Apoptosis Induced by 2-Aryl Benzothiazoles-Mediated Photodynamic

Jun 3, 2014 - Department of Biotechnology, Kaohsiung Medical University, Kaohsiung 807 ... Department of Surgery, School of Medicine, Kaohsiung Medica...
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Apoptosis Induced by 2‑Aryl Benzothiazoles-Mediated Photodynamic Therapy in Melanomas via Mitochondrial Dysfunction Yin-Kai Chen,‡,▽ Gopal Chandru Senadi,†,▽ Chih-Hung Lee,⊥ Yi-Min Tsai,§ Yan-Ren Chen,† Wan-ping Hu,*,‡ Yu-Wei Chou,‡ Kung-Kai Kuo,∥ and Jeh-Jeng Wang*,† †

Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung 807, Taiwan Department of Biotechnology, Kaohsiung Medical University, Kaohsiung 807, Taiwan § School of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan ∥ Department of Surgery, School of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan ⊥ Department of Dermatology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung 833, Taiwan ‡

S Supporting Information *

ABSTRACT: A mild and efficient synthetic development of 2-arylbenzothiazoles 5 mediated by ceric ammonium nitrate (CAN) via intramolecular cyclization of N-phenyl-thiobenzamides 4 was achieved. Further compounds 5 were reduced to corresponding amines 6, and their photodynamic therapy (PDT) effect was evaluated on malignant human melanoma A375 cells. Amine 6l plus ultraviolet A (UVA) induced caspase-3 activity, poly(ADP-ribose)polymerase cleavage, M30 positive CytoDeath staining, and subsequent apoptotic cell death. Our data disclosed that treatment of A375 cells with 6l plus UVA resulted in a decrease in mitochondrial membrane potential (ΔΨmt), oxidative phosphorylation system (OXPHOS) subunits, and adenosine triphosphate (ATP) but an increase in mitochondrial DNA 4977-bp deletion via reactive oxygen species (ROS) generation. Transmission electron microscopy (TEM) observations also showed major ultrastructural alterations of mitochondria. Additionally, 6l plus UVA was also shown to reduce murine melanoma size in a mouse model. The present study supports the hypothesis that 6l-PDT may serve as a potential ancillary modality for the treatment of melanoma.

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cancers, it is responsible for 80% of skin cancer deaths.3,4 Melanoma has the potential to metastasize to lymph nodes and several important internal organs, which leads to a dismal prognosis. The Food and Drug Administration (FDA) have approved IL-2 and dacarbazine as potential agents for treatment of metastatic melanomas. The latter has an overall response rate of 22% with no influence on survival.5 On the other hand, the high dosage of IL-2 results in an overall response rate of 16% with no impact in the survival rate.6,7 Various novel treatment methods are in progress, which include angiogenesis inhibitors, novel cytotoxic agents, and therapeutic cancer vaccines. In particular, BRAF inhibitors and CTLA-4 immunomodulators are being used in clinical trials. However, these treatment methods have shown no superior effects over one another. Therefore, discovery and development of new, more active, selective, and less toxic compounds for the treatment of malignancy are pivotal goals in medicinal chemistry. Photodynamic therapy (PDT), which combines an exogenous photosensitizer and light, has been used as a modality for the treatment of cancer. It is recognized as a potential treatment

INTRODUCTION One of the most important causes of death in Western countries relates to cancer.1 The understandings of the fundamental biology of cancer have been increasing dramatically in recent years.2 Melanoma is a prominent chemoresistant cancer. Even though melanoma accounts for only 4% of all skin

Figure 1. Our previously reported work. Reprinted from ref 14. Copyright 2010, with permission from Elsevier. © 2014 American Chemical Society

Received: March 13, 2014 Published: June 3, 2014 1187

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Scheme 1. General Synthetic Strategy for the Oxidative Cyclization of Thiobenzanilides for the Synthesis of Photosensitizers

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strategy for having the advantages of being minimally invasive and minimally toxic. The three major vital components of PDT are a photosensitizer, a light source, and tissue oxygen. The wavelength used for exciting the photosensitizer should be appropriate to produce oxidative stress and reactive oxygen species (ROS), which are finally responsible for killing the target cells.8 Compared with normal tissues, tumor tissues have higher uptake or retention of photosensitizers, which results in selective destruction of tumor cells after light irradiation. However, there are also some limitations of PDT including penetration depth of the photosensitizers and their activating light source. These limitations have prompted us to synthesize new classes of photosensitizers. Benzothiazoles are one of the most important classes of heterocycles for pharmaceutical purposes; they exist in numerous bioactive molecules such as the clinically used drug zopolrestat,9 a selective fatty acid amide hydrolase inhibitor,10 an aldose reductase inhibitor,11 and antitumor agents,12 as well as a fatty acid oxidation inhibitor.13 Recently, our research group has discovered a series of 2-(4-aminophenyl)benzothiazole derivatives (Figure 1) that absorb light in the UVA region (320−400 nm) as efficient photosensitizers for human basal cell carcinoma (BCC), one of the most common non-melanoma skin cancers (NMSCs).14 Since little research has been done in terms of the PDT effect on malignant melanoma,15−19 we hypothesize that a similar approach using our newly synthesized compounds 6 plus UVA might also be a therapeutic option for treating melanoma. ROS are vital in the regulation of cell death pathways,20 and they are harmful upon excessive production. Mitochondria are notably and actively involved in the production of ROS. The most decisive event in the initiation and execution of apoptosis21 is permeabilization of the outer membrane of mitochondria and consequent pro-apoptotic protein release from the inner membrane space. Previous research has indicated that ROS play a pivotal role in UVA-activated cell damage.22 Subsequently, mitochondrial depolarization might be correlated with cell death induced by UVA-activated compounds. The above considerations prompted us to develop a new synthetic process for 2-aryl benzothiazole scaffolds as photosensitizers (Scheme 1) and to investigate compounds with UVA irradiation on cultured melanoma A375 cells in terms of cell viability, mitochondrial dysfunction, and apoptosis induction. In addition, the in vivo efficacy of PDT using compound 6l in the B16 murine melanoma cell tumorigenesis in skin was also studied.

EXPERIMENTAL SECTION

Chemistry. Materials and Methods. Melting points are uncorrected and determined using a Mel-Temp apparatus. 1H NMR and 13C NMR spectra were recorded on a Varian Mercury Plus 400 spectrometer or a Bruker AC 200 spectrometer using CDCl3 or DMSO as a solvent. 1H NMR chemical shifts are referenced to TMS or CDCl3 (7.26 ppm). 13C NMR was referenced to CDCl3 (77.0 ppm). Multiplicities were determined by the distortionless enhancement by polarization transfer (DEPT) sequence as s, d, t, or q. Mass spectra and high resolution mass spectra (HRMS) were measured using the electron-impact (EI, 70 eV) technique by Taichung Regional Instrument Center of NSC at NCHU. Elemental analyses were performed by Tainan Regional Instrument Center of NSC at NCKU with an Elementar Vario EL III CHN recorder. Flash chromatography was carried out on Silica Gel 60 (E. Merck, 230−400 mesh). All chemicals and reagents were purchased from commercial suppliers and used without any further purifications (Alfa Aesar, Merck, and SigmaAldrich). General Procedure for the Syntheses of 2-Arylbenzothiazoles (5). To a stirred solution of N-phenyl-thiobenzamide (1 mmol) and sodium hydrogen carbonate (504 mg, 6.0 mmol) in a mixture of acetonitrile (20 mL) and water (2 mL) was added CAN (2.31 g, 4.2 mmol) in one portion at 0 °C, and the resulting mixture was stirred at the same temperature for 10 min. The reaction mixture was poured into ice−water (150 mL) and extracted four times with dichloromethane. The combined organic phases were washed with H2O and brine and dried over MgSO4. After removal of the solvent, the residue was purified by flash chromatography (hexane/dichloromethane = 4:1) to give the corresponding 2-arylbenzothiazoles. The spectral characterizations of reported compounds 5d−g, m, q− s, and w and 6d, h, m, q, r, and w are described in Supporting Information14,23 2-Phenylbenzothiazole (5a). White solid; yield 78%; mp 114−115 °C; 1H NMR (CDCl3, 400 MHz) δ 8.12−8.07 (m, 3H), 7.91 (ddd, J = 8.0, 2.8, and 0.8 Hz, 1H), 7.51−7.48 (m, 4H), 7.39 (dt, J = 8.0 and 1.2 Hz, 1H); 13C NMR (CDCl3, 100 MHz) δ 168.0 (s), 154.1 (s), 135.0 (s), 133.6 (s), 131.0 (d), 129.0 (d), 127.5 (d), 126.3 (d), 125.2 (d), 123.2 (d), 121.6 (d). Anal. Calcd for C13H9NS: C, 73.90; H, 4.29; N, 6.63. Found: C, 73.79; H, 4.19; N, 6.81. 6-Fluoro-2-(3-methoxylphenyl)benzothiazole (5b). Light green solid; yield 86%; mp 83−84 °C; 1H NMR (CDCl3, 400 MHz) δ 8.01 (dd, J = 8.8 and 4.8 Hz, 1H), 7.64−7.56 (m, 3H), 7.39 (t, J = 8.0 Hz, 1H), 7.22 (dt, J = 8.8 and 2.4 Hz, 1H), 7.04 (ddd, J = 8.4, 2.8, and 0.8 Hz, 1H), 3.91 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ 167.7 (s), 160.6 (d), 160.4 (s), 150.7 (s), 136.0 (d), 134.6 (s), 130.1 (s), 124.1 (d), 120.1 (s), 117.3 (s), 114.9 (d), 111.9 (s), 107.8 (d), 55.4 (q); HRMS (EI, m/z) for C14H11FNOS, calcd 260.0545, found 260.0544. Anal. Calcd for C14H10FNOS: C, 64.85; H, 3.89; N, 5.40. Found: C, 64.8; H, 4.0; N, 5.6. 6-Chloro-2-(3-methoxylphenyl)benzothiazole (5c). White solid; yield 71%; mp 76−78 °C; 1H NMR (CDCl3, 400 MHz) δ 7.93 (d, J = 8.4 Hz, 1H), 7.85 (d, J = 2.0 Hz, 1H), 7.61 (t, J = 2.2 Hz, 1H), 7.56 (ddd, J = 7.6, 1.2, and 0.8 Hz, 1H), 7.41 (dd, J = 8.8 and 2.4 Hz, 1H), 1188

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MHz) δ 8.10 (d, J = 8.8 Hz, 1H), 8.06 (d, J = 0.4 Hz, 1H), 8.01 (dd, J = 8.8 and 0.4 Hz, 1H), 8.00 (dd, J = 8.8 and 2.0 Hz, 1H), 7.92 (d, J = 2.0 Hz, 1H), 7.50 (dd, J = 8.8 and 2.0 Hz, 1H), 2.71 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ 165.5 (s), 152.5 (s), 150.2 (s), 137.0 (s), 136.5 (s), 134.7 (s), 132.1 (s), 131.5 (s), 127.7 (d), 125.78 (d), 125.58 (d), 124.5 (d), 121.394 (d), 20.6 (q); HRMS (ESI, m/z) for C14H10N2O2SCl, calcd 305.0151, found 305.0152. Anal. Calcd for C14H9N2O2SCl: C, 55.18; H, 2.98; N, 9.19. Found: C, 55.17; H, 3.09; N, 9.12. 6-Bromo-2-(3-methyl-4-nitrophenyl)benzothiazole (5v). Yellow solid; yield 261 mg (75%); mp 176−178 °C; 1H NMR (CDCl3, 400 MHz) δ 8.1−8.07 (m, 3H), 8.01 (ddd, J = 8.4, 2.0, and 0.4 Hz, 1H), 7.96 (d, J = 8.8 Hz, 1H), 7.64 (dd, J = 8.8 and 2.0 Hz, 1H), 2.72 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ 165.6 (s), 152.9 (s), 150.3 (s), 137.0 (s), 136.9 (s), 134.7 (s), 131.5 (d), 130.4 (d), 125.8 (d), 125.6 (d), 124.8 (d), 124.4 (d), 119.8 (s), 20.6 (q); HRMS (ESI, m/z) for C14H10N2O2SBr, calcd 348.9646, found 348.9647. Anal. Calcd for C14H9N2O2SBr: C, 48.15; H, 2.60; N, 8.02. Found; C, 48.12; H, 2.60; N, 8.05. 5-Trifluoromethyl-2-(3-methyl-4-nitrophenyl)benzothiazole (5x). Light yellow solid; yield 219 mg (65%); mp 161−163 °C; 1H NMR (CDCl3, 400 MHz) δ 8.35 (t, J = 0.8 Hz, 1H), 8.08−7.99 (m, 4H), 7.66 (dd, J = 8.4 and 1.2 Hz, 1H), 2.70 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ 167.1 (s), 153.5 (s), 150.4 (s), 138.6 (s), 136.7 (s), 134.7 (s), 131.6 (d), 129.5 (q), 125.9 (d), 125.6 (d), 124.0 (q), 122.5 (d), 122.3 (q), 120.9 (q), 20.5 (q); HRMS (EI, m/z) for C15H9F3N2O2S, calcd 338.0337, found 338.0334. Anal. Calcd for C15H9F3N2O2S: C, 53.25; H, 2.68; N, 8.28. Found; C, 53.23; H, 2.72; N, 8.16. 7-Trifluoromethyl-2-(3-methyl-4-nitrophenyl)benzothiazole (5y). Light yellow solid; yield 23 mg (7%); mp 120−122 °C; 1H NMR (CDCl3, 400 MHz) δ 8.26 (d, J = 8.0 Hz, 1H), 8.09−8.02 (m, 3H), 7.74 (d, J = 7.6 Hz, 1H), 7.64 (dt, J = 8.0 and 0.8 Hz, 1H), 2.70 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ 166.7 (s), 155.2 (s), 150.4 (s), 136.6 (s), 134.7 (s), 131.8 (s), 131.6 (d), 127.1 (d), 126.7 (d), 125.9 (d), 125.6 (d), 124.8 (q), 123.5 (q), 123.4 (q), 20.5 (q); HRMS (ESI, m/z) for C15H10F3N2O2S, calcd 339.0415, found 339.0417. Anal. Calcd for C15H9F3N2O2S: C, 53.25; H, 2.68; N, 8.28. Found; C, 53.26; H, 2.79; N, 8.13. General Procedure for the Syntheses of 2-(4-Aminophenyl)benzothiazoles 6. To a stirred solution of 2-(4-nitrophenyl)benzothiazole (5, 1 mmol) in CH2Cl2 was added 10 mol % of (10% Pd/C) in one portion, and the mixture was stirred under H2 atmosphere for 4 h at room temperature. The completion of reaction was monitored by TLC chromatography. After completion, the reaction mixture was filtered off through a Celite bed, and the filtrate was concentrated. The crude reaction mixture was subjected to flash column chromatography using CH2Cl2 as solvent to get corresponding compound 6. 4-(5,6-Dimethoxybenzo[d]thiazol-2-yl)aniline (6i).54 Yellow solid; 85% yield; mp 220−222 °C; 1H NMR (CDCl3, 400 MHz) δ 7.87 (d, J = 8.4 Hz, 2H), 7.56 (s, 1H), 7.26 (s, 1H), 6.73 (d, J = 8.4 Hz, 2H), 3.98 (s, 3H), 3.96 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 167.21, 165.5, 149.3, 148.9, 148.1, 128.7, 128.1, 125.3, 114.8, 104.6, 104.0, 102.5, 56.3, 56.1; HRMS (ESI, m/z) for C15H14N2O2S, calcd 286.0776, found 286.0778. 2-(4-aminophenyl)-6-fluorobenzothiazole (6j). Yellow solid; 83% yield; mp 201−202 °C; 1H NMR (CDCl3, 400 MHz) δ 7.93−7.89 (m, 1H), 7.85 (d, J = 8.4 Hz, 2H), 7.52 (dd, J = 8.1 and 2.4 Hz, IH), 7.20− 7.13 (m, IH), 6.72 (d, J = 8.4 Hz, 2H), 4.01 (brs, 2H, NH2); 13C NMR (100 MHz, CDCl3) δ 168.21 (d, J = 3.8 Hz), 160.06 (d, J = 242.9 Hz), 150.87 (d, J = 1.8 Hz), 149.26, 135.52 (d, J = 11.1 Hz), 129.01, 123.67, 123.22 (d, J = 9.3 Hz), 114.76, 114.46 (d, J = 24.0 Hz), 107.70 (d, J = 26.6 Hz); HRMS (ESI, m/z) for C13H10FN2S calcd 245.0470, found 245.0472. Anal. Calcd for C13H9FN2S: C, 63.92; H, 3.71; N, 11.47. Found: C, 63.82; H, 3.71; N, 11.44. 4-(6-Chlorobenzothiazol-2-yl)phenylamine (6k). Yellow solid; 93% yield; 1H NMR (CDCl3, 400 MHz) δ 7.88−7.81 (m, 4H), 7.38 (dd, J = 6.4 and 1.6 Hz, 1H), 6.73 (d, J = 8.4 Hz, 2H), 4.06 (brs, NH2, 2H); 13C NMR (100 MHz, CDCl3) δ 168.9, 152.8, 148.3, 135.7,

7.36 (t, J = 8.0 Hz, 1H), 7.02 (ddd, J = 8.0, 2.4, and 0.8 Hz, 1H), 3.88 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ 168.3 (s), 160.0 (s), 152.5 (s), 136.1 (s), 134.4 (s), 131.0 (s), 130.0 (d), 127.0 (d), 123.8 (d), 121.1 (d), 120.1 (d), 117.5 (d), 111.9 (d), 55.4 (q). 5,6-Dimethoxy-2-(4-nitro-phenyl)benzothiazole (5i). Yellow solid; 82% yield; mp 234−236 °C; 1H NMR (CDCl3, 400 MHz) δ 8.32 (d, J = 9.2 Hz, 2H), 8.18 (d, J = 9.2 Hz, 2H), 7.57 (s, 1H), 7.33 (s, 1H), 4.007 (s, 3H), 4.001 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ 162.7, 150.0, 149.5, 148.6, 148.5, 139.4, 127.9, 127.5, 124.3, 104.9, 102.2, 56.3, 56.1, 29.6; HRMS (ESI, m/z) for C15H12N2O4S, calcd 316.0518, found 316.0516. 6-Fluoro-2-(4-nitrophenyl)benzothiazole (5j). Yellow solid; yield 227 mg (83%); mp 191−193 °C; 1H NMR (CDCl3, 400 MHz) δ 8.35 (dt, J = 9.2 and 2.0 Hz, 2H), 8.23 (dt, J = 9.2 and 2.0 Hz, 2H), 8.07 (dd, J = 8.8 and 4.8 Hz, 1H), 7.63 (dd, J = 8.8 and 2.4 Hz, 1H), 7.29 (dt, J = 8.8 and 2.8 Hz, 1H), 7.93 (d, J = 8.4 Hz, 1H), 7.85 (d, J = 2.0 Hz, 1H), 7.61 (t, J = 2.2 Hz, 1H), 7.56 (ddd, J = 7.6, 1.2, and 0.8 Hz, 1H), 7.41 (dd, J = 8.8 and 2.4 Hz, 1H), 7.36 (t, J = 8.0 Hz, 1H), 7.02 (ddd, J = 8.0, 2.4, and 0.8 Hz, 1H), 3.88 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ 168.3 (s), 160.0 (s), 152.5 (s), 136.1 (s), 134.4 (s), 131.0 (s), 130.0 (d), 127.0 (d), 123.8 (d), 121.1 (d), 120.1 (d), 117.5 (d), 111.9 (d), 55.4 (q); HRMS (ESI, m/z) for C13H8N2O2SF, calcd 275.0291, found 275.0289. Anal. Calcd for C13H7FN2O2S: C, 56.93; H, 2.57; N, 10.21. Found: C, 56.98; H, 2.60; N, 10.31. 6-Chloro-2-(4-nitrophenyl)benzothiazole (5k). Yellow solid; yield 247 mg (85%); mp 203−205 °C; 1H NMR (CDCl3, 400 MHz) δ 8.36 (dt, J = 9.2 and 2.0 Hz, 2H), 8.25 (dt, J = 9.2 and 2.0 Hz, 2H), 8.04 (d, J = 8.8 Hz, 1H), 7.94 (d, J = 2.0 Hz, 1H), δ 7.52 (dd, J = 8.8 and 2.0 Hz, 1H); 13C NMR (CDCl3, 100 MHz) δ 165.3 (s), 152.6 (s), 149.1 (s), 138.7 (s), 136.6 (s), 132.3 (s), 128.2 (d), 127.8 (d), 124.6 (d), 124.4 (d), 121.4 (d); HRMS (ESI, m/z) for C13H8N2O2SCl, calcd 290.9995, found 290.9993. 6-Bromo-2-(4-nitrophenyl)benzothiazole (5l). Yellow solid; yield 217 mg (65%); mp 193−194 °C; 1H NMR (CDCl3, 400 MHz) δ 8.36 (dt, J = 9.2 and 2.0 Hz, 2H), 8.25 (dt, J = 9.2 and 2.0 Hz, 2H), 8.10 (d, J = 2.0 Hz, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.66 (dd, J = 8.4 and 2.0 Hz, 1H); 13C NMR (CDCl3, 100 MHz) δ 165.3 (s), 152.9 (s), 149.2 (s), 138.7 (s), 137.0 (s), 136.5 (s), 130.5 (d), 128.3 (d), 125.0 (d), 124.4 (d), 120.0 (s); HRMS (EI, m/z) for C13H8N2O2SBr, calcd 334.9490, found 334.9488. Anal. Calcd for C13H7BrN2O2S: C, 46.58; H, 2.11; N, 8.36. Found: C, 46.72; H, 2. 06; N, 8.38. 7-Trifluoromethyl-2-(4-nitrophenyl)benzothiazole (5n). Light yellow solid; yield 29 mg (9%); mp 156−158 °C; 1H NMR (CDCl3, 400 MHz) δ 8.38 (dt, J = 9.2 and 2.0 Hz,, 2H), 8.32−8.29 (m, 3H), 7.77 (d, J = 7.2 Hz, 1H), 7.67 (t, J = 7.6 Hz, 1H); 13C NMR (CDCl3, 100 MHz) δ 166.6 (s), 155.3 (s), 149.4 (s), 138.4 (s), 132.0 (s), 128.4 (d), 127.3 (d), 126.8 (d), 125.0 (q), 124.4 (d), 123.7 (q), 123.4 (q); HRMS (EI, m/z) for C14H7F3N2O2S, calcd 324.0175, found 324.0177. Anal. Calcd for C14H7F3N2O2S: C, 51.85; H, 2.18; N, 8.64. Found; C, 51.87; H, 2.19; N, 8.60. 5-Trifluoromethyl-2-(4-nitrophenyl)benzothiazole (5o). Yellow solid; yield 207 mg (64%); mp 213-215 °C; 1H NMR (CDCl3, 400 MHz) δ 8.39 (s, 1H),8.37 (dt, J = 9.2 and 2.0 Hz, 2H), 8.28 (dt, J = 9.2 and 2.0 Hz, 2H), 8.08 (d, J = 8.4 Hz, 1H), 7.69 (dd, J = 8.4 and 1.0 Hz, 1H); 13C NMR (CDCl3, 100 MHz) δ 166.9 (s), 153.6 (s), 149.3 (s), 138.7 (s), 138.4 (s), 129.6 (q), 128.4 (d), 124.4 (d), 124.0 (q), 122.5 (d), 122.5 (q), 121.1 (q); HRMS (EI, m/z) for C14H7F3N2O2S, calcd 324.0175, found 324.0177. Anal. Calcd for C14H7F3N2O2S: C, 51.85; H, 2.18; N, 8.64. Found; C, 51.45; H, 2.32; N, 8.48. 6-Fluoro-2-(3-methyl-4-nitrophenyl)benzothiazole (5t). Light yellow solid; yield 244 mg (78%); mp 164−166 °C; 1H NMR (CDCl3, 400 MHz) δ 8.09 (d, 8.4 Hz, 1H), 8.05 (dd, J = 9.2 and 4.8 Hz, 1H), 8.05 (s, 1H), 7.98 (dd, J = 8.4 and 2.0 Hz, 1H), 7.62 (dd, J = 8.0 and 2.8 Hz, 1H), 7.28 (dt, J = 9.2 and 2.8 Hz, 1H), 2.71 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ 164.8 (s), 160.9 (d), 150.7 (s), 150.1 (s), 137.2 (s), 136.4 (d), 134.7 (s), 131.3 (d), 125.8 (d), 125.6 (d), 124.8 (d), 115.7 (d), 108.0 (d), 20.6 (q); HRMS (ESI, m/z) for C14H10N2O2SF, calcd 289.0447, found 289.0446. 6-Chloro-2-(3-methyl-4-nitrophenyl)benzothiazole (5u). Yellow solid; yield 255 mg (84%); mp 171−173 °C; 1H NMR (CDCl3, 400 1189

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Table 1. Synthesis of 2-Arylbenzothiazoles via CAN Mediated Intramolecular C−S Bond Formation Followed by Reduction to Amine Derivativesa

Reaction conditions for step 1: 4 (1 mmol), CAN (4.2 equiv), NaHCO3 (6.0 equiv), CH3CN/H20(10:1), 0 °C, 10 min. Reaction conditions for step 2: 5 (1 mmol), 10% Pd/C (10 mol %), CH2Cl2 (5 mL), H2 balloon, 4 h at rt. a

130.1, 129.2, 126.8, 123.5, 123.1, 121.0, 114.8; HRMS (ESI, m/z) for C13H10ClN2S calcd 260.0175, found 260.0173. 4-(6-Bromo-benzothiazol-2-yl)-phenylamine (6l). Yellow solid; 65% yield; mp 219−220 °C; 1H NMR (CDCl3, 400 MHz) δ 7.98 (d, J = 2.0 Hz, 1H), 7.84 (d, J = 8.4 Hz, 2H), 7.81 (d, J = 8.8 Hz, 1H), 7.54 (dd, J = 8.8 and 2.0 Hz), 7.76 (d, J = 8.8 Hz, 1H); 3C NMR (100 MHz, CDCl3) δ 169.5, 152.6, 150.1, 135.7, 129.3, 128.9, 128.4, 123.7, 122.9, 122.3, 122.2, 117.5, 114.4; HRMS (ESI, m/z) for C13H10BrN2S calcd 304.9670, found 304.9668. Anal. Calcd for C13H9BrN2S: C, 51.16; H, 2.97; N, 9.18. Found: C, 51.04; H, 3.03; N, 9.03. 4-(5-Trifluoromethyl-benzothiazol-2-yl)-phenylamine (6o). Yellow solid; 70% yield; 1H NMR (CDCl3, 400 MHz) δ 8.23 (s, 1H), 7.93 (d, J = 8.4 Hz, 1H), 7.89 (d, J = 8.8 Hz, 2H), 7.54 (d, J = 8.4 Hz, 1H), 6.73 (d, J = 8.8 Hz, 2H); 13C NMR (CDCl3, 100 MHz) δ 170.5

153.9 149.7 138.0, 129.3, 122.9, 120.9, 120.7, 120.6, 119.5, 114.7; HRMS (ESI, m/z) for C14H9F3N2S calcd 294.0433, found 294.0436. 2-(4-Amino-3-methylphenyl)-6-fluorobenzothiazole (6t). Yellow solid; 78% yield; mp 203−205 °C; 1H NMR (DMSO-d6) δ 7.88 (dd, J = 5.9 Hz, 1H), 7.78 (d, J = 1.25 Hz, 1H), 7.70 (dd, J = 8.25 and 1.25 Hz, 1H), 7.51 (dd, J = 8.25 and 2.5 Hz, 1H), 7.16 (dd, J = 9.0 and 2.5 Hz, 1H), 6.71 (d, J = 8.25 Hz, 1H), 3.95 (brs, NH2, 2H), 2.34 (s, 3H); HRMS (ESI, m/z) for C14H12FN2S calcd 259.0627, found 259.0625. Anal. Cald for C14H11FN2S: C, 65.12; H, 4.26; N, 10.85. Found C, 64.81; H, 4.23; N, 10.67. 4-(6-Chloro-benzothiazol-2-yl)-2-methyl-phenylamine (6u). Yellow solid; 91% yield; 1H NMR (CDCl3, 400 MHz) δ 7.87 (d, J = 6.8 Hz, 1H), 7.79 (dd, J = 3.6 and 2.4 Hz, 2H), 7.72 (dd, J = 6.0 and 2.4 Hz, 1H), 7.38 (d, J = 6.4 and 2.0 Hz, 1H), 3.97 (brs. NH2, 2H), 2.24 1190

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(s, 3H); 13C NMR (CDCl3, 100 MHz) δ 169.2, 152.8, 147.8, 135.8, 130.0, 129.7, 126.9, 126.7, 123.4, 123.0, 122.1, 120.9, 114.5, 17.2; HRMS (ESI, m/z) for C14H12ClN2S calcd 274.0331, found 274.0333. Experimental Protocol for Biological Studies. Cell Culture. Two melanoma cell lines, A375 (human) and B16F10 (murine), were purchased from American Type Culture Collection (Manassas, VA), and the methods used were as described previously.24 UVA Irradiation and Cell Viability. The protocols used were as described previously.25 Fluorescence Measurement of Uptake of 6l. Cultured A375 cells were seeded on glass coverslips with a density of 2 × 104 cells/ well in 24-well plate for 24 h until cell attachment. Then the cells were exposed to 6l at 5 μM for indicated times in the dark. The cells were washed twice with PBS and were then fixed with 4% paraformaldehyde at 4 °C for 30 min. The qualitative expression of cell fluorescence was determined using a Leica inverted microscope (Leica DMI6000, Wetzlar Germany). Morphological Observation. A375 cells (5 × 105 cells/well) were seeded in a 6 well plate. The procedure used was as reported previously.14 Determination of Intracellular ROS Level. The methods used were as reported previously.14 Mitochondrial Membrane Potential Assessment (ΔΨmt). A375 cells were used, and the methods utilized were as described previously.14 ATP Content by Bioluminescence Assay. The methods used were as reported previously.14 Quantitative RT-PCR. The methods used were as reported previously.25 The primers used were as follows: mtDNA 4977-bp deletion, forward 5′-ACTACGGTCAATGCTCTG-3′ and reverse, 5′-GGAGGTTGAAGTGAGAGGTATG-3′; GAPDH, forward 5′-CCTCAACTACATGGTTTACATGTTCC-3′ and reverse 5′-ATGGGATTTCCATTGATGACAAG-3′. Transmission Electron Microscopy (TEM). Cells after 6l plus UVA treatment were cultured in a 37 °C incubator for 24 h, harvested, and fixed with 2% paraformadehyde and 2.5% glutaraldehyde for 2 h at 4 °C. Then, cells were washed with PBS, and postfixed in 2% OsO4 for 1 h at 4 °C. Cells were dehydrated with different concentrations of ethanol and propylene oxide and embedded in Epikote. Ultrathin sections were counterstained with uranyl acetate and lead citrate before observation with JEM-2000EXII (JEOL Ltd.). Caspase-3 Colorimetric Assay. The methods used were as reported previously.14 Protein Extraction and Western Blot Analysis. Rabbit polyclonal antibody against poly(ADP-ribose)polymerase (PARP; H250) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse monoclonal antibodies against NDUFS3 and actin were purchased from Novus Biologicals (Littleton, CO, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. Mouse polyclonal antibody against UQCRC2 was purchased from Novus Biologicals (Littleton, CO, USA). Rabbit monoclonal antibody against COX II was purchased from Abcam (Burlingame, CA, USA). The methods used were as reported previously.25 Immunocytochemical Staining. To clarify the role of the tumor apoptosis marker M30 in A375 cells, the mitochondrial marker COX IV, and cytochrome c in B16 cells treated with 6l-PDT their expression was correlated with apoptosis. Cells were seeded on glass coverslips at a density of 1 × 104 cells and were incubated overnight. The cells were treated with different concentrations of compound 6l for 4 h followed by 1 J/cm2 UVA irradiation. Immunocytochemistry was performed on all test samples. Cells were washed several times with PBS, fixed with 4% formaldehyde for 5 min at 4 °C, and then permeabilized with 0.5% Triton X-100 for 5 min. Nonspecific binding was blocked with 5% bovine serum albumin at 37 °C for 45 min. Cells were incubated for 1 h with primary antibodies (mouse anti-M30 at 1:250, rabbit anti-COX IV, and mouse anti-cytochrome c at 1:1000) at RT. Subsequently, slides were incubated with secondary antibodies for 30 min at RT. Finally, about 40 μL of mounting medium containing DAPI was added

to the slides, and the slides were covered with glass coverslips then sealed with nail polish. Animal Experiment. Seven-week-old female ICR strain mice were obtained and maintained as described in our previous study.25 A total of 5 × 106 B16 cells were inoculated into female ICR mice (about 19− 21 g, 7 weeks). The subcutaneous inoculation of tumor cells resulted in tumor generation at the injection site. When tumors reached about 4 × 4 mm2 in diameter, mice were separated into groups. Each group had four mice in each experiment; 4 mg/kg of compound 6l was injected into the tumor site, and then tumor was exposed to different doses of UVA on the day after injection. Tumor volume was measured by calipers every 5 days after agent injection, and tumor volume was calculated by following formula: tumor volume = 1/2 × length × width2. Statistical Analysis. The methods used were as reported previously.24

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RESULTS AND DISCUSSION Chemistry. In general, oxidative cyclization is an efficient approach for the synthesis of benzothiazoles from thiobenza-

Table 2. UV−Visible Absorption Data for Representative Compounds 6

a

compda

λabs, nm

εmax, M−1 cm−1

log εmax

6j 6k 6l 6m 6t 6u 6v 6w

359 357 358 365 352 360 361 367

21480.0 37520.0 39860.0 34250.0 34620.0 35230.0 25730.0 33040.0

4.33 4.57 4.60 4.53 4.54 4.54 4.41 4.51

All compounds were measured at 10 μM using DMSO as solvent.

Table 3. Effect of Compounds plus UVA on A375 Cell Viabilitya compounds + UVA UVA alone 6d 6g 6h 6i 6j 6k 6l 6m 6o 6p 6q 6r 6t 6u 6v 6w 6x

survival (%) 100 76.36 91.81 56.44 99.02 106.02 50.57 15.41 34.91 72.4 94.25 65.09 79.28 100.44 116.17 94.95 95.92 78.77

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.11 0.03 0.04 0.02 0.06 0.36 0.61 0.12 0.74 0.11 0.08 0.03 0.09 0.16 0.28 0.56 0.08 0.02

Cells were cultured with the agents at 5 μM for 4 h before 1 J/cm2 UVA irradiation. Forty-eight hours after irradiation, cell survival was assessed using the MTT assay. The data are expressed as the mean ± SD. a

nilides by using various oxidants.26 Meanwhile, Pd-catalyzed C−S bond formation from thioamides via C−H functionalization has also been realized.27,28 Alternatively, cyclization of 21191

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Figure 3. Uptake of 6l in A375 cells. Cells were treated with 6l at 5 μM for the indicated times in the absence of UVA by immunofluorescence assay. Similar results were obtained in three independent experiments.

Figure 2. Effect of compounds with or without UVA on cell viability. (A) Compounds 6j−m and 6t−w at a concentration of 5 μM were added to the culture media and incubated for 4 h before sham irradiation or irradiated with 1 J/cm2 UVA was applied to the cells. Forty-eight hours after exposure, the MTT reagent was added; the number of living cells is directly proportional to absorbance. (B) Morphological observation was used for the detection of cell viability. Similar results were obtained in three independent experiments.

benzenethiols, we assume that thiobenzamides would undergo intramolecular cyclization faster than intermolecular disulfur bond formation. Herein, we wish to report a novel CANpromoted intramolecular cyclization of 2-substituted benzothiazoles under relatively mild conditions and short reaction times (10 min) (Scheme 1). A similar approach was reported by Jackson and co-workers42 for the synthesis of benzothiazoles using ceric ammonium nitrate in aqueous acetonitrile, but the yields were found to be very poor with benzamides as a major byproduct. As shown in Scheme 1, the benzamides 3 and thiobenzamides 4 were prepared according to our previously reported protocol.14 For complete optimization of reaction conditions to form C−S bonds via oxidative cyclization, see Supporting Information, Table S1. With the optimized conditions in hand, we next explored the scope of the reaction under the best conditions found for 5d (Supporting Information, Table S1). Thus, a variety of substituted N-phenyl-thiobenzamides were prepared for studies of the intramolecular cyclization reaction, and the results are shown in Table 1. The reaction of CAN with the unsubstituted N-phenyl-thiobenzamide produced the desired benzothiazole 5a in good yield. The introduction of electron-withdrawing substituents, like halides and CF3, on the N-phenyl group gave the corresponding products 5j−m and 5p in 65−87% yields. Electron-donating groups, such as Et and OMe, at the para position afforded 93% and 89% yields, respectively (5e,f). Substitution at the meta position resulted in regioisomeric products in a 9:1 ratio, 5g,h. Disubstituted compounds also underwent smooth reaction to achieve the desired compound 5i. Placing methyl or methoxyl groups in the

halophenylthiobenzamides using Pd or Cu as a catalyst provided another approach to benzothiazoles.29−31 However, prefunctionalization of the starting materials in this protocol limited its application. It is of no doubt that the direct oxidative intramolecular C−S bond formation via C−H functionalization would be an attractive approach to synthesize benzothiazoles. 2-Arylbenzothiazoles have also been synthesized by the oxidative reaction of benzenethiols and benzonitriles using cerium(IV) ammonium nitrate (CAN).32 Unfortunately, the reported synthesis of 2-bromophenylthioformamides mediated by CAN is not reproducible. The only products generated are bis(p-tolyl) disulfide and p-tolyl p-toluenethiosulfonate, while benzonitriles were quantitatively recovered after the reaction.33 It was suggested that the reaction likely proceeds through the oxidation of thiol to sulfinyl radical, which rapidly leads to disulfide compounds. CAN is the most notable oxidant among lanthanide reagents for a variety of reactions, such as carbon−heteroatom bond forming reactions, due to its relative abundance, ease of preparation, low cost, and low toxicity.34−37 As part of our program in benzothiazole38 and other heterocyclic derivative synthesis,25,39−41 we became interested as to whether CAN could be applied to the formation of benzothiazoles via oxidative cyclization of N-phenyl-thiobenzamides. Since the oxidative state of sulfur in thiobenzamides is higher than that in 1192

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Figure 4. A375 cells were treated with the agents 6j−m at 5 μM (A) or treated with various concentrations of 6l (B) for 4 h before UVA irradiation and stained with DCFH-DA and analyzed immediately by flow cytometry. Similar results were obtained in three independent experiments.

meta position of thiobenzamides gave 71−91% yield of 5b,c and 5q−w. In the case of cyclization of N-(5-trifluoromethylphenyl)-thiobenzamides, a mixture of the 5- (5o) and 7substituted nitrobenzothiazoles (5n) was formed in a ratio of 7:1; similarly, from N-(5-trifluoromethylphenyl)-3-methoxyl-4nitro-thiobenzamides, a mixture of the 5- (5x) and 7substituted nitrobenzothiazoles (5y) was formed in a ratio of 9:1. It is worth noting that 2-arylbenzothiazoles bearing the nitro group on the aryl ring are obtained in high yields by this method, in contrast to low yields and harsh conditions with Jackson methodology.42 From our previous observations and results, some of the representative benzothiazole scaffolds were taken for reduction according to the previous report from our research group,14 and all the compounds underwent smooth reduction under 10% palladium on carbon using CH2Cl2 as solvent in good to excellent yield of compounds 6 irrespective of electronic and steric factors (Table 1). These synthesized compounds were further evaluated for their PDT efficacy in human melanoma cell lines (A375). We have measured the UV−visible absorption spectrum for some of the representative compounds 6, and data are shown in Table 2. The determination of wavelength and molar

absorption coefficient are important because longer wavelengths and higher molar absorptivity are necessary criteria for photosensitizers. Biology. Cell Viability. UVA-activated effects on A375 cells were evaluated for 17 compounds by the MTT assay. A375 cells were treated with or without 5 μM of compounds for 4 h before 1 J/cm2 UVA irradiation. Forty-eight hours after irradiation, cell viability was measured (Table 3). The results obtained from Table 3 show that compounds with halogen substitution exhibited higher inhibitory activity than nonhalogenated compounds. In this work, based on the results obtained we have compared the inhibitory effect of benzothiazole derivatives 6j−m and 6t−w with or without substitution on the 3′ position. As presented in Figure 2A, our data demonstrate that most of the UVA-activated agents show a higher inhibitory activity than either UVA irradiation alone or treatment with exogenous agent alone. The inhibitory activity of the agent plus UVA on A375 cells was also confirmed by a morphological observation (Figure 2B). The compounds 6j−m without substituent on the 3′ position exhibited a higher inhibitory activity compared with agents 6t−w with substituent on the 3′ position. The probable rationale for these observation could be the higher molar absorption coefficient for 1193

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Figure 6. Effect of 6l plus UVA on cell apoptosis. (A) Caspase-3 in apoptotic cells was determined by colorimetric reaction. (B) After exposure to 6l at different concentrations plus UVA, cell extracts were prepared, and PARP cleavage was analyzed by Western blot analysis. The intact PARP is 116 kDa, and the apoptotic fragment is 85 kDa. For an internal control, the same amounts of protein extract were also probed with antibody against actin. (C) Immunofluorescent staining of M30 in the cytoplasm of A375 cells. Cells were immunostained to detect apoptotic cells, which were stained green with the use of the M30 antibody. Nuclei were stained with DAPI (blue). **p < 0.01 compared with the UVA control group.

compounds 6k−m. Less inhibitory activity of methyl on the 3′ position of compound 6 could be due to formation of a radical under photo-irradiation and another transformation. Hence compounds 6j−m were selected as a model for further studies. Intracellular Accumulation of 6l in A375 Cells. The uptake of 6l in A375 cells was determined by using its fluorescence properties. After being incubated for 0−24 h, the cells showed a significant level of fluorescence (6l uptake) at 4 h, but the fluorescence level decreased afterward (Figure 3). Thus, cells were pretreated with an agent for 4 h followed by UVA irradiation for further mechanistic studies. ROS Generation. The prevailing mechanism for the action of PDT is the local generation of cytotoxic ROS, which produce cell damage and subsequently may lead to cell death by apoptosis or necrosis.43 To determine whether ROS were involved in cell apoptosis, we used UVA-activated agents 6j−m at 5 μM or various concentrations of 6l and measured the

Figure 5. Effect of 6l plus UVA on mitochondrial function. (A) The ΔΨmt of A375 cells after exposure to 6l plus UVA. Cells were treated with 6l at various concentrations for 4 h followed by UVA irradiation (1 J/cm2) and stained with DiOC6, then analyzed by flow cytometry immediately. (B) Immunoblot analysis showed the effect of 6l plus UVA on the protein expression of mitochondrial respiratory enzymes in A375 cells. (C) The mtDNA 4977-bp deletion level in different groups was measured by QPCR. (D) Relative ATP levels were calculated as a percentage of the control group (0 μM and 0 J/cm2) level. (E) Transmission electron micrographs of mitochondria of A375 cells after 24 h incubation with or without 6l, UVA, or 6l plus UVA.*p < 0.05 and **p < 0.01 compared with the control. Similar results were obtained in three independent experiments. 1194

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Figure 7. Effect of 6l plus UVA on cytochrome c release and tumor volume of B16 cells. (A) Immunofluorescent staining with DAPI (nuclei, blue) and the distribution of COX IV (mitochondrial marker, red) and cytochrome c (green) in B16 cells. (B) Tumor was established by injection of 5 × 106 cells/mL of B16 into each sole of feet of the female ICR mice.

determine the expression of the subunits of the mitochondrial oxidative phosphorylation system (OXPHOS). Western blot data showed that expression of NDUFS3 of complex I, UQCRC2 of complex III, and COX II of complex IV was decreased in A375 cells after 6l plus UVA treatment in a dosedependent manner (Figure 5B). Mitochondrial DNA 4977-bp deletion has been implicated in many human diseases and various cancer types and has been used as a mtDNA damage biomarker.45−47 Therefore, the mtDNA 4977-bp deletion level was measured by real-time quantitative PCR. As shown in Figure 5C, comparatively higher levels of 4977-bp deletion were found in A375 cells treated with concentrations higher than 1 μM of 6l plus UVA. Since mitochondrial oxidative phosphorylation is the major ATP synthetic pathway in eukaryotes, we elucidate whether decreased intracellular ATP is due to 6l plus UVA treatment.14 The intracellular ATP contents of A375 cells were decreased by about 20%, 25%, and 32% after 1, 3, and 5 μM, respectively, 6l plus UVA treatment compared with the control group (Figure 5D). Moreover,

production of intracellular H2O2 using the dichloro-dihydrofluorescein diacetate (DCFH-DA) probe. As shown in Figure 4A, compound 6l exhibited higher H2O2 levels compared with those of other agents after UVA irradiation. Hence, 6l was selected as a model for further studies. In addition, the increased H2O2 after treatment with 6l plus UVA exhibited dose-dependence, as compared with the control group (Figure 4B). Mitochondrial Dysfunction. Mitochondrial membrane potential (ΔΨmt) is a vital factor for cellular status. The decline in ΔΨmt is an early event in the process of cell death.44 Therefore; we investigated whether ΔΨmt disruption was involved in PDT-induced apoptosis. A375 cells were treated with 6l at various concentrations for 4 h before receiving UVA irradiation and then analyzed by flow cytometry after DiOC6 dye labeling. As demonstrated in Figure 5A, the ΔΨmt showed no obvious change after UVA irradiation alone, but 6l plus UVA treatment induced a significant decline of ΔΨmt in A375 cells. Another assessment of mitochondrial function was to 1195

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Figure 8. Mechanism for 6l plus UVA leading to apoptotic cell death via mitochondrial dysfunction.

transmission electron microscopy (TEM) observation showed major ultrastructural alterations of the mitochondria after 6l plus UVA treatment (Figure 5E). Apoptosis Detection. The most important cell modulator of apoptosis was found to be caspase-3,48,49 and its activity was determined using colorimetric assay. The caspase-3 level in A375 cells increased about 13%, 32%, and 56% after 1, 3, and 5 μM 6l plus UVA, respectively, compared with that of UVA group (Figure 6A). Poly(ADP-ribose)polymerase (PARP) has been identified as a substrate for caspase-3. Data from Western blot experiments showed that degradation of PARP was observed when A375 cells were exposed to 6l plus UVA compared with the UVA group (Figure 6B). Moreover, one important caspase substrate is cytokeratin 18; caspase-mediated cytokeratin cleavage was assessed by M30 antibody staining.50 As demonstrated in Figure 6C, the number of M30-positive cells was significantly increased by 6l plus UVA in a concentration-dependent manner. Antitumor Activity of 6l Plus UVA in Tumor-Bearing Mice. In order to evaluate the effect of 6l plus UVA on B16 cell lines, we have performed in vitro and in vivo antitumor activity assays of 6l plus UVA (Figure 7). The release of cytochrome c to cytosol shows one more consequence of the mitochondrial permeability transitions. The activation of caspases 3 and 9 has been shown to be initiated by cytosolic cytochrome c (Figure 7A).51,52 These cysteine-aspartate specific proteases play key roles in the downstream events associated with the apoptosis cascade. To address whether PDT using compound 6l has a better effect, we injected mouse melanoma B16 cells into the footpad of the ICR mice. UVA at various doses and in combination with compound 6l was delivered to the mice. We then observed the tumor sizes sequentially. We found that UVA alone did not inhibit tumor growth. However, when compound 6l was used with UVA at 0.5 and 0.75 J/cm2, it induced significant tumor reduction, indicating the usefulness of this novel photosensitizer, compound 6l, in the application of PDT (Figure 7B).

are the major cytotoxic products responsible for PDT-induced cellular damage and death53 Our data indicated that compound 6 plus UVA promotes H2O2 generation. Since mitochondria are known to be a significant source for ROS, mitochondrial function is worth investigating. Our data showed that treatment of A375 cells with 6l plus UVA resulted in a decrease in ΔΨmt, OXPHOS subunits, and ATP and an increase in mtDNA 4977bp deletion via ROS generation. TEM observations also showed major ultrastructural alterations of the mitochondria. Taken together, our studies indicate that 6l plus UVA induces A375 apoptosis through mitochondrial dysfunction, leading to caspase-3 activity, PARP cleavage, M30 positive CytoDeath staining, and consequent apoptotic cell death as shown in Figure 8. Moreover, 6l plus UVA could induce cytochrome c release in B16 cells and also reduce murine melanoma size in the mouse model. This study successfully demonstrated the in vitro and in vivo efficacy of PDT using compound 6l and UVA in the B16 melanoma cell tumorigenesis in ICR mice. Further experiments may be required to see whether the PDT has an in vivo effect on established metastasis and to determine its toxicity profile in vivo.

CONCLUSIONS We have previously reported 2-(4-aminophenyl)benzothiazole derivatives as a photosensitizers for PDT on BCC cells.14 In this study, we have demonstrated new synthetic development for the construction of benzothiazole analogues via oxidative cyclization and also provided evidence indicating that compounds containing halogen groups, under UVA light exposure, induced A375 melanoma cell apoptosis. We found that when cells were pretreated with agent for 4 h before 1 J/ cm2 UVA irradiation, compound 6 plus UVA exhibited a cytotoxic effect on A375 cells. Reactive oxygen species (ROS)

Funding

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ASSOCIATED CONTENT

S Supporting Information *

Spectral characterization of the reported compounds, 1H and 13 C NMR spectral data, and detailed biological experimental procedures. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*Dr. Wan-Ping Hu. Phone: (886)-7-312-1101, ext. 2683. Email: [email protected]. *Professor Jeh-Jeng Wang. Phone: (886)-7-312-1101 ext. 2275. Fax: (886)-7-312-5339. E-mail: [email protected]. Author Contributions

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▽

Y.-K.C. and G.C.S. contributed equally.

Financial support for this project was obtained from Ministry of Science and Technology (MOST), Taiwan. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS CAN, ceric ammonium nitrate; PDT, photodynamic therapy; UVA, ultraviolet radiation A; OXPHOS, oxidative phosphorylation; ROS, reactive oxygen species; ATP, adenosine triphosphate; TEM, transmission electron microscopy; PARP, 1196

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poly(ADP-ribose) polymerase; BCC, basal cell carcinoma; FDA, Food and Drug Administration

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