KINETIC STUDIES ON ANTHRALIN PHOTOOXIDATION

Photochemistry and Photobiology Vol. 52 , No. 3, pp. 44-50. 1990 Printed in Great Britain. All rights reserved

0031-8655/90 $03.00+0.00 Copyright @ 1990 Pergamon Press plc

KINETIC STUDIES ON ANTHRALIN PHOTOOXIDATION KLAUS MULLER, RICHARD C. KANNER and CHRISTOPHER S. FOOTE*

Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90024, USA

(Received 30 October 1989; accepted 21 February 1990)

Abstract-The photooxidation of the antipsoriatic drug anthralin (1,8-dihydroxy-9-anthrone) has been studied by several kinetic techniques, including direct observation of lo2 (IAJ luminescence at ~ 1.27 pm. The rate of deactivation of '0, increases at higher pH, demonstrating that the trihydroxyanthracene anion is the reactive species. Direct determination of the rate constant of loz deactivation (k,+k,) in deuterated buffer systems by luminescence quenching gave a value of 3.0 x lo8 M-I s-I

for the anion; the neutral anthrone is unreactive. The rate constant for the neutral anthrone in benzene-d, is 2.8 X 104 M-' s-l. Competition experiments with tetramethylethylene in acetonitrile gave a rate constant for reaction alone (kR) of 2.1 x lo8 M - l s - I f or the anion.

INTRODUCTION

Psoriasis is a widespread, chronic inflammatory and scaling skin disease, mainly characterized by increased cell proliferation of the epidermis (Christopher and Krueger, 1987). Anthralin (dithranol, 1,8-dihydroxy-9-anthrone) has been used as the primary topical agent in the treatment of psoriasis for over 70 years (Shroot et al., 1981). Because of its lack of carcinogenicity or systemic toxicity (Ippen, 1981), it has an outstanding position in the clinician's armamentarium of antipsoriatic regimens (Mahrle, 1988). Unpleasant side effects of anthralin therapy include irritation and staining of the nonaffected skin (Mustakallio, 1988).

Although many mechanisms have been proposed for anthralin-mediated action, the detailed mechanism of its antipsoriatic activity is yet to be estab- lished. Currently, the role of antipsoriatic drugs in the activation and deactivation of free radicals and singlet molecular oxygen ( I A J , and the involvement of these species in therapy has become the focus of attention (Miyachi, 1987; Mustakallio et al . , 1984; Pathak and Carraro, 1987). The sensitivity of anthralin to oxygen and light suggests that neither the activity nor the side effects of this drug are linked to the anthralin molecule itself, but may involve intermediates that are formed during the oxidation process.

Anthralin is quite stable in nonplar aprotic solvents (Miiller et a[ . , 1986; Sa e Melo et a[ . , 1983) where it exists as the anthrone tautomer exclusively (Avdovich and Neville, 1980; Geiger, 1974; Miiller et al., 1986). In polar aprotic solvents and in buffered bovine serum albumin solutions, it is partially converted to the trihydroxyanthracene anion (Wiegrebe et a l . , 1981); some of this form has also been detected in cytoplasm (Kohen et al . , 1986). In basic solutions it exists mainly as the anion (Sa e Melo et a l . , .1983). Oxidation in aqueous solutions

*To whom correspondence should be addressed.

in the dark yields the 1,8,1',8'-tetrahydroxydianthrone (Cavey et al . , 1982; Miiller et a l . , 1987), while electron transfer to oxygen results in the formation of superoxide anion (*02) (Bruce et al . , 1987; Miiller et al., 1987). More importantly, the far more reactive hydroxyl radical ('OH) is formed via the iron-catalyzed Haber-Weiss-reaction (Miiller and Kappus, 1988). In contrast to the reaction in the dark, chemically or photochemically- generated lo2 converts the anthralin anion to danthrone (1,8-dihydroxy-9,10-anthraquinone). More- over, the anthralin anion is a photosensitizer for the production of lo2, and is oxidized in a self-sensitized process (Miiller et al . , 1986). Recently, the triplet state of anthralin produced by pulse radiolysis in benzene has been characterized and its reaction with oxygen to produce singlet oxygen and the anthryl radical described (Bruce et al., 1989). These authors reported no direct photochemical production of triplet anthralin or of singlet oxygen from anthralin in this nonpolar solvent (Fig. 1).

On the other hand, anthralin autoxidation leads to hydrogen atom and electron transfer to give the 1,8-dihydroxy-9-anthrone-l0-yl-radical in a free- radical chain mechanism (Davies et a l . , 1983; Mar- tinmaa et al . , 1978).

Oxygen consumption of psoriatic lesions is about two-fold greater than that of the noninvolved skin (Hammar and Hellerstrom, 1968; Raab, 1981) and oxygen uptake is significantly decreased by anthralin, especially when exposed to UV irradiation. As there is a high concentration of molecular oxygen in blood vessels, often damaged in skin photo- sensitization (Pathak and Carraro, 1987), a "Type 11" process is possible. Unequivocal determination of the r d e of active oxygen species in antipsoriatic action as well as in inflammatory and erythema reactions is a difficult but needed task. In particular, quantitative information about the photooxidation of anthralin is needed to understand the mode of action of this drug at the molecular level and to design new derivatives which may improve its thera-

PAP 52:3-A 445

446

+ OHa

KLAUS MULLER et al.

-HzO

Figure 1. Anthralin and anthralin anion.

peutic efficacy while diminishing its side effects. Because of its dermatological and pharmaceutical

importance and the interest in confirming the involvement of lo2 in the anthralin photooxidation, we have measured the rate of total '02 consumption of anthralin by indirect chemical techniques and by direct observation of the lo2 luminescence at 1.27 pm (Ogilby and Foote, 1983).

MATERIALS AND METHODS

Chemicals. Anthralin (1,8-dihydroxy-9-anthrone) was prepared by reduction of danthrone (Auterhoff and Sch- erff, 1960) and purified by column chromatograph (Si0,l CH,CI,). 2,3-Dimethyl-2-butene (tetramethyletiylene, TME)* Rose Bengal (RB) and 5,10,15,20-tetraphenyl- 21H,23H-porphine (TPP) were obtained from Aldrich (Milwaukee, WI). CH3CN was spectrophotometric grade (Mallinckrodt, Paris, KY). CH3CN-d3, benzene& and deuterium oxide (99.9%) were purchased from Cambridge Isotope Laboratories (Woburn, MA). All buffers were prepared in distilled watet or in deuterium oxide; pH values and apparent pD [measured values in deuterium oxide, which have not been corrected (Glasoe and Long, 1960) for the difference between pH and pD] were checked with a Corning Model 7 pH-meter.

Determination of the concentration of anthralin anion. The amount of anthralin anion in CH3CN was obtained from Eq. (3) by measuring the extinction module (m) (Pestemer and Briick, 1955) at 355 nm (A,,, of the anthrone tautomer) and 392 nm (A,,, of the anion) using E~~~ = 9800 M-' cm-'; = 2900 M-' cm-l for the protonated form; E~~~ = 3300 M-I cm-I, and E~~~ = 25 200 M-' cm-I for the anion. Extinction coefficients of anthralin and anthralin anion were determined in acidified (1-2 x M HCl) and alkaline (1-2 x M KOH) solutions of anthralin (4.64-10.03 x M ) in CH3CN.

e35.5 ,,,392- 392 AnH €.4nHm355

€AnEAnH €An EAnH 'An = 392 35s - 35s 392 . (3)

*Abbreviations: RB, Rose Bengal; TME, tetramethylethylene; TPP, 5,10,15,20-tetraphenyl-21H,23H-porphine.

The amount of anthralin anion in deuterated buffer solutions was calculated from the apparent pK,, determined from a titration curve obtained in the apparent pH range 7.4-11.8 (0.05 M phosphate buffers, 0.1 M borate buffers and 0.05 M carbonate buffers) on a Hewlett-Packard 8450A diode array spectrophotometer. Anthralin stock solution in acetone (50 pC) was added to 5 mC buffer solution (5 x M anthralin), mixed thoroughly, and the absorbance at 385 nm (Amax of the anion in aqueous solution) was monitored immediately; E~~~ (anthralin anion) = 18 100 M-' cm-I.

Direct disappearance of anthralin anion. CH,CN solutions of anthralin (5.69-23.48 X M ) and RB (2 x M) in 16 x 150 mm Pyrex test tubes were photolyzed with a 650 W Sylvania tungsten-halogen DWY Quartzline lamp operated at 45-70 V in a water-cooled immersion well at 22°C. The samples were saturated with oxygen prior to irradiation. A 1% solution of K,Cr,O, in H,O was used as a cutoff filter (550 nm). No sensitizer bleaching occurred during the reaction. The concentrations of anthralin anion before and after the photooxidation were monitored at 355 and 392 nm with a Beck- man Model 25 spectrophotometer and a Cary 2300 spectrophotometer.

Competition experiments. Solutions of anthralin (9.72-12.82 x M ) , TME (2-4 x lo-, M ) and RB (2 x M ) in CH3CN were photolyzed as described above. The loss of anthralin was determined by the change in absorbance by UV spectroscopy immediately after photolysis. Excess triphenylphosphine was added to the reaction mixture to reduce 2,3-dimethyl-l-butene-3-hydro- peroxide, the product of TME photooxidation, and main- tained in the dark for 15 min. The amount of the corresponding alcohol (2,3-dimethyl-l-butene-3-01), which is equal to the disappearance of TME, was measured on a Hewlett-Packard 5880A gas chromatograph equipped with an integrator; capillary column (30 M DB-17); injection T = 250°C; oven T: initial value = 40"C, final value = l W C , program rate = lV/min; toluene was used as inter- nal standard.

Rate constants from the decay rate of the 1.27 p luminescence. '02 was generated by RB in D,O or TPP in benzene-d,. Anthralin samples in D,O (5 x 10-6-4.02 x M ) were made using a small quan- tity of acetone-d, (200 pC per 5 mC of solution) to increase the solubility of anthralin. Each sample was buffered at the appropriate pD. Samples were excited at 355 or 532 nm using a frequency-tripled or-doubled Nd : YAG Laser (Quanta-Ray/Spectra Physics). Luminescence at 1.27 pm was observed using a system consisting of a 2 mm germanium photodiode (Opto-Electronics) operating at 0.7 V bias, with an OP-37 amplifier (Analog Devices) operating in a trans-impedance mode with a 360 k n feed- back resistor ax.-coupled to a Comlinear Model E220 amplifier. The resultant time-resolved luminescence signal was fed into an Analogic Data 6000 transient digitizer and then transferred to a PDP 11/73 for analysis. The digitizer is triggered by laser light scatter collected after the beam- separating optics and fed to a photodiode (Motorola, MRD 510) using a fiber optic coupler (Hewlett-Packard) which is close-coupled to the trigger input of the digitizer. The 1.27 pm light was collected using a curved mirror behind the cell, and imaged to the surface of the detector with an AR-coated lens, while most of the scattered visible light was removed using an high purity silicon filter AR- coated for peak transmittance at 1.27 pm with a 1100 nm cutoff (Infrared Optics), and an RG-850 filter (Schott Glass). The laser beam (355 and 532 nm) was also filtered to remove any 1060 nm fundamental with 1060 nm/355 nm or 1060 nm/532 nm beam splitters (Newport Corp.), fol- lowed by KG-3 heat absorbing filters. Additionally, the 355 nm beam was filtered to remove any 532 nm with 532 nm/355 nm beam splitters (Newport Corp.). The data were analyzed using the Guggenheim method (Moore and

-

Anthralin photooxidation 447

Pearson, 1981) to determine the rate of decay. Each sample was averaged over a maximum of 15 laser pulses. Although there was some loss of starting material during the experiment, the decay traces for each shot were care- fully determined during the experiment and the collection halted if any change in the decay rate became apparent.

RESULTS AND DISCUSSION

Anthralin photooxidation was studied by different kinetic techniques in benzene, CH3CN and deuterated buffer systems. In benzene, the UV spectrum of anthralin shows a single maximum at 355 nm with no further maxima or shoulders at longer wave- lengths, indicating that only the protonated anthrone tautomer was present. The absorption spectrum in CH3CN shows a shoulder at 392 nm, indicating a low concentration of the anionic form. In aqueous buffer solutions, the anthrone and the anion are in equilibrium; the anion dominates above pH 9.5.

Determination of kR + k, by indirect techniques

Solutions of lop4 M anthralin in benzene, equilib- rated with oxygen, showed no measurable change in absorption at 355 nm during the course of photooxidation (4 h) in the presence of RB, confirming the stability of the anthrone form. As it is the anionic form that is the sensitizer and reactive with lo2, experiments had to be carried out in a solvent where anthralin is deprotonated at least partially. Solutions of M anthralin in oxygen-saturated alkaline methanol oxidized at high rates in the dark. Almost 50% of the initial anthralin is converted to the dehydrodimer, 1,8,1',8'-tetrahydroxydianthrone, within 40 min. Further oxidation and polymerization processes yield a dark precipitate (anthralin- brown). In contrast to alkaline methanol, anthralin autoxidation is extremely slow under identical conditions in CH3CN (9% conversion after 24 h). Consequently, competitive radical reactions that lead to dimerization and polymerization products can be excluded under our experimental conditions. The contribution of the anion to the equilibrium of anthralin in CH3CN was determined spectrophoto- metrically; the absorption spectra of the protonated and deprotonated forms are clearly distinguishable. The concentration of the anion was calculated to be

Provided that the rate of physical quenching (k',) of '02 by the anthrone tautomer (AnH), Eq. (5 ) , is comparatively small and negligible (see below) and under conditions where no autoxidation of the aghrone to the anthralin radical (An') and sub- sequent dimerization (kET+ kDIM) occurs [Eqs. (8) and (9)], the kinetic scheme for anthralin anion (An), which can react (kR) and quench (k,) lo2 is given by Eqs. (4), (6) and (7), where kD is the decay rate of lo2 in the solvent:

5.0 ? 0.4%.

kR

k d AnH+'02 -+ AnH+302

kQ An+ lo2 -+ An+302

An+102 -+ products (danthrone) (4)

( 5 )

(6) k D

k~~

k~~~

lo2 -+ 3 0 2 (7)

AnH+302 + '0, (8)

An'+An' + An-An. (9)

The expression for the rate of direct disappearance of anthralin anion under these conditions is:

A plot of the reciprocal of the rate of anthralin anion disappearance against the reciprocal of anthralin anion concentration will give a ratio of slope to intercept equal to kD/(kR+kQ); because kD was determined from laser experiments, substitution yields the sum of kR+kQ (Foote, 1979). The value of kD/(kR+kQ) in CH3CN is 5.9 * 0.8 X M, leading to a value of kR+kQ of 3.1 * 0.4 x 108 M-1 s-l

The rate constant kR was obtained by competition studies against TME, a well-defined '02-acceptor (Wilkinson and Brummer, 1981). Higgins' equation (Higgins et al., 1968) permits the determination of the rate constant for reaction of anthralin anion, independent of whether anthralin also quenches lo2 physically; the subscripts 0 and f stand for initial and final concentrations of anthralin and TME, respectively.

The ratio of k@/kgME in CH3CN determined in this way is 6.1 * 1.3, which leads to a value of kR equal to 2.1 * 0.5 x lo8 M - l s-l , suggesting that reaction of the anion accounts for about two thirds of the total removal of singlet oxygen.

Direct determination of kR+kg from the decay rate of 1.27 Fm luminescence

In addition to these indirect kinetic techniques, the direct measurement of the decay rate of the 1.27 pm luminescence at different substrate concentrations (Foote, 1988) was employed to obtain rate constants of anthralin. From the change in the observed values for the lifetime of '02 with concentration of an added substrate, the rate of its reaction with '02 can be determined. The rate constant of lo2 luminescence decay (koBs) is

koss = k ~ + ( k ~ + kQ)[AnH] (12) where kD is the rate constant for radiationless decay of lo2 and (kR+kQ) is the total rate of lo2 removal

448 KLAUS MULLER et al.

0.8 - 0.6 -

0.4 - 0.2 -

0.0

by anthralin (AnH). If no appreciable photooxidation of the substrate occurs during the reaction period, a plot of the observed rate constant for the decay of lo2 (koBS) vs substrate concentration will be linear (Ogilby and Foote, 1983). With this tech- nique, values of (kR+kQ) for the protonated anthrone tautomer of anthralin [AnH] and the anion [An] were obtained from plots of koss vs [AnH] or [An], respectively.

In order to obtain the quenching rate for the anthrone form of anthralin, time-resolved detection of '02 luminescence was carried out in benzene-& where a benzophenone type absorption spectrum indicates the sole existence of this tautomer. As the anthrone is not reactive with lo2, shown by its complete resistance to photooxidation, the rate constant determined in this way is due to physical quenching (ko) only. The value is 2.80 2 0.18 x lo4 M-' s-l, four powers of ten less than that of the anion, confirming the assumption (see above) that there is negligible interaction of lo2 with the anthrone tautomer in CH3CN.

As the lifetime of '02 is significantly longer in deuterium oxide than in water (Monroe, 1985), rate constants for removal of '02 by anthralin anion were determined in deuterated buffer systems. The pK, of anthralin is found by titration to be 9.5, in good agreement with a previously reported value (Sa e Melo et al., 1983) of 9.4. Because only the apparent pK, in deuterium oxide corresponds to the actual amount of anthralin anion, this value was used for calculations. The titration curve in D 2 0 is 0.8 units higher than in protisted buffers (Fig. 2), a difference that is in accordance with literature (Glasoe and Long, 1960) values for KH/KD ratios of weak acids. Because the measured pH values of deuterated buffers were not corrected to give absolute pD values (Glasoe and Long, 1960), the actual shift is even higher. Base-catalyzed deuterium exchange in the anthralin molecule at C-10 and C- 2,7 (Geiger, 1974) probably occurs at higher pD.

Plots of koss vs anthralin concentration (Fig. 3) show increasing slopes (kR+kQ) at higher pD. A plot of kR+kQ as a function of pD (Fig. 4) is in accordance with the titration curve of anthralin in D 2 0 assuming that only the anion is the reactive species. The value of kR+kQ for the anion, obtained from plots of koBs vs the calculated concentrations of the deprotonated trihydroxyanthracene, is 3.0 * 0.7 X lo8 M-' s-l . Only values of koBs at pD 9.4-11.4 have been taken for this determination, as the concentration of the deprotonated form (11-92% of the total amount of anthralin) over this pD range is high enough to obtain reasonable error limits.

Table 1 summarizes the results from the present study. The parameters obtained from indirect techniques were converted to absolute rate constants by substituting known absolute values. For direct disappearance experiments, a decay rate of

, , , , . , , , .

E Y)

% 4

1 . 0 ,

Figure 2. Titration curve of anthralin (5 x M ) ; 0 water (pK, in H 2 0 = 9 3 , 0 deuterium oxide (apparent

pK, in D 2 0 = 10.3).

4 00ei4

3 50814

-- 30004 - 9 2 5 0 s t 4

2 00814

150ei4 ' I 1 ow-4 2 008.4 3 008.4 4.0oe.4

[Anlhrdinl, M

Figure 3. Reaction and quenching of lo2 by anthralin (0-4.02 X M ) in deuterated buffer systems at different pD values. Each plot of k,,, vs [anthralin] has a slope of (k,+k,) for the interaction of anthralin with '0, at the corresponding pD: 0 pD 7.4; 8.4; 0 9.4; A 10.4;

A 10.9; +11.0; X11.4.

4.00e+8 , I

P I

7 8 12 9 10 11

PD

Figure 4. Rate of total reaction and quenching of '0, by anthralin (0-4.02 x M ) as a function of pD.

1.8 X lo4 s-l for kD in CH$N (Ogilby and Foote, 1983) and in competition experiments a rate constant for TME of kR=2.97 x lo7 M-' s-' (Monroe, 1985; Wilkinson and Brummer, 1981) were used.

The data in Table 1 clearly show that the deprotonated 1,8,9-trihydroxyanthracene is the active species for interaction with lo2 with high rates of reaction and quenching, and that there is no appreciable contribution from the neutral l,&dihydroxy-9-anthrone. It might be expected that under physiological pH conditions there would be only

Anthralin photooxidation 449

Table 1. Rates of reaction ( k R ) and quenching (k , ) for anthralin and anthralin anion

2.8 2 0.2 Benzene-& Anthralin (anthrone) Luminescence

Anthralin Disappearance 3.1 * 0.4 CH,CN (anion) TME competition 2.1 5 0.5 CHiCN

Luminescence 3.0 5 0.7 D20-buffer

small amounts of anion present, but binding to albumin leads to the formation of deprotonated anthralin (Kohen et al . , 1986; Sa e Melo et al . , 1983; Wiegrebe et al., 1981).

Although the anion produces lo2, its rate of reaction with ‘02 is one magnitude .or more larger than those of important potential targets for oxidative degradation such as histidine, methionine, methyl arachidonate, cholesterol, adenosine and guano- sine. As corresponding rate constants (Wilkinson and Brummer, 1981) for these compounds are lo8, 2 x lo7, 2x x105, 7 x lo4, lo6 and lo6 M-ls - ’ , respectively, the anthralin anion should be preferen- tially oxidized. The involvement of ‘02 in the induc- tion of side effects of anthralin therapy such as erythema and irritation of the healthy skin is also improbable for the same reason.

In a recent study (Bruce et al., 1988), the anthralin radical was discounted as an important agent in antipsoriatic activity because of its stability and inertness towards oxygen. In light of these findings, future investigations should probably concentrate on the kinetics and involvement of oxygen radical species in the mechanism of action of anthralin.

Acknowledgements-This work was supported by NIH grant GM20080. K. M. thanks the Deutsche Forschungs- gemeinschaft for a fellowship.

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KINETIC STUDIES ON ANTHRALIN PHOTOOXIDATION