Approaches to a broad range of high performance PDT sensitizers António M. d’A. Rocha Gonsalvesa, Arménio C. Serraa, Marta Pineiroa, and M. Filomena Botelhob

a. Departamento de Química, Universidade de Coimbra, 3049-535, Coimbra, Portugal. b. Instituto de Biofísica/Biomatemática, IBILI, Faculdade de Medicina de Coimbra, 3000-354

Coimbra, Portugal.

ABSTRACT Starting from expertise in the area of chemical synthesis, particularly in tetrapyrrolic macrocycles and an interest in modelling structures for particular objectives, we came to the point of aiming at modelling photochemical sensitizers designed for photodynamic therapy (PDT) purposes. Our endeavours were gratifying when it was proved that our synthetic methodologies allowed for the easy availability of properly halogenated porphyrins with high quantum yield singlet oxygen efficiency. Joining the presence of this heavy atom and other functionalities as substituents in selected positions of macrocyclic structures we were able to generate novel porphyrin structures whose photophysical and photochemical properties, singlet oxygen formation quantum yields, photobleaching and logP were measured. Cellular uptake measurements and cytotoxicity assays on WiDr adenocarcinoma and A375 tumor cell lines were carried out and some of our porphyrins demonstrated very high performance as PDT sensitizers comparatively to known compounds approved for clinical use and in the market. Further developments of our studies allowed for the generation of different and more efficient structures, easily made available by our own synthetic methodologies.

Our studies in this area allowed us to reach a stage which we believe to correspond to a significant knowledge and capacity to synthesise a broad range of simple structures, whose selectivity and efficiency as PDT sensitizers can be modulated for different cellular and tissue specificities.

Our most recent developments in this area will be presented in this communication. KEYWORDS: PDT, photosensitizer, cytotoxicity, phototoxicity, anti-tumor, oncology, porphyrin, singlet oxygen.

1. INTRODUCTION

Diagnostic, therapeutic, and inactivation photodynamic techniques proved to be useful and have high development potential. Significant progress has occurred in the field, but there is a long way to go before full benefit is taken of the potential of these methodologies1. Chemical, physical, biological and clinical knowledge contributions are required and a well knitted approach of efforts and mutual perception of the essentials by the different players is necessary in order to overcome the remaining obstacles and make the techniques more widely used and useful.

Our own expertise and contribution comes from the chemical side, starting from tetrapyrrolic macrocycle synthetic chemistry studies and specific functionalization of such structures. Our strategy of modelling allowed for the convenient and efficient synthesis of porphyrins and clorins generated from simple and easily available compounds having a diversity of structural features allowing: the modulation of photochemical characteristics enhancing the capacity to generate singlet oxygen; the balance of hydrophilic/hydrophobic character of the structures in order to select the capacity of transport by the body fluids and control the molecular interactions both with the cell structures and with transport vehicles. Our methodology apparently proved worthwhile and some of our sensitizers are patented2 and some of our experimental results already published3. The macrocyclic structures we idealized and built are porphyrins, clorins and bacteriochlorins and can be simply described as structural variations of the basic skeleton of meso-tetraphenylporphyrin, TPP. Our methodologies, developed from improvements and variations of the classical roots to TPP4-7, opened the way to significant improvements of synthetic methods for this well known and largely used structure, but we reckon them as important because a wide range of simple but highly diversified tetrapyrrolic structures designed to answer the requirements of PDT and PDI techniques were made available. Having the required synthetic tools, we began by synthesizing structures intended to be high quantum yield oxygen singlet sensitizers. In the first place, we thought that the incorporation of halogen atoms as peripheral substituents would satisfy our objective through heavy atom effect. Photophysical studies on a series of halogenated porphyrins allowed to establish the convenient positions and number of halogen atoms required to maximize the oxygen singlet quantum yield8.

Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applicationsedited by Samuel Achilefu, Ramesh Raghavachari, Proc. of SPIE Vol. 7190, 719005

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These first porphyrins showed very adequate photophysical characteristics to be used in PDT, but the porphyrin macrocycles were of high hydrophobic nature, while to be useful compounds for photodynamic therapy they must solubilize in body fluids and be able to enter cells. To give the desired compounds the adequate solubility it was necessary to introduce hydrophilic groups on the structure. A known and already approved compound which follows this approach is meso(3-hydroxyphenyl)chlorin, Temoporfin. The presence of the hydroxyl in the meta position of the meso phenyl rings seems to enhance the required activity comparatively to its placement in other positions9, 10. Patrice and co-workers reported a promising diaryl sensitizer having four hydroxyl groups (two in each of two meso phenyl groups)11, 12. Banfi et al. studied a series of 5,10,15,20-tetraarylporphyrins and 5,15-diarylporphyrins with different numbers of hydroxyl groups on the phenyl ring concluding that the compounds with hydroxyl groups at the meta position are the most active in PDT13. Taking into account our first results and preceding knowledge, we planned the synthesis of porphyrin structures having halogens and also other substituents, intending to optimize the transport by the body fluids and allow the most favorable interactions with the cell structures. The easiest first approach at exploiting our synthetic methodologies was the preparation of meso-tetra(2-halogen-3-hydroxyphenyl)porphyrins. The respective bromoporphyrin, a similar iodoporphyrin and a more extensively brominated compound were selected. With these compounds we promoted a study of their physical and chemical characteristics attempting to correlate the nature and location of the substituent with their influence on the physical-chemical properties of the compounds and on their photodynamic activity. From this work the photosensitizer PS1 having four bromines and four hydroxyl groups in the phenyl groups is particularly active, showing good photodynamic activity when tested against WiDr adenocarcinoma and A375 melanoma cells3.

Furthering this study, the in vivo activity of PS1 was evaluated in mice bearing implanted WiDr adenocarcinoma and A375 melanoma tumors. The good results obtained in this first study prompted us to further investigate the effect of the presence of bromine and hydroxyl groups in the meso phenyl groups by synthesizing a series of meso-arylporphyrins with different patterns of substitution (Scheme 1). The photodynamic activity of these porphyrins was tested in vitro against the previously mentioned cell lines.

N

HNN

NH

HO

Br

OH

Br

Br

HO

Br

OH

N

HNN

NHH H

HO

Br

Br

OH

N

HNN

NH

Br

OH

HO

Br

N

HNN

NH

HO

Br

OH

HO

Br

OH

N

HNN

NH

OH

HO

HO

Br

Br

OHN

HNN

NH

HO

Br

OH

HO

Br

OH

OH

HO

N

HNN

NH

RHNO2S

Br

SO2NHR

Br

Br

RHNO2S

Br

SO2NHR

PS1 PS2 PS3 PS4

PS5 PS6 PS7

R=(CH2)2O(CH2)2OH

Scheme 1. Structure of photosensitizers PS1-PS7

2.- METHODOLOGY

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2.1 General method for photosensitizers synthesis

PS1 was prepared as previously described3. For the synthesis of PS2 an excess of pyrrole was condensed with paraformaldehyde in the presence of trichloroacetic acid as catalyst to originate the dipyrrilmethane (DP1). This compound was then condensed with 2-bromo-5-hydroxybenzaldehyde, with trifluoroacetic acid as catalysts and oxidized with DDQ to originate PS2.

The synthesis of PS3 to PS6 was achieved from the condensation of an excess of pyrrole with the corresponding aldehydes in the presence of trifluoroacetic acid as catalyst to originate dipyrrilmethanes (DP2-DP4). These compounds were then condensed with the corresponding aldehydes, with p-toluenesulphonic acid as catalyst, and oxidized with DDQ to originate the methylated forms of photosensitizers PS3-PS6. Chromatographic separation on silica gel was needed in order to separate the methylated products from the unwanted products, which were porphyrins with different patterns of substitution. Demethylation with BBr3 originates the PS3 to PS6 photosensitizers. For the synthesis of PS7, porphyrin (1) was prepared as described6. Chlorosulphonation of (1) was done by reaction with chlorosulphonic acid at room temperature and further reaction with 2-(2-aminoethoxy)ethanol originated PS7 following known procedures. 2.2 In vitro studies The human colon carcinoma cell line WiDr and the human melanoma cell line A375 were purchased from American Type Culture Collection. The cell lines were cultured with Dulbecco’s Modified Eagle medium (Sigma–Aldrich, Inc; Sigma D-5648) supplemented with 10% heat-inactivated fetal bovine serum (Gibco Invitrogen Life Technologies; Gibco 2010-04), 1% Penicillin–Streptomycin (Gibco Invitrogen Life Technologies; 100 U/mL penicillin and 10 lg/mL streptomycin – Gibco 15140-122) and 100 lM sodium pyruvate (Gibco Invitrogen Life Technologies; Gibco 1360) at 37 ºC in a humidified incubator with 95% air and 5% CO2. For each experiment, cells were plated in 48 multiwells (Corning Costar Corp), in a concentration of 40.000 cells/mL and kept in the incubator overnight, in order to allow the attachment of the cells. The formulation of these sensitizers consisted in a 1 mg/mL solution in a ternary mixture of H2O/PEG400/EtOH (50:30:20, v/v/v), the desired concentrations being achieved by successive dilutions. The sensitizers were administered and cells were incubated for 24 h. Cells were washed with PBS and new drug-free medium was added. Each plate was then irradiated with a fluence rate of 7.5 mW/cm2 until a total of 10 J was reached. Cell viability was measured 24 h after the photodynamic treatment. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) colorimetric assay. Cytotoxicity was expressed as the percentage of inhibition of cell proliferation correlated with untreated cell cultures. Each experiment corresponds to the mean of 6 sets of experiments. 2.2 In vivo studies The experiments were performed with Balb/c nude mice of 6–8 weeks of age. The xenotransplants were developed in the back region by injection of 2x106 cells of two different human cell lines, WiDr colorectal adenocarcinoma and A375 melanoma. When the tumors had a size between 300-500 mm3, the mice were injected intraperitoneally with PS1 (2 mg/kg) and after different delay times the tumor is subjected to irradiation with a red laser light. The fluence was 100 mW/cm2 and the total energy was 150 Joules. For result analysis, the mice were distributed into the following four groups: Control: no photosensitizer (n = 8); irradiation 24h post injection, (n = 6), irradiation 72 h post injection, (n = 6) and irradiation 96 h post injection (n = 4). Tumor re-growth was monitored every 1 or 2 days following therapy. The size of the tumors was measured with a caliper 3 times a week, until 12 days after irradiation and the tumor volume calculated according to the following formula: V=A2 × B/2, where A is the width and B is the length. The volume of the tumor at the day of treatment was considered V0 and the growth of the tumor evaluated as Vt/V0.

3. RESULTS

3.1. In vivo photodynamic activity of PS1

Photosensitizer PS1 showed good photodynamic activity in vitro against WiDr adenocarcinoma and A375 melanoma cell lines3. The evaluation of the in vivo photodynamic activity of PS1 was done using implanted tumors of the same cell lines in Balb/c nude mice. The results were presented as the ratio between tumor volume at the time t (Vt) and the volume corresponding to the irradiation day (V0) in WiDr adenocarcinoma (Fig.1) and A375 melanoma (Fig. 2).

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The effectiveness of each treatment scheme was determined by comparison of the mean tumor volume doubling time in each group, defined as the number of days the tumor required to double its pre-treatment volume. For WiDr xenotransplats the time necessary in the control group to double the tumor volume was 4 days; when the irradiation was made 24 h after drug administration, the time necessary was 9 days and when the incubation time was increased to 72 h, the time increased to 13 days. In A375 melanoma implants the tumor volume doubling time for the control group was 3 days while for the group irradiated 24 hours after drug administration was 4,5 days and for the group irradiated 96 hours after drug administration the tumor volume doubling time was 10 days.

The effect of the PDT treatment on the delay of the tumor growth is evident. In both cases irradiation after injection of PS1 resulted in a longer tumor volume doubling time compared to controls. For WiDr tumors there is no significant difference of results for the two delay times (24 hours or 72 hours) between injection and irradiation. However, for melanoma tumors, after 96 hours of delay time there is a significant decrease of the growing tumors relatively to 24 hours of delay time.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 150

1

2

3

4

5

6

7

8

Control 24h 72h ExpDecay Control ExpDecay 24h ExpDecay 72h

Vt/V

0

time, d

Figure 1. Rates of tumor growth of WiDr Xenotransplants.

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0 2 4 6 8 10 120

1

2

3

4

5

6

7

8

control 24h 96h ExpDecay Control ExpDecay 24h ExpDecay 96h

Vt/V

0.

Time, d

Figure 2. Rates of tumor growth of A375 melanoma Xenotransplants. 3.2 Synthesis of porphyrins

Photosensitizer PS1 was prepared by the classical two step cyclization/oxidation route from pyrrol and the required

aldehydes5. Photosensitizer PS2, with only two meso-phenyl groups, was prepared from condensation of pyrrole and paraformaldehyde to give dipyrromethane DP1 which was made to react with 2-bromo-5-hydroxybenzaldehyde to originate PS2. meso-Tetraarylporphyrins (PS3-PS6) were prepared by following the sequence of reactions outlined in Scheme 2. Reaction of pyrrole in the presence of an excess of aldehyde catalysed by trifluoroacetic acid14 originated the corresponding dipyrromethanes (DP2-DP4), typical yield being 60 %. Dipyrromethanes are then reacted with the selected aldehyde in the presence of p-toluenosulfonic acid to give the porphyrins which were demethylated with boron tribromide to give the corresponding hydroxyporphyrins (Scheme 2). Photosensitizer PS7 was prepared from brominated porphyrin (1) by chlorosulphonation6 followed by reaction of the chlorosulphonyl derivative with 2-(2-aminoethoxy)ethanol (2) (Scheme 3).

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NH

+TFA

NH HN

R2R1

R1 R2

O H

DP3- R1=OMe, R2=H, R3=H

DP4- R1=OMe, R2=OMe, R3=H

R3

DP2- R1=H, R2=H, R3=Br

OMe

O H

Br

BBr3 PS3

OMe

O H

Br

BBr3 PS4

O H

BBr3 PS5

O H

BBr3 PS6

R3

OMe

Br

NH

+NH HN

(CH2O)nTCA OH

O H

Br

PS2

DP1

Scheme 2. Synthetic methodology used for the preparation of photosensitizers PS2-PS6.

N

NH N

HN

Br

Br

Br

Br(2)

N

NH N

HN SO2R

RO2S

RO2S

SO2R

Br

Br

Br

Br

R= NHCH2CH2OCH2CH2OH

1. ClSO3H

2.NH2(CH2)2O(CH2)2OH

(1) (PS7) Scheme 3. Synthetic methodology followed for the preparation of the photosensitizer PS7

3.3. In vitro photodynamic activity of photosensitizers The phototoxicity of all the photosensitizers PS1-PS7 was tested against WiDr colon adenocarcinoma cells and A375 melanoma cells. The results obtained are presented in figure 3 and figure 4, respectively.

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Control Solvent 50 nM 250 nM 500 nM 1 uM 5 uM 10 uM0

20

40

60

80

100

120C

ell V

iabi

lity,

% Photofrin PS1 PS2 PS3 PS4 PS5 PS6 PS7

Figure 3. Cell viability % for WiDr human colon adenocarcinoma cells using sensitizers PS1-PS7 and Photofrin®.

Control Solvent 50 nM 250 nM 500 nM 1 uM 5 uM 10 uM

0

20

40

60

80

100

Cel

l Via

bilit

y, %

Concentration, M

Photofrin PS1 PS2 PS3 PS4 PS5 PS6

Figure 4. Cell viability % for melanoma A375 cells using sensitizers PS1-PS7 and Photofrin®. From figure 3 the order of phototoxicity of the different photosensitizers against WiDr cancer cells is : PS2 > PS4 ≥ PS1 > PS3 ≥ PS6 > PS7 > Photofrin® > PS5. Comparing the compounds with the same number of hydroxyl groups

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represented by PS1, PS4 and PS5 the influence of the number and position of the halogen atom on the phenyl group could be analyzed. Photosensitizers PS1 and PS4 show similar activity, which indicates that only two bromine atoms are enough to get high photodynamic activity. In PS5 the activity drastically decreased which can be a consequence of the position of the bromine atom or from the placement of the four hydroxyl groups. In order to analyze the influence of the hydroxyl group, PS1 and PS3 were compared, the change from four to two hydroxyl groups resulting in a decrease of the phototoxicity. However, compared to PS3, PS2 demonstrates higher phototoxicity despite being a compound with the same number of hydroxyl and bromine groups. Dismissing shape, volume and symmetry considerations, the last result points out that not only the number of hydroxyl groups influences the phototoxicity but also that the ratio hydroxyl:phenyl groups is significant. With the ratio 1:1 as in PS2, PS1 and PS4 the photoxicity is high, with 0.5/1 ratio (as in PS3) or 1.25:1 ratio (as in PS6) the phototoxicity is reduced. The separation of the hydroxyl group from the phenyl group by a sulfamoilethyleneoxy chain linker did not prove to be very favorable, a reduction of phototoxicity beeing observed. In this case there is a 1/1 ratio of the hydroxyl/bromine groups, but the hydroxyl are not directly linked to the meso phenyls (PS7). The sequence of decreasing phototoxity against A375 cells is: PS2 > PS3 > PS1 > PS4 > PS6 > Photofrin® > PS5 (fig.4). Except for the case of PS3, the order is the same as the one found for the phototoxicity against WiDr cells. The analysis of the influence of the number and position of halogen atoms is generally the same as the one obtained with WiDr cells. The best result was obtained with a hydroxyl:phenyl group ratio of 1:1, although some cellular specificity is apparent from the behavior of PS3. Also, the dose necessary to obtain similar photodynamic action, in WiDr cells is always higher than that for melanoma A375 cells, showing that the sensitizers are more effective with this cell line. Dark toxicity was measured under the same experimental conditions used for the cytotoxicity assay but in the absence of light using WiDr and A375 cell culture. None of the photosensitizers have significant toxicity in the dark. In Figure 5 the cell viability of some representative photosensitizers is presented.

Control Solvent 50 nm 250 nM 500 nM 1 uM 5 uM 10 uM0

20

40

60

80

100

120

Cel

l Via

bilit

y, %

Concentration, M

PS1(WiDr) PS3 (WiDr) PS5 (A375) PS6 (WiDr) PS6 (A375)

Figure 5. Dark toxicity of some representative photosensitizers.

The original idea of exploiting our capacities in the area of porphyrin synthesis to modulate simple structures, capable of giving adequate answer to the preparation of compounds having required physical-chemical characteristics, namely being good singlet oxygen sensitizers and having the ability to interact with the cellular structures and show biological activity concentrated where the physiological action is desired, proved satisfactory. We were able to select comparatively efficient sensitizers as shown in this communication and extend the types of compounds heretofore

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presented. Easy methodologies to get similar structures in different oxidation levels, chlorins and bacteriochlorins, have also been the object of our more recent interests. The door is open to more extensive biological studies and partnerships are welcome in the fields of PDT, PDI and PDD.

4. CONCLUSION Some preliminary studies of relevant properties with an attempted first model, porphyrin PS1, allowed to exploit the expertise of porphyrin synthetic methodologies to carried out a study of the structure/phototoxicity relationship, which is likely to be useful in devising better specific photosensitizers. It was proven that compounds with little structural differences can have drastic effects in the phototoxicity and easy synthetic approaches to such structures were developed. We show that a series of compounds having the same substituents differently placed on the basic structure of the chromophore can have higher and lower phototoxicity than Photofrin®. Playing on the basis of meso phenyl substituted porphyrins, the presence of halogen atoms at the ortho position of the phenyl groups and a ratio of 1:1 of the phenyl:hydrophilic group are the best characteristics to optimize the phototoxicity. ACKNOWLEDGMENTS The authors thank Bruno Nascimento, Nelson Pereira, Catarina Santos, Alexandra Gonsalves, Margarida Abrantes, Mafalda Laranjo, Cristina Santos, who did much of the work which supported the content of this work. Chymiotechnon, Ministério da Economia/ POE/Prime/Proj 3/293/CLARO, Faculdade de Medicina de Coimbra and CIMAGO for financial support. REFERENCES [1] Serra, A. C., Pineiro, M., Pereira, N., Gonsalves, A. M. d. A. R., Laranjo, M., Abrantes, M. and Botelho, F., "A look on clinical applications and developments on photodynamic therapy ", Oncology Reviews, 3, DOI 10.1007/s12156-008-0081-1 (2008). [2] Gonsalves, A. M. d. A. R., Pineiro, M. and Serra, A. C., "Tetrapyrrolic macrocycles as photodynamic agents", WO03/064427A1, (2003) [3] Serra, A. C., Pineiro, M., Rocha Gonsalves, A. M. d. A., Abrantes, M., Laranjo, M., Santos, A. C. and Botelho, M. F., "Halogen atom effect on photophysical and photodynamic characteristics of derivatives of 5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin", J. Photochem. Photobiol. B: Biol, 92, 61-67 (2008). [4] Rocha Gonsalves, A. M. d. A. and Pereira, M. M., "A new look into the Rothemund meso-tetraalkyl and tetraarylporphyrin synthesis", J. Heterocyclic Chem., 22, 931-933 (1985). [5] Johnstone, R. A. W., Nunes, M. L. P. G., Pereira, M. M., Rocha Gonsalves, A. M. d. A. and Serra, A. C., "Improved syntheses of 5,10,15,20-tetrakisaryl- and tetrakisalkylporphyrins", Heterocycles, 43, 1423-1437 (1996). [6] Rocha Gonsalves, A. M. d. A., Johnstone, R. A. W., Pereira, M. M., SantAna, A. M. P., Serra, A. C., Sobral, A. F. N. L. and Stocks, P. A., "New procedures for the synthesis and analysis of 5,10,15,20-tetrakis(sulphophenyl)porphyrins and derivatives through chlorosulphonation", Heterocycles, 43, 829-838 (1996). [7] Nascimento, B. F. O., Pineiro, M., Rocha Gonsalves, A. M. d. A., Ramos Silva, M., Matos Beja, A. and Paixão, J. A., "Microwave-assisted synthesis of porphyrins and metalloporphyrins:A rapid and efficient synthetic method", J. of Porphyrins and Phthalocyanines, 11, 77-84 (2007). [8] Azenha, E. G., Serra, A. C., Pineiro, M., Pereira, M. M., Seixas de Melo, S. J., Arnaut, L. G., Formosinho, S. J. and Rocha Gonsalves, A. M. d. A., "Heavy-atom effects on metalloporphyrins and polyhalogenated porphyrins", Chem. Phys., 280, 177-190 (2002). [9] Berenbaum, M. C., Akande, S. L., Bonnett, R., Kaur, H., Ioannou, S., White, R. D. and Winfield, U. J., "Meso-tetra(hydroxyphenyl)porphyrins, a new class of potent tumour photosensitizers with favourable selectivity", Br. J. Cancer, 54, 717-725 (1986). [10] Bonnett, R., McGarvey, D. J., Harriman, A., Land, E. J., Truscott, T. G. and Winfield, V. J., "Photophysical properties of meso-tetraphenylporphyrins and some meso-tetra(hydroxyphenyl)porphyrins", Photochem. Photobiol., 48, 271 (1988).

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[11] Bourré, L., Thibaut, S., Fimiani, M., Ferrand, Y., Simonneaux, G. and Patrice, T., "In vivo photosensitizing efficiency of a diphenylchlorin sensitizer: Interest of a DMPC liposome formulation", Pharmacological Research, 47, 253-261 (2003). [12] Ferrand, Y., Bourré, L., Simonneaux, G., Thibaut, S., Odobel, F. and Patrice, T., "Hydroporphyrins as tumor photosensitizers: synthesis and photophysical studies of 2,3-dihydro-5,15-di(3,5-dihydroxyphenyl)porphyrin", Bioorg. Med. Chem. Lett., 13, 833-835 (2003). [13] Banfi, S., Caruso, E., Buccafurni, L., Murano, R., Monti, E., Gariboldi, M., Papa, E. and Gramarica, P., "Comparison between 5,10,15,20-tetraaryl- and 5,15-diarylporphyrins as photosensitizers: synthesis, photodynamic activity, and quantitative structure-activity relationship modeling", J. Med. Chem., 49, 3293-3304 (2006). [14] Lee, C. H. and Lindsey, J. S., "One-flask synthesis of meso-substituted dipyrromethenes and their application in the synthesis of trans-substituted porphyrins building blocks", Tetrahedron, 50, 11427-11440 (1994).

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