Mitochondrial Targeting Cationic Purpurinimide–Polyoxometalate Supramolecular Complexes for Enhanced Photodynamic Therapy with Reduced Dark Toxicity

Article abstract of DOI:10.1002/ejic.202100485

Polyoxometalates (POMs), that are well-defined metal-oxygen cluster anions, are functional molecules extensively used in various research applications. The synthesis of neutral purpurinimides, followed by conversion to cationic purpurinimides (CPIs), formed CPI–POM supramolecular complexes via electrostatic interactions between each CPI and the polyanionic POM. The cellular uptake of purpurinimides, CPIs, and their CPI–POMs in A549 and HeLa cell lines was confirmed by confocal laser scanning microscopy. CPIs showed concentration-dependent dark cytotoxicity; however, CPI–POMs exhibited reduced low dark cytotoxicity. After photoirradiation, CPIs and CPI–POMs revealed enhanced photodynamic therapy (PDT) compared to free purpurinimides against A549 and HeLa cells. The CPIs exhibit low cell viability by incorporating their PDT effect with intrinsic dark cytotoxicity; however, the CPI–POMs exhibited a POM delivery effect-based enhanced PDT activity in the supramolecular complex system with low dark cytotoxicity. In particular, the clicked CPI–POM revealed two times higher PDT activity compared with the clicked free CPI, due to the good delivery effect of POM.

Full text of DOI:10.1002/ejic.202100485

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doi.org/10.1002/ejic.202100485
Mitochondrial Targeting Cationic Purpurinimide–
Polyoxometalate Supramolecular Complexes for Enhanced
Photodynamic Therapy with Reduced Dark Toxicity
[
a]
[a]
[a]
[a]
[a]
Tae Heon Lee, Yang Liu, Hye Jeong Kim, Sang Hyeob Lee, Hyeon Ho Song,
[a]
[a]
[a]
Young Key Shim, Woo Kyoung Lee,* and Il Yoon*
Polyoxometalates (POMs), that are well-defined metal-oxygen
cluster anions, are functional molecules extensively used in
various research applications. The synthesis of neutral purpur-
inimides, followed by conversion to cationic purpurinimides
dark cytotoxicity. After photoirradiation, CPIs and CPI–POMs
revealed enhanced photodynamic therapy (PDT) compared to
free purpurinimides against A549 and HeLa cells. The CPIs
exhibit low cell viability by incorporating their PDT effect with
intrinsic dark cytotoxicity; however, the CPI–POMs exhibited a
POM delivery effect-based enhanced PDT activity in the
supramolecular complex system with low dark cytotoxicity. In
particular, the clicked CPI–POM revealed two times higher PDT
activity compared with the clicked free CPI, due to the good
delivery effect of POM.
(CPIs), formed CPI–POM supramolecular complexes via electro-
static interactions between each CPI and the polyanionic POM.
The cellular uptake of purpurinimides, CPIs, and their CPI–POMs
in A549 and HeLa cell lines was confirmed by confocal laser
scanning microscopy. CPIs showed concentration-dependent
dark cytotoxicity; however, CPI–POMs exhibited reduced low
Introduction
Recently, we have developed delivery systems for better
cellular uptake of PSs into tumour cells using gold nanoparticles
[31–35]
Cationic chlorins and porphyrins are expected to exhibit
increased cellular penetration and retention in tumour cells,
making them useful for applications such as DNA binding/
(sphere and rod types)
complexes. POMs, which are metal-oxygen cluster anions, are
functional molecules extensively used in various applications
and polyoxometalate (POM)
[36]
[1–4]
photocleavage
and for use as photosensitizers (PSs) for
owing to their favourable catalytic, photochemical, and elec-
[5–8]
[37–39]
photodynamic therapy (PDT).
A type of click chemistry
trical properties.
Importantly, there are very rare biomate-
reaction, copper (I)-catalysed alkyne-azide cycloaddition, has
attracted much attention because of its straightforwardness,
mild conditions, and high yields, allowing for the synthesis of
functionally modified and conjugated molecules that have wide
rial applications of POM-based supramolecular materials those
[40–44]
[36]
built on electrostatic interactions.
In a previous report,
we demonstrated a chlorin–POM supramolecular complex that
showed enhanced photodynamic activity compared to free
chlorin. This POM complex exhibited excellent PS delivery,
owing to the stable and strong electrostatic interactions
between polyanionic POM and cationic chlorins. Among the
chlorins, purpurinimides have attracted attention because of
their unique advantages, such as long wavelength absorption
[9–12]
applications.
PDT is a promising non-invasive and patient-
specific cancer treatment with minimal side effects, which is
based on the administration and selective accumulation of a PS
in tumour tissue, to generate cytotoxic reactive oxygen species
1
(
ROS), especially singlet oxygen ( O ), after photoirradiation.
2
These cytotoxic species inflict direct cellular damage, destroy
tumour vascularization, and trigger subsequent inflammatory
to permit deeper light penetration at tumour sites, high
1
extinction coefficient at an appropriate wavelength, good O
2
[13–23]
[5,19,22,45–48]
and immune cell destruction.
However, the clinical applica-
photogeneration, and low hydrophobicity.
In the
tion of PDT is hampered by the need to increase cellular
penetration without causing aggregation, light penetration
with long wavelength absorption, and prevention of dark
toxicity of PSs for enhanced PDT activity with few side
supramolecular system, POM plays an important role as a
delivery vehicle (polar attractor) of cationic purpurinimides
(CPIs) into tumour cells via endocytosis (Figure 1).
[49]
However, the disadvantages of the reported chlorin–POM
complex are its short wavelength absorption (maximum
absorption wavelength [λmax] 665 nm in CH Cl ), that prevents
[15,24–30]
effects.
2
2
[
a] T. H. Lee, Dr. Y. Liu, H. J. Kim, S. H. Lee, H. H. Song, Prof. Dr. Y. K. Shim,
Prof. Dr. W. K. Lee, Prof. Dr. I. Yoon
Center for Nano Manufacturing and Department of Nanoscience and
Engineering
Inje University
deep tissue penetration, as well as high IC50 value (6.61 μM at
8 h and 8.60 μM at 24 h incubation times) due to the very low
photodynamic activity of the free cationic chlorin (IC >20 μM)
in the tumour cells. To overcome these limitations, we herein
report the design and synthesis of new CPIs and the
preparation of their POM complexes (CPI–POMs), that show
4
5
0
[36]
1
97 Injero, Gimhae, Gyeongnam 50834, Republic of Korea
E-mail: yoonil71@inje.ac.kr
wlee@inje.ac.kr
long wavelength absorption (λmax 707 nm in CH Cl ), and
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/ejic.202100485
2
2
therefore permit deeper light penetration at tumour sites and
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1
fully characterised by H-NMR, UV-Vis spectroscopy, and
HRFABMS.
To introduce POM, we used the well-known Keggin-type
[36,56]
4À
POM 4,
[α-SiMo O ] , which was prepared according to a
1
2
40
procedure previously described in literature, and was soluble in
common organic solvents such as acetonitrile in its organic salt
form (e.g. tetrabutylammonium). Combining POM and four
equivalents of each CPI (1+, 2+, and 3+) in acetonitrile
afforded the CPI–POM complexes (1+POM, 2+POM, and 3+
POM) as dark green solids (precipitates) in good yield (70%–
7
3%, Scheme 1b), exhibiting the stable and strong electrostatic
interactions between polyanionic POM and CPIs.
1
The structures of all compounds were characterised by H-
NMR, UV-Vis, and FTIR spectroscopy, and HRFABMS (Supporting
1
Figure 1. (a) Formation of a CPI–POM supramolecular complex via electro-
Information, Figures S1–S17). The H-NMR spectrum of NPPME
static interactions. (b) Proposed mechanism for the cellular uptake followed
shows one triplet signal at 2.31 ppm (J=2.4 Hz) and multiplet
by mitochondrial targeting of CPI–POM complexes into tumour cells through
1
signals at 5.35–5.32 and 5.31–5.23 ppm in CDCl , corresponding
3
endocytosis, causing cell death after photogeneration of O
enhanced photodynamic activity.
2
, resulting in
to characteristic alkyne (C�CH) and N-CH protons, respectively
2
[57]
1
(Figure S3).
The H-NMR spectra of neutral purpurinimide
derivatives 1, 2, and 3, their cationic forms 1+, 2+, and 3+,
and their complexes 1+POM, 2+POM, and 3+POM are
shown in Figure 2, Figure 3, and Figure 4, respectively. All
proton signals were fully assigned, and each signal of the N-
alkyl group attached to the six-membered imide ring is high-
lighted in blue. Furthermore, the chemical shift changes are
high photodynamic activity. Purpurinimides, CPIs, and CPI–
POMs accumulated in A549 and HeLa cells, and exhibited
mitochondrial targeting, inducing mitochondria-mediated
[50–54]
apoptosis to destroy the tumour cells.
The CPIs and CPI–
5
POM complexes exhibited significantly enhanced (highly de-
creased) IC50 values compared to the corresponding free neutral
purpurinimides. More importantly, while the CPIs showed
concentration-dependent dark toxicity, CPI–POM complexes
showed highly reduced dark toxicity. These findings indicate
that CPI–POM complexes are potential PS candidates for
enhanced PDT with reduced dark toxicity.
shown as dashed lines. The proton signal of N in 1+ was
significantly downfield-shifted (~0.8 ppm) from 1. All proton
signals on benzyl pyridine in 2+ were shifted downfield
2
3
compared with the neutral form 2. In particular, N and N
signals on the pyridine unit in 2+ were highly downfield
1
shifted. The H-NMR spectrum of 3 featured a key triazole signal
2
(N , singlet) at 8.48 ppm, indicating a successful click reaction,
as well as signals of the pyridine unit at 8.66 and 7.76 ppm. The
1
H-NMR spectrum of clicked CPI 3+ displays the pyridinium salt
Results and Discussion
signal at 4.11 ppm (broad singlet), indicating successful meth-
ylation, as well as two broad singlets of the pyridine unit at 8.74
and 8.31 ppm. No signals of the tetrabutylammonium cation
(present in the starting POM 4, Figure S5) were detected for any
of the POM complexes, indicating complete cation exchange
with each CPI, which leads to a 4:1 CPI:POM ratio (Scheme 1b).
HRFABMS analysis confirmed the formation of 1 (calcd. for
Characterisation of Purpurinimides and their CPIs and CPI–
POM complexes
The synthesis of purpurinimides and their cationic forms is
straightforward (Scheme 1a). Extraction of chlorophyll-a paste
with acidic methanol (5% H SO4 in MeOH) afforded meth-
ylpheophorbide a (MPa) as an important starting material.
MPa was further converted to purpurin-18 methyl ester (P18ME)
under basic conditions, affording the purpurinimide derivatives
+
[M+H] 677.3815; found 677.3818) (Figure S8), 1+ (calcd. for
2
[55]
+
[M] 691.3966; found 691.3977) (Figure S9), 2 (calcd. for [M+
+
+
+
+
H] 669.3189; found 669.3192) (Figure S10), 2+ (calcd. for [M]
683.3340; found 683.3342) (Figure S11), 3 (calcd. for [M+H]
736.3360; found 736.3357) (Figure S12), and 3+ (calcd. for [M]
1
and 2 after reaction with the corresponding amine, N,N-
dimethylbutylamine and 4-picolylamine, respectively. N-Prop-
argyl purpurinimide methyl ester (NPPME) was obtained from
the reaction of P18ME with propargyl amine, and then used in a
click reaction utilising copper iodide (CuI, 10 mol%) and
diisopropylethylamine (DIPEA) in CH Cl , which furnished a
triazole ring to generate clicked purpurinimide 3. Methylation
of each purpurinimide derivative 1–3 with methyl iodide (MeI)
afforded the corresponding CPI derivatives 1+À 3+, as dark
green solids in quantitative yield without further purification.
All reaction steps were monitored by TLC and UV-Vis
absorption spectroscopy. In addition, all the compounds were
750.3511; found 750.3513) (Figure S13). The FTIR spectrum of 4
displayed signals specific to POM, such as Si=O, Mo=O, and
À 1
À 1
À 1
MoÀ OÀ Mo stretches at 951 cm , 899 cm , and 804 cm ,
respectively (Figure S6). The absorption spectra of all purpur-
inimides, their CPIs, and their CPI–POM complexes, were
recorded in DMSO (Figure 5). The maximum absorption wave-
lengths (λmax) were progressively red-shifted for MPa (668 nm),
P18ME (700 nm), NPPME (707 nm), and 1–3 (707 nm) (Figure 5,
Figure S2, and Figure S4). In purpurinimide derivatives with
various alkyl groups, the λmax values of the purpurinimide core
remained unchanged, even in both cationic and POM complex
2
2
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Scheme 1. (a) Synthesis of purpurinimide derivatives 1–3 and their CPI forms (ammonium, pyridinium, and clicked pyridinium) 1+, 2+, and 3+. (b) Synthesis
of CPI–POM complexes 1+POM, 2+POM, and 3+POM.
forms. In addition, TGA analysis (Figures S14–S16) of 1+POM, 2
POM, and 3+POM showed 60–62 wt% of organic content
four CPI units) and 40–38 wt% of inorganic content (one POM
purpurinimide or CPI molecules (red colour) of all compounds
was detected inside the cells, indicating successful cellular
uptake of purpurinimide or CPI in the cytoplasm of the tumour
cells. In particular, all the purpurinimide or CPI molecules
showed mitochondrial targeting (orange colour in merged
images) inducing mitochondria-mediated apoptosis to destroy
+
(
unit), confirming a 4:1 ratio (CPI:POM), as shown in Scheme 1.
[50–54]
Cellular Uptake and Localization
the tumour cells after photoirradiation.
The cellular penetration and localisation of purpurinimides,
CPIs, and CPI–POM complexes in A549 and HeLa cells before
in vitro irradiation were confirmed by confocal laser scanning
[58,59]
microscopy (CLSM, Figure 6, Figure 7).
Fluorescence of the
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3 6
Figure 2. H-NMR spectra of purpurinimide 1 and CPI 1+ in CDCl , and 1+POM complex in DMSO-d (500 MHz, 298 K).
1
Figure 3. H-NMR spectra of purpurinimide 2 and CPI 2+ in CDCl
3
, and 2+POM complex in DMSO-d
6
(500 MHz, 298 K).
Morphological Changes with Photoirradiation
result was confirmed using fluorescence microscopy of live
green)/dead (red) cells stained with calcein AM/PI. Compound
(
After irradiation with a halogen lamp equipped with a band-
pass filter (640–710 nm) (total light dose 2 Jcm , irradiation
3+ (Figure 8e) and complex 3+POM (Figure 8f) killed more
cells than compound 3 (Figure 8d), which was in agreement
with the morphological changes shown in Figures 8a–c.
À 2
time 15 min), the cells treated with 3+ (Figure 8b) and 3+
POM (Figure 8c) exhibited morphologies different from those
treated with 3 (Figure 8a), due to the formation of apoptotic
bodies, owing to the higher photodynamic activity of the
[39,60]
former compared to 3 at a concentration of 1 μM.
This
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Figure 4. H-NMR spectra of purpurinimide 3 and CPI 3+ in CDCl , and 3+POM complex in DMSO-d (500 MHz, 298 K).
concentration-dependent dark cytotoxicity, owing to the char-
[61,62]
acteristic properties of these cationic PSs.
Also, 3 (neutral
form) showed concentration-dependent dark cytotoxicity than
others. However, none of the POM complexes, 1+POM–3+
POM, showed dark cytotoxicity, which indicates that POM
complexes are potential PS candidates. This result is consistent
[36]
with that of our previous study. On the other hand, in HeLa
cells, all neutral and cationic PSs showed concentration-
dependent dark cytotoxicity. However, POM complexes dis-
played highly reduced dark cytotoxicity, possibly because of the
electrostatic interactions between the cationic purpurinimides
and anionic POMs, which mitigated the toxicity of free CPI. 1+
POM and 2+POM showed concentration-dependent dark
toxicity after 3 h incubation, but negligible dark toxicity after
2
4 h incubation. Notably, 3+POM showed no dark toxicity.
Upon photoirradiation, the cell viability decreased for all
Figure 5. UV-Vis absorption spectra (10 μM in DMSO at 25°C) of free PSs,
cationic PSs, and their POM complexes.
compounds, a finding consistent with the observation of
increased concentration and incubation time. Furthermore, cell
viability was dependent on the cell type. In A549 cells, each
cationic form showed better results than the corresponding
free purpurinimides, which might be because of the electro-
static interaction between the CPI and the cell, followed by
accumulation in the cells. Even though the cationic form, 1+
and 2+, showed almost identical therapeutic activities
(0.14 μM and 0.18 μM of IC50 after 3 h of incubation; 0.13 μM
and 0.15 μM of IC50 after 24 h of incubation, respectively)
(Table 1) compared with the POM complex, 1+POM and 2+
POM, (0.14 μM and 0.14 μM of IC50 after 3 h of incubation;
0.13 μM and 0.13 μM of IC50 after 24 h of incubation, respec-
tively) the high photoactivity led to concentration-dependent
dark toxicity. However, the POM complexes showed no dark
toxicity, indicating that the high photoactivity of the POM
complexes is a sign of good delivery effect of the POM in the
POM complexes. Furthermore, 3+POM (0.37 μM and 0.17 μM
Cell Viabilities for Photocytotoxicity and Dark Cytotoxicity
Photo- and dark cytotoxicities of 1–3, 1+À 3+, and 1+POM–3
+
POM were investigated at a concentration range of 0.25–
2
0 μM, and incubation periods of 3 h and 24 h, against A549
4
and HeLa cells (Figure 9). Each cell (10×10 cells/well) was
incubated with PSs for 24 h, and photoirradiated for the
photocytotoxicity test (total light dose 2 Jcm , irradiation time
À 2
1
2
5 min). After photoirradiation of cells incubated for 3 h and
4 h, their viability (%) was estimated based on the mitochon-
drial activity of NADH (reduced form of nicotinamide adenine
dinucleotide) dehydrogenase using the MTT cell viability assay
(summarised in Tables S1–S4).
Dark toxicity was different for different cell types (A549 and
HeLa). In A549 cells, all cationic PSs 1+À 3+ showed
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Figure 6. Cellular uptake using CLSM images of A549 cells before irradiation
at 6 h post-incubation for (a) 1, 1+, 1+POM, (b) 2, 2+, 2+POM, (c) 3, 3+,
+POM (the concentration of PS is 1 μM in each sample): DAPI nuclear dye
blue), PS (red), Mito-Orange mitochondrial dye (green) and merged images.
Figure 7. Cellular uptake using CLSM images of HeLa cells before irradiation
at 6 h post-incubation for (a) 1, 1+, 1+POM, (b) 2, 2+, 2+POM, (c) 3, 3+,
3
(
3
+POM (the concentration of PS is 1 μM in each sample): DAPI nuclear dye
(
blue), PS (red), Mito-Orange mitochondrial dye (green) and merged images.
Scale bar: 20 μm.
Scale bar: 20 μm.
of IC50 after 3 h and 24 h of incubation, respectively) displays
two times higher photoactivity when compared with its cationic
form 3+ (0.69 μM and 0.48 μM of IC50 after 3 h and 24 h of
incubation, respectively) indicating good delivery effect of the
POM complex. Interestingly, the photoactivities (IC ) of 1+ and
24 h of incubation, which could indicate a very fast intracellular
uptake of 1+, possibly because of the high flexibility of the N-
alkyl group. This phenomenon is consistent with the results of
[45]
our previous study.
In HeLa cells, each CPI and its POM
complex showed identical photoactivities after 24 h of incuba-
5
0
1
+POM after 3 h of incubation were the same as those after
tion. In both A549 and HeLa cells, 1+ and 1+POM, and 2+
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indicates that the enhanced photodynamic activity of POM
complexes based-on the good delivery effect of POM results
from the high cellular penetration and localisation of the
purpurinimide moieties of POM complexes in tumour cells via
[41,66]
endocytosis, as compared to free purpurinimides.
Conclusion
In conclusion, we synthesised CPI derivatives, such as
ammonium, pyridinium, and clicked pyridinium salt, which were
then used to prepare CPI–POM supramolecular complexes,
showing long-wavelength absorption (λmax 707 nm) and cellular
uptake followed by mitochondrial targeting, inducing mito-
chondria-mediated apoptosis after photoirradiation to destroy
the tumour cells. The CPIs and their CPI–POM complexes
exhibited a significantly enhanced PDT effect against A549 and
HeLa cells (IC50 0.12–0.48 μM after 24 h of incubation) compared
with neutral purpurinimide derivatives. The CPIs and their CPI–
POM complexes of ammonium (1+ and 1+POM) and
pyridinium (2+ and 2+POM) exhibited almost identical PDT
activities against A549 and HeLa cells, those might be attributed
by high photoactivity of each cationic PS itself. Interestingly,
the clicked CPI–POM complex (3+POM, IC50 0.17 μM) exhibited
two times better PDT activity than the free clicked CPI (3+, IC50
0.48 μM) after 24 h of incubation against A549 cells, revealing
the good delivery effect of POM. Importantly, while the CPIs
displayed concentration-dependent dark cytotoxicity, the CPI–
POM complexes showed very low dark toxicity, making them
potential PS candidates for PDT. Therefore, this CPI–POM
supramolecular complex could not only be a useful PS, but
could also serve as a suitable delivery vector with reduced side
effects in clinical applications. In addition, for further medicinal
application using these CPI–POM complexes, we considering
that it is need to increase low stability of the CPI–POM
complexes in the physiological environment (Table S5 and
Figures S17–S18 in the Supporting Information) as a future
study.
Figure 8. Optical images of A549 cells after treatment with 3 (a), 3+ (b), and
+POM (c); fluorescence images of live (green)/dead (red) cells stained with
calcein AM/PI after 3 h of incubation with 3 (d), 3+ (e), and 3+POM (f) and
photoirradiation. The concentration of each PS was 1 μM.
3
and 2+POM showed almost identical photoactivities after 24 h
of incubation (IC : 0.13 μM and 0.13 μM, and 0.15 μM and
50
0
0
.13 μM in A549; 0.14 μM and 0.14 μM, and 0.14 μM and
.14 μM in HeLa, respectively). On the other hand, 3+ and 3+
POM showed better photoactivities in HeLa cells after 24 h of
incubation, compared with those in A549 cells (IC : 0.12 μM
5
0
and 0.12 μM in HeLa; 0.48 μM and 0.17 μM in A549, respec-
tively). POM 4 displayed low phototoxicity, which almost
equalled the dark cytotoxicity. It is noteworthy that POM
complexes exhibit very high phototoxicity and very low dark
cytotoxicity, making them potential PS candidates for PDT. POM
complexes display advantageous, stable, and strong electro-
static interactions between POM and the four CPI moieties,
playing an important role not only as a delivery vector for CPIs
[39]
(as polar attractor),
but also as a reducer of their dark
cytotoxicity.
Singlet Oxygen Photogeneration
1
Figure 10 highlights the relative differences between O photo-
2
1
generation by all the compounds using DPBF as a selective O
2
[63]
1
acceptor.
Compounds 2, 2+, and 2+POM showed O2 Experimental Section
photogeneration that was almost identical to that of MB
1
[64,65]
(
standard O2 acceptor).
Interestingly, 2+POM and 3+
Materials
1
POM showed slightly lower O photogeneration than 2 and 2
2
All chemical reagents used in this experiment were of analytical
grade and required no further purification, including potassium
hydroxide (KOH), sulfuric acid (H SO ), nitric acid (HNO ), acetic acid
+
and 3 and 3+ in the absence of tumour cells; however, the
POM complexes showed better or comparable PDT activity
compared with free forms (neutral and cationic PSs), which
2
4
3
(AcOH), anhydrous sodium sulfate, tetrabutylammonium chloride
Table 1. IC50 (μM) values of various purpurinimides and their cationic and POM complex forms against A549 and HeLa cells after 3 h and 24 h of incubation
2
after irradiation (total light dose: 2 J/cm with LED for 15 min), respectively.
Cell type
Incubation time
1
1+
1+POM
2
2+
2+POM
3
3+
3+POM
3
2
3
2
h
4 h
h
0.87
0.46
0.34
0.17
0.14
0.13
0.20
0.14
0.14
0.13
0.31
0.14
1.82
0.93
0.31
0.17 (0.19)
0.18
0.15
0.29
0.14
0.14
0.13
0.22
0.14
12.42
0.94
0.48
0.32
0.69
0.48
0.19
0.12
0.37
0.17
0.18
0.12
A549
HeLa
[a]
4 h
[a] Reference [19] by WST-8 assay after 12 h of incubation.
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À 2
Figure 9. Cell viability for photocytotoxicity (light dose of 2 Jcm ) and dark cytotoxicity of (a) and (d) 1, 1+, and 1+POM, against A549 and HeLa cells,
respectively, (b) and (e) 2, 2+, and 2+POM, against A549 and HeLa cells, respectively, (c) and (f) 3, 3+, and 3+POM, against A549 and HeLa cells,
respectively, at concentrations of 0.25–20 μM. The cell viability percentage was determined by an MTT assay after 3 h or 24 h of incubation before (dark) and
after (light) photoirradiation. Error bars represent the standard deviation of three replicate experiments.
(
(
(C H ) N·Cl), sodium molybdate (Na MoO ·2H O), sodium silicate
blue (MB) (TCI Chemical), 1-propanol, pyridine (Burdick & Jackson
Chemical), EZ-View Live/Dead cell staining kit (calcein-AM
(acetoxymethyl ester) and propidium iodide (PI)) (Biomax Chem-
ical).
4
9 4
2
4
2
Na SiO ), dimethyl sulfoxide (DMSO) (Samchun Chemical), dichloro-
2
3
methane (CH Cl ), n-hexane, methanol (MeOH), acetone,
acetonitrile, diethyl ether, ethyl acetate (EtOAc) (SK Chemical), 4-
chloropyridine hydrochloride, 4-azidopyridine, copper(I) iodide
2
2
(
(
(
CuI), diisopropylethylamine (DIPEA), sodium azide, methyl iodide
MeI) (Sigma Aldrich Chemical), 4’,6-diamidino-2-phenylindole
DAPI), N,N-dimethylbutane-1,4-diamine, 4-aminomethyl pyridine,
propargyl amine, 1,3-diphenylisobenzofuran (DPBF), methylene
Eur. J. Inorg. Chem. 2021, 1–14
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8
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(
500 MHz, CDCl ): δ 9.63, 9.40, 8.59 (all s and 1H, 10-H, 5-H, and 20-
3
1
H, respectively), 7.91 (dd, J=18.1, 11.7 Hz, 1H, 3 CH=CH ), 6.34 (d,
J=18.1, 1.1 Hz, 1H, 3 -H), 6.22 (d, J=11.7, 1.1 Hz, 1H, 3 -H), 5.21 (dd,
J=9.4, 2.5 Hz, 1H, 17-H), 4.42 (q, J=7.9 Hz, 1H, 18-H), 3.82 (s, 3H,
1
(
2
3
1
2
2
2
1
2
2-CH ), 3.67 (q, J=7.9 Hz, 2H, 8 -CH ), 3.61 (s, 3H, 17 -OCH ), 3.37
3 2 3
2
s, 3H, 2-CH ), 3.19 (s, 3H, 7-CH ), 2.77–2.73 (m, 1H, 17 -CH ), 2.52–
.44 (m, 2H, 17 -H), 2.05–1.99 (m, 1H, 17 -CH ), 1.77 (d, J=7.6 Hz,
H, 18-CH ), 1.69 (t, J=7.6 Hz, 3H, 8 -CH ), 0.28, À 0.03 (all br s and
3
3
2
1
2
2
2
3
3
H, NH).
Synthesis of N-Propargyl Purpurinimide Methyl Ester (NPPME)
P18ME (500 mg, 0.864 mmol) and propargyl amine (1 mL,
1
5.6 mmol, >18 eq. excess) were dissolved in CH Cl (5 mL), and
2 2
the mixture was stirred at room temperature for 24 h. The
disappearance of the 700 nm band and the appearance of a new
668 nm band in the UV-Vis spectrum indicated completion of the
reaction and was used to monitor its progress. The solvents and
excess propargyl amine were removed under reduced pressure. The
residue was redissolved in CH Cl (5 mL) and mixed with a solution
of diazomethane in diethyl ether was added and the mixture stirred
for 5 min. The reaction mixture was treated with a catalytic amount
of methanolic KOH at room temperature for 3–5 min, diluted with
Figure 10. DPBF absorbance decay (%, 50 μM in DMSO) at 418 nm after
photoirradiation (total light dose 2 Jcm ; irradiation time 15 min) in the
absence (control) and presence of 1 μM 1, 1+, 1+POM, 2, 2+, 2+POM, 3,
2
2
À 2
3
+, 3+POM and MB (with no tumour cells). Error bars represent the
standard deviation of three replicate experiments.
2
00 mL of CH Cl , and immediately washed with 2×400 mL water.
2 2
The organic layer was dried over anhydrous sodium sulfate, filtered,
and evaporated to afford the crude product, which was chromato-
graphed on a silica column eluting with 2% acetone/CH Cl to give
2
2
General
the desired compound NPPME (372 mg, 70%). UV-Vis (CH Cl ) λ
2
2
max
4
(
ɛ×10 ): 412 nm (6.03), 481 nm (1.28), 511 nm (1.63), 551 nm (3.26),
All reactions were monitored by thin-layer chromatography (TLC)
using Merck 60 silica gel F254 pre-coated (0.2 mm thickness) glass-
backed sheets. Silica gel 60 A (230–400 mesh, Merck) was used for
1
6
8
1
1
5
3
3
58 nm (1.67), 707 nm (4.93). H-NMR (500 MHz, CDCl ): δ 9.63, 9.37,
3
.56 (all s and 1H, 10-H, 5-H and 20-H), 7.91 (dd, J=18.1, 11.7 Hz,
1
2
1
H, 3 CH=CH ), 6.31 (d, J=18.0, 1.1 Hz, 1H, 3 -H), 6.18 (d, J=11.8,
column chromatography. H NMR spectra were obtained using a
2
2
.1 Hz, 1H, 3 -H), 5.41–5.36 (dd, J=9.3, 2.4 Hz, 1H, 17-H), 5.35–5.32,
Varian spectrometer (500 MHz) at the Biohealth Products Research
Centre (BPRC) of the Inje University. Chemical shifts (δ) are given in
parts per million (ppm) relative to tetramethylsilane (TMS, 0 ppm).
High resolution fast atom bombardment mass spectrometry
.31–5.23 (all m and 1H, N-CH -alkyne), 4.36 (q, J=6.0 Hz, 1H, 18-H),
2
1
.84 (s, 3H, 12-CH ), 3.66 (m, 2H, 8 -CH ), 3.57 (s, 3H, OCH ), 3.35 (s,
3
2
3
2
H, 2-CH ), 3.17 (s, 3H, 7-CH ), 2.81–2.70 (m, 1H, 17 -CH ), 2.47–2.39
m, 2H, 17 -H), 2.09–1.97 (s, 1H, 17 -CH ), 2.31 (t, J=2.4 Hz, 1H,
3
3
2
1
2
(
(HRFABMS) analyses were conducted using a Jeol JMS700 high
2
2
C�CH), 1.74 (d, J=6.0 Hz, 3H, 18-CH ), 1.62 (t, J=7.6 Hz, 3H, 8 -CH ),
resolution mass spectrometer at the Daegu centre of KBSI (Korea
Basic Science Institute), Kyungpook National University, Korea. UV-
Vis absorption spectra were recorded on a SCINCO S-3100 UV-Vis
spectrophotometer using a 1-cm quartz cuvette. IR spectra were
recorded on a Varian-640 FT-IR spectrometer. Thermogravimetric
analysis (TGA) was performed on a TA Instrument Q600, PH407
3
3
0.11, 0.07 (all br s and 1H, NH).
Preparation of 4-Azidopyridine
-Chloropyridine hydrochloride (50 mg) and sodium azide (1 g)
4
(Pusan KBSI).
were dissolved in H O (50 mL), and the solution was stirred at room
2
temperature for 24 h. The reaction mixture was extracted with
diethyl ether/H O. The organic layer was dried over anhydrous
sodium sulfate, filtered, and evaporated to obtain a yellow oil
2
Synthesis of Purpurin-18 Methyl Ester (P18ME)
(
350 mg, 70%).
Methyl pheophorbide-a (MPa) was prepared according to
a
[55]
[67]
literature procedure. P18ME was synthesized as follows. MPa
1 g) was dissolved in pyridine (5 mL) and diethyl ether (400 mL),
followed by the addition of KOH in 1-propanol (12 g dissolved in
0 mL). Subsequently, air was bubbled into the solution for 3 h. The
(
[
36,56]
Preparation of POM 4
8
To a solution of Na MoO ·2H O (29.03 g, 0.12 mol) in H O (120 mL)
was slowly added HNO (13 M, 37 mL), followed by a solution of
Na SiO (1.3 g, 0.0107 mol) in H O (50 mL). The resulting yellow
solution was heated at 80 C for 30 min, the above period of time
being required for the transformation of the β-form of [SiMo O ]
into the α-isomer. The reaction mixture was treated with a solution
of (C H ) N·Cl in H O (10 mL) at room temperature to allow the
formation of a homogeneous solution in the preparation of POM
complexes containing the each CPI, which is soluble in the organic
solvent system. The yellow precipitate was filtered off, washed with
water and ethanol, and dried in air at room temperature to furnish
the target product 4 (20.1 g, 68%). UV-Vis (DMSO) λmax (ɛ×10 ):
2
7
2
4
2
2
reaction mixture was extracted with water (500 mL); the aqueous
layer was collected and its pH was adjusted to 2 with cold H SO
3
2
4
2
3
2
solution (25%). The acidified phase was then extracted with CH Cl /
2
2
°
H O. Evaporation of the organic layer afforded a purple residue,
4À
2
12
40
which was crystallized from CH Cl /n-hexane, washed with water
2
2
until neutral, and air-dried overnight to furnish the crude purpurin-
8 carboxylic acid (500 mg, 50%, purple powder). This product was
4
9 4
2
1
then reacted with diazomethane to produce crude P18ME, which
was chromatographed on silica gel using 2% acetone/CH Cl as an
2
2
eluent to afford the title compound after crystallization from
CH Cl /n-hexane (410 mg, 80%, purple red crystals). Mp: 267°C. UV-
4
2
2
4
Vis (CH Cl ) λ
(ɛ×10 ): 412 nm (6.22), 488 nm (0.92), 509 nm
2
2
max
91 nm (6.37). FTIR (ATR (attenuated total reflectance)) 943, 897,
1
(
1.33), 547 nm (3.02), 644 nm (1.43), 700 nm (4.74). H NMR
À 1
91 cm .
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Synthesis of Purpurin-18-N-(N,N-dimethylbutyl)imide Methyl
Ester 1
Synthesis of Purpurin-18-N-(pyridine-4-ylmethyl)imide Methyl
[68]
Ester 2
The same method for the synthesis of NPPME except use of the
corresponding amine (N,N-dimethylbutane-1,4-diamine, 1.80 mmol,
The same method for the synthesis of NPPME except use of the
corresponding amine (4-aminomethyl pyridine, 1.80 mmol, >10 eq.
excess) with P18ME (100 mg, 0.173 mmol) was carried out. The
reaction mixture was chromatographed on a silica gel using 50%
EtOAc/n-Hx as an eluent to furnish the target product 2 (98 mg,
>
10 eq. excess) with P18ME (100 mg, 0.173 mmol) was carried out.
The reaction mixture was chromatographed on a silica gel using
0% MeOH/CH Cl as an eluent to furnish the target product 1
1
2
2
(
(
82 mg, 70%). UV-Vis (DMSO, nm) (log ɛ): 421 (4.67), 514 (4.29), 553
85%). UV-Vis (DMSO, nm) (log ɛ): 421 (4.72), 512 (4.19), 551 (4.31),
1
1
4.36), 651 (4.04), 707 (4.40). H-NMR (500 MHz, CDCl ): δ 9.51 (s, 1H,
665 (3.62), 707 (4.40). H-NMR (500 MHz, CDCl ): δ 9.54, 9.28, 8.49
3
3
3
1
1
6
2
1
2
0-H), 9.27 (s, 1H, 5-H), 8.50 (s, 1H, 20-H), 7.86–7.80 (dd, J=17.9,
(each s and 1H, 10-H, 5-H, 20-H), 8.53 (m, 2H, N ), 7.85 (dd, J=17.5,
1
2
1
2
1.5 Hz, 1H, 3 CH=CH ), 6.24–6.20 (dd, J=17.8, 1.1 Hz, 1H, 3 -H),
11.3 Hz, 1H, 3 -H), 7.52 (d, J=4.8 Hz, 2H, N ), 6.24 (dd, J=17.9,
2 2 1
2
2
.10–6.08 (dd, J=11.5, 1.1 Hz, 1H, 3 -H), 5.27–5.24 (dd, J=9.2,
.8 Hz, 1H, 17-H), 4.41 (t, J=7.5 Hz, 2H, N ), 4.30 (q, J=7.9 Hz, 1H,
8-H), 3.87–3.77 (m, 2H, N ), 3.72 (s, 3H, 12-CH ), 3.58 (q, J=7.6 Hz,
H, 8 -CH ), 3.28 (s, 3H, OCH ), 3.08 (s, 3H, 2-CH ), 2.93 (br, 3H, 7-
1.1 Hz, 1H, 3 -H), 6.11 (dd, J=11.6, 1.1 Hz, 1H, 3 -H), 5.62 (s, 2H, N ),
5.26 (d, J=9.1 Hz, 1H, 17-H), 4.27 (q, J=7.6 Hz, 1H, 18-H), 3.74 (s,
3H, 12-CH ), 3.58 (q, J=7.8 Hz, 2H, 8 -CH ), 3.47 (s, 3H, OCH ), 3.27
1
4
1
3
3
2
3
1
2
(s, 3H, 2-CH ), 3.09 (s, 3H, 7-CH ), 2.64–2.55 (m, 1H, 17 -CH ), 2.35–
3 3 2
1 2
2
3
3
5
2
CH ), 2.64–2.58 (m, 6H, N ), 2.38–2.32 (m, 1H, 17 -CH ), 2.28–2.22 (m,
2.25 (m, 2H, 17 -CH ), 2.06 (m, 1H, 17 -CH ), 1.68 (d, J=7.3 Hz, 3H,
2 2
1 2
3
2
2
1
3
1
7
7
H, 17 -CH ), 1.98 (m, 2H, 17 -CH ), 1.91 (m, 2H, N ), 1.69 (d, J=
.5 Hz, 3H, 18-CH ), 1.58 (t, J=7.7 Hz, 3H, 8 -CH ), 1.46–1.38 (m,
.5 Hz, 2H, N ), À 0.06, À 0.15 (all br s and 1H, NH). HRFABMS: Calcd.
18 -CH ), 1.60 (t, J=7.7 Hz, 3H, 8 -CH ), 0.07, À 0.06 (each br s, each
2
2
3
3
2
+
1H, NH). HRFABMS: Calcd. for C H N O [M+H] 669.3189; Found
40 41 6 4
3
3
2
669.3192.
+
for C H N O [M+H] 677.3815; Found 677.3818.
40
49
6
4
Preparation of Purpurin-18-N-(1-methylpyridinium
iodide-4-yl-methyl)-imide Methyl Ester 2+
[69]
Preparation of Purpurin-18-N-(N,N,N-trimethylammonium
iodide-butyl)imide Methyl Ester 1+
Purpurinimide 2 (27 mg, 0.041 mmol) and MeI (1.5 mL, >500 eq.
Purpurinimide 1 (28 mg, 0.041 mmol) and MeI (1.5 mL, >500 eq.
excess) were dissolved in CH Cl (5 mL), and the solution was stirred
2
2
excess) were dissolved in CH Cl (5 mL), and the solution was stirred
at room temperature under N for 24 h. The solvent and excess MeI
2
2
2
at room temperature under N for 24 h. The solvent and excess MeI
were removed under reduced pressure, affording the title com-
pound 1+ in quantitative yield as a purple red solid without
were removed under reduced pressure, affording the title com-
pound 2+ in quantitative yield as a purple red solid without
further purification. UV-Vis (DMSO, nm) (log ɛ): 419 (4.84), 481
2
1
further purification. UV-Vis (DMSO, nm) (log ɛ): 421 (4.84), 481
(4.32), 512 (4.32), 551 (4.51), 651 (4.28), 707 (4.59). H-NMR
1
3
(
4.29), 512 (4.33), 551 (4.61), 648 (4.33), 707 (4.78). H-NMR
(500 MHz, CDCl ): δ 9.16 (br d, J=6.2 Hz, 1H, N , pyridine), 9.12,
3
(
500 MHz, CDCl ): δ 9.37 (s, 1H, 10-H), 9.23 (s, 1H, 5-H), 8.48 (s, 1H,
9.10, 8.45 (each s and 1H, 10-H, 5-H, 20-H), 8.20 (br d, J=6.2 Hz, 2H,
3
1
2
1
2
1
9
3
0-H), 7.84–7.78 (dd, J=17.9, 11.4 Hz, 1H, 3 CH=CH ), 6.24 (d, J=
N , pyridine), 7.82–7.76 (dd, J=17.9, 11.5 Hz, 1H, 3 CH=CH ), 6.24 (d,
2
2 2
2
2
2
7.8 Hz, 1H, 3 -H), 6.11 (d, J=11.6 Hz, 1H, 3 -H), 5.22–5.20 (d, J=
.1 Hz, 1H, 17-H), 4.46 (t, J=7.5 Hz, 2H, N ), 4.32–4.24 (m, 1H, 18-H),
.84–3.79 (m, 1H, N ), 3.79–3.69 (m, 1H, N ), 3.61 (s, 3H, 12-CH ), 3.52
J=17.9 Hz, 1H, 3 -H), 6.12 (d, J=11.5 Hz, 1H, 3 -H), 5.81–5.70 (m,
1
1
4
2H, N ), 5.15 (d, J=8.7 Hz, 1H, 17-H), 4.61 (s, 3H, N , pyridine-CH ),
3
4
4
1
4.26 (q, J=7.4 Hz, 1H, 18-H), 3.85–3.79 (m, 1H, 8 -H), 3.78–3.72 (m,
3
1
5
1
(m, 2H, 8 -CH ), 3.46 (s, 3H, OCH ), 3.41 (s, 9H, N ), 3.28 (s, 3H, 2-
1H, 8 -H), 3.66 (s, 3H, 12-CH , overlapped with solvent), 3.48 (s, 3H,
2
3
3
2
2
CH ), 3.03 (s, 3H, 7-CH ), 2.66–2.59 (m, 1H, 17 -CH ), 2.35–2.25 (m,
OCH ), 3.27 (s, 3H, 2-CH ), 2.92 (s, 3H, 7-CH ), 2.60-2.53 (m, 1H, 17 -
3
3
2
3
3
3
1
2
3
1
2
2
7
0
H, 17 -CH ), 2.23 (m, 1H, 17 -CH ), 1.90–1.75 (m, 2H, N ), 1.69 (d, J=
.6 Hz, 3H, 18-CH ), 1.54 (t, J=7.1 Hz, 3H, 8 -CH ), 1.44 (m, 2H, N ),
CH ), 2.29–2.19 (m, 2H, 17 -CH ), 2.00–1.92 (m, 1H, 17 -CH ), 1.73 (d,
2 2 2
2
2
2
2
2
J=7.3 Hz, 3H, 18-CH ), 1.43 (t, J=7.3 Hz, 3H, 8 -CH ), 0.03, 0.01 (all
3 3
+
3
3
.12, À 0.12 (all s and 1H, NH). HRFABMS: Calcd. for C H N O [M-
br s and 1H, NH). HRFABMS: Calcd. for C H N O [M-I] 683.3340;
41 43 6 4
41
51
6
4
+
I] 691.3966; Found 691.3977.
Found 683.3342.
Preparation of Purpurin-18-N-(N,N,N-trimethyl ammonium)
butyl-imide Methyl Ester-POM Complex 1+POM
Preparation of Purpurin-18-N-(1-methylpyridinium
iodide-4-yl-methyl)-imide Methyl Ester-POM Complex 2
+
POM
A mixture of CPI 1+ (33 mg, 0.04 mmol) and Keggin-type POM 4
(
α-(Bu N) [SiMo O ]) (28 mg, 0.01 mmol) in acetonitrile (7 mL) was
A mixture of CPI 2+ (32 mg, 0.04 mmol) and Keggin-type POM 4
(α-(Bu N) [SiMo O ]) (28 mg, 0.01 mmol) in acetonitrile (7 mL) was
4
4
12 40
stirred at room temperature for 24 h. The resulting dark green
precipitate was filtered off, washed with cold acetonitrile for several
times, and dried to afford the target product 1+POM (34 mg, 73%)
4
4
12 40
stirred at room temperature for 24 h. The resulting dark green
precipitate was filtered off, washed with cold acetonitrile for several
times, and dried to afford 2+POM (32 mg, 70%) without further
purification. UV-Vis (DMSO, nm) (log ɛ): 421 (4.75), 481 (4.10), 514
without further purification. UV-Vis (DMSO, nm) (log ɛ): 421 (4.75),
1
5
13 (4.07), 553 (4.30), 649 (4.10), 707 (4.51). H-NMR (500 MHz,
1
DMSO-d ): δ 9.52 (br, 1H, 10-H), 9.37 (br, 1H, 5-H), 8.91 (s, 1H, 20-H),
(4.11), 551 (4.50), 649 (4.14), 707 (4.63). H-NMR (500 MHz, DMSO-
6
1
8
1
4
.17–8.11 (dd, J=18.3, 12.0 Hz, 1H, 3 CH=CH ), 6.42 (d, J=17.6 Hz,
H, 3 -H), 6.20 (d, J=11.5 Hz, 1H, 3 -H), 5.27 (d, J=8.5 Hz, 1H, 17-H),
.50 (q, J=7.2 Hz, 1H, 18-H), 4.45–4.36 (m, 2H, N ), 3.76–3.71 (m, 1H,
d ): δ 9.80, 9.55, 8.99 (each s and 1H, 10-H, 5-H, 20-H), 8.99 (br, 2H,
2
6
2
2
3
2
N , pyridine, overlapped with 20-H), 8.39 (br d, J=6.6 Hz, 2H, N ,
1 2
1
pyridine), 8.28–8.22 (m, 1H, 3 CH=CH ), 6.51 (d, J=17.9 Hz, 1H, 3 -
2
2 1
4
4
1
N ), 3.67 (s, 3H, 12-CH ), 3.65–3.61 (m, 1H, N ), 3.51 (m, 2H, 8 -CH ),
H), 6.29 (dd, J=11.6 Hz, 1H, 3 -H), 5.91–5.83 (m, 2H, N ), 5.21 (m, 1H,
3
2
4
3
9
2
7
.39 (3H, OCH & 3H, 2-CH , overlapped with DMSO solvent), 3.13 (s,
17-H), 4.53 (m, 1H, 18-H), 4.39 (s, 3H, N , pyridine-CH ), 3.82 (s, 3H,
12-CH ), 3.73 (m, 2H, 8 -H), 3.45 (3H, OCH , overlapped with DMSO
3
3
3
5
2
1
H, N ), 3.06 (s, 3H, 7-CH ), 2.62–2.54 (m, 1H, 17 -CH ), 2.35–2.28 (m,
H, 17 -CH ), 1.98 (m, 1H, 17 -CH ), 1.91 (m, 2H, N ), 1.73 (d, J=
.7 Hz, 3H, 18-CH ), 1.51 (t, J=7.2 Hz, 3H, 8 -CH ), 1.33–1.19 (m, 2H,
3
2
3
3
1
2
3
2
2
2
solvent), 3.31 (s, 3H, 2-CH ), 3.21 (s, 3H, 7-CH ), 2.55 (m, 1H, 17 -CH ,
3 3 2
1
2
3
3
overlapped with DMSO solvent), 2.35 (m, 2H, 17 -CH ), 2.18 (m, 1H,
2
2 2
2
N ), À 0.36, À 0.45 (all br s and 1H, NH). FTIR (ATR) 941, 899,
17 -CH ), 1.76 (d, J=7.6 Hz, 3H, 18-CH ), 1.65 (t, J=7.7 Hz, 3H, 8 -
2 3
À 1
7
92 cm .
CH ), 0.11, À 0.06 (all br s and 1H, NH). FTIR (ATR) 941, 899,
3
À 1
7
92 cm .
Eur. J. Inorg. Chem. 2021, 1–14
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1
1
2
Synthesis of
1H, 17 -CH
2
), 2.29 (m, 1H, 17 -CH
2
), 1.97–1.91 (m, 1H, 17 -CH
2
), 1.74
2
Purpurin-18-N-(1-(Pyridin-4-yl)-1,2,3-triazol-4-ylmethyl)-imide
Methyl Ester 3
(d, J=6.9 Hz, 3H, 18-CH
À 0.39 (all br s and 1H, NH). FTIR (ATR) 941, 899, 792 cm .
3
), 1.50 (br t, J=6.2 Hz, 3H, 8 -CH
), À 0.28,
3
À 1
A solution of NPPME (50 mg, 0.081 mmol, 1 eq.), 4-azidopyridine
(
0.10 mmol, 1.2 eq. per C�CH group), DIPEA (4 mol% per C�CH
Cell Culture and Photoirradiation
group), CuI (2 mol% per C�CH group), and AcOH (4 mol% per
C�CH group) in CH Cl (5 mL) was stirred at room temperature for
The tested cell lines were A549 human lung carcinoma and HeLa
human cervical cells obtained from the cell line bank of Seoul
National University cancer research center, and were grown in
medium RPMI-1640 (Sigma-Aldrich) with 10% fetal bovine serum,
glutamine, and 1% penicillin at 37°C in a humidified atmosphere of
2
2
0
.5–1 h. The reaction mixture was chromatographed on a silica
plate (preparative thin layer chromatography) using 2% MeOH/
CH Cl as an eluent to furnish the target product 3 (42 mg, 70%).
UV-Vis (DMSO, nm) (log ɛ): 421 (4.73), 481 (4.05), 512 (4.04), 551
(
2
2
1
5% CO in air. Phosphate buffer saline (PBS, Sigma-Aldrich), optical
4.26), 649 (3.94), 707 (4.43). H-NMR (500 MHz, CDCl ): δ 9.53, 9.29,
2
3
4
microscope (Olympus, CK40-32 PH), ELISA (enzyme linked immuno-
sorbent assay)-reader (BioTek, SynergyHT), trypsin-EDTA (ethyl-
enedia-minetetraacetic acid) solution (Sigma-Aldrich), and incuba-
8
.49 (each s and 1H, 10-H, 5-H, 20-H), 8.66 (br d, J=5.7 Hz, 2H, N ,
2
pyridine), 8.43 (s, 1H, N , 5-triazole), 7.86–7.80 (dd, J=17.7, 11.7 Hz,
1
3
1
1
1
1
7
7
1
7
H, 3 CH=CH ), 7.76 (d, J=6.1 Hz, 2H, N , pyridine), 6.24 (d, J=
2
2 2
tor (37°C, 5% CO ) were used. PDT was carried out using a diode
7.9 Hz, 1H, 3 -H), 6.11 (d, J=11.9 Hz, 1H, 3 -H), 5.96 (d, J=14.9 Hz,
2
1
1
laser apparatus (BioSpec LED, Russia) equipped with a halogen
lamp, a band-pass filter (640–710 nm), and a fiber optics bundle.
The duration of irradiation in PDT treatment was calculated taking
into account the empirically determined effective dose of light
H, N ), 5.73 (d, J=14.8 Hz, 1H, N ), 5.33–5.31 (dd, J=8.7, 2.4 Hz, 1H,
7-H), 4.28 (q, J=7.4 Hz, 1H, 18-H), 3.76 (s, 3H, 12-CH ), 3.58 (q, J=
3
1
.4 Hz, 2H, 8 -CH ), 3.50 (s, 3H, OCH ), 3.28 (s, 3H, 2-CH ), 3.09 (s, 3H,
2
3
3
2
1
-CH ), 2.71–2.66 (m, 1H, 17 -CH ), 2.41–2.26 (m, 2H, 17 -CH ), 1.91–
3
2
2
À 2
2
energy (total light dose of 2 Jcm , irradiation time 15 min).
.85 (m, 1H, 17 -CH ), 1.70 (d, J=7.2 Hz, 3H, 18-CH ), 1.59 (t, J=
2
3
2
.6 Hz, 3H, 8 -CH ), 0.02, À 0.10 (all br s and 1H, NH). HRFABMS:
3
+
Calcd. for C H N O [M+H] 736.3360; Found 736.3357.
4
2
42
9
4
MTT assay and cell viability
Cells from each cell line were placed in a 48-well, flat-bottomed
microplate at a volume of 200 μL (2×10 cells/well) for stationary
culture. After 24 h of incubation, the medium was removed, the
cultures were washed twice with physiological saline, and purpur-
inimides 1–3, CPIs 1+À 3+, and their CPI–POM complexes 1+
POM, 2+POM and 3+POM (constant concentration of each PI and
CPI) in 200 μL of the mixed medium were added to each well. After
Preparation of Purpurin-18-N-(1-(1-methylpyridinium
iodide-4-yl)-1,2,3-triazol-4-ylmethyl)imide Methyl Ester 3+
4
Clicked purpurinimide 3 (30 mg, 0.041 mmol) and MeI (1.5 mL,
>
500 eq. excess) were dissolved in CH Cl (5 mL), and the solution
2 2
was stirred at room temperature under N for 24 h. The solvent and
2
excess MeI were removed under reduced pressure, affording the
title compound 3+ in quantitative yield as a purple red solid
without further purification. UV-Vis (DMSO, nm) (log ɛ): 421 (4.68),
2
4 h of incubation, the solutions were discarded, and the cultures
were washed 3 times with physiological saline, followed by the
1
addition of medium (200 μL/well). The cultures were then irradiated
5
(
8
3
12 (4.02), 551 (4.17), 707 (4.34). H-NMR (500 MHz, CDCl ): δ 8.94
3
À 2
4
(2 Jcm ) from a distance of 20 cm for 15 min, followed by an MTT
br, 3H, 10-H, 5-H, 20-H, all overlapped), 8.74 (br, 2H, N , pyridine),
2
3
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay
to evaluate their response to PDT. In the assay, MTT solution (EZ-
CYTOX, DOGEN, 10 μL) was added to each cell culture well followed
by culturing in an incubator for 1 h. Detergent solution (TACS™,
Trevigen, 200 μL) was added to the culture, shaken for 10 min, and
the absorbance was measured with an ELISA-reader at 570 nm.
Measurements were performed after 12 h and 24 h of incubation
after photoirradiation. Each group consisted of 3 wells.
.48 (s, 1H, N , 5-triazole), 8.31 (br, 2H, N , pyridine), 7.77 (br m, 1H,
1
2
2
CH=CH ), 6.23 (d, J=17.1 Hz, 1H, 3 -H), 6.10 (d, J=10.7 Hz, 1H, 3 -
2
1
1
H), 5.83 (br, 1H, N ), 5.64 (br, 1H, N ), 5.28 (br, 1H, 17-H), 4.26 (br, 1H,
5
1
2
8-H), 4.11 (br s, 3H, N , pyridine-CH ), 3.56 (s, 3H, 12-CH ), 3.42 (m,
H, 8 -H), 3.28 (br s, 6H, OCH +2-CH , overlapped), 2.69 (br, 4H, 7-
CH +17 -CH ), 2.49–2.38 (m, 2H, 17 -CH ), 1.97 (m, 1H, 17 -CH ),
.82 (br, 3H, 18-CH ), 1.53 (3H, 8 -CH , overlapped with sol-
3 3
+
vent),À 0.55 (br and 2H, NH). HRFABMS: Calcd. for C H N O [M-I]
3
3
1
3
3
1
2
2
3
2
2
2
2
1
43
44
9
4
750.3511; Found 750.3513.
Cellular accumulation study
Preparation of Purpurin-18-N-(1-(1-methylpyridinium
iodide-4-yl)-1,2,3-triazol-4-ylmethyl)imide Methyl Ester-POM
Complex 3+POM
A549 cells or HeLa cells were seeded in confocal dishes at a density
of 2×10 cells/dish. After 24 h of incubation, the medium was
4
removed, and the dishes were washed twice with PBS. Subse-
quently, purpurinimides 1–3, CPIs 1+À 3+, and their CPI–POM
complexes 1+POM, 2+POM and 3+POM solution of 5 μM
concentration were added to each dish. After 24 h of incubation,
the cells were washed with PBS and fixed with 4% formalin (Sigma-
Aldrich) for 30 min. The fixed cells were washed again with PBS.
Samples were also stained with DAPI (200 nM) (BioLegend) or
Mitoorange (500 nM) to identify the site of the nucleus or
mitochondria in the cells. Cellular uptake images were recorded
using a confocal laser scanning microscope (CLSM, LSM 510 META,
Germany) at Inje University.
A mixture of clicked CPI 3+ (35 mg, 0.04 mmol) and Keggin-type
POM 4 (α-(Bu N) [SiMo O ]) (28 mg, 0.01 mmol) in acetonitrile
4
4
12 40
(
7 mL) was stirred at room temperature for 24 h. The resulting dark
green precipitate was filtered off, washed with cold acetonitrile for
several times, and dried to afford 3+POM (36 mg, 75%) without
further purification. UV-Vis (DMSO, nm) (log ɛ): 421 (4.78), 479
1
(
(
4.22), 511 (4.23), 552 (4.52), 651 (4.29), 707 (4.69). H-NMR
500 MHz, DMSO-d ): δ 9.48, 9.35, 9.31 (each br or s and 1H, 10-H, 5-
6
4
2
H, 20-H), 9.10 (br d, J=6.3 Hz, 2H, N , pyridine), 8.91 (s, 1H, N , 5-
triazole), 8.66 (br d, J=6.6 Hz, 2H, N , pyridine), 8.12 (br m, 1H,
3
1
2
2
3
CH=CH ), 6.40 (d, J=17.6 Hz, 1H, 3 -H), 6.20 (d, J=11.7 Hz, 1H, 3 -
2
1
H), 5.76 (br m, 2H, N ), 5.24 (br d, J=8.6 Hz, 1H, 17-H), 4.48 (br q, J=
5
Singlet oxygen photogeneration study
7
3
.0 Hz, 1H, 18-H), 4.32 (s, 3H, N , pyridine-CH ), 3.64 (s, 3H, 12-CH ),
3
3
1
1
.40 (br, 2H, 8 -H & 3H, OCH3 & 3H, 2-CH , overlapped with DMSO
3
DPBF was used as a selective singlet oxygen ( O ) acceptor, being
2
2
1
[63]
solvent), 3.04 (s, 3H, 7-CH ), 2.64-2.55 (m, 1H, 17 -CH ), 2.42–2.36 (m,
3
2
bleached upon reaction with O . Five sample solutions of DPBF
2
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in DMSO (50 μM) containing, respectively, DPBF only (50 μM,
control sample), DPBF+MB (1 μM), DPBF+purpurinimides 1–3
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Manuscript received: June 4, 2021
Revised manuscript received: July 6, 2021
Accepted manuscript online: July 11, 2021
Eur. J. Inorg. Chem. 2021, 1–14
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© 2021 Wiley-VCH GmbH
��
These are not the final page numbers!

FULL PAPERS
T. H. Lee, Dr. Y. Liu, H. J. Kim, S. H.
Lee, H. H. Song, Prof. Dr. Y. K. Shim,
Prof. Dr. W. K. Lee*, Prof. Dr. I. Yoon*
1
– 14
Mitochondrial Targeting Cationic
Purpurinimide–Polyoxometalate
Supramolecular Complexes for
Enhanced Photodynamic Therapy
with Reduced Dark Toxicity
CPI–POM supramolecular complexes,
resulting from the electrostatic inter-
actions between CPI and polyanionic
POM, those exhibited high cellular
uptake followed by mitochondrial
targeting of CPI–POM complexes into
tumour cells through endocytosis,
causing cell death after photogenera-
1
tion of O , resulting in enhanced pho-
2
todynamic activity with reduced dark
toxicity.