Synthesis , Spectroscopy and Photosensitizing Properties of Hydroxynitrophenylporphyrins

As hidroxinitrofenilporfirinas, 5,10,15,20-tetrakis(2-hidroxi-5-nitrofenil)porfirina e 5mono(carboxifenil)-10,15,20-tris(2-hidroxi-5-nitrofenil)porfirina, estudadas neste trabalho, foram preparadas pelo método de Adler e caracterizadas por H RMN e por espectroscopia de absorção e emissão na região espectral visível. A formação de O 2 foi constatada através do teste do ácido úrico (AU), o qual consiste na observação do decréscimo na absorbância da banda do AU em 293 nm, após irradiação da solução, com laser, contendo o fotossenssibilizador e o AU. Os resultado obtidos indicaram que as hidroxinitrofenilporfirinas podem ser consideradas potenciais fotossensibilizadores para terapia fotodinâmica.


Introduction
Photodynamic therapy (PDT) is one of the most promising modalities currently explored for use in tumor control. 1 PDT involves selective retention of a photosensitizer in the tumor, followed by irradiation with light of a particular wavelength, which initiates tumor necrosis presumably through the formation of singlet oxygen.Optimal tissue penetration by light occurs between 650-800 nm.The first generation of sensitizers, represented by Photofrin ® , absorbs only weakly at about 630 nm.This has led to the search for the so-called "second generation" sensitizers, which absorb at longer wavelengths and have well established structures.Porphyrins, and porphyrin-type macrocycles, such as chlorins, bacteriochlorins and purpurins, represent a major class of photosensitizing agents that are useful in an array of medical areas, including oncology, cardiology, ophthalmology, dermatology, immunology, gynecology and urology. 2 One example concerning the ophthalmology area is a recent (November 1999) recommendation by the FDA scientific advisory board for approval of Visudyne  (verteporfin, a benzoporphyrin mono-acid ring A, derivative BPDMA) as a PDT-type photosensitizer for the treatment of the agerelated macular degeneration (AMD).Other porphyrin-type agents such as Purlytin  (SnET2, by Miravant Medical and Pharmacia Upjohn) and Optrin  , a Motexafin luteniun (Lu-Tex, by Pharmacyclics Inc.) are also under current clinical investigation for the treatment of AMD.Chlorin derivatives such as NPe 6 (mono-L-aspartyl chlorin e 6 , developed by Nippon Chemical Co.) and m-THPC (Temoporfin, by Scotia QuantaNova) have been tested as sensitizing agents in the treatment of a number of malignancies for oncological applications. 2 major drawback of most sensitizers systematically used for PDT today is skin photosensitization.The use of the appropriate sensitizer dose and bleaching rates may significantly decrease the irreversible photodynamic injury caused to the normal tissues.In the majority of the studies involving photobleaching in PDT, the mechanistic processes are oxidative, and usually involve singlet oxygen or reactive radicalar oxygen species. 3Additional information about the photobleaching behavior of sensitizers is needed to calculate the optimum PDT dose.The first relevant observation of photobleaching was reported in 1986 by Moan et al, using Photofrin. 4Studies with m-THPC, which has been investigated in both preclinical and preliminary clinical studies with promising results, have attracted interest in recent years. 5It is noteworthy that m-THPC is a very powerful PDT photosensitizer and presents some advantages over HPD and Photofrin in many aspects: (i) it has been selected on the basis of tumor necrotic activity in vivo after examining a considerable number of photosensitizer preparations and because of its selectivity for tumor over normal tissue; (ii) it has strong absorption at 652 nm, and a Φ ∆ value of 0.43; (iii) the synthetic route is convenient and short (iv) it is a single substance; (v) dark toxicity is low and it is not mutagenic.

Experimental
Ultraviolet-visible (UV-Vis) absorption spectra were recorded on a Hewlett-Packard 8453, Diode Array spectrophotometer.Emission spectra were recorded on a SPEX TRIAX FLUOROLOG 3, using spectroscopic grade methanol as solvent.Infrared (IR) spectra were recorded on a Nicolet FT-IR 5ZDX spectrometer. 1H NMR: Nuclear magnetic resonance spectra were recorded on a Brüker DR XC 400 MHz, 9,4 T spectrometer in MeOD using TMS as internal standard.The chemical shifts are expressed in δ (ppm).
The uric acid test was recorded on a Hewlett Packard 8543 spectrophotometer, and irradiation with a Radio Shack LX 1000, 5 mW, 671 nm.
All reagents were purchased from Aldrich or Sigma and used without further purification.Solvents were dried when necessary using standard techniques.Column chromatography was carried out with silica gel 60 (Merck, 230-400 mesh) or aluminum oxide (Merck, 70-230 mesh).

Uric acid test 7
In order to verify the photodynamic activity of T2H5NPPH 2 and MCT2H5NPPH 2 the uric acid test was carried out. 7A mixture (3 mL) of UA (10 -3 mol L -1 ) and porphyrins (10 -5 mol L -1 ) in ethanol was placed into a quartz cuvette.For all measurements, the solutions were magnetically stirred and kept at 25 o C for 1800 s.After irradiation at 671 nm, the absorbance spectra (200-700 nm) were registered every 3 min.
Photodynamic activity scale (PA).Based on the UA test, the data obtained for each porphyrin could be mathematically determined by the modified Fischer expression (1).All laser light is focused on the sample solution.

Synthesis
Synthetic schemes for the syntheses of T2H5NPPH 2 and MCT2H5NPPH 2 are presented in Figure 1.The porphyrins were prepared as described in the literature by Adler et al. 6 This synthesis involves the condensation of pyrrole with benzaldehyde, under reflux in propionic acid at 141 o C. In such reaction, no oxidant was added to the reaction mixture and the porphyrins were directly obtained in their oxidized form by using atmospheric O 2 .This type of reaction is often used for the preparation of pyridyl, carboxyl and hydroxyl substituted tetraphenylporphyrins.
The synthesis of porphyrins bearing four identical meso-phenyl substituents, such as T2H5NPPH 2 , is provided by condensation of 2-hydroxy-5-nitrobenzaldehyde and pyrrole.In this case, a high crude porphyrin yield of 72% was obtained (estimated by UV-Vis), Figure 1a.
A mixed aldehyde condensation using stoichiometric amounts of two different benzaldehydes (A) and (B) and pyrrole affords a mixture of six different porphyrins, which can be separated chromatographically. 8 In order to decrease the number of porphyrins obtained in the synthesis and to avoid extremely tedious chromatographic purification, the system was optimised with small-scale reactions using different rations of the two aldehydes.In this way, in the synthesis of A 3 B porphyrin, the initial ratio of pyrrole: 2-hydroxy-5nitrobenzaldehyde: 4-carboxybenzaldehyde used was 4: 2,7: 1.This synthesis gave a crude yield of 30% (estimated by the absorption of the Soret band in the UV-Vis spectrum of the reaction mixture) corresponding to the mixture of A 4 and A 3 B porphyrins, Figure 1b.The final yield of MCT2H5NPPH 2 after purification was 5%.
Fluorescence emission spectra of T2H5NPPH 2 and MCT2H5NPPH 2 in ethanol are shown in Figure 3.The spectra of these porphyrins exhibited two maxima, a strong and broad Q(0,0) fluorescence band near 652 nm, instead of a weaker one and a weaker emission band in 713 nm.The same values of λ em were obtained by exciting the samples at the wavelength of maximum absorption of the Soret and the Q-bands.The two bands are characteristic of similar porphyrins such as THPPH 2 , 9 and they have been assigned to Q(0,0) and Q(0,1) transitions. 10

Uric acid test
Uric acid, a known singlet oxygen scavenger, has been used as a chemical dosimeter for the determination of the photodynamic action of different photosensitizers (PS).The production of 1 O 2. appears to be associated with the photodynamic action of sensitizers.The process involved in the photodynamic mechanism can be represented in the reactions described below, (where P = photosensitizer, S 0 , S 1 = ground, first excited, singlet states, T 1 first excited triplet state, isc = intersystem crossing, 3  Photosensitizers have a stable electronic ground state.Following absorption of a photon of specific wavelength the molecule is promoted to a short-lived excited state, which is deactivated by emitting a photon (fluorescence) or by internal conversion, with the energy loss as heat.It is also possible that the molecule is converted to the triplet  state via intersystem crossing, which involves a change in the spin.The triplet state has lower energy than the singlet state and a longer lifetime (> 500 ns for photosensitizers), both of which increase the probability of energy transfer to other molecules.It has been shown that oxygen plays a fundamental role in the photochemistry of many photosensitizing drugs used in photodynamic therapy. 7he products of UA photosensitized oxygenation via 1 O 2 in human serum are known 12 to be triuret, sodium oxanate, allantoxaidin and CO 2 (Figure 4).
In order to test the photodynamic action of T2H5NPPH 2 and MCT2H5NPPH 2 modulated by 1 O 2 , we irradiated, at the same constant concentration, porphyrins in the presence of uric acid.The PS absorbance didn't decrease in the absence of uric acid.This test procedure, under the same conditions, was also performed for THPPH 2 to confirm our results.The absorbance versus irradiation time graph of the solutions containing each porphyrin THPPH 2 , T2H5NPPH 2 and MCT2H5NPPH 2 in the presence of UA after laser irradiation (671 nm) are shown in Figure 5.
Irradiation parameters (wavelength, laser power and time) and PS concentration remained constant.After laser irradiation at 671 nm, specific degradation of uric acid was observed in the solution containing the porphyrins, as shown by the decrease in the absorbance of the 293 nm band such decay was used to calculate the Photodynamic Activity (PA). 13These results were compared with THPPH 2 and are shown in the Table 1.
The photosensitizers T2H5NPPH 2 and MCT2H5NPPH 2 , present good stability due to the presence of the NO 2 and OH groups bound to the phenyl ring.T2H5NPPH 2 showed a high PA value (if compared to THPPH 2 ) even though it presents a low absorption coefficient at 671 nm.Based on the UA test, it is possible to define a photodynamic activity scale, which may be a good tool for comparing the PA of different photosensitizers.
PA = ∆A UA 10 5 / W t A PSλirr (1) PA = photodynamic activity; ∆A UA = UA absorbance decrease at 293 nm in UA and PS solution after irradiation; W = laser power (mW); t = irradiation time (s) and A PSλirr = absorbance of PS in UA and PS solution at the irradiation wavelength (671 nm).

Table 1 .
13 values for T2H5NPPH 2 , MCT2H5NPPH 2 and THPPH 2 in ethanol.determinedby the modified Fischer expression13∆A UA = UA absorbance decrease at 293 nm in UA and PS solution after irradiation, A PSλirr = absorbance of PS in UA and PS solution at 671 nm (laser irradiation wavelength), PA = photodynamic activity.