Synthesis and in vitro photocytotoxicity of coumarin derivatives for one- and two-photon excited photodynamic therapy

Article abstract of DOI:10.1021/jm400025g

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.

Full text of DOI:10.1021/jm400025g

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Article
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
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400025g • Publication Date (Web): 13 Jun 2013
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Synthesis and In Vitro Photocytotoxicity of
Coumarin Derivatives for Oneꢀ and Twoꢀphoton
Excited Photodynamic Therapy
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Qianli Zoua, c, Yanyan Fanga, c, Yuxia Zhaoa,*, Hongyou Zhaob, Ying Wangb, Ying
Gub, and Feipeng Wua,*
aTechnical Institute of Physics and Chemistry, Chinese Academy of Sciences,
b
Beijing 100190, P. R. China; Department of Laser Medicine, Chinese PLA
c
General Hospital, Beijing,100853, P. R. China; Graduate University of Chinese
Academy of Sciences, Beijing 100049, P. R. China.
ABSTRACT: Triethylene glycolꢀfunctionalized coumarin derivatives 1ꢀ5 were
synthesized and investigated as photosensitizers for oneꢀ and twoꢀphoton excited
photodynamic therapy (PDT). Their absorption, fluorescence, tripletꢀstate quantum
yields, and singlet oxygen quantum yields were found to be significantly related to
the substituent at the 7ꢀposition 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 structureꢀproperty relationships. The results showed that derivative 5, with a
twoꢀphoton absorption cross section value of 1556 GM, could be a powerful PDT
agent for both superficial diseases and solid tumors.
INTRODUCTION
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Photodynamic therapy (PDT) is a kind of promising choice for cancer therapy
due to its low systemic toxicity and the feasibility to be used repeatedly.1 Since the
first PDT drug, Photofrin, was approved for clinical use in 1993,2 the researches
with the aim of designing new PDT drugs have been rapidly increasing.3ꢀ5 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 nearꢀinfrared lightꢀsensitive photosensitizers is very crucial. Recently
developed twoꢀphoton excited photodynamic therapy (2PEꢀPDT) supplies a
powerful solution for this problem.7ꢀ9 Compared with traditional, oneꢀphoton
absorption (1PA) induced PDT, 2PEꢀPDT has three advantages.10 Firstly, the light
involved in a twoꢀphoton absorption (2PA) process is usually nearꢀinfrared light,11
by which the deeper penetration can be attained. Secondly, 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 Thirdly, the photosensitizers designed for
2PEꢀPDT still maintain their 1PA ability under visible light, which is beneficial for
the treatments of not only solid tumors but also superficial diseases.
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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 Such as, the psoralen plus UVꢀA
light (PUVA) was the most efficient treatment for vitiligo.12 Regretably, some
strong evidences were presented that this treatment with UVꢀA light increased the
risk of malignant melanoma.13 To overcome this shortcoming, preliminary
researches demonstrated the possibility of exciting coumarin derivatives by
2PA.14ꢀ16 However, the 2PA cross section (
σ2) values of these derivatives are too
low to explore the full potential of 2PEꢀPDT, for example, the σ2 of
4’ꢀhydroxymethylꢀ4,5’,8ꢀtrimethylpsoralen is only 3.2 GM (1 GM = 1 × 10−50 cm4
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s photon−1).15 To date, no result about effective photocytotoxicity toward cancer
cells using coumarin derivatives by 2PEꢀPDT has been achieved. Recently we
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reported a series of coumarin derivatives with enhanced σ2 values, up to 1570 GM
at 850 nm.17,18 In this study, through introducing triethylene glycol (TEG) groups
to improve the bioꢀcompatibility of such coumarin derivatives, we synthesize a
series of novel coumarin derivatives, 1ꢀ5 (Scheme 1), specifically designed for
2PEꢀPDT. After the systemic study of the structureꢀproperty relationships of their
waterꢀsolubility, partition coefficient, 1PA, fluorescence, 2PA, tripletꢀstate
lifetimes and quantum yields, singlet oxygen quantum yields, cellular uptake, and
in vitro photocytotoxicity, one photosensitizer was found exhibiting a high
potential as new 2PEꢀPDT candidate.
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Scheme 1. The synthetic routes of photosensitizers 1ꢀ5.c
cConditions: (a) KOH, THF, room temperature, 24 h; (b) glacial acetic acid,
piperidine, nꢀbutanol, reflux, 4 h.
RESULTS AND DISCUSSION
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Synthesis and characterization. The main reaction toward photosensitizers 1ꢀ5 is
a high yield aldol reaction (Scheme 1). In the final structures, the 7ꢀposition of the
coumarin ring was connected to a substituted alkoxy group (1ꢀ3) or a diethylamino
group (4, 5). Both the alkoxy and the diethylamino groups are electroꢀdonor groups
while the later has stronger electroꢀdonating capability. The impact of the
substituents on the photophysical properties will be discussed hereinafter. All the
new photosensitizers were characterized by various spectroscopic methods and
element analysis. More details are in the experimental section.
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Table 1. Solubility in PBS, octanolꢀwater partition coefficient (PC), and
photophysical propertiesa of photosensitizers 1ꢀ5.
Solubility
(mg
εmax
(104
1PA
fl
2PA
max
ꢀꢁ
(ns)
log λmax
PC (nm)
λmax
(nm)
λmax
σ2
Compd
Φf
(nm)
750
760
740
820
820
(GM)
mLꢀ1)
Mꢀ1cmꢀ1)
0.35(99.34%),
2.3(0.66%)
0.31(99.25%),
1.8(0.75%)
0.34(99.30%),
2.3(0.70%)
0.48(96.26%),
2.8(3.74%)
0.52(97.43%),
2.6(2.57%)
fl
0.0011 3.0 454
3.5
4.1
4.4
7.2
7.7
561 0.031
565 0.028
567 0.026
536 0.084
538 0.093
284
1
2
3
4
5
0.70
>10
1.4 448
0.3 450
3.4 464
2.2 465
409
397
0.026
0.75
1544
1556
a The solvent was toluene unless otherwise noted; λmax1PA, λmax 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
fluorescence lifetime;
σ2max is the experimental 2PA cross section maxima in GM.
Solubility and Octanol-Water Partition Coefficient. For the treatment of solid
tumors and microꢀvascular diseases, the drug should be partially waterꢀsoluble to
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ensure the possibility of being delivered by intravenous injection. Meanwhile, the
drug need maintain enough lipidꢀsolubility to enter target tissues through the lipid
membranes of blood vessels. Thus a moderate octanolꢀwater 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 to evaluate a drug. In
photosensitizers 1ꢀ5, various numbers of TEG chain were introduced to improve
the hydrophilicity and regulate the partition coefficient simultaneously.20 After the
modification, photosensitizers 2, 3, and 5 show higher solubility than 0.5 mg mlꢀ1
in PBS (pH = 7.4) while other two photosensitizers are still low hydrophilic (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
waterꢀsolubility and log PC data of 5 showed the best balance between
hydrophilicity and lipophilicity.
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Photophysical properties. The linear spectra of photosensitizers 1ꢀ5 in toluene are
shown in Figure 1 and corresponding data are listed in Table 1. Within the
experimental scope, 1ꢀ3 presented a charge transfer (CT) absorption band with the
peak at around 450 nm and a localized absorption band of coumarin moiety in
300ꢀ400 nm. Due to the enhanced electroꢀdonating ability of the diethylamino
group, the localized absorption band of coumarin moiety in 4 and 5 redꢀshifted and
overlapped with their CT band (SꢀFigure 1 in Supporting information (SI)), which
resulted in only a main 1PA band around 465 nm with enhanced molar extinction
coefficient shown in 4 and 5. Meanwhile, the fluorescence spectra of 4 and 5 were
blueꢀshifted and their fluorescence quantum yields (Φf) were obviously higher
compared with 1ꢀ3.
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Figure 1. 1PA, 2PA (
σ
2), and normalized fluorescence (Fluo.) spectra of
photosensitizers 1ꢀ5 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).
In light of that there are enone groups in the structures of 1ꢀ5, timeꢀresolved
fluorescence were carried out to evaluate the possibility of photoisomerization. The
decay traces for fluorescence of 1ꢀ5 can be well fitted by biexponential function
(SꢀFigure 2 in SI), 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 1ꢀ3.
The σ2 was determined in toluene from 720 nm 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 SꢀFigure 3 in SI. All the studied photosensitizers showed blueꢀshifted
2PA energy bands compared to corresponding 1PA energy bands. This means a
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higher energy state other than the lowest excited state was possibly reached by
twoꢀphoton excitation. The 2PA spectra of 1ꢀ3 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 nm and 820 nm
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max
with the σ2 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 electroꢀdonor group. The σ2max value of 4 and 5 are 1544 GM and
1556 GM, respectively, which are two orders of magnitude larger than the values
of the coumarin derivatives previously reported for 2PEꢀPDT.15 Meanwhile, based
on the simple molecular structures of 4ꢀ5, these values are quite comparable to
other kinds of photosensitizers which have successfully demonstrated their
possibility for 2PEꢀPDT.8,9,23 For example, Starkey et al. synthesized a
max
porphyrinꢀcontaining photosensitizer with σ2 of ~2000 GM and used it to treat
xenograft.8 GaryꢀBobo et al encapsulated a diphenylenemethane derivative (σ2
of 1200 GM) into mesoporous silica nanoparticles and used it in 2PEꢀPDT.9
Anderson’s group reported twoꢀphotonꢀinduced blood vessel closure by a
porphyrin dimer with σ2max of 17000 GM.23
max
Transient properties and photosensitization activity. Singlet oxygen (1O2)
produced by energy transfer from the excited tripletꢀstate 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 tripletꢀstate lifetime and tripletꢀstate quantum of the sensitizer. The transient
absorption spectra for 1ꢀ5 in nitrogenꢀsaturated or airꢀsaturated toluene solutions
were obtained by laser flash photolysis (the nitrogenꢀsaturated ones plus with their
decay profiles were shown in SꢀFigure 4 in SI). The spectra of 1ꢀ3 obtained at 2.8
ꢁs after laser excitation show two transient absorption peaks at 530 nm and 690 nm,
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respectively. Moreover, a new transient absorption band around 560 nm appears in
the spectra of 1ꢀ3 obtained at 25 ꢁs after laser excitation. The signal at 690 nm
decayed by one exponential term with lifetime of ∼7 ꢁs in nitrogenꢀsaturated
solutions and ∼0.15 ꢁs in airꢀsaturated solutions. Assuming the concentration of
oxygen in airꢀsaturated toluene as 2×10ꢀ3 M,25 the oxygen quenching rate constant
(kq) for the peaks at 690 nm was ∼3.2×109 Sꢀ1 Mꢀ1 (Table 2), close to the diffusion
limit26, indicating the triplet nature of the absorption. In nitrogenꢀsaturated solution,
the decay profiles at 560 nm clearly show two components, one shortꢀlived triplet
state and another longꢀlived ketyl radical (details discussed in SI). The decay
profiles at 530 nm also include the two components, but the shortꢀlived 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, the similar
lifetime (~5.0 ꢁs), and the similar kq toward oxygen. Thus, we regard the transient
spectra of 4 and 5 as pure tripletꢀtriplet absorptions.
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The tripletꢀstate quantum yield (ΦT) and singlet oxygen quantum yield (Φꢂ) in
toluene were measured by independent methods (the fitting graph for Φꢂ was
provided in SꢀFigure 6 in SI) and results were tabulated in Table 2. Both the ΦT
and Φꢂ values of 4 and 5 were close to 0.5. The ΦT values of 1ꢀ3 were about 0.25
while the Φꢂ values of 1ꢀ3 were only 0.05. For singlet oxygen derivate from the
sensitizer tripletꢀstate, Φꢀ can be expressed as the following equations:27
ꢅ
ꢃ
ꢃ
ꢆ
∅∆ ꢂ ∅ꢃꢄ ꢇ
(1)
(2)
∆
ꢋꢌꢍ
ꢊ
ꢉ
ꢅ
ꢆ
ꢄ ꢂ 1 ꢈ
ꢎ
ꢆ
ꢃ
ꢉ
ꢊ
where ΦT is the triplet quantum yield, ꢄꢅ is the fraction of tripletꢀstate quenched
ꢆ
ꢃ
by ground state oxygen, and ꢇꢃ defined as sensitization efficiency is the fraction
∆
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of such quenching events that result in the production of singlet oxygen. ꢀꢃꢏ and
ꢀꢃꢐꢑꢒ are the lifetimes of the tripletꢀstate in nitrogenꢀsaturated and airꢀsaturated
solutions, respectively.
ꢆ
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ꢅ
ꢃ
ꢃ
ꢆ
From the equations, values of ꢇ and ꢄ were calculated based on tripletꢀstates’
∆
lifetimes and provided in Table 2. All the ꢄꢅ values are close to 1, meaning that
ꢆ
ꢃ
all the tripletꢀstates of 1-5 can be efficiently quenched by ground state oxygen. For
ꢇꢃ, the values of 1ꢀ3 were only ~0.19, while the values for 4 and 5 were close to 1.
∆
It indicates that the energyꢀtransfer to oxygen is the main quench manner for the
tripletꢀstates of 4ꢀ5 but only contributes a minor part of the quenching of
tripletꢀstates of 1ꢀ3.
Table 2. The tripletꢀstate properties and singlet oxygen quantum yield of
photosensitizers 1ꢀ5 in toluene.
kq
ꢄꢅ
ꢇꢃ
ꢆ
Compd
ΦT
Φꢀ
109 Sꢀ1 Mꢀ1
∆
ꢃ
0.26
0.25
0.25
0.51
0.53
0.048
0.046
0.045
0.48
3.1
3.2
3.2
3.2
3.2
0.98 0.19
0.98 0.19
0.98 0.18
0.97 0.97
0.97 0.95
1
2
3
4
5
0.49
ΦT is the tripletꢀstate quantum yield; Φꢂ is the singlet oxygen quantum yield; kq is
oxygen quenching rate constant toward the tripletꢀstate; ꢄꢅ is the fraction of
ꢆ
ꢃ
tripletꢀstates quenched by ground state oxygen; ꢇꢃ is the fraction of quenching
∆
events that result in the production of singlet oxygen;
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Table 3. Dark cytotoxicity and oneꢀphoton photocytotoxicity of photosensitizers
1ꢀ5 toward HepG2 cells.a
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Compound IC50 (Dark) IC50 (Light)
>50
>50
>50
>50
>50
>50
8.1
>50
>50
3.9
1
2
3
4
4.6
5
3.3
13
a
Values indicate the concentration of a photosensitizer in ꢃM that is necessary to
achieve 50% growth inhibition (IC50) of HepG2 cells under the dark or irradiated
by 532nm light for 20 min (20 mW cmꢀ2). Data shown are values from three
replicate experiments.
One- and two-photon in vitro activity. In vitro activities of the photosensitizers
were carried out on HepG2 cells and were compared with PSDꢀ007 (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 under the dark or oneꢀphoton PDT were
determined and listed in Table 3. For dark toxicity experiments, the cells were
incubated with a 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 ꢃM. In oneꢀphoton PDT, the cells were incubated with the
photosensitizers for 4 h prior to irradiation with a 532 nm laser for 20 min (20 mW
cmꢀ2). Then the cells were incubated in dark for 24 h before the viability test. The
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IC50 value of photosensitizer 1 was 8.1 ꢃM. No obvious photocytotoxicity was
found for 2 and 3, which may due to their low partition coefficient. Under the same
condition, 4 and 5 both showed high photocytotoxicity with IC50 values less than 5
ꢃM, comparable with the IC50 value of 13. The higher activity of 4 and 5 than 1
can be explained by their stronger photosensitization activity and enhanced 1PA.
Noted that, 532 nm is not the best wavelength for these photosensitizers under in
vitro environment (SꢀFigure 7 in SI), their PDT efficacy may be further improved
by optimizing irradiation wavelength.
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Figure 2. Viability of HepG2 cells incubated with photosensitizers (20 ꢁM) for 4 h
and then illuminated under an 800 nm laser (1.64 W cmꢀ2, 1 KHz, 130 fs) for 10
min (2PEꢀPDT series) or no light (dark series). The error bars denote standard
deviation from three replicates.
To evaluate the 2PEꢀPDT activity, the cells were incubated with 20 ꢁM
photosensitizers for 4 h and then were divided into two equal series. One series
were illuminated with 800 nm light for 10 min (1.64 W cmꢀ2, 1 KHz, 130 fs).
Another series was kept in dark as control groups. After the illumination, all cells
were incubated in the dark for 24 h before viability test. The results were shown in
Figure 2. In the blank group, the cells were incubated with photosensitizerꢀfree
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culture media. Viability of the cells in this group was nearly no change after the
irradiation, which confirmed the safety of the laser to the cells. The cells incubated
with 1, 4, and 5 showed the viability close to 70%, 20%, and 30%, respectively
after the irradiation, while no 2PEꢀPDT effects was found for the groups treated
with photosensitizer 2, 3 and 13. The efficiency of the photosensitizers toward
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cells under twoꢀphoton illumination is determined by
coefficient. The higher 2PEꢀPDT activity of 4 and 5 compared with 1 should be
due to their higher photosensitization activity and larger 2. Taking into account
σ2, Φꢂ and partition
σ
that both σ2 and Φꢂ of 5 are slightly higher than the values of 4, the highest
twoꢀphoton 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 oneꢀ and twoꢀphoton excited PDT, its waterꢀsolubility is limited.
In fact, for clinical application, photosensitizer 5 is the most promising one due to
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, 2,7ꢀdichlorodihydrofluorescein diacetate (DCFHꢀDA).30 In the presence of
ROS, such as superoxide anion, hydroxyl radical, hydrogen peroxide, singlet
oxygen or its secondary ROS derivatives, DCFHꢀDA will convert to fluorescent
2,7ꢀdichlorofluorescein31,32. As shown in Figure 3, all the photosensitizers can
generate ROS under irradiation. The amount of ROS follows the order of
4>5>1>2>3, which consists with the sequence of their photocytotoxicity.
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Figure 3 ROS production in HepG2 cells using the photosensitizers in the absence
and presence of laser. The error bars denote standard deviation from three
replicates.
Cellular uptake and subcellular localization. The cellular uptake and subcellular
localization of 1ꢀ5 were also investigated. As shown in Figure 4, the concentration
of the photosensitizers in cells increased rapidly in the initial of the incubation and
nearly reached a plateau after four hours. The final intracellular concentration of 1
was significantly higher than 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 do 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 1ꢀ3 or between 4 and 5, respectively. This is helpful to predict
the cellular uptake ability of new photosensitizers, especially the ones based on the
similar structures.
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Figure 4 Intracellular concentration of 1ꢀ5 at different incubation time with 20 ꢁM
photosensitizers. The error bars denote standard deviation from three replicates.
The intracellular localization of photosensitizer 1, 4, and 5 was found mainly in
mitochondria by confocal fluorescent microscope (SꢀFigure 8 in SI). The effort to
get the intracellular fluorescence of 2 and 3 was failed, which further confirmed
their poor cell uptake ability.
CONCLUSIONS
In summary, five coumarin derivatives modified by various numbers of TEG
chains were synthesized and explored for oneꢀ and twoꢀphoton 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, tripletꢀstate quantum yield, singlet oxygen quantum yield,
in vitro ROS generation ability, and in vitro oneꢀ and twoꢀphoton excited PDT
activity. The enhanced waterꢀsolubility and significant inhibition of cell
proliferation using 532 or 800 nm lasers highlights the great potential of
photosensitizer 5 as a candidate for PDT and 2PEꢀPDT. This work demonstrated
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the feasibility of using coumarin as the scaffold to construct photosensitizers for
both traditional PDT and 2PEꢀPDT.
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EXPERIMENTAL SECTION
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Synthesis.
The synthetic routes of 1ꢀ5 were shown in Scheme 1. The purity of all final
1
compounds was determined to be at least 95% by a combination of HNMR,
HRꢀMS and element analysis.
3ꢀacetylꢀ7ꢀhydroxycoumarin
(6),
(7),
2ꢀ(2ꢀ(2ꢀmethoxyethoxy)ethoxy)ethylꢀ4ꢀmethylbenzenesulfonate
4ꢀ(methyl(2,5,8,11ꢀtetraoxatridecanꢀ13ꢀyl)amino)benzaldehyde
4ꢀ(di(2,5,8,11ꢀtetraoxatridecanꢀ13ꢀyl)amino)benzaldehyde
(10),
and
(11)
3ꢀacetylꢀ7ꢀdiethylaminocoumarin (12) were synthesized according to our previous
reports.17,20
3ꢀacetylꢀ7ꢀ(2ꢀ(2ꢀ(2ꢀmethoxyethoxy)ethoxy)ethoxy)ꢀ 2Hꢀchromenꢀ2ꢀone (8):
To a stirred solution of 6 (2.04g, 10 mmol) and KOH (1.70g, 30 mmol) in THF (30
ml), 7 (4.80g, 15 mmol) in THF (20 ml) was added 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
1
chromatography to get 8 (2.98g, yield 85%). HNMR (400MHz CDCl3): δ (ppm)
2.70 (s, 3H), 3.38 (s, 3H), 3.54ꢀ3.57 (m, 2H), 3.65ꢀ3.69 (m, 4H), 3.74ꢀ3.76 (m,
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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).
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3ꢀ(3ꢀ(4ꢀ(diethylamino)phenyl)acryloyl)ꢀ7ꢀ(2ꢀ(2ꢀ(2ꢀmethoxyethoxy)ethoxy)ethoxy)
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ꢀ2Hꢀchromenꢀ2ꢀone (1):
4ꢀ(diethylamino)benzaldehyde (9) (0.89g, 5 mmol) and 8 (1.75g, 5 mmol) were
dissolved in 20 ml of nꢀbutanol 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 get the
required photosensitizer 1 (yield 65%): 1HNMR (400MHz CDCl3): δ (ppm) 1.21 (t,
6H, J=7.1 Hz), 3.38ꢀ3.45 (m, 7H), 3.54ꢀ3.57 (m, 2H), 3.64ꢀ3.76 (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), 7.53ꢀ7.58 (m, 3H), 7.77ꢀ7.88 (m, 2H),
8.56 (s, 1H). HRꢀMS (ESI): m/z Calcd for C29H36NO7 [M+H]+ 510.24863; found
510.24839. Elem. Anal.: Calcd for C29H35NO7: C, 68.35; H, 6.92; N, 2.75. Found:
C, 68.14; H, 6.99; N, 2.61.
7ꢀ(2ꢀ(2ꢀ(2ꢀmethoxyethoxy)ethoxy)ethoxy)ꢀ3ꢀ(3ꢀ(4ꢀ(methyl(2,5,8,11ꢀtetraoxatridec
anꢀ13ꢀyl)amino)phenyl)acryloyl)ꢀ2Hꢀchromenꢀ2ꢀone
3ꢀ(3ꢀ(4ꢀ(di2,5,8,11ꢀtetraoxatridecanꢀ13ꢀylamino)phenyl)acryloyl)ꢀ7ꢀ(2ꢀ(2ꢀ(2ꢀmetho
xyethoxy)ethoxy)ethoxy)ꢀ 2Hꢀchromenꢀ2ꢀone (3),
7ꢀ(diethylamino)ꢀ3ꢀ(3ꢀ(4ꢀ(methyl(2,5,8,11ꢀtetraoxatridecanꢀ13ꢀyl)amino)phenyl)ac
ryloyl)ꢀ 2Hꢀchromenꢀ2ꢀone (4) and
(2),
3ꢀ(3ꢀ(4ꢀ(di2,5,8,11ꢀtetraoxatridecanꢀ13ꢀylamino)phenyl)acryloyl)ꢀ7ꢀ(diethylamino)
ꢀ 2Hꢀchromenꢀ2ꢀone (5) were obtained by the same procedure described above for
1. The yield, 1HNMR, HRꢀMS and element analysis data are listed as follows:
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2 (yield 70%): HNMR (400MHz CDCl3): δ (ppm) 3.07 (s, 3H), 3.37ꢀ3.38 (m,
6H), 3.52ꢀ3.68 (m, 22H), 3.73ꢀ3.76 (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), 7.53ꢀ7.58 (m, 3H), 7.78ꢀ7.87 (m, 2H), 8.56 (s, 1H). HRꢀMS (ESI): m/z
Calcd for C35H48NO11 [M+H]+ 658.32219; found 658.32231. Elem. Anal.: Calcd
for C35H47NO11: C, 63.91; H, 7.20; N, 2.13. Found: C, 63.79; H, 7.22; N, 2.09.
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1
3 (yield 55%): HNMR (400MHz CDCl3): δ (ppm) 3.37ꢀ3.38 (m, 9H), 3.53ꢀ3.57
(m, 6H), 3.60ꢀ3.72 (m, 32H), 3.74ꢀ3.76 (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), 7.53ꢀ7.56 (m, 3H), 7.78ꢀ7.87 (m, 2H), 8.56 (s, 1H). HRꢀMS (ESI):
m/z Calcd for C43H64NO15 [M+H]+ 834.42705; found 834.42687. Elem. 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%). 1HNMR (400MHz 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), 3.50ꢀ3.55 (m, 2H), 3.60ꢀ3.68 (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). HRꢀMS (ESI): m/z Calcd for C32H43N2O7 [M+H]+
567.30648; found 567.30661. Elem. 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%): 1HNMR (400MHz CDCl3): δ (ppm) 1.26 (t, 6H, J=7.1 Hz), 3.38 (s,
6H), 3.47 (q, 4H, J=7.1Hz), 3.54ꢀ3.56 (m, 4H), 3.61ꢀ3.67 (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). HRꢀMS (ESI): m/z Calcd for C40H59N2O11 [M+H]+
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743.41134; found 743.41145. Elem. Anal.: Calcd for C40H58N2O11: C, 64.67; H,
7.87; N, 3.77. Found: C, 64.49; H, 7.90; N, 3.63.
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Characterization Methods.
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The details of solubility, partition coefficient, spectroscope, and photophysical
measurements are the same with our previous report unless otherwise noted.20 The
details of TPEF method, singlet oxygen quantum yield, photolysis experiments,
oneꢀphoton PDT model, 2PEꢀPDT model, intracellular ROS production, cell
uptake, and subcellular distribution studies were provided in Supporting
Information.
ASSOCIATED CONTENT
Supporting Information
The materials and instruments. The experimental details of TPEF method, singlet
oxygen quantum yield, photolysis experiments, oneꢀphoton PDT model, 2PEꢀPDT
model, intracellular ROS production, cell uptake, and subcellular distribution
studies. The 1PA of 1, 4, 8, and 12 in toluene; the fluorescence decay profiles; the
transient spectra and the decay profile; the Uvꢀvis spectra of 1ꢀ5 in cell culture
media; confocal fluorescence images. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
*Corresponding author
For Yuxia Zhao: Tel, +86 10 82543532; Fax, +86 10 82543491; Eꢀmail,
yuxia.zhao@mail.ipc.ac.cn. For Feipeng Wu: Tel, +86 10 82543569; Fax, +86 10
82543491; Eꢀmail, fpwu@mail.ipc.ac.cn.
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ACKNOWLEDGMENTS
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This work was supported by the National Natural Science Foundation of China
(No. 60978057). The authors are grateful to Dr Meiling Zheng for her expertise
assistance with the confocal fluorescence microscopy.
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ABBREVIATIONS USED
PDT, photodynamic therapy; 2PEꢀPDT, twoꢀphoton excited photodynamic
therapy; 1PA, oneꢀphoton absorption; 2PA, twoꢀphoton absorption; PC, partition
coefficient; TEG, triethylene glycol;
σ2, the 2PA cross section; ΦT , tripletꢀstate
quantum yield; Φꢂ, singlet oxygen quantum yield; Φf, fluorescence quantum yield;
TPEF, twoꢀphoton excited fluorescence; ROS, reactive oxygen species; SI,
Supporting Information.
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