Synthesis of pheophorbide-a conjugates with anticancer drugs as potential cancer diagnostic and therapeutic agents

Article abstract of DOI:10.1016/j.bmc.2011.07.058

Pheophorbide-a, a chlorine based photosensitizer known to be selectively accumulated in cancer cells, was conjugated with anticancer drugs, doxorubicin and paclitaxel in the purpose of selective cancer diagnosis and therapy. Pheophorbide-a was conjugated with anticancer drugs via directly and by the use of selective cleavage linkers in cancer cell. The fluorescence of pheophorbide-a and doxorubicin conjugate by excitation at 420 or 440 nm was greatly diminished possibly by the energy transfer mechanism between two fluorescent groups. However, upon treatment in cancer cells, the conjugate showed to be cleaved to restore each fluorescence of pheophorbide-a and doxorubicin after 48 h of incubation. Also, pheophorbide-a conjugates either with doxorubicin and paclitaxel inhibited the growth of various cancer cells more potently than pheophorbide-a, which displayed very weak inhibitory activity. The results indicated that the pheophorbide-a conjugates with anticancer drugs could be utilized for selective cancer therapy as well as for the fluorescence detection of cancer.

Full text of DOI:10.1016/j.bmc.2011.07.058

Bioorganic & Medicinal Chemistry 19 (2011) 5383–5391
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of pheophorbide-a conjugates with anticancer drugs
as potential cancer diagnostic and therapeutic agents
Hyun You a, Hyo-Eun Yoon c, Jung-Hoon Yoon c, Hyojin Ko a, , Yong-Chul Kim a,b,
⇑
⇑
a Graduate-Program of Medical System Engineering (GMSE), Gwangju Institute of Science and Technology, Gwangju, Republic of Korea
b School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju, Republic of Korea
c Department of Pathology and Research Center for Oral Disease Regulation of the Aged, School of Dentistry, Chosun University, Gwangju, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Pheophorbide-a, a chlorine based photosensitizer known to be selectively accumulated in cancer cells,
was conjugated with anticancer drugs, doxorubicin and paclitaxel in the purpose of selective cancer diag-
nosis and therapy. Pheophorbide-a was conjugated with anticancer drugs via directly and by the use of
selective cleavage linkers in cancer cell. The fluorescence of pheophorbide-a and doxorubicin conjugate
by excitation at 420 or 440 nm was greatly diminished possibly by the energy transfer mechanism
between two fluorescent groups. However, upon treatment in cancer cells, the conjugate showed to be
cleaved to restore each fluorescence of pheophorbide-a and doxorubicin after 48 h of incubation. Also,
pheophorbide-a conjugates either with doxorubicin and paclitaxel inhibited the growth of various cancer
cells more potently than pheophorbide-a, which displayed very weak inhibitory activity. The results indi-
cated that the pheophorbide-a conjugates with anticancer drugs could be utilized for selective cancer
therapy as well as for the fluorescence detection of cancer.
Received 14 June 2011
Revised 28 July 2011
Accepted 29 July 2011
Available online 2 August 2011
Keywords:
Pheophorbide-a
Conjugates
Doxorubicin
Paclitaxel
Cancer diagnosis
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
investigations on the effects of pheophorbide-a conjugates with
anticancer drugs such as doxorubicin (DOX, 2) and paclitaxel
Many drugs are designed based on the prodrug approach as the
reason for improvement of physicochemical, biopharmaceutical or
pharmacokinetic properties.1 Chemical linker, targeting-ligand,
membrane transporter, and polymer have been used as the pro-
drug conjugation motives.2 As one of the targeting-ligand, photo-
sensitizers have been utilized in the development of cancer
diagnostic agents as well as in photodynamic therapy (PDT) which
could treat cancer and non malignant tumor by reactive oxygen
species generated by light, oxygen and photosensitizer.3
(PTX, 3) (Fig. 1) either by direct coupling or via linkers which are
known for the characteristics of selective cleavage in cancer cell.
The analysis of fluorescence spectrum, cellular uptake using confo-
cal microscopy and in vitro anti-cancer activities in various cancer
cell lines of the new pheophorbide-a conjugates are reported.
2. Results and discussion
2.1. Chemistry
Several photosensitizer conjugates have been developed. The
carbohydrate moieties on conjugation with 3-(10-hexyloxyethyl)-
3-devinyl pyropheophorbide-a (HPPH),4 porphyrin conjugates
with monocolonal antibodies5 and with spermine,6 pyropheophor-
bide conjugate with tamoxifen,7 tetra(pentafluorophenyl)porphy-
rin conjugate with thiosaccharide,8 and Chlorin p6 and histamine
conjugate9 were reported to increase cellular accumulation of
photosensitizer in tumor cells. In addition, anticancer drugs such
as doxorubicin have been reported that their conjugated
compounds enhanced selective cellular uptake and reduced the
The general synthetic procedure for pheophorbide-a (Pa, 1) is de-
scribed in Scheme 1. Treatment of the ethanol solution of chloro-
phyll a (4)16 in an acidic condition (1 N HCl, pH 2.5) enabled to
remove the Mg2+ ion easily to afford a crude pheophytin (5) in the
form of precipitate. The pheophytin (5) was subsequently hydro-
lyzed by reacting with 80% TFA in water to afford pheophorbide-a
as a fine powder. The synthesized Pa was conjugated directly or indi-
rectly using self-immolative linkers with doxorubicin (DOX, 2) or
paclitaxel (PTX, 3). The directly conjugated product, Pa–DOX (6)
and Pa–PTX (7) were synthesized by the reaction using EDCI as a
coupling reagent17,18 (Scheme 2).
side effects.10–13 Pheophorbide-a (Pa, 1) is
a chlorine based
photosensitizer derived from chlorophyll-a with photo-dependent
or -independent cytotoxic activity.14,15 Herein we report our
For the synthesis of Pa–DOX conjugates (10 and 13), hydroxy-
cinnamoyl moiety and aminobenzyloxycarbonyl moiety as self-
immolative linkers were employed. Briefly, 2-hydroxycinnamic
acid (8) was first reacted with doxorubicin hydrochloride (2) and
⇑
Corresponding authors. Tel.: +82 62 970 2502; fax: +82 62 970 2484.
E-mail addresses: kohyojin@gist.ac.kr (H. Ko), yongchul@gist.ac.kr (Y.-C. Kim).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.07.058

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Figure 1. Structure of pheophorbide-a (1) as photosensitizer, and doxorubicin (2), paclitaxel (3) as anticancer agents.
Scheme 1. Synthesis of pheophorbide-a. Reagents and conditions: (a) 1 N HCl, acetone, MeOH, rt, 4 h, 88%; (b) 80% aqueous TFA, 0 °C, 1 h, 58%.
Scheme 2. Synthesis of Pa–DOX and Pa–PTX. Reagents and conditions: (a) DOX (2), EDC, HOBt, TEA, DCM, DMF, rt, 4 h, 53%; (b) PTX (3), EDC, DMAP, DCM, rt, 4 h, 54%.

H. You et al. / Bioorg. Med. Chem. 19 (2011) 5383–5391
5385
subsequently reacted with Pa (1) using EDCI as a coupling reagent
to produce the Pa–(hydroxycinnamoyl)linker–DOX compound (10)
(Scheme 3). For the aminobenzyoxycarbonyl linker, Pa (1) was cou-
pled with 4-aminobenzyl alcohol to synthesize compound (11),
which was further reacted with 4-nitrophenyl chloroformate to af-
ford carbonate ester compound (12). Finally, doxorubicin hydro-
chloride (2) and compound (12) were conjugated to synthesize
the Pa–(aminobenzyloxycarbonyl)linker–DOX compound (13)
bearing the carbamate ester (Scheme 4).
fluorescence emission of Pa–DOX conjugate (6, 10, and 13) showed
a peak at 670 nm, the highest fluorescence being generated by
420 nm excitation. The fluorescence intensity of the emission at
670 nm of all conjugates of Pa–DOX was significantly less than that
of Pa when excited at 440 nm. This data suggested that photo-activ-
ity of the conjugates may be suppressed by the self-quenching effect
between Pa and DOX. Therefore, it could be speculated that the back-
ground fluorescence of the conjugates as they are administered in-
vivo, may not be detected until internalization and cleavage of the
conjugates by enzymatic attack in cancer cells are occurred so that
the photoactivity of Pa and DOX may be recovered. In the case of
Pa–PTX conjugate (7), fluorescence quenching effect was not ob-
served as PTX is not a fluorophore (Fig. 3).
2.2. Cell viability study
A cell viability assay was conducted to test the synthesized com-
pounds on various cancer cells including MCF7 (breast adenocarci-
noma), KB (mouth carcinoma), HeLa (cervical cancer), U-87MG
(glioblastoma), A549 (lung adenocarcinoma), AT-84 (oral cancer),
and YD-10B (oral cancer) cells. Briefly, the cells were incubated for
three days with the synthesized compounds (6, 7, 10, and 13) and
the viability was measured by the protocol of SRB assay. The activity
of the compounds is shown in Figure 2. The results showed that
To verify fluorescence quenching due to a close contact effect,
spectral comparison of fluorescence emission of compounds 6
(1 lM) and 7 (0.5 lM) in aqueous and organic solvent was investi-
gated by 420 nm excitation wavelength using fluorescence
spectrophotometer. In contact quenching, two molecules interact
by proton-coupled electron transfer through the formation of
hydrogen bonds. In aqueous solutions, electrostatic, steric and
hydrophobic forces control the formation of hydrogen bonds.
Therefore, the quenching efficiency of conjugates could depend
on the employed solvents. In fact, the intensity of fluorescence of
conjugates (6 and 7) in 50% aqueous MeOH was significantly
reduced compared with that in 100% MeOH. This data suggested
that two molecules of the conjugates stack together by proton-cou-
pled electron transfer through the formation of hydrogen bonds to
form ground state complex, nonfluorescent species as a contact
quenching mechanism because energy transfer was not observed
with the conjugates (Fig. 4).
10 lM of Pa–DOX direct conjugate (6) moderately inhibited the
growth of cancer cells including MCF7, HeLa, U-87MG, and AT-84
cells, but lower activity than DOX itself. Although the tumor specific
self-immolative linkers in the conjugates (10 and 13) would be the-
oretically cleaved to generate Pa and DOX, the Pa–linker–DOX com-
pounds showed generally less inhibitory activity of the cell viability
compared to the same concentration of Pa–DOX direct conjugate.
The results may be interpreted with possibilities that the conjugates
may suffer from entering the cells or being cleaved in the cells. In
fact, the cellular uptake of the conjugates was occurred slower than
Pa itself as shown in the study of Section 2.4.
Since the environment of cancers generally exhibits lower pH,
fluorescence profiles of conjugates, 6 and 7 either in acidic and ba-
sic conditions were explored by measuring the emission spectrum
On the other hand, Pa–PTX conjugate (7) showed potent inhibi-
tory activity of the viability of MCF7, HeLa, KB, and YD-10B cells at
10
lM. Therefore, the ester bond between Pa and PTX in the conju-
and intensity of fluorescence of compounds 6 (1
lM) and 7
gate (7) may be more easily cleaved than the amide bond of Pa–DOX
(0.3 M) at pH 4 and 10 using 420 nm of excitation wavelength.
l
conjugates in those cancer cells.
The emission spectra of fluorescence of 6 and 7 at pH 4 were
blue-shifted about 10 nm from those at pH 10 and the intensity
of fluorescence was increased at pH 4 but decreased at pH 10.
These results suggested that higher fluorescence of the conjugates
at lower pH as in the case of cancer environment may be advanta-
geous in utilizing them for the fluorescence detection of cancers
(Fig. 5).
2.3. Analysis of fluorescence spectrum
To determine fluorescence quenching effect of the conjugates,
the intensity of fluorescence of each compound with same concen-
tration was analyzed using fluorescence spectrophotometer. The
fluorescence emission spectrum of compounds at 1
lM (1, 2, 6, 10,
and 13) and 0.5 M (7) was obtained by excitation wavelengths
l
2.4. Cellular uptake study by confocal fluorescence microscopy
(420 and 440 nm). The fluorescence emission of Pa (1) showed a
peak at 665 nm, the highest fluorescence being generated by
420 nm excitation. The fluorescence emission of DOX (2) showed a
peak at 585 nm either by 420 and 440 nm excitation. The
The cellular uptake of compounds (2, 6, 10, and 13) with differ-
ent structural features was examined using confocal microscopy.
For the measurement of cellular localization of the conjugates
Scheme 3. Synthesis of Pa–(hydroxycinnamoyl)linker–DOX. Reagents and conditions: (a) EDC, HOBt, TEA, DCM, DMF, rt, 4 h, 11%; (b) Pa (1), EDC, DMAP, DCM, DMF, rt,
overnight, 24%.

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H. You et al. / Bioorg. Med. Chem. 19 (2011) 5383–5391
Scheme 4. Synthesis of Pa–(aminobenzyloxycarbonyl)linker–DOX. Reagents and conditions: (a) 4-aminobenzyl alcohol, EDC, HOBt, TEA, DCM, DMF, rt, 4 h, 38%; (b) 4-
nitrophenyl chloroformate, DIPEA, THF, rt, overnight, 41%; (c) DOX (2), TEA, DMF, rt, 6 h, 19%.
when they are cleaved, confocal microscopy was performed in
HeLa cells based on the cell viability assay result. In a preliminary
study, HeLa cells pretreated with compounds at 10 lM exhibited
4. Experimental protocols
4.1. Chemistry
fluorescence at 570–600 nm and 655–755 nm emission when ex-
cited at 440 nm, which are corresponding to those of DOX and
Pa, respectively, according to the fluorescence microscopy data.
The fluorescence confocal images of HeLa cells after incubation
with compounds for 48 h are shown in Figure 6, where the fluores-
cence of the Pa is shown in red and the fluorescence of the DOX is
shown in green, respectively. Although Pa–DOX conjugates incu-
bated with HeLa cells emitted lower fluorescence intensity com-
pared with free Pa and DOX, the florescence corresponding to
DOX and Pa, the components of conjugates was gradually in-
creased up to 48 h, which could be detected only when the cleav-
age occur. Therefore, the conjugates of Pa–DOX system developed
in this study may possess the potential properties to be used in
cancer diagnosis as well as therapeutic treatment such as more
efficient photodynamic therapies. Since Pa–linker–DOX conjugates
showed similar behavior of uptake and cleavage to that of Pa–DOX,
the reported cancer specific linkers seems not to be effective in the
system for the Pa–DOX conjugates.
Starting materials, reagents, and solvents were purchased from
Aldrich Chemical Co. (Milwaukee, WI) and TCI (Tokyo) and used as
supplied without further purification. Proton nuclear magnetic res-
onance spectroscopy was performed on a JEOL JNM-LA 300WB and
400WB spectrometer, and spectra were taken in CDCl3 or DMSO-
d6. Unless otherwise noted, chemical shifts are expressed as ppm
downfield from tetramethylsilane as the internal standard, and J
values are given in Hz. Data are reported as follows: chemical shift,
multiplicity (s, singlet; d, doublet; t, triplet; m, multiplet; b, broad;
app., apparent), coupling constants, and integration. Mass spec-
troscopy was carried out on FAB (fast atom bombardment) instru-
ments. [FAB source: JEOL FAB source and ion gun (Cs ion beam,
30 kV acceleration)]. High-resolution mass spectra (m/z) were re-
corded on a FAB (JEOL: mass range 2600 amu, 10 kV acceleration)
at Korea Basic Science Institute (Daegu).
4.1.1. Procedure for the synthesis of pheophorbide-a (1)
50% ethanol in water (9 L) was treated to chlorella 6 L for
removing polar materials and the residue was extracted twice with
100% ethanol (9 L) to obtain chlorophyll-a (4, 4.5 g). Chlorophyll-a
(4, 4.0 g, 4.38 mmol) was dissolved in acetone (40 mL) and diluted
with MeOH (100 mL). 1 N HCl (aq) (25 mL) was added to adjust
pH 2.5 and reaction mixture was stirred for 4 h at room tempera-
ture. After 4 h, water was added in reaction flask and the mixture
was stored in refrigerator for 1 day. The reaction mixture was fil-
tered and the precipitate was obtained as a black solid, pheophytin
(5, 3.38 g). Yield = 88%.
3. Conclusion
New conjugates of photosensitizer and anticancer drug using
pheophorbide-a and doxorubicin showed potential characteristics
of cancer diagnosis as well as cancer therapeutics by the study of
inhibitory activities of cancer cell viability and background fluores-
cent quenching effect of the conjugates, and cellular uptake and
cleavage monitored by confocal fluorescence microscopy. Also,
dual anticancer effect by the photosensitizer, Pa in photodynamic
therapy and anticancer drug, DOX after cell uptake and cleavage
could be expected and further study on this field is in progress.
Pheophytin (5, 3.3 g, 3.78 mmol) was dissolved in 80% aqueous
TFA (70 mL) at 0 °C, which had been bubbled with nitrogen for

H. You et al. / Bioorg. Med. Chem. 19 (2011) 5383–5391
5387
Figure 2. Cancer cell viability after 3 days in the presence of 10 lM concentrations of Pa (1), DOX (2) and conjugates (6, 7, 10, and 13).
10 min. The resulting solution was protected from light and stirred
chromatography (chloroform:methanol = 50:1) to give 6 as a
under a nitrogen blanket at 0 °C for 1 h. The reaction mixture was
concentrated to remove TFA, added to 4 N HCl (aq) (400 mL) and
extracted with ethyl acetate (3 Â 400 mL) three times. The com-
bined organic layers were dried with Na2SO4 and concentrated.
The residue was purified by silica column chromatography (chloro-
form:methanol = 30:1) to give Pa as a black solid (1, 1.3 g).
Yield = 58%; 1H NMR (400 MHz, CDCl3) d (ppm) 9.51 (s, 1H), 9.40
(s, 1H), 8.62 (s, 1H), 8.00 (dd, J = 11.6, 6.4 Hz, 1H), 6.34–6.16 (m,
3H), 4.48 (m, 1H), 4.42 (m, 1H), 3.83 (s, 3H), 3.69 (m, 2H), 3.61
(s, 3H), 3.38 (s, 3H), 3.21 (s, 3H), 2.66–2.52 (m, 2H), 2.32–2.21
(m, 2H), 1.81 (d, J = 6.8 Hz, 3H), 1.67 (t, J = 7.6 Hz, 3H); ESI
[M+H] = 593.5; HRMS (FAB) (C35H36N4O5): calcd 593.2764, found
592.2690.
brown sticky solid (50.0 mg). Yield: 53%; 1H NMR (400 MHz, CDCl3)
d (ppm) 9.22 (s, 1H), 9.17 (s, 1H), 8.43 (s, 1H), 7.91 (m, 1H), 7.54 (m,
1H), 7.39 (m, 1H), 6.94 (d, J = 8.0 Hz, 1H), 6.14 (m, 3H), 5.90 (s, 1H,
–OH), 4.90 (s, 2H), 4.62 (s, 2H), 4.41 (m, 1H), 4.21 (m, 2H), 4.12 (m,
1H), 4.04 (m, 1H), 3.85 (m, 3H), 3.72 (s, 3H), 3.52 (m, 2H), 3.38 (s,
1H, –OH), 3.23 (s, 6H), 3.14 (s, 3H), 3.02 (m, 1H), 2.86 (d,
J = 18.0 Hz, 1H), 2.60 (m, 2H), 2.36 (m, 2H), 2.04 (m, 2H), 2.01 (m,
1H), 1.85 (d, J = 14.0 Hz, 1H), 1.69 (d, J = 7.2 Hz, 3H), 1.62 (t,
J = 7.6 Hz, 3H), 1.09 (d, J = 6.0 Hz, 3H); ESI [M+H] = 1118.4; HRMS
(FAB) (C62H63N5O15): calcd 1118.4399, found 1118.4403.
4.1.3. Procedure for the synthesis of pheophorbide-a–paclitaxel
(7)
EDC (10.0 mg, 0.06 mmol) and DMAP (0.2 mg, 0.004 mmol)
were added to Pa (1, 10.0 mg, 0.02 mmol) in DCM (2 mL). Then,
paclitaxel (3, 60.0 mg, 0.70 mmol) was added to the resulting solu-
tion and the reaction mixture was stirred for 4 h at room temper-
ature. The reaction mixture was added to NaHCO3 (aq) (20 mL) and
extracted with chloroform (2 Â 20 mL). The combined organic lay-
ers were dried over MgSO4, concentrated and purified by silica gel
column chromatography (chloroform:methanol = 70:1) to give 7 as
a black sticky solid (11.5 mg). Yield: 54%; 1H NMR (400 MHz,
CDCl3) d (ppm) 9.49 (s, 1H), 9.36 (s, 1H), 8.52 (s, 1H), 8.13 (m,
2H), 8.00 (m, 1H), 7.77 (m, 2H), 7.53 (m, 1H), 7.41–7.27 (m,
4.1.2. Procedure for the synthesis of pheophorbide-a–
doxorubicin (6)
EDC (50.0 mg, 0.25 mmol), HOBt (23.0 mg, 0.17 mmol), and TEA
(25.0 lL, 0.17 mmol) were add to Pa (1, 50.0 mg, 0.08 mmol) in
DCM (3 mL). Then, doxorubicin hydrochloride (2, 60.0 mg,
0.10 mmol) in DMF was added to the resulting solution and the
reaction mixture was stirred for 4 h at room temperature. The reac-
tion mixture was added to NaHCO3 (aq) (30 mL) and extracted
with chloroform (2 Â 30 mL). The combined organic layers were
dried over MgSO4, concentrated and purified by silica gel column

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H. You et al. / Bioorg. Med. Chem. 19 (2011) 5383–5391
Figure 3. Fluorescence spectra of Pa (A), DOX (B) and conjugates (C–F). The emission wavelength with 420 and 440 nm excitation wavelength was shown in blue and red,
respectively.
10H), 7.21 (d, J = 7.2 Hz, 1H, –NH), 6.30–6.16 (m, 3H), 6.08 (s, 1H),
6.05 (s, 1H), 5.69 (d, J = 7.2 Hz, 1H), 5.62 (d, J = 3.6 Hz, 1H), 4.97 (d,
J = 9.6 Hz, 1H), 4.45 (m, 1H), 4.30 (d, J = 8.4 Hz, 1H), 4.22 (m, 2H),
3.81 (d, J = 7.2 Hz, 1H), 3.75 (s, 3H), 3.69 (m, 5H), 3.61 (d,
J = 4.8 Hz, 1H), 3.38 (s, 3H), 3.21 (s, 3H), 2.67–2.51 (m, 5H, –OH),
2.45 (s, 3H), 2.42–2.29 (m, 3H), 2.21 (s, 3H), 2.18 (m, 1H), 1.94
(m, 5H), 1.87 (s, 1H, –OH), 1.79 (d, J = 6.8 Hz, 3H), 1.70–1.65 (m,
6H), 1.23 (s, 3H), 1.13 (s, 3H); ESI [M+H] = 1429.4; HRMS (FAB)
(C82H85N18O5): calcd 1428.5968, found 1428.5973.
concentrated and purified by silica gel column chromatography
(chloroform:methanol = 60:1) to give 9 as a brown sticky solid
(137.0 mg). Yield: 11%; 1H NMR (400 MHz, DMSO) d (ppm) 9.92
(s, 1H, –OH), 7.88 (m, 1H), 7.76 (d, J = 8.8 Hz, 1H), 7.62 (m, 1H),
7.56 (d, J = 16.0 Hz, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.13 (t, J = 8.0 Hz,
1H), 6.83 (d, J = 8.4 Hz, 1H), 6.78 (t, J = 7.6 Hz, 1H), 6.71 (d,
J = 16.0 Hz, 1H), 5.47 (s, 1H), 5.22 (s, 1H), 4.93 (s, 1H, –OH), 4.84
(m, 2H), 4.56 (d, J = 5.6 Hz, 2H), 4.18 (m, 1H), 3.94 (s, 3H), 3.41 (s,
1H, –OH), 3.00 (m, 1H), 2.94 (m, 1H), 2.20 (m, 2H), 1.89 (m, 1H),
1.46 (m, 1H), 1.11 (d, J = 6.4 Hz, 3H); ESI [MÀH] = 687.8.
4.1.4. Procedure for the synthesis of pheophorbide-a–linker–
doxorubicin
4.1.4.1. Synthesis of pheophorbide-a–(hydroxycinnamoyl)-
linker–doxorubicin. 4.1.4.1.1. Pheophorbide-a –2-hydroxycin-
namic acid (9). EDC (661.4 mg, 3.45 mmol), HOBt (349.7 mg,
4.1.4.1.2. Pheophorbide-a –2-hydroxycinnamic acid–doxorubicin
(10). EDC (64.4 mg, 0.336 mmol) and DMAP (2.5 mg, 0.02 mmol)
were added to Pa (1, 100.0 mg, 0.17 mmol) in DCM (5 mL). Then,
the solution of 9 (115.9 mg, 0.86 mmol) in DMF (2 mL) was added
to the resulting solution and the reaction mixture was stirred for
overnight at room temperature. The reaction mixture was added
to NH4Cl (aq) (30 mL) and extracted with chloroform
(2 Â 30 mL). The combined organic layers were dried over MgSO4,
concentrated and purified by silica gel column chromatography
(chloroform:methanol = 70:1) to give 10 as a brown sticky solid
(51.5 mg). Yield: 24%; 1H NMR (400 MHz, CDCl3) d (ppm) 9.42 (s,
1H), 8.91 (s, 1H), 8.38 (s, 1H), 7.99 (d, J = 15.2 Hz, 1H), 7.69 (d,
2.59 mmol), and TEA (360.7 lL, 2.59 mmol) were added to 2-
hydroxycinnamic acid (8, 283.2 mg, 1.73 mmol) in DCM (10 mL).
Then, doxorubicin hydrochloride (2, 1.0 g, 1.73 mmol) in DMF
was added to the resulting solution and the reaction mixture was
stirred for 4 h at room temperature. The reaction mixture was
added to NaHCO3 (aq) (100 mL) and extracted with chloroform
(2 Â 100 mL). The combined organic layers were dried over MgSO4,

H. You et al. / Bioorg. Med. Chem. 19 (2011) 5383–5391
5389
Figure 4. Fluorescence spectra of compounds 6 (A) and 7 (B) in organic and aqueous solvent with 420 nm excitation wavelength. The emission wavelength in MeOH and
aqueous solvent was shown in blue and red, respectively.
J = 16.8 Hz, 1H), 7.64 (d, J = 6.7 Hz, 1H), 7.51 (m, 1H), 7.38 (m, 3H),
7.20 (t, J = 7.6 Hz, 1H), 7.06 (d, J = 8.0 Hz, 1H), 6.52 (d, J = 15.6 Hz,
1H), 6.23 (s, 1H), 6.05 (d, J = 8.0 Hz, 1H), 5.78 (m, 2H), 4.82 (s,
1H), 4.67 (m, 1H), 4.54 (m, 1H), 4.46 (m, 1H), 4.33 (m, 2H), 3.97–
3.83 (m, 6H), 3.71–3.59 (m, 6H, –OH(1)), 3.35 (s, 1H), 3.14 (s,
3H), 3.02 (s, 1H), 2.88 (m, 2H), 2.82 (m, 2H), 2.16 (m, 2H), 1.85–
1.75 (m, 6H), 1.63 (s, 4H), 1.27 (d, J = 6.4 Hz, 3H); ESI
[M+H] = 1264.9; HRMS (FAB) (C71H69N5O17): calcd 1264.4767,
found 1264.4763.
(m, 3H), 4.52 (s, 2H), 4.42 (s, 1H), 3.79 (s, 3H), 3.35 (m, 2H), 3.31
(s, 3H), 3.22 (s, 3H), 3.07 (s, 3H), 2.68 (m, 2H), 2.25 (m, 2H), 1.78
(d, J = 7.2 Hz, 3H), 1.59 (t, J = 7.6 Hz, 3H); ESI [MÀH] = 695.6.
4.1.4.2.2. Pheophorbide-a –4-aminobenzyl alcohol–4-nitrophenyl
formate (12). DIPEA (111.5 lL, 0.64 mmol) were added to 11
(226.5 mg, 0.32 mmol) in THF (5 mL). Then, 4-nitrophenyl chloro-
formate (96.7 mg, 0.45 mmol) in THF was dropwised to the result-
ing solution and the reaction mixture was stirred for overnight at
room temperature. The reaction mixture was added to NaHCO3
(aq) (70 mL) and extracted with chloroform (2 Â 70 mL). The com-
bined organic layers were dried over MgSO4, concentrated and
purified by silica gel column chromatography (chloroform:metha-
nol = 130:1) to give 12 as a brown sticky solid (85.5 mg). Yield:
41%; 1H NMR (400 MHz, CDCl3) d (ppm) 9.43 (s, 1H), 9.42 (s, 1H),
8.54 (s, 1H), 8.26 (d, J = 9.2 Hz, 2H), 8.04 (m, 1H), 7.36 (d,
J = 9.2 Hz, 2H), 6.88 (d, J = 8.8 Hz, 2H), 6.33–6.18 (m, 3H), 5.04 (s,
2H), 4.53 (m, 1H), 4.30 (m, 1H), 3.84 (s, 3H), 3.70 (m, 2H), 3.55
(s, 3H), 3.35 (s, 3H), 3.25 (s, 3H), 2.73 (m, 2H), 2.03 (m, 2H), 1.81
(d, J = 7.2 Hz, 3H), 1.70 (t, J = 8.0 Hz, 3H); ESI [M+H] = 862.8.
4.1.4.2.3. Pheophorbide-a –4-aminobenzyl alcohol–4-nitrophenyl
4.1.4.2. Synthesis of pheophorbide-a–(aminobenzyloxycarbon-
yl)linker–doxorubicin. 4.1.4.2.1. Pheophorbide-a–4-aminobenzyl
alcohol (11). EDC (485.4 mg, 2.53 mmol), HOBt (228.1 mg,
1.69 mmol), and TEA (235.3 lL, 2.59 mmol) were added to Pa (1,
500.0 mg, 0.84 mmol) in DCM (7 mL). Then, 4-aminobenzyl alcohol
(104.3 mg, 0.84 mmol) in DMF was added to the resulting solution
and the reaction mixture was stirred for 4 h at room temperature.
The reaction mixture was added to NaHCO3 (aq) (100 mL) and ex-
tracted with chloroform (2 Â 100 mL). The combined organic lay-
ers were dried over MgSO4, concentrated and purified by silica
gel column chromatography (chloroform:methanol = 80:1) to give
11 as a brown sticky solid (226.5 mg). Yield: 38%; 1H NMR
(400 MHz, CDCl3) d (ppm) 9.34 (s, 1H), 9.01 (s, 1H), 8.51 (s, 1H),
7.98 (m, 1H), 7.15 (d, J = 8.4 Hz, 2H), 6.66 (d, J = 8.8 Hz, 2H), 6.29
formate–doxorubicin (13). TEA (25.8 lL, 0.19 mmol) were added
to 12 (80.0 mg, 0.09 mmol) in DMF (4 mL). Then, doxorubicin
hydrochloride (2, 53.7 mg, 0.09 mmol) in DMF (1 mL) was dropw-
ised to the resulting solution and the reaction mixture was stirred

5390
H. You et al. / Bioorg. Med. Chem. 19 (2011) 5383–5391
Figure 5. pH profile of fluorescence spectra of 6 (A) and 7 (B) at pH 4 and 10 with 420 nm excitation wavelength. The emission wavelength at pH 4 and 10 was shown in blue
and red, respectively.
for 6 h at room temperature. The reaction mixture was added to
NaHCO3 (aq) (40 mL) and extracted with chloroform (2 Â 40 mL).
The combined organic layers were dried over MgSO4, concentrated
and purified by silica gel column chromatography (chloro-
4.3. Cell viability assay
4.3.1. Cell culture
The AT-84 is a squamous cell carcinoma, spontaneously arising
from oral mucosa and syngenic to C3H/HeJ mice (which were
kindly provided by Dr. E.J. Shillitoe, State University of New York,
Upstate Medical University). MCF7, KB, HCT116, PC3, U-87MG,
SK-OV-3, AT-84 cells were grown in RPMI 1640. The adherent cell
line HepG2, HeLa, A549, YD-10B cells were cultured in DMEM
medium. Both media were supplemented with 10% FBS (Invitrogen
Co.), and antibiotic–antimycotic (Invitrogen Co.). All cell lines were
incubated in a humidified atmosphere containing 5% CO2 at 37 °C.
The adherent cells were detached from the culture flasks by re-
moval of the growth medium and addition of 1 mL trypsin/EDTA
solution (0.05% w/v trypsin, 0.016% w/v EDTA). After 1–2 min incu-
bation at 37 °C, when the cells had detached from the surface, tryp-
sinization was stopped by the addition of 4 mL of DMEM medium
containing 10% FBS.
form:methanol = 80:1) to give 13 as
a brown sticky solid
(21.4 mg). Yield: 19%; 1H NMR (400 MHz, CDCl3) d (ppm) 9.19 (s,
1H), 9.14 (s, 1H), 8.39 (s, 1H), 7.90 (m, 1H), 7.42 (d, J = 7.2 Hz,
1H), 7.17 (t, J = 8.0 Hz, 1H), 6.87 (m, 4H), 6.64 (d, J = 6.8 Hz, 1H),
6.25 (m, 3H), 6.15 (d, J = 6.8 Hz, 1H), 4.90 (s, 1H), 4.72 (m, 4H),
4.43 (m, 1H), 4.18 (m, 3H), 3.90 (m, 1H), 3.79 (s, 3H), 3.70 (m,
2H), 3.59 (m, 5H), 3.47 (s, 1H), 3.30 (s, 1H), 3.21 (s, 3H), 3.19 (s,
3H), 3.05 (m, 1H), 2.98 (m, 1H), 2.67 (m, 2H), 2.24 (m, 2H), 2.06
(m, 2H), 1.77 (m, 1H), 1.72 (m, 4H), 1.60 (s, 3H), 1.18 (d,
J = 6.0 Hz, 3H); ESI [M+H] = 1268.1; HRMS (FAB) (C70H71N6O17):
calcd 1267.4876, found 1267.4871.
4.2. HPLC analysis
HPLC was used for purification of all final products. The purifi-
cation was performed on a Shimadzu SCL-10A VP HPLC system
4.3.2. SRB assay
using
a
Shimadzu Shim-pack C18 analytical column
m, 100 Å) in isocratic solvent systems. Sol-
The cells were plated at a density of 50,000 cells/mL and well in
96-well plates and incubated with inhibitors for the time indi-
cated. The SRB assay was carried out as described by Skehan
et al.19 The drug incubation period of the cells was stopped by
(250 mm  4.6 mm, 5
l
vent system was 0.1% TFA in H2O:CH3CN = 5:95 over 30 min at a
flow rate = 1 mL/min. Peaks were detected by UV absorption using
a diode array detector.
the addition of 50 lL of ice cold TCA solution into the growth

H. You et al. / Bioorg. Med. Chem. 19 (2011) 5383–5391
5391
of compounds (1, 2, 6, 10, and 13) and 0.5
lM of compound 7 in
MeOH. The samples were illuminated with 420 and 440 nm of light
and fluorescence emission was scanned from 500 to 800 nm. Fluo-
rescence spectra in both aqueous and organic solvent were gener-
ally taken at 1 lM of compound 6 and 0.5 lM of compound 7 in
100% MeOH and 50% aqueous MeOH by illumination with
420 nm of light. The pH profiles of fluorescence spectra were gen-
erally taken at 1 lM of compound 6 and 0.3 lM of compound 7 at
pH 4 and 10 in MeOH by illumination with 420 nm of light.
4.5. Confocal fluorescence microscopy
HeLa Cells were plated in the slide dishes and incubated in RPMI
1640 medium for overnight. The next day, compound of 10 lM in
complete medium was added and incubated for 48 h in a humidi-
fied atmosphere of air/CO2 (95%:5%). After washing three times
with PBS, the cells were viewed under a FluoView™ FV1000 Con-
focal Microscope. Compounds’ fluorescence was observed at
440 nm for an excitation wavelength and at 575–600 nm and
655–755 nm for an emission wavelength. The images of Pa (1)
and DOX (2) were assigned as red and green color.
Acknowledgments
This study was supported by a grant from the Institute of Med-
ical System Engineering (iMSE) in the GIST, Republic of Korea and
by a grant of the Korea Health Technology R&D Project, Ministry of
Health & Welfare, Republic of Korea (A100490).
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Figure 6. Confocal fluorescence microscope images on HeLa cell after incubation
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medium. After 1 h incubation in the refrigerator, the supernatant
was discarded and the dishes were rinsed with deionized water
five times, and dried at room temperature. One hundred microli-
ters of SRB solution was pipetted into each well and incubated at
room temperature for 1 h. Then, the staining solution was dec-
anted, the dishes were washed four times with 1% (v/v) acetic acid
and dried again at room temperature. SRB stain unspecifically
bound to protein was released by adding 1 mL of 10 mM. Tris buf-
fer per well shakes gently for 20 min. Finally the light extinction at
515 nm wavelength was read in an ELISA-reader. The mean values
of four duplicated samples were calculated. These results were ex-
pressed as
incubations.
a percentage, relative to solvent treated control
4.4. Fluorescence spectrometry
Fluorescence measurements were done using a FluoroMate FS-
2. Steady-state fluorescence spectra were generally taken at 1 lM