Discovery of a f-ATP synthase inhibitor for the treatment of non-tuberculous mycobacteria diseases

Abstract

The current invention relates to compounds of formula (I): (I) where R1 and R2 are as defined herein, or a pharmaceutically acceptable salt or solvate thereof, where said compounds of formula (I) are 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ) and may be used in the treatment of a nontuberculous mycobacterial (NTM) infection.

Description

[0001] DISCOVERY OF A F-ATP SYNTHASE INHIBITOR FOR THE TREATMENT OF NON- TUBERCULOUS MYCOBACTERIA DISEASES

[0002] Field of Invention

[0003] The current invention relates to one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ) for use in the treatment of nontuberculous mycobacterial (NTM) infection, uses of one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), methods of treating nontuberculous mycobacterial (NTM) infection, compositions comprising the one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), and uses of the compositions.

[0004] Background

[0005] The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0006] Mycobacteria can be separated into tuberculosis (TB) causing mycobacteria {Mycobacterium tuberculosis, Mtb; Mtb complex), leprotic mycobacteria, and non-tuberculous mycobacteria (NTM). NTM most commonly cause pulmonary infections (90%), especially among patients with structural airway disease (e.g., cystic fibrosis and bronchiectasis), but can also cause lymphadenitis, skin and soft tissue infection (SSTI), cardiac infection, bone and joint infections, and disseminated disease {Annals Americ. Thoracic Soc. 2020, 17, 178-185). Cell wall impermeability, along with biofilm formation, contributes to the antimicrobial, high-temperature, and disinfectant resistance of mycobacteria {Annals Americ. Thoracic Soc. 2020, 17, 178-185).

[0007] NTM infection presents a growing global health problem, complicated by ubiquitous exposure to the organisms, incomplete understanding of the immune susceptibility to disease, increasing numbers of immune compromised patients, and costly, multi-drug treatment regimens that often fail to cure. Therefore, there is a pressing need to identify NTM inhibitors and enzyme targets essential for NTM under various metabolic conditions. It is desirable that NTM inhibitors are non-toxic and have attractive combinatory potency.

[0008] Classically, NTM have been divided into rapid (< 7 days for mature colony formation in solid media) and slow (>7 days for colonization) growers. Of the NTM species known to cause human disease, notable rapid growers include Mycobacterium abscessus complex (Mabs, comprising M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, and M. abscessus subsp. massiliense), Mycobacterium fortuitum, and Mycobacterium chelonae. Slow growers known to cause human disease include Mycobacterium avium complex (MAC, comprising M. avium, M. intracellulare, and M. chimaera), Mycobacterium kansasii, and Mycobacterium xenopi (Nature Rev. Microbiology 2020, 18, 392-407). A profile of NTM species in Singapore shows Mab, M. fortuitum and MAC as the most frequently isolated NTMs (BMC Pulmonary Med. 2018, 18, 85).

[0009] NTM Infections are increasing exponentially in their global prevalence, morbidity and mortality BMC Pulmonary Med. 2018, 18, 85). Cell wall impermeability, along with biofilm formation, contributes to resistance against antimicrobials, high-temperatures, and disinfectants. Other proposed reasons for this increase include higher air pollution levels, a population increasing in age and comorbid conditions, use of immunosuppressive therapies, and chronic use of inhaled corticosteroids (Am Rev Respir Dis. 1992, 145, 271 ; Expert Rev Respir Med. 2017, 11, 977). Treating mycobacterial infections is challenging because of the complexity of the disease and therapeutics used. Furthermore, the genotype of the various subspecies plays a key role in influencing treatment outcomes (BMC Genomics 2016, 17, 553). Even more, NTM strains can exhibit either a smooth (S) or rough (R) morphotype as a consequence of the presence or absence, respectively, of bacterial surface glycopeptidolipids (Nat. Rev. Microbiol. 2020, 18, 392-407; Front Microbiol. 2018, 9, 1 145) and they show difference in virulence. These morphological distinctions are associated with important physiological differences. In addition, NTM harbor intrinsic resistance mechanisms and also quickly acquire resistance and display drug tolerance, which renders most antimicrobial classes ineffective. Cure rates are globally poor, comparable to or worse than those of multidrug-resistant TB (Drug Disc. Today 2018, 23, 1502). Against M. abscessus lung disease, there is no reliable cure. Bactericidal drug regimens containing new drug candidates are sorely needed, and the current NTM drug development pipeline is very thin.

[0010] In the past decade, the oxidative phosphorylation pathway with its final product ATP, generated by the essential F1F0-ATP synthase, has come into the focus as an attractive and rich drug target. Most anti-NTM drugs target enzymes for biomass generation. Importantly, most NTMs are able to enter a metabolically quiescent state (persistence) for instance under low oxygen tension (hypoxia), where the pathogens phenotypically become more tolerant to drug challenge with existing drugs (Drug Disc. Today 2018, 23, 1502). The mycobacterial enzyme is composed of the catalytic a3:p3-headpiece, in which ATP synthesis or ATP hydrolysis occurs, which together with the central stalk subunits y and E form the Frdomain also called Fi-ATPase (FIG. 1 , Prog. Biophys. Mol. Biol. 2019, 152, 56). The membrane embedded Fo-domain is composed of subunit a and nine c subunits, which are organized in a ring (cg-ring). Subunits a and c form a half channel to translocate protons from the inner membrane to the plasma side by rotation of the cS-ring (Nature, 2020, 589, 143). In case of ATP synthesis, such rotation drives the rotational movement of the central y-£ stalk in clockwise direction, which is coupled to the three catalytic centers located at the three ap- interfaces. In contrast to most bacterial and mitochondrial F1F0 ATP synthase, the mycobacterial enzyme shows very low, latent ATP hydrolysis, and therefore does not promote ATP-driven proton-translocation (FEMS Microbiol Lett, 2010, 313, 68; FEBS J, 2016, 283, 1947). This prevents wastage of ATP and a strict regulation of the membrane potential, an important regulation under hypoxia condition (Proc Natl Acad Sci USA, 2008, 105, 1 1945). Evolutionary modifications of structural, mechanistic, and regulatory elements within the Fi Fo- ATP synthase equipped the mycobacterial pathogens with tools to prevent wastage of ATP, maintain their membrane-potential and to regulate ATP formation (Prog. Biophys. Mol. Biol. 2019, 152, 56).

[0011] Being obligate aerobes, NTMs need to regulate the essential metabolic processes of oxidation and recycling of the electron carriers (NADH2 and FADH2) derived from central carbon metabolism under such hypoxia conditions. This include the generation of a proton motive force (pmf) and the formation of sufficient amounts of ATP, which is driven by the oxidative phosphorylation (OXPHOS) pathway, including the electron transport chain (ETC) and the F1F0 ATP synthase converting the harvested energy of the pmf into the synthesis of ADP and inorganic phosphate to ATP (Proc. Natl. Acad. Sci. USA 2008, 105, 1 1945). Since the ATP pool is reduced in such persister cells, they become exquisitely sensitive to any further ATP depletion, and susceptible to drugs targeting the maintenance of an optimal ATP:ADP ratio. The latter is not only essential to provide the energy of life but also to regulate the key enzymes of catalytic processes (glycolysis, Krebs cycle) and anabolic processes (gluconeogenesis). This observation suggests that inhibitors targeting the essential NTM F1F0-ATP synthase (a3:p3:Y:£:b-6:b’:a:c9; FIG. 2A) could overcome the problem of NTM drug tolerance and shortening the duration of therapy for drug-resistant NTM-diseases.

[0012] Despite being obligate aerobes, mycobacteria have the ability to survive under low oxygen tension (hypoxia), by exiting the cell cycle and entering a dormant state. Hypoxic nonreplicating mycobacteria display a reduced pool of ATP, making them exquisitely sensitive to any further ATP depletion, and susceptible to drugs targeting the maintenance of ATP homeostasis (Proc Natl Acad Sci USA, 2008, 105, 1 1945). This observation suggests that drugs that inhibit oxidative phosphorylation could contribute to shortening the duration of therapy for drug-resistant tuberculosis (TB) or NTM-diseases. This working model is supported by the clinical development and approval of the anti-tuberculosis (TB) drug Sirturo® (bedaquiline, BDQ), consisting of a quinoline-, a phenyl-, a naphthyl ring and a dimethyl amino (DMA) tether, and BDQ-derivatives such as TBAJ-876 and TBAJ-5307, in which the phenyl- and naphtyl ring of the parental molecule have been altered (FIG. 3, Bioorg Med Chem, 2019, 27, 1292; Bioorg Med Chem, 2018, 26, 1797), which targeted the M. abscesses F-ATP synthase (Antimicrob. Agents Chemother. 2017, 61, e01225-17) and entered early clinical development.

[0013] While BDQ is used as salvage therapy against M. abscesses lung diseases (Chest 2015, 148, 499), it has been overshadowed by its pharmacokinetic properties and tolerability profiles as well as its inhibition of the mitochondrial F-ATP synthase in human HEK293S cell mitoplasts (Comm. Biol. 2020, 3, 452). Recently, BDQ’s 3,5-dialkoxypyridine (DARQ) analogue TBAJ- 876 with improved pharmacokinetic and tolerability profiles, has shown to be active in vitro and in vivo against the M. abscesses complex (Antimicrob. Agents Chemother. 2020, 64, e02404). However, in contrast to M. tebercelosis, both inhibitors are not bactericidal and have 100-times lower growth-inhibitory potency in Mab, proposed to be correlated in part with cross resistance in Mab efflux pumps (Antimicrob. Agents Chemother. 2017, 61, e01225-17). In addition, the cryo-electron microscopy structure of M. smegmatis F1F0-ATP synthase, a surrogate of the M. tebercelosis enzyme, showed BDQ-binding to three sites of the F0-domain, namely the leading- and lagging sites and the c-ring, which binds five BDQ-molecules (FIG. 2, Natere 2021 , 589, 143). The leading site involves a c subunit that has interacted with subunit a and picked up a proton from the periplasm, while the lagging site involves a c subunit poised to interact with subunit a to deposit a proton into the cytoplasm (Natere 2021 , 589, 143). The subunit a amino acid sequences of the three Mab subspecies differ from that of M. smegmatis, including the residues at the leading- and lagging sites (FIG. 4).

[0014] Furthermore, the BDQ analogs TBAJ-5307, TBAJ-5316, and TBAJ-5366 with improved potency and physio-chemical properties have been described (Bioorg. Med. Chem. 2019, 27, 1292). Racemates of (±)-5307, (±)-5316, and (±)-5366 (CCDC deposition numbers: 1967685, 1967683, 1967684, respectively; FIG. 5) exhibited improved growth- and ATP synthesis inhibition against M. smegmatis, when compared to BDQ (Angew. Chem. Intern. Edi. 2020, 59, 13295). However, a significant reduction in M. smegmatis growth was not observed with either (±)-5316 or (±)-5366 (Angew. Chem. Intern. Edi. 2020, 59, 13295). There is a need for treatments for NTM infections.

[0015] Summary of Invention

[0016] Disclosed herein are compounds for treating nontuberculous mycobacterial (NTM) infection. Disclosed are 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or pharmaceutically acceptable salts or solvates thereof.

[0017] In this invention, we demonstrate anii-Mab activity of the racemates of (±)-5307, (±)-5316, and (±)-5366 to be also active against NTMs, with (±)-5307 being the most potent one. It is surprisingly found that enantiomer TBAJ-5307 displays high nanomolar efficacy against representatives of the fast- and slow NTM growers, making it an attractive broad spectrum anti-NTM inhibitor. TBAJ-5307 displayed nanomolar in vitro activity against the NTM pathogens M. abscessus complex, the M. avium complex and M. fortuitum representing the most relevant NTM pathogens in Singapore (BMC Pulmonary Med. 2018, 18, 85), as well as M. peregrinum, M. chelonae, M. kansasii and against a clinical isolate. Its potency in cell growth and ATP synthesis inhibition is significantly higher in comparison to BDQ and TBAJ- 876. The mechanism of action of the disclosed inhibitors is addressed by molecular dynamics simulations and binding free energy calculations. Combining (±)-5307 with the anti-TB compound, GaMF1 , revealed a significant reduction in M. smegmatis growth, which was not observed with either (±)-5316 or (±)-5366 (Angew. Chem. Intern. Edi. 2020, 59, 13295). The compound is also potent in vivo as demonstrated by a M. abscessus-infection zebrafish model. Drug-drug potency interaction studies with the commonly used anti-NTM antibiotics showed an additive / partial synergistic effect, suggesting that TBAJ-5307 could be co-administered with currently used drugs

[0018] TBAJ-5307 is efficacious ex vivo and in vivo without altering biofilm formation or being toxic, and potentiates the activity of the clinical Mab antibiotics clofazimine (CFZ), amikacin, rifabutin and the oral pair tebipenem (TBP) and avibactam (AVI). These features make TBAJ-5307 an ideal candidate to tackle the issue of NTM drug tolerance, high nanomolar anti-NTM potency, toxicity, pronounced combinatory efficacy with anti- / Wab antibiotics to overcome intrinsic and acquired resistance as well as to design an oral cocktail in future, which reduces the expense and risks of intravenous therapies of today’s anti-NTM drugs.

[0019] Aspects and embodiments of the current invention will now be described by reference to the following numbered clauses. 1. One or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, for use in the treatment of nontuberculous mycobacterial (NTM) infection, wherein each of the one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, are a compound according to formula (I): wherein:

[0020] X represents CH or N;

[0021] Y represents C-OR3 or N;

[0022] R1 represents H or OR4;

[0023] R2 represents Ci to Ce alkyl;

[0024] R3, when present, represents Ci to Ce alkyl, or R2and R3, together form a Ci to C3 alkylene moiety, which together with the atoms they are attached to provides a 5- to 7-membered ring system;

[0025] R4, when present, represents C2to C4 alkyl; or a salt or solvate thereof, provided that at least one of X and Y are N.

[0026] 2. Use of one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, in the manufacture of a medicament for the treatment of nontuberculous mycobacterial (NTM) infection, wherein each of the one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, are a compound according to formula (I): wherein:

[0027] X represents CH or N;

[0028] Y represents C-OR3 or N;

[0029] R1 represents H or OR4;

[0030] R2 represents Ci to Cs alkyl;

[0031] R3, when present, represents Ci to Ce alkyl, or R2and R3, together form a Ci to C3alkylene moiety, which together with the atoms they are attached to provides a 5- to 7-membered ring system;

[0032] R4, when present, represents C2to C4 alkyl; or a salt or solvate thereof, provided that at least one of X and Y are N.

[0033] 3. A method of treating nontuberculous mycobacterial (NTM) infection comprising the step of administering to a subject in need thereof one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, wherein each of the one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, are a compound according to formula (I): wherein:

[0034] X represents CH or N;

[0035] Y represents C-OR3 or N; R1 represents H or OR4;

[0036] R2represents Ci to C6alkyl;

[0037] R3, when present, represents Ci to Ce alkyl, or R2and R3, together form a Ci to C3 alkylene moiety, which together with the atoms they are attached to provides a 5- to 7-membered ring system; R4, when present, represents C2to C4 alkyl; or a salt or solvate thereof, provided that at least one of X and Y are N.

[0038] 4. A composition comprising one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, for use in the treatment of nontuberculous mycobacterial (NTM) infection, wherein each of the one or more

[0039] 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, are a compound according to formula (I):

[0040] wherein:

[0041] X represents CH or N;

[0042] Y represents C-OR3 or N; R1 represents H or OR4;

[0043] R2represents Ci to C6alkyl;

[0044] R3, when present, represents Ci to Ce alkyl, or R2and R3, together form a Ci to C3 alkylene moiety, which together with the atoms they are attached to provides a 5- to 7-membered ring system; R4, when present, represents C2to C4 alkyl; or a salt or solvate thereof, provided that at least one of X and Y are N.

[0045] 5. Use of a composition comprising one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, in the manufacture of a medicament for the treatment of nontuberculous mycobacterial (NTM) infection, wherein each of the one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, are a compound according to formula (I):

[0046] wherein:

[0047] X represents CH or N;

[0048] Y represents C-OR3 or N; R1 represents H or OR4;

[0049] R2represents Ci to C6alkyl;

[0050] R3, when present, represents Ci to Ce alkyl, or R2and R3, together form a Ci to C3 alkylene moiety, which together with the atoms they are attached to provides a 5- to 7-membered ring system; R4, when present, represents C2to C4 alkyl; or a salt or solvate thereof, provided that at least one of X and Y are N.

[0051] 6. A method of treating nontuberculous mycobacterial (NTM) infection comprising the step of administering to a subject in need thereof a composition comprising one or more 3,5- dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, wherein each of the one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, are a compound according to formula (I):

[0052] wherein:

[0053] X represents CH or N;

[0054] Y represents C-OR3 or N;

[0055] R1 represents H or OR4;

[0056] R2represents Ci to C6alkyl;

[0057] R3, when present, represents Ci to Ce alkyl, or R2and R3, together form a Ci to C3 alkylene moiety, which together with the atoms they are attached to provides a 5- to 7-membered ring system;

[0058] R4, when present, represents C2to C4 alkyl; or a salt or solvate thereof, provided that at least one of X and Y are N.

[0059] 7. The composition according to Clause 4, the use according to Clause 5 or the method according to Clause 6, further comprising one or more pharmaceutically acceptable excipients.

[0060] 8. The one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof according to Clause 1 , the use according to Clause 2, the method according to Clause 3, the composition according to Clause 4 or Clause 7, the use according to Clause 5 or Clause 7, or the method according to Clause 6 or Clause 7 wherein the DARQ analogue is one where one or more of the following apply:

[0061] (I) R4, when present, represents C2to C3 alkyl;

[0062] (ii) R2represents Ci to C3alkyl; and

[0063] (iii) when Y represents COR3, then R3represents Ci to C3 alkyl, or R2and R3 together represent -CH2- or -(CH2)2- 9. The one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, the use, the method, the composition, the use, or the method according to Clause 8 wherein the DARQ analogue is one where one or more of the following apply: (ia) Ri represents H or O'Pr;

[0064] (iia) R2represents CH3; and

[0065] (iiia) when Y represents COR3, then R3represents CH3, or R2and R3together represent -(CH2)2-

[0066] 10. The one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof according to any one of Clause 1 and Clauses 8 and 9, the use according to any one of Clause 2 and Clauses 8 and 9, the method according to any one of Clause 3 and Clauses 8 and 9, the composition according to any one of Clause 4 or Clauses 7 to 9, the use according to any one of Clause 5 or Clauses 7 to 9, or the method according to any one of Clause 6 or Clauses 7 to 9 wherein the DARQ analogue is selected from the group consisting of:

[0067] (±)-TBAJ-5307

[0068]

[0069] 11 . The one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof according to any one of Clause 1 and Clauses 8 and 9, the use according to any one of Clause 2 and Clauses 8 and 9, the method according to any one of Clause 3 and Clauses 8 and 9, the composition according to any one of Clause 4 or Clauses 7 to 9, the use according to any one of Clause 5 or Clauses 7 to 9, or the method according to any one of Clause 6 or Clauses 7 to 9 wherein the DARQ analogue is selected from the group consisting of:

[0070] (±)-TBAJ-5316 ; and

[0071] where the compounds are provided in an enantiomerically enriched form based on the enantiomer shown.

[0072] 12. The one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof according to any one of Clause 1 and Clauses 8 and 9, the use according to any one of Clause 2 and Clauses 8 and 9, the method according to any one of Clause 3 and Clauses 8 and 9, the composition according to any one of Clause 4 or Clauses 7 to 9, the use according to any one of Clause 5 or Clauses 7 to 9, or the method according to any one of Clause 6 or Clauses 7 to 9 wherein the DARQ analogue is: or a pharmaceutically acceptable salt or solvate thereof which is provided in an enantiomerically enriched form based on the enantiomer shown.

[0073] 13. A composition comprising one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, for use in the treatment of nontuberculous mycobacterial (NTM) infection, wherein each of the one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, are a compound according to formula (I): wherein:

[0074] X represents CH or N;

[0075] Y represents C-OR3 or N;

[0076] R1 represents H or OR4;

[0077] R2 represents Ci to Ce alkyl;

[0078] R3, when present, represents Ci to Ce alkyl, or R2and R3, together form a Ci to C3 alkylene moiety, which together with the atoms they are attached to provides a 5- to 7-membered ring system;

[0079] R4, when present, represents C2to C4 alkyl; or a salt or solvate thereof, provided that at least one of X and Y are N, and a second antibiotic, optionally wherein the composition further comprises one or more pharmaceutically acceptable excipients.

[0080] 14. A composition according to Clause 13 for use in the treatment of nontuberculous mycobacterial (NTM) infection. 15. Use of a composition according to Clause 13 in the manufacture of a medicament to treat a nontuberculous mycobacterial (NTM) infection.

[0081] 16. A method of treating nontuberculous mycobacterial (NTM) infection comprising the step of administering to a subject in need thereof a composition according to Clause 13.

[0082] 17. The composition according to Clause 13 or Clause 14, the use according to Clause 15, or the method according to Clause 16, wherein the second antibiotic is selected from the group consisting of Clofazimine, Rifabutin and Amikacin, or a pharmaceutically acceptable salt or solvate thereof.

[0083] 18. A composition comprising one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, for use in the treatment of nontuberculous mycobacterial (NTM) infection, wherein each of the one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, are a compound according to formula (I): wherein:

[0084] X represents CH or N;

[0085] Y represents C-OR3 or N;

[0086] R1 represents H or OR4;

[0087] R2 represents Ci to Ce alkyl;

[0088] R3, when present, represents Ci to Ce alkyl, or R2and R3, together form a Ci to C3alkylene moiety, which together with the atoms they are attached to provides a 5- to 7-membered ring system;

[0089] R4, when present, represents C2to C4alkyl; or a salt or solvate thereof, provided that at least one of X and Y are N. for use in the treatment of nontuberculous mycobacterial (NTM) infection, wherein the composition is to be administered sequentially, simultaneously or concomitantly with a second antibiotic.

[0090] 19. Use of a composition according to Clause 18 in the manufacture of a medicament to treat a nontuberculous mycobacterial (NTM) infection wherein the composition is to be administered sequentially, simultaneously or concomitantly with a second antibiotic.

[0091] 20. A method of treating nontuberculous mycobacterial (NTM) infection comprising the step of administering to a subject in need thereof a composition according to Clause 18 in combination with a second antibiotic, wherein the composition is administered sequentially, simultaneously or concomitantly with the second antibiotic.

[0092] 21. The composition according to Clause 18, the use according to Clause 19, or the method according to Clause 20, wherein the second antibiotic is selected from the group consisting of Clofazimine, Rifabutin and Amikacin, or a pharmaceutically acceptable salt or solvate thereof.

[0093] 22. The composition according to Clause 13 or Clause 17, the composition for use according to any one of Clauses 14, 17, 18 or 21 , the use according to any one of Clauses 15, 17, 19 or 21 , or the method according to any one of Clauses 16, 17, 20 or 21 , wherein the DARQ analogue is one where one or more of the following apply:

[0094] (i) R4, when present, represents C2to C3alkyl;

[0095] (ii) R2represents Ci to C3alkyl; and

[0096] (iii) when Y represents COR3, then R3represents Ci to C3alkyl, or R2and R3together represent -CH2- or -(CH2)2-

[0097] 23. The composition, the composition for use, the use, the method, according to Clause 22 wherein the DARQ analogue is one where one or more of the following apply:

[0098] (ia) Ri represents H or O'Pr;

[0099] (iia) R2represents CH3; and

[0100] (iiia) when Y represents COR3, then R3represents CH3, or R2and R3together represent -(CH2)2- 24. The composition according to Clause 13 or Clause 17, the composition for use according to any one of Clauses 14, 17, 18 or 21 , the use according to any one of Clauses 15, 17, 19 or 21 , or the method according to any one of Clauses 16, 17, 20 or 21 wherein the DARQ analogue is selected from the group consisting of:

[0101] (±)-TBAJ-5316 ; and

[0102]

[0103] 25. The composition according to Clause 13 or Clause 17, the composition for use according to any one of Clauses 14, 17, 18 or 21 , the use according to any one of Clauses 15, 17, 19 or 21 , or the method according to any one of Clauses 16, 17, 20 or 21wherein the

[0104] DARQ analogue is selected from the group consisting of:

[0105] where the compounds are provided in an enantiomerically enriched form based on the enantiomer shown.

[0106] 26. The composition according to Clause 13 or Clause 17, the composition for use according to any one of Clauses 14, 17, 18 or 21 , the use according to any one of Clauses 15,

[0107] 17, 19 or 21 , or the method according to any one of Clauses 16, 17, 20 or 21wherein the DARQ analogue is: or a pharmaceutically acceptable salt or solvate thereof which is provided in an enantiomerically enriched form based on the enantiomer shown.

[0108] 27. The composition according to Clause 4, the use according to Clause 5 or the method according to Clause 6, further comprising one or more pharmaceutically acceptable excipients.

[0109] 28. The one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof according to any one of Clauses 1 and 8 to 12, the use according to any one of Clauses 2, 5, 7, 8 to 12 15, 17, 19 and 21 -23, the method according to any one of Clauses 3, 6 to 12, 16, 17, 20 and 21 -23, the composition according to any one of Clauses 4, 7 to 12, 13 and 17, or the composition for use according to any one of Clauses 5, 7, 14, 17, 19 or 21 -23 wherein the NTM infection is caused by one or more of the group consisting of M. chelonae, M. peregrinum, M. kansasii, and more particularly, M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, M. abscessus subsp. massiliense, M. fortuitum, M. avium and M. intracellulare.

[0110] Brief Description of the Drawings

[0111] A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized. Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures. FIG. 1 depicts the structural model of the mycobacterial F1F0-ATP synthase.

[0112] FIG. 2 depicts the M. smegmatis F-ATP synthase cryo-EM structure with bound BDQ (PDB ID: 7JGA). The hexamer forming a and p subunits are shown in green and orange color, respectively. The y-, £-, b-, 6-, b’-, a- subunits and c-ring are shown in surface representation. The seven BDQ molecules that bind in c-ring are also shown in surface representation. BDQ binds five c-only sites, a leading site and a lagging site. For clarity, the c-ring along with subunit a are shown in cartoon representation in two views. In the zoomed in view the residues, which are interacting with BDQ in the leading site and lagging site, are shown.

[0113] FIG. 3 depicts (A) Bedaquiline (BDQ) with its key pharmacophore features (a quinoline-, a phenyl (Ari)-, a naphthyl (Ar2) ring and a dimethyl amino (DMA) tether). (B) BDQ’s quinoline and dimethylamino groups are retained in TBAJ-876 while its phenyl and naphthyl groups are replaced. (C) In comparison, TBAJ-5307 has a 2,3-dimethoxy-pyridin-4-yl moiety as A group and a 2,6-dimethoxy-pyridin-4-yl moiety as Ar2group, respectively.

[0114] FIG. 4 depicts the sequence part of the M. smegmatis subunit a being in proximity to the BDQ- binding aligned with the three M. abscesses subspecies revealing significant differences in amino acid composition. The calculation of the percentage of identity was performed and presented in darker to lighter shades, representing the most homologous to the least homologous. The proposed residues altering BDQ-binding in M. smegmatis and the M. abscesses complex strains are highlighted in the bold-lined box.

[0115] FIG. 5 depicts the structures of (±)-5307, (±)-5316, and (±)-5366.

[0116] FIG. 6 depicts the Anti- / Wab testing of BDQ analogs and racemats. (A) Growth inhibition doseresponse curve of M. abscesses subsp. abscesses ATCC 19977 cells by the racemates (±)- 5307, (±)-5316 and (±)-5366. Experiments were carried out in two biological replicates and each in three technical replicates. Data represent the average of all the experiments. Bacterial growth is expressed as percentage of control / untreated sample. (B) Growth inhibition doseresponse curve of smooth morphotype strains of M. abscesses subsp. abscesses ATCC 19977 by BDQ, TBAJ-876 and TBAJ-5307. In total, two biological replicates were carried out each in three technical replicates. Data represent the average of all the experiments. Bacterial growth is expressed as percentage of control / untreated sample. (C-D) Intracellular ATP synthesis inhibition of smooth (C) morphotype strains of M. abscesses subsp. abscesses and (D) M. abscesses subsp. abscesses IMVs. The ATP content of the cells was measured by adding BacTiter-Glo (Promega) to the cells. The total ATP content is directly proportional to relative luminescence units (RLUs). Experiments were performed in triplicates. Two biological replicates were carried out each in three technical replicates. Data represent the average of all the experiments. ****p < 0.0001 , statistical analysis was carried out using two-way ANOVA test for all the experiments presented. ATCC, American Type Culture Collection; IMV, inverted-membrane vesicle; Mab, Mycobacterium abscessus.

[0117] FIG. 7 depicts (A) Dose-response curves of BDQ, TBAJ-876 and TBAJ-5307 against the rough morphotype strain of M. abscessus subsp. abscessus. Three independent experiments were carried out, each with three technical replicates. (B) In contrast to the high inhibitory potency of the enantiomer (-)-TBAJ5307 in (A), the enantiomer (+)- TBAJ5307, displayed no significant activity. TBAJ-5307 inhibits growth of the isolate M. abscessus Bamboo (C), the M. abscessus subclasses M. bolletii and M. massiliense (D) as well as M. massiliense clinical isolate 1 and M. massiliense clinical isolate 2 (E). Experiments were performed twice in triplicates. (F) Initial four days of untreated and TBAJ-5307 kill kinetics against M. abscessus subsp. abscessus. The bacteria were grown in liquid culture (7H9) in the presence of the indicated concentrations of TBAJ-5307 up to 4 days. Colony-forming unit (CFU) was calculated by plating the culture on 7H10 agar plates. *; P < 0.05, statistical analysis was carried out for the experiment using ordinary one way ANOVA test. Experiments were performed twice. (G) Intracellular ATP synthesis inhibition of rough morphotype strain of M. abscessus subsp. abscessus by TBAJ- 5307. The ATP content of the cells was measured by adding bactiter gio (promega) to the cells. The total ATP content is directly proportional to relative luminescence units (RLU). Experiments were performed thrice in triplicates. (H) Oxygen consumption assay in M. abscessus using methylene blue as an oxygen sensor. TBAJ-5307 did also not affect oxygen consumption over a 72-h period. 7H9 media with dimethyl sulfoxide (DMSO) as compound solvent served as a blank / negative control. DF stands for drug free condition, including DMSO and M. abscessus, which served as a control for bacterial growth.

[0118] FIG. 8 depicts the predicted binding of TBAJ-5307 to the M. abscessus Fo-lagging site. Position of TBAJ-5307 bound to the Fo-domain in the (A) side- and (B) top view, respectively. Zoom into (C) the binding site and (D) the LigPlot+ representation (J. Chem. inf. Mod. 2011 , 51, 2778) are shown. Residues of the c-ring are displayed in grey. Subunit a residues V174 and 1178 are similar to the ones in M. smegmatis, while residues F177 and 1181 differ in the M. smegmatis counterpart. Figures representing molecular representations were produced with VMD (J. Mol. Graph. 1996, 14, 33-38, 27). FIG. 9 depicts the predicted binding of TBAJ-5307 to the M. abscessus F0-leading site. Position of TBAJ-5307 bound to the Fo-domain in the (A) side- and (B) top view, respectively. Zoom into (C) the binding site and (D) the LigPlot+ representation (J. Chem. Inf. Model. 2011 , 51, 2778) are shown. Residues of the c-ring are displayed in grey. Subunit a residues V174 and 1178 are similar to the ones in M. smegmatis, while residues F177 and 1181 differ in the M. smegmatis counterpart. Figures representing molecular representations were produced with VMD (J. Chem. Inf. Model. 2011 , 51, 2778).

[0119] FIG. 10 depicts the activity of 40 or 120 nM of TBAJ-5307 against 3 h preformed biofilm of Pseudomonas aeruginosa. PAO1 WT (A and C) and Escherichia coli UTI 189 (B and D). Treatment was carried out in 1 x PBS media (A and B) or M9 glucose media (C and D). Both planktonic and biofilm cells were collected from the same sample well, with planktonic cells (black circles) referring to suspended cells present in treatment buffer prior to washing and collection of biofilm cells (open blue triangles). No significant differences were observed between TBAJ-5307 and untreated controls. At least two independent experiments were carried out, each with two technical replicates. Each data point represents averaged data of two independent experiments with three technical replicates, and the line represents the means of all data. Colony-forming unit (CFU) counts were analyzed using GraphPad Prism, version 9.3.0 using two-way ANOVA and multiple comparison of column effect (concentration of compound against untreated control) within each row (planktonic versus biofilm samples).

[0120] FIG. 11 the intracellular activity of TBAJ-5307 against M. abscessus CIP104536T infected THP-1 cells. (A) Macrophages were infected with M. abscessus smooth (CIP S; left) or rough (CIP R; right) (MOI of 2:1 ) for 4 h prior to treatment with TBAJ-5307 (5 nM; 50 nM; 250 nM; 2500 nM), Rifabutin (RFB - 12.5 pg / ml), DMSO, or untreated. CFU were determined at 0, 1 and 3 days after infection. Data are mean values ± SD for three independent experiments analyzed using Kruskal-Wallis with Dunn’s multiple comparisons test. **p < 0.01 ; ***p < 0.001 ; ****p < 0.0001. (B) Representative immune-fluorescent fields were taken at 3 days postinfection showing macrophages infected with M. abscessus spe. abscessus expressing Tdtomato in the absence of antibiotics (UNT), or in the presence of TBAJ-5307 (250 nM or 2,500 nM) and rifabutin (RFB, 12.5 pg / ml). The surface and the endosomal system of the macrophages were detected using anti-CD63 antibodies. The nuclei were stained with 4', 6- diamidino-2-phenylindole (DAPI). Solid arrows indicate bacilli and dotted arrows indicate mycobacterial cords. Scale bar, 50 pm. Enlargement scale bar represents 50 pm. DMSO, dimethyl sulfoxide; Mab, Mycobacterium abscessus; MOI, multiplicity of infection. FIG. 12 depicts the therapeutic activity of TBAJ-5307 against Mab subsp. abscessus (R variant) in an embryonic zebrafish infection model. (A) Groups of around 20 uninfected embryos were immersed in water containing increasing concentrations of TBAJ-5307 (ranging from 50 to 2500 nM) for 4 days. The bolded bar on the x-axis indicates the duration of treatment. The graph shows the survival of the TBAJ-5307-treated and -untreated embryos over an 1 1 -day period. (B) Zebrafish embryos (around 35 per group) at 30 h postfertilization were infected with -250 colony-forming unit (CFU) of Mab subsp. abscessus expressing red fluorescent TdTomato via caudal vein injection. A standard PBS injection control (uninfected) was included. At 1 day postinfection, embryos were exposed to increasing concentrations of TBAJ-5307 (250 or 2500 nM) in fish water. Drugs were renewed at a daily basis for 4 days (bolded bar on the x-axis) after which embryos were washed twice in fresh embryo water and maintained in fish water. Survival was monitored daily over 1 1 -day period. Each treatment group was compared against the untreated control group with significant differences determined using the log rank (Mantel-Cox) statistical test for survival curves: ****p < 0.0001 ; ns, non-significant. Three experiments were performed. (C), Mab infection burden was quantified by fluorescent pixel count (FPC) determination using the ImageJ software after 2 (3 dpi) or 4 (5 dpi) days of exposure to TBAJ-5307 (2500 nM). The data are from three experiments (containing around 35 embryos per group), with each data point representing one infected zebrafish larva. The error bar represents the mean and standard deviations of the dataset. Statistical comparison of the difference using a Mann-Whitney’s t test: *p < 0.05, ****p < 0.0001 . (C) and (D), the 2500 nM values are normalized to the values of the untreated control at each time point. (D) Mab infection foci in embryos after 2 (3 dpi) or 4 (5 dpi) days of exposure to TBAJ-5307 (2500 nM) by FPC determination using the ImageJ software. The error bar represents the mean and standard deviations of the dataset. Statistical comparison was done using a Mann-Whitney’s t test: ***p < 0.001 and ****p < 0.0001 . (E) representative embryos from the untreated group (upper panel) and from the group treated with 250 or 2500 nM TBAJ-5307 at 3 dpi (left panels) or 5 dpi (right panels). Scale bars represents 300 pm. * highlights autofluorescence, dpi, day postinfection; Mab, Mycobacterium abscessus.

[0121] FIG. 13 depicts the following. (A) Spatiotemporal visualization of the infection by M. abscessus expressing tdTomato in untreated at TBAJ-5307 treated embryos at 3 dpi (left panels) and 5 dpi (right panels). Representative embryos from the untreated group (upper panels) and from the treated group with 2,500 nM of TBAJ-5307 (lower panels). The representative fluorescence and transmission overlays of whole embryos are shown (scale bar represents 300 pM). (B) Effect of TBAJ-5307 against M. abcessus CIP104536T (R variant (PLoS One. 2009, 4, e5660)) symptoms in an embryonic zebrafish infection model. Evolution of M. abscessus symptoms from cords (at 3 dpi) to abscesses (at 5 dpi) in embryos from the untreated group {upper panels) and from the treated group exposed to 2,500 nM TBAJ-5307 at 3 dpi (left panels) or 5 dpi (right panels). Scale bars in the main figures and insets represent 300 pm and 210 pm, respectively. * highlights autofluorescence.

[0122] FIG. 14 depicts the increased potency of TBAJ-5307 in combination with the antibiotics CFZ, rifabutin and amikacin in 7H9 broth. M. abscessus subsp. abscesses growth inhibition by TBAJ-5307 in combination with increasing concentrations of (A) CFZ, (B) Rifabutin and (C) Amikacin. *“*, P < 0.0001 , statistical analysis was carried out using two-way ANOVA test for all the experiments presented. (D) Dose response growth inhibition of TBAJ-5307 alone and in combination of TBAJ-5307 with TBP and AVI against M. abscessus ATCC 19977. TBAJ-5307 alone showed an MIC50 of 10 nM. In comparison, its MIC50 (0.7 nM) was 14-times lower in combination with TBP (4 pM) plus AVI (14 pM). ATCC, American Type Culture Collection; AVI, avibactam; CFZ, clofazimine; Mab, Mycobacterium abscessus; MIC50, minimum inhibitory concentrations; TBP, tebipenem.

[0123] FIG. 15 depicts the isobolograms of (A) the combination of TBAJ-5307 and CFZ as well as (B) TBAJ-5307 and Rifabutin (RFB) against M. abscessus subsp. abscessus. The dash line at the bottom-left corner indicates ideal isobole, where drugs act in synergy and independently. The dash line at the top-right corner indicates ideal isobole, where drugs act additively and in partial synergy.

[0124] FIG. 16 depicts the dose response growth inhibition of Tebipenam (TBP) with 4 pg / ml avibactam (AVI) against M. abscessus ATCC 19977. An MIC50 of 2 pM was observed in combination of the above two drugs.

[0125] FIG. 17 depicts the dose-response time-kill curves of TBAJ-5307, and TBAJ-5307+TBP+AVI against M. abscessus ATCC 19977. Cultures of M. abscessus ATCC 19977 were treated with 4x MIC (100 pM) TBP in combination with 4 mg / mL AVI for 4 days, and viability of the cultures was monitored by CFU determination.

[0126] FIG. 18 depicts the anti- / W. avium potency of TBAJ-5307. (A) Growth inhibition dose-response curve of M. avium by TBAJ-5307 in comparison to BDQ and TBAJ-876; and (B) TBAJ-5307 inhibiting oxidative phosphorylation in a whole cell ATP synthesis assay better than BDQ or TBAJ-876. Three independent experiments were carried out, each with three technical replicates. Data represent the average of all the experiments. ****p < 0.0001 , statistical analysis was carried out using two-way ANOVA test for all the experiments presented. BDQ, bedaquiline. FIG. 19 depicts (A) TBAJ-5307 inhibits growth of the clinical isolate M. avium 1 1. (B) Growth inhibition dose response curve of M. intracellulare by TBAJ-5307 in comparison to BDQ and TBAJ-876. Three independent experiments were carried out, each with three technical replicates. (C) TBAJ-5307 inhibits oxidative phosphorylation in a whole cell ATP synthesis assay better compared to BDQ and TBAJ-876. The total ATP content is directly proportional to relative luminescence units (RLU). The experiments have been performed in triplicates. (D) M. mucogenicum growth- and ATP synthesis (E) inhibition by TBAJ-5307. (F) Dose response curve of M. fortuitum growth inhibition by TBAJ-5307 in comparison to BDQ and TBAJ-876. Three independent experiments were carried out, each with three technical replicates.

[0127] FIG. 20 depicts the following. (A) Growth inhibition dose response curves of M. peregrinum, M. chelonae and M. kansasii by TBAJ-5307. Two independent experiments were carried out, each with three technical replicates (B) TBAJ-5307 inhibits whole-cell ATP synthesis of M. peregrinum, M. chelonae and M. kansasii. The experiments were performed in triplicates. (C) Since M. kansasii shows the characteristic yellow formation of colonies, due to the accumulation of p-carotene, cell growth inhibition of M. kansasii by TBAJ-5307 was directly observed in 6 well plates in the absence (DF = drug free) or presence of the inhibitor (10X M IC5O = 14 ± 0.4 nM) in three experiments.

[0128] Description

[0129] In a first aspect of the invention there is provided one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, for use in the treatment of nontuberculous mycobacterial (NTM) infection, wherein each of the one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, are a compound according to formula (I):

[0130] wherein:

[0131] X represents CH or N;

[0132] Y represents C-OR3 or N;

[0133] R1 represents H or OR4;

[0134] R2represents Ci to C6alkyl;

[0135] R3, when present, represents Ci to Ce alkyl, or R2and R3, together form a Ci to C3 alkylene moiety, which together with the atoms they are attached to provides a 5- to 7-membered ring system;

[0136] R4, when present, represents C2to C4 alkyl; or a salt or solvate thereof, provided that at least one of X and Y are N.

[0137] In embodiments herein, the word “comprising” may be interpreted as requiring the features mentioned, but not limiting the presence of other features. Alternatively, the word “comprising” may also relate to the situation where only the components / features listed are intended to be present (e.g. the word “comprising” may be replaced by the phrases “consists of” or “consists essentially of”). It is explicitly contemplated that both the broader and narrower interpretations can be applied to all aspects and embodiments of the present invention. In other words, the word “comprising” and synonyms thereof may be replaced by the phrase “consisting of” or the phrase “consists essentially of’ or synonyms thereof and vice versa.

[0138] The phrase, “consists essentially of’ and its pseudonyms may be interpreted herein to refer to a material where minor impurities may be present. For example, the material may be greater than or equal to 90% pure, such as greater than 95% pure, such as greater than 97% pure, such as greater than 99% pure, such as greater than 99.9% pure, such as greater than 99.99% pure, such as greater than 99.999% pure, such as 100% pure.

[0139] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” includes mixtures of two or more such compositions, reference to “an oxygen carrier” includes mixtures of two or more such oxygen carriers, reference to “the catalyst” includes mixtures of two or more such catalysts, and the like.

[0140] References herein (in any aspect or embodiment of the invention) to compounds of formula I includes references to such compounds per se, to tautomers of such compounds, as well as to pharmaceutically acceptable salts or solvates, or pharmaceutically functional derivatives of such compounds.

[0141] Pharmaceutically acceptable salts that may be mentioned include acid addition salts and base addition salts. Such salts may be formed by conventional means, for example by reaction of a free acid or a free base form of a compound of formula I with one or more equivalents of an appropriate acid or base, optionally in a solvent, or in a medium in which the salt is insoluble, followed by removal of said solvent, or said medium, using standard techniques (e.g. in vacuo, by freeze-drying or by filtration). Salts may also be prepared by exchanging a counter-ion of a compound of formula I in the form of a salt with another counter-ion, for example using a suitable ion exchange resin.

[0142] Examples of pharmaceutically acceptable salts include acid addition salts derived from mineral acids and organic acids, and salts derived from metals such as sodium, magnesium, or preferably, potassium and calcium.

[0143] Examples of acid addition salts include acid addition salts formed with acetic, 2,2- dichloroacetic, adipic, alginic, aryl sulphonic acids (e.g. benzenesulphonic, naphthalene-2- sulphonic, naphthalene-1 ,5-disulphonic and p-toluenesulphonic), ascorbic (e.g. L-ascorbic), L-aspartic, benzoic, 4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulphonic, (+)- (1 S)-camphor-10-sulphonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulphuric, ethane-1 ,2-disulphonic, ethanesulphonic, 2-hydroxyethanesulphonic, formic, fumaric, galactaric, gentisic, glucoheptonic, gluconic (e.g. D-gluconic), glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic), a-oxoglutaric, glycolic, hippuric, hydrobromic, hydrochloric, hydriodic, isethionic, lactic (e.g. (+)-L-lactic and (+)-DL-lactic), lactobionic, maleic, malic (e.g. (-)-L-malic), malonic, (±)-DL-mandelic, metaphosphoric, methanesulphonic, 1 - hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, L-pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulphuric, tannic, tartaric (e.g.(+)-L-tartaric), thiocyanic, undecylenic and valeric acids.

[0144] Particular examples of salts are salts derived from mineral acids such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulphuric acids; from organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, arylsulphonic acids; and from metals such as sodium, magnesium, or preferably, potassium and calcium.

[0145] As mentioned above, also encompassed by formula I are any solvates of the compounds and their salts. Preferred solvates are solvates formed by the incorporation into the solid state structure (e.g. crystal structure) of the compounds of the invention of molecules of a non-toxic pharmaceutically acceptable solvent (referred to below as the solvating solvent). Examples of such solvents include water, alcohols (such as ethanol, isopropanol and butanol) and dimethylsulphoxide. Solvates can be prepared by recrystallising the compounds of the invention with a solvent or mixture of solvents containing the solvating solvent. Whether or not a solvate has been formed in any given instance can be determined by subjecting crystals of the compound to analysis using well known and standard techniques such as thermogravimetric analysis (TGE), differential scanning calorimetry (DSC) and X-ray crystallography.

[0146] The solvates can be stoichiometric or non-stoichiometric solvates. Particularly preferred solvates are hydrates, and examples of hydrates include hemihydrates, monohydrates and di hydrates.

[0147] For a more detailed discussion of solvates and the methods used to make and characterise them, see Bryn etal., Solid-State Chemistry of Drugs, Second Edition, published by SSCI, Inc of West Lafayette, IN, USA, 1999, ISBN 0-967-06710-3.

[0148] “Pharmaceutically functional derivatives” of compounds of formula I as defined herein includes ester derivatives and / or derivatives that have, or provide for, the same biological function and / or activity as any relevant compound of the invention. Thus, for the purposes of this invention, the term also includes prodrugs of compounds of formula I.

[0149] The term “prodrug” of a relevant compound of formula I includes any compound that, following oral or parenteral administration, is metabolised in vivo to form that compound in an experimentally-detectable amount, and within a predetermined time (e.g. within a dosing interval of between 6 and 24 hours (i.e. once to four times daily)).

[0150] Prodrugs of compounds of formula I may be prepared by modifying functional groups present on the compound in such a way that the modifications are cleaved, in vivo when such prodrug is administered to a mammalian subject. The modifications typically are achieved by synthesizing the parent compound with a prodrug substituent. Prodrugs include compounds of formula I wherein a hydroxyl, amino, sulfhydryl, carboxyl or carbonyl group in a compound of formula I is bonded to any group that may be cleaved in vivo to regenerate the free hydroxyl, amino, sulfhydryl, carboxyl or carbonyl group, respectively.

[0151] Examples of prodrugs include, but are not limited to, esters and carbamates of hydroxyl functional groups, esters groups of carboxyl functional groups, N-acyl derivatives and N- Mannich bases. General information on prodrugs may be found e.g. in Bundegaard, H. “Design of Prodrugs” p. 1-92, Elsevier, New York-Oxford (1985).

[0152] Compounds of formula I, as well as pharmaceutically acceptable salts, solvates and pharmaceutically functional derivatives of such compounds are, for the sake of brevity, hereinafter referred to together as the “compounds of formula I”.

[0153] Compounds of formula I may contain double bonds and may thus exist as E (entgegen) and Z (zusammen) geometric isomers about each individual double bond. All such isomers and mixtures thereof are included within the scope of the invention.

[0154] Compounds of formula I may exist as regioisomers and may also exhibit tautomerism. All tautomeric forms and mixtures thereof are included within the scope of the invention.

[0155] Compounds of formula I may contain one or more asymmetric carbon atoms and may therefore exhibit optical and / or diastereoisomerism. Diastereoisomers may be separated using conventional techniques, e.g. chromatography or fractional crystallisation. The various stereoisomers may be isolated by separation of a racemic or other mixture of the compounds using conventional, e.g. fractional crystallisation or HPLC, techniques. Alternatively the desired optical isomers may be made by reaction of the appropriate optically active starting materials under conditions which will not cause racemisation or epimerisation (i.e. a ‘chiral pool’ method), by reaction of the appropriate starting material with a ‘chiral auxiliary’ which can subsequently be removed at a suitable stage, by derivatisation (i.e. a resolution, including a dynamic resolution), for example with a homochiral acid followed by separation of the diastereomeric derivatives by conventional means such as chromatography, or by reaction with an appropriate chiral reagent or chiral catalyst all under conditions known to the skilled person. All stereoisomers and mixtures thereof are included within the scope of the invention.

[0156] For the avoidance of doubt, in the context of the present invention, the term “treatment includes references to therapeutic or palliative treatment of patients in need of such treatment, as well as to the prophylactic treatment and / or diagnosis of patients which are susceptible to the relevant disease states.

[0157] The terms “patient and “patients” include references to mammalian (e.g. human) patients. As used herein the terms "subject" or "patient" are well-recognized in the art, and, are used interchangeably herein to refer to a mammal, including dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, and, most preferably, a human. In some embodiments, the subject is a subject in need of treatment or a subject with a disease or disorder. However, in other embodiments, the subject can be a normal subject. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered.

[0158] The term “effective amount” refers to an amount of a compound, which confers a therapeutic effect on the treated patient (e.g. sufficient to treat or prevent the disease). The effect may be objective (i.e. measurable by some test or marker) or subjective (i.e. the subject gives an indication of or feels an effect).

[0159] Unless otherwise stated, the term “alkyl” refers to an unbranched or branched, acyclic or cyclic, saturated or unsaturated (so forming, for example, an alkenyl or alkynyl) hydrocarbyl radical, which may be substituted or unsubstituted (with, for example, one or more halo atoms). Where the term “alkyl” refers to an acyclic group, it is preferably CHO alkyl and, more preferably, 0-6 alkyl (such as ethyl, propyl, (e.g. n-propyl or isopropyl), butyl (e.g. branched or unbranched butyl), pentyl or, more preferably, methyl). Where the term “alkyl” is a cyclic group (which may be where the group “cycloalkyl” is specified), it is preferably C3-12 cycloalkyl and, more preferably, C5-10 (e.g. C5-7) cycloalkyl. In particular embodiments of the invention, the term “alkyl” may refer to an unbranched or branched acyclic hydrocarbyl radical.

[0160] The term “alkylene” refers to a radical having the base formula (CnH2n)i-3. It will be appreciated that one or more of the hydrogen atoms in the radical may be substituted by a C1-3 alkyl group or a halo group. The term “halo”, when used herein, includes references to fluoro, chloro, bromo and iodo.

[0161] Further embodiments of the invention that may be mentioned include those in which the compound of formula I is isotopically labelled. However, other, particular embodiments of the invention that may be mentioned include those in which the compound of formula I is not isotopically labelled.

[0162] The term "isotopically labelled", when used herein includes references to compounds of formula I in which there is a non-natural isotope (or a non-natural distribution of isotopes) at one or more positions in the compound. References herein to "one or more positions in the compound" will be understood by those skilled in the art to refer to one or more of the atoms of the compound of formula I. Thus, the term "isotopically labelled" includes references to compounds of formula I that are isotopically enriched at one or more positions in the compound.

[0163] The isotopic labelling or enrichment of the compound of formula I may be with a radioactive or non -radioactive isotope of any of hydrogen, carbon, nitrogen, oxygen, sulfur, fluorine, chlorine, bromine and / or iodine. Particular isotopes that may be mentioned in this respect include2H,3H,11C,13C,14C,13N,15N,15O,170,180,35S,18F,37CI,77Br,82Br and125l).

[0164] When the compound of formula I is labelled or enriched with a radioactive or nonradioactive isotope, compounds of formula I that may be mentioned include those in which at least one atom in the compound displays an isotopic distribution in which a radioactive or nonradioactive isotope of the atom in question is present in levels at least 10% (e.g. from 10% to 5000%, particularly from 50% to 1000% and more particularly from 100% to 500%) above the natural level of that radioactive or non-radioactive isotope.

[0165] In a further aspect of the invention, there is provided a use of one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, in the manufacture of a medicament for the treatment of nontuberculous mycobacterial (NTM) infection, wherein each of the one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, are a compound according to formula (I):

[0166] wherein:

[0167] X represents CH or N;

[0168] Y represents C-OR3 or N; R1 represents H or OR4;

[0169] R2represents Ci to C6alkyl;

[0170] R3, when present, represents Ci to Ce alkyl, or R2and R3, together form a Ci to C3 alkylene moiety, which together with the atoms they are attached to provides a 5- to 7-membered ring system; R4, when present, represents C2to C4 alkyl; or a salt or solvate thereof, provided that at least one of X and Y are N.

[0171] In a third aspect of the invention, there is provided a method of treating nontuberculous mycobacterial (NTM) infection comprising the step of administering to a subject in need thereof one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, wherein each of the one or more 3,5- dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, are a compound according to formula (I):

[0172] wherein:

[0173] X represents CH or N;

[0174] Y represents C-OR3 or N; R1 represents H or OR4;

[0175] R2represents Ci to C6alkyl;

[0176] R3, when present, represents Ci to Ce alkyl, or R2and R3, together form a Ci to C3 alkylene moiety, which together with the atoms they are attached to provides a 5- to 7-membered ring system; R4, when present, represents C2to C4 alkyl; or a salt or solvate thereof, provided that at least one of X and Y are N.

[0177] In a fourth aspect of the invention, there is provided a composition comprising one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, for use in the treatment of nontuberculous mycobacterial

[0178] (NTM) infection, wherein each of the one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, are a compound according to formula (I):

[0179] wherein:

[0180] X represents CH or N;

[0181] Y represents C-OR3 or N; R1 represents H or OR4;

[0182] R2represents Ci to C6alkyl;

[0183] R3, when present, represents Ci to Ce alkyl, or R2and R3, together form a Ci to C3 alkylene moiety, which together with the atoms they are attached to provides a 5- to 7-membered ring system; R4, when present, represents C2to C4 alkyl; or a salt or solvate thereof, provided that at least one of X and Y are N.

[0184] In a further aspect of the invention, there is provided a use of a composition comprising one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, in the manufacture of a medicament for the treatment of nontuberculous mycobacterial (NTM) infection, wherein each of the one or more 3,5- dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, are a compound according to formula (I):

[0185] wherein:

[0186] X represents CH or N;

[0187] Y represents C-OR3 or N;

[0188] R1 represents H or OR4;

[0189] R2represents Ci to C6alkyl;

[0190] R3, when present, represents Ci to Ce alkyl, or R2and R3, together form a Ci to C3 alkylene moiety, which together with the atoms they are attached to provides a 5- to 7-membered ring system;

[0191] R4, when present, represents C2to C4 alkyl; or a salt or solvate thereof, provided that at least one of X and Y are N.

[0192] As will be appreciated, the composition (and its use in a method of treatment) may include one or more pharmaceutically acceptable excipients.

[0193] Compounds of formula I may be administered by any suitable route, but may particularly be administered orally, intravenously, intramuscularly, cutaneously, subcutaneously, transmucosally (e.g. sublingually or buccally), rectally, transdermally, nasally, pulmonarily (e.g. tracheally or bronchially), topically, by any other parenteral route, in the form of a pharmaceutical preparation comprising the compound in a pharmaceutically acceptable dosage form. Particular modes of administration that may be mentioned include oral, intravenous, cutaneous, subcutaneous, nasal, intramuscular or intraperitoneal administration.

[0194] Compounds of formula I will generally be administered as a pharmaceutical formulation in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier, which may be selected with due regard to the intended route of administration and standard pharmaceutical practice. Such pharmaceutically acceptable carriers may be chemically inert to the active compounds and may have no detrimental side effects or toxicity under the conditions of use. Suitable pharmaceutical formulations may be found in, for example, Remington The Science and Practice of Pharmacy, 19th ed., Mack Printing Company, Easton, Pennsylvania (1995). For parenteral administration, a parenterally acceptable aqueous solution may be employed, which is pyrogen free and has requisite pH, isotonicity, and stability. Suitable solutions will be well known to the skilled person, with numerous methods being described in the literature. A brief review of methods of drug delivery may also be found in e.g. Langer, Science (1990) 249, 1527.

[0195] Otherwise, the preparation of suitable formulations may be achieved routinely by the skilled person using routine techniques and / or in accordance with standard and / or accepted pharmaceutical practice.

[0196] The amount of compound of formula I in any pharmaceutical formulation used in accordance with the present invention will depend on various factors, such as the severity of the condition to be treated, the particular patient to be treated, as well as the compound(s) which is / are employed. In any event, the amount of compound of formula I in the formulation may be determined routinely by the skilled person.

[0197] For example, a solid oral composition such as a tablet or capsule may contain from 1 to 99 % (w / w) active ingredient; from 0 to 99% (w / w) diluent or filler; from 0 to 20% (w / w) of a disintegrant; from 0 to 5% (w / w) of a lubricant; from 0 to 5% (w / w) of a flow aid; from 0 to 50% (w / w) of a granulating agent or binder; from 0 to 5% (w / w) of an antioxidant; and from 0 to 5% (w / w) of a pigment. A controlled release tablet may in addition contain from 0 to 90 % (w / w) of a release-controlling polymer.

[0198] A parenteral formulation (such as a solution or suspension for injection or a solution for infusion) may contain from 1 to 50 % (w / w) active ingredient; and from 50% (w / w) to 99% (w / w) of a liquid or semisolid carrier or vehicle (e.g. a solvent such as water); and 0-20% (w / w) of one or more other excipients such as buffering agents, antioxidants, suspension stabilisers, tonicity adjusting agents and preservatives.

[0199] Depending on the disorder, and the patient, to be treated, as well as the route of administration, compounds of formula I may be administered at varying therapeutically effective doses to a patient in need thereof. However, the dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to effect a therapeutic response in the mammal over a reasonable timeframe. One skilled in the art will recognize that the selection of the exact dose and composition and the most appropriate delivery regimen will also be influenced by inter alia the pharmacological properties of the formulation, the nature and severity of the condition being treated, and the physical condition and mental acuity of the recipient, as well as the potency of the specific compound, the age, condition, body weight, sex and response of the patient to be treated, and the stage / severity of the disease.

[0200] Administration may be continuous or intermittent (e.g. by bolus injection). The dosage may also be determined by the timing and frequency of administration. In the case of oral or parenteral administration the dosage can vary from about 0.01 mg to about 1000 mg per day of a compound of formula I.

[0201] In any event, the medical practitioner, or other skilled person, will be able to determine routinely the actual dosage, which will be most suitable for an individual patient. The above- mentioned dosages are exemplary of the average case; there can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.

[0202] In yet a further aspect of the invention, there is provided a method of treating nontuberculous mycobacterial (NTM) infection comprising the step of administering to a subject in need thereof a composition comprising one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, wherein each of the one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, are a compound according to formula (I):

[0203] wherein:

[0204] X represents CH or N;

[0205] Y represents C-OR3 or N;

[0206] R1 represents H or OR4;

[0207] R2represents Ci to C6alkyl;

[0208] R3, when present, represents Ci to Ce alkyl, or R2and R3, together form a Ci to C3 alkylene moiety, which together with the atoms they are attached to provides a 5- to 7-membered ring system;

[0209] R4, when present, represents C2to C4 alkyl; or a salt or solvate thereof, provided that at least one of X and Y are N.

[0210] In particular embodiments described herein, the DARQ analogue may be one where one or more of the following apply:

[0211] (i) R4, when present, represents C2to C3 alkyl;

[0212] (ii) R2represents Ci to C3alkyl; and

[0213] (iii) when Y represents COR3, then R3represents Ci to C3 alkyl, or R2and R3together represent -CH2- or -(CH2)2-

[0214] For example, the DARQ analogue may be one where one or more of the following apply:

[0215] (ia) R1 represents H or O'Pr;

[0216] (iia) R2represents CH3; and

[0217] (iiia) when Y represents COR3, then R3represents CH3, or R2and R3together represent -(CH2)2- In particular embodiments of the aspects above, the DARQ analogue may be selected from the group consisting of:

[0218] (±)-TBAJ-5316 ; and

[0219]

[0220] In more particular embodiments of the aspects above, the DARQ analogue may be selected from the group consisting of:

[0221] where the compounds are provided in an enantiomerically enriched form based on the enantiomer shown.

[0222] In yet more particular embodiments of the aspects above, the DARQ analogue may be:

[0223] or a pharmaceutically acceptable salt or solvate thereof which is provided in an enantiomerically enriched form based on the enantiomer shown.

[0224] In a further aspect of the invention, there is provided a composition comprising one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, for use in the treatment of nontuberculous mycobacterial (NTM) infection, wherein each of the one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, are a compound according to formula (I): wherein: X represents CH or N;

[0225] Y represents C-OR3or N;

[0226] Ri represents H or OR4;

[0227] R2 represents Ci to Csalkyl;

[0228] R3, when present, represents Ci to Ce alkyl, or R2and R3, together form a Ci to C3alkylene moiety, which together with the atoms they are attached to provides a 5- to 7-membered ring system;

[0229] R4, when present, represents C2to C4alkyl; or a salt or solvate thereof, provided that at least one of X and Y are N, and a second antibiotic, optionally wherein the composition further comprises one or more pharmaceutically acceptable excipients.

[0230] Related aspects of the invention include:

[0231] (A) a composition as described above for use in the treatment of nontuberculous mycobacterial (NTM) infection;

[0232] (B) use of a composition as described above in the manufacture of a medicament to treat a nontuberculous mycobacterial (NTM) infection; and

[0233] (C) a method of treating nontuberculous mycobacterial (NTM) infection comprising the step of administering to a subject in need thereof a composition as described above.

[0234] The second antibiotic may be any suitable antibiotic that may be used to treat NTM infections. The combination may be synergistic, partly synergistic or additive in nature. For example, the second antibiotic may be selected from the group consisting of Clofazimine, Rifabutin and Amikacin, or a pharmaceutically acceptable salt or solvate thereof.

[0235] In yet a further aspect of the invention, there is provided a composition comprising one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, for use in the treatment of nontuberculous mycobacterial (NTM) infection, wherein each of the one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, are a compound according to formula (I): wherein:

[0236] X represents CH or N;

[0237] Y represents C-OR3 or N;

[0238] R1 represents H or OR4;

[0239] R2represents Ci to C6alkyl;

[0240] R3, when present, represents Ci to Ce alkyl, or R2and R3, together form a Ci to C3 alkylene moiety, which together with the atoms they are attached to provides a 5- to 7-membered ring system;

[0241] R4, when present, represents C2to C4 alkyl; or a salt or solvate thereof, provided that at least one of X and Y are N. for use in the treatment of nontuberculous mycobacterial (NTM) infection, wherein the composition is to be administered sequentially, simultaneously or concomitantly with a second antibiotic.

[0242] Similarly, there is provided use of a composition as described immediately above in:

[0243] (i) the manufacture of a medicament to treat a nontuberculous mycobacterial (NTM) infection wherein the composition is to be administered sequentially, simultaneously or concomitantly with a second antibiotic; and

[0244] (ii) a method of treating nontuberculous mycobacterial (NTM) infection comprising the step of administering to a subject in need thereof said composition in combination with a second antibiotic, wherein the composition is administered sequentially, simultaneously or concomitantly with the second antibiotic. As above, the second antibiotic may be selected from the group consisting of Clofazimine, Rifabutin and Amikacin, or a pharmaceutically acceptable salt or solvate thereof.

[0245] It will be appreciated that the DARQ analogue may be selected from those previously described above generically and specifically, and so a repetition of those generic and specific analogues is not included here for brevity.

[0246] The current invention may relates to the treatment of any NTM infection. NTM infections that may be mentioned herein include, but are not limited to M. chelonae, M. peregrinum, M. kansasii, and more particularly, M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, M. abscessus subsp. massiliense, M. fortuitum, M. avium and M. intracellulare, and combinations thereof.

[0247] Here, we identified TBAJ-5307 as a novel anti- / Wab inhibitor targeting the F1F0 ATP synthase, essential for ATP synthesis, regulation of ATP homeostasis and pmf under multiple growth conditions. The inhibitor interacts with the Fo-domain via the c-ring, as well as the subunits a- c lagging- and leading sites, including the M. abscessus specific residues aF177, all 81 , aA218 and al222. TBAJ-5307’s binding to the a-c interfaces inhibits proton translocation via the Fo-domain half-channels by preventing rotation of the c-ring relative to subunit a. The high potency of TBAJ-5307 in growth inhibition correlates with whole cell ATP depletion, reduction in ATP formation of Mab IMVs and free energy calculations. The compound displays higher efficacy compared to BDQ and TBAJ-876. TBAJ-5307 exhibits anti- / Wab potency in macrophages and zebrafish embryos without being toxic, which is essential for further clinical developments. The compound potentiates the anti- / Wab activity of the NADH dehydrogenase inhibitor CFZ, the 16S rRNA targeting amikacin, the RNA polymerase-targeting antibiotic RFB, and the oral pair TBP-AVI, with TBP inhibiting peptidoglycan synthesis, respectively. In addition, the TBAJ-5307-TBP-AVI cocktail is bactericidal.

[0248] The presented combinations of the low nanomolar ATP synthesis inhibitor TBAJ-5307 with antibiotics targeting transcription, protein synthesis or cell wall formation would be a step forward

[0249] (i) to silence major cell processes of the pathogen under different metabolic states,

[0250] (ii) to reduce the emergence of drug resistance, and

[0251] (iii) to reduce the required concentrations, and thus dosing of these existing antibiotics because of the enhanced inhibitory activity of each within the cocktail, leading to reduced toxicity and side-effects, and (iv) in case of an TBAJ-5307-TBP-AVI to aim for an oral drug combination, which would reduce costs and risks compared to intravenous therapies.

[0252] Combining our discoveries with targeting the F1F0-ATP synthase of the NTM pathogens M. abscessus complex, the M. avium complex and M. fortuitum, M. peregrinum, M. chelonae and M. kansasii afford a competitive advantage in the field. The concept is anchored in novel insights of our team into Nature’s paradigms for securing energy inside NTMs and the repurposing of our anti-TB racemates (±)-5307, (±)-5316, and (±)-5366 as well as the anti-TB compound TBAJ-5307. The compounds block ATP synthesis in a whole cell assay, underlining that the molecular engine, F1F0-ATP synthase, is the target.

[0253] TBAJ-5307 displays pronounced nanomolar anti-NTM activity and shows higher potency of ATP synthesis- and cell growth inhibition against the NTM fast growers M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, M. abscessus subsp. massiliense (Mab complex), and Mycobacterium fortuitum, as well as against the slow growers M. avium and M. intracellulare (MAC) and clinical isolates, when compared to its parent molecule BDQ and the BDQ analog TBAJ-876. Its improved potency compared to BDQ and TBAJ-876 makes the compound a superb candidate to eradicate a brought range of NTM infectious disease in future.

[0254] We also demonstrated that TBAJ-5307 is active against M. abscessus infection in zebrafish embryos. Important also is the observation, that TBAJ-5307 shows an additive / partial synergistic effect against M. abscessus when incubated with the drugs clofazimine (CFZ), amikacin and rifabutin. This opens a new avenue for a highly efficient multi-drug combination.

[0255] TBAJ-5307 has been identified as a potent inhibitor of the F1 Fo-ATP synthase of human NTM pathogens (M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, M. abscessus subsp. massiliense, M. fortuitum, M. avium and M. intracellulare, M. peregrinum, M. chelonae, M. kansasii and clinical isolates) and showed that the compound is potent in in vivo experiments. In addition, TBAJ-5307 revealed an additive / partial synergistic effect with anti- NTM drugs, opening the way for in vitro and in vivo profiling and future studies regarding novel regimens fighting NTM-caused infectious diseases.

[0256] Further aspects and embodiments of the invention will now be discussed by reference ot the following non-limiting examples. Examples

[0257] TBAJ-5307 is not commercially available, and thus it was synthesized by following the known protocol {Bioorg Med Chem, 2019, 27, 1292).

[0258] Synthesis of TBAJ-racemates (±)-5307, (±)-5316) and (±)-5366 and TBAJ-5307

[0259] The racemates (±)-5307, (±)-5316) and (±)-5366 were synthesized according to Sutherland et al. {Bioorg Med Chem, 2019, 27, 1292) and Hotra et al. {Angew Chem Int Ed Engl, 2020, 59, 13295). Chiral separation service of (±)-5307 provided by WuXi AppTec (Wuhan) Co., Ltd. The desired chiral isomers were separated by ChiralPAK AD-3 column using supercritical fluid chromatography (SFC) with gradients of IPA / CO2 / Et2NH as eluent. Purity and enantiomeric excess of the two enantiomers of TBAJ-5307 were >94% and >99%. The levorotatory enantiomer showed highest activity and was assigned (1 R, 2S) stereochemistry in accordance with Sutherland et al. {Bioorg Med Chem, 2019, 27, 1292).

[0260] LC-MS m / z [M+H+] 627.4. [a]D25= -6.28 (c = 0.1 , CHCI3). {Bioorg Med Chem, 2019, 27, 1292)

[0261] Bedaquiline and Its 3,5-Dialkoxypyridine Analogue TBAJ-876

[0262] BDQ was purchased from MedChem Express and TBAJ-876 was synthesized as described in Sarathy et al. {Antimicrob. Agents Chemother. 2020, 64, e02404). Both inhibitors were dissolved in 100% dimethyl sulfoxide (DMSO) (MP Biomedicals).

[0263] Biological Procedures and Data Analysis

[0264] Bacterial Strains

[0265] The NTM strains M. abscesses subsp. abscesses, M. abscesses subsp. bolletii, M. abscesses subsp. massiliense, M. fortuitum, M. avium and M. intracellulare, and clinical isolates strains M. abscesses Bamboo and M. avium 11 , M. abscesses subsp. massiliense clinical isolate 1 and M. abscesses subsp. massiliense clinical isolate 2 were used. The clinical isolate M. abscessus Bamboo has been isolated from a patient with amyotrophic lateral sclerosis and bronchiectasis belongs to the subspecies M. abscessus subsp. abscessus and harbors the inactive, clarithromycin-sensitive erm(41 ) C28 sequevar (GenBank accession no. MVDX00000000) (Genome Announc. 2017, 5, e00388). The clinical isolate M. avium has been isolated from bone marrow of an AIDS patient with disseminated MAC infection, including pulmonary infection, and it is classified as M. avium subsp. hominissuis based on the 3' region of the hsp65 gene sequence with 100% identity when aligned with the hsp653' region of M. avium subsp. hominissuis 104 (GenBank accession no. NC 008595) (Genome Announc. 2017, 5, e00766). M. massiliense clinical isolate 1 and M. massiliense clinical isolate 2 were isolated from the sputum of patients with NTM pulmonary infection and subspecies classification was determined by PCR of erm(41 ) which produced a truncated 397 bp product (Microbiol. Immunol. 2010, 54, 347).

[0266] Culture Maintenance

[0267] All NTM strains were maintained in Middlebrook 7H9 medium (BD Difco) supplemented with 0.2% (vol / vol) glycerol (Fisher Scientific), 0.05% (vol / vol) Tween 80 (Sigma-Aldrich), and 10% (vol / vol) Middlebrook albumin-dextrose-catalase (ADC) (BD Difco). Cation-adjusted Mueller- Hinton (CAMH) broth (BD Difco) was also used to culture M. abscessus strains and was prepared according to the manufacturer’s instructions.

[0268] Example 1 . Anti-Mab Activity of Racemates (±)-5307, (±)-5316 and (±)-5366

[0269] Most of the administered anti-NTM drugs possess minimum inhibitory concentrations (MIC50) for growth inhibition in the micromolar range. To extend the poorly populated anti-NTM drug pipeline with a nanomolar potency inhibitor and the energy converter F-ATP synthase of the fast growing NTM M. abscessus as a target, we firstly used a repurposing approach of our TBAJ-racemates (±)-5307, (±)-5316) and (±)-5366, which were synthesized as described above and according to Sutherland et al. (Bioorg. Med. Chem. 2019, 27, 1292) and Hotra et al. (Angew. Chem. Intern. Edi. 2020, 59, 13295), and evaluated the susceptibility of M. abscessus subsp. abscessus ATCC19977 to the compounds in complete Middlebrook 7H9 broth.

[0270] Minimum Inhibitory Concentration Determination

[0271] The growth inhibition dose-response assay was carried out using the broth microdilution method as described previously ( Front Microbiol 2016, 7, 199). Briefly, each well of clear 96- well flat-bottom Costar cell culture plates (Corning) was filled with 100 pl of liquid medium (complete 7H9 medium). TBAJ-5307 / TBAJ-876 / BDQ were added to the first well in each row of the plate to create two times the desired highest final concentration. A 10- point 2-fold serial dilution of TBAJ-5307 / TBAJ-876 / BDQ was carried out starting from this first well. The M. abscessus strains used for this assay were grown to mid-exponential phase and then diluted to an optical density at 600 nm (OD6oo) value of 0.1 using the same liquid medium used to run the assay. One hundred microliters of the diluted culture were added to each well to create a final ODSoo value of 0.05 in each well. The plates were incubated for 3 days at 37 °C. In case of M. peregrinum, M. chelonae, M. kansasii Vhe plates were incubated at 37 °C for 5-7 days. After the incubation period, the cultures in all wells were manually resuspended, and the OD600 of each well was read using a Tecan Infinite Pro 200 plate reader. The MIC50 values reported represent the concentration that inhibits 50% of bacterial growth compared to the untreated culture.

[0272] Results and Discussions

[0273] Here we have firstly used a repurposing approach of our TBAJ-racemates (±)-5307, (±)-5316, and (±)-5366 anti-TB compounds and evaluated the susceptibility of M. abscessus subsp. abscessus ATCC19977 to the compounds in complete Middlebrook 7H9 broth. As shown in FIG. 6A, the three racemates (±)-5307, (±)-5316 and (±)-5366 showed good growth inhibition of M. abscessus with MIC50 values of 150 ± 40 nM, 510 ± 100 nM and 900 ± 20 nM, respectively, and with (±)-5307 being the most potent racemate.

[0274] Example 2. High potency of TBAJ-5307 against Reference and Clinical Strains of the M. abscessus Complex

[0275] The levorotatory enantiomer, (-)-TBAJ5307, called TBAJ-5307, was obtained according to the protocol disclosed in the Synthesis Protocols section above. MIC50 determinations were carried out by following the protocol disclosed in Example 1 .

[0276] TBAJ-5307 was tested for its ability to inhibit the growth of other subspecies that form the M. abscess sus complex in complete Middlebrook 7H9 broth.

[0277] To determine whether TBAJ-5307 is bactericidal against M. abscessus, we also measured the survival of the strain upon drug exposure.

[0278] Whole Cell ATP Measurement

[0279] Whole cell ATP synthesis assays were carried out as described previously by Hotra et al. in 96 well plates (Angew Chem Int Ed Engl, 2020, 59, 13295). Bacterial Killing Assay

[0280] M. abscessus subsp. abscesses culture were grown to exponential phase and diluted to an ODeoo of 0.005 and aliquoted onto T-25 mm2tissue culture flasks. Test compounds were dispensed into each flask and were incubated at 37 °C for 5 days. Around 10 pl of culture was taken out from each flask followed by the serial dilution with Phosphate-buffered saline (PBS). 25 pl of culture of respective dilutions were plated on each quadrant of 7H10 agar plate. The agar plates were incubated at 37 °C for five days. Bacterial viability was determined by counting the colony-forming units (CPU) on the plates.

[0281] Quantification of Intracellular ATP Levels

[0282] 100 pl of complete 7H9 medium were filled in each well of clear 96-well flat-bottom Costar cell culture plates (Corning). TBAJ-5307 / TBAJ-876 / BDQ was added to the first well in each row to create two times the desired highest final concentration. Subsequently, a 16-point 2-fold serial dilution was carried out starting from the first well in each row. The respective M. abscessus strain, which was grown to mid-exponential phase, was diluted to an OD6oo of 0.1 ; 100 pl of the diluted culture was added to each well to create a final OD6oo of 0.05 in all the wells. The plates were incubated at 37 °C for 6-8 h.

[0283] At the end of the incubation period, the samples were measured for their intracellular ATP content by adding the BacTiter-Glo microbial cell viability assay (Promega), which was carried out according to the manufacturer’s instructions as described previously (Antimicrob. Agents Chemother. 2022, 66, e0001822). 50 pl of bacterial sample was mixed with 50 pl of the BacTiter-Glo reagent in each well of an opaque, white, 96-well, flat- bottom Nunc plate. Luminescence was measured with a Tecan Infinite Pro 200 plate reader after 10 min of incubation of the plate in the dark at room temperature. The background luminescence reading was subtracted from the luminescence readings of all the samples. The amount of ATP content is directly proportional to the relative luminescence units. The graph of the results was made using GraphPad Prism 8 software (C. A. Motulsky H, Fitting models to biological data using linear and nonlinear regression: a practical guide to curve fitting. GraphPad software, Inc, San Diego, CA. www.qraDhoad.com. , 2003L

[0284] Methylene Blue Assay

[0285] Aliquots of 1.5 ml (OD6oo = 0.3) log-phase cultures of M. abscessus subsp. abscessus were transferred into clear 2 ml screw-cap glass vials along with test compounds. The cultures were incubated in the presence of the drugs for 6 h at 37 °C before the addition of methylene blue dye at a final concentration of 0.001%. Upon addition of methylene blue, all vials were tightly closed and incubated at 37 °C in a hypoxic jar. Oxygen in the jar was removed by AnaeroGen sachet. The dye decolorizes when the oxygen in the culture is used up. The color changes and the differences in the exponentially growing cultures and broth reflect the relative oxygen consumption, thus indirectly indicate the respiration of each culture.

[0286] Results and Discussions

[0287] Since (±)-5307 was most potent among the three racemates (FIG. 5), the respective enantiomer TBAJ-5307 was synthesized and the enantiomers were resolved. The levorotatory enantiomer, (-)-TBAJ5307, called TBAJ-5307, displayed high potency with MIC50 of 4.5 ± 0.9 nM and 6 ± 1 .2 nM against the M. abscessus subsp. abscessus smooth strain (S variant; FIG. 6B) and rough strain (R) (FIG. 7A), respectively. In contrast, the dextrorotatory enantiomer, (+)-TBAJ5307, showed no significant activity (FIG. 7B). Importantly, TBAJ-5307 showed a 12- times higher potency compared to BDQ (53 ± 8.2 nM and 74 ± 4 nM) or TBAJ-876 (22 ± 2.9 nM and 63 ± 3.8 nM).

[0288] Table 1. Growth-inhibitory- and ATP synthesis potency of BDQ, TBAJ-876 and TBAJ-5307 against NTM stains in 7H9 broth medium. The experiments were carried out three times independently, n.d. stands for not determined.

[0289] MIC50 (nM) ICso (nM)

[0290] NTM strain

[0291] BDQ TBAJ-876 TBAJ-5307 BDQ TBAJ-876 TBAJ-5307

[0292] Mab abscessus (S) 53 22 4.5 60 106 7

[0293] Mab abscessus (R) 74 63 6.0 148 193 15

[0294] Mab. Bamboo 108 143 39 351 496 100

[0295] Mab bolletii 119 187 58 70 73 11

[0296] Mab massilience 141 246 38 230 467 73

[0297] M. avium 184 64 1.8 267 210 12

[0298] M. avium isolate 7Q 29 31 26 7 4.6

[0299] M. intracellulare 60 30 16 55 16 6.7

[0300] M. fortuitum 8.3 1 .7 0.9 n.d. n.d. n.d.

[0301] Since a potent anti- / Wab agent requires potency against clinical isolates, the efficacy of TBAJ- 5307 was also demonstrated against the isolate M. abscessus Bamboo (Genome Announc. 2017, 5, e00388; Genome Announc. 2017, 5, e00766) (MIC50 = 16 + 1.3 nM; FIG. 7C and Table 1 ). TBAJ-5307 also displayed high efficacy against M. bolletii and M. massiliense with MIC50 of 2.7 ± 0.9 nM and 5.2 ± 1.5 nM, respectively, underscoring that the compound is active against representative strains of the entire M. abscesssus complex. (FIG. 7D). In addition, TBAJ-5307 inhibited growth of M. abscessus subsp. massiliense clinical isolate 2 and M. abscessus subsp. massiliense clinical isolate 1 with an MIC50 of 21 .4 ± 1 .8 nM and 36.1 ± 4.2 nM, respectively (FIG. 7E).

[0302] To determine whether M. abscessus growth inhibition by TBAJ-5307 is related to depletion of ATP, intracellular ATP synthesis was measured. As shown in FIG. 6C and FIG. 7G, TBAJ- 5307 depleted ATP synthesis within the bacilli with an half-maximal inhibitory concentration (IC50) of 7 ± 0.3 nM and 15 ± 1 .4 nM in the S and R variants of M. abscessus subsp. abscessus, respectively. In comparison, lower ATP synthesis inhibition of the S and R variants was observed in the presence of BDQ (60 ± 12.5 nM and 148 ± 9.6 nM) or TBAJ-876 (106 ± 20 nM and 193 ± 35 nM). These results underline that the improved growth inhibition by TBAJ- 5307 is consistent with higher intracellular ATP depletion when compared to BDQ or TBAJ- 876. Since oxygen consumption within the respiratory chain of M. abscessus subsp. abscessus is unaffected at 125 nM and 250 nM of TBAJ-5307 (FIG. 7H), the data also indicates that the inhibitor targets the Mab F-ATP synthase.

[0303] Example 3. TBAJ-5307’s Target Specificity and Mechanism of Action

[0304] To test whether growth inhibition by TBAJ-5307 is related to depletion of ATP synthesis. To confirm that the observed whole cell ATP depletion correlates with the inhibition of the Mab F1F0-ATP synthase only, an intracellular ATP synthesis assay was used (Anti microb Agents Chemother 2015 59, 4457) and ATP formation of inverted-membrane vesicles (IMVs) of M. abscessus subsp. abscessus R variant was measured.

[0305] To explore the inhibitory potency at an energetic level and to understand the mechanism of action, we performed molecular dynamics (MD) simulations and free energy calculations as successfully done for BDQ- and TBAJ-876-binding to the M. smegmatis Fo-domain Curr. Res. Struct. Biol. 2022, 4, 278).

[0306] To further confirm TBAJ-5307’s target specificity and to demonstrate that the molecule does not affect human gut biofilm formation or planktonic bacteria, the compound was assayed against Pseudomonas aeruginosa PAO1 or Escherichia coli UTI 189.

[0307] Preparation of M. abscessus subsp abscessus Inverted Membrane Vesicles

[0308] Inverted membrane vesicles (IMVs) of M. abscessus subsp. abscessus R variant were prepared according to a method described previously to Hotra et al. (Angew. Chem. Intern. Edi. 2020, 59, 13295). ATP Synthesis Assay to Determine ATP Formation of M. abscesses IMVs

[0309] The reaction mixture for the ATP synthesis assay contained assay buffer (50 mM MOPS [morpholinepropanesulfonic acid]-NaOH, 10 mM MgCl2, pH 7.5), 10 pM ADP, 250 pM Pi;and 1 mM NADH. KH2PO salt (100 mM) was dissolved in the assay buffer to adjust the concentration of inorganic phosphate (Pi); 25 pl of the reaction mixture was added to each well of an opaque, white, 96-well, flat-bottom Nunc plate (Thermo Scientific). TBAJ-5307 was added to the first well of each row to create 2x the desired highest final concentration. A 16- point 2-fold serial dilution was then carried out starting from the first well. M. abscesses sebsp. abscesses IMVs were then added to create a final concentration of 5 pg of protein / ml in 50 pl per well. Subsequently, the plate was incubated at room temperature for 30 min. At the end of the incubation period, 50 pl of CellTitre-Glo (Promega) was added to each well. The plate was then incubated again for 10 min in the dark at room temperature. Subsequently, luminescence was measured with a Tecan Infinite Pro 200 plate reader (parameters: luminescence, integration time of 500 ms; no attenuation). The level of luminescence correlates with the amount of ATP synthesized by the F-ATP synthase. The graphs of the results were made using GraphPad Prism 8 software (C. A. Motulsky H, Fitting models to biological data esing linear and nonlinear regression: a practical geide to cerve fitting. GraphPad software, Inc, San Diego, CA. www. oraphoad. com.. 2003).

[0310] Conventional MD Simelations

[0311] Applying the program MODELLER {Meth. Enzymol. 2003, 374, 461 ), we constructed the Fo- domain from M. abscesses by using the BDQ bound Fo-domain from M. smegmatis (PDB-ID: 7JGC) {Natere 2021 , 589, 143) as a template. TBAJ-5307 was modelled by a least square fit procedure on BDQ. The protein-inhibitor complex was inserted into a 1 -palmitoyl-2-oleoyl-sn- glycerol-3-phosphocholine (POPC) bilayer applying the membrane builder module (J. Comp. Chem. 2014, 35, 1997) of the CHARMM graphical user interface (GUI) (J. Comp. Chem. 2008, 29, 1859). Due to the presence of a lipid plug in the internal cavity of the c-ring {FEBS Lett. 2001 , 505, 353), we also modelled a lipid plug into the internal cavity of the c-ring as described previously (J. Phys. Chem. B 2020, 124, 7176). A concentration of 0.15 M NaCI was added after solvation of the system and additional counter ions were inserted. To keep temperature and pressure constant at 300 K and 1 bar, we applied the velocity-rescale thermostat (J. Chem. Phys. 2007, 126, 014101 ) and the Parrinello Rahman barostat (J. Appl. Phys. 1981 , 52, 7182). We simulated two sets of the system: 1) TBAJ-5307 and the key- glutamate (E62) were set charged and 2) TBAJ-5307 and E62 were set neutral. In both simulation setups we charged the E62 residue which is interacting with the essential arginine (R193) in subunit a. We used the CHARMM36m force field for proteins {Nat. Meth. 2017, 14, 71 ), the CHARMM36 force field for lipids {J. Phys. Chem. B2010, 114, 7830), the CHARMM General force field (. Comp. Chem. 2010, 31, 671 ) for TBAJ-5307 and the TIP3P water model (J. Chem. Phys. 1983, 79, 926). Simulations were carried out with GROMACS (version 2018), using a 2-fs integration time step. Electrostatic interactions were calculated with the Particle Mesh Ewald approach applying a real- space cut-off of 12 A. Van-der-Waals interactions were switched after 8 A with a cut-off of 12 A. LINCS (J. Comp. Chem. 1997, 18, 1463-1472) was used to restrain bonds involving hydrogen atoms. All conventional MD simulations were carried out for 20 ns equilibration and 100 ns production time in triplicate.

[0312] Free energy calculations

[0313] We calculated the binding free energy of TBAJ-5307 using the thermodynamics integration (Tl) approach. To prevent solvation of the key-glutamate (J. Biol. Chem. 2012, 287, 36536; J. Mol. Biol. 2010, 395, 20) in its charged state and possible artefacts as a result of this solvation, we used the neutral state for E62 and TBAJ-5307 for these calculations. The coupling parameter A was changed from 0 (drug fully present) to 1 (drug fully absent) using 27 windows. We simulated each window for 1 ns, discarding the first 100 ps as equilibration time. Subsequent analysis was performed with the BAR method (J. Comp. Phys. 1976, 22, 245). As TBAJ-5307 was restrained, an entropic correction was applied. Three independent simulations, using the output structures of the three individual conventional MD runs as an input, were carried out.

[0314] Biofilm Testing

[0315] Biofilm assay was performed in a 24-well plate to evaluate the activity of BDQ and TBAJ-5307 on Pseudomonas aeruginosa PAO1 and Escherichia coli UTI 189 biofilms. Overnight cultures of P. aeruginosa PAO1 and E. coli UTI 189 were diluted in M9 glucose media (1 x M9 salts, 2 mM MgSO4, 0.1 mM CaCIz, 0.4% w / v glucose) to a final OD6oo of 0.05. 1 ml of the diluted culture was then added into each well of the 24-well plate and incubated at 37 °C with 100 rpm shaking. Following 3 h of incubation, each well was washed once, and the culture media was replaced with 1 ml of 1 x PBS (phosphate buffered saline; pH 7.4) or fresh M9 glucose medium containing 1 pM of BDQ or 40 and 120 nM of TBAJ-5307, respectively. The treated samples were further incubated under the same conditions for 3 h. At t = 6 h, the samples were collected. The 1 x PBS (pH 7.4) buffer containing suspended bacteria cells with treatment was collected into 1.5 ml Eppendorf tubes and considered to be “planktonic samples”. Subsequently, each well was washed once with 1 ml 1 x PBS (pH 7.4) before resuspending biofilm cells in the same volume of 1 x PBS (pH 7.4). Biofilm cells were dislodged into the buffer by means of a cell scraper and 1 ml of the sample was collected into 1.5 ml Eppendorf tubes and labelled “biofilm samples”. The samples contained in Eppendorf tubes were sonicated in a water bath using the following settings: 5 min degas mode, 37 Hz, 100%, followed by 5 min pulse mode, 37 Hz, 100%. Subsequently, the samples are serially diluted and used for CFU counts. The experiment was repeated independently at least two times, with two technical replicate per independent experiment. CFU counts were analyzed using Graphpad Prism V9.3.0 using 2-way ANOVA and multiple comparison of column effect (concentration of compound against untreated control) within each row (planktonic vs biofilm samples).

[0316] Results and Discussions

[0317] ATP formation of inverted-membrane vesicles (IMVs) of M. abscessus subsp. abscessus R variant was measured and it resulted in an IC50 of 1 .4 ± 0.5 nM (FIG. 6D), which is comparable to the IC50 observed for the whole cell assay described above (7 ± 0.3 nM; Table 1). This indicates, that TBAJ-5307’s high potency of M. abscessus growth inhibition correlates with the inhibition of Mab’s F-ATP synthase.

[0318] To explore the inhibitory potency at an energetic level and to understand the mechanism of action, we also performed molecular dynamics (MD) simulations and free energy calculations as successfully done for BDQ- and TBAJ-876-binding to the M. smegmatis Fo-domain {Curr. Res. Struct. Biol. 2022, 4, 278). Firstly, the structure of the Mab Fo- domain was modelled based on the cryo-EM structure of M. smegmatis Fo-part {Nature 2021 , 589, 143), TBAJ-5307 was fitted on BDQ, which was resolved in the M. smegmatis structure (Protein Data Bank ID (PDB-ID): 7JGC) {Nature 2021 , 589, 143). While the c-ring residues are conserved between M. smegmatis and the three M. abscessus subspecies, differences in subunit a at the leading- and lagging site can be observed (FIG. 4), which in part may explain the 100-times lower potency of BDQ and TBAJ-876 against M. abscessus when compared to M. smegmatis {Antimicrob. Agents Chemother. 2020, 64, e02404; Antimicrob. Agents Chemother. 2017, 61, e01225-17). The MD simulations revealed the binding site of TBAJ-5307 with emphasis on the lagging and leading sites, since they contribute mainly to the proposed inhibitory mechanism when compared to the c-ring only site {Nature 2021 , 589, 143). TBAJ-5307 interacts with the M. abscessus c-ring residues cA25, cV58, cG59, cL60, cE62, cA63, cA64, cY65, cF66, cl67 and cL69 and subunit a residues aV174, aF177, all 78 and all 81 for the lagging site (FIG. 8), while in the leading site residues cA25, cV58, cG59, cL60, cE62, cA63, cA64, cY65, cF66, cl67, cL69 and residues aA218, aP219, al222, aW223 and aF226 interact with the inhibitor (FIG. 9 and Table 2). Interestingly, the M. abscessus specific residues aF177 and all 81 of the lagging site (aP172 and aV176 in M. smegmatis) form hydrophobic interactions with the 2,3-dimethoxy-pyridin-4-yl moiety of TBAJ-5307 (FIG. 8) and contribute to the strong binding of the agent, which hinders rotation of the c-ring turbine and movement of the proton to residue aR193 for further translocation via the second half-channel to the cytoplasmic side, a requirement for ATP formation. In comparison, the 2,6-dimethoxy-pyridin- 4-yl moiety exhibits hydrophobic contacts with the M. abscessus specific amino acids aA218 and al222 (aF213 and aV217 in M. smegmatis) in the leading site (FIG. 9). Table 2. Mean minimum distance of the M. abscessus Fo-domain residues to TBAJ-5307 during simulations. Distances are shown for TBAJ-5307 bound to each site (leading, lagging and c-ring only) when drug and key-glutamate are charged or protonated. Distances are reported in A. For each protonation state, data were calculated over a total of three replicates of 100 ns.

[0319] Free energy calculations resulted in a binding free energy of -9.1 ± 2.7 kcal / mol compared to -12.0 kcal / mol derived from experimental data (FIG. 6D) for the lagging site; the off-set is similar to that derived from our previous work for strong BDQ binding to M. smegmatis {Curr. Res. Struct. Biol. 2022, 4, 278). The free energy calculations are thus in reasonable agreement with the experimental data, thereby confirming the accuracy of the modelled complex. The offset in free energy likely results from the modelled neutral state of the compound and the key cE62; the alternative charged form would lead to artefactual membrane solvation, as discussed previously (J. Mol. Biol. 2010, 395, 20). For the leading site, we obtained a binding free energy of -9.8 ± 3.8 kcal / mol and thus predict a comparable inhibition of the rotation for the leading site.

[0320] To further confirm TBAJ-5307’s target specificity and to demonstrate that the molecule does not affect human gut biofilm formation or planktonic bacteria, the compound was assayed against Pseudomonas aeruginosa PAO1 or Escherichia coli UTI 189. As revealed in FIG. 10, no killing of planktonic or biofilm cells were observed upon addition of 9x and 26x MIC50 (40 and 120 nM) of TBAJ-5307 to batch biofilms of P. aeruginosa PAO1 or E. coli UTI 189 under both nutrient limited conditions, which was simulated by treatment in phosphate buffered saline (PBS), and under growth conditions, which is simulated by addition of fresh growth media. These results confirm TBAJ-5307’s target specificity and underlines that the compound is not a broad-spectrum antibiotic.

[0321] Example 4. Ex Vivo Anti-Mab Potency of TBAJ-5307

[0322] To explore anti- / Wab potency of the compound in macrophages, a THP-1 infection model was used. Macrophages were infected with M. abscessus subsp. abscessus S- and R variants and treated with TBAJ-5307 in a range of 5 nM to 2,500 nM.

[0323] Macrophage Experiments

[0324] THP-1 monocytes were grown in Roswell Park Memorial Institute (RPMI) medium (BD Gibco) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, Saint Quentin Fallavier, France) (RPMIFBS) and incubated at 37 °C with 5% CO2. Human monocytes were differentiated in macrophages with Phorbol 12-myristate 13-acetate (PMA) at 20 ng / ml for 48 h at 37 °C in the presence of 5% CO2. The intracellular activity of TBAJ-5307 was assessed on M. abscessus S or R-infected THP-1 cells. Each well of a 24-well microplate was seeded with 105 differentiated cells. Infection with each variant of M. abscessus was carried out at 37 °C with 5% CO2 for 4 h at a MOI 2:1 . After washes with PBS, an incubation of 2 h with 250 pg / ml of amikacin was performed to eliminate remaining extracellular bacilli prior to the addition of 500 pl of RPMIFBScontaining either TBAJ-5307 (5 nM; 50 nM; 250 nM; 2500 nM), rifabutin (RFB; 15 pM) or DMSO (negative control). Drugs were renewed on a daily basis and lysed with 100 pl of 1 % Triton X-100 after 1 day or 3 days post-infection. Colonies were counted onto LB agar plates to determine intracellular CFU.

[0325] For microscopy-based macrophages infectivity assays, THP-1 cells were prepared and treated (TBAJ-5307 -250 nM or 2500 nM-, RFB -15 pM- or not) in the same manner as described above, grown on coverslips and infected with M. abscessus expressing tdTomato. Cells were fixed at 3 days post-infection with 4% paraformaldehyde in 1x PBS for 20 min. Macrophages were then permeabilized using 0.2% Triton X-100 for 20 min, blocked with 2% BSA in 0.2% Triton X-100. The surface and the endolysosomal system of the macrophages were detected using anti-CD63 antibodies (Beckton Dickinson, dilution 1 :1000) incubated for 1 h and an Alexa Fluor 488- conjugated antimouse secondary antibody (Molecular Probes, Invitrogen, dilution 1 : 1000) incubated for 45 min. The nuclei were stained with DAPI for 5min (dilution 1 :1000). Cells were then mounted on microscope slides with Immu-mount (Calbiochem) and representative immune- fluorescent fields were captured on a Zeiss Axioimager upright microscope equipped with 63x oil objective and processed using ImageJ software. The data were analyzed using the Student’s unpaired two-tailed ttest.

[0326] Results and Discussions

[0327] FIG. 11 A demonstrate that TBAJ-5307 was active against both morphotypes, reflected in decrease in viable bacterial count (CFU / ml) when compared with initial inoculum at 250 and 2500 nM (day 0). Drug-treated macrophages did not show any optical alteration of the membrane, cell form or size as tested microscopically (FIG. 1 1 B). Immunofluorescence imaging also showed reduced numbers of infected macrophages and cords (for M. abscessus R) during treatment with TBAJ-5307 (FIG. 1 1 B), as reported earlier with rifabutin (Antimicrob. Agents Chemother. 2017, 61, e01225-17), in agreement with the reduced intracellular bacterial burden (FIG. 1 1 A).

[0328] Example 5. In Vivo Potency and Non-Toxicity of TBAJ-5307

[0329] In vivo potency and toxicity of TBAJ-5307 has been studied in the zebrafish model of infection, which have arisen as alternative due to the advantages of rapid development, easy and fast chemical administration, visualizing, high-throughput capabilities (Today 2008, 13, 394; Anna. Rev. Pharmacol. Toxicol. 2012, 52, 433) and the possibility to study the dynamics of the infection in the presence of the active compound (Antimicrob. Agents Chemother. 2017, 61, e01225-17).

[0330] Assessment of BDO Efficacy in Infected Zebrafish

[0331] Rough M. abscessus CIP104536T(ATCC 19977T) carrying the pTEC27 (plasmid 30182; Addgene) and expressing the red fluorescent protein tdTomato was prepared and microinjected intravenously into zebrafish embryos, according to procedures described earlier (Antimicrob Agents Chemother 2014, 58, 4054; J Vis Exp 2015, 103, e53130). Zebrafish Infection Experiments

[0332] Experiments were done with M. abscessus CIP104536T(R morpphotype) expressing tdTomato, obtained after transformation with the replicative plasmid pTEC27 (Proc. Natl. Acad. Sci. USA 2014, 111, E943). At 24 h post-fertilization (hpf), embryos were dechorionated manually using tweezers. At 30 hpf, embryos were anesthetized with 200 pg / ml tricaine followed by caudal vein microinjection of 3 nl of bacterial suspension (approximately 80 CFU / nl). The size of the inoculum was verified a posteriori by injecting 3 nl of each bacterial suspension in sterile PBS and plated on 7H10 +). To monitor embryo survival, infected larvae were transferred into 24-well plates (four embryos / well) and incubated at 28.5 °C. Survival curves were determined by counting dead larvae at a daily basis for up to 1 1 days, with the experiment concluded when uninfected embryos started to die. Dead embryos were determined based on the absence of a heartbeat and removed from the well. TBAJ-5307 treatment of infected embryos and uninfected larvae was started at 1 dpi (day post-infection) for 4 days. The drug-containing solution was renewed daily and wash twice at 5 dpi. Statistical analyses were performed on Prism 9.5.1 (Graphpad, La Jolla, CA, USA) and detailed in each figure legend.

[0333] Zebrafish Care and Ethics Statements

[0334] All zebrafish experiments were approved by the Direction Sanitaire et Veterinaire de I’Herault for the ZEFIX-CRBM zebrafish facility (Montpellier) (registration number C-34-172-39). Handling and experiments were approved by “le ministere de I’enseignement superieur, de la recherche et de I’innovation” under the reference APAFIS#24406-2020022815234677 V3. Experiments were done using the golden mutant (Science (New York, N. Y.) 2005, 310, 1782) crossed with wild-type AB zebrafish, maintained as described earlier (J. Vis. Exp. 2015, 103, pp.e53130).

[0335] Microscopy and image analysis

[0336] In order to visualize the infection foci, infected larvae were anesthetized in 0.02% tricaine solution as previously described (Antimicrob. Agents Chemother. 2014, 58, 4054), mounting on 3% (w / v) methylcellulose solution and taking fluorescent images using a Zeiss Axio Zoom.V16 coupled with an Axiocam 503 mono (Zeiss). Fluorescence Pixel Count (FPC) measurements were determined using the ‘Analyse particles’ function in ImageJ. All experiments were completed at least three times independently.

[0337] Results and Discussions

[0338] By analogy with the protocol developed for BDQ (Antimicrob. Agents Chemother. 2017, 61, e01225-17), the activity of TBAJ-5307 against M. abscessus infection in zebrafish larvae was tested using red fluorescent tdTomato- expressing M. abscessus subsp. abscessus (R variant), which was injected in the caudal vein of embryos at 30 h postfertilization (hpf) and transferred to 24-well plates. TBAJ-5307 was then directly added at 1 -day post-infection (dpi) to the water containing the infected zebrafish, and the TBAJ-5307 supplemented water was then changed daily for 4 days. Non-infected embryos were first exposed to increasing concentrations of TBAJ-5307 and observed under a microscope. No signs of toxicity-induced killing or developmental abnormalities were recorded in the range of concentrations tested (FIG. 12A). When infected embryos were exposed for 4 days to 2,500 nM TBAJ-5307, a significant increased survival rate (80%) was observed compared to the untreated group of embryos (FIG. 12B). Decrease in the physiopathological signs of the TBAJ-5307-treated larvae was corroborated by whole-embryo imaging (FIG. 13A). This indicates that TBAJ-5307 is very efficient in vivo against infection and protects the zebrafish from killing by M. abscessus.

[0339] We next monitored the kinetics of the bacterial burden upon intravenous infection of embryos by determination of fluorescent pixel counts (FPC) at 3 and 5 dpi in the presence or absence of TBAJ-5307. The addition of 2,500 nM TBAJ-5307 for 4 days in fish water was accompanied by a significant reduction in the FPCs at both time points (FIG. 12C). Importantly, our results indicate that at both 3 and 5 dpi, treatment with TBAJ-5307 coincided with a significant reduction in the number of infection foci (FIG. 12D). Whole embryo imaging at 3 and 5 dpi clearly highlighted the effect of the compound, with limited infection, particularly evident in the presence of 2,500 nM TBAJ-5307 (FIG. 12E), consistent with bacterial burden quantification (FIG. 12C). This pronounced effect of the compound in reducing the bacterial burden and infection foci is likely to explain the protective efficacy of TBAJ-5307 on embryo survival.

[0340] Abscess formation can be linked to loss of infection control, typically occurring following cord formation and expansion and representing a marker of disease severity {Proc. Natl. Acad. Sci. USA 2014, 111, E943; PLoS pathogens 2016, 12, e1005986; J. Vis. Exp. 2015, 703, e53130), associated with cell debris, tissue destruction and acute infection. We note that treatment with TBAJ-5307 was associated with a decrease in abscess formation and cording (FIG. 12E and FIG. 13B). This indicates that TBAJ-5307 is very efficient in vivo against infection and protects the zebrafish from killing by the pathogen M. abscessus subsp. abscessus.

[0341] Example 6. TBAJ-5307 Enhances Potency of Existing Anti- Mab Drug Candidates

[0342] Since leveraging the potential of synergistic drug interactions in multidrug treatment regimens to accelerate durable cure is a growing field and the treatment of M. abscessus infections requires drug combinations, the growth inhibition activity of TBAJ- 5307 along with clinical M. abscessus antibiotics CFZ, amikacin and the RNA polymerase inhibitor rifabutin were measured using a checker-board titration assay.

[0343] Checker-Board Titration Assay

[0344] A checkerboard titration assay was carried out as described previously (Diagn. Microbiol. Inf. Dis. 1993, 16, 343; Antimicrob. Agents Chemother. 2015, 59, 6561 ). Briefly, TBAJ-5307 and CFZ / Rifabutin / Amikacin were added to complete 7H9 medium-containing 96-well flat-bottom Costar cell culture plates. Two-fold serial dilutions were done to allow 10 different concentrations of TBAJ-5307 (40 nM to 0.03 nM) to be tested for interaction with 7 different concentrations of CFZ (27.2 pM to 0.2 pM). Hence, a total of 70 different concentration combinations were tested between TBAJ-5307 and CFZ. In combination studies with Rifabutin and Amikacin, 10 different concentrations of TBAJ-5307 (40 nM to 0.03 nM) were tested with 7 different concentrations of Rifabutin (18.8 pM to 0.2 pM) and Amikacin (10 pM to 0.15 pM). Each 96-well plate had a 7H9 medium-only control well and a drug-free bacterial culture control well. M. abscessus subsp. abscessus R variant was cultured in complete 7H9 medium and grown to mid-exponential phase. Subsequently, the culture was diluted to an OD6oo of 0.01 using complete 7H9 medium and added to each well in the 96-well plate to create a final ODeoo value of 0.005. The plates were incubated for 4 days at 37 °C. After the incubation period, the culture in each 96-well plate was manually resuspended, and the ODSoo of each well was read using a Tecan Infinite Pro 200 plate reader.

[0345] In order to assess the combined effect of TBAJ-5307 and TBP / AVI, we performed checkerboard analyses using M. abscessus ATCC 19977 and a 96-well plate format was employed. The optical density at 600 nm (OD6oo) was measured as an indicator of growth. We evaluated the impact of varying concentrations of TBP in a serial dilution, spanning from 12.5 to 0.012 pM, in combination with a fixed concentration of 14 pM AVI. TBAJ-5307 were tested at doses ranging from 10 pM to 0.01 nM.

[0346] Calculation of the fractional inhibitory concentration index (FIGI) was done to analyze the results. The FICI is calculated as (MIC of drug A in combination / MIC of drug A alone) + (MIC of DARQ B in combination / MIC of DARQ B alone). This calculation was done only for wells which showed 50% inhibition of bacterial culture growth compared to drug-free bacterial culture wells. A FICI of <0.5 indicates synergy, a FICI of >0.5 to 4 indicates additivity (no interaction), and a FICI of >4 indicates antagonism (F. C. Odds, J. Antimicrob. Chemoth. 2003, 52, 1 ). Results and Discussions

[0347] Drastic reduction of cell growth was observed with all three drugs tested (FIG. 14A-C). The fractional inhibitory concentration index (FICI) was calculated to characterize the interaction between TBAJ-5307 and each of the test drugs (F. C. Odds, J. Antimicrob. Chemoth. 2003, 52, 1 ). FIG. 15 and Table 3 show an additive / partial synergistic growth reduction triggered by CFZ or Rifabutin with TBAJ-5307, achieving a FICI of 0.75 and 0.77, respectively in M. abscessus subsp. abscessus (Table 3). The results demonstrate that TBAJ-5307 enhances the potency of major antibiotics against M. abscessus.

[0348] Table 3. The FICI was calculated as (MIC of drug A in combination / MIC of drug A alone) + (MIC of drug B in combination / MIC of drug B alone). An FICI of <0.5 indicates synergy, a FICI of >0.5 to 4 indicates additivity (no interaction), and an FICI of >4 indicates antagonism.

[0349] MIC FICIbOutcome

[0350] „ . . . Combination

[0351] I U9with Drug B Drug A Drug A in Drug B Drug B in alone combination alone combination

[0352] TBAJ-5307 Clofazamine 12 nM 5 nM 2.5 pM 0.85 pM 0.75 Additivity

[0353] TBAJ-5307 Rifabutin 12 nM 2.5 nM 1.5 pM 0.5 pM 0.77 Additivity

[0354] Treatment of M. abscessus lung diseases by the aminoglycoside amikacin or the p-lactams imipenem and cefoxitin display modest in vitro activity and is administered by the intravenous route. Given the expense and risks of intravenous therapies, more potent oral combinations would provide huge advantages. Recently the combination of the oral p-lactam TBP, targeting the L,D-transpeptidases LdtMabl and LdtMab2 and D,D-transpeptidases PonA1 , PonA2, and PbpA, which are essential for peptidoglycan synthesis, with the M. abscessus p- lactamase inhibitor AVI have shown an attractive, low pM and strong cidal activity (FIG. 16) (Antimicrob. Agents Chemother. 2020, 65, e01956-20; Antimicrob. Agents Chemother. 2020, 64, e00098-20; Antimicrob. Agents Chemother. 2022, 66, e0079022). Here we tested whether inhibiting the fundamental cell processes of peptidoglycan synthesis in cell wall formation and silencing of the essential mycobacterial enzyme F-ATP synthase by TBAJ-5307 provide a potent combination. As shown in FIG. 14D, combining the TBP-AVI pair with TBAJ-5307 revealed enhanced growth inhibition. The efficacy of TBAJ-5307 increased by a factor of 14 (MIC50 of 0.7 nM) in combination of TBP-AVI. Importantly, TBAJ-5307 does not exert any observable antagonism in killing activity with bactericidal TBP-AVI (FIG. 17), suggesting that TBAJ-5307 could be co-administered with the dual drug. Example 7. TBAJ-5307 is a Broad Spectrum Anti-NTM Antibiotic

[0355] We also explored TBAJ-5307’s efficacy against the NTM fast growers M. mucogenicum, M. fortuitum, M. peregrinum, and M. chelonae, as well as against the slow growers M. avium, M. intracellulare, and M. kansasii. Together with the M. abscessus complex the Mycobacterium avium complex, including M. avium, M. intracellulare, and M. kansasii, are responsible for most cases of pulmonary infections worldwide {Drug Disc. Today 2018, 23, 1502; J Vis Exp 2015, 103, e53130).

[0356] Results and Discussions

[0357] Here we reveal that TBAJ-5307 is highly potent against M. avium with an MIC50 value of 1 .8 ± 0.2 nM, which is 100- and 36-times better than BDQ (184 ± 23 nM) and TBAJ-876, respectively (64 ± 4.8 nM; Table 1 , FIG. 18A). It also inhibits growth of the clinical isolate M. avium 1 1 {Genome Announc. 2017, 5, e00388; Genome Announc. 2017, 5, e00766) with an MIC50 of 31 ± 2.8 nM (FIG. 19A, Table 1 ). Whole cell ATP synthesis studies confirmed that growth inhibition of TBAJ-5307 directly correlated with depletion of ATP and that TBAJ-5307 (IC50 = 12 ± 4 nM) is the strongest inhibitor, when compared to BDQ (IC50 = 267 ± 30 nM) and TBAJ- 876 (IC50 = 210 ± 25 nM) (Table 1 , FIG. 18B). TBAJ-5307 showed slightly better growth inhibition against M. intracellulare (16 ± 3.2 nM) when compared to BDQ (60 ± 2.8 nM) and TBAJ-876 (30 ± 2.5 nM) (Table 1 , FIG. 19B), which is caused by the stronger inhibitory effect of TBAJ-5307 in ATP formation (6.7 ± 2.1 nM; Table 1 , FIG. 19C).

[0358] Growth and whole cell ATP synthesis of the fast grower M. mucogenicum, causing respiratory-, central nervous-, catheter- related-, skin and soft tissue infections {Annals Americ. Thoracic Soc. 2020, 17, 178), was inhibited by TBAJ-5307 with an MIC50 of 1.2 nM ± 0.4 nM and an IC50 of 3.1 ± 0.9 nM, respectively (FIG. 19D-E). As pointed out above, the M. abscessus complex, the M. avium complex and M. fortuitum are reported as the most frequently isolated NTMs in Singapore {BMC Pulmonary Med. 2018, 18, 85). As displayed in FIG. 19F, TBAJ-5307 is also potent against M. fortuitum (MIC50 values of 0.9 ± 0.1 nM), which is mainly isolated from patients with skin and soft tissue infection {Sci. Rep. 2021 , 11, 13011 ), and appears slightly more potent compared to TBAJ-876 (1.7 ± 0.2 nM) or BDQ (8.3 ± 7 nM), confirming that TBAJ-5307 is also very potent against the pathogen M. fortuitum and even more potent compared to TBAJ-876 and BDQ.

[0359] The data demonstrate that TBAJ-5307 is a highly potent anti-NTM agent covering the broad range of fast- and slow NTM growers by targeting the F1 F0-ATP synthase, which converts ADP and Pi to the biological currency ATP. The data also show for the first time, that TBAJ-876, shown to be active against the M. abscessus complex (Antimicrob. Agents Chemother. 2020, 64, e02404), displays attractive in vitro activity against mucogenicum, M. fortuitum, M. avium and M. intracellulare, although with lower potency compared to TBAJ-5307. We also studied TBAJ-5307’s efficacy against the NTM fast growers Mycobacterium peregrinum and Mycobacterium chelonae), opportunistic pathogens mainly isolated from patients with skin and soft tissue infection {Front Immunol 2020, 11, 303; Pathogens 2023, 12, 1446; Akram, S. M., Rathish, B. & Saleh, D. (2024) Mycobacterium chelonae Infection. [Updated 2023 Aug 8]. In: StatPearls [Internet], Treasure Island (FL): StatPearls Publishing; 2024 Jan. Available from: https: / / www.ncbi.nlm.nih.gov / books / NBK430806 / ). As displayed in

[0360] FIG. 20A, TBAJ-5307 is potent against M. peregrinum (MIC50 7.1 ± 0.9 nM and M. chelonae (MIC50 38 ± 7 nM, Table 1 ). Whole cell ATP synthesis inhibition by the compound was in line with growth reduction of M. peregrinum (IC50 of 3.8 ± 0.8 nM, FIG. 20B) and M. chelonae (IC50 of 20 ± 2 nM, FIG. 20B). We also reveal that TBAJ-5307 is potent against the slow grower M. kansasii, one of the most pathogenic and common NTMs isolated from humans worldwide (Johnston, J. C., Chiang, L. & Elwood K. (2017) Mycobacterium kansasii. Microbiol. Spectr. 5). The compound is highly potent, displaying an MIC50 value of 1.4 ± 0.4 nM (FIG. 20A and C) and an IC50 of 1 .7 ± 0.2 nM (FIG. 20B).

Claims

Claims1. One or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, for use in the treatment of nontuberculous mycobacterial (NTM) infection, wherein each of the one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, are a compound according to formula (I):wherein:X represents CH or N;Y represents C-OR3 or N;R1 represents H or OR4;R2 represents Ci to Ce alkyl;R3, when present, represents Ci to Ce alkyl, or R2and R3, together form a Ci to C3 alkylene moiety, which together with the atoms they are attached to provides a 5- to 7-membered ring system;R4, when present, represents C2to C4 alkyl; or a salt or solvate thereof, provided that at least one of X and Y are N.

2. Use of one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, in the manufacture of a medicament for the treatment of nontuberculous mycobacterial (NTM) infection, wherein each of the one ormore 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, are a compound according to formula (I):wherein:X represents CH or N;Y represents C-OR3 or N;R1 represents H or OR4;R2 represents Ci to Cs alkyl;R3, when present, represents Ci to Ce alkyl, or R2and R3, together form a Ci to C3alkylene moiety, which together with the atoms they are attached to provides a 5- to 7-membered ring system;R4, when present, represents C2to C4 alkyl; or a salt or solvate thereof, provided that at least one of X and Y are N.

3. A method of treating nontuberculous mycobacterial (NTM) infection comprising the step of administering to a subject in need thereof one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, wherein each of the one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, are a compound according to formula (I):wherein:X represents CH or N;Y represents C-OR3 or N;R1 represents H or OR4;R2represents Ci to C6alkyl;R3, when present, represents Ci to Ce alkyl, or R2and R3, together form a Ci to C3 alkylene moiety, which together with the atoms they are attached to provides a 5- to 7-membered ring system;R4, when present, represents C2to C4 alkyl; or a salt or solvate thereof, provided that at least one of X and Y are N.

4. A composition comprising one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, for use in the treatment of nontuberculous mycobacterial (NTM) infection, wherein each of the one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, are a compound according to formula (I):wherein:X represents CH or N;Y represents C-OR3 or N;R1 represents H or OR4;R2represents Ci to C6alkyl;R3, when present, represents Ci to Ce alkyl, or R2and R3, together form a Ci to C3 alkylene moiety, which together with the atoms they are attached to provides a 5- to 7-membered ring system;R4, when present, represents C2to C4 alkyl; or a salt or solvate thereof, provided that at least one of X and Y are N.

5. Use of a composition comprising one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, in the manufacture of a medicament for the treatment of nontuberculous mycobacterial (NTM) infection, wherein each of the one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, are a compound according to formula (I):wherein:X represents CH or N;Y represents C-OR3 or N;R1 represents H or OR4;R2represents Ci to C6alkyl;R3, when present, represents Ci to Ce alkyl, or R2and R3, together form a Ci to C3 alkylene moiety, which together with the atoms they are attached to provides a 5- to 7-membered ring system;R4, when present, represents C2to C4 alkyl; or a salt or solvate thereof, provided that at least one of X and Y are N.

6. A method of treating nontuberculous mycobacterial (NTM) infection comprising the step of administering to a subject in need thereof a composition comprising one or more 3,5- dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, wherein each of the one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, are a compound according to formula (I):wherein:X represents CH or N;Y represents C-OR3 or N;R1 represents H or OR4;R2represents Ci to C6alkyl;R3, when present, represents Ci to Ce alkyl, or R2and R3, together form a Ci to C3 alkylene moiety, which together with the atoms they are attached to provides a 5- to 7-membered ring system;R4, when present, represents C2to C4 alkyl; or a salt or solvate thereof, provided that at least one of X and Y are N.

7. The composition according to Claim 4, the use according to Claim 5 or the method according to Claim 6, further comprising one or more pharmaceutically acceptable excipients.

8. The one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof according to Claim 1 , the use according to Claim 2, the method according to Claim 3, the composition according to Claim 4 or Claim 7, the use according to Claim 5 or Claim 7, or the method according to Claim 6 or Claim 7 wherein the DARQ analogue is one where one or more of the following apply:(i) R4, when present, represents C2to C3 alkyl;(ii) R2represents Ci to C3alkyl; and(iii) when Y represents COR3, then R3represents Ci to C3 alkyl, or R2and R3 together represent -CH2- or -(CH2)2-9. The one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, the use, the method, the composition, the use, or the method according to Claim 8 wherein the DARQ analogue is one where one or more of the following apply:(ia) Ri represents H or O'Pr;(iia) R2represents CH3; and(iiia) when Y represents COR3, then R3represents CH3, or R2and R3together represent -(CH2)2-10. The one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof according to any one of Claim 1 and Claims 8 and 9, the use according to any one of Claim 2 and Claims 8 and 9, the method according to any one of Claim 3 and Claims 8 and 9, the composition according to any one of Claim 4 or Claims 7 to 9, the use according to any one of Claim 5 or Claims 7 to 9, or the method according to any one of Claim 6 or Claims 7 to 9 wherein the DARQ analogue is selected from the group consisting of:(±)-TBAJ-530711 . The one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof according to any one of Claim 1 and Claims 8 and 9, the use according to any one of Claim 2 and Claims 8 and 9, the method according to any one of Claim 3 and Claims 8 and 9, the composition according to any one of Claim 4 or Claims 7 to 9, the use according to any one of Claim 5 or Claims 7 to 9, or themethod according to any one of Claim 6 or Claims 7 to 9 wherein the DARQ analogue is selected from the group consisting of:(±)-TBAJ-5316 ; andwhere the compounds are provided in an enantiomerically enriched form based on the enantiomer shown.

12. The one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof according to any one of Claim 1 and Claims 8 and 9, the use according to any one of Claim 2 and Claims 8 and 9, the method according to any one of Claim 3 and Claims 8 and 9, the composition according to any one of Claim 4 or Claims 7 to 9, the use according to any one of Claim 5 or Claims 7 to 9, or the method according to any one of Claim 6 or Claims 7 to 9 wherein the DARQ analogue is:or a pharmaceutically acceptable salt or solvate thereof which is provided in an enantiomerically enriched form based on the enantiomer shown.

13. A composition comprising one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, for use in the treatment of nontuberculous mycobacterial (NTM) infection, wherein each of the one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, are a compound according to formula (I):wherein:X represents CH or N;Y represents C-OR3 or N;R1 represents H or OR4;R2 represents Ci to Ce alkyl;R3, when present, represents Ci to Ce alkyl, or R2and R3, together form a Ci to C3 alkylene moiety, which together with the atoms they are attached to provides a 5- to 7-membered ring system;R4, when present, represents C2to C4alkyl; or a salt or solvate thereof, provided that at least one of X and Y are N, and a second antibiotic, optionally wherein the composition further comprises one or more pharmaceutically acceptable excipients.

14. A composition according to Claim 13 for use in the treatment of nontuberculous mycobacterial (NTM) infection.

15. Use of a composition according to Claim 13 in the manufacture of a medicament to treat a nontuberculous mycobacterial (NTM) infection.

16. A method of treating nontuberculous mycobacterial (NTM) infection comprising the step of administering to a subject in need thereof a composition according to Claim 13.

17. The composition according to Claim 13 or Claim 14, the use according to Claim 15, or the method according to Claim 16, wherein the second antibiotic is selected from the group consisting of Clofazimine, Rifabutin and Amikacin, or a pharmaceutically acceptable salt or solvate thereof.

18. A composition comprising one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, for use in the treatment of nontuberculous mycobacterial (NTM) infection, wherein each of the one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof, are a compound according to formula (I):wherein:X represents CH or N;Y represents C-OR3 or N;R1 represents H or OR4;R2 represents Ci to Ce alkyl;R3, when present, represents Ci to Ce alkyl,or R2and R3, together form a Ci to C3alkylene moiety, which together with the atoms they are attached to provides a 5- to 7-membered ring system;R4, when present, represents C2to C4alkyl; or a salt or solvate thereof, provided that at least one of X and Y are N. for use in the treatment of nontuberculous mycobacterial (NTM) infection, wherein the composition is to be administered sequentially, simultaneously or concomitantly with a second antibiotic.

19. Use of a composition according to Claim 18 in the manufacture of a medicament to treat a nontuberculous mycobacterial (NTM) infection wherein the composition is to be administered sequentially, simultaneously or concomitantly with a second antibiotic.

20. A method of treating nontuberculous mycobacterial (NTM) infection comprising the step of administering to a subject in need thereof a composition according to Claim 18 in combination with a second antibiotic, wherein the composition is administered sequentially, simultaneously or concomitantly with the second antibiotic.21 . The composition according to Claim 18, the use according to Claim 19, or the method according to Claim 20, wherein the second antibiotic is selected from the group consisting of Clofazimine, Rifabutin and Amikacin, or a pharmaceutically acceptable salt or solvate thereof.

22. The composition according to Claim 13 or Claim 17, the composition for use according to any one of Claims 14, 17, 18 or 21 , the use according to any one of Claims 15, 17, 19 or21 , or the method according to any one of Claims 16, 17, 20 or 21 , wherein the DARQ analogue is one where one or more of the following apply:(i) R4, when present, represents C2to C3alkyl;(ii) R2represents Ci to C3alkyl; and(iii) when Y represents COR3, then R3represents Ci to C3alkyl, or R2and R3together represent -CH2- or -(CH2)2-23. The composition, the composition for use, the use, the method, according to Claim 22 wherein the DARQ analogue is one where one or more of the following apply:(ia) Ri represents H or O'Pr;(iia) R2represents CH3; and(iiia) when Y represents COR3, then R3represents CH3, or R2and R3together represent -(CH2)2-4. The composition according to Claim 13 or Claim 17, the composition for use according any one of Claims 14, 17, 18 or 21 , the use according to any one of Claims 15, 17, 19 or1 , or the method according to any one of Claims 16, 17, 20 or 21 wherein the DARQ analogue selected from the group consisting of:

5. The composition according to Claim 13 or Claim 17, the composition for use according any one of Claims 14, 17, 18 or 21 , the use according to any one of Claims 15, 17, 19 or1 , or the method according to any one of Claims 16, 17, 20 or 21 wherein the DARQ analogue selected from the group consisting of:where the compounds are provided in an enantiomerically enriched form based on the enantiomer shown.

26. The composition according to Claim 13 or Claim 17, the composition for use according to any one of Claims 14, 17, 18 or 21 , the use according to any one of Claims 15, 17, 19 or 21 , or the method according to any one of Claims 16, 17, 20 or 21 wherein the DARQ analogue is:or a pharmaceutically acceptable salt or solvate thereof which is provided in an enantiomerically enriched form based on the enantiomer shown.

27. The composition according to Claim 4, the use according to Claim 5 or the method according to Claim 6, further comprising one or more pharmaceutically acceptable excipients.

28. The one or more 3,5-dialkoxypyridine (DARQ) analogues of bedaquiline (BDQ), or a pharmaceutically acceptable salt or solvate thereof according to any one of Claims 1 and 8 to 12, the use according to any one of Claims 2, 5, 7, 8 to 12 15, 17, 19 and 21 -23, the method according to any one of Claims 3, 6 to 12, 16, 17, 20 and 21-23, the composition according to any one of Claims 4, 7 to 12, 13 and 17, or the composition for use according to any one of Claims 5, 7, 14, 17, 19 or 21 -23 wherein the NTM infection is caused by one or more of the group consisting of M. chelonae, M. peregrinum, M. kansasii, and more particularly, M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, M. abscessus subsp. massiliense, M. fortuitum, M. avium and M. intracellulare.

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