4.1.3. General Ugi Reaction Protocol followed by Alkylation (Method B)

To a solution of piperidin-4-one (0.18 mmol) in methanol (2 mL) were added 2 equivalents of benzylamine, 2 equivalents of benzyl isocyanide and the corresponding amino acid (2 equivalents). After stirring the reaction for four days at room temperature, HCl in MeOH (1.2 M) was added and stirred at room temperature for 30 additional minutes. The solvent was removed, the residue redissolved in ethyl acetate and then successively washed with saturated NaHCO₃ (3 × 10 mL) and brine (3 × 10 mL). The organic phase was dried (MgSO₄), filtered and then evaporated to dryness. The Ugi adduct, thus obtained, was used in the next step without further purification. It was dissolved in DMF (5 mL) and alkylated by reaction with 1.2 equivalents of the corresponding alkyl bromide in the presence of 1.5 equivalents of K₂CO₃. The reaction mixture was stirred at 50 °C for 3 h. It was then neutralized with AcOH (0.3 mL), washed with brine, and dried over anhydrous MgSO₄ and filtered. The final crude was purified by flash column chromatography using a mixture of Hexane/EtOAc (1:1 to 0:1) to give the novel N-benzyl 4,4-disubstituted piperidine analogues 54–56 and 60–63.

Methyl(S)-4-(benzyl(4-benzylcarbamoyl-1-(4-methylphenethyl)piperidin-4-yl)amino)-3-benzyloxycarbonylamino-4-oxobutanoate (54): Following Method B, 4-piperidone monohydrate hydrochloride (0.18 mmol, 28 mg), benzylamine (0.36 mmol, 0.058 mL), Cbz-Asp(OMe)-OH (0.36 mmol, 101 mg) and benzyl isocyanide (0.36 mmol, 0.054 mL) in MeOH (2 mL) were reacted. Next, after treating the Ugi adduct with 4-methylphenethyl bromide (0.22 mmol, 44 mg) and with K₂CO₃ (0.27 mmol, 37 mg) in DMF followed by purification, 54 (30 mg, 24%) was obtained as a white foam. ¹H NMR (400 MHz, CDCl₃) δ 7.25 (m, 11H), 7.15 (m, 4H), 7.02 (d, J = 7.8 Hz, 2H), 6.97 (d, J = 7.8 Hz, 2H), 6.73 (s, 1H), 4.99 (d, J = 12.1 Hz, 1H), 4.89 (d, J = 12.1 Hz, 1H), 4.83 (m, 1H), 4.74 (d, J = 18.0 Hz, 1H), 4.30 (dd, J = 15.1, 5.8 Hz, 1H), 4.17 (m, 2H), 3.77 (m, 1H), 3.45 (s, 3H), 3.22 (m, 3H), 2.91 (m, 4H), 2.59 (dd, J = 16.4, 4.8 Hz, 1H), 2.42 (s, 1H), 2.25 (d, J = 7.3 Hz, 1H), 2.22 (s, 3H), 1.99 (s, 4H). ¹³C NMR (101 MHz, CDCl₃) δ 178.1, 175.9, 172.9, 171.6, 155.4, 138.4, 137.7, 136.6, 135.7, 129.4, 129.3, 128.8, 128.6, 128.5, 128.4, 128.2, 127.8, 127.3, 127.2, 126.5, 67.5, 58.3, 52.0, 49.4, 47.9, 43.5, 37.1, 36.4, 30.4, 29.7, 21.0, 18.5. MS (ES+) m/z: 705.57 (M+1)⁺. HPLC retention time = 8.70 min (99% analytical purity) (H₂O/CH₃CN from 15/85 to 0/100 in 5 min flow rate of 1 mL/min). Anal. calculated for C₄₂H₄₈N₄O₆ (C, H, N): C, 71.57; H, 6.86; N, 7.95; O, 13.62. Found: C, 71.84; H, 6.91; N, 7.70; O, 13.49.

Methyl(S)-4-(benzyl(4-benzylcarbamoyl-1-(4-nitrophenethyl)piperidin-4-yl)amino)-3-benzyloxycarbonylamino-4-oxobutanoate (55): According to general method B, 4-piperidone monohydrate hydrochloride (0.18 mmol, 28 mg), benzylamine (0.36 mmol, 0.058 mL), Cbz-Asp(OMe)-OH (0.36 mmol, 101 mg) and benzyl isocyanide (0.36 mmol, 0.054 mL) in MeOH (2 mL) were reacted. To the Ugi adduct, intermediate 4-nitrophenethyl bromide (0.22 mmol, 51 mg) and K₂CO₃ (0.27 mmol, 37 mg) in DMF were added. Purification of the final residue yielded compound 55 (34 mg, 26%) as a white foam. ¹H NMR (500 MHz, CDCl₃) δ 8.13 (d, J = 8.4 Hz, 2H), 7.35 (m, 15H), 7.24 (d, J = 7.8 Hz, 3H), 6.79 (s, 1H), 5.33 (s, 1H), 5.05 (d, J = 12.2 Hz, 1H), 4.98 (d, J = 12.2 Hz, 1H), 4.87 (m, 1H), 4.81 (d, J = 17.7 Hz, 1H), 4.38 (m, 2H), 3.54 (s, 3H), 2.82 (s, 4H), 2.59 (dd, J = 47.7, 13.6 Hz, 4H), 1.73 (s, 4H), 1.26 (s, 2H). 1³C NMR (126 MHz, CDCl₃) δ 172.8, 171.4, 155.3, 138.6, 137.8, 135.8, 129.5, 129.2, 128.7, 128.6, 128.5, 128.4, 128.2, 127.7, 127.5, 127.3, 126.3, 123.8, 77.3, 77.3, 77.2, 77.0, 76.9, 76.8, 67.4, 52.0, 50.3, 49.7, 49.4, 48.1, 43.5, 37.3, 29.7. MS (ES+) m/z: 736.73 (M + 1)⁺. HPLC retention time = 8.42 min (99% analytical purity) (H₂O/CH₃CN from 15/85 to 0/100 in 5 min flow rate of 1 mL/min). Anal. calculated for C₄₁H₄₅N₅O₈ (C, H, N): C, 66.92; H, 6.16; N, 9.52. Found: C, 66.79; H, 6.22; N, 9.65.

Methyl(S)-4-(benzyl(4-benzylcarbamoyl-1-(4-fluorophenethyl)piperidin-4-yl)amino)-3-benzyloxycarbonylamino-4-oxobutanoate (56): Reactants: benzylamine (0.36 mmol, 0.058 mL), a solution of 4-piperidone monohydrate hydrochloride (0.18 mmol, 28 mg), Cbz-Asp(OMe)-OH (0.36 mmol, 101 mg) and benzyl isocyanide (0.36 mmol, 0.054 mL) in MeOH (2 mL). Subsequent treatment of adduct with 4-fluorophenethyl bromide (0.22 mmol, 45 mg) and with K₂CO₃ (0.27 mmol, 37 mg) in DMF afforded, after chromatography, a white foam that was compound 56 (28 mg, 22%). ¹H NMR (400 MHz, CDCl₃) δ 7.29 (m, 15H), 7.07 (dd, J = 8.7, 5.5 Hz, 2H), 6.92 (t, J = 8.7 Hz, 2H), 6.76 (t, J = 5.8 Hz, 1H), 5.37 (d, J = 8.9 Hz, 1H), 4.98 (q, J = 12.2 Hz, 2H), 4.80 (m, 3H), 4.40 (dd, J = 14.8, 6.0 Hz, 1H), 4.33 (dd, J = 14.8, 5.7 Hz, 1H), 3.52 (s, 3H), 2.75 (m, 6H), 2.61 (d, J = 5.2 Hz, 1H), 2.54 (m, 4H), 2.36 (m, 1H), 1.98 (m, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 172.9, 172.6, 171.4, 161.5 (d, J_C-F = 243.3 Hz), 155.3, 138.7, 137.9, 135.9, 130.1 (d, J_C-F = 7.7 Hz), 129.1, 128.7, 128.6, 128.4, 128.2, 127.7, 127.7, 127.3, 126.3, 115.2 (d, J_C-F = 20.9 Hz), 77.4, 77.3, 77.1, 76.8, 67.4, 59.9, 52.0, 50.3, 49.9, 49.6, 48.1, 43.6, 37.5. MS (ES+) m/z: 709.70 (M + 1)⁺. HPLC retention time = 8.47 min (97% analytical purity) (H₂O/CH₃CN from 15/85 to 0/100 in 5 min flowrate of 1 mL/min). Anal. calculated for C₄₁H₄₅FN₄O₆ (C, H, F, N): C, 69.47; H, 6.40; F, 2.68; N, 7.90. Found: C, 69.19; H, 6.55; F, 2.59; N, 7.73.

Tert-butyl(1-(benzyl(4-benzylcarbamoyl-1-(4-fluorobenzyl)piperidin-4-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (60): The reagents 4-piperidone monohydrate hydrochloride (0.18 mmol, 28 mg), benzylamine (0.36 mmol, 0.058 mL), Boc-Phe-OH (0.36 mmol, 96 mg) and benzyl isocyanide (0.36 mmol, 0.054 mL) in MeOH (2 mL) were reacted. 4-fluorobenzyl bromide (0.22 mmol, 42 mg) and K₂CO₃ (0.27 mmol, 37 mg) in DMF were then added to the obtained crude of reaction. Finally, 60 (57 mg, 47%) was obtained, by purification of the crude, as a white foam. ¹H NMR (500 MHz, DMSO-d6) δ 7.83 (t, J = 5.9 Hz, 1H), 7.23 (m, 16H), 7.05 (m, 2H), 7.01 (d, J = 7.2 Hz, 2H), 4.89 (d, J = 18.5 Hz, 1H), 4.78 (d, J = 18.5 Hz, 1H), 4.32 (m, 1H), 3.27 (m, 2H), 4.26 (m, 2H), 2.91 (dd, J = 13.9, 4.0 Hz, 1H), 2.68 (dd, J = 13.9, 9.7 Hz, 1H), 2.55 (m, 1H), 2.40 (m, 4H), 2.24 (t, J = 11.6 Hz, 1H), 1.76 (td, J = 12.5, 4.0 Hz, 1H), 1.53 (m, 1H), 1.22 (s, 9H). ¹³C NMR (126 MHz, DMSO-d6) δ 173.8, 172.7, 161.6 (d, J_C-F = 242.1 Hz), 155.8, 140.4, 139.4, 138.4, 134.9 (d, J = 3.2 Hz), 130.9 (d, J_C-F = 7.9 Hz), 129.8, 129.7, 129.0, 128.5, 128.4, 127.6, 127.5, 127.4, 126.9, 126.9, 126.7, 124.9, 115.2 (d, J_C-F = 21.0 Hz), 78.5, 78.4, 63.9, 61.2, 54.5, 49.9, 49.7, 48.1, 42.8, 37.8, 32.6, 32.2, 28.5. MS (ES+) m/z: 679.59 (M + 1)⁺. HPLC retention time = 9.13 min (96% analytical purity) (H₂O/CH₃CN from 15/85 to 0/100 in 5 min flowrate of 1 mL/min). Anal. calculated for C₄₁H₄₇FN₄O₄ (C, H, F, N): C, 72.54; H, 6.98; F, 2.80; N, 8.25. Found: C, 72.33; H, 6.71; F, 2.95; N, 8.01.

Tert-butyl(1-(benzyl(4-benzylcarbamoyl-1-(4-chlorobenzyl)piperidin-4-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (61): General procedure B was followed with a solution of Boc-Phe-OH (0.36 mmol, 96 mg), benzylamine (0.36 mmol, 0.058 mL), benzyl isocyanide (0.36 mmol, 0.054 mL) and 4-piperidone monohydrate hydrochloride (0.18 mmol, 28 mg), in MeOH (2 mL). The Ugi intermediate was treated with 4-chlorobenzyl bromide (0.22 mmol, 41 mg) and with K₂CO₃ (0.27 mmol, 37 mg) in DMF. The final residue was purified to give a white foam that resulted in compound 61 (68 mg, 54%). ¹H NMR (500 MHz, DMSO-d6) δ 7.84 (t, J = 5.9 Hz, 1H), 7.34 – 7.23 (m, 11H), 7.18 (m, 7H), 7.01 (d, J = 7.2 Hz, 2H), 4.90 (d, J = 18.5 Hz, 1H), 4.78 (d, J = 18.5 Hz, 1H), 4.31(m, 1H), 4.22 (m, 2H), 3.28 (s, 2H), 2.92 (dd, J = 13.8, 4.0 Hz, 1H), 2.69 (dd, J = 13.8, 9.7 Hz, 1H), 2.56 (m, 1H), 2.40 (m, 4H), 2.25 (t, J = 11.6 Hz, 1H), 1.77 (m, 1H), 1.53 (m, 1H), 1.22 (s, 9H). ¹³C NMR (126 MHz, DMSO-d6) δ 173.8, 172.7, 155.8, 140.4, 139.4, 139.1, 138.4, 137.9, 131.8, 130.9, 129.8, 129.7, 129.0, 128.5, 128.5, 128.4, 127.6, 127.5, 127.4, 126.9, 126.9, 126.7, 124.9, 78.5, 78.4, 63.9, 61.2, 55.4, 54.5, 49.9, 49.7, 48.1, 42.8, 37.8, 32.6, 32.2, 28.5. MS (ES+) m/z: 695.46 (M)⁺. HPLC 9.34 min (98%) (H₂O/CH₃CN from 15/85 to 0/100 in 5 min flowrate of 1 mL/min). Anal. for C₄₁H₄₇ClN₄O₄ (C, H, Cl, N C, 70.83; H, 6.81; Cl, 5.10; N, 8.06. Found: C, 70.99; H, 6.65; Cl, 5.39; N, 8.34.

Tert-butyl(1-(benzyl(4-benzylcarbamoyl-1-(4-methoxybenzyl)piperidin-4-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (62): Method B was used with 4-piperidone monohydrate hydrochloride (0.18 mmol, 28 mg), benzylamine (0.36 mmol, 0.058 mL), Boc-Phe-OH (0.36 mmol, 96 mg) and benzyl isocyanide (0.36 mmol, 0.054 mL) in MeOH (2 mL). Next, the Ugi adduct intermediate was treated with 4-methoxybenzyl bromide (0.22 mmol, 44 mg) and with K₂CO₃ (0.27 mmol, 37 mg) in DMF. The residue was chromatographed to afford 62 (46 mg, 37%) as a white foam. ¹H NMR (500 MHz, DMSO-d6) δ 7.82 (t, J = 5.9 Hz, 1H), 7.35 – 7.24 (m, 9H), 7.22 – 7.11 (m, 5H), 7.03 (m, 4H), 6.80 (d, J = 8.6 Hz, 2H), 4.89 (d, J = 18.5 Hz, 1H), 4.77 (d, J = 18.5 Hz, 1H), 4.30 (m, 1H), 4.25 (m, 2H), 3.69 (s, 3H), 3.21 (s, 2H), 2.91 (dd, J = 13.9, 4.0 Hz, 1H), 2.68 (dd, J = 13.9, 9.6 Hz, 1H), 2.54 (m, 1H), 2.38 (m, 4H), 2.22 (m, 1H), 1.73 (m, 1H), 1.51 (m, 1H), 1.22 (s, 9H). ¹³C NMR (126 MHz, DMSO-d6) δ 173.7, 172.8, 158.6, 155.8, 140.4, 139.5, 138.4, 130.6, 130.4, 129.7, 129.0, 128.5, 128.4, 127.5, 127.4, 126.9, 126.9, 126.7, 113.9, 78.5, 78.4, 63.9, 61.6, 55.4, 54.5, 49.9, 49.7, 48.1, 42.8, 37.9, 32.6, 32.2, 28.5. MS (ES+) m/z: 691.47 (M)⁺. HPLC retention time = 9.15 min (96% analytical purity) (H₂O/CH₃CN from 15/85 to 0/100 in 5 min flow rate of 1 mL/min). Anal. calculated for C₄₂H₅₀N₄O₅ (C, H, N): C, 73.02; H, 7.29; N, 8.11. Found: C, 73.21; H, 7.52; N, 8.03

Tert-butyl(1-(benzyl(4-benzylcarbamoyl-1-(4-nitrobenzyl)piperidin-4-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (63): Benzylamine (0.36 mmol, 0.058 mL), Boc-Phe-OH (0.36 mmol, 96 mg), benzyl isocyanide (0.36 mmol, 0.054 mL) and 4-piperidone monohydrate hydrochloride (0.18 mmol, 28 mg) were reacted in MeOH (2 mL) and then treated with 4-nitrobenzyl bromide (0.22 mmol, 47 mg) and with K₂CO₃ (0.27 mmol, 37 mg) in DMF. Finally, after work-up and purification, compound 63 (71 mg, 56%) was obtained as a white foam. ¹H NMR (500 MHz, DMSO-d6) δ 8.11 (d, J = 8.5 Hz, 2H), 7.85 (d, J = 5.9 Hz, 1H), 7.43 (d, J = 8.5 Hz, 2H), 7.35 – 7.24 (m, 9H), 7.22 – 7.11 (m, 5H), 7.02 (d, J = 7.2 Hz, 2H), 4.91 (d, J = 18.4 Hz, 1H), 4.80 (d, J = 18.4 Hz, 1H), 4.34 (m, 1H), 4.27 (t, J = 6.3 Hz, 2H), 3.43 (s, 2H), 2.92 (dd, J = 13.9, 4.0 Hz, 1H), 2.69 (dd, J = 13.9, 9.7 Hz, 1H), 2.53 (m, 1H), 2.41 (m, 4H), 2.30 (m, 1H), 1.85 (m, 1H), 1.56 (m, 1H), 1.22 (s, 9H). ¹³C NMR (126 MHz, DMSO-d6) δ 173.8, 172.7, 155.8, 147.3, 146.9, 140.4, 139.4, 138.4, 130.1, 129.7, 129.0, 128.5, 128.4, 127.5, 127.4, 126.9, 126.9, 126.7, 123.7, 78.5, 78.4, 63.8, 61.2, 54.5, 50.1, 49.84, 42.8, 37.8, 32.7, 32.2, 28.5. MS (ES+) m/z: 706.41 (M + 1)⁺. HPLC retention time = 9.06 min (95% analytical purity) (H₂O/CH₃CN from 15/85 to 0/100 in 5 min flow rate of 1 mL/min). Anal. calculated for C₄₁H₄₇N₅O₆ (C, H, N): C, 69.77; H, 6.71; N, 9.92. Found: C, 69.91; H, 6.49; N, 9.81.