319
J. Photochem. Photobiol. B: Biol., 14 (1992) 319-328
Excited singlet state properties photosensitizers
of anthracenedione
Alessandra Andreoni+ Centro Endoctinologia ed Oncologia Sperimentale C.E.O.S.-C.N.R.
and Dipatiimento di Biolc@a e Patologia Cellulare e Molecolare, University of Naples, 80131 Napoli (Italy)
Albert0
Colasanti
Dipatiimento di Biologia e Patologia Cellulare e Molecolare, University of Naples, 80131 Napoli (Italy)
Giuseppe
Roberti
Dipartimento di Scierue Fisiche, University of Naples, 80125 Napoli (Italy)
(Received November 27, 1991; accepted January 21, 1992)
Abstract The absorption, fluorescence and S, state kinetics of anthracycline antitumour drugs (e.g. daunomycin, adriamycin) and several imino- and/or amino-substituted derivatives are investigated. The study, which includes all anthracyclines which possess photocytocidal activity, is extended to the disubstituted aminoanthracenedione, mitoxantrone, a red-lightabsorbing antitumour drug whose activity, both in vitro and in vivo, is enhanced by photoactivation. The S, state of the anthracycline imino and amino derivatives, in aqueous buffer at pH 1.4, is characterized by bi-exponential decay kinetics which indicates the presence of two ground state populations differing in the extent of hydrogen bonding. The ammonium group of the sugar moiety of anthracyclines contributes to the quenching of the S, state population through a prototropic mechanism.
Keywords: Anthracenediones,
anthracyclines,
mitoxantrone,
absorption,
fluorescence,
S,
lifetimes, photosensitizers.
1. Introduction
During the last few years we have reported that anthracyclines, such as daunomycin (daunorubicin, 1) and 4-demethoxydaunomycin (2), as well as both imino- and aminosubstituted analogues (Fig. l), efficiently photosensitize cytotoxic reactions in vitro on photoexcitation [l-3]. The main reason why we have devoted our efforts to these derivatives of 1 and 2 is that both imino and amino substituents in the anthraquinone chromophoric moiety cause a red shift in the absorption spectrum compared with the parent drugs whose peaks overlap in the blue wavelength region. For the same reason, we also investigated the cell photosensitizing properties of a different antitumour drug, i.e. the disubstituted aminoanthracenedione, mitoxantrone, whose absorption spectrum fAuthor to whom correspondence should be addressed.
Elsevier Sequoia
320
has two peaks at 610 and 660 nm [2, 41, i.e. in a spectral region of relatively high tissue transparency. We recently demonstrated that photoactivation with light in this wavelength range enhances the response to mitoxantrone of B16 melanoma in viva [5]. It is worth noting that, in contrast with other photosensitizers such as porphyrins and phthalocyanines, all molecules that we have demonstrated to be photocytocidal possess cytotoxic activity even in the absence of light [6-121, so much so that some of them are used as chemotherapeutic drugs or considered for future applications in oncology [13-161; their mechanisms of action have been extensively studied [B, 10, 13, 14, 17, 181. Therefore, in principle, photoactivation can either increase the rate of the mechanisms that are activated by enzymes in the dark [17, 181 or initiate other reactions that eventually enhance the cytotoxicity. Owing to the problems of providing in vim evidence of photophysical and/or photochemical pathways that lead to cell damage (e.g. generation of singlet oxygen, i.e. type II photoreaction, or occurrence of type I reactions), it is difficult to establish correlations between drug structures and the photophysical, photochemical and kinetic properties of the excited states relevant to cell damage by photosensitization. There has been little reported work on the lowest excited triplet states of photosensitizing anthracenediones, and parameters such as triplet lifetimes, extinction coefficients and quantum yields have been reported either in pure solvents or micellar systems for only a few compounds [19-211. However, the available data seem to indicate that singlet oxygen sensitized by triplet anthracenediones does not play a relevant role [22]. Furthermore, even less work has been reported on the first excited singlet states of these drugs, yet it has been shown that both the spectral and kinetic properties of fluorescence are strongly affected by the binding to selected biomolecules [23-281. Since it has been suggested that drug photoreduction is probably the first photochemical event in the light-activated pathways that lead to cell death, and it can occur via the Sr state [22], we have investigated both the spectral and kinetic properties of this state. This paper presents the first measurements of the fluorescence lifetimes of a number of amino- and imino-substituted derivatives of 1 and 2. The study is extended to similar derivatives of daunomycinone 3, i.e. the aglywne of 1 in which the daunosamine sugar group is replaced by hydrogen, in an attempt to understand the effects of different structures and substituents.
2. Materials
and methods
The chemical structures of the investigated molecules are shown in Fig. 1. Compounds 1 and 2, as well as their water-soluble derivatives i.e. ll-deoxy-llaminodaunomycin (S), 4-demethoxy-ll-aminodaunomycin (6), 4-demethoxy-6-deoxy-6aminodaunomycin (lo), doxorubicin (adriamycin, 11) and 4’-deoxy-4’-iododoxorubicin (12), were obtained from Farmitalia-Carlo Erba (Milano, Italy) as hydrochloride salts; S-iminodaunomycin (9) hydrochloride was prepared by Dr. V. Malatesta (Istituto “G. Donegani”, Novara, Italy) according to literature methods [29]. Daunomycinone (3) and its derivatives 5-imino-6-aminodaunomycinone (7) and 5-imino-ll-aminodaunomycinone (8) were also from Farmitalia-Carlo Erba. Mitoxantrone (4) was obtained from Lederle (Wayne, NJ). All other chemicals and solvents were Farmitalia-Carlo Erba products. Absorption and fluorescence spectra were recorded on a Perkin-Elmer 554 spectrophotometer (bandwidth, 2 nm) and a Perkin-Elmer LS 50 fluorometer (5 nm slits in both excitation and observation monochromators) respectively.
321
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Excited singlet state lifetimes were measured with a single-photon (SP) timing apparatus using either a mode-locked argon-ion laser tuned at 364 nm or, when wavelengths in the range 580-610 nm were necessary to match the absorption spectra of the compounds to be investigated, a picosecond rhodamine 6G dye laser. Both systems are described in detail in ref. 30. Only the main features will be mentioned here. The fluorescence emitted by the sample was passed through a Jarrel-Ash 0.5 m monochromator equipped with 0.1 mm slits so that the fluorescence decay could be detected at various wavelengths in the emission spectrum of each compound. The time resolution of the apparatus was such that the detected excitation pulse, averaged for a time (5 min) of the order of that typically required to measure fluorescence decays, had a full width at half-maximum (FWHM) duration of 180 ps in the case of excitation at 364 nm and 45 ps when excitation was provided by the dye laser. Occasionally, we repeated some measurements using a PTI LS-1 nanosecond lifetime system equipped with a nitrogen lamp as the excitation source. The following excitation wavelengths were used in this case: 316, 358 and 405 nm. The measurements of the absorption and fluorescence spectra and the time-resolved fluorescence were carried out in aqueous phosphate buffer solutions (lo-’ M, pH 7.4) of the water-soluble compounds and in methanol solutions of compounds 3, I and 8. We also studied compound 1 in methanol. The reverse phase high performance liquid chromatography (HPLC) measurements were carried out with an HP1090 system on Microbore columns (100 mmx2.1 mm) maintained at 40 “C (phosphate buffer, pH 3.5; acetonitrile). 3. Experimental
results
Table 1 shows the absorption peak wavelengths A,, of the first (long-wavelength) absorption bands and the corresponding molar extinction coefficients E,.,,_ determined for compounds 1, 2, 4-6, !)-12 in phosphate buffer and for compounds 1, 3, 7 and 8 in methanol. The visible absorption spectra of the water-soluble anthracyclines were found to exhibit Lambert-Beer behaviour over the range of concentrations used in this study (up to about 2 x 10m5 M). We also measured the fluorescence spectra on excitation at the wavelength of maximum absorption for each compound and obtained spectra peaking at the wavelengths listed in the last column of Table 1. For most compounds we repeated the measurement choosing excitation wavelengths in the second (UV) absorption bands. In the case of daunomycin (1) in methanol, for example, on excitation at either 295 or 357 nm, emission spectra were observed to be of the same shape as that obtained with excitation at 472 nm. However, the 596 nm fluorescence excitation spectrum had a maximum at 472 nm. The same applied for 3 in methanol for excitation at either 336 or 377 nm compared with 472 nm. Compound 4, on excitation at either of the visible absorption peak wavelengths, emits a weak fluorescence with a peak at 683 nm, whereas excitation at 250 nm produces a similarly weak fluorescence at shorter wavelengths (peak at 394 nm). The 11-amino derivatives of 1 and 2, i.e. compounds 5 and 6 respectively, exhibit red-shifted absorption peaks compared with the parent molecules (Table 1) and shoulders at longer wavelengths. Their fluorescence spectra are peaked at the wavelengths reported in the table for any excitation wavelength in the corresponding visible absorption bands. However, excitation of 5 at shorter wavelengths, e.g. 290 and 380 nm, produces blue-shifted emission bands, peaking at 374 and 450 nm respectively. In particular, the emission band at 374 nm predominates over that at 617 nm for excitation at 290 nm. A similar behaviour is observed for compound 6 such that, on excitation at 358 nm, an emission band peaking at 440 nm, stronger that that at 612 nm, is detected. Compounds 7 and 8 in methanol
323 TABLE 1 Summary of spectral Compound
absorption
and fluorescence
data Fluorescence
Absorption
A,,,
1 la 2 3’ 4 5 6 7p 8’ 9
10 11 12
(nm)
A,
(nm)
lmpx(M-l
472, 472, 464, 472, 610, 542, 532, 558, 558, 548, 530,
488 492 482 492 660 580b 562b 602 600 595 565b
11300, 11800 Not determined 11300, 11800 19300, 19600 20300, 23200 7280, 54OOb 8220, 6610b 9250, 12700 17180, 23640 5920, 6050 7970, 66OOb
562’. 595 596 568,61V 592 683 617 612 626-630 626-630 644 620
11300, 11800 2700, 2820
562’, 595 563’, 597
472, 488 472, 488
cm-‘)
%olvent; methanol; buffered (pH 7.4) phosphate solution in all other cases. bShoulder. ‘Secondary peak.
exhibit two well-resolved absorption peaks (Table 1). Excitation at either peak produces the same emission spectrum with a maximum at 626-630 nm for both compounds. Excitation at shorter wavelengths, e.g. in the range 32w80 nm, predominantly yields emission at shorter wavelength with a maximum at about 500 nm. The 5-imino derivative of 1, compound 9, also has two well-resolved peaks in its long-wavelength absorption spectrum when dissolved in phosphate buffer and an emission maximum at 644 nm when it is excited in the range 305-595 nm. Compound 10, which is similar to 6 except for the position of the amino substituent (Fig. l), exhibits absorption and fluorescence spectra very similar to those of 6. Doxorubicin (ll),on excitation in the visible band, emits at A,,- -595 nm and has a weaker emission at 562 nm. The excitation spectra of both the main and secondary peaks coincide with the absorption spectrum for A>370 nm. In contrast, excitation at 275 nm produces an emission spectrum peaking at about 370 nm. The emission spectrum of 12 is similar to that of 11.It should be noted that, although for compounds 1, 11 and 12 the main emission peaks occur at longer wavelengths than the secondary peaks, the reverse applies in the case of 2. The fluorescence decays of 1 and 2 in phosphate buffer were measured in a range of concentrations (10m6 to 1.88 X 10m5 M) with the PTI instrument with excitation at either 316 or 358 nm. In all cases, the decays could be fitted by single exponentials with decay time constants of 1.05 f 0.01 ns for 1 and 1.86 f 0.01 ns for 2. The fluorescence decay times were virtually independent of the detected wavelength, which was varied in the range 590-630 nm. The measurement was repeated for 1 at 620 nm with the SP apparatus. With both dye-laser (580 nm) and argon-ion (364 nm) excitations, we observed, independent of concentration, single-exponential decays of time constant 1.04f0.02 ns. Compound 3 was measured in methanol at a single concentration value, 2X lo-’ M. The decay, as measured at 620 nm with the SP apparatus and excitation at 580 nm, could be fitted with the sum of two decaying exponentials of time constants
324 71= 2.66 ns and r2= 1.18 ns and relative amplitudes Al =0.74 and A2= 0.26. For comparison, we dissolved compound 1 in methanol at the same concentration as the solution of 3 and obtained, under identical excitation and detection conditions, a biexponential decay (rr = 2.44 ns, TV= 1.16 ns, Al = 0.65, A2=0.35). The fluorescence decay of the only diamino-substituted anthracenedione compound examined, mitoxantrone (4), was measured in phosphate buffer over a range of concentrations and at a number of wavelengths available from either the PTI instrument or the modelocked dye laser. All measurements gave first-order decays with very short time constants (0.25 ns). The fluorescence decays of compounds 5 and 6 in buffer (580 nm excitation, SP apparatus) were found to be rather independent of the observation wavelength over the emission range investigated (610-680 nm) and could be fitted by bi-exponential decays with almost equal relative weights (0.5fO.l). The decay time constants were r1 = 0.42 f 0.01 ns and 72= 0.31 f 0.00 ns for 5 and r1 = 0.60 k 0.02 ns and r2 = 0.42 f 0.02 ns for 6. The fluorescence of both imino-amino-derivatives of the water-insoluble daunomycinone (3), 7 and 8, was measured at 620 nm for concentrations of the order of 10m5 M in methanol with the SP system and dye laser excitation at 590 nm. For both compounds relatively fast (0.34f0.04 ns) single-exponential decays were found. The fluorescence decay of compound 9 in phosphate buffer was previously measured by Malatesta and Andreoni [25] and reported to be bi-exponential. We thus carried out further measurements at only two concentration values, namely 1.73X10e5 and 8.64~ lo-’ M, with 580 nm excitation and the SP apparatus and detected fluorescence at wavelengths in a rather limited interval, i.e. 620-650 nm. We found bi-exponential decays in all cases (r1=0.59f0.01 ns, r2=0.42f0.01 ns) with the relative initial amplitude Al decreasing from 0.40 to 0.26 and A2 increasing from 0.60 to 0.74, for both increasing observation wavelengths and on going from high to low concentrations. The fluorescence decays of buffered solutions of compound 10 at different concentrations (from 2~ 10m6 to 4~ lo-’ M) were measured at wavelengths between 620 and 685 nm with the SP apparatus and excitation at 580 nm. All decays could be fitted with bi-exponential functions. Both the time constants (rr = 0.61 rt 0.04 ns and 72= 0.40 f 0.01 ns) and the relative initial amplitudes (Al = 0.27& 0.10 and A2= 0.73 ~fr0.10) were independent of concentration and wavelength. Compound 11 was measured with the SP system and argon-ion excitation and yielded single-exponential decays identical with those of compound 1 under the same experimental conditions. Similar results were obtained for the fluorescence decay of 12 at 465 nm (r= 0.98 * 0.01 ns) measured with the PTI apparatus and excitation at 405 nm. The best-fit parameters for a set of typical fluorescence decays, together with the corresponding x2 values, are listed in Table 2. The experimental conditions under which the measurements were carried out are also indicated. All anthracycline compounds exhibiting double-exponential decays were checked after the fluorescence decay measurements by HPLC. The eluted peaks were monitored by absorption at both the visible wavelength of maximum absorption of the compound under study (Table 1) and at 254 nm. No modifications of the elution profile could be detected.
4. Discussion Inspection of the absorption data for the water-soluble anthracyclines in Table 1 reveals that the spectrum of the anthraquinone chromophore is insensitive to both the removal of the methoxy group at C4 (c$ 2 and 1, 6 and 5) and the structure of
325 TABLE 2 Time constants 7, and 72 and relative initial amplitudes A1 and AZ of the best-fitting mono- or bi-exponential decays for typical time-resolved fluorescence measurement? Compound
1 lb 2 3b 4 5 6 ;: 9 10 11 12
7, (ns)
A, (%)
1.05 2.44 1.86 2.66 0.24 0.42 0.58 0.38 0.31 0.60 0.57 1.04 0.98
100 65 100 74 100 58 50 100 100 29 28 100 100
72
A2
(ns)
(“ro)
1.16
35
1.18
26
0.31 0.42
42 50
0.42 0.40
71 72
x2
1.00 1.34 0.96 1.15 1.31 1.11 1.18 1.01 1.02 1.22 1.00 1.02 1.05
A cxc
Lb,
(nm)
(nm)
350 580 358 580 316 580 580 590 590 580 580 364 405
500 620 550 620 630 617 613 620 620 632 685 550 465
System
C W)
PTI SP PTI SP PTI SP SP SP SP SP SP SP PTI
1.78 x 2.00 x 1.88 x 2.00 x 4.50 x 1.00x 8.60 x 1.00x 1.10x 1.73 x 4.41 x 1.00 x 1.45 x
1O-5 10-S 10-S 10-S 10-S 10-S lo+ 10-s 10-S 10-5 10-6 10-s lo+
‘Fluorescence decays at &,brwere measured by either the PTI or SP system with excitation at h,,, for solutions at the indicated concentrations C. bSolvent; methanol. Buffered (pH 7.4) phosphate solution in ah other cases. the side chain at C9 (cf. 11 and 1, and that the presence of a heavy atom in the sugar moiety simply affects the value of E (cf. 12 and 11).Similarly, substitution with the amino group at Cl1 seems to have the same effect on the visible absorption band of the anthraquinone chromophore, independent of the presence of the methoxy group at Cl. Substitution with the imino group at C5 causes separation of peak wavelengths and a red shift (cf. 9 and 1). The different structure of the side chain of doxorubicin (11)compared with 1, i.e. COCH,OH VS. COCH3, has no influence on the excited singlet state lifetime in aqueous buffer which, together with the previously reported result indicating that these anthracyclines have the same triplet yield value [19, 201, shows that both radiative and non-radiative S1 decay pathways have virtually identical rates for 1 and 11.Similarly the observation that the substitution of iodine in the sugar moiety of 11 brings about a negligible shortening in lifetime, eg. T= 0.98 rt 0.01 ns for 12 W. T= 1.04f0.02 ns for 11, agrees with the absence of any heavy atom effect, as already ascertained from comparative measurements of relative fluorescence yield values [3]. For 4-demethoxydaunomycin (2), we observe a substantially longer fluorescence decay time than for 1 which might originate from the removal of the basic ethereal OCHa group [31], whose presence in 1 may contribute to fluorescence quenching via an intramolecular H+ transfer from the protonated amino sugar. The decay kinetics of daunomycinone (3) are very similar to those of the parent glycoside (1) in methanol; the rationale for the appearance of a long lifetime (2.66 ns and 2.44 ns for 3 and 1 respectively) may parallel our interpretation of the lengthening of both S1 and T1 lifetimes when the protonated amino group in the sugar moiety is either removed or neutralized [19, 20,251. For instance, we have measured a fluorescence decay component with a lifetime of 2.73rt0.22 ns for 1 externally bound to DNA, when the sugar ammonium ion of daunomycin is engaged in an ionic bond with a DNA backbone phosphate group. The T1 lifetime of 1, which is equal to 1.80f0.09 ps in NTflushed phosphate buffer at
326 pH 7.4, becomes three times longer at pH 9, i.e. on deprotonation of the sugar nitrogen. Furthermore, we determined the Ti lifetime of 3 which, being the aglycone of 1, obviously lacks the ammonium ion, and found the relatively long value of 7.00 * 0.35 ps in Nz-flushed benzene. Under the same conditions, we measured a similar value for daunomycin-N-trifluoro-acetamide in which the sugar amino group is substituted by the less basic -NHCOCF3. From all of these observations, we concluded that a sugar-to-anthraquinone proton transfer must be an efficient quenching mechanism acting on both excited singlet and triplet states. The concomitant presence of imino and amino substituents in the daunomycinone chromophore results in a dramatic quenching of the Sr state, as revealed by the short fluorescence decay times measured for compounds 7 and 8: 0.3450.04 ns. Before speculating on the possible mechanism of quenching, it is worthwhile to analyse the effects of the two substituents separately. The bi-exponential fluorescence decays of the 11-amino and 6-amino derivatives of 4demethoxydaunomycin, i.e. 6 and 10 respectively, are well fitted by components with very similar lifetimes (see Table 2 for typical results) which are both much shorter than that of the single-exponential decay of the parent compound (2). The same lifetimes also fit the fluorescence decay of compound 5 with initial amplitudes that, for all compounds excited at 580 nm, do not depend on the observation wavelength. These properties strongly suggest that the two lifetimes reflect a mixture of two ground state populations which do not interact in either the So or Sr state, and which we may tentatively identify as two structures differing by the extent of hydrogen bonding between NH2 and the adjacent carbonyl group. The observation that UV excitation of compounds 5, 6 and 10 produces fluorescence peaking at the wavelengths listed in Table 1 and blue-shifted fluorescence also agrees with the presence of two ground state populations. Furthermore, the fact that the relative weights of the two decay components are similar for compounds 5 and 6 indicates that, whatever the reason for the splitting of the ground state population, it is not affected by the methoxy group at C4. Since the only difference between the fluorescence decays of 6 and 10 is in the pre-exponential factors Ai and AZ, we believe that the position of the amino substituent simply affects the relative balance between the two populations [32] but not the Si state quenching mechanisms. Compound 9 is the only compound which exhibits dependence of the relative initial amplitudes of the two decay components on the observation wavelength, which agrees with our previous interpretation based on the acid-base properties of the imino group at C5 [25]. Further measurements of time-resolved fluorescence should be carried out on compounds 5, 6, 9 and 11 at different pH values to substantiate the interpretation of their fluorescence decay data. Finally, it is worth noting that the fluorescence decay time measured for the diaminosubstituted anthracenedione (4) agrees with that recently reported, though at different excitation and observation wavelengths, by Lin and Struve [28]; in addition the Sr quenching mechanisms invoked to interpret our data are based on interactions between the anthraquinone chromophore and either nitrogen substituents or hydrogen-bonding solvents which all previous reports on the S&i, spectral and kinetic properties of anthracenediones have demonstrated [25-281.
Acknowledgments
We are indebted to Dr. V. Malatesta (Istituto “G. Donegani”, Novara, Italy) for useful discussions and HPLC measurements and to Dr. A. Suarato (Farmitalia-Carlo Erba, Milano, Italy) for supplying the amino-substituted anthracyclines. We are also
327
very grateful to PTI (London, Ont., Canada) and to Dr. R. H. Pottier for cooperation in the use of the IS-1 nanosecond lifetime system which was installed at the Royal Military College (Kingston, Ont., Canada) on the occasion of the NATO-AS “Photobiological Techniques”, 1-14 July, 1990. This work was partially supported by the targeted projects “Electra-optical Technologies” and “Bio-technologies and Bioinstrumentation” of the Italian National Research Council.
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328 18 B. K. Sinha, A. G. Motten and K. W. Hanck, The electrochemical reduction of 1,4-bis(2((2~hydroxyethyl)-amino)ethylamino) anthracenedione and daunomycin: biochemical significance in superoxide formation, Chem.-Biol. Interact., 43 (1983) 371-377. 19 E. J. Land, A. J. McLean and T. G. Truscott, Laser flash photolysis and singlet oxygen quantum yields of anthracyclines, in G. Moreno, R. H. Pottier and T. G. Truscott (eds.), Photosensitization. Molecular, Cellular and Medical Aspects, NATO AS1 Series H, Vol. 15, Springer, Berlin, 1988, pp. 69-72. 20 A. Andreoni, E. J. Land, V. Malatesta, A. J. McLean and T. G. Truscott, Triplet state characteristics and singlet oxygen generation properties of anthracyclines, B&hem. Biophys. Acta, 990 (1989) 190-197. 21 P. Charlesworth, C. Lambert and T. G. Truscott, The mitoxantrone-singlet oxygen interaction, 4th Congress of the European Sociely for Photobiology, September I-6, 1991, Amsterdam BA1, the Netherlands, Abstract H38, Elsevier, Lausanne, 1991, p. 143. 22 A. Andreoni, A. Colasanti, A. Kisslinger, V. Malatesta, M. Mastrocinque and G. Roberti, Anti-tumor drugs as photochemotherapeutic agents, in L 0. Svaasand (ed.), Future Trends in Biomedical Applications of Lasers, Vol. 1525, SPIE, Bellingham, 1992, pp. 351-366. 23 E. Goormaghtight, P. Chatelain, J. Caspers and J. M. Ruysschaert, Evidence for a complex between adriamycin derivatives and cardiolipin: possible role in cardiotoxicity, Biochem. Phannacol., 2 (1980) 3003-3010. 24 J. B. Chaires, N. Dattagupta and D. M. Crothers, Kinetics of the daunomycin-DNA interaction, Biochemistry, 24 (1985) 260-267. 25 V. Malatesta and A. Andreoni, Dynamics of anthracyclines/DNA interaction: a laser timeresolved fluorescence study, Photochem. Photobiol., 48 (1988) 4O!J-415. 26 V. Malatesta and A. Andreoni, Laser time-resolved fluorescence study of the interaction between anthracyclines and cardiolipin, J. Photochem. Photobiol. B: Biol., 3 (1989) 157-164. 27 B. S. Lee and P. K. Dutta, Optical spectroscopic studies of the antitumor drug 1,4-dihydroxy5,8-bis[[2-[(2-hydroxyethyl)amino]ethyl]a~no]-9,lO-anthracenedione (mitoxantrone), J. Phys. Chem;, 93 (1989) 5665-5672. 28 S. Lin and W. S. Struve, Solvatochromism and time-resolved fluorescence of the antitumor agent mitoxantrone and its analogues in solution and in DNA, I. Phys. Chem., 95 (1991) 2251-2256. 29 G. L. Tong, D. W. Henry and E. M. Acton, 5-Iminodaunorubicin. Reduced cardiotoxic properties in an antitumor anthracycline, J. Med. Chem., 22 (1979) 36-39. 30 A. Andreoni, Fluorescence lifetimes of chromophores interacting with biomolecules, in G. Moreno, R. H. Pottier and T. G. Truscott (eds.), Photosensitization. Molecular, Cellular and Medical Aspects, NATO AS1 Series H, Vol. 15, Springer, Berlin, 1988, pp. 29-38. 31 D. D. Perrin, B. Dempsey and E. P. Sejeant, pK, &dictions for Organic Acids and Bases, Chapman and Hall, London, 1981, p. 3. 32 V. Malatesta, G. Ranghino and F. Moraxxoni, Aminoanthracyclines: physicochemical properties as predicted by ab-initio molecular orbital calculations, J. Mot! Struct. (Theochem.), 205 (1990) 169-175.
J. Photochem. Photobiol. B: Biol., 14 (1992) 319-328
Excited singlet state properties photosensitizers
of anthracenedione
Alessandra Andreoni+ Centro Endoctinologia ed Oncologia Sperimentale C.E.O.S.-C.N.R.
and Dipatiimento di Biolc@a e Patologia Cellulare e Molecolare, University of Naples, 80131 Napoli (Italy)
Albert0
Colasanti
Dipatiimento di Biologia e Patologia Cellulare e Molecolare, University of Naples, 80131 Napoli (Italy)
Giuseppe
Roberti
Dipartimento di Scierue Fisiche, University of Naples, 80125 Napoli (Italy)
(Received November 27, 1991; accepted January 21, 1992)
Abstract The absorption, fluorescence and S, state kinetics of anthracycline antitumour drugs (e.g. daunomycin, adriamycin) and several imino- and/or amino-substituted derivatives are investigated. The study, which includes all anthracyclines which possess photocytocidal activity, is extended to the disubstituted aminoanthracenedione, mitoxantrone, a red-lightabsorbing antitumour drug whose activity, both in vitro and in vivo, is enhanced by photoactivation. The S, state of the anthracycline imino and amino derivatives, in aqueous buffer at pH 1.4, is characterized by bi-exponential decay kinetics which indicates the presence of two ground state populations differing in the extent of hydrogen bonding. The ammonium group of the sugar moiety of anthracyclines contributes to the quenching of the S, state population through a prototropic mechanism.
Keywords: Anthracenediones,
anthracyclines,
mitoxantrone,
absorption,
fluorescence,
S,
lifetimes, photosensitizers.
1. Introduction
During the last few years we have reported that anthracyclines, such as daunomycin (daunorubicin, 1) and 4-demethoxydaunomycin (2), as well as both imino- and aminosubstituted analogues (Fig. l), efficiently photosensitize cytotoxic reactions in vitro on photoexcitation [l-3]. The main reason why we have devoted our efforts to these derivatives of 1 and 2 is that both imino and amino substituents in the anthraquinone chromophoric moiety cause a red shift in the absorption spectrum compared with the parent drugs whose peaks overlap in the blue wavelength region. For the same reason, we also investigated the cell photosensitizing properties of a different antitumour drug, i.e. the disubstituted aminoanthracenedione, mitoxantrone, whose absorption spectrum fAuthor to whom correspondence should be addressed.
Elsevier Sequoia
320
has two peaks at 610 and 660 nm [2, 41, i.e. in a spectral region of relatively high tissue transparency. We recently demonstrated that photoactivation with light in this wavelength range enhances the response to mitoxantrone of B16 melanoma in viva [5]. It is worth noting that, in contrast with other photosensitizers such as porphyrins and phthalocyanines, all molecules that we have demonstrated to be photocytocidal possess cytotoxic activity even in the absence of light [6-121, so much so that some of them are used as chemotherapeutic drugs or considered for future applications in oncology [13-161; their mechanisms of action have been extensively studied [B, 10, 13, 14, 17, 181. Therefore, in principle, photoactivation can either increase the rate of the mechanisms that are activated by enzymes in the dark [17, 181 or initiate other reactions that eventually enhance the cytotoxicity. Owing to the problems of providing in vim evidence of photophysical and/or photochemical pathways that lead to cell damage (e.g. generation of singlet oxygen, i.e. type II photoreaction, or occurrence of type I reactions), it is difficult to establish correlations between drug structures and the photophysical, photochemical and kinetic properties of the excited states relevant to cell damage by photosensitization. There has been little reported work on the lowest excited triplet states of photosensitizing anthracenediones, and parameters such as triplet lifetimes, extinction coefficients and quantum yields have been reported either in pure solvents or micellar systems for only a few compounds [19-211. However, the available data seem to indicate that singlet oxygen sensitized by triplet anthracenediones does not play a relevant role [22]. Furthermore, even less work has been reported on the first excited singlet states of these drugs, yet it has been shown that both the spectral and kinetic properties of fluorescence are strongly affected by the binding to selected biomolecules [23-281. Since it has been suggested that drug photoreduction is probably the first photochemical event in the light-activated pathways that lead to cell death, and it can occur via the Sr state [22], we have investigated both the spectral and kinetic properties of this state. This paper presents the first measurements of the fluorescence lifetimes of a number of amino- and imino-substituted derivatives of 1 and 2. The study is extended to similar derivatives of daunomycinone 3, i.e. the aglywne of 1 in which the daunosamine sugar group is replaced by hydrogen, in an attempt to understand the effects of different structures and substituents.
2. Materials
and methods
The chemical structures of the investigated molecules are shown in Fig. 1. Compounds 1 and 2, as well as their water-soluble derivatives i.e. ll-deoxy-llaminodaunomycin (S), 4-demethoxy-ll-aminodaunomycin (6), 4-demethoxy-6-deoxy-6aminodaunomycin (lo), doxorubicin (adriamycin, 11) and 4’-deoxy-4’-iododoxorubicin (12), were obtained from Farmitalia-Carlo Erba (Milano, Italy) as hydrochloride salts; S-iminodaunomycin (9) hydrochloride was prepared by Dr. V. Malatesta (Istituto “G. Donegani”, Novara, Italy) according to literature methods [29]. Daunomycinone (3) and its derivatives 5-imino-6-aminodaunomycinone (7) and 5-imino-ll-aminodaunomycinone (8) were also from Farmitalia-Carlo Erba. Mitoxantrone (4) was obtained from Lederle (Wayne, NJ). All other chemicals and solvents were Farmitalia-Carlo Erba products. Absorption and fluorescence spectra were recorded on a Perkin-Elmer 554 spectrophotometer (bandwidth, 2 nm) and a Perkin-Elmer LS 50 fluorometer (5 nm slits in both excitation and observation monochromators) respectively.
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Excited singlet state lifetimes were measured with a single-photon (SP) timing apparatus using either a mode-locked argon-ion laser tuned at 364 nm or, when wavelengths in the range 580-610 nm were necessary to match the absorption spectra of the compounds to be investigated, a picosecond rhodamine 6G dye laser. Both systems are described in detail in ref. 30. Only the main features will be mentioned here. The fluorescence emitted by the sample was passed through a Jarrel-Ash 0.5 m monochromator equipped with 0.1 mm slits so that the fluorescence decay could be detected at various wavelengths in the emission spectrum of each compound. The time resolution of the apparatus was such that the detected excitation pulse, averaged for a time (5 min) of the order of that typically required to measure fluorescence decays, had a full width at half-maximum (FWHM) duration of 180 ps in the case of excitation at 364 nm and 45 ps when excitation was provided by the dye laser. Occasionally, we repeated some measurements using a PTI LS-1 nanosecond lifetime system equipped with a nitrogen lamp as the excitation source. The following excitation wavelengths were used in this case: 316, 358 and 405 nm. The measurements of the absorption and fluorescence spectra and the time-resolved fluorescence were carried out in aqueous phosphate buffer solutions (lo-’ M, pH 7.4) of the water-soluble compounds and in methanol solutions of compounds 3, I and 8. We also studied compound 1 in methanol. The reverse phase high performance liquid chromatography (HPLC) measurements were carried out with an HP1090 system on Microbore columns (100 mmx2.1 mm) maintained at 40 “C (phosphate buffer, pH 3.5; acetonitrile). 3. Experimental
results
Table 1 shows the absorption peak wavelengths A,, of the first (long-wavelength) absorption bands and the corresponding molar extinction coefficients E,.,,_ determined for compounds 1, 2, 4-6, !)-12 in phosphate buffer and for compounds 1, 3, 7 and 8 in methanol. The visible absorption spectra of the water-soluble anthracyclines were found to exhibit Lambert-Beer behaviour over the range of concentrations used in this study (up to about 2 x 10m5 M). We also measured the fluorescence spectra on excitation at the wavelength of maximum absorption for each compound and obtained spectra peaking at the wavelengths listed in the last column of Table 1. For most compounds we repeated the measurement choosing excitation wavelengths in the second (UV) absorption bands. In the case of daunomycin (1) in methanol, for example, on excitation at either 295 or 357 nm, emission spectra were observed to be of the same shape as that obtained with excitation at 472 nm. However, the 596 nm fluorescence excitation spectrum had a maximum at 472 nm. The same applied for 3 in methanol for excitation at either 336 or 377 nm compared with 472 nm. Compound 4, on excitation at either of the visible absorption peak wavelengths, emits a weak fluorescence with a peak at 683 nm, whereas excitation at 250 nm produces a similarly weak fluorescence at shorter wavelengths (peak at 394 nm). The 11-amino derivatives of 1 and 2, i.e. compounds 5 and 6 respectively, exhibit red-shifted absorption peaks compared with the parent molecules (Table 1) and shoulders at longer wavelengths. Their fluorescence spectra are peaked at the wavelengths reported in the table for any excitation wavelength in the corresponding visible absorption bands. However, excitation of 5 at shorter wavelengths, e.g. 290 and 380 nm, produces blue-shifted emission bands, peaking at 374 and 450 nm respectively. In particular, the emission band at 374 nm predominates over that at 617 nm for excitation at 290 nm. A similar behaviour is observed for compound 6 such that, on excitation at 358 nm, an emission band peaking at 440 nm, stronger that that at 612 nm, is detected. Compounds 7 and 8 in methanol
323 TABLE 1 Summary of spectral Compound
absorption
and fluorescence
data Fluorescence
Absorption
A,,,
1 la 2 3’ 4 5 6 7p 8’ 9
10 11 12
(nm)
A,
(nm)
lmpx(M-l
472, 472, 464, 472, 610, 542, 532, 558, 558, 548, 530,
488 492 482 492 660 580b 562b 602 600 595 565b
11300, 11800 Not determined 11300, 11800 19300, 19600 20300, 23200 7280, 54OOb 8220, 6610b 9250, 12700 17180, 23640 5920, 6050 7970, 66OOb
562’. 595 596 568,61V 592 683 617 612 626-630 626-630 644 620
11300, 11800 2700, 2820
562’, 595 563’, 597
472, 488 472, 488
cm-‘)
%olvent; methanol; buffered (pH 7.4) phosphate solution in all other cases. bShoulder. ‘Secondary peak.
exhibit two well-resolved absorption peaks (Table 1). Excitation at either peak produces the same emission spectrum with a maximum at 626-630 nm for both compounds. Excitation at shorter wavelengths, e.g. in the range 32w80 nm, predominantly yields emission at shorter wavelength with a maximum at about 500 nm. The 5-imino derivative of 1, compound 9, also has two well-resolved peaks in its long-wavelength absorption spectrum when dissolved in phosphate buffer and an emission maximum at 644 nm when it is excited in the range 305-595 nm. Compound 10, which is similar to 6 except for the position of the amino substituent (Fig. l), exhibits absorption and fluorescence spectra very similar to those of 6. Doxorubicin (ll),on excitation in the visible band, emits at A,,- -595 nm and has a weaker emission at 562 nm. The excitation spectra of both the main and secondary peaks coincide with the absorption spectrum for A>370 nm. In contrast, excitation at 275 nm produces an emission spectrum peaking at about 370 nm. The emission spectrum of 12 is similar to that of 11.It should be noted that, although for compounds 1, 11 and 12 the main emission peaks occur at longer wavelengths than the secondary peaks, the reverse applies in the case of 2. The fluorescence decays of 1 and 2 in phosphate buffer were measured in a range of concentrations (10m6 to 1.88 X 10m5 M) with the PTI instrument with excitation at either 316 or 358 nm. In all cases, the decays could be fitted by single exponentials with decay time constants of 1.05 f 0.01 ns for 1 and 1.86 f 0.01 ns for 2. The fluorescence decay times were virtually independent of the detected wavelength, which was varied in the range 590-630 nm. The measurement was repeated for 1 at 620 nm with the SP apparatus. With both dye-laser (580 nm) and argon-ion (364 nm) excitations, we observed, independent of concentration, single-exponential decays of time constant 1.04f0.02 ns. Compound 3 was measured in methanol at a single concentration value, 2X lo-’ M. The decay, as measured at 620 nm with the SP apparatus and excitation at 580 nm, could be fitted with the sum of two decaying exponentials of time constants
324 71= 2.66 ns and r2= 1.18 ns and relative amplitudes Al =0.74 and A2= 0.26. For comparison, we dissolved compound 1 in methanol at the same concentration as the solution of 3 and obtained, under identical excitation and detection conditions, a biexponential decay (rr = 2.44 ns, TV= 1.16 ns, Al = 0.65, A2=0.35). The fluorescence decay of the only diamino-substituted anthracenedione compound examined, mitoxantrone (4), was measured in phosphate buffer over a range of concentrations and at a number of wavelengths available from either the PTI instrument or the modelocked dye laser. All measurements gave first-order decays with very short time constants (0.25 ns). The fluorescence decays of compounds 5 and 6 in buffer (580 nm excitation, SP apparatus) were found to be rather independent of the observation wavelength over the emission range investigated (610-680 nm) and could be fitted by bi-exponential decays with almost equal relative weights (0.5fO.l). The decay time constants were r1 = 0.42 f 0.01 ns and 72= 0.31 f 0.00 ns for 5 and r1 = 0.60 k 0.02 ns and r2 = 0.42 f 0.02 ns for 6. The fluorescence of both imino-amino-derivatives of the water-insoluble daunomycinone (3), 7 and 8, was measured at 620 nm for concentrations of the order of 10m5 M in methanol with the SP system and dye laser excitation at 590 nm. For both compounds relatively fast (0.34f0.04 ns) single-exponential decays were found. The fluorescence decay of compound 9 in phosphate buffer was previously measured by Malatesta and Andreoni [25] and reported to be bi-exponential. We thus carried out further measurements at only two concentration values, namely 1.73X10e5 and 8.64~ lo-’ M, with 580 nm excitation and the SP apparatus and detected fluorescence at wavelengths in a rather limited interval, i.e. 620-650 nm. We found bi-exponential decays in all cases (r1=0.59f0.01 ns, r2=0.42f0.01 ns) with the relative initial amplitude Al decreasing from 0.40 to 0.26 and A2 increasing from 0.60 to 0.74, for both increasing observation wavelengths and on going from high to low concentrations. The fluorescence decays of buffered solutions of compound 10 at different concentrations (from 2~ 10m6 to 4~ lo-’ M) were measured at wavelengths between 620 and 685 nm with the SP apparatus and excitation at 580 nm. All decays could be fitted with bi-exponential functions. Both the time constants (rr = 0.61 rt 0.04 ns and 72= 0.40 f 0.01 ns) and the relative initial amplitudes (Al = 0.27& 0.10 and A2= 0.73 ~fr0.10) were independent of concentration and wavelength. Compound 11 was measured with the SP system and argon-ion excitation and yielded single-exponential decays identical with those of compound 1 under the same experimental conditions. Similar results were obtained for the fluorescence decay of 12 at 465 nm (r= 0.98 * 0.01 ns) measured with the PTI apparatus and excitation at 405 nm. The best-fit parameters for a set of typical fluorescence decays, together with the corresponding x2 values, are listed in Table 2. The experimental conditions under which the measurements were carried out are also indicated. All anthracycline compounds exhibiting double-exponential decays were checked after the fluorescence decay measurements by HPLC. The eluted peaks were monitored by absorption at both the visible wavelength of maximum absorption of the compound under study (Table 1) and at 254 nm. No modifications of the elution profile could be detected.
4. Discussion Inspection of the absorption data for the water-soluble anthracyclines in Table 1 reveals that the spectrum of the anthraquinone chromophore is insensitive to both the removal of the methoxy group at C4 (c$ 2 and 1, 6 and 5) and the structure of
325 TABLE 2 Time constants 7, and 72 and relative initial amplitudes A1 and AZ of the best-fitting mono- or bi-exponential decays for typical time-resolved fluorescence measurement? Compound
1 lb 2 3b 4 5 6 ;: 9 10 11 12
7, (ns)
A, (%)
1.05 2.44 1.86 2.66 0.24 0.42 0.58 0.38 0.31 0.60 0.57 1.04 0.98
100 65 100 74 100 58 50 100 100 29 28 100 100
72
A2
(ns)
(“ro)
1.16
35
1.18
26
0.31 0.42
42 50
0.42 0.40
71 72
x2
1.00 1.34 0.96 1.15 1.31 1.11 1.18 1.01 1.02 1.22 1.00 1.02 1.05
A cxc
Lb,
(nm)
(nm)
350 580 358 580 316 580 580 590 590 580 580 364 405
500 620 550 620 630 617 613 620 620 632 685 550 465
System
C W)
PTI SP PTI SP PTI SP SP SP SP SP SP SP PTI
1.78 x 2.00 x 1.88 x 2.00 x 4.50 x 1.00x 8.60 x 1.00x 1.10x 1.73 x 4.41 x 1.00 x 1.45 x
1O-5 10-S 10-S 10-S 10-S 10-S lo+ 10-s 10-S 10-5 10-6 10-s lo+
‘Fluorescence decays at &,brwere measured by either the PTI or SP system with excitation at h,,, for solutions at the indicated concentrations C. bSolvent; methanol. Buffered (pH 7.4) phosphate solution in ah other cases. the side chain at C9 (cf. 11 and 1, and that the presence of a heavy atom in the sugar moiety simply affects the value of E (cf. 12 and 11).Similarly, substitution with the amino group at Cl1 seems to have the same effect on the visible absorption band of the anthraquinone chromophore, independent of the presence of the methoxy group at Cl. Substitution with the imino group at C5 causes separation of peak wavelengths and a red shift (cf. 9 and 1). The different structure of the side chain of doxorubicin (11)compared with 1, i.e. COCH,OH VS. COCH3, has no influence on the excited singlet state lifetime in aqueous buffer which, together with the previously reported result indicating that these anthracyclines have the same triplet yield value [19, 201, shows that both radiative and non-radiative S1 decay pathways have virtually identical rates for 1 and 11.Similarly the observation that the substitution of iodine in the sugar moiety of 11 brings about a negligible shortening in lifetime, eg. T= 0.98 rt 0.01 ns for 12 W. T= 1.04f0.02 ns for 11, agrees with the absence of any heavy atom effect, as already ascertained from comparative measurements of relative fluorescence yield values [3]. For 4-demethoxydaunomycin (2), we observe a substantially longer fluorescence decay time than for 1 which might originate from the removal of the basic ethereal OCHa group [31], whose presence in 1 may contribute to fluorescence quenching via an intramolecular H+ transfer from the protonated amino sugar. The decay kinetics of daunomycinone (3) are very similar to those of the parent glycoside (1) in methanol; the rationale for the appearance of a long lifetime (2.66 ns and 2.44 ns for 3 and 1 respectively) may parallel our interpretation of the lengthening of both S1 and T1 lifetimes when the protonated amino group in the sugar moiety is either removed or neutralized [19, 20,251. For instance, we have measured a fluorescence decay component with a lifetime of 2.73rt0.22 ns for 1 externally bound to DNA, when the sugar ammonium ion of daunomycin is engaged in an ionic bond with a DNA backbone phosphate group. The T1 lifetime of 1, which is equal to 1.80f0.09 ps in NTflushed phosphate buffer at
326 pH 7.4, becomes three times longer at pH 9, i.e. on deprotonation of the sugar nitrogen. Furthermore, we determined the Ti lifetime of 3 which, being the aglycone of 1, obviously lacks the ammonium ion, and found the relatively long value of 7.00 * 0.35 ps in Nz-flushed benzene. Under the same conditions, we measured a similar value for daunomycin-N-trifluoro-acetamide in which the sugar amino group is substituted by the less basic -NHCOCF3. From all of these observations, we concluded that a sugar-to-anthraquinone proton transfer must be an efficient quenching mechanism acting on both excited singlet and triplet states. The concomitant presence of imino and amino substituents in the daunomycinone chromophore results in a dramatic quenching of the Sr state, as revealed by the short fluorescence decay times measured for compounds 7 and 8: 0.3450.04 ns. Before speculating on the possible mechanism of quenching, it is worthwhile to analyse the effects of the two substituents separately. The bi-exponential fluorescence decays of the 11-amino and 6-amino derivatives of 4demethoxydaunomycin, i.e. 6 and 10 respectively, are well fitted by components with very similar lifetimes (see Table 2 for typical results) which are both much shorter than that of the single-exponential decay of the parent compound (2). The same lifetimes also fit the fluorescence decay of compound 5 with initial amplitudes that, for all compounds excited at 580 nm, do not depend on the observation wavelength. These properties strongly suggest that the two lifetimes reflect a mixture of two ground state populations which do not interact in either the So or Sr state, and which we may tentatively identify as two structures differing by the extent of hydrogen bonding between NH2 and the adjacent carbonyl group. The observation that UV excitation of compounds 5, 6 and 10 produces fluorescence peaking at the wavelengths listed in Table 1 and blue-shifted fluorescence also agrees with the presence of two ground state populations. Furthermore, the fact that the relative weights of the two decay components are similar for compounds 5 and 6 indicates that, whatever the reason for the splitting of the ground state population, it is not affected by the methoxy group at C4. Since the only difference between the fluorescence decays of 6 and 10 is in the pre-exponential factors Ai and AZ, we believe that the position of the amino substituent simply affects the relative balance between the two populations [32] but not the Si state quenching mechanisms. Compound 9 is the only compound which exhibits dependence of the relative initial amplitudes of the two decay components on the observation wavelength, which agrees with our previous interpretation based on the acid-base properties of the imino group at C5 [25]. Further measurements of time-resolved fluorescence should be carried out on compounds 5, 6, 9 and 11 at different pH values to substantiate the interpretation of their fluorescence decay data. Finally, it is worth noting that the fluorescence decay time measured for the diaminosubstituted anthracenedione (4) agrees with that recently reported, though at different excitation and observation wavelengths, by Lin and Struve [28]; in addition the Sr quenching mechanisms invoked to interpret our data are based on interactions between the anthraquinone chromophore and either nitrogen substituents or hydrogen-bonding solvents which all previous reports on the S&i, spectral and kinetic properties of anthracenediones have demonstrated [25-281.
Acknowledgments
We are indebted to Dr. V. Malatesta (Istituto “G. Donegani”, Novara, Italy) for useful discussions and HPLC measurements and to Dr. A. Suarato (Farmitalia-Carlo Erba, Milano, Italy) for supplying the amino-substituted anthracyclines. We are also
327
very grateful to PTI (London, Ont., Canada) and to Dr. R. H. Pottier for cooperation in the use of the IS-1 nanosecond lifetime system which was installed at the Royal Military College (Kingston, Ont., Canada) on the occasion of the NATO-AS “Photobiological Techniques”, 1-14 July, 1990. This work was partially supported by the targeted projects “Electra-optical Technologies” and “Bio-technologies and Bioinstrumentation” of the Italian National Research Council.
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