A model for light-triggered porphyrin anticancer prodrugs based on an o-nitrobenzyl photolabile group

Article abstract of DOI:10.1002/ejoc.200700972

A model for light-triggered porphyrin anticancer prodrug 1 was designed and synthesized. Upon photolysis, prodrug 1 can efficiently liberate the anticancer drug tegafur. The MTT assay demonstrates that prodrug 1 is significantly less toxic than its parent drug tegafur, and 1 can release tegafur upon photoactivation in vitro. The light-triggered porphyrin anti-cancer prodrug technique developed herein may find useful applications in chemotherapy to minimize the side effects of anticancer drugs because of the tumor affinity property of porphyrin and the light-controllable anticancer drug dosing. Wiley-VCH Verlag GmbH & Co. KGaA, 2008.

Full text of DOI:10.1002/ejoc.200700972

SHORT COMMUNICATION
DOI: 10.1002/ejoc.200700972
A Model for Light-Triggered Porphyrin Anticancer Prodrugs Based on an
o-Nitrobenzyl Photolabile Group
Weiying Lin,*[a] Daiwei Peng,[a] Bin Wang,[a] Lingliang Long,[a] Cancheng Guo,[a] and
Jinbin Yuan[a]
Keywords: Anticancer / Prodrugs / Porphyrinoids / Light-triggered compounds / Cytotoxicity
A model for light-triggered porphyrin anticancer prodrug 1
was designed and synthesized. Upon photolysis, prodrug 1
can efficiently liberate the anticancer drug tegafur. The MTT
assay demonstrates that prodrug 1 is significantly less toxic
than its parent drug tegafur, and 1 can release tegafur upon
photoactivation in vitro. The light-triggered porphyrin anti-
cancer prodrug technique developed herein may find useful
applications in chemotherapy to minimize the side effects of
anticancer drugs because of the tumor affinity property of
porphyrin and the light-controllable anticancer drug dosing.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2008)
rivatives for biomedical release applications,[15] Reinhard
developed a series of o-nitrobenzyl-based photosensitive
phosphoramide mustards,[16] Gong exploited a model of a
photoactivated prodrug,[17] and Kehayova synthesized a
caged hydrophobic inhibitor of carbonic anhydrase II.[18]
Very recently, the group of McCoy provided an interesting
example of drug dosing with precise control by photolysis
of light-triggered compounds with the use of UV light.[19]
The major concern of the application of a photochemical
system in medicine is the tissue-damaging effect of UV
light. However, long-wavelength UV light generally imparts
minimal damage to tissues.[19,22] In addition, electromag-
netic radiation can be transferred by a fiber optic to further
minimize potential damage in medicinal applications.[19,17]
To the best of our knowledge, no examples of porphyrin
anticancer prodrugs that are able to release anticancer
drugs have ever been disclosed in the literature. In addition,
although a light-triggered strategy has been applied in the
design of the prodrug,[16–19] there is no report of light-trig-
gered anticancer prodrugs with a porphyrin. To continue
our efforts in the development of porphyrin anticancer pro-
drugs,[10,11] in this communication, we present the first
paradigm of an anticancer prodrug: it contains a porphyrin,
Introduction
Porphyrins are a class of photosensitizers used in photo-
dynamic therapy. Upon irradiation by red light (around
650 nm), porphyrins may be initially excited and further re-
sult in the formation of cytotoxic species such as singlet
oxygen. One important feature of porphyrins as photodyn-
amic therapy agents is that they tend to accumulate in neo-
plastic tissue to higher concentrations than in surrounding
normal tissue.[1–6] The tumor-affinity property of porphy-
rins has been exploited in anticancer drug design by inte-
grating porphyrins with drugs to prepare porphyrin–drug
conjugates: Brunner et al. created a series of porphyrin–
platinum conjugates by combining porphyrin with cytotoxic
platinum complexes;[7,8] Zhou reported some porphyrin–
DNA-alkylation-agent conjugates;[9] We also developed a
series of porphyrin conjugates as anticancer agents by link-
ing porphyrin with nitrogen heterocyclic species.[10,11] How-
ever, all the porphyrin–drug conjugates reported so far lack
a drug releasing mechanism, so they could not release drugs
from the conjugates. Obviously, a true prodrug should be
able to liberate the biologically active drug from the pro-
drug. There is a need to develop porphyrin anticancer pro-
drugs that are able to release the anticancer drug.
As photolysis of light-triggered (“caged”) compounds
can be temporally and spatially controlled,[12,13] light-trig-
gered compounds have been employed as a significant tool
in biological and medicinal studies.[12–22] For example, Dia-
mond reported the photocleavage of o-nitrobenzyl ether de-
[a] College of Chemistry and Chemical Engineering, Hunan Uni-
versity
Changsha, Hunan 410082, P. R. China
Fax: +86-731-8821464
E-mail: weiyinglin@hnu.cn
Supporting information for this article is available on the
WWW under http://www.eurjoc.org/ or from the author.
Figure 1. Schematic representation of the light-triggered porphyrin
anticancer prodrug.
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W. Lin, D. Peng, B. Wang, L. Long, C. Guo, J. Yuan
SHORT COMMUNICATION
but it is also able to release anticancer drugs upon photoly- H2SO4 smoothly produced key intermediate 4 in 78.6%
sis by long-wavelength UV light. The porphyrin anticancer yield. Porphyrin derivative 5 was prepared in 10.5% yield
prodrug developed herein is composed of three parts: a por- by treating aldehyde
4 (1 equiv.) with benzaldehyde
phyrin, a photocleavable o-nitrobenzyl moiety as a light- (4 equiv.) and pyrrole (5 equiv.) under standard Lindsey
triggered group, and a parent anticancer drug (Figure 1). conditions.[26] A statistical mixture of porphyrins was pro-
As a proof of concept, compound 1 (Scheme 1) was chosen duced by this procedure, and desired compound 5 was sepa-
as the model for the light-triggered porphyrin anticancer rated from the other porphyrin products by flash
prodrugs. We describe herein the synthesis, photochemistry, chromatography over silica gel. With compound 5 in hand,
and cytotoxic evaluation of porphyrin anticancer prodrug the stage was set to prepare target compound 1. Alkylation
1.
of intermediate 5 with anticancer drug tegafur (widely used
in cancer treatment), in the presence of potassium carbon-
ate at 70 °C afforded 1 in 92.6% yield. Uracil, a noncyto-
toxic compound, was used to replace anticancer drug te-
gafur in control 2. Similar nucleophilic substitution reac-
tion conditions were employed to prepare control 2 by
treating intermediate 5 with uracil. For uracil, the nucleo-
philic substitution reaction preferably occurred at the N-1
position.[27,28] After initial purification by silica-gel
chromatography, target compound 1 and control 2 were
further purified by preparative HPLC to finally afford a
single peak in the HPLC chromatogram. Compound 1 and
control 2 were both characterized by NMR, mass, and UV
spectroscopy and elemental analysis.
Preliminary assessment of the light-triggered conversion
of anticancer prodrug 1 to anticancer drug tegafur was per-
formed. The solution of prodrug 1 was irradiated with long-
wavelength UV light (350 nm). Aliquots were subsequently
analyzed by using reverse-phase HPLC. The conversion of
prodrug 1 into tegafur was followed as a function of time,
as shown in Figure 2. Prodrug 1 displayed rapid photoliber-
ation to tegafur. For example, after irradiation for 12 min,
approximately 50% of prodrug 1 was photolyzed to release
tegafur. The photolysis byproduct was identified as 5-(3-
nitroso-4-formylphenyl)-10,15,20-triphenylporphyrin {MS
(ESI): m/z = 672.3 [M + H]+}, which is consistent with the
photolysis mechanism of the o-nitrobenzyl group.[29] The
photochemical quantum yield (φ = 0.032) of the photolysis
was determined by ferrioxalate actinometry.[30] Although
the o-nitrobenzyl moiety is linked to the N-1 position of
uracil in control 2, according to the photolysis mechanism
of o-nitrobenzyl compounds,[29] control 2 should release
uracil (the same way as prodrug 1 releases anticancer drug
Scheme 1. Synthetic route to prodrug 1 and control 2. Reagents
and conditions: (a) DIBAL-H, toluene, 0 °C, 2.5 h; (b) KNO3,
H2SO4, 0 °C, 2.5 h; (c) benzaldehyde, pyrrole, BF3·Et2O, CHCl3,
room temp., 5 h, p-chloranil, 60 °C, 2 h; (d) K2CO3, anhydrous
DMF, 70 °C, 10 h; (e) K2CO3, anhydrous DMF, 50 °C, 8 h.
Results and Discussion
A versatile synthetic route to light-triggered prodrug 1
was developed (Scheme 1). We employed 4-bromometh-
ylbenzonitrile as the starting material to prepare key inter-
mediate 4. 4-Bromomethylbenzonitrile was chemoselec-
tively reduced by diisobutylaluminiumhydride (DIBAL-H)
in toluene under a nitrogen atmosphere at 0 °C to give 4-
bromomethylbenzaldehyde (3).[23–25] Toluene instead of
chlorobenzene was chosen as the solvent for easier
Figure 2. Photolysis conversion of prodrug 1 (᭹) and formation of
workup.[24] Nitration of 3 with a mixture of KNO3 and tegafur (ᮀ).
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A Model for Light-Triggered Porphyrin Anticancer Prodrugs
tegafur) upon photolysis. Indeed, control 2 was also pho-
tolyzed to release uracil efficiently.
Conclusions
A versatile synthetic route was developed to prepare anti-
cancer prodrug 1, which upon photolysis can be efficiently
converted into the anticancer drug tegafur. The MTT assay
demonstrated that prodrug 1 is significantly less toxic than
its parent anticancer drug tegafur, and prodrug 1 can re-
lease the cytotoxic anticancer drug tegafur upon photoacti-
vation in vitro. Furthermore, the control experiments also
demonstrated that the photolysis-induced cytotoxicity
(photolysis at 350 nm) observed for prodrug 1 is due to the
release of the anticancer drug. As we are aware, prodrug 1
developed herein represents the first paradigm of a porphy-
rin anticancer prodrug that is able to release anticancer
drugs and the first example of light-triggered anticancer
prodrug with a porphyrin. We envision that, combined with
the up-to-date medical fiber optic technique, the light-trig-
gered porphyrin anticancer prodrug may find useful appli-
cations in chemotherapy to minimize side effects of antican-
cer drugs because of the tumor-affinity property of porphy-
rin[1–6] and the light-controllable anticancer drug dosing.[19]
In addition, the light-triggered porphyrin anticancer pro-
drug technique created herein may potentially be employed
as binary cancer treatment in both chemotherapy and pho-
todynamic therapy to potentiate the efficacy of anticancer
drugs. Because two-photon excitation has better tissue
penetration than UV excitation, and because it could also
minimize the phototoxicity to cells and tissues,[35–38] for
practical use of this light-triggered porphyrin anticancer
prodrug technique, the UV photolabile o-nitrobenzyl moi-
ety will be replaced with a two-photon photolabile species
such as a 7-hydroxycoumarin moiety,[35] and the results will
be reported in due course.
As the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide] assay has been widely used to evaluate
the cytotoxicity of drugs,[10,11,31,32] we decided to also em-
ploy the MTT assay to examine the cytotoxicity of antican-
cer prodrug 1 in the absence or presence of long-wavelength
UV light (350 nm). MCF-7 mammary cancer cells were in-
cubated with anticancer prodrug 1 or the anticancer drug
tegafur. As expected, tegafur induced up to 91% cell death
(Figure 3). By contrast, prodrug 1 induced only 7% cell de-
ath; this indicated that prodrug 1 has significantly less cell
toxicity than tegafur. However, when MCF-7 mammary
cancer cells were incubated with prodrug 1 preirradiated
with UV light for 25 min, approximately 67% cell death was
observed. Similarly, irradiation of cells treated with prodrug
1 for 25 min induced 69% cell death. This is consistent with
the fact that irradiation for 25 min converted roughly 70%
of prodrug 1 into tegafur. A control experiment of UV light
indicated that irradiation for 25 min by long-wavelength
UV light alone (in the absence of prodrug 1 or tegafur) did
not induce marked effects on cell viability. As porphyrins
may produce cytotoxic singlet oxygen when irradiated by
red light (around 650 nm) in photodynamic therapy, it is
necessary to examine whether the photolysis-induced cyto-
toxicity (photolysis at 350 nm) of prodrug 1 is due to the
production of singlet oxygen or the release of the anticancer
drug. Towards this end, control 2, which contains noncyto-
toxic uracil instead of an anticancer drug, was incubated
with cells and irradiated at 350 nm for 25 min, but only ap-
proximately 6% cell death was observed. Therefore, the
cytotoxicity of control 2 upon photolysis at 350 nm UV
light is similar to that with 350 nm UV light alone. These
results unambiguously demonstrated that the photolysis-in-
duced cytotoxicity (photolysis at 350 nm) observed for pro-
drug 1 was due to the release of the anticancer drug, and
the photolysis byproduct, aromatic nitroso, did not appear
to induce apparent deleterious effects on the viability of the
cell (probably due to the high intracellular concentration of
reduced glutathione, which may neutralize the byprod-
uct).[13,33,34]
Supporting Information (see footnote on the first page of this arti-
cle): Procedures for the preparation, photolysis, and in vitro cyto-
toxicity tests for compound 1.
Acknowledgments
Funding was partially provided by the Scientific Research Founda-
tion for the Returned Overseas Chinese Scholars, State Education
Ministry (2007-24), the Key Project of Chinese Ministry of Educa-
tion, and the Hunan University research funds.
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Received: October 16, 2007
Published Online: January 7, 2008
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