A chemoproteomic approach to elucidate the mechanism of action of 6-azasteroids with unique activity in mycobacteria

Abstract

The present invention provides a compound having the structure:

Description

A CHEMOPROTEOMIC APPROACH TO ELUCIDATE THE MECHANISM OF ACTION OF 6- AZASTEROIDS WITH UNIQUE ACTIVITY IN MYCOBACTERIA

[0001] This application claims the benefit of U.S. Provisional Application No.63 / 565,825, filed March 15, 2024, and U.S. Provisional Application No.63 / 581,309, filed September 8, 2023, the contents of each of which are hereby incorporated by reference.

[0002] Throughout this application, various publications are referenced, including referenced in parenthesis. The disclosures of all publications mentioned in this application in their entireties are hereby incorporated by reference into this application in order to provide additional description of the art to which this invention pertains and of the features in the art which can be employed with this invention. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] This invention was made with government support under AI034054 awarded by National Institutes of Health. The government has certain rights in the invention. BACKGROUND OF THE INVENTION

[0004] By illuminating key 6-azasteroid–protein interactions in both Mycobacterium tuberculosis (Mtb) and the closely related model organism Mycobacterium marinum (Mm), the current invention sought to improve the antimycobacterial potency of 6-azasteroids and further understanding of the mechanisms responsible for their potentiation of the antituberculosis drug bedaquiline. Newly developed 6-azasteroid analog and an analog reported previously (ACS Infect Dis 2019, 5 (7), 1239–1251) were selected to study their phenotypic effects on Mtb and Mm, both alone and in combination with bedaquiline. The 6-azasteroid analog, 17β-[N-(4-trifluoromethoxy-diphenylmethyl)carbamoyl]-6-propyl-azaandrostan-3-one, robustly potentiated bedaquiline-mediated antimycobacterial activity, with a nearly 8-fold reduction in Mm bedaquiline minimal inhibitory concentration (85 nM alone versus 11 nM with 20 μM 6-azasteroid). This analog displayed minimal inhibitory activity against recombinant mycobacterial 3β-hydroxysteroid dehydrogenase, a previously identified target of several 6-azasteroids. Dose-dependent potentiation of bedaquiline by this analog reduced mycobacterial intracellular ATP levels and impeded the ability of Mtb to neutralize exogenous oxidative stress in culture. Two 6-azasteroid photoaffinity probes were developed to investigate azasteroid–protein interactions in Mm whole cells. Using bottom-up mass spectrometric profiling of the cross-linked proteins, eight potential Mm / Mtb protein targets for 6-azasteroids were identified. The nature of these potential targets indicates that proteins related to oxidative stress resistance play a key role in the BDQ-potentiating activity of azasteroids and highlights the potential impact of inhibition of these targets on the generation of drug sensitivity.BRIEF SUMMARY OF THE INVENTION

[0005] The present invention provides a compound having the structure: , whereinα is a bond that is present or absent; β is a bond that is present or absent; and γ is a bond that is present or absent, wherein when β is present, γ is absent and R12 is absent, and wherein when β is absent, γ is present and R12 is present; X is C, N, or O; R1is -H, alkyl, alkenyl, alkynyl, aryl, heteroaryl, -C(O)-alkyl, -C(O)-haloalkyl, -C(O)-cycloalkyl, -C(O)-alkenyl, -C(O)-alkynyl, -C(O)-aryl, -C(O)-heteroaryl, -CO2-alkyl, -CO2-alkenyl, -CO2- alkynyl, -CO2-aryl, -CO2-heteroaryl, cycloalkyl, -alkyl-OH, -alkyl-N(alkyl)2, -alkyl-NH2, -alkyl- haloalkyl, -alkyl-CF3, or -alkyl-cycloalkyl; R2is H, halo, alkyl, alkenyl, alkynyl, aryl, heteroaryl, -CO2-alkyl, -CO2-alkenyl, -CO2-alkynyl, - CO2-aryl, -CO2-heteroaryl, cycloalkyl, -alkyl-OH, -alkyl-NH2or -alkyl-N(alkyl)2; R3is -H, halo, alkyl, alkenyl, alkynyl, aryl, heteroaryl, -CO2-alkyl, -CO2-alkenyl, -CO2-alkynyl, -CO2-aryl, -CO2-heteroaryl, cycloalkyl, -alkyl-OH, -alkyl-NH2or -alkyl-N(alkyl)2; R4 is -H, halo, alkyl, alkenyl, alkynyl, aryl, heteroaryl, -CO2-alkyl, -CO2-alkenyl, -CO2-alkynyl, -CO2-aryl, -CO2-heteroaryl, cycloalkyl, -alkyl-OH, -alkyl-NH2, or -alkyl-N(alkyl)2; R5and R6are each independently, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, or a substituted or unsubstituted alkylaryl; and R12is H, OH, NH2, -C(O)-alkyl, -O-C(O)-alkyl-aryl or-O-C(O)-alkyl-heteroaryl; wherein when R5and R6are both fluorophenyl or both chlorophenyl, either α is absent or R2is not H; or a salt or ester thereof.

[0006] The present invention provides a compound having the following structure: , whereinα is a bond that is present or absent; X is C, N, or O; R1is -H, alkyl, alkenyl, alkynyl, aryl, heteroaryl, -C(O)-alkyl, -C(O)-haloalkyl, -C(O)-cycloalkyl, -C(O)-alkenyl, -C(O)-alkynyl, -C(O)-aryl, -C(O)-heteroaryl, -CO2-alkenyl, -CO2-alkynyl, -CO2- aryl, -CO2-heteroaryl, cycloalkyl, -alkyl-OH, -alkyl-N(alkyl)2, -alkyl-NH2, -alkyl-haloalkyl, -alkyl- CF3, or -alkyl-cycloalkyl; R2is H, halo, alkyl, alkenyl, alkynyl, aryl, heteroaryl, -CO2-alkyl, -CO2-alkenyl, -CO2-alkynyl, - CO2-aryl, -CO2-heteroaryl, cycloalkyl, -alkyl-OH, -alkyl-NH2or -alkyl-N(alkyl)2; R3is -H, halo, alkyl, alkenyl, alkynyl, aryl, heteroaryl, -CO2-alkyl, -CO2-alkenyl, -CO2-alkynyl, - CO2-aryl, -CO2-heteroaryl, cycloalkyl, -alkyl-OH, -alkyl-NH2or -alkyl-N(alkyl)2; R4 is -H, halo, alkyl, alkenyl, alkynyl, aryl, heteroaryl, -CO2-alkyl, -CO2-alkenyl, -CO2-alkynyl, - CO2-aryl, -CO2-heteroaryl, cycloalkyl, -alkyl-OH, -alkyl-NH2, or -alkyl-N(alkyl)2; and R5is a substituted aryl or substituted or unsubstituted cycloalkyl; wherein the substitution is H, alkyl, alkenyl, alkynyl carbonyl, amine; C(O)-alkyl, C(O)-haloalkyl, or CO2-alkyl; wherein when is present, R1is not H;wherein when is present, R1is not -(CH2)2CH3;wherein when is present, R1is not CH3;wherein when is present, R1is not H;wherein when H;wherein when R5is and α is present, R1is not H or CH3; wherein when α is present and R5is , , , ,, , notor a salt or ester thereof.

[0007] The present invention provides a method of treating a subject infected with M. tuberculosis comprising administering to the subject an amount of the compound having the structure:, whereinα is a bond that is present or absent; β is a bond that is present or absent; and γ is a bond that is present or absent, wherein when β is present, γ is absent and R12 is absent, and wherein when β is absent, γ is present and R12 is present; X is C, N, or O; R1is -H, alkyl, alkenyl, alkynyl, aryl, heteroaryl, -C(O)-alkyl, -C(O)-haloalkyl, -C(O)-cycloalkyl, -C(O)-alkenyl, -C(O)-alkynyl, -C(O)-aryl, -C(O)-heteroaryl, -CO2-alkyl, -CO2-alkenyl, -CO2- alkynyl, -CO2-aryl, -CO2-heteroaryl, cycloalkyl, -alkyl-OH, -alkyl-N(alkyl)2, -alkyl-NH2, -alkyl- haloalkyl, -alkyl-CF3, or -alkyl-cycloalkyl; R2is H, halo, alkyl, alkenyl, alkynyl, aryl, heteroaryl, -CO2-alkyl, -CO2-alkenyl, -CO2-alkynyl, - CO2-aryl, -CO2-heteroaryl, cycloalkyl, -alkyl-OH, -alkyl-NH2or -alkyl-N(alkyl)2; R3is -H, halo, alkyl, alkenyl, alkynyl, aryl, heteroaryl, -CO2-alkyl, -CO2-alkenyl, -CO2-alkynyl, - CO2-aryl, -CO2-heteroaryl, cycloalkyl, -alkyl-OH, -alkyl-NH2or -alkyl-N(alkyl)2; R4 is -H, halo, alkyl, alkenyl, alkynyl, aryl, heteroaryl, -CO2-alkyl, -CO2-alkenyl, -CO2-alkynyl, - CO2-aryl, -CO2-heteroaryl, cycloalkyl, -alkyl-OH, -alkyl-NH2, or -alkyl-N(alkyl)2; R5and R6are each independently, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, or a substituted or unsubstituted alkylaryl; and R12 is H, OH, NH2, -C(O)-alkyl, -O-C(O)-alkyl-aryl or-O-C(O)-alkyl-heteroaryl; or a salt or ester thereof, so as to thereby treat the subject.

[0008] The present invention provides a method of treating a subject infected with M. tuberculosis comprising administering to the subject an amount of the compound having the structure:, whereinα is a bond that is present or absent; X is C, N, or O; R1is -H, alkyl, alkenyl, alkynyl, aryl, heteroaryl, -C(O)-alkyl, -C(O)-haloalkyl, -C(O)-cycloalkyl, -C(O)-alkenyl, -C(O)-alkynyl, -C(O)-aryl, -C(O)-heteroaryl, -CO2-alkenyl, -CO2-alkynyl, -CO2- aryl, -CO2-heteroaryl, cycloalkyl, -alkyl-OH, -alkyl-N(alkyl)2, -alkyl-NH2, -alkyl-haloalkyl, -alkyl- CF3, or -alkyl-cycloalkyl; R2is H, halo, alkyl, alkenyl, alkynyl, aryl, heteroaryl, -CO2-alkyl, -CO2-alkenyl, -CO2-alkynyl, - CO2-aryl, -CO2-heteroaryl, cycloalkyl, -alkyl-OH, -alkyl-NH2or -alkyl-N(alkyl)2; R3is -H, halo, alkyl, alkenyl, alkynyl, aryl, heteroaryl, -CO2-alkyl, -CO2-alkenyl, -CO2-alkynyl, - CO2-aryl, -CO2-heteroaryl, cycloalkyl, -alkyl-OH, -alkyl-NH2or -alkyl-N(alkyl)2; R4 is -H, halo, alkyl, alkenyl, alkynyl, aryl, heteroaryl, -CO2-alkyl, -CO2-alkenyl, -CO2-alkynyl, - CO2-aryl, -CO2-heteroaryl, cycloalkyl, -alkyl-OH, -alkyl-NH2, or -alkyl-N(alkyl)2; and R5is a substituted or unsubstituted aryl or cycloalkyl; wherein when is present, R1is not H;H3;wherein when is present, R1is not H;wherein when is present, R1is not H;or a salt or ester thereof, so as to thereby treat the subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1. 6-Azasteroid Scaffold and Overview of 6-Azasteroid Analog Activity. (A, B) 6- Azasteroid activity against Mycobacterium tuberculosis (Mtb) and Mycobacterium marinum (Mm) and cytotoxicity to HepG2 and THP-1 cells. For potentiation assays, Mm and Mtb were cultured separately in Middlebrook 7H9 broth supplemented with bovine serum albumin and glycerol as the sole carbon source. Growth inhibition was measured by visual inspection or by staining with Alamar Blue reagent. The fold change in bedaquiline (BDQ) minimal inhibitory concentration (MIC99) upon azasteroid co-treatment is relative to the MIC99 of BDQ without azasteroid co-treatment. For cytotoxicity assays, HepG2 and THP-1 cells were grown in Minimum Eagle Medium and Roswell Park Memorial Institute medium, respectively; and cell viability was measured by staining with Alamar Blue reagent. N.D., BDQ co-MIC not determined. Open circle, cytotoxicity not determined. (C) 6-Azasteroid scaffold and sites of structural modifications indicated in color. (D) Structure of 2. (E) Structure of 35.

[0010] Figure 2. 6-Azasteroid Phenotypic Data. (A) Inhibition of recombinant Mtb 3β-hydroxysteroid dehydrogenase. Inhibition by the 6-azasteroids was determined at a 1 µM enzyme concentration in the presence of 50 µM of each 6-azasteroid. The concentration of NAD+was held fixed at 350 µM, and that of dehydroepiandrosterone was held fixed at 220 µM.8(B) 6-Azasteroid impact on reactive oxygen species (ROS). Mtb (CDC1551) cultures grown to mid-logarithmic phase (OD600= 0.6) were pretreated with 2 or 35 (20 µM) for 6 h. Then the cells were washed to remove excess 6-azasteroid and treated with 5 mM cumene hydroperoxide (CHP) for 30 min. Oxidative stress was quantified and normalized to colony forming unit (CFU) counts. NT, no treatment. (C, D) Impact of 6-azasteroids 2 (C) and 35 (D) on bedaquiline (BDQ)–mediated inhibition of ATP production. RFU, relative fluorescence units. Error bars indicate standard deviation of the mean of three replicates; *, p < 0.05; **, p < 0.01.

[0011] Figure 3. Whole-Cell Lysate Interaction Assay, Azido-PEG3-TAMRA-Biotin Enrichment. (A) Compound 40 (Diazirine PA) and (B) compound 44 (Benzophenone PA). The nonprobe inhibitor 43 is shown in Fig. 6. (C) Fluorescence image of SDS-PAGE gel–Mm lysates incubated with 40 (lane 1), 44 (lane 2), 44 plus excess 43 (lane 3), or DMSO (lane 4) after click conjugation with azido-PEG3-TAMRA- biotin and streptavidin–agarose-bead-mediated enrichment. The ladder (L) is shown separated from the fluorescence gel to depict only the enriched samples and not the samples visualized directly after the click reaction; the full image can be found in supporting documents (Fig.7B).

[0012] Figure 4. Binding Poses of 6-Azasteroid 35 in Hit Protein Structures, Calculated Using AutoDock Vina and LigPlot+. Compound docking in the binding site as determined by homology modeling or with the binding-site software P2Rank: (A) TrxB2 (Rv3913), (B) Mtr (Rv2855), and (C) CydA (Rv1623c). Binding poses are visualized in 2D using LigPlot+ software.

[0013] Figure 5. Synthesis of 6-azasteroids.

[0014] Figure 6. Structures of 6-azasteroids tested for inhibition of Mtb 3β-hydroxysteroid dehydrogenase.

[0015] Figure 7.(A) Full western blot & (B) full fluorescence image of whole cell lysate interaction assay, proteins enriched via azido-PEG3-TAMRA-biotin. Probe 40 (Lane 1 & 5), 44 (Lane 2 & 6), 44 with an excess of 43 (Lane 3 & 7), or DMSO (Lane 4 & 8) after click conjugation with azido-PEG3-TAMRA- biotin and streptavidin agarose bead-mediated enrichment.

[0016] Figure 8. KEGG ID functional analysis of the entire Mm proteome (A).

[0017] Figure 9. KEGG ID functional analysis of the entire Mm proteome (A) and the entire proteomics data set (B) by top 20 functional categories.

[0018] Figure 10. (C) KEGG ID Functional Analysis of Mm Azasteroid Probe Enriched Interactome Proteins (top 20 functional categories).

[0019] Figure 11. Enrichment methods comparison by Venn diagram. Enrichment Methods: AA: Azide Agarose Bead, MA: Azido-functionalized Magnetic Nanoparticles, SM: Streptavidin-functionalized Magnetic Nanoparticles. First, number: Probe, 1: Diazirine PA, 2: Benzophenone PA. Second, letter: 1:DMSO Control, 2:Linker Control. Third, number: 1: First Biological Replicate, 2: Second Biological Replicate.

[0020] Figure 12. CRISPRi negative screen of selected genes against BDQ, INH, and RIF treatment which were hits of probes. Data was mined from Li et al. and visualized heat map plots prepared inGraphpad Prism. Conditions which had a False Discovery Rate of 1 are denoted with an X. Conditions which had an FDR < 0.01 are demarcated with a square in the center of its position (Li et al., 2022).

[0021] Figure 13. CRISPRi negative screen of selected genes against BDQ, INH, and RIF treatment which were hits of probes. Data was mined from Li et al. and visualized heat map plots prepared in Graphpad Prism. Conditions which had a False Discovery Rate of 1 are denoted with an X. Conditions which had an FDR < 0.01 are demarcated with a square in the center of its position (Li et al., 2022).

[0022] Figure 14. CRISPRi negative screen of selected genes against BDQ, INH, and RIF treatment which were hits of probes. Data was mined from Li et al. and visualized heat map plots prepared in Graphpad Prism. Conditions which had a False Discovery Rate of 1 are denoted with an X. Conditions which had an FDR < 0.01 are demarcated with a square in the center of its position (Li et al., 2022).

[0023] Figure 15. CRISPRi negative screen of selected genes against BDQ, INH, and RIF treatment which were hits of probes. Data was mined from Li et al. and visualized heat map plots prepared in Graphpad Prism. Conditions which had a False Discovery Rate of 1 are denoted with an X. Conditions which had an FDR < 0.01 are demarcated with a square in the center of its position (Li et al., 2022).

[0024] Figure 16. Overview of pull-down assay data analysis. The filtering methodology utilized to obtain the list of prioritized hits as targets of 6-azasteroid probes.

[0025] Figure 17.Docking of azasteroids to CydA (Rv1623c). (A) Compound 35. (B) DiazirinePA 40. (C) Compound 43 (D) BenzophenonePA 44. (E) Menaquinone-9 (MQ9).

[0026] Figure 18. Docking of azasteroids to Mtr (Rv2855). (A) Compound 35. (B) DiazirinePA 40. (C) Compound 43 (D) BenzophenonePA 44.

[0027] Figure 19.Docking of azasteroids to TrxB2 (Rv3913). (A) Compound 35. (B) DiazirinePA 40. (C) Compound 43 (D) BenzophenonePA 44.

[0028] Figure 20. Survival of Mtb cultures treated with peroxide alone and in combination with 6- azasteroid. Mtb cultures were pre-incubated with 6-azasteroid or DMSO for 6 hours. Cultures were exposed to cumene hydroperoxide stress for 30 minutes and after workup were plated on 7H11 agarose plates for CFU counting to determine impacts on survival. Data are the mean of three technical replicates ± standard deviation.

[0029] Figure 21. Synthesis of 6-azasteroid probes.

[0030] Figure 22. Nuclear magnetic resonance of compounds 40.

[0031] Figure 23. Nuclear magnetic resonance of compounds 44.DETAILED DESCRIPTION OF THE INVENTION

[0032] The present invention provides a compound having the structure: , whereinα is a bond that is present or absent; β is a bond that is present or absent; and γ is a bond that is present or absent, wherein when β is present, γ is absent and R12 is absent, and wherein when β is absent, γ is present and R12 is present; X is C, N, or O; R1is -H, alkyl, alkenyl, alkynyl, aryl, heteroaryl, -C(O)-alkyl, -C(O)-haloalkyl, -C(O)-cycloalkyl, -C(O)-alkenyl, -C(O)-alkynyl, -C(O)-aryl, -C(O)-heteroaryl, -CO2-alkyl, -CO2-alkenyl, -CO2- alkynyl, -CO2-aryl, -CO2-heteroaryl, cycloalkyl, -alkyl-OH, -alkyl-N(alkyl)2, -alkyl-NH2, -alkyl- haloalkyl, -alkyl-CF3, or -alkyl-cycloalkyl; R2is H, halo, alkyl, alkenyl, alkynyl, aryl, heteroaryl, -CO2-alkyl, -CO2-alkenyl, -CO2-alkynyl, - CO2-aryl, -CO2-heteroaryl, cycloalkyl, -alkyl-OH, -alkyl-NH2or -alkyl-N(alkyl)2; R3is -H, halo, alkyl, alkenyl, alkynyl, aryl, heteroaryl, -CO2-alkyl, -CO2-alkenyl, -CO2-alkynyl, - CO2-aryl, -CO2-heteroaryl, cycloalkyl, -alkyl-OH, -alkyl-NH2or -alkyl-N(alkyl)2; R4 is -H, halo, alkyl, alkenyl, alkynyl, aryl, heteroaryl, -CO2-alkyl, -CO2-alkenyl, -CO2-alkynyl, - CO2-aryl, -CO2-heteroaryl, cycloalkyl, -alkyl-OH, -alkyl-NH2, or -alkyl-N(alkyl)2; R5and R6are each independently, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, or a substituted or unsubstituted alkylaryl; and R12is H, OH, NH2, -C(O)-alkyl, -O-C(O)-alkyl-aryl or-O-C(O)-alkyl-heteroaryl; wherein when R5and R6are both fluorophenyl or both chlorophenyl, either α is absent or R2is not H; or a salt or ester thereof.

[0033] In some embodiments, when γ is present and R12is present, α is absent.

[0034] In some embodiments, the compound has the following structure: , whereinα is a bond that is present or absent; R1is -H, alkyl, alkyl-haloalkyl, alkyl-cycloalkyl, -C(O)-alkyl, -C(O)-haloalkyl, -C(O)-cycloalkyl, cycloalkyl, -alkyl-N(alkyl)2, or -alkyl-NH2; R2is H, halo, alkyl, -CO2-alkyl, cycloalkyl, -alkyl-OH, -alkyl-NH2, or -alkyl-N(alkyl)2; R3is H, halo, alkyl, -CO2-alkyl, -cycloalkyl, -alkyl-OH, -alkyl-NH2, or -alkyl-N(alkyl)2; R4is H, halo, alkyl, -CO2-alkyl, -cycloalkyl, -alkyl-OH, -alkyl-NH2, or -alkyl-N(alkyl)2; and R5and R6are each independently, a substituted or unsubstituted aryl or a substituted or unsubstituted heteroaryl; or a salt or ester thereof.

[0035] In some embodiments, when R5and R6are both fluorophenyl or both chlorophenyl, α is absent.

[0036] In some embodiments, when R5and R6are both fluorophenyl or both chlorophenyl, R2is not H.

[0037] In some embodiments, R1is -H, alkyl, -C(O)-alkyl, alkyl-haloalkyl, -alkyl-CF3, -alkyl- cycloalkyl, -C(O)-alkyl, or -alkyl-N(alkyl)2.

[0038] In some embodiments, R1is -H, C1-C6alkyl, -C(O)-C1-C6alkyl, -C1-C6alkyl-haloalkyl, -C1-C6alkyl-CF3, -C1-C6alkyl-cycloalkyl, -C(O)-C1-C6alkyl, or -C1-C6alkyl-N(alkyl)2.

[0039] In some embodiments, R1is -H, propyl, hexanyl, -C(O)-CH3, -(CH2)2-N-(CH3)2, -CH2- cyclopropyl, -(CH2)2-CH2F, or -(CH2)2-CF3.

[0040] In some embodiments, X is C or N.

[0041] In some embodiments, when X is C, R1is H.

[0042] In some embodiments, R5and R6are each independently a substituted or unsubstituted aryl or a substituted or unsubstituted heteroaryl.

[0043] In some embodiments, R5and R6are each independently a substituted aryl or unsubstituted heteroaryl.

[0044] In some embodiments, the aryl is phenyl.

[0045] In some embodiments, the phenyl is substituted with halo, -CN, -CF3, -OCF3, -OCHF2, -alkyl, - alkenyl, -alkynyl, -aryl, -heteroaryl, -NH2, -NH-alkyl, -NH-alkenyl, -NH-alkynyl, -NH-aryl, -NH- heteroaryl, -OH, -OAc, -O-C(O)alkyl, -O-alkyl, -O-alkylaryl, -O-alkenyl, -O-alkynyl, -O-(aryl), -O- (heteroaryl), -S-(alkyl), -S-(alkenyl), -S-(alkynyl), -S-(aryl), -S-(heteroaryl), -S(O)-(alkyl), -S(O)-(aryl), - S(O)-(heteroaryl), -SO2-(alkyl), -SO2-(aryl), -SO2-(heteroaryl), alkyl-OH, alkyl-O-alkyl, alkyl-NH2, alkyl- NH-alkyl, alkyl-N(alkyl)2, cycloalkyl or alkyl-cycloalkyl.

[0046] In some embodiments, the phenyl is substituted with -CN, -OCHF2, F, -CF3, or -OCF3.

[0047] In some embodiments, the heteroaryl is pyridine.

[0048] In some embodiments, R5and R6are each independently having the following structure: ; wherein R7, R8, R9, R10a, halo, -CN, -CF3, -OCF3, -OCHF2, - alkyl, -alkenyl, -alkynyl, -aryl, -heteroaryl, -NH2, -NH-alkyl, -NH-alkenyl, -NH-alkynyl, -NH-aryl, -NH-heteroaryl, -OH, -OAc, -O-C(O)alkyl, -O-alkyl, -O-alkylaryl, -O-alkenyl, -O-alkynyl, -O- (aryl), -O-(heteroaryl), -S-(alkyl), -S-(alkenyl), -S-(alkynyl), -S-(aryl), -S-(heteroaryl), -S(O)- (alkyl), -S(O)-(aryl), -S(O)-(heteroaryl), -SO2-(alkyl), -SO2-(aryl), -SO2-(heteroaryl), alkyl-OH, alkyl-O-alkyl, alkyl-NH2, alkyl-NH-alkyl, alkyl-N(alkyl)2, cycloalkyl or alkyl-cycloalkyl.

[0049] In some embodiments, at least one of R7, R8, R9, R10and R11is not H.

[0050] In some embodiments, at least two of R7, R8, R9, R10and R11are not H.

[0051] In some embodiments, at least three of R7, R8, R9, R10and R11are not H.

[0052] In some embodiments, at least four of R7, R8, R9, R10and R11are not H.

[0053] In some embodiments, R7is H; R8, R9, R10and R11are not H.

[0054] In some embodiments, R8is H; R7, R9, R10and R11are not H.

[0055] In some embodiments, R9is H; R8, R9, R10and R11are not H.

[0056] In some embodiments, R10is H; R7, R9, R8and R11are not H.

[0057] In some embodiments, R11is H; R7, R9, R8and R10are not H.

[0058] In some embodiments, R5is , orR5and R6are each independentl .

[0059] In some embodiments, R9is H, , -OCF3, -OCHF2, -alkyl, -alkenyl, -alkynyl, -aryl,-heteroaryl, -NH2, -NH-alkyl, -NH-alkenyl, -NH-alkynyl, -NH-aryl, -NH-heteroaryl, -OH, -OAc, -O- C(O)alkyl, -O-alkyl, -O-alkylaryl, -O-alkenyl, -O-alkynyl, -O-(aryl), -O-(heteroaryl), -S-(alkyl), -S- (alkenyl), -S-(alkynyl), -S-(aryl), -S-(heteroaryl), -S(O)-(alkyl), -S(O)-(aryl), -S(O)-(heteroaryl), -SO2- (alkyl), -SO2-(aryl), -SO2-(heteroaryl), alkyl-OH, alkyl-O-alkyl, alkyl-NH2, alkyl-NH-alkyl, alkyl- N(alkyl)2, cycloalkyl or alkyl-cycloalkyl.

[0060] In some embodiments, R9is halo, -CN, -CF3, -OCF3, -alkyl, -alkenyl, -alkynyl, -aryl, -heteroaryl, -NH2, -NH-alkyl, -NH-alkenyl, -NH-alkynyl, -NH-aryl, -NH-heteroaryl, -OH, or -OAc.

[0061] In some embodiments, R9is halo, -CN, -CF3, -OCF3, -OCHF2, -alkyl, -alkenyl, -alkynyl, -aryl, or -heteroaryl.

[0062] In some embodiments, R9is halo, -CN, -CF3, -OCF3, or -OCHF2.

[0063] In some embodiments, R9is F, -CF3, -OCF3, or -OCHF2.

[0064] In some embodiments, R5and R6are each independently: ,

[0065] In some embodiments, R5and R6are each independent ,.

[0067] In some embodiments, R1is -H or alkyl.

[0068] In some embodiments, R1is -H or propyl. ,,,

[0072] In some embodiments, R5and R6are each independently, a substituted aryl or substituted or unsubstituted heteroaryl.

[0073] In some embodiments, R5and R6are each independently having the following structure:; wherein R7, R8, R9, R10a , halo, -CN, -CF3, -OCF3, -OCHF2, - alkyl, -alkenyl, -alkynyl, -aryl, -heteroaryl, -NH2, -NH-alkyl, -NH-alkenyl, -NH-alkynyl, -NH-aryl, -NH-heteroaryl, -OH, -OAc, -O-C(O)alkyl, -O-alkyl, -O-alkylaryl, -O-alkenyl, -O-alkynyl, -O- (aryl), -O-(heteroaryl), -S-(alkyl), -S-(alkenyl), -S-(alkynyl), -S-(aryl), -S-(heteroaryl), -S(O)- (alkyl), -S(O)-(aryl), -S(O)-(heteroaryl), -SO2-(alkyl), -SO2-(aryl), -SO2-(heteroaryl), alkyl-OH, alkyl-O-alkyl, alkyl-NH2, alkyl-NH-alkyl, alkyl-N(alkyl)2, cycloalkyl or alkyl-cycloalkyl.

[0074] In some embodiments, at least one of R7, R8, R9, R10and R11is not H.

[0075] In some embodiments, at least two of R7, R8, R9, R10and R11are not H.

[0076] In some embodiments, at least three of R7, R8, R9, R10and R11are not H.

[0077] In some embodiments, at least four of R7, R8, R9, R10and R11are not H.

[0078] In some embodiments, R7is H; R8, R9, R10and R11are not H.

[0079] In some embodiments, R8is H; R7, R9, R10and R11are not H.

[0080] In some embodiments, R9is H; R8, R9, R10and R11are not H.

[0081] In some embodiments, R10is H; R7, R9, R8and R11are not H.

[0082] In some embodiments, R11is H; R7, R9, R8and R10are not H.

[0083] In some embodiments, R5is and R6is , or R5and R6are each independentl .

[0084] In some embodiments, R9is hao, -CN, -CF3, -OCF3, -OCHF2, -alkyl, -alkenyl, -alkynyl, -aryl, - heteroaryl, -NH2, -NH-alkyl, -NH-alkenyl, -NH-alkynyl, -NH-aryl, -NH-heteroaryl, -OH, -OAc, -O- C(O)alkyl, -O-alkyl, -O-alkylaryl, -O-alkenyl, -O-alkynyl, -O-(aryl), -O-(heteroaryl), -S-(alkyl), -S-(alkenyl), -S-(alkynyl), -S-(aryl), -S-(heteroaryl), -S(O)-(alkyl), -S(O)-(aryl), -S(O)-(heteroaryl), -SO2- (alkyl), -SO2-(aryl), -SO2-(heteroaryl), alkyl-OH, alkyl-O-alkyl, alkyl-NH2, alkyl-NH-alkyl, alkyl- N(alkyl)2, cycloalkyl or alkyl-cycloalkyl.

[0085] In some embodiments, R9is halo, -CN, -CF3, -OCF3, -alkyl, -alkenyl, -alkynyl, -aryl, -heteroaryl, -NH2, -NH-alkyl, -NH-alkenyl, -NH-alkynyl, -NH-aryl, -NH-heteroaryl, -OH, or -OAc,

[0086] In some embodiments, R9is halo, -CN, -CF3, -OCF3, -OCHF2, -alkyl, -alkenyl, -alkynyl, -aryl, or -heteroaryl.

[0087] In some embodiments, R9is halo, -CN, -CF3, -OCF3, or -OCHF2.

[0088] In some embodiments, R9is F, -CF3, -OCF3, or -OCHF2,

[0089] In some embodiments, R5and R6are each independently: .

[0090] In some embodiments, α is absent.

[0091] In some embodiments, R1is -H, alkyl, -C(O)-alkyl, -alkyl-N(alkyl)2, alkyl-haloalkyl, or alkyl- cycloalkyl.

[0092] In some embodiments, R1is -H, propyl, hexanyl, -C(O)-CH3, -(CH2)2-N-(CH3)2, -CH2- cyclopropyl, -(CH2)2-CH2F, or -(CH2)2-CF3. , ,, orunsubstituted heteroaryl.

[0097] In some embodiments, R5and R6are each independently having the following structure: ; wherein R7, R8, R9, R10a , halo, -CN, -CF3, -OCF3, -OCHF2, -alkyl, -alkenyl, -alkynyl, -aryl, -heteroaryl, -NH2, -NH-alkyl, -NH-alkenyl, -NH-alkynyl, -NH-aryl, -NH-heteroaryl, -OH, -OAc, -O-C(O)alkyl, -O-alkyl, -O-alkylaryl, -O-alkenyl, -O-alkynyl, -O- (aryl), -O-(heteroaryl), -S-(alkyl), -S-(alkenyl), -S-(alkynyl), -S-(aryl), -S-(heteroaryl), -S(O)- (alkyl), -S(O)-(aryl), -S(O)-(heteroaryl), -SO2-(alkyl), -SO2-(aryl), -SO2-(heteroaryl), alkyl-OH, alkyl-O-alkyl, alkyl-NH2, alkyl-NH-alkyl, alkyl-N(alkyl)2, cycloalkyl or alkyl-cycloalkyl.

[0098] In some embodiments, at least one of R7, R8, R9, R10and R11is not H.

[0099] In some embodiments, at least two of R7, R8, R9, R10and R11are not H.

[0100] In some embodiments, at least three of R7, R8, R9, R10and R11are not H.

[0101] In some embodiments, at least four of R7, R8, R9, R10and R11are not H.

[0102] In some embodiments, R7is H; R8, R9, R10and R11are not H.

[0103] In some embodiments, R8is H; R7, R9, R10and R11are not H.

[0104] In some embodiments, R9is H; R8, R9, R10and R11are not H.

[0105] In some embodiments, R10is H; R7, R9, R8and R11are not H.

[0106] In some embodiments, R11is H; R7, R9, R8and R10are not H.

[0107] In some embodiments, R5is and R6is . or R5and R6are each independently .

[0108] In some embodiments, R9is halo, -CN, -CF3, -OCF3, -OCHF2, -alkyl, -alkenyl, -alkynyl, -aryl, - heteroaryl, -NH2, -NH-alkyl, -NH-alkenyl, -NH-alkynyl, -NH-aryl, -NH-heteroaryl, -OH, -OAc, -O- C(O)alkyl, -O-alkyl, -O-alkylaryl, -O-alkenyl, -O-alkynyl, -O-(aryl), -O-(heteroaryl), -S-(alkyl), -S- (alkenyl), -S-(alkynyl), -S-(aryl), -S-(heteroaryl), -S(O)-(alkyl), -S(O)-(aryl), -S(O)-(heteroaryl), -SO2- (alkyl), -SO2-(aryl), -SO2-(heteroaryl), alkyl-OH, alkyl-O-alkyl, alkyl-NH2, alkyl-NH-alkyl, alkyl- N(alkyl)2, cycloalkyl or alkyl-cycloalkyl.

[0109] In some embodiments, R9is halo, -CN, -CF3, -OCF3, -alkyl, -alkenyl, -alkynyl, -aryl, -heteroaryl, -NH2, -NH-alkyl, -NH-alkenyl, -NH-alkynyl, -NH-aryl, -NH-heteroaryl, -OH, or -OAc.

[0110] In some embodiments, R9is halo, -CN, -CF3, -OCF3, -OCHF2, -alkyl, -alkenyl, -alkynyl, -aryl, or -heteroaryl.

[0111] In some embodiments, R9is halo, -CN, -CF3, -OCF3, or -OCHF2.

[0112] In some embodiments, R9is F, -CF3, -OCF3, or -OCHF2.

[0113] In some embodiments, R5and R6are each independently: .

[0114] , whereinα is a bond that is present or absent; R1is -H, alkyl, alkyl-haloalkyl, alkyl-cycloalkyl, -C(O)-alkyl, -C(O)-haloalkyl, -C(O)- cycloalkyl, cycloalkyl, -alkyl-N(alkyl)2, or -alkyl-NH2; R2is H, halo, alkyl, -CO2-alkyl, cycloalkyl, -alkyl-OH, -alkyl-NH2, or -alkyl-N(alkyl)2; R3is H, halo, alkyl, -CO2-alkyl, -cycloalkyl, -alkyl-OH, -alkyl-NH2, or -alkyl-N(alkyl)2; R4 is H, halo, alkyl, -CO2-alkyl, -cycloalkyl, -alkyl-OH, -alkyl-NH2, or -alkyl-N(alkyl)2; R5and R6are each independently, a substituted or unsubstituted aryl or a substituted or unsubstituted heteroaryl; and R12 is H, OH, NH2, -C(O)-alkyl, -O-C(O)-alkyl-aryl or-O-C(O)-alkyl-heteroaryl or a salt or ester thereof.

[0115] In some embodiments, α is absent.

[0116] In some embodiments, R1is -H, alkyl, -C(O)-alkyl, -alkyl-N(alkyl)2, alkyl-haloalkyl, or alkyl- cycloalkyl. ,

[0118] In some embodiments R1is H , ,

[0121] In some embodiments, R5and R6are each independently, a substituted aryl or substituted or unsubstituted heteroaryl.

[0122] In some embodiments, R5and R6are each independently having the following structure: ; wherein R7, R8, R9, R10a, halo, -CN, -CF3, -OCF3, -OCHF2, - alkyl, -alkenyl, -alkynyl, -aryl, -heteroaryl, -NH2, -NH-alkyl, -NH-alkenyl, -NH-alkynyl, -NH-aryl, -NH-heteroaryl, -OH, -OAc, -O-C(O)alkyl, -O-alkyl, -O-alkylaryl, -O-alkenyl, -O-alkynyl, -O- (aryl), -O-(heteroaryl), -S-(alkyl), -S-(alkenyl), -S-(alkynyl), -S-(aryl), -S-(heteroaryl), -S(O)- (alkyl), -S(O)-(aryl), -S(O)-(heteroaryl), -SO2-(alkyl), -SO2-(aryl), -SO2-(heteroaryl), alkyl-OH, alkyl-O-alkyl, alkyl-NH2, alkyl-NH-alkyl, alkyl-N(alkyl)2, cycloalkyl or alkyl-cycloalkyl.

[0123] In some embodiments, at least one of R7, R8, R9, R10and R11is not H.

[0124] In some embodiments, at least two of R7, R8, R9, R10and R11are not H.

[0125] In some embodiments, at least three of R7, R8, R9, R10and R11are not H.

[0126] In some embodiments, at least four of R7, R8, R9, R10and R11are not H.

[0127] In some embodiments, R7is H; R8, R9, R10and R11are not H.

[0128] In some embodiments, R8is H; R7, R9, R10and R11are not H.

[0129] In some embodiments, R9is H; R8, R9, R10and R11are not H.

[0130] In some embodiments, R10is H; R7, R9, R8and R11are not H.

[0131] In some embodiments, R11is H; R7, R9, R8and R10are not H.

[0132] In some embodiments, R5and R6are each independentl .

[0133] In some embodiments, R9is halo, -CN, -CF3, -OCF3, -O-alkenyl, -alkynyl, -aryl, - heteroaryl, -NH2, -NH-alkyl, -NH-alkenyl, -NH-alkynyl, -NH-aryl, -NH-heteroaryl, -OH, -OAc, -O- C(O)alkyl, -O-alkyl, -O-alkylaryl, -O-alkenyl, -O-alkynyl, -O-(aryl), -O-(heteroaryl), -S-(alkyl), -S- (alkenyl), -S-(alkynyl), -S-(aryl), -S-(heteroaryl), -S(O)-(alkyl), -S(O)-(aryl), -S(O)-(heteroaryl), -SO2- (alkyl), -SO2-(aryl), -SO2-(heteroaryl), alkyl-OH, alkyl-O-alkyl, alkyl-NH2, alkyl-NH-alkyl, alkyl- N(alkyl)2, cycloalkyl or alkyl-cycloalkyl.

[0134] In some embodiments, R9is halo, -CN, -CF3, -OCF3, -alkyl, -alkenyl, -alkynyl, -aryl, -heteroaryl, -NH2, -NH-alkyl, -NH-alkenyl, -NH-alkynyl, -NH-aryl, -NH-heteroaryl, -OH, or -OAc.

[0135] In some embodiments, R9is halo, -CN, -CF3, -OCF3, -OCHF2, -alkyl, -alkenyl, -alkynyl, -aryl, or -heteroaryl.

[0136] In some embodiments, R9is halo, -CN, -CF3, -OCF3, or -OCHF2.

[0137] In some embodiments, R9is F, or -OCF3.

[0138] In some embodiments .

[0139] In some embodiments, an are eac n epen enty: .

[0140] s a compound having the following structure:, , , , ,, , , , ,or, whereinα is a bond that is present or absent; X is C, N, or O; R1is -H, alkyl, alkenyl, alkynyl, aryl, heteroaryl, -C(O)-alkyl, -C(O)-haloalkyl, -C(O)-cycloalkyl, -C(O)-alkenyl, -C(O)-alkynyl, -C(O)-aryl, -C(O)-heteroaryl, -CO2-alkenyl, -CO2-alkynyl, -CO2- aryl, -CO2-heteroaryl, cycloalkyl, -alkyl-OH, -alkyl-N(alkyl)2, -alkyl-NH2, -alkyl-haloalkyl, -alkyl- CF3, or -alkyl-cycloalkyl; R2is H, halo, alkyl, alkenyl, alkynyl, aryl, heteroaryl, -CO2-alkyl, -CO2-alkenyl, -CO2-alkynyl, - CO2-aryl, -CO2-heteroaryl, cycloalkyl, -alkyl-OH, -alkyl-NH2or -alkyl-N(alkyl)2; R3is -H, halo, alkyl, alkenyl, alkynyl, aryl, heteroaryl, -CO2-alkyl, -CO2-alkenyl, -CO2-alkynyl, - CO2-aryl, -CO2-heteroaryl, cycloalkyl, -alkyl-OH, -alkyl-NH2or -alkyl-N(alkyl)2;R4is -H, halo, alkyl, alkenyl, alkynyl, aryl, heteroaryl, -CO2-alkyl, -CO2-alkenyl, -CO2-alkynyl, - CO2-aryl, -CO2-heteroaryl, cycloalkyl, -alkyl-OH, -alkyl-NH2, or -alkyl-N(alkyl)2; and R5is a substituted aryl or substituted or unsubstituted cycloalkyl; wherein the substitution is H, alkyl, alkenyl, alkynyl carbonyl, amine; C(O)-alkyl, C(O)-haloalkyl, or CO2-alkyl; wherein when is present, R1is not H;H3;wherein when is present, R1is not H;wherein when R5i and α is present, R1is not H or CH3; wherein when α iss, , , , ,, notor a salt or ester thereof.

[0142] In some embodiments, α is absent. ,,,

[0147] In some embodiments, R1is -H or propyl.

[0148] In some embodiments, R5is a substituted aryl or cycloalkyl.

[0149] In some embodiments, the cycloalkyl is adamantane.

[0150] In some embodiments, R5has the following structure: ,wherein R7, R8, R9, R10and R11are each, independently, -H, halo, -CN, -CF3, -OCF3, -OCHF2, - alkyl, -alkenyl, -alkynyl, -aryl, -heteroaryl, -NH2, -NH-alkyl, -NH-alkenyl, -NH-alkynyl, -NH-aryl, -NH-heteroaryl, -OH, -OAc, -O-C(O)alkyl, -O-alkyl, -O-alkylaryl, -O-alkenyl, -O-alkynyl, -O- (aryl), -O-(heteroaryl), -S-(alkyl), -S-(alkenyl), -S-(alkynyl), -S-(aryl), -S-(heteroaryl), -S(O)- (alkyl), -S(O)-(aryl), -S(O)-(heteroaryl), -SO2-(alkyl), -SO2-(aryl), -SO2-(heteroaryl), alkyl-OH, alkyl-O-alkyl, alkyl-NH2, alkyl-NH-alkyl, alkyl-N(alkyl)2, cycloalkyl or alkyl-cycloalkyl.

[0151] In some embodiments, at least one of R7, R8, R9, R10and R11is not H.

[0152] In some embodiments, at least two of R7, R8, R9, R10and R11are not H.

[0153] In some embodiments, at least three of R7, R8, R9, R10and R11are not H.

[0154] In some embodiments, at least four of R7, R8, R9, R10and R11are not H.

[0155] In some embodiments, R7is H; R8, R9, R10and R11are not H.

[0156] In some embodiments, R8is H; R7, R9, R10and R11are not H.

[0157] In some embodiments, R9is H; R8, R9, R10and R11are not H.

[0158] In some embodiments, R10is H; R7, R9, R8and R11are not H.

[0159] In some embodiments, R11is H; R7, R9, R8and R10are not H.

[0160] In some embodiments, R7and R10are each independently halo, -CN, -CF3, -OCF3, -OCHF2, - alkyl, -alkenyl, -alkynyl, -aryl, -heteroaryl, -NH2, -NH-alkyl, -NH-alkenyl, -NH-alkynyl, -NH-aryl, -NH- heteroaryl, -OH, -OAc, -O-C(O)alkyl, -O-alkyl, -O-alkylaryl, -O-alkenyl, -O-alkynyl, -O-(aryl), -O- (heteroaryl), -S-(alkyl), -S-(alkenyl), -S-(alkynyl), -S-(aryl), -S-(heteroaryl), -S(O)-(alkyl), -S(O)-(aryl), - S(O)-(heteroaryl), -SO2-(alkyl), -SO2-(aryl), -SO2-(heteroaryl), alkyl-OH, alkyl-O-alkyl, alkyl-NH2, alkyl- NH-alkyl, alkyl-N(alkyl)2, cycloalkyl or alkyl-cycloalkyl.

[0161] In some embodiments, R7and R10are each independently halo, -CN, -CF3, -OCF3, -alkyl, - alkenyl, -alkynyl, -aryl, -heteroaryl, -NH2, -NH-alkyl, -NH-alkenyl, -NH-alkynyl, -NH-aryl, -NH- heteroaryl, -OH, or -OAc.

[0162] In some embodiments, R7and R10are each independently halo, branched alky, -CN, -CF3, -OCF3, -OCHF2, -alkyl, -alkenyl, -alkynyl, -aryl, or -heteroaryl.

[0163] In some embodiments, R7and R10are each independently halo, branched alky, -CN, -CF3, -OCF3, or -OCHF2.

[0164] In some embodiments, R7and R10are each independently Br, tert-butyl, or -CF3.

[0165] In some embodiments, R5is: .

[0166]

[0167] In some embodiments, R1is -H, alkyl, or alkyl-haloalkyl.

[0168] In some embodiments, R1is -H, propyl, -(CH2)2-CH2F, or -(CH2)2-CF3.

[0169] In some embodiments, R5is a substituted aryl.

[0170] In some embodiments, R5has the following structure: , wherein R7, R8, R9, R10and R11areeach, independently, -H, halo, -CN, -CF3, -OCF3, -OCHF2, - alkyl, -alkenyl, -alkynyl, -aryl, -heteroaryl, -NH2, -NH-alkyl, -NH-alkenyl, -NH-alkynyl, -NH-aryl, -NH-heteroaryl, -OH, -OAc, -O-C(O)alkyl, -O-alkyl, -O-alkylaryl, -O-alkenyl, -O-alkynyl, -O-(aryl), -O-(heteroaryl), -S-(alkyl), -S-(alkenyl), -S-(alkynyl), -S-(aryl), -S-(heteroaryl), -S(O)- (alkyl), -S(O)-(aryl), -S(O)-(heteroaryl), -SO2-(alkyl), -SO2-(aryl), -SO2-(heteroaryl), alkyl-OH, alkyl-O-alkyl, alkyl-NH2, alkyl-NH-alkyl, alkyl-N(alkyl)2, cycloalkyl or alkyl-cycloalkyl.

[0171] In some embodiments, at least one of R7, R8, R9, R10and R11is not H.

[0172] In some embodiments, at least two of R7, R8, R9, R10and R11are not H.

[0173] In some embodiments, at least three of R7, R8, R9, R10and R11are not H.

[0174] In some embodiments, at least four of R7, R8, R9, R10and R11are not H.

[0175] In some embodiments, R7is H; R8, R9, R10and R11are not H.

[0176] In some embodiments, R8is H; R7, R9, R10and R11are not H.

[0177] In some embodiments, R9is H; R8, R9, R10and R11are not H.

[0178] In some embodiments, R10is H; R7, R9, R8and R11are not H.

[0179] In some embodiments, R11is H; R7, R9, R8and R10are not H.

[0180] In some embodiments, R7and R10are each independently halo, -CN, -CF3, -OCF3, -OCHF2, - alkyl, -alkenyl, -alkynyl, -aryl, -heteroaryl, -NH2, -NH-alkyl, -NH-alkenyl, -NH-alkynyl, -NH-aryl, -NH- heteroaryl, -OH, -OAc, -O-C(O)alkyl, -O-alkyl, -O-alkylaryl, -O-alkenyl, -O-alkynyl, -O-(aryl), -O- (heteroaryl), -S-(alkyl), -S-(alkenyl), -S-(alkynyl), -S-(aryl), -S-(heteroaryl), -S(O)-(alkyl), -S(O)-(aryl), - S(O)-(heteroaryl), -SO2-(alkyl), -SO2-(aryl), -SO2-(heteroaryl), alkyl-OH, alkyl-O-alkyl, alkyl-NH2, alkyl- NH-alkyl, alkyl-N(alkyl)2, cycloalkyl or alkyl-cycloalkyl.

[0181] In some embodiments, R7and R10are each independently halo, -CN, -CF3, -OCF3, -alkyl, - alkenyl, -alkynyl, -aryl, -heteroaryl, -NH2, -NH-alkyl, -NH-alkenyl, -NH-alkynyl, -NH-aryl, -NH- heteroaryl, -OH, or -Oac.

[0182] In some embodiments, R7and R10are each independently halo, branched alky, -CN, -CF3, -OCF3, -OCHF2, -alkyl, -alkenyl, -alkynyl, -aryl, or -heteroaryl.

[0183] In some embodiments, R7and R10are each independently halo, branched alky, -CN, -CF3, -OCF3, or -OCHF2.

[0184] In some embodiments, R7and R10are each independently Br, tert-butyl, or -CF3., , , ,, the compounddisclosed in the present invention and a pharmaceutically acceptable carrier.

[0188] In some embodiments, the pharmaceutical composition further comprises an anti-tuberculosis drug and a pharmaceutically acceptable carrier.

[0189] In some embodiments, the pharmaceutical composition further comprises compound disclosed in the U.S. Application No.16 / 097,558, now patented as U.S. Patent No.11,072,633 and a pharmaceutically acceptable carrier.

[0190] In some embodiments, the anti-tuberculosis drug is isoniazid, ethionamide, pretomanid (PA- 824), linezolid, clofazimine, pyrazinamide, ethambutol, rifabutin, kanamycin, amikacin, capreomycin, streptomycin, levofloxacin, moxifloxacin, ofloxacin, para–aminosalicylic acid, cycloserine, terizidone, thionamide, protionamide, rifapentine, rifampicin, delamanid or bedaquiline or combinations thereof.

[0191] In some embodiments, the anti-tuberculosis drug is pyrazinamide, ethambutol, rifabutin, kanamycin, amikacin, capreomycin, streptomycin, levofloxacin, ofloxacin, para–aminosalicylic acid, cycloserine, terizidone, thionamide, protionamide, delamanid or bedaquiline, or combinations thereof.

[0192] In some embodiments, the anti-tuberculosis drug is the anti-tuberculosis drug isOCF3O H NN,, ,, ,

[0193] In some embodiments, the anti-tuberculosis drug is isoniazid or ethionamide.

[0194] The present invention provides a method of inhibiting M. tuberculosis comprises administering the compound disclosed in the present invention.

[0195] The present invention provides a method of controlling the population of M. tuberculosis comprises administering the compound disclosed in the present invention.

[0196] The present invention provides a method of inhibiting reproduction and growth of M. tuberculosis comprises administering the compound disclosed in the present invention.

[0197] The present invention provides a method of treating a subject infected with M. tuberculosis comprising administering to the subject an amount of the compound disclosed in the present invention so as to thereby treat the subject.

[0198] In some embodiments, the M. tuberculosis infection is latent, active, drug-resistant, extensively drug-resistant or multi-drug resistant.

[0199] In some embodiments, the infected subject is afflicted with pulmonary tuberculosis or extrapulmonary tuberculosis.

[0200] In some embodiments, the infected subject is human.

[0201] In some embodiments, the amount of the compound and the amount of the anti-tuberculosis drug when taken together is more effective to treat the M. tuberculosis infection than the anti-tuberculosis drug alone.

[0202] In some embodiments, the amount of the compound is lower than the amount of the anti- tuberculosis drug that is effective to achieve the same level of treatment when administered alone.

[0203] In some embodiments, the amount of the compound and the amount of the anti-tuberculosis drug when administered together are more effective to treat the subject than when each agent at the same amount is administered alone.

[0204] In some embodiments, the amount of the compound and the amount of the anti-tuberculosis drug is administered simultaneously, contemporaneously or concomitantly.

[0205] In some embodiments, the amount of the compound causes the M. tuberculosis in the subject to be more susceptible to treatment with the anti-tuberculosis drug.

[0206] In some embodiments, the amount of the compound enhances the anti-tuberculosis effect of the anti-tuberculosis drug.

[0207] In some embodiments, the method further comprises administering an amount of an anti- tuberculosis drug to the subject.

[0208] In some embodiments, the amount of the compound enhances the anti-tuberculosis effect of the anti-tuberculosis drug.

[0209] In some embodiments, the anti-tuberculosis drug is isoniazid, rifampicin, pyrazinamide, ethambutol or streptomycin or combinations thereof.

[0210] In some embodiments, the anti-tuberculosis drug is pretomanid (PA-824), pyrazinamide, ethambutol, rifabutin, kanamycin, amikacin, capreomycin, streptomycin, levofloxacin, moxifloxacin, ofloxacin, para–aminosalicylic acid, cycloserine, terizidone, thionamide, protionamide, rifapentine, rifampicin, delamanid or bedaquiline or combinations thereof.

[0211] In some embodiments, the anti-tuberculosis drug is pyrazinamide, ethambutol, rifabutin, kanamycin, amikacin, capreomycin, streptomycin, levofloxacin, ofloxacin, para–aminosalicylic acid, cycloserine, terizidone, thionamide, protionamide, delamanid or bedaquiline, or combinations thereof.

[0212] The compounds of the present invention include all hydrates, solvates, and complexes of the compounds used by this invention. If a chiral center or another form of an isomeric center is present in a compound of the present invention, all forms of such isomer or isomers, including enantiomers and diastereomers, are intended to be covered herein. Compounds containing a chiral center may be used as a racemic mixture, an enantiomerically enriched mixture, or the racemic mixture may be separated using well-known techniques and an individual enantiomer may be used alone. The compounds described in the present invention are in racemic form or as individual enantiomers. The enantiomers can be separated using known techniques, such as those described in Pure and Applied Chemistry 69, 1469–1474, (1997) IUPAC. In cases in which compounds have unsaturated carbon-carbon double bonds, both the cis (Z) and trans (E) isomers are within the scope of this invention.

[0213] The compounds of the subject invention may have spontaneous tautomeric forms. In cases wherein compounds may exist in tautomeric forms, such as keto-enol tautomers, each tautomeric form is contemplated as being included within this invention whether existing in equilibrium or predominantly in one form.

[0214] In the compound structures depicted herein, hydrogen atoms are not shown for carbon atoms having less than four bonds to non-hydrogen atoms. However, it is understood that enough hydrogen atoms exist on said carbon atoms to satisfy the octet rule.

[0215] This invention also provides isotopic variants of the compounds disclosed herein, including wherein the isotopic atom is2H and / or wherein the isotopic atom13C. Accordingly, in the compounds provided herein hydrogen can be enriched in the deuterium isotope. It is to be understood that the invention encompasses all such isotopic forms.

[0216] It is understood that the structures described in the embodiments of the methods hereinabove can be the same as the structures of the compounds described hereinabove.

[0217] It is understood that where a numerical range is recited herein, the present invention contemplates each integer between, and including, the upper and lower limits, unless otherwise stated.

[0218] Except where otherwise specified, if the structure of a compound of this invention includes an asymmetric carbon atom, it is understood that the compound occurs as a racemate, racemic mixture, and isolated single enantiomer. All such isomeric forms of these compounds are expressly included in this invention. Except where otherwise specified, each stereogenic carbon may be of the R or S configuration. It is to be understood accordingly that the isomers arising from such asymmetry (e.g., all enantiomers and diastereomers) are included within the scope of this invention, unless indicated otherwise. Such isomers can be obtained in substantially pure form by classical separation techniques and by stereochemically controlled synthesis, such as those described in "Enantiomers, Racemates and Resolutions" by J. Jacques, A. Collet and S. Wilen, Pub. John Wiley & Sons, NY, 1981. For example, the resolution may be carried out by preparative chromatography on a chiral column.

[0219] The subject invention is also intended to include all isotopes of atoms occurring on the compounds disclosed herein. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium. Isotopes of carbon include C-13 and C-14.

[0220] It will be noted that any notation of a carbon in structures throughout this application, when used without further notation, are intended to represent all isotopes of carbon, such as12C,13C, or14C. Furthermore, any compounds containing13C or14C may specifically have the structure of any of the compounds disclosed herein.

[0221] It will also be noted that any notation of a hydrogen in structures throughout this application, when used without further notation, are intended to represent all isotopes of hydrogen, such as1H,2H, or3H.

[0222] Furthermore, any compounds containing2H or3H may specifically have the structure of any of the compounds disclosed herein.

[0223] Isotopically-labeled compounds can generally be prepared by conventional techniques known to those skilled in the art using appropriate isotopically-labeled reagents in place of the non-labeled reagents employed.

[0224] In the compounds used in the method of the present invention, the substituents may be substituted or unsubstituted, unless specifically defined otherwise.

[0225] In the compounds used in the method of the present invention, alkyl, heteroalkyl, monocycle, bicycle, aryl, heteroaryl and heterocycle groups can be further substituted by replacing one or more hydrogen atoms with alternative non-hydrogen groups. These include, but are not limited to, halo, hydroxy, mercapto, amino, carboxy, cyano, carbamoyl and aminocarbonyl and aminothiocarbonyl.

[0226] It is understood that substituents and substitution patterns on the compounds used in the method of the present invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure result.

[0227] In choosing the compounds used in the method of the present invention, one of ordinary skill in the art will recognize that the various substituents, i.e. R1, R2, etc. are to be chosen in conformity with well- known principles of chemical structure connectivity.

[0228] As used herein, "alkyl" is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms. Thus, C1-Cn as in “C1–Cn alkyl" is defined to include groups having 1, 2......, n-1 or n carbons in a linear or branched arrangement, and specifically includes methyl, ethyl, pro yl, butyl, pentyl, hexyl, heptyl, isopropyl, isobutyl, sec-butyl and so on. An embodiment can be C1-C12 alkyl, C2-C12 alkyl, C3-C12 alkyl, C4-C12 alkyl and so on. “Alkoxy" represents an alkyl group as described above attached through an oxygen bridge.

[0229] The term "alkenyl" refers to a non-aromatic hydrocarbon radical, straight or branched, containing at least 1 carbon to carbon double bond, and up to the maximum possible number of non-aromatic carbon- carbon double bonds may be present. Thus, C2-Cn alkenyl is defined to include groups having 1, 2...., n-1 or n carbons. For example, "C2-C6 alkenyl" means an alkenyl radical having 2, 3, 4, 5, or 6 carbon atoms, and at least 1 carbon-carbon double bond, and up to, for example, 3 carbon-carbon double bonds in the case of a C6 alkenyl, respectively. Alkenyl groups include ethenyl, propenyl, butenyl and cyclohexenyl. As described above with respect to alkyl, the straight, branched or cyclic portion of the alkenyl group may contain double bonds and may be substituted if a substituted alkenyl group is indicated. An embodiment can be C2-C12 alkenyl, C3-C12 alkenyl, C4-C12 alkenyl and so on.

[0230] The term "alkynyl" refers to a hydrocarbon radical straight or branched, containing at least 1 carbon to carbon triple bond, and up to the maximum possible number of non-aromatic carbon-carbon triple bonds may be present. Thus, C2-Cnalkynyl is defined to include groups having 1, 2...., n-1 or n carbons. For example, "C2-C6alkynyl" means an alkynyl radical having 2 or 3 carbon atoms, and 1 carbon-carbon triple bond, or having 4 or 5 carbon atoms, and up to 2 carbon-carbon triple bonds, or having 6 carbonatoms, and up to 3 carbon-carbon triple bonds. Alkynyl groups include ethynyl, propynyl and butynyl. As described above with respect to alkyl, the straight or branched portion of the alkynyl group may contain triple bonds and may be substituted if a substituted alkynyl group is indicated. An embodiment can be a C2- Cnalkynyl. An embodiment can be C2-C12alkynyl, C3-C12alkynyl, C4-C12alkynyl and so on

[0231] “Alkylene”, “alkenylene” and “alkynylene” shall mean, respectively, a divalent alkane, alkene and alkyne radical, respectively. It is understood that an alkylene, alkenylene, and alkynylene may be straight or branched. An alkylene, alkenylene, and alkynylene may be unsubstituted or substituted.

[0232] As used herein, "heteroalkyl" includes both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms and at least 1 heteroatom within the chain or branch.

[0233] As herein, "cycloalkyl" shall mean cyclic rings of alkanes of three to eight total carbon atoms, or any number within this range (i.e., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl).

[0234] As used herein, "monocycle" includes any stable polyatomic carbon ring of up to 10 atoms and may be unsubstituted or substituted. Examples of such non-aromatic monocycle elements include but are not limited to: cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. Examples of such aromatic monocycle elements include but are not limited to: phenyl.

[0235] As used herein, "bicycle" includes any stable polyatomic carbon ring of up to 10 atoms that is fused to a polyatomic carbon ring of up to 10 atoms with each ring being independently unsubstituted or substituted. Examples of such non-aromatic bicycle elements include but are not limited to: decahydronaphthalene. Examples of such aromatic bicycle elements include but are not limited to: naphthalene.

[0236] As used herein, "aryl" is intended to mean any stable monocyclic, bicyclic or polycyclic carbon ring of up to 10 atoms in each ring, wherein at least one ring is aromatic, and may be unsubstituted or substituted. Examples of such aryl elements include phenyl, p-toluenyl (4-methylphenyl), naphthyl, tetrahydro-naphthyl, indanyl, biphenyl, phenanthryl, anthryl or acenaphthyl. In cases where the aryl substituent is bicyclic and one ring is non-aromatic, it is understood that attachment is via the aromatic ring.

[0237] As used herein, the term “polycyclic” refers to unsaturated or partially unsaturated multiple fused ring structures, which may be unsubstituted or substituted.

[0238] The term “arylalkyl” refers to alkyl groups as described above wherein one or more bonds to hydrogen contained therein are replaced by a bond to an aryl group as described above. It is understood thatan “arylalkyl” group is connected to a core molecule through a bond from the alkyl group and that the aryl group acts as a substituent on the alkyl group. Examples of arylalkyl moieties include, but are not limited to, benzyl (phenylmethyl), p-trifluoromethylbenzyl (4-trifluoromethylphenylmethyl), 1-phenylethyl, 2- phenylethyl, 3-phenylpropyl, 2-phenylpropyl and the like.

[0239] The term "heteroaryl", as used herein, represents a stable monocyclic, bicyclic or polycyclic ring of up to 10 atoms in each ring, wherein at least one ring is aromatic and contains from 1 to 4 heteroatoms selected from the group consisting of O, N and S. Bicyclic aromatic heteroaryl groups include phenyl, pyridine, pyrimidine or pyridizine rings that are (a) fused to a 6-membered aromatic (unsaturated) heterocyclic ring having one nitrogen atom; (b) fused to a 5- or 6-membered aromatic (unsaturated) heterocyclic ring having two nitrogen atoms; (c) fused to a 5-membered aromatic (unsaturated) heterocyclic ring having one nitrogen atom together with either one oxygen or one sulfur atom; or (d) fused to a 5- membered aromatic (unsaturated) heterocyclic ring having one heteroatom selected from O, N or S. Heteroaryl groups within the scope of this definition include but are not limited to: benzimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, quinolyl, furanyl, indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl, oxazolyl, isoxazoline, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyrimidyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, acridinyl, carbazolyl, quinoxalinyl, pyrrazolyl, indolyl, benzotriazolyl, benzothiazolyl, benzoxazolyl, isoxazolyl, isothiazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, or pyrrolyl. In cases where the heteroaryl substituent is bicyclic and one ring is non-aromatic or contains no heteroatoms, it is understood that attachment is via the aromatic ring or via the heteroatom containing ring, respectively. If the heteroaryl contains nitrogen atoms, it is understood that the corresponding N-oxides thereof are also encompassed by this definition.

[0240] The term “heteroarylalkyl” refers to alkyl groups as described above wherein one or more bonds to hydrogen contained therein are replaced by a bond to an heteroaryl group as described above. It is understood that an “heteroarylalkyl” group is connected to a core molecule through a bond from the alkyl group and that the heteroaryl group acts as a substituent on the alkyl group. Examples of heteroarylalkylmoieties include, but are not limited to, -CH2-(C5H4N), -CH2-CH2-(C5H4N) and the like.

[0241] The term "heterocycle" or “heterocyclyl” refers to a mono- or poly-cyclic ring system which can be saturated or contains one or more degrees of unsaturation and contains one or more heteroatoms. Preferred heteroatoms include N, O, and / or S, including N-oxides, sulfur oxides, and dioxides. Preferably the ring is three to ten-membered and is either saturated or has one or more degrees of unsaturation. Morepreferably the ring is three to four-membered and has one or more degrees of unsaturation. The heterocycle may be unsubstituted or substituted, with multiple degrees of substitution being allowed. Such rings may be optionally fused to one or more of another "heterocyclic" ring(s), heteroaryl ring(s), aryl ring(s), or cycloalkyl ring(s). Examples of heterocycles include, but are not limited to, aziridine, azirine, diazirine, oxirane, thiirane, azetidine, oxetane, thetane, tetrahydrofuran, pyran, 1,4-dioxane, 1,3-dioxane, piperidine, piperazine, pyrrolidine, morpholine, thiomorpholine, tetrahydrothiopyran, tetrahydrothiophene, 1,3- oxathiolane, and the like.

[0242] The alkyl, alkenyl, alkynyl, aryl, heteroaryl and heterocyclyl substituents may be substituted or unsubstituted, unless specifically defined otherwise. In the compounds of the present invention, alkyl, alkenyl, alkynyl, aryl, heterocyclyl and heteroaryl groups can be further substituted by replacing one or more hydrogen atoms with alternative non-hydrogen groups. These include, but are not limited to, halo, hydroxy, mercapto, amino, carboxy, cyano and carbamoyl.

[0243] As used herein, the term “halogen” refers to F, Cl, Br, and I.

[0244] The terms “substitution”, “substituted” and “substituent” refer to a functional group as described above in which one or more bonds to a hydrogen atom contained therein are replaced by a bond to non- hydrogen or non-carbon atoms, provided that normal valencies are maintained and that the substitution results in a stable compound. Substituted groups also include groups in which one or more bonds to a carbon(s) or hydrogen(s) atom are replaced by one or more bonds, including double or triple bonds, to a heteroatom. Examples of substituent groups include the functional groups described above, and halogens (i.e., F, Cl, Br, and I); alkyl groups, such as methyl, ethyl, n-propyl, isopropryl, n-butyl, tert-butyl, and trifluoromethyl; hydroxyl; alkoxy groups, such as methoxy, ethoxy, n-propoxy, and isopropoxy; aryloxy groups, such as phenoxy; arylalkyloxy, such as benzyloxy (phenylmethoxy) and p- trifluoromethylbenzyloxy (4-trifluoromethylphenylmethoxy); heteroaryloxy groups; sulfonyl groups, such as trifluoromethanesulfonyl, methanesulfonyl, and p-toluenesulfonyl; nitro, nitrosyl; mercapto; sulfanyl groups, such as methylsulfanyl, ethylsulfanyl and propylsulfanyl; cyano; amino groups, such as amino, methylamino, dimethylamino, ethylamino, and diethylamino; and carboxyl. Where multiple substituent moieties are disclosed or claimed, the substituted compound can be independently substituted by one or more of the disclosed or claimed substituent moieties, singly or pluraly. By independently substituted, it is meant that the (two or more) substituents can be the same or different.

[0245] It is understood that substituents and substitution patterns on the compounds of the instant invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art, as well as those methods set forth below, from readily available starting materials. If a substituent is itself substituted with more than one group, it isunderstood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure result.

[0246] In choosing the compounds of the present invention, one of ordinary skill in the art will recognize that the various substituents, i.e. R1, R2, etc. are to be chosen in conformity with well-known principles of chemical structure connectivity.

[0247] The various R groups attached to the aromatic rings of the compounds disclosed herein may be added to the rings by standard procedures, for example those set forth in Advanced Organic Chemistry: Part B: Reaction and Synthesis, Francis Carey and Richard Sundberg, (Springer) 5th ed. Edition. (2007), the content of which is hereby incorporated by reference.

[0248] The compounds used in the method of the present invention may be prepared by techniques well known in organic synthesis and familiar to a practitioner ordinarily skilled in the art. However, these may not be the only means by which to synthesize or obtain the desired compounds.

[0249] The compounds used in the method of the present invention may be prepared by techniques described in Vogel’s Textbook of Practical Organic Chemistry, A.I. Vogel, A.R. Tatchell, B.S. Furnis, A.J. Hannaford, P.W.G. Smith, (Prentice Hall) 5thEdition (1996), March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Michael B. Smith, Jerry March, (Wiley-Interscience) 5thEdition (2007), and references therein, which are incorporated by reference herein. However, these may not be the only means by which to synthesize or obtain the desired compounds.

[0250] Another aspect of the invention comprises a compound used in the method of the present invention as a pharmaceutical composition.

[0251] In some embodiments, a pharmaceutical composition comprising the compound of the present invention and a pharmaceutically acceptable carrier.

[0252] As used herein, the term “pharmaceutically active agent” means any substance or compound suitable for administration to a subject and furnishes biological activity or other direct effect in the treatment, cure, mitigation, diagnosis, or prevention of disease, or affects the structure or any function of the subject. Pharmaceutically active agents include, but are not limited to, substances and compounds described in the Physicians’ Desk Reference (PDR Network, LLC; 64th edition; November 15, 2009) and “Approved Drug Products with Therapeutic Equivalence Evaluations” (U.S. Department Of Health And Human Services, 30thedition, 2010), which are hereby incorporated by reference. Pharmaceutically active agents which have pendant carboxylic acid groups may be modified in accordance with the present invention using standard esterification reactions and methods readily available and known to those having ordinary skill in the art of chemical synthesis. Where a pharmaceutically active agent does not possess acarboxylic acid group, the ordinarily skilled artisan will be able to design and incorporate a carboxylic acid group into the pharmaceutically active agent where esterification may subsequently be carried out so long as the modification does not interfere with the pharmaceutically active agent’s biological activity or effect.

[0253] The compounds used in the method of the present invention may be in a salt form. As used herein, a “salt” is a salt of the instant compounds which has been modified by making acid or base salts of the compounds. In the case of compounds used to treat an infection or disease caused by a pathogen, the salt is pharmaceutically acceptable. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as phenols. The salts can be made using an organic or inorganic acid. Such acid salts are chlorides, bromides, sulfates, nitrates, phosphates, sulfonates, formates, tartrates, maleates, malates, citrates, benzoates, salicylates, ascorbates, and the like. Phenolate salts are the alkaline earth metal salts, sodium, potassium or lithium. The term "pharmaceutically acceptable salt" in this respect, refers to the relatively non-toxic, inorganic and organic acid or base addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or by separately reacting a purified compound of the invention in its free base or free acid form with a suitable organic or inorganic acid or base, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like. (See, e.g., Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci.66:1-19).

[0254] The compounds of the present invention may also form salts with basic amino acids such a lysine, arginine, etc. and with basic sugars such as N-methylglucamine, 2-amino-2-deoxyglucose, etc. and any other physiologically non-toxic basic substance.

[0255] As used herein, “administering” an agent may be performed using any of the various methods or delivery systems well known to those skilled in the art. The administering can be performed, for example, orally, parenterally, intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectally, transbuccally, intranasally, liposomally, via inhalation, vaginally, intraoccularly, via local delivery, subcutaneously, intraadiposally, intraarticularly, intrathecally, into a cerebral ventricle, intraventicularly, intratumorally, into cerebral parenchyma or intraparenchchymally.

[0256] The compounds used in the method of the present invention may be administered in various forms, including those detailed herein. The treatment with the compound may be a component of a combination therapy or an adjunct therapy, i.e. the subject or patient in need of the drug is treated or given another drug for the disease in conjunction with one or more of the instant compounds. This combinationtherapy can be sequential therapy where the patient is treated first with one drug and then the other or the two drugs are given simultaneously. These can be administered independently by the same route or by two or more different routes of administration depending on the dosage forms employed.

[0257] As used herein, a "pharmaceutically acceptable carrier" is a pharmaceutically acceptable solvent, suspending agent or vehicle, for delivering the instant compounds to the animal or human. The carrier may be liquid or solid and is selected with the planned manner of administration in mind. Liposomes are also a pharmaceutically acceptable carrier as are slow-release vehicles.

[0258] The dosage of the compounds administered in treatment will vary depending upon factors such as the pharmacodynamic characteristics of a specific chemotherapeutic agent and its mode and route of administration; the age, sex, metabolic rate, absorptive efficiency, health and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment being administered; the frequency of treatment with; and the desired therapeutic effect.

[0259] A dosage unit of the compounds used in the method of the present invention may comprise a single compound or mixtures thereof with additional antitumor agents. The compounds can be administered in oral dosage forms as tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. The compounds may also be administered in intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular form, or introduced directly, e.g. by injection, topical application, or other methods, into or topically onto a site of disease or lesion, all using dosage forms well known to those of ordinary skill in the pharmaceutical arts.

[0260] The compounds used in the method of the present invention can be administered in admixture with suitable pharmaceutical diluents, extenders, excipients, or in carriers such as the novel programmable sustained-release multi-compartmental nanospheres (collectively referred to herein as a pharmaceutically acceptable carrier) suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices. The unit will be in a form suitable for oral, nasal, rectal, topical, intravenous or direct injection or parenteral administration. The compounds can be administered alone or mixed with a pharmaceutically acceptable carrier. This carrier can be a solid or liquid, and the type of carrier is generally chosen based on the type of administration being used. The active agent can be co-administered in the form of a tablet or capsule, liposome, as an agglomerated powder or in a liquid form. Examples of suitable solid carriers include lactose, sucrose, gelatin and agar. Capsule or tablets can be easily formulated and can be made easy to swallow or chew; other solid forms include granules, and bulk powders. Tablets may contain suitable binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents. Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents,including esters, emulsions, syrups or elixirs, suspensions, solutions and / or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents. Oral dosage forms optionally contain flavorants and coloring agents. Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.

[0261] Techniques and compositions for making dosage forms useful in the present invention are described in the following references: 7 Modern Pharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors, 1979); Pharmaceutical Dosage Forms: Tablets (Lieberman et al., 1981); Ansel, Introduction to Pharmaceutical Dosage Forms 2nd Edition (1976); Remington's Pharmaceutical Sciences, 17th ed. (Mack Publishing Company, Easton, Pa., 1985); Advances in Pharmaceutical Sciences (David Ganderton, Trevor Jones, Eds., 1992); Advances in Pharmaceutical Sciences Vol. 7. (David Ganderton, Trevor Jones, James McGinity, Eds., 1995); Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms (Drugs and the Pharmaceutical Sciences, Series 36 (James McGinity, Ed., 1989); Pharmaceutical Particulate Carriers: Therapeutic Applications: Drugs and the Pharmaceutical Sciences, Vol 61 (Alain Rolland, Ed., 1993); Drug Delivery to the Gastrointestinal Tract (Ellis Horwood Books in the Biological Sciences. Series in Pharmaceutical Technology; J. G. Hardy, S. S. Davis, Clive G. Wilson, Eds.); Modem Pharmaceutics Drugs and the Pharmaceutical Sciences, Vol 40 (Gilbert S. Banker, Christopher T. Rhodes, Eds.). All of the aforementioned publications are incorporated by reference herein.

[0262] Tablets may contain suitable binders, lubricants, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents. For instance, for oral administration in the dosage unit form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, gelatin, agar, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

[0263] The compounds used in the method of the present invention may also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids such as lecithin,sphingomyelin, proteolipids, protein-encapsulated vesicles or from cholesterol, stearylamine, or phosphatidylcholines. The compounds may be administered as components of tissue-targeted emulsions.

[0264] The compounds used in the method of the present invention may also be coupled to soluble polymers as targetable drug carriers or as a prodrug. Such polymers include polyvinylpyrrolidone, pyran copolymer, polyhydroxylpropylmethacrylamide-phenol, polyhydroxyethylasparta-midephenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues. Furthermore, the compounds may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, and crosslinked or amphipathic block copolymers of hydrogels.

[0265] Gelatin capsules may contain the active ingredient compounds and powdered carriers, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as immediate release products or as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar-coated or film-coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract.

[0266] For oral administration in liquid dosage form, the oral drug components are combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and / or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents.

[0267] Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance. In general, water, asuitable oil, saline, aqueous dextrose (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions. Solutions for parenteral administration preferably contain a water-soluble salt of the active ingredient, suitable stabilizing agents, and if necessary, buffer substances. Antioxidizing agents such as sodium bisulfite, sodium sulfite, or ascorbic acid, either alone or combined, are suitable stabilizing agents. Also used are citric acid and its salts and sodium EDTA. In addition, parenteral solutions can contain preservatives, such as benzalkonium chloride, methyl- or propyl-paraben, and chlorobactene. Suitablepharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field.

[0268] The compounds used in the method of the present invention may also be administered in intranasal form via use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will generally be continuous rather than intermittent throughout the dosage regimen.

[0269] Parenteral and intravenous forms may also include minerals and other materials such as solutol and / or ethanol to make them compatible with the type of injection or delivery system chosen.

[0270] The compounds and compositions of the present invention can be administered in oral dosage forms as tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. The compounds may also be administered in intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular form, or introduced directly, e.g. by topical administration, injection or other methods, to the afflicted area, such as a wound, including ulcers of the skin, all using dosage forms well known to those of ordinary skill in the pharmaceutical arts.

[0271] Specific examples of pharmaceutically acceptable carriers and excipients that may be used to formulate oral dosage forms of the present invention are described in U.S. Pat. No. 3,903,297 to Robert, issued Sept.2, 1975. Techniques and compositions for making dosage forms useful in the present invention are described-in the following references: 7 Modern Pharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors, 1979); Pharmaceutical Dosage Forms: Tablets (Lieberman et al., 1981); Ansel, Introduction to Pharmaceutical Dosage Forms 2nd Edition (1976); Remington's Pharmaceutical Sciences, 17th ed. (Mack Publishing Company, Easton, Pa., 1985); Advances in Pharmaceutical Sciences (David Ganderton, Trevor Jones, Eds., 1992); Advances in Pharmaceutical Sciences Vol 7. (David Ganderton, Trevor Jones, James McGinity, Eds., 1995); Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms (Drugs and the Pharmaceutical Sciences, Series 36 (James McGinity, Ed., 1989); Pharmaceutical Particulate Carriers: Therapeutic Applications: Drugs and the Pharmaceutical Sciences, Vol 61 (Alain Rolland, Ed., 1993); Drug Delivery to the Gastrointestinal Tract (Ellis Horwood Books in the Biological Sciences. Series in Pharmaceutical Technology; J. G. Hardy, S. S. Davis, Clive G. Wilson, Eds.); Modem Pharmaceutics Drugs and the Pharmaceutical Sciences, Vol 40 (Gilbert S. Banker, Christopher T. Rhodes, Eds.). All of the aforementioned publications are incorporated by reference herein.

[0272] The active ingredient can be administered orally in solid dosage forms, such as capsules, tablets, powders, and chewing gum; or in liquid dosage forms, such as elixirs, syrups, and suspensions, including,but not limited to, mouthwash and toothpaste. It can also be administered parentally, in sterile liquid dosage forms.

[0273] Solid dosage forms, such as capsules and tablets, may be enteric-coated to prevent release of the active ingredient compounds before they reach the small intestine. Materials that may be used as enteric coatings include, but are not limited to, sugars, fatty acids, proteinaceous substances such as gelatin, waxes, shellac, cellulose acetate phthalate (CAP), methyl acrylate-methacrylic acid copolymers, cellulose acetate succinate, hydroxy propyl methyl cellulose phthalate, hydroxy propyl methyl cellulose acetate succinate (hypromellose acetate succinate), polyvinyl acetate phthalate (PVAP), and methyl methacrylate- methacrylic acid copolymers.

[0274] The compounds and compositions of the invention can be coated onto stents for temporary or permanent implantation into the cardiovascular system of a subject.

[0275] Variations on those general synthetic methods will be readily apparent to those of ordinary skill in the art and are deemed to be within the scope of the present invention.

[0276] Each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiments. Thus, all combinations of the various elements described herein are within the scope of the invention.

[0277] This invention will be better understood by reference to the Experimental Details which follow, but those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention as described more fully in the claims which follow thereafter. EXAMPLES METHODS Synthesis of 6-Azasteroid Probes

[0278] 6-Azasteroid inhibitors were synthesized as previously described (Frye et al., 1994). The diazirine linker [2-(3-butyn-1-yl)-3H-diaziren-3-yl]acetic acid (CAS No. 2049109-24-0) was purchased from AmBeed (Li, et al., 2013).

[0279] 17β-[N-(4-Trifluoromethoxy-diphenylmethyl)carbamoyl]-6-propyl-azaandrostan-3-[2-(3- but-3-ynyldiazirin-3-yl) acetate] (40, Diazirine PA). 17β-[N-(4-Trifluoromethoxy- diphenylmethyl)carbamoyl]-6-propyl-azaandrostan-3-one (35, 50 mg, 1 equiv) was added to a round- bottom flask along with 5 mL of anhydrous methanol, and the resulting solution was stirred at room temperature. Then NaBH4(1.1 equiv) was added slowly, and the reaction was allowed to proceed for at least 2 h, or until completion, with monitoring by TLC. Excess ethyl acetate was added to quench thereaction, and the solvent was removed in vacuo. The resulting 3-hydroxy-6-azasteroid was dissolved in DCM, the solution was washed with water, and the aqueous phase was extracted with 3 × 10 mL of DCM. The DCM extracts containing the reduced 3-hydroxy-6-azasteroid were dried with anhydrous MgSO4, and the solvent was removed in vacuo.

[0280] The diazirine linker [2-(3-butyn-1-yl)-3H-diaziren-3-yl]acetic acid was dissolved in anhydrous DCM, and the solution was cooled to 0 °C in an ice–water bath. To the linker suspension was added 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide (1.2 equiv) and 10% dimethylaminopyridine (DMAP, 0.12 equiv), and the reaction mixture was stirred for 1–2 h. Then the 3-hydroxy-6-azasteroid intermediate (2 equiv) was added, and the resulting mixture was allowed to come to room temperature and stir overnight. The progress of the reaction was monitored by TLC. Upon completion, the reaction mixture was washed with excess water to remove urea byproduct, and the organic phase was dried over anhydrous MgSO4 and filtered. The solvent was removed from the filtrate by rotary evaporation, the residue was purified by flash chromatography (0–20 % methanol in DCM) to afford 40 (Fig.21A)

[0281] 17β-[N-[4-(4-Aminobenzoyl)phenyl]pent-4-ynamide]-6-azaandrost-4-en-3-one (44, Benzophenone PA). Pent-4-ynoic acid (Alfa Aesar, CAS No. 6089-09-4) was dissolved in anhydrous toluene (5 mL). Pyridine (2.6 equiv, Sigma Aldrich) and one drop of DMF (~5 µL, Sigma Aldrich) were added, and the mixture was stirred over an ice–water bath for 5 min. Thionyl chloride (1.7 equiv, Alfa Aesar) was added in a dropwise fashion. After the mixture was stirred for 1–2 h at 0 °C, toluene was removed by rotary evaporation, and the resulting acid chloride intermediate was resuspended in anhydrous DCM. A solution of (4,4′-diaminobenzophenone (AmBeed, CAS No. 611-98-3) in anhydrous DMF containing TEA (1.5 equiv) was added dropwise to the acid chloride intermediate over an ice–water bath. The resulting mixture was stirred for 1 h at 0 °C and then at room temperature overnight with monitoring by TLC. Upon completion of the reaction, the solvent was removed in vacuo, and the residue was purified by flash chromatography to afford N-(4-(4′-aminobenzoyl)phenyl)pent-4-ynamide.

[0282] 17β-[6-tert-Butyloxycarbonyl-azaandrost-4-en-3-one]carboxylic acid was suspended in anhydrous toluene (5 mL). Pyridine (2.6 equiv, Sigma Aldrich) and one drop of DMF (~5 µL, Sigma Aldrich) were added, and the mixture was stirred over an ice–water bath for 5 min. Thionyl chloride (1.7 equiv, Alfa Aesar) was added in a dropwise fashion, and the reaction was stirred for 1–2 h at 0 ºC over an ice–water bath. Toluene was removed by rotary evaporation, and the intermediate acid chloride was resuspended in anhydrous DCM. A solution of N-(4-(4′-aminobenzoyl)phenyl)pent-4-ynamide in anhydrous DMF containing TEA (1.5 equiv) was added dropwise to the acid chloride over an ice–water bath. The resulting mixture was stirred for 1 h at 0 °C and then at room temperature overnight with monitoring by TLC. Upon completion of the reaction, the solvent was removed in vacuo, and the residuewas purified by flash chromatography. To a DCM solution of the resulting 17β-[N-[4-(4- aminobenzoyl)phenyl]pent-4-ynamide]-6-tert-butyloxycarbonyl-azaandrost-4-en-3-one was added 1–2 mL of TFA. The reaction was stirred at room temperature for 2 h with monitoring by TLC. Upon completion of the reaction, the TFA was quenched with saturated aqueous NaHCO3. The aqueous phase was extracted with 3 × 10 mL of DCM and dried over anhydrous MgSO4, and the solvent was removed in vacuo to afford 44 (Fig. S12A).

[0283] Compounds 40 and 44 were assessed to be greater than 95% pure by1H NMR spectroscopy. Bacterial Strains and Culturing

[0284] Mycobacterial cultures were grown in poly(ethylene terephthalate glycol) Erlenmeyer flasks of varying sizes (Grenier Bio-One). Both Mtb (CDC1551) and Mm (BAA-535) cultures were grown in Middlebrook 7H9 (g / L: 2.5 g disodium phosphate, 1.0 g monopotassium phosphate, 0.5 g monosodium glutamate, 0.5 g ammonium sulfate, 0.1 g sodium citrate, 0.05 g magnesium sulfate, 0.04 g ferric ammonium citrate, 1.0 mg copper sulfate, 1.0 mg pyridoxine HCl, 1.0 mg zinc sulfate, 0.5 mg biotin, 0.5 mg calcium chloride; pH 6.6 at 25 °C). MIC99 Assays of 6-Azasteroids

[0285] 6-Azasteroids were assayed for their ability to potentiate the inhibition of mycobacterial growth upon treatment with BDQ. In non-tissue-culture-treated 96-well plates, the 6-azasteroids were held at a fixed concentration of 20 µM, whereas BDQ was added to wells by serial dilution in Middlebrook 7H9 media. Mtb (CDC1551) or Mm (BAA-535) was cultured in 7H9 supplemented with glycerol and was diluted to an OD600 of 0.02. Diluted mycobacterium culture (50 µL) was added to the combined drug solutions (50 µL) in wells, and the plates were incubated at 36 °C for 2 weeks (Mtb) or at 30 °C for 5 days (Mm). Mycobacterial growth inhibition was measured by Alamar Blue staining (Mm) or by visual inspection (Mtb). Results were plotted in a dose–response curve against the log of the BDQ concentration and fitted with a nonlinear fit Gompertz curve. MIC99 values are reported as micromolar drug concentrations. Cytotoxicity Assay

[0286] HepG2 or THP-1 cells were utilized to assess the cytotoxicity of 6-azasteroids using the method outlined by Miret et al. Cells purchased from ATCC (cat. Nos. HB-8065 and TIB-202) were cultured from frozen stock grown on Minimum Eagle Medium (Corning) or Roswell Park Memorial Institute 1640 medium (Corning) supplemented with 10% fetal bovine serum and 0.1% penicillin / streptomycin. Cells were plated in 96-well plates at 104cells / well. Azasteroids were dissolved in DMSO to yield a 20 mM stock solution, and serial dilutions were prepared. Assays were performed in technical triplicates. 6-Azasteroids were incubated with cells for 72 h, after which Alamar Blue staining was performed. Data were analyzedby determining the concentration of oxidized Alamar Blue in each well. Results were plotted in a dose– response curve against the log of 6-azasteroid concentration and were fitted with a nonlinear fit curve. IC50values are reported as micromolar drug concentrations. Assessment of 3β-Hsd Inhibition

[0287] Recombinant Mtb 3β-Hsd (Rv1106c) was cloned from genomic DNA and expressed and purified as previously described.10The activity of the recombinant protein was assayed at 25 °C for the linear portion of the reaction (<10% conversion) by monitoring the absorbance of NADH at 340 nm.3β-Hsd was diluted with [tris(hydroxymethyl)methylamino]propanesulfonic acid buffer (100 mM, pH 8.5) to a final concentration of 0.5 µM, and the buffer was supplemented with 30 mM MgCl2 and 150 mM NaCl. A 10 mM stock solution of NAD+cofactor was diluted to a final concentration of 350 µM, which is 1.75 times the reported Km (200 µM). A 3 mM stock solution of dehydroepiandrosterone was prepared in ethanol. For each reaction, the final volume of ethanol was kept constant at 5%; the final concentration was 220 µM, which is 1.83 times the reported Km (120 µM). 6-Azasteroids reconstituted with DMSO were equilibrated with 3β-Hsd, and the reactions were initiated by the addition of NAD+. Reactions were carried out in quartz cuvettes and monitored at 340 nm. ROS Sensitivity

[0288] Exponentially growing Mtb WT CDC1551 or Mm WT BAA-535 strains cultured in 7H9 medium supplemented with 0.4% of glycerol or propionate were treated with 6-azasteroid for 6 h prior to exposure to CHP for 30 min. Cultures were treated with CellROX Green (Life Technologies) at a final concentration of 5 μM for 30 min at 37 °C. The cells were pelleted, the supernatant was discarded, and the pelleted cells were washed with 7H9 medium to remove any extracellular CellROX Green. The washed cells were resuspended in phosphate buffered saline and analyzed on a fluorescence plate reader at excitation / emission wavelengths of 485 / 565 nm. Measurement of ATP Concentration

[0289] Mtb or Mm in the exponential phase of growth was exposed to 0.4, 0.1, 0.025, or 0 µM BDQ in combination with 20, 5, or 0 µM 2 or 35 for 96 h. Aliquots (1.5 mL) of bacterial suspension were removed and mixed with 3 mL of boiling Tris-EDTA reagent (100 mM Tris, 4 mM EDTA, pH 7.75); and cells were lysed for 2 min with glass beads, heated at 100 °C for 5 min, and cooled on ice. Cell debris was removed by centrifugation. Supernatants were collected, an equal volume of luciferase reagent (ATP Bioluminescence Assay Kit HS II, Roche) was added to the supernatants, and luminescence was measured. ATP was measured with an ATP Colorimetric / Fluorometric Assay Kit (BioVision Research Products) according to the manufacturer’s protocol. The survival of mycobacteria was measured by plating them on7H11 agar plates and counting CFUs to normalize ATP concentration to the number of live mycobacterial cells. Protein Target Identification by Affinity Capture

[0290] Mm was cultured on 7H9 media (100 mL) supplemented with glycerol (2 mL / L) or cholesterol (0.5 mM); grown to OD600= 1; and concentrated 10-fold by centrifugation. The concentrated culture was treated for 6 h at 30 °C with 50 µM of a probe (40 or 44), either alone or with a 10-fold excess of the nonprobe analog (43). Treated cultures were irradiated over an ice–water bath with a 100-W UV lamp (Spectroline SB100P, rated 4800 µW / cm2at 38 cm) followed by bead beating lysis in the presence of detergent (10% v / v NP-40 and 5% w / v sodium deoxycholate). Lysates were depleted of endogenous biotin and biotin-binding proteins by incubation with streptavidin–agarose for 30 min. Protein concentration was quantified by means of a bicinchonic acid assay and adjusted to 1.0–2.0 mg / mL with phosphate buffered saline (Smith et al, 1985). The UV-cross-linked lysates were then subjected to click reaction: azido-PEG3- TAMRA-biotin (100 µM) or azide agarose beads or azido-functionalized magnetic nanoparticles, sodium D-ascorbate (2.5 mM), copper(II) sulfate pentahydrate (1 mM), tris(benzyltriazolylmethyl)amine (2 mM). Streptavidin bead enrichment was carried out by means of click reactions with azido-PEG3-TAMRA- biotin, and full-length proteins captured via biotin–streptavidin interaction were eluted from the beads by boiling them in 2× Laemmli buffer. Captured proteins were analyzed via SDS-PAGE and then by antistreptavidin western blot. Western blotting was performed by blocking with 0.22-µm-filtered 3% bovine serum albumin followed by streptavidin-conjugated horseradish peroxidase (Genscript, cat. no. M00091) incubation overnight at 1:1000 dilution in Tris-buffered saline containing 0.1% Tween 20. For proteomic analysis, azide agarose beads or azido-functionalized magnetic nanoparticles were thoroughly washed with 6 M urea and phosphate buffered saline, and the protein-bearing beads or nanoparticles were subjected to on-bead trypsin digestion in 50 mM tetraethylammonium bromide buffer (pH 8) overnight at 37 °C. Proteomic Analysis

[0291] The resulting peptides were dried under vacuum and resuspended in 0.1% formic acid. Peptides were analyzed by C18 reverse-phase liquid chromatography–tandem mass spectrometry. HPLC C18 columns were prepared using a P-2000 CO2laser puller (Sutter Instruments) and silica tubing (100 µm inner diameter × 20 cm) and were self-packed with 3-µm Reprosil resin. Peptides typically were separated at a flow rate of 300 nL / min and a gradient elution step changing from 0.1% formic acid to 40% acetonitrile over 90 min followed by 90% acetonitrile wash and re-equilibration steps. Parent peptide masses and collision-induced fragment masses were collected using an orbital trap instrument (Q-Exactive HF, Thermo), and protein databases were searched with Proteome Discoverer software (ver.2.4, ThermoFisher Scientific). Electrospray ionization was achieved at a spray voltage of ~2.3 kV. Information-dependentmass spectrometry and tandem mass spectrometry acquisitions were made using a 100-ms survey scan (m / z 375–1400) at 60,000 resolution followed typically by “top 20” consecutive second product ion scans at 15,000 resolution. False discovery rates for protein levels and spectra were set to 0.01 (1%) cutoffs. Proteome Discoverer (ver.2.4) was used for data analysis of peptide sequences that were annotated to the proteome of Mm strain ATCC BAA-535 (Proteome ID: UP000001190). In Silico Molecular Docking

[0292] AutoDock Vina was utilized for molecular receptor–ligand docking of 6-azasteroids with X-ray crystallographic structures or AlphaFold-modeled structures of the hit proteins identified in proteomics experiments (Jendele et al., 2019). Two-dimensional inhibitor structures were developed using ChemDraw (ver.19.1) software and were exported as .mol files for use with visualization software. The UCSF Chimera extensible molecular modeling system was used as the visualization software for AutoDock Vina. The Autodock Tools plugin was used to prepare the ligand and receptor for docking operations, including to convert inhibitors to three-dimensional structures. The search box was situated over the active site of each protein in various sizes. Active sites were predicted by the PrankWeb server;34and the top 10 binding poses were predicted.

[0293] Table 1. Overview of 6-azasteroid modifications. Compo13 (4-fluoro)diphenylmethyl[carbamoyl] propyl alkene 14 (4-fluoro)diphenylmethyl[carbamoyl] H alkyl(4-trifluoromethoxy)- H alkyl 30 diphenylmethyl[carbamoyl]

[0294] Table 2. Overview of 6-azasteroids – activity & cytotoxicity ompo Mtb MIC Mammalian Cell IC50 Structure Fold ∆ of BDQ MIC und Alone (µM)RESULTS

[0295] Screening of 6-Azasteroid Analogs for Activity in Mtb and Mycobacterium marinum

[0296] It was previously demonstrated that 6-azasteroid 2, which has a (2,5-di-tert-butyl)anilide group at R1and propyl group at R2, potentiates the activity of isoniazid and BDQ against both Mtb and Mycobacterium marinum (Mm) by 8-fold under normoxic conditions (Yang et al., 2019). Other similarcompounds were disclosed in the U.S. Application No. 16 / 097,558, now patented as U.S. Patent No. 11,072,633, the disclosures of which are incorporated by reference herein in their entirety.

[0297] In the present invention described herein, guided by activity measurements, a small library of 6- azasteroid analogs with an R1side chain with a single anilide group was synthesized, regardless of R2or C4–C5 saturation. A second group of analogs contain a (diphenylmethyl)carbamoyl R1side chain, a saturated or unsaturated A ring (C4–C5), and a hydrogen or a propyl group at R2. Additional analogs contain either a (4-trifluoromethoxy-diphenylmethyl)carbamoyl or a benzophenone R1side chain, a saturated or unsaturated A ring (C4–C5), and various modifications at R2(Figs.1C and 5).

[0298] The antimicrobial activities of the synthesized 6-azasteroids were determined at a fixed concentration of 20 µM and serial dilution of BDQ, and growth inhibition was measured by Alamar Blue staining or by visual inspection in microbroth assays. Efficacy is reported as the fold-change reduction of BDQ minimal inhibitory concentration (MIC99) in the presence of the 6-azasteroid relative to that in its absence (Fig. 1A, 1B). In addition, the metabolic viabilities of cultured HepG2 and THP1 cells after incubation for 72 h with individual 6-azasteroids were determined by Alamar Blue staining to investigate the cytotoxicity of the analogs to eukaryotes (Fig. 1A, 1B). 6-Azasteroid antimycobacterial activity was improved through iterative medicinal chemistry efforts guided by analysis of structure–activity relationships. It was observed that compounds with an ortho-functionalized (diphenylmethyl)carbamoyl side chain at R1displayed impressive BDQ potentiation and low cytotoxicity, leading to a wide therapeutic window. Activity was also influenced by the saturation state of the A ring and the functionality at the 6- position nitrogen. Although most of the 6-azasteroids with an unsaturated A ring had moderate cytotoxicity (generally with an IC50 between 20 and 80 µM), saturated-A-ring azasteroids showed low-micromolar cytotoxicity. The nature of the substitution at the 6-position nitrogen affected cytotoxicity. For example, acylation ablated both activity and cytotoxicity, but propylation effectively tempered cytotoxicity while maintaining activity. Compound 35 was designated as a lead compound because of its large therapeutic window and impressive potentiation of BDQ, and it was compared with compound 2 (Fig. 1D, 1E). At 20 µM, 2 and 35 displayed greater than 8-fold reductions in BDQ MIC99, and their IC50 values in mammalian cells were 25 and >100 µM, respectively. Subsequent phenotypic experiments were conducted with 2 and 35.

[0299] Inhibition of Mycobacterial 3β-Hydroxysteroid Dehydrogenase by 6-Azasteroids

[0300] 6-Azasteroids inhibit both human adrenal 3β-hydroxysteroid dehydrogenase (3β-Hsd) and mycobacterial 3β-Hsd, which shares less than 40% identity with its mammalian counterpart (Frye et al., 1994 and Thomas et al.2011). Considering these observations, 6-azasteroids were screened for potentiation activity with isoniazid and BDQ against Mtb 3β-Hsd knock-out mutants, and potentiation activity was foundto be maintained, indicating that 3β-Hsd is not a key target of 6-azasteroids (Yang et al., 2019). More recently, 6-azasteroids have been shown to inhibit Mycobacterium leprae, which has no cholesterol catabolism genes other than a gene for a 3β-Hsd homolog that shares greater than 75% amino acid identity with Mtb 3β-Hsd (Rosa et al., 2021).

[0301] Given the conflicting evidence for the role of 6-azasteroids in inhibition of Mtb 3β-Hsd, several 6-azasteroids (at 50 µM) for inhibitory activity against recombinant Mtb 3β-Hsd was screened with dehydroepiandrosterone as a substrate fixed at a concentration 1.8 times Kmand NAD+at a fixed concentration 1.75 times Km(Yang et al.2007). The rates of dehydroepiandrosterone dehydrogenation were determined with or without inhibitor by monitoring the NADH formation at 340 nm. Inhibition percentages were determined from the ratios of the rates of dehydrogenation of dehydroepiandrosterone to androstenedione with inhibitor versus the rates without inhibitor.

[0302] Compounds with a saturated A ring generally showed lower inhibitory activity than compounds with an unsaturated A ring, whereas there was no direct correlation between activity and alkylation of the 6-position nitrogen or replacement of an anilide side chain with a (diphenylmethyl)carbamoyl side chain (Fig.2A). Lead compound 35 and its derivative (compound 51) showed insignificant inhibition of 3β-Hsd activity, a result that is consistent with prior observations that 6-azasteroid potentiation of BDQ is unrelated to 3β-Hsd inhibition.

[0303] Impact of 6-Azasteroids 2 and 35 on Reactive Oxygen Species in Mtb and Mm

[0304] It was previously established that azasteroid activity requires the Mce3R regulon, which has been implicated in Mtb persistence in macrophages through resistance to oxidative stress (Yang et al., 2019). To better understand the relationship between azasteroids and oxidative stress, the effects of treatment with 2 or 35 on the generation of reactive oxygen species (ROS) were measured in the presence and absence of peroxide stress in Mtb. Specifically, the ROS levels upon treatment of wild-type (WT) Mtb cultures were measured with 5 mM of an organic peroxide, cumene hydroperoxide (CHP), for 30 min with or without preincubation of the cultures with 6-azasteroid 2 or 35 for 6 h. It was found that 6-azasteroid treatment alone had a negligible effect on intracellular ROS levels (Fig.2B). In comparison, WT Mtb pretreated with the 6-azasteroids was sensitized to oxidative stress generated by the addition of exogenous CHP. Under these conditions, 6-azasteroid pretreatment resulted in a greater accumulation of intracellular oxidants than that observed in mycobacteria that had not been pretreated with a 6-azasteroid. These data show that the 6- azasteroids interfered with the ability of Mtb to neutralize oxidative stress in vitro.

[0305] Impact of 6-Azasteroids 2 and 35 on BDQ-Mediated Inhibition of ATP Production

[0306] The primary mechanism of BDQ antimycobacterial activity is targeting of the ε subunit of ATP synthase, resulting in decreased ATP production. To assess BDQ potentiation by 6-azasteroids 2 and 35, intracellular ATP levels were assayed under co-treatment as a function of BDQ concentration. CDC1551 Mtb culture grown to OD600= 0.6 in 7H9 glycerol-containing media was exposed to 0.4, 0.1, 0.025, or 0 µM BDQ in combination with 20, 5, or 0 µM 2 or 35 for 96 h; and then ATP levels were measured (Fig. 2C,D). ATP production was not greatly affected by either 2 or 35 alone, indicating that their activity is likely unrelated to direct interaction with ATP synthase. When 2 or 35 was administered in combination with BDQ, a dose-dependent reduction in ATP concentration in the Mtb CDC1551 cultures was observed: specifically, the cellular ATP concentration was reduced to less than 50% of that seen upon treatment with BDQ. This effect was observed for both 6-azasteroids at concentrations of 5 and 20 µM.

[0307] Identification of 6-Azasteroid Protein Targets

[0308] To identify potential protein targets of the 6-azasteroids, two 6-azasteroid photochemical probes were developed that contain a terminal alkyne moiety for covalent attachment of affinity tags to enable the isolation of target proteins, as well as a diazirine or benzophenone photoreactive cross-linker moiety to enable covalent attachment to protein targets upon irradiation with UV light (Duckworth et al., 2012). Like their parent compounds, the two hydrophobic probes, designated Diazirine PA (40, Fig. 3A) and Benzophenone PA (44, Figs.3B), showed BDQ potentiation activity in Mm (Fig.1B). The diazirine moiety was attached to the azasteroid scaffold through a short linker to the A ring, and the benzophenone moiety was attached as a side chain on the D ring. The location of photo-cross-linking moieties on opposite hemispheres of the two probes was a crucial feature of the experimental design that favored elimination of proteins that bound nonspecifically to the probes from our downstream data analyses. A terminal alkyne adjacent to the cross-linking functional groups allowed attachment of an affinity tag by means of azide– alkyne click chemistry.

[0309] Protein-enrichment strategies were employed that involved copper-catalyzed azide–alkyne cycloaddition (CuAAC) click reactions with azido-PEG3-biotin or azido-PEG3-TAMRA-biotin reagents for streptavidin capture and visualization by streptavidin-conjugated horseradish peroxidase blots or direct fluorescence imaging of SDS-PAGE gels.

[0310] For proteomic analysis, CuAAC click reactions with azido-functionalized agarose beads or azido-functionalized magnetic nanoparticles (which eliminated the need for streptavidin and thus the accompanying background of natively biotinylated proteins) were utilized, in addition to streptavidin- functionalized magnetic nanoparticles to ensure consistency between proteomic and western blot methodologies.

[0311] In brief, 6-azasteroids or control probes were incubated with Mm whole cells, which were then washed, irradiated with UV light (365 nm), and lysed. Lysates were subjected to click reaction conditions with one of the aforementioned azido reagents, and the products were analyzed by western blotting or direct fluorescence imaging of SDS-PAGE, or used directly for proteomic studies.

[0312] By visual analysis of western blots and SDS-PAGE fluorescence images resulting from pull- down interaction studies, we observed two enriched protein bands of interest in the molecular weight range of 35–44 kDa and one enriched protein band of interest in the range of 48–55 kDa (Fig.3C). In results that were validated by replicate studies, the proteins between 35 and 44 kDa and between 48 and 55 kDa were captured by both Diazirine PA and Benzophenone PA but were not captured in the DMSO control sample or when protein capture by 44 was challenged by competition with a 10-fold excess of 43 (Fig. S2), an analog of 44 that is not functionalized with a terminal alkyne linker and cannot participate in the CuAAC click reactions required to enrich and visualize interacting proteins (Fig. 3). The molecular weight ranges indicated above were used to filter proteins identified in subsequent proteomic analyses.

[0313] A large number of proteins across the three different enrichment strategies was initially detected, a result that was expected given the high potential for nonspecific interactions related to reactive carbene- intermediate-based photo-cross-linkers and nonspecific protein binding by the bead matrices utilized to immobilize the captured proteins. A pool of 494 unique Mm proteins (only 289 of which had Mtb orthologs) was identified that were enriched in at least one data group for either the diazirine-based probe or the benzophenone-based probe and that fell in the molecular weight ranges determined by means of fluorescence visualization of captured proteins (Fig.3C). From a functional perspective, when characterized by KEGG pathway ID (KEGG = Kyoto Encyclopedia of Genes and Genomes), the pool of captured proteins showed no significant functional pathway enrichment relative to the makeup of the Mm proteome, indicating a lack of apparent functional biases under the experimental conditions (Figs.8&9).

[0314] Hit candidates were prioritized by identification of proteins under multiple enrichment conditions across two biological replicates (Fig. 11). The 494 Mm hit proteins were further filtered by including only proteins with Mtb orthologs or proteins with >75% amino acid sequence similarity in subsequent analyses. In this way, 77 unique prioritized proteins were identified across both probes. To further filter these proteins, data from validated mycobacterial platforms were mined that elucidated gene–drug sensitivity. Li et al. employed a genome-wide CRISPR interference library to identify genes mediating drug potency in the presence of nine different anti-TB drugs (Li et al.2022). This data was leveraged to elucidate which of our hit proteins conferred sensitivity to three drugs—BDQ, isoniazid, and rifampicin—that show enhanced activity in the presence of 6-azasteroids (Yang et al.2019).

[0315] Using the two probes described above, eight proteins that were enriched in proteomic analyses were identified; that caused sensitivity to BDQ, isoniazid, and rifampicin when the gene that coded for the protein was knocked down by CRISPR interference (Figs. 12-15 and S16); and that were within the molecular weight ranges identified by fluorescence gel imaging (Fig.17).

[0316] Table 3. Overview of Protein Hits from Proteomic Analyses of 6-Azasteroid Interaction Assays. Molecular R MMAR ID G D i ti i htthought to be involved in detoxification reactions

[0317] The identified proteins have a variety of functions, which are described in Table 3. Several proteins were selected for further study which have been annotated to catalyze antioxidant-related functions: Rv2855 (Mtr), Rv3913 (TrxB2), and Rv1623c (CydA). Antioxidant biological pathways, namely Mce3R-regulated genes, have previously been shown to be affected by azasteroids, warranting the emphasis on the three highlighted antioxidant related targets (Yang et al., 2019). The remaining five proteins have functions related to cell wall biosynthesis, drug detoxification, protein translocation, and virulence. These five proteins are worthy of follow-up, but because of the connection to antioxidant function, docking studies were only performed with the three antioxidant-related targets. To assess the validity of the hits identified by proteomic analysis, in silico molecular docking studies were performed with each of these three proteins using two affinity probes (40 and 44) and the azasteroid analogs from which they were derived (35 and 43) (Figs.17, 18, and 19).

[0318] In Silico Molecular Docking Studies

[0319] In silico molecular docking studies were performed using AutoDock Vina software (ver. 1.2.0) to model protein–azasteroid interactions. The azasteroids were screened by placing the search box over binding sites that were determined by homology modeling or by P2Rank (https: / / prankweb.cz / ), a tool for predicting protein–ligand binding sites and acquired calculated affinities for each protein–ligand pair (Table 4) .

[0320] Table 4. Binding Affinities of 6-Azasteroids 35 and 43 for Hit Proteins, Calculated Using AutoDock Vina.aProtein TrxB2 (Rv3913) Mtr (Rv2855) CydA (Rv1623c)

[0322] The docking studies revealed that the azasteroids categorically fit within the binding sites of the hit proteins. Figure 4 depicts the lowest energy states of 35 docked into three of the prioritized hit protein structures. In each simulation, the ligand is docked within the cavity predicted as the most probable binding pocket by P2Rank software.

[0323] The simulation showed that 35 binds to TrxB2 by partially blocking the NADP+binding site. In the simulation involving the co-crystal structure of TrxB2 with Mg2+, NADP+, and flavin adenine dinucleotide (PDB: 2A87), 35 blocks the interaction of NADP+with the metal cofactor and occupies a portion of the proposed binding channel for mycothiol, potentially preventing the interaction of mycothiol with the catalytic site. Key residues involved in NADP+binding include S166 (potential H-bonding to the side chain amide), Y268 (potential H-bonding to one of the trifluoromethoxy groups), and R295 (potential H-bonding to the other trifluoromethoxy group) (Fig.4A).

[0324] In the analysis of the interaction of 35 with Mtr, a homology modeled Mtr structure from AlphaFold (AF-P9WHH3-F1) was aligned with the structure of a glutathione reductase from Escherichia coli (PDB: 1GET) co-crystallized with NADP+and flavin adenine dinucleotide, to determine the binding poses of the endogenous ligands in Mtr. The amino acid sequences of the two enzymes showed 92% sequence coverage and 30% sequence identity, and both secondary and tertiary structural homology were high. The side chain of 35 overlapped with electron density corresponding to the nicotinamide moiety ofNADP+, indicating that 6-azasteroids likely prevent electron donation mediated by the nicotinamide cofactor. The side chain of 35 forms close contacts with F182 (π–π interaction), I183 (van der Waals interaction), and shows potential for interaction with R286 (H-bonding) (Fig.4B).

[0325] Compound 35 did not exhibit close contacts with the conserved NADP+-binding residues in either TrxB2 or Mtr, thereby indicating that the binding interactions between 35 and the enzymes are attributable to nonconserved residues unique to each enzyme.

[0326] Aurachin D, an experimentally validated inhibitor of CydA, has been modeled to bind in the space between the catalytic heme and menaquinone-9, interacting with W9 and R8, thereby preventing electron transfer and inhibiting the enzyme (Jeffreys et al.2023). When 35 was docked, its steroid scaffold overlapped with menaquinone-9, and the side chain of 35 protruded deep into the binding pocket and occupied a location similar to that occupied by aurachin D (Fig.4C). DISCUSSION

[0327] Tuberculosis (TB), a result of infection by the pathogen Mycobacterium tuberculosis (Mtb), is an ancient endemic disease that has managed to elude eradication by modern medicine. TB presents an enormous global burden; in 2022, it was the second most deadly infectious disease after COVID-19.1TB disproportionately affects the developing world because Mtb is an opportunistic pathogen that spreads easily under conditions of poverty, overcrowding, and malnutrition. Although rates of TB transmission are slowly decreasing in regions where the disease is pervasive, a new threat looms: TB that is resistant to traditional frontline therapies, referred to as multi-drug-resistant TB (MDR-TB). MDR-TB reduces the number of treatment options available, and worse overall outcomes are observed with MDR-TB.

[0328] Globally in 2018, only 59% of patients with MDR-TB experienced favorable outcomes, compared to 86% of patients that started on first-line TB treatment (WHO, 2021).Thus there is still an unmet need to develop improved treatment regimens to shorten treatment duration, prevent relapse and resistance, and improve outcomes. Despite continued interest in the discovery of new TB drugs and the ever-expanding clinical and preclinical pipeline, only a few new TB drugs have been approved over the past five decades. Furthermore, many clinical and preclinical anti-TB drugs have redundant mechanisms of action, targeting ATP synthesis, protein synthesis, redox stress generation, DNA replication, or cell wall synthesis (Yuan, T.; Sampson, N. S., 2018). Antibiotics belonging to the same mechanistic family are susceptible to cross-resistance and thus may not be useful for addressing complications resulting from resistant TB strains (Palomino, J. C.; Martin, A., 2014). TB therapeutics that take advantage of well- validated and novel biological pathways are integral to addressing the global TB problem. Furthermore, therapeutics that function synergistically with current anti-TB treatments are positioned to address MDR-TB by more rapidly and effectively clearing mycobacterial infection, reducing the time during which resistant mutants can arise.4

[0329] The discovery of new TB antibiotics with mechanisms outside those commonly targeted is no simple feat, in part because Mtb is so readily adaptable. It maintains the capacity to reside in diverse environments in hosts, in either active or latent replicative states, making many classically indispensable biological pathways dispensable. To date, most of the anti-TB drugs that are newly approved or under clinical testing were identified from whole-cell-based approaches (Yuan, T.; Sampson, N. S., 2018). Such approaches provide little in the way of mechanistic insight, and therefore, upon the discovery of compounds with antimycobacterial activity, further study is required to elucidate the mechanism of action (Yuan et al., 2012).

[0330] It was previously reported the utility of a series of 6-azasteroids as adjuvants to frontline anti-TB therapeutics, a promising strategy to combat innate drug resistance in Mtb. These compounds were originally hypothesized to target cholesterol metabolism, but there are previously uncovered associations between 6-azasteroid activity and several non-cholesterol-metabolizing pathways, including the Mce3R- regulated oxidative stress pathway, which is required for 6-azasteroid activity (Yang et al., 2019).

[0331] In this invention, the phenotypic effects of several 6-azasteroids alone and in combination with the ATP synthase inhibitor bedaquiline (BDQ) were explored to better understand the mechanisms of BDQ potentiation by 6-azasteroids. Furthermore, this invention sought to directly characterize the biomolecular interactions of the 6-azasteroids by leveraging two photoactivatable cross-linking probes that would enable labeling, enrichment, and proteomic analysis of the molecular target(s) of 6-azasteroids. Although previous attempts to untangle the azasteroid mechanisms of action have not produced conclusive results, a direct- interaction assay had the potential to identify proteins that contribute to azasteroid activity. Using the two probes, eight proteins were identified that mediate responses to oxidative stress in mycothiol-related pathways as well as in the electron transport chain. The potential of these proteins to be targets of 6- azasteroids was corroborated by computational molecular docking studies and by a substantial amount of experimental evidence from the literature. The potential polypharmacological properties of 6-azasteroids make it an attractive chemical class for further development as anti-TB agents.

[0332] In this invention, the phenotypic effects and the mechanism of antimycobacterial 6-azasteroids were explored. Developed by means of drug optimization efforts, lead compound 35 strongly potentiated BDQ, reducing its MIC99from 85 to 11 nM, and showed no signs of toxicity at up to 100 µM in two mammalian cell lines. Previous attempts to elucidate the mechanism of action of 6-azasteroids did not yield direct drug interactors. Therefore, this invention sought to characterize the activity of azasteroids phenotypically and by chemical-interaction assays.

[0333] 6-Azasteroids have previously been shown to have affinity for mammalian 3β-Hsd (Thomas et al., 2011) and, more recently, have been proposed to inhibit the M. leprae 3β-Hsd ortholog (Rosa et al., 2021), which shares over 75% sequence similarity with the mammalian enzyme. In M. leprae, 6-azasteroids inhibit the dehydrogenation of cholesterol to cholestenone and the concomitant reduction of NAD+to generate NADH (Rosa et al., 2021). This dehydrogenation reaction is hypothesized to be a key pathway for NAD+reduction in M. leprae in vivo because, like Mtb, this pathogen can induce lipid droplet formation and utilize host lipids, specifically cholesterol. Several analogs synthesized in the current invention (Figs. 2A and 6) showed inhibition against mycobacterial 3β-Hsd at 50 µM, but only 16 (Fig.6) inhibited activity by greater than 50%. Compound 35 showed insignificant inhibitory activity against 3β-Hsd at 50 µM, indicating that its anti-TB action is likely not due to this activity.

[0334] After 3β-Hsd inhibition was excluded as the primary mechanism of action of 35, 6-azasteroid– protein interactions utilizing a nitrene analog of 35 (Diazirine PA, 40) and another 6-azasteroid with a benzophenone side chain were identified (Benzophenone PA, 44, Fig. 3B), both of which are capable of photo-cross-linking and which maintain potentiation activity in mycobacteria. Although this methodology captured a large number of proteins as identified by mass spectrometry, this invention was able to select hit proteins by comparing data sets from biological replicates and multiple strategies for bead matrix enrichment. Filtering efforts were facilitated by robust CRISPR interference library data that have elucidated Mtb genes whose knockdown confers sensitivity to several anti-TB drugs. On the basis of the lack of activity of 6-azasteroids alone in Mtb or Mm, it was reasoned that the vulnerability of protein targets, or gene essentiality, does not clearly correlate with azasteroid activity. 6-Azasteroids were active only in pairwise combinations with several individual anti-TB compounds; combinations with 6-azasteroids conferred enhanced mycobacterial sensitivity to each anti-TB compound. Therefore, CRISPR interference data were mined that uncovered drug sensitivity and not gene essentiality. Elucidating genes that are responsible for conferring sensitivity to approved anti-TB therapeutics is relevant for designing novel compounds, such as 6-azasteroids, that function by potentiating anti-TB therapies. Combining gene–drug sensitivity data with proteomic data enabled the identification of a small list of prioritized target proteins that interacted with 6-azasteroids and helped us to elucidate how 6-azasteroids may function. On the basis of validated functional annotations, the prioritized proteins provided strong evidence that one aspect of 6- azasteroid-mediated activity arises from the phenotypic impact of these compounds on ROS neutralization.

[0335] 6-Azasteroids contribute to sensitization of Mm and Mtb to ROS or limit the ability of these mycobacteria to neutralize oxidative stress (Fig. 2B). Two proposed targets, Mtr and TrxB2, have been experimentally assessed to contribute to the thiol antioxidant–mediated detoxification of oxidative stress (Lin et al., 2016 and Si et al., 2016). One additional target, CydA, plays a role in maintaining the cellularproton motive force, which is required for ATP generation; and proton motive force uncoupling has been implicated in oxidative stress generation (Rao et al., 2008 and Mascolo et al., 2020).

[0336] Thioredoxin reductase (TrxB2 or TrxR) plays multiple roles in oxidative stress resistance and in essential processes such as the metabolism of sulfur and DNA. Thioredoxin reductases are highly conserved across bacteria and are required for growth in Mtb both in vitro and in mice. In the context of oxidative stress resistance, trxB2 knockdown resulted in a small but statistically significant reduction in mycobacterial survival, as indicated by colony forming unit (CFU) counts, in the presence of hydrogen peroxide. In MIC99assays with hydrogen peroxide, only a 2-fold change is observed, indicating that the role of TrxB2 in detoxification is not as prominent as that of other proteins within the antioxidant system (Lin et al., 2016). These data correlate to results observed for treatment of Mm and Mtb with combinations of 6-azasteroids 2 or 35 and oxidants such as CHP (Fig.20).

[0337] Mycothiol reductase (Mtr) is responsible for reducing mycothione to mycothiol, an integral molecule in the antioxidant system of mycobacteria. The mtr gene has been shown to be actively transcribed during mycobacterial growth, and exposure to isoniazid, even at nonlethal doses, strongly upregulates mtr expression. In an mtr knockout Mycobacterium smegmatis strain, mycothiol levels were quantified by liquid chromatography–mass spectrometry (Holsclaw et al., 2020). Interestingly, the levels were found to be the same in the mtr knockout as in the WT under standard aerobic conditions, but intracellular mycothiol concentration decreased upon the addition of peroxide stress. Intracellular levels of mycothiol in Mtb are known to be in the millimolar range (Bhaskar et al., 2014). Under standard conditions, the abundance of mycothiol may remain stable, but upon the introduction of oxidants, mycothiol represents a first-line strategy for detoxification. In the presence of mtr knockout, mycobacterial recycling of mycothiol is disabled, which leads to a decrease in the quantity of the reduced species. In Corynebacterium glutamicum, an actinobacterial species related to M. smegmatis, treatment of WT cultures with 10 mM CHP severely impacts survival, but this diminished-survival phenotype can be rescued when mtr is overexpressed by 5- fold. This reverse strategy, of using overexpression mutants rather than knockout or deletion mutants, elucidates the integral role that mtr plays in actinobacterial antioxidant systems (Si et al., 2016).

[0338] The function of these two antioxidant-mediating targets, TrxB2 and Mtr, and experimental data obtained with knockout, knockdown, or overexpression mutants support their potential as targets of 6- azasteroids (such as 35) when analyzed in the context of phenotypic data obtained upon treatment of Mm and Mtb with 6-azasteroids. The role of mycothiol during antibiotic treatment supports the observed synergies between 6-azasteroids and BDQ. BDQ inhibits mycobacterial ATP synthesis within the electron transport chain, leading to respiratory poison via the generation of ROS. Considering that the key function of mycothiol is neutralization of oxidative stress, inhibition of Mtr and TrxB2 proteins, which mediatemycothiol levels, will prevent Mtb from responding appropriately to oxidation, resulting in synergy with ROS produced by BDQ treatment (Torfs et al., 2019).

[0339] Mtb has two terminal oxidases that contribute to aerobic respiration: cytochrome bc1-aa3and cytochrome bd oxidase. The cyt bc1-aa3supercomplex is energy efficient in that it pumps four protons; whereas cyt bd oxidase is less energetically efficient than cyt bc1-aa3; it aids in proton motive force generation by charge separation, but it does not pump protons (Bajeli et al., 2020 and Borisov et al., 2011). In vitro, CydA is a non-essential respiratory chain protein that constitutes one-half of the cyt bd oxidase heterodimer complex, the other half being CydB. CydA is the catalytically active component of cyt bd oxidase and binds electron-transporting quinols (Safarian et al., 2021). Cyt bd oxidase is important for mycobacterial survival during infection because it maintains aerobic respiration under oxygen-limiting conditions, which are prevalent during pathogenesis (Shi et al., 2005). Deletion mutants of cyt bd oxidase show no growth defect under standard aerobic conditions. However, upon the addition of oxidative stress or antibiotics, mycobacterial killing is accentuated (Berney et al., 2014 and Lu et al., 2015). In nonmycobacterial species, cyt bd oxidase has been shown to protect against oxidative stress under aerobic conditions based upon the ability of bacteria to reduce dioxygen. In M. thermoacetica, cyt bd oxidase activity is required for neutralization of oxidative stress upon conversion from a hypoxic to a normoxic environment; this neutralization enables respiration (Das et al., 2005). In M. smegmatis, peroxide treatment contributes to more significant killing of a cyt bd oxidase mutant than WT, resulting in nearly no loss of CFUs in WT and almost complete bactericidal effect in a cyt bd oxidase deletion mutant (Lu et al., 2015).

[0340] When treated with BDQ, WT Mtb enters bacteriostasis by remodeling metabolism before BDQ exerts its bactericidal effects (Koul et al., 2014). In M. smegmatis, BDQ treatment results only in bacteriostasis and does not lead to cell death. However, in a cyt bd oxidase M. smegmatis deletion mutant, BDQ treatment has a bactericidal effect. Cyt bd oxidase–deficient Mtb strains treated with BDQ exhibit hypersensitivity in the form of more rapid kill kinetics (in that there is no longer a bacteriostasis period) and greater CFU reduction than in WT (Borisov et al., 2011). Experiments in M. smegmatis, Mtb, and other bacteria highlight the impact of cyt bd oxidase in relation to bioenergetics-targeting antibiotics and oxidative stress protection. The phenotypic impact of 6-azasteroid treatment on Mtb and Mm under oxidative stress conditions, as well as in combination treatment with BDQ, mirrors the effects observed in a cyt bd oxidase deletion mutant and therefore provides evidence for a 6-azasteroid–CydA / cyt bd oxidase interaction.

[0341] Azasteroid activity increases under low-oxygen conditions, such that 6-azasteroids show bacteriostatic activity when used alone (Palomino, J. C.; Martin, A., 2014). These findings agree with several studies on differentially expressed genes that play a role in the ability of Mtb to persist duringhypoxia. Rustad et al. observed increased expression of trxB2 and cydA after 4 and 7 days of hypoxia, respectively (Rustad et al., 2008). Analyses of an oxygen depletion time-course study by Peterson et al. showed that both cydA and trxB2 are upregulated during mid and late hypoxia, respectively (Peterson et al., 2020). The upregulation of these genes highlights the potential importance of the functions of their respective proteins, which enable Mtb to persist during the maintenance of the hypoxic response. Further experimentation will be required to experimentally validate the roles of these proteins in the context of the antimycobacterial activity of 6-azasteroids both in vitro and in vivo.

[0342] Previously, several Mce3R-regulated genes within the mel2 operon, genes that are reported to contribute to oxidative stress resistance, were implicated in the potentiation activity of 6-azasteroids (Shubbian et al., 2007). Interestingly, none of the protein products of these genes were identified as prioritized hits for 6-azasteroid–probe interactions in the current study.

[0343] Sequencing of 6-azasteroid-resistant mutants did not identify mce3R itself, Mce3R-regulated genes, or related genes, instead identifying mutations in PE / PPE genes, which are highly variable in Mtb. Although proteins that are regulated by Mce3R were not detected in interaction assays, it has previously shown that these proteins play a supplementary role in mediating antibiotic stress under 6-azasteroid treatment (Yang et al., 2019). Results from the current study indicate that the mechanisms of mel2-mediated azasteroid activity may be indirect and not a consequence of 6-azasteroid–Mel2 binding. Further experimentation will be necessary to fully elucidate the role of Mce3R-regulated proteins in mediating 6- azasteroid activity.

[0344] As demonstrated in the invention described herein, whole-cell-based phenotypic screens require subsequent work to uncover how molecules interact with the pathogen to generate an antibacterial effect (Yang et al., 2021). These mechanistic studies are required for optimizing and advancing lead compounds into further development. The difficulty of elucidating drug mechanism of action represents a major bottleneck in the phenotypic-screening approach because drug–target interactions can be complex. As indicated by the current investigation into the mechanism of action of 6-azasteroids, including the identification of several enriched proteins in interaction assays, and our previous discovery that Mce3R- regulated genes are required for 6-azasteroid activity and our prior inability to isolate single-gene resistance mutants, the sensitization of mycobacteria to antibiotics by 6-azasteroids is likely due to complex polypharmacology in oxidative stress-related systems.REFERENCES 1. 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Claims

CLAIMS 1. A compound having the structure: , whereinα is a bond that is present or absent; β is a bond that is present or absent; and γ is a bond that is present or absent, wherein when β is present, γ is absent and R12 is absent, and wherein when β is absent, γ is present and R12 is present; X is C, N, or O; R1is -H, alkyl, alkenyl, alkynyl, aryl, heteroaryl, -C(O)-alkyl, -C(O)-haloalkyl, -C(O)-cycloalkyl, -C(O)-alkenyl, -C(O)-alkynyl, -C(O)-aryl, -C(O)-heteroaryl, -CO2-alkyl, -CO2-alkenyl, -CO2- alkynyl, -CO2-aryl, -CO2-heteroaryl, cycloalkyl, -alkyl-OH, -alkyl-N(alkyl)2, -alkyl-NH2, -alkyl- haloalkyl, -alkyl-CF3, or -alkyl-cycloalkyl; R2is H, halo, alkyl, alkenyl, alkynyl, aryl, heteroaryl, -CO2-alkyl, -CO2-alkenyl, -CO2-alkynyl, - CO2-aryl, -CO2-heteroaryl, cycloalkyl, -alkyl-OH, -alkyl-NH2or -alkyl-N(alkyl)2; R3is -H, halo, alkyl, alkenyl, alkynyl, aryl, heteroaryl, -CO2-alkyl, -CO2-alkenyl, -CO2-alkynyl, - CO2-aryl, -CO2-heteroaryl, cycloalkyl, -alkyl-OH, -alkyl-NH2or -alkyl-N(alkyl)2; R4 is -H, halo, alkyl, alkenyl, alkynyl, aryl, heteroaryl, -CO2-alkyl, -CO2-alkenyl, -CO2-alkynyl, - CO2-aryl, -CO2-heteroaryl, cycloalkyl, -alkyl-OH, -alkyl-NH2, or -alkyl-N(alkyl)2; R5and R6are each independently, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, or a substituted or unsubstituted alkylaryl; and R12 is H, OH, NH2, -C(O)-alkyl, -O-C(O)-alkyl-aryl or-O-C(O)-alkyl-heteroaryl; wherein when R5and R6are both fluorophenyl or both chlorophenyl, either α is absent or R2is not H; or a salt or ester thereof.

2. The compound of claim 1 having the structure:, whereinα is a bond that is present or absent; R1is -H, alkyl, alkyl-haloalkyl, alkyl-cycloalkyl, -C(O)-alkyl, -C(O)-haloalkyl, -C(O)-cycloalkyl, cycloalkyl, -alkyl-N(alkyl)2, or -alkyl-NH2; R2is H, halo, alkyl, -CO2-alkyl, cycloalkyl, -alkyl-OH, -alkyl-NH2, or -alkyl-N(alkyl)2; R3is H, halo, alkyl, -CO2-alkyl, -cycloalkyl, -alkyl-OH, -alkyl-NH2, or -alkyl-N(alkyl)2; R4is H, halo, alkyl, -CO2-alkyl, -cycloalkyl, -alkyl-OH, -alkyl-NH2, or -alkyl-N(alkyl)2; and R5and R6are each independently, a substituted or unsubstituted aryl or a substituted or unsubstituted heteroaryl; preferably, R1is -H, alkyl, -C(O)-alkyl, alkyl-haloalkyl, -alkyl-CF3, -alkyl-cycloalkyl, -C(O)-alkyl, or -alkyl-N(alkyl)2; preferably R1is -H, C1-C6alkyl, -C(O)-C1-C6alkyl, -C1-C6alkyl-haloalkyl, - C1-C6alkyl-CF3, -C1-C6alkyl-cycloalkyl, -C(O)-C1-C6alkyl, or -C1-C6alkyl-N(alkyl)2,preferably, R1is -H, propyl, hexanyl, -C(O)-CH3, -(CH2)2-N-(CH3)2, -CH2-cyclopropyl, -(CH2)2-CH2F, or - (CH2)2-CF3; or a salt or ester thereof.

3. The compound of claim 1, wherein when γ is present and R12is present, α is absent.

4. The compound of any one of claims 1-3, wherein X is C or N, preferably when X is C, R1is H.

5. The compound of any one of claims 1-4, wherein R5and R6are each independently a substituted or unsubstituted aryl or a substituted or unsubstituted heteroaryl; preferably, R5and R6are each independently a substituted aryl or unsubstituted heteroaryl; more preferably, the aryl is phenyl; more preferably, the phenyl is substituted with halo, -CN, -CF3, -OCF3, -OCHF2, -alkyl, -alkenyl, -alkynyl, -aryl, -heteroaryl, -NH2, -NH-alkyl, -NH-alkenyl, -NH-alkynyl, -NH-aryl, -NH- heteroaryl, -OH, -OAc, -O-C(O)alkyl, -O-alkyl, -O-alkylaryl, -O-alkenyl, -O-alkynyl, -O-(aryl), - O-(heteroaryl), -S-(alkyl), -S-(alkenyl), -S-(alkynyl), -S-(aryl), -S-(heteroaryl), -S(O)-(alkyl), - S(O)-(aryl), -S(O)-(heteroaryl), -SO2-(alkyl), -SO2-(aryl), -SO2-(heteroaryl), alkyl-OH, alkyl-O- alkyl, alkyl-NH2, alkyl-NH-alkyl, alkyl-N(alkyl)2, cycloalkyl or alkyl-cycloalkyl; more preferably,the phenyl is substituted with -CN, -OCHF2, F, -CF3, or -OCF3; more preferably ,the heteroaryl is pyridine; preferably R5and R6are each independently having the following structure: ; wherein R7, R8, R9, R10a , halo, -CN, -CF3, -OCF3, -OCHF2, - alkyl, -alkenyl, -alkynyl, -aryl, -heteroaryl, -NH2, -NH-alkyl, -NH-alkenyl, -NH-alkynyl, -NH-aryl, -NH-heteroaryl, -OH, -OAc, -O-C(O)alkyl, -O-alkyl, -O-alkylaryl, -O-alkenyl, -O-alkynyl, -O- (aryl), -O-(heteroaryl), -S-(alkyl), -S-(alkenyl), -S-(alkynyl), -S-(aryl), -S-(heteroaryl), -S(O)- (alkyl), -S(O)-(aryl), -S(O)-(heteroaryl), -SO2-(alkyl), -SO2-(aryl), -SO2-(heteroaryl), alkyl-OH, alkyl-O-alkyl, alkyl-NH2, alkyl-NH-alkyl, alkyl-N(alkyl)2, cycloalkyl or alkyl-cycloalkyl.

6. The compound of claim 5, wherein (a) at least one of R7, R8, R9, R10and R11is not H; (b) at least two of R7, R8, R9, R10and R11are not H; (c) at least three of R7, R8, R9, R10and R11are not H; or (d) at least four of R7, R8, R9, R10and R11are not H; preferably; (a) R7is H; R8, R9, R10and R11are not H; (b) R8is H; R7, R9, R10and R11are not H; (c) R9is H; R8, R9, R10and R11are not H; (d) R10is H; R7, R9, R8and R11are not H; or (e) R11is H; R7, R9, R8and R10are not H .

7. The compound of claim 6, wherein orR5and R6are each independentl ; preferably,R9is H, halo, -CN, -CF3, -OCF3, -OCHF2, -alkyl, -alkenyl, -alkynyl, -aryl, -heteroaryl, -NH2, -NH- alkyl, -NH-alkenyl, -NH-alkynyl, -NH-aryl, -NH-heteroaryl, -OH, -OAc, -O-C(O)alkyl, -O-alkyl, -O-alkylaryl, -O-alkenyl, -O-alkynyl, -O-(aryl), -O-(heteroaryl), -S-(alkyl), -S-(alkenyl), -S- (alkynyl), -S-(aryl), -S-(heteroaryl), -S(O)-(alkyl), -S(O)-(aryl), -S(O)-(heteroaryl), -SO2-(alkyl), - SO2-(aryl), -SO2-(heteroaryl), alkyl-OH, alkyl-O-alkyl, alkyl-NH2, alkyl-NH-alkyl, alkyl- N(alkyl)2, cycloalkyl or alkyl-cycloalkyl; preferably, R9is halo, -CN, -CF3, -OCF3, -alkyl, -alkenyl, -alkynyl, -aryl, -heteroaryl, -NH2, -NH-alkyl, -NH-alkenyl, -NH-alkynyl, -NH-aryl, -NH- heteroaryl, -OH, or -OAc; more preferably, R9is halo, -CN, -CF3, -OCF3, -OCHF2, -alkyl, -alkenyl, -alkynyl, -aryl, or -heteroaryl; more preferably, R9is halo, -CN, -CF3, -OCF3, or -OCHF2; more preferably, R9is F, -CF3, -OCF3, or -OCHF2.

8. The compound of claim 7, wherein R5and R6are each independently: , , 9., .

10. The compound of claim 9, wherein R1is -H or alkyl; preferably, alkyl is proyl .

11. The compound of claim 1, wherein R5and R6are each independently, a substituted aryl or substituted or unsubstituted heteroaryl; preferably, R5and R6are each independently having the following structure:; wherein R7, R8, R9, R10a , halo, -CN, -CF3, -OCF3, -OCHF2, - alkyl, -alkenyl, -alkynyl, -aryl, -heteroaryl, -NH2, -NH-alkyl, -NH-alkenyl, -NH-alkynyl, -NH-aryl, -NH-heteroaryl, -OH, -OAc, -O-C(O)alkyl, -O-alkyl, -O-alkylaryl, -O-alkenyl, -O-alkynyl, -O- (aryl), -O-(heteroaryl), -S-(alkyl), -S-(alkenyl), -S-(alkynyl), -S-(aryl), -S-(heteroaryl), -S(O)- (alkyl), -S(O)-(aryl), -S(O)-(heteroaryl), -SO2-(alkyl), -SO2-(aryl), -SO2-(heteroaryl), alkyl-OH, alkyl-O-alkyl, alkyl-NH2, alkyl-NH-alkyl, alkyl-N(alkyl)2, cycloalkyl or alkyl-cycloalkyl.

12. The compound of claim 11, wherein (a) at least one of R7, R8, R9, R10and R11is not H; (b) at least two of R7, R8, R9, R10and R11are not H; (c) at least three of R7, R8, R9, R10and R11are not H; or (d) at least four of R7, R8, R9, R10and R11are not H; preferably, (a) R7is H; R8, R9, R10and R11is not H; (b) R8is H; R7, R9, R10and R11are not H; (c) R9is H; R8, R9, R10and R11are not H; (d) R10is H; R7, R9, R8and R11are not H; or (e) R11is H; R7, R9, R8and R10are not H.

13. The compound of claim 12, wherein orR5and R6are each independently ,preferably, R9is halo, -CN, -CF3, -OCF3, -OCHF2, -alkyl, -alkenyl, -alkynyl, -aryl, -heteroaryl, - NH2, -NH-alkyl, -NH-alkenyl, -NH-alkynyl, -NH-aryl, -NH-heteroaryl, -OH, -OAc, -O-C(O)alkyl, -O-alkyl, -O-alkylaryl, -O-alkenyl, -O-alkynyl, -O-(aryl), -O-(heteroaryl), -S-(alkyl), -S-(alkenyl), -S-(alkynyl), -S-(aryl), -S-(heteroaryl), -S(O)-(alkyl), -S(O)-(aryl), -S(O)-(heteroaryl), -SO2- (alkyl), -SO2-(aryl), -SO2-(heteroaryl), alkyl-OH, alkyl-O-alkyl, alkyl-NH2, alkyl-NH-alkyl, alkyl- N(alkyl)2, cycloalkyl or alkyl-cycloalkyl; preferably, R9is halo, -CN, -CF3, -OCF3, -alkyl, -alkenyl, -alkynyl, -aryl, -heteroaryl, -NH2, -NH-alkyl, -NH-alkenyl, -NH-alkynyl, -NH-aryl, -NH- heteroaryl, -OH, or -OAc; more preferably, R9is halo, -CN, -CF3, -OCF3, -OCHF2, -alkyl, -alkenyl, -alkynyl, -aryl, or -heteroaryl; more preferably, R9is halo, -CN, -CF3, -OCF3, or -OCHF2; more preferably, R9is F, -CF3, -OCF3, or -OCHF2.

14. The compound of claim 13, wherein R5and R6are each independently: . 15.

16. The compound of claim 15, wherein R1is -H, alkyl, -C(O)-alkyl, -alkyl-N(alkyl)2, alkyl-haloalkyl, or alkyl-cycloalkyl; preferably, R1is -H, propyl, hexanyl, -C(O)-CH3, -(CH2)2-N-(CH3)2, -CH2- cyclopropyl, -(CH2)2-CH2F, or -(CH2)2-CF3; R1is H, ,, 17.uted aryl or substituted or unsubstituted heteroaryl; preferably, R5and R6are each independently having the following structure: ;wherein R7, R8, R9, R10and R11are each, independently, -H, halo, -CN, -CF3, -OCF3, -OCHF2, - alkyl, -alkenyl, -alkynyl, -aryl, -heteroaryl, -NH2, -NH-alkyl, -NH-alkenyl, -NH-alkynyl, -NH-aryl, -NH-heteroaryl, -OH, -OAc, -O-C(O)alkyl, -O-alkyl, -O-alkylaryl, -O-alkenyl, -O-alkynyl, -O- (aryl), -O-(heteroaryl), -S-(alkyl), -S-(alkenyl), -S-(alkynyl), -S-(aryl), -S-(heteroaryl), -S(O)- (alkyl), -S(O)-(aryl), -S(O)-(heteroaryl), -SO2-(alkyl), -SO2-(aryl), -SO2-(heteroaryl), alkyl-OH, alkyl-O-alkyl, alkyl-NH2, alkyl-NH-alkyl, alkyl-N(alkyl)2, cycloalkyl or alkyl-cycloalkyl.

18. The compound of claim 17, wherein (a) at least one of R7, R8, R9, R10and R11is not H; (b) at least two of R7, R8, R9, R10and R11are not H; (c) at least three of R7, R8, R9, R10and R11are not H; or (d) at least four of R7, R8, R9, R10and R11are not H; preferably (a) R7is H; R8, R9, R10and R11are not H; (b) R8is H; R7, R9, R10and R11are not H; (c) R9is H; R8, R9, R10and R11are not H; (d) R10is H; R7, R9, R8and R11are not H; or (e) R11is H; R7, R9, R8and R10are not H.

19. The compound claim 18, wherein orR5and R6are each independentl ; preferably,R9is halo, -CN, -CF3, -OCF3, -OCHF2, -alkyl, -alkenyl, -alkynyl, -aryl, -heteroaryl, -NH2, -NH- alkyl, -NH-alkenyl, -NH-alkynyl, -NH-aryl, -NH-heteroaryl, -OH, -OAc, -O-C(O)alkyl, -O-alkyl, -O-alkylaryl, -O-alkenyl, -O-alkynyl, -O-(aryl), -O-(heteroaryl), -S-(alkyl), -S-(alkenyl), -S- (alkynyl), -S-(aryl), -S-(heteroaryl), -S(O)-(alkyl), -S(O)-(aryl), -S(O)-(heteroaryl), -SO2-(alkyl), - SO2-(aryl), -SO2-(heteroaryl), alkyl-OH, alkyl-O-alkyl, alkyl-NH2, alkyl-NH-alkyl, alkyl- N(alkyl)2, cycloalkyl or alkyl-cycloalkyl; preferably, R9is halo, -CN, -CF3, -OCF3, -alkyl, -alkenyl, -alkynyl, -aryl, -heteroaryl, -NH2, -NH-alkyl, -NH-alkenyl, -NH-alkynyl, -NH-aryl, -NH-heteroaryl, -OH, or -OAc; more preferably, R9is halo, -CN, -CF3, -OCF3, -OCHF2, -alkyl, -alkenyl, -alkynyl, -aryl, or -heteroaryl; more preferably, R9is halo, -CN, -CF3, -OCF3, or -OCHF2; more preferably, R9is F, -CF3, -OCF3, or -OCHF2.

20. The compound of claim 19, wherein R5and R6are each independently: . whα is a bond that is present or absent; R1is -H, alkyl, alkyl-haloalkyl, alkyl-cycloalkyl, -C(O)-alkyl, -C(O)-haloalkyl, -C(O)-cycloalkyl, cycloalkyl, -alkyl-N(alkyl)2, or -alkyl-NH2; R2is H, halo, alkyl, -CO2-alkyl, cycloalkyl, -alkyl-OH, -alkyl-NH2, or -alkyl-N(alkyl)2; R3is H, halo, alkyl, -CO2-alkyl, -cycloalkyl, -alkyl-OH, -alkyl-NH2, or -alkyl-N(alkyl)2; R4 is H, halo, alkyl, -CO2-alkyl, -cycloalkyl, -alkyl-OH, -alkyl-NH2, or -alkyl-N(alkyl)2; R5and R6are each independently, a substituted or unsubstituted aryl or a substituted or unsubstituted heteroaryl; and R12 is H, OH, NH2, -C(O)-alkyl, -O-C(O)-alkyl-aryl or-O-C(O)-alkyl-heteroaryl; or a salt or ester thereof.

22. The compound of claim 21, wherein α is absent.

23. The compound of any one of claims 21-22, wherein (a) R1is -H, alkyl, -C(O)-alkyl, -alkyl- N(alkyl)2, alkyl-haloalkyl, or alkyl-cycloalkyl; preferably R1is -H or propyl; (b) R12is H, OH, NH2,; or (c) R5and R6are each independently, a substituted aryl or substitutedoaryl .

24. The compound of any one of claims 21-23, wherein R5and R6are each independently having the following structure: ; wherein R7, R8, R9, R10a , halo, -CN, -CF3, -OCF3, -OCHF2, - alkyl, -alkenyl, -alkynyl, -aryl, -heteroaryl, -NH2, -NH-alkyl, -NH-alkenyl, -NH-alkynyl, -NH-aryl, -NH-heteroaryl, -OH, -OAc, -O-C(O)alkyl, -O-alkyl, -O-alkylaryl, -O-alkenyl, -O-alkynyl, -O- (aryl), -O-(heteroaryl), -S-(alkyl), -S-(alkenyl), -S-(alkynyl), -S-(aryl), -S-(heteroaryl), -S(O)- (alkyl), -S(O)-(aryl), -S(O)-(heteroaryl), -SO2-(alkyl), -SO2-(aryl), -SO2-(heteroaryl), alkyl-OH, alkyl-O-alkyl, alkyl-NH2, alkyl-NH-alkyl, alkyl-N(alkyl)2, cycloalkyl or alkyl-cycloalkyl; preferably, R5and R6are each independentl ;preferably, R9is halo, -CN, -CF3, -OCF3, -OCHF2, -alkyl, -alkenyl, -alkynyl, -aryl, -heteroaryl, -NH2, -NH- alkyl, -NH-alkenyl, -NH-alkynyl, -NH-aryl, -NH-heteroaryl, -OH, -OAc, -O-C(O)alkyl, -O-alkyl, -O-alkylaryl, -O-alkenyl, -O-alkynyl, -O-(aryl), -O-(heteroaryl), -S-(alkyl), -S-(alkenyl), -S- (alkynyl), -S-(aryl), -S-(heteroaryl), -S(O)-(alkyl), -S(O)-(aryl), -S(O)-(heteroaryl), -SO2-(alkyl), - SO2-(aryl), -SO2-(heteroaryl), alkyl-OH, alkyl-O-alkyl, alkyl-NH2, alkyl-NH-alkyl, alkyl- N(alkyl)2, cycloalkyl or alkyl-cycloalkyl; preferably, R9is halo, -CN, -CF3, -OCF3, -alkyl, -alkenyl, -alkynyl, -aryl, -heteroaryl, -NH2, -NH-alkyl, -NH-alkenyl, -NH-alkynyl, -NH-aryl, -NH- heteroaryl, -OH, or -OAc; more preferably, R9is halo, -CN, -CF3, -OCF3, -OCHF2, -alkyl, -alkenyl, -alkynyl, -aryl, or -heteroaryl; more preferably, R9is halo, -CN, -CF3, -OCF3, or -OCHF2; more preferably, R9is F, or -OCF3.

25. The compound of claim 24, wherein (a) at least one of R7, R8, R9, R10and R11is not H; (b) at least two of R7, R8, R9, R10and R11are not H;(c) at least three of R7, R8, R9, R10and R11are not H; or (d) at least four of R7, R8, R9, R10and R11are not H; preferably, (a) R7is H; R8, R9, R10and R11are not H; (b) R8is H; R7, R9, R10and R11are not H; (c) R9is H; R8, R9, R10and R11are not H; (d) R10is H; R7, R9, R8and R11are not H; or (e) R11is H; R7, R9, R8and R10are not H.

26. The compound of any one of claims 24-25, wherein R5and R6are each independently: .

27. ng the structure:, , ,, , , , ,, , or. compoun avng e srucure:, whereinα is a bond that is present or absent; X is C, N, or O; R1is -H, alkyl, alkenyl, alkynyl, aryl, heteroaryl, -C(O)-alkyl, -C(O)-haloalkyl, -C(O)-cycloalkyl, -C(O)-alkenyl, -C(O)-alkynyl, -C(O)-aryl, -C(O)-heteroaryl, -CO2-alkenyl, -CO2-alkynyl, -CO2- aryl, -CO2-heteroaryl, cycloalkyl, -alkyl-OH, -alkyl-N(alkyl)2, -alkyl-NH2, -alkyl-haloalkyl, -alkyl- CF3, or -alkyl-cycloalkyl; R2is H, halo, alkyl, alkenyl, alkynyl, aryl, heteroaryl, -CO2-alkyl, -CO2-alkenyl, -CO2-alkynyl, - CO2-aryl, -CO2-heteroaryl, cycloalkyl, -alkyl-OH, -alkyl-NH2or -alkyl-N(alkyl)2; R3is -H, halo, alkyl, alkenyl, alkynyl, aryl, heteroaryl, -CO2-alkyl, -CO2-alkenyl, -CO2-alkynyl, - CO2-aryl, -CO2-heteroaryl, cycloalkyl, -alkyl-OH, -alkyl-NH2or -alkyl-N(alkyl)2; R4 is -H, halo, alkyl, alkenyl, alkynyl, aryl, heteroaryl, -CO2-alkyl, -CO2-alkenyl, -CO2-alkynyl, - CO2-aryl, -CO2-heteroaryl, cycloalkyl, -alkyl-OH, -alkyl-NH2, or -alkyl-N(alkyl)2; and R5is a substituted aryl or substituted or unsubstituted cycloalkyl; wherein the substitution is H, alkyl, alkenyl, alkynyl carbonyl, amine; C(O)-alkyl, C(O)-haloalkyl, or CO2-alkyl; wherein when is present, R1is not H;wherein when is present, R1is not -(CH2)2CH3;wherein when is present, R1is not CH3;wherein when is present, R1is not H;wherein when H;wherein when R5is and α is present, R1is not H or CH3; wherein when α is present and R5is , , , ,, , notor a salt or ester thereof.

29. The compound of claim 28, wherein α is absent.

30. The compound of any one of 28-29, wherein R1is -H ,; preferably, R1is -H or propyl; or R5is a substituted aryl yl; preferably, cycloalkyl is adamantane.

31. The compound of any one of claims 28-30, wherein R5has the following structure: , wherein R7, R8, R9, R10and R11ardently, -H, halo, -CN, -CF3, -OCF3, -OCHF2, - alkyl, -alkenyl, -alkynyl, -aryl, -heteroaryl, -NH2, -NH-alkyl, -NH-alkenyl, -NH-alkynyl, -NH-aryl, -NH-heteroaryl, -OH, -OAc, -O-C(O)alkyl, -O-alkyl, -O-alkylaryl, -O-alkenyl, -O-alkynyl, -O- (aryl), -O-(heteroaryl), -S-(alkyl), -S-(alkenyl), -S-(alkynyl), -S-(aryl), -S-(heteroaryl), -S(O)- (alkyl), -S(O)-(aryl), -S(O)-(heteroaryl), -SO2-(alkyl), -SO2-(aryl), -SO2-(heteroaryl), alkyl-OH, alkyl-O-alkyl, alkyl-NH2, alkyl-NH-alkyl, alkyl-N(alkyl)2, cycloalkyl or alkyl-cycloalkyl; preferably. R7and R10are each independently halo, -CN, -CF3, -OCF3, -OCHF2, -alkyl, -alkenyl, -alkynyl, - aryl, -heteroaryl, -NH2, -NH-alkyl, -NH-alkenyl, -NH-alkynyl, -NH-aryl, -NH-heteroaryl, -OH, - OAc, -O-C(O)alkyl, -O-alkyl, -O-alkylaryl, -O-alkenyl, -O-alkynyl, -O-(aryl), -O-(heteroaryl), -S- (alkyl), -S-(alkenyl), -S-(alkynyl), -S-(aryl), -S-(heteroaryl), -S(O)-(alkyl), -S(O)-(aryl), -S(O)- (heteroaryl), -SO2-(alkyl), -SO2-(aryl), -SO2-(heteroaryl), alkyl-OH, alkyl-O-alkyl, alkyl-NH2, alkyl-NH-alkyl, alkyl-N(alkyl)2, cycloalkyl or alkyl-cycloalkyl; preferably, R7and R10are each independently halo, -CN, -CF3, -OCF3, -alkyl, -alkenyl, -alkynyl, -aryl, -heteroaryl, -NH2, -NH- alkyl, -NH-alkenyl, -NH-alkynyl, -NH-aryl, -NH-heteroaryl, -OH, or -OAc; more preferably, R7and R10are each independently halo, branched alky, -CN, -CF3, -OCF3, -OCHF2, -alkyl, -alkenyl, -alkynyl, -aryl, or -heteroaryl; more preferably, R7and R10are each independently halo, branched alky, -CN, -CF3, -OCF3, or -OCHF2; more preferably, R7and R10are each independently Br, tert- butyl, or -CF3.

32. The compound of claim 31, wherein (a) at least one of R7, R8, R9, R10and R11is not H; (b) at least two of R7, R8, R9, R10and R11are not H; (c) at least three of R7, R8, R9, R10and R11are not H; or (d) at least four of R7, R8, R9, R10and R11are not H; preferably (a) R9, R10and R11are H; R7and R8are not H; (b) R8, R10and R11are H; R7and R9are not H; (c) R8, R9and R11are H; R7and R10are not H; or (d) R8, R9and R10are H; R7and R11are not H, .

33. The compound of any one of claims 31-32, wherein R5is: . 34.

35. The compound of claim 34, wherein R1is -H, alkyl, or alkyl-haloalkyl; preferably, R1is -H, propyl, -(CH2)2-CH2F, or -(CH2)2-CF3.

36. The compound of any one of claims 34-35, wherein R5is a substituted aryl; preferably, R5has the following structure: , wherein R7, R8, R9, R10and R11arndently, -H, halo, -CN, -CF3, -OCF3, -OCHF2, - alkyl, -alkenyl, -alkynyl, -aryl, -heteroaryl, -NH2, -NH-alkyl, -NH-alkenyl, -NH-alkynyl, -NH-aryl, -NH-heteroaryl, -OH, -OAc, -O-C(O)alkyl, -O-alkyl, -O-alkylaryl, -O-alkenyl, -O-alkynyl, -O- (aryl), -O-(heteroaryl), -S-(alkyl), -S-(alkenyl), -S-(alkynyl), -S-(aryl), -S-(heteroaryl), -S(O)- (alkyl), -S(O)-(aryl), -S(O)-(heteroaryl), -SO2-(alkyl), -SO2-(aryl), -SO2-(heteroaryl), alkyl-OH, alkyl-O-alkyl, alkyl-NH2, alkyl-NH-alkyl, alkyl-N(alkyl)2, cycloalkyl or alkyl-cycloalkyl; preferably, R7and R10are each independently halo, -CN, -CF3, -OCF3, -OCHF2, -alkyl, -alkenyl, - alkynyl, -aryl, -heteroaryl, -NH2, -NH-alkyl, -NH-alkenyl, -NH-alkynyl, -NH-aryl, -NH-heteroaryl, -OH, -OAc, -O-C(O)alkyl, -O-alkyl, -O-alkylaryl, -O-alkenyl, -O-alkynyl, -O-(aryl), -O- (heteroaryl), -S-(alkyl), -S-(alkenyl), -S-(alkynyl), -S-(aryl), -S-(heteroaryl), -S(O)-(alkyl), -S(O)-(aryl), -S(O)-(heteroaryl), -SO2-(alkyl), -SO2-(aryl), -SO2-(heteroaryl), alkyl-OH, alkyl-O-alkyl, alkyl-NH2, alkyl-NH-alkyl, alkyl-N(alkyl)2, cycloalkyl or alkyl-cycloalkyl; preferably, R7and R10are each independently halo, -CN, -CF3, -OCF3, -alkyl, -alkenyl, -alkynyl, -aryl, -heteroaryl, -NH2, -NH-alkyl, -NH-alkenyl, -NH-alkynyl, -NH-aryl, -NH-heteroaryl, -OH, -OAc; more preferably, R7and R10are each independently halo, branched alky, -CN, -CF3, -OCF3, -OCHF2, -alkyl, -alkenyl, -alkynyl, -aryl, or -heteroaryl; more preferably, R7and R10are each independently halo, branched alky, -CN, -CF3, -OCF3, or -OCHF2; more preferably, R7and R10are each independently Br, tert- butyl, or -CF3.

37. The compound of claim 36, wherein (a) at least one of R7, R8, R9, R10and R11is not H; (b) at least two of R7, R8, R9, R10and R11are not H; (c) at least three of R7, R8, R9, R10and R11are not H; or (d) at least four of R7, R8, R9, R10and R11are not H; preferably, (a) R9, R10and R11are H; R7and R8are not H; (b) R8, R10and R11are H; R7and R9are not H; (c) R8, R9and R11are H; R7and R10are not H; or (d) R8, R9and R10are H; R7and R11are not H.

38. The compound of claim 28, wherein R5is: or. p g g, , , ,40. A pharmaceutical composition comprising the compound of any one of claims 1-39 and a pharmaceutically acceptable carrier.

41. The pharmaceutical composition of claim 40, further comprising an anti-tuberculosis drug and a pharmaceutically acceptable carrier; preferably, the anti-tuberculosis drug is isoniazid, ethionamide, pretomanid (PA-824), pyrazinamide, ethambutol, rifabutin, kanamycin, amikacin, capreomycin, streptomycin, levofloxacin, moxifloxacin, ofloxacin, para–aminosalicylic acid, cycloserine, terizidone, thionamide, protionamide, linezolid, clofazimine, delamanid or bedaquiline or combinations thereof; preferably, the anti-tuberculosis drug is isoniazid, ethionamide, pretomanid (PA-824), pyrazinamide, ethambutol, rifabutin, kanamycin, amikacin, capreomycin, streptomycin, levofloxacin, ofloxacin, para–aminosalicylic acid, cycloserine, terizidone, thionamide, protionamide, delamanid or bedaquiline; preferably, the anti-tuberculosis drug is bedaquiline.

42. A method of inhibiting M. tuberculosis, the method comprising administering the compound of any one of claims 1-39 or the pharmaceutical composition of any one of claims 40-41.

43. A method of treating a subject infected with M. tuberculosis, the method comprising administering to the subject an amount of the compound of any one of claims 1-39 so as to thereby treat the subject.

44. The method of claim 43, wherein (a)the M. tuberculosis infection is latent, active, drug-resistant, extensively drug-resistant or multi-drug resistant; or (b) the infected subject is afflicted with pulmonary tuberculosis or extrapulmonary tuberculosis .

45. The method of any one of claims 42-44, further comprising administering an amount of an anti- tuberculosis drug to the subject; preferably, (a)the amount of the compound and the amount of the anti-tuberculosis drug when taken together is more effective to treat the M. tuberculosis infection than the anti-tuberculosis drug alone; (b) the amount of the compound is lower than the amount of the anti-tuberculosis drug that is effective to achieve the same level of treatment when administered alone; or (c) the amount of the compound and the amount of the anti-tuberculosis drug when administered together are more effective to treat the subject than when each agent at the same amount is administered alone .

46. The method of any one of claims 42-44, wherein (a) the amount of the compound and the amount of the anti-tuberculosis drug is administered simultaneously, contemporaneously or concomitantly; (b) the amount of the compound causes the M. tuberculosis in the subject to be more susceptible to treatment with the anti-tuberculosis drug; or (c) the amount of the compound enhances the anti-tuberculosis effect of the anti-tuberculosis drug.

47. The method of any one of claims 42-46, wherein the anti-tuberculosis drug is pretomanid (PA- 824), pyrazinamide, ethambutol, rifabutin, kanamycin, amikacin, capreomycin, streptomycin, levofloxacin, moxifloxacin, ofloxacin, para–aminosalicylic acid, cycloserine, clofazimine, terizidone, thionamide, protionamide, rifapentine, rifampicin, delamanid, or bedaquiline or combinations thereof; preferably, the anti-tuberculosis drug is isoniazid, ethionamide, pretomanid (PA-824), pyrazinamide, ethambutol, rifabutin, kanamycin, amikacin, capreomycin, streptomycin, levofloxacin, ofloxacin, para–aminosalicylic acid, cycloserine, terizidone, thionamide, protionamide, delamanid or bedaquiline; preferably, the anti-tuberculosis drug is isoniazid, rifampicin, pyrazinamide, ethambutol or streptomycin or combinations thereof .

48. The method of claims 47, wherein the anti-tuberculosis drug is OCF3O H N ,,,, , N Cl,

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