WO2011161065A1 - Macrocyclic tetrapyrrolic compound of the family of porphyrins, chlorins and bacteriochlorins as photosensitizers for photodynamic therapy - Google Patents

Abstract

The present invention relates to macrocyclic tetrapyrrolic compounds of the family of porphyrins, chlorins and bacteriochlorins, having substitution in positions 5 and 15 of the porphyrin macrocycle with two phenyl groups having bromine, or chlorine atoms in the ortho position and a hydroxyl group in the meta position of the phenyl ring; and wherein positions 2, 3, 7, 8, 12, 13, 17 and/or 18 of the macrocycle are substituted with hydrogen or halogen atoms or alkyl, vinyl, carboxy, carboxyalkyl, alkoxycarbonyl, alkoxycarbonylalkyl, alkoxycarbonylaryl, acetoxy or hydroxyalkyl groups. The compounds of this invention proved to be useful in PDT against human cancer cells.

Description

Macrocyciic tetrapyrrolic compound of the family of porphyrins, chlorins and bacteriochlorins as photosensitizers for photodynamic therapy

This invention discloses new macrocyciic pyrrolic compounds with appropriate amphiphilicity and halogen substituents which have proved to optimize the photo- physical, photochemical and pharmacological properties of these compounds so that they may be used and are particularly efficient in photodynamic therapy (PDT) and photodiagnosis. The compounds of this invention proved to be useful in PDT against human cancer cells. The present invention also includes a new advantageous synthetic process for the preparation of the dypyrrylmethanes, which are the precursors of this kind of macrocyciic.

The included structures are 5,15-bisphenylchlorins and 5,15- bisphenylbacteriochlortns with a hydroxy! group in the meta position and bromine or chlorine in the ortho positions of the phenyl rings. The substituents in positions 2,3,7,8,12,13,17 and 18 may be hydrogen, halogen atoms or alkyl, vinyl, carboxyl, carboxylalkyi, alkoxycarbonyl, aikoxycarbonylalkyl, alkoxycarbonyiaryl, acetoxy or hydroxyalkyl groups. The invention also comprises the use of these compounds in photodynamic therapy and photodiagnosis.

Background of the invention Photodynamic therapy (PDT) is presently a well established technique allowing very useful oncological and non-oncological therapeutics involving the irradiation of the affected areas with visible light after a sensitizer that has been administered localizes itself preferentially in the target cells. The photosensitizer (PS) is a molecule with the capacity to absorb energy from the radiation and transfer it to gener- ate active species responsible for cellular death or necrosis, that contribute to the cure process. The sensitizer is therefore a species without pharmacological activity and its function consists in the production of true active species from inactive species present in the medium. Normally, the referred species is oxygen which is converted from its natural triplet state into harmful oxygen species, mainly the oxygen singlet state. Thus, the sensitizer does not have the function of a common pharmaceutical compound but, through the interaction with light, basically generates the species that will carry out the pharmacological function. In order to carry out this role as a suitable sensitizer for PDT, some specific properties must be present, such as:

1. be easily transported by the body fluids and be selectively retained by target cellular structures;

2. efficiently absorb light from the visible region designated as the therapeutic window (600-800 nm), preferentially at the highest wavelength, and transfer the energy with high quantum yield to generate singlet oxygen.

3. be eliminated by the organism in an adequate short time to avoid side effects.

Review papers recently published disclose the state of the art of the chemical, physico-chemical, biological and therapeutic studies with respect to the use of tetrapyrrolic macrocycles in PDT ^1"14.

The potential for the application of the tetrapyrrolic macrocycles as photosensitiz- ers (PS) in photodynamic therapy and complementary therapies comprises sev- era! situations:

Non-oncologicai applications include vascular applications of photochemotherapy: an example is the ophthalmologic problem of age related macular degeneration (AMD) which requires sensitizers with absorption values at wavelengths higher than 700 nm; another example is the use of PDT for the prevention of restenosis. Relatively to oncological applications the use of PDT in the destruction of large tumours appears to have unsatisfactory results except in a few cases of brain tumors in which the blood-brain barrier favors the concentration of the sensitizer in the tumor tissue relatively to the healthy tissues. In this case light diffusion through brain tissue also seems to be better than through others. Particularly im- portant is the case of glyoblastoms, an "orphelin" pathology that has a very short survival prognosis, about one year or less. As examples of destruction of tumors by PDT with small area and thickness are the mouth leucodisplasies, very common in developing countries, which can be promptly and easily treated by PDT contrary to the present situation where treatment is not made for being too expen- sive; pre-cancerous lesions such as Barrett mucosa, with increasing cases in industrialized countries, can be cured by PDT with minimal invasive treatments; prostate and bladder cancer and actinic keratosis, with a growing number of cases due to aging of the population and increasing solar exposure, showed complete cure by PDT therapy.

One key characteristic of the PS is the singlet oxygen quantum yield (Φ_Δ) which should to be as high as possible. The presence of halogen atoms as substituents on the PS causes an increase in ΦΔ¹⁵ but this effect can not be directly correlated to an increase in photodynamic activity¹⁶. The chemical structure of the PS and its interaction with cell structures also has a major role in defining the the efficiency of a PS. Having appropriate photophysical characteristics and affinity for cancer cells, porphyrin derivatives are frequently used in PDT studies. The easily prepared 5,10,15,20-tetraphenylporphyrins (TPP) are very commonly used struc- tures, those having hydroxy! substituents in the phenyls and particularly the 3- hydroxyl derivative being the most active¹⁷. However, the structures of such compounds are very different from the natural porphyrins having unsubstituted me- thylenic positions. This structural characteristic can be an obstacle, namely for the required elimination of the sensitizer by the body. A compromise between photo- dynamic activity, synthetic simplicity and a more "natural" structure can be the preparation of diaryl chlorin macrocycles, also having the minimum number of groups that can confer convenient hydrophy!icity to the structure. Another advantage of these macrocycles is their less voluminous structure and low molecular weight, increasing the possibility of having better characteristics for passive diffu- sion across the cellular barriers¹⁸. Examples of the use of this kind of macrocycles in PDT are described by Banfi et al.¹⁹, Bourre^' et al.²⁰ and Senge et al.²¹.

WO 03/064427 A1 describes 5,15-bis(2-bromo-5-hydroxyphenyl)porphyrin used as photodynamic agent..

The photochemistry studies that support this invention allowed for the modulation of the macrocycle structures with the purpose of getting the maximum singlet oxygen quantum yield or the maximum fluorescence quantum yield. This double feature of the invention allows for the synthesis of efficient sensitizers or, alternatively good photodlagnostic compounds through appropriate balance of the required features. The molecules must also have a strong tumor tropism and a very short lifetime in the body. Summary of the invention

The invention relates to macrocyclic tetrapyrrolic compounds of the family of porphyrins, chlorins and bacteriochlorins of the general formula (I)

wherein R¹ and R² are a substituted phenyl group, wherein the substituent at the phenyl group is a halogen atom in ortho or para position of the phenyl ring and a hydroxyl group in meta position of the phenyl ring;

is a single or a double bond;

and wherein positions 2, 3, 7, 8, 12, 13, 17 and/or 18 of the macrocycle are substituted with a group selected from hydrogen atom, halogen atom, alky! group, vinyl group, carboxy group, carboxyalkyl group, alkoxycarbonyl group, alkoxycar- bonylalkyl group, alkoxycarbonylaryl group, acetoxy group or hydroxyalkyl group; wherein the compound 5,15-bis (2-bromo-5-hydroxyphenyl) porphyrin is excluded.

In a preferred embodiment the macrocyclic tetrapyrrolic is a chlorin having a double bond between position 7 and 8 and a single bond between position 17 and18 of the porphyrin ring. In a further preferred embodiment the macrocyclic tetrapyrrolic compounds is a bacteriochlorin having a single bond between position 7 and 8 and a single bond between position17 and 18 of the porphyrin ring. The most preferred compounds are:

5,15-b/s(2-bromo-5-hydroxyphenyl)chlorine,

5,15-o s(2-chloro-5-hydroxyphenyl)chlorine,

5,15-i?/s(2-bromo-5-hydroxyphenyl)bactenochlorine, and

5,15-i)/s(2-chloro-5-hydroxyphenyl) bacteriochiorine.

The compounds according to the invention are used as a medicament, preferably for use in the photodynamic therapy and photodiagnosis. In addition the medicament according to the invention can contain appropriate additives.

Disclosure of the invention This invention is based on the modulation of the structure of the photosensitizer relatively to its photophysical and pharmacological properties. The type of sub- stituents that have been incorporated into the macrocycles was selected with the aim of maximizing the required properties for PDT application. The selection of the adequate pattern of substitution is sustained by our own studies and knowl- edge of photochemical, photophysical and biological properties and the capacity to exploit our expertise in organic synthesis^22"26. The synthetic methods were specifically established to allow efficient preparation of the required sensitizers. The synthetic methods were specifically established to allow efficient preparation of the required sensitizers.

From our studies we concluded that a certain number of halogen atoms located at specific positions in the periphery of the porphyrin macrocycle is a crucial factor in order to maximize the singlet oxygen formation quantum yield by the sensitizer (ΦΔ) and this is an essential part of the invention. Phenyl groups in positions 5 and 15 of the macrocycle with halogen atoms in ortho are particularly efficient, bromine being especially active. The presence of a hydroxyl group, particularly in the meta positions of the mentioned phenyl groups, gives the macrocycle hydro- philic properties, the utility of which is already known²⁷. The specific conditions that allow halogen atoms to affect the singlet oxygen formation quantum yield of these diaryl porphyrins, that turn them into efficient PDT sensitizers is appropriately exploited being part of this invention. This new knowledge is the core of this invention. The type of halogen atom attached to the meso phenyl groups and its most appropriate location can play an essential role, being also part of this inven- tion.

The number of halogen atoms and their location on the macrocycle require a specific tuning to ensure the maximum singlet oxygen quantum yield. Other positions of the porphyrin macrocycle that lack halogen atoms can accommodate substitu- ents such as alkyl, vinyl, carboxyl, carboxyalkyl, alkoxycarbonyl, alkoxycarbon- ylalkyl, alkoxycarbonylaryl, acetoxy or hydroxyalkyl groups in order to grant am- phiphilic characteristics to the porphyrins. Groups that are present in natural porphyrins will be chosen in order to improve the selectivity for different kinds of tumor cells and cellular components and to provide the correct lifetime in order to allow enough time for light treatment, but not so long as to make elimination difficult. The time that the photosensitizer is located in the specific cellular structures has to be long enough to ensure the success of the treatment and short enough to guarantee the elimination from the body and avoid unnecessary tissue accumulation.

The correlation between the structure of the photosensitizer and the ability to interact with light is decisive for the success of the photodynamic treatment. Light to be used in the photodynamic processes must have a wavelength higher than 600 nm. At these wavelengths the tissues are more light-transparent and light has more capacity of tissue penetration⁸. Therefore it is essential that macrocycles present absorption bands with strong absorption coefficients in this region of the spectrum. The photosensitizers object of this patent, besides the presence of halogen and hydroxyl groups to maximize photophysical characteristics and biological requirements, belong to the chiorin and bacteriochlorin type macrocycles which have absorption bands in the red zone of the spectrum with much higher absorption coefficients than porphyrins.

The present invention discloses the use of compounds of the diarylchlorin type (PS1 ,PS2) and corresponding diarylbacteriochlorins for application in phototherapy of tumors and other lesions, having the general formula (II) and (III) described below.

The synthesis of chlorin and bacteriochlorin photosensitizers requires first of ail the preparation of the corresponding porphyrins PSp1 and PSp2 as described below and follows general synthetic processes known by those skilled in porphyrin

chlorins PS1 and PS2 and bacteriochlorins PSbd and PSbc2 which are new compounds. The advantage of these classes of compounds is that they present an intense absorption band at higher wavelengths which is a favorable situation to clinical photodynamic applications.

The above and other objects, features and advantages of the present invention will become apparent from the following description supported by the accompanying figures.

Besides the intense absorption bands at 650 nm for the chlorins PS1 and PS2 and at 740 nm for the bacteriochlorins, PS1 and PS2 also have favorable photo- physical characteristics as illustrated in Table 1.

Table 1. Absorption and fluorescence data, fluorescence quantum yield and singlet oxygen formation quantum yield of photosensitizers PS1 and PS2.

PS Absorption Fluorescence

B(0-0) Qx(1-0) Qx(0-0) Qy{0-0) Q(0-0) Q(0-1 ) Φρ ΦΛ

Amax(nm)e( ^" cm^{" 1}) Amax(^nm)

PS 408 503 596 646 649 695 0.016 0.98

1 7.3x10⁴ 7.3x10^s 2.1x10³ 2.9x10

4

PS 407 502 593 646 648 696 0.048 0.88

2 8.6x10⁴ 7.2x10² 2.6x10² 1.9x10

4

The absorption spectra of photosensitizers PS1 and PS2 show the typical Soret (B(0-0) band) at 408 nm and three Q-bands with the Qy(0-0) band of 5,15- diarylsubstituted chlorins at 646 nm. Relatively to porphyrins PSp1 and PSp2²⁸, these chlorins present a 20 nm shift to the red region and an increase of one hundred times in the absorption coefficient of the band with λ>600 nm, which is a much more favorable situation considering the application of these compounds in PDT. The higher wavelength absorption of the bacteriochlorins PSbd and PSbc2 while maintaining the other characteristic properties of the compounds of this invention, makes them more adequate for specific therapeutic applications. The fluorescence quantum yields for the 5,15-diarylchlorins PS1 and PS2 (Φ_Ρ) have the same order of magnitude of those of the corresponding porphyrins²⁸. The more favorable characteristics of PS1 and PS2 is that the maximum wave- length of the fluorescence band occurs at ca. 650 nm which is a very encouraging property for the use of PS1 and PS2 in photodiagnosis applications.

Another distinct aspect observed in the photophysical characteristics of PS1 and PS2 that is not so predictable by theory is that they present a higher value for the singlet oxygen formation quantum yield (ΦΔ)· For comparison, the ΦΔ of the corresponding porphyrins are about half of these. The presence of a halogen atom also has a favorable influence over the Φ_Λ. Meso-Jb/s-arylporphyrin without halogen has low Φ_Δ ³⁰. The modulation of the physical-chemical properties by halogen introduction in these macrocyc!es is one of the objects of this patent and origi- nates compounds that can be more efficient for PDT due to a higher capacity for generation of singlet oxygen.

The absorption, emission (λ_βχο= 645nm), and fluorescence excitation {λ_βχ0= 649nm) spectra of PS1 is disclosed in figure 1.

The photodynamic activity of compounds PS1 and PS2 were proved against melanoma A375 and WiDr adenocarcinoma human cancer cell cultures. Figures 2 and 3 demonstrate the in vitro phototoxicity of compounds PS1 and PS2 against those human cancer cell lines. The control carried out for each experimental set, in the absence of sensitizer, reveals no cytotoxicity of the solvent mixture used to administer the photosensitizers. Similar experiments carried out in the absence of light and in the presence the photosensitizers reveal no dark cytotoxicity in the range of concentrations used. With respect to Photofrin^R, the results show a much higher phototoxicity of the PS1 and PS2 compounds and prove the advantages of these compounds relatively to other prior art photosensitizers. Figure 2 shows the A375 melanoma cancer cells viability after photodynamic treatment when incubated with different concentrations of photosensitizers PS1 PS2 and photofrin. Figure 3 shows the WiDr adenocarcinoma cells viability after photodynamic treatment when incubated with different concentrations of photosensitizers PS1 , PS2 and photofrin.

Photofrin^R is a sodium profimer of the following formula shown in Fig 4.

Phototoxicity of compounds PS1 and PS2 is clear from the analysis of the very low cell viability observed after the photodynamic treatment when incubated at nanomolar level with the disclosed photosensitizers. The compounds described in this invention can be used for the preparation of pharmaceutical compositions which can be administered to humans or animals. These compositions can be run via enteric, parenteric or transdermic way, and can also be presented as tablets, pills, capsules, suspensions or solutions (oral and intravenous), dermic ointment or transdermic band-aid, in which the com- pound can be associated with additives and/or excipients usually employed in the pharmaceutical art. The doses can be comprised between 0.1 and 20 mg/kg body weight.

EXAMPLES

The following examples are intended to illustrate the invention but not to limit its scope.

Example 1

In vitro studies of phototoxicity Biological studies. The cell culture conditions are as follows. 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 Dul- becco's modified Eagle medium (Sigma D-5648; Sigma-Aldrich, Inc.) supplemented with 10% heat-inactivated fetal bovine serum (Gibco 2010-04; Gibco Invi- trogen Life Technologies), 1% penicillin-streptomycin (Gibco Invitrogen Life Technologies; 100 U/mL penicillin and 10 g/mL streptomycin-Gibco 15140-122) and 100 μΜ Sodium Piruvate (Gibco 1360; Gibco Invitrogen Life Technologies) at 37°C, in a humidified incubator with 95% air and 5% C0₂. Photodynamic treatment: For each experiment, cells were plated in 48 muftiwells (Corning Costar Corp) at 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 H₂0:PEG₄oo:Ethanol (50:30:20, v v), the desired concentrations being achieved by successive dilutions. The sensitizers were administered in several concentrations (50 nM, 250 nM, 500 nM, 1 μΜ, 5 μΜ and 10 μΜ) and cells were incubated for 24 h. Cells were washed with PBS and new drug-free medium was added. Each plate was irradiated at a fluence rate of 7.5 mW cm^"2 until a total of 10 J was reached. Cell viability was measured 24 h after this photodynamic treatment by using the MTT, (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide col- orimetric assay. Cytotoxicity was expressed as the percentage of inhibition of cell proliferation correlated with untreated cultures. Example 2

Preparation of 2,2'-dipyrrylmethane (DP). To a round-bottom flask, pyrrole (20 ml_, 288 mmol), dichloromethane (20 mL), acetic acid (20 mL) and trichloroacetic acid (0.3 g, 18 mmol) were added. The solution was heated to reflux and a mixture of pyrrole (10 mL, 144 mmol) and paraformaldehyde (0.7 g, 23 mmol) in dichloromethane (20 mL) was added. The mixture was left in reflux for 1 h giving a dark-green solution. After cooling, the or- ganic phase was carefully washed with a NaOH 10% solution followed by water and dried with anhydrous sodium sulfate. The solution was concentrated in vacuum and the excess of pyrrole removed by vacuum distillation. Dipyrrylmethane (DP) was purified by flash chromatography (using dichloromethane with a few drops of triethylamine as eluent). A white solid (0.977 g, 29%) was obtained with mp: 73-74°C (lit. 74°C); ¹H NMR (CDCI₃, 400 MHz): δ = 3.88 and 3.96 (s, 2H, methylene bridge-H), 6.03 (m, 2H, pyrrole-H), 6.14 (m, 2H, pyrrole-H), 6.55 (m, 2H, pyrrole-H), 7,83 (bs, 2H, NH); MS (El): rr z 146 (100%, M⁺).

Example 3

Preparation of 5, 15-b/s(2-bromo-5-hydroxyphenyl)porphyrin (PSp1 ).

In a 1 L round-bottom flask with dichloromethane (650 mL), the compound prepared in example 2(DP) (0.50 g, 3.4 mmol) and 2-bromo-5- hydroxybenzaldehyde¹⁶ (0.82 g, 4.1 mmol) were added. The solution was stirred at room temperature for 20 min under nitrogen and then trifluoroacetic acid (0.26 mL, 3.5 mmol) was added. The mixture was left overnight. The mixture was neutralized with triethylamine and DDQ (1.17 g, 5.1 mmol) was added. After 2 h, the solution was evaporated to dryness and the porphyrin purified by dry flash chromatography (CH₂CI₂ / ethyl acetate, 9 :1 ). A violet solid was obtained (0.24 g, 11 %). ¹H NMR (DMSOd₆, 400 MHz): δ = 7.24 (2H, ArH), 7.70 (d, J = 3.03 Hz, 2H, ArH), 7.87 (d, J = 8.7 Hz, 2H, ArH), 8.95 (d, J = 4.6 Hz, 4H, pyrrole-H), 9.38 (d, J = 4.6 Hz, 4H, pyrrole-H) 10.29 (s. 2H, methylene bridge-H). MS (ESI): rr z 653 [(M + 1)*]. HRMS (FAB): rr z calc. for C₃2H2iBr₂N₄02 (M+H)⁺, 653.0054; found, 652.99809. Example 4

Preparation of 5_r15-6/s(2-chloro-5-hydroxyphenyl)porphyrin (PSp2)

According to the procedure described in example 3 with dipyrrylmethane (0.92 g, 6.3 mmol) prepared in example 2 and 2-chloro-5-methoxybenzaldehyde²⁸ (1 .08 g, 6.3 mmol) in dichloromethane (750 ml_) and trifluoroacetic acid (0.50 mL, 6.7 mmol) as catalyst. A solid was isolated (0.21 g, 23%) from silica chromatography (CH₂C!₂) which was identified as the 5,15-jb/s(2-chloro-5-methoxyphenyl)porphyrin by mass spectrometry. MS (ESI): rr z 591 [(M + 1 )*]. The demethylation was carried out as follows. The porphyrin (100 mg, 0.13 mmol) was dissolved in dichloromethane (15 mL), the solution cooled to -25°C and 1 mL of BBr₃ solution was slowly added. The mixture was left overnight at room temperature and was diluted with ethyl acetate and treated with methanol and triethylamine. The organic phase was carefully washed with water, dried with sodium sulfate and evaporated in vacuum to give 80 mg (84%) of 5,15-i>/s(2-chloro-5- hydroxyphenyl)porphyrin (PSp2). ¹H NMR (DMSOd6, 400 MHz): δ = 7.24 (dd, J = 2.9, 8.7 Hz, 2H, ArH), 7.55-7.64 (m, 4H, ArH), 8.93 (d, J = 4.6 Hz, 4H, pyrrole-H), 9.31 (d, J = 4.6 Hz, 4H, pyrrole-H), 10.21 (s. 2H, methylene bridge-H),10.26 (s. 2H_T methylene bridge-H). MS (ESI): rr z 563 [(M + 1 )⁺]. HRMS (FAB): rr z calc. For C-₃₂H2iCI₂N₄0₂ (M+H)⁺, 563.10361 ; found, 563.10151 .

Example 5

Preparation of 5,15-£>/s(2-bromo-5-hydroxyphenyl)chlorin (PS1 )

5,15-ib/s(2-bromo-5-hydroxyphenyl)porphyrin (90 mg, 0.138 mmol), anhydrous K2CO3 (360 mg) and anhydrous pyridine (7 mL) were heated at 1 10°C for 5 min under N₂. A solution of 350 mg of p-toluenesulfonylhydrazide in 1.5 mL of pyridine was added in three portions during 30 minutes. The solution was kept at this temperature until the maximum for the bacteriochlorin band (734 nm) is reached (1 -2 hour). After cooling, the pyridine was evaporated under vacuum. The resulting solid product was redissolved in ethyl acetate and the organic phase was washed with water and dried over anhydrous Na₂S0₄. After filtration, p-cloranil (80 mg) was added in small portions to the stirred organic solution at room temperature until the absorption peak of the bacteriochlorin had disappeared. The solution was washed with NaHS0₄ (5%), distilled water and saturated bicarbon- ate. The resulting solution was stirred with a solution of 2M HCI for 30 min. The organic layer was separated, dried (MgS04) and concentrated on a rotary evaporator. The residue was chromatographed (silica-gel, CH₂CI₂/ethyl ace- tate/triethylamine, 60:39:1 ) to give 60 mg of 5,15-jb/s(2-bromo-5- hydroxyphenyi)chlorin as a dark green solid. Vis/UV (ethyl acetate)(A_maxnm): 407, 503, 529, 590, 645; ¹H NMR (CDCI₃, 400 MHz): δ - 9.83 (s, 1 H, meso-H), 9.10 (d, 1 H, J = 4.5 Hz, pyrrole-H ), 9.00 (s, 1 H, meso-H), 8.94 (d, 1H, J = 4.2 Hz, pyrrole- H), 8.79 (d, 1 H, J = 4.5 Hz, pyrrole-H), 8.66 (d, 1 H, J = 4.6 Hz, pyrrole-H), 8.48 (d, 1 H, J - 4.2 Hz, pyrrole-H), 8.29 (d, 1 H, J = 4.6 Hz, pyrrole-H), 7.90-7.0 (m, 6H), 4.69 (m, 2H), 4.28 (m, 6H), -1.94 (s, 1 H). MS (MALDI-TOF): rrvt 654 [M ⁺].

Example 6

Preparation of 5,15-£)/5(2-chloro-5-hydroxyphenyl)chiorin (PS2)

According to the procedure described in example 7, 43 mg of 5, 5-£>/s(2-chloro-5- hydroxyphenyl)chlorine was obtained. Vis/UV (ethyl acetate)(A_ma_Xnm): 403, 500, 526, 589, 643; ¹H NMR (CDCI_3> 300 MHz): 6 = 9.81 (m, 1 H, meso-H), 9.39 (m, 1 H, pyrrole-H ), 9.09 (m, 1 H, meso-H), 8.95 (m, 1 H, pyrrole-H), 8.78 (d m, 1 H, pyrrole-H), 8.64 (m, 1H,pyrrole-H), 8.48 (m, 1 H, pyrrole-H), 8.26 (m, 1 H, pyrrole- H), 7.90-7.0 (m, 6H), 4.68 (m, 2H), 4.26 (m, 6H), -2.01 (s, 1 H). MS (MALDI-TOF): rryz 564 [M ⁺].

References

1) Bonnett, R. Chem. Soc. Rev. 1995, 24, 19-33.

2) Bonnett, R. Chemical Aspects of Photodynamic Therapy; Gordon and Breach science Publishers: Amsterdam, 2000; Vol. 1.

3) Dougherty, T. J. Photochem. Photobiol. 1993, 58, 895-900.

4) Jori, G.J. Photochem. Photobiol. A: Chem. 1992, 62, 371.

5) acDonald, I. J.; Dougherty, T. J. J. of Porphyrins and Phthalocyanines 2001 , 5, 105-129.

6) Abels, C; Goezt, A. E. A Clinical Protocol for Photodynamic Therapy; Abels, C; Goezt, A. E., Ed.; OEMF spa: Milano, 1996, pp 265-281.

7) Reddi, E. Strategies Adopted for Optimizing the Photodynamic Therapy of Tumors; Reddi, E., Ed.; OEMF spa: Milano, 1996, pp 247-264.

8) Plaetzer, K., Krammer, B., Berlanda, J., Berr, F., Kiesslich, T. Lasers Med. Sci. 2009, 24, 259-268.

9) Nyman, E. S., Hynninen, P. H., J. Photochem. Photobiol. B. 2004, 73, -28

10) Juzeniene, A., Peng, Q., Moan, J. Photochem. Photobiol. Sci. 2007, 6, 1234- 1245

11) Detty, M. R., Gibson, S. L, Wagner, S. J. J. Med. Chem. 2004, 47, 3897- 3915

12) Marcus, S. J., Mclntyre, W. R. Expert Opin. Emerging Drugs 2002, 7, 321-334

13) Serra, M., Pineiro, M., Pereira N., Rocha Goncalves A., Laranjo M.,

Margarida A., Botelho F., Oncol. Rev. 2008, 2, 189-203.

14) Pineiro, M., Serra, A. C, Rocha Gonsalves, A. M. d'A., Recent Patents on Chemical Engineering 2009, 2, 98-122.

15) Azenha, E. G., Serra A. C, Pineiro, M., Pereira, M. M., Seixas de Melo, J. S., Arnaut, L. G., Formosinho, S. J., Rocha Gonsalves, A. M.d. A., Chem. Phys. 2002, 280, 177-190.

16) Serra, A. C, Pineiro M., Rocha Gonsalves, A. M. d. A., Abrantes, M., Laranjo, M., Santos, A. C. Botelho, M. F. J. Photochem. Photobiol. B 2008, 92, 61-67,

17) Berenbaum, M. C, Akande, S. L., Bonnett, R., Kaur, H., loannou, S., White, R. D. & Winfield, U. J. Br. J. Cancer 1986, 54, 712-725.

18) Fischer, H., Gottschlich, R., Seeling, A., J. Membrane Biol. 1998, 165, 201- 211. 19) Banfi, S., Caruso, E., Buccafurni, L, Murano, R. Monti, E. Gariboldi, M., Papa, E., Gramarica, P. J. Med. Chem 2006, 49, 3293-3304.

20) Bourre, L, Thibaut, S. Fimiani, M., Ferrand, Y., Simonneaux, G., Patrice, T., Pharmacol. Res. 2003, 47, 253-26 .

21 ) Wiehe, A., Shaker, Y. M., Brandt, J. C, Mebs, S., Senge, M. O. Tetrahedron 2005, 61 , 5535-5564.

22) Johnstone, R. A. W.; Nunes, M. L. P. G.; Pereira, M. M.; Rocha Gonsalves, A. M. d'A.; Serra, A. C, Heterocycles 1996, 43, 1423-1437.

23) 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., Heterocycles 1996,

43, 829-838

24) Nascimento, B. F. O., Pineiro, M., Rocha Gonsalves, A. M. d. A., Ramos Silva, M., Matos Beja, A. and Paixao, J. A, J. of Porphyrins and Phthalocyanines 2007, 11 , 77-84.

25) Rocha Gonsalves, A. M. d'A., Serra A C, Pineiro M., J. of Porphyrins and Phthalocyanines 2007, 13, 429-445.

26) Pineiro, M.; Carvalho, A. L; Pereira, M. M.; Rocha Gonsalves, A. M. d'A.; Arnaut, L. G.; Formosinho, S. J., Chem. Eur. J. 1998, 4, 2299-2307.

27) Bonnett, R., Berembaum, M. C, US-4,837,221 (1989)

28) Serra, A., Pineiro, M., Santos, C. I., Rocha Gonsalves, A. M. d'A., Abrantes, M., Laranjo, M., Bote!ho, M. F., Photochem. Photobiol., 2010, 86, 206-212.

29) Whitlock, H. W.; Hanauer, R.; Oester, M. Y.; e B. K. Bower, B. K. J. Am. Chem. Soc. 1969, 91, 7485-7489.

30) Bourre, L, Simonneaux, G., Ferrand, Y._t Thibaut, S., Lajat, Y., Patrice, T., J. Photochem. Photobiol., B.: 2003, 69, 179-192.

Claims

Claims

1. Macrocyclic tetrapyrrolic compound of the family of porphyrins, chlorins and bacteriochlorins of the general formula (I)

wherein R¹ and R² are a substituted phenyl group, wherein the substituent at the phenyl group is a halogen atom in ortho or para position of the phenyl ring and a hydroxyl group in meta position of the phenyl ring;

is a single or a double bond;

and wherein positions 2, 3, 7, 8, 12, 3, 17 and/or 18 of the macrocycle are substituted with a group selected from hydrogen atom, halogen atom, alkyl group, vinyl group, carboxy group, carboxyalkyl group, alkoxycarbonyl group, alkoxycar- bonylalkyl group, alkoxycarbonylaryl group, acetoxy group or hydroxyalkyl group; wherein the compound 5,15-bis (2-bromo-5-hydroxyphenyl) porphyrin is excluded.

2. Macrocyclic tetrapyrrolic compounds according to claim 1 , wherein the compound is a chlorin having a double bond between position 7 and 8 and a single bond between position 17 and 18 of the porphyrin ring.

3. Macrocyclic tetrapyrrolic compounds according to claim 1 , wherein the compound is a bacteriochlorin having a single bond between position 7 and 8 and a single bond between position 17 and 18 of the porphyrin ring.

4. Compound according to claims 1 or 2, characterized in that the compound is 5,15-b;s(2-bromo-5-hydroxyphenyl)chlorin.

5. Compound according to claims 1 or 2, characterized in that the compound is 5,15-b/s(2-chloro-5-hydroxyphenyl)chlorin.

6. Compound according to claims 1 and 3, characterized in that the compound is 5, 5-jb/s(2-bromo-5-hydroxyphenyl)bacteriochlorin.

7. Compound according to claims 1 and 3, characterized in that the compound is 5,15-£w^'s(2-chloro-5-hydroxyphenyl) bacteriochlorin.

8. The compounds according to claims 1 to 7 for use as a medicament.

9. The medicament according to claim 7, characterized in that the medicament further comprises an appropriate additive.

10. Medicament comprising the compounds according to any of the claims 1 to 7 for use in the photodynamic therapy and photodiagnosis.