Journal of Medicinal Chemistry 2.6 mmol) in dichloromethane (8 mL) at 0 °C, and N- (benzyloxycarbonyloxy)succinimide (648 mg, 2.6 mmol) was added in portions. The reaction mixture was stirred at room temperature for 17 h, diluted with dichloromethane (30 mL), and washed with brine (2 × 15 mL). The organic phase was dried (Na2SO4), filtered, and concentrated in vacuo. Purification by flash chromatography (heptane/ ethyl acetate 2:1, Rf = 0.23) gave (S)-4 as a transparent oil (0.94 g, 84%). Analytical chiral HPLC (hexane/2-propanol 95:5) tR 18.7 min, 98% purity, ee 100%. 1H NMR (rotamers) δ 7.48−7.19 (m, 5H), 5.18 and 5.11 (rot, s, 2H), 4.31−3.79 (m, 6H), 2.80−2.41 (m, 3H), 2.04− 1.89 (m, 1H), 1.80−1.32 (m, 12H), 1.27 and 1.17 (rot, t, J = 7.1 Hz, 3H). 13C NMR δ 169.8, 155.9, 155.1, 154.5, and 154.4 (rot), 136.2, 128.3, and 128.2 (rot), 127.8, 127.6, 79.5, 67.5, and 67.1 (rot), 61.0, 53.2, and 53.0 (rot), 47.1 and 46.5 (rot), 45.6 and 45.4 (rot), 43.3, 28.7, 28.2, 24.4, 13.9; [α]D20 +14.0 (c 1.0, CHCl3). IR (neat) v 3499, 2976, 2863, 1747, 1693, 1415, 1152 cm−1; HRMS (FT-ICR-MS) calcd for C22H32N2O6 [M + H]+ 421.2333; found 421.2334. (−)-Ethyl 2-[(Benzyloxycarbonyl)(1-(tert-butoxycarbonyl-L- leucinyl)-(R)-piperidin-3-yl)amino]acetate ((R)-5). Deprotection of (R)-4 (870 mg, 2.07 mmol) with TFA (2.0 mL, 27.0 mmol) in dichloromethane (8 mL) was performed as described for (S)-5 to give the Boc-deprotected intermediate. The crude intermediate and Boc-L- Leu × H2O (551 mg, 2.21 mmol) were coupled as described for (S)-5 using EDC (424 mg, 2.21 mmol), HOBt (299 mg, 2.21 mmol), and Et3N (813 mg, 8.04 mmol) in dichloromethane (11 mL) with molecular sieves (600 mg). Purification by flash chromatography (heptane/ethyl acetate 1:1, Rf = 0.2) gave (R)-5 as a transparent oil (1.0 g, 91% yield over two steps). Analytical chiral HPLC (hexane/ ethanol 70:30) tR 7.5 min, 99% purity, de 97%. 1H NMR (CD3OD) δ 7.41−7.25 (m, 5H), 5.19−5.10 (m, 1H), 5.06 (s, 2H), 4.64−4.38 (m, 2H), 4.23−3.84 (m, 6H), 3.22−3.04 (m, 1H), 3.03−2.91 (m, 1H), 2.76−2.61 (m, 1H), 2.55−2.41 (m, 1H), 1.99−1.56 (m, 2H), 1.55− 1.07 (m, 14H), 1.00−0.72 (m, 6H). 13C NMR (CD3OD) δ 173.7 and 173.4 (rot), 171.8 and 171.7 (rot), 157.6, 157.2, 137.8, 129.5, 129.1, 128.8, 80.5, 68.6, 62.3, 55.0, 50.2, 46.7, 46.0, 43.1, 42.4, and 42.1 (rot), 29.0, 28.7, 26.0, 25.4, 23.6, 22.2, 14.4; [α]D20 −31.5 (c 1.0, CH3OH). IR (KBr) v 3423, 2959, 1753, 1708, 1642, 1445, 1205 cm−1. HRMS (FT-ICR-MS) calcd for C28H43N3O7 [M + H]+ 534.3174; found 534.3176. (+)-Ethyl 2-[(Benzyloxycarbonyl)(1-(tert-butoxycarbonyl-L- leucinyl)-(S)-piperidin-3-yl)amino]acetate ((S)-5). TFA (2.0 mL, 27.0 mmol) was added to (S)-4 (950 mg, 2.26 mmol) in dichloromethane (8 mL) and stirred at room temperature for 16 h. The solvent and excess of reagents were removed in vacuo to give the Boc-deprotected intermediate. Boc-L-Leu × H2O (620 mg, 2.49 mmol), EDC (477 mg, 2.49 mmol), and HOBt (336 mg, 2.49 mmol) were stirred in dichloromethane (6 mL) with molecular sieves (600 mg) at 0 °C for 15 min. Into this solution, the Boc-deprotected intermediate and Et3N (9.14 mg, 9.04 mmol) in dichloromethane (5 mL) were added and the reaction mixture was stirred at room temperature for 15 h. The mixture was diluted with dichloromethane (20 mL) and washed with aqueous citric acid (10%) (2 × 10 mL), saturated aqueous NaHCO3 (2 × 10 mL), and brine (1 × 10 mL). The organic phase was dried (Na2SO4) and filtered. The solvent was removed in vacuo. Purification by flash chromatography (heptane/ ethyl acetate 1:1, Rf = 0.18) gave (S)-5 as a transparent oil (950 mg, 79% yield over two steps). Analytical chiral HPLC (hexane/ethanol 70:30) tR 6.4 min, purity 100%. 1H NMR (CD3OD) δ 7.49−7.22 (m, 5H), 5.44−5.15 (m, 1H), 5.07 (s, 2H), 4.72−4.47 (m, 2H), 4.24−3.65 (m, 6H), 3.58−3.30 (m, 1H), 3.12−2.81 (m, 1H), 2.69−2.48 (m, 1H), 2.47−2.23 (m, 1H), 2.12−1.90 (m, 2H), 1.89−1.10 (m, 15H), 1.06− 0.72 (m, 6H). 13C NMR (CD3OD) δ (174.0, 173.8 and 173.4, rot), 171.8, 171.6, (157.7, 157.0 and 156.8, rot), 137.8 and 137.6 (rot), 129.5 and 129.4 (rot), 129.2 and 129.1 (rot), 128.8 and 128.7 (rot), 80.4 and 80.1 (rot), 68.5 and 68.3 (rot), 62.2, 54.5, and 54.3 (rot), 50.4, 48.0, 46.4, and 46.2 (rot), 45.8, 30.1, 29.7, and 29.5 (rot), 28.8, 26.2, 25.9, and 25.7 (rot), 23.7, 22.0, 14.5; [α]D20 +25.7 (c 1.0, CH3OH). IR (KBr) v 3425, 2958, 1754, 1720, 1640, 1442, 1204 cm−1. HRMS (FT-ICR-MS) calcd for C28H43N3O7 [M + H]+ 534.3174; found 534.3174. Article (−)-2-[(Benzyloxycarbonyl)(1-(tert-butoxycarbonyl-L-leucin- yl)-(R)-piperidin-3-yl)amino]acetamide ((R)-6). Compound (R)-6 was synthesized from (R)-5 (200 mg, 0.38 mmol) and NH3 in methanol (7 N) (4.0 mL) as described for (S)-2. Purification by flash chromatography (dichloromethane/methanol 95:5, Rf = 0.09) gave (R)-6 as a white solid (122 mg, 65%). Analytical chiral HPLC (hexane/ethanol 60:40) tR 12.3 min, 95% purity, de 90%. 1H NMR (CD3OD, 50 °C) δ 7.39−7.23 (m, 5H), 5.12 (br s, 2H), 4.49−4.44 (m, 1H), 4.11−3.80 (m, 3H), 3.18−3.04 (m, 1H), 3.03−2.89 (m, 1H), 2.77−2.61 (m, 1H), 2.56−2.40 (m, 1H), 2.03−1.91 (m, 1H), 1.85− 1.27 (m, 15H), 1.02−0.73 (m, 6H). 13C NMR (CD3OD, 50 °C) δ 174.4, 173.7, and 173.4 (rot), 157.6, 137.8, 129.5, 129.1, 128.8, 80.5, 68.7, 54.9, 50.2, 47.6, 46.7, and 46.4 (rot), 43.1, 42.4, 28.8, 26.6, 25.9, 25.5, 23.5, 22.2; [α]D20 −33.9 (c 1.0, CH3OH). IR (KBr) v 3414, 2956, 1707, 1640, 1446 cm−1. HRMS (FT-ICR-MS) Calcd for C26H40N4O6 [M + H]+ 505.3021; found 505.3021. (+)-2-[(Benzyloxycarbonyl)(1-(tert-butoxycarbonyl-L-leucin- yl)-(S)-piperidin-3-yl)amino]acetamide ((S)-6). Compound (S)-6 was synthesized from (S)-5 (156 mg, 0.29 mmol) and NH3 in methanol (7 N) (4.0 mL) as described for (S)-2. Purification by flash chromatography (dichloromethane/methanol 95:5, Rf = 0.07) gave the product as a white solid (108 mg, 73%). Analytical chiral HPLC (hexane/ethanol 60:40) tR 8.5 min, 100% purity. 1H NMR (CD3OD, 50 °C) δ 7.46−7.23 (m, 5H), 5.12 (br s, 2H), 4.49−4.38 (m, 1H), 4.20−3.79 (m, 3H), 3.26−3.10 (m, 1H), 3.09−2.93 (m, 1H), 2.77− 2.62 (m, 1H), 2.56−2.38 (m, 1H), 2.05−1.61 (m, 4H), 1.60−1.24 (12H), 1.00−0.75 (m, 6H). 13C NMR (CD3OD, 50 °C) δ 174.4, 173.8, and 173.5 (rot), 157.7, 137.8, 129.5, 129.0, 128.8, 80.4, 68.7, 54.7, 50.6, 47.4, 46.4, 43.6, 42.8, and 42.2 (rot), 28.8, 26.3, 26.0, 25.6, 23.6, 22.1; [α]D20 +13.2 (c 1.0, CH3OH). IR (KBr) v 3421, 2957, 1703, 1638, 1456 cm−1. HRMS (FT-ICR-MS) calcd for C26H40N4O6 [M + H]+ 505.3021; found 505.3022. (−)-2-[(1-L-Leucinyl-(R)-piperidin-3-yl)amino]acetamide × 2TFA ((R)-LpipG) ((R)-7). Compound (R)-6 (115 mg, 0.23 mmol) was deprotected as described for (R)-LLpipG ((R)-10) to give the white TFA salt of (R)-LpipG ((R)-7) (95 mg, 84%). Analytical reversed phase HPLC (the mobile phase was 6.4−32.8% CH3CN in water during 30 min, 0.085% TFA throughout) tR 6.2 min, 100% purity. 1H (CD3OD) NMR δ 4.75 (app d, 1H), 4.48−4.38 (m, 1H), 3.98−3.86 (m, 2H), 3.74 (app d, 1H), 3.23−3.08 (m, 2H), 2.85 (app t, 1H), 2.32−2.18 (m, 1H), 1.98−1.51 (m, 6H), 1.05−0.95 (m, 6H). 13C NMR (CD3OD) δ 169.9, 168.5, 54.7, 50.5, 46.6, 46.4, 44.5, 41.0, 28.0, 25.3, 25.0, 23.5, 21.6; [α]D20 −11.2 (c 1.0, CH3OH). IR (KBr) v 3420, 2967, 1676, 1202, 1134 cm−1. HRMS (FT-ICR-MS) calcd for C13H26N4O2 [M + H]+ 271.2129; found 271.2128. For NMR spectral assignment, see the Supporting Information. (+)-2-[(1-L-Leucinyl-(S)-piperidin-3-yl)amino]acetamide × 2TFA ((S)-LpipG) ((S)-7). Compound (S)-6 (117 mg, 0.23 mmol) was deprotected as described for (R)-LLpipG ((R)-10) but with stirring for 14 h for the first deprotection. The second deprotection gave the white TFA salt of (S)-LpipG ((S)-7) (87 mg, 75%). Analytical reversed phase HPLC (the mobile phase was 6.4−32.8% CH3CN in H2O for 30 min, 0.085% TFA throughout) tR 6.6 min, 100% purity. 1H (CD3OD) NMR δ 4.55−4.40 (m, 1H), 4.25 (app d, 1H), 4.00−3.85 (m, 2H), 3.66−3.57 (m, 1H), 3.50−3.42 (m, 1H), 2.63 (app t, 1H), 2.32−2.16 (m, 1H), 2.03−1.93 (m, 1H), 1.90−1.48 (m, 6H), 1.07−0.94 (m, 6H). 13C NMR (CD3OD) δ 170.3, 168.5, 54.9, 50.5, 46.7, 46,6, 43.9, 41.1, 27.3, 25.3, 23.5, 23.1, 21,7; [α]D20 +14.8 (c 1.0, CH3OH). IR (KBr) v 3418, 2965, 1676, 1201, 1134 cm−1. HRMS (FT-ICR-MS) calcd for C13H26N4O2 [M + H]+ 271.2129; found 271.2128. (−)-Ethyl 2-[(Benzyloxycarbonyl)(1-((tert-butoxycarbonyl-L- leucinyl)-L-leucinyl)-(R)-piperidin-3-yl)amino]acetate ((R)-8). Deprotection of (R)-5 (649 mg, 1.22 mmol) by TFA (1.0 mL, 13 mmol) in dichloromethane (6 mL) were performed as described for (S)-5 to give the Boc-deprotected intermediate. The intermediate and the second Boc-L-Leu × H2O (334 mg, 1.34 mmol) were coupled as described for (S)-5 with EDC (257 mg, 1.34 mmol), HOBt (181 mg, 1.34 mmol), Et3N (493 mg, 4.87 mmol), and molecular sieves (200 mg) in dichloromethane (11 mL). Purification by flash chromatog- 2557 dx.doi.org/10.1021/jm201517a | J. Med. Chem. 2012, 55, 2549−2560