Preparation and in Vitro Photodynamic Activities of Folate-Conjugated

Oct 2, 2013 - Functional aza-boron dipyrromethenes for subcellular imaging and organelle-specific photodynamic therapy. Qiong Wang , Dennis K. P. Ng ...
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Preparation and in Vitro Photodynamic Activities of FolateConjugated Distyryl Boron Dipyrromethene Based Photosensitizers Mei-Rong Ke,† Sin-Lui Yeung,‡ Dennis K. P. Ng,*,† Wing-Ping Fong,‡ and Pui-Chi Lo*,† †

Department of Chemistry and ‡School of Life Sciences, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China S Supporting Information *

ABSTRACT: Two folate-conjugated diiododistyryl boron dipyrromethenes have been prepared and characterized with various spectroscopic methods. These conjugates exhibit higher photocytotoxicity toward the KB human nasopharyngeal carcinoma cells, which have high expression of folate receptors when compared with the MCF-7 human breast adenocarcinoma cells, which have low expression of folate receptors. The difference in photocytotoxicity for these two cell lines is particularly large for the conjugate with a shorter oligoethylene glycol linker (compound 11a) as a result of its higher cellular uptake and slightly lower aggregation tendency. Its IC50 value toward KB cells (0.06 μM) is 43-fold lower than that for MCF-7 cells, while the difference is only 6-fold for the analogue with a longer linker (compound 11b). The length of the spacer also affects their subcellular localization. While compound 11a shows high affinity toward the endoplasmic reticulum of KB cells, conjugate 11b is mainly localized in the lysosomes.

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INTRODUCTION There has been considerable interest in the development of novel and efficient photosensitizers for photodynamic therapy (PDT).1−4 Apart from the classical tetrapyrrole derivatives, such as porphyrins, chlorins, pheophorbides, and phthalocyanines, boron dipyrromethene (BODIPY) derivatives are also promising photosensitizers owing to their desirable optical and photophysical properties that can be tuned readily through chemical modification of the π skeleton5 and their relatively high stability even in aqueous media. This field of study, however, has not been fully developed. As described in the two recent review articles,6,7 most of the BODIPY-based photosensitizers are confined to the aza-BODIPYs, which intrinsically absorb in the near-infrared region. The photobiological properties of non-aza analogues have been little studied despite the fact that these compounds, after suitable modification, can also absorb in the near-infrared region and exhibit superior photodynamic activities, and the meso position can also be used to introduce an additional functionality. We have recently reported a series of pegylated distyryl BODIPY derivatives.8,9 Their cellular uptake, subcellular localization, and photocytotoxicity are greatly affected by the styryl substituents. The analogue with five triethylene glycol chains shows the highest potency with an IC50 of 7 nM against HT29 human colorectal carcinoma. As an extension of this study, we report herein two analogues that are conjugated with a folate group at the meso position via a triazole-linked oligoethylene glycol chain, including their preparation and in vitro photodynamic activities. The effects of the chain length on these properties were also investigated. © 2013 American Chemical Society

In the development of highly efficient photosensitizers, a major challenge is to enhance their selectivity so that they can preferentially localize and function at the tumor site. A common strategy is through direct conjugation with targeting ligands, such as monoclonal antibodies, proteins, peptides, steroids, and folates.10−12 Among these vectors for active drug targeting, folates are of particular interest because of their low molecular weight, high stability, high tissue permeability, and nonimmunogenicity. More importantly, these pterin-based vitamins required by eukaryotic cells for the biosynthesis of nucleotides have high binding affinity to folate receptors, which are overexpressed on most epithelial cancers but not on normal tissues. As a result, folate-mediated delivery of various imaging and therapeutic agents has been widely studied as an effective approach to enhance their tumor selectivity.13−16 For potential application in PDT, a number of folate-conjugated photosensitizers, including porphyrins,17,18 pheophorbides,19 chlorins,20,21 bacteriochlorophyll,22 and quantum dots,23 and nanoparticles entrapping with photosensitizers24−29 have been reported. We believed that by connection of a folate group to the distyryl BODIPY-based photosensitizers, the resulting conjugates could exhibit enhanced tumor selectivity and therefore would be useful for targeted PDT.

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RESULTS AND DISCUSSION Synthesis and Characterization. Scheme 1 shows the synthetic route used to prepare these conjugates. The hydroxy Received: June 19, 2013 Published: October 2, 2013 8475

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Scheme 1. Synthesis of folate-conjugated distyryl BODIPYs 11a and 11b

BODIPY 130 was first treated with propargyl bromide (2) or alkynyl tosylate 331 in the presence of K2CO3 in acetone to give the respective substituted product 4a and 4b. To promote the intersystem crossing and the formation of singlet oxygen through the heavy-atom effect, compounds 4a and 4b were iodinated using a mixture of iodine and iodic acid in ethanol, giving the diiodo analogues 5a and 5b, respectively. These compounds then underwent Knoevenagel condensation with triethylene glycol substituted benzaldehyde 632 to afford the distyryl BODIPYs 7a and 7b. This modification could extend the π conjugation and shift the absorption to the red, thereby facilitating the light penetration into tissue. The ethylene glycol chains were introduced to enhance the hydrophilicity, biocompatibility, and cellular uptake of the dyes. To prepare the folate component, folic acid (8) was treated with 2-[2-(2azidoethoxy)ethoxy]ethanamine (9) in the presence of N,N′dicyclohexylcarbodiimide (DCC) and pyridine in dimethylsulfoxide (DMSO) to give the azido folate 10 as a mixture of α- and γ-isomers depending on the position of the amide formed. As revealed by HPLC analysis (Figure S1a in the Supporting Information), these two isomers were obtained in a ratio of

approximately 3:7. Because of the less hindered environment, the γ-carboxy group of folic acid reacted favorably giving the γisomer (as shown in Scheme 1) as the major product. This result was in accord with the literature reports of other folate conjugation reactions.20,33,34 This mixture of α- and γ-isomers of 10, without further separation, was then clicked with the alkynyl distyryl BODIPYs 7a and 7b to afford the conjugates 11a and 11b, respectively. Both compounds were purified by HPLC and characterized with UV−vis spectroscopy and highresolution electrospray ionization (ESI) mass spectrometry. As shown in Figure S1b and S1c in the Supporting Information, the signals for the α- and γ-isomers were less well-resolved compared with the case of 10, particularly for 11b. All the other new compounds were fully characterized with various spectroscopic methods and elemental analysis. Electronic Absorption and Photophysical Properties. The UV−vis spectra of 11a and 11b were measured in N,Ndimethylformamide (DMF). Both spectra showed an intense Q-band at 662 nm, which strictly followed the Lambert−Beer law (Figure S2 in the Supporting Information), suggesting that these two conjugates are not significantly aggregated in DMF. 8476

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Upon excitation at 610 nm, these conjugates showed a fluorescence emission at 687 nm (for 11a) or 689 nm (for 11b) with a fluorescence quantum yield (ΦF) of 0.20 relative to the unsubstituted zinc(II) phthalocyanine (ZnPc) (ΦF = 0.28).35 The relatively weak fluorescence emission of these compounds can be attributed to the presence of two iodo groups, which promote the intersystem crossing and reduce the probability of relaxation via fluorescence emission.36 The electronic absorption and fluorescence data are listed in Table 1.

the photodynamic action than the MCF-7 cells. It is worth noting that the difference in photocytotoxicity for these two cell lines is particularly large for 11a. The IC50 value of 11a toward KB cells is 43-fold lower than that for MCF-7 cells, while the difference is only 6-fold for 11b and 3-fold for 7b. These results suggest that both folate-conjugated photosensitizers 11a and 11b exhibit higher affinity toward the FR+ KB cells. The selectivity and photocytotoxicity depend on the length of the oligoethylene glycol linker between the photosensitizer and the folate moiety. Conjugate 11a, which has a shorter linker, exhibits enhanced targeting property and higher photocytotoxicity against the KB cells. The effects of linker have also been examined for some other folate-conjugated antitumor agents.17,39,40 For example, Guaragna et al. prepared two folate conjugates connected to the anticancer drug chlorambucil via either an aminoether or a pseudo-β-dipeptide linker and evaluated their cell specificity and cytotoxicity using three FR+ and FR− leukemic cell lines.39 The results showed that both conjugates exhibited high specificity toward the FR+ cells and their antitumor activity was comparable with that of chlorambucil in its free form. By contrast, in a series of folate conjugates bearing a dinitrophenyl group as an antigenic hapten, their antitumor activity and allergic potential varied depending on the linker despite showing similar affinities for the folate receptors.40 For the two folate−porphyrin conjugates reported by Schneider et al.,17 the one with an ethylene glycol linker was found to be more photocytotoxic than the analogue with a hydrocarbon spacer. Their light dose values leading to 50% growth inhibition showed a 3-fold difference. Our results showed that even with the same nature, a different length of the linker could also exert a significant effect on the selectivity and cytotoxicity of the folate conjugates. To further reveal the targeting effect of the folate group, we also performed a competition study. FR+ KB cells were coincubated with 11a (0.06 μM, i.e., its IC50 value) and free folic acid (0.50 μM) for 24 h before the photodynamic treatment. It was found that the cell viability increased from 50% to 94%. The result clearly suggested that addition of free folic acid would inhibit the cellular uptake of 11a, thereby reducing its photocytotoxicity. To account for the different photocytotoxicity of 11a and 11b, the aggregation behavior of these conjugates in the RPMI culture medium was examined using UV−vis and fluorescence

Table 1. Electronic Absorption and Fluorescence Data for 11a and 11b in DMF

a

compd

λabs (nm) (log ε)

λem (nm)a

ΦF b

11a 11b

378 (4.47), 447 (4.01), 662 (4.69) 377 (4.57), 450 (4.10), 662 (4.79)

687 689

0.20 0.20

Excited at 610 nm. bRelative to ZnPc in DMF (ΦF = 0.28).

To evaluate the photosensitizing efficiency of these conjugates, their singlet oxygen generation efficiency was studied in DMF using 1,3-diphenylisobenzofuran (DPBF) as the singlet oxygen scavenger. The rate of photodegradation of this quencher was measured by monitoring the decrease in absorbance at 415 nm with time. As shown in Figure S3 in the Supporting Information, both conjugates can generate singlet oxygen and the efficiency is comparable with that of ZnPc, which is a common reference compound. In Vitro Studies. The in vitro photodynamic activities of 11a and 11b (formulated with Tween 80) were investigated against the KB human nasopharyngeal carcinoma cells, which have high expression of folate receptors (FR+),37 and the MCF-7 human breast adenocarcinoma cells, which have low expression of folate receptors (FR−).38 Figure 1 shows the dose-dependent survival curves for these conjugates. Both compounds were essentially noncytotoxic in the absence of light. However, upon illumination, both 11a and 11b became cytotoxic. The photocytotoxicity of 11a (IC50 = 0.06 μM for KB cells and IC50 = 2.56 μM for MCF-7 cells) and 11b (IC50 = 0.18 μM for KB cells and IC50 = 1.00 μM for MCF-7 cells) varied significantly for different cells. By contrast, the difference was relatively small for the non-folate-conjugated analogue 7b (IC50 = 0.02 μM for KB cells and IC50 = 0.06 μM for MCF-7 cells), which was more potent than the folate-conjugated counterparts. Generally, the KB cells were more susceptible to

Figure 1. Comparison of the cytotoxic effects of (a) 11a and (b) 11b on KB (squares) and MCF-7 (triangles) cells in the absence (closed symbol) and presence (open symbol) of light (λ > 610 nm, 40 mW cm−2, 48 J cm−2). Data are expressed as the mean value ± standard error of the mean value of three independent experiments, each performed in quadruplicate. 8477

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Figure 2. (a) UV−vis and (b) fluorescence spectra (excited at 610 nm) of 11a and 11b (both at 8 μM) formulated with Tween 80 (0.1% by volume) in the RPMI medium.

Figure 3. (a) Bright field (left) and fluorescence (right) images of KB cells after incubation with 11a or 11b (1.0 μM) in a folic acid free medium for 24 h. (b) Comparison of the average intracellular fluorescence intensity of 11a and 11b in KB cells. Data are expressed as the mean ± standard deviation (number of cells = 50).

Figure 4. (a) Bright field (left) and fluorescence (right) images of KB and MCF-7 cells after incubation with 11a (5.0 μM) in a folic acid free medium for 24 h. (b) Comparison of the average intracellular fluorescence intensity of 11a in KB and MCF-7 cells. Data are expressed as the mean ± standard deviation (number of cells = 24). 8478

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Figure 5. Visualization of the intracellular fluorescence of KB cells by using filter sets specific for 11a (in red, column 3) and ER-Tracker, Lyso-Tracker, or Mito-Tracker (in green, column 2). The corresponding superimposed images and the bright field images are given in column 4 and column 1, respectively.

Figure 6. Visualization of the intracellular fluorescence of KB cells by using filter sets specific for 11b (in red, column 3) and ER-Tracker, LysoTracker, or Mito-Tracker (in green, column 2). The corresponding superimposed images and the bright field images are given in column 4 and column 1, respectively.

spectroscopic methods. As shown in Figure 2, the Q-band of 11b is slightly broadened with an increase in intensity in the blue-shifted shoulder and its fluorescence emission is also slightly weaker compared with those of 11a. This indicates that 11b is slightly more aggregated than 11a in the culture medium, probably because of the extra triethylene glycol chain which may induce dipole−dipole interactions with the neighboring oligoethylene glycol chains.

The photocytotoxicity results could be further addressed by examining the uptake of these conjugates by KB cells using fluorescence microscopy. The cells were incubated with 11a or 11b for 24 h. Then the bright field and fluorescence images of the cells were captured (Figure 3a), and the average intracellular fluorescence intensities were also determined (Figure 3b). It can be seen that 11a shows much stronger intracellular fluorescence than 11b (about 3-fold in intensity), 8479

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Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, China. UV−vis and steady-state fluorescence spectra were taken on a Cary 5G UV−vis−NIR spectrophotometer and a Hitachi F-7000 spectrofluorometer, respectively. The fluorescence quantum yields (ΦF) of the samples were determined by the equation: ΦF(sample) = (Fsample/Fref)(Aref/Asample)(n2sample/n2ref)ΦF(ref),41 where F, A, and n are the measured fluorescence (area under the emission peak), the absorbance at the excitation position (610 nm), and the refractive index of the solvent, respectively. ZnPc in DMF was used as the reference [ΦF(ref) = 0.28].35 To minimize reabsorption of radiation by the ground-state species, the emission spectra were obtained in very dilute solutions of which the absorbance at 610 nm was about 0.04. Reverse-phase HPLC experiments were performed on a Apollo-C18 column (5 μm, 4.6 mm × 250 mm) or on a XBridge-C18 column (5 μm, 10 mm × 250 mm) using a Shimadzu CBM-20A controller with a SPD-M20A diode array detector. The conditions were set as follows: solvent A = 0.1% trifluoroacetic acid (TFA) in distilled water; solvent B = acetonitrile. For purification, the parameters were the following: 90% A + 10% B to 50% A + 50% B in the first 7 min, then changed to 0% A + 100% B in 23 min and maintained at this condition for 3 min, then changed to 90% A +10% B in 2 min, and finally maintained at this condition for a further 5 min. The flow rate was fixed at 4.0 mL min−1. For analysis, the parameters were the following: 90% A + 10% B to 0% A + 100% B in the first 35 min and maintained at this condition for 15 min, then changed to 90% A + 10% B in 2 min, and finally maintained at this condition for 3 min. The flow rate was fixed at 0.8 mL min−1. 4,4-Difluoro-1,3,5,7-tetramethyl-8-(4-propargyloxyphenyl)4-bora-3a,4a-diaza-s-indacene (4a). A mixture of BODIPY 1 (0.50 g, 1.47 mmol), propargyl bromide (2) (0.87 g, 7.35 mmol), and K2CO3 (1.02 g, 7.38 mmol) in acetone (30 mL) was heated at reflux for 7 h. The volatiles were then removed in vacuo. The residue was mixed with water (100 mL), and then the mixture was extracted with CH2Cl2 (50 mL × 3). The combined organic layer was dried over anhydrous Na2SO4 and then evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography using hexane/CHCl3 (3:2, v/v) as the eluent to give an orange solid (0.51 g, 91%). 1H NMR (CDCl3): δ 7.20 (d, J = 8.8 Hz, 2 H, ArH), 7.09 (d, J = 8.8 Hz, 2 H, ArH), 5.98 (s, 2 H, pyrrole-H), 4.76 (d, J = 2.4 Hz, 2 H, CH2), 2.56 (s, 7 H, CH3 and CH), 1.42 (s, 6 H, CH3). 13C{1H} NMR (CDCl3): δ 158.2, 155.5, 143.2, 141.6, 131.9, 129.3, 128.1, 121.3, 115.7, 78.1, 76.0, 56.1, 14.7. MS (ESI): m/z 401 [M + Na]+ (100%). HRMS (ESI): m/z calcd for C22H21BF2N2NaO [M + Na]+ 401.1607, found 401.1614. Anal. Calcd for C22H21BF2N2O: C, 69.86; H, 5.60; N, 7.41. Found: C, 69.72; H, 5.46; N, 7.42. 4,4-Difluoro-1,3,5,7-tetramethyl-8-(4-(3,6,9-trioxa-9-propargylnonoxy)phenyl)-4-bora-3a,4a-diaza-s-indacene (4b). According to the procedure described for 4a, BODIPY 1 (0.15 g, 0.44 mmol) was treated with the alkynyl tosylate 3 (0.30 g, 0.88 mmol) and K2CO3 (0.18 g, 1.32 mmol) in acetone (30 mL) to give 4b, which was purified by silica gel column chromatography using hexane/ethyl acetate (3:2, v/v) as the eluent. The product was obtained as an orange sticky solid (0.14 g, 63%). 1H NMR (CDCl3): δ 7.14 (d, J = 8.8 Hz, 2 H, ArH), 7.01 (d, J = 8.8 Hz, 2 H, ArH), 5.96 (s, 2 H, pyrrole-H), 4.20 (d, J = 2.4 Hz, 2 H, CH2), 4.17 (t, J = 4.4 Hz, 2 H, CH2), 3.90 (t, J = 4.4 Hz, 2 H, CH2), 3.75−3.77 (m, 2 H, CH2), 3.69−3.73 (m, 6 H, CH2), 2.54 (s, 6 H, CH3), 2.42 (t, J = 2.4 Hz, 1 H, CH), 1.41 (s, 6 H, CH3). 13 C{1H} NMR (CDCl3): δ 159.3, 155.1, 143.1, 141.8, 131.7, 129.0, 127.0, 121.0, 115.1, 79.6, 74.6, 70.7, 70.5, 70.3, 69.6, 69.0, 67.4, 58.3, 14.5, 14.4. MS (ESI): m/z 533 [M + Na]+ (100%). HRMS (ESI): m/z calcd for C28H33BF2N2NaO4 [M + Na]+ 533.2399, found 533.2391. Anal. Calcd for C28H33BF2N2O4: C, 65.89; H, 6.52; N, 5.49. Found: C, 65.59; H, 6.54; N, 5.45. 4,4-Difluoro-2,6-diiodo-1,3,5,7-tetramethyl-8-(4-propargyloxyphenyl)-4-bora-3a,4a-diaza-s-indacene (5a). Iodic acid (0.47 g, 2.64 mmol) dissolved in a minimum amount of water was added dropwise to a mixture of BODIPY 4a (0.50 g, 1.32 mmol) and iodine (0.84 g, 3.31 mmol) in EtOH (40 mL). The mixture was heated at 50 °C for 2 h. After cooling, the mixture was evaporated under

which suggests a higher cellular uptake for this conjugate. Hence, the higher photocytotoxicity of 11a against KB cells can be attributed to its higher cellular uptake and lower aggregation tendency in the biological environment, which can facilitate the formation of singlet oxygen.8,9 To evaluate the targeting property of the folate moiety in 11a, its cellular uptake was further examined using the FR+ KB and the FR− MCF-7 cells. After incubation with 11a for 24 h, the KB cells showed a strong intracellular fluorescence, while the fluorescence was significantly weaker in the MCF-7 cells (Figure 4a). The average fluorescence intensity in KB cells is about 5-fold higher than that in MCF-7 cells (Figure 4b). The results indicate that the folate moiety in conjugate 11a can assist the delivery of the distyryl BODIPY-based photosensitizer into the cancer cells which overexpress folate receptors. The subcellular localization of 11a and 11b in KB cells was also investigated. The cells were first incubated with these conjugates in a folic acid free medium for 24 h, then stained with Lyso-Tracker DND 26, Mito-Tracker Green FM, or ER-Tracker Green (for 15 min), which are specific fluorescent dyes for lysosomes, mitochondria, or endoplasmic reticulum, respectively. As shown in Figure 5, the fluorescence caused by 11a can superimpose with the fluorescence caused by the ER-Tracker and partially overlap with that caused by the Lyso-Tracker but not with that caused by Mito-Tracker. The results suggest that 11a has high affinity to the endoplasmic reticulum and can also bind to the lysosomes. For conjugate 11b, it accumulates preferentially in lysosomes and partially in endoplasmic reticulum (Figure 6). Hence, the length of the linker also affects the subcellular localization property of these conjugates.

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CONCLUSIONS We have prepared and characterized two folate-conjugated distyryl BODIPYs and evaluated their in vitro photodynamic activities. Both conjugates exhibit higher photocytotoxicity toward the FR+ KB cells compared with the FR− MCF-7 cells. The difference is particularly large for 11a, of which the IC50 for KB cells is 43-fold lower than that for MCF-7 cells. In addition to the photocytotoxicity, the length of the linker between the distyryl BODIPY-based photosensitizer and the folate moiety also affects the aggregation tendency, cellular uptake, and subcellular localization. Conjugate 11a, which has a shorter linker, is more potent and can serve as a promising photosensitizer for targeted PDT.

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EXPERIMENTAL SECTION

General. All the reactions were performed under an atmosphere of nitrogen. DMSO was dried over calcium hydride and distilled under reduced pressure. Tetrahydrofuran (THF) and toluene were distilled from sodium benzophenone ketyl and sodium, respectively. Chromatographic purifications were performed on silica gel (MachereyNagel, 70−230 mesh) columns with the indicated eluents. Size exclusion chromatography was carried out on Bio-Rad Bio-Beads S-X1 beads (200−400 mesh) using THF as the eluent. All other solvents and reagents were of reagent grade and used as received. Compounds 1,30 3,31 and 632 were prepared as described. 1 H and 13C{1H} NMR spectra were recorded on a Bruker AVANCE III 400 spectrometer (1H, 400; 13C, 100.6 MHz) in CDCl3. Spectra were referenced internally by using the residual solvent (1H, δ = 7.26) or solvent (13C, δ = 77.2) resonances relative to SiMe4. ESI mass spectra were recorded on a Thermo Finnigan MAT 95 XL mass spectrometer. Elemental analyses were performed by the 8480

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8.12 (d, J = 16.8 Hz, 2 H, CCH), 7.59 (d, J = 8.8 Hz, 4 H, ArH), 7.58 (d, J = 16.8 Hz, 2 H, CCH), 7.15 (d, J = 8.8 Hz, 2 H, ArH), 7.05 (d, J = 8.8 Hz, 2 H, ArH), 6.96 (d, J = 8.8 Hz, 4 H, ArH), 4.22 (d, J = 2.4 Hz, 2 H, CH2), 4.17−4.21 (m, 6 H, CH2), 3.92 (vt, J = 4.8 Hz, 2 H, CH2), 3.89 (vt, J = 4.8 Hz, 4 H, CH2), 3.65−3.79 (m, 20 H, CH2), 3.55−3.57 (m, 4 H, CH2), 3.38 (s, 6 H, CH3), 2.44 (t, J = 2.4 Hz, 1 H, CH), 1.49 (s, 6 H, CH3). 13C{1H} NMR (CDCl3): δ 160.0, 159.8, 150.4, 145.8, 139.1, 138.7, 133.3, 129.8, 129.7, 129.3, 127.5, 116.9, 115.6, 115.1, 82.7, 79.7, 77.4, 74.7, 72.0, 71.0, 70.7, 70.6, 69.8, 69.2, 67.7, 67.6, 59.1, 58.5, 17.8 (some of the signals are overlapped). MS (ESI): m/z 1285 [M + Na]+ (100%). HRMS (ESI): m/z calcd for C56H67BF2I2N2NaO12 [M + Na]+ 1285.2746, found 1285.2732. Anal. Calcd for C56H67BF2I2N2O12: C, 53.27; H, 5.35; N, 2.22. Found: C, 53.16; H, 5.26; N, 2.25. α- and γ-2-(2-(2-Azidoethoxy)ethoxy)ethylfolamide (10). A mixture of folic acid (0.50 g, 1.13 mmol), DCC (0.47 g, 2.27 mmol), and pyridine (5 mL) in DMSO (25 mL) was stirred at room temperature for 1 h. 2-[2-(2-Azidoethoxy)ethoxy]ethanamine (9) (0.20 g, 1.13 mmol) was added to the mixture, which was then stirred for a further 20 h in the dark. The resulting mixture was filtered, and the filtrate was poured into cold diethyl ether (200 mL) with vigorous stirring. The orange solid formed was collected by filtration and redissolved in acetone (60 mL), then precipitated again by the addition of diethyl ether (140 mL). The product was collected as an orange solid (0.20 g, 30%), which was used for the conjugation with distyryl BODIPYs 7a and 7b without further purification. HPLC analysis showed that the α- and γ-isomers were in a ratio of about 3:7. Part of the product was taken out and purified by HPLC for characterization. α-Isomer: MS (ESI): m/z 620 [M + Na]+ (90%) and 636 [M + K]+ (100%). HRMS (ESI): m/z calcd for C25H31N11O7K [M + K]+ 636.2040, found 636.2038. γ-Isomer: MS (ESI): m/z 620 [M + Na]+ (100%) and 636 [M + K]+ (72%). HRMS (ESI): m/z calcd for C25H31N11O7Na [M + Na]+ 620.2300, found 620.2302. Folate-Conjugated Distyryl BODIPY 11a. A mixture of BODIPY 7a (20.0 mg, 17.7 μmol), azido folate 10 (15.9 mg, 26.6 μmol), CuSO4·5H2O (0.7 mg, 2.8 μmol), and sodium ascorbate (1.1 mg, 5.6 μmol) in a mixture of DMSO and water (15:1, v/v, 1.6 mL) was stirred at room temperature for 7 h. The mixture was precipitated by the addition of diethyl ether (150 mL). After centrifugation, the supernatant was removed and dried in vacuo. The green solid was redissolved in DMSO (0.5 mL) and then precipitated by the addition of water. After centrifugation, the solid was washed with MeOH and then dried in vacuo. The crude product was further purified by HPLC to afford a dark-green solid (9.8 mg, 32%). MS (ESI): m/z 1750 [M + Na]+ (100%). HRMS (ESI): m/z calcd for C75H86BF2I2N13NaO16 [M + Na]+ 1750.4370, found 1750.4381. The purity was found to be ∼96% by HPLC analysis. Folate-Conjugated Distyryl BODIPY 11b. According to the procedure described for 11a, BODIPY 7b (30.0 mg, 23.8 μmol) was treated with azido folate 10 (21.3 mg, 35.6 μmol), CuSO4·5H2O (0.9 mg, 3.6 μmol), and sodium ascorbate (1.4 mg, 7.1 μmol) in a mixture of DMSO and water (15:1, v/v, 1.6 mL) to give 11b (12 mg, 27%). MS (ESI): m/z 1883 [M + Na]+ (100%). HRMS (ESI): m/z calcd for C81H98BF2I2N13NaO19 [M + Na]+ 1882.5157, found 1882.5101. The purity was found to be ∼97% by HPLC analysis. Cell Lines and Culture Conditions. The KB (ATCC, no. CCL17) and MCF-7 (ATCC, no. HTB-22) cells were maintained in RPMI 1640 (Invitrogen, no. 23400-021) supplemented with fetal calf serum (10%) and penicillin−streptomycin solution (100 units mL−1 and 100 μg mL−1, respectively). Approximately 1 × 104 (for KB) or 2 × 104 (for MCF-7) cells per well were seeded in 96-multiwell plates and incubated overnight at 37 °C in a humidified atmosphere with 5% CO2. Photocytotoxicity Assay. Compounds 11a and 11b were first dissolved in DMF to give 1.6 mM solutions, which were diluted to 80 μM with an aqueous solution of Tween 80 (Arcos, 0.5% by volume in these 80 μM solutions). The solutions were diluted with folic acid free RPMI 1640 (Invitrogen, no. 27016-021) to various concentrations. Both the Tween 80 solution and the culture medium were found to be noncytotoxic in both the absence and presence of light.

reduced pressure. The crude product was purified by silica gel column chromatography using hexane/CHCl3 (3:2, v/v) as the eluent to afford a bright red solid (0.64 g, 77%). 1H NMR (CDCl3): δ 7.17 (d, J = 8.8 Hz, 2 H, ArH), 7.12 (d, J = 8.8 Hz, 2 H, ArH), 4.78 (d, J = 2.4 Hz, 2 H, CH2), 2.64 (s, 6 H, CH3), 2.57 (t, J = 2.4 Hz, 1 H, CH), 1.44 (s, 6 H, CH3). 13C{1H} NMR (CDCl3): δ 158.6, 156.8, 145.5, 141.3, 131.8, 129.2, 127.7, 116.1, 85.8, 78.0, 76.2, 56.2, 17.3, 16.2. MS (ESI): m/z 630 [M+] (48%) and 611 [M − F]+ (70%). HRMS (ESI): m/z calcd for C22H19BF2I2N2O [M]+ 629.9646, found 629.9650. Anal. Calcd for C22H19BF2I2N2O: C, 41.94; H, 3.04; N, 4.45. Found: C, 41.70; H, 3.01; N, 4.45. 4,4-Difluoro-2,6-diiodo-1,3,5,7-tetramethyl-8-(4-(3,6,9trioxa-9-propargylnonoxy)-phenyl)-4-bora-3a,4a-diaza-s-indacene (5b). According to the procedure described for 5a, BODIPY 4b (0.40 g, 0.78 mmol) was treated with iodic acid (0.28 g, 1.57 mmol) and iodine (0.50 g, 1.96 mmol) in EtOH (50 mL) to give 5b, which was purified by silica gel column chromatography using CH2Cl2/ CHCl3 (1:1, v/v) as the eluent. The product was obtained as a red sticky solid (0.48 g, 80%). 1H NMR (CDCl3): δ 7.12 (d, J = 8.8 Hz, 2 H, ArH), 7.04 (d, J = 8.8 Hz, 2 H, ArH), 4.21 (d, J = 2.4 Hz, 2 H, CH2), 4.20 (t, J = 4.8 Hz, 2 H, CH2), 3.92 (t, J = 4.8 Hz, 2 H, CH2), 3.76−3.79 (m, 2 H, CH2), 3.71−3.73 (m, 6 H, CH2), 2.64 (s, 6 H, CH3), 2.43 (t, J = 2.4 Hz, 1 H, CH), 1.43 (s, 6 H, CH3). 13C{1H} NMR (CDCl3): δ 159.7, 156.4, 145.3, 141.5, 131.6, 128.9, 126.6, 115.4, 85.6, 79.6, 74.7, 70.8, 70.5, 70.4, 69.6, 69.0, 67.5, 58.3, 17.2, 16.0. MS (ESI): m/z 785 [M + Na]+ (100%). HRMS (ESI): m/z calcd for C28H31BF2I2N2NaO4 [M + Na]+ 785.0343, found 785.0343. Anal. Calcd for C28H31BF2I2N2O4: C, 44.12; H, 4.10; N, 3.68. Found: C, 44.08; H, 4.10; N, 3.69. 4,4-Difluoro-2,6-diiodo-1,7-dimethyl-3,5-bis(4-(3,6,9trioxadecoxy)styryl)-8-(4-propargyloxyphenyl)-4-bora-3a,4adiaza-s-indacene (7a). A mixture of 5a (0.45 g, 0.71 mmol), benzaldehyde 6 (0.46 g, 1.71 mmol), glacial acetic acid (2.0 mL, 34.9 mmol), piperidine (2.4 mL, 24.3 mmol), and a small amount of Mg(ClO4)2 in toluene (60 mL) was heated at reflux overnight. The water formed during the reaction was removed with a Dean−Stark apparatus. The mixture was concentrated under reduced pressure. The residue was then purified by silica gel column chromatography using CH2Cl2/MeOH (100:1, v/v) as the eluent. The green fraction was collected and rotary-evaporated. Then it was further purified by size exclusion chromatography with Bio-beads S-X1 beads using THF as the eluent. The crude product was further purified by silica gel column chromatography using CH2Cl2/MeOH (100:1, v/v) as the eluent to give a dark-green solid (0.20 g, 25%). 1H NMR (CDCl3): δ 8.13 (d, J = 16.8 Hz, 2 H, CCH), 7.60 (d, J = 8.8 Hz, 4 H, ArH), 7.58 (d, J = 16.8 Hz, 2 H, CCH), 7.20 (d, J = 8.8 Hz, 2 H, ArH), 7.13 (d, J = 8.8 Hz, 2 H, ArH), 6.96 (d, J = 8.8 Hz, 4 H, ArH), 4.79 (d, J = 2.4 Hz, 2 H, CH2), 4.19 (t, J = 4.8 Hz, 4 H, CH2), 3.89 (t, J = 4.8 Hz, 4 H, CH2), 3.75−3.78 (m, 4 H, CH2), 3.66−3.72 (m, 8 H, CH2), 3.55−3.58 (m, 4 H, CH2), 3.39 (s, 6 H, CH3), 2.58 (t, J = 2.4 Hz, 1 H, CH), 1.50 (s, 6 H, CH3). 13C{1H} NMR (CDCl3): δ 160.0, 158.5, 150.5, 145.8, 139.2, 138.3, 133.3, 129.8, 129.3, 128.3, 116.8, 116.0, 115.1, 82.8, 78.0, 76.2, 72.0, 71.0, 70.8, 70.7, 69.8, 67.6, 59.2, 56.2, 17.8 (some of the signals are overlapped). MS (ESI): m/z 1153 [M + Na]+ (55%). HRMS (ESI): m/z calcd for C50H55BF2I2N2NaO9 [M + Na]+ 1153.1959, found 1153.1970. Anal. Calcd for C50H55BF2I2N2O9: C, 53.12; H, 4.90; N, 2.48. Found: C, 52.73; H, 4.88; N, 2.50. 4,4-Difluoro-2,6-diiodo-1,7-dimethyl-3,5-bis(4-(3,6,9trioxadecoxy)styryl)-8-(4-(3,6,9-trioxa-9-propargylnonoxy)phenyl)-4-bora-3a,4a-diaza-s-indacene (7b). According to the procedure described for 7a, BODIPY 5b (0.20 g, 0.26 mmol) was treated with benzaldehyde 6 (0.17 g, 0.63 mmol), glacial acetic acid (1.0 mL, 17.5 mmol), piperidine (1.2 mL, 12.1 mmol), and a small amount of Mg(ClO4)2 in toluene (50 mL) to give 7b, which was purified by silica gel column chromatography using CH2Cl2/MeOH (100:1, v/v) as the eluent followed by size exclusion chromatography with Bio-beads S-X1 beads using THF as the eluent. The crude product was purified by silica gel column chromatography again using CH2Cl2/MeOH (100:1, v/v) as the eluent. The product was obtained as a dark-green solid (0.13 g, 40%). 1H NMR (CDCl3): δ 8481

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The cells, after being rinsed with phosphate buffered saline (PBS), were incubated with 100 μL of these distyryl BODIPY solutions for 24 h at 37 °C under 5% CO2. The cells were then rinsed again with PBS and refilled with 100 μL of the culture medium before being illuminated at ambient temperature. The light source consisted of a 300 W halogen lamp, a water tank for cooling, and a color glass filter (Newport) with cuton at 610 nm. The fluence rate (λ > 610 nm) was 40 mW cm−2. Illumination of 20 min led to a total fluence of 48 J cm−2. After illumination, the cells were incubated at 37 °C under 5% CO2 overnight. Cell viability was determined by means of the colorimetric MTT assay.42 An MTT (USB) solution in PBS (3 mg mL−1, 50 μL) was added to each well followed by incubation for 3 h under the same environment at 37 °C. A solution of sodium dodecyl sulfate (USB, 10% by weight, 50 μL) was then added to each well. The plate was incubated in an oven at 60 °C for 30 min. Then 80 μL of isopropanol was added to each well. The plate was agitated on a Bio-Rad microplate reader at ambient temperature with 10 s of shaking before the absorbance at 540 nm for each well was taken. The average absorbance of the blank wells, which did not contain the cells, was subtracted from the readings of the other wells. The cell viability was then determined by the following equation: % viability = {∑[(Ai/Acontrol) × 100]}/n, where Ai is the absorbance of the ith data (i = 1, 2, ..., n), Acontrol is the average absorbance of the control wells in which the distyryl BODIPY was absent, and n (=4) is the number of the data points. Fluorescence Microscopic Studies. About 2 × 105 KB or MCF-7 cells in the culture medium (2 mL) were seeded on a coverslip and incubated overnight at 37 °C under 5% CO2. The medium was then removed. The cells, after being rinsed with PBS, were incubated with a solution of 11a in the folic acid free medium (5.0 μM, 2 mL) for 24 h under the same conditions. The cells were then rinsed with PBS and viewed with an Olympus IX 70 inverted microscope. The excitation light source at 630 nm was provided by a multiwavelength illuminator (Polychrome IV, TILL Photonics). The emitted fluorescence at >660 nm was collected, digitized, and analyzed using MetaFluor V 6.3 (Universal Imaging). The average intracellular fluorescence intensities (for a total of 24 cells for each sample) were also determined. For the cellular uptake study of 11a and 11b in KB cells, after incubation with 11a or 11b in the folic acid free medium (1.0 μM, 2 mL) for 24 h, the cells were rinsed with PBS and viewed with a Leica SP5 confocal microscope equipped with a 633 nm helium neon laser. The emission signals at 650−750 nm were collected, and the images were digitized and analyzed by Leica Application Suite Advanced Fluorescence software. The average intracellular fluorescence intensities (for a total of 50 cells in each sample) were also determined. Subcellular Localization Studies. About 1 × 105 KB cells in the culture medium (2 mL) were seeded on a coverslip and incubated overnight at 37 °C with 5% CO2. The medium was removed, and then the cells were rinsed with PBS. The cells were incubated with a solution of 11a or 11b in the folic acid free medium (1 μM, 2 mL) for 24 h under the same conditions. For the study using Lyso-Tracker, the cells were incubated with Lyso-Tracker Green DND 26 (Molecular Probes, 4.0 μM in the medium) for a further 15 min. For the study using Mito-Tracker and ER-Tracker, the cells were incubated with Mito-Tracker Green FM (Molecular Probes, 0.2 μM in the medium) or ER-Tracker Green (Molecular Probes, 1.0 μM in PBS) for a further 15 min. For all the cases, the cells were then rinsed with PBS and viewed with a Leica SP5 confocal microscope equipped with a 488 nm argon laser and a 633 nm helium neon laser. All the Trackers were excited at 488 nm and monitored at 510−560 nm, while 11a and 11b were excited at 633 nm and monitored at 650−800 nm. The images were digitized and analyzed using the Leica Application Suite Advanced Fluorescence software. The subcellular localization of 11a and 11b was revealed by comparing the intracellular fluorescence images caused by the Lyso-Tracker, Mito-Tracker, or ER-Tracker and the dyes.

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of DPBF in DMF using ZnPc, 11a, or 11b as the photosensitizer, and 1H and 13C{1H} NMR spectra of 4a, 4b, 5a, 5b, 7a, and 7b in CDCl3. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*For D.K.P.N.: phone, +852-3943-6375; fax, +852-2603-5057; e-mail, [email protected]. *For P.-C.L.: phone, +852-3943-1375; fax, +852-3943-1326; e-mail, [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Direct Grant for Research (for 2009/10 and 2010/11) of The Chinese University of Hong Kong.

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ABBREVIATIONS USED BODIPY, boron dipyrromethene; DCC, N,N′-dicyclohexylcarbodiimide; DMF, N,N-dimethylformamide; DMSO, dimethylsulfoxide; DPBF, 1,3-diphenylisobenzofuran; ESI, electrospray ionization; PBS, phosphate buffered saline; PDT, photodynamic therapy; TFA, trifluoroacetic acid; THF, tetrahydrofuran; ZnPc, zinc(II) phthalocyanine; ΦF, fluorescence quantum yield

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

HPLC chromatograms of 10, 11a, and 11b, UV−vis spectra of 11a and 11b in DMF, comparison of the rate of photodegradation 8482

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