Zinc(II) and copper(II) complexes of β-substituted hydroxylporphyrins as tumor photosensitizers

Article abstract of DOI:10.1016/j.bmcl.2005.02.094

Novel photosensitizers β-(hydroquinon-2-yl)-5,10,15,20-tetra(4-hydroxylphenyl)porphyrinato zinc(II) (Zn(II)P) and β-(hydroquinon-2-yl)-5,10,15,20-tetra(4-hydroxylphenyl)porphyrinato copper(II) (Cu(II)P) were synthesized and characterized. Their ability of

Full text of DOI:10.1016/j.bmcl.2005.02.094

Bioorganic & Medicinal Chemistry Letters 16 (2006) 3030–3033
Zinc(II) and copper(II) complexes of b-substituted
hydroxylporphyrins as tumor photosensitizers
Qimao Huang,a Zhiquan Pan,a,* Ping Wang,b Zhangping Chen,b
Xiaolian Zhangc and Hansheng Xub
aHubei Key Lab of Novel Reactor & Green Chemical Technology, Wuhan 430073, PR China
bCollege of Chemistry and Molecular Sciences, Wuhan University, Hubei Wuhan 430072, PR China
cSchool of Medicine, Wuhan University, Hubei Wuhan 430072, PR China
Received 1 February 2005; revised 4 February 2006; accepted 21 February 2006
Available online 15 March 2006
Abstract—Novel photosensitizers b-(hydroquinon-2-yl)-5,10,15,20-tetra(4-hydroxylphenyl)porphyrinato zinc(II) (Zn(II)P) and b-
(hydroquinon-2-yl)-5,10,15,20-tetra(4-hydroxylphenyl)porphyrinato copper(II) (Cu(II)P) were synthesized and characterized. Their
ability of producing singlet oxygen under irradiation was detected by the measurement of decomposition of 1,3-diphenylisobenzofu-
ran (DPBF). The preliminary biological activity studies show that the Zn(II)P has photo-toxicity on human chronic myelogenous
leukemia cell (K562) and could cleave supercoiled DNA (pBR 322 DNA), while the Cu(II)P has inferior biological activity. Results
showed Zn(II)P having high anti-tumor activity, which presents a promising photosensitizer for photodynamic therapy.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Photodynamic therapy (PDT) is an attractive method
for treating tumors.1 The curative activity of PDT is
based on the interaction between photosensitizers and
light.2 Porphyrins have been developed as promising
photosensitizers in recent years,3 among these photosen-
sitizers, hydroxyl-substituted porphyrins show excellent
activity.4 To our knowledge, few b-substituted porphy-
rins are synthesized as potential tumor photosensitiz-
ers,5 however the sterically encumbered structure of
b-substituted porphyrins is more close to that of natural
porphyrin than that of meso-substituted porphyrins,
and it ought to be more widely used in biological stud-
ies.6 On the basis of these advisement by this informa-
tion, we now report the synthesis and the anti-tumor
activity studies of novel b-substituted hydroxylporphy-
rins which bear four hydroxyl group on meso position
and two hydroxyl group on beta position, with the pur-
pose of improving the hydrophilicity and anti-tumor
activity. The compounds Zn(II)P and Cu(II)P were first
synthesized. The ability of producing singlet oxygen was
detected by the measurement of decomposition of 1,3-
diphenylisobenzofuran (DPBF) for compounds Zn(II)P
and Cu(II)P under irradiation. The photosensitizing
activity toward supercoiled DNA (pBR 322 DNA) and
K562 human chronic myelogenous leukemia cell line
of these hydroxylporphyrin photosensitizers was
investigated. The results show that Zn(II)P could cleave
supercoiled DNA (pBR 322 DNA) and induce necrosis
or apoptosis of K562 human chronic myelogenous
leukemia cells under irradiation. While Cu(II)P and
5,10,15,20-tetra(4-hydroxylphenyl)porphyrin
exhibit
inferior photosensitizing activity and lower singlet
oxygen yield. The anti-tumor activity testing results
are corresponding with their ability of producing singlet
oxygen under irradiation.
The synthetic route of the b-(hydroquinon-2-yl)-
5,10,15,20-tetra(4-hydroxylphenyl)porphyrins is shown
in Scheme 1. The condensation of pyrrole with 4-meth-
oxylbenzaldehyde gives compound 1 (22% yield after
purification by silica gel column chromatography).
Metallization of 1 obtains 2 in high yield. The regiose-
lective synthesis of 3 was realized by the reaction be-
tween 2 and copper(II) nitrate in the presence of
acetic acid/acetic anhydride.7 Demethylation of 3 offers
b-NO2-5,10,15,20-tetra(4-hydroxylphenyl)porphyrinato
copper(II) 5. Cu(II)P was synthesized by the direct
reaction of 5 with neutral hydroquinone in 93% yields.8
Because the regioselective nitration of porphyrinato
zinc(II) was not efficacious as porphyrinato copper(II),
b-NO2-5,10,15,20-tetra(4-hydroxylphenyl)porphyrinato
zinc(II) 7 has to be synthesized by an indirect way:
*
Corresponding author. Tel.: +86 27 87194980; fax: +86 27
87194465; e-mail: huangqim@hotmail.com
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.02.094

Q. Huang et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3030–3033
3031
OMe
As expected from presenting the six hydroxyl group, the
b-substituted hydroxylporphyrins Cu(II)P and Zn(II)P
are well soluble in methanol or component solvent of
methanol and water, and insoluble in chloroform or
CH2Cl2, while original material porphyrins(1–4) repre-
sent reverse solubility. Porphyrins(5–7) which bear a
four hydroxyl group are weakly soluble in chloroform,
but well soluble in acetone. These results reveal that
the hydrophilic or hydrophobic property of the porphy-
rins achieves excellent changes, which surmount partly
hindrance of water insolubility for photosensitizers.
CHO
OMe
NH
N
N
a
MeO
OMe
OMe
OMe
+
HN
N
OMe
OMe
OMe
b
NO
2
It is well known that scientific basis for photodynamic
therapy (PDT) is the photodynamic effect of certain
photosensitizers: the photodynamic reactions lead to
the production of many reactive oxygen species (ROS)
that induced the tumor necrosis or apoptosis.10 In order
to determine the photosensitive activity of the novel b-
substituted porphyrins Zn(II)P and Cu(II)P, we investi-
gated their cleaving of DNA (supercoiled pBR322) by
using agarose gel electrophoresis. All experiments were
performed in buffer solutions (pH 8.0, 10 mM
Tris–HCl, 1 mM EDTA, and 4% DMF). High-pressure
mercury lamp (50 W) was used when irradiation was
needed. The distance from the sample to the lamp was
ca. 25 cm. A control containing photosensitizers kept
in darkness was analyzed at the same time (Fig. 1, lanes
2, 4, and 6). The photo-assisted DNA-cleaving activity
of 5,10,15,20-tetra(4-hydroxylphenyl)porphyrinato zin-
c(II) was also performed as a control compound
(Fig. 2, lane 8). The results reveal that the novel hydrox-
ylporphyrin showed cleaving ability only when they irra-
diated with light (Fig. 1, lanes 3, 5, and 7; Fig. 2, lane 6),
while there was almost no observed cleavage of DNA
when Cu(II)P and 5,10,15,20-tetra(4-hydroxylphe-
N
N
N
N
N
Cu
MeO
OMe
MeO
Cu
N
N
N
c
OMe
OH
OMe
OMe
e
d
NO
NO
2
2
NH
N
N
N
N
N
Cu
MeO
HO
OH
HN
N
g
OMe
OH
OH
OH
d
HO
NO
2
OH
N
N
N
NH
N
N
Cu
HO
OH
HO
OH
N
HN
Cu(II)P
1
2
3
4
5
6
7
OH
OH
OH
OH
f
Form II
Form I
HO
NO
2
OH
N
N
N
N
N
Figure 1. Cleavage of supercoiled pBR322 DNA by compound
Zn(II)P. [Lane 1, 0.1 lg pBR322 + 2 mL DMF, ht (2.5 h); lane 2,
0.1 lg pBR322 + Zn(II)P (50 lM); lane 3, 0.1 lg pBR322 + Zn(II)P
(50 lM), ht (2.5 h); lane 4, 0.1 lg pBR322 + Zn(II)P (100 lM); lane 5,
0.1 lg pBR322 + Zn(II)P (100 lM), ht (2.5 h); lane 6, 0.1 lg
pBR322 + Zn(II)P (200 lM); lane 7, 0.1 lg pBR322 + Zn(II)P
(200 lM), ht (2.5 h).]
Zn
Zn
HO
OH
HO
OH
N
N
N
g
Zn(II)P
OH
OH
Scheme 1. Synthesis of b-hydroquinone-5,10,15,20-tetra(4-hydroxyl-
phenyl)porphyrin and its derivatives. (a) Propanoic acid, reflux for
2.0 h (22%); (b) Cu(OAc)2, CHCl3, methanol, reflux for 1 h (97%); (c)
(CH3CO)2O/CH3COOH, CHCl3, Cu(NO3)2, 45 ꢁC, 5 min (97%); (d)
BBr3, CH2Cl2, ꢀ10 ꢁC, 24 h (75%); (e) CF3COOH, CHCl3, rt, 30 min
(95%); (f) Zn(OAc)2, CHCl3, methanol, reflux for 1 h (97%); (g)
hydroquinone, reflux under argon protection for 1.0 h (93%).
Form II
Form I
1
2
3
4
5
6
7
8
Figure 2. Cleavage of supercoiled pBR322 DNA by compound
Zn(II)P and 5,10,15,20-tetra(4-hydroxylphenyl)porphyrinato zinc(II)
(Zn(II)P4 [lane 1, 0.1 lg pBR322; lane 2, 0.1 lg pBR322, ht (2.5 h);
lane 3, 0.1 lg pBR322 + 1 mL DMF; lane 4, 0.1 lg pBR322 + 1 mL
DMF, ht (2.5 h); lane 5, 0.1 lg pBR322 + Zn(II)P (200 lM); lane 6,
0.1 lg pBR322 + Zn(II)P (200 lM), ht (2.5 h); lane 7, 0.1 lg
pBR322 + Zn(II)P4 (200 lM); lane 8, 0.1 lg pBR322 + Zn(II)P4
(200 lM), ht (2.5 h).]
demetallization of 3 with CF3COOH gives porphyrin 4,
then demethylates with BBr3 in CH2Cl2 at ꢀ10 ꢁC for
20 h to give 6, compound 6 metallized with Zn(OAc)2
in methanol gaining 7. Zn(II)P was obtained by the
reaction of 7 with hydroquinone in 93% yield.8 The
new compounds were fully characterized by 1H
NMR, FAB-MS, and UV.9

3032
Q. Huang et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3030–3033
nyl)porphyrinato zinc(II) replaced Zn(II)P even with
irradiation (Fig. 2, lane 8). It means that the porphyrins
did not chemically modify the target DNA, but photo-
dynamically. As increasing the concentration of the
Zn(II)P, the cleavage efficiency to DNA increased
(Fig. 1, lanes 3, 5, and 7). In respect to that with increas-
ing the concentration of porphyrin under irradiation,
the concentration of reactive oxygen increased. Their
ability of producing singlet oxygen under irradiation
was detected by the measurement of decomposition of
1,3-diphenylisobenzofuran (DPBF) (Fig. 5). DPBF is
an excellent quencher of singlet oxygen. Relative
reduction percentage of DPBF corresponds to the abil-
ity of photosensitizers to produce singlet oxygen. The
results reveal that singlet oxygen producing ability of
b-substituted hydroxylporphyrins Zn(II)P is superior
to that of 5,10,15,20-tetra(4-hydroxylphenyl)porphyri-
nato zinc(II). Cu(II)P has inferior ability to produce
singlet oxygen, which may be owing to that Cu(II) as
a quencher could inhibit the production of singlet
oxygen.11 Antibacterial activity of Cu(II)P and Zn(II)P
exhibits similar tendency to be able to produce singlet
oxygen: the compound Zn(II)P possesses a higher
anti-microbial activity under illumination for Staphylo-
coccus aureus ATCC 25923 than that of Cu(II)P. It is
believed that major reactive oxygen species originated
from this photodynamic course is singlet oxygen.
3.0
2.5
2.0
1.5
1.0
0.5
0.0
0
32 64 96 128 160 192 224 256 288 320
Concentration of porphyrin(nM)
Figure 4. Inactivation of the human chronic myelogenous leukemia
cell (K562) incubated with Zn(II)P in different concentrations irradi-
ated with high-pressure mercury lamp.
80
70
60
ZnP,1mg/mL
CuP,1mg/mL
ZnP4,1mg/mL
50
40
The compounds Zn(II)P and Cu(II)P were used to test
in vitro photosensitizing activity on human chronic mye-
logenous leukemia cell (K562). Trypan blue exclusion
assay was employed to evaluate the effect of the concen-
tration on the phototoxic potential of Zn(II)P and
Cu(II)P. The cells were treated with photosensitizers
(16 nmol/L, 80 nmol/L, 160 nmol/L, and 320 nnmol/L)
for 24 h at 37 ꢁC. Irradiation experiments (high-pressure
mercury lamp) and a control containing photosensitizers
kept in darkness were analyzed at the same time. Cell
cultures not being treated with Zn(II)P or Cu(II)P were
irradiated under similar conditions. Survival rate was
100% for cells under irradiation in the absence of photo-
sensitizers (Fig. 3a). Result reveals that cytotoxicity
which is induced by Zn(II)P with illumination is signifi-
cantly stronger than that of Cu(II)P. The cytotoxicity of
photosensitizers with irradiation is also stronger than
that of those maintained in dark. The Zn(II)P induces
30
20
10
0
20
40
60
80
100
t/s
Figure 5. Decomposition of DPBF by compounds Zn(II)P, Cu(II)P,
and
(Zn(II)P4); solvent: ethanol/water, 1:1 (v/v); DPBF (1.0 · 10ꢀ4 M),
porphyrin: 1 mg/mL.
5,10,15,20-tetra(4-hydroxylphenyl)porphyrinato
zinc(II)
more than 95% cell death even if the concentration of
Zn(II)P were 0.32 · 10ꢀ6mol/L (Fig. 3b). Figure 4 shows
the survival rate of the human chronic myelogenous leu-
kemia cell (K562) that was incubated with irradiated
Figure 3. Trypan blue staining of human chronic myelogenous leukemia cell (K562). (a) Control cultures, cell incubated for 16 h and illuminated for
30 min with 50 W high-pressure mercury lamp (wavelength >400 nm); (b) Cell incubated with Zn(II)P 320 nmol/L for 16 h and illuminated for
30 min with 50 W high-pressure mercury lamp (wavelength >400 nm).

Q. Huang et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3030–3033
3033
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Zn(II)P in different concentrations. Further studies are
in progress using other b-substituted hydroxyporphyrins
and various metalloporphyrins, and extensive biological
studies are ongoing.
Acknowledgments
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The authors are grateful to National Nature Science
Foundation of China for financial support (Nos.
20471045, 29872033).
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References and notes
9. Selected data: Cu(II)P UV–vis (CH3OH) kmax: 417, 517,
541 nm; FAB-MS: Anal. Calcd for C50H32N4O6Cu: 848.
Found: 848 (M+) Zn(II)P 1H NMR (300 MHz,
CD3COCD3, ppm) d: 8.9 (2H, s, 10,15-meso-Ar-OH),
8.6–8.4 (6H, m, b-pyrrole-H), 8.1–8.0 (8H, m, 10,15-meso-
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(1H, s, 2-60-H), 7.2–7.1 (2H, m, 2-30-H,2-40-H), 3.6–3.5
(4H, m, 2-20-OH,2-50-OH, 5, 20-meso-Ar-OH); UV–vis
(CH3OH) kmax: 426, 548, 589 nm; FAB-MS: Anal. Calcd
for C50H32N4O6Zn: 850. Found: 850 (M+).
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