Synthesis of meso -substituted cationic porphyrins as potential photodynamic agents

Novel meso -substituted cationic porphyrins have been synthesized as potential photodynamic agents. 5,10-bis(4-Acetamidophenyl)-15,20-bis(4-methylphenyl)porphyrin 1 was prepared using a modification of the Alder-Longo procedure. However, two different approaches were compared in the synthesis of 5-(4-trifluorophenyl)-10,15,20-tris(4-acetamidophenyl)porphyrin 2 . One involved a condensation of binary mixture of aldehydes and pyrrole under equilibrium conditions and the other a binary mixture of aldehydes and meso -(4-acetamidophenyl)dipyrromethane 3 . The last synthetic pathway was advantageous mainly for an easier reaction work up and a higher yield. Both amido porphyrin 1 and 2 were hydrolyzed in basic media and treated with methyl iodide to form 5,10-di(4-methylphenyl)-15,20-di(4-trimethylammonium phenyl)porphyrin iodide 4 and 5-(4-trifluorophenyl)-10,15,20-tris(4-trimethyl ammoniumphenyl)porphyrin iodide 5 , respectively. Porphyrin 5 bears a highly lipophilic trifluoromethyl group, which increases the amphiphilic character of the structure. On the other hand, the photodynamic properties of porphyrin 5 were changed forming metal complex with Pd(II), porphyrin 6 . Absorption and fluorescence spectroscopic studies of these porphyrins were compared in different solvents. These amphiphilic cationic porphyrins are promising photosensitizers with potential applications in bacteria inactivation by photodynamic therapy.


Introduction
Tetrapyrrolic macrocycles occupy a central place in bioorganic chemistry. 1Meso-substituted porphyrins have recently found specific biomedical applications, particularly in the field of detection and treatment of neoplastic tissues. 2,3Photodynamic therapy (PDT) consists of the administration of a photosensitizer, which is selectively retained by the neoplastic tissue.The subsequent irradiation with visible light in the presence of oxygen specifically inactivates tumor cells. 4,5Adequate photosensitizers are deemed to have a high absorption coefficient in the visible region of the spectrum and have a triplet excited state, with a long lifetime, to efficiently produce O 2 ( 1 ∆ g ). 6][9][10][11] These porphyrins are able to interact with DNA bases, inducing DNA lesions upon photoactivation. 12,13Recently, cationic porphyrins have also shown important applications as sensitizers to photoinduce direct inactivation of multidrug resistant microorganisms. 14Gram-positive bacteria are susceptible to the photodynamic effect produced by neutral and anionic sensitizers.Gram-negative bacteria have only been inactivated in these conditions in the presence of an agent, which stimulates the membrane translocation of the porphyrins. 15][18] The combination of hydrophobic and hydrophilic substituents in the sensitizer structure results in an intramolecular polarity axis, which can facilitate membrane penetration and produces a better accumulation in subcellular compartments, enhancing the effective photosensitization. 19,20This sensitizer architecture required the formation of asymmetrically meso-substituted porphyrins.These porphyrins containing two different types of mesosubstituents (A and B) can be prepared by a binary mixed aldehyde and pyrrole condensation. 21,22 his approach is statistical in nature and usually six porphyrins are formed, which involve AABB-and AB 3 -porphyrin patterns. 23However, the isolation of the desired porphyrin requires slow and careful chromatography for purification, the yield is very poor and obtaining pure porphyrin is not always possible.A convenient approach for the synthesis of meso-substituted trans-porphyrins (ABAB-porphyrins) has been developed from the condensation of dipyrromethane with an aldehyde in a MacDonald-type 2+2 condensation. 24,25lso, condensation of a dipyrromethane with a binary mixture of aldehydes was used to obtain meso-substituted porphyrins bearing three identical substituents, B and one different A (AB 3porphyrins). 26,27In these cases, the structure A bears a functional group, which can be used to link the porphyrin with other molecules, while B was used to change the macrocycle properties.
5-(4-Trifluoromethylphenyl)-10,15,20-tris(4-acetamidophenyl) porphyrin 2 was synthesized by two different methods.In one case, the AB 3 -porphyrin 2 was obtained by a condensation of a binary mixture of aldehydes and pyrrole catalyzed by trifluoroacetic acid, TFA, at room temperature (Scheme 2).After treatment with DDQ, the mixture produced porphyrin 2, which was separated by column chromatographic with a yield of 3.6 %.

Scheme 2
The alternative method to synthesize porphyrin 2 involved two steps: 1) formation of meso-(4-acetamidophenyl)dipyrromethane 3 (Scheme 3) and 2) condensation of dipyrromethane 3 with the appropriate benzaldehyde mixture (Scheme 4).Thus, dipyrromethane 3 was obtained from the condensation of 4-acetamidobenzaldehyde with a large excess of pyrrole (1:38 aldehyde/pyrrole mol ratio) catalyzed by TFA (Scheme 3).The reaction mixture was stirred for 20 min at room temperature resulting in the complete disappearance of aldehyde.In these reaction conditions, pyrrole serves as the reactant in excess and as the solvent for the reaction, giving direct formation of dipyrromethane.The brown crude solution was washed with dilute aqueous NaOH.The dipyrromethane 3 was isolated (70 %) by flash chromatography on silica gel in a mildly basic medium, using ethyl acetate/triethylamine (100:1) as eluent.Under this condition, triethylamine prevents decomposition of the dipyrromethane on silica column, which is slightly acidic.Dipyrromethane 3 is stable in the purified form upon storage at 0°C in nitrogen atmosphere and absence of light.Then, dipyrromethane 3 was condensed with 4acetamidobenzaldehyde and 4-trifluoromethylbenzaldehyde [2:0.9:1.1 mol ratio, respectively] under catalysis by TFA and at room temperature to form a mixture of porphyrins.The reaction was catalyzed by TFA at room temperature.After oxidation with DDQ, the desired amido porphyrin 2 (15 %) was separated by flash chromatography.Porphyrin properties and spectroscopic studies Porphyrin 4 bears two 4-methylphenyl groups and two cationic 4-trimethylammoniumphenyl groups with a AABB-porphyrin symmetry, while porphyrin 5 contains one 4trifluoromethylphenyl group and three 4-trimethylammoniumphenyl groups.To evaluate the effect produced by this distribution of different polarity groups upon the intramolecular polarity, the dipole moments of the porphyrins were estimated.The semi-empirical method for molecular modeling (AM1) was used in structure geometry optimization calculations.Values of 39.7 D and 39.9 D were found for 4 and 5, respectively.These values are approximately 10 times higher that of the corresponding porphyrin models substituted by amino and methyl groups.As expected, the presence of cationic groups in the periphery of porphyrins considerably enhances the dipole moment with respect to the non-charged structure.In particular, for porphyrin 5, a trifluoromethyl group produces an increase from 25.4 D to 39.9 D with respect to a methyl group.
The absorption spectrum of porphyrins 4, 5 and 6 in methanol show the typical Soret and Qbands characteristic of free-base porphyrin derivatives and their metal-complex (Figure 1A). 37he spectra of porphyrins were also recorded in different solvents.The absorption maxima are summarized in Table 1.The solvatochromic effect on the location of Soret absorption band shows a slight blue shift upon solubilization in methanol.The steady-state fluorescence emission spectra of porphyrins 4 and 5 in methanol are shown in Figure 1B.No emission from the Pd(II) porphyrin 6 was detected.Two bands are characteristic for porphyrin derivatives and they have been assigned to Q(0-0) and Q(0-1) transitions.Also, a small Stokes shift is expected for tetraphenylporphyrin derivatives indicating that the spectroscopic energy is nearly identical to the relaxed energy of the singlet state. 38

Conclusions
Two different approaches were compared in the synthesis of 5-(4-trifluorophenyl)-10,15,20tris(4-acetamidophenyl)porphyrin 2. One involved a condensation of binary mixture of aldehydes and pyrrole and the other a binary mixture of aldehydes and meso-(4acetamidophenyl)dipyrromethane 3 catalyzed by acid.The last synthetic pathway was advantageous mainly for a relatively simpler reaction work up and higher yield (~4 times) than the reaction in the presence of pyrrole.However, this approach can not be used to synthesize AABB-porphyrin patterns, such as 1.Thus, porphyrin 1 was obtained by the first method with a low yield.Both amido porphyrin 1 and 2 were hydrolyzed in basic media and treated with methyl iodide to form dicationic 4 and tricationic 5 porphyrin derivatives, respectively.Porphyrin 5 bears a highly lipophilic trifluoromethyl group, which increases the amphiphilic character of the structure.On the other hand, the photodynamic properties of porphyrin 5 were changed by forming metal complex with Pd(II), porphyrin 6.These amphiphilic cationic porphyrins offer a promising molecular architecture for photosensitizer agents with potential applications in bacteria inactivation by photodynamic treatment.Studies of photosensitization in vitro, using Gram-negative bacteria Escherichia coli, are presently in progress in our laboratory.

Experimental Section
General Procedures.UV-visible and fluorescence spectra were recorded on a Shimadzu UV-2401PC spectrometer and on a Spex FluoroMax fluorometer, respectively.Proton nuclear magnetic resonance ( 1 H NMR) spectra were recorded on a Bruker ARX 300 multinuclear spectrometer at 300 MHz.Mass Spectra were taken with a Varian Matt 312 operating in EI mode at 70 eV.Silica gel (230-400 mesh, Merk) was used for column chromatography.Alumina neutral (150 mesh, Aldrich) was employed.Silica gel thin-layer chromatography (TLC) plates 250 microns from Aldrich (Milwaukee, WI, USA) were used.All the chemicals from Aldrich were used without further purification.Solvents (GR grade) from Merck were distilled.Semiempirical molecular orbital calculations (AM1) were carried out using HyperChem software.

5,10-bis(4-Acetamidophenyl)-15,20-bis(4-methylphenyl)porphyrin (1).
A solution of 4methylbenzaldehyde (3.42 g, 21 mmol) and 4-acetamidobenzaldehyde (2.52 g, 21 mmol) in propionic acid (150 mL) was stirred at 90 °C and pyrrole (3.0 mL, 42 mmol) was slowly added.The resulting mixture was heated to reflux for 40 min.The mixture was poured into of water (500 mL), containing of NaCl (20 g).The precipitated material was collected by vacuum filtration and dried under vacuum at 100 ºC for 48 hours to remove propionic acid.The resulting material was dissolved in chloroform/methanol (5%) and filtered through a short column of alumina (5 cm).This procedure was repeated using silica gel.The solvents were removed under reduced pressure and the residue treated with DDQ (2.27 g, 10 mmol) in 200 mL of chloroform for 3 hours.The solvent was evaporated and the residue was pulverized to a fine powder.Slow chromatographic column (silica gel, chloroform/methanol gradient) afforded porphyrin 1 (purple solid, 111 mg, 1.4 %) as second moving band.TLC analysis (dichloromethane/methanol 5%) R f 0.35.