| 97% |
With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In dichloromethane for 2h; Inert atmosphere; |
|
| 96% |
With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In dichloromethane for 2h; |
|
| 89% |
With dicyclohexyl-carbodiimide In tetrahydrofuran at 0 - 20℃; for 2.75h; |
|
| 87% |
With dmap; 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In dichloromethane at 20℃; |
2,5-dioxopyrrolidin-1-yl pent-4-ynoate (13)
2 (500 mg, 9.12 mmol) was dissolved in 15 mL of DCM. N-hydroxysuccinimide (1 g, 10.19 mmol), DMAP (1.57 g, 10.28 mmol) and EDC.HCl (2.69 g, 14.05 mmol) were added and the reaction was stirred overnight at rt. The solvent was removed under reduced pressure and the crude product purified by flash chromatography using hexane:EtOAc (3:2). 1.55 g of a white solid were obtained, yield 87%. 1H-NMR (300 MHz, DMSO-d6) δ: 2.90-2.83 (6H, m), 2.60 (2H, dt, Jd=2.4, Jt=7.8 Hz), 2.05 (1H, t, J=2.6 Hz). 13C-NMR (300 MHz, DMSO-d6) δ: 169.11 (2C), 167.18, 81.03, 70.26, 30.46, 25.75 (2C), 14.26. MS (ESI) [M+H3O+]+: observed m/z 214 |
| 87% |
With dmap; 1,2-dichloro-ethane In dichloromethane at 20℃; |
|
| 86% |
With dicyclohexyl-carbodiimide In tetrahydrofuran at 0 - 20℃; for 2.66h; |
|
| 85% |
With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In dichloromethane at 20℃; |
1
2.0 g (20 mmol) of 4-pentynoic acid was dissolved in 80 ml CH2Cl2. 2.54 g (22 mmol) of N- hydroxysuccinimide (NHS) was added. Then, l-ethyl-3- (3- dimethylaminopropyl) carbodiimide hydrochloride (EDC) was added (4.22 g, 22 mmol). The reaction was stirred at room temperature overnight. The reaction mixture was concentrated and the pure product was separated by silica gel column (hexane : ethyl acetate = 2:1) . Yield: 85%. 1H NMR (500 Hz, CDCl3) δ 2.88-2.83 (m, 6 H), 2.60 (td, J1 = 2.44 Hz, J2 = 7.81 Hz, 2H), 2.04 (t, J= 2.44 Hz, 1 H) ppm. |
| 84% |
With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In N,N-dimethyl-formamide at 20℃; |
1.c
4-pentynoic acid (1 g, 10 mmol, 1 equiv), was dissolved in DMF (20 mL) in presence of Nhydroxysuccinimide (1.77 g, 15 mmol, 1.5 equiv) and EDCJ (2.93, 15 mmol, 1.5 equiv). Thereaction mixture was stirred overnight at rt, concentrated under reduced pressure and finallypurified by flash column chromatography (EP/AcOEt 3:2 then 1:1) to give the desiredintermediate (mass: 1.67 g, yield: 84 %). 1-(pent-4-ynoyloxy)pyrrolidine-2,5-dione (74 mg,0.38 mmol, 1 equiv) and Cyclo(Arg(Pbf)-Gly-Asp(OtBu)-D-Phe-Lys) (230 mg, 0.38 mmol, 1equiv) were dissolved in DMF. Triethylamine (79 il, 0.57 mmol, 1.5 equiv) was added andthe reaction mixture was stuffed until total conversion of the starting material. The productwas concentrated under reduced pressure, dissolved in MeOH followed by precipitation byslow adding of diethyl ether to afford the pure product (mass : 181 mg, yield: 70 %). JR(film): 3265, 2935, 1635, 1540, 1435, 1370, 1200, 1080, 840, 650, 600 cm-’. HRMS (ESJ):(m/z): calcd for C32H45N908+H: 684.3469, found: 684.3489. [diD25 = -4.8 (c=0.001, MeOH). |
| 84% |
With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In N,N-dimethyl-formamide at 20℃; |
1.c
Example 1.c Synthesis of intermediate (19) Cyclo(L-arginylglycyl-L-α-aspartyl-D-phenylalanyl-N6-pent-4-ynoyl-L-lysyl) [0115] 4-pentynoic acid (1 g, 10 mmol, 1 equiv), was dissolved in DMF (20 mL) in presence of N-hydroxysuccinimide (1.77 g, 15 mmol, 1.5 equiv) and EDCI (2.93, 15 mmol, 1.5 equiv). The reaction mixture was stirred overnight at rt, concentrated under reduced pressure and finally purified by flash column chromatography (EP/AcOEt 3:2 then 1:1) to give the desired intermediate (mass: 1.67 g, yield: 84 %). 1-(pent-4-ynoyloxy)pyrrolidine-2,5-dione (74 mg, 0.38 mmol, 1 equiv) and Cyclo(Arg(Pbf)-Gly-Asp(OtBu)-D-Phe-Lys) (230 mg, 0.38 mmol, 1 equiv) were dissolved in DMF. Triethylamine (79 µl, 0.57 mmol, 1.5 equiv) was added and the reaction mixture was stirred until total conversion of the starting material. The product was concentrated under reduced pressure, dissolved in MeOH followed by precipitation by slow adding of diethyl ether to afford the pure product (mass : 181 mg, yield : 70 %). IR (film): 3265, 2935, 1635, 1540, 1435, 1370, 1200, 1080, 840, 650, 600 cm-1. HRMS (ESI): (m/z): calcd for C32H45N9O8+H: 684.3469, found: 684.3489. [α]D25 = -4.8 (c=0.001, MeOH). |
| 83% |
Stage #1: 1-hydroxy-pyrrolidine-2,5-dione; 4-pentynoic acid With dmap; 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In dichloromethane at 0 - 20℃; Inert atmosphere;
Stage #2: Inert atmosphere; |
|
| 83% |
With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In N,N-dimethyl-formamide at 20℃; for 16h; |
|
| 83% |
With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In N,N-dimethyl-formamide at 20℃; for 16h; |
1 Preparation of 2, 5-dioxopyrrolidin- 1 -yl pent-4-ynoate
Preparation of 2, 5-dioxopyrrolidin- 1 -yl pent-4-ynoate 4-Pentynoic acid (2.00 g, 20.36 mmol) was dissolved in DMF (40 mL) and EDCI (5.84 g, 29.84 mmol, 1 .5 equiv.) and NHS (4.68 g, 40.76 mmol, 2.0 equiv.) were added. The mixture was stirred for 16 hours at RT. After evaporation of DMF the residue was diluted in EtOAc (120 mL) and washed two times with NH4CI (1 M, 120 mL) and two times with saturated NaHC03 (120 mL). The organic layer was dried with Na2S04 and the product was purified with flash column chromatography (EtOAc: PE) to obtain the desired activated ester as a white powder (3.3 g, 83 %). 1H NMR (400 MHz, CDCI3): δ 2.89-2.83 (m, 4H), 2.63-2.59 (m, 4H), 1.55 (bs, 1 H); 13C NMR (100 MHz, CDCI3): δ 168.8, 167.0, 80.8, 70.0, 30.3, 25.5, 14.1. |
| 80% |
With dicyclohexyl-carbodiimide In 1,4-dioxane; ethyl acetate at 0 - 23℃; for 20h; |
1.1.1.3 Synthesis of N-hydroxysuccinimide ester (21 )
[00161 ] To a solution of 500 mg (5.096 mmol, 1 .0 eq) 3-Butynic acid and 587 mg (5.096 mmol, 1 .0 eq) N-hydroxysuccinimide in 64 mL of 1 :1 -mixture of dry EtOAc and dry dioxane was added 1 .051 g (5.096 mmol, 1 .0 eq) DCC in one portion at 0°C. The mixture was stirred at 0°C for 5 h and additional 15 h at 23°C, before it was filtered and the filtrate was concentrated under reduced pressure. The residue was recrystallized from CH2CI2/Pentane obtaining 800 mg (4.100 mmol, 80%) of 21 as a white amorphous solid. (0238) [00162] N-hydroxysuccinimide ester (21): 1H-NMR (500 MHz, CDCI3) δ [ppm]: 2.86 (dd, J = 8.2, 6.7 Hz, 2H), 2.82 (s, 4H), 2.60 (ddd, J = 8.6, 6.7, 2.7 Hz, 2H), 2.03 (t, J = 2.7 Hz, 1 H). 13C-NMR (126 MHz, CDCI3) δ [ppm] 169.27, 167.39, 81.23, 70.43, 30.70, 25.97, 14.49. |
| 78% |
With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In dichloromethane at 15 - 25℃; for 16h; Inert atmosphere; |
4 2,5-dioxopyrrolidin-l-yl pent-4-ynoate (1)
To 4-pentynoic acid (80.0 mg, 0.815 mmol) in CH2CI2 (7 mL) was added EDC.HC1 (234 mg, 1.22 mmol), followed by /V-hydroxysuccinimide (140 mg, 1.22 mmol). The resultant mixture was stirred at RT for 16 h, under argon. The solvent was then removed in vacuo and purification by column chromatography (gradient elution from pet. ether to 50% EtOAc in pet. ether) afforded the target compound as a white solid (124 mg, 0.635 mmol, 78%). (0181) mp 65-67 °C; 1H NMR (600 MHz, CDCl3) d H 2.86 (t, 2H, J= 7.2, OC(O)CH2), 2.81 (br s, (0182) 4H, 2 x NC(O)CH2), 2.59 (td, 2H, 7= 7.5, 2.6, CHCCH2), 2.04 (1H, t, J= 2.7, CH); 13C NMR (150 MHz, CDCl3) d c 169.2 (NC(O)), 167.2 (OC(O)), 81.0 (C), 70.2 (CH), 30.4 (NC(O)CH2), 25.7 (OC(O)CH2), 14.2 (CHCCH2); IR (solid) vmax/cm-1 3262 (m), 2955 (w), 2917 (w), 2849 (w), 1811 (m), 1786 (m), 1723 (s); LRMS (ES+) m/z 196 ([M+H]+, 100), 176 (23), 149 (76), 132 (45), 116 (23); HRMS (ES+) calcd for [C10H9NO4]+ [M+H]+ 196.0610, observed 196.0614. |
| 72% |
With dicyclohexyl-carbodiimide In 1,4-dioxane; ethyl acetate Cooling with ice; |
|
| 72% |
Stage #1: 4-pentynoic acid With dicyclohexyl-carbodiimide In dichloromethane at 20℃; for 0.0833333h;
Stage #2: 1-hydroxy-pyrrolidine-2,5-dione In dichloromethane at 20℃; for 3h; |
|
| 72% |
Stage #1: 4-pentynoic acid With dicyclohexyl-carbodiimide In dichloromethane for 0.0833333h;
Stage #2: 1-hydroxy-pyrrolidine-2,5-dione In dichloromethane at 20℃; for 3h; |
1 Pentynoic NHS-ester
To a solution of pentynoic acid (210 mg, 2.14 mmol) in DCM (8ml_) was added N, N’-dicyclohexylcarbodiimide (480 mg, 2.3 mmol). The mixture was stirred for 5 mins and then N-hydroxysuccinimide (260 mg, 2.3 mmol) was added. The reaction was continuously stirred at r.t for 3h. Afterwards, the precipitated dicyclohexylcarbamide was filtered off with Celite and the filter cake was washed with cold DCM (2x1 OmL). The filtrate was collected and DCM was removed in vacuo. The product was redissolved in EtOAc (10 ml_) and cooled in a refrigerator at 0°C for 20 mins. The precipitate was filtered off again with Celite and the filtrate was washed with saturated NaHC03 (2x50 ml_) and brine (2x50 ml_), dried over anhydrous MgS04, filtered and concentrated in vacuo to give crude product as colorless oil. Purification of the residue by silica gel column chromatography (petroleum ether: EtOAc= 2:1 ) yield pentynoic NHS-ester as a white solid (300 mg, 72% yield). 1 H NMR (400 MHz, CDCI3): d 2.90-2.82 (m, 6H, COCH2CH2CO, OCOCH2CH2), 2.62 (ddd, 2H, CH2CCH), 2.05 (t, 1 H, CC H). 13C NMR (101 MHz, CDCI3): d 168.89 (COCH2CH2CO), 167.02 (OCOCH2), 80.84 (CH2CCH), 70.04 (CH2CCH), 30.31 (OCOCH2), 25.57 (COCH2CH2CO), 14.09 (CH2CCH). |
| 69.9% |
With dicyclohexyl-carbodiimide In tetrahydrofuran at 0 - 20℃; for 5.5h; |
3.1.3. Preparation of SCT-Asn-Alkyne
First, N-(4-pentynoyloxy)-succinimide was synthesized [41]. 4-Pentynoic acid (392.0 mg,1 eq) and N-hydroxysuccinimide (466.0 mg, 1 eq) were dissolved in dry THF (7 mL) andcooled to 0 C. Next, N’-dicyclohexylcarbodiimide (852.0 mg, 1 eq) dissolved in dry THF(2 mL) was added dropwise. The mixture was stirred for 1.5 h at 0 C, slowly warmedto room temperature, and then stirred for an additional 4 h. The precipitate was filtered,and the solvent was evaporated at a reduced pressure. The residue was dissolved in ethylacetate, washed with saturated NaHCO3 solution and brine, and dried over Na2SO4. Thesolvent was evaporated at a reduced pressure, and the residue dissolved in MeOH wassubject to gel filtration with a Sephadex LH-20 column. The column was eluted with MeOH,and product fractions were combined and concentrated in vacuo to yield pent-4-ynoicacid 1-oxysuccinimidyl ester as a white powder (545.1 mg, yield = 69.9%). EI-MS (m/z):195 [M]+, C9H9NO4. 1H-NMR (600 MHz, CD3OD) 2.87 (t, J = 7.2 Hz, 2H, H-4), 2.83 (s,4H, H-7), 2.58 (td, J = 7.2, 2.7 Hz, 2H, H-3), 2.33 (t, J = 2.7 Hz, 1H, H-1). 13C-NMR (101 MHz,CD3OD) 171.7, 168.9, 82.2, 70.9, 31.2, 26.5, 14.7. |
| 66.8% |
With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride at 25 - 30℃; for 1h; |
Preparation of BCY9297
To a solution of compound 1 (2 g, 20.4 mmol) in DCM (50 ml.) was added EDCI (7.82 g, 40.8 mmol) and compound 2 (2.58 g, 22.43 mmol, 1.1 eq). The mixture was stirred at 25-30 °C for 1 hr. TLC (Eluent: Petroleum ether: Dichloromethane = 0:1 , Color Developing Reagent: Bromocresol green, Rf = 0.44) indicated Reactant 1 was consumed completely and one new spot formed. The reaction was clean according to TLC. The reaction mixture was then concentrated under reduced pressure to give a residue, following by purification through flash silica gel chromatography (ISCO; 40 g SepaFlash Silica Flash Column, Eluent of 0~50% Ethyl acetate/Petroleum ether gradient 40 mL/min) to give compound 3 (2.8 g, 13.63 mmol, 66.8% yield) as a white solid. |
| 62.33% |
With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In dichloromethane at 20℃; for 1h; |
General Procedure for Preparation of Compound 3
To a solution of compound 1 (500 mg, 5.10 mmol, 1.0 eq) in DCM (25 mL) were added compound 2 (645.2 mg, 5.61 mmol, 1.1 eq) and EDCl (1.95 g, 10.19 mmol, 2.0 eq). The mixture was stirred at 20° C. for 1 hr. TLC (PE: DCM=0:1, Rf=0.43, Color Developing Reagent: Bromocresol green) indicated compound 1 was consumed completely and one new spot was formed. The reaction was clean according to TLC. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=2/1 to 1:1) to give compound 3 (620 mg, 3.18 mmol, 62.33% yield) as a white solid. (0238) 1H NMR: 400 MHz CDCl3 (0239) δ 2.80-2.95 (m, 6H), 2.55-2.70 (m, 2H), 2.05-2.10 (t, 1H) |
| 61.6% |
With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In dichloromethane at 25℃; for 1h; |
6.2 Procedure for preparation of compound B2
To a solution of compound 1 (1 g, 10.19 mmol, 1 eq.) in DCM (50 mL) was added EDCI (3.91 g, 20.39 mmol, 2 eq.) and compound 2 (1.29 g, 1 1.21 mmol, 1.1 eq.). The reaction mixture was stirred at 25 °C for 1 h. TLC indicated compound 1 was consumed completely and one new spot was formed. The reaction mixture was directly purified by flash silica gel chromatography (ISCO; 40 g SepaFlash Silica Flash Column, Eluent of 0-50% Ethyl acetate/Petroleum ether gradient 40 mL/min) to give compound 3 (1.29 g, 6.28 umol, 61.6% yield) as a colorless solid. |
| 61.6% |
With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In dichloromethane at 25℃; for 1h; |
23.2.1 Procedure for preparation of compound 3
To a solution of compound 1 (1 g, 10.19 mmol, 1 eq.) in DCM (50 mL) was added EDCI (3.91 g, 20.39 mmol, 2 eq.) and compound 2 (1.29 g, 11.21 mmol, 1.1 eq.). The reaction mixture was stirred at 25 °C for 1 h. TLC indicated compound 1 was consumed completely and one new spot was formed. The reaction mixture was directly purified by flash silica gel chromatography (ISCO; 40 g SepaFlash Silica Flash Column, Eluent of 0-50% Ethyl acetate/Petroleum ether gradient 40 mL/min) to give compound 3 (1.29 g, 6.28 umol, 61.6% yield) as a colorless solid. |
| 57% |
With dicyclohexyl-carbodiimide In 1,4-dioxane; ethyl acetate at 20℃; for 5h; |
|
| 50% |
With dmap; 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In dichloromethane for 72h; |
|
| 40% |
Stage #1: 1-hydroxy-pyrrolidine-2,5-dione; 4-pentynoic acid With dmap In dichloromethane for 0.5h;
Stage #2: With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In dichloromethane at 22℃; for 4h; |
|
| 24% |
With dicyclohexyl-carbodiimide In dichloromethane at 20℃; |
|
|
With dicyclohexyl-carbodiimide In dichloromethane at 20℃; |
18
4-ρentynoic acid (2.943 g, 3.0 mmol) was dissolved in CH2Cl2 (25 niL). N- hydroxysuccinimide (3.80 g, 3.3 mmol) and DCC (4.66 g, 3.0 mmol) were added and the solution was stirred overnight at room temperature. The resulting crude NHS ester 7 was used in the following reaction without further purification.[555] mPEG-NH2 with a molecular weight of 5,000 Da (mPEG-NH2, 1 g, Sunbio) was dissolved in THF (50 mL) and the mixture was cooled to 4 0C. NHS ester 7 (400 mg, 0.4 mmol) was added portion-wise with vigorous stirring. The mixture was allowed to stir for 3 hours while warming to room temperature. Water (2 mL) was then added and the solvent was removed under vacuum. To the residue was added CH2Cl2 (50 mL) and the organic layer was separated, dried over anhydrous Na2SO4, and the volume was reduced to approximately 2 mL. This CH2Cl2 solution was added to ether (150 mL) drop-wise. The resulting precipitate was collected and dried in vacuo. |
|
With dicyclohexyl-carbodiimide In tetrahydrofuran at 0 - 20℃; for 6.08333h; |
|
|
With dicyclohexyl-carbodiimide |
|
|
With dicyclohexyl-carbodiimide In dichloromethane at 20℃; |
18
4-pentynoic acid (2.943 g, 3.0 mmol) was dissolved in CH2Cl2 (25 mL). N-hydroxysuccinimide (3.80 g, 3.3 mmol) and DCC (4.66 g, 3.0 mmol) were added and the solution was stirred overnight at room temperature. The resulting crude NHS ester 7 was used in the following reaction without further purification. |
|
With dicyclohexyl-carbodiimide In dichloromethane at 20℃; |
6
4-pentynoic acid (2.943 g, 3.0 mmol) was dissolved in CH2Cl2 (25 mL). N-hydroxysuccinimide (3.80 g, 3.3 mmol) and DCC (4.66 g, 3.0 mmol) were added and the solution was stirred overnight at room temperature. The resulting crude NHS ester 7 was used in the following reaction without further purification. |
|
With dmap; dicyclohexyl-carbodiimide In dichloromethane at 20℃; for 12h; |
|
|
With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In dichloromethane at 20℃; for 24h; |
|
|
With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In dichloromethane at 20℃; |
|
| 0.83 g |
With dicyclohexyl-carbodiimide In 1,4-dioxane; ethyl acetate at 0 - 20℃; for 6h; |
|
|
With dicyclohexyl-carbodiimide In tetrahydrofuran at 0 - 20℃; for 5.08333h; Inert atmosphere; |
|
|
With diisopropyl-carbodiimide In dichloromethane at 20℃; Inert atmosphere; |
|
|
With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In tetrahydrofuran at 0℃; for 22h; |
|
| 1.62 g |
With dicyclohexyl-carbodiimide; 4-(dimethylamino)pyridinium tosylate In acetonitrile at 20℃; |
61 Preparation of 4-Pentyn-1-ol, NHS ester
Example 61 Preparation of 4-Pentyn-1-ol, NHS ester A solution of 1.02 grams of 4-pentynoic acid and 1.20 grams of N-hydroxysuccinimide in 20 ml of dry acetonitrile was treated with 300 mg of DPTS, followed by 2.8 grams of DCC, and the reaction was stirred at room temperature overnight. The reaction was filtered and concentrated to give a residue, which was subjected to flash column chromatography on silica gel with 30% ethyl acetate in hexane. The product containing fractions were combined and concentrated to give a 1.62 grams of the desired product as a white solid. NMR (CDCl3): δ 2.89 (d of d, 2H, CH2C=O, J=7.9, 6.4 Hz), 2.85 (s, 4H, O=CCH2CH2C=O), 2.62 (app d of d of d, 2H, CHCCH2, J=8.6, 6.9, 2.7 Hz), 2.06 (t, 1H, CH, J=2.7 Hz). |
|
With dicyclohexyl-carbodiimide In dichloromethane at 20℃; |
18.1
4-pentynoic acid (2.943 g, 3.0 mmol) was dissolved in CH2C12 (25 mL). N- hydroxysuccinimide (3.80 g, 3.3 mmol) and DCC (4.66 g, 3.0 mmol) were added and thesolution was stirred overnight at room temperature. The resulting crude NHS ester 7 was used in the following reaction without further purification |
|
With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In dichloromethane at 20℃; for 4h; Inert atmosphere; |
|
|
With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In tetrahydrofuran; dichloromethane at 0 - 20℃; |
Synthesis of 2,5-dioxopyrrolidin-1-yl pent-4-ynoate (S2)
To a solution of S1 (2.4 g, 24.5 mmol) and EDCHCl (9.39 g, 49.0 mmol) in THF/CH2Cl2 (1:2,total 246 mL), HOSu (5.63 g, 49.0 mmol) was added at 0 C. After stirring for overnight at room temperature, aqueous NaHCO3 was added at 0 C. The mixture was extracted with EtOAc for threetimes, washed with brine, and dried over Na2SO4, sequentially. After the desiccant was filtered off,the filtrate was concentrated under reduced pressure to give the product S2 as yellow oil. Half of S2was used for the next step without purification and the other half was stored at -30 C until use. |
|
With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In dichloromethane at 20℃; for 3.5h; |
|
|
With dicyclohexyl-carbodiimide In 1,4-dioxane; ethyl acetate at 0 - 20℃; for 12h; |
4-Pentynoic acid succinimidyl ester
4-Pentynoic acid succinimidyl ester: A solution of 4-pentynoic acid (0.5 g, 5.1 mmol) and /V-Hydroxysuccinimide (0.59 g, 1 eq) in EtOAc-Dioxane (1 : 1 , 50 mL) was stirred at 0°C and DCC (1.0g, 1 eq) was added allowing the mixture to reach room temperature (rt) and kept at these conditions for 12 h. The DCU formed was filtrated and the filtrate concentrated under vacuum. EtOAc (100 mL) was added and washed with 5% NaHC03 (2x40 mL), water (40 mL) and brine (40 mL). After drying over anhNa2S04 and concentrating it was recrystallized from DCM/Hexane to obtain a white solid that was used in the next step without further purification. 1H-NMR (400 MHz, CDCI3) δ: 2.90 (t, 2H, J 7.0 Hz), 2.86 (s, 4H), 2.64 (td, 2H, J 7.0, 2.7 Hz), 2.07 (t, 1 H, J 2.7 Hz). |
|
With dicyclohexyl-carbodiimide In dichloromethane at 20℃; |
2 4-pentynoic acid succinimidyl ester:
Pentynoic acid (0.98 g, 10.0 mmol) and N-Hydroxysuccinimide (1.15 g, 10 mmol) were suspended in 200 mL of dry dichloromethane, and then N,N′ - Dicyclohexylcarbodiimide (2.06 g, 10 mmol) was added. The reaction mixture was stirred at room temperature overnight, and then the white precipitate was filtered off. The filtrate was concentrated to afford the crude product. Pure 4-pentynoic acid succinimidyl ester was obtained as white solid after recrystallization from dichloromethane/diethylether. 111 NMR complies with the reported value. |
|
With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In dichloromethane at 0 - 20℃; Inert atmosphere; |
4-Pentynoic acid NHS ester (17)
4-Pentynoic acid (10 mg, 0.100 mmol, 1 equiv) was dissolved in 2 mL of dry DCM under argonand cooled to 0 °C. EDC·HCl (23 mg, 0.120 mmol, 1.2 equiv) and NHS (14 mg, 0.120 mmol,1.2 equiv) were sequentially added to the solution and the reaction mixture was stirred overnightat room temperature. The mixture was diluted in 20 mL of DCM and the organic phase waswashed with water (3 × 7 mL), dried over MgSO4, filtrated and concentrated under reducedpressure to give the product as a yellowish oil that was used in next step without furthermodifications. |
|
With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride |
|
|
With dicyclohexyl-carbodiimide In dichloromethane at 20℃; |
24
4-pentynoic acid (2.943 g, 3.0 mmol) was dissolvedin CH2C12 (25 mE). N-hydroxysuccinimide (3.80 g, 3.3mmol) and DCC (4.66 g, 3.0 mmol) were added and thesolution was stirred overnight at room temperature. Theresulting crude NHS ester 7 was used in the following reaction without further purification. |
|
With dicyclohexyl-carbodiimide In dichloromethane at 20℃; |
18.1
[646] 4-pentynoic acid (2.943 g, 3.0 mmol) was dissolved in CH2Cl2 (25 niL). N- hydroxysuccinimide (3.80 g, 3.3 mmol) and DCC (4.66 g, 3.0 mmol) were added and the solution was stirred overnight at room temperature. The resulting crude NHS ester 7 was used in the following reaction without further purification.[647] HiPEG-NH2 with a molecular weight of 5,000 Da (mPEG-NH2, 1 g, Sunbio) was dissolved in THF (50 mL) and the mixture was cooled to 4 0C. NHS ester 7 (400 mg, 0.4 mmol) was added portion-wise with vigorous stirring. The mixture was allowed to stir for 3 hours while warming to room temperature. Water (2 mL) was then added and the solvent was removed under vacuum. To the residue was added CH2Cl2 (50 mL) and the organic layer was separated, dried over anhydrous Na2SO4, and the volume was reduced to approximately 2 mL. This CH2Cl2 solution was added to ether (150 mL) drop-wise. The resulting precipitate was collected and dried in vacuo. |
|
With dicyclohexyl-carbodiimide In 1,4-dioxane; ethyl acetate at 0 - 20℃; for 12h; |
|
| 76.4 mg |
With dmap; dicyclohexyl-carbodiimide In dichloromethane at 0 - 20℃; for 2h; |
2.2.4. Synthesis of compound 6
4-Pentynoic acid (5.00 g, 51.0 mmol), NHS (6.40 g, 55.6 mmol), andDMAP (1.24 g, 10.1 mmol) were dissolved in 100 mL CH2Cl2 at 0 °Cfollowed by the addition of DCC (12.6 g, 61.2 mmol) in 50 mL CH2Cl2.The reaction mixture was then warmed to room temperature and stirredfor another 2 h. After filtration, the solution was washed with 1 N HCl,saturated NaHCO3 aqueous solution, and brine. The organic phase wasdried over anhydrous Na2SO4, filtered, and concentrated. The crudeproduct was recrystallized in ethyl acetate/hexane to give PA-NHS as a white crystal with a yield of 76.4%. |
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With dicyclohexyl-carbodiimide In dichloromethane at 0 - 20℃; for 16h; |
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With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In dichloromethane |
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