4
60
Chemistry Letters Vol.33, No.4 (2004)
Synthesis and Photocytotoxicity of Nitroxyl Radical-substituted Porphyrin
ꢀ
Jian Wu, Weimin Shi, and Di Wu
Department of Chemistry, Zhejiang University, Yuquan Compus 310027, Hangzhou, P. R. China
(Received December 18, 2003; CL-031252)
ꢁ
A novel tetraphenylporphyrin derivative bearing nitroxyl
Instead of the fuming nitric acid (d ¼ 1:5 g mL 1) which was
radical moiety was efficiently synthesized by improved prepara-
tion of monoamino-substituted tetraphenylporphyrin as the pre-
cursor. Preliminary photocytotoxicity investigation of the spin
labeled porphyrin against SPC-A1 adenocarcinoma cell lines
was tested.
used in the Kruper’s method, less-concentrated nitric acid
(d ¼ 1:4 g mL ) was found to be much preferable for this reac-
ꢁ1
tion in the yield high as 77%.
Table 1. Regiospecific nitration of TPP with nitric acida
Stoichiometry of
Nitric Acid
b
Nitration Reagents
Yield of 2
In the past decades, a wide variety of structurally modified
porphyrins have been synthesized for their potential application
17
19
23
25
50
49
64
77
71
62
1
as photosensitizer in photodynamic therapy (PDT) of cancer. In
attempt to improve tumor cell targeting, efforts have been direct-
ed at covalently attaching porphyrins to a number of biomole-
Concentrated nitric acid
(d ¼ 1:4 g mLꢁ1)
2
,3
4
cules including sugar and peptide. It has been reported that
a number of nitroxyl spin labeled derivatives of antitumor com-
pounds had superior pharmacological properties and marked de-
1
7
37
45
29
3
Fuming nitric acid
1
19
23
(d ¼ 1:5 g mLꢁ
)
5
,6
crease in toxicity compared to their parent molecules. Al-
though the exact mechanism is unknown, nitroxyl radical
derivatives was thought to be beneficial for the drug to permeate
3
0
a
ꢂ
b
In CH Cl at 0–5 C under N for 3 h. Percent isolated
2
2
2
7
through the cell membrane to reach the DNA. Thus, it is of in-
terest to attain nitroxyl radical-containing porphyrin for biolog-
ical investigation.
yield was determined by silica gel chromatography.
Usual reduction of the nitro group with SnCl /HCl was ap-
2
plied to 2 to give 5-(4-aminophehyl)-10,15,20-triphenylpor-
phyrin 3. Followed by the condensation reaction with 1-oxyl-
2,2,5,5-tetramethylpyrrolidine-3-carboxylic acid in the pres-
ence of dicyclohexylcarbodiimide (DCC) in CH Cl , porphyrin
3 was converted to the nitroxyl radical-substituted porphyrin 4.12
The photocytotoxicity of porphyrin 4 was tested against
SPC-A1 adenocarcinona cell line. A normal cell line, L929
mouse fibroblast cell line, was also applied to the test for com-
parison. Cells were suspended in a RPMI medium containing
8
meso-Tetraphenylporphyrin (TPP, 1) is one of the most
readily available synthetic porphyrin. Its monofunctinalized de-
rivatives such as 3 could serve as useful synthetic precursors for
further modification. However, significant difficulty in prepara-
tion of 3 was caused by the low yield and tedious separation with
classical procedure of condensation between pyrrole and two
1
1
2
2
9
10
different aldehydes. Previously Kruper et al. reported mononi-
tration at phenyl ring of TPP with excess of fuming nitric acid.
Now we report our exploitation of this reaction to synthesize
mono-nitroxyl radical-substituted tetraphenylporphyrin 4. Its in
vitro photocytotoxicity against SPC-A1 adenocarcinoma cell
line was tested.
ꢁ
4
10 M porphyrin. The suspension was irradiated with fluores-
cent light (ꢀ ¼ 600 nm, fluence rate = 60 W m ) for a certain
ꢁ2
ꢂ
period of time. After further 24 h incubation in dark at 37 C,
The synthesis procedure was shown in Scheme 1. The start-
ing porphyrin TPP was subjected to regiospecific nitration to
give 5-(4-nitrophenyl)-10,15,20-triphenylporphyrin 2. It was
found that the reaction mainly depends on the concentration
and stoichiometry of the nitric acid used, as shown in Table 1.
the dead cells were identified as propidium iodide (PI) permea-
ble ones, and the counts were measured by flow cytometry.
Figure 1 displays dead cell counts in function of irradiation
time with porphyrins 3 and 4 against SPC-A1 cells and L929
mouse fibroblast cells, respectively. The dead cell percentage in-
creased with augmentation of irradiation time for both of the
porphyrins. The dead SPC-A1 cell counts were always higher
than that of L929 cells in the same irradiation time. In compar-
ison with its precursor 3, the nitroxyl radical-substituted porphy-
rin 4 exhibited enhanced photocytotoxicity and selectivity
against tumor cell, and the maximum of dead SPC-A1 cell per-
centage (87%, 140 min) was over twice of that of L929 cell. It
might be attributed to the superior membrane permeability aris-
R
1
2
3
R =
H
i
R = NO2
ii
HOOC
R = NH2
NH
N
N
O
iii
N
O
Ph
Ph
4 R = HNC
HN
NO-radical acid
N
O
7
ing from the nitroxyl radical-substitution.
Ph
In conclusion, a novel tetraphenylporphyrin derivative bear-
ing nitroxyl radical moiety was synthesized through three steps
with high yield. Introduction of nitroxyl radical group to porphy-
rin was found to be beneficial for the photocytotoxicity against
Scheme 1. Synthesis of nitroxyl radical-substituted porphyrin.
i) concentrated nitric acid (d ¼ 1:4 g mL ), 77%; ii) SnCl2,
HCl, 83%; iii) NO- radical acid, DCC, CH2Cl2, 90%.
ꢁ1
Copyright Ó 2004 The Chemical Society of Japan
Wu, Jian
