2.1 Chemistry

A Varian Prostar liquid chromatograph (Varian Scientific Instruments, Palo Alto, CA, USA) was used for the purification and purity assessment of the peptides. Preparative reversed-phase HPLC was performed on a Vydac 218-TP column (PROTO 300 C18 10 μm, 20 mm x 250 mm) with a linear gradient of 15 – 50 % MeOH in 0.1% trifluoroacetic acid (TFA) over 40 min at a flow rate of 12 mL/min, absorption being measured at 220 and 280 nm. Analytical reversed-phase HPLC was carried out on a Vydac 218-TP column (PROTO 300 C18 5 μm, 10 mm x 250 mm) with a linear gradient of 20 – 80% MeOH in 0.1 % TFA over 30 min at a flow rate of 1 mL/min. All compounds had >95% purity. The same column was used for the determination of the capacity factor K′ under the same conditions. Precoated plates (silica gel 60 F₂₅₄, 250 μm, Merck, Darmstadt, Germany) were used for ascending thin layer chromatography (TLC) in the following systems: (I) n-BuOH/AcOH/H₂O (4:1:1); (II) n-BuOH/pyridine/AcOH/H₂O (15:10:3:2). Molecular masses of compounds were determined by electrospray mass spectrometry on a hybrid Q-Tof mass spectrometer interfaced to a Mass Lynx 4.0 data system. ¹H NMR spectra were recorded on a Varian INOVA 500 MHz spectrometer and referenced with respect to residual signals of the solvent. The following abbreviations were used: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet. Specific optical rotations were determined on an AA-5 automatic polarimeter.

The compounds were prepared by cyclization between Lys and Phe or D-Lys and D-Phe of linear precursor peptides according to a synthetic scheme previously used for the preparation of cyclic peptide 5.^[15] The linear precursor peptides were synthesized by the manual solid-phase technique using a 2-chlorotrityl resin (200 – 400 mesh, substitution 1.66 mequiv/g, Bachem Bioscience, King of Prussia, PA). 9-Fluorenylmethyloxycarbonyl (Fmoc) protection was used for the α-amino group of amino acids, and the side chains of Arg and Lys were protected with tert-butyloxycarbonyl (Boc) and 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc), respectively. N,N-dicyclohexylcarbodiimide (DIC)/6-chloro-N-hydroxybenzotriazole (6-Cl-HOBt) were used as coupling agents. Peptides were cleaved from the resin by treatment with 1% TFA in dichloromethane (DCM), followed by N-terminal Fmoc deprotection with 10% diethylamine (DEA) in DMF. After solvent evaporation the residue was triturated with anhydrous Et₂O, yielding the peptides in solid form. The peptides were ≥90% pure as established by analytical HPLC and were used in the cyclization reaction without further purification.

For peptide cyclization, a solution of the side chain protected linear peptide precursor (0.1 mmol) and triethylamine (TEA) (11.4 μL, 0.1 mmol) in 30 mL of anhydrous DMF was slowly added to a solution of benzotriazole-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP) (282 mg,0.6 mmol) and 4-dimethylaminopyridine (DMAP) (102 mg, 0.8 mmol) in 300 mL of anhydrous DMF. The solution was stirred in the dark, and progress of the cyclization was monitored by analytical reversed-phase HPLC (linear gradient of 50 – 90 % MeOH in 0.1% TFA over 30 min at a flow rate of 1 mL/min). The time required for completion of the cyclization reaction varied from 2 to 24 h. After completion of the reaction the solvent was removed by vacuum distillation and Boc and Pmc side chain protection was removed by treatment with 95% TFA/H₂O for 4 h. After evaporation of the solvent and trituration of the residue with anhydrous Et₂O the peptides were obtained in solid form. Purification by preparative reversed-phase HPLC yielded the pure cyclic peptides. Yields for cyclization of the linear precursors were determined after side chain deprotection and final purification by reversed-phase HPLC.

Cyclization yield 50%; HPLC K′ 5.38; TLC R_f 0.43 (I), R_f 0.73(II); HRMS (ESI) m/z calcd for C₃₀H₄₃N₈O₅ 595.3358, found 595.3356; [α]_D²⁰ −37.0° (c 0.5, 7% AcOH ); ¹H NMR (500 MHz, DMSO-d₆) δ 9.20 (s, 1H), 8.00 (d, J = 9.1 Hz, 1H), 7.87 (d, J = 9.0 Hz, 1H), 7.66 (d, J = 8.9 Hz, 2H), 7.48 (t, J = 5.2 Hz, 1H), 7.37 (d, J = 9.5 Hz, 1H), 7.32 – 7.26 (m, 3H), 7.25 – 7.19 (m, 4H), 6.96 (d, J = 8.3 Hz, 2H), 6.64 (d, J = 8.4 Hz, 2H), 4.46 (q, J = 8.8 Hz, 1H), 4.35 (dd, J = 16.6, 7.8 Hz, 1H), 4.26 (dd, J = 15.4, 8.6 Hz, 1H), 3.93 (q, J = 8.7 Hz, 1H), 3.04 – 2.88 (m, 7H), 2.76 – 2.68 (m, 4H), 1.71 – 1.04 (m, 12H).

Cyclization yield 35%; HPLC K′ 5.36; TLC R_f 0.43 (I), R_f 0.73(II); HRMS (ESI) m/z calc. for C₃₀H₄₃N₈O₅ 595.3358, found 595.3349; [α]_D²⁰ +37.0° ( c 0.5, 7% AcOH ); ¹H NMR (500 MHz, DMSO-d₆) δ 9.19 (s, 1H), 8.01 (d, J = 9.2 Hz, 1H), 7.87 (d, J = 9.1 Hz, 1H), 7.66 (d, J = 9.0 Hz, 2H), 7.48 (t, J = 5.6 Hz, 1H), 7.36 (d, J = 9.4 Hz, 1H), 7.32 – 7.27 (m, 3H), 7.23 - 7.19 (m, 4H), 6.96 (d, J = 8.5 Hz, 2H), 6.64 (d, J = 8.5 Hz, 2H), 4.46 (q, J = 8.6 Hz, 1H), 4.35 (dd, J = 16.6, 7.8 Hz, 1H), 4.26 (dd, J = 15.3, 9.0 Hz, 1H), 3.93 (q, J = 8.4 Hz, 1H), 3.04- 2.88 (m, 7H), 2.75-2.68 (m, 4H), 1.74 – 1.04 (m, 12H).

Cyclization yield 10%; HPLC K′ 4.10; TLC R_f 0.33 (I), R_f 0.62(II); HRMS (ESI) m/z calcd for C₃₀H₄₃N₈O₅ 595.3358, found 595.3354; [α]_D²⁰ +60.8^° (c 0.5, 7% AcOH); ¹H NMR (500 MHz, DMSO-d₆) δ 9.25 (s, 1H), 7.93 (dd, J = 16.0, 10.0 Hz, 2H), 7.68 (dd, J = 13.6, 8.2 Hz, 3H), 7.48 (m, 3H), 7.29 (t, J = 7.5 Hz, 3H), 7.25 – 7.11 (m, 6H), 6.98 (d, J = 8.5 Hz, 2H), 6.67 (d, J = 8.2 Hz, 2H), 4.47 (dd, J = 17.7, 10.1 Hz, 1H), 4.38 (dd, J = 17.6, 8.9 Hz, 1H), 3.98 (dt, J = 15.4, 7.5 Hz, 2H), 3.08 (dd, J = 13.2, 7.1 Hz, 3H), 1.66 – 1.09 (m, 10H).

Cyclization yield 10%; HPLC K′ 4.20; TLC R_f 0.34 (I), R_f 0.65(II); HRMS (ESI) m/z calcd for C₃₀H₄₃N₈O₅ 595.3358, found 595.3354; [α]_D²⁰ -60.3^° (c 0.5, 7%, AcOH); ¹H NMR (500 MHz, DMSO-d₆) δ 9.25 (s, 1H), 7.93 (dd, J = 16.1, 10.1 Hz, 2H), 7.68 (dd, J = 13.8, 8.5 Hz, 3H), 7.48 (m, 3H), 7.29 (t, J = 7.5 Hz, 3H), 7.26 – 7.11 (m, 6H), 6.98 (d, J = 8.5 Hz, 2H), 6.67 (d, J = 8.2 Hz, 2H), 4.47 (dd, J = 17.7, 10.1 Hz, 1H), 4.38 (dd, J = 17.1, 9.6 Hz, 1H), 3.98 (td, J = 15.2, 7.7 Hz, 2H), 3.08 (dd, J = 13.2, 7.1 Hz, 3H), 1.66 – 1.05 (m, 10H).

The compound was prepared according to a synthetic scheme previously used for the synthesis of cyclic peptide 7.^[15] The linear tripeptide H-D-Dmt-Arg(Tos)-D-Phe (Tos = p-toluenesulfonyl) was assembled by the manual solid-phase technique on a Sasrin resin (0.65 equiv/g, Bachem). To introduce the reduced peptide bond between the D-Lys and D-Dmt residues, a reductive alkylation reaction^[16] between Boc-D-Lys(Z-2-Cl)-aldehyde (Z-2-Cl = 2-chlorobenzyloxycarbonyl) and the α-amino group of the resin-bound tripeptide was performed. Boc-D-Lys(Z-2-Cl)-aldehyde was synthesized via preparation of Boc-D-Lys(Z-2-Cl)-N-methoxy-N-methylamide by using a published procedure.^[17] The peptide was then cleaved from the resin and N-terminally deprotected by treatment with a TFA/H₂O/TIS (9.5:0.25:0.25) (TIS = triisopropylsilane) solution for 3 h. After filtration and evaporation of the solvent the residue was triturated with Et₂O, yielding the crude, side chain protected tetrapeptide in solid form. The product was purified by preparative HPLC using a Vydac 218-TP column PROTO 300 C18 10 μm, 20 mm x 250 mm) with a linear gradient of 50 – 90% MeOH in 0.1% TFA over 40 min at a flow rate of 12 mL/min. Its structural identity was confirmed by mass spectrometry ([M+H⁺] = 949). Cyclization was performed as described above for the preparation of compounds 1 – 4. The cyclization was complete after 4 h, as determined by analytical reversed-phase HPLC. After evaporation of the DMF, the Z-2-Cl and Tos protection groups were removed by treatment with HF/anisole at 0 °C for 60 min. Following HF evaporation the residue was taken up in 7% AcOH, and after lyophilisation the target peptide was purified by preparative reversed-phase HPLC.

Cyclization yield 35%; HPLC K′ 5.38; TLC R_f 0.13(I), R_f 0.66(II); HRMS (ESI) m/z calcd for C₃₂H₄₉N₈O₄ 609.3877, found 609.3861; [α]_D²⁰ + 51.2° (c 0.5, 7% AcOH); ¹H NMR (500 MHz, DMSO-d₆) δ 9.07 (s, 1H), 7.78 (s, 3H), 7.47 (t, J = 5.5 Hz, 1H), 7.27-7.21 (m, 6H), 7.14 (d, J=7.3 Hz, 1H), 6.41 (s, 2H), 4.26 (dd, J = 15.1, 7.8 Hz, 1H), 4.20 (dd, J = 15.5, 8.0 Hz, 1H), 3.01- 2.88 (m, 5H), 2.82-2.72 (m, 3H), 2.17 (s, 6H), 1.65 – 1.42 (m, 5H), 1.37-1.21 (m, 3H), 1.04 (m, 2H).

Cyclization yield 40%; HPLC K′ 5.38; TLC R_f 0.13(I), R_f 0.66(II); HRMS (ESI) m/z calcd for C₃₂H₄₉N₈O₄ 609.3877, found 609.3862; [α]_D²⁰ – 51.0° (c 0.5, 7% AcOH); ¹H NMR (500 MHz, DMSO-d₆) δ 9.04 (s, 1H), 7.74 (s, 3H), 7.45 (t, J = 5.5 Hz, 1H), 7.26 – 7.21 (m, 6H), 7.14 (d, J = 7.3 Hz, 1H), 6.49 (s, 2H), 4.26 (dd, J = 14.7, 8.4 Hz, 1H), 4.20 (dd, J = 15.5, 8.0 Hz, 1H), 2.94-2.89 (m, 5H), 2.78-2.72 (m, 3H), 2.20 (s, 6H), 1.65 – 1.42 (m, 5H), 1.36- 1.26 (m, 3H), 1.09-1.05 (m, 2H).