(2S)-3-hydroxy-2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoic acid

Conditions: temp:0.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a solution of (2S)-3-hydroxy-2-t-butoxycarbonylaminopropanoic acid (10.11 g) in dimethylformamide (200 ml, abbreviated as DMF), sodium hydride (60%, 3.95 g) was added under cooling with ice. The mixture was stirred for 30 minutes at 0° C. To the reaction mixture, (bromomethyl)cyclohexane (9.0 ml) was added at a dropwise under cooling with ice. Tetra-n-butylammonium iodide (910 mg) was added thereto. The mixture was stirred for 23 hours at room temperature. In addition, to the reaction mixture, (bromomethyl)cyclohexane (2.1 ml) was added at a dropwise. The mixture was stirred for 4 hours. Again, (bromomethyl)cyclohexane (2.1 ml) was added at a dropwise thereto. The mixture was stirred for 25 hours at room temperature. After the concentration of reaction mixture, the residue was diluted with 1N—HCl and extracted with ethyl acetate. The extract solution was washed by water and saturated solution of sodium chloride successively, dried over anhydrous sodium sulfate and concentrated. The residue was purified with column chromatography on silica gel (chloroform:methanol=97:3) to obtain the title compound (2.52 g) having the following physical data. under cooling with ice under cooling with ice The mixture was stirred for 23 hours at room temperature The mixture was stirred for 4 hours The mixture was stirred for 25 hours at room temperature After the concentration of reaction mixture extracted with ethyl acetate The extract solution was washed by water and saturated solution of sodium chloride successively dried over anhydrous sodium sulfate concentrated The residue was purified with column chromatography on silica gel (chloroform:methanol=97:3)

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

Procedure: In a 100 mL round bottom flask, N-Boc-L-serine (1.0 mmol), coupling reagent (520 mg of PBOP or 380 mg of HATU, 1 mmol) and N,N-diisopropyl-ethyl-amine (650 mg, 5 mmol) were mixed in CH2Cl2 (10 mL). The reaction mixture was stirred at room temperature for 30 minutes. S-trans, trans-farnesyl-L-cysteine (325 mg, 1 mmol) was added to the reaction mixture. The reaction mixture was stirred at room temperature overnight and the CH2Cl2 was removed by rotary evaporation. The remaining residue was dissolved in ethyl acetate (50 mL). The resulting organic solution was washed with an NH4Cl saturated solution (50 mL), dried over Na2SO4, and concentrated to afford a crude mixture. The crude mixture was purified by preparative HPLC (51 mg, 10%) to yield Compound N-55. 1H-NMR (500 MHz, CD3OD): δ 1.47 (s, 9H), 1.62 (bs, 6H), 1.69 (s, 3H), 1.71 (s, 3H), 1.99 (t, J=7 Hz, 2H), 2.05-2.22 (m, 6H), 2.81 (dd, J=7.5, 14 Hz, 1H), 3.01 (dd, J=4, 14.5 Hz, 1H), 3.17 (dd, J=7, 13 Hz, 1H), 3.27 (dd, J=8, 12.5 Hz, 1H), 3.33 (bs, 2H), 3.74-3.80 (m, 2H), 4.22 (t, J=5 Hz, 1H), 4.63 (dd, J=5, 7.5 Hz, 1H), 5.11 (m, 2H), 5.23 (t, J=7.5 Hz, 1H). 13C-NMR (125 MHz, CDCl3): δ 16.2, 16.3, 17.8, 26.0, 27.4, 27.8, 28.7, 30.4, 33.6, 40.8, 40.9, 53.3, 58.0, 59.6, 63.4, 80.9, 121.6, 125.2, 125.5, 132.1, 136.3, 140.6, 157.8, 173.0, 173.8; ES-MS: mass calcd for Chemical Formula: C26H44N2O6S 512.7. Found (M+Na) m/z 535.4. The reaction mixture was stirred at room temperature overnight the CH2Cl2 was removed by rotary evaporation The remaining residue was dissolved in ethyl acetate (50 mL) The resulting organic solution was washed with an NH4Cl saturated solution (50 mL) dried over Na2SO4 concentrated to afford a crude mixture The crude mixture was purified by preparative HPLC (51 mg, 10%)

Conditions: temp:0.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a mixture of 4.10 g (20 mmol) of N-Boc-L-serine, 2.97 g (22 mmol) of HOBT, 2.42 mL (22 mmol) of NMM, and 2.40 mL (22 mmol) of benzylamine in 50 mL of anhydrous DMF at 0° C. was added 4.21 g of WSC. The mixture was stirred at 0° C. for 1 h and at room temperature for 3 h. This was then taken up in 300 mL of EtOAc and washed with 100 mL of H2O (2×) and brine (1×). The combined organic layers were dried over MgSO4 and concentrated. After drying under vacuum overnight, the residue was dissolved in 20 mL of CH2Cl2 and treated with 20 mL of CF3CO2H at 0-10° C. for ca. 7 h. The solvent and CF3CO2H were removed under vacuum. The trace CF3CO2H was removed azeotropically with CHCl3 at the end. The residue was purified by preparative HPLC, eluting with 8% (10% conc. NH4OH in MeOH)--CH2Cl2 to afford 3.57 g (92%) of the serinamide: mp 97-98° C.; 1H NMR (CDCl3) δ7.82 (br s, 1H), 7.26 (m, 5H), 4.42 (ddd, J=16.7, 15.0, 6.0 Hz, 2H), 3.85 (dd, J=10.8, 5.2 Hz, 1H), 3. 70 (dd, J=10. 8, 5.3 Hz, 1H), 3.44 (m, 1H), 2.22 (br s, 3H); FDMS 195 (M+1). Anal. (C10H14N2O2) C, H, N. washed with 100 mL of H2O (2×) and brine (1×) The combined organic layers were dried over MgSO4 concentrated After drying under vacuum overnight the residue was dissolved in 20 mL of CH2Cl2 treated with 20 mL of CF3CO2H at 0-10° C. for ca. 7 h The solvent and CF3CO2H were removed under vacuum The trace CF3CO2H was removed azeotropically with CHCl3 at the end The residue was purified by preparative HPLC eluting with 8% (10% conc. NH4OH in MeOH)

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

Procedure: Briefly, 1.1 mmol N-BOC-L-Serine (Fluka) is dissolved in 2 ml methylene chloride. Then 1.1 mmol 7-Amino-4-methylcoumarin and 1.2 mmol hydroxybenztriazole are added with stirring. Over a period of 30 minutes, a solution of 1.3 mmol dicyclohexyl carbodiimide in 1.4 ml methylene chloride is added dropwise. The reaction is stirred at room temperature for 20 hours, during which time a precipitate of dicyclohexylurea forms. The precipitate is removed by filtration and washed with methylene chloride. The combined solutions (filtrates) are evaporated under reduced pressure and the residue chromatographed over silica gel with methylene chloride--ethylacetate as the eluent yielding 2-((tert-butoxy)carbonylamino)-3-hydroxy-N-(4-methyl-2-oxo(2H-chromen-7-yl))propanamide. The precipitate is removed by filtration washed with methylene chloride The combined solutions (filtrates) are evaporated under reduced pressure the residue chromatographed over silica gel with methylene chloride

Conditions: temp:0.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a solution of 61 mg of methyl (4R, 7S)-7-amino-12,14-bis(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylate and 21 mg N-(tert-butoxycarbonyl)-L-serine in 2 ml of acetonitrile, cooled to 0° C., were added 20 mg of N-(dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride. The mixture was stirred at 0° C. for 4 hours, then diluted with 30 ml of ethyl acetate, and washed successively with 0.5N hydrochloric acid, water, 5% sodium bicarbonate solution, and with brine. The organic layer was dried over sodium sulfate and evaporated in vacuo. The residue was dissolved in 2 ml of methanol, 20 mg of ammonium fluoride were added, and the mixture was stirred for 30 minutes at room temperature. The mixture was diluted with 40 ml of ethyl acetate and washed with 30 ml of water. The organic layer was dried over sodium sulfate, and the solvent was evaporated in vacuo. The solid residue was triturated with 2 ml of ether, then hexane was added, and the white solid was isolated by filtration to give 22 mg of methyl (4R, 7S)-7-[(S)-2-(1-tert-butoxyformamido)-3-hydroxypropionylamino]-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylate. washed successively with 0.5N hydrochloric acid, water, 5% sodium bicarbonate solution The organic layer was dried over sodium sulfate evaporated in vacuo The residue was dissolved in 2 ml of methanol 20 mg of ammonium fluoride were added the mixture was stirred for 30 minutes at room temperature The mixture was diluted with 40 ml of ethyl acetate washed with 30 ml of water The organic layer was dried over sodium sulfate the solvent was evaporated in vacuo The solid residue was triturated with 2 ml of ether hexane was added the white solid was isolated by filtration

Conditions: temp:0.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A suspension of 17.2 g of L-cysteine methyl ester hydrochloride and 20.5 g of N-(t-butoxycarbonyl)-L-serine in 300 ml of acetonitrile was treated at 0° C. with 10.1 g of 4-methylmorpholine. To the stirred solution was added dropwise at 10° C. within 30 minutes a solution of 20.6 g N,N-dicyclohexylcarbodiimide in 300 ml of acetonitrile. After stirring the reaction mixture for 3 hours at 0° C. the precipitate formed was filtered off, and the filtrate was evaporated in vacuo. The oily residue was dissolved in 200 ml of ethyl acetate and the solution washed consecutively with 0.5N hydrochloric acid, water, 5% aqueous sodium bicarbonate solution and brine. The organic layer was dried over sodium sulfate and evaporated in vacuo. The crude product was chromatographed on silica gel using ethyl acetate/n-hexane as eluent, and the so purified product was crystallized from diethyl ether/n-hexane to give N-[N-(t-butoxycarbonyl)-L-seryl]-L-cysteine methyl ester as white crystals with mp 72°-74° C. formed was filtered off the filtrate was evaporated in vacuo The oily residue was dissolved in 200 ml of ethyl acetate the solution washed consecutively with 0.5N hydrochloric acid, water, 5% aqueous sodium bicarbonate solution and brine The organic layer was dried over sodium sulfate evaporated in vacuo The crude product was chromatographed on silica gel the so purified product was crystallized from diethyl ether/n-hexane

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

Procedure: A solution of N-[(1,1-dimethylethoxy)carbonyl]-L-serine (24.3 g., 0.118 mole) in dry dimethylformamide (25 ml.) was added dropwise over a period of 1.0 hour to a cooled (0°, ice-salt bath) suspension of 60% sodium hydride (10.1 g., 0.25 mole) in dry dimethylformamide (200 ml.) and stirring was continued at 0° until the frothing subsided (about 2.0 hours). The reaction mixture was treated dropwise with 1-fluoro-2-nitrobenzene (14.3 ml., 0.13 mole) over a period of 20 minutes, stirred at 0° under argon for 4.0 hours then poured into ice-water (750 ml.) and extracted with ethyl acetate (2×100 ml.). The aqueous phase was brought to pH 1.0 with 6 N hydrochloric acid (70 ml.), extracted with ethyl acetate (3×500 ml.) and the combined organic extracts were washed with brine (100 ml.), dried (anhydrous sodium sulfate), filtered, evaporated to dryness and dried in vacuo. The crude product mixture was chromatographed on a silica gel column (Merck), eluting the column with methylene chloride:methanol:acetic acid (100:5:0.2) to give 27.22 g. of product as a thick yellow syrup; Rf =0.27 (methylene chloride:methanol:acetic acid, 100:5:0.5). a cooled was continued at 0° until the frothing (about 2.0 hours) stirred at 0° under argon for 4.0 hours extracted with ethyl acetate (2×100 ml.) extracted with ethyl acetate (3×500 ml.) the combined organic extracts were washed with brine (100 ml.) dried (anhydrous sodium sulfate) filtered evaporated to dryness dried in vacuo The crude product mixture was chromatographed on a silica gel column (Merck) eluting the column with methylene chloride:methanol:acetic acid (100:5:0.2) to give 27.22 g

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

Procedure: A solution of N-[(1,1-dimethylethoxy)carbonyl]-L-serine (24.3 g., 0.118 mole) in dry dimethylformamide (25 ml.) was added dropwise over a period of 1.0 hour to a cooled (0°, ice-salt bath) suspension of 60% sodium hydride (10.1 g., 0.25 mole) in dry dimethylformamide (200 ml.) and stirring was continued at 0° until the frothing subsided (about 2.0 hours). The reaction mixture was treated dropwise with 1-fluoro-2-nitrobenzene (14.3 ml., 0.13 mole) over a period of 20 minutes, stirred at 0° under argon for 4.0 hours then poured into ice-water (750 ml.) and extracted with ethyl acetate (2×100 ml.). The aqueous phase was brought to pH 1.0 with 6N hydrochloric acid (70 ml.), extracted with ethyl acetate (3×500 ml.) and the combined organic extracts were washed with brine (100 ml.), dried (anhydrous sodium sulfate), filtered, evaporated to dryness and dried in vacuo. The crude product mixture was chromatographed on a silica gel column (Merck), eluting the column with methylene chloride:methanol:acetic acid (100:5:0.2) to give 27.22 g. of product as a thick yellow syrup; Rf =0.27 (methylene chloride:methanol:acetic acid, 100:5:0.5). a cooled was continued at 0° until the frothing (about 2.0 hours) stirred at 0° under argon for 4.0 hours extracted with ethyl acetate (2×100 ml.) extracted with ethyl acetate (3×500 ml.) the combined organic extracts were washed with brine (100 ml.) dried (anhydrous sodium sulfate) filtered evaporated to dryness dried in vacuo The crude product mixture was chromatographed on a silica gel column (Merck) eluting the column with methylene chloride:methanol:acetic acid (100:5:0.2) to give 27.22 g

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

Procedure: A solution of N-[(1,1-dimethylethoxy)carbonyl]-L-serine (24.3 g., 0.118 mole) in dry dimethylformamide (25 ml.) was added dropwise over a period of 1.0 hour to a cooled (0°, ice-salt bath) suspension of 60% sodium hydride (10.1 g., 0.25 mole) in dry dimethylformamide (200 ml.) and stirring was continued at 0° until the frothing subsided (about 2.0 hours). The reaction mixture was treated dropwise with 1-fluoro-2-nitrobenzene (14.3 ml., 0.13 mole) over a period of 20 minutes, stirred at 0° under argon for 4.0 hours then poured into ice-water (750 ml.) and extracted with ethyl acetate (2×100 ml.). The aqueous phase was brought to pH 1.0 with 6N hydrochloric acid (70 ml.), extracted with ethyl acetate (3×500 ml.) and the combined organic extracts were washed with brine (100 ml.), dried (anhydrous sodium sulfate), filtered, evaporated to dryness and dried in vacuo. The crude product mixture was chromatographed on a silica gel column (Merck), eluting the column with methylene chloride:methanol:acetic acid (100:5:0.2) to give 27.22 g. of product as a thick yellow syrup; Rf =0.27 (methylene chloride:methanol:acetic acid, 100:5:0.5). a cooled was continued at 0° until the frothing (about 2.0 hours) stirred at 0° under argon for 4.0 hours extracted with ethyl acetate (2×100 ml.) extracted with ethyl acetate (3×500 ml.) the combined organic extracts were washed with brine (100 ml.) dried (anhydrous sodium sulfate) filtered evaporated to dryness dried in vacuo The crude product mixture was chromatographed on a silica gel column (Merck) eluting the column with methylene chloride:methanol:acetic acid (100:5:0.2) to give 27.22 g

Conditions: temp:0.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Tetrapeptide H-Ser(Bzl)-Gly-Leu-Gly-OCH3 : HCl.H-Leu-Gly-OCH3 (12.2 g, 51.3 mmol) was dissolved in DMF (200 mL). DIEA (24 mL, 139.8 mmol) was added to the solution. This first solution was cooled to 0° C. A second solution of Boc-Ser-(Bzl)-Gly-OH (16.42 g, 46.6 mmol) and DPPA (14.1 g, 51.26 mmol) in DMF (122 mL) was added with stirring to the first solution. The mixture was stirred for 20 h while the temperature of the mixture gradually came to room temperature. Concentration of the reaction mixture under reduced pressure gave an oil. The oil was dissolved in EtOAc. The solution was washed with 1N HCl, 1N NaHCO3 and H2O, dried (Na2SO4 /MgSO4) and concentrated to dryness to give an oil. Trituration of the latter oil with Et2O-hexane yielded Boc-Ser(Bzl-Gly-Leu-Gly-OCH3 as a solid (23.82 g). The latter compound (28.82 g) was dissolved in 1N HCl in glacial acetic acid (100 mL). The mixture was stirred at room temperature for 1 h and then concentrated under reduced vacuum to give an oil. Trituration of the oil with Et2O afforded the tetrapeptide as a solid (19.71 g, HCl salt). The mixture was stirred for 20 h while the temperature of the mixture gradually came to room temperature Concentration of the reaction mixture under reduced pressure gave an oil The solution was washed with 1N HCl, 1N NaHCO3 and H2O dried (Na2SO4 /MgSO4) concentrated to dryness to give an oil

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

Procedure: In 200 ml of N,N-dimethylformamide is suspended 10.1 g of 60% sodium hydride (oily), and a solution of 25 g of Boc-L-serine in 10 ml of N,N-dimethylformamide is added dropwise to the suspension in a stream of nitrogen at 0° C. with stirring. After stirring is continued at 0° C. until the evolution of hydrogen stops, 19 g of o-fluoronitrobenzene is added dropwise to the mixture. After the stirring at room temperature for 4 hours, the reaction mixture is poured in ice-cooled water containing dilute hydrochloric acid, followed by extraction with ethyl acetate. The organic layer is washed with water and dried, and then the solvent is evaporated off under reduced pressure. The resulting residue is purified by silica gel column chromatography (hexane-ethyl acetate=1:1) to give 30 g of O-(o-nitrophenyl)-Boc-L-serine as a colorless oil. After stirring the reaction mixture is poured in ice- followed by extraction with ethyl acetate The organic layer is washed with water dried the solvent is evaporated off under reduced pressure The resulting residue is purified by silica gel column chromatography (hexane-ethyl acetate=1:1)

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

Procedure: In 200 ml of N,N-dimethylformamide is suspended 10.1 g of 60% sodium hydride (oily), and a solution of 25 g of Boc-L-serine in 10 ml of N,N-dimethylformamide is added dropwise to the suspension in a stream of nitrogen at 0° C. with stirring. After stirring is continued at 0° C. until the evolution of hydrogen stops, 19 g of o-fluoronitrobenzene is added dropwise to the mixture. After the stirring at room temperature for 4 hours, the reaction mixture is poured in ice-cooled water containing dilute hydrochloric acid, followed by extraction with ethyl acetate. The organic layer is washed with water and dried, and then the solvent is evaporated off under reduced pressure. The resulting residue is purified by silica gel column chromatography (hexane-ethyl acetate=1:1) to give 30 g of O-(o-nitrophenyl)-Boc-L-serine as a colorless oil. After stirring the reaction mixture is poured in ice- followed by extraction with ethyl acetate The organic layer is washed with water dried the solvent is evaporated off under reduced pressure The resulting residue is purified by silica gel column chromatography (hexane-ethyl acetate=1:1)