# Investigation of Sonosensitizers Based on Phenothiazzinium Photosensitizers
## 2 Materials and Methods  
### Materials  
Methylene blue (MB) was purchased from Alfa Aesar. 1,3-Diphenylisobenzofuran (DPBF) was purchased from Aldrich Chemical Co. All of the solvents were of spectrometric grade. In this study, the highest-grade general chemicals were employed that obtained from Acros Organic Co., Merck Ltd., or Aldrich Chemical Co. and used without further purification. All compound and DPBF stock solutions (10\({}^{-2}\) M) were kept in the dark at 4 \({}^{\circ}\)C until use. Molecular 3,7-bis(di(2-hydroxyethyl)amino)-phenothiazzin-5-ium iodide (MBOH) and 3,7-bis(dihexylamino)-phenothiazzin-5-ium iodide (MB6C) were synthesized as shown in Scheme 1. All the spectra for identifying compounds are listed in the supporting information (Supplementary Materials Figures S1-S6)  
_Synthesis of phenothiazzinum tetraiodide hydrate 1_[41]. To a solution of phenothiazze (1.0 g, 0.005 mol) in chloroform (35 mL) contained in a 250 mL round-bottom flask, an iodine (3.8 g, 0.015 mol)-containing chloroform (115 mL) was added dropwise in an ice bath and then kept at room temperature for 4 h. The solution was filtered, and the solid was washed with a large amount of chloroform (50 mL \(\times\) 4) to remove the excess iodine. After drying, a dark green powder (3.1 g, 88%) was obtained. \({}^{1}\)H NMR (400 MHz, DMSO-\(d_{6}\)): \(\delta\) (ppm) = 7.82-8.05 (m, 4H), 7.60-7.75 (m, 4H). Mass (ESI\({}^{*}\)): calculated for C\({}_{12}\)H\({}_{8}\)NS\({}^{*}\)\(m/z\) = 198.04, found 198.0.  
_Synthesis of 3,7-bis(dihexylamino)-phenothiazzn-5-ium iodide (MB6C)_[42]. Tetraio-diide hydrate salt **1** (1.44 g, 0.002 mol) was dissolved in 40 mL of methanol at room temperature. An excess amount of \(N\),\(N\)-dihexylamine (8 mmol) in methanol (10 mL) was added dropwise, and the system was stirred for 4 h. The product tetraalkylamino phenothiazzinium precipitated and was filtered and recrystallized three times by a CH\({}_{2}\)Cl\({}_{2}\)-MeOH system. The final product was collected as a dark purple solid (yield 68%) after drying overnight in an oven. \({}^{1}\)H NMR (400 MHz, DMSO-\(d_{6}\)) \(\delta\) (ppm) = 7.92 (d, \(J\) = 8.8 Hz, 2 Ha), 7.50 (dd, \(J\) = 8.8, 2.4 Hz, 2 Hb)-7.47 (d, \(J\) = 2.4 Hz, 4 Hc), 3.68 (t, 8H), 2.99-2.75 (m, 8H), 1.7-1.45 (m, 8H), 1.4-1.2 (m, 8H), 0.8-1.0 (m, 20H). MS (ESI\({}^{*}\)): calculated for C\({}_{28}\)H\({}_{42}\)N\({}_{3}\)S\({}^{*}\)\(m/z\) = 564.0, found \(m/z\) = 564.5.  
_Synthesis of 3,7-bis(di(2-hydroxyethyl)amino)-phenothiazzn-5-ium iodide (MBOH)._ Similar to _MB6C_[43], a solution of the appropriate \(N\),\(N\)-di(2-hydroxyethyl)amine (8 mmol) in methanol (10 mL) was added to the solution containing compound 1 and stirring was continued for 3 h by monitoring with thin-layer chromatography. The methanol was removed under vacuum and purified by flash chromatography (eluent CH\({}_{2}\)Cl\({}_{2}\)/MeOH 95/5 (\(v/v\))) to yield a dark green-purple solid (52%). \({}^{1}\)H NMR (400 MHz, DMSO-\(d_{6}\)) \(\delta\) (ppm) = 7.87 (d, \(J\) = 8.8 Hz, 2 Ha), 7.53-7.56 (m, 2 Hb and 2 \(\rm{Hc}\)), 5.03 (t, 4H, CCOH) 3.84 (m, 8 H, CH\({}_{2}\)CH\({}_{2}\)OH), 3.71 (m, 8 H, CH\({}_{2}\)CH\({}_{2}\)OH). MS (ESI\({}^{*}\)): calcd. for C\({}_{20}\)H\({}_{26}\)N\({}_{3}\)O\({}_{4}\)S\({}^{*}\)\(m/z\) = 404.16, found \(m/z\) = 404.2.