The photochemistry of flutamide and its inclusion complex with β-cyclodextrin. Dramatic effect of the microenvironment on the nature and on the efficiency of the photodegradation pathways

Article abstract of DOI:10.1562/0031-8655(2001)073<0006:TPOFAI>2.0.CO;2

The photochemistry of the anticancer drug flutamide (FM), 2-methyl-N-[4-nitro-3-(trifluoromethyl)phenyl]propanamide, in homogeneous media and in the β-cyclodextrin (β-CD) cavity has been investigated. The photoreactivity of the free molecule has been rationalized on the basis of an intramolecular nitro to nitrite rearrangement followed by cleavage of the nitrite intermediate. The twisted geometry of the nitro group with respect to the aromatic plane plays a key role in triggering such a photoprocess. Incorporation of FM in the β-CD cavity leads to dramatic effects on both the efficiency and the nature of the photochemical deactivation pathways of the guest molecule. A 20-fold increase in the FM photodecomposition quantum yield and the formation of photoproducts originated by both reduction of the nitro group and cleavage of the amide bond were observed in the presence of the macrocycle. Such a behavior cannot be attributed exclusively to the micropolarity of β-CD and/or to its role as a reactant. The induced circular dichroism spectra and the nature of the photoproducts formed in these experimental conditions provide indications that the photoreacrivity in the β-CD microenvironment could likely be mediated by structural changes of FM upon complexation.

Full text of DOI:10.1562/0031-8655(2001)073<0006:TPOFAI>2.0.CO;2

The Photochemistry of Flutamide and its Inclusion Complex with β-Cyclodextrin.
Dramatic Effect of the Microenvironment on the Nature and on the Efficiency of
the Photodegradation Pathways
Author(s): S. Sortino, S. Giuffrida, G. De Guidi, R. Chillemi, S. Petralia, G. Marconi, G. Condorelli, and
S. Sciuto
Source: Photochemistry and Photobiology, 73(1):6-13.
Published By: American Society for Photobiology
DOI: http://dx.doi.org/10.1562/0031-8655(2001)073<0006:TPOFAI>2.0.CO;2
URL: http://www.bioone.org/doi/full/10.1562/0031-8655%282001%29073%3C0006%3ATPOFAI
%3E2.0.CO%3B2
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Photochemistry and Photobiology, 2001, 73(1): 6–13
The Photochemistry of Flutamide and its Inclusion Complex with
␤-Cyclodextrin. Dramatic Effect of the Microenvironment on the Nature
and on the Efficiency of the Photodegradation Pathways¶
S. Sortino*1, S. Giuffrida1, G. De Guidi1, R. Chillemi1, S. Petralia1, G. Marconi2, G. Condorelli1 and
S. Sciuto1
1Dipartimento di Scienze Chimiche, Universita` di Catania, Catania, Italy and
2Istituto di Fotochimica e Radiazioni d’Alta Energia, Bologna, Italy
Received 26 June 2000; accepted 11 October 2000
ABSTRACT
effects upon UVA light excitation (4–9). Despite these well-
documented cases of liver phototoxicity, the complex issue
concerning the mechanism of FM photosensitization is still
unclear. This is probably due to the lack of data concerning
the photochemical behavior of FM. To the best of our
knowledge, no reports on the photochemistry of this drug
appear in the literature up to now (10). It is generally ac-
cepted that shedding light on drug photochemical behavior
is a prerequisite to understand the molecular basis of the
drug-photoinduced biological damage. In this connection, a
sequential approach consisting in extending the knowledge
concerning the photobehavior in homogeneous media to bi-
ological mimicking systems of increasing complexity, rep-
resents an adequate strategy for a more appropriate corre-
lation between phototoxicity and photochemical properties.
Actually, properties such as hydrophobicity, geometry, size,
shape and charge often lead to the formation of supramolec-
ular aggregates between photosensitizer and biomolecule.
These aggregates can display different photobehavior when
compared to that of the ‘‘free molecule.’’ Among the several
biological mimetic systems, cyclodextrins (CD) represent
one of the most simple ones. Photochemical investigation in
such a kind of microenvironment is an extremely active area
of research for both fundamental studies and for practical
purposes (11). The most important property of these mole-
cules is the ability to form host–guest inclusion complexes
with a great variety of organic and inorganic substrates (12–
18). A number of factors influence complexation. Among
them, the ‘‘goodness of fit’’ between the host and the guest
and the hydrophobic effects are probably the most important.
The weak binding forces responsible for association to the
constrained microenvironment of CD provide a useful model
to mimic the interactions of drugs with hydrophobic pockets
of biological substrates. As already reported for a large va-
riety of systems, CD have extensively proven their potential
as media for controlling chemical and photochemical reac-
tions (19–21). The nature of the lowest excited states of the
guest molecule, the efficiency of its deactivation pathways,
the fate of the reaction intermediates and the opening of new
photoreactive channels, are some of the parameters that may
be modulated by the CD microenvironment.
The photochemistry of the anticancer drug flutamide (FM),
2-methyl-N-[4-nitro-3-(trifluoromethyl)phenyl]propan-
amide, in homogeneous media and in the ␤-cyclodextrin (␤-
CD) cavity has been investigated. The photoreactivity of the
free molecule has been rationalized on the basis of an in-
tramolecular nitro to nitrite rearrangement followed by
cleavage of the nitrite intermediate. The twisted geometry
of the nitro group with respect to the aromatic plane plays
a key role in triggering such a photoprocess. Incorporation
of FM in the ␤-CD cavity leads to dramatic effects on both
the efficiency and the nature of the photochemical deacti-
vation pathways of the guest molecule. A 20-fold increase
in the FM photodecomposition quantum yield and the for-
mation of photoproducts originated by both reduction of
the nitro group and cleavage of the amide bond were ob-
served in the presence of the macrocycle. Such a behavior
cannot be attributed exclusively to the micropolarity of ␤-
CD and/or to its role as a reactant. The induced circular
dichroism spectra and the nature of the photoproducts
formed in these experimental conditions provide indications
that the photoreactivity in the
could likely be mediated by structural changes of FM upon
complexation.
␤-CD microenvironment
INTRODUCTION
Flutamide (FM)†, 2-methyl-N-[4-nitro-3-(trifluorome-
thyl)phenyl]propanamide, is a nonsteroidal antiandrogen
drug that blocks androgen receptor sites; it is commonly
used in advanced prostate cancer (1–3). Recent reports have
shown the capability of FM to induce in vivo phototoxic
¶Posted on the website on 22 November 2000.
*To whom correspondence should be addressed at: Dipartimento di
Scienze Chimiche, Universita` di Catania, Viale Andrea Doria, 8
I-95125 Catania, Italy. E-mail: ssortino@dipchi.unict.it
†Abbreviations:
␤-CD, ␤-cyclodextrin; EI, electron impact; FAB,
fast atom bombardment; FM, flutamide; H-abstraction, hydrogen
abstraction; HPLC, high performance liquid chromatography;
ICD, induced circular dichroism; MS, mass spectrometry; NMR,
nuclear magnetic resonance; TLC, thin-layer chromatography.
Due to their molecular structure, many drugs are suitable
guests for CD receptors. In this regard, photophysics and
᭧
2001 American Society for Photobiology 0031-8655/00
$5.00ϩ0.00
6

Photochemistry and Photobiology, 2001, 73(1)
7
equipped with a variable number of ‘‘black light’’ phosphor lamps
with an emission in the 310–390 nm range with a maximum at 350
nm.
The quantum yields related to the FM photodegradation and to
the formation of its photoproducts were calculated at 325 nm, below
the 15% conversion of the starting compound, from the following
relation:
photochemistry of drug–CD inclusion complexes have re-
ceived considerable attention, especially in the last few years
(22–26). The use of CD as a drug complexing agent can
represent in fact not only a suitable biological mimicking
environment but also a valid tool to minimize the drug-pho-
toinduced biological damage as well as to increase the drug
photostability (24,27–29).
⌽ ϭ (d[X]/dt)v/FI
(1)
This report centers on the photoreactivity of FM both in
the absence and in the presence of ␤-CD in order to con-
where d[X]/dt is the initial rate of disappearance of FM or the initial
rate of formation of the single photoproducts, v is the volume of the
tribute to the general picture of drug photochemistry. Be-
yond this strict photochemical point of view, in light of the
reasons described above, the present investigation may rep-
resent a step forward in the understanding of the phototoxic
effects induced by FM. Finally, it is worth to mention that,
due to the poor water solubility of FM, efforts have been
made to improve the bioavailability of the drug by using
suitable carrier systems based on the formation of supra-
molecular complexes (30). Thus, the widely spread appli-
cation of CD in pharmaceutical formulations to enhance
drug solubilization (31) further reinforces our interest in the
present topic.
A
irradiated sample, F
ϭ
1–10Ϫ is the fraction of photons absorbed
by FM at the excitation wavelength and I is the light intensity (mol
of photons minϪ1).
Photodegradation of FM
4
FM solutions (200 mL, 10Ϫ M) in phosphate buffer in the absence
2
or in the presence of 10Ϫ
M ␤-CD were irradiated in the Rayonet
reactor under nitrogen bubbling and continuous stirring. The irra-
diated solutions (ca 15% FM transformation, determined by HPLC)
were extracted with diethyl ether (3
tracts were dried over anhydrous sodium sulfate and evaporated in
vacuo at 40 C. An analysis of the extracted mixture was performed
ϫ 20 mL); the combined ex-
Њ
by thin layer chromatography (TLC), on precoated silica gel 60 F254
plates (Merck) using dichloromethane : n-propanol 99:1 (vol/vol)
and dichloromethane:cyclohexane 80:20 (vol/vol) for the experi-
MATERIALS AND METHODS
Chemicals
ments carried out in the absence and in the presence of
spectively. In the former case the TLC analysis showed a main pho-
toproduct, 1 (Rf 0.13), and the starting compound (Rf 0.80). In
the latter case the photoproduct 1 (Rf 0.11) was accompanied by
the presence of two other photoproducts, 2 (Rf 0.28) and 3 (Rf
0.38) (starting compound, Rf 0.2). On a preparative scale, the
photoproduct 1 was obtained, from the organic extracts of FM so-
␤-CD, re-
ϭ
ϭ
FM (molecular weight 250.2) was purchased from Sigma Chemical
Co. (St. Louis, MO) and used as received. Phosphate buffer 10Ϫ2 M,
pH 7.4 was prepared from reagent grade products. pH of the solu-
tions were measured with a glass electrode. All solvents used (from
Carlo Erba, Milan) in the isolation of the photoproducts and in the
photochemical experiments were high performance liquid chroma-
tography (HPLC) and analytical grade, respectively.
ϭ
ϭ
ϭ
ϭ
4
lutions (10Ϫ M, 5000 mL) irradiated in the absence of
preparative liquid chromatography on a silica gel column (40–63
m; 1.0
18 cm2) using dichloromethane:n-propanol 99:1 (vol/vol)
␤-CD, by
␮
ϫ
as eluent. The photoproduct 2 and 3 were recovered from the organic
4
extracts of FM solutions (10Ϫ M, 2000 mL) irradiated in the pres-
Instrumentation
ence of
gel column (40–63
cyclohexane 60:40 (vol/vol) (3, Rf
0.13; 1, Rf 0.0).
␤
-CD, carrying out a chromatographic separation on a silica
m; 1.0
20 cm2) eluting with dichloromethane:
0.24; 2, Rf 0.18; FM, Rf
Steady state absorption and induced circular dichroism spectra (ICD)
were recorded with a Beckman 650 DU spectrophotometer and a
Jasco J-615 dichrograph, respectively. The 1H-, 19F- and 13C-nuclear
magnetic resonance (NMR) spectra were recorded on a Varian Inova
␮
ϫ
ϭ
ϭ
ϭ
ϭ
The photolysis of FM in the organic solvent was performed by
1
spectrometer at 499.9, 470.3 and 125.7 MHz, respectively. The H
4
irradiating 10Ϫ M solutions of FM under nitrogen bubbling and
and 13C experiments were obtained in CD3OD and/or CDCl3 using
tetramethylsilane as an internal reference; definitive assignments of
continuous stirring. The quantitative analysis of the irradiated mix-
ture was performed by means of HPLC under the same experimental
conditions described above.
1
individual H and 13C resonances of the photoproducts were sup-
1
ported by H–1H-correlated spectroscopy and pulsed field gradient
heteronuclear single-quantum correlation experiments, together with
the analysis of 13C–19F coupling constants. Fast atom bombardment
(FAB) and electron impact (EI) mass spectra were obtained on a
Fisons ZAB 2SE spectrometer.
HPLC was performed on a Hewlett–Packard 1100 chromatograph
equipped with on-line photodiode array detector. The quantitative
analysis of FM and its photoproducts was achieved on a LiChroCart
Characterization of the photoproducts
Photoproduct 1: 2-methyl-N-[4-hydroxy-3-(trifluoromethyl)phenyl]
propanamide. 1H-NMR (CD3OD):
␦
1.19 (d, J
7.0 Hz, 1H, CH[CH3]2), 6.88 (d, JH-5,H-6
8.7 Hz, 1H, H-5), 7.54 (dd, JH-6,H-5 8.7 Hz, JH-6,H-2 2.2 Hz, 1H,
2.2 Hz, 1H, H-2); selected 1H-NMR (CDCl3)
ϭ 7.0 Hz, 6H,
CH[CH3]2), 2.59 (septet, J
ϭ
ϭ
ϭ
ϭ
H-6), 7.72 (d, JH-2,H-6
ϭ
RP-18 column (5 ␮m packing, 4 ϫ 250 mm Hewlett–Packard) elut-
data:
(CD3OD):
Hz, C-3), 117.9 (C-5), 120.2 (q, JCCCF
␦
5.63 (br s, 1H, OH), 7.09 (br s, 1H, CONH). 13C-NMR
ing with a linear gradient of CH3CN in 0.01 M phosphate buffer
(pH 7) from 0 to 75% in 25 min, at a flow rate of 1 mL minϪ1. The
retention times of FM and the photoproducts 1, 2 and 3 were 16.7,
13.7, 17.0 and 15.0 min, respectively.
␦
19.8 (CH[CH3]2), 36.9 (CH[CH3]2), 117.8 (q, JCCF ϭ 30.8
5.2 Hz, C-2), 125.2 (q, JCF
ϭ
ϭ
271.0 Hz, CF3), 126.7 (C-6), 131.6 (C-1), 153.6 (C-4), 178.5
(CONH). 19F-NMR (CDCl3):
␦
15.61 (s, CF3). FAB-mass spectrom-
Retention times in HPLC analysis for the compounds in the pres-
etry (MS) (
Photoproduct 2: 2-methyl-N-[4-nitroso-3-(trifluoromethyl)phenyl]
propanamide. 1H-NMR (CD3OD):
1.22 (d, J 6.9 Hz, 6H,
CH[CH3]2), 2.69 (septet, J 6.9 Hz, 1H, CH[CH3]2), 6.41 (d, JH-5,H-6
9.0 Hz, 1H, H-5), 7.83 (dd, JH-6,H-5 9.0 Hz, JH-6,H-2 2.2 Hz, 1H,
2.2 Hz, 1H, H-2); selected 1H-NMR (CDCl3)
19.6 (CH[CH3]2),
6.0 Hz, C-2), 122.2
273.4 Hz, CF3), 134.9 (q, JCCF 32.5 Hz, C-
3), 147.5 (C-1), 158.9 (C-4), 179.2 (CONH). 19F-NMR (CDCl3):
21.10 (s, CF3). FAB-MS ( ) (3-nitrobenzyl alcohol as matrix): m/z
261 [M
H]ϩ.
Photoproduct 3: 4-nitro-3-(trifluoromethyl)aniline. 1H-NMR (CDCl3):
ϩ) (3-nitrobenzyl alcohol as matrix): m/z 248 [M ϩ
H]ϩ.
2
ence of 10Ϫ
M ␤-CD were the same as described above. Moreover,
a quantitative evaluation of the injected material assured that no
inclusion products were retained by the column (see Table 2).
␦
ϭ
ϭ
ϭ
ϭ
ϭ
Irradiation conditions
H-6), 8.46 (d, JH-2,H-6
ϭ
Irradiations of FM were performed using monochromatic irradiation
obtained from a Series 200 He–Cd 325 nm laser (Liconix, Santa
Clara, CA). The incident photon flux on 3 mL quartz cuvettes was
data:
37.3 (CH[CH3]2), 109.3 (C-5), 117.3 (q, JCCCF
(C-6), 125.9 (q, JCF
␦ ␦
7.56 (br s, CONH). 13C-NMR (CD3OD):
ϭ
ϭ
ϭ
ca 5
ϫ
1015 quanta sϪ1. The experimental procedures of irradiation
␦
and the light intensity measurements have been described previously
(32). On a preparative scale, the irradiation of FM was performed
in 200 mL quartz vessel using a Rayonet photochemical reactor
ϩ
ϩ

8
S. Sortino et al.
␦
4.46 (br s, 2H,NH2), 6.78 (dd, JH-6,H-5
ϭ
8.8 Hz, JH-6,H-2
ϭ
2.6 Hz,
8.8
16.34 (s, CF3). FAB-MS (
H]ϩ. EI-MS (70 eV, 60
C): m/
z (rel. int. %) 206 [M] (25.5), 176 [M
NO]•ϩ (28.9), 167 (18.9),
Table 1. Calculated energies, oscillator strengths and nature of the
excited states of FM*
1H, H-6), 6.98 (d, JH-2,H-6
Hz, 1H, H-5). 19F-NMR (CDCl3):
(glycerol as matrix): m/z 207 [M
ϭ
2.6 Hz, 1H, H-2), 7.97 (d, JH-5,H-6
ϭ
␦
ϩ)
ϩ
Њ
Energy
Oscillator
strength
•ϩ
Ϫ
1
State
(kcal molϪ
)
Nature
•
•
160 [M
(100).
Ϫ Ϫ
NO2 ]ϩ (21.1), 125 [176 CHF2 ]ϩ (20.0), 69 [CF3]ϩ
T1
T2
T3
T4
S1
S2
S3
44.7
56.6
58.7
67.3
67.7
70.1
91.3
␲,␲*
n,
␲
*
*
RESULTS AND DISCUSSION
n,␲
␲,␲*
Absorption spectra and electronic states of FM
0.042
0.001
0.263
n,
␲
*
*
n,␲
The absorption spectrum of FM in phosphate buffer solution
pH 7.4 (Fig. 1) is characterized by a broad band with max-
␲,␲*
Ϫ
1
Ϫ1
imum at 300 nm (⑀ ϭ 6200 M cm ) and a weak tail
extending beyond 400 nm. A significant shift toward shorter
wavelengths was observed as the solvent polarity decreased.
In particular, the main band shifted to 296 nm in methanol,
294 nm in isopropanol and 278 nm in cyclohexane. In order
to asses the nature of the excited states of FM, we performed
semiempirical quantum mechanical calculations by means of
the Zerner intermediate neglect of differential overlap/spec-
troscopic method (Hyperchem software package), starting
*FM
ϭ
flutamide; T
ϭ
triplet; S
ϭ
singlet.
was always the main photoproduct. At a first glance, the lack
in these solvents of clear evidence for any product originated
by either an expected photoreduction of the nitro group or
predictable cleavage of the amide bond might seem quite
surprising. Indeed, although formed with low quantum effi-
ciency, such photoproducts are commonly observed in the
case of nitro aromatic (34–39) and aryl amide derivatives
(38–42). This apparent incongruity can be understood on the
basis of the chemical structure of FM. As discussed above,
because of the presence in ortho of the CF3 substituent the
nitro group is placed almost perpendicularly to the aromatic
plane and, as a consequence, not conjugated with the latter.
As pictorially represented in Scheme 1, such a twisted ge-
ometry makes the p orbital of the oxygen atom having a
constructive overlap with the adjacent p orbital of the aro-
matic ring in the ground state. This geometry is believed to
from a ground-state geometry optimized through the MM
ϩ
molecular mechanics routine (33). The results obtained are
reported in Table 1.
The first sizeable singlet, responsible for the absorption
band at 300 nm in aqueous solution, is calculated at 91.3
Ϫ
1
kcal mol as S3 (
vent with decreasing polarity is in agreement with the fact
that the main band has contributions from a * state which
is more polar than the ground state. As it can be noticed
from Table 1, the lowest excited triplet state T1 has a
nature and lies ca 10 kcal below the n, * triplet T2 contrary
to nitrobenzene, chosen as a comparison compound, where
the lowest excited triplet has an n, * character.
␲,␲*). The blueshift observed in the sol-
␲,␲
␲,␲*
␲
determine an n,␲* excited state characterized by a low bir-
adical character and, as a consequence, by a very low pho-
toreactivity towards hydrogen abstraction (H-abstraction)
even in hydrogen-donating solvents.
␲
The presence of the CF3 substituent plays a key role in
determining the geometry of the FM ground state as well as
the energies of the excited states. As reported in Fig. 2 the
In light of this view, the formation of 1 can be rationalized
on the basis of the well-known nitro to nitrite rearrangement
followed by rupture of the O–N bond to generate a phenoxy
radical (Scheme 1). According to literature data (43–46) the
out of plane geometry of the nitro group and the consequent
orbital overlap are prerequisites to observe such a type of
intramolecular photorearrangement. In this connection, it
should be noted that although the nitrite isomer has never
been identified it is widely regarded as a logical intermediate
(45). Elegant studies performed by Hamanoue et al. (46)
FM structure optimized through MMϩ calculations shows
clearly that the nitro group is placed almost perpendicularly
to the aromatic ring and, as a consequence, not conjugated
with the latter. Both steric and electronic factors, due to the
presence of the CF3 substituent, can play dominant roles in
determining such geometry. The lack of conjugation be-
tween the nitro group and the aromatic plane was further
confirmed by the marked blueshift (ca 40 nm) of the ab-
sorption maximum of FM if compared to that of the analog
compound without the CF3 group.
Photoreactivity in homogeneous media
Figure 3 shows the spectral changes observed in nitrogen
Ϫ
4
saturated solution of FM 10 M in phosphate buffer pH 7.4
at different irradiation times upon 325 nm light excitation.
As confirmed by the analysis of the irradiated mixture (see
‘‘Experimental’’) the spectral changes observed are consis-
tent with the almost exclusive formation of 1 (see Scheme
1) as the main stable photoproduct. The photodegradation
quantum yield of FM in these experimental conditions, cal-
Ϫ3
culated through HPLC analysis, was
⌽
-FM
ϭ 3.5 ϫ 10 .
Even when the irradiation of FM was performed in an-
aerobic conditions in solvents with hydrogen-donating abil-
ity better than water such as methanol and isopropanol, 1
2
Figure 1. Absorption spectrum of FM in 10Ϫ M phosphate buffer,
pH 7.4.

Photochemistry and Photobiology, 2001, 73(1)
9
Figure 2. Optimized structure of FM obtained by MM
ϩ calcula-
tions.
demonstrated that the nitro to nitrite photorearrangement oc-
curs likely from an upper excited triplet state with an n,
character located at higher energy with respect to the lowest
* triplet state. This picture accords well with our results
␲*
Scheme 1
␲,␲
concerning both the nature and the energies of the low-lying
triplet states of FM (see previous section).
some hints on the structure of the complex formed even in
absence of more refined calculations (47).‡
In particular the effect of geometry of the NO2 group (pla-
nar or perpendicular to the ring) on the sign of the ICD of
the first band was investigated through a calculation of the
FM–␤-CD inclusion complex
The absorption spectrum of FM was slightly affected by the
m-␮
term (48,49), which appears relevant for an isolated
addition of -CD (data not shown). A weak hypochromic
␤
n,
␲* transition centered on this group. It was found that the
effect accompanied by the lack of any clear isosbestic point
was in fact observed upon addition of the macrocycle up to
10 M. Although this observation was indicative for some
type of association, the spectral changes were too small to
obtain any quantitative information on the host–guest system
through this spectroscopic technique. A much more clear
different symmetry of the magnetic moment in the two dif-
ferent situations plays a decisive role, giving rise to a fairly
large negative rotatory power for the planar geometry and
to a small but positive term for the perpendicular one. On
Ϫ2
the other hand, the ICD band of the
the phenyl ring, is calculated positive through the
tric dipole–dipole term. This argument support well the mod-
␲,␲* state, centered on
␮–␮
elec-
confirmation for the incorporation of FM in the ␤-CD cavity
was provided by ICD. As shown in Fig. 4, the ICD signal
arises as a consequence of the complexation of the nonchiral
drug with the optically active host molecule. The ICD spec-
el invoked to explain the photoreactivity of FM in
below).
␤-CD (see
The nonlinear analysis of the dependence of the ICD sig-
nals on the -CD concentration (Fig. 4, insets) was per-
formed according to (Eq. 2) (corresponding to conditions
where the -CD concentrations are in excess with respect to
the guest concentration):
trum clearly shows a negative signal centered at
and a larger, positive signal at 350 nm. On the basis of
the calculated energies, one can attribute the negative signal
to the lowest n, * states, centered mainly on the NO2 group,
whereas the positive signal arises from the first allowed
ϳ425 nm
␤
ϳ
␤
␲
␲,␲*
ICD 10 c0Kass
ϭ
␪
[␤-CD]/(1
ϩ
Kass
[␤-CD])
(2)
transition. Inspection of the sign of the ICD bands affords
where c0 is the initial concentration of FM, is the molar
ellipticity of the complex and Kass is the association constant.
We have preferred to apply a nonlinear fit because in this
way the data are weighted in a manner that reflects better
its quality. The results of the plot are consistent with a 1:1
Ϫ
1
association constant, Kass
ϭ 300 Ϯ 50 M . This value com-
pares well with those reported both for similar aryl amide
(50–54) and nitro aromatic derivatives (55). In this regard,
it should be noted that although the ICD data fit the equation
that considers only a 1:1 complex, the lack of any isosbestic
point observed in the absorption spectra upon addition of ␤-
CD indicate that more than one type of host–guest complex
could be formed. Thus, the association constant obtained
might be related to different complexes that cannot be dis-
Figure 3. Spectral changes observed in nitrogen-flushed 10Ϫ4 M FM
solution in phosphate buffer upon regular irradiation time intervals
of 30 min.
‡Unfortunately, we could not perform Monte Carlo theoretical cal-
culation of the inclusion complex due to problems related to the
parametrization of the NO2 group.

10 S. Sortino et al.
Figure 4. ICD of 10Ϫ4 M FM in the presence of 10Ϫ2 M
␤-CD. Cell
path: 1 cm. Inset: dependence of the ICD signal at 350 nm on [
␤-
CD] and nonlinear fit according to (Eq. 2).
tinguished by either absorption or ICD spectroscopy. This
hypothesis is not unprecedented. Actually, the formation of
different inclusion complexes with ␤-CD characterized by 1:
1 stoichiometry but different inclusion geometry is quite
common for both aryl amide (51–54) and nitro aromatic de-
rivatives (55).
Scheme 2
At first sight, we were surprised to observe the formation
of the photoreduction product 2 in these experimental con-
ditions. Indeed, the origin of such a compound cannot be
roughly rationalized on the basis of either the presence of
the easily abstractable hydrogen atoms of the macrocycle or
the low polarity of the cavity when compared with water.
As outlined earlier, the irradiation of FM performed in sol-
vents such as either methanol or isopropanol, characterized
by hydrogen-donating properties better than water and po-
Photoreactivity in
␤-CD environment
Figure 5 shows the spectral changes observed in a nitrogen-
saturated solution of FM in the presence of 10
Ϫ
2
M ␤-CD
upon 325 nm light excitation. A remarkably different spec-
tral behavior is evident with respect to that observed in the
absence of the macrocycle (Fig. 3). As confirmed by both
quantitative and qualitative analyses of the irradiated mix-
ture, the observed spectral variations are consistent with very
significant changes in the nature and in the efficiency of the
photodegradation pathways of FM. The formation of 1 was
indeed accompanied by two new photoproducts, 2 and 3,
resulted from photoreduction of the nitro group and photo-
cleavage of the amide bond, respectively (Scheme 2). Fur-
thermore, the photodegradation quantum yield of FM in the
presence of the macrocycle increased by ca 20-fold with
respect to that found in its absence. The overall results con-
cerning the photoreactivity of FM in aqueous solution in the
larity not too much different than
␤-CD, gave indeed the
almost exclusive formation of 1. Moreover, only this pho-
toproduct was also observed when the irradiation of FM was
performed in aqueous solution in the presence of 0.1 M glu-
cose.
A possible hypothesis to explain the photogeneration of 2
could be consistent with ‘‘cage photoreactions’’ triggered by
structural changes of FM upon incorporation in the
␤-CD
cavity. A less perpendicular geometry of the nitro group with
respect to the aromatic ring, caused by steric constrains and
specific weak interactions (i.e. H-bond involving the CF3
presence of
␤-CD and, for the sake of comparison, in its
absence are summarized in Table 2.
and/or NO2) with ␤-CD, would account well for the forma-
tion of the nitroso derivative 2. Such changes in the perpen-
dicularity of the nitro group would lead in fact to a less-
extended overlap of the p atomic orbital of oxygen with the
adjacent orbital of the aromatic ring. The consequence of
this type of conformation is reflected in a logical increase of
the biradical character of the n,␲* triplet and a consequent
higher ability of this latter in abstracting hydrogen in the
presence of a suitable H-donor. Under these conditions, an
intermolecular H-abstraction photoprocess involving the ni-
tro group and the available hydrogen atoms of the
␤-CD
cavity could become a significant photoprocess, thus giving
rise to the formation of 2 according to the very well-known
mechanistic pathways (34–39). Moreover, a reasonable de-
crease in the energy gap between the two low-lying n,
and * triplet states due to the low polarity of the -CD
cavity might also play a key role for the increased photo-
␲*
4
Figure 5. Spectral changes observed in a nitrogen flushed 10Ϫ
M
2
FM solution in phosphate buffer in the presence of 10Ϫ
upon regular irradiation time intervals of 10 min.
M ␤-CD
␲,␲
␤

Photochemistry and Photobiology, 2001, 73(1) 11
Table 2. Quantum yields related to the photodegradation of FM and formation of its photoproducts in aqueous buffered solution pH 7.4
under anaerobic conditions in the absence and in the presence of
␤-CD*†
␤-CD (M)
⌽
Ϫ
⌽
1
⌽
2
⌽
3
FM
3
2
3
0
0.01
(3.5
(6.5
Ϯ
Ϯ
0.3)
0.7)
ϫ
ϫ
10Ϫ
10Ϫ
(3.0
(6.0
Ϯ
Ϯ
0.4)
0.8)
ϫ
ϫ
10Ϫ
0
0.4)
0
0.3)
3
2
2
10Ϫ
(3.5
Ϯ
ϫ
10Ϫ
(2.0
Ϯ
ϫ
10Ϫ
*
†
␭
␤
ϭ
325 nm.
exc
-CD ϭ ␤-cyclodextrin.
reactivity. Finally, it is noteworthy that the quantum yield
related to the formation of 2 (Table 2) accords well with the
values commonly observed in the case of H-abstraction pro-
mation of this photoproduct would have been expected. Ac-
cording to literature data regarding the formation of similar
products in CD cages (51–54), the change of polarity may
cesses from the n,␲* triplet of the nitroaromatic compounds
be responsible only in part for the increase of ⌽1. Then, we
in good hydrogen-donating solvents (34,36).
believe that an important part of 1 is formed from the re-
combination of the phenoxy radicals that are not in the sol-
vent cage but trapped in the cavity of the macrocycle. As
proposed by Veglia and de Rossi (52), under these condi-
tions the competition with H-abstraction becomes more fa-
vorable than in aqueous solution given that in the interior of
NMR and UV spectroscopic investigations do not provide
unambiguous information to ascertain the structure of the
inclusion complex. Nevertheless, the sequence of signs
shown by the ICD spectrum, discussed in the previous sec-
tion, leads to conclude that a more coplanar structure of FM
in the
␤-CD appears highly probable.
the ␤-CD cavity there are 14 available hydrogen atoms bond-
As reported in Scheme 2, formation of photoproduct 3, in
addition to 2 and 1, occurred when FM was irradiated in the
ed to secondary carbons and close to the radical center.
This result stimulates a further comment. Since the for-
presence of
␤
-CD. Taking into account that the photocleav-
mation of 1 also takes place in the ␤-CD environment, a
age of the amide bond is favored by a decrease of the solvent
polarity (51,52,56), it would be conceivable to attribute the
constructive overlap between the p orbital of the oxygen
with the adjacent p orbital of the aromatic ring could still
be present even in the included compound. In this regard,
the photoreduction pathway to form 2 and the nitro–nitrite
photorearrangement to form 1 may reflect competitive pho-
toprocesses, occurring from the same host–guest complex,
independent photoprocesses, taking place from two different
complexes with different inclusion geometry, or both. As
reported in the case of similar aryl amide and nitro aromatic
derivatives (53–55), the proposed scenario is quite common
when more than one type of inclusion complex is formed.
In these cases, the reactivity of complexed substrates is very
much determined by the structure of the complexes whose
orientation within the cavity is of great importance.
formation of 3 to the low polarity of the
␤-CD cavity if
compared to the aqueous solution. The absolute lack of 3
noticed in solvents with lower polarity than water, such as
methanol and isopropanol (see ‘‘Photoreactivity in homo-
geneous media’’), rules out this hypothesis. Further, a hy-
drolytic generation of 3 due to a potential catalytic effect of
the
carded. No degradation of FM at all was in fact observed
after stirring a FM– -CD solution for 5 h at 40 C. On the
basis of these results, the formation of 3 in the presence of
-CD might also reflect the conformational changes of the
nitro group proposed earlier. Indeed, its tendency toward a
less-perpendicular geometry in the -CD environment,
␤-CD in cleaving the amide bond (16,55) was also dis-
␤
Њ
␤
␤
would make this group much more conjugated with the ar-
omatic ring, therefore leading to a weakening of the amide
bond and favoring the cleavage of the latter. As ascertained
well, a singlet excited state is the more likely responsible
for these types of photoreaction (40,41,51–54). It is also
noteworthy that no formation of isopropyl 2-amino-5-nitro-
4-(trifluoromethyl)phenyl ketone, the ortho isomer of FM
expected by photo-Fries rearrangement after the amide bond
photocleavage, was noticed. Since the formation of ortho/
para isomeric products is quite a common process for sim-
ilar compounds (38,39,50–54) our results suggest that the
photocleavage reaction occurs mainly in the cavity. Indeed,
the acyl radical produced by the amide cleavage is big
enough to be sterically hindered by the macrocycle in giving
the ortho isomer of FM.
CONCLUSIONS
The photochemistry of the FM in homogeneous media pro-
vides an example of nitro to nitrite intramolecular photo-
rearrangement triggered by the twisted conformation of the
nitro group. Such an out of plane geometry is responsible
for the low biradical character of the n,␲* triplet state of the
drug reflected in the negligible photoreactivity towards H-
abstraction even in hydrogen-donating solvents.
FM incorporates into the ␤-CD cavity. From a strict chem-
ical point of view the overall photochemistry observed in the
presence of the macrocycle represents an example of how
the localization of a guest molecule in a restricted micro-
environment can lead to dramatic changes in both the effi-
ciency and the nature of the photochemical deactivation
pathways. The photoreactivity of the guest molecule cannot
be simply rationalized on the basis of the polarity of the host
molecule and on its role as a reactant. The occurrence of a
structural modification, as suggested by the ICD spectrum
and by the nature of the photoproducts, appears more prob-
able.
As shown in Table 2, the formation of 1 in the experiment
performed in the presence of ␤-CD occurs with a quantum
yield higher than that observed in water solution. This fact
clearly suggests that the molecules of FM not included in
the cavity (ca 30%) cannot be the only ones responsible for
the formation of 1, otherwise a lower efficiency in the for-

12 S. Sortino et al.
16. Saenger, W. (1980) Cyclodextrin inclusion compounds in re-
search and industry. Angew. Chem. Int. Ed. Engl. 19, 344–362.
17. Wenz, G. (1994) Cyclodextrins as building blocks for supra-
molecular structures and functional units. Angew. Chem. Int. Ed.
Engl. 33, 803–822.
The present report can also be of biological relevance.
Actually, given the potential of CD in providing an useful
model to mimic the interactions of the drug with hydropho-
bic biological sites and by taking into account that all the
18. Uekama, K., F. Hirayama and T. Ire (1998) Cyclodextrin drug
carrier systems. Chem Rev. 98, 2045–2076.
19. Ramamurthy, V., R. G. Weiss and G. S. Hammond (1993) A
model for the influence of organized media on photochemical
reactions. Adv. Photochem. 18, 67–234.
20. Ramamurthy, V. (1991) Photochemistry in Organized and Con-
strained Media. VCH Publishers, New York.
21. Kalyanasundaram, K. (1987) Photochemistry in Microhetero-
geneous Systems. Academic Press. Orlando.
22. Monti, S., S. Sortino, G. De Guidi and G. Marconi (1998) Su-
pramolecular photochemistry of 2-(3-benzoylphenyl)propionic
acid (Ketoprofen). A study in the -cyclodextrin cavity. New J.
Chem. 22, 599–604.
23. Sortino, S., G. De Guidi, G. Marconi and S. Monti (1998) Trip-
let photochemistry of suprofen in aqueous environment and in
photoproducts formed in ␤-CD are mediated by radical path-
ways, the obtained results represent a good step forward in
understanding the recently reported phototoxic effects dis-
played by FM. Incorporation or localization of the drug in
specific pockets of a biosubstrate with particular hydropho-
bicity and in the presence of steric constrains and specific
interactions, could lead in fact to new and more efficient
photodegradation pathways, with a significant increase in the
photoproduction of radical species.
Finally, in light of the much lower photostability of the
FM–␤-CD inclusion complex if compared with the free mol-
ecule, the study presented herein suggests that the use of
CD, or similar kinds of drug/carrier system, could not be the
more appropriate approach to enhance the drug solubility.
the
␤-cyclodextrin inclusion complex. Photochem. Photobiol.
67, 603–611.
24. Sortino, S., J. C. Scaiano, G. De Guidi and S. Monti (1999)
Effect of ␤-cyclodextrin complexation on the photochemical and
photosensitizing properties of tolmetin: a steady state and time
resolved study. Photochem. Photobiol. 70, 549–556.
Acknowledgements Partial financial supports from MURST in the
framework of the ‘‘Programmi di Ricerca di Rilevante Interesse Na-
zionale’’ (project: Mechanisms of Photoinduced Processes in Ho-
mogeneous Media and in Complex Matrices) and from the Progetto
‘‘Proprieta` Chimico-Fisiche dei medicamenti e loro sieurezze
d’uso’’ of the ISS is gratefully acknowledged. We wish to thank Dr.
F. Vargas for sharing with us a preprint of his manuscript.
25. Jimenez, M. C., M. A. Miranda and R. Tormos (1995) Photo-
decarboxylation of 2-phenylpropionic acid in solution and in-
duced within ␤-cyclodextrin. Tetrahedron 51, 2953–2958.
26. Monti, S., S. Sortino, S. Encinas, G. Marconi, G. De Guidi and
M. A. Miranda (1998) Photoprocesses in photosensitizing drugs
containing a benzophenone-like chromophore. In: Drugs Pho-
tochemistry and Photostability (Edited by A. Albini and E. Fa-
sani), pp. 150–161. The Royal Society of Chemistry, Cam-
bridge.
27. De Guidi, G., G. Condorelli, L. L. Costanzo, S. Giuffrida, S.
Monti, and S. Sortino (1998) Molecular mechanisms of photo-
sensitization induced by drugs on biological systems and design
of photoprotective systems. In: Drugs Photochemistry and Pho-
tostability (Edited by A. Albini and E. Fasani), pp. 194–210.
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