L-Methioninol

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A 25 mL round-bottomed flask was charged with 2-(6-fluoro-1-methyl-1H-indazol-3-yl)-5-(2-trimethylsilanyl-ethoxymethyl)-5H-pyrrolo[2,3-b]pyrazine-7-carboxylic acid (100 mg, 0.23 mmol), (S)-2-amino-4-(methylthio)butan-1-ol (32 mg, 0.24 mmol), and HATU (90 mg, 0.24 mmol). Then added acetonitrile (4 mL) followed by N,N-diisopropylethylamine (0.19 mL, 1.1 mmol). The reaction mixture was stirred at room temperature overnight then quenched with water. The mixture was extracted with ethyl acetate (3×). The organic layers were combined, dried over Na2SO4, and concentrated. The residue was purified by chromatography over SiO2 with EtOAc/heptanes (gradient: 5-50% EtOAc) to afford 105 mg (83%) of 2-(6-fluoro-1-methyl-1H-indazol-3-yl)-5-(2-trimethylsilanyl-ethoxymethyl)-5H-pyrrolo[2,3-b]pyrazine-7-carboxylic acid ((S)-1-hydroxymethyl-3-methylsulfanyl-propyl)-amide as an off-white foam. MS: [M+H]+=559. then quenched with water The mixture was extracted with ethyl acetate (3×) dried over Na2SO4 concentrated The residue was purified by chromatography over SiO2 with EtOAc/heptanes (gradient: 5-50% EtOAc)

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A 25 mL round-bottomed flask was charged with 2-(6-fluoro-1-methyl-1H-indazol-3-yl)-5-(2-trimethylsilanyl-ethoxymethyl)-5H-pyrrolo[2,3-b]pyrazine-7-carboxylic acid (100 mg, 0.23 mmol), (S)-2-amino-4-(methylthio)butan-1-ol (32 mg, 0.24 mmol), and HATU (90 mg, 0.24 mmol). Then added acetonitrile (4 mL) followed by N,N-diisopropylethylamine (0.19 mL, 1.1 mmol). The reaction mixture was stirred at room temperature overnight then quenched with water. The mixture was extracted with ethyl acetate (3×). The organic layers were combined, dried over Na2SO4, and concentrated. The residue was purified by chromatography over SiO2 with EtOAc/heptanes (gradient: 5-50% EtOAc) to afford 105 mg (83%) of 2-(6-fluoro-1-methyl-1H-indazol-3-yl)-5-(2-trimethylsilanyl-ethoxymethyl)-5H-pyrrolo[2,3-b]pyrazine-7-carboxylic acid ((S)-1-hydroxymethyl-3-methylsulfanyl-propyl)-amide as an off-white foam. MS: [M+H]+=559. then quenched with water The mixture was extracted with ethyl acetate (3×) dried over Na2SO4 concentrated The residue was purified by chromatography over SiO2 with EtOAc/heptanes (gradient: 5-50% EtOAc)

Conditions: temp:80.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: 100 mg (0.74 mmol) of L-methioninol was added to a solution of 250 mg (0.68 mmol) of 2-[4-[2-(4-methyl-1-piperazinyl)-4-thiazolyl]phenyl]-3-oxa-1-azaspiro[4.5]dec-1-en-4-one in 20 ml of dimethylformamide. After the mixture was stirred at 80° C. for 15 hours, the reaction solution was concentrated under reduced pressure, ethyl acetate was added thereto, and the mixture was successively washed with water, a saturated aqueous sodium hydrogencarbonate solution and saturated brine, followed by drying with anhydrous sodium sulfate. After the solvent was distilled off under reduced pressure, and diisopropyl ether was added to the residue to wash the crystal to obtain 319 mg (86%) of the title compound. the reaction solution was concentrated under reduced pressure, ethyl acetate was added the mixture was successively washed with water by drying with anhydrous sodium sulfate After the solvent was distilled off under reduced pressure, and diisopropyl ether was added to the residue to wash the crystal

Conditions: temp:80.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: 100 mg (0.74 mmol) of L-methioninol was added to a solution of 250 mg (0.68 mmol) of 2-[4-[2-(4-methyl-1-piperazinyl)-4-thiazolyl]phenyl]-3-oxa-1-azaspiro[4.5]dec-1-en-4-one in 20 ml of dimethylformamide. After the mixture was stirred at 80° C. for 15 hours, the reaction solution was concentrated under reduced pressure, ethyl acetate was added thereto, and the mixture was successively washed with water, a saturated aqueous sodium hydrogencarbonate solution and saturated brine, followed by drying with anhydrous sodium sulfate. After the solvent was distilled off under reduced pressure, and diisopropyl ether was added to the residue to wash the crystal to obtain 319 mg (86%) of the title compound. the reaction solution was concentrated under reduced pressure, ethyl acetate was added the mixture was successively washed with water by drying with anhydrous sodium sulfate After the solvent was distilled off under reduced pressure, and diisopropyl ether was added to the residue to wash the crystal

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: 43 mg (0.32 mmol) of L-methioninol was added to a solution of 100 mg (0.29 mmol) of 2-[4-(4-morpholinylmethyl)phenyl]-3-oxa-1-azaspiro[4.5]dec-1-en-4-one in 3 ml of N,N-dimethylformamide, the mixture was stirred overnight. Water was slowly added, and the precipitated crystal was collected by filtration. The obtained crystal was dried under reduced pressure to obtain 65 mg (49%) of the title compound. the precipitated crystal was collected by filtration The obtained crystal was dried under reduced pressure

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: 284 mg (2.1 mmol) of L-methioninol was added to a solution of 500 mg (1.75 mmol) of 2-(2-benzothienyl)-3-oxa-1-azaspiro[4.5]dec-1-en-4-one in 3 ml of N,N-dimethylformamide, and the mixture was stirred overnight. Water was slowly added, and the precipitated crystal was collected by filtration. The obtained crystal was dried under reduced pressure to obtain 722 mg (98%) of the title compound. the precipitated crystal was collected by filtration The obtained crystal was dried under reduced pressure

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: 3.1 g of Boc-MePhe-OH are dissolved in 30 ml of tetrahydrofuran, cooled to -20°, and 1.45 ml of N-ethylmorpholine are added with stirring followed by 1.45 ml of chloroformic acid iso-butyl ester and the mixture stirred for a further 5 minutes at -20°. A cold solution of 1.80 g of methioninol in 1 ml of tetrahydrofuran is added, the mixture left to stand for 2 hours at a temperature of from -5° to 0° and then stirred for 2 hours at room temperature. The reaction mixture is diluted with ca. 350 ml of acetic acid and washed repeatedly with water, 1 N citric acid, 10% KHCO3 and 30% NaCl solution. The organic phase is dried over Na2SO4 and evaporated, whereby Boc-MePhe-methioninol is obtained as an amorphous product. cooled to -20° the mixture stirred for a further 5 minutes at -20° the mixture left stirred for 2 hours at room temperature washed repeatedly with water, 1 N citric acid, 10% KHCO3 and 30% NaCl solution The organic phase is dried over Na2SO4 evaporated

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A 25 mL round-bottomed flask was charged with 2-(6-fluoro-1-methyl-1H-indazol-3-yl)-5-(2-trimethylsilanyl-ethoxymethyl)-5H-pyrrolo[2,3-b]pyrazine-7-carboxylic acid (100 mg, 0.23 mmol), (S)-2-amino-4-(methylthio)butan-1-ol (32 mg, 0.24 mmol), and HATU (90 mg, 0.24 mmol). Then added acetonitrile (4 mL) followed by N,N-diisopropylethylamine (0.19 mL, 1.1 mmol). The reaction mixture was stirred at room temperature overnight then quenched with water. The mixture was extracted with ethyl acetate (3×). The organic layers were combined, dried over Na2SO4, and concentrated. The residue was purified by chromatography over SiO2 with EtOAc/heptanes (gradient: 5-50% EtOAc) to afford 105 mg (83%) of 2-(6-fluoro-1-methyl-1H-indazol-3-yl)-5-(2-trimethylsilanyl-ethoxymethyl)-5H-pyrrolo[2,3-b]pyrazine-7-carboxylic acid ((S)-1-hydroxymethyl-3-methylsulfanyl-propyl)-amide as an off-white foam. MS: [M+H]+=559. then quenched with water The mixture was extracted with ethyl acetate (3×) dried over Na2SO4 concentrated The residue was purified by chromatography over SiO2 with EtOAc/heptanes (gradient: 5-50% EtOAc)

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Boc-methionine was esterified to hydroxymethylpolystyrene-divinylbenzene (1% crosslinked) using dicyclohexylcarbodiimide and a p-dimethylaminopyridine catalyst; amino acid analysis showed a substitution of 0.316 mmoles Met/g resin. Excess hydroxyl groups were acetylated, and the peptide was assembled on the resin in the standard fashion, using the Beckman 990 synthesizer. The hydroxyl group of tyrosine was blocked during synthesis with the bromocarbobenzoxy group. Next, 2 g of peptide resin, H-Tyr(Brz)-DAla-Phe-Met-OCH2 -resin, was placed into a 100 ml roundbottomed flask. Then, 133 mg LiBH4 (ALFA) (about 10 equiv. excess over resin substitution value for Met) dissolved in 75 ml tetrahydrofuran (THF, peroxidefree) was added. A drying tube was placed on the flask and the reaction mixture stirred at room temperature for 2 hours. Excess LiBH4 was destroyed with glacial HOAc and the mixture then filtered through a coarse sinteredglass funnel. After several washes of resin with glacial HOAc, the solvents were evaporated under reduced pressure and freeze-dried. The crude lyophilized peptide (Pauly+) and remaining Li salts were then subjected to countercurrent distribution in nBAW, 4:1:5, for 100 transfers. CCD cuts were made to maximize purity rather than yield. Some 355 mg peptide was recovered from the various cuts and all cuts contained the same major product in excess over any impurities. A particularly homogeneous cut, k=2.70, yielded 55 mg of essentially pure peptide, H-Tyr-DAla-Phe-Met-ol. (TLC: nBAW 12:3:5, silica gel, Rf =0.70, Nin+, Pauly+; HVE: 1 M HOAc, pH 2.8, 60 min at 1 KV, Elys =0.44, Nin+, Pauly+; amino acid analysis: Tyr, 0.99; Ala, 1.01; Phe, 1.01; Met, 0.0; Met-ol was not detectable on analyzer).

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Boc-methionine was esterified to hydroxymethylpolystyrene-divinylbenzene (1% crosslinked) using dicyclohexylcarbodiimide and a p-dimethylaminopyridine catalyst; amino acid analysis showed a substitution of 0.316 mmoles Met/g resin. Excess hydroxyl groups were acetylated, and the peptide was assembled on the resin in the standard fashion, using the Beckman 990 synthesizer. The hydroxyl group of tyrosine was blocked during synthesis with the bromocarbobenzoxy group. Next, 2 g of peptide resin, H-Tyr(Brz)-DAla-Phe-Met-OCH2 -resin, was placed into a 100 ml roundbottomed flask. Then, 133 mg LiBH4 (ALFA) (about 10 equiv. excess over resin substitution value for Met) dissolved in 75 ml tetrahydrofuran (THF, peroxidefree) was added. A drying tube was placed on the flask and the reaction mixture stirred at room temperature for 2 hours. Excess LiBH4 was destroyed with glacial HOAc and the mixture then filtered through a coarse sinteredglass funnel. After several washes of resin with glacial HOAc, the solvents were evaporated under reduced pressure and freeze-dried. The crude lyophilized peptide (Pauly+) and remaining Li salts were then subjected to countercurrent distribution in nBAW, 4:1:5, for 100 transfers. CCD cuts were made to maximize purity rather than yield. Some 355 mg peptide was recovered from the various cuts and all cuts contained the same major product in excess over any impurities. A particularly homogeneous cut, k=2.70, yielded 55 mg of essentially pure peptide, H-Tyr-DAla-Phe-Met-ol. (TLC: nBAW 12:3:5, silica gel, Rf =0.70, Nin+, Pauly+; HVE: 1 M HOAc, pH 2.8, 60 min at 1 KV, Elys =0.44, Nin+, Pauly+; amino acid analysis: Tyr, 0.99; Ala, 1.01; Phe, 1.01; Met, 0.0; Met-ol was not detectable on analyzer).

Conditions: temp:134.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A mixture of 2-(4-chloro-2,6-dimethyl-phenoxy)-4-chloro-6-methyl-nicotinic acid methyl ester and L-methioninol in N-methyl-2-pyrodone (NMP) was heated in a 134° C. oil bath for 3 hr. Standard work-up procedure and purification provided the title compound. 1H NMR(CDCl3) d 8.25(d,1H), 7.02(s,2H), 6.30(s,1H), 3.85(s,3H), 3.6-3.9(m,3H), 2.5-2.7(m,2H), 2.14(s,3H), 2.10(s,3H), 2.06(s,6H), 1.8-2.1 (m,2H)ppm. Standard work-up procedure and purification

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: The title compound was prepared using 5-cyano-1-(3-trifluoromethoxybenzyl)-1H-indole-2-carboxylic acid [prepared from 3-(trifluoromethoxy)benzyl bromide and ethyl 5-cyanoindole-2-carboxylate] and L-(−)-methioninol in a manner similar to that described in Example 11.