IDEBENONE DERIVATIVES AND THEIR USE IN TREATING PLANTS

Abstract

The invention relates to a compound of formula (I) comprising a mitochondrial targeting group linked to a group capable of releasing hydrogen sulphide for use in the treatment of the human or animal body or tissues and cells derived therefrom and to the use in the treatment of plants and to novel related compounds.

##STR00001##

Claims

1. A compound of formula (I): ##STR00050## wherein R.sup.1 and R.sup.2 are independently selected from a C.sub.1-6 alkyl group, a C.sub.1-6 alkoxy group or together form a cycloalkyl or aryl ring; wherein R.sup.3 is an C.sub.1-6 alkyl or C.sub.1-6 alkoxy group; wherein L is a linker group; and wherein A is a group capable of releasing hydrogen sulphide, or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1, wherein group A is selected from: ##STR00051## wherein X is S, O or NOH and R.sup.4, R.sup.5 and R.sup.6 are independently selected from H or C.sub.1-7 alkyl groups.

3. The compound according to claim 1, wherein A is selected from a thiocarbamoyl group, a 5-thioxo-5H-1,2-dithiol-3-yl group, a 5-thioxo-5H-1,2-dithiol-4-yl group, a 5-oxo-5H-1,2-dithiol-3-yl group, a 5-oxo-5H-1,2-dithiol-4-yl group, a 5-hydroxyimino-5H-1,2-dithiol-3-yl group, a 5-hydroxyimino-5H-1,2-dithiol-4-yl group, a phosphinodithioate group or a phosphinodithioic acid group.

4. The compound according to claim 1, wherein L comprises a group B which is an optionally substituted alkyl chain, optionally substituted alkenyl chain, or optionally substituted alkynyl chain.

5. The compound according to claim 1, wherein L comprises a group Z selected from a direct bond, C(O)NH, NHC(O), O, S, S(O).sub.2NH, NHS(O).sub.2, OC(O), OC(O)CH.sub.2O and C(O)O.

6. The compound according to claim 1, wherein L comprises a group Y which is an optionally substituted 5 or 6 membered cycloalkyl or aryl ring.

7. The compound according to claim 1, having the formula (II): ##STR00052## wherein R.sup.1, R.sup.2 and R.sup.3 are as defined in claim 1; wherein B is an optionally substituted alkyl chain, optionally substituted alkenyl chain, or optionally substituted alkynyl chain; wherein Z is selected from a direct bond, C(O)NH, NHC(O), O, S, S(O).sub.2NH, NHS(O).sub.2, OC(O), OC(O)CHO and C(O)O; wherein Y is an optionally substituted 5 or 6 membered cycloalkyl or aryl ring; and wherein A is a group capable of releasing hydrogen sulphide, or a pharmaceutically acceptable salt thereof.

8. The compound according to claim 1, having the formula (III): ##STR00053## wherein R.sub.1 and R.sub.2 are both C.sub.1-6 alkoxy groups or together form a 6-membered aryl ring; wherein R.sup.3 is an C.sub.1-6 alkyl group. wherein B is an optionally substituted a C.sub.6-14 alkyl chain; wherein Z is a group selected from C(O)NH, NHC(O), O, OC(O), OC(O)CH.sub.2O, OCH.sub.2C(0)O andC(O)O; wherein Y is an optionally substituted phenyl group wherein groups Z and A are attached para to each other on the phenyl group; wherein A is selected from: ##STR00054## and wherein X is S, O or NOH and R.sup.4, R.sup.5 and R.sup.6 are independently selected from H or C.sub.1-7alkyl groups; or a pharmaceutically acceptable salt thereof.

9. The compound according to claim 7 wherein A is selected from a thiocarbamoyl group, a 5-thioxo-5H-1,2-dithiol-3-yl group, a 5-thioxo-5H-1,2-dithiol-4-yl group, a 5-oxo-5H-1,2-dithiol-3-yl group, a 5-oxo-5H-1,2-dithiol-4-yl group, a 5-hydroxyimino-5H-1,2-dithiol-3-yl group, a 5-hydroxyimino-5H-1,2-dithiol-4-yl group, a phosphinodithioate group and a phosphinodithioic acid group.

10. The compound according to claim 1, wherein R.sup.1 and R.sup.2 are both OMe and R.sup.3 is an C.sub.1-3 alkyl group.

11. The compound according to claim 1, wherein R.sup.1 and R.sup.2 form a 5-or 6-membered aryl ring.

12. The compound according to claim 7, wherein B is an unsubstituted C.sub.1-20alkyl group.

13. The compound according to claim 7, wherein Z is C(O)O orOC(O)CH.sub.2O.

14. The compound according to claim 7, wherein Y is an optionally substituted phenyl group and wherein groups Z and A are attached para to each other on the phenyl group.

15. The compound according to claim 1 selected from a group consisting of: ##STR00055## ##STR00056##

16. The compound according to claim 1 selected from a group consisting of: ##STR00057##

17. (canceled)

18. A method of treating- a neuromuscular or muscular condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound according to claim 1.

19. The method of claim 18, wherein the neuromuscular or muscular condition is mediated by mitochondrial H.sub.2S donors.

20. The method of claim 18, wherein the neuromuscular or muscular condition is selected from Duchenne Muscular dystrophy, COPD, Leigh syndrome, primary mitochondrial disease, Pancreatic islet transplant, Pre-eclampsia, Cardiac transplant, Renal transplant, Cardiovascular dysfunction, Blunt chest trauma and haemorrhagic shock, Necrotizing enterocolitis, Myocardial reperfusion injury, Burn injury, Diabetic vascular disease, Alzheimer's disease, Acute renal injury, Neurological damage post cardiac arrest and Hypertension.

21. A pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof according to claim 1, and a pharmaceutically acceptable carrier, excipient, or diluent.

22. (canceled)

23. (canceled)

Description

EXAMPLES

Synthesis of Example 1

[0343] Example 1 was prepared in three steps, with an overall yield of 25% (from ADTOH), using the following reaction scheme.

##STR00027##

[0344] Scheme 1. Example 1 synthetic route. Firstly, 2 (90%) was prepared by a reaction between ADTOH(5-(4-hydroxyphenyl) -3H-1,2-dithiole-3-thione) and tert-butyl bromoacetate (1.5 eq.) with caesium carbonate (2 eq.) in acetone. Following that, the ester protecting group was removed with trifluoroacetic acid (10 eq.) (46% yield). The coupling reaction with idebenone (1 eq.) was carried out using EDCI (1.5 eq.) and DMAP (0.1 eq.) thus, Example 1 was obtained (60% yield).

Example 2, 3, 4 Syntheses

[0345] A more efficient, alternative synthetic approach for bonding ADTOH to idebenone was also carried out. The alcohol functionality in idebenone was oxidised, as previously reported [U.S. Pat. No. 8,263,094], into a carboxylic acid using the Jones reagent (chromic acid made by chromium trioxide or a dichromate salt with sulfuric acid) and a coupling reaction was carried out between ADTOH and the idebenone carboxylic acid to produce Example 2.

##STR00028##

[0346] Scheme 2: Example 2 synthetic route. The idebenone alcohol functionality was oxidised into carboxylic acid (94% yield), using Jones' reagent (16 eq.) made with sodium dichromate dihydrate and sulfuric acid. The product obtained was linked to ADTOH (1 eq.), using EDCl (1.5 eq.) and DMAP (0.1 eq.) to give Example 2 (45% yield).

[0347] In an analogous manner, coupling reactions between idebenone carboxylic acid and both HTB (4-hydroxythiobenzamide) and intermediate RT02 were carried out. Example 3 and Example 4 (Scheme 3) were obtained with 45% and 66% overall yields, respectively (from idebenone).

##STR00029##

[0348] Scheme 3. Example 3 and Example 4 synthetic routes. Idebenone carboxylic acid (1 eq.) was linked to HTB or RT02 (5-(4-hydroxyphenyl) -3H-1,2-dithiol-3-one) using DCCl (N,N-Dicyclohexylcarbodiimide) (1.5eq.) and DMAP (4-dimethylaminopyridine) (0.1 eq.), producing Example 3 (48% yield) and Example 4 (70% yield).

[0349] Example 1 (10-(4,5-dimethoxy-2-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl) decyl (4-3-thioxo-3H-1,2- dithiol-5yl) phenoxy) acetate). In the synthesis of Example 1 silica gel flash chromatography was carried out using a solvent mixture of petroleum ether/ethyl acetate 1/1, which gave RT154 as a red oil (363 mg, 60%, 0.60 mmol) (cLogP=6.23). IR spectrum V.sub.max/cm.sup.1=2851 (m), 1760 (CO) (m), 1607 (CO) (s), 1523(s), 1588 (w), 1487 (m), 1436 (m), 1412 (w), 1203 (w), 1184 (m), 1166 (m), 1024 (m), 945 (w), 835 (w), 743(w). .sup.1H-NMR .sub.H(400 MHZ, CDCl.sub.3)=7.55 (2H, d, part of AABB, J=8 Hz, aryl CH), 7.31 (1H, s, alkene CH), 6.92 (2H, d, part of AABB, J=12 Hz, aryl CH), 4.63 (2H, s, OCCH.sub.2O), 4.14 (2H, t, J=8 Hz, CH.sub.2O), 3.92 (6H, s, 2CH.sub.3O), 2.37 (2H, t, J=8 Hz, CH.sub.2), 1.94 (3H, s, CH.sub.3), 1.58 (2H, t, J=8 Hz, CH.sub.2), 1.27-1.19(14H, m, 7CH.sub.2). .sup.13C-NMR .sub.C (100 MHZ, CDCl.sub.3)=215.20 (CS), 184.72 (CO), 184.17 (CO), 172.57(COO), 168.19 (SC=CH), 160.99 (aryl CO), 144.30 (CC), 143.03 (CC), 138.70 (CC), 134.94 (alkene CH), 128.64 (aryl CH), 125.15 (aryl CC), 115.59 (aryl CH), 65.80 (OCCH.sub.2), 65.23 (CH.sub.2O), 61.17 (CH.sub.3O), 29.81 (CH.sub.2), 29.46 (CH.sub.2), 29.40 (CH.sub.2), 29.33 (CH.sub.2), 29.14 (CH.sub.2), 28.72 (CH.sub.2), 28.50 (CH.sub.2), 26.39 (CH.sub.2), 25.77 (CH.sub.2), 11.95 (CH.sub.3).

[0350] Example 2 ((4-(3-thioxo-3H-1,2-dithiol-5yl) phenoxy)10-(4,5-dimethoxy-2-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl) decanoate). In the synthesis of Example 2 silica gel flash chromatography was carried out using a solvent mixture of petroleum ether/ethyl acetate 2/1, which gave the product as a red oil (252 mg, 45%, 0.45 mmol) (cLogP=6.10). HRMS(ES).sup.+ found m/z (rel. intensity)561.1445 (MH.sup.+; 30), C.sub.28H.sub.33O.sub.6S.sub.3 requires 561.1439, 226.9663 (MH-idebenone.sup.+; 100). IR spectrum V.sub.max/cm.sup.1=1762 (CO) (w), 1705 (CO) (m), 1642 (s), 1605 (s), 1546 (w), 1436 (m), 1379 (w), 1263 (m), 1204 (m), 1172 (m), 1094 (w), 1025 (w), 836 (w). .sup.1H-NMR .sub.H(400 MHZ, CDCl.sub.3)=7.70 (2H, d, part of AABB, J=8 Hz, aryl CH), 7.42 (1H, s, alkene CH), 7.25 (2H, d, part of AABB, J=8 Hz, aryl CH), 4.01 (6H, s, 2CH.sub.3O), 2.60 (2H, t, J=8 Hz, CH.sub.2), 2.47 (2H, t, J=8 Hz, CH.sub.2), 2.03 (3H, s, CH.sub.3), 1.77 (2H, m, CH.sub.2), 1.42-1.34 (12H, m, 6CH.sub.2). 13C-NMR .sub.C (100 MHZ, CDCl.sub.3)=215.51 (CS), 184.72 (CO), 184.18 (CO), 171.78 (COO), 171.72 (SCCH), 153.71 (aryl CO), 144.30 (CC), 143.02 (CC), 138.71 (CC), 136.01 (alkene CH), 129.10 (aryl CC), 128.21 (aryl CH), 122.96 (aryl CH), 61.19 (CH.sub.3O), 34.36 (CH.sub.2), 29.79 (CH.sub.2), 29.28 (CH.sub.2), 29.19 (CH.sub.2), 28.72 (CH.sub.2), 26.40 (CH.sub.2), 24.79 (CH.sub.2), 11.95 (CH.sub.3).

[0351] Example 3 ((4-carbamothioylphenoxy) l0-(4,5-dimethoxy-2-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)decanoate). In the synthesis of Example 3, silica gel flash chromatography was carried out using a solvent mixture of ether/ethyl acetate 1/1 and RTI64 was obtained as an orange solid (234 mg, 48%, 0.48 mmol) (cLogP=5.59). HRMS(ES).sup.+ found m/z (rel. intensity)488.2095 (MH.sup.+; 100), C.sub.26H.sub.34NO6S requires 488.2107. 1H-NMR .sub.H(400 MHZ, CDCl.sub.3)=7.90 (2H, d, part of AABB, J=8 Hz, aryl CH), 7.84 (1H, br s, NH), 7.55 (1H, br s, NH), 7.10 (2H, d, part of AABB, J=8 Hz, aryl CH), 3.98 (6H, s, 2CH.sub.3O), 2.57 (2H, t, J=8 Hz, CH.sub.2), 2.44 (2H, t, J=8 Hz, CH.sub.2), 2.01 (3H, s, CH.sub.3), 1.75 (2H, m, CH.sub.2), 1.40-1.32 (12H, m, 6CH.sub.2). .sup.13C-NMR .sub.C (100 MHZ, CDCl.sub.3)=201.45 (CS), 184.76 (CO), 184.23 (CO), 171.99 (COO), 153.58 (aryl CO), 144.29 (CC), 144.26 (CC), 143.05 (CC), 138.76 (CC), 136.60 (aryl CC), 128.53 (aryl CH), 121.54 (aryl CH), 61.18 (CH.sub.3O), 34.36 (CH.sub.2), 29.78 (CH.sub.2), 29.24 (CH.sub.2), 29.12 (CH.sub.2), 28.97 (CH.sub.2), 28.72 (CH.sub.2), 26.40 (CH.sub.2), 24.79 (CH.sub.2), 11.95 (CH.sub.3).

[0352] Example 4 ((4-(3-oxo-3H-1,2-dithiol-5-yl) phenoxy) l0-(4,5-dimethoxy-2-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl) decanoate). In the synthesis of Example 4, silica gel flash chromatography was carried out using a solvent mixture of petroleum ether/ethyl acetate 2/1. Example 4 was obtained as an orange solid (381 mg, 70%, 0.70 mmol) (cLogP=5.40). HRMS(ES).sup.+ found m/z (rel. intensity)545.1663 (MH.sup.+; 20), C.sub.28H.sub.3307S2 requires 545.1662, 210.9913 (MH-idebenone+; 100). IR spectrum V.sub.max/cm 1=3295 (m), 1760 (CO) (w), 1734 (CO) (w), 1706 (CO) (w), 1641 (CO) (s), 1604 (s), 1585 (s), 1457 (m), 1436 (m), 1373 (w), 1263 (m), 1126 (m), 1073 (m), 828 (w), 800 (w), 742 (w). .sup.1H-NMR .sub.H(400 MHZ, CDCl.sub.3)=7.58 (2H, d, part of AABB, J=8 Hz, aryl CH), 7.16 (2H, d, part of AABB, J=12 Hz, aryl CH), 6.75 (1H, s, alkene CH), 3.92 (6H, s, 2CH.sub.3O), 2.52 (2H, t, J=8 Hz, CH.sub.2), 2.38 (2H, t, J=8 Hz, CH.sub.2), 1.94 (3H, s, CH.sub.3), 1.69 (2H, m, CH.sub.2), 1.33-1.25 (12H, m, 6CH.sub.2). 13C-NMR .sub.C (100 MHZ, CDCl.sub.3)=194.02 (SCO), 184.73 (CO), 184.19 (CO), 171.80 (COO), 169.25 (SC=CH), 153.41 (aryl CO), 144.30 (CC), 143.03 (CC), 138.72 (CC), 130.03 (aryl CC), 127.82 (aryl CH), 122.76 (aryl CH), 118.03 (1H, s, alkene CH), 61.18 (CH.sub.3O), 34.35 (CH.sub.2), 29.80 (CH.sub.2), 29.29 (CH.sub.2), 29.19 (CH.sub.2), 29.04 (CH.sub.2), 28.72 (CH.sub.2), 26.40 (CH.sub.2), 24.80 (CH.sub.2), 11.95 (CH.sub.3).

[0353] General procedure for synthesis of Examples 5, 6 and 7. These syntheses were carried out modifying a reported literature protocol [Ger et al 2016]. 10-(3-methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)decanoic acid (292 mg; 0.853 mmol) was dissolved in dichloromethane (8 ml). The mixture was stirred at room temperature and ADTOH(193 mg; 0.853 mmol) or HTB (131 mg; 0.853 mmol) or 5-(4-hydroxyphenyl)-3H-1,2-dithiol-3-one (179 mg; 0.853 mmol) was added to it. 4-Dimethylaminopyridine (10 mg; 0.085 mmol) and N,N-dicyclohexylcarbodiimide (264 mg; 1.28 mmol) (for Examples 5 and 7) or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (164 mg; 0.853 mmol) (for Example 6) were added to the initial solution, which was stirred at room temperature for 18 h. The solution was filtered to remove the precipitate formed and the solvent was evaporated in vacuo (for Examples 5 and 7). Alternatively, the reaction mixture was washed with deionised water (615 ml), the organic phase was dried over MgSO4 and the solvent was removed under reduced pressure (for Example 6). The crude product obtained was loaded onto a silica gel flash chromatography column.

[0354] Synthesis of 10-(3-methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl) decanoic acid (2-methyl-1,4-naphthoquinone acid derivative). The acid derivative synthesis was carried out by slightly modifying a reported literature protocol [SalmonChemin et al 2001]. To a stirred solution containing 2-methyl-1,4-naphthoquinone (300 mg; 1.74 mmol) and undecanedioic acid (1.129 g; 5.22 mmol) in 50 ml of degassed 30% aqueous acetonitrile, silver nitrate (88 mg; 0.522 mmol) was added. A solution of ammonium peroxodisulfate (516 mg; 2.26 mmol) in 12 ml of degassed 30% aqueous acetonitrile was added dropwise to the stirred solution over a 15 minutes period. The resulting solution was stirred under nitrogen atmosphere at 70 C. for 3 h. After cooling the solution to room temperature, the residue was extracted with dichloromethane (350 ml) and the organic phases were combined and washed with deionised water (350 ml). The organic solution was dried over MgSO4 and the solvent removed under reduced pressure. The crude product was loaded onto a silica gel flash chromatography column, which was eluted with an initial solvent mixture of 3/1 petroleum ether (bp 40-60 C.) /ethyl acetate, followed by 2/1 petroleum ether (bp 40-60 C.) /ethyl acetate solvent mixture to give the title product as yellow solid (292 mg; 49%; 0.853 mmol). 10-(3-methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl) decanoic acid .sup.1H-NMR OH(400 MHZ, CDCl.sub.3)=8.01-7.99 (2H, m, aryl CH), 7.63-7.60 (2H, m, aryl CH), 2.55 (2H, t, J=8.0 Hz, CH.sub.2C(O) ), 2.27(2H, t, J=8.0 Hz, CH.sub.2C=C), 2.12 (3H, s, CH.sub.3), 1.58-1.53 (2H, m, CH.sub.2), 1.40-1.23 (12H, m, 6CH.sub.2). 13C-NMR .sub.C (100 MHZ, CDCl.sub.3)=185.42 (CO), 184.75 (CO), 179.86 (COOH), 147.55 (aryl CC), 143.12 (aryl CC), 133.33 (aryl CH), 133.30 (aryl CH), 132.20 (aryl CC), 126.28 (aryl CH), 126.18 (aryl CH), 34.00 (CH.sub.2), 29.94 (CH.sub.2), 29.33 (CH.sub.2), 29.29 (CH.sub.2), 29.18 (CH.sub.2), 29.01 (CH.sub.2), 28.73 (CH.sub.2), 27.10 (CH.sub.2), 24.64 (CH.sub.2), 12.66 (CH.sub.3).

[0355] Example 5 (4-(3-Thioxo-3H-1,2-dithiol-5yl) phenoxy) l0-(1,4-dihydronaphthalen-2-yl) decanoate). In the synthesis of Example 5, the silica gel flash chromatography was carried out using a solvent mixture of petroleum ether (bp 40-60 C.) /ethyl acetate 3/1 and RTK-46 was obtained as an orange waxy solid (179 mg; 38%; 0.324 mmol) (cLogP=7.23). .sup.1H-NMR .sub.H(400 MHZ, CDCl.sub.3)=8.02-7.99 (2H, m, aryl CH), 7.63-7.60 (4H, m, aryl CH), 7.33 (1H, s, alkene CH), 7.16 (2H, d, part of AABB, J=8.0 Hz, aryl CH), 2.58-2.50 (4H, m, CH.sub.2C(O) and CH.sub.2C=C), 2.12 (3H, s, CH.sub.3), 1.69-1.67 (2H, m, CH.sub.2), 1.40-1.27 (12H, m, 6CH.sub.2). .sup.13C-NMR .sub.C(100 MHZ, CDCl.sub.3)=215.52 (CS), 185.40 (CO), 184.77 (CO), 171.76 (COO), 171.71 (aryl CC), 153.71 (aryl CC), 147.51 (aryl CC), 143.14 (aryl CC), 136.00 (alkene CH), 133.36 (aryl CH), 133.34 (aryl CH), 132.20 (aryl CC), 132.17 (aryl CC), 129.09 (aryl CC), 128.20 (aryl CH), 126.28 (aryl CH), 126.21 (aryl CH), 122.95 (aryl CH), 34.36 (CH.sub.2), 29.95 (CH.sub.2), 29.33 (CH.sub.2), 29.30 (CH.sub.2), 29.18 (CH.sub.2), 29.02 (CH.sub.2), 28.74(CH.sub.2), 27.10 (CH.sub.2), 24.79 (CH.sub.2), 12.68 (CH.sub.3).

[0356] Example 6 (4-Carbamothioylphenoxy)10-(1,4-dihydronaphthalen-2-yl) decanoate). In the synthesis of Example 6, the silica gel flash chromatography was carried out starting with a solvent mixture of petroleum ether (bp 40-60 C.) /ethyl acetate 2/1 followed by a solvent mixture of petroleum ether (bp 40-60 C.)/ethyl acetate 1/1 and Example 6 was obtained as a yellow solid (98 mg; 24%; 0.205 mmol) (cLogP=6.35). .sup.1H-NMR .sub.H(400 MHZ, CDCl.sub.3)=8.00-7.96 (2H, m, aryl CH), 7.81 (2H, d, part of AABB, J=8.0 Hz, aryl CH), 7.75 (1H, br s, NH), 7.61-7.60 (2H, m, aryl CH), 7.41 (1H, br s, NH), 7.02 (2H, d, part of AABB, J=8.0 Hz, aryl CH), 2.56-2.47 (4H, m, CH.sub.2C(O) and CH.sub.2C=C), 2.10 (3H, s, CH.sub.3), 1.69-1.65 (2H, m, CH.sub.2), 1.40-1.26 (12H, m, 6CH.sub.2). 13C-NMR .sub.C(100 MHZ, CDCl.sub.3)=201.49 (CS), 184.76 (CO), 184.78 (CO), 171.98 (COO), 171.26 (aryl CC), 153.59 (aryl CC), 147.53 (aryl CC), 143.15 aryl (CC), 136.61 (aryl CC), 133.67 (aryl CH), 133.38 (aryl CC), 128.49 (aryl CH), 126.27 (aryl CH), 126.19 (aryl CH)121.56 (aryl CH), 34.36 (CH.sub.2), 29.93 (CH.sub.2), 29.31 (CH.sub.2), 29.28 (CH.sub.2), 29.15 (CH.sub.2), 29.01 (CH.sub.2), 28.74 (CH.sub.2), 27.09 (CH.sub.2), 24.79 (CH.sub.2), 12.67 (CH.sub.3).

[0357] Example 7 (4-(3Oxo-3H-1,2-dithiol-5-yl) phenoxy) l0-(1,4-dihydronaphthalen-2-yl) decanoate). In the synthesis of Example 7, the silica gel flash chromatography was carried out using a solvent mixture of petroleum ether (bp 40-60 C.) /ethyl acetate 4/1 and RTK-48 was obtained as a yellow solid (128 mg; 28%; 0.239 mmol) (cLogP=6.75). .sup.1H-NMR .sub.H(400 MHZ, CDCl.sub.3)=8.00-7.99 (2H, m, aryl CH), 7.63-7.56 (4H, m, aryl CH), 7.15 (2H, d, part of AABB, J=8.0 Hz, aryl CH), 6.74 (1H, s, alkene CH), 2.56-2.50 (4H, m, CH.sub.2C(O) and CH.sub.2CC), 2.12 (3H, s, CH.sub.3), 1.69-1.57 (2H, m, CH.sub.2), 1.34-1.27 (12H, m, 6CH.sub.2). .sup.13C-NMR .sub.C(100 MHZ, CDCl.sub.3)=193.99 (SCO), 185.39 (CO), 184.76 (CO), 171.79 (COO), 169.23 (aryl CC), 153.41 (aryl CC), 147.51 (aryl CC), 143.13 (aryl CC), 133.36 (aryl CH), 133.33 (aryl CH), 132.20 (aryl CC), 132.16 (aryl CC), 130.02 (aryl CC), 127.81 (aryl CH), 126.27 (aryl CH), 126.20 (aryl CH), 122.76 (aryl CH), 118.02 (alkene CH), 34.36 (CH.sub.2), 29.95 (CH.sub.2), 29.33 (CH.sub.2), 29.30 (CH.sub.2), 29.18 (CH.sub.2), 29.02 (CH.sub.2), 28.74 (CH.sub.2), 27.10 (CH.sub.2), 24.80 (CH.sub.2), 12.68 (CH.sub.3).

[0358] Synthesis of 2,3-dimethoxy-5-methyl-6-{10-[4-(3-sulfanylidene-3H-1,2-dithiol-5-yl)phenoxy]decyl} cyclohexa-2,5-diene-1,4-dione (Example 8).

##STR00030##

[0359] 2-(10-hydroxydecyl) -5,6-dimethoxy-3-methyl-1,4-benzoquinone (1.00 g, 0.00295 mol), and TPP (triphenyl phosphate) (0.775 g, 0.00295 mol) were dissolved in dry THF (12 mL) under nitrogen. DEAD (diethyl azodicarboxylate) (2.2M in toluene) (2.20 mol/L, 1.34 mL, 0.00295 mol) was added dropwise (slightly exotherm noted) and stirred at room temperature for 5 minutes. 5-(4-hydroxyphenyl) dithiole-3-thione (0.669g, 0.00295 mol) was added and stirred at room temperature overnight. The reaction mixture was evaporated to dryness and purified by column chromatography, eluting with DCM. Most impurities were removed. Re-columned in 0-30% EtOAc in hexane to give the product in, Example 8, 18% yield. 1H NMR (400 MHZ, CDCl.sub.3) 7.64-7.56 (m, 2H), 7.39 (s, 1H), 7.00-6.92 (m, 2H), 4.02 (t, J=6.5 Hz, 2H), 3.99 (d, J=1.2 Hz, 6H), 2.49-2.39 (m, 2H), 2.01 (d, J=0.7 Hz, 3H), 1.81 (dt, J=14.6, 6.7 Hz, 2H), 1.46 (p, J=6.8 Hz, 2H), 1.42-1.24 (m, 13H). .sup.13C NMR (101 MHZ, CDCl.sub.3) 215.12 (C), 184.71 (C), 184.17 (C), 173.17 (C), 162.61 (C), 144.34 (C), 144.32 (C), 143.05 (C), 138.69 (C), 134.55 (CH), 128.58 (CH), 123.94 (C), 115.47 (CH), 68.48 (CH.sub.2), 61.15 (CH), 29.81 (CH.sub.2), 29.48 (CH.sub.2), 29.40 (CH.sub.2), 29.33 (CH.sub.2), 29.29 (CH.sub.2), 29.04 (CH.sub.2), 28.72 (CH.sub.2), 26.40 (CH.sub.2), 25.94 (CH.sub.2), 11.91 (CH.sub.3). LCMS 80.5%, @ 225nm (+/50), MH+547.0

Synthesis of Example 9

##STR00031##

[0360] Step 1: synthesis of 4-{[10-(4,5-dimethoxy-2-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)decyl]oxy}benzonitrile, Idebenone (1g, 2.9mol) and 4-hydroxybenzonitrile (352 mg, 2.9 mol, 1 eq) were dissolved in THF (20 ml). After dissolution TPP (852 mg, 3.3 mmol, 1.1 eq) was added, followed by DEAD (2.2 mol sol in toluene, 1.48 ml, 0.00325 mol, 1.1 eq) was added dropwise and the reaction mixture was stirred overnight. The solution was concentrated before purification with ethyl acetate: hexane system to give the product as a red viscous oil which solidified on standing to give the intermediate product 3 as an orange solid, 0.831g, 64% yield. Rf=0.22 (20% ethyl acetate: hexane). .sup.1H NMR (400 MHZ, CDCl.sub.3) 7.61-7.51 (m, 2H), 6.97-6.89 (m, 2H), 3.99 (d, J=1.2 Hz, 9H), 2.49-2.41 (m, 2H), 2.009 (s, 3H), 1.84-1.74 (m, 2H), 1.42-1.23 (m, 14H). LCMS 75%@225nm (+/50), MH+ 440, 441.

[0361] Step 2: Synthesis of 4-{[10-(4,5-dimethoxy-2-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)decyl]oxy}benzene-1-carbothioamide (compound 9). Magnesium chloride hexahydrate (189 mg, 1.8mmol, 1 eq) and sodium hydrogen sulfide (173 mg, 1.8 mmol, 2 eq) were stirred in THF (5 ml) and to it added intermediate 3 (408 mg, 0.93mmol, 1eq in 5ml THF). This was heated to 30 C., after 1 hour TLC showed complete reaction, and the reaction was concentrated to an oil. The organic phase was then taken up in DCM and eluted through a plug of silica and the relevant spots concentrated. This was then eluted down a Biotage system using 0 to 25% ethyl acetate: hexane to give after concentration the product as an orange solid Example 9, 235 mg, 53% yield. Rf=0.75 (10% ethyl acetate: DCM), 0.13 (20% ethyl acetate: hexane). 1H NMR (400 MHZ, CDCl.sub.3) 7.63-7.48 (m, 2H), 7.02-6.85 (m, 2H), 5.28 (d, J=22.5 Hz, 2H), 3.99 (t, J=6.5 Hz, 2H), 3.89 (d, J=1.1 Hz, 6H), 2.64-2.49 (m, 2H), 2.15 (s, 3H), 1.79 (dt, J=14.6, 6.6 Hz, 2H), 1.38 (d, J=53.7 Hz, 14H). .sup.13C NMR (CDCl.sub.3, 101 MHZ) 162.49 (C), 140.03 (C), 129.83 (C), 136.64 (C), 136.62 (C), 133.96 (CH), 123.21 (C), 119.33 (C), 117.63 (C), 115.20 (CH), 103.64 (C), 68.45 (CH.sub.2), 60.79 (CH.sub.3), 60.73 (CH.sub.3), 29.88 (CH.sub.2), 29.51 (CH.sub.2), 29.48 (CH.sub.2), 29.29 (CH.sub.2), 28.97 (CH.sub.2), 26.34 (CH.sub.2), 25.91 (CH.sub.2), 11.15 (CH.sub.3). LCMS 95% @ 225nm (+/50), MH.sup. 472.

[0362] Example compounds 10-17 were prepared using analogous procedures to those described above for compounds 1-9.

[0363] The structures of Example compounds 1-15 are given below.

TABLE-US-00003 Ex- am- ple Structure Name 1 [00032] 10-(4,5-dimethoxy-2- methyl-3,6- dioxocyclohexa-1,4- dien-1-yl)decyl 2-[4-(3-sulfanylidene-3H-1,2- dithiol-5-yl)phenoxy]acetate 2 [00033] 4-(3-sulfanylidene-3H-1,2- dithiol-5-yl)phenyl) 10-(4,5-dimethoxy-2- methyl-2,6-dioxocyclohexa- 1,4-dien-1-yl)decanoate 3 [00034] 4-(3-oxo-3H-1,2-dithiol- 5-yl)phenyl 10- (4,5-dimethoxy-2-methyl-3,6- dioxocyclohexa-1,4-dien-1- yl)decanonate 4 [00035] 4-carbamothioylphenyl 10-(4,5- dimethoxy-2-methyl-3,6- dioxocyclohexa-1,4-dien-1- yl)decanoate 5 [00036] 4-(3-sulfanylidene-3H- 1,2-dithiol-5-yl) phenyl10-(3-methyl-1,4-dioxo- 1,4-dihydronaphthalen-2- yl)decanoate 6 [00037] 4-carbamothioylphenyl 10-(3- methyl-1,4-dioxo-1,4- dihydronaphthalen-2-yl) decanoate 7 [00038] 4-(3-oxo-3H-1,2-dithiol-5- yl)phenyl 10-(3-methyl-1,4-dioxo-1,4- dihydronaphthalen-2-yl) decanoate 8 [00039] 2,3-dimethoxy-5-methyl- 6-{10-[4- (3-sulfanylidene-3H- 1,2-dithiol-5-yl) phenoxy]decyl}cyclohexa- 2,5-diene-1,4-dione. 9 [00040] 4-{[10-(4,5-dimethoxy- 2-methyl- 3,6-dioxocyclohexa-1,4-dien-1- yl)decyl]oxy}benzene-1- carbothioamide 10 [00041] 10-(4,5-dimethoxy-2-methyl- 3,6-dioxocyclohexa-1,4- dien-1-yl)-N-[4-(3-oxo-3H-1,2- dithiol-5- yl)phenyl]decanamide 12 [00042] N-(4-carbamothioylphenyl)- 10-(4,5- dimethoxy-2-methyl-3,6- dioxocyclohexa-1,4-dien-1- yl)decanamide 13 [00043] 10-(4,5-dimethoxy-2-methyl- 3,6- dioxocyclohex-1,4-dien-1- yl)-N-[4-(3- sulfanylidene-3H-1,2-dithiol-5- yl)phenyl]decanamide 14 [00044] 10-(3-methyl-1,4-dioxo-1,4- dihydronaphthalen-2-yl)-N- [4-(3-sulfanylidene-3H- 1,2-dithiol-5- yl)phenyl]decanamide 15 [00045] 10-(3-methyl-1,4-dioxo-1,4- dihydronaphthalen-2-yl)-N- [4-(3-oxo-3H-1,2-dithiol-5- yl)phenyl]decanamide 16 [00046] 2,3-dimethoxy-5- methyl-6-{10-[4- (3-oxo-3H-1,2-dithiol-5-yl) phenoxy]decyl}cyclohexa- 2,5-diene-1,4-dione 17 [00047] N-(4-carbamothioylphenyl)- 10-(3-methyl-1,4-dioxo-1,4- dihydronaphthalen-2- yl)decanamide

Biological Screening Methods And Results

Cell Culture

[0364] b.End3 cells were split by trypsinisation when they reached or slightly exceeded 90% of confluence (usually two times per week). The following procedure was followed: firstly, the old cell medium was removed and the cells were washed with approximately 10 ml of PBS without calcium and magnesium salts. 1 ml of trypsin-EDTA was added to cells, which were rinsed twice with it and then the trypsin solution was removed from the cell flask. Cells were incubated for 2-3 minutes at 37 C., until they detached from the flask surface and finally, fresh supplemented DMEM (4 or 5 ml) was added to the cell flask. The resulting cell-containing mixture was removed from the original flask and it was divided in different flasks with a ratio of 1/4 or 1/5 of the volume. In each flask, more DMEM was added, in order to bring the volume to 12-13 ml. Cell growth and eventual traces of contamination were checked with an inverted microscope every day or every two days.

[0365] In order to store the cells for a longer time period and to avoid having to culture them when they were not immediately needed, cells were maintained frozen in liquid nitrogen however, cells were not frozen after passage 27. In order to freeze the cells, the old cell medium was removed and after trypsinisation, they were re-suspended in a solution of cold (4 C.) supplemented DMEM containing 5% of DMSO(Cell Culture Grade) following that, they were transferred (10.sup.6 cells/ml) into cryogenic vials (1.5 ml per each vial) which were placed in a freezing container (Mr. Frosty) filled with isopropyl alcohol to achieve a cooling rate of- 1 C./minute. The freezing container was placed inside a 80 C. freezer, where cells were kept overnight and the following morning, they were transferred to a liquid nitrogen tank. When needed, frozen cells were thawed by warming at 37 C. in a water bath for 1-2 minutes and subsequently, they were gently transferred into a pre-incubated (5 minutes) T75 flask, containing 12 ml of supplemented DMEM. In order to allow the cells to attach to the flask surface, they were incubated overnight and afterwards, the old cell medium was removed and fresh DMEM was added in the flask.

Hyperglycaemia (HG)-Induced Mitochondrial Dysfunction

[0366] b.End3 cell line was used as a model for oxidative stress because in vivo the vascular endothelium was found to be a primary target for oxidative stress. Moreover, in cardiovascular diseases such as hypertension and diabetes a loss of endothelial function and cell death were observed [Poredos et al, 2021]. Also, diabetic microvascular complications (i.e. neuropathy, nephropathy, retinopathy) are mainly caused by an extended exposure of tissue to excessive glucose concentration, which results in an increase of mitochondrial ROS production, changes in mitochondrial membrane potential and loss of ATP synthesis [Vincent et al 2002; Kiritoshi et al, 2003; Manea et al 2004]

[0367] In order to induce hyperglycemia in b.End3 cells, a literature reported protocol Lorenzi et al 1985; Qu et al 2014] was followed. After trypsinisation, cells were diluted with supplemented DMEM (approximately 2 ml per flask) and consequently, cells from 3 or 4 different flasks were transferred in a 15 ml sterile conical centrifuge tube and centrifuged for 5 minutes, in order to separate the cells from the medium. Following that, the supernatant medium was removed and the cells were gently re-suspended and mixed with approximately 4 ml of fresh supplemented DMEM. 20 l of this solution was added to 40 l of trypan blue and 10 l of the final solution was loaded onto a cell counting slide dual-chamber. Trypan blue allows to distinguish between live and dead cells. Indeed it is impermeable in live cells, while it is absorbed by dead cells. The number of cells per ml was measured using an automated cell counter. Commonly, the value found was 10.sup.6 cell/ml and consequently, the cell solution was diluted, in order to obtain a concentration of 20,000 cells/well in the minimum volume necessary to fill a 96-well plate (200 l/well, 13 ml for one plate, since the two external columns and rows were loaded with cell-free medium because they are susceptible to evaporation thus, the cell medium in these wells prevented the external cell-containing wells from evaporating). Cells were cultured overnight in a humidified incubator, in order to allow them to attach to the plate surface. Afterwards, the medium was removed (a part of the plate was kept with normal glucose DMEM as a positive control) and supplemented high-glucose DMEM was added. Cells were cultured for 8 days since, in previous studies, it was determined that this is the minimum amount of time required to induce extensive mitochondrial dysfunction, such as excessive oxidant production and mitochondrial membrane hyperpolarisation Lorenzi et al 1985; Qu et al 2014]

[0368] On day 8, 10 l/well (1/20 dilution factor) of a solution of an H.sub.2S donor or of a control compound or a PBS vehicle solution (with calcium and magnesium salts and 10% DMSO) was administered to cells. The final concentration of DMSO in cells was 0.5%: higher DMSO doses may be toxic for the cells. The cells were incubated for a further two days in the presence of the compound and after ten days of hyperglycaemia exposure, the amount of mitochondrial oxidant and mitochondrial membrane hyperpolarisation were determined. Noticeably in this procedure, in order to evaluate the cytoprotective activity of the novel compounds, the drugs were added only after the mitochondrial dysfunction in cells had already occurred. The protocol described is summarised in the scheme below:

##STR00048##

[0369] On day 0, cells (20,000 cell/ml) were plated in a 96-well plate. Cells were cultured overnight at 37 C. On day 1, supplemented DMEM was removed and high-glucose supplemented DMEM was added. On day 8, an H.sub.2S donor compound (Examples 1-7), a control compound or altematively a vehicle solution was added to cells. On day 10, mitochondrial dysfunction were determined Lorenzi et al 1985; Qu et al 2014].

Mitochondrial Superoxide Production

[0370] The determination of the mitochondrial superoxide generation was performed with Mitosox Red as previously described in the literature [Mukhopadhyay, 2007]. Mitosox Red is selectively and rapidly taken up by the mitochondria, due to its positive, delocalised charge and its polarity. Inside the organelle, the dye can be easily oxidised by superoxide exhibiting a highly red fluorescence. The dye is selective towards superoxide, and it is not oxidised by nitrogen oxidative species [300].

[0371] Oxidation reaction of Mitosox Red by superoxide The oxidised form of Mitosox Red binds to DNA and produces fluorescence [Mukhopadhyay, 2007]:

##STR00049##

[0372] The Mitosox Red protocol was executed as follows: after 10 days of hyperglycemia exposure and two with the test compounds, the medium was removed and endothelial cells were washed twice with 100 l/well of PBS(with calcium and magnesium salts). They were incubated at 37 C. with 50 l/well of a 5 M Mitosox Red solution (5 l from a 5 mM stock solution in DMSO, added to 5 ml of calcium/magnesium-containing PBS) for 25 minutes. Following that, the cells were washed three times with 100 l/well of PBS(with calcium/magnesium salts) and loaded with 100 l/well of reading medium (PBS with calcium/magnesium supplemented, with 10% of FBS). Following that, the oxidation of Mitosox Red (Ex/Em: 510/580 nm) was recorded kinetically on a Pherastar microplate reader at 37 C. using a ROX filter (Ex/Em: 575/610 nm) for 60 minutes. Mitochondrial ROS production is determined as the Vmax value of the fluorescence probe oxidation.

Results

[0373] Table 1 shows the results of this mitochondrial dysfunction screening for the comparative example AP39 and Examples 1-4.

TABLE-US-00004 TABLE 1 H.sub.2S donor biologically relevant properties. IC.sub.50 IC.sub.25 H-bond (Mitochondrial (Mitochondrial donor/ Compound potential ROS level TC.sub.50 H-bond name restore) (nM) restore) (nM) (M) cLogP* acceptor AP39 4 10 8 9.48 0 2 Example 1 0.4 6.5 147 6.23 0 7 Example 2 6 10 130 6.10 0 6 Example 3 5 10 76 5.40 0 7 Example 4 1 5 100 5.59 2 7

[0374] Table 1 shows that the compounds of the invention are as potent, and sometimes more potent, than AP39, and have the advantage of being less toxic. They have a lower clogP which is a predictor of increased aqueous solubility allowing them to be more easily formulated into a medicament. In contrast, AP39 suffers from high hygroscopicity which makes formulation much more difficult than the compounds of the invention.

Worm screening

Strains and Culture Conditions

[0375] C. elegans strains were cultured at 20 C. on Petri dishes containing nematode growth medium (NGM) agar and a lawn of Escherichia coli OP50, unless stated otherwise. Animals for the study were age-synchronized by gravity synchronization from the L1 stage and allowed to grow to the desired day of adulthood. The C. elegans strains used in this study were Bristol strain N2 (WT) provided by the Caenorhabditis Genetics Center.

Mitochondrial and Cell Death Imaging

[0376] Mitochondrial imaging was used in day 1 adults, with or without treatment, to examine the mitochondrial network. Worms were cultured on test compounds as described. Approximately 20 day 1 adults were picked into 20 L of M9 buffer on a microscope slide with a coverslip applied. Worms were imaged at 40 magnification using a Nikon Eclipse 50i microscope. The protocol used was as described by Oh and Kim. Briefly animals were assessed at day 4 and day 8 of adulthood, where the number of dead muscle cells was determined by quantifying the number of muscle cells that had lost their distinct circular nuclear GFP signal. Approximately 30 animals were picked into 20 L of M9 buffer on a microscope slide with a coverslip applied. Worms were imaged at 10 magnification by using a Nikon Eclipse 50i microscope.

Results

[0377] FIGS. 1-3 show the results obtained from the worm screening of the test compounds.

[0378] FIG. 1 shows the fragmentation of the mitochondrial network in C. elegans caused by ageing: fragmentation is inhibited by Example 2, but not by the constituent parts of the molecule, idebenone and ADTOH.

[0379] FIG. 2 shows the reduction of networked mitochondria in C. elegans caused by aging. This network is preserved by Example 2 and not by the constituent parts of the molecule idebenone and ADTOH.

[0380] FIG. 3 shows the damage caused to mitochondrial network in aging C. elegans which can be inhibited by Example 2 but not its constituent parts idebenone and ADTOH.

[0381] These results clearly show the therapeutic effect of compounds of the invention in the C. elegans model of mitochondrial aging. Thus the compounds may find utility in indications where maintenance of mitochondrial health is therapeutic. In particular C. elegans muscle aging is a well validated animal model for human sarcopenia (reviewed in [Christian and Benian, 2020]).

Improvement Cellular Bioenergetics in Senescent Primary Human Lung Fibroblasts

[0382] Human primary lung fibroblasts were isolated from healthy volunteers and cultured in DMEM supplemented with 10% fetal calf serum and 1% pen/strep and 2 nM L-glutamine, at 37 C., 5% CO.sub.2. To induce senescence, cells were seeded at a density of 50,000 cells per well in 250 l of cell culture media onto Seahorse V7 plates and treated daily with fresh cell culture media containing 100 nM H.sub.2O2 for 5 days, cells were then cultured with normal medium for a further 9 days. Senescence was confirmed by microscopy and staining for the senescence marker, senescence-associated--galactosidase (SA-gal; Cell Signalling kit #9860). Cells were then treated with compounds (0-300 nM) for a further 24 h (controls are *SIP cells without compounds) and after this time, cellular bioenergetics determined using a Seahorse XFe24 extracellular flux analyser performed as described below. * SIP-senescence-induced phenotype

Assessment of Cellular Metabolism (Seahorse)Lung Fibroblasts

[0383] For the mitochondrial stress and glycolytic stress tests (Agilent, UK)Seahorse XFe24 sensor cartridges were hydrated with Seahorse XF calibrant solution and maintained at 37 C. in a non-CO.sub.2 incubator overnight. After confirmation of cellular senescence, media was replaced with low buffered Seahorse XF medium supplemented with test example compounds (*0-300 nM) and cells incubated for 1 hr in non-CO.sub.2 incubator, at 37 C. for 1 hr. After incubation, plates were loaded onto Seahorse XFe24 Analyser and basal oxygen consumption rate (OCR) measured for 3 cycles. After basal measurements, cells were injected with the following every 3 cycles: Oligomycin (final concentration 1 M), FCCP (final concentration 1 M) and Rotenone/Antimycin A (1:1 ratio, final concentration 0.5 M). Measurements were taken every 8 minutes on a 3 minute mix, 2 minute wait, 3 minute measure cycle. Extracellular acidification rate (ECAR) and Oxygen consumption rate (OCR) were measured for 3 baseline cycles and injection strategy initiated. Following completion of all assays, media was removed and cells lysed with sodium hydroxide (100 L per well of 50 mM NaOH). Protein concentrations were quantified using the Bradford method. OCR and ECAR readings were normalised to total protein concentration in each well.

[0384] FIG. 4 shows the results for the test compound Example 17. The reduction in the loss of OCR provided by the test compound compared to the control shows that the dysfunction of the mitochondria in these senescent cells is reversed by Example 17. This demonstrates that the compounds of the present invention may provide benefits in ageing cells, in particular ageing airway muscle cells and therefore may provide benefits in the treatment of COPD.

C2C12 Cell Culture And Differentiation

Cell Culture

[0385] C2C12 myoblast media requirements and seeding densities in Tables 2 and 3, respectively. Cells were maintained sub-confluent (60-70%) in culture in growth medium. For differentiation, cells were plated according to seeding densities and volumes on Table 3, in plating medium, and incubated for 48 hrs at 37 C., 5% CO.sub.2. After 48 hrs (100% confluence) medium was replaced with differentiation medium. Medium was changed every day for 6 consecutive days. Overnight treatments were conducted in serum-deprived amino acid poor medium (Table 2). All experiments were conducted in serum-deprived medium.

TABLE-US-00005 TABLE 1 Media composition for C2C12 myoblast culture and differentiation Medium Components Growth medium High glucose (25 mM) DMEM supplemented with 10% FBS, 4% (v/v) L-glutamine, 2% penicillin/streptomycin (pen/strep) Plating medium Glucose-free DMEM with 4% L-glutamine supplemented with 5.5 mM glucose, 10% FBS and 2% pen/strep Differentiation medium Glucose-free DMEM with 4% L-glutamine supplemented with 5.5 mM glucose, 2% horse serum and 2% pen/strep Amino acid-deprived medium Earl's balanced salt solution 1 x, supplemented with sodium bicarbonate (EBSS) (2.2 g/L) and 0.34 mM AA solution. Seahorse XF medium - XF DMEM with 5.5 mM glucose, 2.5 mM sodium pyruvate, 2 mM mitochondrial stress test L-glutamine, pH 7.4

TABLE-US-00006 TABLE 2 Seeding density and volumes for C2C12 myoblast culture Culture Density per Plating Differentiation Experimental platform well/dish volume volume (mL) volume (mL) T75 flask 1 10.sup.6 10 mL 60 mm dish 2 10.sup.5 5 mL 3 mL 1.5 mL F-bottom 96 5 10.sup.3 200 L 200 L 50-200 L well plate Seahorse 3 10.sup.3 200 L 200 L 180 L XFe96 microplate

Assessment of Cellular Metabolism (Seahorse)Murine C2C12 Skeletal Muscle Myotubes

[0386] For the mitochondrial stress tests (Agilent, UK), C2C12 myoblasts were differentiated as described above in Seahorse XFe96 microplates at 310.sup.3 cells/well. Seahorse XFe96 sensor cartridges were hydrated with Seahorse XF calibrant solution and maintained at 37 C. in a non-CO.sub.2 incubator overnight. On day 7, medium was replaced with low buffered Seahorse XF medium (Table 3) supplemented with test example compounds (*0-300 nM) and cells incubated for 1 hr in non-CO.sub.2 incubator, at 37 C. for 1 hr. After incubation, plates were loaded onto Seahorse XFe96 Analyser and basal oxygen consumption rate (OCR) measured for 4 cycles. After basal measurements, cells were injected with the following every 4 cycles:

[0387] Oligomycin (final concentration 2 M), FCCP (final concentration 1 M) and Rotenone/Antimycin A (1:1 ratio, final concentration 1 M). Measurements were taken every 6 minutes on a 3 minute mix, 3 minute measure cycle. Extracellular acidification rate (ECAR) was measured for 4 baseline cycles and injection strategy initiated. Following completion of all assays, media was removed and cells lysed with sodium hydroxide (100 L per well of 50 mM NaOH). Protein concentrations were quantified using the Bradford method. OCR and ECAR readings were normalised to total protein concentration in each well.

REFERENCES

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