Spectroscopic and Photochemical Properties of some Annulated Boldine Derivatives

O objetivo deste estudo foi determinar a influência dos grupos substituintes na fotoestabilidade de oito derivados de boldina, incluindo alguns derivados de oxazol e oxazinona, com o propósito de aumentar as propriedades de proteção solar previamente demonstradas para boldina. Tais derivados apresentaram bandas similares à boldina nos espectros de absorção e emissão. As propriedades espectroscópicas são dependentes do pH. A fotoestabilidade dos derivados foi maior que a da boldina, em conformidade com o baixo valor obtido para o rendimento de fotoconsumo quântico. Os baixos valores de rendimento quântico de fluorescência observados para a boldina 1 e 4, tanto na sua forma de base como hidrocloreto, possibilitam postular que esses compostos são desativados por um mecanismo diferente ao da fluorescência, provavelmente devido ao efeito retirador de elétron do grupo NO para a boldina 1 e ao aumento da aromaticidade para a boldina 4.


Introduction
UV solar radiation has been implicated in cancer probably due to cellular damage that begins with proteins and DNA as the primary targets.][6] Recently, the increasing cosmetic use of solar filters has put in evidence the side effects associated with molecules used as photoprotectors.Such effects include photosensitivity reactions, such as allergies and phototoxicity, and constitute a strong limitation for the massive use of these filters; 7 nevertheless, these limitations have encouraged the search for new molecules that have some basic characteristics: a high molar extinction coefficient in the UVA/UVB zone, photostability and innocuousness in topical use.In this context, natural products may be an important source for research into new active compounds. 8S)-(+)-Boldine, 9 the major alkaloid present in leaves and bark of the Chilean Boldo tree (Peumus boldus Molina, Monimiaceae), has been characterized in the past few years as an antioxidant that effectively protects different systems against free-radical-induced lipid peroxidation or enzyme inactivation. Independently of these properties, boldine is a slightly selective R1A-adrenergic antagonist in vascular tissue and a nonselective D1-and D2-dopaminergic antagonist in the central nervous system.13 It therefore seemed interesting to study whether, by introducing substituents in the available boldine molecule, more favorable pharmacokinetics could be obtained, while maintaining or improving some of its useful pharmacological activities.14 Structure-antioxidative activity studies on boldine and related compounds suggest that both the phenol groups bonded to the aporphine ring system and the basic benzylic amine function contribute to these properties.15 Such structural features should be retained in boldine derivatives to be considered as potential antioxidants.On the other hand, there is presently no way of predicting the effects of derivatization of boldine upon its scantily known monoaminergic properties. 16Therefore, it seemed reasonable that variations on the boldine motif should aim for increased lipophilicity without affecting much the key phenolic and amine functional groups.One way of achieving this would be to replace the aromatic ring hydrogens with not very bulky substituents that would be expected to make a positive contribution to the overall lipophilicity.17 An important part of the deleterious effects of exposing skin to solar UV radiation is attributed to the generation of reactive oxygen species (i.e. siglet oxygen, superoxide and hydroxyl radicals).From a mechanistic perspective, the free radicals generated can cause cellular damage through oxidative modifications of lipids and/or membrane proteins (functional-structural), or through the alterations of nucleics acid.At the present time, a clear participation in the beginning and/or evolution of skin alterations, such as premature aging, dermatitis and skin cancer, is attributed to free radicals.As with the oxidative effects associated with UV radiation, the use of chemical agents such as boldine, that are able to scavenge free radicals 18 and to interfere with oxidative processes, arises today as an interesting and promissory alternative of photoprotection.
The photoprotection capacity of boldine was evaluated before and after irradiation in a previous paper.The photostability and photoprotection factor of boldine and glaucine were compared, demonstrating that both are photoreactive under irradiation conditions.However, the photoproducts preserved the aporphine structure and showed a higher photoprotection factor (SPF). 19 This is important for a future application of the alkaloid as a sunscreen.
All the synthesized boldine derivatives were capable of scavenging ROS (reactive oxygen species) generated by the hypoxanthine-xanthine oxidase system in a concentrationdependent manner. 18In this sense, phenanthrene alkaloids (boldine derivatives) may become promising candidates for the development of anti-inflammatory agents due to their strong ROS scavenging activity.In this paper, the influence of substituents groups and annelated systems such as oxazole and oxazinone rings against the photostability in eight boldine derivatives was evaluated.

Experimental
5][16][17] Hydrochloride salts were prepared by dissolving the compounds in isopropanol and subsequently adding HCl to precipitate the hydrochloride with ethyl ether.

Absorption and emission spectra
Absorption spectra were recorded in a Cecil C-550 spectrophotometer (Cambridge, England).Fluorescence spectra and fluorescence yields were measured in a Shimadzu RF-540 spectrofluorimeter (Kyoto, Japan).

Fluorescence quantum yield
Fluorescence were evaluated by comparing the fluorescence spectra of the compounds with that of anthracene in ethanol (φ Fl = 0.27). 20

Solubility and pKa determination
The solubility of the compounds was estimated from the absorbance of the saturated solution (after evaluating the extinction coefficient) using the break in absorbance versus the added concentration plot.The pKa values were obtained from plots of absorbance versus pH (titration curve).
Due to the possible use of bases as sunscreen filters and their low solubility in water, photoconsumption quantum yields (φ c ) were evaluated in methanol solutions (derivatives 1-3, 2 × 10 -5 mol L -1 ) and in tetrahydrofuran (THF, derivative 4, 1 × 10 -5 mol L -1 ) and were irradiated during 2 h in a photochemical reactor (Rayonet Southern New England Ultraviolet Company, England, CT, USA) with an irradiance of 3100 μW cm -2 .The photoconsumption was evaluated from changes in the absorption spectra using the high performance thin layer chromatography (HPTLC) method (mobile phase chloroform-methanol-water; 65:40:2, v/v/v).

Absorption spectra
In Figure 2 the influence of pH on the absorption spectra of boldine derivatives (Bol 1 -Bol 4) base form was shown.In all spectra, there is a slight bathochromic shift in both bands at pH 3 with respect to boldine in methanol, possibly due to the solvation of the non-bonding electrons from auxochrome groups, which would diminish the n orbital energy, therefore affecting the n-π * transitions and producing the observed shift. 21t pH 10, the first band shows a bathochromic shift only in derivatives in Bol 2 and Bol 4 bases and a hypsochromic shift in Bol 1 and 3 bases in the second band, presumably due to the stabilization of the π* excited state in the auxochrome groups by the solvent effect. 21

Fluorescence spectra, fluorescence quantum yield
The fluorescence quantum yields (Table 1) of all compounds present low values and Bol 1 and Bol 4 bases are the lowest.This behavior may be due to the presence of paramagnetic substituents that could allow other deactivation paths such as intersystem crossing. 21,22he extending of the aromatic structure could enhance fluorescence due to the π-π * energy levels.The biphenyl system without conjugation (independent phenolic systems in boldine) is converted into a phenolic system conjugated with an oxazol or oxazinone in the derivates.

pKa determination
All boldine derivatives except Bol 2 base have two pKa values near 5 and 9 (Table 2).However, we must mention that the methodology shows limitations at certain pH values, where the absorbances may overlap.Nevertheless, values are in accordance with those reported previously by Shao-Wen et al., 23 for molecules that have an aporphinic structure.From a structural point of view, a pKa around 4 or 5 corresponds to one of the hydroxyl groups, while a pKa between 8 and 9 would correspond to the amino group at C6 in ring B.

Photostability determinations
The consumption percentages of boldine derivatives plotted as ln(C 0 /C) (Figure 3) showed that, in the case of Bol 1 base, the initial concentration decreased 50% when the solution was irradiated during approximately 40 min, reaching a maximum disappearance of around 90% at 80 min and then remaining constant at a maximum around 90%, and then it remained constant (Figure 4).Consumption percentages of derivatives Bol 2, 3 and 4 bases were in the range from 10 to 30%.At larger times, no variation in the concentration was observed.
By comparing the results obtained in the photostability kinetics of the different derivatives (Figure 3), the behavior of Bol 1 base is different to all other compounds studied, since it was first consumed and then reappeared.This behavior could be explained by the presence of the NO group in Bol 1, which could act as scavenger 24,25 for radicals generated by irradiation, followed by release at longer times of photolysis; Bol 4 and Bol 3 bases had the lowest degree of decomposition, 16.5 and 15.9%, respectively, after 2 h of irradiation (Figure 4) with a normal kinetic behavior.For instance, using these molecules the hydrochloride improved the solubility of boldine derivatives in most of the solvents used, producing no significant changes in the spectral properties, as observed in boldine. 19hen comparing the photoconsumption quantum yield of derivatives with boldine (Table 3), the higher photostability of the substituted aporphinic structures is confirmed.Boldine     has a biphenyl system, which allows the existence of a double phenolic system which explains the stability of free radicals on the framework system.Furthermore, the nitroso and amine derivatives, as well as the incorporation of oxazole and oxazinone heterocycles, provided greater photostability, possibly due to electron-withdrawing and electron-releasing groups represented by nitroso-oxazinone and amine-oxazole that could stabilize possible radical intermediaries formed during irradiation.

Conclusions
All boldine base derivatives in this study showed lower photoconsumption quantum yields and photodecomposition rates than boldine.Thus, boldine derivatives bearing substituent groups and annelated heterocycles with the aporphinic structures have a higher photostability against UVB radiation.
The compounds derived from boldine bearing oxazole and oxazinone rings have promising photophysical and photochemical properties that would allow their use as possible sunscreens.Further studies of sun protection factor and phototoxicity are currently in progress.

Figure 2 .
Figure 2. Absorption spectra at different pH values and in methanol.