Phthalocyanines and Tetrapyrazinoporphyrazines with Two Cationic Donuts: High Photodynamic Activity as a Result of Rigid Spatial Arrangement of Peripheral Substituents

Article abstract of DOI:10.1021/acs.jmedchem.7b00272

High photodynamic activity was observed for hexadeca-cationic zinc, magnesium, and metal-free phthalocyanines (Pcs) and tetrapyrazinoporphyrazines with EC₅₀ values as low as 5 nM (MCF-7 cells) for the best compound; this activity was several times better than that of clinically established photosensitizers verteporfin, temoporfin, S₃AlOHPc, or protoporphyrin IX. This lead compound was characterized by low dark toxicity (TC₅₀ = 369 μM), high efficiency against other cell lines (HCT 116 and HeLa), and possible activation by light above 680 nm. The excellent photodynamic activity resulted from the rigid spatial arrangement of the quaternized triazole moieties above and below the Pc core, as confirmed by X-ray crystallography. The triazole moieties thus formed two cationic donuts that protected the hydrophobic core against aggregation in water. The lysosomes were found to be the site of subcellular localization and were consequently the primary targets of photodynamic injury, resulting in predominantly necrotic cell death.

Full text of DOI:10.1021/acs.jmedchem.7b00272

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Article
Phthalocyanines and tetrapyrazinoporphyrazines with two
cationic donuts: high photodynamic activity as a result
of rigid spatial arrangement of peripheral substituents
Basma Ghazal, Miloslav Machacek, Mona Abbas Shalaby,
Veronika Novakova, Petr Zimcik, and Saad Makhseed
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 30 May 2017
Downloaded from http://pubs.acs.org on May 30, 2017
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Phthalocyanines
and
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tetrapyrazinoporphyrazines with two cationic
donuts: high photodynamic activity as a result
of rigid spatial arrangement of peripheral
substituents
Basma Ghazal,† Miloslav Machacek,‡ Mona Abbas Shalaby,† Veronika Novakova,§
Petr Zimcik*,ǁ and Saad Makhseed*,†
†Department of Chemistry, Kuwait University, P.O. Box 5969, Safat, 13060, Kuwait
‡
Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Kralove,
Charles University in Prague, Heyrovskeho 1203, Hradec Kralove, 500 05, Czech
Republic
§Department of Biophysics and Physical Chemistry, Faculty of Pharmacy in Hradec
Kralove, Charles University in Prague, Heyrovskeho 1203, Hradec Kralove, 500 05,
Czech Republic
ǁDepartment of Pharmaceutical Chemistry and Pharmaceutical Analysis, Faculty of
Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203,
Hradec Kralove, 500 05, Czech Republic
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ABSTRACT
High photodynamic activity was observed for hexadecaꢀcationic zinc, magnesium and
metalꢀfree phthalocyanines (Pcs) and tetrapyrazinoporphyrazines with EC50 values as
low as 5 nM (MCFꢀ7 cells) for the best compound; this activity was several times
better than that of clinically established photosensitizers verteporfin, temoporfin,
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S AlOHPc or protoporphyrin IX. This lead compound was characterized by low dark
toxicity (TC50 = 369 ꢁM), high efficiency against other cell lines (HCT 116 and
HeLa) and possible activation by light above 680 nm. The excellent photodynamic
activity resulted from the rigid spatial arrangement of the quaternized triazole
moieties above and below the Pc core, as confirmed by Xꢀray crystallography. The
triazole moieties thus formed two “cationic donuts” that protected the hydrophobic
core against aggregation in water. The lysosomes were found to be the site of
subcellular localization and were consequently the primary targets of photodynamic
injury, resulting in predominantly necrotic cell death.
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INTRODUCTION
Photodynamic therapy (PDT) is a relatively modern cancer treatment used as
an alternative to chemotherapy, radiotherapy and surgical intervention. PDT combines
three potentially nontoxic components—light, oxygen, and a photosensitizer (PS)—
that are able to destroy target cells by the production of toxic species, the most
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important being singlet oxygen. PSs from various structural groups have been
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approved for use in clinical practice or are under investigation in clinical trials. The
key photophysical characteristics of an efficient PS are good production of singlet
oxygen and strong absorption of light in the optical window of tissues (or
phototherapeutic window, i.e., 650ꢀ850 nm), where the penetration of light is the
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deepest. From this point of view, phthalocyanines (Pcs), which typically possess very
5
ꢀ1
ꢀ1
good singlet oxygen production and extinction coefficients over 2 × 10 M cm are
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interesting molecular structures. Indeed, sulfonated hydroxyaluminum Pc with an
average degree of sulfonation n = 3 (S AlOHPc) has been approved in Russia since
3
5,6
2001. On the other hand, the Pc core is planar and strongly hydrophobic; as a result,
it tends to form strong aggregates in water and waterꢀbased media, which can
substantially reduce or completely eliminate the photophysical pathways important
for PDT. Several strategies on how to resolve this obstacle have been developed by
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,8
various research groups. The strategies include the use of surfactants/coꢀsolvents or
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delivery systems
to dissolve the lipophilic compounds or substitution by highly
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14ꢀ17
hydrophilic substituents axially on the central metal
or on the Pc core.
Recently, we identified Pcs with rigid cationic substituents bearing
quaternized imidazolyl moieties, which led to highly waterꢀsoluble Pcs that did not
18
display any aggregation in water.
Applying the same substituents to
tetrapyridoporphyrazines resulted in a compound that belongs among the most potent
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PSs reported to date. Considering the promising anticancer photodynamic activity of
these PSs bearing imidazolyl moieties, we applied the same protocol to prepare
triazoleꢀcontaining Pc complexes and anticipated obtaining new Pcs with enhanced
activity. Pcs and their aza analogs, tetrapyrazinoporphyrazines (TPyzPzs), with
different central metals bearing either quaternized or nonꢀquaternized triazoles were
included in this study to evaluate the effects of the macrocyclic core, central metal
and quaternization on photophysical properties and photodynamic activity. The
bulkiness of the peripheral 2,6ꢀbis((1Hꢀ1,2,4ꢀtriazolꢀ1ꢀyl)methyl)ꢀ4ꢀmethylphenoxy
substituents was expected to place the triazoles (quaternized or not) above and below
the plane of the core. Steric hindrance makes the substituents very rigid; thus, the
triazoles cannot bend out of their position and should efficiently protect the core from
undesirable aggregation.
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RESULTS AND DISCUSSION
Synthesis. The synthetic routes for the target Pc (6-10) and TPyzPz (11ꢀ16)
macrocycles bearing eight sterically hindered peripheral phenoxy substituents are
shown in Scheme 1. The reaction pathway was started by the synthesis of phenol 1 by
the condensation of triazole with 2,6ꢀbis(hydroxymethyl)ꢀ4ꢀmethylphenol following a
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previously reported procedure. The initial phthalonitrile 2 and pyrazineꢀ2,3ꢀ
dicarbonitrile 3 were prepared in good yields from an aromatic nucleophilic
substitution reaction between 4,5ꢀdichlorophthalonitrile or 5,6ꢀdichloropyrazineꢀ2,3ꢀ
dicarbonitrile, respectively, and triazoleꢀcontaining phenol 1. To further support the
structural identification of the targeted quaternized complexes, quaternized precursors
4
and 5 were prepared from the reaction of 2 or 3 with methyl iodide in DMF at 80
°C.
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Scheme 1. Synthetic routes of the studied compounds and their quaternized
a
derivatives
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a
Reagents and conditions: i) CsF, DMF, 80 °C, 24 h; ii) Mg(OBu)
2 2
, BuOH, I ,
reflux, 3 h; iii) TsOH, THF, rt, overnight; iv) anhydrous Zn(OAc)
h; v) DMF, CH I, 80 °C, 24 h; vi) acetone, pyridine, rt, 24 h.
2
, pyridine, reflux, 3
3
The initial attempts to prepare the proposed metalꢀfree Pc and TPyzPz macrocycles
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using lithium as an initiator in pentanol (i.e., the Linstead method) failed and gave
unidentified brown products instead. A metalꢀionꢀmediated cyclotetramerization
procedure was successfully used to prepare zinc TPyzPz 13 by the reaction of 3 with
anhydrous zinc acetate in dry quinoline under a nitrogen atmosphere, although it gave
a
very low yield (≤10%). In contrast, magnesium butoxideꢀinduced
cyclotetramerization gave Mg complexes 6 and 11 in high yields of 90% and 50%,
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respectively. Subsequently, demetallation of Mg complexes using pꢀtoluenesulfonic
acid (TsOH) gave metalꢀfree ligands 7 and 12, which, in turn, were complexed with a
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zinc cation using zinc acetate in pyridine to yield Zn complexes of Pc and TPyzPz 8
and 13. Both demetallation and zinc complexation proceeded in high yields, typically
over 90%. Subsequently, all these Pcs and TPyzPzs were quaternized with methyl
iodide to give completely waterꢀsoluble quaternized derivatives 9, 10, and 14-16.
Notably, the preparation of the quaternized Mg complex of Pc 6 failed using the
aforementioned conditions, and the reaction instead gave only the quaternized metalꢀ
free Pc 9. Other attempts using different procedures (in chloroform or DMF at room
temperature) also failed. All the synthesized complexes were fully characterized by
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elemental analysis, IR, H NMR and C NMR. Elemental and Xꢀray analyses showed
a variable number of water molecules coordinated to the triazole moieties in all of the
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synthesized complexes, similar to a previous report.
The intrinsic aggregation behavior of the Pcs and their analogs represent the
primary obstacle for researchers due to the efficiency limitation of using these
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materials in PDT applications. Therefore, UVꢀVis (see below) and H NMR
spectroscopic techniques were employed to evaluate the aggregation behavior of the
1
prepared complexes in either organic solvents or water. The H NMR spectra of all
the prepared complexes were recorded primarily in deuterated DMSO and gave wellꢀ
resolved spectra with sharp peaks in both the aromatic and aliphatic regions
(Supporting Information, Figures S3, S4, S9 and S10). No broadening or peak shifts
were observed over a broad concentration range, and the high quality of the spectra
remained unchanged with wellꢀresolved peaks, even at high concentrations (ranging
from 3–20 mM), indicating the absence of aggregation. In addition, the spectra of
both starting precursors 2 and 3 and their corresponding complexes were almost
identical and showed exactly the same excellent quality. The metalꢀfree ligands 7, 9,
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12 and 15 also showed inner core protons that exhibited no chemical shifts at variable
concentrations.
Since the prepared polycationic complexes (9, 10 and 14-16) were all well soluble
in water, their aggregation behavior in an aqueous medium was evaluated by
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recording their H NMR spectra in D
2
O. The excellent quality of the NMR spectra
obtained in D O was similar in terms of features (i.e., wellꢀresolved and sharp peaks)
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to those obtained in DMSO for the same complexes, as well as to those of the
corresponding quaternized precursors 4 and 5, apart from the chemical shifts resulting
from the solvent effect (Supporting Information, Figures S9 and S10). These findings
suggested that the aggregation behavior was totally absent in water and thus implied
that promising results could be achieved in terms of the photodynamic activity.
Notably, the chemical shift for the more acidic proton of the triazole moiety was
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2
missing due to D O exchange. The C APT spectrum of 10 confirmed that the
triazole proton had been exchanged with a D atom because the signal appeared as a
triplet in the even carbon phase with J = 33 Hz (Supporting Information, Figure S12).
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Figure 1. H NMR spectra of 10 at variable temperatures from 5 to 60 °C in D
2
O.
Interestingly, the two aliphatic protons belonging to methylene groups in complexes
9
and 10 were surprisingly not magnetically equivalent and displayed doubletꢀofꢀ
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doublet resonances with J = 15 Hz in D
2
O (not in DMSO, Supporting Information
Figure S14), as shown in Figure 1 and Supporting Information, Figure S13. The
coupling constant of 15 Hz indicated geminal coupling between the two protons (Hꢀ
CꢀH), consequently suggesting the existence of diastereotopic protons. This result
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could be explained by the presence of a nearby chiral center, presumably the sp
nitrogen of the triazole moiety, due to the greatly reduced dynamic of the nitrogen
lone pair as a result of the solvent effect and the high rigidity of the complexes
themselves. Therefore, variableꢀtemperature studies (5ꢀ60 °C) were performed and
showed that the coalescence of the methylene peaks (i.e., they became a single peak)
in complexes 9 and 10 occurred at 50 °C (Figure 1 and Supporting Information,
Figures S13 and S14). The conventional explanation could be related to the loss of N
chirality due to the increase in the kinetic energy of its lone pair at higher
1
temperatures. As revealed by the temperatureꢀdependent H NMR spectra (Figure 1
and Supporting Information, Figures S13 and S14), the excellent quality, including
the sharp and wellꢀresolved peaks, remained unchanged; the aromatic peaks shifted
only slightly into the highꢀfield region.
X-ray single-crystal structures. The unique structural features of the triazoleꢀ
substituted Pc and TPyzPz analogs were confirmed by singleꢀcrystal Xꢀray diffraction
analysis. This technique helped to verify the relative orientations of the various
triazole moieties or their quaternized counterparts with respect to the macrocycle ring
in the solid state, and it provided valuable information regarding their aggregation
behavior and intermolecular interactions. The crystal structures of both triazoleꢀ
substituted phenoxy phthalonitrile and the corresponding pyrazine precursors 2ꢀ5
were also generated (by solvent diffusion techniques) and compared to those of the
novel, structurally modified Pcs and TPyzPzs. The crystal structures (Supporting
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Information, Figures S33ꢀS36) of these starting compounds confirmed that the plane
of the phenoxy rings containing the triazole or methyl triazole moieties was projected
almost perpendicular to the aromatic plane containing the nitrile groups and that the
rotation of the phenoxy moieties was completely blocked by the presence of bulky
substituents.
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Figure 2. Crystal structures of 7 (7A: top view; 7B: side view) and 9 (9A: top view;
9
B side view). Color code: grayꢀcarbon, redꢀoxygen, blueꢀnitrogen, violetꢀiodide and
blackꢀhydrogen. In the top view, the hydrogen atoms are hidden for clarity.
Many attempts were made to generate goodꢀquality single crystals of the various
triazoleꢀsubstituted Pcs and their corresponding TPyzPz derivatives. Metalꢀfree Pc 7
and its quaternized analog 9 as well as the Mg and Zn complexes of TPyzPzs 11 and
1
3 were crystallized in good quality for diffraction analysis. The molecular structures
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of these four Pc systems obtained from Xꢀray diffraction analysis are depicted in
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Figures 2 and 3. Similar to most of their metalꢀfree analogs, the cores of both 7 and
9
were observed to be planar in their crystal structures. At the same time, the
magnesium and zinc ions in 11 and 13 were both positioned slightly above the plane
of the corresponding TPyzPz units, and one water molecule was observed to be
coordinated to the core metal. As a result, both 11 and 13 had domed shapes with
coordinated water at the apex position. The magnesium ion and its coordinated water
molecule in 11 exhibited positional disorder and appeared on both sides of the Pc
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plane. Both of these MgꢀH O fractions were found to possess half occupancies each;
hence, the overall MgꢀH
2
O occupancy was unity.
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Figure 3. Crystal structures of 11 (11A: top view; 11B: side view) and 13 (13A: top
view; 13B side view). Color code: grayꢀcarbon, redꢀoxygen, blueꢀnitrogen, pinkꢀ
magnesium, greenꢀzinc and blackꢀhydrogen. In the top view, the hydrogen atoms are
hidden for clarity. In the case of 11, the occupancy of MgꢀH O at the opposite side
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due to positional disorder is hidden.
Similar to the case of precursors 2 and 3, the peripheral phenoxy units of all these
Pcs and TPyzPzs were positioned orthogonally with respect to the plane of the Pc
ring. The triazole functional units, which were present at the ortho positions of the
phenoxy moieties in all of these crystals, were thus oriented just above or below the
Pc plane, as depicted in Figures 2 and 3 (all hydrogen atoms and solvent molecules
are hidden for clarity). However, their relative orientations with respect to the
macromolecular plane were found to be slightly different in each type of crystal,
presumably due to steric reasons. This phenomenon was more evident in the case of 9,
where most of the triazole moieties were projected outward from the macrocycle ring.
The positioning of triazole moieties either upward or downward with respect to the Pc
planes, as revealed from the crystal structures, clearly dismissed the possibility of any
faceꢀtoꢀface Pc aggregation among these moieties. The distances (coreꢀtoꢀcore)
between two Pc or TPyzPz molecules in all these crystal networks were observed to
be greater than 10 Å, which was too far away to cause any appreciable faceꢀtoꢀface πꢀ
stacking. Additionally, in the case of crystals of 11 and 13, the phenoxy units
orthogonal to the TPyzPz plane were capable of engaging in intermolecular πꢀπ
interactions with the adjacent phenoxy fragment of the neighboring TPyzPz units.
Moreover, some triazole substituents in the crystal networks that were positioned
above or below the ring could also engage in intramolecular πꢀπ interactions with the
TPyzPz macrocycle or in intermolecular triazoleꢀtriazole πꢀbonding between adjacent
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2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
TPyzPzs. Figure 4 illustrates all such πꢀπ interactions that were observed in the case
of the crystals of 11. In the case of 7, such triazoleꢀmediated πꢀbonding was very
limited, and these interactions were not observed at all for 9. Although goodꢀquality
single crystals could not be obtained from the other studied Pcs and TPyzPzs to verify
their structural features, we could presume that these complexes would also adopt the
same orthogonal orientation to effectively avoid cofacial selfꢀassociation.
Transmission electron microscopy was used to observe morphology of the
compounds (Supporting Information, Figure S41). The images revealed that the nonꢀ
quaternized Pcs 7 and 8 are cubic in shape. After quaternization, the morphology of
complexes 9 and 10 converted to the perfect spherical with very narrow size
distribution.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Packing pattern of molecules of 11 in their crystal network showing the
possibility of πꢀπ interactions among the adjacent molecules. A: possible πꢀπ
interactions of adjacent phenoxy moieties. B: πꢀπ interactions (both intramolecular
and intermolecular) among triazole substituents. Hydrogen atoms are hidden for
clarity.
Absorption spectra. The UVꢀVis spectroscopic technique was used to assess the
absorption spectra and further evaluate the aggregation behavior in either organic or
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aqueous media. As anticipated, a sharp, unperturbed single Qꢀband, typical of
monomeric Pcs and TPyzPzs with D4h symmetry, was the characteristic feature of all
the synthesized metalꢀcontaining complexes in DMF (Figure 5 and Supporting
Information, Figures S15ꢀS18). The absorption spectra of metalꢀfree ligands 7, 9, 12
and 15 were slightly complicated by the full or partial deprotonation of the central NH
and will be discussed below. Since the position of the Qꢀband represents an essential
factor for excitation in PDT, we should indicate that the Qꢀbands of the prepared
complexes were located above 678 nm for the Pcs and 630 nm for the TPyzPzs (Table
10
11
12
13
14
15
16
17
18
19
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47
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49
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53
54
55
56
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58
59
60
1
). The blue shift of the Qꢀbands of the TPyzPzs by approximately 50 nm compared
with the corresponding Pc derivatives was in good agreement with previously
24
observed trends for these types of dyes. This result makes the Pc derivatives more
suitable for application in PDT since light of longer wavelengths typically penetrates
3
more deeply into human tissues. Further assessment was performed to confirm the
absence of aggregation by recording the absorption spectra in DMF for each
derivative at different concentrations ranging from 1 to 9 µM. As expected, there were
no spectral changes as the concentration increased, and the monomeric characteristics
were the prominent features for all the inspected complexes, which obeyed the Beerꢀ
Lambert law in the studied concentration range (Supporting Information, Figures S15ꢀ
S17). Furthermore, the solutions were strongly fluorescent (Table 1).
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0
1
2
3
4
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6
7
8
9
0
1
2
3
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5
6
7
8
9
0
1
2
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Absorption spectra of the studied Pc derivatives in DMF (black) and in cell
culture medium (red) at a concentration of 1 ꢁM. Spectra were normalized to the same
absorption in the Bꢀband. Dashed lines represent spectra collected 1 h after sample
preparation. A) 6, B) 7, C) 8, D) 9, E) timeꢀlapse spectral changes of 9 in cell culture
medium over 3 h, F) 10. Samples of waterꢀinsoluble compounds 6ꢀ8 in cell culture
medium were prepared from a 300 ꢁM DMF stock solution.
The photophysical properties of PSs can be greatly affected by their aggregation
behavior in water or cell culture medium and consequently may determine their
activity in PDT applications. All the tested compounds (6ꢀ16) (the solutions of the
nonꢀquaternized derivatives were prepared from a DMF stock solution) displayed
absorption spectra in water or in cell culture medium that were typical of
predominantly monomeric species. While the Qꢀband of the quaternized derivatives
was sharp (sometimes even sharper than in DMF) and the solutions exerted strong
fluorescence, the spectra of the nonꢀquaternized derivatives were slightly broader, and
the Qꢀband was less intense (Figures 5 and S18) and was accompanied by a loss of
F
fluorescence, with Φ typically below 0.01 in water (Table 1). These data suggested
that the polycationic species remained fully nonꢀaggregated in waterꢀbased media,
while atypical types of aggregates with absorption spectra with a predominantly
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monomeric character were observed for the nonꢀquaternized derivatives in water,
similar to previous results for hydrophilic Pcs and TPyzPzs with extremely bulky
25
substituents. We believed that these aggregates could be formed based on the
intermolecular πꢀπ interactions between phenoxy substituents and/or triazole units that
were observed in the crystal structure of nonꢀquaternized 11 (Figure 4). No such
interactions were observed in quaternized 9. At the same time, the distance between
the Pc cores in 11 (more than 10 Å) was not sufficient for faceꢀtoꢀface aggregation
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
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50
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55
56
57
58
59
60
(which would change absorption spectra), as indicated by the crystal structure.
However, this distance was within the range of energy resonance transfer between
two Pc molecules, which could deactivate the excited state without changing the
absorption spectra. Although we are aware that the situation in water can be different
from that in the solid state in crystal, the presence of such intermolecular interactions
seems to be a plausible explanation.
The Qꢀband absorption maxima (i.e., shape and position) of the polycationic
compounds in water were independent of concentration and followed the Beerꢀ
Lambert law with a constant extinction coefficient in the studied concentration range
(1 to 9 µM, Supporting Information, Figures S16 and S17). Additionally, the UVꢀVis
spectra of Zn complexes 10 and 16 were assessed under different conditions. The
shape of the Qꢀband remained fully constant (Supporting Information, Figure S19),
with strong fluorescence of the solutions in water, in buffers with pH values of 2, 7,
and 11, and in cell culture medium at a concentration up to 120 ꢁM, suggesting that
there was no effect of dye concentration, pH, ion concentrations or serum proteins on
the monomeric character of these species. The absence of aggregation in aqueous
solution exhibited by the prepared polycationic complexes appeared to be intrinsically
related to the orthogonal conformation adopted by the eight peripheral bulky phenoxy
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5
5
6
substituents, as clearly demonstrated in the singleꢀcrystal Xꢀray structures (Figure 2).
The quaternized nitrogens formed two “cationic donuts” of positive charges that
efficiently protected the hydrophobic core from cofacial interactions by electrostatic
repulsive forces. This excellent monomeric character in water under different
conditions is extremely rare in Pcs and TPyzPzs and is a valuable prerequisite for high
photodynamic activity.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
As briefly mentioned above, metalꢀfree ligands 7, 9, 12 and 15 exhibited atypical
26
behavior in that they were deprotonated at the acidic central NH. The Qꢀbands of 7
and 12 remained typically split due to the reduced D2h symmetry (Supporting
Information, Figure S20) in nonꢀbasic organic solvents (THF and CHCl ). However,
3
the deprotonation of the central NH in DMF or pyridine restored (sometimes only
partially) the D4h symmetry of the macrocycle, and the Qꢀband was found to be single
2
7
and unsplit. TPyzPzs are typically more acidic, which was why the absorption
spectra of both 12 and 15 were predominantly deprotonated shortly after dissolution
in DMF, water or cell culture medium (Figure 6). In the case of Pcs 7 and 9, the
splitting was observed in DMF, water and cell culture medium shortly after
dissolution, although the spectrum gradually changed with time, reaching the fully
deprotonated form approximately 3 h after dissolution in one experiment (Figure 5 C,
D, F). Notably, the extent and rate of NH deprotonation changed from batch to batch,
which meant that it could not be properly characterized.
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a
Table 1. Photophysical data for the target complexes in DMF and water.
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
b
b
b
λ
max (log ε) λmax (log ε)
Φ
F
Φ
F
λ
F
λ
F
τ
F
/ ns
τ
F
/ ns
Φ
ꢂ
ꢂ F
Φ + Φ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
Cpd.
DMF
679 (5.31)
water
ꢀ
DMF water DMF water
0.537 0.011 685 689
DMF
5.57
water
ꢀ
DMF
0.224
DMF
0.760
6
6
7
69 (5.13),
00 (4.95)
7
8
9
ꢀ
ꢀ
0.276 0.005 705 708
0.269 0.0025 684 700
4.78
2.81
ꢀ
0.258
0.588
0.270
0.534
0.857
0.500
678 (5.41)
ꢀ
c
670 (5.08),
697 (5.03)
c
c
677 (5.33)
0.230 0.157 686 706
2.33 (33%), 4.91 (67%)
1.09 (26%), 3.70 (74%) (1.50)
10
11
12
680 (5.33) 681 (5.49) 0.146 0.187 687 690
1.68 (17%), 2.89 (83%)
1.07 (4%), 4.51 (96%)
1.30 (35%), 2.69 (65%)
1.18 (19%), 2.26 (81%)
1.20 (8%), 4.82 (92%)
0.93 (20%), 3.47 (80%)
0.96 (13%), 2.26 (87%)
1.22 (6%), 2.76 (94%) (1.19)
0.478
0.336
0.420
0.600
0.082
0.091
0.137
0.625
0.685
0.562
0.761
0.156
0.162
0.170
630 (5.12)
ꢀ
ꢀ
ꢀ
0.349 0.010 637 637
ꢀ
c
c
c
630 (5.11)
0.142 0.011 638 655
0.162 0.0055 636 636
ꢀ
13 632 (5.16)
ꢀ
14
15
16
633 (5.22) 638 (5.00) 0.074 0.216 664 651
1.18 (3%), 5.09 (97%)
0.56 (38%), 2.63 (62%)
0.80 (4%), 2.71 (96%)
c
c
c
c
632 (4.90) 633 (5.31) 0.071 0.028 638 660
634 (5.20) 643 (4.95) 0.033 0.124 657 647
a
F F
Qꢀband absorption maximum (λmax); extinction coefficient (ε); emission maximum (λ ); fluorescence lifetime (τ ); fluorescence quantum yield
); singlet oxygen quantum yield (Φ
Predominantly deprotonated species.
b
28
21
(
Φ
F
ꢂ
ꢂ F
). Unsubstituted ZnPc was used as the reference compound (Φ = 0.56 in DMF , Φ = 0.32 in THF ).
c
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0
1
2
3
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9
0
1
2
3
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5
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9
0
1
2
3
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 6. Normalized absorption (black, dashed), excitation (orange) and emission
(blue) spectra in DMF (AꢀC) and water (DꢀF) of the studied TPyzPzs: A) 11, B) 12,
C) 13, D) 14, E) 15, and F) 16.
Photophysical characterization. Singlet oxygen is believed to be the primary
cytotoxic species in PDT, and fluorescence emission is often used to localize PSs at
the subcellular level or in animal and human tissues. The quantum yield of singlet
ꢂ F F
oxygen (Φ ), quantum yield of fluorescence (Φ ) and fluorescence lifetimes (τ ) were
therefore determined in DMF and in water (for fluorescence) by the comparative
method using unsubstituted zinc phthalocyanine (ZnPc) as the reference compound.
The photophysical data are summarized in Table 1. The results in Table 1 were in
good agreement with previously published data in terms of the presence of a central
2
9
metal and its heavyꢀatom effect. The zinc complexes of these particular ligands were
always stronger producers of singlet oxygen, while the magnesium complexes
exhibited much stronger fluorescence. The properties of the metalꢀfree ligands could
not be properly compared to the others because of different levels of deprotonation
during measurement and because both forms (protonated and deprotonated) may
3
0
exhibit different photophysical behaviors. Nevertheless, it seemed that both singlet
oxygen production and fluorescence were reduced in these derivatives, in agreement
21
with previous observations of similar metalꢀfree Pcs and TPyzPzs. Interestingly,
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both quantum yields of the polycationic TPyzPzs (14ꢀ16) in DMF were substantially
lowered, as seen from the sum of these most important relaxation pathways of the
F
excited state (Table 1). However, their Φ values in water were substantially
increased. At the same time, the fluorescence decays were best characterized by two
lifetimes for compounds 14ꢀ16 but also for 9ꢀ15. This fact could indicate the
induction of some nonꢀradiative relaxation pathway (most likely photoinduced
electron transfer between deprotonized acidic CH in triazole and macrocyclic core)
and may be of interest in future research.
10
11
12
13
14
15
16
17
18
19
20
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22
23
24
25
26
27
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41
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44
45
46
47
48
49
50
51
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55
56
57
58
59
60
As briefly mentioned above, the polycationic compounds were monomeric in
aqueous solution. This observation was accompanied by comparable or even greater
Φ
F
values in water media than in DMF, further emphasizing their monomeric
character. On the other hand, the nonꢀquaternized derivatives, despite their seemingly
monomeric absorption spectra, were almost nonꢀfluorescent in water (Φ typically
F
≤0.01), indicating that they were losing their photodynamic potential. Their
fluorescence was slightly improved (typically by up to 3ꢀfold, data not shown) upon
dissolution in cell culture medium, which suggested interactions with serum
(lipo)proteins and separation of the molecules. This result was further confirmed by
the titration of selected compounds with fetal bovine serum (FBS, see below).
The emission spectra of all the studied Pcs and TPyzPzs were typically mirror
images of the Qꢀband and were characterized by a small Stokes shift. The excitation
spectra of the metal complexes were in perfect agreement with the absorption spectra,
even for the polycationic compounds in water media, further confirming the presence
of monomeric species in solution (Figure 6 and Supporting Information, Figure S21).
In the case of the metalꢀfree ligands, the excitation spectra sometimes did not
perfectly match the absorption spectra due to the different levels of deprotonation and
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1
1
1
1
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
the apparently different photophysical (i.e., fluorescence) properties of the protonated
and deprotonated species. Specifically, in the spectra of metalꢀfree Pc 9 in water, the
Qꢀband in the absorption spectrum indicated the presence of the predominantly
protonated form, while the excitation spectrum suggested the presence of the
prevalently deprotonated form, which seemed to be the more fluorescent species in
solution (Supporting Information, Figure S21).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Log
P. Lipophilicity, expressed as log P (P is the partition coefficient between
octanol and water), is an important parameter for drugs. The log P values for the
studied Pcs and TPyzPzs were determined experimentally (Table 2). Quaternized
compounds 9, 10, and 14ꢀ16 were detected primarily (or exclusively in the case of 10;
no signal was detected in octanol) in the water phase because of their polycationic
character. The nonꢀquaternized triazoles in 6 and 8 did not make the compounds very
hydrophilic, and they were found preferentially in the octanol phase. Log P could not
be determined for some of the nonꢀquaternized derivatives (7 and 11ꢀ13) since they
precipitated in the interphase, and the amounts detected in each phase were not
significant.
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a
Table 2. Comparison of the log P, dark toxicity (TC50 values), and photodynamic activity (EC50 values) of the studied PSs in different cell lines.
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
Cpd.
log P
EC50 (ꢁM)
HCT 116
TC50 (ꢁM)
HaCaT
TC50/EC50
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
HeLa
MCFꢀ7
HaCaT
HeLa
HeLa
n/a
HaCaT
b
b
b
b
b
b
b
b
6
7
8
9
1.34
>1
>1
>1
>1
>1
>1
>1
>1
>1
n/a
c
b
b
b
b
b
n/a
>1
>1
>1
>1
n/a
n/a
b
0.35
0.53 ± 0.099
0.44 ± 0.011
0.64 ± 0.038
0.15 ± 0.061
0.61 ± 0.027
0.081 ± 0.006
>1
>2
>2
ꢀ0.77
<ꢀ3.0
0.042 ± 0.016 0.051 ± 0.028
276 ± 21
369 ± 53
550 ± 22
874 ± 20
6571
30750
n/a
6790
134500
n/a
d
10
11
12
13
0.012 ± 0.004 0.0053 ± 0.0008 0.0087 ± 0.0002 0.0065 ± 0.0013
c
b
b
b
b
b
b
b
b
b
b
b
b
b
b
n/a
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
c
b
b
n/a
>1
>1
n/a
n/a
c
b
b
n/a
>1
>1
n/a
n/a
e
e
e
e
e
e
14
ꢀ2.00
1.18
22.4
3.16
2.71
433 ± 24
1081
125
±
367
400
1
5
6
ꢀ1.53
ꢀ1.53
1.56
1.15
0.06
>100
>100
>100
>100
363 ± 27
436 ± 44
18.6 ± 4.2
20.6 ± 2.5
>10
979 ± 44
637 ± 28
n/a
n/a
1
0.064 ± 0.013 0.038 ± 0.004
0.036 ± 0.010
0.039 ± 0.003
0.026 ± 0.0012
6813
510
460
>10
24500
f
f
verteporfin
temoporfin
0.045 ± 0.007
f
Pp IX
0.80 ± 0.13
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1
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3
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3
3
3
3
4
4
4
4
4
4
4
4
4
4
f
S
3
AlOHPc
ꢀ2.12
2.07 ± 0.29
127 ± 7.8
60
a
−2
−2
Data are presented as the TC or EC values ± standard deviations. Irradiation conditions: λ > 570 nm, 12.4 mW cm , 15 min, 11.2 J cm .
The compounds precipitated at concentrations above the listed values. Compound precipitated during determination. Compound detected only
in water phase. Ambiguous data (viability decreased only approx. up to 50% and then remained constant up to a concentration of 50 µM; 100%
decrease in viability was not obtained). Data from ref.
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In vitro evaluation of dark toxicity and photodynamic activity. Promising
photophysical data with high singlet oxygen production for the zinc complexes and
excellent water solubility with a fully monomeric character in water, even at high
concentrations, for polycationic macrocycles are valuable prerequisites for potentially
successful PSs. All Pcs and TPyzPzs were therefore evaluated (Table 2) for their
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photodynamic activity after irradiation (λ > 570 nm, 11.2 J cm , expressed as the
halfꢀmaximal effective concentration, EC50) in human cervical carcinoma (HeLa),
human breast carcinoma (MCFꢀ7), and colorectal carcinoma (HCT 116) cell lines as
well as human keratinocytes (HaCaT). Inherent toxicity without activation (expressed
as the halfꢀmaximal toxic concentration, TC ) was assessed in HeLa and HaCaT cells
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as representatives of cancerous and normal cell lines, respectively. Stock solutions of
the polycationic compounds (9, 10 and 14ꢀ16) were prepared in cell culture medium
without any additives, while the nonꢀquaternized derivatives (6ꢀ8 and 11ꢀ13) were
prepared in DMSO and diluted to the required concentration with cell culture
medium.
The inherent toxicity of polycationic Pcs 9 and 10 and TPyzPzs 14ꢀ16 without
activation was very low, with TC50 values typically greater than 300 ꢁM (Table 2,
Figure 7 and Supporting Information, Figure S22) in HeLa cells; TPyzPzs were
slightly less toxic than Pcs. This low toxicity seems to be a general property of
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similarly substituted Pcs and TPyzPzs.
The other apparent difference arose from
the obviously lower toxicity against normal (HaCaT) cells compared with that against
cancerous (HeLa) cells: the TC50 values were greater 500 ꢁM, at times reaching even
millimolar concentrations (Table 2 and Supporting Information, Figure S23). Routine
phaseꢀcontrast microscopy observation of the cells during treatment revealed that nonꢀ
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quaternized compounds 6ꢀ8 and 11ꢀ13 precipitated from the solution at micromolar
concentrations, which was why their TC50 values could not be determined.
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Figure 7. Photodynamic activity (λ > 570 nm, 12.4 mW cm , 15 min, 11.2 J cm
;
full lines and full symbols) and dark toxicity (dashed lines and open symbols) in HeLa
cells of 8 (orange, rhombus), 9 (blue, circle), 10 (green, square), 14 (magenta, reverse
triangle), 15 (black, asterisk) and 16 (red, triangle). At least five independent
experiments, each in quadruplicate, were typically performed.
After activation by light, some of the compounds induced a lethal photodynamic
effect on the cells (Figure 7 and Supporting Information, Figures S22 and S23). Very
high activity was observed for polycationic 9, 10 and 16 in all tested cell lines with
low EC50 values. The most susceptible cell line to PDT treatment seemed to be MCFꢀ
7, which generally had the lowest EC50 values for all the active compounds. No
significant difference was detected between the cancerous and normal cell lines,
indicating the rather nonspecific effect of singlet oxygen on the cells. Selectivity in
PDT in vivo is, however, achieved by the irradiation of the selected area only.
Considering the structureꢀactivity relationships, the Zn complex of cationic Pc 10
appeared to be extraordinary active, with better activity than structurally similar
hexadecaꢀcationic ZnPc with quaternized imidazolyl moieties (EC50 = 37 nM, HeLa,
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λ > 570 nm, 11.2 J cm ), followed by zinc TPyzPz 16 and metalꢀfree Pc 9, thus
indicating superior performance of the zinc complexes and Pc core over the TPyzPz
core and other central metals. Unusual behavior following the photodynamic
treatment was observed for polycationic magnesium TPyzPz 14. Viability decreased
in a concentrationꢀdependent manner (Figure 7 and Supporting Information, Figures
S22 and S23), reaching approximately 50% viability and then remaining at this level,
even at high concentrations (50 ꢁM). Currently, we do not have a clear explanation
for this atypical result, which was obtained in all tested cell lines. The cationic
charges on the triazoles seemed to be crucial for efficient photodynamic activity. With
exception of Zn complex of Pc 8 with rather modest activity (EC ~ 0.50 ꢁM), no
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photodynamic effect on HeLa cells was observed for the nonꢀquaternized derivatives
, 7, and 11ꢀ13 up to the maximum possible tested concentration. The most likely
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explanation for the low activity of these derivatives was the aforementioned formation
of aggregates in water and cell culture medium, which did not occur for the cationic
derivatives.
Evaluations of the photodynamic activity of novel PSs are always hampered by low
transferability of the published results. The different cell lines, light sources and
irradiation conditions used by different research groups make comparisons of their
results difficult. For this reason, we recently assessed four clinically approved PSs
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verteporfin, temoporfin, protoporphyrin IX (Pp IX) and S AlOHPc ) under the
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same conditions utilized in this study. As seen from the photodynamic results listed
in Table 2, the obtained polycationic compounds performed comparably (9 and 16) or
even much better (10) than the best clinically approved PSs tested under our
conditions. Additionally, the Pcs and TPyzPzs were at least an order of magnitude
less toxic without activation, and the “therapeutic ratio” (TC /EC ) for the most
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promising compound (10) was approximately two orders of magnitude better than the
ratios for verteporfin and temoporfin.
Photodynamic treatment is not only drugꢀdose dependent; the amount of activation
light may also play a significant role. Thus, the EC50 values for active compounds 8ꢀ
10 and 16 in HeLa cells were determined with longer and shorter irradiation times of
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0 min and 5 min, which resulted in total fluences of 22.4 J cm and 3.7 J cm ,
respectively. With the increased light dose, the EC50 values significantly improved to
96 nM, 25 nM, 6 nM and 20 nM for 8ꢀ10 and 16, respectively (Figure 8 and
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Supporting Information, Figure S22). As expected, a radical decrease in the light dose
resulted in a reduction in the photodynamic effectiveness, with increases in EC50
values to 702 nM, 269 nM, 32 nM and 71 nM for 8-10 and 16, respectively (Figure 8
and Supporting Information, Figure S22). This experiment clearly demonstrated that
the efficacy of PDT was also a function of the light dose. At the same time, it
confirmed the low comparability of published photodynamic results to each other,
unless the same irradiation source and conditions are used.
.
Figure 8. Dependence of EC values in HeLa cells on irradiation time (in min) for
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compounds 8 (A, orange), 9 (B, blue), 10 (C, green) and 16 (D, red). The irradiation
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Figure 9. Cellular uptake of compounds 8 (orange, rhombus), 10 (green, square) and
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6 (red, triangle) by HeLa cells after incubation with a concentration of 4 ꢁM (for 10
and 16) or 1 ꢁM (for 8) of the dye. The experiments were performed in duplicate.
Uptake. The amount of the dye inside the cells could influence the final
photodynamic effect. The uptake profiles in HeLa cells were therefore determined for
active compounds 8, 10 and 16. Metalꢀfree 9 could not be tested due to different
amounts of the deprotonated form during testing, which did not allow a determination
of the clear dependence of the fluorescence signal on incubation time. As seen from
Figure 9, the uptake of all compounds started relatively quickly within the first few
hours, reaching a relatively steady level at 12 h, which was used for incubation before
photodynamic treatment. The amount of the studied PSs in cells after 12 h was almost
the same: 0.26 nmol/mg of protein and 0.28 nmol/mg of protein for cationic
derivatives 10 and 16, respectively. Nonꢀquaternized 8 was taken up by cells to a
slightly greater extent (0.34 nmol/mg of protein). Its uptake by cells was most likely
even higher since the concentration used for the incubation of HeLa cells with this
compound was only 1 ꢁM, while a concentration of 4 ꢁM (used routinely in our
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laboratory for these experiments
) was applied for 10 and 16. The lower
concentration of 8 was used to avoid the possible precipitation of this compound in
cell culture medium, which could lead to the misinterpretation of the obtained results.
Subcellular localization. The short lifetime of singlet oxygen is the main reason
why the assessment of the subcellular localization of novel compounds is crucial—the
position within the cell indicates the primary target of the photodamage induced by
PSs. For subcellular localization, specific fluorescent probes for mitochondria
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(
MitoTracker) and lysosomes (LysoTracker) were used, along with the intrinsic
fluorescence of the Pcs and TPyzPzs. All of the studied quaternized PSs (9, 10 and
14-16) were detected in the acidic endolysosomal compartment as punctate
fluorescence colocalized with the lysosomal probe (Figure 10 and Supporting
Information, Figures S25, S27 and S28). For the nonꢀquaternized derivatives (6-8 and
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1-13), some aggregates were visibly attached to the outside of the cellular membrane
(clearly observed as clumps by routine phaseꢀcontrast microscopy and by differential
interference contrast (DIC) microscopy; Supporting Information, Figures S24 E, K, Q
and S26 E, K, Q). No colocalization of these clumps with intracellular organelles was
detected by either MitoTracker or LysoTracker. Only the zinc nonꢀquaternized
derivatives (8 and 13) were detected as punctate fluorescence colocalized with the
lysosomal probe (Supporting Information, Figure S24 M and 26 M) in addition to
their presence in the clumps.
The localization was further confirmed for the most active Pc 10 by staining with
another specific probe (ERꢀTracker) together with various organelleꢀspecific protein
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transfections (Lamp1ꢀRFP for lysosomes, Rab7aꢀGFP for late endosomes and
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GALNT2ꢀRFP for the Golgi apparatus ) (Figure 10). The punctuate fluorescence of
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(Lamp1 and Rab7a; Figure 10 E, F). The endoplasmic reticulum typically appeared as
a fine tubular network throughout the whole cell that was very dense in the
perinuclear area and well resolved in the peripheral parts of the cytoplasm (Figure 10
C, green). The Golgi apparatus, on the other hand, was localized to the perinuclear
area only and was characterized by its typical cisternaeꢀlike morphology (Figure 10 C,
blue). No colocalization with the endoplasmic reticulum or the Golgi apparatus was
observed (Figure 10 C, D).
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Figure 10. Subcellular localization of compound 10 (red) in HeLa cells. A –
MitoTracker (green) and LysoTracker (blue). C – ERꢀTracker (green) and GALNT2ꢀ
RFP (blue). E – Proteins specific for the endolysosomal compartment, Rab7aꢀGFP
(green) and Lamp1ꢀRFP (blue). B, D, F – corresponding fluorescence intensity
profiles.
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