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Stage #1: N-(9-fluorenylmethoxycarbonyl)-D-alanine With benzotriazol-1-ol; diisopropyl-carbodiimide In N,N-dimethyl-formamide at 0 - 10℃; for 2h;
Stage #2: With 3-azapentane-1,5-diamine In N,N-dimethyl-formamide for 0.5h;
Stage #3: Fmoc-Leu-OH; Fmoc-L-Lys(iPr,Boc)-OH; 9-fluorenylmethyloxycarbonyl-N(4)-(L-hydroorotyl)-4-aminophenylalanine; 9-fluorenylmethyloxycarbonyl-N(4)-(t-butylcarbamoyl)-D-4-aminophenylalanine; Fmoc-Pro-OH; Fmoc-Ser(tBu)-OH; 9-fluorenylmethyloxycarbonyl-D-2-naphthylalanine; acetic anhydride; 9-fluorenylmethyloxycarbonyl-D-4-chlorophenylalanine; (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(pyridin-3-yl)propanoic acid Further stages; |
1-12 Step 1 Rink Amide Resin Activation
Fmoc-Rink amide AM resin (0.5 g) was placed in a peptide vessel and the resin was washed with 5 ml DMF 2 times and kept in DMF (5 ml) for 45 min and the solvent was then removed by applying vacuum. The washed resin was then deprotected with 5% diethylenetriamine in DMF 5 ml for 15 min. The vessel was emptied by applying vacuum and treated with 5% diethylenetriamine in DMF (5 ml) for 15 min. The vessel was emptied by applying vacuum and the resin was washed with DMF (5 ml). The vessel was further washed with methanol (5 ml*2 times) and DMF (5 ml*2 times). A solution of 0.326 gm Fmoc-Ala-OH (3.0 eq), 0.14 gm HOBT.H2O (3.0 eq) and 0.165 ml diisopropylcarbodimide (3.0 eq) was dissolved in DMF at 0-10° C. and allowed to activate for 10 min. This solution was charged into the peptide vessel, stirred for 2 hrs. and deprotected with 5% diethylenetriamine in DMF (5 ml) for 15 min. The vessel was emptied by applying vacuum and a second treatment 5% diethylenetriamine in DMF 5 ml for 15 min. The vessel was emptied by applying vacuum and the resin was washed with DMF (5 ml). The vessel was further washed with methanol (5 ml*2 times) and DMF (5 ml*2 times). Step 2 (0104) A solution of 0.354 gm Fmoc-Pro-OH (3.0 eq), 0.14 gm HOBT.H2O (3.0 eq) and 0.165 ml diisopropylcarbodimide (3.0 eq) was dissolved in DMF at 0-10° C. and allowed to activate for 10 min. This solution was then charged into the peptide vessel, stirred for 2 hrs. and washed with 5 ml DMF (4 times). Then it was deprotected with 5% diethylenetriamine in DMF 5 ml for 15 min. The vessel was emptied by applying vacuum and a second treatment 5% diethylenetriamine in DMF 5 ml for 15 min. The vessel was emptied by applied vacuum and the resin washed with DMF (5 ml). The vessel was further washed with methanol (5 ml*2 times) and DMF (5 ml*2 times). (0105) Step 3 (0106) A solution of 0.536 gm Fmoc-Lys (ipr,Boc)-OH (3.0 eq), 0.14 gm HOBT.H2O (3.0 eq) and 0.165 ml diisopropylcarbodimide (3.0 eq) was dissolved in DMF at 0-10° C. and allowed to activate for 10 min and this solution charged into peptide vessel , stirred for 2 hrs., and washed with 5 ml DMF 4 times, and deprotected with 5% diethylenetriamine in DMF 5 ml for 15 min. The vessel was emptied by applying a vacuum and a second treatment 5% diethylenetriamine in DMF 5 ml for 15 min. The vessel was emptied by applied vacuum and the resin washed with DMF (5 ml). The vessel was further washed with methanol (5 ml*2 times) and DMF (5 ml*2 times). (0107) Step 4 (0108) A solution of 0.371 gm Fmoc-Leu-OH (3.0 eq), 0.14 gm HOBT.H2O (3.0 eq) and 0.165 ml diisopropylcarbodimide (3.0 eq) was dissolved in DMF at 0-10° C. and allowed to activate for 10 min and this solution charged into a peptide vessel, stirred for 2 hrs., washed with 5 ml DMF 4 times, and deprotected with 5% diethylenetriamine in DMF 5 ml for 15 min. The vessel was emptied by applying a vacuum and second treatment 5% diethylenetriamine in DMF 5 ml for 15 min. The vessel was emptied by applied vacuum and resin washed with DMF (5 ml). The vessel was further washed with methanol (5 ml*2 times) and DMF (5 ml*2 times). (0109) Step 5 (0110) A solution of 0.351 gm Fmoc-DAph(tBuCbm)-OH (2.0 eq),0.107 gm HOBT.H2O (2.0 eq) and 0.11 ml diisopropylcarbodimide (2.0 eq) was dissolved in DMF at 0-10° C. and allowed to activate for 10 min and this solution charged into the peptide vessel, stirred for 2 hrs., washed with 5 ml DMF 4 times, and deprotected with 5% diethylenetriamine in DMF 5 ml for 15 min. The vessel was emptied by applying a vacuum and a second treatment of 5% diethylenetriamine in DMF 5 ml for 15 min. The vessel was emptied by applying a vacuum and the resin washed with DMF (5 ml). the vessel was further washed with methanol (5 ml*2 times) and DMF (5 ml*2 times). (0111) Step 6 (0112) A solution of 0.379 gm Fmoc-Aph(Hor)-OH (2.0 eq),0.107 gm HOBT.H2O (2.0 eq) and 0.11 ml diisopropylcarbodimide (2.0 eq) was dissolved in DMF at 0-10° C. and allowed to activate for 10 min and this solution charged into the peptide vessel, stirred for 2 hrs. and deprotected with 5% diethylenetriamine in DMF 5 ml for 15 min. The vessel was emptied by applying a vacuum and a second treatment 5% diethylenetriamine in DMF 5 ml for 15 min. The vessel was emptied by applying a vacuum and the resin washed with DMF (5 ml). The vessel was further washed with methanol (5 ml*2 times) and DMF (5 ml*2 times). (0113) Step 7 (0114) A solution of 0.402 gm Fmoc-Ser(tBu)-OH (3.0 eq),0.14 gm HOBT.H2O (3.0eq) and 0.165 ml diisopropylcarbodimide (3.0 eq) was dissolved in DMF at 0-10° C. and allowed to activate for 10 min and this solution charged into peptide vessel, stirred for 2 hrs., washed with 5 ml DMF 4 times, and deprotected with 5% diethylenetriamine in DMF 5 ml for 15 min. The vessel was emptied by applying a vacuum and a second treatment 5% diethylenetriamine in DMF 5 ml for 15 min. The vessel was emptied by applying a vacuum and the resin washed with DMF (5 ml). The vessel was further washed with methanol (5 ml*2 times) and DMF (5 ml*2 times). (0115) Step 8 (0116) A solution of 0.407 gm Fmoc-D-3-Pal-OH (3.0 eq), 0.14 gm HOBT.H2O (3.0 eq) and 0.165 ml diisopropylcarbodimide (3.0 eq) was dissolved in DMF at 0-10° C. and allowed to activate for 10 min and this solution charged into peptide vessel, stirred for 2 hrs., and washed with 5 ml DMF 4 times and deprotected with 5% diethylenetriamine in DMF 5 ml for 15 min. The vessel was emptied by applying a vacuum and a second treatment 5% diethylenetriamine in DMF 5 ml for 15 min. The vessel was emptied by applying a vacuum and the resin washed with DMF (5 ml). A vessel was further washed with methanol (5 ml*2 times) and DMF (5 ml*2 times). (0117) Step 9 (0118) A solution of 0.442 gm Fmoc-D-Phe (4-Cl)-OH (3.0 eq), 0.14 gm HOBT.H2O (3.0eq) and 0.165 ml diisopropylcarbodimide (3.0 eq) was dissolved in DMF at 0-10° C. and allowed to activate for 10 min and this solution charged in to peptide vessel, stirred for 2 hrs. washed with 5 ml DMF 4 times, and deprotected with 5% diethylenetriamine in DMF 5 ml for 15 min. The vessel was emptied by applying a vacuum and a second treatment 5% diethylenetriamine in DMF 5 ml for 15 min. The vessel was emptied by applying a vacuum and the resin washed with DMF (5 ml). The vessel was further washed with methanol (5 ml*2 times) and DMF (5 ml*2 times). (0119) Step 10 (0120) A solution of 0.459 gm Fmoc-D-2-Nal-OH (3.0 eq), 0.14 gm HOBT.H2O (3.0 eq) and 0.165 ml diisopropylcarbodimide (3.0 eq) was dissolved in DMF at 0-10° C. and allowed to activate for 10 min and this solution charged into peptide vessel, stirred for 2 hrs., washed with 5 ml DMF 4 times, and deprotected with 5% diethylenetriamine in DMF 5 ml for 15 min. The vessel was emptied by applying a vacuum and second treatment 5% diethylenetriamine in DMF 5 ml for 15 min. The vessel was emptied by applying a vacuum and resin washed with DMF (5 ml). A vessel was further washed with methanol (5 ml*2 times) and DMF (5 ml*2 times). (0121) Step 11 (0122) Acetic anhydride (0.125 gm) was dissolved in dichloromethane (MDC) at 0-10° C. and charged into peptide vessel. The vessel was stirred for 3 hours and washed with dichloromethane (MDC-5 ml*4 times) and Methyltertiarybutylether (MTBE-5 ml*2 times) simultaneously. (0123) Step 12 (0124) TFA (5 ml) was taken in RBF and cooled to 0-10° C. Above the resin bed was then charged into TFA solution. Reaction mixture was then stirred for 24 hrs. at room temperature. Solution was filtered and then washed with TFA (1 ml). Filtrate ml was then distilled out up to one volume under vacuum and below 35° C. Methyltertiarybutylether (MTBE) (30 ml) was taken in another RBF and cooled to 0-10° C. Residue was then charged in this solution and then stirred for 1 hr. and filtered. The compound was dried under vacuum at 35° C. for 6-8 hr. Purity of the crude was about 85%-95%. The compound was further purified using preparative HPLC to get highly pure compound, greater than about 85%-95%. |