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Dyes and Pigments 99 (2013) 144e153

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Dyes and Pigments

journal homepage: www.elsevier .com/locate/dyepig

Novel meso-phenothiazinylporphyrin dyes: Synthesis, optical,electrochemical properties and PDT assay

Emese Gal a, Balazs Brema, Iani Pereteanu a, Luiza G�ain�a a, Tamas Lovasz a,Maria Perde-Schrepler b, Laura Silaghi-Dumitrescu c, Castelia Cristea a,*,Luminita Silaghi-Dumitrescu a

aBabes-Bolyai University, Faculty of Chemistry and Chemical Engineering, RO-400028 Cluj-Napoca, Romaniab “Ion Chiricuta” Oncology Institute Cluj-Napoca, Radiobiology and Tumour Biology Department, RomaniacBabes-Bolyai University, Raluca Ripan Institute for Research in Chemistry, Romania

a r t i c l e i n f o

Article history:Received 23 February 2013Received in revised form25 April 2013Accepted 29 April 2013Available online 13 May 2013

Keywords:PhenothiazinePorphyrinFluorescencePDTSpectroscopic and electrochemicalpropertiesFrontier molecular orbitals

* Corresponding author. Tel.: þ40 264 593833; faxE-mail address: castelia@chem.ubbcluj.ro (C. Crist

0143-7208/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.dyepig.2013.04.034

a b s t r a c t

The synthesis of new chromophore architectures based on arrays of phenothiazine and (hetero)aromaticunits joint in the meso positions of a porphyrin core (phenothiazinyl A4, phenothiazinyl and (hetero)aryl:AB3 trans-A2B2 and A3B), is described. The structures were assigned based on HRMS and NMR spectra.Their optical properties were evaluated according UVeVis spectroscopic data, which emphasizedabsorption maxima around 420 nm with molar absorptivities 105 M�1cm�1 and emission characterizedin solution by large Stokes shifts (3800e4500 cm�1) and fluorescence quantum yields ranging from 0.01to 0.06. The electron transfer properties of the compounds were estimated using cyclic voltammetrywhich pointed out three quasi-reversible one-electron oxidations potentials ranging between 800 and1400 mV (vs. Ag/AgCl). Selected meso-phenothiazinylporphyrins were evaluated as photosensitizers forphotodynamic therapy (PDT) by in vitro irradiation of two human skin cell lines (HaCaT and A431) usingred and blue light.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

A tremendous evolution in the chemistry and applications ofvarious functional dyes characterized by specific photo-, thermo- andelectrochemical properties was recorded lately [1]. Porphyrin andphenothiazine are two azaheterocyclic systems, which may beidentified as the core of many derivatives with important biologicalactivity and noticeable capacity for mediation of the energy/electrontransfer processes. The properties of the very potent pharmacophoricphenothiazine core have been largely exploited in medicinal appli-cations [2], while recent literature reports demonstrate progress inthe identification of promising new biological applications [3,4].

The typical redox processes of the phenothiazine core encryptedin various compounds containing its neutral or oxidized state (withemphasis on the well known phenothiazinium dyes e.g. methyleneblue, thionine, toluidine blue, etc.) laid the groundwork for detailedphotochemical [5] and electrochemical [6,7] studies.

: þ40 264 590818.ea).

All rights reserved.

Porphyrins have been extensively exploited as platforms for thestudy of theoretical principles and applications in a wide variety offields such as chemistry, physics, material science, engineering,biology and medicine [8]. Most of the currently approved clinicalphotosensitizers for photodynamic therapy belong to the porphyrinfamily [9]. The porphine macrocycle is hydrophobic, with a ten-dency to aggregate in body fluids and tissues, leading to suboptimalconcentration in target tissues and poor photodynamic activity [10].These reasons explain the continuous interest for the design of newporphyrin derivatives with improved biocompatibility, reducedtoxicity and enhanced photodynamic effect on target cells [11].

Similarities between the photochemical properties of porphyrinand phenothiazine derivatives in the oxidized state enabledcomparative studies regarding their potential as photosensitizers inphototherapeutic [12] or photobactericidal [13] applications. Suit-able candidates for photoinduced energy transfer systems andfluorescence-emitting materials were obtained by attachingvarious substituents to the peripheral positions of the porphyrincore. Star shaped meso-tetraarylporphyrins with neutral pheno-thiazine arms extending the linear p-conjugation [14], showed

S

N CN HN

NC

C

NH

C

R

S

NR

S

NR

CH=O

S

NR

S

NR

NH

+

i) or ii)R= Me, Et

1a: R = Me1b: R = Et

Scheme 1. Synthetic route to meso-tetrakis(10-alkyl-phenothiazin-3yl)porphyrins.Reaction conditions: i) CHCl3 BF3-Et2O r.t. 2 h, chloranil 60 �C 2 h, TEA; ii) Et-COOHAc2O 110 �C 4 h.

E. Gal et al. / Dyes and Pigments 99 (2013) 144e153 145

emission of intense red light with fluorescent quantum yieldshigher than most porphyrins reported.

The meso-phenothiazinylporphyrins (MPP) described below arenew chromophore architectures based on arrays of phenothiazineand (hetero)aromatic units directly linked to a porphyrin core.A transform based strategy applied in the retrosynthetic analysis ofour target MPP, pointed out the advantages of the reported meth-odology describing the condensation of carbaldehydes with pyrrole[15]. A comparison between the synthetic protocols developed forthe preparation of meso-tetraarylporphyrins, using Brönsted acidcatalysts and atmospheric oxygen conditions [16] vs. Lewis acidcatalysts and oxidizing agents [17], emphasize low yields of por-phyrins generated by a simple work up, while improved yieldsrequired more elaborated reaction conditions. In this paper wepropose convenient synthetic protocols for the preparation of MPPtogether with their UVeVis absorption/emission spectroscopic andelectrochemical characterization. The in vitro evaluation of theirpotential as photosensitizers for photodynamic therapy describedbelow, completed by the specific photo- and electrochemicalproperties recollected by joining the individual properties of thetwo major heterocyclic units in molecular compounds, may opennew perspectives for further applications as functional dyes.

2. Experimental

2.1. Measurements

10H-Phenothiazine, alkyl bromides, pyrrole, aromatic carbal-dehydes and propionic acid were purchased from Merck. Pyrrolewas redistilled before use. CH2Cl2 was dehydrated All reactions aswell as column chromatography were followed by TLC analysisusingMerck pre-coated silica gel 60 F254 aluminium sheets. Columnchromatography was performed on silica (particle size 0.063e0.2 mm).

2.1.1. Spectral measurements1D, DEPT, 2D-COSY, 2D-HSQC and 2D-HMBC NMR spectra were

recorded at room temperature on Bruker Avance instruments(1H/13C: 400 MHz/100 MHz or 300 MHz/75 MHz) in solution(deuterated solvents chloroform (CDCl3), or DMSO).

The mass spectra were recorded on a HRMS spectrometer LTQOrbitrap XL-Thermo-Scientific.

UVeVis spectra were recorded in CH2Cl2 with a Perkin ElmerLambda 35 spectrometer, Fluorescence spectra were recorded inCH2Cl2 with a Perkin Elmer PL 55 spectrophotometer. The fluo-rescence quantumyield was calculated using tetraphenylporphyrin(TPP) standard (F ¼ 0.13 in CH2Cl2 [18].

DFTcalculationswerecarriedoutusingB3LYP/6-311Gmethod [19].

2.1.2. Electrochemical measurementsThe oxidation potentials were determined by cyclic voltamme-

try measurements using a Potentiostat/Galvanostat/ZRA Reference600, with a typical three-electrode setup composed of platinum(1 mm) working electrode, platinum and Ag/AgCl as auxiliary andreference electrodes. The electrolyte was 0.1 M tetrabutylammo-nium hexafluorophosphate in dry dichloromethane purged withargon prior to a measurement. Ferrocene/ferrocenium (Fc/Fcþ) wasemployed as internal standard.

2.2. Synthesis

2.2.1. Condensation of 10-alkyl-phenothiazine-3-carbaldehyde andpyrrole

The procedure described by Lindsey et al. [20], and Adler et al.[16a] were adapted to the synthesis of MPP 1a,b (Scheme 1).

10-Alkyl-phenothiazine-3-carbaldehydes were prepared accordingthe previously described procedures [21].

2.2.1.1. General procedure i). Dichloromethane (750 ml), 10-alkyl-10H-phenothiazine-3-carbaldehyde (4 mmol) and pyrrole (0.28 ml,4 mmol) were mixed under an inert atmosphere (N2), thenBF3$OEt2 (0.1 ml, 2.5 M) was added to the stirred solution. To thereaction mixture, kept in darkness at room temperature for 2 h,p-Chloranil (1.10 g, 4 mmol) was added and the reaction mixturewas heated at 60 �C for 2 h, when triethylamine (0.3 ml) was added.The reaction progress was monitored by TLC. The solvent wasevaporated under reduced pressure and the residue was driedunder vacuum. The black solid obtained was further purified bycolumn chromatography on silica (eluent: CH2Cl2/petrol ether (b.p.40e60 �C): 10/1).

2.2.1.2. General procedure ii). Propionic acid (100 ml) and aceticanhydride (3 ml) were mixed and heated at 110 �C for 1 h. Aftercooling at room temperature, 10-alkyl-10H-phenothiazine-3-carbaldehyde (10 mmol), and pyrrole (10 mmol) were added andthe reaction mixture was heated at 110 �C for 4 h. After cooling atroom temperature, purple crystals were formed; in the case whenthe compound did not precipitate, the propionic acid was removedby vacuum distillation and the dark precipitate was washed withwater (3 � 25 ml) to remove the traces of propionic acid. The crudemixture was filtered and washed with methanol (3 � 5ml). Thefurther purification by column chromatography on silica usingCH2Cl2/petrol ether (40e60 �C) 10/1 gave the final products.

Characteristics of the synthesized MPP are presented in Table 1.

Table 1NMR and MS data for the synthesized MPP.

Cpd Method/yield

1H, 13C NMR d[ppm] and HRMS (ESIþ) data

1a i/8%ii/20%

1H NMR (300 MHz, CDCl3): �2.80 (s, 2H), 3.51 (s, 3H), 6.99 (d, 1H, 3J¼ 7.3 Hz), 7.04 (d, 1H, 3J¼ 8 Hz), 7.26 (m, 2H), 7.28 (d, 1H), 7.96 (d, 2H), 8.90 (s, 8H);13C NMR 35.6 (4NCH3), 112.3 (4CH), 114.5 (4CH), 122.7 (4CH), 123.8 (4C), 124.2 (4C), 126.9 (4CH), 127.1 (4CH), 127.6 (4CH), 132.8 (4C), 133.9 (4CH),135.8 (8C), 141.8 (8CH),145.2 (4C), 146.2 (4C); HRMS Calcd for: C72H51N8S4 [M þ H]þ 1155.3114, Found: 1155.3118.

2a i/10%ii/14%

1HNMR (400MHz, CDCl3):�2.72 (s, 2H), 3.61 (s, 3H, CH3), 7.00 (d, 1H, 3J¼ 8 Hz), 7.04 (t, 1H, 3J¼ 7.6 Hz), 7.14 (d, 1H, 3J¼ 8 Hz), 7.28 (m, 2H), 7.77 (m, 9H),7.98 (dd, 1H, 4J ¼ 1.6 Hz, 3J ¼ 8 Hz), 8.03 (d, 1H, 4J ¼ 1.6 Hz), 8.24 (d, 6H, 3J ¼ 6 Hz), 8.86 (s, 6H), 8.92 (d, 2H, 3J ¼ 4.4 Hz); 13C NMR 35.6 (CH3), 112,3 (CH),114.3 (CH), 119.0 (C), 120.1 (2C), 121.9 (C), 122.8 (C), 123.3 (C), 126.6 (CH), 127.4 (C) 127.7 (CH), 131.0 (CH), 132.9 (CH), 133.9 (CH), 134.5 (CH), 136.5 (C),145.5 (C), 145.8 (C); HRMS Calcd for: C51H35N5S [M þ H]þ 750.2686, Found: 750.2677.

2b i/10%ii/17%17%

1HNMR (400MHz, CDCl3):�2.85 (s, 2H), 3.67 (s, 3H), 4.10 (s, 3H), 7.13 (m, 2H), 7.21 (d, 6H), 7.33 (m, 2H), 7.35 (d, 1H, 3J¼ 8.4 Hz), 7.96 (d, 1H, 3J¼ 8.4 Hz),7,98 (s, 1H), 8.12 (d, 6H 3J¼ 8 Hz), 8.87 (d, 8H 3J¼ 4.2 Hz); 13C NMR: 35.7 (NCH3), 55.6 (OCH3), 113.9 (CH), 114.4 (6CH), 115.8 (CH), 118.8 (2C), 120.2 (2C),120.8 (8C), 122.2 (CH), 123.4 (C), 125.1 (CH), 128.2 (CH), 131.2 (8CH), 133.0 (CH), 134.5 (CH), 136.5 (6CH), 145.3 (C), 145.9 (C), 159.4 (3C); HRMS Calcdfor: C54H41O3N5S [M þ H]þ 840.2937, Found: 840.2965.

2c i/12%ii/19%

1HNMR (300MHz, CDCl3):�2.78 (s, 2H), 3.16 (s, 3H), 3.61 (s, 3H), 6.99e7.07 (m, 2H), 7.12 (d, 1H, 3J¼ 8.2 Hz), 7.28e7.33 (m, 2H), 7.47 (d, 2H, 3J¼ 7.7 Hz),7.96 (dd, 1H, 4J ¼ 1.6 Hz, 3J ¼ 8.2 Hz), 8.02 (s, 1H, 4J ¼ 1.6 Hz), 8.18 (d, 2H, 3J ¼ 8.2 Hz), 8.84 (s, 6H), 8.94 (d, 2H, 3J ¼ 4.7 Hz); 13C NMR: 35.8 (NCH3), 112.5(CH), 113.7 (CH), 114.0 (CH), 119.0 (2C), 119.6 (2C), 120.8 (8C), 122.2 (CH), 122.7 (CH), 123 (C), 123.4 (C), 127.4 (6CH), 127.6 (CH), 127.9 (CH), 131.2 (8CH),133.1 (CH), 134 (6CH), 136.4 (CH), 145.9 (C), 145.7 (C), 14164.6 (3C); HRMS: Calcd for: C51H32F3N5S [M þ H]þ 804.2403, Found: 804.2404.

2d i/11%ii/18%

1H NMR (300 MHz, CDCl3): �2.83 (s, 2H), 3.64 (s, 3H), 8.85 (s, 6H), 7.05 (t, 1H, 3J ¼ 7.4 Hz, 3J ¼ 7.4 Hz), 7.07 (d, 1H, 3J ¼ 8 Hz), 7.3 (t, 1H, 3J ¼ 7.4 Hz,3J¼ 8 Hz), 7.35 (d, 1H, 3J¼ 7.8 Hz), 7.4 (d, 1H, 3J¼ 8.4 Hz), 7.76 (d, 6H, 3J¼ 7.8 Hz), 7.99 (d, 1H, 3J¼ 8.4 Hz), 8.02 (s, 1H), 8.15 (d, 6H, 3J¼ 7.8 Hz), 8.95 (d, 2H,3J ¼ 4.5 Hz); 13C NMR 35.9 (NCH3), 112.5 (CH), 114.5 (CH), 119.0 (2C), 120.0 (2C), 120.2 (8C), 122.2 (CH), 122.7 (CH), 122.8 (C), 123.3 (C), 127.2 (6CH),127.6 (CH), 127.9 (CH), 128.7 (C), 131.0 (8CH), 132.9 (CH), 133.1 (CH), 134.0 (6CH), 136.5 (CH), 142.1 (3C), 145.9 (C), 145.7 (C); HRMS Calcd for:C51H32Cl3N5S [M þ H]þ 852.1517/854.1487, Found: 852.1495/854.1470

c2e i/10%ii/17%

1H NMR (400 MHz, CDCl3): �2.80 (s, 2H), 3.57 (s, 3H), 7.04 (d, 1H, 3J ¼ 8.1 Hz), 7.06 (t, 1H, 3J ¼ 8.1 Hz), 7.20 (d, 1H, 3J ¼ 8.1 Hz), 7.30 (t, 1H, 3J ¼ 8.1 Hz),7.35 (d, 1H, 3J ¼ 7.8 Hz), 7.92 (d, 6H, 3J ¼ 7.8 Hz), 7.98 (dd, 1H, 3J ¼ 8.1 Hz), 8.01 (s, 1H), 8.05 (d, 6H, 3J ¼ 7.8 Hz), 8.84 (d, 6H, 3J ¼ 4.8 Hz), 8.94 (d, 2H,3J¼ 4.8 Hz); 13C NMR 35.7 (NCH3), 112.4 (CH), 114.4 (CH), 118.7 (CH), 118.9 (2C), 119.8 (2C), 120.3 (8C), 122.1 (CH), 122.6 (CH), 122.9 (C), 123.2 (C), 127.5(6CH), 127.6 (CH), 127.8 (CH), 131.3 (8CH), 132.9 (CH), 133.8 (CH), 134.5 (6CH), 136.2 (CH), 141 (3C), 145.7 (C), 145.9 (C); HRMS Calcd for: C51H32Br3N5S[M þ H]þ 987.9960/985.9981, Found: 987.9952/985.9967

2f i/11%ii/20%

1H NMR (300 MHz, CDCl3) 3.60 (s, 3H), 7.1e7.13 (m, 3H), 7.3e7.36 (m, 2H), 7.4 (d, 1H, 3J ¼ 8 Hz), 7.97 (d, 1H, 3J ¼ 8.2 Hz), 8.06 (m, 7H), 8.36 (d, 6H,3J ¼ 7.5 Hz), 8.85 (d, 6H, 3J ¼ 4.8 Hz), 9.02 (d, 2H, 3J ¼ 4.8 Hz), �2.70 (s, 2H); 13C NMR 35.6 (NCH3), 112.5 (CH), 114.5 (CH), 118.6 (CH), 118.8 (2C), 120.4(2C), 120.8 (8C), 122.3 (CH), 123.1 (C), 123.2 (C), 126.4(3CF3), 127.6 (6CH), 127.9 (CH), 130.1 (CH), 130.5 (8CH), 133.1 (CH), 133.9 (CH), 134.7 (6CH), 136.1(CH), 145.7 (3C), 145.8 (C), 145.9 (C); HRMS Calcd for: C54H32F9N5S [M þ H]þ 954.2307, Found: 954.2307.

3a i/8%ii/14%

1H NMR (300 MHz, CDCl3) 3.62 (s, 6H), 7.00 (d, 1H, 3J ¼ 8 Hz), 7.04 (t, 1H, 3J¼ 7.6 Hz), 7.26 (d, 1H, 3J¼ 7.4 Hz), 7.3 (t, 1H, 3J¼ 8 Hz), 7.4 (d, 1H, 3J¼ 8 Hz),7.76 (m, 6H), 7.98 (dd, 2H, 4J¼ 1.2 Hz, 3J¼ 8 Hz), 8.02 (d, 2H, 4J¼ 1.2 Hz), 8.22 (d, 4H, 3J¼ 6 Hz), 8.85 (d, 4H, 3J¼ 4.6 Hz), 8.9 (d, 4H, 3J¼ 4.6 Hz),�2.75 (s,2H); 13C NMR 35.3 (2NCH3), 112.3 (2CH), 114.1 (2CH), 119.9 (2CH), 120.2 (4C), 122.6 (2CH), 123.0 (2C), 123.5 (2C), 127.2 (2CH), 127.5 (2CH), 131 (8C),132.8 (2CH), 133.6 (2C), 134.4 (2CH), 136.3 (4CH), 142.1 (2C), 145.3 (2C), 145.6 (2C); HRMS Calcd for: C58H40N6S2 [M þ H]þ 885.2829, Found: 885.2834.

3b i<2% ii<2% HRMS Calcd for: C60H44O2N6S2 [M þ H]þ 945.3000, Found: 945.29923c i/9%

ii/18%

1H NMR (300 MHz, CDCl3) 3.66 (s, 6H), 7.00e7.07 (m, 4H), 7.16 (d, 1H, 3J ¼ 8.2 Hz), 7.28e7.33 (m, 4H), 7.45 (dd, 4H, 3J ¼ 8.5 Hz), 8.00 (dd, 4H), 8.17 (dd,4H, 3J¼ 8.5 Hz), 8.80 (d, 4H, 3J¼ 5 Hz), d 8.91 (d, 4H, 3J¼ 5 Hz),�2.79 (s, 2H),; 13C NMR 35.7 (2NCH3), 112.5 (2CH), 113.7 (2CH), 114.5 (2CH), 118.9 (4CH),119.5 (2CH), 122.9 (2CH), 123.3 (2C), 123.4 (2C), 127.6 (2CH), 127.8 (2CH), 131.9 (8C), 133.0 (2CH), 133.6 (2C), 134.1 (2CH), 135.8 (2CH), 136.4 (4CH),138.1 (2C), 145.7 (2C), 145.9 (2C), 161.6 (2C); HRMS Calcd for: C58H38F2N6S2 [M þ H]þ 921.2640, Found: 921.2641.

3d i/8%ii/16%

1HNMR (300MHz, CDCl3) 3.67 (s, 6H), 7.03 (d, 2H, 3J¼ 8 Hz), 7.04 (t, 2H, 3J¼ 8.1 Hz), 7.19 (d, 2H, 3J¼ 8.1 Hz), 7.33 (t, 2H, 3J¼ 8 Hz), 7.36 (d, 2H, 3J¼ 8 Hz),7.98 (d, 2H, 3J¼ 8 Hz), 8.00 (d, 4H, 3J¼ 9.6 Hz), 8.02 (s, 2H), 8.15 (d, 4H, 3J¼ 7.8 Hz), 8.83 (d, 4H, 3J¼ 6 Hz), 8.93 (d, 4H, 3J¼ 5.4 Hz),�2.80 (s, 2H); 13C NMR35.7 (2NCH3), 112.4 (2CH), 114.5 (2CH), 118.8 (4CH), 119.6 (2CH), 120 (2C), 122.1 (2C), 122.9 (2CH), 123.2 (2C), 124.5 (2C), 127.6 (2CH), 127.8 (2CH),128.7 (2CH), 132.9 (8C), 133.8 (2CH), 134.2 (4CH), 136.2 (4CH), 140.5 (2C), 145.6 (2C), 145.9 (2C); HRMS Calcd for: C58H38Cl2N6S2 [M þ H]þ 953.2049/955.2021, Found: 953.2023/955.2001

3e i/9%ii/18%

1H NMR (300 MHz, CDCl3) 3.55 (s, 6H), 6.99 (d, 2H, 3J ¼ 8.4 Hz), 7.06 (d, 2H, 3J ¼ 6.9 Hz), 7.10 (t, 2H, 3J ¼ 7.5 Hz), 7.24 (d, 2H, 3J ¼ 7.4 Hz), 7.33 (t, 2H,3J¼ 7.5 Hz), 7.88 (m, 4H, 3J¼ 7.5 Hz), 7.95 (dd, 2H, 4J¼ 1.5 Hz, 3J¼ 6.9 Hz), 8.04 (d, 2H, 4J¼ 1.5 Hz) 8.04 (d, 4H, 3J¼ 7.5 Hz), 8.83 (d, 4H, 3J¼ 4.2 Hz), 8.93 (d,4H, 3J¼ 4.2 Hz),�2.76 (s, 2H); 13C NMR 35.6 (2NCH3), 112.4 (2CH), 114.4 (2CH), 118.8 (4CH), 119.7 (2CH), 122.1 (4C), 122.9 (2CH), 123.2 (2C), 126.6 (2C),127.3 (2CH), 127.5 (2CH), 127.8 (2CH), 128.7 (2CH), 132.9 (8C), 133.8 (4CH), 134.5 (4CH), 136.3 (2CH), 142.1 (2C), 145.8 (2C), 145.6 (2C); HRMS Calcd for:C58H38Br2N6S2 [M þ H]þ 1041.1039/1043.1018, Found: 1041.1028/1043.0983.

3f i/10%ii/20%

1H NMR (300 MHz, CDCl3) 3.59 (s, 6H), 6.99e7.34 (m, 10H), 7.4 (d, 1H, 3J ¼ 8 Hz), 7.99 (m, 8H), 8.37 (d, 4H), 8.95 (d, 8H), �2.69 (s, 2H); 13C NMR 35.6(2NCH3), 112.5 (2CH), 114.5 (2CH), 118.3 (4CH), 120.1 (2C), 122.2 (2C), 122.9 (2CH), 123.3 (2C), 123.8 (2C), 126.5 (2C), 127.6 (2CH), 127.9 (2CH), 130.4(2C), 133.1 (2CH), 134.7 (4CH), 136.2 (4CH), 145.7 (2C), 145.8 (2C), 145.9 (2C); HRMS Calcd for: C60H38F6N6S2 [M þ H]þ 1021.2576, Found: 1021.2566

4a ii/7% 1H NMR (300 MHz, acetone-D6) 3.62 (s, 3H), 7.03 (t, 1H, 3J ¼ 7.7 Hz), 7.13 (d, 1H, 3J ¼ 8.20 Hz), 7.21 (d, 2H, 3J ¼ 7.35 Hz), 7.36e7.31 (m, 2H), 7.86 (t, 3H,3J¼ 7.2 Hz), 8.00 (m, 2H), 8.51 (d, 6H, 3J¼ 6.5 Hz), 8.64 (d, 2H, 3J¼ 4.5 Hz), 9.04 (t, 6H, 3J¼ 4.5 Hz), 9.08 (d, 3H, 3J¼ 6.2 Hz), 9.39 (s, 3H),�2.78 (s, 2H); 13CNMR 36.3 (NCH3), 117.9 (CH), 118.2 (CH), 120.1 (2C), 122.8 (CH), 123.1 (6CH), 123.8 (C), 124.0 (C), 128.4 (CH), 127.9 (CH), 131.9 (4CH), 133.7 (CH), 135.1(6CH), 136.4 (6CH), 136.6 (C), 139.7 (C), 146.6 (C), 148.3 (C), 150.5 (3CH), 152.9 (3CH); HRMS Calcd for: C48H32N8S [M þ H]þ 753.2543, Found: 753.2573.

4b ii/6% 1H NMR (300 MHz, DMSO-D6) 3.60 (s, 3H), 7.16 (t, 1H, 3J ¼ 7.15 Hz, 3J ¼ 7.7 Hz), 7.18 (d, 1H, 2J ¼ 7.8 Hz), 7.42e7.27 (m, 3H), 8.03 (m, 2H), 8.27 (d, 2H,3J ¼ 5.3 Hz), 9.05 (t, 6H, 3J ¼ 5.3 Hz), �2.94 (s, 2H); 13C NMR 36.3 (NCH3), 117.9 (CH), 118.2 (CH), 120.1 (6CH), 120.5 (4C), 122.8 (C), 123.1 (C), 124.0 (C),127.9 (CH), 128.4 (CH), 131.9 (8CH), 135.1 (CH), 136.4 (CH), 136.6 (CH), 139.7 (4C), 146.6 (C), 148.3 (C), 149.3 (6CH);HRMS Calcd for: C48H32N8S [MþH]þ

753.2543, Found: 753.2524.5a ii/12% 1H NMR (300 MHz, DMSO-D6) �2.74 (s, 2H), 3.58 (s, 6H), 6.96e7.04 (m, 6H), 7.13e7.03 (m, 4H), 7.74 (m, 2H), 7.92 (d, 2H), 8.03 (s, 2H), 8.51 (d, 2H,

3J¼ 6.2 Hz), 8.84 (d, 4H, 3J¼ 4.3 Hz), 8.97 (d, 4H, 3J¼ 4.3 Hz), 9.00 (d, 2H, 3J¼ 6.2 Hz), 9.46 (s, 2H); 13CMNR 35.6 (2NCH3), 112.4 (2CH), 114.4 (2CH), 119.7(2CH), 121.0 (2CH), 121.9 (4CH), 122.5 (2C), 122.9 (2C), 127.1 (2CH), 128.1 (2CH), 130.6 (2CH), 132.3 (2C), 134.1 (2CH), 135.0 (2CH), 137.1 (2CH), 140.8(2C), 140.1 (4CH), 141.2 (4CH), 142.3 (2C), 145.7 (2C), 153.3 (4C); HRMS Calcd for: C56H38N8S2 [M þ H]þ 887.2734, Found: 887.2731.

5b ii/13% 1HNMR (300MHz, CDCl3)�2.86 (s, 2H), 3.66 (s, 6H); 7.02e7.08 (m, 4H), 7.20 (d, 2H, 3J¼ 8.7 Hz), 7.27e7.31 (m, 4H), 7.98 (m, 4H), 8.16 (d, 4H, 3J¼ 5.2 Hz),8.89 (d, 4H, 3J ¼ 4.7 Hz), 8.95 (d, 4H, 3J ¼ 4.7 Hz), 9.03 (d, 2H, 3J ¼ 5.2 Hz); 13C NMR 36.3 (2NCH3), 112.3 (2CH), 114.8 (2CH), 119.8 (2CH), 120.1 (2CH),121.6 (4CH), 122.8 (2C), 123.3 (2C), 127.4 (2CH), 127.7 (2CH), 132.9 (2CH), 133.9 (2CH), 136.5 (4C), 142.9 (4C), 145.5 (2C), 145.8 (2C), 148.8 (4C), 149.5(4CH), 153.2 (4C); HRMS Calcd for: C56H38N8S2 [M þ H]þ 887.2734, Found: 887.2728.

6a ii/20% 1H NMR (300 MHz, CDCl3) �2.77 (s, 2H), 3.61 (s, 9H), 6.98e7.06 (m, 6H), 8.77 (d, 2H, 3J ¼ 4.8 Hz), 7.14 (d, 3H, 3J¼ 7.3 Hz), 7.25e7.32 (m, 6H), 7.75 (t, 1H,3J¼ 7.2 Hz), 7.98 (m, 6H), 8.51 (d, 1H, 3J¼ 7.2 Hz), 8.93 (t, 6H, 3J¼ 4.8 Hz), 9.03 (d, 1H, 3J¼ 7.2 Hz), 9.46 (s, 1H); 13C NMR 35.7 (3NCH3), 112.5 (3CH), 114.6(3CH), 115.2, 119.6 (3C), 122.1, 122.2 (3C), 123.3 (3C), 123.7 (C), 127.5 (3CH), 127.8 (3CH), 133.1 (8CH), 134.1 (CH), 139.4 (CH), 141.4 (CH), 145.7 (3C),145.9 (3C), 148.6 (2C), 152.5 (6C); HRMS Calcd for: C64H44N8S3 [M þ H]þ 1021.2924, Found: 1021.2902.

6b ii/21% 1HNMR (300MHz, CDCl3)�2.82 (s, 2H), 3.66 (s, 9H), 7.02e7.09 (m, 6H), 7.19 (d, 3H, 3J¼ 7.9 Hz), 7.30e7.35 (m, 6H), 7.99 (m, 6H), 8.20 (d, 2H, 3J¼ 5.6 Hz),8.76 (d, 2H, 3J ¼ 4.8 Hz), 8.93 (t, 6H, 3J ¼ 4.8 Hz), 9.06 (d, 2H, 3J ¼ 5.6 Hz); 13C NMR 35.6 (3NCH3), 114.3 (3CH), 115.3 (3CH), 119.2 (3C), 120.2 (3C), 121.6(3CH), 122.2 (3CH), 122.6 (3C), 123.8 (C), 124.8 (3CH), 127.2 (3CH), 127.7 (3CH), 128.4 (3CH), 131.2 (8CH), 143.3 (3C), 144.5 (3C), 144.8 (3C), 149.5 (2C),153.2 (6C); HRMS Calcd for: C64H44N8S3 [M þ H]þ 1021.2924, Found: 1021.2882.

E. Gal et al. / Dyes and Pigments 99 (2013) 144e153146

S

N

CH=ONH N

CH=O

C

N

+ +

ii)

E. Gal et al. / Dyes and Pigments 99 (2013) 144e153 147

2.2.2. One pot mixed condensation of 10-alkyl-phenothiazine-3-carbaldehyde and (hetero)aromatic carbaldehydes with pyrrole

General procedure i) using 10-methyl-10H-phenothiazine-3-carbaldehyde (4 mmol), (hetero)aromatic carbaldehyde (12 mmol)and pyrrole (16 mmol), was applied for the synthesis of MPP 2aef,3aef (Scheme 2).

General procedure ii) using 10-methyl-10H-phenothiazine-3-carbaldehyde (4 mmol), (hetero)aromatic carbaldehyde(12 mmol) and pyrrole (16 mmol), was applied for the synthesis ofMPP 2aef, 3aef (Scheme 2), 4a,b, 5a,b, and 6a,b (Scheme 3).

2.3. Photodynamic therapy experiments

2.3.1. Cell lines, cultureTwo human skin cell lines were used:

S

N

CH=ONH

CH=O

R

S

N CN HN

NC

C

NH

C

N

S

R

R

CN HN

NC

C

NH

C

N

S

R

R

R

+ +

R = -HR = -OCH3R = -ClR = -Br

2a: R = H2b: R = OCH32c R = F2d: R = Cl2e: R = Br2f: R= CF3

i) or ii)

3a: R = H3b: R = OCH33c R = F3d: R = Cl3e: R = Br3f: R= CF3

+

Scheme 2. Synthesis of aryl-MPP by mixed condensation of phenothiazinecarbalde-hyde, para-substituted benzaldehyde and pyrrole/Reaction conditions: i) CHCl3 BF3-Et2O r.t, 2 h, chloranil 60 �C 2 h, TEA; ii) Et-COOH Ac2O 110 �C 4 h.

S

N CN HN

NC

C

NH

C

N

N

S

N

N CN HN

NC

NH

C

N

S

N

4a: 3-Py4b: 4-Py

5a: 3-Py5b: 4-Py

S

N CN HN

NC

C

NH

C

N

N

S

6a: 3-Py6b: 4-Py

SN

+

+

Scheme 3. Synthesis of pyridyl-MPP by mixed condensation of phenothiazine-carbaldehyde, pyridine carbaldehyde, and pyrrole. Reaction conditions: ii) Et-COOH,Ac2O, 110 �C, 4 h.

Table 2Optical properties of MPP dyes. The values in bold were employed in the calculation of Stokes shift values (listed in the eight column of the same table).

Cpd. labs [nm] Stokes shift cm�1 FFb lem [nm]

Phenothiazine Soret ( 3mol�1 cm�1) Q4a Q3 Q2 Q1

1a 254 429 (87,923) 522 564 592 654 4209 0.05 6692a 255 420 (170,931) 519 557 592 650 4116 0.05 6602b 250 423 (10,494) 519 556 594 651 4093 0.01 6592c 254 417 (168,627) 516 552 591 650 4228 0.02 6602d 255 420 (356,977) 517 554 591 649 3958 0.05 6502e 255 420 (328,529) 517 554 592 650 4528 0.03 6752f 256 416 (297,100) 516 553 591 649 3995 0.02 6503a 255 421 (206,151) 519 558 594 651 4342 0.06 6703c 255 420 (122,345) 519 557 593 651 4342 0.02 6703d 255 420 (216,570) 519 558 593 651 4001 0.06 6553e 255 420 (241,878) 519 558 593 651 4342 0.03 6703f 255 420 (166,545) 519 558 592 650 4453 0.02 6754a 256 417 (168,843) 517 554 591 650 4122 0.02 6574b 256 417 (156,925) 516 554 590 647 3809 0.04 6505a 256 420 (136,924) 519 558 592 652 4305 0.01 6705b 255 422 (99,677) 521 562 598 653 4042 0.05 6606a 255 424 (106,595) 521 562 590 654 4228 0.06 6606b 255 424 (150,991) 520 560 593 652 4125 0.04 662

a l employed for excitation in fluorescence experiments.b Quantum yields against TPP standard.

E. Gal et al. / Dyes and Pigments 99 (2013) 144e153148

a) the spontaneously immortalized human keratinocyte cell lineHaCaT purchased from the Cell Line Service of the GermanCancer Research Centre in Heidelberg and maintained inDMEM high glucose with 10% foetal bovine serum, 1% gluta-mine, antibiotic;

b) the epidermoid carcinoma cell line A431 purchased from theEuropean cell culture collection and maintained in DMEMwith10% FCS, 1% glutamine, 1% NEA, antibiotic. All media werepurchased from Sigma.

Cells were detached from the growth surface using trypsin-EDTA (Sigma), and reseeded on 96-well plates (cell density e

2 � 104 cells/well in 200 ml medium) and 24-well plates(5 � 104 cells/well in 1.5 ml medium).

2a, 2c, 2,d 2e and 3c MPP were evaluated as photosensitizersusing initial concentrations of: 2a: 1.9 mg/ml; 2c: 1.5 mg/ml; 2d:3.9 mg/ml; 2e: 3.7 mg/ml; 3c: 3.6 mg/ml. For each tested compoundfive serial dilutions were prepared as follows: 5�, 10�, 20�, 30�and 40�.

Fig. 1. UVeVis absorption spectra of MPP 1a, 4a, 5a, 6a (10�4 M in CH2Cl2).

2.3.2. Photosensitizing treatmentCells seeded on 96 and 24-well plates were treated with MPP.

For the evaluation of dark toxicity (cytotoxicity without lightexposure) one of the plates was kept for 24 h in the dark (eachconcentration at least in threewells), while another was exposed tovisible light.

2.3.2.1. Irradiation. Red light irradiation was delivered by a LEDsystem emitting at l ¼ 615e630 nm with 5 mW/cm2 intensity atthe surface of the cells (measured with LM2 PowerMeter, Carl ZeissJena). Cells were irradiated for 30 min, corresponding to an energylevel of 9 J/cm2. After irradiation, complete medium was added tothe cells, which were then incubated for 24 h.

Blue light (l ¼ 400e505 nm) was delivered by an Optilux 501curing unit for the polymerization of light curedmaterials, emittingwith 850 mW/cm2 intensity. Exposure time was 5 s, in which cellswere delivered 4.25 J/cm2.

Fig. 2. Excitation and emission spectra of MPP 1a, 3a, 3d and 6a (10�5 M in CH2Cl2).

Fig. 3. Fluorescence spectra of MPP 2a (10�5 M in CH2Cl2 solution) excited at (a)255 nm; (b) 520 nm.

E. Gal et al. / Dyes and Pigments 99 (2013) 144e153 149

2.3.2.2. Determination of cell survival. The cytotoxic effect oftreatments was assessed using the MTT assay with minor modifi-cations [22]. This method is based on the reduction of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] to acoloured formazan compound (lmax 492 nm) only by living cellswith active mitochondrial hydrogenase, which represents a part ofthe respiratory chain. Briefly, 24 h after treatments, 100 ml of MTT(Sigma) were added and cells were incubated for 1 h. After thatinterval, formazan crystals were dissolved by adding 150 ml ofDMSO and absorbancewas thenmeasured using a Biotek Synergy 2microplate reader at 492 nm. Statistic analysis was performed andgraphs were plotted using Graph Pad Prism 5 software.

3. Results and discussion

3.1. Synthesis of meso-phenothiazinylporphyrins (MPP)

Our goal was to develop a convenient and robust procedure forthe condensation of 10-alkyl-phenothiazine-3-carbaldehyde withpyrrole. The benefits induced by the variation of solvent (chloro-form, acetic or propanoic acid), catalyst (BF3 or carboxylic acid) andoxidizing agent (chloranil, air, or nitrobenzene) were explored byapplying the Lindsay [20], Adler [16a] or Gonsalves [23] procedures.Best yields (15e20%) of meso-tetrakis(10-alkyl-phenothiazin-3yl)-porphyrins 1 were obtained in chloroform solution, using BF3catalyst and chloranil (Scheme 1).

For the mixed condensation of pyrrole with 10-methylphe-nothiazine-3-carbaldehyde and several (hetero)arylcarbaldehydeswe developed efficient one-pot procedures which enabled us toobtain new MPP dyes containing up to three phenothiazinyl unitsdirectly linked to the porphyrin chromophore core.

Table 3Computed relative energies of the frontier orbitals of MPP.

Cpd. E [kJ/mol] DE HOMO�LUMO

[kJ/mol]HOMO LUMO HOMO�1 LUMOþ1

1a �452.2800 �207.2984 �484.4874 �207.6653 244.98162a �469.6902 �215.1717 �492.0825 �212.2785 254.51852c �491.8215 �243.7867 �511.3785 �241.2125 248.03472e �491.8215 �243.7867 �513.6491 �244.7735 248.03474a �489.6925 �238.8368 �513.8450 �247.3111 250.85574b �505.3169 �253.7504 �521.0232 �253.3511 251.5664

Two synthetic protocols proved to be successful for themixed condensation of 10-methyl-phenothiazine-3-carbaldehydeand para-substituted benzaldehyde derivatives with pyrrole(Scheme 2). A3B 2aef and trans A2B2 3aef were obtained as majorreaction products in raw 1:1 M ratio in each case after columnchromatography separation of the resulting mixture of porphyrins,excepting the case of 3b which was identified only in traces. Nomajor influence of the electronic effect of the para substituent inthe benzaldehyde reagent was observed, the results mainly point-ing towards a steric control induced by the folded structure ofphenothiazinecarbaldehyde.

Similar one-pot procedures entail mixing 10-methylpheno-thiazine-3-carbaldehyde and pyridine-3(4)-carbaldehyde withpyrrole in the presence of acid catalysts (Scheme 3). Tris-pyridyl-MPP 4a,b, bis-pyridyl-MPP 5a,b and mono-pyridyl-MPP 6a,b werefound in 1:2:4 M ratio after column chromatography separation ofthe resulting mixture of porphyrins. These experimental results areconsistent with a low competitivity of the pyridine carbaldehyde tothe condensation reaction.

The structural assignments were based on high resolution1H and 13C NMR spectroscopy and mass spectrometry as shown inTable 1. The symmetrical structure of bis-aryl-MPP is unambigu-ously sustained by 1H NMR spectra of 3aef: in each case, the eightprotons situated in the beta positions of the pyrrole units encryptedin the macrocyclic structure revealed four isochrone AB spin sys-tems situated as the most deshielded signals (8.6e8.9 ppm), withvicinal coupling constants of 4.2e5.4 Hz.

3.2. Optical properties of MPP

An intense UV absorption band situated around 255 nmgenerated by pep* transitions in the phenothiazine chromophore[24] and a strong near-UV Soret band situated at 417e429 nm,followed by four low intensity Q bands located in the visiblespectral region at 516e654 nm, characteristic of free-base etio-typeporphyrin chromophore were present in the UVeVis absorptionspectrum of each MPP (Table 2).

A slight bathochromic shift (9e12 nm) of the Soret band( 3¼ 0.9e3 � 105) responsible for the dark purple colour of allcompounds) is observable in the series 4a (417 nm) 5a (420 nm) 6a(424 nm) 1a (429 nm) illustrated in Fig. 1, as well as in the series 2a(420 nm), 3a (421 nm),1a (429 nm), or 4a (417 nm) 5a (420 nm) 6a(424 nm) 1a (429 nm), respectively and can be directly correlatedwith the increasing number of electron-donating phenothiazineunits in the MPP. The symmetrical MPP 1a exhibit the lowest molarabsorptivity pointing towards a less favoured electronic transition.The para substitution of the phenyl rings in the MPP 2bef and 3befwith halogen, electron donor metoxy, or electron withdrawingtrifluoromethyl groups does not produce noticeable effects uponthe absorption properties of MPP.

All compounds show red-orange daylight fluorescence. Uponirradiation at the lmax corresponding to Q4 band, in solution, MPPs

Table 4Half-cell potentials of halogen-containing MPP as determined by cyclicvoltammetry.

Cpd. E0=þ11=2 [V] Eþ1=þ2

1=2 [V] Eþ2=þ31=2 [V]

2c 0.800 1.080 1.3502d 0.800 1.115 1.3852e 0.820 1.130 1.4002f 0.820 1.120 1.4503c 0.820 1.120 1.3203d 0.830 1.110 1.4103e 0.870 1.175 1.4353f 0.830 1.230 1.420

Fig. 4. Energy levels and contour plots of simulated frontier molecular orbitals of 2e.

E. Gal et al. / Dyes and Pigments 99 (2013) 144e153150

display fluorescence with noticeable Stokes shifts (3809e4528 cm�1) and quantum yields between 0.01 and 0.06 whenmeasured against TPP standard. Fig. 2 illustrates the emissionspectra of MPP 3a, 3d and 6awhich displayed the highest emissionin solution. The presence of para eF, eOeCH3 or eCF3 substituentin the phenyl rings diminishes the MPP emission properties, as wellas the increasing number of pyridinyl substituents in the MPPfluorophore.

Similar emission maxima were recorded upon excitation withthe lmax corresponding to the electronic excitation of the pheno-thiazine chromophore (255 nm), or the Q4 absorption band of theporphyrin chromophore (520 nm) thus supporting a large conju-gation between the two chromophore units. Fig. 3 showsthe emission spectra of 2a upon excitation at two differentwavelengths.

1.5 1.0 0.5 0.0-20

0

20

40

Ι [μ

Α]

E [V ]

-

1

1

1

1

Ι [μ

Α]

Fig. 5. Cyclic voltammograms of 2e and 3e (recorded in CH2Cl2, 20 �C, v ¼ 100 mV/s, elelectrode) using ferrocene/ferrocenium (Fc/Fcþ) as internal standard.

Quantummechanical semi-empirical, and DFTcalculations wereperformed using the SPARTAN package [19] (Table 3). Fig. 3 showsthe contour plot of the frontier orbitals of 2e, where a largecontribution of atomic orbitals in the phenothiazine unit to theHOMO can be seen, along with a LUMO centred on the porphyrinstructural unit, thus supporting the typical emission properties ofthe porphyrin chromophore.

3.3. Electrochemical properties of MPP

Electrochemical data obtainedby cyclic voltammetryonhalogen-containingMPP 2cef and 3cef in dichloromethanewith scanning inthe anodic (up to 1.5 V) and cathodic (up to �0.2 V) region aresummarized in Table 4. The typical CV pattern of MPP consists of afirst quasi-irreversible oxidation process ðE0=þ1

1=2 ¼ 800� 870 mVÞ,

1.5 1.0 0.5 0.0

20

0

20

40

60

80

00

20

40

60

E [V ]

ectrolyte: nBu4NþPF�6 , Pt working electrode, Pt counter electrode, Ag/AgCl reference

Fig. 6. MTT assay of epidermoid carcinoma A431 cell survival after photodynamic effect initiated by blue light irradiation (l ¼ 400e505 nm) upon: (a) photosensitizer 2a 1.9 mg/mlin serial dilutions (***p < 0.0001 two-way ANOVA); (b) comparative IC50 calculation for 2a and 2e.

Fig. 7. MTT assay of keratinocyte HaCaT cell survival in PDT experiments withphotosensitizer 2a 1.9 mg/ml in serial dilutions, dark toxicity and photodynamic effectupon blue light irradiation (l ¼ 400e505 nm) (***p < 0.001, two-way ANOVA).

E. Gal et al. / Dyes and Pigments 99 (2013) 144e153 151

followed by a second and a third irreversible ones situated atincreasing anodic potentials (Eþ1=þ2

1=2 ¼ 1080� 1230 mV andEþ2=þ31=2 ¼ 1320� 1450 mV). The first oxidation process observable

in the CV pattern of MPP can be particularly assigned to thephenothiazine structural unit by analogy with the recorded CV ofparent 10-Me-10H-phenothiazine ðE0=þ1

0 ¼ 767 mVÞ and the MPPcomputational data indicating a HOMO largely centred on thephenothiazine unit (Fig. 4). The noticeable anodic shift may suggestan electron donor effect of the phenothiazine unit in conjugationwith the porphyrin core. A comparison between the CV patterns ofhalogenated MPP 2cef and their counterparts, 3cef (e.g. 2e, 3e inFig. 5), revealed anodic shifts of the oxidation potentials andincreased current intensities in the case of MPP 3cef containing twophenothiazine units.

Fig. 8. MTT assay of cell survival after PDT with photosensitizer 2a 1.9 mg/ml in serial dilutionlines (***p < 0.001, two-way ANOVA); (b) keratinocyte HaCaT cell lines (*p < 0.05; **p < 0.

3.4. MPP as photosensitizers for photodynamic therapy

In vitro photosensitization experiments were performed on twohuman skin cell lines: normal keratinocyte HaCaT and epidermoidcarcinoma A431 respectively. The cells seeded on well plates weretreated with tris-aryl-MPP 2a, 2c, 2d, 2e and bis-aryl-MPP 3c inserial dilutions. The cytotoxic effect of treatments was assessedusing the spectrophotometric MTT method, which indicated thatnone of the tested MPP were dark toxic to the two cell lines. Theexposure to visible light of the cells treated with MPP led to somecytotoxic effects.

The cells lines treated with MPP were irradiated for 30 minwithred light and for 5 s with blue light respectively. Bis-aryl-MPP 3chad a slight photodynamic effect on A431 cells when exposed toblue light, but this effect was not statistically significant. MPP 2dand 3c showed no photosensitizer properties upon irradiationwithneither blue nor red light.

Compounds 2a, 2c and 2e showed variable photodynamic ef-fects in correlation with the concentrations, cell lines and irradia-tion wavelengths employed.

Upon irradiation with blue light, both MPP 2a and 2e reducedsignificantly the viability of tumoral A431 cells, in a dose dependentmanner (Fig. 6a). 2a appears to be a more potent photosensitizer(IC50 ¼ 0.079 mg/ml) compared to 2e (IC50 ¼ 0.148 mg/ml) as shownin Fig. 6b. 2a had a statistically significant phototoxic effect at allconcentrations on normal keratinocyte HaCaT cells (**p < 0.001,two-way ANOVA) (Fig. 7), while 2e did not affect the normal ker-atinocytes (data not shown). This fact could represent an importantdrawback in the use of compound 2a in clinic, because normal cellscould be affected as well. Therefore, other characteristics of thiscompound should be investigated. Ideally, photosensitizers should

s using red light irradiation (l ¼ 615e630 nm) on: (a) epidermoid carcinoma A431 cell01, two-way ANOVA).

Fig. 9. MTT assay of epidermoid carcinoma A431 cell survival after PDT with photo-sensitizer 2c 1.5 mg/ml in serial dilutions, using red light irradiation (l ¼ 615e630 nm)(*p < 0.05, **p < 0.001, two-way ANOVA).

E. Gal et al. / Dyes and Pigments 99 (2013) 144e153152

be retained preferentially by the diseased tissue and for longerperiods of time compared to the healthy tissue, thus, light exposureshould be timed in order to minimize damage in normal cells. Anenhanced therapeutic effect can also be achieved by the specificillumination of the target tissue and also by topical application ofthe photosensitizer. The fact that compound 2e showed alsoincreased toxicity towards tumour cells when activated by bluelight makes it attractive as a potential photosensitizer which isworth further investigation.

After exposure to red light 2a showed a more potent cytotoxiceffect on A431 cells (IC50 ¼ 0.046 mg/ml) compared to the one ob-tained when activated with blue light, (IC50 ¼ 0.079 mg/ml). Thisproperty is also an argument in considering 2a a potential candi-date for photodynamic therapy, having in mind the fact that longerwavelength penetrates the tissues better, thus making PDT suitablefor deeper-seated/larger lesions. Photosensitizer 2a determinessignificant reduction of cellular viability in both epidermoid carci-noma cells (Fig. 8a) and normal HaCaT cells (Fig. 8b) in a dose-dependent manner. This reduction was highly significant in A431cells at all concentrations (p < 0.001), while in HaCaT cells viabilitywas significantly reduced (p < 0.5) only beginning with dilution10�, showing higher toxicity towards tumour cells.

Compound 2c initiated a photodynamic effect on A431 cellswhen exposed to red light. The phototoxic effect was significant atall concentrations while the highest concentration employed hadalso dark toxicity (two-way ANOVA, Fig. 9). This fact reduces thepotential of compound 2c to be employed as a photosensitizer inPDT (one of themain characteristics of a good photosensitizer beingthe absence of dark toxicity).

4. Conclusions

Convenient one-pot procedures were developed for the statis-tical synthesis of new MPP dyes containing up to four phenothia-zinyl units attached in the meso positions of the porphyrin core.

The UVeVis absorption spectra of the dark purple MPP dyescomprises the strong UV absorption typical of the phenothiazinemoiety as well as the strong near-UV Soret band accompanied bythe low-Vis Q absorption bands characteristic of free-base etio-typeporphyrin chromophores.

All MPP showed red-orange daylight fluorescence. Upon irra-diation with the absorption maxima corresponding to eitherporphyrin or phenothiazine unit, MPPs display fluorescence withlow quantum yields, but noticeable Stokes shifts, recommendingthem as suitable functional dyes candidates.

The electron transfer ability of MPP, as estimated by cyclic vol-tammetry, suggest their possible implication in redox processesrequiring oxidation potentials ranging between 800 and 1400 mV(vs. Ag/AgCl).

An insight view of the electron distribution in the frontier mo-lecular orbitals, as predicted by quantum mechanical calculations,indicate a large contribution of atomic orbitals located in thephenothiazine unit to the HOMO orbital, while the LUMO appearscentred on the porphyrin core, thus supporting the observedfluorescence and electrochemical properties.

In vitro PDT experiments demonstrated the photosensitizingproperties of MPP: 2a showed increased phototoxicity when acti-vated by red light (longer wavelength which penetrates tissuesdeeper) and also a slightly stronger phototoxic effect on tumourcells compared to normal keratinocytes; 2e had also photoxic ef-fects, but only when activated by blue light and it showed a higherselectivity against tumour cells. All these findings let us emphasizethat these MPP are good candidates to be used in PDT, but theirpromising behaviour needs to be further investigated by bothin vitro and in vivo experiments.

Acknowledgements

Financial support from the Romanian Ministry of Education,Research Youth and Sports (Grant ID PCCE 140/2008), as well asfrom the Sectorial Operational Programme for Human ResourcesDevelopment 2007e2013, co-financed by the European Social Fund(POSDRU 89/1.5/S/60189 with the title “Postdoctoral Programs forSustainable Development in a Knowledge Based Society” and by aPhD scholarship to IP Contract POSDRU/88/1.5/S/60185 e “Inno-vative doctoral studies in a knowledge based society”) is gratefullyacknowledged.

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