Spectroscopic and photochemical properties of some annulated boldine derivatives

Article abstract of DOI:10.1590/S0103-50532010001200006

The aim of this study was to determine the influence of the substituent groups on the photostability of eight boldine derivatives, including some oxazole and oxazinone derivatives, in order to increase the sunscreen protection properties previously demonstrated for boldine. Such derivatives showed bands similar to those of boldine in the absorption and emission spectrum. The spectroscopic properties are pH dependent. The photostability of the derivative compounds was higher than that of boldine, in accordance with the low value of the photoconsumption quantum yield determined. The low fluorescence quantum yield values observed for boldine 1 and 4 as both base and hydrochloride derivative compounds led us to postulate that these compounds are deactivated by a mechanism different to the fluorescence pathway, probably due to the electron-withdrawing inductive effect of NO for boldine 1, and to the increase of the aromatic framework for boldine 4.

Full text of DOI:10.1590/S0103-50532010001200006

J. Braz. Chem. Soc., Vol. 21, No. 12, 2205-2210, 2010.
Printed in Brazil - ©2010 Sociedade Brasileira de Química
0103 - 5053 $6.00+0.00
Spectroscopic and Photochemical Properties of some Annulated Boldine Derivatives
María E Hidalgo,*,a Myriam G. Alarcón,b Juan R. Ojeda,b Ernesto C. Fernández,b,†
Eduardo M. Sobarzo-Sánchezc andJulio R. De la Fuented
aFacultad de Ciencias, Universidad de Valparaíso, Avenida Gran Bretaña 1111, Valparaíso, Chile
bFacultad de Farmacia, Universidad de Valparaíso, Avenida Gran Bretaña 1093, Valparaíso, Chile
cDepartamento de Farmacia y Tecnologia Farmacéutica, Facultad de Farmacia, Universidad de Santiago
de Compostela, Praza Seminario de Estudos Galegos s/n, 15782 Santiago de Compostela, España
dFacultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Vicuña Mackenna 20,
Santiago, Chile
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.
The aim of this study was to determine the influence of the substituent groups on the
photostability of eight boldine derivatives, including some oxazole and oxazinone derivatives, in
order to increase the sunscreen protection properties previously demonstrated for boldine. Such
derivatives showed bands similar to those of boldine in the absorption and emission spectrum. The
spectroscopic properties are pH dependent. The photostability of the derivative compounds was
higher than that of boldine, in accordance with the low value of the photoconsumption quantum yield
determined. The low fluorescence quantum yield values observed for boldine 1 and 4 as both base
and hydrochloride derivative compounds led us to postulate that these compounds are deactivated
by a mechanism different to the fluorescence pathway, probably due to the electron-withdrawing
inductive effect of NO for boldine 1, and to the increase of the aromatic framework for boldine 4.
Keywords: boldine, oxazol-oxazinone derivatives, photostability
Introduction
deactivated by electron transfer, hydrogen abstraction
and/or the generation of singlet oxygen.1-3
UV solar radiation has been implicated in cancer
probably due to cellular damage that begins with proteins
and DNA as the primary targets. This damage could be
initiated by two mechanisms: (i) direct absorption of the
incident light by the ground state or (ii) photosensitization,
where the light is absorbed by an endogenous or exogenous
photosensitizer that, excited to its triplet state, can be
With the purpose of reducing the effects of UV
radiation, diverse molecules have been synthesized to act
as radiation filters, diminishing the radiation dose (UVA/
UVB) received by the skin in topical applications.4-6
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
*e-mail: maria.hidalgo@uv.cl

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Spectroscopic and Photochemical Properties of some Annulated Boldine Derivatives
J. Braz. Chem. Soc.
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.8
(S)-(+)-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.10 This activity presumably underlies
the hepatoprotective and cytoprotective effects recently
demonstrated for this alkaloid11and may also be related to
its antipyretic and anti-inflammatory behavior and to its
protective effect in experimental colitis.12
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
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).19This 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 concentration-
dependent manner.18 In 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
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. singlet 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 radicals18 and to interfere
Boldine derivatives (1-4) (Figure 1) were synthesized
and characterized by standard methods.14-17 Hydrochloride
salts were prepared by dissolving the compounds in
isopropanol and subsequently adding HCl to precipitate
the hydrochloride with ethyl ether.
The reagents Brij 35 (Aldrich Laboratories, Milwaukee,
WI, USA), dimethylsulfoxide and ethanol, methanol,
tetrahydrofuran, acetonitrile (Merck, Santiago de Chile,
Chile) were used. Distilled water adjusted to pH 3 and 10
with HCl and NaOH, respectively (Merck) was used.
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 (φFl = 0.27).20
Solubility and pKa determination
The solubility of the compounds was estimated from the
absorbance of the saturated solution (after evaluating the

Vol. 21, No. 12, 2010
Hidalgo et al.
2207
(THF, derivative 4, 1 × 10-5 mol L-1) and were irradiated
during 2 h in a photochemical reactor (Rayonet Southern
NewEnglandUltravioletCompany, England, CT, USA)with
an irradiance of 3100 μW cm-2. The photoconsumption was
evaluatedfromchangesintheabsorptionspectrausingthehigh
performance thin layer chromatography (HPTLC) method
(mobile phase chloroform-methanol-water; 65:40:2, v/v/v).
Results and Discussion
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.21
At 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.21,22
The extending of the aromatic structure could enhance
Figure 1. Boldine derivatives’ structures.
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).
*
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.
Photostability determinations
Photolyses were performed in standard solutions of
oxygen and nitrogen (10-4 mol L-1 derivatives 1-4) using a
medium pressure mercury lamp (Hanovia Union, NJ, USA),
monochromatic UVB light at 313 nm with 1460 μW cm-2
irradiance. The photodegradation was analyzed by high
performance liquid chromatography (HPLC) using a Merck
Hitachi Lachrom HPLC (Tokyo, Japan) (mobile phase
ammoniumphosphatepH4.3;acetonitrile-methanol;45:12:1
(v/v/v); Merck RP 18e LiChroCART® 125-4 column).
Due to the possible use of bases as sunscreen filters
and their low solubility in water, photoconsumption
quantum yields (φc) were evaluated in methanol solutions
(derivatives 1-3, 2 × 10-5 mol L-1) and in tetrahydrofuran
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.

2208
Spectroscopic and Photochemical Properties of some Annulated Boldine Derivatives
J. Braz. Chem. Soc.
Figure 2. Absorption spectra at different pH values and in methanol.
Photostability determinations
Table 1. Fluorescence quantum yields
λ / nm
The consumption percentages of boldine derivatives
plotted as ln(C0/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.
Compound
Φf
excitation
288
emission
360-470
414
Bol 1 Base/MeOH
0.0034
0.2445
0.2903
0.0022
0.0026
0.3150
0.3864
0.0022
Bol 2 Base/MeOH
296
Bol 3 Base/MeOH
284
359
Bol 4 Base/MeOH
296
412
Bol 1 hydrochloride/MeOH
Bol 2 hydrochloride/MeOH
Bol 3 hydrochloride/MeOH
Bol 4 hydrochloride/MeOH
282
406
297
418
286
408
294
410
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 scavenger24,25 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.19
Table 2. pKa values
Compound
pka
λ1*
λ2*
Boldine
4.98-9.02
4.97-8.97
3.99-8.00
4.98-9.00
4.49-8.50
4.00-8.00
4.00-8.03
3.97-8.98
3.99-8.98
4.98-9.00
5.03-9.03
----
Bol 1 Base
Bol 2 Base
Bol 3 Base
5.01-9.00
4.49-8.50
5.00-7.98
3.97-7.98
3.99-8.00
4.02-9.00
Bol 4 Base
Bol 1 hydrochloride
Bol 2 hydrochloride
Bol 3 hydrochloride
Bol 4 hydrochloride
When comparing the photoconsumption quantum yield
ofderivativeswithboldine(Table3), thehigherphotostability
of the substituted aporphinic structures is confirmed. Boldine
*λ1 acid solution (pH 3) and λ2 basic solution (pH 10).

Vol. 21, No. 12, 2010
Hidalgo et al.
2209
Figure 3. Photoconsumption kinetics determination.
Figure 4. Consumption percentages of Bol 2, 3 and 4 bases.

2210
Spectroscopic and Photochemical Properties of some Annulated Boldine Derivatives
J. Braz. Chem. Soc.
hasabiphenylsystem, whichallowstheexistenceofadouble
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.
4. Naylor, M. F.; Boyd,A.; Smith, D.W.; Cameron, G. S.; Hubbard,
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6. Lautenschlager, S.; Wulf, H. C.; Pittelkow, M. R; The Lancet
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Pharmacol. 1991, 41, 1575.
Table 3. Photoconsumption quantum yields
Compound
Boldine
k air / min-1
0.043 0.006
0.016 0.005
0.003 0.001
0.001 0.0004
0.002 0.0003
Φc air (102)
11. Jiménez, I.; Speisky, H.; Phytother. Res. 2000, 14, 254.
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3.8
2.5
0.4
0.2
0.1
Bol 1 base
Bol 2 base
Bol 3 base
Bol 4 base
Conclusions
15. Schmeda-Hirschmann, G.; Rodríguez, J. A.; Theoduloz, C.;
Astudillo, S. L.; Feresin, G. E; Tapia,A.; Free Radic. Res. 2003,
37, 447.
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.
16. Martínez, S.; Madrero, Y.; Elorriaga, M.; Noguera, M. A.;
Cassels, B.; Sobarzo, E.; D’Ocon, P.; Ivorra, M. D.; Life Sci.
1999, 64, 1205.
17. Sobarzo-Sánchez, E.; Jullian, C.; Cassels, K. B.; Saitz, C.;
Magn. Reson. Chem. 2001, 39, 361.
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.
18. Milián, L.; Estellés, R.; Abarca, B.; Ballesteros, R.; Sanz, M.
J.; Blázquez, M. A.; Chem. Pharm. Bull. 2004, 52, 696.
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CRC Press: Boca Raton, Florida, 1989.
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E. S. thanks Dr. B. K. Cassels for his unconditional
support and encouraging comments to continue this
research.
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Submitted: October 14, 2008
Published online: September 23, 2010