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Synthesis of chlorin-based unsaturated fatty acid conjugates: Their in vitro phototoxicity on TC-1 cancer cell line
Journal of Photochemistry and Photobiology B: Biology 110 (2012) 50–57
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Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Synthesis of chlorin-based unsaturated fatty acid conjugates: Their in vitro phototoxicity on TC-1 cancer cell line Gantumur Battogtokh a, Hai-Bo Liu a, Su-Mi Bae a, Pankaj Kumar Chaturvedi a, Yong-Wan Kim a, In-Wook Kim a, Woong Shick Ahn a,b,⇑ a b
Cancer Research Institute, Catholic Research Institute of Medical Science, The Catholic University of Korea, Republic of Korea Department of Obstetrics and Gynecology, Seoul St. Mary’s Hospital, The Catholic University of Korea, Republic of Korea
a r t i c l e
i n f o
Article history: Received 15 December 2011 Received in revised form 2 March 2012 Accepted 13 March 2012 Available online 22 March 2012 Keywords: PDT Methyl pyropheophorbide-a Unsaturated fatty acid Photosensitizer Phototoxicity Synthesis
a b s t r a c t Chlorin-based photosensitizers in photodynamic therapy are the promising anticancer agents, but some of their properties such as specific-targeting to tumor need to be improved. The aim of this study was to synthesize chlorin-based unsaturated fatty acid conjugates to obtain an optimal photosensitizers. Thus four chlorin-based fatty acid conjugates were successfully synthesized through an esterification reaction using carbodiimide coupling reagents in enough yields. Then, structures of these conjugates were confirmed by 1H NMR, MALDI-MS, and UV–vis spectroscopy. Furthermore, their in vitro phototoxicity and cellular uptake were evaluated on TC-1 lung cancer cell line and HeLa cell line. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction Photodynamic therapy (PDT) is a promising cancer treatment that requires a combination of a photosensitizer (PS), tissue oxygen, and light to produce cytotoxic reactive oxygen species, mostly singlet oxygen [1,2]. During the last few years, a number of dyes which strongly absorb a light in the range of 650–800 nm have been reported as potential photosensitizers for PDT [3,4]. Of these, chlorin- and bacteriochlorin-based photosensitizers are monomeric compounds, efficient generators of singlet oxygen, and may be particularly effective in treating large and/or deeply seated tumors by absorbing long wavelengths [5]. Although most first and second-generation PSs studied for PDT display only a slight preference for malignant cells, they often lead to significant skin photosensitivity (Photofrin) and high uptake by healthy cells and tissues [6,7]. In order to overcome these side effects, third-generation PSs are being designed and synthesized to actively target diseased tissue [6]. In general, there are two pathways in this strategy: (i) modification of the photosensitizing agent moiety through its
⇑ Corresponding author at: Department of Obstetrics and Gynecology, Seoul St. Mary’s Hospital, The Catholic University of Korea, 505 Banpo-dong, Seocho-ku, Seoul 137-040, Republic of Korea. Tel.: +82 2 2258 6946; fax: +82 2 599 4120. E-mail address: [email protected] (W.S. Ahn). 1011-1344/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jphotobiol.2012.03.004
physicochemical properties or (ii) improving the targeting by conjugation of a photosensitizer to a molecule such as antibody, polymer, peptide scaffold, and carbohydrate [8,9]. Recently, poly unsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA), have been investigated for its potential for treating cancers and neurodegenerative disorders [10]. Some PUFAs have also been known to be rapidly taken up by tumor cells from the arterial blood, presumably for use as biochemical precursors and energy sources [11]. In addition, PUFAs are readily incorporated into the lipid bilayer of cells, resulting in the distraction of membrane structure and fluidity [11,12]. Bradley et al. [13] have obtained a paclitaxel–DHA conjugate that had greater antitumor activity compared to paclitaxel. As reported by Siddiqui et al. [14], long chain PUFAs (DHA) have been known to be highly susceptible to both enzymatic and non-enzymatic peroxidations since their discovery. Interestingly, reactive oxygen substrates (super oxide and hydrogen peroxide) produced from a PS upon irradiation might lead to the initiation of the peroxidation chain reaction of PUFAs [15]. As encouraged via above reports, we aimed to conjugate the UFAs to the chlorin derivatives through an esterification reaction. UFAs were linked to the different sites of PSs to observe their activity. Here, we report the characterization and synthesis of four kinds of chlorin-based fatty acid conjugates, and their phototoxicity in vitro against TC-1 lung cancer cell line.
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2. Materials and methods
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2.3. 3-Hydroxymethyl-3-devinylpyropheophorbide-a-methyl ester and docosahexaenoic acid conjugate (5)
2.1. General All reagents were purchased from Sigma, Aldrich, Fluka, Alfa Aesar and Daihan companies. If necessary, anhydrous solvents were distilled according to standard procedures [16]. Other commercially available reagents were used without further purification. Column chromatographic separations were performed over silica gel 60 (63–200 and 200–400 mesh, Merck). Analytical thin layer chromatography (TLC) was carried out on sheets precoated with silica gel F254 (0.2 mm thick, Merck). All reactions were carried out under an argon atmosphere in darkness. The progress of reactions was monitored by TLC and detection was carried out by means of an UV – lamp at 265 nm or 365 nm wavelengths. Electronic absorption spectra were recorded on an UltrospecÒ3000 (Pharmacia Biotech, Cambridge, England) spectrophotometer. 1H NMR spectra were recorded on Unity Inova 500 (UI 500, Varian Inc., USA) spectrometer at 500 MHz; chemical shifts are expressed in parts per million (ppm) relative to tetramethylsilane (TMS, 0.00). MALDI mass spectra were obtained with a Voyager-DE™ STR Biospectrometry Workstation spectrometer. Fluorescence spectra were obtained using a spectrofluorophotometer (RF-5301 PC; Shimadzu, Japan) with a xenon short-arc lamp (XBO 150; Ushino, Japan). Compound 7 was prepared from methyl pyropheophorbide-a (2), which was obtained via methyl pheophorbide-a (1) from Spirulina maxima algae according to the previously reported methods [17]. Compound 4 was prepared from methyl pyropheophorbide-d (3), which was obtained from methyl pyropheophorbide-a (2). 2.2. Preparation of pyropheophorbide-a-173-N-hexanol (8) Pyropheophorbide-a (7) (100 mg, 0.186 mmol), N,N0 -dicyclohexylcarbodiimide (DCC) (77 mg, 0.37 mmol) and N-hydroxysuccinimide (NHS) (26 mg, 0.226 mmol) were dissolved in 3 mL of anhydrous dichloromethane. Three drops of triethylamine were added and stirred under argon gas at room temperature. After 4 h, 6-hexanolamine (22 mg, 0.188 mmol) in methanol (2 mL) was added to the reaction mixture and stirred for 1.5 h. Next, 10 ml of water was added to the solution and stirred for 10 min, and diluted with dichloromethane (30 mL). The organic layer was joined, washed twice with water (100 mL), and dried over anhydrous sodium sulfate. After evaporation, the residue was dissolved in 5 mL of chloroform or ethylacetate and filtered through cold (10 °C) to remove side-mixtures. Subsequently, the residue was purified on silica gel (70–230 mesh) by column chromatography using 5% methanol in dichloromethane as an eluant. After purification, pure product gave as a pink–blue solid. Yield: 140.2 mg (71%), Rf 0.31 (in dichloromethane: methanol/95:5). UV–vis (CH2Cl2): kmax, nm (e, M1 cm1) 668.4 (47360.0), 610.5 (20446.5), 540.0 (22443.7), 414.8 (90725.8). 1H NMR (500 MHz, CDCl3, Me4Si): dH, ppm 9.60 (1H, s, 5-meso-H), 9.34 (1H, s, 10-meso-H), 8.86 (1H, s, 20-meso-H), 8.15 (1H, dd, J = 11.5, 17.8 Hz, 31-CH,), 7.70 (1H, t, J = 5.5 Hz, 174-NH), 6.35 and 6.18 (2H, each d, J = 18.0; 11.5 Hz, 32-CHa, 32-CHb), 5.22 and 5.08 (2H, each d, J = 19.8; 19.7 Hz, 132CH2), 4.55 (1H, q, J = 7.2 Hz, 18-CH), 4.27 (1H, s, 1710-OH), 4.26 (1H, d, J = 8.5 Hz, 17-CH), 3.61 (2H, q, J = 7.5 Hz, 81-CH2), 3.57 (3H, s, 121-CH3), 3.41 (3H, s, 21-CH3), 3.30 (2H, q, J = 6.4 Hz, 1710CH2), 3.14 (3H, s, 71-CH3), 2.95 (2H, m, 174-CH2), 2.60–2.32 (2H, m, 171-CH2), 2.16–2.06 (2H, m, 172-CH2), 1.79 (3H, d, J = 7.3 Hz, 181-CH3), 1.58 (3H, t, J = 7.5 Hz, 82-CH3), 1.30 (2H, p, J = 6.9 Hz, 179-CH2), 1.22 (2H, p, J = 6.9 Hz, 176-CH2), 1.15 (4H, m, 177 and 178-CH2), 0.14 and 1.67 (2H, each br s, 21-, 23-NH). MS (MALDI-TOF): m/z 634.2467 [M + H]+ (calcd. for C39H47N5O3 633.8222).
4 (30 mg, 0.054 mmol), DCC (22.4 mg, 0.108 mmol), and 4(dimethylamino) pyridine (DMAP) (9.9 mg, 0.081 mmol) in dichloromethane (5 mL) were stirred and 17.8 mg (0.054 mmol) of docosahexaenoic acid (DHA) was added. The reaction was carried out under argon gas at room temperature for 2 h. Next, 10 mL of water was added to the reaction mixture, stirred for 10 min, and diluted with dichloromethane (20 mL). The organic layer was joined and washed with water two times, and dried over anhydrous sodium sulfate. After evaporation, the residue was dissolved in chloroform or ethyl-acetate and filtered by suction to remove N,N0 -dicyclohexylurea (DCU) (formed from DCC). Then, the residue was purified on silica gel (230–400 mesh) by column chromatography using 2% methanol in dichloromethane as an eluant. The product was dried at 25 °C in a vacuum oven. The structure of the compound was characterized by 1H NMR and UV–vis spectroscopy. Yield: 39.78 mg (85%); pink–blue melt; Rf 0.55 (in dichloromethane:methanol/ 98:2). UV–vis (CH2Cl2): kmax, nm (e, M1 cm1) 665.1 (59547.2), 606.8 (22369.0), 537.1 (25165.0), 411.2 (102007.0). 1H NMR (500 MHz, CDCl3, Me4Si): dH, ppm 9.53 (1H, s, 5-meso-H), 9.42 (1H, s, 10-meso-H), 8.6 (1H, s, 20-meso-H), 6.38 (2H, s, 31-CH2), 5.30 (13H, m, 35;6;8;9;11;12;14;15;17;18;20;21, 132-CHa, overlapped) and 5.11 (1H, d, J = 19.5 Hz, 132-CHb), 4.5 (1H, q, J = 7.3 Hz, 18CH), 4.31 (1H, d, J = 8.5 Hz, 17-CH), 3.70 (2H, q, J = 7.6 Hz, 81-CH2), 3.68 (3H, s, 174-CH3), 3.6 (3H, s, 121-CH3), 3.44 (3H, s, 21-CH3), 3.26 (3H, s, 71-CH3), 2.74 (10H, m, 37,10,13,16,19-CH2), 2.71–2.52 (2H, m, 171-CH2), 2.50 (2H, m, 34-CH2), 2.46 (2H, t, J = 6.8 Hz, 33-CH2), 2.29 (2H, m, 172-CH2), 2.03 (2H, p, J = 7.3 Hz, 322-CH2), 1.81 (3H, d, J = 7.3 Hz, 181-CH3), 1.70 (3H, t, J = 7.6 Hz, 82-CH3), 0.93 (3H, t, J = 7.5 Hz, 323-CH3), 0.31 and 1.70 (2H, each br s, 21-, 23-NH). MS (MALDI-TOF): m/z 863.4110 [M + H]+ (calcd. for C55H66N4O5 863.1363). 2.4. 3-Hydroxymethyl-3-devinylpyropheophorbide-a-methyl ester and oleic acid conjugate (6) 4 (25 mg, 0.045 mmol), DCC (22 mg, 0.09 mmol), and DMAP (6.1 mg, 0.05 mmol) in DCM were stirred together and 12.77 mg (0.045 mmol) of oleic acid (OA) was added to the reaction mixture. The reaction was carried out according to the procedure above. Yield: 30.9 mg (84%); pink–blue solid; Rf 0.42 (in dichloromethane: methanol/98:2). UV–vis (CH2Cl2): kmax, nm (e, M1 cm1) 665.0 (72059.5), 607.0 (24895.2), 537.0 (28046.5), 411.1 (126787.0). 1H NMR (500 MHz, CDCl3, Me4Si): dH, ppm 9.44 (1H, s, 5-meso-H), 9.42 (1H, s, 10-meso-H), 8.61 (1H, s, 20-meso-H), 6.38 (2H, s, 31CH2), 5.28 and 5.13 (2H, each d, J = 19.5; 19.5 Hz, 132-CH2), 5.18 (2H, m, 310,11-CH), 4.50 (1H, q, J = 7.3 Hz, 18-CH), 4.31 (1H, d, J = 8.5 Hz, 17-CH), 3.7 (2H, q, J = 7.6 Hz, 81-CH2), 3.68 (3H, s, 174CH3), 3.60 (3H, s, 121-CH3), 3.44 (3H, s, 21-CH3), 3.27 (3H, s, 71CH3), 2.7–2.56 (2H, m, 171-CH2), 2.43 (2H, t, J = 7.5 Hz, 33-CH2), 2.29 (2H, m, 172-CH2), 1.88 (4H, m, 39,12-CH2), 1.80 (3H, d, J = 7.3 Hz, 181-CH3), 1.70 (3H, t, J = 7.6 Hz, 82-CH3), 1.67 (2H, m, 34-CH2), 1.13 (20H, m, 35;6;7;8;13;14;15;16;17;18-CH2), 0.83 (3H, t, J = 2.8 Hz, 319-CH3), 0.31 and 1.79 (2H, each br s, 21-, 23-NH). MS (MALDI-TOF): m/z 816.3974 [M + H]+. (calcd. for C51H68N4O5 817.1094). 2.5. Pyropheophorbide-a 173-N-(4-hexanol) and docosahexaenoic acid conjugate (9) 8 (30 mg, 0.047 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC-HCl) (19.5 mg, 0.094 mmol), and DMAP (6.9 mg, 0.056 mmol) in 3 mL of chloroform were stirred together and 15.5 mg (0.047 mmol) of docosahexaenoic acid (DHA) was
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added to the reaction mixture. The reaction was carried out under argon gas at room temperature for 3 h. Next, the reaction mixture was diluted with dichloromethane (DCM) (20 mL), the organic layer was joined and washed with water two times, and dried over anhydrous sodium sulfate. After evaporation, the residue was purified on silica gel by preparative TLC (20 20) using 3% methanol in dichloromethane as an eluant. The product was dried with phosphorus pentoxide at 25 °C in a vacuum oven. Yield: 33.5 mg (75%); pink–blue solid; Rf 0.17 (in dichloromethane: methanol/ 98:2). UV–vis (CH2Cl2): kmax, nm (e, M1 cm1) 668.5 (56923.5), 610.0 (28036.9), 539.5 (31010.6), 414.9 (105067.8). 1H NMR (500 MHz, CDCl3, Me4Si): dH, ppm 9.5 (1H, s, 5-meso-H), 9.41 (1H, s, 10-meso-H), 8.56 (1H, s, 20-meso-H), 8.03 (1H, dd, J = 11.5; 17.8 Hz, 31-CH,), 6.29 and 6.18 (2H, dd, J = 17.9; 11.5 Hz, 32a-, 32b-CH), 5.34 (12H, m, 1714;15;17;18;20;21;23;24;26;27;29;30-CH), 5.18 (2H, dd, J = 19.5; 19.5 Hz, 132-CH2), 4.77 (1H, t, J = 5.5 Hz, 174-NH), 4.51 (1H, q, J = 7.3 Hz, 18-CH), 4.36 (1H, d, J = 8.5 Hz, 17-CH), 3.93 (2H, t, J = 6.7 Hz, 1710-CH2), 3.70 (2H, q, J = 7.6 Hz, 81-CH2), 3.66 (3H, s, 121-CH3), 3.41 (3H, s, 21-CH3), 3.25 (3H, s, 71-CH3), 2.93 (2H, m, 175-CH2), 2.79 (10H, m, 1716,19,22,25,28-CH2), 2.7–2.4 (2H, m, 171-CH2), 2.30 (2H, m, 1713-CH2), 2.06–1.82 (2H, m, 172-CH2), 2.04 (2H, p, J = 7.4 Hz, 1731-CH2), 1.80 (3H, d, J = 7.3 Hz, 181-CH3), 1.70 (3H, t, J = 7.6 Hz, 82-CH3), 1.61 (2H, m, 1712-CH2), 1.42 (2H, p, J = 7.2 Hz, 179-CH2), 1.05 (6H, m, 17 6,7,8CH2), 0.95 (3H, t, J = 7.5 Hz, 1732-CH3), 0.47 and 1.68 (2H, each br s, 21-, 23-NH). MS (MALDI-TOF): m/z 944.4903 [M]+ (calcd. for C61H77N5O4 944.2952). 2.6. Pyropheophorbide-a-173-N-(4-hexanol) and oleic acid conjugate (10) 8 (30 mg, 0.047 mmol), EDC-HCl (19.5 mg, 0.094 mmol), and DMAP (6.9 mg, 0.056 mmol) in 3 mL of chloroform were stirred together and 13.4 mg (0.047 mmol) of oleic acid (OA) was added to the reaction mixture. This reaction was carried out according to the procedure above. Yield: 34 mg (80%), Rf 0.13 (in dichloromethane:methanol/98:2). UV–vis (CH2Cl2): kmax, nm (e, M1 cm1) 668.6 (110061.6), 610.2 (53819.8), 539.6 (59201.8), 414.9 (202093.5). 1H NMR (500 MHz, CDCl3, Me4Si): dH, ppm 9.49 (1H, s, 5-meso-H), 9.4 (1H, s, 10-meso-H), 8.56 (1H, s, 20-meso-H), 8.02 (1H, dd, J = 11.5; 17.8 Hz, 31-CH,), 6.29 and 6.18 (2H, each d, J = 17.9; 11.6 Hz, 32a-; 32b-CH), 5.32 (2H, m, 1719,20-CH), 5.24 and 5.11 (2H, dd, J = 19.5; 19.5 Hz, 132-CH2), 4.8 (1H, m, 174-NH), 4.51 (1H, q, J = 7.2 Hz, 18-CH), 4.36 (1H, d, J = 8.5 Hz, 17-CH), 3.91 (2H, t, J = 6.7 Hz, 1710-CH2), 3.7 (2H, q, J = 7.7 Hz, 81-CH2), 3.64 (3H, s, 121-CH3), 3.41 (3H, s, 21-CH3), 3.25 (3H, s, 71-CH3), 2.89 (2H, m, 175-CH2), 2.65 and 2.45 (2H, each m, 171-CH2), 2.18 (2H, t, J = 7.6 Hz, 1712-CH2), 2.15 and 1.86 (2H, each m, 172-CH2), 1.97 (4H, m, 1718,21-CH2), 1.80 (3H, d, J = 7.3 Hz, 181-CH3), 1.70 (3H, t, J = 7.6 Hz, 82-CH3), 1.51 (2H, m, 1713-CH2), 1.41 (2H, p, J = 7 Hz, 179-CH2), 1.24 (20H, m, 1714;15;16;17;22;23;24;25;26;27-CH2), 1.05 (6H, m, 176,7,8-CH2), 0.86 (3H, t, J = 6.7 Hz, 1728-CH3), 0.47 and 1.69 (2H, each br s, 21-, 23-NH). MS (MALDI-TOF): m/z 898.4696 [M]+ (calcd. for C57H79N5O4 898.2682).
source. A collimated laser beam was directed to the sample cuvette through an optical fiber. The decrease in fluorescence intensity of DMA (excitation, 360 nm) as a result of the photosensitization reaction was monitored using a spectrofluorophotometer controlled by a PC. SOQ yield was calculated by the following equation [18b]:
uDðUÞ ¼ uDðStÞ SðUÞ=SðStÞ where U and St denote unknown and standard, respectively, and S is the slope of bleaching of the probe absorbance with irradiation time. The SOQ yield (u4) of each sample was calculated from standard u4 value (PPa-0.52). 2.8. Cell culture and in vitro phototoxicity Human lung cancer cells (TC-1), which were cotransformed by HPV-16 E6/E7 oncoproteins and c-Has-Ras, were cultured in RPMI 1640 supplemented with 5% fetal bovine serum. 1% Penicillin– Streptomycin (Penicillin G, sodium salt; streptomycin sulfate) and 0.4 mg/mL G418 disulfate (Duchefa, Netherlands) were added to the medium. The cell line was maintained at 37 °C in humidified 5% CO2 atmosphere. For viable cell counting, TC-1 cells (3 103 cells) per well (96 well plate) were treated with free PS or conjugates (5, 6, 9 and 10), ranging 0.25 and 0.5 lM, after incubation for 12 h (37 °C, 5% CO2) in RPMI 1640 containing 5% FBS, then laser irradiation (662 ± 3 nm, 6.25 J/cm2) was performed. Cell growth inhibition was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) 24 h after irradiation. For the MTT assay, 20 lL of 5 mg/mL MTT solution was added to each cell-culture well and cultured for 4 h. Next 100 lL of dimethylsulfoxide was added to the culture, shaken for 10 s, and the absorbance was measured with an ELISA-reader (Spectra Max 340/Molecular Devices, USA) at 570 nm. Measurements were performed every 24 h after laser irradiation. Each group consisted of three wells; the means of their values were used as the measured values. 2.9. Cellular uptake The 24 h after incubation with compounds 4, 5, 6, 8, 9 and 10 (1 lM) at 37 °C, cells (5 104 cells/well in 6-well plate) were washed twice with PBS buffer. After that, 1 mL of paraformaldehyde (PFA) (1%) was loaded into 6-well plates for fixation. After 15 min, the supernatant was discarded and the cover glasses holding cells were putted on a slide glass where a drop of mounting solution was placed. After drying for 4 h, fluorescence was visualized under a confocal spectral microscopy (Leica, model TCS SP2). Fluorescence images were taken at the emission 545 nm. 3. Results and discussion 3.1. Preparation of the pyropheophorbide derivatives and their fatty acid conjugates
2.7. Singlet oxygen quantum yields Singlet oxygen quantum (SOQ) yields were measured by using a modification of the technique described by Bae and Na [18a]. In brief, to determine the SOQY of PSs, singlet oxygen trap was prepared using 20 mM 9,10-dimethylanthracene (DMA). 50 lM PSs were prepared in N,N-dimethylformamide (DMF) and added to DMA stock solution to give a final concentration of 20 lM DMA. After a 10 min period of equilibration, an appropriate amount of DMA was added. Samples containing sensitizers and DMA were irradiated at a light intensity of 5 mW/cm2 using a 670 nm laser
As showed in Scheme 1, in this study, methyl pyropheophorbide-a was used as a starting material. To prepare the starting material, compound 1 was obtained from Spirulina maxima algae according to previously reported methodologies by Smith et al. [17a] and Galindev et al. [17b], which was then used to obtain compound 2. Compounds 3 (methyl pyropheophorbide-d), 4 (3-devinyl-3-(10 -hydroxyl) pyropheophorbide-a-methyl ester), and 7 (pyropheophorbide-a) were prepared by following known methodologies [19,21]. Compound 8 was first prepared from compound 7 in high yield using a DCC and NHS coupling reagents
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Scheme 1. Synthesis of the chlorin-based UFA conjugates. Reaction conditions and reagents: (a) extraction and acidic treatment; (b) reflux in collidine for 1.5 h; (c) NaIO4, OsO4 and acetic acid in THF under N2 at room temperature for 14.5 h; (d) tert-butyl borane complex in DCM at room temperature for 20 min; (e) DHA or OA, DCC/DMAP in DCM under Ar at room temperature for 2 h; (f) H2SO4 (50%) for 2 h; (g) 6-hexanolamine, DCC/NHS, in DCM; (k) DHA or OA, EDC-HCI, in DCM under Ar at room temperature for 3 h.
based on procedures reported by Ronsin et al. and Yoo and Park [20]. To prepare polyunsaturated fatty acids (PUFAs) and photosensitizer conjugates, docosahexaenoic acid was linked with 3-devinyl-3-(10 -hydroxyl) pyropheophorbide-a-methyl ether (4) and pyropheophorbide-a-17-(4-N-hexanol) (8), respectively. The related monounsaturated acid (MUFA), oleic acid, was also similarly conjugated to compounds 4 and 8 in order to observe any differences between polyunsaturated and monounsaturated bonds in the acids. To synthesize these compounds, we decided to establish
the linkage using an esterification reaction between the reactive carboxyl groups of those acids and the reactive hydroxyl groups of the PSs. The esterification reactions were performed using carbodiimide mediated (DCC or EDC-HCl) coupling reagents in the presence of DMAP [22]. In organic synthesis, coupling reagents containing the carbodiimide functionality are often used to activate carboxylic acids towards ester or amide formation in high yields [23]. A byproduct N,N0 -dicyclohexylurea (DCU) was removed from ethyl acetate or cold chloroform solution via suction filtration. Then, targeted compounds were purified using column
Fig. 1. Molecular structures of the chlorin-based fatty acid conjugates. All carbon atoms in these conjugates are numbered.
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Fig. 2. 1H NMR spectra of chlorin derivatives and chlorin-based fatty acid conjugates. (a) 3-Devinyl-3-(10 -hydroxyl) pyropheophorbide-a-methyl ester (4); (b) 3-devinyl-3OA-PPa-methyl ester (OA-Mpp) (6); (c) 3-devinyl-3-DHA-PPa-methyl ester (DHA-Mpp) (5); (d) pyropheophorbide-a-174-N-hexanol (PPa-He, 8); (e) PPa-He-1710-OA (PpaOA) (10); (f) PPa-He-101-DHA (Ppa-DHA) (9).
chromatography on silica gel (merck-60) and afforded in high yields. Compounds 6 and 10 were purified using a preparative TLC (Merck) under same condition. The structures of all compounds were fully characterized using 1H NMR, 1H–1H COSY NMR, MALDI-TOF-MS, and UV–vis spectroscopy. 3.2. Characterization of the pyropheophorbide derivatives and their fatty acid conjugates The 1H NMR spectrum of compound 8 confirmed its formation by the appearance of the triplet, singlet, multiplet, quartet, pentat, and multiplets peaks (at 7.7, 4.27, 3.30, 2.95, 1.22, and 1.15 ppm)
corresponding to protons at 174-NH, 1710-OH, 1710-CH2, 175-CH2, 176-CH2, and 177-, 178-CH2 positions, respectively. In addition to 1 H NMR, MALDI-MS data showed a molecular ion peak at m/z 634.2467 (100%) for (M + H)+. In the 1H NMR spectra (Fig. 2) of compounds 5, 6, 9 and 10 (Fig. 1), the structures of these compounds was confirmed by the appearance of following significant signals: the signals of six cisdouble bonds (12 H) and methylene groups (10 H) between of two double bonds in DHA moieties of compounds 5 and 9 appeared as multiplets at 5.30–5.34 ppm and 2.74–2.79 ppm, respectively. In the 1H NMR of compounds 6 and 10, the signals of protons (2 H) of a double bond and methylene protons (20 H) in long chain of OA
Fig. 3. Fluorescence spectra of PS (50 lM) in DMF at exitation wavelength 500 nm. (a) Fluorescence peaks of compounds 4, 5, and 6 at 673, 676, and 675 nm respectively and (b) fluorescence peaks of compounds 8, 9, and 10 at 685, 681, and 678 nm, respectively.
G. Battogtokh et al. / Journal of Photochemistry and Photobiology B: Biology 110 (2012) 50–57 Table 1 Singlet oxygen quantum yields of the PS and conjugates detected by indirect method.
a
Compounds
u4 a
3-Hydroxyl methyl pyropheophorbide-a (HO-MPPa) (4) DHA-MPP (5) OA-MPP (6) Pyropheophorbide-a-173-(NH) hexanol (PPa-he) (8) PPa-DHA (9) PPa-OA (10)
0.26 0.24 0.25 0.28 0.24 0.21
States singlet oxygen quantum yield.
moiety were observed as mutiplets at 5.18–5.32 ppm and 1.13– 1.20 ppm, respectively. Moreover, singlet peaks of protons at 31CH2 of 5 and 6 shifted downfield as 0.49 and 0.47 ppm in each case, than that of 4 because of the formation of an ester bond at the 31 position. No signal for the proton 1710-hydroxyl group in the PPa moiety of compounds 9 and 10 was evident. In addition, a triplet at 1710-CH2 and a pentat at 179-CH2 of 9 and 10 shifted downfield as 0.63; 0.61 and 0.12; 0.11 ppm, respectively. Furthermore, a triplet at 174-NH of 9 greatly shifted upfield as 2.93 ppm. All other protons’ peaks were clearly revealed at corresponding regions. As for mass spectrometer (MALDI-TOF), data of compounds 5, 6, 9, and 10 showed the molecular ion peaks (100%) at m/z 863.4110 þ + + for C55 H66 N4 Oþ 5 (M + H) , 816.3974 for C51 H68 N4 O5 (M + H) , þ + 944.4903 for C61 H77 N5 Oþ (M) and 898.4696 for C H N O 57 79 5 4 4 + (M) , respectively. The UV spectra of all the derivatives showed characteristic absorptions for the chlorin related compounds. Indeed, there was no significant difference between the electronic absorption spectra for all the chlorin derivatives. However, the UV spectrum of compound 8 in the same solvent revealed an absorption peak at 669 nm, 4 nm more than that of compound 7. This absorption was also observed for compounds 9 and 10. In addition, extinction coefficients of all conjugates slightly increased than those of parent derivatives. In the fluorescence spectra (Fig. 3) of conjugates, compounds 4, 5, and 6 showed the emission peaks at 673, 676, and 675 nm, respectively. While, compounds 8, 9, and 10 revealed the emission peaks at 685, 681, and 678 nm, respectively. In both case, oleic acid-based conjugates exhibited higher intensity peaks, whereas DHA-based conjugates showed one had lower intensity and the other had higher intensity peaks. There were slight blue (for 5 and 6) and red (for 9 and 10) shifts between emission wavelengths of free PS and conjugates. Furthermore, as shown in Table 1, singlet oxygen quantum yields (SOQYs) (u4) were calculated for each PS with 9,10-dimethylanthracene (DMA) as the probe molecule in N,N-dimethylformamide (DMF). For these measurements, DMA was employed as a 1 O2 trap, since it reacts selectively with 1O2 to form the 9,10-endoperoxide (DMAO2).
55
Therefore, the SOQY for each compound was obtained by comparing the slope of the decrease in fluorescence intensity versus time for the molecule of interest against that of a standard. The kinetic traces of the photosensitization reactions were fitted to an analytical function, and the UD values were evaluated. At 2 mL solution of 20 lM DMA in DMF containing 50 lM PS, 8 was found to have the highest value of UD (0.28), followed by 4 (0.26), 6 (0.25), 5 (0.24) and 9 (0.24) as well as 10 (0.21). The UD of PS-fatty acid conjugates obtained from 3-hydroxyl methyl pyropheophorbide-a were not decreased. However, in case of conjugates obtained from pyropheophorbide-a-(173-NH)-hexanol were slightly decreased. Although the calculated UD values were a slightly lower than that of the standard PPa (7), they were close to that of MPPa (2) in the literature (UD = 0.2; in ethanol) [18]. Since our obtained PSs are quite lipophilic, they are not soluble in water. Thus, the conjugates can be aggregated in aqueous solution, as a result they can show reduced fluorescence intensities and singlet oxygen quantum yield in aqueous solution [24]. This might be a barrier for animal experiment, because hydrophobic PSs can be aggregated during circulation in blood [25]; however, it can be solved using a drug carrier and reducing concentration of PS [25,26]. Contrarily, hydrophilic PSs are difficult to penetrate the phospholipid bilayer of the cell’s plasma membrane compared to lipophilic PSs [25]. 3.3. Phototoxicity Phototoxicity of the unsaturated fatty acid–chlorophyll derivatives prepared above was evaluated against TC-1 cancer cell line. Among these conjugates, conjugates 5 and 6 at concentration of 0.25 and 0.5 lM showed higher phototoxicity than conjugates 9 and 10 as shown in Fig. 4. In other case, conjugates 9 and 10 revealed a slightly darktoxicity. Actually, the conjugates were established at two different positions (3rd for 5 or 6 and 17th for 9 or 10) of PS through an esterification reaction between a hydroxyl group of PS and a carboxyl group of fatty acids. Based on above, the conjugation at 17th position may help to happen a quenching between PS and unsaturated fatty acid and it might lead to decrease the singlet oxygen yield, as well. Subsequently, a position of conjugation may possess a major role for activity of conjugates. We also checked the time-dependent cytotoxicity of the conjugates at period of 1–24 h on TC-1 cell line. Interestingly, the result showed that conjugates 5 and 6 were able to show 100% cytotoxicity against TC1 cells after 3 h, while the other two revealed around 40% cell cytotoxicity and no significant change observed even after 24 h (data not shown). 3.4. Cellular uptake We examined the cellular uptake of these PS in TC-1 and HeLa cell lines at 24 h after incubation with PS by confocal microscopy
Fig. 4. Cell test results. In vitro phototoxicity and darktoxicity of compounds 4, 5, 6, 8, 9, and 10 at concentrations of 0.25 and 0.5 lM in TC-1 cells. The data are expressed as mean of three experiments.
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Fig. 5. Cellular uptake of photosensitizers (1 lM) on TC-1 cell line (a) and HeLa cell line (b) by confocal microscopy after 24 h incubation.
to compare uptake of the PSs into two cell lines. The results (Fig. 5a and b) showed that the conjugates have similar cellular uptake. Even though conjugates 9 and 10 did not reveal higher PDT effect in TC-1 cell line at given concentrations, they showed slightly higher uptake compared to the others. In case of HeLa cells, the PSs showed a similar cellular uptake as shown in Fig. 5b. 4. Conclusion In the summary, we have synthesized four kinds of chlorin (pyropheophorbide)-based fatty acid conjugates and their structures were fully characterized by 2D 1H NMR, MALDI-TOF mass spectrometer and UV–vis spectroscopy. The UD value for each conjugate was calculated for better understanding of the kinetic mechanism of the photosensitization. Their phototoxicity in vitro was evaluated on TC-1 cancer cell line. And the TC-1 cell test data
indicated that (5 and 6) both can be potential photosensitizers compared to the other two conjugates (9 and 10), even though their phototoxicity was similar to that of free PS used in this study. In fact, as previously reported [13,27], in most cases the in vitro efficacy of fatty acid conjugates linked with anticancer drug was lower than that of free drug but in vivo efficacy of those conjugates was higher than that of free drug used in the study. In case of us, it might be similar. However, our further work will be focused on more detail in vitro experiment especially a mechanism and localization of PS in different cell lines. References [1] (a) T.J. Dougherty, C.J. Gomer, B.W. Henderson, G. Jori, D. Kessel, M. Korbelik, J. Moan, Q. Peng, Photodynamic therapy, J. Natl. Cancer Inst. 90 (1998) 889–905; (b) I.J. Macdonald, T.J. Dougherthy, Basic principles of photodynamic therapy, J. Porphyr. Phthalocya. 5 (2001) 105–129.
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Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Synthesis of chlorin-based unsaturated fatty acid conjugates: Their in vitro phototoxicity on TC-1 cancer cell line Gantumur Battogtokh a, Hai-Bo Liu a, Su-Mi Bae a, Pankaj Kumar Chaturvedi a, Yong-Wan Kim a, In-Wook Kim a, Woong Shick Ahn a,b,⇑ a b
Cancer Research Institute, Catholic Research Institute of Medical Science, The Catholic University of Korea, Republic of Korea Department of Obstetrics and Gynecology, Seoul St. Mary’s Hospital, The Catholic University of Korea, Republic of Korea
a r t i c l e
i n f o
Article history: Received 15 December 2011 Received in revised form 2 March 2012 Accepted 13 March 2012 Available online 22 March 2012 Keywords: PDT Methyl pyropheophorbide-a Unsaturated fatty acid Photosensitizer Phototoxicity Synthesis
a b s t r a c t Chlorin-based photosensitizers in photodynamic therapy are the promising anticancer agents, but some of their properties such as specific-targeting to tumor need to be improved. The aim of this study was to synthesize chlorin-based unsaturated fatty acid conjugates to obtain an optimal photosensitizers. Thus four chlorin-based fatty acid conjugates were successfully synthesized through an esterification reaction using carbodiimide coupling reagents in enough yields. Then, structures of these conjugates were confirmed by 1H NMR, MALDI-MS, and UV–vis spectroscopy. Furthermore, their in vitro phototoxicity and cellular uptake were evaluated on TC-1 lung cancer cell line and HeLa cell line. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction Photodynamic therapy (PDT) is a promising cancer treatment that requires a combination of a photosensitizer (PS), tissue oxygen, and light to produce cytotoxic reactive oxygen species, mostly singlet oxygen [1,2]. During the last few years, a number of dyes which strongly absorb a light in the range of 650–800 nm have been reported as potential photosensitizers for PDT [3,4]. Of these, chlorin- and bacteriochlorin-based photosensitizers are monomeric compounds, efficient generators of singlet oxygen, and may be particularly effective in treating large and/or deeply seated tumors by absorbing long wavelengths [5]. Although most first and second-generation PSs studied for PDT display only a slight preference for malignant cells, they often lead to significant skin photosensitivity (Photofrin) and high uptake by healthy cells and tissues [6,7]. In order to overcome these side effects, third-generation PSs are being designed and synthesized to actively target diseased tissue [6]. In general, there are two pathways in this strategy: (i) modification of the photosensitizing agent moiety through its
⇑ Corresponding author at: Department of Obstetrics and Gynecology, Seoul St. Mary’s Hospital, The Catholic University of Korea, 505 Banpo-dong, Seocho-ku, Seoul 137-040, Republic of Korea. Tel.: +82 2 2258 6946; fax: +82 2 599 4120. E-mail address: [email protected] (W.S. Ahn). 1011-1344/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jphotobiol.2012.03.004
physicochemical properties or (ii) improving the targeting by conjugation of a photosensitizer to a molecule such as antibody, polymer, peptide scaffold, and carbohydrate [8,9]. Recently, poly unsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA), have been investigated for its potential for treating cancers and neurodegenerative disorders [10]. Some PUFAs have also been known to be rapidly taken up by tumor cells from the arterial blood, presumably for use as biochemical precursors and energy sources [11]. In addition, PUFAs are readily incorporated into the lipid bilayer of cells, resulting in the distraction of membrane structure and fluidity [11,12]. Bradley et al. [13] have obtained a paclitaxel–DHA conjugate that had greater antitumor activity compared to paclitaxel. As reported by Siddiqui et al. [14], long chain PUFAs (DHA) have been known to be highly susceptible to both enzymatic and non-enzymatic peroxidations since their discovery. Interestingly, reactive oxygen substrates (super oxide and hydrogen peroxide) produced from a PS upon irradiation might lead to the initiation of the peroxidation chain reaction of PUFAs [15]. As encouraged via above reports, we aimed to conjugate the UFAs to the chlorin derivatives through an esterification reaction. UFAs were linked to the different sites of PSs to observe their activity. Here, we report the characterization and synthesis of four kinds of chlorin-based fatty acid conjugates, and their phototoxicity in vitro against TC-1 lung cancer cell line.
G. Battogtokh et al. / Journal of Photochemistry and Photobiology B: Biology 110 (2012) 50–57
2. Materials and methods
51
2.3. 3-Hydroxymethyl-3-devinylpyropheophorbide-a-methyl ester and docosahexaenoic acid conjugate (5)
2.1. General All reagents were purchased from Sigma, Aldrich, Fluka, Alfa Aesar and Daihan companies. If necessary, anhydrous solvents were distilled according to standard procedures [16]. Other commercially available reagents were used without further purification. Column chromatographic separations were performed over silica gel 60 (63–200 and 200–400 mesh, Merck). Analytical thin layer chromatography (TLC) was carried out on sheets precoated with silica gel F254 (0.2 mm thick, Merck). All reactions were carried out under an argon atmosphere in darkness. The progress of reactions was monitored by TLC and detection was carried out by means of an UV – lamp at 265 nm or 365 nm wavelengths. Electronic absorption spectra were recorded on an UltrospecÒ3000 (Pharmacia Biotech, Cambridge, England) spectrophotometer. 1H NMR spectra were recorded on Unity Inova 500 (UI 500, Varian Inc., USA) spectrometer at 500 MHz; chemical shifts are expressed in parts per million (ppm) relative to tetramethylsilane (TMS, 0.00). MALDI mass spectra were obtained with a Voyager-DE™ STR Biospectrometry Workstation spectrometer. Fluorescence spectra were obtained using a spectrofluorophotometer (RF-5301 PC; Shimadzu, Japan) with a xenon short-arc lamp (XBO 150; Ushino, Japan). Compound 7 was prepared from methyl pyropheophorbide-a (2), which was obtained via methyl pheophorbide-a (1) from Spirulina maxima algae according to the previously reported methods [17]. Compound 4 was prepared from methyl pyropheophorbide-d (3), which was obtained from methyl pyropheophorbide-a (2). 2.2. Preparation of pyropheophorbide-a-173-N-hexanol (8) Pyropheophorbide-a (7) (100 mg, 0.186 mmol), N,N0 -dicyclohexylcarbodiimide (DCC) (77 mg, 0.37 mmol) and N-hydroxysuccinimide (NHS) (26 mg, 0.226 mmol) were dissolved in 3 mL of anhydrous dichloromethane. Three drops of triethylamine were added and stirred under argon gas at room temperature. After 4 h, 6-hexanolamine (22 mg, 0.188 mmol) in methanol (2 mL) was added to the reaction mixture and stirred for 1.5 h. Next, 10 ml of water was added to the solution and stirred for 10 min, and diluted with dichloromethane (30 mL). The organic layer was joined, washed twice with water (100 mL), and dried over anhydrous sodium sulfate. After evaporation, the residue was dissolved in 5 mL of chloroform or ethylacetate and filtered through cold (10 °C) to remove side-mixtures. Subsequently, the residue was purified on silica gel (70–230 mesh) by column chromatography using 5% methanol in dichloromethane as an eluant. After purification, pure product gave as a pink–blue solid. Yield: 140.2 mg (71%), Rf 0.31 (in dichloromethane: methanol/95:5). UV–vis (CH2Cl2): kmax, nm (e, M1 cm1) 668.4 (47360.0), 610.5 (20446.5), 540.0 (22443.7), 414.8 (90725.8). 1H NMR (500 MHz, CDCl3, Me4Si): dH, ppm 9.60 (1H, s, 5-meso-H), 9.34 (1H, s, 10-meso-H), 8.86 (1H, s, 20-meso-H), 8.15 (1H, dd, J = 11.5, 17.8 Hz, 31-CH,), 7.70 (1H, t, J = 5.5 Hz, 174-NH), 6.35 and 6.18 (2H, each d, J = 18.0; 11.5 Hz, 32-CHa, 32-CHb), 5.22 and 5.08 (2H, each d, J = 19.8; 19.7 Hz, 132CH2), 4.55 (1H, q, J = 7.2 Hz, 18-CH), 4.27 (1H, s, 1710-OH), 4.26 (1H, d, J = 8.5 Hz, 17-CH), 3.61 (2H, q, J = 7.5 Hz, 81-CH2), 3.57 (3H, s, 121-CH3), 3.41 (3H, s, 21-CH3), 3.30 (2H, q, J = 6.4 Hz, 1710CH2), 3.14 (3H, s, 71-CH3), 2.95 (2H, m, 174-CH2), 2.60–2.32 (2H, m, 171-CH2), 2.16–2.06 (2H, m, 172-CH2), 1.79 (3H, d, J = 7.3 Hz, 181-CH3), 1.58 (3H, t, J = 7.5 Hz, 82-CH3), 1.30 (2H, p, J = 6.9 Hz, 179-CH2), 1.22 (2H, p, J = 6.9 Hz, 176-CH2), 1.15 (4H, m, 177 and 178-CH2), 0.14 and 1.67 (2H, each br s, 21-, 23-NH). MS (MALDI-TOF): m/z 634.2467 [M + H]+ (calcd. for C39H47N5O3 633.8222).
4 (30 mg, 0.054 mmol), DCC (22.4 mg, 0.108 mmol), and 4(dimethylamino) pyridine (DMAP) (9.9 mg, 0.081 mmol) in dichloromethane (5 mL) were stirred and 17.8 mg (0.054 mmol) of docosahexaenoic acid (DHA) was added. The reaction was carried out under argon gas at room temperature for 2 h. Next, 10 mL of water was added to the reaction mixture, stirred for 10 min, and diluted with dichloromethane (20 mL). The organic layer was joined and washed with water two times, and dried over anhydrous sodium sulfate. After evaporation, the residue was dissolved in chloroform or ethyl-acetate and filtered by suction to remove N,N0 -dicyclohexylurea (DCU) (formed from DCC). Then, the residue was purified on silica gel (230–400 mesh) by column chromatography using 2% methanol in dichloromethane as an eluant. The product was dried at 25 °C in a vacuum oven. The structure of the compound was characterized by 1H NMR and UV–vis spectroscopy. Yield: 39.78 mg (85%); pink–blue melt; Rf 0.55 (in dichloromethane:methanol/ 98:2). UV–vis (CH2Cl2): kmax, nm (e, M1 cm1) 665.1 (59547.2), 606.8 (22369.0), 537.1 (25165.0), 411.2 (102007.0). 1H NMR (500 MHz, CDCl3, Me4Si): dH, ppm 9.53 (1H, s, 5-meso-H), 9.42 (1H, s, 10-meso-H), 8.6 (1H, s, 20-meso-H), 6.38 (2H, s, 31-CH2), 5.30 (13H, m, 35;6;8;9;11;12;14;15;17;18;20;21, 132-CHa, overlapped) and 5.11 (1H, d, J = 19.5 Hz, 132-CHb), 4.5 (1H, q, J = 7.3 Hz, 18CH), 4.31 (1H, d, J = 8.5 Hz, 17-CH), 3.70 (2H, q, J = 7.6 Hz, 81-CH2), 3.68 (3H, s, 174-CH3), 3.6 (3H, s, 121-CH3), 3.44 (3H, s, 21-CH3), 3.26 (3H, s, 71-CH3), 2.74 (10H, m, 37,10,13,16,19-CH2), 2.71–2.52 (2H, m, 171-CH2), 2.50 (2H, m, 34-CH2), 2.46 (2H, t, J = 6.8 Hz, 33-CH2), 2.29 (2H, m, 172-CH2), 2.03 (2H, p, J = 7.3 Hz, 322-CH2), 1.81 (3H, d, J = 7.3 Hz, 181-CH3), 1.70 (3H, t, J = 7.6 Hz, 82-CH3), 0.93 (3H, t, J = 7.5 Hz, 323-CH3), 0.31 and 1.70 (2H, each br s, 21-, 23-NH). MS (MALDI-TOF): m/z 863.4110 [M + H]+ (calcd. for C55H66N4O5 863.1363). 2.4. 3-Hydroxymethyl-3-devinylpyropheophorbide-a-methyl ester and oleic acid conjugate (6) 4 (25 mg, 0.045 mmol), DCC (22 mg, 0.09 mmol), and DMAP (6.1 mg, 0.05 mmol) in DCM were stirred together and 12.77 mg (0.045 mmol) of oleic acid (OA) was added to the reaction mixture. The reaction was carried out according to the procedure above. Yield: 30.9 mg (84%); pink–blue solid; Rf 0.42 (in dichloromethane: methanol/98:2). UV–vis (CH2Cl2): kmax, nm (e, M1 cm1) 665.0 (72059.5), 607.0 (24895.2), 537.0 (28046.5), 411.1 (126787.0). 1H NMR (500 MHz, CDCl3, Me4Si): dH, ppm 9.44 (1H, s, 5-meso-H), 9.42 (1H, s, 10-meso-H), 8.61 (1H, s, 20-meso-H), 6.38 (2H, s, 31CH2), 5.28 and 5.13 (2H, each d, J = 19.5; 19.5 Hz, 132-CH2), 5.18 (2H, m, 310,11-CH), 4.50 (1H, q, J = 7.3 Hz, 18-CH), 4.31 (1H, d, J = 8.5 Hz, 17-CH), 3.7 (2H, q, J = 7.6 Hz, 81-CH2), 3.68 (3H, s, 174CH3), 3.60 (3H, s, 121-CH3), 3.44 (3H, s, 21-CH3), 3.27 (3H, s, 71CH3), 2.7–2.56 (2H, m, 171-CH2), 2.43 (2H, t, J = 7.5 Hz, 33-CH2), 2.29 (2H, m, 172-CH2), 1.88 (4H, m, 39,12-CH2), 1.80 (3H, d, J = 7.3 Hz, 181-CH3), 1.70 (3H, t, J = 7.6 Hz, 82-CH3), 1.67 (2H, m, 34-CH2), 1.13 (20H, m, 35;6;7;8;13;14;15;16;17;18-CH2), 0.83 (3H, t, J = 2.8 Hz, 319-CH3), 0.31 and 1.79 (2H, each br s, 21-, 23-NH). MS (MALDI-TOF): m/z 816.3974 [M + H]+. (calcd. for C51H68N4O5 817.1094). 2.5. Pyropheophorbide-a 173-N-(4-hexanol) and docosahexaenoic acid conjugate (9) 8 (30 mg, 0.047 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC-HCl) (19.5 mg, 0.094 mmol), and DMAP (6.9 mg, 0.056 mmol) in 3 mL of chloroform were stirred together and 15.5 mg (0.047 mmol) of docosahexaenoic acid (DHA) was
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added to the reaction mixture. The reaction was carried out under argon gas at room temperature for 3 h. Next, the reaction mixture was diluted with dichloromethane (DCM) (20 mL), the organic layer was joined and washed with water two times, and dried over anhydrous sodium sulfate. After evaporation, the residue was purified on silica gel by preparative TLC (20 20) using 3% methanol in dichloromethane as an eluant. The product was dried with phosphorus pentoxide at 25 °C in a vacuum oven. Yield: 33.5 mg (75%); pink–blue solid; Rf 0.17 (in dichloromethane: methanol/ 98:2). UV–vis (CH2Cl2): kmax, nm (e, M1 cm1) 668.5 (56923.5), 610.0 (28036.9), 539.5 (31010.6), 414.9 (105067.8). 1H NMR (500 MHz, CDCl3, Me4Si): dH, ppm 9.5 (1H, s, 5-meso-H), 9.41 (1H, s, 10-meso-H), 8.56 (1H, s, 20-meso-H), 8.03 (1H, dd, J = 11.5; 17.8 Hz, 31-CH,), 6.29 and 6.18 (2H, dd, J = 17.9; 11.5 Hz, 32a-, 32b-CH), 5.34 (12H, m, 1714;15;17;18;20;21;23;24;26;27;29;30-CH), 5.18 (2H, dd, J = 19.5; 19.5 Hz, 132-CH2), 4.77 (1H, t, J = 5.5 Hz, 174-NH), 4.51 (1H, q, J = 7.3 Hz, 18-CH), 4.36 (1H, d, J = 8.5 Hz, 17-CH), 3.93 (2H, t, J = 6.7 Hz, 1710-CH2), 3.70 (2H, q, J = 7.6 Hz, 81-CH2), 3.66 (3H, s, 121-CH3), 3.41 (3H, s, 21-CH3), 3.25 (3H, s, 71-CH3), 2.93 (2H, m, 175-CH2), 2.79 (10H, m, 1716,19,22,25,28-CH2), 2.7–2.4 (2H, m, 171-CH2), 2.30 (2H, m, 1713-CH2), 2.06–1.82 (2H, m, 172-CH2), 2.04 (2H, p, J = 7.4 Hz, 1731-CH2), 1.80 (3H, d, J = 7.3 Hz, 181-CH3), 1.70 (3H, t, J = 7.6 Hz, 82-CH3), 1.61 (2H, m, 1712-CH2), 1.42 (2H, p, J = 7.2 Hz, 179-CH2), 1.05 (6H, m, 17 6,7,8CH2), 0.95 (3H, t, J = 7.5 Hz, 1732-CH3), 0.47 and 1.68 (2H, each br s, 21-, 23-NH). MS (MALDI-TOF): m/z 944.4903 [M]+ (calcd. for C61H77N5O4 944.2952). 2.6. Pyropheophorbide-a-173-N-(4-hexanol) and oleic acid conjugate (10) 8 (30 mg, 0.047 mmol), EDC-HCl (19.5 mg, 0.094 mmol), and DMAP (6.9 mg, 0.056 mmol) in 3 mL of chloroform were stirred together and 13.4 mg (0.047 mmol) of oleic acid (OA) was added to the reaction mixture. This reaction was carried out according to the procedure above. Yield: 34 mg (80%), Rf 0.13 (in dichloromethane:methanol/98:2). UV–vis (CH2Cl2): kmax, nm (e, M1 cm1) 668.6 (110061.6), 610.2 (53819.8), 539.6 (59201.8), 414.9 (202093.5). 1H NMR (500 MHz, CDCl3, Me4Si): dH, ppm 9.49 (1H, s, 5-meso-H), 9.4 (1H, s, 10-meso-H), 8.56 (1H, s, 20-meso-H), 8.02 (1H, dd, J = 11.5; 17.8 Hz, 31-CH,), 6.29 and 6.18 (2H, each d, J = 17.9; 11.6 Hz, 32a-; 32b-CH), 5.32 (2H, m, 1719,20-CH), 5.24 and 5.11 (2H, dd, J = 19.5; 19.5 Hz, 132-CH2), 4.8 (1H, m, 174-NH), 4.51 (1H, q, J = 7.2 Hz, 18-CH), 4.36 (1H, d, J = 8.5 Hz, 17-CH), 3.91 (2H, t, J = 6.7 Hz, 1710-CH2), 3.7 (2H, q, J = 7.7 Hz, 81-CH2), 3.64 (3H, s, 121-CH3), 3.41 (3H, s, 21-CH3), 3.25 (3H, s, 71-CH3), 2.89 (2H, m, 175-CH2), 2.65 and 2.45 (2H, each m, 171-CH2), 2.18 (2H, t, J = 7.6 Hz, 1712-CH2), 2.15 and 1.86 (2H, each m, 172-CH2), 1.97 (4H, m, 1718,21-CH2), 1.80 (3H, d, J = 7.3 Hz, 181-CH3), 1.70 (3H, t, J = 7.6 Hz, 82-CH3), 1.51 (2H, m, 1713-CH2), 1.41 (2H, p, J = 7 Hz, 179-CH2), 1.24 (20H, m, 1714;15;16;17;22;23;24;25;26;27-CH2), 1.05 (6H, m, 176,7,8-CH2), 0.86 (3H, t, J = 6.7 Hz, 1728-CH3), 0.47 and 1.69 (2H, each br s, 21-, 23-NH). MS (MALDI-TOF): m/z 898.4696 [M]+ (calcd. for C57H79N5O4 898.2682).
source. A collimated laser beam was directed to the sample cuvette through an optical fiber. The decrease in fluorescence intensity of DMA (excitation, 360 nm) as a result of the photosensitization reaction was monitored using a spectrofluorophotometer controlled by a PC. SOQ yield was calculated by the following equation [18b]:
uDðUÞ ¼ uDðStÞ SðUÞ=SðStÞ where U and St denote unknown and standard, respectively, and S is the slope of bleaching of the probe absorbance with irradiation time. The SOQ yield (u4) of each sample was calculated from standard u4 value (PPa-0.52). 2.8. Cell culture and in vitro phototoxicity Human lung cancer cells (TC-1), which were cotransformed by HPV-16 E6/E7 oncoproteins and c-Has-Ras, were cultured in RPMI 1640 supplemented with 5% fetal bovine serum. 1% Penicillin– Streptomycin (Penicillin G, sodium salt; streptomycin sulfate) and 0.4 mg/mL G418 disulfate (Duchefa, Netherlands) were added to the medium. The cell line was maintained at 37 °C in humidified 5% CO2 atmosphere. For viable cell counting, TC-1 cells (3 103 cells) per well (96 well plate) were treated with free PS or conjugates (5, 6, 9 and 10), ranging 0.25 and 0.5 lM, after incubation for 12 h (37 °C, 5% CO2) in RPMI 1640 containing 5% FBS, then laser irradiation (662 ± 3 nm, 6.25 J/cm2) was performed. Cell growth inhibition was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) 24 h after irradiation. For the MTT assay, 20 lL of 5 mg/mL MTT solution was added to each cell-culture well and cultured for 4 h. Next 100 lL of dimethylsulfoxide was added to the culture, shaken for 10 s, and the absorbance was measured with an ELISA-reader (Spectra Max 340/Molecular Devices, USA) at 570 nm. Measurements were performed every 24 h after laser irradiation. Each group consisted of three wells; the means of their values were used as the measured values. 2.9. Cellular uptake The 24 h after incubation with compounds 4, 5, 6, 8, 9 and 10 (1 lM) at 37 °C, cells (5 104 cells/well in 6-well plate) were washed twice with PBS buffer. After that, 1 mL of paraformaldehyde (PFA) (1%) was loaded into 6-well plates for fixation. After 15 min, the supernatant was discarded and the cover glasses holding cells were putted on a slide glass where a drop of mounting solution was placed. After drying for 4 h, fluorescence was visualized under a confocal spectral microscopy (Leica, model TCS SP2). Fluorescence images were taken at the emission 545 nm. 3. Results and discussion 3.1. Preparation of the pyropheophorbide derivatives and their fatty acid conjugates
2.7. Singlet oxygen quantum yields Singlet oxygen quantum (SOQ) yields were measured by using a modification of the technique described by Bae and Na [18a]. In brief, to determine the SOQY of PSs, singlet oxygen trap was prepared using 20 mM 9,10-dimethylanthracene (DMA). 50 lM PSs were prepared in N,N-dimethylformamide (DMF) and added to DMA stock solution to give a final concentration of 20 lM DMA. After a 10 min period of equilibration, an appropriate amount of DMA was added. Samples containing sensitizers and DMA were irradiated at a light intensity of 5 mW/cm2 using a 670 nm laser
As showed in Scheme 1, in this study, methyl pyropheophorbide-a was used as a starting material. To prepare the starting material, compound 1 was obtained from Spirulina maxima algae according to previously reported methodologies by Smith et al. [17a] and Galindev et al. [17b], which was then used to obtain compound 2. Compounds 3 (methyl pyropheophorbide-d), 4 (3-devinyl-3-(10 -hydroxyl) pyropheophorbide-a-methyl ester), and 7 (pyropheophorbide-a) were prepared by following known methodologies [19,21]. Compound 8 was first prepared from compound 7 in high yield using a DCC and NHS coupling reagents
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Scheme 1. Synthesis of the chlorin-based UFA conjugates. Reaction conditions and reagents: (a) extraction and acidic treatment; (b) reflux in collidine for 1.5 h; (c) NaIO4, OsO4 and acetic acid in THF under N2 at room temperature for 14.5 h; (d) tert-butyl borane complex in DCM at room temperature for 20 min; (e) DHA or OA, DCC/DMAP in DCM under Ar at room temperature for 2 h; (f) H2SO4 (50%) for 2 h; (g) 6-hexanolamine, DCC/NHS, in DCM; (k) DHA or OA, EDC-HCI, in DCM under Ar at room temperature for 3 h.
based on procedures reported by Ronsin et al. and Yoo and Park [20]. To prepare polyunsaturated fatty acids (PUFAs) and photosensitizer conjugates, docosahexaenoic acid was linked with 3-devinyl-3-(10 -hydroxyl) pyropheophorbide-a-methyl ether (4) and pyropheophorbide-a-17-(4-N-hexanol) (8), respectively. The related monounsaturated acid (MUFA), oleic acid, was also similarly conjugated to compounds 4 and 8 in order to observe any differences between polyunsaturated and monounsaturated bonds in the acids. To synthesize these compounds, we decided to establish
the linkage using an esterification reaction between the reactive carboxyl groups of those acids and the reactive hydroxyl groups of the PSs. The esterification reactions were performed using carbodiimide mediated (DCC or EDC-HCl) coupling reagents in the presence of DMAP [22]. In organic synthesis, coupling reagents containing the carbodiimide functionality are often used to activate carboxylic acids towards ester or amide formation in high yields [23]. A byproduct N,N0 -dicyclohexylurea (DCU) was removed from ethyl acetate or cold chloroform solution via suction filtration. Then, targeted compounds were purified using column
Fig. 1. Molecular structures of the chlorin-based fatty acid conjugates. All carbon atoms in these conjugates are numbered.
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Fig. 2. 1H NMR spectra of chlorin derivatives and chlorin-based fatty acid conjugates. (a) 3-Devinyl-3-(10 -hydroxyl) pyropheophorbide-a-methyl ester (4); (b) 3-devinyl-3OA-PPa-methyl ester (OA-Mpp) (6); (c) 3-devinyl-3-DHA-PPa-methyl ester (DHA-Mpp) (5); (d) pyropheophorbide-a-174-N-hexanol (PPa-He, 8); (e) PPa-He-1710-OA (PpaOA) (10); (f) PPa-He-101-DHA (Ppa-DHA) (9).
chromatography on silica gel (merck-60) and afforded in high yields. Compounds 6 and 10 were purified using a preparative TLC (Merck) under same condition. The structures of all compounds were fully characterized using 1H NMR, 1H–1H COSY NMR, MALDI-TOF-MS, and UV–vis spectroscopy. 3.2. Characterization of the pyropheophorbide derivatives and their fatty acid conjugates The 1H NMR spectrum of compound 8 confirmed its formation by the appearance of the triplet, singlet, multiplet, quartet, pentat, and multiplets peaks (at 7.7, 4.27, 3.30, 2.95, 1.22, and 1.15 ppm)
corresponding to protons at 174-NH, 1710-OH, 1710-CH2, 175-CH2, 176-CH2, and 177-, 178-CH2 positions, respectively. In addition to 1 H NMR, MALDI-MS data showed a molecular ion peak at m/z 634.2467 (100%) for (M + H)+. In the 1H NMR spectra (Fig. 2) of compounds 5, 6, 9 and 10 (Fig. 1), the structures of these compounds was confirmed by the appearance of following significant signals: the signals of six cisdouble bonds (12 H) and methylene groups (10 H) between of two double bonds in DHA moieties of compounds 5 and 9 appeared as multiplets at 5.30–5.34 ppm and 2.74–2.79 ppm, respectively. In the 1H NMR of compounds 6 and 10, the signals of protons (2 H) of a double bond and methylene protons (20 H) in long chain of OA
Fig. 3. Fluorescence spectra of PS (50 lM) in DMF at exitation wavelength 500 nm. (a) Fluorescence peaks of compounds 4, 5, and 6 at 673, 676, and 675 nm respectively and (b) fluorescence peaks of compounds 8, 9, and 10 at 685, 681, and 678 nm, respectively.
G. Battogtokh et al. / Journal of Photochemistry and Photobiology B: Biology 110 (2012) 50–57 Table 1 Singlet oxygen quantum yields of the PS and conjugates detected by indirect method.
a
Compounds
u4 a
3-Hydroxyl methyl pyropheophorbide-a (HO-MPPa) (4) DHA-MPP (5) OA-MPP (6) Pyropheophorbide-a-173-(NH) hexanol (PPa-he) (8) PPa-DHA (9) PPa-OA (10)
0.26 0.24 0.25 0.28 0.24 0.21
States singlet oxygen quantum yield.
moiety were observed as mutiplets at 5.18–5.32 ppm and 1.13– 1.20 ppm, respectively. Moreover, singlet peaks of protons at 31CH2 of 5 and 6 shifted downfield as 0.49 and 0.47 ppm in each case, than that of 4 because of the formation of an ester bond at the 31 position. No signal for the proton 1710-hydroxyl group in the PPa moiety of compounds 9 and 10 was evident. In addition, a triplet at 1710-CH2 and a pentat at 179-CH2 of 9 and 10 shifted downfield as 0.63; 0.61 and 0.12; 0.11 ppm, respectively. Furthermore, a triplet at 174-NH of 9 greatly shifted upfield as 2.93 ppm. All other protons’ peaks were clearly revealed at corresponding regions. As for mass spectrometer (MALDI-TOF), data of compounds 5, 6, 9, and 10 showed the molecular ion peaks (100%) at m/z 863.4110 þ + + for C55 H66 N4 Oþ 5 (M + H) , 816.3974 for C51 H68 N4 O5 (M + H) , þ + 944.4903 for C61 H77 N5 Oþ (M) and 898.4696 for C H N O 57 79 5 4 4 + (M) , respectively. The UV spectra of all the derivatives showed characteristic absorptions for the chlorin related compounds. Indeed, there was no significant difference between the electronic absorption spectra for all the chlorin derivatives. However, the UV spectrum of compound 8 in the same solvent revealed an absorption peak at 669 nm, 4 nm more than that of compound 7. This absorption was also observed for compounds 9 and 10. In addition, extinction coefficients of all conjugates slightly increased than those of parent derivatives. In the fluorescence spectra (Fig. 3) of conjugates, compounds 4, 5, and 6 showed the emission peaks at 673, 676, and 675 nm, respectively. While, compounds 8, 9, and 10 revealed the emission peaks at 685, 681, and 678 nm, respectively. In both case, oleic acid-based conjugates exhibited higher intensity peaks, whereas DHA-based conjugates showed one had lower intensity and the other had higher intensity peaks. There were slight blue (for 5 and 6) and red (for 9 and 10) shifts between emission wavelengths of free PS and conjugates. Furthermore, as shown in Table 1, singlet oxygen quantum yields (SOQYs) (u4) were calculated for each PS with 9,10-dimethylanthracene (DMA) as the probe molecule in N,N-dimethylformamide (DMF). For these measurements, DMA was employed as a 1 O2 trap, since it reacts selectively with 1O2 to form the 9,10-endoperoxide (DMAO2).
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Therefore, the SOQY for each compound was obtained by comparing the slope of the decrease in fluorescence intensity versus time for the molecule of interest against that of a standard. The kinetic traces of the photosensitization reactions were fitted to an analytical function, and the UD values were evaluated. At 2 mL solution of 20 lM DMA in DMF containing 50 lM PS, 8 was found to have the highest value of UD (0.28), followed by 4 (0.26), 6 (0.25), 5 (0.24) and 9 (0.24) as well as 10 (0.21). The UD of PS-fatty acid conjugates obtained from 3-hydroxyl methyl pyropheophorbide-a were not decreased. However, in case of conjugates obtained from pyropheophorbide-a-(173-NH)-hexanol were slightly decreased. Although the calculated UD values were a slightly lower than that of the standard PPa (7), they were close to that of MPPa (2) in the literature (UD = 0.2; in ethanol) [18]. Since our obtained PSs are quite lipophilic, they are not soluble in water. Thus, the conjugates can be aggregated in aqueous solution, as a result they can show reduced fluorescence intensities and singlet oxygen quantum yield in aqueous solution [24]. This might be a barrier for animal experiment, because hydrophobic PSs can be aggregated during circulation in blood [25]; however, it can be solved using a drug carrier and reducing concentration of PS [25,26]. Contrarily, hydrophilic PSs are difficult to penetrate the phospholipid bilayer of the cell’s plasma membrane compared to lipophilic PSs [25]. 3.3. Phototoxicity Phototoxicity of the unsaturated fatty acid–chlorophyll derivatives prepared above was evaluated against TC-1 cancer cell line. Among these conjugates, conjugates 5 and 6 at concentration of 0.25 and 0.5 lM showed higher phototoxicity than conjugates 9 and 10 as shown in Fig. 4. In other case, conjugates 9 and 10 revealed a slightly darktoxicity. Actually, the conjugates were established at two different positions (3rd for 5 or 6 and 17th for 9 or 10) of PS through an esterification reaction between a hydroxyl group of PS and a carboxyl group of fatty acids. Based on above, the conjugation at 17th position may help to happen a quenching between PS and unsaturated fatty acid and it might lead to decrease the singlet oxygen yield, as well. Subsequently, a position of conjugation may possess a major role for activity of conjugates. We also checked the time-dependent cytotoxicity of the conjugates at period of 1–24 h on TC-1 cell line. Interestingly, the result showed that conjugates 5 and 6 were able to show 100% cytotoxicity against TC1 cells after 3 h, while the other two revealed around 40% cell cytotoxicity and no significant change observed even after 24 h (data not shown). 3.4. Cellular uptake We examined the cellular uptake of these PS in TC-1 and HeLa cell lines at 24 h after incubation with PS by confocal microscopy
Fig. 4. Cell test results. In vitro phototoxicity and darktoxicity of compounds 4, 5, 6, 8, 9, and 10 at concentrations of 0.25 and 0.5 lM in TC-1 cells. The data are expressed as mean of three experiments.
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Fig. 5. Cellular uptake of photosensitizers (1 lM) on TC-1 cell line (a) and HeLa cell line (b) by confocal microscopy after 24 h incubation.
to compare uptake of the PSs into two cell lines. The results (Fig. 5a and b) showed that the conjugates have similar cellular uptake. Even though conjugates 9 and 10 did not reveal higher PDT effect in TC-1 cell line at given concentrations, they showed slightly higher uptake compared to the others. In case of HeLa cells, the PSs showed a similar cellular uptake as shown in Fig. 5b. 4. Conclusion In the summary, we have synthesized four kinds of chlorin (pyropheophorbide)-based fatty acid conjugates and their structures were fully characterized by 2D 1H NMR, MALDI-TOF mass spectrometer and UV–vis spectroscopy. The UD value for each conjugate was calculated for better understanding of the kinetic mechanism of the photosensitization. Their phototoxicity in vitro was evaluated on TC-1 cancer cell line. And the TC-1 cell test data
indicated that (5 and 6) both can be potential photosensitizers compared to the other two conjugates (9 and 10), even though their phototoxicity was similar to that of free PS used in this study. In fact, as previously reported [13,27], in most cases the in vitro efficacy of fatty acid conjugates linked with anticancer drug was lower than that of free drug but in vivo efficacy of those conjugates was higher than that of free drug used in the study. In case of us, it might be similar. However, our further work will be focused on more detail in vitro experiment especially a mechanism and localization of PS in different cell lines. References [1] (a) T.J. Dougherty, C.J. Gomer, B.W. Henderson, G. Jori, D. Kessel, M. Korbelik, J. Moan, Q. Peng, Photodynamic therapy, J. Natl. Cancer Inst. 90 (1998) 889–905; (b) I.J. Macdonald, T.J. Dougherthy, Basic principles of photodynamic therapy, J. Porphyr. Phthalocya. 5 (2001) 105–129.
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