Cuquerella et al.
yield decreased with pH (from 0.12 at pH ) 7.4 to less
than 0.001 at pH ca. 12). This fact was previously
observed and rationalized as due to different intrinsic
radiative constants of the fluoroquinolone anion and the
zwitterionic form.21 In view of the present results, this
effect can be better understood by means of the above-
mentioned intramolecular electron transfer from the 4′-N
piperazinyl group to the fluoroquinolone ring system in
its singlet excited state.
tion of fluoride anion. This process is explained by direct
attack of OH to the excited triplet state, to give a
-
cyclohexadienyl anion, with subsequent release of fluo-
ride. Inhibition of this process by addition of a triplet
quencher is in agreement with the involvement of the
ANFX triplet excited state. On the other hand, the 4′-N
of the piperazinyl ring plays an important role in the
photochemistry and the photophysics of these com-
pounds. When its electron lone pair is fully available, the
singlet excited state of the fluoroquinolone system is
quenched through intramolecular electron transfer. This
precludes observation of fluorescence or any LFP tran-
sient and leads to photostability.
On the other hand, it has been postulated that deflu-
orination of NFX in aqueous solutions at neutral pH
occurs from its triplet excited state. This is in agreement
with the fact that oxygen and several anions, such as
phosphate buffer, are able to quench the NFX triplet
excited state and decrease NFX photodegradation rates.24
In this context, two reaction mechanisms have been
proposed: (i) heterolytic cleavage of triplet NFX, followed
by nucleophilic trapping of the resulting aryl cation, or
Exp er im en ta l Section
Syn th esis of 7-(4-Acetyl-1-piper azin yl)-1-eth yl-6-flu or o-
1
,4-d ih yd r o-4-oxoqu in olin e-3-ca r boxylic a cid (ANF X). A
solution of 1-ethyl-6-fluoro-7-(1-piperazinyl)-1,4-dihydro-4-oxo-
quinoline-3-carboxylic acid (NFX) (150 mg, 0.47 mmol) in Ac
25 mL) was refluxed for 5 h. The solution was cooled to room
-
3
(
2
ii) direct nucleophilic attack of OH or H O to NFX, to
2
O
give subsequent release of fluoride. The lack of NFX
signal in alkaline medium precluded the possibility of
providing direct LFP evidence in favor of the second
reaction pathway. The use of ANFX could be a valuable
tool for this purpose, as both fluorescence and T-T
absorption are observable at higher pH values.
(
temperature and concentrated. The residue was dissolved in
water, neutralized to pH ∼7.4, and extracted with CH Cl . The
2
2
1
organic phase was concentrated to dryness. The H NMR
spectrum was essentially coincident with that previously
reported by Koga et al.29
Ir r a d ia tion P r oced u r es. Irradiations were performed in
a multilamp photochemical reactor equipped with six lamps
emitting in the 310-390 nm range, with a maximum at 350
nm. Samples of different pH were obtained by dropwise
addition of some stock solutions of NaOH (from 10 to 0.01 M);
pH values were measured by means of a glass electrode.
In the LFP experiments with ANFX from pH ) 7.4 to
3.4, a transient absorption spectrum was detected that
1
was assigned to the triplet excited state based on its
quenching not only by oxygen but also by NP. The fact
that similar intensities of the T-T band of ANFX were
observed in the whole range indicates the formation of
ANFX triplet with similar quantum yields. Although the
hydrated electron was also observed as transient, its
origin is assumed to be biphotonic;27 thus, this species
should not play any role in the photoreaction mechanism
upon lamp irradiation.
4
NaClO (0.1 M) was present in all the solutions. The photo-
reactions were performed under anaerobic conditions and
monitored by HPLC on an analytical C18 column (25 × 0.4
cm, mean particle size 5 µm) with a flow rate of 0.7 mL/min
and a mixture of acetonitrile/water/trifluoracetic acid 15/84.9/
0
.1 as eluent. Photolysis of aqueous solutions of (A)NFX (60
µM) at pH 7.4 was performed with and without PB (10-100
mM). Moreover, irradiations of (A)NFX in aqueous solutions
at pH ∼ 12 were carried out.
On the other hand, the photoreaction was faster at
-
higher pH values, and the triplet was quenched by OH
anions with a measurable rate constant of (0.3 ( 0.1) ×
For quantum yield measurements, irradiations were per-
formed at different pH values (7.2-13.2) and the photodeg-
radation of ANFX was also followed by HPLC. The quantum
yields were obtained by comparison with the value reported
6
-1 -1
1
0 M
s . This is in agreement with photosubstitution
following pathway ii, involving direct attack of the
nucleophile to the excited triplet. Accordingly, photolysis
of ANFX in the presence of the triplet quencher NP
proceeded much more slowly than in the absence of this
quencher. The fact that the photodegradation rates follow
a plateau behavior in the pH range 7-10 suggests that,
under these conditions, water is the actual nucleophile.
29
for NFX photodegradation (0.06), which was used as acti-
nometer.
On the other hand, irradiation of deaerated solutions of
ANFX in 10 mM PB in the presence and absence of NP (0.5
mM) was performed with the laser beam at 355 nm as the
light source. Under these conditions there is no remarkable
quenching of the ANFX fluorescence by NP (less than 5%) and
99% of the light is absorbed by the ANFX ground state.
Ir r a d ia tion of ANF X: Isola tion of 1-Eth yl-1,4-d ih yd r o-
2
Taking into account the relative concentrations of H O
-
-4
and OH at pH ) 10 (55 M vs 10 M, respectively), the
rate constants for water attack to triplet ANFX must be
at least 3 orders of magnitude lower than that of
hydroxide.
6
-h yd r oxy-4-oxo-7-(4-a cet yl-1-p ip er a zin yl)q u in olin e-3-
ca r boxylic Acid (2). ANFX (80 mg, 0.21 mmol) was dissolved
in water (2 L) and the solution was irradiated for 6 h. The
resulting solution was extracted with CH Cl and the organic
Finally, the mechanistic insight gained in the present
work allows a better understanding of previous observa-
tions on the photochemistry and photophysics of nor-
floxacin, enoxacin, and related monofluorinated quino-
lones, which lead also to 6-hydroxyquinolones by photosub-
stitution of fluoride anion in neutral media and are
unreactive under basic conditions.
2
2
layer was concentrated down to 50 mL. After the organic layer
was cooled in an ice bath, a solution of diazomethane (216 mg,
4
mmol) in diethyl ether (100 mL) was added dropwise. The
mixture was stirred at room temperature overnight. Then it
was concentrated under vacuum, and the residue was purified
by silica gel chromatography (eluting with 85/15 CH
solution) and subsequently by semipreparative HPLC, with
4.9/0.01/25 H O/CF COOH/CH CH as eluent. In this way,
compound 2 was characterized as its methyl ester. H NMR
2 2
Cl /MeOH
7
2
3
3
1
Con clu sion s
The photodegradation of ANFX in basic media, under
anaerobic conditions, involves photonucleophilic substitu-
(
29) Koga, H.; Itoh, A.; Murayama, S.; Suzue, S.; Irikura, T. J . Med.
Chem. 1980, 23, 1358.
7
260 J . Org. Chem., Vol. 69, No. 21, 2004