Synthesis and in vitro photodynamic activity of mono-substituted amphiphilic zinc(II) phthalocyanines

Article abstract of DOI:10.1016/j.bmcl.2006.11.017

A series of novel zinc(II) phthalocyanines mono-substituted with a 1,3-bis(dimethylamino)-2-propoxy group at the α- or β-position, and the corresponding di-N-methylated derivatives, have been synthesized. All these compounds can generate singlet oxygen ef

Full text of DOI:10.1016/j.bmcl.2006.11.017

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1073–1077
Synthesis and in vitro photodynamic activity of mono-substituted
amphiphilic zinc(II) phthalocyanines
Pui-Chi Lo,a Baozhong Zhao,a Wubiao Duan,a Wing-Ping Fong,b Wing-Hung Koc and
Dennis K. P. Nga,*
aDepartment of Chemistry and Center of Novel Functional Molecules, The Chinese University of Hong Kong,
Shatin, N.T., Hong Kong, China
bDepartment of Biochemistry and Center of Novel Functional Molecules, The Chinese University of Hong Kong,
Shatin, N.T., Hong Kong, China
cDepartment of Physiology, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China
Received 7 September 2006; revised 4 November 2006; accepted 8 November 2006
Available online 10 November 2006
Abstract—A series of novel zinc(II) phthalocyanines mono-substituted with a 1,3-bis(dimethylamino)-2-propoxy group at the a- or
b-position, and the corresponding di-N-methylated derivatives, have been synthesized. All these compounds can generate singlet
oxygen effectively and exhibit high in vitro photodynamic activities toward HT29 human colorectal carcinoma cells with IC50 values
down to 0.08 lM. The dicationic derivatives have a higher affinity to the cell membrane compared with the non-ionic counterparts.
Ó 2006 Elsevier Ltd. All rights reserved.
Photodynamic therapy (PDT) is an innovative and
promising modality to treat localized and superficial tu-
mors.1 The treatment utilizes the combined action of a
photosensitizer, light, and molecular oxygen to cause cel-
lular and tissue damage, in which singlet oxygen, gener-
ated through a series of photo-induced processes, is
believed to be the major cytotoxic agent. Among the var-
ious considerations to improve the overall efficacy of this
therapy, the behavior of the photosensitizer is of para-
mount importance. Over the last decade, a substantial ef-
fort has been put in the development of various classes of
photosensitizers which have better absorption and pho-
tophysical properties, greater tumor specificity, and less
cutaneous photosensitivity compared with the first-gen-
eration photosensitizer Photofrin.2 We are particularly
interested in phthalocyanine-based photosensitizers be-
cause of their strong absorption in the red visible region,
which allows a deeper light penetration, high efficiency to
generate singlet oxygen, and ease of chemical modifica-
tion to facilitate the tailoring of properties such as aggre-
gation and cellular uptake.2,3 Recently, we have prepared
a series of silicon(IV) phthalocyanines with 1,3-bis(dim-
ethylamino)-2-propoxy group or its di-N-methylated
counterpart as the axial substituent. These compounds
exhibit very high photocytotoxicities toward a range of
cancer cell lines with IC50 values (defined as the dye con-
centration required to kill 50% of the cells) down to
0.02 lM.4 The most potent compound SiPc[OC3H5(N-
Me2)2](OMe) has a high and selective affinity to the mito-
chondria of cells and induces apoptosis.5 We report
herein an extension of this work to zinc(II) phthalocya-
nines which in general have higher stability than the sil-
icon(IV) counterparts. Four mono-substituted zinc(II)
phthalocyanines with these substituents at the peripheral
a or b-position have been synthesized and evaluated for
their in vitro photodynamic activity against HT29 hu-
man colorectal carcinoma cells. It is expected that these
mono-substituted phthalocyanines, having a high amph-
iphilicity, can have a high cellular uptake, which in turn
may increase the photodynamic activity. Zinc(II)
phthalocyanines with four of these substituents at the
b-positions have been reported previously,6 while the
mono-substituted analogues reported herein have only
been briefly mentioned in patents,7 all for the study of
photo-inactivation of bacteria.
Keywords: Phthalocyanines; Photodynamic therapy; Photosensitizers;
Colorectal carcinoma.
The synthesis of these mono-substituted phthalocya-
nines is shown in Scheme 1. Treatment of 3- or
*
Corresponding author. Tel.: +852 2609 6375; fax: +852 2603 5057;
e-mail: dkpn@cuhk.edu.hk
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.11.017

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P.-C. Lo et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1073–1077
CN
CN
CN
N
N
K2CO3
DMF
+
CN
O
O2N
N
OH
N
1α -substituted (34 %)
2 β-substituted (37 %)
CN
CN
N
N
MeI
NMP
N
N
N
N
N
Zn
N
N
Zn
N
.
Zn(OAc)2 2H2O
DBU, C5H11OH
N
N
N
+
I-
N
N
O
N
N
O
N
+
N
N
I-
5α -substituted (42 %)
6 β-substituted (90 %)
3α -substituted (15 %)
4 β-substituted (14 %)
Scheme 1. Preparation of phthalocyanines 3–6.
4-nitrophthalonitrile with 1,3-bis(dimethylamino)-2-
propanol in the presence of K2CO3 in DMF gave the
corresponding substituted products 1 and 2. These
precursors were then treated with the unsubstituted pht-
halonitrile in the presence of Zn(OAc)2Æ2H2O and 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU) in n-pentanol to
give the desired ‘3+1’ products 3 and 4, which could
be isolated and purified by column chromatography in
14–15% yield. To enhance the amphiphilicity, com-
pounds 3 and 4 were methylated using methyl iodide
in N-methyl-2-pyrrolidinone (NMP). The resulting
dicationic compounds 5 and 6 were isolated by precipi-
tation with diethyl ether followed by extensive washing
with diethyl ether, hexane, and dichloromethane.8
gated in aqueous media as shown by absorption spec-
troscopy. As shown in Figure 1, the absorption
spectrum of 5 in water shows a broad signal peaking at
ca. 620 and 670 nm in the Q band region. The former
band is characteristic for face-to-face or H-aggregates
of phthalocyanines.9 This spectral feature is very differ-
ent from that recorded in DMF, in which the phthalocy-
anine exists mainly in monomeric state (Fig. 1).
0.4
0.3
0.2
0.1
0.0
The electronic absorption and photophysical data of 3–6
measured in DMF are summarized in Table 1. The
absorption spectra of 3–6 in DMF are typical for non-ag-
gregated phthalocyanines, showing a B (or Soret) band
at 334–346 nm, an intense and sharp Q band at 672–
679 nm, together with a vibronic band at 606–612 nm.
The Q band straightly follows the Lambert–Beer law
up to 10 lM. Upon excitation at 610 nm, these com-
pounds show a fluorescence emission at 676–682 nm with
a fluorescence quantum yield (UF) of 0.22–0.31. Both the
absorption position of the Q-band and the fluorescence
emission are slightly red-shifted for the a-substituted
analogues (3 and 5) compared with the b-substituted
counterparts (4 and 6). The dicationic analogues 5 and
6 are readily soluble in water, but they are highly aggre-
300
400
500
600
700
800
Wavelength (nm)
Figure 1. Electronic absorption spectra of 5 in water (---) and in DMF
(—) (both at 2 lM).
Table 1. Electronic absorption and photophysical data for 3–6 in DMF
a
b
UF
c
UD
Compound
kmax (nm) (log e)
kem (nm)
3
4
5
6
339 (4.69), 612 (4.53), 679 (5.31)
346 (4.74), 606 (4.52), 673 (5.29)
334 (4.63), 609 (4.48), 676 (5.28)
344 (4.67), 606 (4.46), 672 (5.25)
682
677
682
676
0.22
0.31
0.24
0.26
0.50
0.52
0.57
0.53
a Excited at 610 nm.
b Using unsubstituted zinc(II) phthalocyanine (ZnPc) in 1-chloronaphthalene as the reference [fluorescence quantum yield (UF) = 0.30].
c Using ZnPc as the reference [singlet oxygen quantum yield (UD) = 0.56 in DMF].

P.-C. Lo et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1073–1077
1075
Table 2. Comparison of the IC50 and IC90 values of 3-6 against HT29
To evaluate the photosensitizing efficiency of these com-
pounds, their singlet oxygen quantum yields (UD) were
determined by a steady-state method using 1,3-diph-
enylisobenzofuran as the scavenger. The concentration
of the quencher was monitored spectroscopically at
411 nm along with time, from which the values of UD
could be determined.10 As shown in Table 1, all the pht-
halocyanines have similar UD values (0.50–0.57) and are
as efficient as the unsubstituted zinc(II) phthalocyanine
(ZnPc) (UD = 0.56) in sensitizing the formation of singlet
oxygen in DMF.
Compound
IC50 (lM)
IC90 (lM)
3
4
5
6
0.48
0.15
0.64
0.08
1.67
0.67
1.04
0.12
the b-di-N-methylated phthalocyanine 6 is particularly
potent with an IC50 value of 0.08 lM. Apparently,
its photocytotoxicity is much higher than that of the clas-
sical photosensitizer Photofrin (IC50 = 7.5 lgmLÀ1 vs
0.08 lgmLÀ1 for 6),11 pheophorbide a (IC50 = 0.5 lM),11
and several other chlorin-based photosensitizers.12
The photodynamic activities of compounds 3–6 in
Cremophor EL emulsions were investigated against
HT29 human colorectal carcinoma cells. Figure 2, which
shows the effects of 4 on HT29, is a typical dose-dependent
survival curve for all these phthalocyanines. While all
these compounds are essentially non-cytotoxic in the
absence of light, they exhibit a high photocytotoxicity.
As shown in Table 2, which summarizes the IC50 and
IC90 values of these compounds, the b-substituted
phthalocyanines (4 and 6) are generally more photocyto-
toxic than the a-substituted counterparts (3 and 5), and
To have a better understanding of the in vitro photody-
namic action of these compounds, we examined their
absorption and photophysical properties in the culture
medium.13 While a strong and sharp Q band was re-
tained for 4 and 6 in the medium, only a weak Q band
was observed for 3 and 5. The fluorescence emission
for the former two compounds was also substantially
higher (see Figures S1 and S2 in Supporting informa-
tion). These observations seemed to suggest that the
b-substituted analogues are less aggregated than the a-
substituted counterparts in the culture medium. Howev-
er, their singlet-oxygen generation efficiency did not
seem to have a great difference. We employed imidaz-
ole/4-nitrosodimethylaniline (RNO)14 as a probe to
monitor the singlet oxygen formation. It was found that
the rates of decay of RNO (as monitored by the decrease
in absorbance at 440 nm) induced by 4 and 6 were only
marginally faster than those by using 3 and 5 as the
photosensitizers (see Figure S3 in Supporting
information).
100
80
60
40
20
0
We also employed fluorescence microscopy to study the
cellular uptake and subcellular localization. After incu-
bation with compounds 3–6 for 2 h and upon excitation
at 630 nm, the HT29 cells showed a strong intracellular
fluorescence (see Figure S4 in Supporting information),
indicating that there were substantial uptakes of the
dyes. As mitochondria are believed to be the targets
for the initiation of apoptosis by PDT,15 it would be
important to reveal whether the dyes have a selective
affinity to these subcellular components. We stained
the HT29 cells with MitoTracker Green FM, which is
0.0
0.5
1.0
1.5
2.0
[4] (μM)
Figure 2. Effects of 4 on HT29 in the absence (j) and presence (h) of
light. For the latter, the cells were illuminated with a red light
(k > 610 nm, 40 mW cmÀ2, 48 J cmÀ2). Data are expressed as mean
values S.E.M. of three independent experiments, each performed in
triplicate.
Figure 3. Visualization of intracellular fluorescence of HT29 using filter sets specific for (a) the MitoTracker (in red) and (b) phthalocyanine 4 (in
blue). (c) The corresponding superimposed image.

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P.-C. Lo et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1073–1077
Figure 4. Visualization of intracellular fluorescence of HT29 using filter sets specific for (a) the MitoTracker (in red) and (b) phthalocyanine 6 (in
blue). (c) The corresponding superimposed image.
a specific fluorescence dye for mitochondria, prior to the
treatment with 3–6. Figures 3 and 4 show the images for
compounds 4 and 6, which are similar to those for the a-
substituted analogues 3 and 5, respectively (see Figures
S5 and S6 in Supporting information). It can be seen
that the fluorescence caused by the phthalocyanines
(excited at 630 nm, monitored at >660 nm) is diffused
throughout the cytoplasm and cannot be perfectly
superimposed with the fluorescence caused by the Mito-
Tracker (excited at 490 nm, monitored at 500–575 nm).
This observation indicates that these compounds are
not exclusively localized in the mitochondria. A close
examination of these figures reveals that compound 6,
as well as 5, gives a brighter emission in the cell mem-
brane region compared with the non-ionic counterparts.
It is likely that due to the dicationic nature of these com-
pounds, they are preferentially retained in the mem-
brane’s lipid bilayer.
References and notes
1. (a) Dolmans, D. E. J. G. J.; Fukumura, D.; Jain, R. K.
Nat. Rev. Cancer 2003, 3, 380; (b) Brown, S. B.; Brown, E.
A.; Walker, I. Lancet Oncol. 2004, 5, 497.
2. (a) Ali, H.; van Lier, J. E. Chem. Rev. 1999, 99, 2379; (b)
Nyman, E. S.; Hynninen, P. H. J. Photochem. Photobiol.,
B 2004, 73, 1; (c) Detty, M. R.; Gibson, S. L.; Wagner, S.
J. J. Med. Chem. 2004, 47, 3897.
3. (a) Lukyanets, E. A. J. Porphyrins Phthalocyanines 1999,
3, 424; (b) Allen, C. M.; Sharman, W. M.; van Lier, J. E. J.
Porphyrins Phthalocyanines 2001, 5, 161.
4. (a) Lo, P.-C.; Huang, J.-D.; Cheng, D. Y. Y.; Chan, E. Y.
M.; Fong, W.-P.; Ko, W.-H.; Ng, D. K. P. Chem. Eur. J.
2004, 10, 4831; (b) Huang, J.-D.; Lo, P.-C.; Chen, Y.-M.;
Lai, J. C.; Fong, W.-P.; Ng, D. K. P. J. Inorg. Biochem.
2006, 100, 946; (c) Lo, P.-C.; Leung, C.-H.; Chan, E. Y.
M.; Fong, W.-P.; Ko, W.-H.; Ng, D. K. P. Photodiag.
Photodyn. Ther. in press.
5. Lai, J. C.; Lo, P.-C.; Ng, D. K. P.; Ko, W.-H.; Leung, S.
C. H.; Fung, K.-P.; Fong, W.-P. Cancer Biol. Ther. 2006,
5, 413.
6. (a) De Filippis, M. P.; Dei, D.; Fantetti, L.; Roncucci, G.
Tetrahedron Lett. 2000, 41, 9143; (b) Segalla, A.; Borsar-
elli, C. D.; Braslavsky, S. E.; Spikes, J. D.; Roncucci, G.;
Dei, D.; Chiti, G.; Jori, G.; Reddi, E. Photochem.
Photobiol. Sci. 2002, 1, 641.
7. (a) Roncucci, G.; Fantetti, L.; De Filippis, M. P.; Dei, D.;
Jori, G. Eur. Pat. Appl. EP 1 164 135 A1, 2001; (b)
Roncucci, G.; Fantetti, L.; De Filippis, M. P.; Dei, D.;
Jori, G. US Pat. Appl. US 0170178 A1, 2003.
In summary, we have prepared four mono-substituted
zinc(II) phthalocyanines with an amphiphilic character.
These compounds are highly photodynamically active
toward HT29 cells. The dicationic phthalocyanines 5
and 6 have a higher affinity to the cell membrane com-
pared to their neutral counterparts.
Acknowledgments
8. The experimental procedure and full characterization
data for these compounds are given in Supporting
information.
9. Tai, S.; Hayashi, N. J. Chem. Soc., Perkin Trans. 2 1991,
1275.
10. (a) Spiller, W.; Kliesch, H.; Wo¨hrle, D.; Hackbarth, S.;
Ro¨der, B.; Schnurpfeil, G. J. Porphyrins Phthalocyanines
1998, 2, 145; (b) Maree, M. D.; Kuznetsova, N.; Nyokong,
T. J. Photochem. Photobiol., A 2001, 140, 117.
We thank Wallace C. Y. Yip for technical support in the
imaging study. This work was supported by a grant
from the Research Grants Council of the Hong Kong
Special Administrative Region, China (Project No.
402103), and a strategic investments scheme adminis-
tered by The Chinese University of Hong Kong.
11. Hajri, A.; Wack, S.; Meyer, C.; Smith, M. K.; Leberquier,
C.; Kedinger, M.; Aprahamian, M. Photochem. Photobiol.
2002, 75, 140, In this work, the light (640 nm for Photofrin
and 670 nm for pheophorbide a) was delivered by a
halogen lamp fitted with specific 10 nm-width band-pass
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.
2006.11.017.
filters with a total fluence of 5 J cmÀ2
.

P.-C. Lo et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1073–1077
12. Laville, I.; Figueiredo, T.; Loock, B.; Pigaglio, S.; Mail- (10%),
1077
and
penicillin–streptomycin
(100 U mLÀ1
lard, Ph.; Grierson, D. S.; Carrez, D.; Croisy, A.; Blais, J.
Bioorg. Med. Chem. 2003, 11, 1643, In this work, the cells
were irradiated with an Ar+ laser at 514 nm with a light
fluence of 5–25 J cmÀ2. By using 1 lM of the photosensi-
tizers, the cell survival fraction was found to be ca. 5–30%.
13. Dulbecco’s modified Eagle’s medium (DMEM) (Invitro-
gen, cat no. 10313-021) supplemented with fetal calf serum
100 lg mLÀ1, respectively), L-glutamine (2 mM), and
transferrin (10 lg mLÀ1).
14. Kraljic, I.; El Mohsni, S. Photochem. Photobiol. 1978, 28,
577.
15. Oleinick, N. L.; Morris, R. L.; Belichenko, I. Photochem.
Photobiol. Sci. 2002, 1, 21.