Chemical synthesis of lipophilic methylene blue analogues which increase mitochondrial biogenesis and frataxin levels

As part of an ongoing program to develop potential therapeutic agents for the treatment of the neurodegenerative disease Friedreich׳s ataxia (FRDA), we have prepared a number of lipophilic methylene blue analogues. Some of these compounds significantly increase mitochondrial biogenesis and frataxin levels in cultured Friedreich’s ataxia cells [1]. This data article describes the chemical synthesis and full physicochemical characterization of the new analogues.


Synthesis of the methylene blue analogues
2.2.1. tert-Butyl 2-Cyano-10H-phenothiazine-10-carboxylate (8) A sample of 2.00 g (8.90 mmol) of 2-cyanophenothiazine was dissolved in 25 mL of anhydrous DMF. The reaction mixture was cooled to 0°C and 0.53 g (13.3 mmol) of 60% NaH was added. The dark reaction mixture was stirred at 0°C for an additional 15 min and 2.33 g (10.6 mmol) of di-tert-butyl dicarbonate was added. The reaction mixture was stirred at room temperature for 18 h and was then diluted with 60 mL of brine. The aqueous layer was extracted with three 25-mL portions of ethyl acetate. The combined organic extract was dried over anhydrous MgSO 4 and concentrated under diminished pressure. The crude product was purified on a silica gel column (20 Â 3 cm). Elution with 9:1 hexanes-ethyl acetate gave 8 as a pale yellow solid: yield 2.10 g (72%); silica gel TLC R f 0.26 (9:1 hexanes-ethyl acetate); 1

tert-Butyl 2-Formyl-10H-phenothiazine-10-carboxylate (9)
To a solution of 2.30 g (7.10 mmol) of 8 in 25 mL of anhydrous CH 2 Cl 2 was added dropwise at À78°C 8.50 mL (8.50 mmol) of 1 M DIBAL-H in toluene. The reaction mixture was stirred at À 78°C for 3 h and was then diluted with 30 mL of brine. The aqueous layer was extracted with three 30-mL portions of CH 2 Cl 2 . The combined organic extract was dried over anhydrous MgSO 4 and then concentrated under diminished pressure. The residue was purified on a silica gel column (20 Â 3 cm). Elution with 9:1 hexane-ethyl acetate afforded 9 as a yellow solid: yield 1.88 g (81%); silica gel TLC R f 0.17 (9:1 hexane-ethyl acetate); 1  A sample containing 2.30 g (5.81 mmol) of (1-butyl)triphenylphosphonium bromide was dissolved in 25 mL of anhydrous tetrahydrofuran. The cooled ( À 78°C) reaction mixture was treated dropwise with 5.81 mL (5.81 mmol) of sodium bis(trimethylsilyl) amide. The reaction mixture was stirred at 0°C for 3 h. The reaction mixture was cooled to À 78°C and 1.90 g (5.81 mmol) of 9, dissolved in 15 mL of anhydrous tetrahydrofuran, was added. The combined reaction mixture was stirred at 0°C for 18 h. The reaction mixture was extracted with two 30-mL portions of dichloromethane. The combined organic phase was washed with 20 mL of brine, dried over anhydrous Na 2 SO 4 and concentrated under diminished pressure. The residue was purified on a silica gel column (20 Â 3 cm). Elution with 4:1 hexane-dichloromethane afforded 10 as a yellow solid: yield 1.3 g (62%); silica gel TLC R f 0.66 (9:1 hexane-dichloromethane); 1  A sample containing 1.90 g (5.31 mmol) of 10 was dissolved in 20 mL of 7:3 ethanoldichloromethane and purged with argon for 20 min. To the resulting solution was added 110 mg of 10% palladium-on-carbon. The suspension was stirred at room temperature under an atmosphere of H 2 (40 psi) for 2 h. The reaction mixture was then filtered through a Celite pad. The filtrate was concentrated under diminished pressure. The residue was purified on a silica gel column (20 Â 3 cm). Elution with 4:1 hexane-dichloromethane afforded 16 as a colorless oil: yield 1.7 g (87%); silica gel TLC R f 0.62 (9:1 hexane-dichloromethane); 1

N-(7-(Dimethylamino)-2-pentyl-3H-phenothiazin-3-ylidene)-N-methylmethanaminium Iodide (1)
A sample containing 1.70 g (4.60 mmol) of 16 was dissolved in 20 mL of anhydrous dichloromethane. To the resulting solution was added dropwise 2.81 mL (36.8 mmol) of trifluoroacetic acid. The reaction mixture was stirred at room temperature for 18 h. The reaction was quenched with 20 mL of saturated sodium bicarbonate solution, extracted with two 30-mL portions of dichloromethane, dried over anhydrous Na 2 SO 4 and then concentrated under diminished pressure. The crude residue was utilized for the next step without further purification.
To a solution containing 180 mg (0.66 mmol) of the crude residue in 5 mL of dichloromethane was added 543 mg (2.14 mmol) of iodine and the reaction mixture was stirred in the dark for 15 min. To the resulting solution was added dropwise 1.70 mL (3.34 mmol) of 2 M dimethylamine in THF, and the reaction mixture was stirred at room temperature for 4 h. The greenish blue mixture was purified on a silica gel column (20 Â 3 cm). Elution with at 1:1 methanol-acetonitrile afforded 1 as a blue solid: 2.2.6. tert-Butyl (E)-2-(Undec-1-enyl)-10H-phenothiazine-10-carboxylate (11) A sample containing 792 mg (1.64 mmol) of (1-decyl)triphenylphosphonium bromide was dissolved in 12 mL of anhydrous tetrahydrofuran. The cooled ( À 78°C) reaction mixture was treated dropwise with 1.64 mL (1.64 mmol) of sodium bis(trimethylsilyl) amide. The reaction mixture was stirred at 0°C for 3 h. The reaction mixture was cooled to À 78°C and 537 mg (1.64 mmol) of 9, dissolved in 8 mL of anhydrous tetrahydrofuran, was added. The reaction mixture was stirred at 0°C for 18 h. The reaction mixture was extracted with two 15-mL portions of dichloromethane. The organic phase was washed with 15 mL of brine, dried over anhydrous Na 2 SO 4 and concentrated under diminished pressure. The residue was purified on a silica gel column (20 Â 1 cm). Elution with 4:1 hexane-dichloromethane afforded 11 as a yellow solid: yield 145 mg (20%); silica gel TLC R f 0.68 (1:1 hexane-dichloromethane); 1

tert-Butyl 2-Undecyl-10H-phenothiazine-10-carboxylate (17)
A sample containing 850 mg (1.88 mmol) of 11 was dissolved in 10 mL of 7:3 ethanoldichloromethane and purged with argon for 20 min. To the resulting solution was added 40 mg of 10% palladium-on-carbon. The suspension was stirred at room temperature under an atmosphere of H 2 (40 psi) for 2 h. The reaction mixture was then filtered through a Celite pad. The filtrate was concentrated under diminished pressure. The residue was purified on a silica gel column (20 Â 1 cm).

N-(7-(Dimethylamino)-3H-phenothiazin-3-ylidene-2-undecyl)-N-methyl methanaminium Iodide (2)
A sample containing 820 mg (1.81 mmol) of 17 was dissolved in 12 mL of anhydrous dichloromethane. To the resulting solution was added dropwise 1.10 mL (14.5 mmol) of trifluoroacetic acid. The reaction mixture was stirred at room temperature for 18 h. The reaction was quenched with 10 mL of saturated sodium bicarbonate solution, extracted with two 20-mL portions of dichloromethane, dried over anhydrous Na 2 SO 4 and then concentrated under diminished pressure. The crude residue was utilized for the next step without further purification.
To a solution containing 1.40 g (4.00 mmol) of the crude residue in 20 mL of dichloromethane was added 3.20 g (12.9 mmol) of iodine and the reaction mixture was stirred in the dark for 15 min. To the resulting solution was added dropwise 10.1 mL (20.3 mmol) of 2 M dimethylamine in THF and the reaction mixture was stirred at room temperature for 4 h. The greenish blue mixture was purified on a silica gel column (20 Â 3 cm). Elution with methanol afforded 2 as a blue-green solid: yield 102 mg (25%); silica gel TLC R f 0.07 (methanol); 1  ultraviolet/visible spectrum λ max 670 nm (CH 2 Cl 2 ), λ max 665 nm (MeOH).

tert-Butyl (E)-2-(Tridec-1-enyl)-10H-phenothiazine-10-carboxylate (12)
A sample containing 2.65 g (5.19 mmol) of (1-dodecyl)triphenylphosphonium bromide was dissolved in 25 mL of anhydrous tetrahydrofuran. The cooled ( À 78°C) reaction mixture was treated dropwise with 5.19 mL (5.19 mmol) of sodium bis(trimethylsilyl) amide. The reaction mixture was stirred at 0°C for 3 h. The reaction mixture was cooled to À 78°C and 1.70 g (5.19 mmol) of 9, dissolved in 15 mL of anhydrous tetrahydrofuran was added. The reaction mixture was stirred at 0°C for 18 h. The product was extracted with two 30-mL portions of dichloromethane. The organic phase was washed with 20 mL of brine, dried over anhydrous Na 2 SO 4 and concentrated under diminished pressure. The residue was purified on a silica gel column (20 Â 3 cm). Elution with 4:1 hexanedichloromethane afforded 12 as a yellow solid: yield 1.  2.2.11. N-(7-(Dimethylamino)-3H-phenothiazin-3-ylidene-2-tridecyl)-N-methylmethanaminium Iodide (3) A sample containing 1.50 g (3.11 mmol) of 18 was dissolved in 20 mL of anhydrous dichloromethane. To the resulting solution was added dropwise 1.90 mL (24.9 mmol) of trifluoroacetic acid. The reaction mixture was stirred at room temperature for 18 h. The reaction was then quenched with 20 mL of saturated sodium bicarbonate solution, extracted with two 30-mL portions of dichloromethane, dried over anhydrous Na 2 SO 4 and then concentrated under diminished pressure. The crude residue was utilized for the next step without further purification.
To a solution containing 1.40 g (3.67 mmol) of the crude residue in 20 mL of dichloromethane was added 3.00 g (11.7 mmol) of iodine and the reaction mixture was stirred in the dark for 15 min. To the resulting solution was added dropwise 9.20 mL (18.3 mmol) of 2 M dimethylamine in THF and the reaction mixture was stirred at room temperature for 4 h. The greenish blue mixture was purified on a silica gel column (20 Â 3 cm). Elution with 1:1 ethyl acetate-methanol afforded 3 as a blue solid: yield 934 mg (43%); silica gel TLC R f 0.03 (methanol); 1

tert-Butyl-2-pentadecyl-10H-phenothiazine-10-carboxylate (19)
A sample containing 2.00 g (3.94 mmol) of 13 was dissolved in 22 mL of 7:3 ethanoldichloromethane and purged with argon for 20 min. To the resulting solution was added 80 mg of 10% of palladium-on-carbon. The suspension was stirred at room temperature under an atmosphere of H 2 (40 psi) for 2 h. The reaction mixture was filtered through a Celite pad. The filtrate was then concentrated under diminished pressure. The residue was purified on a silica gel column (20 Â 3 cm). Elution with 4:1 hexane-dichloromethane afforded 19 as a colorless oil: yield 1.91 g (97%); silica gel

N-(7-(Dimethylamino)-2-pentadecyl-3H-phenothiazin-3-ylidene)-N-methylmethanaminium Iodide (4)
A sample containing 1.89 g (3.71 mmol) of 19 was dissolved in 25 mL of anhydrous dichloromethane. To the resulting solution was added dropwise 2.30 mL (24.7 mmol) of trifluoroacetic acid. The reaction mixture was stirred at room temperature for 18 h. The reaction was then quenched with 20 mL of saturated sodium bicarbonate solution, extracted with two 30-mL portions of dichloromethane, dried over anhydrous Na 2 SO 4 and then concentrated under diminished pressure. The crude residue was utilized for the next step without further purification.

N-(7-(Dimethylamino)-2-hexadecyl-3H-phenothiazin-3-ylidene)-N-methylmethanaminium Iodide (5)
A sample containing 1.23 g (2.35 mmol) of 20 was dissolved in 20 mL of anhydrous dichloromethane. To the resulting solution was added dropwise 1.43 mL (18.8 mmol) of trifluoroacetic acid. The reaction mixture was stirred at room temperature for 18 h. The reaction was then quenched with 20 mL of saturated sodium bicarbonate solution, extracted with two 30-mL portions of dichloromethane, dried over anhydrous Na 2 SO 4 and then concentrated under diminished pressure. The crude residue was utilized for the next step without further purification.
To a solution containing 1.15 g (2.71 mmol) of the crude residue in 20 mL of dichloromethane was added 2.20 g (8.67 mmol) of iodine and the reaction mixture was stirred in the dark for 15 min. To the resulting solution was added dropwise 6.70 mL (13.5 mmol) of 2 M dimethylamine in THF and the reaction mixture was stirred at room temperature for 4 h. The greenish blue mixture was purified on a silica gel column (20 Â 3 cm). Elution with 1:1 ethyl acetate-methanol afforded 5 as a blue solid: yield 0.36 g (24%); silica gel TLC R f 0.06 (methanol); 1  A sample containing 174 mg (0.30 mmol) of (1-hexadecyl)triphenylphosphonium bromide was dissolved in 10 mL of anhydrous tetrahydrofuran. The cooled ( À78°C) reaction mixture was treated dropwise with 0.30 mL (0.30 mmol) of sodium bis(trimethylsilyl) amide. The reaction mixture was stirred at 0°C for 3 h. The mixture was cooled to À 78°C and 100 mg (0.30 mmol) of 9, dissolved in 7 mL of anhydrous tetrahydrofuran was added. The reaction mixture was stirred at 0°C for 18 h. The product was extracted with two 10-mL portions of dichloromethane. The organic phase was washed with 10 mL of brine, dried over anhydrous Na 2 SO 4 and concentrated under diminished pressure. The residue was purified on a silica gel column (20 Â 1 cm). Elution with 4:1 hexane-dichloromethane afforded 15 as a yellow solid: yield 117 mg (72%); silica gel TLC R f 0.27 (9:1 hexane-dichloromethane); 1

N-(7-(Dimethylamino)-2-heptadecyl-3H-phenothiazin-3-ylidene)-N-methylmethanaminium Iodide (6)
To a solution of 0.23 g (0.43 mmol) of 21 in 8 mL of anhydrous CH 2 Cl 2 was added dropwise 0.26 mL (3.44 mmol) of trifluoroacetic acid. The reaction mixture was stirred at room temperature for 12 h under an argon atmosphere and quenched with 50 mL of saturated NaHCO 3 solution. The aqueous layer was extracted with three 30-mL portions of CH 2 Cl 2 . The combined organic layer was dried over anhydrous MgSO 4 and then concentrated under diminished pressure. The crude residue was utilized in the next step without further purification.

4-(2-Heptadecyl-7-morpholino-3H-phenothiazin-3-ylidene)morpholin-4-ium Iodide (7)
To a solution of 0.23 g (0.43 mmol) of 21 in 8 mL of anhydrous CH 2 Cl 2 was added dropwise 0.26 mL (3.44 mmol) of trifluoroacetic acid. The reaction mixture was stirred at room temperature for 12 h under an argon atmosphere and then neutralized with 50 mL of saturated NaHCO 3 solution. The aqueous layer was extracted with three 30-mL portions of CH 2 Cl 2 . The combined organic layer was dried over anhydrous MgSO 4 and concentrated under diminished pressure. The crude product was utilized in the next step without further purification.
To a solution of 82.0 mg of the crude product in 8 mL of CH 2 Cl 2 was added 144 mg (0.57 mmol) of iodine followed by 61.0 mL (0.72 mmol) of morpholine. The reaction mixture was stirred at room temperature under an argon atmosphere for 3 h. The greenish blue mixture was purified on a silica gel column (20 Â 1 cm). Elution with at 1:1 ethyl acetate-methanol afforded 7 as a dark green solid: