Bioorganic & Medicinal Chemistry 23 (2015) 1453–1462

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Pheophorbide-a conjugates with cancer-targeting moieties for targeted photodynamic cancer therapy Hyun You a, Hyo-Eun Yoon c, Pyeong-Hwa Jeong a, Hyojin Ko b,⇑, Jung-Hoon Yoon c,⇑, Yong-Chul Kim a,b,⇑ a

Department of Medical System Engineering (DMSE), Gwangju Institute of Science and Technology, 123 Cheomdangwagi-ro, Buk-gu, Gwangju 500-712, Republic of Korea School of Life Science, Gwangju Institute of Science and Technology, 123 Cheomdangwagi-ro, Buk-gu, Gwangju 500-712, Republic of Korea c Department of Oral & Maxillofacial Pathology, College of Dentistry, Deajeon Dental Hospital, Wonkang University, 77, Dunsan-ro, Seo-gu, Daejeon 302-120, Republic of Korea b

a r t i c l e

i n f o

Article history: Received 23 January 2015 Accepted 9 February 2015 Available online 16 February 2015 Keywords: Pheophorbide-a Conjugate Fluorescence Apoptosis PDT

a b s t r a c t Pheophorbide-a, a non-selective photosensitizer, was conjugated with cancer-targeting moieties, such as folic acid, the CRGDLASLC peptide, the cRGDfK peptide and leuprorelin, for the purpose of targeted photodynamic cancer therapy. The cellular uptake of pheophorbide-a conjugates in cancer cells overexpressing the corresponding receptors of the targeting moieties was largely enhanced compared with that in the receptor-negative cells. In the study of in vitro photodynamic activity and selectivity of pheophorbide-a conjugates in the receptor-positive and receptor-negative cells, a pheophorbide-a conjugate, (14) with an avb6 ligand (CRGDLASLC) exhibited the highest selectivity in the positive FaDu cells. Targeted PDT with 14 induced cell death through apoptosis and morphological apoptosis-like characteristics. These results suggest that pheophorbide-a conjugate 14 could be utilized in selective photodynamic therapy for oral cancers primarily expressing the avb6 receptor. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Traditional cancer chemotherapy can result in serious side effects caused by the inability to deliver the correct amount of drug directly to cancer cells without affecting normal cells.1 Therefore, anti-cancer strategies for selective drug targeting to cancer cells has been investigated to improve the efficacy and circumvent the side effects of therapeutic anticancer agents.2 Photodynamic therapy (PDT) has been regarded as a promising new cancer treatment modality, which typically includes the intravenous injection of a photosensitizer followed by illumination with visible light at the appropriate wavelength for the activation of the photosensitizer taken up in the cancer cells; this induces phototoxicity by generating reactive oxygen species.3 The primary drawback of current PDT with respect to oncology is low selectivity of the uptake of photosensitizers in cancers over normal tissues, which can result in acute damage to surrounding normal tissues and prolonged skin phototoxicity.4 To increase the selectivity of photosensitizers for cancer tissues, several new photosensitizers conjugated with cancer-targeting moieties have been investigated. One approach is through the conjugation of photosensitizers to monoclonal antibodies (mAbs) that ⇑ Corresponding authors. Tel.: +82 62 970 2502; fax: +82 62 970 2484. E-mail addresses: [email protected] (H. Ko), [email protected] (J.-H. Yoon), [email protected] (Y.-C. Kim). http://dx.doi.org/10.1016/j.bmc.2015.02.014 0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

are specific for tumor-associated antigens often termed photoimmunotherapy (PIT).5–7 Additionally, various efforts have been directed toward using targeting moieties, such as small molecules or peptides, that are specific for receptors overexpressed in tumors. For example, the meta-tetra (hydroxyphenyl) chlorin (m-THPC) and folic acid conjugate was proposed for improved selectivity in PDT against folate receptor-positive tumors.8 The 3-(10-hexyloxyethyl)-3-devinylpyropheophorbide-a (HPPH) and cyclic RGD peptide conjugate exhibited a remarkable impact on PDT efficacy due to increased uptake, intracellular localization, and clearance from tumor tissues.9 cRGDfK conjugated to zinc phthalocyanine was recently reported to improve intracellular fluorescence intensity and cytotoxicity.10 In this study, we investigated new conjugates of pheophorbidea (1, Fig. 1), an efficient chlorine-based photosensitizer, for targeted photodynamic cancer therapy using several cancer targeting moieties, such as folic acid (2), the CRGDLASLC peptide (3), cRGDfK (4) and leuprorelin (5).11–14 Folic acid, a member of the vitamin B family, is a ligand that can target the folate receptor, which is overexpressed on the surface of many malignant cells for survival and proliferation.15,16 The CRGDLASLC peptide, a type of cyclic RGD peptide linked by a bisulfide bridge, binds to the avb6 integrin receptor which is overexpressed in head and neck squamous cancer cells and plays roles in tumor migration and invasion.17 cRGDfK, another cyclic RGD peptide, recognizes the avb3 integrin receptor, which is an important cell adhesion

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Figure 1. Structures of pheophorbide-a, folic acid, cRGDfK, CRGDLASLC and leuprorelin.

molecule for tumor angiogenesis, progression, and metastasis in various cancer cells.18,19 Leuprorelin, an approved drug for the treatment of advanced prostate cancer, could be used as a targeting moiety for the luteinizing hormone-releasing hormone (LHRH) receptor, which is overexpressed in ovarian, breast and prostate cancer cells.20,21 In our experiments, the phototoxicity of pheophorbide-a was non-selective toward several cancer cells, such as FaDu, KB, A549, MCF7, MDA-MB 231 and HCT116 cells, after PDT. Because there have been no attempts to investigate pheophorbide-a conjugates with cancer-targeting moieties for selective PDT of cancer cells, we synthesized and evaluated new pheophorbide-a-based photosensitizers with the above targeting moieties. 2. Results and discussion

pheophorbide-a and 11, which was monoprotected from 10, produced 12. After the deprotection of the Boc group in 12, the resulting free amine of 13 was coupled with the CRGDLASLC peptide (3) via treatment with EDC and HOBt. For the synthesis of the conjugate of pheophorbide-a and cRGDfK (17), the aminobutanoyl moiety was employed (Scheme 3). Pheophorbide-a was reacted with N-hydroxysuccinimide to afford the activated compound 15, which was subsequently reacted with c-amino butyric acid (GABA) to produce compound 16. Finally, cRGDfK and compound 16 were coupled to synthesize the conjugate 17 bearing two amide bonds. Similarly, the EDC-mediated coupling of pheophorbide-a and leuprorelin afforded the conjugate 18, as shown in Scheme 3. All final compounds were purified via RP-HPLC and identified via LC–MS and HRMS (see Supplementary data).

2.1. Chemistry

2.2. Cellular uptake study

The synthetic procedure for the conjugate of pheophorbide-a and folic acid (9) is described in Scheme 1. Pheophorbide-a (1) was linked to Boc-monoprotected 2,20 -(ethylenedioxy)-bis(ethylamine) (6) to produce 7 through an EDC-mediated coupling reaction. The deprotection of the Boc group with TFA, followed by EDC-mediated coupling of 8 with folic acid (2), afforded the conjugate of pheophorbide-a and folic acid (9). Although folic acid has two possible coupling sites, pheophorbide-a coupled primarily with the c-carboxyl group of the glutamate residue of folic acid. The reactivity of the c-carboxyl group of the glutamate residue of folic acid is known to be stronger than the a-carboxyl group for amide coupling reactions,22 and only the c-conjugate is capable of binding to the folate receptor.23 Scheme 2 shows the synthetic approach for the conjugate of pheophorbide-a and CRGDLASLC (14). The EDC coupling of

To verify the selectivity of the conjugates toward the corresponding targeting moieties, the cellular uptake of 9, 14, 17, and 18 was determined every 0.5 h for up to 2 h post incubation with 2 lM conjugates as a function of time (see Supplementary data). Each compound exhibited significantly higher cellular uptake in cells with the corresponding receptor of the targeting moiety compared with the targeting receptor-negative cells, as determined via confocal microscopy. Figure 2 shows the intracellular confocal images of four pheophorbide-a conjugates at the time of the highest cellular uptake in the targeting receptor-positive cells. The cellular uptake of the pheophorbide-a conjugate of folic acid (9) in KB cells, which overexpress folate receptors, was significantly enhanced after 0.5 h incubation, whereas there was negligible uptake in A549 cells, a folate receptor-negative cell line (Fig. 2a).8 In the case of the pheophorbide-a conjugate of the

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Scheme 1. Synthesis of the conjugate of pheophorbide-a and folic acid (9).

CRGDLASLC peptide (14), 14 showed significantly higher levels of cellular uptake in avb6 integrin-overexpressing FaDu cells after 1 h incubation; however, very low levels of 14 were observed in avb6-negative A549 cells (Fig. 2b).24–26 Similarly, the cellular uptake of the pheophorbide-a conjugate of cRGDfK (17) was substantially higher in avb3 integrin-overexpressing U87MG cells than in avb3-negative A549 cells at 1 h post incubation (Fig. 2c).27 Additionally, the fluorescence confocal images for the uptake of the pheophorbide-a conjugate of leuprorelin (18) (Fig. 2d) after incubation for 1.5 h exhibited substantial selectivity for the LHRH receptor-overexpressing MCF7 cells vs. the LHRH receptornegative SKOV3 cells.11 2.3. In vitro photodynamic activity The in vitro photodynamic activities of the conjugates were evaluated against positive cells overexpressing the corresponding receptors of the targeting moieties and targeting receptor-negative cells based on the profiles of cellular uptake as shown in Figure 2. The conjugates were exposed to a light dose of 1.25 J/cm2 from a red light-emitting diode (LED), which was the source of light at wavelength of 613–645 nm wavelengths. As shown in Figure 3, the pheophorbide-a conjugate of folic acid (9) showed no photodynamic effect in A549 cells but a moderate effect in KB cells, which was correlated with the results of Figure 2a (Fig. 3a). Remarkably, the pheophorbide-a conjugate of CRGDLASLC (14) exhibited a very potent and selective photodynamic effect in avb6 integrinoverexpressing FaDu cells versus receptor-negative A549 cells. The distinguished selective photodynamic effect of 14 in FaDu

cells, particularly at 2 lM, is parallel to the larger cellular uptake of 14 in FaDu cells over A549 cells (Fig. 3b). However, in the case of the pheophorbide-a conjugate of cRGDfK (17), there is no difference in the photodynamic effect in both the receptor-positive U87MG and receptor-negative A549 cells, respectively, despite the higher cellular uptake of 17 in U87MG cells than in A549 cells (Fig. 3c). In the literature, similar results were reported for the conjugates of pyropheophorbide-a and phthalocyanine with cRGDfK.9,10 Additionally, the pheophorbide-a conjugate of leuprorelin (18) showed no selective photodynamic effects when evaluated in MCF7 and SKOV-3 cells (Fig. 3d). These in vitro PDT data indicate that 14 could possess specificity of RGD to avb6 integrin receptor-positive FaDu cells, as we predicted. Additionally, 9 showed moderate specificity of folic acid to the folate receptor positive-KB cells. However, 17 and 18 appeared to have no specificity to the corresponding receptors of targeting moieties. 2.4. Mechanism of cell death via PDT Further biological studies were performed to determine whether the growth-inhibition effects of 14 by PDT in A549 and FaDu cells were associated with the induction of apoptosis or necrosis. The type of cell death induced after PDT was analyzed by Annexin-V-FITC and PI staining using flow cytometry. As shown in Figure 4a, the population eliminated by Annexin-V-positive apoptotic cell death increased by up to 31.4% (in a dose-dependent manner) in FaDu cells compared with the control whereas this rate in A549 cells slightly increased to 19.8%. To confirm apoptosis

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Scheme 2. Synthesis of the conjugate of pheophorbide-a and CRGDLASLC (14).

Scheme 3. Synthesis of pheophorbide-a conjugates with cRGDfK (17) and leuprorelin (18).

due to the photodynamic effect of 14, morphological changes of the cells were monitored under an inversion microscope, resulting in the observation of decreased cell density and morphological apoptotic-like characteristics, such as cell rounding, cellular shrinkage, the condensation and the fragmentation of nuclear structure after DAPI staining (Fig. 4b). Moreover, the protein level after the cleavage of PARP, a known endogenous substrate for caspases that plays important roles in

apoptosis, was substantially increased in FaDu cells compared with A549 cells after PDT with 14. Additionally, the expression levels of procaspase-3 (proenzyme of caspase, a hallmark of apoptosis) and anti-apoptotic Bcl-2 were reduced by the PDT with 14, and proapoptotic Bax levels were remarkably increased with selectivity for FaDu cells (Fig. 4c). Taken together, these findings suggest that PDT with 14 induces substantially higher growth inhibition through apoptosis in FaDu cells than in A549 cells.

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Figure 2. Intracellular confocal images of receptor-positive and receptor-negative cells after incubation with 2 lM pheophorbide-a conjugates; (a) KB and A549 cells after incubation with 9 for 0.5 h; (b) FaDu and A549 cells after incubation with 14 for 1 h; (c) U87MG and A549 cells after incubation with 17 for 1 h; (d) MCF7 and SKOV3 cells after incubation with 18 for 1.5 h.

3. Conclusion We have designed and synthesized pheophorbide-a conjugates with 4 different cancer-targeting moieties, including folic acid, the cRGDfK peptide, the CRGDLASLC peptide and leuprorelin. These conjugates exhibited significantly enhanced cellular uptake in the cells expressing the corresponding receptors of the targeting moieties compared with those in negative cells. In particular, compounds 9 and 14 targeting the folate and avb6 integrin receptors respectively, exhibited photodynamic selectivity with receptormediated selective uptake and activation due to LED irradiation. Additionally, PDT with 14, the most selective targeted photosensitizer in this study, exhibited apoptotic cell death and morphological apoptosis-like characteristics. These results suggest that targeted cancer dynamic therapy using 14 may be applied for the treatment of oral cancers primarily expressing the avb6 receptor. 4. Experimental protocols 4.1. Chemistry Pheophorbide-a was purchased from Frontier Scientific Inc. (Logan, UT, USA). The cyclic (RGDfK) peptide, the CRGDLASLC peptide, and leuprorelin were purchased from Anygen Inc (Korea), and they were used as received. Starting materials, reagents, and solvents were purchased from Aldrich Chemical Co. (Milwaukee, WI) and TCI (Tokyo) and they used as supplied without further purification. Proton nuclear magnetic resonance spectroscopy was performed on a JEOL JNM-LA 400WB spectrometer, and spectra were taken in CDCl3. Unless otherwise noted, chemical shifts are expressed as ppm downfield from tetramethylsilane as the internal standard, and J values are given in Hz. Data are reported as follows: chemical shift, multiplicity (s, singlet; d, doublet; t, triplet; m, multiplet; br, broad; app., apparent), coupling constants, and integration. Mass spectroscopy was carried out on FAB (fast atom bombardment) instruments. [FAB source: JEOL FAB source and ion gun (Cs ion beam, 30 kV acceleration)]. High-resolution mass spectra (m/z) were recorded on a FAB (JEOL: mass range 2600 amu, 10 kV acceleration) at Korea Basic Science Institute (Daegu).

4.1.1. Procedure for synthesis of the conjugate of pheophorbide-a-folic acid (9) 4.1.1.1. Pheophorbide-a-Boc-2,20 -(ethylenedioxy)-bis-ethylamine (7). Pheophorbide-a (1, 650.0 mg, 1.10 mmol) and Boc-protected 2,20 -(ethylenedioxy)-bis-ethylamine (6, 60.0 mg, 1.10 mmol) were dissolved in anhydrous DCM (30.0 mL). TEA (0.31 mL, 2.19 mmol) and EDC (630.3 mg, 3.29 mmol) were then added, and the mixture was stirred for 6 h at rt. The reaction mixture was added to satdNaHCO3 (aq) (300 mL) and it was extracted with chloroform (2  300 mL). The combined organic layer was dried over MgSO4, concentrated and purified by silica gel column chromatography (chloroform/methanol = 80:1) to give 7 as a brown sticky solid (270.0 mg). Yield: 30%; 1H NMR (400 MHz, CDCl3) d (ppm) 9.48 (s, 1H), 9.37 (s, 1H), 8.56 (s, 1H), 8.01 (m, 1H), 6.30 (m, 2H), 6.18 (m, 1H), 5.67 (s, 1H, –NH), 4.74 (s, 1H, –NH), 4.51 (m, 1H), 4.22 (m, 1H), 3.86 (s, 3H), 3.67 (m, 2H), 3.65 (s, 3H), 3.39 (m, 5H), 3.25 (m, 6H), 3.20 (m, 5H), 3.07 (q, J = 5.2 Hz, 2H), 2.64 (m, 1H), 2.46 (m, 2H), 2.26 (m, 1H), 1.81 (d, J = 7.6 Hz, 3H), 1.69 (t, J = 8.8 Hz, 3H), 1.33 (s, 9H); ESI [M+H] = 823.9. 4.1.1.2. Pheophorbide-a-2,20 -(ethylenedioxy)-bis-ethylamine (8). Compound 7 (270.0 mg, 0.33 mmol) was treated with TFA/DCM (1:1, 5.0 mL) at 0 °C. After being stirred for 2 h, TFA and DCM were evaporated. The green residue was taken up and purified by silica gel column chromatography (chloroform/ methanol = 5:1) to give 8 as a brown sticky solid (73.0 mg). Yield: 32%; 1H NMR (400 MHz, CDCl3) d (ppm) 9.29 (s, 1H), 9.27 (s, 1H), 8.50 (s, 1H), 7.94 (m, 1H), 6.25 (m, 3H), 4.49 (m, 1H), 4.13 (m, 1H), 3.82 (s, 3H), 3.63 (m, 2H), 3.47 (s, 3H), 3.43 (m, 6H), 3.32 (m, 4H), 3.17 (s, 3H), 3.14 (s, 3H), 3.00 (q, J = 7.2 Hz, 2H), 2.68 (m, 2H), 2.39 (m, 2H), 1.79 (d, J = 7.2 Hz, 3H), 1.63 (t, J = 7.6 Hz, 3H); ESI [M+H] = 723.4. 4.1.1.3. Pheophorbide-a-2,20 -(ethylenedioxy)-bis-ethylaminefolic acid conjugate (9). Folic acid (44.0 mg, 0.10 mmol) and EDC (39.0 mg, 0.20 mmol) were dissolved in anhydrous DMF (5.0 mL) and pyridine (2.0 mL) and the mixture was stirred for 15 min at rt. Compound 8 (72.0 mg, 0.10 mmol) was then added and the reaction mixture was further stirred for 24 h at rt. The

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Figure 3. In vitro PDT efficacy of pheophorbide-a conjugates. (a) 9 in KB and A549 cells. (b) 14 in FaDu and A549 cells. (c) 17 in U87MG and A549 cells. (d) 18 in PC-3 and SKOV3 cells. The conjugates were exposed to a 1.25 J/cm2 light dose from a red light-emitting diode (LED). This source emitted light at wavelengths of 613–645 nm. The peak at 635 nm (Philips Luxeon, San Jose, CA) was characterized as 35 mW/cm2 as measured with a Delta Ohm DO 9721 quantum photo-radiometer and temperature data logger.

reaction mixture was added to 1 N HCl (aq) (50 mL) and it was extracted with ether (2  100 mL). The combined organic layer was filtered and the dark precipitate was obtained. Generated black solid was washed with DCM and it was purified by RPHPLC on a C18 semi-preparative column (9, 25.0 mg). Yield: 22%; 9.48 (s, 1H), 9.37 (s, 1H), 8.56 (s, 1H), 8.01 (m, 1H), 6.30 (m, 2H), 6.18 (m, 1H), 5.67 (s, 1H, –NH), 4.74 (s, 1H, –NH), 4.51 (m, 1H), 4.22 (m, 1H), 3.86 (s, 3H), 3.67 (m, 2H), 3.65 (s, 3H), 3.39 (m, 5H), 3.25 (m, 6H), 3.20 (m, 5H), 3.07 (q, J = 5.2 Hz, 2H), 2.64 (m, 1H), 2.46 (m, 2H), 2.26 (m, 1H), 1.81 (d, J = 7.6 Hz, 3H), 1.69 (t, J = 8.8 Hz, 3H), 1.33 (s, 9H); ESI [M+H] = 1146.6; HRMS (FAB) calcd for C60H68N13O11 [M+H]+: 1146.5161, found: 1146.5166. 4.1.2. Procedure for synthesis of the conjugate of pheophorbide-a-CRGDLASLC peptide (14) 4.1.2.1. N-Boc-1,3-propanediamine (11). To a solution of 1,3-propanediamine (10, 23.0 lL) in DCM (2.0 mL) at 0 °C was added Boc2O (5.9 mg, 0.03 mmol) dissolved in DCM (0.1 mL) dropwise for 0.5 h. The reaction mixture was allowed to warm to

ambient temperature and stirred for 48 h. After filtering of the mixture, the filtrate was concentrated and the oily residue was dissolved in EtOAc (1 mL). The solution was washed with brine, dried over MgSO4 and concentrated to give 11 (10.0 mg) as a white solid. Yield 21%, 1H NMR (400 MHz, CDCl3) d (ppm) 4.88 (br s, 1H), 3.22 (q, J = 6.0 Hz, 2H), 2.78 (t, J = 6.8 Hz, 2H), 1.64 (tt, J = 6.8, 6.4 Hz, 2H), 1.43 (s, 9H); ESI [M+H] = 175.3. 4.1.2.2. Pheophorbide-a-N-Boc-1,3-propanediamine Pheophorbide-a (1, 10.0 mg, 0.02 mmol) and compound (12). 11 (3.0 mg, 0.02 mmol) were dissolved in anhydrous DCM (1.0 mL). TEA (4.7 lL 0.04 mmol) and EDC (9.7 mg, 0.06 mmol) were then added, and the mixture was stirred for 12 h at rt. The reaction mixture was added to sat.NaHCO3 (aq) (30 mL) and it was extracted with chloroform (2  30 mL). The combined organic layer was dried over MgSO4, concentrated and purified by silica gel column chromatography (chloroform/methanol = 80:1) to give 12 as a brown sticky solid (4.0 mg). Yield: 31%; 1H NMR (400 MHz, CDCl3) d (ppm) 9.46 (s, 1H), 9.37 (s, 1H), 8.55 (s, 1H), 8.02 (m, 1H),

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Figure 4. Apoptotic cell death via PDT with 14 in A549 and FaDu Cells. Cells were treated with the indicated concentration of 14 and then irradiated under a light dose of 1.25 J/cm2. (a) Flow cytometry analysis via staining with Annexin-V-FITC and PI for 24 h of incubation after PDT with 14. The left lower quadrant represents live cells, the right lower quadrant contains apoptotic cells, and the upper left includes necrotic cells. (b) DAPI staining for 24 h of incubation after PDT with 14. The morphological changes in nuclear chromatin were then observed with a fluorescence microscope. The arrows point to the apoptotic bodies in PDT-treated cells. (Magnification, 400). (c) Expression of the apoptosis-related proteins for 24 h of incubation after PDT with 14. The protein fraction was prepared and resolved using SDS–PAGE and the expression of apoptosisrelated proteins was detected using western blot analysis. The protein levels were normalized by a comparison with the actin levels. The representative bands from three independent experiments are shown.

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6.30 (m, 2H), 6.19 (m, 1H), 5.91 (br s, 1H, –NH of diaminopropane), 4.71 (s, 1H, –NH of diaminopropane), 4.51 (m, 1H), 4.21 (m, 1H), 3.85 (s, 3H), 3.68 (m, 2H), 3.61 (s, 3H), 3.39 (s, 3H), 3.22 (s, 3H), 3.09 (m, 2H), 2.89 (d, J = 5.6 Hz, 2H), 2.85 (m, 1H), 2.42 (m, 3H), 2.05 (m, 2H), 1.80 (d, J = 7.2 Hz, 3H), 1.69 (t, J = 7.6 Hz, 3H), 1.30 (s, 9H); ESI [M+H] = 649.7 (Mass of deprotection of Boc Group). 4.1.2.3. Pheophorbide-a-1,3-propanediamine (13). Compound 12 (4.0 mg, 0.005 mmol) was treated with TFA/DCM (1:1, 2.0 mL) at 0 °C. After being stirred for 2 h, TFA and DCM were evaporated. The green residue was taken up and purified by silica gel column chromatography (chloroform/methanol = 5:1) to give 13 as a brown sticky solid (3.0 mg). Yield: 87%; 1H NMR (400 MHz, CDCl3) d (ppm) 9.33 (s, 1H), 9.29 (s, 1H), 8.50 (s, 1H), 7.95 (m, 1H), 6.26 (m, 2H), 6.15 (m, 1H), 4.54 (m, 1H), 4.10 (m, 1H), 3.76 (s, 3H), 3.62 (m, 2H), 3.48 (s, 3H), 3.33 (s, 3H), 3.17 (s, 3H), 3.09 (m, 2H), 2.83 (m, 3H), 2.34 (m. 3H), 2.12 (m, 2H), 1.75 (d, J = 7.2 Hz, 3H), 1.64 (t, J = 7.2 Hz, 3H); ESI [M+H] = 649.6. 4.1.2.4. Pheophorbide-a-1,3-propanediamine-CRGDLASLC peptide conjugate (14). To a solution of compound 13 (3.0 mg, 0.004 mmol) in anhydrous DMF (1.0 mL), CRGDLASLC (4.0 mg, 0.04 mmol), HOBt (1.1 mg, 0.008 mmol), DMAP (1.0 mg, 0.008 mmol), and EDC (2.3 mg, 0.012 mmol) were added and stirred under N2 at rt for 12 h. DMF was removed under high vacuum pump. The residue was filtered with water and the precipitate was obtained. It was purified by silica gel column chromatography (chloroform/methanol = 3:1) and dried to yield 3.0 mg of black solid. The crude product was purified by RP-HPLC on a C18 semipreparative column (14, 2.0 mg). Yield: 28%; ESI [M+H] = 1548.4; HRMS (FAB) calcd for C74H103N18O15S2 [M+H]+: 1547.7292, found: 1547.7299. 4.1.3. Procedure for synthesis of the conjugate of pheophorbide-a-cRGDfK peptide (17) 4.1.3.1. Pheophorbide-a-NHS (15). Pheophorbide-a (1, 50.0 mg, 0.08 mmol) and N-hydroxysuccinimide (15.0 mg, 0.013 mmol) were dissolved in anhydrous DCM (10.0 mL). DMAP (10.3 mg 0.08 mmol) and EDC (48.0 mg, 0.25 mmol) were then added, and the mixture was stirred for 4 h at rt. The reaction mixture was added to satdNH4Cl (aq) (50 mL) and it was extracted with chloroform (2  50 mL). The combined organic layer was dried over MgSO4, concentrated and purified by silica gel column chromatography (chloroform/methanol = 100:1) to give 15 as a brown sticky solid (23.0 mg). Yield: 40%; 1H NMR (400 MHz, CDCl3) d (ppm) 9.51 (s, 1H), 9.36 (s, 1H), 8.58 (s, 1H), 8.01 (m, 1H), 6.30 (m, 3H), 4.50 (m, 1H), 4.30 (m, 1H), 3.87 (s, 3H), 3.68 (s, 3H), 3.64 (m, 2H), 3.40 (s, 3H), 3.20 (s, 3H), 2.78 (s, 4H), 2.62 (m, 1H), 2.51 (m, 2H), 2.27 (m, 1H), 1.82 (d, J = 7.2 Hz, 3H), 1.70 (t, J = 7.6 Hz, 3H); ESI [M+H] = 823.9. 4.1.3.2. Pheophorbide-a-GABA (16). Compound 15 (23.0 mg, 0.03 mmol) and Gamma-amino butyric acid (5.0 mg, 0.05 mmol) were dissolved in anhydrous DMF (3.0 mL). TEA (10.0 lL, 0.06 mmol) was then added, and the mixture was stirred for 4 h at rt. The reaction mixture was added to sat.NaHCO3 (aq) (30 mL) and it was extracted with ethyl acetate (2  30 mL). The combined organic layers were dried over MgSO4, concentrated and purified by silica gel column chromatography (chloroform/methanol = 10:1) to give 16 as a brown sticky solid (13.0 mg). Yield: 60%; 1H NMR (400 MHz, CDCl3) d (ppm) 9.28 (s, 1H), 9.23 (s, 1H), 8.48 (s, 1H), 7.91 (m, 1H), 6.23 (m, 3H), 4.47 (m, 1H), 4.14 (m, 1H), 3.80 (s, 3H), 3.58 (m, 2H), 3.37 (s, 3H), 3.30 (s, 3H), 3.18 (s, 3H), 3.15 (t, J = 6.8 Hz, 2H), 2.86 (m, 2H), 2.42 (m, 2H), 2.32 (t, J = 6.0 Hz, 2H), 1.80 (d, J = 7.6 Hz, 3H), 1.68 (t, J = 6.0 Hz, 2H), 1.63 (t, J = 7.2 Hz, 3H); ESI [M+H] = 678.4.

4.1.3.3. Pheophorbide-a-GABA-cRGDfK peptide conjugate (17). To a solution of compound 16 (10.0 mg, 0.02 mmol) in anhydrous DMF (1.0 mL), c(RGDfK) (9.0 mg, 0.02 mmol), HOBt (5.0 mg, 0.04 mmol), DMAP (2.3 mg, 0.02 mmol), and EDC (7.1 mg, 0.04 mmol) were added. The reaction mixture was stirred for 12 h at rt. After DMF was removed under high vacuum pump, the residue was filtered with water and the precipitate was obtained. It was purified by silica gel column chromatography (chloroform/methanol = 3:1) and dried to yield 3.0 mg of black solid. The crude product was purified by RP-HPLC on a C18 semipreparative column (17, 2.0 mg). Yield: 11%; ESI [MH] = 1263.5; HRMS (FAB) calcd for C60H83N14O12 [M+H]+: 1263.6315, found: 1263.6320. 4.1.4. Procedure for synthesis of the conjugate of pheophorbide-a-Leuprorelin (18) To a solution of compound 16 (5.6 mg, 0.008 mmol) in anhydrous DMF (1.0 mL), leuprorelin (10.0 mg, 0.008 mmol), HOBt (2.8 mg, 0.02 mmol), DMAP (3.0 mg, 0.02 mmol), and EDC (4.7 mg, 0.02 mmol) were added. The reaction mixture was stirred for 12 h at rt. After DMF was removed under high vacuum pump, the residue was filtered with water and the precipitate was obtained. It was purified by silica gel column chromatography (chloroform/methanol = 3:1) and dried to yield 3.0 mg of black solid crude. The crude was purified by RP-HPLC on a C18 semipreparative column (18, 1.8 mg). Yield: 12%; ESI [M+H] = 1870.2; HRMS (FAB) calcd for C98H126N21O17 [M+H]+: 1169.9671, found: 1869.9878. 4.2. HPLC analysis HPLC was used for purification of all final products. The purification was performed on a Shimadzu SCL-10A VP HPLC system using a Luna Phenomenex RP-HPLC on C18 semi-preparative column (250  10.0 mm, 10 lM, 100 Å) using 0.1% TFA in water/ acetonitrile gradient, monitored by both absorbance at 420 nm on a diode array detector (Shimadzu). The eluting solvent was 0.1% TFA in water/acetonitrile (70:30, v/v) for 30 min followed by 95% acetonitrile. 4.3. Procedure of the biological experiments 4.3.1. Cell culture KB, human oral carcinoma cell, U87MG, human glioblastoma cell, SKOV3, human ovary cancer cell, FaDu, hypopharyngeal carcinoma cell and LNCap, human prostate adenocarcinoma cell were cultured in RPMI 1640. A549, human lung adenocarcinoma epithelial cell was cultured in Dulbecco0 s modified Eagle’s medium (DMEM, GIBCO, Rockville, MD). Both media were supplemented with 10% FBS (Invitrogen Co.), and antibiotic antimycotic (Invitrogen Co.). All cell lines were incubated in a humidified atmosphere containing 5% CO2 at 37 °C. The adherent cells were detached from the culture flasks by removal of the growth medium and addition of 1 mL trypsin/EDTA solution (0.05% w/v trypsin, 0.016% w/v EDTA). After 1–2 min incubation at 37 °C, when the cells had detached from the surface, trypsinization was stopped by the addition of 4 mL of DMEM medium containing 10% FBS. 4.3.2. Confocal fluorescence microscopy Various cancer cells were plated in the slide dishes and incubated in RPMI 1640 and DMEM medium for overnight. The next day, conjugates of 2 lM to the medium were added and they were incubated for 30 min or 1 h in a humidified atmosphere of air/CO2 (95%: 5%). After being washed three times with PBS, the cells were viewed under a FluoViewTM FV1000 Confocal Microscope.

H. You et al. / Bioorg. Med. Chem. 23 (2015) 1453–1462

Conjugates’ fluorescence was observed at 440 nm for an excitation wavelength and at 655–755 nm for an emission wavelength. 4.3.3. In vitro PDT 4.3.3.1. Photochemical treatment. The various cancer cells were seeded at the density of 1  105 cells/mL. After overnight, the cells were washed with PBS and were incubated serum-free culture medium, containing photosensitizers. A stock solution of photosensitizers (1 mM) was prepared in dimethyl sulfoxide (DMSO), and it was stored at 20 °C in dark. Two hours after photosensitizers’ administration, the medium was removed and then exposed to light dose 1.25 J/cm2 from a red light-emitting diode (LED). This source emitted light at 613–645 nm wavelengths. The peak at 635 nm (Philips Luxeon, San Jose, CA) was characterized by 35 mW/cm2 as measured with a Delta Ohm DO 9721 quantum photo-radiometer and thermometer data logger (Model DO9721, Padua, Italy). During the incubation of cell with photosensitizers, light exposure was limited. 4.3.3.2. Measurement of cell viability. The cells were treated PDT for 24 h and the cell viability was assessed using MTT assay. Briefly, the PDT treated cells were washed twice with ice-cold PBS, and then 0.5 mL cell culture medium with 50 lL of 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma, St. Louis, MO, USA) (5 mg/mL in PBS) were added to each well of the 12-well plate (containing 500 lL medium and cells) and were incubated for 3 h at 37 °C. After the supernatant was discarded, the resulting insoluble purple formazan crystal was dissolved in 250 lL of acid-isopropanol (0.04 M HCl in isopropanol). The optical density (OD) was measured by a Microplate Autoreader ELISA (Bio-Tek Instruments, Inc., Winooski, VT) at a wavelength of 570 nm. Background value was subtracted from sample readings and cell viability is expressed as the percentage of control cells. The results are reported as the mean ± SD of three separate experiments. 4.3.4. Cell proliferation assay FaDu cells were maintained in Minimum Essential medium (MEM) containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin at 37 °C in a humidified atmosphere containing 5% CO2. The cells (1  105 cells/well) were cultured in 12 well plates overnight, and treated with various concentrations of 14. After 1 h incubation with 14, the cells were exposed to light dose of 1.25 J/cm2 from a red light-emitting diode (LED). This source emitted light at 613–645 nm wavelengths. The peak at 635 nm (Philips Luxeon Lumileds, San Jose, CA) was characterized by 35 mW/cm2 as measured with a Delta Ohm DO 9721 quantum photo-radiometer and thermometer data logger (Model DO9721, Padua, Italy). Immediately after PDT, the cells were exposed to fresh medium followed by incubation for 24 h. Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)2,5-diphenylte-trazolium Bromide (MTT) assays. Briefly, the cells were washed twice with ice-cold PBS, and were incubated with MTT reagent (5 mg/mL in PBS) for 3 h at 37 °C. After the medium was removed, a acid-isopropanol (0.04 M HCl in isopropanol) of 250 lL was added. The optical density was measured by a Microplate Autoreader ELISA (Bio-Tek Instruments, Inc., Winooski, VT) at a wavelength of 570 nm. 4.3.5. Annexin V-FITC/PI double staining A549 and FaDu cells seeded in 6 cm2 plates (5  105 cells/well) were treated with photodynamic therapy with 14 for 24 h as previously described. The cells were harvested and were washed with cold PBS. The specific binding of Annexin V-FITC/PI was performed by incubating the cells for 15 min at rt in a binding

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buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4) containing saturated concentrations of Annexin V-FITC and PI. Following incubation, the cells were pelleted and analyzed in a FACScan analyzer (Beckman Coulter Inc., Fullerton, CA, USA). 4.3.6. Analysis of cell morphology For studies of morphology changes, the A549 and FaDu cells (1  105 cells/well) were seeded in a 6-well plate and were cultured overnight in a growth medium. The cells were treated with photodynamic therapy with 14 at 1 lM for 24 h as previously described and cellular morphology was observed by using phase contrast microscopy (Olympus, CKX41, Japan). 4.3.7. DAPI staining A549 and FaDu cells seeded in 6 cm2 plates (5  105 cells/well) were treated with photodynamic therapy with 14 for 24 h as previously described. The cells were fixed in 4% paraformaldehyde and were incubated in 4,6-diamidino-2-phenylindole (DAPI) solution of 1 lg/mL for 5 min in the dark at rt. After the cells were washed with an ice-cold PBS, the cellular fluorescence intensity changes were observed using conventional fluorescence microscope (Olympus IX 71, Olympus America, Melville, NY). 4.3.8. Western blotting A549 and FaDu cells seeded in 6-well plates (2  105 cells/well) were treated via photodynamic therapy with 14 for 24 h as previously described. The cells were then washed with PBS and harvested in lysis buffer. Samples containing equal amounts of protein were loaded onto each lane of an SDS–polyacrylamide gel for electrophoresis and subsequently transferred onto a polyvinylidene difluoride membrane. The membranes were blocked and then incubated with antibodies. Antibodies against PARP were purchased from Cell Signaling Technology (Beverly, MA, USA); Bax, Bcl-2, pro-caspase3, Actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Acknowledgments This research was supported by a grant of the Korea Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A100490) and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF2014R1A2A1A11052300). Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmc.2015.02.014. References and notes 1. Peng, X.; Jincan, C.; Zhuo, C.; Shanyong, Z.; Ping, H.; Xueyuan, C.; Mingdong, H. PLoS ONE 2012, 7, 1. 2. Fiorenza, R.; Matthias, H.; Andreas, M.; Eugeny, A.; Norbert, J.; Beate, R.; Andreas, H.; Fritz, B. Bioconjugate Chem. 2007, 18, 1078. 3. Wesley, M.; Cynthia, M.; Johan, E. Drug Discovery Today 1999, 4, 507. 4. Stanley, B.; Elizabeth, A.; Ian, W. Lancet Oncol. 2004, 5, 497. 5. Nela, M.; Karen, S.; Huguette, S.; John, G.; Ross, W. Int. J. Oncol. 2006, 28, 1561. 6. Hudson, R.; Carcenac, M.; Smith, K.; Madden, L.; Clarke, O. J.; Pelegrin, A.; Greenman, J.; Boyle, R. W. Br. J. Cancer 2005, 92, 1442. 7. Del Governatore, M.; Hamblin, M. R.; Piccinini, E. E.; Ugolini, G.; Hasan, T. Br. J. Cancer 2000, 82, 56. 8. Julien, G.; Raphael, S.; Celine, F.; Thierry, B.; Frederic, S.; Jacques, D.; Francois, G.; Muriel, B. J. Med. Chem. 2008, 51, 3867. 9. Avinash, S.; Manivannan, E.; Suresh, K. P.; Shiprp, D.; Xiang, Z.; Ting-Hsiu, L.; Masayuki, S.; Joseph, M.; Janet, M.; Ravindra, K. P. Mol. Pharm. 2011, 8, 1186. 10. Mei-Rong, K.; Ng Dennis, K. P.; Pui-Chi, L. Chem. Asian J. 2014, 9, 554. 11. Ritu, B.; Nandana, S.; Uma, S.; Girjesh, G. J. Mol. Struct. 1994, 327, 201.

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Pheophorbide-a conjugates with cancer-targeting moieties for targeted photodynamic cancer therapy.

Pheophorbide-a, a non-selective photosensitizer, was conjugated with cancer-targeting moieties, such as folic acid, the CRGDLASLC peptide, the cRGDfK ...
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