General chemistry

All reagents and solvents were purchased as ACS/HPLC grade. All reactions were monitored by TLC and/or by LC/MS using a Shimadzu 2010 series HPLC and/or an Agilent 1200 series HPLC coupled with an Agilent G6140A Quadrupole MS system (Santa Clara, CA). LC/MS analyses were achieved using 0.1% formic acid in water and 0.1% formic acid in methanol as mobile phase A and B, respectively. The column temperature was maintained at 40 °C, and the UV wavelength for detection was set at 280 nm. The gradient was set from 10 to 90% B over 5 min at a flow rate of 0.8 mL/min. Compound purification was carried out using flash column chromatography on a Teledyne ISCO CombiFlash system (Thousand Oaks, CA). Lyophilization of final compounds was performed on a VirTis freezemobile freeze dryer (SP Scientific, Warminster, PA). 1H (400 MHz) and 13C (100 MHz) NMR spectra were recorded at ambient temperature on a Bruker UltrashieldTM 400 MHz Multinuclear NMR spectrometer (Billerica, MA) and calibrated using residual undeuterated solvents (chloroform, DMSO, water) as the internal reference. All chemical shifts are reported in parts per million (ppm). All coupling constants are reported in Hz. Spin multiplicities were abbreviated as s (singlet), d (doublet), dd (doublet of doublets), t (triplet), m (multiplet), and br (broad). High-resolution mass spectrometry (HRMS) experiments were conducted by the Center for Integrative Proteomics Research (CIPR) at Rutgers University using a Thermo LTQ Orbitrap Velos ETD with Dionex UltiMate 3000 nano-flow 2D LC system (Thermo Fischer Scientific Inc., Waltham, MA).

General amide coupling procedure for the synthesis of intermediates 7a-j and 8a-h

To a suspended mixture of Boc-L-cystine-OH in anhydrous dichloromethane was added DIPEA (6 equiv.), leading to a clear solution. Subsequently, the amine (3 equiv.) and PyAOP (2 equiv.) were added to the solution, and the reaction mixture was stirred at room temperature for 30 min. The reaction mixture was diluted with water and extracted with dichloromethane three times. The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by flash chromatography using pre-packed normal phase silica gel column.

N,N′-Bis(tert-butoxycarbonyl)-L-cystine bispiperazide (7a)

The compound was eluted with 2% methanol in dichloromethane with 0.1% triethylamine and concentrated under reduced pressure to give a light yellow solid (74 mg, yield: 60%). 1H NMR (400 MHz, CDCl3): δ 5.44 (d, 2H, J = 8 Hz), 4.93 (t, 2H, J = 8 Hz), 3.67-3.40 (m, 16H), 3.01 (br, 4H), 1.47 (s, 18H), 1.43 (s, 18H); 13C NMR (100 MHz, CDCl3): δ 169.2, 155.0, 154.4, 80.4, 49.1, 45.8, 42.2, 41.5, 28.3; LC-MS (ESI+): m/z 777.1 [M + H]+.

N,N′-Bis(tert-butoxycarbonyl)-L-cystine bis(N′-tert-butylpiperazide) (7b)

The compound was eluted with 5% methanol in dichloromethane with 0.1% triethylamine and concentrated under reduced pressure to give a light yellow solid (80 mg, yield: 73%). 1H NMR (400 MHz, CDCl3): δ 5.46 (d, 2H, J = 8 Hz), 4.93 (br, 2H), 3.64 (br, 8H), 3.02-2.94 (m, 4H), 2.56 (br, 8H), 1.42 (s, 18H), 1.06 (s, 18H); 13C NMR (100 MHz, CDCl3): δ 168.6, 155.0, 80.0, 49.1, 46.4, 45.5, 42.9, 41.8, 28.4, 25.8; LC-MS (ESI+): m/z 689.2 [M + H]+.

N,N′-Bis(tert-butoxycarbonyl)-L-cystine bis(N′-cyclopropylpiperazide) (7c)

The compound was eluted with 1.2% methanol in dichloromethane with 0.1% triethylamine and concentrated under reduced pressure to give a yellow solid (70 mg, yield: 67%). 1H NMR (400 MHz, CDCl3): δ 5.47 (d, 2H, J = 8 Hz), 4.93 (t, 2H, J = 8 Hz), 3.56 (br, 8H), 3.03-2.96 (m, 4H), 2.62-2.57 (m, 8H), 1.60 (br, 2H), 1.41 (s, 18H), 0.46-0.40 (m, 8H); 13C NMR (100 MHz, CDCl3): δ 168.8, 155.0, 80.1, 53.1, 49.1, 45.8, 41.8, 38.2, 28.4, 5.9; LC-MS (ESI+): m/z 657.1 [M + H]+.

N,N′-Bis(tert-butoxycarbonyl)-L-cystine bis(N′-cyclopentylpiperazide) (7d)

The compound was eluted with 5% methanol in dichloromethane with 0.1% triethylamine and concentrated under reduced pressure to give a light yellow solid (85 mg, yield: 75%). 1H NMR (400 MHz, CDCl3): δ 5.44 (d, 2H, J = 8 Hz), 4.91 (br, 2H), 3.75 (br, 8H), 3.15-3.01 (m, 2H), 2.98 (br, 4H), 2.68 (br, 8H), 1.89 (br, 4H), 1.74 (br, 4H), 1.58–1.48 (m, 8H), 1.41 (s, 18H); 13C NMR (100 MHz, CDCl3): δ 168.8, 155.1, 80.3, 67.6, 52.1, 51.6, 49.0, 45.8, 41.5, 29.7, 28.3, 23.9; LC-MS (ESI+): m/z 713.2 [M + H]+.

N,N′-Bis(tert-butoxycarbonyl)-L-cystine bis(N′-phenylpiperazide) (7e)

The compound was eluted with 1.7% methanol in dichloromethane with 0.1% triethylamine and concentrated under reduced pressure to give a dark yellow solid (84 mg, yield: 73%). 1H NMR (400 MHz, CDCl3): δ 7.27-7.23 (m, 4H), 6.89 (d, 6H, J = 8 Hz), 5.48 (d, 2H, J = 8 Hz), 5.00 (br, 2H), 3.77 (br, 8H), 3.17 (br, 8H), 3.02 (br, 4H), 1.41 (s, 18H); 13C NMR (100 MHz, CDCl3): δ 169.0, 155.0, 150.8, 129.3, 120.6, 116.6, 80.2, 55.9, 49.8, 45.8, 42.3, 41.7, 28.4; LC-MS (ESI+): m/z 729.1 [M + H]+.

N,N′-Bis(tert-butoxycarbonyl)-L-cystine bis(4′-tert-butoxycarbonyl-2’,2’-dimethylpiperazide) (7f)

The compound was eluted with 2.2% methanol in dichloromethane with 0.1% triethylamine and concentrated under reduced pressure to give a dark yellow solid (80 mg, yield: 63%). 1H NMR (400 MHz, CDCl3): δ 5.32 (br, 2H), 4.88 (br, 2H), 3.88–3.48 (m, 12H), 2.99 (br, 4H), 1.46 (s, 18H), 1.41 (s, 18H), 1.36 (s, 12H); 13C NMR (100 MHz, CDCl3): δ 170.9, 155.1, 154.8, 80.3, 56.1, 54.1, 50.6, 49.3, 45.3, 44.0, 39.8, 28.4, 25.0; LC-MS (ESI+): m/z 833.0 [M + H]+.

N,N′-Bis(tert-butoxycarbonyl)-L-cystine bis(4’-(dimethylamino)piperidineamide) (7g)

The compound was eluted with 7% methanol in dichloromethane with 0.3 M ammonia and concentrated under reduced pressure to give a colorless oil (68 mg, yield: 94%). 1H NMR (400 MHz, CDCl3): δ 5.46 (br, 2H), 4.93 (br, 2H), 4.54 (br, 2H), 4.07 (br, 2H), 3.09-2.92 (m, 6H), 2.67-2.58 (m, 2H), 2.34 (br, 2H), 2.24 (s, 12H), 1.85 (br, 4H), 1.40 (s, 22H); 13C NMR (100 MHz, CDCl3): δ 168.6, 155.0, 80.0, 74.8, 61.8, 53.4, 49.1, 44.8, 41.6, 29.0, 28.3; LC-MS (ESI+): m/z 661.4 [M + H]+.

N,N′-Bis(tert-butoxycarbonyl)-L-cystine bis(4’-(1”-pyrrolidinyl)piperidine) (7h)

The compound was eluted with 6% methanol in dichloromethane with 0.3 M ammonia and concentrated under reduced pressure to give a colorless oil (70 mg, yield: 90%). 1H NMR (400 MHz, CDCl3): δ 5.50 (br, 2H), 4.94 (br, 2H), 4.40 (br, 2H), 4.01 (br, 2H), 3.15-2.76 (m, 8H), 2.55 (br, 8H), 2.25 (br, 4H), 1.92 (br, 4H), 1.76 (br, 8H), 1.40 (s, 20H); 13C NMR (100 MHz, CDCl3): δ 168.6, 155.1, 80.0, 74.8, 61.4, 51.4, 49.1, 44.2, 41.1, 31.9, 31.0, 28.3, 23.2.

N,N′-Bis(tert-butoxycarbonyl)-1-l-cystinyl bis(1,8-diazaspiro[4.5]decane)-8-carboxylate tert-butylester (7j)

The compound was eluted with 5% methanol in dichloromethane with 0.3 M ammonia and concentrated under reduced pressure to give a colorless oil (80 mg, yield: 33%). 1H NMR (400 MHz, CDCl3): δ 5.34 (br, 2H), 4.65 (br, 2H), 4.10 (br, 2H), 4.01 (br, 2H), 3.70 (br, 2H), 3.61 (br, 2H), 2.98–2.70 (m, 12H), 2.05-1.86 (m, 8H), 1.41 (d, 36H, J = 8 Hz), 1.25-1.21 (m, 4H); 13C NMR (100 MHz, CDCl3): δ 168.9, 155.2, 154.5, 80.0, 79.4, 65.2, 52.4, 48.7, 41.8, 41.0, 35.0, 32.4, 28.5, 28.3, 22.7.

N,N′-Bis(tert-butoxycarbonyl)-L-cystine bis(N′,N’-dimethylethyleneamide) (8a)

The compound was eluted with 14% methanol in dichloromethane with 0.1% triethylamine and concentrated under reduced pressure to give a yellowish-white solid (250 mg, yield: 95%). 1H NMR (400 MHz, DMSO-d6): δ 8.01 (br, 2H), 7.05 (d, 2H, J = 8 Hz), 4.14 (br, 2H), 3.24 (br, 4H), 3.08 (dd, 2H, J = 12, 4 Hz), 2.83 (dd, 2H, J = 12, 8 Hz), 2.64 (t, 4H, J = 4 Hz), 2.40 (s, 12H), 1.36 (s, 18H); 13C NMR (100 MHz, DMSO-d6): δ 170.4, 155.2, 78.4, 56.8, 53.6, 43.8, 40.5, 35.6, 28.1; LC-MS (ESI+): m/z 581.2 [M + H]+.

N,N′-Bis(tert-butoxycarbonyl)-L-cystine bis(N′-pyrrolidinoethyleneamide) (8b)

The compound was eluted with 6% methanol in dichloromethane with 0.1% triethylamine and concentrated under reduced pressure to give a white solid (82 mg, yield: 81%). 1H NMR (400 MHz, CDCl3): δ 7.64 (br, 2H), 5.65 (br, 2H), 4.47 (br, 2H), 3.30 (br, 4H), 3.10-2.86 (m, 4H), 2.60 (br, 12H), 1.68 (br, 8H), 1.26 (s, 18H); 13C NMR (100 MHz, CDCl3): δ 170.5, 155.5, 79.9, 54.7, 54.2, 54.0, 44.2, 37.9, 28.3, 23.3; LC-MS (ESI+): m/z 633.3 [M + H]+.

N,N′-Bis(tert-butoxycarbonyl)-L-cystine bis(N′-pyrrolidin-2-oneethyleneamide) (8c)

The compound was eluted with 6% methanol in dichloromethane with 0.1% triethylamine and concentrated under reduced pressure to give a white solid (82 mg, yield: 81%). 1H NMR (400 MHz, CDCl3): δ 7.94 (br, 2H), 5.58 (br, 2H), 4.60 (br, 2H), 3.52–3.34 (m, 12H), 2.98 (br, 4H), 2.34 (t, 4H, J = 8 Hz), 2.01–1.95 (m, 4H), 1.43 (s, 18H); 13C NMR (100 MHz, CDCl3): δ 175.9, 170.7, 155.6, 80.2, 54.5, 47.3, 44.8, 42.1, 37.1, 31.0, 28.4, 18.0 ppm. LC-MS (ESI+): m/z 661.4 [M + H]+.

N,N′-Bis(tert-butoxycarbonyl)-L-cystine bis(N′-piperidinoethyleneamide) (8d)

The compound was eluted with 4% methanol in dichloromethane with 0.1% triethylamine and concentrated under reduced pressure to give a yellowish-white solid (95 mg, yield: 90%). 1H NMR (400 MHz, CDCl3): δ 7.65 (br, 2H), 5.65 (d, 2H, J = 8 Hz), 4.53 (br, 2H), 3.38 (br, 4H), 3.10-2.96 (m, 4H), 2.54 (br, 12H), 1.59 (br, 8H), 1.37 (s, 22H) ppm. 13C NMR (100 MHz, CDCl3): δ 170.4, 155.5, 80.0, 57.2, 55.2, 54.2, 43.9, 36.1, 28.3, 25.1, 23.6; LC-MS (ESI+): m/z 661.3 [M + H]+.

N,N′-Bis(tert-butoxycarbonyl)-L-cystine bis(N′-morpholinoethyleneamide) (8e)

The compound was eluted with 7% methanol in dichloromethane with 0.1% triethylamine and concentrated under reduced pressure to give a white solid (85 mg, yield: 80%). 1H NMR (400 MHz, CDCl3): δ 7.51 (br, 2H), 5.63 (d, 2H, J = 12 Hz), 4.66 (br, 2H), 3.67 (t, 8H, J = 4 Hz), 3.45–3.29 (m, 4H), 3.01-2.97 (m, 4H), 2.50-2.44 (m, 12H), 1.44 (s, 18H); 13C NMR (100 MHz, CDCl3): δ 170.2, 155.7, 80.2, 66.9, 57.5, 54.5, 53.5, 45.3, 36.4, 28.4; LC-MS (ESI+): m/z 665.3 [M + H]+.

N,N′-Bis(tert-butoxycarbonyl)-L-cystine-bis(N′,N’-dimethyl-1’,3’-diaminopropaneamide) (8f)

The compound was eluted with 15% methanol in dichloromethane with 0.1% triethylamine and concentrated under reduced pressure to give a light yellow solid (55 mg, yield: 58%). 1H NMR (400 MHz, CDCl3): δ 7.90 (br, 2H), 5.61 (d, 2H, J = 8 Hz), 4.69 (br, 2H), 3.33-3.25 (m, 4H), 3.02-2.94 (m, 4H), 2.31 (t, 4H, J = 8 Hz), 2.20 (s, 12H), 1.72-1.64 (m, 4H), 1.45 (s, 18H); 13C NMR (100 MHz, DMSO-d6): δ 170.1, 155.8, 80.1, 57.7, 54.5, 45.9, 45.4, 38.5, 28.4, 27.2; LC-MS (ESI+): m/z 609.2 [M + H]+.

N,N′-Bis(tert-butoxycarbonyl)-L-cystine bis(1’-pyrrolidine-3’-aminopropaneamide) (8g)

The compound was eluted with 16% methanol in dichloromethane with 0.1% triethylamine and concentrated under reduced pressure to give a yellow solid (85 mg, yield: 81%). 1H NMR (400 MHz, CDCl3): δ 8.35 (br, 2H), 5.78 (br, 2H), 4.40 (br, 2H), 3.60 (br, 4H), 3.30 (br, 4H), 3.18 (br, 4H), 2.93 (br, 4H), 2.02 (br, 12H), 1.31 (s, 18H); 13C NMR (100 MHz, DMSO-d6): δ 171.2, 155.3, 79.7, 54.1, 53.6, 52.9, 42.7, 36.6, 28.3, 25.6, 23.2; LC-MS (ESI+): m/z 661.3 [M + H]+.

N,N′-Bis(tert-butoxycarbonyl)-L-cystine bis(1’-morpholine-3’-aminopropaneamide) (8h)

The compound was eluted with 16% methanol in dichloromethane with 0.1% triethylamine and concentrated under reduced pressure to give a white solid (85 mg, yield: 80%). 1H NMR (400 MHz, CDCl3): δ 7.76 (br, 2H), 5.60 (br, 2H), 4.49 (br, 2H), 3.61 (br, 8H), 3.20-3.15 (m, 4H), 2.88 (br, 4H), 2.40 (br, 12H), 1.68-1.60 (m, 4H), 1.30 (s, 18H); 13C NMR (100 MHz, CDCl3): δ 170.3, 155.6, 80.0, 66.3, 56.5, 54.5, 53.5, 45.0, 38.1, 28.3, 25. 6; LC-MS (ESI+): m/z 693.3 [M + H]+.

General procedure for the synthesis of L-cystine diamides 3a-3i, 4a-4h, and 5a-5i

Respective intermediates 7a-7i, 8a-8h, and 17a-17i were dissolved in methanol and cooled to 0 °C, to which 4 N HCl in 1,4-dioxane (at least 20 equiv.) was added in portions every half an hour over 1–3 h. The reaction mixture was gradually warmed up to room temperature after the last addition of 4 N HCl and stirred at room temperature for 0.5–5 h. The reaction mixture was concentrated under reduced pressure and then purified by recrystallization.

L-Cystine bis(piperazide) tetrahydrochloride (3a)

The compound was recrystallized three times with diethyl ether/methanol to give a pale yellow solid (6 mg, yield: 85%). 1H NMR (400 MHz, D2O): δ 3.87-4.14 (m, 7H), 3.80–3.69 (m, 2H), 3.52 − 3.28 (m, 13H); 13C NMR (100 MHz, D2O): δ 166.6, 49.7, 42.9, 42.7, 42.5, 39.4, 37.9; LC/MS (ESI+): m/z 377.2 [M + H]+; HRMS (ESI+): m/z calculated for C14H29N6O2S2+ [M + H]+ 377.1788, found 377.1799.

L-Cystine bis(N′-tert-butylpiperazide) tetrahydrochloride (3b)

The compound was recrystallized three times with diethyl ether/methanol to give a pale yellow solid (21 mg, yield: 84%). 1H NMR (400 MHz, D2O): δ 4.30-4.24 (m, 2H), 3.98-3.57 (m, 9H), 3.57–3.01 (m, 11H), 1.44 (s, 9H), 1.43 (s, 9H); 13C NMR (100 MHz, D2O): δ 166.3, 64.8, 49.7, 45.7, 45.4, 42.9, 39.9, 23.6; LC/MS (ESI+): m/z 489.2 [M + H]+; HRMS (ESI+): m/z calculated for C22H45N6O2S2+ [M + H]+ 489.3040, found 489.3075.

L-Cystine bis(N′-cyclopropylpiperazide) tetrahydrochloride (3c)

The compound was recrystallized three times with diethyl ether/methanol to give a pale yellow solid (19 mg, yield: 80%). 1H NMR (400 MHz, D2O): δ 4.69 (b, 2H), 3.30–3.86 (m, 20H), 2.91 (b, 2H), 1.03 (s, 8H); 13C NMR (100 MHz, D2O): δ 166.5, 52.0, 51.9, 49.8, 42.6, 39.5, 37.9, 3.6; LC/MS (ESI+): m/z 457.3 [M + H]+; HRMS (ESI+): m/z calculated for C20H37N6O2S2+ [M + H]+ 457.2414, found 457.2444.

L-Cystine bis(N′-cyclopentylpiperazide) tetrahydrochloride (3d)

The compound was recrystallized three times with diethyl ether/methanol to give a pale yellow solid (25 mg, yield: 76%). 1H NMR (400 MHz, D2O): δ 5.01 – 4.92 (m, 2H), 4.69 – 4.53 (m, 2H), 4.24 (d, 2H, J = 14.7 Hz), 3.84 – 3.57 (m, 8H), 3.46 – 3.11 (m, 10H), 2.28 – 2.09 (m, 4H), 1.85 – 1.61 (m, 12H); 13C NMR (100 MHz, D2O): δ 166.2, 68.2, 50.2, 50.1, 49.8, 49.7, 42.8, 39.7, 27.9, 23.3; LC/MS (ESI+): m/z 513.3 [M + H]+; HRMS (ESI+): m/z calculated for C24H45N6O2S2+ [M + H]+ 513.3040, found 513.3069.

L-Cystine bis(N′-phenylpiperazide) tetrahydrochloride (3e)

The compound was recrystallized three times with diethyl ether/methanol to give a pale yellow solid (23 mg, yield: 88%). 1H NMR (400 MHz, D2O): δ 7.55 – 7.45 (m, 4H), 7.45–7.28 (m, 6H), 4.94 (t, 2H, J = 6.0 Hz), 4.12–3.94 (m, 6H), 3.94 – 3.83 (m, 2H), 3.69–3.49 (m, 8H), 3.46 – 3.27 (m, 4H); 13C NMR (100 MHz, D2O): δ 166.4, 144.7, 130.3, 127.1, 119.5, 52.5, 50.0, 49.8, 44.2, 41.1, 37.9; LC/MS (ESI+): m/z 529.3 [M + H]+; HRMS (ESI+): m/z calculated for C26H37N6O2S2+ [M + H]+ 529.2414, found 529.2446.

L-Cystine bis(2′,2′-dimethylpiperazide) tetrahydrochloride (3f)

The compound was recrystallized three times with diethyl ether/methanol to give a white solid (4 mg, yield: 80%). 1H NMR (400 MHz, D2O): δ 4.35–4.19 (m, 1H), 4.06 – 3.89 (m, 3H), 3.88–3.74 (m, 2H), 3.72 – 3.61 (m, 1H), 3.57–3.27 (m, 11H), 1.55 – 1.37 (m, 12H); 13C NMR (100 MHz, D2O): δ 167.1, 55.1, 54.5, 52.5, 49.6, 42.3, 39.1, 38.9, 38.3, 37.8, 22.8, 22.5, 20.7, 20.1; LC/MS (ESI+): m/z 433.3 [M + H]+; HRMS (ESI+): m/z calculated for C18H37N6O2S2+ [M + H]+ 433.2414, found 433.2444.

L-Cystine bis(4′-(dimethylamino)piperidineamide) tetrahydrochloride (3g)

The compound was recrystallized with diethyl ether/methanol to give a white solid (22 mg, yield: 100%). 1H NMR (400 MHz, D2O): δ 4.99 – 4.89 (m, 2H), 4.70 – 4.55 (m, 2H), 4.17 (t, 2H, J = 12.2 Hz), 3.70 – 3.54 (m, 2H), 3.48 – 3.18 (m, 6H), 3.05 – 2.80 (m, 14H), 2.40 – 2.17 (m, 4H), 1.96 – 1.62 (m, 4H); 13C NMR (100 MHz, D2O): δ 166.2, 62.6, 49.9, 44.1, 41.3, 39.8, 26.3, 25.5; LC/MS (ESI+): m/z 461.3 [M + H]+; HRMS (ESI+): m/z calculated for C20H41N6O2S2+ [M + H]+ 461.2727, found 461.2757.

L-Cystine bis(4′-(1′′-pyrrolidinyl)piperidineamide) tetrahydrochloride (3h)

The compound was recrystallized with diethyl ether/methanol to give a white solid (20 mg, yield: 100%). 1H NMR (400 MHz, D2O): δ 5.00–4.89 (m, 2H), 4.65 – 4.53 (m, 2H), 4.21–4.05 (m, 2H), 3.79 – 3.66 (m, 4H), 3.60 – 3.50 (m, 2H), 3.47 – 3.15 (m, 10H), 2.98–2.83 (m, 2H), 2.45–2.30 (m, 4H), 2.26 – 2.14 (m, 4H), 2.11 – 1.95 (m, 4H), 1.95 – 1.61 (m, 4H); 13C NMR (100 MHz, D2O): δ 165.9, 60.9, 51.8, 49.9, 43.9, 41.2, 28.5, 27.7, 22.5; LC/MS (ESI+): m/z 513.3 [M + H]+; HRMS (ESI+): m/z calculated for C24H45N6O2S2+ [M + H]+ 513.3040, found 513.3070.

L-Cystine bis(1,8-diazaspiro[4.5]decan-1-yl) tetrahydrochloride (3j)

The compound was recrystallized three times with diethyl ether/methanol to give a white solid (6 mg, yield: 100%). 1H NMR (400 MHz, D2O): δ 4.59 (dd, 2H, J = 8.4, 4.3 Hz), 3.79 (dt, 2H, J = 9.8, 6.2 Hz), 3.69 (dt, 2H, J = 9.8, 7.0 Hz), 3.55 (q, 2H, J = 7.1 Hz), 3.51–3.36 (m, 6H), 3.21–3.04 (m, 6H), 3.00 – 2.87 (m, 4H), 2.19 (dt, 2H, J = 12.5, 6.2 Hz), 2.07 (q, 2H, J = 6.9 Hz), 1.99 (p, 4H, J = 5.7 Hz), 1.75 (d, 4H, J = 13.9 Hz); 13C NMR (100 MHz, D2O): δ 165.9, 64.1, 51.9, 49.0, 42.2, 36.8, 34.6, 28.7, 28.1, 22.3; LC/MS (ESI+): m/z 485.3 [M + H]+; HRMS (ESI+): m/z calculated for C22H41N6O2S2+ [M + H]+ 485.2727, found 485.2761.

L-Cystine bis(N′,N’-dimethylethyleneamide) tetrahydrochloride (4a)

The compound was recrystallized three times with diethyl ether/methanol to give a pale yellow solid (160 mg, yield: 73%). 1H NMR (400 MHz, D2O): δ 4.44 (dd, 2H, J = 8.0, 5.2 Hz), 3.80 (m, 2H), 3.69–3.58 (m, 2H), 3.48 (m, 2H), 3.45–3.38 (m, 4H), 3.26 (m, 2H), 2.99 (s, 12H); 13C NMR (100 MHz, D2O): δ 168.8, 56.1, 52.0, 43.3, 43.1, 37.2, 35.0; LC/MS (ESI+): m/z 381.2 [M + H]+; HRMS (ESI+): m/z calculated for C14H33N6O2S2+ [M + H]+ 381.2101, found 381.2125.

L-Cystine bis(N′-pyrrolidinoethyleneamide) tetrahydrochloride (4b)

The compound was recrystallized six times with diethyl ether/methanol to give a yellow solid (25 mg, yield: 86%). 1H NMR (400 MHz, D2O): δ 4.38 (dd, 2H, J = 8.4, 4.7 Hz), 3.72 – 3.57 (m, 6H), 3.55–3.46 (m, 2H), 3.42–3.27 (m, 6H), 3.20–2.96 (m, 6H), 2.14–2.01 (m, 4H), 2.00 – 1.90 (m, 4H); 13C NMR (100 MHz, D2O): δ 168.7, 54.7, 54.5, 53.4, 52.0, 37.1, 36.1, 22.7; LC/MS (ESI+): m/z 433.3 [M + H]+; HRMS (ESI+): m/z calculated for C18H37N6O2S2+ [M + H]+ 433.2414, found 433.2439.

L-Cystine bis(N′-piperidinoethyleneamide) tetrahydrochloride (4c)

The compound was recrystallized six times with diethyl ether/methanol to give a yellow solid (31 mg, yield: 86%). 1H NMR (400 MHz, D2O): δ 4.45 – 4.36 (m, 2H), 3.80–3.69 (m, 2H), 3.66–3.53 (m, 6H), 3.50 – 3.40 (m, 2H), 3.37–3.28 (m, 4H), 3.27–3.18 (m, 2H), 3.06 – 2.94 (m, 4H), 2.01–1.90 (m, 4H), 1.86 – 1.69 (m, 6H), 1.57 – 1.41 (m, 2H); 13C NMR (100 MHz, D2O): δ 168.6, 55.1, 53.7, 53.6, 52.0, 37.1, 34.5, 22.8, 21.0; LC/MS (ESI + ): m/z 461.4 [M + H]+; HRMS (ESI+): m/z calculated for C20H41N6O2S2+ [M + H]+ 461.2000, found 461.2028.

L-Cystine bis(N′-morpholinoethyleneamide) tetrahydrochloride (4d)

The compound was recrystallized four times with diethyl ether/methanol to give a yellow solid (49 mg, yield: 100%). 1H NMR (400 MHz, D2O): δ 4.47 – 4.40 (m, 2H), 4.22 – 4.04 (m, 4H), 3.93 – 3.76 (m, 6H), 3.71–3.56 (m, 6H), 3.52–3.40 (m, 6H), 3.36–3.18 (m, 6H); 13C NMR (100 MHz, D2O): δ 168.7, 63.7, 55.6, 52.1, 52.0, 37.0, 34.1; LC/MS (ESI+): m/z 465.4 [M + H]+; HRMS (ESI+): m/z calculated for C18H37N6O4S2+ [M + H]+ 465.2313, found 465.2340.

L-Cystine bis(N′-pyrrolidin-2-one ethyleneamide) dihydrochloride (4e)

The compound was recrystallized three times with diethyl ether/methanol to give a pale yellow solid (67 mg, yield: 99%). 1H NMR (400 MHz, D2O): δ 4.32 (b, 2H), 3.28-3.61 (m, 14H), 3.12–3.19 (m, 2H), 2.44 (b, 4H), 2.04 (b, 4H); 13C NMR (100 MHz, D2O): δ 178.9, 167.9, 51.9, 47.9, 41.9, 37.2, 36.9, 31.1, 17.4; LC/MS (ESI+): m/z 461.4 [M + H]+; HRMS (ESI+): m/z calculated for C18H33N6O4S2+ [M + H]+ 461.2000, found 461.2028.

L-Cystine bis(N′,N′-dimethyl-1′,3′-diaminopropaneamide) tetrahydrochloride (4f)

The compound was recrystallized three times with diethyl ether/methanol to give a pale yellow solid (11 mg, yield: 68%). 1H NMR (400 MHz, D2O): δ 4.36 (dd, 2H, J = 7.5, 5.7 Hz), 3.46 – 3.30 (m, 6H), 3.30–3.15 (m, 6H), 2.91 (s, 12H), 2.21 – 1.85 (m, 4H) ppm. 13 C NMR (100 MHz, D2O): δ 168.11, 55.34, 52.07, 42.90, 42.85, 37.40, 36.68, 23.91; LC/MS (ESI+): m/z 409.3 [M + H]+; HRMS (ESI+): m/z calculated for C16H37N6O2S2+ [M + H]+ 409.2414, found 409.2438.

L-Cystine bis(1′-pyrrolidine-3′-aminopropaneamide) tetrahydrochloride (4g)

The compound was recrystallized six times with diethyl ether/methanol to give a yellow solid (16 mg, yield: 45%). 1H NMR (400 MHz, D2O): δ 4.40 (t, 2H, J = 6.6 Hz), 3.80–3.65 (m, 4H), 3.50 – 3.35 (m, 6H), 3.30 (ddd, 6H, J = 8.1, 6.4, 3.3 Hz), 3.14 (dt, 4H, J = 10.3, 7.5 Hz), 2.26 – 2.14 (m, 4H), 2.06 (ttd, 8H, J = 9.6, 7.6, 2.4 Hz); 13C NMR (100 MHz, D2O): δ 168.1, 54.3, 52.6, 52.2, 37.5, 36.9, 25.3, 22.7; LC/MS (ESI+): m/z 461.5 [M + H]+; HRMS (ESI+): m/z calculated for C20H41N6O2S2+ [M + H]+ 461.2727, found 461.2757.

L-Cystine bis(1′-morpholine-3′-aminopropaneamide) tetrahydrochloride (4h)

The compound was recrystallized six times with diethyl ether/methanol to give a yellow solid (40 mg, yield: 85%). 1H NMR (400 MHz, D2O): δ 4.41 (t, 2H, J = 6.7 Hz), 4.19 (d, 4H, J = 13.5 Hz), 3.89 (t, 4H, J = 12.8 Hz), 3.70 – 3.55 (m, 4H), 3.50 – 3.37 (m, 6H), 3.37–3.19 (m, 10H), 2.17–2.01 (m, 4H); 13C NMR (100 MHz, D2O): δ 168.2, 63.9, 54.9, 51.9, 37.5, 36.9, 23.1; LC/MS (ESI+): m/z 493.5 [M + H]+; HRMS (ESI+): m/z calculated for C20H41N6O4S2+ [M + H]+ 493.2625, found 493.2662.

Methyl N-(tert-butoxycarbonyl)-O-tosyl-L-serinate (14a)

Boc-Ser-OMe (13a, 2.5 g, 11.4 mmol) was dissolved in 50 mL of dry dichloromethane and cooled to 0 °C. Pyridine (2 mL, 23 mmol) was added to the chilled solution followed by para-toluenesulfonyl chloride (6.6 g, 34.2 mmol) and the reaction mixture was left to stir at 0 °C for 15 min, then warmed to room temperature, and left to stir for 45 min. The solvent was evaporated under reduced pressure and the white residue was purified via ISCO (ethyl acetate/hexane) and the product was eluted at 15%. The solvent was evaporated under reduced pressure to give a colorless oil (4180 mg, 98% yield). 1H NMR (400 MHz, CDCl3) δ 1.39 (s, 9H), 2.43 (s, 3H), 3.67 (s, 3H), 4.25-4.39 (m, 2H), 4.49 (b, 1H), 5.29 (b, 1H), 7.33 (d, 2H, J = 8 Hz), 7.74 (d, 2H, J = 8 Hz); 13C NMR (100 MHz, CDCl3) δ 169.0, 155.0, 145.2, 132.4, 129.9, 128.0, 80.5, 69.5, 53.0, 52.9, 28.2, 21.0; LC-MS (ESI+) m/z 374.49 [M + H]+.

Methyl N-(tert-butoxycarbonyl)-S-((R)-2-((tert-butoxycarbonyl)amino)-3-methoxy-3-oxo-propyl)-L-homocysteinate (15a)

Boc-L-Hcy-OMe was prepared from the L-Hcy in three steps of esterification, Boc protection, and reduction. Boc-L-Hcy-OMe (11, 255 mg, 0.7 mmol) and Boc-Ser(OTs)-OMe (14a, 209.4 mg, 0.84 mmol) were dissolved in 9 mL of degassed anhydrous N,N-dimethylformamide and cooled to 0 °C. NaH (28.6 mg, 1.19 mmol) and the reaction mixture was left to stir at 0 °C for 30 m then was brought to room temperature and left to stir under argon for 4 h. The mixture was diluted with 10 mL of water. The organic layer was washed with (3 ×40 mL) of dichloromethane. The combined organic layers were washed with (20 mL) of brine and dried over sodium sulfate. The solvent was evaporated under reduced pressure and the brown residue was purified via ISCO (ethyl acetate/hexane) and the product was eluted at 25%. The solvent was evaporated under reduced pressure to give a yellow solid (150 mg, 48% yield). 1H NMR (400 MHz, CDCl3) δ 1.44 (s, 18H), 1.88-2.12 (m, 2H), 2.54-2.58 (m, 2H), 2.94 (b, 2H), 3.75 (s, 6H), 4.38 (b, 1H), 4.51 (b, 1H), 5.13 (b, 1H), 5.36 (b, 1H); 13C NMR (100 MHz, CDCl3) δ 172.6, 171.5, 155.1, 80.2, 53.2, 52.6, 52.4, 34.6, 32.6, 28.3; LC-MS (ESI+) m/z 451.33 [M + H]+.

N-(tert-Butoxycarbonyl)-S-((R)-2-((tert-butoxycarbonyl)amino)-2-carboxyethyl)-L-homocysteine (16a)

Compound 15a (60 mg, 0.13 mmol) was dissolved in 900 µL of tetrahydrofuran and cooled to 0 °C. Lithium hydroxide (7 mg, 0.28 mmol) was dissolved in 900 µL of water then was added to the chilled solution and the reaction mixture was left to stir at room temperature for 1 h. The reaction mixture was diluted with 5 mL of water and was acidified by 0.5 N potassium hydrogen sulfate to pH 2 then was extracted with ethyl acetate (3 ×10 mL). The combined organic layers were washed with brine (5 mL) and dried over sodium sulfate. The solvent was evaporated under reduced pressure to give a yellow solid (56 mg, 100% yield). 1H NMR (400 MHz, CDCl3) δ 1.44 (s, 18H), 1.86-2.10 (m, 2H), 2.63 (b, 2H), 3.03 (b, 2H), 4.42 (b, 1H), 4.57 (b, 1H), 5.45 (b, 1H), 5.56 (b, 1H), 9.20 (b, 2H); 13C NMR (100 MHz, DMSO-d6) δ 173.7, 172.5, 155.3, 78.16, 53.8, 52.5, 32.7, 30.9, 28.2; LC-MS (ESI+) m/z 423.17 [M + H]+.

tert-Butyl ((R)-3-(((S)-3-((tert-butoxycarbonyl)amino)-4-(4-methylpiperazin-1-yl)-4-oxobutyl)-thio)-1-(4-methylpiperazin-1-yl)-1-oxopropan-2-yl)carbamate (17a)

To a solution of compound 16a (55 mg, 0.13 mmol) in 300 µL of anhydrous N,N-dimethylformamide, HATU (104 mg, 0.273 mmol) and DIPEA (140 µL, 0.8 mmol) were added. Then 1-methyl piperazine (36 µL, 0.32 mmol) was added to the mixture and the reaction mixture was left to stir at room temperature for 3 h. The mixture was diluted with ethyl acetate (20 mL) and washed with water (3 ×10 mL), brine (5 mL), and dried over sodium sulfate. The solvent was evaporated under reduced pressure and the yellow residue was purified via ISCO (20% MeOH:DCM + 0.1% TEA/DCM) and the product was eluted at 55%. The solvent was evaporated under reduced pressure to give a yellow oil (28 mg, 37% yield). 1H NMR (400 MHz, CDCl3) δ 1.43 (s, 18H), 1.74-1.81 (m, 2H), 2.38 (s, 6H), 2.57 (b, 10H), 2.73-2.88 (m, 2H), 3.48 (b, 8H), 4.72 (b, 2H), 5.40 (b, 2H); 13C NMR (100 MHz, CDCl3) δ 170.1, 155.6, 79.9, 54.9, 54.4, 49.5, 48.9, 45.6, 41.7, 35.3, 33.6, 28.3; LC-MS (ESI+) m/z 587.18 [M + H]+.

(S)-2-Amino-4-(((R)-2-amino-3-(4-methylpiperazin-1-yl)-3-oxopropyl)thio)-1-(4-methyl-piperazin-1-yl)butan-1-one tetrahydrochloride (5a)

Compound 17a (23 mg, 0.04 mmol) was dissolved in 200 µL of methanol and cooled to 0 °C. To the chilled solution was added 4 N HCl in dioxane (90 µL, 0.36 mmol) in four portions every half an hour and the solution was warmed to room temperature after each addition. There was still some mono-deprotected product so additional 4 N HCl in dioxane (45 µL, 0.18 mmol) was added in two portions to the solution at 0 °C and the solution was warmed to room temperature and left to stir for 5 h then in fridge for overnight. The solvent was evaporated under reduced pressure and the residue was dried under vacuum. The residue then was dissolved in a minimal amount of methanol and recrystallized with an excess of diethyl ether then the vial was centrifuged and the solvent was decanted; this purification step was repeated three times. The residue was dried to give a white solid (18 mg, 85% yield). 1H NMR (400 MHz, D2O) δ 2.19 (b, 2H), 2.78 (b, 2H), 2.97 (s, 6H), 3.10-3.25 (m, 10H), 3.66 (b, 8H), 4.19 (b, 2H); 13C NMR (100 MHz, D2O) δ 167.8, 166.9, 52.7, 49.7, 43.1, 42.8, 39.7, 31.9, 29.8, 27.1; LC-MS (ESI+) m/z 387.19 [M + H]+; HRMS (ESI+) m/z calculated for C17H35N6O2S+ [M + H]+ 387.2537, found 387.2562.

Di-tert-butyl ((7 R,12 S)-2,17-dimethyl-6,13-dioxo-9-thia-2,5,14,17-tetraazaocta-decane-7,12-di-yl)dicarbamate (17f)

To a solution of 16a (250 mg, 0.59 mmol) in 2.5 mL of anhydrous N,N-dimethylformamide, HATU (718 mg, 1.89 mmol) and DIPEA (924 µL, 5.31 mmol) were added. Then N,N-dimethylethylenediamine (216 µL, 2.1 mmol) was added to the mixture and the reaction mixture was left to stir at room temperature for 3 h. The mixture was diluted with ethyl acetate (20 mL), washed with water (3 ×10 mL) and brine (5 mL), and dried over sodium sulfate. The solvent was evaporated under reduced pressure and the light brown oil was purified via ISCO (20% MeOH:DCM + 0.1% TEA/DCM) and the product was eluted at 80%. The solvent was evaporated under reduced pressure to give white crystals (189 mg, 57% yield). 1H NMR (400 MHz, CDCl3) δ 1.42 (s, 18H), 1.93-2.03 (m, 2H), 2.34 (s, 12H), 2.56 (b, 4H), 2.67-2.95 (m, 2H), 3.02-3.08 (m, 2H), 3.40 (b, 4H), 4.26 (b, 2H), 5.61 (b, 2H); 13C NMR (100 MHz, CDCl3) δ 171.8, 170.8, 155.6, 79.9, 57.7, 53.4, 50.3, 44.8, 36.7, 34.9, 34.2, 33.1, 28.5; LC-MS (ESI+) m/z 563.19 [M + H]+.

(S)-2-Amino-4-(((R)-2-amino-3-((2-(dimethylamino)ethyl)amino)-3-oxopropyl)thio)-N-(2-(di-methylamino)ethyl)butanamide tetrahydrochloride (5f)

Compound 17 f (165 mg, 0.3 mmol) was dissolved in 1.5 mL of MeOH and cooled to 0 °C. To the chilled solution was added 4 N HCl in dioxane (900 µL, 3.6 mmol) in four portions every half an hour and the solution was warmed to room temperature after each addition. The solvent was evaporated under reduced pressure and the residue was dried under vacuum. The residue then was dissolved in a minimal amount of methanol and recrystallized with an excess of diethyl ether then the vial was centrifuged and the solvent was decanted; this purification step was repeated five times. The residue was dried to give a white solid (133 mg, 87% yield). 1H NMR (400 MHz, D2O) δ 2.18-2.26 (m, 2H), 2.75 (t, 2H, J = 8 Hz), 2.97 (s, 12H), 3.05-3.22 (m, 2H), 3.38 (t, 4H, J = 8 Hz), 3.55–3.85 (m, 4H), 4.15 (t, 1H, J = 8 Hz), 4.24 (t, 1H, J = 8 Hz); 13C NMR (100 MHz, D2O) δ 170.8, 56.3, 51.9, 43.7, 34.5, 32.8, 29.9, 26.6; LC-MS (ESI+) m/z 363.32 [M + H]+; HRMS (ESI+) m/z calculated for C15H35N6O2S+ [M + H]+ 363.2537, found 363.2567.

Methyl (tert-butoxycarbonyl)-L-homocysteinate (11)

Compound 10 (838 mg, 1.69 mmol), triphenyl phosphine (499 mg, 1.9 mmol), sodium acetate (58 mg, 0.7 mmol) were suspended in a mixture of 8 mL of methanol, 3.5 mL of water, and 58 µL of glacial acetic acid and heated to 60 °C for 75 min. The mixture was diluted with ethyl acetate (80 mL), washed with water (2 ×40 mL) and brine (10 mL), and dried over sodium sulfate. The solvent was evaporated under reduced pressure and the colorless oil was purified via ISCO (ethyl acetate/ hexane) and the product was eluted at 15%. The solvent was evaporated under reduced pressure to give a colorless oil (778 mg, 92% yield). 1H NMR (400 MHz, CDCl3) δ 1.41 (s, 9H), 1.55 (t, 1H, J = 8 Hz), 1.84-2.08 (m, 2H), 2.55 (t, 2H, J = 8 Hz), 3.72 (s, 3H), 4.42 (b, 1H), 5.12 (b, 1H); 13C NMR (100 MHz, CDCl3) δ 172.8, 155.4, 80.1, 52.4, 52.2, 37.2, 28.3, 20.7; LC-MS (ESI+) m/z 250.21 [M + H]+.

Methyl (tert-butoxycarbonyl)-L-homoserinate (13b)

A flask was charged with L-homoserine (12b, 600 mg, 5.04 mmol) and 5 mL of water and 5 mL of MeCN were added then the solution was cooled to 0 °C. Sodium hydroxide (202 mg, 5.04 mmol) was added followed by di-tert-butyl carbonate (1430 mg, 6.55 mmol) and the reaction mixture was brought to room temperature and left to stir for 1.5 h. The reaction mixture was concentrated as possible under reduced pressure and then the water was lyophilized to give a white foam (1121 mg, 92% yield). 1H NMR (400 MHz, DMSO-d6) δ 1.37 (s, 9H), 1.64-1.71 (m, 2H), 3.64 (b, 4H), 5.98 (b, 1H); 13C NMR (100 MHz, DMSO-d6) δ 173.6, 154.6, 79.1, 59.7, 54.6, 36.5, 28.2.

The Boc-homoserine intermediate obtained above (1121 mg, 4.65 mmol) was dissolved in 10 mL of anhydrous N,N-dimethylformamide. Iodomethane (350 µL, 5.6 mmol) was added at 0 °C then the reaction mixture was brought to room temperature and left to stir for 5 h. The reaction mixture was diluted with ethyl acetate (150 mL), washed with water (2 ×25 mL), sodium bicarbonate (2 ×25 mL), 0.5 N potassium hydrogen sulfate (25 mL), and brine (15 mL), and dried over sodium sulfate. The solvent was evaporated under reduced pressure to give a colorless oil (487 mg, 45% yield). 1H NMR (400 MHz, CDCl3) δ 1.33 (s, 9H), 1.59-1.65 (m, 1H), 1.99 (b, 1H), 3.42 (b, 1H), 3.59 (b, 2H), 3.64 (s, 3H), 4.34 (b, 1H), 5.51 (b, 1H); 13C NMR (100 MHz, CDCl3) δ 173.3, 156.2, 80.1, 58.3, 52.3, 50.8, 35.4, 28.2; LC-MS (ESI+) m/z 234.72 [M + H]+.

Methyl N-(tert-butoxycarbonyl)-O-tosyl-L-homoserinate (14b)

Compound 13b (487 mg, 2.09 mmol) was dissolved in 12 mL of dry dichloromethane and cooled to 0 °C. Pyridine (337 µL, 4.18 mmol) was added to the chilled solution followed by para-toluenesulfonyl chloride (1196 mg, 6.27 mmol) and the reaction mixture was left to stir at 0 °C for 15 min, then was warmed to room temperature and left to stir for 1 h. The solvent was evaporated under reduced pressure and the yellow residue was purified via ISCO (ethyl acetate/hexane) and the product was eluted at 30%. The solvent was evaporated under reduced pressure to give a colorless oil (561 mg, 69% yield). 1H NMR (400 MHz, CDCl3) δ 1.40 (s, 9H), 2.08-2.24 (m, 2H), 2.43 (s, 3H), 3.70 (s, 3H), 4.07 (b, 2H), 4.31 (b, 1H), 5.12 (b, 1H), 7.34 (d, 2H, J = 8 Hz), 7.77 (d, 2H, J = 8 Hz); 13C NMR (100 MHz, CDCl3) δ 172.0, 155.5, 144.9, 132.7, 129.9, 128.0, 80.2, 66.2, 52.6, 50.5, 31.6, 28.2, 21.6; LC-MS (ESI+) m/z 388.57 [M + H]+.

Dimethyl 4,4’-thio(2 S,2’S)-bis(2-((tert-butoxycarbonyl)amino)butanoate) (15b)

Compound 14b (550 mg, 1.42 mmol) was dissolved in 10 mL of degassed anhydrous N,N-dimethylformamide and cooled to 0 °C. Compound 11 (354 mg, 1.42 mmol) was added to the chilled solution followed by NaH (58 mg, 2.4 mmol) and the reaction mixture was left to stir at 0 °C for 30 m then was brought to room temperature and left to stir under argon for 3 h. The mixture was quenched with water (2 mL) and diluted with ethyl acetate (150 mL). The organic layer was washed with water (2 ×50 mL) and brine (20 mL), and dried over sodium sulfate. The solvent was evaporated under reduced pressure and the brown residue was purified via ISCO (ethyl acetate/hexane) and the product was eluted at 30%. The solvent was evaporated under reduced pressure to give a light yellow oil (443 mg, 67% yield). 1H NMR (400 MHz, CDCl3) δ 1.41 (s, 18H), 1.84-1.91 (m, 2H), 2.03 (b, 2H), 2.52 (t, 4H, J = 8 Hz), 3.71 (s, 6H), 4.36 (b, 2H), 5.16 (b, 2H); 13C NMR (100 MHz, CDCl3) δ 172.7, 155.3, 80.0, 60.3, 52.5, 52.4, 32.6, 28.3; LC-MS (ESI+) m/z 465.29 [M + H]+.

(2 S,2’S)-4,4’-Thiobis(2-((tert-butoxycarbonyl)amino)butanoic acid) (16b)

Compound 15b (443 mg, 0.95 mmol) was dissolved in 6 mL of tetrahydrofuran and cooled to 0 °C. Lithium hydroxide (48 mg, 2 mmol) was dissolved in 6 mL of water then was added to the chilled solution and the reaction mixture was left to stir at room temperature for 2 h. The reaction mixture was diluted with water (20 mL) and washed with dichloromethane (3 ×20 mL). Then the aqueous layer was acidified by 0.5 N potassium hydrogen sulfate to pH 2 and then was extracted with dichloromethane (3 ×40 mL). The combined organic layers were washed with brine (10 mL) and dried over sodium sulfate. The solvent was evaporated under reduced pressure to give a pale yellow solid (361 mg, 87% yield). 1H NMR (400 MHz, CDCl3) δ 1.45 (s, 18H), 2.11 (b, 4H), 2.45–2.69 (m, 4H), 4.31 (b, 2H), 5.43 (b, 1H), 7.59 (b, 1H), 10.81 (b, 2H); 13C NMR (100 MHz, CDCl3) δ 174.2, 157.1, 82.0, 54.1, 32.7, 32.2, 28.3; LC-MS (ESI+) m/z 437.13 [M + H]+.

Di-tert-butyl ((2 S,2’S)-thiobis(1-(4-methylpiperazin-1-yl)-1-oxobutane-4,2-diyl))di-carbamate (17b)

To a solution of compound 16b (150 mg, 0.344 mmol) in 1 mL of anhydrous N,N-dimethylformamide, HATU (274 mg, 0.72 mmol) and DIPEA (360 µL, 2.1 mmol) were added. Then 1-methylpiperazine (96 µL, 0.86 mmol) was added to the mixture and the reaction mixture was left to stir at room temperature for 3 h. The mixture was diluted with ethyl acetate (25 mL), washed with water (3 ×10 mL) and brine (5 mL), and dried over sodium sulfate. The solvent was evaporated under reduced pressure and the light brown oil was purified via ISCO (20% MeOH:DCM + 0.1% TEA/DCM) and the product was eluted at 65%. The solvent was evaporated under reduced pressure to give white crystals (155 mg, 75% yield). 1H NMR (400 MHz, CDCl3) δ 1.41 (s, 18H), 1.75-1.93 (m, 4H), 2.55 (b, 4H), 2.63 (s, 6H), 2.90 (b, 8H), 3.86 (b, 8H), 4.70 (b, 2H), 5.36 (b, 2H); 13C NMR (100 MHz, CDCl3) δ 170.5, 155.6, 80.1, 54.1, 48.9, 45.9, 44.5, 43.8, 40.3, 33.3, 28.3; LC-MS (ESI+) m/z 601.16 [M + H]+.

(2 S,2’S)-4,4’-Thiobis(2-amino-1-(4-methylpiperazin-1-yl)butan-1-one) tetrahydro-chloride (5b)

Compound 17b (23 mg, 0.04 mmol) was dissolved in 250 µL of MeOH and cooled to 0 °C. To the chilled solution was added 4 N HCl in dioxane (42 µL, 0.17 mmol) in two portions every half an hour and the solution was warmed to room temperature after each addition. There was still starting material and mono-deprotected product so additional 4 N HCl in dioxane (42 µL, 0.17 mmol) was added in four portions to the solution at 0 °C and the solution was warmed to room temperature and left to stir for 5 h then in fridge for overnight. The solvent was evaporated under reduced pressure and the residue was dried under vacuum. The residue then was dissolved in a minimal amount of methanol and recrystallized with an excess of diethyl ether then the vial was centrifuged and the solvent was decanted; this purification step was repeated three times. The residue was dried to give a white solid (6 mg, 29% yield). 1H NMR (400 MHz, D2O) δ 2.18 (b, 4H), 2.72 (b, 4H), 2.96 (s, 6H), 3.14-3.26 (m, 8H), 3.64 (b, 8H), 4.19 (b, 2H); 13C NMR (100 MHz, D2O) δ 167.9, 52.6, 49.7, 43.1, 42.6, 39.6, 29.7, 26.1; LC-MS (ESI+) m/z 401.33 [M + H]+; HRMS (ESI+) m/z calculated for C18H37N6O2S+ [M + H]+ 401.2693, found 401.2722.

Di-tert-butyl-((7 S,13 S)-2,18-dimethyl-6,14-dioxo-10-thia-2,5,15,18-tetraazanona-decane-7,13-diyl)dicarbamate (17g)

To a solution of compound 16b (361 mg, 0.83 mmol) in 1.5 mL of anhydrous N,N-dimethylformamide, HATU (663 mg, 1.74 mmol) and DIPEA (868 µL, 4.98 mmol) were added. The reaction mixture was left to stir at room temperature for 15 min. N,N-Dimethylethylenediamine (271 µL, 2.48 mmol) was added to the mixture and the reaction mixture was left to stir at room temperature for 2 h. The mixture was diluted with ethyl acetate (120 mL), washed with water (2 ×20 mL) and brine (20 mL), and dried over sodium sulfate. The solvent was evaporated under reduced pressure and the dark yellow oil was purified via ISCO (20% MeOH:DCM + 0.1% TEA/DCM) and the product was eluted at 65%. The solvent was evaporated under reduced pressure to give a light yellow oil (208 mg, 44% yield). 1H NMR (400 MHz, CDCl3) δ 1.40 (s, 18H), 1.84-1.89 (m, 2H), 1.99-2.03 (m, 2H), 2.20 (s, 12H), 2.40 (t, 4H, J = 8 Hz), 2.53 (b, 4H), 3.29 (b, 4H), 4.22 (b, 2H), 5.46 (d, 2H, J = 4 Hz), 6.87 (b, 2H); 13C NMR (100 MHz, CDCl3) δ 171.5, 155.5, 79.9, 57.8, 53.5, 46.0, 45.1, 36.9, 32.7, 28.3, 27.8; LC-MS (ESI+) m/z 577.24 [M + H]+.

(2 S,2’S)-4,4’-Thiobis(2-amino-N-(2-(dimethylamino)ethyl)butanamide) tetrahydro-chloride (5g)

Compound 17 g (190 mg, 0.33 mmol) was dissolved in 400 µL of MeOH and cooled to 0 °C. To the chilled solution was added 4 N HCl in dioxane (989 µL, 3.96 mmol) in four portions every half an hour and the solution was warmed to room temperature after each addition. The solvent was evaporated under reduced pressure and the residue was dried under vacuum. The residue then was dissolved in a minimal amount of methanol and recrystallized with an excess of diethyl ether then the vial was centrifuged and the solvent was decanted; this purification step was repeated three times. The residue was dried to give a white solid (104 mg, 84% yield). 1H NMR (400 MHz, D2O) δ 2.23-230 (m, 4H), 2.77 (t, 4H, J = 8 Hz), 3.02 (s, 12H), 3.40-3.45 (m, 4H), 3.61-3.66 (m, 2H), 3.81-3.89 (m, 2H), 4.21–4.24 (m, 2H); 13C NMR (100 MHz, D2O) δ 170.2, 56.3, 52.7, 43.4, 35.0, 30.4, 26.3; LC-MS (ESI+) m/z 377.33 [M + H]+; HRMS (ESI+) m/z calculated for C16H37N6O2S+ [M + H]+ 377.2693, found 377.2724.

Methyl (S)-3-azido-2-((tert-butoxycarbonyl)amino)propanoate (18)

Compound 14a (4100 mg, 10.98 mmol) was dissolved in 6.5 mL of anhydrous N,N-dimethylformamide. Sodium azide (1785 mg, 27.5 mmol) was added slowly to the solution and the reaction mixture was shaken at 50 °C for 3 h. The reaction mixture was diluted with (100 mL) of cold water and extracted with dichloromethane (2 ×50 mL). The combined organic layers were washed with brine (5 mL) and dried over sodium sulfate. The solvent was evaporated under reduced pressure and the colorless residue was purified via ISCO (ethyl acetate/hexane) and the product was eluted at 10%. The solvent was evaporated under reduced pressure to give a colorless oil (1150 mg, 43% yield). 1H NMR (400 MHz, CDCl3) δ 1.42 (s, 9H), 3.69 (b, 2H), 3.76 (s, 3H), 4.45 (b, 1H), 5.37 (b, 1H); 13C NMR (100 MHz, CDCl3) δ 170.2, 155.1, 80.5, 53.5, 52.8, 52.7, 28.3; LC-MS (ESI+) m/z 245.05 [M + H]+.

Methyl (S)-3-amino-2-((tert-butoxycarbonyl)amino)propanoate (19)

Compound 63 (1150 mg, 4.7 mmol) was dissolved in 15 mL of methanol and 10% palladium on carbon (64 mg) was added and the reaction mixture was deoxygenated and stirred at room temperature under H2 at balloon pressure for 3 h. The mixture was filtered through a celite layer and the filtrate was dried under reduced pressure to get a pale yellow oil (1024 mg, 100% yield). 1H NMR (400 MHz, CDCl3) δ 1.24 (b, 2H), 1.41 (s, 9H), 3.01 (b, 2H), 3.73 (s, 3H), 4.27 (b, 1H), 5.42 (b, 1H); 13C NMR (100 MHz, CDCl3) δ 172.1, 155.5, 80.0, 55.9, 52.4, 43.9, 28.3; LC-MS (ESI+) m/z 219.1 [M + H]+.

Methyl-N2-(tert-butoxycarbonyl)-N4-((S)-2-((tert-butoxycarbonyl)amino)-3-methoxy-3-oxo-propyl)-L-asparaginate (15c)

To a solution of Boc-Asp-OMe (866 mg, 3.5 mmol) in 3 mL of anhydrous N,N-dimethylformamide, HATU (1472 mg, 3.87 mmol) and DIPEA (1.9 mL, 10.62 mmol) were added. The reaction mixture was left to stir at room temperature for 10 min. Compound 19 (769 mg, 3.5 mmol) was dissolved in 2 mL of anhydrous N,N-dimethylformamide and then was added to the mixture and the reaction mixture was left to stir at room temperature for 2 h. The mixture was diluted with ethyl acetate (100 mL), washed with water (3 ×50 mL) and brine (5 mL), and dried over sodium sulfate. The solvent was evaporated under reduced pressure and the yellow residue was purified via ISCO (ethyl acetate/hexane) and the product was eluted at 40%. The solvent was evaporated under reduced pressure to give a colorless oil (630 mg, 41% yield). 1H NMR (400 MHz, CDCl3) δ 1.40 (s, 18H), 2.60 (b, 1H), 3.02-3.09 (m, 1H), 3.75 (s, 6H), 3.85-3.93 (m, 2H), 4.33 (b, 1H), 4.60 (b, 1H), 5.45 (b, 2H), 7.22 (b, 1H); 13C NMR (100 MHz, CDCl3) δ 175.8, 174.5, 170.1, 155.6, 80.3, 52.9, 51.7, 49.8, 40.8, 36.5, 28.3; LC-MS (ESI+) m/z 448.18 [M + H]+.

N2-(tert-Butoxycarbonyl)-N4-((S)-2-((tert-butoxycarbonyl)amino)-2-carboxyethyl)-L-asparagine (16c)

Compound 15c (600 mg, 1.34 mmol) was dissolved in 9 mL of tetrahydrofuran and cooled to 0 °C. Lithium hydroxide (81 mg, 3.35 mmol) was dissolved in 9 mL of water then was added to the chilled solution and the reaction mixture was left to stir at room temperature for 1 h. The reaction mixture was diluted with water (60 mL) and washed with dichloromethane (3 ×20 mL). Then the aqueous layer was acidified by 0.5 N potassium hydrogen sulfate to pH 2 and then was extracted with ethyl acetate (3 ×120 mL). The combined organic layers were washed with brine (20 mL) and dried over sodium sulfate. The solvent was evaporated under reduced pressure to give a pale yellow solid (439 mg, 79% yield). 1H NMR (400 MHz, DMSO-d6) δ 1.37 (s, 18H), 2.45–2.49 (m, 2H), 3.21–3.58 (m, 2H), 4.00 (b, 1H), 4.22 (b, 1H), 6.80 (b, 1H), 6.90 (b, 1H), 7.92 (b, 1H), 12.46 (b, 2H); 13C NMR (100 MHz, DMSO-d6) δ 173.2, 172.1, 169.8, 155.3, 78.2, 53.4, 50.1, 48.6, 36.9, 28.1; LC-MS (ESI+) m/z 420.42 [M + H]+.

tert-Butyl ((S)-3-((S)-3-((tert-butoxycarbonyl)amino)-4-(4-methylpiperazin-1-yl)-4-oxobutan-amido)-1-(4-methylpiperazin-1-yl)-1-oxopropan-2-yl)carbamate (17c)

Compound 16c (206 mg, 0.5 mmol) was suspended in 6 mL of dry dichloromethane. DIPEA (523 µL, 3 mmol) was added and gave a clear solution. Then 1-methyl piperazine (164 µL, 1.5 mmol) and PyAOP (548 mg, 1 mmol) were added to the solution and the reaction mixture was left to stir at room temperature for 1.5 h. The mixture was diluted with (40 mL) of water and extracted with dichloromethane (7 ×100 mL). The combined organic layers were washed with brine (20 mL) and dried over sodium sulfate. The solvent was evaporated under reduced pressure and the yellow residue was purified via ISCO (20% MeOH:DCM + 0.1% TEA/DCM) and the product was eluted at 60%. The solvent was evaporated under reduced pressure to give a pale yellow solid (160 mg, 55% yield). 1H NMR (400 MHz, DMSO-d6) δ 1.35 (s, 18H), 2.18 (s, 6H), 2.26 (b, 10H), 3.02-3.52 (m, 10H), 4.48 (b, 1H), 4.69 (b, 1H), 6.84 (b, 1H), 7.03 (b, 1H), 7.88 (b, 1H); 13C NMR (100 MHz, DMSO-d6) δ 169.9, 169.2, 168.3, 155.0, 79.1, 54.6, 54.2, 49.6, 46.9, 45.5, 44.5, 41.4, 37.6, 28.1; LC-MS (ESI+) m/z 584.22 [M + H]+.

(S)-3-Amino-N-((S)-2-amino-3-(4-methylpiperazin-1-yl)-3-oxopropyl)-4-(4-methylpiperazin-1-yl)-4-oxobutanamide tetrahydrochloride (5c)

Compound 17c (140 mg, 0.24 mmol) was dissolved in 550 µL of MeOH and cooled to 0 °C. To the chilled solution was added 4 N HCl in dioxane (725 µL, 2.9 mmol) in four portions every half an hour and the solution was warmed to room temperature after each addition. There was still some mono-deprotected product so additional 4 N HCl in dioxane (363 µL, 1.45 mmol) was added in two portions to the solution at 0 °C and the solution was warmed to room temperature and left to stir for 1 h. The solvent was evaporated under reduced pressure and the residue was dried under vacuum. The residue then was dissolved in a minimal amount of methanol and recrystallized with an excess of diethyl ether then the vial was centrifuged and the solvent was decanted; this purification step was repeated five times. The residue was dried to give a yellowish white solid (125 mg, 98% yield). 1H NMR (400 MHz, D2O) δ 2.97 (s, 6H), 3.19-3.43 (m, 8H), 3.66 (b, 12H), 4.30 (b, 2H); 13C NMR (100 MHz, D2O) δ 173.0, 166.1, 52.6, 51.1, 50.1, 43.3, 42.5, 40.9, 39.6, 35.1; LC-MS (ESI+) m/z 384.29 [M + H]+; HRMS (ESI+) m/z calculated for C17H34N7O3+ [M + H]+ 384.2718, found 384.2755.

Di-tert-butyl ((7 S,12 S)-2,17-dimethyl-6,10,13-trioxo-2,5,9,14,17-pentaazaoctadecane-7,12-di-yl)dicarbamate (17h)

Compound 16c (208 mg, 0.5 mmol) was suspended in 6 mL of dry dichloromethane. DIPEA (523 µL, 3 mmol) was added and gave a clear solution. Then N,N-dimethylethylenediamine (164 µL, 1.5 mmol) and PyAOP (548 mg, 1 mmol) were added to the solution and the reaction mixture was left to stir at room temperature for 45 min. The mixture was diluted with water (40 mL) and extracted with dichloromethane (12 ×80 mL). The combined organic layers were washed with brine (20 mL) and dried over sodium sulfate. The solvent was evaporated under reduced pressure and the yellow residue was purified via ISCO (20% MeOH:DCM + 0.1% TEA/DCM) and the product was eluted at 75%. The solvent was evaporated under reduced pressure to give a pale yellow solid (156 mg, 56% yield). 1H NMR (400 MHz, DMSO-d6) δ 1.36 (s, 18H), 2.23 (b, 2H), 2.81 (b, 4H), 3.11 (b, 16H), 3.71 (b, 1H), 3.99 (b, 1H), 6.55 (b, 2H), 8.03 (b, 3H); 13C NMR (100 MHz, CDCl3) δ 171.8, 170.8, 155.1, 79.2, 55.7, 5.17, 51.5, 45.3, 42.4, 37.5, 34.1, 28.1; LC-MS (ESI+) m/z 560.29 [M + H]+.

(S)-2-Amino-N4-((S)-2-amino-3-((2-(dimethylamino)ethyl)amino)-3-oxopropyl)-N1-(2-(di-methylamino)ethyl)succinamide tetrahydrochloride (5h)

Compound 17 h (130 mg, 0.23 mmol) was dissolved in 550 µL of MeOH and cooled to 0 °C. To the chilled solution was added 4 N HCl in dioxane (700 µL, 2.8 mmol) in four portions every half an hour and the solution was warmed to room temperature after each addition. There was still some mono-deprotected product so additional 4 N HCl in dioxane (175 µL, 0.7 mmol) was added to the solution at 0 °C and the solution was warmed to room temperature and left to stir for 1 h. The solvent was evaporated under reduced pressure and the residue was dried under vacuum. The residue then was dissolved in a minimal amount of methanol and recrystallized with an excess of diethyl ether then the vial was centrifuged and the solvent was decanted; this purification step was repeated three times. The residue was dried to give a yellowish white solid (75 mg, 65% yield). 1H NMR (400 MHz, D2O) δ 2.97 (s, 6H), 3.19-3.43 (m, 8H), 3.66 (b, 12H), 4.30 (b, 2H); 13C NMR (100 MHz, D2O) δ 171.6, 169.5, 168.5, 56.3, 53.1, 50.0, 43.2, 39.9, 35.3, 34.9; LC-MS (ESI+) m/z 360.29 [M + H]+; HRMS (ESI+) m/z calculated for C15H34N7O3+ [M + H]+ 360.2718, found 360.2748.

Methyl-N2-(tert-butoxycarbonyl)-N5-((S)-2-((tert-butoxycarbonyl)amino)-3-methoxy-3-oxo-propyl)-L-glutaminate (15d)

Compound 19 (200 mg, 0.92 mmol) was dissolved in 6 mL of dry dichloromethane. DIPEA (481 µL, 2.76 mmol) was added to give a clear solution. Then Boc-Glu-OMe (240 mg, 0.92 mmol) and PyAOP (528 mg, 1 mmol) were added to the solution and the reaction mixture was left to stir at room temperature for 1.5 h. The mixture was diluted with water (40 mL) and extracted with dichloromethane (3 ×80 mL). The combined organic layers were washed with brine (10 mL) and dried over sodium sulfate. The solvent was evaporated under reduced pressure and the residue was purified via ISCO (ethyl acetate/hexane) and the product was eluted at 50%. The solvent was evaporated under reduced pressure to give a white solid (176 mg, 42% yield). 1H NMR (400 MHz, CDCl3) δ 1.41 (s, 18H), 1.81 (b, 1H), 2.12-2.26 (m, 3H), 3.51 (b, 1H), 3.72 (s, 6H), 3.82 (b, 1H), 4.29 (b, 1H), 4.40 (b, 1H), 5.35 (b, 1H), 5.78 (b, 1H), 6.73 (b, 1H); 13C NMR (100 MHz, CDCl3) δ 172.7, 171.2, 155.1, 80.4, 53.8, 52.6, 41.7, 32.6, 29.7, 28.3; LC-MS (ESI+) m/z 462.31 [M + H]+.

N2-(tert-Butoxycarbonyl)-N5-((S)-2-((tert-butoxycarbonyl)amino)-2-carboxyethyl)-L-glutamine (16d)

Compound 15d (170 mg, 0.37 mmol) was dissolved in 3 mL of tetrahydrofuran and cooled to 0 °C. Lithium hydroxide (23 mg, 0.93 mmol) was dissolved in 3 mL of water, which was then added to the chilled solution and the reaction mixture was left to stir at room temperature for 3 h. The reaction mixture was concentrated under reduced pressure and then diluted with water (30 mL) and washed with dichloromethane (3 ×10 mL). Then the aqueous layer was acidified by 0.5 N potassium hydrogen sulfate to pH 2 and then was extracted with ethyl acetate (6 ×60 mL). The combined organic layers were washed with (20 mL