Fluorescence of 1,4-Dihydropyridine Derivatives Relevant to Age Pigments. KIYOMI KIKUGAWA,* TAKAMI NAKAHARA and KEIZO SAKURAI

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1 4656 Vol. 35 (1987) Chem. Pharm. Bull. 35(11) (1987) Fluorescence of 1,4-Dihydropyridine Derivatives Relevant to Age Pigments KIYOMI KIKUGAWA,* TAKAMI NAKAHARA and KEIZO SAKURAI Tokyo College of Pharmacy, Horinouchi, Hachioji, Tokyo , Japan (Received May 6, 1987) 1,4-Disubstituted-1,4-dihydropyridine-3,5-dicarbaldehydes, models of fluorogens in age pigments, show a high intensity of fluorescence comparable to that of quinine sulfate. Relationships between the structures and fluorescence emission of the 1,4-dihydropyridine derivatives were studied. Loss of substituents at the 1- or 4-positions had little effect on the fluorescence. Reduction of one of the aldehyde groups to an alcoholic group greatly decreased the fluorescence intensity. When the aldehyde groups were replaced by acetyl groups, the fluorescence was greatly decreased. A derivative without aldehyde or carbonyl groups at the 3- and 5-positions was virtually nonfluorescent. Thus, the aldehyde groups at the 3- and 5-positions are requisite for the high intensity of fluorescence of the 1,4-dihydropyridines. Keywords \1,4-dihydropyridine; 1,4-dihydropyridine-3,5-dicarbaldehyde; 3-hydroxymethyl-1,4-dihydropyridine-5-carbaldehyde; 3,5-diacetyl-1,4-dihydrolutidine; fluorescence; age pigment Fluorescent lipofuscin granules develop in cells or tissues as they age.1,2) It has been assumed that accumulation of age pigments is due to lipid oxidation in cells or subcellular organisms.34) Lipid oxidation produces lipid hydroperoxides which in turn degrade into many secondary products including malonaldehyde and monofunctional aldehydes.51 Among the secondary products, malonaldehyde produces fluorescence by reaction with primary amines or proteins. Two models have been proposed for the fluorogens: conjugated Schiff bases') and 1,4-disubstituted-1,4-dihydropyridine-3,5-dicarbaldehydes.7) The latter class of compounds may include the fluorogens produced in biological systems, since they can be produced under physiological conditions:7) 1,4-Disubstituted-1,4-dihydropyridine-3,5-dicarbaldehydes (I: R1, R2 = alkyl) formed by reaction of malonaldehyde and primary amines R2NH2 in the absence and presence of monofunctional aldehydes R1CHO exhibit high intensity of fluorescence, comparable to that of quinine sulfate.81 In order to obtain information of the relationship between the structures and fluorescence emission of 1,4-dihydropyridines, we prepared several 1,4-dihydropyridine derivatives to compare their fluorescence characteristics. Experimental Methods \Ultraviolet (UV) absorption spectra were measured with a Shimadzu UV-200S double beam spectrophotometer. Fluorescence spectra were taken with a Hitachi fluorescence spectrophotometer. Fluorescence intensity was measured at the concentration range in which the fluorescence linearly increased, and was compared with the intensity of 0.1 ƒêm quinine sulfate in 0.1 N sulfuric acid (excitation at 350 nm and emission at 450 nm). Relative molar intensity of fluorescence was expressed with respect to quinine sulfate. Mass spectra (MS) were taken with a Hitachi M-80 double focusing mass spectrometer. Nuclear magnetic resonance (NMR) spectra were taken in CDCl3 or d6-dimethyl sulfoxide on a JEOL PS-100 machine with tetramethylsilane as an internal standard. Column chromatographies were done with silicic acid (Mallinkrodt) or silica gel for chromatography (Wako Pure Chemical Industries, Ltd.). Thin-layer chromatography was carried out by the use of Wakogel B-O (Wako Pure Chemical Industries, Ltd.). Malonaldehyde (TMP hydrolysate) was prepared from tetramethoxypropane (TMP) according to the method described elsewhere.8)

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$4658 Vol. 35 (1987) 1,4-Dihydropyridine-3,5-dicarbaldehydes (Ia-c) \Compounds Ia \c were synthesized according to the method previously described.8d) Thus, a mixture of 0.1 M TMP hydrolysate, 0.$

3 4658 Vol. 35 (1987) 1,4-Dihydropyridine-3,5-dicarbaldehydes (Ia-c) \Compounds Ia \c were synthesized according to the method previously described.8d) Thus, a mixture of 0.1 M TMP hydrolysate, 0.05 m R1CHO and 0.05 m R2NH2 in 1 1 of 0.1 M phosphate buffer (ph 7)-methanol (1 : 1) was incubated at 37 Ž for 50 h. The mixture was extracted with chloroform (for Ia and Ic) or ethyl acetate (for Ib) in the presence of sodium chloride. The extract was evaporated to dryness in vacuo, and the residue was crystallized (Ia) or purified by column chromatography (Ib and Ic). For column chromatography, the residue was applied to a column (3.2 ~ 35 cm) of silica gel and eluted with chloroform or a chloroform-methanol system. The fluorescent fractions were collected and evaporated in vacuo, and the residue was crystallized (Table I). 1,4-Dimethyl-3-hydroxymethyl-1,4-dihydropyridine-5-carbaldehyde (Ha) A mixture of 3.0 g of I (R1= 11.2 = CH3)8a) and 3.0 g of sodium borohydride in 300 ml of methanol was kept at room temperature overnight. The mixture was evaporated to a small volume and extracted with 200 ml of chloroform twice. The extract was evaporated to dryness in vacuo and the residue was applied to a column (2.8 ~ 10 cm) of silicic acid. The column was eluted with chloroform and successively with chloroform-methanol (49 : 1). The green fluorescent fractions were collected and evaporated to dryness in vacuo. The residue was dissolved in 5 ml of methanol and applied to 15 thinlayer plates (20 ~ 20 cm). The plates were developed with chloroform-methanol (9 : 1). The green fluorescent zones, whose RI. value (0.65) was smaller than that of the starting compound (Rf 0.80), were excised and extracted with chloroform-methanol (8 : 2). The extract was evaporated to dryness and kept at - 20 Ž. The solid material IIa was obtained (Table I). 3,5-Diacetyl-1,4-dihydrolutidines (IIIa-d) Compounds I IIa \d were prepared by Nash's method.9) A mixture of 0.1 M acetylacetone, 0.05 m ammonium acetate and 0.05 M R, CHO in 1 1 of 0.08 M phosphate buffer (ph 7) was incubated at 37 Ž for 50 h. The products were extracted with ethyl acetate (for I IIa and I I1b) or chloroform (I I Ic and IIId), the extract was evaporated to dryness in vacuo and the residue was crystallized (Table I). 1-Phenyl-2-propyl-3,5-diethy1-1,4-dihydropyridine (IVa) \ \Compound IVa was synthesized by the reaction of 1- butanal and aniline in acetic acid-water according to the method described previously." by 2.5 mmhg C (ref. 10; by 0.5 mmhg 125 Ž). MS m/z (rel intensity); M (4), M [propyl] 212 (100). Results and Discussion The structures of 1,4-dihydropyridine derivatives prepared for comparison of fluorescence are shown in Chart 1. 1,4-Dihydropyridine-3,5-dicarbaldehydes without a substituent at the 4-position (Ia) or the 1-position (Ib), and the derivative in which the alkyl substituent at the 1-position was replaced by an aryl substituent (Ic) were prepared according to the previously reported method8d) (Table I). Compound Ic revealed the same fluorescence spectrum as the previously reported 1,4-disubstituted derivatives I8c,d) (Table II). While Ib showed the same fluorescence spectrum, Ia showed excitation and emission maxima shifted to slightly higher wavelength. The fluorescence intensities of Ia \c were comparable to that of quinine sulfate as well as those of the previously reported 1,4-disubstituted compounds I,8c,d) although the intensity of Ia was somewhat lower. When 1,4-dimethyl-1,4-dihydropyridine- 3,5-dicarbaldehyde I (R, = R2 = CH) was reduced with sodium borohydride, compound IIa in which one of the aldehyde groups was converted into an alcoholic group was obtained (Table I). The fluorescence intensity of IIa was one-thousandth of that of the original compound, although the wavelengths of maximum excitation and emission were little changed (Table II). IVa Chart 1

$No. 11 4659 TABLE II. Spectral Data for I, II, III and IV 3,5-Diacetyl-1,4-difiydrolutidines (IIIa \d) were prepared according to the method of Nash9) (Table I).$

4 No TABLE II. Spectral Data for I, II, III and IV 3,5-Diacetyl-1,4-difiydrolutidines (IIIa \d) were prepared according to the method of Nash9) (Table I). Among them, IIIa has no substituents at the 1- and 4-positions, and IIIb \d have an alkyl group at the 4-position and no substituent at the 1-position. Compounds IIIa \d emitted weaker fluorescence than I did. While IIIa showed fluorescence similar to those of I, III \d exhibited excitation and emission maxima shifted to slightly higher wavelength. The fluorescence intensity of IIIa was about 5% of that of quinine sulfate, but those of IIIb \d were less than 0.3% of the intensity of quinine sulfate (Table II). The 1,4-dihydropyridines without carbonyl or aldehyde groups at the 3- and 5-positions (IVa)10) emitted virtually no fluorescence (Table II). From these results, it is concluded that the high intensity of fluorescence of I was caused by the presence of aldehyde groups at the 3- and 5-positions, and the substituents at the 1- or 4-position did not greatly affect the fluorescence intensity. In earlier studies it has been shown that 1,4-dihydropyridine derivatives usually fluoresce.13) 3,5-Diacetyl-l,4-dihydrolutidines9) and 3,5-diacety1-1,4-dihydropyridines14) are fluorescent. From the present experiments, it was found that 1,4-dihydropyridine-3,5- dicarbaldehydes I showed much higher intensity of fluorescence than other 1,4-dihydropyridines. The aldehyde and carbonyl groups at the 3- and 5-positions of 1,4-dihydropyridines contributed to the fluorescence emission, and in particular the aldehyde groups were requisite for the high intensity of fluorescence. It is interesting that the fluorescence intensity of I was diminished by borohydride treatment,8c,d) which resulted in the formation of II with much lower intensity of fluorescence. The fluorescence of polylysine modified with malonaldehyde was lost after treatment with borohydride,15) which presumably reduced the aldehyde group of the 1,4-dihydropyridine-3,5-dicarbaldehyde fluorogens. References 1) B. L. Strehler, "Adv. Gerontol. Res.," Vol. 1, ed. by B. L. Strehler, Academic Press, New York, 1964, p ) M. Elleder, "Age Pigments," ed. by R. S. Sohal, Elsevier/North-Holland Biomedical Press, Amsterdam, 1981, p ) K. S. Chio, R. Reiss, B. Fletcher and A. L. Tappel, Science, 166, 1535 (1969).

4660 Vol. 35 (1987) 4) C. J. Dillard and A. L. Tappel, Lipids, 6, 715 (1971). 5) E. N. Frankel, Prog. Lipid Res., 22, 1 (1982). 6) A. L. Tappel, "Free Radicals in Biology," Vol. IV, ed. by W. A. Pryor, Academic Press, New York, 1980, p.

5 4660 Vol. 35 (1987) 4) C. J. Dillard and A. L. Tappel, Lipids, 6, 715 (1971). 5) E. N. Frankel, Prog. Lipid Res., 22, 1 (1982). 6) A. L. Tappel, "Free Radicals in Biology," Vol. IV, ed. by W. A. Pryor, Academic Press, New York, 1980, p. 1. 7) K. Kikugawa, Adv. Free Radical Biol. Med., 2, 389 (1986). 8) a) K. Kikugawa, T. Maruyama, Y. Machida and T. Kurechi, Chem. Pharm. Bull., 29, 1423 (1981); b) K. Kikugawa, Y. Machida, M. Kida and T. Kurechi, Chem. Pharm. Bull., 29, 3003 (1981); c) K. Kikugawa and Y. Ido, Lipids, 19, 600 (1984); d) K. Kikugawa, Y. Ido and A. Mikami, J. Am. Oil Chem. Soc., 61, 1574 (1984). 9) T. Nash, Biochem. J., 55, 416 (1953). 10) D. Craig, L. Schaefgen and W. P. Tyler, J. Am. Chem. Soc., 70, 1624 (1948). 11) J. M. Erikson and H. G. Biggs, J. Chem. Educ., 50, 631 (1973). 12) B. J. Compton and W. C. Purdy, Can. J. Chem., 58, 2207 (1980). 13) U. Eisner and J. Kuthan, Chem. Rev., 72, 1 (1972). 14) a) N. Sugiyama, G. Inouye and K. Ito, Bull. Chem. Soc. Jpn., 35, 927 (1962); b) Y. Kurabayashi, K. Kubota, Y. Omote and N. Sugiyama, Nippon Kagaku Zasshi, 86, 106 (1965). 15) K. Kikugawa, K. Takayanagi and S. Watanabe, Chem. Pharm. Bull., 33, 5437 (1985).

Antitumor Principles from Ginkgo biloba L.

3016 Vol. 35 (1987) Chem. Pharm. Bull. 35( 7 )3016-3020(19871 Antitumor Principles from Ginkgo biloba L. HIDER ITOKAWA,*, a NOBUO TOTSUKA,a KEISUKE NAKAHARA,a KOICHI TAKEYA,a JEAN-PIERRE LEPOITTEVIN,b