Molecular-target-based anticancer photosensitizer: Synthesis and in vitro photodynamic activity of erlotinib-zinc(II) phthalocyanine conjugates

Article abstract of DOI:10.1002/cmdc.201402373

Targeted photodynamic therapy is a new promising therapeutic strategy to overcome growing problems in contemporary medicine, such as drug toxicity and drug resistance. A series of erlotinib-zinc(II) phthalocyanine conjugates were designed and synthesized. Compared with unsubstituted zinc(II) phthalocyanine, these conjugates can successfully target EGFR-overexpressing cancer cells owing to the presence of the small mo-lecular- target-based anticancer agent erlotinib. All conjugates were found to be essentially non-cytotoxic in the absence of light (up to 50 mm), but upon illumination, they show significantly high photo-cytotoxicity toward HepG2 cells, with IC₅₀ values as low as 9.61-91.77 nm under a rather low light dose (λ = 670 nm, 1.5 J cm^-2). Structure-activity relationships for these conjugates were assessed by determining their photophysical/ photochemical properties, cellular uptake, and in vitro photodynamic activities. The results show that these conjugates are highly promising antitumor agents for molecular-target-based photodynamic therapy.

Full text of DOI:10.1002/cmdc.201402373

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DOI: 10.1002/cmdc.201402373
Molecular-Target-Based Anticancer Photosensitizer:
Synthesis and in vitro Photodynamic Activity of Erlotinib–
Zinc(II) Phthalocyanine Conjugates
[a]
[a]
[a]
[b]
[a]
Feng-Ling Zhang, Qi Huang, Jian-Yong Liu, Ming-Dong Huang, and Jin-Ping Xue*
Targeted photodynamic therapy is a new promising therapeu-
tic strategy to overcome growing problems in contemporary
medicine, such as drug toxicity and drug resistance. A series of
erlotinib–zinc(II) phthalocyanine conjugates were designed and
synthesized. Compared with unsubstituted zinc(II) phthalocya-
nine, these conjugates can successfully target EGFR-over-
expressing cancer cells owing to the presence of the small mo-
lecular-target-based anticancer agent erlotinib. All conjugates
were found to be essentially non-cytotoxic in the absence of
light (up to 50 mm), but upon illumination, they show signifi-
cantly high photo-cytotoxicity toward HepG2 cells, with IC50
values as low as 9.61–91.77 nm under a rather low light dose
(l=670 nm, 1.5 Jcm ). Structure–activity relationships for
these conjugates were assessed by determining their photo-
physical/photochemical properties, cellular uptake, and in vitro
photodynamic activities. The results show that these conju-
gates are highly promising antitumor agents for molecular-
target-based photodynamic therapy.
ꢀ
2
1
. Introduction
Photodynamic therapy (PDT) is a minimally invasive therapeu-
tic modality for several types of cancer and non-oncological
disorders. In PDT, reactive oxygen species (ROS), generated
through a series of photoinduced processes by the combina-
tion of a photosensitizer, light, and molecular oxygen, are be-
For the latter, the complex structure and low stability of bio-
macromolecules consistently imposes difficulties in the prepa-
ration process, as well as changes—even losses—in biological
[22]
activities during the modification process. For these reasons,
a small molecular-target-based anticancer agent with a simple
molecular structure and high stability would be a more suita-
ble moiety for conjugation with a photosensitizer to achieve
targeted PDT. However, covalent conjugation of photosensitiz-
ers with small molecular-target-based anticancer agents has
[
1–3]
lieved to be the major cytotoxic agents.
Conventional PDT
has been less commonly used for cancer therapy because the
current non-targeted photosensitizers are also taken up in
normal tissues, causing serious side effects, although the exci-
[4]
[22]
tation light itself is harmless in the near-infrared (NIR) range.
only rarely been reported within PDT studies.
The development of specifically targeted photosensitizers
could greatly decrease the side effects of PDT. Various ap-
proaches have been explored, including the use of nanoparti-
cles modified with target moieties as delivery vehicles for pho-
tosensitizers, or conjugating photosensitizers to bio-macromo-
Small molecular-target-based anticancer therapies with spe-
cific affinities toward cancer cells, achieved by modulating the
aberrant molecular pathways that underlie tumor growth and
progression, have shown tremendous success in recent de-
[23,24]
cades.
Many small molecular-target-based anticancer
[
5–10]
[11–13]
lecules with targeting functions (antibodies,
peptides,
agents that target epidermal growth factor receptors (EGFR)
and their signaling pathways have been developed and sub-
[
14–16]
[17–19]
[20,21]
proteins,
and aptamers,
etc.).
Although these two
[25]
strategies have yielded promising results, they still face chal-
lenges. For the former, the complex level of control required
for nanoparticles in terms of their size, shape, stability, drug
loading and release capacity renders the accuracy—a basic
demand for drug administration—much lower than that of the
jected to clinical trials. Such target-based anticancer agents
can pass into the cytoplasm, and can therefore be designed to
[26]
target any molecule regardless of subcellular localization.
Most tumors, particularly solid tumors, are multifactorial and
are frequently linked to defects in more than one signaling
[22]
[27]
photosensitizer-conjugated targeting biomolecule approach.
pathway.
It was therefore considered feasible to design
a conjugate in which a small target-based anticancer drug is
bound with a photosensitizer in order to greatly increase the
targeting efficiency of PDT.
[
a] Dr. F.-L. Zhang, Q. Huang, Prof. J.-Y. Liu, Prof. J.-P. Xue
Fujian Engineering Research Center for Drugs, Diagnoses and Treatment of
Photodynamic Therapy, College of Chemistry, Fuzhou University, Fuzhou,
Fujian, 350108 (P.R. China)
Erlotinib, a small molecular-target-based anticancer drug
that can target the ATP binding domain of tyrosine kinase in
epidermal growth factor receptor (EGFR)-overexpressing
tumors, has shown specific affinity for tumor cells, thereby
E-mail: xuejinping66@fzu.edu.cn
[b] Prof. M.-D. Huang
Fujian Institute of Research on the Structure of Matter
Chinese Academy of Sciences, Fuzhou, Fujian, 350002 (China)
[28]
leading to molecular targeting anticancer therapy. Phthalo-
cyanines are promising second-generation photosensitizers for
PDT, given their strong absorption of tissue-penetrating red
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201402373.
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light and high efficiency in gen-
[
29]
erating singlet oxygen.
Over
the past few years, we have
been interested in developing
efficient photosensitizers for
PDT, especially zinc(II) phthalo-
[
30,31]
cyanine derivatives.
We re-
cently reported two erlotinib–
zinc(II) phthalocyanine conju-
gates; erlotinib was chosen as
the targeting moiety to conju-
gate with a zinc(II) phthalocya-
nine core at the a-position via
an oligoethylene glycol spacer,
which can improve the amphi-
philicity and biocompatibility of
the conjugate. By introduction
of the erlotinib moiety, the two
conjugates exhibited high specif-
icity toward HepG2 cancer cells
and A431 tumor tissues in nude
mice. Meanwhile, the high pho- Scheme 1. Synthesis of erlotinib–zinc(II) phthalocyanine conjugates. Reagents and conditions: a) azido-oligoethy-
lene glycol; b) CuSO
3
1
4
·5H
,4-dicyano-1-nitrobenzene; d) DMF, K
508C, 5 h.
2
O, sodium ascorbate, THF/tBuOH/H
2
O (2:1:2), 608C, 3 h; c) 2,3-dicyano-1-nitrobenzene or
O, n-pentanol, DBU,
todynamic activities of these
conjugates were observed to be
similar to that of non-conjugated
2
CO , 508C, 5 h; e) 1,2-dicyanobenzene; f) Zn(OAc)·2H
3
2
[
22]
phthalocyanine.
In this work, we extended the study to a series of erlotinib–
zinc(II) phthalocyanine conjugates with various lengths of oli-
goethylene glycol chain as linker at different peripheral substi-
tuted positions of phthalocyanine. It was expected that the oli-
goethylene glycol linker would enhance the hydrophilicity and
decrease the tendency for aggregation of the phthalocyanine
core, while maintaining the maximum targeting capacity of er-
lotinib. The effects of linker length and peripheral substituents
on photophysical and photochemical properties, aggregation
behavior, cellular uptake, and in vitro photodynamic activities
of these conjugates were also examined.
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in n-pentanol afford-
ed the corresponding “3+1” unsymmetrical phthalocyanines
(a-1c)–(a-5c) or (b-1c)–(b-5c). All phthalocyanine derivatives
could be purified readily by silica gel column chromatography
in 11–19% yield. All new compounds were characterized by
1
H NMR, HRMS, and IR.
2.2. Photophysical and photochemical properties
The electronic absorption spectra of erlotinib–zinc(II) phthalo-
cyanine conjugates in DMF were recorded. The spectra
showed typical features of non-aggregated phthalocyanine
(Figures S1a–S6a in the Supporting Information). They dis-
played a Soret band at 320–350 nm, an intense and sharp
Q band at 678 nm for (a-1b)–(a-5b) and 672 nm for (b-1b)–
(b-5b), respectively, which strictly followed the Beer–Lambert
law. Upon excitation at 610 nm, corresponding to a shoulder in
the electronic absorption spectra of phthalocyanine conju-
gates, these conjugates show a fluorescence emission at 686
and 680 nm (Figures S1b–S6b in the Supporting Information).
The electronic absorption and fluorescence emission spectral
data for all conjugates are summarized in Table 1. Compared
2
. Results and Discussion
2.1. Molecular design and synthesis
In the title conjugates, a zinc(II) phthalocyanine was used as
the photosensitizing unit due to its high photodynamic activi-
ty. Given its specific affinity for cancer cells, erlotinib was se-
lected as the target-based moiety. The two components were
linked by an oligoethylene glycol chain, which can enhance
the amphiphilicity and biocompatibility of the system. The syn-
thesis of erlotinib–zinc(II) phthalocyanine conjugates is shown
in Scheme 1. Firstly, azido-oligoethylene glycol underwent 1,3-
dipolar cycloaddition with erlotinib in the presence of sodium
ascorbate and CuSO ·5H O to afford 1a–5a in tetrahydrofuran,
II
with unsubstituted Zn phthalocyanine (ZnPc, 670 nm), all con-
jugates exhibit a red-shifted Q band. The a-substituted phtha-
locyanine further shifts its Q band to 678 nm, while the b-sub-
stituted phthalocyanine has its Q band at 672 nm. The respec-
4
2
water, and tert-butanol. Then 1a–5a underwent nucleophilic
substitution with 3- or 4-nitrophthalonitrile to give correspond-
ing phthalonitriles (a-1b)–(a-5b) or (b-1b)–(b-5b). Mixed cycli-
zation of these phthalonitriles with excess unsubstituted
phthalonitrile (9 equiv) in the presence of Zn(OAc) ·2H O and
tive F values for (a-1c)–(a-5c) and (b-1b)–(b-5b) are 0.26
F
and 0.27; it is likely that the introduction of an electron-donat-
ing alkoxy group at the phthalocyanine macrocycle does not
influence the F value of ZnPc.
2
2
F
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Table 1. Photophysical/photochemical data for conjugates in DMF.
[
a]
[b]
[c]
Conjugate
l
max [nm] (loge)
l
em [nm]
F
F
F
D
a-1c
678 (5.49)
678 (5.25)
678 (5.37)
678 (5.28)
672 (5.32)
672 (5.35)
672 (5.49)
672 (5.45)
686
686
686
686
680
680
680
680
0.26
0.26
0.26
0.26
0.27
0.27
0.27
0.27
0.63
0.66
0.63
0.57
0.53
0.54
0.53
0.50
[
[
22]
22]
a-3c
a-4c
a-5c
b-1c
b-3c
b-4c
b-5c
[
33]
[a] Excitation at 610 nm. [b] Relative to ZnPc (F
F
=0.28 in DMF). [c] Rel-
ative to ZnPc (F
D
=0.56 in DMF). Data listed are from a single experi-
ment.
To evaluate the photosensitizing efficiency of these conju-
gates, their singlet oxygen generation quantum yield (F ) was
D
examined in DMF by a steady-state method using 1,3-dipheny-
[
32]
lisobenzofuran (DPBF) as the scavenger. All the FD values
are summarized in Table 1. The results show that a-substituted
Figure 1. Confocal fluorescence images of mixed HepG2 (regions 4–6) and
HELF (regions 1–3) cells after incubation with a-4c, b-4c, and ZnPc for 24 h
phthalocyanines, with similar quantum yields (F =0.57-0.66),
D
are slightly better singlet oxygen generators than b-substituted
phthalocyanines (F =0.50–0.54) and ZnPc (F =0.56). The
(
all at 10 mm). Scale bar: 40 mm.
D
D
data listed in Table 1 also show that the length of the oligo-
ethylene glycol chain (with n=0, 2, 3, and 4) does not overtly
affect the photophysical and photochemical properties of the
conjugates.
2.3. In vitro specificity toward cancer cells
To assess the specificity of these conjugates for cancer cells,
we mixed HELF cells (human embryo lung fibroblasts, a cell
line with low expression of EGFR) and HepG2 cells (human
[34]
hepatocarcinoma cells, an EGFR-overexpressing cell line ),
which show significant morphological differences from each
other in a cell culture dish. Unsubstituted ZnPc was used as
control. After incubation with conjugate or ZnPc in the culture
medium for 24 h, the fluorescence emitted (l : 633 nm, moni-
ex
tored at lem: 650–750 nm) in these two cell lines was deter-
mined by confocal laser scanning microscope. As shown by
the images captured (Figure 1, and Figures S7–S9 of the Sup-
porting Information), all conjugates clearly showed higher fluo-
rescence in HepG2 cancer cells than in HELF cells. In contrast,
the fluorescence intensity of ZnPc in both cell types showed
nearly no difference; the relative intracellular fluorescence in-
tensity of ZnPc in HepG2 cells is equal to that in HELF cells.
The results indicate that all erlotinib–zinc(II) phthalocyanine
conjugates can successfully target cancer cells with high EGFR
expression due to the erlotinib moiety.
Figure 2. Cellular uptake of conjugates and ZnPc at 10 mm by HepG2 and
HELF cells. Data are expressed as the meanꢁSD of three independent ex-
periments, each performed in sextuple. Statistical significance: **p<0.01,
*
p<0.05.
conjugates by HepG2 cancer cells is clearly higher than that by
HELF cells, whereas for the reference compound ZnPc, uptake
by both cell types is nearly identical. Highly significant differen-
ces between HELF and HepG2 cells were observed in the
uptake of a-1c, b-1c, a-3c, b-3c, a-4c, b-4c, and b-5c (p<
0.01), and a significant difference for a-5c (p<0.05) was ob-
served. It appears that the length of the oligoethylene glycol
chain influences the cellular uptake of these conjugates for
HepG2 cancer cells. Interestingly, the cellular uptake of conju-
gates with linker n=3 (a-4c and b-4c) by HepG2 cancer cells
To further test their targeting ability, the quantitative cellular
uptake rates of these conjugates and ZnPc by HepG2 and
HELF cells was also examined by fluorescence spectrometry.
After incubation with these compounds, 1% SDS was used to
lyse the cells and extract the dyes. The dye concentrations
inside the cells were quantified by measuring their fluores-
cence at 686 or 680 nm (l : 610 nm). The results are depicted
ex
in Figure 2, which shows that the cellular uptake of the title
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is clearly higher than for other conjugates, and the uptake
ratio of for these two between HepG2 and HELF cells is signifi-
cantly higher than for other conjugates. This indicates that the
oligoethylene glycol chain can affect the targeting capacity of
these conjugates.
[
a]
Table 2. Cytotoxicity of conjugates against HepG2 cells.
Conjugate
IC50 [nm]
Conjugate
IC50 [nm]
a-1c
12.88ꢁ0.68
9.61ꢁ2.49
27.79ꢁ2.61
44.50ꢁ3.28
43.30ꢁ4.72
b-1c
b-3c
b-4c
b-5c
33.97ꢁ3.84
44.83ꢁ5.68
41.86ꢁ3.91
91.77ꢁ10.58
[
22]
22]
a-3c
a-4c
a-5c
[
ZnPc
2.4. In vitro photodynamic activities
ꢀ2
ꢀ2
[
a] l=670 nm, 80 mWcm , 60 s, 1.5 Jcm . Data are the meanꢁSEM of
three independent experiments, each performed in sextuple.
The in vitro photodynamic activities of these conjugates and
ZnPc were investigated against HepG2 cells. The dose-depen-
dent survival curves for all conjugates are shown in Figure 3;
the cytotoxicity of ZnPc was not investigated due to its insolu-
bility above 5 mm in the dark in RPMI 1640. It can be seen that
Compared with ZnPc, with an IC50 value of 43.30 nm upon il-
lumination, all conjugates retained the high photodynamic ac-
tivity of the phthalocyanine core. It can also be seen that the
photo-cytotoxicities of a-substituted phthalocyanines (9.61–
44.50 nm) tend to be slightly higher than those of the corre-
sponding b-substituted conjugates (33.97–91.77 nm). Evidently,
the photo-cytotoxicity of the conjugates decreases slightly
with increasing oligoethylene glycol chain length.
The aggregation state of photosensitizers is an important
factor relating to their photodynamic activities. Aggregation
usually leads to a significant decrease in, or complete suppres-
sion of, photodynamic activity. The aggregation behavior of
the title conjugates and ZnPc in formulation with Cremophor
EL in RPMI 1640 was examined by absorption spectroscopy. As
is the case with ZnPc, the conjugates show a relatively sharp
and intense Q band, as shown in Figure 4. The results suggest
Figure 4. Electronic absorption spectra of title conjugates in RPMI 1640 cell
culture medium (all at 10 mm).
Figure 3. Cytotoxic effects of title conjugates toward HepG2 cells in the
ꢀ
2
ꢀ2
a) presence and b) absence of light (l=670 nm, 80 mWcm , 1.5 Jcm ).
Data are the meanꢁSEM of three independent experiments, each per-
formed in sextuple.
that the conjugates are not significantly aggregated under
these conditions, which seems to be in accord with their high
photo-cytotoxicity.
all these conjugates are essentially non-cytotoxic in the ab-
sence of light up to 50 mm (Figure 3b). However, upon illumi-
nation, they all show significantly high photo-cytotoxicity
toward HepG2 cells with IC50 values (defined as the dye con-
centration required to kill 50% of the cells) as low as 9.61–
The subcellular localization of these conjugates was also in-
vestigated by confocal laser scanning microscopy. The cells
were first incubated with the conjugate at 10 mm in the culture
medium for 24 h, and then stained with LysoTracker DND 26 or
MitoTracker Green FM (30 min), which are specific dyes for ly-
sosomes and mitochondria, respectively. As shown in Figure 5a
9
1.77 nm (Figure 3a, Table 2) under a relatively low light dose
ꢀ
2
ꢀ2
(
l=670 nm, 80 mWcm , 1.5 Jcm ).
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Figure 5. a) Confocal fluorescence images of HepG2 cells after incubation with b-4c (10 mm) for 24 h. Scale bar: 30 mm. b) Fluorescence intensity profiles of b-
c (c) and Tracker (c) traced along the white lines in the micrograph images.
4
and Figures S10a–S14a (in the Supporting Information), the
fluorescence caused by LysoTracker (excitation at 488 nm,
monitored at 510–570 nm) superimposes well with the fluores-
cence caused by conjugates (excitation at 633 nm, monitored
at 650–750 nm). The very similar fluorescence intensity line
profiles of conjugates (red) and LysoTracker (black) traced
along the white line in the merged images of Figure 5b and
Figures S10b–S14b (in the Supporting Information) also con-
firmed that conjugates can localize in the cell lysosomes. By
contrast, the fluorescence images of conjugates could be
slightly merged with that of MitoTracker (excitation at 633 nm,
monitored at 650–750 nm), indicating that these conjugates
are partly localized in the mitochondria. Compared with these
conjugates, ZnPc exhibits different subcellular localization: it
can localize both in the lysosomes and mitochondria (Fig-
ure S15, Supporting Information). The difference between ZnPc
and conjugates in subcellular localization may be explained by
the varying degree of protonation caused by lower lysosomal
pH, and by the conjugates becoming more water soluble
through the presence of erlotinib; in this way they would be
detained in the lysosomes due to greater difficulty in crossing
the lysosomal membrane.
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Compound 1a: According to the general procedure, a mixture of
erlotinib (3.00 g, 7.63 mmol) was treated with azido-oligoethylene
glycol (1.00 g, 11.49 mmol), CuSO ·5H O (0.20 g, 0.80 mmol), and
3
. Conclusions
In summary, we have synthesized and characterized a series of
novel erlotinib–zinc(II) phthalocyanine conjugates and evaluat-
ed their specific affinity for HepG2 cancer cells and in vitro
photodynamic activities. With a photodynamic group covalent-
ly tethered to a small-molecule targeting agent, these conju-
gates function cooperatively as potential anticancer agents. In-
troduction of the erlotinib moiety enhanced the cellular
uptake and specificity for HepG2 cancer cells of the phthalo-
cyanine unit. The length of the oligoethylene glycol chain can
affect the targeting ability of these conjugates; compounds
with linker of n=3 (a-4c and b-4c) exhibited the highest tar-
geting capacity. All erlotinib–zinc(II) phthalocyanine conjugates
retained the high photo-cytotoxicity of the phthalocyanine
core toward HepG2 cells (9.61–91.77 nm). It seems that both
the peripheral substituted position of the phthalocyanine core
and the length the linker have a slight effect on the photo-cy-
totoxicity of these conjugates. a-Substituted phthalocyanines
4
2
sodium ascorbate (0.30 g, 1.53 mmol) in THF (10 mL), H O (5 mL),
2
and tBuOH (10 mL) to give 1a as a white solid (1.98 g, 54%).
1
H NMR (400 MHz, [D ]DMSO): d=3.39 (s, 3H, CH ), 3.41 (s, 3H,
6
3
CH ), 3.78 (virt.t, J=4.4 Hz, 2H, CH ), 3.82 (virt.t, J=4.8 Hz, 2H,
3
2
CH ), 3.85–3.89 (m, 2H, CH ), 4.31–4.36 (m, 4H, CH ), 4.49 (t, J=
2
2
2
5.6 Hz, 2H, CH ), 5.13 (virt.t, J=5.6 Hz, 1H, OH), 7.26 (s, 1H, Ar-H),
2
7.49 (t, J=8.0 Hz, 1H, Ar-H), 7.59 (d, J=8.0 Hz, 1H, Ar-H), 7.93 (d,
J=8.4 Hz, 1H, Ar-H), 7.96 (s, 1H, Ar-H), 8.30 (s, 1H, Ar-H), 8.52 (s,
1
H, Ar-H), 8.58 (s, 1H, Ar-H), 9.60 ppm (s, 1H, N-H); HRMS (ESI): m/z
+
calcd for C H N O [M+H] , 481.2193, found 481.2236; IR: u
24
28
6
5
as
(
(
-CH ) 2924, u (-CH ) 1384, u (Ar) 1667, 1622, 1529, 1504, 1449, u
3 s 3
CꢀOꢀC) 1249, 1203, 1113, 1066, 1034 cm .
as
ꢀ1
Compound 4a: According to the general procedure, a mixture of
erlotinib (3.00 g, 7.63 mmol) was treated with azido-oligoethylene
glycol (2.00 g, 9.13 mmol), CuSO ·5H O (0.20 g, 0.80 mmol), and
4
2
sodium ascorbate (0.30 g, 1.53 mmol) in THF (10 mL), H O (5 mL),
2
and tBuOH (10 mL) to give 4a as a white solid (4.27 g, 91%).
1
H NMR (400 MHz, CDCl ): d=3.43 (s, 3H, CH ), 3.44 (s, 3H, CH ),
3
3
3
(
IC : 9.61–44.50 nm) tend to slightly more potent than the cor-
3.53 (virt.t, J=4.8 Hz, 2H, CH ), 3.57 (s, 4H, CH ), 3.59 (s, 4H, CH ),
2 2 2
50
responding b-substituted phthalocyanines (IC : 33.97–
3.69 (virt.t, J=4.4 Hz, 2H, CH ), 3.80–3.87 (m, 6H, CH ), 4.25 (t, J=
50
2
2
9
1.77 nm), and the photo-cytotoxicity of the conjugates slight-
4.4 Hz, 2H, CH
2
), 4.29 (t, J=4.4 Hz, 2H, CH
2
), 4.54 (t, J=4.8 Hz, 2H,
CH ), 7.20 (s, 1H, Ar-H), 7.41 (virt.t, J=8.6 Hz, 1H, Ar-H), 7.49 (s, 1H,
ly decreases with increasing oligoethylene glycol chain length.
These conjugates also show preferential localization in lyso-
somes. Overall, our results show that these erlotinib–zinc(II)
phthalocyanine conjugates are highly promising antitumor
agents for molecular-target-based PDT.
2
Ar-H), 7.58 (d, J=8.0 Hz, 1H, Ar-H), 7.92 (d, J=8.0 Hz, 1H, Ar-H),
7
.99 (s, 1H, Ar-H), 8.00 (s, 1H, Ar-H), 8.31 (brs, 1H, N-H), 8.62 ppm
+
(s, 1H, Ar-H); HRMS (ESI): m/z calcd for C H N O [M+H] ,
30 40 6 8
6
13.2980, found 613.2988; IR: uas (-CH ) 2927, 2874, u (-CH ) 1382,
3 s 3
u (Ar) 1628, 1575, 1505, 1426, u (CꢀOꢀC) 1241, 1200, 1124,
as
ꢀ
1
1
062 cm .
General procedure for the preparation of phthalonitriles a-1b,
a-4b, b-1b, b-3b, b-4b, and b-5b: Compounds 1a–5a (1 equiv),
Experimental Section
Chemistry
2
,3-dicyano-1-nitrobenzene (1.2 equiv), or 3,4-dicyano-1-nitroben-
zene (1.2 equiv) were completely dissolved in DMF followed by the
General: All reactions were performed under an atmosphere of ni-
trogen. n-Pentanol was distilled from sodium. Azido-oligoethylene
glycol was prepared as described. All other solvents and reagents
addition of K CO (4 equiv). The mixture was stirred at 508C under
2
3
an atmosphere of nitrogen for 5 h. DMF was evaporated under re-
duced pressure, and then the residue was mixed with a saturated
aqueous solution of NaCl and extracted with CH Cl . The obtained
2 2
1
were of reagent grade and were used as received. H NMR spectra
1
were recorded on an AVANCE III 400 ( H, 400 MHz) spectrometer in
organic extracts were dried in vacuo. The residue was purified by
CDCl or [D ]DMSO. Chemical shifts (d) are expressed in ppm rela-
3
6
silica gel column chromatography using CH Cl /CH OH (30:1 v/v)
2
2
3
tive to TMS (0 ppm). HRMS analyses were carried out on an ion
trap MS DECAX-30000 LCQ Deca XP mass spectrometer. IR spectra
were measured on a PerkinElmer SP2000 instrument. Electronic ad-
sorption spectra were measured on a Beijing PuXi Tu-1901 spec-
trometer. Fluorescence spectra were obtained on a Varian Cary
eclipse spectrometer. Subcellular location was determined with an
Olympus FV1000 confocal laser scanning microscope. Compounds
azido-oligoethylene glycol, 3a, 5a, a-3b, a-5b, a-3c, and a-5c
as eluent.
Phthalonitrile a-1b: According to the general procedure, a mixture
of 1a (2.00 g, 4.17 mmol) and 2,3-dicyano-1-nitrobenzene (0.87 g,
5.03 mmol) was treated with K CO (2.30 g, 16.68 mmol) in 15 mL
2
3
1
DMF to give a brown solid (1.50 g, 58%). H NMR (400 MHz, CDCl ):
3
d=3.45 (s, 3H, CH ), 3.47 (s, 3H, CH ), 3.85–3.87 (m, 4H, CH ), 4.28
3
3
2
(virt.t, J=4.4 Hz, 2H, CH ), 4.31 (virt.t, J=4.4 Hz, 2H, CH ), 4.45
2
2
[22]
were prepared described in our previous work. The purity of all
new compounds was determined by HPLC analysis and was found
to be ꢂ95%.
(virt.t, J=4.8 Hz, 2H, CH ), 4.85 (virt.t, J=4.4 Hz, 2H, CH ), 7.15 (d,
2 2
J=8.8 Hz, 1H, Ar-H), 7.21 (s, 1H, Ar-H), 7.35–7.38 (m, 2H, Ar-H),
7.43 (virt.t, J=8.0 Hz, 1H, Ar-H), 7.57–7.64 (m, 2H, Ar-H), 7.92 (brs,
1
1
H, N-H), 7.99 (s, 1H, Ar-H), 8.05 (d, J=8.0 Hz, 1H, Ar-H), 8.15 (s,
General procedure for the preparation of compounds 1a and
H, Ar-H), 8.65 ppm (s, 1H, Ar-H); HRMS (ESI): m/z calcd for
4
a: A mixture of erlotinib (1 equiv), azido-oligoethylene glycol (1.2
+
C H N O [M+H] , 607.2412; found: 607.2435; IR: u (-CN) 2238,
32
30
8
5
or 1.5 equiv), CuSO ·5H O (0.1 equiv), and sodium ascorbate
4
2
uas (-CH ) 2927, u (-CH ) 1396, u (Ar) 1631, 1575, 1508, 1452, 1411,
3
s
3
(
0.2 equiv) in THF/H O/tBuOH (2:1:2 v/v/v) was stirred at 608C
2
ꢀ1
uas (CꢀOꢀC) 1220, 1184, 1407 cm .
under an atmosphere of nitrogen for 3 h. The volatiles were re-
moved in vacuo. The residue was the mixed with H O (100 mL)
Phthalonitrile a-4b: According to the general procedure, a mixture
of 4a (2.00 g, 3.27 mmol) and 2,3-dicyano-1-nitrobenzene (0.68 g,
3.93 mmol) was treated with K CO (1.81 g, 13.08 mmol) in 15 mL
2
and extracted with CH Cl2 (3ꢁ80 mL). The obtained organic ex-
2
tracts were dried over anhydrous MgSO . After evaporation of the
4
2
3
1
solvent under reduced pressure, the residue was purified by silica
gel column chromatography using CH Cl /EtOAc/CH OH (20:1:1
DMF to give a brown solid (1.36 g, 56%). H NMR (400 MHz, CDCl ):
3
2
2
3
d=3.46 (s, 3H, CH ), 3.47 (s, 3H, CH ), 3.60–3.67 (m, 8H, CH ), 3.75
3
3
2
v/v/v) as eluent.
ꢀ
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 10
&
6
&
ÞÞ
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CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
(
(
(
(
virt.t, J=4.4 Hz, 2H, CH ), 3.85 (virt.t, J=4.8 Hz, 4H, CH ), 3.90
(m, 2H, CH ), 3.83–3.90 (m, 6H, CH ), 4.05–4.08 (m, 2H, CH ), 4.28
(virt.t, J=4.8 Hz, 2H, CH ), 4.31 (virt.t, J=4.4 Hz, 2H, CH ), 4.56 (t,
2 2
2
2
2
2
2
virt.t, J=5.2 Hz, 2H, CH ), 4.10 (virt.t, J=4.0 Hz, 2H, CH ), 4.27
2
2
virt.t, J=4.4 Hz, 2H, CH ), 4.43 (virt.t, J=4.8 Hz, 2H, CH ), 4.56
J=4.8 Hz, 2H, CH ), 7.01–7.04 (m, 1H, Ar-H), 7.17 (d, J=2.4 Hz, 1H,
2
2
2
virt.t, J=4.8 Hz, 2H, CH ), 7.09 (d, J=8.8 Hz, 1H, Ar-H), 7.21 (s, 1H,
Ar-H), 7.22 (brs, 1H, Ar-H), 7.43 (virt.t, J=8.0 Hz, 2H, Ar-H), 7.57–
7.60 (m, 2H, Ar-H), 7.96 (d, J=8.0 Hz, 1H, Ar-H), 8.06 (s, 2H, Ar-H),
8.62 ppm (brs, 1H, Ar-H); HRMS (ESI): m/z calcd for C H N O
9
2
Ar-H), 7.28 (s, 1H, Ar-H), 7.38–7.441 (m, 2H, Ar-H), 7.50 (d, J=
.0 Hz, 1H, Ar-H), 7.53–7.56 (m, 1H, Ar-H), 7.89 (d, J=9.2 Hz, 1H,
8
4
0
46
8
+
Ar-H), 8.03 (s, 1H, Ar-H), 8.06 (s, 1H, Ar-H), 8.61 ppm (s, 1H, Ar-H);
HRMS (ESI): m/z calcd for C H N O [M+H] , 739.3198, found
7
1
1
[M+H] , 783.3460, found 783.3483; IR: u (CꢀN) 1243, u (CN) 2230,
+
u (-CH -) 2877, u (CꢀOꢀC) 1122, 1095, 1047, u (ArꢀOꢀR) 1293,
38
42
8
8
s
2
as
ꢀ
1
39.3166; IR: u (-CN) 2360, uas (-CH ) 2918, u (-CH ) 1334, u (Ar)
u (C=C) 1621, u (Ar) 1597, 1581, 1541, g (Ar-H) 788, 865 cm .
3
s
3
664, 1623, 1582, 1504, 1427, u (CꢀOꢀC) 1293, 1240, 1206, 1123,
as
ꢀ
1
General procedure for the preparation of phthalocyanines a-1c,
a-4c, b-1c, b-3c, b-4c, and b-5c: A mixture of the above phthalo-
064 cm .
Phthalonitrile b-1b: According to the general procedure, a mixture
of 1a (2.00 g, 4.16 mmol) and 3,4-dicyano-1-nitrobenzene (0.86 g,
nitriles (1 equiv), 1,2-dicyanobenzene (9 equiv), and Zn(OAc)·2H O
2
(5 equiv) in n-pentanol (10 mL) was heated at 1008C under an at-
mosphere of nitrogen, then 0.5 mL DBU was added. The mixture
was stirred at 1508C under an atmosphere of nitrogen for 5 h. The
volatiles were removed in vacuo, and then the residue was mixed
with H O and extracted with CH Cl . The obtained organic extracts
4
.99 mmol) was treated with K CO (2.30 g, 16.68 mmol) in 15 mL
2 3
1
DMF to give a brown solid (1.57 g, 62%). H NMR (400 MHz, CDCl ):
3
d=3.46 (s, 6H, CH ), 3.82–3.86 (m, 4H, CH ), 3.94 (s, 1H, N-H),
3
2
4
.27–4.30 (m, 4H, CH ), 4.52 (virt.t, J=5.2 Hz, 2H, CH ), 4.84 (virt.t,
2 2
2
2
2
J=5.2 Hz, 2H, CH ), 7.17 (d, J=8.1 Hz 1H, Ar-H), 7.23 (s, 1H, Ar-H),
were dried under reduced pressure. The residue was purified by
2
7
.43 (virt.t, J=7.6 Hz, 1H, Ar-H), 7.50 (d, J=8.0 Hz, 1H, Ar-H), 7.58
silica gel column chromatography using CH Cl /CH OH (30:1 v/v)
2
2
3
(
brs, 1H, Ar-H), 7.68 (d, J=8.4 Hz, 1H, Ar-H), 7.85 (d, J=7.6 Hz, 1H,
as eluent.
Ar-H), 7.91 (s, 1H, Ar-H), 8.14 (s, 1H, Ar-H), 8.63 ppm (s, 1H, Ar-H);
+
Phthalocyanine a-1c: According to the general procedure, a mix-
ture of a-1b (0.25 g, 0.40 mmol) and 1,2-dicyanobenzene (0.46 g,
HRMS (ESI): m/z calcd for C H N O [M+H] , 607.2412, found
32
30
8
5
6
07.2451; IR: u (CN) 2229, u (CꢀN) 1251, u (-CH -) 2927, u (CꢀOꢀ
as
2
as
3
.59 mmol) was treated with Zn(OAc)·2H O (0.44 g, 2.01 mmol) in
2
C) 1153, 1123, 1068, u (ArꢀOꢀR) 1205, u (C=C) 1620, u (Ar) 1579,
509, 1490, d (CH ) 1385, g (Ar-H) 786, 833, 862, 891 cm .
ꢀ1
10 mL n-pentanol at 1008C under an atmosphere of nitrogen, then
.5 mL DBU was added, and the mixture was stirred at 1508C
1
3
0
Phthalonitrile b-3b: According to the general procedure, a mixture
of 3a (2.00 g, 3.52 mmol) and 3,4-dicyano-1-nitrobenzene (0.73 g,
under an atmosphere of nitrogen for 5 h to give a blue solid
1
(48 mg, 11%). H NMR (400 MHz, [D ]DMSO): d=3.26 (s, 3H, CH ),
6
3
4
.22 mmol) was treated with K CO (2.00 g, 14.49 mmol) in 15 mL
3.43 (s, 3H, CH ), 3.46–3.47 (m, 2H, CH ), 3.55 (brs, 2H, CH ), 3.81
3 2 2
(brs, 2H, CH ), 4.12 (brs, 2H, CH ), 5.12 (brs, 2H, CH ), 5.51 (brs,
2 2 2
2
3
1
DMF to give a brown solid (1.25 g, 51%). H NMR (400 MHz, CDCl ):
3
d=3.46 (s, 6H, CH ), 3.65 (s, 4H, CH ), 3.73 (virt.t, J=4.8 Hz, 2H,
2H, CH ), 5.67 (s, 1H, Ar-H), 6.47 (s, 1H, Ar-H), 6.67 (s, 1H, Ar-H),
3
2
2
CH ), 3.84 (virt.t, J=4.8 Hz, 4H, CH ), 3.92 (t, J=4.8 Hz, 2H, CH ),
6.72 (t, J=7.6 Hz, 1H, Ar-H), 6.93 (d, J=8.0 Hz, 1H, Ar-H), 7.04 (d,
J=7.2 Hz, 1H, Ar-H), 7.49 (s, 1H, Ar-H), 7.70 (d, J=8.0 Hz, 1H, Ar-
H), 7.90 (virt.t, J=7.6 Hz, 1H, Pc-H ), 8.00 (t, J=7.6 Hz, 1H, Pc-H ),
2
2
2
4
4
.00 (virt.t, J=4.0 Hz, 2H, CH ), 4.26 (virt.t, J=4.4 Hz, 2H, CH ),
2 2
.31 (t, J=4.4 Hz, 2H, CH ), 4.57 (t, J=4.8 Hz, 2H, CH ), 7.00–7.03
2
2
b
b
(
m, 1H, Ar-H), 7.06 (d, J=2.4 Hz, 1H, Ar-H), 7.21 (s, 1H, Ar-H), 7.35–
8.08 (t, J=7.6 Hz, 1H, Pc-H ), 8.23–8.24 (m, 4H, Pc-H ), 8.37 (s, 1H,
b b
7
1
1
.39 (m, 2H, Ar-H), 7.48 (d, J=8.0 Hz, 1H, Ar-H), 7.56 (d, J=8.8 Hz,
H, Ar-H), 7.77 (d, J=8.4 Hz, 1H, Ar-H), 7.96 (s, 2H, Ar-H), 8.23 (s,
Pc-H ), 8.94 (d, J=7.2 Hz, 1H, Pc-H ), 9.14 (d, J=7.6 Hz, 1H, Pc-H ),
b a a
9.19 (d, J=7.6 Hz, 1H, Pc-H ), 9.24–9.25 (m, 1H, Pc-H ), 9.29–9.31
a
a
H, N-H), 8.61 ppm (s, 1H, Ar-H); HRMS (ESI): m/z calcd for
(m, 3H, Pc-H ), 10.46 ppm (s, 1H, N-H); HRMS (ESI): m/z calcd for
a
+
+
C H N O [M+H] , 695.2936, found 695.2993; IR: u (CN) 2228, u
C H N O Zn [M+H] , 1055.2827, found 1055.2882; IR: u (Pc)
3
6
38
8
7
as
56 42 14
5
(
-CH -) 2925, u (CꢀOꢀC) 1122, 1095, 1031, u (ArꢀOꢀR) 1206, u (C=
1622, 1578, 1493, 1428, u (CꢀN) 1335, u (ArꢀOꢀR) 1258, uas (Cꢀ
2
as
as
ꢀ1
C) 1621, u (Ar) 1598, 1503, 1425, d (CH ) 1384, g (Ar-H) 659, 691,
7
OꢀC) 1121, 1083, 1053 cm .
3
ꢀ
1
86, 862 cm .
Phthalocyanine a-4c: A mixture of a-4b (0.25 g, 0.34 mmol) and
Phthalonitrile b-4b: According to the general procedure, a mixture
of 4a (2.00 g, 3.27 mmol) and 3,4-dicyano-1-nitrobenzene (0.68 g,
1,2-dicyanobenzene (0.39 g, 3.59 mmol) was treated with Zn-
(OAc)·2H O (0.37 g, 1.70 mmol) in 10 mL n-pentanol at 1008C
2
3
.92 mmol) was treated with K CO (1.81 g, 13.08 mmol) in 15 mL
under an atmosphere of nitrogen, and then 0.5 mL DBU was
2
3
1
DMF to give a brown solid (1.30 g, 53%). H NMR (400 MHz, CDCl ):
d=3.46 (s, 6H, CH ), 3.62 (s, 8H, CH ), 3.75–3.77 (m, 2H, CH ),
added, and the mixture was stirred at 1508C under an atmosphere
3
1
of nitrogen for 5 h to give a blue solid (46.95 mg, 14%). H NMR
3
2
2
3
.83–3.90 (m, 6H, CH ), 4.06 (virt.t, J=4.0 Hz, 2H, CH ), 4.27 (virt.t,
(400 MHz, [D ]DMSO): d=3.29 (s, 3H, CH ), 3.32 (s, 3H, CH ), 3.52–
2
2
6
3
3
J=3.6 Hz, 2H, CH ), 4.32 (virt.t, J=4.0 Hz, 2H, CH ), 4.55 (virt.t, J=
3.53 (m, 2H, CH ), 3.55–3.56 (m, 2H, CH ), 3.64–3.66 (m, 2H, CH ),
2 2 2
2
2
4
.4 Hz, 2H, CH ), 7.04–7.06 (m, 1H, Ar-H), 7.16 (d, J=2.4 Hz, 1H, Ar-
3.67–3.70 (m, 4H, CH ), 3.82 (t, J=5.2 Hz, 2H, CH ), 3.97 (t, J=
2 2
2
H), 7.22 (s, 1H, Ar-H), 7.40 (virt.t, J=8.0 Hz, 2H, Ar-H), 7.51 (d, J=
4.4 Hz, 2H, CH ), 4.12–4.16 (m, 4H, CH ), 4.24–4.26 (m, 2H, CH ),
2 2 2
7
.6 Hz, 1H, Ar-H), 7.60 (d, J=8.8 Hz, 1H, Ar-H), 7.91 (d, J=7.6 Hz,
4.51 (t, J=4.8 Hz, 2H, CH ), 4.68 (brs, 2H, CH ), 6.91 (s, 1H, N-H),
2 2
1
H, Ar-H), 7.97 (s, 1H, Ar-H), 8.10 (s, 1H, Ar-H), 8.61 ppm (s, 1H, Ar-
7.39 (virt.t, J=8.0 Hz, 1H, Ar-H), 7.44 (d, J=8.0 Hz, 1H, Ar-H), 7.50
(d, J=7.6 Hz, 1H, Ar-H), 7.71 (s, 1H, Ar-H), 7.81 (d, J=8.8 Hz, 1H,
Ar-H), 7.87 (t, J=7.6 Hz, 1H, Ar-H), 8.05 (t, J=7.2 Hz,1H, Ar-H), 8.10
+
H); HRMS (ESI): m/z calcd for C H N O [M+H] , 739.3198, found
38
42
8
8
7
39.3198; IR: u (CꢀN) 1240, u (CN) 2228, u (-CH -) 2918, u (CꢀOꢀ
as
2
as
C) 1063, 1123, u (ArꢀOꢀR) 1207, u (C=C) 1621, u (Ar) 1575, 1505,
(d, J=7.2 Hz, 1H, Ar-H), 8.13–8.19 (m, 4H, Pc-H ), 8.22–8.23 (m, 2H,
b
ꢀ
1
1
486, g (Ar-H) 659, 688, 790, 860 cm .
Pc-H ), 8.47 (s, 1H, Pc-H ), 8.64 (d, J=7.6 Hz, 1H, Pc-H ), 8.98 (d, J=
b
b
b
7
.2 Hz, 1H, Pc-H ), 9.12–9.16 (m, 2H, Pc-H ), 9.20–9.22 (m, 3H, Pc-
a a
Phthalonitrile b-5b: According to the general procedure, a mixture
of 5a (2.00 g, 3.05 mmol) and 3,4-dicyano-1-nitrobenzene (0.63 g,
Ha), 9.40 ppm (s, 1H, Pc-Ha); HRMS (ESI): m/z calcd for
C H N O Zn [M+H] , 1187.3613, found 1187.3690; IR: u (Pc)
+
62
54 14
8
3
.66 mmol) was treated with K CO (1.68 g, 12.20 mmol) in 15 mL
2 3
1
625, 1578, 1490, 1431, uas (CꢀN) 1329, u (ArꢀOꢀR) 1238, uas (Cꢀ
1
DMF to give a brown solid (1.21 g, 50%). H NMR (400 MHz, CDCl ):
ꢀ1
3
OꢀC) 1127, 1088, 1047 cm .
d=3.47 (s, 6H, CH ), 3.61 (s, 8H, CH ), 3.65 (s, 4H, CH ), 3.74–3.76
3
2
2
ꢀ
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 10
&
7
&
ÞÞ
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CHEMMEDCHEM
FULL PAPERS
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Phthalocyanine b-1c: According to the general procedure, a mix-
ture of b-1b (0.25 g, 0.41 mmol) and 1,2-dicyanobenzene (0.47 g,
3.57 (m, 8H, CH ), 3.69–3.66 (m, 6H, CH ), 3.76 (virt.t, J=4.4 Hz,
2
2
2H, CH ), 3.90 (t, J=4.8 Hz, 2H, CH ), 4.02 (virt.t, J=4.0 Hz, 2H,
2
2
3
1
.69 mmol) was treated with Zn(OAc)·2H O (0.38 g, 2.05 mmol) in
0 mL n-pentanol at 1008C under an atmosphere of nitrogen, and
CH ), 4.17–4.12 (m, 4H, CH ), 4.54 (s, 2H, CH ), 4.59 (t, J=5.2 Hz,
2 2 2
2
2H, CH ), 6.90 (s, 1H, N-H), 7.44 (t, J=7.6 Hz, 1H, Ar-H), 7.51 (d, J=
2
then 0.5 mL DBU was added, and the mixture was stirred at 1508C
8.4 Hz, 1H, Ar-H), 7.55 (d, J=7.6 Hz, 1H, Ar-H), 7.72 (s, 1H, Ar-H),
7.86 (d, J=7.6 Hz, 1H, Ar-H), 8.09–8.07 (m, 1H, Ar-H), 8.11–8.14 (m,
2H, Ar-H), 8.16–8.18 (m, 3H, Pc-H ), 8.24 (s, 2H, Pc-H ), 8.35 (s, 1H,
under an atmosphere of nitrogen for 5 h to give a blue solid
1
(
59.13 mg, 13%). H NMR (400 MHz, [D ]DMSO): d=3.34–3.38 (m,
6
b
b
6
H, CH ), 3.74 (brs, 4H, CH ), 4.27 (brs, 4H, CH ), 4.99 (brs, 2H,
Pc-H ), 8.54 (s, 1H, Pc-H ), 8.79 (d, J=8.0 Hz, 1H, Pc-H ), 9.06 (t, J=
b b a
3
2
2
CH ), 5.22 (brs, 2H, CH ), 7.16 (brs, 1H, N-H), 7.42–7.44 (d, J=
8.4 Hz, 2H, Pc-H ), 9.14 (d, J=7.6 Hz, 1H, Pc-H ), 9.23–9.18 (m, 3H,
a a
2
2
8
1
3
8
.8 Hz, 1H, Ar-H), 7.53 (t, J=8.0 Hz, 1H, Ar-H), 7.76 (d, J=7.6 Hz,
H, Ar-H), 7.90 (brs, 1H, Ar-H), 7.95 (brs, 1H, Ar-H), 8.02–8.08 (m,
H, Ar-H), 8.11 (d, J=7.2 Hz, 1H, Pc- H ) 8.13–8.17 (m, 2H, Pc-H ),
Pc-Ha), 9.43 ppm (s, 1H, Pc-Ha); HRMS (ESI): m/z calcd for
+
C H N O Zn [M+H] , 1231.3875, found 1231.3870; IR: u (CꢀN)
64
58 14
9
1240, u (-CH -) 2863, u (CꢀOꢀC) 1113, 1089, 1058, u (ArꢀOꢀR)
b
b
s
2
as
.20 (brs, 1H, Pc-H ), 8.46 (brs, 2H, Pc-H ), 8.61 (d, J=8.8 Hz, 1H,
1283, u (C=C) 1606, u (Pc)1575, 1505, 1486, g (Ar-H) 697, 722, 737,
b
b
ꢀ
1
Pc-H ), 8.91 (t, J=7.6 Hz, 2H, Pc-H ), 9.00–9.03 (m, 2H, Pc-H ), 9.11–
750, 774, 886 cm .
b
a
a
9
.18 (m, 3H, Pc-H ), 9.54–9.57 ppm (m, 1H, Pc-H ); HRMS (ESI): m/z
a a
+
Photophysical and photochemical studies: Fluorescence quan-
calcd for C H N O Zn [M+H] , 1055.2827, found 1055.3060; IR:
5
6
42 14
5
tum yields (F ) were measured according to Equation (1), in which
F
u (CꢀN) 1239, u (CꢀOꢀC) 1117, 1093, 1162, u (ArꢀOꢀR) 1208,
as
F, A, and h are the integrated fluorescence area (E =610 nm, area
x
u (C=C) 1609, u (Pc) 1558, 1577, 1487, g (Ar-H) 722, 751, 735,
ꢀ
1
under the emission peak), the absorbance at the excitation wave-
length (610 nm), and the refractive index of the solvent, respective-
ly. ZnPc in DMF was used as the reference (F =0.28). The F
8
87 cm .
Phthalocyanine b-3c: According to the general procedure, a mix-
ture of b-3b (0.25 g, 0.36 mmol) and 1,2-dicyanobenzene (0.42 g,
F
F
measurements were performed using diluted solutions (absorb-
ance values of photosensitizers at 610 nm are 0.04–0.06). Singlet
3
.24 mmol) was treated with Zn(OAc)·2H O (0.39 g, 1.80 mmol) in
2
1
0 mL n-pentanol at 1008C under an atmosphere of nitrogen, and
oxygen quantum yields (F ) were measured using 1,3-diphenyliso-
D
then 0.5 mL DBU was added and the mixture was stirred at 1508C
benzofuran (DPBF) as the scavenger. Laser light (670 nm,
80 mWcm ) was used as the light source, and ZnPc was selected
ꢀ2
under an atmosphere of nitrogen for 5 h to give a blue solid
1
(
80.20 mg, 19%). H NMR (400 MHz, [D ]DMSO): d=3.24 (s, 3H,
as the reference (F =0.56 in DMF).
6
D
CH ), 3.27 (s, 3H, CH ), 3.53 (s, 2H, CH ), 3.59 (s, 2H, CH ), 3.78 (s,
3
3
2
2
2
2
2
H, CH ), 3.81 (s, 2H, CH ), 3.95–4.01 (m, 8H, CH ), 4.49 (brs, 2H,
F
FðsampleÞ ¼ ðFsample=FrefÞ ðAref=AsampleÞ ðhsample =href Þ FFðrefÞ
ð1Þ
2
2
2
CH ), 4.70 (brs, 2H, CH ), 6.60 (brs, 1H, Ar-H), 7.40 (brs, 1H, Ar-H),
2
2
7
7
8
1
.46–7.48 (m, 2H, Ar-H), 7.59 (d, J=7.6 Hz, 1H, Ar-H), 7.82 (d, J=
.6 Hz, 1H, Ar-H), 8.01 (brs, 1H, Ar-H), 8.09–8.13 (m, 2H, Pc-Hb),
.18 (brs, 3H, Pc-H ), 8.27 (s, 1H, Pc-H ), 8.37 (s, 1H, N-H), 8.64 (s,
Biological methods
Cellular uptake: Approximately 3.0ꢁ10 HepG2 or HELF cells in
b
b
H, Pc-H ), 8.82 (d, J=7.6 Hz, 1H, Pc-H ), 9.12 (d, J=6.4 Hz, 2H, Pc-
4
b
a
H ), 9.19–9.24 ppm (m, 5H, Pc-H ); HRMS (ESI): m/z calcd for
a
a
1
00 mL culture medium (per well) were seeded on 96-multiwell
+
C H N O Zn [M+H] , 1143.3351, found 1143.3308; IR: u (CꢀN)
6
0
50 14
7
plates and incubated overnight at 378C under 5% CO . The old
2
1
239, u (-CH -) 2972, u (CꢀOꢀC) 1091, 1051, u (ArꢀOꢀR) 1115,
as
2
as
culture medium was removed, and then cells were incubated with
a solution of phthalocyanine in fresh medium (10 mm, containing
1% DMSO) for 24 h under the same conditions. The solution was
then removed, and the cells were rinsed with PBS three times and
then lysed by 1% SDS solution to give a homogenous solution.
The fluorescence of phthalocyanine in the cell extract was mea-
sured on a Bio-Tek microplate reader (lex: 610 nm, lem: 680 nm).
Cellular protein content was determined with a BCA Protein Assay
Kit (Beyotime Institute of Biotechnology Co., Ltd., China). Results
are expressed as nmol photosensitizer per mg cell protein.
ꢀ1
u (C=C) 1618, u (Pc) 1488, 1578, g (Ar-H) 737, 884 cm
.
Phthalocyanine b-4c: A mixture of b-4b (0.25 g, 0.34 mmol), 1,2-
dicyanobenzene (0.39 g, 3.06 mmol) was treated with Zn-
(
OAc)·2H O (0.37 g, 1.70 mmol) in 10 mL n-pentanol at 1008C
2
under an atmosphere of nitrogen, and then 0.5 mL DBU was
added and the mixture was stirred at 1508C under an atmosphere
of nitrogen for 5 h to give a blue solid (54.63 mg, 14%). H NMR
1
(
400 MHz, [D ]DMSO): d=3.27 (s, 6H, CH ), 3.59 (virt.t, J=4.4 Hz,
6 3
4
3
H, CH ), 3.65–3.68 (m, 6H, CH ), 3.76 (virt.t, J=3.6 Hz, 2H, CH ),
2 2 2
.94–4.02 (m, 8H, CH ), 4.48 (s, 2H, CH ), 4.64 (t, J=4.8 Hz, 2H,
2
2
For the targeting test, HELF and HepG2 cell suspensions were
mixed first, and then plated on a culture dish (50000:50000 cells).
CH ), 6.68 (s, 1H, N-H), 7.45–7.51 (m, 3H, Ar-H), 7.58 (d, J=7.6 Hz,
2
1
8
8
2
9
H, Ar-H), 7.88 (d, J=8.0 Hz, 1H, Ar-H), 8.08–8.14 (m, 3H, Ar-H),
After overnight incubation at 378C under 5% CO , the cells were
2
.20–8.16 (m, 4H, Pc-H ), 8.26 (s, 1H, Pc-H ), 8.37 (s, 1H, Pc-H ),
.58 (s, 1H, Pc-H ), 8.83 (d, J=8.0 Hz, 1H, Pc-H ), 9.11 (t, J=5.2 Hz,
b
b
b
exposed to 10 mm phthalocyanine and incubated for 24 h. After in-
cubation, the cells were rinsed with PBS three times, and the intra-
cellular fluorescence caused by phthalocyanines was recorded and
statistically analyzed by confocal laser scanning microscopy.
b
a
H, Pc-H ), 9.17 (d, J=7.2 Hz, 1H, Pc-H ), 9.22–9.26 (m, 3H, Pc-H ),
a
a
a
.31 ppm (s, 1H, Pc-H ); HRMS (ESI): m/z calcd for C H N O Zn
a
62 54 14
8
+
[
2
M+H] , 1187.3613, found 1187.3677; IR: u (CꢀN) 1239, u (-CH -)
s
2
877, u (CꢀOꢀC) 1058, 1089, 1112, u (ArꢀOꢀR) 1283, u (C=C)
Cytotoxicity assays: HepG2 cells were seeded onto 96-well plates at
7000 cells per well and incubated overnight. Conjugates were di-
luted to the required concentration and added to six replicate
wells. After 24 h incubation, the old medium containing drugs was
replaced with fresh medium, and the cells were exposed to a light
as
ꢀ
1
1
607, u (Pc)1575, 1505, 1486, g (Ar-H) 737, 721 cm
.
Phthalocyanine b-5c: A mixture of b-5b (0.25 g, 0.32 mmol) and
,2-dicyanobenzene (0.37 g, 2.88 mmol) was treated with Zn-
OAc)·2H O (0.35 g, 1.60 mmol) in 10 mL n-pentanol at 1008C
1
(
2
ꢀ2
dose of 1.5 Jcm . Cells were then incubated for another 24 h, and
under an atmosphere of nitrogen, and then 0.5 mL DBU was
added, and the mixture was stirred at 1508C under an atmosphere
of nitrogen for 5 h to give a blue solid (48.75 mg, 12%). H NMR
ꢀ1
then a solution of MTT in PBS (10 mL, 4 mgmL ) was added to
each well followed by incubation for 3–4 h. DMSO (100 mL) was
then added to each well. The plate was incubated at room temper-
ature for 30 min. The absorbance at 570 nm at each well was re-
1
(
400 MHz, [D ]DMSO): d=3.29 (s, 3H, CH ), 3.31 (s, 3H, CH ), 3.60–
6 3 3
ꢀ
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 10
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CHEMMEDCHEM
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corded by a microplate reader. For dark toxicity, the procedures
are almost the same as above, except for the absence of irradia-
tion. The survival curves were plotted as a function of dye concen-
tration, and IC50 values were calculated.
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Received: September 3, 2014
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Published online on && &&, 0000
ꢀ
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 10
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These are not the final page numbers!

FULL PAPERS
F.-L. Zhang, Q. Huang, J.-Y. Liu,
M.-D. Huang, J.-P. Xue*
Waging war on cancer: Herein we
report a series of conjugates with
phthalocyanine as the “warhead” and
erlotinib, a molecular-target-based anti-
cancer drug, as the “guidance system”.
These conjugates combine the high
activity of photodynamic therapy with
the highly effective targeting capacity
of molecular-target-based anticancer
therapy, thereby acting as “missile-style”
target-directed photosensitizers for
potential use in cancer treatments.
&
& – &&
Molecular-Target-Based Anticancer
Photosensitizer: Synthesis and in vitro
Photodynamic Activity of Erlotinib–
Zinc(II) Phthalocyanine Conjugates
ꢀ
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 10
&10
&
ÞÞ
These are not the final page numbers!