The benzophenothiazinium dye EtNBS has previously been tested as a photosensitizer to mediate photodynamic therapy (PDT). used to mediate PDT destruction of Leishmaniasis parasites . EtNBS derivative compounds to date have largely been created through modifications to the amine side groups Bay 60-7550 or through replacement of the central ring chalcogen. These modifications are functionally orthogonal with the heavier chalcogen (Se) derivative leading to increased intersystem crossing and subsequent singlet oxygen production . Side chain modifications on the other hand enable the tuning of properties Bay 60-7550 such as molecular hydrophobicity and charge. Side chain derivatives were found to alter photosensitizer uptake localization and ultimately PDT efficacy both in the treatment of microorganisms and cancer . Nile blue derivatives such as EtNBS have unique properties that make Bay 60-7550 them good photosensitizers for anti-microbial therapy. Though EtNBS is a lipophilic compound it also has a single charge delocalized throughout its ring system making the molecule readily water soluble. Containing only a single sulfur heteroatom in the ring EtNBS actually has a relatively low singlet oxygen quantum yield of 3% . Nevertheless EtNBS has been observed to be a potent photosensitizer. Recent studies investigating EtNBS and structural derivatives have found that the molecule can act via both Type I and Type II photochemical pathways. This consideration allows EtNBS to operate over a wide range of oxygen tensions even in severely hypoxic environments [13 21 This property could be of significant benefit especially in the treatment of complex infections and biofilms where oxygen is not always present in high concentrations. The high PDT activity of EtNBS and its TSPAN11 derivatives and the broad-spectrum of microbial targets observed in the previous reports taken together support more studies on new derivatives to better understand the structural features needed for improved efficacy of PDT. For this reason in the present study we aimed to evaluate the effect of EtNBS and a panel of novel derivatives on two Gram-positive bacterial strains and and two Gram-negative strains and Initially we tested three derivatives with electron-withdrawing (nitro EtNBS-N) or electron-donating (amino EtNBS-A and acetamido EtNBS-Ac) groups prepared by substitution at the 1-position of the benzene ring. We then tested the hypothesis that 1-substitution distorts the planar structure of the conjugated rings by comparing two compounds substituted with N-ethylpropylsulfonamido either at the 1-position (EtNBS-So) or at the 4-position (EtNBS-Sp). Finally as small cationic molecules such as EtNBS have been found potent against microorganisms the arginine side-chain (EtNBS-G) derivative was included in this study to examine the role of increased charge on photosensitizer uptake and activity. All the structures are shown in Table 1. Table 1 Structures of the seven benzophenothiazinium dyes LogP and TPSA values. Materials and methods Synthesis of compounds 5 chloride (EtNBS) and (sodium 2-amino-5-diethylaminophenylthiosulfuric acid) were prepared according to previously published procedures . General method for synthesis of the EtNBS derivatives A refluxing mixture of derivatized naphthylamine (1.0 mmol) and Bunte salt (138 mg 0.5 mmol) in 25 mL of methanol was charged with silver carbonate (303 mg 1.1 mmol) and allowed to stir for about 30 min. The deep blue reaction mixture Bay 60-7550 was then filtered through a pad of celite and concentrated to near-dryness with the aid of a rotary evaporator. The resulting solid was portioned between 25 mL dichloromethane and 25 mL saturated sodium carbonate solution. The organic layer was separated acidified with ~0.25 mL conc. HCl and allowed to dry overnight in a fume hood. The crude product was purified by silica-gel chromatography using 5 – 10% methanol in dichloromethane as the mobile phase. Derivatives EtNBS-A EtNBS-Ac and EtNBS-G were further purified in small batches by reversed-phase high-performance liquid chromatography. N-(9-(diethylamino)-1-nitro-5H-benzo[a]phenothiazin-5-ylidene)ethanaminium (EtNBS-N) This compound was synthesized starting with N-ethyl-5-nitronaphthalen-1-amine. 1H NMR (DMSO-d6) δ 9.93 (m 1 8.82 (d = 8.7 Hz 1 8.3 (d = 7.8 Hz 1 8.07 (t = 7.8 Hz 1 7.7 (m 4 3.77 (m 6 1.42 (t = 7.2 Hz 3 1.29 (t = 7.2 Hz 6 MALDI-MS calculated for C22H23N4O2S+: 407.1; found 407.0. HPLC retention time = 22.3 min conditions: C-18 reversed-phase column (4.6 mm × 250 mm) eluting with CH3CN in water (20% to 100% in 25 min) with a flow.