3.1. Chemical Synthesis General Methods
Mass spectra were recorded using a MicrOTOF-QII mass spectrometer (Bruker Daltonics, Bremen, Germany) coupled with a KD Scientific syringe pump, with analysis using Bruker Compass DataAnalysis v 4.1 software. Infrared spectra were recorded on a Perkin Elmer Spectrum 100 Fourier Transform infrared spectrometer equipped with a universal ATR accessory. Optical rotations were obtained with a Rudolph Analytical Autopol IV automatic polarimeter using a 0.1 dm cell (concentration units of g/100 mL). Electronic circular dichroism readings were obtained with a Chirascan circular dichroism spectrometer using a 1 mm cuvette (concentration units of mol/L). All NMR spectra were recorded using a Bruker (Karlsruhe, Germany) Avance 400 spectrometer operating at 400.13 for 1H nuclei and 100.62 for 13C nuclei. Chemical shifts are expressed in parts per million (ppm) relative to the solvent peaks (DMSO-d6: 1H 2.50, 13C 39.52 ppm; CD3OD: 1H 3.31, 13C 49.00 ppm). Assignments are based on 1- and 2-dimensional NMR experiments and analogue comparisons. Standard Bruker pulse sequences were utilized. Reversed-phase flash column chromatography was carried out using LiChroPrep RP-8 (40–63 μm) (Merck Millipore, Darmstadt, Germany). Analytical thin layer chromatography (TLC) was carried out on 0.2 mm thick plates of Merck DC Kieselgel 60 RP-18 F254S plates. All solvents were of analytical grade or better and/or purified according to standard procedures. Chemical reagents used were purchased from standard chemical suppliers and used as purchased.
3.1.1. General Procedure A: Amide Bond Formation
HBTU was added to a stirred solution of Nα-Boc-Arg-OH hydrochloride (1.05 eq), Trp-OMe (1.0 eq.), HOBt (3.6 eq.), and diisopropylethylamine (DIPEA) (4.8 eq.) in anhydrous DMF (2 mL) at 0 °C (1.2 eq.). The reaction mixture was stirred for 1.5 h under N2 atmosphere and then ethyl acetate (50 mL) was added and the organic layer was washed with citric acid (100 mL), sat. NaHCO3 (100 mL) and brine (100 mL), then dried with anhydrous MgSO4. The organic layer was then dried in vacuo before being taken to the next step without further purification.
3.1.2. General Procedure B: Boc Deprotection
A solution of the tert-butyl-carbamate derivative was stirred in CH2Cl2 (2 mL) with TFA (0.2 mL) at room temperature under N2 for 2 h, then dried in vacuo. The crude product was purified using C8 reversed-phase column chromatography (MeOH (+0.05% TFA):H2O (+0.05% TFA), 0:100 ⟶ 1:3) to afford the product as the di-TFA salt.
3.1.3. General Procedure C: Diketopiperazine Formation
NH4OH was added dropwise to a solution of deprotected dipeptide in MeOH (0.25 M) at 0 °C (28–30% in H2O, 1 mL per 6 mL MeOH). The reaction mixture was stirred for 24 h after which the crude product was purified using C8 reversed-phase column chromatography eluting with water.
3.1.4. Methyl (tert-butoxycarbonyl)-l-arginyl-l-tryptophanate hydrochloride (3a)
Following general procedure A, reaction of Nα-Boc-l-Arg-OH (56 mg, 0.206 mmol), l-tryptophan methyl ester hydrochloride (50 mg, 0.196 mmol), HOBt (96 mg, 0.706 mmol), DIPEA (0.16 mL, 0.941 mmol), and HBTU (89 mg, 0.235 mmol) afforded the hydrochloride salt of dipeptide 3a as a clear oil/gum (91 mg, 91%). [α]21D +0.77 (c 0.130, MeOH); Rf = 0.09 (MeOH); IR (ATR) vmax 3362, 2952, 2844, 1738, 1659, 1524, 1162, 1016, 824 cm−1; 1H NMR (400 MHz, DMSO-d6) δ 10.92–10.87 (1H, m, NH-5), 8.20 (1H, d, J = 7.2 Hz, NH-11), 7.72–7.62 (1H, m, NH-17), 7.48 (1H, d, J = 7.5 Hz, H-9), 7.34 (1H, d, J = 7.5 Hz, H-6), 7.18–7.14 (1H, m, H-4), 7.09–7.03 (1H, m, H-7), 7.01–6.95 (1H, m, H-8), 6.88 (1H, d, J = 8.1 Hz, NH-21), 4.53 (1H, dt, J = 6.8, 6.8 Hz, H-1), 4.02–3.94 (1H, m, H-13), 3.54 (3H, br s, OMe), 3.15–3.10 (1H, m, H-2a), 3.10–3.06 (1H, m, H-2b), 3.08–3.02 (2H, m, H2-16), 1.67–1.55 (1H, m, H-14a), 1.53–1.41 (3H, m, H-14b, H2-15), 1.37 (9H, br s, Boc); 13C NMR (100 MHz, DMSO-d6) δ 172.1 (C-10/C-12), 172.0 (C-10/C-12), 156.7 (C-18), 155.2 (Boc), 136.1 (C-5a), 127.0 (C-9a), 123.7 (C-4), 121.0 (C-7), 118.4 (C-8), 117.9 (C-9), 111.4 (C-6), 109.1 (C-3), 78.2 (Boc), 53.6 (C-13), 53.0 (C-1), 51.8 (OMe), 40.4 (C-16), 29.1 (C-14), 28.2 (Boc), 27.0 (C-2), 25.0 (C-15); (+)-HRESIMS [M + H]+ m/z 475.2647 (calcd for C23H35N6O5, 475.2663).
3.1.5. Methyl (tert-butoxycarbonyl)-d-arginyl-d-tryptophanate hydrochloride (3b)
Following general procedure A, reaction of Nα-Boc-d-Arg-OH hydrochloride (64 mg, 0.206 mmol), d-Trp-OMe hydrochloride (50 mg, 0.196 mmol), HOBt (95 mg, 0.706 mmol), DIPEA (0.16 mL, 0.941 mmol), and HBTU (89 mg, 0.235 mmol) afforded the hydrochloride salt of dipeptide 3b as a clear oil/gum (56 mg, 56%). [α]21D −0.74 (c 0.136, MeOH); Rf = 0.09 (MeOH); IR (ATR) vmax 3364, 2953, 2837, 1738, 1658, 1520, 1161, 839 cm−1; 1H NMR (400 MHz, DMSO-d6) δ 10.92–10.82 (1H, m, NH-5), 8.20 (1H, d, J = 7.0 Hz, NH-11), 7.49 (1H, d, J = 8.1 Hz, H-9), 7.35 (1H, d, J = 8.1 Hz, H-6), 7.20–7.16 (1H, m, H-4), 7.11–7.05 (1H, m, H-7), 7.03–6.97 (1H, m, H-8), 6.88 (1H, d, J = 8.1 Hz, NH-21), 4.59–4.50 (1H, m, H-1), 4.05–3.96 (1H, m, H-13), 3.56 (3H, br s, OMe), 3.16–3.11 (1H, m, H-2a), 3.11–3.04 (3H, m, H-2b, H2-16), 1.68–1.57 (1H, m, H-14a), 1.55–1.42 (3H, m, H-14b, H2-15), 1.39 (9H, br s, Boc); 13C NMR (100 MHz, DMSO-d6) δ 172.1 (C-10/C-12), 172.0 (C-10/C-12), 156.7 (C-18), 155.2 (Boc), 136.1 (C-5a), 127.0 (C-9a), 123.7 (C-4), 121.0 (C-7), 118.4 (C-8), 117.9 (C-9), 111.4 (C-6), 109.1 (C-3), 78.2 (Boc), 53.6 (C-13), 53.0 (C-1), 51.8 (OMe), 40.4 (C-16), 29.1 (C-14), 28.2 (Boc), 27.0 (C-2), 25.0 (C-15); (+)-HRESIMS [M + H]+ m/z 475.2650 (calcd for C23H35N6O5, 475.2663).
3.1.6. Methyl (tert-butoxycarbonyl)-d-arginyl-l-tryptophanate hydrochloride (3c)
Following general procedure A, reaction of Nα-Boc-d-Arg-OH hydrochloride (64 mg, 0.206 mmol), l-Trp-OMe hydrochloride (50 mg, 0.196 mmol), HOBt (95 mg, 0.706 mmol), DIPEA (0.16 mL, 0.941 mmol), and HBTU (89 mg, 0.235 mmol) afforded the hydrochloride salt of dipeptide 3c as a clear oil/gum (67 mg, 67%). [α]21D +0.98 (c 0.102, MeOH); Rf = 0.09 (MeOH); IR (ATR) vmax 3357, 2954, 1731, 1653, 1516, 1368, 1161, 837 cm−1; 1H NMR (400 MHz, DMSO-d6) δ 10.86 (1H, d, J = 2.0 Hz, NH-5), 8.17 (1H, d, J = 7.9 Hz, NH-11), 7.48 (1H, d, J = 7.9 Hz, H-9), 7.40 (1H, t, J = 5.2 Hz, NH-17), 7.34 (1H, d, J = 7.9 Hz, H-6), 7.12 (1H, d, J = 2.0 Hz, H-4), 7.07 (1H, ddd, J = 8.2, 7.9, 1.0 Hz, H-7), 6.99 (1H, ddd, J = 8.2, 7.9, 1.0 Hz, H-8), 6.84 (1H, d, J = 8.2 Hz, NH-21), 4.51 (1H, dt, J = 8.6, 6.2 Hz, H-1), 3.97 (1H, dt, J = 9.2, 5.9 Hz, H-13), 3.58 (3H, br s, OMe), 3.20–3.11 (1H, m, H-2a), 3.10–3.04 (1H, m, H-2b), 3.02–2.96 (2H, m, H2-16), 1.58–1.47 (1H, m, H-14a), 1.47–1.37 (1H, m, H-14b), 1.38 (9H, br s, Boc), 1.37–1.28 (2H, m, H2-15); 13C NMR (100 MHz, DMSO-d6) δ 172.1 (C-10/C-12), 171.8 (C-10/C-12), 156.6 (C-18/Boc), 155.2 (C-18/Boc), 136.1 (C-5a), 127.0 (C-9a), 123.7 (C-4), 121.0 (C-7), 118.4 (C-8), 117.9 (C-9), 111.4 (C-6), 109.2 (C-3), 78.2 (Boc), 53.6 (C-13), 52.9 (C-1), 51.8 (OMe), 40.4 (C-16), 29.1 (C-14), 28.2 (Boc), 27.2 (C-2), 24.9 (C-15); (+)-HRESIMS [M + H]+ m/z 475.2651 (calcd for C23H35N6O5, 475.2663).
3.1.7. Methyl (tert-butoxycarbonyl)-l-arginyl-d-tryptophanate hydrochloride (3d)
Following general procedure A, reaction of Nα-Boc-l-Arg-OH (113 mg, 0.413 mmol), d-Trp-OMe hydrochloride (100 mg, 0.393 mmol), HOBt (190 mg, 1.41 mmol), DIPEA (0.33 mL, 1.89 mmol), and HBTU (179 mg, 0.472 mmol) afforded the hydrochloride salt of dipeptide 3d as a clear oil/gum (164 mg, 82%). [α]19D +3.0 (c 0.10, MeOH); Rf = 0.11 (MeOH); IR (ATR) vmax 3363, 2952, 2834, 1738, 1654, 1520, 1440, 1392, 1017, 841 cm−1; 1H NMR (400 MHz, DMSO-d6) δ 10.88 (1H, d, J = 1.9 Hz, NH-5), 8.18 (1H, d, J = 7.9 Hz, NH-11), 7.48 (1H, d, J = 7.5 Hz, H-9), 7.48–7.42 (1H, m, NH-17), 7.34 (1H, d, J = 7.5 Hz, H-6), 7.12 (1H, d, J = 1.9 Hz, H-4), 7.07 (1H, ddd, J = 8.4, 7.5, 1.2 Hz, H-7), 6.99 (1H, ddd, J = 8.4, 7.5, 1.2 Hz, H-8), 6.83 (1H, d, J = 8.3 Hz, NH-21), 4.51 (1H, dt, J = 7.5, 6.4 Hz, H-1), 3.98 (1H, dt, J = 6.2, 5.3 Hz, H-13), 3.58 (3H, br s, OMe), 3.19–3.12 (1H, m, H-2a), 3.10–3.03 (1H, m, H-2b), 3.03–2.97 (2H, m, H2-16), 1.58–1.46 (1H, m, H-14a), 1.46–1.36 (1H, m, H-14b), 1.40–1.28 (2H, m, H2-15), 1.38 (9H, br s, Boc); 13C NMR (100 MHz, DMSO-d6) δ 172.1 (C-10/C-12), 171.8 (C-10/C-12), 156.6 (C-18), 155.2 (Boc), 136.1 (C-5a), 127.0 (C-9a), 123.7 (C-4), 121.0 (C-7), 118.4 (C-8), 117.9 (C-9), 111.4 (C-6), 109.2 (C-3), 78.2 (Boc), 53.6 (C-13), 52.9 (C-1), 51.8 (OMe), 40.4 (C-16), 28.3 (C-14, Boc), 27.2 (C-2), 24.9 (C-15); (+)-HRESIMS [M + H]+ m/z 475.2652 (calcd for C23H35N6O5, 475.2663).
3.1.8. Methyl l-arginyl-l-tryptophanate bis(2,2,2-trifluoroacetate) (4a)
Following general procedure B, dipeptide 3a (49 mg, 0.096 mmol) was reacted with TFA (0.2 mL) in CH2Cl2 (2 mL) to afford the di-TFA salt of dipeptide 4a a pale yellow oil/gum (33 mg, 57%). [α]21D +1.5 (c 0.135, MeOH); Rf = 0.35 (MeOH); IR (ATR) vmax 3350, 3199, 3067, 2958, 2879, 1667, 1630, 1532, 1200, 1135 cm−1; 1H NMR (400 MHz, DMSO-d6) δ 10.95 (1H, d, J = 2.4 Hz, NH-5), 8.95 (1H, d, J = 7.4 Hz, NH-11), 8.20 (3H, d, J = 4.0 Hz, NH3-21), 7.85 (1H, t, J = 5.8 Hz, NH-17), 7.50 (1H, d, J = 8.0 Hz, H-9), 7.36 (1H, d, J = 8.0 Hz, H-6), 7.20 (1H, d, J = 2.4 Hz, H-4), 7.08 (1H, ddd, J = 8.5, 8.0, 1.0 Hz, H-7), 7.00 (1H, ddd, J = 8.5, 8.0, 1.0 Hz, H-8), 4.61 (1H, dt, J = 8.8, 6.5 Hz, H-1), 3.89–3.80 (1H, m, H-13), 3.60 (3H, br s, OMe), 3.24–3.17 (1H, m, H-2a), 3.16–3.07 (3H, m, H-2b, H2-16), 1.78–1.69 (2H, m, H2-14), 1.59–1.48 (2H, m, H2-15); 13C NMR (100 MHz, DMSO-d6) δ 171.8 (C-10), 168.8 (C-12), 156.9 (C-18), 136.2 (C-5a), 127.0 (C-9a), 124.0 (C-4), 121.1 (C-7), 118.6 (C-8), 117.9 (C-9), 111.6 (C-6), 108.9 (C-3), 53.4 (C-1), 52.0 (OMe), 51.7 (C-13), 40.3 (C-16), 28.5 (C-14), 27.0 (C-2), 24.0 (C-15); (+)-HRESIMS [M + H]+ m/z 375.2124 (calcd for C18H27N6O3, 375.2139).
3.1.9. Methyl d-arginyl-d-tryptophanate bis(2,2,2-trifluoroacetate) (4b)
Following general procedure B, Boc-protected dipeptide 3b (52 mg, 0.110 mmol) was reacted with TFA (0.2 mL) in CH2Cl2 (2 mL) to afford the di-TFA salt of dipeptide 4b as a pale yellow oil/gum (42 mg, 69%). [α]19D −1.2 (c 0.424, MeOH); Rf = 0.35 (MeOH); IR (ATR) vmax 3358, 3200, 3072, 2956, 2879, 1671, 1638, 1545, 1202, 1136 cm−1; 1H NMR (400 MHz, DMSO-d6) δ 10.94 (1H, d, J = 2.4 Hz, NH-5), 8.93 (1H, d, J = 7.1 Hz, NH-11), 8.18 (3H, d, J = 5.0 Hz, NH3-21), 7.80–7.74 (1H, m, NH-17), 7.49 (1H, d, J = 8.0 Hz, H-9), 7.36 (1H, d, J = 8.0 Hz, H-6), 7.20 (1H, d, J = 2.4 Hz, H-4), 7.11–7.05 (1H, m, H-7), 7.03–6.98 (1H, m, H-8), 4.64–4.57 (1H, m, H-1), 3.87–3.78 (1H, m, H-13), 3.60 (3H, br s, OMe), 3.23–3.16 (1H, m, H-2a), 3.15–3.07 (3H, m, H-2b, H2-16), 1.77–1.67 (2H, m, H2-14), 1.58–1.48 (2H, m, H2-15); 13C NMR (100 MHz, DMSO-d6) δ 171.7 (C-10), 168.7 (C-12), 156.8 (C-18), 136.2 (C-5a), 126.9 (C-9a), 123.9 (C-4), 121.1 (C-7), 118.5 (C-8), 117.9 (C-9), 111.5 (C-6), 108.8 (C-3), 53.3 (C-1), 52.0 (OMe), 51.6 (C-13), 40.2 (C-16), 28.4 (C-14), 27.0 (C-2), 23.9 (C-15); (+)-HRESIMS [M + H]+ m/z 375.2133 (calcd for C18H27N6O3, 375.2139).
3.1.10. Methyl d-arginyl-l-tryptophanate bis(2,2,2-trifluoroacetate) (4c)
Following general procedure B, Boc-protected dipeptide 3c (48 mg, 0.094 mmol) was reacted with TFA in CH2Cl2 to afford the di-TFA salt of dipeptide 4c as a pale yellow oil/gum (31 mg, 55%). [α]21D -11.2 (c 0.143, MeOH); Rf = 0.09 (MeOH); IR (ATR) vmax 3350, 3197, 3077, 2961, 1663, 1624, 1556, 1435, 1356, 1182, 1131 cm−1; 1H NMR (400 MHz, DMSO-d6) δ 10.90–10.86 (1H, m, NH-5), 8.95 (1H, d, J = 8.0 Hz, NH-11), 8.20–8.11 (3H, m, NH3-21), 7.78–7.71 (1H, m, NH-17), 7.50 (1H, d, J = 7.8 Hz, H-9), 7.35 (1H, d, J = 7.8 Hz, H-6), 7.17–7.15 (1H, m, H-4), 7.10–7.04 (1H, m, H-7), 7.02–7.00 (1H, m, H-8), 4.68–4.61 (1H, m, H-1), 3.86–3.80 (1H, m, H-13), 3.62 (3H, br s, OMe), 3.24–3.17 (1H, m, H-2a), 3.11–3.02 (1H, m, H-2b), 3.01–2.92 (2H, m, H2-16), 1.62–1.49 (2H, m, H2-15), 1.36–1.25 (1H, m, H-14a), 1.25–1.12 (1H, m, 14b); 13C NMR (100 MHz, DMSO-d6) δ 171.8 (C-10), 168.4 (C-12), 156.8 (C-18), 136.1 (C-5a), 126.9 (C-9a), 124.0 (C-4), 121.1 (C-7), 118.5 (C-8), 117.9 (C-9), 111.5 (C-6), 109.0 (C-3), 53.0 (C-1), 52.1 (OMe), 51.6 (C-13), 40.0 (C-16), 28.3 (C-15), 27.4 (C-2), 23.7 (C-14); (+)-HRESIMS [M + H]+ m/z 375.2139 (calcd for C18H27N6O3, 375.2139).
3.1.11. Methyl l-arginyl-d-tryptophanate bis(2,2,2-trifluoroacetate) (4d)
Following general procedure B, Boc-protected dipeptide 3d (35 mg, 0.068 mmol) was reacted with TFA in CH2Cl2 to afford the di-TFA salt of dipeptide 4d as a pale-yellow oil/gum (23 mg, 56%). [α]19D +27 (c 0.10, MeOH); Rf = 0.35 (MeOH); IR (ATR) vmax 3347, 3195, 3080, 2948, 2871, 1666, 1624, 1590, 1439, 1200, 1184, 1134 cm−1; 1H NMR (400 MHz, DMSO-d6) δ 10.91–10.87 (1H, m, NH-5), 8.95 (1H, d, J = 7.8 Hz, NH-11), 7.86–7.23 (1H, m, NH-17), 7.50 (1H, d, J = 7.8 Hz, H-9), 7.35 (1H, d, J = 7.8 Hz, H-6), 7.18–7.13 (1H, m, H-4), 7.11–7.04 (1H, m, H-7), 7.03–6.97 (1H, m, H-8), 4.64 (1H, dt, J = 9.2, 6.7 Hz, H-1), 3.86–3.77 (1H, m, H-13), 3.62 (3H, br s, OMe), 3.25–3.17 (1H, m, H-2a), 3.11–3.02 (1H, m, H-2b), 3.02–2.91 (2H, m, H2-16), 1.65–1.49 (2H, m, H2-15), 1.37–1.25 (1H, m, H-14a), 1.25–1.14 (1H, m, H-14b); 13C NMR (100 MHz, DMSO-d6) δ 171.8 (C-10), 168.4 (C-12), 156.9 (C-18), 136.1 (C-5a), 126.9 (C-9a), 124.0 (C-4), 121.1 (C-7), 118.5 (C-8), 117.9 (C-9), 111.5 (C-6), 108.9 (C-3), 53.0 (C-1), 52.0 (OMe), 51.7 (C-13), 40.0 (C-16), 28.3 (C-15), 27.4 (C-2), 23.7 (C-14); (+)-HRESIMS [M + H]+ m/z 375.2134 (calcd for C18H27N6O3, 375.2139).
3.1.12. Cyclo(l-Trp-l-Arg) 2,2,2-trifluoroacetate (5a)
Following general procedure C, dipeptide 4a (113 mg, 0.189 mmol) was reacted with NH4OH (0.13 mL) in MeOH (0.76 mL) to afford the TFA salt of cyclo(l-Trp-l-Arg) (5a) as a pale-yellow oil/gum (47 mg, 55%). [α]24D -10.5 (c 0.105, MeOH); ECD (c 0.00035, MeOH) λ (Δε) 194 (0), 210 (−17.4), 223 (0), 231 (+10.2); Rf = 0.60 (MeOH); IR (ATR) vmax 3363, 3233, 2969, 1659, 1648, 1457, 1137, 1106, 748 cm−1; 1H NMR (400 MHz, DMSO-d6) δ 10.88–10.84 (1H, m, NH-17), 8.09 (1H, d, J = 2.0 Hz, NH-4), 8.01–7.97 (1H, m, NH-1), 7.57 (1H, d, J = 7.9 Hz, H-21), 7.33 (1H, d, J = 7.9 Hz, H-18), 7.27–7.19 (1H, m, NH-10), 7.08–7.04 (1H, m, H-16), 7.05–7.00 (1H, m, H-19), 7.00–6.91 (1H, m, H-20), 4.14–4.09 (1H, br m, H-3), 3.59–3.53 (1H, br m, H-6), 3.23 (1H, dd, J = 14.5, 4.5 Hz, H-14a), 3.04 (1H, dd, J = 14.5, 4.5 Hz, H-14b), 2.72–2.64 (2H, m, H2-9), 1.08–0.95 (1H, m, H-7a), 0.95–0.78 (2H, m, H2-8), 0.67–0.55 (1H, m, H-7b); 13C NMR (100 MHz, DMSO-d6) δ 167.2 (C-2), 166.9 (C-5), 156.7 (C-11), 135.9 (C-17a), 127.8 (C-21a), 124.6 (C-16), 120.8 (C-19), 119.0 (C-21), 118.4 (C-20), 111.2 (C-18), 108.6 (C-15), 55.5 (C-3), 53.4 (C-6), 40.1 (C-9), 30.6 (C-7), 29.0 (C-14), 23.4 (C-8); (+)-HRESIMS [M + H]+ m/z 343.1874 (calcd for C17H23N6O2, 343.1877).
1H NMR (400 MHz, CD3OD) δ 7.63 (1H, d, J = 8.0 Hz, H-21), 7.35 (1H, d, J = 8.0 Hz, H-18), 7.12–7.07 (1H, m, H-19), 7.08 (1H, br s, H-16), 7.04–6.98 (1H, m, H-20), 4.31 (1H, ddd, J = 4.6, 4.0, 1.2 Hz, H-3), 3.68 (1H, ddd, J = 7.4, 6.5, 1.2 Hz, H-6), 3.49 (1H, dd, J = 14.6, 3.7 Hz, H-14a), 3.14 (1H, dd, J = 14.6, 4.6 Hz, H-14b), 2.62 (2H, t, J = 7.1 Hz, H2-9), 0.93–0.80 (2H, m, H-7a, H-8a), 0.77–0.65 (1H, m, H-8b), 0.56–0.45 (1H, m, H-7b); 13C NMR (100 MHz, CD3OD) δ 169.9 (C-2), 169.5 (C-5), 158.5 (C-11), 137.8 (C-17a), 129.4 (C-21a), 126.1 (C-16), 122.5 (C-19), 120.3 (C-20), 120.2 (C-21), 112.2 (C-18), 109.6 (C-15), 57.5 (C-3), 55.2 (C-6), 41.7 (C-9), 32.1 (C-7), 30.5 (C-14), 24.5 (C-8).
3.1.13. Cyclo(d-Trp-d-Arg) 2,2,2-trifluoroacetate (5b)
Following general procedure C, dipeptide 4b (27 mg, 0.045 mmol) was reacted with NH4OH (0.03 mL) in MeOH (0.18 mL) to afford the TFA salt of cyclo(d-trp-d-Arg) (5b) as a pale-yellow oil/gum (15 mg, 71%). [α]24D +10.7 (c 0.103, MeOH); ECD (c 0.00037, MeOH) λ (Δε) 195 (0), 213 (+18.7), 223 (0), 230 (-9.83); Rf = 0.60 (MeOH); IR (ATR) vmax 3350, 3216, 2979, 1659, 1654, 1457, 1201, 1137 cm−1; 1H NMR (400 MHz, DMSO-d6) δ 10.87–10.83 (1H, m, NH-17), 8.09 (1H, d, J = 2.2 Hz, NH-4), 8.00 (1H, d, J = 2.2 Hz, NH-1), 7.57 (1H, d, J = 8.0 Hz, H-21), 7.32 (1H, d, J = 8.0 Hz, H-18), 7.25–7.12 (1H, m, NH-10), 7.07–7.05 (1H, m, H-16), 7.06–7.01 (1H, m, H-19), 6.97–6.91 (1H, m, H-20), 4.14–4.09 (1H, br m, H-3), 3.59–3.53 (1H, br m, H-6), 3.23 (1H, dd, J = 14.6, 4.4 Hz, H-14a), 3.04 (1H, dd, J = 14.6, 4.6 Hz, H-14b), 2.74–2.64 (2H, m, H2-9), 1.08–0.96 (1H, m, H-7a), 0.96–0.79 (2H, m, H2-8), 0.66–0.54 (1H, m, H-7b); 13C NMR (100 MHz, DMSO-d6) δ 167.2 (C-2), 166.9 (C-5), 156.6 (C-11), 135.9 (C-17a), 127.8 (C-21a), 124.6 (C-16), 120.8 (C-19), 119.0 (C-21), 118.4 (C-20), 111.1 (C-18), 108.7 (C-15), 55.4 (C-3), 53.4 (C-6), 40.1 (C-9), 30.6 (C-7), 29.0 (C-14), 23.4 (C-8); (+)-HRESIMS [M + H]+ m/z 343.1867 (calcd for C17H23N6O2, 343.1877).
1H NMR (400 MHz, CD3OD) δ 7.63 (1H, d, J = 8.0 Hz, H-21), 7.35 (1H, d, J = 8.0 Hz, H-18), 7.09 (1H, ddd, J = 8.2, 8.0, 1.0 Hz, H-19), 7.08 (1H, br s, H-16), 7.01 (1H, ddd, J = 8.2, 8.0, 1.0 Hz, H-20), 4.31 (1H, ddd, J = 5.0, 4.0, 1.0 Hz, H-3), 3.68 (1H, ddd, J = 7.5, 6.2, 1.2 Hz, H-6), 3.48 (1H, dd, J = 14.6, 3.8 Hz, H-14a), 3.14 (1H dd, J = 14.6, 4.6 Hz, H-14b), 2.62 (2H, t, J = 7.1 Hz, H2-9), 0.94–0.79 (2H, m, H-7a, H-8a), 0.77–0.64 (1H, m, H-8b), 0.54–0.43 (1H, m, H-7b); 13C NMR (100 MHz, CD3OD) δ 169.9 (C-2), 169.5 (C-5), 158.4 (C-11), 137.8 (C-17a), 129.4 (C-21a), 126.0 (C-16), 122.5 (C-19), 120.2 (C-20, C-21), 112.2 (C-18), 109.6 (C-15), 57.5 (C-3), 55.2 (C-6), 41.7 (C-9), 32.0 (C-7), 30.4 (C-14), 24.5 (C-8).
3.1.14. Cyclo(l-Trp-d-Arg) 2,2,2-trifluoroacetate (5c)
Following general procedure C, dipeptide 4c (118.1 mg, 0.196 mmol) was reacted with NH4OH (0.13 mL) in MeOH (0.78 mL) to afford the TFA salt of cyclo(l-Trp-d-Arg) (5c) as a pale-yellow oil/gum (48 mg, 54%). [α]21D +26.0 (c 0.131, MeOH); ECD (c 0.00035, MeOH) λ (Δε) 204 (0), 216 (−10.6), 227 (0), 235 (+2.95); Rf = 0.49 (MeOH); IR (ATR) vmax 3356, 3215, 2961, 2903, 1662, 1458, 1202, 1138 cm−1; 1H NMR (400 MHz, DMSO-d6) δ 10.91 (1H, d, J = 2.0 Hz, NH-17), 8.10 (1H, d, J = 2.2 Hz, NH-4), 7.91–7.90 (1H, m, NH-1), 7.57 (1H, d, J = 7.9 Hz, H-21), 7.47 (1H, t, J = 5.2 Hz, NH-10), 7.32 (1H, d, J = 7.9 Hz, H-18), 7.07 (1H, d, J = 2.0 Hz, H-16), 7.04 (1H, ddd, J = 8.4, 7.9, 1.0 Hz, H-19), 6.95 (1H, ddd, J = 8.4, 7.9, 1.0 Hz, H-20), 4.10–4.05 (1H, br m, H-3), 3.29–3.22 (1H, dd, J = 14.5, 4.5 Hz, H-14a), 3.10–3.01 (1H, m, H-14b), 3.08–3.03 (1H, m, H-6), 3.02–3.00 (2H, m, H2-9), 1.61–1.50 (1H, m, H-7a), 1.50–1.42 (1H, m, H-7b), 1.42–1.30 (2H, m, H2-8); 13C NMR (100 MHz, DMSO-d6) δ 168.2 (C-2), 167.5 (C-5), 156.7 (C-11), 135.9 (C-17a), 127.6 (C-21a), 124.6 (C-16), 120.9 (C-19), 118.8 (C-21), 118.4 (C-20), 111.2 (C-18), 108.4 (C-15), 55.4 (C-3), 52.9 (C-6), 40.4 (C-9), 29.1 (C-7), 28.9 (C-14), 23.5 (C-8); (+)-HRESIMS [M + H]+ m/z 343.1874 (calcd for C17H23N6O2, 343.1877).
1H NMR (400 MHz, CD3OD) δ 7.60 (1H, d, J = 8.0 Hz, H-21), 7.33 (1H, d, J = 8.0 Hz, H-18), 7.11–7.05 (1H, m, H-19), 7.06 (1H, br s, H-16), 7.00 (1H, ddd, J = 8.5, 8.0, 1.0 Hz, H-20), 4.22 (1H, t, J = 4.0 Hz, H-3), 3.46 (1H, dd, J = 14.6, 4.0 Hz, H-14a), 3.15 (1H, dd, J = 14.6, 4.4 Hz, H-14b), 3.04 (2H, t, J = 6.8 Hz, H2-9), 2.77 (1H, t, J = 4.0 Hz, H-6), 1.73–1.62 (1H, m, H-7a), 1.54–1.32 (3H, m, H-7b, H2-8); 13C NMR (100 MHz, CD3OD) δ 171.4 (C-2), 170.3 (C-5), 158.6 (C-11), 137.9 (C-17a), 128.8 (C-21a), 126.1 (C-16), 122.6 (C-19), 120.2 (C-20), 119.8 (C-21), 112.2 (C-18), 109.0 (C-15), 57.7 (C-3), 54.3 (C-6), 42.0 (C-9), 31.1 (C-14), 29.6 (C-7), 24.3 (C-8).
3.1.15. Cyclo(d-Trp-l-Arg) 2,2,2-trifluoroacetate (5d)
Following general procedure C, dipeptide 4d (52 mg, 0.086 mmol) was reacted with NH4OH (0.057 mL) in MeOH (0.344 mL) to afford the TFA salt of cyclo(d-Trp-l-Arg) (5d) as a pale-yellow gum (18 mg, 47%). [α]19D -26.4, (c 0.421, MeOH); ECD (c 0.00035, MeOH) λ (Δε) 204 (0), 216 (+11.8), 227 (0), 234 (-1.75); Rf = 0.49 (MeOH); IR (ATR) vmax 3342, 3214, 2964, 1662, 1651, 1456, 1431, 1201, 1135 cm−1; 1H NMR (400 MHz, DMSO-d6) δ 10.93 (1H, d, J = 2.0 Hz, NH-17), 8.11–8.09 (1H, m, NH-4), 7.92–7.90 (1H, m, NH-1), 7.63 (1H, t, J = 5.5 Hz, NH-10), 7.57 (1H, d, J = 8.1 Hz, H-21), 7.32 (1H, d, J = 8.1 Hz, H-18), 7.07 (1H, d, J = 2.0 Hz, H-16), 7.04 (1H, ddd, J = 8.1, 7.5, 1.0 Hz, H-19), 6.94 (1H, ddd, J = 8.1, 7.5, 1.0 Hz, H-20), 4.10–4.04 (1H, br m, H-3), 3.29–3.21 (1H, dd, J = 14.5, 4.4 Hz, H-14a), 3.09–3.03 (1H, m, H-6), 3.08–3.01 (1H, m, H-14b), 3.02–3.00 (2H, m, H2-9), 1.61–1.45 (2H, m, H2-7), 1.43–1.31 (2H, m, H2-8); 13C NMR (100 MHz, DMSO-d6) δ 168.2 (C-2), 167.5 (C-5), 156.8 (C-11), 135.9 (C-17a), 127.6 (C-21a), 124.6 (C-16), 120.9 (C-19), 118.8 (C-21), 118.4 (C-20), 111.2 (C-18), 108.4 (C-15), 55.4 (C-3), 52.9 (C-6), 40.5 (C-9), 29.1 (C-7), 28.9 (C-14), 23.5 (C-8); (+)-HRESIMS [M + H]+ m/z 343.1870 (calcd for C17H23N6O2, 343.1877).
1H NMR (400 MHz, CD3OD) δ 7.60 (1H, d, J = 8.0 Hz, H-21), 7.33 (1H, d, J = 8.0 Hz, H-18), 7.11–7.05 (1H, m, H-19), 7.06 (1H, br s, H-16), 7.03–6.98 (1H, m, H-20), 4.23 (1H, t, J = 4.1 Hz, H-3), 3.46 (1H, dd, J = 14.6, 4.0 Hz, H-14a), 3.15 (1H, dd, J = 14.6, 4.5 Hz, H-14b), 3.04 (2H, t, J = 6.7 Hz, H-9), 2.78 (1H, t, J = 3.9 Hz, H-6), 1.73–1.61 (1H, m, H-7a), 1.54–1.33 (3H, m, H-7b, H2-8); 13C NMR (100 MHz, CD3OD) δ 171.4 (C-2), 170.3 (C-5), 158.6 (C-11), 137.9 (C-17a), 128.8 (C-21a), 126.1 (C-16), 122.6 (C-19), 120.2 (C-20), 119.8 (C-21), 112.2 (C-18), 109.0 (C-15), 57.7 (C-3), 54.3 (C-6), 42.0 (C-9), 31.1 (C-14), 29.6 (C-7), 24.3 (C-8).