NOVEL PLECOMACROLIDE DERIVATIVES FOR MHC-I NEF DOWNMODULATION

BACKGROUND

FIELD OF THE DISCLOSURE

[0001] This disclosure relates generally to inhibitors of MHC-I downmodulation, and methods of treating or preventing an HIV infection by administering the inhibitors to a patient in need of treatment thereof.

BRIEF DESCRIPTION OF RELATED TECHNOLOGY

[0002] The development of combination antiretroviral therapy (cART) has improved the outcome for individuals with human immunodeficiency virus (HIV). However, HIV is able to persist even during cART by establishing a latent reservoir in a subset of infected cells (Siliciano et al., 2003). To fully eliminate HIV infected cells and cure the infection, a two-pronged approach is necessary to first reactivate this viral reservoir and then stimulate CTL-mediated clearance of virally infected cells. One challenge to this approach is that HIV infected cells evade clearance by anti-HIV cytotoxic T-lymphocytes (CTLs) due in part to the activity of the HIV accessory protein Nef (Collins et al., 1998). CTL recognition and killing of infected cells depends on expression of host MHC-I proteins on the cell surface. Following HIV infection, the HIV-Nef protein downmodulates MHC-I HLA-A and -B, allowing the virus to evade recognition (Collins et al., 1998). To downmodulate MHC-I, Nef binds to the cytoplasmic tail of MHC-1 and redirects it to the lysosome for degradation thereby blocking the transport of antigens to the cell membrane (Robert-Guroff et al., 1990; Roeth et al., 2004). Concanamycin A (CMA, 1) reverses this activity of Nef, normalizing MHC-I expression and allowing anti-HIV CTLs to effectively eliminate HIV infected cells in vitro (Painter et al., 2020). Thus far, it has been found that CMA acts by reducing the interaction between Nef, MHC-I, and clathrin adaptor protein 1 (AP-1) that is necessary to alter MHC-I trafficking in HIV infected cells (Painter et al., 2020). However, the precise molecular mechanism by which CMA reduces Nef-MHC-l-AP-1 complex formation and whether V-ATPase plays a role in this bioactivity remains unknown.

[0003] While CMA inhibits Nef activity at sub-nanomolar (nM) concentrations, its utility as a potential anti-HIV therapeutic requires identifying a derivative with lower toxicity. Because CMA has a much lower IC50 for Nef inhibition than lysosomal neutralization, we hypothesized the existence of separable CMA inhibitory targets. If this hypothesis is true, it should be possible to reduce toxicity by generating derivatives with maximal anti-Nef potency and minimal lysosomal neutralization. Therefore, a need remains for chemically related molecules with Nef inhibitory activity, such as derivatives of CMA, having lower toxicity.

SUMMARY

[0004] The present disclosure generally relates to methods of treating HIV, to methods of inhibiting the replication of HIV viruses, to methods of reducing the amount of HIV viruses in a patient, and to compounds and compositions that can be employed for such methods.

[0005] In one aspect, the disclosure provides compounds of Formula (I) and pharmaceutically acceptable salts thereof:

C(0)Ci-8 alkyl, or 0C(0)Ci-8 alkyl, or R ^H and R ¹ together with the carbon to which they are attached form an oxo (=0) group; R ² is H, OH, or OCH3; R ³ is CHO, C(0)Ci-8 alkyl, 0C(0)R ⁶ or a modified sugar; each R ⁴ is independently H or C1-6alkyl; R ⁵ is C1-6alkyl or C2-6alkenyl; and R ⁶ is H, C1-6haloalkyl, or C2-6 alkynyl. In some instances, is or or represents a direct bond; R ^H is H; R ¹ is OH or 0C(0)Ci-8 alkyl, or R ^H and R ¹ together with the carbon to which they are attached form an oxo (=0) group; R ² is H, OH, or OCH3;

R ³ is 0C(0)R ⁶ or a modified sugar; each R ⁴ is independently H or C1-6alkyl; R ⁵ is C1-6alkyl or C2-6alkenyl; and R ⁶ is H, C1-6haloalkyl, or C2-6 alkynyl.

[0006] In some cases, the compounds are compounds of Formula (la), (lb), or (Ic): both R ^5' and R ^5” are Ci-2alkyl. [0007] Further provided are methods of administering to a patient a safe and effective amount of a compound disclosed herein, e.g., as represented by Formulas (I), (la), (lb), (lb), or a compound of Table A, and pharmaceutically acceptable salts thereof.

[0008] Also provided are methods of modulating HIV Nef in a subject in need thereof by contacting said HIV Nef with a safe and effective amount of a compound as disclosed herein, e.g., as represented by Formulas (I),

(la), (lb), (lb), or a compound of Table A, and pharmaceutically acceptable salts thereof. In some cases, modulating HIV Nef includes administering to a patient a safe and effective amount of a compound as disclosed herein e.g., as represented by Formulas (I), (la), (lb), (lb), or a compound of Table A, and pharmaceutically acceptable salts thereof.

[0009] Further provided are methods of treating an HIV Nef-associated disorder in a host by administering a safe and effective amount of a compound as disclosed herein, e.g., as represented by Formulas (I), (la), (lb),

(lb), or a compound of Table A, and pharmaceutically acceptable salts thereof.

[0010] Further provided are methods of treating HIV infection in a patient, comprising administering to said patient a safe and effective amount of a compound as disclosed herein, e.g., as represented by Formulas (I), (la), (lb), (lb), or a compound of Table A, and pharmaceutically acceptable salts thereof.

[0011] Further provided are methods of reducing an HIV reservoir in a patient, comprising administering to said patient a safe and effective amount of a compound as disclosed herein, e.g., as represented by Formulas (I), (la), (lb), (lb), or a compound of Table A, and pharmaceutically acceptable salts thereof.. Also provided are methods of eliminating an HIV reservoir in a patient, comprising administering to said patient a safe and effective amount of a compound as disclosed herein, e.g., as represented by Formulas (I), (la), (lb), (lb), or a compound of Table A, and pharmaceutically acceptable salts thereof.

[0012] Also provided are pharmaceutical compositions comprising a compound as disclosed herein, e.g., as represented by any of Formulas (I), (la), (lb), (lb), or a compound of Table A, and pharmaceutically acceptable salts thereof., or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, carrier, adjuvant or vehicle.

[0013] Further provided herein are uses of a compound described herein for the manufacture of a medicament for treating HIV infection in a patient, for reducing an HIV reservoir in a patient, or for eliminating an HIV reservoir in a patient.

DETAILED DESCRIPTION

[0014] Provided herein are analogs of plecomacrolides, such as analogs of Bafilomycins (Bafs) and Concanamycins. For the current structure-activity relationship analysis, a panel of macrolactones, macrolides, and plecomacrolides was screened against Nef expressing primary T cells to perform an in-depth analysis of functional group patterns needed for separating Nef and V-ATPase inhibitory activities (expanding neutralization window). In embodiments, the analogs of plecomacrolides disclosed herein comprise modified 18-membered ring plecomacrolides that retain single-digit nM potency and provide substantive reduction in V-ATPase inhibition, pointing the way toward final lead optimization.

[0015] Certain compounds are referred to herein by numbers, which are explained below.

[0016] Table 2

[0017] Table 3

[0018] Table S1

[0019] Table S4

Screening for Nef and V-ATPase inhibition reveals the importance of macrolactone ring size

[0020] To investigate structure activity relationships (SAR) for Nef inhibitory activity across a variety of related macrocyclic compounds, a panel of 74 molecules with varying ring sizes and additional select structural modifications was tested, along with two known V-ATPase inhibitors, diphy II in and archazolid A, that are structurally unrelated to plecomacrolides but share this "off target” (V-ATPase) inhibitory activity (Sorensen et al., 2007). Inhibition of HIV-Nef was measured by assessing its capacity to downmodulate MHC-I HLA-A2 on the surface of primary CD4+ T cells 24-hours after treatment with each compound. V-ATPase inhibition was assessed at the same time point using lysosomal acidification as an indicator of V-ATPase activity. This was measured using a flow cytometric assay that quantified Lysotracker mean fluorescence intensity as previously described (Painter et al., 2020). Cell viability was measured at 24-hour and 72-hour time points using a previously described flow cytometric assay (Painter et al., 2020).

[0021] All compounds tested with relatively small macrocyclic rings (12- and 14-membered macrolactones) failed to show potent Nef or V-ATPase inhibitory activity at concentrations below 1000 nM, which was the upper limit of consideration in this SAR study. In addition, none of the non-macrolide compounds tested, including the V-ATPase inhibitor diphyllin, had a Nef IC50 below 1000nM. A small number of compounds, such as borrelidin, were toxic at 24-hours making any determination of Nef inhibitory activity unreliable. Of the two 24-membered macrolactones that were tested, only archazolid A (3), an inhibitor that binds to the same subunit of V-ATPase as the plecomacrolides, had a Net IC50 less than 1000nM (Table B) (Keen et al., 2022). Indeed, 3 was more potent than 2, achieving the sub nM potency observed with 1 (Table B). In comparison to 1, compound 3 had a smaller therapeutic window separating Net IC50 and TC50 (6.6 & 1 .4-fold, respectively) as well as a smaller neutralization window between Nef and V-ATPase IC50S (9.4 & 2.9-fold, respectively; Table B). Nevertheless, 3 is notable in that this 24-membered macrocycle was the only structurally distinct compound from the plecomacrolides tested that displayed sub-nM potency (Nef IC50 = 0.47nM).

[0022] A detailed comparison between 1 and 2 showed that 1 displays over 60-fold more potent Nef inhibitory activity relative to 2 and has a larger therapeutic window (Table B) (Painter et al., 2020). A comparative assessment of lysosomal neutralization was conducted between these two molecules, and it was found that 1 had a greater neutralization window than 2 (9.4 & 2.9-fold respectively; Table B).

Semi-synthetic modification of 16-membered plecomacrolides identifies critical moieties required for Nef inhibition

The role of the pyran ring at C-19 in bafilomycin A1

[0023] To better understand which structural variations within the plecomacrolide family members determine toxicity, Nef and V-ATPase inhibition, synthetic modifications were tested first with 2, a more readily accessible plecomacrolide, leading to a total of 15 semi-synthetic derivatives (Li et al., 2017). It has previously been reported that bafilomycin D (lacking a pyran) displayed very poor potency as a Nef inhibitor, which similarly shows low level V-ATPase inhibition (Drose et al., 2001; Osteresch et al., 2012; Painter et al., 2020; Wang et al., 2021). An investigation of the impact of pyran ring modifications showed that C-19 methylation, deoxygenation, and/or C-21 methylation all decreased potency (increased Nef IC50) with dehydration of C-20 (S40) having the greatest effect. To directly confirm the requirement of the hemiketal functionality in a semi-synthetic derivative, Mitsunobu conditions yielded "ring-opened” bafilomycin A1 (S8; Table B) (Bowman et al., 1988). This alteration led to a 26-fold decrease in potency compared to 2 (Nef IC50 = 310nM; p< 0.001; Table B).

Enhancement of Nef and V-ATPase inhibition through modifications at C-21 in 16-membered ring plecomacrolides

[0024] Based on observations that the pyran ring was more amenable to modifications, development efforts focused on derivatizing the C-21 -OH to assess if chain extensions at this position (increasing the overall size of 2), would mimic the larger concanamycin analogs and improve potency. Interestingly, modifying C-21 -OH with an acetyl group (S9) led to a 2.5-fold increase in potency compared to 2 (Nef IC50 = 4.5nM vs 12nM, p< 0.05; Table B) (Bender et al., 2007). This improvement was not accompanied by a significant change in V-ATPase inhibition as the neutralization window for S9 was almost 2-fold larger than 2 (p> 0.05; Table B). Without wishing to be bound by any particular theory, introduction of esters with longer acyl chains (S10 & S11, or an unsaturated chain at C-21 -OH) resulted in decreased potency (Nef IC50 = 37nM and 130nM, respectively) and worsening of the neutralization window, however, incorporation of an unsaturated chain (S12) restored potency to parent compound levels (Nef IC50 = 17nM vs 12nM; Table B). Again without wishing to be bound by any particular theory, incorporation of an aromatic substituent (S13) led to a decrease in potency (Nef IC50 = 92nM) highlighting that steric hindrance plays an important role at the C-21-OH position and compromises the biological activity of bafilomycin analogs.

[0025] In addition to the larger macrocyclic ring, 1 contains a 4β-D-rhamnose at C-23, instead of a hydroxyl at the analogous C-21 position in 2. To determine the extent to which this sugar moiety is required for Nef and V- ATPase inhibition, the activity of three 16-membered ring glycosylated plecomacrolides was examined: leucanicidin (S14), PC-766B (S15) and elaiophylin (S16) (Table B). Interestingly, S14 and S15 were found to have single digit nM potency (Nef IC50 = 1.2nM and 1.2nM, respectively), however, the increase in potency was paired with an increase in toxicity (TC50 = 1.4nM and 1.3nM, respectively; Table B). Despite the presence of a £- D-rhamnose at C-21 , S16 displayed a 20-fold decrease in potency compared to 2 (Nef IC50 = 250. OnM vs 12nM; p< 0.001 ; Table B).

The bafilomycin A1 C-7 hydroxyl group is essential for both Nef and V-ATPase inhibition

[0026] A recently reported cryo-EM structure of bafilomycin A1 (2) bound to the Vo domain of V-ATPase highlights the importance of the hydrogen bond interaction between the C-7-OH on the macrocyclic ring and the corresponding tyrosine residue at position 144 of the c subunit (Wang et al., 2021). To investigate whether this interaction also plays a role in Nef inhibition and to confirm its requirement for V-ATPase inhibition, a Corey- Suggs oxidation was used to generate a new bafilomycin A1 derivative bearing a keto group at C-7 as well as the corresponding C-7 and C-21 diketo derivative (S17 & S18, respectively; Table B) (Gatti et al., 1996). Without wishing to be bound by any particular theory, oxidation at C-7 was sufficient to entirely abolish both Nef and V- ATPase inhibition with IC50S above the activity threshold (Table B). We also found there was a notable decrease in activities for 7,21 -diacetyl bafilomycin (S19), supporting the possibility that there are steric limitations at this position.

Moieties in 18-membered ring plecomacrolides required for Nef inhibition

The role of the Concanamycin macrolactone C-8 position

[0027] Along with 1, there are two additional naturally occurring concanamycin analogs that are obtained concurrently by fermentation, including concanamycin B (4) that differs from 1 by a single carbon at the C-8 position and concanamycin C (5), which lacks the 4' -carbamoyl group on the sugar (Table 3; (Kinashi et al., 1984)). Without wishing to be bound by any particular theory, the ethyl vs methyl difference between 1 and 4 resulted in a 10-fold decrease in potency, however, 4 maintained sub-nM potency (Nef IC50 = 1.2 nM vs. 0.18 nM; p <xxx; Table B). Although both compounds had similar therapeutic windows, 4 exhibited an improved neutralization window compared to 1 (24-fold vs. 9.4-fold, respectively; Table B). By contrast, 5, which lacks the 4'-carbamoyl on the P-D-rhamnose, displayed decreased potency compared to 1 (Nef IC50 = 0.71 nM vs. 0.18 nM; p < 0.01) and a concomitant decrease in the therapeutic and neutralization windows.

Role of the 4'-carbamoyl-8-D-rhamnose at C-23 of CMA

[0028] To further explore the role of the 4'-carbamoyl-β-D-rhamnose in 1, an aglycone derivative was synthesized and tested, concanamycin F (6) that was accessible through acid-catalyzed hydrolysis of the sugar moiety (Table B) (Bindseil and Zeeck, 1993). Without wishing to be bound by any particular theory, lack of the 4'- carbamoyl-P-D-rhamnose led to a decrease in potency compared to 1 (Nef IC50 = 1.6nM vs 0.18nM; p< 0.001). This also led to 6 having a smaller neutralization window compared to 1 (4-fold vs 9.4-fold; Table B). Further highlighting the importance that the 4'-carbamoyl-β-D-rhamnose plays in Nef inhibition, the aglycone of 4, CMX (7), also displayed a reduction in potency (Nef IC50 = 9.1 nM vs 1.2nM; p < 0.0001 ; Table B). Though 6 showed a decrease in V-ATPase inhibition compared to 1 (V-ATPase IC50 = 5.8nM vs 1.7nM; p< 0.001), the neutralization window for both aglycones was smaller compared to their parent compounds, since the absence of the 4'- carbamoyl-P-D-rhamnose had a greater impact on decreasing potency.

Enhancement of Nef and V-ATPase inhibition by modifications at C-3' in 18-membered ring plecomacrolides

[0029] A series of acyl chains were selected to modify 1 and 4 to assess changes in inhibitory activities. Addition of longer, flexible chains to C-3', such as a pentanoate group to each parent (8 and 9), resulted in a significant increase in the Nef IC50 (0.18 to 0.75nM and 1.2 to 3.4nM, respectively). A similar increase in the neutralization window of 8 was also observed (9.4-fold to 15-fold).. Addition of a second pentanoate group to the C-9-OH (10) did not significantly alter potency and caused no change in the neutralization window compared to 9 (V-ATPase IC50 = 110nM & 130nM respectively; Table B). However, when compared to the parent compound 4, the second addition at C-9-OH decreased V-ATPase inhibition by 5-fold (V-ATPase IC50 = 130nM vs 26nM; p< 0.001 ; Table B). Extension of the chain in the form of a 3'-nonanoate CMB derivative (23) resulted in small but statistically significant increases in Nef IC50 (6.3 and 9.1 nM respectively). In contrast, a single C-3'-OH acetylation (12) improved the Nef I C50 of 4 (0.66nM vs 1 ,2nM) and slightly increased the therapeutic window (8-fold vs 5- fold). Importantly, double acetylation at C-3’ -OH and C-9-OH (13) maintained Nef potency (Nef IC50 = 0.99nM) while widening the therapeutic window (8.8-fold vs 5.5-fold), without significantly changing the neutralization window. This compound had the largest neutralization and therapeutic windows, motivating our synthesis of corresponding single and multiple acetylations of 1, 6, and 5 (yielding 14/15, S20/S21, and S22/S23, respectively). Semi-synthetic derivative 3',9-diacetyl CMA (15) positively impacted its parent compound (1) the most, displaying a greater than two-fold improvement in the neutralization window (21 -fold vs 9.4-fold, p<0.001) while maintaining a sub-single digit nanomolar Nef IC50 (0.39 nM).

The 16-methoxy in 18 membered ring plecomacrolides

[0030] Finally, after noting that relatively small structural variations in the macrolactone core can alter potency, analogs that had a modification on the right side of the macrocycle were tested. Without wishing to be bound by any particular theory, it has been suggested that the concanamycin C-16-OMe position is crucial for maintaining the active macrolactone conformation and retaining potent V-ATPase inhibitory properties (Drose etal., 2001). This hypothesis was examined by converting the C-16-OMe to the C-16-OH derivative from the previously synthesized 21 -deoxy concanamycin F (S24), as deoxygenation at the C-21 showed no change in potency, to form 21-deoxy-16-hydroxy concanamycin F (S25; Table B). The change at C-2 led to a complete loss of activity (over 200-fold reduction) compared to 6 (p < 0.001; Table B).

Compounds [0031] The present disclosure provides compounds of Formula I, and pharmaceutically acceptable salts thereof:

R ¹ is OH, C(O)C1-8 alkyl, or OC(O)C1-8 alkyl, or R ^H and R ¹ together with the carbon to which they are attached form an oxo (=0) group;

R ² is H, OH, or OCH ₃ ;

R ³ is OHO, C(0)Ci-8 alkyl, 0C(0)R ⁶ or a modified sugar; each R ⁴ is independently H or C1-6alkyl;

R ⁵ is C1-6alkyl or C2-6alkenyl; and

R ⁶ is H, C1-6haloalky I , or C2-6 alkynyl. In some instances, or represents a direct bond;

R ^H is H;

R ¹ is OH or OC(O)C1-8 alkyl, or R ^H and R ¹ together with the carbon to which they are attached form an oxo (=0) group;

R ² is H, OH, or OCH ₃ ;

R ³ is 0C(0)R ⁶ or a modified sugar; each R ⁴ is independently H or C1-6alkyl;

R ⁵ is C1-6alkyl or C2-6alkenyl; and

R ⁶ is H, C1-6haloalkyl, or C2-6 alkynyl. [0032] In some cases, is or , or represents a direct bond. In some cases, is In some cases, is In some cases, is In some cases, represents a direct bond. In some cases, the compound has a structure of Formula (la), (lb), or

(Ic): (Ic), wherein (i) R ^5' is C2-5alkenyl and R ^5” is H, or (ii) both R ^5' and R ^5” are Ci-2alkyl . In some cases, the compound has a structure of Formula la (la). In some cases, the compound has a structure of

[0033] In some cases, R ^H is H. In some cases, R ¹ is C(O)Ci-8 alkyl. In some cases, R ¹ is OH or OC(O)Ci-8 alkyl. In some cases, R ¹ is OH. In some cases, R ¹ is OC(O)Ci-8 alkyl. In some cases, R ^H and R ¹ together with the carbon to which they are attached form an oxo (=0) group. In some cases, R ¹ is or In some cases, R ¹ is In some cases, R ¹ is

[0034] In some cases, R ² is H or OH. In some cases, R ² is OH or OCH3. In some cases, R ² is H or OCH3. In some cases, R ² is H. In some cases, R ² is OH. In some cases, R ² is OCH3.

[0035] In some cases, R ³ is CHO. In some cases, R ³ is C(O)Ci-8 alkyl. In some cases, R ³ is OC(O)R ⁶ or a modified sugar. In some cases, R ³ is OC(O)R ⁶ . In some cases, R ⁶ is H. In some cases, R ⁶ is C1-6haloalkyl. In some cases, R ³ is 2-fluoroacetyl, 2-chloroacetyl, 2-bromoacetyl, or 2-bromopropanoate. In some cases, R ³ is 2- fluoroacetyl. In some cases, R ³ is 2-chloroacetyl. In some cases, R ³ is 2-bromoacetyl. In some cases, R ³ is 2- bromopropanoate. In some cases, R ⁶ is C2-6 alkynyl. In some cases, R ³ is In some cases, R ³ is a modified sugar. In some cases, R ³ is a modified rhamnose. In some cases, R ³ is a modified deoxyrhamnose. In some cases, R ³ is a modified p-D-deoxyrhamnose. In some cases, R ³ is

, wherein R ⁷ is H, C(0)Ci-8alkyl, C(O)C2-8alkenyl, or C(O)C2-8alkynyl, or OR ⁷ and R ⁹ together with the carbon to which they are attached form an oxo (=0) group; R ⁸ is H, C(0)Ci-8alkyl, C(O)C2-8alkenyl, C(O)C2-8alkynyl, or

C(O)NH2; and R ⁹ is H; wherein C^alkynyl is optionally substituted with a fused 3-membered heterocycloalkyl ring comprising two nitrogen atoms. In some cases, R ³ is In some cases, R ³ is In some cases, R ⁷ is H. In some cases, R ⁷ is C(O)Ci-8alkyl, C(O)C2-8alkenyl, or C(O)C2-

8alkynyl. In some cases, R ⁷ is C(O)C1-8alkyl . In some cases, R ⁷ is C(O)C2-8alkenyl. In some cases, R ⁷ is

In some cases, R ⁷ is N-N . in some cases, R ⁹ is H. In some cases, OR ⁷ and R ⁹ together with the carbon to which they are attached form an oxo (=0) group. In some cases, R ⁸ is H. In some cases, R ⁸ is C(O)C1-6alkyl. In some cases, R ⁸ is In some cases, R ⁸ is C(O)NH2.

[0036] In some cases, at least one R ⁴ is H. In some cases, each R ⁴ is H. In some cases, at least one R ⁴ is C1-6alkyl. In some cases, each R ⁴ is C1-6alkyl. In some cases, at least one R ⁴ is methyl. In some cases, each R ⁴ is methyl.

[0037] In some cases, R ⁵ is C1-6alkyl. In some cases, R ⁵ is C3alkyl. In some cases, R ⁵ is isopropyl. In some cases, R ⁵ is C2-6alkenyl. In some cases, R ⁵ is In some cases,

[0038] In some cases, R ^5' is C1-5alkyl. In some cases, In some cases, R ^5' is methyl. In some cases, R ^5' is C2- 5alkenyl . In some cases, R ^5' is C3aakenyl. In some cases, R ^5' is allyl. In some cases, R ^5” is H. In some cases, R ^5” is C1-5alkyl. In some cases, R ^5” is methyl. In some case, R ^5' is C2-5alkenyl and R ^5” is H. In some cases, In some cases, R ^5' is C2alkenyl and R ^5” is H. In some cases, both R ^5' and R ^5” are Ci-2alkyl. In some cases, R ^5' and R ^5” are methyl.

[0039] Unless otherwise indicated, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, cis-trans, conformational, and rotational) forms of the structure. For example, the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers are included in this disclosure, unless only one of the isomers is drawn specifically.

[0040] Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, cis/trans, conformational, and rotational mixtures of the present compounds are within the scope of the disclosure. [0041] Unless otherwise indicated, all tautomeric forms of the compounds described herein are within the scope of the disclosure.

[0042] Additionally, unless otherwise indicated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopical ly enriched atoms. Discussion of an element is intended to include all isotopes of that element. For example, a substituent shown as a hydrogen includes where that hydrogen is in the deuterium or tritium isotope form, and a carbon atom can be present as a ¹³ C- or ¹⁴ C- carbon isotope.

[0043] It is understood that selections of values of each variable are those that result in the formation of stable or chemically feasible compounds.

[0044] Also provided are compounds listed in Table A, and pharmaceutically acceptable salts thereof:

Table A

[0045] As used herein, the term "alkyl” refers to straight chained and branched saturated hydrocarbon groups containing one to eight carbon atoms, for example, one to eight carbon atoms, or one to six carbon atoms, or one to four carbon atoms. The term C _n means the alkyl group has “n” carbon atoms. For example, C4 alkyl refers to an alkyl group that has 4 carbon atoms. C1-8 alkyl and C1-C8 alkyl refer to an alkyl group having a number of carbon atoms encompassing the entire range (i.e., 1 to 8 carbon atoms), as well as all subgroups (e.g, 1-8, 2-8, 3-8, 4-8, 5-8, 6-8, 7-8, 1-7, 2-7, 3-7, 4-7, 5-7, 6-7, 1-6, 2-6, 3-6, 4-6, 5-6, 1-5, 2-5, 3-5, 4-5, 1-4, 2- 4, 3-4, 1-3, 2-3, 1-2, 1, 2, 3, 4, 5, 6, 7, and 8 carbon atoms). Nonlimiting examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl (2-methylpropyl), t-butyl (1,1 -dimethylethyl), 3,3- dimethylpentyl, and 2-ethylhexyl. Unless otherwise indicated, an alkyl group can be an unsubstituted alkyl group or a substituted alkyl group.

[0046] The term "haloalkyl" refers to an alkyl group as defined herein which is substituted with one or more halogen atoms, e.g., 1 to 4, 1 to 3, 1 to 2, 1, 2, 3, or 4 halogen atoms. Nonlimiting examples of haloalkyl groups include fluoromethyl, difluoromethyl, trifluoromethyl, pentafluoroethyl, 1,1 -difluoroethyl, chloromethyl, chlorofluoromethyl and trichloromethyl groups.

[0047] The term "alkenyl" as used herein means a straight or branched chain hydrocarbon comprising one or more double bonds. [0048] The term "alkynyl" as used herein means a straight or branched chain hydrocarbon comprising one or more triple bonds.

[0049] As used herein, the term "cycloalkyl” refers to an aliphatic cyclic hydrocarbon group containing five to eight carbon atoms (e.g., 5, 6, 7, or 8 carbon atoms). The term C _n means the cycloalkyl group has “n” carbon atoms. For example, C5 cycloalkyl refers to a cycloalkyl group that has 5 carbon atoms in the ring. C5-8 cycloalkyl and C5-C8 cycloalkyl refer to cycloalkyl groups having a number of carbon atoms encompassing the entire range (i.e., 5 to 8 carbon atoms), as well as all subgroups (e.g., 5-6, 6-8, 7-8, 5-7, 5, 6, 7, and 8 carbon atoms). Nonlimiting examples of cycloalkyl groups include cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Unless otherwise indicated, a cycloalkyl group can be an unsubstituted cycloalkyl group or a substituted cycloalkyl group. The cycloalkyl groups described herein can be isolated or fused to another cycloalkyl group, a heterocycloalkyl group, an aryl group and/or a heteroaryl group.

[0050] As used herein, the term “heterocycloalkyl” is defined similarly as cycloalkyl, except the ring contains one to three heteroatoms independently selected from oxygen, nitrogen, and sulfur. In particular, the term “heterocycloal ky I” refers to a ring containing a total of two to 3 atoms, of which 1 or 2 of those atoms are heteroatoms independently selected from the group consisting of oxygen, nitrogen, and sulfur, and the remaining atoms in the ring are carbon atoms. Nonlimiting examples of heterocycloalkyl groups include azirines, diazirines, oxiranes, thiiranes, and the like. Heterocycloalkyl groups can be saturated or partially unsaturated ring systems. Heterocycloalkyl groups optionally can be fused to an alkyl chain as described herein, such as in a spiro configuration.

[0051] In some embodiments, R ³ is a modified sugar. As used herein the term "modified sugar" refers to a sugar wherein a hydroxyl group is replaced by another group, such as an H, an alkyl group, an alkoxy group, an acyl group, a benzyl group, and the like. A sugar can be a pentose, hexose, heptose, or an amino sugar (e.g., aminopentose, aminohexose, aminoheptose, or a neuraminic acid), for example. In some embodiments, a modified sugar is a modified rhamnose or a modified 2-deoxy-p-D-rhamnose, and the like. In some cases, a modified sugar is an unnatural sugar. Nonlimiting examples of unnatural sugars include halogenated sugars (e.g., fluorinated sugars), XXX. For the avoidance of doubt, the terms "carbohydrate,” "sugar,” and "saccharide” are all used interchangeably.

[0052] As described herein, compounds described herein may optionally be substituted with one or more substituents, such as illustrated generally below, or as exemplified by particular classes, subclasses, and species described herein. It will be appreciated that the phrase "optionally substituted" is used interchangeably with the phrase "substituted or unsubstituted." In general, the term "substituted", whether preceded by the term "optionally" or not, refers to the replacement of one or more hydrogen radicals in a given structure with the radical of a specified substituent. Unless otherwise indicated, an optionally substituted group may have a substituent at each substitutable position of the group. When more than one position in a given structure can be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at each position. When the term "optionally substituted" precedes a list, said term refers to all of the subsequent substitutable groups in that list. If a substituent radical or structure is not identified or defined as "optionally substituted", the substituent radical or structure is unsubstituted. In some cases, the substituent is selected from group A: halo, CN, OH, CO2H, CHO, NH2, oxo, NO2, C1-6alkyl, C1-6haloalkyl, C1-6alkoxy, C1- 6alkylthio, C1-6 alkyl-OH, C3-10carbocyclyl, 3-7 membered heterocyclyl, C3-10carbocyclyl-C1-6alkoxy, C3- 10carbocyclyl-0-C1-6alkylene, C3-10carbocyclyl-C1-6 alkoxy-C1-6alkylene, 3-7 membered heterocyclyl-C1-6 alkoxy, 3- 7 membered heterocyclyl-O-C alkylene, 3-7 membered heterocyclyl-C1-6alkoxy-C1-6alkylene, C1-6haloalkoxy, Ci. 6alkoxy-C1-6alkylene, C1-6alkoxy-C1-6alkoxy, C1-6alkyl-C(O)-, C1-6alkyl-C(O)O-, NHC1-6alkyl, C1-6alkyl-C(O)NH-, Ci. ₆ haloalkyl-C(O)NH, C1-6alkyl-NHC(O)-, C1-6alkyl-SO ₂ -, C1-6alkyl-SO-, and C1-6alkylSO ₂ NH-.

[0053] Selection of substituents and combinations of substituents contemplated herein are those that result in the formation of stable or chemically feasible compounds. The term "stable", as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, specifically, their recovery, purification, and use for one or more of the purposes disclosed herein. In some embodiments, a stable compound or chemically feasible compound is one that is not substantially altered when kept at a temperature of 40 °C or less, in the absence of moisture or other chemically reactive conditions, for at least a week. Only those choices and combinations of substituents that result in a stable structure are contemplated. Such choices and combinations will be apparent to those of ordinary skill in the art and may be determined without undue experimentation.

[0054] Unless otherwise indicated, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, cis-trans, conformational, and rotational) forms of the structure. For example, the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers are included in this disclosure, unless only one of the isomers is drawn specifically.

[0055] Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, cis/trans, conformational, and rotational mixtures of the present compounds are within the scope of the disclosure.

[0056] Unless otherwise indicated, all tautomeric forms of the compounds described herein are within the scope of the disclosure.

[0057] Additionally, unless otherwise indicated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopical ly enriched atoms. Discussion of an element is intended to include all isotopes of that element. For example, a substituent shown as a hydrogen includes where that hydrogen is in the deuterium or tritium isotope form, and a carbon atom can be present as a ¹³ C- or ¹⁴ C- carbon isotope.

[0058] It is understood that selections of values of each variable are those that result in the formation of stable or chemically feasible compounds.

Methods of Use

[0059] The analogs described herein or pharmaceutically acceptable salts thereof can be used to modulate an HIV Nef. Modulating an HIV Nef includes inhibiting an HIV Nef. [0060] The term "HIV Nef-associated disorder” is used herein to mean diseases or disorders whose status or progression is influenced by the expression of HIV Nef in a patient. A non-limiting example of an HIV Nef- associated disorder is HIV infection, e.g., HIV-1 infection.

[0061] As used herein, "HIV" refers to the human immunodeficiency virus. HIV includes, without limitation, HIV-1 . HIV-1 includes but is not limited to extracellular virus particles and the forms of HIV-1 associated with HIV-1 infected cells. The human immunodeficiency virus (HIV) may be either of the two known types of HIV (HIV-1 or HIV-2). As used herein, HIV-1 refers to any of the known major subtypes (classes A, B, C, D, E, F, G, H, or J), outlying subtype (Group O), yet to be determined subtypes of HIV-1, and recombinations thereof.

[0062] As used herein, "HIV infection” refers to infection of a subject with HIV.

[0063] The terms, "disease", "disorder", and "condition" may be used interchangeably herein to refer to an HIV Nef-associated medical or pathological condition, such as HIV infection.

[0064] As used herein, the terms "subject", "host”, and "patient" are used interchangeably. The terms "subject", "host”, and "patient" refer to an animal (e.g., a bird such as a chicken, quail or turkey, or a mammal), specifically a "mammal" including a non-primate (e.g., a cow, pig, horse, sheep, rabbit, guinea pig, rat, cat, dog, or mouse) and a primate (e.g., a monkey, chimpanzee, or human), and more specifically a human. In some embodiments, the subject is a non-human animal such as a farm animal (e.g., a horse, cow, pig or sheep), or a pet (e.g., a dog, cat, guinea pig or rabbit). In a preferred embodiment, the subject is a "human".

[0065] As used herein, the terms "treat", "treatment," and "treating" refer to therapeutic treatment and/or prophylactic treatments. For example, therapeutic treatments include the reduction or amelioration of the progression, severity and/or duration of HIV infection, or the amelioration of one or more symptoms (specifically, one or more discernible symptoms) of HIV infection, resulting from the administration of one or more therapies (e.g., one or more therapeutic agents such as a compound or composition described herein). In specific embodiments, the therapeutic treatment includes the amelioration of at least one measurable physical parameter of an HIV infection. In other embodiments, the therapeutic treatment includes the inhibition of the progression of an HIV infection, either physically by, e.g., stabilization of a discernible symptom, physiologically by, e.g., stabilization of a physical parameter, or both. In other embodiments, the therapeutic treatment includes the reduction or stabilization of HIV infections. Antiviral drugs can be used in the community setting to treat people who already have HIV infection to reduce the severity of symptoms and suppress the infection. Treating and HIV infection includes reducing or eliminating an HIV reservoir in a patient.

[0066] As used herein, the term "HIV reservoir” refers to a group of cells in a patient that are infected with HIV but have not produced new HIV (i.e., are in a latent stage of infection) for many months or years. Very early during acute HIV infection, a latent reservoir is established and despite effective combination anti-retroviral therapy (cART), HIV persists in latently infected cells. If a patient having a latent HIV infection stops treatment with cART, the presence of an HIV reservoir in a patient can allow an active HIV infection to become reestablished in the patient. [0067] The terms "prophylaxis", "prophylactic”, "prophylactic use", and "prophylactic treatment" as used herein, refer to any medical or public health procedure whose purpose is to prevent, rather than treat or cure a disease. As used herein, the terms "prevent", "prevention" and "preventing" refer to the reduction in the risk of acquiring or developing a given condition, or the reduction or inhibition of the recurrence or said condition in a subject who is not ill, but who has been or may be near a person with the disease.

[0068] As used herein, prophylactic use includes use to prevent contagion or spread of the infection in populations or individuals at high risk of HIV infection. Prophylactic use may also include treating a person who is not ill with HIV or not considered at high risk for contracting HIV, in order to reduce the chances of becoming infected with HIV and passing it on to another pereson.

[0069] In some embodiments, the methods of the disclosure are applied as a prophylactic measure to members of a community or population group, specifically humans, in order to prevent the spread of infection.

[0070] As used herein, an "effective amount" refers to an amount sufficient to elicit the desired biological response. In the present disclosure the desired biological response is to inhibit the replication of HIV, to reduce the amount of HIV, or to reduce or ameliorate the severity, duration, progression, or onset of an HIV infection, prevent the advancement of an HIV infection, prevent the recurrence, development, onset or progression of a symptom associated with an HIV infection, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy used against HIV infections. The precise amount of compound administered to a subject will depend on the mode of administration, the type and severity of the infection and on the characteristics of the subject, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. When co-administered with other antiviral agents, e.g., when co-administered with an anti-HIV medication, an effective amount of the second agent will depend on the type of drug used. A safe amount is one with minimal side effects, as can readily be determined by those skilled in the art. Suitable dosages are known for approved agents and can be adjusted by the skilled artisan according to the condition of the subject, the type of condition(s) being treated and the amount of a compound described herein being used. In cases where no amount is expressly noted, a safe and effective amount should be assumed. For example, compounds described herein can be administered to a subject in a dosage range from between approximately 0.01 to 100 mg/kg body weight/day for therapeutic or prophylactic treatment.

[0071] As used herein, a "safe and effective amount” of a compound or composition described herein is an effective amount of the compound or composition which does not cause excessive or deleterious side effects in a patient.

[0072] Generally, dosage regimens can be selected in accordance with a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the renal and hepatic function of the subject; and the particular compound or salt thereof employed, the duration of the treatment; drugs used in combination or coincidental with the specific compound employed, and like factors well known in the medical arts. The skilled artisan can readily determine and prescribe a safe and effective amount of the compounds described herein required to treat, to prevent, inhibit (fully or partially) or arrest the progress of the disease.

[0073] Dosages of the compounds described herein can range from between about 0.01 to about 100 mg/kg body weight/day, about 0.01 to about 50 mg/kg body weight/day, about 0.1 to about 50 mg/kg body weight/day, or about 1 to about 25 mg/kg body weight/day. It is understood that the total amount per day can be administered in a single dose or can be administered in multiple dosing, such as twice a day (e.g., every 12 hours), three times a day (e.g., every 8 hours), or four times a day (e.g., every 6 hours).

[0074] For therapeutic treatment, the compounds described herein can be administered to a patient within, for example, 48 hours (or within 40 hours, or less than 2 days, or less than 1 .5 days, or within 24 hours) of onset of symptoms (e.g., nasal congestion, sore throat, cough, aches, fatigue, headaches, and chills/sweats). The therapeutic treatment can last for any suitable duration, for example, for 5 days, 7 days, 10 days, 14 days, etc.

Pharmaceutically Acceptable Salts

[0075] The analogs described herein can exist in free form, or, where appropriate, as salts. Those salts that are pharmaceutically acceptable are of particular interest since they are useful in administering the analogs described below for medical purposes. Salts that are not pharmaceutically acceptable are useful in manufacturing processes, for isolation and purification purposes, and in some instances, for use in separating stereoisomeric forms of the compounds described herein or intermediates thereof.

[0076] As used herein, the term "pharmaceutically acceptable salt" refers to salts of a compound which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue side effects, such as, toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.

[0077] Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference. Pharmaceutically acceptable salts of the analogs described herein include those derived from suitable inorganic and organic acids and bases. These salts can be prepared in situ during the final isolation and purification of the compounds.

[0078] Where the compound described herein contains a basic group, or a sufficiently basic bioisostere, acid addition salts can be prepared by 1) reacting the purified compound in its free-base form with a suitable organic or inorganic acid and 2) isolating the salt thus formed. In practice, acid addition salts might be a more convenient form for use and use of the salt amounts to use of the free basic form.

[0079] Examples of pharmaceutically acceptable, non-toxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, glycolate, gluconate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3- phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like.

[0080] Where the compound described herein contains a carboxylic acid group or a sufficiently acidic bioisostere, base addition salts can be prepared by 1) reacting the purified compound in its acid form with a suitable organic or inorganic base and 2) isolating the salt thus formed. In practice, use of the base addition salt might be more convenient and use of the salt form inherently amounts to use of the free acid form. Salts derived from appropriate bases include alkali metal (e.g., sodium, lithium, and potassium), alkaline earth metal (e.g., magnesium and calcium), ammonium and N ⁺ (Ci-4alky 1)4 salts. This disclosure also envisions the quaternization of any basic nitrogen-containing groups of the compounds disclosed herein. Water or oil-soluble or dispersible products may be obtained by such quaternization.

[0081] Basic addition salts include pharmaceutically acceptable metal and amine salts. Suitable metal salts include the sodium, potassium, calcium, barium, zinc, magnesium, and aluminum. The sodium and potassium salts are usually preferred. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate. Suitable inorganic base addition salts are prepared from metal bases which include sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide and the like. Suitable amine base addition salts are prepared from amines which are frequently used in medicinal chemistry because of their low toxicity and acceptability for medical use. Ammonia, ethylenediamine, N-methyl-glucamine, lysine, arginine, ornithine, choline, N, N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N- benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, triethylamine, dibenzylamine, ephenamine, dehydroabietylamine, N-ethylpiperidine, benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, basic amino acids, dicyclohexylamine and the like.

[0082] Other acids and bases, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds described herein and their pharmaceutically acceptable acid or base addition salts.

[0083] It should be understood that this disclosure includes mixtures/combinations of different pharmaceutically acceptable salts and also mixtures/combinations of compounds in free form and pharmaceutically acceptable salts. Pharmaceutical Compositions

[0084] The analogs described herein can be formulated into pharmaceutical compositions that further comprise a pharmaceutically acceptable carrier, diluent, adjuvant or vehicle. In some embodiments, the present disclosure relates to a pharmaceutical composition comprising an analog described herein, and a pharmaceutically acceptable carrier, diluent, adjuvant or vehicle. In some embodiments, the present disclosure includes a pharmaceutical composition comprising a safe and effective amount of a compound described herein or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, diluent, adjuvant or vehicle. Pharmaceutically acceptable carriers include, for example, pharmaceutical diluents, excipients or carriers suitably selected with respect to the intended form of administration, and consistent with conventional pharmaceutical practices.

[0085] An "effective amount" includes a "therapeutically effective amount" and a "prophylactically effective amount". The term "therapeutically effective amount" refers to an amount effective in treating and/or ameliorating an HIV infection in a patient.

[0086] A pharmaceutically acceptable carrier may contain inert ingredients which do not unduly inhibit the biological activity of the compounds. The pharmaceutically acceptable carriers should be biocompatible, e.g., non-toxic, non-inflammatory, non-immunogenic or devoid of other undesired reactions or side-effects upon the administration to a subject. Standard pharmaceutical formulation techniques can be employed.

[0087] The pharmaceutically acceptable carrier, adjuvant, or vehicle, as used herein, includes any solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in formulating pharmaceutically acceptable compositions and known techniques for the preparation thereof. Except insofar as any conventional carrier medium is incompatible with the compounds described herein, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutically acceptable composition, its use is contemplated to be within the scope of this disclosure. As used herein, the phrase "side effects" encompasses unwanted and adverse effects of a therapy (e.g., a prophylactic or therapeutic agent). Side effects are always unwanted, but unwanted effects are not necessarily adverse. An adverse effect from a therapy (e.g., prophylactic or therapeutic agent) might be harmful or uncomfortable or risky. Side effects include, but are not limited to fever, chills, lethargy, gastrointestinal toxicities (including gastric and intestinal ulcerations and erosions), nausea, vomiting, neurotoxicities, nephrotoxicities, renal toxicities (including such conditions as papillary necrosis and chronic interstitial nephritis), hepatic toxicities (including elevated serum liver enzyme levels), myelotoxicities (including leukopenia, myelosuppression, thrombocytopenia and anemia), dry mouth, metallic taste, prolongation of gestation, weakness, somnolence, pain (including muscle pain, bone pain and headache), hair loss, asthenia, dizziness, extra-pyramidal symptoms, akathisia, cardiovascular disturbances and sexual dysfunction.

[0088] Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as twin 80, phosphates, glycine, sorbic acid, or potassium sorbate), partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, or zinc salts), colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, poly acrylates, waxes, polyethylene-polyoxypropylene-block polymers, methylcellulose, hydroxypropyl methylcellulose, wool fat, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols; such a propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

Administration Methods

[0089] The analogs and pharmaceutically acceptable compositions described above can be administered to humans and other animals orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, or drops), bucally, as an oral or nasal spray, to the pulmonary system, such as by using an inhaler, such as a metered dose inhaler (MDI), or the like, depending on the severity of the infection being treated.

[0090] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, EtOAc, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0091] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1 ,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. [0092] The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0093] In order to prolong the effect of an analog described herein, it is often desirable to slow the absorption of the compound from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the compound then depends upon its rate of dissolution that, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered compound form is accomplished by dissolving or suspending the compound in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the compound in biodegradable polymers such as poly lactide-polyglycol ide. Depending upon the ratio of compound to polymer and the nature of the particular polymer employed, the rate of compound release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the compound in liposomes or microemulsions that are compatible with body tissues.

[0094] Compositions for rectal or vaginal administration are specifically suppositories which can be prepared by mixing the compounds described herein with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.

[0095] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

[0096] Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

[0097] The active compounds can also be in microencapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

[0098] Dosage forms for topical or transdermal administration of a compound described herein include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulation, eardrops, and eye drops are also contemplated as being within the scope of this disclosure. Additionally, the present disclosure contemplates the use of transdermal patches, which have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

[0099] The compositions described herein may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term "parenteral" as used herein includes, but is not limited to, subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Specifically, the compositions are administered orally, intraperitoneally or intravenously.

[0100] Sterile injectable forms of the compositions described herein may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1 ,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as polysorbates, sorbitan esters, and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.

[0101] The pharmaceutical compositions described herein may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers commonly used include, but are not limited to, lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.

[0102] Alternatively, the pharmaceutical compositions described herein may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient which is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug. Such materials include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.

[0103] The pharmaceutical compositions described herein may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs.

[0104] Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Topically-transdermal patches may also be used.

[0105] For topical applications, the pharmaceutical compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds described herein include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2 octyldodecanol, benzyl alcohol and water.

[0106] For ophthalmic use, the pharmaceutical compositions may be formulated as micronized suspensions in isotonic, pH adjusted sterile saline, or, specifically, as solutions in isotonic, pH adjusted sterile saline, either with or without a preservative such as benzalkonium chloride. Alternatively, for ophthalmic uses, the pharmaceutical compositions may be formulated in an ointment such as petrolatum. [0107] The compounds for use in the methods described herein can be formulated in unit dosage form. The term "unit dosage form" refers to physically discrete units suitable as unitary dosage for subjects undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier. The unit dosage form can be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form can be the same or different for each dose.

[0108] The disclosure will be more fully understood by reference to the examples described herein which detail exemplary embodiments. These examples should not, however, be construed as limiting the scope of the disclosure. All citations throughout the disclosure are hereby expressly incorporated by reference.

COMPARATIVE EXAMPLES

[0109] Several compounds were synthesized for comparative purposes with the compounds of Formula (I).

[0110] Comparative Example 1 : Structure-Activity Relationship (SAR) Study

[0111] The identification of a potent Nef inhibitor that enhances CTL-mediated clearance of active and latent reservoirs of HIV is an important goal toward developing a cure. Through the curation and testing of a SAR panel with 74 macrocycles, it was determined that microbially derived 16-, 18-, or 24-membered ring compounds are potent Nef inhibitors. Prior to the discovery of the plecomacrolides as potent Nef inhibitors, compounds 1 and 2 were primarily known for their highly specific and potent V-ATPase inhibitory activity in eukaryotic cells (Bowman et al., 1988; Osteresch et al., 2012; Painter et al., 2020). Moreover, the 24-membered ring macrocycle archazolid A (3) that potently inhibits Nef is also an effective V-ATPase inhibitor (Osteresch et al., 2012). Recently, cryo-EM structures revealed that compounds 2 and 3 have distinct binding sites on the V0 domain of V-ATPase. Without wishing to be bound by any particular theory, the C-7-OH of 2 and 3 forms a critical hydrogen bond with a tyrosine residue that mediates binding to the c-subunit (Keon et al., 2022). In the instant disclosure, a semisynthetic derivative of 2 that has been oxidized at C-7-OH to a keto group (S17) led to abrogation of activity at all concentrations tested, highlighting the role of the hydrogen bond donating abilities of the C-7-OH to achieve Nef and V-ATPase inhibition. Without wishing to be bound by any particular theory, it appears that Nef-mediated MHC-I downmodulation is activated through a V-ATPase-dependent step. However, currently, there is no structure available detailing how 1 induces V-ATPase inhibition, only a hypothesis that it binds the c-subunit similarly to its 16-membered ring family member, 2 (Osteresch et al., 2012; Yoshimori et al., 1991). The SAR study disclosed herein highlights how 1, and its semi-synthetic derivatives act differently than 2 or 3 by inhibiting Nef at low concentrations with minimal effect on lysosomal neutralization. This ability to separate Nef inhibition while limiting V-ATPase inhibition supports the hypothesis that distinct pathways exist for downmodulation of MHC-1 and V-ATPase dependent lysosomal acidification (Table B). To visualize these two inhibitory pathways, the neutralization window (the fold-change between Nef and V-ATPase IC50S) between the parent compounds (CMA/CMB) was compared with that for their respective semi-synthetic derivatives with the aim of developing an improved candidate for future in vivo studies. [0112] One of the most important factors for determining potency of Nef and V-ATPase inhibition was macrolactone ring-size. Comparing 16- versus 18-membered-ring plecomacrolides (2 vs. 1) revealed that the latter has 100-fold greater Nef inhibitory activity. In addition, the 24-membered macrolactone (3) had potent Nef inhibitory activity, however others such as rifampicin, did not, indicating that sub-nM Nef inhibitory activity is not a general feature of large macrocyclic compounds (Table B). Smaller rings with 10-, 12-, and 14-membered macrolactones failed to display inhibitory activity at all concentrations tested (Table B). For molecules 1-3, the trend observed for Nef IC50 corresponded to that observed for lysosomal neutralization (1 < 3 < 2; Table B). However, there were distinct differences in the neutralization window (9.4-, 3.6-, & 2.9-fold for 1, 3, & 2, respectively) supporting the hypothesis that alternative pathways are employed to inhibit Nef-dependent MHC-I downmodulation versus lysosomal acidification for each of these compounds. Without wishing to be bound by any particular theory, the level of potency for lysosomal neutralization may relate to differential binding to V- ATPase based on ring size and other structural feature. Indeed, previous studies have noted that the size of the plecomacrolide macrocycle impacts binding orientation to the c-subunit of V-ATPase (Osteresch et al., 2012; Wang et al., 2021). Identification of the direct target and structural studies are required to explain the relative potencies of 1-3 for Nef inhibition.

[0113] In addition to the macrolactone ring size, it was found that the pyran ring is important for both Nef and V-ATPase inhibition, as conversion to a linear system drastically reduced both inhibitory activities (2 vs. S8). Within the pyran ring, modifications at C-19 of 2 had little effect (S5), whereas alterations at C-21 of 2 either increased or decreased potency (Table B). For example, acetylation (S9) improved potency for both inhibitory activities whereas the addition of longer acyl chains (S10, S11) had an opposing effect. We similarly observed both positive and negative effects on potency for changes at C-21 of 19-deoxy bafilomycin (S5). Conversion of C-21-OH to an allyl-ester (S27) decreased potency whereas addition of a longer chain with a terminal alkyne improved potency (S65, Table B). This trend was reinforced by the C-21-penta-3-enoate (S29) and C-21-penta- 2,4-dienoate (S12) derivatives of 2 where addition of acyl chains bearing variant degrees of unsaturation improved potency compared to S10 or S11 (Table B). Without wishing to be bound by any particular theory, it is possible that hydrophobic interactions mediated by these linear substituents may be important for target binding especially because they parallel the effects of hydrophobic substituents previously reported to play a role in V- ATPase binding (Wang et al., 2021).

[0114] CMA (1) contains a 4'-carbamoyl-β-D-rhamnose moiety at the pyranyl C-23 position that is absent from the analogous C-21 position of 2. Two microbially derived analogs, S14 and S15 were tested as they contained modified P-D-rhamnose moieties (Table B). When C-21 -OH is glycosylated, the 16-membered scaffold becomes a significantly more potent Nef inhibitor with single-digit nM IC50S (S14 and S15; Table B). However, neutralization and therapeutic windows were relatively poor. Thus, although identifying semi-synthetic derivatives of 2 have helped guide synthetic modification strategies, the bafilomycin system has largely been ruled out as a lead scaffold for generating improved HIV Nef inhibitor drug candidates.

[0115] The same trend was observed with 18-membered ring plecomacrolides as comparison of Nef IC50S amongst concanamycin F (6) and concanamycin C (5) revealed that the P-D-rhamnose moiety (6 vs 5) improved potency (Table B). Addition of the 4'-carbamoyl-β-D-rhamnose moiety onto the pyran ring (5 vs. 1) led to a 14- fold improvement in potency (Table B). This was further supported by the testing of an additional aglycone of concanamycin B [concanamycin X (7)], lacking the 4'-carbamoyl-β-D-rhamnose, which also suffered a reduction in potency (Table B). It is also important to note that both aglycones displayed significantly smaller neutralization windows compared to their parent compounds, especially for 4. These results confirm, using the 18-membered ring plecomacrolide scaffold, that the β-D-rhamnose and 4'-carbamoyl moieties are integral motifs for nM inhibition of HIV Nef and inhibition of V-ATPase.

[0116] For both 1 and 2, it was observed that the pyran ring is vital for Nef inhibition and lysosomal neutralization, as conversion to a linear system (S8) drastically reduced both inhibitory activities for 2. More limited changes, including modification at C-19 of 2 reduced Nef inhibitory potency while other changes, such as esterification of C-21-OH of 2 yielded results that depended on chain length [small acyl chains (S9) improved potency for both inhibitory activities whereas the addition of longer acyl chains (S10, S11) had the opposite effect]. It was similarly observed both positive and negative effects on potency for changes at C-21 of 19-deoxy bafilomycin (S5); conversion of C-21 -OH to an allyl-ester (S27) decreased potency whereas addition of a longer chain with a terminal alkyne improved potency (S28). This trend was reinforced by the C-21-penta-3-enoate (S29) and C-21-penta-2,4-dienoate (S12) derivatives of 2 where addition of acyl chains bearing variant degrees of unsaturation improved potency compared to S10 or S11. Without wishing to be bound by any particular theory, it is thought that hydrophobic interactions mediated by these linear substituents may be important for target binding especially because they parallel the effects of hydrophobic substituents previously reported to play a role in V-ATPase bindingfu.

[0117] The key results of the instantly-disclosed study reveal that concanamycin B (4), which bears a C-8 methyl compared to ethyl in 1 represents the most compelling starting point to identify lead compounds for clinical development. Although 4 has 10-fold lower potency for Nef inhibition compared to 1, it has an expanded neutralization window (approximately 24-fold vs 10-fold for 1: Table B). Further semi-synthetic modification of 4 revealed that compound 13 (3',9-diacetylated concanamycin B) demonstrated both an expanded therapeutic and neutralization window and has good overall characteristics for further development (Table B). Expansion of the neutralization window was not observed with a single acetylation at 3’ -OH (12), highlighting that the C-9 position on 4 is critical for distinguishing Nef versus V-ATPase inhibitory activity.

[0118] The most compelling 18-membered macrolactone derivatives were obtained by esterifying the C-9-OH position, and motivated synthesis of acetyl and diacetyl derivatives 14/15, S22/S23, and S20/S21 (derived from 1, 5, and 6, respectively). The most notable compound from the acetylation panel was 3',9-diacetyl CM A (15), as it displayed a significantly increased neutralization window of the CMA scaffold, approaching neutralization windows that had only been observed with CMB and its derivatives, while maintaining both potent Nef inhibition and the therapeutic window. The increase in magnitude of the neutralization window for analogs of 1 and therapeutic windows for 4 when esterifying C-9-OH (which was observed to a lesser degree in the monoacetylated compounds (14 & 12)), suggests that diversifying C-9 is critical for distinguishing Nef inhibition from lysosomal acidification and cellular toxicity. Further studies are needed to assess whether improving the neutralization window in this manner will also reduce toxicity in a more significant way.

[0119] Through this SAR study it was shown that 18-membered plecomacrolides with an intact pyran ring system, a C-8 methyl group, 4'-carbamoyl- β-D-rhamnose, and addition of short acyl ester chains represent good opportunities for identifying improved Nef inhibitors. Modifications to the macrocycle, the pyran ring, or substituents off the ring impact potencies in both 16- and 18-membered ring compounds and further sugar diversification should be considered moving forward to gain a deeper understanding of the role this key moiety plays in nM potency and the mechanism of action. Differences in the way these compounds inhibit Nef- dependent MHC-I downmodulation versus lysosomal neutralization suggest there are structural parameters in the respective molecule(s) for each inhibitory pathway that remain to be identified.

[0120] Comparative Example 2: Synthesis of Bafilomycin A2 (Compound 1 , also called S4)

[0122] Bafilomycin A1 (74 mg, .090 mmol) was dissolved in MeOH (2 mL) under anhydrous conditions and cooled to 0 °C. FeCl3 (2.9 mg, 0.018 mmol, 0.2 eq) was added to the solution. The FeCl3 was added to the reaction after first dissolving it in a separate flask with MeOH. The reaction was ran at room temperature for 15- 30 minutes, checking TLC (55% ethyl acetate/hexane) for consumption of starting material. The reaction was quenched with phosphate buffer (1.0 M, 7.1 pH). The reaction was dissolved in DCM, washed with water, brine, dried over MgSO4, filtered, and concentrated. The crude reaction was then purified by flash column chromatography (30-60% ethyl acetate/hexane) to yield the product. S4 ¹ H NMR (599 MHz, Acetone) 5 6.69 (d, J = 0.9 Hz, 1 H), 6.66 (dd, J = 15.0, 10.8 Hz, 1 H), 5.96 (dt, J = 8.9, 1.2 Hz, 1 H), 5.81 (d, J = 10.8 Hz, 1 H), 5.17 (dd, J = 15.0, 9.0 Hz, 1 H), 5.09 (dd, J = 8.1 , 1 .5 Hz, 1 H), 4.05 (d, J = 8.5 Hz, 1 H), 4.04 (d, J = 5.3 Hz, 1 H), 3.93 - 3.89 (m, 1 H), 3.66 (s, 3H), 3.52 - 3.49 (m, 1 H), 3.46 (q, J = 4.1 , 3.1 Hz, 1 H), 3.32 (dp, J = 8.2, 3.0, 2.5 Hz, 1 H), 3.23 (s, 3H), 3.11 - 3.05 (m, 1 H), 3.04 (s, 3H), 2.59 - 2.46 (m, 1 H), 2.19 (dd, J = 13.3, 4.3 Hz, 1 H), 2.12 - 2.06 (m, 1 H), 2.05 (p, J = 2.2 Hz, 2H), 2.04 - 1 .99 (m, 1 H), 1 .98 (d, J = 1 .2 Hz, 3H), 1 .97 - 1 .93 (m, 1 H), 1 .92 (d, J = 1.4 Hz, 3H), 1.88 (p, J = 7.1 Hz, 1 H), 1.48 (dd, J = 13.4, 10.6 Hz, 1 H), 1.27 (dd, J = 10.0, 6.6 Hz, 1 H), 1.08 (dd, J = 14.1 , 7.2 Hz, 1 H), 1.05 (d, J = 7.0 Hz, 3H), 1.03 (d, J = 6.9 Hz, 3H), 0.97 (d, J = 7.0 Hz, 3H), 0.95 (d, J = 6.9 Hz, 3H), 0.92 (t, J = 7.0 Hz, 6H), 0.89 (d, J = 6.8 Hz, 3H). ¹³ C NMR (151 MHz, Acetone) 5 167.23, 145.31 , 144.50, 142.13, 134.06, 133.61 , 132.68, 127.27, 125.32, 103.84, 83.84, 80.45, 78.18, 77.65, 70.39, 70.34, 60.19, 55.72, 46.68, 42.32, 41.52, 41.24, 40.06, 39.84, 39.17, 38.05, 29.14, 22.40, 21.12, 20.21 , 17.80, 14.63, 14.09, 12.63, 11.11 , 7.90.

[0123] Comparative Example 3: Synthesis of 19-deoxy Bafilomycin (S5)

[0124] Bafilomycin A2 (45 mg, 70.7 μmol, was dissolved in EtOH (6 mL) and stirred under nitrogen. NaBHsCN (26.6 mg, 424 μmol, 6.0 eq) and HCI (11.1 mg, 304 μmol, 4.3 eq) were added to the reaction flask that was then left to run for approximately 1 hour, checking by TLC to determine the reaction's completeness. The crude reaction mixture was extracted with DCM (x 4). The organics were combined, washed with water, brine, dried over NaSO4, filtered and concentrated. The crude material was purified further by flash column chromatography (12-100% ethyl acetate/hexane) to yield S5 as the major product (40. mg, 70.7 μmol, 93%) as a pale-yellow solid, with small amounts of two minor products, 19-Deoxy-20-ene Bafilomycin A1 (S7) and 19-Deoxy-21- Methoxy Bafilomycin A1 (S6). 19-deoxy Bafilomycin ¹ H NMR (599 MHz, Acetone) 5 6.68 (s, 1 H), 6.64 (dd, J= 15.0, 10.8 Hz, 1 H), 5.94 (d, J = 8.9 Hz, 1 H), 5.80 (d, J = 10.8 Hz, 1 H), 5.22 - 5.18 (m, 1 H), 5.17 (dd, J = 14.6, 8.4 Hz, 1 H), 4.04 (t, J = 8.3 Hz, 1 H), 4.00 (d, J = 5.4 Hz, 1 H), 3.81 - 3.76 (m, 1 H), 3.64 (t, J = 4.1 Hz, 1 H), 3.62 (s, 3H), 3.37 (ddd, J = 11 .5, 8.2, 1 .8 Hz, 1 H), 3.32 (s, 1 H), 3.27 (td, J = 10.2, 4.6 Hz, 1 H), 3.23 (s, 3H), 2.79 (dd, J = 10.0, 2.2 Hz, 1 H), 2.60 (s, 1 H), 2.54 (ddd, J = 9.0, 7.0, 2.0 Hz, 1 H), 2.14 (s, 1 H), 2.07 - 2.00 (m, 3H), 1.97 (d, J = 1.3 Hz, 3H), 1.90 (s, 3H), 1.89 - 1.82 (m, 2H), 1.69 - 1.61 (m, 1 H), 1.26 (s, 1 H), 1.15 (q, J = 11.5 Hz, 1 H), 1.05 (d, J = 7.1 Hz, 3H), 0.95 (d, J = 6.8 Hz, 3H), 0.93 (d, J = 6.9 Hz, 3H), 0.91 (d, J = 6.5 Hz, 3H), 0.87 (d, J = 6.9 Hz, 3H), 0.84 (d, J = 7.0 Hz, 3H), 0.79 (d, J = 6.8 Hz, 3H). ¹³ C NMR (151 MHz, Acetone) 5 166.63, 144.72, 144.14, 142.34, 133.80, 133.15, 132.64, 127.27, 125.35, 85.14, 84.03, 80.50, 77.68, 77.62, 74.22, 70.45, 59.97, 55.70, 42.31 , 42.06, 41.26, 40.83, 40.71 , 38.76, 38.12, 29.23, 22.52, 21.58, 20.06, 17.91 , 14.83, 14.19, 12.67, 10.64, 8.89.

[0125] Comparative Example 4: Synthesis of 19-Deoxy-20-ene Bafilomycin A1 (S7)

[0126] ¹ H NMR (599 MHz, acetone) 5 6.66 (d, J = 0.9 Hz, 1 H), 6.63 (dd, J = 15.1 , 10.9 Hz, 1 H), 5.94 (dt, J =

8.9, 1.2 Hz, 1 H), 5.83 - 5.79 (m, 2H), 5.62 (dt, J = 10.3, 2.1 Hz, 1 H), 5.24 (dd, J = 7.4, 2.0 Hz, 1 H), 5.20 (dd, J =

15.1 , 8.6 Hz, 1 H), 4.07 - 4.00 (m, 3H), 3.77 (ddd, J = 10.3, 3.8, 1.7 Hz, 1 H), 3.64 (s, 1 H), 3.62 (d, J = 3.8 Hz, 1 H), 3.33 (ddd, J = 7.6, 5.7, 2.0 Hz, 1 H), 3.22 (s, 3H), 2.95 (dd, J = 9.5, 2.1 Hz, 1 H), 2.53 (tt, J = 7.9, 6.0 Hz, 1 H), 2.28 - 2.20 (m, OH), 2.09 (dd, J = 14.1 , 11 .3 Hz, 1 H), 2.04 - 1 .99 (m, 1 H), 1 .97 (d, J = 1.3 Hz, 3H), 1 .92 - 1 .85 (m, 5H), 1 .74 - 1 .68 (m, 1 H), 1 .05 (d, J = 7.0 Hz, 3H), 1 .01 (d, J = 6.9 Hz, 3H), 0.94 (dd, J = 7.9, 6.9 Hz, 6H), 0.88 (d, J = 7.1 Hz, 3H), 0.86 (d, J = 6.9 Hz, 2H), 0.83 (d, J = 6.8 Hz, 3H). ¹³ C NMR (151 MHz, acetone) 5 166.20, 144.43, 143.88, 142.45, 133.58, 133.33, 132.76, 132.53, 129.34, 127.16, 125.39, 85.05, 84.13, 80.48, 78.72, 77.24, 71.12, 59.91 , 55.74, 42.32, 41.04, 39.91 , 38.89, 38.22, 32.26, 22.71 , 21.11, 19.85, 18.02, 17.43, 15.21 , 14.18, 10.73, 8.97.

[0127] Comparative Example 5: 19-Deoxy-21 -Methoxy B Bafilomycin A1 (S6)

19-deoxy Bafilomycin 19-deoxy-21-methoxy-Bafilomycin

[0128] ¹ H NMR (599 MHz, acetone) 5 6.68 (d, J = 0.9 Hz, 1 H), 6.64 (dd, J = 15.0, 10.8 Hz, 1 H), 5.94 (dt, J = 9.0, 1.2 Hz, 1 H), 5.81 (d, J = 10.8 Hz, 1 H), 5.20 - 5.14 (m, 2H), 4.03 (dd, J = 10.4, 6.8 Hz, 2H), 3.78 (ddd, J = 10.4, 4.5, 1.7 Hz, 1 H), 3.66 (d, J = 4.5 Hz, 1 H), 3.62 (s, 3H), 3.37 - 3.30 (m, 5H), 3.23 (s, 3H), 2.92 (td, J = 10.4, 4.6 Hz, 1 H), 2.82 - 2.80 (m, 1 H), 2.54 (dqd, J = 9.0, 7.0, 1.9 Hz, 1 H), 2.27 (ddd, J = 12.1 , 4.6, 1.9 Hz, 1 H), 1.97 (d, J = 1.2 Hz, 3H), 1.90 (d, J = 1.3 Hz, 3H), 1.86 (dtt, J = 13.8, 6.9, 3.8 Hz, 1 H), 1.67 (tt, J = 7.3, 5.6 Hz, 1 H), 1 .33 (dtd, J = 9.9, 6.5, 3.5 Hz, 1 H), 1 .05 (d, J = 7.0 Hz, 3H), 0.95 (d, J = 6.8 Hz, 3H), 0.92 (d, J = 6.9 Hz, 3H), 0.88 - 0.84 (m, 8H), 0.78 (d, J = 6.8 Hz, 3H). ¹³ C NMR (151 MHz, acetone) 5 166.67, 144.78, 144.19, 142.31 , 133.84, 133.18, 132.64, 127.25, 125.33, 85.09, 83.98, 83.73, 80.47, 77.63, 77.48, 70.41 , 59.95, 56.16, 55.69, 42.28, 41.30, 40.95, 40.10, 38.72, 38.09, 35.57, 22.48, 21.51 , 20.09, 17.89, 14.74, 14.19, 12.61 , 10.61, 8.86.

[0129] Comparative Example 6: Synthesis of 19-Deoxy-21 -allylester Bafilomycin A1 (S42)

[0130] To a solution of S5 (7.4 mg, 12 μmol) in DCM (2 mL) was added crotonic anhydride (4.7 mg, 30 μmol, 2.5 eq.) and DBU (5.5 μL, 36 μmol, 3 eq.) at 0°C. The reaction was allowed to warm to room temperature and proceed for 16 h. The reaction was diluted to ~20 mL with DCM and washed with an equal volume of a 3% (w/v) citric acid solution, followed by PBS buffer (pH 7.4) and brine. The organic layer was dried in vacuo and dissolved in HPLC acetone (300 μL). Purification was accomplished using a semi-preparative reversed phase C18 (2) column (Phenomenex, 250 x 10 mm), running a gradient of water (solvent A) and acetonitrile (solvent B) at 8 mLmin-1 . The gradient was run using an initial 60% Solvent B isocratic step for 2 min, followed by a linear increasing gradient from 60% to 95% solvent B over 38 min (40 min total). This was followed by a second isocratic step using 95% solvent B for 20 min (60 min total). The compound of interest eluted at -45.5 min, and dried in vacuo yielding 3.2 mg (41 %) of a white solid. Subsequent NMR characterization identified the compound as 19-deoxy bafilomycin A1 allyl ester, suggesting a spontaneous conversion from the crotonyl ester to the allyl ester during derivatization. ¹ H NMR (599 MHz, acetone) 5 6.68 (d, J = 0.9 Hz, 1 H), 6.64 (dd, J = 15.0, 10.8 Hz, 1 H), 6.03 - 5.86 (m, 2H), 5.80 (d, J = 10.8 Hz, 1 H), 5.21 - 5.10 (m, 4H), 4.63 (td, J = 10.7, 4.8 Hz, 1 H), 4.04 (d, J = 8.4 Hz, 1 H), 4.02 (d, J = 5.1 Hz, 1 H), 3.77 (dtd, J = 9.0, 4.5, 1 .6 Hz, 1 H), 3.70 (d, J = 4.6 Hz, 1 H), 3.63 (s, 3H), 3.45 (ddd, J = 11.5, 8.3, 1.9 Hz, 1 H), 3.32 (ddd, J = 7.4, 5.7, 2.0 Hz, 1 H), 3.23 (s, 3H), 3.17 - 3.08 (m, 2H), 2.94 (dd, J = 10.0, 2.2 Hz, 1 H), 2.54 (tt, J = 8.1, 6.1 Hz, 1 H), 2.13 (ddd, J = 12.1 , 4.8, 2.0 Hz, 1 H), 1.97 (d, J = 1.2 Hz, 3H), 1.91 (d, J = 1.2 Hz, 3H), 1.88 (qd, J = 6.8, 5.7, 3.8 Hz, 2H), 1.72 - 1.66 (m, 1 H), 1.56 (tq, J = 10.2, 6.5 Hz, 1 H), 1.19 (q, J = 11.5 Hz, 1 H), 1.05 (d, J = 7.0 Hz, 3H), 0.97 (d, J = 6.9 Hz, 3H), 0.93 (d, J = 6.9 Hz, 3H), 0.87 (d, J = 6.9 Hz, 3H), 0.83 (d, J = 7.1 Hz, 3H), 0.81 (dd, J = 6.7, 3.4 Hz, 6H). ¹³ C NMR (151 MHz, acetone) 5 171.29, 166.72, 144.83, 144.23, 142.29, 133.88, 133.23, 132.66, 132.02, 127.25, 125.33, 118.28, 84.74, 83.95, 80.48, 77.63, 77.22, 77.20, 70.36, 59.98, 55.69, 42.29, 41.33, 40.83, 39.74, 39.02, 38.69, 38.09, 36.72, 29.18, 22.47, 21.40, 20.11 , 17.88, 14.67, 14.19, 12.53, 10.58, 8.68.

[0132] In a flame dried flask, S5 (7.9 mg, 13 mmol), pentynoic acid (1.9 mg, 20. mmol, 1.5 eq), and N,N'- dicyclohexylcarbodiimide (DCC) (4.0 mg, 20. mmol, 1 .5 eq) was dissolved in dry DCM (260 pL, 0.05 M) at 0 °C. DMAP was added to the solution (tip of spatula approx. 5 mol%) before the reaction was allowed to warm to RT and run for 24 hrs. The reaction was washed with sat. NaHCO3, back extracted with DCM (1 mL x3), dried over Na2SO4, filtered, and concentrate in vacuo to give 8.0 mg of material. The material was purified by RP HPLC to yield 2.5 mg (3.6 mmol, 28%) of product. ¹ H NMR (700 MHz, acetone) 5 6.68 (s, 1 H), 6.64 (dd, J = 15.0, 10.8 Hz, 1 H), 5.94 (d, J = 8.9 Hz, 1 H), 5.80 (d, J = 10.8 Hz, 1 H), 5.21 - 5.14 (m, 2H), 4.66 (td, J = 10.7, 4.8 Hz, 1 H), 4.03 (t, J = 8.4 Hz, 1 H), 4.00 (d, J = 5.6 Hz, 1 H), 3.81 - 3.75 (m, 1 H), 3.69 (d, J = 4.6 Hz, 1 H), 3.63 (s, 3H), 3.45 (ddd, J = 10.6, 8.4, 1.9 Hz, 1 H), 3.32 (p, J = 3.3, 2.6 Hz, 1 H), 3.23 (s, 3H), 2.94 (dd, J = 10.0, 2.2 Hz, 1 H), 2.62 - 2.51 (m, 3H), 2.49 (td, J = 6.8, 2.0 Hz, 2H), 2.38 (t, J = 2.6 Hz, 1 H), 2.13 (ddd, J = 12.1 , 4.9, 1.9 Hz, 1 H), 1.97 (d, J = 1.3 Hz, 3H), 1.90 (s, 3H), 1.89 - 1.85 (m, 2H), 1.69 (p, J = 7.3 Hz, 1 H), 1.56 (ddt, J = 16.9, 10.3, 6.5 Hz, 1 H), 1 .20 (q, J = 11 .5 Hz, 1 H), 1 .05 (d, J = 7.0 Hz, 3H), 0.97 (d, J = 6.8 Hz, 3H), 0.93 (d, J = 6.8 Hz, 3H), 0.87 (d, J = 6.9 Hz, 3H), 0.84 - 0.80 (m, 9H). ¹³ C NMR (176 MHz, acetone) 5 171.92, 166.88, 144.96, 144.37, 142.46, 134.02, 133.38, 132.82, 127.42, 125.50, 84.94, 84.12, 83.64, 80.65, 77.80, 77.48, 77.37, 70.56, 70.53, 60.14, 55.85, 55.11 , 42.46, 41.47, 41.00, 39.13, 38.86, 38.26, 36.92, 34.40, 29.35, 22.63, 21.56, 20.25, 18.03, 15.03, 14.83, 14.33, 12.75, 10.74, 8.85.

[0133] Bafilomycin esterification general procedure.

[0134] DMAP, EDC, and carboxylic acid were added to a solution of bafilomycin Ai in DCM under anhydrous conditions. The reaction was stirred overnight then concentrated under reduced pressure. The crude reaction mix was purified by preparative TLC (pTLC).

[0135] Comparative Example 8: Synthesis of 21-Acetyl Bafilomycin (S9)

[0136] Amounts: DMAP (6.47 mg, 53.0 μmol, 2.1 eq), EDC (13.9 mg, 55.5 μmol, 2.2 eq), acetic acid (1.0 eq, approximately 0.25 mL of a 1.44 μg/mL solution), bafilomycin Ai (15.0 mg, 25.2 μmol), and DCM (2.92 mL). Purified by pTLC (20% acetone in hexanes) to yield the product. 21-Acetyl Bafilomycin ¹ H NMR (599 MHz, Acetone) 5 6.71 (d, J = 0.8 Hz, 1 H), 6.68 (dd, J = 15.0, 10.8 Hz, 1 H), 5.96 (dt, J = 8.9, 1 .2 Hz, 1 H), 5.80 (d, J = 10.9 Hz, 1 H), 5.38 (d, J = 2.1 Hz, 1 H), 5.14 (dd, J = 15.0, 9.2 Hz, 1 H), 4.97 (dd, J = 8.4, 1.4 Hz, 1 H), 4.91 (td, J = 10.9, 4.8 Hz, 1 H), 4.76 (dd, J = 4.4, 1.1 Hz, 1 H), 4.18 (ddd, J = 10.7, 4.4, 1.9 Hz, 1 H), 4.09 - 4.03 (m, 2H), 3.64 (s, 3H), 3.58 (dd, J = 10.4, 2.2 Hz, 1 H), 3.31 (td, J = 6.5, 5.7, 2.0 Hz, 1 H), 3.24 (s, 3H), 2.55 (tt, J = 9.2, 6.5 Hz, 1 H), 2.25 (dd, J = 11.8, 4.9 Hz, 1 H), 2.20 - 2.12 (m, 1 H), 2.08 - 1.99 (m, 2H), 2.00 (s, 3H), 1.98 (d, J = 1.2 Hz, 3H), 1.97 - 1.84 (m, 5H), 1.83 (dt, J = 8.8, 6.4 Hz, 1 H), 1.52 (tdd, J = 13.0, 8.5, 5.2 Hz, 1 H), 1.21 (td, J = 11.5, 2.1 Hz, 1 H), 1.05 (d, J = 7.0 Hz, 3H), 0.99 (d, J = 7.2 Hz, 3H), 0.94 (d, J = 6.8 Hz, 3H), 0.92 (d, J = 6.8 Hz, 3H), 0.87 (d, J = 6.9 Hz, 3H), 0.81 (d, J = 3.7 Hz, 3H), 0.80 (d, J = 4.1 Hz, 3H). ¹³ C NMR (151 MHz, Acetone) 5 170.62, 167.80, 145.72, 144.83, 141.95, 134.45, 134.20, 132.78, 127.02, 125.23, 99.71 , 83.36, 80.41 , 77.41 , 76.57, 74.14, 71.57, 60.17, 55.67, 42.96, 42.27, 41.70, 40.78, 38.99, 38.29, 38.01 , 28.74, 22.25, 21.62, 21.07, 20.35, 17.73, 14.59, 14.15, 12.53, 10.30, 7.36.

[0137] Comparative Example 9: Synthesis of 7,21 -Diacetyl Bafilomycin Ai

[0138] Amounts: DMAP (6.0 mg, 50. μmol, 2.1 eq), EDC (9.9 mg, 52 μmol, 2.2 eq), acetic acid (2.7 μL, 47 μL, 2.0 eq), 21 -acetyl bafilomycin Ai (15.0 mg, 24 μmol), and DCM (2.9 mL). Purified by pTLC (20% acetone in hexanes) to yield the product. Diacetyl Baf ¹ H NMR (599 MHz, Acetone) 56.72 (dd, J= 15.0, 10.6 Hz, 1H), 6.70 (d, J = 0.9 Hz, 1H), 5.91 (d, J= 10.7 Hz, 1H), 5.81 (dp, J = 9.0, 1.2 Hz, 1H), 5.36 (d, J = 2.2 Hz, 1H), 5.24 (dd, J = 15.1, 9.2 Hz, 1H), 4.99 (dd, J = 8.3, 1.5 Hz, 1H), 4.92 (td, J= 10.9, 4.8 Hz, 1H), 4.74 (dd, J= 6.5, 2.5 Hz, 1H), 4.71 (dd, J= 4.4, 1.1 Hz, 1H), 4.19 (ddd, J= 10.7, 4.4, 1.9 Hz, 1H), 4.09 (t, J = 8.7 Hz, 1H), 3.65 (s, 3H), 3.58 (dd, J= 10.4, 2.2 Hz, 1H), 3.25 (s, 3H), 2.80-2.74 (m, 1H), 2.26 (dd, J= 11.8, 4.8 Hz, 1H), 2.21 -2.15 (m, 1H), 2.15 (s, 3H), 2.04-2.01 (m, 1H), 2.00 (d, J= 1.7 Hz, 6H), 1.93 (td, J= 6.8, 2.2 Hz, 1H), 1.91 (d, J= 1.3 Hz, 3H), 1.86-1.81 (m, 1H), 1.73 (dd, J= 14.9, 11.5 Hz, 1H), 1.53 (tq, J = 10.4, 6.5 Hz, 1H), 1.21 (td, J= 11.5, 2.1 Hz, 1 H), 1.04 (d, J = 6.9 Hz, 3H), 0.99 (d, J = 7.2 Hz, 3H), 0.95 (d, J = 6.8 Hz, 3H), 0.92 (d, J = 7.0 Hz, 3H), 0.89 (d, J = 6.9 Hz, 3H), 0.81 (d, J = 2.5 Hz, 3H), 0.80 (d, J= 2.8 Hz, 3H). ¹³ C NMR (151 MHz, Acetone) 5171.53, 170.60, 167.58, 143.39, 142.72, 142.01, 133.93, 133.20, 128.29, 126.00, 99.71, 83.35, 81.87, 77.59, 76.59, 74.13, 71.57, 60.16, 55.80, 42.99, 42.05, 40.79, 39.29, 39.00, 38.38, 36.98, 28.74, 22.36, 21.63, 21.25, 21.07, 20.63, 17.16, 14.60, 14.12, 12.54, 10.37, 7.36.

[0139] Comparative Example 10: Synthesis of 21-Pentanoate Bafilomycin Ai (S10)

[0140] Amounts: DMAP (4.9 mg, 41 μmol, 2.1 eq), EDC (8.1 mg, 42 μmol, 2.2 eq), pentanoic acid (3.9 mg, 39 μmol, 2 eq), bafilomycin Ai (12 mg, 20 μmol), and DCM (2.5 mL). Purified by pTLC (20% acetone in hexanes) to yield the product.21-pentanone Bafilomycin ¹ H NMR (599 MHz, Acetone) 56.71 (s, 1H), 6.68 (dd, J = 15.0, 10.8 Hz, 1H), 5.96 (d, J= 8.8 Hz, 1H), 5.80 (d, J= 10.8 Hz, 1H), 5.38 (d, J= 2.1 Hz, 1H), 5.15 (dd, J= 15.0, 9.2 Hz, 1H), 4.97 (dd, J = 8.4, 1.4 Hz, 1H), 4.93 (td, J = 10.8, 4.8 Hz, 1H), 4.76 (dd, J = 4.3, 1.0 Hz, 1H), 4.18 (ddd, J = 10.7, 4.3, 1.8 Hz, 1H), 4.09-4.03 (m, 2H), 3.64 (s, 3H), 3.59 (dd, J= 10.4, 2.2 Hz, 1H), 3.31 (ddd, J= 7.2, 5.5, 2.0 Hz, 1 H), 3.24 (s, 3H), 2.56 (pd, J = 7.0, 3.5 Hz, 1 H), 2.30 (td, J = 7.5, 4.0 Hz, 2H), 2.25 (dd, J = 11.7, 4.8 Hz, 1H), 2.21 -2.12 (m, 1H), 2.09-1.98 (m, 2H), 1.98 (d, J= 1.2 Hz, 3H), 1.93 (s, 3H), 1.89 (dtd, J = 18.0, 7.0, 3.1 Hz, 2H), 1.83 (dd, J = 8.1 , 6.3 Hz, 1 H), 1.63 - 1.55 (m, 2H), 1.58 - 1.49 (m, 1 H), 1.35 (h, J = 7.4 Hz, 2H), 1.21 (td, J = 11.5, 2.1 Hz, 1 H), 1.05 (d, J = 7.0 Hz, 3H), 0.99 (d, J = 7.1 Hz, 3H), 0.95 (d, J = 6.9 Hz, 3H), 0.92 (d, J = 5.0 Hz, 3H), 0.91 (t, J = 6.4 Hz, 3H), 0.88 (d, J = 6.8 Hz, 3H), 0.81 (d, J = 3.7 Hz, 3H), 0.80 (d, J = 3.9 Hz, 3H). ¹³ C NMR (151 MHz, Acetone) 5173.28, 167.82, 145.71, 144.82, 141.95, 134.45, 134.20, 132.79, 127.04, 125.24, 99.72, 83.34, 80.42, 77.42, 76.57, 73.97, 71.58, 60.17, 55.67, 42.95, 42.27, 41.70, 40.82, 39.00, 38.28, 38.01, 34.66, 28.75, 27.90, 22.88, 22.25, 21.63, 20.36, 17.73, 14.60, 14.16, 14.01, 12.58, 10.30, 7.38.

[0141] Comparative Example 11: Synthesis of 21 -Nonanoate Bafilomycin Ai (S11)

[0142] Amounts: DMAP (4.9 mg, 41 μmol, 2.1 eq), EDC (8.1 mg, 42 μmol, 2.2 eq), nonanoic acid (6.10 mg, 38.5 μmol, 2 eq), bafilomycin Ai (12 mg, 20 μmol), and DCM (2.5 mL). Purified by pTLC (20% acetone in hexanes) to yield the product.21-nonanoate Bafilomycin ¹ H NMR (599 MHz, CDCI3) 56.67 (s, 1H), 6.50 (dd, J = 15.0, 10.6 Hz, 1H), 5.81 (d, J= 10.6 Hz, 1H), 5.77 (d, J = 9.1 Hz, 1H), 5.47 (d, J = 2.0 Hz, 1H), 5.16 (dd, J = 15.0, 9.3 Hz, 1H), 5.01 -4.93 (m, 2H), 4.61 (d, J = 4.1 Hz, 1H), 4.15-4.09 (m, 1H), 3.88 (t, J = 9.0Hz, 1H), 3.64 (s, 3H), 3.60 (dd, J= 10.4, 2.2 Hz, 1H), 3.30 (dd, J = 7.1, 3.9 Hz, 1H), 3.24 (s, 3H), 2.58-2.50 (m, 1H), 2.32 (dd, J= 11.8, 4.8 Hz, 1H), 2.28 (t, J= 7.5 Hz, 2H), 2.13 (td, J = 13.6, 12.3, 5.3 Hz, 2H), 1.99 (s, 3H), 1.94 (s, 4H), 1.93-1.84 (m, 2H), 1.75 (q, J = 7.2 Hz, 1H), 1.62 (q, J = 7.2, 5.2 Hz, 2H), 1.54 (q, J= 4.1 Hz, 1H), 1.34-1.23 (m, 12H), 1.17 (td, J= 11.4, 2.1 Hz, 1H), 1.10 (s, OH), 1.07 (d, J = 7.0 Hz, 3H), 1.02 (d, J = 7.1 Hz, 3H), 0.94 (d, J = 6.4 Hz, 3H), 0.91 (d, J = 6.8 Hz, 2H), 0.88 (t, J = 6.9 Hz, 3H), 0.82 (t, J = 6.5 Hz, 6H), 0.77 (d, J = 6.7 Hz, 3H). ¹³ C NMR (151 MHz, CDCI3) 5173.18, 167.28, 142.98, 142.70, 141.29, 133.48, 133.02, 132.98, 127.26, 125.31, 98.81, 82.23, 81.22, 76.85, 75.58, 73.60, 70.61, 59.94, 55.52, 42.06, 41.21, 40.20, 40.01, 38.21, 37.17, 36.69, 34.74, 31.81, 29.25-29.13 (m), 27.92, 25.16, 22.65, 21.66, 21.10, 20.17, 17.28, 14.29, 14.07 (d, J= 9.3 Hz), 12.30, 9.84, 7.08.

[0143] Comparative Example 12: Synthesis of 21-penta-3-enoate Bafilomycin A1 (S29)

[0144] Amounts: DMAP (5.4 mg, 44 μmol, 2.1 eq), EDC (8.8 mg, 46 μmol, 2.2 eq), (E)-pent-3-enoic acid (4.2 mg, 42 μmol, 2 eq), bafilomycin A1 (13 mg, 21 μmol), and DCM (2.4 mL). Purified by pTLC (20% acetone in hexanes) to yield the product.21-pent-3-enoate Bafilomycin ¹ H NMR (599 MHz, Acetone) 56.71 (s, 1H), 6.68 (dd, J = 14.9, 10.8 Hz, 1H), 5.96 (d, J = 8.8 Hz, 1H), 5.80 (d, J = 10.7 Hz, 1H), 5.65-5.51 (m, 2H), 5.38 (d, J = 2.1 Hz, 1H), 5.14 (dd, J = 15.0, 9.1 Hz, 1H), 4.97 (dd, J = 8.4, 1.5 Hz, 1H), 4.92 (td, J = 10.9, 4.8 Hz, 1H), 4.76 (dd, J = 4.3, 1.2 Hz, 1H), 4.18 (ddd, J= 10.8, 4.4, 1.8 Hz, 1H), 4.10-4.03 (m, 2H), 3.64 (d, J= 1.2 Hz, 3H), 3.58 (dd, J = 10.4, 2.2 Hz, 1 H), 3.35 - 3.28 (m, 1 H), 3.24 (d, J = 1.2 Hz, 3H), 3.06 - 2.96 (m, 2H), 2.84 (s, 1 H), 2.60 - 2.52 (m, 1 H), 2.25 (dd, J = 11.7, 4.8 Hz, 1 H), 2.20 - 2.12 (m, 1 H), 2.02 (d, J = 11.0 Hz, 2H), 1.98 (d, J = 1.4 Hz, 3H), 1.93 (s, 3H), 1.96 - 1.86 (m, 1 H), 1.83 (q, J = 7.1 Hz, 1 H), 1.68 - 1.64 (m, 3H), 1.59 - 1.49 (m, 1 H), 1.25 - 1.18 (m, 1H), 1.05 (d, J= 6.9 Hz, 3H), 0.99 (d, J = 7.2 Hz, 3H), 0.95 (d, J= 6.8 Hz, 3H), 0.92 (d, J = 6.7 Hz, 3H), 0.88 (d, J= 6.8 Hz, 3H), 0.80 (dd, J = 6.7, 1.5 Hz, 6H). ¹³ C NMR (151 MHz, Acetone) 5171.66, 167.81, 145.71, 144.82, 141.95, 134.45, 134.20, 132.79, 129.21, 127.03, 125.24, 124.41, 99.72, 83.34, 80.42, 77.41, 76.55, 74.33, 71.57, 60.17, 55.67, 42.94, 42.27, 41.70, 40.76, 39.02, 38.67, 38.28, 38.01, 28.73, 22.25, 21.62, 20.36, 18.00, 17.73, 14.60, 14.16, 12.53, 10.29, 7.38.

[0145] Comparative Example 13: Synthesis of 21-penta-2,4-dienoate Bafilomycin Ai (S12)

[0146] Amounts: DMAP (6.2 mg, 51 μmol, 2.1 eq), EDC (10 mg, 53 μmol, 2.2 eq), (E)-penta-2,4-dienoic acid (4.7 mg, 48 μmol, 2 eq), bafilomycin Ai (15 mg, 24 μmol), and DCM (2.9 mL). Purified by pTLC (20% acetone in hexanes) to yield the product.21-penta-2,4-dienoate Bafilomycin ¹ H NMR (599 MHz, Acetone) 57.27 (dd, J = 15.4, 11.0 Hz, 1H), 6.72 (d, J= 1.0 Hz, 1H), 6.68 (dd, J= 15.0, 10.8 Hz, 1H), 6.58 (dt, J = 17.0, 10.5 Hz, 1H), 6.02-5.96 (m, 1H), 5.96 (d, J = 9.1 Hz, 1H), 5.80 (d, J= 10.8 Hz, 1H), 5.73-5.67 (m, 1H), 5.52 (dd, J= 10.2, 1.6 Hz, 1H), 5.41 (t, J= 1.8 Hz, 1H), 5.15 (dd, J = 15.0, 9.2 Hz, 1H), 5.01 (td, J= 10.9, 4.8 Hz, 1H), 4.97 (dd, J = 8.3, 1.5 Hz, 1H), 4.77 (dd, J = 4.4, 1.1 Hz, 1H), 4.19 (ddd, J = 10.8, 4.5, 1.8 Hz, 1H), 4.09-4.03 (m, 2H), 3.64 (d, J = 1.3 Hz, 3H), 3.63 - 3.59 (m, 1 H), 3.31 (td, J = 6.4, 5.5, 1.7 Hz, 1 H), 3.24 (d, J = 1.3 Hz, 3H), 2.56 (p, J = 7.9, 7.5 Hz, 1H), 2.30 (dd, J= 11.8, 4.9 Hz, 1H), 2.21 - 2.13 (m, 1H), 2.02 (s, 2H), 1.98 (d, J = 1.3 Hz, 3H), 1.94 (s, 1 H), 1.93 (d, J = 1.4 Hz, 3H), 1.85 (q, J = 7.3 Hz, 1 H), 1.60 (td, J = 10.4, 6.4 Hz, 1 H), 1.30 - 1.23 (m, 1 H), 1.05 (d, J = 6.9 Hz, 3H), 1.00 (d, J = 7.1 Hz, 3H), 0.96 (d, J = 6.8 Hz, 2H), 0.92 (d, J = 6.7 Hz, 3H), 0.88 (d, J = 6.8 Hz, 3H), 0.82 (dd, J= 6.7, 3.5 Hz, 6H). ¹³ C NMR (151 MHz, Acetone) 5167.82, 166.52, 145.71, 145.34, 144.82, 141.95, 135.91, 134.45, 134.21, 132.79, 127.04, 125.95, 125.24, 123.44, 99.75, 83.35, 80.42, 77.42, 76.56, 74.38, 71.59, 60.17, 55.67, 42.98, 42.27, 41.70, 40.82, 39.09, 38.29, 38.01, 28.76, 22.25, 21.63, 20.36, 17.73, 14.61, 14.16, 12.58, 10.30, 7.37.

[0147] 21-benzoate Bafilomycin Ai (S13). Amounts: DMAP (4.9 mg, 41 μmol, 2.1 eq), EDC (8.1 mg, 42 μmol, 2.2 eq), benzoic acid (4.71 mg, 38.5 μmol, 2 eq), bafilomycin Ai (12 mg, 20 μmol), and DCM (2.4 mL). Purified by pTLC (20% acetone in hexanes) to yield 8.0 mg of a white solid (57%).21-benzyl Bafilomycin ¹ H NMR (599 MHz, Acetone) 58.07 - 8.02 (m, 2H), 7.66 - 7.61 (m, 1 H), 7.56 - 7.49 (m, 2H), 6.72 (d, J = 0.9 Hz, 1H), 6.68 (dd, J= 15.0, 10.8 Hz, 1H), 5.97 (dt, J = 8.9, 1.2 Hz, 1H), 5.81 (d, J= 10.8 Hz, 1H), 5.45 (d, J = 2.0 Hz, 1H), 5.20 (dt, J= 10.8, 5.4 Hz, 1H), 5.15 (dd, J= 14.8, 9.0 Hz, 1H), 4.98 (dd, J= 8.4, 1.4 Hz, 1H), 4.79 (dd, J = 4.3, 1.0 Hz, 1H), 4.21 (ddd, J= 10.7, 4.4, 1.8 Hz, 1H), 4.10-4.03 (m, 2H), 3.68 (dd, J= 10.4, 2.3 Hz, 1H), 3.65 (s, 3H), 3.31 (td, J = 6.8, 6.1, 2.1 Hz, 1H), 3.24 (s, 3H), 2.56 (tt, J = 9.0, 7.0 Hz, 1H), 2.42 (dd, J= 11.8, 4.9 Hz, 1 H), 2.22 - 2.13 (m, 1 H), 2.03 (d, J = 11.0 Hz, 2H), 1.99 (d, J = 1.2 Hz, 3H), 1.99 - 1.95 (m, 1 H), 1.93 (d, J = 1.2 Hz, 3H), 1.88 (qd, J = 7.5, 2.7 Hz, 2H), 1.76 (ddt, J= 16.9, 10.4, 6.5 Hz, 1H), 1.39 (td, J= 11.5, 2.0 Hz, 1H), 1.06 (d, J = 7.0 Hz, 3H), 1.02 (d, J = 7.1 Hz, 3H), 0.98 (d, J = 6.8 Hz, 3H), 0.92 (d, J = 6.8 Hz, 3H), 0.89 (dd, J = 6.7, 1.9 Hz, 6H), 0.85 (d, J= 6.8 Hz, 3H). ¹³ C NMR (151 MHz, Acetone) 5167.8, 166.4, 145.7, 144.8, 142.0, 134.5, 134.2, 133.8, 132.8, 131.7, 130.2, 129.4, 127.1, 125.3, 99.8, 83.4, 80.4, 77.4, 76.6, 75.3, 71.6, 60.2, 55.7, 43.0, 42.3, 41.7, 40.8, 39.2, 38.3, 38.0, 28.8, 22.3, 21.7, 20.4, 17.7, 14.7, 14.2, 12.7, 10.3, 7.4. LC-MS (IT): calcd for C42H62O10 [MAOH ₃ OH+H] ⁺ 759.6; found, 760.5.

[0148] Comparative Example 14: Synthesis of 21-penta-4-ene Bafilomycin

Bafilomycin A ₁ 21-penta-4-ene Bafilomycin

[0149] 21-pent-4-ene Baf 1H NMR (599 MHz, Acetone) 56.74 (s, 1H), 6.71 (dd, J = 15.0, 10.9 Hz, 1H), 5.99 (d, J = 8.8 Hz, 1H), 5.93-5.81 (m, 2H), 5.41 (d, J = 2.2 Hz, 1H), 5.17 (dd, J = 15.1, 9.3 Hz, 1H), 5.09 (dt, J = 17.1, 1.7 Hz, 1H), 5.02- 4.94 (m, 3H), 4.79 (d, J = 4.3 Hz, 1H), 4.21 (dd, J = 11.1, 4.1 Hz, 1H), 4.12-4.06 (m, 2H), 3.67 (d, J = 1.4 Hz, 3H), 3.61 (d, J = 10.6 Hz, 1H), 3.36-3.31 (m, 1H), 3.27 (d, J = 1.4 Hz, 3H), 2.81 (d, J = 1.4 Hz, 1 H), 2.62 - 2.53 (m, 1 H), 2.43 (dd, J = 8.0, 5.3 Hz, 2H), 2.40 - 2.32 (m, 3H), 2.28 (dd, J = 11.7, 4.6 Hz, 1H), 2.19 (dq, J = 13.6, 7.1 Hz, 1H), 2.04 (s, 1H), 2.01 (d, J = 1.4 Hz, 3H), 1.96 (s, 4H), 1.86 (t, J = 7.3 Hz, 1H), 1.57 (h, J = 8.9, 8.2 Hz, 1H), 1.24 (t, J = 11.4 Hz, 1H), 1.08 (d, J = 7.2 Hz, 3H), 1.01 (dd, J = 7.1, 1.4 Hz, 3H), 0.98 (d, J = 7.0 Hz, 2H), 0.95 (d, J = 6.8 Hz, 2H), 0.93 - 0.88 (m, 4H), 0.86 - 0.81 (m, 7H). 13C NMR (151 MHz, Acetone) 5172.63, 167.82, 145.72, 144.83, 141.96, 138.03, 134.46, 134.21, 132.79, 127.04, 125.25, 115.64, 99.72, 83.35, 80.43, 77.42, 76.58, 74.21, 71.58, 60.17, 55.67, 42.96, 42.28, 41.71, 40.84, 38.98, 38.28, 38.01, 34.27, 28.75, 22.25, 21.63, 20.36, 17.73, 14.60, 14.16, 12.60, 10.30, 7.38.

[0150] Comparative Example 15: Synthesis of 21-(3-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)propanoate)

Bafilomycin

[0151] ¹ H NMR (599 MHz, Acetone) 56.71 (d, J= 0.9 Hz, 1H), 6.68 (dd, J= 15.0, 10.8 Hz, 1H), 5.96 (dt, J = 8.8, 1.2 Hz, 1H), 5.83-5.78 (m, 1H), 5.39 (d, J = 2.1 Hz, 1H), 5.14 (dd, J= 15.0, 9.2 Hz, 1H), 4.99-4.94 (m, 1 H), 4.97 - 4.91 (m, 1 H), 4.76 (dd, J = 4.4, 1.1 Hz, 1 H), 4.18 (ddd, J = 10.7, 4.4, 1.9 Hz, 1 H), 4.09 - 4.02 (m, 2H), 3.64 (s, 3H), 3.58 (dd, J= 10.4, 2.2 Hz, 1H), 3.31 (ddd, J = 7.3, 5.5, 2.0 Hz, 1H), 3.24 (s, 3H), 2.60-2.51 (m, 1 H), 2.39 (t, J = 2.7 Hz, 1 H), 2.28 (dd, J = 11.8, 4.8 Hz, 1H), 2.18 (td, J = 7.5, 3.5 Hz, 2H), 2.15 (ddt, J = 6.7, 4.3, 2.0 Hz, 1 H), 2.09 - 2.05 (m, 2H), 2.05 - 2.00 (m, 2H), 1.98 (d, J = 1.2 Hz, 3H), 1.96 - 1.85 (m, 4H), 1.85 - 1.81 (m, 1H), 1.81 -1.77 (m, 2H), 1.66 (t, J= 7.5 Hz, 2H), 1.54 (tdd, J = 13.1, 8.6, 5.3 Hz, 1H), 1.23 (td, J= 11.5, 2.1 Hz, 1H), 1.05 (d, J= 7.0 Hz, 3H), 0.99 (d, J = 7.2 Hz, 3H), 0.95 (d, J = 6.8 Hz, 3H), 0.92 (d, J= 6.8 Hz, 3H), 0.87 (dd, J= 7.0, 2.8 Hz, 3H), 0.82 (d, J = 6.5 Hz, 3H), 0.80 (d, J= 6.8 Hz, 3H). ¹³ C NMR (151 MHz, Acetone) 5 172.27, 167.95, 145.85, 144.96, 142.08, 134.59, 134.34, 132.92, 127.17, 125.37, 99.86, 83.66, 83.47, 80.56, 77.55, 76.70, 74.75, 71.71, 70.73, 60.30, 55.80, 43.08, 42.41, 41.84, 40.87, 39.11, 38.40, 38.14, 33.10, 29.22, 28.87, 28.83, 28.70, 22.39, 21.76, 20.50, 17.86, 14.73, 14.30, 13.72, 12.72, 10.43, 7.53.

[0152] Comparative Example 16: Synthesis of 21-Pent-4-ynoate-Bafilomycin A1

Bafilomycin A ₁ 21 -pent-4-y noate bafilomycin

[0153] ¹ H NMR (599 MHz, Acetone) 56.71 (s, 1H), 6.68 (dd, J= 15.0, 10.8 Hz, 1H), 5.96 (d, J = 8.9 Hz, 1H), 5.80 (d, J = 10.9 Hz, 1H), 5.39 (d, J = 2.1 Hz, 1H), 5.14 (dd, J= 15.0, 9.2 Hz, 1H), 5.01 -4.93 (m, 2H), 4.76 (dd, J= 4.4, 1.1 Hz, 1H), 4.18 (ddd, J= 10.7, 4.4, 1.8 Hz, 1H), 4.09-4.03 (m, 2H), 3.64 (d, J = 0.7 Hz, 3H), 3.59 (dd, J = 10.3, 2.3 Hz, 1 H), 3.31 (td, J = 6.3, 5.4, 1.8 Hz, 1 H), 3.24 (d, J = 0.7 Hz, 3H), 2.59 - 2.51 (m, 3H), 2.51 - 2.44 (m, 2H), 2.37 (t, J = 2.6 Hz, 1H), 2.27 (dd, J= 11.8, 4.8 Hz, 1H), 2.16 (dd, J= 10.5, 6.9 Hz, 1H), 2.09-2.00 (m, 2H), 1.98 (d, J= 1.2 Hz, 3H), 1.93 (s, 3H), 1.93-1.86 (m, 2H), 1.84 (q, J= 7.7, 7.1 Hz, 1H), 1.55 (tdd, J= 12.9, 8.4, 5.2 Hz, 1 H), 1.24 (td, J = 11.5, 2.1 Hz, 1 H), 1.05 (d, J = 7.0 Hz, 3H), 0.99 (d, J = 7.1 Hz, 3H), 0.95 (d, J = 6.9 Hz, 3H), 0.92 (d, J = 6.8 Hz, 3H), 0.88 (d, J= 6.8 Hz, 3H), 0.83 (d, J = 6.5 Hz, 3H), 0.80 (d, J= 6.8 Hz, 3H). ¹³ C NMR (151 MHz, Acetone) 5171.65, 167.82, 145.72, 144.83, 141.95, 134.46, 134.21, 132.79, 127.03, 125.24, 99.73, 83.51, 83.35, 80.42, 77.41, 76.58, 74.60, 71.58, 70.40, 60.17, 55.67, 42.95, 42.27, 41.71, 40.79, 38.98, 38.29, 38.01, 34.26, 28.74, 22.25, 21.63, 20.36, 17.73, 14.88, 14.59, 14.16, 12.59, 10.30, 7.38.

[0154] Comparative Example 17: Synthesis of 21 -pent-4-ynoate-7-(3-(trifluoromethyl)-3H-diazirin-3- yl)benzoate Bafilomycin

[0155] ¹ H NMR (599 MHz, Acetone) 58.21 -8.16 (m, 2H), 7.47 (d, J = 8.2 Hz, 2H), 6.79 (s, 1H), 6.69 (dd, J =

15.1, 10.6 Hz, 1H), 6.05 (d, J = 9.0 Hz, 1H), 5.96 (d, J= 10.6 Hz, 1H), 5.38 (d, J = 2.0 Hz, 1H), 5.28 (dd, J = 15.0, 9.1 Hz, 1H), 5.02 (dd, J = 8.1, 1.4 Hz, 1H), 4.98 (td, J= 11.0, 4.9 Hz, 1H), 4.95 (dd, J= 6.7, 2.3 Hz, 1H), 4.72 (d, J= 4.4 Hz, 1H), 4.22 (ddd, J= 10.7, 4.5, 1.8 Hz, 1H), 4.09 (t, J = 8.6 Hz, 1H), 3.68 (s, 3H), 3.60 (dd, J = 10.3, 2.2 Hz, 1H), 3.31 (d, J= 3.5 Hz, 1H), 3.22 (s, 3H), 3.12 - 3.08 (m, 1H), 2.94 (tt, J = 9.2, 7.0 Hz, 1H), 2.78 (s, 1 H), 2.58 - 2.52 (m, 2H), 2.51 - 2.45 (m, 2H), 2.38 (t, J = 2.6 Hz, 1 H), 2.28 (dd, J = 11.8, 4.8 Hz, 1 H), 2.25 - 2.13 (m, 3H), 2.06 (s, 3H), 1.98-1.90 (m, 1H), 1.90-1.83 (m, 1H), 1.77 (dd, J = 15.3, 11.5 Hz, 1H), 1.62-1.53 (m, 1H), 1.52 (s, 3H), 1.25 (td, J= 11.5, 2.0 Hz, 1H), 1.14 (d, J = 6.8 Hz, 3H), 1.00 (dd, J = 8.8, 7.0 Hz, 6H), 0.96 (d, J = 6.8 Hz, 3H), 0.93 (d, J = 6.8 Hz, 3H), 0.83 (dd, J = 9.2, 6.6 Hz, 6H). ¹³ C NMR (151 MHz, Acetone) 5 171.65, 167.59, 166.53, 142.98, 142.79, 141.52, 134.33, 134.17, 133.59, 133.17, 132.63, 131.24, 128.72, 127.73, 126.08, 123.85, 122.03, 99.74, 84.12, 83.52, 83.20, 77.80, 76.60, 74.59, 71.59, 70.41, 60.19, 55.76, 42.99, 41.82, 40.79, 39.11, 38.99, 38.45, 37.13, 34.27, 28.75, 22.03, 21.64, 20.67, 17.48, 14.89, 14.61, 14.24, 12.60, 10.43, 7.36.

[0156] Comparative Example 18: Synthesis of 21 -(3-(trifluoromethyl)-3H-diazirin-3-yl)benzoate Bafilomycin

21-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzoate Bafilomycin

[0157] ¹ H NMR (599 MHz, Acetone) 58.18-8.13 (m, 2H), 7.44 (d, J = 8.2 Hz, 2H), 6.72 (d, J= 0.9 Hz, 1H), 6.68 (dd, J= 15.0, 10.8 Hz, 1H), 5.97 (dt, J= 8.9, 1.2 Hz, 1H), 5.81 (d, J= 10.8 Hz, 1H), 5.47 (d, J= 2.0 Hz, 1H), 5.20 (td, J= 10.9, 4.8 Hz, 1H), 5.15 (dd, J= 15.0, 9.2 Hz, 1H), 4.98 (dd, J = 8.4, 1.4 Hz, 1H), 4.79 (dd, J= 4.4, 1.0 Hz, 1 H), 4.21 (ddd, J = 10.7, 4.4, 1.9 Hz, 1 H), 4.09 - 4.03 (m, 2H), 3.70 - 3.62 (m, 1 H), 3.65 (s, 3H), 3.34 - 3.28 (m, 1H), 3.24 (s, 3H), 2.56 (tt, J = 7.2, 5.1 Hz, 1H), 2.41 (dd, J= 11.8, 4.9 Hz, 1H), 2.22 -2.14 (m, 1H), 2.03 (d, J= 11.0 Hz, 2H), 1.99 (d, J= 1.2 Hz, 3H), 1.97 (td, J= 6.7, 2.1 Hz, 1H), 1.93 (d, J= 1.2 Hz, 3H), 1.92-1.85 (m, 2H), 1.81 - 1.71 (m, 1 H), 1.40 (td, J = 11.5, 2.1 Hz, 1 H), 1.06 (d, J = 7.0 Hz, 3H), 1.01 (d, J = 7.2 Hz, 3H), 0.98 (d, J = 6.9 Hz, 3H), 0.92 (d, J = 6.8 Hz, 3H), 0.89 (d, J = 4.2 Hz, 3H), 0.88 (d, J = 3.8 Hz, 3H), 0.85 (d, J = 6.8 Hz, 3H). ¹³ C NMR (151 MHz, Acetone) 5167.97, 165.54, 145.87, 144.97, 142.09, 134.60, 134.37, 133.95, 133.23, 132.93, 131.04, 127.70, 127.17, 125.38, 124.00, 122.18, 99.97, 83.48, 80.56, 77.56, 76.69, 76.12, 71.75, 60.32, 55.81, 49.93, 43.11, 42.41, 41.85, 40.85, 39.24, 38.42, 38.15, 28.92, 22.39, 21.77, 20.50, 17.87, 14.77, 14.30, 12.81, 10.44, 7.52.

[0158] Comparative Example 19: Synthesis of 21 -(3-(trifluoromethyl)-3H-diazirin-3-yl)benzoate-7-pent-4- ynoate-Bafilomycin

[0159] ¹ H NMR (599 MHz, Acetone) 58.18 (dq, J= 8.5, 1.9 Hz, 2H), 7.50-7.45 (m, 2H), 6.81 -6.77 (m, 1H), 6.68 (dd, J = 15.1, 10.6 Hz, 1H), 6.05 (dt, J= 9.0, 1.3 Hz, 1H), 5.96 (d, J = 10.5 Hz, 1H), 5.38 (d, J= 2.1 Hz, 1H), 5.28 (dd, J= 15.0, 9.0 Hz, 1H), 5.02 (dt, J= 8.3, 1.6 Hz, 1H), 4.98 (td, J= 11.0, 4.9 Hz, 1H), 4.95 (dd, J = 6.8, 2.2 Hz, 1 H), 4.74 - 4.70 (m, 1 H), 4.25 - 4.19 (m, 1 H), 4.09 (t, J = 8.7 Hz, 1 H), 3.68 (d, J = 1.6 Hz, 3H), 3.60 (dd, J = 10.4, 2.2 Hz, 1 H), 3.22 (s, 3H), 2.94 (ddd, J = 9.1 , 7.0, 2.2 Hz, 1 H), 2.58 - 2.51 (m, 2H), 2.52 - 2.45 (m, 2H), 2.38 (t, J = 2.6 Hz, 1H), 2.28 (ddd, J= 11.8, 4.9, 1.7 Hz, 1H), 2.26-2.13 (m, 3H), 2.04 (s, 3H), 1.98-1.89 (m, 1 H), 1.86 (q, J = 6.9 Hz, 1 H), 1.77 (dd, J = 15.4, 11.6 Hz, 1 H), 1.62 - 1.53 (m, 1 H), 1.52 (d, J = 1.4 Hz, 3H), 1.25 (td, J= 11.5, 2.0 Hz, 1H), 1.14 (d, J = 6.8 Hz, 3H), 1.02-0.98 (m, 3H), 1.01 -0.96 (m, 3H), 0.96 (dd, J = 6.9, 1.6 Hz, 3H), 0.93 (d, J = 6.9 Hz, 3H), 0.86-0.82 (m, 3H), 0.82 (d, J= 6.8 Hz, 3H). ¹³ C NMR (151 MHz, Acetone) 5 171.66, 167.59, 166.53, 142.98, 142.79, 141.53, 134.33, 134.17, 133.58, 133.17, 132.63, 131.24, 128.71, 127.73, 126.07, 123.85, 122.31, 99.74, 84.12, 83.52, 83.20, 77.80, 76.61, 74.59, 71.59, 70.41, 60.19, 55.76, 42.99, 41.82, 40.79, 39.11, 38.99, 38.45, 37.13, 34.27, 28.75, 22.03, 21.64, 20.67, 17.47, 14.89, 14.61, 14.23, 12.60, 10.43, 7.36.

[0160] Comparative Example 20: Synthesis of 21 -Methoxy CMC

[0161] CMC (55 mg, 67 μmol) was dissolved in methanol (5.5 mL) under anhydrous conditions. The reaction flask was cooled to 0 °C before the addition of Iron (III) chloride (0.1 mL from a 40 mg/mL solution in methanol, 2.17 mg, 13.4 μmol, 0.2 eq). The reaction was left to stir for 1-3 hours, checking regularly by TLC (12% IPA/CHCI3). The mixture was quenched with NaHCOs then slightly concentrated before it was extracted with ethyl acetate (x3), washed with brine, dried over MgSO4, filtered and concentrated. The crude material was purified by flash column chromatography using the standard IPA/Hexane/Chloroform conditions (12% IPA with 22% Hexane and 66% CHCI3) yielding the product. Methyl CMC ¹ H NMR (599 MHz, CDCI3) 56.52 (dd, J = 15.1, 10.7 Hz, 1H), 6.47 (s, 1H), 5.80 (d, J = 10.6 Hz, 1H), 5.72-5.64 (m, 1H), 5.42 (ddd, J = 15.2, 8.3, 1.7 Hz, 1 H), 5.28 - 5.22 (m, 1 H), 5.14 (s, 1 H), 4.59 (dd, J = 9.6, 1.9 Hz, 1 H), 3.82 (t, J = 8.9 Hz, 1 H), 3.67 (s, 3H), 3.70 - 3.59 (m, 2H), 3.51 (dd, J= 10.3, 8.3 Hz, 1H), 3.44 (dd, J= 10.2, 5.0 Hz, 1H), 3.29 (dq, J = 9.1, 6.1 Hz, 1H), 3.25 (s, 3H), 3.10 (td, J = 9.0, 3.2 Hz, 1H), 3.04 (s, 3H), 2.75 (s, 1H), 2.33 (dd, J= 13.6, 5.0 Hz, 1H), 2.28 (s, 1H), 2.23 (d, J = 3.7 Hz, 1H), 2.17 (s, 2H), 2.19-2.12 (m, 2H), 2.06 (dd, J = 15.1, 7.7 Hz, 2H), 2.02-1.99 (m, 4H), 1.96 (s, 1 H), 1 .85 (s, 4H), 1 .72 (dd, J = 6.5, 1 .6 Hz, 3H), 1 .64 - 1 .57 (m, 1 H), 1 .53 (dd, J = 13.5, 11 .1 Hz, 1 H), 1 .35 - 1 .27 (m, 4H), 1 .27 - 1 .22 (m, 3H), 1 .21 (s, 2H), 1 .07 (s, 5H), 1 .00 (d, J = 7.0 Hz, 3H), 0.87 (dd, J = 9.6, 6.7 Hz, 6H).

[0162] Comparative Example 21 : Synthesis of 21 -Deoxy CMC (S62)

[0163] 21 -methoxy CMC (45 mg, 54 μmol) was dissolved in EtOH (4.68 mL). NaBHsCN (20 mg, 323 μmol, 6 eq) was added to the solution, followed by addition of 0.5 M HCI (0.47 mL, 4.4 eq). The reaction was allowed to run for 4 hours before neutralized with phosphate buffer. The reaction mixture was then extracted with chloroform twice, washed with water, brine, dried over sodium sulfate, filtered and concentrated. The crude reaction mixture was purified first by flash column chromatography (0-100% IPA in hexane/CHCh mixture (25% hexane in CHCI3)). The spot corresponding to 21-deoxy CMC was further purified by pTLC (8% IPA/CHCI3) to yield the product. ¹ H NMR (599 MHz, 278 K, CDCI3) 5 6.57 - 6.50 (m, 1 H), 6.40 (s, 1 H), 5.78 (d, J = 10.7 Hz, 1 H), 5.67 (d, J = 9.9 Hz, 1 H), 5.57 (dq, J = 12.9, 6.2 Hz, 1 H), 5.34 (dd, J = 15.4, 7.6 Hz, 1 H), 5.25 (dd, J = 15.1 , 8.9 Hz, 1 H), 5.19 (d, J = 8.7 Hz, 1 H), 4.64 - 4.59 (m, 1 H), 3.87 - 3.77 (m, 3H), 3.68 - 3.56 (m, 2H), 3.59 (s, 3H), 3.49 (td, J = 10.5, 4.5 Hz, 1 H), 3.45 (s, 1 H), 3.32 (dd, J = 10.4, 7.2 Hz, 1 H), 3.29 - 3.26 (m, 1 H), 3.26 (s, 3H), 3.25 - 3.19 (m, 1 H), 3.11 (t, J = 8.9 Hz, 1 H), 2.73 (s, 1 H), 2.57 (s, 2H), 2.29 (s, 1 H), 2.21 - 2.05 (m, 3H), 2.03 (s, 1 H), 1 .97 (s, 3H), 1.95 (s, 2H), 1.86 (s, 3H), 1.72 (dd, J = 16.2, 7.4 Hz, 1 H), 1.68 - 1.59 (m, 4H), 1.56 - 1.45 (m, 1 H), 1.33 (d, J = 6.0 Hz, 3H), 1.32 - 1.23 (m, 3H), 1.24 - 1.15 (m, 3H), 1.10 - 1.07 (m, 3H), 1.05 (d, J = 6.9 Hz, 3H), 0.96 (s, 1 H), 0.90 (d, J = 7.1 Hz, 3H), 0.87 (d, J = 6.5 Hz, 5H), 0.83 (d, J = 8.0 Hz, 3H). ¹³ C NMR (151 MHz, 278 K, CDCI3) 5 165.97, 142.10, 141.65, 139.13, 132.81 , 132.20, 130.57, 130.27, 128.50, 127.40, 123.10, 95.74, 82.99, 82.03, 79.60, 78.38, 77.63, 76.81 , 76.07, 74.51 , 71.82, 71.41 , 69.56, 59.29, 55.78, 44.61, 43.40, 41.26, 39.37, 39.23, 37.47, 36.46, 34.80, 34.55, 22.86, 21.50, 17.84, 17.67, 16.86, 16.47, 14.19, 13.57, 11.66, 9.62, 8.24.

Concanamycin F (6). Concanamycin C (CMC; 60.0 mg, 72.9 pimol) was dissolved in MeCN (7.61 mL) and water (1.78 mL) before the addition of pTsOH (55.5, 292 pimol, 4 eq). The reaction was ran overnight (between 15-20 hrs) before being cooled to 0°C and quenched with a saturated solution of sodium bicarbonate. The crude product was extracted with chloroform (3 times), washed with water, dried over Na2SO4, filtered and concentrated. The crude material was purified by FCC using 4-12% isopropanol in a 25% hexanes/chloroform solution. To fully wash the column, it was flushed with 100% isopropanol. The material collected was further purified by pTLC with 4% isopropanol/chloroform to yield 10 mg (19.8%) of 6 as a white solid. Starting material (30 mg, 30%) was also recovered. Concanamycin F ¹ H NMR (599 MHz, 278 K, CDCI3) 5 6.56 (dd, J= 15.1 , 10.7 Hz, 1 H), 6.40 (s, 1 H), 5.85 - 5.82 (m, 1 H), 5.78 (d, J = 10.5 Hz, 1 H), 5.68 (d, J = 9.6 Hz, 1 H), 5.55 (dt, J = 13.3, 6.8 Hz, 1 H), 5.32 - 5.25 (m, 1 H), 5.22 (dd, J = 15.2, 9.0 Hz, 1 H), 5.02 (d, J = 9.0 Hz, 1 H), 4.67 (d, J = 4.3 Hz, 1 H), 4.02 (d, J = 11 .6 Hz, 1 H), 3.97 (t, J = 9.2 Hz, 1 H), 3.86 (t, J = 9.1 Hz, 1 H), 3.85 - 3.80 (m, 1 H), 3.73 (td, J = 10.6, 4.5 Hz, 1 H), 3.56 (s, 3H), 3.26 (s, 3H), 3.22 (d, J = 10.3 Hz, 1 H), 2.73 (s, 1 H), 2.32 (dt, J = 12.2, 6.1 Hz, 2H), 2.22 - 2.15 (m, 1 H), 1.97 (s, 3H), 1.95 (s, 2H), 1.87 (s, 3H), 1.76 (d, J = 7.3 Hz, 1 H), 1.63 - 1.57 (m, 3H), 1.52 (dd, J = 10.8, 5.4 Hz, 1 H), 1.19 (s, 2H), 1.19 - 1.12 (m, 2H), 1.09 (d, J = 6.7 Hz, 3H), 1.05 (dd, J = 7.1 , 3.5 Hz, 6H), 0.92 (d, J = 6.5 Hz, 3H), 0.87 - 0.82 (m, 3H), 0.82 (d, J = 6.9 Hz, 3H). ¹³ C NMR (151 MHz, 278 K, CDCI ₃ ) 5 166.6, 142.1 , 141.9, 139.6, 133.3, 132.2, 130.8, 130.6, 127.9, 127.1 , 123.0, 99.5, 81.3, 79.7, 75.5,

75.2, 74.3, 70.5, 70.0, 59.1 , 55.7, 44.7, 43.5, 43.3, 43.0, 41.3, 36.9, 36.3, 34.6, 22.8, 21.7, 17.8, 16.8, 16.4, 14.2,

13.2, 11.7, 9.3, 7.1. LC-MS (IT): calcd for C^O [M+Fa-H]- 737.5; found, 737.5.

[0164] Comparative Example 22: Synthesis of Concanamycin X

[0165] Concanamycin B (CMA; 80. mg, 94 μmol) was dissolved in MeCN (6.6 mL) and water (1.32 mL) before the addition of pTsOH (35.7 mg, 188 μmol, 2 eq). The reaction was ran overnight (between 15-20 hrs) before being cooled to 0 °C and quenched with a saturated solution of NaHCO3. The crude product was extracted with CHCI3 (x3), washed with water, dried over Na2SO4, filtered and concentrated. The crude material was purified by flash column chromatography using 4-12% isopropanol in a 25% hexanes/chloroform solution. To fully wash the column, it was flushed with 100% isopropanol. The material collected was further purified by pTLC with 4% isopropanol/chloroform to yield the product. Concanamycin X ¹ H NMR (600 MHz, CDCI3) 5 6.59 - 6.51 (m, 1 H), 6.36 (d, J = 72.1 Hz, 1 H), 5.93 - 5.71 (m, 2H), 5.66 (d, J = 9.3 Hz, 1 H), 5.55 (dt, J = 15.0, 6.6 Hz, 1 H), 5.28 (dt, J = 14.9, 6.9 Hz, 1 H), 5.21 (dd, J = 15.0, 9.4 Hz, 1 H), 5.04 (dd, J = 41.4, 9.4 Hz, 1 H), 4.72 - 4.38 (m, 1 H), 4.04 (dt, J = 12.5, 6.3 Hz, 1 H), 3.97 (t, J = 9.5 Hz, 2H), 3.85 (t, J = 9.4 Hz, 1 H), 3.73 (td, J = 10.9, 4.9 Hz, 1 H), 3.56 (s, 3H), 3.26 (d, J = 5.2 Hz, 3H), 3.39 - 3.06 (m, 1 H), 2.89 (t, J = 7.9 Hz, 1 H), 2.33 (dd, J = 12.1 , 4.7 Hz, 1 H), 2.19 (dq, J = 14.0, 7.3 Hz, 1 H), 2.12 (t, J = 8.0 Hz, 1 H), 2.01 (d, J = 37.0 Hz, 4H), 1.83 (d, J = 61.9 Hz, 3H), 1.75 (d, J = 7.5 Hz, 4H), 1 .59 (d, J = 6.5 Hz, 3H), 1 .22 (d, J = 6.2 Hz, 3H), 1 .19 (d, J = 11 .2 Hz, 2H), 1 .05 (p, J = 7.4, 5.9 Hz, 6H), 0.91 (d, J = 6.4 Hz, 3H), 0.83 (dt, J = 20.7, 10.1 Hz, 7H). ¹³ C NMR (151 MHz, CDCI3) 5 166.4, 142.6, 142.1 , 139.6, 133.8, 133.0, 130.9, 129.8, 128.1 , 127.6, 125.8, 123.7, 99.7, 84.6, 81.3, 80.7, 75.4, 75.3, 70.6, 70.2, 64.6, 59.5, 59.2, 55.9, 45.4, 44.9, 43.7, 43.4, 43.2, 41.4, 37.3, 36.8, 36.5, 31.6, 25.5, 18.0, 17.7, 17.2, 16.3, 15.9, 14.9, 14.3, 13.4, 9.4, 7.2. LC-MS (IT): calcd for C38H ₆ 2Oi ₀ [M+Fa-H]- 723.4; found, 723.4.

[0166] Comparative Example 23: Synthesis of 21-deoxy CMF & 21-deoxy-16-hydroxy CMF (S24 and 25)

[0167] MeCN (11.6 mL) was added to a vial containing a mixture of 21 -deoxy CMC (140 mg 165 μmol) before water (2.32 mL) was added. The reaction was stirred and pTsOH (72.0 mg, 379 μmol) was added before heating to 38°C. The reaction was run overnight (~16 hrs) then cooled to 0°C. Sodium bicarbonate was added to the reaction mix to basify and before concentrating. The water mix that was left over was washed with chloroform 3 times. The organics were combined, washed with brine, dried over sodium sulfate, filtered, and concentrated. The crude, yellow foam was first purified by FCC (12% acetone/DCM) and fractions 12/13 were collected and found to contain majority of the product. Starting material was also recovered when the gradient was increased to 100% acetone. Fractions 12/13 were further purified by pTLC (12% acetone/DCM) to yield 21 -deoxy concanamycin F (CMF) and 21-deoxy-16-hydroxy-Concanamycin F.21-Deoxy CMF ¹ H NMR (600 MHz, CDCI3) 56.53 (q, J = 15.5, 14.2 Hz, 1H), 6.35 (d, J =38.6 Hz, 1H), 5.89-5.75 (m, 1H), 5.67 (d, J =9.8 Hz, 1H), 5.56 (dt, J = 14.3, 7.3 Hz, 1H), 5.37-5.30 (m, 1H), 5.28-5.21 (m, 1H), 5.20 (d, J = 8.5 Hz, 1H), 3.83 (q, J = 10.6, 9.8 Hz, 2H), 3.75 (s, 1H), 3.62 (d, J = 10.4 Hz, 1H), 3.58 (s, 3H), 3.49 (d, J = 10.3 Hz, 1H), 3.43-3.36 (m, 1H), 3.33 (t, J= 8.9 Hz, 1H), 3.26 (d, J = 2.8 Hz, 3H), 3.22 (d, J = 10.6 Hz, 1H), 2.72 (t, J= 8.9 Hz, 1H), 2.28 (s, 1H), 2.11 (dt, J = 17.8, 9.7 Hz, 2H), 2.05-1.93 (m, 5H), 1.86 (s, 3H), 1.69 (s, 1H), 1.62 (d, J =6.6 Hz, 3H), 1.51 (s, 1H),

I.31 -1.19 (m, 2H), 1.20- 1.15 (m, 2H), 1.13-1.06 (m, 3H), 1.05 (d, J = 6.9 Hz, 3H), 0.93-0.81 (m, 12H). ¹³ C NMR (151 MHz, CDCI3) 5165.9, 142.2, 141.6, 139.0, 132.8, 132.2, 130.4, 130.1, 128.5, 127.5, 123.1, 82.5, 82.0,

79.6, 76.8, 76.0, 74.5, 74.2, 69.2, 59.2, 55.8, 44.6, 43.8, 43.4, 39.2, 38.8, 37.4, 36.5, 34.5, 22.9, 21.5, 17.8, 16.9,

16.5, 14.2, 13.4, 11.7, 9.6, 8.2. LC-MS (IT): calcd for C39H64O9 [M+Fa-H]- 721.5; found, 721.4. 21-Deoxy-16- hydroxy-CMF ¹ H NMR (600 MHz, CDCI3) 56.43 (d, J = 3.4 Hz, 1 H), 6.32 (t, J = 13.1 Hz, 1 H), 5.94 (d, J = 9.7 Hz, 1H), 5.79 (d, J= 10.8 Hz, 1H), 5.70 (h, J= 5.4 Hz, 1H), 5.43 (dd, J= 15.9, 7.8 Hz, 2H), 5.09 (q, J= 12.2, 9.0 Hz, 1 H), 4.60 (d, J = 9.8 Hz, 1 H), 3.84 (d, J = 10.3 Hz, 1 H), 3.64 (d, J = 23.2 Hz, 4H), 3.41 (dq, J = 23.9, 13.1 ,

II.3 Hz, 3H), 3.25 (d, J= 9.9 Hz, 1H), 2.68-2.62 (m, 1H), 2.33 (s, 1H), 2.21 -2.14 (m, 2H), 2.14 (s, 1H), 1.95 (s, 3H), 1.90 - 1.76 (m, 2H), 1.73 (d, J = 6.1 Hz, 3H), 1.67 (s, 3H), 1.54 (s, 1 H), 1.39 (s, 1 H), 1.25 (d, J = 55.4 Hz, 4H), 1.14 (t, J = 4.5 Hz, 3H), 1.04 (d, J = 6.1 Hz, 3H), 0.99 (d, J = 6.6 Hz, 2H), 0.97 - 0.87 (m, 9H). ¹³ C NMR (151 MHz, CDCI3) 5164.5, 142.2, 140.1, 137.7, 132.4, 130.8, 130.2, 129.2, 128.5, 128.0, 121.7, 82.3, 81.8, 81.7,

78.6, 78.4, 77.5, 77.0, 74.1, 59.8, 45.1, 43.7, 42.1, 40.9, 39.5, 38.7, 38.2, 35.0, 24.6, 19.2, 18.2, 18.0, 16.8, 14.4,

13.6, 13.3, 12.2, 8.2. LC-MS (QTOF): calcd for C38H 64O9 [M+H]* 663.45; found, 663.45.

[0168] Comparative Example 24: Synthesis of 3'-nonanoate Concanamycin B (11) [0169] CMB (30 mg, 35 μmol), nonanoic acid (12 μL, 70, μmol, 2.0 eq), DMAP (9.0 mg, 74 μmol, 2.1 eq), EDC (15 mg, 78 μmol, 2.2 eq), DCM (5 mL). 3’-nonanoate CMB ¹ H NMR (600 MHz, CDCI ₃ ) 5 6.54 (h, J = 14.0, 13.0 Hz, 1 H), 6.34 (d, J = 72.0 Hz, 1 H), 5.87 (d, J = 12.3 Hz, 1 H), 5.81 (d, J = 19.8 Hz, 1 H), 5.64 (d, J = 9.9 Hz, OH), 5.53 (dt, J = 14.9, 6.8 Hz, 1 H), 5.26 (dd, J = 15.5, 7.7 Hz, 1 H), 5.19 (dd, J = 15.0, 9.4 Hz, 1 H), 5.03 (dd, J = 41.1 , 9.4 Hz, 1 H), 4.93 (td, J = 10.8, 5.2 Hz, 1 H), 4.75 (s, 1 H), 4.70 (s, 1 H), 4.63 - 4.56 (m, 2H), 4.06 - 3.92 (m, 2H), 3.84 (t, J = 9.4 Hz, 1 H), 3.76 (td, J = 11.1 , 4.8 Hz, 1 H), 3.55 (s, 3H), 3.39 (dd, J = 9.7, 6.1 Hz, 1 H), 3.25 (d, J = 5.4 Hz, 3H), 2.87 (t, J = 7.7 Hz, 1 H), 2.32 (td, J = 13.7, 12.5, 6.1 Hz, 1 H), 2.28 (t, J = 7.6 Hz, 2H), 2.19 (dq, J = 12.5, 7.3, 6.3 Hz, 2H), 2.11 (d, J = 13.5 Hz, 1 H), 2.02 (s, 2H), 1.96 (s, 2H), 1.86 (d, J = 12.2 Hz, 2H), 1.75 (d, J = 15.0 Hz, 3H), 1 .67 (q, J = 11.1 Hz, 1 H), 1 .57 (d, J = 6.5 Hz, 5H), 1 .23 (dd, J = 13.4, 6.6 Hz, 19H), 1 .11 (d, J = 8.5 Hz, 1 H), 1 .03 (dd, J = 23.3, 6.9 Hz, 5H), 0.94 (d, J = 7.2 Hz, 2H), 0.86 (t, J = 6.9 Hz, 7H), 0.82 (d, J = 6.7 Hz, 3H).

[0170] Comparative Example 25: Synthesis of 3'-pent-4-ynoate Concanamycin A

[0171] Comparative Example 26: Synthesis of 3'-pent-4-ynoate Concanamycin B

[0172] Comparative Example 27: Synthesis of 3'-pent-4-ynoate Concanamycin C

EXAMPLES

[0173] Example 1 : Bafilomycin esterification general procedure.

[0174] DMAP, EDC, and carboxylic acid were added to a solution of bafilomycin Ai in DCM under anhydrous conditions. The reaction was stirred overnight then concentrated under reduced pressure. The crude reaction mix was purified by preparative TLC (pTLC). [0175] Example 2: 21 -Formyl Bafilomycin Ai (Compound A1, also called S61)

[0176] Amounts: DMAP (5.1 mg, 42.0 μmol, 2.1 eq), EDC (8.5 mg, 44 μmol, 2.2 eq), formic acid (10.95 μL, 25 μmol, 1.25 eq), bafilomycin Ai (12 mg, 20 μmol), and DCM (2.5 mL). Purified by pTLC (20% acetone in hexanes) to yield the product. S36 ¹ H NMR (599 MHz, Acetone) 58.17 (d, J= 1.2 Hz, 1H), 6.71 (s, 1H), 6.68 (dd, J = 15.0, 10.8 Hz, 1H), 5.96 (d, J = 8.8 Hz, 1H), 5.80 (d, J = 10.7 Hz, 1H), 5.43 (t, J = 1.6 Hz, 1H), 5.14 (dd, J = 15.0, 9.2 Hz, 1H), 5.02 (td, J = 10.9, 4.8 Hz, 1H), 4.96 (dq, J = 8.5, 1.5 Hz, 1H), 4.78 (d, J = 4.3 Hz, 1H), 4.18 (ddt, J = 9.4, 5.2, 1.7 Hz, 1H), 4.09- 4.03 (m, 2H), 3.64 (s, 3H), 3.60 (dd, J = 10.3, 2.1 Hz, 1H), 3.31 (tt, J =5.1,

2.3 Hz, 1 H), 3.24 (s, 3H), 2.55 (dt, J = 8.9, 7.0 Hz, 1 H), 2.28 (ddd, J = 11.7, 4.9, 1.3 Hz, 1 H), 2.21 - 2.13 (m, 1 H), 2.10-1.99 (m, 2H), 1.98 (s, 3H), 1.93 (d, J = 1.4 Hz, 3H), 1.93-1.86 (m, 2H), 1.84 (t, J = 7.4 Hz, 1H), 1.62-

1.53 (m, 1 H), 1.29 (s, 1 H), 1.05 (dd, J = 7.1 , 1.3 Hz, 3H), 1.00 (dd, J = 7.2, 1.3 Hz, 3H), 0.95 (dd, J = 6.9, 1.3 Hz, 3H), 0.92 (dd, J = 6.8, 1.3 Hz, 3H), 0.88 (dd, J = 6.9, 1.3 Hz, 3H), 0.83 (dd, J = 6.5, 1.3 Hz, 3H), 0.81 (dd, J = 6.7,

1.4 Hz, 3H). ¹³ C NMR (151 MHz, Acetone) 5167.84, 161.73, 145.74, 144.84, 141.94, 134.47, 134.24, 132.79, 127.04, 125.24, 99.72, 83.34, 80.43, 77.42, 76.50, 74.26, 71.59, 60.18, 55.67, 42.89, 42.27, 41.71, 40.87, 38.86, 38.28, 38.01, 28.75, 22.25, 21.62, 20.37, 17.73, 14.58, 14.16, 12.48, 10.29, 7.37.

[0177] Example 3: Synthesis of 21-(2-fluoro)acetate Baf (Compound A2)

[0178] Amounts: DMAP (4.31 mg, 35 μmol, 2.1 eq), EDC (7.1 mg, 37 μmol, 2.2 eq), fluoroacetic acid (1.9 μL, 34 μM, 2.0 eq), bafilomycin A1 (10. mg, 17 μmol), and DCM (2.1 mL). Purified by pTLC (20% acetone in hexanes) to yield the product.21-(2-fluoro)acetate Baf 1H NMR (599 MHz, Acetone) 56.76-6.72 (m, 1H), 6.74 -6.66 (m, 1H), 5.99 (d, J = 8.9 Hz, 1H), 5.83 (d, J = 10.8 Hz, 1H), 5.48-5.44 (m, 1H), 5.20 -5.13 (m, 1H), 5.11 -5.06 (m, 1H), 5.06 (s, 1H), 5.06-4.90 (m, 3H), 4.80 (dt, J = 4.5, 1.3 Hz, 1H), 4.20 (d, J = 10.2 Hz, 1H), 4.08 (tt, J = 8.1, 1.9 Hz, 2H), 3.68-3.65 (m, 3H), 3.63 (dd, J = 10.5, 2.4 Hz, 1H), 3.33 (q, J = 4.7, 2.9 Hz, 1H), 3.30- 3.24 (m, 3H), 2.62 - 2.53 (m, 1 H), 2.34 (ddd, J = 11.9, 5.1 , 2.0 Hz, 1 H), 2.22 - 2.16 (m, 1 H), 2.01 (q, J = 1.3 Hz, 3H), 2.00- 1.94 (m, 3H), 1.90 (s, 2H), 1.95-1.84 (m, 1H), 1.61 (dt, J = 12.8, 5.3 Hz, 2H), 1.09- 1.06 (m, 3H), 1.03 - 1.00 (m, 3H), 0.99 - 0.96 (m, 3H), 0.94 (dt, J = 6.8, 1.4 Hz, 3H), 0.92 - 0.88 (m, 3H), 0.88 - 0.84 (m, 3H), 0.84-0.81 (m, 3H). 13C NMR (151 MHz, Acetone) 5168.22 (d, J = 21.7 Hz), 167.83, 145.74, 144.84, 141.95, 134.47, 134.23, 132.79, 127.03, 125.24, 99.77, 83.34, 80.42, 78.46 (d, J = 178.2 Hz), 77.41, 76.51, 75.68, 71.59, 60.18, 55.67, 42.91, 42.27, 41.71, 40.65, 38.96, 38.28, 38.01, 28.72, 22.25, 21.60, 20.36, 17.73, 14.57, 14.16, 12.48, 10.29, 7.37.

[0179] Example 4: Synthesis of 21-(2-chloro)acetate Baf (Compound A3)

[0180] Synthesized in an analogous fashion to 21-(2-fluoro)acetate Baf (Compound A2).

[0181] Example 5: Synthesis of 21-(2-bromo)acetate Baf (Compound A4)

Bafilomycin A ₁ 21 -(2-bromo)acetate Baf

[0182] 21-(2-bromo)acetate Baf ¹ H NMR (599 MHz, Acetone) 56.75-6.66 (m, 2H), 5.98 (d, J = 8.9 Hz, 1H), 5.82 (d, J= 11.0 Hz, 1H), 5.47-5.41 (m, 1H), 5.17 (dd, J = 15.1, 9.1 Hz, 1H), 5.05-4.96 (m, 2H), 4.80 (d, J = 4.3 Hz, 1 H), 4.34 - 4.28 (m, 1 H), 4.30 - 4.23 (m, 1 H), 4.19 (t, J = 8.8 Hz, 1 H), 4.14 - 4.02 (m, 2H), 3.69 - 3.64 (m, 3H), 3.62 (d, J = 10.0 Hz, 1H), 3.33 (ddd, J = 5.5, 3.3, 2.1 Hz, 3H), 3.29- 3.24 (m, 3H), 2.62-2.52 (m, 1H), 2.33 (dt, J= 11.7, 3.0 Hz, 1H), 2.03 (d, J= 7.3 Hz, 1H), 2.00 (q, J= 1.2 Hz, 3H), 1.95 (s, 4H), 1.89 (dd, J= 17.8, 9.7 Hz, 2H), 1.61 (td, J= 10.4, 5.3 Hz, 1H), 1.07 (dt, J = 7.1, 1.2 Hz, 3H), 1.01 (dt, J = 7.2, 1.2 Hz, 3H), 0.98- 0.95 (m, 3H), 0.94 (dt, J = 6.7, 1.2 Hz, 3H), 0.90 (dt, J = 6.9, 1.2 Hz, 3H), 0.86 (dt, J = 6.6, 1.2 Hz, 3H), 0.84 - 0.82 (m, 3H). ¹³ C NMR (151 MHz, Acetone) 5167.61, 145.75, 144.84, 141.94, 134.47, 134.23, 132.79, 127.03, 125.24, 99.78, 99.68, 83.34, 80.35 (d, J = 20.5 Hz), 77.41, 76.53 (d, J= 8.7 Hz), 71.53 (d, J = 18.4 Hz), 60.18, 55.67, 52.38 (d, J = 780.9 Hz), 42.90 (d, J = 4.1 Hz), 42.27, 41.86, 41.71, 40.54, 38.99, 38.28, 38.00 (d, J = 3.2 Hz), 28.71, 22.25, 21.60, 20.36, 17.73, 14.57, 14.16, 12.45, 10.29, 7.38.

[0183] Example 6: Synthesis of 21-(2-(S)-bromo)propanoate baf (Compound A5)

Bafilomycin A ₁ 21 -(2-(S)-bromo)propanoate baf

[0184] 21-(2-(S)-bromo)propanoate Baf 1H NMR (599 MHz, Acetone) 56.74 (q, J = 0.9 Hz, 1H), 6.74-6.67 (m, 1H), 5.99 (d, J = 8.8 Hz, 1H), 5.86-5.81 (m, 1H), 5.45 (t, J = 1.9 Hz, 1H), 5.17 (dd, J = 15.1, 9.2 Hz, 1H), 5.04-4.96 (m, 2H), 4.80 (dt, J = 4.4, 1.4 Hz, 1H), 4.59 (qd, J = 6.9, 1.6 Hz, 1H), 4.24-4.18 (m, 1H), 4.14-4.06 (m, 2H), 3.71 -3.63 (m, 3H), 3.63 (dd, J = 10.4, 2.0 Hz, 1H), 3.33 (ddd, J = 7.1, 4.7, 1.9 Hz, 1H), 3.31 -3.25 (m, 3H), 2.58 (dtd, J = 9.2, 7.2, 5.3 Hz, 1 H), 2.35 - 2.27 (m, 1 H), 2.23 - 2.15 (m, 1 H), 2.08 - 2.02 (m, 1 H), 2.03 - 1.96 (m, 3H), 1.98 - 1.94 (m, 3H), 1.91 (s, 1 H), 1.88 (q, J = 7.2 Hz, 1 H), 1.84 - 1.79 (m, 3H), 1.74 - 1.60 (m, 1 H), 1.34 - 1.25 (m, 4H), 1.08 (dd, J = 7.1 , 1.6 Hz, 3H), 1.04- 1.00 (m, 3H), 0.98 (dd, J = 7.0, 1.7 Hz, 3H), 0.95 (dd, J = 6.8, 1.6 Hz, 3H), 0.91 (dt, J = 7.0, 2.0 Hz, 6H), 0.84 (dd, J = 6.9, 1.7 Hz, 3H). 13C NMR (151 MHz, Acetone) 5170.28, 167.84, 145.74, 144.84, 141.95, 134.47, 134.23, 132.80, 127.03, 125.24, 99.76, 83.34, 80.42, 77.42, 76.54, 76.13, 71.59, 60.18, 55.67, 42.91, 42.28, 41.73 (d, J = 2.8 Hz), 40.47, 39.14, 38.28, 38.01, 28.67, 22.25, 21.94, 21.61, 20.37, 17.73, 14.58, 14.16, 12.48, 10.29, 7.40.

[0185] Example 7: Synthesis of 3' -one CM A (Compounds A6 and A7, also called S47 and S48)

[0186] CMA (49 mg, 57 μmol) was dissolved in dry DCM (3.0 mL) and activated molecular sieves (4A, 8-12 mesh) were added.4-Methylmorpholine N-oxide monohydrate (31 mg, ) was added to the reaction, followed by TRAP () in two increments. The mixture was stirred at RT for 2 hours, with the solution changing from a grey- green to black over the course. To remove some of the catalyst, the reaction mixture was filtered over a bed of celite. Residual catalyst was removed upon flash column chromatography (5%-40% I PA in a hexane/CHCl3 mixture (25% Hexane in CHCI3) to yield S66 and S63. S66 and S63 were further purified separately by pTLC (5% IPA/CHCI3) to yield the product. S63 ¹ H NMR (599 MHz, 278 K, CDCI3) 56.56 (dd, J= 15.1, 10.7 Hz, 1H), 6.40 (s, 1H), 5.89 (s, 1H), 5.78 (d, J = 10.7 Hz, 1H), 5.68 (d, J= 9.6 Hz, 1H), 5.57 (dd, J = 15.1, 6.8 Hz, 1H), 5.29 (dd, J= 15.1, 8.3 Hz, 1H), 5.22 (dd, J = 15.1, 9.0 Hz, 1H), 5.01 (d, J = 9.1 Hz, 1H), 4.82 (d, J= 10.1 Hz, 1H), 4.75 (dd, J = 7.2, 4.7 Hz, 1 H), 4.70 (d, J = 4.1 Hz, 1 H), 4.00 (q, J = 9.6, 8.7 Hz, 2H), 3.89 - 3.80 (m, 3H), 3.63 - 3.56 (m, 1H), 3.57 (s, 3H), 3.26 (s, 3H), 3.25-3.20 (m, 1H), 2.77-2.68 (m, 3H), 2.30 (dd, J= 12.0, 4.8 Hz, 2H), 2.18 (s, 1H), 1.98 (s, 5H), 1.87 (s, 3H), 1.76 (d, J = 7.2 Hz, 2H), 1.59 (d, J= 6.5 Hz, 4H), 1.51 (s, 1H), 1.39 (d, J = 6.0 Hz, 3H), 1.35 (d, J = 6.1 Hz, 1 H), 1.13 (t, J = 10.6 Hz, 2H), 1.09 (d, J = 6.7 Hz, 3H), 1.04 (d, J = 7.1 Hz, 6H), 0.93 (d, J= 6.4 Hz, 4H), 0.87 (dt, J= 16.5, 7.1 Hz, 3H), 0.82 (d, J = 6.8 Hz, 3H). ¹³ C NMR (151 MHz, 278 K, CDCI3) 5199.57, 165.63, 154.18, 141.12, 140.79, 138.60, 132.29, 131.18, 129.69, 129.11, 126.97, 126.08, 121.95, 98.48, 96.04, 80.25, 78.63, 77.91, 75.52, 74.53, 74.41, 73.26, 69.06, 68.64, 58.05, 54.73, 47.55, 43.69, 42.29, 40.24, 40.15, 38.57, 35.81, 35.31, 33.54, 21.71 (d, J= 6.9 Hz), 20.65, 17.41, 16.79, 15.76, 15.37, 13.17, 12.41, 10.63, 8.28, 6.10.

[0187] Example 8: Concanamycin esterification general procedure.

[0188] DMAP, EDC, and carboxylic acid were added to a solution of concanamycin of choice in DCM under anhydrous conditions. After stirring for 30 minutes a TLC was taken (5% IPA in CHCI3) and every 15 minutes afterwards. Once the TLC showed two distinct spots above the starting material, the reaction was concentrated (usually < 2.5 hrs total). The crude material was first purified by flash column chromatography, yielding 2 products and purified residual starting material (-40% IPA/ 15% Hex/ 55% CHCI3). The two products were further purified by pTLC.

[0189] Example 9: 3' -Acetyl Concanamycin B (Compound A8, also called 24) & 3',9-Diacetyl CMB

(Compound A9, also called 25)

[0190] CMB (30 mg, 35 μmol), acetic acid (3.0 μL, 53 μmol, 1.5 eq), DMAP (9.0 mg, 74 μmol, 2.1 eq), EDO (14.8 mg, 78 μmol, 2.2 eq), DCM (5.0 mL). isolated 12 and 13. Acetyl CMB ¹ H NMR (600 MHz, CDCI3) 56.62 - 6.48 (m, 1H), 6.36 (d, J = 71.2 Hz, 1H), 5.91 -5.78 (m, 2H), 5.66 (d, J = 10.0 Hz, 1H), 5.55 (dt, J= 14.8, 6.7 Hz, 1H), 5.28 (dt, J= 15.3, 7.3 Hz, 1H), 5.21 (dd, J= 15.0, 9.4 Hz, 1H), 5.04 (dd, J= 40.8, 9.5 Hz, 1H), 4.94 (ddd, J = 11.6, 9.4, 5.2 Hz, 1H), 4.60 (q, J= 9.6, 9.1 Hz, 2H), 4.44 (d, J = 4.3 Hz, 1H), 3.98 (q, J= 14.2, 9.4 Hz, 2H), 3.85 (t, J= 9.4 Hz, 1H), 3.77 (td, J= 10.6, 4.4 Hz, 1H), 3.73-3.67 (m, 1H), 3.56 (s, 3H), 3.40 (dq, J= 12.2, 6.2 Hz, 1 H), 3.26 (d, J = 5.1 Hz, 3H), 3.35 - 3.07 (m, 1 H), 2.93 - 2.51 (m, 1 H), 2.38 (q, J = 7.6 Hz, 1 H), 2.32 (dd, J = 12.1, 4.7 Hz, 1H), 2.21 (dt, J= 11.4, 6.1 Hz, 2H), 2.06 (s, 5H), 2.00 (d, J = 36.7 Hz, 3H), 1.83 (d, J = 62.0 Hz, 3H), 1.75 (d, J = 7.2 Hz, 1 H), 1.73 - 1.67 (m, 1 H), 1.58 (d, J = 6.5 Hz, 3H), 1.30 - 1.22 (m, 4H), 1.15- 0.77 (m, 20H). ¹³ C NMR (151 MHz, CDCI3) 5169.8, 165.1, 154.8, 141.5, 138.5, 134.6, 132.6, 131.8, 131.2, 130.0, 129.8,

128.5, 126.9, 126.2, 124.9, 124.7, 122.3, 98.5, 94.9, 83.4, 83.1, 80.1, 79.5, 75.0, 74.7, 74.5, 74.2, 74.0, 69.9, 69.0, 68.8, 58.4, 54.7, 44.2, 43.7, 40.3, 40.2, 38.7, 36.1, 35.9, 35.6, 35.3, 34.3, 33.8, 30.4, 21.0, 20.1, 18.0, 16.8,

16.5, 16.4, 15.7, 14.9, 14.7, 13.7, 13.2, 12.4, 11.6, 11.5, 8.3, 6.1. LC-MS (IT): calcd for C ₄ 7H7 ₅ NOI ₅ [M+Fa-H]- 938.5; found, 938.5. . Diacetyl CMB ¹ H NMR (600 MHz, CDCI3) 56.59 (dd, J= 15.0, 10.8 Hz, 1H), 6.40 (s, 1H), 5.90 (d, J= 2.3 Hz, 1H), 5.86 (d, J = 10.7 Hz, 1H), 5.60 (d, J = 9.9 Hz, 1H), 5.55 (dq, J = 13.1, 6.2 Hz, 1H), 5.28 (dd, J= 15.3, 8.1 Hz, 1H), 5.23 (dd, J= 15.0, 9.3 Hz, 1H), 4.99 (d, J = 9.5 Hz, 1H), 4.94 (td, J= 10.6, 5.1 Hz,

1 H), 4.73 (d, J = 4.3 Hz, 1 H), 4.66 (d, J = 11.0 Hz, 1 H), 4.64 - 4.56 (m, 2H), 4.06 - 4.00 (m, 1 H), 3.98 (t, J = 9.2 Hz, 1H), 3.84 (t, J= 9.4 Hz, 1H), 3.77 (td, J= 10.8, 4.7 Hz, 1H), 3.61 -3.57 (m, 1H), 3.59-3.55 (m, 3H), 3.40 (dq, J = 12.1, 6.1 Hz, 1H), 3.25 (d, J= 1.3 Hz, 3H), 2.68 (t, J = 9.0 Hz, 1H), 2.55 (t, J= 8.3 Hz, 1H), 2.32 (dd, J = 12.1, 4.7 Hz, 1H), 2.22 (dq, J= 12.0, 6.2, 4.5 Hz, 2H), 2.15-2.09 (m, 4H), 2.08-2.05 (m, 4H), 1.95 (s, 3H), 1.88 (s, 3H), 1.84 (dd, J = 9.7, 6.9 Hz, 1H), 1.75 (dt, J= 16.7, 8.5 Hz, 1H), 1.69 (q, J= 11.3 Hz, 1H), 1.58 (d, J = 6.4 Hz, 3H), 1.24 (d, J= 7.3 Hz, 4H), 1.12 (t, J= 11.6 Hz, 1H), 1.07 (d, J= 7.1 Hz, 3H), 1.03 (d, J = 6.9 Hz, 3H), 0.88 (d, J= 6.4 Hz, 6H), 0.82 (d, J = 6.9 Hz, 3H), 0.69 (d, J = 7.1 Hz, 3H). ¹³ C NMR (151 MHz, CDCI3) 5170.99, 170.77, 166.57, 155.75, 141.87, 141.72, 138.90, 133.72, 132.44, 130.94, 130.80, 127.90, 127.55, 123.73, 99.57, 95.92, 84.66, 81.05, 76.06, 75.47, 75.32, 75.02, 70.96, 69.99, 69.83, 59.00, 55.66, 45.90, 41.30, 41.19, 39.75, 37.01, 36.92, 36.48, 34.85, 33.21, 21.50, 21.15, 21.09, 17.81, 17.42, 16.90, 16.76, 16.04, 14.15, 13.40, 9.20, 7.14.

[0191] Example 10: Synthesis of 3'-pentanoate Concanamycin B & 3',9-Dipentanoate Concanamycin B (Compounds A10 and A11 , also called 21 and 22)

[0192] CMB (45 mg, 53 μmol), pentanoic acid (8.6 μL, 79 μmol, 1.5 eq), DMAP (14 mg, 111 μmol), EDC (22 mg, 116 μmol, 2.2 eq), DCM (7.5 mL). CMB pent ¹ H NMR (599 MHz, CDCI ₃ ) 56.57 - 6.49 (m, 1 H), 6.34 (d, J = 73.4 Hz, 1H), 5.86 (d, J = 10.7 Hz, 1H), 5.83 (s, 1H), 5.64 (d, J = 9.5 Hz, OH), 5.52 (dq, J= 17.2, 6.3 Hz, 1H), 5.29 - 5.23 (m, 1 H), 5.25 - 5.16 (m, 1 H), 5.02 (dd, J = 41.0, 9.4 Hz, 1 H), 4.93 (ddd, J = 11.8, 9.4, 5.2 Hz, 1 H), 4.77 (s, 1 H), 4.69 (s, 2H), 4.62 - 4.55 (m, 2H), 4.43 (s, 1 H), 3.96 (q, J = 9.5, 8.4 Hz, 2H), 3.84 (t, J = 9.4 Hz, 1 H), 3.75 (td, J= 10.8, 4.7 Hz, 1H), 3.55 (s, 3H), 3.38 (dq, J= 12.3, 6.2 Hz, 1H), 3.30 (d, J= 8.8 Hz, 1H), 3.24 (d, J = 4.6 Hz, 3H), 2.90-2.84 (m, 1H), 2.60 (s, 1H), 2.34-2.28 (m, 1H), 2.28 (t, J = 7.5 Hz, 2H), 2.19 (dt, J= 11.0, 5.7 Hz, 2H), 2.11 (d, J = 12.6 Hz, 1H), 2.02 (s, 3H), 1.97 (d, J= 11.5 Hz, 2H), 1.87 (s, 3H), 1.74 (s, 1H), 1.65 (ddd, J = 19.0, 11.1 , 8.4 Hz, 1 H), 1.56 (p, J = 7.7 Hz, 5H), 1.30 (h, J = 7.4 Hz, 2H), 1.23 (s, 1 H), 1.22 (d, J = 6.1 Hz, 3H), 1.20 (d, J =6.2 Hz, 3H), 1.13-1.08 (m, 1H), 1.09-0.99 (m, 6H), 0.94 (d, J =7.1 Hz, 1H), 0.87 (q, J = 6.5, 5.7 Hz, 6H), 0.86-0.76 (m, 4H). ¹³ C NMR (151 MHz, CDCI3) 5173.66, 166.20, 155.92, 142.67, 142.48, 141.78, 139.67 (d, J= 17.0 Hz), 133.72, 132.95, 132.37, 131.19, 130.93, 129.61, 128.34, 128.05, 127.32, 125.82, 123.41, 99.69, 96.05, 84.61, 80.67, 76.17, 75.66 (d, J= 11.3 Hz), 75.35, 75.09, 70.79, 70.21, 70.01, 59.37 (d, J = 52.1 Hz), 55.88, 45.63, 44.86, 43.40, 41.46, 41.33, 39.89, 37.27, 37.13, 36.79, 36.51, 36.06, 34.98, 34.26, 31.56, 27.11, 25.46, 22.25, 17.94, 17.68, 17.55, 16.92, 16.31, 15.89, 14.88, 14.28, 13.88, 13.52, 12.82, 9.43, 7.26. dipentanoate CMB ¹ H NMR (600 MHz, CDCI3) 56.52 (dd, J = 15.1, 10.7 Hz, 1H), 6.33 (s, 1H), 5.83 (d, J = 2.0 Hz, 1H), 5.79 (d, J= 10.8 Hz, 1H), 5.54 (d, J= 9.8 Hz, 1H), 5.48 (dq, J= 13.2, 6.4 Hz, 1H), 5.21 (ddd, J= 15.3, 8.1, 1.8 Hz, 1H), 5.15 (dd, J= 15.0, 9.3 Hz, 1H), 4.92 (dd, J= 9.5, 1.6 Hz, 1H), 4.88 (ddd, J= 11.9, 9.5, 5.3 Hz, 1 H), 4.66 (d, J = 4.3 Hz, 1 H), 4.60 (d, J = 11.0 Hz, 1 H), 4.57 - 4.50 (m, 2H), 4.48 (s, 1 H), 3.96 (dt, J = 10.5, 3.3 Hz, 1H), 3.90 (dd, J= 10.2, 8.0 Hz, 1H), 3.77 (t, J = 9.4 Hz, 1H), 3.70 (td, J= 10.8, 4.8 Hz, 1H), 3.53-3.46 (m, 1H), 3.49 (s, 3H), 3.33 (dq, J = 9.7, 6.2 Hz, 1H), 3.18 (s, 3H), 2.60 (dd, J= 11.3, 6.0 Hz, 1H), 2.48 (ddd, J = 9.7, 6.9, 2.5 Hz, 1H), 2.30 (t, J = 7.6 Hz, 2H), 2.28-2.24 (m, 1H), 2.23 (t, J = 7.5 Hz, 2H), 2.15 (ddd, J= 12.2, 9.9, 6.3 Hz, 2H), 2.05 (d, J = 15.6 Hz, 1H), 2.02-1.92 (m, 1H), 1.92-1.83 (m, 3H), 1.81 (s, 3H), 1.80-1.73 (m, 1 H), 1.73 - 1.66 (m, 1 H), 1.65 - 1.59 (m, OH), 1.58 - 1.54 (m, 2H), 1.55 - 1.48 (m, 5H), 1.34 - 1.26 (m, 2H), 1.23 (dt, J= 14.6, 7.4Hz, 2H), 1.19- 1.14 (m, 4H), 1.08-1.01 (m, 1H), 1.00 (d, J = 7.1 Hz, 3H), 0.96 (d, J = 7.0 Hz, 3H), 0.86 (t, J= 7.4 Hz, 3H), 0.81 (dt, J= 10.6, 7.6 Hz, 10H), 0.75 (d, J = 6.9 Hz, 3H), 0.61 (d, J= 7.0 Hz, 3H). ¹³ C NMR (151 MHz, CDCI3) 5172.49, 165.55, 154.66, 140.87, 140.67, 137.91, 132.68, 131.40, 129.93, 129.77, 126.86, 126.50, 122.68, 98.54, 94.88, 83.20, 80.03, 75.01, 74.42, 73.92, 69.62, 68.97, 68.83, 57.98, 54.63, 44.87, 40.27, 40.17, 38.73, 36.05, 35.95, 35.47, 33.82, 33.18, 33.10, 32.23, 26.18, 25.95, 21.35, 21.09, 20.49, 16.78, 16.39, 15.93, 15.79, 15.02, 13.12, 12.79, 12.72, 12.36, 8.18, 6.11.

[0193] Example 11 : Synthesis of 3'-pentanoate Concanamycin A (Compound A12)

[0194] CMA (25 mg, 29 μmol), pentanoic acid (6.3 μL, 58 μmol, 2 eq), DMAP (7.4 mg, 61 μmol, 2.1 eq), EDC (12 mg, 64 μmol, 2.2 eq), DCM (4.09 mL). isolated the product. 3’-pentanoate CMA ¹ H NMR (599 MHz, CDCI3) 56.56 (dd, J = 15.1, 10.7 Hz, 1H), 6.40 (s, 1H), 5.87 (s, 1H), 5.81 -5.75 (m, 1H), 5.68 (d, J= 9.8 Hz, 1H), 5.55 (dq, J= 13.1, 6.6 Hz, 1H), 5.28 (dd, J= 14.9, 8.0 Hz, 1H), 5.22 (dd, J= 15.1, 9.0 Hz, 1H), 5.01 (dd, J = 9.0, 6.2 Hz, 1 H), 5.00 - 4.91 (m, 1 H), 4.73 - 4.66 (m, 2H), 4.64 - 4.57 (m, 2H), 4.02 (d, J = 10.3 Hz, 1 H), 3.97 (t, J = 9.2 Hz, 1 H), 3.86 (t, J = 9.2 Hz, 1 H), 3.84 - 3.81 (m, 1 H), 3.77 (td, J = 10.8, 4.7 Hz, 1 H), 3.56 (s, 3H), 3.44 - 3.36 (m, 1H), 3.26 (s, 3H), 3.22 (d, J= 10.3 Hz, 1H), 2.73 (s, 1H), 2.31 (dt, J= 10.7, 7.5 Hz, 4H), 2.22 (dd, J = 5.3, 1.9 Hz, 1H), 2.22- 2.14 (m, 1H), 2.05-1.91 (m, 5H), 1.87 (s, 3H), 1.75 (s, 1H), 1.75-1.63 (m, 1H), 1.63-1.53 (m, 5H), 1.55-1.44 (m, 1H), 1.32 (p, J = 7.5 Hz, 2H), 1.25 (dd, J= 11.1, 6.2 Hz, 4H), 1.23- 1.10 (m, 3H), 1.07 (td, J = 14.0, 12.9, 7.2 Hz, 9H), 0.89 (td, J= 7.5, 2.8 Hz, 9H), 0.84 (dd, J = 21.0, 7.1 Hz, 3H). ¹³ C NMR (151 MHz, CDCI3) 5173.52, 166.63, 155.80, 142.11, 141.83, 139.57, 133.29, 132.22, 130.81, 130.64, 127.83, 127.13, 122.99, 99.55, 95.92, 81.29, 79.66, 76.04, 75.55, 75.48, 74.98, 74.29, 70.65, 70.09, 69.86, 59.07, 55.75, 44.72, 43.32, 41.32, 41.20, 39.75, 37.00, 36.85, 36.33, 34.56, 34.13, 26.98, 25.82, 22.76, 22.12, 21.68, 17.80, 17.42, 16.78, 16.39, 14.18, 13.74, 11.66, 9.32, 7.12.

[0195] Example 12: Synthesis of 3' -Acetyl CMA & 3',9-Diacetyl CMA (Compounds A14 and A15)

[0196] CMA (35 mg, 40 μmol), pentanoic acid (3.5 μL, 61 μmol, 1.5 eq), DMAP (10. mg, 85 μmol, 2.1 eq), EDC (17 mg, 89 μmol, 2.2 eq), DCM (4.9 mL). Acetyl CMA ¹ H NMR (600 MHz, CDCI3) 56.57 (dd, J= 15.0, 10.6 Hz, 1 H), 6.38 (d, J = 21.5 Hz, 1 H), 5.88 (s, 1 H), 5.82 - 5.75 (m, 1 H), 5.68 (d, J = 9.9 Hz, 1 H), 5.55 (dt, J = 13.6, 6.8 Hz, 1H), 5.28 (dd, J = 15.3, 8.0 Hz, 1H), 5.21 (dt, J = 18.9, 9.4 Hz, 1H), 5.01 (d, J = 9.2 Hz, 1H), 4.98-4.91 (m, 1 H), 4.70 (d, J = 4.2 Hz, 1 H), 4.60 (q, J = 9.4 Hz, 2H), 4.05 - 4.00 (m, 1 H), 3.97 (t, J = 9.2 Hz, 1 H), 3.86 (t, J = 9.2 Hz, 1H), 3.82 (d, J = 10.4 Hz, 1H), 3.77 (td, J= 10.8, 4.6 Hz, 1H), 3.56 (s, 3H), 3.40 (dq, J= 12.3, 6.3 Hz, 1 H), 3.26 (s, 3H), 3.22 (d, J = 10.3 Hz, 1H), 2.75 (dt, J = 36.6, 8.1 Hz, 1H), 2.32 (dt, J= 14.5, 7.2 Hz, 2H), 2.20 (ddd, J = 17.5, 11.6, 6.3 Hz, 2H), 2.06 (s, 3H), 2.04 - 1.93 (m, 5H), 1.87 (s, 3H), 1.74 (dd, J = 20.2, 6.1 Hz, 1 H), 1.67 (dd, J = 22.8, 11.3 Hz, 1 H), 1.58 (d, J = 6.4 Hz, 3H), 1.51 (dt, J = 11.1 , 5.8 Hz, 1 H), 1.24 (d, J = 6.5 Hz, 4H),

I.15 (dd, J = 22.7, 8.6 Hz, 3H), 1.07 (td, J = 15.1, 13.3, 6.9 Hz, 9H), 0.88 (d, J = 6.4 Hz, 3H), 0.88-0.83 (m, 3H), 0.82 (d, J=6.7 Hz, 3H). ¹³ C NMR (151 MHz, CDCI ₃ ) 5170.78, 166.63, 155.80, 142.13, 141.80, 139.61, 133.30, 132.19, 130.79, 130.67, 127.85, 127.08, 122.96, 99.53, 95.91, 81.28, 79.63, 76.04, 75.53, 75.47, 75.01, 74.25, 70.95, 70.07, 69.82, 59.07, 55.75, 44.72, 43.30, 41.30, 41.17, 39.73, 36.92, 36.84, 36.30, 34.56, 22.73, 21.69, 21.15, 17.81, 17.41, 16.78, 16.38, 14.17, 13.39, 11.66, 9.31,7.12. Diacetyl CMA ¹ H NMR (600 MHz, CDCI3) 5 6.58 (dd, J = 15.1, 10.7 Hz, 1H), 6.40 (s, 1H), 5.89 (s, 1H), 5.79 (d, J = 10.7 Hz, 1H), 5.63 (d, J = 9.8 Hz, 1H), 5.56 (dq, J= 13.3, 6.5 Hz, 1H), 5.28 (dd, J= 15.3, 8.0 Hz, 1H), 5.22 (dd, J = 15.1, 9.2 Hz, 1H), 5.00 (d, J = 9.3 Hz, 1H), 4.94 (ddd, J= 11.4, 9.4, 5.2 Hz, 1H), 4.85 (d, J = 10.9 Hz, 1H), 4.72 (d, J =4.2 Hz, 1H), 4.64-4.56 (m, 2H), 4.05-4.00 (m, 1H), 3.98 (t, J= 9.1 Hz, 1H), 3.85 (t, J= 9.3 Hz, 1H), 3.77 (td, J= 10.8, 4.7 Hz, 1H), 3.68 (dt, J= 10.3, 3.5 Hz, 1H), 3.56 (s, 3H), 3.40 (dq, J= 12.1, 6.2 Hz, 1H), 3.25 (s, 3H), 2.71 -2.61 (m, 2H), 2.33 (dd, J = 12.1, 4.8 Hz, 1H), 2.21 (dt, J= 10.7, 5.6 Hz, 2H), 2.12 (s, 3H), 2.08 (s, 1H), 2.06 (s, 3H), 2.01 (dd, J= 15.6, 6.8 Hz, 1 H), 1.95 (s, 3H), 1.88 (s, 3H), 1.76 (q, J = 7.3 Hz, 1 H), 1.73 - 1.64 (m, 1 H), 1.58 (d, J = 6.6 Hz, 4H), 1.28 (dd, J = 11.6, 5.4 Hz, 1 H), 1.24 (d, J = 6.6 Hz, 3H), 1.12 (t, J = 11.3 Hz, 1 H), 1.07 (d, J = 7.2 Hz, 3H), 1.04 (d, J = 6.9 Hz, 3H), 0.89 (td, J= 10.8, 5.2 Hz, 9H), 0.82 (d, J= 6.9 Hz, 3H). ¹³ C NMR (151 MHz, CDCI3) 5170.99, 170.77, 166.61, 155.74, 141.81, 141.78, 139.13, 133.55, 132.30, 130.79, 130.66, 127.85, 127.39, 123.51, 99.55, 95.92, 81.22, 79.54, 76.05, 75.45, 75.01, 74.59, 70.96, 70.02, 69.82, 59.03, 55.72, 45.27, 43.64, 41.32, 41.19, 39.75, 36.92, 36.62, 34.69, 33.54, 21.30, 21.23, 21.15 (d, J = 1.8 Hz), 17.82, 17.41, 16.76, 16.15, 14.14, 13.39,

II.96, 9.25, 7.13.

[0197] Example 13: Synthesis of 3'-penta-2,4-diene CMA (Compound A16)

[0198] CMA (20 mg, 35 μmol), nonanoic acid (3.5 μL, 35, μmol, 1.5 eq), DMAP (6.0 mg, 49 μmol, 2.1 eq), EDC (9.9 mg, 52 μmol, 2.2 eq), DCM (2.87 mL).*NMR resolution was incredibly pour for the compound. Confirmation of structure was done by both NMR analysis and MS analysis. ¹³ C NMR (151 MHz, Acetone) 5 160.03, 149.51, 139.37, 128.35, 124.57, 120.15, 115.29, 93.32, 89.73, 74.28, 71.00, 70.79, 70.57, 69.81, 68.74, 64.77, 63.83, 63.62, 49.52, 35.08, 34.95, 33.52, 23.48, 16.48, 11.59, 11.21, 8.53, 7.95, 7.17, 3.05, -6.23.

[0199]

[0200] Example 14: Synthesis of 3', 4' -diacetyl CMC & 3',4',9-triacetyl CMC (Compounds A17 and A18)

[0201] CMC (30 mg, 36 μmol), acetic acid (5.2 μL, 91, μmol, 2.5 eq), DMAP (14 mg, 113 μmol, 3.1 eq), EDC (22.4 mg, 117 μmol, 3.2 eq), DCM (4.5 mL). Diacetyl CMC ¹ H NMR (600 MHz, CDCI ₃ ) 56.56 (dt, J= 20.2, 10.1 Hz, 1 H), 6.38 (d, J = 21.0 Hz, 1 H), 5.88 (s, 1 H), 5.83 - 5.75 (m, 1 H), 5.68 (d, J = 9.8 Hz, 1 H), 5.55 (dq, J = 13.3, 6.4 Hz, 1H), 5.28 (dd, J= 15.3, 8.0 Hz, 1H), 5.22 (dd, J = 15.1, 9.0 Hz, 1H), 5.10-4.98 (m, 1H), 4.93 (ddd, J = 11.9, 9.5, 5.3 Hz, 1 H), 4.75 (t, J = 9.5 Hz, 1 H), 4.70 (d, J = 4.3 Hz, 1 H), 4.62 (dd, J = 9.8, 2.1 Hz, 1 H), 4.05 - 3.99 (m, 1 H), 3.97 (t, J = 9.2 Hz, 1 H), 3.86 (t, J = 9.3 Hz, 1 H), 3.83 (d, J = 9.9 Hz, 1 H), 3.77 (td, J = 10.7, 4.5 Hz, 1 H), 3.56 (s, 3H), 3.47 - 3.39 (m, 1 H), 3.26 (s, 3H), 3.22 (d, J = 10.3 Hz, 1 H), 2.82 - 2.68 (m, 1 H), 2.32 (td, J =

12.3, 4.2 Hz, 2H), 2.26-2.20 (m, 1H), 2.19 (dd, J= 10.7, 6.6 Hz, 1H), 2.14-2.09 (m, 1H), 2.07 (s, 3H), 2.03 (s, 3H), 1.97 (s, 3H), 1.96 (d, J = 5.3 Hz, 1 H), 1.85 (d, J = 23.5 Hz, 3H), 1.76 (d, J = 8.2 Hz, 1 H), 1.68 (d, J = 34.6 Hz, 1H), 1.58 (d, J = 6.4 Hz, 3H), 1.51 (dt, J= 11.1, 5.5 Hz, 1H), 1.27 (d, J= 7.5 Hz, 1H), 1.19 (d, J = 6.2 Hz, 3H), 1.15 (d, J = 6.8 Hz, 2H), 1.13 (d, J= 10.6 Hz, 1H), 1.07 (td, J= 15.5, 13.3, 6.9 Hz, 9H), 0.90-0.80 (m, 9H). ¹³ C NMR (151 MHz, CDCI3) 5170.61, 170.29, 166.63, 142.12, 141.81, 139.57, 133.29, 132.21, 130.79, 130.66, 127.86, 127.10, 122.97, 99.54, 95.88, 81.28, 79.63, 76.04, 75.54, 75.45, 74.28, 73.99, 71.03, 70.07, 69.62, 59.07, 55.75, 44.70, 43.30, 41.30, 41.17, 39.71, 36.87, 36.32, 34.55, 22.74, 21.67, 21.09, 20.96, 17.81, 17.47, 16.77, 16.38, 14.17, 13.39, 11.66, 9.31, 7.13. ¹³ CNMR(151 MHz, CDCI3) 5170.61, 170.29, 166.63, 142.12, 141.81, 139.57, 133.29, 132.21, 130.79, 130.66, 127.86, 127.10, 122.97, 99.54, 95.88, 81.28, 79.63, 76.04,

75.54, 75.45, 74.28, 73.99, 71.03, 70.07, 69.62, 59.07, 55.75, 44.70, 43.30, 41.30, 41.17, 39.71, 36.87, 36.32,

34.55, 22.74, 21.67, 21.09, 20.96, 17.81, 17.47, 16.77, 16.38, 14.17, 13.39, 11.66, 9.31, 7.13. Triacetyl CMC ¹ H NMR (600 MHz, CDCI3) 56.58 (dd, J= 15.0, 10.7 Hz, 1H), 6.40 (s, 1H), 5.89 (d, J= 2.1 Hz, 1H), 5.79 (d, J = 10.7 Hz, 1H), 5.63 (d, J= 9.8 Hz, 1H), 5.56 (dq, J= 13.2, 6.5 Hz, 1H), 5.28 (ddd, J= 15.2, 8.0, 1.8 Hz, 1H), 5.22 (dd, J= 15.0, 9.2 Hz, 1H), 5.00 (dd, J = 9.3, 1.6 Hz, 1H), 4.93 (ddd, J= 11.9, 9.5, 5.3 Hz, 1H), 4.85 (d, J= 11.0 Hz, 1H), 4.75 (t, J= 9.5 Hz, 1H), 4.72 (d, J = 4.2 Hz, 1H), 4.62 (dd, J= 9.8, 1.9 Hz, 1H), 4.02 (ddd, J= 10.9, 4.4, 2.2 Hz, 1H), 3.98 (dd, J= 10.3, 8.0 Hz, 1H), 3.85 (t, J= 9.3 Hz, 1H), 3.78 (td, J= 10.7, 4.7 Hz, 1H), 3.68 (ddt, J =

7.3, 4.5, 2.6 Hz, 1 H), 3.56 (s, 3H), 3.42 (dq, J = 9.9, 6.1 Hz, 1 H), 3.25 (s, 3H), 2.73 - 2.62 (m, 2H), 2.33 (dd, J = 12.1, 4.8 Hz, 1H), 2.22 (tdd, J= 15.5, 6.8, 3.5 Hz, 2H), 2.12 (s, 3H), 2.10 (s, 1H), 2.07 (s, 3H), 2.03 (s, 3H), 2.02 - 1.97 (m, 1 H), 1.96 - 1.94 (m, 3H), 1.88 (s, 3H), 1.79 - 1.71 (m, 1 H), 1.71 - 1.65 (m, 1 H), 1.58 (dd, J = 6.6, 1.6 Hz, 4H), 1.31 - 1.25 (m, 1H), 1.19 (d, J= 6.2 Hz, 3H), 1.15-1.09 (m, 2H), 1.07 (d, J = 7.2 Hz, 3H), 1.04 (d, J = 7.0 Hz, 3H), 0.88 (td, J= 10.8, 10.0, 5.1 Hz, 10H), 0.82 (d, J = 6.9 Hz, 3H). ¹³ C NMR (151 MHz, CDCI3) 5170.98, 170.61, 170.29, 166.61, 141.80, 141.78, 139.12, 133.54, 132.30, 130.79, 130.66, 127.86, 127.39, 123.51, 99.55, 95.88, 81.22, 79.54, 76.05, 75.44, 74.60, 73.99, 71.03, 70.02, 69.62, 59.03, 55.72, 45.27, 43.64, 41.32, 41.18, 39.73, 36.88, 36.62, 34.69, 33.54, 21.30, 21.23, 21.16, 21.09, 20.96, 17.82, 17.47, 16.76, 16.15, 14.14, 13.40, 11.97, 9.25, 7.13.

[0202] Example 15: Synthesis of 23-acetyl CMF & 3',9-diacteyl CMF (Compounds A19 and A20)

[0203] CMF (20 mg, 29 μmol), acetic acid (2.5 pL, 43, μmol, 1.5 eq), DMAP (7.4 mg, 61 μmol, 2.1 eq), EDC (12 mg, 64 μmol, 2.2 eq), DCM (4.1 mL). Isolated 23-acetyl CMF and 9,23-diacetyl CMF. Acetyl CMF ¹ H NMR (600 MHz, CDCI ₃ ) 56.56 (dd, J= 15.2, 10.9 Hz, 1H), 6.37 (d, J = 20.5 Hz, 1H), 5.81 (s, 1H), 5.80-5.72 (m, 1H), 5.68 (d, J= 9.7 Hz, 1H), 5.58 (dd, J= 14.3, 7.0 Hz, 1H), 5.28 (dd, J= 15.4, 8.2 Hz, 1H), 5.22 (dd, J = 15.1, 9.1 Hz, 1H), 5.04-4.95 (m, 2H), 4.64 (d, J =4.4 Hz, 1H), 4.07 (t, J =9.1 Hz, 1H), 4.00 (d, J= 11.5 Hz, 1H), 3.84 (q, J= 10.2, 9.7 Hz, 2H), 3.56 (d, J = 2.2 Hz, 3H), 3.25 (d, J = 2.2 Hz, 3H), 3.22 (d, J = 10.6 Hz, 1H), 2.72 (t, J= 8.2 Hz, 1H), 2.37 (dd, J= 12.0, 4.8 Hz, 1H), 2.30 (s, 1H), 2.20-2.14 (m, 1H), 2.05 (d, J = 2.2 Hz, 3H), 2.04-1.93 (m, 5H), 1.87 (s, 3H), 1.74 (q, J = 7.0, 5.4 Hz, 1 H), 1.59 (d, J = 6.4 Hz, 3H), 1.53 - 1.48 (m, 1 H), 1.40 (d, J = 8.0 Hz, 1H), 1.23-1.20 (m, 1H), 1.15 (td, J = 15.7, 7.7 Hz, 2H), 1.09 (d, J =6.6 Hz, 3H), 1.04 (t, J =7.9 Hz, 6H), 0.86 (q, J= 7.9, 7.4 Hz, 3H), 0.80 (t, J = 8.4 Hz, 6H). ¹³ C NMR (151 MHz, CDCI ₃ ) 5170.53, 166.55, 142.07, 141.86, 139.50, 133.25, 132.23, 130.52, 130.46, 128.29, 127.14, 122.99, 99.42, 81.28, 79.62, 75.54, 75.01, 74.30, 73.21, 70.00, 59.04, 55.73, 44.68, 43.31, 41.23, 40.66, 39.81, 36.83, 36.35, 34.54, 22.75, 21.65, 21.33, 17.81, 16.79, 16.40, 14.19, 13.30, 11.66, 9.33, 7.07. Diacetyl CMF ¹ H NMR (600 MHz, CDCI3) 56.58 (dd, J = 15.1, 10.7 Hz, 1H), 6.39 (s, 1H), 5.82 (d, J = 2.0 Hz, 1H), 5.79 (d, J = 10.7 Hz, 1H), 5.63 (d, J = 9.9 Hz, 1H), 5.63 -5.54 (m, 1H), 5.29 (ddd, J= 15.4, 8.2, 1.8 Hz, 1H), 5.22 (dd, J= 15.0, 9.2 Hz, 1H), 4.99 (td, J= 10.6, 3.3 Hz, 2H), 4.85 (d, J= 10.9 Hz, 1H), 4.67 (d, J= 4.3 Hz, 1H), 4.08 (dd, J= 10.3, 8.1 Hz, 1H), 4.01 (ddd, J= 10.9, 4.4, 2.2 Hz, 1 H), 3.84 (t, J = 9.3 Hz, 1 H), 3.70 - 3.63 (m, 1 H), 3.56 (s, 3H), 3.24 (s, 3H), 2.72 - 2.61 (m, 2H), 2.37 (dd, J= 11.8, 4.8 Hz, 1H), 2.23-2.14 (m, 1H), 2.12 (s, 3H), 2.09 (d, J = 15.3 Hz, 1H), 2.06 (s, 3H), 2.01 (dd, J = 15.5, 6.7 Hz, 1H), 1.95 (s, 3H), 1.88 (s, 3H), 1.77-1.71 (m, 1H), 1.60 (dd, J = 6.5, 1.6 Hz, 3H), 1.59-1.54 (m, 1H), 1.40 (tdd, J= 13.0, 8.4, 5.3 Hz, 1H), 1.24- 1.20 (m, 1H), 1.10-1.07 (m, 1H), 1.04 (dd, J= 7.1, 5.5 Hz, 6H), 0.90 (d, J= 6.9 Hz, 3H), 0.87 (d, J = 3.5 Hz, 4H), 0.80 (t, J = 6.6 Hz, 6H). ¹³ C NMR (151 MHz, CDCI3) 5170.98,

170.54, 166.54, 141.86, 141.74, 139.06, 133.52, 132.32, 130.54, 130.46, 128.30, 127.42, 123.52, 99.44, 81.23,

79.54, 75.44, 75.00, 74.62, 73.22, 69.96, 59.01, 55.70, 45.25, 43.65, 41.25, 40.68, 39.83, 36.61, 34.69, 33.57, 21.33, 21.30, 21.25, 21.16, 17.82, 16.77, 16.16, 14.15, 13.31, 11.97, 9.27, 7.08.

[0204] Example 16: Synthesis of DLP CMA & DLP, DLP CMA

[0205] CMA (30 mg, 34 μmol), 3-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)propanoic acid (8.5 pL, 51 μmol, 1.5 eq), DMAP (8.7 mg, 72 μmol, 2.1 eq), EDC (14 mg, 75 μmol, 2.2 eq), DCM (4.8 mL). DLP CMA ¹ H NMR (600 MHz, CDCI3) 56.55 (td, J = 22.1, 18.1, 10.9 Hz, 1H), 6.43-6.35 (m, 1H), 5.86 (d, J= 17.9 Hz, 1H), 5.83-5.75 (m, 1H), 5.68 (d, J = 9.8 Hz, 1H), 5.55 (dd, J= 14.5, 6.8 Hz, 1H), 5.28 (dd, J= 15.3, 8.0 Hz, 1H), 5.22 (dd, J= 15.3, 9.2 Hz, 1 H), 5.01 (d, J = 9.1 Hz, 1 H), 4.95 (tq, J = 9.4, 3.7, 2.2 Hz, 1 H), 4.70 (d, J = 4.3 Hz, 1 H), 4.64 - 4.55 (m, 2H), 4.05 - 3.98 (m, 1 H), 3.97 (d, J = 9.3 Hz, 1 H), 3.86 (dt, J = 9.3, 4.6 Hz, 1 H), 3.84 - 3.81 (m, 1 H), 3.81 - 3.73 (m, 1 H), 3.56 (d, J = 2.2 Hz, 3H), 3.43 - 3.34 (m, 1 H), 3.26 (d, J = 2.2 Hz, 3H), 3.22 (d, J = 10.3 Hz, 1 H), 2.82 - 2.69 (m, 1H), 2.32 (ddd, J= 12.5, 5.2, 2.6 Hz, 2H), 2.26-2.19 (m, 1H), 2.19 (t, J = 8.8 Hz, 1H), 2.13 (td, J = 7.0, 6.2, 4.0 Hz, 2H), 2.06 - 1.99 (m, 4H), 1.97 (s, 4H), 1.88 (s, 3H), 1.86 - 1.75 (m, 2H), 1.78 - 1.73 (m, 1 H), 1.73 - 1.65 (m, 1 H), 1.68 - 1.63 (m, 2H), 1.59 (d, J = 6.5 Hz, 3H), 1.54 - 1.48 (m, 1 H), 1.24 (td, J = 7.7, 6.4, 3.7 Hz, 4H), 1.19- 1.11 (m, 3H), 1.12-1.02 (m, 9H), 0.91 -0.80 (m, 9H). ¹³ C NMR (151 MHz, CDCI3) 5171.75, 166.63, 155.72, 142.14, 141.79, 139.62, 133.31, 132.19, 130.78, 130.68, 127.85, 127.08, 122.96, 99.53, 95.87, 82.61, 81.27, 79.62, 76.08, 75.53, 75.47, 74.88, 74.25, 71.31, 70.07, 69.80, 69.33, 59.06, 55.75, 44.72, 43.30, 41.29,

41.17, 39.73, 36.88, 36.84, 36.30, 34.56, 31.93, 28.54, 27.72, 27.59, 22.73, 21.69, 17.81, 17.40, 16.78, 16.38,

14.17, 13.39, 13.23, 11.67, 9.31, 7.12.

[0206] DLP, DLP CMA ¹ H NMR (600 MHz, CDCI ₃ ) 66.59 (dd, J= 15.0, 10.8 Hz, 1H), 6.39 (s, 1H), 5.89 (d, J = 2.1 Hz, 1H), 5.78 (d, J = 10.7 Hz, 1H), 5.62 (d, J = 9.8 Hz, 1H), 5.56 (dq, J= 13.2, 6.5 Hz, 1H), 5.28 (ddd, J = 15.3, 8.1, 1.8 Hz, 1H), 5.22 (dd, J= 15.0, 9.2 Hz, 1H), 5.00 (dd, J= 9.3, 1.6 Hz, 1H), 4.95 (ddd, J= 11.9, 9.5, 5.2 Hz, 1 H), 4.85 (d, J = 11.0 Hz, 1 H), 4.72 (d, J = 4.3 Hz, 1 H), 4.64 - 4.56 (m, 2H), 4.02 (ddd, J = 10.8, 4.4, 2.2 Hz, 1H), 3.98 (dd, J= 10.2, 8.0 Hz, 1H), 3.85 (t, J = 9.3 Hz, 1H), 3.77 (td, J= 10.7, 4.7 Hz, 1H), 3.64 (ddd, J= 10.3, 4.5, 2.8 Hz, 1H), 3.56 (s, 3H), 3.40 (dq, J = 9.5, 6.2 Hz, 1H), 3.25 (s, 3H), 2.67 (ddd, J= 16.7, 8.0, 4.6 Hz, 2H), 2.32 (dd, J= 12.1, 4.7 Hz, 1H), 2.26-2.18 (m, 2H), 2.13 (dd, J= 8.0, 6.6 Hz, 3H), 2.11 -2.07 (m, 1H), 2.04 (dd, J = 7 A, 2.7 Hz, 1 H), 2.02 (d, J = 4.9 Hz, 6H), 2.01 - 1.97 (m, 1 H), 1.95 (d, J = 1.3 Hz, 3H), 1.88 (s, 3H), 1.86 (dd, J = 7.6, 1.5 Hz, 1 H), 1.77 - 1.73 (m, 1 H), 1.71 -1.68 (m, 4H), 1.58 (dd, J = 6.4, 1.6 Hz, 3H), 1.56 (d, J = 7.8 Hz, 1H), 1.27 (s, 1H), 1.24 (d, J = 6.2 Hz, 3H), 1.12 (td, J= 10.5, 9.3, 2.6 Hz, 1H), 1.11 - 1.07 (m, 1H), 1.07 (d, J = 7.2 Hz, 3H), 1.04 (d, J= 6.9 Hz, 3H), 0.93-0.84 (m, 10H), 0.82 (d, J= 6.9 Hz, 3H). ¹³ C NMR (151 MHz, CDCI3) 5172.06, 171.75, 166.62, 155.67, 141.80, 141.75, 139.08, 133.58, 132.30, 130.79, 130.66, 127.86, 127.42, 123.54, 99.55, 95.87, 82.61, 82.57, 81.21, 79.98, 76.08, 75.45, 75.43, 74.88, 74.49, 71.32, 70.01, 69.80, 69.33, 69.28, 59.02, 55.72, 45.34, 43.70, 41.32, 41.19, 39.75, 36.88, 36.58, 34.73, 33.38, 32.20, 31.94, 28.54, 28.26, 27.73, 27.70, 27.64, 27.59, 21.38, 21.11, 17.82, 17.40, 16.75, 16.12, 14.14, 13.39, 13.28, 13.23, 12.02, 9.24, 7.12.

[0207] Example 17: Synthesis of DLP CMB (Compound A21)

[0208] ¹ H NMR (599 MHz, CDCI3, 323 K) 56.53 (dd, J= 15.1, 10.6 Hz, 1H), 6.37 (s, 1H), 5.80 (d, J= 10.6 Hz, 1H), 5.65 (s, 1H), 5.55 (dq, J = 13.1, 6.4 Hz, 1H), 5.34-5.27 (m, 1H), 5.23 (dd, J= 15.2, 8.9 Hz, 1H), 5.03 (d, J = 9.1 Hz, 1H), 4.96 (ddd, J = 12.1, 9.3, 5.2 Hz, 1H), 4.63-4.53 (m, 4H), 4.07-4.01 (m, 1H), 3.97 (dd, J= 10.3, 7.6 Hz, 1H), 3.85 (q, J= 9.4 Hz, 2H), 3.77 (td, J= 10.7, 4.6 Hz, 1H), 3.57 (s, 2H), 3.40 (dq, J= 12.4, 6.3 Hz, 1H), 3.28 (s, 1H), 3.25 (s, 3H), 2.76-2.69 (m, 1H), 2.30 (dd, J= 12.0, 4.8 Hz, 1H), 2.20 (dt, J= 15.4, 7.3 Hz, 2H), 2.12 (t, J = 7.6 Hz, 2H), 2.00 (dt, J = 17.1, 4.8 Hz, 8H), 1.82 (d, J= 10.8 Hz, 3H), 1.82 - 1.73 (m, 2H), 1.76-1.71 (m, 1H), 1.73-1.65 (m, 1H), 1.63 (t, J = 7.4 Hz, 2H), 1.59 (d, J = 6.5 Hz, 3H), 1.38- 1.16 (m, 5H), 1.09 (s, OH), 1.06 (t, J = 7.1 Hz, 7H), 0.88 (t, J = 7.9 Hz, 6H), 0.82 (d, J = 6.9 Hz, 3H).

[0209] Example 18: Synthesis of DLP CMC (Compound A22)

[0210] ¹ H NMR (599 MHz, CDCI ₃ ) 56.58 -6.51 (m, 1H), 6.38 (s, 1H), 5.76 (s, 1H), 5.68 (s, 1 H), 5.56 (tt, J = 13.0, 7.2 Hz, 1H), 5.33-5.26 (m, 1H), 5.22 (dd, J= 15.4, 9.2 Hz, 1H), 5.02 (d, J = 8.9 Hz, 1H), 4.81 (ddd, J = 12.7, 8.1,5.2 Hz, 1H), 4.64-4.59 (m, 2H), 4.03 (s, 2H), 4.01 -3.95 (m, 1H), 3.91 -3.83 (m, 1H), 3.82 (s, 2H), 3.78 (td, J= 10.7, 4.8 Hz, 1H), 3.56 (d, J= 1.1 Hz, 3H), 3.50-3.45 (m, 1H), 3.31 (ddt, J= 11.0, 8.2, 3.9 Hz, 2H), 3.29-3.24 (m, 3H), 2.73 (s, 1H), 2.63-2.46 (m, 4H), 2.35-2.29 (m, 1H), 2.31 (s, 2H), 2.24-2.15 (m, 2H), 2.00 (td, J = 2.5, 1.0 Hz, 1 H), 1.98 (s, 5H), 1.96 - 1.91 (m, 3H), 1.86 (s, 2H), 1.78 - 1.59 (m, 5H), 1.40 - 1.32 (m, 3H), 1.34-1.30 (m, 3H), 1.25 (h, J = 6.5, 5.9 Hz, 3H), 1.18 (dt, J= 12.1, 4.8 Hz, 1H), 1.17-1.09 (m, 2H), 1.10- 1.01 (m, 7H), 0.96 - 0.84 (m, 3H), 0.86 (s, 5H), 0.82 (d, J = 6.9 Hz, 2H).

[0211] Example 19: Cell Based Assay

Preparation of Primary CD4 T Lymphocytes

[0212] Anonymized leukocytes isolated by apheresis were obtained from New York Blood Center and peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque Plus (17144002; GE Healthcare) centrifugation using SepMate tubes (85450; StemCell Technologies) according to the manufacturers protocol. CD8 ⁺ lymphocytes were depleted with Dynabeads according to the manufacturer's protocol (11147D; Invitrogen) and the remaining cells were incubated at a density of 1 x10 ⁶ cells per milliliter in 20 milliliters of R10 medium and stimulated with 10ug/mL phytohemagglutinin-L (PHA-L) (431784; Millipore Sigma) in 70mL T25 cell culture flasks. Then, 20-24 hours post-PHA activation, 15mL of R10/PHA medium is removed and replaced with 5mL of R10 + 100u/mL Recombinant Human IL-2 (202IL010; R&D Systems).48 hours after IL-2 stimulation, primary CD4 ⁺ T cells were infected via spinoculation with previously described HIV AGPE construct.

Viral Construct & Infections

[0213] Infectious supernatants for the HIV AGPE construct were prepared by cotransfection of HEK-293T cells using pelyethyenimine (PEI) as previously described [2] with the AGPE construct, the HIV packaging plasmid pCMV-HIV and the broad tropism envelope glycoprotein G of Vesicular Stomatitis Virus (VSV-G) at a mass ratio of 1 :1 :1. Infections were performed by spinoculation. For spinoculation, cells are plated at a density of 1x10 ⁶ cells/well in a 24 well plate and spun for 2 hours at 1050xg in 1mL of viral supernatant + 4ug/mL hexadimethrine bromide (polybrene, H9268, Sigma-Aldrich). After spinoculation, cells are resuspended in 2mL R10+50u/mL IL-2 per well.

Compound Preparation

[0214] All compounds were transferred to pre-weighed vials (weighed in triplicate) for dilution calculations prior to being dissolved in sterile DMSO (Sigma-Aldrich Cat#: 276885). 5mM or 10mM solutions were made depending on the overall mass of the material and serial dilutions (in DMSO) were completed to achieve a 10OμM solution. Serial dilutions were vortexed after each step. 50μL of the 10Oμ M solution was transferred to a glass vial insert (will add brand later) and stored at -20 °C, if necessary, before testing. Thermo Scientific™ E1- ClipTip Bluetooth electronic single channel pipettes were used to throughout the entire sample preparation to avoid carry over of errors due to miscalibration or pipette tips variation.

Compound Treatments of Primary CD4 ⁺ T Cells

[0215] 48 hours after spinoculation, cells were pooled and plated in flat bottom 96-well plate. Mock cells were plated at 1 x10 ⁵ cells/well in 10OμL R10+100u/mL IL-2 while HIV AGPE infected cells were plated at 1 x10 ⁵ cells/well in 50μL R10+100u/mL IL-2. Compounds were solubilized in sterile DMSO (Sigma-Aldrich Cat#: 276885) to a concentration of 1 OOμ M and frozen at -20°C for storage. Compounds were thawed at room temperature and diluted in R10 medium to 2x starting concentration. Compounds were then serially diluted in a 96-well round bottom plate in R10. Dilutions of compounds were then added to respective AGPE and Mock plates to bring final concentration of compounds to 1x and IL-2 concentration to 50u/mL.

Flow Cytometry Staining

[0216] All AGPE infected cells were collected 24 hours after treatment. Cells were suspended in Lysotracker Red DND-99 (L7528; Invitrogen, 1 :5000) and HLA-A2 (BB7.2 from HB-82 hybridoma as previously described [3], 0.5μg/mL) in phosphate buffered saline (PBS) for 1 hr at 37°. The cells were washed once with FACS buffer (2% fetal bovine serum (26140079, Gibco), 1% human antibody serum (BP2525, Fisher), 2mM HEPES (151630080, Gibco) 0.025% sodium azide (S8032, Sigma-Aldrich) in PBS). After washing, the cells were resuspended in 4ng/mL DAPI (4’,6-diamidino-2-phenylindole,62248, Thermo Scientific) for viability and 1 :1000 goat anti-mouse lgG2b-Alexa Fluor 647 (A21242, Invitrogen) in FACS buffer for 5 minutes at room temperature and after, washed with FACS buffer, then finally fixed in 2% paraformaldehyde for 30 minutes at room temperature.

[0217] All Mock cells were collected 72 hours after treatment. Cells were suspended in 4ng/mL DAPI in FACS buffer for 20 minutes on ice, washed once with FACS buffer and fixed in 2% paraformaldehyde for 30 minutes at room temperature. [0218] In all experiments, flow cytometry data was collected with a BioRad Ze5 cytometer and data was analyzed using FlowJo software. Cells were gated sequentially by forward scatter vs. side scatter for cells, doublet exclusion (forward scatter area vs. height), and exclusion of DAPI for viable cells.

Calculation and Statistical Analysis:

[0219] MFI = median fluorescence intensity of MHC-I

[0220] Fold downmodulation = MFI /MFI

[0221] Normalized Nef activity = Fold downmodulation MHC-I _sampie / Fold downmodulation MHC-l _Solvent

[0222] All statistical analyses were performed using GraphPad Prism software. Curves were generated using GraphPad Prism software using [Inhibitor] vs. response with variable slope (four parameters).

[0223] The results of the anti-Nef activity, toxicity, and lysosomal acidification assays are summarized in T able B, below.

Table B

Name No. Nef Lysotracker Viability Viability:Nef Lys:Nef

CMA 1 0.18 1.7 1.3 6.6 9.4

Bafilomycin Ai 2 12 35 16 1.4 2.3

Archazolid A 3 0.47 1.6 .74 1.6 3.6

Ring Open

S8 310 >1000 520 1.7 — Bafilomycin Ai

21 -acetyl Bafilomycin

S9 4.5 23 10. 2.4 5.1 Ai

21-pentanoate

S10 37 57 51 1.4 1.5 Bafilomycin Ai

21 -nonanoate

S11 130 150 140 1.1 1.2 Bafilomycin Ai

21-penta-2,4-dienoate

S12 17 24 22 1.4 1.5 Bafilomycin Ai

21- benzoateBafilomycin S13 92 140 130 1.4 1.5 Ai

Leucanicidin S14 1.2 2.5 1.4 1.3 2.1

PC-766B S15 1.2 2.5 1.3 1.1 2.1

Elaiophylin S16 250 ND 160 N/A 0.55

7-one Bafilomycin Ai S17 >1000 >1000 >1000 — —

7,21 -one Bafilomycin — —

S18 >1000 >1000 >1000 Ai

CMF 6 1.6 5.8 2.9 1.8 4.0

CMX 7 9.1 38 14 1.6 4.1

CMC 5 0.71 3.7 1.3 2.3 7.1

21-Deoxy-16-Hydroxy

S25 400 >1000 >1000 — — CMF

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