Sulfanyl porphyrazines: Molecular barrel-like self-assembly in crystals, optical properties and in vitro photodynamic activity towards cancer cells

Accepted Manuscript Sulfanyl porphyrazines: Molecular barrel-like self-assembly in crystals, optical properties and in vitro photodynamic activity towards cancer cells Jaroslaw Piskorz, Sebastian Lijewski, Mateusz Gierszewski, Karolina Gorniak, Lukasz Sobotta, Barbara Wicher, Ewa Tykarska, Nejat Düzgüneş, Krystyna Konopka, Marek Sikorski, Maria Gdaniec, Jadwiga Mielcarek, Tomasz Goslinski PII:

S0143-7208(16)30800-2

DOI:

10.1016/j.dyepig.2016.09.054

Reference:

DYPI 5506

To appear in:

Dyes and Pigments

Received Date: 7 July 2016 Revised Date:

19 September 2016

Accepted Date: 22 September 2016

Please cite this article as: Piskorz J, Lijewski S, Gierszewski M, Gorniak K, Sobotta L, Wicher B, Tykarska E, Düzgüneş N, Konopka K, Sikorski M, Gdaniec M, Mielcarek J, Goslinski T, Sulfanyl porphyrazines: Molecular barrel-like self-assembly in crystals, optical properties and in vitro photodynamic activity towards cancer cells, Dyes and Pigments (2016), doi: 10.1016/ j.dyepig.2016.09.054. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT Sulfanyl porphyrazines: molecular barrel-like self-assembly in crystals, optical properties and in vitro photodynamic activity towards cancer cells

Jaroslaw Piskorza,*, Sebastian Lijewskib, Mateusz Gierszewskic, Karolina Gorniakb, Lukasz

RI PT

Sobottaa, Barbara Wicherb, Ewa Tykarskab, Nejat Düzgüneşd, Krystyna Konopkad, Marek Sikorskic, Maria Gdaniecc, Jadwiga Mielcareka, Tomasz Goslinskib

Department of Inorganic and Analytical Chemistry, Poznan University of Medical Sciences,

SC

a

Grunwaldzka 6, 60-780 Poznan, Poland

Department of Chemical Technology of Drugs, Poznan University of Medical Sciences,

Grunwaldzka 6, 60-780 Poznan, Poland c

Faculty of Chemistry, Adam Mickiewicz University in Poznan, Umultowska 89b, 61-614

Poznan, Poland

USA

Abstract

TE D

Department of Biomedical Sciences University of the Pacific,155 Fifth Street, San Francisco,

EP

d

M AN U

b

Novel sulfanyl porphyrazines with peripheral 4-bromobenzyl and 4-biphenylylmethyl

AC C

substituents were synthesized, characterized by photochemical methods and evaluated as sensitizers for photodynamic therapy (PDT). The X-ray crystallography study performed for sulfanyl porphyrazine with 4-biphenylylmethyl substituents revealed that the biphenyl residues from two consecutive molecules form the barrel-like cages with a macrocyclic core constituting a bottom and a top of the barrel and pyridine molecules enclosed inside. The optical properties of both porphyrazines were evaluated in various protic and aprotic solvents. To complement the conventional fluorescence measurements excitation-emission maps were

1

ACCEPTED MANUSCRIPT recorded. The potential photosensitizing efficiency of the novel sulfanyl porphyrazines was evaluated by assessing the quantum yields of photosensitized singlet oxygen production. For this purpose the emission of light specific for the transition of singlet oxygen to ground state oxygen was measured. Photodynamic activities of porphyrazines and their liposomal

RI PT

formulations with different surface charge toward two oral squamous cell carcinoma cells (CAL 27, HSC-3) and human cervical epithelial adenocarcinoma cells (HeLa) were determined. Sulfanyl porphyrazines incorporated in cationic liposomes showed high activity,

SC

in contrast to the lack of activity of the free form porphyrazines in solution. Thus, cationic liposomes composed of 1,2-dioleoyl-3-trimethylammonium-propane and 1-palmitoyl-2-

M AN U

oleoyl-sn-glycero-3-phosphocholine can be considered as a promising drug delivery system for the sulfanyl porphyrazines for the photodynamic therapy of cancers.

Keywords: porphyrazine, crystal structure, singlet oxygen, cytotoxicity, photodynamic

*Correspondence to:

TE D

therapy

EP

J. Piskorz, Department of Inorganic and Analytical Chemistry, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780, Poznan, Poland. Tel.: +48 61 8546606, fax: +48

AC C

618546609. E-mail: [email protected]

2

ACCEPTED MANUSCRIPT 1. Introduction Porphyrazines (Pzs) belong to the macrocyclic class of compounds called porphyrinoids and consist of four pyrrole rings linked by aza bridges. The expansion of the periphery of Pzs with various sulfur moieties led to libraries of macrocycles of distinct physico-chemical

RI PT

properties, including solubility, aggregation tendency, absorption and emission properties, as well as electrochemical behaviour. Examples of Pzs equipped with peripheral sulfanyl substituents in their β-positions include ether [1-5], benzyl [6-8], fluorinated units [1,6,9,10], [5,10],

alkenyl

[11,12],

carboranyl

[13,14],

dibenzazepine,

[15]

SC

hydroxyalkyl

tetrathiafulvalene [16] and triphenylphosphonium [17] derivatives.

M AN U

The spectroscopic and photochemical properties of sulfanyl porphyrazines have been explored [8,16,18-21] and the numerous potential applications of these compounds have been recognized [22-27]. Self-organized Pz films have been developed for molecular electronics and nanotechnology [22-24]. Moreover, sulfanyl Pzs have been applied to the preparation of

TE D

Langmuir–Blodgett thin films and used as environmental gas probes [28]. In medicine, Pzs with peripheral sulfanyl substituents have been considered as potential photosensitizers in photodynamic therapy (PDT), as they possess the ability to generate singlet oxygen upon

EP

excitation with light of the appropriate wavelength, with promising photocytotoxicity. The photocytotoxic effect of various sulfanyl porphyrazines can be modified by appropriate

AC C

peripheral sulfur modifications with various groups, which affect their hydrophilic–lipophilic balance [1,4,26,29,30].

The main

drawbacks

of currently used

and

researched

photosensitizers, including Pzs, are poor solubility and the tendency to form aggregates. These issues have been addressed by developing drug delivery systems of which liposomes were found to be the most useful. For various groups of porphyrinoid photosensitizers, different forms of carriers such as liposomes allow the increase in the efficiency and safety of the therapy [31]. A relationship was found between the structure of sulfanyl Pzs and their

3

ACCEPTED MANUSCRIPT ability to bind liposomes and generate singlet oxygen [4]. It was also demonstrated that poly(carbonylalkylthio)porphyrazine inserted into liposomes improves the potential of multiple-approach cancer therapy [13]. We reported previously the syntheses, and physicochemical and photocytotoxic properties

RI PT

of sulfanyl Pzs possessing peripheral systems of fluoroalkyl, diether [1] nitroimidazobutyl [32], isophthaloxyalkyl [33,34] and dendrimeric substituents [35]. In this present study we assessed the effects of both heavy atoms and conjugated biaryl substituents on the optical

SC

properties and photocytotoxicities of sulfanyl porphyrazines, utilizing 4-bromobenzyl and 4biphenylylmethyl substituents. It is known that the presence of heavy atoms such as iodine

M AN U

and bromine in the structure of photosensitizers can increase the level of intersystem crossing from an excited singlet to a triplet state. A higher level of intersystem crossing results in an increased efficiency of singlet oxygen generation and a decreased intensity of fluorescence. Studies performed on the BODIPY-based photosensitizers revealed that the greatest heavy

TE D

atom effect was present when bromine atoms were directly connected directly to the photosensitizer core. The introduction of bromine to the peripheral substituents using e.g. 4bromophenyl groups resulted in enhanced singlet oxygen generation, whereas other spectral

EP

properties, including absorption and fluorescence, were not affected significantly [36,37]. In addition, the studies performed on phthalocyanines showed that the bromination can lead to

AC C

the red shift of the Q-band absorption and emission maxima [38,39]. The red-shifted Q-band absorptions are desirable for PDT candidates, because light of longer wavelengths is able to penetrate deeper into the irradiated tissues [40]. Recently, biphenyl substituents have gained much attention, because of their geometric and electronic characteristics and possible applications as sensors [41-44]. Moreover, porphyrazines with bulky 4-biphenyl groups revealed interesting photophysical properties and a low tendency to form aggregates [45,46].

4

ACCEPTED MANUSCRIPT 2. Experimental 2.1. General procedures All reactions were conducted in oven dried glassware under an argon atmosphere using Radleys Heat-On heating system. All solvents were rotary evaporated at or below 50ºC under

RI PT

reduced pressure. Dry flash column chromatography was carried out on Merck silica gel, particle size 40-63 µm. Thin layer chromatography (TLC) was performed on silica gel Merck Kieselgel 60 F254 plates and visualized with UV illumination (λmax 254 or 365 nm). UV–Vis 13

C NMR

SC

spectra were recorded on a Hitachi UV/VIS U-1900 spectrophotometer. 1H NMR,

spectra were prepared using a Bruker Avance spectrometer operating at 600.1 MHz for 1H

M AN U

and 150.9 MHz for 13C. Chemical shifts (δ) are quoted in parts per million (ppm) and referred to a residual solvent peak. Coupling constants (J) are quoted in Hertz (Hz). The abbreviations s, d, t refer to singlet, doublet and triplet, respectively. Mass spectra (ES, MALDI TOF) and elementary analysis were carried out by the Advanced Chemical Equipment and

TE D

Instrumentation Facility at the Faculty of Chemistry, Adam Mickiewicz University in Poznan.

2.2. Synthetic procedures

EP

Dimercaptomaleonitrile disodium salt and 4-bromomethylbiphenyl were purchased from TCI Chemicals (Tokyo, Japan). 4-Bromobenzyl bromide was purchased from Sigma-Aldrich

AC C

(Saint Louis, Missouri, USA). 2.2.1. 2,3-Bis(4-bromobenzylsulfanyl)maleonitrile (1) Dimercaptomaleodinitrile disodium salt (465 mg, 2.50 mmol) and 4-bromobenzyl bromide (1.56 g; 6.25 mmol) were dissolved in anhydrous methanol (35.0 mL) and stirred under reflux for 4 h. Next, the reaction mixture was cooled, the solvent was evaporated and a crude brown oil was chromatographed (hexane/ethyl acetate, 7:3, v/v) to give 1 as yellow crystals (998 mg; 83%); m.p. 91-93 °C; Rf (hexane/ethyl acetate; 7:1, v/v) = 0.40; UV–Vis (dichloromethane):

5

ACCEPTED MANUSCRIPT λmax, nm (log ε) 345 (4.14); FT-IR (KBr), v (cm-1): 3028, 2211, 1488, 1173, 1008, 836, 697; 1

H NMR (600.1 MHz, DMSO-d6) δ 7.55 (d, 4H, 3J = 8 Hz, C2C6, ArH), 7.30 (d, 4H, 3J = 8

Hz, C3C5, ArH), 4.43 (s, 4H, CH2);

13

C NMR (150.9 MHz, DMSO-d6) δ 135.1 (C1, ArC),

131.6 (C3C5, ArC), 131.1 (C2C6, ArC), 121.7 (C4, ArC), 121.1 (CN), 112.4 (NCCS), 37.3

RI PT

(CH2); MS (ES) m/z 503 [M+Na]+, 519 [M+K]+, 515 [M+Cl]-. Anal. Calc. for C18H12Br2N2S2: C, 45.02; H, 2.52; N, 5.83; S, 13.35. Found: C, 45.00; H, 2.46; N, 5.79, S, 13.26.

Crystal Data: C18H12Br2N2S2 (M = 480.24 g/mol), monoclinic, space group P21/n, a =

SC

8.1346(1) Å, b = 23.4423(4) Å, c = 9.8566(2) Å, β = 91.997(1)°, V = 1878.45(5) Å3, Z = 4, T = 130(2) K, µ(CuKα) = 7.56 mm-1, Dcalc = 1.698 g/cm3, 10440 reflections measured (7.54° ≤

M AN U

2Θ ≤ 133.15°), 3319 unique (Rint = 0.023, Rsigma = 0.019) which were used in all calculations. The final R1 = 0.026 (I > 2σ(I)) and wR2 = 0.069 (all data), number of restraints – 0

2.2.2. 2,3-Bis(biphenyl-4-ylmethylsulfanyl)maleonitrile (2)

TE D

Dimercaptomaleodinitrile disodium salt (186 mg, 1.00 mmol) and 4-bromomethylbiphenyl (593 mg, 2.40 mmol) were dissolved in anhydrous methanol (12.5 mL) and stirred under reflux for 4 h. Next, the reaction mixture was cooled, the solvent was evaporated and the

EP

resulting crude brown oil was chromatographed (hexane/ethyl acetate, 7:1, v/v) to give 2 as yellow crystals (389 mg; 82%); m.p. 140-141 °C; Rf (hexane/ethyl acetate, 7:1, v/v) = 0.4;

AC C

UV–Vis (dichloromethane): λmax, nm (log ε) 259 (4.64), 347 (4.23); FT-IR (KBr), v (cm-1): 3051, 2211, 1489, 1265, 1174, 1072, 1013, 738; 1H NMR (600.1 MHz, DMSO-d6) δ 7.63 (t, 8H, 3J = 6 Hz, ArH), 7.43 (m, 8H, ArH), 7.37 (t, 2H, 3J = 6 Hz; ArH ), 4.51 (s, 4H, CH2); 13C NMR (150.9 MHz, DMSO-d6) δ 139.6, 139.3, 134.7, 129.5, 128.9, 127.6, 126.9, 126.5, 121.7, 112.5, 37.8 (CH2); MS (ES) m/z 475 [M-H]-, 497 [M+Na]+. Anal. Calc. for C30H22N2S2: C, 75.91; H, 4.67; N, 5.90; S, 13.51. Found: C, 75.26; H, 4.63; N, 5.86; S, 14.04.

6

ACCEPTED MANUSCRIPT Crystal Data: C30H22N2S2 (M = 474.61 g/mol), monoclinic, space group P21/n, a = 14.7200(7) Å, b = 10.5650(5) Å, c = 16.0640(7) Å, β = 103.301(4)°, V = 2431.2(2) Å3, Z = 4, T = 130(2) K, µ(MoKα) = 0.24 mm-1, Dcalc = 1.297 g/cm3, 15693 reflections measured (5.76° ≤ 2Θ ≤ 51.36°), 4618 unique (Rint = 0.043, Rsigma = 0.040) which were used in all

RI PT

calculations. The final R1 = 0.038 (I > 2σ(I)) and wR2 = 0.097 (all data), number of restraints 0.

SC

2.2.3. 2,3,7,8,12,13,17,18–Octakis[4-bromobenzylsulfanyl]porphyrazinatomagnesium(II) (3)

M AN U

Magnesium turnings (20 mg, 0.83 mmol) and a small iodine crystal were refluxed for 4.5 h in n-butanol (6.5 mL). After the mixture was cooled to room temperature, 1 (400 mg, 0.83 mmol) was added, and the reaction mixture was refluxed for 23 h. After n-butanol was evaporated with toluene, a dark blue residue was dissolved in dichloromethane, Celite filtered,

TE D

chromatographed (dichloromethane/methanol, 50:1, v/v; then n-hexane/ethyl acetate, 7:1 to 7:3, v/v) to give porphyrazine 3 as a blue film (99 mg, 25%); Rf (n-hexane/ethyl acetate, 7:5, v/v) = 0.4. UV–vis (dichloromethane): λmax, nm (log ε) 226 (4.81), 377 (4.56), 668 (4.58); FT-

1

(KBr),

v

(cm-1):

2925,

EP

IR

1653,

1487,

1224,

1071,

1012,

835;

H NMR (600.1 MHz, pyridine–d5) δ 7.51 (d, 16H,3J = 8.5 Hz, C2C6, ArH), 7.35 (d, 16H,3J

AC C

= 8.5 Hz, C3C5, ArH), 5.52 (s, 16H, CH2);

13

C NMR (150.9 MHz, pyridine–d5) δ 158.2,

(pyrrole-C1C4), 141.7 (pyrrole-C2C3), 138.9 (C1, ArC), 132.4 (C3C5, ArC), 132.0 (C2C6, ArC), 121.9 (C4, ArC), 39.6 (CH2); MS (MALDI) m/z 1947 [M+Na]+. 98.94-100.00% purity by HPLC (see Supporting Information). 2.2.4.

2,3,7,8,12,13,17,18–Octakis(biphenyl-4-ylmethylsulfanyl)porphyrazinato-

magnesium(II) (4)

7

ACCEPTED MANUSCRIPT Magnesium turnings (7.0 mg, 0.29 mmol) and a small iodine crystal were refluxed for 2.5 h in n-butanol (3.0 mL). After the mixture was cooled to room temperature, 2 (127 mg, 0.27 mmol) was added and the reaction mixture was refluxed for 23 h. After n-butanol was evaporated with toluene, the dark blue residue was dissolved in dichloromethane, Celite

RI PT

filtered and chromatographed (dichloromethane/methanol, 35:1, v/v) to give porphyrazine 4 as a blue film (38 mg, 29%); Rf (dichloromethane/methanol, 50:1, v/v) = 0.47. UV–vis (dichloromethane): λmax, nm (log ε) 256 (4.85), 379 (4.45), 678 (4.55). FT-IR (KBr),

SC

v (cm-1): 3028, 2927, 1487, 1403, 1202, 1008, 739; 1H NMR (600.1 MHz, pyridine–d5) δ 7.80 (d, 16H, 3J = 6 Hz, ArH); 7.51 (d, 16H, 3J = 6 Hz, ArH); 7.47 (d, 16H, 3J = 16H, ArH); 7.32

M AN U

(t, 16H, 3J = 6 Hz, ArH); 7.28 (t, 8H, 3J = 12 Hz, ArH); 5.74 (s, 16H, 8×CH2);

13

C NMR

(150.9 MHz, pyridine–d5) δ 158.5, 141.9, 141.2, 140.7, 138.8, 130.8, 129.7, 128.1, 128.0, 127.6, 40.3; MS (MALDI) Found: [M+H]+ 1921.4823; C120H89MgN8S8 requires [M+H]+ 1921.4826. 99.06-100.00% purity by HPLC (see Supporting Information).

TE D

Crystal Data: C120H88MgN8S8·2.34(C5H5N) (M = 2107.60 g/mol): triclinic, space group P-1, a = 12.4455(3) Å, b = 14.1500(3) Å, c = 16.6838(5) Å, α = 106.259(2)°, β = 104.706(2)°, γ = 98.808(2)°, V = 2647.5(1) Å3, Z = 1, T = 130(2) K, µ(CuKα) = 2.08 mm-1, Dcalc = 1.322

EP

g/cm3, 28634 reflections measured (12.73° ≤ 2Θ ≤ 136.49°), 9652 unique (Rint = 0.038, Rsigma = 0.031) which were used in all calculations. The final R1 = 0.079 (I > 2σ(I)) and wR2 = 0.214

AC C

(all data), number of restraints - 102.

2.3. Single crystal X-ray structure determination Maleonitrile

derivatives

1,

2

and

porphyrazine

4

were

dissolved

in

dichloromethane/methanol (1:1, v/v) and pyridine respectively. Crystals suitable for X-ray analysis were obtained by slow evaporation of the solution at room temperature. All data were processed with CrystAlisPro software [47]. The structures were solved by direct methods with

8

ACCEPTED MANUSCRIPT SIR2004 [48] and refined by full matrix least-squares based on F2 with SHELXL-2014 [49]. The detailed discussion of the X-ray experimental work is given in supplementary materials. A summary of the data collection/refinement and selected torsion angles for 1, 2 and 4 are given in Tables 1S and 2S, respectively. Fractional atomic coordinates, anisotropic

RI PT

displacement parameters, the full list of bond lengths and angles in CIF format have been deposited at the Cambridge Crystallographic Data Centre with accession code CCDC 1481355-1481357 for 1, 2, and 4.

SC

2.4. Spectral and photophysical measurements

Absorption spectra of Pz 3 and 4 in different organic solvents were recorded in the range

M AN U

from 300 nm to 800 nm on a JASCO V-650 spectrophotometer. Emission measurements (steady-state fluorescence excitation and emission spectra, the excitation-emission matrix) were recorded on a Jobin Yvon-Spex Fluorolog 3-221 spectrofluorometer. Fluorescence quantum yields were calculated using two different standards: (i) quinine sulfate in 0.05 M H2SO4 as a reference for the S2 →S0 emission ( Φ F = 0.55) [50] and (ii) zinc phthalocyanine

TE D

st

(ZnPc) reference for the S1 →S0 emission ( Φ F = 0.17) [51]. The values of fluorescence st

(1 − 10 − Ast ) ( n X ) 2 Fk 2 − AX ) (nst ) st (1 − 10

∫F ∫F

X

(Eq. 1)

AC C

ΦF = Φ

st F

EP

quantum yields were calculated according to the equation below (Eq. 1):

Here, ∫FX is the area under the emission curve of the sample, ∫Fst is the area under the emission curve of the standard, AX and Ast are the absorbance of the sample and standard at the excitation wavelength, respectively. nX is the solvent refractive index for the sample, nst the solvent refractive index for the standard, Fk the constant describing the instrumental factors, including geometry and other parameters, and

Φ stF is the value of the fluorescence quantum

yield of the standard. The contour maps of the emission-excitation of 3 and 4 were obtained in

9

ACCEPTED MANUSCRIPT acetonitrile by recording the emission spectra in the 350-820 nm range, using excitation wavelengths from 300 nm to 420 nm, spaced by 5 nm intervals in the excitation domain. 2.5. Photosensitized production of singlet oxygen The Jobin Yvon-Spex Fluorolog 3-221 spectrofluorometer with the H10330B-75 NIR-

RI PT

PMT module was used to determine the quantum yield of singlet oxygen production (Ф∆) and the singlet oxygen lifetimes (τ∆) of 3 and 4. Steady-state measurements were performed using a xenon lamp as an excitation source, whereas phosphorescence decay kinetics was measured

SC

with a SpectraLed diode (λex = 371 nm or 625 nm; λem = 1270 nm). The quantum yields of singlet oxygen production in the presence of 3 and 4 in dichloromethane were calculated

M AN U

using two different standards: perinaphthenone (Ф∆ = 0.95) [52] and methylene blue (Ф∆ = 0.57) [53]. In the first step, two solutions were prepared: (i) studied porphyrazines and (ii) standard samples in the same solvent (dichloromethane), for which values of absorbance at the excitation wavelength were practically identical (with a difference of ± 0.005). Further,

TE D

the characteristic emission spectra of singlet oxygen were recorded for 3 or 4 and standard samples: perinaphthenone or methylene blue. The slight difference in absorbance for porphyrazine and standard samples at a given excitation wavelength was included in

 

EP

calculations according to the equation (Eq. 2):   

Ф = Ф       

(Eq. 2)

AC C



Here, Ф∆ is the quantum yield of singlet oxygen production by Pz 3 or 4; ФST is the same value for the reference sample (perinaphthenone or methylene blue); AreaX is the area under the characteristic singlet oxygen emission spectrum of Pz 3 or 4; AreaST is the area under the characteristic singlet oxygen emission spectrum of the reference sample; AST is the absorbance at the excitation wavelength for the standard sample; and AX is the absorbance at the excitation wavelength of Pz 3 or 4. 2.6. Biological activity in cultured cells 10

ACCEPTED MANUSCRIPT 2.6.1. Cell Culture Oral squamous cell carcinoma cell lines derived from the tongue (CAL 27, HSC-3) were purchased from ATCC (Manassas, VA, USA) (CAL 27) or provided by Dr. R. Kramer (University of California, San Francisco, UCSF, USA) (HSC-3) [54]. Human cervical

RI PT

epithelial adenocarcinoma cells (HeLa) were purchased from ATCC. Cells were cultured in Dulbecco’s modified Eagle medium (DMEM), supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), penicillin (100 U/mL), streptomycin (100 µg/mL) and L-glutamine

SC

(4 mM) (DMEM/10). Cells were incubated in tissue culture flasks at 37 °C in a humidified atmosphere containing 5% CO2, and were passaged 1:6 (HSC-3, HeLa) or 1:4 (CAL 27) twice

M AN U

a week, using a Trypsin-EDTA solution to detach the cells. All media, penicillin-streptomycin solution, L-glutamine, FBS, Trypsin-EDTA, phosphate buffered saline (PBS), Dulbecco's phosphate buffered saline (DPBS), were obtained from the UCSF Cell Culture Facility (San Francisco, CA, USA). Photosensitizers were dissolved in dimethyl sulfoxide (Sigma-Aldrich)

TE D

and subsequently diluted in DMEM (without FBS and phenol red) to obtain the desirable concentration of photosensitizer used in the experiments. The DMSO concentration in the final solution did not exceed 0.5%.

EP

2.6.2. Dark Toxicity

One day before the experiment, cells were seeded in 48-well plates at a density of 1.8×105

AC C

cells in 1 mL of medium (with FBS and phenol red), and used at approximately 80% confluence. Subsequently, cells were washed with PBS (1.0 mL) and 1 mL of medium without FBS and phenol red, containing photosensitizer at a given concentration, was added to each well, except controls. The FBS-free media were used to avoid binding of photosensitizers to serum proteins. After a 24 h incubation at 37 °C, cells were washed with PBS, 1 mL of complete medium was added to each well, and the cells were incubated for 24 h

11

ACCEPTED MANUSCRIPT at 37 °C. Cell viability was quantified by the Alamar Blue assay. Cells incubated either with medium alone or medium with 0.5% DMSO served as controls. 2.6.3. Light-dependent Toxicity One day before the experiment CAL 27, HSC-3 or HeLa cells were seeded in 48-well

RI PT

plates at a density of 1.8×105 cells per well, respectively, in 1 mL of complete medium, and used at approximately 80% confluence. Cells were washed with PBS (1.0 mL), and medium without FBS and phenol red (1.0 mL), containing photosensitizer, was added to each well,

SC

except controls. The cells were incubated for 24 h at 37 °C, washed with PBS and irradiated for 20 min with light of wavelength 690 nm from a High Power LED Multi Chip Emitter

M AN U

(Roithner Lasertechnik, 9.8V). The light intensity at the surface of the plate was set to 3.0 mW/cm2 measured by a Thorlabs TM100A Optical Power Meter and the total light dose was 3.6 J/cm2. One plate from each experiment was not exposed to light and served as a control. Directly after light exposure, the medium without FBS and phenol red was replaced with 1.0

TE D

mL of complete medium and the cells were incubated for 24 h at 37 °C. Cell viability was quantified by the Alamar Blue assay. 2.6.4. Cell Viability

EP

Cell morphology was evaluated using a Nikon TMS inverted phase contrast microscope at 100× magnification. The number of viable cells used for the experiments was determined by

AC C

the Trypan Blue exclusion assay (Gibco-Invitrogen Corporation). Cell viability was quantified by a modified Alamar Blue assay [55,56]. Briefly, 1.0 mL of 10% (v/v) Alamar Blue dye in the appropriate complete medium was added to each well. After incubation at 37 °C for 2–3 h, 200 µL of the supernatant was assayed by measuring the absorbance at 570 nm and 600 nm. Cell viability (as a percentage of control cells) was calculated according to the formula: Cell viability = [(A570 – A600) of test cells] × 100 / [(A570 – A600) of control cells] 2.6.5. Statistical Analysis

12

ACCEPTED MANUSCRIPT Statistical analyses were performed with one-way analysis of variance ANOVA followed by Dunnett's multiple comparison test using GraphPad Prism version 6.07 for Windows, GraphPad Software, San Diego California, USA. The results are presented as the means ± SD from experiments performed in triplicate. A probability value (p) of less than 0.05 was

RI PT

considered statistically significant. 2.6.6. Liposome preparation

1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), L-α-phosphatidyl-DL-glycerol

SC

(chicken egg, PG) and 1,2-dioleoyl-3-trimethylammoniumpropane (chloride salt, DOTAP) were purchased from Avanti Polar Lipids Inc. Two different liposome formulations were

M AN U

prepared by a thin-film hydration method [57,58]. Appropriate amounts of the lipid stock solutions in chloroform (POPC – 25 mg/mL, PG – 25 mg/mL, DOTAP – 10 mg/mL) and photosensitizer (0.4 mg/mL) were placed in glass tubes, mixed and evaporated to dryness using a rotary evaporator. Films formed on the bottom of the glass tubes were dried overnight

TE D

in a vacuum oven at room temperature to evaporate any remaining chloroform. Subsequently, the dried films were hydrated with HEPES buffered saline (10 mM HEPES (N-(2Hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid)), 140 mM NaCl, pH = 7.5) and dispersed

EP

by vortexing for 5–10 min. The resulting liposome suspensions were passed 21 times through polycarbonate membranes with a pore diameter of 100 nm, using a syringe extruder (Avanti

AC C

Polar Lipids) to obtain unilamellar liposomes with a uniform size distribution. The molar ratios of ingredients in final liposome formulations were: (i) Pzs 3, 4 (0.1), PG (2), POPC (8); (ii) Pzs 3, 4 (0.1), DOTAP (2), POPC (8). The liposome size was determined by dynamic light scattering measurements using a Coulter N4 Plus Particle Size Analyzer (Beckman) (Table 10S in Supporting Information). Samples were stored at 2–8 °C under argon and were protected from light. The final concentration of photosensitizer achieved in the liposome suspensions was 100 µM. These suspensions were diluted with DME medium without FBS to

13

ACCEPTED MANUSCRIPT achieve the appropriate concentration for biological activity evaluation on HSC-3 cells. Free liposomes without photosensitizers were prepared as controls.

3. Results and Discussion

RI PT

3.1. Synthesis and characterization

The alkylation reaction of dimercaptomaleonitrile disodium salt with 4-bromobenzyl and 4-biphenylylmethyl bromides led to novel maleonitriles 1 and 2, respectively. Next, the

SC

obtained compounds were subjected to the Linstead macrocyclization reactions using magnesium n-butanolate in n-butanol to give porphyrazines 3 and 4 with 4-

EP

TE D

M AN U

bromobenzylsulfanyl and 4-biphenylylmethylsulfanyl substituents, respectively (Scheme 1).

Scheme 1. Synthesis of compounds 1–4. Reagents and conditions: (i) CH3OH, reflux, 4 h; (ii)

AC C

Mg(O-nC4H9)2, nC4H9OH, reflux, 23 h. Synthesized compounds were characterized using UV-Vis, MS and NMR techniques (see Supporting Information). In addition, the structures of maleonitriles 1 and 2 as well as porphyrazine 4 were determined by X-ray crystallography. Both maleonitriles crystallize in the P21/n space group with one molecule in the asymmetric unit (Fig. 1). In the crystals, the molecules of 1 and 2 assume asymmetric conformations (Table 2S). In 2, the biphenyl substituents that are slightly twisted about their central C-C bond are located at the same side of the maleonitrile group forming a U-shaped molecule (Fig. 1b). Inversion-center related 14

ACCEPTED MANUSCRIPT molecules of 2 pack into columns extended along the b axis (Fig. 2a). Columns related by translation along the [101] direction form (-1 0 1) layers. Biphenyl substituents are located inside the layer, whereas maleonitrile fragments with the attached S atoms form the layer

TE D

M AN U

SC

RI PT

surfaces (Fig 2b).

Fig. 1. The asymmetric unit of (a) 1 and (b) 2 showing the labeling scheme. The thermal

AC C

EP

ellipsoids are drawn at the 50% probability level.

15

SC

RI PT

ACCEPTED MANUSCRIPT

M AN U

Fig. 2. The assembly mode of 2 in crystals: (a) columns; (b) (-101) layers. H atoms are omitted for clarity.

The porphyrazine 4 crystallizes in the P-1 space group and the asymmetric unit consists of one half of the Mg-Pz complex as well as 1.17 coordinating (0.5) and solvent (0.67) pyridine

TE D

molecules. The Mg(II) ion is disordered over two equally occupied positions related by an inversion center. It is coordinated by four isoindoline nitrogen atoms of the Pz core and one N

EP

atom of the pyridine ligand and exhibits distorted square-pyramidal coordination geometry (Fig. 3). The N−Mg distances are 2.122(4) and 2.150(4) Å for the coordinating Pz ion and

AC C

2.14(1) and 2.16(1) Å for disordered pyridine ligands. The Mg2+ cation is displaced by 0.818(5) Å from the best plane of the 24-atom Pz core. This is the largest shift found in PzMg complexes. A survey of the Cambridge Structural Database [59] resulted in 15 Pz-Mg structures with shifts ranging from 0.429 to 0.790 Å. In 4, the peripheral biphenyl substituents of the B unit are located at the same side of the isoindoline ring, whereas in the A unit they are alternatively oriented relative to the ring (Fig. 3). This, in connection with the presence of an inversion center in the middle of the Pz ion, results in four adjacent biphenyls at the Pz-core being directed upwards and the remaining 16

ACCEPTED MANUSCRIPT four downwards from the Pz planar fragment. In the crystal, the Pz-Mg molecules are arranged into columns extended along the a-axis (Fig. 4). The analysis of disorder shows that within one column, the Mg ion is always located at the same side of the Pz-core. The distance between two neighboring 24-atom Pz planes is 12.26 Å (centroid-centroid distance of 12.47

RI PT

Å). The biphenyl residues from two consecutive Pz molecules along the column form the barrel-like cages with the Pz core constituting the bottom and the top of the barrel, and the

EP

TE D

M AN U

SC

pyridine molecules enclosed inside the barrel (Fig 5).

AC C

Fig. 3. The molecule of 4. The units A and B of Pz are marked with different shades of grey. Pyridine C atoms are shown in red. H atoms are omitted for clarity.

17

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

Fig. 4. The column formed by Pz-Mg molecules in 4. The neighboring molecules are drawn in different shades of grey.

18

RI PT

ACCEPTED MANUSCRIPT

SC

Fig. 5. The space-filling representation of the barrel formed by two neighboring molecules of 4, viewed along the column direction. The Pz rings from the bottom and top of the barrel are

M AN U

removed. Some of the pyridine molecules enclosed in the barrel are shown in ball and stick representation with C atoms drawn in red.

3.2. Spectral and photophysical studies

TE D

3.2.1. Absorption and emission properties

Solvents with different properties were chosen for spectral and photophysical studies. The absorption spectra of Pz 3 and 4 in selected organic solvents are shown in Fig. 6. Table 3S

EP

included in the Supplementary Information presents the spectral and photophysical data for the singlet states, including the absorption maxima of the Soret- and Q-bands (λ2 and λ1). The

AC C

structural differences between Pz 3 and 4 and the applied solvent affected the maxima positions of the Soret- and Q-bands in the UV–Vis spectra. In the case of Pz 3, for the majority of the solvents, the maxima of the Soret bands were found between 373 nm and 383 nm, whereas for Pz 4 they were between 377 nm and 385 nm. Note that in the same solvent both the Soret band and the Q-band maxima of Pz 4 appeared at longer wavelengths compared to Pz 3. Protic or aprotic properties of solvents did not affect significantly the Q-

19

ACCEPTED MANUSCRIPT band maxima localization (Table 3S in Supplementary Information). On the contrary

TE D

M AN U

SC

RI PT

chlorinated solvents red-shifted both Q- and Soret-bands compared to the other solvents used.

EP

Fig. 6. Absorption spectra of 3 (A) and 4 (B) in selected organic solvents. Pz 3 and 4 had interesting fluorescence properties in organic solvents. Fig. 7 presents the

AC C

emission spectra recorded at different excitation wavelengths (λex = 380 nm and 610 nm) in dichloromethane and acetonitrile. The S2 → S0 (λF2) and S1 → S0 (λF1) fluorescence emission maxima are included in Table 3S (Supporting Information). Low fluorescence intensity was observed for Pz 3 and 4, independently of the solvents and derivatives used. When the excitation wavelength was 380 nm, two emission bands were observed in all solvents for both Pz 3 and 4. The short-wavelength, broad bands appeared with their maxima between 475530 nm for Pz 3 and 500-523 nm for Pz 4, whereas the narrow long-wavelength bands were located with their maxima between 685-694 nm (Pz 3) and 690-707 nm (Pz 4). The short20

ACCEPTED MANUSCRIPT wavelength emission bands were correlated to the S2 → S0 and the long-wavelength emission bands to the S1 → S0 radiative transitions. However, it is worth pointing out that Pz 3 and 4 had relatively low intensity short-wavelength emission bands in protic solvents (alcohols). For example it was impossible to observe the S2 → S0 emissions in methanol and ethanol, while in

RI PT

other alcohols the maxima of the S2 → S0 emissions could be estimated. However, when the 610 nm excitation was used, a single long-wavelength emission band was observed at the same wavelength as that obtained for 380 nm excitation. The calculated fluorescence quantum

SC

yield values (ФF) for the S2 → S0 and S1 → S0 transitions indicated that fluorescence is not a primary channel of deactivation of the singlet excited state for both porphyrazines. Typically,

M AN U

the ФF values for the S2 → S0 transition were lower than that for the S1 → S0 transition in all of the solvents used. The S2 → S0 emission was rather weak in all of the solvents, with quantum yields of about 10-5. The highest values for ФF (S2 → S0) were measured in DMSO for Pz 3 and in dichloromethane for Pz 4. The lowest ФF (S2 → S0) were obtained for Pz 3 and

TE D

4 in acetonitrile and 1-pentanol, respectively. The S1 → S0 emissions had quantum yields of about 10-4 for both porphyrazines in all solvents. The highest ФF (S1 → S0) were obtained for Pz 3 and 4 in DMF and in acetone, respectively. The lowest ФF (S1 → S0) were obtained for

AC C

EP

both porphyrazines in 2-propanol (Table 3S in Supporting Information).

21

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Fig. 7. Emission spectra of Pz 3 (A) and 4 (B) (λex = 380 nm and 610 nm) in acetonitrile or

TE D

dichloromethane.

Moreover, the excitation spectra of Pz 3 and 4 were recorded in all solvents. This approach aimed to confirm that the short-wavelength emission bands in the fluorescence spectra

EP

originated from higher singlet exited states, and not from the impurities. Fig. 2S (Supporting Information) includes the absorption and excitation spectra (λem = 550 nm and 740 nm) of Pz

AC C

3 and 4 in acetonitrile, with similar results obtained in other solvents. For both compounds, a satisfactory correspondence between the Soret band absorption spectra and the excitation spectra was observed, irrespective of the emission wavelength. This observation confirmed that the short-wavelength emission band is generated by the radiative electronic transitions between the S2 and S0 states, independently of the solvent used (Fig. 7). Monitoring the emission at 740 nm, a satisfactory correlation was observed for the Q-bands of Pz 3 and 4 (Fig. 2S in Supporting Information).

22

ACCEPTED MANUSCRIPT As a complement to conventional fluorescence measurements discussed above, the excitation-emission maps were recorded for Pz 3 and 4 in acetonitrile (Fig. 5S in Supporting Information). The excitation-emission maps showed more details of the fluorescence of Pz 3 and 4 compared to classical fluorescence spectra, and may be used as a "fingerprint" for

RI PT

molecules in biological systems. In addition, the excitation-emission maps revealed the emission region of compounds as a function of the excitation wavelength. The excitationemission maps presented in Fig. 5S in the Supporting Information were all recorded in the

SC

same way. The samples were excited between 300-420 nm and the emission was monitored between 350-820 nm. For each of the compounds, two distinct emission regions were found at

M AN U

350-580 nm and 650-820, with results being consistent with those of the emission study of Pz 3 and 4 in acetonitrile presented in Fig. 7.

3.2.2. Singlet oxygen generation measurements

The photophysical process between the excited photosensitizer and oxygen to generate

TE D

singlet oxygen is the main mechanism responsible for the photocytotoxic effect in photodynamic therapy [40]. Thus, the potential efficacy of Pz 3 and 4 in PDT was assessed by measuring the quantum yield of singlet oxygen generation. The spectroscopic method

EP

employing the characteristic luminescence of singlet oxygen in the NIR spectral range was used. The emission maximum at about 1270 nm is highly specific for the transition of singlet

AC C

oxygen (1∆g) to the ground-state oxygen (triplet state: 3Σg-). The measurements were performed in dichloromethane using both steady-state and time-resolved approaches. Porphyrazines 3 and 4 were excited with light at two different wavelengths 371 and 660 nm. Perinaphthenone was used as a reference for the 371 nm excitation, whereas methylene blue was used at 660 nm. The characteristic singlet oxygen phosphorescence spectra produced in the presence of Pz 3 and 4 are shown in Fig. 3S (Supporting Information), and the quantum yield values of singlet oxygen production (Ф∆) are presented in Table 1. The calculated Ф∆

23

ACCEPTED MANUSCRIPT values were almost identical for 371 nm and 660 nm excitation, for each of the porphyrazines wavelengths. Pz 3 was a better photosensitizer for singlet oxygen generation than Pz 4 with the Ф∆ values of about 0.06 and 0.03 for Pz 3 and Pz 4, respectively. The linear correlations between the emission areas and the absorbances proved the absence of singlet oxygen

RI PT

quenching by either Pz 3 or Pz 4 in their ground states (Fig. 4S in Supporting Information). Additionally, time-resolved measurements indicated singlet oxygen lifetimes τ∆ at λex = 371 nm or 625 nm, with λem = 1270 nm. Porphyrazine derivatives and reference samples were

SC

studied in air-equilibrated dichloromethane solutions with mono-exponential phosphorescence decays. The resulting singlet oxygen lifetimes were very similar for both porphyrazines. The

M AN U

τ∆ values at λex = 371 and 625 nm were 82 µs and 81 µs for Pz 3 and 83 µs and 84 µs for Pz 4, respectively (Table 1).

Table 1. Quantum yields of singlet oxygen photosensitized production (Ф∆) and the singlet

Compound

TE D

oxygen lifetimes (τ∆) for Pz 3 and 4 in dichloromethane

3

λex = 371 nm

0.06 0.03

EP

λem = 1270 nm

4

Ф∆

0.05 0.02

AC C

λex = 660 nm

λem = 1270 nm λex = 371 nm

λem = 1270 nm

82

83

81

84

τ∆ [µs]

λex = 625 nm λem = 1270 nm

3.3. Biological activity in cultured cells 3.3.1. Cytotoxic activity 24

ACCEPTED MANUSCRIPT The photodynamic activity of Pz 3 and 4 was investigated in vitro using two oral squamous cell carcinoma cell lines derived from the tongue (CAL 27, HSC-3) and human cervical epithelial adenocarcinoma cells (HeLa). Dark toxicity of Pz 3, 4 was evaluated within the limited concentration range of 0.1 to 10.0 µM because of the aggregation of photosensitizers

RI PT

observed at higher concentrations. There was no significant dark toxicity found on CAL 27 and HSC-3 cell lines. Pz 3 caused a significant decrease in the HeLa cells viability by almost 20% at 10.0 µM. Pz 3 at lower concentrations and Pz 4 did not cause significant cytotoxicity

SC

to HeLa cells (Fig. 8 and 9, Tables 4S-6S in Supporting Information). The light-induced toxicity was examined at the same concentrations as the dark toxicity (0.1, 1.0 and 10.0 µM).

M AN U

The cells were incubated for 24 h in a medium containing either dissolved Pz 3 or 4 at the given concentrations and subsequently irradiated for 20 min with 690 nm light from a High Power LED Multi Chip Emitter (Roithner Lasertechnik). Cell viability was quantified by the Alamar Blue assay. Neither of the compounds demonstrated any significant light-induced

TE D

cytotoxicity on any of the cells tested (Fig.8 and 9, Tables 4S-6S in Supporting Information). 3.3.2. Liposomal formulations and their in vitro photodynamic activity The photocytotoxicity results obtained for Pz 3 or 4 prompted us to develop a drug

EP

delivery system. Liposomal nanoparticles have been used already as a drug delivery system for photosensitizers, enhancing their solubility in water and cell uptake [31]. Two different

AC C

liposome formulations were prepared by a thin-film hydration method: (i) negatively charged liposomes composed of L-α-phosphatidyl-DL-glycerol (PG) and 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine (POPC) and (ii) positively charged liposomes containing 1,2dioleoyl-3-trimethylammonium-propane (chloride salt, DOTAP) and POPC [57,58]. All liposomes were extruded through polycarbonate membranes to produce a uniform size distribution. The mean diameter of the extruded liposomes containing Pz 3 varied from 0.23 to 0.24 µm and with Pz 4 from 0.29 to 0.30 µm, respectively (Table 8S in Supporting

25

ACCEPTED MANUSCRIPT Information). Cytotoxicities of the liposomes with incorporated Pz 3 and 4 against cancer cells (CAL27, HSC-3 and HeLa) were examined at the same concentrations as those of the free photosensitizers in solution (0.1, 1.0 and 10.0 µM). None of the liposomes showed any toxicity without exposure to light (Fig. 8 and 9, Tables 7S-9S in Supporting Information).

RI PT

Positively charged liposomes composed of Pz 3, DOTAP and POPC at 10.0 µM produced photocytotoxicities of 93% for CAL 27 and HSC-3 cells, and 87% for HeLa cells. At 1.0 µM concentration the viability of CAL 27 was reduced by 47% and HSC-3 by 36%, but there was

SC

no significant change in the viability of HeLa cells. Finally, at the lowest concentration (0.1 µM) the photocytotoxic effect of Pz 3 was observed only for HSC-3 cells with a reduction of

M AN U

cell viability by 30%. The negatively charged liposomes consisting of Pz 3 : PG : POPC were less active and showed photocytotoxicity only at 10.0 µM against HSC-3 cells (Fig. 8). Liposomes containing Pz 4 revealed a generally lower photosensitizing efficiency. Cationic liposomes composed of Pz 4 : DOTAP : POPC decreased the viability of HSC-3 cells by 18%

TE D

and 30% at 1.0 and 10.0 µM concentrations, respectively, but the viabilities of CAL 27 and HeLa cells were unaffected. Negatively charged Pz 4 : PG : POPC liposomes had no significant viability effects on any of the cells (Fig. 9). Noteworthy is that a higher sensitivity

EP

of HSC-3 as compared to CAL 27 cells to photodynamic treatment was also observed in our previous studies [35]. In addition, the higher photosensitizing activity of Pz 3 and 4 in cationic

AC C

liposomes based on DOTAP and POPC than in PG : POPC liposomes, corresponds well with the results presented by Altieri et al. [60]. They reported on a symmetrical sulfanyl porphyrazine with peripheral carboranyl substituents inserted into cationic, anionic and zwitterionic liposomes. They found that cationic liposomes containing DOTAP were taken up by rat colon carcinoma (DHD/K12/TRb) and murine melanoma cells (B16-F10) more efficiently than anionic or zwitterionic liposomes. These authors suggest that this could be due to electrostatic interactions of positively charged cationic liposomes with the negatively

26

ACCEPTED MANUSCRIPT charged surface of mammalian plasma membranes. However, these data contradict our previous study on liposomal formulations containing tribenzoporphyrazine with an annulated diazepine ring. The encapsulation of this photosensitizer into negatively charged liposomes resulted in almost three times higher photocytotoxicity as compared to positively charged

RI PT

ones [61]. These observations suggest that the effectiveness of liposomes as a drug delivery

TE D

M AN U

SC

system is strongly dependent upon the structure of incorporated compounds.

EP

Fig. 8. Light toxicity (and dark control) of Pz 3 against: a) CAL 27, b) HSC-3, c) HeLa cells. Data represent the mean ± standard deviation obtained from experiments performed in

AC C

triplicate. Statistical significance is indicated with asterisks: *p<0.050, **p<0.010, ***p<0.001.

27

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Fig. 9. Light toxicity (and dark control) of Pz 4 against: a) CAL 27, b) HSC-3, c) HeLa cells. Data represent the mean ± standard deviation obtained from experiments performed in triplicate. Statistical significance is indicated with asterisks: *p<0.050, **p<0.010,

4. Conclusions

TE D

***p<0.001.

EP

Novel sulfanyl porphyrazines with peripheral 4-bromobenzyl and 4-biphenylylmethyl substituents were synthesized and characterized using UV-Vis spectrophotometry, mass

AC C

spectrometry and NMR spectroscopy. The X-ray crystallography revealed barrel-like selfassemblies in crystals of porphyrazine with 4-biphenylylmethyl substituents. Photophysical studies of the obtained porhyrazines indicated two interesting aspects: (i) relationship between the solvent properties and the spectral and photophysical parameters of the tested porphyrazines and (ii) relationship between the peripheral group structure and the absorption and fluorescence properties of the molecules. Moreover, the excitation-emission maps showed the emission range of the studied compounds in function of the excitation wavelengths. The

28

ACCEPTED MANUSCRIPT potential photosensitizing efficiency of novel sulfanyl porphyrazines was evaluated by measuring singlet oxygen generation quantum yields using a spectroscopic method based on the light emission at about 1270 nm, specific for the transition of the singlet oxygen to the ground-state oxygen. We found that porphyrazine with peripheral 4-bromobenzylsulfanyl

biphenylylmethylsulfanyl

substituents.

Examination

of

RI PT

groups is a better photosensitizer of singlet oxygen than its analog with 4the

cytotoxicity

of

novel

porphyrazines was performed on CAL 27, HSC-3 and HeLa cells. Neither compound free in

SC

solution showed any significant light-induced cytotoxicity on the cells. However, delivery of the porphyrazine containing 4-bromphenylsulfanyl groups in positively charged liposomes

M AN U

caused significant photocytotoxicity on all the tested cell lines, with a reduction of cell viability exceeding 85% at 10.0 µM concentration of the compound. Negatively charged liposomes were less effective for this porphyrazine and showed low photocytotoxicity only against HSC-3 cells. Our results indicate high activity of sulfanyl porphyrazines after their

TE D

incorporation in cationic liposomes, in contrast to the lack of activity of the free form porphyrazines in solution. Thus cationic liposomes composed of 1,2-dioleoyl-3trimethylammonium-propane

and

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

EP

constitute a promising drug delivery system for sulfanyl porphyrazines.

AC C

Acknowledgements

This study was supported by the Polish National Science Centre under grant No. 2012/05/N/NZ7/00624 and funds from the University of the Pacific, Arthur A. Dugoni School of Dentistry. Jaroslaw Piskorz was a scholarship holder within the project of Polish National Science Centre “Etiuda” for Ph.D. students under fellowship 2013/08/T/NZ7/00241. The authors thank Beata Kwiatkowska, Rita Kuba, Jennifer Cheung and Ryan Yu for excellent technical assistance.

29

ACCEPTED MANUSCRIPT

Appendix A. Supplementary data Supplementary data associated with this article can be found in the online version, at

RI PT

doi:...........

References

[1] Piskorz J, Skupin P, Lijewski S, Korpusinski M, Sciepura M, Konopka K, Sobiak S,

SC

Goslinski T, Mielcarek J. Synthesis, physical-chemical properties and in vitro photodynamic activity against oral cancer cells of novel porphyrazines possessing fluoroalkylthio and

M AN U

dietherthio substituents. J Fluor Chem 2012;135:265-71.

[2] Kabay N, Söyleyici S, Gök Y. The synthesis and characterization of novel metalloporphyrazine containing crown ether linked calix[4]arene moieties. Inorg Chem Commun 2009;12:304-7.

TE D

[3] Bilgin A, Ertem B. Synthesis and characterization of novel supercryptands-fused porphyrazines. Inorg Chem Commun 2008;11:1113-6. [4] Sholto A, Lee S, Hoffman BM, Barrett AGM, Ehrenberg B. Spectroscopy, binding to

EP

liposomes and production of singlet oxygen by porphyrazines with modularly variable water solubility. Photochem Photobiol 2008;84:764-73.

AC C

[5] Kandaz M, Özkaya AR, Koca A, Salih B. Water and alcohol-soluble octakismetalloporphyrazines bearing sulfanyl polyetherol substituents: Synthesis, spectroscopy and electrochemistry. Dyes Pigm 2007;74:483-9. [6] Gonca E, Highly symmetrical polyfluorinated porphyrazines. J Fluorine Chem 2013;149:65-71. [7] Keskin B, Köseoğlu Y, Avcıata U, Gül A. Synthesis and EPR studies of porphyrazines with bulky substituents. Polyhedron 2008;27:1155-60.

30

ACCEPTED MANUSCRIPT [8] Prasad R, Kumar A. Spectroscopic and electrochemical investigation of coordination complexes of octakis(benzylthio)tetraazaporphyrincobalt(II). Inorganica Chimica Acta 2005;358:3201-10. [9] Şimşek T, Gonca E. Enrichment of solubility by esterification: Metal-free and metallo-

RI PT

porphyrazines with polyfluorinated units. Transit Met Chem 2013;38:37-43.

[10] Kunt H Gonca E. Synthesis and characterization of novel metal-free and metalloporphyrazines with eight 3-thiopropylpentafluorobenzoate units. Polyhedron 2012;38:218-23.

SC

[11] Tuncer S, Koca A, Gül A, Avcıata U. Synthesis, characterization, electrochemistry and

Dyes Pigm 2011;92:610-8.

M AN U

spectroelectrochemistry of novel soluble porphyrazines bearing unsaturated functional groups.

[12] Belviso S, Ricciardi G, Lelj F. Inter-ring interactions and peripheral tail effects on the discotic mesomorphism of ‘free-base’ and Co(II), Ni(II) and Cu(II) alkenyl(sulfanyl) porphyrazines. J Mater Chem 2000;10:297-304.

TE D

[13] Ristori S, Salvati A, Martini G, Spalla O, Pietrangeli D, Rosa A, Ricciardi G. Synthesis and liposome insertion of a new poly(carboranylalkylthio)porphyrazine to improve potentiality in multiple-approach cancer therapy. J Am Chem Soc 2007;129:2728-9.

EP

[14] Pietrangeli D, Ricciardi G. Neutral and polyanionic carboranylporphyrazines: Synthesis and physico-chemical properties Appl Rad Isotopes 2009;67:97-100.

AC C

[15] Karadeniz H, Gök Y. The synthesis and characterization of novel porphyrazines containing 5H-dibenz[b,f]azepine units. Dyes Pigm 2008;77:351-6. [16] Leng F, Wang X, Jin L, Yin B. Synthesis, characterization and electrochemistry of the novel metalloporphyrazines annulated with tetrathiafulvalene having pentoxycarbonyl substituents. Dyes Pigm 2010;87:89-94.

31

ACCEPTED MANUSCRIPT [17] Gonca E, Gül A. Magnesium porphyrazinate with eight triphenylphosphonium moieties attached through (2-sulfanyl-ethoxycarbonyl-2-propyl) bridges. Inorg Chem Commun 2005;8:343-6.

Porphyr Phthalocya 2004;8:1129-65.

RI PT

[18] Rodríguez-Morgade MS, Stuzhin PA. The chemistry of porphyrazines: an overview. J

[19] Michel SLJ, Hoffman BM, Baum SM, Barrett AGM. Progress in Inorganic Chemistry. New York: John Wiley & Sons; 2000, 473-590.

SC

[20] Dubinina TV, Borisova NE, Sedova MV, Tomilova LG, Furuyama T, Kobayashi N. Synthesis and spectral properties of nonclassical binuclear thienoporphyrazines. Dyes Pigm

M AN U

2015;117:1-6.

[21] Keskin B, Altindal A, Avciata U, Gül A. A.c. and d.c. conduction processes in octakis[(4-tert-butylbenzylthio)-porphyrazinato]Cu(II) thin films with gold electrodes. Bull Mater Sci 2014;37:461-8.

TE D

[22] Fuchter MJ, Zhong C, Zong H, Hoffman BM, Barrett AGM. Porphyrazines: Designer macrocycles by peripheral substituent change. Aust J Chem 2008;61:235-55. [23] Sun P, Zong H, Salaita K, Ketter JB, Barrett AGM, Hoffman BM, Mirkin CA. Probing

EP

surface-porphyrazine reduction potentials by molecular design. J Phys Chem B 2006;110:18151-3.

AC C

[24] Vesper BJ, Salaita K, Zong H, Mirkin CA, Barrett AGM, Hoffman BM. Surface-bound porphyrazines: Controlling reduction potentials of self-assembled monolayers through molecular proximity/orientation to a metal surface. J Am Chem Soc 2004;126:16653-8. [25] Pietrangeli D, Rosa A, Ristori S, Salvati A, Altieri S, Ricciardi G. Carboranylporphyrazines and derivatives for boron neutron capture therapy: From synthesis to in vitro tests. Coord Chem Rev 2013;257:2213-31.

32

ACCEPTED MANUSCRIPT [26] Lee S, Vesper BJ, Zong H, Hammer ND, Elseth KM, Barrett AGM, Radosevich JA. Synthesis and biological analysis of thiotetra(ethylene glycol) monomethyl etherfunctionalized porphyrazines: Cellular uptake and toxicity studies. Met Based Drugs 2008;2008:1-13.

RI PT

[27] Koca A, Gonca E, Gül A. Voltammetric and spectroelectrochemical characterization of porphyrazines: Electrochemical metal sensor. J Electroanal Chem 2008;612:231-40. [28] Bonosi F, Ricciardi G, Lelj F, Martini G. J Phys Chem 1994;98:10613-20.

SC

[29] Hammer ND, Lee S, Vesper BJ, Elseth KM, Hoffman BM, Barrett AGM, Radosevich

Med Chem 2005;48:8125-33.

M AN U

JA. Charge dependence of cellular uptake and selective antitumor activity of porphyrazines. J

[30] Vesper BJ, Lee S, Hammer ND, Elseth KM, Barrett AGM, Hoffman BM, Radosevich JA. Developing a structure–function relationship for anionic porphyrazines exhibiting selective anti-tumor activity. J Photochem Photobiol B 2006;82:180-6.

TE D

[31] Skupin-Mrugalska P, Piskorz J, Goslinski T, Mielcarek J, Konopka K, Düzgüneş N. Current status of liposomal porphyrinoid photosensitizers, Drug Discov Today 2013;18:77684.

EP

[32] Lijewski S, Piskorz J, Kucinska M, Wierzchowski M, Czerniak K, Billert H, Murias M, Mielcarek M, Goslinski T. Synthesis, characterization, photochemical properties and

AC C

cytotoxicity of the novel porphyrazine functionalized with nitroimidazolylbutylsulfanyl groups. Inorg Chem Commun 2013;29:97-100. [33] Gierszewski M, Falkowski M, Sobotta L, Stolarska M, Popenda L, Lijewski S, Wicher B, Burdzinski G, Karolczak J, Jurga S, Gdaniec M, Tykarska E, Sikorski M, Mielcarek J, Goslinski T. Porphyrazines with peripheral isophthaloxyalkylsulfanyl substituents and their optical properties. J Photochem Photobiol A 2015;307-308:54-67. [34]

Rebis T, Lijewski S, Nowicka J, Popenda L, Sobotta L, Jurga S, Mielcarek J,

33

ACCEPTED MANUSCRIPT Milczarek G, Goslinski T. Electrochemical properties of metallated porphyrazines possessing isophthaloxybutylsulfanyl substituents: Application in the electrocatalytic oxidation of hydrazine. Electrochim Acta 2015;168:216-24. [35] Mlynarczyk DT, Lijewski S, Falkowski M, Piskorz J, Szczolko W, Sobotta L, Stolarska

RI PT

M, Popenda L, Jurga S, Konopka K, Düzgüneş N, Mielcarek J. Goslinski T. Dendrimeric sulfanyl porphyrazines: Synthesis, physico-chemical characterization, and biological activity for potential applications in photodynamic therapy. ChemPlusChem 2016;81:460-70.

SC

[36] Wang Z, Hong X, Zong S, Tang C, Cui Y, Zheng Q. BODIPY-doped silica nanoparticles with reduced dye leakage and enhanced singlet oxygen generation. Sci Rep 2015;5:12602.

M AN U

[37] Gorman A, Killoran J, O’Shea C, Kenna T, Gallagher WM, O’Shea DF. In vitro demonstration of the heavy-atom effect for photodynamic therapy. J Am Chem Soc 2004;126:10619-31.

[38] Wu Y, Gu D, Gan F. Bromine and alkoxyl substituted copper phthalocyanine derivative

TE D

thin films studied with spectroscopic ellipsometry, Opt Mater 2003;24:477-82. [39] Özçeşmeci I. Synthesis and fluorescence properties of phthalocyanines with dibromoand tribromo-phenoxy functionalities, Synthetic Met 2013;176:128-33.

EP

[40] Plaetzer K, Krammer B, Berlanda J, Berr F, Kiesslich T. Photophysics and

68.

AC C

photochemistry of photodynamic therapy: fundamental aspects. Lasers Med Sci 2009;24:259-

[41] Wang J, Cooper G, Tulumello D, Hitchcock AP. Inner shell excitation spectroscopy of biphenyl and substituted biphenyls: probing ring-ring delocalization. J. Phys. Chem. A 2005;109:10886-96 [42] Zhang L, Ye M, Zheng L, Li H, Li Y, Fan X. Twist effects on the electronic and photophysical characters of biphenyl and 4,4-bis(phenylsulfonyl) biphenyl. Comput Theor Chem 2016;1085:1-6.

34

ACCEPTED MANUSCRIPT [43] Zharnikov M, Grunze M. Spectroscopic characterization of thiol-derived self-assembling monolayers. J Phys Condens Matter 2001;13:11333-65. [44] Frey S, Stadler V, Heister K, Eck W, Zharnikov M, Grunze M. Structure of thioaromatic self-assembled monolayers on gold and silver, Langmuir 2001;17:2408-215.

RI PT

[45] Gonca E, Baklaci UG, Dinçer HA. Synthesis and spectral properties of novel secoporphyrazines with eight 4-biphenyl groups. Polyhedron 2008;27:2431-5

[46] Szczolko W, Wzgarda A, Koczorowski T, Wicher B, Sobotta L, Gdaniec Z, Gdaniec M,

SC

Mielcarek J, Tykarska E, Goslinski T. The Suzuki cross-coupling reaction for the synthesis of

Polyhedron 2015;102:462-8.

M AN U

porphyrazine possessing bulky 2,5-(biphenyl-4-yl)pyrrol-1-yl substituents in the periphery,

[47] CrysAlis PRO. Agilent Technologies Ltd, Yarnton, Oxfordshire, England. [48] Burla MC, Caliandro R, Camalli M, Carrozzini B, Cascarano GL, De Caro L, Giacovazzo C, Polidori G, Spagna R SIR2004: an improved tool for crystal structure

TE D

determination and refinement. J Appl Cryst 2005;38:381-8.

[49] Sheldrick GM. Crystal Structure Refinement with SHELXL. Acta Cryst 2015;C71:3-8. [50] Krystkowiak E, Dobek K, Maciejewski A. An intermolecular hydrogen-bonding effect

EP

on spectral and photophysical properties of 6-aminocoumarin in protic solvents. Photochem Photobiol Sci 2013;12:446-55.

AC C

[51] Ogunsipe A, Maree D, Nyokong T. Solvents effects on the photochemical and fluorescence properties of zinc phthalocyanine derivatives. J Mol Struct 2003;650:131-40. [52]

Schmidt R, Tanielian C, Dunsbach R, Wolff C. Phenalenone, a universal reference

compound for the determination of quantum yields of singlet oxygen O2 (1∆g) sensitization. J Photochem Photobiol A 1994;79:11-7. [53]

Chin K, Trevithick-Sutton C, McCallum J, Jockusch S, Turro NJ, Scaiano JC, Foote

CS, Garcia-Garibay MA. Quantitative determination of singlet oxygen generated by excited

35

ACCEPTED MANUSCRIPT state aromatic amino acids, proteins, and immunoglobulins. J Am Chem Soc 2008;130:69123. [54]

Matsumoto K, Horikoshi M, Rikimaru K, Enomoto S. A study of an in vitro model for

invasion of oral squamous cell carcinoma. J Oral Pathol Med 1989;18:498-501. Fields

RD,

Lancaster

MV.

Dual-attribute

continuous

proliferation/cytotoxicity. Am Biotechnol Lab 1993;11:48-50. [56]

monitoring

of

RI PT

[55]

cell

Konopka K, Pretzer E, Felgner PL, Düzgüneş N. Human immunodeficiency virus

SC

type-1 (HIV-1) infection increases the sensitivity of macrophages and THP-1 cells to cytotoxicity by cationic liposomes. Biochim Biophy Acta 1996;1312:186-96.

M AN U

[57] Düzgüneş N. Preparation and quantitation of small unilamellar liposomes and large unilamellar reverse-phase evaporation liposomes. Methods Enzymol 2003;367:23-7. [58] Kryjewski, M.; Tykarska, E.; Rebis, T.; Dlugaszewska, J.; Ratajczak, M.; Teubert, A.; Gapinski, J.; Patkowski, A.; Piskorz, J.; Milczarek, M.; Gdaniec, M.; Goslinski, T.;

TE D

Mielcarek, J. Polyhedron 2015;98:217-223.

[59] Groom CR, Allen FH. The Cambridge Structural Database in retrospect and prospect.

EP

Angew Chem Int Ed 2014;53:662-71.

[60] Altieri S, Balzi M, Bortolussi S, Bruschi P, Ciani L, Clerici AM, Faraoni P, Ferrari C,

AC C

Gadan MA, Panza L, Pietrangeli D, Ricciardi G, Ristori S. Carborane derivatives loaded into liposomes as efficient delivery systems for boron neutron capture therapy. J Med Chem 2009;52:7829-35. [61]

Piskorz J, Konopka K, Düzgüneş N, Gdaniec Z, Mielcarek J, Goslinski T.

Diazepinoporphyrazines containing peripheral styryl substituents and their promising nanomolar photodynamic activity against oral cancer cells in liposomal formulations ChemMedChem 2014;9:1775-82.

36

ACCEPTED MANUSCRIPT Scheme and Figures captions: Scheme 1. Synthesis of compounds 1–4. Reagents and conditions: (i) CH3OH, reflux, 4 h; (ii) Mg(O-nC4H9)2, nC4H9OH, reflux, 23 h.

ellipsoids are drawn at the 50% probability level.

RI PT

Fig. 1. The asymmetric unit of (a) 1 and (b) 2 showing the labeling scheme. The thermal

Fig. 2. The assembly mode of 2 in crystals: (a) columns; (b) (-101) layers. H atoms are

SC

omitted for clarity.

M AN U

Fig. 3. The molecule of 4. The units A and B of Pz are marked with different shades of grey. Pyridine C atoms are shown in red. H atoms are omitted for clarity. Fig. 4. The column formed by Pz-Mg molecules in 4. The neighboring molecules are drawn in different shades of grey.

TE D

Fig. 5. The space-filling representation of the barrel formed by two neighboring molecules of 4, viewed along the column direction. The Pz rings from the bottom and top of the barrel are removed. Some of the pyridine molecules enclosed in the barrel are shown in ball and stick

EP

representation with C atoms drawn in red.

AC C

Fig. 6. Absorption spectra of 3 (A) and 4 (B) in selected organic solvents. Fig. 7. Emission spectra of Pz 3 (A) and 4 (B) (λex = 380 nm and 610 nm) in acetonitrile or dichloromethane.

Fig. 8. Light toxicity (and dark control) of Pz 3 against: a) CAL 27, b) HSC-3, c) HeLa cells. Data represent the mean ± standard deviation obtained from experiments performed in triplicate. Statistical significance is indicated with asterisks: *p<0.050, **p<0.010, ***p<0.001.

37

ACCEPTED MANUSCRIPT Fig. 9. Light toxicity (and dark control) of Pz 4 against: a) CAL 27, b) HSC-3, c) HeLa cells. Data represent the mean ± standard deviation obtained from experiments performed in triplicate. Statistical significance is indicated with asterisks: *p<0.050, **p<0.010,

Table captions:

RI PT

***p<0.001.

Table 1. Quantum yields of singlet oxygen photosensitized production (Ф∆) and the singlet

AC C

EP

TE D

M AN U

SC

oxygen lifetimes (τ∆) for Pz 3 and 4 in dichloromethane

38

ACCEPTED MANUSCRIPT Highlights

•

Sulfanyl porphyrazines with bromobenzyl or biphenylylmethyl groups were synthesized Porphyrazne with biphenylyl groups forms barrel-like self-assemblies in crystals

•

Porphyrazines incorporated in cationic liposomes showed high photodynamic activity

AC C

EP

TE D

M AN U

SC

RI PT

•