well and analyzed by UPLC-MS (Waters, Milford, Mass.) with compounds detected by SIR detection on a single quadrupole mass spectrometer. Additionally, to the remaining material at each time point, acetonitrile was added to force dissolution of compound (to test for recovery of compound). An aliquot of this was also analyzed by UPLC-MS.

(277) 3.3. GSH Stability

(278) Stability was determined in the presence of PBS pH 7.4 M and 50 M glutathione with 0.1% DMSO. Each compound was prepared in duplicate on six separate plates and allowed to equilibrate at room temperature with a 250-rpm orbital shake for 48 hours. One plate was removed at each time point (0, 2, 4, 8, 24, and 48 hours). An aliquot was removed from each well and analyzed by UPLC-MS (Waters, Milford, Mass.) with compounds detected by SIR detection on a single quadrupole mass spectrometer. Additionally, to the remaining material at each time point, acetonitrile was added to force dissolution of compound (to test for recovery of compound). An aliquot of this was also analyzed by UPLC-MS.

(279) 3.4. Plasma Protein Binding

(280) Plasma protein binding was determined by equilibrium dialysis using the Rapid Equilibrium Dialysis (RED) device (Pierce Biotechnology, Rockford, Ill.) for both human and mouse plasma. Each compound was prepared in duplicate at 5 M in plasma (0.95% acetonitrile, 0.05% DMSO) and added to one side of the membrane (200 L) with PBS pH 7.4 added to the other side (350 L). Compounds were incubated at 37 C. for 5 hours with a 250-rpm orbital shake. After incubation, samples were analyzed by UPLC-MS (Waters, Milford, Mass.) with compounds detected by SIR detection on a single quadrupole mass spectrometer.

(281) 3.5. Plasma Stability

(282) Plasma stability was determined at 37 C. at 5 hours in both human and mouse plasma. Each compound was prepared in duplicate at 5 M in plasma diluted 50/50 (v/v) with PBS pH 7.4 (0.95% acetonitrile, 0.05% DMSO). Compounds were incubated at 37 C. for 5 hours with a 250-rpm orbital shake with time points taken at 0 hours and 5 hours. Samples were analyzed by UPLC-MS (Waters, Milford, Mass.) with compounds detected by SIR detection on a single quadrupole mass spectrometer.

(283) 3.6. Results

(284) The solubility of the probe compound I-B-4 (ML316) was experimentally determined to be 1.1 M in phosphate buffered saline with 1% (v/v) DMSO. The probe is reasonably stable in PBS solution (>60% remaining after a 48-hour incubation). The data from the PBS stability assay is provided in FIG. 2. The probe compound I-B-4 is unstable in human and murine plasma with less than 1% remaining after incubation at 37 C. for 5 hours. The ethyl carbamate appears to be the primary source of instability since replacing the carbamate with a methyl group (compound I-A-10, CID56604835) significantly increases plasma stability (Table 1). Unfortunately, the stable analog compound I-A-10 (CID56604835) shows no activity against the C. albicans test strains (see Section 3.4 for full details).

(285) TABLE-US-00001 TABLE 1 Plasma Stability of Compound I-B-4 and Compounds I-A-1 and I-A-10. Compound # I-B-4 I-A-1 I-A-10 PubChem CID 56604860 3889161 56604835 ML# ML316 Not applicable Not applicable Plasma stability Human <1.0% <1.0% 95.7% (% remaining Murine <1.0% <1.0% 96.6% after 5 hours) Plasma protein Human N/A N/A 98.6% binding Murine N/A N/A 99.9% (% bound)

(286) The physical properties of compound I-B-4 (ML316) are summarized below in Table 2.

(287) TABLE-US-00002 TABLE 2 Computed Properties of the Probe Compound I-B-4 (ML316) ethyl 1-(4-fluorophenyl)-4-oxo-2- thioxo-1,3-diazaspiro[4.5]decane-3- IUPAC chemical name carboxylate PubChem CID 56604860 Molecular Formula C.sub.17H.sub.19FN.sub.2O.sub.3S Molecular Weight (g/mol) 350.41 Exact Mass (amu) 350.1100 ClogP* 4.57 Topological Polar Surface 49.85 Area* H-Bond Donors 0 H-Bond Acceptors 3 Rotatable Bond Count 4 *Calculated with ChemBioDraw Ultra, version 12.0.

Example 4

Biological Assays of the Compounds

(288) 4.1. Materials and Methods

(289) Materials and Reagents. Ilicicolin H was previously purchased from Analyticon Discovery (Catalog No. NP-005728) and was purified by column chromatography prior to use. Alamar Blue was purchased from Invitrogen (Catalog No. DAL1100). Amphotericin B was purchased from Sigma-Aldrich (Catalog No. A2411). Geldanamycin was obtained from AG Scientific (Catalog No. G-1047). JC-1 dye was obtained from Invitrogen (Catalog No. T-3168). Antimycin A was obtained from Sigma (Catalog No. A8674).

(290) Cell Lines. Candida albicans CaCi-2; a drug-resistant clinical isolate was provided by the Whitehead Institute (Redding et al., Clin. Infect. Dis. 1994, 18(2):240-2). This strain was used for the primary assay. Candida albicans CaCi-17; a strongly drug-resistant clinical isolate was provided by the Whitehead Institute (Redding et al., Clin. Infect. Dis. 1994, 18(2):240-2). This strain was used for the primary assay Saccharyomyces cerevisiae W303-1a (ATCC 208352); a well-characterized Saccharomyces strain used for determination of glycolytic vs. respiratory metabolism, as well as in the mitochondrial disruption assay. Candida glabrata (ATCC 2001); used for determination of glycolytic vs. respiratory metabolism. NIH-3T3 mammalian fibroblasts (ATCC; CRL no.1658); used for mammalian toxicity assays.

(291) A summary listing completed assays and corresponding PubChem AID numbers is provided in Table 11.

(292) TABLE-US-00003 TABLE 11 Summary of Completed Assays and AIDs PubChem Concentration Samples AID Type Target Range (M) Tested 558529 Primary CaCi-2 growth inhibition 26-0.01 39 588530 Primary CaCi-17 growth 26-0.01 39 inhibition 602190 Confirmatory CaCi-2 growth inhibition 26-0.1 24 (Powder) 602189 Confirmatory CaCi-17 growth inhibition 26-0.1 24 (Powder) 623899 Analogs CaCi-2 growth inhibition 26-0.01 55 623897 Analogs CaCi-17 growth 26-0.01 55 inhibition 623981, Analogs (low dose) CaCi-2 growth inhibition 3-0.000006 7 624006 623982 Analogs (low dose) CaCi-17 growth 3-0.000006 7 inhibition 588634 Secondary Fibroblast Toxicity 26-0.01 39 602394 Secondary (Analogs) Fibroblast Toxicity 26-0.01 55 623967, Secondary (Analogs) C glabrata on glucose 3-0.000006 7 624013 623965, Secondary (Analogs) C glabrata on glycerol 3-0.000006 7 624014 623971, Secondary (Analogs) S cerevisiae on glucose 3-0.000006 7 624025 623969, Secondary (Analogs) S cerevisiae on glycerol 3-0.000006 7 624012 624062 Secondary (Analogs) C. glabrata mitochondria 1.0 7 inhibition 624068 Secondary (Analogs) S. cerevisiae 1.0 7 mitochondria inhibition
4.2. Primary CaCi-2 (AID No. 588529) and CaCi-17 (588530), CaCi-2 Dose-Response Retest (AID Nos. 602190, 623899, 623981, 624006) and CaCi-17 Dose-Response Retest (AID Nos. 602189, 623897, 623982)

(293) The primary assay measures reduction of viability (as measured by reduced fluorescence in the presence of Alamar Blue) by potential inhibitors. The assay was performed using two Candida albicans clinical isolates to determine the range of the antifungal activity. In this assay, a fungal inoculum is dispensed into 384-well assay plates in which 100 nL of compounds have been pinned. After 48 hours of incubation at 30 C., Alamar Blue is added and fluorescence output is read. Compounds possessing an IC.sub.50 value less than 10 M were selected for further studies.

(294) Materials and Reagents

(295) Clear, flat bottom, black, 384-well plates (Corning, Catalog No. 3712BC Lot No. 35808016); Amphotericin (Sigma, Catalog No. A2411) 15 mM stock solution in DMSO; Pen/Strep (Gibco, Catalog No. 10378-016; Lot No. 21040170) 100 in PBS; Alamar Blue (AG Scientific, Catalog no.DAL 1100; Lot No. 151016SA); PBS without Calcium and Magnesium (Cellgro, Catalog No. 21-040-CV).

(296) Synthetic defined growth medium. RPMI 1640 medium, (powder without sodium bicarbonate; Invitrogen, Catalog No. 31800-089; Lot No. 648072); Uridine 8 mg/mL in water (Sigma, Catalog No. U3750, Lot No. 028KO760); Glucose 40% (w/v) in water (Sigma, Catalog No. G-5400); MOPS Buffer (Sigma, Catalog No. M-1254; Lot No. 098K0033). Prepare 1RPMI medium by dissolving 10.4 grams powdered medium in 800 mL water. Add 34.52 g MOPS. While stirring, adjust pH to 7.0 with 10 N NaOH. Add 10 mL uridine solution and 50 mL glucose solution; adjust final volume to 1000 mL. Filter sterilize.

(297) Fungal inoculum. Test Strains: C. albicans CaCi-2 and C. albicans CaCi-17. Inoculate 500 L of strain from cryopreserved stock into 250 mL shaker flask containing 30 mL growth medium. Shake at 30 C. overnight. Read OD 600 of 1 mL fungal culture in a cuvette using a standard optical density reader (Eppendorf BioPhotometer Plus), with growth medium as a background blank. Dilute to desired volume of fungal inoculums according to the following formula: (1/OD measured)(Desired Final Volume of Inoculum)0.3=Volume of fungal culture (L) to add to desired volume of growth medium. When added to media in wells, this yields a calculated starting OD of the fungal inoculum of 0.00015.

(298) Procedures Add Pen/Strep at 0.1 mL per 10 ml media (1% v/v). Use a Thermo Combi nL to dispense 20 L/well of assay media into all wells. Pin 100 nL test compound from compound plates into assay plates using CyBi-Well pin tool. Dispense 20 L/well of culture into the assay media in all wells. Incubate plates in a humidified (90% humidity) Liconic incubator at 37 C. without agitation for 48 hours. Dilute Alamar Blue Reagent 1:40 in Ca/Mg-free PBS. To all plates, add 5 L/well of the diluted Alamar to a final dilution factor of 1:200. Incubate the plates for an additional 2 hours. Read the Relative Fluorescence Intensity (RFU) of wells on a standard plate reader as a measure of relative fungal growth. Envision (Perkin Elmer) plate reader set-up: Ex 544 nm, Em 590 nm, Bandwidth 12 nm, Top read.
4.3. Counterscreen Mammalian Cell Toxicity Assay (AID Nos. 588634, 602394)

(299) Quiescent murine fibroblasts were subjected to incubation with compounds of interest for 48 hours. Subsequent cell viability was determined via measuring Alamar Blue fluorescence. Toxicity at less than 10 times the effective CaCi-2 IC.sub.50 or less than 20 M was an indication of a poor therapeutic index and resulted in exclusion of the associated compound from further consideration.

(300) Materials and Reagents

(301) Clear, flat bottom, black 384-well plates (Corning, Catalog No. 3712BC; Lot No. 35808016); Geldanamycin (AG Scientific, Catalog No. G-1047) 15 mM stock solution in DMSO; Alamar Blue (AG Scientific, Catalog No. DAL1100, Lot No. 151016SA); PBS without Calcium and Magnesium (Cellgro, Catalog No. 21-040-CV).

(302) Assay medium. Optimem medium (Invitrogen, Catalog No. 31985-070; Lot No. 548536); 2.5% (v/v) Fetal Bovine Serum (Hyclone, Catalog No. 30071.03; Lot No. ARF26748); 1% (v/v) Pen/Strep solution (Invitrogen, Catalog No. 15140-122; Lot No. 529891).

(303) Cell inoculum. Test Strain: NIH-3T3 mammalian fibroblasts (ATCC CRL No. 1658). Plate cells in 384-well plates at 1,000 cells/well in 40 L assay medium. Incubate plates overnight at 37 C. under 5% CO.sub.2.

(304) Procedures After overnight culture, pin compounds into wells at 100 nL/well using the CyBio CyBi-Well pinning instrument. Return the plates to the tissue culture incubator and incubate the culture for an additional 48 hours at 37 C. under 5% CO.sub.2. At the completion of this incubation, add Alamar Blue solution diluted 1:40 in PBS to each well (10 L/well) to achieve a final dilution of 1:200. Incubate the plates for an additional 2-3 hours at 37 C. under 5% CO.sub.2. Read the Relative Fluorescence Intensity (RFU) of wells on a standard plate reader as a measure of relative cell growth. Envision (Perkin Elmer) plate reader set-up: Ex 544 nm, Em 590 m, Bandwidth12 nm, Top read.
4.4. Secondary S. cerevisiae Inhibition in Glucose Media (AID Nos. 623971, 624025), S. cerevisiae Inhibition in Glycerol Media (AID Nos. 623969, 624012), C. glabrata Inhibition in Glucose Media (AID Nos. 623967, 624013), and C. glabrata Inhibition in Glycerol Media (AID Nos. 623965, 624014)

(305) Saccharomyces, while not a major human pathogen, is a useful lab model with well-developed genetic tools available. Candida glabrata is an increasingly prominent clinical problem for immune-compromised patients but with fewer genetic tools available with which to study it. These organisms can utilize alternative metabolic pathways to support growth and survival depending on the carbon source. By comparing the concentration-dependent inhibition of yeast growth in glycerol relative to glucose media, a quantitative assessment of a compound's ability to disrupt respiration can be determined. It is preferred that compounds in this assay have an IC.sub.50 of less than 10 M in both organisms when grown in glycerol media. It is also preferred that there is at least a 10-fold loss in potency against both organisms when they are grown in glucose media, thus indicating respiratory disruption.

(306) Secondary Glycerol Assay Protocol (AID 623969, 623965, 624014, 624012)

(307) Materials and Reagents

(308) Clear, flat bottom, black 384-well plates (Corning Catalog no. 3712BC; Lot no. 35808016); Amphotericin B (Sigma Catalog no. A2411) 15 mM stock solution in DMSO; Pen/Strep (Gibco Catalog no. 10378-016; Lot no21040170) 100 in PBS.

(309) Synthetic defined growth medium. Complete Supplement Mixture; (Sunrise Science, Catalog No. 1001-100); Yeast Nitrogen Base without ammonium sulfate (MP Biomedicals, Catalog No. 4027-012); Glycerol (Sigma Catalog No. G-9012). Prepare medium by dissolving 0.79 g Complete Supplement Mixture and 1.7 g Yeast Nitrogen Base in 800 mL water. Add 20 mL of glycerol. Adjust final volume to 1000 mL. Filter sterilize.

(310) Fungal Inoculum. Test Strains: C. glabrata (ATCC 2001) and Saccharomyces cerevisiae W303. Inoculate 100 L of yeast from cryopreserved stock into 250 mL shaker flask containing 20 mL growth medium. Shake at 30 C. overnight (about 16 hours). Read OD 600 of 1 mL of fungal culture in a cuvette using a standard optical density reader (Eppendorf BioPhotometer Plus), with growth medium as a background blank. Dilute to desired volume of fungal inoculum to produce an OD600 reading of 0.005 (S. cerevisiae) or 0.004 (C. glabrata).

(311) Procedures Use a Thermo Combi nL to dispense 20 L/well of assay media into all wells. Add 2 L of Amphotericin B to control wells. Pin 100 nL of test compound from compound plates into assay plates using a CyBi-Well pin tool. Dispense 20 L/well of culture into the assay media in all wells. Incubate the plates in a humidified (90% humidity) Liconic incubator at 37 C. without agitation for 48 hours. Shake plates briefly. Read OD600 of wells on a standard plate reader as measure of relative fungal growth. Envision (Perkin Elmer) plate reader settings: Ex Photometric 600, Top read.
Secondary Glucose Assay Protocol (AID 623971, 623967, 624013, 624025)
Materials and Reagents

(312) Clear, flat bottom, black 384-well plates (Corning, Catalog No. 3712BC; Lot No. 35808016); Amphotericin B (Sigma, Catalog no. A2411) 15 mM stock solution in DMSO; Pen/Strep (Gibco, Catalog No. 10378-016; Lot no21040170) 100 in PBS.

(313) Synthetic defined growth medium. Complete Supplement Mixture; (Sunrise Science, Catalog No. 1001-100); Yeast Nitrogen Base without ammonium sulfate (MP Biomedicals, Catalog No. 4027-012); Glucose (Sigma, Catalog No. G7021). Prepare medium by dissolving 0.79 g of Complete Supplement Mixture and 1.7 g of Yeast Nitrogen Base in 800 mL of water. Add 20 g of glucose. Adjust final volume to 1000 mL. Filter sterilize.

(314) Fungal Inoculum. Test Strains: C. glabrata (ATCC 2001) and Saccharomyces cerevisiae W303. Inoculate 100 L of yeast from cryopreserved stock into a 250 mL shaker flask containing 20 mL of growth medium. Shake at 30 C. overnight (about 16 hours). Read OD 600 of 1 mL of fungal culture in a cuvette using a standard optical density reader (Eppendorf BioPhotometer Plus), with growth medium as a background blank. Dilute to desired volume of fungal inoculum to produce an OD600 reading of 0.005 (S. cerevisiae) or 0.004 (C. glabrata).

(315) Procedures Use a Thermo Combi nL to dispense 20 L/well of assay media into all wells. Add 2 L of Amphotericin B to control wells. Pin 100 nL of test compound from compound plates into assay plates using a CyBi-Well pin tool. Dispense 20 L/well of culture into the assay media in all wells. Incubate the plates in a humidified (90% humidity) Liconic incubator at 37 C. without agitation for 48 hours. Shake plates briefly. Read OD600 of wells on a standard plate reader as measure of relative fungal growth. Envision (Perkin Elmer) plate reader settings: Ex Photometric 600, Top read.
4.5. Secondary Mitochondrial Disruption Assay (AID No. 624062 and 624068)

(316) The fluorescent dye JC-1 was used to quantify the ability of compounds to disrupt the mitochondrial membrane potential of fungi. Mitochondrial membrane depolarization is indicated by a decrease in the yellow/green fluorescence intensity ratio. This is a non-gating, binning assay that establishes whether inhibition of fungal growth in glycerol results from direct compromise of mitochondrial function or alternative components of fungal respiratory metabolism. Probes that disrupt mitochondrial function as well as those that perturb other metabolic components are desirable.

(317) Materials and Reagents

(318) JC-1 Dye (Invitrogen, Catalog No. T-3168); Antimycin A (Sigma, Catalog No. A8674).

(319) Synthetic defined growth medium. Complete Supplement Mixture; (Sunrise Science, Catalog No. 1001-010); Yeast Nitrogen Base without ammonium sulfate (BD, Catalog No. 239210); Glucose (Sigma, Catalog No. G7021). Prepare medium by dissolving 0.79 g of Complete Supplement Mixture and 6.7 g of Yeast Nitrogen Base in 800 mL water. Add 20 g of glucose. Adjust final volume to 1000 ml. Filter sterilize.

(320) Test Strains. Saccharomyces cerevisiae strain W303-1a (ATCC#208352) and Candida glabrata (ATCC#2001).

(321) Procedures Grow a 2 mL of overnight culture of W303 strain at 30 C. Dilute to an OD600 of 0.1 in 2 mL of fresh media (one tube per compound replicate). Incubate at 30 OC with agitation for 90 minutes. Add compounds at desired concentrations. Incubate 4 hr at 30 C. with agitation. Aliquot 1 mL of culture into a microcentrifuge tube. Add JC-1 dye stock to tube at a final concentration of 1 g/mL. Incubate 30 minutes in the dark with agitation at 30 C. Wash out dye by centrifuging and resuspending in SD-CSM media twice. Analyze by flow cytometry using a 488 nM excitation laser and detect emission on FL1 and FL2 channels. Polarized mitochondria will emit strongly at about 590 nm in the FL2 channel; while depolarized mitochondria will emit mainly at about 525 nm in the FL1 channel. The ratio of FL2 to FL1 fluorescence thus reports on polarization, which must be initially calibrated with a negative DMSO control.
4.6. Data Analysis

(322) For the primary screen and other assays, negative-control (NC) wells and positive-control (PC) wells were included on every plate. The raw signals of the plate wells were normalized using the Stimulators Minus Neutral Controls method in Genedata Assay Analyzer (v7.0.3). The median raw signal of the intra-plate NC wells was set to a normalized activity value of 0, while the median raw signal of the intra-plate PC wells was set to a normalized activity value of 100. Experimental wells were scaled to this range, resulting in an activity score representing the percent change in signal relative to the intra-plate controls. The mean of the replicate percent activities were presented as the final Pubchem Activity Score. The Pubchem Activity Outcome class was assigned as described below, based on an activity threshold of 70%: Activity_Outcome=1 (inactive), less than half of the replicates fell outside the threshold. Activity_Outcome=2 (active), all of the replicates fell outside the threshold, OR at least half of the replicates fell outside the threshold AND the Pubchem Activity Score fell outside the threshold. Activity_Outcome=3 (inconclusive), at least half of the replicates fell outside the threshold AND the Pubchem Activity Score did not fall outside the threshold.
4.7. Results

(323) Probe Attributes Inhibits growth in primary screen cell line C. albicans CaCi-2 at an IC.sub.5010 M. Inhibits growth in resistant cell line C. albicans CaCi-17 at an IC.sub.5010 M. Inhibits growth in S. cerevisiae grown on glycerol at an IC.sub.5010 M. Inhibits growth in C. glabrata grown on glycerol at an IC.sub.5010 M. Shows at least 10-fold selectivity between the CaCi-2 test strain and murine 3T3 cells. Shows at least 10-fold selectivity between S. cerevisiae grown on glycerol and S. cerevisiae grown on glucose. Show at least 10-fold selectivity between C. glabrata grown on glycerol and C. glabrata grown on glucose.

(324) An earlier MLPCN effort has identified compounds capable of reversing antifungal drug-resistance, thereby providing probes to interrogate the mechanisms responsible for drug-resistance in medically relevant fungi (PubChem summary AID 2007). Here, a primary assay is used to identify compounds that directly inhibit fungal growth, and a secondary assay is used to measure a compound's intrinsic antifungal activity (AID 2387), rather than its ability to reverse resistance to the widely used antimycotic fluconazole. This secondary assay identified a number of compounds that possessed potent single agent activity against a range of opportunistic human fungal pathogens, preferably under culture conditions that require mitochondrial respiratory metabolism for growth and survival.

(325) Data reported in PubChem for growth inhibition of Candida albicans as well as mammalian fibroblasts (AID 2387 and 2327, respectively) have been analyzed. Analysis of this data identified 43 compounds that inhibited C. albicans growth with IC.sub.5010 M. Approximately 75% (30 of 43) of these single-agent antimycotics showed modest selectivity against fibroblasts (IC.sub.5020 M). Based on this data mining, seventeen dry powders were obtained and evaluated for C. albicans and fibroblast growth inhibition. After examining the re-test data, a spirocyclic thiohydantoin derivative I-A-1 (CID3889161) was identified for further development.

(326) The initial hit compound I-A-1 (CID3889161, FIG. 3) is a potent inhibitor of C. albicans growth (IC.sub.50=0.3 M), with insignificant activity against murine fibroblasts (IC.sub.50>26 M). Analogs of I-A-1 (CID3889161) were designed and prepared accordingly, resulting in the identification of the more potent probe compound I-B-4 (ML316). The biological properties of compound I-B-4 (ML316) are summarized in Tables 12-13.

(327) TABLE-US-00004 TABLE 12 Biological Properties of Compound I-B-4 (ML316) CID/ML IC.sub.50 (M) Anti- IC.sub.50 (M) Fold No. Targets [SID, AID] Target [SID, AID] Selective* 56604860 ML316 CaCi-2 0.04 Fibroblast >26 >650x growth [134356650, 623899] toxicity [134356650, 602394] inhibition CaCi-17 1.0 >37x growth [134356650, 623897] inhibition *Selectivity = Anti-target IC.sub.50/Target IC.sub.50

(328) TABLE-US-00005 TABLE 13 Biological Properties of Compound I-B-4 (ML316) CID/ML IC.sub.50 (M) Fold No. Secondary Assays [SID, AID] Selective.sup. 56604860 Inhibition of S. cerevisiae 0.004 >750x ML316 growth on glycerol [134356650, 623969] Inhibition of S. cerevisiae >3.0 growth on glucose [134356650, 623971] Inhibition of C. glabrata 0.011 >272x growth on glycerol [134356650, 623965] Inhibition of C. glabrata >3.0 growth on glucose [134356650, 623967] .sup.Selectivity = Glucose IC.sub.50/Glycerol IC.sub.50.
Summary of Screening Results

(329) An earlier high-throughput screen evaluated 302,509 compounds for their ability to chemosensitize the fluconazole-resistant Candida albicans strain CaCi-2 to a sublethal concentration of fluconazole (PubChem AID 1979). A secondary screen of the primary assay hits was included to identify any intrinsic antimycotic activity, and 342 compounds were evaluated in this assay. 43 compounds displayed inhibitory activity against CaCi-2 with IC.sub.50 values below 10 M (AID 2387). While their antifungal activity disqualified these compounds from the prior project, this set of 43 compounds was re-purposed for the current campaign to identify potent single-agent antimycotics.

(330) DMSO stocks of only 39 compounds were available for re-test against the C. albicans strain CaCi-2 (PubChem AID 588529) and the more highly drug-resistant CaCi-17 (PubChem AID 588530). Of the 39 compounds, 23 surpassed the less than 10 M IC.sub.50 screening cutoff for both assays. Unfortunately, the cytotoxicity of many compounds appeared to be non-specific as determined by a murine 3T3 fibroblast counterscreen (AID 588634). Of the 23 antifungal compounds, only two compounds (CID26662188 and CID3889161) displayed greater than 10-fold selectivity towards fungal cells. One compound (CID26662188) was deemed chemically intractable and subsequently discarded.

(331) In order to identify additional candidates, the PubChem data for AID 2387 was revisited. The CaCi-2 inhibitory activity threshold was raised from 10 M to 15 M to produce a list of 67 compounds. This hit list was cross-referenced against the 39 compounds previously evaluated as described above and also checked for fibroblast toxicity using available PubChem data (AID 2327). After applying these filters, an additional 17 compounds were obtained.

(332) Dry powders for these 17 compounds, as well as for CID3889161, were procured for re-testing. In addition, several analogs of promising compounds were obtained to provide preliminary SAR data and validate these scaffolds. After purity analysis and structural confirmation by NMR spectroscopy, a total 24 substances was screened according to the workflow outlined below in FIG. 4. The screens in this pathway consist of CaCi-2 and CaCi-17 inhibitory activity, mammalian toxicity, and growth media-dependent activity against C. glabrata and S. cerevisiae. The associated PubChem AIDs for these assays are provided in FIG. 4. CID3889161 emerged as the most promising candidate and was subsequently prioritized for further investigation. A number of analogs related to this scaffold were prepared and assayed for fungal selective respiratory inhibition.

(333) Dose Response Curves for Probe Compound I-B-4 (ML316)

(334) The dose response curves for probe compound I-B-4 (ML316) are shown in FIGS. 5-6.

(335) SAR Tables

(336) In order to investigate the activity of the hit (CID3889161), a collection of 51 structurally related analogs were synthesized and evaluated for their ability to inhibit growth in the C. albicans test strains. The biological assay data of these analogs are presented in Tables 3-8 (PubChem AIDs 623899, 623897, 602394).

(337) The hit compound (CID3889161, Table 3, Entry 1) displayed significant activity against the C. albicans CaCi-2 strain (IC.sub.50=0.3 M), but was considerably less effective at inhibiting the growth of the more resistant CaCi-17 strain (IC.sub.50=8.5 M). There was also no noticeable toxicity to fibroblasts (IC.sub.50>26 M).

(338) The importance of the ethyl carbamate was examined first, and the results are presented in Table 3. Alternative alkyl carbamates displayed moderate activity against CaCi-2 (Table 3, entries 2-5). Potency against this strain correlated with the size of the carbamate, and smaller alkyl chains were more effective. Except for the methyl carbamate derivative which only showed marginal activity, these analogs had no effect on CaCi-17 growth. Phenyl carbamate (Table 3, entry 6) showed no activity against either C. albicans strain. Replacing the nitrogen cap with other functionalities such as methyl sulfonamide, butyl amide, and N-alkyl chains did not confer additional antifungal activity (Table 3, entries 7-10). Similarly, the unsubstituted nitrogen failed to inhibit C. albicans growth (Table 3, entry 11).

(339) While the substituent of the imide nitrogen seemed resistant to modification, the neighboring N-tolyl group was considerably more pliant (Table 4). Substitution at the phenyl ring's 4-position was not required for activity, and removing the methyl group appeared to improve potency against the more resistant CaCi-17 line (Table 4, entry 2). Conversely, incorporating methyl ether at that position attenuated the compound's effect on both C. albicans strains (Table 4, entry 3). Progressing to electron-deficient phenyl rings provided the most significant gains in potency (Table 4, entries 4-7). While the trifluoromethyl was the worst performer among the electron-withdrawing substituents examined, this analog was still only 2-fold weaker than the original hit. In comparison, the 4-fluoro, chloro, and cyano replacements were all 4 to 8-fold more effective against both CaCi-2 and CaCi-17. Comparison of positional effects showed that the location of the substituent has only a marginal influence on the compound's potency (Table 4, entries 8-9), although minor gains in PBS solubility were observed. The 4-ethyl and 4-propyl derivatives retain potency against CaCi-2 (Table 4, entries 10-11), but potency diminishes rapidly with larger substituents at this position (Table 4, entries 12-14). However, significantly smaller substituents are not effective growth inhibitors either (Table 4, entries 15-16). The N-benzyl analog showed a 10-fold drop in activity (Table 4, entry 17). The activity trends of this subset suggest the N-tolyl cap may lie within a sizeable pocket. While excessively large substituents attenuate activity, smaller functionalities (e.g. isopropyl or NH) seem unable occupy this space effectively and therefore lower cellular activity.

(340) Modification of the spirocyclic cyclohexane led to drastic reductions in activity (Table 5). Incorporating oxygen into the ring system provided a large increase in solubility, but this was accompanied by a 100-fold drop in potency (Table 5, entry 2). Substituting smaller aliphatic rings reiterated this trend; gains in solubility were offset by the inability to affect C. albicans growth (Table 5, entries 3-5).

(341) Table 6 summarizes attempts to alter the underlying thiohydantoin core. Exchanging any of the chalcogens for other elements (O.fwdarw.N or S.fwdarw.O) eliminated cellular activity (Table 6, entries 2-3). Similarly, switching the nitrogen substituents produced an inactive derivative (Table 6, entry 4). N-alkyl hydantoins showed no ability to inhibit C. albicans growth (Table 6, entries 5-6) and neither did N-alkylated 4-thiohydantoins (Table 6, entries 7-8). Many intermediates prepared were also evaluated for antifungal activity, but none of these compounds were able to affect C. albicans (Table 7). The results summarized in Table 7 clearly demonstrate the importance of the carbamate (cf. Table 7, entries 7-8 & 10).

(342) Six of the most potent inhibitors of C. albicans, including hit CID3889161, were then evaluated against C. glabrata and S. cerevisiae under different growth conditions to investigate their ability to perturb cellular respiration pathways in fungi (Table 8). All of the compounds tested were able to deter growth in both fungal strains at nanomolar concentrations (IC.sub.50 's 0.006-0.058 M) when the yeast were cultured on non-fermentable media. Conversely, yeast grown on a glucose-derived media showed considerable resistance to the test compounds (IC.sub.50>3M). Amphotericin B significantly inhibited C. glabrata and S. cerevisiae growth regardless of the growth media (Table 8, entry 7).

(343) Of the analogs evaluated, the 4-fluoro derivative (CID56604860) demonstrated the most activity against the C. albicans test strains CaCi-2 (IC.sub.50=0.04 M) and CaCi-17 (IC.sub.50=1.0 M) (Table 4, entry 4). In addition, CID56604860 appears to disrupt cellular respiration processes of the fermenting yeast C. glabrata and S. cerevisiae. CID56604860 is a potent growth inhibitor of these species when only non-fermentable carbon sources are available (IC.sub.50=0.011 and 0.004 M, respectively), but its antifungal properties can be mitigated by permitting the yeast to ferment glucose (IC.sub.50>3.0 M) (Table 8, entry 3). Because of its impressive performance in these cellular assays, CID56604860 was nominated as the probe (compound I-B-4 (ML316)) for the respiration-selective growth inhibition of pathogenic fungi.

(344) TABLE-US-00006 TABLE 3 SAR Analysis of Exemplary Compounds of Formula (I) Growth Inhibition Activity, IC.sub.50 (M).sup. CID PBS C. albicans 3T3 Entry SID Solubility (n = 3) Fibroblasts No. Broad ID R (M) CaCi-2 CaCi-17 (n = 1) 1 3889161 131404760 BRD-K97464451-001-02-7 0 <1.0 0.3 8.5 Inactive 2 3951437 134356653 BRD-K26272324-001-01-7 1.2 1.9 17.1 Inactive 3 56604849 134356659 BRD-K19100570-001-01-0 <1.0 1.2 inactive Inactive 4 56604859 134356634 BRD-K65907975-001-01-2 <1.0 5.9 inactive Inactive 5 656604866 134356630 BRD-K98528213-001-01-9 <1.0 inactive inactive 7.0 6 56604875 134356666 BRD-K53670532-001-01- <1.0 inactive inactive Inactive 7 56604886 134356649 BRD-K03429132-001-01-2 29 inactive inactive Inactive 8 56604869 134356667 BRD-K80955814-001-01-7 2.2 inactive inactive 18.8 9 56604814 134356625 BRD-K89849897-001-01-3 <1.0 inactive inactive 24.2 10 56604835 134356654 BRD-K32391448-001-01-8 6.6 inactive inactive Inactive 11 56604892 134356619 BRD-K14153519-001-01-8 0 7.8 inactive inactive Inactive .sup.Inactive compounds showed no significant activity when tested below 26 M.

(345) TABLE-US-00007 TABLE 4 SAR Analysis of Exemplary Compounds of Formula (I) Growth Inhibition Activity, IC.sub.50 (M).sup. CID PBS C. albicans 3T3 Entry SID Solubility (n = 3) Fibroblasts No. Broad ID R (M) CaCi-2 CaCi-17 (n = 1) 1 3889161 131404760 BRD-K97464451-001-02-7 <1.0 0.3 8.5 Inactive 2 56604821 134356644 BRD-K49254495-001-01-1 1.6 0.2 4.2 Inactive 3 56604819 134356620 BRD-K86579583-001-01-8 <1.0 1.8 inactive Inactive 4 56604860 134356650 BRD-K83072125-001-01-2 1.1 0.04.sup. 1.0 Inactive 5 56604865 134356623 BRD-K39763022-001-01-5 <1.0 0.06.sup. 1.4 22.3 6 56604813 134356635 BRD-K52387572-001-01-9 <1.0 0.8 16.2 12.5 7 56604847 134356662 BRD-K50804393-001-01-2 <1.0 0.08.sup. 1.8 Inactive 8 24052146 134356624 BRD-K36592023-001-01-0 18 1.2 23.9 Inactive 9 56604823 134356628 BRD-K16218093-001-01-1 00 2.6 0.2 8.2 24.2 10 56604836 134356632 BRD-K00266879-001-01-8 01 <1.0 1.9 inactive 19.5 11 56604858 134356652 BRD-K16262788-001-01-9 02 <1.0 6.9 inactive Inactive 12 56604817 134356638 BRD-K05712817-001-01-1 03 <1.0 inactive inactive 13.8 13 56604837 134356629 BRD-K05074617-001-01-8 04 <1.0 inactive inactive 16.5 14 56604882 134356647 BRD-K01156547-001-01-2 05 <1.0 inactive inactive 9.8 15 56604830 134356618 BRD-K23441366-001-01-0 06 61 inactive inactive Inactive 16 56604896 134356656 BRD-K23097153-001-01-7 07 12 inactive inactive Inactive 17 56604878 134356658 BRD-K43828902-001-01-5 08 7.2 3.8 22.8 Inactive .sup.Inactive compounds showed no significant activity when tested below 26 M. .sup.For this compound, n = 6. The IC.sub.50 value was determined to be less than 0.10 M from three independent runs, performed in duplicate (n = 3, PubChem AID 623899). Subsequent re-tests at lower concentrations were used to calculate the reported IC.sub.50 value (n = 3, PubChem AIDs 623981, 624006).

(346) TABLE-US-00008 TABLE 5 SAR Analysis of Exemplary Compounds of Formula (I) 09 Growth Inhibition Activity, IC.sub.50 (M).sup. CID PBS C. albicans 3T3 Entry SID Solubility (n = 3) Fibroblasts No. Broad ID R (M) CaCi-2 CaCi-17 (n = 1) 1 3889161 131404760 BRD-K97464451-001-02-7 0 <1.0 0.3 8.5 Inactive 2 56604818 134356640 BRD-K45116687-001-01-4 28.5 20.6 inactive Inactive 3 56604843 134356657 BRD-K27513748-001-01-1 9.2 9.8 inactive Inactive 4 56604894 134356661 BRD-K83367276-001-01-3 9.9 Inactive inactive Inactive 5 56604873 134356665 BRD-K82164803-001-01-7 45.3 inactive inactive Inactive .sup.Inactive compounds showed no significant activity when tested below 26 M.

(347) TABLE-US-00009 TABLE 6 Modifications of the Thiohydantoin Core Growth Inhibition Activity, IC.sub.50 (M).sup. CID PBS C. albicans 3T3 Entry SID Solubility (n = 3) Fibroblasts No. Broad ID Structure (M) CaCi-2 CaCi-17 (n = 1) 1 3889161 131404760 BRD-K97464451-001-02-7 <1.0 0.3 8.5 Inactive 2 56604827 134356639 BRD-K51126930-001-01-9 55 inactive inactive Inactive 3 56604867 134356626 BRD-K04998372-001-01-4 26 inactive inactive Inactive 4 56604852 134356655 BRD-K81345344-001-01-9 <1.0 inactive inactive Inactive 5 56604845 134356664 BRD-K57101886-001-01-0 100 inactive inactive Inactive 6 56604877 134356636 BRD-K60902532-001-01-4 0 64 inactive inactive Inactive 7 56604857 134356617 BRD-K41190721-001-01-1 <1.0 inactive inactive 20.9 .sup.Inactive compounds showed no significant activity when tested below 26 M.

(348) TABLE-US-00010 TABLE 7 Bioactivity of Various Intermediates Growth Inhibition Activity, IC.sub.50 (M).sup. CID PBS C. albicans 3T3 Entry SID Solubility (n = 3) Fibroblasts No. Broad ID Structure (M) CaCi-2 CaCi-17 (n = 1) 1 3889161 131404760 BRD-K97464451-001-02-7 <1.0 0.3 8.5 Inactive 2 56604883 134356663 BRD-K15707586-001-01-8 14 inactive inactive Inactive 3 56604884 134356642 BRD-K51157407-001-01-8 21 inactive inactive Inactive 4 56604829 134356622 BRD-K69099108-001-01-4 >100 inactive inactive Inactive 5 56604856 134356646 BRD-K54655126-001-01-8 5.9 inactive inactive Inactive 6 56604841 134356648 BRD-K28436532-001-01-4 >100 inactive inactive Inactive 7 56604862 134356631 BRD-K16604305-001-01-6 43 inactive inactive Inactive 8 823120 134356621 BRD-K77788822-001-01-7 79 inactive inactive Inactive 9 56604820 134356637 BRD-K41378710-001-01-5 0 32 inactive inactive Inactive 10 14297244 134356660 BRD-K97666538-001-01-6 >100 inactive inactive Inactive 11 56604885 134356641 BRD-K48843907-001-01-4 >100 inactive inactive Inactive 12 56604876 134356668 BRD-K01920745-001-01-1 72 inactive inactive 32.6 13 56604811 134356643 BRD-K83470897-001-01-4 62 inactive inactive Inactive 14 56604879 134356651 BRD-K33397992-001-01-4 55 inactive inactive Inactive 15 56604844 134356645 BRD-K10211459-001-01-0 <1.0 inactive inactive 20.7 16 759335 134356627 BRD-K28884125-001-01-8 >100 inactive inactive Inactive .sup.Inactive compounds showed no significant activity when tested below 26 M.

(349) TABLE-US-00011 TABLE 8 Growth Media-Dependent Activity of C. Albicans Active Analogs Growth Inhibition Activity, IC.sub.50 (M) CID C. glabrata S. cerevisiae Entry SID (n = 3) (n = 3) No. Broad ID Structure On glycerol On glucose On glycerol On glucose 1 3889161 131404760 BRD-K97464451- 001-02-7 0.041 >3.0 0.011 >3.0 2 56604821 134356644 BRD-K49254495- 001-01-1 0.026 >3.0 0.015 >3.0 3 56604860 134356650 BRD-K83072125- 001-01-2 0 0.011 >3.0 0.004 >3.0 4 56604865 134356623 BRD-K39763022- 001-01-5 0.022 >3.0 0.006 >3.0 5 56604847 134356662 BRD-K50804393- 001-01-2 0.033 >3.0 0.004 >3.0 6 56604823 134356628 BRD-K16218093- 001-01-1 0.034 >3.0 0.019 >3.0 7 46897897 99351062 BRD-A42129474- 001-02-7 Amphotericin B 0.62 1.2 0.26 0.38
Cellular Activity

(350) Both primary assays were performed with whole cells. One secondary screen evaluating toxicity utilized murine 3T3 fibroblast cells, and growth inhibition assays of whole S. cerevisiae and C. glabrata on different media were included as additional secondary assays. An overview of the assays and full experimental details are described herein. The probe (CID56604860/compound I-B-4 (ML316)) met the established cellular activity criteria specified for this project (Table 9).

(351) TABLE-US-00012 TABLE 9 Comparison of Probe Compound I-B-4 (ML316) to Project Criteria CPDP I-B-4 No. Property Requirement (ML316) 1 C. albicans CaCi-2 growth inhibition IC.sub.50 10 M 0.04 M 2 C. albicans CaCi-17 growth IC.sub.50 10 M 1.0 M inhibition 3 Growth inhibition of S. cerevisiae on IC.sub.50 10 M 0.004 M glycerol 4 Growth inhibition of S. cerevisiae on 10X glycerol >3.0 M glucose IC.sub.50 5 Growth inhibition of C. glabrata on IC.sub.50 10 M 0.011 M glycerol 6 Growth inhibition of C. glabrata on >10X glycerol >3.0 M glucose IC.sub.50 7 Fibroblast growth inhibition IC.sub.50 20 M >26 M
4.8. Discussion

(352) Publicly available data from the NIH's PubChem repository facilitated an abbreviated screening campaign for the current project. 302,509 compounds from the NIH's screening collection had been previously evaluated for inhibitory activity against C. albicans CaCi-2 (AIDs 1979, 2387) as well as 3T3 mammalian fibroblasts (AID 2327), and cross-referencing this data identified 67 compounds demonstrating acceptable potency against CaCi-2 that were also reasonably non-toxic towards fibroblasts. After dry powder validation of these candidates, thiohydantoin CID3889161 was selected as a priority scaffold for further investigation.

(353) Over 50 derivatives of CID3889161 were synthetically prepared and evaluated for antifungal activity against two Candida albicans clinical isolates, CaCi-2 and CaCi-17. Both strains have demonstrated resistance towards the commonly prescribed antimycotic fluconazole, with CaCi-17 being significantly more resistant than CaCi-2. In addition to fungal growth inhibition, these analogs were screened for non-specific toxicity using murine 3T3 fibroblasts. FIG. 7 summarizes the various modifications explored during SAR studies. Three primary diversity points were investigated, and the number of analogs prepared for each location is reported in FIG. 7. Several analogs do not fit into these three clusters and are classified as miscellaneous analogs.

(354) From the SAR study, it was determined that carbamates were the preferred substituent for the imide nitrogen and that the ethyl carbamate was optimal. Electron-deficient phenyl rings are superior to the original N-tolyl group, while ortho- and meta-substituents do not appear to significantly influence cellular activity. Contracting the cyclohexyl ring system provides increases in solubility while diminishing antifungal potency. While some variation of the nitrogen substituents is tolerated, the modification of the underlying thiohydantoin motif is not. The 4-fluorophenyl analog I-B-4 (CID56604860) emerged from these studies as the most potent antifungal of this class. Subsequent investigation of compound I-B-4 revealed that its antifungal properties could be modulated by the specific growth media given to the fungal test strains, as desired by the project criteria. Consequently, compound I-B-4 was nominated as the probe compound I-B-4 (ML316).

(355) Based on the project goals summarized below in Table 9, the probe compound I-B-4 clearly surpasses all of the specified criteria. Compound I-B-4 possesses potent activity against the C. albicans test strains CaCi-2 (IC.sub.50=0.04 M) and CaCi-17 (IC.sub.50=1.0 M). In addition, compound I-B-4 is a potent growth inhibitor of C. glabrata and S. cerevisiae when only non-fermentable carbon sources are available (IC.sub.50=0.011 and 0.004 M, respectively), but its antifungal properties are lost when the yeast is supplied glucose for fermentation (IC.sub.50>3.0 M). There is no apparent toxicity against mammalian fibroblasts at concentrations up to 26 M, indicating that compound I-B-4 may be a fungal selective, respiratory inhibitor.

(356) According to PubChem, compound I-B-4 (ML316, CID56604860) has not been tested in any other assay reported to its database. The original hit, compound I-A-1 (CID3889161), has been evaluated in seven assays. All seven assays are associated with the original screening campaign (AID 2007) from which the current project is derived. The cellular activity of ompound I-A-1 (CID3889161) described herein recapitulates the published PubChem data.

(357) A search of the available literature identified several compounds capable of disrupting fungal respiration (von Jagow et al., Methods Enzymol. 1986, 126:253-71; Ueki et al., Curr. Opin. Anti-Infective Invest. Drugs 2000, 2(4):387-398; Sridhara et al., J. Pharmaceutical. Res. 2011, 4(2):496-500; Mathre, Pest Biochem. Physiol. 1971, 1(2):216-224). With the exception of ilicicolin H, these compounds do not show appreciable selectivity towards fungi over mammalian targets or cells. Hence, it was decided to compare the probe compound I-B-4 (ML316) with ilicicolin H.

(358) Previous work with the antifungal antibacterial ilicicolin H has shown that this natural product is a potent inhibitor of the mitochondrial cytochrome bc.sub.1 complex (Gutierrez-Cirlos et al., J. Biol. Chem. 2004, 279(10):8708-14; Rotsaert et al., Biochim. Biophys. Acta 2008, 1777(2):211-9). Using purified proteins, Trumpower et al. determined that ilicicolin H binds to the S. cerevisiae enzyme complex with almost 100-fold greater selectivity than the corresponding bovine homolog. This selectivity has not been reproduced within a cell-based assay.

(359) A commercial sample of ilicicolin H was procured from Analyticon Discovery and determined to be approximately 85% pure by HPLC. The commercial sample was purified by column chtomatography over silica gel prior to testing in cellular assays. It was recently attempted to obtain additional quantities of ilicicolin H but discovered that this compound is no longer commercially available.

(360) Probe compound I-B-4 (ML316) was tested alongside ilicicolin H in a direct comparison of biological activity. The results are summarized below in Table 10. Compound I-B-4 (ML316) clearly shows a stronger inhibitory effect upon Candida albicans growth than ilicicolin H. While both compounds show differential activity relative to growth media, the distinction is far less pronounced with ilicicolin H regardless of the fungal test strain.

(361) Given the complex structure of ilicicolin H, optimization of biological and physical properties through synthetic methods will be difficult. Despite having been isolated 40 years ago (Hayakawa et al., J. Antibiot. 1971, 24(9):653-4), the first and only total synthesis of the ilicicolin H was reported in 1985 (Williams et al., J. Org. Chem. 1985, 50(15):2807-9). That this pioneering synthesis produced racemic material further underscores the inherent difficulties of preparing analogs of this compound. While semi-synthetic analogs are possible (Liu et al., Tetrahedron Lett. 2005, 46(46): 8009-12; Singh et al., Tetrahedron Lett. 2011, 52(46): 6190-1), the lack of a commercial feedstock renders this option untenable at the present time.

(362) As a small molecule probe, compound I-B-4 (ML316) is the superior compound because of its cellular potency and selectivity, and its simple preparation and chemical tractability facilitate further optimization.

(363) TABLE-US-00013 TABLE 10 Comparison of Probe Compound I-B-4 (ML316) to Prior Art Compound Ilicicolin H ML316 No. Property (I-B-4) ilicicolin H 1 C. albicans CaCi-2 growth inhibition 0.04 M >3.0 M 2 C. albicans CaCi-17 growth inhibition 1.0 M >3.0 M 3 Growth inhibition of S. cerevisiae on 0.004 M 0.03 M glycerol 4 Growth inhibition of S. cerevisiae on >3.0 M >3.0 M glucose 5 Growth inhibition of C. glabrata on 0.011 M 2.6 M glycerol 6 Growth inhibition of C. glabrata on >3.0 M >3.0 M glucose 7 Fibroblast growth inhibition >26 M 10.0 M 8 PBS solubility (with 1% v/v DMSO) 1.1 M <1.0 M
Mechanism of Action Studies

(364) A series of secondary assays were performed to interrogate the possible mode of action. Compound I-B-4 (ML316) appears to disrupt cellular respiration of fungal organisms as demonstrated by the distinctly different growth patterns of C. glabrata and S. cerevisiae when they are fed fermentable and non-fermentable carbon sources. In both species, compound I-B-4 (ML316) is most effective when the yeast are given glycerol and displays a greater than 250-fold reduction in potency when glucose is available.

(365) Additionally, direct impairment of mitochondrial function in C. glabrata and S. cerevisiae was assessed using a fluorescence assay. Application of JC-1 dye to living S. cerevisiae treated with 1 M compound I-B-4 (ML316) determined that depolarization of the mitochondrial membrane does not occur (PubChem AIDs 624062, 624068). Comparison of the DMSO controls with compound I-B-4 suggests the yeast can still maintain their normal mitochondrial membrane potential; it appears that the molecular target of ML316 may be integrated within an alternative metabolic pathway that fungi utilize for respiration (Ferrari et al., PLoS Pathog. 2009, 5(1):e1000268).

Example 5

Biological Assays of Compound I-B-4 (ML316)

(366) 5.1. Materials and Methods

(367) Materials and Reagents.

(368) Cell Lines. Candida albicans CaCi-2; a drug-resistant clinical isolate was provided by the Whitehead Institute (Redding et al., Clin. Infect. Dis. 1994, 18(2):240-2). This strain was used for the primary assay. Candida albicans strain SC5314 Candida albicans ATCC 200950 S. cerevisiae strain BY4743; BY4743+pRS316-MIR1; BY4743 MIR1/+; BY4743+pRS316-MIR1N184T; BY4741 mir1

(369) Procedures

(370) Growth inhibition of I-B-4 (ML316) against Candida albicans CaCi-2 in the presence or absence of fluconazole was assessed using the broth microdilution method with RPMI media. FIG. 8 summarizes the inhibitory activities (IC.sub.50=120 nM for I-B-4 (ML316) alone; IC50=10 nM for I-B-4 (ML316) with FCZ).

(371) Growth of Candida albicans CaCi-2 and derived I-B-4-R strain were assessed on solid agar media containing yeast nitrogen base with either 2% glucose or 2% glycerol as carbon source; I-B-4 was included as indicated at 100 nM; fluconazole was included at 32 g/mL. Photos were taken after 2 days of growth at 37 C. FIG. 9 shows the results of the growth assessment.

(372) Whole genome sequence of ATCC 200950 and three I-B-4-resistant mutants was determined using Illumina HiSeq sequencing system. Sequences were aligned to C. albicans reference genome to identify unique mutations in resistant strains. FIG. 10 shows the results of the genome sequencing. FIG. 11 shows the protein sequence alignment of a region of MIR1 homologs from fungi and humans to highlight the N184T polymorphism.

(373) The inhibitory activities of I-B-4 (ML316) on the growth of S. cerevisiae strain BY4743; BY4743+pRS316-MIR1; BY4743 MIR1/+; BY4743+pRS316-MIR1N184T strains were performed in synthetic glycerol media with varying concentrations of I-B-4. Strains were grown in liquid media in 96-well plates. Growth was measured by OD600 after 3 days (see FIG. 12).

(374) The growth of untransformed and transformed S. cerevisiae mir1+ cells in both glycerol and glucose media was conducted. BY4741 mir1 was transformed with empty pRS316 vector or pRS316 vector containing S. cerevisiae MIR1 under the TEF promoter, or human homolog SLC25A3 under the TEF, GPD, or ADH promoters. Strains were grown on solid agar yeast synthetic media containing 2% glucose or 2% glycerol (FIG. 13).

(375) Compound I-B-4 (ML316) was studied in fluconazole-sensitive Candida albicans strain SC5314. The SC5314 strain was inoculated into RPMI media at 10^3 CFU/mL in the presence or absence of 5 M I-B-4 (ML316) and 32 ug/mL fluconazole. Colonies were plated from the cultures after 24 and 48 hours and counted. FIG. 14 shows the cell proliferation data over time.

(376) Compound I-B-4 (ML316) was also studied in fluconazole-sensitive Candida albicans CaCi-2. The CaCi-2 strain was inoculated into RPMI media at 10^3 CFU/mL in the presence or absence of 5 M I-B-4 (ML316) and 32 ug/mL fluconazole. Colonies were plated from the cultures after 24 and 48 hours and counted. FIG. 15 shows the cell proliferation data over time.

EQUIVALENTS AND SCOPE

(377) In the claims articles such as a, an, and the may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include or between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

(378) Furthermore, the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive termsfrom one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements and/or features, certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms comprising and containing are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

(379) This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any claim, for any reason, whether or not related to the existence of prior art.

(380) Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.