TW201217348A - Synthesis of 2-(4-aminophenyl)benzothiazole derivatives and use thereof - Google Patents

Abstract

The present invention relates to a method of preparing a compound of 2-(4-aminophenyl)benzothiazoles. The present invention also relates to a pharmaceutical composition for providing photodynamic therapy to a patient having at least one tumor to inhibit growth of the tumor.

Description

201217348 VI. Description of the invention: [Technical field to which the invention pertains] The present invention relates to a method for preparing a 2_(4·filament) shot. The invention also relates to a pharmaceutical composition for providing photodynamic therapy to a patient having at least one tumor to inhibit growth of the tumor. [Prior Art] Non-melanoma skin cancer (NMSC) is a growing problem in the world. Basal cell carcinoma (BCC) is the most common type of development in NMSC and most commonly occurs in white-skinned populations (Marks, R. Cancer 1995, 75, 607). Photodynamic therapy (pdt) is a non-invasive treatment with good cosmetic effects for the prevention and treatment of basal cell carcinoma cells (Gmpengiesser, S.; Ericson, Grade; Gudmundsson, F. (3⁄4. and; 7. Such as &gt;/· 2002, 27, 493), this therapy uses a combination of sensitizing agents and light (Fantasy, T.; Krammer, B.; Plaetzer, K. Curr. 尬 2006, 13, 2189). Photoactivation accumulates in the tumor and causes the production of reactive oxygen species (ROS) in the presence of oxygen molecules. This material eventually kills the target cells (D〇lmafls, DE; Fukumura, D.; Jain, RK Qm (10) 2003, 3 , 380). However, the published cure rates are not the same, and the photosensitizer and the light source that activates the photosensitizer, both of which are listed as limiting factors, promote the new photosensitizer. Studies 2. 2-Phenylbenzothiazoles (Figure 1) represent a powerful and selective new class of anti-tumor drugs (Bradshaw, TD; Wrigley, S.; Shi, DF.; 3 201217348 hultz, RJ} Pauli, KD; Stevens, MFG Br. J. Cancer 199 8 77 745). For example, in a project to design a tyrosine kinase inhibitor, 2_(4-aminophenyl)benzothiazole (2_(4_amin〇phenyl)) is a coffee (5) (Formula 6) , Scheme 1) was prepared as a synthetic intermediate by mimicking the structural comparison of flavonoid quercetin and isoflavones, which competed with ATP binding sites on tyrosine kinases (Stevens, MFG; McCall, CJ) Lelieveld, P.; Alexander, P.; Richter, A.; Davies, DEJ Med CTzem· 1994, 37, 1689). However, other biological properties may also be involved in this complex biological phenomenon, so further research is needed to resolve This problem may be harmful in the production of reactive oxygen species, and the active participation of mitochondria in the production of reactive oxygen species is well known and critically involved in the regulation of cell death pathways (Gogvadze; Zhivotovsky, BJ calendar owemftr 2007, 39, 23). Permeability of the outer membrane of the mitochondria and pro-apoptotic proteins released from the membrane mesenchyme are considered to be decisive events in the initiation and/or execution of cells/weeks (Crompton, M. Biochem J. 1999, 341, 233), this 'Current research indicates that reactive oxygen species play an important role in cell damage caused by uva (Valencia, A.; Kochevar, IE FreeiJWc. </ br> Med 2006, 40, 641) 'Therefore induced by activated UVA formula 6 Cell death may be associated with mitochondrial depolarization. The mitogen-activated protein prune (MAPK) family consists of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein, JNK and p38. The MAPK pathway is known to be activated by various environmental stresses and chemicals (Roulston, A.; Reinhard, C.; Amiri, P.; Williams, LT/. CTzew. 1998, 273, 10232), and ERK sequence (cascade) It is activated by mitotic stimulation and is important for proliferation and survival (Nagata, Y.; 201217348)

Todokoro, Κ·5/〇〇ίΠ999, 94, 853). However, ERK signaling has been thought to have an apoptosis-promoting effect during cell apoptosis (Xiao, D.; Singh, S. V. 2002, position 3615). SUMMARY OF THE INVENTION The object of the present invention is to use a newly synthesized compound of the formula 6 as a photosensitizer and to study the mechanism of apoptosis induced by 6-UVA on BCC cells. φ The present invention carries out the synthesis of a compound of the formula 6 (2-(4-aminophenyl)benzothiazole derivative) and a biological assay for use as a photosensitizer. The synthesis of the compound of formula 6 requires only four steps and the materials required are relatively inexpensive. Thus, a large scale synthesis of the compound of formula 6 can be achieved by the present invention. Because these compounds have a chromogenic structure and have light absorptivity in the ultraviolet A line (UVA) range (320-400 nm), the photosensitivity of uva-activated Formula 6 in BCC cells was analyzed in an in vitro (plus v) study. It can be speculated that UVA may cause side effects such as carcinogenesis or photoaging, but these side effects do not occur in the system of the present invention because the exposure time required for the activation of the formula 6 is very short 'no long-term exposure to UVA. problem. Unless otherwise defined, the terms used herein have their meaning as commonly understood by those of ordinary skill in the art. As used throughout this application, the following terms have the following meanings: The term "UVA" means ultraviolet radiation of relatively long wavelengths. The term "6-UVA" means a compound of formula 6 which is UVA activated. The term "6f-UVA" means a UVA activated compound of formula 6f. 201217348 Accordingly, the present invention provides a method of preparing a compound of formula 6

Containing: a. Compound of formula 1

NHz and compound of formula 2

Reacting to form a compound of formula 3 ^hVt&quot;2 k^N02 3 ; wherein X is Cl or OH of formula 2; b. treating compound of formula 3 with Lawesson's reagent to form compound 4 201217348

R2 N〇2 C. reacting a compound of formula 4 with potassium ferricyanide to produce a compound of formula 5

And d. catalytic reduction of the nitro group of the compound of formula 5 using a palladium on carbon catalyst (pauadium 〇ncharc〇al) to produce a compound of formula 6, wherein R1 in formulae 1 to 6 is fluorene, Cl-10, and Cl. 10 alkoxy or Cl 1 〇 基 ' and R 2 in the formula 1 to formula 6 is hydrazine or CM0 alkyl. • In a preferred embodiment, R1 in Equations 1 to 6 is Η, 6-Et, 6-OMe, 6-OCH3, 6-Me, 7-OMe or 6-CF3' and Equations 1 to 6 The r2 in is η or CH3. Preferably, when &amp; is H in Formulas 1 to 6, Formulas 1 to 6 are H, 6_Et, 6-OMe, 6-Me, 7-OMe or 6-CF3; When r2 in Formula 6 is CH3, the formulae in Formulae 1 to 6 are oxime, 6-OCH3, 6-Et or 6-CF3. More preferably, R! in Formula 1 to Formula 为 is 6-CF3' and R2 in Formulae 1 to 6 is Η. The invention also provides a pharmaceutical composition for providing photodynamic therapy to a patient having at least one tumor to inhibit the growth of the tumor, comprising a compound of formula 6 (wherein Ri is η, (^-10 201217348 alkyl, Cw oxygenated) Base or Cw draws a base!, and R2 is H or Ci 〇 〇; preferably, R! is Η, 6-Et, 6-OMe, 6-OCH3, 6-Me, 7-OMe or 6- CF3, and the formula 2 to the formula 2 is H or CH3; more preferably, when it is H, &amp; is H, 6 Et, 6 〇 Me, 6-Me, 7-〇Me or 6- CF3, when R2 is CH3, R is η, 6-OCH3, 6-Et or 6-CF3; more preferably, R1 is 6_Cf3 and &amp; is η). In a preferred embodiment, the medicine The composition comprises at least one compound of formula 6 or a pharmaceutically acceptable salt thereof. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier or diluent. In a preferred embodiment, the photodynamic therapy comprises the following steps : administering the composition to the patient; waiting for sufficient time for the administered composition to be absorbed by the target tissue having at least one tumor; and illuminating the patient with the region containing the target tissue. In a preferred embodiment The patient has skin cancer. Preferably, the patient has basal cell carcinoma. In a preferred embodiment, the compound of formula 6 is activated by UVA. Preferably, the growth of the tumor is by tumor cells. Apoptosis is inhibited. More preferably, the intracellular Η2 〇 2 level of the tumor cells rises, and the intracellular mitochondrial membrane potential (Δ Δ) of the tumor cells decreases. [Embodiment] The present invention may be in different forms. The examples are not limited to the examples mentioned below. The following examples are merely representative of the different aspects and features of the present invention. Example 1 Method and Materials 201217348 2-(4-Aminobenzene)benzothiazole (Formula 6) The preparation is shown in Figure 2. 4-Nitro-phenylbenzothiazole was obtained by reacting aniline ((10) mnes (Formula 1) with nitrochloride (Formula 2) in a bite for 4 hours (Method A). (4_Nitr〇_#_phenyl benzamides) (Formula 3). Alternatively, in the presence of sulfene gas in benzene, aniline (formula) is coupled with nitrobenzoic acid (Formula 2) under reflux conditions. High yield of benzamide (formula 3) (method B). 3) Treatment with a Lawson's reagent in a gas phase under reflux conditions to form 4-nitro-iV-phenylthiobenzamide (4_nitr〇-N_Phenylthi〇benzamide S) (Formula 4). The cyclization of Formula 4 is promoted by ferricyanide to form 2-(4-nitrophenyl) benzothiazoles (Formula 5) (Hutchinson, I.; Chua, MS; Browne, HL; Trapani, V.;

Bradshaw, TD; Westwell, AD; Stevens, MF G J. Med. Chem. 2001, #,1446) 'Next, the nitro group of formula 5 is catalytically reduced with a palladium on carbon catalyst in methanol, yielding a good yield target Compound 6. The obtained chemical reagent was not further purified and was distilled without solvent (s〇lvents free) φ before use. Thin layer chromatography was performed using a thin layer chromatography chip Merck plates 60 F254 to monitor the reaction&apos; rapid color layer analysis using a solvent indicated using Merck Silica Gel 60 (40-63 lm). The refining points were calibrated using a Fargo MP-2D melting point tester, and the 13C NMR spectra were recorded on a Varian UNITY plus-400 at 4 〇〇 MHz and 100 ί^, respectively, using CDCI3 as the solvent. The 1 NMR magnetic resonance shift is referenced to TMS or CDC13 (7.26 ppm) and the 13C NMR is referenced to CDCi3 (77.0 Ppm). Mass spectra were recorded on a Bruker APEX II mass spectrometer. The elemental analysis was performed with the elemental analyzer Elemental* vario EL III and the result would be ±0.4% theoretical

S 9 201217348 Fan Park. The purity of the compound tested was &gt; 95%. General procedure for the synthesis of benzamide (3a-3f) (Method a) In a stirred solution of stilbamide (225 mmol '1.2 eq.) in pyridine (45 Torr), 4-nitrobenzidine was added at room temperature under nitrogen. Chlorine (186 mm 〇 1), and the mixture was refluxed for 4 hours. After cooling to room temperature, the solution was poured into ice/water. The resulting precipitate was filtered and recrystallized from dioxane to give the corresponding compound 3. 4-Nitro-N-phenylbenzamide (3a). White solid; Yield 91%; mp 214-216 〇 C; NMR (CDC 13, 400 MHz) 59.42 (bs, NH), 8.38 (dt, J = 2.4 and 3.6 Hz, 2H), 8.23 (dt, J =2.4 and 3.6 Hz, 2H), 7.85-7.82 (m, 2H), 7.40-7.35 (m, 2H), 7.17-7.13 (m, 1H); 13C NMR (CDC13, 100 MHz) 5165.3, 151.2, 142.6, </ RTI> </ RTI> </ RTI> <RTIgt; N, 11.56. Found, C, 64.72; H, 4.25; N, 11.54. N-(4-methylphenyl)-4-nitrobenzamide (3b). White solid; Yield 93%; mp. 201-203°C; W NMR (CDC13, 400 ΜΗζ) δ9·37 (bs, NH), 8.26 (d, J = 8.8 Hz, 2H), 8.11 (d, J = 8.4 Hz, 2H), 7.58 (d, J = 8.0 Hz, 2H), 7.16 (d, J = 8.0 Hz, 2H), 2.34 (s, 3H); 13C NMR (CDC13, 100 MHz) 5163.8, 149.3, 140.7 , 135.3, 134.4, 129.3, 128.7, 123.4, 120.8, 20.8; HRMS (ESI, m/z) for Ci4Hi2N2〇3Na calcd 279.0746, found 279.0744; Anal, calcd for C14H12N2〇3: C, 65.62; H, 4.72; N , 10.93. Found C, 65.71; H, 5.00; N, 10.92. N-(4-ethylphenyl)-4-nitrobenzamide (3c). White solid; yield 96%; mp 186-188 ° C; NMR (CDCL 400 ΜΗζ) δ 8.32 (dd, J = 5.2 and 1.6 Hz, 201217348 2H), 8.03 (d, J = 8.4 Hz, 2H), 7.92 (bs, NH), 8.23 (d, J = 8.0 Hz, 2H), 7.22 (d, J = 8.4 Hz, 2H), 2.65 (q, J = 7.6Hz, 2H), 1.24 (t, J = 7.2 Hz, 3H); 13C NMR (CDC13, 100 MHz) 6163.6, 149.6, 141.5, 140.6, 134.8, 128.6, 128.2, 124.0, 120.6, 28.4, 15.6; HRMS (ESI, m/z) for C15H15N2O3 calcd 271.1083, found 271.1084 Anal, calcd for Ci5Hi4N2〇3: C, 66.66; H, 5.22; N, 10.36. Found C, 66.48; H, 5.41; N, 10.15. N-(3-methoxyphenyl)-4-nitrobenzene Formamide (3d). Yellow solid; Yield 80%; mp 158-160°C; W NMR (CDC13, 400 ΜΗζ) δ 9.98 (bs, NH), 8.31 to 8.28

(m, 2H), 8.18 (d, J = 8.8 Hz, 2H), 7.94 (s, 1H), 7.32-7.23 (m, 2H), 6.72-6.69 (m, 1H), 3.82 (s, 3H); 13C NMR (CDC13, 100 MHz) 6163.8, 159.5, 149.0, 140.5, 139.2, 129.1, 128.7, 123.0, 112.8, 109.9, 106.3, 54.9; HRMS (ESI, m/z) for Ci4Hi2N2〇4Na calcd 295.0695, found 295.0694; Anal. Calcd for C14H12N2O4: C, 61.76; H, 4.44; N, 10.29. Found C, 61.49; H, 4.59; N, 10.33. N-(4-methoxyphenyl)-4-nitrobenzamide (3e). Yellow solid; Yield 80%; mp 196-197 ° C; W NMR (CDC13, 400 ΜΗζ) δ 9.35 (bs, NH), 8.22 (d, J = 8.0 Hz, 2H), 8.07 (d, J = 8.0 Hz, 2H), 7.68 (d, J = 8.4 Hz, 1H), 6.95 (d, J = 8.4 Hz, 2H), 3.75 (s, 3H); 13C NMR (CDC13, 100 MHz) 5163.7, 156.6, 149.2 , 140.7, 130.9, 128.6, 123.3, 122.5, 113.9, 55.3; HRMS (ESI, m/z) for C14H13N2O4 calcl 273.0875, found 273.0873; Anal. Calcd for C14H12N2O4: C, 61.76; H, 4.44; N, 10.29. C, 61.88; H, 4.65; N, 10.14. N-(4-trifluoromethylphenyl)-4-nitrobenzamide (3f). White solid; yield 95%;

Melting point 194-196°C; iHNMR (DMSO, 400 ΜΗζ) δ 10.87 (bs, NH), 8.37 (d, J = 8.4 Hz, 2H), 8.14 (d, J = 8.4 Hz, 2H), 7.97 (d , J = 8.0 Hz, 2H), 7.70 (d, J = 8.4 Hz, 2H); 13C NMR (DMSO, 100 MHz) 6164.9, 149.6, 142.5, 140.3, 129.6, 126.3, 124.5 (q, J = 140 Hz, 1C), 123.9, 120.7, 114.1; HRMS (El,

S 11 201217348 m/z) for C14H9F3N2O3 calcd 310.0560, found 310.0563; Anal, calcd for C14H9F3N2O3: C, 54.20; H, 2.92; N, 9.03. Found C, 54.50; H, 2.84; N, 9.25. Synthetic benzamidine General procedure for the amine (3g-3j) (Method b) A stirred solution of 4-nitrobenzoic acid (82 mmol) in benzene (165 ml) at room temperature under nitrogen at room temperature with sulphur chloride (58 mL ' 820 mmol, 10 Equivalent) treatment, then the mixture was refluxed for 4 hours. After removal of the solvent, the phenylhydrazine chloride intermediate was slowly added to a solution of the appropriately substituted succinimide (123 mmol &lt; The mixture was refluxed at room temperature under nitrogen for 4 hr. After cooling to room temperature, the solution was poured into ice/water. The resulting precipitate was filtered and recrystallized from dichloromethane to give the corresponding compound 3. N-(phenyl)_3·methyl-based benzalkonium (3 g). White solid; Yield 82%; mp 149-151 ° C; NMR (CDC13, 400 ΜΗζ) δ 8.01 (d, J = 8.4 Hz, 1H), 7.98 (bs, NH), 7.84 (s, lH), 7.78 (d, J = 8.4 Hz, 1H), 7.63 (d, J = 7.6 Hz, 2H), 7.40-7.36 (m, 2H), 7.21-7.17(m, 1H), 2.64 (s, 3H); 13C NMR (CDC13, 100 MHz) 6163.9, 150.9, 138.8, 137.27, 134.2, 131.8, 139.2, 125.4, 125.3, 125.0, 120.4, 20.3; HRMS (ESI, m/z) for C14H13N2〇3 calcd 257.0926, found 257.0925; Anal , calcd for C14Hi2N2〇3: C, 65.62; H, 4.72; N, 10.93. Found C, 65.53; H, 4.60; N, 11.00. N-(4-Hexylphenyl)-3-methyl-4-nitro Benzobenzamide (3h&gt; white solid; yield 80%; mp 137-139°C; 4 NMR (CDC13, 400 ΜΗζ) δ 8.03 (bs, NH), 7.97 (d, J = 8.4 Hz, 1H), 7.82 (s, 1H), 7.75 (d. J = 8.4 Hz, 1H), 7.52 (d, J = 8.4 Hz, 2H), 7.29 (d, J = 8.0 Hz, 2H), 2.67-2.61 ( m, 5H), 1.24 (t, J = 8.4 Hz, 3H); 13C NMR (CDCI3, 100 MHz) 5163.8, 150.8, 141.4, 138.9, 134.9, 134.1, 131.8, 128.5, 125.4, 125.0, 120.6, 28.3, 20.3 , 15.6; HRMS (ESI, m/s) for C16H17N2O3 calcd 285.1239, found 285.1241; Anal, Calcd for 201217348

Ci6H16N2〇3: C, 67.59; H, 5.67; N, 9.85. Found C, 67.50; H, 5.45; N, 9.85. N-(4-methoxyphenyl)-3-methyl-4-nitrobenzene Formamide (3i). White solid; Yield 71%; mp 152-154 ° C; NMR (CDC13, 400 ΜΗζ) δ 7.98 (d, J = 8.4 Hz, 1H), 7.94 (bs, NH), 7.81 (s, 1H), 7.75 (d. J = 8.0 Hz, 1H), 7.52 (d, J = 8.8 Hz, 2H), 6.89 (d, J = 9.2 Hz, 2H), 3.81 (s, 3H), 2.62 (s, 3H); 13C NMR (CDC13, 100 MHz) 5163.7, 157.0, 150.8, 138.8, 134.1, 131.7, 130.2, 125.3, 124.9, 122.3, 114.3, 114.2, 55.4, 20.2; HRMS (ESI, m/z) for C15H15N2O4 calcd 287.1032, found Anal, calcd for C15H14N2O4: C, 62.93; H, 4.93; N, 9.79. Found C, 62.77; H, 5.17; N, 9.82. 3-Methyl-4-nitro-N-(4-trifluoro) Tolyl) benzamide (3j). White solid; Yield 75%; mp 154-156°C; NMR (CDC13, 400 ΜΗζ) δ 9.38 (bs, NH), 8.01 (d, J = 8.8 Hz, 1H), 7.92 (s, 1H), 7.87-7.83 (m, 2H), 7.61 (d, J = 8.4 Hz, 2H), 2.64 (s, 3H); 13C NMR (CDC13, 100 MHz) 5164.8, 150.8, 141.0,

138.4, 133.8, 132.1, 126.1 (q, J = 38Hz, CF3-C), 125.8, 124.7, 123.2 (q, J = 140Hz, CF3), 120.3, 120.2 20.0; HRMS (EI, m/z) for C15H„ N202F3 calcd 324.0722, found 324.0725; Anal, calcd for Ci5HuF3N2〇3: C, 55.56; H, 3.42; N, 8.64. Found, C, 55.50; H, 3.48; N, 8.64. Synthetic thiobenzamide (4a- 4j) General procedure 4-Vinyl-N-phenylbenzamide (41 mmol) and a mixture of Lawson's reagent (8.52 g, 21 mmol '0.51 equivalent) in chlorobenzene (30 ml) are heated under reflux 4 to After 6 hours, it was concentrated and purified by column chromatography (CHbCVhexane = 2:3) to give the corresponding compound 4. 4-nitro-N-phenylthiobenzamide (4a). Yellow solid; Yield 60 〇 / 〇; Happiness 154-156 ° C; !H NMR (CDC13, 400 MHz) 89.08 (bs, NH), 8.26 (d, J = 8 4

S 13 201217348

Hz, 2H), 7.95 (d, J = 8.4 Hz, 2H), 7.77 (d, J = 7.6 Hz, 2H), 7.47 (t, J = 8.0 Hz, 2H), 7.34 (t, J = 8.0 Hz, 1H); 13C NMR (CDC13, 100 MHz) 5195.5, 148.9, 148.2, 138.5, 129.2, 127.7, 127.5, 123.9, 123.5; HRMS (El, m/z) for C13H10N2O2S calcd 258.0463, found 258.0463; Anal, calcd for Ci3H10N2 〇2S, C, 60.45; H, 3.90; N, 10.85. Found C, 60.44; H, 4.06; N, 10.75. N-(4-methylphenyl)-4-nitrothiobenzamide (4b). Yellow solid; yield 60%; melting point 195-196 ° C; 4^^(000:13,400^02) 39.15 (bs, NH), 8.21 (d, J = 8.4 Hz, 2H), 7.91 (d, J = 8.8 Hz, 2H), 7.60 (d, J = 8.4 Hz, 2H), 7.26-7.23 (m, 2H), 2.38 (s, 3H); 13C NMR (CDC13, 100 MHz) 5195.27, 148.8, 148.0, 137.6, 135.9, 129.7, 127.7, 123.8, 123.6, 21.2; HRMS (ESI, m/z) for Ci4Hi2N2〇2SNa calcd 295.0517, found 295.0516; Anal, calcd for C14Hi2N2〇2S: C, 61.75; H, .4.44; N, 10.29 Found C, 61.81; H, 4.55; N, 10.29. N-(4-Ethylphenyl)-4-nitrothiobenzamide (4c). Yellow solid; yield 65%; mp 142-144 ° C; NMR (CDC13, 400 ΜΗζ) δ 9.07 (bs, NH), 8.25 (d, J = 8.4 Hz, 2H), 7.94 (d, J = 8.8 Hz, 2H), 7.66 (d, J = 8.4 Hz, 2H), 7.28 (d, J = 8.0 Hz, 2H), 2.69 (q, J = 8.0 Hz 2H), 1.27 (t, J = 7.6 Hz, 3H); 13C NMR (CDCI3, 100 MHz) 5195.2, 148.9, 148.2, 143.9, 136.1, 128.9, 128.6, 127.7, 123.9, 123.5, 28.5, 15.3; HRMS (ESI, m/z) for C15H15N2〇2S calcd 287.0854, Found 287.0856; Anal·calcd for C15H14N2〇2S: C, 62.92; H, .4.93; N, 9.78. Found C, 62.91; H, 4.95; N, 9.60. N-(3-methoxyphenyl)-4- Nitrothiobenzamide (4d). Yellow solid; yield 64%;

Melting point 138-140 ° C; 4 NMR (CDC13, 400 ΜΗζ) δ10·61 (bs, NH), 8.23 (d, J = 8.8 Hz, 2H), 7.96 (d, J = 8.8 Hz, 2H), 7.60 (s , 1H), 7.36-7.30 (m, 2H), 6.86-6.83 (m, 1H), 3.83 (s, 3H); 13C NMR (CDC13, 100 MHz) 6195.3, 159.8, 148.8, 148.2, 146.2, 129.6, 128.2 , 123.3, 115.8, 112.7, 109.2, 55.4; 201217348 HRMS (ESI, m/z) for Ci4H12N2〇3SNa calcd 311.0466, found 311.0464; Anal, calcd for C14H12N2O3S: C, 58.32; H, .4.20; N, 9.72. , C, 58.25; H, 4.36; N, 9.61. N-(4-methoxyphenyl)-4-nitrothiobenzamide (4e). Yellow solid; yield 62%; m.p. 174-175°C; 4 NMR (CDC13, 400 ΜΗζ) δ 9.98 (bs, :NH), 8.31-8.28 (m, 2H), 8.18 (d, J = 8.8 Hz , 2H), 7.94 (s, 1H), 7.32-7.23 (m, 2H), 6.72-6.69 (m, 1H), 3.82 (s, 3H); 13C NMR (CDC13) 100 MHz) 5163.8, 159.5, 149.0, 140.5, 139.2, 129.1, 128.7, 123.0, 112.8, 109.9, 106.3, 54.85; HRMS (El, m/z) for C14H12N2O3S calcd 288.0569, found 288.0571; Anal. Called cald'for Q4H12N2O3S: C, 58'32; H, .4.20; N, 9.72. 'Found C, 58.29; H, 4.42; N, 9.47. N-(4-Trifluoromethylphenyl)-4-nitrothiobenzamide (4f). Yellow solid; Yield 71%; m.p. 174- 175°C; b NMR (CDC13, 400 ΜΗζ) δ11.49 (bs, NH), 8.25 (d, J = 8.4 Hz, 2H), 8.07 (d, J = 8.8 Hz , 2H), 7.99 (d, J = 8.4 Hz, 2H), 7.68 (d, J = 8.0 Hz, 2H); 13C NMR (CDC13} 100 MHz) 6196.1, 148.5, 148.0, 142.4, 128.4, 125.8, 125.8, 123.5, 123.1, 122.3 (q, J = 140Hz, CF3); HRMS(ESI, m/z) for C14H10N2〇2F3S calcd 326.0337,found 326.0335. ^ Anal, calcd for C14H10N2O2F3S: C, 51.53; H, 2.78; N, 8.59. Found C, 51.90; H, 2.86; N, 8.63. N-(phenyl)-3-methyl_4-nitrothiobenzamide (4g). Yellow solid; Yield 72%; mp 118-120°C; W NMR (CDC13, 400 ΜΗζ) δ9·09 (bs, NH), 7.99 (d, J = 8.4 Hz, 1H), 7.78-7.71 (m, 4H), 7.46 (t, J = 8.0 Hz, 2H), 7.33 (t, J = 7.6 Hz, 1H), 2.64 (s, 3H); 13C NMR (CDC13, 100 MHz) 6195.7, 146.6, 138.5, 134.2, </ RTI> </ RTI> </ RTI> <RTIgt; Found C, 62.01; H, 4.39; N, 10.26. § 15 201217348 N-(4-Ethylphenyl)-3-methyl-4-nitrothiobenzamide (4h). Yellow solid; yield 70%; m.p. 160-162°C; iHNMR^CDCls/OOMHzMAtB (bs, NH), 7.99 (d, J = 8.4 Hz, 1H), 7.78-7.65 (m, 3H), 7.28 (d , J = 8.4 Hz, 2H), 2.72-2.64 (m, 5H), 1.26 (t, J = 7.6 Hz, 3H); 13C NMR (CDC13, 100 MHz) 5195.4, 150.1.146.6.143.8.136.1.134.2. </ RTI> </ RTI> <RTIgt; N, 9.33. Found C, 63.99; H, 5.39; N, 9.30. N-(4-methoxyphenyl)-3-methyl-4-jingylthiobenzamide (4i). Yellow solid; yield 61%; melting point 160-162° (:; 111)^^(€00:13,40〇]'^) 510.81 (^, _, 7.96 (d, J = 8.1 Hz, 1H), 7.83 (dd, J = 1.6 and 0.8 Hz, 1H), 7.77 (dd, J = 8.4 and 2.0 Hz, 1H), 7.69 (dt, J = 8.8 and 3.2 Hz, 2H), 6.94 (dt, J = 8.8 and 3.2 Hz, 2H), 3.83 (s, 3H), 2.63 (s, 3H); 13C NMR (CDC13, 100 MHz) 5195.0, 157.9, 149.5, 146.2, 133.3, 132.2, 131.7, 125.4, 124.3, 113.7, 55.2, 20.2; HRMS (ESI, m/z) for C15H15N2O3S calcd 303.0803, found 303.0804; Anal, calcd for C15H15N2O3S: C, 59.59; H, 4.67; N, 9.27. Found C, 59.95; H, 4.90; N, 9.07. -Methyl-4-nitro-N-(4-trifluoromethylphenyl)thiobenzamide (4j). Yellow solid; Yield 74%; m.p. 170-172 ° C; 1H NMR (CDC13, 400 ΜΗζ Δ8.04-7.98 (m, 3H), 7.81-7.68 (m, 4H), 2.65 (s, 3H); 13C NMR (CDC13, 100 MHz) 5208.5, 196.6, 149.8, 146.4, 142.2, 133.5, 131.5, 125.8, 125.3, 124.4, 123.5, 122.3 (q, J = 140Hz, CF3), 29.7, 20.0; HRMS (ESI, m/z) for C15H11N202SF3 calcd 340.0493, found 340.0494; Anal, calcd for C15H11N202SF3: C, 52.94; H , 3.26; N, 8.23. Found C, 52.68; H, 3.47; N, 8.27. General procedure for the synthesis of 2-(4-nitrophenyl)benzothiazole (5a-5j) 201217348 Substituted 4* nitrothiobenzamide ( 50 mmol) A solution of 3 drops of 95% EtOH and 30% aqueous sodium nitrite (1 〇 6 mL, 8 eq.) was added dropwise at 9 〇〇C to add potassium iron hydride (13.16 g, 4 eq.) to water ( 5 〇 mL) of the solution. The reaction mixture was heated for an additional 1 hour and then cooled in ice. The resulting precipitate was filtered and washed with water, followed by flash chromatography (CHzCyhexane = 1 : 4) to give the corresponding compound 5. 2-(4-Nitylphenyl)benzothiazole (5a). White solid; yield 68%; m.p.: 228-230 〇C; !H NMR (CDC13, 400 MHz) 58.35 (dt, J = 9.2 and 2.0 Hz, 2H), 8.27 (dt, J = 9.2 and 2.0 Hz, 2H), 8.13 (d, J = 8.0 Hz, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.56 (dt, J = 8.0 and 1.2 Hz, 1H), 7.47 (dt, J = 8.0 and 1.2 Hz, 1H); 13CNMR (CDC13, 100 MHz) 6164.8, 154.1, 149.0, 139.1, 135.4, 128.2, 126.9, 126.2, 124.3, 123.9, 121.8; HRMS (El, m/z) for Ci3H8N2〇2S calcd 256.0306, found 256.0308; Anal, calcd for Ci3H8N2〇2S: C, 60.93; H, 3.15; N, 10.93. Found C, 60.89; H, 3.34; N, 10.75. 6-methyl-2-(4-nitrophenyl)benzene And thiazole (5b). White solid; yield 65%; mp 148-150 〇C; lU NMR (CDC13, 400 MHz) 58.31 (s, 1H), 8.23 (s, 2H), 7.99 (d, J = 6.4 Hz, 1H), 7.72 (s, 1H), 7.36 (s, 1H), 2.52 (s, 3H); 13C NMR (CDC13j 100 MHz) 5163.7, 152.2, 148.8, 139.3, 136.6, 135.6, 128.5, 128.0, 124.2, 123.3, 121.4, 21.6; HRMS (EI, m/z) for C14H12N202S calcd 295.0517, found 295.0516; Anal, calcd for Ci4Hi〇N2〇2S: C, 62.21; H, 3.73; N, 10.36. Found, C, 62.37; H, 3.91; N, 10.31. 6-Ethyl-2-(4-nitrophenyl)benzothiazole (5c). White solid; Yield 72%; mp 151-152 〇 C; NMR (CDC13, 400 MHz) 68.34 (dd, J = .8 and 2.0 Hz, 2H), 8.25 (dd, J = 4.8 and 2.0 Hz, 2H), 8.02 (d, J = 8.4 Hz, 1H), 7.76 (d, J = 0.8

S 17 201217348

Hz, 1H), 7.40 (dd, J = 8.4 and 1.6 Hz, 1H), 2.28 (q, J = 7.6 Hz, 2H), 1.33 (t, J = 7.6 Hz, 3H); 13C NMR (CDC13) 100 MHz ) 5163.8, 152.4, 148.8, 143.0, 139.3, 135.7, 128.0, 127.6, 124.3, 123.5, 120.3, 29.0, 15.7; HRMS (ESI, m/z) for C15H12N202S calcd 285.0698, found 285.0695; Anal, calcd for Ci5H12N2〇2S : C, 63.36; H, 4.25; N, 9.85. Found C, 63.40; H, 4.55; N, 9.53. 7-Methoxy-2-(4-phenylene)benzothiazole (5d). White solid; Yield 25%; mp 228-230 ° C; NMR (CDC13, 400 ΜΗζ), δ 8.33 (d, J 8.8 Hz, 2H), 8.25 (d, J = 8.8 Hz, 2H), 7.75 -7.73 (m, 1H), 7.49 (t, J = 8.4 Hz, 1H), 6.89 (d, J = 8.4 Hz, 1H), 4.03 (s, 3H); , 3C NMR (CDC13, 100 MHz) 5165.4, 155.6, 154.3, 148.9, 139.2, 128.2, 127.8, 124.3, 116.4, 105.9, 56.0; HRMS (ESI, m/z) for Ci4Hi〇N2〇3SNa calcd 309.0310, found 309.0308; Anal, calcd for C14H10N2O3S: C, 58.73; H, 3.52; N, 9.78. Found C, 58.94; H, 3.70; N, 9.65. 6-decyloxy-2-(4-nitrophenyl)benzothiazole (5e). Yellow solid; yield 63%; melting point 214-216. . ; towel NMR (CDC13, 400 ΜΗζ) δ8.33 (dt, J = 9.2 and 2·0 Hz, 2H), 8.20 (dt, J = 9.2 and 2.0 Hz, 2H), 8.00 (d, J = 9.2 Hz, 1H), 7.38 (d, J = 2.4 Hz, 1H), 7.15 (dd, J= 9.2 and 2.4 Hz, 1H), 3.92(s, 3H); 13C NMR (CDCI3, 100 MHz) 5162.2, 158.5, 148.7, And calcd for C14H10N2O3S: C, 58.73; H, 3.52; N, 9.78. Found C, 58.68; H, 3.47; N, 9.75. 2-(4-Nitylphenyl)-6-trifluoromethylbenzothiazole (5f). Yellow solid; Yield 61%; mp 149-151 ° C; 4 NMR (CDC13, 400 MHz) δ 8.35 (dt, J = 9.2 and 2.0 Hz, 2H), 8.27-8.23 (m, 3H), 8.19 ( d, J = 8.4 Hz, 1H), 7.77 (dd, J = 8.4 and 2.0 Hz, 1H); 13C NMR (CDC13, 100 MHz) 5167.8, 155.8, 149.4, 138.3, 135.4, 128.4, 128.2, 124.3, 124.2, </ RTI> </ RTI> <RTIgt; N, 8.69. 2-(3-Methyl-4-nitrophenyl)benzothiazole (5 g). White solid; Yield 71%; mp 163-165 〇 C; * NMR (CDC13, 400 MHz) 58.13-8.08 (m, 3H), 8.02 (dd, J =

8.0 and 2.0 Hz, 1H), 7.95 (dd, J = 8.0 and 1.2 Hz, 1H), 7.55 (dt, J = 8.0 and 1.2 Hz, 1H), 7.45 (dt, J = 8.0 and 1.2 Hz, 1H), 2.71 (s, 3H); 13C NMR (CDCI3, 100 MHz) 5165.1, 153.9, 150.1, 137.4, 135.4, 134.7, 131.4, 126.8, 126.0, 125.8, 125.5, 123.7, 121.8, 20.6; HRMS (ESI, m/z For C14H11N2O2S calcd 271.0541, found 271.0541. Anal, calcd for Ci4HnN202S: C, 62.21; H, 3.73; N, 10.36. Found C, 62.07; H, 3.88; N, 10.26. 6-ethyl-2-(3- Methyl-4-nitrophenyl)benzothiazole (5h). Yellow solid; yield 75%; mp llS-Uirc/HNMR CCDCIsJOOMHzWS.HHcUsS.SHz, 1H), 8.08 (s, 1H), 8.02 (d, J = 8.4 Hz, 1H), 8.01 (dd, J = 8.4 and 2.0 Hz, 1H), 7.75 (d, J = 2.0 Hz, 1H), 7.38 (dd, J = 8.4 and 2.0 Hz, 1H), 2.83 (q, J = 7.6 Hz, 2H), 2.71 (s, 3H), 1.33 (t, J = 7.6 Hz, 3H); 13C NMR (CDC13, 100 MHz) 8164.1, 152.4, 149.9, 142.8, 137.7, 135.6, 134.6, 131.3, 127.4, 125.6, 125.5, 123.3, 120.3, 29.0, 20.6, 15.7; HRMS (ESI, m/z) for C16H15N2O2S calcd 299.0854, found 299.0856; Anal, calcd for Ci6H15N202S: C, 64.41; H, 4.73; N, 9.39. Found C, 64.20; H, 4.80; N, 9.14. -Methoxy-2-(3-methyl-4-nitrophenyl)benzothiazole (5i). Yellow solid; yield 64%; m.p. 195-197.: NMR (CDC13, 400 ΜΗζ) δ 8.07 (d, J = 8.8 Hz, 1H), 8.02 (d, J = 0.8 Hz, 1H), 7.98 (d , J = 9.2 Hz, 1H), 7.95 (dd, J = 8.4 and 2.0 Hz, 1H), 7.36 (d, J = 2.4 Hz, 1H), 7.14 (dd, J = 9.2 and 2.4 Hz, 1H), 3.90 (s, 3H), 2.71 (s, 3H); 13C NMR (CDC13, 100 MHz) 5162.3, 158.4, 201217348 149.6, 148.6, 137.6, 136.8, 134.6, 130.9, 125.5, 125.3, 124.3, 116.4, 103.9, 55.8, 20.6; HEMS (ESI, m/z) for C15H14N203S calcd 301.0647, found 301.0648. Anal, calcd for C15H14N203S: C, 59.93; H, 4.03; N, 9.33. Found C, 60.37 H, 4.13; N, 9.36. (3-Methyl-4-nitrophenyl)-6-trifluoromethylbenzothiazole (5j). Yellow solid; Yield 30%; mp 99-101 ° C; 4 NMR (CDC13, 400 ΜΗζ) δ8·21-8,15 (m,2H), 8.08-8.06 (m, 2H), 8.00 (dd, J = 8.8 and 1.6 Hz, 1H), 7.76 (dd, J = 8.8 and 1.6 Hz, 1H), 2.69 (s, 3H); 13C NMR (CDC13, 100 MHz) 5168.0, 155.7, 150.4, 136.6, 135.2134.6, 131.60, 127.9 (q, J = 32.6 Hz, 1C), 125.9 (d, J = 43.2 Hz, 1C), 125.49, 124.08, 123.8, 122.2, 119.4 (q, J = 4.5 Hz, 1C), 20.4; HRMS ( ESI, m/z) for C15H1()N202F3S calcd 339.0415, found 339.0414; Anal, calcd for C15H9N202F3S: C, 53.25; H, 2.68; N, 8.28. Found C, 53.21; H, 2.73; N, 8.14. -(4-Aminophenyl)benzothiazole (6a-6j) in a general procedure in 2-(4-aminophenyl)benzothiazole (lg, 3.9 mmol) in CH2C12 (30 mL) 10% Pd/C (O.lg) was added for 4 hours. The resulting solution was concentrated and subjected to flash chromatography (CH2C12) to give the corresponding compound 6. 2-(4-Aminophenyl)benzothiazole (6a). Yellow solid; Yield 94%; mp 130-132 ° C; lU NMR (CDC13, 400 MHz) 67.99 (m, 1H), 7.89 (m, 2H), 7.84 (m, 1H), 7.44 (m, 1H) , 7.32 (m, 1H), 6.72 (dt, J = 4.2 and 2.0 Hz, 2H), 4.00 (bs, NH2); 13C NMR (CDC13, 100 MHz) 6168.5, 154.2, 149.2, 134.6, 129.1, 126.0, 124.4 , H.sub.2, s. H, 4.69; N, 12.29. 2-(4-Aminophenyl)-6-methylbenzothiazole (6b). Yellow solid; yield 950/. ; melting point 201217348 181-183〇C; 'HNMR^DCla, 400 MHz) 57.88-7.85 (m, 3H), 7.63 (s, 1H), 7.25 (dd, J = 6.8 and 1.6 Hz, 1H), 6.71 (dd , J = 4.4 and 2.4 Hz, 1H), 7.32 (m, 1H), 6.72 (dt, J = 4.2 and 2.0 Hz, 2H), 4.00 (bs, NH2), 2.46 (s, 3H); 13C NMR (CDC13 , 100 MHz) 5167.5, 152.3, 149.0, 134.7, 134.5, 129.5, 129.0, 127.6, 126.3, 124.1, 122.0, 121.2, 114.8, 21.5; HRMS(ESI, m/z) for C14H13N2S calcd 241.0799, found 241.0798; Anal. Calcd for C14H12N2S: C, 69.97; H, 5.03; N, 11.66. Found C, 69.84; H, 5.01; N, 11.70. 2-(4-Aminophenyl)-6-ethylbenzothiazole (6c). Yellow solid; Yield 92%; mp 154-156 〇 C; *H NMR (CDC13, 400 MHz) 57.90-7.86 (m, 3H), 7.65 (t, J = 0.4 Hz, 1H), 7.27 (dd, J = 7.6 and 2.0 Hz, 1H), 6.81 (dt, J = 4.8 and 2.0 Hz, 2H), 3.98 (bs, NH2), 2.76 (q, J = 7.6 Hz, 2H), 1.29 (t, J = 7.6 Hz , 3H); 13C NMR (CDC13, 100 MHz) 6167.6, 152.5, 149.0, 141.0, 134.7, 129.0, 126.5, 124.1, 122.1, 114.7, 28.87, 15.8; HRMS (ESI, m/z) for Ci5H14N2OSNa calcd 277.0775, found 277.0776. Anal, calcd for Ci5Hi4N2S: C, 70.83; H, 5.55; H 11.01. Found C, 70.62; H, 5.29; N, 10.90. 2-(4-Aminophenyl K7-methoxybenzothiazole (6d) Yellow solid; yield 95%; melting point 142-144°C; W NMR (CDC13, 400 ΜΗζ) δ7·90 (d, J = 8.4 Hz, 2H),

7.62 (d, J = 8.0 Hz, 1H), 7.39 (t, J = 8.0 Hz, 1H), 6.78 (d, J = 8.0 Hz, 1H), 6.73 (d, J = 8.4 Hz, 2H), 3.99 ( s, 5H); 13C NMR (CDC13, 100 MHz) δ169.1, 15.9, 154.2, 149.2, 129.1, 126.9, 124.0, 123.0, 115.3, 114.8, 104.7, 55.9; HRMS (ESI, m/z) for Ci4H13N2OS calcd 257.0749, found 257.0748; Anal, calcd for cl4H12N2〇S: C, 65.60; H, 4.72; N, 10.93. Found C, 65.66; H, 4.86; N, 10.92. 2-(4-Aminophenyl)-6- Methoxybenzopyrene (6e). Yellow solid; Yield 96% melting point 174-176°C; 4 NMR (CDC13, 400 ΜΗζ) δ7·88 (s, 1H), 7.84 (dt, J = 4.8

And 2.8 Hz, 2H), 7.32 (d, J = 2.4 Hz, 1H), 7.04 (dd, J = 4.8 and 2.4 Hz, 1H), 7.73 (dt, J = 4.8 and 2.8 Hz, 2H), 3.98 (bs , NH2), 3.87 (s, 3H); 13C 21 201217348 NMR (CDC13, 100 MHz) 6166.1, 157.3, 148.8, 148.8, 135.9, 135.9, 128.8, 124.2, 123.0, 115.1, 114.8, 104.3, 55.8; HRMS (ESI m, calcd for C14H12N2OS: C, 65.60; H, 4.72; N, 10.93. Found C, 65.35; H, 4.90; N, 10.67. 2-(4-amine Phenyl)-6-trifluoromethylbenzothiazole (6f). Yellow solid; Yield 91%; mp 181-183 ° C; W NMR (CDC13, 400 ΜΗζ) δ8'11 (s, 1H), 8.03 (d, J = 8.4 Hz, 1H), 7.90 (dt, J = 4.8 and 2.4 Hz, 2H), 7.67 (dd, J = 6.8 and 1.6 Hz, 1H), 6.73 (dt, J = 4.8 and 2.4 Hz, 2H), 4.08 (bs, NH2); 13C NMR (CDGI3, 100 MHz ) 5169.9, 158.1, 149.9, 132.3, 129.4, 123.1, 123.0, 122.5, 119.0, 119.0, 114.7; HRMS (ESI, m/z) for C14H9F3N2S calcd 294.0439, found 294.0438; Anal, calcd for C14H9F3N2S: C, 57.14; , N, 9.52. Found C, 57.20; H, 3.18; N, 9.49. 2-(4-Amino-3-tolyl)benzothiazole (6 g). Yellow solid; Yield 94%; mp 147-149 〇 C; NMR (CDC13, 400 MHz) 57.99 (d, J = 8.0 Hz, 1H), 7.85-7.83 (m, 2H), 7.75 (dd, J = 8.0 and 2.0 Hz, 1H), 7.44 (td, J = 8.0 and 1.2 Hz, 1H), 7.31 (td, J = 8.0 and 1.2 Hz? 1H)? 6.71 (d? J = 8.0 Hz, 1H), 3.94 (bs, NH2), 2.23 (s, 3H); 13C NMR (CDC13, 100 MHz) 6168.7, 154.2, 147.5, 134.5, 129.7, 126.9, 126.0, 124.3, 123.8, 122.4, 122.1, 121.3, 114.5, 17.1; HRMS (ESI, m/z) for Ci4Hi2N2OSNa calcd 263.0619, found 263.0618. Anal, calcd for C14H12N2OS: C, 69.97; H, 5.03; N, 11.66. Found C, 69.84; H, 5.01; N, 11.70. 2-(4 -Amino-3-tolyl)-6-ethylbenzothiazole (6h). Yellow solid; yield 93%; melting point 171-173 ° C; ^1^111 (00 (:13,4001^) 57.90 ((1,1 = 8.413⁄4 1H), 7.80 (d, J =1.6 Hz, 1H ), 7.32 (dd, J = 6.0 and 2.4 Hz, 1H), 7.65 (d, J = 0.8 Hz, 1H), 7.27 (dd, J = 6.8 and 1.6 Hz, 1H), 6.70 (d, J = 8.4 Hz) , 1H), 3.92 (bs, NH2), 2.76 (q, J = 8.0 Hz, 2H), 2.22 (s, 3H), 1.29 (t, J - 7.6 Hz, 201217348 3H); 13C NMR (CDC13, 100 MHz ) 6167.8, 152.5, 147.4, 140.9, 140.9, 134.7, 129.6, 126.8, 126.5, 124.1, 122.1, 122.0, 120.0, 114.6, 28.9, 17.2, 15.8; HRMS(ESI, m/z) for Ci6Hi7N2OS calcd 269.1112, found 269.1110 Anal, calcd for Ci6H16N2S: C, 71.61; H, 6.01; N, 10.44. Found C, 72.01; H, 6.36; N, 10.52. 2-(4-Amino-3-tolyl)-6-methoxy Benzothiazole (6i). Yellow solid; Yield 95%; mp 151-153 ° C; 4^111 (000:13,400^4112) 57.87 (41 = 8.813⁄4

1H), 7.77 (d, J = 2.0 Hz, 1H), 7.69 (dd, J = 6.0 and 2.4 Hz, 1H), 7.30 (d, J = 2.8 Hz, 1H), 7.03 (dd, J = 6.0 and 2.4 Hz, 1H), 6.69 (d, J = 8.4 Hz, 1H), 3.86 (s, 5H), 2.22 (s, 3H); 13C NMR (CDC13, 100 MHz) 5166.4, 157.2, 148.7, 147.2, 135.8, 129.4 , 126.6, 124.0, 122.9, 122.2, 115.0, 114.6, 104.3, 55.8, 17.2; HRMS (ESI, m/z) for C15H15N2OS calcd271.0905, found 271.0906; Anal, calcd for C15H14N2OS: C, 66.64; H, 5.22; N, 10.36. Found C, 66.68; H, 5.38; N, 10.48. 2-(4-Amino-3-tolyl)-6-trifluoromethylbenzothiazole (6j). Yellow solid; Yield 91%; mp 152-154 ° C; NMR </ RTI> CDOb, 400 ΜΗζ) δ 8.12 (s, 1H), 8.04 (d, J = 8.8 Hz, 1H), 7.83 (d, J = 1.2 Hz, 1H), 7.77 (dd, J = 6.0 and 2.4 Hz, 1H), 7.66 (dd, J = 6.8 and 1.6 Hz, 1H), 6.72 (d, J = 8 Hz, 1H), 4.20 (bs, NH2), 2.24 (s, 3H); 13C NMR (CDC13, 100 MHz) 5171.1, 156.3, 148.3, 134.6, 130.0, 127.3, 126.4 (q, J = 65.5 Hz, 1C), 123.0 (q, J = 5.6 Hz , 1C), 122.4, 122.1, 118.9 (q, J = 5.6 Hz, 1C), 114.5, 17.2; HRMS (ESI, m/z) for C15H12F3N2S calcd 308.0595, found 308.0597; Anal, calcd for C15H„F3N2S: C, 58.43; H, 3.60; N, 9.09. Found C, 58.50; H, 3.60; N, 9.09. Cell culture As mentioned above, fibroblasts were taken from adult foreskin samples (Yang, CC; Lin, SD; Yu, HS) J. Dermaio/. 1997, 162), Human Basal Cell Carcinoma (BCC) purchased $23 201217348 ^ (American Type Culture Collection) (Manassas, VA) 'Save in rpmi· medium towel and supplement i〇% fetal bovine serum , i〇〇u site Qing Huisu G and 100 _„ L sulfuric acid streptomycin (Gibc〇, BRL). After treatment with 5 edta (Gibco, BRL), BCC cells were confluent and passaged in 5% CO 2 in a humidified environment of 37 °c. Ultraviolet Irradiation Ultraviolet exposure is described in previous studies (Kao, c s; Yu, H s j, 1992, Cai, 734). For UV irradiation, a specific UV lamp is used to emit a peak wavelength of 365 G.75 mW/em2 (vilber Μ_

La Vallee, Cedex, France). The cells cultured before the ultraviolet irradiation were treated for 4 hours (4) with different chemicals or at different concentrations (〇_4 μΜ). The cultured cells were washed with phosphate buffered saline (PBS) and then irradiated with monovalent melon 2 UVA in pBS to prevent the formation of toxic light products derived from the medium by ultraviolet irradiation. The measurement of the illumination was measured using a UVX radiometer (UVP, San Gabriel, CA, USA). Immediately after the light treatment, the PBS was removed and the medium was added to the cells. All subsequent experiments will be performed in triplicate. Cell viability Cell viability is analyzed by the MTT assay, a mitochondrial functional assay based on the reduction of the redox indicator MTT to non-soluble alpha crystals by living cell mitochondrial dehydrogenase. Briefly, cells were seeded in 96-well plates at a density of 2500 cells/well. 201217348 After overnight incubation, cells are treated with 4 μL of compound and cultured for 24 hours. The medium was then discarded&apos; and replaced with 10 ML of dye. The plate will be at 37. (: culture for 2 hours. The resulting crystals will be dissolved in 100 dimethyl hydrazine (DMS (1) and read at 540 nm with a micro-disc analyzer (MRX-II, Dynex technology, Chantilly, VA)). Density. Sub-G1 (Sub-Gl) regional analysis

BCC cells were treated with 4 μΜ compound and 1 J/cm 2 UVA. After 24 hours of irradiation, the cells were collected by centrifugation and centrifugation. The pellets of the pellets were suspended in 50 ° / 〇 cold ethanol and fixed at _2 ° ° C. After fixation, the cells were washed once with cold PBS and in 0.5 mL PBS containing 100 pg / mL RNase A Incubate at 37 ° C for 20 minutes. The cells were collected by centrifugation at 400 g for 5 minutes, and PBS 250 pL containing 50 gg/mL propidium iodide (PI) was added to the cell pellet. Thirty minutes later, the DNA content of 1 〇 000 cells was measured by a FACSscan flow cytometer (Elite ESP, Beckman Coulter, Brea, CA). The histogram is analyzed by the Windows Multiple Document Interface (WinMDI) software. Cells with less DNA content than untreated and in G0/G1 phase are considered to be cells > weeks of death. Caspase-3 colorimetric analysis After 24 hours of irradiation, the cells were collected by centrifugation and washed once with PBS, and the cell pellets were counted and 25 μ!71 X 1〇6 cells. The concentration is suspended in a cold lysis buffer and homogenized. According to the manufacturer's instructions (R&amp;D Systems,

S 25 201217348

Minneap〇lis, MN) 'Homogenate was centrifuged at 12, (8) 〇ipm for 1 min at 4 t, and the activity of thiosporin protein _3 was measured using the supernatant and using an ELISA-based assay. The results are shown in the form of mean soil standard deviation. The combination of Aimexin V and pr〇pidine i〇dide (PI) was divided into two groups to evaluate the early apoptosis and necrosis. BCC cells were treated with different concentrations before irradiation (0-4). The molecular formula 6f of μΜ) was treated for 4 hours. After 24 hours of irradiation, cells were trypsinized and collected by centrifugation, and each sample was added to annexin V_FITC protein to 106 cells and measured by cytometry according to the manufacturer's instructions (Bender MedSystems, Vienna, Austria). At the same time, the cells were stained with ρι. Flow cytometry data was analyzed using WinMDI software. Type observation BCC or fibroblasts (5xl〇5 cells/well) were grown on 6-well plates. The cells were treated with 4 μΜ of the formula 6f for 4 hours and then irradiated with 1 j/cm 2 uva. After exposure for 24 hours, the microscope was photographed at 200 times. Determination of the degree of intracellular reactive oxygen species In order to assess the extent of intracellular reactive oxygen species, 2',7'-dichlorofluorescein diacetate (DCFH-DA, Molecular Probes) fluorescence was used. Dyes are used to clarify this issue. In cells, when non-polar DCFH-DA is converted to polar 201217348-derived DCFH, it is carried out by vinegar. DCFH is non-fluorescent but is rapidly oxidized to high fluorescing dcf by intracellular hydrogen peroxide or nitrogen oxides. In addition, catalase (Sigma), an effective hydrogen peroxide scavenger, was also used in this study. Cells were pretreated with catalase (10) u/mL before treatment with formula 6f (〇-4 μΜ). After the specified irradiation is carried out, DCFH_DA (1 μ μΜ) is immediately added to the cultured cells at 37 ° C for 3 G minutes. Fluorescence of the sample is measured by flow cytometry. 2, 7, 7, Dichloro Fluorescent Yellow (DCF) will be recorded using a FL-1 photomultiplier. Evaluation of mitochondrial membrane potential (ΔΨηιί) BCC cells were cultured in 35-mm culture dishes and allowed to grow exponentially for an hour before treatment. The cells were treated with 6 sputum, 2 and 4M before irradiation with 1 J/cm2 UVA. Cells that have been removed and adhered to the medium will be trypsinized. The cells were pelleted by centrifugation at 400 g for 5 minutes and stained for 30 minutes at room temperature in 100 nM/ml DiOC6 stain (Molecular Probes, Eugene, OR), washed twice with PBS and suspended in PBS. The samples were immediately subjected to fluorescence analysis (FL-1 detector, bandpass filter 530/30 nm) using a FACScan flow cytometer (Elite ESP, Beckman Coulter, Brea, CA). The histogram is analyzed with the Windows Multiple Document Interface (WinMDI) software. Contents of the ATP bioluminescence assay The amount of intracellular ATP is determined by bioluminescence assay based on the output of the luciferin-luciferase reaction. Immediately after treatment with different concentrations of 6f-UVA, all cells were extracted from the cultured BBC cells with cell lysate. After centrifugation to remove cell debris, the supernatant of 2012 2012348 was collected to measure ATP. The total amount of intracellular ATP will be based on the ATpUte* cluster (Perkin

The process provided by Elmer, Boston, ΜΑ) is judged. Protein extraction and Western blotting method, containing 100 pg/mL PMSF, 2 pg/mL aprotinin (apr〇tinin),

2 pg/mL leupeptin and 100 pg/mL sodium fluoride in ice-cold dumb eight buffer (! xPBS buffer, i%-40, 0.5% sodium deoxycholate, 〇1〇/ In 〇SDS), cultured BBC cells were lysed to obtain whole cell extracts. After centrifugation for 3 minutes, the protein in the supernatant was quantified by the Bradford method (Bio-Rad), and 4 〇mg of protein per liter of runway (lane) in 1% of SDS_polypropylene. Performed in an amide gel. After electrophoresis, the protein was transferred from the electrosonic to the polyvinylidene-oxygen (PVDF) membrane (Millipore, Bedford, MA), and the membrane was incubated at room temperature in pbs + 0.1% Tween 20 (PBS containing 5 °/〇 skim milk). -T) Blocking - hours. After simple rinsing with PBS-T, the dots were detected at room temperature for two hours with respective primary antibodies, or overnight at 4 °C. Anti-jnk (46 k〇a), p38 (38kDa), ERK (42, 44kDa) rabbit polyclonal antibodies and anti-p-JNK, p p38, p-ERK mouse monoclonal antibodies from Santa Cruz Biotechnology ( Santa Cruz, CA) purchased. An anti-actin mouse monoclonal antibody system was purchased from chemic〇n Int Inc (Temecula, CA). 25 μΜ oligomycin' (Sigma, granule-specific performance of FoF! ATP synthesis inhibitor) or 1 io io^m ATP (Sigma) was added to the medium separately for the blocking test before 6f-UVA treatment. The membrane was incubated with the corresponding horseradish peroxidase-labeled primary antibody (Santa Cruz Biotechnology) for 1 hour at room temperature. The membrane was washed 4 times with PBS-T for 15 minutes and treated with Western blotting chemiluminescent agent (Western Lightning 28 s 201217348)

Chemiluminescence Reagent Plus) (Perkin Elmer Life Sciences, Boston, MA) visualizes protein dots. The relative amount of specific protein is determined by the density of the x-ray film and analyzed by the Eagle Eye Image System (Stratagene, La Jolla, CA). Statistical analysis results were expressed as mean soil standard deviation and analyzed by statistical analysis system (SPSS, SPSS Inc" Chicago, IL). Differences between groups were analyzed by Student's occlusion. P values were &lt;0.05 for all tests. The results are considered to be significant.Example 2 Results Cell viability The effect of Formula 6 on UVA activation on BCC cell activity is assessed by MTT assay. BCC cells are treated at different concentrations (ie, Equation 6, where r^ 6-CF3 and R2 is H) treatment for 4 hours, followed by irradiation. After 24 hours exposure, cell viability was determined. As shown in Fig. 3A, the inhibition effect depends on the drug concentration. The concentration of formula 6f is 4 _, J/ The cell viability after cm2UVA irradiation was higher than 5%. To confirm whether 丨J/cm2 was still the most influential dose in this study, the cells were pretreated with 4 μΜ6 before treatment with UVA. Compared with the untreated control group, the BCC cells were irradiated with 〇.25, 〇5 and 丨J/em2 UVA, and the cell viability was 71, 65 and 45%, respectively. In addition, this data also showed that compared with only the UVA irradiation or only exogenous 6 treatment, 6_uyA showed higher inhibition '' and '6f' is not cytotoxic to BCC cells (Fig. 3B). 29 201217348 Cell G1 accumulation In order to investigate the effect of compound 6 on the cell cycle of BCC cells, flow cytometry analysis DNC contains 4. _ feed to the sub-diploid (hypodipbid) DNA (sub_G1 material) 'material domain and reflect the DNA of the broken mBBC cells treated with Na reagent for 4 hours, and then with 2 wa After 24 hours of irradiation, the rib reagent was further added. Each group was divided into cap cells and analyzed by FACScan flow cytometry to obtain the fineness of the secondary G1 DNA peaks: 2.2% (control group), 6.5% ( 6a), 13.4% (6b), 29.2% (6c), 24.4% (6d), 6.5% (6e), 35.4% (6f), 13.6% (6g), 13 8% (6h), 14.8% (6i And 6.4% (6j) (% means the percentage of cell content). Since the compound of formula 6f exhibits the most sub-G1 accumulation in BCC cells, the formula was selected as a model for further study. Apoptosis detection of cystein Protein plum-3 has been shown to be one of the most important cell death performers in apoptosis (Shi, Y. M?/. Ce// 2002, 9, 459). Thiocyanine The activity of peptone_3 was determined by colorimetric method. Compared with the untreated control group, the activity of thiosporin-3 in BCC cells increased after 6f-UVA treatment (p&lt;〇. 〇l) (Fig. 4A). Furthermore, BBC cells treated with 0, 2, and 4 μΜ of the formula 6 under 1 J/cm2 UVA irradiation induced apoptosis (annexin ν7ΡΓ) levels of 0.2% and 19.5 °/, respectively. And 27.5%. In contrast, necrotic cells did not change significantly (annexinV4/?!4) (Fig. 4B). In addition, 201217348 It is interesting to understand the apoptotic activity of 6f-UVA on normal cells. Therefore, human skin fibroblasts are used to clarify this issue. Analysis of the morphology data and annexin ν/ρι analysis showed no significant apoptosis effects after 6f_UYA treatment compared with the control group (Fig. 4C). These results have led to further studies of the mechanism of apoptosis induced by the photosensitizing effects involved in BCC cells. Granulocyte dysfunction _ More and more evidence suggests that mitochondrial dysfunction plays an important role in oxidative stress (Hail, N. Jr., 0, 0, 2005, Sichuan, 687), which induces activity Production of oxygen species and cell death in apoptotic mode (Hiideman, d. A.; Mitchell, T.; Teague, T. Κ.; Henson, Ρ.; Day, Β. J.; Kappler, J .; Marrack, Ρ. C. Immunity. 1999, Sichuan, 735). To determine whether the reactive oxygen species involved mitochondria-dependent apoptosis induced by 6ί1υνΑ, the production of η2〇2 in the cells was measured using a DCFH-DA detector. The results showed that 6f-UVA significantly increased the intracellular 〇2. In addition, catalase significantly inhibited the production of increased reactive oxygen species in B BC cells treated with 4 φ W 6f_UVA (Fig. 5A). The mitochondrial membrane potential (△ less (4)) is not only an important parameter for the granulosa, but also for the state of the cells. This reduction is an early event in the course of cell death, and a decrease in fluorescence intensity reflects the collapse of the MV, which usually indicates an early stage of apoptosis that has been irreversible. Therefore, the present invention tests whether the initial active oxygen species formation changes ΔΨπή after 6f-UVA irradiation. After treatment with f' 2 and 4 μΜ of 6f-UVA, BBC cells were harvested and stained with 〇1〇 (:6 staining followed by flow cytometry. The dye binds to the inner membrane of the mitochondria The outer membrane, and undergoes redshift during fluorescence during membrane depolarization. As shown in Figure 5B,

31 201217348 2 Μ or more dose-treated cells showed a significant reduction in Δψ^ in BCC cells. ATP is a central parameter of cellular energy, metabolic regulation, and cellular signaling, so determining intracellular ATP is valuable for cellular physiological properties. The intracellular ATP content of BCC cells treated with 2 and 4 μΜ of 6f-UVA decreased by about 30% and 45%, respectively, compared to the untreated control group (Fig. 5C). Activation of the mitogen-activated protein kinase (MAPKs) pathway In order to determine the possibility of involvement of various protein kinase pathways in 6f-UVA-induced apoptosis, mitogen-activated protein-induced mei was evaluated by measuring MAPK subfamilies Activity. Compared with the untreated control group, BBC cells increased the acidified extracellular signal-regulated kinase (p_ERK) and lindated p38 (p-p38) after treatment with 4 μΜ of 6f-UVA. BBC cells treated with 6f-UVA compared to ERK and p38' did not stimulate phosphorylation of JNK MAPK (Fig. 6A). Furthermore, in order to clarify whether the activation of MAPK is due to a decrease in cellular ATP synthesis, cells treated with 4 μΜ 6f-UVA will first be treated with oligomycin (a granulocyte-specific FqF! ATP synthesis inhibitor). Or ATP pretreatment. The results showed an increase in the activation of ERK with oligomycin but not pretreated with ATP (Fig. 6B). Those skilled in the art will readily appreciate that the present invention can readily achieve the objectives and achieve the results and advantages mentioned, as well as those that are present therein. The compositions of the present invention, and the processes and methods of making the same, are representative of the preferred embodiments, which are exemplary and not limited to the field of the invention. Those skilled in the art will appreciate the modifications and other uses of 201217348. These modifications are intended to be within the spirit of the invention and are defined in the scope of the claims. The present invention has been described in detail with reference to the embodiments of the present invention, and the invention may be Spirit and scope. All patents and publications mentioned in the specification are subject to the general skill of the art in the field of the invention. All patents and publications are hereby incorporated to the same extent as if each individual publication is specifically and individually indicated. The invention, as exemplified herein, may be practiced in the absence of any element, or limitation, or the specific limitations disclosed herein. The name (5) used and the expression 疋 as a description of the specification are non-restrictive and are not intended to be used to exclude the expression of any such emblem and its part, but it is necessary to recognize This issue _ special wealth please _ there may be a variety of different changes. Therefore, the present invention has been described in terms of a preferred embodiment and is intended to be modified and varied, and such modifications and variations are still within the scope of the invention. Within the scope of the patent application. [Simple diagram of the diagram]. S 1 shows the structure diagram of 2-phenyibenzothiaz〇les (2-Phenyibenzothiaz〇les) showing 2_(4-amine) Benzopyrene (2-(4-aminophenyl) Complete synthesis of benzothiazoles) (Formula 6). Reagents and conditions: (丨) Method a: χ = C1, pyridine, 33 201217348 reflux, method b: X = OH 'SOCl2, benzene, reflux. (2) Lawson's reagent, chlorine stupid, i35 ° C. (3) K3Fe(CN)6 aqueous solution, NaOH, 90 °C. (4) H2/Pd/C 10%, CH2C12, 25 °C. Figure 3 shows the effect of 6-UVA cell viability. (A) A dose response curve of BCC cells to mixture 6f was tested. The cells were seeded in 96-well plates of 2500 cells per well and cultured overnight until the cells were attached. 6f-UVA was added to the culture solution at the specified concentration (three repetitions) and irradiated with 1 J/cm 2 of ultraviolet light after 4 hours of culture. MTT reagent was added to each well after 24 hours of UV irradiation. The absorbance is directly proportional to the number of viable cells. (B) For the sake of clearing the phase, whether 6-UVA is more cytotoxic than the treatment with UVA alone or with the formula 6, the cells are cultured or not cultured with 4 μΜ of the formula 6f before being irradiated with different doses of ultraviolet light. Compared with the control group, *p &lt; 0.05, **p &lt; 〇.〇1. Figure 4 shows the effect of 6f-UVA treatment on apoptosis. (a) In apoptosis, the enzyme activity of caspase-3 is determined by a colorimetric reaction. The peptide cleavage by cysteinospergic acid releases the chromophore p-nitroaniline (pNA, p-nitroaniline), which can be quantified at a wavelength of 405 mm. (b) BBC cell dot plots at various concentrations of 6 £:UVA treatment. BBC cells are stained with pi and then the PS remaining on the cell surface is specifically detected by binding to annexin v _ FITC. (C) Detection of apoptosis in fibroblasts using morphological observation and annexin v/PI double staining. About 10,000 cells were taken from each group and analyzed by flow cytometry. The data shown is representative of the experimental results, which were repeated three times and the results were similar. Compared with the control group 201217348, ** p &lt; 0.01. Figure 5 shows the effect of 6f-UVA on mitochondrial function. (a) 6f-UVA results in the production of reactive oxygen species in BCC cells, as shown by the number of cells in BBC cells treated with different concentrations of 6f_uyA and stained with DCFH-DA. ROS was determined as a control in the presence of catalase, a h2〇2 scavenger. (B) Mitochondrial membrane potential (ΔΨ^) of BBC cells after exposure to 6f-UVA. The cells were treated with 〇, 2 and 4 μΜ of 6f for 4 hours, then irradiated with 1 J/cm 2 UVA, and then analyzed by DiOQ and immediately analyzed by flow cytometry of the materials and methods described below. The number in Ml indicates the percentage of cells with reduced mitochondrial membrane potential. Approximately 10,000 cells per group will be analyzed by a flow analyzer. (c) The relative amount of ATP calculated as a percentage, based on the amount of 0 μΜ. In three unrelated experiments, the results were similar. Compared with the control group, ** ρ < 〇 〇 1. Figure 6 shows an immunoblot analysis showing that 6f-UyA has an effect on the protein expression of _kinase on BCC cells. (A) After exposing the cells to different concentrations of 6f_WA, collect the cell lysate and perform immunoblotting with specific antibodies to indicate Q. For internal control, the same amount of protein extract is also detected by agonist antibodies. (B) The amount of extracellular signal-regulated kinase (p-ERK) phosphorylated in the presence of oligomydn (25 μM) and ATP (1 X 10_4 M) was measured as a control.

S 35

Claims (1)

201217348 VII. Patent application scope 1. A method for preparing compound of formula 6 6 , comprising: a. compound of formula 1 Compound with formula 2 COX no2 2 Reaction to form compound of formula 3 Wherein X of the formula 2 is C1 or OH; b. The compound of formula 3 is treated with Lawesson's reagent to form a compound of formula 4 S 36 201217348 s R2 N〇2 C. reacting a compound of formula 4 with potassium ferricyanide to produce a compound of formula 5 And d. catalytically reducing the Schwanky of the compound of formula 5 using a paiiadium on charcoal to produce a compound of formula 6, wherein the rule 1 in formulae 1 to 6 is 11, C^oalkyl, Cm alkoxy Or Qm haloalkyl, and &amp; in formula 1 to formula 6 is 11 or (:1_10 alkyl. 2. The method of claim 1, wherein the rule 1 in the formula 6 is 11 , 6-Et, 6-OMe, 6_OCH3, 6-Me, 7-OMe or 6-CF3, and R2 in the formula 1 to formula 6 is Η or CH3. 3. The method of claim 2, wherein ^ is in the formula 6 ^^^-Et, 6-〇Me, 6-Me, 7-OMe or 6-CF3 'and r々H in the formula i to the formula 6. 4. The method of item 2, wherein, in the formula i to the formula 6, 6_〇CH3, 0 or 6-CF3, and R2&amp;CH3 in the formula 1 to the formula 6. 5. The method of claim 2, wherein Si to the heart of the formula 6 is 6 (^3, and the formula (5) R2 in the formula 6 is Η » 6. - The species provides photodynamic therapy with at least one _ of the scorpion touch tumor growth 37 201217348 pharmaceutical composition Containing a compound of formula 6 as defined in claim 1 of the patent application. The composition of claim 6, wherein the photodynamic therapy comprises the steps of: administering the composition to a patient; waiting for sufficient time for the administered composition to be absorbed by the target tissue having at least one tumor; and irradiating the patient with the The area of the target tissue. 8. The composition of claim 6 has a skin cancer. 9. The composition of claim 8 wherein the patient has basal cell carcinoma. The composition of the sixth aspect of the invention is wherein the compound of the formula G is activated by the leg. η. The composition of claim 6, wherein the growth of the tumor is caused by apoptosis of the tumor cell. A. For example, a composition for a specific correction, wherein the cell cell level of the tumor cell rises. 13. If the application is specifically for the composition of the 12th item, the cell peak line body membrane of the axis cell The potential (△ is normal) drops.