Microwave-assisted Synthesis of Unsymmetrical Tetrapyrrolic Compounds

RADU SOCOTEANU1, RICA BOSCENCU2*, VERONICA NACEA2, ANABELA SOUSA OLIVEIRA3, LUIS FILIPE VIEIRA FERREIRA3 1 Romanian Academy, Institute of Physical Chemistry “Ilie Murgulescu”, 202 Splaiul Independentei, 060021, Bucharest, Romania 2 "Carol Davila” University of Medicine and Pharmacy, Faculty of Pharmacy, Inorganic Chemistry Department, 6 Traian Vuia, 020956, Bucharest, Romania 3 CQFM-Centro de Química-Física Molecular and IN-Institute Nanoscience and Nanotecnology, Complexo Interdisciplinar, Instituto Superior Técnico, Av. Rovisco Pais 1049-001, Lisbon, Portugal

Porphyrins are a class of naturally occurring macrocyclic compounds, which play a very important role in the metabolism of living organisms [1,2].They have attracted attention for many decades due to their biomedical applications such as photodynamic therapy, treatment of psoriasis, viral and bacterial infections including HIV [3][4][5][6][7][8].
Photodynamic therapy (PDT) has emerged as a useful alternative to chemotherapy or radiotherapy in cancer treatment, offering improved selectivity due to preferential accumulation of the photosensitizing agent and controlled light delivery at the tumour.PDT, as a selective method of antitumor treatment, supposes the administration of the pharmaceutical formulation containing the photosensitizer, its selective location at the level of tumoral cells, laser irradiation of the loaded tissue followed by the generation of a very reactive chemical species -the singlet oxygen ( 1 O 2 ) which destroys the tumour cells [9][10][11][12][13][14].
The most important advantage of photodynamic therapy consists in the use of visible and ultraviolet light, supposing much lower energies than those used in the classical radiologic therapy.
A good PDT photosensitizer has to answer the following demands: it must have absorption coefficients as great as possible in the 630-680 nm field, a great singlet oxygen yield, non-toxicity, photostability, amphiphilic properties (which allow a good location of the photosensitizer at cellular level); the xenobiotic has to be rapidly eliminated from the body after performing the procedure, and its metabolites should not be toxic [15][16][17][18][19][20][21][22].
High-speed synthesis with microwave irradiation has attracted considerable attention in recent years [23].Microwave-assisted reactions are believed to facilitate polarization of the substrates thereby promoting the reactions [24][25][26].Microwave-assisted reactions have become increasingly important in tetrapyrolic compounds synthesis due to advantages such as: significant reduction in reaction times, side reactions, increased yields, ease of purification, and minimization of the amount of solvent used [27,28].
We previously reported the synthesis of unsymmetrical porphyrins by classical methods: these unsymmetrical porphyrins were typically synthesized in reflux of propionic acid, the yields were relatively poor (~ 10%) and the reaction mixture was stirred under reflux for 3h at 125°C [29].

Materials and methods
Commercially available chemicals and solvents were used as received from Aldrich, Merck and Sigma.
The NMR spectra of the synthesized complexes were recorded with a 400 MHz Bruker NMR Spectrometer. 1 H-NMR, 13 C-NMR, DEPT, HMQC, HMBC and COSY spectra were measured.
IR spectra were recorded with a FTIR 400D Nicolet Impact spectrophotometer.The substances under analysis, previously dried for 24 h, at 150 o C, were processed as a KBr pellet of spectroscopic purity.The spectra were recorded in the 4000-500 cm -1 spectral range.
The molecular absorption spectra were recorded with a Specord M400 Carl Zeiss Jena UV-Vis Spectrometer, assisted by an internal computer, within a spectral range of 210-900 nm.All spectra were recorded in single-beam system, in order to eliminate the specific adsorption of the solvent used and the absorption differences caused by the optical pathway.

Synthesis and purification of porphyrinic compounds
Methyl 4-formyl benzoate (0.36 mol), hydroxy substituted benzaldehyde (0.12 mol), fresh distilled pyrrole (0,48 mol) and 10g of Kieselgel 60, 200-500 µm, 35-70 mesh, dry silica was mixed at room temperature in a Pyrex bottle.The mixture reaction was subsequently subjected to microwave irradiation for 12 min.The power of microwave oven was set to 450 W. The extraction of samples for monitoring analysis was performed after every 2 minutes of irradiation.Upon completion of the reaction, the catalyst was separated by filtration and washed thoroughly with 150 ml of dichloromethane.The solvent was removed under reduced pressure to give a viscous residue.
The crude product was then purified by several column chromatography elutions, using dichloromethane as eluent and silica gel (100-200 mesh size) as stationary phase; to give the final substituted porphyrins, preparative TLC (on 2 mm Kieselgel 60 plates) was used.
Following preparation porphyrins were characterized by UV, IR and NMR spectrometry.
The obtained yields are 13% for TCMPOH M and 38% for TCMP.

Infrared spectra
Table 1 presents all the relevant band frequencies and assignments of the porphyrinic compounds herein synthesized.The spectral data are in accordance with the proposed structures for the synthesized porphyrins.In this context, the analysis of the IR spectra of TCMPOH M and TCMP confirms the presence of the characteristic bands of ν N-H and ν H-O in the spectral range 3310-3521 cm -1 .The N-H stretching bands are observed in the spectral range 3310-3440 cm -1 and the presence of the -OH functional group in the TCMPOH M was confirmed by the IR spectra where the band at 3503 cm -1 , can be assign to the O-H vibrations.Other bands observed in the higher wavenumber region (2952 cm -1 -2923 cm -1 ) are due to the stretching vibrational motion of C-H bond of the porphyrin ring.Furthermore the IR spectrum of substituted porphyrinic compounds clearly indicates the presence of the -O-CH 3 group at ~2851 cm -1 and C=O bands at ~ 1717 cm -1 and the bands at ~ 1597-1603 cm -1 and 1543-1560 cm -1 can be assign to C-N stretching vibrations.

UV-Vis spectra
UV-Visible spectra have been studied for TCMP and TCMPOH M in various solvents and the results obtained are presented in figures 3 and 4. The spectroscopic data obtained were in agreement with the proposed structure of the dyes.
The electronic absorption spectrum of these porphyrinic compounds is dominated by the very strong Soret band located in the spectral range of 416.5nm -424.5nm,depending on the solvent were the spectra were measured.The other bands, commonly referred as Q bands, consist of four absorption peaks which are typical of Q x (0,0), Q x (1,0), Q y (0,0), Q y (1,0) transitions in the free base porphyrins D 2h symmetry and appears at the red side of the spectrum, respectively from 513nm to 674 nm.The absorption data of substituted porphyrins recorded in various solvents are listed in table 2.
A slightly bathochromic shift is observed on Soret band, according to the solvent type used as follow: ethanolmethylene chloride -DMSO; at the same measurement parameters was registered the hypochromic displacement of the Q bands: ethanol -DMSO -methylene chloride, as shown in figure 3 and 4 respectively.

NMR experiments
In this study, various NMR experimental approaches (including 13 C-NMR, DEPT 90, DEPT 135, COSY, HMBC, and HMQC) were developed.To clearly illustrate the relation structure-NMR spectra we present the β pyrrole and mesopositions (according to Fischer's rule) and also, the terms of substituents arrangement (fig.5).We present below, the chemical shifts and the multiplicities of the 1 H-NMR signals for TCMPOH M and TCMP, besides important adjacent data provided by 13 C-NMR (table 3).

Conclusions
In this paper we presented the synthesis of substituted porphyrins using microwave irradiation assisted synthesis.Microwave assisted solvent-free chemistry proved to be a technique that has the power to accelerate the generation of porphyrinic compounds.Also, the absence of solvent clearly reduces the reaction time and generally improves the yields.The reduced numbers of adjacent compounds, including chlorine recommend this method also from ecological point of view.In this way, the synthetic process is under complete control, with good yields.
The spectral properties of the porphyrinic compounds were investigated by FTIR, NMR and UV-Vis spectroscopy.Spectroscopic data were in agreement with the desired structure.
The UV-VIS spectra showed the absorption peaks of the Soret bands of the porphyrinic compounds at about 417-424 nm, which was slightly red shifted relatively to the 5,10,15,20-tetraphenyl-porphyrin (TPP, 416nm).The four weak Q bands located from 513 nm to 674 nm confirm the heterocycle formation.
NMR and IR data complete the structural frame.The single peak at about -2,81 ppm in 1 H NMR spectra of the compounds points the presence of the -NH group in porphyrinic core, concluding the successful of the reaction.

Table 2
UV-VIS SPECTRAL CHARACTERISTICS OF THE STUDIED PORPHYRINIC COMPOUNDS

Table 3
DATA FROM 1 H-NMR AND13C-NMR FOR THE SYNTHESIZED PORPHYRINS