Quantitative and site-directed chemical modification of hypocrellins toward direct drug delivery and effective photodynamic activity

Article abstract of DOI:10.1021/jm2017368

For photodynamic therapy (PDT) treatment of microvascular diseases, drugs are delivered via blood circulation and the targets are vasculature endothelial cells, for which the contradictory requirements of hydrophilicity and lipophilicity of the drugs have been achieved by liposome preparations. Herein, it is demonstrated that the drug delivery and target affinity are achieved by a single chemical compound, hypocrellin B (HB) derivative 6 selected from three novel aminoalkanesulfonic acid HB derivatives, 5-7. 6 exhibits a much higher PDT activity (IC₅₀ = 22 nM) on human gastric carcinoma BGC823 cells than HB, while it has no cellular toxicity in the dark. On the basis of estimation of the clinically required concentration according to relative PDT activity and clinical criteria, it can be predicted that 6 is directly deliverable to and PDT effective on target cells. The enhanced red absorption and superhigh photoactivity suggest that 6 is more powerful for PDT of tumors than HB.

Full text of DOI:10.1021/jm2017368

Article
pubs.acs.org/jmc
Quantitative and Site-Directed Chemical Modification of Hypocrellins
toward Direct Drug Delivery and Effective Photodynamic Activity
Hong Deng,† Xin Liu,† Jie Xie,*,† Rong Yin,‡ Naiyan Huang,‡ Ying Gu,*,‡ and Jingquan Zhao*,†
†Beijing National Laboratory for Molecular Sciences (BNLMS), Key Laboratory of Photochemistry, Institute of Chemistry, Chinese
Academy of Sciences, Beijing 100080, People’s Republic of China
‡Department of Laser Medicine, Chinese PLA General Hospital, Beijing 100853, People’s Republic of China
S
* Supporting Information
ABSTRACT: For photodynamic therapy (PDT) treatment of
microvascular diseases, drugs are delivered via blood
circulation and the targets are vasculature endothelial cells,
for which the contradictory requirements of hydrophilicity and
lipophilicity of the drugs have been achieved by liposome
preparations. Herein, it is demonstrated that the drug delivery
and target affinity are achieved by a single chemical compound,
hypocrellin B (HB) derivative 6 selected from three novel
aminoalkanesulfonic acid HB derivatives, 5−7. 6 exhibits a
much higher PDT activity (IC50 = 22 nM) on human gastric
carcinoma BGC823 cells than HB, while it has no cellular
toxicity in the dark. On the basis of estimation of the clinically required concentration according to relative PDT activity and
clinical criteria, it can be predicted that 6 is directly deliverable to and PDT effective on target cells. The enhanced red absorption
and superhigh photoactivity suggest that 6 is more powerful for PDT of tumors than HB.
Chart 1. Structures of 1−7
INTRODUCTION
■
In recent years, photodynamic therapy (PDT) has become a
powerful clinical treatment of various diseases.1,2 In principle,
PDT depends on selective destruction of the targets by singlet
oxygen or other reactive oxygen species (ROS) generated
jointly by photosensitizers (drugs), light (usually a laser), and
oxygen. Compared to well-developed lasers, clinically usable
photosensitizers (drugs) specifically aimed at individual disease
are seriously deficient.3 An ideal PDT drug should satisfy some
criteria, including strong absorption during the “photo-
therapeutic window”, high photoactivity but low dark toxicity,
high affinity for target cells but fast clearance from tissues, and
ready deliverability in vivo.4 Hypocrellins, including hypocrellin
A (1) and hypocrellin B (2) (shown in Chart 1), photo-
sensitizers isolated originally from wild Hypocrella bambusae but
recently synthesized chemically or biologically,5,6 exhibit most
of these advantages;7−10 however, low absorption during the
phototherapeutic window (600−900 nm) is a serious drawback
for PDT of solid tumors,11,12 while strong lipophilicity restricts
drug delivery. Compared to solid tumors, some microvascular
diseases, such as age-related macular degeneration (AMD) and
port wine stains (PWSs), commonly occur on shallow surfaces
(no more than 1 mm), which just coincides with the tissue-
penetration depth of light from 500 to 600 nm.13,14 Therefore,
hypocrellins may be characteristic photosensitizers specifically
suitable for PDT of microvascular diseases. Aqueous solubility
is essential for bioavailability of a drug,15 and even more
important for those specifically aimed at microvascular diseases
because the drug is delivered via intravenous injection.
Lipophilic 1 and 2 are favorable for cellular uptake, but they
may aggregate in blood and therefore block vascular nets. On
the other hand, water-soluble derivatives are readily deliverable
but commonly exhibit low PDT activity due to poor cellular
uptake.12 To satisfy the contradictory requirements of hydro-
Received: June 23, 2011
Published: February 21, 2012
© 2012 American Chemical Society
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Figure 1. Absorption (left) and fluorescence emission (right, excited at 470 nm and normalized to the maximum) spectra of compounds 2 and 5−7
at the same concentration of 50 μM in dimethyl sulfoxide.
philicity and lipophilicity, a pharmaceutical strategy is to use
drug-delivery vehicles such as liposomes or water-soluble
particles.16−18 However, a large amount of low-density
lipoproteins or other photoinactive materials may burden
blood circulation and reduce the effective drug concentration.
Alternatively, a chemical strategy is to find a compromise
between the lipophilicity and hydrophilicityamphiphilicity. In
the past two decades, about 50 hypocrellin derivatives were
synthesized by introducing polar groups, and most of them
were announced to be “amphiphilic”, but the amphiphilicity
had only relative significance. Furthermore, it was found that
any improvement in the aqueous solubility would unexcep-
tionally lead to the PDT activity being sacrificed.12,19 From a
medicinal chemistry view, amphiphilicity or aqueous solubility
should be quantitative rather than qualitative, which can be
defined as the aqueous solubility being equal to or larger than
the clinically required concentration while the lipophilicity
remains as high as possible. Noticeably, sulfonated hypocrellin
is completely water soluble,20 but loses biological PDT activity,
while taurine- or aminopropanesulfonic acid-substituted hypo-
crellin B (3 and 4, Chart 1) shows a sequential decrease in
aqueous solubility but an increase in PDT activity,21,22 implying
that the aqueous solubility may be quantitatively regulated by a
step lengthening the alkyl chain in the substitutent.
Besides cellular uptake, photosensitization activity, mainly
evaluated by the singlet oxygen yield, is another key important
factor for the PDT effect. It was reported that the PDT activity
of pegylated distyrylboron dipyrromethene derivatives depend
greatly on the types of substituents,23 which may be a complex
result of cellular uptake and photosensitizing activity. Besides
what has been mentioned, another advantage of hypocrellins is
that the chemical structures are readily modifiable by
introducing various substituent(s) to site 2, 5, 8, 11, 13, 14,
or 17, but singlet oxygen yields of the derivatives vary greatly.
Generally, 2-, 5-, or 8-substituted derivatives exhibit a low
singlet oxygen (1O2) yield24−27 and 13-, 14-, or 17-substituted
derivatives a higher 1O2 yield.22,28,29 Especially the singlet
oxygen yield for 17-(3-amino-1-propanesulfonic acid)-substi-
tuted hypocrellin B Schiff base (4) is almost comparable to that
of the parent 2;22 however, it was synthesized in strong alkali
solution in a yield of no more than 10%.
anesulfonic acid)-substituted hypocrellin B (5−7, Chart 1),
novel aminoalkanesulfonic acid-substituted hypocrellin B
derivatives with a step increase in the carbon atom number
of the alkyl chain, were synthesized in much higher yield (30%).
Remarkably, the three derivatives exhibit much higher singlet
oxygen yields (0.94 0.01, 0.98 0.01, 1.01 0.01) than 2
(0.76). In vitro biological experiments confirmed that the PDT
activity of 6 was far higher than those of 5 and 2. On the basis
of the relative PDT activity, aqueous solubility, and clinically
required concentration, it was estimated that just 6 achieved the
optimized amphiphilicity and could be directly deliverable via
intravenous injection without the need for any drug-delivery
vehicle.
RESULTS AND DISCUSSION
■
pH-Dependent Yield. Up to now, 17-substituted Schiff
base derivatives of hypocrellins were all synthesized in strong
alkali solution (pH > 12), which was necessary to prevent the
amino group, with an isoelectric point at around 9.0,30 from
protonating, but the yields were very low (no more than
10%).22,31 In fact, at pH > 12, 2 presents mainly in the form of
a double negative for its two isoelectric points at pH 8.4 and
11.0,32 which is favorable for the electrophilic addition of an
amino group to site 2 but not for the Schiff base reaction. To
optimize the yield, 6 was synthesized at a series of pH values
from 6.0 to 14.0, from which the dependence of the yield on
the pH was derived as shown in Figure S1 (in the Supporting
Information). It was indicated that the yield was maximum
(30%) at pH 9.5, 2 times more than what was ever reported,
greatly promoting the pharmaceutical significance of the Schiff
base derivatives.
Spectral Properties. Figure 1 shows the absorption spectra
and fluorescence emission for 2 and 5−7 in dimethyl sulfoxide
at the same concentration. Compared to 2, a remarkable feature
of the Schiff base derivatives is enhancement of the absorption
of green (500−530 nm), orange (580 nm), and red (640 nm)
light, and the emission peak shifts to 675 nm. The absorption of
green or orange light matching a copper vapor laser (510, 580
nm) or Nd:YAG laser (532 nm) is favorable for PDT of
microvascular diseases,33−36 while the red absorption peak
suggests that the derivatives are more favorable for PDT of
tumors than their parent.22,28,29
In the current work, 17-(4-amino-1-butanesulfonic acid)-, 17-
(5-amino-1-pentanesulfonic acid)-, and 17-(6-amino-1- hex-
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Solubility and Partition Coefficient. The solubility in
phosphate-buffered saline (PBS) and partition coefficient (PC)
were determined by the methods described in the Experimental
Section. Table 1 lists the carbon atom number of the alkyl chain
Table 1. Partition Coefficient (PC) and Solubility in PBS (S)
a
b
compd
carbon atom number
PC
S in PBS (mg/mL)
2
3
4
5
6
7
0
2
3
4
5
6
28.6 0.6
2.2 0.4
4.8 0.3
7.5 0.5
9.0 0.4
11.6 0.4
(4.6 0.4) × 10−3
9.2 0.4
6.6 0.4
3.9 0.4
1.7 0.5
0.2 0.1
a
PC is the mean partition coefficient SEM of three independent
b
experiments. S is the mean solubility
SEM in PBS of three
independent experiments.
Figure 2. Transient absorption spectra recorded from 0 to 50 μs for 2
(A), 3 (B), and 6 (C) in deaerated cyclohexane. (D) Time-dependent
absorbance at 560 nm for 2 and 6 and at 510 nm for 3 within 1 μs. (E)
Transient decay trace of the absorbance at 560 nm for 2 and 6 and at
510 nm for 3.
in the substituent, PC, and solubility for 5−7 along with those
for 2−4 as references.
Plots of the solubility and PC versus the carbon atom
number are shown in Figure S2 in the Supporting Information.
The linearity of the plots indicates that the quantitative
solubility or optimized amphiphilicity can be determined as
long as the clinically required concentration is known, which
will be estimated in the last section.
Singlet Oxygen (1O2) Yields. Photosensitization activity is
mainly evaluated by the singlet oxygen (1O2) yield for it is the
most important species for the PDT effect.2,37,38 Singlet oxygen
yields were determined by the 9,10-diphenylanthracene (DPA)
bleaching method for the derivatives with identical absorption
under various light conditions. On the basis of the plots of
decreases of DPA absorbance at 376 nm with irradiation time,
the 1O2 yield for 5−7 was estimated to be 0.94 0.01, 0.98
0.01, and 1.01
0.01, respectively, with 0.76 for 2 as a
reference,39 and invariable under blue, orange, or red light or
light of wavelengths larger than 470 nm (shown in Figure S3 in
the Supporting Information), suggesting that a high 1O2 yield is
an intrinsic feature of the Schiff base derivatives.
Triplet-Excited-State Yields. Singlet oxygen may form via
an energy transfer from a triplet excited photosensitizer to a
ground-state oxygen molecule.40,41 To search for the origin of
the singlet oxygen, transient absorption spectra were selectively
measured for structurally different 2, 3, and 6 after a pulse
excitation, as shown parts A−C, respectively, in Figure 2. The
positive peaks appearing at around 560 nm for 2 and 6 but 510
nm for 3 were ascribed to the triplet absorption, and plots of
the peak amplitudes versus time are electively shown for the
period of 0−1000 ns in Figure 2D. It can be seen that the
maximum absorbance appears commonly at around 300 ns,
ascribed to intersystem crossing. Figure 2E shows the decay
trace of the peak absorbance for 2, 3, and 6, from which the
lifetime was estimated to be 9, 6, and 12 μs, respectively. By a
comparative method42 described on p S4 in the Supporting
Information, the triplet-state yields for 3 and 6 were estimated
to be 0.31 0.01 and 1.00 0.03, respectively, with 0.77 for 2
as a reference,43 indicating that a high triplet-excited-state yield
is a feature of the Schiff base derivatives.
Figure 3. BGC823 cell images in bright field (a, c) and cellular
fluorescence (b, d) in the absence and presence of 6, respectively.
apparent fluorescence intensity is not necessarily equivalent
to the amount of photosensitizer in various regions of the cell
for it varies sensitively to the microenvironment polarity.44−47
Alternatively, liposomes were used as a mimic biomem-
brane48,49 to monitor the uptake quantity and kinetics. First, the
absorption spectra in PBS were measured for 2, 3, and 6 in
concentrations of 0.0−8.0 μM, which is less than the maximum
solubility of 2 (8.8 μM47). Second, a plot of the absorbance
versus concentration was derived as shown in Figure 4. Third,
the slope value of each plot, reflecting the character of the
derivative, was calculated and is listed in Table 2.
Cellular and Liposome-Mimic Uptakes. Cellular uptake
of 6 by human gastric carcinoma BGC823 cells was detected by
a laser confocal fluorescence microscope, as shown in Figure 3.
After the cells were incubated with 6 (1 μM) for 5 h, the red
fluorescence image indicated uptake of 6. However, the
To monitor the molecular transportation of a derivative in
PBS to liposomes, absorption spectra were measured at a series
of times after 2, 3, or 6 in PBS (8 μM) was added to the
liposome solution in PBS, from which a plot of the absorbance
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Figure 4. Maximum absorbance for 6 (A), 2 (B), and 3 (C) at a series of concentrations (from 0.0 to 8.0 μM) in PBS (pH 7.4). Insets: absorption
spectra.
Table 2. Slopes of the Absorbance−Concentration Plots for 6, 2, and 3 in PBS (pH 7.4) and Those ahead of or behind the
Intersections in Figure 6 as Well as the Correlative Coefficient (R)
compd
slope in PBS
R
slope ahead of the intersection
R
slope behind the intersection
R
2
3
6
0.0071
0.0080
0.0101
0.9999
0.9999
0.9999
0.0156
0.0100
0.0161
0.9999
0.9998
0.9999
0.0072
0.0081
0.0101
0.9999
0.9998
0.9999
Figure 5. Time-dependent peak absorbance after addition of 6 (A), 2 (B), or 3 (C) (8 μM) in PBS to liposomes (0.08 g/L) in PBS (pH 7.4). Insets:
absorption spectra measured at a series of times.
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Figure 6. Maximum absorbance versus the concentration of 6 (A), 2 (B), and 3 (C) in PBS solution (pH 7.4) derived from absorption spectra
(insets) measured 4, 5, and 2 h after addition to a PBS solution of liposomes.
Figure 7. (A) Percent survival of human gastric cancer BGC823 cells in the presence of 2, 5, or 6 (0, 19, 38, 57, or 76 nM) and light (532 nm laser,
20 mW/cm2, 1000 s). (B) Same as (A) but in the absence of light. Data were averaged from three independent experiments with the error range as
indicated.
versus time was derived, as shown in Figure 5. The figure shows
that the absorbance for 2, 3, and 6 increased linearly until 5, 2,
and 4 h, respectively, suggesting the time at which the
derivative reached equilibrium distribution between PBS and
liposomes. It also indicates that a more hydrophilic derivative
reaches equilibration faster. The saturation time is indicative of
the cell incubation in the in vitro experiments.
To measure the maximum uptake for various derivatives,
absorption spectra were measured after 2, 3, or 6 in a series of
concentrations in PBS was added to liposomes at 5, 2, or 4 h,
respectively, from which a plot of the absorbance versus
concentration was derived, as shown in Figure 6. It can be seen
that the plots for 2, 3, and 6 undergo a slope change at 7.0, 3.0,
and 6.5 μM, respectively, and the slope values on both sides of
the intersection point are listed in Table 2. The slope value
behind the intersection point is exactly identical to that in PBS,
while that ahead of the intersection point indicates the
absorption feature in liposomes. This suggests that the
maximum liposomal uptake of 2, 3, and 6 is 7.0, 3.0, and 6.5
μM in the experimental conditions. Reasonably, the uptake of 6
is nearly comparable to that of the parent 2, while the most
hydrophilic 3 exhibits the lowest uptake.
PDT Activity on Human Gastric Carcinoma BGC823
Cells. The human gastric carcinoma BGC823 cells were
incubated for 5 h in the presence of 2, 5, or 6 (0−76 nM)
followed by irradiation with a 532 nm laser of 20 J/cm2 (20
mW/cm2) for 1000 s or no irradiation. Parts A and B of Figure
7 show the estimated cell survivals with and without irradiation,
respectively. It was confirmed that 2, 5, and 6 in the
concentrations used had no dark toxicity to the cells and
light did not result in any cell death independently, while the
cell death was completely ascribed to the PDT effect. Estimated
from the data in Figure 7A, IC50, defined as the photosensitizer
concentration required to kill 50% of the cells, is 40, 54, and 22
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Scheme 1. Procedure for the Synthesis of 4-Amino-1-butanesulfonic Acid (n = 4), 5-Amino-1-pentanesulfonic Acid (n = 5), and
6-Amino-1-hexanesulfonic Acid (n = 6)
nM for 2, 5, and 6, respectively. It can be seen from Figure 1
that the absorbances of 2 and 6 at 532 nm are almost identical;
therefore, the higher PDT activity for 6 can be ascribed to
photosensitization activity and cellular uptake. In fact, 6 is the
first hypocrellin derivative to exhibit a higher PDT activity than
its parent. At a certain concentration (76 nM) of 2, 5, and 6,
the cell survival is also a function of light dose, as shown in
Figure S5 (in the Supporting Information), suggesting that light
delivery is a key factor for the PDT activity.
criteria, it can be concluded that 6 achieves quantitative
solubility and optimized photosensitization activity at the same
time and it is predicted that it can be directly deliverable
without the need for any drug-delivery vehicles. The ideas
presented in this work possess a common significance in
medicinal chemistry.
EXPERIMENTAL SECTION
■
Materials. 1 was isolated from the fungus sacs of Hypocrella
bambusae and recrystallized three times from acetone before use. 2 was
prepared by dehydration of 1 in alkaline solution and purified by
recrystallization twice from acetone.62 Potassium phthalimide
derivative was purchased from Acros Organics, and 1,4-dibromobu-
tane, 1,5-dibromopentane, and 1,6-dibromohexane were purchased
from Alfa Aesar. DPA was purchased from Aldrich Chemical Co. 1,4-
Diazabicyclo[2.2.2]octane (DABCO) and dimethyl sulfoxide (DMSO)
were purchased from Merck Co. Cyclohexane, dimethylformamide
(DMF), deuterated solvents for 1H NMR, and other agents of
analytical grade were purchased from the Beijing Chemical Plant. PBS
solution (pH 7.4) was composed of 1.4 mM KH2PO4, 6.4 mM
Na2HPO4, 137 mM NaCl, and 2.6 mM KCl. Phosphate buffer,
carbohydrate buffer, K2CO3−NaOH, and NaOH were used for pH
ranges of 6.0−8.5, 8.5−10.5, 10.5−12.0, and >12.0, respectively. Water
and DMF were redistilled, and the working solutions were prepared
immediately before use.
On the basis of the relative PDT activity and 2 dosage in
animal experiments as well as some clinical criteria, the
clinically required concentration (C) was estimated to be
around 0.8 mg/mL (p S2 in the Supporting Information). As
listed in Table 1, the solubilities of 3−6 are all larger than the
concentration; however, the largest PC for 6 predicts the largest
cellular uptake. Consequently, it is concluded that 6 achieves
optimized amphiphilicity. Interestingly, as shown in Figure S2
(Supporting Information), the linearly descending aqueous
solubility or ascending PC illustrates a general significance for
the optimized amphiphilicity or quantitative aqueous solubility.
It is a general rule that biological PDT activity in vitro
correlates positively to that in vivo,50−54 especially for
microvascular diseases;55,56 therefore, it is predictable that 6
is more PDT active in vivo than 2. In vitro experiments should
also have been done by the use of vasculature endothelial cells;
however, it is expected that the result would be similar because
the cellular structures are not so different from a photodynamic
therapy point of view.57,58 PDT could result in cell death via
destruction of the membranes, cores, and organs or
apoptosis;59−61 however, the current work mainly concerns
the photosensitizer efficiency to induce cell death but not the
cell-death mechanism. Besides, red light is necessary for
effective PDT of solid tumors but not of the cells. In fact,
the much higher singlet oxygen yield for 6 under red light (in
the Supporting Information) allows prediction of a higher
cellular PDT activity than that of 2; on the other hand,
evaluating the PDT activity under red light is not equitable
because the absorptions of 2 and 6 are incomparable.
Preparation of 5. 4-Amino-1-butanesulfonic acid (1200 mg, 7.84
mmol) and K2CO3 (1.7 g, 0.0123 mol) in 10 mL of water were mixed
with 2 (100 mg, 0.189 mmol) in 10 mL of DMF and allowed to react
at pH 9.5 under stirring and refluxing at 120 °C for 3 h in the dark.
The solvent was removed under reduced-pressure evaporation. The
residue was applied to a 1% citric acid−silica gel plate with
dichloromethane/methanol (v/v = 5/1) used as the eluent. The
gray-brown constituent was collected and further applied to a 1% citric
acid−silica gel plate with ethyl acetate/methanol (v/v = 3/1) as the
eluent, from which the gray-green component was collected and dried
in vacuum to give a solid product (37 mg, 30%): UV−vis (DMSO,
λmax, log ε) 500 nm (4.31), 595 nm (3.90), 645 nm (4.13); IR (KBr,
1
νmax, cm−1) 3443, 2940, 1711, 1616; H NMR (400 MHz, MeOD-d6,
δ) 6.74, 6.52 (s, 2H, 5,8-H), 3.95−4.16 (m, 12H, 2,6,7,11-OCH3),
3.66 (m, 2H, 19-CH2), 3.52 (m, 2H, 22-CH2), 3.14, 2.52 (d, 2H, 13-
CH2), 2.96 (s, 3H, 18-CH3), 2.34 (s, 3H, 16-CH3), 2.03−2.08 (m, 4H,
20,21-CH2); ESI-MS (m/z) 662.3 (M − 1); HRMS (ESI, m/z) calcd
for C34H34O11NS (M + H) 664.1847, found: 664.1847; HPLC
(methol/water = 9/1) retention time 3.632 min. Anal. Calcd for
C34H33NO11S: C, 61.53; H, 5.01; N, 2.11; S, 4.83; Found: C, 61.38; H,
5.19; N, 2.04; S, 4.71.
Preparation of 6. Similar to the procedure described for 5, a
redistilled DMF solution (10 mL) of 2 (100 mg, 0.189 mmol) was
mixed with a distilled water solution (10 mL) of 5-amino-1-
pentanesulfonic acid (1000 mg, 5.98 mmol) and K2CO3 (1.5 g,
0.0109 mol) and allowed to react at pH 9.5. The product 6 was
CONCLUSIONS
■
Enlightened by quantitative aqueous solubility and the site-
dependent photosensitization activity, three novel 17-substi-
tuted hypocrellin derivatives, 5−7, were synthesized in a much
higher yield than what was ever reported. 5−7 exhibit singlet
oxygen yields of 0.94 0.01, 0.98 0.01, and 1.01 0.01 and
aqueous solubilities of 3.9, 1.7, and 0.2 mg/mL, respectively. 2,
5, and 6 exhibit IC50 values of 40, 22, and 54 nM, with no dark
toxicity to human gastric carcinoma BGC823 cells. On the basis
of the solubility, the relative PDT activity, and some clinical
obtained as a gray-green solid (39 mg, 30%).: UV−vis (DMSO, λmax
log ε) 499 nm (4.32), 593 nm (3.93), 640 nm (4.14); IR (KBr, νmax
,
,
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1
cm−1) 3494, 2939, 1699, 1610; H NMR (400 MHz, MeOD-d6, δ)
the Beer−Lambert law, PC = (AoctanolεPBS)/(εoctanolAPBS). Here, Aoctanol
and APBS are the maximum absorptions of photosensitizer in 1-octanol
and PBS and εoctanol and εPBS are the molar extinction coefficients of
photosensitizer in 1-octanol and PBS. εoctanol/εPBS for 5−7 was
determined to be 1.95, 1.96, and 1.98, respectively, according to the
Beer−Lambert law, that is, the absorbance in the same concentration
in the two types of solvents.
Solubility in PBS Solution. 5−7 dissolved in PBS solution (pH
7.4) in a series of concentrations were stored in the dark for 12 h, and
then the absorption spectra were measured, from which a plot of the
absorbance versus the concentration for each photosenstizer was
derived. The solubility was determined by the maximum value in the
linear range of the plot.
Determination of the Singlet Oxygen Quantum Yield. The
1O2 yields were determined by the DPA-bleaching method39 under
irradiation with a medium-pressure sodium lamp (450 W) with light of
wavelengths longer than 470 nm, blue (400−480 nm), orange (580−
600 nm), or red (600−700 nm) light. The photobleaching of DPA was
monitored by the decrease in the absorbance at 376 nm. To make the
results comparable, the concentrations of all of the photosensitizers
were adjusted to give identical integrate area of the absorption.
Laser Flash Photolysis. Laser flash photolysis experiments were
carried out on an Edinburgh LP920-laser flash photolysis spectrometer
equipped with a Q-switched Nd:YAG laser to provide 532 nm light of
35 mJ and a 7 ns pulse. The analyzing beam source was generated by a
pulsed xenon arc lamp (450 W, ozone free), and a photomultiplier
tube was used as the detector. The kinetic curves of the transient
absorption in the 400−700 nm range were recorded after the pulse
excitation. Photosensitizer solution in cyclohexane was freshly
prepared, and the concentration was adjusted to give the same optical
density (0.58) at 532 nm. The solution was carefully deaerated by
bubbling high-purity N2 for at least 20 min before measurement at 20
°C under pure N2.
Cellular Fluorescence Images. Exponentially grown human
gastric carcinoma cells (BGC823 cells) in the culture medium (2 mL)
were seeded on a coverslip at a density of 1 × 105 and incubated
overnight at 37 °C under humidified air containing 5% CO2. The
medium was removed. Then the cells were incubated with 6 (1 μM)
for 5 h at 37 °C containing 5% CO2. The cells were rinsed three times
with PBS and then observed with an Olympus FV1000 confocal
microscope equipped with a 488 nm argon ion laser. Emission signals
from 650 to 750 nm were collected. The control experiment was done
under the same conditions but without photosensitizers.
Preparation of Photosensitizers in Liposomes. Liposomes
were prepared by egg L-phosphatidylcholine (EPC) and cholesterol by
using the film-ultrasonic technique.63 3 was prepared as described
previously.21 First, concentrated solutions of 2, 3, and 6 in DMSO (2
mg/mL) were slowly added to PBS (pH 7.4) to give a final
concentration from 0 to 8 μM, and the absorbance spectra were
measured. 2, 3, or 6 in each concentration in PBS was slowly added
into the liposomal solution with stirring, and the absorption spectra
were measured at a series of times.
Assessment of Cell Survival. The human gastric carcinoma cell
line (BGC823 cells) used in this study was cultured in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with 5% (v/v) fetal
bovine serum (FBS), 1% (v/v) nonessential amino acids, penicillin
(100 U/mL), and streptomycin (100 μg/mL) in 75 cm2 flasks at 37
°C under humidified air containing 5% CO2. The cells in logarithmic
phase were detached by treatment with trypsin−ethylenediaminetetra-
acetic acid (EDTA)−PBS and diluted to a density of 5 × 105 cells/mL
in the presence of DMEM supplemented with 5% FBS. The cell
suspension was seeded in wells (100 μL each) in 96-well microplates
and incubated under 5% CO2 at 37 °C for 24 h. One column of wells
did not receive cells to serve as the blank. Then the cells were changed
to DMEM (FBS-free) medium containing photosensitizers in a series
of concentrations (0, 19, 38, 57, and 76 nM) and incubated at 37 °C in
the dark for 5 h. After removal of the above medium containing
photosensitizers and addition of the fresh DMEM medium, the cells
were irradiated with a 532 nm laser of 0−30 Mw/cm2 for 1000 s and
then incubated in the dark for 24 h before survival assessment.
6.83, 6.77 (s, 2H, 5,8-H), 4.04−4.17 (m, 12H, 2,6,7,11-OCH3), 3.65
(m, 2H, 19-CH2), 3.49 (m, 2H, 23-CH2), 3.14, 2.39 (d, 2H, 13-CH2),
2.78 (s, 3H, 18-CH3), 2.20 (s, 3H, 16-CH3), 1.74−1.82 (m, 6H,
20,21,22-CH2); ESI-MS (m/z) 676.3 (M − 1); HRMS (ESI, m/z)
calcd for C35H36O11NS (M + H) 678.2004, found 678.2005; HPLC
(methol/water = 9/1) retention time 3.687 min. Anal. Calcd for
C35H35NO11S: C, 62.03; H, 5.21; N, 2.07; S, 4.73 Found: C, 61.84; H,
5.48; N, 1.89; S, 4.63.
Preparation of 7. Similar to the procedures described for 5 and 6,
2 (100 mg, 0.189 mmol) dissolved in 10 mL of redistilled DMF was
mixed with 6-amino-1-hexanesulfonic acid (1200 mg, 6.63 mmol) and
K2CO3 (1.5 g, 0.0109 mol) in 10 mL of distilled water and allowed to
react at pH 9.5. 7 was obtained as a gray-green solid (39 mg, 30%):
UV−vis (DMSO, λmax, log ε) 498 nm (4.33), 593 nm (3.95), 642 nm
(4.14); IR (KBr, νmax, cm−1) 3459, 2939, 1689, 1610; H NMR (400
1
MHz, MeOD-d6, δ) 6.75, 6.65 (s, 2H, 5,8-H), 3.93−4.18 (m, 12H,
2,6,7,11-OCH3), 3.78 (m, 2H, 19-CH2), 3.66 (m, 2H, 24-CH2), 3.50,
2.06 (d, 2H, 13-CH2), 2.87 (s, 3H, 18-CH3), 2.37 (s, 3H, 16-CH3),
1.65−1.77 (m, 8H, 20,21,22,23-CH2); MALDI TOF MS (m/z) 690.4
(M − 1); HRMS (ESI, m/z) calcd for C36H38O11NS (M + H)
692.2160, found 692.2167; HPLC (methol/water = 9/1) retention
time 4.008 min. Anal. Calcd for C36H37NO11S: C, 62.51; H, 5.39; N,
2.02; S, 4.64; Found: C, 62.15; H, 5.63; N, 1.92; S, 4.50.
Preparation of Substituents. Because merchant 4-amino-1-
butanesulfonic acid, 5-amino-1-pentanesulfonic acid, and 6-amino-1-
hexanesulfonic acid are not available, the substituents were prepared
according to Scheme 1. Generally, potassium phthalimide (32.30
mmol) and dibromoalkane (64.50 mmol) were mixed in DMF (75
mL) and allowed to react at room temperature for 18 h under stirring.
KBr formed in the reaction was precipitated by adding acetone (75
mL) and removed by filtration, and then the solution was dried under
reduced pressure. The residue was further purified by silica gel column
chromatography (eluent hexanes/ethyl acetate = 10/1 (v/v)) to
obtain a white solid, compound 9 (yield 72%). Compound 9 (23.15
mmol) and Na2SO3 (46.83 mmol) were dissolved in a mixed solvent
of H2O (140 mL)−95% ethyl acetate (85 mL) and allowed to react at
95 °C for 20 h with stirring. The reacted solution was dried under
reduced pressure, and the residue was dissolved in water and washed
three times with ethyl acetate. The aqueous layer was isolated and
dried to obtain solid 10. The solid was added to 37.5% HCl (73 mL)
and allowed to react at 110 °C for 18 h with stirring. To get rid of the
precipitates by filtration, the solution was washed three times with
ethyl acetate. The aqueous layer was dried under reduced pressure to
obtain a yellow solid which was further purified by recrystallization in
water−95% ethanol to obtain the product (yield 50%). 1H NMR for 4-
amino-1-butanesulfonic acid (400 MHz, D2O, δ): 3.50 (t, 2H), 2.68 (t,
2H), 1.50−1.65 (m, 4H). 1H NMR for 5-amino-1-pentanesulfonic acid
(400 MHz, D2O, δ): 2.87 (t, 2H), 2.79 (t, 2H), 1.66−1.36 (m, 6H).
1H NMR for 6-amino-1-hexanesulfonic acid (400 MHz, D2O, δ): 2.93
(t, 2H), 2.86 (t, 2H), 1.35−1.75 (m, 8H).
Spectral Measurements. Steady-state absorption spectra were
recorded on a Shimadzu UV-1601 spectrophotometer at room
temperature. Infrared spectra were measured with a Bruker RF100/S
spectrometer. Mass spectra were measured with a Fourier transform
ion cyclotron resonance (FT-ICR) mass spectrometer, and proton
nuclear magnetic resonance (1H NMR) spectra were recorded with a
Bruker Avance 400 spectrometer. The purities of the final compounds
were determined using a Shimadzu LCsolution high-performance
liquid chromatography (HPLC) system with a C-18 column (Shim-
pack VP-ODS, 4.6 × 150 mm, 4.6 μm). High-resolution mass spectra
were recorded with an APEXII FT-ICR mass spectrometer from
Bruker Daltonics Inc. Elemental analyses were determined with a Flash
EA 1112.
Partition Coefficients. Photosensitizers were dissolved in 1-
octanol (2 mL) to a concentration of 20 μM, and then the same
volume (2 mL) of PBS (pH 7.4) was added. The mixture was
dispersed ultrasonically for 2 min and then centrifuged for 10 min to
separate the two phases. The PC was determined by the ratio of
photosensitizer concentration in 1-octanol to that in PBS solution via
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Journal of Medicinal Chemistry
Article
Meanwhile, dark toxicity was measured under the same conditions
without irradiation in parallel. The cell survival was estimated by the 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay.64,65 Briefly, after 20 μL of MTT (5 mg/mL) was added to
each well, the cells were further incubated for 90 min. MTT solution
was removed, and 200 μL of DMSO was added to dissolve the
formazan crystals. Then the plates were shaken at room temperature
for 10 min, and the optical density at 570 nm was read on a microplate
reader, from which cell survivals were derived.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Dependence of the yields of 6 on the pH values, estimation of
the clinically required concentration, linear plots of the PC or
the solubility versus the carbon atom number in the substituent,
dependence of the DPA absorbance decreases on the
illumination time under various light conditions, estimation of
the triplet-state yield ΦT; light-dose-dependent cell survivals in
the presence of a certain concentration (76 nM) of 2, 5, or 6,
1H NMR, MS, HRMS, and IR spectra of 5−7, 1H NMR spectra
of 4-amino-1-butanesulfonic acid, 5-amino-1-pentanesulfonic
acid, and 6-amino-1-hexanesulfonic acid, and HPLC analysis of
5−7. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: +86-10-82617053 (J.Z.); +86-10-66939794 (Y.G.);
+86-10-82617053 (J.X.). Fax: +86-10-82617315 (J.Z.); +86-10-
68212522 (Y.G.); +86-10-82617315 (J.X.). E-mail: zhaojq@
iccas.ac.cn (J.Z.); guyinglaser@sina.com (Y.G.); xiejie@iccas.ac.
cn (J.X.).
■
(12) Xu, S. J.; Zhang, X. X.; Chen, S.; Zhang, M. H.; Shen, T.; Wang,
Z. P. Novel phototherapeutic agents: Investigation and progress of
hypocrellin derivatives. Chin. Sci. Bull. 2003, 48, 1775−1785.
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This project was supported by the National Natural Science
Foundation of China (Grants 20872144 and 31170614).
■
ABBREVIATIONS USED
■
PDT, photodynamic therapy; HB, hypocrellin B; ROS, reactive
oxygen species; AMD, age-related macular degeneration; PWS,
1
port wine stain; O2, singlet oxygen; PBS, phosphate-buffered
saline; DPA, 9,10-diphenylanthracene; DABCO, 1,4-
diazabicyclo[2.2.2]octane; DMSO, dimethyl sulfoxide; DMF,
dimethylformamide; NMR, nuclear magnetic resonance; MS,
mass spectrometry; HRMS, high-resolution mass spectrometry;
HPLC, high-performance liquid chromatography; FBS, fetal
bovine serum; EPC, egg L-phosphatidylcholine; DMEM,
Dulbecco’s modified Eagle’s medium; EDTA, ethylenediami-
netetraacetic acid; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-di-
phenyltetrazolium bromide; PC, partition coefficient
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