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A series of nucleosides was phosphitylated using [O-ss-CYANOETHYL-N,] N, N', N'- [TETRAISOPROPYLPHOSPHORAMIDITE] and the N-methylimidazole salt of saccharin as activator. General method: In an appropriate sized flask was added the nucleoside (1.5 [MMOL)] and the solid was dried azeotropically by distilling (rotary evaporator) two times with 20 mL of pyridine. The flask was purged with Ar and to the flask was added 15 mL of acetonitrile. The mixture was stirred at room temperature until a clear solution was obtained. To the mixture was added [0-P-CYANOETHYL-N,] N, N', N'-tetraisopropylphosphoramidite (Tetraphos) followed by the addition of N-methylimidazole salt of saccharin. The mixture was stirred at room temperature while the reaction was monitored for end of reaction by HPLC. At the end of the reaction, the mixture was diluted with 30 mL of ethyl acetate and the organic mixture was washed with 2 x 25 mL of saturated aqueous sodium bicarbonate and 25 mL of saturated aqueous sodium chloride. The organic layer was separated and dried over [MGS04.] The suspension was filtered and the solvent was removed using a rotary evaporator. The residue was dried under vacuum to give a foam.
75%
Stage #1: for 0.0833333 h; Stage #2: at 20℃; for 18 h;
In a 500 mL round bottom flask was added 5'-DMT-N-Bz-2'-deoxyadenosine (18.00 g, 27.37 [MMOL)] and the solid was dried [AZEOTROPICALLY] by the addition and evaporation (rotary evaporator) of 2 x 200 mL of toluene. The residue was dried under vacuum for 16 h. The residue was dissolved in acetonitrile (180 mL) under an argon atmosphere and [O-ss-] cyanoethyl-N, N, N', N'-tetraisopropylphosphorodiamidite (9.90 g, 32.84 [MMOL)] was added. The mixture was stirred for 5 minutes and solid N-methylimidazole salt of saccharin (3.63 g, 13.69 [MMOL)] was added. The mixture was stirred at room temperature while the reaction was monitored by HPLC. After 18h, no further reaction was observed. To the reaction mixture was added ethyl acetate (200 [ML)] and the organic solution was washed with saturated aqueous sodium bicarbonate (2 x 150 mL) and saturated aqueous sodium chloride (150 mL). The organic layer was separated and dried over [MGS04.] The suspension was filtered and the solvent was removed using a rotary evaporator. The residue was dried under vacuum for 16h to give a white foam. Crude yield : 23.70 g HPLC : 92.5percent The crude material (23.70 g) was chromatographed using a silica gel (230 g) column. The column was loaded using 30percent ethyl acetate/hexanes containing 0.5percent triethylamine. The column was washed with 2 column volumes of 30percent ethyl acetate/hexanes. The crude material was loaded and the column was eluted using 2 column volumes of 30percent ethyl acetate/hexanes, 2 column volumes of 40percent ethyl acetate/hexanes, 2 column volumes of 50percent ethyl acetate/hexanes and finally 3 column volumes of 70percent ethyl acetate/hexanes. Fractions were collected when the product was detected by TLC (8: 3 ethyl acetate: hexanes). Fractions containing the desired product were combined and the solvent was removed using a rotary evaporator. The residue was dried under vacuum for 16 h to give a white foam. Yield : 17.55 g (75percent) HPLC: 97.5percent [31 P] NMR: 99.3percent
Stage #1: at 20℃; Stage #2: With 5-Phenyl-1H-tetrazole In acetonitrile at 20℃; for 2 - 24 h;
Example 2; Synthesis of 5'-O-(4,4'-dimethoxytrityl)-N6-benzoyl-2'-deoxyadenosine 3'-O-(2-cyanoethyl N,N-diisopropylphosphoroamidite) ; 2.0 g of 5'-O-(4,4'-dimethoxytrityl)-N6-benzoyl-2'-deoxyadenosine was mixed with 10 mL of dehydrated acetonitrile and 1.10 g of 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (1.2 equivalent to the molar number of a raw material) was dropped to a suspension stirred at a room temperature, followed by further stirring. Then, 0.53 g of 5-phenyl-1H-tetrazole (1.2 equivalent to the molar number of a raw material) was added thereto and the resulting mixture was stirred at a room temperature for 2 hours. The reaction solution was analyzed according to the high performance liquid chromatography (reverse phase column, eluent: water/acetonitrile 5/5 (TEAA 250 mM), detection wavelength: 254 nm). As a result, the yield was 96percent. The reaction selectivity (HPLC area percent of the entitled compound / HPLC area percent of the by-product) represented by the ratio of the entitled compound to the by-product represented by the general formula [5c] was 152, [] wherein R1 represents a 2-cyanoethyl group; R4 represents a 4,4'-dimethoxytrityl group; R5 represents a hydrogen atom; and B represents N6-benzoyl-9-adenine. Further, the reaction solution after 96 hours was analyzed. As a result, the yield was 99percent and the reaction selectivity was 187.; Example 3; Synthesis of 5'-O-(4,4'-dimethoxytrityl)-N6-benzoyl-2'-deoxyadenosine 3'-O-(2-cyanoethyl N,N-diisopropylphosphoroamidite); The reaction was conducted in the same manner as in Example 2, except that the equivalent of 5-phenyl-1H-tetrazole was changed. 0.04 g of 5-phenyl-1 H-tetrazole (0.1 equivalent to the molar number of a raw material) was used and the resulting mixture was stirred for 24 hours. The reaction solution was analyzed according to the high performance liquid chromatography (reverse phase column, eluent: water/acetonitrile 5/5 (TEAA 250 mM), detection wavelength: 254 nm). As a result, the yield was 99percent. The reaction selectivity (HPLC area percent of the entitled compound / HPLC area percent of the by-product) represented by the ratio of the entitled compound to the by-product represented by the general formula [5c] was 699.; Example 4; Synthesis of 5'-O-(4,4'-dimethoxytrityl)-N6-benzoyl-2'-deoxyadenosine 3'-O-(2-cyanoethyl N,N-diisopropylphosphoroamidite) ; The reaction was conducted in the same manner as in Example 2, except that the equivalent of 5-phenyl-1 H-tetrazole was changed. 0.22 g of 5-phenyl-1 H-tetrazole (0.5 equivalent to the molar number of a raw material) was used and the resulting mixture was stirred for 8 hours. The reaction solution was analyzed according to the high performance liquid chromatography (reverse phase column, eluent: water/acetonitrile 5/5 (TEAA 250 mM), detection wavelength: 254 nm). As a result, the yield was 99percent. The reaction selectivity (HPLC area percent of the entitled compound / HPLC area percent of the by-product) represented by the ratio of the entitled compound to the by-product represented by the general formula [5c] was 785.
90%
With pyridine; trifluoroacetic acid In acetic acid methyl ester at 20℃; for 12 h;
Comparative Examples 1-3; Three comparative phosphitylation reactions (C1-C3) comprising reacting a protected nucleoside reagent with 2-Cyanoethyl-N,N,N',N'-tetraisopropylphosphordiamidite in the presence of an pyridine-TFA activator were conducted, and the product yields of each calculated, according to the General Procedure described above for Examples 12-18. The various combinations of protected nucleoside, solvent, and yield for each of the 3 reactions are listed in Table 3. As illustrated by the yields in Table 3 (as compared to those of Tables 1 and 2), the yields associated with the methods of the present invention surprisingly tend to be at least as good, and in many embodiments, better, than those associated with comparable reactions using conventional activators comprising significantly less-hindered salts of unsubstituted pyridine.
83%
With N-ethyl-N,N-diisopropylamine; trifluoroacetic acid In tetrahydrofuran at 20℃; for 12 h;
Example 19; This example illustrates the phosphitylation of N6-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyAdenosine (Bz-DMT-dA) with diisopropylethyl ammonium trifluoroacetate and 2-Cyanoethyl-N,N,N',N'-tetraisopropylphosphor-diamidite according of the present invention. Diisopropylethylamine 6.4g (49.4 mmol) is dissolved in 20 ml of dry THF in a reaction vessel. Trifluoroacetic acid 4.9g (43.6 mmol) is added to the THF mixture at ambient temperature to form an activator solution for use in the following reaction step. Bz-DMT-dA 30g (45 mmol) is dissolved in 185 ml of dry THF in a reaction vessel and 50 ml of the THF is then distilled off under reduced pressure to form a reaction mixture. To the reaction mixture is added 14.7g (47.2 mmol) of 2-Cyanoethyl-N,N,N',N'-tetraisopropylphosphordiamidite at ambient temperature. The activator solution prepared above is then added to the reaction mixture at ambient temperature with vigorous stirring. After 12 hours, the reaction mixture is diluted with 80 ml toluene and washed with 50 ml of water. The organic layer is separated and concentrated under reduced pressure. The resulting product is purified on a short silica gel column using methylacetate/toluene (80/20). The appropriate product fractions are concentrated under reduced pressure and solvent until an approximately 50percent solution of 5'-O-Dimethoxytrityl-2-deoxyAdenosine-(N6-benzoyl)-3'-N,N-diisopropylamino-O-(2-cyanoethyl)phosphoramidite (PAm-Bz-DMT-dA) is obtained. The approximately 50percent solution is added, with vigorous stirring (approximately 500-600 rpm), to a 1-L stainless steel reactor equipped with a mechanical stirrer and containing 500 ml hexane at ambient temperature. After 3 hours the resulting precipitate is filtered, washed with 50 ml hexane and dried yielding 32 g (83percent) Pam-Bz-DMT-dA.
79%
Stage #1: at 20℃; Stage #2: With 1H-tetrazole; 5-(4-nitrophenyl)-1H-1,2,3,4-tetrazole; pyridinium trifluroacetate In acetonitrile at 20℃; for 2 - 8 h;
Comparative Examples 3 to 5 ; The reaction was conducted in the same manner as in Example 2, except that 0.256 g of tetrazole (1.2 equivalent to the molar number of a raw material: Comparative Example 3), 0.707 g of pyridinium trifluoroacetate (1.2 equivalent to the molar number of a raw material. Comparative Example 4) and 0.689 g of 5-(4-nitrophenyl)-1H-tetrazole (1.2 equivalent to the molar number of a raw material: Comparative Example 5) were respectively used, instead of 5-phenyl-1 H-tetrazole. The reaction solution after 96 hours was analyzed according to the high performance liquid chromatography (reverse phase column, eluent: water/acetonitrile 5/5 (TEAA 250 mM), detection wavelength: 254 nm). The results are shown in Table 2.; Comparative Examples 6 to 8 ; The reaction was conducted in the same manner as in Example 4, except that 0.107 g of tetrazole (0.5 equivalent to the molar number of a raw material: Comparative Example 6), 0.295 g of pyridinium trifluoroacetate (0.5 equivalent to the molar number of a raw material: Comparative Example 7) and 0.291 g of 5-(4-nitrophenyl)-1H-tetrazole (0.5 equivalent to the molar number of a raw material: Comparative Example 8) were respectively used, instead of 5-phenyl-1 H-tetrazole. The reaction solution after 96 hours was analyzed according to the high performance liquid chromatography (reverse phase column, eluent: water/acetonitrile 5/5 (TEAA 250 mM), detection wavelength: 254 nm). The results are shown in Table 3.
95 %Chromat.
With α-picoline; trifluoroacetic acid In propanoate at 20℃; for 12 h;
Examples 1-11; These Examples illustrate the phosphitylation of several protected nucleoside reagents with 2-Cyanoethyl-N,N,N',N'-tetraisopropylphosphordiamidite in the presence of several activators according to the present invention. Eleven phosphitylation reactions (1-11) comprising reacting a protected nucleoside reagent with 2-Cyanoethyl-N,N,N',N'-tetraisopropylphosphordiamidite in the presence of an acid-base activator according to the present invention were conducted, and the product yields of each calculated, as described in the General Procedure, below. The various combinations of protected nucleoside, activator base, activator acid, solvent, and yield for each of the 11 reactions are listed in Table 1. General Procedure: The activator base (1.1 to 1.2 equivalents) is added to the solvent and 0.95 to 1.1 equivalents of activator acid is subsequently added thereto at ambient temperature to form the activator solution. About 1 equivalent of the protected nucleoside is dissolved in about 10 equivalents of the solvent in a separate vessel and about 3 equivalents of the solvent is then distilled off under reduced pressure. About 1 to 1.2 equivalents of 2-Cyanoethyl-N,N,N',N'-tetraisopropylphosphordiamidite is added to the nucleoside mixture at ambient temperature, and the activator solution prepared previously is then added to the nucleoside mixture at ambient temperature with vigorous stirring. After 12 hours, the reaction mixture is diluted with toluene and washed with water. The organic layer is separated, dried over sodium sulfate if necessary, and concentrated under reduced pressure. The yield of the desired amidite is then calculated using HPLC techniques, that is, the resulting product mixture is run through an HPLC column using an appropriate eluent, and the area under the HPLC peaks used to determine the percentyield of product in the mixture.
89 %Chromat.
With 2,4,6-trimethyl-pyridine; trifluoroacetic acid In propanoate at 20℃; for 12 h;
Examples 1-11; These Examples illustrate the phosphitylation of several protected nucleoside reagents with 2-Cyanoethyl-N,N,N',N'-tetraisopropylphosphordiamidite in the presence of several activators according to the present invention. Eleven phosphitylation reactions (1-11) comprising reacting a protected nucleoside reagent with 2-Cyanoethyl-N,N,N',N'-tetraisopropylphosphordiamidite in the presence of an acid-base activator according to the present invention were conducted, and the product yields of each calculated, as described in the General Procedure, below. The various combinations of protected nucleoside, activator base, activator acid, solvent, and yield for each of the 11 reactions are listed in Table 1. General Procedure: The activator base (1.1 to 1.2 equivalents) is added to the solvent and 0.95 to 1.1 equivalents of activator acid is subsequently added thereto at ambient temperature to form the activator solution. About 1 equivalent of the protected nucleoside is dissolved in about 10 equivalents of the solvent in a separate vessel and about 3 equivalents of the solvent is then distilled off under reduced pressure. About 1 to 1.2 equivalents of 2-Cyanoethyl-N,N,N',N'-tetraisopropylphosphordiamidite is added to the nucleoside mixture at ambient temperature, and the activator solution prepared previously is then added to the nucleoside mixture at ambient temperature with vigorous stirring. After 12 hours, the reaction mixture is diluted with toluene and washed with water. The organic layer is separated, dried over sodium sulfate if necessary, and concentrated under reduced pressure. The yield of the desired amidite is then calculated using HPLC techniques, that is, the resulting product mixture is run through an HPLC column using an appropriate eluent, and the area under the HPLC peaks used to determine the percentyield of product in the mixture.
98 %Chromat.
With N-ethyl-N,N-diisopropylamine; trifluoroacetic acid In propanoate at 20℃; for 12 h;
Examples 1-11; These Examples illustrate the phosphitylation of several protected nucleoside reagents with 2-Cyanoethyl-N,N,N',N'-tetraisopropylphosphordiamidite in the presence of several activators according to the present invention. Eleven phosphitylation reactions (1-11) comprising reacting a protected nucleoside reagent with 2-Cyanoethyl-N,N,N',N'-tetraisopropylphosphordiamidite in the presence of an acid-base activator according to the present invention were conducted, and the product yields of each calculated, as described in the General Procedure, below. The various combinations of protected nucleoside, activator base, activator acid, solvent, and yield for each of the 11 reactions are listed in Table 1. General Procedure: The activator base (1.1 to 1.2 equivalents) is added to the solvent and 0.95 to 1.1 equivalents of activator acid is subsequently added thereto at ambient temperature to form the activator solution. About 1 equivalent of the protected nucleoside is dissolved in about 10 equivalents of the solvent in a separate vessel and about 3 equivalents of the solvent is then distilled off under reduced pressure. About 1 to 1.2 equivalents of 2-Cyanoethyl-N,N,N',N'-tetraisopropylphosphordiamidite is added to the nucleoside mixture at ambient temperature, and the activator solution prepared previously is then added to the nucleoside mixture at ambient temperature with vigorous stirring. After 12 hours, the reaction mixture is diluted with toluene and washed with water. The organic layer is separated, dried over sodium sulfate if necessary, and concentrated under reduced pressure. The yield of the desired amidite is then calculated using HPLC techniques, that is, the resulting product mixture is run through an HPLC column using an appropriate eluent, and the area under the HPLC peaks used to determine the percentyield of product in the mixture.
Reference：
[1] Chemical Communications, 1997, # 9, p. 877 - 878
[2] Patent: EP1582528, 2005, A1, . Location in patent: Page/Page column 9
[3] Patent: US2003/232980, 2003, A1, . Location in patent: Page 7
[4] Organic Process Research and Development, 2000, vol. 4, # 3, p. 175 - 181
[5] Patent: US2003/232980, 2003, A1, . Location in patent: Page 7
[6] Chemical Communications, 2014, vol. 50, # 56, p. 7463 - 7465
[7] Patent: EP1582528, 2005, A1, . Location in patent: Page/Page column 10
[8] Bulletin of the Polish Academy of Sciences, Chemistry, 1987, vol. 35, # 11-12, p. 507 - 516
[9] Recueil des Travaux Chimiques des Pays-Bas, 1986, vol. 105, # 1, p. 33 - 34
[10] Nucleosides and Nucleotides, 1998, vol. 17, # 9-11, p. 1639 - 1644
[11] Patent: US2003/232980, 2003, A1, . Location in patent: Page 6
[12] Patent: US2003/232980, 2003, A1, . Location in patent: Page 6
[13] Patent: US2003/232980, 2003, A1, . Location in patent: Page 6
3
[ 124482-92-4 ]
[ 64325-78-6 ]
[ 98796-53-3 ]
Reference：
[1] Green Chemistry, 2009, vol. 11, # 9, p. 1391 - 1396
[2] Chemical Communications, 2011, vol. 47, # 20, p. 5846 - 5848
[3] Tetrahedron Letters, 1983, vol. 24, # 52, p. 5843 - 5846
[4] Tetrahedron Letters, 1998, vol. 39, # 41, p. 7455 - 7458
4
[ 133728-84-4 ]
[ 109-78-4 ]
[ 98796-53-3 ]
Reference：
[1] Patent: US6335439, 2002, B1, . Location in patent: Example 14
Stage #1: N6-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine With N-ethyl-N,N-diisopropylamine In dichloromethane for 0.166667h;
Stage #2: 2-cyanoethyl-N,N-(diisopropylamino)chlorophosphine With 1-hexyl-1-methylimidazoliumtris(pentafluoroethyl)trifluorophosphate for 1.75h;
80%
With N-ethyl-N,N-diisopropylamine for 0.5h; Ionic liquid; ball miling;
61.2%
Stage #1: N6-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine With dmap; N-ethyl-N,N-diisopropylamine In tetrahydrofuran at 15℃; for 0.333333h; Molecular sieve;
Stage #2: 2-cyanoethyl-N,N-(diisopropylamino)chlorophosphine In tetrahydrofuran at 15℃; for 0.666667h;
5.5.10 Procedure for preparation of compound 3
To a mixture of compound 2 (1.88 g, 2.86 mmol, 1 eq) and Molecular sieve 3A (1 g) in 30 mL of dry THF was added DIEA (1.48 g, 11.4 mmol, 2.0 mL, 4 eq) and DMAP (34.9 mg, 285.8μmol , 0.1 eq) at 15°C. The mixture was stirred at 15°C for 20 min. Then 3-[chloro- (diisopropylamino)phosphanyl]oxypropanenitrile (1.0 g, 4.3 mmol, 1.5 eq) was added to the mixture at 15°C. And the mixture was stirred at 15°C for 40 min. LCMS showed compound 2 consumed completely, and the desired mass was detected. The reaction mixture was filtered, and the filtrate was concentrated in vacuum. The residue was purified by flash silica gel chromatography (ISCO; 40 g SepaLlash Silica Plash Column, Eluent of 30-80% Ethyl acetate/Petroleum ether gradient 80 mL/min) to give compound 3 (1.5 g, 1.75 mmol, 61.2% yield) as a white foam. MS (ESI) m/z: calcd. for [M+H]+, 858.37; found, 858.4 (M/1+H)+.
In tetrahydrofuran according to ref 3) with modifications;
With N-ethyl-N,N-diisopropylamine In tetrahydrofuran for 0.583333h; Ambient temperature;
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