WO2025178571A1 - Anti-non-tuberculous mycobacteria inhibitors - Google Patents

Abstract

The present invention provides anti-NTM compounds that target the NTM F₁F_O-ATP synthase. The present invention provides the use of the anti-NTM compounds as a medicament to treat NTM. The present invention also provides the use of the anti-NTM compounds as a medicament to treat NTM with a second antibiotic.

Description

ANTI-NON-TUBERCULOUS MYCOBACTERIA INHIBITORS

FIELD OF INVENTION

The present invention provides anti-non-tuberculous mycobacteria (NTM) inhibitors and more particularly relates to anti-NTM inhibitors for enhanced ATP synthesis inhibition and anti-NTM activity.

BACKGROUND

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.

Non-tuberculous mycobacteria (NTM) cause a wide range of serious illnesses, including lymphadenitis, soft and skin tissue, cardiac, bone, joint and, most commonly, pulmonary infections. The number of pulmonary infections due to NTM within developed countries has become even higher than those of TB, which is caused by the cousin Mycobacterium tuberculosis. The cure rate of NTM treatment is low, lengthy and costly, which in part is the result of drug resistance, low potency, poor clinical response and severe side effects. NTM have been grouped into rapid and slow growers. Mycobacterium chelonae, Mycobacterium peregrinum, Mycobacterium fortuitum, Mycobacterium mucogenicum and the Mycobacterium abscessus complex are representatives of rapid growers. The M. abscessus complex is divided into M. abscessus subsp. abscessus, M. abscessus subsp. bolletii and M. abscessus subsp. massitiense, which show different drug susceptibilities to anti - M. abscessus drugs and clinical outcomes. The varied response to some antibiotic treatments in these subspecies can, at least partly, be attributed to the presence or absence of surface-associated glycopeptidolipids in smooth (S) or rough (R) morphotypes, respectively. The Mycobacterium avium complex, including M. avium and Mycobacterium intracellulare, Mycobacterium kansasii and Mycobacterium xenopi, are representatives of slow growers causing human diseases.

Non-fermentative NTMs depend on oxidative phosphorylation (OXPHOS) to generate ATP and to sustain their energy requirements under normal and hypoxic conditions. This is underscored by the ATP-forming F1F0 ATP synthase (F-ATP synthase) being essential for mycobacteria and serves as a new anti-/W. abscessus drug target. The mycobacterial F-ATP synthase consists of nine subunits, forming a water-soluble Fi domain with the subunits a₃:p₃:y:£, a membrane-embedded F_o domain with subunits a: C₉ and a peripheral stalk, including subunits b:b':5. Protons derived from the proton motive force (pmf) of the electron transport chain (ETC) complexes at the intermembrane space of NTMs are translocated via two proton half-channels of the interface of the a:c subunits, combined with rotation of the eg ring (Figure 1 a). Such rotational movements trigger the central stalk subunits in an anticlockwise rotation, which alters the conformation in the a₃:p₃ headpiece for the synthesis of ADP and inorganic phosphate to ATP. The peripheral stalk (b:b':δ) facilitates flexible coupling of the Fo and Fi sector, making it an important regulator in ATP formation. Differences in amino acid composition, as well as structural elements, allows special regulatory control in the functioning of the mycobacterial enzyme in comparison with its human and bacterial counterparts. These traits paved the way for new inhibitors targeting the central stalk subunits y:e and the design and screening for novel inhibitors such as GaMF1 and EpM abF1 . The anti- TB drug bedaquiline, and its analogues TB Alliance-Janssen (TBAJ)-876 and TBAJ-5307 with improved potency and physiochemical properties, have shown high efficacy against NTM in vitro and in vivo. This class of inhibitors binds to five single c subunits of the turbine and two high-affinity sites of different subunit a-c interfaces. These hydrophobic binding sites indicate the low hydrophilicity of the three drugs with a calculated log octanol-water partition coefficient (cLogP) of 7.25 for bedaquiline, as well as 5.15 and 4.7 for the improved analogues TBAJ-876 and TBAJ-5307, respectively.

In comparison, the squaramide and anti-TB inhibitor SQ31f (FIG. 1 b), which inhibits M. tuberculosis with an MIC₅₀ of 6.2 μM, has a favourable cLogP of 1.02 and low cytotoxicity. SQ31f is highly potent against M. tuberculosis in vitro and in vivo by specifically binding to the proton half-channel of subunits a:c on the cytosolic side (FIG. 1a), where the proton becomes released during ATP formation, as visualized by the cryo-electron microscopy (EM) structure of the Mycobacterium smegmatis F-ATP synthase. This half-channel is filled with water and this fact indicates why SQ31 f, with its lower cLogP, is binding in such a distinct cavity in comparison with bedaquiline. Besides attractive hydrophilicity, an important additional aspect of this attractive inhibitor resides in its short and operationally simple synthesis for high production rates and low cost.

Thus, there is a need for alternative and/or improved compounds suitable for the treatment of NTM. SUMMARY

Aspects and embodiments of this invention will be described by reference to the following numbered clauses.

1. A compound of formula I: where:

R₁ represents, -(CH₂)_n-N(R_4aR_4b)₂, -(CH₂)_n-piperidyl, -(CH₂)_n-2-pyridyl, -(CH₂)_n-quinolinyl, -(CH₂)_n-pyrrolidinyl , or -(CH₂)_r-morpholinyl, which (CH₂)_n-piperidyl, -(CH₂)_n-2-pyridyl, -(CH₂)_n- quinolinyl, -(C H₂)_n-pyrrolidiny I , and -(CH₂)_n-morpholinyl are unsubstituted or substituted by one or more groups selected from the group consisting of halo, and CN; n is 0, 1 or 2;

R₂ and R3 are each independently selected from H, Ar, C_1-6 alkyl, -(CH₂)_m-CO₂R_4c, -(CH₂)_m-Ar, -(CH₂)_m-Het¹, where Ar, C_1-6 alkyl, -(CH₂)_m-Ar, and -(CH₂)_m-Het¹ are unsubstituted or are substituted by one or more substituents selected from the group consisting of halo, and OR_4d, each Ar is independently a C_6-10 aromatic group;

Het¹ is a 5- to 10 heteroaromatic group having from 1 to 3 heteroatoms; m is 0, 1 , or 2;

R_4C is H, C_1-5 alkyl or phenyl, where the C_1-6 alkyl or phenyl groups are unsubstituted or substituted by one or more substituents selected from the group consisting of halo, OH, and OC_1-3 alkyl; R_4d is H or C_1-6 alkyl, where the C_1-6 alkyl group is unsubstituted or substituted by one or more substituents selected from the group consisting of halo, OH, and OC_1-3 alkyl; and Pharmaceutically acceptable salts or solvates thereof, provided that, when Ph represents unsubstituted -(CH₂)_r-2-pyridyl, then one or R₂ and R₃ are not H.

2. The compound according to Clause 1 , or pharmaceutically acceptable salts or solvates thereof, wherein R₂ and R₃ are each independently selected from H, Ph, CF₃, Me, Et, ⁿPr, 'Pr, cyclopropyl, ⁿBu, -CH₂-OH, -CH₂-O-Me, -CH₂-O-Et, -CH₂-O-ⁿPr, -CHs-O-iPr, -CHs-O-cyclopropyl, -CO₂H, -CH₂-CO₂H, -CH₂-CH₂-CO₂H, -CH₂-CH₂-CO₂Me, -CH₂-CH₂-CO₂Et, -CH₂-CH₂-CO₂ ⁿPr, -CH^CHs-CCyPr, -CH₂-CH₂-C0₂cyclopropyl, -CH₂-CH₂- CO₂Ph, -CH₂-Ph, -CH₂-quinoline, -CH₂-isoquinolene, -CH₂-quinazoline, -CH₂-pthalazine.

3. The compound according to Clause 2, or pharmaceutically acceptable salts or solvates thereof, wherein R₂ and R₃ are each independently selected from H, Me, Et, ⁿPr, 'Pr, cyclopropyl, -CH₂-OH, -CH₂-O-Me, -CH₂-O-Et, -CO₂H, -CH₂-CH₂-CO₂H, -CH₂-CH₂-CO₂Me, - CH₂-CH₂-CO₂Et, -CH₂-CH₂-CO₂Ph, -CH₂-Ph, -CH₂-quinoline, -CH₂-isoquinolene, -CH₂- quinazoline, -CH₂-pthalazine.

4. The compound according to any one of the preceding clauses, or pharmaceutically acceptable salts or solvates thereof, wherein R₂ represents CH₃ or CP₃ and R₃ represents CP₃, Me, -CH₂-OH, -CH₂-O-Me, -CO₂H, or-CH₂-C0₂H, optionally wherein Ri represents -(CH₂)_n-2- pyridyl, which -(CH₂)_n-2-pyridyl group is unsubstituted or substituted by one or more substituents selected from halo and CN.

5. The compound according to any one of the preceding clauses, or pharmaceutically acceptable salts or solvates thereof, wherein R₁ represents, -(CH₂)-N(Me)₂, -(CH₂)_n-N(Et)₂, -(CH₂)_n-piperidyl, -(CH₂)_n-2-pyridyl, quinolinyl, -CH₂pyrrolidinyl, or -CH₂morpholinyl, where - (CH₂)_n-piperidyl, quinolinyl, -CH₂pyrrolidinyl, and -CH₂morpholinyl are unsubstituted and - (CH₂)_n-2-pyridyl is unsubstituted or substituted by one or more substituents selected from the group consisting of F, Cl, Br and CN. 6. The compound according to any one of the preceding clauses, or pharmaceutically acceptable salts or solvates thereof, wherein Ri represents,

7. The compound according to any one of Clauses 1 to 5, or pharmaceutically acceptable salts or solvates thereof, wherein: Ri represents, -(CH₂)_n-N(Et)₂, -(CH2)_n-piperidyl, -(CH₂)_n-2-pyridyl: and n is 1 or 2.

8. The compound according to any one of the preceding clauses, wherein the compound of formula I is selected from the list: or a pharmaceutically acceptable salt or solvate thereof.

9. The compound according to Clause 8, wherein the compound of formula I is

, or a pharmaceutically acceptable salt or solvate thereof

10. A compound of formula I as defined in any one of Clauses 1 to 9, or a pharmaceutically acceptable salt or solvate thereof, for use in the treatment of nontuberculous mycobacterial (NTM) infection.

11. Use of a compound of formula I as defined in any one of Clauses 1 to 9, or a pharmaceutically acceptable salt or solvate thereof, in the manufacture of a medicament for the treatment of nontuberculous mycobacterial (NTM) infection.

12. A method of treating a nontuberculous mycobacterial (NTM) infection comprising the step of administering to a subject in need thereof a compound of formula I as defined in any one of Clauses 1 to 9, or a pharmaceutically acceptable salt or solvate thereof.

13. A composition comprising a compound of formula I as defined in any one of Clauses 1 to 9, or a pharmaceutically acceptable salt or solvate thereof, and one or more pharmaceutically acceptable excipients. 14. Use of a compound of formula I as defined in any one of Clauses 1 to 9, or a pharmaceutically acceptable salt or solvate thereof, and a further antibiotic compound, or a pharmaceutically acceptable salt or solvate thereof, in the manufacture of a medicament for the treatment of a nontuberculous mycobacterial (NTM) infection wherein the compound of formula I, or a pharmaceutically acceptable salt or solvate thereof, is to be administered sequentially, simultaneously or concomitantly with the second antibiotic.

15. A compound of formula I as defined in any one of Clauses 1 to 9, or a pharmaceutically acceptable salt or solvate thereof, and a further antibiotic compound, or a pharmaceutically acceptable salt or solvate thereof, for use in the treatment of a nontuberculous mycobacterial (NTM) infection wherein the compound of formula I, or a pharmaceutically acceptable salt or solvate thereof, is to be administered sequentially, simultaneously or concomitantly with the second antibiotic.

16. A method of treating a nontuberculous mycobacterial (NTM) infection comprising the step of administering to a subject in need thereof a compound of formula I as defined in any one of Clauses 1 to 9, or a pharmaceutically acceptable salt or solvate thereof and a second antibiotic, or a pharmaceutically acceptable salt or solvate thereof, wherein the compound of formula I, or a pharmaceutically acceptable salt or solvate thereof, is administered sequentially, simultaneously or concomitantly with the second antibiotic.

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

18. The use according to Clause 1 1 or Clause 14, the method of Clause 12 or Clause 15, the compound according to Clause 13 or Clause 16, wherein the NTM infection is caused by one or more of the group consisting of M. chelonae, M. peregrinum, M. kansasii, and more particularly, M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, M. abscessus subsp. massiliense, M. fortuitum, M. avium and M. intracellulare.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 depicts (a) Structural model of the M. smegmatis F-ATP synthase derived from cryo- EM studies (PDB: 8G07), composed of the subunits forming the Fi domain, the membrane-embedded and proton-translocating subunits a and c, and subunits b, b' and 6 forming the peripheral stalk, (b) SQ31f consists of a 4-amino-3-phenyl-cyclobut-3-ene-1 ,2- dione as the core nucleus (highlighted in the box) plus phenyl, morpholine and pyridylmethyl groups.

FIG. 2 depicts a two-step synthesis of SQ31f, showing chemical formulae for compounds 1 , 2, 3 and 4.

FIG. 3 includes (a) Growth inhibition dose-response curve of the S and R forms of M. abscessus subsp. abscessus, M. bolletii and M. massiliense, respectively, and (b) the clinical isolate M. abscessus Bamboo, (c) ATP synthesis inhibition by SQ31f in whole-cell assays using the S and R variants of M. abscessus subsp. abscessus, as well as M. bolletii and M. massiliense, respectively, and (d) the clinical isolate M. abscessus Bamboo. Data represented are the average of three independent experiments, each performed in triplicate. Error bars represent the standard deviation (SD). (e) Oxygen consumption assay in M. abscessus using methylene blue as an oxygen sensor. SQ31 f did not affect oxygen consumption over a 72-h period. 7H9 media with DMSO as compound solvent served as a blank/negative control. The anti-TB drug bedaquiline was used as a comparison, (f) Testing the possible effect of SQ31 f on ATP synthesis of E. coli IMVs. ATP synthesis of E. coli IMVs in the presence of SQ31f, and quercetin as a control. Two-fold serial dilution was done for SQ31f, starting from 100 μM and from 1 mM for quercetin, accordingly. The data reveal the specificity of SQ31 f as a mycobacterial F-ATP synthase inhibitor. P < 0.0001 ; statistical analysis was carried out using one-way analysis of variance (ANOVA). The experiment was carried out three times in triplicate.

FIG. 4 depicts (a) Cytotoxicity of SQ31f and rifabutin on THP-1 macrophages. THP-1 cells were differentiated with phorbol 12-myristate 13-acetate (PMA) for 48 h, exposed to increasing concentrations of either SQ31f or rifabutin for an additional 72 h at 37°C with 5% CO₂. The experiments were performed in three experiments, each in quadruplicate, (b) Macrophages were infected with the M. abscessus S variant expressing tdTomato (moi of 2:1 ) for 4 h prior to treatment with 25 or 50 μM SQ31f. cfu were determined at 4 hpi and 3 dpi, respectively. Data are mean values ± SD for three independent experiments. Data were analysed using the one-tailed Mann-Whitney f-test; **** P < 0.0001. (c) Three immunofluorescent fields were taken on 72 dpi at 63x magnification showing macrophages infected with fluorescent M. abscessus. The surface of the macrophages was detected using anti-CD43 antibodies. The nuclei were stained with DAPI. White arrows depict intracellular mycobacteria. The scale bar represents 20 pm and the enlargement scale bar represents 5 pm. FIG. 5 depicts (a) Binding mode of SQ31f at the M. abscessus/M. avium Fo domain. The squaramide moiety was engaged in a multitude of polar/ hydrogen interactions with R188 (3.2 A), Y249 and Q243 residues of the a subunit. The amino-methyl-pyridine fills in the shallow cavity of the c ring and maintains the essential interactions with its ‘NH’ and aza (N) atom of pyridine with COOH atoms of the c subunit E65 residue. The bridging phenyl groups maintain TT-TT interactions with the F68 residue of the c ring (not shown for clarity), while the morpholine is juxtaposed near the vicinity of H166 and engaged in electrostatic interaction with the N174 side chain (not shown for clarity), (b) Growth inhibition dose-response curve of M. smegmatis WT and M. smegmatis harbouring an I66M mutation subunit c. The cl66M mutant was tested with bedaquiline alone or in combination with SQ31f. Three independent experiments were carried out, each with three technical replicates.

FIG. 6 depicts increased potency of SQ31f in combination with the antibiotic amikacin, clofazimine (CFZ) and rifabutin in 7H9 broth, respectively. M. abscesses subsp. abscesses growth inhibition by SQ31 f in combination with increasing concentrations of amikacin (a), CFZ (b), rifabutin (c), linezolid (d), clarithromycin (e) and tebipenem plus avibactam (2 μM) (f) against M. abscesses ATCC 19977. P< 0.0001 ; statistical analysis was carried out using the two-way ANOVA test for all the experiments presented.

FIG. 7 includes ¹H (top) and ¹³C NMR spectra of SQ31f 1 (400 and 100 MHz, CDCI₃).

FIG. 8 is a growth inhibition dose response curve of M abscesses by SQ31 f. Two biological replicates were carried out, each with three technical replicates. The bacteria were grown in liquid culture (7H9) with different concentrations of SQ31f in 96-well microtiter plate and were incubated for 14 days. OD₆₀₀ of the bacterial plates were measured on day 3 and day 14.

FIG. 9 depicts initial five days of untreated and SQ31 f kill kinetics against M. abscesses subsp. abscesses. The bacteria were grown in liquid culture (7H9) in the presence of the indicated concentration of SQ31f up to 5 days. CFU was calculated by plating the culture on 7H10 agar plates. P< 0.05, statistical analysis was carried out for the experiment using ordinary one-way ANOVA test. Experiments were performed twice.

FIG. 10 depicts inhibition of ATP synthesis by SQ31 f of mycobacterial IMVs using the electron donor succinate and NADH demonstrating that the inhibitory profile of SQ31f on IMVs did not alter. Experiments were conducted in triplicates. FIG. 11 includes growth inhibition dose response curve of M. fortuitum and M. peregrinum (A) as well as M. mucogenicum and M. chelonae (B) by SQ31f. Three independent experiments were carried out, each with three technical replicates. SQ31 inhibits oxidative phosphorylation M. fortuitum and M. peregrinum (C) as well as M. mucogenicum and M. chelonae (D) in a whole cell ATP synthesis assay. The experiments were performed in triplicates.

FIG. 12 includes (A) Growth inhibition dose response curve of M. avium subsp. avium, M. intracellulare and M. kansasii by SQ31 f. Three independent experiments were carried out, each with three technical replicates. (B) Since M. kansasii shows the characteristic yellow formation of colonies, due to the accumulation of [3-carotene, cell growth inhibition of M. kansasii by SQ31f was directly observed in 6 well plates in the absence (DF = drug free) or presence of the inhibitor (1 OX MIC₅₀- 1 6 μM) in three experiments. The plates were incubated at for 7- 10 days. (C) SQ31f inhibits oxidative phosphorylation of M. avium subsp. avium, M. intracellulare and M. kansasii in a whole cell ATP synthesis assay (adding bactiter gio (promega). The total ATP content is directly proportional to relative luminescence units (RLU). The experiments were performed in triplicates.

FIG. 13 includes sequence alignments of mycobacterial, Escherichia coli and human F-ATP synthase a- and c subunits. (A) Sequence alignment of M. smegmatis-, M. tuberculosis-, M. abscessus, M. avium, E. coli and human subunit a amino acids with SQ31f-binding residues highlighted by an *. (B) Similarly, amino acids sequence alignment of M. smegmatis-, M. tuberculosis-, M. abscessus, M. avium, E. coli and human c-subunits with the SQ31 f-binding residues highlighted by an *.

FIG. 14 includes growth inhibition dose response curve of the clinical isolates (A) TelM aba- 003; (B) TelM abm-001 , TelM abm-004, TelM abm-005, TelM abm-011 and TelM abm-012; (C) ATP synthesis inhibition by SQ31f in whole-cell assays using TelM abm-001 , TelM abm-004, TelM abm-005, TelM abm-011 and TelM abm-012; (D) growth inhibition dose response curve of the clinical isolate TelM abm-008; and (E) ATP synthesis inhibition by SQ31f in whole-cell assays using TelM abm-008.

FIG. 15 includes the structures of the SQ31f analogues according to Example 6 of the present disclosure.

FIG. 16 includes the structure of TMN-01 and growth inhibition dose response curve of M. abscessus subsp. massiliense (A), M. abscessus subsp. bolletii (B) and M. bovis BCG (C) by SQ31f (□) and TMN-01 (o). The experiments have been performed in triplicates. FIG. 17 includes the structures of the planned analogues of SQ31f according to Example 6 of the present disclosure.

FIG. 18 depicts the reaction scheme for the synthesis of substituted pyridine analogues of SQ31f according to Example 6 of the present disclosure.

FIG. 19 depicts the reaction scheme for the synthesis of the substituted morpholine analogues of squaramide according to Example 6 of the present disclosure.

Description

The present inventors have developed compounds against NTM. The compounds inhibit growth of a wide spectrum of NTM, including clinical isolates, and show anti-M . abscesses activity in macrophages. Growth inhibition is in line with depletion of whole-cell ATP formation. ATP synthesis studies on Escherichia coli inverted-membrane vesicles (IMVs) confirm the species specificity of the compounds. The compounds are proposed to bind to a cavity within the a-c interface of the NTM F-ATP synthase. Furthermore, the compounds enhance the potency of a variety of NTM antibiotics.

Thus, in a first aspect of the invention, there is provided a compound of formula I: where:

R₁ represents, -(CH₂)_n-N(R_4aR_4b)₂, -(CH₂)_n-piperidyl, -(CH₂)_n-2-pyridyl, -(CH₂)_n-quinolinyl, -(CH₂)_n-pyrrolidinyl, or -(CH₂)_r-morpholinyl, which -(CH₂)_n-piperidyl, -(CH₂)_n-2-pyridyl, -(CH₂)_n- quinolinyl, -(C H₂)_n-pyrrolidiny I , and -(CH₂)_n-morpholinyl are unsubstituted or substituted by one or more groups selected from the group consisting of halo, and CN; n is 0, 1 or 2;

R₂ and R₃ are each independently selected from H, Ar, C_1-6 alkyl, -(CH₂)_m-CO₂R4_C, -(CH₂)_m-Ar, -(CH₂)m-Het¹, where Ar, C_1-6 alkyl, -(CH₂)_m-Ar, and -(CH₂)_m-Het¹ are unsubstituted or are substituted by one or more substituents selected from the group consisting of halo, and OR4d, each Ar is independently a Cs 10 aromatic group;

Het¹ is a 5- to 10 heteroaromatic group having from 1 to 3 heteroatoms; m is 0, 1 , or 2; is H, C_1-6 alkyl or phenyl, where the C_1-6 alkyl or phenyl groups are unsubstituted or substituted by one or more substituents selected from the group consisting of halo, OH, and OC_1-3 alkyl;

R_4d is H or C_1-6 alkyl, where the C1 6 alkyl group is unsubstituted or substituted by one or more substituents selected from the group consisting of halo, OH, and OC_1-3 alkyl; and Pharmaceutically acceptable salts or solvates thereof, provided that, when R₁ represents unsubstituted -(CH₂)_n-2-pyridyl, then one or R₂ and R₃ are not H.

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.

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.

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.

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

The term “halo”, when used herein, includes references to fluoro, chloro, bromo and iodo.

Unless otherwise stated, the term “alkyl” refers to an unbranched or branched acyclic, saturated or unsaturated (so forming, for example, an alkenyl or alkynyl) hydrocarbyl radical, which may be substituted or unsubstituted (with, for example, one or more halo atoms). Where the term “alkyl” refers to an acyclic group, it is preferably C_1-w alkyl and, more preferably, C_1-6 alkyl (such as ethyl, propyl, (e.g. n-propyl or isopropyl), butyl (e.g. branched or unbranched butyl), pentyl or, more preferably, methyl).

Unless otherwise stated, the term “aromatic group” when used herein includes Ce-io aryl groups. Such groups may be monocyclic, bicyclic or tricyclic and have between 6 and 10 ring carbon atoms, in which at least one ring is aromatic. The point of attachment of aryl groups may be via any atom of the ring system. However, when aryl groups are bicyclic or tricyclic, they may be linked to the rest of the molecule via an aromatic ring. Cs -10 aryl groups include phenyl, naphthyl and the like, such as 1 ,2,3,4-tetrahydronaphthyl, indanyl, and indenyl. Embodiments of the invention that may be mentioned include those in which aryl is phenyl.

The term “heterocyclic ring system” when used herein refers to an aromatic group containing one or more heteroatom(s) (e.g. one to three heteroatoms) preferably selected from N, O and S (so forming, for example, a mono-, bi-, or tricyclic heteroaromatic group). Heterocyclic ring system include those which have between 5 and 10 members and may be monocyclic, bicyclic or tricyclic, provided that at least one of the rings is aromatic. However, when heteroaryl groups are bicyclic or tricyclic, they may be linked to the rest of the molecule via an aromatic ring. Heterocyclic groups that may be mentioned include benzothiadiazolyl (including 2,1 ,3- benzothiadiazolyl), isothiochromanyl and, more preferably, benzimidazolyl, benzodioxanyl, benzodioxolyl (including 1 ,3-benzodioxolyl), benzofuranyl, benzofurazanyl, benzothiazolyl, benzoxadiazolyl (including 2,1 ,3-benzoxadiazolyl), benzoxazinyl (including 3,4-dihydro-2H-

1.4-benzoxazinyl), benzoxazolyl, benzomorpholinyl, benzoselenadiazolyl (including 2,1 ,3- benzoselenadiazolyl), benzothienyl, carbazolyl, chromanyl, cinnolinyl, furanyl, imidazolyl, imidazo[1 ,2-a]pyridyl, indazolyl, indolinyl, indolyl, isobenzofuranyl, isochromanyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiaziolyl, isoxazolyl, naphthyridinyl (including 1 ,6-naphthyridinyl or, preferably, 1 ,5-naphthyridinyl and 1 ,8-naphthyridinyl), oxadiazolyl (including 1 ,2,3-oxadiazolyl,

1.2.4-oxadiazolyl and 1 ,3,4-oxadiazolyl), oxazolyl, phthalazinyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolinyl, quinolizinyl, quinoxalinyl, tetrahydroisoquinolinyl (including 1 ,2,3,4-tetrahydroisoquinolinyl and 5,6,7,8-tetrahydroisoquinolinyl), tetrahydroquinolinyl (including 1 ,2,3,4-tetrahydroquinolinyl and 5,6,7,8-tetrahydroquinolinyl), tetrazolyl, thiadiazolyl (including 1 ,2,3-thiadiazolyl, 1 ,2,4- thiadiazolyl and 1 ,3,4-thiadiazolyl), thiazolyl, thiochromanyl, thiophenetyl, thienyl, triazolyl (including 1 ,2,3-triazolyl, 1 ,2,4-triazolyl and 1 ,3,4-triazolyl) and the like. Substituents on heteroaryl groups may, where appropriate, be located on any atom in the ring system including a heteroatom. The point of attachment of heteroaryl groups may be via any atom in the ring system including (where appropriate) a heteroatom (such as a nitrogen atom), or an atom on any fused carbocyclic ring that may be present as part of the ring system. Heteroaryl groups may also be in the N- or S-oxidised form. Particularly preferred heteroaryl groups include pyridyl, pyrrolyl, quinolinyl, furanyl, thienyl, oxadiazolyl, thiadiazolyl, thiazolyl, oxazolyl, pyrazolyl, triazolyl, tetrazolyl, isoxazolyl, isothiazolyl, imidazolyl, pyrimidinyl, indolyl, pyrazinyl, indazolyl, pyrimidinyl, thiophenetyl, thiophenyl, pyranyl, carbazolyl, acridinyl, quinolinyl, benzoimidazolyl, benzthiazolyl, purinyl, cinnolinyl and pterdinyl. Particularly preferred heteroaryl groups include monocylic heteroaryl groups.

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.

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.

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.

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.

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

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

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

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

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

Compounds of formula I, as well as pharmaceutically acceptable salts, solvates and pharmaceutically functional derivatives of such compounds are, for the sake of brevity, hereinafter referred to together as the “compounds of formula I”. Compounds of formula I may contain double bonds and may thus exist as E (entgegen) and Z (zusammen geometric isomers about each individual double bond. All such isomers and mixtures thereof are included within the scope of the invention.

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.

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

In certain embodiments of the invention that may be mentioned herein n may be 1 or 2. This may apply to any of the embodiments mentioned herein.

In certain embodiments of the invention that may be mentioned herein, the compound of formula I or pharmaceutically acceptable salts or solvates thereof, may be one where R2 and R3 may each be independently selected from H, Ph, CF3, Me, Et, ⁿPr, 'Pr, cyclopropyl, ⁿBu, - CH2-OH, -CH₂-O-Me, -CH₂-O-Et, -CH₂-O-ⁿPr, -CH₂-O^JPr, -CH₂-O-cyclopropyl, -CO₂H, -CH2-CO₂H, -CH2-CH2-CO₂H, -CH2-CH2-CO₂Me, -CH₂-CH₂-CO₂Et, -CH₂-CH₂-CO₂ ⁿPr, -CH₂-CH₂-CO₂Pr, -CH₂-CH₂-C0₂cyclopropyl, -CH₂-CH₂- CO₂Ph, -CH2-Ph, -CH2-quinoline, -CH₂-isoquinolene, -CH₂-quinazoline, -CH₂-pthalazine. For example, R₂ and R3 may each be independently selected from H, Me, Et, ⁿPr, Pr, cyclopropyl, -CH2-OH, -CH₂-O-Me, -CH₂-O-Et, -CO₂H, -CH₂-CH₂-CO₂H, -CH₂-CH₂-CO₂Me, -CH₂-CH₂- CO₂Et, -CH₂-CH₂-CO₂Ph, -CH₂-Ph, -CH₂-quinoline, -CH₂-isoquinolene, -CH₂-quinazoline, - CH₂-pthalazine. In more particular embodiments of the invention that may be mentioned herein, R₂ may represent CH₃ or CF₃ and R3 may represent CF₃, Me, -CH₂-OH, -CH₂-O-Me, -CO₂H, or-CH₂-CO₂H, optionally wherein R1 represents -(CH₂)_n-2-pyridyl, which - (CH₂)_r-2-pyridyl group is unsubstituted or substituted by one or more substituents selected from halo and CN.

In certain embodiments of the invention that may be mentioned herein, the compound of formula I, or pharmaceutically acceptable salts or solvates thereof, may be one where Ri represents, -(CH₂)-N(Me)₂, -(CH₂)_n-N(Et)₂, -(CH₂)_n-piperidyl, -(CH₂)_n-2-pyridyl, quinolinyl, - CH₂pyrrolidinyl, or -CH₂morpholinyl, where -(CH₂)_n-piperidyl, quinolinyl, -CH₂pyrrolidinyl, and -CH₂morpholinyl are unsubstituted and -(CH₂)_n-2-pyridyl is unsubstituted or substituted by one or more substituents selected from the group consisting of F, Cl, Br and CN.

In certain embodiments of the invention that may be mentioned herein, the compound of formula I, or pharmaceutically acceptable salts or solvates thereof, may be one where Ri

In certain embodiments of the invention that may be mentioned herein, the compound of formula I, or pharmaceutically acceptable salts or solvates thereof, may be one where:

Ri represents, -(CH₂)_n-N(Et)₂, -(CH₂)_n-piperidyl, -(CH₂)n-2-pyridyl: and n is 1 or 2. In certain embodiments of the invention that may be mentioned herein, the compound of formula I, or pharmaceutically acceptable salts or solvates thereof, may be one where the

or a pharmaceutically acceptable salt or solvate thereof.

For example, the compound of formula I, or pharmaceutically acceptable salt or solvate thereof, may be one where it pharmaceutically acceptable salt or solvate thereof

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.

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

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 include ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷0, ¹⁸0, ³⁵S, ¹⁸F, ³⁷CI, ⁷⁷Br, ⁸²Br and ¹²⁵l).

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.

The compounds disclosed herein are particularly useful for the treatment of patients that have a nontuberculous mycobacterial (NTM) infection.

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.

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

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).

In a further aspect of the invention, there is provided a compound of formula I as defined hereinbefore, or a pharmaceutically acceptable salt or solvate thereof, for use in the treatment of nontuberculous mycobacterial (NTM) infection.

In a further aspect of the invention, there is provided a use of a compound of formula I as defined hereinbefore, or a pharmaceutically acceptable salt or solvate thereof, in the manufacture of a medicament for the treatment of nontuberculous mycobacterial (NTM) infection. In a further aspect of the invention, there is provided a method of treating a nontuberculous mycobacterial (NTM) infection comprising the step of administering to a subject in need thereof a compound of formula I as defined hereinbefore, or a pharmaceutically acceptable salt or solvate thereof.

In a further aspect of the invention, there is provided a composition comprising a compound of formula I as defined hereinbefore, or a pharmaceutically acceptable salt or solvate thereof, and one or more pharmaceutically acceptable excipients.

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.

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 (1 995). 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.

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.

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.

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.

Depending on the disorder, and the patient, to be treated, as well as the route of administration, compounds of formula I may be administered at varying therapeutically effective doses to a patient in need thereof.

However, the dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to effect a therapeutic response in the mammal over a reasonable timeframe. One skilled in the art will recognize that the selection of the exact dose and composition and the most appropriate delivery regimen will also be influenced by inter alia the pharmacological properties of the formulation, the nature and severity of the condition being treated, and the physical condition and mental acuity of the recipient, as well as the potency of the specific compound, the age, condition, body weight, sex and response of the patient to be treated, and the stage/severity of the disease.

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.

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.

It will be appreciated that the compounds of formula I may be provided alone or in combination with other agents, where they may show additive or synergistic activity.

Thus, in a further aspect of the invention, there is also provided a use of a compound of formula I as defined hereinbefore, or a pharmaceutically acceptable salt or solvate thereof, and a further antibiotic compound, or a pharmaceutically acceptable salt or solvate thereof, in the manufacture of a medicament for the treatment of a nontuberculous mycobacterial (NTM) infection wherein the compound of formula I, or a pharmaceutically acceptable salt or solvate thereof, is to be administered sequentially, simultaneously or concomitantly with the second antibiotic.

In a further aspect of the invention, there is also provided a compound of formula I as defined hereinbefore, or a pharmaceutically acceptable salt or solvate thereof, and a further antibiotic compound, or a pharmaceutically acceptable salt or solvate thereof, for use in the treatment of a nontuberculous mycobacterial (NTM) infection wherein the compound of formula I, or a pharmaceutically acceptable salt or solvate thereof, is to be administered sequentially, simultaneously or concomitantly with the second antibiotic.

In a further aspect of the invention, there is also provided a method of treating a nontuberculous mycobacterial (NTM) infection comprising the step of administering to a subject in need thereof a compound of formula I as defined hereinbefore, or a pharmaceutically acceptable salt or solvate thereof and a second antibiotic, or a pharmaceutically acceptable salt or solvate thereof, wherein the compound of formula I, or a pharmaceutically acceptable salt or solvate thereof, is administered sequentially, simultaneously or concomitantly with the second antibiotic.

In the above combinations, the second antibiotic may be selected from any suitable compound that can be used in the treatment of an NTM infection, provided that the compounds provide an additive or synergistic effect when used in combination. For example, in certain embodiments, the second antibiotic may be selected from the group consisting of Clofazimine, Rifabutin and Amikacin, or a pharmaceutically acceptable salt or solvate thereof.

It will be appreciated that the second antibiotic may be a single further compound or it may be two or more (e.g. 2, 3 or 4) other compound. When used herein, the term “administered sequentially, simultaneously or concomitantly" includes references to: 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 component (A) in conjunction with 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)).

The compounds of formula I, whether used alone or in combination with a second antibiotic, are used to treat an NTM infection. This may be caused by any non-tuberculosis mycobacterium. For example, the NTM infection may be caused by one or more of the group consisting of M. chelonae, M. peregrinum, M. kansasii, and more particularly, M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, M. abscessus subsp. massiliense, M. fortuitum, M. avium and M. intracellulare.

Thus, there is further provided:

(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

(II) a kit of parts comprising components:

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

(ii) a pharmaceutical formulation including another therapeutic agent, in admixture with a pharmaceutically-acceptable adjuvant, diluent or carrier, which components (i) and (ii) are each provided in a form that is suitable for administration in conjunction with the other. 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.

Further aspects and embodiments of the invention will now be described by reference to the following non-limiting examples.

EXAMPLES

Materials and Methods

Bacterial strains and culture media

The NTM strains M. abscessus subsp. bolletii, M. abscesses subsp. massiliense, M. abscessus subsp. abscessus, M. mucogenicum, M. fortuitum, M. avium, M. chelonae, Mycobacterium peregrinum, M. kansasii, M. intracellulare, Mycobacterium smegmatis and its BDQ-resistant mutant strain I66M as well as the clinical isolates strain M. abscessus Bamboo were used. The clinical isolate M. abscessus Bamboo has been isolated from a patient with amyotrophic lateral sclerosis and bronchiectasis, belongs to the subspecies M. abscessus subsp. abscessus. M. mucogenicum, M. fortuitum, M. avium, M. chelonae, Mycobacterium peregrinum, M. kansasii, and M. intracellulare strains were provided by the Department of Medical Sciences, Hackensack Meridian School of Medicine, Nutley, New Jersey, USA. The clinical isolates TelM abm-001 , TelM abm-004, TelM abm-005, TelM abm-01 1 , TelM abm-012, TelA4abb-008 and TelM aba-003 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 M. abscessus 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).

M. bovis BCG (ATCC 700084) cultures were maintained in complete Middlebrook 7H9 medium (Sigma-Aldrich) supplemented with 0.5% (vol/vol) glycerol (Promega), 0.05% Tween- 80 (Sigma-Aldrich), and 10% Middlebrook albumin-dextrose-catalase (ADC) (Sigma-Aldrich). Corning T-25 mm² tissue culture flasks, 96-well clear flat bottom polystyrene microplates and 96-well white half area microplate were used for the experiments. Bacterial colonies were grown on Middlebrook 7H10 agar (Sigma-Aldrich) supplemented with 0.5% (vol/vol) glycerol (Promega), 0.05% Tween-80 (Sigma-Aldrich), and 10% Middlebrook oleic acid-albumin- dextrose-catalase (OADC) (Sigma-Aldrich).

Growth inhibition dose-response assay MIC₅₀ values against specific strains were determined using the broth microdilution method. All the wells of clear 96-well flat bottom cell culture plates (Corning) were filled with 100 pl of liquid medium (complete 7H9 medium). SQ31f 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 SQ31f was carried out starting from this first well (500 μM - 0.015 μM). NTM cultures were grown to mid-exponential phase and further diluted to an optical density at 600 nm (OD₆₀₀) value of 0.1 in complete 7H9 medium. In order to create a final ODeoo 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. chelonae, M. peregrinum, M. kansasii the plates were incubated at 37 °C for 5-7 days. For M. smegmatis and I66M mutant, plates were incubated at 37 °C for 2-3 days. The cultures in the wells were manually suspended upon completion of the incubation time and the ODeoo of each well was read using a Tecan Infinite Pro 200 plate reader. The reported MIC₅₀ values represent the concentration that inhibits 50% of bacterial growth compared to the untreated culture.

Determination of intracellular ATP formation

All the wells of clear 96-well plate (Corning) were filled with 100 pl of complete 7H9 medium. SQ31 f was added at two times the desired highest final concentration to the first well in each column. Subsequently, a two-fold serial dilution was done starting from the first well for 16 points to attain a concentration range from 500 μM to 0.015 μM. The respective NTM strain, which was grown to mid-exponential phase, was diluted to an ODeoo of 0.1; 100 pl of the diluted culture was added to each well to create a final OD₅₀₀ of 0.05 in all the wells. The plates were incubated at 37 °C for 6-8 hrs. Upon completion of the incubation period, the intracellular ATP content of the samples was measured by adding the BacTiter-Glo microbial cell viability reagent (Promega), which was carried out according to the manufacturer’s instructions. Equal volumes (50 pl) of bacterial sample and BacTiter-Glo reagent were mixed in each well of an opaque, half-area, white, 96-well, flat-bottom Corning plate. Luminescence was measured with a Biotek Synergy HTX multimode plate reader after 10 min of incubation of the plate in the dark at room temperature. The background luminescence reading was subtracted from the luminescence readings of all the samples. The amount of ATP content is directly proportional to the relative luminescence units. The resulting graph was made using Graph Pad Prism 8 software. Checker-board titration assay

A checkerboard titration assay was carried out as described previously (Hsieh MH, Yu CM, Yu VL et al. Synergy assessed by checkerboard. A critical analysis. Diagn Microbiol Infect Dis 1993; 16: 343-9; Kaushik A, Makkar N, Pandey P et al. Carbapenems and Rifampin Exhibit Synergy against Mycobacterium tuberculosis and Mycobacterium abscessus. Antimicrob Agents ChemotherZOl 5; 59: 6561 -7). SQ31 f and CFZ/RFB/amikacin were added to complete 7H9 medium-containing 96-well flat-bottom Corning cell culture plates. Ten distinct doses of SQ31f (100 μM to 0.25 μM) were examined for interaction with seven different concentrations of CFZ (50 μM to 0.75 μM), amikacin (10 μM to 0.15 μM), or RFB (5 μM to 0.075 μM) using two-fold serial dilutions. A drug-free bacterial culture control well and a control well using only 7H9 media were included in every 96-well plate. The M. abscessus subsp. abscessus R variant was cultivated in complete 7H9 medium and grown to mid-exponential phase. The culture was then added to each well in the 96-well plate upon being diluted to an OD₆₀₀ of 0.01 using 7H9, yielding a final OD₆₀₀ value of 0.005. Plates were kept at 37 °C for four days. Following incubation, the culture was manually resuspended in each 96-well plate, and the OD₆₀₀ of each well was measured with a Biotek Synergy HTX multimode plate reader.

To assess the combined effect of SQ31f and linezolid, TBP/AVI, or clarithromycin, we used M. abscessus ATCC 19977 and a 96-well plate format. The OD₆₀₀ was measured as an indicator of growth. We evaluated the impact of varying concentrations of TBP in a serial dilution, spanning from 12.5 to 0.012 μM, in combination with a fixed concentration of 14 μM AVI. Linezolid was tested at three different concentrations (100 μM, 10 μM and 1 μM). Similarly, three different concentrations of clarithromycin (5 μM, 0.5 μM and 0.05 μM) were tested along with SQ31 f. SQ31 f were tested at doses ranging from 100 μM to 0.25 μM. Calculations of the fractional inhibitory concentration index (FICI) was done to analyze the results. The FICI is calculated as (MIC of drug A in combination/MIC of drug A alone) + (MIC of drug B in combination/MIC of drug B alone). This calculation was done only for wells which showed 50% inhibition of bacterial culture growth compared to drug-free bacterial culture wells. A FICI of <0.5 indicates synergy, a FICI of >0.5 to 4 indicates additive, and a FICI of >4 indicates antagonism.

Bacterial killing assay

M. abscessus subsp. abscessus cultures were aliquoted onto T-25 mm² tissue culture flasks after being grown to the exponential phase and diluted to an OD₆₀₀ of 0.005. Each flask was filled with test compounds, which were then incubated for five days at 37 °C. Around 10 pl of culture was taken out from each flask followed by the serial dilution with Phosphate-buffered saline (PBS). On each quadrant of the 7H10 agar plate, 25 pl of the corresponding dilutions of the culture were plated. For five days, the agar plates were incubated at 37 °C. Colonyforming units (CFU) on the plates were counted to measure the viability of the bacteria.

Methylene blue assay

Test compounds were added to 1.5 ml (OD₆₀₀ = 0.3) aliquots of log-phase cultures of M. abscessus subsp. abscesses that were placed into transparent 2 ml screw-cap glass vials. The medications were added to the cultures and allowed to incubate for 6 hrs at 37 °C. Next, methylene blue dye was added, at a final concentration of 0.001%. All vials were securely sealed after adding methylene blue, and they were kept in a hypoxic jar at 37 °C for 72 hrs. Using an AnaeroGen sachet, the oxygen in the jar was removed. When the culture's oxygen supply runs out, the dye becomes less colorful. The color changes and the differences in the exponentially growing cultures and broth reflect the relative oxygen consumption, thus indirectly indicate the respiration of each culture.

Macrophage experiments

Cytotoxicity assay. Human THP-1 monocytes were differentiated with Phorbol Myristate Acetate (PMA) for 48 hrs and exposed to decreasing concentrations of SQ31f or RFB 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.

Intracellular growth inhibition assay.

THP-1 cells were grown in RPMI medium supplemented with 10% Fetal bovine serum (Sigma Aldrich) (RPMI^FBS) 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 (10⁵ cells/ml) and incubated for 48 hrs at 37 °C with 5% CO₂. Infection with M. abscessus harboring pTEC27 fluorescent tdTomato was carried out at 37 °C in the presence of 5% CO₂ hrs at a MOI 2:1 . After extensive washing with 1 X PBS, cells were incubated with RPMI^FBS containing 250 pg/ml amikacin for 2 hrs and washed again with PBS prior to the addition of 500 pl RPMI^FBS containing DMSO (negative control) or 500 pl RPMI^FBS containing 25 or 50 pg/ml SQ31f. Macrophages were washed and lysed with 100 pl of 1 % Triton X-100 at 4 and 72 hpi. Serial dilutions of macrophage lysates were plated onto LB agar plates and colonies were counted to determine intracellular CPUs.

Microscopy-based infectivity assays. Differentiated macrophages were grown on coverslips in 24-well plates at a density of 10⁵ cells/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), 25 or 50 pg/ml SQ31f, and fixed at 3 dpi 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 :1000) 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 :1000), cells were mounted onto microscope slides using Immu-mount (Calbiochem) and examined with a confocal microscope using a 63X objective.

Preparation of inverted membrane vesicles and ATP synthesis

To prepare E. coli IMVs, cells were grown overnight at 37 °C in LB (lysogeny broth) medium until they reached an OD₅₀₀ of 0.6-0.7. This culture was used to inoculate a 500 ml culture that was then grown overnight in 2 I shake flasks (180 rpm) until it reached an OD₆₀₀ of 0.6- 0.7. Approximately 5 g (wet weight) of E. coli cells were resuspended in 20 ml membrane preparation buffer (50 mM MOPS, 2 mM MgCI₂, pH 7.5) containing EDTA-free protease inhibitor cocktail (one tablet per 20 ml buffer, Roche) and 1 .2 mg/ml lysozyme. The suspension was stirred at room temperature for 45 min, and additionally supplemented with 300 pl of 1 M MgCI₂ and 50 pl DNase I, and stirring was continued for another 15 min at room temperature. All subsequent steps were performed on ice. Cells were lysed by three passages through an ice-cooled microfluidizer (model M-110L, Microfluidics, Westwood, MA, USA) at 13 000 psi. The suspension containing lysed cells was centrifuged at 4,200 g at 4 °C for 20 min. The supernatant containing the membrane fraction was further subjected to ultracentrifugation at 45,000 g at 4 °C for 1 h. The supernatant was discarded, and the precipitated membrane fraction was resuspended in membrane preparation buffer containing 15% glycerol, separated into aliquots, snap-frozen and stored at -80 °C. The concentrations of the proteins in the vesicles were determined by the bicinchonic acid assay (BCA; Pierce, Rockford, IL, USA). IMVs were stored at -80 °C. Mycobacterial IMVs were prepared according to Krah et al. (Krah A, Ragunathan P, Bond PJ, Gruber G. Variations of the Mycobacterium abscessus F-ATP synthase subunit a-c interface alter binding and potency of the anti-TB drug bedaquiline. Biochem Biophys Res Commun 2024; 690: 149249).

ATP synthesis was measured in flat-bottomed white 96-well microtiter plates (Corning). The reaction mix (50 pl) comprised assay buffer (50 mM MOPS, pH 7.5, 10 mM MgCI₂) containing 10 μM ADP, 250 μM Pi and 1 mM NADH. The concentration of Pi was adjusted by addition of 100 mM KH₂PO₄ to the assay buffer. ATP synthesis was started by adding E. coli IMVs to a final concentration of 5 pg/ml . The reaction mixture was incubated at room temperature for 30 min before adding 50 pl CellTiter-Glo reagent, followed by incubation for another 10 min in the dark at room temperature. The luminescence produced, which correlates with the amount of ATP synthesized, was measured by an Infinite 200 Pro plate reader (Tecan), using the following parameters: luminescence; integration time, 500 ms; attenuation, none.

Synthesis of 3,4-Dimethoxycyclobut-3-ene- 1,2-dione

A procedure by Moore et al. (Schrodinger LLC. Schrodinger Release 2020-4: Maestro, Force Fields, Macromodel, Prime, Protein Preparation Wizard, LigPrep, Glide. 2019) was adapted. Trimethyl orthoformate (3 mL, 0.02 mol) and trifluoroacetic acid (0.15 mL, 0.87 mmol) were added to a solution of squaric acid (1 g, 8.76 mmol) in methanol (MeOH) (20 mL). The reaction mixture was heated at reflux for 12-16 h. The resulting mixture was cooled to room temperature and concentrated in vacuo. Water (30 mL) was added, the aqueous layer was extracted with ethyl acetate (3 x 30 mL), and the combined organic layers were washed with brine (30 mL), dried over anhydrous Na2SC>4, filtered and concentrated in vacuo to afford the title compound (1 g, 80%) as an off-white solid. The title compound was used directly in the next step without further purification. ¹ H NMR (400 MHz, CDCI3) 64.37 (s) spectroscopic data (FIG. 7) were in good agreement with literature values (Tantry SJ, Markad SD, Shinde V etal. Discovery of imidazo[1 ,2-a] pyridine ethers and squaramides as selective and potent inhibitors of mycobacterial adenosine triphosphate (ATP) synthesis. J Med Chem 2017; 60: 1379-99).

Synthesis of 3-(4-Morpholinophenyl)-4-((pyridin-2-ylmethyl)amino) cyclobut-3-ene- 1,2-dione (SQ31f)

A solution of 4-(4-bromophenyl)morpholine (compound 2 in FIG. 2) (200 mg, 0.82 mmol) in tetrahydrofuran (THF) (2 mL) was added over 1 min to a solution of n-butyllithium (n-BuLi) (1 .6 M in cyclohexane, 0.52 mL, 0.82 mmol) in THF (5 mL) at -78°C. After 30 min, 3,4- dimethoxycyclobut-3-ene-1 ,2-dione (1 17 mg, 0.82 mmol) in THF (2 mL) was added over 10 min followed after 30 min by trifluoroacetic anhydride (0.14 mL, 0.99 mmol). After a further 10 min, saturated aqueous NH₄CI (10 mL) was added, and the resulting mixture was allowed to warm to room temperature. The phases were separated, and the aqueous layer was extracted with ethyl acetate (3 x 20 mL). The combined organic layers were washed with brine (30 mL), dried (Na2SO4) and concentrated in vacuo to give aryl squarate (compound 3 in FIG. 2), which was used directly in the next step without further purification. 2-Aminomethylpyridine (compound 4 in FIG. 2) (16.4 mg, 0.15 mmol) was added to a solution of the crude aryl squarate (83.1 mg, 0.15 mmol) in anhydrous MeOH (10 mL) at 0°C. The mixture was allowed to warm to room temperature and stirred for 12 h. The bright yellow solid was isolated by filtration, washed with MeOH (20 mL) and ethyl acetate (10 mL) to give the SQ31f (compound 1 in FIG. 2) as a yellow solid (26 mg, 49% yield over two steps). ¹H NMR (400 MHz, CDCI₃) 5 8.62 (d, J = 4.4 Hz, 1 H), 7.84 (d, J = 8.4 Hz, 2H), 7.81-7.78 (m, 1 H), 7.68 (brs, 1 H), 7.43 (d, J= 7.6 Hz, 1 H), 7.34 (t, J= 5.2 Hz, 1 H), 6.95 (d, J= 8.8 Hz, 2H), 5.16 (d, J = 4.4 Hz, 2H), 3.86 (t, J = 4.8 Hz, 4H), 3.30 (t, J = 4.8 Hz, 4H) and ¹³C NMR (100 MHz, CDCh) 5 191.6, 189.7, 178.1 , 164.6, 154.6, 152.6, 148.5, 137.9, 128.1 , 123.3, 122.6, 120.1 , 1 14.3, 66.5, 47.9, 47.6; MS (ESI) m/z 350.2 [M + H]+ spectroscopic data (FIG. 7) were in good agreement with literature values (Tantry SJ, Markad SD, Shinde V et al. Discovery of imidazo[1 ,2-a] pyridine ethers and squaramides as selective and potent inhibitors of mycobacterial adenosine triphosphate (ATP) synthesis. J Med Chem 2017; 60: 1379-99).

Synthesis of SQ31 f analogues

Synthesis of substituted pyridine analogs of squaramide is depicted in FIG.18. A 4-step synthetic pathway was employed to provide substituted pyridine amines. Firstly, in the presence of tert-butyl nitrite and potassium tert-butoxide base, the formation of the nitroso methyl intermediate was formed which will tautomerize to give the oximes (Caravez, J. C., Hu, Y., Oftadeh, E., Mamo, K. T., & Lipshutz, B. H. (2024). Preparation of a key intermediate en route to the anti-HIV drug lenacapavir. The Journal of Organic Chemistry, 89(6), 3995-4000). Then, utilizing zinc in acetic acid, the reduction of oximes to amines were formed in good yields. Amines were converted to carbamates. The carbamate was cleaved using trifluoracetic acid (TFA) and the TFA salt was then used for the substitution reaction with 3-methoxy-4-(4- morpholinophenyl)cyclobut-3-ene-1 , 2-dione.

Subsequently, 3, 4-dimethoxycyclobut-3-ene-1 , 2-dione was treated with the in situ prepared lithium reagent derived from 4-(4-bromophenyl)morpholine, followed by addition of trifluoroacetic anhydride to the reaction solution to give the intermediate 3-methoxy-4-(4- morpholinophenyl)cyclobut-3-ene-1 , 2-dione. Then, the monoketal was reacted with the TFA salt of substituted 2-(aminomethyl)pyridine in the presence of triethylamine in MeOH to yield substituted 2-(aminomethyl)pyridine analogs of squaramide in moderate yield. The ethylenediamine analogs of squaramide were synthesised through a straightforward synthetic approach in one step by treating the commercially available ethylenediamines with the intermediate 3-methoxy-4-(4-morpholinophenyl)cyclobut-3-ene-1 , 2-dione intermediate in MeOH.

The substituted morpholine analogs of squaramide were synthesised as described in FIG. 19. AZ-alkylation of the 4-bromoaniline with methyl 2-bromoacetate provided methyl (4- bromophenyl)glycinate, which was lactonized in the presence of the appropriate epoxide and lithium perchlorate to provide the lactone in moderate yield. The cyclic lactone was reduced to the acetal, followed by Kursanov deoxygenation under acidic conditions to the corresponding morpholine. Subsequently, 3, 4-dimethoxycyclobut-3-ene-1 ,2-dione was treated with the in situ prepared lithium reagent derived from 4-(4-bromophenyl)morpholine, followed by addition of trifluoroacetic anhydride to the reaction solution to give the intermediate 3-methoxy-4-(4-morpholinophenyl)cyclobut-3-ene-1 ,2-dione. Then, the monoketal was reacted with 2-(aminomethyl)pyridine in MeOH to yield substituted 2-(aminomethyl)pyridine analogs of squaramide in moderate yield. in silica studies

Homology models of both the M. abscessus and M. avium Fo domains were generated using the SQ31f-bound F_o domain of the M. smegmatis F-ATP synthase (PDB: 8G07) as a reference template and the resulting model was energy minimized using prime/macromodel tools with the Schrodinger suite of programs. SQ31f was sketched using 2D-sketcher and prepared using a ligand preparation tool for docking studies to analyse the molecular interaction at the a-c interface. A docking grid was generated using the bound coordinates of SQ31f and the same was used for standard and extra-precision (XP) glide docking studies.

Example 1 : Modified synthesis of SQ31f

The fast-growing NTM M. abscessus complex is of increasing clinical concern. To extend the low-populated anti- M. abscessus drug pipeline with a soluble and potent inhibitor targeting the OXPHOS pathway of the M. abscessus complex, a repurposing approach of the anti-TB inhibitor SQ31 f was first used. This compound was originally synthesized by Tantry et al. (Tantry SJ, Markad SD, Shinde V et al. Discovery of imidazo[1 ,2-a] pyridine ethers and squaramides as selective and potent inhibitors of mycobacterial adenosine triphosphate (ATP) synthesis. J Med Chem 2017; 60: 1379-99). Their route, however, included a low-yielding carbon-carbon bond-forming step (16%). We therefore modified the procedure of Reed et al. (Reed MW, Pollart DJ, Perri ST et al. Synthesis of 4-substituted- 3-alkoxy-3-cyclobutene-1 ,2- diones. J Org Chem 1988; 53: 2477-82). The reaction between the lithium reagent derived from bromide (compound 2) and dimethyl squarate generated the arylated derivative (compound 3). Treatment of the derivative (compound 3) with 2-aminomethylpyridine (compound 4) in MeOH cleanly gave SQ31f (compound 1 ) with a 26% yield over two steps (FIG. 2). Additionally, this procedure delivered pure material without the need for column chromatography (FIG. 7).

Example 2: In vitro profiling of SQ31f on NTM The susceptibility of M. abscessus subsp. abscessus ATCC 19977 to the synthesized SQ31 f was evaluated in complete Middlebrook 7H9 broth. The compound displayed good potency with an MIC₅₀ of 5.0 ± 0.9 and 6.2 ± 1.1 μM against the M. abscessus subsp. abscessus S strain (FIG. 3a) and R strain (FIG. 3a), respectively. All MIC₅₀ and MIC₉₀ values are provided in Table 1. To test the inhibitor under more clinically relevant settings, the MIC of the M. abscessus subsp. abscessus S strain was determined after 14 days. As shown in FIG. 8, the MIC remained largely unchanged compared with the 3-day treatment, mirroring the results observed with the bedaquiline control. An important requirement of a potent anti - M. abscessus agent is its efficacy against clinical isolates. FIG. 3b shows SQ31f’s efficacy against the isolate M. abscessus Bamboo, with an MIC₅₀ of 9.2 ± 0.7 μM (Table 1) and the clinical isolate TelM aba-003 2.2 ± 0.9 μM (FIG. 14A and Table 1 ). The compound also displayed high efficacy against Mycobacterium bolletii and Mycobacterium massiliense with MIC₅₀ values of 2.0 ± 0.4 and 5.9 ± 0.4 μM (FIG. 3a), respectively, and the clinical strains Tel Mabm-00l , TelM abm-004, TelMabm-005, TelM abm-01 1 and TelM abm-012 with MIC₅₀ values of 5.6 ± 2.1 μM, 2.7 ± 1 μM, 2.2 ± 0.7 μM, 0.7 ± 0.2, and 1 .4 ± 0.5, respectively (FIG. 14B and Table 1 ) which belong to the subspecies M. abscessus subsp. massiliense, as well as the M. bolletti clinical isolate TelM abb-008 with MIC₅₀ values of 0.9 ± 0.3 μM (FIG. 14D and Table 1 ), indicating that the inhibitor is active against the three subspecies and clinical isolates of the M. abscessus complex.

Table 1. Growth and ATP synthesis inhibitory potency of SQ31f against NTM and the isolate

M. abscessus Bamboo in 7H9 media

To test whether SQ31 f is bactericidal or bacteriostatic against M. abscessus, the survival of the bacterium upon drug exposure was studied. As revealed in FIG. 9, the compound was bacteriostatic against M. abscessus subsp. abscessus at 10-fold its MIC₅₀ in 7H9 broth.

We next tested whether M. abscessus growth inhibition by SQ31f is related to depletion of ATP by measuring intracellular ATP synthesis in the presence of the inhibitor. As presented in FIG. 3c, SQ31f inhibited ATP synthesis within the bacterium with an IC50 of 8.3 ± 0.3 and 8.3 ± 1.4 μM in M. abscessus subsp. abscessus S and R variants, respectively, as well as in the isolate M. abscessus Bamboo (IC50 = 3.6 ± 0.8 μM; FIG. 3c and Table 1 ), TelM abm-001 , -004, -005, -01 1 and -012 with IC50 values of 24 ± 9 μM, 7.5 ± 3 μM, 3.15 ± 1.2 μM,1.9 ± 0.3 μM and 1.3 ± 0.4 μM respectively (FIG. 14C, Table 1). SQ31f inhibited ATP synthesis of TelM abb-008 with IC50 values of 2.5 ± 0.6 μM (FIG. 14E). These results underscore that the SQ31f-mediated growth inhibition is in line with intracellular ATP depletion and that OXPHOS of the pathogen is the main target. To exclude the possibility that SQ31f may affect oxygen consumption within the ETC, a methylene blue assay was performed. FIG. 3e underlines the fact that oxygen consumption of M. abscessus subsp. abscessus was unaffected at 12 and 24 μM SQ31f and suggests that SQ31f targets the F-ATP synthase of M. abscessus. SQ31 f reduced ATP synthesis of IMVs, demonstrating that the whole-cell ATP depletion observed is only caused by inhibition of OXPHOS and not by substrate-level phosphorylation in glycolysis. In addition, ATP inhibition was similar in the presence of the electron donors NADH and succinate (FIG. 10), indicating that the compound targets the mycobacterial F-ATP synthase. To underscore the target specificity of SQ31f, ATP formation of E. coli IMVs was measured. As revealed in FIG. 3f, SQ31f does not affect ATP formation of E. coli IMVs, while quercetin — known to inhibit the E. coli F-ATP synthase — reduced ATP synthesis of E. coli IMVs. We also studied SQ31f’s efficacy against fast-growing NTM species, including M. fortuitum, M. peregrinum, M. mucogenicum and M. chelonae. As displayed in FIG 11a and FIG. 11 b), SQ31f exhibited potent activity against M. fortuitum (MIC₅₀ = 1.3 ± 0.1 μM), M. peregrinum (MIC₅₀ = 4.2 ± 1.3 μM) and M. chelonae (MIC₅₀ = 0.8 ± 0.1 μM), all opportunistic pathogens mainly isolated from patients with skin and soft tissue infection. Additionally, M. mucogenicum, which is associated with respiratory, CNS, catheter-related, and skin and soft tissue infections was inhibited by SQ31f, with an MIC₅₀ of 0.6 ± 0.4 μM (FIG. 11 b). The whole-cell ATP synthesis inhibition assays were consistent with the observed growth reduction of M. fortuitum (MIC₅₀ = 14.0 ± 2.3 μM), M. peregrinum (MIC₅₀ = 1-7 ± 0.8 μM, FIG. 11c), M. mucogenicum (MIC₅₀ = 1.0 ± 0.4 μM, FIG. l id, Table 1 ) and M. chelonae (MIC₅₀ = 2.3 ± 0.9 μM, FIG. l id, Table 1 ).

In addition, we investigated the effect of SQ31f against the slow-growing M. avium subsp avium, M. intracellulare and M. kansasii, which are major causes of pulmonary infections worldwide. SQ31f demonstrated potent activity against M. avium (MIC₅₀ = 1.1 ± 0.3 μM), M. intracellulare (MIC₅₀ = 13.0 ± 1.7 μM) and M. kansasii (MIC₅₀ = 0.37 ± 0.2 μM) (FIG. 12a and FIG. 12b, Table 1 ). Whole-cell ATP synthesis inhibition assays further corroborated these findings, with MIC₅₀ values of 3.7 ± 1 .1 μM for M. avium, 29 ± 3.9 μM for M. intracellulare and 0.36 ± 0.1 μM for M. kansasii (FIG. 12c, Table 1 ), underscoring a strong correlation between ATP depletion and growth inhibition.

Example 3: Ex vivo anti-M. abscessus potency of SQ31f

To investigate the anti-/W. abscessus potency of the inhibitor in macrophages, we used a THP- 1 infection model, as previously reported (Johansen MD, Daher W, Roquet-Baneres F et al. Rifabutin is bactericidal against intracellular and extracellular forms of Mycobacterium abscessus. Antimicrob Agents Chemother 2020; 64: e00363-20). Initially, the cytotoxicity of SQ31f against THP-1 cells during a 3-day exposure period was assessed. FIG 4a suggests that the compound exhibits cytotoxicity only at high concentrations (CC50 = 160 μM) and the rate of macrophage killing occurred at lower concentrations with rifabutin compared with SQ31f.

Given the observed low cytotoxicity, the intracellular efficacy of SQ31 f in THP-1 cells infected with M. abscessus CIP104536⁷ (S variant) expressing tdTomato was evaluated. Following infection, macrophages were initially exposed to 250 pg/mL amikacin for 2 h to kill and eliminate extracellular bacteria and then treated with SQ31f (25 and 50 μM). Untreated infected macrophages served as a negative control for intracellular bacterial replication. At 4 h post-infection (hpi) and 3 days post-infection (dpi), macrophages were lysed and plated to determine the intracellular bacterial loads. As depicted in FIG. 4b, SQ31 f significantly impeded M. abscessus growth, as evidenced by the reduction in viable bacterial counts (cfu/mL) at both concentrations. By 3 dpi, there was an approximately 1.5 log decrease in the number of intracellular M. abscessus when infected macrophages were treated with 50 μM SQ31f (FIG. 4b). The bacterial loads being similar to the initial inoculum (untreated, 4 hpi) suggests that SQ31f is bacteriostatic, confirming the in vitro killing experiments (FIG. 9). Concurrently, macrophages were stained with anti-CD43 and DAPI and examined under a confocal microscope. SQ31 f-treated macrophages did not exhibit any visual alterations in membrane integrity, cell morphology or size upon microscopic examination (FIG. 4c) but showed a notable reduction in the number of bacilli inside the macrophages treated with SQ31f (FIG. 4c), consistent with the decreased intracellular bacterial burden (FIG. 4b). Collectively, these findings demonstrate that SQ31f enters human macrophages and inhibits bacterial replication.

Example 4: SQ31f binding to a proton half-channel

To understand SQ31f’s mechanism of action, we generated a structural model of the M. abscessus/M. avium Fo domain (FIG. 5a) based on the homology of the Fo subunits (FIG. 13) and performed docking studies. Firstly, the structure of the M. abscessus/M. avium Fo domain was modelled based on the cryo-EM structure of the M. smegmatis Fo part, wherein the SQ31 f ligand was seen to bind to a distinct site at the lagging interface of M. abscessus/M. avium’s subunits a:c, in comparison with diarylquinolines (bedaquiline, TBAJ-876 and TBAJ-5307), which are described to bind to leading and lagging sites of the a-ce interface as well as to the c ring. SQ31 f was predicted to dock tightly with an XP glide score of -6.3 kcal/mol in the cavity corresponding to the cytosolic proton half-channel, being solvent exposed. These data corroborate and explain the high potency of aqueous soluble SQ31f (cLogP = 1 .02) in contrast to bedaquiline (cLogP = 7.25), TBAJ-876 (cLogP = 5.15) or TBAJ-5307 (cLogP = 4.7). The molecular docking results predicted the binding pose of SQ31f with a root mean square deviation of 0.7 A to the cryo-EM binding pose. The squaramide scaffold was involved in a multitude of polar interactions, including the two carbonyl groups, which form polar/hydrogen bonding interactions with subunit a residues R188 (3.2 A), Y240 (1 .8 A) and Q243 (2.1 A), respectively, while the pyridyl methyl group on the amide group of the squaramide was oriented into the shallow cavity of the eg ring (FIG. 5a). The ‘NIT atoms from the amide group of the squaramide and the ‘N’ atom of the pyridyl group maintain the essential H-bonding (2.0 A)/polar interactions (3.2 A) with the COOH group of the E65 side chain, respectively (FIG. 5a). Interestingly, the morpholine group, which affords the highest potency in this squaramide series, was seen to be anchored between subunit a residues H166/1173 and engaged in electrostatic interaction with N174 side chain atoms (data not shown) (FIG. 5a). In addition, the phenyl ring of SQ31f mediates TT-TT interaction with subunit c residue F68, as well as van der Waals contacts with subunit a residues Q243, K181 and E177 and was totally occluded into the cavity of subunit a:c₉ helices.

The difference between the binding sites of SQ31 f and bedaquiline is nicely illustrated by the additive effect of SQ31f in the bedaquiline-resistant F-ATP synthase subunit c mutant I66M of M. smegmatis, where 0.3 μM SQ31f in combination with bedaquiline prevents growth of the mutant strain (FIG. 5b).

Example 5: SQ31f potentiates existing antibiotics

To accelerate durable cure, multidrug regimens are required with potentiating inhibitors, we studied growth inhibition activity of SQ31f along with the clinical M. abscessus antibiotic clofazimine, which inhibits electron transfer from NADH to menaquinone in the ETC, amikacin, clarithromycin or linezolid, targeting the ribosome, rifabutin, binding to the RNA polymerase, or the oral pair tebipenem and avibactam, which are important for cell wall synthesis, in a chequerboard titration assay. Significant cell growth inhibition was observed with all the drugs tested (FIG. 6a-f). The FIC indices (FICIs), which describe the interaction between SQ31 f and each of the test inhibitors, are listed in Table 2. The calculated FICI values of 0.55, 0.93, 0.86, 0.62, 0.94 and 0.55 indicate additive growth inhibition in M. abscessus subsp. abscessus triggered by amikacin, clofazimine, rifabutin, clarithromycin, linezolid or tebipenem/avibactam with SQ31f, respectively. Overall, these data indicate that SQ31f is a potential inhibitor for combinatory approaches with major anti-A4. abscessus drugs.

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

Example 6: SQ31f analogues

Our first synthetic effort leading to the analogues of SQ31 f aimed to modify the 2 -pyridyl methyl substitution on the right-hand side (RHS) (FIG. 15). Further, we focused on the derivatives with an additional alkyl and/or phenyl group on the morpholine moiety, where was found to be the best by Tantry et al. (Tantry, S.J., Markad, S.D., Shinde, V., Bhat, J., et al. (2017) Discovery of lmidazo[1 ,2-a]pyridine Ethers and Squaramides as Selective and Potent Inhibitors of Mycobacterial Adenosine Triphosphate (ATP) Synthesis. J Med Chem 60:1379- 1399). Therefore, we were determined to explore further possibilities on the RHS and extend the structure-activity relationship (SAR) study of SQ31f. For evaluating SAR, the extended carbon chain disubstituted amine derivatives were designed to investigate the role of 2-pyridyl methyl substitution on the RHS of SQ31f which interacts with E62 residue of c-ring. This hydrophilic cavity also highlights the possibility to enhance interaction and potency of inhibitor with E62 residue. Due to their potential to facilitate better hydrogen bonding interaction with the NTM F-ATP synthase, we designed substituted pyridine analogues of SQ31f, having a set of halogen group (F, Cl, and Br) and CN that are installed at the 5 or 6 position of pyridine moiety. We also implemented quinoline moiety to our structure mainly from the synthetic point of view to observe the relationship between structure and reactivity. In this study, 14 squaramide analogues were designed and synthesised. While the potency of analogue TMN- 01 will be described below, the biological properties of the novel analogues will be evaluated and a subset of SQ31f analogues will be attentively selected for further investigation based on their antimycobacterial activity.

Extension of a methyl on the morpholine moiety in the analogue TMN-01 (FIG. 16A; cLogP = 1.54) resulted in a similar growth inhibition profile against M. abscesses subsp. massiliense and M. abscesses sebsp. bolletii (FIG. 16B-C). Interestingly, TMN-01 revealed a better potency against Mycobacteriem bovis BCG (ATCC 700084), which is 100% homolog to the tuberculosis causing Mycobacteriem tebercelosls counterpart (FIG. 16D). Structural modelling of the SQ31f-bound Mab F₀-domain shed light into the binding epitope and cavities around the SQ31f morpholine- and phenyl ring, providing insights for further structure activity relationship studies (SAR) to even optimize the potency of the inhibitor. The planned analogues of SQ31 f are shown in FIG. 17.

Conclusion

Diseases caused by NTMs are increasing worldwide. To tackle these difficult-to-manage opportunistic pathogens, new potent and soluble inhibitors with good pharmacophores and combinatory properties are needed to design potent multidrug treatment regimens. Here, we present SQ31f as a novel anti-NTM inhibitor binding to the FO domain of the F1 FO-ATP synthase and preventing translocation of protons from the intermembrane to the cytosolic site. SQ31f binds at an interface of subunits a and c, which is very different to the binding site of bedaquiline and other diarylquinolines like TBAJ-876 or TBAJ-5307. SQ31f fits tightly in the cavity corresponding to the cytosolic proton half-channel, which is expected to be filled with water, consistent with the favourable high aqueous solubility of SQ31 f compared with bedaquiline, TBAJ-876 or TBAJ-5307. SQ31 f reveals specificity for mycobacterial F-ATP synthase, as indicated by its lack of inhibitory effect on the E. coli F-ATP synthase in IMVs (FIG. 3f) and the distinct interacting residues in the E. co// subunits a and c (FIG. 13). Similarly, the amino acid sequences of the human a and c subunits differ from their mycobacterial counterparts (FIG. 13), indicating that SQ31 f is unlikely to bind to the human enzyme, thereby preventing inhibition of ATPase activity and ATP synthesis, as observed with bedaquiline. These are critical characteristics of SQ31f, contributing to its reduced toxicity in humans.

Inhibiting the NTM F-ATP synthase affects not only the pathogen’s production of the currency of life but also ATP homeostasis, regulation of pmf under multiple growth conditions, ATP- dependent replication or mediation of mycobacterial stringent response by the Rel protein. Importantly, this compound does not only overcome the problem of bedaquiline resistance, which is on the rise, but it also complements the anti-/W. abscessus activity of clofazimine, amikacin, clarithromycin, linezolid, rifabutin or the oral combination tebipenem/avibactam, which are targeting electron transport, transcription, protein synthesis or cell wall formation. Such drug associations would open the door to inhibit major cell processes of NTM, to reduce the emergence of drug resistance, to reduce the inhibitor concentrations of each within the cocktail, and finally to lower toxicity and side effects. This makes SQ31f a potentially cost- effective and attractive drug for pharma, healthcare and, most importantly, improving treatment outcomes for NTM patients.

Claims

Claims

1. A compound of formula I: where:

R₁ represents, -(CH₂)_n-N(R_4aR_4b)₂, -(CH₂)_n-piperidyl, -(CH₂)_n-2-pyridyl, -(CH₂)_n-quinolinyl, -(CH₂)_n-pyrrolidinyl , or morpholinyl, which -(CH₂)_n-piperidyl, -(CH₂)_n-2-pyridyl, -(CH₂)_n- quinolinyl, -(C H₂)_n-pyrrolidiny I , and -(CH₂)_n-morpholinyl are unsubstituted or substituted by one or more groups selected from the group consisting of halo, and CN; n is 0, 1 or 2;

R₂ and R₃ are each independently selected from H, Ar, C_1-6 alkyl, -(CH₂)_m-CO₂R_4c, -(CH₂)_m-Ar, -(CH₂)m-Het¹, where Ar, C_1-a alkyl, -(CH₂)_m-Ar, and -(CH₂)_m-Het¹ are unsubstituted or are substituted by one or more substituents selected from the group consisting of halo, and OR_4d, each Ar is independently a C_6-10 aromatic group;

Het¹ is a 5- to 10 heteroaromatic group having from 1 to 3 heteroatoms; m is 0, 1 , or 2;

R_4C is H, C_1-5 alkyl or phenyl, where the C_1-6 alkyl or phenyl groups are unsubstituted or substituted by one or more substituents selected from the group consisting of halo, OH, and OC_1-3 alkyl;

R_4d is H or C_1-6 alkyl, where the C_{1 6} alkyl group is unsubstituted or substituted by one or more substituents selected from the group consisting of halo, OH, and OC_1-3 alkyl; and pharmaceutically acceptable salts or solvates thereof, provided that, when R₁ represents unsubstituted -(CH₂)_r-2-pyridyl, then one or R₂ and R₃ are not H.

2. The compound according to Claim 1 , or pharmaceutically acceptable salts or solvates thereof, wherein R₂ and R₃ are each independently selected from H, Ph, CF₃, Me, Et, ⁿPr, 'Pr, cyclopropyl, ⁿBu, -CH₂-OH, -CH₂-O-Me, -CH₂-O-Et, -CH₂-O-ⁿPr, -CHs-O-'Pr, -CH₂-O-cyclopropyl, -CO₂H, -CH₂-CO₂H, -CH₂-CH₂-CO₂H, -CH₂-CH₂-CO₂Me, -CH₂-CH₂-CO₂Et, -CH₂-CH₂-CO₂ ⁿPr, -CH₂-CH₂-CO₂ ⁱPr, -CH₂-CH₂-C0₂cyclopropyl, -CH₂-CH₂- CO₂Ph, -CH₂-Ph, -CH₂-quinoline, -CH₂-isoquinolene, -CH₂-quinazoline, -CH₂-pthalazine.

3. The compound according to Claim 2, or pharmaceutically acceptable salts or solvates thereof, wherein R₂ and Rs are each independently selected from H, Me, Et, ⁿPr, 'Pr, cyclopropyl, -CH₂-OH, -CH₂-O-Me, -CH₂-O-Et, -CO₂H, -CH₂-CH₂-CO₂H, -CH₂-CH₂-CO₂Me, - CH₂-CH₂-CO₂Et, -CH₂-CH₂-CO₂Ph, -CH₂-Ph, -CH₂-quinoline, -CH₂-isoquinolene, -CH₂- quinazoline, -CH₂-pthalazine.

4. The compound according to any one of the preceding claims, or pharmaceutically acceptable salts or solvates thereof, wherein R₂ represents CH₃ or CF₃ and R₃ represents CF₃, Me, -CH₂-OH, -CH₂-O-Me, -CO₂H, or-CH₂-C0₂H, optionally wherein Ri represents -(CH₂)n-2- pyridyl, which -(CH₂)_n-2-pyridyl group is unsubstituted or substituted by one or more substituents selected from halo and CN.

5. The compound according to any one of the preceding claims, or pharmaceutically acceptable salts or solvates thereof, wherein Ri represents, -(CH₂)-N(Me)₂, -(CH₂)_n-N(Et)₂, -(CH₂)_n-piperidyl, -(CH₂)_n-2-pyridyl, quinolinyl, -CH₂pyrrolidinyl, or -CH₂morpholinyl, where - (CH₂)_n-piperidyl, quinolinyl, -CH₂pyrrolidinyl, and -CH₂morpholinyl are unsubstituted and - (CH₂)_n-2-pyridyl is unsubstituted or substituted by one or more substituents selected from the group consisting of F, Cl, Br and CN.

6. The compound according to any one of the preceding claims, or pharmaceutically acceptable salts or solvates thereof, wherein R₁ represents,

7. The compound according to any one of Claims 1 to 5, or pharmaceutically acceptable salts or solvates thereof, wherein:

R₁ represents, -(CH₂)_n-N(Et)2, -(CH₂)_n-piperidyl, -(CH₂)_n-2-pyridyl: and n is 1 or 2.

8. The compound according to any one of the preceding claims, wherein the compound of formula I is selected from the list:

or a pharmaceutically acceptable salt or solvate thereof.

9. The compound according to Claim 8, wherein the compound of formula I is

, or a pharmaceutically acceptable salt or solvate thereof

10. A compound of formula I as defined in any one of Claims 1 to 9, or a pharmaceutically acceptable salt or solvate thereof, for use in the treatment of nontuberculous mycobacterial (NTM) infection.

11. Use of a compound of formula I as defined in any one of Claims 1 to 9, or a pharmaceutically acceptable salt or solvate thereof, in the manufacture of a medicament for the treatment of nontuberculous mycobacterial (NTM) infection.

12. A method of treating a nontuberculous mycobacterial (NTM) infection comprising the step of administering to a subject in need thereof a compound of formula I as defined in any one of Claims 1 to 9, or a pharmaceutically acceptable salt or solvate thereof.

13. A composition comprising a compound of formula I as defined in any one of Claims 1 to 9, or a pharmaceutically acceptable salt or solvate thereof, and one or more pharmaceutically acceptable excipients. pharmaceutically acceptable salt or solvate thereof, and a further antibiotic compound, or a pharmaceutically acceptable salt or solvate thereof, in the manufacture of a medicament for the treatment of a nontuberculous mycobacterial (NTM) infection wherein the compound of formula I, or a pharmaceutically acceptable salt or solvate thereof, is to be administered sequentially, simultaneously or concomitantly with the second antibiotic.

15. A compound of formula I as defined in any one of Claims 1 to 9, or a pharmaceutically acceptable salt or solvate thereof, and a further antibiotic compound, or a pharmaceutically acceptable salt or solvate thereof, for use in the treatment of a nontuberculous mycobacterial (NTM) infection wherein the compound of formula I, or a pharmaceutically acceptable salt or solvate thereof, is to be administered sequentially, simultaneously or concomitantly with the second antibiotic.

16. A method of treating a nontuberculous mycobacterial (NTM) infection comprising the step of administering to a subject in need thereof a compound of formula I as defined in any one of Claims 1 to 9, or a pharmaceutically acceptable salt or solvate thereof and a second antibiotic, or a pharmaceutically acceptable salt or solvate thereof, wherein the compound of formula I, or a pharmaceutically acceptable salt or solvate thereof, is administered sequentially, simultaneously or concomitantly with the second antibiotic.

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

18. The use according to Claim 1 1 or Claim 14, the method of Claim 12 or Claim 15, the compound according to Claim 13 or Claim 16, wherein the NTM infection is caused by one or more of the group consisting of M. chelonae, M. peregrinum, M. kansasii, and more particularly, M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, M. abscessus subsp. massiliense, M. fortuitum, M. avium and M. intracellulare.