Inhibitor of the f-ATP synthase for the treatment of nontuberculous mycobacteria diseases

Abstract

Disclosed herein are compounds of formula I, where R1; R2, X, Y and n are defined herein. The compounds of formula I and their pharmaceutical formulations particularly relate to inhibitors of the F-ATP synthase and their medical uses for the treatment of nontuberculous mycobacterial diseases such as mycobacterium avium, mycobacterium peregrinum, mycobacterium kansasii, mycobacterium marinum and mycobacterium chelonae.

Description

[0001] INHIBITOR OF THE F-ATP SYNTHASE FOR THE TREATMENT OF NONTUBERCULOUS MYCOBACTERIA DISEASES

[0002] Field of Invention

[0003] The present invention generally relates to inhibitors for the treatment of nontuberculous mycobacteria diseases, and more particularly relates to inhibitors of the F-ATP synthase for the treatment of nontuberculous mycobacteria diseases.

[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] Nontuberculous mycobacteria (NTM) cause a wide range of serious illnesses, including lymphadenitis, soft and skin tissue infection, cardiac-, bone-, joint and most commonly pulmonary infections. In developed countries, the number of pulmonary infections due to NTM is higher than that of tuberculosis (TB), which is caused by NTM’s cousin Mycobacterium tuberculosis (Mtb). In general, patient numbers are rising globally. NTM species present in municipal water systems have been enriched through decades of broad chlorine use, to which they are resistant, unlike most bacterial species. Natural disasters and global warming have also been proposed as potential drivers of the rising numbers. The cure rate of NTM treatment goes along with high pill burden, long duration, unsatisfactory cure rates, toxicity, the use of injectable agents, and drug-drug interactions. NTM have been grouped into rapid (e.g. Mycobacterium peregrinum, Mycobacterium abscessus) and slow growers (e.g. Mycobacterium, avium, Mycobacterium intracellulare, Mycobacterium kansasii and Mycobacterium marinum).

[0007] Non-fermentative NTM depend on oxidative phosphorylation to generate the biological currency ATP and to sustain their energy requirements under normal and hypoxia conditions. This is underscored by the ATP forming F1FoATP synthase (F-ATP synthase) being essential for mycobacteria and serves as a new anti-Mab drug target. Differences in amino acid composition as well as structural elements allowed specific regulatory control in the functioning of the mycobacterial F-ATP synthase in comparison to its human and bacterial counterparts. These traits paved the way for the design of new inhibitors targeting the central stalk subunits γ-ε and the screen for novel inhibitors, such as GaMF1 and EpMabF1. The anti-TB drugbedaquiline (BDQ), a known inhibitor of the mycobacterial F-ATP synthase, has shown efficacy against NTM and is used as salvage therapy against M. abscessus lung diseases. However, the successful advance of BDQ has been overshadowed by its lipophilicity, leading to a very long terminal half-life and toxicity, potential cardiotoxicity, as well as by results showing BDQ-inhibition of mitochondrial F-ATP synthase in human HEK293S cell mitoplasts and its binding to the human F-ATP synthase as visualized by cryo-EM most recently.

[0008] Therefore, there exists a need for new inhibitors of the F-ATP synthase for the treatment of nontuberculous mycobacteria diseases.

[0009] Summary of Invention

[0010] Aspects and embodiments of the invention are provided in the following numbered clauses.

[0011] 1. A compound according to formula I:

[0012]

[0013] where:

[0014] R1represents C1-5alkyl;

[0015] R2represents H, F or OCF3;

[0016] X and Y independently represent O, S or CH2; and

[0017] n is a number from 1 to 5,

[0018] or a salt or solvate thereof, provided that the compound of formula I is not:

[0019]

[0020] 2. The compound or a salt or solvate thereof, according to Clause 1, wherein R2represents H.

[0021] 3. The compound or a salt or solvate thereof, according to Clause 1 or Clause 2, wherein Ri is CH3 or CH2CH3.

[0022] 4. The compound or a salt or solvate thereof, according to any one of the preceding clauses, wherein n is 2 or 3.

[0023] 5. The compound or a salt or solvate thereof, according to any one of the preceding clauses, wherein X is O or S.

[0024] 6. The compound or a salt or solvate thereof, according to Clause 5, wherein X is O.

[0025] 7. The compound or a salt or solvate thereof, according to any one of the preceding clauses, wherein Y is O or S.

[0026] 8. The compound or a salt or solvate thereof, according to Clause 7, wherein Y is O.

[0027] 9. The compound or a salt or solvate thereof, according to any one of the preceding clauses, wherein:

[0028] R1is CH3or CH2CH3;R2is H;

[0029] n is 2 or 3;

[0030] X is O; and

[0031] Y is O.

[0032] 10. The compound or a salt or solvate thereof, according to Clause 1, wherein the compound of formula I is selected from the list:

[0033]

[0034] and

[0035]

[0036]

[0037] 11. The compound or a salt or solvate thereof, according to Clause 10, wherein the compound of formula I is selected from the list:

[0038] and

[0039]

[0040]

[0041] 12. Use of a compound according to formula I:

[0042]

[0043] where:

[0044] R1represents C1-5alkyl;R2represents H, F or OCF3;

[0045] X and Y independently represent O, S or CH2; and

[0046] n is a number from 1 to 5,

[0047] or a salt or solvate thereof,

[0048] in the manufacture of a medicament for the treatment of a nontuberculosis mycobacterial infection.

[0049] 13. The use according to Clause 12, wherein R2represents H.

[0050] 14. The use according to Clause 12 or Clause 13, wherein Ri is CH3or CH2CH3.

[0051] 15. The use according to any one of Clauses 12 to 14, wherein n is 2 or 3.

[0052] 16. The use according to any one of Clauses 12 to 15, wherein X is O or S, optionally wherein X is O.

[0053] 17. The use according to any one of Clauses 12 to 16, wherein Y is O or S, optionally wherein Y is O.

[0054] 18. The use according to any one of Clauses 12 to 17, wherein:

[0055] Ri is CH3or CH2CH3;

[0056] R2is H;

[0057] n is 2 or 3;

[0058] X is O; and

[0059] Y is O.

[0060] 19. The use according to Clause 12, wherein the compound of formula I is selected from the list:

[0061]

[0062]

[0063]

[0064] 20. The use according to Clause 19, wherein the compound of formula I is selected from the list:

[0065] and

[0066]

[0067]

[0068] 21. A compound according to formula I or a salt or solvate thereof, as described in any one of Clauses 12 to 20 for use in the treatment of a nontuberculosis mycobacterial infection.

[0069] 22. A method of treating a nontuberculosis mycobacterial infection comprising the steps of providing a compound of formula I or or a salt or solvate thereof, as described in any one of Clauses 12 to 20, to a subject in need thereof.

[0070] 23. The use according to any one of Clauses 12 to 20, the compound for use according to Clause 21 or the method according to Clause 22, wherein the nontuberculosis mycobacterial infection is caused by one of more of mycobacterium avium, mycobacterium peregrinum, mycobacterium kansasii, mycobacterium marinum, and mycobacterium chelonae.

[0071] 24. Use of a compound of formula I, or a salt or solvate thereof, as described in any one of Clauses 12 to 20 and a second antibiotic in the manufacture of a medicament for the treatment of nontuberculosis mycobacterial infection, wherein the compound of formula I or a salt or solvate thereof, is to be administered sequentially, simultaneously or concomitantly with the second antibiotic.25. A compound according to formula I or a salt or solvate thereof, as described in any one of Clauses 12 to 20 and a second antibiotic for use in the treatment of a nontuberculosis mycobacterial infection wherein the compound of formula I or a salt or solvate thereof, is to be administered sequentially, simultaneously or concomitantly with the second antibiotic.

[0072] 26. A method of treating a nontuberculosis mycobacterial infection comprising the steps of providing a compound of formula I or or a salt or solvate thereof, as described in any one of Clauses 12 to 20 and a second antibiotic, to a subject in need thereof, wherein the compound of formula I or a salt or solvate thereof, is to be administered sequentially, simultaneously or concomitantly with the second antibiotic.

[0073] 27. The use according to Clause 24, the compound for use according to Clause 25 or the method according to Clause 26, wherein the nontuberculosis mycobacterial infection is caused by one of more of mycobacterium avium, mycobacterium peregrinum, mycobacterium kansasii, mycobacterium marinum, and mycobacterium chelonae.

[0074] 28. The use according to Clause 24, the compound for use according to Clause 25 or the method according to Clause 26, wherein the second antibiotic is selected from one or more of the group consisting of clofazimine, rifabutin and amikacin.

[0075] 29. A formulation comprising a compound according to any one of Clauses 1 to 11, or a pharmaceutically acceptable salt or solvent thereof, and a pharmaceutically acceptable carrier.

[0076] 30. A formulation comprising a compound according to any one of Clauses 1 to 11, or a pharmaceutically acceptable salt or solvent thereof, and an acceptable carrier suitable to prevent growth of mycobacteria in an aqueous environment that is useful for the farming of fish.

[0077] Drawings

[0078] FIG. 1 depicts structural model of the BDQ and M. avium F-ATP synthase. (A) Electrostatic potential surface view of BDQ bound to the M. avium Fo domain with a cartoon view of the F₁-domain, the peripheral stalk domains which holds the F1-Fodomains together (LHS). Shown in middle is the zoomed view of electrostatic potential surface at the leading site of a-c halfchannel which clearly highlight the prominent crevice, which can be utilized to grow the fragment on meta- position(s) (highlighted by arrows) on phenyl (Ar₁) group. 2D-structure of BDQ (RHS). (B) Electrostatic potential surface view of TBAJ-876 bound to M. smegmatis doeshighlight the additional crevices highlighted in yellow dots circles as well as black dotted circles that become available due to F213A substitution (black dotted circle) in M. avium F-ATP synthase. (C) Electrostatic view of binding pose of C118 on M. avium F-ATP synthase. Shown in the middle section is the C118 docked pose which highlights, that the bromo-quinoline core of BDQ scaffold was oriented deeply towards the a-c interface as well as the dimethylamine (DMA) moiety interaction with amino acid E63 of subunit c were intact and maintain the essential interactions as was seen with BDQ / TBAJ-876 analogs. Most importantly, the newly extended 3-propoxy-thio-ethyl fragment (Ar₁ group) gets filled into the additional pocket (black dotted circle). The thio-ethyl fragment of the propoxy group anchored the additional sulphur-TT interactions, alkyl interactions of ethyl moiety with subunit a residues aI210 (4.4 Å), aW212 (4.0 Å) and aP214 that form the crevice on the a-c interface. (RHS) 2D-structure of C118.

[0079] FIG. 2 depicts six step synthesis of C118.

[0080] FIG. 3 depicts synthetic scheme of acNTMF0-1.

[0081] FIG. 4 depicts synthetic scheme of acNTMF0-2.

[0082] FIG. 5 depicts synthetic scheme of acNTMF0-3.

[0083] FIG. 6 depicts synthetic scheme of acNTMF0-4.

[0084] FIG. 7 depicts (A) growth inhibition dose response curve and whole cell ATP synthesis depletion (B) of M. avium (o) in comparison to BDQ (•), and an M. avium clinical isolate (A). Three independent experiments were carried out, each with three technical replicates. The total ATP content is directly proportional to relative luminescence units (RLU). The experiments have been performed in two biological replicates, each with three triplicates replicates triplicates.

[0085] FIG.8 depicts increased potency of C118 in combination with the antibiotics amikacin, rifabutin or CFZ in 7H9 broth. Mav growth inhibition by 0118 in combination with increasing concentrations of Amikacin (A), Rifabutin (B), and CFZ (C). The experiments have been performed in triplicates.

[0086] FIG. 9 depicts (A) growth inhibition dose response curve of M. kansasii and M. peregrinum by 0118. Two independent experiments were carried out, each with three technical replicates. (B) Since M. kansasii shows the characteristic yellow formation of colonies, due to theaccumulation of β-carotene, cell growth inhibition of M. kansasii by C118 was directly observed in 6 well plates in the absence (DF = drug free) or presence of the inhibitor (10X MIC50– 56 ± 0.4 nM) in three wells. The plates were incubated at 37 ºC for 7-10 days. (C) C118 inhibits oxidative phosphorylation of M. kansasii and M. peregrinum in a whole cell ATP synthesis assay (adding bactiter gio (promega). The experiments were performed in triplicates.

[0087] FIG. 10 depicts (A) growth inhibition dose response curve of M. marinum by C118. Two independent experiments were carried out, each with three technical replicates. (B) M. marinum reveals yellow formation of colonies, caused by the accumulation of p-carotene. This allows cell growth inhibition of M. marinum by C118 to visualize in 6-well plates in the absence (DF = drug free) or presence of the inhibitor (10X MIC50– 43 ± 1.5 nM). The plates were incubated at 30 ºC for 7-10 days. (C) C118 inhibits oxidative phosphorylation of M. marinum studied by a whole cell ATP synthesis assay. The experiments were performed in triplicates.

[0088] FIG. 11 depicts three independent experiments were carried out, each with three technical replicates. The total ATP content is directly proportional to relative luminescence units (RLU). The experiments have been performed in two biological replicates, each with three triplicates replicates triplicates.

[0089] FIG. 12 depicts in vivo efficacy of C118 against M. marinum infected zebrafish larvae. (A) Schematic representation of the experimental design. (B) Groups of 24 uninfected embryos were immersed in water containing increasing concentrations of C118 (ranging from 3.5 to 14 pM) for 4 days. The orange bar indicates the duration of treatment. The graph shows the survival of the C118-treated and untreated larvae over a 12-day period. Two experiments were performed. (C) At 30 hours post-fertilization, embryos were infected with approximately 200 colony-forming units (CFU) of M. marinum expressing mScarlet via caudal vein injection. A standard uninfected control was included for each experiment. At 1-day post-infection (dpi), larvae were randomly split into equal groups of approximately 24 individuals, and C118 (at 7 and 3.5 pM) was directly added to the water. The drug was renewed daily for 4 days (indicated in orange along the X axis). After treatment, larvae were washed twice in E3 and maintained in E3. Embryo survival was monitored daily over an 11 -days period (3 replicates). (B, C) Each C118-treatment group was compared against the untreated control group with significant differences calculated using the log-rank (Mantel-Cox) statistical test for survival curves: *** P ≤ 0.001; **** P ≤ 0.0001. (D) The M. marinum burden was quantified after 2 days of exposure (3 dpi) to 3.5 pM of C118, following FPC quantification. The data are the mean of three independent experiments (approximately 20 embryos per group). The error bar represents the mean and standard deviations of the dataset. Mann-Whitney test, ““ P <0.0001. (E) Representative larvae from the untreated group (upper panel) or the C118-treated group for 4 days (lower panel), imaged at 5. Arrows indicate small infection foci or individual bacilli.

[0090] FIG. 13 depicts (A) cytotoxicity of C118 and rifabutin on THP-1 macrophages. THP-1 cells were differentiated with PMA for 48 hrs, exposed to increasing concentrations of either C118 or rifabutin for an additional 72 hrs at 37 °C with 5% CO2. The experiments were performed in three experiments, each in quadruplicate. Data represented are the average of three independent experiments, each performed in quadruplicate. (B) Macrophages were infected with the M. abscessus expressing tdTomato (MOI of 2:1) for 4 hrs. Data are mean values ± SD for three independent experiments. Data were analyzed using the one-tailed Mann-Whitney t-test, **** p ≤0.0001. (C) Three immunofluorescent fields were taken 72 dpi at 63× magnification showing macrophages infected with fluorescent M. abscessus (red). The surface of the macrophages was detected using anti-CD43 antibodies (green). The nuclei were stained with DAPI (blue). Scale bar represents 20 pm and the enlargement scale bar represents 5 pm.

[0091] Description

[0092] NTM diseases are on the rise and is largely neglected, as reflected by the remarkably thin clinical development pipeline, populated by only a few repurposed antibiotics. The treatment of NTM diseases is problematic and goes along with high pill burden, long duration, unsatisfactory cure rates, toxicity, the use of injectable agents with serious side effects and drug-drug interactions. Therefore, there is a pressing need to identify inhibitors with high nanomolar potency and enzyme targets being essential for the pathogen under various metabolic conditions, high target specificity, and with attractive combinatory potency. Here, we designed and synthesized the analog of BDQ labelled C118.

[0093] It has been surprisingly found that certain compounds may be useful in the treatment of a nontuberculosis mycobacterial infection. Thus, in a first aspect of the invention, there is provided a compound according to formula I:

[0094]

[0095] where:

[0096] R1represents C1-5alkyl;

[0097] R2represents H, F or OCF3;

[0098] X and Y independently represent O, S or CH2; and

[0099] n is a number from 1 to 5,

[0100] or a salt or solvate thereof, provided that the compound of formula I is not:

[0101]

[0102] 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.

[0103] 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.

[0104] 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.

[0105] For the avoidance of doubt, it is explicitly contemplated that where a number of numerical ranges related to the same feature are cited herein, that the end points for each range are intended to be combined in any order to provide further contemplated (and implicitly disclosed) ranges.

[0106] 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.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.

[0107] 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.

[0108] 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.

[0109] 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.The solvates can be stoichiometric or non-stoichiometric solvates. Particularly preferred solvates are hydrates, and examples of hydrates include hemihydrates, monohydrates and di hydrates.

[0110] For a more detailed discussion of solvates and the methods used to make and characterise them, see Bryn et al., Solid-State Chemistry of Drugs, Second Edition, published by SSCI, Inc of West Lafayette, IN, USA, 1999, ISBN 0-967-06710-3.

[0111] As will be appreciated, the compounds of formula I may be presented as a salt that is solvated.

[0112] 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.

[0113] Compounds of formula I 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 poof 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. Nevertheless, the absolute stereochemistry depicted in the compound of formula I is preferred above all other possible stereochemical options.

[0114] The preferred absolute stereochemistry has been confirmed for one or more enantiomers / diasteroeisomers by experiment. For example, the relative stereochemistry of Racemate C118-A was confirmed by X-ray crystallography. The assigned pure enantiomer (-)-(1 R,2S)-C118 as (1R,2S) and (+)-(1S,2R)-C118 as (1S,2R) were confirmed by ECD (Electronic Circular Dichroism) stereochemistry analysis.

[0115] 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.

[0116] 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 vertebrate, such as a fish or 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.

[0117] 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).

[0118] 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, but preferably a saturated unbranched or branched) 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 C1-5 alkyl 5 (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, C3-12 cycloalkyl and, more preferably, C5-10(e.g. C5-7) cycloalkyl.

[0119] 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.

[0120] 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 tocompounds of formula I that are isotopically enriched at one or more positions in the compound.

[0121] 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,17O,18O,35S,18F,37CI,77Br,82Br and125I).

[0122] 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.

[0123] 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.

[0124] 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.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.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] 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.

[0129] 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.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.

[0130] 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.

[0131] The aspects of the invention described herein (e.g. the above-mentioned compounds, combinations, methods and uses) may have the advantage that, in the treatment of the conditions described herein, they may be more convenient for the physician and / or patient than, be more efficacious than, be less toxic than, have better selectivity over, have a broader range of activity than, be more potent than, produce fewer side effects than, or may have other useful pharmacological properties over, similar compounds, combinations, methods (treatments) or uses known in the prior art for use in the treatment of those conditions or otherwise.

[0132] In certain embodiments of the invention, one or more of the following may apply:

[0133] R2 may represent H;

[0134] R1 may be CH3or CH2CH3;

[0135] n may be 2 or 3;

[0136] X may be O or S (e.g. X is O); and

[0137] Y may be O or S (e.g. Y is O).

[0138] For the avoidance of doubt, any combination of the above variables may be used herein.

[0139] In more particular embodiments that may be mentioned herein, the following may apply: R1is CH3or CH2CH3;

[0140] R2is H;

[0141] n is 2 or 3;

[0142] X is O; and

[0143] Y is O.

[0144] That the compounds of formula I in the embodiment above must be selected only from the listed variables for R1, R2, n, X and O.In yet more particular embodiments of the invention, the compound of formula I may be selected from the list:

[0145]

[0146]

[0147] In yet more particular embodiments of the invention, the compound of formula I may be selected from the list:

[0148]

[0149] For the avoidance of doubt, the specific compounds mentioned above may be provided as the free base, or as a salt or solvate thereof. In addition, the absolute stereochemistry of the compounds may be that depicted.

[0150] In a second aspect of the invention, there is provided a use of a compound according to formula I:

[0151]

[0152] where:

[0153] R1represents C1-5alkyl;

[0154] R2represents H, F or OCF3;

[0155] X and Y independently represent O, S or CH2; and

[0156] n is a number from 1 to 5,

[0157] or a salt or solvate thereof,

[0158] in the manufacture of a medicament for the treatment of a nontuberculosis mycobacterial infection.

[0159] In certain embodiments of this aspect, that the compound of formula I is not:

[0160]

[0161] In certain embodiments of the invention, one or more of the following may apply:

[0162] R2 may represent H;

[0163] R1 may be CH3or CH2CH3;

[0164] n may be 2 or 3;

[0165] X may be O or S (e.g. X is O); and

[0166] Y may be O or S (e.g. Y is O).

[0167] For the avoidance of doubt, any combination of the above variables may be used herein.

[0168] In more particular embodiments that may be mentioned herein, the following may apply: R1 is CH3or CH2CH3;

[0169] R2is H;

[0170] n is 2 or 3;

[0171] X is O; and

[0172] Y is O.

[0173] That the compounds of formula I in the embodiment above must be selected only from the listed variables for R1, R2, n, X and O.

[0174] In yet more particular embodiments of the invention, the compound of formula I may be selected from the list:

[0175]

[0176]

[0177]

[0178] In yet more particular embodiments of the invention, the compound of formula I may be selected from the list:

[0179] and

[0180]

[0181]

[0182] In a third aspect of the invention, there is provided a compound according to formula I or a salt or solvate thereof, as described in the second aspect of the invention and embodiments thereof, for use in the treatment of a nontuberculosis mycobacterial infection.

[0183] In a fourth aspect of the invention, there is provided a method of treating a nontuberculosis mycobacterial infection comprising the steps of providing a compound of formula I or a salt or solvate thereof, as described in the second aspect of the invention and embodiments thereof, to a subject in need thereof.

[0184] In embodiments of the invention, the use according to the second aspect of the invention, the compound for use according to the third aspect of the invention or the method according to the fourth aspect of the invention, may be one where the nontuberculosis mycobacterial infection is caused by one of more of mycobacterium avium, mycobacterium peregrinum, mycobacterium kansasii, mycobacterium marinum, and mycobacterium chelonae.

[0185] In accordance with the invention, compounds of formula I may be administered alone (i.e. as a monotherapy). In alternative embodiments of the invention, however, compounds of formula I may be administered in combination with another therapeutic agent. Thus, in a fifth aspectof the invention, there is provided a use of a compound of formula I, or a salt or solvate thereof, as described in as described in the second aspect of the invention and embodiments thereof, and a second antibiotic in the manufacture of a medicament for the treatment of nontuberculosis mycobacterial infection, wherein the compound of formula I or a salt or solvate thereof, is to be administered sequentially, simultaneously or concomitantly with the second antibiotic.

[0186] In a sixth aspect of the invention, there is provided a compound according to formula I or a salt or solvate thereof, as described in as described in the second aspect of the invention and embodiments thereof, and a second antibiotic for use in the treatment of a nontuberculosis mycobacterial infection wherein the compound of formula I or a salt or solvate thereof, is to be administered sequentially, simultaneously or concomitantly with the second antibiotic.

[0187] In a seventh aspect of the invention, there is provided a method of treating a nontuberculosis mycobacterial infection comprising the steps of providing a compound of formula I or or a salt or solvate thereof, as described in as described in the second aspect of the invention and embodiments thereof, and a second antibiotic, to a subject in need thereof, wherein the compound of formula I or a salt or solvate thereof, is to be administered sequentially, simultaneously or concomitantly with the second antibiotic.

[0188] In embodiments of the invention, the use according to the fifth aspect of the invention, the compound for use according to the sixth aspect of the invention or the method according to the seventh aspect of the invention, may be one where the nontuberculosis mycobacterial infection is caused by one of more of mycobacterium avium, mycobacterium peregrinum, mycobacterium kansasii, mycobacterium marinum, and mycobacterium chelonae.

[0189] In embodiments of the invention, the use according to the fifth aspect of the invention, the compound for use according to the sixth aspect of the invention or the method according to the seventh aspect of the invention, may be one where the second antibiotic is selected from one or more of the group consisting of clofazimine, rifabutin and amikacin.

[0190] When used herein, the term “administered sequentially, simultaneously or concomitantly’’ includes references to:

[0191] administration of separate pharmaceutical formulations (one containing the compound of formula I and one or more others containing the one or more other therapeutic agents); and administration of a single pharmaceutical formulation containing the compound of formula I and the other therapeutic agent(s).The combination product described above provides for the administration of the compound of formula I (component (A)) in conjunction with the second antibiotic (component (B)), and may thus be presented either as separate formulations, wherein at least one of those formulations comprises component (A) and at least one comprises component (B), or may be presented (i.e. formulated) as a combined preparation (i.e. presented as a single formulation including component (A) and component (B)).

[0192] Thus, there is further provided:

[0193] (I) a pharmaceutical formulation including a compound of formula I, as hereinbefore defined and another therapeutic agent, in admixture with a pharmaceutically-acceptable adjuvant, diluent or carrier (which formulation is hereinafter referred to as a “combined preparation”); and

[0194] (II) a kit of parts comprising components:

[0195] (i) a pharmaceutical formulation including a compound of formula I, as hereinbefore defined, in admixture with a pharmaceutically-acceptable adjuvant, diluent or carrier; and

[0196] (ii) a pharmaceutical formulation including the second antibiotic, in admixture with a pharmaceutically-acceptable adjuvant, diluent or carrier,

[0197] which components (i) and (ii) are each provided in a form that is suitable for administration in conjunction with the other.

[0198] Component (i) of the kit of parts is thus component (A) in admixture with a pharmaceutically-acceptable adjuvant, diluent or carrier. Similarly, component (ii) is component (B) in admixture with a pharmaceutically-acceptable adjuvant, diluent or carrier.

[0199] A further aspect of the invention may also include formulation comprising a compound according to the first aspect of the invention and embodiments thereof, or a pharmaceutically acceptable salt or solvent thereof, and a pharmaceutically acceptable carrier.

[0200] A yet further aspect of the invention may also relate to a formulation comprising a compound according to any one of Claims 1 to 11, or a pharmaceutically acceptable salt or solvent thereof, and an acceptable carrier suitable to prevent growth of mycobacteria in an aqueous environment that is useful for the farming of fish.

[0201] Advantages of the present invention may include the following, which may or may not be described elsewhere herein.The presently disclosed novel anti-NTM inhibitors target the mycobacterial F-ATP synthase, which is essential for ATP synthesis, regulation of ATP homeostasis and proton motive force under multiple growth conditions. C118 reveals nanomolar efficacy against M. avium, M. kansasii, M. peregrinum, M. marinum and M. chelonae clinical isolates, respectively, and is potent against M. abscessus THP-1 cells. The observed cell growth inhibition correlates with high depletion of ATP synthesis and drastic inhibition of the target enzyme, F-ATP synthase. For example, the compound C118 potentiates the anti- Mav activity of the drugs Amikacin, CFZ and Rifabutin which target the 16S rRNA, the NADH dehydrogenase and the RNA polymerase, respectively.

[0202] Importantly, compounds disclosed herein, such as C118, may inhibit growth of M. marinum a pathogen, responsible for cutaneous infections in human and diseases in fish, including aquarium, marine, brackish, and freshwater fish is very common. Such infection can affect virtually any fish organ system but especially the spleen, kidneys, and liver. To date, there are no universally accepted treatments for mycobacteriosis in fish. The high potency of C118 against M. marinum infected zebrafish embryos (FIG. 12) makes the compound an effective inhibitor to prevent M. marinum infections of fish in artificial farms and aquariums, leading to economic and health benefits and improved food-safety and -quality.

[0203] Mycobacterium marinum infections are water borne as well as Zoonotic infections possessing the ability to infect both fresh water and marine fish species leading to a chronic and progressive diseases known as piscine mycobacteriasis. As a consequence, the M marinum infections not only pose a health hazard for fish and impacts aquaculture industry productivity but also a public health hazard for people working in aquaculture handling infected fish.

[0204] In particular, the M. marinum infections in recirculating aquaculture systems (RAS) or industrial settings are often diagnosed by uncoordinated swimming, weight loss, abdominal swelling, skin ulceration, and granuloma formation in organs like the liver, kidney, and spleen. This eventually leads to increased morbidity / mortality rates of cultured fish populations, affecting reliable fish farming productivity, causing massive economic losses and reduced food availability for human consumption posing food security issues to humans.

[0205] Further aspects and embodiments of the invention may relate to the following numbered embodiments.In the first aspect of the invention, there is provided an analogue of bedaquiline (BDQ) having the formula I:

[0206]

[0207] Where Ri = alkyl of a length up to 5 carbons, R2 = F, OCF3, X, Y = O, S or CH2, and n is a number between 1 to 5.

[0208] In the second aspect of the invention, there is provided a composition comprising the analogue of the first aspect of the invention for use in the treatment of nontuberculous mycobacterial (NTM) infection.

[0209] In the third aspect of the invention, there is provided a composition comprising the analogue of the first aspect of the invention; and a second antibiotic for use in the treatment of nontuberculous mycobacterial (NTM) infection.

[0210] In various embodiments, the second antibiotic is selected from the group consisting of Clofazimine, Rifabutin or Amikacin.

[0211] In various embodiments, the analogue of the first, second and third aspects of the invention is

[0212]

[0213] Further aspects and embodiments of the invention will now be discussed by reference to the following non-limiting examples.Examples

[0214] Materials

[0215] All reagents were obtained from commercial suppliers and used without further purification unless where indicated otherwise. Reactions requiring anhydrous or air free conditions were performed under an atmosphere of nitrogen. Glassware was oven dried at 120 °C and cooled under vacuum. Anhydrous THF and ether were distilled from sodium metal and benzophenone under nitrogen atmosphere. Anhydrous toluene was distilled from sodium and anhydrous dichloromethane was distilled from calcium hydride.

[0216] Analytical techniques

[0217] 1H and13C NMR spectra were recorded on a Bruker Avance III 400 spectrometer and a JEOL ECA400 UltraShield in CDCl3, DMSO-d6, and MeOD. Chemical shifts are given in parts per million (ppm) with residual protic solvent as the internal standard. Coupling constants are given in Hertz. Low resolution mass spectra were recorded on a (LRMS) Thermo Scientific LTQ XL with a quadrupole ion trap equipped with Thermo Ultimate LC. High resolution mass spectra were recorded on a Waters Xevo G2-X2 MS in either positive or negative mode equipped with Waters Acquity UPLC. Analytical thin layer chromatography was performed on Merck DC precoated TLC plates with 0.25 mm Kieselgel 60 F254. The plates were visualised with a 254 nm UV lamp, or by staining with ammonium molybdate or potassium permanganate. Flash chromatography was performed on silica gel 230-400 mesh.

[0218] Example 1.3-Hydroxy-2-methoxybenzaldehyde

[0219] CHO

[0220] ^L -OMe

[0221]

[0222] To a stirred solution of 2,3 dihydroxybenzaldehyde (2.0 g,14.5 mmol) and potassium carbonate (2.2 g,14.5 mmol) in DMF (20 mL), was added iodomethane (1.0 mL,15.9 mmol) dropwise. The reaction mixture was stirred at room temperature for 18 h and then quenched with water (40 mL). The mixture was extracted with EtOAc (3x100 mL) and the combined organic layers were dried over anhydrous Na2SO4, concentrated in vacuo, and purified by flash column chromatography (silica gel, 30% EtOAc / Hexanes) to afford 3-hydroxy-2-methoxybenzaldehyde as a colorless solid (0.84 g, 38% yield).1H NMR (400MHz, CDCl3) 5 10.29 (s, 1H), 7.41 - 7.17 (m, 3H), 5.81 (brs, 1H), 4.00 (s, 3H).Example 2. 3-(3-Bromopropoxy)-2-methoxybenzaldehyde

[0223] CHO

[0224]

[0225] To a stirred solution of 3-hydroxy-2-methoxybenzaldehyde (0.18 g, 1.17 mmol), potassium carbonate (0.36 g, 2.57 mmol), benzyltriethylammonium chloride (27 mg, 0.12 mmol) in CH3CN (5 mL), was added dibromopropane (0.36 mL, 3.51 mmol). The reaction mixture was stirred at room temperature for 18 h and then quenched with water (20 mL). The mixture was extracted with EtOAc (3x50 mL) and the combined organic layers were dried over anhydrous Na2SO4, concentrated in vacuo, and purified by flash column chromatography (silica gel, 10% EtOAc / Hexanes) to afford the title compound 3-(3-bromopropoxy)-2-methoxybenzaldehyde as a colorless oil (0.23 g, 73% yield).1H NMR (400MHz, CDCl3) δ 10.45 (s, 1H), 7.47 – 7.44 (m, 1H), 7.22 - 7.13 (m, 2H), 4.22 (t, J = 5.6 Hz, 2H), 4.01 (s, 3H), 3.67 (t, J = 5.6 Hz, 2H), 2.41 (quint, J = 5.6 Hz, 2H);13C NMR (100 MHz, CDCl3) 8 190.0, 152.9, 152.1, 129.9, 124.2, 119.7, 119.5, 66.5, 62.3, 32.1, 29.7; HRMS (ESI-Quadrupole) m / z calc’d for [C11H13BrO3+ H]+273.0126, found 273.0124.

[0226] Example 3. 3-(3-(Ethylthio)propoxy)-2-methoxybenzaldehyde

[0227] CHO

[0228]

[0229] To a stirred solution of 3-(3-bromopropoxy)-2-methoxybenzaldehyde (0.23 g, 0.85 mmol) and cesium carbonate (0.61 g, 1.87 mmol) in DMF (5 mL), was added ethanethiol (0.08 mL, 1.02 mmol). The reaction mixture was stirred at room temperature for 18 h and then quenched with water (20 mL). The mixture was extracted with EtOAc (3x30 mL) and the combined organic layers were dried over anhydrous Na2SO4, concentrated in vacuo, and purified by flash column chromatography (silica gel, 10% EtOAc / Hexanes) to afford 3-(3-(ethylthio)propoxy)-2-methoxybenzaldehyde as a colorless oil (0.19 g, 89% yield).1H NMR (400 MHz, CDCl3) 8 10.45 (s, 1H), 7.45 - 7.42 (m, 1H), 7.28 - 7.13 (m, 2H), 4.17 (t, J= 6.0 Hz, 2H), 4.01 (s, 3H), 2.78 (t, J = 6.0 Hz, 2H), 2.59 (q, J = 7.2 Hz, 2H), 2.17 (quint, J= 6.0 Hz, 2H), 1.30 (t, J = 7.2 Hz, 3H);13C NMR (100 MHz, CDCl3) 8 190.0, 152.7, 152.2, 129.7, 124.0, 119.2, 119.2, 67.3, 62.2, 29.1, 28.0, 25.9, 14.7; HRMS (ESI-Quadrupole) m / z calc’d for [C13H18O3S + H]+255.0155, found 255.1049.

[0230] Example 4. (6-Bromo-2-methoxyquinolin-3-yl)(3-(3-(ethylthio)propoxy)-2-methoxyphenyl)methanolOMe

[0231]

[0232] A solution of 2,2,6,6-tetramethylpiperidine (0.25 mL, 1.47 mmol) in anhydrous THF (2 mL) was cooled to -40 °C, and n-BuLi (0.7 mL of a 2.0 N solution in hexane, 1.25 mmol) was added and the reaction mixture was stirred at -40 °C for 15 min, then cooled to -78 °C. A solution of 6-bromo-2-methoxyquinoline (0.1 g, 0.39 mmol) in anhydrous THF (2 mL) was added dropwise, and the orange solution was stirred at -78 °C for 1 h, then a solution of (3) (0.1 g, 0.39 mmol) in anhydrous THF (2 mL) was added. The mixture was stirred at -78 °C for 4 h, then acetic acid (0.1 mL, 1.47 mmol) was added and the solution was allowed to warm to room temperature, then quenched with water (10 mL). The resulting mixture was extracted with EtOAc (3x30 mL) and the combined organic layers were dried over anhydrous Na2SO4, concentrated in vacuo, and purified by flash column chromatography (silica gel, 15% EtOAc / Hexanes) to afford (6-bromo-2-methoxyquinolin-3-yl)(3-(3-(ethylthio)propoxy)-2-methoxy-phenyl)methanol as a colourless oil (54 mg, 28% yield).1H NMR (400 MHz, CDCl3) 8 7.90 - 7.86 (m, 2H), 7.72 - 7.65 (m, 2H), 7.04 - 6.85 (m, 3H), 6.35 (s, 1H), 4.13 (t, J = 6.4 Hz, 2H), 4.06 (s, 3H), 3.82 (s, 3H), 3.37 (brs, 1H), 2.77 (t, J= 7.2 Hz, 2H), 2.58 (q, J= 7.2 Hz, 2H), 2.14 (quint, J= 7.2 Hz, 2H), 1.29 (t, J = 7.2 Hz, 3H);13C NMR (100 MHz, CDCI3) 8 160.0, 151.8, 146.7, 144.4, 135.2, 134.4, 132.4, 129.6, 128.5, 128.4, 126.5, 124.0, 119.7, 117.3, 113.2, 67.0, 66.8, 60.8, 53.6, 29.3, 28.2, 25.9, 14.7; HRMS (ESI-Quadrupole) m / z calc’d for [C23H26BrNO4S + H]+492.0844, found 492.0847.

[0233] Example 5.6-Bromo-3-(3-(3-(ethylthio)propoxy)-2-methoxybenzyl)-2-methoxyquinoline

[0234] OMe

[0235]

[0236] To a stirred solution of ((6-bromo-2-methoxyquinolin-3-yl)(3-(3-(ethylthio)propoxy)-2-methoxyphenyl)-methanol (54.1 mg, 0.10 mmol) in CH2CI2 (5 mL) was cooled to 0 °C. Triethylsilane (0.4 mL, 2.20 mmol) and trifluoroacetic acid (0.1 mL, 1.10 mmol) were added. The reaction mixture was stirred for 30 min at 0 °C, then heated to 40 °C for 18 h. The resulting mixture was cooled to 0 °C, quenched with saturated aqueous NaHCO3(20 mL) and partitioned between CH2CI2and water. The aqueous layer was extracted with CH2CI2(3x30 mL) and the combined organic layers were dried over anhydrous Na2SO4, concentrated invacuo, and purified by flash column chromatography (silica gel, 5% EtOAc / hexanes) to afford 6-bromo-3-(3-(3-(ethylthio)propoxy)-2-methoxybenzyl)-2-methoxyquinoline as a colourless oil (48 mg, 93% yield).1H NMR (400 MHz, CDCI3) δ 7.75 – 7.60 (m, 3H), 7.45 (s, 1 H), 7.03 - 6.99 (m, 1 H), 6.89 - 6.87 (m, 1 H), 6.79 - 6.76 (m,1 H), 4.14 (t, J = 6.4 Hz, 2H), 4.12 (s, 3H), 4.05 (s, 2H), 3.79 (s, 3H), 2.78 (t, J= 7.2 Hz, 2H), 2.59 (q, J = 7.2 Hz, 2H), 2.15 (quint, J= 6.8 Hz, 2H), 1.29 (t, J= 7.2 Hz, 3H);13C NMR (100 MHz, CDCI3) δ 161.1, 152.1, 147.6, 144.0,135.6, 132.6, 131.6, 129.0, 128.5, 126.8, 126.7, 123.9, 122.8, 116.9, 112.1, 67.0, 60.5, 53.6, 30.1, 29.4, 28.2, 25.9, 14.7; HRMS (ESI-Quadrupole) m / z calc’d for [C23H26BrNO3S + H]+476.0895, found 476.0894.

[0237] Example 6. 1-(6-Bromo-2-methoxyquinolin-3-yl)-2-(2,6-dimethoxypyridin-4-yl)-4-(dimethylamino)-1-(3-(3-(ethylthio)propoxy)-2-methoxyphenyl)butan-2-ol

[0238]

[0239] A solution of 2,2,6,6-tetramethylpiperidine (78.6 JLLL, 0.46 mmol) in anhydrous THF (1 mL) was cooled to -40 °C, and n-BuLi (0.2 mL of a 2.0 N solution in cyclohexane, 0.41 mmol) was added and the solution was stirred at -40 °C for 15 min, then cooled to -78 °C. A solution of (5) (88 mg, 0.18 mmol) in anhydrous THF (2 mL) was added dropwise, and the orange solution was stirred at -78 °C for 1 h, then a solution of 1-(2,6-dimethoxypyridin-4-yl)-3-(dimethylamino)propan-l-one (44 mg, 0.18 mmol) in anhydrous THF (2 mL) was added. The mixture was stirred at -78 °C for 4 h, then acetic acid (26.4 p. L, 0.46 mmol) was added and the solution was allowed to warm to r.t., then quenched with water (20 mL). The resulting mixture was extracted with EtOAc (3x30 mL) and the combined organic layers were dried over anhydrous NazSC, concentrated in vacuo, and purified by flash column chromatography (silica gel, 1-20% MeOH / CHzCIz) to afford 1-(6-bromo-2-methoxyquinolin-3-yl)-2-(2,6-dimethoxypyridin-4-yl)-4-(dimethylamino)-1-(3-(3-(ethylthio)propoxy)-2-methoxyphenyl)butan-2-ol, racemate C118-A as an off-white solid (9.6 mg, 7% yield) and racemate C118-B as an off-white solid (7.3 mg, 5% yield).

[0240] Racemate C118-A:1H NMR (400 MHz, CDCI3) δ 8.14 (s, 1 H), 7.82 - 7.80 (m, 2H), 7.69 - 7.58 (m, 2H), 6.87 - 6.82 (m, 1 H), 6.64 - 6.58 (m, 1 H), 6.55 (brs, 2H), 5.56 (s, 1 H), 4.23 (s, 3H),3.88 (t, J= 6.4 Hz, 2H), 3.85 (s, 2x3H), 3.51 (s, 3H), 2.65 (t, J= 7.2 Hz, 2H), 2.53 (q, J = 7.2 Hz, 2H), 2.28 (quint, = 7.2 Hz, 2H), 2.03 (m, 2H), 2.01 (s, 2x3H), 1.83 (m, 2H), 1.23 (t, J= 7.2 Hz, 3H);13C NMR (100 MHz, CDCI3) δ 163.1, 162.7, 161.4, 151.2, 147.0, 143.7, 139.7, 136.2, 131.8, 129.7, 128.3, 127.6, 126.6, 123.1, 122.8, 116.7, 111.6, 99.4, 99.0, 81.2, 66.7, 59.9, 55.6, 53.9, 53.4, 44.7, 43.3, 37.0, 35.5, 29.3, 28.2, 25.8, 16.0, 14.6; HRMS (ESI-Quadrupole) m / z calc’d for [C35H44BrN3O6S + H]+714.2212, found 714.2253.

[0241] Racemate C118-B:1H NMR (400 MHz, CDCI3) δ 8.77 (s, 1 H), 7.84 - 7.80 (m, 1 H), 7.51 - 7.58 (m, 2H), 7.23 - 7.21 (m, 1 H), 6.96 - 6.92 (m, 1 H), 6.87 - 6.80 (m, 1 H), 6.64 (brs, 2H), 5.50 (s, 1H), 4.23 - 4.12 (m, 2H), 4.05 (s, 3H), 3.84 (s, 3H), 3.84 (s, 2x3H), 2.63 (t, J = 7.2 Hz, 2H), 2.63 (q, J = 7.2 Hz, 2H), 2.21 - 2.17 (m, 2H), 2.00 (s, 2x3H), 1.75 - 1.70 (m, 2H), 1.28 (t, J = 7.2 Hz, 3H).13C NMR (100 MHz, CDCI3) δ 171.1, 163.1, 160.4, 151.4, 148.0, 143.4, 139.7, 138.1, 133.2, 131.8, 129.7, 127.6, 126.7, 123.1, 122.8, 116.5, 111.4, 100.0, 99.0, 81.4, 66.8, 60.9, 60.4, 55.6, 53.9, 53.3, 44.6, 43.2, 35.1, 29.4, 28.3, 21.0, 14.6, 14.2; HRMS (ESI-Quadrupole) m / z calc’d for [C35H44BrN3O6S + H]+714.2212, found 714.2202.

[0242] Racemate C118-A were separated by supercritical fluid chromatography (SFC); DAICEL CHIRALPAK AD column; mobile phase: [CO2-iPrOH (0.1% NH4OH)]; 35%, isocratic elution mode) to give enantiomer pure (+)-(1S,2fi)-C118 as an off-white solid (51.3 mg, >99% purity, >95% ee) and enantiomer pure (-)-(1 R,2S)-C118 as an off-white solid (64.5 mg, >99% purity, >95% ee). The levorotatory enantiomer showed highest activity against M. avium, M. kansasii, M. peregrinum, M. abscessus and M. avium (see Example 37) and was assigned (1 R, 2S) stereochemistry in accordance with Sutherland et al. (Sutherland, H. S. et al., Bioorg. Med. Chem. 2018, 26, 1797-1809) and confirmed by ECD spectroscopy.

[0243] Example 7. (1 R,2S)-1 -(6-Bromo-2-methoxyquinolin-3-yl)-2-(2,6-dimethoxypyridin-4-yl)-4-(dimethylamino)-1-(3-(3-(ethylthio)propoxy)-2-methoxyphenyl)butan-2-ol

[0244]

[0245] (-)-(1 R,2S)-C118:1H NMR (400 MHz, MeOD) δ 8.23 (s, 1H), 7.87(s, 1 H), 7.79 (d, J= 8.0 Hz, 1 H), 7.71 (d, J = 8.8 Hz, 1 H), 7.64 (d, J = 8.8 Hz, 1 H), 6.86 (t, J = 8.0 Hz, 1 H), 6.70 (d, J = 8.0Hz, 1 H), 6.50 (s, 2H), 5.63 (s, 1 H), 4.25 (s, 3H), 3.94 - 3.91 (m, 2H), 3.85 (s, 6H), 3.53 (s, 3H), 2.64 (t, J = 6.8 Hz, 2H), 2.48 (q, J = 7.2 Hz, 2H), 2.31 - 2.27 (m, 1 H), 1.95 (s, 6H), 1.95 - 1.94 (m, 4H), 1.82 - 1.76 (m, 1H), 1.19 (t, J = 7.2 Hz, 3H);13C NMR (100 MHz, MeOD) δ 163.2, 162.1, 161.4, 151.3, 147.0, 143.7, 139.7, 135.8, 131.8, 129.2, 128.1, 127.3, 126.6, 123.0, 122.7, 116.7, 112.0, 98.8, 80.3, 66.7, 59.3, 54.9, 53.1, 52.4, 43.6, 43.2, 36.5, 29.2, 27.5, 25.1, 13.7.

[0246] [a]o22= -21.95 (c = 0.23, MeOH)

[0247] HRMS (ESI-Quadrupole) m / z calc’d for [Css^ BrNsOeS + H]+ 714.2212, found 714.2201.

[0248] Example 8. 3-(3-Ethoxypropoxy)-2-methoxybenzaldehyde

[0249] OMe

[0250]

[0251] O' O Me

[0252] To a stirred solution of 3-hydroxy-2-methoxy-benzaldehyde (6.2 g, 40.7 mmol, 1.0 eq.) and potassium carbonate (11.2 g, 81.5 mmol, 2.0 eq.) in DMF (60 mL) was added 1 -chloro-3-ethoxy-propane (5.0 g, 40.7 mmol, 1.0 eq.) dropwise. The reaction mixture was heated to 100 °C and stirred for 8 h and then quenched with water (60 mL). The reaction mixture was diluted with EtOAc (150 mL) and extracted with water (3x150 mL). The combined organic layers were washed with brine (150 mL), dried over anhydrous Na2SC>4, filtered, and concentrated under reduced pressure to give 3-(3-ethoxypropoxy)-2-methoxybenzaldehyde as a colorless oil (8.0 g, 82% yield).1H NMR (400 MHz, CDCI3) δ 10.43 (s, 1 H), 7.40 (dd, J = 7.6, 1.6 Hz, 1H), 7.20 - 7.14 (m, 1 H), 7.13 - 7.07 (m, 1 H), 4.14 (t, J = 6.4 Hz, 2H), 3.99 (s, 3H), 3.64 (t, J= 6.0 Hz, 2H), 3.50 (q, J= 7.2 Hz, 2H), 2.12 (quint, J= 6.4 Hz, 2H), 1.20 (t, J = 7.2 Hz, 3H). LCMS (ESI) m / z 239.0 [M + H]+

[0253] Example 9. (E)-W-(3-(3-Ethoxypropoxy)-2-methoxybenzylidene)-4-methyl benzenesulfonohydrazide

[0254] Me

[0255] OMe

[0256]

[0257] To a stirred solution of 3-(3-ethoxypropoxy)-2-methoxybenzaldehyde (10.0 g, 41.9 mmol, 1.0 eq.) in 1,4-dioxane (100 mL) was added pTsOH (7.23 g, 41.9 mmol, 1.0 eq.) and 4-methylbenzenesulfono-hydrazide (7.82 g, 41.9 mmol, 1.0 eq.). The mixture was stirred at 60 °C for 1 h. The reaction mixture was diluted with EtOAc (300 mL) and extracted with water (3x300 mL). The combined organic layers were washed with brine (300 mL), dried over anhydrous Na2SC>4, filtered, and concentrated under reduced pressure to give a residue which was purified by column chromatography (silica gel, 10-67% EtOAc / hexanes) to give (E)- / V-(3-(3-ethoxypropoxy)-2-methoxybenzylidene)-4-methylbenzenesulfono-hydrazide as a colorless oil (15 g, 88% yield).1H NMR (400 MHz, CDCI3) δ 8.22 (brs, 1 H), 8.12 (s, 1H), 7.87 (d, J= 8.4 Hz, 2H), 7.40 (dd, J = 8.0, 1.2 Hz, 1 H), 7.29 (d, J = 8.0 Hz, 2H), 7.03 - 6.97 (m, 1 H), 6.95 -6.90 (m, 1H), 4.08 (t, J= 6.4 Hz, 2H), 3.80 (s, 3H), 3.63 - 3.57 (m, 2H), 3.49 (q, J= 7.2 Hz, 2H), 2.39 (s, 3H), 2.08 (t, J = 6.0 Hz, 2H), 1.19 (t, J = 7.2 Hz, 3H). LCMS (ESI) m / z 407.5 [M + H]+.

[0258] Example 10. 6-Bromo-3-(3-(3-ethoxypropoxy)-2-methoxybenzyl)-2-methoxyquinoline

[0259] OMe

[0260]

[0261] To a stirred solution of (E)- / V-(3-(3-ethoxypropoxy)-2-methoxybenzylidene)-4-methylbenzenesulfono-hydrazide (2.1 g, 5.17 mmol, 1.0 eq.) in 1,4-dioxane (20 mL) was added potassium carbonate (2.14 g, 15.5 mmol, 3.0 eq.) and (6-bromo-2-methoxy-3-quinolyl)boronic acid (1.46 g, 5.17 mmol, 1.0 eq.). The reaction mixture was stirred at 110 °C for 8 h. The reaction mixture was diluted with EtOAc (200 mL) and extracted with water (3x200 mL). The combined organic layers were washed with brine (200 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give a residue which was purified by column chromatography (silica gel, 5-50% EtOAc / petroleum ether) to give 6-bromo-3-(3-(3-ethoxypropoxy)-2-methoxybenzyl)-2-methoxyquinoline as a colorless oil (1.0 g, 40% yield).1H NMR (400 MHz, CDCl3) 87.74 (d, J= 2.4 Hz, 1 H), 7.71 - 7.66 (m, 1H), 7.63 -7.58 (m, 1 H), 7.44 (s, 1 H), 7.06 - 6.96 (m, 1 H), 6.88 (dd, J = 8.0, 1.6 Hz, 1 H), 6.76 (dd, J = 7.6, 1.6 Hz, 1 H), 4.13 (t, J = 6.0 Hz, 2H), 4.11 (s, 3H), 4.04 (s, 2H), 3.78 (s, 3H), 3.65 (t, J = 6.0 Hz, 2H), 3.51 (q, J= 7.2 Hz, 2H), 2.13 (t, J= 6.0 Hz, 2H), 1.22 (t, J= 7.2 Hz, 3H). LCMS (ESI) m / z 460.0 [M + H]+.

[0262] Example 11. 1-(6-Bromo-2-methoxyquinolin-3-yl)-2-(2,6-dimethoxypyridin-4-yl)-4-(dimethylamino)-1-(3-(3-ethoxypropoxy)-2-methoxyphenyl)butan-2-ol

[0263]

[0264] A solution of 2,2,6,6-tetramethylpiperidine (0.18 mL, 1.08 mmol) in anhydrous THF (2 mL) was cooled to -40 °C, and n-BuLi (0.54 mL of a 2.0 N solution in cyclohexane, 1.08 mmol) was added and the solution was stirred at -40 °C for 15 min, then cooled to -78 °C. A solution of 6-bromo-3-(3-(3-ethoxypropoxy)-2-methoxybenzyl)-2-methoxyquinoline (200 mg, 0.43 mmol, 1.0 eq.) in anhydrous THF (2 mL) was added dropwise, and the orange solution was stirred at -78 °C for 1 h, then a solution of 1-(2,6-dimethoxypyridin-4-yl)-3-(dimethylamino)propan-1-one (155 mg, 0.65 mmol, 1.5 eq.) in anhydrous THF (2 mL) was added dropwise. The reaction mixture was stirred at -78 °C for 4 h, then water (1 mL) was added and the solution was allowed to warm to room temperature, then quenched with water (20 mL). The resulting mixture was extracted with EtOAc (3x30 mL) and the combined organic layers were washed with brine (20 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give the residue mixture which was purified by preparative HPLC (38-58% MeCN / H2O; 0.2% formic acid) to afford racemate acNTMF0-1A as an off-white solid (27 mg, 9% yield) and racemate acNTMFO-1 B as an off-white solid (28 mg, 9% yield).

[0265] Racemate acNTMF0-1 A:1H NMR (400 MHz, DMSO-d6) 8 8.19 (s, 1H), 8.11 (s, 1 H), 7.74 (d, J = 8.0 Hz, 1 H), 7.70 (d, J = 1.2 Hz, 2H), 7.39 (brs, 1 H), 6.86 - 6.80 (m, 1 H), 6.69 (d, J = 7.2 Hz, 1 H), 6.43 (s, 2H), 5.49 (s, 1 H), 4.17 (s, 3H), 3.91 - 3.87 (m, 1 H), 3.80 (brs, 1 H), 3.78 (s, 6H), 3.44 - 3.40 (m, 5H), 3.37 - 3.33 (m, 2H), 2.16 - 2.08 (m, 1 H), 1.88 (s, 6H), 1.86 - 1.80 (m, 4H), 1.65 - 1.56 (m, 1 H), 1.03 (t, J = 7.2 Hz, 3H); LCMS (ESI) m / z 698.2 [M + H]+.

[0266] Racemate acNTMF0-1 B:1H NMR (400 MHz, DMSO-d6) 8 8.83 (s, 1H), 8.32 (s, 1 H), 8.09 (d, J= 2.0 Hz, 1 H), 7.64 (dd, J= 8.8, 2.4 Hz, 1H), 7.52 (d, J= 8.8 Hz, 1 H), 7.19 (dd, J= 6.8, 2.8 Hz, 1 H), 6.95 - 6.88 (m, 2H), 6.43 (s, 1 H), 5.42 (s, 1 H), 4.11 - 4.06 (m, 1 H), 4.03 - 3.99 (m, 1H), 3.94 (s, 3H), 3.82 (s, 3H), 3.73 (s, 6H), 3.57 (s, 2H), 3.44 (s, 2H), 2.17 - 2.11 (m, 1 H), 2.03 - 1.97 (m, 2H), 1.94 (s, 6H), 1.92 (brs, 1 H), 1.77 - 1.69 (m, 2H), 1.10 (t, J = 7.2 Hz, 3H); LCMS (ESI) m / z 698.2 [M + H]+.

[0267] Racemate acNTMF0-1 A were separated by supercritical fluid chromatography (SFC); DAICEL CHIRALPAK IG column; mobile phase: [CO2-MeOH(0.1% NH4OH)]; 5-30%, isocraticelution mode) to give enantiomer pure acNTMF0-1A-P1 as an off-white solid (19.5 mg, >99% purity, >95% ee) and enantiomer pure acNTMF0-1A-P2 as an off-white solid (12.9 mg, >99% purity, >95% ee).

[0268] (1R*,2S*)-1-(6-Bromo-2-methoxyquinolin-3-yl)-2-(2,6-dimethoxypyridin-4-yl)-4-(dimethylamino)-1-(3-(3-ethoxypropoxy)-2-methoxyphenyl)butan-2-ol

[0269]

[0270] acNTMFO-1 A-P1:1H NMR (400 MHz, MeOD) δ 8.57 (s, 1 H), 8.22 (s, 1 H), 7.88 (brs, 1 H), 7.79 (d, J= 8.0 Hz, 1H), 7.73 -7.64 (m, 2H), 6.86 (t, J= 8.0 Hz, 1 H), 6.72 (dd, J = 8.4, 1.6 Hz, 1H), 6.50 (s, 2H), 5.63 (s, 1H), 4.25 (s, 3H), 3.97 -3.88 (m, 2H), 3.84 (s, 6H), 3.56 -3.53 (m, 5H), 3.44 (q, J = 7.2 Hz, 2H), 2.34 - 2.27 (m, 1 H), 2.00 -1.92 (m,10H), 1.91 -1.75 (m, 1 H), 1.13 (t, J= 7.2 Hz, 3H);13C NMR (100 MHz, MeOD) δ 168.9, 163.2, 162.0, 161.4, 151.4, 147.0, 143.7, 139.7, 135.8, 131.8, 129.2, 128.1, 127.3, 126.6, 123.0, 122.6, 116.7, 111.9, 98.8, 80.3, 66.8, 65.8, 65.2, 59.3, 54.9, 53.1, 52.4, 43.6, 43.2, 36.5, 29.4, 14.0.

[0271] [a]D22= +44.85 (c= 0.61, MeOH)

[0272] HRMS (ESI-Quadrupole) m / z calc’d for [C35H44BrN3O7+ H]+698.2441, found 698.2438.

[0273] Example 12. 2-Methoxy-3-(2-methoxyethoxy)benzaldehyde

[0274]

[0275] To a stirred solution of 3-hydroxy-2-methoxy-benzaldehyde (4.5 g, 29.6 mmol, 1.0 eq.) and potassium carbonate (6.13 g, 44.4 mmol, 1.5 eq.) in DMF (20 mL) was added 1 -bromo-2-methoxyethane (4.93 g, 35.5 mmol, 3.34 mL, 1.2 eq.) dropwise. The reaction mixture was heated to 90 °C for 1 h and then quenched with water (50 mL). The reaction mixture was diluted with EtOAc (150 mL) and extracted with water (3x150 mL). The combined organic layers were washed with brine (150 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give the crude residue which was purified by column chromatography (silica gel, 9-25% EtOAc / hexanes) to afford 2-methoxy-3-(2-methoxyethoxy)benzaldehyde as a yellow oil (6 g, 91% yield). LCMS (ESI) m / z 211.0 [M + H]+.Example 13. (6-Bromo-2-methoxyquinolin-3-yl)(2-methoxy-3-(2-methoxyethoxy)phenyl)methanol

[0276]

[0277] A solution of 2,2,6,6-tetramethylpiperidine (2.64 mL, 15.5 mmol, 2.5 eq.) in anhydrous THF (10 mL) was cooled to -40 °C, and n-BuLi (6.2 mL of a 2.0 N solution in hexane, 12.4 mmol, 2.0 eq.) was added and the reaction mixture was stirred at -40 °C for 15 min, then cooled to -78 °C. A solution of 6-bromo-2-methoxy-quinoline (1.47 g, 6.18 mmol, 1.3 eq.) in anhydrous THF (20 mL) was added dropwise, and the orange solution was stirred at -78 °C for 1 h, then a solution of 2-methoxy-3-(2-methoxy-ethoxy)benzaldehyde (1.0 g, 4.76 mmol, 1.0 eq.) in anhydrous THF (20 mL) was added. The mixture was stirred at -78 °C for 4 h, then water (1 mL) was added and the solution was allowed to warm to room temperature, then quenched with water (10 mL). The resulting mixture was extracted with EtOAc (3x30 mL) and the combined organic layers were washed with brine (30 mL), dried over anhydrous Na2SC>4, filtered, and concentrated under reduce pressure to provide the crude mixture. The residue was purified by column chromatography (silica gel, 10-25% EtOAc / hexanes) to afford to afford (6-bromo-2-methoxy-3-quinolyl)-[2-methoxy-3-(2-methoxyethoxy)phenyl]methanol as a yellow solid (1.5 g, 71% yield). LCMS (ESI) m / z 448.0 [M + H]+.

[0278] Example 14.6-Bromo-2-methoxy-3-(2-methoxy-3-(2-methoxyethoxy)benzyl)quinoline

[0279]

[0280] To a stirred solution of 6-bromo-2-methoxyquinolin-3-yl)(2-methoxy-3-(2-methoxyethoxy)phenyl)me-thanol (1.00 g, 2.23 mmol, 1.0 eq.) anhydrous in CH2CI2 (10 mL) was cooled to 0 °C. Trifluoroacetic acid (0.5 mL, 6.69 mmol, 3.0 eq.) and triethylsilane (3.56 mL, 22.3 mmol, 10 eq.) were added. The reaction mixture was stirred for 30 min at 0 °C, then warmed to room temperature for 2 h. The reaction mixture was concentrated in vacuo to provide the crude mixture which was purified by flash column chromatography (silica gel, 10-25% EtOAc / hexanes) to afford 6-bromo-2-methoxy-3-(2-methoxy-3-(2-methoxyethoxy)benzyl)quinolone as a red oil (0.8 g, 57% yield). LCMS (ESI) m / z 432.9 [M + H]+.

[0281] Example 15. 1-(6-Bromo-2-methoxyquinolin-3-yl)-2-(2,6-dimethoxypyridin-4-yl)-4-(dimethylamino)-1-(2-methoxy-3-(2-methoxyethoxy)phenyl)butan-2-ol

[0282]

[0283] A solution of 2,2,6,6-tetramethylpiperidine (0.29 mL, 1.73 mmol, 2.5 eq.) in anhydrous THF (2 mL) was cooled to -40 °C, and n-BuLi (0.69 mL of a 2.0 N solution in cyclohexane, 1.38 mmol, 2.0 eq.) was added and the solution was stirred at -40 °C for 15 min, then cooled to -78 °C. A solution of 6-bromo-2-methoxy-3-(2-methoxy-3-(2-methoxyethoxy)benzyl)quinoline (300 mg, 0.69 mmol, 1.0 eq.) in anhydrous THF (5 mL) was added dropwise, and the orange solution was stirred at -78 °C for 1 h, then a solution of 1-(2,6-dimethoxypyridin-4-yl)-3-(dimethylamino)propan-l-one (248 mg, 1.04 mmol, 1.5 eq.) in anhydrous THF (5 mL) was added dropwise. The reaction mixture was stirred at -78 °C for 4 h, then water (1 mL) was added and the solution was allowed to warm to room temperature, then quenched with water (20 mL). The resulting mixture was extracted with EtOAc (3x20 mL) and the combined organic layers were washed with brine (20 mL), dried over anhydrous Na2SC>4, filtered, and concentrated under reduced pressure to give the residue mixture which was purified by preparative HPLC (25-55% MeCN / H2O; 0.2% formic acid) to afford racemate acNTMF0-2A as an off-white solid (55 mg, 12% yield) and racemate acNTMF0-2B as an off-white solid (50 mg, 10% yield).

[0284] Racemate acNTMF0-2A:1H NMR (400 MHz, DMSO-d6) 5 8.21 (s, 1H), 8.12 (s, 1 H), 7.74 (d, J = 7.8 Hz, 1 H), 7.70 (s, 2H), 6.87 - 6.80 (m, 1 H), 6.70 (d, J = 8.0 Hz, 1 H), 6.43 (s, 2H), 5.50 (s, 1 H), 4.17 (s, 3H), 3.98 - 3.93 (m, 1 H), 3.90 - 3.85 (m, 1 H), 3.78 (s, 6H), 3.55 (t, J = 5.6 Hz, 2H), 3.42 (s, 3H), 3.23 (s, 3H), 2.17 - 2.09 (m, 1 H), 1.88 (s, 6H), 1.86 - 1.80 (m, 2H), 1.67 -1.57 (m, 1 H); LCMS (ESI) m / z 670.1 [M + H]+.

[0285] Racemate acNTMF0-2B:1H NMR (400 MHz, DMSO-d6) δ 8.86 (s, 1 H), 8.10 (d, J = 2.0 Hz, 1H), 7.76 (s, 1 H), 7.65 (dd, J= 8.8, 2.0 Hz, 1H), 7.54 (d, J = 8.8 Hz, 1 H), 7.19 (t, J = 4.8 Hz, 1H), 6.93 (d, J= 4.8 Hz, 2H), 6.45 (s, 2H), 5.45 (s, 1 H), 4.18 (dd, J= 6.4, 4.8 Hz, 1H), 4.12 -4.06 (m, 1H), 3.98 (s, 3H), 3.84 (s, 3H), 3.75 (s, 6H), 3.73 (brs, 2H), 3.36 (s, 3H), 2.18 - 2.10(m, 1 H), 1.96 (s, 6H), 1.95 - 1.90 (m, 1 H), 1.80 - 1.72 (m, 2H); LCMS (ESI) m / z 670.1 [M + H]+.

[0286] Racemate acNTMF0-2A were separated by supercritical fluid chromatography (SFC); DAICEL CHIRALPAK IG column; mobile phase: [CO2-MeOH(0.1% NH4OH)]; 5-25%, isocratic elution mode) to give enantiomer pure acNTMF0-2A-P1 as an off-white solid (40.8 mg, >99% purity, >95% ee) and enantiomer pure acNTMF0-2A-P2 as an off-white solid (41.8 mg, >99% purity, >95% ee).

[0287] (1R*,2S*)-1-(6-Bromo-2-methoxyquinolin-3-yl)-2-(2,6-dimethoxypyridin-4-yl)-4-(dimethylamino)-1-(2-methoxy-3-(2-methoxyethoxy)phenyl)butan-2-ol

[0288]

[0289] acNTMF0-2A-P1:1H NMR (400 MHz, MeOD) δ 8.24 (s, 1H), 7.86 (d, J= 2.0 Hz, 1 H), 7.81 (d, J = 7.2 Hz, 1H), 7.71 (d, J = 9.2 Hz, 1 H), 7.64 (dd, J = 8.8, 2.0 Hz, 1 H), 6.85 (t, J = 8.0 Hz, 1 H), 6.70 (d, J = 8.4 Hz, 1 H), 6.50 (s, 2H), 5.64 (s, 1 H), 4.25 (s, 3H), 4.02 -3.91 (m, 2H), 3.84 (s, 6H), 3.65 - 3.62 (m, 2H), 3.54 (s, 3H), 3.34 (s, 3H), 2.32 - 2.26 (m, 1 H), 1.99 (s, 6H), 1.98 - 1.92 (m, 2H), 1.83 -1.77 (m, 1 H);13C NMR (100 MHz, MeOD) δ 163.2, 162.1, 161.4, 151.2, 147.2, 143.7, 139.7, 135.8, 131.8, 129.2, 128.1, 127.3, 126.6, 123.0, 122.9, 116.7, 112.4, 98.9, 80.3, 70.8, 67.7, 59.3, 57.7, 54.9, 53.1, 52.4, 43.6, 43.2, 36.4.

[0290] [a]D22= +23.69 (c= 0.89, MeOH)

[0291] HRMS (ESI-Quadrupole) m / z calc’d for [CssH^BrNsOy + H]+670.2128, found 670.2119.

[0292] Example 16. 2-(3-Bromo-5-fluoro-2-methoxyphenyl)-1,3-dioxolane

[0293] OMe O-A

[0294]

[0295] F

[0296] To a solution of 3-bromo-5-fluoro-2-methoxy-benzaldehyde (5.00 g, 21.4 mmol, 1.0 eq.) and pTsOH (369 mg, 2.15 mmol, 0.1 eq.) in toluene (50 mL) was added ethylene glycol (4.00 g, 64.4 mmol, 3.59 mL, 3.0 eq.). The reaction mixture was heated under reflux for 5 h. Upon completion, the mixture was cooled to room temperature, diluted with H2O (20 mL), extracted with EtOAC (2x20 mL), washed with saturated aqueous NaHCOs solution (20 mL) and brine(20 mL), concentrated in vacuo to give the crude mixture. The crude product was purified by column chromatography (silica gel, 5% EtOAc / hexanes) to give 2-(3-bromo- 5-fluoro-2-methoxyphenyl)-1,3-dioxolane as a colorless oil (5.4 g, 88% yield).1H NMR (400 MHz, MeOD) 8 7.41 (dd, J= 8.0, 3.2 Hz, 1 H), 7.24 (dd, J = 9.2, 3.2 Hz, 1H), 6.01 (d, J= 1.2 Hz, 1 H), 4.15 - 4.08 (m, 2H), 4.07 - 4.00 (m, 2H), 3.85 (s, 3H); LCMS (ESI) m / z 277.0 [M + H]+.

[0297] Example 17. 2-(3-(1,3-Dioxolan-2-yl)-5-fluoro-2-methoxyphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane

[0298]

[0299] F

[0300] To a solution of 2-(3-bromo-5-fluoro-2-methoxy-phenyl)-1,3-dioxolane (5.40 g, 19.5 mmol, 1.0 eq.), KOAc (3.83 g, 39.0 mmol, 2.0 eq.), and B2Pin2(5.94 g, 23.4 mmol, 1.2 eq.) in anhydrous 1,4-dioxane (60 mL) was added PdCI2(dppf) (1.43 g, 1.95 mmol, 0.1 eq.) under nitrogen atmosphere. The reaction mixture was heated to 100 °C for 5 h and cooled to room temperature. The precipitate was filtered through a plug of Celite®, washed with EtOAc (3x60 mL). The filtrate was concentrated under reduced pressure to give the crude 2-(3-(1,3-dioxolan-2-yl)-5-fluoro-2-methoxyphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (5.80 g) as a yellow oil which was used directly in the next step without further purification.

[0301] Example 18. 3-(1,3-Dioxolan-2-yl)-5-fluoro-2-methoxyphenol

[0302] OMe O-A

[0303]

[0304] F

[0305] To a solution of 2-(3-(1,3-dioxolan-2-yl)-5-fluoro-2-methoxyphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxa-borolane (5.70 g, 17.6 mmol, 1.0 eq.) and NaOH (2.11 g, 52.7 mmol, 3.0 eq.) in THF / H2O (2:1, 90 mL) was added 30% H2O2in H2O (9.97 g, 87.9 mmol, 8.45 mL, 5.0 eq.) at 0 °C. The reaction mixture was warmed to room temperature and stirred for 3 h. The reaction mixture was quenched with saturated aqueous Na2SOs solution (500 mL) and extracted with CH2CI2(2X100 mL). The combined organic layers was washed with brine (100 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give the crude product. The crude residue was purified by column chromatography (silica gel, 10% EtOAc / hexanes) to give 3-(1,3-dioxolan- 2-yl)-5-fluoro-2-methoxyphenol as a colorless oil (3.50 g, 93% yield over 2 steps).1H NMR (400 MHz, MeOD) δ 6.65 (dd, J= 8.8, 2.8 Hz, 1 H),6.59 (td, J= 9.6, 1.6 Hz, 1 H), 5.99 (s, 1H), 4.13 - 4.06 (m, 2H), 4.02 - 3.97 (m, 2H), 3.78 (s, 3H); LCMS (ESI) m / z 213.0 [M - H]

[0306] Example 19. 2-(3-(3-Bromopropoxy)-5-fluoro-2-methoxyphenyl)-1,3-dioxolane OMe O"\

[0307]

[0308] To a solution of 3-(1,3-dioxolan-2-yl)-5-fluoro-2-methoxy-phenol (100 mg, 0.47 mmol, 1.0 eq.), K2CO3 (142 mg, 1.03 mmol, 2.2 eq.), benzyltriethylammonium chloride (10.6 mg, 0.05 mmol, 0.1 eq.) in anhydrous CH3CN (10 mL) was added 1,3-dibromopropane (0.14 mL, 1.41 mmol, 3.0 eq.) dropwise. The reaction mixture was stirred at 25 °C for 3 h and quenched with H2O (20 mL). The mixture was extracted with EtOAc (3x10 mL) and the combined organic layers were washed with brine (10 mL), dried over anhydrous Na2SC>4, filtered, and concentrated under reduced pressure to provide 2-(3-(3-bromopropoxy)-5- fluoro-2-methoxyphenyl)-1,3-dioxolane (150 mg, crude) as a yellow oil which was used directly in the next step without further purification.

[0309] Example 20. 2-(3-(3-(Ethylthio)propoxy)-5-fluoro-2-methoxyphenyl)-1,3-dioxolane

[0310]

[0311] To a solution of 2-(3-(3-bromopropoxy)-5-fluoro-2-methoxyphenyl)-1,3- dioxolane (150 mg, 0.45 mmol, 1.0 eq.) and CS2CO3 (320 mg, 0.98 mmol, 2.2 eq.) in anhydrous DMF (2 mL) was added ethanethiol (83.4 mg, 1.34 mmol, 3.0 eq.). After stirred at 25 °C for 1 h, the reaction mixture was diluted with water (20 mL), extracted with EtOAc (3x10 mL), and the combined organic layers were washed with brine (10 mL), dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated in vacuo to give the crude product which was purified by preparative-TLC (silica gel, 20% EtOAc / hexanes) to provide 2-(3-(3- (ethylthio)propoxy)-5-fluoro-2-methoxyphenyl)-1,3-dioxolane as a yellow oil (50 mg, 35% yield over 2 steps).1H NMR (400 MHz, MeOD) δ 6.84 (dd, J = 10.4, 2.8 Hz, 1 H), 6.75 (dd, J = 8.8, 2.8 Hz, 1 H), 6.01 (s, 1H), 4.14 - 4.08 (m, 4H), 4.02 - 3.97 (m, 2H), 3.81 (s, 3H), 2.75 (t, J = 7.2 Hz, 2H), 2.56 (q, J= 7.6 Hz, 2H), 2.08 (quint, J= 6.4 Hz, 2H), 1.25 (t, J= 7.6 Hz, 3H). LCMS (ESI) m / z 317.4 [M + H]+.

[0312] Example 21. 3-(3-(Ethylthio)propoxy)-5-fluoro-2-methoxybenzaldehydeOMe O

[0313]

[0314] F

[0315] To a solution of 2-(3-(3-(ethylthio)propoxy)-5-fluoro-2-methoxyphenyl)-1,3-dioxolane (1.40 g, 4.42 mmol, 1.0 eq.) in anhydrous THF (7 mL) was added 2.0 M HCI in 1,4-dioxane (7 mL) at 0 °C and stirred for 2 h. The resultant mixture was warmed to room temperature and quenched with H2O (10 mL), basified with Na2COs to pH 8 at 0 °C, followed by extraction with EtOAc (3x30 mL). The combined organic layers was washed with brine (30 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give the crude mixture. The residue was purified by column chromatography (silica gel, 20% EtOAc / hexanes) to give 3-(3-(ethylthio)propoxy)-5-fluoro-2-methoxy-benzaldehyde as a colorless oil (1.00 g, 72% yield).1H NMR (400 MHz, CDCI3) 5 10.38 (d, J = 3.2 Hz, 1 H), 7.08 (dd, J = 8.0, 3.2 Hz, 1 H), 6.90 (dd, J = 9.6, 3.2 Hz, 1H), 4.14 (t, J = 6.0 Hz, 2H), 3.96 (s, 3H), 2.77 (t, J= 7.2 Hz, 2H), 2.58 (q, J = 7.2 Hz, 2H), 2.16 (quint, J = 6.4 Hz, 2H), 1.29 (t, J = 7.6 Hz, 3H). LCMS (ESI) m / z 273.3 [M + H]+.

[0316] Example 22. (E)-W-(3-(3-(Ethylthio)propoxy)-5-fluoro-2-methoxybenzylidene)-4-methyl benzenesulfono-hydrazide

[0317]

[0318] F

[0319] To a solution of 3-(3-ethylsulfanylpropoxy)-5-fluoro-2-methoxy-benzaldehyde (1.00 g, 3.19 mmol, 1.0 eq.) in anhydrous MeOH (10 mL) was added 4-methylbenzene sulfonohydrazide (594 mg, 3.19 mmol, 1.0 eq.). The reaction mixture was stirred at room temperature for 60 min. The resultant mixture was concentrated under reduced pressure to give the crude mixture which was purified by column chromatography (silica gel, 20% EtOAc / hexanes) to give (£)- / V-(3-(3-(ethylthio)propoxy)-5-fluoro-2-methoxybenzylidene)-4 methylbenzenesulfonohydrazide as a colorless oil (1.40 g, 95% yield).1H NMR (400 MHz, CDCh) 58.04 (d, J = 2.0 Hz, 1 H), 7.94 - 7.83 (m, 3H), 7.33 (d, J = 8.0 Hz, 2H), 7.08 (dd, J = 9.2, 3.2 Hz, 1 H), 6.66 (dd, J= 9.6, 2.8 Hz, 1 H), 4.07 (t, J= 6.0 Hz, 2H), 3.77 (s, 3H), 2.73 (t, J = 7.2 Hz, 2H), 2.56 (q, J = 7.2 Hz, 2H), 2.43 (s, 3H), 2.11 (t, J = 6.8 Hz, 2H), 1.27 (d, J = 1.2 Hz, 3H). LCMS (ESI) m / z 441.5 [M + H]+.

[0320] Example 23. 6-Bromo-3-(3-(3-(ethylthio)propoxy)-5-fluoro-2-methoxybenzyl)-2-methoxyquinoline

[0321]

[0322] To a solution of (E)- / V-(3-(3-(ethylthio)propoxy)-5-fluoro-2-methoxybenzylidene)-4-methylbenzene-sulfonohydrazide (0.68 g, 1.53 mmol, 1.0 eq.), K2CO3 (423 mg, 3.06 mmol, 2.0 eq.) and (6-bromo-2-methoxy-3-quinolyl)boronic acid (648 mg, 2.30 mmol, 1.5 eq.) in anhydrous 1,4-dioxane (7 mL) was heated to 110 °C and stirred for 3 h. Upon completion, the reaction mixture was cooled to room temperature, diluted with water (20 mL), and extracted with ethyl acetate (3x50 mL). The combined organic layers was washed with brine (50 mL), dried over anhydrous Na2SO, filtered, and concentrated under reduced pressure to afford the crude mixture which was purified by column chromatography (silica gel, 5% EtOAc / hexanes) to give 6-bromo-3-(3-(3-(ethylthio)propoxy)-5-fluoro-2-methoxybenzyl)-2-methoxyquinoline as an off-white solid (160 mg, 10% yield).1H NMR (400 MHz, CDCh) 57.77 (d, J= 2.0 Hz, 1 H), 7.72 - 7.66 (m, 1 H), 7.65 - 7.59 (m, 1 H), 7.50 (s, 1 H), 6.59 (dd, J = 2.8, 10.0 Hz, 1 H), 6.46 (dd, J = 2.8, 8.8 Hz, 1 H), 4.13 - 4.06 (m, 5H), 4.01 (s, 2H), 3.75 (s, 3H), 2.76 (t, J= 7.2 Hz, 2H), 2.58 (q, J = 7.2 Hz, 2H), 2.14 (quint, J = 6.4 Hz, 2H), 1.31 - 1.27 (m, 3H); LCMS (ESI) m / z 494.4 [M + H]+.

[0323] Example 24. 1-(6-Bromo-2-methoxyquinolin-3-yl)-2-(2,6-dimethoxypyridin-4-yl)-4-(dimethylamino)-1-(3-(3-(ethylthio)propoxy)-5-fluoro-2-methoxyphenyl)butan-2-ol

[0324]

[0325] A solution of 2,2,6,6-tetramethylpiperidine (0.1 mL, 0.70 mmol, 2.5 eq.) in anhydrous THE (2 mL) was cooled to -40 °C, and n-BuLi (0.28 mL of a 2.0 N solution in cyclohexane, 0.56 mmol, 2.0 eq.) was added and the solution was stirred at -40 °C for 15 min, then cooled to -78 °C. A solution of 6-bromo-3-(3-(3-(ethylthio)propoxy)-5-fluoro-2-methoxybenzyl)-2-methoxyquinoline (140 mg, 0.28 mmol, 1.0 eq.) in anhydrous THF (3 mL) was added dropwise, and the orange solution was stirred at -78 °C for 1 h, then a solution of 1-(2,6-dimethoxypyridin-4-yl)-3-(dimethylamino)propan-1-one (101 mg, 0.43 mmol, 1.5 eq.) in anhydrous THF (3 mL) was added dropwise. The reaction mixture was stirred at -78 °C for 1h, then water (1 mL) was added and the solution was allowed to warm to room temperature, then quenched with water (20 mL). The resulting mixture was extracted with EtOAc (3x30 mL) and the combined organic layers were washed with brine (20 mL), dried over anhydrous Na2SC>4, filtered, and concentrated under reduced pressure to give the residue mixture which was purified by reversed-phase flash (5-95% CH3CN / H2O; 0.1% formic acid) and preparative HPLC (42-62% MeCN / H2O; 0.1% formic acid) to afford racemate acNTMF0-3A as an off-white solid (20 mg, 9% yield) and racemate acNTMF0-3B as an off-white solid (14 mg, 6% yield).

[0326] Racemate acNTMF0-3A:1H NMR (400 MHz, DMSO-d6) 5 8.25 - 8.17 (m, 2H), 7.73 - 7.69 (m, 2H), 7.59 - 7.52 (m, 1H), 6.64 (dd, J = 3.2, 10.4 Hz, 1 H), 6.44 (s, 2H), 5.48 (s, 1 H), 4.17 (s, 3H), 3.96 - 3.83 (m, 2H), 3.80 (s, 6H), 3.38 (s, 3H), 2.58 - 2.53 (m, 2H), 2.45 (q, J = 7.6 Hz, 2H), 2.18 - 2.06 (m, 1 H), 1.88 (s, 6H), 1.87 - 1.78 (m, 4H), 1.64 - 1.52 (m, 1 H), 1.11 (t, J = 7.2 Hz, 3H); LCMS (ESI) m / z 732.3 [M + H]+.

[0327] Racemate acNTMF0-3B:1H NMR (400 MHz, DMSO-d6) 5 8.80 (s, 1 H), 8.13 (d, J = 2.0 Hz, 1H), 7.83 (brs, 1 H), 7.65 (dd, J= 2.4, 8.8 Hz, 1H), 7.55 - 7.50 (m, 1 H), 6.98 (dd, J= 3.2, 10.4 Hz, 1 H), 6.86 (dd, J = 2.8, 10.4 Hz, 1 H), 6.41 (s, 2H), 5.41 (s, 1 H), 4.16 - 4.04 (m, 2H), 3.90 (s, 3H), 3.82 (s, 3H), 3.73 (s, 6H), 2.75 - 2.65 (m, 2H), 2.57 - 2.53 (m, 2H), 2.20 - 2.10 (m, 1 H), 2.07 - 2.00 (m, 2H), 1.97 (s, 6H), 1.78 - 1.69 (m, 2H), 1.23 (brs, 1 H), 1.19 (t, J = 7.2 Hz, 3H); LCMS (ESI) m / z 732.3 [M + H]+.

[0328] Racemate acNTMF0-3A were separated by supercritical fluid chromatography (SFC); DAICEL CHIRALPAK AD column; mobile phase: [CO2-iPrOH(0.1% NH4OH)]; 40%, isocratic elution mode) to give enantiomer pure acNTMF0-3A-P1 as an off-white solid (12.1 mg, >99% purity, >95% ee) and enantiomer pure acNTMF0-3A-P2 as an off-white solid (13.6 mg, >99% purity, >95% ee).

[0329] (1R*,2S*)-1-(6-Bromo-2-methoxyquinolin-3-yl)-2-(2,6-dimethoxypyridin-4-yl)-4-(dimethylamino)-1-(3-(3-(ethylthio)propoxy)-5-fluoro-2-methoxyphenyl)butan-2-ol

[0330]

[0331] acNTMF0-3A-P2:1H NMR (400 MHz, MeOD) δ 8.21 (s, 1H), 7.92 (s, 1H), 7.72 – 7.59 (m, 3H), 6.51 (brs, 3H), 5.61 (s, 1H), 4.26, 3.94 – 3.86 (m, 2H), 3.86 (s, 6H), 3.50 (s, 3H), 2.62 (t, J = 6.4 Hz, 2H), 2.48 (q, J = 7.2 Hz, 2H), 2.31 -2.27 (m, 1 H), 1.99 (s, 6H), 1.99 - 1.94 (m, 4H),1.79 - 1.73 (m, 1 H), 1.18 (t, J = 7.2 Hz, 3H);13C NMR (100 MHz, MeOD) 5163.3, 161.8, 161.3, 159.5, 157.2, 152.1, 152.0, 143.8, 143.2, 143.1, 139.5, 136.7, 136.6, 131.9, 129.3, 128.1, 126.6, 126.5, 129.3, 128.1, 126.6, 126.5, 116.8, 108.3, 108.1, 99.7, 99.4, 98.7, 80.2, 66.9, 59.5, 54.9, 53.2, 52.4, 43.6, 43.2, 36.2, 28.9, 27.3, 25.1, 13.7

[0332] [a]o22= +60.75 (c= 0.43, MeOH)

[0333] HRMS (ESI-Quadrupole) m / z calc’d for [Css^sBrSFNsOe + H]+732.2118, found 732.2104.

[0334] Example 25. 3-Bromo-2-hydroxy-5-(trifluoromethoxy)benzoic acid

[0335] CK. OH

[0336] 1 1

[0337]

[0338] F3CO'^^'Br

[0339] A mixture of 2-hydroxy-5-(trifluoromethoxy)benzoic acid (8.40 g, 37.8 mmol, 1.0 eq.) and NBS (6.73 g, 37.8 mmol, 1.0 eq.) in anhydrous DMF (50 mL) was heated to 70 °C for 2 h. The reaction mixture was cooled to room temperature and diluted with H2O (300 mL), and extracted with EtOAc (3x300 mL). The combined organic layers were washed with H2O (3x300 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give the resultant mixture. The residue was purified by reversed phase flash chromatography (5-95% MeCN / H2O; 0.1% formic acid) to give 3-bromo-2-hydroxy-5-(trifluoromethoxy)benzoic acid (8.90 g, 77% yield) as a yellow solid.1H NMR (400 MHz, DMSO-d6) δ 13.80 – 9.21 (m, 2H), 7.96 (d, J = 2.8 Hz, 1 H), 7.73 - 7.70 (m, 1 H); LCMS (ESI) m / z 301.1 [M + H]+.

[0340] Example 26. Methyl 3-bromo-2-methoxy-5-(trifluoromethoxy)benzoate

[0341] Ck JDMe

[0342] ^L. OMe

[0343] JQ

[0344] F

[0345]

[0346] 3CO'^^'Br

[0347] To a mixture of 3-bromo-2-hydroxy-5-(trifluoromethoxy)benzoic acid (8.90 g, 29.6 mmol, 1.0 eq.) and K2CO3 (12.3 g, 88.7 mmol, 3.0 eq.) in anhydrous DMF (90 mL) was added dimethyl sulfate (18.7 g, 148 mmol, 14.0 mL, 5.0 eq.) in one portion at 0 °C under nitrogen atmosphere. The reaction mixture was warmed and stirred at 25 °C for 2 h. Upon completion, the reaction mixture was cooled to 0 °C, then quenched by cautious addition water (30 mL) under argon atmosphere. The layers were separated and the aqueous layer was extracted with EtOAc (3x30 mL). The combined organic layers were washed with the saturated aqueous sodium chloride solution (30 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give the crude mixture. The residue was purified by column chromatography (silica gel, 5% EtOAc / petroleum ether) to give methyl 3-bromo-2-methoxy-5-(trifluoromethoxy)benzoate as a yellow solid (8.60 g, 88% yield).1H NMR (400 MHz, DMSO-d6) 88.03 (d, J= 2.8 Hz, 1H), 7.71 (d, J= 2.4 Hz, 1 H), 3.88 (s, 3H), 3.83 (s, 3H); LCMS (ESI) m / z 329.1 [M + H]+.

[0348] Example 27. (2-Methoxy-3-(methoxycarbonyl)-5-(trifluoromethoxy)phenyl)boronic acid O^OMe

[0349] X, OMe

[0350] F

[0351]

[0352] 3CO'XX^B(OH)2

[0353] To a mixture of methyl 3-bromo-2-methoxy-5-(trifluoromethoxy)ben-zoate (4.0 g, 12.2 mmol, 1.0 eq.) and 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane (3.40 g, 13.4 mmol, 1.1 eq.) in anhydrous dioxane (40 mL) was added potassium acetate (2.39 g, 24.3 mmol, 2.0 eq.) and Pd(dppf)CI2(889 mg, 1.22 mmol, 0.1 eq.) in one portion under nitrogen atmosphere. The reaction mixture was heated to 100 °C for 1 h. Upon completion, the reaction mixture was cooled to room temperature and filtered through a plug of Celite®, washed with EtOAc (3x30 mL), and the combined organic layers were concentrated under reduced pressure to get the resultant mixture. The residue was purified by reversed phase flash chromatography (5-95% MeCN / H2O; 0.1% formic acid) to give 2-methoxy -3-methoxycarbonyl-5-(trifluoromethoxy)phenylboronic acid as a yellow oil (2.60 g, 73% yield).1H NMR (400 MHz, DMSO-d6) 8 8.41 (s, 2H), 7.59 (d, J= 2.4 Hz, 1H), 7.51 (d, J = 2.4 Hz, 1H), 3.84 (s, 3H), 3.78 (s, 3H).

[0354] Example 28. Methyl 3-hydroxy-2-methoxy-5-(trifluoromethoxy)benzoate

[0355] (X ^OMe

[0356] I zOMe

[0357] 1 JL

[0358] F

[0359]

[0360] 3CO^^^OH

[0361] To a mixture of 2-methoxy-3-methoxycarbonyl-5-(trifluoromethoxy) phenyl boronic acid (4.50 g, 15.3 mmol, 1.0 eq.) in anhydrous THF (45 mL) was added Oxone (3.86 g, 22.9 mmol, 1.5 eq.) in H2O (45 mL). After stirred at 25 °C for 1 h, the reaction mixture was diluted with water (150 mL) and extracted with ethyl acetate (3x150 mL). The combined organic layers were washed with sodium bisulfite solution (150 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (silica gel, 10-50% EtOAc / petroleum ether) to give methyl 3-hydroxy-2-methoxy-5-(trifluoromethoxy)benzoate as a yellow oil (2.95 g, 56% yield).1H NMR (400 MHz, DMSO-d6) 8 10.44 (brs, 1 H), 7.01 (d, J = 1.2 Hz, 2H), 3.82 (s, 3H), 3.76 (s, 3H); LCMS (ESI) m / z 267.1 [M + H]+.Example 29. Methyl 3-(3-bromopropoxy)-2-methoxy-5-(trifluoromethoxy)benzoate CK ^OMe

[0362] ±, OMe

[0363] F

[0364]

[0365] 3CO' 1^ A5^O'^X-^XBr

[0366] To a solution of methyl 3-hydroxy-2-methoxy-5-(trifluoromethoxy) benzoate (2.65 g, 9.96 mmol, 1.0 eq.), K2CO3 (4.13 g, 29.9 mmol, 3.0 eq.), benzyl-triethylammonium chloride (227 mg, 996 pmol, 0.1 eq.) in anhydrous CH3CN (53 mL) was added 1,3-dibromopropane (7.03 g, 34.9 mmol, 3.55 mL, 3.5 eq.). The reaction mixture was stirred at 25 °C for 2 h. The reaction mixture was diluted with water (80 mL) and extracted with ethyl acetate (2x250 mL). The combined organic layers were washed with brine (90 mL), dried over anhydrous Na2SC>4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (silica gel, 5-70% EtOAc / hexanes) to give methyl 3-(3-bromopro-poxy)-2-methoxy-5(trifluoromethoxy)benzoate as a yellow oil (3.32 g, 73% yield).1H NMR (400 MHz, CDCl3) δ 7.22 (d, J = 1.6 Hz, 1H), 6.94 (d, J= 2.8 Hz, 1 H), 4.17 (t, J= 5.6 Hz, 2H), 3.93 (s, 3H), 3.90 (s, 3H), 3.65 (t, J= 6.4 Hz, 2H), 2.39 (quint, J = 6.0 Hz, 2H); LCMS (ESI) m / z 387.1 [M + H]+.

[0367] Example 30. Methyl 3-(3-(ethylthio)propoxy)-2-methoxy-5-(trifluoromethoxy)benzoate

[0368]

[0369] To a solution of methyl 3-(3-bromopropoxy)-2-methoxy-5(trifluoro-methoxy) benzoate (3.00 g, 7.75 mmol, 1.0 eq.) in anhydrous DMF (30 mL) was added CS2CO3 (6.31 g, 19.4 mmol, 2.5 eq.) and ethanethiol (2.41 g, 38.8 mmol, 5.0 eq.) in anhydrous DMF (30 mL). The reaction mixture was stirred at 25 °C for 2 h. The mixture was diluted with ethyl acetate (40 mL), washed with water (3x60 mL). The combined organic layers were washed with brine (60 mL), dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated in vacuo to give the crude mixture. The residue was purified by column chromatography (silica gel, 5-50% EtOAc / hexanes) to afford methyl 3-(3-(ethylthio)propoxy)-2-methoxy -5-(trifluoromethoxy)benzoate as a yellow oil (2.10 g, 66% yield).1H NMR (400 MHz, CDCl3) 8 7.60 (dd, J= 2.8, 1.2 Hz, 1 H), 7.01 (d, J = 2.8 Hz, 1 H), 4.18 (t, J= 6.0 Hz, 2H), 4.10 (s, 3H), 2.76 (t, J= 7.2 Hz, 2H), 2.59 (q, J= 7.2 Hz, 2H), 2.17 (quint, J= 6.4 Hz, 2H), 1.32 - 1.27 (m, 3H); LCMS (ESI) m / z 369.3 [M + H]+.Example 31. (3-(3-(Ethylthio)propoxy)-2-methoxy-5-(trifluoromethoxy)phenyl)methanol OH

[0370] .. OMe

[0371]

[0372] To a solution of methyl 3-(3-ethylsulfanylpropoxy)-2-methoxy-5-(tri-fluoromethoxy)benzoate (1.90 g, 5.16 mmol, 1.0 eq.) in anhydrous THF (20 mL) was added LiAIHU (2.5 M in THF, 4.13 mL, 2.0 eq.) at 0 °C. The reaction mixture was stirred at 25 °C for 1 hour. The mixture was acidified with diluted hydrochloric acid to pH < 3, diluted with EtOAc (30 mL), washed with water (30 mL) and brine (30 mL), filtered, and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (silica gel, 5-50% EtOAc / hexanes) to afford (3-(3-(ethylthio) propoxy)-2-methoxy-5-(trifluoromethoxy)phenyl)methanol as a yellow oil (1.65 g, 85% yield).1H NMR (400 MHz, CDCl3) δ 6.85 (d, J = 1.6 Hz, 1H), 6.74 (d, J = 2.4 Hz, 1 H), 4.70 (s, 2H), 4.11 (t, J = 6.0 Hz, 2H), 3.88 (s, 3H), 2.75 (t, J= 7.2 Hz, 2H), 2.60 -2.55 (m, 2H), 2.13 (t, J= 6.8 Hz, 2H), 1.30 -1.26 (m, 3H); LCMS (ESI) m / z 340.3 [M + H]+.

[0373] Example 32.3-(3-(Ethylthio)propoxy)-2-methoxy-5-(trifluoromethoxy)benzaldehyde

[0374] OMe

[0375]

[0376] O’

[0377] To a solution of [3-(3-ethylsulfanylpropoxy)-2-methoxy-5-(trifluoro-methoxy)phenyl]methanol (1.55 g, 4.55 mmol, 1.0 eq.) in anhydrous CH2CI2(40 mL) was added MnO2(3.96 g, 45.5 mmol, 10.0 eq.). The reaction mixture was stirred at 25 °C for 2 h. After completion, the reaction mixture was filtered through a plug of Celite®, washed with CH2CI2(2x40 mL), and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (silica gel, 5-50% EtOAc / hexanes) to afford 3-(3-ethylsulfanylpropoxy)-2-methoxy-5 (trifluoromethoxy) benzaldehyde (1.45 g, 85% yield) as a yellow oil.1H NMR (400 MHz, CDCl3) 8 10.40 (s, 1H), 7.29 - 7.27 (m, 1 H), 6.99 (d, J= 2.4 Hz, 1H), 4.16 (t, J = 6.0 Hz, 2H), 4.01 (s, 3H), 2.77 (t, J = 7.2 Hz, 2H), 2.59 (q, J = 7.2 Hz, 2H), 2.17 (quint, J= 6.4 Hz, 2H), 1.29 (t, J= 7.2 Hz, 3H); LCMS (ESI) m / z 339.3 [M + H]+.

[0378] Example 33. (E)-A / '-(3-(3-(Ethylthio)propoxy)-2-methoxy-5- (trifluoromethoxy)benzylidene)-4-methylbenzene-sulfonohydrazide

[0379]

[0380] OCF3

[0381] To a solution of 3-(3-ethylsulfanylpropoxy)-2-methoxy-5 (trifluorome-thoxy)benzaldehyde (1.45 g, 4.29 mmol, 1.0 eq.) and 4-methylbenzenesulfono-hydrazide (798 mg, 4.29 mmol, 1.0 eq.) in anhydrous 1,4-dioxane (15 mL) was added pTsOH (369 mg, 2.14 mmol, 0.5 eq.). The mixture was stirred at 60 °C for 2 h. The reaction mixture was diluted with water (50 mL) and extracted with ethyl acetate (2x50 mL). The combined organic layers were washed with brine (70 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (silica gel, 2-50% EtOAc / hexanes) to give A / -[(E)-[3-(3-ethylsulfanylpropoxy)-2-metho-xy-5-(trifluoro-methoxy)phenyl]methyleneamino]-4-methylbenzene-sulfonamide as a yellow oil (1.72 g, 73% yield).1H NMR (400 MHz, DMSO-d6) δ 11.60 (brs, 1H), 8.11 (s, 1 H), 7.74 (d, J= 8.4 Hz, 2H), 7.40 (d, J = 8.0 Hz, 2H), 7.15 - 7.00 (m, 2H), 4.11 (t, J = 6.0 Hz, 2H), 3.74 (s, 3H), 2.65 (t, J = 7.2 Hz, 2H), 2.53 (d, J = 6.8 Hz, 2H), 2.36 (s, 3H), 2.00 - 1.96 (m, 2H), 1.17 (t, J = 7.2 Hz, 3H); LCMS (ESI) m / z 507.1 [M + H]+.

[0382] Example 34. 6-Bromo-3-(3-(3-(ethylthio)propoxy)-2-methoxy-5- (trifluoromethoxy)benzyl)-2-methoxyquino-line

[0383] OMe

[0384]

[0385] OCF3

[0386] To a solution of / V-[(E)-[3-(3-ethylsulfanylpropoxy)-2-methoxy-5 (tri-fluoromethoxy)phenyl]methyl-eneamino]-4-methyl-benzenesulfonamide (1.60 g, 3.16 mmol, 1.0 eq.) and (6-bromo-2-methoxy-3-quinolyl)boronic acid (1.34 g, 4.74 mmol, 1.5 eq.) in anhydrous dioxane (30 mL) was added K2CO3(1.75 g, 12.6 mmol, 4.0 eq.). The mixture was stirred at 100 °C for 3 h. The reaction mixture was diluted with water (80 mL) and extracted with ethyl acetate (2x50 mL). The combined organic layers were washed with brine (90 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The crude product was purified by reversed-phase HPLC (5-95% MeCN / H2O; 0.1% formic acid) to give 6-bromo-3-(3-(3-(ethylthio)-propoxy)-2-methoxy-5-(trifluoro-methoxy)benzyl)-2-methoxyquino-line as a white solid (490 mg, 25% yield).1H NMR (400 MHz, DMSO-d6) δ 8.12 (d, J = 1.6 Hz, 1H), 7.84 (s, 1 H), 7.73 - 7.67 (m, 2H), 6.99 (d, J = 2.4 Hz, 1H), 6.75 (d, J= 1.6 Hz, 1 H), 4.10 (t, J= 6.0 Hz, 2H), 4.01 (brs, 2H), 3.99 (s, 3H), 3.69 (s, 3H),2.66 (t, J = 7.2 Hz, 2H), 2.53 (d, J = 5.6 Hz, 2H), 2.01 - 1.97 (m, 2H), 1.19 - 1.15 (m, 3H); LCMS (ESI) m / z 560.1 [M + H]+.

[0387] Example 35. 1-(6-Bromo-2-methoxyquinolin-3-yl)-2-(2,6-dimethoxypyridin-4-yl)-4-(dimethylamino)-1-(3-(3-(ethylthio)propoxy)-2-methoxy-5-(trifluoromethoxy)phenyl)butan-2-ol

[0388]

[0389] A solution of 2,2,6,6-tetramethylpiperidine (0.19 mL, 1.13 mmol, 2.5 eq.) in anhydrous THF (2 mL) was cooled to -40 °C, and n-BuLi (0.45 mL of a 2.0 N solution in cyclohexane, 0.9 mmol, 2.0 eq.) was added and the solution was stirred at -40 °C for 15 min, then cooled to -78 °C. A solution of 6-bromo-3-[[3-(3-ethylsulfanylpropoxy)-2-methoxy-5-(trifluoromethoxy)phenyl]methyl]-2-methoxyquinoline (250 mg, 0.45 mmol, 1.0 eq.) in anhydrous THF (5 mL) was added dropwise, and the orange solution was stirred at -78 °C for 30 min, then a solution of 1-(2,6-dimethoxypyridin-4-yl)-3-(dimethylamino)propan-1-one (159 mg, 0.67 mmol, 1.5 eq.) in anhydrous THF (3 mL) was added dropwise. The reaction mixture was stirred at -78 °C for 1 h, then water (1 mL) was added and the solution was allowed to warm to room temperature, then quenched with water (20 mL). The resulting mixture was extracted with EtOAc (3x30 mL) and the combined organic layers were washed with brine (20 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give the residue mixture which was purified by reversed-phase flash (5-95% CH3CN / H2O; 0.1% formic acid) to afford racemate acNTMF0-4A as an off-white solid (26 mg, 7% yield) and racemate acNTMF0-4B as an off-white solid (30 mg, 8% yield).

[0390] Racemate acNTMF0-4A:1H NMR (400 MHz, DMSO-d6) δ 8.13 (d, J = 11.2 Hz, 2H), 7.80 (brs, 1H), 7.73 - 7.69 (m, 2H), 6.74 (d, J= 2.8 Hz, 1H), 6.43 (brs, 2H), 5.46 (s, 1H), 4.18 (s, 3H), 3.96 - 3.92 (m, 1 H), 3.90 - 3.86 (m, 1 H), 3.78 (s, 6H), 3.41 (s, 3H), 2.57 - 2.53 (m, 2H), 2.47 - 2.41 (m, 2H), 2.28 - 2.16 (m, 1 H), 1.97 (d, J= 0.8 Hz, 6H), 1.94 - 1.89 (m, 2H), 1.88 - 1.83 (m, 2H), 1.65 - 1.57 (m, 1 H), 1.11 (t, J = 7.2 Hz, 3H); LCMS (ESI) m / z 798.3 [M + H]+.

[0391] Racemate acNTMF0-4B:1H NMR (400 MHz, DMSO-d6) 5 8.78 (s, 1H), 8.34 (s, 1 H), 8.14 (d, J= 2.4 Hz, 1H), 7.65 (dd, J = 8.8, 2.4 Hz, 1 H), 7.53 (d, J = 8.8 Hz, 1H), 7.27 (d, J= 1.6 Hz, 1 H), 6.97 (d, J = 2.4 Hz, 1 H), 6.42 (s, 2H), 5.43 (s, 1 H), 4.15 - 4.08 (m, 2H), 3.94 (s, 3H), 3.82(s, 3H), 3.73 (s, 6H), 2.72 - 2.67 (m, 2H), 2.56 - 2.52 (m, 2H), 2.19 - 2.11 (m, 1 H), 2.07 -2.02 (m, 1 H), 2.02 - 1.98 (m, 2H), 1.96 (s, 6H), 1.74 - 1.68 (m, 2H), 1.19 (t, J = 7.2 Hz, 3H); LCMS (ESI) m / z 798.3 [M + H]+.

[0392] Racemate acNTMF0-4A were separated by supercritical fluid chromatography (SFC); DAICEL CHIRALPAK IG column; mobile phase: [CO2-EtOH(0.1% NH4OH)]; 15%, isocratic elution mode) to give enantiomer pure acNTMF0-4A-P1 as an off-white solid (11.5 mg, >99% purity, >95% ee) and enantiomer pure acNTMF0-4A-P2 as an off-white solid (10.5 mg, >99% purity, >95% ee).

[0393] (1F?'.2S*)-1 -(6-Bromo-2-methoxyquinolin-3-yl)-2-(2,6-dimethoxypyridin-4-yl)-4-(dimethylamino)-1-(3-(3-(ethylthio)propoxy)-2-methoxy-5-(trifluoromethoxy)phenyl)butan-2-ol

[0394]

[0395] acNTMF0-4A-P1:1H NMR (400 MHz, MeOD) S 8.14 (s, 1H), 7.87 (d, J= 1.6 Hz, 1 H), 7.84 (d, J = 1.2 Hz, 1 H), 7.73 (d, J = 9.2 Hz, 1 H), 7.67 (d, J = 9.2 Hz, 1 H), 6.63 (d, J = 2.8 Hz, 1 H), 6.50 (brs, 2H), 5.60 (s, 1H), 4.26 (s, 3H), 4.00 -3.91 (m, 2H), 3.86 (s, 6H), 3.53 (s, 3H), 2.63 (t, J = 7.2 Hz, 2H), 2.48 (q, J = 7.6 Hz, 2H), 2.32 – 2.27 (m, 1 H), 2.00 (s, 6H), 2.00 - 1.92 (m, 4H), 1.80 - 1.73 (m, 1 H), 1.18 (t, J = 7.2 Hz, 3H);13C NMR (100 MHz, MeOD) 8 163.4, 161.6, 161.3, 151.9, 145.7, 144.2, 143.8, 139.5, 136.7, 132.0, 129.2, 128.2, 126.5, 116.8, 114.8, 105.4, 98.7, 80.2, 67.0, 59.4, 54.9, 53.2, 52.4, 43.6, 43.4, 36.1, 28.9, 27.3, 25.1, 13.7 [α]D22= +61.59 (c = 0.51, MeOH)

[0396] HRMS (ESI-Quadrupole) m / z calc’d for [Cse^sBrSFsNsOy + H]+ 798.2035, found 798.2048.

[0397] Example 36. Novel C118 analogs

[0398] Computational method

[0399] Ligand preparation: BDQ and its derivative C118 were sketched using 2D-sketcher and 2D structures were converted into 3D structures and energy minimised using OPLS3e force field in ligand prep tool in Maestro 12.5 Schrodinger suite of programs (Schrodinger Release 2020-4: Maestro, force fields, macromodel, prime, protein preparation wizard, ligprep, glide Schrodinger, LLC, New York, NY, 2019).Protein and grid preparation: The M. smegmatis Fo-domain coordinates (PDB (Protein Data Bank): 7JGC (Zhang, Y. etal., Nature 2024, 631, 409-414)) were utilised for molecular docking studies. Both the Fo-domain coordinates are evaluated for missing side chain atoms and the same were rebuilt using Prime during pre-processing step of protein preparation. Afterwards, any overlapping H-atoms were optimized during refinement step and finally the energy minimization of the prepared protein was carried out using restrained minimization using OPLS3e forcefield. Next, the lead site bound BDQ grids were prepared on lead / lag sites bound BDQ, using the default parameters within the receptor grid generation tool of maestro Schrodinger suite of programs.

[0400] Docking: Standard precision and Extra precision settings were used with option to add Epik state penalties to docking score, reward intra-molecular hydrogen bonds, enhance planarity of conjugated π groups and to output 20 poses for each of the ligand.

[0401] Results and discussion

[0402] The BDQ-saturated cryo-EM structure of the M. smegmatis F1F0 ATP synthase has highlighted that the compound docks at the leading and lagging sites of half channels of Fo-domain and blocks rotation during ATP synthesis and ATP hydrolysis, respectively (Guo, H. etal., Nature 2021, 589, 143-147).

[0403] To further optimize BDQ against M. avium, a structural model of the M. avium Fo-domain (FIG.

[0404] 1 A) was generated based on the homology of the Fo subunits and performed docking studies. The structure of the M. avium Fo-domain was modelled based on the cryo-EM structure of M. smegmatis Fo-part (Guo, H. et al., Nature 2021, 589, 143-147), wherein the BDQ was seen to bind to the leading and lagging sites of the subunits a-cg interface as well as to the c-ring, as described for the BDQ-bound structure of the M. smegmatis F1F0 ATP synthase (Guo, H. et al., Nature 2021, 589, 143-147). The electrostatic surface potential surface mapping (FIG. 1A) has highlighted a very deep cavity in the lead interface (marked in dots of FIG. 1 A) near the 3rdposition of BDQ’s phenyl ring (An) and a cavity near the 5thposition of An, which could be exploited for extending new BDQ analogs into the half-channels of the a-c interface, being responsible for proton translocation and therefore ATP synthesis. Contrastingly, the leading site of the a-cg interface of the M. tuberculosis / M. smegmatis half-channel had a shallow or narrow space with a phenylalanine (aF213) residue anchoring the a-c interface and might not tolerate any more substitutions on the 3rdposition of the phenyl ring (aF213, not shown for clarity), as the phenyl (An) ring of BDQ appear to dock tightly at residue aF213 (FIG. 1 B). However, this cavity was much wider due to the variation of aF213 into an Alanine residue(A213, not shown for clarity) of the M. avium a subunit, providing additional pocket to anchor fragment growth in M. avium (FIG. 1A). With these observations, we have used the 3rd(or meta)-position of phenyl (An) as ‘R group’ optimization point and grown the ligand into additional pocket at leading a-cg interface of the half channel and obtained a focused library of fragment hits (data not shown).

[0405] We have also modelled the TBAJ-876 bound form, which has substitution on the phenyl (An), naphthyl (Ar2) rings of the parental BDQ molecule (FIG. 1 B, RHS). The docked poses also maintain the posing as seen in the cryo-EM structures and possibilities to target the additional crevices that were available in the M. avium F-ATP synthase (FIG. 1 B). Because of the of improved pharmacokinetic properties compared to BDQ, lower cytotoxicity and a similar efficacy to that of BDQ against M. avium, structure-based ligand design, including in silico screening and free energy calculations have been performed. Among the best fragments identified, a linear fragment, 3-propoxy-thio-ethyl-2-methoxy-phenyl was predicted to bind with a glide score of -8.3 kcal / mol (FIG. 1C). This novel analog was called C118. As shown in FIG.

[0406] 1C, the crucial interaction of the BDQ dimethylamine (DMA) moiety with amino acid E63 of subunit c as well as 6-bromo-quioline were maintained intact in most of the docked C118 poses. As envisaged, the newly extended 3-propoxy-thio-ethyl fragment on 2-methoxy-phenyl (Ari group) was seen to nicely fit into the additional pocket and was engaged in additional sulphur-π interactions, alkyl interactions of the ethyl moiety with residues al210 (4.4 A), aW212 (4.0 A) and aP214 forming the crevice on the a-c interface.

[0407] A protocol was developed to synthesize C118, which is summarized in FIG. 2.

[0408] Structural modelling of the C118-bound M. avium Fo-domain shed light into the binding epitope and cavities around the C118 phenyl ring, which provided insights for the design and synthesis of the new analogs acNTMF0-1 to acNTMF0-4 with improved properties.

[0409] The sulfur of the thioethyl fragment was substituted by an oxygen to increase metabolic stability, enhance polar binding interactions and improve solubility. To improve chemical and metabolic stability of C118, acNTMFo-1 with C-3-(3-ethoxypropoxy) and a lower ClogP and shorter spacer acNTMFo-2 with C-3-(2-methoxyethoxy) were designed and synthesized. While the unique ability of fluorine and to broaden the chemical space of C118, fluoro and trifluoromethoxy substitution of C-5 phenyl acNTMFo-3 and acNTMFo-4 were also included.

[0410] In a convergent strategy similar fashion to our previous synthesis of C118, the desired compound acNTMF0-1 was synthesized by a synthetic sequence involving coupling of ahydrazone with a quinolinyl boronic acid fragment, followed by lithiation of the resulting biarylmethane and addition to the known ketone, following the normal BDQ procedure as shown in FIG. 3.

[0411] Compound acNTMFO-2 was prepared in a similar way (FIG. 4), but using addition of a lithiated quinoline to the corresponding aldehyde, followed by Kursanov reduction to access the biarylmethane precursor.

[0412] Compound acNTMFO-3 and acNTMFO-4 were prepared using the hydrazone-quinolinyl boronic acid method as described for acNTMFO-2 (FIGS. 5 and 6).

[0413] Example 37. Potency of C118 and four analogs acNTMFo-1, acNTMFo-2, acNTMFo-3 and acNTMFo-4

[0414] Bacterial strains and culture media

[0415] The NTM strains M. avium, M. kansasii, M. peregrinum, and M. marinum as well as the M. avium clinical isolate strain were used. The M. avium clinical isolate has been isolated from a 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) (Yee, M. et al., Gen. Announc. 2017, 5(32)). The clinical isolates Tel / Mabc-013, Tel / Mabc-019 and Tel / Mabc-020 were obtained from sputum samples of patients infected with NTM at Tan Tock Seng Hospital, Singapore. This study was approved by human biomedical research regulated by the human Biomedical research Act (HBRA) and by NHG domain specific review board (#2023 / 00896). 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).

[0416] Growth inhibition dose-response assay

[0417] MIC50 values against specific strains were determined using the broth microdilution method (Clinical and Laboratory Standards Institute. 2018. Performance standards for susceptibility testing of mycobacteria, Nocardia spp., and other aerobic actinomycetes, 1st ed CLSI supplement M62 Clinical and Laboratory Standards Institute, Wayne, PA.). All the wells of clear 96-well flat bottom cell culture plates (Corning) were filled with 100 pl of liquid medium (complete 7H9 medium). C118 at two times the desired highest final concentration was added to the first well of each column. A 16-point 2-fold serial dilution of C118 was carried out startingfrom this first well (500 pM - 0.015 pM). NTM cultures were grown to mid-exponential phase and further diluted to an optical density at 600 nm (OD6oo) value of 0.1 in complete 7H9 medium. In order to create a final OD6oo value of 0.05 in each well, 100 pl of the diluted culture were added to each well. The plates were incubated for 3 days at 37 °C. In case of M. peregrinum, M. kansasiiVhe plates were incubated at 37 °C for 5-7 days. M. marinum plates were incubated at 30 °C for 5-7 days. The cultures in the wells were manually suspended upon completion of the incubation time and the OD60o of each well was read using a Tecan Infinite Pro 200 plate reader. The reported MIC50 and MIC90 values represent the concentration that inhibits 50% of bacterial growth compared to the untreated culture.

[0418] Whole cell ATP measurement

[0419] Whole cell ATP synthesis assays were carried out in 96 well plates, as described previously in Hotra, A. etal., Angew. Chem. Int. Ed. Engl. 2020, 59, 13295-304.

[0420] Results and discussion

[0421] To test the potency of C118, growth of the slow grower M. avium subspecies avium (Mav) in complete Middlebrook 7H9 broth was studied. C118 displayed good growth inhibition of Mav with a minimum inhibitory concentration (MIC50) of 3.2 ± 0.7 nM, which is of 64- and 20-times higher potency compared to BDQ (206 ± 30 nM; FIG. 7A, Table 1 ) or TBAJ-876 (64 ± 4.8 nM (Ragunathan, P. et al., J. Biol. Chem. 2024, 300, 105618)), respectively. An important requirement of a potent anti-Mav agent is its efficacy against clinical isolates. FIG. 7B shows C118’s efficacy against the clinical isolate M. avium with an IC50 of 2.5 ± 0.4 (Table 1) and around 12-times better than TBAJ-5307 (31 ± 2.8 nM (Ragunathan, P. et al., J. Biol. Chem.

[0422] 2024, 300, 105618)).

[0423] Table 1. Growth and ATP synthesis inhibitory potency of C118 against NTM, the M. avium clinical isolate and three clinical isolates of M. chelonae in 7H9 media.

[0424] Strains Antimicrobial Antimicrobial Intracellular ATP activity (MIC50, nM) activity (MIC90, nM) synthesis inhibition (IC50, nM)

[0425] M. avium 3.2 ± 0.7 5.76 ± 0.7 3.5 ± 1

[0426] M. avium clinical 2.5 ± 0.4 4.5 ± 0.4 2.9 ± 1.2

[0427] isolate

[0428] M. peregrinum 6.5 ± 0.8 11.7 ± 0.8 14 ± 3M. kansasii 5.6 ± 0.4 10 ± 0.4 3.8 ± 1.2

[0429] M. marinum 4.3 ± 1.5 7.7 ± 1.5 43 ± 9

[0430] M. che / onae clinical isolates

[0431]

[0432] Tel / Wabc013 6.0 ± 0.7 10.8 ±0.7 6.6 ± 1

[0433] Tel / Wabc019 5.7 ± 1.0 10.3 ± 1.0 7.4 ± 1.4

[0434] 1e\Mabc 020 5.3 ± 0.8 9.5 ± 0.8 3.9 ± 0.4

[0435] The experiments were carried out in two biological replicates, each with three technical replicates.

[0436] We explored whether growth inhibition of M. avium and its clinical isolate by C118 is related to depletion of ATP by measuring intracellular ATP synthesis in the presence of the inhibitor. As presented in FIG. 7B, C118 very potently inhibited ATP synthesis of M. avium and its clinical isolate with an half-maximal inhibitory concentration (IC50) of 3.5 ± 1 and 2.9 ± 1.2, respectively. These results indicate that the C118-mediated growth inhibition is in line with intracellular ATP depletion and that oxidative phosphorylation of the pathogen is the main target.

[0437] The four analogs acNTMFo-1, acNTMFo-2, acNTMFo-3 and acNTMFo-4 are active against M. avium as indicated by the MIC50 values of 1.7 ± 0.4 nM, 0.9 ± 0.15 nM, 5.0 ± 1.1 nM and 6.9 ± 1.3 nM, respectively (Table 2). Growth inhibition is in line with whole cell ATP formation as underscored by the determined IC50 values of 2.1 ± 0.3 nM, 1.5 ± 0.2 nM, 8.2 ± 1.2 nM and 15.3 ± 1.4 nM, respectively (Table 2).

[0438] Table 2. Growth and ATP synthesis inhibitory potency of C118 analogs against M. avium in 7H9 media.

[0439] Compound ID Antimicrobial Intracellular ATP synthesis inhibition activity (MIC50, nM) (IC50, nM)

[0440] C118 3.2 ± 0.7 3.5 ± 1

[0441] acNTMF0-1 1.7 ± 0.4 2.1 ± 0.3acNTMF0-2 0.9 ± 0.2 1.5 ± 0.2 acNTMFo-3 5.0 ± 1.1 8.2 ± 1.2

[0442] acNTMF0-4 6.9 ± 1.3 15.3 ± 1.4

[0443] The experiments were carried out in two biological replicates, each with three technical replicates.

[0444] Compared to the parental molecule, the improved potency of acNTMF0-1 and acNTMF0-2 as well as their lower calculated clogP (calculated using ChemDraw v22.0.0.22; CambridgeSoft) of 4.044 and 3.272, respectively, prompted to test their ability as broader anti-NTM compounds. Table 3 reveals the high potency of both new analogs against M. peregrinum. M. kansasii, M chelonae and its clinical isolates.

[0445] Table 3. Potency of C118, acNTMF0-1 and acNTMF0-2 against M. peregrinum. M. kansasii, M chelonae and its clinical isolates.

[0446] C118 acNTMFo-1 acNTMFo-2 MICso ICso MICso ICso MICso ICso Strains (nM) (nM) (nM) (nM) (nM) (nM) M. avium 3.2 ± 3.5 ± 1 1.7 ± 0.4 2.1 ± 0.9 ± 0.2 1.5 ± 0.2 0.7 0.3

[0447] M. 6.5 ± 14 ± 3 3.0 ± 0.4 2.1 ± 0.6 ± 0.1 0.7 ± 0.1 peregrinum 0.8 0.6

[0448] M. kansasii 5.6 ± 3.8 ± 0.4 ± 2.5 ± 0.3 ± 0.02 1.6 ± 0.3 0.4 1.2 0.06 0.6

[0449] M. marinum 4.3 ± 43 ± 9 2.8 ± 0.4 3.8 ± 4.8 ± 0.7 7.2 ± 0.7

[0450] 1.5 0.7

[0451] M. chelonae clin.

[0452] isolates

[0453] Tel / Wabc013 6.0 ± 6.6 ± 1 5.9 ± 0.5 3.2 ± 3.5 ± 0.6 2.6 ± 0.4 0.7 0.4

[0454] Tel / Wabc019 5.7 ± 1 7.4 ± 3.1 ± 1 7.1 ± 2.6 ± 0.2 7.3 ± 1.2

[0455] 1.4 0.8Tel / Made 020 5.3 ± 3.9 ± 3.2 ± 0.4 5.2 ± 2.6 ± 0.4 5.4 ± 1.9 0.8 0.4 1.2

[0456] The experiments were carried out in two biological replicates, each with three technical replicates.

[0457] To accelerate durable cure, multidrug regimens are required with potentiating inhibitors. We studied growth inhibition activity of C118 along with the clinical M. avium antibiotic amikacin, targeting the ribosome, rifabutin, binding to the RNA polymerase, or clofazimine (CFZ), which inhibits electron transfer from NADH to menaquinone in the ETC, in a checker-board titration assay. Significant cell growth inhibition was observed with all the drugs tested (FIG. 8). The curves indicate potentiated growth inhibition in / Wavtriggered by amikacin, CFZ, and rifabutin, respectively. Overall, these data indicate that C118 is a potential inhibitor for combinatory approaches with major anti- Mav drugs.

[0458] We also showed that C118 is potent against slow grower Mycobacterium kansasii, one of the most pathogenic and common NTM isolated from humans worldwide. The compound is highly potent, displaying an MIC50 value of 5.6 ± 0.4 nM (FIGS. 9A-B, Table 1). The whole cell ATP synthesis assay underscores the correlation between ATP depletion and growth inhibition of M. kansasiiby C118, resulting in an IC50 = 3.8 ± 1.2 nM (FIG. 9C, Table 1).

[0459] We also studied C118’s efficacy against the NTM fast grower Mycobacterium peregrinum and Mycobacterium chelonae clinical isolates. As displayed in FIG. 9A, C118 is potent against M. peregrinum (MIC50 6.5 ± 0.8 nM, Table 1), an opportunistic pathogen mainly isolated from patients with skin and soft tissue infection. Whole cell ATP synthesis inhibition by the compound were in line with growth reduction of M. peregrinum (14 ± 3 nM, FIG. 9C). M. chelonae is known to cause skin- or catheter-related infections. As presented in Table 1, the compound was potent against the M. chelonae clinical strains Tel Mabc-013, Tel / Wabc-019 and Tel / Wabc-020 with MIC50 values of 6.0 ± 0.7 nM, 5.7 ± 1 nM and 5.3 ± 0.8 nM, respectively (Table 1). Growth inhibition correlated with whole cell ATP depletion, reflected by the IC50 values of 6.6 ± 1 nM, 7.4 ± 1.4 nM and 3.9 ± 0.4 nM, respectively (Table 1).

[0460] While the aforementioned Mycobacteria are mostly related to human infections, the slow grower Mycobacterium marinum is also associated with fishes and sea food. M. marinum is prevalent all over the world affecting natural and artificial farming populations of marine, brackish, and freshwater fish. It naturally infects more than 150 species of fish, frog, freshwater eels and oyster with economic and health consequences to food-safety and -quality. To date,there are no universally accepted treatments for mycobacteriosis in fish. In addition, M. marinum strains infecting humans were isolated from swimming pools, wells, rivers and hobby fish tanks, making M. marinum the leading cause of extra respiratory human infections among NTM worldwide, including skin and soft tissue infections. Like other mycobacteria, M. marinum is a strict aerobe bacterium, making it dependent on oxidative phosphorylation catalyzed by the M. marinum F-ATP synthase. Here, we investigated the potency of C188 against M. marinum. As presented in FIGS. 10A-B, the compound inhibits growths with an MIC50 value of 4.3 ± 1.5 nM, which is caused by depletion of ATP (IC50 of 43 ± 9 nM), and a 65-times better potency when compared to BDQ (MIC50 = 276 ± 9 nM; FIG. 11).

[0461] Table 4. Growth and ATP synthesis inhibitory potency of (+)-(1S,1 R)-C118 and (-)-(1R,2S)-C118 in 7H9 media.

[0462] Strains (+)-(1S,1R)-C118 (-)-(l R,2S)-C118

[0463] (pM) (nM) Antimicrobial Intracellular Antimicrobial Intracellular activity (MIC50) ATP synthesis activity (MIC50) ATP synthesis inhibition inhibition (IC50) (IC50) M. avium >10 >10 3.2 ± 0.7 3.5 ± 1 M. peregrinum >10 >10 6.5 ± 0.8 14 ± 3 M. kansasii >10 >10 5.6 ± 0.4 3.8 ± 1.2 M. marinum >10 >10 4.3 ± 1.5 43 ± 9

[0464]

[0465] Example 38. In vitro pharmacokinetic and toxicity of C118, acNTMF0-1 and acNTMF0-2

[0466] in vitro ADME profiling revealed that C118 and acNTMFO-1 and acNTMFO-2 were stable in mouse plasma and simulated gastric, with an improved stability of the two analogs (Table 5); experimental services were performed by BioDuro LLC (Irvine, CA, USA) in accordance with the company’s established protocol. The analogs show an improved clearance compared to C118 with acNTMFO-2 representing a moderate value. Importantly, none of the three inhibitors show no toxicity against the family of cytochrome P450 enzymes.

[0467] Table 5. In vitro pharmacokinetic and toxicity of C118, acNTMF0-1 and acNTMF0-2.C118 acNTMFo-1 acNTMFo-2 Stability in mouse plasma (t1 / 2min) >372.8 >372.8 >372,8 Stability in simulated gastric (t1 / 2min) 104 >372.8 >3728 Metabolic stability

[0468] Microsomal stability ClintμL min-1mg-1129.1 95.4 51.8 In human

[0469] CYP Inhibition 1A2, 2C9, 2C19. 2D6, 2B6, 2C8 (iCw) >50 >50 >50

[0470]

[0471] Determination

[0472] Example 39. In vivo potency and toxicity of C118

[0473] In vivo potency and toxicity of C118 against M. marinum infected zebrafish larvae was studied (FIG. 12), using M. marinum expressing red fluorescent mScarlet via caudal vein infection.

[0474] C118 was then added to the water containing the infected zebrafish as shown in FIG. 12A. No signs of toxicity-induced killing or developmental abnormalities were recorded in the presence of C118 (FIGS. 12B-C). When infected embryos were exposed for 5 days to 3.5 μnM concentration of C118 tested, a significant increased survival rate (close to 100%) was observed compared to the untreated group of embryos (FIG. 12C). Decrease in the physiopathological signs of the C118-treated larvae was corroborated by whole-embryo imaging (FIGS. 12D-E). This indicates that C118 is very efficient in vivo against infection and protects the zebrafish from killing by M. marinum.

[0475] Example 40. Ex vivo anti-NTM potency of C118

[0476] Macrophage experiments

[0477] Cytotoxicity assay. Human THP-1 monocytes were differentiated with Phorbol Myristate Acetate (PMA) for 48 hrs and exposed to decreasing concentrations of C118 or rifabutin for an additional 72 hrs at 37 °C with 5% CO2. Following incubation, 10% (vol / vol) resazurin dye was added to each well and left to incubate for 4 hrs at 37 °C and 5% CO2. Data was acquired using a fluorescent plate reader (excitation 540 nm, emission 590 nm). Dimethylsulfoxide (DMSO) was included as a negative control.

[0478] Intracellular growth inhibition assay

[0479] THP-1 cells were grown in RPMI medium supplemented with 10% Fetal bovine serum (Sigma Aldrich) (RPMIFBS) and incubated at 37 °C in the presence of 5% CO2. Cells were differentiated into macrophages in the presence of 20 ng / ml PMA in 24-well flat-bottom tissue culture microplates (105cells / ml) and incubated for 48 hrs at 37 °C with 5% CO2. Infection with M.abscessus harboring the pTEC27 (for expression of the red fluorescent marker tdTomato) (Takaki, K. etal. Nat. Protoc. 2013, 8, 1114-1124) was carried out at 37 °C in the presence of 5% CO2for 4 hrs at a MOI 2:1. After extensive washing with 1X PBS, cells were incubated with RPMIFBScontaining 250 pg / ml amikacin for 2 hrs and washed again with PBS prior to the addition of 500 pl RPMIFBScontaining DMSO (negative control) or 500 pl RPMIFBScontaining 7 pM or 14 pM C118. Macrophages were washed and lysed with 100 pl of 1% Triton X-100 at 4-, 24- and 72 hours post-infection. Serial dilutions of macrophage lysates were plated onto LB agar plates, incubated at 37 °C and colonies were counted to determine intracellular CFUs.

[0480] Microscopy-based infectivity assays

[0481] Differentiated macrophages were grown on coverslips in 24-well plates at a density of 105cells / ml for 48 hrs at 37 °C with 5% CO2 prior to infection with tdTomato-expressing M. abscessus for 4 hrs at a MOI of 2:1. After washing and amikacin treatment to remove the extracellular bacilli, macrophages were exposed to DMSO (negative control), 7 pM or 14 pM C118, and fixed at 3 days post-infection with 4% paraformaldehyde in PBS for 20 min. Cells were then permeabilized using 0.2% Triton X-100 for 20 min, blocked with 2% BSA in PBS supplemented with 0.2% Triton X-100 for 20 min, incubated with anti-CD43 antibodies (Becton Dickinson); dilution 1:1,000) for 1 hr and with an Alexa Fluor 488-conjugated anti-mouse secondary antibody (Molecular Probes, Invitrogen). After 5 min of incubation with DAPI (dilution 1:1,000), cells were mounted onto microscope slides using Immu-mount (Calbiochem) and examined with a confocal microscope using a 63X objective.

[0482] Results and discussion

[0483] To investigate the anti-NTM potency of the inhibitor in macrophages, the established THP-1 infection model, as previously reported (Johansen, M. D. etal., Antimicrob. Agents Chemother.

[0484] 2020, 64, e00363-20), was used. Initially, we assessed the cytotoxicity of C118 against THP-1 cells during a 3-day exposure period. FIG. 13A suggests that the compound exhibits cytotoxicity only at high concentrations and the rate of macrophage killing occurred at lower concentrations with rifabutin compared to C118.

[0485] Given the observed low cytotoxicity, we evaluated the intracellular efficacy of C118 in THP-1 cells infected with the Mycobacterium abscessus strain CIP104536T(S variant) expressing tdTomato. This approach should also provide information about C118 potency against M. abscessus, which together with M. avium accounts for up to 95% of NTM-caused pulmonary infections, and underscoring that C118 is indeed a potent anti-NTM agent. Tests in M. abscessus-infected macrophages demonstrated that C118 is efficiently active just after 24 hours of treatment, and at 72 hpi, we observed a drastic reduction in intracellular bacterialload when compared to the untreated infected macrophages (FIG. 13B). Concurrently, macrophages were stained with anti-CD43 and DAPI and examined under a confocal microscope. C118-treated macrophages did not exhibit any visual alterations in membrane integrity, cell morphology, or size upon microscopic examination (FIG. 13C) but showed a notable reduction in the number of bacilli residing inside the macrophages treated with C118 (FIG. 13C), consistent with the decreased intracellular bacterial burden (FIG. 13B). Collectively, these findings demonstrate that C118, inhibits also M. abscessus growth, enters human macrophages and inhibits bacterial replication.

[0486] General discussion

[0487] The present disclosure describes the design, synthesis as well as the in vitro and ex vivo potency of the new BDQ analog C118 against NTM. C118 represents an attractive inhibitor to tackle the issues associated with NTM drug tolerance and toxicity. Its combinatory potency with anti- / Wab drugs holds potential for overcoming resistance.

[0488] Combining the discoveries disclosed herein with targeting of the F1F0-ATP synthase of the NTM pathogens M. avium, M. kansasii, M. peregrinum, M. abscessus, M. marinum and M. chelonae clinical isolates afford a competitive advantage in the field. Without wishing to be bound by theory, the concept is anchored in nature’s paradigms for securing energy inside NTM, and in the design, synthesis and evaluation of the BDQ analog, C118. The compound blocks ATP synthesis in a whole cell assay, underlining that the molecular engine, F1F0-ATP synthase, is the target. C118 shows high potency of ATP synthesis- and cell growth inhibition against the NTM slow growers M. avium, M. kansasii, and M. marinum, as well as against the fast growers M. peregrinum, M. chelonae and M. abscessus. The compound also shows high efficacy against intracellular mycobacteria and in in vivo studies of infected zebrafish embryos. It is demonstrated that C118 potentiates the effect against M. avium when incubated with the drugs Amikacin, CFZ and Rifabutin.

Claims

Claims1. A compound according to formula I:where:R1represents C1-5alkyl;R2represents H, F or OCF3;X and Y independently represent O, S or CH2; andn is a number from 1 to 5,or a salt or solvate thereof, provided that the compound of formula I is not:

2. The compound or a salt or solvate thereof, according to Claim 1, wherein R2represents H.

3. The compound or a salt or solvate thereof, according to Claim 1 or Claim 2, wherein Ri is CH3 or CH2CH3.

4. The compound or a salt or solvate thereof, according to any one of the preceding claims, wherein n is 2 or 3.

5. The compound or a salt or solvate thereof, according to any one of the preceding claims, wherein X is O or S.

6. The compound or a salt or solvate thereof, according to Claim 5, wherein X is O.

7. The compound or a salt or solvate thereof, according to any one of the preceding claims, wherein Y is O or S.

8. The compound or a salt or solvate thereof, according to Claim 7, wherein Y is O.

9. The compound or a salt or solvate thereof, according to any one of the preceding claims, wherein:R1is CH3or CH2CH3;R2is H;n is 2 or 3;X is O; andY is O.

10. The compound or a salt or solvate thereof, according to Claim 1, wherein the compound of formula I is selected from the list:and11. The compound or a salt or solvate thereof, according to Claim 10, wherein the compound of formula I is selected from the list:and12. Use of a compound according to formula I:where:R1represents C1-5alkyl;R2represents H, F or OCF3;X and Y independently represent O, S or CH2; andn is a number from 1 to 5,or a salt or solvate thereof,in the manufacture of a medicament for the treatment of a nontuberculosis mycobacterial infection.

13. The use according to Claim 12, wherein R2represents H.

14. The use according to Claim 12 or Claim 13, wherein Ri is CH3or CH2CH3.

15. The use according to any one of Claims 12 to 14, wherein n is 2 or 3.

16. The use according to any one of Claims 12 to 15, wherein X is O or S, optionally wherein X is O.

17. The use according to any one of Claims 12 to 16, wherein Y is O or S, optionally wherein Y is O.

18. The use according to any one of Claims 12 to 17, wherein:Ri is CH3or CH2CH3;R2is H;n is 2 or 3;X is O; andY is O.

19. The use according to Claim 12, wherein the compound of formula I is selected from the list:

20. The use according to Claim 19, wherein the compound of formula I is selected from the list:and21. A compound according to formula I or a salt or solvate thereof, as described in any one of Claims 12 to 20 for use in the treatment of a nontuberculosis mycobacterial infection.

22. A method of treating a nontuberculosis mycobacterial infection comprising the steps of providing a compound of formula I or or a salt or solvate thereof, as described in any one of Claims 12 to 20, to a subject in need thereof.

23. The use according to any one of Claims 12 to 20, the compound for use according to Claim 21 or the method according to Claim 22, wherein the nontuberculosis mycobacterial infection is caused by one of more of mycobacterium avium, mycobacterium peregrinum, mycobacterium kansasii, mycobacterium marinum, and mycobacterium chelonae.

24. Use of a compound of formula I, or a salt or solvate thereof, as described in any one of Claims 12 to 20 and a second antibiotic in the manufacture of a medicament for the treatment of nontuberculosis mycobacterial infection, wherein the compound of formula I or a salt or solvate thereof, is to be administered sequentially, simultaneously or concomitantly with the second antibiotic.

25. A compound according to formula I or a salt or solvate thereof, as described in any one of Claims 12 to 20 and a second antibiotic for use in the treatment of a nontuberculosis mycobacterial infection wherein the compound of formula I or a salt or solvate thereof, is to be administered sequentially, simultaneously or concomitantly with the second antibiotic.

26. A method of treating a nontuberculosis mycobacterial infection comprising the steps of providing a compound of formula I or or a salt or solvate thereof, as described in any one of Claims 12 to 20 and a second antibiotic, to a subject in need thereof, wherein the compound of formula I or a salt or solvate thereof, is to be administered sequentially, simultaneously or concomitantly with the second antibiotic.

27. The use according to Claim 24, the compound for use according to Claim 25 or the method according to Claim 26, wherein the nontuberculosis mycobacterial infection is caused by one of more of mycobacterium avium, mycobacterium peregrinum, mycobacterium kansasii, mycobacterium marinum, and mycobacterium chelonae.

28. The use according to Claim 24, the compound for use according to Claim 25 or the method according to Claim 26, wherein the second antibiotic is selected from one or more of the group consisting of clofazimine, rifabutin and amikacin.

29. A formulation comprising a compound according to any one of Claims 1 to 11, or a pharmaceutically acceptable salt or solvent thereof, and a pharmaceutically acceptable carrier.

30. A formulation comprising a compound according to any one of Claims 1 to 11, or a pharmaceutically acceptable salt or solvent thereof, and an acceptable carrier suitable to prevent growth of mycobacteria in an aqueous environment that is useful for the farming of fish.

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