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Keywords:

  • chemical structure;
  • heterocycles;
  • peptide;
  • steroids;
  • terpenes

Abstract

  1. Top of page
  2. Abstract
  3. Methods and Materials
  4. Results and Discussion
  5. Conclusion
  6. Acknowledgments
  7. References
  8. Supporting Information

Cbz-(protected)-tri- and tetrapeptide conjugates with steroids, sugars, terpenes, and heterocycles were prepared using Cbz-(protected)-tri- and tetrapeptidoylbenzotriazoles as active intermediates.

Peptides and their derivatives such as hormones, neurotransmitters, and neuromodulators act as signal molecules in diverse biological and medicinal applications and thus have attracted considerable synthetic attention (1,2). Esterifications of amino acids and peptides for the protection of carboxylic acid functionality and for their activation to make peptide conjugates are well known (3–5).

Peptidyl steroids, with amino acid or peptide units linked to the steroidal frame, are important in living systems; examples such as cholyl glycine and cholyl taurine are found in the human body (6). Amino acid esters of hydroxylic terpenes, a class of peptide conjugate, are effective medicinal agents, for example, for atherosclerosis (7). N-protected α-aminoacyl esters derived from long-chain alkanols containing 12–22 carbon atoms and α-amino acids or peptides possess medicinal, nutritional and industrial utility (8). The combination of sugar moieties with peptides is important in biochemical processes ranging from cell growth regulation, immune responses, and binding of pathogens to intercellular communication, intercellular targeting, cancer cell metastasis, and inflammation (9–11).

Reported preparative methods for peptides and their conjugates include (i) activating the C-terminus with coupling reagents such as carbodiimides, (4) HOBt, and HOAt (3)-based uronium (12), phosphonium (13), and immonium salts (3) and (ii) procedures involving the isolated C-terminus-activated intermediates such as acyl halides (14–16), acylimidazoles (17), active alkyl and phenolic esters (18), and acyloxy boron intermediates (19).

Peptide conjugates with sugar, steroid, and heterocyclic moieties have previously been prepared (i) by solid-phase syntheses (20,21) and (ii) in solution phase by stepwise synthesis using regular coupling reagents like EDC, (22) TBTU, (23) DCC (24), and BOP (25). However, such methods suffer from difficulties in product purification and analysis by (HPLC): High Performance Liquid Chromatography, (20) coupling steps being sensitive to the environment and water, (20) and low yields (23), especially with glycine units (24).

We have already utilized N-acylbenzotriazoles extensively for N- (26,27), C- (28,29), O- (30), and S-acylations (31). Previously, we reported the facile synthesis of amino acid conjugates with steroids, terpenes, and sugars. (32) Herein, we present the extension of our methodology for the convenient and efficient formation of Cbz-protected tri- and tetrapeptide conjugates with sugars, steroids, terpenes, and heterocyclic nuclei of biological importance.

Methods and Materials

  1. Top of page
  2. Abstract
  3. Methods and Materials
  4. Results and Discussion
  5. Conclusion
  6. Acknowledgments
  7. References
  8. Supporting Information

Melting points were determined on a hot-stage apparatus and are uncorrected. All reactions were carried out under nitrogen unless otherwise specified. All microwave-assisted reactions were carried out with a single-mode cavity Discover Microwave Synthesizer (CEM Corporation, Matthews, NC, USA). Column chromatography was conducted on flash silica gel (200–425 mesh). Visualization of TLC plates was via UV and phosphomolybdic acid staining. 1H NMR (300 MHz) and 13C NMR (75 MHz) spectra were determined in CDCl3 with TMS as the internal standard, (CD3)2CO or DMSO-d6.

General procedure for the synthesis of N-Cbz-(dipeptidoyl)benzotriazoles (1a–c)

The compounds were synthesized following our established procedure (32).

Benzyl((S)-1-(((S)-1-(1H-benzo[d][1,2,3]triazol-1-yl)-1-oxo-3-phenylpropan-2-yl)amino)-1-oxopropan-2-yl)carbamate (Cbz-Ala-Phe-Bt) (1a)

White microcrystals (93%); mp 148–150 °C; Lit mp 148–149 °C.

Benzyl((S)-1-(((S)-1-(1H-benzo[d][1,2,3]triazol-1-yl)-1-oxo-3-phenylpropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (Cbz-Val-Phe-Bt) (1b)

White microcrystals (93%); mp 188–190 °C; 1H NMR (CDCl3) δ 8.22 (d, J = 8.7 Hz, 1H), 8.15 (d, J = 8.4 Hz, 1H), 7.68 (t, J = 7.7 Hz, 1H), 7.54 (t, J = 7.7 Hz, 1H), 7.41–7.27 (m, 5H), 7.24–7.17 (m, 3H), 7.13–7.07 (m, 2H), 6.70 (d, J = 7.2 Hz, 1H), 6.30–6.20 (m, 1H), 5.31 (d, J = 8.4 Hz, 1H), 5.17–5.06 (m, 2H), 4.08 (t, J = 7.2 Hz, 1H), 3.47 (dd, J = 14.1, 5.1 Hz, 1H), 3.24 (dd, J = 14.0, 7.7 Hz, 1H), 2.19–2.02 (m, 1H), 0.94 (d, J = 6.6 Hz, 3H), 0.87 (d, J = 6.3 Hz, 3H); 13C NMR (CDCl3) δ 171.3, 170.4, 156.5, 146.2, 136.4, 135.0, 131.2, 131.0, 129.4, 129.0, 128.7, 128.4, 128.3, 127.7, 126.8, 120.6, 114.5, 67.4, 60.4, 54.2, 38.8, 31.1, 19.3, 17.9. Anal. Calcd for C28H29N5O4: C, 67.32; H, 5.85; N, 14.02; found: C, 67.44; H, 5.86; N, 14.09.

(S)-Benzyl(1-((2-(1H-benzo[d][1,2,3]triazol-1-yl)-2-oxoethyl)amino)-1-oxo-3-phenyl-propan-2-yl)carbamate (Cbz-Phe-Gly-Bt) (1c)

White microcrystals (85%); mp 169–171 °C; 1H NMR (DMSO-d6) δ 8.89 (t, J = 5.5 Hz, 1H), 8.28 (d, J = 8.4 Hz, 1H), 8.23 (d, J = 8.1 Hz, 1H), 7.80 (t, J = 7.6 Hz, 1H), 7.70–7.60 (m, 2H), 7.35–7.19 (m, 10H), 5.08–4.91 (m, 4H), 4.50–4.42 (m, 1H), 3.16 (dd, J = 13.5, 3.0 Hz, 1H), 2.88–2.78 (m, 1H); 13C NMR (DMSO-d6) δ 172.7, 168.5, 155.9, 145.3, 138.1, 137.0, 131.0, 130.6, 129.2, 128.3, 128.1, 127.6, 127.4, 126.6, 126.3, 120.1, 113.7, 65.2, 56.1, 42.7, 37.6. Anal. Calcd for C25H23N5O4: C, 65.64; H, 5.07; N, 15.31; found: C, 65.67; H, 5.00; N, 15.10.

General procedure for the synthesis of N-Cbz-tripeptides (2a–c)

The compounds were synthesized following our established procedure (33).

(5S,8S)-8-Benzyl-5-methyl-3,6,9-trioxo-1-phenyl-2-oxa-4,7,10-triazadodecan-12-oic acid (Cbz-Ala-Phe-Gly-OH) (2a)

White microcrystals (78%); mp 108–110 °C; 1H NMR (DMSO-d6) δ 8.35 (t, J = 5.7 Hz, 1H), 7.93–7.88 (m, 1H), 7.46–7.43 (m, 1H), 7.42–7.25 (m, 5H), 7.24–7.16 (m, 5H), 5.00 (s, 2H), 4.60–4.52 (m, 1H), 4.01 (q, J = 6.9 Hz, 1H), 3.78 (d, J = 5.7 Hz, 2H), 3.10–2.98 (m, 1H), 2.88–2.76 (m, 1H), 1.12 (d, J = 6.9 Hz, 3H); 13C NMR (DMSO-d6) δ 172.2, 171.3, 171.1, 155.2, 137.1, 136.5, 128.8, 127.9, 127.5, 127.3, 125.7, 65.0, 53.0, 49.8, 40.2, 37.2, 17.7. Anal. Calcd for C22H25N3O6. 0.5 H2O: C, 60.54; H, 6.00; N, 9.63; found: C, 60.89; H, 5.83; N, 9.73.

(5S,8S)-8-Benzyl-5-isopropyl-3,6,9-trioxo-1-phenyl-2-oxa-4,7,10-triazadodecan-12-oic acid (Cbz-Val-Phe-Gly-OH) (2b)

White microcrystals (91%); mp 212–215 °C; 1H NMR (DMSO-d6) δ 8.36 (t, J = 5.7 Hz, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.40–7.28 (m, 5H), 7.27–7.11 (m, 6H), 5.02 (br s, 2H), 4.68–4.55 (m, 1H), 3.86–3.71 (m, 3H), 3.02 (dd, J = 13.5, 3.9 Hz, 1H), 2.78 (dd, J = 13.8, 9.9 Hz, 1H), 1.91–1.78 (m, 1H), 0.78–0.64 (m, 6H); 13C NMR (DMSO-d6) δ 171.4, 171.0, 170.8, 156.0, 137.6, 137.0, 129.2, 128.4, 128.0, 127.8, 127.7, 126.2, 65.5, 60.4, 53.5, 40.7, 37.8, 30.4, 19.2, 18.1. Anal. Calcd for C24H29N3O6: C, 63.28; H, 6.42; N, 9.22; found: C, 63.48; H, 6.47; N, 9.20.

(5S,11S)-5-Benzyl-11-isobutyl-3,6,9-trioxo-1-phenyl-2-oxa-4,7,10-triazadodecan-12-oic acid (Cbz-Phe-Gly-Leu-OH) (2c)

White microcrystals (80%); mp 79–81 °C; 1H NMR (DMSO-d6) δ 8.34–8.30 (m, 1H), 7.99 (d, J = 7.8 Hz, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.33–7.19 (m, 10H), 4.99–4.90 (m, 2H), 4.32–4.22 (m, 2H), 3.77 (d, J = 7.8 Hz, 2H), 3.08–3.00 (m, 1H), 2.80–2.71 (m, 1H), 1.68–1.60 (m, 1H), 1.55–1.50 (m, 2H), 0.89 (d, J = 6.6 Hz, 3H), 0.85 (d, J = 6.3 Hz, 3H); 13C NMR (DMSO-d6) δ 174.0, 171.9, 168.6, 156.0, 138.2, 137.0, 129.2, 128.3, 128.1, 127.7, 127.5, 126.3, 65.3, 56.3, 50.2, 41.8, 40.3, 40.1, 37.3, 24.2, 22.8, 21.4. Anal. Calcd for C25H31N3O6: C, 63.95; H, 6.65; N, 8.95; found: C, 64.06; H, 6.75; N, 8.98.

General procedure for the synthesis of N-Cbz-tripeptidoylbenzotriazole (3a–c)

The compounds were synthesized following our established procedure (33).

Benzyl((S)-1-(((S)-1-((2-(1H-benzo[d][1,2,3]triazol-1-yl)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-1-oxopropan-2-yl)carbamate(Cbz-Ala-Phe-Gly-Bt) (3a)

White microcrystals (92%); mp 182–185 °C; 1H NMR (DMSO-d6) δ 8.83–8.81 (m,1H), 8.30 (d, J = 8.4 Hz, 1H), 8.23 (d, J = 8.1 Hz, 1H), 8.02 (d, J = 7.8 Hz, 1H), 7.81 (t, J = 7.8 Hz, 1H), 7.64 (t, J = 7.8 Hz, 1H), 7.45 (d, J = 7.5 Hz, 1H), 7.35–7.22 (m, 10 H), 5.02–4.96 (m, 4H), 4.70–4.68 (m, 1H), 4.03 (t, J = 6.9 Hz, 1H), 3.14–3.08 (m, 1H), 2.96–2.78 (m, 1H), 1.13 (d, J = 6.9 Hz, 3H); 13C NMR (DMSO-d6) δ 172.2, 168.4, 155.6, 145.2, 137.5, 131.0, 130.5, 129.2, 128.3, 128.0, 127.7, 126.6, 126.2, 120.1, 113.7, 65.4, 53.5, 50.2, 42.6, 37.7, 18.2. Anal. Calcd for C28H28N6O5: C, 63.63; H, 5.34; N, 15.90; found: C, 63.22; H, 5.37; N, 15.79.

Benzyl((S)-1-(((S)-1-((2-(1H-benzo[d][1,2,3]triazol-1-yl)-2-oxoethyl)amino)-1-oxo-3-phenyl-propan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (Cbz-Val-Phe-Gly-Bt) (3b)

White microcrystals (93%); mp 214–215 °C; 1H NMR (DMSO-d6) δ 8.84 (brs, 1H), 8.29 (d, J = 8.1 Hz, 1H), 8.22 (d, J = 8.4 Hz, 1H), 8.07 (d, J = 8.1 Hz, 1H), 7.81 (t, J = 7.5 Hz, 1H), 7.64 (t, J = 7.5 Hz, 1H), 7.25–7.15 (m, 11H), 5.10–4.90 (m, 4H), 4.85–4.68 (m, 1H), 3.84 (t, J = 7.5 Hz, 1H), 3.19–3.05 (m, 1H), 2.95–2.80 (m, 1H), 1.98–1.79 (m, 1H), 0.80–0.60 (m, 6H); 13C NMR (DMSO-d6) δ 172.1, 170.9, 168.4, 156.0, 145.3, 137,6, 137.0, 131.0, 130.6, 129.2, 128.3, 128.0, 127.6, 126.2, 120.1, 113.7, 65.4, 60.4, 53.4, 42.6, 37.9, 30.4, 19.1, 18.1. Anal. Calcd for C30H32N6O5: C, 64.73; H, 5.79; N, 15.10; found: C, 64.60; H, 5.79; N, 15.01.

Benzyl((S)-1-((2-(((S)-1-(1H-Benzo[d][1,2,3]triazol-1-yl)-4-methyl-1-oxopentan-2-yl)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (Cbz-Phe-Gly-Leu-Bt) (3c)

White microcrystals (84%); mp 144–146 °C; 1H NMR (DMSO-d6) δ 8.81–8.77 (m, 1H), 8.42–8.38 (m, 1H), 8.28 (d, J = 7.8 Hz, 1H), 8.20 (d, J = 7.2 Hz, 1H), 8.10–8.06 (m, 1H), 7.80–7.77 (m, 1H), 7.65–7.59 (m, 1H), 7.39–7.12 (m, 10H), 4.94 (s, 2H), 4.51–4.45 (m, 1H), 4.29–4.25 (m, 1H), 3.88–3.74 (m, 2H), 3.08–3.03 (m, 1H), 2.80–2.72 (m, 1H), 1.80–1.65 (m, 1H), 1.60–1.50 (m, 2H), 0.99–0.87 (m, 6H); 13C NMR (DMSO-d6) δ 173.7, 172.6, 169.3, 169.1, 156.6, 146.0, 138.9, 137.6, 131.7, 129.9, 129.0, 128.7, 128.4, 128.1, 127.3, 126.9, 120.9 114.5, 65.9, 56.9, 51.5, 43.2, 42.8, 38.0, 24.8, 23.8, 22.2. Anal. Calcd for C31H34N6O5: C, 65.25; H, 6.01; N, 14.73; found: C, 65.21; H, 5.85; N, 14.56.

General procedure for the synthesis of N-Cbz-tetrapeptides (4a–c)

The compounds were synthesized following our established procedure (33).

(5S,8S,14S)-8-Benzyl-5,14-dimethyl-3,6,9,12-tetraoxo-1-phenyl-2-oxa-4,7,10,13-tetraazapentadecan-15-oic acid (Cbz-Ala-Phe-Gly-Ala-OH) (4a)

White microcrystals (74%); mp 184–186 °C; 1H NMR (DMSO-d6) δ 8.26 (t, J = 6.0 Hz, 1H), 8.08 (d, J = 6.9 Hz, 1H), 7.98 (d, J = 6.9 Hz, 1H), 7.41 (d, J = 7.8 Hz, 1H), 7.36–7.30 (m, 5H), 7.24–7.16 (m, 5H), 5.08–4.94 (m, 2H), 4.52–4.42 (m, 1H), 4.21 (quintet, J = 7.2 Hz, 1H), 4.08–3.96 (m, 1H), 3.73 (d, J = 5.7 Hz, 2H), 3.04 (dd, J = 13.5, 4.2 Hz, 1H), 2.88–2.78 (m, 1H), 1.28 (d, J = 7.2 Hz, 3H), 1.12 (d, J = 7.2 Hz, 3H); 13C NMR (DMSO-d6) δ 173.9, 172.4, 171.1, 168.3, 155.6, 137.6, 136.9, 129.2, 128.3, 128.0, 127.7, 126.2, 65.4, 53.9, 50.1, 47.5, 41.7, 37.3, 18.0, 17.3. Anal. Calcd for C25H30N4O7: C, 60.23; H, 6.07; N, 11.24; found: C, 59.95; H, 6.04; N, 11.07.

(5S,8S)-8-Benzyl-5-methyl-3,6,9,12-tetraoxo-1-phenyl-2-oxa-4,7,10,13-tetraaza-penta decan-15-oic acid (Cbz-Ala-Phe-Gly-Gly-OH) (4b)

White microcrystals (85%); mp 148–150 °C; 1H NMR (DMSO-d6) δ 8.88 (t, J = 5.4 Hz, 1H), 8.68 (t, J = 5.8 Hz, 1H), 8.57 (d, J = 7.5 Hz, 1H), 8.04 (d, J = 8.4 Hz, 1H), 8.04–7.94 (m, 5H), 7.88–7.78 (m, 5H), 5.63 (d, J = 12.6 Hz, A part of AB system, 1H), 5.57 (d, J = 12.9 Hz, B part of AB system, 1H), 5.16–5.04 (m, 1H), 4.68–4.57 (m, 1H), 4.38–4.24 (m, 4H), 3.66–3.61 (m, 1H), 3.46–3.22 (m, 1H), 1.71 (d, J = 6.9 Hz, 3H); 13C NMR (DMSO-d6) δ 172.4, 171.1, 171.1, 169.0, 155.7, 137.6, 136.9, 129.3, 128.3, 128.0, 127.8, 126.2, 65.5, 53.8, 50.2, 41.8, 40.6, 37.4, 18.1. Anal. Calcd for C24H28N4O7: C, 59.50; H, 5.82; N, 11.56; found: C, 59.12; H, 5.84; N, 11.88.

(5S,11S)-5-Benzyl-11-isobutyl-3,6,9,12-tetraoxo-1-phenyl-2-oxa-4,7,10,13-tetraazapenta decan-15-oic acid (Cbz-Phe-Gly-leu-Gly-OH) (4c)

White microcrystals (88%); mp 109–110 °C; 1H NMR (DMSO-d6) δ 8.32–8.26 (m, 2H), 7.94 (d, J = 8.4 Hz, 1H), 7.59 (d, J = 8.7 Hz, 1H), 7.35–7.17 (m, 10H), 5.10–4.90 (m, 2H), 4.38–4.32 (m, 1H), 4.30–4.23 (m,1H), 3.80–3.72 (m, 4H), 3.10–3.00 (m, 1H), 2.80–2.70 (m, 1H), 1.65–1.60 (m, 1H), 1.51–1.45 (m, 2H), 0.88 (d, J = 6.6 Hz, 3H), 0.84 (d, J = 6.6 Hz, 3H); 13C NMR (DMSO-d6) δ 172.3, 171.8, 171.1, 168.4, 155.9, 138.2, 136.9, 129.2, 128.3, 128.0, 127.7, 127.4, 126.2, 65.3, 56.2, 50.7, 42.0, 41.0, 40.6, 37.3, 24.1, 23.1, 21.6. Anal. Calcd for C27H34N4O7: C, 61.58; H, 6.51; N, 10.64; found: C, 61.69; H, 6.62; N, 10.48.

General procedure for the synthesis of N-Cbz-tetrapeptidoylbenzotriazole (5a–c)

The compounds were synthesized following our established procedure (33) (Table 1).

Table 1.   Preparation of N-Pg–tripeptidoylbenzotriazoles 3a–c and tetrapeptidoylbenzotriazoles 5a–c
EntryReactantProductYield (%)Mp(°C)
  1. aLit mp 186–187 °C (33), bLit mp 212–214 °C (33).

1Z–L–Ala–L–Phe–Gly–OH 2aZ–L–Ala–L–Phe–Gly–Bt 3a92180–182a
2Z–L–Val–L–Phe–Gly–OH 2bZ–L–Val–L–Phe–Gly–Bt 3b93214–215
3Z–L–Phe–Gly-L–Leu-OH 2cZ–L–Phe–Gly-L–Leu-Bt 3c84144–146
4Z–L–Ala–L–Phe–Gly–L-Ala-OH 4aZ–L–Ala–L–Phe–Gly–L-Ala-Bt 5a72200–202b
5Z–L–Ala–L–Phe–Gly–Gly-OH 4bZ–L–Ala–L–Phe–Gly–Gly-Bt 5b62144–145
6Z–L–Phe–Gly–L-Leu–Gly-OH 4cZ–L–Phe–Gly–L–Leu–Gly-Bt 5c79280–282
Benzyl((S)-1-(((S)-1-((2-(((S)-1-(1H-benzo[d][1,2,3]triazol-1-yl)-1-oxopropan-2-yl)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-1-oxopropan-2-yl)carbamate (Cbz-Ala-Phe-Gly-Ala-Bt) (5a)

White microcrystals (72%); mp 200–202 °C; 1H NMR (DMSO-d6) δ 8.72–8.70 (m, 1H), 8.30 (d, J = 7.8 Hz, 2H), 8.22 (d, J = 8.4 Hz, 1H), 8.00 (d, J = 6.9 Hz, 1H), 7.80 (t, J = 7.5 Hz, 1H), 7.64 (t, J = 7.7 Hz, 1H), 7.46–7.10 (m, 11H), 5.66–5.60 (m, 1H), 5.02–4.96 (m, 2H), 4.48–4.45 (m, 1H), 4.02–3.95 (m, 1H), 3.82 (d, J = 5.4 Hz, 2H), 3.10–2.97 (m, 1H), 2.86–2.80 (m, 1H), 1.56 (d, J = 6.9 Hz, 3H), 1.10 (d, J = 8.1 Hz, 3H); 13C NMR (DMSO-d6) δ 172.5, 171.8, 171.2, 169.1, 155.6, 145.3, 137.6, 136.9, 131.1, 130.6, 129.2, 128.3, 127.9, 127.7, 126.7, 126.2, 125.3, 120.2, 114.9, 113.9, 65.4, 53.9, 50.2, 48.5, 41.5, 37.3, 18.0, 16.7. Anal. Calcd for C31H33N7O6: C, 62.09; H, 5.55; N, 16.35; found: C, 62.12; H, 5.56; N, 15.98.

Benzyl((S)-1-(((S)-1-((2-((2-(1H-benzo[d][1,2,3]triazol-1-yl)-2-oxoethyl)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-1-oxopropan-2-yl)carbamate (Cbz-Ala-Phe-Gly-Gly-Bt) (5b)

White microcrystals (62%); mp 144–145 °C; 1H NMR (DMSO-d6) δ 8.62–8.55 (m, 1H), 8.42–8.36 (m, 1H), 8.29 (d, J = 8.1 Hz, 1H), 8.20 (d, J = 9.0 Hz, 1H), 8.03 (d, J = 8.2 Hz, 1H), 7.80 (t, J = 7.5 Hz, 1H), 7.63 (t, J = 7.8 Hz, 1H), 7.45 (d, J = 6.9 Hz, 1H), 7.43–7.30 (m, 5H), 7.24–7.22 (m, 5H), 5.02–4.96 (m, 4H), 4.62–4.50 (m, 1H), 4.10–3.99 (m, 1H), 3.94–3.86 (m, 2H), 3.10–3.05 (m, 1H), 2.87–2.80 (m, 1H), 1.13 (d, J = 7.2 Hz, 3H); 13C NMR (DMSO-d6) δ 172.5, 171.3, 169.7, 168.5, 155.7, 145.3, 137.6, 136.9, 131.0, 130.6, 129.3, 128.3, 128.0, 127.8, 126.6, 126.2, 120.2, 113.7, 65.5, 53.8, 50.2, 42.6, 41.8, 37.3, 18.0. Anal. Calcd for C30H31N7O6.0.5 H2O: C, 60.60; H, 5.42; N, 16.49; found: C, 60.70; H, 5.26; N, 16.22.

Benzyl((S)-1-((2-(((S)-1-((2-(1H-benzo[d][1,2,3]triazol-1-yl)-2-oxoethyl)amino)-4-methyl-1-oxopentan-2-yl)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (Cbz-Phe-Gly-Leu-Gly-Bt) (5c)

White microcrystals (79%); mp 194–195 °C; 1H NMR (DMSO-d6) δ 8.80–8.70 (m, 1H), 8.35–8.30 (m, 1H), 8.28 (d, J = 8.4 Hz, 1H), 8.21 (d, J = 8.4 Hz, 1H), 8.04 (d, J = 8.1 Hz, 1H), 7.79 (t, J = 7.2 Hz, 1H), 7.66–7.55 (m, 2H), 7.33–7.19 (m, 10H), 4.97–4.90 (m, 4H), 4.55–4.46 (m, 1H), 4.32–4.22 (m, 1H), 3.89–3.72 (m, 2H), 3.08–3.02 (m, 1H), 2.80–2.50 (m, 1H), 1.70–1.66 (m, 1H), 1.60–1.56 (m, 2H), 0.92 (d, J = 6.3 Hz, 3H), 0.89 (d, J = 6.3 Hz, 3H); 13C NMR (DMSO-d6) δ 172.9, 171.8, 168.5, 168.4, 155.9, 145.2, 138.1, 136.9, 131.0, 130.5, 129.2, 128.2, 128.0, 127.6, 127.4, 126.6, 126.2, 120.1, 113.7, 65.2, 56.2, 50.7, 42.5, 42.0, 41.0, 37.3, 24.1, 23.1, 21.6. Anal. Calcd for C33H37N7O6: C, 63.14; H, 5.94; N, 15.62; found: C, 62.88; H, 5.92; N, 15.46.

General procedure for O-acylation: synthesis of compounds (6a–f)

Compounds were prepared by using similar microwave conditions reported for O-acylation. (34) A dried heavy-walled Pyrex tube containing a small stir bar was charged with benzotriazole intermediate 3a–c or 5b–c (1 eq.), O-nucleophile (1.5 eq.), and base dimethylaminopyridine (DMAP) (0.1 eq.) dissolved in THF. The reaction mixture was exposed to microwave irradiation (100 W) at 70 °C for specified times. Each mixture was allowed to cool through an inbuilt system until the temperature had fallen below 30 °C (ca. 10 min). The reaction mixture was quenched with water, extracted with EtOAc, and the extracts were washed with (10%) Na2CO3, water, and dried over MgSO4. The solvent was removed under reduced pressure, and the residue was subjected to silica gel column using EtOAc/hexane as an eluent to give the corresponding compound 6a–f.

(5S,8S)-(8S,9S,10R,13R,14S,17R)-10,13-Dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 8-benzyl-5-methyl-3,6,9-trioxo-1-phenyl-2-oxa-4,7,10-triazadodecan-12-oate (Cbz-Ala-Phe-Gly-O-Cholesterol) (6a)

White microcrystals (45%); mp 162–163 °C; 1H NMR (CDCl3) δ 7.40–7.30 (m, 5H), 7.29–7.15 (m, 5H), 6.76 (d, J = 7.2 Hz, 1H), 6.70–6.60 (m, 1H), 5.38–5.34 (m,1H), 5.27 (d, J = 6.6 Hz, 1H), 5.14–5.00 (m, 2H), 4.80–4.70 (m,1H), 4.66–4.58 (m, 1H), 4.22–4.16 (m, 1H), 4.08–3.98 (m, 1H), 3.90–3.80 (m, 1H), 3.22–3.10 (m, 1H), 3.09–3.00 (m, 1H), 2.30 (d, J = 7.8 Hz, 2H), 2.01–1.93 (m, 2H), 1.90–1.78 (m, 4H), 1.62–1.40 (m, 8H), 1.36–1.28 (m, 2H), 1.27 (d, J = 7.2 Hz, 6H), 1.17–1.07 (m, 4H), 1.06–1.00 (m, 6H), 0.91 (d, J = 6.6 Hz, 3H), 0.89 (d, J = 7.5 Hz, 6H), 0.67 (s, 3H); 13C NMR (CDCl3) δ 172.4, 171.0, 169.0, 156.4, 139.5, 136.6, 136.2, 129.4, 128.8, 128.5, 128.3, 127.2, 123.2, 75.6, 67.4, 56.9, 56.4, 54.3, 51.1, 50.2, 42.5, 41.7, 39.9, 39.7, 38.2, 37.1, 36.8, 36.4, 36.0, 32.1, 29.9, 28.4, 28.2, 27.9, 24.5, 24.1, 23.0, 22.8, 21.2, 19.5, 18.9, 18.4, 12.1. HRMS Calcd for C49H69N3O6Na [M + Na]+ 818.5079; found 818.5089.

(5S,8S)-(E)-3,7-Dimethylocta-2,6-dien-1-yl-8-benzyl-5-methyl-3,6,9-trioxo-1-phenyl-2-oxa-4,7,10-triazadodecan-12-oate (Cbz-Ala-Phe-Gly-O-Nerol) (6b)

White microcrystals (50%); mp 114–116 °C; 1H NMR (CDCl3) δ 7.36–7.28 (m, 5H), 7.26–7.12 (m, 5H), 7.02 (d, J = 7.5 Hz, 1H), 6.92–6.82 (m, 1H), 5.58 (d, J = 7.2 Hz, 1H), 5.30 (t, J = 7.2 Hz, 1H), 5.12–5.00 (m, 3H), 4.80 (q, J = 7.5 Hz, 1H), 4.59 (d, J = 7.5 Hz, 2H), 4.30–4.20 (m, 1H), 4.08–3.80 (m, 2H), 3.13 (dd, J = 13.8, 6.6 Hz, 1H), 3.01 (dd, J = 13.8, 7.2 Hz, 1H), 2.14–2.00 (m, 4H), 1.74 (s, 3H), 1.67 (s, 3H), 1.58 (s, 3H), 1.26 (d, J = 6.9 Hz, 3H); 13C NMR (CDCl3) δ 172.7, 171.2, 169.6, 156.3, 143.4, 136.6, 136.3, 132.4, 129.4, 128.7, 128.6, 128.3, 128.2, 127.0, 123.6, 118.7, 67.2, 62.1, 54.2, 50.9, 41.4, 38.3, 32.3, 26.7, 25.8, 23.6, 18.6, 17.8. Anal. Calcd for C32H41N3O6: C, 68.18; H, 7.33; N, 7.45; found: C, 68.28; H, 7.57; N, 7.50.

(5S,8S)-((3aR,5R,5aS,8aS,8bR)-2,2,7,7-Tetramethyltetrahydro-3aH-bis([1,3]dioxolo) [4,5-b:4′,5′-d]pyran-5-yl)methyl-8-benzyl-5-isopropyl-3,6,9-trioxo-1-phenyl-2-oxa-4,7,10-triazadodecan-12-oate (Cbz-Val-Phe-Gly-O-Glactopyranose) (6c)

White microcrystals (47%); mp 129–131 °C; 1H NMR (CDCl3) δ. 7.45–7.30 (m, 5H), 7.28–7.15 (m, 5H), 6.68–6.58 (m, 2H), 5.53 (d, J = 4.8 Hz, 1H), 5.27 (d, J = 7.5 Hz, 1H), 5.14–5.03 (m, 2H), 4.82–4.71 (m, 1H), 4.62 (dd, J = 8.0, 2.3 Hz, 1H), 4.35–4.19 (m, 4H), 4.11–3.88 (m, 4H), 3.20–3.10 (m, 1H), 3.08–2.98 (m, 1H), 2.14–2.00 (m, 1H), 1.51 (s, 3H), 1.43 (s, 3H), 1.33 (s, 6H), 0.88 (d, J = 6.9 Hz, 3H), 0.77 (d, J = 6.6 Hz, 3H); 13C NMR (CDCl3) δ 171.3, 171.0, 169.4, 156.7, 136.6, 136.2, 129.4, 128.8, 128.5, 128.4, 127.2, 109.9, 109.0, 96.4, 71.1, 70.8, 70.6, 67.5, 66.0, 64.4, 60.9, 54.2, 41.5, 38.2, 30.8, 26.3, 26.1, 25.1, 24.7, 19.4, 17.7. Anal. Calcd for C36H47N3O11: C, 61.97; H, 6.79; N, 6.02; found: C, 61.78; H, 6.94; N, 5.91.

(5S)-(8R,9S,13S,14S)-13-Methyl-17-oxo-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-3-yl-8-benzyl-5-isopropyl-3,6,9-trioxo-1-phenyl-2-oxa-4,7,10-triazadodecan-12-oate (Cbz-Val-Phe-Gly-O-estrone) (6d)

White flakes (35%); mp 151–153 °C; 1H NMR (CDCl3) δ 7.38–7.30 (m, 6H), 7.28–7.22 (m, 2H), 7.22–7.16 (m, 4H), 6.87–6.78 (m, 2H), 6.60 (d, J = 7.8 Hz, 1H), 5.30–5.20 (m, 1H), 5.14–4.98 (m, 3H), 4.85–4.71 (m, 1H), 4.34–3.80 (m, 4H), 3.23–2.98 (m, 3H), 2.87 (dd, J = 8.9, 4.1 Hz, 1H), 2.58–1.91 (m, 4H), 1.70–1.35 (m, 5H), 0.90 (s, 3H), 0.87 (dd, J = 6.9, 3.3 Hz, 3H), 0.76 (dd, J = 6.6, 3.0 Hz, 3H); 13C NMR (CDCl3) δ 171.3, 170.9, 169.5, 168.5, 156.9, 148.3, 138.3, 137.9, 136.6, 136.2, 129.4, 128.9, 128.8, 128.5, 128.4, 127.3, 126.7, 121.5, 118.6, 61.7, 61.1, 54.3, 50.6, 48.1, 44.3, 41.5, 38.2, 36.1, 31.8, 30.6, 29.6, 26.5, 26.0, 21.8, 19.4, 14.4, 14.1. HRMS Calcd for C42H49N3O7Na [M + Na]+: 730.3463; found: 730.3472.

(5S,8S)-(3aR,5R,6R,6aR)-5-((R)-2,2-Dimethyl-1,3-dioxolan-4-yl)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-6-yl-8-benzyl-5-methyl-3,6,9,12-tetraoxo-1-phenyl-2-oxa -4,7,10,13-tetraazapentadecan-15-oate (Cbz-Ala-Phe-Gly-Ala-O-diacetoneglucose) (6e)

White microcrystals (58%); mp 102–104 °C; 1H NMR (CDCl3) δ 7.40–7.29 (m, 7H), 7.26–7.09 (m, 6H), 5.86 (d, J = 3.6 Hz, 1H), 5.76 (d, J = 6.1 Hz, 1H), 5.27–5.25 (m, 1H), 5.10–5.00 (m, 2H), 4.78 (q, J = 7.2 Hz, 1H), 4.49 (d, J = 3.6 Hz, 1H), 4.40–4.34 (m, 1H), 4.24–4.1.8 (m, 2H), 4.16–3.99 (m, 4H), 3.98–3.80 (m, 2H), 3.12–2.95 (m, 2H), 1.50 (s, 3H), 1.38 (s, 3H), 1.30–1.25 (m, 9H), 0.92–0.82 (m, 1H); 13C NMR (CDCl3) δ 173.1, 171.4, 169.4, 168.8, 156.5, 136.5, 136.3, 129.4, 128.8, 128.5, 128.2, 127.3, 112.5, 109.6, 105.2, 83.3, 79.8, 72.5, 67.4, 54.8, 51.1, 43.0, 41.4, 38.3, 27.1, 26.9, 26.4, 25.4, 18.6. Anal. Calcd for C36H46N4O12: C, 59.49; H, 6.38; N, 7.71; found: C, 59.26; H, 6.56; N, 7.35.

(5S,11S)-(1S,2S)-2-Isopropyl-5-methylcyclohexyl 5-benzyl-11-isobutyl-3,6,9,12-tetra oxo-1-phenyl-2-oxa-4,7,10,13-tetraazapentadecan-15-oate (Cbz-Phe-Gly-Leu-Gly-O-menthol) (6f)

White microcrystals (42%); mp 104–106 °C; 1H NMR (CDCl3) δ. 7.40–7.06 (m, 13H), 5.79 (d, J = 6.6 Hz, 1H), 5.06 (d, A part of AB system, J = 12 .0 Hz, 1H), 4.98 (d, B part of AB system, J = 12.3 Hz, 1H), 4.76–4.51 (m, 3H), 4.04–3.83 (m, 4H), 3.14 (dd, J = 15.0, 5.4 Hz, 1H), 3.00–2.92 (m, 1H), 2.18–2.10 (m, 1H), 1.98–1.91 (m, 1H), 1.89–1.78 (m, 1H), 1.68–1.50 (m, 5H), 1.49–1.31 (m, 2H), 1.03–0.88 (m, 14), 0.71 (d, J = 6.9 Hz, 3H); 13C NMR (CDCl3) δ 172.5, 172.3, 169.7, 169.1, 156.5, 136.6, 136.2, 129.4, 128.8, 128.7, 128.4, 128.2, 127.2, 75.9, 67.3, 56.5, 51.9, 47.1, 43.4, 41.6, 41.2, 40.9, 38.4, 34.3, 31.5, 26.4, 24.9, 23.6, 23.1, 22.2, 22.1, 20.9, 16.5. HRMS Calcd for C37H52N4O7Na [M + Na]+: 687.3728; found: 687.3743.

General procedure for S-acylation: synthesis of compounds (7a–d)

Mercapto nucleophile (1 eq.) was dissolved in THF and triethyl amine (1.5 eq.). Benzotriazole intermediate (1 eq.) was added to the solution, and the mixture was stirred at room temperature for 1–2 h. The mixture was then acidified with 6 N HCl, concentrated, and then diluted with ethyl acetate. The organic layer was washed with 6 N HCl and dried over anhydrous MgSO4, filtered, and then evaporated to give the desired compound.

(5R,8R)-S-Phenyl 8-benzyl-5-methyl-3,6,9-trioxo-1-phenyl-2-oxa-4,7,10-triaza-dodecane-12-thioate (7a)

White microcrystals (78%); mp 146–147 °C; 1H NMR (DMSO-d6) δ 8.89–8.86 (m, 1H), 8.11 (d, J = 8.4 Hz, 1H), 7.47–7.25 (m, 16H), 5.02–4.90 (m, 2H), 4.63–4.59 (m, 1H), 4.13–4.05 (m, 2H), 4.06–4.01 (m, 1H), 3.19–3.08 (m, 1H), 2.91–2.82 (m, 1H), 1.12 (d, J = 6.6 Hz, 3H); 13C NMR (DMSO-d6) δ 196.2, 172.3, 171.9, 155.6, 137.6, 137.0, 134.5, 129.5, 129.4, 129.2, 128.3, 128.1, 127.7, 126.9, 126.3, 65.4, 53.7, 50.2, 48.8, 37.3, 18.2. Anal. Calcd for C28H29N3O5S.H2O: C, 62.55; H, 5.81; N, 7.82; found: C, 62.85; H, 5.49; N, 7.46.

(5S,8S)-Methyl-8-benzyl-5-methyl-3,6,9,12-tetraoxo-1-phenyl-2-oxa-13-thia-4,7,10-triazapentadecan-15-oate (7b)

White powder (83%); mp 158–160 °C; 1H NMR (DMSO-d6) δ 8.85–8.82 (m, 1H), 8.07 (d, J = 8.1 Hz, 1H), 7.42–7.18 (m, 11H), 5.03–4.99 (m, 2H), 4.62–4.59 (m, 1H), 4.09–3.98 (m, 3H), 3.76 (s, 2H), 3.64 (s, 3H), 3.14–3.06 (m, 1H), 2.91–2.85 (m, 1H), 1.14 (d, J = 6.9 Hz, 3H); 13C NMR (DMSO-d6) δ 197.6, 172.3, 171.9, 168.9, 155.6, 137.6, 137.0, 129.2, 128.3, 128.1, 127.7, 126.3, 65.4, 53.7, 52.4, 50.1, 48.5, 37.3, 30.2, 18.2. Anal. Calcd for C25H29N3O7S: C, 58.24; H, 5.67; N 8.15; found: C, 58.08; H, 5.63; N, 8.04.

(5S,8S,14R)-8-Benzyl-5,14-dimethyl-3,6,9,12,15-pentaoxo-1-phenyl-2-oxa-16-thia-4,7,10,13-tetraazaoctadecan-18-oic acid (7c)

Off-white microcrystals (70%); mp 103–105 °C; 1H NMR (DMSO-d6) δ. 8.52 (d, J = 7.2 Hz, 1H), 8.36–8.28 (m, 1H), 7.99 (d, J = 7.8 Hz, 1H), 7.42 (d, J = 7.2 Hz, 1H), 7.38–7.30 (m, 5H), 7.25–7.18 (m, 5H), 5.07–4.94 (m, 2H), 4.55–4.38 (m, 2H), 4.07–3.97 (m, 1H), 3.78 (t, J = 5.6 Hz, 1H), 3.62 (s, 2H), 3.08–3.00 (m, 1H), 2.88–2.78 (m, 1H), 1.28 (d, J = 6.9 Hz, 3H), 1.11 (d, J = 6.9 Hz, 3H); 13C NMR (DMSO-d6) δ 200.9, 172.4, 171.2, 169.6, 169.0, 155.6, 137.6, 136.9, 129.2, 128.3, 128.0, 127.7, 126.2, 65.4, 54.5, 53.9, 50.1, 41.8, 37.3, 30.9, 18.0, 17.3. HRMS Calcd for C27H32N4O8NaS [M + Na]+: 595.1833; found: 595.1835.

(5S,11S)-S-Benzyl-5-benzyl-11-isobutyl-3,6,9,12-tetraoxo-1-phenyl-2-oxa-4,7,10,13-tetraazapentadecane-15-thioate (7d)

White microcrystals (94%); mp 186–187 °C; 1H NMR (DMSO-d6) δ 8.67 (t, J = 5.56 Hz, 1H), 8.32 (t, J = 5.1 Hz, 1H), 8.03 (d, J = 8.7 Hz, 1H), 7.60 (d, J = 8.4 Hz, 1H), 7.39–7.19 (m, 15H), 4.95 (s, 2H), 4.42–4.22 (m, 2H), 4.10 (s, 2H), 4.05–4.00 (m, 2H), 3.81–3.74 (m, 2H), 3.09–3.00 (m, 1H), 2.81–2.70 (m, 1H), 170–1.60 (m, 1H), 1.59–1.49 (m, 2H), 0.89 (d, J = 6.3 Hz, 3H), 0.85 (d, J = 6.0 Hz, 3H); 13C NMR (DMSO-d6) δ 197.8, 172.7, 171.8, 168.6, 155.9, 138.2, 137.7, 136.9, 129.2, 128.7, 128.5, 128.3, 128.0, 127.7, 127.4, 127.1, 126.2, 65.3, 56.2, 50.9, 48.7, 42.0, 40.6, 37.3, 31.7, 24.1, 23.0, 21.5. Anal. Calcd for C34H40N4O6S: C, 64.54; H, 6.37; N 8.85; found: C, 64.50; H, 6.50; N, 8.48.

General procedure for N-acylation: synthesis of compounds (8a, 8d)

N-nucleophile (1 eq.) was dissolved in THF and triethylamine (1.5 eq.). The benzotriazole intermediate (1 eq.) was added to the solution, and the mixture was stirred at room temperature for 1 h. The mixture was acidified with 6 N HCl and then diluted with ethyl acetate. The organic layer was washed with 6 N HCl and dried over anhydrous MgSO4, and filtered and evaporated to give the desired compound.

Benzyl((S)-1-(((S)-1-((2-((3-(1H-imidazol-1-yl)propyl)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-1-oxopropan-2-yl)carbamate (8a)

White microcrystals (75%); mp 134–137 °C; 1H NMR (DMSO-d6) δ 8.30–8.20 (m, 1H), 8.08 (d, J = 7.2 Hz, 1H), 7.78–7.72 (m, 1H), 7.61 (s, 1H), 7.48 (d, J = 7.5 Hz, 1H), 7.38–7.30 (m, 5H), 7.23–7.20 (m, 5H), 7.16 (s, 1H), 6.88 (s, 1H), 5.02–4.88 (m, 2H), 4.52–4.40 (m, 1H), 4.08–3.90 (m, 3H), 3.79–3.68 (m, 1H), 3.66–3.54 (m, 1H), 3.08–2.99 (m, 3H), 2.92–2.78 (m, 1H), 1.92–1.78 (m, 2H), 1.12 (d, J = 6.9 Hz, 3H); 13C NMR (DMSO-d6) δ 172.7, 171.2, 168.6, 155.7, 137.6, 137.3, 136.9, 129.2, 128.3, 128.0, 127.7, 126.2, 119.3, 65.5, 54.2, 50.2, 43.4, 42.2, 37.1, 35.7, 30.7, 17.9. Anal. Calcd for C28H34N6O5: C, 62.91; H, 6.41; N 15.72; found: C, 62.56; H, 6.46; N, 15.52.

Benzyl ((S)-1-((2-(((S)-4-methyl-1-((2-(4-methylpiperazin-1-yl)-2-oxoethyl)amino)-1-oxopentan-2-yl)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (8d)

White microcrystals (68%); mp 181–183 °C; 1H NMR (DMSO-d6) δ 8.29 (t, J = 5.7 Hz, 1H), 8.01 (t, J = 5.3 Hz, 1H), 7.93 (d, J = 8.4 Hz, 1H), 7.56 (d, J = 8.4 Hz, 1H), 7.34–7.18 (m, 10H), 4.95–4.89 (m, 2H), 4.42–4.34 (m, 1H), 4.30–4.22 (m, 1H), 3.91 (d, J = 5.4 Hz, 2H), 3.76 (t, J = 5.7 Hz, 2H), 3.41–3.36 (m, 4H), 3.04 (dd, J = 13.6, 3.9 Hz, 1H), 2.75 (dd, J = 13.5, 11.1 Hz, 1H), 2.27–2.22 (m, 4H), 2.15 (s, 3H), 1.65–1.58 (m, 1H), 1.51–1.45 (m, 2H), 0.87 (dd, J = 6.3 Hz, 3H), 0.83 (d, J = 6.3 Hz, 3H); 13C NMR (DMSO-d6) δ 172.0, 171.8, 168.5, 166.6, 156.0, 138.2, 137.0, 129.2, 128.3, 128.1, 127.7, 127.5, 126.3, 65.3, 56.3, 54.6, 54.2, 51.0, 45.7, 43.9, 42.1, 41.3, 41.0, 37.3, 24.1, 23.1, 21.6. Anal. Calcd for C32H44N6O6: C, 63.14; H, 7.29; N 13.81; found: C, 62.84; H, 7.38; N, 13.68.

General procedure for N-acylation: synthesis of compounds (8b–c)

Compounds were prepared by using similar reaction condition reported for (α-aminoacyl)benzotriazoles. (35) A dried heavy-walled Pyrex tube containing a small stir bar was charged with benzotriazole intermediate (1 eq.), and N-nucleophile (1 eq.) was dissolved in DMF. The reaction mixture was exposed to microwave irradiation (60 W) at 65 °C temperature for specified times. Each mixture was allowed to cool through an inbuilt system until the temperature had fallen below 30 °C (ca. 10 min). Each reaction mixture was quenched with water, and the solid obtained was filtered and washed with 10% Na2CO3 and water to give the desired compound.

Benzyl((S)-3-methyl-1-oxo-1-(((S)-1-oxo-1-((2-oxo-2-(pyridin-2-ylamino)ethyl)amino)-3-phenylpropan-2-yl)amino)butan-2-yl)carbamate (8b)

White microcrystals (68%); mp 214–215 °C; 1H NMR (DMSO-d6) δ 10.47 (s, 1H), 8.50–8.30 (m, 2H), 8.05 (d, J = 8.1 Hz, 2H), 7.85–7.72 (m, 1H), 7.50–7.05 (m, 12H), 5.02 (s, 2H), 4.72–4.58 (m, 1H), 4.10–3.90 (m, 2H), 3.82 (t, J = 7.2 Hz, 1H), 3.15–3.00 (m, 1H), 2.90–2.75 (m, 1H), 1.99–1.72 (m, 1H), 0.80–0.61 (m, 6H); 13C NMR (DMSO-d6) δ 171.5, 170.9, 168.3, 156.0, 151.7, 148.0, 138.2, 137.7, 137.0, 129.2, 128.3, 128.0, 127.6, 126.2, 119.4, 113.4, 65.5, 60.4, 53.6, 42.7, 37.7, 30.4, 19.1, 18.0. HRMS Calcd for C29H34N5O5 [M + H]+: 532.2554; found: 532.2572.

Benzyl ((S)-1-(((S)-1-((2-(((S)-1-((6-methoxybenzo[d]thiazol-2-yl)amino)-1-oxo propan -2-yl)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-1-oxopropan-2-yl)carbamate (8c)

White microcrystals (60%); mp 148–150 °C; 1H NMR (DMSO-d6) δ 8.28–8.22 (m, 1H), 8.00 (d, J = 7.2 Hz, 1H), 7.63 (d, J = 8.7 Hz, 1H), 7.58–7.52 (m, 1H), 7.41 (d, J = 8.4 Hz, 2H), 7.36–7.30 (m, 5H), 7.26–7.18 (m, 6H), 7.06–6.90 (m, 1H), 5.02–4.92 (m, 2H), 4.58–4.42 (m, 2H), 4.01 (t, J = 7.2 Hz, 1H), 3.84–3.62 (m, 5H), 3.08–2.98 (m, 1H), 2.88–2.68 (m, 1H), 1.35 (d, J = 7.2 Hz, 3H), 1.11 (d, J = 7.2 Hz, 3H); 13C NMR (DMSO-d6) δ 172.1, 172.0, 171.2, 168.7, 156.1, 142.6, 137.6, 137.0, 132.8, 129.2, 128.3, 128.0, 127.7, 126.2, 121.1, 114.9, 104.7, 65.5, 55.6, 53.9, 50.7, 48.6, 41.7, 37.3, 22.7, 18.1, 17.6 Anal. Calcd for C33H36N6O7S: C, 59.99; H, 5.49; N 12.72; found: C, 59.61; H, 5.50; N, 12.50.

General procedure for C-acylation: synthesis of compounds (9a–b)

A solution of benzotriazole intermediate 3a, 5c (1eq.), C-nucleophile (1eq.), and DMAP (1eq.) in THF (5 mL), was added to a dried heavy-walled Pyrex tube with a small stir bar. This reaction mixture was exposed to microwave irradiation (70 °C, 50 W) for specified time with a simultaneous cooling. After the reaction was completed (monitored by TLC), the THF was evaporated, and the solution was acidified and then extracted with ethyl acetate. The solvent was removed under reduced pressure, and the residue was purified by recrystallization from ethyl acetate to give the corresponding product 9a and 9b.

Benzyl-((S)-1-(((S)-1-((2-(4,4-dimethyl-2,6-dioxocyclohexylidene)-2-hydroxyethyl) amino)-1-oxo-3-phenylpropan-2-yl)amino)-1-oxopropan-2-yl)carbamate (9a)

White microcrystals (69%); mp 103–105 °C; 1H NMR (DMSO-d6) δ 8.78–8.60 (m, 1H), 8.40–8.26 (m, 1H), 7.84–7.55 (m, 11H), 5.38 (br s, 2H), 5.05–4.82 (m, 2H), 4.94–4.81 (m, 2H), 4.43–4.40 (m, 1H), 4.20–4.16 (m, 1H), 3.45–3.38 (m, 1H), 3.26–3.18 (m, 1H), 2.89 (s, 4H), 1.50 (d, J = 7.5 Hz, 3H), 1.40–1.30 (m, 6H); 13C NMR (DMSO-d6) δ 200.6, 195.2, 172.1, 171.3, 171.1, 155.5, 137.6, 136.9, 129.2, 128.3, 127.9, 127.7, 126.2, 111.1, 65.4, 53.5, 50.2, 47.5, 37.7, 30.7, 27.5, 18.1. HRMS Calcd for C30H35N3O7Na [M + Na]+ 572.2367; found 572.2377.

Benzyl-((S)-1-((2-(((S)-1-((2-(2,6-dioxocyclohexyl)-2-oxoethyl)amino)-4-methyl-1-oxo-pentan-2-yl)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (9b)

White microcrystals (69%); mp 103–105 °C; 1H NMR (DMSO-d6) δ 8.37–8.26 (m, 1H), 8.22–8.15 (m, 1H), 7.96 (d, J = 8.4 Hz, 1H), 7.58 (d, J = 8.4 Hz, 1H), 7.36–7.18 (m, 10H), 5.02–4.88 (m, 2H), 4.50–4.20 (m, 4H), 3.88–3.67 (m, 2H), 3.07–2.98 (m, 1H), 2.80–2.65 (m, 2H), 2.55–2.27 (m, 3H), 1.95–1.44 (m, 5H), 0.89 (d, J = 6.0 Hz, 3H), 0.86 (d, J = 6.3 Hz, 3H); 13C NMR (DMSO-d6) δ 201.1, 172.3, 171.8, 168.5, 155.9, 138.2, 136.9, 129.2, 128.3, 128.0, 127.7, 127.4, 126.2, 112.1, 65.2, 56.2, 50.8, 47.6, 42.0, 41.0, 37.3, 24.1, 23.1, 21.6, 18.7. HRMS Calcd for C33H41N4O8 [M + H]+ 621.2919; found 621.2931. HRMS Calcd for C33H41N4O8Na [M + Na]+ 643.2738; found 643.2757.

Results and Discussion

  1. Top of page
  2. Abstract
  3. Methods and Materials
  4. Results and Discussion
  5. Conclusion
  6. Acknowledgments
  7. References
  8. Supporting Information

Syntheses of Cbz-protected-tripeptidoylbenzotriazole and tetrapeptidoylbenzotriazole

The Cbz-protected tripeptides 2a–c and tetrapeptides 4ac were synthesized by our previously reported method by stepwise coupling (33) of amino acids with subsequent benzotriazole-activated N-protected amino acid analogs in solution phase in good yields (67–91%). Compounds 2a–c and 4ac were further activated by benzotriazole to obtain known intermediates 3a, 5a and novel intermediates 3b–c and 5b–c in good yields (62–93%). These tri-peptidoylbenzotriazoles 3a–c and tetrapeptidoylbenzotriazoles 5a–c were used as active intermediates to prepare peptide conjugates (Scheme 1, Table 1).

image

Figure Scheme 1:.  Syntheses of benzotriazole derivatives of tri- and tetrapeptides.

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O-Acylations carried out by tri- and tetrapeptidoylbenzotriazoles

Compounds 3a–b, 5b, and 5c were reacted with a variety of steroids, terpenes, and sugar derivatives in the presence of catalytic amount of DMAP under microwave irradiations at 70 °C and 65 W power for 1.5–3 h to afford novel peptide conjugates 6a–f in yields of 35–58% (Scheme 2, Table 2).

image

Figure Scheme 2:. O-Acylation of N–Pg-tri- and tetrapeptidoylbenzotriazoles.

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Table 2. O-Acylation with N–Pg-tri- and tetrapeptidoylbenzotriazoles
ProductReactantReactantTimeYield (%)Mp(°C)
6aZ–L–Ala–L–Phe–Gly–Bt 3aCholesterol1 h 45 min45162–163
6bZ–L–Ala–L–Phe–Gly–Bt 3aNerol1 h 30 min50114–116
6cZ–L–Val–L–Phe–Gly–Bt 3bGalactopyranose1 h 30 min47129–132
6dZ–L–Val–L–Phe–Gly–Bt 3bEstrone2 h 15 min35151–153
6eZ–L–Ala–L–Phe–Gly–Gly-Bt 5bDiacetone glucose1 h 45 min58102–104
6fZ–L–Phe–Gly–L–Leu–Gly-Bt 5cMenthol1 h 45 min42104–106

Compounds 6a–f were fully characterized by 1H NMR and 13C NMR and elemental analysis. The tri- and tetrapeptide conjugates are at least 95% chirally pure as supported by the NMR spectra of 6a–f, which showed no duplication of signals (Appendix S1).

S-Acylation with tri- and tetrapeptidoylbenzotriazoles

Compounds 3a, 5a and 5c were reacted with S-nucleophiles in the presence of triethylamine at room temperature for 2 h to give their corresponding S-acylated tripeptide conjugates 7a–b (78–83%) and tetrapeptide conjugates 7cd (68–94%) (Scheme 3, Table 3). The chiral purity of 7a–d was supported by their 1H NMR and 13C NMR spectra, which showed no evidence of epimerization in their S-acylated tri- and tetrapeptide conjugates (Appendix S1).

image

Figure Scheme 3:. S-Acylation of N–Pg-tri- and tetrapeptidoylbenzotriazoles.

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Table 3. S-Acylation with N–Pg-tri- and tetrapeptidoylbenzotriazoles
ProductReactantReactantYield (%)Mpt(°C)
7aZ–L–Ala–L–Phe–Gly–Bt 3aThiophenol78146–148
7bZ–L–Ala–L–Phe–Gly–Bt 3aMethylmercaptoacetate83158–160
7cZ–L–Ala–L–Phe–Gly–L-Ala-Bt 5aMercaptoacetic acid68103–105
7dZ–L–Phe–Gly–L–Leu–Gly-Bt 5cBenzyl mercaptan94186–187

The 1H NMR spectra of compounds 7a and 7b showed prominent doublets for the methyl protons of alanine unit at 1.14 ppm, and 13C NMR showed prominent signals for all carbonyls of the tripeptide unit in the range of 196.2–155.6 ppm. Duplicate signals are absent in the 13C NMR spectra of compounds 7a–b. Compound 7c showed sharp singlets of CH2 protons of mercaptoacetic acid unit and Cbz group at 3.62 and 4.99 ppm, respectively, with no other repetition of signals in the NMR spectrum. Compound 7d also showed singlet of CH2 protons of benzyl group and Cbz group at 4.10 and 4.95 ppm, respectively, with no other repeated signals confirming the formation of chirally pure 7c and 7d.

N-Acylation with tri- and tetrapeptidoylbenzotriazoles

Compounds 3a–b and 5a, 5c were reacted with different N-nucleophiles in the presence of base to give the corresponding chirally pure N-acylated tripeptides 8a–b and tetrapeptides 8c–d (Scheme 4, Table 4). Compounds 3a and 5c were reacted with N-nucleophiles in the presence of triethylamine at 20 °C for 1 h to give the corresponding tripeptide conjugate 8a (75%) and tetrapeptide conjugate 8d (68%). Compounds 3b and 5b reacted with heterocyclic N-nucleophiles under microwave irradiation at 65 °C for 30 min in DMF to give the corresponding chirally pure peptide conjugates 8b (68%) and 8c (60%). Compounds 8a–d were fully characterized by NMR spectroscopy and elemental analysis (Appendix S1).

image

Figure Scheme 4:. N-Acylation of N–Pg-tri- and tetrapeptidoylbenzotriazoles.

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Table 4. N-Acylation with N–Pg-tri- and tetrapeptidoylbenzotriazoles
ProductReactantReactantConditionsYield (%)Mpt(°C)
8aZ–L–Ala–L–Phe–Gly–Bt 3aN-(3-Amino-propyl)-imidazoleTHF, TEA, 1 h, RT7565–69
8bZ–L–Val–L–Phe–Gly–Bt 3b2-AminopyridineDMF, 65°, MW, 0.5 h68214–215
8cZ–L–Ala–L–Phe–Gly–L-Ala-Bt 5a2-Amino-6-methoxy-benzothiazoleDMF, 65°, MW, 0.5 h60148–150
8dZ–L–Phe–Gly–L–Leu–Gly-Bt 5cN-MethylpiperazineTHF, TEA, 1 h, RT68181–183

The chiral purity of compounds 8a–d was confirmed by 1H NMR and 13C NMR. Tripeptide conjugate 8a and tetrapeptide conjugate 8c showed doublets for the methyl protons of the alanine unit at δ 1.12 ppm (8a) and 1.35, 1.12 ppm (8c) with no duplication of signals in 1H NMR and 13C NMR spectra. Compound 8b showed prominent singlet of NH proton of aminopyridine unit at 10.47 ppm, and compound 8d showed sharp singlet for the methyl protons of the N-methylpiperazine unit at 2.15 ppm with no other duplication of signals in 1H NMR and 13C NMR spectra, which confirms the formation of chirally pure 8b and 8d.

C-Acylation using tri- and tetrapeptidoylbenzotriazoles

Compounds 3a and 5a were reacted with different C-nucleophiles in the presence of 1 equivalent of DMAP in MW at 70 °C under 50 W irradiation power to give the corresponding tripeptide conjugate 9a (69%) and tetrapeptide conjugate 9b (61%) (Scheme 5, Table 5). Compounds 9a and 9b were fully characterized by NMR and HRMS analysis. No evidence for epimerization was found in the 1H NMR and 13C NMR spectra of 9a and 9b.

image

Figure Scheme 5:. C-Acylation of N–Pg-tri- and tetrapeptidoylbenzotriazoles.

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Table 5. C-Acylation with N–Pg-tri- and tetrapeptidoylbenzotriazoles
ProductReactantReactantYield (%)Mpt(°C)
9aZ–L–Ala–L–Phe–Gly–Bt 3bDimedone69135–137
9bZ–L–Phe–Gly–L–Leu–Gly-Bt 5c1,3 Cyclohexanedione61188–190

Conclusion

  1. Top of page
  2. Abstract
  3. Methods and Materials
  4. Results and Discussion
  5. Conclusion
  6. Acknowledgments
  7. References
  8. Supporting Information

In conclusion, N-(Pg-α-tripeptidoyl)benzotriazoles and N-(Pg-α-tetrapeptidoyl)benzotriazoles are convenient coupling reagents, sufficiently reactive to form amide and ester bonds at ambient temperature. They offer an efficient preparation of chirally pure N-protected tri- and tetrapeptide conjugates with sugars, steroids, terpenes, and different heterocycles by O-, S-, N-, and C-acylations in synthetically useful yields without detectable racemization.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Methods and Materials
  4. Results and Discussion
  5. Conclusion
  6. Acknowledgments
  7. References
  8. Supporting Information

We thank the University of Florida, The Kenan Foundation, and King Abdulaziz University, Jeddah, Saudi Arabia, for financial support.

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  3. Methods and Materials
  4. Results and Discussion
  5. Conclusion
  6. Acknowledgments
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Methods and Materials
  4. Results and Discussion
  5. Conclusion
  6. Acknowledgments
  7. References
  8. Supporting Information

Appendix S1. 1H NMR and 13C NMR spectra for 1b–c, 2a–c, 3a–c, 4a–c, 5a–c, 6a–f, 7a–d, 8a–d, 9a–b.

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