Conjugation of 2-(1′-hexyloxyethyl)-2-devinylpyropheophorbide-a (HPPH) to carbohydrates changes its subcellular distribution and enhances photodynamic activity in vivo

Article abstract of DOI:10.1021/jm9001617

The carbohydrate moieties on conjugating with 3-(1′-hexyloxyethyl)-3- devinyl pyropeophorbide-α (HPPH) altered the uptake and intracellular localization from mitochondria to lysosomes. In vitro, HPPH-Gal 9 PDT showed increased PDT efficacy over HPPH-PDT as detectable by the oxidative cross-linking of nonphosphorylated STAT3 and cell killing in ABCG2-expressing RIF cells but not in ABCG2-negative Colon26 cells. This increased efficacy in RIF cells could at least partially be attributed to increased cellular accumulation of 9, suggesting a role of the ABCG2 transporter for which HPPH is a substrate. While such differences in the accumulation in HPPH derivatives by tumor tissue in vivo were not detectable, 9 still showed an elevated light dose-dependent activity compared to HPPH in mice bearing RIF as well as Colon26 tumors. Further optimization of the carbohydrate conjugates at variable treatment parameters in vivo is currently underway.

Full text of DOI:10.1021/jm9001617

4306 J. Med. Chem. 2009, 52, 4306–4318
DOI: 10.1021/jm9001617
Conjugation of 2-(10-Hexyloxyethyl)-2-devinylpyropheophorbide-a (HPPH) to Carbohydrates
Changes its Subcellular Distribution and Enhances Photodynamic Activity in Vivo†
Xiang Zheng,‡ Janet Morgan,§ Suresh K. Pandey,‡ Yihui Chen,‡ Erin Tracy, Heinz Baumann,*, Joseph R. Missert,‡
Carrie Batt,‡ Jennifer Jackson,§ David A. Bellnier,‡ Barbara W. Henderson,‡ and Ravindra K. Pandey*,‡
‡PDT Center, Department of Cell Stress Biology and §Department of Dermatology, and Department of Molecular and Cellular
Biology, Roswell Park Cancer Institute, Buffalo, New York 14263
Received February 7, 2009
The carbohydrate moieties on conjugating with 3-(10-hexyloxyethyl)-3-devinyl pyropeophorbide-a
(HPPH) altered the uptake and intracellular localization from mitochondria to lysosomes. In vitro,
HPPH-Gal 9 PDT showed increased PDT efficacy over HPPH-PDT as detectable by the oxidative
cross-linking of nonphosphorylated STAT3 and cell killing in ABCG2-expressing RIF cells but not in
ABCG2-negative Colon26 cells. This increased efficacy in RIF cells could at least partially be attributed
to increased cellular accumulation of 9, suggesting a role of the ABCG2 transporter for which HPPH is a
substrate. While such differences in the accumulation in HPPH derivatives by tumor tissue in vivo were
not detectable, 9 still showed an elevated light dose-dependent activity compared to HPPH in mice
bearing RIF as well as Colon26 tumors. Further optimization of the carbohydrate conjugates at variable
treatment parameters in vivo is currently underway.
Introduction
characteristics are promising for the improvement of clinical
applications, enhanced selectivity toward tumors is still a
desirable goal.
Photodynamic therapy (PDTa) is a noninvasive antitumor
treatment that requires the combination of photosensitizer
(PS), tissue oxygen, and light to produce cytotoxic reactive
oxygen species, predominantly singlet oxygen. PDT has po-
tential advantages over surgery and radiotherapy due to its
tissue-sparing properties.1 Various PS have different pharma-
cokinetics, but effective treatment relies in part on greater
accumulation of the sensitizer in the tumor extracellular
matrix or tumor cells, via undefined binding, relative to
normal tissue. Porfimer sodium is the only PS that has
received worldwide regulatory approval for the treatment of
cancer in humans.2 Porfimer sodium has important limita-
tions in that it (i) consists of a complex mixture of monomers,
dimers, and oligomers, (ii) has a short-wavelength absorption
(λmax = 630 nm, ε = 5000) that can restrict applications that
require deep penetration of excitation light, and (iii) can cause
prolonged (4-6 weeks) skin photosensitivity due to its phar-
macokinetic and tissue-retention properties. These shortcom-
ings have spurred the development of a plethora of second-
and third-generation PS. Of these, chlorin- and bacteriochlor-
in-based PS are monomeric compounds, efficient generators
of singlet oxygen (Φ = 45-60%), and may be particularly
effective in treating large and/or deeply seated tumors due to
their long wavelength (650-800 nm) absorption.3 While these
Our laboratory has extensively applied synthetic ap-
proaches for developing PS with improved photosensitizing
efficacy. One avenue has been a classical QSAR approach in
which the overall lipophilicity of the molecule is altered by
varying the length of the aliphatic chain or by replacing the
substituents at the peripheral positions of the tetrapyrrole
system.4 A more challenging approach is to design PS con-
jugates that specifically target cell-surface proteins or recep-
tors overexpressed by tumors.5 In recent years,6 a variety
of porphyrins and nonporphyrin compounds have been
conjugated with carbohydrates to serve as substrates for
cellular lectins (e.g., galectins),7 known for their higher ex-
pression in many tumor cells. Such conjugates have shown
some potential in tumor imaging and PDT.8 Encouraged by
these reports and results from our group,9,10 we conjugated
PS with carbohydrates known for their high affinity to
galectin-3. We selected the rigidified multivalent lactose deri-
vatives reported by Vrasidas et al.11 that display significantly
different binding to galectin-1 and galectin-3. For example,
derivatives with rigidified multivalent lactose bound with
4300-fold higher affinity to galectin-3 than to galectin-1.
Therefore, to determine the effect of multivalent lactose in
adding tumor-specificity, we have linked such carbohydrate
moieties to our PS12 with the expectation of enhancing
specificity for galectin-3-expressing tumors.
† Dedicated to (late) Dr. Allan R. Oseroff, Chief, Department of
Dermatology, RPCI, Buffalo, New York.
*To whom correspondence should be addressed. For H.B.: Phone:
716-845-4587. Fax: 716-845-5908. E-mail:Heinz.baumann@roswell-
park.org. For R.K.P.: Phone: 716-845-3203. Fax: 716-845-8920. E-mail:
ravindra.pandey@roswellpark.org.
a Abbreviations: PDT, photodynamic therapy; QSAR, quantitative
structure-activity relationship; PS, photosensitizer; ABCG2, ATP-
binding cassette, subfamily G, member 2; STAT3, signal transducers
and activator of transcription proteins.
A second aspect that could potentially influence and even
limit the antitumor efficacy of porphyrin PS is the presence
or absence in the tumor tissue of ATP-dependent transporters,
such as ABCG2, also known as BCRP (breast cancer resis-
tance protein).12 Such transporters have been recognized to
export a diverse array of compounds, including certain PS.13-15
r
pubs.acs.org/jmc
Published on Web 06/09/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4307
Scheme 1. Synthesis of Carbohydrate Scheme 4
Figure 1. IC50 (μM) of PS in RIF cells (derived from Figure 3A) as a
function of log P (determined using computer software PALLAS):
9 (HPPH-Gal), 10 (HPPH-Glu), 11 (HPPH-Lac), 12 (HPPH-Cell),
21 (HPPH-Monolac). The log P values for compounds 22 (HPPH-
Dilac) and 23 (HPPH-Tetralac) could not be directly calculated due
to software limitations (see text).
However, by conjugation with carbohydrates, porphyrins
appear to lose their substrate property for ABCG2 and, hence,
may be retained more effectively by cells thereby contributing
to improved PDT.16
The present manuscript describes a detailed investigation
showing the synthesis and in vitro and in vivo photodynamic
effects of a series of carbohydrate conjugates of 2-(10-hexylox-
yethyl) pyropheophorbide-a (HPPH), by itself an effective
photosensitizer currently under phase I/II clinical trials at
Roswell Park Cancer Institute, Buffalo NY.17-20 We discov-
ered that carbohydrate modifications altered the cell biology
of PS uptake and PDT action, and that these changes
appeared to be independent of galectin-3 expression by the
target cells.
Reagents: (a) HBr/AcOH; (b) NaN3, Bu4NHSO4; (c) H2, Pd/C.
reagent produced the corresponding intermediates 5-8
which under standard deprotection conditions (NaOMe in
dichloromethane/methanol) afforded the final conjugates
9-12 (Scheme 2).
For the preparation of the mono- and multivalent rigidi-
fied lactose conjugates, the acetyl protected lactose with a
rigidified linker was synthesized first by following known
methodology.11 In brief, intermediate 13, which upon stir-
ring with TFA yielded 14, which was then coupled with the
rigidified lactose moieties (15-17) using (benzotriazol-1-
yloxy)tris(dimethylamino) phosphonium hexafluoro-phos-
phate (BOP) as a coupling reagent to produce the intermedi-
ates 18-20, which on subsequent deacetylation afforded the
conjugates 21-23, respectively (see Scheme 3). The electro-
nic absorption spectra of all the conjugates were similar to
that of HPPH.
Rationale for the Synthesis of Carbohydrate Conjugates
with Similar Lipophilicity. The HPPH-carbohydrate con-
jugates were designed to evaluate the importance of the
carbohydrate moieties to enhance target-specificity. For
example, in conjugates 9 and 10, HPPH was linked to
galactose and glucose moieties, respectively. Both com-
pounds exhibit similar lipophilicity (Figure 1) but differ in
the orientation of the hydroxy-group at position-4 of the
hexose that determines the binding to lectins. If lipophilicity
is the predominant criterion that defines cellular uptake and
action, then both conjugates might be expected to produce
similar PDT efficacy. Similarly, the conjugates 11 and 12,
containing the lactose (galactose-glucose) and cellobiose
(glucose-glucose), respectively, were prepared. These dis-
accharide conjugates were used to determine the effects of
the orientation of the 4-OH group when presented in the
context of a glycosidically linked galactose in a disaccharide
and also as a part of a compound with reduced overall
Results and Discussion
Synthesis of the HPPH-Carbohydrate Conjugates. To
investigate the impact of β-galactose carbohydrates on tu-
mor-selectivity and photosensitizing efficacy, galactose and
lactose (Galβ1f4Glc) moieties were individually conju-
gated with 3-devinyl-3-(10-hexyloxy)ethyl-pyropheophor-
bide-a (HPPH). The related non-β-galactose carbohydrates,
glucose and cellobiose (Glcβ1f4Glc), were similarly con-
jugated to HPPH in order to obtain reagents to assess
the relative contribution of galectin binding to uptake and
action. The pairs of HPPH conjugates with either mono- or
disaccharides possess similar overall lipophilicity (Figure 1).
To determine the impact of linker(s) with multivalent carbo-
hydrate units in the conjugates, certain rigidified multivalent
lactose molecules were also synthesized. For the preparation
of HPPH-carbohydrate conjugates without a rigid linker,
the acetoxy- group at position-1 (anomeric carbon atom)
of the commercially available acetyl protected carbohydrate
was converted into the corresponding 1-bromo analogue,
which was then converted into the azido derivative by
following known methodology.21 Hydrogenation of the
azido analogues in the presence of Pd/C at room tempera-
ture produced the required 1-aminotetraacetogalactose
(1), 1-aminotetraacetoglucose (2), 1-aminoheptaacetolac-
tose (3), and 1-aminoheptaacetocellobiose (4) in good yields.
A typical example is illustrated in Scheme 1.
Reaction of carbohydrates 1-4 with HPPH using 1-ethyl-3-
(30-dimethylaminopropyl) carbodiimide (EDCI) as a coupling

4308 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Scheme 2. Synthesis of HPPH-Carbohydrate Conjugatesa
Zheng et al.
a Reagents: (a) EDCI, DMAP; (b) NaOMe/MeOH.
Scheme 3. HPPH Linked with Various Carbohydrates with Rigid Linkersa
a Reagents and conditions: (a) N-Boc-ethylenediamine, BOP, Et3N; (b) TFA; (c) BOP, Et3N; (d) NaOMe/MeOH.
lipophilicity when compared to the mono- (9 and 10) and
disaccharide-HPPH conjugates (11 and 12).
In general, lipophilicity has proven to be an important
factor in directing the pharmacokinetic and pharmacody-
namic properties of many PS because it influences the
biodistribution and clearance and thus the bioactivity of
drugs.4 Lipophilicity is indicated by the logarithm of the par-
tition coefficient, log P, which reflects the equilibrium parti-
tioning of a molecule between a nonpolar and a polar phase,
such as n-octanol and water.22 Recently, several theoretical

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4309
In contrast, Colon26 cells, analyzed for their response to
HPPH, the corresponding galactose analogue 9 and glucose
derivative 10 only showed equal photosensitivity to all three
agents when exposed to them for 24 h. When exposure
conditions were reduced to 4 h, HPPH, 9, and 10 showed
similar responses to the 24 h incubation in RIF cells.
In our earlier QSAR study in a congeneric series of HPPH
analogues without carbohydrate modifications, we showed
the importance of lipophilicity for in vitro and in vivo
PS efficacy, with HPPH possessing optimal values with a
log P of ∼6.4 Although HPPH and its carbohydrate con-
jugates are not strictly a congeneric series, a number of
observations can be made with regard to lipophilicity
(Figure 1). HPPH, as well as compounds 9 and 10, which
differ by less than one log value in lipophilicity, displayed
equivalent activity in Colon26 cells after 24 h of drug uptake.
In RIF cells, compounds 9 and 10 showed similar activity to
that seen in Colon26 cells; however, relatively lower activity
was detected for HPPH in RIF cells, suggesting that either
cellular uptake and accumulation, or the photoreaction in
these cells, are altered. Altered cellular accumulation, in part,
could be due to the action of the ABCG2 pump (Figure 2) for
which HPPH is a substrate.16
The lower activity of the compounds 11 and 12, and in
particular of the compounds 21, 22, and 23, is not clearly
attributable to lipophilicity alone and may be influenced by
the presence of a larger carbohydrate moiety on the mole-
cules and the rigidity of the linkage, either of which could
potentially affect the mode by which the compounds are
taken up and intracellularly distributed
In Vitro Uptake of PS. To assess whether carbohydrate-
dependent changes in phototoxicity were due to altered PS
levels in cells at the time of light treatment, PS uptake was
determined. Initial measurements by fluorescence spectros-
copy of the PS uptake in RIF cells indicated increased
accumulation over 3 or 24 h of the mono- and disaccharide
analogues (9-12) over HPPH. However, the conjugates with
an increasing number of lactose moieties with an extended
linker showed uptake similar to HPPH for 21 and minimal
uptake for 22 and 23. The failure to be bound and/or be
internalized explains the inactivity for compounds 22 and 23
in the cytotoxity assay (Figure 3A). The same distributions,
albeit with slightly lower levels, were observed after 3 h PS
exposure. To compare the PS uptake in RIF and Colon26
cells and to avoid potential pitfalls of fluorescence measure-
ments such as PS aggregation etc., 14C-labeled HPPH and
HPPH-Gal (9), as the representative carbohydrate conju-
gate, were prepared by following the methodology shown in
Scheme 2 in which 14C was introduced into HPPH by
reacting pyropheophorbide with 14C-labeled hexanol23 (for
synthetic details see Experimental Section). It is apparent
from Figures 4B,C that accumulation of 9 in RIF cells
exceeded that of HPPH at both exposure periods, while
there was no significant difference in accumulation between
the two PS in Colon26 cells. These observations suggest a
role for the ABCG2 transporter in determining intracellular
level of HPPH in RIF cells.
Figure 2. Expression of galectin-1, galectin-3, and ABCG2 in RIF
and Colon26 cells in vitro. Equal amounts of cell extracts were
analyzed by Western blotting for the indicated proteins.
methods have been developed to predict lipophilicity. We
applied a program module of PALLAS to determine the
lipophilicity of the compounds synthesized, and the results
are shown in Figure 1. The log P values of conjugates 22 and
23 could not be calculated because their molecular weight
exceeded the program’s limitation but must be below that of
conjugates 21 because (i) of the additive-constitutive nature
of substituted functional groups and (ii) the very strongly
negative estimated (Pallas) log P of moieties 15 (log P ∼ -3)
and 16 (log P ∼ -5).
In Vitro Photosensitizing Activity. HPPH and the corre-
sponding carbohydrate conjugates were evaluated for in
vitro photosensitizing efficacy in the murine radiation-
induced fibrosarcoma (RIF) and colon carcinoma (Colon26)
cell lines. These cell lines express closely similar levels of the
β-galactose-recognizing proteins galectin-1 and galectin-3
(Figure 2). They differ, however, in immune-detectable levels
of the ATP-dependent transporter ABCG2 that is able to
mediate cellular export of HPPH,16 with RIF cells expressing
high levels while Colon26 cells are essentially devoid of
ABCG2 expression.
Cells were incubated with increasing concentrations of
the PS for 24 h, followed by exposure to 665 nm light and
MTT assays performed 48 h later. Neither RIF nor Colon26
cells showed any significant dark cytotoxicity up to 1 μM PS
(Figure 3A,B, upper panels). RIF cells showed drug- and
concentration-dependent phototoxicity (Figure 3A, lower
panel). All mono- and disaccharide conjugates (9, 10, 11, 12)
produced higher PDT efficacy than HPPH. Among the
conjugates, those with galactose (9) and glucose (10) were
approximately twice as effective as those with lactose (11)
and cellobiose (12). Compared to the lactose analogue (11),
which was linked to HPPH with an amide bond, the lactose
derivative with a rigid linker (21) was several-fold less
effective and was comparable to HPPH. The conjugates
with the same rigid linker but with an increasing number
of saccharide moieties, e.g., the tetramer (22) and the octa-
mer (23), were ineffective. No appreciable differences
were observed between the conjugates, which could be
attributed to the differing orientation of the 4-OH group in
the hexose (galactose and glucose), thus ruling out a measur-
able contribution of galectin-3 to the PS efficacy of the
galactose conjugates.
We have shown earlier that HPPH is a substrate for the
ABCG2 transporter.16 Retention of HPPH by RIF cells
is improved by treatment of the cells with the tyrosine
kinase inhibitor imatinib mesylate that inhibits, among
others, the ATP-dependent transport function of ABCG2
and thus HPPH efflux. We have further reported that 9 does
not appear to be a substrate for ABCG2. Therefore, 9

4310 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Zheng et al.
Figure 3. In vitro photoreaction with HPPH, 9 (HPPH-Gal), 10 (HPPH-Glu), 11 (HPPH-Lac), 12 (HPPH-Cel), 21 (HPPH-Monolac),
22 (HPPH-Dilac), and 23 (HPPH-Tetralac) in RIF (A) and Colon26 cells (B). Cells were incubated with the indicated PS in 10% serum-
containing medium for 24 h and then exposed to light (665 nm at 3.2 mW/cm2) for a total fluence of 0.5 J/cm2 (lower panels). Cells exposed to
the PS without light are shown in the upper panels. After treatment, the cells were incubated in growth medium for 48 h. Surviving cell
population was measured by MTT assay. The data are expressed as mean of three experiments. Standard deviations are <6.1% and for most
values are not indicated for clarity.
Figure 4. In vitro uptake of HPPH and HPPH carbohydrate conjugates. (A) Replicate cultures were incubated with 0.25 μM PS in
medium containing 10% fetal calf serum for varying time periods at 37 °C; PS = HPPH, 9 (HPPH-Gal), 10 (HPPH-Glu), 11 (HPPH-Lac),
12 (HPPH-Cel), 21 (HPPH-Monolac), 22 (HPPH-Dilac), and 23 (HPPH-Tetralac). PS content was determined by fluorescence spectroscopy
and expressed as fluorescent units per mg protein; each determination was done in triplicate. (B,C) Uptake of [14C]-HPPH and [14C]-HPPH-Gal
by RIF and Colon26 cells; Replicate cultures were incubated in medium containing 10% fetal calf serum and 0.8 μM 14C-labeled compounds at
37 °C. At the times indicated, triplicate cultures were washed, counted, and the cell-associated radioactivity determined. The results are
expressed as mean and standard deviation.
accumulation is not measurably affected by imatinib mesy-
late in RIF cells.16 Neither HPPH nor 9 accumulation are
affected by imatinib mesylate in ABCG2 nonexpressing
Colon26 cells (data not shown).
(11) deviated somewhat from the above pattern in that also a
fraction overlapped with the Golgi marker. The low level
accumulation of the tetra- and octasaccharide conjugates
appeared to have broad localization with fluorescence
detectable in the three subcellular compartments tested:
mitochondria, lysosomes, and golgi (summarized in Table 3,
Supporting Information). A similar localization was found
for each PS when monitored after 3 and 24 h of incubation.
In the case of carbohydrate conjugates, an initial binding to
the cell surface followed by internalization and progressive
lysosomal accumulation was evident. Lysosomal localiza-
tion became most pronounced after 24 h of incubation,
probably in part due to sequestration into the organelle.
The change from mitochondria to lysosomes suggests that
Subcellular Localization. Previous studies with various
types of PS, including the alkyl ether analogues of pyro-
pheophorbide-a, showed that the most effective forms loca-
lized to mitochondria.24,25 Fluorescence microscopy
confirmed the predominant mitochondrial location of
HPPH (Figure 5). In contrast, the subcellular localization
of the representative galactose-conjugated HPPH in RIF
cells differed from HPPH by being predominantly lysosomal
(Figure 5). Similarly, all other mono- and disaccharide
conjugates were located in lysosomes. The lactose conjugate

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4311
Figure 6. Covalent cross-linking of STAT-3 as a function of PS
action. RIF and Colon26 cells were incubated with the indicated
concentrations of HPPH or HPPH conjugates in medium contain-
ing 10% FCS. After 4 h of drug incubation, the cells were exposed to
665 nm light at 3 J/cm2 and immediately extracted. STAT-3 cross-
linking was identified by Western blotting. The relative level of
cross-linking was determined by densitometry and the values in-
dicated at the top of the immunoblot images.
STAT-3 cross-linking itself is not causative in cell death. The
STAT-3 cross-linking reactions in RIF and Colon26 cells
(Figure 6) reported a PS type and dose dependence that
mirrored the relative activities of the PS to cause cell killing
(Figure 3), namely a several-fold difference between HPPH
and both galactose and glucose-conjugated HPPH, a differ-
ence that was characteristic for RIF cells but not for Colon26
cells. Furthermore, the results correspond well with the PS
uptake data shown in Figure 4, with RIF cells accumulating
significantly greater amounts of 9 and 10 than HPPH, while
Colon26 cells contained nearly equal levels of HPPH and 9.
Also, RIF cells appear less sensitive to HPPH than Colon26
cells despite nearly equal HPPH content (Figure 4). These
results indicate that not only the relative amount of total PS
taken up by cells but also the cell type and local production of
singlet oxygen determines the level of oxidative reactions
within the PS-harboring organelles as well as the cytoplasmic
compartment in which subsequent propagation of redox
reactions occur.
In Vivo Biodistribution of HPPH and HPPH-Galactose.
The PS efficacy of HPPH conjugates was evaluated in vivo
using tumor bearing mice. The biodistribution of the PS was
determined with the same preparations of [14C]-HPPH and
[14C]-HPPH-galactose derivative 9 as applied in vitro (Fig-
ure 4). The relative uptake of the PS by the different organs in
C3H mice bearing RIF tumor and BALB/c mice bearing
Colon26 tumor were comparable (Figure 7). The uptake of
PS by the tumors was in the range of major organs with a
tendency for tumor values to be ∼2-fold higher than skin.
HPPH values in tumors tended slightly higher than 9 with
appreciable retention of both PS lasting at least 48 h. The
notable exception in the biodistribution was the several-fold
higher level for 9 in the liver. The prominent liver localization
of 9 was expected and might be attributed to clearance
through the galactose receptor system on hepatic parenchy-
mal cells.
Figure 5. Subcellular localization of HPPH and 9 (HPPH-Gal)
in RIF cells. The cells were incubated for 3 h in medium containing
10% FCS and 2 μM PS (red) and the indicated trackers (green) for
mitochondria, lysosomes, or golgi. Images were merged to indicate
the overlap in fluorescence. (See Table 1 and Figure 3 of Supporting
Information) for a summary of localization of other HPPH-
carbohydrate conjugates.)
with the carbohydrate conjugation, HPPH has been sub-
jected to a different mode of uptake and intracellular
distribution. The predominance of the compounds in lyso-
somes suggests the involvement of endocytosis, i.e., through
phagocytosis/pinocytosis. However, neither the β-galactose
conjugates (9, 11) nor the related non-β-galactose carbohy-
drates (10, 12) showed specificity for galectin-3 that could
be inhibited by inclusion of even >100-fold excess of
corresponding free carbohydrates during in vitro or in vivo
evaluation of the conjugates (data not shown).
From this we conclude that the photosensitizing action of
HPPH and its carbohydrate conjugates is appreciably deter-
mined by the subcellular location. This suggests that the
altered subcellular localization, i.e., to the lysosome, triggers
mechanisms that differ from those of the predominantly
mitochondrially localizing HPPH and that the effectiveness
of these mechanisms shows considerable cell type specificity.
STAT-3 Dimerization in RIF and Colon26 Cells. An im-
mediate consequence of the PDT reaction is the generation
of singlet oxygen, which, through propagation of redox
reactions, results in the oxidation of intracellular proteins.26
One of the specific targets of the photoreaction is the
covalent cross-linking of the latent signal-transducing pro-
tein STAT-3 in the cytoplasm.27
In Vivo Photosensitizing Activity of HPPH and HPPH
Conjugates. The in vivo photosensitizing efficacy of HP-
PH and selected HPPH-carbohydrate conjugates was
The degree of STAT-3 cross-linking is a direct measure of
the PS-dependent reaction and in part correlates with the
level of stress reactions leading to cell killing. Of note is that

4312 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Zheng et al.
Figure 7. The in vivo biodistribution of 14C-labeled HPPH and 9 (HPPH-Gal) in C3H and Balb/c mice: the 14C-labeled photosensitizers
(0.2 mL/5 μCi) were administered to 6 mice/group (Balb/c mice bearing Colon26 tumors and C3H mice bearing RIF tumors). At 24 and
48 h after injection, three mice/time point were sacrificed. The organs of interest (skin, muscle, tumor, heart, lung, liver, kidney, and spleen)
were removed and the radioactivity was measured (see the text). The raw data were then converted to counts/g tissue weight and plotted using
SigmaPlot 10.0 software.
determined in RIF and Colon26 tumors. To maximize the
detection of potential PDT improvement with carbohydrate
conjugates, a low fluence regimen of 48 J/cm2 was first
chosen that produces no tumor cures with an HPPH dose
of 0.47 μmol/kg (Figure 8A,B). In contrast to HPPH, 9,
under the same conditions, achieved long-term tumor con-
trol of ∼40% of RIF and 30% of Colon26 tumors. To
validate the in vitro results for inactive carbohydrate con-
jugates, the dilactose conjugate (22) was tested in RIF
tumors and also was found to be inactive in vivo. With
increased fluence (135 J/cm2), the efficacy of the active PS
tested in RIF tumors improved such that HPPH yielded
approximately 50% cures and cure rates with the galactose
derivative 9 were raised to ∼90% (data not shown).
The similarity of cure data in Figure 8 and tumor drug
distribution data in Figure 7 between the ABCG2 positive
RIF and ABCG2 negative Colon26 tumor lines suggests
that in vivo drug delivery to the tumor cells and tumor
response may be governed primarily by conditions in the
tumor microenvironment such as coresident stromal cells
and the prevalent vascular leakiness rather than tumor
cell-specific uptake mechanisms. Nevertheless, the func-
tional properties identified for HPPH and HPPH conju-
gates in RIF and Colon26 cells also are likely to apply
to stromal and endothelial cell types that contribute to the
tumor tissue.
Figure 8. In vivo photosensitizing efficacy of HPPH and 9 (HPPH-
Skin Phototoxicity of HPPH and the Corresponding Ga-
lactose Derivative (9). One of the problems with most por-
Gal) (drug dose: 0.47 μmol/kg; fluence: 48 J/cm2) in C3H (RIF
tumors) and Balb/c (Colon26 tumors) mice (10 mice/group,
phyrin-based compounds is long-lasting skin phototoxicity.
Therefore, the skin phototoxicity of HPPH-Gal was com-
pared with HPPH in C3H mice bearing RIF tumors at the
therapeutic doses of PS. At various times after PS adminis-
tration, mice were restrained without anesthesia in Plexiglas
holders designed to allow one hind leg be exposed to
therapeutic light. The contralateral hind leg served as a
control.28 The results are summarized in Figure 9. While
normal skin phototoxicity subsided within 4 days with both
light delivered 24 h post PS injection); 22 (HPPH-Dilac).
PS, as indicated by a lack of response when feet were exposed
to light at 96 h after drug injection, the initial erythema
responses were stronger with HPPH than 9. In part, the skin
reaction was in agreement with the detectable presence of
HPPH and conjugates in skin tissue (Figure 7). In no case did
the response exceed pronounced erythema and/or progress
to exudation and/or necrosis.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4313
Synthesis of 1-r-Bromo-cellobiose Pentaacetate (4b). Cello-
biose octaacetate (1 g, 1.47 mmol) was treated with HBr/AcOH
(5 mL) under ice-cold conditions for 4 h. The reaction mixture
was poured into aqueous saturated sodium bicarbonate solu-
tion, and the precipitate so obtained was filtered and washed
with cold water (50 mL) to yield 997 mg (97%) of the title
product 4b and was used as such in the next step without any
further purification; mp 185-187 °C, reported 184 °C.29
Synthesis of 1-β-Azido-cellobiose Heptaacetate (4c). To a
suspension of the 1-R-bromo cellobiose heptaacetate 4b (997
mg), sodium azide (465 mg, 7.15 mmol) and tetrabutyl ammo-
nium hydrogen sulfate (486 mg, 1.43 mmol) in ethyl acetate (10
mL) and aqueous saturated sodium bicarbonate solution (10
mL) were added. The reaction mixture was stirred vigorously at
room temperature overnight. It was then diluted with ethyl
acetate (40 mL) and washed with water (3 ꢀ 50 mL). The
organic layer was dried over anhydrous sodium sulfate and
filtered. The solvents were evaporated, and the residue was
purified over a silica column using 50% ethyl acetate/hexane
as eluant to yield 789 mg (84%) of pure product 4c. 1H NMR
(600 MHz, CDCl3, δ ppm): 5.15 (m, 1H, 3-H), 5.11 (m, 1H, 30-
H), 5.02 (m, 1H, 40-H), 4.88 (m, 1H, 20-H), 4.82 (m, 1H, 2-H),
4.59 (d, J = 8.56 Hz, 1H, 10-H), 4.49 (m, 2H, 1-H and 6-Ha), 4.32
(m, 1H, 60-Ha), 4.09 (m, 1H, 6-Hb), 4.01 (m, 1H, 60-Hb), 3.76 (t,
J = 9.30 Hz, 1H, 4-H), 3.65 (m, 2H, 40-H and 5-H), 2.10, 2.05,
2.02, 1.99, 1.98, 1.97, and 1.94 (each s, 3H, 6 ꢀ CH3CO2); mp
180-182 °C, reported 182-182.5 °C.30
Synthesis of 1-β-Amino-cellobiose Heptaacetate (4). The 1-β-
azido-cellobiose heptaacetate (789 mg) in methanol (10 mL) was
hydrogenated in the presence of palladium/carbon (10% w/w) at
room temperature for 2 h. The reaction mixture was filtered over
celite to remove palladium/carbon particles, which upon eva-
porating the solvent afforded 4 in quantitative yield. 1H NMR
(600 MHz, CDCl3, δ ppm): 5.21 (m, 1H, 3-H), 5.14 (m, 1H,
30-H), 5.06 (m, 1H, 40-H), 4.92 (m, 1H, 20-H), 4.73 (m, 1H, 2-H),
4.50 (m, 2H, 10-H and 6-Ha), 4.36 (m, 1H, 60-Ha), 4.32 (m, 1H,
1-H), 4.06 (m, 2H, 6-Hb and 60-Hb), 3.71 (m, 1H, 4-H), 3.65
(m, 1H, 5-H), 3.58 (m, 1H, 40-H), 2.16, 2.08, 2.05, 2.02, 2.01,
2.00, and 1.98 (each s, 3H, 7ꢀCH3CO2). Mass calcd for C26H38-
NO17: 636.2139 (MH+); HRMS found: 636.2142.
Figure 9. Foot response (skin phototoxicity) of HPPH and 9
(HPPH-Gal) was measured in C3H mice bearing RIF tumors at
the therapeutic dose. Foot response was judged using a 0-3 scale.28
0-0.1 = no apparent difference from normal, 0.3 = slight edema,
0.5 = (moderate edema), 0.75 = large edema, 1.0-large erythema
with exudate, 1.2 = moderate edema with slight scaly or crusty
appearance, 1.5 = definite erythema and definite scaly or crusty
appearance.
Conclusions
In an effort to enhance the efficacy and selectivity of
photosensitizers currently under development by our group,
we conjugated the clinically effective antitumor photo-
sensitizer 3-devinyl-3-(10-hexyloxyethyl) pyropheophorbide-a
(HPPH) with mono-, di-, or tetrasaccharides. We hoped that
these conjugates would target carbohydrate-binding mole-
cules known to be differentially expressed on the surface of
many tumor cells such as the carbohydrate-binding lectins
galectin-1 and -3. Carbohydrate conjugates and unconjugated
HPPH had similar singlet oxygen yields. Several of the
resulting carbohydrate conjugates exhibited superior antitu-
mor activity. This could not be explained by enhanced
photosensitizer binding to cellular galectins. Drug lipophili-
city and lack of conjugate transport by the ABCG2 trans-
porter may be contributing factors. PDT efficacy of carbo-
hydrate-conjugated HPPH may be determined by subcellular
localization and the signaling mechanisms initiated by the
photoreaction at the site of photosensitizer accumulation.
Further definition of these signaling mechanisms as well as
optimization of the carbohydrate conjugates at variable treat-
ment parameters in vivo are currently underway.
By following a similar synthetic approach other carbohydrate
analogues 1-β-amino-galactose tetraacetate, 1-β-amino-glucose
tetraacetate and 1-β-amino-lactose pentaacetate were synthe-
sized in good yields from galactose pentaacetate, glucose pen-
taacetate, and lactose octaacetate, respectively.
General Procedure for the Synthesis of Peracetylated Carbo-
hydrate-HPPH Conjugates Linked with Amide Bond (5-8).
Methyl3-(10-hexyloxyethyl)-3-devinylpyropheophorbide-a(HPPH)
was synthesized by following the methodology developed in our
laboratory.17 To a solution of HPPH (1 equiv) and 1-β-amino-
carbohydrates 1-4 (2 equiv) in 10 mL of dry dichloromethane,
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDCI, 2 equiv),
and 4-(dimethylamino)pyridine (DMAP, 2 equiv) were added
and the reaction mixture was stirred at room temperature under
N2 atmosphere overnight. It was then diluted with dichloromethane
(40 mL), washed with water (3 ꢀ 50 mL), dried over anhydrous
sodium sulfate, and concentrated to yield a crude product, which
was first partially purified by alumina column using 2% methanol in
dichloromethane as eluant and finally by silica column using 3%
methanol in dichloromethane to obtain pure final precursors 5-8 in
fair to good yield.
Experimental Section
All the chemicals and reagents were purchased from chemical
vendors and used as received. The solvents were dried before
using in various reactions by following the standard procedures.
UV-visible spectra were recorded in dichloromethane. NMR
spectra were recorded in deuterated chloroform (CDCl3) or
pyridine-d5 containing tetramethylsilane (TMS) as an internal
standard at 303 K. Chemical shifts are reported as parts per
million (ppm) with respect to TMS (δ 0.00) or most deshielded
peak of pyridine-d5 (δ 8.74). High-resolution mass spectroscopy
(HRMS) analyses were performed at the Michigan State Uni-
versity (MSU)-NIH Mass Spectrometry Facility, East Lansing,
MI. The purity of the compounds was ascertained by HPLC
analyses and was >95%. The reactions were monitored using
small strips of precoated silica thin layer chromatography (TLC)
(250 μm thickness) plates. Preparative TLC was performed on
20 cm ꢀ 20 cm silica gel TLC plates (1 mm thickness).
Peracetylated Galactose-HPPH Conjugate (5). The title
compound was obtained in 66% yield. H NMR (600 MHz,
1
CDCl3, δ ppm): 9.80 (d, J = 2.1 Hz, 1H, meso-H), 9.48 and 8.53
(each s, 1H, meso-H), 6.11 (m, 1H, CONH), 5.92 (m, 1H, 31-CH-
(O-hexyl)CH3), 5.40 (m, galactose-H), 5.30-4.96 (m, total 5H,
132-CH2, 3 ꢀ galactose-H), 4.49 and 4.37 (each m, 1H, 17- and
18-H), 4.10-3.96 (m, 3H, galactose-H), 3.70 (m, 4H, 81-CH2
and 32-OCH2CH2CH2CH2CH2CH3), 3.62, 3.40, and 3.28 (each
s, 3H, 2-,7- and 12-CH3), 2.69, 2.38, and 2.18 (each m, total 4H,

4314 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Zheng et al.
171- and 172-CH2), 2.13 (m, 3H, 31-CH(O-hexyl)CH3), 2.06,
2.00, 1.96, and 1.93 (each s, 3H, 4 ꢀ galactose CH3CO2), 1.82
(d, J = 6.40 Hz, 3H, 18-CH3), 1.73 (m, 3H, 82-CH3), 1.50-
1.20(m, 8H, 32-OCH2CH2CH2CH2CH2CH3), 0.79 (m, 3H,
32-OCH2CH2CH2CH2CH2CH3), 0.46 and -1.69 (each br,
1H, NH). Mass calcd for C53H68N5O12: 966.4864 (MH+);
HRMS found: 966.4869.
Galactose-HPPH Conjugate (9). The title compound was
obtained in 94% yield. UV-vis λmax (THF, nm): 661 (4.75 ꢀ
104), 604 (0.71 ꢀ 104), 536 (0.88 ꢀ 104), 505 (0.87 ꢀ 104), 410
(8.37 ꢀ 104), 317 (1.89 ꢀ 104). 1H NMR (400 MHz, pyridine-d5,
δ ppm): 10.25 (d, J = 5.6 Hz, 1H, meso-H), 9.95 (s, 1H, meso-
H), 9.62 (d, J = 9.2, 1H, CONH), 8.80 (s, 1H, meso-H), 6.14 (m,
1H, 31-CH(O-hexyl)CH3), 5.94 (t, J = 9.0, 1H, NHCO), 5.27
(ABX, 2H, 132-CH2), 4.89 (brs, 4H, 4 ꢀ galactose-OH), 4.60-
4.15 (m, total 9H, 7 ꢀ galactose-H, 17- and 18-H), 3.85-3.80 (m,
4H, 81-CH2 and 31-OCH2CH2CH2CH2CH2CH3), 3.75, 3.46,
and 3.36 (each s, 3H, 2-, 7- and 12-CH3), 2.96-2.32 (each m,
total 4H, 171- and 172-CH2), 2.28 (d, J = 6.6 Hz, 3H, 31-CH(O-
hexyl)CH3), 1.85 (m, 2H, 32-OCH2CH2CH2CH2CH2CH3), 1.82
(d, J = 7.2 Hz, 3H, 18-CH3), 1.74 (t, J = 7.6 Hz, 3H, 82-CH3),
1.60-1.20 (m, 6H, 32-OCH2CH2CH2CH2CH2CH3), 0.75 (m,
3H, 32-OCH2CH2CH2CH2CH2CH3), 0.43 and -1.65 (each br,
1H, NH). Mass calcd for C45H59N5O8: 798.4442 (MH+);
HRMS found: 798.4440.
Peracetylated Glucose-HPPH Conjugate (6). The title com-
pound was obtained in 73% yield. 1H NMR (400 MHz, CDCl3,
δ ppm): 9.79 (s, 1H, meso-H), 9.46 (d, J = 2.2 Hz, 1H, meso-H)
and 8.51 (s, 1H, meso-H), 6.07 (m, 1H, CONH), 5.90 (q, J = 6.7,
1H, 31-CH(O-hexyl)CH3), 5.30-4.95 (m, total 4H, 132-CH2 and
2 ꢀ glucose-H), 4.82 and 4.75 (each m, 1H, 2 ꢀ glucose-H), 4.46
and 4.35 (each m, 1H, 17- and 18-H), 4.30-4.00 (m, 3H, 3 ꢀ
glucose-H), 3.76 (m, 1H, glucose-H), 3.68 (m, 4H, 81-CH2 and
32-OCH2(CH2)4CH3), 3.58, 3.39, and 3.27 (each s, 3H, 2-, 7- and
12-CH3), 2.64, 2.36, and 2.16 (each m, total 4H, 171- and 172-
CH2), 2.11 (m, 3H, 31-CH(O-hexyl)CH3), 2.09, 2.07, 2.03, and
2.01 (each s, 3H, 4 ꢀ glucose CH3CO2), 1.80 (d, J = 7.30 Hz, 3H,
18-CH3), 1.67 (m, 3H, 82-CH3), 1.42-1.22 (m, 8H, 32-OCH2-
CH2CH2CH2CH2CH3), 0.75 (m, 3H, 32-OCH2CH2CH2CH2-
CH2CH3), 0.45 and -1.71 (each br, 1H, NH). Mass calcd for
C53H68N5O12: 966.4864 (MH+); HRMS found: 966.4867.
Peracetylated Lactose-HPPH Conjugate (7). The title com-
pound was obtained in 70% yield. UV-vis λmax (CH2Cl2, nm):
660 (4.75 ꢀ 104), 605 (0.78 ꢀ 104), 537 (0.95 ꢀ 104), 505 (0.94 ꢀ
Glucose-HPPH Conjugate (10). The title compound was
obtained in 96% yield. UV-vis (THF, nm): 661 (4.75 ꢀ 104),
604 (0.68 ꢀ 104), 536 (0.85 ꢀ 104), 506 (0.84 ꢀ 104), 411 (9.19 ꢀ
104), 318 (1.84 ꢀ 104). 1H NMR (400 MHz, pyridine-d5, δ ppm):
10.25 (d, J = 5.2 Hz, 1H, meso-H), 9.96 and 8.80 (each s, 1H,
meso-H), 9.59 (d, J = 9.0, 1H, CONH), 6.14 (m, 1H, 31-CH(O-
hexyl)CH3), 6.02 (t, J = 9.0 Hz, 1H, NHCO), 5.36 (br, 6H, 132-
CH2 and 4 ꢀ glucose-OH), 3.84-3.79 (m, 5H, 81-CH2, 32-
OCH2CH2CH2CH2CH2CH3), 3.76, 3.46, and 3.37 (each s, 3H,
2-, 7-, and 12-CH3), 2.97-2.45 (each m, total 4H, 171- and 172-
CH2), 2.28 (d, J = 6.6 Hz, 3H, 31-CH(O-hexyl)CH3), 1.87 (m,
2H, 32-OCH2CH2CH2CH2CH2CH3), 1.85 (d, J = 7.1 Hz, 3H,
18-CH3), 1.75 (t, J = 7.4, 3H, 82-CH3), 1.55-1.18 (m, 6H, 32-
OCH2CH2CH2CH2CH2CH3), 0.75 (m, 3H, 32-OCH2CH2CH2-
CH2CH2CH3), 0.43 and -1.64 (each br, 1H, NH). Mass calcd
for C45H59N5O8: 798.4442 (MH+); HRMS found: 798.4441.
Lactose-HPPH Conjugate (11). The title compound was
obtained in 85% yield. UV-vis (THF, nm): 661 (4.75 ꢀ 104),
536 (0.95 ꢀ 104), 505 (0.94 ꢀ 104), 409 (9.84 ꢀ 104). 1H NMR (400
MHz, pyridine-d5, δppm): 10.24 (d, J= 4.7 Hz, meso-H), 9.94 and
8.78 (each s, 1H, meso-H), 9.66 (d, J = 9.0 Hz, 1H, NHCO), 6.13
(m, 1H, 31-CH(O-hexyl)CH3), 5.94 (t, J = 9.0 Hz, 1H, NHCO),
5.24 (ABX, 2H, 132-CH2), 4.94 (br, 12H, 17-H, 18-H, 5 ꢀ lactose-
H and 7 ꢀ lactose-OH), 4.55-3.95 (m, 11H, 17-H, 18-H and 9 ꢀ
lactose-H), 3.83-3.76 (m, 4H, 81-CH3 and 31-OCH2CH2CH2-
CH2CH2CH3), 3.74, 3.45, and 3.36 (each s, 3H, 2-, 7-, and 12-
CH3), 3.00-2.43 (each m, total 4H, 171-and172-CH2), 2.27 (d, J=
6.8 Hz, 3H, 31-CH(O-hexyl)CH3), 1.84 (m, 2H, 32-OCH2CH2-
CH2CH2CH2CH3), 1.81 (d, J = 7.4 Hz, 3H, 18-CH3), 1.73 (t, J =
7.5 Hz, 3H, 18-CH3), 1.55-1.20 (m, 6H, 32-OCH2CH2CH2CH2-
CH2CH3), 0.74 (m, 3H, 32-OCH2CH2CH2CH2CH2CH3), 0.48
and -1.70 (each br, 1H, NH). Mass calcd for C51H69N5O13:
960.4970 (MH+); HRMS found: 960.4966.
1
104), 410 (10.39 ꢀ 104). H NMR (400 MHz, CDCl3, δ ppm):
9.79 (s, 1H, meso-H), 9.41 (d, J = 3.2 Hz, 1H, meso-H) and 8.51
(s, 1H, meso-H), 6.05 (m, 1H, CONH), 5.91 (q, J = 6.6 Hz, 1H,
31-CH(O-hexyl)CH3), 5.33 (m, 1H, lactose-H), 5.29-5.05 (m,
total 4H, 132-CH2 and 2 ꢀ lactose -H), 4.92 (m, 1H, lactose-H),
4.68(m, 1H, lactose-H), 4.50-4.34 (m, total 5H, 17-H, 18-H and
3 ꢀ lactose-H), 4.15-4.00 (m, 3H, 3 ꢀ lactose-H), 3.83 (t, J =
6.6, 1H, lactose-H), 3.65-3.60 (m, total 4H, 81-CH2 and 32-
OCH2CH2CH2CH2CH2CH3), 3.68, 3.39, and 3.27 (each s,
3H, 2-, 7- and 12-CH3), 2.67 and 2.36 (each m, total 3H,
171- and 172-CH2), 2.18-1.95 (m, total 22H, 171-CH2, 31-CH
(O-hexyl)CH3, 6 ꢀ lactose CH3CO2), 1.90 (s, 3H, 18-CH3),
1.79 (m, 3H, lactose CH3CO2), 1.73-1.66 (m, total 6H, 82-CH3
and 32-OCH2CH2CH2CH2CH2CH3), 1.20 (m, total 8H, 32-
OCH2CH2CH2CH2CH2CH3), 0.78(m, 3H, 32-OCH2CH2CH2-
CH2CH2CH3), 0.48 and -1.70 (each br, 1H, NH). Mass calcd
for C65H84N5O20: 1254.5709 (MH+); HRMS found: 1254.5712.
Peracetylated Cellobiose-HPPH Conjugate (8). The title
1
compound was obtained in 38% yield. H NMR (600 MHz,
CDCl3, δ ppm): 9.80 (s, 1H, meso-H), 9.26 (d, J = 10.3 Hz,
meso-H) and 8.53 (s, 1H, meso-H), 6.21 (m, 1H, CONH), 5.93
(m, 1H, 31-CH(O-hexyl)CH3), 5.30-5.04 (m, 5H, 132-CH2, 3 ꢀ
cellobiose-H), 4.90 (m, 1H, cellobiose-H), 4.70 (m, 1H, cello-
biose-H), 4.52-4.30 (m, 6H, 17-H, 18-H and 4 ꢀ cellobiose-H),
4.07 (m, 1H, cellobiose-H), 4.01 (m, 1H, cellobiose-H), 3.71-
3.57 (m, total 7H, 32-OCH2CH2CH2CH2CH2CH3, 81-CH2 and
3 ꢀ cellobiose-H), 3.69, 3.39, and 3.28 (each s, 3H, 2-, 7- and 12-
CH3), 2.68 and 2.38 (each m, total 3H, 171- and 172-CH2), 2.13
(m, 3H, 31-CH(O-hexyl)CH3), 2.11 (m, 1H, 171-CH2), 2.07-
1.93 (each s, 3H, 7 ꢀ cellobiose CH3CO2), 1.81 (d, J = 7.90 Hz,
3H, 18-CH3), 1.61 (m, 3H, 82-CH3), 1.50-1.20 (m, 8H, 32-
OCH2CH2CH2CH2CH2CH3), 0.78(m, 3H, 32-OCH2CH2CH2-
CH2CH2CH3), 0.48 and -1.70 (each br, 1H, NH). Mass calcd.
for C65H84N5O20: 1254.5709 (MH+); HRMS found: 1254.5711.
General Procedure for the Deacetylation of Peracetylated
Carbohydrate-HPPH Conjugates Linked with Amide Bond
(9-12). To a solution of 5-8 in dry dichloromethane (10 mL)
and methanol (1 mL), sodium methoxide in methanol (1 M, 200
μL) was added and the reaction mixture was stirred for 30 min
under N2 atmosphere at room temperature. It was then neu-
tralized with acetic acid, evaporated under vacuum, and the
crude product thus obtained was purified by silica column using
gradient 2-8% methanol in dichloromethane as eluant to
obtain compounds 9-12 in good yield.
Cellobiose-HPPH Conjugate (12). The title compound
was obtained in 90% yield. UV-vis (THF, nm): 661 (4.75 ꢀ
104), 604 (0.72 ꢀ 104), 536 (0.89 ꢀ 104), 505 (0.89 ꢀ 104), 409
(9.34 ꢀ 104), 318 (1.97 ꢀ 104). 1H NMR (400 MHz, pyridine-d5,
δ ppm): 10.25 (d, J = 4.9 Hz, 1H, meso-H), 9.96 and 8.79
(each s, 1H, meso-H), 9.67(dd, J = 2.0 and 9.2, 1H, NHCO),
6.14 (m, 1H, 31-CH(O-hexyl)CH3), 5.96 (t, J = 9.1, 1H,
NHCO), 5.35 (m, 1H, 132-CH2), 5.15 (m, 2H, 132-CH2 and
cellobiose-H), 4.88 (brs, 7H, 7 ꢀ cellobiose-OH), 4.58-3.96
(m, 14H, 17-H, 18-H and 12 ꢀ cellobiose-H), 3.86-3.76 (m, 4H,
81-CH2 and 32-OCH2CH2CH2CH2CH2CH3), 3.77, 3.46, and
3.37 (each s, 3H, 2-, 7-, and 12-CH3), 3.00-2.43 (each m, total
4H, 171- and 172-CH2), 2.28 (d, J = 6.5 Hz, 3H, 31-CH(O-hexyl)
CH3), 1.85 (m, 2H, 32-OCH2 CH2CH2CH2CH2CH3), 1.82 (d,
J = 7.2 Hz, 3H, 18-CH3), 1.75 (t, J = 7.6 Hz, 3H, 82-CH3),
1.50-1.20 (m, 6H, 32-OCH2CH2CH2CH2CH2CH3), 0.74 (m,
3H, 32-OCH2CH2CH2CH2CH2CH3), 0.46 and -1.69 (each br,
1H, NH). Mass calcd for C51H69N5O13: 960.4970 (MH+);
HRMS found: 960.4972.

Article
HPPH with Boc Protected Amino Side Chain (13). To a stirred
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4315
Dowex resin. After removal of the resin by filtration, the
solvent was evaporated under vacuum to dryness and
washed with dichloromethane. Yield 10 mg (79%) of the
title compound. UV-vis λmax (THF, nm): 663 (1.475 ꢀ 104),
540 (0.42 ꢀ 1040.164), 508 (0.41 ꢀ 104), 398 (3.31 ꢀ 104). Mass
calcd for C132H168N16O46S4: 2842.0259 (MH+); HRMS found:
2842.0202.
Synthesis of 14C HPPH Methyl Ester. Methyl pyropheophor-
bide-a 50 mg (0.091 mmol) was reacted with 33%HBr/acetic
acid (3.0 mL) for 2 h at room temperature. Evaporation of the
acids under high vacuum gave a residue, which was not isolated
and was immediately reacted with n-hexanol [1-14C] (250 μL,
2 mmol, specific activity-5 mCi /mmol, American Radiolabeled
Chemicals, cat. no. ARC0225) in dry dichloromethane (3 mL).
Anhydrous potassium carbonate (20 mg) was added, and the
reaction mixture was stirred for 45 min under a nitrogen atmo-
sphere. It was then diluted with dichloromethane (20 mL) and
washed with water (2 ꢀ 50 mL). The organic layer was separated
and dried over anhydrous sodium sulfate. The crude residue
obtained after evaporating the solvent was purified by prepara-
tive plates (Silica), eluting with 2% methanol/dichloromethane.
Evaporation of the solvents gave the title compound in 33%
yield (20 mg).
Synthesis of 14C-HPPH. The foregoing 14C HPPH methyl
ester was dissolved in a solution of methanol (4 mL), distilled
peroxide-free tetrahydrofuran (3 mL), and lithium hydroxide
(32 mg in 3 mL water) and stirred for 2 h until the hydrolysis was
complete (monitored by TLC). After the standard workup, the
desired product was purified by silica prep plate (1000 um),
eluted with 7% methanol/dichloromethane).Yield (determined
spectrophotometrically): 17 mg (87%), specific activity: 5.0 mCi /
mmol. Purity was ascertained by HPLC (98% pure).
Synthesis of 14C-Labeled Galactose-HPPH Conjugate. To a
solution of aminogalactose pentaacetate (5.6 mg), 14C labeled
HPPH (5 mg, 0.00517 mmol), and BOP (6.6 mg) in dry DMF
(2 mL), 40 μL of triethylamine were added. The reaction mixture
was stirred at room temperature under the N2 atmosphere for
7 h. Evaporation of the solvent under vacuum gave a crude
product, which was purified by silica plates (5% MeOH in
dichloromethane). The product was dissolved in a mixture of
dichloromethane (5 mL) and 20 μL of NaOMe (in MeOH) was
added, and the reaction mixture was stirred for 30 min, then 0.8 g
of Dowex resin was added to quench the reaction. The solution
was collected after removing the resin, concentrated and pur-
ified by a silica column (eluent: 1-10% MeOH in dichloro-
methane). The appropriate fractions were collected. Evapora-
tion of the solvent gave pure 14C labeled galactose-HPPH
conjugate in quantitative yield (determined spectrophoto-
metrically: 6.3 mg (quantitative yield) with specific activity
4.6 mCi/ mmol.
solution of HPPH (600 mg) and N-Boc-ethylenediamine (200
μL) in dry dichloromethane (10 mL), BOP (440 mg), and dry
triethylamine (1 mL) were added and the reaction mixture was
stirred under N2 atmosphere overnight. Solvents were removed
under reduced pressure, and the crude residue was purified by
silica column using 4% methanol in dichloromethane as eluant
to obtain title compound 13 in 95% yield. 1H NMR (400 MHz,
CDCl3, δ ppm): 9.79 (d, J = 3.2 Hz, 1H, meso-H), 9.15 (d, J =
9.8 Hz, 1H, meso-H), 8.52 (s, 3H, meso-H), 6.19 (br, 1H,
CONH), 5.92 (m, 1H, 31-CH(O-hexyl)CH3), 5.16 (ABX, 2H,
132-CH2), 4.89 (brs, 1H, NHBoc), 4.52 and 4.30 (each m, 1H, 17-
and 18-H), 3.72-3.52 (m, 4H, 81-CH2 and 32-OCH2CH2-
CH2CH2CH2CH3), 3.39 and 3.28 (each s, 3H, 2- and 7-CH3),
3.24-3.09 (m, 7H, 12-CH3 and CONHCH2CH2NHBoc), 2.70-
2.32 (each m, total 4H, 171- and 172-CH2), 2.13 (d, J = 6.6 Hz,
3H, 31-CH(O-hexyl)CH3), 1.79 (d, J = 7.7 Hz, 3H, 18-CH3),
1.75 (m, 2H, 32-OCH2CH2CH2CH2CH2CH3), 1.62 (m, 3H, 82-
CH3), 1.26-1.20 (m, 15H, (CH3)3COO and 32-OCH2-
CH2CH2CH2CH2CH3), 0.78 (m, 3H, OCH2CH2CH2CH2 CH2-
CH3), 0.50 and -1.64 (each br, 1H, NH). Mass calcd for
C46H63N6O5: 779.4860 (MH+); HRMS found: 779.4863.
HPPH with Free Amino Side Chain (14). Compound 13
(36.8 mg) in dichloromethane (5 mL) was treated with trifluor-
oacetic acid (1 mL). After 30 min, solvents were removed under
reduced pressure and the residue was used as such in the next
step without further purification.
General Approach for the Synthesis of Peracetylated Carbohy-
drate-HPPH Conjugates Linked with Rigidified Linker (18-
20). Peracetylated multivalent lactose analogues 15-17 were
prepared by following the reported procedure. For the synthesis
of the title compounds, compound 14 (2 equiv), BOP (2 equiv),
and triethylamine (3 equiv) were dissolved in 10 mL of dry
dichloromethane under N2 atmosphere and reacted individually
to multivalent lactose analogues 15-17 at room temperature
overnight. After the standard workup, the residue was purified
by silica column chromatography. The major band was col-
lected (eluted with 2-4% methanol in dichloromethane) to give
18-20 in modest yields. The NMR data were similar to those
reported in the literature.
Monovalent Lactose-HPPH Conjugate (21). To a solution of
18 (60 mg) in dry dichloromethane (10 mL) and methanol
(1 mL), sodium methoxide (1 M, 200 μL) in methanol was
added and the reaction mixture was stirred for 30 min under the
N2 atmosphere. It was then neutralized by strong cationic resin
(Dowex), and after filtering out the resin, the reaction mixture
was concentrated to dryness and the crude solid thus obtained
was chromatographed on silica column using 10% methanol in
dichloromethane as eluant to yield 40 mg (83%) of title com-
pound. UV-vis λmax (THF, nm): 661 (4.75 ꢀ 104), 604 (0.79 ꢀ
104), 536 (0.95 ꢀ 104), 506 (0.99 ꢀ 104), 410 (10.00 ꢀ 104), 317
(3.17 ꢀ 104). Mass calcd for C64H82N8O14S: 1219.5749 (MH+);
HRMS found: 1219.5740.
Divalent Lactose-HPPH Conjugate (22). To a solution of 19
(40 mg) in dry dichloromethane (10 mL) and methanol (1 mL),
1 M NaOMe in methanol (200 μL) was added and the reaction
mixture was stirred for 1 h under a N2 atmosphere. It was then
neutralized by strong cationic resin (Dowex), and after filtering
out the resin, the reaction mixture was evaporated to dryness.
The residue was washed with dichloromethane (2 ꢀ 50 mL) and
water (2 ꢀ 50 mL). Evaporation of the solvent gave the title
compound in 8%% (25 mg) yield. UV-vis λmax (THF, nm): 664
(4.615 ꢀ 104), 606 (0.99 ꢀ 104), 541 (1.13 ꢀ 104), 509 (1.13 ꢀ 104),
414 (9.70 ꢀ 104). Mass calcd for C80H106N10O24S2: 1655.6900
(MH+); HRMS found: 1655.6910.
HPLC Analysis. The purity of the final products was ascer-
tained by HPLC using a Waters Delta 600 system consisting of
the 600 controller, 600 fluid handling unit, and 996 photodiode
array detector equipped with a Waters Symmetry C18 column,
5 μm particle size, with dimensions of 4.6 mm ꢀ 150 mm. The
mobile phase composition was 95% methanol/5% water, run
isocratically at a flow of 1.0 mL/min. The retention time and
percent purity of each compound is tabulated in Table 2
(Supporting Information); the component percentages are
based on the area counts of the peaks from the 408 nm channel.
Under these HPLC conditions, the conjugates 22 and 24 could
not be eluted. However, their corresponding acetoxy-derivatives
were >95% pure.
In Vitro Photocytotoxicity Assay. RIF and Colon26 cells were
grown in R-MEM with 10% fetal calf serum, L-glutamine,
penicillin, and streptomycin at 37 °C in 5% CO2, 95% air, and
100% humidity. Cells were seeded in 96-well plates at a 5 ꢀ 103
cells per well (five replicates) in complete medium. After over-
night incubation at 37 °C, photosensitizers were added at
variable concentrations for 24 h in the dark. Drug-containing
Tetravalent Lactose-HPPH Conjugate (23). To a solution of
20 (18 mg) in dry dichloromethane (10 mL) and methanol
(1 mL), NaOMe (1 M) in methanol (250 μL) was added and
the reaction mixture was stirred for 4 h under a N2 atmosphere.
A homogeneous solution was obtained after neutralization with

4316 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Zheng et al.
medium was replaced with fresh medium and cells were illumi-
nated with 0.5 J/cm2 delivered by an argon-pumped dye laser
(665 nm at a dose rate of 3.2 mW/cm2). After PDT, the cells
were incubated for 48 h at 37 °C in the dark and phototoxicity
(relative growth compared to untreated cells of each compound
tested) was determined by the MTT (3-[4,5-dimethyl-
thiazol-2-yl]-2,5-diphenyltetrazoliumbromide) assay. Dose-
response cell survival curves were generated (using Microcal
Origin 6.0), and the LD50 values were determined based on
Gaussian and Sigmoidal fit. Each data point represents the
mean from three separate experiments, and the error bars are
the standard deviation. Similar experiments were later per-
formed with Colon-26 with HPPH and 9 for comparison and
for both cell types with drugs (HPPH and the galactose deriva-
tive 9) incubated for 4 h.
unlabeled PS. For flowcytometric analyses of PS accumulation,
the cells incubated with nonradioactive PS were released from
culture wells and resuspended in cytometric tubes in cold
medium with 2% FCS. Tubes were thereafter kept in the dark
on ice to inhibit ABCG2 efflux activity. Fluorescence of the
photosensitizer was measured by flow cytometry on a FACS-
Caliber flow cytometer, BD Invitrogen), with excitation at
488 nm and emission at 680/20 nm. After subtracting autofluor-
escence (untreated or inhibitor only cells), the (percentage)
change in the fluorescence mediated by the inhibitor was
calculated. An increase of more than 15% fluorescence was
considered significant (p < 0.05, students 2-tailed t test) and
indicated that the photosensitizer was a substrate of the ABCG2
pump.
In Vivo Photosensitizing Efficacy. The in vivo antitumor
photosensitizing efficacies of HPPH-carbohydrate conjugates
were evaluated in mice with tumors implanted on the shoulder.
C3H mice were implanted with RIF tumors and BALB/c mice
with Colon26 tumors. Groups of five C3H or 10 BALB/c mice
were injected with the compounds (0.47 μmol/kg) intravenously.
The tumors were exposed to laser light (665 nm) at various
fluence and fluence rates at 24 h post injection. Two light dose
conditions were applied 135 J/cm2, 75 mW/cm2, and 48 J/cm2,
7 mW/cm2). After in vivo PDT, the tumor volume V= (Lꢀ W2)ꢀ
0.5 (L = length (the longest dimension) and W = width
(perpendicular to the long axis) was measured with calipers.
The hours-to-end point (HTE) the time for the tumor to grow to
400 mm3 was calculated by linear interpolation using Microsoft
Excel-based software. Mice were sacrificed just after their
tumors reach the end point. For each experimental group, a
Kaplan-Meier hours-to-end point (HTE) curve was generated
(using Prism 4, GraphPad Software, Inc.), and the median
Kaplan-Meier tumor growth HTE time was estimated. To test
for significant differences between pairs of HTE curves, the
Cox-Mantel test was used.31
In Vitro PS Uptake Determined by Fluorescence. RIF cells
were seeded at 1.5 ꢀ 105 cells per well in 6-well plates. After
overnight incubation at 37 °C, PS at 0.25 μM were added to
triplicate wells and incubated in the dark at 37 °C for 3 or 24 h.
Drug-containing medium was removed and cells were washed
with PBS and were solubilized by incubating with gentle rocking
in the dark at 37 °C for 2 h with 1 mL Solvable (PerkinElmer Life
and Analytical Sciences). The cell solutions were diluted with
2 mL of ddH2O, and the fluorescence of each sample was
measured (excitation 415 nm; emission 667 nm) on a Fluoro-
Max-2 (ISA, JOBIN YVON-SPEX, Horiba Group). The pro-
tein content of the sample was determined using the Bio-Rad
DC Assay. The data was plotted as fluorescence units/mL
of protein.
Intracellular Localization. Cells were grown in 6-well plates
on poly-L-lysine coated coverslips, coincubated with PS and
organelle-specific fluorescent dyes (3-4 h and 24 h), washed
with PBS, and imaged at 40ꢀ on an inverted fluorescence
microscope (Zeiss Axiovert 200W, Germany) with a charged-
coupled device camera (Dage Zeiss AxioCam MRm) using an
AxioCam MR-MRGrab Framegrabber and AxioVision LE 4.1
imaging software. For organelle staining, cells were incubated
with MitoTracker Green (1 μM for 1 h, mitochondria), Lyso-
Tracker Green, or carboxylate-modified 0.1 μm diameter
yellow-green Fluospheres (0.5 μM for 0.5 h or 1/100000 dilution
overnight, respectively, lysosomes), or Bodipy C5 ceramide-
albumin complexes (5 μM, Golgi apparatus) according to
manufacturer’s directions (Invitrogen). Filter combinations
were as follows: for HPPH-containing compounds Ex BP D410/
40 nm, BeamSplitter FT 505dcxvu, and Em BP 675/50 nm;
for LysoTracker Green and Fluospheres Ex 390/22 nm,
FT 420 nm and Em BP4 60/50 nm; and for Bodipy C5 cera-
mide-albumin Ex BP 565/30 nm, BeamSplitter FT 585 nm
and Em BP 520/60.
Uptake of 14C-Labeled HPPH or the Corresponding Galactose
Derivative (9). For uptake by 14C-labeled HPPH or HPPH-Gal
(specific activity 5 Ci/mol and 4.6 Ci/mol, respectively), RIF and
Colon26 cells were seeded at 3 ꢀ 104 cells per cm2 culture area in
24-well or 6-well cluster plates. After one day, the medium was
replaced by 0.2 mL per cm2 culture area with medium containing
either 0.5% or 10% fetal calf serum and 0.8 μM 14C-labeled PS
and incubated in the dark at 37 °C. At different time points up to
24 h, the drug-containing medium was removed, cells were
washed with phosphate buffered saline and either released by
trypsin digestion and counted in a hematocytometer or solubi-
lized within the culture well. The solubilized cell samples were
transferred to scintillation vials, UniverSol scintillation fluid
added, and the radioactivity counted. The uptake of radio-
activity was corrected for background counts and expressed in
pmol PS per 1 ꢀ 106 cells. Experiments were carried out in
triplicate.
In Vivo Biodistribution Studies. The 14C-labeled photosensi-
tizers (0.2 mL/5 μCi) were individually administered to mice
through a lateral tail vein (three mice/group (BALB/c mice
bearing Colon26 tumors and C3H mice bearing RIF tumors)).
At 24, 48 h after injection, three mice/time point were sacrificed
using CO2 followed by cervical dislocation as per Institute’s
approved protocol. The blood was collected into heparinized
syringes, kept ice cold, and immediately centrifuged to obtain
plasma. The plasma was transferred to a scintillation vial for
C-14 counting. The organs of interest (skin, muscle, tumor,
heart, lung, liver, kidney, and spleen) were removed. Sample
weights were recorded, and 1 mL of Solvable was added to each
sample and incubated overnight at 53 °C. The samples were
allowed to dissolve before adding 80 μL of peroxide (20 μL at a
time). After samples were sufficiently bleached, 15 mL of Ultima
Gold scintillation fluid was added to each and the samples were
run on a Beckman LS 6000LL scintillation counter. The raw
data were then converted to count s/g tissue weight and plotted
using SigmaPlot 10.0 software.
Acknowledgment. Financial assistance from the NIH (CA
55791) and shared resources of the RPCI support grant
(P30CA16056) is highly appreciated. We thank Adam Sumlin
for his assistance in evaluating HPPH and HPPH-Gal for skin
phototoxicity.
Supporting Information Available: NMR and the HPLC
analysis of the final products and the intracellular localization
characteristics of compounds 9-12 and 21-23. This material is
available free of charge via the Internet at http://pubs.acs.org.
Test for ABCG2 Pump Specificity. In the same experimental
setting as used for PS uptake measurements, RIF or Colon26
were preincubated at 37 °C with or without 10 μM imatinib
mesylate for 0.5 h, followed by addition of 14C-labeled PS or
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