Synthesis and in Vitro Photocytotoxicity of Coumarin Derivatives for

Jun 13, 2013 - Graduate University of Chinese Academy of Sciences, Beijing 100049, P. R. China. •S Supporting Information. ABSTRACT: Triethylene gly...
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Synthesis and in Vitro Photocytotoxicity of Coumarin Derivatives for One- and Two-Photon Excited Photodynamic Therapy Qianli Zou,†,§ Yanyan Fang,†,§ Yuxia Zhao,*,† Hongyou Zhao,‡ Ying Wang,‡ Ying Gu,‡ and Feipeng Wu*,† †

Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China Department of Laser Medicine, Chinese PLA General Hospital, Beijing 100853, P. R. China § Graduate University of Chinese Academy of Sciences, Beijing 100049, P. R. China. ‡

S Supporting Information *

ABSTRACT: Triethylene glycol functionalized coumarin derivatives 1−5 were synthesized and investigated as photosensitizers for one- and two-photon excited photodynamic therapy (PDT). Their absorption, fluorescence, triplet-state quantum yields, and singlet oxygen quantum yields were found to be significantly related to the substituent at the 7-position of the coumarin ring. In vitro photocytotoxicity of these derivatives toward HepG2 cells was examined and compared with a clinical drug. In vitro generation of reactive oxygen species (ROS), cellular uptake, and intracellular distribution of these derivatives were also characterized to fully reveal their structure−property relationships. The results showed that derivative 5, with a two-photon absorption cross section value of 1556 GM, could be a powerful PDT agent for both superficial diseases and solid tumors.

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melanoma.13 To overcome this shortcoming, preliminary research demonstrated the possibility of exciting coumarin derivatives by 2PA.14−16 However, the 2PA cross section (σ2) values of these derivatives are too low to explore the full potential of 2PE-PDT; for example, the σ2 of 4′-hydroxymethyl-4,5′,8-trimethylpsoralen is only 3.2 GM (1 GM = 1 × 10−50 cm4 s photon−1).15 To date, no result about effective photocytotoxicity toward cancer cells using coumarin derivatives by 2PE-PDT has been achieved. Recently we reported a series of coumarin derivatives with enhanced σ2 values, up to 1570 GM at 850 nm.17,18 In this study, through introduction of triethylene glycol (TEG) groups to improve the biocompatibility of such coumarin derivatives, we synthesized a series of novel coumarin derivatives, 1−5 (Scheme 1), specifically designed for 2PE-PDT. After the systemic study of the structure−property relationships of their water solubility, partition coefficient, 1PA, fluorescence, 2PA, triplet-state lifetimes and quantum yields, singlet oxygen quantum yields, cellular uptake, and in vitro photocytotoxicity, one photosensitizer was found exhibiting a high potential as a new 2PEPDT candidate.

INTRODUCTION Photodynamic therapy (PDT) is a promising choice for cancer therapy because of its low systemic toxicity and the feasibility of being used repeatedly.1 Since the first PDT drug, Photofrin, was approved for clinical use in 1993,2 the amount of research with the aim of designing new PDT drugs has been rapidly increasing.3−5 Though the optimal window for PDT is from 700 to 900 nm,6 the absorption of Photofrin is very low in this range.3 For promoting the application of PDT in oncology, designing near-infrared light-sensitive photosensitizers is very crucial. Recently developed two-photon excited photodynamic therapy (2PE-PDT) supplies a powerful solution for this problem.7−9 Compared with traditional, one-photon absorption (1PA) induced PDT, 2PE-PDT has three advantages.10 First, the light involved in a two-photon absorption (2PA) process is usually near-infrared light,11 by which the deeper penetration can be attained. Second, the 2PA only occurs under high illumination intensity, so the 2PA induced damage will only happen in a focused spot of the laser beam.10 Third, the photosensitizers designed for 2PE-PDT still maintain their 1PA ability under visible light, which is beneficial for the treatments of not only solid tumors but also superficial diseases. The photocytotoxicity of coumarin derivatives have drawn much attention for decades. One branch of these derivatives, psoralen, had been successfully used for clinical treatment of dermatological disorders.12 For example, the psoralen plus UVA light (PUVA) was the most efficient treatment for vitiligo.12 Regrettably, some strong evidence was presented that this treatment with UV-A light increased the risk of malignant © 2013 American Chemical Society

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RESULTS AND DISCUSSION Synthesis and Characterization. The main reaction toward photosensitizers 1−5 is a high yield aldol reaction (Scheme 1). In the final structures, the 7-position of the

Received: January 4, 2013 Published: June 13, 2013 5288

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Scheme 1. Synthetic Routes of Photosensitizers 1−5a

a

Conditions: (a) KOH, THF, room temperature, 24 h; (b) glacial acetic acid, piperidine, n-butanol, reflux, 4 h.

Table 1. Solubility in PBS, Octanol−Water Partition Coefficient (PC), and Photophysical Propertiesa of Photosensitizers 1−5 compd

solubility (mg mL−1)

1 2 3 4 5

0.0011 0.70 >10 0.026 0.75

log PC λmax1PA (nm) 3.0 1.4 0.3 3.4 2.2

454 448 450 464 465

εmax (104 M−1 cm−1)

λmaxfl (nm)

Φf

3.5 4.1 4.4 7.2 7.7

561 565 567 536 538

0.031 0.028 0.026 0.084 0.093

τf (ns) 0.35 0.31 0.34 0.48 0.52

(99.34%), (99.25%), (99.30%), (96.26%), (97.43%),

2.3 1.8 2.3 2.8 2.6

(0.66%) (0.75%) (0.70%) (3.74%) (2.57%)

λmax2PA (nm)

σ2max (GM)

750 760 740 820 820

284 409 397 1544 1556

a The solvent was toluene unless otherwise noted. λmax1PA, λmaxfl, and λmax2PA are the 1PA, fluorescence emission, and 2PA maxima, respectively. εmax is the molar extinction coefficient at λmax1PA. Φf is the fluorescence quantum yield. τf is the fluorescence lifetime. σ2max is the experimental 2PA cross section maximum in GM.

neously.20 After the modification, photosensitizers 2, 3, and 5 show higher solubility than 0.5 mg mL−1 in PBS (pH 7.4) while the other two photosensitizers have low hydrophilicity (Table 1). For the partition coefficient, the log PC value of 3 is significantly low (Table 1), which will limit its cellular uptake ability. Among the five photosensitizers, the water solubility and log PC data of 5 showed the best balance between hydrophilicity and lipophilicity. Photophysical Properties. The linear spectra of photosensitizers 1−5 in toluene are shown in Figure 1, and corresponding data are listed in Table 1. Within the experimental scope, 1−3 presented a charge transfer (CT) absorption band with a peak at around 450 nm and a localized absorption band of coumarin moiety at 300−400 nm. Because of the enhanced electrodonating ability of the diethylamino group, the localized absorption band of the coumarin moiety in 4 and 5 red-shifted and overlapped with their CT band (SFigure 1 in Supporting Information), which resulted in only a main 1PA band around 465 nm with enhanced molar extinction coefficient shown in 4 and 5. Meanwhile, the fluorescence

coumarin ring was connected to a substituted alkoxy group (1− 3) or a diethylamino group (4, 5). Both the alkoxy and the diethylamino groups are electrodonor groups, while the latter has stronger electrodonating capability. The impact of the substituents on the photophysical properties will be discussed below. All the new photosensitizers were characterized by various spectroscopic methods and element analyses. More details are in the Experimental Section. Solubility and Octanol−Water Partition Coefficient. For the treatment of solid tumors and microvascular diseases, the drug should be partially water-soluble to ensure the possibility of being delivered by intravenous injection. Meanwhile, the drug needs to maintain enough lipid solubility to enter target tissues through the lipid membranes of blood vessels. Thus, a moderate octanol−water partition coefficient (PC), which is defined as the ratio of the concentration of a drug in a mixture of octanol and water at equilibrium,19 is important for evaluating a drug. In photosensitizers 1−5, various numbers of TEG chain were introduced to improve the hydrophilicity and regulate the partition coefficient simulta5289

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Figure 1. 1PA, 2PA (σ2), and normalized fluorescence (Fluo) spectra of photosensitizers 1−5 in toluene. The excitation wavelength for the fluorescence was 460 nm. 2PA cross sections are given in GM (1 GM = 1 × 10−50 cm4 s photon−1).

spectra of 4 and 5 were blue-shifted and their fluorescence quantum yields (Φf) were obviously higher compared with those for 1−3. Because there are enone groups in the structures of 1−5, time-resolved fluorescence was carried out to evaluate the possibility of photoisomerization. The decay traces for fluorescence of 1−5 can be well fitted by a biexponential function (S-Figure 2 in Supporting Information), in which the components with short fluorescence lifetime (τf) are the majority (more than 96%, Table 1). This result indicates that photoisomerization is involved within our experimental conditions but the contributions from minor isomers are very limited, especially for 1−3. The σ2 was determined in toluene from 720 to 880 nm in 10 nm intervals by the TPEF method21 using Rhodamine B in methanol (10−4 M) as the reference.22 The results were summarized in Table 1 and Figure 1 with the viability of the tests provided in S-Figure 3 in Supporting Information. All the studied photosensitizers showed blue-shifted 2PA energy bands compared to corresponding 1PA energy bands. This means that a higher energy state other than the lowest excited state was possibly reached by two-photon excitation. The 2PA spectra of 1−3 showed two absorption bands, which is similar to their 1PA spectra, but the maxima 2PA peaks are in the shorter wavelength band; for example, 3 showed two 2PA peaks at 740 and 820 nm with a σ2max value of 397 GM at 740 nm. The intensity and shape of the 2PA spectra of 4 and 5 are similar. The σ2 values of 4 and 5 were strongly enhanced by the change of the electrodonor group. The σ2max values of 4 and 5 are 1544 and 1556 GM, respectively, which are 2 orders of magnitude larger than the values of the coumarin derivatives previously reported for 2PE-PDT.15 Meanwhile, on the basis of the simple molecular structures of 4 and 5, these values are quite comparable to values of other kinds of photosensitizers that have successfully demonstrated their possibility for 2PEPDT. 8,9,23 For example, Starkey et al. synthesized a porphyrin-containing photosensitizer with σ2max of ∼2000 GM and used it to treat xenograft.8 Gary-Bobo et al.

encapsulated a diphenylenemethane derivative (σ2max of 1200 GM) into mesoporous silica nanoparticles and used it in 2PEPDT.9 Anderson’s group reported two-photon-induced blood vessel closure by a porphyrin dimer with σ2max of 17 000 GM.23 Transient Properties and Photosensitization Activity. Singlet oxygen (1O2) produced by energy transfer from the excited triplet state of a sensitizer to the ground state of an oxygen is considered as the main cytotoxic agent involved in PDT.24 The probability of the energy transfer process is positively correlative to the triplet-state lifetime and triplet-state quantum yield of the sensitizer. The transient absorption spectra for 1−5 in nitrogen-saturated or air-saturated toluene solutions were obtained by laser flash photolysis (the nitrogensaturated ones plus with their decay profiles are shown in SFigure 4 in Supporting Information). The spectra of 1−3 obtained at 2.8 μs after laser excitation show two transient absorption peaks at 530 and 690 nm, respectively. Moreover, a new transient absorption band at around 560 nm appears in the spectra of 1−3 obtained at 25 μs after laser excitation. The signal at 690 nm decayed by one exponential term with a lifetime of ∼7 μs in nitrogen-saturated solutions and ∼0.15 μs in air-saturated solutions. Assuming the concentration of oxygen in air-saturated toluene to be 2 × 10−3 M,25 the oxygen quenching rate constant (kq) for the peaks at 690 nm was ∼3.2 × 109 s−1 M−1 (Table 2), close to the diffusion limit,26 indicating the triplet nature of the absorption. In nitrogensaturated solution, the decay profiles at 560 nm clearly show two components, one short-lived triplet state and another longlived ketyl radical (details discussed in Supporting Information). The decay profiles at 530 nm also include the two components, but the short-lived one is the majority. For 4 or 5, two peaks, at 520 and 770 nm, respectively, were found in the photolysis process. The two peaks showed the same decrease manner, a similar lifetime (∼5.0 μs), and a similar kq toward oxygen. Thus, we regard the transient spectra of 4 and 5 as pure triplet−triplet absorptions. The triplet-state quantum yield (ΦT) and singlet oxygen quantum yield (ΦΔ) in toluene were measured by independent 5290

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Table 2. Triplet-State Properties and Singlet Oxygen Quantum Yields of Photosensitizers 1−5 in Toluenea compd

ΦT

ΦΔ

kq (109 s−1 M−1)

PT 2

O

fTΔ

1 2 3 4 5

0.26 0.25 0.25 0.51 0.53

0.048 0.046 0.045 0.48 0.49

3.1 3.2 3.2 3.2 3.2

0.98 0.98 0.98 0.97 0.97

0.19 0.19 0.18 0.97 0.95

Table 3. Dark Cytotoxicity and One-Photon Photocytotoxicity of Photosensitizers 1−5 toward HepG2 Cellsa

ΦT is the triplet-state quantum yield. ΦΔ is the singlet oxygen quantum yield. kq is oxygen quenching rate constant toward the tripletO state. PT 2 is the fraction of triplet states quenched by ground state oxygen. fTΔ is the fraction of quenching events that result in the production of singlet oxygen. a

PTO2 = 1 −

IC50 (light)

1 2 3 4 5 13

>50 >50 >50 >50 >50 >50

8.1 >50 >50 3.9 4.6 3.3

Values indicate the concentration of a photosensitizer in μM that is necessary to achieve 50% growth inhibition (IC50) of HepG2 cells in the dark or irradiated by 532 nm light for 20 min (20 mW cm−2). Data shown are values from three replicate experiments.

explained by their stronger photosensitization activity and enhanced 1PA. Note that 532 nm is not the best wavelength for these photosensitizers under an in vitro environment (S-Figure 7 in Supporting Information); their PDT efficacy may be further improved by optimizing irradiation wavelength. To evaluate the 2PE-PDT activity, the cells were incubated with 20 μM photosensitizers for 4 h and then were divided into two equal series. One series was illuminated with 800 nm light for 10 min (1.64 W cm−2, 1 kHz, 130 fs). Another series was kept in the dark as control groups. After the illumination, all cells were incubated in the dark for 24 h before the viability test. The results are shown in Figure 2. In the blank group, the

(1)

τTair τTN2

IC50 (dark)

a

methods (the fitting graph for ΦΔ is provided in S-Figure 6 in Supporting Information), and results are tabulated in Table 2. Both the ΦT and ΦΔ values of 4 and 5 were close to 0.5. The ΦT values of 1−3 were about 0.25, while the ΦΔ values of 1−3 were only 0.05. For the singlet oxygen derivate from the sensitizer triplet state, ΦΔ can be expressed by the following equations:27 ΦΔ = ΦTPTO2fΔT

compd

(2) O PT 2

where ΦT is the triplet quantum yield, is the fraction of triplet state quenched by ground state oxygen, and fTΔ defined as sensitization efficiency is the fraction of such quenching events that result in the production of singlet oxygen. τNT 2 and τair T are the lifetimes of the triplet state in nitrogen-saturated and airsaturated solutions, respectively. From the equations, values of fTΔ and POT 2 were calculated based on triplet-state lifetimes and are provided in Table 2. All the POT 2 values are close to 1, meaning that all the triplet states of 1−5 can be efficiently quenched by ground state oxygen. For fTΔ, the values of 1−3 were only ∼0.19 while the values for 4 and 5 were close to 1. It indicates that the energy transfer to oxygen is the main quench manner for the triplet states of 4 and 5 but only contributes a minor part of the quenching of triplet states of 1−3. One- and Two-Photon in Vitro Activity. In vitro activities of the photosensitizers were carried out on HepG2 cells and were compared with PSD-007 (a mixture of porphyrins, 13),28 which is a clinical drug for the treatment of superficial lesions using the 532 nm laser. The concentrations of the photosensitizers to achieve 50% growth inhibition (IC50) of HepG2 cells in the dark or with onephoton PDT were determined and are listed in Table 3. For dark toxicity experiments, the cells were incubated with various concentrations of photosensitizers for 24 h before the viability test. For the assay, all the photosensitizers had negligible or undetectable dark toxicity up to 50 μM. In one-photon PDT, the cells were incubated with the photosensitizers for 4 h prior to irradiation with a 532 nm laser for 20 min (20 mW cm−2). Then the cells were incubated in dark for 24 h before the viability test. The IC50 value of photosensitizer 1 was 8.1 μM. No obvious photocytotoxicity was found for 2 and 3, which may due to their low partition coefficient. Under the same conditions, 4 and 5 both showed high photocytotoxicity with IC50 values less than 5 μM, comparable with the IC50 value of 13. The higher activity of 4 and 5 compared with 1 can be

Figure 2. Viability of HepG2 cells incubated with photosensitizers (20 μM) for 4 h and then illuminated under an 800 nm laser (1.64 W cm−2, 1 kHz, 130 fs) for 10 min (2PE-PDT series) or no light (dark series). The error bars denote standard deviation of three replicates.

cells were incubated with photosensitizer-free culture medium. Viability of the cells in this group was nearly unchanged after the irradiation, which confirmed the safety of the laser to the cells. The cells incubated with 1, 4, and 5 showed a viability close to 70%, 20%, and 30%, respectively, after the irradiation, while no 2PE-PDT effects were found for the groups treated with photosensitizers 2, 3, and 13. The efficiency of the photosensitizers toward cells under two-photon illumination is determined by σ2, ΦΔ, and the partition coefficient. The higher 2PE-PDT activity of 4 and 5 compared with 1 should be due to their higher photosensitization activity and larger σ2. Taking into account that both σ2 and ΦΔ of 5 are slightly higher than the values of 4, the highest two-photon photocytotoxicity of 4 should be coming from its higher partition coefficient. Since 13 is a mixture of several porphyrin monomers,28 it is not surprising that it is invalid under current light dose.29 Though 4 showed the best efficiency in both one- and two-photon excited PDT, its water solubility is limited. In fact, for clinical application, photosensitizer 5 is the most promising one 5291

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cellular uptake ability of new photosensitizers, especially the ones based on similar structures. The intracellular localization of photosensitizers 1, 4, and 5 was found mainly in mitochondria by confocal fluorescent microscope (S-Figure 8 in Supporting Information). The effort to get the intracellular fluorescence of 2 and 3 failed, which further confirmed their poor cell uptake ability.

because of its appreciable water solubility and remarkable photocytotoxicity. Intracellular ROS Generation. To further understand the photocytotoxicity, the intracellular production of ROS (reactive oxygen species) was studied using a ROS probe, 2,7dichlorodihydrofluorescein diacetate (DCFH-DA).30 In the presence of ROS, such as superoxide anion, hydroxyl radical, hydrogen peroxide, singlet oxygen, or its secondary ROS derivatives, DCFH-DA will convert to fluorescent 2,7dichlorofluorescein.31,32 As shown in Figure 3, all the

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CONCLUSIONS In summary, five coumarin derivatives modified by various numbers of TEG chains were synthesized and explored for oneand two-photon excited PDT. The increasing numbers of TEG chains are beneficial for the aqueous solubility but adverse to the cell uptake ability of these photosensitizers. All these photosensitizers exhibited low dark toxicity toward the HepG2 cells. Two photosensitizers, 4 and 5, with diethylamino groups in the 7 position of coumarin showed high 1PA, 2PA, tripletstate quantum yield, singlet oxygen quantum yield, in vitro ROS generation ability, and in vitro one- and two-photon excited PDT activity. The enhanced water solubility and significant inhibition of cell proliferation using 532 or 800 nm lasers highlight the great potential of photosensitizer 5 as a candidate for PDT and 2PE-PDT. This work demonstrated the feasibility of using coumarin as the scaffold to construct photosensitizers for traditional PDT and 2PE-PDT.

Figure 3. ROS production in HepG2 cells using the photosensitizers in the absence and presence of laser. The error bars denote standard deviation of three replicates.

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EXPERIMENTAL SECTION

Synthesis. The synthetic routes of 1−5 are shown in Scheme 1. The purity of all final compounds was determined to be at least 95% by a combination of 1H NMR, HR-MS, and element analysis. 3-Acetyl-7-hydroxycoumarin (6), 2-(2-(2-methoxyethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (7), 4-(methyl(2,5,8,11-tetraoxatridecan-13-yl)amino)benzaldehyde (10), 4-(di(2,5,8,11-tetraoxatridecan13-yl)amino)benzaldehyde (11), and 3-acetyl-7-diethylaminocoumarin (12) were synthesized according to our previous reports.17,20 3-Acetyl-7-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-2Hchromen-2-one (8). To a stirred solution of 6 (2.04g, 10 mmol) and KOH (1.70g, 30 mmol) in THF (30 mL) was added 7 (4.80g, 15 mmol) in THF (20 mL) dropwise over a period of 1 h. After the addition, the reaction mixture was stirred for 24 h at room temperature. Then the solvent was removed under reduced pressure. The obtained residue was dissolved in water and extracted with dichloromethane (3 × 100 mL). The organic phase was collected and dried over anhydrous MgSO4. The solvent was evaporated under reduced pressure and the residue was further purified by silica gel column chromatography to give 8 (2.98g, yield 85%). 1HNMR (400 MHz CDCl3): δ (ppm) 2.70 (s, 3H), 3.38 (s, 3H), 3.54−3.57 (m, 2H), 3.65−3.69 (m, 4H), 3.74−3.76 (m, 2H), 3.90 (t, 2H, J = 4.8 Hz), 4.22 (t, 2H, J = 4.6 Hz), 6.84 (d, 1H, J = 2.3 Hz), 6.93 (dd, 1H, J1 = 8.7 Hz, J2 = 2.4 Hz), 7.54 (d, 1H, J = 8.7 Hz), 8.49 (s, 1H). 3-(3-(4-(Diethylamino)phenyl)acryloyl)-7-(2-(2-(2methoxyethoxy)ethoxy)ethoxy)-2H-chromen-2-one (1). 4(Diethylamino)benzaldehyde (9) (0.89g, 5 mmol) and 8 (1.75g, 5 mmol) were dissolved in 20 mL of n-butanol under heating. Then 0.6 mL of glacial acetic acid and 0.6 mL of piperidine were added to the solution. The reaction mixture was refluxed for 4 h under nitrogen atmosphere, and then the solvent was removed in vacuum. The residue was purified by silica gel column chromatography to give the required photosensitizer 1 (yield 65%). 1HNMR (400 MHz CDCl3): δ (ppm) 1.21 (t, 6H, J = 7.1 Hz), 3.38−3.45 (m, 7H), 3.54−3.57 (m, 2H), 3.64−3.76 (m, 6H), 3.91 (t, 2H, J = 4.8 Hz), 4.23 (t, 2H, J = 4.8 Hz), 6.66 (d, 2H, J = 8.9 Hz), 6.86 (d, 1H, J = 2.2 Hz), 6.92 (dd, 1H, J1 = 9.0 Hz, J2 = 2.5 Hz), 7.53−7.58 (m, 3H), 7.77−7.88 (m, 2H), 8.56 (s, 1H). HR-MS (ESI): m/z calcd for C29H36NO7 [M + H]+ 510.248 63; found 510.248 39. Anal. Calcd for C29H35NO7: C, 68.35; H, 6.92; N, 2.75. Found: C, 68.14; H, 6.99; N, 2.61.

photosensitizers can generate ROS under irradiation. The amount of ROS follows the order of 4 > 5 > 1 > 2 > 3, which is consistent with the sequence of their photocytotoxicity. Cellular Uptake and Subcellular Localization. The cellular uptake and subcellular localization of 1−5 were also investigated. As shown in Figure 4, the concentration of the

Figure 4. Intracellular concentrations of 1−5 at different incubation times with 20 μM photosensitizers. The error bars denote standard deviation of three replicates.

photosensitizers in cells increased rapidly in the initial stage of the incubation and nearly reached a plateau after 4 h. The final intracellular concentration of 1 was significantly higher than those of other photosensitizers. The uptake efficiency of 2 and 3 was relatively low, which was responsible for their low photocytotoxicity. Moreover, although the order (1 > 4 > 5 > 2 > 3) of the intracellular concentration of these photosensitizers does not totally coincide with the sequence of their partition coefficients (4 > 1 > 5 > 2 > 3), the positive correlation between cellular uptake and PC can be found among 1−3 or between 4 and 5, respectively. This is helpful for predicting the 5292

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7-(2-(2-(2-Methoxyethoxy)ethoxy)ethoxy)-3-(3-(4-(methyl(2,5,8,11-tetraoxatridecan-13-yl)amino)phenyl)acryloyl)-2Hchromen-2-one (2), 3-(3-(4-(Di-2,5,8,11-tetraoxatridecan-13ylamino)phenyl)acryloyl)-7-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-2H-chromen-2-one (3), 7-(Diethylamino)-3-(3-(4(methyl(2,5,8,11-tetraoxatridecan-13-yl)amino)phenyl)acryloyl)-2H-chromen-2-one (4), and 3-(3-(4-(Di-2,5,8,11-tetraoxatridecan-13-ylamino)phenyl)acryloyl)-7-(diethylamino)2H-chromen-2-one (5). Compounds 2−5 were obtained by a similar procedure as described above for 1. The yield, 1H NMR, HR-MS, and element analysis data are listed as follows. 2. Yield 70%. 1H NMR (400 MHz CDCl3): δ (ppm) 3.07 (s, 3H), 3.37−3.38 (m, 6H), 3.52−3.68 (m, 22H), 3.73−3.76 (m, 2H), 3.91 (t, 2H, J = 4.6 Hz), 4.23 (t, 2H, J = 4.6 Hz), 6.70 (d, 2H, J = 8.9 Hz), 6.86 (d, 1H, J = 2.2 Hz), 6.92 (dd, 1H, J1 = 8.7 Hz, J2 = 2.4 Hz), 7.53−7.58 (m, 3H), 7.78−7.87 (m, 2H), 8.56 (s, 1H). HR-MS (ESI): m/z calcd for C35H48NO11 [M + H]+ 658.322 19; found 658.322 31. Anal. Calcd for C35H47NO11: C, 63.91; H, 7.20; N, 2.13. Found: C, 63.79; H, 7.22; N, 2.09. 3: Yield 55%. 1H NMR (400 MHz CDCl3): δ (ppm) 3.37−3.38 (m, 9H), 3.53−3.57 (m, 6H), 3.60−3.72 (m, 32H), 3.74−3.76 (m, 2H), 3.91 (t, 2H, J = 4.5 Hz), 4.23 (t, 2H, J = 4.4 Hz), 6.70 (d, 2H, J = 8.9 Hz), 6.85 (d, 1H, J = 2.2 Hz), 6.92 (dd, 1H, J1 = 8.7 Hz, J2 = 2.3 Hz), 7.53−7.56 (m, 3H), 7.78−7.87 (m, 2H), 8.56 (s, 1H). HR-MS (ESI): m/z calcd for C43H64NO15 [M + H]+ 834.427 05; found 834.426 87. Anal. Calcd for C43H63NO15: C, 61.93; H, 7.61; N, 1.68. Found: C, 62.05; H, 7.60; N, 1.67. 4. Yield 62%. 1H NMR (400 MHz CDCl3): δ (ppm) 1.25 (t, 6H, J = 7.1 Hz), 3.06 (s, 3H), 3.37 (s, 3H), 3.46 (q, 4H, J = 7.1 Hz), 3.50− 3.55 (m, 2H), 3.60−3.68 (m, 14H), 6.49 (d, 1H, J = 2.3 Hz), 6.62 (dd, 1H, J1 = 8.9 Hz, J2 = 2.4 Hz), 6.69 (d, 2H, J = 8.8 Hz), 7.42 (d, 1H, J = 9.0 Hz), 7.57 (d, 2H, J = 8.8 Hz), 7.82 (d, 1H, J = 15.5 Hz), 7.95 (d, 1H, J = 15.5 Hz), 8.54 (s, 1H). HR-MS (ESI): m/z calcd for C32H43N2O7 [M + H]+ 567.306 48; found 567.306 61. Anal. Calcd for C32H42N2O7: C, 67.82; H, 7.47; N, 4.94. Found: C, 67.56; H, 7.51; N, 4.81. 5. Yield 56%. 1H NMR (400 MHz CDCl3): δ (ppm) 1.26 (t, 6H, J = 7.1 Hz), 3.38 (s, 6H), 3.47 (q, 4H, J = 7.1 Hz), 3.54−3.56 (m, 4H), 3.61−3.67 (m, 28H), 6.50 (d, 1H, J = 2.3 Hz), 6.62 (dd, 1H, J1 = 9.0 Hz, J2 = 2.5 Hz), 6.71 (d, 2H, J = 8.9 Hz), 7.43 (d, 1H, J = 9.0 Hz), 7.56 (d, 2H, J = 8.8 Hz), 7.82 (d, 1H, J = 15.5 Hz), 7.96 (d, 1H, J = 15.5 Hz), 8.55 (s, 1H). HR-MS (ESI): m/z calcd for C40H59N2O11 [M + H]+ 743.411 34; found 743.411 45. Anal. Calcd for C40H58N2O11: C, 64.67; H, 7.87; N, 3.77. Found: C, 64.49; H, 7.90; N, 3.63. Characterization Methods. The details of solubility, partition coefficient, spectroscope, and photophysical measurements are the same as with our previous report unless otherwise noted.20 The details of TPEF method, singlet oxygen quantum yield, photolysis experiments, one-photon PDT model, 2PE-PDT model, intracellular ROS production, cell uptake, and subcellular distribution studies are provided in Supporting Information.

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AUTHOR INFORMATION

Corresponding Author

*For Y.Z.: phone, +86 10 82543532; fax, +86 10 82543491; email, [email protected]. For F.W.: phone, +86 10 82543569; fax, +86 10 82543491; e-mail, [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grant No. 60978057). The authors are grateful to Dr. Meiling Zheng for her expertise and assistance with the confocal fluorescence microscopy.

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ABBREVIATIONS USED PDT, photodynamic therapy; 2PE-PDT, two-photon excited photodynamic therapy; 1PA, one-photon absorption; 2PA, twophoton absorption; PC, partition coefficient; TEG, triethylene glycol; σ2, the two-photon absorption cross section; ΦT, tripletstate quantum yield; ΦΔ, singlet oxygen quantum yield; Φf, fluorescence quantum yield; TPEF, two-photon excited fluorescence; ROS, reactive oxygen species

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ASSOCIATED CONTENT

S Supporting Information *

Materials and instruments; experimental details of TPEF method; singlet oxygen quantum yield; photolysis experiments; one-photon PDT model; 2PE-PDT model; intracellular ROS production, cell uptake, and subcellular distribution studies; 1PA of 1, 4, 8, and 12 in toluene; fluorescence decay profiles; transient spectra and the decay profile; UV−vis spectra of 1−5 in cell culture media; confocal fluorescence images. This material is available free of charge via the Internet at http:// pubs.acs.org. 5293

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Journal of Medicinal Chemistry

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