2.1. Chemistry

Unless otherwise specified, all chemicals used throughout this work were purchased from Sigma-Aldrich Srl (Milan, Italy). ¹H-NMR spectra were recorded at 400 MHz on a Bruker AC 400 spectrometer (Bruker, Billerica, MA, USA); reporting chemical shifts in δ (ppm) units relative to the internal reference tetramethylsilane (Me₄Si). All compounds were routinely checked by TLC and ¹H-NMR. TLC was performed on aluminium-backed silica gel plates (Merck DC, Alufolien Kieselgel 60 F254, Kenilworth, NJ, USA) with spots visualised by UV light. Yields of all reactions refer to the purified products. All chemicals were of the highest purity. Mass spectra were recorded on an API-TOF Mariner by Perspective Biosystem; samples were injected by a Harvard pump using a flow rate of 5–10 µL/min, infused in the Electrospray system. Elemental analyses were performed by a PE 2400 (Perkin-Elmer, Waltham, MA) analyser and have been used to determine purity of the described compounds, which is >95%. Analytical results are within ±0.40% of the theoretical values.

Melting points were determined on a Buchi 530 melting point apparatus (Flawil, Switzerland) and are uncorrected.

Succinic anhydride (196 mg, 1.96 mmol, 2.0 eq.) was added to a solution of 6-((7-nitrobenzo[c][1,2,5] oxadiazol-4-yl)thio)hexan-1-ol (5) (260 mg, 0.98 mmol, 1 eq.) and 4-(dimethylamino)pyridine (DMAP) (120 mg, 0.98 mmol, 1.0 eq.) in dry dichloromethane (DCM) (9 ml) and the resulting mixture was stirred at reflux for 4 h. Upon completion, the reaction mixture was poured into a solution of 2 N HCl (22 ml) and extracted with ethyl acetate (4 × 25 ml). The organic phase was then washed with saturated sodium chloride solution (25 ml), dried over anhydrous sodium sulphate, filtered, and concentrated under vacuum. Finally, the crude residue was purified by silica gel column chromatography eluting with the appropriate mixture CHCl₃/MeOH 25:1 to provide the final compound as a yellow powder. Yield: 70%. M.p.: 90–93 °C. ¹H-NMR (DMSO) δ 1.809–1.838 (m, 4H, –SCH₂CH₂CH₂CH₂O–), 2.478–2.509 (m, 4H, –OCOCH₂CH₂COOH), 3.408 (t, 2H, –SCH₂CH₂CH₂CH₂O–), 4.100 (t, 2H, –SCH₂CH₂CH₂CH₂O–), 7.534 (d, 1H, CH benzoxadiazole ring), 8.574 (d, 1H, CH benzoxadiazole ring), 12.126–12.266 (br s, 1H, COOH). MS (ESI), m/z: 368.1 [M – H]^-. Anal. (C₁₄H₁₅N₃O₇S) Calcd. (%): C, 45.53; H, 4.09; -N, 11.38; S, 8.68. Found (%) C, 45.68; H, 4.11; N, 11.30; S, 8.64.

Succinic anhydride (277.8 mg, 2.77 mmol, 3.0 eq.) was added to a solution of 2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)ethoxy)ethan-1-ol (264 mg, 0.93 mmol, 1 eq.) (2) and 4-(dimethylamino)pyridine (DMAP) (169.5 mg, 1.39 mmol, 1.5 eq.) in dry dichloromethane (DCM) (9 ml) and the resulting mixture was stirred at reflux for 4 h. Upon completion, the reaction mixture was poured into a solution of 2 N HCl (22 ml) and extracted with ethyl acetate (4 × 30 ml). The organic phase was then washed with saturated sodium chloride solution (30 ml), dried over anhydrous sodium sulphate, filtered, and concentrated under vacuum. Finally, the crude residue was purified by silica gel column chromatography eluting with the mixture CHCl₃/MeOH 20:1 to provide the final compound. Yield: 51%. M.p.: 74–76 °C. ¹H-NMR (DMSO) δ 2.461–2.509 (m, 4H, COCH₂CH₂COOH), 3.598 (t, 2H, –SCH₂CH₂OCH₂CH₂O–), 3.673 (t, 2H, –SCH₂CH₂OCH₂CH₂O–), 3.835 (t, 2H, –SCH₂CH₂OCH₂CH₂O–), 4.148 (t, 2H, –OCH₂CH₂O–), 7.583 (d, 1H, CH benzoxadiazole ring), 8.579 (d, 1H, CH benzoxadiazole ring), 12.170–12.2174 (br s, 1H, COOH). MS (ESI), m/z: 384.1 [M - H]^-. Anal. (C₁₄H₁₅N₃O₈S) Calcd. (%): C, 43.64; H, 3.92; N, 10.90; S, 8.32. Found (%) C, 43.80; H, 3.94; N, 10.82; S, 8.27.

Pyridine (0.69 ml, 8.62 mmol, 3.5 eq.) and 2-mercaptopyrimidine (275.9 mg, 2.46 mmol, 1.0 eq.) were added to a solution of 4-chloro-7-nitrobenzo[c][1,2,5]oxadiazole (491.8 mg, 2.46 mmol, 1.0 eq.) in a mixture (18 ml) EtOH:H₂O (0.3:1 v/v). The mixture was stirred at room temperature for 16 h. Subsequently, the suspension was filtered, and the crude solid was washed on the filter with H₂O. The compound obtained was first purified by trituration with CHCl₃ and then by column chromatography on SiO₂ gel eluting with a mixture CHCl₃/MeOH (90:1). Yield: 76%. M.p.: 144–147 °C. ¹H-NMR (DMSO): δ 7.416 (t, 1H, CH pyrimidine ring), 8.288 (d, 1H, CH benzoxadiazole ring), 8.683–8.696 (m, 2H, CH pyrimidine ring), 8.719 (d, 1H, CH benzoxadiazole ring). MS (ESI), m/z: 276.0 [M + H]⁺. Anal. (C₁₀H₅N₅O₃S) Calcd. (%): C, 43.64; H, 1.83; N, 25.44; S, 11.65. Found (%) C, 43.76; H, 1.85; N, 25.37; S, 11.59.

Yield: 78%. M.p.: 166–168 °C. ¹H-NMR (DMSO): δ 1.857 (s, 3H, –NHCOCH₃), 3.575–3.630 (dd, 1H, –SCHHCHNH–), 3.792–3.837 (dd, 1H, –SCHHCHNH–), 4.631–4.685 (m, 1H, CH₂CHNH), 7.597 (d, 1H, CH benzoxadiazole ring), 8.540 (d, 1H, CHNHCOCH₃), 8.601 (d, 1H, CH benzoxadiazole ring), 13.107–13.271 (br s, 1H, COOH). MS (ESI), m/z: 325.0 [M - H]^-. Anal. (C₁₁H₁₀N₄O₆S) Calcd. (%): C, 40.49; H, 3.09; N, 17.17; S, 9.83. Found (%) C, 40.60; H, 3.11; N, 17.11; S, 9.76.

Yield: 81%. M.p.: 206–208 °C. ¹H-NMR (DMSO): δ 7.136 − 7.417 (br m, 2H, CH benzimidazole ring), 7.430 − 7.716 (m, 3H, CH benzimidazole and benzoxadiazole rings), 8.582–8.633 (d, 1H, CH benzoxadiazole ring), 13.475 (br s, NH). MS (ESI), m/z: 314.0 [M + H]⁺. Anal. (C₁₃H₇N₅O₃S) Calcd. (%): C, 49.84; H, 2.25; N, 22.35; S, 10.23. Found (%) C, 49.90; H, 2.27; N, 22.28; S, 10.17.