## 2 Materials and Methods
### General Procedures for Synthesis of TPZs  
_3-Amino-1,2,4-benzotriazine 1,4-dioxide_ (TPZ **(1)**). The starting 3-amino-1,2,4-benzotriazine 1-oxide was synthesized by the condensation of 2-nitroaniline (2.07 g, 1.5 mM) with cyanamide (2.5 g, 15 mmol) suspended in a CF\({}_{3}\)COOH/CH\({}_{3}\)COOH mixture (1:1, 50 mL) andheated to 50 \({}^{\circ}\)C. Hydrogen peroxide (50 mL, 35% conc.) was introduced, stirred and heated for approximately 12 h, and neutralized by solid KHCO\({}_{3}\). The final product was filtered, washed with distilled H\({}_{2}\)O, dried and purified by flash-column chromatography on silica gel (eluent: ethylacetate). Yield: 78%. \({}^{1}\)H NMR (CD\({}_{3}\))\({}_{2}\)SO) \(\delta\) 9.83 (bs, 2H, NH\({}_{2}\)); 8.43 (d, 2H, J = 9.6 Hz); 8.15 (t, IH, J = 7.8 Hz,); 7.86 (t, IH, J = 7.7 Hz). HRMS (ESI) \(m\)/\(z\)[M+H]\({}^{+}\) = 179.0564 (calculated: 179.0563). _3-Acetylamino-1,2,4-benzotriazine 1,4-dioxide_ (compound **2**) was obtained from TPZ **(1)** (10 mM) dissolved in 10 mL of an acetic acid/acetic anhydride mixture (1:1) heated over a boiling water bath for approximately 10 h. All volatiles were evaporated in vacuo and the remaining solid was re-crystallized from 2-propylacetate.Yield: 76%. Melting point: 201 \({}^{\circ}\)C. \({}^{1}\)H NMR (CDCl\({}_{3}\)): \(\delta\) 8.62 (d, 2H, H5 and H8,  
Figure 1: The formulae of 3-amino-1,2,4-benzotriazine-1,4-dioxide (tirapazamine (TPZ **(1)**, SR4233) and its derivatives used in the current work. J = 4.9 Hz), 8.29 (t, 1H, H6, J = 4.9 Hz) 7.99 (t, 1H, H7, J = 4.9 Hz), 6.52 (bs, 1H, NH), 2.51 (s, 3H, CH3). HRMS (ESI) _m/z_ [M+H]\({}^{+}\) = 207.0878 (calculated: 207.0875). _3-Propionylamino-1,2,4-benzotriazine 1,4-dioxide_ (compound **3**) was synthesized following a similar procedure as described for TPZ (**1**). Yield 48%. Melting point: 167 \({}^{\circ}\)C. \({}^{1}\)H NMR (CDC\({}_{3}\)): \(\delta\) 8.96 (bs, 1H, NH), 8.61 (d, 2H, H5 and H8, J = 4.9 Hz), 8.48 (t, 1H, H6, J = 4.9 Hz), 7.48 (t, 1H, H6, J = 4.9 Hz), 2.80 (q, 2H, CH\({}_{2}\)), 1.24 (t, 3H, CH\({}_{3}\)); HRMS (ESI) _m/z_ [M+H]\({}^{+}\) = 235.0829 (calculated: 235.0826). _3-Methoxycarbonylamino-1,2,4-benzotriazine 1,4-dioxide_ (compound **4**) was obtained following theSchotten- Baumann synthetic procedure by the acylation reaction of TPZ (**1**) (10 mM) in basic medium (10 mL of 10% solution of Cs\({}_{2}\)CO\({}_{3}\)) with methoxycarbonylchloride (13 mM) using aqueous dioxane (50%) as the best reaction medium. The reaction was neutralized by the drop-wise addition of acetic acid. The final product was filtered, washed with distilled H\({}_{2}\)O, dried and re-crystallized from acetonitrile. Yield 54%. Melting point: 194 \({}^{\circ}\)C. \({}^{1}\)H NMR (CDC\({}_{3}\)): \(\delta\) 8.92 (bs, 1H, NH), 8.58 (d, 2H, H5 and H8, J = 7.9 Hz), 8.43 (t, 1H, H6, J = 4.7 Hz), 7.44 (t, 1H, H6, J = 4.7 Hz); 0.84 (s, 3H, OCH\({}_{3}\)); HRMS (ESI) _m/z_ [M+H]\({}^{+}\) = 237.0618 (calculated: 237.0617). _Acylated and sulfonylated TPZs_ (compounds **5**, 7-9**) were obtained following the Schotten-Baumann synthetic procedure by means of the acylation of TPZ (**1**) (10 mM) in basic medium (10% solution of Cs\({}_{2}\)CO\({}_{3}\)) with carbonylchlorides or sulfochlorides (13 mM) in aqueous dioxane (50%), and the final products were crystallized from ethyl acetate. _3-Methoxyacetylamino-1,2,4-benzotriazine 1,4-dioxide_ (compound **5**): yield 58%, melting point 170 \({}^{\circ}\)C, \({}^{1}\)H NMR (CDC\({}_{3}\)): \(\delta\)8.71 (bs, 1H, NH), 8.42 (d, 2H, H5 and H8, J = 9.4 Hz), 8.09 (t, 1H, H6, J = 4.6 Hz), 7.28 (t, 1H, H6, J = 4.6 Hz),2.31 (s, 2H, CH\({}_{2}\)), 0.88 (s, 3H, OCH\({}_{3}\)); HRMS (ESI) _m/z_ [M+H]\({}^{+}\) = 251.0776 (calculated: 251.0774). _3-Ethoxycarbonylamino-1,2,4-benzotriazine 1,4-dioxide_ (compound **6**): yield: 62%, melting point: 183 \({}^{\circ}\)C, \({}^{1}\)H NMR (CDC\({}_{3}\)): \(\delta\)8.66 (bs, 1H, NH), 8.51 (d, 2H, H5 and H8, J = 7.9 Hz), 8.35 (t, 1H, H6, J = 4.7 Hz), 7.18 (t, 1H, H6, J = 4.7 Hz), 2.30 (q, 2H, OCH\({}_{2}\)), 1.02 (t, 3H, CH\({}_{3}\)).