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With methanol; N-ethyl-N,N-diisopropylamine; In dichloromethane; for 3h; |
EXAMPLE 4 This Example describes the synthesis of Compounds 1 and 13 to 16. Synthesis of Compound 1 al Synthesis of Intermediate A Intermediate A TFA (2 mL) was added to Boc-D-Lys (Fmoc) -OAllyl (2.86 g, 5.6 mmol) and left to stand for 5 min. The TFA was then removed by a stream of dry nitrogen to afford H-D- Lys (Fmoc) -OAllyl which was dried on a high vacuum line for 2 h to remove all traces of TFA. 2-Chlorotrityl resin (1 g, 1.4 mmol) was pre-swelled in DCM (10 mL) for 1 h. The resin was drained and a solution of H-D-Lys (Fmoc) -OAllyl (2.30 g, 5.64 mmol) and DIEA (729 mg, 982 82 LL, 5.64 mmol) in DCM (10 mL) was added and the reaction mixture shaken for 1 h. Further DIEA (1.46 g, 1.95 mL, 11.3 mmol) was added to the resin and the reaction mixture shaken for a further Ih. Methanol (1 mL) was added to end-cap any unreacted resin and the reaction mixture shaken for a further 1 h. The resin was filtered and washed with DMF (2 x 5 mL), DCM (2 x 5 mL) and DMF (2 x 5 mL). The resin was subjected to Fmoc- solid phase peptide synthesis (SPPS) using the following conditions: (i) Fmoc deprotection: 20 % piperidine in DMF (2 x 10 mL) for 2 min followed by washing with DMF (4 x 5 mL), DCM (4 x 5 mL) and DMF (4 x 5mL). (ii) Coupling conditions: In all couplings the solution of the coupling reagent in DMF is added to the Fmoc-amino acid. This solution is added to the resin followed by DIEA. (a) Fmoc-Trp (Boc) -OH (2.95 g, 5.6 mmol), HBTU (0.5 M solution, 11.2 mL) and DIEA (0.975 mL, 5.6 mmol) 20 min. (b) Fmoc-N-Me- Leu-OH (2.06 g, 5.6 mmol), HBTU (0.5 M solution, 11.2 mL) and DIEA (0.975 mL, 5.6 mmol) 20 min. (c) Fmoc-Leu-OH (1.98 g, 5.6 mmol), HOBt (756 mg, 5.6 mmol), HATU (2.13 g, 5.6 mmol) and DIEA (314 pLL, 1.8 mmol) in DMF (10 mL) 3 h. (d) Fmoc-Ala-OH (1.74 g, 5.6 mmol), HBTU (0.5 M solution, 11.2 mL) and DIEA (0.975 mL, 5.6 mmol) 20 min. Following all couplings the resin was filtered and washed with DMF (4 x 5 mL), DCM (4 x 5 mL) and DMF (4 x 5mL). All couplings except for (c) were monitored using the ninhydrin test, coupling (c) was monitored using a bromophenol blue test. All couplings were also monitored by MS by cleaving a small amount of resin (5 mg) with 100 % TFA for 5 min, the filtrate from the resin was then analysed by MS. A solution of Pd (PPh3) 4 (1.62 g, 1.4 mmol) and dimedone (1.96 g, 14 mmol) in THF: DCM (1: 1, 50 mL) was sparged with nitrogen gas for 10 min. , added to the resin and the mixture shaken for 16 h. The reaction mixture was filtered and washed with DCM (3 x 5 mL), DMF (3 x 5 mL) a solution of 0.5% DIEA and 0.5% diethyldithiocarbamic acid sodium salt in DMF (3 x 5 mL) and DMF (3 x 5mL). The resin was treated with 20 % piperidine in DMF (2 x 10 mL) for 2 min. followed by washing with DMF (4 x 5 mL), DCM (4 x 5 mL), 10% pyridinium hydrochloride in DCM: DMF (1: 1, 4 x 5 mL) and DMF (4 x 5 mL). A solution of PyBroP (718 mg, 1.54 mmol) and DIEA (1 mL, 5.74 mmol) in DCM: DMF (1: 1,10 mL) was added to the resin and the mixture shaken for 3 h after which a ninhydrin test was negative. The cyclic peptide was cleaved from the resin by treatment with 50% TFA in DCM (20 mL) for 1 h. The resin was filtered, washed with TFA (2 x 5 mL) and DCM (2 x 5 mL), concentrated to dryness, re-dissolved in MeCN: H20 (0. 1% TFA) and lyophilised to afford crude Intermediate A (435 mg, 50% based on the 2-chlorotrityl resin). Purification by RPHPLC (95: 5 H20 (1% TFA): MeCN (1% TFA) to 2: 3 H20 (1% TFA): MeCN (1% TFA)) over 60 min afforded Intermediate A (0.417 g, 3.6 %). b) lyl-N2-[(9H-fluoren-9-ylmethoxy)carbonyl]-N5-{imino[(2,2,4,6,7-pentamethyl-2,3- dihvdro-1-benzofuran-5-yl) aminolmethvl ornithinate N2-[(9H-fluoren-9-ylmethoxy) carbonyl]-N5- {imino [ (2, 2,4, 6,7-pentamethyl-2, 3- dihydro-l-benzofuran-5-yl) amino] methyl} ornithine (l. Og, 1.54 mmol) was dissolved in DMF (5 mL). Caesium carbonate (377 mg, 1.16 mmol) was added and the reaction mixture stirred for 1 h. Allyl bromide (0.913 mL, 10.8 mmol) was then added and stirring was continued for a further 1 h resulting in a milky white solution. Water (25 mL) was added and the reaction mixture acidified with 2M KHS04. DCM (50 mL) was added and the phases separated. The aqueous phase was washed with DCM (2 x 50 mL) and the combined organics washed with brine (50 mL), dried (MgSO4), filtered and concentrated to dryness to afford allyl-N2- [ (9H- fluoren-9-ylmethoxy) carbonyl]-N5- {imino [(2, 2,4, 6, 7-pentamethyl-2, -dihydro-l-benzofuran- 5-yl) amino] methyl} ornithinate as colourless foam (857 mg, 81%). lHNMR (CDC13, 500 MHz): D 1.43 (s, 6H), 1.59 (m, 2H), 1.73 (m, 1H), 1.86 (m, 1H), 2.09 (s, 3H), 2.52 (s, 3H), 2.61 (s, 3H), 2.91 (s, 2H), 3.22 (m, 2H), 4.17 (t, J7 Hz, 1H), 4.32 (m, 1H), 4. 37 (m, 1H), 4. 59 (br d, J4. 5 Hz, 2H), 5.21 (d, J 10.5 Hz, 1H), 5. 30 (d, J 17 Hz, 1H), 5.83 (m, 1H), 5.88 (m, 1H), 6.26 (br s, 1H), 6.35 (br s, 2H), 7.26 (t, J 7. 5 Hz, 2H), 7.37 (t, J 7.5 Hz, 2H), 7.57 (m, 2H), 7.74 (d, J7. 5 Hz, 2H). 13CNMR (CDC13,125 MHz): 0 12.68, 18.... |
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