2,3-diaminophenol

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

Procedure: 2,3-Diaminophenol (2.48 g, 20 mmol) was dissolved in methylene chloride (200 mL) and the solution was cooled to ice-water bath temperature. Aqueous 5 N sodium hydroxide solution (126 mL) was added followed by 4-methoxybenzoyl chloride (8.6 mL, 63 mmol). The mixture was allowed to warm to room temperature and stirred for 18 h. The organic layer was separated and washed with dilute aqueous sodium hydroxide solution, dilute aqueous hydrochloric acid, and saturated aqueous sodium chloride solution. The organic layer was dried (sodium sulfate), filtered, and concentrated in vacuo to give 7.75 g (74%) of the title compound as a brown foam. This material was used without further purification. the solution was cooled to ice-water bath temperature The organic layer was separated washed with dilute aqueous sodium hydroxide solution, dilute aqueous hydrochloric acid, and saturated aqueous sodium chloride solution The organic layer was dried (sodium sulfate) filtered concentrated in vacuo

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

Procedure: Forty mg of Met-NT-3 having methionine at its N-terminal obtained in Reference Example 3 was dissolved in 4 ml of 3 M urea solution. To the mixture was added a solution containing 50 mM copper sulfate 0.5 ml, glyoxylic acid 0.25 g and pyridine 0.5 ml and allowed to stand at 25 ° C. for 1 hour. The reaction solution was passed through Sephadex G-25 column (25 mmID×600 mmL) equilibrated with 2.5 M urea and 50 mM phosphate buffer solution (pH 6.0) and the column was washed with the same buffer solution at 6 ml/minute of flow rate to collect the fraction of diketone derivative of Met-NT-3. To this fraction was added the same volume of 4 M acetic acid-4 M sodium acetate solution and then added 2,3-diaminophenol to give a final concentration of 40 mM, followed by vacuum and sealing with nitrogen gas, and the reaction was proceeded at 37° C. for 15 hours. The reaction solution was purified by the same method as described in Working Example 37 to obtain a purified NT-3. obtained in Reference Example 3 To the mixture was added a solution the column was washed with the same buffer solution at 6 ml/minute of flow rate to collect the fraction of diketone derivative of Met-NT-3 To this fraction was added the same volume of 4 M acetic acid-4 M sodium acetate solution

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

Procedure: Forty mg of Met-BTC having methionine at its N-terminal obtained in Reference Example 4 was dissolved in 4 ml of 3 M urea solution. To the mixture was added a solution containing 50 mM copper sulfate 0.5 ml, glyoxylic acid 0.25 g and pyridine 0.5 ml and allowed to stand at 25° C. for 1 hour. The reaction solution was passed through Sephadex G-25 column (25 mmID×600 mmL) equilibrated with 2.5 M urea and 50 mM phosphate buffer solution (pH 6.0) and the column was washed with the same buffer solution at 6 ml/minute of flow rate to collect the fraction of diketone derivative of Met-BTC. To this fraction was added the same volume of 4 M acetic acid-4 M sodium acetate solution and then added 2,3-diaminophenol to give a final concentration of 40 mM, followed by vacuum and sealing with nitrogen gas, and the reaction was proceeded at 37° C. for 15 hours. The reaction solution was purified by the same method as described in Working Example 23 to obtain a purified BTC. obtained in Reference Example 4 To the mixture was added a solution the column was washed with the same buffer solution at 6 ml/minute of flow rate to collect the fraction of diketone derivative of Met-BTC To this fraction was added the same volume of 4 M acetic acid-4 M sodium acetate solution

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

Procedure: To a solution of 3-nitro-4-aminophenol (4.6 g, 30 mmol) in methanol is added 500 mg of Pd/C. A hydrogenation reaction is performed at room temperature for 2 hours and then terminated. The Pd/C is removed by filtration to obtain 4.1 g of 3-hydroxy-o-phenylenediamine, with a yield of 95%. A hydrogenation reaction terminated The Pd/C is removed by filtration

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

Procedure: To a solution of 3-nitro-4-aminophenol (4.6 g, 30 mmol) in methanol is added 500 mg of Pd/C. A hydrogenation reaction is performed at room temperature for 2 hours and then terminated. The Pd/C is removed by filtration to obtain 4.1 g of 3-hydroxy-o-phenylenediamine, with a yield of 95%. A hydrogenation reaction terminated The Pd/C is removed by filtration

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

Procedure: Forty mg of Met-NT-3 having methionine at its N-terminal obtained in Reference Example 3 was dissolved in 4 ml of 3 M urea solution. To the mixture was added a solution containing 50 mM copper sulfate 0.5 ml, glyoxylic acid 0.25 g and pyridine 0.5 ml and allowed to stand at 25° C. for 1 hour. The reaction solution was passed through Sephadex G-25 column (25 mmID×600 mmL) equilibrated with 2.5 M urea and 50 mM phosphate buffer solution (pH 6.0) and the column was washed with the same buffer solution at 6 ml/minute of flow rate to collect the fraction of diketone derivative of Met-NT-3. To this fraction was added the same volume of 4 M acetic acid-4 M sodium acetate solution and then added 2,3-diaminophenol to give a final concentration of 40 mM, followed by vacuum and sealing with nitrogen gas, and the reaction was proceeded at 37° C. for 15 hours. The reaction solution was purified by the same method as described in Working Example 37 to obtain a purified NT-3. obtained in Reference Example 3 To the mixture was added a solution the column was washed with the same buffer solution at 6 ml/minute of flow rate to collect the fraction of diketone derivative of Met-NT-3 To this fraction was added the same volume of 4 M acetic acid-4 M sodium acetate solution

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

Procedure: Forty mg of Met-NT-3 having methionine at its N-terminal obtained in Reference Example 3 was dissolved in 4 ml of 3 M urea solution. To the mixture was added a solution containing 50 mM copper sulfate 0.5 ml, glyoxylic acid 0.25 g and pyridine 0.5 ml and allowed to stand at 25° C. for 1 hour. The reaction solution was passed through Sephadex G-25 column (25 mmID×600 mmL) equilibrated with 2.5 M urea and 50 mM phosphate buffer solution (pH 6.0) and the column was washed with the same buffer solution at 6 ml/minute of flow rate to collect the fraction of diketone derivative of Met-NT-3. To this fraction was added the same volume of 4 M acetic acid-4 M sodium acetate solution and then added 2,3-diaminophenol to give a final concentration of 40 mM, followed by vacuum and sealing with nitrogen gas, and the reaction was proceeded at 37° C. for 15 hours. The reaction solution was purified by the same method as described in Working Example 37 to obtain a purified NT-3. obtained in Reference Example 3 To the mixture was added a solution the column was washed with the same buffer solution at 6 ml/minute of flow rate to collect the fraction of diketone derivative of Met-NT-3 To this fraction was added the same volume of 4 M acetic acid-4 M sodium acetate solution

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

Procedure: Forty mg of Met-BTC having methionine at its N-terminal obtained in Reference Example 4 was dissolved in 4 ml of 3 M urea solution. To the mixture was added a solution containing 50 mM copper sulfate 0.5 ml, glyoxylic acid 0.25 g and pyridine 0.5 ml and allowed to stand at 25° C. for 1 hour. The reaction solution was passed through Sephadex G-25 column (25 mmID×600 mmL) equilibrated with 2.5 M urea and 50 mM phosphate buffer solution (pH 6.0) and the column was washed with the same buffer solution at 6 ml/minute of flow rate to collect the fraction of diketone derivative of Met-BTC. To this fraction was added the same volume of 4 M acetic acid-4 M sodium acetate solution and then added 2,3-diaminophenol to give a final concentration of 40 mM, followed by vacuum and sealing with nitrogen gas, and the reaction was proceeded at 37° C. for 15 hours. The reaction solution was purified by the same method as described in Working Example 23 to obtain a purified BTC. obtained in Reference Example 4 To the mixture was added a solution the column was washed with the same buffer solution at 6 ml/minute of flow rate to collect the fraction of diketone derivative of Met-BTC To this fraction was added the same volume of 4 M acetic acid-4 M sodium acetate solution

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

Procedure: To a solution of 40 ml 4M CHl 1.1 g (0.0087 mol) of 2,3-diaminophenol and 2 g (0.026 mol) of glycolic acid were added and the solution was heated under reflux for 20 h. Upon cooling the reaction mixture was alkalized with 10M NaOH to pH 8.5. The volatiles were removed under reduced pressure and the residual oil was suspended in methanol. The suspension was filtered and evaporated to dryness in vacuo. Chromatography on silica gel and elution with methylene chloride:methanol (10:2) gave 0.78 g of the title compound. the solution was heated under reflux for 20 h Upon cooling the reaction mixture The volatiles were removed under reduced pressure The suspension was filtered evaporated to dryness in vacuo Chromatography on silica gel and elution with methylene chloride:methanol (10:2)

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

Procedure: 2,3-dinitrophenol (80%) 25 g (0.11 mol) was dissolved in 700 ml ethanol and 0.5 g Pd/C was added. The mixture was hydrogenated at room temperature until the uptake of hydrogen ceased (4 h). The solution was filtered (celite) in N2 -atmosphere and evaporated to dryness in vacuo to give the title compound as an unstable oil (18 g), which was used immediately for the next step. (4 h) The solution was filtered (celite) in N2 evaporated to dryness in vacuo

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

Procedure: 1.0 equivalent of 2,6-dichlorophenyl isothiocyanate was treated with 1.0 equivalent of 2,3-diaminophenol and heated to reflux for 1 h. After cooling to RT, the precipitate was filtered off with suction, washed with ether, and dried. The desired thiourea was obtained in a yield of 61%. M.p.: 202-204° C. heated to reflux for 1 h the precipitate was filtered off with suction washed with ether dried

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

Procedure: 1.0 equivalent of 2,6-dichlorophenyl isothiocyanate was treated with 1.0 equivalent of 2,3-diaminophenol and heated to reflux for 1 h. After cooling to RT, the precipitate was filtered off with suction, washed with ether, and dried. The desired thiourea was obtained in a yield of 61%. M.p.: 202-204° C. heated to reflux for 1 h the precipitate was filtered off with suction washed with ether dried