3.2.7. General Method G: Solid Phase Peptide Coupling:
Swelling: The resin was bubbled for 30 min in a long-necked glass sinter in a solvent (DMF or DCM) with a volume 3x that of the bed volume. Subsequently the solvent was removed by filtration.
Washing off: The resin was washed with DMF (3 × 8 mL for 30 s), IPA (2 × 8 mL for 30 s) and finally with petroleum ether (40–60 °C) (2 × 8 mL for 30 s). The resin was air-dried, then dried in a vacuum oven at 40 °C overnight. The solvents were removed by filtration after each step.
Attachment to 2-chlorotrityl chloride resin: After swelling the resin in DCM the solution of Fmoc-1-aminoalkyltetrazole 4 and DIPEA in DCM was added to the resin, and the mixture was bubbled for 2 h. After filtration, the resin was bubbled with DCM, DIPEA and methanol mixture for 1 h. Later the resin was washed with DMF (3 × 8 mL for 30 s), methanol (3 × 8 mL for 30 s) then with DCM or petroleum ether (40–60 °C) (3 × 8 mL for 30 s) depending on whether the resin needed to be swelled or dried. The solvents and solutions were removed by filtration after each step.
Fmoc deprotection: The resin swelled in DMF was treated with a mixture of piperidine and DMF (1:4 ratio, 10 mL/g resin, 1 × 5 min and 3 × 10 min), then it was alternately washed with DMF and IPA (2 × 8 mL for 30 s) finished with washing with DMF (8 mL for 30 s). The solvents and solutions were removed by filtration after each step.
Fmoc test: A sample consisting of a few resin beads was washed with DMF (4 × 1 mL for 30 s), IPA (5 × 1 mL for 30 s) and with methanol (2 × 1 mL for 30 s), followed by transfer into a separate flask by washing with 1% TFA in DCM (2 × 1 mL for 30 s). The filtrate was placed on a TLC plate with a capillary and observed under UV light (254 nm). A positive test shows fluorescence on the TLC plate. The solvents and solutions were removed by filtration after each step.
Coupling: DIPEA (1 eq) was added to the solution of HBTU (3 eq) and the protected amino acid (3 eq) in DMF (10 mL). The solution was stirred for 8 min and then it was added to the swelled resins. After 20 min bubbling witH-Nitrogen, more DIPEA (0.5 eq) was added to the mixture, then it was bubbled until the TNBS test showed a negative result. After the reaction was complete the resin was washed off using the procedure mentioned above.
Coupling with succinic anhydride: The solution of succinic anhydride (3 eq) in DMF (10 mL) was added to the swelled resins, followed by the addition of DIPEA (1 eq). It was bubbled until the TNBS test showed a negative result (typically 1 h). After the reaction was over the resin was washed off using the procedure mentioned above.
TNBS test: The solution of DIPEA in DMF (10%, 3 drops) and the aqueous solution of TNBS (1 M, 3 drops) were mixed with sample consisting of a few resin beads. After a waiting period of 10 min, the resin beads were inspected under a light microscope (magnification 4×). The result was positive if the beads were coloured red, orange or yellow and it was negative if the beads were colourless.
Cleavage from resin: The resin swelled in DCM was treated with 1% TIS containing TFA, DMF mixture (5:95 ratio, 10 mL/g) for 1 h. After washing the resin with the same TFA, DCM mixture, the filtrate was evaporated under reduced pressure. (
Supplementary Materials).
HPLC method A: column: ACE 5 C18 (150 × 3 mm i.d.); eluent: isocratic: water (0.1% formic acid) - methanol (0.1% formic acid) 95:5 ratio. The purity of all the compounds tested using this method were ≥95%.
HPLC method B: column: ACE 5 C18 (150 × 3 mm i.d.); eluent: isocratic: water (0.1% formic acid) - methanol (0.1% formic acid) 80:20 ratio. The purity of all the compounds tested using this method were ≥95%.
HPLC method C: column: ACE 5 C18 (150 × 3 mm i.d.); eluent: isocratic: water (0.1% formic acid) - methanol (0.1% formic acid) 50:50 ratio. The purity of all the compounds tested using this method were ≥95%.
HPLC method D: column: ACE 5 C18 (150 × 3 mm i.d.); eluent: isocratic: water (0.1% formic acid) - methanol (0.1% formic acid) 20:80 ratio. The purity of all the compounds tested using this method were ≥95%.
Prep HPLC method A: column: Vydac protein and peptide 218TP510; eluent: gradient: water (0.1% formic acid) - methanol (0.1% formic acid) (0–90%).
Methyl-(2S or R)-2-{[(benzyloxy)carbonyl]amino}propanoate (
6a) [
29]: Cbz-
l/
d-alanine
5a (10.0 g, 45.0 mmol) was esterified and worked up using general method A, yielding the desired product as a white powder (10.6 g, 99%). The reaction and the work up were monitored by TLC (eluent: petroleum ether (40–60 °C) - ethyl acetate, 2:1 ratio, R
f: 0.51). Mp: 41–43 °C (literature: 43–44 °C, light petrol)[
30];
1H-NMR (300 MHz, DMSO-
d6) δ
H 7.74 (1H, d,
J = 7.5 Hz, 5-H), 7.34 (5H, m, 9,10,11-H), 5.03 (2H, s, 7-H), 4.11 (1H, quint,
J = 7.5 Hz, 2-H), 3.63 (3H, s, 4-H), 1.27 (3H, d,
J = 7.2 Hz, 3-H);
13C-NMR (75.5 MHz, DMSO-
d6) δ
C 173.8 (1-C), 156.3 (6-C), 137.4 (8-C), 128.8 (2C, 10-C), 128.3 (11-C), 128.2 (2C, 9-C), 65.9 (7-C), 52.3 (4-C), 49.8 (2-C), 17.4 (3-C); ν
max/cm
−1 3335 (N-H), 1703 (C=O), 1668 (C=O), 1524 (N-H), 1209 (C-O); MS(ESI)
m/
z 260.1 (M + Na)
+.
Benzyl-N-[(1S or R)-1-carbamoylethyl]carbamate (
7a)
[9]: Cbz-
l/
d-alanine methyl ester
6a (10.0 g, 42 mmol) was reacted and worked up using general method B, to give the product as a white powder (9.40 g, 99%). The reaction and the work up were monitored by TLC (eluent: petroleum ether (40–60 °C) - ethyl acetate, 1:1 ratio, R
f: 0.11). Mp: 131–132 °C (literature: 130–131 °C, methanol–diethyl ether)[
31];
1H-NMR (300 MHz, DMSO-
d6) δ
H 7.35 (6H, m, 5,9,10,11-H), 7.26 (1H, s, 4-H
a), 6.93 (1H, s, 4-H
b), 5.02 (2H, s, 7-H), 3.98 (1H, quint,
J = 7.2 Hz, 2-H), 1.21 (3H, d,
J = 7.2 Hz, 3-H);
13C-NMR (75.5 MHz, DMSO-
d6) δ
C 174.9 (1-C), 156.1 (6-C), 137.5 (8-C), 128.76 (2C, 10-C), 128.2 (11-C), 128.1 (2C, 9-C), 65.8 (7-C), 50.4 (2-C), 18.7 (3-C); ν
max/cm
−1 3386 (N-H), 3308 (N-H), 3196 (N-H), 1651 (C=O), 1537 (N-H), 1247 (C-O); MS(ESI)
m/
z 223.1 (M + H)
+.
Benzyl-N-[(1S or R)-1-cyanoethyl]carbamate (
8a) [
9]
: Cbz-
l/
d-alanine amide
7a (3.48 g, 15.7 mmol) was reacted and worked up using general method C. The crude product was washed with heptane to give the product as white crystals (3.12 g, 97%). The reaction and the work up were monitored by TLC (eluent: petroleum ether (40–60 °C) - ethyl acetate 1:1 ratio, R
f: 0.41). Mp: 79–80 °C (literature: 82–83 °C [
32]);
1H-NMR (300 MHz, DMSO-
d6) δ
H 8.14 (1H, d,
J = 5.4 Hz, 4-H), 7.37 (5H, m, 8,9,10-H), 5.08 (2H, s, 6-H), 4.59 (1H, quint,
J = 7.2 Hz, 2-H), 1.43 (3H, d,
J = 7.2 Hz, 3-H);
13C-NMR (75.5 MHz, DMSO-
d6) δ
C 155.7 (5-C), 137.0 (7-C), 128.9 (9-C), 128.5 (10-C), 128.4 (8-C), 120.9 (1-C), 66.5 (6-C), 38.2 (2-C), 18.8 (3-C); ν
max/cm
−1 3332 (N-H), 2356 (C≡N), 1686 (C=O), 1521 (N-H), 1253 (C-O); MS(ESI)
m/
z 227.1 (M + Na)
+.
Benzyl-N-[(1S or R)-1-(1H-1,2,3,4-tetrazol-5-yl)ethyl]carbamate (
9a)
[9]: Cbz-
l/
d-alanyl nitrile (
8a, 7.80 g, 38.0 mmol) was reacted and worked up using general method D. The product was gained as a white solid (7.55 g, 80%). The reaction and the work up were monitored by TLC (eluent: petroleum ether (40–60 °C) - ethyl acetate 1:1, R
f: 0.09). Mp: 134–138 °C (literature: 139–141 °C [
33]);
1H-NMR (300 MHz, DMSO-
d6) δ
H 7.99 (1H, d,
J = 6.9 Hz, 4-H), 7.33 (5H, m, 9,10,11-H), 5.01 (3H, m, 2,7-H), 1.49 (3H, d,
J = 7,2 Hz, 3-H);
13C-NMR (75.5 MHz, DMSO-
d6) δ
C 158.6 (1-C), 155.7 (6-C), 136.8 (8-C), 128.31 (2C, 10-C), 127.8 (3C, 9,11-C), 65.7 (7-C), 41.9 (2-C), 19.3 (3-C); ν
max/cm
–1 3308 (N-H), 2980, 2866, 2738, 2614 (tetrazole), 1686 (C=O), 1524 (N-H), 1255 (C-O); MS(ESI)
m/
z 248.1 (M + H)
+.
(1S or R)-1-(1H-1,2,3,4-tetrazol-5-yl)ethan-1-amine (
4a) [
9]: Cbz-
l/
d-aminoethyltetrazole (
9a, 0.75 g, 3.0 mmol) was deprotected using general method E. Following filtration, the solid residue was washed with ethanol and the filtrate was evaporated under reduced pressure to give the product as a white solid (0.33 g, 97%). The reaction and the work up were monitored by TLC (eluent: ethyl acetate - ethanol 8:1 ratio, R
f: 0.03). Mp: 230 °C (decomposed) (literature: 268–270 °C, (decomposed)
[9]).
1H-NMR (300 MHz, DMSO-
d6) δ
H 4.55 (1H, quart,
J = 6.6 Hz, 2-H), 1.54 (3H, d,
J = 6.9 Hz, 3-H);
13C-NMR (75.5 MHz, DMSO-
d6) δ
C 160.4 (1-C), 44.1 (2-C), 19.8 (3-C); ν
max/cm
–1 3388 (N-H), 2916, 2719, 2634, 2522 (tetrazole); MS(ESI)
m/
z 114.1 (M + H)
+.
Methyl-(2S)-2-{[(benzyloxy)carbonyl]amino}butanoate (
6b-L) [
34]: Cbz-
l-ethylglycine (
5b-L, 5.0 g, 21.0 mmol) was reacted and worked up using general method A, yielding the product as colourless oil (5.1 g, 97%). The reaction and the work up were monitored by TLC (eluent: petroleum ether (40–60 °C) - ethyl acetate, 2:1 ratio, R
f: 0.77).
1H-NMR (300 MHz, DMSO-
d6) δ
H 7.69 (1H, d,
J = 7.5 Hz, 6-H), 7.36 (5H, m, 10,11,12-H), 5.04 (2H, s, 8-H), 3.97 (1H, td,
J = 5.4,
J = 8.4 Hz, 2-H), 3.63 (1H, s, 5-H), 1.66 (2H, m, 3-H), 0.89 (3H, t,
J = 7.2 Hz, 4-H);
13C-NMR (75.5 MHz, DMSO-
d6) δ
C 173.2 (1-C), 156.6 (7-C), 137.4 (9-C), 128.8 (11-C), 128.3 (12-C), 128.2 (10-C), 65.9 (8-C), 55.8 (2-C), 52.2 (5-C), 24.6 (3-C), 10.9 (4-C); ν
max/cm
–1 3345 (N-H), 1703 (C=O), 1520 (N-H), 1206 (C-O); MS(ESI)
m/
z 250.8 (M – H)
-.
Benzyl-N-[(1S)-1-carbamoylpropyl]carbamate (
7b-L) [
35]: Cbz-
l-ethylglycine methyl ester (
6b-L, 2.0 g, 8.0 mmol) was reacted and worked up using general method B. The crude product was washed with diethyl ether to give the product as white crystals (1.72 g, 91%). The reaction and the work up were monitored by TLC (eluent: petroleum ether (40–60 °C) - ethyl acetate, 1:1 ratio, R
f: 0.07). Mp: 140–142 °C (literature: 141 °C [
36]);
1H-NMR (300 MHz, DMSO-
d6) δ
H 7.36 (5H, m, 10,11,12-H), 7.29 (1H, s, 5a-H), 7.20 (1H, d,
J = 8.4 Hz, 6-H), 6.95 (1H, s, 5b-H), 5.03 (2H, s, 8-H), 3.87 (1H, td,
J = 5.4,
J = 8.4 Hz, 2H), 1.67 (1H, m, 3a-H), 1.55 (1H, m, 3b-H), 0.86 (3H, t,
J = 7.2 Hz, 4-H);
13C-NMR (75.5 MHz, DMSO-
d6) δ
C 174. 2 (1-C), 156.4 (7-C), 137.6 (9-C), 128.8 (11-C), 128.2 (12-C), 128.1 (10-C), 65.8 (8-C), 56.3 (2-C), 25.6 (3-C), 10.8 (4-C); ν
max/cm
–1 3380 (N-H), 3308 (N-H), 3196 (N-H), 1649 (C=O), 1535 (N-H), 1240 (C-O); MS(ESI)
m/
z 237.1 (M + H)
+.
Benzyl-N-[(1S)-1-cyanopropyl]carbamate (
8b-L) [
37]: Cbz-
l-ethylglycine amide (
7b-L, 4.25 g, 18.0 mmol) was reacted and worked up using general method C to give the product as white crystals (3.88 g, 99%). The reaction and the work up were monitored by TLC (eluent: petroleum ether (40–60 °C) - ethyl acetate 1:2 ratio, R
f: 0.64). Mp: 43–45 °C;
1H-NMR (300 MHz, DMSO-
d6) δ
H 8.15 (1H, d,
J = 7.8 Hz, 5-H), 7.37 (5H, m, 9,10,11-H), 5.09 (2H, s, 7-H), 4.48 (1H, quart,
J = 7.8 Hz, 2-H), 1.78 (2H, pentd,
J = 1.5,
J = 7.5 Hz, 3-H), 0.96 (3H, t,
J = 7.2 Hz, 4-H);
13C-NMR (75.5 MHz, DMSO-
d6) δ
C 156.0 (6-C), 137.0 (8-C), 128.9 (10-C), 128.5 (11-C), 128.5 (9-C), 120.1 (1-C), 66.6 (7-C), 44.2 (2-C), 25.8 (3-C), 10.3 (4-C); ν
max/cm
–1 3312 (N-H), 1694 (C=O), 1524 (N-H), 1263 (C-O); MS(ESI)
m/
z 219.1 (M + H)
+.
Benzyl-N-[(1S)-1-(1H-1,2,3,4-tetrazol-5-yl)propyl]carbamate (
9b-L) [
38]: Cbz-
l-ethylglycyl nitrile (
8b-L, 3.9 g, 18 mmol) was reacted and worked up using general method D. The product was gained as a white solid (3.83 g, 81%). The reaction and the work up were monitored by TLC (eluent: petroleum ether (40–60 °C) - ethyl acetate 1:2, R
f: 0.20). Mp: 142–144 °C ;
1H-NMR (300 MHz, DMSO-
d6) δ
H 7.98 (1H, d,
J = 7.2 Hz, 6-H), 7.36 (5H, m, 10,11,12-H), 5.08 (1H, d,
J = 12.3 Hz, 8a-H), 5.02 (1H, d,
J = 12.6 Hz, 8b-H), 4.83 (1H, quart,
J = 8.1 Hz, 2-H), 1.89 (2H, m, 3-H), 0.89 (3H, t,
J = 7.2 Hz, 4-H);
13C-NMR (75.5 MHz, DMSO-
d6) δ
C 158.4 (1-C), 156.4 (7-C), 137.3 (9-C), 128.8 (11-C), 128.3 (12-C), 128.2 (10-C), 66.1 (8-C), 48.2 (2-C), 26.7 (3-C), 10.7 (4-C); ν
max/cm
–1 3296 (N-H), 2976, 2880, 2714, 2615, (tetrazole), 1686 (C=O), 1528 (N-H), 1263 (C-O); MS(ESI)
m/
z 262.1 (M + H)
+.
(1S)-1-(1H-1,2,3,4-tetrazol-5-yl)propane-1-amine (
4b-L) [
39]: Cbz-
l-aminopropyltetrazole (
9b-L, 3.72 g, 14.0 mmol) was deprotected using general method E. Following filtration, the solid residue was washed with ethanol and the filtrate was evaporated under reduced pressure to give the crude, which was washed to gain the pure product as a white solid (1.60 g, 88%). The reaction and the work up were monitored by TLC (eluent: ethanol, R
f: 0.18). Mp: 260 °C, decomposed (literature: 273–274 °C [
39]).
1H-NMR (300 MHz, D
2O) δ
H 4.62 (1H, t,
J = 7.2 Hz, 2-H), 2.05 (2H, quint,
J = 7.5 Hz, 3-H), 0.82 (3H, t,
J = 7.5 Hz, 4-H);
13C-NMR (75.5 MHz, D
2O) δ
C 159.6 (1-C), 48.7 (2-C), 26.0 (3-C), 8.9 (4-C); ν
max/cm
–1 2820, 2716, 2613, 2561 (tetrazole), 1530 (N-H); MS(ESI)
m/
z 128.1 (M + H)
+.
9H-Fluoren-9-ylmethyl-N-[(1S or R)-1-(1H-1,2,3,4-tetrazol-5-yl)ethyl]carbamate (
10a) [
40]:
l/
d-1-Aminoethyltetrazole (
4a, 2.00 g, 17.7 mmol) was reacted using general method F. The crude product was purified by recrystallization from acetonitrile to give the pure product as white crystals (0.92 g, 56%). The reaction and the work up was monitored by TLC (eluent: dichloromethane – ethanol 20:1 ratio, R
f: 0.07). Mp: 197–199 °C (literature: 200–202 °C (decomposed) [
40]);
1H-NMR (300 MHz, DMSO-
d6) δ
H 8.05 (1H, d,
J = 7.5 Hz, 4-H), 7.88 (2H, d,
J = 7.5 Hz, 13-H), 7.71 (2H, t,
J = 6.6 Hz, 10-H), 7.41 (2H, t,
J = 7.2 Hz, 12-C), 7.31 (2H, m, 11-H), 5.01 (1H, quint,
J = 7.2 Hz, 2-H), 4.32 (1H, m, 8-H), 4.24 (2H, m, 7-H), 1.51 (3H, d,
J = 6.9 Hz, 3-H);
13C-NMR (75.5 MHz, DMSO-
d6) δ
C 159.0 (1-C), 156.0 (6-C), 144.3 (9a-C), 144.2 (9b-C), 141.2 (14-C), 128.1 (12-C), 127.5 (11-C), 125.7 (10-C), 120.6 (13-C), 66.2 (7-C), 47.1 (8-C), 42.3 (2-C), 19.8 (3-C); ν
max/cm
–1 3310 (N-H), 2965, 2868, 2740, 2617 (tetrazole), 1686 (C=O), 1523 (N-H), 1258 (C-O); MS(ESI)
m/
z 336.2 (M + H)
+.
9H-Fluoren-9-ylmethyl-N-[(1S)-1-(1H-1,2,3,4-tetrazol-5-yl)propyl]carbamate (10b-L): l-1-Amino-ethyltetrazole (4b-L, 1.79 g, 6.9 mmol) was reacted using general method F. The crude product was purified by recrystallization in 2 generations from acetonitrile to give the pure product as white crystals (1.66 g, 75%). The reaction and the work up were monitored by TLC (eluent: dichloromethane – ethanol 10:1 ratio, Rf: 0.21). Mp: 209–211 °C (melted and decomposed); 1H-NMR (300 MHz, DMSO-d6) δH 16.26 (6-H), 8.04 (1H, d, J = 7.8 Hz, 5-H), 7.89 (2H, d, J = 7.5 Hz, 14-H), 7.72 (2H, t, J = 7.2 Hz, 11-H), 7.42 (2H, t, J = 7.5 Hz, 13-H), 7.33 (2H, m, 12-H), 4.81 (1H, quart, J = 8.1 Hz, 2-H), 4.34 (2H, m, 8-H), 4.23 (1H, m, 9-H), 1.91 (2H, m, 3-H), 0.88 (3H, t, J = 7.2 Hz, 4-H); 13C-NMR (75.5 MHz, DMSO-d6) δC 156.4 (7-C), 144.3 (10a-C), 144.2 (10b-C), 141.2 (15-C), 128.1 (13-C), 127.5 (12-C), 125.7 (11-C), 120.6 (14-C), 66.2 (8-C), 48.1 (2-C), 47.1 (9-C), 26.6 (3-C), 10.7 (4-C); νmax/cm–1 3300 (N-H), 2978, 2876, 2717, 2617 (tetrazole), 1682 (C=O), 1533 (N-H), 1265 (C-O); MS(ESI) m/z 350.1 (M + H)+; CHN [Found: C, 65.08; H, 5.53; N, 19.86. C19H19N5O2 requires C, 65.32; H, 5.48; N, 19.86%].
Benzyl-N-({[(1S)-1-(1H-1,2,3,4-tetrazol-5-yl)ethyl]carbamoyl}methyl)carbamate (13g-L): Prepared using general method G. The Fmoc group of Fmoc-1-l-aminoethyltetrazole attached to 2-chlorotrityl chloride resin (resin:loading; 1:1 ratio; 1.3 mmol) was removed, this was monitored by the Fmoc and TNBS tests. After the coupling of Cbz-glycine with the resin, the title compound was cleaved off. The cleaving mixture (TFA/DCM/TIS, ratio 5:95:1 eq) was removed by evaporation under reduced pressure. The residue washed with petroleum ether (40 – 60 °C) to obtain the product as white crystals (0.24 g, 59%). The reaction and the work up were monitored by TLC (eluent: ethanol, Rf: 0.56). Mp: 134–136 °C; 1H-NMR (300 MHz, DMSO-d6) δH 8.56 (1H, d, J = 7.2 Hz, 4-H), 7.35 (6H, m, 7,12,13,14-H), 5.24 (1H, quint, J = 7.2 Hz, 2-H), 5.03 (2H, s, 10-H), 3.68 (2H, d, J = 6.3 Hz, 6-H), 1.49 (3H, d, J = 6.9 Hz, 3-H); 13C-NMR (75.5 MHz, DMSO-d6) δC 169.3 (5-C), 158.9 (1-C), 156.9 (9-C), 137.5 (11-C), 128.8 (13-C), 128.2 (14-C), 128.1 (12-C), 65.9 (10-C), 43.8 (6-C), 40.3 (2-C), 19.8 (3-C); νmax/cm–1 3289 (N-H), 3269 (N-H), 1697 (C=O), 1661 (C=O), 1530 (N-H), 1244 (C-O); MS(ESI) m/z 305.1 (M + H)+; HRMS (Found (M + H)+ 305.1360. Calcd. for C13H17O3N6: (M + H)+ 305.1357.); (Purity test: HPLC method D).
2-Amino-N-[(1S)-1-(1H-1,2,3,4-tetrazol-5-yl)ethyl]acetamide (14g-L): Cbz-glycyl-1-l-aminoethyl-tetrazole (13g-L, 0.18 g, 0.57 mmol) was deprotected by general method E. Following filtration, the solid residue was washed with 30 mL methanol, which was discarded. The solid residue was washed into a separate flask with water, which was freeze dried to give the product as a white solid (0.01 g, 99%). Further purification was performed by Prep HPLC method A. Mp: 180 °C (decomposed); 1H-NMR (300 MHz, D2O) δH 5.27 (1H, quart, J = 6.9 Hz, 2-H), 3.75 (2H, s, 6-H), 1.52 (3H, d, J = 6.9 Hz, 3-H); 13C-NMR (75.5 MHz, D2O) δC 167.3 (5-C), 164.3 (1-C), 42.3 (2-C), 41.0 (6-C), 19.3 (3-C); νmax/cm–1 3215 (N-H), 1672 (C=O) 1547 (N-H); MS(ESI) m/z 171.1 (M + H)+; HRMS (Found (M + H)+ 171.0987. Calcd. for C5H11ON6: (M + H)+ 171.0989.); (Purity test: HPLC method A).
Benzyl-N-[(1S)-1-{[(1S)-1-(1H-1,2,3,4-tetrazol-5-yl)ethyl]carbamoyl}ethyl]carbamate (13a-LL): Prepared using general method G. The Fmoc group of Fmoc-1-l-aminoethyltetrazole attached to 2-chlorotrityl chloride resin (resin:loading; 1:1 ratio; 1.3 mmol) was removed, this was monitored by the Fmoc and TNBS tests. After the coupling of Cbz-l-alanine with the resin, the title compound was cleaved off. The cleaving mixture (TFA/DCM/TIS, ratio 5:95:1 eq) was removed by evaporation under reduced pressure and washed with petroleum ether (40–60 °C) to obtain the product as white crystals (0.23 g, 58%). The reaction and the work up were monitored by TLC (eluent: ethyl acetate and 1 drop of glacial acetic acid, Rf: 0.07). Mp: 186–187 °C; 1H-NMR (300 MHz, DMSO-d6) δH 16.18 (1H, br s, 9-H), 8.54 (1H, d, J = 7.5 Hz, 4-H), 7.35 (6H, m, 8,13-15-H), 5.23 (1H, quint, J = 7.2 Hz, 2-H), 5.05 (1H, d, J = 12.6 Hz, 11-Ha), 5.00 (1H, d, J = 12.6 Hz, 11-Hb), 4.09 (1H, quint, J = 7.2 Hz, 6-H), 1.50 (3H, d, J = 7.2 Hz, 3-H), 1.20 (3H, d, J = 7.2 Hz, 7-H); 13C-NMR (75.5 MHz, DMSO-d6) δC 172.1 (5-C), 158.3 (1-C), 155.6 (10-C), 137.0 (12-C), 128.3 (14-C), 127.7 (15-C), 127.7 (13-C), 65.3 (11-C), 49.8 (6-C), 40.3 (2-C), 19.2 (7-C), 17.9 (3-C); νmax/cm–1 3287 (N-H), 3264 (N-H), 1684 (C=O), 1655 (C=O), 1535 (N-H), 1230 (C-O); MS(ESI) m/z 319.2 (M + H)+; CHN [Found: C, 53.06; H, 5.64; N, 26.58. C14H18N6O3 requires C, 52.82; H, 5.70; N, 26.40%].
(2S)-2-amino-N-[(1S)-1-(1H-1,2,3,4-tetrazol-5-yl)ethyl]propanamide (
14a-LL) [19]: Cbz-
l-alanyl-1-
l-aminoethyltetrazole (
13a-LL, 0.58 g, 1.83 mmol) was deprotected by general method E. Following filtration, the solid residue was washed with methanol, which was discarded. The solid residue was washed into a separate flask with water, then freeze dried to give the product as a white solid (0.29 g, 86%). Further purification was performed by Prep HPLC method A. The reaction and the work up were monitored by TLC (eluent: ethanol, R
f: 0.07). Mp: 200 °C (decomposed);
1H-NMR (300 MHz, D
2O) δ
H 5.25 (1H, quart,
J = 6.9 Hz, 2-H), 3.57 (1H, quart,
J = 6.9 Hz, 6-H), 1.52 (3H, d,
J = 7.2 Hz, 3-H), 1.25 (3H, d,
J = 7.2 Hz, 7-H);
13C-NMR (75.5 MHz, D
2O) δ
C 176.2 (5-C), 164.3 (1-C), 49.9 (6-C), 42.0 (2-C), 19.4 (3-C), 19.2 (7-C); ν
max/cm
–1 3247 (N-H), 1643 (C=O) 1561 (N-H); MS(ESI)
m/
z 185.1 (M + H)
+; HRMS (Found (M+H)
+ 185.1145. Calcd. for C
6H
13ON
6: (M + H)
+ 185.1145.); (Purity test: HPLC method A).
Benzyl-N-[(1S)-1-{[(1R)-1-(1H-1,2,3,4-tetrazol-5-yl)ethyl]carbamoyl}ethyl]carbamate (
13a-LD)
[18]: Prepared using general method G. The Fmoc group of Fmoc-1-
d-aminoethyltetrazole attached to 2-chlorotrityl chloride resin (resin:loading; 1:1 ratio; 1.2 mmol) was removed, this was monitored by the Fmoc and TNBS tests. After the coupling of Cbz-
l-alanine with the resin, the title compound was cleaved off. The cleaving mixture (TFA/DCM/TIS, ratio 5:95:1 eq) was removed by evaporation under reduced pressure, and the residue washed with petroleum ether (40–60 °C) to obtain the product as white crystals (0.34 g, 89%). The reaction and the work up were monitored by TLC (eluent: ethyl acetate and 1 drop of glacial acetic acid, R
f: 0.07). Mp: 166–168 °C;
1H-NMR (300 MHz, DMSO-d
6) δ
H 8.56 (1H, d,
J = 7.5 Hz, 4-H), 7.35 (6H, m, 8,13-15-H), 5.23 (1H, quint,
J = 7.2 Hz, 2-H), 5.04 (1H, d,
J = 12.9 Hz, 11-H
a), 5.00 (1H, d,
J = 12.6 Hz, 11-H
b), 4.10 (1H, quint,
J = 7.5 Hz, 6-H), 1.50 (3H, d,
J = 7.2 Hz, 3-H), 1.23 (3H, d,
J = 6.9 Hz, 7-H);
13C-NMR (75.5 MHz, DMSO-d
6) δ
C 172.6 (5-C), 158.0 (1-C), 156.1 (10-C), 137.4 (12-C), 128.8 (14-C), 128.2 (15-C), 128.2 (13-C), 65.9 (11-C), 50.4 (6-C), 40.3 (2-C), 19.6 (3-C), 18.8 (7-C); ν
max/cm
–1 3289 (N-H), 2988, 2874, 2756, 2612 (tetrazole), 1682 (C=O), 1651 (C=O), 1528 (N-H), 1224 (C-O); MS(ESI)
m/
z 319.1 (M + H)
+; CHN [Found: C, 53.10; H, 5.78; N, 26.01. C
14H
18N
6O
3 requires C, 52.82; H, 5.70; N, 26.40%].
(2S)-2-Amino-N-[(1R)-1-(1H-1,2,3,4-tetrazol-5-yl)ethyl]propanamide (
14a-LD) [
18]: Cbz-
l-alanyl-1-
d-aminoethyltetrazole (
13a-LD, 0.58 g, 1.83 mmol) was deprotected by general method E. Following filtration, the solid residue was washed with ethanol, which was discarded. The solid residue was washed into a separate flask with water, which was freeze dried and purified by column chromatography (eluent: ethyl acetate – ethanol 4:1, 2:1, 1:1 then 0:1 ratio) to give the pure product as white solid (98 mg, 58%). The reaction and the work up were monitored by TLC (eluent: ethanol, R
f: 0.13). Mp: 69 °C (phase change), 177 °C (melted);
1H-NMR (300 MHz, D
2O) δ
H 5.24 (
1H, quart,
J = 6.6 Hz, 2-H), 4.00 (1H, quart,
J = 6.6 Hz, 6-H), 1.52 (3H, d,
J = 7.5 Hz, 3-H), 1.48 (3H, d,
J = 7.8 Hz, 7-H);
13C-NMR (75.5 MHz, D
2O) δ
C 170.5 (5-C), 164.0 (1-C), 49.3 (6-C), 42.3 (2-C), 19.1 (3-C), 16.7 (7-C); ν
max/cm
–1 3204 (N-H), 1667 (C=O), 1539 (N-H); MS(ESI)
m/
z 185.1 (M + H)
+; HRMS (Found (M + H)
+ 185.1145. Calcd. for C
6H
13ON
6: (M + H)
+ 185.1145.); (Purity test: HPLC method A).
(S)-Benzyl-(3-((1-(1H-tetrazol-5-yl)ethyl)amino)-3-oxopropyl)carbamate (13d-L): Prepared using general method G. The Fmoc group of Fmoc-1-l-aminoethyltetrazole attached to the 2-chlorotrityl chloride resin (resin:loading; 1:1 ratio; 1.3 mmol) was removed, this was monitored by the Fmoc and TNBS tests. After the coupling of Cbz-β-alanine with the resin, the title compound was cleaved off. The cleaving mixture (TFA/DCM/TIS, ratio 5:95:1 eq) was removed by evaporation under reduced pressure, and the residue washed with petroleum ether (40–60 °C) to obtained the product as white crystals (0.28 g, 67%). The reaction and the work up were monitored by TLC (eluent: ethyl acetate and 1 drop of glacial acetic acid, Rf: 0.39). Mp: 147–148 °C; 1H-NMR (300 MHz, DMSO-d6) δH 16.08 (1H, brs, 9-H), 8.56 (1H, d, J = 7.5 Hz, 4-H), 7.35 (5H, m, 13-15-H), 7.21 (1H, m, 8-H), 5.22 (1H, quint, J = 7.2 Hz, 2-H), 5.02 (2H, s, 11-H), 3.24 (2H, quart, J = 6.9Hz, 7-H), 2.34 (2H, t, J = 6.9 Hz, 6-H), 1.48 (3H, d, J = 7.2 Hz, 3-H); 13C-NMR (75,5 MHz, DMSO-d6) δC 170.6 (5-C), 158.8 (1-C), 156.5 (10-C), 137.6 (12-C), 128.8 (2C, 14-C), 128.2 (15-C), 128.1 (2C, 13-C), 65.7 (11-C), 40.9 (2-C), 37.3 (7-C), 35.9 (6-C), 19.6 (3-C); νmax/cm–1 3323 (N-H), 3273 (N-H), 2994, 2882, 2746, 2615 (tetrazole), 1692 (C=O), 1645 (C=O); MS(ESI) (M + Na)+ found 341.2 m/z; HRMS (Found (M + H)+ 319.1516. Calcd. for C14H19O3N6: (M + H)+ 319.1513.); (Purity test: HPLC method C).
3-Amino-N-[(1S)-1-(1H-1,2,3,4-tetrazol-5-yl)ethyl]propanamide (14d-L): Cbz-β-alanyl-1-l-amino-ethyltetrazole (13d-L, 0.20 g, 0.63 mmol) was deprotected by general method E. Following filtration, the solid residue was washed with methanol, which was discarded. The solid residue was washed into a separate flask with water, which was freeze dried to give the product as a light brown hygroscopic amorphous solid (0.09 g, 78%). Further purification was performed by Prep HPLC method A. 1H-NMR (300 MHz, D2O) δH 5.21 (1H, quart, J = 6.9 Hz, 2-H), 3.17 (2H, m, 7-H), 2.63 (2H, t, J = 6.6 Hz, 6-H), 1.48 (3H, d, J = 6.9 Hz, 3-H); 13C-NMR (75,5 MHz, DMSO-d6) δC 171.6 (5-C), 164.5 (1-C), 40.0 (2-C), 35.8 (7-C), 32.5 (6-C), 19.3 (3-C); νmax/cm–1 3232 (N-H), 1641 (C=O); MS(ESI) (M+H)+ found 185.1 m/z; HRMS (Found (M + H)+ 185.1145. Calcd. for C6H13ON6: (M + H)+ 185.1145.); (Purity test: HPLC method A).
Benzyl-N-[(1S)-1-{[(1S)-1-(1H-1,2,3,4-tetrazol-5-yl)ethyl]carbamoyl}butyl]carbamate (13e-LL): Prepared using general method G. The Fmoc group of Fmoc-1-l-aminoethyltetrazole attached to 2-chlorotrityl chloride resin (resin:loading; 1:1 ratio; 1.2 mmol) was removed, this was monitored by the Fmoc and TNBS tests. After the coupling of Cbz-L-norvaline with the resin, the title compound was cleaved off. The cleaving mixture (TFA/DCM/TIS, ratio 5:95:1 eq) was removed by evaporation under reduced pressure, and the residue washed with petroleum ether (40–60 °C) to obtain the product as white crystals (0.21, 51%). The reaction and the work up were monitored by TLC (eluent: ethyl acetate and 1 drop of glacial acetic acid, Rf: 0.2). Mp: 175–178 °C; 1H-NMR (300 MHz, DMSO-d6) δH 16.22 (1H, brs, 11-H), 8.55 (1H, d, J = 7.5, 4-H), 7.35 (6H, m, 10, 15-17-H), 5.23 (1H, quint, J = 7.2 Hz, 2-H), 5.02 (2H, s, 13-H), 4.04 (1H, m, 6-H), 1.59 (2H, m, 7-H), 1.49 (3H, d, J = 6.9 Hz, 3-H), 1.27 (2H, m, 8-H), 0.84 (3H, t, J = 7.2 Hz, 9-H); 13C-NMR (75.5 MHz, DMSO-d6) δC 172.2 (5-C), 159.1 (1-C), 156.4 (12-C), 137.5 (14-C), 128.8 (2C, 16-C), 128.2 (17-C), 128.1 (2C, 15-C), 65.8 (13-C), 54.6 (6-C), 40.9 (2-C), 34.3 (7-C), 19.7 (3-C), 19.1 (8-C), 14.0 (9-C); νmax/cm–1 3310 (N-H), 3271 (N-H), 2959, 2877, 2752, 2609 (tetrazole), 1682 (C=O), 1651 (C=O), 1533 (N-H), 1234 (C-O); MS(ESI) m/z 369.2 (M + Na)+; HRMS (Found (M + H)+ 347.1830. Calcd. for C16H23O3N6: (M + H)+ 347.1826.); (Purity test: HPLC method D)
(S)-N-((S)-1-(1H-tetrazol-5-yl)ethyl)-2-aminopentanamide (14e-LL): Cbz-l-norvalyl-1-l-amino-ethyltetrazole (13e-LL, 0.10 g, 0.29 mmol) was deprotected by general method E. Following filtration, the solid residue was washed with methanol, which was discarded. The residue was washed with water, then freeze dried to give the product as a white solid (47 mg, 77%). Further purification was performed by Prep HPLC method A. The reaction and the work up were monitored by TLC (eluent: ethanol, Rf: 0.16). Mp: 251–254 °C; 1H-NMR (300 MHz, D2O) δH 5.28 (1H, quart, J = 7.2 Hz, 2-H), 3.79 (1H, t, J = 6.6 Hz, 6-H), 1.70 (2H, m, 7-H), 1.54 (3H, d, J = 6.9 Hz, 3-H), 1.21 (2H, m, 8-H), 0.81 (3H, t, J = 7.2 Hz, 9-H); 13C-NMR (75.5 MHz, DMSO-d6) δC 171.2 (5-C), 164.7 (1-C), 53.5 (6-C), 42.0 (2-C), 33.8 (7-C), 19.1 (3-C), 17.6 (8-C), 12.8 (9-C); νmax/cm–1 3210 (N-H), 1687 (C=O), 1551 (N-H); MS(ESI) m/z 213.1 (M + H)+; HRMS (Found (M + H)+ 213.1458. Calcd. for C8H17ON6: (M + H)+ 213.1460.); (Purity test: HPLC method A).
Benzyl-N-[(1S)-3-methyl-1-{[(1S)-1-(1H-1,2,3,4-tetrazol-5-yl)ethyl]carbamoyl}butyl]carbamate (13h-LL): Prepared using general method G. The Fmoc group of Fmoc-1-l-aminoethyltetrazole attached to 2-chlorotrityl chloride resin (resin:loading; 1:2 ratio; 1.5 mmol) was removed, this was monitored by the Fmoc and TNBS tests. After the coupling of Cbz-l-leucine with the resin, the title compound was cleaved off. The cleaving mixture (TFA/DCM/TIS, ratio 5:95:1 eq) was removed by evaporation under reduced pressure, and the residue washed with petroleum ether (40–60 °C) to obtain the product as white crystals (0.45, 84%). The reaction and the work up were monitored by TLC (eluent: ethyl acetate – ethanol – drop of glacial acetic acid 1:1 ratio, Rf: 0.64). Mp: 149–150 °C; 1H-NMR (300 MHz, DMSO-d6) δH 16.1 (1H, brs, 11-H), 8.55 (1H, d, J = 7.5 Hz, 4-H), 7.36 (6H, m, 10,15,16,17-H), 5.22 (1H, quint, J = 6.9 Hz, 2-H), 5.04 (1H, d, J = 12.6, 13a-H), 5.00 (1H, d, J = 12.6 Hz, 13a-H), 4.07 (1H, m, 6-H), 1.60 (1H, m, 8-H), 1.49 (3H, d, J = 7.2 Hz, 3-H), 1.42 (2H, m, 7-H), 0.85 (3H, d, J = 6.6 Hz, 9a-H), 0.84 (3H, d, J = 6.6 Hz, 9b-H); 13C-NMR (75.5 MHz, DMSO-d6) δC 172.6 (5-C), 156.4 (12-C), 137.5 (14-C), 128.8 (16-C), 128.2 (17-C), 128.1 (15-C), 65.8 (13-C), 53.3 (6-C), 41.0 (2-C), 40.3 (7-C), 24.6 (8-C), 23.6 (9a-C), 21.8 (9b-C), 19.6 (3-C); νmax/cm–1 3316 (N-H), 3271 (N-H), 2961, 2876, 2743, 2615 (tetrazole), 1682 (C=O), 1653 (C=O), 1530 (N-H), 1233 (C-O); MS(ESI) m/z 360.8 (M + H)+; CHN [Found: C, 56.82.; H, 6.68; N, 23.05. C17H24N6O3 requires C, 56.65; H, 6.71; N, 23.32%].
(2S)-2-Amino-4-methyl-N-[(1S)-1-(1H-1,2,3,4-tetrazol-5-yl)ethyl]pentanamide (14h-LL): Cbz-l-leucyl-1-l-aminoethyltetrazole (13h-LL, 0.95 g, 2.6 mmol) was deprotected by general method E. Following filtration, the solid residue was washed with ethanol, which was discarded. The residue was washed with water, then freeze dried and washed with diethyl ether to give the product as a white solid (0.48 g, 81%). Further purification was performed by Prep HPLC method A. The reaction and the work up were monitored by TLC (eluent: ethanol, Rf: 0.3). Mp: 210 °C (decomposed); 1H-NMR (300 MHz, D2O) δH 5.28 (1H, quart, J = 7.2 Hz, 2-H), 3.49 (1H, t, J = 7.2Hz, 6-H), 1.53 (3H, d, J = 7.2 Hz, 3-H), 1.44 (3H, m, 7,8-H), 0.82 (3H, d, J = 4.8 Hz, 9a-H), 0.80 (3H, d, J = 5.1 Hz, 9b-H); 13C-NMR (75.5 MHz, DMSO-d6) δC 175.6 (5-C), 169.7 (1-C), 52.9 (6-C), 42.7 (7-C), 41.8 (2-C), 24.0 (8-C), 21.9 (9a-C), 21.4 (9b-C), 16.8 (3-C); νmax/cm–1 3219 (N-H), 1653 (C=O), 1562 (N-H); MS(ESI) m/z 227.2 (M + H)+; HRMS (Found (M + Na)+ 249.1436. Calcd. for C9H18ON6Na: (M + Na)+ 249.1434.); (Purity test: HPLC method A).
Benzyl-N-[(1S)-3-methyl-1-{[(1R)-1-(1H-1,2,3,4-tetrazol-5-yl)ethyl]carbamoyl}butyl]carbamate (13h-LD): Prepared using general method G. The Fmoc group of Fmoc-1-d-aminoethyltetrazole attached to 2-chlorotrityl chloride resin (resin:loading; 1:1 ratio; 1.2 mmol) was removed, this was monitored by Fmoc and TNBS tests. After the coupling of Cbz-l-leucine with the resin, the title compound was cleaved off. The cleaving mixture (TFA/DCM/TIS, ratio 5:95:1 eq) was removed by evaporation under reduced pressure, and washed with petroleum ether (40–60 °C) to obtain the product as white crystals (0.30 g, 69%). The reaction and the work up were monitored by TLC (eluent: ethyl acetate – ethanol 1:1 ratio, Rf: 0.63). Mp: 143–144 °C; 1H-NMR (300 MHz, DMSO-d6) δH 16.2 (1H, brs, 11-H), 8.61 (1H, d, J = 7.5 Hz, 4-H), 7.35 (6H, m, 10,15,16,17-H), 5.26 (1H, quint, J = 7.5 Hz, 2-H), 5.04 (1H, d, J = 13.2, 13a-H), 5.00 (1H, d, J = 12.9 Hz, 13a-H), 4.11 (1H, m, 6-H), 1.61 (1H, m, 8-H), 1.45 (3H, d, J = 6.9 Hz, 3-H), 1.42 (2H, m, 7-H), 0.88 (3H, d, J = 6.0 Hz, 9a-H), 0.86 (3H, d, J = 5.7 Hz, 9b-H); 13C-NMR (75.5 MHz, DMSO-d6) δC 172.4 (5-C), 158.5 (1-C), 156.4 (12-C), 137.5 (14-C), 128.8 (16-C), 128.2 (17-C), 128.1 (15-C), 65.9 (13-C), 53.4 (6-C), 41.4 (2-C), 40.3 (7-C), 24.7 (8-C), 23.5 (9a-C), 21.9 (9b-C), 19.6 (3-C); νmax/cm–1 3312 (N-H), 3269 (N-H), 2959, 2876, 2756, 2621 (tetrazole) 1690 (C=O), 1649 (C=), 1528 (N-H), 1234 (C-O); MS(ESI) m/z 361.2 (M + H); HRMS (Found (M + H)+ 361.1987. Calcd. for C17H25O3N6: (M + H)+ 361.1983.); (Purity test: HPLC method D).
(2S)-2-Amino-4-methyl-N-[(1R)-1-(1H-1,2,3,4-tetrazol-5-yl)ethyl]pentanamide (14h-LD): Cbz-l-leucyl-1-d-aminoethyltetrazole (13h-LD, 0.25 g, 0.7 mmol) was deprotected by general method E. Following filtration, the solid residue was washed with ethanol, which was discarded. The residue was washed with water, which was freeze dried and purified by column chromatography (eluent: ethyl acetate – ethanol 1:1 – 1:2 – 0:1) to give the pure product as a white solid (83 mg, 53%). The reaction and the work up were monitored by TLC (eluent: ethanol, Rf: 0.21). Mp: 136 °C (decomposed), 161 °C (melted); 1H-NMR (300 MHz, D2O) δH 5.27 (1H, quart, J = 6.9 Hz, 2-H), 3.95 (1H, t, J = 6.9Hz, 6-H), 1.71 (3H, m, 7,8-H), 1.53 (3H, d, J = 7.2 Hz, 3-H), 0.93 (3H, d, J = 6.0 Hz, 9a-H), 0.91 (3H, d, J = 6.0 Hz, 9b-H); 13C-NMR (75.5 MHz, DMSO-d6) δC 169.8 (5-C), 164.7 (1-C), 52.2 (6-C), 42.4 (2-C), 39.9 (7-C), 24.0 (8-C), 21.6 (9a-C), 21.3 (9b-C), 19.1 (3-C); νmax/cm–1 3194 (N-H), 1667 (C=O), 1543 (N-H); MS(ESI) m/z 227.2 (M + H)+; HRMS (Found (M + H)+ 227.1625 Calcd. for C9H19ON6: (M + H)+ 227.1620.); (Purity test: HPLC method A).
Benzyl-N-[(1S,2S)-2-methyl-1-{[(1S)-1-(1H-1,2,3,4-tetrazol-5-yl)ethyl]carbamoyl}butyl]carbamate (13i-LL): Prepared using general method G. The Fmoc group of Fmoc-1-l-aminoethyltetrazole attached to 2-chlorotrityl chloride resin (resin:loading; 1:1 ratio; 1.8 mmol) was removed, this was monitored by the Fmoc and TNBS tests. After the coupling of Cbz-L-isoleucine with the resin, the title compound was cleaved off. The cleaving mixture (TFA/DCM/TIS, ratio 5:95:1 eq) was removed by evaporation under reduced pressure, and washed with petroleum ether (40–60 °C) to obtain the product as white crystals (0.40 g, 62%). The reaction and the work up were monitored by TLC (eluent: ethyl acetate – ethanol 10:1 ratio, Rf: 0.13). Mp: 186–189 °C; 1H-NMR (300 MHz, DMSO-d6) δH 8.58 (1H, d, J = 7.2 Hz, 4-H), 7.35 (5H, m, 16,17,18-H), 7.24 (1H, d, J = 9.0 Hz, 11-H), 5.24 (1H, quint, J = 6.9 Hz, 2-H), 5.05 (1H, d, J = 12.6 Hz, 14a-H), 5.00 (1H, d, J = 12.6 Hz, 14b-H), 3.92 (1H, t, J = 8.1 Hz, 6-H), 1.70 (1H, m, 7-H), 1.50 (3H, d, J = 6.9 Hz, 3-H), 1.35 (1H, m, 9a-H), 1.07 (1H, m, 9b-H), 0.77 (3H, t, J = 7.5 Hz, 10-H), 0.75 (3H, d, J = 7.5 Hz, 8-H); 13C-NMR (75.5 MHz, DMSO-d6) δC 171.4 (5-C), 158.3 (1-C), 156.5 (13-C), 137.5 (15-C), 128.8 (17-C), 128.2 (18-C), 128.1 (16-C), 65.9 (14-C), 59.3 (6-C), 39.9 (2-C), 36.9 (7-C), 24.7 (9-C), 19.5 (3-C), 15.7 (8-C), 11.3 (10-C); νmax/cm–1 3279 (N-H), 2963, 2878, 2693, 2608 (tetrazole), 1690 (C=O), 1651 (C=O), 1535 (N-H), 1234 (C-O); MS(ESI) m/z 361.1 (M + H)+; CHN [Found: C, 56.61.; H, 6.77; N, 23.01. C17H24N6O3 requires C, 56.65; H, 6.71; N, 23.32%].
(2S,3S)-2-amino-3-methyl-N-[(1S)-1-(1H-1,2,3,4-tetrazol-5-yl)ethyl]pentanamide (14i-LL): Cbz-l-isoleucyl-1-l-aminoethyltetrazole (13i-LL, 0.35 g, 0.9 mmol) was deprotected by general method E. Following filtration, the solid residue was washed with ethanol, which was discarded. The residue was washed with water, which was freeze dried to give the product as white solid (190 mg, 86%). Due to hygroscopicity, the corresponding hydrochloride salt was made with 2M HCl in diethyl ether solution (20 mL) for characterization. Further purification was performed by Prep HPLC method A. The reaction and the work up were monitored by TLC (eluent: ethanol, Rf: 0.31). Mp: 164 - 167 °C (melted and decomposed); 1H-NMR (300 MHz, D2O) δH 5.39 (1H, quart, J = 7.2 Hz, 2-H), 3.85 (1H, d, J = 5.7 Hz, 6-H), 1.92 (1H, m, 7-H), 1.65 (3H, d, J = 7.2 Hz, 3-H), 1.31 (1H, m, 9a-H), 1.14 (1H, m, 9b-H), 0.89 (3H, d, J = 6.9 Hz, 8-H), 0.83 (3H, t, J = 7.5 Hz, 10-H); 13C-NMR (75.5 MHz, DMSO-d6) δC 169.1 (5-C), 158.1 (1-C), 57.7 (6-C), 40.7 (2-C), 36.3 (7-C), 23.9 (9-C), 17.6 (3-C), 14.1 (8-C), 10.4 (10-C); νmax/cm–1 3240 (N-H), 1682 (C=O), 1574 (N-H); MS(ESI) m/z 227.2 (M + H)+; HRMS (Found (M + Na)+ 249.1436. Calcd. for C9H18ON6Na: (M + Na)+ 249.1434.); (Purity test: HPLC method A).
Benzyl-(5S)-2-oxo-5-{[(1S)-1-(1H-1,2,3,4-tetrazol-5-yl)ethyl]carbamoyl}pyrrolidine-1-carboxylate (13f-LL): Prepared using general method G. The Fmoc group of the Fmoc-1-l-aminoethyltetrazole attached to 2-chlorotrityl chloride resin (resin:loading; 1:2 ratio; 2.0 mmol) was removed. After coupling of Cbz-L-pyroglutamine with the resin, the title compound was cleaved off. The cleaving mixture (TFA/DCM/TIS, ratio 5:95:1 eq) was subsequently removed by evaporation under reduced pressure. The crude product was purified by column chromatography (eluent: ethyl acetate – ethanol 10:1, 6:1 then 1:1 ratio) to obtain the product as white crystals (0.46g, 65%). The reaction and the work up were monitored by TLC (eluent: ethyl acetate – ethanol 10:1, Rf: 0.09). Mp: 195 °C (decomposed); 1H-NMR (300 MHz, DMSO-d6) δH 16.05 (1H, brs, 11-H), 8.93 (1H, d, J = 7.5 Hz, 4-H), 7.36 (5H, m, 15-17-H), 5.17 (3H, m, 2,13-H), 4.64 (1H, dd, J = 2.7 Hz, J = 9.0 Hz, 6-H), 2.41 (2H, m, 8-H), 2.26 (1H, m, 7-Ha), 1.94 (1H, m, 7-Hb), 1.42 (3H, d, J = 6.9 Hz, 3-H); 13C-NMR (75.5 MHz, DMSO-d6) δC 174.1 (9-C), 171.0 (5-C), 158.3 (1-C), 150.9 (12-C), 136.0 (14-C), 128.8 (2C, 16-C), 128.5 (17-C), 128.0 (15-C), 67.5 (13-C), 59.3 (6-C), 40.2 (2-C), 31.3 (8-C), 22.3 (7-C), 19.6 (3-C); νmax/cm–1 3289 (N-H), 2993, 2868, 2739, 2615 (tetrazole), 1780 (C=O), 1701 (C=O), 1655 (C=O), 1545 (N-H), 1236 (C-O); MS(ESI) m/z 381.2 (M + Na)+. CHN [Found: C, 53.63; H, 5.10; N, 23.18. C16H18N6O4 requires C, 53.63; H, 5.06; N, 23.45%].
(S)-N-((S)-1-(1H-tetrazol-5-yl)ethyl)-5-oxopyrrolidine-2-carboxamide (14f-LL): Cbz-l-pyroglutamyl-1-l-aminoethyltetrazole (13f-LL, 0.44 g, 1.2 mmol) was reacted by general method E. Following filtration, the solid residue was washed with methanol and the filtrate was evaporated under reduced pressure. The residual oil was triturated with diethyl ether to give the title compound as a white solid (0.23 g, 84%). Further purification was performed by Prep HPLC method A. The reaction and the work up were monitored by TLC (ethanol, Rf: 0.3). Mp: 160 °C (decomposed); 1H-NMR (300 MHz, D2O) δH 5.35 (1H, quart, J = 7.2 Hz, 2-H), 4.34 (1H, dd, J = 4.8 Hz, J = 8.4 Hz, 6-H), 2.52 (1H, m, 7-Ha), 2.40 (2H, m, 8-H) 2.04 (1H, m, 7-Hb), 1.61 (3H, d, J = 6.9 Hz, 3-H); 13C-NMR (75.5 MHz, D2O) δC 182.3 (9-C), 174.6 (5-C), 159.0 (1-C), 56.9 (6-C), 41.0 (2-C), 29.3 (8-C), 25.0 (7-C), 18.1 (3-C); νmax/cm–1 3262 (N-H), 1649 (C=O), 1547 (N-H); MS(ESI) m/z 225.1 (M + H)+. HRMS (Found (M + H)+ 225.1096. Calcd. for C8H13O2N6: (M + H)+ 225.1095.); (Purity test: HPLC method A).
Benzyl-N-[(1S)-2-phenyl-1-{[(1S)-1-(1H-1,2,3,4-tetrazol-5-yl)ethyl]carbamoyl}ethyl]carbamate (13j-LL): Prepared using general method G. The Fmoc group of Fmoc-1-l-aminoethyltetrazole attached to 2-chlorotrityl chloride resin (resin:loading; 1:1 ratio; 1.0 mmol) was removed, this was monitored by the Fmoc and TNBS tests. After the coupling of Cbz-l-phenylalanine with the resin, the title compound was cleaved off. The cleaving mixture (TFA/DCM/TIS, ratio 5:95:1 eq) was removed by evaporation under reduced pressure, and washed with petroleum ether (40–60 °C) to obtain the product as white crystals (0.32 g, 79%). Further purification was performed using a CombiFlash EZ Prep system (Teledyne-ISCO, Lincoln, NE, USA, eluent: gradient: dichloromethane – methanol 0–30%). The reaction and the work up were monitored by TLC (eluent: ethyl acetate, Rf: 0.11). Mp: 191–193 °C; 1H-NMR (300 MHz, DMSO-d6) δH 16.21 (1H, brs, 8.72 (1H, d, J = 7.5 Hz, 4-H), 7.44 (1H, d, J = 8.7 Hz, 12-H), 7.33 - 7.24 (10H, m, 9,10,11,17,18,19-H), 5.27 (1H, quint, 6.9 Hz, 2-H), 4.96 (1H, d, J = 13.5 Hz, 15a-H), 4.92 (1H, d, J = 13.5 Hz, 15b-H), 4.30 (1H, m, 6-H), 3.03 (1H, dd, J = 3.3 Hz, J = 13.8 Hz, 7a-H), 2.72 (1H, dd, J = 10.8 Hz, J = 13.5 Hz, 7b-H), 1.52 (3H, d, J = 6.9 Hz, 3-H); 13C-NMR (75.5 MHz, DMSO-d6) δC 171.7 (5-C), 158.7 (1-C), 156.3 (14-C), 138.5 (8-C), 137.5 (16-C), 129.6, 128.7, 128.5, 128.1, 127.9, 126.7 (9,10,11,17,18,19-C), 65.7 (15-C), 56.5 (6-C), 40.0 (2-C), 37.8 (7-C), 19.7 (3-C); νmax/cm–1 3300 (N-H), 2984, 2874, 2741, 2615 (tetrazole), 1684 (C=O), 1651 (C=O), 1531 (N-H), 1234 (C-O); MS(ESI) m/z 417.2 (M + Na)+; CHN [Found: C, 60.78; H, 5.68; N, 20.92. C20H22N6O3 requires C, 60.90; H, 5.62; N, 21.31%].
(2S)-2-Amino-3-phenyl-N-[(1S)-1-(1H-1,2,3,4-tetrazol-5-yl)ethyl]propanamide (14j-LL): Cbz-l-phenylalanyl-1-l-aminoethyltetrazole (13j-LL, 0.30 g, 0.8 mmol) was deprotected by general method E. Following filtration, the solid residue was washed with ethanol, which was discarded. The residue was washed with water, which was freeze dried, then purified by column chromatography (eluent: dichloromethane – ethanol 10:1, 8:1, 6:1, 1:1 then 0:1 ratio) to give the pure product as a white solid (40 mg, 20%). Further purification was performed by Prep HPLC method A. The reaction and the work up were monitored by TLC (eluent: ethanol, Rf: 0.39). Mp: 152–155 °C; 1H-NMR (300 MHz, D2O) δH 7.15 (3H, m, 10,11-H), 7.01 (2H, m, 9-H), 5.24 (1H, quart, J = 6.9 Hz, 2-H), 3.77 (1H, m, 6-H), 2.97 (1H, dd, J = 6.0, J = 13.5 Hz, 7a-H), 2.89 (1H, dd, J = 7.8, J = 13.5 Hz, 7b-H), 1.43 (3H, d, J = 7.2 Hz, 3-H); 13C-NMR (75.5 MHz, D2O) δC 163.5 (1-C), 150.6 (5-C), 135.5 (8-C), 129.2 (9-C), 128.6 (10-C), 127.1 (11-C), 55.5 (6-C), 41.3 (2-C), 39.3 (7-C), 19.3 (3-C); νmax/cm–1 3217 (N-H), 1651 (C=O), 1528 (N-H); MS(ESI) m/z 283.2 (M + Na)+. HRMS (Found (M + H)+ 261.1462. Calcd. for C12H17ON6: (M + H)+ 261.1458.); (Purity test: HPLC method A).
Benzyl-N-[(1S)-1-{[(1S)-1-(1H-1,2,3,4-tetrazol-5-yl)propyl]carbamoyl}ethyl]carbamate (13b-LL): Prepared using general method G. The Fmoc group of the Fmoc-1-l-aminopropyltetrazole attached to 2-chlorotrityl chloride resin (resin:loading; 1:1 ratio; 2.9 mmol) was removed. After coupling of Cbz-l-alanine with the resin, the title compound was cleaved off. The cleaving mixture (TFA/DCM/TIS, ratio 5:95:1 eq) was removed by evaporation under reduced pressure. The crude product was subsequently washed with petroleum ether (40–60 °C) to obtain the product as white crystals (0.49 g, 52%). Mp: 174–175 °C; 1H-NMR (300 MHz, DMSO-d6) δH 8.44 (1H, d, J = 7.5 Hz, 5-H), 7.41 (1H, d, J = 7.2 Hz, 9-H), 7.34 (5H, m, 14,15,16-H), 5.06 (1H, m, 2-H), 5.02 (2H, s, 12-H), 4.10 (1H, quint, J = 6.9 Hz, 7-H), 1.88 (2H, m, 3-H), 1.19 (3H, d, J = 7.2 Hz, 8-H), 0.87 (3H, t, J = 7.2 Hz, 4-H); 13C-NMR (75.5 MHz, DMSO-d6) δC 173.0 (6-C), 156.8 (1-C), 156.1 (11-C), 137.5 (13-C), 128.8 (15-C), 128.2 (16-C), 128.1 (14-C), 65.8 (12-C), 50.4 (7-C), 45.8 (2-C), 26.8 (3-C), 18.4 (8-C), 10.6 (4-C); νmax/cm–1 3300 (N-H), 3265 (N-H), 2976, 2876, 2735, 2617 (tetrazole), 1691 (C=O), 1651 (C=O), 1533 (N-H), 1246 (C-O); MS(ESI) m/z 333.1 (M + H)+; CHN [Found: C, 54.20; H, 6.11; N, 24.91. C15H20N6O3 requires C, 54.21; H, 6.07; N, 25.29%].
(2S)-2-Amino-N-[(1S)-1-(1H-1,2,3,4-tetrazol-5-yl)propyl]propanamide (14b-LL): Cbz-l-alanyl-1-l-aminopropyltetrazole (13b-LL, 0.40 g, 1.2 mmol) was deprotected by general method E. Following filtration, the solid residue was washed with ethanol, which was discarded. The residue was washed with water, which was freeze dried to give the product as a white solid (206 mg, 86%). Due to poor solubility, the corresponding hydrochloride salt was prepared using 2M HCl in diethyl ether solution (20 mL) for characterization (hygroscopic solid). Further purification was performed by Prep HPLC method A. The reaction and the work up were monitored by TLC (eluent: ethanol, Rf: 0.12). 1H-NMR (300 MHz, DMSO-d6) δH 9.17 (1H, d, J = 7.6 Hz, 5-H), 8.34 (3H, brs, 9-H), 5.09 (1H, quart, J = 7.8 Hz, 2-H), 3.93 (1H, quart, J = 6.7 Hz, 7-H), 1.92 (2H, m, 3-H), 1.38 (3H, d, J = 7.0 Hz, 8-H), 0.90 (3H, t, J = 7.4 Hz, 4-H); 13C-NMR (75.5 MHz, DMSO-d6) δC 170.0 (6-C), 158.0 (1-C), 48.6 (7-C), 46.4 (2-C), 26.8 (3-C), 17.4 (8-C), 10.6 (4-C); IR; MS(ESI) m/z 199.1 (M + H)+; HRMS (Found (M + H)+ 199.1302. Calcd. for C7H15ON6: (M + H)+ 199.1302.); (Purity test: HPLC method A).
N-[(1S)-1-{[(1S)-1-{[(1S)-1-(1H-1,2,3,4-tetrazol-5-yl)ethyl]carbamoyl}ethyl]carbamoyl} ethyl]carbamate (13-LLL): Prepared using general method G. The Fmoc group of Fmoc-1-l-aminoethyltetrazole attached to the 2-chlorotrityl chloride resin (resin:loading; 1:1 ratio; 1.0 mmol) was removed, this was monitored by the Fmoc and TNBS tests. After the coupling of Fmoc-l-alanine with the resin, the Fmoc group was removed again. Finally, Cbz-l-alanine was attached to the resin, and the title compound was cleaved off. The cleaving mixture (TFA/DCM/TIS, ratio 5:95:1 eq) was removed by evaporation under reduced pressure, and washed with petroleum ether (40–60 °C) to obtain the product as white crystals (0.19 g, 47%). Further purification was performed by Prep HPLC method A. Mp: 199–201 °C (melted and decomposed); 1H-NMR (300 MHz, DMSO-d6) δH 16.09 (13-H), 8.48 (1H, d, J = 6.9 Hz, 4-H), 7.93 (1H, d, J = 7.5 Hz, 8-H), 7.42 (1H, d, J = 7.2 Hz, 12-H), 7.35 (5H, m, 17-19-H), 5.21 (1H, quint, J = 6.9 Hz, 2-H), 5.02 (2H, s, 15-H), 4.29 (1H, quint, J = 7.2 Hz, 6-H), 4.06 (1H, quint, J = 7.2 Hz, 10-H), 1.49 (3H, d, J = 6.9 Hz, 3-H), 1.19 (6H, d, J = 6.9 Hz, 7,11-H); 13C-NMR (75.5 MHz, DMSO-d6) δC 172.6 (9-C), 172.3 (5-C), 159.1 (1-C), 156.1 (14-C), 137.5 (16-C), 128.8 (18-C), 128.2 (19-C), 128.1 (17-C), 65.8 (15-C), 50.4 (10-C), 48.4 (6-C), 40.9 (2-C), 19.7 (3-C), 18.5 (7-C), 18.4 (11-C); νmax/cm–1 3298(N-H), 1694 (C=O), 1643 (C=O), 1526 (N-H), 1219 (C-O); MS(ESI) m/z 390.2 (M + H)+; HRMS (Found (M + H)+ 390.1883. Calcd. for C17H24O4N7: (M + H)+ 390.1884.); (Purity test: HPLC method C).
(S)-N-((S)-1-(1H-tetrazol-5-yl)ethyl)-2-((S)-2-aminopropanamido)propanamide (14c-LLL): Cbz-l-alanyl-l-alanyl-1-l-aminoethyltetrazole (13c-LLL, 0.12 g, 0.31 mmol) was reacted by the general method E. Following the filtration, the solid residue was washed with methanol, then the filtrate was evaporated under reduced pressure to give the crude product (1.11 g, 95%), further recrystallization from a methanol - ethyl acetate mixture provided the pure product as white crystals (0.477 g, 40%). Further purification was performed by Prep HPLC method A. The reaction and the work up were monitored by TLC (eluent: ethanol, Rf: 0.13). Mp: 135 °C (melted and decomposed); 1H-NMR (300 MHz, D2O) δH 5.25 (1H, quart, J = 7.2 Hz, 2-H), 4.30 (1H, quart, J = 7.2 Hz, 6-H), 3.95 (1H, quart, J = 6.9 Hz, 10-H), 1.52 (3H, d, J = 7.2 Hz, 3-H), 1.43 (3H, d, J = 7.2 Hz, 11-H), 1.33 (3H, d, J = 7.2 Hz, 7-H); 13C-NMR (75.5 MHz, D2O) δC 173.6 (5-C), 171.7 (9-C), 164.1 (1-C), 49.8 (6-C), 49.0 (10-C), 42.0 (2-C), 19.4 (3-C), 17.0 (11-C), 16.5 (7-C); νmax/cm–1 3252 (N-H), 1639 (C=O), 1539 (N-H); MS(ESI) m/z 256.1 (M + H)+; HRMS (Found (M + H)+ 256.1520. Calcd. for C9H18O2N7: (M + H)+ 256.1516.); (Purity test: HPLC method A).
3-{[(1S)-1-{[(1S)-1-(1H-1,2,3,4-tetrazol-5-yl)ethyl]carbamoyl}ethyl]carbamoyl}propanoic acid (16-LL): Prepared using general method G. The Fmoc group of Fmoc-1-l-aminoethyltetrazole attached to the 2-chlorotrityl chloride resin (resin:loading; 1:2 ratio; 1.4 mmol) was removed. After the coupling of Fmoc-L-alanine with the resin, the Fmoc group was removed again. Finally, a succinyl group was attached to the resin using succinic anhydride, and the title compound was cleaved off. The cleaving mixture (TFA/water/TIS, ratio 95:2.5:2.5) was removed by evaporation under reduced pressure, and recrystallized from a water – acetone solvent mixture to obtain the product as white crystals (0.16 g, 40%). The reaction and the work up were monitored by TLC (eluent: ethyl acetate – ethanol – drop of glacial acetic acid 1:1 ratio, Rf: 0.21). Mp: 170–171 °C; 1H-NMR (300 MHz, D2O) δH 5.32 (1H, quart, J = 7.2 Hz, 2-H), 4.28 (1H, quart, J = 7.2 Hz, 6-H), 2.64 (2H, m, 11-H), 2.54 (2H, m, 10-H), 1.62 (3H, d, J = 6.9 Hz, 3-H), 1.35 (3H, d, J = 7.2 Hz, 7-H); 13C-NMR (75.5 MHz, D2O) δC 176.9 (12-C), 175.0 (9-C), 174.9 (5-C), 158.5 (1-C), 49.7 (6-C), 40.7 (2-C), 29.9 (10-C), 29.0 (11-C), 17.8 (3-C), 16.4 (7-C); νmax/cm–1 3358 (N-H), 3254 (O-H), 2992, 2945, 2886, 2778, 2712 (tetrazole), 1713 (C=O), 1636 (C=O), 1543 (N-H), 1526 (N-H), 1231 (C-O); MS(MALDI) m/z 285.30 (M + H)+; HRMS (Found (M + H)+ 285.1310. Calcd. for C10H17O4N6: (M + H)+ 285.1306.); (Purity test: HPLC method B).
3-{[(1S)-1-{[(1S)-1-{[(1S)-1-(1H-1,2,3,4-tetrazol-5-yl)ethyl]carbamoyl}ethyl]carbamoyl}ethyl]-carbamoyl}propanoic acid (17-LLL): Prepared using general method G. The Fmoc group of Fmoc-1-l-aminoethyltetrazole attached to the 2-chlorotrityl chloride resin (resin:loading; 1:2 ratio; 2.8 mmol) was removed. After the coupling of Fmoc-l-alanine with the resin, the Fmoc group was removed, and the free amino group was coupled with Fmoc-l-alanine again. After removing the Fmoc protection, a succinyl group was attached to the resin using succinic anhydride, and the title compound was cleaved off. The cleaving mixture (TFA/water/TIS, ratio 95:2.5:2.5) was removed by evaporation under reduced pressure, and triturated with diethyl ether to obtain the product as white crystals (0.39 g, 79%). Further purification was performed by Prep HPLC method A. The reaction and the work up were monitored by TLC (eluent: ethyl acetate – ethanol – drop of glacial acetic acid 1:1 ratio, Rf: 0.22). Mp: 186 °C (melted and decomposed); 1H-NMR (300 MHz, D2O) δH 5.32 (1H, quart, J = 7.2 Hz, 2-H), 4.29 (1H, quart, J = 7.2 Hz, 6-H), 4.21 (1H, quart, J = 7.2 Hz, 10-H), 2.66 (2H, m, 16-H), 2.55 (2H, m, 15-H), 1.62 (3H, d, J = 6.9 Hz, 3-H), 1.35 (3H, d, J = 7.2 Hz, 7-H), 1.34 (3H, d, J = 7.2 Hz, 11-H); 13C-NMR (75.5 MHz, D2O) δC 177.0 (17-C), 175.3 (14-C), 175.2 (9-C), 174.6 (5-C), 158.1 (1-C), 50.0 (10-C), 49.6 (6-C), 40.7 (2-C), 29.8 (16-C), 29.0 (15-C), 17.8 (3-C), 16.3 (7-C), 16.2 (11-C); νmax/cm–1 3343 (N-H), 3381 (O-H), 1707 (C=O), 1651 (C=O), 1634 (C=O), 1539 (N-H), 1201 (C-O); MS(MALDI) m/z 356.30 (M + H)+. HRMS (Found (M + H)+ 356.1682. Calcd. for C13H22O5N7: (M + H)+ 356.1677.) (Purity test: HPLC method A).
3-{[(1S)-1-{[(1S)-1-{[(1S)-1-{[(1S)-1-(1H-1,2,3,4-tetrazol-5-yl)ethyl]carbamoyl}ethyl]carbamoyl}ethyl]-carbamoyl}ethyl]carbamoyl}propanoic acid (18-LLLL): Prepared using general method G. The Fmoc group of Fmoc-1-l-aminoethyltetrazole attached to the 2-chlorotrityl chloride resin (resin:loading; 1:1 ratio; 1.5 mmol) was removed, and resin was coupled with Fmoc-l-alanine, the Fmoc deprotection and coupling with Fmoc-l-alanine procedure was repeated 2 more times. After removing the Fmoc protection for the final time, a succinyl group was attached to the resin using succinic anhydride, and the title compound was cleaved off. The cleaving mixture (TFA/water/TIS, ratio 95:2.5:2.5) was removed by evaporation under reduced pressure. The residue was triturated with diethyl ether to obtain the product as a white solid (0.44 g, 68%). Further purification was carried out by column chromatography (eluent: ethyl acetate – ethanol 4:1, 1:1 then 0:1 ratio) and by Prep HPLC method A. The reaction and the work up were monitored by TLC (eluent: ethanol, Rf: 0.21). Mp: 231 °C (decomposed); 1H-NMR (300 MHz, D2O) δH 5.32 (1H, quart, J = 7.0 Hz, 2-H), 4.26 (3H, m, 6,10,14-H), 2.64 (2H, m, 19-H), 2.58 (2H, m, 18-H), 1.63 (3H, d, J = 7.0 Hz, 3-H), 1.36 (9H, d, J = 7.2 Hz, 7,11,15-H); 13C-NMR (75.5 MHz, D2O) δC 177.9 (20-C), 175.5 (17-C), 175.3, 174.9, 174.4 (5,9,13-C), 158.7 (1-C), 50.1, 49.8, 49.6 (6,10,14-C), 40.9 (2-C), 29.9 (18-H), 29.2 (19-H), 18.0 (3-C), 16.4, 16.3, 16.2 (7, 11, 15-C); νmax/cm–1 3265 (N-H), 1690 (C=O), 1653 (C=O), 1531 (N-H), 1223 (C-O); MS(ESI) m/z 425.1 (M-H)-; HRMS (Found (M + H)+ 427.2047. Calcd. for C16H27O6N8: (M + H)+ 427.2048.); (Purity test: HPLC method B).