5-ethyl-5-phenyl-1,3-diazinane-2,4,6-trione

Conditions: temp:0.00; pressure:0.00; stir:

Conditions: temp:0.00; pressure:0.00; stir:

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

Procedure: A solution of phenobarbital (69.6 g, 0.3 mole) in dimethylformamide (60 ml) containing triethylamine (20.2 g, 0.2 mole) was stirred with 1-chloro-3-n-butoxy-2-propanol (33.2 g, 0.2 mole) for 6 hours at 70° C. The reaction mixture was then poured into 3% sulphuric acid (500 ml) and the precipitated product was extracted with toluene (200 ml) at 70° C. The toluene layer was separated, washed with 3% aqueous sulphuric acid (200 ml) and left overnight at 4° C. The precipitated phenobarbital was filtered off and filtrate extracted with 5% aqueous sodium carbonate (5×100 ml). The organic layer was then extracted with 1M aqueous sodium hydroxide (2×200 ml), the aqueous layers collected, washed with toluene (100 ml) and the pH was adjusted to 3 with sulphuric acid. The precipitated product was extracted with toluene (150 ml), the organic layer was separated, dried and evaporated under reduced pressure to give the title compound (38.5 g, 53%); purity by HPLC 96.5% . the precipitated product was extracted with toluene (200 ml) at 70° C The toluene layer was separated washed with 3% aqueous sulphuric acid (200 ml) The precipitated phenobarbital was filtered off filtrate extracted with 5% aqueous sodium carbonate (5×100 ml) The organic layer was then extracted with 1M aqueous sodium hydroxide (2×200 ml) the aqueous layers collected washed with toluene (100 ml) The precipitated product was extracted with toluene (150 ml) the organic layer was separated dried evaporated under reduced pressure

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

Procedure: A solution of phenobarbital (69.6 g, 0.3 mole) in dimethylformamide (60 ml) containing triethylamine (20.2 g, 0.2 mole) was stirred with 1-chloro-3-n-butoxy-2-propanol (33.2 g, 0.2 mole) for 6 hours at 70° C. The reaction mixture was then poured into 3% sulphuric acid (500 ml) and the precipitated product was extracted with toluene (200 ml) at 70° C. The toluene layer was separated, washed with 3% aqueous sulphuric acid (200 ml) and left overnight at 4° C. The precipitated phenobarbital was filtered off and filtrate extracted with 5% aqueous sodium carbonate (5×100 ml). The organic layer was then extracted with 1M aqueous sodium hydroxide (2×200 ml), the aqueous layers collected, washed with toluene (100 ml) and the pH was adjusted to 3 with sulphuric acid. The precipitated product was extracted with toluene (150 ml), the organic layer was separated, dried and evaporated under reduced pressure to give the title compound (38.5 g, 53%); purity by HPLC 96.5% . the precipitated product was extracted with toluene (200 ml) at 70° C The toluene layer was separated washed with 3% aqueous sulphuric acid (200 ml) The precipitated phenobarbital was filtered off filtrate extracted with 5% aqueous sodium carbonate (5×100 ml) The organic layer was then extracted with 1M aqueous sodium hydroxide (2×200 ml) the aqueous layers collected washed with toluene (100 ml) The precipitated product was extracted with toluene (150 ml) the organic layer was separated dried evaporated under reduced pressure

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

Procedure: A solution of phenobarbital (69.6 g, 0.3 mole) in ethanol (150 ml) containing triethylamine (2.02 g, 0.02 mole) was stirred with butyl glycidyl ether (26.0 g, 0.2 mole) (available from Merck & Co.) for 6 hours at 70° C. The reaction mixture was then poured into water (500 ml) and the precipitated product was extracted with toluene (200 ml) at 70° C. The toluene layer was separated, washed with 3% aqueous sulphuric acid (200 ml) and left overnight at 4° C. The precipitated phenobarbital was filtered off and the filtrate extracted with 5% aqueous sodium carbonate (5×100 ml). The organic layer was then extracted with 1M aqueous sodium hydroxide (2×200 ml), the aqueous layers collected, washed with toluene (100 ml) and the pH was adjusted to 3 with sulphuric acid. The precipitated product was extracted with toluene (150 ml), the organic layer was separated, dried and evaporated under reduced pressure to give the title compound (40.1 g, 55%); purity by HPLC 99% m.p. 92° C. the precipitated product was extracted with toluene (200 ml) at 70° C The toluene layer was separated washed with 3% aqueous sulphuric acid (200 ml) The precipitated phenobarbital was filtered off the filtrate extracted with 5% aqueous sodium carbonate (5×100 ml) The organic layer was then extracted with 1M aqueous sodium hydroxide (2×200 ml) the aqueous layers collected washed with toluene (100 ml) The precipitated product was extracted with toluene (150 ml) the organic layer was separated dried evaporated under reduced pressure

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

Procedure: A solution of phenobarbital (69.6 g, 0.3 mole) in ethanol (150 ml) containing triethylamine (2.02 g, 0.02 mole) was stirred with butyl glycidyl ether (26.0 g, 0.2 mole) (available from Merck & Co.) for 6 hours at 70° C. The reaction mixture was then poured into water (500 ml) and the precipitated product was extracted with toluene (200 ml) at 70° C. The toluene layer was separated, washed with 3% aqueous sulphuric acid (200 ml) and left overnight at 4° C. The precipitated phenobarbital was filtered off and the filtrate extracted with 5% aqueous sodium carbonate (5×100 ml). The organic layer was then extracted with 1M aqueous sodium hydroxide (2×200 ml), the aqueous layers collected, washed with toluene (100 ml) and the pH was adjusted to 3 with sulphuric acid. The precipitated product was extracted with toluene (150 ml), the organic layer was separated, dried and evaporated under reduced pressure to give the title compound (40.1 g, 55%); purity by HPLC 99% m.p. 92° C. the precipitated product was extracted with toluene (200 ml) at 70° C The toluene layer was separated washed with 3% aqueous sulphuric acid (200 ml) The precipitated phenobarbital was filtered off the filtrate extracted with 5% aqueous sodium carbonate (5×100 ml) The organic layer was then extracted with 1M aqueous sodium hydroxide (2×200 ml) the aqueous layers collected washed with toluene (100 ml) The precipitated product was extracted with toluene (150 ml) the organic layer was separated dried evaporated under reduced pressure

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

Procedure: Ten percent (w/v) latex microparticles (number of carboxy groups approx. 0.21 mmol/g latex, mean microparticle diameter 0.2 μm, Seradyne Inc., Indianapolis, Ind.) were diluted to one percent (w/v) concentration with 10 mM 2-morpholino-ethanesulfonic acid (MES), pH 5.3 containing 0.09% (w/v) ascorbic acid sodium salt. The desired volume of 1% microparticles was measured out and the microparticles activated by the addition of N-hydroxysulfosuccinimide (sulfo-NHS) followed by the addition of N-ethyl-N′-(3-dimethyl-aminopropyl)carbodiimide hydrochloride (EDC.HCl). Both sulfo-NHS and EDC were added at a ratio of 10 moles of each reagent per mole of carboxylates present on the surface of the microparticles. After stirring for about 1 hour at room temperature, the microparticles were washed, concentrated and resuspended to a concentration of 2% by exchange of buffers into 50 mM 3-morpholinopropanesulfonic acid (MOPS), pH 5.7, containing 0.09% (w/v) of ascorbic acid sodium salt, in a hollow-fiber system. A solution of phenobarbital monoclonal antibodies (MAK<Pheba>M-29D4-IgG) in 50 mM MOPS buffer, pH 5.7, also containing 0.09% (w/v) of ascorbic acid sodium salt and 10 g/L of BSA, was added to the resuspended microparticles such that there was 0.2 mg antibody/mL of microparticle solution. The antibody-latex mixture was stirred for about 1 hour at 23-27° C. A solution of 90 mg/mL of BSA in 50 mM MOPS, pH 5.7, containing 0.09% (w/v) of ascorbic acid sodium salt, was then added to the latex mixture (1.125 mg BSA/mg latex). After stirring for 0.5-1.5 hours at 23-27° C., a solution of 11% (w/w) 2-(2-amino-ethoxy)ethanol (AEO) in water adjusted to pH 9 with HCl, was added to the latex mixture. After stirring overnight at 40-45° C., the microparticles were washed with and exchanged into storage buffer (50 mM MOPS, pH 7.4, 0.1% (w/v) BSA, 0.09% sodium azide) to a final concentration of 1% latex microparticles (w/v) which was then stored at 2-8° C. until used. the microparticles were washed concentrated containing 0.09% (w/v) of ascorbic acid sodium salt A solution of phenobarbital monoclonal antibodies (MAK<Pheba>M-29D4-IgG) in 50 mM MOPS buffer, pH 5.7, also containing 0.09% (w/v) of ascorbic acid sodium salt and 10 g/L of BSA was added to the resuspended microparticles such that there The antibody-latex mixture was stirred for about 1 hour at 23-27° C A solution of 90 mg/mL of BSA in 50 mM MOPS, pH 5.7, containing 0.09% (w/v) of ascorbic acid sodium salt was then added to the latex mixture (1.125 mg BSA/mg latex) After stirring for 0.5-1.5 hours at 23-27° C. was added to the latex mixture After stirring overnight at 40-45° C. the microparticles were washed with and was then stored at 2-8° C.

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

Procedure: Ten percent (w/v) latex microparticles (number of carboxy groups approx. 0.21 mmol/g latex, mean microparticle diameter 0.2 μm, Seradyne Inc., Indianapolis, Ind.) were diluted to one percent (w/v) concentration with 10 mM 2-morpholino-ethanesulfonic acid (MES), pH 5.3 containing 0.09% (w/v) ascorbic acid sodium salt. The desired volume of 1% microparticles was measured out and the microparticles activated by the addition of N-hydroxysulfosuccinimide (sulfo-NHS) followed by the addition of N-ethyl-N′-(3-dimethyl-aminopropyl)carbodiimide hydrochloride (EDC.HCl). Both sulfo-NHS and EDC were added at a ratio of 10 moles of each reagent per mole of carboxylates present on the surface of the microparticles. After stirring for about 1 hour at room temperature, the microparticles were washed, concentrated and resuspended to a concentration of 2% by exchange of buffers into 50 mM 3-morpholinopropanesulfonic acid (MOPS), pH 5.7, containing 0.09% (w/v) of ascorbic acid sodium salt, in a hollow-fiber system. A solution of phenobarbital monoclonal antibodies (MAK<Pheba>M-29D4-IgG) in 50 mM MOPS buffer, pH 5.7, also containing 0.09% (w/v) of ascorbic acid sodium salt and 10 g/L of BSA, was added to the resuspended microparticles such that there was 0.2 mg antibody/mL of microparticle solution. The antibody-latex mixture was stirred for about 1 hour at 23-27° C. A solution of 90 mg/mL of BSA in 50 mM MOPS, pH 5.7, containing 0.09% (w/v) of ascorbic acid sodium salt, was then added to the latex mixture (1.125 mg BSA/mg latex). After stirring for 0.5-1.5 hours at 23-27° C., a solution of 11% (w/w) 2-(2-amino-ethoxy)ethanol (AEO) in water adjusted to pH 9 with HCl, was added to the latex mixture. After stirring overnight at 40-45° C., the microparticles were washed with and exchanged into storage buffer (50 mM MOPS, pH 7.4, 0.1% (w/v) BSA, 0.09% sodium azide) to a final concentration of 1% latex microparticles (w/v) which was then stored at 2-8° C. until used. the microparticles were washed concentrated containing 0.09% (w/v) of ascorbic acid sodium salt A solution of phenobarbital monoclonal antibodies (MAK<Pheba>M-29D4-IgG) in 50 mM MOPS buffer, pH 5.7, also containing 0.09% (w/v) of ascorbic acid sodium salt and 10 g/L of BSA was added to the resuspended microparticles such that there The antibody-latex mixture was stirred for about 1 hour at 23-27° C A solution of 90 mg/mL of BSA in 50 mM MOPS, pH 5.7, containing 0.09% (w/v) of ascorbic acid sodium salt was then added to the latex mixture (1.125 mg BSA/mg latex) After stirring for 0.5-1.5 hours at 23-27° C. was added to the latex mixture After stirring overnight at 40-45° C. the microparticles were washed with and was then stored at 2-8° C.

Conditions: temp:120.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a suspension of estrone (11, 8.11 g, 30 mmol) in acetic acid (250 mL) was heated to 120° C. and cooled to 50° C. To the reaction mixture was added 70% nitric acid (2.27 mL, 36 nunol, in an acetic acid (8.0 mL) solution) at 50° C., and stirred for 20 h at room temperature. The precipitate was filtered and washed with acetic acid, H2O, Et2O to afford 1.18 g of 76a (13% yield) mp: >250° C. To the filtrate was added H2O; it was then extracted with Et2O. The combined organic layers were washed with H2O, saturated aqueous NaCl, and then dried (Na2SO4). The desiccant was filtered and the solvent was evaporated at reduced pressure. The residue was purified by column chromatography (silica gel) using n-hexane:EtOAc (2:1, v/v) to afford 3.25 g of 76b (34% yield) mp: 178-180° C. cooled to 50° C The precipitate was filtered washed with acetic acid, H2O, Et2O

Conditions: temp:120.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a suspension of estrone (11, 8.11 g, 30 mmol) in acetic acid (250 mL) was heated to 120° C. and cooled to 50° C. To the reaction mixture was added 70% nitric acid (2.27 mL, 36 nunol, in an acetic acid (8.0 mL) solution) at 50° C., and stirred for 20 h at room temperature. The precipitate was filtered and washed with acetic acid, H2O, Et2O to afford 1.18 g of 76a (13% yield) mp: >250° C. To the filtrate was added H2O; it was then extracted with Et2O. The combined organic layers were washed with H2O, saturated aqueous NaCl, and then dried (Na2SO4). The desiccant was filtered and the solvent was evaporated at reduced pressure. The residue was purified by column chromatography (silica gel) using n-hexane:EtOAc (2:1, v/v) to afford 3.25 g of 76b (34% yield) mp: 178-180° C. cooled to 50° C The precipitate was filtered washed with acetic acid, H2O, Et2O

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

Procedure: A mixture of phenobarbital (46.5 g, 0.2 mole) and tetrabutylammonium hydroxide (500 mL, 0.2 mole of 0.4M in water) in dichloromethane (500mL) was prepared and to it was added methyl 5-bromovalerate (39.0 g, 0.2 mole). The reaction mixture was stirred vigorously overnight (20 hrs). To this mixture was added saturated sodium chloride solution (100 mL), the organic layer was separated, and the aqueous solution was washed with dichloromethane (2×100 mL). The combined organic solution was washed with saturated sodium chloride solution (100 mL), dried over anhydrous MgSO4, filtered, and the solvent removed. was prepared and to it the organic layer was separated the aqueous solution was washed with dichloromethane (2×100 mL) The combined organic solution was washed with saturated sodium chloride solution (100 mL) dried over anhydrous MgSO4 filtered the solvent removed

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

Procedure: A mixture of phenobarbital (46.5 g, 0.2 mole) and tetrabutylammonium hydroxide (500 mL, 0.2 mole of 0.4M in water) in dichloromethane (500mL) was prepared and to it was added methyl 5-bromovalerate (39.0 g, 0.2 mole). The reaction mixture was stirred vigorously overnight (20 hrs). To this mixture was added saturated sodium chloride solution (100 mL), the organic layer was separated, and the aqueous solution was washed with dichloromethane (2×100 mL). The combined organic solution was washed with saturated sodium chloride solution (100 mL), dried over anhydrous MgSO4, filtered, and the solvent removed. was prepared and to it the organic layer was separated the aqueous solution was washed with dichloromethane (2×100 mL) The combined organic solution was washed with saturated sodium chloride solution (100 mL) dried over anhydrous MgSO4 filtered the solvent removed