Delayed release singlet oxygen sensitizers based on pyridone-appended porphyrins

Article abstract of DOI:10.1039/c7pp00244k

A new type of porphyrin photosensitizer capable of generating singlet oxygen upon irradiation, storing it through binding to pyridone subunits, followed by slow release at 20-40 °C, is reported. The timescale of singlet oxygen release can be varied depend

Full text of DOI:10.1039/c7pp00244k

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Delayed release singlet oxygen sensitizers based on pyridone-
appended porphyrins
Susan Callaghan,a Mikhail A. Filatov,a,* Elisabeth Sitte,a Huguette Savoie,b Ross W. Boyle,b Keith J.
Flanagana and Mathias O. Sengea,c,*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
A new type of porphyrin photosensitizer capable of generating release this species in target cells. Materials designed for this
singlet oxygen upon irradiation, storing it through binding to purpose, e.g., based on inorganic peroxides combined with
pyridone subunits, followed by slow release at 20-40 °C is biodegradable polymeric carriers showed promise in tissue
reported. The timescale of singlet oxygen release can be varied engineering, but suffer from poor selectivity of the release
depending on the pyridone group substitution pattern, forming process.4 The only chemical process of reversible binding of
endoperoxides of different stability. Modified tetra- and octa- singlet oxygen which can be remotely controlled relies on
substituted pyridone-porphyrins showed solubility in water, [4+2] cycloaddition to aromatic organic molecules. First
allowing for straightforward delivery into cells. The effect of reported by Dufraisse in 1926,5 it has been actively studied for
delayed singlet oxygen formation due to endoperoxide almost a century and guidelines for the design of such systems
decomposition was demonstrated on cancer cells in vitro.
have recently been formulated.6 Release of singlet oxygen
from such adducts has been shown to induce cellular damage
in malignant tissue for the deactivation of bacteria, viruses,
and parasites.7
Photodynamic therapy (PDT) relies on the generation of highly
reactive singlet oxygen (1O2) through a photosensitizer (PS)
excited state interaction with ground state triplet oxygen (3O2),
resulting in oxidative stress and cell death.1 One of the
limitations associated with this method is skin sensitivity to
strong light exposure. To minimize damage of healthy tissue
during the treatment, techniques including precise delivery of
the PS into diseased cells, spatiotemporal control of light
treatment, special formulations and chemical activation of PS
in the target cells have been developed.2
A
combination of standard photosensitizers with
endoperoxide-forming moieties could provide a significant
improvement to current PDT methods. To apply this approach
to conventional PDT treatments, PS bearing several
endoperoxide-trapping subunits are required to ensure that
significant amount of singlet oxygen is conserved in
a
chemically bound form within the irradiation period.
Previously reported systems based on naphthalene and
anthracene derivatives are not suitable as models for in vitro
studies, due to their hydrophobic character.8 However, 2-
pyridone derivatives are particularly well-suited for biomedical
applications due to their facile synthesis and inherent polarity
which enhances water solubility.9 Additionally, the respective
endoperoxide stability is strongly affected by the substitution
pattern: 2-pyridone endoperoxide possesses a half-life time of
30 min at 37 °C, while the 3-methyl-2-pyridone-endoperoxide
The need for prolonged irradiation of tissue to cause a
significant cytotoxic effect is, in part, associated with the short
lifetime of 1O2 in biological media. It is susceptible to
quenching by water molecules and antioxidants, causing decay
back to the ground state on a timescale of a few microseconds
and thus a reduced therapeutic effect.3 This near instant
deactivation of singlet oxygen upon photosensitization could
be overcome by using chemical sources of 1O2 which slowly
is considerably more stable (t1/2 ̴
8 h) (Scheme 1).9b Recently,
such 2-pyridones endoperoxides were demonstrated to cause
cytotoxicity in hypoxic cancer tissues.10
Herein we report a new type of porphyrin photosensitizers
modified with different numbers of 2-pyridone subunits (four
subunits in P1, P2, P5 and P6; eight subunits in P3 and P4) on
the periphery of the macrocycle. Upon light irradiation the
1
system produces O2, similar to conventional porphyrin PS. A
fraction of the 1O2 formed is bound to the pyridine subunits via
a Diels-Alder-type process. Photosensitized 1O2 is responsible
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for an immediate therapeutic response of the PDT agent, i.e. system was initiated upon heating to 40 °C and was complete
facilitates oxidation reactions in the cells. Following this initial within 7 h at this temperature.
response, a “slow” phase of the therapeutic effect occurs,
whereby a retro-Diels-Alder process evolves 1O2 from the
trapping moieties, which can last up to several hours,
depending on the substitution pattern of the trapping subunits
(Scheme 1).
R1
R1
O
O
t1/2, h
0.5
8.5
1O2
O
O
O
R2 =
R1 = H
Me
O
3
N
O
N
Figure 1. SOSG assay to detect 1O2 released from endoperoxides formed by P1 and P2.
Fluorescence of the sensor was excited at 505 nm. A) Rise of SOSG fluorescence in a
THF solution of P1 at 20 °C (inset: 525 nm emission changes with time). B) Rise of SOSG
fluorescence in solution of P2 at 40 °C.
R2
R2
1O2
3O2
R
N
R
O
EnT
O
Porphyrins P1 (Fig. 2)‡,§ and P2 were found to be insoluble in
water, despite the polar nature of pyridone substituents.
Hence, two approaches to increase solubility for cytotoxicity
studies were attempted: i) introduction of more pyridone
moieties (octasubstituted pyridone-porphyrins P3 and P4) and ii)
introduction of carboxyl groups in the meso-aryl substituents
N
NH
N
N
HN
N
O
P1
, R = H
P2, R = Me
R
O
N
R
R
N
O
or -O-O-
(
P5 and P6
involved bromination of 3,5-dimethylbenzoate
compound (Scheme 2). This was followed by reduction to the
alcohol with DIBAL and its oxidation to the aldehyde with
MnO2. A subsequent pyrrole condensation reaction gave the
octa((4-(bromomethyl)phenyl)porphyrin in 39 yield.
Treatment of with excess 2-pyridone or 3-methyl-2-pyridone
)
.
The synthesis of the octapyridone-porphyrins
O
N
O
O
1
to yield
NH
N
N
HN
2
N
3
4
O
R
O
1O2
N
5
%
or -O-O-
or -O-O-
R
5
Delayedrelease
in the presence of NaH gave the target systems P3 and P4 in
Scheme 1. Reversible addition of singlet oxygen to 2-pyridones and pyridone-
porphyrins.
58% and 65% yield, respectively.
Porphyrins P1 and P2 bearing four pyridone subunits were
prepared by reacting a 5,10,15,20-tetrakis(4-bromomethyl-
phenyl)porphyrin precursor (TBMPP)‡ with 2-hydroxypyridine
and 3-methyl-2-hydroxypyridine, respectively (see supporting
information (SI)).11 To prove the idea of delayed singlet oxygen
release from the pyridone endoperoxides formed upon
binding of self-sensitized 1O2 to P1 and P2, a Singlet Oxygen
Sensor Green (SOSG) assay was applied. In SOSG a singlet
oxygen responsive anthracene moiety is attached to
a
fluorophore and quenches its fluorescence via PeT
a
mechanism.12 Reaction of the anthracene unit with singlet
oxygen results in an increase in the probe fluorescence. Note,
that such probes can overestimate the level of 1O2 through
self-sensitization of 1O2.13 However, here the fluorescent
response of the probe was examined without light irradiation
of the sample excluding self-sensitization effects.
Figure 2. Molecular structure of P1 (thermal displacement 50%) showing the water
bridges between pyridone substituents. Hydrogen bonds are indicated by dotted lines.
1
To determine the timescale of thermal O2 release, P1 and P2
The second approach required selective monooxidation of
were irradiated with broadband light (400-700 nm) in THF at 0
°C for 1 h to ensure complete conversion of the pyridone
moieties into the endoperoxides. After irradiation, SOSG (0.1
eq.) was added and its fluorescence was monitored over time
while warming the sample to room temperature. As shown in
Fig. 1A, 1O2 release from P1 took place at 20 °C and was
complete within 5 h. In contrast, using P2 under the same
conditions, no detectable increase in the fluorescence was
observed within 24 h. However, release of 1O2 from this
dibromide
aldehyde
2
using bis(tetrabutylammonium)dichromate to
6
which was used to access the tetrasubstituted
pyridone-porphyrins P5 and P6 fashioned with carboxylic
groups (Scheme 3). Condensation of with pyrrole provided
the brominated porphyrin which was used in substitution
reactions with the corresponding pyridones to yield or
Finally, saponification of the ester using excess KOH gave P5
6
7
8
9.
and P6
.
All of the aforementioned pyridone-substituted
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porphyrins were purified by recrystallization as degradation difference (Fig. 3). However, P3 and P4, carrying eight
during silica column chromatography was observed and were pyridone/methyl pyridone units showed clear differences with
characterized by NMR and mass-spectrometry (see SI).
80% cell inactivation for P4 versus 23% for P3 at the highest
concentration used (200 M). Taking the structural similarities
µ
of P3 and P4 into account this result may be explained by
CHO
CH2OH
CO2Me
CO2Me
c
a
b
enhanced cell killing due to slow 1O2 release post irradiation.
+
N
62%
30%
81%
BrH2C
CH2Br
H
BrH2C
CH2Br
BrH2C
CH2Br
4
2
3
1
R
39%
d
O
O
N
R
R
N
N
BrH2C
CH2Br
O
N
R
O
N
NH
N
CH2Br
BrH2C
BrH2C
e
NH
N
58-65%
HN
N
O
N
HN
N
CH2Br
O
R
R
N
R
R
+
O
N
BrH2C
CH2Br
O
N
O
H
(15 eq.)
5
P3, R = H
P4,
R
R = Me
Scheme 2: Synthesis of octapyridone-porphyrins. a) NBS (2 eq.), benzoyl peroxide, CCl4,
reflux; b) DIBAL (2 eq.), toluene, 70 °C; c) MnO2 (10 eq.), DCM, rt; d) BF3·Et2O, DCM, rt,
then DDQ (0.75 eq.), rt; e) NaH (15 eq.), THF, reflux.
BrH2C
CO2Me
2
11 %
a
CH2Br
R
O
CHO
MeO2C
BrH2C
Figure 3. HT29 cell viabilities after 1 h incubation with porphyrins P3-6 with and
without light irradiation (400-700 nm, 20 J·cm-2).
NH
N
N
b
+
+
N
H
42%
N
HN
BrH2C
CO2Me
H
(15 eq.)
CO2Me
6
7
c
Conclusions
MeO2C
CH2Br
55-77%
CO2Me
R
O
In summary, we have synthesized and studied porphyrin
photosensitizers bearing endoperoxide-forming 2-pyridone
substituents on the periphery of the macrocycle.
Photosensitization of oxygen by the porphyrin yields 1O2, a
fraction of which is stored due to [4+2] cycloaddition with the
2-pyridone subunits. The effect of the substitution pattern on
the stability of endoperoxide gives access to systems which
either release 1O2 at room temperature or are completely
stable and require moderate heating to trigger endoperoxide
decomposition.
N
O
N
R
R
O
MeO2C
N
NH
N
COOH
HN
N
CO2Me
N
O
O
N
R
R
HOOC
N
NH
d
MeO2C
8, R = H
72-88%
N
O
R
HN
N
COOH
N
O
9
, R = Me
R
HOOC
Further synthetic modifications of the sensitizers gave water-
soluble derivatives, which can be used in vitro and in vivo. The
differences observed in the cytotoxicities of the systems
studied illustrate a viable concept for delayed singlet oxygen
release from endoperoxide moieties formed upon irradiation
of tissue/cell-localized sensitizers.
N
O
P5
, R = H
P6, R = Me
R
Scheme 3: Synthesis of tetrapyridone-porphyrins with carboxylic acid functionalities. a)
(Bu4N)2Cr2O7 (0.66 eq.), CHCl3, 70 °C; b) BF3·Et2O, DCM, rt, then DDQ (0.75 eq.), rt; c)
NaH (15 eq.), THF, reflux; d) KOH (30 eq.), THF, reflux.
In order to ascertain whether the presence of pyridone versus While this study gives proof-of-concept for using a photo- and
methylpyridone units on the porphyrin core resulted in chemodynamic approach for 1O2 production, storage, and
measurable differences, in terms of the compound’s ability to release, further studies and optimizations are needed to
cause
photosensitized
damage
to
cells,
human achieve a significant improvement over conventional PDT. In
P4 P5 addition to uptake and intracellular localization studies these
adenocarcinoma cells (HT-29) were incubated with P3
,
,
and P6. After 1 h the cells were washed, fresh medium was include a significant shortening of the irradiation time to
added, and the cells were irradiated with broadband visible provide a cytotoxic effect and require materials with a larger
light (400–700 nm, 20 J·cm-2). On completion of irradiation number of oxygen traps located in the proximity of the PS
cells were returned to the incubator for 24 h, after which cell (such as polymeric endoperoxide carriers) which are currently
viability was determined using the MTT assay.14
under development.
A comparison of the photocytotoxicity results for P5 and P6
,
which bear four pyridone/methyl pyridone units revealed little
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Acknowledgements
This work was supported by grants from Science Foundation Ireland
(IvP 13/IA/1894), the European Commission (CONSORT, Grant No.
655142) and the Irish Research Council (GOIPG/2016/1250).
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