Photoreactivity of fluoroquinolones: Nature of aryl cations generated in water

Article abstract of DOI:10.1021/ol301694p

The nature of stabilized aryl cations generated from photodehalogenations of fluoroquinolones in aqueous media has been studied by comparing the photophysical and photochemical behavior of lomefloxacin (LFX) and its N(4′)-acetylated form (ALFX). Photoproduct studies, laser flash photolysis, and emission measurements have shown that this small peripheral modification produces important changes in the properties of the singlet aryl cations generated. Also, in basic medium, a new photodehalogenation pathway for 6,8-dihalogenated fluoroquinolones has been observed.

Full text of DOI:10.1021/ol301694p

ORGANIC
LETTERS
2012
Vol. 14, No. 15
3940–3943
Photoreactivity of Fluoroquinolones:
Nature of Aryl Cations Generated in Water
Sonia Soldevila and Francisco Bosca*
´
Instituto de Tecnologıa Quımica, Consejo Superior de Investigaciones Cientıficas/Universidad
Politecnica de Valencia (CSIC/UPV), Avd. Los Naranjos s/n, 46022-Valencia, Spain
´
´
ꢀ
fbosca@itq.upv.es
Received June 20, 2012
ABSTRACT
The nature of stabilized aryl cations generated from photodehalogenations of fluoroquinolones in aqueous media has been studied by comparing
the photophysical and photochemical behavior of lomefloxacin (LFX) and its N(40)-acetylated form (ALFX). Photoproduct studies, laser flash
photolysis, and emission measurements have shown that this small peripheral modification produces important changes in the properties of the
singlet aryl cations generated. Also, in basic medium, a new photodehalogenation pathway for 6,8-dihalogenated fluoroquinolones has been
observed.
Fluoroquinolones (FQs) are antibacterial agents that
develop their pharmacological activity through the
inhibition of the bacterial topoisomerase II, an enzyme
involved in the replication and repair of bacterial
DNA.1 In recent years FQs have drawn much attention
due to their antitumoral activity.2 In vitro and in vivo
studies have corroborated the anticancer effects of
quinolone antibiotics supporting the observation that
FQs reduce all-cause mortality among cancer patients.3
The mechanism suggested for the direct FQ antitumor
effect has been the inhibition of mammalian topo-
isomerases (I and II) and DNA polymerase. In this
context, the genotoxic effects exhibited by FQs in
eukaryotic systems are enhanced by UV irradiation,
which confers to these molecules a potential property
as photochemotherapeutic agents.4 This photoinduced
genotoxicity has remarkably been detected in dihalo-
genated FQs such as fleroxacin (FLX,), BAY y3118
(BAY), and lomefloxacin (LFX), a compound proposed
as a standard for photomutagenic action (structures are
shown in Scheme 1).5
The DNA photoinduced damage has been associated
with the alkylating reactivity of an intermediate arising
from FQ photodehalogenation.6 In fact, formation of
covalent bindings between LFX and biomolecules such
as guanosine monophosphate has been observed.7a
(4) Perrone, C. E.; Takahashi, K. C.; Williams, G. M. Toxicol. Sci.
2002, 69, 16–22.
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Photobiol. 2009, 85, 861–868. (b) Marrot, L.; Belaidi, J. P.; Jones, C.;
Perez, P.; Riou, L.; Sarasin, A.; Meunier, J. R. J. Invest. Dermatol. 2003,
121, 596–606. (c) Meunier, J. R.; Sarasin, A.; Marrot, L. Photochem.
Photobiol. 2002, 75, 437–447. (d) Martinez, L. J.; Li, G.; Chignell,
C. F. Photochem. Photobiol. 1997, 65, 599–602. (e) Chignell, C. F.;
Haseman, J. K.; Sik, R. H.; Tennant, R. W.; Trempus, C. S. Photochem.
Photobiol. 2003, 77, 77–80. (f) Fasani, E.; Profumo, A.; Albini, A.
Photochem. Photobiol. 1998, 68, 666–674. (g) Jeffrey, A. M.; Shao, L.;
Brendler-Schwaab, S. Y.; Schluter, G.; Williams, G. M. Arch. Toxicol.
2000, 74, 555–559.
(1) Domagala, J. M.; Hanna, L. D.; Heifetz, C. L.; Hutt, M. P.; Mich,
T. F.; Sanchez, J. P.; Solomon, M. J. Med. Chem. 1986, 29, 394–404.
(2) (a) Cullen, M.; Baijal, S. Br. J. Cancer 2009, 101, S11–S14.
(b) Kim, K.; Pollard, J. M.; Norris, A. J.; McDonald, J. T.; Sun,
Y. L.; Micewicz, E.; Pettijohn, K.; Damoiseaux, R.; et al. Clin. Cancer
Res. 2009, 15, 7238–7245. (c) Al-Trawneh, S. A.; Zahra, J. A.; Kamal,
M. R.; El-Abadelah, M. M.; Zani, F.; Incerti, M.; Cavazzoni, A.; et al.
Bioorg. Med. Chem. 2010, 18, 5873–5884.
(3) Paul, M.; Gafter-Gvili, A.; Fraser, A.; Leibovici, L. Eur J. Clin.
Microbiol. Infect. Dis. 2007, 26, 825–831.
(6) Albini, A.; Monti, S. Chem. Soc. Rev. 2003, 32, 238–250.
r
10.1021/ol301694p
Published on Web 07/25/2012
2012 American Chemical Society

Scheme 1. Structure of Photogenotoxic Fluoroquinolones and
the Model Compound AFQ
Scheme 2. Photodegradation of LFX in Neutral Aqueous
Media with and without Halides
In order to determine the involved mechanisms of
this new class of photoactivated drugs, a large number of
studies concerning the photophysical and photochemical
properties of FQs bearing a further halogen atom at posi-
tion 8 of the quinolinic ring have been carried out dur-
ing the past few years.5d,7 All of them have shown an
unusual photodehalogenation by heterolysis of the strong
C8ꢀhalogen bond.5d,7 Generation of an aryl cation seems
to be the key intermediate of the FQs photobinding
properties.7h,g However, controversial interpretations
about the nature and reactivity of this type of intermediate
have been reported in the literature.7a,f,h In fact, in studies
performed with LFX, the nature of the aryl cation detected
was attributed not only to a charge delocalized species
with a carbene character 3LFXþ (triplet ground state
multiplicity)7f but also to a carbocation with singlet multi-
With this background, and considering that acetyla-
tion of the piperazinyl ring of norfloxacin as a peripheral
change of the heterocyclic skeleton of this FQ produces
photophysical changes in the singlet and triplet excited
states,8 the whole aminoalkyl substituent (R1R2Nꢀ) is
expected to be very influential in the photophysical and/
or photochemical behavior of 6,8-dihalogenated fluoro-
quinolones. Thus, the calculations performed for AFQ
photodehalogenation should not be extrapolated to FQs
with this type of substituent such as LFX, FLX, or BAY.
Accordingly, it appeared reasonable to check this hypo-
thesis using LFX and its N(40)-acetylated derivative (ALFX)
to obtain further evidence of the involvement of singlet
aryl cations in FQ photodegradation. This issue has been
addressed by performing both steady-state and time-
resolved studies in aqueous media at pH ca. 7.4 as well as
under basic conditions. Lomefloxacin is commercially
available, and the preparation of its N-acetyl derivative
(ALFX) was achieved following standard acetylation
procedures,9 and its structure was unambiguously assigned
on the basis of its NMR spectroscopic data (Supporting
Information).
3
plicity (1LFXþ).7h The involvement of LFXþ was sup-
ported by a lack of reactivity between the aryl cation and
water as a neutral nucleophile and by theoretical calcula-
tions, claiming that the triplet carbene generated from
photodefluorination of an aminofluoroquinolone AFQ
(Scheme 1) is the lowest transient state.7f By contrast,
the high reactivity of the intermediate toward Brꢀ and
Clꢀ and the absence of quenching by molecular oxygen
were the most important results supporting the participa-
1
tion of LFXþ.7h Moreover, although in LFX photo-
The study was initiated by analyzing the singlet excited
state properties of both compounds. Acetylation induced a
red shift in the emission (Figure 1) and a slightly different
fluorescence lifetime (τ = 1.2 and 1.7 ns for LFX and
ALFX respectively). Besides, when the pH was increased
to 12, the fluorescence faintly changed for ALFX but
almost disappeared in the case of LFX. Therefore, at this
degradation it was established that product 1 is generated
by an intramolecular reaction of 3LFXþ with a neighbor-
ing CꢀH bond (position β in the N-ethyl group), the
intermediate involved in the formation of 1Cl and 1Br
remains unclear (Scheme 2).7f,h
1
pH, a new reaction pathway from LFX must be occur-
(7) (a) Fasani, E.; Manet, I.; Capobianco, M. L.; Monti, S.; Pretali,
L.; Albini, A. Org. Biomol. Chem. 2010, 8, 3621–3623. (b) Fasani, E.;
Mella, M.; Albini, A. Eur. J. Org. Chem. 2004, 5075–5082. (c) Fasani, E.;
Mella, M.; Caccia, D.; Tassi, S.; Fagnoni, M.; Albini, A. Chem.
Commun. 1997, 1329–1330. (d) Fasani, E.; Mella, M.; Monti, S.; Albini,
A. Eur. J. Org. Chem. 2001, 391–397. (e) Fasani, E.; Rampi, M.; Albini,
A. J. Chem. Soc., Perkin Trans. 2 1999, 1901–1907. (f) Freccero, M.;
Fasani, E.; Mella, M.; Manet, I.; Monti, S.; Albini, A. Chem.;Eur. J.
2008, 14, 653–663. (g) Monti, S.; Sortino, S.; Fasani, E.; Albini, A.
Chem.;Eur. J. 2001, 7, 2185–2196. (h) Cuquerella, M. C.; Miranda,
M. A.; Bosca, F. J. Phys. Chem. B 2006, 110, 6441–6443. (i) Fasani, E.;
Monti, S.; Manet, I.; Tilocca, F.; Pretali, L.; Mella, M.; Albini, A. Org.
Lett. 2009, 11, 1875–1878.
ring. An intramolecular electron transfer between the lone
pair of N(40) and the quinolinic ring (path I, Scheme 3), as
previously described for norfloxacin,8a would explain this
result.
Flash photolysis of ALFX and LFX in water also
revealed the influence of the N-acetylation of the piper-
azinyl ring on the spectroscopic and kinetics properties of
the detected intermediates arising from LFX. In fact, both
FQs under a N2O atmosphere at pH = 7.4 showed very
(8) (a) Cuquerella, M. C.; Miranda, M. A.; Bosca, F. J. Phys. Chem.
A 2006, 110, 2607–2612. (b) Bosca, F.; Lhiaubet-Vallet, V.; Cuquerella,
M. C.; Castell, J. V.; Miranda, M. A. J. Am. Chem. Soc. 2006, 128, 6318–
6319. (c) Lhiaubet-Vallet, V.; Cuquerella, M. C.; Castell, J. V.; Bosca, F.;
Miranda, M. A. J. Phys. Chem. B 2007, 111, 7409–7414.
(9) Cuquerella, M. C.; Bosca, F.; Miranda, M. A. J. Org. Chem. 2004,
69, 7256–7261.
Org. Lett., Vol. 14, No. 15, 2012
3941

Scheme 3. Photodegradation Pathways of (A)LFX in Aqueous Media
different transient absorption spectra (Figure 2A and 2B).
In the case of LFX, two successive short-lived transients
species were detected. The first one, absorbing at λmax
=
370 nm and with a τ of ca. 30 ns, has been claimed as
the LFX triplet excited state (3LFX).7f Nevertheless,
3
this transient species could also be attributed to LFXþ
3
because all FQ show absorption maxima at long wave-
lengths6,8c and there is an important similarity between the
absorption spectrum of this LFX transient species and
those observed with other triplet carbenes (see path II,
Scheme 3).10
The second species detected in LFX experiments (λmax
490 nm and τ = 200 ns) corresponds to an aryl cation.
=
However, as mentioned above, the nature of this tran-
sient is still an open matter (1LFXþ or 3LFXþ).7f,h
Analysis of the results obtained using ALFX also
showed two successive intermediates but with absorptions
at λmax = 600 nm (the first intermediate τ = 340 ns) and at
Figure 2. Transient absorption spectra of LFX (A) and ALFX
(B) aqueous solutions in N2O at different times after excitation
at 355 nm. Kinetics of ALFX aqueous solutions in the absence
and presence of NaCl at 600 nm (C) and 480 nm (D).
λmax = 480 nm (the second one with τ = 440 ns). To
characterize these transient species, similar quenching
studies to those performed with the intermediates arising
from LFX were performed. Thus, it was observed that
the two bands are not appreciably quenched or modified
by the presence of an O2 atmosphere. This means that both
transient species can be neither a triplet excited state nor an
aryl cation with carbene character because it appears to be
well established that both types of intermediates are very
reactive to O2.10,11
(10) (a) Grabner, G.; Richard, C.; Kohler, G. J. Am. Chem. Soc.
1994, 116, 11470–11480. (b) Othmen, K.; Boule, P.; Szczepanik, B.;
Rotkiewicz, K.; Grabner, G. J. Phys. Chem. A 2000, 104, 9525–9534.
(11) Ortica, F.; Pohlers, G.; Scaiano, J. C.; Cameron, J. F.; Zampini,
A. Org. Lett. 2000, 2, 1357–1360.
Figure 1. Emission spectra of LFX and ALFX in aqueous media
at pH = 7.4 and 12.
3942
Org. Lett., Vol. 14, No. 15, 2012

analysis of LFX and ALFX photolysis showed mainly
the formation of 112 and 2 respectively, which arise
from their triplet aryl cations (Scheme 3). Nevertheless,
under basic conditions, the φD values of LFX and
ALFX showed important differences (Table 1). More-
over, while 1B1 and 1B2 were the photoproducts de-
tected for LFX,7b 2 and 2OH were obtained from
the photolysis of ALFX. The structures of 2 and 2OH
were unambiguously assigned (key analytic and spectro-
scopic data are listed in the Supporting Information).
In this context, in agreement with path IIA in Scheme 3,
the increase of pH produces the formation of higher
amounts of photoproduct 2OH and a rapid quenching
of 1ALFXþ. By contrast, the different type of com-
pounds obtained from LFX at pH = 12 would be
produced by the intramolecular electron transfer of the
N(40) lone pair of the piperazinyl substituent and the
quinolinic ring (path I in Scheme 3), which clearly
discards the suggested involvement of the LFX triplet
excited state or any aryl cation.7b
In summary, the contribution of the aryl cations 1LFXþ
and 1ALFXþ to the (A)LFX photodehalogenations
has clearly been demonstrated in the present investiga-
tion and all findings are in agreement with the photo-
physical and photochemical pathways shown in Scheme 3.
Additionally, with these processes taken into account, the
photochemistry of other 6,8-dihalogenated quinolones
such as fleroxacin7i and BAY y31187h as well as the
alkylating properties described for this type of FQ can be
better understood through the involvement of aryl cations
with singlet multiplicity.
Table 1. Quantum Yields of Fluorescence and Photodegrada-
tion of LFX and ALFX Aqueous Solutions at pH 7.4 and 12
LFX
ALFX
pH
ΦF
ΦD
ΦF
ΦD
7.4
12
0.08a
0.55a
0.25
0.11
0.6
0.4
<0.002
0.095
a Values from the literature.7h Experimental data and results analysis
in the Supporting Information.
When ALFX was submitted to LFP in the presence of
halides, the first intermediate was found to be highly
reactive. Bimolecular rate constants (kq) of 0.5 ꢁ 108,
0.5 ꢁ 109, and 0.7 ꢁ 109 Mꢀ1 sꢀ1 were determined for its
interaction with Clꢀ, Brꢀ, and Iꢀ respectively (Figure 2 C).
With these results it can be asserted that the species
absorbing at λmax = 600 nm is a singlet aryl cation
1ALFXþ (path II, Scheme 3). Besides, the fact that cation
1ALFXþ is quenched by halides with kq ca. 10 times lower
1
than those reported in the literature for LFXþ (kq with
Clꢀ, Brꢀ, and Iꢀ are 4 ꢁ 108, 4 ꢁ 109, and 7 ꢁ 109 Mꢀ1 sꢀ1
respectively)7f,h is evidence of the importance of the
acetylation of the piperazinyl ring in the reactivity of this
type of aryl cation.
The LFP experiments performed using LFX aqueous
solutions at pH = 12 did not show any transient species.
Nevertheless, in the case of ALFX, the intermediates
absorbing at λmax = 600 nm (1ALFXþ) and at λmax
=
480 nm were detected (transient absorption spectra are
shown in the Supporting Information). This important
difference observed between both FQs under basic condi-
tions clearly confirmed the intramolecular pathway de-
On the other hand, results have revealed the impact of
N-alkyl peripheral substituents on the photophysical and
photochemical properties of 6,8-dihalogenated quinolones.
Thus, by changing the substituents of fluoroquinolones, it is
possible to modulate not only their pharmacological prop-
erties but also the reactivity of their aryl cations, which may
be used to design a new family of photoactivated drugs for
photochemotherapy.
1
1
scribed above for LFX, which is blocked for ALFX
(path I, Scheme 3). Under basic conditions it was also
observed that the hydroxyl anion quenches 1ALFXþ with
a rate constant of 0.2 ꢁ 109 Mꢀ1 sꢀ1. Moreover, increasing
amountsofOHꢀ aswellasofhalidesatneutral pH resultin
rapid generation of the second intermediate but with a
decrease of its absorbance (Figure 2C and 2D). Therefore,
the structure of this species would be attributed to the
diradical cation XH but not to the cation XHþ because the
latter would be also quenched by OHꢀ and this fact does
not take place (path IIB, Scheme 3).
Acknowledgment. Financial support from Spanish
government (CTQ2010-19909) and the Generalitat
Valenciana (PROMETEO program, ref 2008/090) isgrate-
fully acknowledged.
Supporting Information Available. Spectroscopic data
and experimental details. This material is available free of
charge via Internet http://pubs.acs.org.
The photophysical results were complemented by photo-
product studies in order to confirm the intermediates
involved in the photolysis of ALFX. Irradiations of ALFX
and LFX in deaerated aqueous solutions at pH ca. 7.4 and
12 were performed in a multilamp photoreactor using
UVA light at λmax = 350 nm. Kinetics studies revealed
that photodegradation quantum yields (φD) of LFX and
ALFX are very similar at pH 7.4 (Table 1). Product
(12) Fasani, E.; Barberis Negra, F. F.; Mella, M.; Monti, S.; Albini, A.
J. Org. Chem. 1999, 64, 5388–5395.
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 15, 2012
3943