Photodynamic inactivation of Enterococcus faecalis by conjugates of zinc(II) phthalocyanines with thymol and carvacrol loaded into lipid vesicles

Article abstract of DOI:10.1016/j.ica.2019.02.031

Terpene-macrocycle conjugates, consisting of thymol or carvacrol and phthalocyanine were synthesized, characterized, and subjected to detailed optical and biological studies. The macrocyclization reactions of terpene-substituted o-phthalonitrile derivativ

Full text of DOI:10.1016/j.ica.2019.02.031

InorganicaChimicaActa489(2019)180–190
Contents lists available at ScienceDirect
Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Research paper
Photodynamic inactivation of Enterococcus faecalis by conjugates of zinc(II)
phthalocyanines with thymol and carvacrol loaded into lipid vesicles
⁎
Lukasz Sobottaa, , Sebastian Lijewskib, Jolanta Dlugaszewskac, Joanna Nowickaa,
Jadwiga Mielcareka, Tomasz Goslinskib
a Department of Inorganic and Analytical Chemistry, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Poznan, Poland
b Department of Chemical Technology of Drugs, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Poznan, Poland
c Department of Genetics and Pharmaceutical Microbiology, Poznan University of Medical Sciences, Swiecickiego 4, 60-781 Poznan, Poland
A R T I C L E I N F O
A B S T R A C T
Keywords:
Terpene-macrocycle conjugates, consisting of thymol or carvacrol and phthalocyanine were synthesized, char-
acterized, and subjected to detailed optical and biological studies. The macrocyclization reactions of terpene-
substituted o-phthalonitrile derivatives were performed in a microwave reactor to give thymol- and carvacrol-
phthalocyanine conjugates, which were carefully purified by column chromatography and subsequently ana-
lyzed using HPLC and characterized using UV–Vis, NMR and MS. Both isolated phthalocyanine derivatives re-
present isomers of C2v symmetry, which was confirmed by NMR study. The absorption UV–Vis spectra of studied
thymol- and carvacrol-phthalocyanine conjugates with the Soret and the Q band possesses the same profile. Both
molecules reveal emission in the red region of the spectrum with fluorescence quantum yields about two-fold
lower in DMF and significantly lower in DMSO than that observed for zinc(II) phthalocyanine. The analyzed
phthalocyanine derivatives generate singlet oxygen upon excitation with light at ca. three-fold lower level then
that noticed for zinc(II) phthalocyanine. In comparison to zinc(II) phthalocyanine, both molecules also reveal
two-fold lower photostability in DMF and similar stability in DMSO. Thymol- and carvacrol-phthalocyanine
conjugates, zinc(II) phthalocyanine, thymol, and carvacrol were loaded into modified liposomes and subjected to
biological activity study. Thymol-phthalocyanine conjugate at both 100 and 10 µM revealed high, ca. 5 log
photoinactivation potential on the growth of Enterococcus faecalis, similarly to reference zinc(II) phthalocyanine.
For carvacrol-phthalocyanine conjugate, an increase of photoinactivation activity was observed at only 100 µM
in comparison to zinc(II) phthalocyanine, whereas a decrease at the concentration of 10 µM was noticed. It is
crucial that both studied phthalocyanines cross the border of 3 log photoinactivation potential, which indicates
their bactericidal potential. In the Microtox® bioassay both conjugates revealed significantly decreased dark
toxicity in comparison to thymol and carvacrol only. Interestingly, the carvacrol-phthalocyanine conjugate was
found to be more toxic in comparison to thymol-phthalocyanine conjugate and zinc(II) phthalocyanine.
Phthalocyanines
Singlet oxygen
PACT
Terpenes
Enterococcus faecalis
1. Introduction
as a new strategy to destroy antibiotic-resistant microorganisms as the
Golden Age of antibiotics seems to be coming dusk [6,7]. Many
Phthalocyanines (Pcs) are tetrapyrrolic macrocycles possessing a
vast spectrum of applications [1,2]. Pcs exhibit the ability to generate
reactive oxygen species, including extremely reactive singlet oxygen,
upon irradiation with visible light. They have been considered as pro-
mising photosensitizers in photodynamic therapy (PDT) and photo-
dynamic antimicrobial chemotherapy (PACT) [3–5]. In PDT, phthalo-
cyanines underwent clinical trials for the treatment of tumors and age-
related macular degeneration, for example, aluminum(III) sulfonated
phthalocyanine applied as Photosens®. PACT has also been considered
phthalocyanines have revealed photodynamic antimicrobial activity in
a plethora of preclinical trials. Pcs have been considered as very effi-
cient and stable photosensitizers for PACT. The limitations which
should be overcome to broaden their more extensive usage in therapy
are their hydrophobicity and tendency to form aggregates in solution
[8]. Many studies revealed that the connection of phthalocyanines with
novel drug delivery systems or nanostructures, as well as proper per-
ipheral functionalization or conjugation with other molecules could
improve the effectiveness of PACT. Some phthalocyanines, including
⁎ Corresponding author.
E-mail address: lsobotta@ump.edu.pl (L. Sobotta).
https://doi.org/10.1016/j.ica.2019.02.031
Received 8 January 2019; Received in revised form 21 February 2019; Accepted 22 February 2019
Availableonline23February2019
0020-1693/©2019PublishedbyElsevierB.V.

L. Sobotta, et al.
InorganicaChimicaActa489(2019)180–190
amphiphilic sulphonated zinc(II), cationic methylpyridinium aluminum
(III), cationic indium(III) derivatives revealed differentiated anti-
bacterial activity against Staphylococcus aureus, Escherichia coli and
Candida albicans [9,10]. Especially, PACT with cationic phthalocya-
nines has been found effective against various bacteria planktonic and
biofilm cultures, including E. coli [11] and S. aureus [12]. Silicon(IV)
phthalocyanines have revealed photoinactivation potential against E.
coli cultures [13] and C. albicans [14,15]. Also, various zinc(II) phtha-
locyanines conjugated with an oligolysine chains showed interesting
PACT potential against different strains of S. aureus, E. coli and P. aer-
uginosa [16,17]. PACT potential can also be significantly strengthened
by proper pharmaceutical formulation, using emulsions and liposomes.
For example, photodynamic inactivation of Cryptococcus neoformans
melanized cells up to 6-log in survival was achieved with chlor-
oaluminum phthalocyanine emulsion in the in vitro study [18]. In our
latest studies, we have also demonstrated in vitro photodynamic activity
potential of zinc(II) phthalocyanine substituted with 2-propoxy sub-
stituents and pyrrolyl-substituted zinc(II) porphyrazine and chlorins
incorporated into modified liposome vesicles against Enterococcus fae-
calis [19–23].
concentrations well above its solubility limit using the emulsion-eva-
poration technique were developed [37]. Also, the antimicrobial ac-
tivity of nanodispersed thymol in tryptic soy broth was analyzed in
comparison to free thymol. The study demonstrated that transparent
nanodispersions of thymol have promising antimicrobial activity
against a broad spectrum of foodborne pathogens [38]. In a similar
study, nanodispersed eugenol has revealed improved antimicrobial
activity against E. coli O157:H7 and Listeria monocytogenes in bovine
milk [39]. Mutual prodrugs of ibuprofen with menthol, thymol, and
eugenol have been selected with the aim of getting a synergistic effect.
These prodrugs have showed increased anti-inflammatory activity that
has been attributed to the synergistic effect as ibuprofen conjugates to
natural analgesics [40]. Moreover, some sulfamethoxazole conjugates
with monocyclic terpenes, like thymol and eugenol have revealed po-
tent antibacterial activity and have been more active than individual
terpenes, against multidrug-resistant (MDR) gruesome pathogenic
bacteria [41]. In a similar study, the thymol-sulfadiazine conjugate has
been found to be effective antibacterial against gruesome MDR bacteria
[42]. There are also known some photosensitizer-antibiotic conjugates
with antibacterial activity, including Rose bengal-penicillanic acid,
Rose bengal-linker- penicillanic acid and Rose bengal-linker-kanamycin
against Staphylococcus aureus and Escherichia coli. The highest eradica-
tion of S. aureus and E. coli was observed for Rose bengal-linker-kana-
mycin [43]. Hence, conjugation of phthalocyanine macrocycle with
terpene moiety could result with a compound of improved physical-
chemical properties and a high potency in photodynamic therapy,
especially against microorganisms (PACT) we decided to obtain and
characterize novel terpene-phthalocyanine analogs in this study.
The genus Enterococcus comprises a ubiquitous group of Gram-po-
sitive bacteria that inhabit the gastrointestinal tract, oral cavity, and
vagina of mammal’s [24]. Moreover, these microorganisms are
common in the environment, as well as in dairy and meat products
[25,26]. The majority Enterococcal infections are caused by En-
terococcus faecalis (80–90%) followed Enterococcus faecium (10%). En-
terococci cause a range of infections in the community setting (including
pelvic, neonatal and urinary tract infections), infective endocarditis,
hospital-acquired infections, as well as dental infections. In dentistry,
Enterococcus species, in particular, Enterococcus faecalis, are associated
with chronic periodontitis and failed root canal treatments [27,28]. It is
believed that enterococci can survive harsh conditions such as a wide pH
range and nutrient-limited conditions and persist within the root canal
system after treatment. Moreover, enterococci demonstrate intrinsic
resistance to common antibiotics, including all generations of cepha-
losporins, clindamycin, trimethoprim-sulfamethoxazole, and ami-
noglycosides at low concentrations. Also, they can acquire resistance to
glycopeptides, quinupristin-dalfopristin, daptomycin, linezolid, as well
as high-level aminoglycosides and ampicillin resistance [29]. As tradi-
tional antibiotic therapy is not always effective, novel approaches, in-
cluding antimicrobial photodynamic therapy are worthy of further in-
vestigations.
2. Experimental
2.1. General procedures
All reactions were conducted in oven-dried glassware under an
argon atmosphere using the Radleys Heat-On heating system.
Microwave-assisted synthesis of macrocycles was conducted in an
Anton Paar Monowave 400 reactor. All solvents were rotary evaporated
at or below 50 °C under reduced pressure. Flash column chromato-
graphy was carried out on Merck silica gel, particle size 40–63 µm.
Reversed-phase chromatography was carried out on Fluka C18 silica gel
90 (particle size 40–63). Thin layer chromatography (TLC) was per-
formed on silica gel Merck Kieselgel 60 F254 plates and visualized with
UV illumination (λmax 254 or 365 nm). UV–Vis spectra were recorded
In our current study, we decided to obtain terpene-phthalocyanine
conjugates. The rationale for such approach is related to known phar-
macological and physicochemical properties of terpenes. Terpenes
constitute a large family of organic compounds produced mainly by
plants. In nature, they serve as plant protection from microorganisms,
parasites, and herbivores. Terpenes are also biologically crucial since
e.g. carotenoids or vitamin A belong to terpenes. Herbs containing
terpenes have been known and used in traditional medicine since an-
cient times. e.g., chamomile is used in many soothing creams, oint-
ments, and cosmetics. Among many properties like anti-inflammatory
or antimicrobial activity, terpenes (e.g. azulene) have been proved to
possess photosensitizing potential [30,31]. Thymol is a naturally oc-
curring phenol monoterpene derivative constituting one of the major
constituents of essential oils of medicinal plant thyme (Thymus vulgaris)
[32]. Carvacrol is a phenolic monoterpenoid found in essential oils of
oregano (Origanum vulgare), thyme (Thymus vulgaris), wild bergamot
(Citrus aurantium bergamia) and other plants [33]. Thymol and carva-
crol have well known antimicrobial and antifungal activity. Also, they
have been considered as alternative antimicrobial agents against anti-
biotic-resistant pathogenic bacteria [34], and as a possible food pre-
servative or components of polymer films for antimicrobial food
packaging [35,36]. The interest is related in developing technology to
produce nanoscale systems for delivering lipophilic components in
aqueous foods, e.g. in this way nanodispersions containing thymol at
on
a Hitachi UV/VIS U-1900 and Shimadzu UV-160A spectro-
photometers. 1H NMR, 13C NMR spectra were prepared using a Bruker
Avance II spectrometer operating at 400.13 MHz for 1H and
100.61 MHz for 13C and Bruker Avance III spectrometer operating at
500.25 MHz for 1H and 125.79 MHz for 13C at the Institute of
Bioorganic Chemistry Polish Academy of Sciences in Poznan. Chemical
shifts (δ) are quoted in parts per million (ppm) and referred to a re-
sidual solvent peak. Coupling constants (J) are quoted in Hertz (Hz).
The abbreviations s, d, t, m refer to a singlet, doublet, triplet and
multiplet, respectively. Mass spectra (ESI) were recorded at Bruker
Impact HD ESI-Q-TOF mass spectrometer at the Wielkopolska Centre
for Advanced Technologies in Poznan.
2.2. Synthetic procedures
All reagents were purchased from commercial suppliers and used
without additional purification. 1-Azulenecarbaldehyde was synthe-
sized adopting literature method [44].
2.2.1. (2Z)-2-Amino-3-{[azulen-1-ylmethylidene]amino}but-2-enedinitrile
(2)
1-Azulenecarbaldehyde (849 mg, 5.4 mmol) and diaminomaleoni-
trile 1 (486 mg, 4.5 mmol) were dissolved in anhydrous methanol
181

L. Sobotta, et al.
InorganicaChimicaActa489(2019)180–190
(20 mL). Careful addition of trifluoroacetic acid (0.4 mL) resulted with
precipitation of brown solid. Next, the obtained solid was filtered,
washed with methanol and chromatographed (CH2Cl2:methanol, 50:1,
v/v) to give 2 as a dark brown solid (0.969 g, 90% yield); m.p. 205 °C. Rf
(CH2Cl2:methanol, 50:1, v/v) 0.33. UV–Vis (CH2Cl2) λmax nm (log
ε) = 289 (3.51), 337 (3.64), 439 (4.02), 613 (2.85). 1H NMR
(400.15 MHz, DMSO-d6) δ: 9.30 (d, 3J = 10.0 Hz, 1H), 8.77 (s, 1H),
8.54 (d, 3J = 9.5 Hz, 1H), 8.50 (d, 3J = 4.0 Hz, 1H), 7.91 (t,
3J = 10.0 Hz, 1H), 7.62–7.43 (m, 5H). 13C NMR (100.63 MHz, DMSO-
d6) δ: 150.3, 145.3, 140.1, 139.3, 138.4, 138.4, 136.8, 127.7, 127.3,
124.6, 123.9, 119.9, 115.2, 114.1, 105.4. MS (ES pos): m/z 247 [M
UV–Vis (CH2Cl2) λmax nm (log ε) = 228 (3.69), 319 (4.15). 1H NMR
(400.13 MHz, DMSO-d6) δ: 7.80 (s, 1H), 7.79 (s, 1H), 7.32 (d,
3J = 8.0 Hz, 1H), 7.15 (d, 3J = 8.0 Hz, 1H), 7.06 (s, 1H), 7.02 (m, 1H),
2.88 (m, 1H), 2.10 (s, 3H), 1.18 (d, 3J = 7.0 Hz, 6H). 13C NMR
(100.61 MHz, DMSO-d6) δ: 160.1, 151.3, 149.0, 136.1, 131.9, 127.5,
126.7, 124.4, 120.1, 118.7, 115.9, 115.6, 113.3, 103.8, 32.9, 23.7,
15.0. MS (ESI): m/z 277 [M+H]+, 299 [M+Na]+. HRMS (ESI): m/z
calc. for (C18H16N2O): 277.1341 [M+H]+; found: 277.1332 [M+H]+
.
2.2.5. {1,8,18,25-Tetrakis[5-methyl-2-(propan-2-yl)phenoxy]
phthalocyanine}zinc(II) (7)
+H]+, 269 [M+Na]+, 285 [M+K]+. MS (ES neg): m/z 245 [M+H]−
.
n-Pentanol (1 mL), zinc acetate (0.18 g, 1 mmol) and 5 (0.55 g,
2 mmol) were placed in a G10 vial. The vial was filled with argon and
sealed. Reaction was conducted in a microwave reactor at 200 °C for
15 min. Next, reaction mixture was filtered, the solid was washed with
toluene and the combined filtrates were evaporated. The dark green
residue was chromatographed using silica gel (CH2Cl2:methanol, 50:1,
v/v) and C-18 reversed-phase gel (methanol, then CH2Cl2) to give 7 as a
dark green solid (0.11 g, 19% yield); m.p. 235 °C. Rf (CH2Cl2:methanol,
50:1, v/v) 0.23. UV–Vis (CH2Cl2) λmax nm (log ε) = 227 (4.41), 327
(4.39), 633 (4.29), 703 (5.04), 747 (4.24). 1H NMR (500.25 MHz,
pyridine-d5) δ: 9.62 (d, 3J = 7.5 Hz), 9.55 (m), 9.35 (m), 8.2 (t,
3J = 7.5 Hz), 8.09 (m), 7.98 (t, 3J = 7.5 Hz), 7.66 (m), 7.42 (s), 7.36
(s), 7.35 (s), 7.25 (s), 4.07 (m, 1H), 3.69 (s, 1H), 2.24 (m, 6H), 1.64 (m,
6H), 1.14 (m, 6H). 13C NMR (125.79 MHz, pyridine-d5) δ: 158.2, 158.1,
156.1, 156.0, 156.0, 155.3, 155.2, 155.2, 155.1, 155.0, 154.9, 154.9,
154.8, 154.8, 154.7, 154.5, 154.3, 142.7, 142.6, 142.6, 142.5, 142.4,
142.3, 139.1, 138.0, 138.0, 137.9, 137.9, 137.8, 131.8, 131.6, 131.4,
128.3, 128.2, 128.1, 127.9, 127.5, 127.4, 127.2, 127.2, 126.9, 126.8,
126.3, 126.2, 121.7, 121.6, 121.6, 118.7, 118.6, 118.5, 118.4, 117.5,
117.3, 117.2, 117.1, 28.7, 27.6, 24.0, 23.8, 21.4, 21,3. MS (ESI): m/z
1169 [M+H]+. HRMS (ESI): m/z calc. for (C72H64N8O4Zn): 1169.4420
2.2.2. (2Z)-2-[(Azulen-1-ylmethyl)(methyl)amino]-3-(dimethylamino)
but-2-enedinitrile (3)
Compound 2 (738 mg, 3 mmol) was suspended in methanol (50 mL)
and sodium borohydride (456 mg, 12 mmol) was added in small por-
tions until substrate dissolved completely. Mixture was poured on water
with ice mixture (1:1). Resulting precipitate was washed with water,
dried and chromatographed (CH2Cl2:methanol, 50:1, v/v) to give dark
brown solid (677 mg, 91% yield); Rf (CH2Cl2:methanol, 50:1, v/v) 0.38;
UV–Vis (CH2Cl2) λmax nm 289, 439. Resulting amine was highly un-
stable and was immediately applied in the next step. Sodium hydride
(60% dispersion in mineral oil, 87 mg, 3.62 mmol) was suspended in
anhydrous dimethylformamide (7.5 mL) at −18 °C. After 30 min the
amine substrate (409 mg, 1.65 mmol) dissolved in dimethylformamide
(2.5 mL) was added dropwise during 30 min. Next, dimethyl sulfate
(349 µL, 3.62 mmol) dissolved in dimethylformamide (0.5 mL) was
added dropwise. After 2 h mixture was poured on water with ice, and
the resulting precipitate was washed with water, dried and chromato-
graphed (CH2Cl2:methanol, 50:1, v/v) to give 3 as a dark brown solid
(174 mg, 36% yield); m.p. 205 °C. Rf (n-hexane:ethyl acetate, 7:2, v/v)
0.3. UV–Vis (CH2Cl2) λmax nm 232, 288, 452, MS (ES) m/z 275
[M−CH3]+, 313 [M+Na]+. Unfortunately, compound 3 was found to
be very unstable during physicochemical characterization and sub-
sequent macrocyclization reaction (magnesium n-butanolate in n-bu-
tanol).
[M+H]+
;
found: 1169.4404 [M+H]+
. HPLC (see Supporting
Information).
2.2.6. {1,8,18,25-Tetrakis[2-methyl-5-(propan-2-yl)phenoxy]
phthalocyanine}zinc(II) (8)
n-Pentanol (1 mL), zinc acetate (0.18 g, 1 mmol) and 6 (0.55 g,
2 mmol) were placed in a G10 vial. The vial was filled with argon and
sealed. Reaction was conducted in a microwave reactor at 200 °C for
15 min. Next, reaction mixture was filtered, the solid was washed with
toluene and filtrates were evaporated. Dark green residue was chro-
matographed using silica gel (CH2Cl2:methanol, 50:1, v/v) and C-18
reversed-phase gel (methanol, then CH2Cl2) to give 8 as a dark green
solid (0.08 g, 13% yield); m.p. 227 °C. Rf (CH2Cl2:methanol, 50:1, v/v)
0.34. UV–Vis (CH2Cl2) λmax nm (log ε) = 228 (4.60), 327 (4.51), 632
(4.40), 702 (4.99), 745 (4.37). 1H NMR (500.25 MHz, pyridine-d5) δ:
9.63 (m), 9.58 – 9.50 (m), 9.37 (t, 3J = 7.5 Hz), 9.27 (t, 3J = 7.0 Hz),
8.20 (m), 8.10 (m), 8.03 (m), 7.92 (t, 3J = 7.6 Hz), 7.73 (d,
3J = 7.8 Hz), 7.68 – 7.60 (m), 7.56 (s), 7.50 (m), 7.43 – 7.37 (m), 7.33
(s), 7.19 (m), 7.17 – 7.09 (m), 2.91 – 2.82 (m), 2.82 – 2.74 (m), 2.35 –
2.24 (m), 1.27 – 1.18 (m), 1.09 (m). 13C NMR (125.79 MHz, pyridine-
d5) δ: 158.1, 156.1, 156.0, 156.0, 155.3, 155.2, 155.2, 155.1, 155.0,
154.9, 154.9, 154.8, 154.8, 154.7, 154.5, 154.3, 142.7, 142.6, 142.6,
142.5, 142.4, 142.3, 139.1, 138.0, 138.0, 137.9, 137.9, 137.8, 131.8,
131.6, 131.4, 128.3, 128.2, 128.1, 127.9, 127.5, 127.4, 127.2, 127.2,
126.9, 126.8, 126.3, 126.2, 121.7, 121.6, 121.6, 118.7, 118.6, 118.5,
118.4, 117.5, 117.3, 117.2, 117.1, 28.7, 27.6, 24.0, 23.8, 21.4, 21.3.
2.2.3. 3-[5-Methyl-2-(propan-2-yl)phenoxy]benzene-1,2-dicarbonitrile (5)
(modification of the literature approach [45])
Anhydrous K2CO3 (4.1 g, 30 mmol), was added to a well stirred
slurry of thymol (1.35 g, 9 mmol) and 3-nitrophtalonitrile 4 (0.43 g,
3 mmol) in DMF (8 mL) and heated at 70 °C for 24 h. After cooling to
room temperature the reaction mixture was poured into water and ice
mixture (1:1, 300 mL). The resulting precipitate was filtrated, washed
with
distilled
water
(3 × 100 mL)
and
chromatographed
(CH2Cl2:methanol, 50:1, v/v) to give 5 as light yellow solid (0.69 g, 83%
yield); m.p. 95 °C. Rf (CH2Cl2) 0.56. UV–Vis (CH2Cl2) λmax nm (log ε):
228 (3.79), 319 (4.29). 1H NMR (400.13 MHz, DMSO-d6) δ: 7.80 (s,
1H), 7.79 (d, 3J = 1.5 Hz, 1H), 7.34 (d, 3J = 8.0 Hz, 1H), 7.14 (m, 1H),
7.11 (m, 1H), 6.93 (s, 1H), 2.98 (m, 1H), 2.27 (s, 3H), 1.14 (d,
3J = 7.0 Hz, 6H). 13C NMR (100.61 MHz, DMSO-d6) δ: 160.3, 150.5,
137.5, 136.5, 136.1, 127.7, 127.5, 127.3, 120.9, 120.7, 115.9, 115.6,
113.4, 104.2, 26.7, 22.8, 20.3. MS (ESI): m/z 277 [M+H]+, 299 [M
+Na]+. HRMS (ESI): m/z calcd. for (C18H16N2O): 277.1341 [M+H]+
;
found: 277.1328 [M+H]+
.
2.2.4. 3-[2-Methyl-5-(propan-2-yl)phenoxy]benzene-1,2-dicarbonitrile (6)
Anhydrous K2CO3 (4.1 g, 30 mmol), was added to a well stirred
slurry of carvacrol (1.35 g, 9 mmol) and 3-nitrophtalonitrile 4 (0.43 g,
3 mmol) in DMF (8 mL) and heated at 70 °C for 24 h. After cooling to
room temperature the reaction contents were poured into water and ice
mixture (1:1, 300 mL). The resulting precipitate was filtrated, washed
MS (ESI): m/z 1169 [M+H]+
. HRMS (ESI): m/z calc. for
(C72H64N8O4Zn): 1169.4420 [M+H]+; found: 1169.4426 [M+H]+
HPLC (see Supporting Information).
.
2.3. Spectral properties
with
distilled
water
(3 × 100 mL)
and
chromatographed
(CH2Cl2:methanol, 50:1, v/v) to give 6 as a light yellow solid (0.56 g,
68% yield); m.p. 63 °C. Rf (n-hexane:ethyl acetate, 7:2, v/v) 0.48.
Absorption spectra were recorded with Hitachi UV/VIS U-1900 and
Shimadzu UV-160A spectrophotometers in DMF and DMSO at ambient
182

L. Sobotta, et al.
InorganicaChimicaActa489(2019)180–190
temperature. Emission spectra were recorded with a Jasco 6200 spec-
trofluorometer in DMF and DMSO at ambient temperature.
Fluorescence quantum yields were determined according to the
methods described earlier. As a reference it was used unsubstituted zinc
(II) phthalocyanine (ZnPc) with known fluorescence quantum yields
equal 0.20 and 0.17 for DMF and DMSO respectively [46–49].
tryptic soy agar (TSA) plates. After an incubation period (20 h at
36
1 °C), the number of colony was counted and the number of vi-
able bacteria (colony forming units; CFUs) in 1 mL was calculated. A
control study in darkness without studied compounds was performed
simultaneously. The results were expressed as log10[CFU/ml]. The re-
duction of living bacteria in each sample was determined by the cal-
culation log reduction factors (RF) as follows:
2.4. Singlet oxygen formation
RF = Log L(−)P(−) − Log L(−)P(+), where L(−)P(−) – no irra-
diation, no Pc+ (negative control); L(−)P(+) – no irradiation, Pc+
(dark control).
Singlet oxygen generation quantum yields were evaluated in DMF
and DMSO solutions at ambient temperature. Measurements were
conducted according to the comparative method using a chemical
quencher of singlet oxygen (DPBF) and a reference compound (ZnPc).
The mixture of DPBF and a photosensitizer was irradiated with light
adjusted to the maximum absorbance in the Q band of Pc spectrum.
Spectrum changes were monitored with OceanOptics Flame
Spectrometer and light source DT-MINI-2-GS. The quantum yields of
singlet oxygen formation were calculated according to equation pre-
sented earlier [46,48–51].
2.9. Light-dependent activity
Light activity evaluation was performed similarly to the dark one,
with irradiation of the culture after an incubation time of 20 min.
Bacteria were irradiated at room temperature with high power LED
MultiChip Emitters (60 high-efficiency AlGaAs diode chips, Roithner
LaserTechnik GmbH, Vienna, Austria) with maximum wavelength
690
15 nm at a fixed total light dose 30 J/cm2. The reduction of
living bacteria in each sample was determined by the calculation log
reduction factors as follows:
2.5. Photostability determination
RF = Log L(−)P(−) − Log L(+)P(+), where L(−)P(−) – no irra-
diation, no Pc+ (negative control); L(+)P(+) – irradiation, Pc+.
Photostability determination was performed in DMF and DMSO in
aerobic condition at ambient temperature according to the method
previously described. A xenon lamp (150 W, Optel) was used as a light
source. Visible light was separated with cutting filter. Spectrum changes
were monitored with OceanOptics Flame Spectrometer and light source
DT-MINI-2-GS. The quantum yields of photodecomposition process
were calculated according to the earlier presented equation
[46,50,52,53].
2.10. Microtox® analysis
Experiments were performed according to 81.9% Microtox® test
protocol with Microtox Model 500 analyzer and MicrotoxOmni soft-
ware (Modern Water, Inc.) [55,56].
3. Results and discussion
2.6. Modified lipid vesicles preparation
3.1. Synthesis and characteristics
Lipid vesicles were obtained by the method described by Dragicevic-
Curic and co-workers [54]. POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-
phosphocholine) and DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-
trimethylammonium chloride, 25 mg/ml) purchased from Avanti Polar
Lipids Inc. (Alabaster, AL, USA) were dissolved in chloroform (Aldrich)
and mixed in a molar ratio of 8:2. In the next step 7, 8, thymol and
carvacrol chloroform solutions at the concentration of 1 mg/ml were
added in a proper amount to achieve the final concentration in the
vesicle 100 and 10 µM. Simultaneously, there were prepared “empty”
liposomes. Then, the obtained mixture was evaporated under reduced
pressure to give thin lipid film. Next, ethanol (3.3% w/v) was added,
and vortexing for 5 min was performed. Further, the saline buffer was
added, and vortexing for 5 min continued. The unification was per-
formed by the extrusion of the mixture through the polycarbonate filter
(200 nm). The size of obtained vesicles was measured with NanoSight
LM10 (Malvern).
An attempt towards novel terpene-macrocycle conjugate was per-
formed. In the first approach, the azulene-diaminomaleonitrile con-
jugate was successfully synthesized using 1-azulenecarbaldehyde and
diaminomaleonitrile (Scheme 1). The resulting Schiff base compound 2
was carefully characterized and subsequently reduced and methylated
to the novel derivative 3. Unfortunately, conjugate 3 turned out to be
very unstable during characterization (access to air). Moreover, even if
compound 3 was quickly purified and immediately subjected to mac-
rocyclization reaction following the conditions: magnesium n-butano-
late in n-butanol, no progress of the reaction was observed. It seems that
the proposed azulene-diaminomaleonitrile conjugate is not stable en-
ough as the chemical entity thus it cannot be considered as an inter-
mediate in the macrocyclization reaction conditions. As the continua-
tion of the study, another approach towards terpene-macrocycle
conjugate was proposed. Namely, 3-nitrobenzene-1,2-dicarbonitrile
was combined with thymol or carvacrol using nitro-group displacement
reaction conditions (K2CO3 as a base in DMF at 50 °C for 24 h) (Scheme
1) [57]. The resulting conjugates 5 and 6 were subjected to macro-
cyclization reactions in a microwave reactor to give phthalocyanines 7
and 8, which were carefully purified by column chromatography and
their purity was subsequently analyzed using HPLC (see Supporting
Information). It is worth to note that both macrocyclization reactions
were conducted without addition of DBU as a base. Experiments with
DBU either failed to result in the macrocyclic product, or the reaction
yields were very low. It seems that the basicity of substrates, like zinc
(II) acetate was enough for the occurrence of cyclotetramerization re-
action.
2.7. In vitro photodynamic antibacterial activity
Enterococcus faecalis strain was purchased from American Type
Culture Collection (ATCC; E. faecalis ATCC 29212) and cultured aero-
bically in BHI broth at 36
1 °C for 18–20 h. After this time micro-
organisms were harvested by centrifugation (3000G for 15 min) and re-
suspended in 10 mM phosphate buffered saline (PBS, pH = 7.0) and
diluted in PBS to a final concentration of ca. 107 colony forming units
(CFU)/mL.
2.8. Dark activity
Thymol-phthalonitrile conjugate 5 has been previously synthesized
by Ma et al. [45], and also subjected to the macrocyclization reaction.
Since 5 is unsymmetrical, it can condensate in two ways. That is why
Ma et al. [45] obtained an inseparable mixture of four constitutional
isomers (randomers) D2h:Cs:C2v:C4h, which were formed in different
Aliquots of a microbial suspension were placed in the microtitration
plate and then solutions containing compounds incorporated in lipo-
somal formulations were added and incubated for 20 min. Next bac-
terial suspension from each well, after dilution, was inoculated on
183

L. Sobotta, et al.
InorganicaChimicaActa489(2019)180–190
N
N
N
N
N
N
N
NH2
NH2
N
(ii)
(i)
(iii)
N
NH2
1
2
3 (unstable)
R
O
R1 =
O
O
N
R
N
N
R
R
N
N
N
N
(iv)
(v)
N
Zn
N
N
N
N
N
5 R = R1
6 R = R2
4
R2 =
R
O
7 R = R1
8 R = R2
Scheme 1. Synthesis of terpene-phthalocyanine conjugates 7 and 8. Reagents and conditions: (i) azulenecarbaldehyde, TFA, rt; (ii) NaBH4, MeOH, rt; (iii) Me2SO4,
DMF, −18 °C to rt; (iv) thymol or carvacrol, K2CO3, DMF, 50 °C, 24 h; (v) Zn(CH3COO)2, n-pentanol, MW, 200 °C, 15 min.
proportions 1:4:2:1 (Scheme 1S, Supporting Information). It is chal-
lenging to isolate specific isomer after macrocyclization reaction during
typical flash column chromatography. HPLC was proved to be the best
and most successful method of separation, which has been presented by
Hanack and his co-workers [58]. They successfully separated for the
first time the C4h and the D2h isomers, which was possible due to the
developed HPLC phases based on π-π interactions. Surprisingly, in our
work, for both substrates 5 and 6, only one macrocyclic product was
found in the reaction mixture. It is worth noting that, no other mac-
rocyclic compounds were found during thin layer chromatography and
HPLC analysis. Moreover, both isolated phthalocyanines 7 and 8 were
found to be isomers of C2v symmetry, which was confirmed by NMR
studies. Similar situations when only one product was isolated after the
macrocyclization reaction with unsymmetrical phthalonitrile have not
been often reported in the literature. It has happened only for phtha-
lonitriles with bulky substituents in the 3rd or 4th position. One of the
most interesting examples was published by Lv et al. [59], who suc-
cessfully isolated “pinwheel-like” menthol-substituted phthalocyanine
of C4h symmetry. They have performed macrocyclization reaction
procedure with (D)- and (L)-3-(2-isopropyl-5-methylcyclohexoxyl)-1,2-
dicyanobenzene in refluxing n-pentanol in the presence of lithium n-
pentanolate followed by treatment with acetic acid. Noteworthy, in our
earlier work, a metronidazole-phthalocyanine conjugate of C2v sym-
metry has been the only isomer isolated after applying the Linstead
macrocyclization procedure starting from 1-[2-(2,3-dicyanophenoxy)
ethyl]-2-methyl-5-nitro-1H-imidazole. That time only a tiny amount of
another macrocyclic product was found (presumably one of the other
isomers Cs, D2h, C4h) in the crude reaction mixture analyzed by TLC, not
allowing for separation [57].
belong to methyl groups directly substituted to the aromatic ring of
thymol moiety. The 1H NMR spectrum of pc 8 represents a similar
pattern. Nevertheless, protons of 2-methyl groups (F, Fig. 1) at ca. 2.28
and 2.87 ppm are separated. Also, one of these signals at 2.87 ppm
overlaps with isopropyl proton signal marked as D. To sum up, in the 1H
NMR spectra of pc 7 and 8 a split of certain signals is strongly marked.
Especially diagnostic are separated signals belonging to methyl groups
(C and F, Fig. 1). In the case of pc 7 one slightly split signal can be
observed in the 1H NMR spectrum, whereas in the case of pc 8 two
separated signals appear. We stipulate that this may be the result of
steric hindrance between two terpenoid groups oriented to the same
side of symmetry axis, which limits free rotation of terpenoid moieties
around phenolic OeC bonds. Also, the isopropyl groups in ortho posi-
tions of pc 7, as well as the methyl groups in ortho positions of pc 8, can
also hinder the rotation around phenolic OeC bonds in a very different
manner due to the steric hindrance (phthalocyanine ring).
3.2. Spectroscopic and photochemical properties
Absorption spectra of studied compounds 7 and 8 are typical for
phthalocyanines (Fig. 2). In the UV–Vis spectra of both molecules there
can be recognized the Soret bands in the range of 200–400 nm and the
Q bands in the range of 600–800 nm. Interestingly, phthalocyanine
substituted with thymol (7) possesses a similar absorption profile to
phthalocyanine substituted with carvacrol (8) presumably to the same
electron donating/withdrawing properties of these groups. The terpene
electron donating groups of phthalocyanines 7 and 8 (λQmax = 709 nm)
cause red-shift of the Q band in comparison to ZnPc (λQmax = 670 nm
in DMF). This effect has been recently discussed by Topal and co-
workers [60]. Phthalocyanine derivatives 7 and 8 reveal emission in the
red region of the UV–Vis spectrum (Fig. 1). Their excitation spectrum is
comparable to the absorption one. Thus it can be concluded that in the
excited state the molecules are not deformed. Moreover, this observa-
tion gives the evidence for the purity of the studied samples. The cal-
culated fluorescence quantum yields of 7 and 8 are 2-fold lower than
those measured for ZnPc (Table 1). A similar data were presented by
Aliosman and co-workers for compound I in DMF [61]. Interestingly,
compounds 7 and 8 dissolved in DMSO reveal a dramatic drop in the
light emission (ΦFL = 0.02 for 7 and ΦFL = 0.03 for 8) in comparison to
In the 1H NMR spectrum of phthalocyanine 7, two characteristic
multiplets at 4.07 and 3.69 ppm were assigned to two pairs of CH iso-
propyl groups protons (marked as A in Fig. 1). This fact suggests that
the macrocyclic molecule possesses one axis of symmetry. Considering
all possible isomers (see Scheme 1S, Supporting Information), only C2v
isomer reveals this characteristic feature in the 1H NMR spectrum. In
the 1H–1H COSY spectrum both CH isopropyl peaks at 4.07 and
3.69 ppm correlate with another two aliphatic CH3 isopropyl group
protons (marked as B) at 1.64 and 1.14 ppm. Unlike, two signals at 2.24
and 2.27 ppm do not correlate with other signals, indicating that they
184

L. Sobotta, et al.
InorganicaChimicaActa489(2019)180–190
Fig. 1. 1H NMR and 1H–1H COSY spectra expansions of 7 and 8 in pyridine-d5 in the region 1.10–4.10 ppm.
Table 1
Quantum yields of fluorescence, photodecomposition and singlet oxygen for-
mation for 7, 8 and reference compound ZnPc.
Compound
Solvent
ΦFL
106 ΦP
ΦΔ
7
DMF
0.11
17.40
3.06
0.15
DMSO
DMF
0.02
0.18
8
0.08
23.90
4.01
0.15
DMSO
DMF
0.03
0.13
ZnPc
0.20 [40]
0.17 [40]
10.20
3.50
0.56 [41]
0.67 [41]
DMSO
steric hindrance appearing between the macrocyclic ring and bulky
rotating terpene substituents. This steric hindrance can also block ac-
cess of molecular oxygen to the phthalocyanine core and as a con-
sequence the singlet oxygen formation. It is known that the collision of
molecular oxygen with the porphyrinoid core is essential for the for-
mation of singlet oxygen [62]. It was proved that the periphery of the
phthalocyanine can hamper access of molecular oxygen to the macro-
cyclic core efficiently [63]. Interestingly, Aliosman and co-workers
have presented compound I (Fig. 3) with singlet oxygen formation
quantum yield ΦΔ = 0.31 in DMF [61]. The phthalocyanine derivatives
7, 8 and phthalocyanine I possess phenyloxy substituents. The periph-
eral substituents of I are bulkier and exhibit much stronger electron
withdrawing properties, which are essential for the enhancement of
singlet oxygen formation abilities [5]. Thus, for 7 and 8 it was observed
a decrease of ΦΔ in comparison to unsubstituted zinc(II) phthalocyanine
as a result of steric hindrance. Moreover, steric hindrance impacts the
singlet oxygen formation which was indicated by Erdoğmuş and co-
workers [64]. Also, for compound II a 2-fold decrease of ΦΔ has been
Fig. 2. Absorption, emission and excitation spectra of 7 in DMF.
ZnPc (ΦFL = 0.17). A similar effect has been observed by Güzel and co-
workers for substituted zinc(II) phthalocyanine [5].
Both studied macrocycles generate singlet oxygen. Thus, they can be
considered as potential photosensitizers for photodynamic therapy,
including bacteria photoinactivation. Phthalocyanine derivative
7
forms singlet oxygen slightly better than 8. In comparison to un-
substituted zinc(II) phthalocyanine both studied compounds (7, 8)
present a dramatic drop in singlet oxygen formation quantum yields –
see Table 1. This phenomenon may be linked with the above discussed
185

L. Sobotta, et al.
InorganicaChimicaActa489(2019)180–190
OH
O
O
O
O
O
NH2
NH
HO
O
O
O
O
O
O
O
O
O
O
NH2
N
N
N
N
N
NH
N
O
O
N
N
O
O
O
N
O
O
O
O
Zn
N
N
Zn
N
N
Zn
O
O
N
N
O
O
N
N
N
N
N
N
N
O
N
N
NH
H2
O
O
O
O
O
N
O
O
O
O
III
O
I
II
O
O
OH
HN
R
O
O
H2N
O
O
O
O
HO
O
N
N
N
O
O
O
N
N
Zn
N
N
N
N
R
O
O
O
O
Zn
N
N
N
N
N
O
N
N
R
O
O
R = Br,
R = Cl,
R = F,
IV
V
VI
O
VII
O
R
Fig. 3. Structures of phthalocyanines I-VII.
observed as compared to III [64]. On the other hand, a 2-fold higher ΦΔ
of I in comparison to 7 and 8 can be explained by its stronger effect of
electron-donating substituents [5,60]. Kirbaç and co-workers have de-
monstrated the influence of electron donating/withdrawing strength of
substituents for phthalocyanines IV-VI in DMF [65]. The substituents of
compound IV have exhibited lowest electron withdrawing potential
(highest donating) and revealed ΦΔ = 0.70, whereas V’s ΦΔ = 0.72 and
finally VI’s ΦΔ = 0.95 with highest withdrawing properties (lowest
donating) within studied series. Interestingly, this tendency has not
been observed in DMSO and THF solutions, which can be linked with
solvent-molecule interactions [65]. Peripherally substituted phthalo-
cyanine (VII) with phenoxy groups has been studied by Ogunsipe and
co-workers. This molecule generated singlet oxygen with quantum yield
values of 0.60 and 0.53 in DMSO and DMF respectively [48]. In com-
parison to 7 and 8, compound VII differs slightly in the structure.
Compounds 7 and 8 represent phthalocyanines substituted in the non-
peripheral positions and possess alkyl groups in the bulky peripheral
substituents, which potentiates steric hindrance effect, whereas
phthalocyanine VII is substituted at peripheral positions, which gen-
erates lower steric hindrance effect. Thus, compared to 7 and 8 are
weaker singlet oxygen generators than VII.
was presented for samples dissolved in DMF. Compound I (presented in
Fig. 1), in comparison to 7 and 8, has shown much higher photo-
decomposition rate in DMF equal ΦP = 2.54·10−4 [61]. A similar ten-
dency was observed for compounds II and III. In DMSO solutions
compound III has revealed ΦP = 1.02·10−6 quantum yield and II
ΦP = 8.53·10−6 [64]. Compounds IV, V and VI in comparison to ZnPc
(ΦP = 10.2·10−6) have revealed higher photodecomposition rates with
quantum yields in DMF equal ΦP = 20.0·10−4 ΦP = 4.0·10−4 and
ΦP = 73.0·10−4 respectively [65]. Considering the above observations,
it seems that the higher number or the bigger size of substituents can be
responsible for more intensive tensions in the excited state of the mo-
lecule and stimulate the decomposition. This hypothesis needs further
studies, mainly conducted with molecular calculations. Otherwise,
compounds revealed similar photostability in DMSO, what can be
connected to the coordination of DMSO to the central metal ion
[48,52,64,65].
Terpene-phthalocyanine derivatives 7 and 8 were loaded into the
modified liposomes. Also, there were prepared formulations containing
unsubstituted zinc(II) phthalocyanine (ZnPc), thymol and carvacrol.
The mean size and the size distribution of the formulation are shown in
Table 2.
Irradiated phthalocyanines can undergo phototransformation or
photobleaching process [66]. Compounds 7 and 8 under continuous
illumination with visible light lose their colour. This observation en-
ables to classify photodecomposition of studied phthalocyanines as
photobleaching. Both phthalocyanines underwent photodecomposition
with similar photodecomposition quantum yields, see Table 1. Inter-
estingly, in comparison to the unsubstituted zinc(II) phthalocyanine,
both phthalocyanine derivatives 7 and 8 revealed 2-fold lower photo-
stability. It is worth mentioning that their singlet oxygen formation
abilities were 3-fold lower than reference ZnPc. Mentioned dependence
Thymol-phthalocyanine conjugate 7 at both 100 and 10 µM con-
centrations revealed high photoinactivation of Enterococcus faecalis of
ca. 5-log growth reduction. Similar observations were made for re-
ference compound ZnPc. Thus, it can be concluded that attachment of
the thymol to the phthalocyanine ring does not increase its activity.
Interestingly, for carvacrol-phthalocyanine conjugate 8, an increase of
its activity at 100 µM was observed in comparison to ZnPc and a de-
crease of the activity at the concentration of 10 µM – see Table 2. The
drug-free liposomes, thymol and carvacrol loaded ones did not reveal
significant activity. It is important that both studied phthalocyanines
186

L. Sobotta, et al.
InorganicaChimicaActa489(2019)180–190
Table 2
Size of the lipid vesicles and their potential for photoinactivation of Enterococcus faecalis.
Photoinactivation of E. faecalis
Size of the modified liposomes
RF*
conditions
100 µM
10 µM
Mean diameter [nm]
Dv10
136
120
62
Dv50
209
152
106
123
159
163
Dv90
529
200
139
161
193
249
7
light
dark
light
dark
light
dark
light
dark
light
dark
light
dark
4.72
0.08
6.02
0.00
5.24
0.00
0.34
0.00
0.13
0.06
0.39
−0.08
5.02
282
161
107
122
166
177
0.01
8
3.71
−0.03
4.85
ZnPc
0.10
empty liposomes
thymol
−0.05
0.00
74
−0.02
−0.05
0.39
131
132
carvacrol
−0.07
RF - * logarithmic reduction factor.
O
O
N+
O
N+
O
I-
I-
O
O
N+
O
O
N+ -
I
I-
O
O
O
O
O
O
O
N
N
N
N
N
N
N
N
N
Zn
N
Mg
N
N
O
O
I-
N+
N
N
N+
N
N
O
I-
I-
O
O
O
O
O
N+
I-
N+
O
VIII
IX
Fig. 4. Chemical structures of compounds published earlier VIII, and IX [70,71]
cross the border of 3-log photoinactivation, which is characteristic for
the bactericidal agents following FDA (Food and Drug Administration)
regulations [7,67,68]. Some authors have also suggested the level of 4-
log for bactericidal compounds [69], which was also achieved by 7 and
8. Both phthalocyanines 7 and 8 similarly to the compounds VIII and IX
(recently published by our group) revealed high photoactivity against
E. faecalis [70,71] (Fig. 4).
The ideal photosensitizer for PACT should be inactive in the dark
conditions, what is crucial for the treatment selectivity [72–74].
Therefore, Microtox® bioassay for 7 and 8 was performed. This fast and
easy protocol enables to detect the acute and prolonged toxic effect of
the examined agent. It is based on bioluminescent bacteria Vibrio fi-
scheri, which under exposition to the toxic molecules lose its lumines-
cent ability [55,56,75].
Terpene-phthalocyanine conjugates 7 and 8 revealed a decreased
dark toxicity in comparison to the pure thymol and carvacrol (Fig. 5).
Thymol and carvacrol are known to increase the permeability of the
bacterial membrane leading to bacteria death [36]. As mentioned
above, 7 and 8 revealed only reduced dark toxicity, which can be ex-
plained by lower bacterial membrane penetration of big phthalocyanine
molecules in comparison to higher permeability of terpenes. Also, 7
revealed decreased toxicity in comparison to ZnPc and 8.
Fig. 5. Vibrio fischeri luminescence inhibition in Microtox® analysis.
187

L. Sobotta, et al.
InorganicaChimicaActa489(2019)180–190
4. Conclusion
Acknowledgments
In our initial study an attempt towards novel terpene-macrocycle
conjugate, consisting of phthalocyanine and azulene was performed.
This attempt was abandoned on the stage of azulene-maleonitrile de-
rivative substrate macrocyclization reaction, which turned out to be
very unstable during characterization and macrocyclization reaction
reconnaissance study. Another approach towards terpene-macrocycle
conjugate was successfully performed when 3-nitrobenzene-1,2-di-
carbonitrile was conjugated with thymol or carvacrol using nitro-group
displacement procedure and when the resulting conjugates were sub-
sequently subjected to macrocyclization reactions in a microwave re-
actor towards desired terpene-phthalocyanine conjugates. It is worth to
note that both reactions were conducted without addition of DBU as a
base. Experiments with DBU either failed to result with the macrocyclic
product or the reaction yields were very low. It seems that the basicity
of substrates, like zinc(II) acetate was enough for the occurrence of
cyclotetramerization reaction. The obtained compounds were char-
acterized by UV–Vis, NMR, MS, and HPLC. It is interesting that in the
referenced approach by Ma et al. [45], a thymol-phthalonitrile con-
jugate when subjected to the macrocyclization reaction led to the
mixture of thymol-phthalocyanine randomers. Surprisingly, in our
work, for both phthalonitrile derivatives, only one macrocyclic product
of C2v symmetry was found in the reaction mixture. The structure of the
macrocyclic product was unambiguously confirmed in the NMR study.
For both compounds, diagnostic signals belonging to isopropyl and
methyl groups of terpene substituents were identified.
This work has been supported by the National Science Centre,
Poland through grant No. 2015/19/N/NZ7/01342.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.ica.2019.02.031.
References
[1] D. Wöhrle, G. Schnurpfeil, S.G. Makarov, A. Kazarin, O.N. Suvorova, Practical ap-
plications of phthalocyanines – from dyes and pigments to materials for optical
electronic and photo-electronic devices, Macroheterocycles 5 (2012) 191–202,
https://doi.org/10.6060/mhc2012.120990w.
[2] A. Günsel, E. Güzel, A.T. Bilgiçli, İ. Şişman, M.N. Yarasir, Synthesis of non-per-
ipheral thioanisole-substituted phthalocyanines: photophysical, electrochemical,
photovoltaic, and sensing properties, J. Photochem. Photobiol. Chem. 348 (2017)
57–67, https://doi.org/10.1016/j.jphotochem.2017.07.040.
[3] S. Yano, S. Hirohara, M. Obata, Y. Hagiya, S. Ogura, A. Ikeda, H. Kataoka,
M. Tanaka, T. Joh, Current states and future views in photodynamic therapy, J.
Photochem. Photobiol. C Photochem. Rev. 12 (2011) 46–67, https://doi.org/10.
1016/j.jphotochemrev.2011.06.001.
[4] C.M. Cassidy, M.M. Tunney, P.A. McCarron, R.F. Donnelly, Drug delivery strategies
for photodynamic antimicrobial chemotherapy: from benchtop to clinical practice,
J. Photochem. Photobiol. B 95 (2009) 71–80, https://doi.org/10.1016/j.jphotobiol.
2009.01.005.
[5] E. Güzel, A. Günsel, A.T. Bilgiçli, G.Y. Atmaca, A. Erdoğmuş, M.N. Yarasir, Synthesis
and photophysicochemical properties of novel thiadiazole-substituted zinc (II),
gallium (III) and silicon (IV) phthalocyanines for photodynamic therapy, Inorg.
Chim. Acta 467 (2017) 169–176, https://doi.org/10.1016/j.ica.2017.07.058.
[6] T. Maisch, A new strategy to destroy antibiotic resistant microorganisms: anti-
microbial photodynamic treatment, Mini Rev. Med. Chem. 9 (2009) 974–983,
https://doi.org/10.2174/138955709788681582.
Absorption spectra of studied compounds are typical for phthalo-
cyanines with the Soret the Q band and possess the same profile. This
phenomenon is linked to the same electron donating/withdrawing
properties of substituents. Studied phthalocyanines reveal emission in
the red region of UV–Vis spectrum with calculated fluorescence
quantum yields about 2-fold lower than that for ZnPc. Interestingly,
compounds dissolved in DMSO presented a dramatic drop in light
emission. Among studied compounds thymol-phthalocyanine conjugate
generated singlet oxygen slightly more efficiently than carvacrol-
phthalocyanine conjugate. Nevertheless, in comparison with un-
substituted zinc(II) phthalocyanine both studied compounds presented
a decrease in singlet oxygen formation quantum yields. This phenom-
enon may be linked with steric hindrance formed by rotating sub-
stituents. Both terpene-phthalocyanine conjugates under illumination
with visible light continuously lose their colour, which enables to
classify photodecomposition of studied molecules as photobleaching.
Both phthalocyanines underwent photodecomposition with similar
photodecomposition quantum yield. Interestingly, in comparison to the
unsubstituted zinc(II) phthalocyanine, both terpene-phthalocyanine
conjugates revealed 2-fold lower photostability and 3-fold lower singlet
oxygen formation ability in DMF. It seems that higher number or bigger
size of substituents is responsible for more intensive tensions in the
excited state of the molecule and stimulates the decomposition.
Otherwise, in DMSO compounds revealed similar photostability, which
can be the result of coordination of DMSO to the central metal ion. Both
phthalocyanine analogs were loaded to the modified liposomes.
Formulations containing unsubstituted zinc(II) phthalocyanine, thymol
and carvacrol were obtained as well. Thymol-phthalocyanine conjugate
at both 100 and 10 µM revealed high photoinactivation growth of
Enterococcus faecalis at ca. 5-log, similarly to reference ZnPc. For car-
vacrol-phthalocyanine conjugate, an increase of activity at 100 µM in
comparison to ZnPc was observed and a decrease at 10 µM. Both stu-
died conjugates cross the border of 3-log, which indicates their bac-
tericidal potential according to the FDA regulations. The Microtox®
bioassay was performed for both conjugates revealing their low dark
toxicity, which was significantly below the dark toxicity measured for
the pure thymol and carvacrol.
[7] L. Sobotta, P. Skupin-Mrugalska, J. Piskorz, J. Mielcarek, Non-porphyrinoid pho-
tosensitizers mediated photodynamic inactivation against bacteria, Dyes Pigm. 163
(2019) 337–355, https://doi.org/10.1016/j.dyepig.2018.12.014.
[8] A. Tillo, M. Stolarska, M. Kryjewski, L. Popenda, L. Sobotta, S. Jurga, J. Mielcarek,
T. Goslinski, Phthalocyanines with bulky substituents at non-peripheral positions –
synthesis and physico-chemical properties, Dyes Pig. 127 (2016) 110–115, https://
doi.org/10.1016/j.dyepig.2015.12.017.
[9] L. Ryskova, V. Buchta, M. Karaskova, J. Rakusan, J. Cerny, R. Slezak, In vitro an-
timicrobial activity of light-activated phthalocyanines, Cent. Eur. J. Biol. 8 (2013)
168–177, https://doi.org/10.2478/s11535-013-0118-0.
[10] A. Sindelo, O.L. Osifeko, T. Nyokong, Synthesis, photophysicochemical and pho-
todynamic antimicrobial chemotherapy studies of indium pyridyl phthalocyanines:
charge versus bridging atom, Inorg. Chim. Acta 476 (2018) 68–76, https://doi.org/
10.1016/j.ica.2018.02.020.
[11] M. Li, B. Mai, A. Wang, Y. Gao, X. Wang, X. Liu, S. Song, Q. Liu, S. Wei, P. Wang,
Photodynamic antimicrobial chemotherapy with cationic phthalocyanines against
Escherichia coli planktonic and biofilm cultures, RSC Adv. 7 (2017) 40734–40744,
https://doi.org/10.1039/C7RA06073D.
[12] Y. Gao, B. Mai, A. Wang, M. Li, X. Wang, K. Zhang, Q. Liu, S. Wei, P. Wang,
Antimicrobial properties of a new type of photosensitizer derived from phthalo-
cyanine against planktonic and biofilm forms of Staphylococcus aureus,
Photodiagn. Photodyn. Ther. 21 (2018) 316–326, https://doi.org/10.1016/j.pdpdt.
2018.01.003.
[13] C. Uslan, N.D. İşleyen, Y. Öztürk, B.T. Yıldız, Z.P. Çakar, M. Göksel, M. Durmuş,
Y.H. Gürsel, B.Ş. Sesalan, A novel of PEG-conjugated phthalocyanine and evaluation
of its photocytotoxicity and antibacterial properties for photodynamic therapy, J.
Porphyr. Phthalocyanines 22 (2018) 10–24, https://doi.org/10.1142/
S1088424617500729.
[14] B.-Y. Zheng, X.-J. Jiang, T. Lin, M.-R. Ke, J.-D. Huang, Novel silicon(IV) phthalo-
cyanines containing piperidinyl moieties: synthesis and in vitro antifungal photo-
dynamic activities, Dyes Pigments 112 (2015) 311–316, https://doi.org/10.1016/j.
dyepig.2014.07.029.
[15] B.-Y. Zheng, T. Lin, H.-H. Yang, J.-D. Huang, Photodynamic inactivation of Candida
albicans sensitized by a series of novel axially di-substituted silicon (IV) phthalo-
cyanines, Dyes Pigm. 96 (2013) 547–553, https://doi.org/10.1016/j.dyepig.2012.
09.013.
[16] M.-R. Ke, J.M. Eastel, K.L.K. Ngai, Y.-Y. Cheung, P.K.S. Chan, M. Hui, D.K.P. Ng, P.-
C. Lo, Oligolysine-conjugated zinc(II) phthalocyanines as efficient photosensitizers
for antimicrobial photodynamic therapy, Chem. - Asian J. 9 (2014) 1868–1875,
https://doi.org/10.1002/asia.201402025.
[17] Z. Chen, S. Zhou, J. Chen, L. Li, P. Hu, S. Chen, M. Huang, An effective zinc
phthalocyanine derivative for photodynamic antimicrobial chemotherapy, J.
Lumin. 152 (2014) 103–107, https://doi.org/10.1016/j.jlumin.2013.10.067.
[18] G.B. Rodrigues, F.L. Primo, A.C. Tedesco, G.U.L. Braga, In vitro photodynamic
188

L. Sobotta, et al.
InorganicaChimicaActa489(2019)180–190
inactivation of cryptococcus neoformans melanized cells with chloroaluminum
[41] S.S. Swain, S.K. Paidesetty, R.N. Padhy, Development of antibacterial conjugates
using sulfamethoxazole with monocyclic terpenes: a systematic medicinal chem-
istry based computational approach, Comput. Methods Programs Biomed. 140
(2017) 185–194, https://doi.org/10.1016/j.cmpb.2016.12.013.
phthalocyanine nanoemulsion, Photochem. Photobiol. 88 (2012) 440–447, https://
doi.org/10.1111/j.1751-1097.2011.01055.x.
[19] L. Sobotta, J. Dlugaszewska, P. Kasprzycki, S. Lijewski, A. Teubert, J. Mielcarek,
M. Gdaniec, T. Goslinski, P. Fita, E. Tykarska, In vitro photodynamic activity of
lipid vesicles with zinc phthalocyanine derivative against Enterococcus faecalis, J.
Photochem. Photobiol. B 183 (2018) 111–118, https://doi.org/10.1016/j.
jphotobiol.2018.04.025.
[42] S.S. Swain, S.K. Paidesetty, R.N. Padhy, Antibacterial activity, computational ana-
lysis and host toxicity study of thymol-sulfonamide conjugates, Biomed.
Pharmacother. 88 (2017) 181–193, https://doi.org/10.1016/j.biopha.2017.01.
036.
[20] L. Sobotta, J. Sniechowska, D. Ziental, J. Dlugaszewska, M.J. Potrzebowski,
Chlorins with (trifluoromethyl)phenyl substituents – synthesis, lipid formulation
and photodynamic activity against bacteria, Dyes Pigm. 160 (2019) 292–300,
https://doi.org/10.1016/j.dyepig.2018.08.004.
[43] R. Cahan, N. Swissa, G. Gellerman, Y. Nitzan, Photosensitizer-antibiotic conjugates:
a novel class of antibacterial molecules, Photochem. Photobiol. 86 (2010) 418–425,
https://doi.org/10.1111/j.1751-1097.2009.00674.x.
[44] Z. Zhang, G.M. Ferrence, T.D. Lash, adj-Diazuliporphyrins, a New Family of
Dicarbaporphyrinoids with Unprecedented Mesoionic Characteristics †, Org. Lett.
11 (2009) 101–104, https://doi.org/10.1021/ol802406f.
[21] L. Sobotta, J. Dlugaszewska, M. Gierszewski, A. Tillo, M. Sikorski, E. Tykarska,
J. Mielcarek, T. Goslinski, Photodynamic inactivation of Enterococcus faecalis by
non-peripherally substituted magnesium phthalocyanines entrapped in lipid ve-
sicles, J. Photochem. Photobiol. B 188 (2018) 100–106, https://doi.org/10.1016/j.
jphotobiol.2018.09.003.
[45] C. Ma, D. Tian, X. Hou, Y. Chang, F. Cong, H. Yu, X. Du, G. Du, Synthesis and
characterization of several soluble tetraphenoxy-substituted copper and zinc
phthalocyanines, ChemInform 36 (2005), https://doi.org/10.1002/chin.
200533201.
[22] L. Sobotta, J. Dlugaszewska, D. Ziental, W. Szczolko, T. Koczorowski, T. Goslinski,
J. Mielcarek, Optical properties of a series of pyrrolyl-substituted porphyrazines and
their photoinactivation potential against Enterococcus faecalis after incorporation
into liposomes, J. Photochem. Photobiol. Chem. 368 (2019) 104–109, https://doi.
org/10.1016/j.jphotochem.2018.09.015.
[46] L. Sobotta, P. Fita, W. Szczolko, M. Wrotynski, M. Wierzchowski, T. Goslinski,
J. Mielcarek, Functional singlet oxygen generators based on porphyrazines with
peripheral 2,5-dimethylpyrrol-1-yl and dimethylamino groups, J. Photochem.
Photobiol. Chem. 269 (2013) 9–16, https://doi.org/10.1016/j.jphotochem.2013.
06.018.
[23] L. Sobotta, D. Ziental, J. Sniechowska, J. Dlugaszewska, M.J. Potrzebowski, Lipid
vesicle-loaded meso -substituted chlorins of high in vitro antimicrobial photo-
dynamic activity, Photochem. Photobiol. Sci. (2019), https://doi.org/10.1039/
C8PP00258D.
[47] V. Chauke, A. Ogunsipe, M. Durmuş, T. Nyokong, Novel gallium(III) phthalocyanine
derivatives – synthesis, photophysics and photochemistry, Polyhedron 26 (2007)
2663–2671, https://doi.org/10.1016/j.poly.2007.01.016.
[24] S. Koch, M. Hufnagel, C. Theilacker, J. Huebner, Enterococcal infections: host re-
sponse, therapeutic, and prophylactic possibilities, Vaccine 22 (2004) 822–830,
https://doi.org/10.1016/j.vaccine.2003.11.027.
[48] A. Ogunsipe, D. Maree, T. Nyokong, Solvent effects on the photochemical and
fluorescence properties of zinc phthalocyanine derivatives, J. Mol. Struct. 650
(2003) 131–140, https://doi.org/10.1016/S0022-2860(03)00155-8.
[49] A. Ogunsipe, M. Durmuş, D. Atilla, A.G. Gürek, V. Ahsen, T. Nyokong, Synthesis,
photophysical and photochemical studies on long chain zinc phthalocyanine deri-
vatives, Synth. Met. 158 (2008) 839–847, https://doi.org/10.1016/j.synthmet.
2008.06.007.
[25] C.M.A.P. Franz, A.B. Muscholl-Silberhorn, N.M.K. Yousif, M. Vancanneyt, J. Swings,
W.H. Holzapfel, Incidence of virulence factors and antibiotic resistance among
enterococci isolated from food, Appl. Environ. Microbiol. 67 (2001) 4385–4389,
https://doi.org/10.1128/AEM.67.9.4385-4389.2001.
[26] I. Kühn, Comparison of enterococcal populations in animals, humans, and the en-
vironment – a European study, Int. J. Food Microbiol. 88 (2003) 133–145, https://
doi.org/10.1016/S0168-1605(03)00176-4.
[50] I. Seotsanyana-Mokhosi, N. Kuznetsova, T. Nyokong, Photochemical studies of
tetra-2, 3-pyridinoporphyrazines, J. Photochem. Photobiol. Chem. 140 (2001)
215–222.
[27] R. Souto, A.P.V. Colombo, Prevalence of Enterococcus faecalis in subgingival bio-
film and saliva of subjects with chronic periodontal infection, Arch. Oral Biol. 53
(2008) 155–160, https://doi.org/10.1016/j.archoralbio.2007.08.004.
[28] R.M. Love, Enterococcus faecalis - a mechanism for its role in endodontic failure,
Int. Endod. J. 34 (2001) 399–405, https://doi.org/10.1046/j.1365-2591.2001.
00437.x.
[51] X. Shen, W. Lu, G. Feng, Y. Yao, W. Chen, Preparation and photoactivity of a novel
water-soluble, polymerizable zinc phthalocyanine, J. Mol. Catal. Chem. 298 (2009)
17–22, https://doi.org/10.1016/j.molcata.2008.09.023.
[52] N.A. Kuznetsova, V.V. Okunchikov, V.M. Derkacheva, O.L. Kaliya, E.A. Lukyanets,
Photooxidation of metallophthalocyanines: the effects of singlet oxygen and PcM-O
2 complex formation, J. Porphyr. Phthalocyanines 9 (2005) 393–397.
[53] N.A. Kuznetsova, D.A. Makarov, O.A. Yuzhakova, L.I. Solovieva, O.L. Kaliya, Study
on the photostability of water-soluble Zn(II) and Al(III) phthalocyanines in aqueous
solution, J. Porphyr. Phthalocyanines 14 (2010) 968–974, https://doi.org/10.
1142/S1088424610002835.
[29] C.A. Arias, B.E. Murray, The rise of the Enterococcus: beyond vancomycin re-
sistance, Nat. Rev. Microbiol. 10 (2012) 266–278, https://doi.org/10.1038/
nrmicro2761.
[30] H.-M. Chiang, J.-J. Yin, Q. Xia, Y. Zhao, P.P. Fu, K.-C. Wen, H. Yu, Photoirradiation
of azulene and guaiazulene—formation of reactive oxygen species and induction of
lipid peroxidation, J. Photochem. Photobiol. Chem. 211 (2010) 123–128, https://
doi.org/10.1016/j.jphotochem.2010.02.007.
[54] N. Dragicevic-Curic, D. Scheglmann, V. Albrecht, A. Fahr, Development of different
temoporfin-loaded invasomes—novel nanocarriers of temoporfin: characterization,
stability and in vitro skin penetration studies, Colloids Surf. B Biointerfaces 70
(2009) 198–206, https://doi.org/10.1016/j.colsurfb.2008.12.030.
[55] T.X.H. Le, T.V. Nguyen, Z. Amadou Yacouba, L. Zoungrana, F. Avril, D.L. Nguyen,
E. Petit, J. Mendret, V. Bonniol, M. Bechelany, S. Lacour, G. Lesage, M. Cretin,
Correlation between degradation pathway and toxicity of acetaminophen and its
by-products by using the electro-Fenton process in aqueous media, Chemosphere
172 (2017) 1–9, https://doi.org/10.1016/j.chemosphere.2016.12.060.
[56] R. Weltens, K. Deprez, L. Michiels, Validation of Microtox as a first screening tool
for waste classification, Waste Manage. 34 (2014) 2427–2433, https://doi.org/10.
1016/j.wasman.2014.08.001.
[31] R.R.A. Hayek, N.S. Araújo, M.A. Gioso, J. Ferreira, C.A. Baptista-Sobrinho,
A.M. Yamada, M.S. Ribeiro, Comparative study between the effects of photo-
dynamic therapy and conventional therapy on microbial reduction in ligature-in-
duced peri-implantitis in dogs, J. Periodontol. 76 (2005) 1275–1281, https://doi.
org/10.1902/jop.2005.76.8.1275.
[32] B. Salehi, A.P. Mishra, I. Shukla, M. Sharifi-Rad, M. del M. Contreras, A. Segura-
Carretero, H. Fathi, N.N. Nasrabadi, F. Kobarfard, J. Sharifi-Rad, Thymol, thyme,
and other plant sources: health and potential uses: thymol, health and potential
uses, Phytother. Res. 32 (2018) 1688–1706, https://doi.org/10.1002/ptr.6109.
[33] M. Sharifi-Rad, E.M. Varoni, M. Iriti, M. Martorell, W.N. Setzer, M. del Mar
Contreras, B. Salehi, A. Soltani-Nejad, S. Rajabi, M. Tajbakhsh, J. Sharifi-Rad,
Carvacrol and human health: a comprehensive review: carvacrol and Human
Health, Phytother. Res. 32 (2018) 1675–1687, https://doi.org/10.1002/ptr.6103.
[34] M.Y. Memar, P. Raei, N. Alizadeh, M. Akbari Aghdam, H.S. Kafil, Carvacrol and
thymol: strong antimicrobial agents against resistant isolates, Rev. Med. Microbiol.
28 (2017) 63–68, https://doi.org/10.1097/MRM.0000000000000100.
[35] A. Marchese, I.E. Orhan, M. Daglia, R. Barbieri, A. Di Lorenzo, S.F. Nabavi,
O. Gortzi, M. Izadi, S.M. Nabavi, Antibacterial and antifungal activities of thymol: a
brief review of the literature, Food Chem. 210 (2016) 402–414, https://doi.org/10.
1016/j.foodchem.2016.04.111.
[57] M. Wierzchowski, L. Sobotta, P. Skupin-Mrugalska, J. Kruk, W. Jusiak, M. Yee,
K. Konopka, N. Düzgüneş, E. Tykarska, M. Gdaniec, J. Mielcarek, T. Goslinski,
Phthalocyanines functionalized with 2-methyl-5-nitro-1H-imidazolylethoxy and
1,4,7-trioxanonyl moieties and the effect of metronidazole substitution on photo-
cytotoxicity, J. Inorg. Biochem. 127 (2013) 62–72, https://doi.org/10.1016/j.
jinorgbio.2013.06.012.
[58] M. Sommerauer, C. Rager, M. Hanack, Separation of 2(3),9(10),16(17),23(24)-
tetrasubstituted phthalocyanines with newly developed HPLC phases, J. Am. Chem.
Soc. 118 (1996) 10085–10093, https://doi.org/10.1021/ja961009x.
[59] W. Lv, J. Duan, S. Ai, “Pinwheel-like” phthalocyanines with four non-peripheral
chiral menthol units: Synthesis, spectroscopy, and electrochemistry, Chin. Chem.
Lett. 30 (2019) 389–391, https://doi.org/10.1016/j.cclet.2018.06.002.
[60] S.Z. Topal, Ü. İşci, U. Kumru, D. Atilla, A.G. Gürek, C. Hirel, M. Durmuş, J.-
B. Tommasino, D. Luneau, S. Berber, F. Dumoulin, V. Ahsen, Modulation of the
electronic and spectroscopic properties of Zn(ii) phthalocyanines by their sub-
stitution pattern, Dalton Trans. 43 (2014) 6897, https://doi.org/10.1039/
c3dt53410c.
[36] A. Guarda, J.F. Rubilar, J. Miltz, M.J. Galotto, The antimicrobial activity of mi-
croencapsulated thymol and carvacrol, Int. J. Food Microbiol. 146 (2011) 144–150,
https://doi.org/10.1016/j.ijfoodmicro.2011.02.011.
[37] B. Shah, S. Ikeda, P. Michael Davidson, Q. Zhong, Nanodispersing thymol in whey
protein isolate-maltodextrin conjugate capsules produced using the emulsion–eva-
poration technique, J. Food Eng. 113 (2012) 79–86, https://doi.org/10.1016/j.
jfoodeng.2012.05.019.
[61] M. Aliosman, M. Göksel, V. Mantareva, I. Stoineva, M. Durmuş, Tyrosine conjugated
zinc(II) phthalocyanine for photodynamic therapy: synthesis and photo-
physicochemical properties, J. Photochem. Photobiol. Chem. 334 (2017) 101–106,
https://doi.org/10.1016/j.jphotochem.2016.10.037.
[38] B. Shah, P.M. Davidson, Q. Zhong, Antimicrobial activity of nanodispersed thymol
in tryptic soy broth, J. Food Prot. 76 (2013) 440–447, https://doi.org/10.4315/
0362-028X.JFP-12-354.
[39] B. Shah, P.M. Davidson, Q. Zhong, Nanodispersed eugenol has improved anti-
microbial activity against Escherichia coli O157:H7 and Listeria monocytogenes in
bovine milk, Int. J. Food Microbiol. 161 (2013) 53–59, https://doi.org/10.1016/j.
ijfoodmicro.2012.11.020.
[62] M.C. DeRosa, R.J. Crutchley, Photosensitized singlet oxygen and its applications,
Coord. Chem. Rev. 233 (2002) 351–371.
[63] G. Cheng, X. Peng, G. Hao, V.O. Kennedy, I.N. Ivanov, K. Knappenberger, T.J. Hill,
M.A.J. Rodgers, M.E. Kenney, Synthesis, photochemistry, and electrochemistry of a
series of phthalocyanines with graded steric hindrance, J. Phys. Chem. A 107
(2003) 3503–3514, https://doi.org/10.1021/jp027006q.
[40] V.K. Redasani, S.B. Bari, Synthesis and evaluation of mutual prodrugs of ibuprofen
with menthol, thymol and eugenol, Eur. J. Med. Chem. 56 (2012) 134–138, https://
doi.org/10.1016/j.ejmech.2012.08.030.
[64] A. Erdoğmuş, T. Nyokong, New soluble methylendioxy-phenoxy-substituted zinc
189

L. Sobotta, et al.
InorganicaChimicaActa489(2019)180–190
phthalocyanine derivatives: synthesis, photophysical and photochemical studies,
025.
Polyhedron 28 (2009) 2855–2862, https://doi.org/10.1016/j.poly.2009.06.019.
[65] E. Kırbaç, G.Y. Atmaca, A. Erdoğmuş, Novel highly soluble fluoro, chloro, bromo-
phenoxy-phenoxy substituted zinc phthalocyanines; synthesis, characterization and
photophysicochemical properties, J. Organomet. Chem. 752 (2014) 115–122,
https://doi.org/10.1016/j.jorganchem.2013.12.005.
[71] J. Dlugaszewska, W. Szczolko, T. Koczorowski, P. Skupin-Mrugalska, A. Teubert,
K. Konopka, M. Kucinska, M. Murias, N. Düzgüneş, J. Mielcarek, T. Goslinski,
Antimicrobial and anticancer photodynamic activity of a phthalocyanine photo-
sensitizer with N-methyl morpholiniumethoxy substituents in non-peripheral posi-
tions, J. Inorg. Biochem. 172 (2017) 67–79, https://doi.org/10.1016/j.jinorgbio.
2017.04.009.
[66] R. Bonnett, G. Martınez, Photobleaching of sensitisers used in photodynamic
therapy, Tetrahedron 57 (2001) 9513–9547.
[72] R.R. Allison, G.H. Downie, R. Cuenca, X.-H. Hu, C.J. Childs, C.H. Sibata,
Photosensitizers in clinical PDT, Photodiagnosis Photodyn. Ther. 1 (2004) 27–42,
https://doi.org/10.1016/S1572-1000(04)00007-9.
[67] E. Alves, M.A. Faustino, M.G. Neves, A. Cunha, J. Tome, A. Almeida, An insight on
bacterial cellular targets of photodynamic inactivation, Future Med. Chem. 6 (2014)
141–164, https://doi.org/10.4155/fmc.13.211.
[73] P. Agostinis, K. Berg, K.A. Cengel, T.H. Foster, A.W. Girotti, S.O. Gollnick,
S.M. Hahn, M.R. Hamblin, A. Juzeniene, D. Kessel, M. Korbelik, J. Moan, P. Mroz,
D. Nowis, J. Piette, B.C. Wilson, J. Golab, Photodynamic therapy of cancer: an
update, CA Cancer J. Clin. 61 (2011) 250–281, https://doi.org/10.3322/caac.
20114.
[68] A. Spaeth, A. Graeler, T. Maisch, K. Plaetzer, CureCuma–cationic curcuminoids with
improved properties and enhanced antimicrobial photodynamic activity, Eur. J.
Med. Chem. (2017), https://doi.org/10.1016/j.ejmech.2017.09.072.
[69] G. Jori, C. Fabris, M. Soncin, S. Ferro, O. Coppellotti, D. Dei, L. Fantetti, G. Chiti,
G. Roncucci, Photodynamic therapy in the treatment of microbial infections: basic
principles and perspective applications, Lasers Surg. Med. 38 (2006) 468–481,
https://doi.org/10.1002/lsm.20361.
[74] J.F. Lovell, T.W.B. Liu, J. Chen, G. Zheng, Activatable photosensitizers for imaging
and therapy, Chem. Rev. 110 (2010) 2839–2857, https://doi.org/10.1021/
cr900236h.
[70] L. Sobotta, J. Dlugaszewska, P. Kasprzycki, S. Lijewski, A. Teubert, J. Mielcarek,
M. Gdaniec, T. Goslinski, P. Fita, E. Tykarska, In vitro photodynamic activity of
lipid vesicles with zinc phthalocyanine derivative against Enterococcus faecalis, J.
Photochem. Photobiol. B (2018), https://doi.org/10.1016/j.jphotobiol.2018.04.
[75] V.R. Urbano, M.G. Maniero, M. Pérez-Moya, J.R. Guimarães, Influence of pH and
ozone dose on sulfaquinoxaline ozonation, J. Environ. Manage. 195 (2017)
224–231, https://doi.org/10.1016/j.jenvman.2016.08.019.
190