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Laser-flash photolysis studies of benoxaprofen and its analogues 1. Yields of triplet states and singlet oxygen in acetonitrile solutions
97
L Photochem. Phobsbiol. A: Chem., 73 (1993) 97-103
Laser-flash photolysis studies of benoxaprofen and its analogues 1. Yields of triplet states and singlet oxygen in acetonitrile solutions Suppiah
Navaratnam,
Barry
J. Parsons
and
John
Ll. Hughes
Multidisciplinay Research and Innovation Centre, 7he North East Wales In.&& (Received
November
30, 19W, accepted
February
Deeside, Chvyd CH5 4BR (UK)
11, 1993)
Abstract The photochemistry of benoxaprofen(BP) and four of its analogues in acetonitrile has been investigated using both steady-state and laser-flash photolysis. On steady-state photolysis, all compounds decarboxylated, albeit in small quantum yields. The fluorescence yields were found to be in the range 0.4-0.6. The triplet-triplet absorption spectra of all the compounds studied show absorption maxima at 430 nm with extinction coefficients of around 2.5 000 dm3 mol-1 cm-‘. Furthermore, all the compounds underwent intersystem crossing with efficiencies of around 0.4. Direct measurements of the time-resolved luminescence from singlet oxygen (‘4) using benzophenone as a triplet-state sensitizer in aerated solutions of benoxaprofen and its analogues, indicated that the efficiency of singlet oxygen formation (S,) was close to unity. These results imply that the singlet-oxygen quantum yields are close to 0.4.
1. Introduction Benoxaprofen (BP) (2-(4-chlorophenyl)-rrmethyl5benzoxazole acetic acid) is a non-steroidal anti-inflammatory agent. It is known to cause a number of phototoxic side effects including erythema, weal and flare response, as well as stinging and burning sensations [l, 21. BP is also known to cause photohaemolysis of human erythrocytes [3,4] in which both oxygen-dependent and oxygen independent effects were observed [5]. Using electron-spin resonance (ESR) direct photolysis of BP in alcoholic solvents containing trapping agents, was shown to yield singlet oxygen, alkoxyl radicals and superoxide anion radicals [6]. In other photochemical studies in aqueous solution, it was concluded from oxygen uptake measurements that only singlet oxygen was formed in a relatively high yield, 4=0.18 [7, 81. It would seem therefore, that in alcoholic solutions, superoxide anion radicals are probably formed in an elimination process involving alcohol peroxy radicals. In the latter studies, it was also apparent that the only other significant process, i.e. apart from the production of BP triplet states and subsequent singlet-oxygen formation, was decarboxylation, the yield of which was also relatively high (4 =0.18). In a more recent study, it was demonstrated that the fluorescence quantum yield of BP varies markedly with solvent, i.e. from 0.01 in water to 0.65 in cyclohexane [9].
lOlO-6030/93/%6.00
Such a high value, e.g. in cyclohexane, would imply lower yields for the triplet state, the decarboxylation process and/or non-radiative processes. The influence of solvent on the triplet-state yield was investigated recently using laser-flash photolysis techniques [lo], where it was concluded that there was little effect. This view was, however, based on the assumption that the BP triplet-state extinction coefficient does not vary greatly with solvent. In any event, extinction coefficients were not measured in that study. In addition, no comparison of yields was made with that measured for the BP triplet state in aqueous solution in the earlier laser-flash photolysis study [S]. The molecular mechanism(s) by which BP causes cutaneous photoxicity may involve both oxygendependent and oxygen-independent pathways, as demonstrated already in the photohaemolysis of erythrocytes. It may therefore be that decarboxylation is the effective process when no oxygen is present [ll], and that singlet oxygen is the active species under aerobic conditions [S]. Since there are now several BP derivatives for which cutaneous photosensitivity data are known, it is appropriate to investigate whether there is any correlation of this data with the yields of singlet oxygen, triplet states and decarboxylation from the photoexcited BP derivatives in aqueous solution. All these measurements can now be made relatively easily in
0 1993 - Elsevier Sequoia. All rights reserved
98
S. Navarahzam et al. I Laser-jiash photo&sir studies of benomprofen
solution, particularly since the yield of singlet oxygen (‘AZ) can now be measured directly using techniques for measuring the time-resolved nearIR luminescence from’ singlet oxygen [ 121. Singlet oxygen yields are more readily measurable in solvents such as cyclohexane and acetonitrile, where standard energy-transfer reagents such as benzophenone and naphthalene are easily soluble, and for which both triplet-state and singlet-oxygen yields are known [13]. It is the purpose of this study, therefore, to measure the singlet oxygen, triplet state and decarboxylation yields of BP and several of its derivatives in acetonitrile solution, as a basis for analogous measurements of those parameters in aqueous solution. The latter measurements can be made from a knowledge of the comparative photophysical properties of singlet oxygen in water and in acetonitrile [13]. Such data are essential for the understanding of the molecular basis of the phototoxicity of benoxaprofen and its analogues.
2. Materials
and methods
Benoxaprofen and its derivatives were supplied by Dista Products Ltd. - the derivatives being chlorobenoxaprofen (CIBP), dichlorobenoxaprofen (2,4-Cl,BP), difluorobenoxaprofen (2,4-F,BP), ethylbenoxaprofen (EtBP) and hydroxyethyl benoxaprofen (OHEtBP) (see Scheme 1). Acetonitrile was obtained from Romil Chemicals Ltd. Solutions were either saturated with ‘white spot’ nitrogen, oxygen or air as appropriate.
Hydroxy-ethyl BP (OHEtBPl
2.4 Bdluora-BP
2,4 D,chloro-BP
(2.4.F%BP)
(2.4.CI,BPl
Scheme 1. Benoxaprofen
and derivatives.
Fluorescence measurements were made on a Perkin Elmer MPF-43A spectrofluorimeter. Photolysis of samples was reduced using slit widths less than 3 nm for excitation. Dilute solutions having optical density of 0.1 at the excitation wavelength (320 nm) were used. The emission spectra were recorded in the 320-500 nm region using a right-angle configuration. Fluorescence quantum yields were determined by comparison with tryptophan (af=0.13 [14]) as standard. Steady-state photolysis experiments to measure decarboxylation yields and BP (or derivative) loss were carried out using a Philips 400 W High Pressure Hg vapour lamp HPA 400. A water filter was placed between the lamp and the reaction cell to cut off infra-red radiation. A Barr and Stroud broad-band filter UG5 with a transmittance maximum at 340 run was also used to limit the wavelength range of excitation. The irradiation cell was a 2 cm quartz cell with a second identical cell placed behind it for actinometric measurements. Samples 6 cm3 in size, of a 5 X lop4 mol dmm3 BP (or derivative) solution in acetonitrile were placed in the reaction vessel, saturated with nitrogen and subsequently irradiated for different periods up to 120 min. The solutions were continuously stirred. The products were analysed by HPLC (high pressure liquid chromatography) using equipment made by Pye Unicam, consisting of a model PU 4011 pump, PU 4030 controller, PU 4031 oven and PU 4040 solvent module. Samples were injected onto the reverse phase Spherisorb SSCN column via a Rheodyne Model 7125 injector port. The eluant was a vacuum-degassed mixture of acetonitrile, water and methanol (1:2:2) at a flow rate of 1 ml min-‘. The detector system consisted on a PU 4020 UV detector set at 310 nm. Data were captured on a Nelson Analytical Interface Model 950 and transferred to an Amstrad PC 1640 for analysis using integrator software supplied by Nelson Analytical. Quantum yield measurements were performed using ferrioxalate actinometry where the photon flux was calculated using a difference method in which solutions of BP or derivatives were placed in the front cell and that of ferrioxalate in the rear cell [15]. By comparing the reduction of ferrioxalate in this way, with identical measurements made using only acctonitrile in the front cell, the photon flux absorbed by the BP samples could be calculated. The laser-flash photolysis experiments were carried out with a J. K. Lasers System 2000 Neodymium/YAG oscillator with a Neodymium-glass amplifier. The laser delivers up to 100 mJ of 266 nm or 355 nm radiation in single pulses of 12 ns
S. Navaratnam et al. I Laser-flash photolysis studies of bemxapmfen
duration. A quartz prism was used to separate the 266 nm from the fundamental radiation and other harmonics produced by the laser system. The detection system consisted of a Xenon arc lamp and Applied Photophysics pulsing unit, and monochromator and quartz optics. Optical transmissions (I cm path length) at various wavelengths selected with the monochromator (bandwidths l-10 nm) were measured as a function of time before and after the pulse using photoelectric detection. The output of the photomultiplier RCA IP28A) was displayed on a Philips Digital Storage Oscilloscope PM 3311. Data acquisition and processing were carried out using a Hewlett Packard 9000 Series 300 computer [16]. All recorded traces were taken on fresh samples using a flow system in order to avoid interference from photoproducts. The luminescence (1270 nm) from excited singlet oxygen (I%) produced by photoexcitation of BP and its derivatives in oxygenated solution was detected by a Judson 516 germanium photodiode (5 mm’) closely coupled to the laser photolysis cell in right-angle geometry [17]. A 5 mm thick (5 cm diameter) piece of Ar-coated silicon metal (II-IV Inc.) was placed between the diode and cell to act as a narrow band filter for the 1270 nm luminescence. The photodiode output current was amplified with a Judson 000 50 dB amplifier with a 50 fi resistor connected across its + 15 V pin, and ground in order to reverse bias the diode effectively. The output from the amplifier was fed into the Philips Digital Storage Oscilloscope PM 3311 via a Cornlinear 150 MHz, 20 dB amplifier,
Wovelength
(nm)
Fig. 1. Absorption (-) and uncorrected spectra of BP in acetonitrile.
TABLE 1. Fluorescence quantum its analogues in acetonitrile.
fluorescence
(--
yields of benoxaprofen
Compound
Quantum
BP ClBP 2,4-CIJSP 2,4-FzBP EtBP OHEtBP
0.62 0.41 0.45 0.58 0.62 0.59
-)
and
yield
TABLE 2. Quantum yields for the degradation of BP (or analogue) and for the formation of the decarboxylated product in acetonitrile.
3. Results 3.1. Fluorescence measurements Uncorrected fluorescence spectra of BP (Fig. 1) and its analogues in acetonitrile show a band centred around 350 nm. The spectrum for BP agrees well with that previously measured [9]. The fluorescence quantum yields of BP and its analogues were also determined in acetonitrile using tryptophan as standard, and are shown in Table 1. The value for BP found in this study (0.62) is comparable to that obtained in a previous study (0.57) [9]. 3.2. Steady-state photolpti Photolysis of BP in acetonitrile solution produced decarboxylated BP, i.e. ethylbenoxaprofen (EtBP) as confirmed by co-injection in the HPLC experiments of authentic EtBP. In addition, there were indications of two other photoproducts present in
Compound
+(degadation)
4(formation)
BP ClBP 2,4-t&BP 2.4.F,BP
0.01 0.013 0.005 0.008
0.003 0.003 0.004
0.008
smaller yields. No attempt was made to identify these. Similar HPLC photoproduct patterns were also found for the BP analogues. By analysing samples irradiated up to 120 min (see Section 2), it was possible to determine the quantum yields for the loss of BP itself (or analogue) as well as for the formation of the decarboqlated product. In a typical experimerit the absorbance at 330 nm was four. The maximum conversion of BP (or analogue) was less than 5%. In both cases, the yields are very low and are summarised in Table 2. Moreover, similar yields of decarboxylated prod-
S. Navamtnam ef aI. I Laser-flash photoiysis studies of benoxaprofeti
100
ucts were oxygenated
obtained solutions.
both
in deoxygenated
and
3.3. Triplet-state measurements Upon laser-flash photolysis of deaerated solutions of BP and its derivatives in acetonitrile solution, transient absorption spectra were detected following the laser pulse. All spectra showed maxima at about 410 nm and 500 nm, and by comparison with spectra measured in an earlier study in aqueous solution [8] these can be assigned to the triplet states. The spectrum is shown for 2,4-Fe,BP in Fig. 2. Spectra (not shown) were also measured for BP, ClBP, 2,4-&BP, EtBP and OHEtBP, which exhibited the same maxima and are similar to that previously observed (410 nm) for the BP triplet state in acetonitrile [lo]. In measuring the decay of these spectra, it was noticed that the apparent first-order decay constant measured at 410 nm decreased significantly as the intensity of the laser pulse was decreased. For BP itself, for example, the half-life varied from several microseconds at high-pulse intensities to about 40 us at the lowest pulse energy employed. This effect is attributable to the contribution of second-order triplet-state self-reaction processes that can be minimised by using low-pulse energies. By extrapolation of the apparent first-order decay constant therefore to law laser-pulse energies, a value for the intrinsic first-order decay process of the triplet state was estimated. These values are shown for the benoxaprofen analogues in Table 3. The extinction coefficients of the triplet-states of BP and its analogues in acetonitrile solution
TABLE 3. First-order rate constants for the disappearance of transient absorbance at 410 nm following laser-flash photolysis of benoxaprofen analogues in N+aturated acetonitrile solutions. Compound
k (s-l)
ClBP 2,4-C1,BP 2,6-&BP 2,4-F,BP 2,6-F,BP
2.0 x 2.3 x 1.7x 2.3 x 2.0 x
were estimated by monitoring the energy transfer reaction between the benzophenone triplet-state and ground-state BP: 3BzPh* + ‘BP -
0D(3BP*(410 250
350
450 Wavelength
550
650
Cnm)
Fig. 2. Transient difference absorption spectra measured at 2.2 ps (U) and 7.6 fis (-+-) after delivering a laser puke (266 nm, 10 ntJ) to a nitrogen-saturated solution of 5 X lo-’ mol dm-’ 2,4-F,BP in acetonitrile.
3BP* -t ‘BzPh
(1)
In these experiments, deaerated solutions of benzophenone (3 X lop3 mol dmd3) containing in ace10-4-10-3 mol dm- 3 BP (or analogue) tonitrile were given a 355 nm laser pulse. Under these conditions, more than 99% of the absorbed light is taken up by benzophenone. The triplet state of benzophenone (3BzPh*) was thus produced within the laser-pulse lifetime, and was identified by its absorption spectrum (h,,=520 nm [18]). The extent of reaction 1 was estimated by comparing the first-order decay rate constants of the benzophenone triplet state in these experiments the rate (k=1.1X10”s-‘tok=1.0X10’s-‘)with constant @=9.2x 104 s-l) measured in similar experiments carried out in the absence of benoxaprofen (or its analogues). Its half-life in the presence of 10m3 mol dme3 BP (or analogue) was typically about 70 ns (k = LOX 10” dm3 mol-’ s-‘) indicating that more than 99% of the triplet state had undergone energy transfer as in reaction 1. The half-life of the BP triplet state so formed was typically about 30 /_Ls.It was important in these experiments to make these comparisons at identical laser-pulse energies because the overall decay constant for the benzophenone triplet state contains a contribution from second-order triplet-triplet annihilation processes [18]. The extinction coefficients of the triplet state of benoxaprofen (or analogues) were then calculated using the equation: ODCBzPh*(520
-10000
lo4 lo4 lo4 10’ lo4
nm)) nm))
= e(‘BzPh*(520 nm)) ~(~BP*(410 nm))
(2) where the OD values refer to the absorbances of the benzophenone triplet state at the beginning of reaction 1 and of the benoxaprofen triplet state at the end of reaction 1. The extinction coefficient of the benzophenone triplet state in acetonitrile
S. Navarainam
et al. I Laser-fish
was taken to be 6500 dm3 mol- ’ cm- ’ [18]. Care was taken in these and other laser-flash photolysis experiments to work with solutions having relatively low absorbance at the excitation wavelength (lypically the absorbance was less than 0.5) so as to provide as homogeneous a distribution of excited states in the optical detection path as possible. Similarly, the lowest laser-pulse energies were employed (typically 3 mJ or less) to avoid unwanted biphotonic processes or unnecessarily high triplet-triplet annihilation rates of reaction. In this way, by using a range of benoxaprofen concentrations (1 X 10e4 mol dme3 - 1 x 10e3 mol dme3) and making several repeats of each measurement, the extinction coefficient of the benoxaprofen triplet in acetonitrile solution was found to be 26000~2000 dm3 mol-l cm-’ at 410 nm. This, and values for other BP analogues, are shown in Table 4. The extinction coefficient values in Table 4 can be used to calculate the quantum yields for intersystem (&,,) crossing for BP and its analogues in acetonitrile solutions by giving laser pulses to deaerated solutions of benzophenone (approximately 4~10~~ mol dmp3) and comparing the maximum optical density observed at 520 nm attributable to the triplet state with the maximum optical density observed at 410 nm after giving an identical laser pulse to a deaerated solution of benoxaprofen. The latter transient absorbance is attributable to the triplet state of benoxaprofen and can be used to calculate its quantum yield by application of the following formula:
photo&
studies of benoxaprojen
101
The comparisons were made using benzophenone and benoxaprofen solutions of identical optical density at the excitation wavelength. Taking I& for benzophenone to be 1.0 [18], the quantum yields for intersystem crossing for BP and its analogues were calculated from such comparative experiments. These are summarised in Table 4. 3.4. Singlet oxygen (‘4) yields The inset in Fig. 3 shows a typical oscilloscope trace obtained on exciting an oxygenated acetonitrile solution of benoxaprofen (or its analogues) with a 266 nm laser pulse. This shows the formation of excited singlet oxygen (‘A.& via the reaction: 3BP* -I-‘0, -
‘BP + lo,*
(4) As indicated already in Section 1, the timeresolved luminescence from singlet oxygen can be measured at 1270 nm using an appropriate diode as a detector [12, 131, and the technique thus provides a means of measuring the quantum yields of singlet oxygen formation because the detection system can be calibrated. Alternatively, a comparative technique as employed for intersystem crossing may be used. However, there are no standards for this method when the excitation wavelength is 266 nm, as in this case. Under this circumstance it is necessary to measure the efficiency of singlet oxygen formation (S,) before quantum yields (43 may be calculated from the equation:
501
0D(3BzPh*(520 nm)) 0D(3BP*(410 nm))
p’
= E(3BzPh*(520 nm))+&BzPh) l (~BP*(410 nm))+iXE(BP)
(3)
TABLE 4. Extinction coefficients at 410 nm (dm3 mol-’ cm-‘) and quantum yields of the triplet states of benoxaprofen and its analogues. Compound
E”
4%cb
SAC
BP ClBP 2,4-Cl,BP 2,4-F,BP EtBP OHEtBP
26000 22000 26000 2ooOo 26000 23600
0.38 0.44 0.44 0.40 0.40 0.41
1 1 1 1 _
‘Error limits are ~2000 bError limits are f0.04; ‘Error limits are f0.1.
dm” mol-’ cn-‘;
l/
oy 0
I
2
3
4
,
,
5
6
Energy (rnJ)
Fig. 3. The effect of laser-pulse energy (mJ) on J, (mv) for naphthalene (0) and benoxaprofen (+) in acetonitrile solutions containing benzophenone (see text for experimental conditions). The insert shows the decay of emission at 1270 nm measured in the benoxaprofen experiments. (Horizontal scale 20 ps/div.; vertical scale 5 mV/div.)
S. Navaratnam et al. I Laser-flash photo/ysis studies of Lmoxaprofen
102
dA
=
(5)
d&A
S, values for BP and its analogues in acetonitrile solution were measured in this work using a technique developed by Gorman et al. [13]. Here, a standard solution of aerated benzophenone naphthalene (N) (3 X 10e3 mol dme3) containing (1 x 10-l mol dmm3) in acetonitrile was given laser pulses (355 nm) of various energies up to 3 mJ, and the resulting time-resolved emissions from singlet oxygen were monitored. Under these conditions, only benzophenone is excited by the laser pulse to form its triplet state with unit efficiency [18]. Energy transfer to naphthalene is also efficient under these conditions and produces the naphthalene triplet state [13]: 3BzPh* + N -
3N* + BzPh
(6)
In this way, 3N* was produced within the laser pulse, and in the presence of air it disappeared within about 100 ns to produce singlet oxygen in a very efficient reaction, i.e. it can be estimated from a comparison with the lifetime of 3N* in an air-free solution that more than 99% of the naphthalene triplet states was scavenged by oxygen in these experiments. It was concluded in an earlier study that Sb is unity for the reaction between 3N* and oxygen [13]. Thus emission from singlet oxygen in this system could be detected over several microseconds following the laser pulse. This emission was found to decay exponentially with a typical half-life of 50 ps. Extrapolation of the emission signal enabled the maximum intensity 1, (mV) to Ia was then measured in further be estimated. separate experiments as a function of laser-pulse intensity (mJ). The resulting plot for naphthalene showed no curvature in the employed range of laser-pulse energies of up to 3 m-J (see Fig. 3). In identical experiments using benzophenone (3 x 10pJ mol dm-3)-benoxaprofen (1 X 10-l mol mixtures instead of benzophendme3) one-naphthalene, I, values were also measured as a function of laser-pulse energy. This plot was found to be coincident with that measured for naphthalene (see Fig. 3) and indicates that SA is also unity for benoxaprofen in acetonitriIe solutions. S, values of unity were also found for ClBP, 2,4-C1,BP and 2,4-F,BP. From the data in Table 4 and eqn. (5), &, was found to be close to 0.4 for all the BP derivatives studied.
studied in acetonitrile is close to unity and indicates that all other photochemical and photophysical processes are negligible. This is corroborated by the very low (less than 0.01) quantum yield for the photodecarboxylation of the compounds. Quantum yields for both decarboxylation and degradation in the absence and in the presence of oxygen are the same, and this fact implies that the excited singlet state is the precursor for the decarboqlation reaction. The SA values of unity for all the derivatives that were measured indicate that the main reaction of the triplet state in the presence of oxygen is the formation of excited singlet oxygen, and not decarboxylation or any other reaction. Furthermore, since the lifetime (half-life of 50 ps) of excited singlet oxygen in acetonitrile in the absence and presence of benoxaprofen (or its analogues) remains the same, it is concluded that the excited singlet oxygen does not react with benoxaprofen (or its analogues). This is corroborated by the absence of any degradation of BP (or its analogues) in an oxygenated solution under steady-state photolysis. The excited singlet oxygen has been implicated in cell damage in a number of photobiological reactions, such as photodynamic therapy. Phototoxic reactions resulting from benoxaprofen may also be initiated by excited singlet oxygen. Relatively straightforward measurements of the yields of fluorescence, intersystem crossing and singlet oxygen provide a good basis for a comparative measurement of the singlet oxygen yields in aqueous solutions that can be expected to be of greater relevance to the understanding of phototoxicity of benoxaprofen in viva. If similar values for I$~ in aqueous solutions are obtained then we would expect a high incidence of phototoxicity because of these compounds.
Acknowledgment We are grateful to Lilly Research for their support in this work.
Centre
Ltd.
References
4. Discussion
M. C. Griest, I. I. Ozols, A. S. Ridolfo and J. C. Muus, Eur. J. Rheum. Influmm., 5 (1982) 138. G. F. Webster, K. H. Kaidbey and M. Klipan, Photochem. Ptwrobiol., 36 (1982) 59. I. E. Kochevar, IL W. Hoover and M. Yoon, I. InveSr. Der-
The sum of the quantum yields for fluorescence and intersystem crossing for all the compounds
moro1., 80 (1983) 318. I. E. Kochevar, K. W. Hoover and M. Gawienowski, I. Invest. Dermarol., 82 (1984) 214.
S. Navaratnam
et
al. I Laser-flash photolysb studies c# bmoxaprofen
5 R. H. Sik, C. S. Paschal1 and C. F. Chignell, Photo&m Photobiol., 38 (1983) 411. 6 K. Rezka, and C. F. Chignell, Photo&em. Phofobiol., 38 (1983) 281. B. .I. Parsons and G. 0. Phillips in W. Bors, 7 S. Navaratnam, M. Saran and D. Tait teds.), Oxygen Radicals in Chemistry and Biology, Walter de Gruyter, Berlin, 1984, p. 479. 8 S. Navaratnam, J. Ll. Hughes, B. J. Parsons and G. 0. Phillips. Photochem. PhotobioL, 41 (1985) 375. 9 M. Yoon and K. H. Lee, L Chem. SW, Perkin Trans., II, (1988) 1943. 10 R. S. Becker, S. Chakravorti and M. Yoon, Photochem. Photobiol., 51 (1990) 151. 11 I. E. Kochevar, Mechanisms of drug photosensitization, Photo&em. Photobiol., 45 (1987) 891.
103
12 M. A. J. Rodgers and P. Snowden, L Am. Chem. Sot., 104 (1982) 5541. 13 A. A. Gorman, I. Hamblett, C. Lambert, A. L. Prescott, M. A. I. Rodgers and H. M. Spence, I. Am. Gem. Sot., 109 (1987) 3091. 14 I. Tatischeff and R. Klein in J. Birks (ed.), Excited States in Biological Molecules, Wiley, London, 1976, p. 375. 15 A. K. Davies, S. Navaratnam and G. 0. Phillips, L Chem. Sot., Perkin Trans., II, (1976) 25. 16 S. Navaratnam and G. 0. Phillips, in F. Lcnci, F. Ghetti, G. Colombetti, D-P. Hader and Pill-Soon Song (eds.), Biophysics of Photoreceptors and Photomovements in Micworgan&u, Plenum, New York, 1991, p. 139. 17 M. A. J. Rodgers, J. Am. Chem. Sot., 105 (1983) 6201. 18 R. V. Bensasson and J. C. Gramain, J. Chem. Sot. Faraday Trans. I, 76 (1980) 1801.
L Photochem. Phobsbiol. A: Chem., 73 (1993) 97-103
Laser-flash photolysis studies of benoxaprofen and its analogues 1. Yields of triplet states and singlet oxygen in acetonitrile solutions Suppiah
Navaratnam,
Barry
J. Parsons
and
John
Ll. Hughes
Multidisciplinay Research and Innovation Centre, 7he North East Wales In.&& (Received
November
30, 19W, accepted
February
Deeside, Chvyd CH5 4BR (UK)
11, 1993)
Abstract The photochemistry of benoxaprofen(BP) and four of its analogues in acetonitrile has been investigated using both steady-state and laser-flash photolysis. On steady-state photolysis, all compounds decarboxylated, albeit in small quantum yields. The fluorescence yields were found to be in the range 0.4-0.6. The triplet-triplet absorption spectra of all the compounds studied show absorption maxima at 430 nm with extinction coefficients of around 2.5 000 dm3 mol-1 cm-‘. Furthermore, all the compounds underwent intersystem crossing with efficiencies of around 0.4. Direct measurements of the time-resolved luminescence from singlet oxygen (‘4) using benzophenone as a triplet-state sensitizer in aerated solutions of benoxaprofen and its analogues, indicated that the efficiency of singlet oxygen formation (S,) was close to unity. These results imply that the singlet-oxygen quantum yields are close to 0.4.
1. Introduction Benoxaprofen (BP) (2-(4-chlorophenyl)-rrmethyl5benzoxazole acetic acid) is a non-steroidal anti-inflammatory agent. It is known to cause a number of phototoxic side effects including erythema, weal and flare response, as well as stinging and burning sensations [l, 21. BP is also known to cause photohaemolysis of human erythrocytes [3,4] in which both oxygen-dependent and oxygen independent effects were observed [5]. Using electron-spin resonance (ESR) direct photolysis of BP in alcoholic solvents containing trapping agents, was shown to yield singlet oxygen, alkoxyl radicals and superoxide anion radicals [6]. In other photochemical studies in aqueous solution, it was concluded from oxygen uptake measurements that only singlet oxygen was formed in a relatively high yield, 4=0.18 [7, 81. It would seem therefore, that in alcoholic solutions, superoxide anion radicals are probably formed in an elimination process involving alcohol peroxy radicals. In the latter studies, it was also apparent that the only other significant process, i.e. apart from the production of BP triplet states and subsequent singlet-oxygen formation, was decarboxylation, the yield of which was also relatively high (4 =0.18). In a more recent study, it was demonstrated that the fluorescence quantum yield of BP varies markedly with solvent, i.e. from 0.01 in water to 0.65 in cyclohexane [9].
lOlO-6030/93/%6.00
Such a high value, e.g. in cyclohexane, would imply lower yields for the triplet state, the decarboxylation process and/or non-radiative processes. The influence of solvent on the triplet-state yield was investigated recently using laser-flash photolysis techniques [lo], where it was concluded that there was little effect. This view was, however, based on the assumption that the BP triplet-state extinction coefficient does not vary greatly with solvent. In any event, extinction coefficients were not measured in that study. In addition, no comparison of yields was made with that measured for the BP triplet state in aqueous solution in the earlier laser-flash photolysis study [S]. The molecular mechanism(s) by which BP causes cutaneous photoxicity may involve both oxygendependent and oxygen-independent pathways, as demonstrated already in the photohaemolysis of erythrocytes. It may therefore be that decarboxylation is the effective process when no oxygen is present [ll], and that singlet oxygen is the active species under aerobic conditions [S]. Since there are now several BP derivatives for which cutaneous photosensitivity data are known, it is appropriate to investigate whether there is any correlation of this data with the yields of singlet oxygen, triplet states and decarboxylation from the photoexcited BP derivatives in aqueous solution. All these measurements can now be made relatively easily in
0 1993 - Elsevier Sequoia. All rights reserved
98
S. Navarahzam et al. I Laser-jiash photo&sir studies of benomprofen
solution, particularly since the yield of singlet oxygen (‘AZ) can now be measured directly using techniques for measuring the time-resolved nearIR luminescence from’ singlet oxygen [ 121. Singlet oxygen yields are more readily measurable in solvents such as cyclohexane and acetonitrile, where standard energy-transfer reagents such as benzophenone and naphthalene are easily soluble, and for which both triplet-state and singlet-oxygen yields are known [13]. It is the purpose of this study, therefore, to measure the singlet oxygen, triplet state and decarboxylation yields of BP and several of its derivatives in acetonitrile solution, as a basis for analogous measurements of those parameters in aqueous solution. The latter measurements can be made from a knowledge of the comparative photophysical properties of singlet oxygen in water and in acetonitrile [13]. Such data are essential for the understanding of the molecular basis of the phototoxicity of benoxaprofen and its analogues.
2. Materials
and methods
Benoxaprofen and its derivatives were supplied by Dista Products Ltd. - the derivatives being chlorobenoxaprofen (CIBP), dichlorobenoxaprofen (2,4-Cl,BP), difluorobenoxaprofen (2,4-F,BP), ethylbenoxaprofen (EtBP) and hydroxyethyl benoxaprofen (OHEtBP) (see Scheme 1). Acetonitrile was obtained from Romil Chemicals Ltd. Solutions were either saturated with ‘white spot’ nitrogen, oxygen or air as appropriate.
Hydroxy-ethyl BP (OHEtBPl
2.4 Bdluora-BP
2,4 D,chloro-BP
(2.4.F%BP)
(2.4.CI,BPl
Scheme 1. Benoxaprofen
and derivatives.
Fluorescence measurements were made on a Perkin Elmer MPF-43A spectrofluorimeter. Photolysis of samples was reduced using slit widths less than 3 nm for excitation. Dilute solutions having optical density of 0.1 at the excitation wavelength (320 nm) were used. The emission spectra were recorded in the 320-500 nm region using a right-angle configuration. Fluorescence quantum yields were determined by comparison with tryptophan (af=0.13 [14]) as standard. Steady-state photolysis experiments to measure decarboxylation yields and BP (or derivative) loss were carried out using a Philips 400 W High Pressure Hg vapour lamp HPA 400. A water filter was placed between the lamp and the reaction cell to cut off infra-red radiation. A Barr and Stroud broad-band filter UG5 with a transmittance maximum at 340 run was also used to limit the wavelength range of excitation. The irradiation cell was a 2 cm quartz cell with a second identical cell placed behind it for actinometric measurements. Samples 6 cm3 in size, of a 5 X lop4 mol dmm3 BP (or derivative) solution in acetonitrile were placed in the reaction vessel, saturated with nitrogen and subsequently irradiated for different periods up to 120 min. The solutions were continuously stirred. The products were analysed by HPLC (high pressure liquid chromatography) using equipment made by Pye Unicam, consisting of a model PU 4011 pump, PU 4030 controller, PU 4031 oven and PU 4040 solvent module. Samples were injected onto the reverse phase Spherisorb SSCN column via a Rheodyne Model 7125 injector port. The eluant was a vacuum-degassed mixture of acetonitrile, water and methanol (1:2:2) at a flow rate of 1 ml min-‘. The detector system consisted on a PU 4020 UV detector set at 310 nm. Data were captured on a Nelson Analytical Interface Model 950 and transferred to an Amstrad PC 1640 for analysis using integrator software supplied by Nelson Analytical. Quantum yield measurements were performed using ferrioxalate actinometry where the photon flux was calculated using a difference method in which solutions of BP or derivatives were placed in the front cell and that of ferrioxalate in the rear cell [15]. By comparing the reduction of ferrioxalate in this way, with identical measurements made using only acctonitrile in the front cell, the photon flux absorbed by the BP samples could be calculated. The laser-flash photolysis experiments were carried out with a J. K. Lasers System 2000 Neodymium/YAG oscillator with a Neodymium-glass amplifier. The laser delivers up to 100 mJ of 266 nm or 355 nm radiation in single pulses of 12 ns
S. Navaratnam et al. I Laser-flash photolysis studies of bemxapmfen
duration. A quartz prism was used to separate the 266 nm from the fundamental radiation and other harmonics produced by the laser system. The detection system consisted of a Xenon arc lamp and Applied Photophysics pulsing unit, and monochromator and quartz optics. Optical transmissions (I cm path length) at various wavelengths selected with the monochromator (bandwidths l-10 nm) were measured as a function of time before and after the pulse using photoelectric detection. The output of the photomultiplier RCA IP28A) was displayed on a Philips Digital Storage Oscilloscope PM 3311. Data acquisition and processing were carried out using a Hewlett Packard 9000 Series 300 computer [16]. All recorded traces were taken on fresh samples using a flow system in order to avoid interference from photoproducts. The luminescence (1270 nm) from excited singlet oxygen (I%) produced by photoexcitation of BP and its derivatives in oxygenated solution was detected by a Judson 516 germanium photodiode (5 mm’) closely coupled to the laser photolysis cell in right-angle geometry [17]. A 5 mm thick (5 cm diameter) piece of Ar-coated silicon metal (II-IV Inc.) was placed between the diode and cell to act as a narrow band filter for the 1270 nm luminescence. The photodiode output current was amplified with a Judson 000 50 dB amplifier with a 50 fi resistor connected across its + 15 V pin, and ground in order to reverse bias the diode effectively. The output from the amplifier was fed into the Philips Digital Storage Oscilloscope PM 3311 via a Cornlinear 150 MHz, 20 dB amplifier,
Wovelength
(nm)
Fig. 1. Absorption (-) and uncorrected spectra of BP in acetonitrile.
TABLE 1. Fluorescence quantum its analogues in acetonitrile.
fluorescence
(--
yields of benoxaprofen
Compound
Quantum
BP ClBP 2,4-CIJSP 2,4-FzBP EtBP OHEtBP
0.62 0.41 0.45 0.58 0.62 0.59
-)
and
yield
TABLE 2. Quantum yields for the degradation of BP (or analogue) and for the formation of the decarboxylated product in acetonitrile.
3. Results 3.1. Fluorescence measurements Uncorrected fluorescence spectra of BP (Fig. 1) and its analogues in acetonitrile show a band centred around 350 nm. The spectrum for BP agrees well with that previously measured [9]. The fluorescence quantum yields of BP and its analogues were also determined in acetonitrile using tryptophan as standard, and are shown in Table 1. The value for BP found in this study (0.62) is comparable to that obtained in a previous study (0.57) [9]. 3.2. Steady-state photolpti Photolysis of BP in acetonitrile solution produced decarboxylated BP, i.e. ethylbenoxaprofen (EtBP) as confirmed by co-injection in the HPLC experiments of authentic EtBP. In addition, there were indications of two other photoproducts present in
Compound
+(degadation)
4(formation)
BP ClBP 2,4-t&BP 2.4.F,BP
0.01 0.013 0.005 0.008
0.003 0.003 0.004
0.008
smaller yields. No attempt was made to identify these. Similar HPLC photoproduct patterns were also found for the BP analogues. By analysing samples irradiated up to 120 min (see Section 2), it was possible to determine the quantum yields for the loss of BP itself (or analogue) as well as for the formation of the decarboqlated product. In a typical experimerit the absorbance at 330 nm was four. The maximum conversion of BP (or analogue) was less than 5%. In both cases, the yields are very low and are summarised in Table 2. Moreover, similar yields of decarboxylated prod-
S. Navamtnam ef aI. I Laser-flash photoiysis studies of benoxaprofeti
100
ucts were oxygenated
obtained solutions.
both
in deoxygenated
and
3.3. Triplet-state measurements Upon laser-flash photolysis of deaerated solutions of BP and its derivatives in acetonitrile solution, transient absorption spectra were detected following the laser pulse. All spectra showed maxima at about 410 nm and 500 nm, and by comparison with spectra measured in an earlier study in aqueous solution [8] these can be assigned to the triplet states. The spectrum is shown for 2,4-Fe,BP in Fig. 2. Spectra (not shown) were also measured for BP, ClBP, 2,4-&BP, EtBP and OHEtBP, which exhibited the same maxima and are similar to that previously observed (410 nm) for the BP triplet state in acetonitrile [lo]. In measuring the decay of these spectra, it was noticed that the apparent first-order decay constant measured at 410 nm decreased significantly as the intensity of the laser pulse was decreased. For BP itself, for example, the half-life varied from several microseconds at high-pulse intensities to about 40 us at the lowest pulse energy employed. This effect is attributable to the contribution of second-order triplet-state self-reaction processes that can be minimised by using low-pulse energies. By extrapolation of the apparent first-order decay constant therefore to law laser-pulse energies, a value for the intrinsic first-order decay process of the triplet state was estimated. These values are shown for the benoxaprofen analogues in Table 3. The extinction coefficients of the triplet-states of BP and its analogues in acetonitrile solution
TABLE 3. First-order rate constants for the disappearance of transient absorbance at 410 nm following laser-flash photolysis of benoxaprofen analogues in N+aturated acetonitrile solutions. Compound
k (s-l)
ClBP 2,4-C1,BP 2,6-&BP 2,4-F,BP 2,6-F,BP
2.0 x 2.3 x 1.7x 2.3 x 2.0 x
were estimated by monitoring the energy transfer reaction between the benzophenone triplet-state and ground-state BP: 3BzPh* + ‘BP -
0D(3BP*(410 250
350
450 Wavelength
550
650
Cnm)
Fig. 2. Transient difference absorption spectra measured at 2.2 ps (U) and 7.6 fis (-+-) after delivering a laser puke (266 nm, 10 ntJ) to a nitrogen-saturated solution of 5 X lo-’ mol dm-’ 2,4-F,BP in acetonitrile.
3BP* -t ‘BzPh
(1)
In these experiments, deaerated solutions of benzophenone (3 X lop3 mol dmd3) containing in ace10-4-10-3 mol dm- 3 BP (or analogue) tonitrile were given a 355 nm laser pulse. Under these conditions, more than 99% of the absorbed light is taken up by benzophenone. The triplet state of benzophenone (3BzPh*) was thus produced within the laser-pulse lifetime, and was identified by its absorption spectrum (h,,=520 nm [18]). The extent of reaction 1 was estimated by comparing the first-order decay rate constants of the benzophenone triplet state in these experiments the rate (k=1.1X10”s-‘tok=1.0X10’s-‘)with constant @=9.2x 104 s-l) measured in similar experiments carried out in the absence of benoxaprofen (or its analogues). Its half-life in the presence of 10m3 mol dme3 BP (or analogue) was typically about 70 ns (k = LOX 10” dm3 mol-’ s-‘) indicating that more than 99% of the triplet state had undergone energy transfer as in reaction 1. The half-life of the BP triplet state so formed was typically about 30 /_Ls.It was important in these experiments to make these comparisons at identical laser-pulse energies because the overall decay constant for the benzophenone triplet state contains a contribution from second-order triplet-triplet annihilation processes [18]. The extinction coefficients of the triplet state of benoxaprofen (or analogues) were then calculated using the equation: ODCBzPh*(520
-10000
lo4 lo4 lo4 10’ lo4
nm)) nm))
= e(‘BzPh*(520 nm)) ~(~BP*(410 nm))
(2) where the OD values refer to the absorbances of the benzophenone triplet state at the beginning of reaction 1 and of the benoxaprofen triplet state at the end of reaction 1. The extinction coefficient of the benzophenone triplet state in acetonitrile
S. Navarainam
et al. I Laser-fish
was taken to be 6500 dm3 mol- ’ cm- ’ [18]. Care was taken in these and other laser-flash photolysis experiments to work with solutions having relatively low absorbance at the excitation wavelength (lypically the absorbance was less than 0.5) so as to provide as homogeneous a distribution of excited states in the optical detection path as possible. Similarly, the lowest laser-pulse energies were employed (typically 3 mJ or less) to avoid unwanted biphotonic processes or unnecessarily high triplet-triplet annihilation rates of reaction. In this way, by using a range of benoxaprofen concentrations (1 X 10e4 mol dme3 - 1 x 10e3 mol dme3) and making several repeats of each measurement, the extinction coefficient of the benoxaprofen triplet in acetonitrile solution was found to be 26000~2000 dm3 mol-l cm-’ at 410 nm. This, and values for other BP analogues, are shown in Table 4. The extinction coefficient values in Table 4 can be used to calculate the quantum yields for intersystem (&,,) crossing for BP and its analogues in acetonitrile solutions by giving laser pulses to deaerated solutions of benzophenone (approximately 4~10~~ mol dmp3) and comparing the maximum optical density observed at 520 nm attributable to the triplet state with the maximum optical density observed at 410 nm after giving an identical laser pulse to a deaerated solution of benoxaprofen. The latter transient absorbance is attributable to the triplet state of benoxaprofen and can be used to calculate its quantum yield by application of the following formula:
photo&
studies of benoxaprojen
101
The comparisons were made using benzophenone and benoxaprofen solutions of identical optical density at the excitation wavelength. Taking I& for benzophenone to be 1.0 [18], the quantum yields for intersystem crossing for BP and its analogues were calculated from such comparative experiments. These are summarised in Table 4. 3.4. Singlet oxygen (‘4) yields The inset in Fig. 3 shows a typical oscilloscope trace obtained on exciting an oxygenated acetonitrile solution of benoxaprofen (or its analogues) with a 266 nm laser pulse. This shows the formation of excited singlet oxygen (‘A.& via the reaction: 3BP* -I-‘0, -
‘BP + lo,*
(4) As indicated already in Section 1, the timeresolved luminescence from singlet oxygen can be measured at 1270 nm using an appropriate diode as a detector [12, 131, and the technique thus provides a means of measuring the quantum yields of singlet oxygen formation because the detection system can be calibrated. Alternatively, a comparative technique as employed for intersystem crossing may be used. However, there are no standards for this method when the excitation wavelength is 266 nm, as in this case. Under this circumstance it is necessary to measure the efficiency of singlet oxygen formation (S,) before quantum yields (43 may be calculated from the equation:
501
0D(3BzPh*(520 nm)) 0D(3BP*(410 nm))
p’
= E(3BzPh*(520 nm))+&BzPh) l (~BP*(410 nm))+iXE(BP)
(3)
TABLE 4. Extinction coefficients at 410 nm (dm3 mol-’ cm-‘) and quantum yields of the triplet states of benoxaprofen and its analogues. Compound
E”
4%cb
SAC
BP ClBP 2,4-Cl,BP 2,4-F,BP EtBP OHEtBP
26000 22000 26000 2ooOo 26000 23600
0.38 0.44 0.44 0.40 0.40 0.41
1 1 1 1 _
‘Error limits are ~2000 bError limits are f0.04; ‘Error limits are f0.1.
dm” mol-’ cn-‘;
l/
oy 0
I
2
3
4
,
,
5
6
Energy (rnJ)
Fig. 3. The effect of laser-pulse energy (mJ) on J, (mv) for naphthalene (0) and benoxaprofen (+) in acetonitrile solutions containing benzophenone (see text for experimental conditions). The insert shows the decay of emission at 1270 nm measured in the benoxaprofen experiments. (Horizontal scale 20 ps/div.; vertical scale 5 mV/div.)
S. Navaratnam et al. I Laser-flash photo/ysis studies of Lmoxaprofen
102
dA
=
(5)
d&A
S, values for BP and its analogues in acetonitrile solution were measured in this work using a technique developed by Gorman et al. [13]. Here, a standard solution of aerated benzophenone naphthalene (N) (3 X 10e3 mol dme3) containing (1 x 10-l mol dmm3) in acetonitrile was given laser pulses (355 nm) of various energies up to 3 mJ, and the resulting time-resolved emissions from singlet oxygen were monitored. Under these conditions, only benzophenone is excited by the laser pulse to form its triplet state with unit efficiency [18]. Energy transfer to naphthalene is also efficient under these conditions and produces the naphthalene triplet state [13]: 3BzPh* + N -
3N* + BzPh
(6)
In this way, 3N* was produced within the laser pulse, and in the presence of air it disappeared within about 100 ns to produce singlet oxygen in a very efficient reaction, i.e. it can be estimated from a comparison with the lifetime of 3N* in an air-free solution that more than 99% of the naphthalene triplet states was scavenged by oxygen in these experiments. It was concluded in an earlier study that Sb is unity for the reaction between 3N* and oxygen [13]. Thus emission from singlet oxygen in this system could be detected over several microseconds following the laser pulse. This emission was found to decay exponentially with a typical half-life of 50 ps. Extrapolation of the emission signal enabled the maximum intensity 1, (mV) to Ia was then measured in further be estimated. separate experiments as a function of laser-pulse intensity (mJ). The resulting plot for naphthalene showed no curvature in the employed range of laser-pulse energies of up to 3 m-J (see Fig. 3). In identical experiments using benzophenone (3 x 10pJ mol dm-3)-benoxaprofen (1 X 10-l mol mixtures instead of benzophendme3) one-naphthalene, I, values were also measured as a function of laser-pulse energy. This plot was found to be coincident with that measured for naphthalene (see Fig. 3) and indicates that SA is also unity for benoxaprofen in acetonitriIe solutions. S, values of unity were also found for ClBP, 2,4-C1,BP and 2,4-F,BP. From the data in Table 4 and eqn. (5), &, was found to be close to 0.4 for all the BP derivatives studied.
studied in acetonitrile is close to unity and indicates that all other photochemical and photophysical processes are negligible. This is corroborated by the very low (less than 0.01) quantum yield for the photodecarboxylation of the compounds. Quantum yields for both decarboxylation and degradation in the absence and in the presence of oxygen are the same, and this fact implies that the excited singlet state is the precursor for the decarboqlation reaction. The SA values of unity for all the derivatives that were measured indicate that the main reaction of the triplet state in the presence of oxygen is the formation of excited singlet oxygen, and not decarboxylation or any other reaction. Furthermore, since the lifetime (half-life of 50 ps) of excited singlet oxygen in acetonitrile in the absence and presence of benoxaprofen (or its analogues) remains the same, it is concluded that the excited singlet oxygen does not react with benoxaprofen (or its analogues). This is corroborated by the absence of any degradation of BP (or its analogues) in an oxygenated solution under steady-state photolysis. The excited singlet oxygen has been implicated in cell damage in a number of photobiological reactions, such as photodynamic therapy. Phototoxic reactions resulting from benoxaprofen may also be initiated by excited singlet oxygen. Relatively straightforward measurements of the yields of fluorescence, intersystem crossing and singlet oxygen provide a good basis for a comparative measurement of the singlet oxygen yields in aqueous solutions that can be expected to be of greater relevance to the understanding of phototoxicity of benoxaprofen in viva. If similar values for I$~ in aqueous solutions are obtained then we would expect a high incidence of phototoxicity because of these compounds.
Acknowledgment We are grateful to Lilly Research for their support in this work.
Centre
Ltd.
References
4. Discussion
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S. Navaratnam
et
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