Porphyrins containing nitric oxide donors: Synthesis and cancer cell-oriented NO release

Article abstract of DOI:10.1016/j.bmcl.2009.02.005

Four novel porphyrins containing nitric oxide (NO) donors were synthesized, and the structures of all the products were characterized by IR, UV-vis, ¹H NMR, and elementary analysis. Interestingly, these new compounds not only were able to relea

Full text of DOI:10.1016/j.bmcl.2009.02.005

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1647–1649
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Porphyrins containing nitric oxide donors: Synthesis and cancer
cell-oriented NO release
*
Wukun Liu, Chaozhou Liu, Changjun Gong, Weiying Lin, Cancheng Guo
College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Four novel porphyrins containing nitric oxide (NO) donors were synthesized, and the structures of all the
products were characterized by IR, UV–vis, 1H NMR, and elementary analysis. Interestingly, these new
compounds not only were able to release NO, but also showed cancer cell-oriented accumulation. Higher
accumulation of these new porphyrins containing NO donors in BEL-7402 liver cancer cells than in L-02
liver normal cells was corroborated by UV–vis spectroscopy. The biological activity of these porphyrins
against BEL-7402 liver cancer cells was tested with a MTT assay. The studies indicated that they had more
effective killing of BEL-7402 liver cancer cells than that of L-02 liver normal cells, and they had similar
activity against MCF-7 breast cancer cells when compared to 5-fluorouracil in the absence of light.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 24 October 2008
Revised 15 January 2009
Accepted 2 February 2009
Available online 7 February 2009
Keywords:
Porphyrin
Nitric oxide donors
Synthesis
Biological evaluation
Nitric oxide (NO) is a key signaling molecule involved in the
regulation of many biological processes, such as regulation of
blood pressure, and neurotransmission.1,2 NO is generated either
In this Letter, we described the synthesis of four new porphy-
rins containing NO donors and their cancer cell-oriented release
of NO. Furthermore, we also investigated the accumulation around
the cancer cells and the preliminary anticancer activity of these
new compounds.
The synthetic route to porphyrins bearing NO donors was out-
lined in Scheme 1. Parahydroxybenzaldehyde 1 was treated with
dibromoalkanes bearing two to three carbons in K2CO3 and acetone
at 56 °C to generate compound 2 in 63–67% yields22, which was
then reacted with AgNO3 in CH3CN to afford the corresponding ni-
trates 3 in good yields (70–78%).23
Initially, an attempt was made to synthesize porphyrins 4a–d
via the Adler–Longo method24 by refluxing reactants in propionic
acid. Different acids, such as HCOOH, CH3COOH, and CH3CH2COOH
were employed as refluxing solvents, and zinc acetate was used as
template to stabilize the cyclic tetramer structure of intermediates.
However, no product was detected. LC/MS analysis showed that
the organic nitrates were decomposed in the presence of acid with
high temperature. This indicates that the Adler–Longo method is
not suitable for the synthesis of NO releasing porphyrins. In addi-
tion, we also found that the original reaction conditions of the
Lindsey method25 also could not afford porphyrin 4a–d. Fortu-
nately, the products were obtained when CF3COOH was used as
catalyst. The reaction of 3a or 3b with benzaldehyde or p-tolualde-
hyde with pyrrole in the ratio of 1:3:4 catalyzed by CF3COOH
smoothly afforded porphyrin 4a–d in 6.7–8.2% yield.26
from L-arginine under the catalysis of NO synthase (NOS) or from
synthetic releasing NO compounds, such as nitrate, furoxan,
hydroxyguanidine, S-nitrosothiol, diazeniumdiolate, and others.3,4
Recent studies have showed that NO appears to be critical for the
tumoricidal activity of the immune system. High concentration of
NO was cytotoxic and could induce the apoptosis of tumor cells,
preventing tumors from metastasizing and assisting macrophage
to kill tumor cells.5–10 Increased scientific evidence supports that
NO deficiency is implicated in many physiological and pathological
processes within the mammalian body.4 This fact provides a solid
biologic basis for the application of NO replacement therapy in
clinic. One of such use is to make a specific tissue-targeted delivery
of NO releasing drugs to tumor cells apoptosis.3–5 However, the
development of NO donors with good tissue specificity is still very
challenging.11–13
As porphyrin can selectively accumulate in tumor tissues,14–17
it may serve as an ideal base for the design of new NO releasing
compounds for the production of NO specifically in tumor tissues.
We reasoned that porphyrins bearing moieties releasing nitric
oxide could make the concentration of NO in tumor tissue higher
than in surrounding normal tissue by the transport of porphy-
rins.18,19 In addition, we have recently reported that some hybrid
porphyrins have a better anticancer activity in the absence of
light.20,21
To examine whether the newly synthesized porphyrins contain-
ing NO donors could indeed release NO, the percentage of NO re-
leased in vitro from 4a to 4d upon incubation with
L-cysteine for
* Corresponding author. Tel.: +86 731 8821314; fax: +86 731 8821488.
E-mail address: ccguo@hnu.cn (C. Guo).
1.5 h at 37 °C, was determined (Table 1).27
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.02.005

1648
W. Liu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1647–1649
R
CHO
CHO
CHO
CHO
N
H
NH
N
N
R
R
R
iii
ii
HN
OH
O(CH2) ONO2
n
O(CH2) Br
n
4a n = 2 R = H
4b n = 2 R = CH3
4c n = 3 R = H
4d n = 3 R = CH3
1
2
n = 2, 3
3 n = 2, 3
O(CH2) ONO2
n
Scheme 1. Synthetic routes of porphyrins bearing NO donor outlined. Reagents and conditions: (i) Br (CH2)nBr, K2CO3, CH3COCH3, 56 °C, 20 h (2a 63%, 2b 67%); (ii) CH3CN,
AgNO3, reflux (3a 70%, 3b 78%). (iii) (a) CF3COOH, CH2Cl2, room temperature 12 h; (b) p-chloranil reflux 30 min (6.7–8.2%).
Table 1
contrast, tetraphenylporphyrin was significantly less active against
tested cancer cell lines in the absence of light. This result is in
Nitric oxide released from 4a to 4da
Compound
% NO release in PBSb
% NO release in 18 lM L
-cysteinec
accordance with the fact that tetraphenylporphyrin has low dark
cytotoxicity.21 Furthermore, these results suggested the cytotoxic-
ity activity of these new compounds might come from the part of
NO donors.
An enhanced accumulation of compound 4a in BEL-7402 liver
cancer cells compared to L-02 liver normal cells was detected by
UV–vis spectroscopy.31 The absorbance of the cell culture fluid
4a
4b
4c
4d
0.43 0.01
0.55 0.03
0.47 0.03
1.10 0.06
5.67 0.08
3.12 0.04
3.95 0.06
6.25 0.07
a
Percentage of nitric oxide released (mean value, n = 3) relative to a theoretical
maximum release of 1 mol of NO/mol of test compound was determined using the
Griess reaction.
was detected after incubation of 4a (100 lmol/L) for 30 min
b
Incubated in phosphate buffer solution (PBS, pH 7.4) at 37 °C for 1.5 h.
Incubated in the presence of 18 lM L-cysteine in phosphate buffer solution (pH
(Fig. 1). The absorbance of the cell culture fluid in BEL-7402 liver
cancer cells decreased more when compared to the absorbance
of the cell culture fluid in L-02 liver normal cells. These results
showed that the new porphyrins selectively release NO in tumor
cells.
To determine whether NO releasing derivatives of porphyrin ex-
hibit different toxicity between tumor and normal cells, 4a–d and
tetraphenylporphyrin were evaluated toward L-02 liver normal
cells and Bel 7402 liver cancer cells using a MTT based cell viability
assay. As shown in Figure 2, 4a–d showed more effective killing of
BEL-7402 liver cancer cells than that of L-02 liver normal cells at
c
7.4) at 37 °C for 1.5 h.
It has been reported that a reduced thiol group such as
teine, -cysteamine, or glutathione is required for the release of
NO from certain NO donor agents such as those containing an or-
ganic nitrates moiety.28 The data of the percentage of NO release
acquired in this study are consistent with the literature since the
percentage of NO released from 4a to 4d were higher upon incuba-
L-cys-
L
tion in the presence of L-cysteine (3.12–6.67%) compared to that
determined in phosphate buffer solution (PBS) at pH 7.4 (0.43–
1.10%). The superior NO release of these compounds is in good
agreement with the reported results that high dose of NO showed
potent cytotoxicity on tumor cells.6,7
the concentration of 1.4
lmol/L after 72 h of incubation. Compared
with TPP, 4a–d showed
a
statistical difference in viability
(p < 0.001). Tetraphenylporphyrin exhibited less activity against
tested cell lines in the absence of light and compounds 3a and
3b exhibited more toxicity toward L-02 liver normal cells than to-
ward BEL-7402 liver cancer cells. This indicates that new porphy-
rins release more NO in the circumstance of cancer cells than in
MTT cytotoxicity assay29 was widely used to estimate the bio-
activity of a drug molecule. Compounds 4a–d and the reference
compounds tetraphenylporphyrin and 5-fluorouracil, were evalu-
ated for their tumor cell cytotoxicity using a MTT cytotoxicity as-
say30 (Table 2). All compounds showed higher activity against
BEL-7402 liver cancer cells when compared to 5-fluorouracil in
the absence of light. Compound 4a showed similar activity against
MCF-7 breast cancer cells when compared to 5-fluorouracil. In
1.2
0 min
30 min L02
30 min BEL7402
1.0
0.8
0.6
0.4
0.2
0.0
Table 2
Cytotoxicity of the target compounds against BEL-7402 and MCF-7 cells in vitro
Compound
Cytotoxicity (IC50
BEL-7402
,
l
M)a,b
MCF-7
4a
4b
4c
4d
0.8 0.04
1.8 0.2
3.0 0.1
1.4 0.06
350 18.3
4.3 0.7
8.7 0.6
55 4.9
30 3.2
46 3.1
245 21.8
4.7 0.4
Tetraphenylporphyrin
5-Fluorouracil
400
500
600
Wavelength, nm
a
The IC50 values represent the concentration which results in a 50% decrease in
cell growth after 72 h incubation.
Figure 1. The absorbance of cell culture fluid after incubation of 100 lmol/L 4a for
30 min.
b
Values are the means of three experiments.

W. Liu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1647–1649
1649
26. Procedure of synthesis of porphyrin 4a–d. The reactions were performed in a
250 mL three neck round bottomed flask fitted with a gas inlet port. The flask
was charged with 100 mL of distilled CH2Cl2, 1.0 mmol of substituted
benzaldehyde 3a or 3b, 3.0 mmol of benzaldehyde or p-tolualdehyde and
4 mmol of pyrrole. The resulting solution was magnetically stirred at room
temperature and purged by nitrogen gas for 20 min. After addition of 2.5 mmol
of CF3COOH into the solution, the reaction flask was shielded from light for
12 h, then 1.6 mmol of p-chloranil was added at once. The solution was stirred
for another 2–3 min before it was refluxed for 30 min in water bath. After
cooling down to room temperature, 2 mmol of Et3N was added to neutralize
the acid. The solvent (CH2Cl2) was removed by rotary evaporation under
vacuum. The crude product was purified by column chromatography with
mixture eluent of CHCl3 and petroleum ether. The second band was collected.
The solvent was removed under vacuum to obtain the desired products. meso-
5-(20-Nitrooxyethoxyphenyl)-10,15,20-triphenylporphine (4a). Yield 8.2%;
mp > 300 °C; MS m/z: 720 (M+H)+; 1H NMR (CDCl3, 400 MHz): d 8.84 (8H,
pyrrolic), 7.27–8.23 (19H, ArH), 4.99 (t, 2H, OCH2, J = 4.4 Hz), 4.52 (t, 2H, OCH2,
J = 4.4 Hz), ꢀ2.79 (2H, NH) ppm. Anal. Calcd for C46H33N5O4: C, 76.76; H, 4.62;
N, 9.73; O, 8.89. Found: C, 76.72; H, 4.62; N, 9.70; O, 8.92; IR (KBr, cmꢀ1): 3317
BEL7402
L02
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
3a
3b
4a
4b
44
4d
TPP
Figure 2. Cytotoxicity (data were subjected to one-way analysis of variance
(ANOVA), followed by multiple comparisons with least significant differences (LSD)
test. Statistical significance was considered with P < 0.05) of the target compounds
between tumor and normal cells at the concentration of 1.4 lmol/L.
(N–H), 1607, 1506, 1473 (C@C) cmꢀ1
;
UV–vis [kmax
,
nm
(e
ꢁ 10ꢀ3 cmꢀ1
normal cells, and it is consistent with the finding that new com-
pounds accumulate more in the circumstance of cancer cells than
in normal cells.
In summary, a series of novel porphyrins releasing NO were
synthesized and their biological activities were evaluated. All
new compounds can release a high percentage of NO and have bet-
ter accumulation toward cancer cells than toward normal cells.
Consequently, they showed better activities towards cancer cells.
We believe that they could be employed as promising agents in
chemotherapy.
molꢀ1 L)] in CH2Cl2: 417.5, 515, 550, 589, 647.5. meso-5-(20-Nitrooxy-
ethoxyphenyl)-10,15,20-tri-(methylphenyl)porphyrin
(4b).
Yield
6.7%;
mp > 300 °C; MS m/z: 762 (M+H)+; 1H NMR (CDCl3, 400 MHz): d 8.85 (8H,
pyrrolic), 7.27–8.10 (16H, ArH), 5.01 (t, 2H, OCH2, J = 4.4 Hz), 4.54 (t, 2H,
OCH2,J = 4.4 Hz), 2.70 (s, 9H, CH3), ꢀ2.81 (2H, NH) ppm. Anal. Calcd for
C49H39N5O4: C, 77.25; H, 5.16; N, 9.19; O, 8.40. Found: C, 77.14; H, 5.11; N,
9.16; O, 8.43; IR (KBr, cmꢀ1): 3315 (N–H) 1627, 1505, 1463 (C@C) cmꢀ1; UV–
vis [kmax, nm (
e
ꢁ 10ꢀ3 cmꢀ1 molꢀ1 L)] in CH2Cl2: 418.5, 515, 552,591.5, 647.
meso-5-(30-Nitrooxypropoxyphenyl)-10,15,20-triphenylporphine (4c). Yield
7.2%; mp > 300 °C; MS m/z: 734 (M+H)+; 1H NMR (CDCl3, 400 MHz): d 8.83
(8H, pyrrolic), 7.27–8.22 (19H, ArH), 4.86 (t, 2H, OCH2, J = 6.4 Hz), 4.37 (t, 2H,
OCH2, J = 6.0 Hz), 2.41 (m, 2H, –CH2–, J = 6.0 Hz), ꢀ2.77 (2H, NH) ppm. Anal.
Calcd for C47H35N5O4: C, 76.93; H, 4.81; N, 9.54; O, 8.72. Found: 76.90; H, 4.83;
N, 9.56; O, 8.71; IR (KBr, cmꢀ1): 3317 (N–H), 1635, 1508, 1467 (C@C) cmꢀ1
UV–vis [kmax, nm (
;
Acknowledgment
e
ꢁ 10ꢀ3 cmꢀ1 molꢀ1 L)] in CH2Cl2: 417.5, 515, 550.5, 589,
646.
meso-5-(30-Nitrooxypropoxyphenyl)-10,15,20-tri-(methylphenyl)por-
phyrin (4d). Yield 7.8%; mp > 300 °C; MS m/z: 776 (M+H)+; 1H NMR (CDCl3,
400 MHz): d 8.85 (8H, pyrrolic), 7.27–8.13 (16H, ArH), 4.85 (t, 2H, OCH2,
J = 6.4 Hz), 4.36 (t, 2H, OCH2, J = 5.6 Hz), 2.70 (s, 9H, CH3), 2.40 (m, 2H, –CH2–,
J = 6.0 Hz), ꢀ2.77 (2H, NH) ppm. Anal. Calcd for C50H41N5O4: C, 77.40; H, 5.33;
N, 9.03; O, 8.24. Found: C, 77.46; H, 5.28; N, 9.00; O, 8.26; IR (KBr, cmꢀ1): 3311
The authors gratefully thank the financial supports of National
Natural Science Foundation of China (Grants CN J0830415).
References and notes
(N–H) 1630, 1508, 1472 (C@C) cmꢀ1
cmꢀ1 molꢀ1 L)] in CH2Cl2: 419, 481, 517.5, 552, 647.
27. In vitro nitric oxide release assays. (1) Incubation with 18 mM
;
UV–vis [kmax
,
nm
(
e
ꢁ 10ꢀ3
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2002, 102, 1091.
L
-cysteine in
phosphate buffer (pH 7.4). A solution of the test compound (1 mL of a 0.2 mM
solution in 0.1 M phosphate buffer, pH 7.4) was mixed thoroughly with a
freshly prepared solution of L-cysteine (1 mL of a 3.6 mM solution in 0.1 M
phosphate buffer, pH 7.4), and the mixture was incubated at 37 °C for 1.5 h in
the absence of air. After exposure to air for 10 min at 25 °C, an aliquot of the
Griess reagent (1 mL) (freshly prepared by mixing equal volumes of 1.0%
sulfanilamide and 0.1% N-naphthylethylenediamine dihydrochloride in water)
was added to an equal volume (1 mL) of each test compound’s incubation
solution with mixing. After 10 min had elapsed, absorbance was measured at
5. Ekmekcioglu, S.; Tang, C. H.; Grimm, E. A. Curr. Cancer Drug Targets 2005, 5, 103.
6. Kerwin, J. J.; Heller, M. Med. Res. Rev. 1994, 14, 23.
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8. Cai, T. B.; Wang, P. G. Ther. Pat. 2004, 12, 849.
9. Hirst, D.; Robson, T. J. Pharm. Pharmacol. 2007, 59, 3.
540 nm using
Solutions of 0–100
a
Simadzu UV 2100 UV–vis scanning spectrophotometer.
sodium nitrite were used to prepare nitrite
10. Sullivan, R.; Graham, C. H. Curr. Pharm. Des. 2008, 14, 1113.
11. Praneeth, V. K.; Näther, C.; Peters, G.; Lehnert, N. Inorg. Chem. 2006, 45, 2795.
12. Praneeth, V. K.; Neese, F.; Lehnert, N. Inorg. Chem. 2005, 44, 2570.
13. Lehnert, N.; Praneeth, V. K.; Paulat, F. J. Comput. Chem. 2006, 27, 1338.
14. Winkelman, J.; Slater, G.; Grossman, J. Cancer Res. 1967, 27, 2060.
15. Tronconi, M.; Colombo, A.; De Cesare, M.; Marchesini, R.; Woodburn, K. W.;
Reiss, J. A.; Phillips, D. R.; Zunino, F. Cancer Lett. 1995, 88, 41.
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17. Dolmans, D. E.; Fukumura, D.; Jain, R. K. Nat. Rev. Cancer 2003, 3, 380.
18. Zheng, X.; Pandey, R. K. Anticancer Agents Med. Chem. 2008, 8, 241.
19. Fukumura, D.; Kashiwagi, S.; Jain, R. K. Nat. Rev. Cancer 2006, 6, 521.
20. Guo, C. C.; Li, H. P.; Zhang, X. B. Bioorg. Med. Chem. 2003, 11, 1745.
21. Guo, C. C.; Tong, R. B.; Li, K. L. Bioorg. Med. Chem. 2004, 12, 2469.
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Med. Chem. 2006, 14, 866.
23. Procedure of synthesis of substituted benzaldehyde 3. A mixture of 2 (5 mmol),
silver nitrate (10 mmol), and acetonitrile (10 ml) was stirred at refluxed
temperature for 8 h, and then filtered and concentrated. The product was
purified with column chromatography (silica gel, 1:1, petroleum ether/ethyl
acetate). 4-(20-Nitrooxyethoxy)-benzaldehyde 3a. Yield 70%; MS m/z: 212
(M+H)+; 1H NMR (CDCl3, 400 MHz): d 9.90 (1H, CHO), 7.86–7.84 (d, 2H, ArH),
7.02–7.00 (d, 2H, ArH), 4.86 (t, 2H, OCH2, J = 4.4 Hz), 4.34 (t, 2H, OCH2,
J = 4.4 Hz), ppm. Anal. Calcd for C9H9NO5: C, 51.19; H, 4.30; N, 6.63; O, 37.88.
Found: C, 51.07; H, 4.44; N, 6.35; O, 37.54. 4-(30-Nitrooxypropoxy)-
benzaldehyde 3b. Yield 78%; MS m/z: 226 (M+H)+; 1H NMR (CDCl3,
400 MHz): d 9.88 (1H, CHO), 7.85–7.82 (d, 2H, ArH), 7.01–6.99 (d, 2H, ArH),
4.68 (t, 2H, OCH2, J = 6.4 Hz), 4.16 (t, 2H, OCH2, J = 5.6 Hz), 2.26 (m, 2H, –CH2–,
J = 6.0 Hz), ppm. Anal. Calcd for C10H11NO5: C, 53.33; H, 4.92; N, 6.22; O, 35.53.
Found: C, 53.53; H, 4.72; N, 6.05; O, 37.50.
lM
a
absorbance versus concentration curve under the same experimental
conditions. The percent nitric oxide released (quantitated as nitrite ion) was
calculated ( SEM, n = 3) from the standard nitrite versus concentration curve.
(2) Incubation with phosphate buffer (pH 7.4). This assay was performed as
described under procedure 1 above except that a solution of the test compound
(2 mL of a 2 mM solution in 0.1 M phosphate buffer pH 7.4) was used and no
cysteine was added.
L-
28. Civelli, M.; Caruso, P.; Giossi, M.; Bergamaschi, M.; Razzetti, R.; Bongrani, S.;
Gasco, A. Br. J. Pharmacol. 1996, 118, 923.
29. Carystinos, G. D.; Alaoui-Jamali, M. A.; Batist, G. Cancer Chemother. Pharmacol.
2001, 47, 126.
30. In vitro cell cytotoxicity test (MTT assay). The cytotoxic effects of the compounds
on Bel-7402, MCF-7 and L-02 cells were determined by using the MTT assay.
Cells were planted in 100
well in 96-well microtiter plates. Plates had been incubated for 24 h at 37 °C
l
L medium at a concentration of 1 ꢁ 103 cells per
under an atmosphere of air containing 5% CO2. Medium (100
test drugs were added to quadruplicate wells and incubated for additional
72 h. The medium was then removed from the wells and 200 L MTT (1 g/mL
in complete medium) was added to each well, and then incubated for another
4 h. The formazan crystals were dissolved in 100 dimethylsulfoxide
buffered with 25 L glycine–NaCl solution (0.1 M glycine, 0.1 M NaCl, pH
lL) containing the
l
l
lL
l
10.5). The absorbance was measured in an enzyme-linked immunoabsorbent
assay plate reader (Bio-Rad) at a wavelength of 570 nm. The concentration
required for 50% inhibition of cell viability (IC50) was determined for the
various compounds tested.
31. In vitro accumulation assays test. BEL-7402 and L-02 cell cultures were grown in
24-well plates at 37 °C in 5% CO2 atmosphere until at least 70% confluency. The
medium was removed and replaced by a 100
lmol/L drug-containing one
24. Adler, A. D.; Longo, F. R.; Finarelli, J. D.; Goldmacher, J.; Assour, J.; Korsakoff, L. J.
Org. Chem. 1967, 32, 476.
25. Lindsey, J. S.; Schreiman, I. C.; Hsu, H. C.; Kearney, P. C.; Marguerettaz, A. M. J.
Org. Chem. 1987, 52, 827.
(500 L). After 0 and 30 min incubation at 37 °C in 5% CO2 atmosphere, the
l
drug-containing medium was removed and their absorbance was measured
using a Simadzu UV 2100 UV–vis scanning spectrophotometer.