Rifamycins against nontuberculous mycobacteria

Abstract

Disclosed herein are compounds of formula I and methods of using same to treat or prevent infections caused by mycobacteria in a subject in need thereof, comprising administering to the subject a compound of formula I, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a compound of formula I. JPEG2025536245000185.jpg61150

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 413,472, filed October 5, 2022, the contents of which are incorporated by reference in their entirety. Statement Regarding Federally Sponsored Research This invention was made with government support under grants AI132374, AI142731, and AI177342 awarded by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0002] Nontuberculous mycobacteria (NTM) can cause pulmonary and extrapulmonary infections. Mycobacterium abscessus (M. abscessus), first isolated in 1952, is the second most common pathogenic NTM and is recognized as the most common etiologic agent of pulmonary infections caused by rapidly proliferating mycobacteria. Current treatment outcomes for mycobacteria, such as M. abscessus infections, remain unstable and vary among subspecies. Subspecies abscessus is the most lethal, with treatment success rates typically below 50%, ranging from 20% to 40%. The poor treatment outcomes for mycobacterial infections are largely related to the intrinsic resistance of this pathogen to most existing antibiotics. Therefore, there is a need to develop new therapeutic agents that can be used as potent and safe antibiotics against mycobacteria, particularly non-tuberculous mycobacteria. Summary of the Invention

[0003] Disclosed herein are compounds and methods for treating or preventing bacterial infections, such as mycobacterial infections. One aspect of this technology is a compound of Formula I: [ka] I or a pharmaceutically acceptable salt thereof.

[0004] In compounds of formula I, R 1 is an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl, an optionally substituted cycloalkyl, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted heterocycloalkyl, -NR 4 R 5 , and -NHSO2R 6 and R 4 and R 5 are independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, or R 4 and R 5 together with the nitrogen atom to which they are attached, form N, O, and S(O) 0~2 and R forms an optionally substituted 4- to 8-membered heterocycloalkyl containing one or more heteroatoms selected from the group consisting of 6 is selected from the group consisting of optionally substituted alkyl, optionally substituted aryl, and optionally substituted heteroaryl; [ka] is the formula (a), the formula (b), or the formula (c), [ka] and In the formula, N1 and N2 are independently an integer of 1 to 3, and Y 1 is N, O, or CR i and 2is selected from hydrogen, optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, and -(CH) n R 3 and R i is hydrogen, halo, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, or R i and R 2 together with the carbon atom to which they are attached form an optionally substituted 4- to 8-membered heterocycloalkyl, n is an integer from 1 to 6, and R 3 -OR 7 , -NR 8 R 9 , -C(O)OR 10 , -S(O)2OR 11 and heteroaryl, R 7 , R 8 , R 9 , R 10 , and R 11 is independently selected from the group consisting of hydrogen, alkyl, and haloalkyl; R 12 is heteroaryl, N3 is an integer of 1 to 2, and R 1 is not methyl. Exemplary embodiments of the compounds are disclosed herein.

[0005] Another aspect of this technology provides a method for treating or preventing an infection caused by a mycobacterium in a subject in need thereof, comprising administering to the subject one or more of the compounds disclosed herein or pharmaceutically acceptable salts thereof. Preferably, the mycobacterium is Mycobacterium abscessus, Mycobacterium simiae, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium avium complex, Mycobacterium avium subsp. hominisuis, Mycobacterium intracellulare, Mycobacterium chimaera, Mycobacterium kansasii, Mycobacterium turugai, Mycobacterium szulgai, Mycobacterium xenopi, and combinations thereof. In some embodiments, the mycobacterium is Mycobacterium abscessus, which may optionally be selected from the group consisting of Mycobacterium abscessus subsp. abscessus, Mycobacterium abscessus subsp. bolletii, Mycobacterium abscessus subsp. massiliense, and combinations thereof.

[0006] Another aspect of this technology provides a method for treating an infection caused by bacteria in a subject in need thereof. The method includes administering to the subject one or more of the compounds disclosed herein or pharmaceutically acceptable salts thereof. The compound can be administered to a subject whose bacteria is resistant to rifampicin, rifabutin, or rifapentine, and the bacteria express an enzyme that catalyzes the ADP-ribosylation of rifampicin, rifabutin, or rifapentine. The compound can be administered to a subject whose bacteria expresses an enzyme that catalyzes the ADP-ribosylation of rifampicin, rifabutin, or rifapentine. In some embodiments, the enzyme is rifamycin ADP-ribosyltransferase. Preferably, the bacterium is a mycobacterium, which can optionally be selected from any of the mycobacteria disclosed herein.

[0007] Another aspect of this technology provides a method for treating or preventing an infection in a subject in need of an antibacterial compound. The method includes administering to the subject one or more of the compounds disclosed herein or pharmaceutically acceptable salts thereof. The administered compound may have a lower induction effect on cytochrome P450 enzymes than any one or more of rifampicin, rifabutin, and rifapentine. The cytochrome P450 enzyme may be P450 3A4. In some embodiments, the subject is receiving treatment with a compound that is metabolized by a cytochrome P450 enzyme or is in need of treatment with a compound that is metabolized by a cytochrome P450 enzyme. In some embodiments, the subject is receiving treatment with a compound that is sensitive to co-administration of an inducer of a cytochrome P450 enzyme or is in need of treatment with a compound that is sensitive to co-administration of an inducer of a cytochrome P450 enzyme. Preferably, the subject is infected with a mycobacterium, which may optionally be selected from any of the mycobacteria disclosed herein. Also provided are pharmaceutical compositions comprising one or more compounds disclosed herein for use in any of the methods disclosed herein. Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying drawings, which are schematic and are not intended to be drawn to scale. In the drawings, each identical or nearly identical component shown is typically represented by a single numeral. For clarity, not every component is labeled in every drawing, and not every component of every embodiment of the present invention is shown, and illustrations are not necessarily necessary to enable those skilled in the art to understand the present invention. [Brief explanation of the drawings]

[0008] [Figure 1] 1 shows that rifabutin is inactivated in M. abscessus. M. abscessus Arr catalyzes the formation of an ADP-ribosyl-oxocarbenium intermediate from NAD+ and subsequent ADP-ribosylation at C23-OH. [Figure 2A] Rifamycin design based on homology modeling shows that C-25 modifications prevent interaction with ArrMab. Figure 2A shows the structure of the prototypical compound 25-O-benzoylrifabutin (5a), in which the C25 acetyl group of rifabutin is replaced with a benzoyl group. [Figure 2B] Figure 2B shows the overall structure of the ArrMab homology model in which the ADP-ribosyl-oxocarbenium intermediate (molecule on the right) is complexed with 5a (molecule on the left). [Figure 2C] Figure 2C shows the proposed binding modes of rifabutin (left panel) and 5a (right panel) with the ArrMab homology model. The distance between C23-OH and oxocarbenium-C1' is measured and indicated by the dashed line. [Figure 3A] Active site structure of 5a (middle molecule) bound to RNAPMtb. Residues involved in key hydrogen bonds are shown as stick figures. H-bonds are shown as dashed lines. [Figure 3B] FIG. 1 shows that the C-25 benzoate (C25-OBz) of 5a (left) and F439 (right) form π-stacking interactions. [Figure 3C] Figure 1 shows that the C-25 benzoate of 5a (bottom left molecule) is located in the cavity formed by F439 and R173, which is indicated by the darker shading towards the right side of 5a. [Figure 4] Figure 7 shows in vitro characterization of ADP-ribosylation of rifamycin using overexpressed ArrMab. All peaks were identified by MS (Table 7). The extension time differences of the peaks are labeled. Rifabutin was completely converted to the ADP-ribosyl adduct over a 40-minute incubation period. In contrast, ArrMab-catalyzed conversion of synthetic compound 5a was not observed under the same incubation conditions. [Figure 5] Analog 5j exhibited bactericidal in vivo efficacy at 10 mg / kg. Animals infected with M. abscessus received drug treatment for 10 consecutive days. Drugs were administered once daily to groups of 6 mice per study group. On day 11 post-infection, organ homogenates were plated on agar medium to determine bacterial burden. Results were analyzed using one-way analysis of variance (ANOVA) multiple comparisons and Dunnett's post-hoc test. *, P<0.05; **, P<0.01; ***, P<0.001. D1: day 1. D11: day 11. CLR: clarithromycin. RBT: rifabutin. [Figure 6] Binding mode of rifampicin (middle molecule) in RNAPMtb (PDB:5UHB). The binding pocket is indicated by the lighter shading around the molecule. The space accommodating C25-OAc is outlined by a dashed line. [Figure 7] Figure 1 shows the proposed binding modes of rifabutin (a molecule without a six-membered ring on top) and 5a (a molecule with a six-membered ring on top) in the RNAPMab homology model. A high degree of overlap was observed between the two molecules. [Figure 8]Figure 1 shows a comparison of the co-crystal structures of 5a-RNAPMtb and rifampicin-RNAPMtb (PDB: 5UHB). Residues that directly interact with the ligand are labeled. The three-dimensional structure revealed that 5a adopts a very similar binding mode at the same binding site as rifampicin. [Figure 9] 1 shows the proposed binding modes and affinities of analogs 5b-5g in the RNA PMab homology model. A high degree of overlap was observed for these molecules. [Figures 10A-10G] Figure 10 shows in vitro characterization of ADP-ribosylation of rifamycin and synthetic compounds using overexpressed ArrMab. All peaks were further confirmed by MS (Table 6). The peak retention time differences are labeled. Control rifampicin (Figure 10A) and rifabutin (Figure 10B) were completely ADP-ribosylated after 40 min of incubation. No ADP-ribosylated adducts were observed with synthetic compounds 5a (Figure 10C), 5b (Figure 10D), and 5m (Figure 10E), which have bulky C-25 substituents. Synthetic compounds 5k (Figure 10F) and 5l (Figure 10G), which have small alkyl substituents at C-25, were unable to inhibit ADP-ribosylation and were converted to their respective ADP-ribosylated adducts over a 40 min time course of incubation. [Figure 11] FIG. 1 shows 1H NMR and 13C NMR data of compound 5a. [Figure 12] FIG. 1 shows 1H NMR and 13C NMR data of compound 5b. [Figure 13] FIG. 1 shows 1H NMR and 13C NMR data of compound 5c. [Figure 14] FIG. 1 shows 1H NMR and 13C NMR data of compound 5d. [Figure 15] FIG. 1 shows 1H NMR and 13C NMR data of compound 5e. [Figure 16] FIG. 1 shows 1H NMR and 13C NMR data of compound 5f. [Figure 17]FIG. 1 shows 1H NMR and 13C NMR data of compound 5g. [Figure 18] FIG. 1 shows 1H NMR and 13C NMR data of compound 5h. [Figure 19] FIG. 1 shows 1H NMR and 13C NMR data of compound 5i. [Figure 20] FIG. 1 shows 1H NMR and 13C NMR data of compound 5j. [Figure 21] FIG. 1 shows 1H NMR and 13C NMR data of compound 5k. [Figure 22] FIG. 1 shows 1H NMR and 13C NMR data of compound 5l. [Figure 23] FIG. 1 shows 1H NMR and 13C NMR data of compound 5m. [Figure 24] FIG. 1 shows 1H NMR and 13C NMR data of compound 5n. [Figure 25] FIG. 1 shows 1H NMR and 13C NMR data of compound 5o. [Figure 26] Figure 1 shows that the acylation of compound 3 using only anhydride or anhydride with a substoichiometric amount of DMAP resulted in little conversion of the starting material (cases 1 and 2). In certain cases, the relatively low yields were due to very slow conversion and extensive side reactions upon heating (cases 3 and 4). [Figure 27] FIG. 1 shows a plot of cLogP versus plasma unbound fraction. [Figure 28]Growth of M. abscessus Bamboo on caseum surrogate. The surrogate matrix was generated from cultured THP-1 cells (ATCC TIB-202) as previously described (mBio, 2023, Vol. 14, e0059823). Exponential cultures (OD600 0.6-0.9) of M. abscessus grown in Middlebrook 7H9 broth (Sigma-Aldrich) were centrifuged and resuspended in water to OD600 of 7, 0.7, and 0.07. As described in the M. tuberculosis caseum surrogate assay (mBio, 2023, Vol. 14, p. e0059823), bacterial suspensions (three different dilutions resulting in approximately 10, 10, and 10 starting CFU / mL, represented by circle, square, and triangle symbols, respectively) were added to caseum surrogate at a 2:1 (volume / mass) ratio, briefly homogenized with 1.4 mm zirconia beads, equally divided into nine 1.5 mL microcentrifuge tubes, and incubated at 37°C as static cultures. At designated time points, tubes were removed and used for CFU counting by plating on Middlebrook 7H11 agar (Sigma-Aldrich). Separate tubes were used for each time point. A culture with 10 starting CFU / mL was used to determine the kill curve (resulting in the middle curve with square symbols). The arrows indicate the time points at which drugs were added and the end of treatment. The experiment was independently repeated three times with similar results. A representative example is shown. The points and error bars represent the mean and standard deviation of three technical replicates, respectively. [Figures 29A-29M]Figure 28 shows dose-response kill curves for M. abscessus bamboo with caseum surrogate. M. abscessus cultures were prepared as described in the legend of Figure 28. Bacterial cell suspensions were added to a starting CFU of 10 / mL (Figure 28, middle growth curve with square symbols). On day 5, after the cultures entered stationary phase (Figure 28, first arrow), 50 μL of the mixture (culture with caseum surrogate) was exposed to drug (1 μL in DMSO). Amikacin, clarithromycin, clofazimine, imipenem, rifabutin, and tigecycline were purchased from Sigma-Aldrich; moxifloxacin and linezolid were purchased from Sequoia Research Products; and cefoxitin and bedaquiline were purchased from MedChemExpress. Rifabutin analogs were synthesized as described (doi:10.1002 / anie.202211498) and administered at doses ranging from 0.125 to 512 μM (128 μM for clofazimine and rifabutin analogs 5a, 5m, and 5n) for 5 days (10 days for bedaquiline, as described in Table 16) after which CFU were counted. The addition of 2% vehicle DMSO did not affect viable bacterial counts. The shaded area indicates the drug concentration range achieved in vivo. (Antimicrob.Agents Chemother., 2021, Vol. 65, p. e0050621; Antimicrob.Agents Chemother., 2022, Vol. 66, p. e0221221; PLoS Med., 2019, Vol. 16, p. e1002773; ACS Infect. Dis., 2016, Vol. 2, pp. 251-267). Experiments were independently repeated twice with similar results. Representative examples are shown. Points and error bars represent the mean and standard deviation of three technical replicates, respectively. The horizontal dotted line indicates the cutoff value for a 1-log reduction in CFU compared to drug-free control cultures for 10 days. The cMBC90 values ​​shown in Table 16 are the drug concentrations that reduce CFU by 90% compared to the CFU of the drug-free control at day 10. Because the cultures were in stationary phase on day 5 when drug treatment was initiated, the CFU of the drug-free cultures on day 10 were similar to the CFU of the drug-free cultures on day 5 (Figure 28). [Figure 30]More than 150 compounds were synthesized and their MIC data against Mycobacterium abscessus were evaluated. [Figure 31] This figure shows that 88 compounds met the activity criterion (MIC<100 nM against Mycobacterium abscessus). A comprehensive SAR was developed for the rational design of active compounds. [Figure 32] Figure 1 shows a strong correlation between fu and clogP. clogP and logfu form a linear correlation with r2 = 0.66. [Figure 33]

[0023] Figure 1 shows that UMN22 and UMN34 show significantly better efficacy compared to RFB, both showing a ~2 log reduction in lung CFU burden compared to the RFB and untreated (UNRX) groups. DETAILED DESCRIPTION OF THE INVENTION

[0009] The present technology provides rifabutin analogs and methods of using them to treat infections caused by nontuberculous mycobacteria. The disclosed compounds are derivatized at the ansa chain of rifabutin, specifically at C25, to increase their potency against M. abscessus by directly addressing the problem of rifamycin inactivation by ADP-ribosylation. Rifabutin analogs include, but are not limited to, C25 carbamates, sulfonamides, and carboxylic acid esters. The disclosed compounds restore the inherent low-nanomolar antimycobacterial activity of rifamycin antibiotics and have been shown to possess significant in vitro and in vivo antibacterial efficacy. Furthermore, the disclosed compounds exhibit substantially lower CYP induction than rifampicin or rifabutin.

[0010] One aspect of this technology is a compound of formula I: [ka] I or a pharmaceutically acceptable salt thereof; During the ceremony, R 1 is an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl, an optionally substituted cycloalkyl, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted heterocycloalkyl, -NR 4 R 5 , and -NHSO2R 6 is selected from the group consisting of R 4 and R 5 are independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl; or R 4 and R 5 together with the nitrogen atom to which they are attached, form N, O, and S(O) 0~2 forming an optionally substituted 4- to 8-membered heterocycloalkyl containing one or more heteroatoms selected from the group consisting of R 6 is selected from the group consisting of optionally substituted alkyl, optionally substituted aryl, and optionally substituted heteroaryl; [ka] is the formula (a), the formula (b), or the formula (c), [ka] and During the ceremony, N1 and N2 are independently integers of 1 to 3, Y 1 is N, O, or CR i and R 2is selected from hydrogen, optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, and -(CH) n R 3 is selected from the group consisting of R i is hydrogen, halo, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, or R i and R 2 together with the carbon atom to which they are attached form an optionally substituted 4- to 8-membered heterocycloalkyl; n is an integer from 1 to 6, R 3 -OR 7 , -NR 8 R 9 , -C(O)OR 10 , -S(O)2OR 11 and heteroaryl; R 7 , R 8 , R 9 , R 10 , and R 11 is independently selected from the group consisting of hydrogen, alkyl, and haloalkyl; R 12 is heteroaryl, N3 is an integer from 1 to 2, The compound is not rifabutin.

[0011] In some embodiments, R 1 is not methyl, ethyl, or propyl, or substituted methyl, ethyl, or propyl. In some embodiments, R 1 is not an alkyl substituted with a carbonyl-containing group such as a carboxylic acid (-COOH), an ester (-COOR), or an amide (-CONRR'). The term "alkyl" includes all isomeric forms of straight or branched chain alkyl groups, such as straight or branched chain groups of 1 to 12, 1 to 10, or 1 to 6 carbon atoms, and is defined herein as C1 to C6, respectively. 12 Alkyl, C1-C 10 It is called alkyl, and C1-C6 alkyl. The term "aryl" refers to a carbocyclic aromatic group. Representative aryl groups include phenyl, naphthyl, anthracenyl, and the like. The term "aryl" includes polycyclic ring systems having two or more carbocyclic rings where two or more carbons are common to two adjacent rings (the rings are "fused rings"), where at least one of the rings is aromatic and the other rings may be, for example, cycloalkyl, cycloalkenyl, cycloalkynyl, and / or aryl. In some embodiments, aryl groups are 6-10 membered ring structures (i.e., C6-C8 10 aryl). The term "heteroaryl" refers to an aromatic ring structure containing a specified number of ring atoms, at least one of which is a heteroatom (i.e., oxygen, nitrogen, or S(O)). 0~2 ) and the remaining ring atoms are C(O) 0~1 , oxygen, nitrogen, and sulfur. A 5-6 membered heteroaryl has 5 or 6 ring atoms, at least one of which is N, O, or S(O). 0~2 Similarly, a 5-10 membered heteroaryl has 5-10 ring atoms, at least one of which is N, O, or S(O). 0~2 Heteroaryls are aromatic ring systems in which: Heteroaryls may contain two or more fused rings.

[0012] Examples of heteroaryl substituents include 6-membered ring substituents such as pyridinyl, pyrazinyl, pyrimidinyl, and pyridazinyl; 5-membered ring substituents such as triazolyl, imidazolyl, furanyl, thiophenyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, 1,2,3-, 1,2,4-, 1,2,5-, or 1,3,4-oxadiazolyl, and isothiazolyl; 6 / 5-membered fused ring substituents such as benzothiofuranyl, isobenzothiofuranyl, benzisoxazolyl, benzoxazolyl, purinyl, and anthranilyl; and 6 / 6-membered fused rings such as quinolinyl, isoquinolinyl, cinnolinyl, quinazolinyl, and 1,4-benzoxazinyl. In groups having a heteroaryl substituent, the ring atoms of the heteroaryl substituent bonded to the group may be at least one heteroatom or ring carbon atom, and the ring carbon atom may be in the same ring as the at least one heteroatom, or the ring carbon atom may be in a different ring than the at least one heteroatom. Similarly, when a heteroaryl substituent is in turn substituted with a group or substituent, the group or substituent may be bonded to at least one heteroatom or ring carbon atom, and the ring carbon atom may be in the same ring as the at least one heteroatom, or the ring carbon atom may be in a different ring than the at least one heteroatom. Examples of 2-fused ring heteroaryls include indolizinyl, pyranopyrrolyl, 4H-quinolidinyl, purinyl, naphthyridinyl, pyridopyridinyl (including pyrido[3,4-b]-pyridinyl, pyrido[3,2-b]-pyridinyl, or pyrido[4,3-5]-pyridinyl), pyrrolopyridinyl, pyrazolopyridinyl, and imidazothiazolyl, and pteridinyl.

[0013] Other examples of fused ring heteroaryls include benzo-fused heteroaryls such as indolyl, isoindolyl, indoleninyl, isoindazolyl, benzazinyl (including quinolinyl or isoquinolinyl), phthalazinyl, quinoxalinyl, benzodiazinyl (including cinnolinyl or quinazolinyl), benzopyranyl, benzothiopyranyl, benzoxazolyl, indoxazinyl, anthranilyl, benzodioxolyl, benzodioxanyl, benzoxadiazolyl, benzofuranyl, isobenzofuranyl, benzothienyl, isobenzothienyl, benzothiazolyl, benzothiadiazolyl, benzimidazolyl, benzotriazolyl, benzoxazinyl, benzisoxazinyl, and the like. The term "optionally substituted" refers to groups (e.g., alkyl, aryl, and heteroaryl) that are unsubstituted or substituted with one or more substituents independently selected from the group consisting of halo, azido, alkyl, alkenyl, alkynyl, alkylaryl, cycloalkyl, heterocycloalkyl, hydroxyl, alkoxy, amino, nitro, amido, -C(O)H, -C(O)-alkyl, -C(O)O-alkyl, carboxyl, alkylthio, sulfonamido, -S(O)-alkyl, aryl, heteroaryl, haloalkyl, cyano, carboxylic ester, and hydroxyalkyl. The term "halo" refers to a halogen atom or halogen radical (e.g., -F, -Cl, -Br, or -I).

[0014] The term "haloalkyl" refers to an alkyl group substituted with at least one halogen, such as, for example, -CH2F, -CHF2, -CF3, -CH2CF3, and -CF2CF3. The term "azide" refers to the radical -N=N + =N - (i.e., -N3). The term "alkenyl," as used herein, refers to C2 to C6 alkyl groups, such as straight or branched chain groups of 2 to 12, 2 to 10, or 2 to 6 carbon atoms, respectively. 12 Alkenyl, C2-C10 Alkenyl, and also referred to as C2-C6 alkenyl, refers to an unsaturated straight or branched chain hydrocarbon having at least one carbon-carbon double bond. The term "alkynyl" as used herein refers to C2 to C6 alkyl groups, such as straight or branched chain groups of 2 to 12, 2 to 10, or 2 to 6 carbon atoms, respectively. 12 Alkynyl, C2-C 10 Alkynyl, and C2-C6 alkynyl, refers to an unsaturated straight or branched chain hydrocarbon having at least one carbon-carbon triple bond. The term "alkylaryl" refers to an alkyl substituted with an aryl. In some embodiments, the alkylaryl group is benzyl.

[0015] The term "alkylheteroaryl" refers to an alkyl substituted with a heteroaryl. The term "cycloalkyl" refers to a carbocyclic substituent having a specified number of carbon atoms derived by removing a hydrogen atom from a saturated carbocyclic molecule. In one embodiment, a cycloalkyl substituent has from 3 to 7 carbon atoms (i.e., C3-C7 cycloalkyl). Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. The term "cycloalkyl" includes mono-, bi-, and tricyclic saturated carbocycles, as well as bridged and fused ring carbocycles, and spiro-fused ring systems. The term "heterocycloalkyl" refers to a monovalent saturated cyclic, bicyclic, or bridged cyclic hydrocarbon group of 3 to 12, 3 to 8, 4 to 8, or 4 to 6 carbons, wherein at least one carbon of the cycloalkane is replaced by, for example, N, O, and / or S(O). nand the like, where n is an integer of 0 to 2. "4- to 7-membered heterocycloalkyl" refers to a heterocycloalkyl containing 4 to 7 atoms, including one or more heteroatoms, in the cyclic portion of the heterocycloalkyl. Examples of single-ring heterocycloalkyls include azetidinyl, oxetanyl, thietanyl, dihydrofuranyl, tetrahydrofuranyl, dihydrothiophenyl, tetrahydrothiophenyl, pyrrolinyl, pyrrolidinyl, imidazolinyl, imidazolidinyl, pyrazolinyl, pyrazolidinyl, thiazolinyl, isothiazolinyl, thiazolidinyl, isothiazolidinyl, dihydropyranyl, piperidinyl, morpholinyl, piperazinyl, azepinyl, oxepinyl, and diazepinyl. In some embodiments, the heterocycloalkyls described herein may be fused to a cycloalkyl, aryl, or heteroaryl described herein.

[0016] The term "hydroxyl" refers to an "-OH" substituent. The term "hydroxyalkyl" refers to an alkyl substituted with a hydroxyl group. The term "cyano" refers to a "-CN" substituent. The terms "alkoxy" or "alkoxyl" refer to an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxy groups include methoxy, ethoxy, and tert-butoxy. The terms "amine" and "amino" refer to both unsubstituted and substituted amines (e.g., mono- or di-substituted amines), where the substituents can include, for example, alkyl, cycloalkyl, heterocyclyl, alkenyl, aryl, and amino. The term "nitro" refers to a "-NO2" substituent. The term "amide," as used herein, refers to the radical --C(O)NRR' or --NR--C(O)R', where R and R' may independently be hydrogen, alkyl, aryl, arylalkyl, cycloalkyl, formyl, haloalkyl, heteroaryl, heterocyclyl, or amino.

[0017] The terms "carboxy" or "carboxyl" as used herein refer to the radical --COOH. The term "carboxylic acid ester," as used herein, refers to the radical -C(O)OR or -OC(O)R, where R is a non-hydrogen group including, but not limited to, alkyl, aryl, arylalkyl, cycloalkyl, haloalkyl, heteroaryl, or heterocyclyl. The term "alkylthio" refers to the radical -S-alkyl. The term "sulfonamide," as used herein, refers to the radical -S(O)NRR' or -NR-S(O)R', where R and R' can be the same or different. For example, R and R' can independently be hydrogen, alkyl, aryl, arylalkyl, cycloalkyl, formyl, haloalkyl, heteroaryl, or heterocyclyl.

[0018] The term "pyridyl" as used herein refers to the following radical: [ka] Refers to... The term "pyrimidyl" as used herein refers to the following radical: [ka] Refers to... The term "thiazolyl" as used herein refers to the radical [ka] Refers to...

[0019] The term "pharmaceutically acceptable salt" refers to a salt of a compound disclosed herein that is substantially non-toxic to living organisms. Typical pharmaceutically acceptable salts include salts prepared by reacting a compound disclosed herein with an organic / inorganic acid or base. Such salts are known as acid addition salts and base addition salts. The skilled reader will understand that all compounds disclosed herein can form salts, and that salt forms of pharmaceuticals are commonly used because they are more easily crystallized and purified than the free acid or base. R 1 is an aryl optionally substituted with one or more alkyls, an alkoxy optionally substituted with halo or hydroxyl, a hydroxyl, a hydroxyalkyl, a halo, a haloalkyl, a haloalkoxy, a cyano, an aryl, -C(O)OZ 1 , -C(O)NZ 2 Z 3 , -S(O)Z 4 , -S(O)Z 5 , -S(O)2NZ 6 Z 7 or an amide, wherein Z 1 , Z 2 , Z 3 , Z 4 , Z 5 , Z 6 , and Z 7 is independently hydrogen, haloalkyl, alkyl, or —C(O)-alkyl.

[0020] R 1 may be heteroaryl optionally substituted with one or more hydroxyl, halo, optionally substituted amino, amido, haloalkyl, or carboxylic acid ester. R 1 Ha-NR 4 R 5 and R 4 is hydrogen and R 5is alkyl optionally substituted with one or more alkynyls or heteroaryls, wherein said heteroaryl is alkyl optionally substituted with one or more aryls, or one or more alkyls optionally substituted with one or more aryls or carboxylic acid esters. R 1 Ha-NR 4 R 5 R may be 4 is hydrogen and R 5 is one or more of alkylaryl, alkoxy, amino, aryl, -C(O)OZ 8 or alkyl optionally substituted with one or more haloalkyl or hydroxyl. R 1 is -NR 4 R 5 R may be 4 is hydrogen and R 5 is one or more of aryl, alkoxy, alkyl, -C(O)OZ 8 , -C(O)NZ 9 Z 10 , -S(O)Z 11 , or -S(O)2NZ 12 Z 13 and optionally substituted aryl. R 1 Ha-NR 4 R 5 R may be 4 is hydrogen and R 5 is one or more of alkyl, halo, haloalkyl, amino, hydroxyl, -OC(O)Z 8 or heteroaryl which may be substituted with an aryl which may be substituted with an aryl.

[0021] In the above, Z 8 , Z 9 , Z 10 , Z 11 , Z 12 , and Z 13may independently be hydrogen, alkyl, or —C(O)-alkyl. R 1 is R 4 and R 5 together with the nitrogen atom to which they are attached, N, O, and S(O) 0~2 and forming a 4- to 8-membered heterocycloalkyl containing one or more heteroatoms selected from the group consisting of: wherein the heterocycloalkyl is optionally substituted with one or more aryl, amino, haloalkyl, hydroxyalkyl, alkoxy, carboxylic acid ester, or alkyl. R 1 is -NHSO2R 6 R may be 6 is heteroaryl or aryl optionally substituted with one or more halo, alkoxy, or haloalkyl. R 1 may be alkyl. R 1 may be alkynyl. R 1 may be cycloalkyl. In some embodiments, R of the compound of formula I 1 is an optionally substituted aryl or an optionally substituted heteroaryl.

[0022] In some embodiments, the compound of Formula I has the formula I(a): [ka] I(a) and In the formula, R 1 is alkyl, -NR 4 R 5 , -NHSO2R 6, cycloalkyl, heteroaryl optionally substituted with one or more hydroxyl, halo, optionally substituted amino, amido, haloalkyl, or carboxylic acid ester, and alkoxy optionally substituted with one or more alkyl, halo, or hydroxyl, hydroxyl, hydroxyalkyl, halo, haloalkyl, haloalkoxy, cyano, aryl, -C(O)OZ 1 , -C(O)NZ 2 Z 3 , S(O)Z 4 , S(O)2Z 5 , -S(O)2NZ 6 Z 7 or aryl optionally substituted with an amide, wherein Z 1 , Z 2 , Z 3 , Z 4 , Z 5 , Z 6 , and Z 7 is independently hydrogen, alkyl, or —C(O)-alkyl.

[0023] In some embodiments, R of the compound of formula I 1 is as follows: [ka] [ka] [ka]

[0024] In some embodiments, the compound of formula I is: [ka] [ka] [ka]

[0025] In some embodiments, R of the compound of formula I 1 Ha-NR 4 R 5 In some such embodiments, R 4 and R 5 is hydrogen, alkylheteroaryl optionally substituted with aryl, one or more alkylaryl, alkoxy, amino, aryl, -C(O)OZ 8 or heterocycloalkyl optionally substituted with one or more haloalkyl or alkyl optionally substituted with hydroxyl, one or more aryl, alkoxy, alkyl, -C(O)OZ 8 , -C(O)NZ 9 Z 10 , -S(O)Z 11 , or -S(O)2NZ 12 Z 13 and one or more aryl, halo, haloalkyl, -OC(O)Z 8 , amino, or heteroaryl optionally substituted with hydroxyl, or R 4 and R 5 together with the nitrogen atom to which they are attached, form N, O, and S(O) 0~2 and forming a 4-8 membered heterocycloalkyl containing one or more heteroatoms selected from the group consisting of: 8 , Z 9 , Z 10 , Z 11 , Z 12, and Z 13 is independently hydrogen, alkyl, or —C(O)-alkyl.

[0026] In some embodiments, R of the compound of formula I 1 is as follows: [ka] [ka] [ka]

[0027] In some embodiments, the compound of formula I is: [ka] [ka] [ka] [ka]

[0028] In some embodiments, R of the compound of formula I 1 is NHS(O)2R 6 and R 6 is heteroaryl or aryl optionally substituted with one or more halo, alkoxy, or haloalkyl.

[0029] In some such embodiments, the compound of formula I is: [ka] is.

[0030] In some embodiments, formula (b) is: [ka] is.

[0031] In some embodiments, formula (c) is: [ka] is.

[0032] In some embodiments, R of the compound of formula I 1 is an optionally substituted aryl or an optionally substituted heteroaryl, [ka] is formula (a), N1 and N2 are 2, Y 1 is N, R 2 is selected from the group consisting of hydrogen and alkyl optionally substituted with one or more hydroxyl, amino, carboxyl, or heteroaryl.

[0033] In some such embodiments, R 2 teeth, [ka] is selected from the group consisting of:

[0034] In some embodiments, R of the compound of formula I 1is an optionally substituted aryl or an optionally substituted heteroaryl, N1 and N2 are independently 1 or 3, N3 is 1, and Y 1 is N and R 2 is alkyl.

[0035] In some such embodiments, [ka] teeth, [ka] is selected from the group consisting of:

[0036] In some embodiments, R of the compound of formula I 1 teeth, [ka] is selected from the group consisting of [ka] teeth, [ka] is selected from the group consisting of:

[0037] In some embodiments, the compound of formula I is [ka] is.

[0038] In some embodiments, the compounds of Formula I are antibacterial. The term "antibacterial" refers to the ability of a compound to prevent the growth or spread of bacteria. The antibacterial properties of the compounds disclosed herein can be assessed by the minimum inhibitory concentration (MIC) that results in 90% growth inhibition (MIC) of nontuberculous mycobacteria, including, but not limited to, Mycobacterium abscessus, Mycobacterium simiae, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium avium complex, Mycobacterium avium subsp. hominisuis, Mycobacterium intracellulare, Mycobacterium chimera, Mycobacterium kansasii, Mycobacterium turgai, Mycobacterium xenopi, and combinations thereof. In some embodiments, the Mycobacterium abscessus is selected from the group consisting of Mycobacterium abscessus subsp. abscessus, Mycobacterium abscessus subsp. bollettii, Mycobacterium abscessus subsp. massiliense, and combinations thereof.

[0039] In some embodiments, compounds disclosed herein have an MIC of less than 1200 nM. In some embodiments, compounds disclosed herein have an MIC of 1 nM to 1150 nM, 1 nM to 1100 nM, 1 nM to 600 nM, 1 nM to 300 nM, 1 nM to 200 nM, 1 nM to 100 nM, 1 nM to 75 nM, 1 nM to 70 nM, 1 nM to 65 nM, 1 nM to 60 nM, 1 nM to 55 nM, 1 nM to 50 nM, 1 nM to 45 nM, 1 nM to 40 nM, 1 nM to 35 nM, 1 nM to 30 nM, or 1 nM to 25 nM. In some embodiments, the compounds of Formula I have a lower induction effect on cytochrome P450 (CYP) enzymes than rifampicin, rifabutin, or rifapentine. Cytochrome P450 is a family of isozymes responsible for the biotransformation of several drugs. Cytochrome P450 is a family of isozymes responsible for the oxidative biotransformation of numerous drugs. Drug metabolism by the cytochrome P450 system has emerged as an important determinant in the occurrence of several drug interactions that can result in drug toxicity, reduced pharmacological efficacy, and adverse drug reactions.

[0040] CYP enzyme inducers increase the amount of CYP enzymes and thus the rate of metabolism of CYP substrates. Increased metabolism can affect a patient's response to a particular drug, potentially rendering it ineffective. CYP enzyme inducers can be characterized as strong inducers, i.e., compounds that cause a ≥80% reduction in the area under the curve (AUC) of the substrate; moderate inducers, i.e., compounds that cause a ≥50% to <80% reduction in the area under the curve (AUC) of the substrate; or weak inducers, i.e., compounds that cause a <50% reduction in the area under the curve (AUC) of the substrate. Rifampicin, rifabutin, and rifapentine are examples of CYP enzyme inducers that decrease the plasma concentrations of coadministered CYP substrates. Rifampicin is a strong inducer of CYP3A4, CYP3A5, and CYP2C8 and a moderate inducer of CYP1A2, CYP2B6, CYP2C9, and CYP2C19. Rifabutin is a moderate potentiator of CYP3A4 and CYP3A5. Rifapentine is a strong inducer of CYP3A4. In some embodiments, the compounds disclosed herein have a CYP3A4 gene induction fold of less than 20, as determined by the assay described in Example 9. Suitably, the compounds have a CYP3A4 gene induction fold of 0.5 and 20, 0.5 and 10, 0.5 and 5, 0.5 and 4, 0.5 and 3, 0.5 and 2, or 0.5 and 1.

[0041] Another aspect of the present technology is a pharmaceutical composition comprising a therapeutically effective amount of a compound disclosed herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient, or diluent. The phrase "therapeutically effective amount" refers to a dosage of a compound that provides a specific pharmacological response for administration to a significant number of subjects in need of such treatment. The effective amount of a drug administered to a particular subject in a particular case will not always be effective in treating the conditions / diseases described herein, even if such a dosage is considered to be a therapeutically effective amount by those skilled in the art. The phrase "pharmaceutically acceptable carrier, excipient, or diluent" refers to a carrier, excipient, or diluent that is not biologically or otherwise undesirable and is generally non-toxic, useful in preparing a pharmaceutical composition. The pharmaceutical compositions disclosed herein may include carriers, excipients, or diluents that are acceptable for veterinary use as well as human pharmaceutical use. Examples of "pharmaceutically acceptable carriers" include proteins, carbohydrates, sugars, talc, magnesium stearate, cellulose, calcium carbonate, and / or starch-gelatin paste.

[0042] Examples of "pharmaceutically acceptable excipients" include binders, fillers, lubricants, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, and effervescent agents. Fillers include lactose monohydrate, lactose anhydrous, and various starches. Examples of binders include various celluloses and cross-linked polyvinylpyrrolidone, microcrystalline cellulose such as Avicel® PH101 and Avicel® PH102, microcrystalline cellulose, and silicified microcrystalline cellulose (ProSolv SMCC™). Suitable lubricants, including agents that affect the flowability of the powder to be compressed, include colloidal silicon dioxide, such as Aerosil® 200, talc, stearic acid, magnesium stearate, calcium stearate, and silica gel. Examples of sweeteners include any natural or artificial sweetener, such as sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acesulfame. Examples of flavoring agents include Magnasweet® (a trademark of MAFCO), bubble gum flavor, and fruit flavors. Examples of preservatives include potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of parahydroxybenzoic acid, such as butylparaben, alcohols, such as ethyl alcohol or benzyl alcohol, phenolic compounds, such as phenol, or quaternary compounds, such as benzalkonium chloride. Examples of effervescent agents include effervescent couples, such as organic acids and carbonates or bicarbonates. Suitable organic acids include, for example, citric acid, tartaric acid, malic acid, fumaric acid, adipic acid, succinic acid, and alginic acid, as well as anhydrides and acid salts. Suitable carbonates and bicarbonates include, for example, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium glycine carbonate, L-lysine carbonate, and arginine carbonate. Alternatively, only the sodium bicarbonate component of the effervescent couple may be present.Suitable disintegrants include lightly cross-linked polyvinylpyrrolidone, corn starch, potato starch, maize starch, and modified starches, croscarmellose sodium, cros-povidone, sodium starch glycolate, and mixtures thereof.

[0043] Examples of "pharmaceutically acceptable diluents" include pharmaceutically acceptable inert fillers such as microcrystalline cellulose, lactose, calcium hydrogen phosphate, saccharides, and mixtures of any of the foregoing. Examples of diluents include microcrystalline cellulose such as Avicel® PH101 and Avicel® PH102, lactose such as lactose monohydrate, lactose anhydrous, and Pharmatose® DCL21, calcium hydrogen phosphate such as Emcompress®, mannitol, starch, sorbitol, sucrose, and glucose. Another aspect of this technology provides a method for treating or preventing an infection caused by a mycobacterium in a subject in need thereof, comprising administering to the subject a compound disclosed herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition disclosed herein.

[0044] In some embodiments, the nontuberculous mycobacterium is selected from the group consisting of Mycobacterium abscessus, Mycobacterium simiae, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium chimaera, Mycobacterium kansasii, Mycobacterium turgai, Mycobacterium xenopi, and combinations thereof. In some embodiments, the mycobacterium is Mycobacterium abscessus. In some embodiments, the Mycobacterium abscessus is selected from the group consisting of Mycobacterium abscessus subsp. abscessus, Mycobacterium abscessus subsp. bollettii, Mycobacterium abscessus subsp. massiliense, and combinations thereof. In some embodiments, the Mycobacterium is Mycobacterium avium. In some embodiments, the Mycobacterium avium may be Mycobacterium avium subspecies hominisuis. In some embodiments, the compounds disclosed herein, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions disclosed herein are administered orally or intravenously. As used herein, the terms "treat," "treating," and "treatment" refer to eliminating, reducing, or ameliorating an infection, disease, or disorder and / or symptoms associated therewith. Although not exclusive, treating an infection, disease, or disorder need not completely eliminate the infection, disease, disorder, or symptoms associated therewith.

[0045] As used herein, the term "prevent" or "preventing" refers to reducing the chance of developing or re-developing an infection, disease, or disorder, or the chance of a previously controlled disease or condition recurring, in a subject who does not have the infection, disease, or disorder but who is at risk or susceptible to re-development of the infection, disease, or disorder or recurrence of the infection, disease, or disorder. The term "subject" refers to an animal, such as a mammal (e.g., a human), that has been the target of treatment, observation, or experiment. In some embodiments, the subject has an infection caused by any of the bacteria disclosed herein, such as a Mycobacterium. In some embodiments, the subject has an infection caused by a bacterium that is resistant to rifampicin, rifabutin, or rifapentine. In some embodiments, the subject has an infection caused by a bacterium that expresses an enzyme that catalyzes the ADP-ribosylation of rifampicin, rifabutin, or rifapentine, e.g., rifamycin ADP-ribosyltransferase. In some embodiments, the subject has an infection in which the bacterium is present in the subject's caseum. In some embodiments, the subject has caseous necrosis.

[0046] Another aspect of the present technology is a method for treating or preventing an infection caused by bacteria in a subject in need thereof. The method includes administering to the subject a compound disclosed herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition disclosed herein. The compound of the present disclosure may be suitable for treating a subject for bacteria resistant to rifampicin, rifabutin, or rifapentine. Rifamycin resistance in M. abscessus is caused by a group transfer inactivation mechanism mediated by rifamycin ADP-ribosyltransferase (Arr). Compounds of the present disclosure have demonstrated the ability to overcome Arr-mediated resistance in bacteria expressing rifamycin ADP-ribosyltransferase. Bacteria include, but are not limited to, mycobacteria, such as Mycobacterium abscessus, Mycobacterium simiae, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium avium complex, Mycobacterium avium subsp. hominisuis, Mycobacterium intracellulare, Mycobacterium chimaera, Mycobacterium kansasii, Mycobacterium turgai, Mycobacterium xenopi, Mycobacterium obuense, Mycobacterium phlei, Mycobacterium gilvum, Mycobacterium marinum, Mycobacterium marinum, or Mycobacterium scrofulaceum.

[0047] The therapeutic methods disclosed herein may also be useful for treating or preventing infections caused by bacteria other than mycobacteria. Examples of bacteria that express rifamycin ADP-ribosyltransferase include, but are not limited to, Gordonia bronchialis, Gordonia terrae, Tsukamurella paurometabolum, Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Streptomyces coelicolor, Clostridium bolteae, and Klebsiella oxytoca.

[0048] Another aspect of the present technology is a method for treating or preventing an infection in a subject requiring an antibacterial compound, comprising administering to the subject a compound according to claim 1 or a pharmaceutically acceptable salt thereof. The compound administered to the subject can be selected to reduce the possibility of drug-drug interactions compared to rifampicin, rifabutin, or rifapentine. Preferably, the antibacterial compound administered to the subject has a lower induction effect on cytochrome P450 enzymes, such as CYP3A4, than rifampicin, rifabutin, or rifapentine. In some cases, the subject is undergoing treatment with another therapeutic agent or compound that is metabolized by cytochrome P450 enzymes or is sensitive to co-administration of an inducer of cytochrome P450 enzymes. The compounds of Formula I described herein may contain one or more chiral centers and / or double bonds and therefore exist as stereoisomers, such as geometric isomers, enantiomers, or diastereomers. The term "stereoisomer," as used herein, consists of all geometric isomers, enantiomers, or diastereomers. Such compounds can be designated with the symbols "R" or "S," or "+" or "-," depending on the configuration of substituents around an asymmetric carbon atom or the observed optical rotation. Compounds of Formula I can encompass various stereoisomers and mixtures thereof. Stereoisomers include enantiomers and diastereomers. Mixtures of enantiomers or diastereomers can be designated (±) in the nomenclature, although those skilled in the art will recognize that chiral centers may be implicit by the structure. Unless otherwise indicated, graphical representations of chemical structures, e.g., basic chemical structures, are understood to encompass all stereoisomers of the specified compound. Compositions comprising, consisting essentially of, or consisting of enantiopure compounds are also contemplated herein, which may comprise, consist essentially of, or consist of at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of a single enantiomer of a given compound (e.g., at least about 99% R enantiomer of a given compound).

[0049] others Unless otherwise specified or dictated by context, the terms "a," "an," and "the" mean "one or more." For example, "a molecule" should be interpreted as meaning "one or more molecules."

[0050] As used herein, "about," "approximately," "substantially," and "significantly" will be understood by those of ordinary skill in the art and will vary to some extent depending on the context in which they are used. If there are uses of such terms that are not clear to persons of ordinary skill in the art given the context in which they are used, "about" and "approximately" will mean plus or minus ≦10% of the particular term, and "substantially" and "significantly" will mean plus or minus >10% of the particular term.

[0051] As used herein, "include" and "including" have the same meaning as the terms "comprise" and "comprising." The terms "comprise" and "comprising" should be interpreted as "open" transitional terms that allow for the inclusion of additional elements beyond those recited in a claim. The terms "consist" and "consisting of" should be interpreted as "closed" transitional terms that do not allow for the inclusion of additional elements beyond those recited in a claim. The term "consisting essentially of" should be interpreted as partially closed, allowing for the inclusion of only additional elements that do not fundamentally alter the nature of the claimed subject matter. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to better illustrate the invention and does not impose limitations on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0052] All references, including publications, patent applications, and patents, cited in this specification are herein incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein. Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of these preferred embodiments will become apparent to those of skill in the art upon reading the foregoing description. The inventors anticipate that skilled artisans will employ such variations as appropriate, and the inventors intend that the invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Furthermore, this invention includes any combination of the above-described elements in all possible variations unless otherwise indicated herein or otherwise clearly contradicted by circumstances. [Example]

[0053] Rifamycin antibiotics are a class of antimicrobial agents useful for treating infections caused by mycobacteria and other persistent bacteria due to their potent bactericidal activity against replicating and non-replicating pathogens. However, the clinical utility of rifamycins against Mycobacterium abscessus is severely compromised by a novel resistance mechanism: inactivation of rifamycins by ADP-ribosylation. Using a structure-based approach, we rationally redesigned rifamycins by strategically modifying the ansa chain to prevent ADP-ribosylation while preserving on-target activity. Validated through a combination of biochemical, structural, and microbiological studies, the most potent analogs overcame ADP-ribosylation, restored their original low-nanomolar activity, and demonstrated significant in vivo antibacterial efficacy. Further optimization by tuning the drug's pharmacokinetic properties yielded preclinical candidates that exhibited remarkable potency and excellent pharmacokinetic profiles. The compounds of the present disclosure exhibit a lower induction effect on cytochrome p450 enzymes than rifampicin, rifabutin, or rifapentine.

[0054] Example 1 Antimicrobial resistance (AMR) represents a significant and ever-increasing public health burden, resulting in approximately 2 to 5 million deaths worldwide each year. Among the resistance mechanisms used by microorganisms, enzymatic inactivation of antibiotics was one of the earliest identified, dating back to 1940, when β-lactamases were first reported to "destroy penicillin." While microorganisms can degrade antibiotics through hydrolysis or redox transformations, antibiotic modification by transgroup modification is the most chemically diverse drug inactivation mechanism. Numerous antibiotics, including aminoglycosides, macrolides, and lincosamides, are susceptible to transgroup inactivation through transformations such as acylation, phosphorylation, and glycosylation. Within this family of antibiotic modification chemistry, ADP-ribosylation of rifamycins is a particularly novel and noteworthy member. Natural product-derived rifamycin antibiotics function as inhibitors of bacterial transcription by allosterically binding to bacterial DNA-dependent RNA polymerase (RNAP) and preventing the elongation of nascent RNA. They exhibit potent bactericidal activity against Mycobacterium tuberculosis and numerous other pathogens. Due to their nanomolar antibacterial activity, rifamycins are known to be of clinical value, particularly in the treatment of persistent bacterial and mycobacterial infections, due to their remarkable efficacy against bacterial persisters, often in a dormant state within biofilms, which are highly tolerant to most antibiotics. However, rifamycin drugs are not clinically effective against Mycobacterium abscessus. M. abscessus is an emerging nontuberculous mycobacterium causing often fatal pulmonary infections, lacking reliable treatment options due to its inherent drug resistance to virtually all antibacterial classes. Rifamycin resistance in M. abscessus is caused by a transinactivation mechanism mediated by rifamycin ADP-ribosyltransferase (Arr). Arr is NAD +As a donor, the enzyme catalyzes the formation of an ADP-ribosyl-oxocarbenium intermediate, which is then regioselectively transferred to the C23-OH of the rifamycin polyketide ansa chain. This, in turn, prevents binding to bacterial RNAP and significantly reduces rifamycin efficacy (Figure 1). Consequently, for example, the semisynthetic rifamycin drug rifabutin exhibits low nanomolar activity against M. tuberculosis and numerous Gram-positive pathogens, but only micromolar activity against M. abscessus. Although Arr in M. abscessus and some other bacteria is the only known ADP-ribosyltransferase to date that targets small molecules, most bacterial ADP-ribosyltransferases are protein toxins that function by post-translational ADP-ribosylation of host proteins and are important virulence factors in Corynebacterium diphtheriae, Vibrio cholerae, Bordetella pertussis, and Clostridium botulinum. Thus, rifamycin ADP-ribosylation represents an unprecedented and novel mechanism of antimicrobial resistance.

[0055] To determine possible modification sites of rifamycins, we analyzed M. abscessus RNAP (RNAP Mab M. tuberculosis RNAP (RNAP) complexed with rifampicin, which shares 97% sequence identity with the rifamycin-binding site Mtb The 3D structure of rifamycin-binding domain (PDB: 5UHB, Figure 6) was used to investigate the rifamycin-binding mode in mycobacterial RNAP. The C23-OH, C21-OH, and C22-CH3 residues around the inactivating position are closely surrounded by the binding pocket, while the C24-CH3 and C25-OAc residues face the larger space of the pocket.

[0056] Results and Discussion C25 modification is RNAP MabTo explore whether rifamycin-binding to RNAP affects its binding, Mtb RNAP using structure 5UHB as a template Mab Molecular docking studies were performed against a homology model of rifabutin. Binding of the prototypical ligand 25-O-benzoylrifabutin (5a, Figure 2A), in which the bulkier benzoyl moiety was replaced with a C25 acetyl group, was investigated and showed a highly consistent binding mode compared to unmodified rifabutin at the conserved rifamycin binding site (Figure 7).

[0057] C25 modification of rifamycin inhibits the activity of M. abscessus Arr (Arr Mab To determine whether Arr disrupts the interaction with Arr, we used the Arr gene from M. smegmatis (sequence identity 64%, PDB: 2HW2). Mab A homology model of Arr was generated. The ADP-ribosyl-oxocarbenium intermediate was placed into this model, and the complex was further optimized using molecular dynamics simulations (Figure 2B). Mab Molecular docking analysis of 5a with rifabutin using the model revealed that 5a, in contrast to rifabutin, has a C23-OH and NAD + The distance between Arr and Cl' increased from 4.9 Å (rifabutin) to 7.1 Å (5a), suggesting the formation of a catalytically incompetent complex (Figure 2C). Based on these results, we hypothesized that rifamycin analogs with modifications at C25 would be better suited to RNAP but would be unable to undergo ADP-ribosylation by Arr, thereby restoring their high potency against M. abscessus.

[0058] A series of C-25 substituted rifamycin analogs containing a wide range of stereodiverse alkyl, aryl, and heteroaryl esters was synthesized (Scheme 1). Rifabutin was chosen as the template because it is the most potent rifamycin against M. abscessus, has the lowest P450 induction potential, and possesses the most favorable pharmacokinetic profile among clinically approved rifamycin antibiotics. Prior to initiating modifications at C25, the C21,23-diol was protected to give the acetonide-containing rifabutin 2. Carefully controlled methanolysis of 2 using potassium carbonate afforded the deacetylated intermediate 3, minimizing competitive lactam ring-opening by the electron-withdrawing naphthoquinone core, which enhances the reactivity of the amide linkage. Acylation of the newly liberated C25-OH proved extremely difficult. Steric hindrance of the adjacent acetonide group prevented acylation with less reactive reagents, whereas under stronger acylation conditions, the nucleophilic spiroimidazopiperidine N-3 amine also reacted, reducing regioselectivity. After extensive experimentation (Table 3), we found that regioselective esterification of the C25-OH could only be achieved by using a large excess of the acid anhydride or mixed anhydride formed in situ from the acid and pivaloyl chloride. The acetonide group was successfully cleaved using CSA in methanol to give the final rifabutin analogs. For the most sterically demanding substrates, the less sterically hindered 25-O-desacetylrifabutin 6 was obtained. [ was directly acylated using an acid anhydride to give the desired product after careful isolation. The structures of all final products were verified by 1D and 2D NMR. Representative examples of analogs 5e, 5g, and 5i are shown below. 1 H- 1 H NOESY spectra were acquired (see Table 1) to ensure that the configuration of the chiral centers, particularly C25, was maintained throughout the synthesis. [ka]

[0059] Scheme 1. Synthesis of 25-O-acyl rifabutin analogs: a) 2,2-dimethoxypropane, CSA, acetone, room temperature, 2 h, 68%; b) KCO, MeOH, 50 °C, 48 h, 59%; c) RC(O)OC(O)R, DMAP, 1,2-dichloroethane, room temperature or 50 °C, 72–96 h; d) RCOOH, pivaloyl chloride, triethylamine, DMAP, DCM, 0 °C–room temperature, 4 h; e) CSA, MeOH, room temperature, 0.5 h; f) NaOH, ZnCl, MeOH, room temperature, overnight, 71%. For 5a–5h and 5j–5o, the yields over two steps (c or d and then e) were 7–72%. For 5i, the yield from 25-O-desacetyl rifabutin 6 in step c was 6%. CSA = camphorsulfonic acid.

[0060] [Table 1]

[0061] To clarify the effect of C-25 modification on the potency of synthetic rifabutin analogs, we determined the minimum inhibitory concentrations (MICs) of these analogs that resulted in 90% growth inhibition against M. abscessus (Table 1). Wild-type (WT) M. abscessus ATCC 19977 strains and isogenic arr-deleted (Δarr) M. abscessus strains were used in parallel to assess the resistance phenotype. Clarithromycin, rifampicin, and rifabutin were included as controls. Compound 5a, which contains a 25-O-benzoyl (benzyol) group, had an MIC of 53 nM, which is 20- and 100-fold lower than that of rifabutin and rifampicin, respectively. More importantly, 5a was equally potent against WT and Δarr M. abscessus strains, indicating that this compound was no longer inactivated by Arr. Compounds with ortho-, meta-, and para-methylbenzoates (5b–5d) demonstrated that ortho-substitution was favorable. Further investigation of various ortho-substituted C-25 benzoates clearly demonstrated the effect of steric bulk on activity. Small groups (F, Cl, OMe 5e–5g) could be added without loss of activity, whereas bulkier groups (CF3, Ph 5h and 5i) caused a 2-fold and 5-fold decrease in potency compared to 5a, respectively. Analog 5j, which possesses a small F at the meta position of the C25-benzoate, also retained activity. On the other hand, analogs 5k and 5l, which possess small alkyl groups, exhibited only micromolar MIC values ​​against wild-type M. abscessus. Potent activity was only achieved against an isogenic Δarr deletion strain, indicating that 5k and 5l are inactivated by Arr. Furthermore, compounds 5a, 5b, and 5j were shown to maintain potent activity against a panel of drug-resistant M. abscessus clinical isolates (Table 4). Collectively, these results demonstrate that C-25 modification of rifabutin significantly increases its potency and is a viable strategy to effectively prevent rifamycin inactivation by ADP-ribosylation.

[0062] [Table 2]

[0063] [Table 3]

[0064] In the following activity screening using the rifamycin-resistant M. abscessus mutant RFB-R1, which harbors an RNAP point mutation, analogs 5a, 5b, and 5j lost detectable activity (Table 5), suggesting on-target activity through RNAP inhibition. To further understand how the C-25 modification of rifabutin affects binding to RNAP, we performed a rifabutin-specific ... Mtb The crystal structure of a representative compound, 5a, complexed with rifampicin was solved at 3.9 Å resolution (see Table 6 for data collection and refinement statistics). The three-dimensional structure reveals that 5a adopts a highly similar binding mode to rifampicin at the same binding site (Figure 8), preserving all essential hydrogen bonds with C1-O, C8-OH, C21-OH, and C23-OH (Figure 3A). A notable difference in the binding of 5a is that the C-25 benzoate forms a unique π-stacking interaction with Phe439, with the two phenyl rings exhibiting a "proximal translation" geometry (Figure 3B). These results suggest that molecular targeting RNAP can accommodate the bulky C-25 modification by forming a novel ligand-target interaction. A small cavity formed by Phe439 and Arg173 was found to encase the C-25 benzoate of 5a (Figure 3C), a phenomenon not observed in previous structures of RNAP-rifampicin complexes. This cavity may explain the lower potency of analogs with phenyl and trifluoromethyl substituted benzoates, since these very large substituents may not fit into this small cavity, thereby reducing overall binding affinity. Analogs 5b-5g bind to the RNAPs mentioned above. MabIt was proposed to bind to RNAP in a highly similar mode to 5a, as assessed by molecular docking studies using a homology model (Fig. 9 ).

[0065] [Table 4]

[0066] [Table 5]

[0067] To biochemically verify the effect of C-25 modification on ADP-ribosylation, we used recombinant Arr Mab We cloned, expressed, purified, and developed a liquid chromatography-mass spectrometry (LC-MS) enzymatic assay to quantify rifamycin and ADP-ribosylation adducts. Mab and NAD + When incubated with Δarr, rifampicin and rifabutin were completely converted within 40 min (Figure 4 and Figures 10A-10G), and a new peak was identified as the ADP-ribosyl adduct (ADP-ribosyl rifabutin m / z = 694.90; see Table 7 for MS identification of all LC signals). In contrast, no conversion was observed for 5a and 5b, which exhibited no loss of activity against WT M. abscessus with a functional antibiotic-inactivated Arr (Figure 4 and Figures 10A-10G). On the other hand, the small alkyl-substituted analogs 5k and 5l, which exhibited significantly shifted MICs against WT and Δarr M. abscessus strains, were completely converted by ADP-ribosylation (Figures 10A-10G). These results suggest that the 25-O-benzoyl rifabutin compound is a 25-O-benzoyl rifabutin compound with Arr. Mab It further shows that the compound cannot be modified by

[0068] [Table 6]

[0069] The promising in vitro activity of the rifabutin analogs led to an investigation of their in vivo PK properties. Candidates 5b and 5j were administered intravenously (iv) and orally (po) to CD1 mice to assess their PK parameters and determine optimal dose regimens for future efficacy studies (Table 2). Both compounds exhibited a smaller volume of distribution (V d ) and reduced clearance (CL), and 1 / 2 ) and increased in vivo drug exposure as measured by the area under the concentration-time curve (AUC). C25 deacetylation by esterases is the major metabolic pathway for rifamycin drugs. To assess whether candidate compounds 5b and 5j are also susceptible to enzymatic ester hydrolysis, we assessed the amount of 25-O-desacetylrifabutin 6 in mouse plasma after intravenous and oral administration. Both 5b and 5j exhibited high metabolic stability against potential hydrolysis in plasma or the intestine / liver, with less than 0.2% of 6 detected in both candidates via both administration routes (Table 2). Hydrolysis of rifabutin C-25 acetate was not evident in this mouse model, with only 0.4% of 6 detected via both administration routes.

[0070] [Table 7]

[0071] [Table 8]

[0072] Given the favorable PK profile, we next characterized the in vivo efficacy of the candidate compounds using an infected mouse model. Compound 5j, whose PK parameters were validated, was selected as a candidate. 5j, clarithromycin, rifabutin, and vehicle were orally administered once daily to M. abscessus-infected mice for 10 consecutive days, and then the bacterial burden in the lungs and spleen was assessed. Drug efficacy was defined as a statistically significant reduction in colony-forming units (CFU) in the test group relative to the vehicle control at the end of the experiment (Figure 5). In this model, candidate compound 5j significantly reduced the bacterial burden in the lungs by 10-fold and achieved a similar level of in vivo bactericidal activity to the positive control clarithromycin, a widely used anti-M. abscessus drug. The comparable efficacy of 5j and clarithromycin was also reflected in the reduction of spleen CFU. However, 5j did not exhibit improved efficacy compared to rifabutin, as both compounds reduced lung and spleen CFU counts to similar extents.

[0073] Further investigation of the pharmacokinetic / pharmacodynamic (PK / PD) profiles of the candidate compounds revealed that plasma protein binding (PPB) was likely a limiting factor. Both 5b and 5j were highly protein-bound as measured by the PPB assay, and the percentage of unbound drug in plasma was significantly lower than that of rifabutin (126-fold and 380-fold lower, respectively; Table 2). Given that the free concentration of antimycobacterial drugs correlates highly with their in vivo efficacy, we selected the ratio of the area under the unbound drug concentration-time profile to the MIC (fAUC / MIC) as a PK / PD index to determine efficacy, and sought rifabutin analogs with higher fAUC / MIC in subsequent compound optimization.

[0074] Characterization of synthetic rifabutin analogs revealed that switching from C25-acetate to benzoate increased the lipophilicity of the molecule, as reflected by longer LC retention times. Therefore, we suspected that the lipophilicity of the analogs may contribute to their PPB. To further optimize the candidate compounds with the goal of reducing the lipophilicity of the C-25 substituent while maintaining favorable potency, the phenyl group of 5a was replaced with bioisosteric heterocycles to generate analogs bearing 3-pyridyl (5m), 5-pyrimidyl (5n), and 2-thiazolyl (5o) groups. All heterocyclic analogs were found to be even more potent, with MICs as low as 17 nM, and insensitive to ADP-ribosylation (Table 1). PK characterization revealed that candidate compound 5m exhibited favorable V d Not only did 5m maintain its low clearance and high stability against C-25 ester cleavage, it also showed at least a 100-fold increase in the unbound fraction in plasma. The combination of the dramatic increase in plasma-free fraction and the low MIC of 5m led to an impressive fAUC / MIC value of 28.2, which is 128-fold higher than that of rifabutin (Table 2). Furthermore, 5m exhibits antibacterial activity against Mycobacterium simiae (M. simiae), Mycobacterium chelonae (M. chelonae), Mycobacterium fortuitum (M. fortuitum), and other mycobacteria, including M. abscessus subsp. abscessus, massiliense, and bollettii. See Table 9 for detailed data.

[0075] [Table 9]

[0076] In conclusion, we rationally redesigned rifabutin to restore the low-nanomolar antimycobacterial activity of rifamycin antibiotics and extend their clinical utility against intrinsically multidrug-resistant M. abscessus by circumventing a novel rifamycin resistance mechanism. Structure-based derivatization at C-25 of rifabutin yielded an analog that is more than 100-fold more potent than the widely used rifampicin and no longer susceptible to the primary rifamycin resistance of M. abscessus via ADP-ribosylation. X-ray crystallography and molecular docking studies suggest that additional ligand-target interactions contribute to favorable on-target activity. The ability to overcome Arr-mediated resistance was verified using an in vitro biochemical assay that directly detects ADP-ribosylation, and the results demonstrated that wild-type and Δ arr The microbiological activity was consistent with that observed using M. abscessus strains. One representative compound also demonstrated potent in vivo efficacy comparable to that of the anti-M. abscessus drug clarithromycin. In a further stage of modification, three heterocyclic C-25 ester analogs were strategically designed based on the key driving factor of the compound's free fraction in plasma. Compound 5m emerged as an exemplary candidate with potent in vitro antibacterial activity, excellent pharmacokinetics, and significantly improved in vivo PK properties.

[0077] References JPEG2025536245000046.jpg146170 JPEG2025536245000047.jpg175170 JPEG2025536245000048.jpg64170 JPEG2025536245000049.jpg168170

[0078] Biological Procedures Statement of ethical approval for animal experiments All experiments using live mice were approved by the Institutional Animal Care and Use Committee of the Center for Discovery and Innovation, Hackensack Meridian Health. Approval number: A4278-01. IACUC numbers: 269.00 (in vivo pharmacokinetic studies); 287.00 (in vivo animal efficacy studies).

[0079] Bacterial strains, culture media, and compounds M. abscessus bambu was isolated from the sputum of a patient with amyotrophic lateral sclerosis and bronchiectasis and was provided by Wei Chang Huang of Taichung Veterans General Hospital, Taichung, Taiwan. Whole genome sequencing of M. abscessus bambu demonstrated that this strain belongs to the M. abscessus subsp. abscessus possesses an inactive clarithromycin-susceptible erm(41)C28 sequevar. [6~7] erm(41)T28 sequevar, which confers inducible clarithromycin resistance [8] Mycobacterium abscessus subsp. abscessus ATCC19977, which carries the genotype 1, was purchased from the American Type Culture Collection (ATCC). M. abscessus subsp. abscessus K21 was isolated from a patient and provided by Sung Jae Shin (Department of Microbiology, Yonsei University College of Medicine, Seoul, Korea) and Won-Jung Koh (Division of Pulmonary and Critical Care Medicine, Samsung Medical Center, Seoul, Korea). This strain was used as previously determined. [9] , has inactive clarithromycin-susceptible erm(41)C28 sequevar. Δarr in the M. abscessus ATCC19977 genetic background by recombination Mab The generation of

[10] . Selection and characterization of the rifamycin-resistant M. abscessus mutant RFB-R1 was previously described.

[10] RFB-R1 harbors the rpoB (RNAP) c1339t nucleotide mutation, which corresponds to the H447Y missense mutation previously reported in M. tuberculosis. [11~12].

[0080] For basic bacterial culture and MIC experiments, Middlebrook 7H9 broth (BD Difco) was supplemented with 0.5% albumin, 0.2% glucose, 0.085% sodium chloride, 0.0003% catalase, 0.2% glycerol, and 0.05% Tween 80. Clarithromycin was purchased from Sigma-Aldrich. Rifampicin was purchased from GoldBio. Rifabutin was purchased from Acros Organics. All drugs were prepared as 10 mM stocks in 100% DMSO.

[0081] MIC assay in 96-well plate format MIC determinations were performed in a 96-well plate format as previously described [13~14] First, a 96-well plate was prepared with 100 μL of 7H9 per well. For each compound, 10-point 2-fold serial dilutions, starting from 2x the highest desired concentration, were dispensed into the 96-well plate using a Tecan D300e digital dispenser, and the DMSO concentration was normalized to 2%. The cells were cultured in mid-logarithmic growth phase (OD ). 600 M. abscessus cultures grown to an OD (OD = 0.4–0.6) were analyzed by 600 =0.1(1×10 7 100 μL of the resulting bacterial suspension was dispensed into 96-well plates containing compounds in a final volume of 200 μL per well, and the initial OD was measured. 600 =0.05(5×10 6The concentrations were measured in 100 ml of culture medium (CFU / mL) with a final DMSO concentration of 1%. Final compound concentration ranges were typically 50–0.098 μM, 6.25–0.012 μM, 0.006–3.13 μM, or 0.003–1.56 μM. Each plate included an untreated control well containing bacterial suspension and 1% DMSO. Plates were sealed with parafilm, stored in a box with wet paper towels, and incubated at 37°C with shaking (110 RPM). Plates were incubated for 3 days. OD was measured on days 0 and 3 using a Tecan Infinite M200 plate reader to determine growth. 600 Two biological replicates were performed. Each experiment included clarithromycin as a positive control. For each well of the 96-well plate, measure the OD on day 3. 600 OD on day 0 from the value 600 The bacterial growth was calculated by subtracting the values. For each compound series, the bacterial growth values ​​of the untreated control wells were averaged to calculate the mean drug-free bacterial growth. For compound-containing wells, the growth value was divided by the mean drug-free bacterial growth of the compound series and multiplied by 100 to calculate the growth percentage. We plotted the growth percentage versus compound concentration for each compound series. We determined the MIC of the compound by visually inspecting the dose-response curves as the compound concentration that would result in 90% growth inhibition.

[0082] Pharmacokinetic studies For oral pharmacokinetic studies, CD-1 female mice (22–25 g) were used. Rifabutin, 5b, 5j, and 5m were administered intravenously (IV) at a single dose of 10 mg / kg in a solution formulation composed of 5% DMSO:95% (4% Cremophor EL) or orally (PO) by gavage. For oral administration, 50 μL aliquots of blood were collected from each mouse (n = 3 per route and dose) by puncturing the lateral tail vein at 30 min, 1, 3, 5, 7, and 24 h after administration, and at 1 min, 15 min, 1, 3, 7, and 24 h after IV administration. Blood was collected in CB300 blood collection tubes containing K2EDTA and stored on ice. Plasma was collected after centrifugation and stored at -80 °C until analysis by high-pressure liquid chromatography coupled to tandem mass spectrometry (LC-MS / MS). Pharmacokinetic parameters were calculated using noncompartmental pharmacokinetic analysis.

[0083] LC-MS / MS analytical methods for pharmacokinetic studies Neat 1 mg / mL DMSO stocks of rifabutin, C25-desacetylrifabutin, 5b, 5j, and 5m were serially diluted with 50 / 50 acetonitrile (ACN) / Milli-Q water to create neat standard solutions. Plasma standards were generated by adding 10 μL of spiked solution to 90 μL of drug-free plasma (CD-1 K2EDTA mouse, Bioreclamation IVT). Five μL of control, standard, or research sample was added to 100 μL of ACN protein precipitation solvent containing 10 ng / mL of the internal standards verapamil (Sigma-Aldrich) and rifabutin-d7 (Toronto Research Chemical). The extract was vortexed for 5 minutes and centrifuged at 4000 RPM for 5 minutes. 75 μL of the supernatant was transferred and diluted with 75 μL of Milli-Q deionized water for LC-MS / MS analysis. Rifabutin was purchased from Carbosynth, Inc. C25-desacetylrifabutin and rifabutin-d7 were purchased from Toronto Research Chemical Co. Verapamil was purchased from Sigma-Aldrich.

[0084] LC-MS / MS analysis was performed on a Sciex Applied Biosystems Qtrap 6500+ triple quadrupole mass spectrometer coupled to a Shimadzu Nexera X2 UHPLC system to quantify each drug in plasma. Chromatography was performed on an Agilent SB-C8 (2.1 x 30 mm, 3.5 μm particle size) using a reversed-phase gradient. The aqueous mobile phase was Milli-Q deionized water with 0.1% formic acid, and the organic mobile phase was 0.1% formic acid in ACN. All analytes were quantified using multiple reaction monitoring of parent / daughter transitions in electrospray positive ionization mode. The following MRM transitions were used for rifabutin (847.60 / 755.60), rifabutin-d7 (854.60 / 762.60), C25-desacetylrifabutin (805.48 / 773.50), 5b (923.48 / 891.40), 5j (927.39 / 895.30), 5m (910.45 / 878.40), and verapamil (455.40 / 165.00). Samples were accepted if the concentrations of quality control samples were within 20% of the nominal concentration. Data processing was performed using Analyst software (version 1.6.2, Applied Biosystems Sciex).

[0085] Plasma protein binding assay DMSO stock was spiked into plasma to a concentration of 10,000 ng / mL. 200 μL of spiked plasma was pipetted into the sample chamber of a rapid equilibrium dialysis (RED) cartridge. 350 μL of PBS was added to the adjacent cartridge. The plate containing the RED was sealed and incubated at 37°C for 4 hours at 300 RPM in a thermomixer. After incubation, a 50 μL aliquot of plasma was removed and mixed (1:1) with 50 μL of blank plasma in a deep-well plate. Similarly, a 50 μL aliquot of PBS was removed and added to 50 μL of blank plasma. This created an identical matrix between the buffered and non-buffered samples. Samples were processed and quantified as specified in the LC-MS / MS analytical method.

[0086] Calculation of physicochemical properties For the physicochemical properties disclosed in the examples, clogP was calculated using ChemDraw 21.0.0.28. The remaining physicochemical properties were calculated using the "SwissADME" online service (http: / / www.swissadme.ch / ).

[0087] Crystal structure determination M. tuberculosis RNAP (RNAP Mtb ) M. tuberculosis σ A Crystals of RPo were prepared as described

[15] Crystals were immersed overnight at 23 °C in cryoprotectant solution (20 mM Tris-HCl, pH 8.2, 200 mM potassium chloride, 20 mM magnesium chloride, 7% (wt / vol) PEG-3350, 20% (vol / vol) (2R,3R)-(-)-2,3-butanediol, 1 mM CHAPSO) supplemented with 0.5 mM 5a and then flash-frozen in liquid nitrogen. X-ray diffraction data were collected at the Stanford Synchrotron Radiation Lightsource (SSRL) beamline 12-2 and processed using HKL3000.

[16] The structure of M. tuberculosis σ A RPo(PDB:5UHA)

[15] The structure of Coot was solved by molecular replacement using it as a search model.

[17] and Phenix Refine

[18] Iterative cycles of model building and refinement were performed using the NMR spectroscopy. The final model was obtained by refinement using secondary structure constraints and individual and group B-factors. The atomic model and structure factors have been deposited in the Protein Data Bank (PDB) under accession number 7U22.

[0088] Arr Mab Expression and purification Arr Mabwas codon-optimized for E. coli and cloned into the pET-28b(+) expression vector. Clones were transformed into E. coli BL21(DE3) cells. Single colonies were picked and allowed to grow overnight in 50 mL of LB broth with the desired antibiotic at 37°C with shaking at 250 RPM. The overnight-grown primary culture was transferred to 1 L of LB broth with 50 μg / mL kanamycin and grown at 37°C with shaking at 250 RPM until the OD (600 nm) reached 0.6. Protein expression was induced by adding 0.5 mM IPTG and reducing the temperature to 20°C for 16 hours. Cells were harvested by centrifugation at 8000 g for 10 min and resuspended in lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, and 1.0 M sorbitol) containing complete protease inhibitor tablets, PMSF (5 mM), and hen egg white lysozyme (0.5 mg / mL). Cells were lysed in an Avastin C3 ultra-high pressure lysis device at 15,000-20,000 psi for 15 min. The supernatant was obtained after high-speed centrifugation and incubated with equilibrated Ni-NTA beads on a rotator for 90 min at 4°C. Both proteins were eluted with a gradient of 50-200 mM imidazole.

[0089] HPLC analysis for in vitro verification of ADP-ribosylation Endpoint reactions for rifampicin, rifabutin, and rifabutin analogs were set up in the presence of 5 μM enzyme in 50 mM HEPES buffer. Reactions were quenched with methanol and analyzed by HPLC. Reversed-phase LC was performed on a Kinetex C8 column (100 mm × 2.1 mm, 2.6 μm, Phenomenex, Torrance, CA) using an LC1200 Infinity Series Agilent Technologies instrument, monitored at wavelengths of 260 nm (for rifampicin) and 277 nm (for rifabutin and rifabutin analogs). The elution gradient was performed with a binary solvent system consisting of 0.1% formic acid in HO (solvent A) and 0.1% formic acid in MeCN (solvent B). A linear gradient profile (t (min), % B) of solvent B with the following proportions (volume / volume): (0, 5), (0.5, 5), (9, 100), (10.5, 100), (12, 10) was applied, with a total run time of 17 min, including 5 min of re-equilibration. The flow rate was 0.3 mL / min, and the column oven was maintained at 28 °C. The injection volume was 10 μL.

[0090] Efficacy evaluation in M. abscessus mouse infection model 8-week-old female NOD.CB17-Prkdc scid / NCrCrl(NOD SCID) mice (Charles River Laboratories) were treated with IFN-γ-α (IFN-γ) as previously described. [9] , M. abscessus subsp. abscessus K21 approximately 10 6Mice were infected intranasally with 10 CFU. Acute infection was achieved within 1 day. Starting 1 day after infection, drugs or vehicle control were administered by oral gavage once daily for 10 consecutive days. Clarithromycin (250 mg / kg, Sandoz Clinical Tablets), rifabutin (10 mg / kg, Carbosynth), and 5j (10 mg / kg) were formulated in 0.5% carboxymethylcellulose / 0.5% Tween 80 at a dose volume of 8 mL / kg. All mice were euthanized 24 hours after the last dose, and lungs and spleens were aseptically removed before homogenization. Bacterial burden in these organs was determined by plating serial dilutions of organ homogenates on Middlebrook 7H11 agar (BD Difco) supplemented with 0.2% (v / v) glycerol and 10% (v / v) OADC. The agar plates were incubated at 37°C for 5 days before colonies were counted.

[0091] computational procedures RNAP Mab and Arr Mab Sequence acquisition and homology modeling RNAP Mab and Arr Mab The sequence of was obtained from the UniProt database (entries B1MH62 and B1MH05). A protein-protein BLAST (Blastp) search was performed to find suitable homologous sequences (templates) with known 3D structures for this amino acid sequence.

[19] Then, the best template is RNAP. Mtb (PDB:5UHB) and Arr Msm The sequences of (PDB:2HW2) and the study target were aligned using Clustal Omega

[20] RNAP based on selected templates Mab and Arr Mab An initial homology model of the nucleotide sequence was generated using the automated homology modeling software MODELLER (version 9). This program is based on comparative structural modeling.

[21] Arr MabThe catalytic domain of Arr was further aligned to the similar poly ADP-ribose polymerase (PARP) domains of Pseudomonas aeruginosa exotoxin (PDB: 1AER) and Gallus gallus poly ADP-ribose polymerase (PDB: 1A26), revealing that Arr Mab NAD + The binding site was predicted. The ADP-ribosyl-oxocarbenium intermediate was Mab The ADP-ribosyl-oxocarbenium intermediate was docked into the predicted binding site of Arr and the best binding mode was selected. Mab A homology model was constructed. Mab Model and Arr Mab Both models were further energy minimized in Discovery Studio 3.5 using the CHARMM force field. The quality and stability of the homology models were verified by checking the stereochemical parameters using PROCHECK, VERIFY3D, and ERRAT on the SAVES server (http: / / nihserver.mbi.ucla.edu / SAVES). Arr Mab Rationalization of the ADP-ribosyl-oxocarbenium intermediate in models NAD + After confirming that both compounds showed better binding affinity values ​​when the nicotinamide moiety of Arr was not present in the model, the ADP-ribosyl-oxocarbenium intermediate Arr Mab A homology model was used to dock 5a and rifabutin, in which the hydroxyl groups at C23 of rifabutin and 5a interact with NAD + Join Arr Mab Compared with the homology model, it is predicted to be closer to C1' of the ribose ring, and this prediction supports the proposed mechanism in which an oxocarbenium transition state allows the hydroxyl group at position 23 of the antibiotic to attack C1' of the ribose.

[22] Therefore, Arr with ADP-ribosyl-oxocarbenium intermediate Mab The homology model shows the rifabutin analogue and Arr MabIt was a better model for predicting the interaction between

[0092] Ligand generation The 2D structures of rifabutin and synthetic rifabutin analogs were drawn in Chemdraw and their SMILES names were obtained. The 3D structures were obtained after energy minimization with Discovery Studio 3.5 and converted into SDF files.

[0093] In silico molecular docking analysis Molecular docking was performed in Pyrx using the Autodock Vina engine. The AutoDockTools package was used to generate pdbqt docking input files. Both the protein target and ligand were opened in the PyRx virtual screening tool as starting protein structures in pdbqt format. Docking was performed using RNAP Mab and Arr Mab The rifampicin binding site residues were placed within a grid box with X, Y, and Z coordinates conforming to the following dimensions: 162.8 Å × 163.38 Å × 20.22 Å, 20 Å × 20 Å × 20 Å, 66.89 Å × 66.62 Å × 10.54 Å, and 20 Å × 20 Å × 20 Å, respectively. The Lamarckian genetic algorithm (LGA) was used for the ligand conformation search process, with other parameters set to default. The quality of the docking was verified by redocking the ligand to observe the accuracy of the docking conditions. All docking simulations using various ligands were performed with an exhaustiveness value of 24. The best binding mode and affinity values ​​were obtained in the PyRx virtual screening GUI and log file.

[0094] Example 2 Basic methods of chemical synthesis Reagents and solvents were purchased from commercial suppliers (Fisher Scientific, MilliporeSigma, A2B Chem, Oakwood Chemical) and used as received unless otherwise noted. Rifabutin was purchased from WuXi AppTec Co., Ltd. (Tianjin). Reactions were monitored using Macherey-Nagel® ALUGRAM® SIL G / UV254 aluminum TLC plates. LC was visualized under visible light or UV fluorescence (254 nm). Flash chromatography was performed using Sorbtech® silica gel (porosity: 60 Å, particle size: 40-63 μm (230 × 400 mesh)). Preparative TLC was performed on Silicyle® glass-backed TLC plates (thickness: 1000 μm, indicator: F-254). NMR spectra were recorded on a Bruker 600 MHz Avance NEO. 1 The H frequency is 601MHz. 13 The C frequency is 151 MHz. The chemical shift (δ) is the residual solvent peak [CDCl3( 1 H:7.26, 13 C:77.2) or CD2Cl2 ( 1 H:5.32, 13 The concentrations were reported in parts per million (ppm) relative to the mean (C: 53.8). Peak multiplicities were indicated as follows: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and br (broad). High-resolution mass spectra (HRMS) were recorded on a Bruker BioTOF II ESI / TOF-MS. Analytical HPLC analysis was performed on an Agilent® 1260 Infinity Quaternary LC system with a reversed-phase C18 column (Gemini-Nx 5 micron, 150 × 4.60 mm, Phenomenex).

[0095] Experimental procedures and characterization data Synthesis of 25-O-acyl rifabutin analogues [ka] 21,23-O-Isopropylidenerifabutin (2). To a solution of rifabutin 1 (5.01 g, 5.90 mmol, 1.00 equiv.) and camphorsulfonic acid (1.66 g, 7.15 mmol, 1.20 equiv.) in acetone (59.0 mL, dried over 4 Å molecular sieves) was added 2,2-dimethoxypropane (15.0 mL, 122 mmol, 20.7 equiv.). The reaction mixture was stirred at 23 °C for 2 h, then NaHCO (1.8 g, 21 mmol, 3.60 equiv.) was added, and the mixture was stirred at room temperature for an additional 30 min. The reaction was then partitioned between CHCl (80 mL) and HO (80 mL). The aqueous phase was back-extracted with CHCl (3 × 50 mL). The combined organic layers were dried (NaSO) and concentrated under reduced pressure. Purification by silica gel flash chromatography (CH2Cl2:MeOH 50:1) gave the title compound (3.94 g, 75%) as a purple solid. f =0.13(50:1 CH2Cl2:MeOH); 1H NMR(601MHz、CD2Cl2)δ14.88(s、1H)、8.80(s、1H)、7.77(s、1H)、6.29(dd、J=15.8、10.8Hz、1H)、6.18~6.14(m、1H)、6.06(dd、J=15.8、7.1Hz、1H)、5.91(dd、J=12.2、1.1Hz、1H)、5.04(dd、J=12.2、6.6Hz、1H)、4.89(dd、J=7.9、1.5Hz、1H)、3.59(dd、J=10.7、3.4Hz、1H)、3.38(ddd、J=6.6、2.3、1.1Hz、1H)、3.03(dd、J=10.3、5.2Hz、1H)、3.01~2.87(m、2H)、2.82(s、3H)、2.68~2.53(m、2H)、2.27(s、3H)、2.32~2.19(m、3H)、2.03(s、2H)、1.93(s、3H)、1.89~1.79(m、1H)、1.79~1.75(m、1H)、1.74(s、3H)、1.56~1.48(m、1H)、1.44(pd、J=7.1、2.2Hz、1H)、1.20(s、1H)、1.17(s、3H)、0.94(d、J=6.6Hz、6H)、0.86(s、3H)、0.85(d、J=6.6Hz、2H)、0.82(d、J=6.8Hz、3H)、0.69(d、J=7.2Hz、3H)、0.36(d、J=7.1Hz、3H); 13 C NMR(151MHz、CD2Cl2)δ192.6、181.6、172.4、170.4、169.0、168.5、155.4、142.7、141.2、141.0、132.5、131.9、125.8、124.2、115.5、113.8、112.1、109.0、106.3、104.9、100.2、100.2、95.4、79.0、77.3、74.5、71.3、69.7、66.8、56.4、51.9、51.8、41.5、41.0、36.6、36.5、35.9、34.7、32.0、29.5、26.2、26.0、24.0、21.0、21.0、21.0、20.4、20.4、17.9、13.0、9.9、9.6、7.8;MS(ESI):C 49 H 66 N4O 11 のm / z[M+H] +Theoretical value: 887.4801; Measured value: 887.4786 (error 1.7 ppm). [ka]

[0096] 25-Desacetyl-21,23-O-isopropylidene erifabutin (3). A mixture of 2 (3.56 g, 4.01 mmol, 1.00 equiv.) and KCO (3.33 g, 24.1 mmol, 6.00 equiv.) in MeOH (40 mL) was heated at 50 °C for 48 h. The reaction was cooled to room temperature and partitioned between CHCl (50 mL) and brine (50 mL). The organic layer was separated, and the aqueous phase was extracted with CHCl (3 × 40 mL). The combined organic extracts were dried (NaSO) and concentrated under reduced pressure. Purification by flash chromatography on silica gel (CHCl:MeOH 60:1) afforded the title compound (2.17 g, 64%, containing traces of starting material) as a purple solid. R f =0.19 (Hexane: EtOAc: MeOH: Et3N 8:1:1:0.1); 1H NMR(600MHz、CD2Cl2)δ14.93(s、1H)、8.72(s、1H)、7.81(s、1H)、6.28(dd、J=15.8、10.3Hz、1H)、6.16(dd、J=11.4、1.6Hz、1H)、6.15(d、J=11.2Hz、1H)、5.96(dd、J=15.8、6.6Hz、1H)、4.95(dd、J=12.2、9.1Hz、1H)、3.60(dd、J=9.1、3.4Hz、1H)、3.52(dd、J=10.4、3.2Hz、1H)、3.44(dt、J=8.8、3.0Hz、1H)、3.15(d、J=3.7Hz、1H)、3.14(s、3H)、3.11(dd、J=9.1、5.0Hz、1H)、2.99~2.86(m、1H)、2.67~2.50(m、2H)、2.25(dd、J=7.4、1.9Hz、2H)、2.23(s、3H)、2.03(s、3H)、2.09~1.87(bs、4H)、1.84(dq、J=13.6、6.7Hz、1H)、1.75(s、3H)、1.66~1.58(m、1H)、1.59~1.52(m、1H)、1.35(ddd、J=9.5、6.8、2.7Hz、1H)、1.02(s、3H)、0.94(d、J=6.6Hz、6H)、0.85(d、J=7.0Hz、3H)、0.82(d、J=6.8Hz、3H)、0.80(s、3H)、0.72(d、J=6.8Hz、3H)、0.49(d、J=7.1Hz、3H); 13 C NMR(151MHz、CD2Cl2)δ191.3、181.9、171.6、169.2、168.5、155.5、143.0、142.9、140.6、132.5、132.2、125.9、124.5、114.4、112.2、112.1、108.9、105.9、105.2、99.7、95.4、83.2、76.0、71.6、71.1、66.8、56.4、52.1、51.8、41.2、40.1、36.7、35.8、35.2、35.1、26.2、25.7、24.5、21.0、21.0、20.0、20.0、17.8、13.0、13.0、8.5、7.8;MS(ESI):C 49 H 65 N4O 11 Naのm / z[M+Na] +Theoretical value: 867.4515; Measured value: 867.4538 (error 2.6 ppm). [ka]

[0097] Acylation Procedure A: To a solution of 3 (1.00 equiv.) in 1,2-dichloroethane (0.1 M) at room temperature or 50 °C, add the respective acid [1] Acid anhydride (3.00–5.00 equiv.) prepared from and DMAP (0.50–0.80 equiv.) were added every 12 h until most of 3 was converted. In most cases, a total of 10.0–20.0 equiv. of anhydride and 2.00–3.00 equiv. of DMAP were required over 2–3 days. The reaction mixture was then poured into CHCl (10 mL) and saturated aqueous NaHCO (10 mL), and the biphasic mixture was stirred at 23 °C for 16 h to quench any remaining anhydride. The organic layer was separated, and the aqueous phase was extracted with CHCl (2 × 10 mL). The combined organic layers were dried (NaSO) and concentrated under reduced pressure. Purification by preparative silica gel TLC afforded 25-O-acyl-21,23-isopropylidene rifabutin analog 4, which was deprotected according to the general deprotection procedure.

[0098] Acylation Procedure B. To a solution of acid (4.00 equiv.), triethylamine (5.00 equiv.), and DMAP (2.00 equiv.) in CHCl (0.4 M relative to acid) at 0 °C, pivaloyl chloride (4.00 equiv.) was added and the reaction was allowed to warm to 23 °C over 1 h. 3 (1.00 equiv.) was then added, and the resulting solution was further stirred at 23 °C for 4 h. The reaction mixture was poured into CHCl (10 mL) and saturated aqueous NaHCO (10 mL), and the biphasic mixture was stirred at 23 °C for 16 h to quench any remaining mixed anhydride. The organic layer was separated, and the aqueous layer was extracted with CHCl (2 × 10 mL). The combined organic layers were dried (NaSO) and concentrated under reduced pressure. Purification by preparative silica gel TLC gave 25-O-acyl-21,23-isopropylidene rifabutin analog 4, which was deprotected according to the general deprotection procedure.

[0099] Basic deprotection procedure. A solution of 25-O-acyl-21,23-isopropylidene rifabutin analog (1.00 equiv.) prepared using acylation procedure A or B and camphorsulfonic acid (2.00 equiv.) in methanol (0.1 M) was stirred at room temperature for 30 minutes, and the reaction was then partitioned between CHCl (10 mL) and saturated aqueous NaHCO (10 mL). The aqueous phase was then back-extracted with CHCl (2 × 10 mL). The combined organic layers were dried (NaSO) and concentrated under reduced pressure. The resulting crude product was purified by preparative TLC (hexane:ethyl acetate:methanol:triethylamine = 8:1:1:0.1, or hexane:ethyl acetate:methanol = 7:2:1) to give 25-O-acyl rifabutin analog 5 (see below for detailed characterization).

[0100] Synthesis of 25-O-carbamoyl and carbamoylsulfonamide rifabutin (carbamate and sulfonamide analogs) Basic Scheme [ka] Basic Scheme 1. Synthesis of rifabutin analogs with C25 carbamate and sulfonamide modifications Synthesis of 25-O-carbamoyl rifabutin (carbamate analogue) Carbamate formation via acylimidazolidate intermediate LT-I-00F (Carbamate formation procedure A) CDI Coupling Method I: [ka] To a solution of LT-I-00B (3.5 g, 4.14 mmol) in toluene (30 mL) was added CDI (3.36 g, 20.71 mmol). The mixture was stirred at 100 °C for 12 h. LC-MS showed that the reactant was completely consumed and the desired compound was detected. The residue was concentrated and purified by flash silica gel chromatography (eluent: 0–6% dichloromethane:methanol gradient @ 100 mL / min) to give LT-I-00F (1.8 g, 46% yield) as a purple solid.

[0101] CDI Coupling Method II: [ka] A mixture of 3 (1.02 g, 1.21 mmol, 1.00 equiv) and 1,1'-carbonyldiimidazole (CDI) (0.59 g, 3.64 mmol, 3.00 equiv) in 1,2-dichloroethane (8 mL) was heated at 50 °C for 24 h. CDI (0.20 g, 1.21 mmol, 1.00 equiv) was then added, and the reaction was further stirred at 50 °C for 24 h. The reaction was then cooled to room temperature and partitioned between CHCl (20 mL) and water (20 mL). The organic layer was separated, and the aqueous phase was extracted with CHCl (3 × 20 mL). The combined organic extracts were dried (NaSO) and concentrated under reduced pressure. Purification by flash chromatography on silica gel (CHCl:MeOH 50:1) afforded the title compound (774.0 mg, 68%) as a purple solid. Rf = 0.08 (hexane: EtOAc: MeOH: Et3N 8: 1: 1: 0.1); 1H NMR(600MHz、CD2Cl2)δ14.91(s、1H)、8.81(s、1H)、8.03(s、1H)、7.90(s、1H)、7.36(t、J=1.5Hz、1H)、6.99(dd、J=1.7、0.9Hz、1H)、6.29(dd、J=15.9、10.7Hz、1H)、6.16(d、J=10.7Hz、1H)、6.05(dd、J=15.8、7.0Hz、1H)、5.98(dd、J=12.2、0.9Hz、1H)、5.13(dd、J=8.1、1.6Hz、1H)、5.06(dd、J=12.2、7.3Hz、1H)、3.59(dd、J=10.6、3.4Hz、1H)、3.42~3.38(m、1H)、3.05(dd、J=10.2、5.3Hz、1H)、3.02~2.95(m、1H)、2.95~2.89(m、1H)、2.76(s、3H)、2.69~2.55(m、2H)、2.27(s、3H)、2.33~2.21(m、3H)、2.03(s、3H)、2.09~1.89(m、4H)、1.90~1.80(m、1H)、1.75(s、3H)、1.78~1.70(m、3H)、1.59~1.51(m、1H)、1.13(s、3H)、0.95(d、J=6.6Hz、6H)、0.85(d、J=4.3Hz、3H)、0.85(d、J=4.3Hz、3H)、0.83(d、J=7.2Hz、3H)、0.83(d、J=6.6Hz、3H)、0.49(d、J=7.1Hz、3H); 13 C NMR(151MHz、CD2Cl2)δ192.3、181.7、172.2、169.0、168.5、155.5、148.6、142.8、142.1、140.7、137.3、132.4、132.2、130.6、125.8、124.3、117.4、114.1、113.8、112.1、108.9、106.2、105.0、100.3、95.4、79.7、79.3、76.8、71.3、66.8、56.2、51.9、51.8、41.0、40.8、36.7、36.6、35.8、34.6、26.2、25.9、23.8、21.00、20.98、20.97、20.3、17.8、13.0、10.6、9.8、7.8;MS(ESI):C 51 H 67 N6O 11 のm / z[M+H]+ Theoretical value: 939.4862; Measured value: 939.4780 (error 8.7 ppm). [ka]

[0102] To a solution of LT-I-00F (1.00 equiv.) in dichloromethane or 1,2-dichloroethane (0.1 M) was added methyl trifluoromethanesulfonate (1.25–1.50 equiv.). After stirring the mixture at room temperature for 10–15 min, the activated intermediate LT-I-00F-A was concentrated under reduced pressure for further use. Alternatively, an amine was added to the same pot, and the reaction mixture was further stirred at room temperature until complete conversion of the starting material. The reaction mixture was then partitioned between dichloromethane and HO. The aqueous phase was then back-extracted with dichloromethane. The combined organic layers were concentrated under reduced pressure, and the resulting crude product was purified by preparative TLC (hexane:ethyl acetate:methanol:triethylamine=8:1:1:0.1) or preparative HPLC to afford the respective acetonide-protected carbamate analogs.

[0103] Direct Synthesis of Acetonide-Protected Carbamates (Carbamate Formation Procedure B) [ka] To a solution of each acid (5.00 equiv.) and DMAP (5.00 equiv.) in 1,2-dichloroethane (0.5 M relative to the acid) was added DPPA (5.00 equiv.). After stirring the mixture at 50 °C for 24 h, 3 (1.00 equiv.) was added, and the mixture was further stirred at 50 °C until 3 was completely consumed. The reaction mixture was then partitioned between CHCl and HO. The aqueous phase was back-extracted twice with CHCl. ​​The combined organic phases were concentrated under reduced pressure, and the resulting crude product was purified by preparative TLC (hexane:ethyl acetate:methanol:triethylamine=7:2:1:0.1) or flash chromatography (0-10% MeOH in CHCl) to give the acetonide-protected 25-O-aminoacyl rifabutin intermediate. [ka]

[0104] A solution of 25-O-carbamoyl-21,23-isopropylidene rifabutin analog (1.00 equiv.) and camphorsulfonic acid (2.00–5.00 equiv.) in methanol (0.1 M) was stirred at room temperature for 30 minutes. The reaction was then partitioned between dichloromethane (10 mL) and saturated aqueous NaHCO3 (10 mL). The aqueous phase was then back-extracted with dichloromethane (2 × 10 mL). The combined organic layers were dried (Na2SO4) and concentrated under reduced pressure. The resulting crude product was purified by preparative TLC (hexane:ethyl acetate:methanol:triethylamine = 8:1:1:0.1 or dichloromethane:methanol = 15:1) or preparative HPLC to give the 25-O-carbamoyl rifabutin analog.

[0105] NOTE: For analogs derivatized with basic heteroaromatic substituents, 5 equivalents of CSA resulted in a more efficient reaction, while for the remaining deprotection, 2 equivalents of CSA was sufficient for complete conversion within 30 min.

[0106] Synthesis of 5i [ka] To a solution of NaOH (0.28 g, 6.00 equiv.) and ZnCl (0.16 g, 1.00 equiv.) in MeOH (12 mL) was added 1 (1.00 g, 1.00 equiv.), and the reaction mixture was stirred at room temperature overnight. The mixture was then partitioned between CHCl (30 mL) and brine (30 mL). The aqueous phase was extracted with CHCl (3 × 30 mL). The combined organic phases were dried (NaSO) and concentrated under reduced pressure. Purification by flash column chromatography (CHCl:MeOH = 9:1) afforded the title compound (0.68 g, 71%). 1H NMR (601MHz, CDCl3) δ14.57 (s, 1H), 9.68 (s, 1H), 8.26 (s, 1H), 6.34 (d, J=12 .8Hz, 1H), 6.30~6.23(m, 2H), 5.94~5.85(m, 1H), 5.18(dd, J=12.8, 10.1Hz, 1 H), 4.15 (s, 1H), 3.70 (d, J=9.8Hz, 1H), 3.55 (ddd, J=10.3, 7.9, 2.4Hz, 1H), 3 .39(dd, J=10.3, 4.2Hz, 1H), 3.32(dd, J=10.0, 2.1Hz, 1H), 3.16(s, 3H), 3.03 ~2.92(m, 3H), 2.87(dq, J=8.1, 2.6Hz, 1H), 2.68(br, 2H), 2.42(dt, J=9.6, 6 .6Hz, 1H), 2.32(s, 2H), 2.28(s, 3H), 2.04(s, 3H), 1.90~1.82(m, 2H), 1.80~1 .74(m, 1H), 1.72(s, 3H), 1.24(s, 4H), 1.08(d, J=7.0Hz, 3H), 0.95(d, J=6.5H z, 6H), 0.83 (d, J=7.0Hz, 3H), 0.55 (d, J=6.8Hz, 3H), ~0.14 (d, J=7.0Hz, 3H). 13 C NMR (151MHz, CDCl3) δ192.1, 180.5, 171.0, 168.3, 168.2, 155.0, 147.5, 141.6, 1 41.0, 132.9, 132.6, 124.9, 123.3, 114.7, 114.2, 111.5, 109.5, 108.1, 104.4, 94 .8, 85.5, 76.8, 71.6, 70.8, 66.4, 56.1, 51.6, 51.6, 39.5, 38.9, 37.9, 36.1, 35.2 ,32.8,29.8,25.9,22.9,21.0,21.0,20.2,17.2,12.2,10.9,8.4,7.9;MS(ESI):C 49 H 65 N4O 11 Naのm / z[M+Na] + Theoretical value: 827.4202; measured value: 827.4198 (error 0.5ppm).

[0107]

change

[0108] 25-O-Benzoyl-25-O-desacetylifabutin (5a). [ka] Prepared from 3 (50.0 mg) using acylation procedure A and the basic deprotection procedure to give the title compound (30.6 mg, 57% over two steps) as a purple solid. 51 H 65 N4O 11 HRMS (ESI-TOF) m / z [M+H] + Theoretical value: 909.4644, measured value: 909.4665 (error 2.3 ppm). 1 H NMR and 13 For C NMR data, see Figure 11.

[0109] 25-O-(2-methylbenzoyl)-25-O-desacetylifabutin (5b). [ka] Prepared from 3 (30.0 mg) using acylation procedure A and the basic deprotection procedure to give 5b (3.9 mg, 12% over two steps) as a purple solid. 52 H 67 N4O 11 HRMS (ESI-TOF) m / z [M+H] + Theoretical value: 923.4801, measured value: 923.4829 (error 3.0 ppm). 1 H NMR and 13 For C NMR data, see Figure 12.

[0110] 25-O-(3-methylbenzoyl)-25-O-desacetylifabutin (5c). [ka] Prepared from 3 (25.0 mg) using acylation procedure A and the basic deprotection procedure to give 5c (2.5 mg, 9% over two steps) as a purple solid. 52 H 67 N4O 11 HRMS (ESI-TOF) m / z [M+H] + Theoretical value: 923.4801, measured value: 923.4815 (error 1.5 ppm). 1 H NMR and 13 For C NMR data, see Figure 13.

[0111] 25-O-(4-methylbenzoyl)-25-O-desacetylifabutin (5d). [ka] Prepared from 3 (18.9 mg) using acylation procedure A and the basic deprotection procedure to give 5d (8.1 mg, 39% over two steps) as a purple solid. 52 H 67 N4O 11 HRMS (ESI-TOF) m / z [M+H] + Theoretical value: 923.4801, measured value: 923.4821 (error 2.2 ppm). 1 H NMR and 13 For C NMR data, see Figure 14.

[0112] 25-O-(2-fluorobenzoyl)-25-O-desacetylifabutin (5e). [ka] Prepared from 3 (30.0 mg) using acylation procedure A and the basic deprotection procedure to give 5e (17.5 mg, 53% over two steps) as a purple solid. 51 H 63FN4O 11 HRMS (ESI-TOF) m / z [M+H] of Na + Theoretical value: 949.4370, measured value: 949.4341 (error 3.0 ppm). 1 H NMR and 13 For C NMR data, see Figure 15.

[0113] 25-O-(2-chlorobenzoyl)-25-O-desacetylifabutin (5f). [ka] Prepared from 3 (25.0 mg) using acylation procedure A and the basic deprotection procedure to give 5f (19.0 mg, 68% over two steps) as a purple solid. 51 H 64 ClNO 11 HRMS (ESI-TOF) m / z [M+H] + Theoretical value: 943.4255, measured value: 943.4245 (error 1.0 ppm). 1 H NMR and 13 For C NMR data, see Figure 16.

[0114] 25-O-(2-Methoxybenzoyl)-25-O-desacetylifabutin (5g). [ka] Prepared from 3 (600 mg) using acylation procedure A and the basic deprotection procedure to give 5g (116.2 mg, 26% over two steps) as a purple solid. 52 H 67 N4O 12 HRMS (ESI-TOF) m / z [M+H] + Theoretical value: 939.4750, measured value: 939.4777 (error 2.9 ppm). 1 H NMR and 13 For C NMR data, see Figure 17.

[0115] 25-O-(2-trifluoromethylbenzoyl)-25-O-desacetylifabutin (5h). [ka] Prepared from 3 (30.0 mg) using acylation procedure A and the basic deprotection procedure to give 5h (10.4 mg, 30% over two steps) as a purple solid. 52 H 64 F3N4O 11 HRMS (ESI-TOF) m / z [M+H] + Theoretical value: 977.4518, measured value: 977.4519 (error 0.1 ppm). 1 H NMR and 13 For C NMR data, see Figure 18.

[0116] 25-O-(2-phenylbenzoyl)-25-O-desacetylifabutin (5i). [ka] Prepared from 6 (415.5 mg) using acylation procedure C to give 5i (29.8 mg, 6% over two steps) as a purple solid. 57 H 69 N4O 11 HRMS (ESI-TOF) m / z [M+H] + Theoretical value: 985.4957, measured value: 985.4952 (error 0.5 ppm). 1 H NMR and 13 For C NMR data, see Figure 19.

[0117] 25-O-(3-fluorobenzoyl)-25-O-desacetylifabutin (5j). [ka] Prepared from 3 (20.0 mg) using acylation procedure A and the basic deprotection procedure to give 5j (12.0 mg, 55% over two steps) as a purple solid. 49 H 69 N4O 11 HRMS (ESI-TOF) m / z [M+H] + Theoretical value: 927.4550, measured value: 927.4556 (error 0.6 ppm). 1H NMR and 13 For C NMR data, see Figure 20.

[0118] 25-O-(2-methylbutyl)-25-O-desacetylifabutin (5k). [ka] Prepared from 3 (32.8 mg) using acylation procedure A and the basic deprotection procedure to give 5k (22.2 mg, 64% over two steps) as a purple solid. 49 H 69 N4O 11 HRMS (ESI-TOF) m / z [M+H] + Theoretical value: 889.4957, measured value: 889.4995 (error 4.3 ppm). 1 H NMR and 13 For C NMR data, see Figure 21.

[0119] 25-O-(2-ethylbutyl)-25-O-desacetylifabutin (5l). [ka] Prepared from 3 (20.9 mg) using acylation procedure A and the basic deprotection procedure to give 5l (16.0 mg, 72% over two steps) as a purple solid. 50 H 71 N4O 11 HRMS (ESI-TOF) m / z [M+H] + Theoretical value: 903.5114, measured value: 903.5105 (error 1.0 ppm). 1 H NMR and 13 For C NMR data, see Figure 22.

[0120] 25-O-(3-Pyridinecarbonyl)-25-O-desacetylifabutin (5m). [ka] Prepared from 3 (59.7 mg) using acylation procedure A and the basic deprotection procedure to give 5m (29.3 mg, 46% over two steps) as a purple solid. 50 H 64 N5O 11 HRMS (ESI-TOF) m / z [M+H] + Theoretical value: 910.4597, measured value: 910.4606 (error 1.0 ppm). 1 H NMR and 13 For C NMR data, see Figure 23.

[0121] 25-O-(5-pyrimidinecarbonyl)-25-O-desacetylifabutin (5n). [ka] Prepared from 3 (40.0 mg) using acylation procedure B and the basic deprotection procedure to give 5n (29.3 mg, 56% over two steps) as a purple solid. 49 H 62 NO 11 HRMS (ESI-TOF) m / z of Na [M+Na] + Theoretical value: 933.4369, measured value: 933.4395 (error 2.8 ppm). 1 H NMR and 13 For C NMR data, see Figure 24.

[0122] 25-O-(2-Thiazolecarbonyl)-25-O-desacetylifabutin (5o). [ka] Prepared from 3 (40.0 mg) using acylation procedure B and the basic deprotection procedure to give 5o (29.3 mg, 51% over two steps) as a purple solid. 48 H 62 N5O 11 HRMS (ESI-TOF) m / z [M+H] of S + Theoretical value: 916.4161, measured value: 916.4204 (error: 4.7 ppm). 1 H NMR and 13For C NMR data, see Figure 25.

[0123] Notes on the acylation process of compound synthesis The acylation procedure reported above, using excess amounts of anhydride and DMAP, is the only method we have found that can yield the desired C25-OH acylated product. Under conditions where the anhydride was used alone or with a substoichiometric amount of DMAP, little conversion of the starting material was observed (see Figure 26, Cases 1 and 2). This method proved effective and gave reasonable to high yields in most of our syntheses. In certain cases, the relatively low yields were due to very slow conversion and extensive side reactions upon heating (Cases 3 and 4).

[0124] 25-O-(3-Methoxybenzoyl)-25-O-desacetylifabutin [ka] Prepared from 3 (25.7 mg) using acylation procedure A and the basic deprotection procedure to give the title compound (15.0 mg, 52% over two steps) as a purple solid. 1H NMR(601MHz、CD2Cl2)δ14.87(s、1H)、9.01(s、1H)、8.21(s、1H)、7.56(dt、J=7.6、1.2Hz、1H)、7.50(dd、J=2.7、1.5Hz、1H)、7.34(t、J=8.0Hz、1H)、7.10(ddd、J=8.3、2.7、1.0Hz、1H)、6.39(dd、J=15.8、10.6Hz、1H)、6.24(dd、J=10.4、2.5Hz、1H)、6.10(dd、J=12.5、1.2Hz、1H)、6.05(dd、J=15.9、6.8Hz、1H)、5.09(dd、J=12.5、6.4Hz、1H)、5.07(dd、J=9.2、1.5Hz、1H)、3.90(br、1H)、3.82(s、3H)、3.72(dd、J=9.9、1.8Hz、1H)、3.42(ddd、J=6.4、2.7、1.2Hz、1H)、3.31(br、1H)、3.05(d、J=10.3Hz、1H)、3.03~2.98(m、1H)、2.97~2.92(m、1H)、2.91(s、3H)、2.67 (br、2H)、2.36~2.32(m、1H)、2.31(s、3H)、2.30(s、2H)、2.04(s、3H)、2.14~1.90(m、4H)、1.88(m、1H)、1.80(ddd、J=10.3、7.1、2.8Hz、1H)、1.78~1.74(m、1H)、1.73(s、3H)、1.60(ddd、J=10.3、6.9、1.7Hz、1H)、0.96(d、J=6.4Hz、9H)、0.84(d、J=7.0Hz、3H)、0.69(d、J=6.9Hz、3H)、-0.05(d、J=7.1Hz、3H); 13C NMR (151MHz, CD2Cl2) δ192.7, 181.7, 172.0, 168.6, 168.5, 167.6, 160.0, 155.6, 143.4, 1 42.9, 141.5, 133.1, 131.7, 131.6, 129.8, 125.7, 124.5, 122.4, 119.5, 116.8, 115.0, 114. 3, 112.2, 109.2, 107.3, 105.1, 95.1, 79.6, 77.3, 74.6, 73.3, 66.7, 57.1, 55.8, 51.8, 51.8 ,38.5,38.4,38.2,36.5,35.7,33.5,26.2,21.7,21.0,20.5,17.7,11.3,10.6,9.1,7.7;C 52 H 67 N4O 12 HRMS (ESI-TOF) m / z [M+H] + Theoretical value: 939.4750, measured value: 939.4772 (error 2.3 ppm).

[0125] 25-O-(4-Methoxybenzoyl)-25-O-desacetylifabutin [ka] Prepared from 3 (65.2 mg) using acylation procedure A and the basic deprotection procedure to give the title compound (5.2 mg, 7% over two steps) as a purple solid. 1H NMR(601MHz、CD2Cl2)δ14.89(s、1H)、8.99(s、1H)、8.11(s、1H)、7.97~7.91(m、2H)、6.91(d、J=8.9Hz、1H)、6.40(dd、J=15.9、10.5Hz、1H)、6.24(dd、J=10.8、1.3Hz、1H)、6.09(dd、J=12.4、1.2Hz、1H)、6.06(dd、J=15.8、6.9Hz、1H)、5.04(dd、J=12.5、6.2Hz、1H)、5.01(dd、J=10.5、1.5Hz、1H)、4.07~4.03(br、1H)、3.84(s、3H)、3.70(dd、J=9.9、1.5Hz、1H)、3.43(ddd、J=6.3、2.7、1.3Hz、1H)、3.33(br、1H)、3.06~2.99(m、1H)、3.04~2.92(m、2H)、2.90(s、3H)、2.71~2.60(br、2H)、2.31(s、3H)、2.35~2.26(m、3H)、2.04(s、3H)、2.08~1.90(m、4H)、1.90~1.82(m、1H)、1.82~1.73(m、3H)、1.72(s、3H)、1.61(ddd、J=10.1、6.7、1.6Hz、1H)、0.96(d、J=3.4Hz、5H)、0.95(d、J=3.9Hz、5H)、0.83(d、J=6.9Hz、3H)、0.67(d、J=6.9Hz、3H)、-0.04(d、J=7.1Hz、3H); 13 C NMR(151MHz、CD2Cl2)δ192.7、181.7、172.0、168.7、168.5、167.7、164.1、155.7、143.8、142.8、141.7、133.2、132.3、131.5、125.8、124.5、122.3、116.7、114.3、114.0、112.3、109.2、107.3、105.2、95.0、79.5、77.4、74.2、73.5、66.7、57.2、55.9、51.8、51.8、38.6、38.3、38.1、36.6、35.8、33.6、30.5、26.2、21.7、21.0、20.5、17.7、11.4、10.6、9.1、7.7;C 52 H 67 N4O 12 のHRMS(ESI-TOF)m / z[M+H] +Theoretical value: 939.4750, measured value: 939.4756 (error 0.6 ppm).

[0126] 25-O-(4-pyrimidinecarbonyl)-25-O-desacetylifabutin [ka] Prepared from 3 (50.0 mg) using acylation procedure B and the basic deprotection procedure to give the title compound (36.2 mg, 67% over two steps) as a purple solid. 1 H NMR(601MHz,CD2Cl2)δ14.68(s, 1H), 9.45(s, 1H), 9.31(d, J=1.4Hz, 1H), 8.91(d, J=5.0Hz, 1H), 8.1 8(s, 1H), 7.89(dd, J=5.0, 1.4Hz, 1H), 6.33(dd, J=15.4, 10.1Hz, 1H), 6.26(dd, J=10.3, 1.7Hz, 1H), 6.19(d, J=12.7Hz, 1H), 5.99(dd, J=15.7, 6.4Hz, 1H), 5.34(dd, J=12.7, 8.6Hz, 1H), 5.12(dd, J=10. 7, 1.8Hz, 1H), 3.73(d, J=9.9Hz, 1H), 3.39(d, J=7.8Hz, 1H), 3.29(dd, J=8.5, 3.2Hz, 1H), 3.07(ddd, J=10.2, 7.6, 2.5Hz, 1H), 3.00~2.90(m, 3H), 2.82(s, 3H), 2.65(br, 2H), 2.42~2.36(m, 1H), 2.34(s, 3H), 2.29(d, J=7.4Hz, 2H), 2.11~1.94(m, 4H), 2.04(s, 3H), 1.95~1.88(m, 1H), 1.88~1.81(m, 1H), 1 .79(dtd, J=9.3, 6.7, 2.1Hz, 1H), 1.76~1.73(m, 1H), 1.72(s, 3H), 1.50~1.40(m, 1H), 0.99(d, J=7.0 Hz, 3H), 0.95(d, J=6.6Hz, 7H), 0.84(d, J=7.0Hz, 3H), 0.72(d, J=6.9Hz, 3H), -0.06(d, J=7.1Hz, 3H); 13C NMR (151MHz, CD2Cl2) δ192.5, 181.1, 171.8, 168.6, 168.3, 164.4, 159.5, 159.2, 155.8, 1 55.4, 145.5, 142.5, 141.1, 132.9, 132.8, 125.4, 124.0, 121.4, 116.8, 115.0, 112.0, 109 .7, 107.8, 104.9, 95.2, 82.2, 76.8, 75.4, 72.5, 66.8, 56.4, 51.9, 51.8, 39.2, 38.8, 37.8 , 36.5, 35.6, 33.3, 26.3, 22.3, 21.0, 20.3, 17.5, 11.9, 11.0, 9.1, 7.7;MS(ESI)m / z[M+H] + 911.4, [MH] - 909.3.

[0127] 25-O-(2-pyrazinecarbonyl)-25-O-desacetylifabutin [ka] Prepared from 3 (50.0 mg) using acylation procedure B and the basic deprotection procedure to give the title compound (36.1 mg, 67% over two steps) as a purple solid. 1H NMR(601MHz、CD2Cl2)δ14.72(s、1H)、9.36(s、1H)、9.17(d、J=1.5Hz、1H)、8.69(d、J=2.5Hz、1H)、8.67(dd、J=2.4、1.5Hz、1H)、8.19(s、1H)、6.35(dd、J=15.8、10.3Hz、1H)、6.26(dd、J=10.4、1.7Hz、1H)、6.18(dd、J=12.6、0.8Hz、1H)、6.00(dd、J=15.8、6.5Hz、1H)、5.30(dd、J=12.3、7.8Hz、1H)、5.15(dd、J=10.7、1.8Hz、1H)、3.73(d、J=9.9Hz、1H)、3.46(d、J=7.4Hz、1H)、3.33(dd、J=8.0、2.9Hz、1H)、3.08(ddd、J=10.0、7.2、2.4Hz、1H)、3.01(s、1H)、3.00~2.89(m、2H)、2.84(s、3H)、2.65(br、2H)、2.38(dt、J=10.0、7.0Hz、1H)、2.34(s、3H)、2.29(d、J=7.4Hz、2H)、2.10~1.94(m、4H)、2.04(s、3H)、1.95~1.89(m、1H)、1.89~1.81(m、1H)、1.81~1.75(m、1H)、1.72(s、3H)、1.54~1.46(m、1H)、0.98(d、J=7.0Hz、3H)、0.95(d、J=6.5Hz、6H)、0.84(d、J=7.0Hz、3H)、0.72(d、J=6.9Hz、3H)、-0.04(d、J=7.1Hz、3H); 13 C NMR(151MHz、CD2Cl2)δ192.6、181.3、171.9、168.6、168.3、164.5、155.4、147.8、146.7、145.1、144.8、144.3、142.6、141.2、132.9、132.6、125.5、124.1、116.7、114.8、112.0、109.6、107.7、104.9、95.2、81.7、76.9、75.2、72.7、66.8、56.6、51.9、51.8、39.1、38.7、38.0、36.6、35.7、33.3、26.3、22.2、21.0、20.4、17.5、11.7、11.0、9.2、7.8;MS(ESI)m / z[M+H] + 911.4、[M-H]- 909.4.

[0128] 25-O-(6-carboxyl-2,5-dichlorobenzoyl)-25-O-desacetylifabutin [ka] Prepared from 3 (100.0 mg) using acylation procedure A and the basic deprotection procedure to give the title compound (14.5 mg, 12% over two steps) as a purple solid. 1 H NMR (400MHz, methanol-d4) δ=7.52(d,J=8.6Hz, 1H), 7.41(d,J=8.6Hz, 1H), 6.70(dd,J=11.0, 15.9Hz, 1H), 6.45(d,J=11.0Hz, 1H) ), 6.25(dd,J=7.2, 15.9Hz, 1H), 6.11(dd,J=1.3, 12.5Hz, 1H), 5.69(d,J=10.1Hz, 1H), 5.10(dd,J=4.9, 12.5Hz, 1H), 4.03(br d,J=10.3Hz, 2H), 3.95(br d,J=4.9Hz, 2H), 3.85~3.72(m, 3H), 3.30(br d,J=7.0Hz, 3H), 3.26(s, 3H), 3.22(s, 1H), 2.85~2.66(m, 2H), 2.52~2.31(m , 6H), 2.15(s, 4H), 2.07~1.92(m, 3H), 1.87~1.70(m, 6H), 1.43~1.32(m, 1H), 1.23(d,J=1.8Hz, 3H), 1.22(d,J=1.9Hz, 3H), 1.14(d,J=7.0Hz, 3H), 0.99(d ,J=6.9Hz, 3H), 0.83~0.78(m, 4H), -0.01(d,J=7.2Hz, 3H);MS(ESI)m / z[M+H] + 1021.4.

[0129] 25-O-(6-carboxyl-3,4-dichlorobenzoyl)-25-O-desacetylifabutin (UMN99) [ka] Prepared from 3 (150.0 mg) using acylation procedure A and the basic deprotection procedure to give the title compound (14.2 mg, 8% over two steps) as a purple solid. 1 H NMR (400MHz, methanol-d4) δ=7.94(s, 1H), 7.64(s, 1H), 6.69(dd, J=11.0, 15.9Hz, 1H), 6.45(br d. s, 4H), 4.04~3.88(m, 3H), 3.83~3.70(m, 2H), 3.66(br d, J=5.1Hz, 1H), 3.25(br d,J=8.6Hz, 2H), 3.15(s, 3H), 2.82~2.67(m, 2H), 2.52~2.34(m, 6H), 2.16(s, 3H), 2.08~1.90(m, 3H), 1.86~1.75(m, 6H), 1.53~1.43(m, 1) H), 1.24(d, J=6.4Hz, 6H), 1.10(d, J=6.9Hz, 3H), 0.98(d, J=6.9Hz, 3H), 0.83(d, J=6.9Hz, 3H), -0.01(d, J=7.0Hz, 3H);MS(ESI)m / z[M+H] + 1021.4.

[0130] The ester form of UMN99 with a free carboxyl group can be prepared by the following procedure. [ka] To a solution of 3 (300 mg, 355.02 μmol) in DCE (3 mL) were added 5,6-dichloroisobenzofuran-1,3-dione (385.20 mg, 1.78 mmol) and DMAP (34.70 mg, 284.01 μmol) at 20°C. The mixture was stirred at 50°C for 16 h. LC-MS indicated that approximately 3% of reactant 1 remained and the desired compound was detected. Then, 5,6-dichloroisobenzofuran-1,3-dione (385.20 mg, 1.78 mmol) and DMAP (34.70 mg, 284.01 μmol) were added at 20°C, and the mixture was stirred at 50°C for 16 h. LC-MS indicated that reactant 1 was completely consumed and the desired compound was detected. The reaction mixture was diluted with DCM (10 mL), and a portion of saturated NaHCO3 solution (30 mL) was added. The biphasic mixture was stirred at room temperature for an additional 16 h. The biphasic mixture was then separated using a separatory funnel, and the aqueous layer was back-extracted with DCM (3 × 30 mL). The organic layers were then combined, dried (NaSO), concentrated in vacuo, and separated to give a residue. The residue was purified by flash silica gel chromatography (ISCO®, 4 g SepaFlash® silica flash column, 0–6% methanol / dichloromethane gradient @ 75 mL / min) (dichloromethane / methanol = 10, P1 Rf = 0.46) to give target 83 (300 mg, 282.48 μmol, 79.57% yield) as a purple solid.

[0131] [ka] To a solution of target 83 (200 mg, 188.32 μmol) in acetone (2 mL) was added MeI (53.46 mg, 376.64 μmol, 23.45 μL) and Na2CO3 (39.92 mg, 376.64 μmol). The mixture was stirred at 20 °C for 12 h. LC-MS showed that the reactants were completely consumed and the desired mass was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative TLC (SiO2, dichloromethane / methanol = 10 / 1, P1 Rf = 0.67) to give target 83-M (55 mg, 51.11 μmol, 27.14% yield) as a purple solid.

[0132] [ka] To a solution of target 83-M (55 mg, 51.11 umol, 1 equiv.) in MeOH (1 mL) was added CSA (25.59 mg, 102.22 umol). The mixture was stirred at 20 °C for 1 h. LC-MS showed that reactant 1 was completely consumed and the desired mass was detected. NaHCO (30 mg) was added, and then the mixture was partitioned between DCM (3 mL) and water (3 mL). The aqueous phase was back-extracted with DCM (3 * 5 mL). The organic layers were then combined, dried (NaSO), and concentrated in vacuo. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative HPLC (neutral conditions) column: Waters Xbridge BEH C18 100*30mm*10um; mobile phase: [water (NH4HCO3)-ACN]; B%: 25%~65%, 8 min) to give target 89 (14.6 mg, 13.37 umol, yield 26.16%, purity 94.88%) as a purple solid. 1H NMR (400 MHz, chloroform-d) δ = 14.69 (s, 1H), 9.14 (brs, 1H), 8.21 (s, 1H), 7.80 (s, 1H), 7.65 (s, 1H), 6.31–6.23 (m, 1H), 6.17 (d, J = 12.4 Hz, 2H), 5.80 (dd, J = 6.7, 15.7 Hz, 1H), 5.10 (dd, J = 9.0, 12.7 Hz, 1H), 4.74–4.66 (m, 1H), 4.29–4.19 (m, 1H), 3.82 (s, 3H), 3 .54~3.48(m, 2H), 3.27(d, J=6.0Hz, 1H), 3.20(dd, J=2.8, 9.0Hz, 1H), 3.17~3.10(m, 1H), 2.96(s, 3H), 2.91~2.82(m, 2H), 2.64 ~2.49(m, 2H), 2.21(s, 4H), 2.05~1.96(m, 2H), 1.94(s, 4H), 1.79~1.72(m, 3H), 1.65(brd, J=7.0Hz, 2H), 1.61(s, 3H), 1.33(br dd, J=6.4, 14.4Hz, 1H), 0.92(d, J=7.0Hz, 3H), 0.85(d, J=6.5Hz, 6H), 0.72(d, J=6.9Hz, 3H), 0.56(d, J=6.9Hz, 3H), 0.01(d, J=7.0Hz, 3H);MS(ESI)m / z[M+H] + 1035.3.

[0133] [ka] To a solution of target 83 (300 mg, 282.48 μmol, 1 equiv) in acetone (2 mL) was added EtI (88.11 mg, 564.95 μmol, 45.19 μL, 2 equiv) and Na2CO3 (59.88 mg, 564.95 μmol, 2 equiv). The mixture was stirred at 20 °C for 12 h. LC-MS showed that reactant 1 was completely consumed and the desired mass was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative TLC (SiO2, dichloromethane / methanol = 10 / 1, P1 Rf = 0.67) to give target 83-E (100 mg, 91.74 μmol, 32.48% yield) as a purple solid.

[0134] [ka] To a solution of target 83-E (100 mg, 91.74 μmol) in MeOH (1 mL) was added CSA (45.93 mg, 183.47 μmol). The mixture was stirred at 20 °C for 1 h. NaHCO (30 mg) was added, and then the mixture was partitioned between DCM (3 mL) and water (3 mL). The aqueous phase was back-extracted with DCM (3 * 5 mL). The organic layers were combined, dried (Na SO ), and concentrated in vacuo to give a residue. The residue was purified by preparative HPLC (neutral conditions) column: Column: Waters Xbridge BEH C18 100 * 30 mm * 10 μm; Mobile phase: [water (NH 4 HCO 3 )-ACN]; B%: 25% to 65%, 8 min) to give target 94 (11.8 mg, 10.90 μmol, 11.88% yield, 97% purity) as a purple solid. 1 H NMR (400MHz, chloroform-d) δ=14.87(br s, 1H), 8.77(br s, 1H), 8.19(s, 1H), 7.51~7.41(m, 2H), 6.59~6.47(m, 1H), 6.28(br d, J=10.6Hz, 1H), 6.19(dd, J=7.0, 15.9Hz, 1H), 6.06(dd, J=1.2, 12.3Hz, 1H), 5.5 0(d, J=10.1Hz, 1H), 5.09(dd, J=4.7, 12.3Hz, 1H), 4.42(q, J=7.1Hz, 2H), 3.84(br d. 30(m, 6H), 2.12(s, 4H), 1.83(s, 5H), 1.76~1.67(m, 6H), 1.60~1.54(m, 1H), 1.43(t, J=7.1Hz, 3H), 1.09(d, J=7.0Hz, 3H), 1.00(br MS(ESI)m / z[M+H] + 1049.7.

[0135] Synthesis of 25-O-carbamoylsulfonamide rifabutin (sulfonamide analogue) [ka] To a solution of LT-I-00F (40.0 mg, 0.043 mmol) in dichloromethane (0.5 mL) was added methyl trifluoromethanesulfonate (0.064 mmol, 7 μL). After stirring the mixture at room temperature for 10 minutes, benzenesulfonamide (33.5 mg, 0.21 mmol) and triethylamine (0.22 mmol, 30 μL) were added, and the reaction mixture was further stirred at room temperature for 1 hour. The reaction mixture was then partitioned between dichloromethane (5 mL) and HO (5 mL). The aqueous phase was then back-extracted with dichloromethane (2 × 5 mL). The combined organic layers were concentrated under reduced pressure, and the resulting crude product was purified by preparative TLC (hexane:ethyl acetate:methanol:triethylamine=7:2:1:0.1) to give BSFA-acetonide rifabutin (33.4 mg, 76%). MS (ESI): C 54 H 69 N5O 13 m / z of S [M+H] + Theoretical value: 1028.4685; Measured value: 1028.4637 (error 4.7 ppm). 1H NMR(600MHz、CDCl3)δ14.73(s、1H)、8.67(s、1H)、7.95~7.86(m、4H)、7.70(s、1H)、7.58~7.53(m、1H)、7.52~7.46(m、3H)、7.42(t、J=7.7Hz、2H)、7.03(t、J=1.1Hz、1H)、6.87(t、J=1.3Hz、1H)、6.27(dd、J=15.7、10.8Hz、1H)、6.16(dd、J=10.7、1.6Hz、1H)、6.05(dd、J=15.7、7.0Hz、1H)、5.85~5.80(m、1H)、4.96(dd、J=12.2、6.6Hz、1H)、4.79(dd、J=8.2、1.7Hz、1H)、3.69(s、2H)、3.53(dd、J=10.6、3.2Hz、1H)、3.26(s、1H)、3.12(h、J=11.4Hz、4H)、2.94(dd、J=10.2、5.1Hz、1H)、2.66(s、3H)、2.58(s、2H)、2.30(s、3H)、2.23(dt、J=10.6、6.9Hz、1H)、2.06~1.95(m、4H)、1.75(s、3H)、1.63(t、J=8.2Hz、1H)、1.49~1.41(m、1H)、1.36~1.29(m、1H)、1.08(s、3H)、1.01(d、J=6.5Hz、6H)、0.81(d、J=6.0Hz、6H)、0.75(d、J=6.6Hz、3H)、0.55(d、J=7.1Hz、3H)、0.22(d、J=6.8Hz、3H)。 13 C NMR(151MHz、CDCl3)δ192.8、181.7、172.1、168.8、168.2、155.6、142.4、142.2、141.2、140.9、140.8、137.9、132.9、132.7、132.6、131.2、129.2、129.0、128.7、127.8、126.5、125.3、123.9、120.2、114.8、114.0、111.7、108.7、106.1、104.8、100.0、93.8、70.9、65.7、56.1、51.6、51.3、40.8、40.6、36.1、35.2、34.5、34.4、33.5、25.8、25.4、23.8、21.1、21.1、20.3、20.3、17.9、12.9、9.7、9.6、7.9。

[0136] Synthesis of carbamate 1 [ka] To a solution of LT-I-00F-A (200 mg, 209.61 μmol) in DCM (0.5 mL) was added (3-phenylisoxazol-5-yl)methanamine (365.14 mg, 2.10 mmol). The mixture was stirred at 20 °C for 2 h. LC-MS showed that LT-I-00F-A was completely consumed and the desired compound was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative TLC (SiO, petroleum ether / ethyl acetate / methanol = 7 / 2 / 1, P1 Rf = 0.31) to give 1-P (70 mg, 66.97 μmol, 31.95% yield) as a purple solid.

[0137] [ka] To a solution of 1-P (70 mg, 66.97 μmol) in MeOH (1 mL) was added CSA (33.53 mg, 133.94 μmol). The mixture was stirred at 20° C. for 0.5 h. LC-MS showed that 1-P was completely consumed and the desired mass was detected. NaHCO (30 mg) was added, and then the mixture was partitioned between DCM (3 mL) and water (3 mL). The aqueous phase was back-extracted with DCM (3*5 mL). The organic layers were then combined, dried (NaSO), and concentrated in vacuo. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative HPLC (neutral conditions) using a Waters Xbridge BEH C18 column (100*30mm*10um); mobile phase: [water (NH4HCO3)-ACN]; B%: 70%-100% in 8 minutes to give carbamate 1 (11.4mg, 10.74umol, yield 16.04%, purity 94.74%) as a purple solid. 1H NMR Chloroform-d 400MHz δ=14.81(br s, 1H), 9.02(br s, 1H), 8.16(s, 1H), 7.80(br d, J=3.6Hz, 2H), 7.48~7.43(m, 3H), 6.52(s, 1H), 6.38(br dd, J=10.1, 15.7Hz, 1H), 6.28~6.14(m, 2H), 5.98(br dd, J=6.7, 15.4Hz, 1H), 5.33~5.26(m, 1H), 5.03(br dd, J=7.4, 12.3Hz, 1H), 4.62~4.48(m, 2H), 4.40(br dd, J=5.4, 16.2Hz, 1H), 4.22(br d, J=4.0Hz, 1H), 3.70~3.56(m, 2H), 3.35(br d, J=5.6Hz, 1H), 3.15~2.93(m, 6H), 2.77~2.55(m, 2H), 2.39~2.24(m, 6H), 2. 04(s, 4H), 1.89~1.80(m, 2H), 1.78~1.67(m, 6H), 1.51~1.40(m, 2H), 1.00(br d, J=6.9Hz, 3H), 0.94(br d, J=5.9Hz, 6H), 0.83(br d, J=6.8Hz, 3H), 0.58(br d, J=6.6Hz, 3H), -0.01(d, J=7.1Hz, 3H)

[0138] Synthesis of carbamate 2 [ka] To a solution of LT-I-00F-A (200 mg, 212.74 μmol) in ACN (2 mL) was added benzylpiperidin-4-amine (404.81 mg, 2.13 mmol). The mixture was stirred at 20 °C for 2 h. LC-MS showed that LT-I-00F-A was completely consumed and the desired mass was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative TLC (SiO, petroleum ether / ethyl acetate / methanol = 7 / 2 / 1, P1 Rf = 0.46) to give 2-P (100 mg, 94.22 μmol, 44.29% yield) as a purple solid.

[0139] [ka] To a solution of 2-P (100 mg, 94.22 μmol) in MeOH (2 mL) was added CSA (47.17 mg, 188.45 μmol). The mixture was stirred at 20° C. for 0.5 h. LC-MS showed that 2-P was completely consumed, with approximately 80% of the desired mass detected. NaHCO (30 mg) was added, and then the mixture was partitioned between DCM (3 mL) and water (3 mL). The aqueous phase was back-extracted with DCM (3*5 mL). The organic layers were combined, dried (NaSO), and concentrated to give a residue. The residue was purified by preparative HPLC (neutral column: Waters Xbridge BEH C18 100*30mm*10um; mobile phase: [water (NH4HCO3)-ACN]; B%: 75%-95%, 8 min) to give carbamate 2 (54.6 mg, 50.26 umol, yield 53.34%, purity 94%) as a purple solid. 1H NMR Chloroform-d 400MHz δ=14.88(s, 1H), 8.92(br s, 1H), 8.21(s, 1H), 7.37~7.33(m, 2H), 7.32~7.26(m, 3H), 6.42(br dd, J=10.3, 15.6Hz, 1H), 6.27(br d, J=10.1Hz, 1H), 6.16(d, J=12.4Hz, 1H), 6.07(br dd, J=6.9, 15.7Hz, 1H), 5.04(dd, J=6.5, 12.4Hz, 1H), 4.68(br d, J=8.1Hz, 1H), 4.61~4.50(m, 2H), 3.80(s, 1H), 3.67(br d, J=9.8Hz, 1H), 3.53~3.48(m, 3H), 3.43(br d, J=5.6Hz, 1H), 3.11(s, 4H), 2.99(br d, J=4.1Hz, 2H), 2.82(br d, J=10.4Hz, 2H), 2.64(br dd, J=10.5, 12.0Hz, 2H), 2.38~2.29(m, 6H), 2.16~2.06(m, 7H), 1.93~1.75(m, 9H), 1.69~1.61(m, 1 H), 1.51~1.40(m, 3H), 1.05(d, J=7.0Hz, 3H), 0.97(d, J=6.5Hz, 6H), 0.86(d, J=6.9Hz, 3H), 0.59(br d, J=6.8Hz, 3H), -0.01(d, J=7.0Hz, 3H).

[0140] Synthesis of carbamate 3 [ka] To a solution of LT-I-00F-A (200 mg, 212.74 μmol) in DCM (2 mL) was added 2-methylaniline (227.96 mg, 2.13 mmol). The mixture was stirred at 20 °C for 2 h. LC-MS showed that LT-I-00F-A was completely consumed and the desired mass was detected. The reaction mixture was concentrated and purified by preparative TLC (SiO, petroleum ether / ethyl acetate / methanol = 7 / 2 / 1, P1 Rf = 0.46) to give 3-P (90 mg, 34.60% yield, 80% purity) as a purple solid.

[0141] [ka] To a solution of 3-P (90 mg, 92.01 umol) in MeOH (2 mL) was added CSA (46.06 mg, 184.02 umol). The mixture was stirred at 20 °C for 0.5 h. LC-MS showed that 3-P was completely consumed and the desired mass was detected. NaHCO (30 mg) was added, and then the mixture was partitioned between DCM (3 mL) and water (3 mL). The aqueous phase was back-extracted with DCM (3 * 5 mL). The organic layers were combined, dried (NaSO), and concentrated to give a residue. The residue was purified by preparative HPLC (neutral column: Waters Xbridge BEH C18 100*30mm*10um; mobile phase: [water (NH4HCO3)-ACN]; B%: 75%-95%, 8 min) to give carbamate 3 (53.2 mg, yield 56.09%, purity 91%) as a purple solid. 1 H NMR Chloroform-d 400MHz, δ=14.82(s, 1H), 8.97(br s, 1H), 8.18(br s, 1H), 7.70(br s, 1H), 7.19(br t, J=7.8Hz, 1H), 7.14(d, J=7.4Hz, 1H), 7.06~7.00(m, 1H), 6.47~6.35(m, 2H), 6.23(br d, J=10.3Hz, 1H), 6.16(br d, J=12.4Hz, 1H), 6.01(br dd, J=6.9, 15.8Hz, 1H), 5.05(dd, J=6.9, 12.4Hz, 1H), 4.62(br d, J=9.8Hz, 1H), 4.26(br d, J=3.6Hz, 1H), 3.71~3.61(m, 2H), 3.42(br d, J=6.0Hz, 1H), 3.15(br dd, J=3.7, 9.4Hz, 1H), 3.09(s, 3H), 2.95(br d, J=5.1Hz, 2H), 2.61(br s, 2H), 2.36~2.25(m, 6H), 2.21(s, 3H), 2.15~1.91(m, 7H), 1.80~1.71(m, 6H), 1.56~1. 46(m, 1H), 1.01(d, J=7.0Hz, 3H), 0.92(d, J=6.5Hz, 6H), 0.83(d, J=6.9Hz, 3H), 0.61(br d, J=6.6Hz, 3H), -0.01(br d, J=6.8Hz, 3H).

[0142] Synthesis of carbamate 4 [ka] To a solution of LT-I-00F-A (200 mg, 212.74 μmol) in DCM (2 mL) was added 2-methoxyaniline (262.00 mg, 2.13 mmol). The mixture was stirred at 20 °C for 2 h. LC-MS showed that LT-I-00F-A was completely consumed and the desired mass was detected. The reaction mixture was concentrated and purified by preparative TLC (SiO, petroleum ether / ethyl acetate / methanol = 7 / 2 / 1, P1 Rf = 0.46) to give 4-P (90 mg, 34.04% yield, 80% purity) as a purple solid.

[0143] [ka] To a solution of 4-P (90 mg, 90.53 umol) in MeOH (2 mL) was added CSA (45.32 mg, 181.05 umol). The mixture was stirred at 20 °C for 0.5 h. LC-MS showed that 4-P was completely consumed, with approximately 85% of the desired mass detected. NaHCO (32 mg) was added, and then the mixture was partitioned between DCM (3 mL) and water (3 mL). The aqueous phase was back-extracted with DCM (3 * 5 mL). The organic layers were combined, dried (NaSO), and concentrated in vacuo. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative HPLC (neutral column: Waters Xbridge BEH C18 100*30mm*10um; mobile phase: [water (NH4HCO3)-ACN]; B%: 75%~95%, 8 min, and preparative HPLC (neutral column): Phenomenex Luna C18 75*30mm*3um; mobile phase: [water (FA)-ACN]; B%: 30%~70%, 8 min) to give carbamate 4 (22.7 mg, 25.76% yield, 98% purity) as a purple solid. 1H NMR Chloroform-d 400MHz δ=14.81(s, 1H), 8.89(br s, 1H), 8.15(s, 1H), 8.04(br d, J=5.5Hz, 1H), 7.24(br s, 1H), 7.08~6.92(m, 2H), 6.86(d, J=8.1Hz, 1H), 6.47~6.35(m, 1H), 6.25(br d, J=10.1Hz, 1H), 6.14(d, J=12.5Hz, 1H), 6.04(dd, J=6.9, 15.7Hz, 1H), 5.04(dd, J=6.6, 12.5Hz, 1H), 4.67(br d, J=9.3Hz, 1H), 4.33(br s, 1H), 3.85(s, 3H), 3.78~3.63(m, 2H), 3.46(br d, J=5.3Hz, 1H), 3.17(br d, J=10.1Hz, 1H), 3.07(s, 5H), 2.73(br d. d, J=6.5Hz, 6H), 0.84(d, J=6.8Hz, 3H), 0.63(d, J=6.8Hz, 3H), -0.01(d, J=7.1Hz, 3H)

[0144] Synthesis of carbamate 5 [ka] To a solution of LT-I-00F-A (250 mg, 262.02 μmol) in DCM (2 mL) was added 1-isobutylpiperazine dihydrochloride (169.13 mg, 786.06 μmol). The mixture was stirred at 20 °C for 3 h. LC-MS showed that LT-I-00F-A was completely consumed and the desired mass was detected. The reaction mixture was concentrated and purified by preparative TLC (SiO, petroleum ether / ethyl acetate / methanol = 7 / 2 / 1, P1 Rf = 0.31) to give 5-P (66 mg, 65.14 μmol, 24.86% yield) as a purple solid.

[0145] [ka] To a solution of 5-P (66 mg, 65.14 umol) in MeOH (1 mL) was added CSA (32.61 mg, 130.27 umol). The mixture was stirred at 20 °C for 0.5 h. LC-MS showed that 5-P was completely consumed and the desired mass was detected. NaHCO (30 mg) was added, and then the mixture was partitioned between DCM (3 mL) and water (3 mL). The aqueous phase was back-extracted with DCM (3 * 5 mL). The organic layers were combined, dried (NaSO), and concentrated to give a residue. The residue was purified by preparative HPLC (neutral column: Waters Xbridge BEH C18 100*30mm*10um; mobile phase: [water (NH4HCO3)-ACN]; B%: 75%-95%, 10 min) to give carbamate 5 (22.27 mg, 33.73% yield, 96% purity) as a purple solid. 1 H NMR chloroform-d 400 MHz δ = 14.86 (s, 1H), 8.97 (br s, 1H), 8.12 (s, 1H), 6.45 (br dd, J = 10.4, 15.8 Hz, 1H), 6.27 (br d, J = 10.5 Hz, 1H), 6.19–6.05 (m, 2H), 5.05 (dd, J = 6.3, 12.4 Hz, 1H), 4.76 (br d, J = 3.6 Hz, 1H), 4.62 (br d, J = 10.6 Hz, 1H), 3.82 (s, 1H), 3.69 (br d, J=9.8Hz, 1H), 3.52~3.39(m, 5H), 3.10(s, 3H), 3.08~2.94(m, 3H), 2.71~2.58(m, 2H), 2.41~2.25(m, 11H), 2.20~2.15(m, 1H), 2.08(br s, 5H), 1.97(br d, J=15.3Hz, 1H), 1.89~1.83(m, 2H), 1.79(s, 5H), 1.71~1.64(m, 1H), 1.58~1.52(m, 1H), 1.06(br d, J=6.9Hz, 3H), 0.96(br d, J=6.3Hz, 6H), 0.91(d, J=6.4Hz, 6H), 0.87(br d, J=6.9Hz, 3H), 0.60(br d, J=6.6Hz, 3H), -0.01(br d, J=7.0Hz, 3H).

[0146] Synthesis of carbamate 6 [ka] To a solution of LT-I-00F-A (250 mg, 262.02 μmol) in DCM (2 mL) was added pyrimidin-4-amine (74.76 mg, 786.06 mmol). The mixture was stirred at 20 °C for 2 h. LC-MS showed that LT-I-00F-A was completely consumed and the desired mass was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative TLC (SiO, petroleum ether / ethyl acetate / methanol = 7 / 2 / 1, P1 Rf = 0.31) to give 6-P (70 mg, 23.78% yield, 86% purity) as a purple solid.

[0147] [ka] To a solution of 6-P (70 mg, 72.45 μmol) in MeOH (1 mL) was added CSA (36.27 mg, 144.91 μmol). The mixture was stirred at 20 °C for 0.5 h. LC-MS showed that 6-P was completely consumed and the desired mass was detected. NaHCO (30 mg) was added, and then the mixture was partitioned between DCM (3 mL) and water (3 mL). The aqueous phase was back-extracted with DCM (3 * 5 mL). The organic layers were combined, dried (NaSO), and concentrated to give a residue. The residue was purified by preparative HPLC (neutral condition column: Waters Xbridge BEH C18 100*30mm*10um; mobile phase: [water (NH4HCO3)-ACN]; B%: 50%-70%, 8 min) to give carbamate 6 (34.2 mg, yield 50.97%, purity 100%) as a purple solid. 1H NMR Chloroform-d 400MHz δ=14.71(br s, 1H), 9.36(br s, 1H), 9.15(br s, 1H), 8.86(s, 1H), 8.59(d, J=5.9Hz, 1H), 8.23(s, 1H), 8.01(d, J=5.5Hz, 1H), 6.3 9~6.21(m, 3H), 5.96(dd, J=6.6, 15.4Hz, 1H), 5.27(dd, J=8.5, 12.6Hz, 1H), 4.76(br d, J=10.6Hz, 1H), 3.70~3.62(m, 2H), 3.32(br dd. d. d, J=7.0Hz, 3H), 0.96(d, J=6.5Hz, 6H), 0.84(br d, J=6.9Hz, 3H), 0.65(br d, J=6.9Hz, 3H), -0.01(br d, J=7.0Hz, 3H).

[0148] Synthesis of carbamate 7 [ka] To a solution of LT-I-00F-A (250 mg, 262.02 μmol) in DCM (2 mL) was added pyridin-3-amine (73.98 mg, 786.06 μmol). The mixture was stirred at 20 °C for 3 h. LC-MS showed that LT-I-00F-A was completely consumed and the desired mass was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative TLC (SiO, petroleum ether / ethyl acetate / methanol = 7 / 2 / 1, P1 Rf = 0.31) to give 7-P (130 mg, 51.41% yield) as a purple solid.

[0149] [ka] To a solution of 7-P (120 mg, 124.33 umol) in MeOH (1 mL) was added CSA (62.24 mg, 248.67 umol). The mixture was stirred at 20 °C for 0.5 h. LC-MS showed that 7-P was completely consumed and the desired mass was detected. NaHCO (30 mg) was added, and then the mixture was partitioned between DCM (3 mL) and water (3 mL). The aqueous phase was back-extracted with DCM (3 * 5 mL). The organic layers were combined, dried (NaSO), and concentrated to give a residue. The residue was purified by preparative HPLC (neutral condition column: Waters Xbridge BEH C18 100*30mm*10um; mobile phase: [water (NH4HCO3)-ACN]; B%: 60%-80%, 8 min) to give carbamate 7 (30.91 mg, 33.41 umol, 26.87% yield) as a purple solid. 1H NMR Chloroform-d 400MHz δ=14.77(s, 1H), 9.13(br s, 1H), 8.42(br d, J=1.5Hz, 1H), 8.31(br d, J=4.3Hz, 1H), 8.19(s, 1H), 8.00(br s, 1H), 7.26~7.22(m, 1H), 6.90(br s, 1H), 6.42~6.33(m, 1H), 6.28~6.13(m, 2H), 5.99(dd, J=6.8, 15.8Hz, 1H), 5.14(dd, J=7.6, 12.5Hz, 1H), 4.69(br d, J=10.5Hz, 1H), 3.94(br d, J=5.3Hz, 1H), 3.65(br d, J=9.9Hz, 1H), 3.48(s, 1H), 3.40(br d, J=5.8Hz, 1H), 3.16(br dd, J=5.3, 9.0Hz, 1H), 3.05(s, 3H), 2.96(br s, 2H), 2.62(br s, 2H), 2.41~2.31(m, 4H), 2.27(br d, J=6.6Hz, 2H), 2.11(br d, J=5.9Hz, 1H), 2.04(s, 3H), 1.95(br s, 2H), 1.86~1.72(m, 7H), 1.52~1.41(m, 1H), 1.03(d, J=6.9Hz, 3H), 0.93(br d, J=6.4Hz, 6H), 0.83(d, J=6.9Hz, 3H), 0.62(d, J=6.9Hz, 3H), -0.01(d, J=7.0Hz, 3H)

[0150] Synthesis of carbamate 8 [ka] To a solution of LT-I-00F-A (250 mg, 262.02 μmol) in DCM (2 mL) was added 4-phenylthiazol-2-amine (138.53 mg, 786.06 μmol). The mixture was stirred at 20 °C for 2 h. LC-MS showed that LT-I-00F-A was completely consumed and the desired mass was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative TLC (SiO, petroleum ether / ethyl acetate / methanol = 7 / 2 / 1, P1 Rf = 0.31) to give 8-P (100 mg, 30.98% yield, 85% purity) as a purple solid.

[0151] [ka] To a solution of 8-P (100 mg, 95.9 μmol) in MeOH (1 mL) was added CSA (47.80 mg, 190.97 μmol). The mixture was stirred at 20 °C for 0.5 h. LC-MS showed that 8-P was completely consumed and approximately 52% of the desired mass was detected. NaHCO (30 mg) was added, and then the mixture was partitioned between DCM (3 mL) and water (3 mL). The aqueous phase was back-extracted with DCM (3 * 5 mL). The organic layers were combined, dried (Na SO ), and concentrated to give a residue. The residue was purified by preparative HPLC (neutral column: Waters Xbridge BEH C18 100*30mm*10um; mobile phase: [water (NH4HCO3)-ACN]; B%: 60%-80%, 8 min) to give carbamate 8 (15.6 mg, 15.57% yield) as a purple solid. 1H NMR Chloroform-d 400MHz δ=14.79(s, 1H), 9.15(br s, 1H), 8.82~8.66(m, 1H), 8.19(s, 1H), 7.82(d, J=7.4Hz, 2H), 7.44~7.39(m, 2H), 7.36~7.31 (m, 1H), 7.14(s, 1H), 6.46~6.36(m, 1H), 6.33~6.26(m, 1H), 6.20(d, J=12.5Hz, 1H), 6.04(br dd, J=6.7, 15.7Hz, 1H), 5.20(dd, J=7.4, 12.5Hz, 1H), 4.82(br d, J=9.0Hz, 1H), 3.77~3.66(m, 2H), 3.45(s, 1H), 3.39(br d, J=5.4Hz, 1H), 3.21~3.14(m, 1H), 3.05(s, 3H), 3.03~2.96(m, 2H), 2.70(br s, 2H), 2.41(s, 4H), 2.35(br dd, J=2.9, 7.1Hz, 2H), 2.18~2.14(m, 1H), 2.09(s, 3H), 1.89(td, J=6.3, 13.1Hz, 2H), 1.84~1.71(m, 7H), 1.57~1.45(m, 1H), 1.07(d, J=7.0Hz, 3H), 0.98(d, J=6.4Hz, 6H), 0.87(d, J=6.9Hz, 3H), 0.59(br d, J=4.8Hz, 3H), -0.01(d, J=7.0Hz, 3H)

[0152] Synthesis of carbamate 9 [ka] To a solution of LT-I-00F-A (250 mg, 262.02 μmol) in DCM (2 mL) was added 1,3,4-thiadiazol-2-amine (79.49 mg, 786.06 μmol). The mixture was stirred at 20 °C for 3 h. LC-MS showed that LT-I-00F-A was completely consumed and the desired mass was detected. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by preparative TLC (SiO, petroleum ether / ethyl acetate / methanol = 7 / 2 / 1, P1 Rf = 0.31) to give 9-P (70 mg, 27.48% yield) as a purple solid.

[0153] [ka] To a solution of 9-P (60 mg, 61.72 μmol) in MeOH (1 mL) was added CSA (30.90 mg, 123.44 μmol). The mixture was stirred at 20° C. for 0.5 h. LC-MS showed that 9-P was completely consumed and the desired mass was detected. NaHCO (30 mg) was added, and then the mixture was partitioned between DCM (3 mL) and water (3 mL). The aqueous phase was back-extracted with DCM (3*5 mL). The organic layers were combined, dried (NaSO), and concentrated in vacuo to give a residue. The residue was purified by preparative HPLC (neutral condition column: Waters Xbridge Prep OBD C18 150*40mm*10um; mobile phase: [water (NH4HCO3)-ACN]; B%: 45%-75%, 8 min) to give carbamate 9 (14.92 mg, 15.53 umol, 25.16% yield, 97% purity) as a purple solid. 1 H NMR Chloroform-d 400MHz δ=14.74(s, 1H), 9.31(br s, 1H), 8.79(s, 1H), 8.23(s, 1H), 6.42~6.18(m, 3H), 6.02(br dd, J=6.6, 15.6Hz, 1H), 5.33(dd, J=8.0, 12.5Hz, 1H), 4.90(br d, J=10.5Hz, 1H), 3.72(br d, J=9.4Hz, 2H), 3.46(br d, J=5.9Hz, 1H), 3.32(br s, 1H), 3.27~3.17(m, 1H), 3.00(s, 5H), 2.72(br s, 2H), 2.49~2.30(m, 7H), 2.09(s, 5H), 1.98~1.84(m, 4H), 1.79(s, 4H), 1.53~1.42(m, 1H), 1.07(br d, J=6.9Hz, 3H), 0.97(br dd, J=2.9, 5.7Hz, 6H), 0.88(br d, J=6.9Hz, 3H), 0.68(br d, J=6.6Hz, 3H), -0.01(br d, J=7.0Hz, 3H)

[0154] Synthesis of carbamate 10 [ka] To a solution of LT-I-00F-A (200 mg, 209.61 μmol) in DCM (2 mL) was added 1-tert-butylpiperazine (89.45 mg, 628.84 μmol). The mixture was stirred at 20 °C for 3 h. LC-MS showed that LT-I-00F-A was completely consumed and the desired mass was detected. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by preparative TLC (SiO, petroleum ether / ethyl acetate / methanol = 7 / 2 / 1, P1 Rf = 0.31) to give 10-P (100 mg, 44.26% yield, 94% purity) as a purple solid.

[0155] [ka] To a solution of 10-P (100 mg, 98.68 μmol) in MeOH (1 mL) was added CSA (49.41 mg, 197.38 μmol). The mixture was stirred at 20 °C for 0.5 h. LC-MS showed that 10-P was completely consumed and the desired mass was detected. NaHCO (30 mg) was added, and then the mixture was partitioned between DCM (3 mL) and water (3 mL). The aqueous phase was back-extracted with DCM (3 * 5 mL). The organic layers were combined, dried (NaSO), and concentrated in vacuo to give a residue. The residue was purified by preparative HPLC (neutral condition column: Phenomenex C18 75*30mm*3um; mobile phase: [water (NH4HCO3)-ACN]; B%: 60% to 95%, 10 min) to give carbamate 10 (46.7 mg, yield 45.71%, purity 94%) as a purple solid. 1H NMR Chloroform-d 400MHz δ=14.87(s, 1H), 8.95(br s, 1H), 8.16(s, 1H), 6.50~6.41(m, 1H), 6.27(d, J=10.5Hz, 1H), 6.18~6.07(m, 2H), 5.06(dd, J=6.3, 12.4Hz, 1H), 4.77 (d, J=4.0Hz, 1H), 4.64(d, J=10.4Hz, 1H), 3.84(s, 1H), 3.70(d, J=9.8Hz, 1H), 3.52~3.37(m, 5H), 3.10(s, 3H), 3.07(br dd, J=2.8, 10.3Hz, 1H), 2.99(br dd, J=3.5, 7.4Hz, 2H), 2.71~2.59(m, 2H), 2.50(br d, J=5.0Hz, 4H), 2.39(br dd, J=7.0, 16.4Hz, 1H), 2.35~2.29(m, 5H), 2.24~2.14(m, 1H), 2.08(s, 3H) ), 2.03~1.93(m, 2H), 1.89~1.82(m, 2H), 1.80(s, 4H), 1.67(ddd, J=2.4, 7 .3, 10.2Hz, 1H), 1.59~1.53(m, 1H), 1.09~1.04(m, 12H), 0.97(d, J=6.5Hz , 6H), 0.87(d, J=6.9Hz, 3H), 0.61(d, J=6.9Hz, 3H), -0.01(d, J=7.1Hz, 3H)

[0156] Synthesis of carbamate 11 [ka] To a solution of LT-I-00F-A (250 mg, 262.02 μmol) in DCM (2 mL) was added 1-phenylpiperazine (127.52 mg, 786.06 μmol). The mixture was stirred at 20 °C for 3 h. LC-MS showed that LT-I-00F-A was completely consumed and the desired mass was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative TLC (SiO, petroleum ether / ethyl acetate / methanol = 7 / 2 / 1, P1 Rf = 0.31) to give 11-P (100 mg, 32.50% yield, 88% purity) as a purple solid.

[0157] [ka] To a solution of 11-P (100 mg, 96.78 μmol) in MeOH (1 mL) was added CSA (48.45 mg, 193.56 μmol). The mixture was stirred at 20 °C for 0.5 h. LC-MS showed that 11-P was completely consumed and the desired mass was detected. NaHCO (30 mg) was added, and then the mixture was partitioned between DCM (3 mL) and water (3 mL). The aqueous phase was back-extracted with DCM (3 * 5 mL). The organic layers were combined, dried (NaSO), and concentrated to give a residue. The residue was purified by preparative HPLC (neutral column: Waters Xbridge BEH C18 100*30mm*10um; mobile phase: [water (NH4HCO3)-ACN]; B%: 50%-80%, 8 min) to give carbamate 11 (46.9 mg, 45.33 umol, 46.84% yield, 96% purity) as a purple solid. 1 H NMR chloroform-d 400MHz δ = 14.83 (s, 1H), 9.00 (br s, 1H), 8.14 (s, 1H), 7.30-7.26 (m, 2H), 6.93-6.87 (m, 3H), 6.47-6.35 (m, 1H), 6.25 (br d, J=10.1Hz, 1H), 6.16(d, J=12.3Hz, 1H), 6.03(dd, J=6.9, 15.8Hz, 1H), 5.04(dd, J=6.9, 12.4Hz, 1H), 4. 62~4.55(m, 2H), 3.75(s, 1H), 3.68~3.51(m, 5H), 3.36(dd, J=1.4, 6.8Hz, 1H), 3.16~3.01(m, 8H), 2.96(br d, J=4.0Hz, 2H), 2.74~2.53(m, 2H), 2.40~2.26(m, 6H), 2.19~2.09(m, 1H), 2.05(s, 3H), 1.98(br d, J=15.4Hz, 2H), 1.88~1.80(m, 2H), 1.75(s, 5H), 1.56~1.44(m, 1H), 1.03(d, J=7.0Hz, 3H) , 0.94(d, J=6.5Hz, 6H), 0.83(d, J=6.9Hz, 3H), 0.60(d, J=6.8Hz, 3H), -0.01(d, J=7.1Hz, 3H)

[0158] Synthesis of carbamate 12 [ka] To a solution of LT-I-00F-A (250 mg, 262.02 μmol) in DCM (2 mL) was added pyridazin-3-amine (74.76 mg, 786.06 mmol). The mixture was stirred at 20 °C for 3 h. LC-MS showed that LT-I-00F-A was completely consumed and the desired mass was detected. The reaction mixture was concentrated and purified by preparative TLC (SiO, petroleum ether / ethyl acetate / methanol = 7 / 2 / 1, P1 Rf = 0.31) to give 12-P (80 mg, 23.07% yield, 73% purity) as a purple solid.

[0159] [ka] To a solution of 12-P (80 mg, 82.80 μmol) in MeOH (1 mL) was added CSA (41.45 mg, 165.61 μmol). The mixture was stirred at 20° C. for 0.5 h. LC-MS showed that 12-P was completely consumed and the desired mass was detected. NaHCO (30 mg) was added, and then the mixture was partitioned between DCM (3 mL) and water (3 mL). The aqueous phase was back-extracted with DCM (3*5 mL). The organic layers were combined, dried (NaSO), and concentrated to give a residue. The residue was purified by preparative HPLC (neutral condition column: Waters Xbridge BEH C18 100*30mm*10um; mobile phase: [water (NH4HCO3)-ACN]; B%: 60%-80%, 8 min) to give carbamate 12 (26.9 mg, yield 35.08%, purity 100%) as a purple solid. 1H NMR Chloroform-d 400MHz δ=14.74(s, 1H), 9.28(br s, 1H), 8.90(br d, J=3.9Hz, 1H), 8.29~8.21(m, 2H), 7.99(s, 1H), 7.48(dd, J=4.6, 9.0Hz, 1H), 6.41~6.33(m, 1H), 6.30~6.20(m, 2H), 6.02(dd, J=6.5, 15.6Hz, 1H), 5.28(dd, J=8.1, 12.6Hz, 1H), 4.80(br d, J=10.3Hz, 1H), 3.79~3.69(m, 2H), 3.45~3.38(m, 1H), 3.34(s, 1H), 3.26~3.17(m, 1H), 3.10~2.95(m, 5H), 2.67(br s, 2H), 2.46~2.37(m, 4H), 2.32(br d, J=7.3Hz, 2H), 2.20~2.12(m, 1H), 2.08(s, 3H), 2.04~1.96(m, 2H), 1.93(br d, J=2.9Hz, 4H), 1.78(s, 3H), 1.53~1.42(m, 1H), 1.07(d, J=7.0Hz, 3H), 0.97(d, J=6.5Hz, 6H), 0.87(br d, J=6.9Hz, 3H), 0.68(d, J=6.9Hz, 3H), -0.01(d, J=7.1Hz, 3H).

[0160] 25-O-(1-methylpiperidine-4-aminoacyl)-25-O-desacetylifabutin [ka] Prepared from LT-I-00F (200.0 mg) using carbamate formation procedure A and the basic deprotection procedure to afford the title compound (21.6 mg, 10% over three steps) as a purple solid. 1H NMR (400MHz, chloroform-d) δppm -0.03(d, J=7.13Hz, 3H)0.57(d, J=6.88Hz, 3H)0.84(d, J=7.00Hz, 3H)0.94(d, J=6.50Hz, 6H)1.03(d, J=7.00Hz, 3H)1.39~1.5 2(m, 3H) 1.58~1.67(m, 1H) 1.74~1.79(m, 4H) 1.81~1.99(m, 7H) 2.03~2.13(m, 6H) 2.26~2.43(m, 9H) 2.54~2.70(m, 2H) 2.79(br d, J=10.13Hz, 2H)2.98(br d, J=5.25Hz, 2H) 3.09 (s, 4H) 3.32~3.84 (m, 4H) 4.46~4.57 (m, 2H) 4.66 (br d, J=8.13Hz, 1H)5.03(dd, J=12.44, 6.57Hz, 1H)6.04(dd, J=15.70, 6.94Hz, 1H)6.15(d, J=12.51Hz, 1H)6.25(br d, J=10.13Hz, 1H)6.32~6.45(m, 1H)8.19(s, 1H)8.91(br s, 1H)14.85(s, 1H);C 51 H 73 NO 11 HRMS (ESI-TOF) m / z [M+H] + Theoretical value: 945.5332, measured value: 945.5368 (error 3.8 ppm).

[0161] 25-O-(1-ethylpiperidine-4-aminoacyl)-25-O-desacetylifabutin [ka] Prepared from LT-I-00F (300.0 mg) using carbamate formation procedure A and the basic deprotection procedure to afford the title compound (35.0 mg, 10% over three steps) as a purple solid. 1H NMR (400MHz, chloroform-d) δppm -0.05 (br d, J=7.00Hz, 3H) 0.56 (br d, J=6.75Hz, 3H) 0.84(d, J=6.88Hz, 3H) 0.95(d, J=6.50Hz, 6H) 1.03(d, J=7.00Hz, 3H) 1.14(br s, 3H) 1.40~1.67 (m, 4H) 1.83~1.99 (m, 5H) 2.00~2.12 (m, 5H) 2.19 (br d. C 52 H 75 NO 11 HRMS (ESI-TOF) m / z [M+H] + Theoretical value: 959.5488, measured value: 959.5521 (error 3.4 ppm).

[0162] 25-O-(1-Isopropylpiperidine-4-aminoacyl)-25-O-desacetylifabutin [ka] Prepared from LT-I-00F (200.0 mg) using carbamate formation procedure A and the basic deprotection procedure to afford the title compound (19.8 mg, 10% over three steps) as a purple solid. 1H NMR (400MHz, chloroform-d) δppm -0.03 (d, J=7.00Hz, 3H) 0.57 (br d, J=6.88Hz, 3H) 0.84(d, J=6.88Hz, 3H) 0.94(d, J=6.50Hz, 6H) 1.00~1.07(m, 9H) 1.26~1.55(m, 4H) 1.62~1.79 (m, 7H) 1.81~2.00 (m, 7H) 2.06 (s, 5H) 2.22~2.40 (m, 8H) 2.55~2.78 (m, 3H) 2.84 (br d, J=10.88Hz, 2H)2.97(br d, J=4.25Hz, 2H) 3.36~3.52(m, 2H) 3.64(br d, J=9.76Hz, 1H) 3.77(s, 1H) 4.46~4.59(m, 2H) 4.65(br d. dd, J=15.63, 10.26Hz, 1H)8.17(s, 1H)8.91(br s, 1H)14.86(s, 1H);C 53 H 77 NO 11 HRMS (ESI-TOF) m / z [M+H] + Theoretical value: 973.5645, measured value: 973.5668 (error 2.4 ppm).

[0163] 25-O-(1-isobutylpiperidine-4-aminoacyl)-25-O-desacetylifabutin [ka] Prepared from LT-I-00F (200.0 mg) using carbamate formation procedure A and the basic deprotection procedure to give the title compound (35.2 mg, 15% over three steps) as a purple solid. 1H NMR (400 MHz, chloroform-d) δ ppm -0.03 (br d, J = 7.00 Hz, 3H), 0.57 (br d, J = 6.75 Hz, 3H), 0.84 (d, J = 6.88 Hz, 3H), 0.89 (br d, J=6.25Hz, 6H) 0.95(d, J=6.50Hz, 6H)1.03(d, J=7.00Hz, 3H)1.37~1.53(m, 3H)1 .55~1.68(m, 1H)1.70~1.80(m, 5H)1.81~1.98(m, 5H)2.00~2.13(m, 8H)2.26~2.43( m, 6H) 2.63~2.89 (m, 4H) 2.95~3.15 (m, 6H) 3.31~3.54 (m, 3H) 3.58~3.96 (m, 2H) 4.38 ~4.76(m, 3H)5.02(dd, J=12.51, 6.38Hz, 1H)5.90~6.72(m, 4H)8.15(s, 1H)8.87(br s, 1H)14.84(s, 1H);C 54 H 79 NO 11 HRMS (ESI-TOF) m / z [M+H] + Theoretical value: 987.5801, measured value: 987.5798 (error 0.3 ppm).

[0164] 25-O-(pyrimidine-4-aminoacyl)-25-O-desacetylifabutin [ka] Prepared from 3 (29.9 mg) using carbamate formation procedure B and the basic deprotection procedure to give the title compound (16.0 mg, 49% over two steps) as a purple solid. 1H NMR(601MHz、CDCl3)δ14.67(s、1H)、9.44~9.20(br、1H)、8.82(d、J=1.3Hz、1H)、8.57(d、J=5.8Hz、1H)、8.48(s、1H)、8.18(s、1H)、7.97(dd、J=5.9、1.3Hz、1H)、6.33(dd、J=15.7、10.1Hz、1H)、6.26(dd、J=10.1、1.7Hz、1H)、6.22(d、J=12.6Hz、1H)、5.95(dd、J=15.7、6.6Hz、1H)、5.25(dd、J=12.6、8.7Hz、1H)、4.75(dd、J=10.7、1.9Hz、1H)、3.65(d、J=10.0Hz、1H)、3.59(d、J=6.7Hz、1H)、3.31(dd、J=8.5、3.2Hz、1H)、3.21(s、1H)、3.13(ddd、J=9.7、6.7、2.2Hz、1H)、3.05~2.89(m、1H)、2.97(s、3H)、2.73~2.55(m、2H)、2.38(s、3H)、2.43~2.22(m、4H)、2.05(d、J=1.5Hz、3H)、2.00~1.91(m、2H)、1.90~1.79(m、2H)、1.74(s、3H)、1.78~1.70(m、1H)、1.44~1.36(m、1H)、1.03(d、J=7.0Hz、3H)、1.00~0.89(br、6H)、0.83(d、J=6.9Hz、3H)、0.64(d、J=6.9Hz、3H)、-0.03(d、J=7.1Hz、3H); 13 C NMR(151MHz、CDCl3)δ192.5、181.0、171.5、168.4、168.2、158.3、158.2、157.9、153.5、145.6、141.9、140.8、133.0、131.9、125.1、124.0、115.4、114.7、111.8、109.3、109.2、107.7、104.7、94.9、82.1、76.8、75.2、72.4、66.4、56.5、51.6、51.6、48.5、43.6、38.8、38.3、37.8、36.4、35.6、33.1、26.0、24.1、22.4、21.0、20.4、17.4、11.8、11.3、9.0、7.8;MS(ESI)m / z[M+H] + 926.4、[M-H] -924.4.

[0165] 25-O-(pyrazine-2-aminoacyl)-25-O-desacetylifabutin [ka] Prepared from 3 (40.0 mg) using carbamate formation procedure B and the basic deprotection procedure to give the title compound (28.2 mg, 64% over two steps) as a purple solid. 1 H NMR (601MHz, CDCl3) δ14.70(s, 1H), 9.30(s, 1H), 8.29(d, J=2.6Hz, 1H), 8.21~8.18(m, 2H), 7.86(s, 1H), 6.36(dd, J=15.8, 10.1Hz, 1H), 6.25(dd, J=10.1, 1.8Hz, 1H), 6.21(d , J=12.6Hz, 1H), 5.96(dd, J=15.8, 6.7Hz, 1H), 5.20(dd, J=12.6, 8.1Hz, 1H), 4.73(dd, J=10.6, 1.8Hz, 1H), 3.73(d, J=6.1Hz, 1H), 3.65(d, J=9.8Hz, 1H), 3.36(s, 1H), 3.35(d, J=3.0Hz, 1H), 3.05~2.90(br, 2H), 3.01(s, 3H), 2.75~2.51(br, 2H), 2.41~2.36(m, 1H) , 2.36(s, 3H), 2.34~2.20(m, 2H), 2.04(s, 3H), 2.15~1.94(m, 3H), 1.95~1.87(m, 2H), 1. 87~1.80(m, 1H), 1.78~1.74(m, 3H), 1.74(s, 3H), 1.48~1.40(m, 1H), 1.03(d, J=7.2Hz, 3H), 0.95(s, 6H), 0.83(d, J=6.9Hz, 3H), 0.63(d, J=6.9Hz, 3H), -0.00(d, J=7.1Hz, 3H); 13C NMR (151MHz, CDCl3) δ192.5, 181.1, 171.5, 168.5, 168.3, 155.3, 153.7, 148.3, 145.3 , 142.0, 141.8, 140.9, 139.5, 136.2, 133.1, 131.6, 125.1, 124.2, 115.3, 114.7, 111. 9, 109.1, 107.6, 104.8, 94.7, 81.7, 75.4, 72.6, 66.4, 56.6, 51.6, 38.6, 38.1, 38.0, 3 6.3, 35.5, 33.2, 25.9, 22.3, 21.0, 20.4, 17.4, 11.6, 11.4, 9.0, 7.8;MS(ESI)m / z[M+H] + 926.5, [MH] - 924.4.

[0166] 25-O-(thiazole-5-aminoacyl)-25-O-desacetylifabutin [ka] Prepared from 3 (40.0 mg) using carbamate formation procedure B and the basic deprotection procedure to give the title compound (36.0 mg, 82% over two steps) as a purple solid. 1H NMR(601MHz、CD2Cl2)δ14.83(s、1H)、9.13(s、1H)、8.39(s、1H)、8.16(s、1H)、7.45(s、1H)、6.35(dd、J=15.8、10.3Hz、1H)、6.24(dd、J=10.4、1.9Hz、1H)、6.15(d、J=12.5Hz、1H)、6.01(dd、J=15.8、6.8Hz、1H)、5.11(dd、J=12.6、7.3Hz、1H)、4.77(d、J=10.5Hz、1H)、3.86(d、J=6.0Hz、1H)、3.68(d、J=9.8Hz、1H)、3.45(s、1H)、3.23(s、1H)、3.15(br、1H)、3.02(s、3H)、2.97~2.89(m、2H)、2.70~2.50(br、2H)、2.38~2.32(m、1H)、2.31(s、3H)、2.26(d、J=7.4Hz、2H)、2.10~1.88(m、4H)、2.03(s、3H)、1.88~1.80(m、1H)、1.80~1.73(m、1H)、1.73~1.70(m、1H)、1.69(s、3H)、1.53~1.44(m、1H)、1.01(d、J=7.0Hz、3H)、0.94(d、J=6.3Hz、5H)、0.84(d、J=7.0Hz、3H)、0.60(d、J=6.8Hz、3H)、-0.06(d、J=7.0Hz、3H); 13 C NMR(151MHz、CD2Cl2)δ192.9、181.5、171.9、168.6、168.6、155.6、154.9、146.2、144.5、142.6、141.4、136.8、133.0、132.0、128.6、125.6、124.4、116.2、114.6、112.3、109.3、107.6、105.1、95.1、80.5、77.2、76.3、73.1、66.8、57.0、51.9、51.7、38.7、38.7、38.2、36.5、35.7、33.5、26.2、21.9、20.99、20.98、20.5、17.6、11.4、11.0、9.0、7.8;MS(ESI)m / z[M+H] + 931.4、[M-H] - 929.4。

[0167] 25-O-(5,7-dimethylpyrazolo[1,5-a]pyrimidine-3-aminoacyl)-25-O-desacetylifabutin [ka] Prepared from 3 (135.0 mg) using carbamate formation procedure B and the basic deprotection procedure to give the title compound (25.3 mg, 15% over two steps) as a purple solid. 1 H NMR (400MHz, chloroform-d) δ=14.85(s, 1H), 8.94(br s, 1H), 8.42(s, 1H), 8.12(s, 1H), 6.92(s, 1H), 6.59~6.51(m, 1H), 6.48~6.37(m, 1H), 6.25(br d, J=10.3Hz, 1H), 6.18~6.02(m, 2H), 5.09(dd, J=6.3, 12.4Hz, 1H), 4.76(br d, J=10.6Hz, 1H), 4.40(br d, J=4.4Hz, 1H), 3.78~3.65(m, 2H), 3.53(br d, J=4.8Hz, 1H), 3.23(br dd, J=3.8, 9.9Hz, 1H), 3.10(s, 3H), 2.97(br s, 2H), 2.71(s, 3H), 2.66~2.49(m, 5H), 2.40~2.25(m, 5H), 2.21~2.10(m, 1H), 2.06(s, 3H), 1.96(br s, 1H), 1.89~1.67(m, 7H), 1.64~1.52(m, 1H), 1.03(br d, J=6.9Hz, 3H), 0.95(br d, J=6.4Hz, 6H), 0.86(br d, J=6.9Hz, 3H), 0.64(br d, J=6.8Hz, 3H), 0.00(br d, J=7.1Hz, 3H);C 53 H 69 N8O 11 HRMS (ESI-TOF) m / z [M+H] + Theoretical value: 993.5080, measured value: 993.5084 (error 3.7 ppm).

[0168] 25-O-(5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidine-2-aminoacyl)-25-O-desacetylifabutin [ka] Prepared from 3 (90.0 mg) using carbamate formation procedure B and the basic deprotection procedure to give the title compound (29.5 mg, 14% over two steps) as a purple solid. 1 H NMR (400MHz, chloroform-d) δ=14.83(s, 1H), 8.96(s, 1H), 8.11(s, 2H), 6.76(s, 1H), 6.45~6.36(m, 1H), 6.25(br d, J=10.3Hz, 1H), 6.15~6.03(m, 2H), 5.07(dd, J=6.6, 12.4Hz, 1H), 4.85(d, J=10.5Hz) , 1H), 3.97(d, J=5.1Hz, 1H), 3.71~3.64(m, 2H), 3.54(dd, J=1.7, 6.3Hz, 1H), 3.25(br dd, J=4.6, 9.4Hz, 1H), 3.08(s, 3H), 3.04~2.91(m, 2H), 2.78(s, 3H), 2.70~2.53(m, 5H), 2.41~2 .33(m, 4H), 2.29(d, J=7.3Hz, 2H), 2.20~1.93(m, 6H), 1.88~1.77(m, 4H), 1.75(s, 3H), 1.58(br C 52 H 68 N9O 11 HRMS (ESI-TOF) m / z [M+H] + Theoretical value: 994.5033, measured value: 994.4996 (error 3.7 ppm).

[0169] C3, C4-modification chemistry [ka] Basic Scheme 2: Synthesis of C3, C4-modified rifamycin analogues. For reported procedures for C3 and C4 modifications, see (1) 10.1002 / hlca.19730560720; (2) 10.7164 / antibiotics.34.1033. [ka]

[0170] Basic Scheme 3: Synthesis of C3, C4-modified thiazolorifamycins. [ka] To a solution of rifamycin S (5.00 g, 7.17 mmol) in DMF (50 mL) was added NBS (1.50 g, 8.43 mmol). The mixture was stirred at 20 °C for 2 h. LC-MS showed that rifamycin S was completely consumed and the desired mass was detected. The reaction mixture was diluted with 100 mL of HO and extracted with DCM (100 mL × 3). The combined organic phases were dried over anhydrous NaSO, the mixture was filtered, and the filtrate was concentrated under vacuum. The residue was purified by flash silica gel chromatography (ISCO®, 40 g SepaFlash® silica flash column, elution with 0–44% ethyl acetate / petroleum ether gradient @ 100 mL / min) (petroleum ether / ethyl acetate = 1 / 1, P1 Rf = 0.23) to give 3-bromomycin S (2 g, 2.49 mmol, 34.79% yield, 96.55% purity) as a yellow solid.

[0171] [ka] To a solution of 3-bromo- and diaminidase S (50.0 mg, 64.55 μmol) in EtOH (2 mL) was added 2-pyridylthiourea (10.88 mg, 71.00 μmol). The mixture was stirred at 0°C for 2 hours. LC-MS showed the desired compound. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative HPLC FA conditions: column: Phenomenex luna C18 100*40 mm*5 μm, mobile phase: [HO (0.2% FA)-ACN]; gradient: 40% to 70% B over 8.0 min to give thiazolorifamycin 1 (40.6 mg, 44.68 μmol, yield 69.21%, purity 96.39%, FA salt) as an orange solid.1 H NMR (400MHz, chloroform-d) δppm -0.71 (br d, J=6.75Hz, 3H) 0.13 (br d, J=6.75Hz, 3H) 0.82~1.00 (m, 7H) 1.20 (br dd, J=9.57, 7.19Hz, 1H) 1.56 (br d, J=6.50Hz, 1H) 1.87~2.09(m, 9H)2.26(s, 3H)2.35(br dd, J=15.76, 7.13Hz, 1H)2.45~3.10(m, 6H)3.32(br d, J=6.00Hz, 1H) 3.49~3.77 (m, 3H) 4.64~4.75 (m, 1H) 4.76~4.94 (m, 1H) 5.90 ~6.05(m, 1H)6.20~6.42(m, 2H)6.51~6.75(m, 2H)7.03~7.12(m, 1H)7.59(br t, J=7.25Hz, 1H)7.66~7.79(m, 1H)8.46~8.63(m, 1H)15.95~16.18(m, 1H);C 43 H 51 N4O 11 HRMS (ESI-TOF) m / z [M+H] of S + Theoretical value: 831.3270, measured value: 831.3279 (error 1.1 ppm).

[0172] Example 3 Biological data for certain exemplified compounds are shown in Tables 10 and 11. For cLogP vs. plasma fraction unbound plots, see Figure 27. For pharmacokinetic (PK) parameters for certain exemplified compounds, see Table 12. [ka] [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4] [Table 10-5] [Table 10-6] nd

[0173] [Table 11-1] [Table 11-2]

[0174] [Table 12]

[0175] Example 4 In vitro activity, calculated physicochemical properties, and f u was determined using the method disclosed in Example 1 and is shown in Table 13. [Table 13-1] [Table 13-2] [Table 13-3]

[0176] In vitro activity, cLogP, and f of rifabutin carbamate analogs u are shown in Table 14. [Table 14]

[0177] In vitro activity, cLogP, and f of heterocyclic aromatic carbamate analogues of rifabutin u are shown in Table 15. [Table 15-1] [Table 15-2]

[0178] Example 5 Broth MICs and broth MBCs of clinically used antibiotics and a novel ADP-ribosylation-resistant rifabutin analogue against M. abscessus bamboo 90 (bMBC 90 ), and caseum substitute MBC 90 (cMBC 90 ) were evaluated. Rifamycin analogs 5a, 5m, and 5n showed improved bactericidal activity against M. abscessus in surrogate caseum. See Table 16. [ka]

[0179] [Table 16]

[0180] Biological Procedures Substitute matrices were generated from cultured THP-1 cells (ATCC TIB-202) as previously described (doi:10.1128 / mbio.00598-23). ​​Exponential-phase cultures (OD ) of M. abscessus grown in Middlebrook 7H9 broth (Sigma-Aldrich) were used. 600 0.6-0.9) was centrifuged and the OD 600 Bacterial suspensions (approximately 10) were resuspended in water to pH 7, 0.7, and 0.07. 9 , 10 8 , 10 7 The 100 ml of 100% PBS containing 100% PBS (three different dilutions resulting in starting CFU / mL) was added to casein surrogate in a 2:1 ratio (volume / mass), briefly homogenized with 1.4 mm zirconia beads, divided equally into nine 1.5 mL microcentrifuge tubes, and incubated as static cultures at 37°C. At the designated time points, tubes were removed and used for CFU enumeration by plating on Middlebrook 7H11 agar (Sigma-Aldrich). A separate tube was used for each time point. Drug kill curves were determined by 100 ml of PBS containing 100% PBS. 8 Cultures with a starting CFU / mL of 0.01 were used. The experiment was repeated three times independently with similar results.

[0181] On day 5, after the cultures had entered stationary phase, 50 μL of the mixture (cultured with casein surrogate) was exposed to drugs ranging from 0.125 to 512 μM (128 μM for clofazimine and rifabutin analogs 5a, 5m, and 5n) for 5 days (or 10 days for bedaquiline). (Amikacin, clarithromycin, clofazimine, imipenem, rifabutin, and tigecycline were purchased from Sigma-Aldrich, moxifloxacin and linezolid from Sequoia Research Products, and cefoxitin and bedaquiline from MedChemExpress. Rifabutin analogs were synthesized as described (doi:10.1002 / anie.202211498).) for 5 days (or 10 days for bedaquiline). CFU were then counted, and the casein surrogate MBC90 (cMBC90) was calculated. The addition of 2% vehicle DMSO did not affect the viable cell count. The experiment was repeated independently twice with similar results. A representative example is shown. The cMBC90 values ​​shown in Table 16 are the drug concentrations that reduced CFU by 90% compared to the CFU of the drug-free control at day 10. Because the cultures were in stationary phase on day 5 when drug treatment was initiated, the CFU of the drug-free cultures on day 10 was similar to the CFU of the drug-free cultures on day 5.

[0182] Example 6 The broad-spectrum antimycobacterial profiling of compound 5m is shown in Table 17. MIC data was determined using the method disclosed in Example 1. The data suggested that the rifamycin analog had an extended antimycobacterial spectrum. [ka] [Table 17]

[0183] Example 7 From the analog collection, the important pharmacokinetic parameter, plasma unbound fraction (f u) was found to correlate with molecular lipophilicity as characterized by clogP. See Figure 32.

[0184] Additional pharmacokinetic data for some selected rifabutin analogs are shown in Table 18. Pharmacokinetic optimization has also been performed. See Tables 19 and 20. Optimized parameters are shown in italics. [Table 18]

[0185] [ka] [Table 19]

[0186] [Table 20]

[0187] Example 8 The in vivo efficacy of compounds UMN22 and UMN34 was evaluated in an acute Mab mouse infection model. See Table 21. Both resulted in an approximately 2 log reduction in lung CFU burden compared to the RFB and untreated (UNRX) groups. 10 See Figure 33. [Table 21]

[0188] Example 9 Rifamycins induce human CYPs, accelerating the metabolism of co-administered drugs. CYP induction can reduce the effectiveness of other drugs and is particularly problematic for patients receiving polypharmacy. Newer rifabutin analogs can reduce or completely eliminate CYP induction. See Table 22. [Table 22-1] [Table 22-2]

[0189] Determination of induction factor The threshold cycle (CT) of each isoform cDNA was measured by real-time qRT-PCR. The mRNA levels of each isoform were assessed by relative quantification using a reference gene (GAPDH) as a normalization method. Normalized fold induction was calculated using 0.1% DMSO as a reference (vehicle control). -ΔΔCT The induction potential of each test compound was calculated by the α method. The induction potential of each test compound was analyzed by comparing its fold induction to a predetermined cutoff value for each hepatocyte lot. The hepatocyte lots used in this study were validated using 10 known inducers and 5 known non-inducers for each isoenzyme. The cutoff value was determined based on the validation study for each hepatocyte lot. In accordance with the FDA industry guideline "In Vitro Drug Interaction Studies - Cytochrome P450 Enzyme- and Transporter-Mediated Drug Interactions" (January 2020), if the fold induction of a test compound at any test concentration is greater than the cutoff value for that CYP isoform in at least one donor within the test concentration range, the compound is considered to be an in vitro inducer of that CYP isoform.

[0190] References JPEG2025536245000152.jpg95170 JPEG2025536245000153.jpg80170 JPEG2025536245000154.jpg95170 JPEG2025536245000155.jpg71170 JPEG2025536245000156.jpg16170

Claims

1. Compounds of Formula I: 【Chemistry 1】 or a pharmaceutically acceptable salt thereof, During the ceremony, R 1 is an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl, an optionally substituted cycloalkyl, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted heterocycloalkyl, —NR 4 R 5 , and -NHSO 2 R 6 is selected from the group consisting of R 4 and R 5 are independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl; or R 4 and R 5 together with the nitrogen atom to which they are attached form N, O, and S(O) 0~2 forming an optionally substituted 4- to 8-membered heterocycloalkyl containing one or more heteroatoms selected from the group consisting of: R 6 is selected from the group consisting of optionally substituted alkyl, optionally substituted aryl, and optionally substituted heteroaryl; 【Chemistry 2】 is represented by formula (a), formula (b), or formula (c), 【Transformation 3】 and During the ceremony, N1 and N2 are independently integers from 1 to 3; Y 1 is N, O, or CR i and R 2 is selected from hydrogen, optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, and -(CH 2 ) n R 3 is selected from the group consisting of R i is hydrogen, halo, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, or R i and R 2 together with the carbon atom to which they are attached form an optionally substituted 4- to 8-membered heterocycloalkyl; n is an integer from 1 to 6, R 3 is -OR 7 , -NR 8 R 9 , -C(O)OR 10 , -S(O) 2 OR 11 and heteroaryl; R 7 , R 8 , R 9 , R 10 , and R 11 is independently selected from the group consisting of hydrogen, alkyl, and haloalkyl; R 12 is heteroaryl, N3 is an integer from 1 to 2, R 1 is not methyl, The compound, or a pharmaceutically acceptable salt thereof.

2. R 1 teeth, (i) Aryl optionally substituted with one or more alkyls, alkoxy optionally substituted with halo or hydroxyl, hydroxyl, hydroxyalkyl, halo, haloalkyl, haloalkoxy, cyano, aryl, —C(O)OZ 1 , -C(O)NZ 2 Z 3 , -S(O)Z 4 , -S(O) 2 Z 5 , -S(O) 2 New Zealand 6 Z 7 or an amide, wherein Z 1 , Z 2 , Z 3 , Z 4 , Z 5 , Z 6 , and Z 7 are independently hydrogen, haloalkyl, alkyl, or —C(O)-alkyl; (ii) heteroaryl optionally substituted with one or more hydroxyl, halo, optionally substituted amino, amido, haloalkyl, or carboxylic acid ester; (iii)-NR 4 R 5 and R 4 is hydrogen, and R 5 teeth, (a) alkyl optionally substituted with one or more alkynyl or heteroaryl, wherein the heteroaryl is: one or more aryls optionally substituted with one or more aryls, where the substituted aryls are halo-substituted aryls; or one or more alkyl groups optionally substituted with one or more aryl groups or carboxylic acid ester groups; alkyl, optionally substituted with (b) one or more of alkylaryl, alkoxy, amino, aryl, —C(O)OZ 8 or heterocycloalkyl optionally substituted with one or more haloalkyl or alkyl optionally substituted with hydroxyl, (c) one or more aryl, alkoxy, alkyl, halo, —C(O)OZ 8 , -C(O)NZ 9 Z 10 , -S(O) 2 Z 11 , or -S(O) 2 New Zealand 12 Z 13 aryl optionally substituted with (d) one or more of alkyl, halo, haloalkyl, amino, hydroxyl, —OC(O)Z 8 or aryl optionally substituted with aryl, heteroaryl optionally substituted with During the ceremony, Z 8 , Z 9 , Z 10 , Z 11 , Z 12 , and Z 13 are independently hydrogen, alkyl, or —C(O)-alkyl; —NR 4 R 5 Or (iv) -NR 4 R 5 and R 4 and R 5 together with the nitrogen atom to which they are attached form N, O, and S(O) 0~2 and forming a 4-8 membered heterocycloalkyl containing one or more heteroatoms selected from the group consisting of: 4 R 5 or (v) -NHSO 2 R 6 and R 6 is heteroaryl or aryl optionally substituted with one or more halo, alkoxy, or haloalkyl, —NHSO 2 R 6 Or (vi) alkyl, (vii) alkynyl, or (viii) is cycloalkyl; The compound of claim 1.

3. R 1 The compound of claim 1 , wherein is optionally substituted aryl or optionally substituted heteroaryl.

4. Formula I(a): 【Chemistry 4】 and In the formula, R 1 is alkyl, —NR 4 R 5 , -NHSO 2 R 6 , cycloalkyl, heteroaryl optionally substituted with one or more hydroxyl, halo, optionally substituted amino, amido, haloalkyl, or carboxylic acid ester, and alkoxy optionally substituted with one or more alkyl, halo, or hydroxyl, hydroxyl, hydroxyalkyl, halo, haloalkyl, haloalkoxy, cyano, aryl, —C(O)OZ 1 , -C(O)NZ 2 Z 3 , S(O)Z 4 , S(O) 2 Z 5 , -S(O) 2 New Zealand 6 Z 7 or aryl optionally substituted with an amide; During the ceremony, Z 1 , Z 2 , Z 3 , Z 4 , Z 5 , Z 6 , and Z 7 are independently hydrogen, alkyl, or —C(O)-alkyl; The compound of claim 1.

5. R 1 teeth, 【Chemistry 5-1】 【Chemistry 5-2】 【Chemistry 5-3】 5. The compound of claim 4, wherein:

6. The compound is 【Chemistry 6-1】 【Chemistry 6-2】 【Transformation 6-3】 5. The compound of claim 4, wherein:

7. R 1 is -NR 4 R 5 and In the formula, R 4 and R 5 is hydrogen, alkyl, heteroaryl optionally substituted with aryl, heterocycloalkyl which is one or more alkylaryl, alkoxy, amino, aryl, -C(O)OZ 8 or heterocycloalkyl optionally substituted with one or more haloalkyl or alkyl optionally substituted with hydroxyl, one or more aryl, alkoxy, alkyl, —C(O)OZ 8 , -C(O)NZ 9 Z 10 , -S(O) 2 Z 11 , or -S(O) 2 New Zealand 12 Z 13 and one or more of aryl, halo, haloalkyl, —OC(O)Z 8 , amino, or heteroaryl optionally substituted with hydroxyl; R 4 and R 5 together with the nitrogen atom to which they are attached form N, O, and S(O) 0~2 forming a 4-8 membered heterocycloalkyl containing one or more heteroatoms selected from the group consisting of: During the ceremony, Z 8 , Z 9 , Z 10 , Z 11 , Z 12 , and Z 13 are independently hydrogen, alkyl, or —C(O)-alkyl; The compound of claim 4.

8. R 1 teeth, 【Chemistry 7-1】 【Chemistry 7-2】 【Transformation 7-3】 8. The compound of claim 7, wherein:

9. The compound is 【Chemistry 8-1】 【Chemistry 8-2】 【Chemistry 8-3】 【Chemistry 8-4】 5. The compound of claim 4, wherein:

10. R 1 is NHS(O) 2 R 6 and R 6 5. The compound of claim 4, wherein is heteroaryl or aryl optionally substituted with one or more halo, alkoxy, or haloalkyl.

11. The compound is 【Chemistry 9】 11. The compound of claim 10, wherein:

12. Formula (b) is 【Chemistry 10】 2. The compound of claim 1, wherein:

13. Formula (c) is 【Chemistry 11】 2. The compound of claim 1, wherein:

14. R 1 is an optionally substituted aryl or an optionally substituted heteroaryl, 【Chemistry 12】 is formula (a), N1 and N2 are 2, Y 1 is N, R 2 is selected from the group consisting of hydrogen and alkyl optionally substituted with one or more hydroxyl, amino, carboxyl, or heteroaryl; The compound of claim 1.

15. R 2 teeth, 【Chemistry 13】 15. The compound of claim 14 selected from the group consisting of:

16. R 1 is an optionally substituted aryl or an optionally substituted heteroaryl, N1 and N2 are independently 1 or 3; N3 is 1, Y 1 is N, R 2 is alkyl, The compound of claim 1. 【Request Item 17】 【Chemistry 14】 teeth, 【Chemistry 15】 17. The compound of claim 16, selected from the group consisting of:

18. R 1 teeth, 【Chemistry 16】 is selected from the group consisting of 【Chemistry 17】 teeth, [Chemistry 18] 2. The compound of claim 1 selected from the group consisting of:

19. The compound is 【Chemistry 19】 2. The compound of claim 1, wherein:

20. A compound according to any one of claims 1 to 19 which is antibacterial.

21. 21. The compound according to any one of claims 1 to 20, which has a lower inductive effect on cytochrome P450 enzymes than rifampicin, rifabutin, or rifapentine.

22. 22. The compound of claim 21, wherein the cytochrome P450 enzyme is cytochrome P450 3A4.

23. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of claims 1 to 22, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient, or diluent.

24. A method for treating or preventing an infection caused by a mycobacterium in a subject in need thereof, comprising administering to the subject a compound of claim 1 or a pharmaceutically acceptable salt thereof.

25. 25. The method of claim 24, wherein the mycobacterium is selected from the group consisting of Mycobacterium abscessus, Mycobacterium simiae, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium avium complex, Mycobacterium avium subsp. hominisuis, Mycobacterium intracellulare, Mycobacterium chimera, Mycobacterium kansasii, Mycobacterium turgai, Mycobacterium xenopi, and combinations thereof.

26. 25. The method of claim 24, wherein the mycobacterium is Mycobacterium abscessus.

27. 27. The method of claim 26, wherein the Mycobacterium abscessus is selected from the group consisting of Mycobacterium abscessus subsp. abscessus, Mycobacterium abscessus subsp. bollettii, Mycobacterium abscessus subsp. massiliense, and combinations thereof.

28. The method of any one of claims 24 to 27, wherein the subject has caseous necrosis.

29. 28. The method of any one of claims 24 to 27, wherein the mycobacterium is present within the caseum of the subject.

30. 10. A method for treating or preventing an infection caused by a bacterium in a subject in need thereof, comprising administering to the subject a compound of claim 1 or a pharmaceutically acceptable salt thereof.

31. 31. The method of claim 30, wherein the bacteria is resistant to rifampicin, rifabutin, or rifapentine.

32. 31. The method of claim 30, wherein the bacterium expresses an enzyme that catalyzes the ADP-ribosylation of rifampicin, rifabutin, or rifapentine.

33. 33. The method of claim 32, wherein the enzyme that catalyzes the ADP-ribosylation of rifampicin, rifabutin, or rifapentine is rifamycin ADP-ribosyltransferase.

34. 34. The method of any one of claims 30 to 33, wherein the bacterium is selected from the group consisting of Mycobacterium abscessus, Mycobacterium simiae, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium avium complex, Mycobacterium avium subsp. hominisuis, Mycobacterium intracellulare, Mycobacterium chimera, Mycobacterium kansasii, Mycobacterium turgai, Mycobacterium xenopi, and combinations thereof.

35. 35. The method of claim 34, wherein the mycobacterium is Mycobacterium abscessus.

36. 36. The method of claim 35, wherein the Mycobacterium abscessus is selected from the group consisting of Mycobacterium abscessus subsp. abscessus, Mycobacterium abscessus subsp. bollettii, Mycobacterium abscessus subsp. massiliense, and combinations thereof.

37. The method of any one of claims 30 to 36, wherein the subject has caseous necrosis.

38. 37. The method of any one of claims 30 to 36, wherein the bacteria is present in the caseum of the subject.

39. 10. A method for treating or preventing an infection in a subject in need of an antibacterial compound, comprising administering to the subject a compound of claim 1 or a pharmaceutically acceptable salt thereof.

40. 40. The method of claim 39, wherein the administered compound has a lower inductive effect on cytochrome P450 enzymes than rifampicin, rifabutin, or rifapentine.

41. 41. The method of claim 40, wherein the cytochrome P450 enzyme is cytochrome P450 3A4.

42. 42. The method of any one of claims 39 to 41, wherein the subject is undergoing treatment with a compound that is metabolized by the cytochrome P450 enzyme.

43. 42. The method of any one of claims 39 to 41, wherein the subject is undergoing treatment with a compound that is sensitive to co-administration of an inducer of the cytochrome P450 enzyme.

44. 42. The method of any one of claims 39 to 41, wherein the subject is infected with a mycobacterium selected from the group consisting of Mycobacterium abscessus, Mycobacterium simiae, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium avium complex, Mycobacterium avium subsp. hominisuis, Mycobacterium intracellulare, Mycobacterium chimera, Mycobacterium kansasii, Mycobacterium turgai, Mycobacterium xenopi, and combinations thereof.

45. 45. The method of claim 44, wherein the mycobacterium is Mycobacterium abscessus.

46. 46. ​​The method of claim 45, wherein the Mycobacterium abscessus is selected from the group consisting of Mycobacterium abscessus subsp. abscessus, Mycobacterium abscessus subsp. bollettii, Mycobacterium abscessus subsp. massiliense, and combinations thereof.

47. The method of any one of claims 39 to 46, wherein the subject has caseous necrosis.

48. 47. The method of any one of claims 39 to 46, wherein the bacteria is present in the caseum of the subject.

49. 49. The method of any one of claims 24 to 48, wherein the compound or a pharmaceutically acceptable salt thereof is administered orally or intravenously.

50. The method of any one of claims 24 to 49, wherein the compound is a compound according to any one of claims 2 to 22.

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