365 Nm) of SOME COUMARINS 1129

PHOTOREACTICITY (365 nm) OF SOME COUMARINS 1129

Photoreactivity (365 nm) of Some Coumarins and 4',5'-dihydro-furocoumarins with Nucleic acids

S. MARCIANI, F. DALL'ACQUA, L. GUELFI, and D. VEDALDI

Institute of Pharmaceutical Chemistry, Cattedra di Chimica farmaceutica applicata, of the Padua University, Padova (Italy)

(Z. Naturforsch. 26 b, 1129—1136 [1971] ; received June 19, 1971)

The Authors have studied the capacity to photoreact with native DNA and r-RNA using six coumarins and four 4',5'-dihydro-furo-coumarins. Six of these substances have shown a definite photoreactivity both with DNA and r-RNA, even if in a very much lower extent in respect to that of skin-photosensitizing furocoumarins. The low capacity to photoreact with nucleic acids of these substances is discussed, also in con- nection with the absence of skin-photosensitizing activity.

Behaviour of coumarins (e. g. coumarin 1) and if^Vî furocoumarins (e. g. psoralen 2) under irradiation ^^ö o ô^ô o with ultraviolet light has been studied for a long } 2 time1-8: they both give C4-cyclo-dimers, in which the 3,4-positions are involved; no dimers of furo- ou F / OH coumarins are known in which the 4 ,5 -positions ^^^ are involved. Furocoumarins irradiated in the pre- H0JJl0I0 CH HO^^XI O HOVÔ c sence of other compounds can give photo- OH adducts9'10; in recent times much study has been 3 4 5 6 given to the photoreactions between furocoumarins and pyrimidine bases, which lead to photoadducts in which either the 3,4 or the 4',5'-double bonds are r^Yî H2C— involved11-14. Formation of these photoadducts oc- ho^ûx) CH30^!j^0'^0 0 curs also when furocoumarins are added to nucleic CH3° CH3° acids (DNA, native or denatured, r-RNA) and then 7 8 9

Fig. 1. Molecular structures of the coumarin and 4',5'-dihy- drofurocoumarin derivatives used. 1 — coumarin; 2 — psoralen; 3— 7-hydroxy-coumarin (umbelliferone); 4— 7-methoxy- ?CH3 coumarin (herniarin) ; 5 — 5,7-dihydroxy-coumarin; 6 — H2C—I^TÎ H29—[Y^l 7,8-dihydroxy-coumarin (daphnetin) ; 7 — 7-hydroxy-8-meth- ^(/yôô 0 oxy-coumarin; 8 — 7,8-dimethoxy-coumarin; 9 — 4',5'-di- CH3 hydro-psoralen; 10 — 4',5'-dihydro-8-methyl-psoralen; 11 — 4',5'-dihydro-bergapten; 12 — 4',5'-dihydro-xanthotoxin. 10 11

Reprints request to Dr. S. MARCIANI, University di Padova, 8 S. MARCIANI, F. DALL'ACQUA, P. RODIGHIERO, G. CAPO- Cattedra di Chimica Farmaceutica Applicata, Via Marzolo, RALE, and G. RODIGHIERO, Gazz. chim. ital. 100, 435 5, Padua (Italien). [1970]. 1 G. CIAMICIAN and P. SILBER, Chem. Ber. 35, 4130 [1902]. 9 C. H. KRAUCH and S. FARID, Chem. Ber. 100, 1685 [1967]. 2 F. WESSELY and J. PLAICHINGER, Chem. Ber. 75, 971 10 S. FARID and C. H. KRAUCH, Radiation Research 1966, p. [1942]. 870, North Hollad, Amsterdam 1967. 3 G. RODIGHIERO and V. CAPPELLINA, Gazz. chim. ital. 91, 11 L. MUSAJO, F. BORDIN, G. CAPORALE, S. MARCIANI, and 103 [1961]. G. RIGATTI, Photochem. Photobiol. 6, 711 [1967]. 4 G. 0. SCHENCK, I. VON WILUCKI, and C. H. KRAUCH, 12 L. MUSAJO, F. BORDIN, and R. BEVILACQUA, Photochem. Chem. Ber. 95,1409 [1962]. Photobiol. 6,927 [1967]. 5 C. H. KRAUCH, S. FARID, and G. O. SCHENCK, Chem. Ber. 13 C. H. KRAUCH, D. M. KRÄMER, and A. WACKER, Photo- 99,625 [1966]. chem. Photobiol. 6, 341 [1967]. 6 H. MORRISON, H. CURTIS, and T. MCDOWELL, J. Amer. 14 F. DALL'ACQUA, S. MARCIANI, F. BORDIN, and R. BEVI- diem. Soc. 88, 5415 [1966]. LACQUA, Ricerca sei. 38, 1094 [1968]. 7 G. RODIGHIERO, F. DALL'ACQUA, and G. CHIMENTI, Ann. Chimica 58, 551 [1968]. 1130 S. MARCIANI, F. DALL'ACQUA, L. GUELFI, AND D. VEDALDI irradiated at 365 nm15_17. Furthermore, in the Coumarins and 4' ,5'-dihydro furocoumarins photoreaction with native DNA, some furocoumarins Six coumarin derivatives (3 — 8) and the 4',5'-dihy- can give also a double photoadduct with two pyri- dro-derivatives of four furocoumarins (9 —12), all pre- pared by synthesis in this Institute, were used. midine bases involving both the 3,4- and 4',5'-double They were tritiated by the WILZBACH method26, bond and forming cross-linkings between the two and the purification was achieved by a way analogous strands of DNA 18~20. to that used for the preparation of several tritiated furocoumarins27. 200 mg of each substance, together The photoreactions between furocoumarins and with 1 Ci of tritium, were sealed in a glass tube and DNA represent the molecular basis of the photobio- kept there for 2 months. After this contact the purifi- logical effects exerted by furocoumarins under ir- cation was performed in the following stages: a) dis- radiation at 365 nm 17'21. solution in 2.7 N NaOH solution and precipitation, after 2 hours, with 2.7 N HCl solution; b) crystallization Coumarins and 4/,5/-dihydrofurocoumarins have (2 times) from boiling water or from boiling water- always been found inactive from a photobiological ethyl alcohol; c) sublimation in high-vacuum and cry- point of view 22-24; on the other hand, they can react stallization from ethyl-alcohol-water 2:8 (v/v) ; d) silica-gel thin-layer preparative chromatography: Merck under irradiation giving dimers and recently PlLL- cat. 5717 plates were used; development with ethyl- SOON SONG et al. 23 suggested on the basis of the acetate-cyclohexane 2:1; elution of the substances luminescence spectroscopy and MO calculations that with absolute ethyl-alcohol and filtration through Mil- the 3,4-double bond of coumarin in the excited state lipore Mitef 5 /u filter. is somewhat more reactive than the 3,4-double bond Ultraviolet spectra and m.p. of the obtained substances were identical to those of the authentic of psoralen, also in the excited state. samples. Control of the radioactive purity was per- It appeared therefore interesting to study the capa- formed using analytical silica-gel thin-layer chromato- plates (Merck cat. 5715; development with ethyl-ace- city of some coumarins to photoreact with nucleic tate-cyclohexane 3:1): the entire radioactivity was acids, in particular with native DNA and with localized in the single fluorescent band (characteristic r-RNA. The results obtained using six coumarin of the substances) which was present in the plates. derivatives (3 — 8) and four 4',5'-dihydro-furocou- Specific radioactivity (dpm/mmole x 109) : 7-hydro- marins (9 —12) showed that these compounds have xy-coumarin 12.3; 7-methoxycoumarin 1.61; 5,7-dihydroxy-coumarin 7.31; 7,8-dihydroxycoumarin 5.69; much less capacity to photoreact both with DNA and 7-hydroxy-8-methoxycoumarin 3.41; 7,8-dimethoxy- with RNA than furocoumarins. This fact is discussed coumarin 5.47; 4',5'-dihydropsoralen 3.32; 4',5'-dihy- from a photochemical and photobiological point of dro-8-methylpsoralen 1.91; 4',5'-dihydro-5-methoxypsoralen 6.08; 4',5'-dihydro-8-methoxypsoralen 3.18. view. Radioactivity measurements A liquid scintillation counting system Beckman Materials and methods LS 150 was used. For determination of the radioactivity of pure coumarins and 4',5'-dihydrofurocoumarins, Nucleic acids substances were used in dioxane solutions which were added in measured amounts to 10 ml of dioxane scintil- Native calf-thymus DNA, highly polimerized, from lator (4 g P.P.O., 0.075 g P.O.P.O.P. and 120 g naph- Mann Research Laboratories (New York) was used; thalene dissolved in dioxane up to 1000 ml of solu- its hypochromicity was higher than 40 percent. tion) . For nucleic acids, 0.2 ml of their aqueous solu- Ribosomal RNA, highly polymerized, from Calbio- tion was diluted with 1 ml of water and then added to chem (Los Angeles), was extracted from yeast. 10 ml of dioxane scintillator.

15 L. MUSAJO, G. RODIGHIERO, and F. DALL'ACQUA, Ex- 21 G. RODIGHIERO, L. MUSAJO, F. DALL'ACQUA, S. MAR- perientia [Basel] 21,24 [1965]. CIANI, G. CAPORALE, and L. CIAVATTA, Biochim. biophy- 16 L. MUSAJO, G. RODIGHIERO, A. BRECCIA, F. DALL'ACQUA, sica Acta [Amsterdam] 217,40 [1970]. and G. MALESANI, Photochem. Photobiol. 5, 739 [1966]. 22 L. MUSAJO. G. RODIGHIERO, and G. CAPORALE, Bull. Soc. 17 L.. MUSAJO and G. RODIGHIERO, Photochem. Photobiol. 11, chim. biol. 36, 1213 [1954]. 27 [1970]. 23 L. MUSAJO and G. RODIGHIERO, Experientia [Basel] 18, 18 F. DALL'ACQUA. S. MARCIANI, and G. RODIGHIERO, FEBS 153 [1962]. Letters 9, 121 [1970]. 24 M. A. PATHAK, J. H. FELLMAN, and K. D. KAUFMAN, J. 19 R. S. COLE, Biochim. biophysica Acta [Amsterdam] 217, Invest. Dermat. 35, 165 [I960]. 30 [1970]. 25 PILL-SOON SONG et al., Photochem. Photobiol. 14, 521 20 F. DALL'ACQUA, S. MARCIANI, L. CIAVATTA, and G. [1971], RODIGHIERO, Z. Naturforsch. 26 b, 561 [1971]. 26 K. E. WILZBACH, J. Amer. diem. Soc. 79, 1013 [1957]. 27 S. MARCIANI, F. DALL'ACQUA, and C. COLOMBINI, Ann. Chimica 59, 1067 [1969]. PHOTOREACTICITY (365 nm) OF SOME COUMARINS 1131

Solubilitiy of the substances in water and in 0.1% After irradiation, nucleic acid was precipitated from aqueous solution of DNA the solution with absolute ethyl alcohol, washed with 80% ethyl alcohol and redissolved in 2 ml of water, as To samples of 8 ml of water (or of a 0.1% aqueous described in previous papers 16' 21> 2fl> 30. The solutions solution of DNA, containing 2 MM NaCl) small amounts so obtained were utilized for the determination of the of concentrated alcoholic solutions of the various label- radioactivity and, from this, for the calculation of the led coumarins and 4',5'-dihydro-furocoumarins were amount of substances linked to the nucleic acids. added until a noticeable precipitation of the substances For the quantum-yield determination, the irradiation occurred. Small amounts (1 — 3 mg) of the solid sub- at 365 nm was performed with a Baush and Lomb high- stances were further added to the samples and then intensity grating monochromator, provided with a they were kept in a thermostatic bath (25°C±0.01) super-pressure short arc lamp. The intensity of the and gently shaken for 5 hours. After this, 4 ml of each radiation beam was determined by use of a thermo- suspension were filtered various times through a Milli- pile Kipp and Zonen "Microva" Type AL 4. The solu- pore SM 5 p membrane and discarded (it is known tions were irradiated into a spectrophotometric quartz in fact that such filtering membranes can adsorb small cuvette having an optical path of 1 cm. Quantum yields amounts of the dissolved substances). The remaining were calculated as the ratio between the number of 4 ml were then filtered through the same saturated furocoumarin molecules linked to the nucleic acid and membrane and utilized for the radioactivity measure- the number of quanta absorbed by the solution in a ments, by means of which the concentrations of the determined period of time (corresponding to the ab- substances were calculated. sorption of 6 x 1018 quanta).

Spectrophotometric determinations Results and discussion The ultraviolet absorption spectra of aqueous solutions of the substances, alone or in the presence of Photoreactions with native DNA and with ribosomal DNA, between 300 and 390 nm were performed using RNA a Perkin Elmer Mod. 124 double beam spectrophoto- meter, provided with a recorder. The extinction values Six oxygenated coumarin derivatives and four of the solutions at the Amax were then more accurately 4',5'-dihydro-furocoumarins were studied; the latter determined using an Optica CF4 single-beam spectro- compounds are only in form similar to furocouma- photometer. rins; actually, saturation of the furanic double bond Photoreactions with native DNA or r-RNA-Irradiation leaves only the coumarin nucleus as the aromatic procedure moiety and therefore their photo-chemical properties will be more likely those of coumarin derivatives To 25 ml of an aqueous 0.1% solution of DNA or RNA containing 2 MM NaCl were added small amounts than those of furocoumarin ones. of concentrated alcoholic solutions of the substances In studying the photoreactions of coumarin- and (coumarins and 4',5'-dihydro-furocoumarins) to give 4/,5'-dihydro-furocoumarin derivatives with nucleic final concentrations of 20 /ig/ml. The solutions were acids (DNA and r-RNA) we have followed the gently shaken at room temperature for 20 min. and then filtered through a Millipore SM 5 p, membrane, procedure at other times already used when stu- discarding the first 10 ml of the filtered solutions. dying the photoreactions between furocoumarins and 2 ml of the solutions so obtained were irradiated DNA26' 21>29' 30. The substances were previously into glass calibrated tubes, 12 mm in diameter, immer- labelled with tritium (see section Materials and sed in a small cell with glass walls, in which thermo- Methods); the tritiated compounds were added statically controlled water circulated. Irradiation was carried out by means of two HPW 125 Philips lamps, (20 yUg/ml) to aqueous solutions (0.1%) of DNA or which emit almost exclusively at 365 nm, placed one RNA and irradiated in standard conditions for in- on each side of the cell at a distance of 3.5 cm. The creasing periods at a controlled temperature; sub- total incident radiation on the 2 ml of the solutions stances which did not bind to the nucleic acids were was equivalent to 2.9 x 1016 quanta/sec [determined by means of 0.15 M potassium ferrioxalate chemical actino- then separated by precipitation of the nucleic acids meter 28]. from the solutions by means of ethyl alcohol *;

28 C. G. HATCHARD and C. A. PARKER, Proc. Roy. Soc. [Lon- * Other researchers have worked out this separation by means don] , Ser. B. 235, 518 [1956]. of gel-filtration 13> 31. Precipitation with ethyl alcohol, much 29 F. DALL'ACQUA, S. MARCIANI, and G. RODIGHIERO, Z. Na- more simple to work out, leads also to a complete separa- turforsch. 24 b, 307 [1969]. tion of the nucleic acid from the non-covalently bound 30 F. DALL'ACQUA, S. MARCIANI, and G. RODIGHIERO, Z. Na- furocoumarin or coumarin derivatives, with constantly re- turforsch. 24 b, 667 [1969]. producible results. 31 M. A. PATHAK and D. M. KRÄMER, Biochim. biophysica Acta [Amsterdam] 195,197 [1969]. 1132 S. MARCIANI, F. DALL'ACQUA, L. GUELFI, AND D. VEDALDI finally the acquired radioactivity of the nucleic acids DNA 29. Therefore, in the present study irradiations was measured and on this basis the amounts of the have always been performed at controlled tempera- bound coumarin derivatives were calculated. tures and we have worked out two series of experi- In a previous study on the photoreactions between ments, one at 22 °C and another at 2 °C. certain furocoumarins and DNA30 we have found The results obtained are reported in Figs. 2, 3 that 365 nm radiation, although not strongly ab- and 4. In these Figs., as a reference, are reported sorbed by furocoumarins, gives the highest quan- also the results obtained with psoralen, of course tum yield; by decreasing the wavelength of the in the same experimental conditions. As is shown, radiation, going to spectral regions which cor- the photoreactivity of the various coumarins and respond to a maximum of absorption of furo- dihydro-furocoumarins is always very much lower coumarins, quantum yield strongly decreases, very than that of psoralen, both in respect to DNA and probably as a consequence of a partial absorption to RNA. of the radiations by DNA. Like furocoumarins, 7-hydroxy-coumarin derivatives (all our compounds belong to this type) also generally have u.v. ab- ,7.8-dimethoxy coumarin sorption spectra which show a large band at wave- (*)< 7.8-dihydroxycoumarin [7-hydroxy-8-methoxycoumarin lengths longer than 300 nm. Therefore, in the present experiments we have used the same 365 nm radiation, already used for studying the photoreactions of furocoumarins. The molar extinction coefficients of the various substances at this wavelength are shown in Table II. Furthermore, we have previously observed that the temperature at which irradiation is performed can have an influence on the rate of the photoreactions, in particular on the photoreactions between some furocoumarins and RNA or denatured

7-hydroxxcqumarjp „ fi "ijVdijwdroxycoumarin |/xf> - 20 isfegase t " 4 L (*> (1) 7-hydroxy-8-methoxycoumarin 20 L0 60 80 time of irradiation (minutes) (2) 7.8-dihydroxycoumarin Fig. 3. Photoreactions (365 nm) of some coumarin and di- hvdro-furocoumarin derivatives with r-RNA by irradiation at "5 15 ,ora*en 22 °C.

Among the substances used, all four 4',5'-dihydro-furocoumarins and moreover 7-hydroxy- and 2 10 7-methoxycoumarin have little, but well-defined photoreactivity, both with DNA and with RNA,

-»«CO««»® while the photoreactivity of the 5,7- and 7,8-dioxy- gfess derivatives is fairly insignificant. In previous studies21'29 we have found that photoreactivity of furocoumarins with DNA is al- - -•^^dimeUjOxyc»'" ways higher than that with RNA. In the present

20 40 60 80 case the situation is not so well-defined; in fact time of irradiation (minutes) 4/,5'-dihydro-psoralen and 4',5'-dihydro-8-methyl- Fig. 2. Photoreactions (365 nm) of some coumarin and 4',5'- psoralen photoreact more with RNA than with (lihydro-furocoumarin derivatives with native DNA by irradia- DNA, while the other substances behave in the op- tion at 22 °C. The results obtained by irradiation at 2 °C were rather the same. posite way. PHOTOREACTICITY (365 nm) OF SOME COUMARINS 1133

Free coumarin + nucleic acid ^ molecular complex a | hv 365 nm b \ hv 365 nm -7,8-dimethoxycou marin Photodimers Covalent binding of (*)

Furthermore, the results obtained show that in Compounds Initial rate constants X 10~3 X min-1 the photoreactions with DNA temperature of irra- DNA RNA RNA diation has practically no influence; they were more at 22°C* at 22°C at 2°C or less the same at 22° and at 2 °C. Towards RNA too, no influence was observed on the photoreacti- 7-hydroxy-coumarin 0.91 0.28 0.28 7-methoxy-coumarin 2.10 1.03 1.04 / vity of 4',5 -dihydro-psoralen, 7-hydroxy- and 4',5'-dihydro-psoralen 0.72 3.26 3.33 7-methoxy-coumarin; by contrast, photoreactivity 4',5'-dihydro-8-methyl- 3.50 4.53 5.25 psoralen / / of 4 ,5'-dihydro-8-methyl-psoralen, 4 ,5'-dihydro- 4',5'-dihydro-bergapten 3.23 2.30 4.22 xanthotoxin and 4',5'-dihydro-bergapten was a little 4',5'-dihydro-xanthotoxin 1.90 0.46 1.54 higher when irradiation was performed at 2 °C than Table I. Initial rate constants of the photoreactions (365 nm) at 22 °C. In a previous study29 it was demonstrated between some coumarins and 4',5'-dihydro-furocoumarins with that the increase of photoreactivity at low tempera- native DNA and with r-RNA. * Rate constant values of the photoreactions with DNA by irradiation at 2 °C were rather ture showed by some furocoumarins towards RNA the same. and denatured DNA was due to an increase of the formation of molecular complexes with these nucleic Quantum yields of the photoreactions acids (see later for other comments). We think that The low photoreactivity with nucleic acids showed in the present case also this fact can be attributed by coumarin derivatives and by the 4',5'-dihydro- to the same cause. furocoumarins is a little surprising after the results

obtained by studying their C4-cyclo-dimerization Rate constants of the photoreactions and the theoretical calculations of PILL-SOON SONG on the coumarin and psoralen molecules 25. 21 In a previous paper we analyzed the system To ascertain the importance of the absorption formed by an aqueous solution of DNA and a furo- factor at 365 nm in determining this low photo- coumarin and the various reactions which can take reactivity, we have determined for the various sub- place when the system is irradiated. In the present stances the molar extinction coefficients at 365 nm case the situation is similar, although not identical, (see Table II) and the quantum yields of their and can be summarized as follows: photobinding reactions to DNA. 1134 S. MARCIANI, F. DALL'ACQUA, L. GUELFI, AND D. VEDALDI

Compounds values at X max e values at 365 nm

A max* e values e values A e in water in 0.1% DNA [%] in water in 0.1% DNA solution solution

7-hydroxy-coumarin 324 14400 13610 5.2 480 650 7-methoxy-coumarin 323 12190 11100 8.9 445 525 5,7-dihydroxy-coumarin 327 14900 12300 17.4 2720 2650 7,8-dihydroxy-coumarin 323 11400 10020 12.2 1085 1090 7-hydroxy-8-methoxy-coumarin 322 12175 11500 5.5 415 430 7,8-dimethoxy-coumarin 320 11600 11460 1.2 365 375 4/,5'-dihydro-psoralen 335 13950 12850 8.0 3140 3160 4',5'-dihydro-8-methyl-psoralen 336 14500 11650 19.6 4200 4380 4',5'-dihydro-bergapten 331 14520 10700 26.2 3541 4130 4/,5/-diliydro-xanthotoxin 334 14700 13190 10.5 2740 2725

Table II. Spectrophotometric properties of coumarins and 4',5'-dihydro-furocoumarins and modifications induced by the presence of DNA. * Practically no shifts of the Amax were observed in the presence of DNA.

The quantum yield values were as follows Formation of molecular complexes ( x 10-4) : 7-hydroxy-coumarin 3.4; 7-methoxy- ooumarin 7.9; 4',5/-dihydro-psoralen 1.1; 4',5'-di- In previous papers we have found that furo- hydro-8-methyl-psoralen 3.7; 4',5'-dihydro-bergap- coumarins can form molecular complexes with ten 3.6; 4/,5'-dihydro-xanthotoxin 2.5. As can we nucleic acids and especially with native DNA 32' 33. see the quantum yield values are always very low, Various experimental results indicate that an inter- in the range of 10~4, that is at least ten times less calation of the planar furocoumarin molecules be- than the quantum yields of the photoreactions between two base pairs of the double stranded DNA tween furocoumarins and native DNA. This indi- takes place. This intercalation appears to be a cates an effectively low ability of the substances to very suitable condition for the photoreaction of photoreact. furocoumarins with the pyrimidine bases of DNA. Concerning the four coumarin-derivatives which Therefore, the extent of complex formation has practitally do not photoreact, for which we did not generally a remarkable influence on the rate of calculate the quantum yields, we can see from photoreaction; very recently we have found that the Table III that only 7-hydroxy-8-methoxy-couma- very much higher photoreactivity with DNA shown rin and 7,8-dihydroxy-coumarin have small values by some methyl-derivatives of psoralen in respect of the molar extinction coefficient at 365 nm; these to psoralen itself, is due, at least in the main part, 20 values, however, are in the same range as those of to a greater ability to form such complexes . 7-hydroxy- and 7-methoxy-coumarin, which have a It has been reported 32 that the viscosity of DNA- significant photoreactivity. solutions increases by addition of some furocouma-

Compounds Solubility Solubilitv Amounts solu- [•B] in water bilized more in 0.1% DNA [A] solution than in water l>g/ml] [^g/ml] l>g/mll [A] [•B]

7-hvdroxy-coumarin 222.4 288.2 65.8 0.295 4',5'-dihydro-psoralen 52.1 55.6 3.5 0.067 4',5'-dihydro-8-methyl-psoralen 38.5 57.1 18.6 0.493 4',5'-dihydro-bergapten 34.0 75.5 41.5 1.220 4',5'-dihydro-xanthotoxin 90.7 145.4 54.7 0.602

Table III. Solubility in water and in 0.1% DNA aqueous solution (25 °C) of 7-hydroxy-coumarin and of the 4',5'-dihydro-furocoumarin derivatives.

32 G. RODIGHIERO, G. CAPORALE, and T. DOLCHER, Rend. 33 F. DALL'ACQUA and G. RODIGHIERO, Rend. Accad. Naz. Accad. Naz. Lincei 30, 84 [1961]. Linczi, 40, 411 [1966]. PHOTOREACTICITY (365 nm) OF SOME COUMARINS 1135 rin and coumarin-derivatives, as a consequence of range as or higher than that obtained for psoralen the complex formation; this increase, however, is (0.49) in the same experimental conditions. definitely smaller with coumarins than with furocoumarins. 0.6 (a) (b) Considering the importance that this factor may exert on the rate of the photoreactions, we have worked out some experiments for studying the complex formation ability of the substances used in the 0.4 . \ present research. • rM We have firstly determined the modifications of \ the spectrophotometric properties of the substances / / \ provoked by the presence of the nucleic acids in the 0.2 solutions. i \\\ - \ - / / \\ Generally, when a small molecule is complexed with a macromolecule, its molar extinction coefficient decreases and there is a shift of the absorp- \ \ tion bands towards longer wavelengths. As appears 1 1 1 i i S— i 300 330 360 390 300 330 360 390 from Fig. 5, in which are reported the u.v. absorp- nm tion spectra of 4',5'-dihydro-bergapten in the ab- Fig. 5. Absorption spectra of 4',5'-dihydrobergapten (8.22 /ug/ sence and in the presence of DNA, the absorption ml) (part a) and of 7,8-dimethoxycoumarin (10.6 pg/ml) (part b) in water and in 0.1% DNA aqueous solu- band at 331 nm is decreased in the presence of DNA; tion . practically no shift at lmax is present. The u.v. spectrum of 7,8-dimethoxy-coumarin in contrast is Therefore we must conclude that the low photo- very much less modified by the presence of DNA. reactivity of coumarin and 4',5'-dihydrofurocouma- In Table II, are reported the variations of the rin derivatives with nucleic acids cannot in general £max for the various substances used in this study find a full explanation in the results now obtained occurring in the presence of DNA. No variations about the complex formation. These results can con-

of the ^max were observed in any case. The extent of tribute to the explanation of only some particular the decrease of the e value can be assumed as an aspects; for instance, the very low photoactivity of indication of the extent of the complex formation. 4',5'-dihydro-psoralen with DNA and the relatively Furthermore, for the more significant substances higher one of 4',5'-dihydro-Jbergapten (it is to be we have determined the solubility in water and in a noted that, by contrast, psoralen is much more 0.1% solution of DNA; this method has already photoreactive than bergapten) find a good parallel frequently been used by other researchers 34-36 and in the complex formation ability of the two sub- by us21'33 for studying the complex formation; in stances, very low in the first case, higher in the fact the amount of substance which is more solubi- second. lized than in water is considered bound to DNA in the molecular complex.. The results obtained are Conclusion reported in Table III. From all these results it appears that only 7,8-di- Binding capacity to the nucleic acids under ir- methoxy-coumarin has a very small ability to form radiation at 365 nm exerted by a group of couma- complexes with DNA; the practical absence of rin- and 4',5'-dihydro-furocoumarin derivatives ap- photoreactivity of this substance could be explained pears to be markedly smaller than that generally in this way. However, all other substances are able exerted by furocoumarins. Although no direct in- to form complexes; the values obtained for the ratio vestigations have as yet been made, we can easily [bound substance]. assume that the photobinding reaction in the case in some cases were in the same [free substance] of coumarins is analogous to that of furocoumarins,

34 E. BOYLAND and B. GREEN, Brit. J. Cancer 16, 347, 507 36 J. K. BOLL, J. A. MCCARTER, and M. F. SMITH, Biochim. [1962]. biophysica Acta [Amsterdam] 103, 275 [1965]. 35 A. M. LIQUORI, B. DE LERMA, F. ASCOLI, C. BOTRE, and M. TRASCIATTI, J. molecular Biol. 5, 521 [1962]. 1136 PHOTOREACTICITY (365 nm) OF SOME COUMARINS 1136 that is a C4-cyclo-addition of the substances to the their 3,4-double bond is so for from the 5,6-double 5,6-double bond of the pyrimidine bases of the bond of a pyrimidine that photo-addition is impos- nucleic acids by means of their 3,4-double bond sible. This possibility showed up clearly from the (furocoumarins can react also with 4',5'-double use of scale molecular models of DNA and couma- bond, but of course this is not the case with the rins. For 4',5'-dihydrofurocoumarins, it is possible substances now studied). The small ability of cou- that hindrance due to the greater volume of the marin-derivatives to photobind to the nucleic acids hydrogenated 4',5'-positions prevents the molecules shown by the results of irradiation experiments and from assuming all the positions possible for furo- by the initial rate constants of the photoreactions, coumarins and suitable for the photoreaction. was confirmed by the low values of the quantum Further studies are necessary to verify these yield of the same photoreactions (in the range hypotheses. 10~4). In any case, from a photobiological point of This fact appears to be in contrast with the re- view, the low photoreactivity of coumarin- and sults obtained in studying the C4-cyclo-dimerization 4',5'-dihydro-furocoumarin derivatives appears to of some coumarins 5 7, which seems to proceed in explain why they were always found inactive when a fairly rapid way and with the results obtained they were placed on the skin and were irradiated. by means of MO calculations, which suggest that the In fact we must assume that a detectable biological 3,4-double bond of coumarin in the excited state is effect can appear only after a minimum degree of more reactive than the same bond of psoralen, also damage is produced to the cellular components. In in the excited state 25. We have tried to find an ex- the tests for evaluating the skin-photosensitizing ac- planation of the low photoreactivity of coumarins tivity this minimum amount of damage is obtained with nucleic acids studying the ability of these sub- by varying the time of irradiation; we have found stances to form complexes with DNA; in fact in the that a good correlation exists between the skin- case of furocoumarins, the preliminary formation photosensitizing activity of furocoumarins and their of such complexes is a very suitable condition for ability to photoreact with DNA. Due to the low the subsequent photoreaction. Actually, the results photoreactivity of coumarins and 4',5'-dihydro- obtained have shown that both coumarins and 4',5'- furocoumarins, very long periods of irradiation dihydrofurocoumarins were able to form complexes, would be necessary to arrive at this minimum in some instances also to a considerable extent, even amount of damage to DNA, very much longer than if the complexed molecules seemed to have a be- those usually employed for testing the activity of haviour somewhat different from that of the com- the substances on human or guinea-pig skin. For plexed furocoumarins; for instance, only a decrease instance, it was previously found that to obtain of the e values of the substances was observed in the erythema in the test conditions used for evaluating presence of DNA, but practically no shift of the the skin-photosensitizing activity on guinea-pigs, a 2max; furthermore, coumarins had a very much less time of irradiation of 27 min.37 is necessary for effect than furocoumarins in increasing the visco- psoralen. sity of DNA-solutions 32. Generally we have observed that irradiation ex- We can suggest two hypotheses, both able to experiments in which guinea-pigs were kept immo- plain the low photoreactivity with nucleic acids: bilized for more than 1 hour were very distressing a) It may be that the complexes formed bv for the animals. For 4',5'-dihydro-8-methyI-psoralen, coumarins and 4',5/-dihydro-furocoumarins with which is the most reactive of the now studied sub- DNA are of different kinds to that formed by furo- stances and in respect to DNA has a photoreaction coumarins, that is they are not due to an inter- rate constant 11 times smaller than that of psora- calation of the molecules between two base pairs len, we can calculate that a time of irradiation of of DNA. 300 min. would be necessary to produce erythema b) If, by contrast, intercalation takes place, it is on guinea-pigs under the same test conditions. possible that due to the smaller size of the coumarin This research was supported by a financial aid of molecules in respect to furocoumarins, they can as- Consiglio Nazionale delle Ricerche, Roma. We are sume a greater number of positions in the space indebted to Prof. GIOVANNI RODIGHIERO for helpful between two base-pairs of DNA, in many of them discussion and for his interest on the research.

37 G. CAPORALE, L. MUSAJO, G. RODIGHIERO, and F. BACCIC TTI, Experientia [Basel] 23,985 [1967].