Inhibitors of the cytochrome BD oxidase for the treatment of tuberculosis and other mycobacterial diseases

Abstract

The present invention provides cytochrome bd oxidase inhibitors. The present invention provides the use of the cytochrome bd oxidase inhibitors as a medicament to treat a bacterial infection. The present invention also provides the use of the cytochrome bd oxidase inhibitors as a medicament to treat a bacterial infection with a QcrB inhibitor.

Description

[0001] INHIBITORS OF THE CYTOCHROME BD OXIDASE FOR THE TREATMENT OF TUBERCULOSIS AND OTHER MYCOBACTERIAL DISEASES

[0002] FIELD OF INVENTION

[0003] The present invention provides cytochrome bd oxidase inhibitors. The present invention provides the use of the cytochrome bd oxidase inhibitors as a medicament to treat a bacterial infection. The present invention also provides the use of the cytochrome bd oxidase inhibitors as a medicament to treat a bacterial infection with a QcrB inhibitor.

[0004] BACKGROUND

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

[0006] Mycobacterium tuberculosis is a leading cause of death worldwide from a single infectious agent. More than 10 million people fall ill and approximately 1 .4 million die of the disease every year. The emergence and spread of cases caused by multidrug resistant (MDR) and extensive drug resistant (XDR) strains is a great concern. While several new drugs such as bedaquiline were approved for clinical use in recent years, additional novel drugs are needed to develop sterilizing drug regimen able to shorten treatment to 4 months or less, associated with a low propensity to elicit drug resistance.

[0007] Besides tuberculosis, several other mycobacterial diseases are either highly prevalent or emerging. For instance, infections caused by non-tuberculosis mycobacteria (NTM) are on the rise globally: NTM lung diseases are already much more prevalent than tuberculosis in many developed countries.

[0008] Oxidative phosphorylation is gaining interest as a drug target space for the treatment of tuberculosis. Following the approval of the FiF0ATP synthase inhibitor bedaquiline (Sirturo®) for MDR and XDR tuberculosis, several series of compounds targeting the terminal cytochrome bcc:aaa oxidase (QcrB inhibitors) have been reported. The QcrB inhibitor Q203 (Telecebec) recently completed a phase 2 clinical trial. Despite their clinical promise, QcrB inhibitors are bacteriostatic, which is a disadvantage since bactericidal agents are necessary to develop a rapidly sterilizing drug regime. A functional redundancy between the cytochrome bcc:aaa and the alternate cytochrome bd oxidase explains the bacteriostatic nature of Q203. The discovery of ND-01 1992 (Lee BS, et al. (2021 ) Dual inhibition of the terminal oxidases eradicates antibiotic-tolerant Mycobacterium tuberculosis. EMBO Mol Med 13(1 ):e13207) represents a proof of concept that well-designed cytochrome bd oxidase inhibitors act synergistically to kill M. tuberculosis.

[0009] Thus, there is a need for alternative and / or improved cytochrome bdoxidase inhibitors suitable for the treating and / or ameliorating M. tuberculosis infections and other mycobacterial infections (e.g., non-tuberculous mycobacteria such as Mycobacterium abscessus or Mycobacterium avium).

[0010] SUMMARY

[0011] Aspects and embodiments of the current invention will now be described by reference to the following numbered clauses.

[0012] 1. A compound of formula I: wherein:

[0013] X represents CF or N;

[0014] Y represents CR5 or N;

[0015] R1 and R2 each independently represent H, C1-6 alkyl, halo, and ORe, where the C1-6 alkyl is unsubstituted or substituted by one or more halo atoms;

[0016] R3 represents:

[0017] where the wiggly line is the point of attachment to the rest of the molecule;

[0018] FU and R5, when present, each independently represent H, halo or CN;

[0019] Re represents H, or C1-6 alkyl, where the C1-6 alkyl is unsubstituted or substituted by one or more halo atoms; each R?a and R?b independently represent H, F, C1-5 alkyl, where each Ci-e alkyl group is unsubstituted or substituted by one or more halo atoms; each R7Cto R7eindependently represent H, halo, OC1-6 alkyl, and CN, where each OC1-6 alkyl group is unsubstituted or substituted by one or more halo atoms; more particularly each R7C’ and R7e’ independently represent H, halo and OC1-6 alkyl, where each OC1-6 alkyl group is unsubstituted or substituted by one or more halo atoms; each R7fto R7h independently represent H, halo, and C1-6 alkyl, where C1-6 alkyl group is unsubstituted or substituted by one or more halo atoms; each R7iand R7j independently represent H, halo, C1-6 alkyl, OC1 6 alkyl, CN, and piperidinyl, where each C1-6 alkyl group and each OC1-6 alkyl group is unsubstituted or substituted by one or more halo atoms;

[0020] R7, represents H or halo;

[0021] R7I represents C1-6 alkyl, where each C1-6 alkyl group is unsubstituted or substituted by one or more halo atoms, or a pharmaceutically acceptable salt or solvate thereof, provided that the compound is not 2. The compound according to Clause 1 or a pharmaceutically acceptable salt or solvate thereof, wherein Ri represents H, CH3, Br, OCH3, or CF3.

[0022] 3. The compound according to Clause 1 or Clause 2, or a pharmaceutically acceptable salt or solvate thereof, wherein R2represents H or Cl.

[0023] 4. The compound according to any one of the preceding clauses, or a pharmaceutically acceptable salt or solvate thereof, wherein R4, when present, represents H, or Cl or more particularly, F.

[0024] 5. The compound according to any one of the preceding clauses, or a pharmaceutically acceptable salt or solvate thereof, wherein R5, when present, represents H, F or Cl.

[0025] 6. The compound according to any one of the preceding clauses, or a pharmaceutically acceptable salt or solvate thereof, wherein R6is H or CH3.

[0026] 7. The compound according to any one of the preceding clauses, or a pharmaceutically acceptable salt or solvate thereof, wherein each R7aand R?b independently represent H, F, CH3or CF3.

[0027] 8. The compound according to any one of the preceding clauses, or a pharmaceutically acceptable salt or solvate thereof, wherein R7crepresents H, F, OCH3, OCF3, Br or Cl and R7arepresents H, Cl or F.

[0028] 9. The compound according to Clause 8, or a pharmaceutically acceptable salt or solvate thereof, wherein R7crepresents H, F, OCH3, OCF3, Br or Cl and R7erepresents H.

[0029] 10. The compound according to any one of the preceding clauses, or a pharmaceutically acceptable salt or solvate thereof, wherein R7d represents H, F, Cl, OCH3, CN, or Br.

[0030] 11 . The compound according to any one of the preceding clauses, or a pharmaceutically acceptable salt or solvate thereof, wherein R7G’ represents H, F, OCH3, or Br and R7e’ represents H.

[0031] 12. The compound according to any one of the preceding clauses, or a pharmaceutically acceptable salt or solvate thereof, wherein one or more of the following apply:

[0032] (a) R7frepresents H or CF3; (b) R7grepresents H or CF3;

[0033] (c) R7hrepresents H or F.

[0034] 13. The compound according to any one of the preceding clauses, or a pharmaceutically acceptable salt or solvate thereof, wherein R7krepresents H or Br.

[0035] 14. The compound according to any one of the preceding clauses, or a pharmaceutically acceptable salt or solvate thereof, wherein:

[0036] (i) when present, R?i represents CF3; or

[0037] (ii) when present:

[0038] R?i represents H, Cl or CN or more particularly, OCH3, OCF3 or CF3; and R7j represents H, F, OCH3, CF3or more particularly, Cl or CN.

[0039] 15. The compound according to any one of the preceding clauses, or a pharmaceutically acceptable salt or solvate thereof, wherein:

[0040] X represents CH, C-CI, C-F, or N; and

[0041] Y represents CH, C-CI, C-F or N, optionally wherein:

[0042] (a) when X represents CH, then Y represents C-CI, C-F or N; or

[0043] (b) when Y represents CH, then X represents C-CI, C-F or N.

[0044] 16. The compound according to any one of the preceding clauses, or a pharmaceutically acceptable salt or solvate thereof, wherein when R3is one or more of the following applies:

[0045] (i) when R7cor R7erepresents F or Cl, then R7drepresents H; and

[0046] (ii) when R7drepresents F or Cl, then R7cand R7erepresents H.

[0047] 17. The compound according to any one of the preceding clauses, or a pharmaceutically acceptable salt or solvate thereof, is a compound selected from the list of:

[0048]

[0049] 18. The compound according to any one of the preceding clauses, or a pharmaceutically acceptable salt or solvate thereof, is a compound selected from the list of:

[0050]

[0051] 19. A pharmaceutical formulation comprising a compound of formula I or a pharmaceutically acceptable salt or solvate thereof as described in any one of Clauses 1 to 18 in combination with one or more of a pharmaceutically acceptable adjuvant, diluent or carrier.

[0052] 20. A compound of Clauses I or a pharmaceutically acceptable salt or solvate thereof as described in any one of Clauses 1 to 18 for use in medicine.

[0053] 21 . Use of a compound of formula I or a pharmaceutically acceptable salt or solvate thereof as described in any one of Clauses 1 to 18 in the preparation of a medicament for use in treating a bacterial infection.

[0054] 22. A compound of formula I or a pharmaceutically acceptable salt or solvate thereof as described in any one of Clauses 1 to 18 for use in the treatment of a bacterial infection.

[0055] 23. A method of treating a bacterial infection comprising the step of administering a pharmaceutically effective amount of a compound of formula I or a pharmaceutically acceptable salt or solvate thereof as described in any one of Clauses 1 to 18 to a subject in need thereof.

[0056] 24. The use according to Clause 21 , the compound for use according to Clause 22, and the method according to Clause 23, wherein the bacterial infection is caused by one or more of Mycobacterium tuberculosis, Mycobacterium abscessus, and Mycobacterium avium.

[0057] 25. A compound of formula I or a pharmaceutically acceptable salt or solvate thereof as described in any one of Clauses 1 to 18 for use in the treatment of a bacterial infection, wherein the compound of formula I is administered sequentially, simultaneously or concomitantly with a QcrB inhibitor.

[0058] 26. Use of a compound of formula I, or a pharmaceutically acceptable salt or solvate thereof as described in any one of Clauses 1 to 18, and QcrB inhibitor for the preparation of a medicament for the treatment of a bacterial infection.

[0059] 27. Use of a compound of formula I, or a pharmaceutically acceptable salt or solvate thereof as described in any one of Clauses 1 to 18, for the preparation of a medicament for the treatment of a bacterial infection, wherein the compound of formula I is administered sequentially, simultaneously or concomitantly with a QcrB inhibitor.

[0060] 28. A method of treating a bacterial infection comprising the step of administering a pharmaceutically effective amount of a compound of formula I or a pharmaceutically acceptable salt or solvate thereof as described in any one of Clauses 1 to 18 to a subject in need thereof, wherein the compound of formula I is administered sequentially, simultaneously or concomitantly with a QcrB inhibitor.

[0061] BRIEF DESCRIPTION OF DRAWINGS

[0062] FIG. 1 depicts a general structure of the exemplified inhibitors in the present disclosure.

[0063] FIG. 2 depicts some of the exemplified inhibitors in the present disclosure.

[0064] FIG. 3 depicts the bactericidal potency of the exemplified inhibitors according to Example 2 of the present dislcosure. M. tuberculosis H37Rv was exposed to 78 (A), 76 (B), or 80 (C) at 6 |1M with or without Q203 (100 nM) for 14 days before CFU enumeration on agar plates. Inoc.: inoculum at day 0, DMSO: untreated control at day 14. **: p-value <0.01

[0065] FIG. 4 depicts (A) Effect of compound 78 on NADH-driven electron transfer in inverted- membrane vesicles (IMVs) from M. bovis BCG::pMV262-cyte4BDC. (B) Subunit CydA of the cyt-bd oxidase with prosthetic groups heme tesa, teas, heme c / and the proposed electron-flow. (C) Effect of compound 78 on NADH-driven electron transfer in IMVs from M. abscessus qcrCAB. The NADH-based difference spectra in the absence (solid line) and presence of 2 pM compound 78 (dashed line) reflect the dominant effect of compound 78 on the reduction of the hemes in both systems. DESCRIPTION

[0066] The present inventors have developed compounds which inhibits the cytochrome bd oxidase. The compounds are useful for treating and / or ameliorating M. tuberculosis infections and other mycobacterial infections (e.g., non-tuberculous mycobacteria such as Mycobacterium abscessus or Mycobacterium avium), in particular when combined with a QcrB inhibitor (such as Q203).

[0067] Thus, in a first aspect of the invention, there is provided a compound of formula I: wherein:

[0068] X represents CF or N;

[0069] Y represents CR5 or N; R1 and R2 each independently represent H, C1-6 alkyl, halo, and ORg, where the Ci-e alkyl is unsubstituted or substituted by one or more halo atoms;

[0070] R3 represents:

[0071] where the wiggly line is the point of attachment to the rest of the molecule;

[0072] FU and R5, when present, each independently represent H, halo or CN;

[0073] Rs represents H, or C1-6 alkyl, where the C1-6 alkyl is unsubstituted or substituted by one or more halo atoms; each R7aand R?b independently represent H, F, C1-6 alkyl, where each C1-6 alkyl group is unsubstituted or substituted by one or more halo atoms; each R7cto R7eindependently represent H, halo, OC1-6 alkyl, and CN, where each OC1-6 alkyl group is unsubstituted or substituted by one or more halo atoms; more particularly, each R7C' and R7e' independently represent H, halo and OCvs alkyl, where each OC1-6 alkyl group is unsubstituted or substituted by one or more halo atoms; each R7fto R-qindependently represent H, halo, and C1-6 alkyl, where C1-6 alkyl group is unsubstituted or substituted by one or more halo atoms; each R7iand R7j independently represent H, halo, C1 6 alkyl, OC1 6 alkyl, CN, and piperidinyl, where each C1-6 alkyl group and each OC1-6 alkyl group is unsubstituted or substituted by one or more halo atoms;

[0074] R7,_ represents H or halo;

[0075] R7I represents C1-6 alkyl, where each C1-6 alkyl group is unsubstituted or substituted by one or more halo atoms, or a pharmaceutically acceptable salt or solvate thereof, provided that the compound is not

[0076] The word “comprising” refers herein 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.

[0077] 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 greaterthan 99.9% pure, such as greater than 99.99% pure, such as greater than 99.999% pure, such as 100% pure.

[0078] 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 formulation” includes mixtures of two or more such formulation, reference to “a pharmaceutically acceptable salt” includes mixtures of two or more such pharmaceutically acceptable salts, and the like.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] The solvates can be stoichiometric or non-stoichiometric solvates. Particularly preferred solvates are hydrates, and examples of hydrates include hemihydrates, monohydrates and di hydrates.

[0086] 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.

[0087] 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)).

[0088] 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.

[0089] Examples of prodrugs include, but are not limited to, esters and carbamates of hydroxyl functional groups, esters groups of carboxyl functional groups, N-acyl derivatives and N- Mannich bases. General information on prodrugs may be found e.g. in Bundegaard, H. “Design of Prodrugs” p. 1-92, Elsevier, New York-Oxford (1985).

[0090] 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”.

[0091] Compounds of formula I may contain double bonds and may thus exist as E entgegen) and Z (zusammeri) geometric isomers about each individual double bond. All such isomers and mixtures thereof are included within the scope of the invention.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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).

[0097] The term “halo”, when used herein, includes references to fluoro, chloro, bromo and iodo.

[0098] Unless otherwise stated, the term “aryl” when used herein includes Ce-14 (such as Ce-io) aryl groups. Such groups may be monocyclic, bicyclic or tricyclic and have between 6 and 14 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 are linked to the rest of the molecule via an aromatic ring. Cs aryl groups include phenyl, naphthyl and the like, such as 1 ,2,3,4-tetrahydronaphthyl, indanyl, indenyl and fluorenyl. Embodiments of the invention that may be mentioned include those in which aryl is phenyl.

[0099] Unless otherwise stated, the term “alkyl” refers to an unbranched or branched, acyclic or cyclic, saturated or unsaturated (so forming, for example, an alkenyl or alkynyl)hydrocarbyl radical, which may be substituted or unsubstituted (with, for example, one or more halo atoms). Where the term “alkyl” refers to an acyclic group, it is preferably CMO alkyl and, more preferably, C1-6 alkyl (such as ethyl, propyl, (e.g. n-propyl or isopropyl), butyl (e.g. branched or unbranched butyl), pentyl or, more preferably, methyl). Where the term “alkyl” is a cyclic group (which may be where the group “cycloalkyl” is specified), it is preferably C3-12 cycloalkyl and, more preferably, C5io (e.g. C57) cycloalkyl.

[0100] The term “heteroaryl” when used herein refers to an aromatic group containing one or more heteroatom(s) (e.g. one to four heteroatoms) preferably selected from N, O and S (so forming, for example, a mono-, bi-, or tricyclic heteroaromatic group). Heteroaryl groups include those which have between 5 and 14 (e.g. 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 are 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, acridinyl, benzimidazolyl, benzodioxanyl, benzodioxepinyl, 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, phenazinyl, phenothiazinyl, 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.

[0101] Unless otherwise specified herein, a “heterocyclic ring system” may be 4- to 14-membered, such as a 5- to 10-membered (e.g. 6- to 10-membered), heterocyclic group that may be aromatic, fully saturated or partially unsaturated, and which contains one or more heteroatoms selected from O, S and N, which heterocyclic group may comprise one or two rings. Examples of hetereocyclic ring systems that may be mentioned herein include, but are not limited to azetidinyl, dihydrofuranyl (e.g. 2,3-dihydrofuranyl, 2,5-dihydrofuranyl), dihydropyranyl (e.g. 3,4-dihydropyranyl, 3,6-dihydropyranyl), 4,5-dihydro-1 H-maleimido, dioxanyl, dioxolanyl, furanyl, furazanyl, hexahydropyrimidinyl, hydantoinyl, imidazolyl, isothiaziolyl, isoxazolidinyl, isoxazolyl, morpholinyl, 1 ,2- or 1 ,3-oxazinanyl, oxazolidinyl, oxazolyl, piperidinyl, piperazinyl, pyranyl, pyrazinyl, pyridazinyl, pyrazolyl, pyridinyl, pyrimidinyl, pyrrolinyl (e.g. 3-pyrrolinyl), pyrrolyl, pyrrolidinyl, pyrrolidinonyl, 3-sulfolenyl, sulfolanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl (e.g. 3,4,5,6-tetrahydropyridinyl), 1 ,2,3,4- tetrahydropyrimidinyl, 3,4,5,6-tetrahydropyrimidinyl, tetrahydrothiophenyl, tetramethylenesulfoxide, tetrazolyl, thiadiazolyl, thiazolyl, thiazolidinyl, thienyl, thiophenethyl, triazolyl and triazinanyl.

[0102] Unless otherwise specified herein, a “carbocyclic ring system” may be 4- to 14-membered, such as a 5- to 10-membered (e.g. 6- to 10-membered, such as a 6-membered or 10- membered), carbocyclic group that may be aromatic, fully saturated or partially unsaturated, which carbocyclic group may comprise one or two rings. Examples of carbocyclic ring systems that may be mentioned herein include, but are not limited to cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, phenyl, naphthyl, decalinyl, tetralinyl, bicyclo[4.2.0]octanyl, and 2, 3, 3a, 4, 5, 6, 7,7a- octahydro-1 H-indanyl. Particularly preferred carbocyclic groups include phenyl, cyclohexyl and naphthyl. For the avoidance of doubt, references herein to compounds of formula I include pharmaceutically acceptable salts or solvates, or pharmaceutically functional derivatives of such compounds.

[0103] 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.

[0104] 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.

[0105] The isotopic labelling or enrichment of the compound of formula I may be with a radioactive or non-radioactive isotope of any of hydrogen, carbon, nitrogen, oxygen, sulfur, fluorine, chlorine, bromine and / or iodine. Particular isotopes that may be mentioned in this respect include2H,3H,11C,13C,14C,13N,15N,15O,170,180,35S,18F,37CI,77Br,82Br and125l).

[0106] 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.

[0107] In certain embodiments that may be mentioned herein, one or more of the following may apply: (Al) Ri represents H, CH3, Br, OCH3, or CF3;

[0108] (AH) R2represents H or Cl;

[0109] (Alii) R4, when present, represents H or Cl or more particularly, F;

[0110] (Aiv) R5, when present, represents H, F or Cl; and

[0111] (Av) R6is H or CH3. In certain embodiments that may be mentioned herein, one or more of the following may apply:

[0112] (Avi) each R7aand R7bindependently represent H, F, CH3or CF3;

[0113] (Avii) R7Crepresents H, F, OCH3, OCF3, Br or Cl and R7erepresents H, Cl or F;

[0114] (Aviii) R7drepresents H, F, Cl, OCH3, CN, or Br; and

[0115] (Aix) more particularly, R7C' represents H, F, OCH3, or Br and R7e' represents H.

[0116] In certain particular embodiments that may be mentioned herein, R7cmay represent H, F, OCH3, OCF3, Br or Cl and R7emay represent H.

[0117] In certain embodiments that may be mentioned herein, one or more of the following may apply:

[0118] (a) R7frepresents H or CF3;

[0119] (b) R7grepresents H or CF3; and

[0120] (c) R7h represents H or F.

[0121] In certain embodiments that may be mentioned herein, R7k may represent H or Br.

[0122] In certain embodiments that may be mentioned herein, one or more of the following may apply:

[0123] (i) when present, R7I represents CF3; or

[0124] (ii) when present:

[0125] R7irepresents H, Cl or CN or more particularly, OCH3, OCF3or CF3; and

[0126] R7j represents H, F, OCH3, CF3or more particularly, Cl or CN.

[0127] In certain embodiments that may be mentioned herein,

[0128] X may represent CH, C-CI, C-F, or N; and

[0129] Y may represent CH, C-CI, C-F or N.

[0130] In certain particular embodiments that may be mentioned herein,

[0131] (a) when X represents CH, then Y may represent C-CI, C-F or N; or

[0132] (b) when Y represents CH, then X may represent C-CI, C-F or N.

[0133] In certain embodiments that may be mentioned herein, when R3is , one or more of the following may apply:

[0134] (I) when R7cor R7erepresents F or Cl, then R7drepresents H; and

[0135] (ii) when R7drepresents F or Cl, then R7cand R7erepresents H. In certain embodiments that may be mentioned herein, the compound may be selected from the list of:

[0136]

[0137]

[0138]

[0139]

[0140] In certain particular embodiments that may be mentioned herein, the compound may be selected from the list of:

[0141]

[0142] As will be appreciated, the compounds of the current invention may be suitable for treating a subject. As such, in a further aspect of the invention, there is provided a pharmaceutical formulation comprising a compound of formula I or a pharmaceutically acceptable salt or solvate thereof as described hereinbefore in combination with one or more of a pharmaceutically acceptable adjuvant, diluent or carrier.

[0143] 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.

[0144] Compounds of formula I will generally be administered as a pharmaceutical formulation in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier, which may be selected with due regard to the intended route of administration and standard pharmaceutical practice. Such pharmaceutically acceptable carriers may be chemically inert to the active compounds and may have no detrimental side effects or toxicity under the conditions of use. Suitable pharmaceutical formulations may be found in, for example, Remington The Science and Practice of Pharmacy, 19th ed., Mack Printing Company, Easton, Pennsylvania (1995). For parenteral administration, a parenterally acceptable aqueous solution may be employed, which is pyrogen free and has requisite pH, isotonicity, and stability. Suitable solutions will be well known to the skilled person, with numerous methods being described in the literature. A brief review of methods of drug delivery may also be found in e.g. Langer, Science (1990) 249, 1527.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] 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.

[0152] 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.

[0153] As will be appreciated, a further aspect of the invention relates to a compound of formula I or a pharmaceutically acceptable salt or solvate thereof as described hereinbefore, for use in medicine.

[0154] Thus, further aspects of the invention relate to the following.

[0155] (a) Use of a compound of formula I or a pharmaceutically acceptable salt or solvate thereof as described hereinbefore in the preparation of a medicament for use in treating a bacterial infection.

[0156] (b) A compound of formula I or a pharmaceutically acceptable salt or solvate thereof as described hereinbefore for use in the treatment of a bacterial infection. (c) A method of treating a bacterial infection comprising the step of administering a pharmaceutically effective amount of a compound of formula I or a pharmaceutically acceptable salt or solvate thereof as described hereinbefore to a subject in need thereof.

[0157] In certain particular embodiments that may be mentioned herein, the bacterial infection may be caused by one or more of Mycobacterium tuberculosis, Mycobacterium abscessus, and Mycobacterium avium.

[0158] In accordance with the invention, compounds of formula I may be administered alone (i.e. as a monotherapy, such as a monotherapy for the treatment of a bacterial infection). In alternative embodiments of the invention, however, compounds of formula I may be administered in combination with another therapeutic agent (e.g. a QcrB inhibitor for the treatment of the bacterial infection).

[0159] Thus, further aspects of the invention relate to the following.

[0160] (a) A compound of formula I or a pharmaceutically acceptable salt or solvate thereof as described hereinbefore for use in the treatment of a bacterial infection, wherein the compound of formula I is administered sequentially, simultaneously or concomitantly with a QcrB inhibitor.

[0161] (b) Use of a compound of formula I, or a pharmaceutically acceptable salt or solvate thereof as described hereinbefore, and QcrB inhibitor for the preparation of a medicament for the treatment of a bacterial infection.

[0162] (c) Use of a compound of formula I, or a pharmaceutically acceptable salt or solvate thereof as described hereinbefore, for the preparation of a medicament for the treatment of a bacterial infection, wherein the compound of formula I is administered sequentially, simultaneously or concomitantly with a QcrB inhibitor.

[0163] (d) A method of treating a bacterial infection comprising the step of administering a pharmaceutically effective amount of a compound of formula I or a pharmaceutically acceptable salt or solvate thereof as described hereinbefore to a subject in need thereof, wherein the compound of formula I is administered sequentially, simultaneously or concomitantly with a QcrB inhibitor.

[0164] The aspects of the invention described herein (e.g. the above-mentioned compounds, combinations, methods and uses) may have the advantage that, in the treatment of the conditions described herein, they may be more convenient for the physician and / or patient than, be more efficacious than, be less toxic than, have better selectivity over, have a broader range of activity than, be more potent than, produce fewer side effects than, or may have other useful pharmacological properties over, similar compounds, combinations, methods (treatments) or uses known in the prior art for use in the treatment of those conditions or otherwise.

[0165] Further aspects and embodiments of the invention will now be discussed by reference to the following non-limiting examples.

[0166] EXAMPLES

[0167] In the present Examples, 80 inhibitors were synthesized and evaluated in a cyt-bd whole-cell assay in Mycobacterium bovis BCG and Mycobacterium tuberculosis - a generalized structure is indicated in FIG. 1 and some of the exemplified inhibitors are indicated in FIG. 2.

[0168] Materials and Methods

[0169] Chemistry. Compounds were synthesized following the procedures described in general synthesis routes A and B. All starting materials, reagents, and solvents were obtained from commercial vendors and used as received. All reactions were carried out under nitrogen atmosphere in dry solvents, unless otherwise mentioned. Reactions were monitored either by thin-layer chromatography (TLC) using Merck silica gel 60, F254 precoated glass plates and visualized with ultraviolet light or by LCMS analysis carried out using an Agilent 1290 Infinity system with a Zorbax Eclipse Plus C18 column (1.8 pm, 50 x 2.1 mm). Compounds were purified either with RediSep® normal-phase silica flash columns on a Teledyne Isco CombiFlash® system or with a Waters Mass-directed HPLC system with an XBridge preparative C18 column (5 pm, 19 x 150 mm). Compound purity was determined using the Shimadzu prominence-i HPLC instrument with a Phenomenex Luna C18 column (5 pm, 150 x 4.6 mm) and mobile phase of 0.1% formic acid in acetonitrile and 0.1% formic acid in water.1H NMR spectra were recorded on a Bruker 400 MHz spectrometer and chemical shifts are reported in ppm with residual undeuterated solvent as internal reference (DMSO-ofe:1H NMR = 2.50 ppm, MeOD:1H NMR = 3.31 ppm). Coupling constants J are recorded in Hz and data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br. = broad, m = multiplet), coupling constants and integration. All final compounds were > 95% pure by HPLC and / or 1 H NMR, unless stated otherwise.

[0170] Chemicals and reagents. Q203 was purchased from Medchem Express. BacTiter-Glo™ (G8233) from Promega. Breatheasy™ membrane (BEM-1 ) from Diversified Biotech. All chemical and reagents were laboratory grade purchased from known companies. Strains and growth conditions. All mycobacteria strains were cultured in Middlebrook 7H9 medium (Becton Dickson and Company Limited, USA) supplemented with 0.05% Tween 80, 0.5% glycerol, and ADS (Albumin, Dextrose, Salt) enrichment, or cultivated on Middlebrook 7H10 agar (Becton Dickson) supplemented with OADC (Oleic Acid, Albumin, Dextrose, Salt supplement; Becton Dickson). The M. tuberculosis clinical isolates used in this study were a gift from Sebastien Gagneux (Swiss Tropical and Public Health Institute, Basel, Switzerland). M. tuberculosis cydAB knockout and corresponding complement strains used in this study were constructed in a previous study (Kalla et al. (2017) Exploiting the synthetic lethality between terminal respiratory oxidases to kill Mycobacterium tuberculosis and clear host infection. PNAS 1 14,28: 7426-7431 ). A M. bovis BCG strain overexpressing cyt-bd was constructed by electroporating plasmid pMV262-cydABDC into the parental M. bovis BCG strain. The plasmid pMV262-cyc / ABDCwas constructed by incorporating the cydABDC operon, including 330bp upstream of the coding region, into the pMV262 vector. Prior to the start of all experiments, replicating cultures were harvested at logarithmic phase, washed to remove glycerol from its media, and diluted to specified cell density according to different experiments.

[0171] Example 1 : Synthesis and characterisation of cyt-bd inhibitors (compound no. 1 to 80)

[0172] There are two general synthesis routes (i.e., General synthesis route A and General synthesis route B) as follows:

[0173] General synthesis route A: Pd(OAc)2, Xantphos, NaOfBu, DMF, 100 °C

[0174] Specific example for the synthesis of compound 3 via synthesis route A

[0175] Synthesis of 2-methylpyrazolo[1 ,5-a]quinazolin-5(4H)-one (A-1): To a cooled mixture containing 3-methyl-1 H-pyrazol-5-amine (105.7 mg, 1 .3 equiv.) and K2CO3 (231.5 mg, 2.0 equiv.) in anhydrous DMF (8.0 mL) was added dropwise a solution of 2-fluorobenzoyl chloride (132.8 mg, 1.0 equiv.) in DMF (2.0 mL). The reaction was stirred at - 10 °C for 10 mins before it was warmed up to room temperature and subsequently heated at 140 °C for 16 hours. Water (10 mL) was added to the reaction mixture and the mixture was extracted with EtOAc (3 x 8 mL). The combined organics were washed with brine, filtered and concentrated under vacuo to give a yellow residue. The crude product was purified by silica gel flash chromatography (0- 100% EtOAc / Hexanes) to afford compound A-1 as a solid (20.0 mg, 12.0% yield);1H NMR (400 MHz, DMSO-c / e) 6 ppm 12.08 (s, 1 H), 8.15-8.07 (m, 1 H), 8.03-7.97 (m, 1 H), 7.89-7.80 (m, 1 H), 7.49-7.39 (m, 1 H), 5.75 (d, J = 2.0 Hz, 1 H), 2.28 (s, 3H); MS (ESI) m / z 200.1 [C11H9N3O + H]+.

[0176] Synthesis of 5-chloro-2-methylpyrazolo[1 ,5-a]quinazoline (A-2): A mixture of A-1 (500 mg, 1.0 equiv.) and POCI3 (4.70 mL, 20.0 equiv.) was heated at 1 10 °C for 3.5 hours before the reaction mixture was cooled to room temperature and added dropwise into an ice-bath (20mL) with stirring. The resultant mixture was stirred for 30 minutes, to allow for the complete precipitation and quenching of excess POCI3, before it was extracted with dichloromethane (5 x 10 mL). The combined organic layers were dried over sodium sulphate, filtered and concentrated under vacuo to its crude. The crude product was purified by silica gel flash chromatography (0-100% EtOAc / Hexanes) to afford compound A-2 as a solid (500 mg, 91 .5% yield);1H NMR (400 MHz, MeOD) 5 ppm 8.35 (d, J = 8.4 Hz, 1 H), 8.26 (d, J = 8.4 Hz, 1 H), 7.99 (t, J = 8.4 Hz, 1 H), 7.62 (t, J = 8.4 Hz, 1 H), 6.54 (s, 1 H), 2.51 (s, 3H); MS (ESI) m / z 218.0 [CH H8CIN3+ H]+.

[0177] Synthesis of N-(4-chlorophenethyl)-2-methylpyrazolo[1 ,5-a]quinazolin-5-amine (3): To a solution of A-2 (10.0 mg, 1.0 equiv.) in DMSO (0.5 mL) was added 2-(4-chlorophenyl)ethan- 1 -amine (21 .5 mg, 3.0 equiv.). The resultant mixture was heated at 90 °C for 2 hours before it was diluted with DMSO (0.6 mL). The crude product was purified by preparative HPLC (20- 95% MeCN / H2O; 0.1% formic acid) to afford compound 3 as an off-white solid upon lyophilization (10.9 mg, 70.4% yield);1H NMR (400 MHz, MeOD) 5 ppm 8.16 (d, J = 8.4 Hz, 1 H), 8.03 (d, J = 7.6 Hz, 1 H), 7.80 (t, J = 7.2 Hz, 1 H), 7.42 (t, J = 7.2 Hz, 1 H), 7.27 (s, 4H), 6.00 (s, 1 H), 3.78 (t, J = 7.2, 2H), 3.03 (t, J = 7.2, 2H), 2.40 (s, 3H); MS (APCI) m / z 337.1 [C19H17N4CI + H]+.

[0178] Specific example for the synthesis of compound 36 via synthesis route A

[0179] Compound 36

[0180] Synthesis of 4-((2-methylpyrazolo[1 ,5-a]quinazolin-5-yl)amino)-2-

[0181] (trifluoromethyl)benzonitrile (36): A mixture of A-2 (10.0 mg, 1.0 equiv.), 4-amino-2- (trifluoromethyl)benzonitrile (17.1 mg, 2.0 1.0 equiv.), sodium tert-butoxide(11 .0 mg, 2.5 1.0 equiv.), Xantphos (5.3 mg, 20 mol %) and Pd(OAc)2(6.3 mg, 15 mol %) in DMF (0.5 mL) was heated to 100 °C for 16 hours. The reaction mixture was diluted with EtOAc (1 mL) and washed with water (1 mL). The aqueous phase was back extracted with EtOAc (3 x 1 mL) and the combined EtOAc layers were dried over Na2SO4, filtered and concentrated under reduced pressure. The crude product was purified by preparative HPLC (20-95% MeCN / H2O; 0.1% formic acid) to afford compound 36 as an off-white solid upon lyophilization (4.0 mg, 23.7% yield);1H NMR (400 MHz, MeOD) 5 ppm 8.56 (s, 1 H), 8.46-8.40 (m, 2H), 8.31 (d, J = 8.4 Hz, 1 H), 7.95-7.91 (m, 2H), 7.57 (t, J = 7.6 Hz, 1 H), 6.24 (s, 1 H), 2.47 (s, 3H); MS (ESI) m / z 368.1 [C19H12N5F3 + H]+.

[0182] General synthesis route B:

[0183] Compound 65

[0184] Synthesis of 2-methylpyrazolo[1 ,5-a]pyrido[4,3-e]pyrimidin-5(4H)-one (B-1): To a mixture of methyl 3-bromopyridine-4-carboxylate (1.4 g, 1.0 equiv.), 5-methyl-1 H-pyrazol-3- amine (661 mg, 1 .1 equiv.), copper(l) iodide (247 mg, 0.2 equiv.) and cesium carbonate (4.22 g, 2.0 equiv.) under inert atmosphere was added water (13.0 mL). The suspension was refluxed at 100 °C overnight. Water (50 mL) was added to the reaction mixture and the aqueous layer was extracted with CH2CI2:IPA (10:1 , 2 x 50 mL). Brine (20 mL) was added and the aqueous layer was extracted with CH2CI2:IPA (10:1 , 2 x 50 mL). A second portion of brine (20 mL) was added to the aqueous layer and the mixture was extracted further with CH2CI2:IPA (10:1 , 4 x 50 mL). The combined organic layers were dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was triturated thrice with MeOH to give B- 1 as a solid (266.6 mg, 27.4% yield);1H NMR (400 MHz, DMSO-cfe) 5 ppm 12.35 (br. s, 1 H), 9.31 (s, 1 H), 8.65 (d, J= 4.8 Hz, 1 H), 7.95 (dd, J= 5.2, 0.8 Hz, 1 H), 5.80 (s, 1 H), 2.30 (s, 3H); MS (ESI) m / z 201 .9 [CioH8N40 + H]+.

[0185] Synthesis of 5-chloro-2-methylpyrazolo[1 ,5-a]pyrido[4,3-e]pyrimidine (B-2): A mixture of B-1 (10.3 mg, 1.0 equiv.) and phosphorus oxychloride (472 pL, 50 equiv.) was refluxed at 1 10 °C overnight. The resultant mixture was cooled, diluted with CH2CI2(10 mL) and concentrated under reduced pressure twice to afford B-2 as a solid (11 .3 mg, 99.9%) in its crude; MS (ESI) m / z 219.8 [C10H7CIN4 + H]+.

[0186] Synthesis of / V-((frans)-2-(4-chlorophenyl)cyclopropyl)-2-methylpyrazolo[1 ,5- a]pyrido[4,3-e]pyrimidin-5-amine (65): To a solution of B-2 (1 1 .3 mg, 1.0 equiv.) in DMSO (1.0 mL) was added / V,A / -diisopropylethylamine (90 pL, 10 equiv.). The solution was stirred at room temperature for 5 mins before (trans)-2-(4-chlorophenyl)cyclopropan-1 -amine hydrochloride (31.5 mg, 3.0 equiv.) was added and the mixture was stirred overnight at 100 °C. Water (20 mL) was added to the reaction mixture and the aqueous layer was extracted with EtOAc (3 x 20 mL). The combined organic layers were dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude material was dissolved in minimal amount of MeOH and purified using Varian preparative HPLC (30-95% ACN in water over 20 min) to afford compound 65 (7.3 mg, 40.6% yield) as a white solid upon lyophilisation;1H NMR (400 MHz, DMSO-cfc) 6 ppm 9.46 (d, J = 0.8 Hz, 1 H), 8.67 (d, J = 5.6 Hz, 1 H), 8.40 (d, J = 3.2 Hz, 1 H), 8.20-8.10 (m, 1 H), 7.40-7.30 (m, 2H), 7.30-7.25 (m, 2H), 6.04 (s, 1 H), 3.15-3.05 (s, 1 H), 2.33 (s, 3H), 2.25-2.15 (s, 1 H), 1.50-1.35 (s, 2H); MS (ESI) m / z 350.1 [CI9HI6CIN5+ H]+.

[0187] Results and Discussion:

[0188] The complete set of compounds synthesized and their corresponding NMR and m / Z data can be found in Table 1 .

[0189] Table 1 : Cvt-bd inhibitors (compound no. 1 to 80) Where absolute stereochemistry has not been indicated, compounds herein are racemic mixtures with relative stereochemistry as drawn.

[0190] Example 2: Biological data for cyt-bd inhibitors (compound no. 1 to 80)

[0191] Intracellular ATP quantification in M. bovis BCG and M. tuberculosis N0145

[0192] M. bovis BCG (Pasteur strain) was cultured in Middlebrook 7H9 medium (Becton Dickson and Company Limited, USA) supplemented with 0.05% Tween 80, 0.5% glycerol, and ADS enrichment (Bovine Serum Albumin, D-glucose, and NaCI). Prior to the start of all experiments, replicating cultures were harvested at logarithmic phase, washed to remove glycerol, and diluted to a cell density of 0.08 OD6oo- 25 pl of culture was aliquoted into each well on a 384- well white plate containing compounds at various test concentrations spotted by Echo acoustic dispenser. DMSO concentration was kept at 0.5% across all wells. The plates were incubated at 37°C for 15 h. Subsequently, 10 pl of BacTiter-Glo reagent (Promega) was added to each well and incubated further for 10 min. The luminescence of each well was measured using Tecan Plate reader. Data analysis was performed on GraphPad Prism software. The assay was performed in the presence of 100 nM Q203. For counter-screen, the exact assay was performed without Q203. Compounds active only in the presence of Q203 were considered cyt-bd inhibitors. A comparable protocol was used to confirm potency in M. tuberculosis N0145, except that the DMSO was kept at 0.1% across all wells and that the compounds were spotted manually. Luminescence was recorded on a Cytation 3 plate reader for M. tuberculosis N0145.

[0193] Bacterial viability assay. Exponentially grown M. tuberculosis H37Rv culture was adjusted to an OD6QO of 0.005 and aliquoted into 24-well plates. Test compounds were dispensed into each well. After 14 days, treated samples were washed once by centrifugation at 3800 rpm for 10 min and resuspending in equal volume of 7H9 medium. Serial ten-fold dilutions were spread on 7H10 agar plates and incubated at 37°C. Bacteria viability was determined by enumerating counting the number of colonies forming units (CFU) after incubation for 14-20 days.

[0194] Minimum inhibitory concentration (MIC50). The MIC was determined using previously established procedure (Kalia et al. (2017) Exploiting the synthetic lethality between terminal respiratory oxidases to kill M. tuberculosis and clear host infection. PNAS 114,28: 7426-7431 ). A total of 2 pL of two serially diluted compounds were added to 96 well plate at 100 times the desired concentration. Exponentially growing culture was washed twice with 7H9 medium and resuspended to an OD80o of 0.005. Culture was dispensed into 96-well flat-bottom plates at 200 pL per well. The corner wells were filled with water as hydrant to avoid evaporation. The plates were then incubated at 37°C for five days followed by measurements of absorbance at 600 nm. The MICso values represent the drug concentrations at which 50% inhibition of bacterial growth was inhibited. Absorbance data was analyzed and plotted using GraphPad Prism latest software.

[0195] Results and Discussion:

[0196] The ATP depletion ICsodata for the cyt-bd inhibitors (compound no. 1 to 80) is summarized in Table 2.

[0197] Table 2: ATP depletion ICsodata for cyt-bd inhibitors (compound no. 1 to 80)

[0198] ND: Not determined +: > 20 pM ++: >2-20 pM +++: 200 nM - 2 pM ++++: < 200 nM

[0199]

[0200] Representatives of the series show chiral differentiation (Table 3), indicating a specific mode of target inhibition. In Table 3, racemate of 63, 58, and 60 as well as their pure enantiomers were tested for their capacity to inhibit ATP levels in M. bovis BCG and M. tuberculosis H37Rv with or without 100 nM Q203. As expected from drugs targeting the cytochrome bd oxidase, the compounds depleted ATP levels only in the presence of Q203. The (1 S,2R) enantiomers were the most potent.

[0201] Table 3: Potency against M. bovis BCG and Mycobacterium tuberculosis H37Rv. The compounds (racemates and pure enantiomers) were tested in an ATP depletion assay in the presence or absence of Q203.

[0202] Potency of 76, 78 and 80 was further validated against a diverse set of M. tuberculosis clinical isolates (Table 4).

[0203] Table 4: Potency of 76. 78, and 80 against M. tuberculosis clinical isolates. The compounds were tested in an ATP depletion assay in the presence or absence of Q203.

[0204] Next, the (1 S,2R) enantiomers (76, 78) and 80 were tested in a bactericidal assay against M. tuberculosis H37Rv. As expected from drugs targeting the cytochrome bd oxidase, 76, 78 and 80 had no effect on M. tuberculosis viability (FIG. 3). However, a bactericidal potency was observed in the presence of Q203 for the three derivatives (FIG. 3).

[0205] In follow-up experiments, the three (1S,2R) enantiomers were tested against the clinically relevant NTM M. avium (ATCC #19977) and M. abscessus (ATCC #19977). Since M. avium is known to be susceptible to the cytochrome bcc:aa3 inhibitor Q203, the potency of 78, 76 and 80 was tested in an ATP depletion assay and in a growth inhibition (MIC50) assay in the presence or absence of Q203. As observed in M. tuberculosis, the three compounds were inactive alone but potently inhibited ATP levels as well as growth in the presence of Q203 (Table 5). Table 5. Potency against Mycobacterium avium. 76, 78, and 80 were evaluated against M. 19977, in the presence or absence of 25 nM Q203, in an ATP depletion assay growth inhibition assay (MICso).

[0206] Since Q203 is inactive against M. abscessus (Sorayah R, et al. (2019) Naturally-Occurring Polymorphisms in QcrB Are Responsible for Resistance to Telacebec in Mycobacterium abscessus. ACS Infect Dis 5(12):2055-2060), a strain deficient for the expression of the cytochrome bcc:aa3 (AqcrCAB) was used to test the potency of 76, 78 and 80. If the compounds target the cytochrome bd oxidase in M. abscessus as well, an ATP depletion and growth inhibition in the AqcrCAB strain is expected, but not in the parental strain. Consistently, 76, 78 and 80 depleted ATP levels in M. abscessus AqcrCAB but not in M. abscessus wildtype (Table 6). The lack of growth inhibitory potency of 80 could possibly be attributed to a low solubility coupled with a higher expression levels of the cytochrome bd oxidase in the AqcrCAB strain.

[0207] Table 6: Potency against Mycobacterium abscessus. 76. 78, and 80 were evaluated against M. abscessus wt (ATCC # 19977) and M. abscessus AgcrCAB in an ATP depletion assay (ATP IC5o) and growth inhibition assay (MIC5Q).

[0208] Example 3: Target Characterisation of Cyt-bd inhibitor

[0209] Heme absorbance spectra of inverted-membrane vesicles

[0210] A sequential transfer of electrons is proposed in mycobacterial cyt-bd from heme 655s to heme b595 and heme d. To further characterize the target engagement of compound 78 and determine the effect of the compound on the electron transfer in cyt-bd, inverted-membrane vesicles were employed in a heme absorbance study. The intrinsically low expression of the cyt-bd, poses a challenge in visualizing the heme bsss, heme bSgs and heme d signature peaks in a wildtype system. Hence the M. bovis BCG::pMV262-cydABDC strain, which overexpresses the cyt-bd, was utilized in the study to validate the target of compound 78 in M. tuberculosis. The study was extended to M. abscessus to understand if compound 78 targets the cyt-bd oxidase and inhibits the enzyme by blocking electron transfer. The IMVs from M. abscessus \qcrCAB strain were selected for the study as the strain overexpresses the cyt-bd and is deficient for the expression of cyt bc?-aa3(Sorayah R, et al. (2019) Naturally-Occurring Polymorphisms in QcrB Are Responsible for Resistance to Telacebec in Mycobacterium abscessus. ACS Infect Dis 5(12):2055-2060), which allows for the cyt-bd heme peaks to be visualised well.

[0211] The spectra were analyzed with an Amersham Biosciences Ultrospec 2100 Pro-UV-Visible absorption spectroscopy (Amersham, Piscataway, NJ). The spectra were recorded from wavelengths of 400 to 700 nm. The inverted-membrane vesicles (IMV) of M. abscessus AqcrCAB and M. bovis BCG::pMV262-cydABDC were generated according to Hotra et al. (Hotra A, Suter M, Biukovic G, Ragunathan P, Kundu S, Dick T & Gruber (2016) Deletion of a unique loop in the mycobacterial F-ATP synthase y subunit sheds light in its inhibitory role in ATP hydrolysis driven H+-pumping. FEBS J 283, 1947-1961) and used at a concentration of 2 mg / mL in the respective experiments. The IMV were oxidized with 100 mM potassium ferricyanide and the oxidized UV spectra was recorded. The sample was subsequently reduced using 2 mM p-Nicotinamide adenine dinucleotide (NADH). The reduced UV spectrum was recorded after 30 minutes following the addition of NADH. The difference spectra were then obtained by subtracting the absorbance value of the reduced state from that of the oxidized state. To study the effect of compound 78, 2 pM of the compound was added after the addition of the oxidant and before the addition of the electron donor NADH.

[0212] Results and Discussion:

[0213] The difference spectrum of the M. bovis BCG::pMV262-cyd4BDC mutant IMV energized with NADH (2 mM final concentration) demonstrated electron transfer followed by the reduction in the cyt-bd hemes (b: 558 and 595 nm; dr 623 nm) (FIG. 4A and FIG. 4B).

[0214] The presence of compound 78 (2 pM) reduced the b hemes and d heme intensities in M. abscessus, postulating that compound 78 has a strong interaction with M. abscessus cyt-bd (FIG. 4C). The slightly stronger reduction of the M. abscessus cyt-bd hemes in the presence of compound 78 compared to the M. tuberculosis can be attributed to the absence of cyt- bcc.aa3in the M. abscessus AqcrCAB strain. The observation nevertheless confirms that compound 78 targets M. tuberculosis and M. abscessus and cyt-bd, respectively, and is able to disrupt electron transfer between the b hemes centers, essential for the final catalytic step of reduction of oxygen to water.

[0215] Together, the results indicate that a promising tricyclic scaffold has been discovered, composed of either pyrazolo quinazoline amine or pyrazolopyridopyrimidine amine that inhibits potently the cytochrome bd oxidase in M. tuberculosis, M. abscessus, and M. avium, and probably all other pathogenic mycobacteria expressing a cytochrome bd oxidase.

Claims

CLAIMS1. A compound of formula I:wherein:X represents CFL or N;Y represents CR5 or N;R1 and R2 each independently represent H, C1-6 alkyl, halo, and ORe, where the C1-6 alkyl is unsubstituted or substituted by one or more halo atoms;R3 represents:where the wiggly line is the point of attachment to the rest of the molecule;FU and R5, when present, each independently represent H, halo or CN;Rs represents H, or C1-6 alkyl, where the C1-6 alkyl is unsubstituted or substituted by one or more halo atoms; each R7aand R?b independently represent H, F, C1-6 alkyl, where each C1-6 alkyl group is unsubstituted or substituted by one or more halo atoms; each R7cto R7eindependently represent H, halo, OC1-6 alkyl, and CN, where each OC1-6 alkyl group is unsubstituted or substituted by one or more halo atoms; more particularly each R7G' and R7e' independently represent H, halo and OC1-6 alkyl, where each OC1-6 alkyl group is unsubstituted or substituted by one or more halo atoms; each R7fto R-qindependently represent H, halo, and C1-6 alkyl, where C1-6 alkyl group is unsubstituted or substituted by one or more halo atoms; each R7iand R7j independently represent H, halo, C1 6 alkyl, OC1 6 alkyl, CN, and piperidinyl, where each C1-6 alkyl group and each OC1-6 alkyl group is unsubstituted or substituted by one or more halo atoms;R7,_ represents H or halo;R7I represents C1-6 alkyl, where each C1-6 alkyl group is unsubstituted or substituted by one or more halo atoms, or a pharmaceutically acceptable salt or solvate thereof, provided that the compound is not2. The compound according to Claim 1 or a pharmaceutically acceptable salt or solvate thereof, wherein R1 represents H, CH3, Br, OCH3, or CF3.

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

4. The compound according to any one of the preceding claims, or a pharmaceutically acceptable salt or solvate thereof, wherein R4, when present, represents H or Cl or more particularly, F.

5. The compound according to any one of the preceding claims, or a pharmaceutically acceptable salt or solvate thereof, wherein R5, when present, represents H, F or Cl.

6. The compound according to any one of the preceding claims, or a pharmaceutically acceptable salt or solvate thereof, wherein R6is H or CH3.

7. The compound according to any one of the preceding claims, or a pharmaceutically acceptable salt or solvate thereof, wherein each R7aand R7b independently represent H, F, CH3or CF3.

8. The compound according to any one of the preceding claims, or a pharmaceutically acceptable salt or solvate thereof, wherein R7crepresents H, F, OCH3, OCF3, Br or Cl and R7erepresents H, Cl or F.

9. The compound according to Claim 8, or a pharmaceutically acceptable salt or solvate thereof, wherein R7crepresents H, F, OCH3, OCF3, Br or Cl and R7erepresents H.

10. The compound according to any one of the preceding claims, or a pharmaceutically acceptable salt or solvate thereof, wherein R7drepresents H, F, Cl, OCH3, CN, or Br.11 . The compound according to any one of the preceding claims, or a pharmaceutically acceptable salt or solvate thereof, wherein R7C’ represents H, F, OCH3, or Br and R7e’ represents H.

12. The compound according to any one of the preceding claims, or a pharmaceutically acceptable salt or solvate thereof, wherein one or more of the following apply:(a) R7f represents H or CF3;(b) R7grepresents H or CF3;(c) R7h represents H or F.

13. The compound according to any one of the preceding claims, or a pharmaceutically acceptable salt or solvate thereof, wherein R7krepresents H or Br.

14. The compound according to any one of the preceding claims, or a pharmaceutically acceptable salt or solvate thereof, wherein:(i) when present, R?i represents CF3; or(ii) when present:R7irepresents H, Cl or CN or more particularly, OCH3, OCF3or CF3; and R7jrepresents H, F, OCH3, CF3or more particularly, Cl or CN.

15. The compound according to any one of the preceding claims, or a pharmaceutically acceptable salt or solvate thereof, wherein:X represents CH, C-CI, C-F, or N; andY represents CH, C-CI, C-F or N, optionally wherein:(a) when X represents CH, then Y represents C-CI, C-F or N; or(b) when Y represents CH, then X represents C-CI, C-F or N.

16. The compound according to any one of the preceding claims, or a pharmaceuticallyacceptable salt or solvate thereof, wherein when R3is one or more of the following applies:(i) when R7Cor R7erepresents F or Cl, then R7drepresents H; and(ii) when R7drepresents F or Cl, then R7cand R7erepresents H.

17. The compound according to any one of the preceding claims, or a pharmaceutically acceptable salt or solvate thereof, is a compound selected from the list of:

18. The compound according to any one of the preceding claims, or a pharmaceutically acceptable salt or solvate thereof, is a compound selected from the list of:

19. A pharmaceutical formulation comprising a compound of formula I or a pharmaceutically acceptable salt or solvate thereof as described in any one of Claims 1 to 18 in combination with one or more of a pharmaceutically acceptable adjuvant, diluent or carrier.

20. A compound of formula I or a pharmaceutically acceptable salt or solvate thereof as described in any one of Claims 1 to 18 for use in medicine.21 . Use of a compound of formula I or a pharmaceutically acceptable salt or solvate thereof as described in any one of Claims 1 to 18 in the preparation of a medicament for use in treating a bacterial infection.

22. A compound of formula I or a pharmaceutically acceptable salt or solvate thereof as described in any one of Claims 1 to 18 for use in the treatment of a bacterial infection.

23. A method of treating a bacterial infection comprising the step of administering a pharmaceutically effective amount of a compound of formula I or a pharmaceutically acceptable salt or solvate thereof as described in any one of Claims 1 to 18 to a subject in need thereof.

24. The use according to Claim 21 , the compound for use according to Claim 22, and the method according to Claim 23, wherein the bacterial infection is caused by one or more of Mycobacterium tuberculosis, Mycobacterium abscessus, and Mycobacterium avium.

25. A compound of formula I or a pharmaceutically acceptable salt or solvate thereof as described in any one of Claims 1 to 18 for use in the treatment of a bacterial infection, wherein the compound of formula I is administered sequentially, simultaneously or concomitantly with a QcrB inhibitor.

26. Use of a compound of formula I, or a pharmaceutically acceptable salt or solvate thereof as described in any one of Claims 1 to 18, and QcrB inhibitor for the preparation of a medicament for the treatment of a bacterial infection.

27. Use of a compound of formula I, or a pharmaceutically acceptable salt or solvate thereof as described in any one of Claims 1 to 18, for the preparation of a medicament for the treatment of a bacterial infection, wherein the compound of formula I is administered sequentially, simultaneously or concomitantly with a QcrB inhibitor.

28. A method of treating a bacterial infection comprising the step of administering a pharmaceutically effective amount of a compound of formula I or a pharmaceutically acceptable salt or solvate thereof as described in any one of Claims 1 to 18 to a subject in need thereof, wherein the compound of formula I is administered sequentially, simultaneously or concomitantly with a QcrB inhibitor.

Images