ANTIBACTERIAL COMPOUNDS

Abstract

The present invention relates to the following compounds (I) wherein the integers are as defined in the description, and where the compounds may be useful as medicaments, for instance for use in the treatment of tuberculosis (e.g. in combination).

##STR00001##

Claims

1. A compound of formula (I): ##STR00021## wherein: R.sup.1 is C.sub.1-6 alkyl, —Br, hydrogen or —C(O)N(R.sup.q1)R.sup.q2; R.sup.q1 and R.sup.q2 independently are hydrogen or C.sub.1-6 alkyl, or are linked together to form a 3-6 membered carbocyclic ring optionally substituted by one or more C.sub.1-3 alkyl substituents; Sub is one or more optional substituents that are halo, —CN, C.sub.1-6 alkyl or —O—C.sub.1-6 alkyl (wherein the latter two alkyl moieties are optionally substituted by one or more fluoro atoms); the two “X” rings together are a 9-membered bicyclic heteroaryl ring containing between one and four heteroatoms, and is optionally substituted by one or more substituents that are halo or C.sub.1-6 alkyl (itself optionally substituted by one or more fluoro atoms); L.sup.1 is an optional linker group; R.sup.x1 and R.sup.x2 independently represent are hydrogen or C.sub.1-3 alkyl; Z.sup.1 is one of the following moieties: ##STR00022## (v) perfluoro C.sub.1-3 alkyl; (vi) —F, —Br, —Cl or —CN; ring A is a 5-membered aromatic ring containing at least one heteroatom and is optionally substituted by one or more substituents independently that are R.sup.f; ring B is a 6-membered aromatic ring containing at least one heteroatom and is optionally substituted by one or more substituents independently that are R.sup.g; Y.sup.b is —CH.sub.2 or NH, and R.sup.h are one or more substituents on the 6-membered N and Y.sup.b-containing ring (which R.sup.h substituents are optionally present on Y.sup.b); R.sup.a, R.sup.b, R.sup.c, R.sup.d and R.sup.e independently are hydrogen or a substituent that is B.sup.1; each R.sup.f, each R.sup.g and each R.sup.h (which are optional substituents), when present, independently are a substituent that is B.sup.1; each B.sup.1 independently is: (i) halo; (ii) —R.sup.d1; (iii) —OR.sup.e1; (iv) —C(O)N(R.sup.e2)R.sup.e3 (v) —SF.sub.5; or (vi) —N(R.sup.e4)S(O).sub.2R.sup.e5; R.sup.d1 is C.sub.1-6 alkyl optionally substituted by one or more halo atoms; R.sup.e1, R.sup.e2, R.sup.e3, R.sup.e4 and R.sup.e5 each independently are hydrogen or C.sub.1-6 alkyl optionally substituted by one or more fluoro atoms, or a pharmaceutically-acceptable salt thereof.

2. The compound of claim 1, wherein R.sup.1 is C.sub.1-3 alkyl.

3. The compound of claim 1, wherein the “X” rings: contains at least one nitrogen atom; and/or contains one, two, three or four heteroatoms in total.

4. The compound of claim 1, wherein the “X” rings is of formula (IA): ##STR00023## wherein: one of X.sup.1 and X.sup.2 is N and the other is C; the other integers X.sup.3, X.sup.4 and X.sup.5 are C, CH, or a heteroatom; or none, any one or two of X.sup.3, X.sup.4 and X.sup.5 is a heteroatom and the other is C (or CH).

5. The compound of claim 1, wherein: L.sup.1 is a direct bond, —O—, —OCH.sub.2—, —C(R.sup.x1)(R.sup.x2)— or —C(O)—N(H)—CH.sub.2—; R.sup.x1 and R.sup.x2 independently are hydrogen.

6. The compound of claim 1, wherein: none, one or two of R.sup.a, R.sup.b, R.sup.c, R.sup.d and R.sup.e is B.sup.1 and the others are hydrogen; or one of R.sup.b, R.sup.c and R.sup.d is B.sup.1 and the others are hydrogen.

7. The compound of claim 1, wherein B.sup.1 is: (i) fluoro; (ii) —OR.sup.e1; (iii) C.sub.1-3 alkyl substituted by one or more fluoro atom; (iv) —C(O)N(R.sup.e2)R.sup.e3; (v) —N(R.sup.e4)S(O).sub.2R.sup.e5; or (vi) —SF.sub.5.

8. The compound of claim 1, wherein: R.sup.e2 and R.sup.e4 independently are hydrogen; R.sup.e1, R.sup.e3 and R.sup.e5 each independently are C.sub.1-3 alkyl optionally substituted by one or more fluoro atoms.

9. (canceled)

10. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and, as active ingredient, a therapeutically effective amount of the compound of claim 1.

11. (canceled)

12. (canceled)

13. A method of treatment of tuberculosis, comprising administration of a therapeutically effective amount of the compound of claim 1.

14. A combination of (a) a compound of claim 1, and (b) one or more other anti-tuberculosis agent.

15. A product containing (a) a compound of claim 1, and (b) one or more other anti-tuberculosis agent, as a combined preparation for simultaneous, separate or sequential use in the treatment of a bacterial infection.

16. (canceled)

17. (canceled)

18. A method of treatment of tuberculosis, comprising administration of a therapeutically effective amount of a combination of claim 14.

19. A method of enhancement of activity of another anti-tuberculosis agent, comprising administering the compound of claim 1 in combination with the another anti-tuberculosis agent.

20. A process for the preparation of a compound of formula (I) of claim 1, comprising: (i) conversion of a compound of formula (II), ##STR00024## by reaction with an appropriate reagent; or (ii) reaction of a compound of formula (III), ##STR00025## with a compound of formula (IV), ##STR00026##

21. The compound of claim 1, wherein: the two “X” rings together form a 6-membered aromatic ring fused to another 5-membered aromatic ring; and/or the 9-membered bicyclic heteroaryl ring contains one to four heteroatoms that are nitrogen oxygen or sulfur; and/or L.sup.1 is a direct bond, —O—, —OCH.sub.2—, —C(R.sup.x1)(R.sup.x2)— or —C(O)—N(H)—CH.sub.2—; and/or Z1 is CF.sub.3; and/or Ring A contains at last one nitrogen atom; and/or Ring B contains at last one nitrogen atom; and/or R.sup.d1 is optionally substituted by one or more fluoro atoms.

22. The compound of claim 2, wherein R.sup.1 is methyl.

23. The compound of claim 3, wherein the “X” rings contain at least one nitrogen at the ring junction.

24. The compound of claim 4 wherein the “X” rings are ##STR00027##

25. The compound of claim 4, wherein: X.sup.3, X.sup.4 and X.sup.5 are C, CH, N, O or S; or one or two of X.sup.3, X.sup.4 and X.sup.5 is a heteroatom; or one or two of X.sup.3, X.sup.4 and X.sup.5 is N, O, or S.

26. The compound of claim 6, wherein one or two of R.sup.a, R.sup.b, R.sup.c, R.sup.d and R.sup.e is B.sup.1; or one of R.sup.a, R.sup.b, R.sup.c, R.sup.d and R.sup.e is B.sup.1; or one of R.sup.b, R.sup.c and R.sup.d is B.sup.1; or R.sup.c is B.sup.1.

27. The compound of claim 8, wherein R.sup.e1, R.sup.e3 and R.sup.e5 are methyl.

28. The combination of claim 14, wherein the other anti-tuberculosis agent is an inhibitor of the electron transport chain of mycobacteria.

29. The combination of claim 28, wherein the other anti-tuberculosis agent is a cytochrome be inhibitor, an ATP synthase inhibitor, a NDH2 inhibitor, or an inhibitor of the menaquinone synthesis pathway.

30. The combination of claim 29, wherein the inhibitor of the menaquinone synthesis pathway is a MenG inhibitor.

31. The product of claim 15, wherein the other anti-tuberculosis agent is an inhibitor of the electron transport chain of mycobacteria.

32. The combination of claim 31, wherein the other anti-tuberculosis agent is a cytochrome be inhibitor, an ATP synthase inhibitor, a NDH2 inhibitor, or an inhibitor of the menaquinone synthesis pathway.

33. The combination of claim 32, wherein the inhibitor of the menaquinone synthesis pathway is a MenG inhibitor.

34. The process of claim 20, wherein the appropriate reagent is BBr.sub.3 or NaSCH.sub.3.

Description

EXPERIMENTAL

Synthesis of Intermediate AA-2

[0152] ##STR00009##

Preparation of Intermediate BA-1

[0153] To a solution of 4-hydroxy-3-methylquinolin-2(1H)-one (CAS [1873-59-2], 2.00 g, 11.4 mmol) and K.sub.2CO.sub.3 (3.15 g, 22.8 mmol) in acetone (150 mL), was added dimethyl sulfate (1.30 mL, 13.7 mmol) at room temperature. The reaction mixture was stirred at 60° C. for 4 h then allowed to cool back to room temperature. The reaction mixture was concentrated to dryness, triturated with water and a white solid was recovered by filtration on a glass frit, washed with water, and vacuum-dried at 50° C. to afford intermediate BA-1 as a beige solid (1.76 g, 82%).

Preparation of Intermediate BA-2

[0154] A mixture of BA-1 (3.08 g, 14.2 mmol) and POCl.sub.3 (13.2 mL, 141 mmol) was stirred at 60° C. for 1 h, then allowed to cool back to room temperature and concentrated under reduced pressure.

[0155] The residue was carefully quenched with ice-water (300 mL) and extracted with DCM (3×100 mL). Combined organic phases were washed with a saturated aqueous solution of NaHCO.sub.3 (100 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure.

[0156] The residue (beige solid, 5 g) was purified by flash chromatography over silica gel (IR-50SI/200G column, cyclohexane/DCM 95:5 to 0:100, 45 min). The product fractions were collected, the solvent was evaporated, and the product was dried under high vacuum to afford intermediate BA-2 as a white solid, 2.38 g (81%).

##STR00010##

Preparation of Intermediate A1

[0157] A mixture of 5-bromo-2-methyl-pyridine (CAS [3430-13-5], 840 mg, 4.88 mmol) and 3-Trifluoromethoxy-benzoic acid methyl ester (CAS [148438-00-0], 2.15 g, 9.77 mmol) in THE (2.2 mL) was cooled at 0° C. under nitrogen atmosphere. Then Lithium bis(trimethylsilyl)amide solution 1M in THE (CAS [4039-32-1], 14.7 mL, 14.7 mmol) was added dropwise at 0° C. and the reaction mixture was stirred at room temperature for 18 h. Ethyl acetate and water were added to the solution, then the organic phase was washed with water (10 mL) and brine (2×10 mL), dried over sodium sulfate, filtered and concentrated to dryness. The crude product was purified by flash chromatography on silica gel (IR-50SI/F0120, Cyclohexane/EtOAc from 100:0 to 90:10) to give intermediate A1 as a yellow solid 1.04 g (58%).

Preparation of Intermediate A2

[0158] Crude solution of MSH (18.6 mL, max. 3.07 mmol) was cooled to 0° C., then a solution of intermediate A1 (745 mg, 2.07 mmol) in DCM (7.2 mL) was added dropwise at 0° C. The reaction mixture was allowed to warm to room temperature and stirred for 19 h. The reaction mixture was filtered then the filtrate was washed with water (25 mL), a saturated aqueous solution of NaHCO.sub.3 (25 mL) and brine (2×25 mL). The organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated to dryness to afford a yellow oil. The crude residue was purified by flash chromatography over silica gel (IR-50SI/F0040, Cyclohexane/EtOAc from 100/0 to 95/5) to give intermediate A2 as a yellow solid, 0.29 g (39%).

[0159] Preparation of a fresh solution of (0-(mesitylsulfonyl)hydroxylamine) (=MSH) in CH.sub.2Cl.sub.2:

[0160] A solution of ethyl O-(2-mesitylenesulfonyl)acethydroxamate (CAS [38202-27-6], 1.22 g, 4.28 mmol) in 1,4-dioxane (9.8 mL) was stirred at 0° C. Perchloric acid (70%, 0.554 mL, 6.43 mmol) was added dropwise and the mixture was allowed to warm to room temperature and stirred for 15 min, then poured into ice water and stirred for 2 h. The precipitate was collected by filtration, washed with water and dissolved in CH.sub.2Cl.sub.2 (100 mL). The solution was dried over Na.sub.2SO.sub.4, filtered and concentrated to a volume of 26 mL. Considering quantitative yield, the freshly prepared crude solution of MSH in CH.sub.2Cl.sub.2 (with a concentration of max. 0.165 M) was used as such for amination. Concentrations of MSH solutions might vary in different experiments.

Preparation of Intermediate A3

[0161] A nitrogen atmosphere purged mixture of intermediate A2 (287 mg, 0.804 mmol), bis(pinacolato)diboron (CAS [73183-34-3), 245 mg, 0.964 mmol), potassium acetate (197 mg, 2.01 mmol) and [1,1′-bis(diphenylphosphino)ferrocene] dichloropalladium (58.8 mg, 0.080 mmol) in 1,4-dioxane (2.5 mL) was stirred at 100° C. for 2 h. The reaction mixture was filtered through Celite®, the filter cake was rinsed with EtOAc (20 mL) and the filtrate was concentrated to dryness to afford intermediate A3 as a black oil, 0.53 g (purity 60%, quantitative). The product was used in the next step.

Preparation of Intermediate A4

[0162] A mixture of BA-2 (111 mg, 0.536 mmol), intermediate A3 (533 mg, max. 0.804 mmol) and potassium phosphate monohydrate (370 mg, 1.61 mmol) in a mixture of 1,4-dioxane (0.8 mL) and water (0.2 mL) was purged with argon, then [1,1′-bis(diphenylphosphino)ferrocene] dichloropalladium (39.2 mg, 0.054 mmol) was then added and the mixture was purged again with argon and stirred at 100° C. for 2 h. The reaction mixture was filtered through Celite®, the filter cake was rinsed with EtOAc (20 mL) and the filtrate was concentrated to dryness to afford a black oil. The crude oil was purified by flash chromatography over silica gel (IR-50SI/F0025, Cyclohexane/EtOAc from 100/0 to 80/20) to give intermediate A4 as a yellow solid, 0.22 g, 90%.

Preparation of Compound 1

[0163] To a solution of intermediate A4 (217 mg, 0.483 mmol) in DMF (2.1 mL) was added sodium thiometoxyde (169 mg, 2.41 mmol) at room temperature and the resulting mixture was stirred at 80° C. for 1 h. The reaction mixture was quenched with water (10 mL), the aqueous layer was filtered on a glass frit and the collected solid was washed with water to give after vacuum-drying a yellow solid, 0.28 g. It was purified by flash chromatography over silica gel (IR-50SI/F0025, DCM/MeOH from 100/0 to 96/4) to give as a yellow solid, 0.13 g.

[0164] The solid was triturated with a mixture of DCM and MeOH 90/10, the mixture was concentrated to dryness to give a yellow solid. Then it was dissolved in refluxing EtOAc (80 mL, stirred for 15 min) and concentrated to dryness to give a white solid, dried under high vacuum at 60° C. to give Compound 1 as a white solid, 0.118 g (56%).

[0165] mp: 243.7° C. (DSC 1 Mettler Toledo 5° C./min).

[0166] 1H NMR (400 MHz, DMSO-d6) δ ppm 11.71 (s, 1H), 9.11 (s, 1H), 8.15 (d, J=8.2 Hz, 1H), 8.09 (d, J=8.0 Hz, 1H), 7.99 (s, 1H), 7.89 (d, J=9.1 Hz, 1H), 7.68-7.57 (m, 3H), 7.45-7.40 (m, 2H), 7.35-7.30 (m, 2H), 2.00 (s, 3H).

##STR00011##

Preparation of Intermediate BA-1

[0167] To a solution of 4-hydroxy-3-methylquinolin-2(1H)-one (CAS [1873-59-2], 2.00 g, 11.4 mmol) and K.sub.2CO.sub.3 (3.15 g, 22.8 mmol) in acetone (150 mL), was added dimethyl sulfate (1.30 mL, 13.7 mmol) at room temperature. The reaction mixture was stirred at 60° C. for 4 h then allowed to cool back to room temperature. The reaction mixture was concentrated to dryness, triturated with water and a white solid was recovered by filtration on a glass frit, washed with water, and vacuum-dried at 50° C. to afford intermediate BA-I as a beige solid (1.76 g, 82%).

Preparation of Intermediate BA-2

[0168] A mixture of intermediate BA-1 (1.45 g, 7.66 mmol) and POCl.sub.3 (7.14 mL, 76.6 mmol) was stirred at 60° C. for 1 h. The resulting mixture was allowed to cool back to room temperature, combined with another reaction mixture obtained from 0.264 mmol of intermediate ZZ-1 and concentrated to dryness. The residue was carefully quenched with ice-water and extracted with CH.sub.2Cl.sub.2. The combined organic layers were washed with a saturated aqueous solution of NaHCO.sub.3, dried over Na.sub.2SO.sub.4 and concentrated to dryness. The crude residue was purified by flash chromatography over silica gel (IR50SI, cyclohexane/CH.sub.2Cl.sub.2 from 95:5 to 40:60) to afford intermediate BA-2 as a white solid (1.34 g, 81%).

Synthesis of Intermediate BB-2

[0169] ##STR00012##

Preparation of Intermediate BB-1

[0170] A solution of intermediate BA-2 (3.00 g, 14.4 mmol) and tributyl(1-ethoxyvinyl)tin (CAS [97674-02-7], 6.35 mL, 18.8 mmol) in toluene (60 mL) was argon-purged bis(triphenylphosphine)palladium(II) dichloride (0.507 g, 0.722 mmol) was added and the mixture was purged again with argon and stirred at 110° C. for 14 h. The reaction mixture was concentrated under reduced pressure to approximately 15 mL, then MeOH (60 mL) and a 12 M aqueous solution of HCl (15 mL) were added and the mixture was stirred at 50° C. for 3.5 h. MeOH was removed under reduced pressure and 3 M aqueous NaOH was added until pH˜ 7. The aqueous layer was extracted with CH.sub.2Cl.sub.2 and the combined organic layers were dried over Na.sub.2SO.sub.4 and concentrated to dryness. The residue was purified by flash chromatography over silica gel (IR50SI, cyclohexane/EtOAc 95:5) to afford intermediate BB-1 as a white solid (2.09 g, 64%).

Preparation of Compound BB-2

[0171] To a solution of intermediate intermediate BB-1 (2.09 g, 9.20 mmol) in AcOH (40 mL) were added successively HBr 33 wt. % in acetic acid (6.50 mL, 37.1 mmol) and bromine (0.498 mL, 9.66 mmol) and the mixture was stirred at room temperature for 4 h. The reaction mixture was concentrated to dryness, then the residue was taken up with CH.sub.2Cl.sub.2 and a saturated aqueous solution of NaHCO.sub.3 and the aqueous layer was extracted with CH.sub.2Cl.sub.2. The combined organic layers were dried over sodium sulfate, filtered and concentrated to dryness. The crude product intermediate BB-2 was considered as quantitative and used as such in the next step (2.84 g containing maximum 9.20 mmol).

Synthesis of Intermediate BC-1

[0172] ##STR00013##

Preparation of Compound BC-1

[0173] An argon-purged mixture of intermediate BA-2 (1.00 g, 4.82 mmol), 2-aminopyridine-5-boronic acid pinacol ester (CAS [827614-64-2], 1.27 g, 5.78 mmol), K.sub.3PO.sub.4.H.sub.2O (3.33 g, 14.4 mmol) and Pd(dppf)Cl.sub.2 (0.352 g, 0.482 mmol) in a mixture of 1,4-dioxane (7 mL) and water (1.75 mL) was stirred at 100° C. for 3 h, then allowed to cool back to room temperature. The reaction mixture was filtered on a pad of Celite® which was rinsed with EtOAc and the filtrate was washed with water, brine, dried over Na.sub.2SO.sub.4 and concentrated to dryness. The crude residue was purified by flash chromatography over silica gel (IR50SI, CH.sub.2Cl.sub.2/MeOH from 100:0 to 94:6) to afford intermediate BC-1 as a brown solid (1.14 g, 89%).

##STR00014##

Preparation of Intermediate BK-1

[0174] To a solution of 4-(trifluoromethoxy)phenylacetic acid (CAS [4315-07-5], 2.00 g, 9.09 mmol) and N,N-dimethylformamide (0.0352 mL, 0.454 mmol) in CH.sub.2Cl.sub.2 (15 mL) was added oxalyl chloride (0.846 mL, 9.99 mmol) dropwise at 0° C. under argon atmosphere. The reaction mixture was stirred at room temperature for 2 h then concentrated to dryness to afford intermediate BK-1 as a yellow liquid (2.09 g, 96%).

Preparation of Intermediate BK-2

[0175] To a solution of intermediate BK-1 (2.09 g, 8.76 mmol) in a mixture of THF (5 mL) and MeCN (5 mL) was added (trimethylsilyl)diazomethane (2 M in Et.sub.2O) (8.76 mL, 17.5 mmol) dropwise at 0° C. under argon atmosphere. The reaction mixture was stirred at room temperature for 1.5 h then concentrated to dryness under reduced pressure. The crude mixture was dissolved in AcOH (35 mL), cooled to 0° C. and 48 wt. % aqueous HBr (1.67 ml, 14.7 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 1.5 h, quenched with a 3M NaOH aqueous solution until pH-7. The aqueous layer was extracted with EtOAc. The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and concentrated to dryness. The crude residue was purified by flash chromatography over silica gel (IR50SI, cyclohexane/CH.sub.2Cl.sub.2 from 100:0 to 70:30) to afford intermediate BK-2 as a brown liquid (1.58 g, 59%).

Preparation of Intermediate BK-3

[0176] A mixture of intermediate BC-1 (0.250 g, 0.942 mmol), intermediate BK-2 (0.509 g, 0.942 mmol) and NaHCO.sub.3 (0.158 g, 1.89 mmol) in EtOH (9 mL) was stirred at 80° C. for 17 h, then allowed to cool back to room temperature. The reaction mixture was then concentrated to dryness and the residue was taken in CH.sub.2Cl.sub.2 and washed with water. The aqueous layer was then extracted with CH.sub.2Cl.sub.2 and the combined organic layers were dried over Na.sub.2SO.sub.4, filtered and concentrated to dryness. The residue was purified by flash chromatography over silica gel (IR50SI, CH.sub.2Cl.sub.2/MeOH from 100:0 to 96:4) to afford intermediate BK-3 as a brown foam (0.293 g, 67%).

Preparation of Compound 2

[0177] A mixture of intermediate BK-3 (0.266 g, 0.574 mmol) and NaSMe (0.201 g, 2.87 mmol) in DMF (1.5 mL) was stirred at 80° C. for 1 h then allowed to cool back to room temperature. The reaction mixture was then diluted with isopropyl acetate and washed with water, a saturated aqueous solution of NH.sub.4Cl and brine. The aqueous layer was then extracted with CH.sub.2Cl.sub.2. The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and concentrated to dryness. The crude residue was purified by flash chromatography over silica gel (IR50SI, CH.sub.2Cl.sub.2/MeOH from 100:0 to 95:5) to afford compound 2 as a beige solid (0.174 g, 67%).

[0178] 1H NMR (400 MHz DMSO-d.sub.6) δ ppm 11.64 (s, 1H), 8.84 (dd, J=1.7, 0.9 Hz, 1H), 8.13 (dd, J=8.2, 1.3 Hz, 1H), 7.81 (s, 1H), 7.66-7.60 (m, 2H), 7.57 (d, J=8.3 Hz, 1H), 7.45 (d, J=8.7 Hz, 2H), 7.37 (dd, J=9.2, 1.8 Hz, 1H), 7.34-7.28 (m, 3H), 4.13 (s, 2H), 1.95 (s, 3H).

##STR00015##

Preparation of Intermediate BL-1

[0179] A mixture of bromo-1-[3-(trifluoromethoxy)phenyl]ethan-1-one (CAS [237386-01-5], 2.00 g, 7.07 mmol) and 5-bromo-2-methylpyridine (CAS [3430-13-5], 1.58 g, 9.19 mmol) in acetone (20 ml) was stirred at 90° C. for 12 h. The formed solid was collected by filtration and diluted in Et.sub.3N (4.92 ml, 35.3 mmol) and MeCN (40 ml). The resulting yellow solution was stirred at 60° C. for 12 h and then concentrated to dryness. The residue was dissolved in dichloromethane, washed with saturated aqueous NaHCO.sub.3, dried over Na.sub.2SO.sub.4, filtered and concentrated to dryness to afford intermediate BL-1 as yellow solid (0.882 g, 35%). The product was used as such for the next step.

Preparation of Intermediate BL-2 and BL-3

[0180] An argon-purged mixture of intermediate BL-1 (441 mg, 1.24 mmol), bis(pinacolato)diboron (377 mg, 1.49 mmol), KOAc (304 mg, 3.10 mmol) and Pd(dppf)Cl.sub.2 (90.6 mg, 0.124 mmol) in 1,4-dioxane (18 ml) was stirred at 100° C. for 7 h. The mixture was filtered through Celite®, the filter cake was rinsed with EtOAc and the filtrate was concentrated to dryness to afford crude intermediate BL-2 (840 mg) as a black oil. A mixture of this crude intermediate BL-2 (840 mg) with intermediate BA-2 (110 mg, 0.531 mmol) and K3PO4.H2O (367 mg, 1.59 mmol) in 1,4-dioxane (18 ml) and water (1 ml) was argon-purged, then Pd(dppf)Cl2 (38.9 mg, 0.053 mmol) was added, the mixture was purged again with argon and stirred at 100° C. for 4 h. The reaction mixture was filtered through a pad of Celite® which was rinsed with EtOAc and the filtrate was washed with water and brine, dried over Na2SO4, filtered and concentrated to dryness. The crude product was purified by flash chromatography over silica gel (IR-50SI, cyclohexane/EtOAc gradient from 100/0 to 90/10) to give intermediate BL-3 as a yellowish solid (82.1 mg).

Preparation of Compound 3

[0181] A 1 M solution of BBr3 in CH2Cl2 (0.892 ml, 0.892 mmol) was added dropwise to a solution of intermediate BL-3 (80.0 mg, 0.178 mmol) in CH2Cl2 (3.8 ml) cooled to −78° C. under argon atmosphere, and the resulting mixture was allowed to warm back to room temperature and stirred for 4.5 h, then quenched with water and extracted with CH2Cl2. The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated to dryness. The residue was combined with additional compound obtained analogously in a second experiment (reaction time 6 h) starting from 75.1 mg (0.167 mmol) BL-3 and purified by reversed phase flash chromatography (IR-50C18; water/MeCN from 70/30 to 0/100), triturated in MeOH and vacuum-dried (50° C.) to afford compound 3 as a yellow solid (22.3 mg, combined yield: 15%).

[0182] 1H NMR (400 MHz, DMSO-d6) □ ppm 11.66 (s, 1H), 8.57 (s, 1H), 8.21 (d, J=1.3 Hz, 1H), 8.13 (dd, J=8.1 Hz, 1 Hz, 1H), 7.82-7.78 (m, 1H), 7.72 (s, 1H), 7.66-7.53 (m, 4H), 7.33-7.29 (m, 1H), 7.26 (d, J=8.4 Hz, 1H), 7.02 (s, 1H), 6.89 (dd, J=9.1 Hz, 1.5 Hz, 1H), 2.00 (s, 3H).

##STR00016##

Preparation of Intermediate BM-1

[0183] Under nitrogen atmosphere, to suspension of 2-Phenylimidazo[1,2-a]pyridine-6-carbonitrile (CAS [214958-29-9], 537 mg, 2.45 mmol) in dry THF (10.7 mL) at −78° C., were added CuBre.SMe2 (20.1 mg, 0.098 mmol) and a 1M solution of ethylmagnesium bromide in THF (3.67 mL, 3.67 mmol). The reaction mixture was then warmed up to room temperature and stirred for 1 h. The reaction mixture was hydrolyzed with water for 1 h. The aqueous phase was extracted with CH2Cl2. The organic phase was washed with brine, dried over MgSO4, filtered and evaporated to dryness to give a brown solid which was purified by flash chromatography over silica gel (irregular SiOH, 15-40 μm, 40 g, Grace, dry loading (silica), mobile phase gradient: from DCM 100% to DCM 90%, MeOH 10% over 15 CV) to give intermediate BM-1 as an orange solid (0.360 g, 59%).

Preparation of Compound 4

[0184] A solution of intermediate BM-1 (209 mg, 1.10 mmol), 2-(2-Aminophenyl)-4,4-dimethyl-2-oxazoline (CAS [63478-10-4], 302 mg, 1.21 mmol) and p-toluenesulfonic acid monohydrate (83 mg, 0.44 mmol) in n-butanol (3 mL) was stirred at 130° C. under nitrogen overnight. The reaction mixture was evaporated to dryness. The residue was taken up in EtOAc and washed with water. The organic phase was washed with brine, dried over MgSO4, filtered and evaporated to dryness to give a residue. The residue was purified by flash chromatography over silica gel (irregular SiOH, 15-40 μm, 40 g, Grace, dry loading (silica), mobile phase gradient: from DCM 100% to DCM 90%, MeOH 10% over 15 CV) to give a yellow solid.

[0185] The solid was combined with another batch, triturated in Et2O, the supernatant was removed with a pipette to give an off-white solid, which was dissolved in EtOH and slowly concentrated to dryness under vacuum to give compound 4 as off-white solid (0.124 g, combined yield 16%).

[0186] 1H NMR (500 MHz, DMSO-d6) □ ppm 11.74 (br s, 1H) 8.90 (s, 1H) 8.52 (s, 1H) 8.15 (d, J=8.0 Hz, 1H) 8.03 (d, J=7.3 Hz, 2H) 7.77 (d, J=9.1 Hz, 1H) 7.65 (t, J=7.7 Hz, 1H) 7.60 (d, J=8.2 Hz, 1H) 7.42-7.51 (m, 3H) 7.30-7.39 (m, 2H) 1.99 (s, 3H).

##STR00017##

Preparation of Intermediate BN-1

[0187] A mixture of 5-bromo-2-aminopyridine (CAS [1072-97-5], 1.8 g, 10.4 mmol), 2-Bromo-1-(3-trifluoromethoxyphenyl)ethanone (CAS [237386-01-5], 3 g, 10.6 mmol) and NaHCO3(1.2 g, 14.3 mmol) in ethanol (14 mL) was refluxed for 17 h. After cooling down to room temperature, water was added and the solid was filtered. The precipitate was washed with water and ethanol then dried under vacuum to give intermediate BN-1 as a light brown solid (3.22 g, 87%).

Preparation of Intermediate BN-2

[0188] In a sealed tube, a mixture of dppf (160 mg, 0.289 mmol) in dry DMF (14 mL) was purged with nitrogen for 2 minutes, then Pd.sub.2(dba).sub.3 (130 mg, 0.142 mmol), Zn(CN).sub.2 (200 mg, 1.70 mmol) and intermediate BN-1 (1 g, 2.8 mmol) were added and the mixture was heated at 100° C. for 2 h. The mixture was filtered off and the cake was washed with EtOAc. The filtrate was evaporated in vacuo to afford a solid which was purified by flash chromatography over silica gel (irregular SiOH, 15-40 μm, 50 g, Merck, dry loading (Celite®), mobile phase gradient: from heptane 90%, EtOAc 10% to Heptane 55%, EtOAc 45% over 12CV) to afford intermediate BN-2 as a yellow solid (0.795 g, 94%).

Preparation of Intermediate BN-3

[0189] In a sealed tube, CuBreSMe.sub.2 (40 mg, 0.195 mmol) and B (3.2 mL, 3.2 mmol) were added to a suspension of intermediate BN-2 (745 mg, 2.46 mmol) in dry Me-THF (7.5 mL) under nitrogen at −78° C. The reaction mixture was then warmed up to room temperature and stirred for 2 h. Aqueous 1M HCl was added and the mixture was stirred at room temperature for 15 minutes. Water and CH.sub.2Cl.sub.2 were added, the layers were separated and the aqueous layer was extracted with CH.sub.2Cl.sub.2 (twice). The combined organic layers were dried over MgSO.sub.4, filtered off and evaporated in vacuo to afford a solid which was purified by flash chromatography over silica gel (irregular SiOH, 15-40 μm, 50 g, Merck, dry loading (Celite®), mobile phase gradient: from Heptane 85%, EtOAc 15% to Heptane 50%, EtOAc 50% over 12 CV) to give intermediate BN-3 as a yellow solid (0.531 g, 65%).

Preparation of Compound 5

[0190] A solution of intermediate BN-3 (225 mg, 0.673 mmol), 2-(2-Aminophenyl)-4,4-dimethyl-2-oxazoline (CAS [63478-10-4], 155 mg, 0.815 mmol) and zirconium (IV) chloride (60 mg, 0.257 mmol) in n-butanol (3 mL) was stirred at 130° C. under nitrogen overnight. The reaction mixture was combined with others batches and filtered through a pad of Celite®. The filtrate was evaporated in vacuo. The residual gum was solubilized in EtOAc then washed with water and brine. The organic layer was dried over MgSO4, filtered off and evaporated in vacuo to give a brown gum. The gum was purified by flash chromatography over silica gel (irregular SiOH, 15-40 μm, 50 g, Merck, dry loading (Celite®), mobile phase gradient: from heptane 100% to heptane 25%, EtOAc 75% over 12 CV) to afford a pale yellow gum which was triturated and sonicated in Et2O/DCM (9:1), filtered over a glass-frit then dried under high vacuum (50° C., 16 h) to give an off-white solid. The solid was co-evaporated with EtOH (twice), triturated in iPr.sub.2O then dried under high vacuum to give compound 5 a white solid (0.103 g, combined yield 14%).

[0191] 1H NMR (500 MHz, DMSO-d.sub.6) δ ppm 11.75 (br s, 1H) 8.92 (s, 1H) 8.65 (s, 1H) 8.15 (br d, J=8.2 Hz, 1H) 8.06 (br d, J=7.9 Hz, 1H) 8.00 (br s, 1H) 7.80 (d, J=9.1 Hz, 1H) 7.57-7.68 (m, 3H) 7.48 (br d, J=9.5 Hz, 1H) 7.30-7.38 (m, 2H) 1.98 (s, 3H)

##STR00018##

Preparation of Intermediate BO-1

[0192] Accordingly, intermediate BO-1 was prepared in the same way as intermediate BN-3 starting from 2-[4-(trifluoromethoxy)phenyl]-imidazo[1,2-a]pyridine-6-carbonitrile, (CAS [1972643-35-8], 770 mg, 2.54 mmol) yielding 0.394 g, yield: 46%.

Preparation of Compound 6

[0193] Accordingly, compound 6 was prepared in the same way as compound 5 starting from intermediate BO-1 (394 mg, 1.18 mmol) and 2-(2-Aminophenyl)-4,4-dimethyl-2-oxazoline (CAS [63478-10-4], 270 mg, 1.42 mmol) yielding 0.103 g, yield: 20%.

[0194] 1H NMR (500 MHz, DMSO-d.sub.6) δ ppm 11.73 (s, 1H) 8.92 (s, 1H) 8.57 (s, 1H) 8.15 (d, J=8.5 Hz, 3H) 7.78 (d, J=9.1 Hz, 1H) 7.65 (t, J=7.7 Hz, 1H) 7.59 (d, J=8.1 Hz, 1 H) 7.44-7.50 (m, 3H) 7.33 (t, J=7.5 Hz, 1H) 1.99 (s, 3H)

##STR00019##

Preparation of Intermediate BP-1

[0195] A sealed tube was loaded with 1-(6-aminopyridin-3-yl)propan-1-one [1355218-29-9](50 mg, 0.33 mmol), NaHCO.sub.3 (56 mg, 0.67 mmol), acetonitrile (0.59 mL), ethyl-3-oxovalerate (0.048 mL, 0.330 mmol), and bromotrichloromethane (0.066 mL, 0.670 mmol). The tube was sealed and the reaction mixture was heated at 80° C. overnight. The reaction mixture was diluted with water (5 mL) and the aqueous layer was extracted with EtOAc (5×15 mL). Combined organic layers were washed with brine, dried (MgSO4) and solvent was removed in vacuo. Purification by flash column chromatography on silica gel (GraceResolv, 4 g, SiOH 25-40 μM, dry loaded on Celite®, heptane:ethyl acetate; 40:60) yielded intermediate BP-1 as an offwhite solid, 24.6 mg, 27% yield.

Preparation of Intermediate BP-2

[0196] To a solution of intermediate BP-1 (44.3 mg, 0.16 mmol) in ethanol (3 mL) was added NaOH (3N solution, 0.16 mL, 0.48 mmol) at rt. The reaction mixture was stirred at room temperature until completion, quenched with HCl (3 N solution, 0.16 mL, 0.48 mmol) and solvent was removed in vacuo. The crude product intermediate BP-2 (39.8 mg, 0.16 mmol) was used in the next step without purification.

Preparation of Intermediate BP-3

[0197] To a solution of intermediate BP-2 (39.8 mg, 0.16 mmol) and HATU (67.5 mg, 0.18 mmol) in DMF (3 mL) was added DIPEA (0.083 mL, 0.48 mmol). The reaction mixture was stirred at room temperature for 30 min prior to add 4-(trifluoromethoxy)benzylamine (0.027 mL, 0.18 mmol). The reaction mixture was stirred overnight and diluted with EtOAc (20 mL). The organic layer was washed with brine (5×10 mL), dried (MgSO4) and solvent was removed in vacuo. Purification by flash column chromatography on silica gel (GraceResolv, 4 g, SiOH 25-40 μM, dry loaded on Celite®, heptane:ethyl acetate; 60:40) yielded intermediate BP-3 as a brow solid, 62.8 g, 93% yield (contaminated with HATU derivatives and used as such in the next step).

Preparation of Compound 7

[0198] To a solution of 2-(2-Aminophenyl)-4,4-dimethyl-2-oxazoline (CAS [63478-10-4], 25.9 mg, 0.14 mmol) and intermediate BP-3 (62.8 mg, 0.15 mmol) in n-butanol (0.32 mL) was added PTSA (14.1 mg, 0.082 mmol) at room temperature. The reaction mixture was heated for 16 h at 140° C. Purification by flash column chromatography on silica gel (GraceResolv, SiOH 25-40 μM, dry loaded on Celite®, CH.sub.2Cl.sub.2:MeOH:AcOH; 95:5:0.1) and subsequent trituration (MeCN:EtOH; 8:2) yielded compound 7 as a colourless solid, 28 mg, 33% (over 3 steps.

[0199] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ ppm 11.72 (br s, 1H), 9.21 (s, 1H), 8.56 (t, J=5.8 Hz, 1H), 8.14 (dd, J=8.0, 1.1 Hz, 1H), 7.79 (d, J=9.1 Hz, 1H), 7.62-7.67 (m, 1H), 7.56-7.61 (m, 2H), 7.51 (d, J=8.8 Hz, 2H), 7.30-7.36 (m, 3H), 4.56 (d, J=5.7 Hz, 2 H), 3.06 (q, J=7.5 Hz, 2H), 1.95 (s, 3H), 1.31 (t, J=7.6 Hz, 3H); .sup.19F NMR (471 MHz, DMSO-d.sub.6) δ ppm −56.84 (s, 1 F).

[0200] The following compounds are/were also prepared in accordance with the methods described herein:

##STR00020##

Abbreviations

[0201] K.sub.2CO.sub.3 Potassium carbonate

[0202] CH.sub.2Cl.sub.2 Dichloromethane

[0203] DCM Dichloromethane

[0204] Na.sub.2SO.sub.4 Sodium Sulfate

[0205] NaHCO.sub.3 Sodium Bicarbonae

[0206] THE Tetrahydrofuran

[0207] EtOAc Ethyl Acetate

[0208] MSH O-Mesitylenesulfonylhydroxylamine

[0209] MeOH Methanol

[0210] Et.sub.2O Diethyl Ether

[0211] NH.sub.4Cl Ammonium Chloride

[0212] EtOH Ethanol

[0213] H.sub.2 Dihydrogen gas

[0214] POCl.sub.3 Phosphoryl chloride

[0215] LiHMDS Lithium bis(trimethylsilyl)amide

[0216] DMF Dimethylformamide

[0217] Analytical Methods

[0218] Reactions

[0219] Were in general carried out in anhydrous solvents under argon atmosphere if no other gas atmosphere was required.

[0220] NMR

[0221] Was carried out on a Bruker 400 MHz spectrometer.

[0222] Melting Points

[0223] Were determined by DSC on a Mettler-Toledo DSC1 instrument (using aluminum standard 40 μL pans with air as purge gas and a thermal gradient between −10° C. and 350° C.) or on a melting point apparatus Buchi M-560, both applying indicated heating rates.

[0224] For flash chromatography, in general the following stationary phases (Interchim) were used:

[0225] Silica gel IR-50SI (irregular, 50 μm), silica gel PF-15SIHP (spherical, 15 μm) or C18-reversed silica gel IR-50C18 (irregular, 50 μm).

[0226] LCMS

[0227] The mass of some compounds was recorded with LCMS (liquid chromatography mass spectrometry). The methods used are described below.

[0228] General Procedure

[0229] The High Performance Liquid Chromatography (HPLC) measurement was performed using a LC pump, a diode-array (DAD) or a UV detector and a column as specified in the respective methods. If necessary, additional detectors were included (see table of methods below).

[0230] Flow from the column was brought to the Mass Spectrometer (MS) which was configured with an atmospheric pressure ion source. It is within the knowledge of the skilled person to set the tune parameters (e.g. scanning range, dwell time, etc) in order to obtain ions allowing the identification of the compound's nominal monoisotopic molecular weight (MW). Data acquisition was performed with appropriate software. Compounds are described by their experimental retention times (Rt) and ions. If not specified differently in the table of data, the reported molecular ion corresponds to the [M+H].sup.+ (protonated molecule) and/or [M−H].sup.− (deprotonated molecule). In case the compound was not directly ionizable the type of adduct is specified (i.e. [M+NH.sub.4].sup.+, [M+HCOO].sup.−, etc). For molecules with multiple isotopic patterns (Br, Cl . . . ), the reported value is the one obtained for the lowest isotope mass. All results were obtained with experimental uncertainties that are commonly associated with the method used.

[0231] Hereinafter, the following abbreviations may be used: “SQD” means Single Quadrupole Detector, “RT” room temperature, “BEH” bridged ethylsiloxane/silica hybrid, “HSS” High Strength Silica, “DAD” Diode Array Detector, “MSD” Mass Selective Detector.

TABLE-US-00001 TABLE LCMS Method codes (Flow expressed in mL/min; column temperature (T) in ° C.; Run time in minutes). Method Flow Run Code Instrument Column Mobile phase gradient Column T time Method Thermo- Agilent: A: HCOOH 98% A for 3 1 28 A scientific Eclipse 0.1% in min, to 0% A 30 Ultimate XDB C18 water/ in 12 min, held 3000 (5 μm, B: for 5 min, back DAD and 4.6 × 150 HCOOH to 98% A in 2 Brucker mm) 0.05% in min, held for 6 HCT CH3CN min ultra Method Thermo- Agilent: A: HCOOH 98% A for 2 1 18.4 B scientific Poroshell 0.1% in min, to 0% A 30 Ultimate EC-C18 water/ in 10 min, held 3000 (4 μm, B: for 3.4 min, DAD and 4.6 × 100 HCOOH back to 98% A Brucker mm) 0.05% in in 1.3 min, held HCT CH3CN for 1.7 min ultra Method Thermo- Agilent: A: HCOOH 50% A for 2 1 18.4 C scientific Poroshell 0.1% in min, to 0% A 30 Ultimate EC-C18 water/ in 10 min, held 3000 (4 μm, B: for 3.4 min, DAD and 4.6 × 100 HCOOH back to 50% A Brucker mm) 0.05% in in 1.3 min, held HCT CH3CN for 1.7 min ultra Method D Waters: Waters: BEH A: 95% 84.2% A for 0.343 6.2 Acquity C18 (1.7 μm, CH.sub.3COONH.sub.4 0.49 min, to 40 UPLC ®- 2.1 × 100 mm) 7 mM/5% 10.5% A in DAD and CH.sub.3CN, B: 2.18 min, held for Quattro CH.sub.3CN 1.94 min, back to Micro ™ 84.2% A in 0.73 min, held for 0.73 min. Method Waters: Waters: BEH A: 95% 84.2% A to 0.343 6.1 E Acquity ® C18 (1.7 μm CH.sub.3COON 10.5% A in 40 H-Class- 2.1 × 100 mm) H.sub.4 7 mM/ 2.18 min, held DAD and 5% CH.sub.3CN, for 1.96 min, SQD2 ™ B: CH.sub.3CN back to 84.2% A in 0.73 min, held for 0.73 min.

[0232] If a compound is a mixture of isomers which give different peaks in the LCMS method, only the retention time of the main component would be given in the LCMS table.

TABLE-US-00002 Characterising Data Table Com- LCMS pound Melting Point Rt UV BPM1/ LCMS No (Kofler or DSC) min Area % MW BPM2 Method 1 243.7° C. 10.7 98.82 435.40 436 Method B 2 244.8° C. (DSC 1 10 99.7 449.1 450 Method A Mettler Toledo 5° C./min 3 257.8° C. (DSC 1 11.2 99.3 434.1 435 Method B Mettler Toledo 5° C./min 4 337.2° C. (DSC 2.23 98.4 351.4 352.5/ Method E Mettler Toledo 350.5 10° C./min 5 229.9° C. (DSC 2.77 98.4 435.4 436.2/ Method D Mettler Toledo 434.2 10° C./min 6 306.4° C. (DSC 2.75 100 435.4 436.2/ Method D Mettler Toledo 434.2 10° C./min 7 2.69 99.8 520.2 521.3/ Method D 519.3

Pharmacological Examples

[0233] In the tests described below, individual compounds of the invention/examples (or combinations containing such compounds, for instance cytochrome bd inhibitors of the invention/examples in combination with one or more other inhibitor(s) of a (different) target of the electron transport chain of mycobacteria, as described herein) may be tested. For instance, in Tests 1 to 4, combinations may be tested (e.g. combinations of test cytochrome bd compounds with known cytochrome be inhibitors, such as Q203 and Compound X). Where a control cytochrome bd compound is employed, then CK-2-63 is employed.

[0234] The compound Q203 (cytochrome bc1 inhibitor) may be prepared in accordance with the procedures in J. Medicinal Chemistry, 2014, 57 (12), pp 5293-5305, as well as, in WO 2011/113606 (see Compound 289 “6-chloro-2-ethyl-N-(4-(4-(4-(trifluoromethoxy)phenyl)piperidin-1-yl)benzyl)imidazo[1,2-a]pyridine-3-carboxamide”).

[0235] Compound X is 6-chloro-2-ethyl-N-({4-[2-(trifluoromethanesulfonyl)-2-azaspiro[3.3]heptan-6-yl]phenyl}methyl)imidazo[1,2-a]pyridine-3-carboxamide, which is described as Compound 154 of WO 2017/001660 and may be prepared according to the procedures described therein.

[0236] CK-2-63 may be prepared in accordance with the procedures disclosed in WO 2017/103615 (see experimental and the disclosures therein, referring to WO 2012/2069856, where an experimental procedure is provided for “3-methyl-2-(4-(4-(trifluoromethoxy)phenoxy)phenyl)quinolin-4(1H)-one”).

[0237] MIC Determination Against M. tuberculosis: Test 1

[0238] Test compounds and reference compounds were dissolved in DMSO and 1 μl of solution was spotted per well in 96 well plates at 200× the final concentration. Column 1 and column 12 were left compound-free, and from column 2 to 11 compound concentration was diluted 3-fold. Frozen stocks of Mycobacterium tuberculosis strain EH4.0 expressing green-fluorescent protein (GFP) were previously prepared and titrated. To prepare the inoculum, 1 vial of frozen bacterial stock was thawed to room temperature and diluted to 5×10 exp5 colony forming units per ml in 7H9 broth. 200 μl of inoculum, which corresponds to 1×10 exp5 colony forming units, were transferred per well to the whole plate, except column 12. 200 μl 7H9 broth were transferred to wells of column 12. Plates were incubated at 37° C. in plastic bags to prevent evaporation. After 7 days, fluorescence was measured on a Gemini EM Microplate Reader with 485 excitation and 538 nm emission wavelengths and IC.sub.50 and/or pIC.sub.50 values (or the like, e.g. IC.sub.50, IC.sub.90, pIC.sub.90, etc) were (or may be) calculated.

[0239] MIC Determination Against M. tuberculosis: Test 2

[0240] Appropriate solutions of experimental and reference compounds were made in 96 well plates with 7H9 medium. Samples of Mycobacterium tuberculosis strain H37Rv were taken from cultures in logarithmic growth phase. These were first diluted to obtain an optical density of 0.3 at 600 nm wavelength and then diluted 1/100, resulting in an inoculum of approximately 5×10 exp5 colony forming units per ml. 100p of inoculum, which corresponds to 5×10 exp4 colony forming units, were transferred per well to the whole plate, except column 12. Plates were incubated at 37° C. in plastic bags to prevent evaporation. After 7 days, resazurin was added to all wells. Two days later, fluorescence was measured on a Gemini EM Microplate Reader with 543 excitation and 590 nm emission wavelengths and MIC.sub.50 and/or pIC.sub.50 values (or the like, e.g. IC.sub.50, IC.sub.90, pIC.sub.90, etc) were (or may be) calculated.

[0241] Time Kill Kinetics Assays: Test 3

[0242] Bactericidal or bacteriostatic activity of the compounds can be determined in a time kill kinetic assay using the broth dilution method. In this assay, the starting inoculum of M. tuberculosis (strain H37Rv and H37Ra) is 10.sup.6 CFU/ml in Middlebrook (1×) 7H9 broth. The test compounds (cyt bd inhibitors) are tested in combination with a cyt be inhibitor (for example Q203 or Compound X) at the concentration ranging from 10-30 μM to 0.9-0.3 μM respectively. Tubes receiving no antibacterial agent constitute the culture growth control. The tubes containing the microorganism and the test compounds are incubated at 37° C. After 0, 1, 4, 7, 14 and 21 days of incubation samples are removed for determination of viable counts by serial dilution (10.sup.0 to 10.sup.−6) in Middlebrook 7H9 medium and plating (100 μl) on Middlebrook 7H111 agar. The plates are incubated at 37° C. for 21 days and the number of colonies are determined. Killing curves can be constructed by plotting the log.sub.10CFU per ml versus time. A bactericidal effect of a cytochrome be and cytochrome bd inhibitor (either alone or in combination) is commonly defined as 2-log.sub.10 decrease (decrease in CFU per ml) compared to Day 0. The potential carryover effect of the drugs is limited by using 0.4% charcoal in the agar plates, and by serial dilutions and counting the colonies at highest dilution possible used for plating.

[0243] Phenotypic Assay to Determine the O.sub.2 Consumption Rate of Mycobacterium tuberculosis: Test 4

[0244] The aim of this assay is to evaluate the O.sub.2 consumption rate of Mycobacterium tuberculosis (Mtb) bacilli after inhibition of cyt bc1 and cyt bd, using extracellular flux technology. Inhibition of cyt bc1 (e.g. using known inhibitors such as Q203 or Compound X) forces the bacillus to use the less energetically efficient terminal oxidase cyt bd. The inhibition of cyt bd will cause a significant decrease O.sub.2 consumption. A sustained decrease of O.sub.2 consumption under membrane potential disrupting conditions, via the addition of the uncoupler CCCP, will show to the efficacy of the cyt bd inhibitor.

[0245] The oxygen consumption rate (OCR) of Mtb (stain H37Ra) bacilli adhered to the bottom of a Cell-Tak (BD Biosciences) coated XF cell culture microplate (Agilent), at 5×10.sup.6 bacilli per well, was measured using the Agilent Seahorse XFe96. Prior to the assay Mtb bacilli are cultured for two days to an OD.sub.600˜0.7-0.9 in liquid medium, using 7H9 supplemented with 10% and 0.02% Tyloxapol. The assay media used is unbuffered 7H9 only supplemented with 0.2% glucose. For this assay the Compound X (final concentration of 0.9 μM, Compound X), is used to inhibit cyt bc1 and the cyt bd inhibitor, CK-2-63 (final concentration of 10 μM), is used as a positive control. The uncoupler CCCP is used at a final concentration of 1 μM.

[0246] In general, four basal OCR measurements are taken before the automatic addition of Compound X, through drug port A of the sensor cartridge, after which seven more OCR measurements are taken to allow enough time for the inhibition of cyt bc1. Next the cyt bd test compounds (final concentration of 10 μM), as well as the positive and negative controls (assay media with a final DMSO concentration of 0.4%), are added (drug port B) followed by seven OCR measurements. Finally, CCCP is added followed by three OCR measurements, this is done twice (drug ports C and D). For the control's measurements are performed in eight replicate wells and for the assay compounds six replicate wells per condition. Compounds are scored for their sustained inhibition of cyt bd in relation to the positive and negative controls.

[0247] Further Phenotypic Assay: Using a Cytochrome Bc Knock-Out TB Strain and MIC Determination Against M. tuberculosis: Test 5

[0248] Appropriate solutions of experimental and reference compounds were made in 384 well plates with 7H9 medium. Samples of Mycobacterium tuberculosis strain H37Rv ΔctaE-ΔqcrCAB (Nat Commun 10, 4970, 2019, https://doi.org/10.1038/s41467-019-12956-2) were taken from cultures in logarithmic growth phase. These were first diluted to obtain an optical density of 0.4 at 600 nm wavelength and then diluted 1/150, resulting in an inoculum of approximately 5×10 exp5 colony forming units per ml. 30 μl of inoculum, which corresponds to 5×10 exp5 colony forming units, were transferred per well to the whole plate, except columns 23-24. Plates were incubated at 37° C., in an extra humidified incubator, in plastic bags to prevent evaporation. After 10 days, optical density at 620 nm wavelength was measured on an EnVision 2105 Multimode Plate Reader with a Photometric 620/8 excitation filter, and MIC.sub.50 and/or pIC.sub.50 values (or the like, e.g. IC.sub.50, IC.sub.90, pIC.sub.90, etc) were (or may be) calculated.

Pharmacological Results

Biological Data—Example A

[0249] Compounds of the invention/examples (or combinations, e.g. compounds of the invention/examples in combination with one or more other inhibitors of a target of the electron transport chain), for example when tested in any of Tests 1 to 3, may display activity.

Biological Data—Example B

[0250] Compounds of the examples were tested in Test 4 described above (in section “Pharmacological Examples”; O.sub.2 consumption rate testing), together with Compound X—a known cytochrome be inhibitor—as described above, and the following results were obtained:

TABLE-US-00003 (i) % OCR after (ii) cyt bd % OCR Example inhibitor after cccp 1 62.4 93.2 2 111.8 112.0 3 98.5; 98.0 196.1; 131.0 4 83.4 144.1 5 80.8 159.3 6 104.1 177.3 7 90.7 110.6 8 62.42 93.125

Biological Data—Example C

[0251] Compounds of the examples are tested in Test 3 (the kill kinetics) described above, obtaining results expressed as a log reduction in CFUs per ml as compared to Day 0

Biological Data—Example D

[0252] Compounds of the examples were re-tested in Test 5 described above, and the following results were obtained:

TABLE-US-00004 Compound number pIC.sub.50 4 <4.000 5 <4.000 2 4.037 3 <4.000 1 4.556

[0253] Further Data

[0254] The compounds of the invention/examples may have advantages associated with in vitro potency, kill kinetics (i.e. bactericidal effect) in vitro, PK properties, food effect, safety/toxicity (including liver toxicity, coagulation, 5-LO oxygenase), metabolic stability, Ames II negativity, MNT negativity, aqueous based solubility (and ability to formulate) and/or cardiovascular effect e.g. on animals (e.g. anesthetized guinea pig). The data below that was generated/calculated may be obtained using standard methods/assays, for instance that are available in the literature or which may be performed by a supplier (e.g. Microsomal Stability Assay—Cyprotex, Mitochondrial toxicity (Glu/Gal) assay—Cyprotex, as well as literature CYP cocktail inhibition assays).

[0255] Mitotoxicity Data:

TABLE-US-00005 Δ cpd IC50, IC50, IC50, glu/ number glu gal IC50, gal Score 8 [x]200 [x]10.82 [x]18.49 positive

[0256] The score for other compounds was as follows:

[0257] Compound 1: positive

[0258] Compound 2: positive

[0259] Compound 3: negative

[0260] Compound 7: negative

[0261] Certain compounds of the invention/examples may be found to be advantageous as no mitotoxicity alerts were observed (e.g. in the Glu/Gal assay).

TABLE-US-00006 Cardio tox Compound rCaCH (pIC50), rNaCH (IC50), hERG(DOF) (IC50) 5 >10, 3.8, >10 2 >10, 2.6, 1.8 3 >10, 1.9, >10 1 3.5, 3.02, 3.98

[0262] Compounds of the invention/examples, may therefore have the advantage that: [0263] No in vitro cardiotoxicity is observed (for example either due to the CVS results or due to the Glu/Gal assay results, for instance low mitotoxicity (<3 in the Glu/Gal assay indicates no mitotoxicity alerts); and/or [0264] No reactive metabolite formation is observed (e.g. GSH);

[0265] for instance as compared to other compounds, for instance prior art compounds.