In vitro cellular uptake and dimerization of signal transducer and activator of transcription-3 (STAT3) identify the photosensitizing and imaging-potential of isomeric photosensitizers derived from chlorophyll-a and bacteriochlorophyll-a

Article abstract of DOI:10.1021/jm200805y

Among the photosensitizers investigated, both ring-D and ring-B reduced chlorins containing the m-iodobenzyloxyethyl group at position-3 and a carboxylic acid functionality at position-17² showed the highest uptake by tumor cells and light-dependent photoreaction that correlated with maximal tumor-imaging [positron emission tomography (PET) and fluorescence] and long-term photodynamic therapy (PDT) efficacy in BALB/c mice bearing Colon26 tumors. However, among the ring-D reduced compounds, the isomer containing the 1′-m-iobenzyloxyethyl group at position-3 was more effective than the corresponding 8-(1′-m-iodobenzyloxyethyl) derivative. All photosensitizers showed maximum uptake by tumor tissue 24 h after injection, and the tumors exposed with light at low fluence and fluence rates (128 J/cm², 14 mW/cm²) produced significantly enhanced tumor eradication than those exposed at higher fluence and fluence rate (135 J/cm², 75 mW/cm ²). Interestingly, dose-dependent cellular uptake of the compounds and light-dependent STAT3 dimerization have emerged as sensitive rapid indicators for PDT efficacy in vitro and in vivo and could be used as in vitro/in vivo biomarkers for evaluating and optimizing the in vivo treatment parameters of the existing and new PDT candidates.

Full text of DOI:10.1021/jm200805y

ARTICLE
pubs.acs.org/jmc
In Vitro Cellular Uptake and Dimerization of Signal Transducer and
Activator of Transcription-3 (STAT3) Identify the Photosensitizing
and Imaging-Potential of Isomeric Photosensitizers Derived
from Chlorophyll-a and Bacteriochlorophyll-a
Avinash Srivatsan,†,# Yanfang Wang,† Penny Joshi,† Munawwar Sajjad,‡ Yihui Chen,† Chao Liu,†
Krishnakumar Thankppan,§ Joseph R. Missert,† Erin Tracy,|| Janet Morgan,^ Nestor Rigual,§
Heinz Baumann,*,|| and Ravindra K. Pandey*,†,#
Departments of †Cell Stress Biology/PDT Center, §Head and Neck/Plastic Surgery, Molecular and Cellular Biology, ^Dermatology,
and #Molecular Pharmacology and Cancer Therapeutics, Roswell Park Cancer Institute, Buffalo, New York 14263, United States
‡Department of Nuclear Medicine, SUNY, Buffalo, New York 14221
S Supporting Information
b
ABSTRACT: Among the photosensitizers investigated, both
ring-D and ring-B reduced chlorins containing the m-iodoben-
zyloxyethyl group at position-3 and a carboxylic acid function-
ality at position-172 showed the highest uptake by tumor cells
and light-dependent photoreaction that correlated with max-
imal tumor-imaging [positron emission tomography (PET) and
fluorescence] and long-term photodynamic therapy (PDT)
efficacy in BALB/c mice bearing Colon26 tumors. However,
among the ring-D reduced compounds, the isomer containing
the 10-m-iobenzyloxyethyl group at position-3 was more effec-
tive than the corresponding 8-(10-m-iodobenzyloxyethyl) deri-
vative. All photosensitizers showed maximum uptake by tumor
tissue 24 h after injection, and the tumors exposed with light at
low fluence and fluence rates (128 J/cm2, 14 mW/cm2) produced significantly enhanced tumor eradication than those exposed at
higher fluence and fluence rate (135 J/cm2, 75 mW/cm2). Interestingly, dose-dependent cellular uptake of the compounds and
light-dependent STAT3 dimerization have emerged as sensitive rapid indicators for PDT efficacy in vitro and in vivo and could be
used as in vitro/in vivo biomarkers for evaluating and optimizing the in vivo treatment parameters of the existing and new PDT
candidates.
’ INTRODUCTION
In PET imaging,18F-FDG (18F-fluoro-2-deoxyglucose) is widely
used in the clinic for the assessment of glucose metabolism in the
heart, lungs, and brain.6ꢀ8 However,18FDG-imaging suffers from
several limitations, such as the reduced specificity for tumor cells due
to FDG uptake in inflammatory lesions, short half-life, etc. The
longer half-life (4 days) of the 124I-product(s) compared to 90 min
for 18F-FDG has a number of additional advantages: (i) patients do
not have to go through another injection after surgery to view its
effectiveness, (ii) imaging centers do not need to order a large
number of vials, (iii) as a result of its long half-life, it can be
transported over long distances and, therefore, could reduce the
number of highly expensive cyclotrons, and, finally, (iv) not only
does this mean an increased benefit to the end user (the patient), it
also provides a significant benefit to insurance companies.
Molecular imaging can be used for early detection and
localization of lesions and for “real time” monitoring of the efficacy
of therapeutic treatments.1ꢀ3 In recent years, PET technology has
been included in drug development, and it has contributed to the
understanding of drug action, drug dose regimens, and treatment
strategies.4 Such studies will eventually provide the means to
accomplish “personalized medicine” by monitoring individual
responses to drug delivery. For diagnostic imaging, the half-life
of the radionuclide must be long enough to carry out the chemistry
of the desired radiopharmaceutical and allow its accumulation in
the target tissue in the patient while the drug clears from the
nontarget organs. Radiometals for pharmaceuticals used in PET
and γ scintigraphy range in half-life from about 10 min (62Cu) to
several days (67Ga). For example, heart or brain perfusion-based
radiopharmaceuticals require shorter half-lives, since they reach
the target quickly whereas tumor-targeted compounds often take
longer to reach the target and to be accumulated for gaining
optimal target-to-background ratios.5
The design of a radiopharmaceutical agent requires optimizing the
balance between specific in vivo targeting (e.g., cancerous tumor),
Received: June 21, 2011
Published: August 15, 2011
r
2011 American Chemical Society
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Scheme 1. Modification of Methyl Ester Functionality
clearance from nontarget tissues/organs, as well as time-depen-
dent application in line with the decay properties of the
radionuclide.9 Several considerations need to be made when
designing selective radiolabeled drugs. These include efficient
drug delivery, optimization of relative accumulation of the drug at
the target over nontarget sites, maximizing the residence time of
the radioactivity at target sites, and prevention of structural
alteration of the drug.10 Because of the multiple parameters that
must be considered, developing effective radiopharmaceuticals
for combined imaging and therapy of cancer is a challenging
undertaking that is not simply solved by attaching a radionuclide
in any fashion to a nonradiolabled targeting compound. At the
very least, the radionuclide should not interfere with pharmaco-
kinetics, binding specificity, or retention by cancer cells. The
selection of the radionuclides and the chemical strategies for
radiolabeling of molecules are critical elements for developing
safe and improved imaging/therapeutic agents.11
and related tetrapyrrolic systems is higher in malignant tumors
than in most normal tissues, and that has been the main reason
to use these molecules as photosensitizer (PS). Some tetra-
pyrrole-based compounds have been effective in treating le-
sions at different organ sites, including skin, lung, bladder, head,
and neck and esophagus.14 The precise mechanism(s) of PDT
and post-PDT reactions are still largely unknown; however, data
from in vitro and in vivo studies suggested that localized oxidative
damage mediated by PS-specified photoreactions determines
direct cell killing, loss of vascular function, and post-PDT
immune reactions, all of which play significant roles in PDT
outcome.15ꢀ20 Superficial lesions, or those that are endoscopi-
cally accessible, e.g. endobronchial or esophageal tumors, are
easily treated. However, the majority of malignant lesions are too
deep to be reached by light of the wavelength required to trigger
singlet oxygen production by the current generation of PSs.
Although the technology to deliver therapeutic light to deep
lesions via optical fibers “capped” by a terminal diffuser is well
developed, a deep lesion is by definition not visible from the skin
surface and the precise PDT of deep tumors, thus far, has been
For the last several years, one of the objectives of our laboratory
has been to use porphyrin-based compounds for the treatment
of cancer by PDT.12,13 The accumulation of certain porphyrins
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Figure 1. Various approaches of structural modification of iodinated PS: (X) modification of methyl ester functionality; (Y) effect of the position of the
(10-m-iodobenzyloxyethyl) substituent; (Z) effect of ring-B vs ring-D reduced PSs in biological efficacy.
impractical. However, nuclear imaging guided implantation of
optical fibers within deep tumors would greatly enhance PDT.
On the basis of a SAR study of a series of alkyl ether analogues
of pyropheophorbide-a, a relatively long wavelength absorbing
PS, the 3-(1-hexyloxyethyl) derivative (HPPH).21,22 has been
developed in our laboratory, proved to be tumor-avid, and is
currently in phase I/II multicenter human clinical trials.23,24
Initially, we investigated the utility of HPPH as a “vehicle” for
delivering the mono- or dibisaminoethanethiols (N2S2 ligand)25
combined with technetium-99 m to tumors. The in vivo biodis-
tribution data indicated that the tumor/nontumor uptake ratio of the
conjugates correlated with time and tumor size. Its clearance from
tumor was slower than that of most of the nontumor tissues.
However, the short 6-h half-life of 99mTc was incompatible with a
24-h PS uptake time, suggesting that longer-lived isotopes, such as
111In or 124I, could provide a useful scanning agent. Among a series of
radiolabeled PSs25ꢀ28 investigated so far, the 124I- labeled methyl
3-(m-iodobenzyloxyethyl)pyropheophorbide-a was found to be an
effective PET/PDT imaging agent.
In our attempt to develop improved PDT agents, we have
shown that (i) overall lipophilicity and (ii) the presence and
position of various substituents in a variety of tetrapyrrolic
systems related to pyropheophorbides, purpurinimides, and
bacteriopurpurinimides have a significant impact on cellular
uptake, intracellular localization, photoreaction, and PDT effi-
cacy. Though the 124I-labeled PS 3 ( Scheme 1) containing a
3-(10-m-iodobenzyloxyethyl) group at position-3 of the molecule
showed promising tumor imaging (PET) ability, we were inter-
ested to determine whether an alternative position of the
iodobenzyl group on the macrocycle or reduction of the B-ring
in place of the D-ring would change the cell biology of the PS,
such as cellular uptake and retention, that could then be exploited
for designing tumor-specific PS delivery. To do so, we synthe-
sized a series of related analogues by (i) modifying the methyl
ester group of the propionic ester side chain, (ii) introducing the
(m-iodobenzyloxyethyl) group at position-8 (instead of position-3),
and (iii) preparing the corresponding ring-B reduced isomer
(instead of ring-D) and investigating the biological consequences
of these both in vitro and in vivo (Figure 1). We discovered that
the position of the iodobenzyl group affects efficacy in uptake and
retention of the PSs, whereas the carboxylic group at position-172
determines not only the quantitative amount of PS internaliza-
tion but also the site of long-term intracellular retention. Inter-
estingly, the mechanism of internalization and subcellular
deposition of the PS radically changed by conjugating polyethy-
leneglycol (mol wt 5000) at position-172.
’ RESULTS AND DISCUSSIONS
Chemistry. The iodinated PS 1 containing the methyl ester
functionality and the corresponding 124I-analogue 3 were prepared
by following the methodology developed in our laboratory26
(Scheme 1). To investigate the effect of the polyethyleneglycol
(PEG) moiety in cellular uptake and PDT efficacy, the PEG
analogue 7 was synthesized in excellent yield. The corresponding
isomer 11 containing a (10-m-iodobenzyloxyethyl) group at posi-
tion-8 and the corresponding carboxylic acid derivative 12 were
prepared from the methyl pyropheophorbide-a 8 by following the
methodology depicted in Scheme 2. During the preparation of the
desired compound, methyl-8-vinylpyropheophorbide-a 16, a by-
product in minor quantity, was also isolated, and the possible
mechanism for the formation of the byproduct 16 is shown in
Scheme 2. To investigate the impact of ring-B vs ring-D reduced
isomers in tumor imaging and PDT efficacy, the desired labeled
analogues containing either a propionic ester or a carboxylic acid
side chain were synthesized from methyl bacteriopyropheophor-
bide-a 17, which in turn was obtained from Rb. sphaeroides.29 The
reaction sequences followed for the preparation of the desired
compounds are shown in Scheme 3. In brief, two approaches were
used for the preparation of intermediate 20. In the first approach,
the bacteriochlorin 17 was reacted with sodium borohydride, and
the resulting hydroxy ethyl derivative 18 was reacted with HBr gas,
the intermediate bromo- analogue was not isolated and, after
drying under vacuum (inert atmosphere), was immediately re-
acted with 3-iodobenzyl alcohol, resulting in bacteriochlorin 19.
For the preparation of ring-B reduced chlorin 20, bacteriochlorin
19 was subjected to ferric chloride oxidation and the desired
analogue was isolated in 70ꢀ75% yield. In the second approach,
chlorin 21 in a sequence of reaction gave the corresponding 3-(m-
iodobenzyloxyethyl) analogue 20 (Scheme 3). Starting from 20,
the 124I-labeled analogues either as methyl ester 26 or carboxylic
acid 27 were synthesized by following the methodology discussed
for other isomers (Schemes 1). The structures of all the inter-
mediate and the final products were confirmed by NMR, mass
spectrometry, and elemental analyses. The purity was ascertained
by HPLC. Radiolabeling efficiency and specificity of the labeled
analogues both as methyl ester and carboxylic acid derivatives was
30ꢀ32%, and the specific activity was greater than 1 Ci/μmol.
Before evaluating these compounds for biological efficacy, the
structures of ring-B vs ring-D reduced isomers were confirmed by
1H NMR. The striking features observed between the ring-D and
ring-B reduced chlorins are shown in Figure 2. In brief, the 132-H
proton observed as an ABX pattern at δ 5.37 ppm in ring-D
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Scheme 2. Synthesis of 8-(10-m-Iodobenzyloxyethyl)pyropheophorbides 11 and 12 and the 8-Vinyl Analogue 16
reduced chlorin 1 appeared as a singlet at 5.82 ppm in ring-B
reduced chlorin 20, suggesting a regioselective oxidation of ring-
D, which was further confirmed by the presence of triplets at δ
4.25 and 3.33 ppm, each integrating for two protons and assigned
to the 171- and 172-CH2 protons of the propionic ester side
chain, whereas, in ring-D reduced chlorin, these protons were
observed at δ 2.51, 2.35, and 2.05 ppm, respectively. The meso-
region of both the chlorins showed interesting features. For
example, the 5-H proton appearing at δ 9.62 for chlorin 1 was
observed at 9.21 ppm for the ring-B reduced isomer 20. The
20-meso proton, which appeared at δ 8.46 ppm in D-ring reduced
chlorin 1, was observed at δ 8.65 in isomer 20. Compared to the
case of the ring-D reduced isomer, the resonances of 20 at δ
9.01 ppm integrated for a single proton assigned to the 10-meso
proton appeared at δ 9.21 ppm in chlorin 1, possibly as a result of
higher electron density at these positions (see the Supporting
Information).
The course of reaction for the preparation of the intermediates
and final products for the synthesis of the isomeric structures 1,
11, and 20 (or the corresponding carboxylic acids 4, 12, and 23,
respectively) could be followed by monitoring changes in the
absorption spectrum. The following interesting differences be-
tween the electronic spectra of the ring-B and ring-D reduced
isomers were observed: As shown in Figure 3, the isomers 4 and
12 containing an iodobenzyloxyethyl group at position-3 or
position-8 of the chlorin system showed almost identical electro-
nic absorption spectra (long wavelength absorption 660 nm
(CH2Cl2)); however, the ring-B reduced chlorin 23 exhibited the
long wavelength absorption at λmax 670 nm. No significant
difference in singlet oxygen yields was detected between the
ring-B and ring-D reduced isomers, and they ranged from 0.45
to 0.48.
The rate of photobleaching of isomers 4, 12, and 23 was also
investigated in 17% bovine calf serum (BCS) in equimolar con-
centration. As can be seen from Figure 3B, all isomers showed
almost 50% photobleaching on exposing the photosensitizers to
light (75 mW/cm2) for 20ꢀ22 min. Interestingly, on further
irradiation, the rate of photobleaching of 12 (ring-D reduced
chlorin containing the 10-benzyloxyethyl group at position-8)
was slightly slower than those of the other two isomers, suggest-
ing its increased photostability.
Biological Studies. The initial functional assessment of the
PSs (cellular uptake, intracellular localization, STAT3 dimeriza-
tion, and PDT efficacy) was carried out in the cell culture model
of human basal cell carcinoma (BCC1). The relative PDT
efficacies determined in these cells were then evaluated in the
mouse colon adenocarcinoma cell line (Colon26) grown in vitro
and as subcutaneous tumors in BALB/c mice.
Correlation between Cellular Uptake, STAT3 Dimerization,
and Cell Killing. The cell biological properties of the iodinated PS
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Scheme 3. Synthetic Approaches for the Preparation of Ring-B Reduced 3-(10-m-Iodobenzyloxyethyl) Chlorins from
Bacteriopyropheophorbide-a
8 (PS 12) or B-ring reduction (PS 23) in the carboxylic acid PS
derivatives. Incubation of BCC1 cells with these PSs for 30 min
allowed assessment of immediate uptake (Figure 4A). The cellular
distribution of the PS fluorescence indicated that PS 4 was
efficiently taken up and deposited into the mitochondrial compart-
ment. Both the B-Ring reduction (PS 23) and placement of the
iodobenzyl group to position-8 (PS 12) in the carboxylic acid
derivatives lowered the cellular uptake by 2-fold and 4-fold,
respectively, relative to the case of PS 4. The subcellular localiza-
tion was identical for all three PSs and was not altered as a function
of continued incubation of the cells in PS-free medium for 4ꢀ24 h
(data not shown). However, as found for other porphyrin-based
PSs, cell-associated PS 4, 12, and 23 were lost with a ∼4ꢀ5-fold
higher rate when the cells were cultured in medium containing
10% serumcompared tocells cultured inserum-free medium(data
not shown). Strikingly different was the >100-fold lower uptake of
Figure 2. 1H NMR spectra of ring-D vs ring-B reduced chlorins 1 and
20, respectively.
1 were evaluated as a function of derivatization at position 172 to
carboxylic acid (PS 4) or conjugated with PEG (polyethylene
glycol, mol wt 5000) (PS 7). The same cellular characterization
determined the impact of the iodobenzyl group added to position
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Figure 3. (A) Electronic absorption spectra of isomers 4, 12, and 23 at equimolar concentration (6.5 μM) in 17% bovine calf serum (BCS). (B) Rate of
photobleaching of bacteriochlorins 4, 12, and 23 in 17% BCS. The photosensitizers at equimolar concentration (6.5 μM) were irradiated
with light (light dose: 75 mW/cm2), and the spectra were taken at variable time points. The normalized long wavelength absorption values were
plotted with time, showing a comparative rate of photobleaching of the photosensitizers by reactive oxygen species (mainly singlet oxygen)
produced in situ.
Figure 4. PS-specific uptake, photoreaction, and post-PDT survival. Replicate 24-well culture plates of BCC1 cells were incubated for 30 min in
serum-free DMEM containing 200 nM (A) or for 2 h with serum-free DMEM containing 2-fold serially diluted preparations (B and C) of the PSs
indicated at the top. The cells were washed free of PS and imaged at 640ꢁ magnification under phase contrast and HPPH fluorescence (A). The
plates treated with the serially diluted PSs were illuminated for 9 min with 665 nm light, yielding a fluence of 3 J/cm2. The cell monolayers in one
culture plate were immediately extracted. Equal aliquots of the extracts were analyzed on Western blots for the levels of immune detectable STAT3,
EGFR, and phosphorylated and total p38 MAPK (B). The oxidative cross-linking of STAT3 was determined by densitometric scanning of the bands
representing monomeric and homodimeric complex I. The percent conversion of STAT3 to the dimeric form is indicated at the top of panel “B”. The
second culture plate was changed to DMEM containing 10% fetal calf serum immediately after light treatment and cultured for an additional 24 h
period. The number of surviving, trypan blue-excluding cells were counted in a hemocytometer and expressed relative to the number of cells in the
control (C).
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were manifested in the level of STAT3 cross-linking, loss of
EGFR protein, and activation of the stress MAPK, p38
(Figure 4B). The same PDT treatment also led to a PS dose-
dependent cell killing as determined by the fraction of the cells
surviving 24 h post-PDT-incubation (Figure 4C). Under the
conditions used in Figure 4, the cellular uptake of PS 1 and PS 7
was barely detectable by fluorescent microscopy, and the low
levels of PS were ineffective in mediating photoreactions that
were quantifiable by the biochemical markers. By increasing the
concentrations of the PSs in cell treatments, we detected that the
two PSs displayed fundamentally different modes of uptake and
subcellular localization compared to the carboxylic acid-contain-
ing PSs. A time course analysis indicated that, within the 30 min
incubation period, a low-level of PS 1 was accumulated in the
mitochondrial compartment, yielding a pattern as observed for
PS 4 (Figure 5A, left panel). However, during a subsequent 4 h
incubation period in PS-free medium, a major fraction of the cell-
associated PS fluorescence was redistributed and retained at new
subcellular sites that include the lysosomal compartment,
whereas PS 4 maintained the initial mitochondrial localization
(Figure 5A, right panel). The uptake of PS 1, as PS 4, was subject
to competition with serum protein, with ∼5-fold lower fluores-
cence recorded for cells incubated with PS 1 in the presence of
10% fetal calf serum (data not shown). The opposite uptake
properties in the presence of serum proteins were detected for
PS 7. The PEGylated PS 7 was synthesized to identify whether
this hydrophilic version of the PS will reduce hepatic clearance
and, thus, enhance the availability for uptake by tumor in vivo.
Short-term incubation of basal cell carcinoma (BCC1) cells with
PS 7 indicated that PS 7 uptake was very low, with levels below
microscopic detection (Figure 4A). Only at higher input con-
centrations and prolonged incubation, i.e., 24 h, could an
increased and time-dependent cellular accumulation be observed
(Figure 5B, right panel). Unexpectedly, the cellular uptake was
several-fold higher in the presence of serum proteins (Figure 5B,
right panel). The subcellular localization, regardless of the in-
cubation conditions, was exclusively the endosomal/lysosomal
compartment. Since PS 7, unlike other lysosomally targeted
PSs, such as HPPH-Gal,30 did not show any binding to cell
surface internalization, we concluded that the uptake of PS 7
was not through the route of first binding to the plasma
membrane followed by endocytosis but largely through fluid
phase pinocytosis.
Differential Functions of the PSs Are Reproducible in
Murine Colon26 Tumor Model. The relative activities of the
PSs determined in BCC1 cells were confirmed in the mouse
tumor cell lines Colon26. The comparative study involved two
separate sets of experiments. In the first set, Colon26 cells were
incubated for 24 h in serum-containing culture medium with
either PS 1, 4, or 7. The duration of PS treatment was extended to
24 h in order to enhance intracellular accumulation of the PSs, in
particular PS 1 and 7. Following the photoreaction at increasing
light doses and culture in growth medium for an additional 24 h,
the surviving Colon26 cells were evaluated by MTT assay
(Figure 6A). The relative PDT efficacy of the PSs followed the
patterns determined in BCC1 cells (Figure 4C), with PS 4 as the
most effective PS. The second experimental set determined the
efficacy of PS 4 with the other isomers, PS 12 and PS 23
(Figure 6B). All three isomers showed comparable in vitro
efficacy that in part was due to the application of higher PS
concentration. Measurement of PS uptake by Colon26 cells
showed comparable values (PS 4 g 23 > 12) as found in
Figure 5. Specific patterns of uptake and subcellular distribution of the
PSs. (A) BCC1 cells were incubated for 30 min in serum-free DMEM
containing 3.2 μM PS 1 or 0.2 μM PS 4. The cells were washed, and
images of the cell monolayers were recorded at 640ꢁ magnification
under phase contrast or HPPH fluorescence. The cultures were in-
cubated in PS- and serum-free DMEM for an additional 4 h period. The
cells were imaged again under identical condition as before. (B) BCC1
cells were incubated for 24 h in either serum-free DMEM or DMEM
with 10% fetal calf serum, both containing 3.2 μM PS 7. The cells were
washed free of PS and imaged at 640ꢁ magnification.
the methyl ester derivative, PS 1, and the PEGylated PS 7 relative
to PS 4 (Figure 4A). The quantitative difference in uptake and
retention was also evident following incubation of the cells with
the PSs for 2 h, although the overall level of cell-associated PSs was
enhanced (data not shown).
Treatment of the PS-incubated cells with therapeutic light
mediated photoreactions that were strictly proportional to the
level of PS as determined by fluorescence. The photoreactions
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Figure 6. (A) In vitro photosensitizing efficacy of PS 1, 4, and 7. (B) PS 4, 12, and 23. Colon26 cells were incubated with 0.3 μM (A) and 0.0625 μM
(B) of PSs, respectively, in growth medium for 24 h. The cells were treated at the peak absorption wavelength with variable light doses and cultured for an
additional 48 h post-treatment. (C) PS uptake of compounds 4, 12, and 23 in Colon26 cells 24 h postincubation was determined by flow cytometry.
Figure 7. (A) Comparative in vivo photosensitizing efficacy of PS 1, PS 4, and PS 7 and (B) PS 4, 12, and 23 in BALB/c mice bearing Colon26 tumors
(10 mice/group). The mice were injected with photosensitizers (1.00 μmol/kg), and the tumors were exposed to laser light at 665 nm for compounds 1,
4, and 7; 674 nm for compound 23 and 660 nm for compound 12 (light dose: 128 J/cm2, 14 mW/cm2) at 24 h postinjection. At day 60, photosensitizers
4 and 23 gave 70% and 60% tumor response, respectively (7/10 and 6/10 mice were tumor free), whereas compound 12 showed limited efficacy.
Figure 8. Whole-body PET images of a BALB/c mouse bearing Colon26 tumor with 124I-PS 6 (A) taken at 24, 48, and 72 h postinjection. The
maximum uptake of the PS was observed at 24 h postinjection. However, the best contrast was obtained at 72 h for compound PS 6 postinjection.
BCC1 cells (Figures 4C and 6C). On the basis of the in vitro
findings, the relative activity of the PSs in mediating Colon26
tumor treatment in vivo was determined. PS 1, 4, and 7 were
administered at a dose of 1.0 μmol/kg to BALB/c mice bearing
subcutaneous Colon26 tumors. After 24 h, the tumors were
exposed to light (128 J/cm2, 14 mW/cm2). The animals were
monitored for 60 days for recurring tumor growth (Figure 7A).
In line with the relative activities determined in cell culture, PS 4
gave 50% tumor cure whereas PS 1 was significantly less effective
and only 20% cure mice were tumor-free on day 60. Under these
treatment parameters, PS 7 was ineffective. We interpret the
substantial in vivo activity of PS 1 to be the result of progressive
PS 1 accumulation in the tumor during the 24 h period postinjec-
tion and to possibly involve the mode of cellular uptake detected in
cultured cells (Figure 5A). A second series of in vivo tumor
treatments tested the activity of PS 23 and PS 12 relative to PS 4
(Figure 7B). PS 4 and PS 23 showed relative efficacy that matches
those detected for these two PSs in vitro (Figures 4C and 6B).
Comparative PET Imaging Ability of Photosensitizers 3
and 6. As evident from the tissue culture experiments and in vivo
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tumor treatments, PS 4 displayed the highest PDT efficacy,
followed by PS 23 and 12. We have previously shown that the
124I-labeled methyl ester derivative of PS 1, PS 3, showed a great
potential for imaging tumors in BALB/c mice bearing Colon26
tumors. In light of the new finding regarding the activity of PS 4
and our goal to develop a “multifunctional agent” based on
optimized PS structures for efficient tumor-imaging and therapy,
we compared the tumor-imaging and biodistribution of PS 3 with
the corresponding 124I-labeled form of PS 4, PS 6. Similar to the
methyl ester derivative,26 PS 6 showed significant tumor uptake
at 24, 48, and 72 h postinjection (Figure 8). Compound PS 27
showed a similar uptake profile as compared to that of PS 6 (data
not shown). The corresponding methyl ester derivative of
compound PS 27 and its 124I-labeld form, PS 26, also exhibited
a similar uptake profile as compared to that of compound PS 3
(data not shown). With both analogues, the maximal tumor
uptake was observed at 24 h postinjection, as determined by
region of interest (ROI) calculations. However, for imaging, 72 h
postinjection for compounds PS 4 and 6 and compound PS 27
(data not shown) produced the best contrast, as the compounds
cleared more rapidly from liver, spleen, and intestine than from
the tumor (Figure 8). The accumulation of label in the thyroid
due to release of free 124I could be blocked by injecting cold
potassium iodide in water 24 h prior to injecting the labeled
photosensitizer (data not shown).
Comparative in Vivo Biodistribution of PS 3, PS 6, and 18F-
FDG. Among the isomers investigated for PDT efficacy, PS 4, the
pyropheophorbide-a containing m-iodobenzyloxyethyl at posi-
tion-3 of the ring-D reduced chlorin system and with a carboxylic
acid group at the 172-position, was found to be most effective.
Interestingly, PS 1, the derivative bearing an ester functionality,
despite its lower PDT efficacy, showed remarkable tumor im-
aging ability.26 For determining the relative tumor uptake and
other organ uptake, we compare the in vivo biodistribution of
124I following injection of both PS 3 and 6 into BALB/c mice
bearing Colon26 tumors. Specifically, a set of nine tumor-bearing
mice were injected with each compound (50 μCi), and three
mice per group were imaged after 24, 48, and 72 h for 30 min with
microPET. Following imaging, the mice were sacrificed and
specific radioactivity values in individual organs (% injected dose
per gram) were determined (Figure 9). Both PSs showed similar
biodistribution and clearance from all the organs, with the
notable exception of slightly higher spleen-uptake of PS 1. 124I-
labeled PS 27 (B-ring isomer) also demonstrated similar tumor
uptake as compared to PS 6 (Figure 9A) but showed significantly
lower spleen uptake (data not shown).
Among the PET imaging agents, 18F-FDG is a current clinical
standard, with the several limitation of short-term uptake pro-
cesses. To assess the usefulness of PS 3 or PS 6 as alternative PET
agents to FDG, we also determined the biodistribution of 18F-
FDG (isotope half-life 110 min) in BALB/c mice bearing
Colon26 tumors. In brief, 18F-FDG (150 μCi) was injected iv
into three tumor-bearing mice, imaged by a PET scanning at 2 h
postinjection. Mice were then sacrificed, and the specific radio-
activity in the various organs was measured (Figure 10). The
results indicated substantially different organ distribution than
those for PS 3 and PS 6, with higher uptake in muscle and heart
relative to tumor.
Isomers 4, 12, and 23 Showed a Direct Correlation be-
tween Tumor-Uptake and Fluorescence Imaging. For inves-
tigating the fluorescence imaging potential of the isomers, the PS
4, 12, and 23 were injected intravenously (1.0 μmol/kg) in
BALB/c mice (3 mice/group) bearing Colon26 tumors. For best
visualization, the tumors were grown subcutaneously on the right
shoulder to a size of 4ꢀ5 mm in diameter. The fluorescence in
intact animals was imaged using the Nuance camera system at 24,
48, and 72 h postinjection. The images were then analyzed by
generating false color images representing the fluorescence
intensity of the drug at the tumor site. Among all the isomers
investigated, the PS 4 (ring-D isomer) and 23 (ring-B isomer)
showed similar drug uptake at 24, 48, and 72 h postinjection (p.i.)
(Figure 11a and c). This could explain the similar in vivo
photosensitizing efficacy of the two PS. Both PSs also showed
Figure 9. Comparative biodistribution of 124I following injection of PS
3 (A) and PS 6 (B) (100 μCi) in BALB/c mice bearing Colon26 tumors
(three mice/time point) at 24, 48, and 72 h postinjection. Replacing the
methyl ester functionality in PS 3 with a carboxylic acid functionality
(PS 6) did not make much difference in uptake of the PS in tumor and
other organs, except the spleen, where the uptake was significantly reduced.
Figure 10. Biodistribution of 18F-FDG in BALB/c mice (3 mice/group) bearing Colon26 tumors at 2 h postinjection. The tumor uptake ratio is also
presented in tabulated form.
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Figure 11. Whole body fluorescence reflectance images (FRI) of representative BALB/c mice implanted with C-26 tumors on the shoulder with
compounds 4, 12, and 23, respectively, at variable time points with a therapeutic dose (1 μmol kgꢀ1): (A) 24 h p.i.; (B) 48 h p.i.; (C) 72 h p.i. (D) average
fluorescent intensity (AFU) of 3 mice ( SD of a ROI (20 mm diameter) over the tumor in AU, arbitrary units.
exposed to light to see if any fluorescence was associated with
skin mass. The results clearly showed the tumor avid nature of
compounds of PS 4 and 23 (Figure 12). Most of the fluorescence
was displayed from the tumor with little fluorescence associated
with the skin. This might be useful to identify tumor mass from
normal tissue mass during surgical removal of tumors.
’ CONCLUSIONS
In summary, the results obtained from a series of iodinated PSs
suggest that a carboxylic acid group at position 172 favors a
significant increase in cellular uptake by diffusion and deposition
into the mitochondrial compartment, thereby increasing in vitro/
in vivo PDT efficacy. In contrast, the same PS but with a methyl
ester at position 172 displays low level cellular uptake and is
subjected to a yet to define intracellular redistribution. Linking
the PEG moiety (mol wt: 5000) to position-172 changes the PS
to a hydrophilic molecule that assumes a behavior common to
plasma constituents that are to be taken up by cells through fluid
Figure 12. Whole body fluorescence reflectance images (FRI) of
phase pinocytosis and deposited in the endosomal/lysosomal
representative BALB/c mice implanted with Colon26 tumors on the
compartment. Interestingly, the isomer 12 in which the m-iodo-
shoulder with compounds 4 and 23 at 24 h postinjection (dose: 1 μmol
kgꢀ1). λex = 665 and 674 nm for PS 4 and 23, respectively; λem
=
benzyloxyethyl group was substituted at position-8 showed
limited PDT efficacy. However, the isomer 23 in which the
m-iodobenzyloxy was present at position-3, but instead of ring-D
(PS 4) the ring-B was reduced, was equally effective. The
function/structure analysis of these photosensitizers demon-
strated the close relationship of PS concentration in cells, PS-
mediated photoreaction resulting in STAT3 dimerization, and
PDT efficacy. These activities defined in vitro proved to a large
extent to be predictive for the PS’s action in vivo and also
confirmed activities reported for other photosensitizers.31 The
124I-analogues of the effective PS also showed a great potential to
function as tumor-imaging agent. PSs 1 and 4 were successfully
applied to image tumors by PET and fluorescence. Detailed
toxicological studies of these compounds in a good laboratory
practice (GLP) facility following the United States Food and Drug
Administration (US-FDA) guidelines are currently in progress.
710 nm). (A) PS 4; (B) PS 23; tumor was removed and skin was flapped
back to expose the residual tumor. The two PSs 4 and 23, exhibiting
the highest in vivo phototoxicity, were injected intravenously into mice
bearing Colon26 tumors to confirm that the recorded fluorescence
coincided with the actual tumor mass. After 24 h, the tumors were
surgically removed from the primary tumor site and imaged separate from
the animal. The skin that covered the tumor was exposed to the camera,
and the site along the tumor was imaged. The results clearly indicate
that most of the fluorescence was confined to the tumor with only low
fluorescence associated with the skin section.
similar clearance rates at 48 and 72 h p.i. (Figure 11d). PS 12
showed lower tumor uptake at 24 h compared to PSs 4 and 23
(Figure 11b), which is in agreement with the low accumulation in
cultured cells (Figure 4) and tumor phototoxicity exhibited by PS
12 (Figure 7).
To study the potential of these agents as tumor imaging
agents, the most effective compounds, PS 4 and 23, were injected
into mice bearing Colon26 tumors at the therapeutic dose of
1 μmol/kg. Twenty four hours post injection, the animals were
sacrificed, and the primary tumor was removed surgically and
imaged for fluorescence. The skin beneath the tumor was also
’ EXPERIMENTAL SECTION
Chemistry. All chemicals were of reagent grade and used as such.
Solvents were dried using standard methods. Reactions were carried out
under nitrogen atmosphere and were monitored by thin-layer chroma-
tography and/or UVꢀvisible spectroscopy. Preparative scale thin-layer
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chromatography was carried out on 20 ꢁ 20 cm2 glass plates coated with
2.50ꢀ2.62, and 2.20ꢀ2.34 (m, 4H, 171-and172-H), 2.14(m, 3H, 32-CH3),
1.81 (d, J = 5.6, 3H, 18-CH3), 1.71 (t, J = 7.6, 3H, 8-CH2CH3), 0.50 (br s,
1H, NH), 0.19 (s, 9H, tert-butyltin), ꢀ1.70 (br s, 1H, NH). 13C NMR
(CDCl3; 400 MHz): δ 197.39, 172.07, 160.96, 155.59, 151.08, 149.12,
145.21, 141.32, 140.68, 138.46, 137.63, 137.03, 136.49, 135.49, 133.11,
130.21, 130.01, 128.37, 127.80, 127.29, 114.66, 105.88, 104.21, 97.77, 94.44,
93.00, 72.08, 70.30, 51.72, 50.21, 48.03, 31.47, 31.11, 30.32, 29.75,
24.52, 23.14, 19.49, 17.44, 14.24, 11.95, 11.33, 11.09, 1.05. Mass: calcd
for C43H50N4O4Sn, 806.3; found, 807.5 (MH+). HRMS: calcd for
C43H51N4O4Sn (MH), 807.2932; found, 807.2995.
Synthesis of Peglate 3-Devinyl-3-{10-(m-iodobenzyoxy)
ethyl}pyropheophoride-a (7). Methyl 3-devinyl-3-{10-(m-iodo-
benzyoxy)ethyl}pyropheophoride-a carboxylic acid (4) (50 mg, 0.065
mmol), PEG (390 mg, 0.078 mmol), DCCI (13.8 mg, 0.072 mmol), and
DMAP (7.93 mg, 0.065 mmol) were dissolved in anhydrous DMF
(4 mL). The reaction mixture was stirred at room temperature for
overnight. The solvent was evaporated. The crude product was purified
by preparative plates. 4% methanol/dichloromethane was used as
the eluting solvent to yield compound 7. It was then crystallized
from dichloromethane/hexane in 51% yield (191 mg). mp >320 ꢀC.
UVꢀvisible, λmax (CH2Cl2), nm (ε): 664 (4.64 ꢁ 104), 607 (9.22 ꢁ
103), 535 (9.43 ꢁ 103), 507 (9.66 ꢁ 103), 412 (9.53 ꢁ 104). 1H NMR
(CDCl3; 400 MHz): δ 9.77, 9.76 (two s, 1H, 5-H), 9.54, and 8.59 (each s
for 1H, 5- and 10-meso-H), 7.75, 7.63, 7.07, 6.89 (each m for 1H, ArH),
7.40 (m, 2H, ArH), 5.96 (q, J = 6.3, 1H, 31-H), 5.28 (d, J = 17.7, 1H, 132-
CH2), 5.12 (d, J = 17.6, 1H, 132-CH2), 4.68 (dd, J = 5.4, 11.3, 1H,
OCH2Ar), 4.57 (m, 2H, 1H for OCH2Ar, 1H for 17-H), 4.35 (m, 1H,
18-H), 4.04ꢀ3.14 (broad and high peak, many protons, including all
protons from PEG group ꢀNHCH2CH2(OCH2CH2)nOCH3, another
11H for 2-CH3, 7-CH3, 12-CH3, and 8-CH2CH3), 2.43 (m, 2H, 171-H),
2.17 (m, 2H, 172-H), 1.82 (d, J = 5.5, 3H, 3-CH3), 1.70 (t, J = 7.6, 3H, 18-
CH2CH3), 1.25 (brs, 1H, NH), ꢀ1.73 (brs, 1H, NH). HRMS: calcd for
C267H496N5O116I, 5756.2111; found, 5756.2132.
1
Merck G 254 silica gel (2 mm thick). H and 13C NMR spectra were
recorded on a Bruker AMX 400-MHz or Varian 400 NMR spectrometer
at 303 K in CDCl3 or ∼10% CD3OD in CDCl3 or DMSO-d6. Proton
chemical shifts (δ) are reported in parts per million (ppm) relative to
CDCl3 (7.26 ppm) or TMS (0.00 ppm). Coupling constants (J) are
reported in Hertz (Hz) and s, d, t, q, p, m, and br refer to singlet, doublet,
triplet, quartet, pentet, multiplet, and broad, respectively. Mass spectral
data (electrospray ionization, ESI by fusion) were obtained from Bioploy-
mer facility, RPCI. The high resolution mass spectrometry analyses were
performed at Mass Spectrometry Facility, Michigan State University, East
Lansing, MI. CHN analyses were done at Midwest Microlab LLC,
Indianapolis, IN. UVꢀvisible spectra were recorded on a Varian Cary 50
Bio UVꢀvisible spectrophotometer using CH2Cl2/MeOH as solvent.
Melting points were uncorrected and were measured on a Thomas/
Bristonline microscopic hot stage apparatus. In general most of the products
obtained after column or preparative chromatography were crystallized/
precipitated from dichloromethane/hexane. The purity of the compounds
was confirmed by HPLC, and all compounds were >95%. The HPLC
profiles of the final products are included in the Supporting Information.
Synthesis of 3-Devinyl-3-{10-(m-iodobenzyoxy)ethyl}pyro-
pheophoride-a (4). Methyl 3-devinyl-3-{10-(m-iodobenzyoxy)ethyl}
pyropheophoride-a (1) (150 mg, 0.19 mmol) obtained following
the procedure developed by our group26 was reacted with 240 mg (10.04
mmol) of aqueous LiOH in THF, and the reaction was stirred at room
temperature overnight. The reaction mixture was neutralized with 2%
AcOH in H2O, and compound was extracted with CH2Cl2. The organic
layer was washed with H2O, dried over Na2SO4, concentrated, and
precipitated with hexanes to yield the proposed compound (yield: 80%),
mp: 138ꢀ140 ꢀC. UVꢀvisible, λmax (MeOH/CH2Cl2), nm (ε): 663 nm
(4.75 ꢁ 104), 605 (6.81 ꢁ 103), 539 (6.81 ꢁ 103), 506 (6.61 ꢁ 103), 411
(9.52 ꢁ 104). 1H NMR(CDCl3; 400 MHz): δ 9.62, 9.21, and 8.46 (three s,
each for 1H, meso-H), 7.67 (s, 1H, ArH), 7.558 (d, J = 8.0, 1H, ArH), 7.183
(m, 1H, ArH), 6.962 (m, 1H, ArH), 5.867 (q, J= 6.8, 1H, 31-H), 5.16 (d, J=
18.6, 1H, 132-CH2), 4.97 (d, J = 18.6, 1H, 132-CH2), 4.62ꢀ4.50 (m, 1H,
18H), 4.49ꢀ4.26 (m, 2H, OCH2Ar), 4.20ꢀ4.00 (m, 1H, 17H), 3.46 (q, J =
8.0, 2H, 8-CH2CH3), 3.35ꢀ2.99 (m, all 3H, for 3 ꢁ ring CH3), 2.40ꢀ2.73,
2.38ꢀ2.18 (m, 4H, 171- and 172-H), 2.08 (s, 3H, 32-CH3), 1.61 (d, J = 7.6,
3H, 18-CH3), 1.48 (s, 3H, 8-CH2CH3), 0.07 (brs, 1H, NH), ꢀ1.82 (brs,
1H, NH). 13C NMR (CDCl3; 400 MHz): δ 196.95, 171.71, 160.67, 155.35,
151.04, 149.13, 145.13, 141.38, 140.87, 138.51, 137.85, 137.11, 136.91,
135.4, 132.99, 130.34, 128.41, 127.94, 127.35, 106.16, 104.13, 97.93, 94.62,
93.00, 72.19, 70.37, 51.74, 50.19, 48.14, 31.24, 29.97, 24.73, 23.29, 19.58,
17.59, 14.21, 12.03, 11.53, 11.26, 1.25. MS for C40H41N4O4I: 768.22
(calcd), 769.2 (found, MH+). Anal. Calcd for C40H41N4O4I: C, 62.50;
H, 5.38; N, 7.29. Found: C, 62.59; H, 5.42; N, 7.20.
Synthesis of 3-Devinyl-3-{10-(m-trimethylstannylbenzyloxy)
ethyl}pyropheophoride-a (5). To the solution of methyl 3-devi-
nyl-3-{10-(m-iodobenzyoxy)ethyl}pyropheophoride-a carboxylic acid
(4) (50 mg, 0.065 mmol) in dry THF were added hexamethydistannane
(0.1 mL, 0.48 mmol) and bis(triphenylphosphine) palladium(II)dichloride
(10 mg, 0.014 mmol), and the reaction mixture was stirred at room
temperature for overnight. The solvent was evaporated. The crude product
was purified by preparative plates. 4% methanol/dichloromethane was used
as the eluting solvent to yield compound 5. The compound obtained after
evaporating the solvents was crystallized from dichloromethane/hexane in
75% yield (39 mg). mp >320 ꢀC .UVꢀvisible, λmax (MeOH/CH2Cl2), nm
(ε): 663 (4.65 ꢁ 104), 606 (9.20 ꢁ 103), 537 (9.42 ꢁ 103), 506 (9.64 ꢁ
103), 411 (9.52 ꢁ 104). 1H NMR (CDCl3; 400 MHz): δ 9.74, 9.50, and
8.53 (all s, 1H, meso-H), 7.50 (m, 2H, ArH), 7.40 (m, 2H, ArH), 6.01 (q,
J = 6.4, 1H, 31-H), 5.27 (d, J = 17.6, 1H, 132-CH2), 5.10 (d, J = 19.6, 1H,
132-CH2), 4.78 (dd, J = 5.4, 11.9, 1H, OCH2Ar), 4.60 (dd, J = 1.7, 12.0, 1H,
OCH2Ar), 4.50 (m, 1H, 18-H), 4.28 (m, 1H, 17-H), 3.70 (q, J = 7.8, 2H,
8-CH2CH3), 3.64, 3.35, and 3.16 (m, all 3H, for 3 ꢁ ring CH3), 2.36ꢀ2.76,
Synthesis of Methyl 3-Devinyl-8-{10-(m-iodobenzyoxy)
ethyl}pyropheophoride-a (11). Methyl 3-devinyl-3-ethyl-7,8-di-
hydroxypyropheophoride-a (10) (100 mg, 0.17 mmol) was reacted with
3-iodobenzyl alcohol in refluxing toluene, and a few drops of HCl was
added. The reaction mixture was neutralized with NaHCO3 in H2O, and
compound was extracted with CH2Cl2. The organic layer was washed
with H2O and dried over Na2SO4. The crude product was purified by
preparative plates, using 5% methanol/dichloromethane as the eluting
solvent. Two bands were separated and identified as follows: the slower
moving band (minor product) was characterized as methyl 3-devinyl-3-
ethyl-8-vinyl pyropheophoride-a (16); the faster moving band (major
product, 54 mg, 40% yield) was identified as the desired product 11, mp:
130ꢀ132 ꢀC. UVꢀvisible, λmax (CH2Cl2), nm (ε): 656 (4.69 ꢁ 104),
1
599 (8.22 ꢁ 103), 539 (8.14 ꢁ 103), 506 (9.91 ꢁ 103). H NMR
(CDCl3; 400 MHz): δ 9.98, 9.26, 8.48 (all s, 1H, meso-H), 7.71 (d, J =
4.0, 1H, ArH), 7.59 (d, J = 8.0, 1H, ArH), 7.20 (t, J = 8.2, 1H, ArH), 7.05
(t, J = 8.2, 1H, ArH), 5.78 (q, 6.8, 1H, 81-H), 5.28 (d, J = 19.8, 1H, 132-
CH2), 5.13 (d, J = 19.8, 1H, 132-CH2), 4.68 (d, J = 13.0, 1H, OCH2Ar),
4.56 (d, J = 11.6, 1H, OCH2Ar), 4.41ꢀ4.48 (m, 1H, 18-H), 4.22ꢀ4.31
(m, 1H, 17-H), 3.84 (q, J = 8.0, 2H, 3-CH2CH3), 3.62, 3.59, 3.30, and
3.28 (all s, all 3H, for 173-COOCH3 and 3 ꢁ ring CH3), 2.61ꢀ2.75,
2.44ꢀ2.58, and 2.17ꢀ2.35 (m, 4H, 171- and 172-H), 2.12 (t, J = 4.8, 3H,
82-CH3), 1.79 (t, J = 10.4, 3H, 18-CH3), 1.73 (t, J = 7.6, 3H,
3-CH2ꢀCH3), 0.41 (brs, 1H, NH), ꢀ1.74 (brs, 1H, NH). 13C NMR
(CDCl3; 400 MHz): δ 196.42, 173.69, 172.16, 160.78, 153.90, 149.58,
148.86, 142.67, 142.19, 141.33, 138.03, 137.24, 136.83, 131.44, 130.68,
129.55, 128.08, 127.38, 119.98, 114.96, 106.67, 105.97, 96.22, 94.65,
92.77, 72.55, 70.20, 51.90, 50.24, 48.22, 31.11, 30.08, 25.39, 23.32, 19.61,
18.70, 17.17, 12.72, 12.41, 11.61, 11.17. MS for C41H43O4N4I: 782.23
(calcd), 805.1 (found, M + 23). Anal. Calcd for C41H43O4N4I: C, 62.90;
H, 5.54; N, 7.16. Found: C, 62.80; H, 5.52; N, 7.11.
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Synthesis of 3-Devinyl-3-ethyl-8-{10-(m-iodobenzyoxy)ethyl}
pyropheophoride-a (12). Methyl 3-devinyl-8-{10-(m-iodobenzyox-
y)ethyl}pyropheophoride-a (11) was reacted with aqueous LiOH by
following the method discussed for the preparation of 3-devinyl-
3-{10-(m-iodobenzyoxy)ethyl}pyropheophoride-a carboxylic acid (4),
and 12 was obtained in 70% yield, mp: 171ꢀ172 ꢀC. UVꢀvisible, λmax
(CH2Cl2), nm (ε): 655 (4.65 ꢁ 104), 600 (8.03 ꢁ 103), 538 (8.00 ꢁ
103), 506 (9.63 ꢁ 103). 1H NMR (CDCl3; 400 MHz): δ 9.90, 9.16, and
8.42 (all s, 1H, meso-H), 7.70 (s, 1H, ArH), 7.55 (d, J = 7.8, 1H, ArH),
7.21 (d, J = 8.0, 1H, ArH), 6.962 (t, J = 8.0, 1H, ArH), 5.73 (q, J = 8.0, 1H,
81-H), 4.95ꢀ5.36 (m, 2H, 132-CH2), 4.57ꢀ4.70 (m, 1H, 18H),
4.44ꢀ4.54 (d, J = 10.4, 1H, OCH2Ar), 4.43ꢀ4.33 (m, 1H, OCH2Ar),
4.20ꢀ4.24 (m, 1H, 17H), 3.75 (q, J = 8.0, 2H, ꢀCH2CH3), 3.56, 3.25,
3.22 (all s, all 3H, for 3 ꢁ ring CH3), 2.47ꢀ2.71, 2.20ꢀ2.37 (m, 4H, 171
and 172-H), 2.09 (s, 3H, 82-CH3), 1.78 (s, 3H, 18-CH3), 1.67 (s, 3H,
3-CH2CH3), 0.85 (brs, 1H, NH), ꢀ1.72 (brs, 1H, NH). 13C NMR
(CDCl3; 400 MHz): δ 197.25, 173.94, 172.50, 160.65, 154.17, 149.36,
148.67, 142.64, 142.01, 140.84, 138.01, 137.28, 137.02, 136.86, 136.60,
131.53, 130.05, 129.80, 129.12, 127.61, 127.22, 114.49, 105.38, 95.81,
94.19, 92.62, 72.27, 69.94, 51.59, 51.42, 50.01, 30.79, 29.79, 29.57, 24.87,
22.85, 19.23, 16.71, 11.89, 11.16 . Mass for C40H41O4N4I: 768.22
(calcd), 769.4 (found, MH+). Anal. Calcd for C40H41O4N4I: C, 62.50;
H, 5.38; N, 7.29. Found: C, 62.39; H, 5.45; N, 7.20.
70%. UVꢀvis (CH2Cl2, λmax, nm, (ε)): 670 (4.69 ꢁ 104), 613(5.54ꢁ 103),
540 (5.11 ꢁ 103), 513 (9.57 ꢁ 103), 413(7.70ꢁ 104). 1HNMR(400MHz,
CDCl3): δ 9.21, 9.00/8.99, 8.65 (all s, 1H, meso-H), 7.75 (d, 1H, ArH), 7.62
(d, J = 8.0 Hz, 1H, ArH), 7.25 (s, 1H, ArH), 7.04 (t, J = 8.0 Hz, 1H, ArH),
5.91 (m, 1H, 31-H), 5.44 (s, 2H, 131-H), 4.64ꢀ4.67 and 4.50ꢀ4.54 (m, 2H,
OCH2Ar), 4.43ꢀ4.45 and 4.21 (m, 2H, 7-H + 8-H), 3.83 (t, J = 8.0 Hz, 2H,
171-CH2), 3.74 (s, 3H, COOCH3), 3.55 (s, 3H, 12-CH3), 3.42 (s, 3H,
2-CH3), 3.21 (s, 3H, 18-CH3), 2.91 (t, J = 8.0 Hz, 2H, 172-CH2), 2.44ꢀ2.49
(m, 1H, 81-H), 2.14ꢀ2.17 (m, 4H, 81-H + 31-CH3), 1.80/1.90 (distorted d,
3H, 7-CH3), 1.16 (t, 3H, 81-CH3). 13C NMR (CDCl3; 400 MHz): δ195.99,
195.98, 173.11, 172.15, 172.06, 166.25, 154.24, 146.54, 145.24, 145.22,
142.43, 140.79, 140.69, 139.77, 139.61, 139.31, 137.31, 136.98, 136.87,
136.75, 136.38, 136.31, 135.19, 135.13, 133.54, 130.12, 127.19, 127.12,
121.73, 121.72, 115.51, 97.08, 97.05, 96.72, 96.68, 94.49, 94.38, 93.85,
93.77, 71.97, 71.71, 70.18, 70.06, 55.55, 55.51, 51.82, 51.81, 48.96, 48.95,
48.90, 36.08, 30.35, 30.30, 24.34, 24.19, 23.36, 23.23, 11.66, 11.64, 11.22,
11.19, 11.18, 11.17, 11.04, 11.00. MS (ESI): m/z 783.4 (M + H+). HRMS
(ESI): calcd for C41H44IN4O4, 783.2407; found, 783.2410 (MH+). Anal.
Calcd for C41H43IN4O4: C, 62.91; H, 5.54; N, 7.16. Found: C, 62.85; H, 5.57;
N, 7.01.
Synthesis of 3-Deacetyl-3-(10-m-iodobenzyloxyethyl)-7,8-
dihydrophylloerythrin (23). Compound 20 (40 mg, 0.05 mol,
1equiv) was dissolved in degassed THF (10 mL). Degassed water and
Synthesis of Methyl 3-(10-(3-Iodobenzyloxy)ethyl)-3-dea-
cetylbacteriopyropheophorbide-a(19). Compound 1829 (100.0 mg,
0.17 mmol, 1.0 equiv) was dissolved in dry CH2Cl2 (15 mL), and HBr gas
was bubbled into the solution for 2 min. The reaction mixture was stirred for 5
min at room temperature. The solvent was evaporated under high vacuum,
the resulting crude was dried, and the entire crude was dissolved in dry
CH2Cl2 (15 mL). A 50.0 mg portion of dry K2CO3 was added to this
mixture, followed by addition of 3-iodobenzyl alcohol (0.25 mL). The
resulting reaction mixture was stirred for 30 min at room temperature and
then worked up. Purification of the crude mixture was done by column
chromatography (silica gel, 50% ethyl acetate in hexanes). Yield: 89 mg, 65%.
1H NMR (400 MHz, CDCl3): δ 8.52/8.53, 8.23, 8.04 (all s, 1H, meso-H),
7.72 (d, 1H, ArH), 7.62 (d, J = 8.0 Hz, 1H, ArH), 7.25 (s, 1H, ArH), 7.04 (t,
1H, ArH), 5.68ꢀ5.73 (m, 1H, 31-H), 4.96 (d, J = 19.6 Hz, 1H, 131-H), 4.79
(d, J = 19.6 Hz, 1H, 131-H), 4.60ꢀ4.63 (m, 1H, OCH2Ar), 4.45ꢀ4.50 (m,
1H, OCH2Ar), 4.14ꢀ4.19 (m, 2H, 7-H + 18-H), 4.00ꢀ4.02 (m, 1H, 17-H),
3.88ꢀ3.90 (m, 1H, 8-H), 3.61 (s, 3H, COOCH3), 3.35 (s, 3H, 12-CH3),
3.14 (s, 3H, 2-CH3), 2.44ꢀ2.59 (m, 2H, 81-H + 171-H), 2.21ꢀ2.43 (m, 4H,
171-H + 171-CH2 + 81-H), 2.01 (d, J = 8.0 Hz, 3H, 31-CH3), 1.65ꢀ1.77
(doublets, 6H, 18-CH3 + 7-CH3), 1.12 (t, 3H, 81-CH3). 13C NMR (CDCl3;
400 MHz): δ 195.56, 173.54, 170.95, 170.85, 170.21, 161.53, 155.22, 147.83,
147.81, 140.78, 140.74, 140.70, 140.62, 138.48, 138.41, 137.37, 137.36,
136.95, 136.84, 136.74, 136.02, 135.99, 133.67, 130.12, 127.13, 127.09,
118.22, 118.18, 108.74, 99.24, 99.21, 95.00, 94.94, 94.35, 93.82, 93.80,
71.72, 71.49, 70.10, 70.00, 54.34, 54.30, 51.65, 50.43, 50.41, 49.89, 49.06,
47.38, 30.78, 30.21, 30.19, 29.88, 24.01, 23.90, 22.78, 22.63, 22.54, 22.51,
11.26, 10.88, 10.84, 10.80. MS (ESI): m/z 785.4 (M + H+). HRMS (ESI):
calcd for C41H46IN4O4, 785.2564; found, 785.2587 (MH+). UVꢀvis
(CH2Cl2, λmax, nm, (ε)): 719 (3.58 ꢁ 104), 659 (1.21 ꢁ 104), 602
(4.71 ꢁ 103), 517 (2.55 ꢁ 104), 486 (6.69 ꢁ 103), 456 (2.81 ꢁ 103), 382
(4.59ꢁ 104), 354 (8.63 ꢁ 104).
a solution of LiOH H2O (50 mg, 1.19 mmol, 23 equiv) in methanol
3
(10 mL, 1:1 v/v) were added. The entire reaction mixture was then
stirred under nitrogen atmosphere at room temperature for 2 h, and the
resulting mixture was diluted with dichloromethane (20 mL). It was
then washed with water (3 ꢁ 200 mL). The organic layer was separated
and dried over Na2SO4, and the solvent was removed under reduced
pressure. The residue obtained was purified by preparative TLC (silica
gel, 5% methanol in CH2Cl2) and crystallized from dichloromethane/
hexane. Yield:22 mg, 55%. UVꢀvis (CH2Cl2, λmax, nm, (ε)): 670 (4.69 ꢁ
104), 613 (5.54 ꢁ 103), 540 (5.11 ꢁ 103), 513 (9.57 ꢁ 103), 413 (7.70 ꢁ
104). 1H NMR (400 MHz, CDCl3): δ 9.21, 8.64 (all s, 1H, meso-H),
7.73 (d, 1H, ArH), 7.60 (d, 1H, ArH), 7.24 (s, 1H, ArH), 6.99ꢀ7.05 (m,
1H, ArH), 5.87ꢀ5.88 (m, 1H, 31-H), 5.40 (s, 2H, 131-H), 4.48ꢀ4.62
and 4.48ꢀ4.53 (m, 2H, OCH2Ar), 4.40ꢀ4.43 and 4.19ꢀ4.21 (m, 2H,
7-H + 8-H), 3.86 (m, 2H, 172-CH2), 3.53 (s, 3H, 12-CH3), 3.30 (s, 3H,
2-CH3), 3.22 (s, 3H, 18-CH3), 2.95 (t, 2H, 171-CH2), 2.43ꢀ2.47 (m,
1H, 81-H), 2.12ꢀ2.17 (m, 4H, 81-H + 31-CH3), 1.75ꢀ1.89 (d, 3H,
7-CH3), 1.13 (m, 3H, 81-CH3). MS(ESI): m/z 769.2 (M + H+). HRMS
(ESI): calcd for C40H42IN4O4, 769.2251; found, 769.2271 (MH+).
Synthesis of Methyl 3-{10-(m-Trimethylstannylbenzyloxy)
ethyl}-7,8-dihydrophylloerythrin (25). To a solution of 20 (50 mg,
0.06 mmol, 1.0 equiv) in dry DMF (10 mL) were added hexamethylditin
(83 mg, 0.25 mmol, 4.0 equiv) and Pd2(dba)3 CHCl3 (6.6 mg, 0.006 mmol,
3
0.1 equiv), and the reaction mixture was stirred at 50 ꢀC for 4 h. After
removal of the solvent under vacuum to dryness, the crude mixture was
purified by preparative plates using 5% acetone/CH2Cl2 to give 25.
Yield = 21 mg, 40% UVꢀvis (CH2Cl2, λmax, nm, (ε)): 669, 613, 540, 513,
413. 1H NMR (400 MHz, CDCl3): δ 9.17, 9.06, 8.65 (all s, 1H, meso-H),
7.26ꢀ7.51 (m, 4H, ArH), 5.92ꢀ5.97 (m, 1H, 31-H), 5.53 (s, 2H, 131-H),
4.69ꢀ4.77 and 4.56ꢀ4.62 (m, 2H, OCH2Ar), 4.42ꢀ4.45 and 4.22 (m,
2H, 7-H+8-H), 3.75 (overlapped, 5H, 2Hof171-CH2, 3HofCOOCH3),
3.56 (s, 3H, 12-CH3), 3.42 (s, 3H, 2-CH3), 3.18 (s, 3H, 18-CH3), 2.88 (t,
J = 8.0 Hz, 2H, 172-CH2), 2.51 (m, 1H, 81-H), 2.14ꢀ2.17 (m, 4H, 81-H +
31-CH3), 1.80/1.89 (d, J = 8.0 Hz, 3H, 7-CH3), 1.16 (t, 3H, 81-CH3), 0.25
(s, 9H, Sn(CH3)3). 13C NMR (CDCl3; 400 MHz): δ 196.05, 173.14,
172.24, 172.13, 166.18, 154.37, 146.55, 145.26, 142.56, 142.46, 142.34,
139.84, 139.79, 139.35, 137.68, 137.62, 137.46, 136.35, 136.24, 135.72,
135.62, 135.53, 135.50, 135.26, 135.19, 133.51, 128.25, 128.19, 128.17,
128.04, 121.60, 115.50, 96.96, 96.92, 96.72, 96.66, 94.02, 93.86, 71.74,
71.39, 71.28, 71.23, 55.55, 55.46, 51.81, 48.94, 36.12, 30.36, 30.30, 24.43,
24.20, 23.33, 23.19, 22.44, 11.64, 11.24, 11.19, 11.15, 10.96, 10.92, ꢀ9.58,
Synthesis of Methyl-3-deacetyl-3-(10-m-iodobenzyloxyethyl)-
7,8-dihydrophylloerythrin (20). Compound 19 (50.0 mg, 0.06 mmol,
1.0 equiv) was dissolved in dichloromethane (50 mL). To this mixture was
added slowly a nitromethane (4 mL) solution of FeCl3 6H2O(68.9mg, 0.25
3
mmol, 4.0 equiv). The resulting reaction mixture was stirred at room
temperature for 30 min, quenched by addition of 20 mL of methanol, and
washed with water three times. The organic layer was separated and dried over
anhydrous Na2SO4, and solvent was removed under vacuum. The product
obtained after evaporating the solvents was purified by preparative plates
(silica gel; eluting solvent, 2% acetone in dichloromethane). Yield: 35.0 mg,
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ꢀ9.59. MS (ESI): m/z 821.5 (M + H+). HRMS (ESI): calcd for
C44H52N4O4Sn, 821.3089; found, 821.3078 (MH+).
Photoreaction was performed by illuminating PS-treated cell cultures
in serum-free medium at 37 ꢀC (within a tissue culture incubator) with
665 nm light of an argon-pumped dye laser for 9 min to a total fluence of
3 J/cm2. Depending upon the experimental design, cells were either
extracted immediately after light treatment (identification of signaling
reaction) or incubated at 37 ꢀC for 24 h (evaluation of PDT-mediated
cell kill).
3-Deacetyl-3-(10-m-trimethylstannylbenzyloxyethyl)-7,8-di-
hydrophylloerythrin (24). To a solution of 23 (9.0 mg, 0.01 mmol,
1.0 equiv) in dry THF was added hexamethydistannane (35.0 μL) and
bis(triphenylphosphine)palladium(II)dichloride (3.2 mg, 0.004 mmol,
0.4 equiv), and the reaction mixture was stirred at room temperature for
overnight. The solvent was evaporated. The crude product was purified by
preparative plates. 4% methanol/dichloromethane was used as the eluting
solvent to yield compound (24). Yield: 6.0 mg (75%), UVꢀvisible,
Cell Analyses. Survival was defined as the percent viable cells
recovered after 24 h of PDT and incubation in full growth medium. Cells
were released by trypsin treatment, and trypan blue-excluding cells were
counted in a hemocytometer. For protein analysis, cells were lysed in
radioimmunoprecipitation assay (RIPA) buffer and subjected to protein
analyses as described.8,9 Briefly, protein extracts (20 μg) were separated
on 6% to 10% SDS-polyacrylamide gels. On all gels, reference protein
markers for loading, molecular size detection, and cross-comparison
among gels were included. After separation, proteins were transferred to
Hybond-P membranes (Amersham Pharmacia Biotech). Nonspecific
interactions were blocked by incubating the membranes with PBS-0.1%
Tween 20ꢀ5% skim milk. Membranes were then reacted overnight at
4 ꢀC with one of the primary antibodies: STAT3 (Santa Cruz Biotech-
nology, Santa Cruz, CA), p38 P-p38, and EGFR (Cell Signaling
Technologies, Inc., Danver, MA). The immune complexes were visua-
lized by reacting with peroxidase-coupled secondary antibodies and
enhanced chemiluminescence detection (ECL) (Pierce Chemicals,
Rockford, IL). ECL images were recorded on X-ray films by various
lengths of exposure to ensure recovery of signals within the linear range
of digital scanning. The net pixel values of each band were integrated by
using the ImageQuant TL program (Amersham Biosciences). The
cross-comparison of separate analyses and immunoblots relied on
coseparated reference markers. STAT3 cross-linking was expressed by
the percent conversion of monomeric STAT3 into the dimer form I of
the STAT3 cross-linked complexes.31
To quantify the uptake of PS by Colon26 cells, the cells were seeded
in six-well plates with 0.33 ꢁ 106 cells per well in 2 mL of growth
medium. The cells were incubated overnight at 37 ꢀC. Four concentra-
tions (100, 200, 400, and 800 nM) of the photosensitizers were added to
triplicate wells and incubated at 37 ꢀC for 4 h. Triplicate wells without
photosensitizers were included to determine the baseline autofluores-
cence. The cells were then harvested by trypsinization, neutralized with
cold medium containing 2% FCS, transferred to 5 mL flow cytometry
tubes and centrifuged for 5 min at 500 g at 4 ꢀC to prevent efflux of the
photosensitizers. The supernatant was removed, the cell pellet resus-
pended with 0.25 mL cold PBS containing 2% FCS, placed on ice in the
dark and immediately analyzed by flow cytometry. Data was acquired with
Cell Quest on an LSRII flow cytometer (Beckton and Dickenson), using
anexcitationwavelength of 633nm and detectingfluorescenceemission at
730( 45 nm of 10,000 events and analyzed with FCS Express (Denovo
software). Histograms were generatedand data was expressed as the mean
fluorescence of triplicate samples after subtracting autofluorescence.
The photosensitizing activity was determined in Colon26 cells using
the colorimetric MTT assay. Colon26 cells were seeded in 96-well plates
at a density of 3 ꢁ 103 cells/well in RPMI with 10% fetal calf serum. After
overnight incubation, the PSs were added at the indicated concentra-
tions and incubated at 37 ꢀC for 24 h in the dark. Before light treatment,
the medium was replaced with drug-free complete medium. Cells were
then illuminated (0ꢀ4 J/cm2) with 665 nm for compounds 1, 4, and 7,
using 660 nm light for compound 12 and 674 nm for compound 23 from
an argon-pumped dye laser at a fluence rate of 3.2 mW/cm2. After PDT,
the cells were incubated for a further 48 h at 37 ꢀC in the dark. During the
last 4 h of incubation, 10 μL of a 4.0 mg/mL solution in PBS of 3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromi (MTT) (Sigma, St.
Louis, MO) was added to each well. After 4 h, the MTT and medium
were removed and 100 μL of DMSO was added to solubilize the
formazan crystals. Absorbances were read on a microtiter plate reader
1
λmax(CH2Cl2), nm (ε): 670, 613, 541, 513, 412. H NMR ((C4D8)O);
400 MHz): δ 9.23, 9.11/9.03, 8.70 (all s, 1H, meso-H), 7.48 (d, 1H, ArH,
J= 7.6 Hz), 7.39ꢀ7.34 (m, 2H, ArH), 7.32ꢀ7.23 (m, 1H, ArH), 6.03ꢀ5.98
(m, 1H, 31-H), 5.25 (s, 2H, 131-H), 4.60ꢀ4.72 (m, 2H, OCH2Ar),
4.42ꢀ4.45 and 4.17ꢀ4.18 (broad m, 2H, 7-H + 8-H), 3.68 (broad m,
2H, 172-CH2), 3.47 (s, 3H, 12-CH3), 3.40 (s, 3H, 2-CH3), 3.15 (s, 3H, 18-
CH3), 2.83 (t, 2H, 171-CH2, J = 8.0 Hz), 2.68 (broad m, 5H, 2H of 81-H +
3H of 31-CH3), 2.11/2.12 (distorted D, 3H, 7-CH3), 1.18(m, 3H, 81-CH3),
0.20 and 0.18 (splitted s, 9H, Sn(CH3)3). MS(ESI): m/z 807.3 (M + H+).
HRMS(ESI):calcdforC43H51N4O4Sn (MH), 807.2932; found, 807.2920.
General Method for the Preparation of 124I-Labeled
Photosensitizers 6, 26, and 27. The trimethyltin analogue (5,
25, or 24, respectively) (60 μg) dissolved in 5% acetic acid in methanol
(50 μL) was added to a tube containing dried Na124I (60 ng). To this
solution was added 10 μL of N-chlorosuccinimide (1 mg/mL in
methanol). The reaction mixture was incubated at room temperature
for 8 min. The reaction mixture was loaded onto a HPLC column
(symmetry C18 5 μm, 4.6 mm ꢁ 150 mm). A mixture of methanol/
water (88:12) was used as eluant. The flow rate was 1 mL/min. The UV
detector was set at 254 nm. The labeled product was collected, and the
solution was evaporated under nitrogen gas flow at 80 ꢀC. The product
was dissolved in 10% ethanol/saline solution for in vivo experiments.
The radiochemical yield was 30%, and the specific activity was greater
than 1 Ci/μmol. The purity was ascertained by HPLC.
HPLC Analyses of the Final Products. HPLC analyses of com-
pounds 4, 12, and 23 were performed using a Waters Delta 600 System
consisting of the 600 Controller, 600 Fluid Handling Unit, and 996
photodiode array detector equipped with a EMD LichroCart RP8, reverse
phase C8 column, 5 μm particle size with dimensions of 4 mm ꢁ 250 mm .
Compounds were eluted at a flow rate of 1.5 mL/min using a gradient
program: solvent A, methanol; solvent B, water; gradient profile: 0 min, 75%
A/25% B; linear grade to 90% A/10% B from 0 to 10 min; maintained at this
composition until 20 min; then linear grade to 100% A. The retention time
and percent purity of each compound were as follows: 4, 10.13 min; 12,
10.48 min; and 23, 9.94 min. The component percentages are based on the
area counts of the peaks from the 408 nm channel.
Cell Cultures. A subclonal line of human basal cell carcinoma cells
(Dicker et al., 2002, J. Invest Dermatol 118: 859-865) (provided by Dr.
Tak-Wah Wang) were cultured in DMEM containing 10% fetal calf
serum. PS uptake was determined in subconfluent cultures established in
24- or 6-well culture plates. The cells were washed twice with serum-free
DMEM, and cells and then incubated in DMEM containing the
concentration of fetal calf serum and photosensitizers as indicated in
the specific experiments. After an incubation period ranging from 30 to
24 h, the cells were washed with serum-free DMEM. Cell-associated PS
was visualized by 640ꢁ fluorescent microscopy on an inverted fluores-
cence microscope (Zeiss Axiovert 200) with an Axio camera. The filters
for HPPH fluorescence were Ex 410/40 nm and Em 675/750 nm.
Internalization of PS was achieved by incubating PS-treated cells in
serum-free or serum-containing medium for 4ꢀ24 h at 37ꢀ. PS amounts
in cells were quantified by extraction in Solvable (PerkinElmer Life and
Analytical Sciences) and measurement of fluorescence (Ex 415 nm; Em
667 nm) in a Varian Cary Eclipse fluorescence spectrophotometer
(Agilent Technologies, Santa Clara, CA).
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(BioTek Instruments, Inc., ELx800 Absorbance Microplate Reader) at
570 nm. The results were plotted as the survival rate of treated cells
against different light doses for each compound tested. Each experiment
was done with four replicate wells and repeated three times indepen-
dently to obtain an accurate photosensitizing efficacy.
The 27 resulting TIFF images were loaded into a single data structure in
memory, forming a spectral stack with a spectrum at every pixel.
Autofluorescence spectra and PS fluorescence spectra were manually
selected from the spectral image using the computer mouse to select
appropriate regions. Spectral unmixing algorithms (available from CRI,
Inc.) were applied to create the unmixed images of pure autofluores-
cence and pure PS fluorescence signals, as shown in Figure 11 and 12. All
the images were reanalyzed using Image J software to show false color
fluorescence reflectance (FRI) images.
Following an IACUC-approved protocol, the BALB/c mice were
subcutaneously injected with 1 ꢁ 106 Colon26 cells in 50 μL of serum
free complete RPMI-1640 media (into the right posterior axilla), and
tumors were grown until they reached 4ꢀ5 mm in diameter. The day
before PS injection, all hairs were removed from the inoculation site and
the mice were imaged under anesthesia (IP injection of ketamine and
xylazine mixture). In a dark box, illumination was provided by fiber optic
lighting at 540/40ꢁ nm (green color), and a long-pass filter was used to
reject scattering and to pass Stoke-shifted PS fluorescence at 667 nm and
676 nm, respectively. Under similar conditions, mice were imaged at 24,
48, and 72 h postinjection of the PSs 4, 12, and 23 (at therapeutic dose
1 μmol/kg).
Animal Models. The photosensitizers were evaluated in mice
(BALB/c, 10 mice/group) bearing Colon26 tumors, implanted sub-
cutaneously, and were allowed to grow to an approximate diameter of
5ꢀ7 mm. All animal procedures were carried out in accordance with
guidelines approved by the Institute Animal Use and Care Committee.
PET Imaging. Mice were imaged in the micropet FOCUS 120, a
dedicated 3D small-animal PET scanner (Concorde Microsystems Inc.)
at State University of New York at Buffalo (South Campus) under the
Institutional Animal Care and Use Committee (IACUC) guidelines. All
BALB/c mice (n = 10 for each compound) tumored with Colon26 cells
were injected via the tail vein with 52ꢀ122 μCi of PS 1, 6, and 27,
respectively. After 24, 48, and 72 h postinjection, the mice were
anesthetized by inhalation of isoflurane/oxygen and placed head first,
prone for imaging, and the acquisition time was set for 30 min. No efforts
were made to block the radioiodine uptake by thyroid or stomach, and
surprisingly, in only a few cases was a little bit of radioiodine uptake
observed in the thyroid, and that too was cleared by 72 h. Some tumored
mice were also imaged using 18F-FDG radiotracer 24 h prior to radio-
active 124I imaging. These mice were administered 150ꢀ200 μCi of 18F-
FDG via the tail vein and were scanned for 20 min in the fashion
described above, beginning 45 min after FDG injection. For biodistribu-
tion studies, the mice were injected with 15ꢀ50 μCi of compounds PS 1,
6, and 27, respectively, via the tail vein, and three mice each at 24, 48, and
72 h time interval were sacrificed, and body organs (tumor, heart, liver,
spleen, kidney, lung, muscle, etc.) were removed immediately. After
weighing, the amount of radioactivity in the tumor (300ꢀ400 mg), body
organs, and blood was measured by a γ-well counter. Radioactivity
uptake was calculated as the percentage of the injected dose per gram of
the tissue (%ID/g). Statistical analyses and data (%ID/g vs time point)
were plotted using GraphPad Prism 5.
After whole-body imaging, the tumor was surgically removed from
the original site (axilla) and placed on a shaved region of skin away from
the original site as a means to image the skin flap (minus the tumor).
Imaging was repeated as described before.
’ ASSOCIATED CONTENT
Supporting Information. 1H and 13C NMR spectra of all
S
b
compounds; HPLC profiles of compounds 4, 7, 12, and 23;
experimental details of known compounds; and spectroscopic
data of compounds 10, 18, 21, and 22. This material is available
free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
In Vivo PDT Efficacy and the Assessment of Response.
Before PDT treatment, Nair was used to remove all hair surrounding the
tumor site. When the tumor reached 4ꢀ5 mm in diameter, the mice
were injected with compounds 1, 4, 7, 12, and 23 at a dose of 1 μmol/kg.
At 24 h postinjection, the mice were restrained in plastic Plexiglass
holders without anesthesia and treated with laser light (665 nm for
compounds 1, 4, and 7; 660 nm for compound 12, and 674 for
compound 23, 128 J/cm2, 14 mW/cm2). The power was monitored
during the entire treatment. Post-PDT, the mice were observed daily for
tumor regrowth or tumor cure. Visible tumors were measured using two
orthogonal measurements L and W (perpendicular to L), and the
volumes were calculated using the Microsoft Excel formula V = LW2/2
and recorded. Mice were considered cured if there was no palpable
tumor by day 60ꢀ90. Once the tumor reached 400 mm3, the mice were
euthanized due to tumor burden as per RPCI IACUC rules.
Corresponding Author
*R.K.P.: telephone, 716-845-3203; fax, 716-845-8920; e-mail,
ravindra.pandey@roswellpark.org. H.B.: telephone, 716-845- 4587;
fax, 716-845-5908; e-mail, heinz.baumann@roswellpark.org.
’ ACKNOWLEDGMENT
The help rendered by Paula Pera in animal studies is highly
appreciated. The authors are thankful to NIH RO1CA127369,
PO1 CA55791 (partial), and the shared resources of the Roswell
Park Cancer Center support grant CA16056 for the financial
assistance.
’ ABBREVIATIONS USED
In Vivo Optical Fluorescence Imaging. In vivo fluorescence
imaging was accomplished by using a Illumination Dual Light System
(Lightools Research, Encinitas, CA), designed for small animal studies.
True-Color fluorescence images were obtained using dielectric long-
pass filters (Chroma Tech) and a digital color camera (Optronics,
Magnafire SP, Olympus America). Wavelength-resolved spectral ima-
ging was carried out by using a Nuance multispectral imaging system
(CRI, Inc., Woburn, MA) comprising an optical head that includes
Varispec liquid crystal tunable filters (LCTFs, with a bandwidth of
20 nm and a scanning wavelength range of 400ꢀ720 nm), an optical
coupler, and a high-resolution scientific-grade CCD camera, along with
image acquisition and analysis software. The tunable filter was auto-
matically stepped in 10-nm increments from 550 to 720 nm while the
camera captured images at each wavelength with a constant exposure.
PS, photosensitizer; PDT, photodynamic therapy; PET, positron
emission tomography; STAT3, signal transducer and activation
of transcription 3; ROI, region of interest; NMR, nuclear mag-
netic resonance; HRMS, high resolution mass spectrometry;
FDG, fluorodeoxyglucose; SAR, structureꢀactivity relationship
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