ANALYST, JANUARY 1992, VOL. 117 77 Relationship Between the Structures of Dihydrofuro- and Di hydropyrano[2,3-b]quinolinium Alkaloids, Their Spectral Properties (Ultraviolet Absorption and Fluorescence) and Their Chromatographic Behaviour Monique Montagu, Genevieve Petit-Paly, Pierre Levillain, Jean-Claude Chenieux and Marc Rideau UFR Pharmacie, 37042 Tours Cedex, France Chromophores of dihydrofuro- and dihydropyrano[2,3-b]quinolinium alkaloids differ from each other only by the position of the electron-donating substituents attached to the aromatic ring. It is shown that there is a connection between the electronic absorption and the fluorescence properties (taking advantage of solvent effects) and the chromophore structure of the alkaloids. The non-chromophore part of these structures also has an influence on the chromatographic behaviour of the molecules.It is shown that the spectral and chromatographic properties not only permit the identification of known structures but are also useful for predicting the structure of new quinolinium alkaloids. An example is given for two alkaloids isolated from Ptelea trifoliata (Rutaceae). Keywords : Electronic absorption; flu o rescence-structu re relationship; 4 ua te rna r y alkaloid; dih ydro fu ro - and dih ydrop yrano[2,3-b] quinolinium The electronic absorption spectra of some dihydrofuro- and dihydropyrano[2,3-b]quinolinium compounds (extracted from a few species of Rutaceael) have been reported previously,'-h but their fluorescence properties have hitherto received little attention.For both absorption and fluor- escence, no relationship was established between spectral properties and the molecular structure in terms of various media. In a previous paper,7 we studied the electronic absorption and fluorescence of two dihydrofuro[2,3-h]quinolinium alka- loids, namely, balfourodinium and platydesminium; in par- ticular, attention was directed to the influence of various solvents and anions, and to the effect of pH on the spectra. It was shown that, although their electronic absorption spectra are, in practice, independent of the medium, their fluor- escence is generally strongly dependent on the medium.7 This investigation led to the development of spectrofluorimetric and spectrofluoridensitometric assays for these two alka- 1 o i ds .7-9 This work was extended to other quinolinium alkaloids that were extracted from Ptelea trifoliata L.and Ruta graveolens L. (Rutaceae). I n this paper it is shown how, with the aid of these spectral properties, it is possible, at an early stage of the isolation and with only a few picomoles of substance, to predict the structure of new dihydrofuro[2,3-b]quinolinium alkaloids. Experimental Equipment Fluorescence measurements were made with a Jobin-Yvon Model JY3CS spectrofluorimeter, equipped with a xenon arc source, with two monochromators, with an R-212 photomulti- plier, and with a 1 X 1 cm quartz cell. Spectra were recorded with a 4 nm bandwidth and a scan speed of 40 nm min-1: the fluorescence data are given without spectral correction. Reference Alkaloids Seven alkaloids were investigated: platydesminium perchlor- ate, balfourodinium chloride, ptelefolonium perchlorate, isoptelefolonium perchlorate, hydroxyluninium chloride, ribalinium perchlorate and rutalinium perchlorate.They had been isolated previously and analysed by proton nuclear magnetic resonance ( * H NMR) spectroscopy and mass spec- trometry (MS). 1 0 Apart from rutalinium, which has a di- hydropyrano[2,3-b]quinolinium structure [Fig. l(b)], these alkaloids have a dihydrofuro[2,3-b]quinolinium structure [Fig. l(a)]. All of these compounds have a methoxy group in position 4 of the quinolinium structure, but differ either in the nature and position of the substituents in positions 6 , 7 and 8, or in the nature of the R group, i.e., R = l-hydroxy-l- methylethyl or 1-methylethenyl (Fig. 1).CH3 OCH? (b) R Z = YCH2 CH3 Fig. 1 ( u ) Structure of dihydrofuro[2.3-b]quinolinium alkaloids: (h) structure of rutalinium; and (c) conjugation effect induced by a rnethoxy group in position 7 of the dihydrofuro[2.3-h]quinolinium struct urc78 ANALYST. JANUARY 1992, VOL. 117 Table 1 Absorption spectra of dihydrofuro- and dihydropyrano- [2.3-b]quinolinium alkaloids. Solvent: methanol Absorption maximumlnm (log E-I) substituent" 'Ch 'Bb ' L', I Lt, Alkaloid and Platydesminium perchlorate (A) R' 2 16(4.49) 236(4.52) 294( 4.04) 3 17( sh)t Balfourodinium chloride (B) R' ; -0CHj (8)s 2 14(4.40) 2S3(4.S 1 ) 30O(3.88) 32O( s h) perchlorate (C) Isoptelefolonium perchlorate (D) H ydrox y 1 unini um chloride (E) Ri balinium perchlorate (F) R1; -OH (6)s 220( 4.52) 246( 4.48) 294( 4.05) 336( 3.89) perchlorate (G) -OH (6)s 2 13(4.46) 248(4.48) 304( 3.97) 347( 3.85) * R1 = l-Hydroxy-l-methylethyl; R' = l-methylethenyl.t E = Molar absorptivity (dm' mol-1 cm-1). .$ sh = Shoulder. 9 Numbers in parentheses indicate the position of the substituent in 220(sh) Ptelefolonium R';-OCH3 (6.8)s 216(4.40) 259(4.56) 299(3.70) 345(3.48) R'; -0CH3 (7.8)s 226(4.38) ZSO(4.80) 322(4.23) R'; -0-CHz-O- (7.8)s 2 H(4.28) ZSs(4.45) 335(3.63) Rutalinium 294(sh) the alkaloid ring system. Results and Discussion Relationship Between the Chemical Structure and the Electronic Absorption of Alkaloids The spectral data (using methanol as solvent) are shown in Table 1. They are similar to those obtained in a 0.1 mol dm-3 HCI04 solution in methanol: only a weak hyperchromic effect is observed in the spectra of balfourodinium and isoptele- folonium (results not shown).Platt's nomenclature11 applied to quinoline and the quinolinium ion" was used to denote the absorptivity bands. From the electronic absorption properties, it is possible to dinstinguish the alkaloids in terms of the substituent in positions 6, 7 and 8 from the non-substituted platydesminium. Monosubstitution on the quinolinium structure in position 6 or 8 induces a bathochromic shift of the IB,,, lLZl and ILb bands. As observed by Zanker,I3 the shift of the longitudinally polarized 'Lb band is much more significant when a group is substituted in position 6 than when a group is substituted in position 8, which affects the transversally polarized IL;, band (Table 1 ; compare B with F and G).However, the influence of the nature of the substituent, all the substituents are electron- donating groups, is position-dependent: this influence is observed on comparing the spectra of balfourodinium (-OCH3 in position 8) and pteleatinium (-OH in position 8), the bathochromic effect being more significant in the latter, but it is not observed on comparing the spectra of ribalinium (-OH in position 6) and methylribalinium (-OCH3 in position 6).14 The bathochromic effect increases with the number of substituents; however, the effect is modified by the relative positions of the various groups. In particular, substitution in positions 7 and 8 induces a coalescence of the *L:, and 'Lb bands (Table 1; D and E).This is not observed on substituting in positions 6 and 8 (Table 1; C). Possible conjugation of the electrons of the methoxy group in position 7 might be responsible for this effect [Fig. l(c)]. The conjugated form can give an nn" transition, but this transition undergoes a hypsochromic shift caused by the methoxy group in position 4; this induces the coalescence of the 1L and nn* bands. Table 2 Fluorescence spectra of dihydrofuro- and dihydropyrano- [2,3-b]quinolinium alkaloids. Medium: methanol, unless indicated otherwise Exci t at ionlemission Alkaloid Substituent* wavelengthlnm (It) Platydcsminium perchlorate (A) RI 238,3041346 (20. 100) Balfourodinium chloride (B) RI 264,312,3361433 (8)s (79, 96. 100) Ptelefolonium perchlorate (C) R' 263,307,348l454 -0CH3 (6.8)s (48,56.100) 263,307,348l4061 (48,56, lOO)'11 Isoptelefolonium perchlorate (D) R' 256.3361478 -0CH3 (7,8)6 (44, 100) 256.33614787 (42. 1OO)y H ydrox y 1 un ini um chloride (E) R' 262,3401368 -0-CH?-O- (7.8) (54, 100) Ribalinium perchlorate (F) R1 250.3O2.3461405 -OH (6)s 250,302,3461520 (30.46, 100) 250,302.3461406~ (34.53, l0o)fl 250. 302,3461500, 5201 (12,19.36)1 (20,44,100) (10.22, SO) Rutalinium perchlorate (G) -OH (6)s 253,307,3561418 253,307,3561526 253,307,3561418~ (20.42, lOO)1 253.3O7.3561525~ (7.14.33)1 * RI and R' as in Table 1 . .i- I = Intensity ratio of the fluorescence bands in %. bSlnm 42 97 106 587 142 1421 128 59 174 608 741 62 70 62'11 169'11 $ 6 = Differcnce between the emission and excitation wave- $ Numbers in parentheses indicate the position of the substituent in 7 Medium: 0.1 mol dm-3 HCI04 in methanol.lengths. the alkaloid ring system. In contrast, the dihydrofuranoid and dihydropyranoid rings, cf. the auxochromic oxygen function, are not part of the chromophore itself. They can behave in the same way as auxochromic groups, and, in this instance, have some effect on the spectrum. As observed previously,15 a bathochromic shift of the ILb band occurs when the dihydrofuranoid group is replaced by the dihydropyranoid group (Table 1; F and G). Relationship Between the Chemical Structure and the Fluorescence of Alkaloids Alkaloids with two fused benzene rings (which give the molecule a certain rigidity) show a fairly high fluorescence, allowing the assay of these alkaloids to within a picomole.Moreover, with these alkaloids, the nitrogen lone pair is localized by methylation, thus preventing internal coupling between a singlet and a triplet state. Hence, phosphorescence, which usually develops at the expense of fluorescence, is inhibited. 16 The influence of four different media on the fluorescence spectra was studied. Table 2 shows that the excitation maxima in methanol, with or without HC104, are identical. (The same data were also obtained in water, with or without HCI04.) Investigation of the excitation spectra allows one to dis- tinguish between three groups of alkaloids, as follows: (1) twoANALYST, JANUARY 1992, VOL. 117 79 i; r”\ I t I \ \ ;\ ’ \ 200 400 600 I I I \ / \ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . , . . . a . . . . . . . . . . . . . . . . . . . * . * . . . . . . . . . . . . . . . . . . :: * . . . . . . . . . . . . . . .-._ a : : .. * : . : . : * . * . . . . * . : : : : *: . *..a .. r.. 200 400 600 200 400 AJnm 600 Fig. 2 Fluorescence spectra of dihydrofuro- and dihydropyrano[2.3-h]quinolinium alkaloids: ( a ) platydesminium; (b) balfourodinium; (c) ptclefolonium; ( d ) isoptelefolonium; (e) hydroxyluninium; (f) ribalinium; (8) rutalinium; and ( A ) and (i), new alkaloids. Solid line shows spectra in methanol medium; broken line shows spectra in 0.1 mol dm-3 HCIOl in methanol medium; and dotted line shows similar spectra in both media narrow well-separated bands are obtained for alkaloids substituted in positions 7 and 8 [Fig. 2 ( d ) and ( e ) ] ; (2) spectra with three bands, with the IL bands very close to each other or almost overlapping, are obtained for non-substituted alkaloids and alkaloids substituted in position 8 [Fig.2(a) and ( b ) ] ; and (3) spectra with three well-separated bands are obtained for alkaloids substituted in position 6 [Fig. 2 0 and (g)] and in positions 6 and 8 [Fig. 2(c)]. The emission maxima of the alkaloid spectra can be very different when using different media (Table 2). In methanol, the molecule with no substituent attached to the aromatic ring (platydesminium) re-emits the quasi-totality of the absorbed energy (see Table 2: the 6 value corresponds to the difference between the absorbed and re-emitted energies; when this value is low, most of the absorbed energy is re-emitted).On the other hand, as soon as the aromatic ring is substituted, the emission band undergoes a marked bathochromic shift. This effect is due to the existence of a very polar excited state, arising from a perturbation in the location of charges in these molecules. The bathochromic shift increases with the number of substituents. However, the non-radiative dissipated energy ( i . e . , the difference between the excitation and emission wavelengths) before re-emission is almost identical for molecules substituted in position 8 as for those substituted in positions 6 and 8 (Table 2; B and C). It increases noticeably for molecules substituted in positions 7 and 8 (Table 2; compare B and C with D and E). A hydroxy group in position 6 (Table 2; F and G) yields the most significant bathochromic shift (emission greater than 500 nm).It might be that the exceptional fluorescence is caused by the creation of a zwitterionic structure, which induces a consider- able lowering of the energy between the absorption and re-emission levels. 17 The energy reduction might be explained by an increase in the solvation effect of the molecule, which is more polar when in an excited state, which lowers the energy of the excited level and leads to a lower frequency re-emission. Derivatives with a hydroxy group in position 6 also have two emission bands, hence the existence of two possible energy levels in an excited state. By using 0.1 mol dm-3 HCI04 in methanol in place of methanol, it is possible to distinguish between two groups of dihydrofuro[2,3-b]quinolinium alkaloids according to their emission spectra: (1) non-substituted alkaloids or alkaloids substituted with a methoxy group in position 8 or in positions 7 and 8: at most a slight hyperchromic effect is observed [Fig.2(a), ( b ) , (d) and ( e ) ] ; and (2) alkaloids substituted in positions 6 and 8 or in position 6: the fluorescence spectra are modified [Fig. 2(c) and 01. However, for ptelefolonium [Fig. 2(c)], this modification is essentially a hypsochromic shift together with the hyperchromic effect of the emission band, while for ribalinium [Fig. 2 0 3 , with an emission spectrum having two maxima, it is the hyperchromic effect of the higher energy band. These exceptional effects can be compared with those observed for indolic compounds with a hydroxy group in the para position with respect to the nitrogen atom.18 These phenomena are strongly linked to the pK,, of the alkaloids in80 ANALYST, JANUARY 1992, VOL.117 the excited state. On the other hand, it is possible to distinguish dihydrofuro[2,3-b]quinolinium alkaloids from dihydropyrano[2,3-b]quinolinium alkaloids. Rutalinium [Fig. 2(g)], a dihydropyrano[2,3-b]quinolinium alkaloid, has a spectrum which is altered in the same way as that of ribalinium [Fig. 2 0 1 in HC104, but to a lesser extent. Indeed, for the spectra of ribalinium and rutalinium, the intensity ratios of the two emission maxima ( 1 4 0 ~ Z s 2 0 ) are increased by a factor of 2.8 and 1.2, respectively. The rigidity of the dihydrofuranoid ring makes the molecule planar, which would allow conjugation of the free electrons on the oxygen atom with the N-methyl- quinolinium structure.This conjugation is less likely to occur with the dihydropyranoid ring, which is not as rigid, whence the less marked difference between the spectra obtained in the two media. It should be noted that the variations of the observed spectra (in the HCI04-methanol medium) are due to the acidity of the medium and not to the C104- anion, as the spectra obtained in a methanolic solution of NaC10, are identical with those obtained in methanol. l y It should also be noted that the fluorescence spectra of all the alkaloids are identical in both water and 0.1 mol dm-3 HCIO, (results not shown). Relationship Between the Structure and the Chromatographic Behaviour of Alkaloids Dihydrofuro- and dihydropyrano[2,3-b]quinolinium alkaloids can be separated by reversed-phase liquid chromatography”) and thin-layer chromatography.8 In the latter instance the behaviour of these compounds on silica with the solvent system ethyl acetate-formic acid-water (10 + 1 + 1) makes it possible to discriminate between two alkaloid groups which separate in different RF zones according to the group present on the dihydrofuranic ring (RI = l-hydroxy-l-methylethyl and R’ = l-methylethenyl). The Rl-substituted alkaloids have lower RF values (RF = 0.20 k 0.05) than the RZ-substituted alkaloids (RF = 0.35 k 0.07).Application to the Determination of the Structures of Two Dihydrofuro[2,3-b]quinolinium Alkaloids Concurrently with this work, two new dihydrofuro[2,3-b]- quinolinium alkaloids were isolated, one from in vitro cultures and the other from leaf stems of Relea trifoliata (Rutaceae).It was possible to determine the structures of these two alkaloids from their absorption and fluorescence spectra without isolating them completely. An examination of the fluorescence spectra (Fig. 2 and Table 3), obtained in methanol, shows, for the alkaloid (H), isolated from cultured cells, an excitation spectrum with three bands; two IL bands are near to each other, and the Table 3 Absorption and fluorescence spectra of the dihydrofuro- [2,3-b]quinolinium alkaloids (H) and (I). Medium: methanol. unless indicated otherwise Absorption maximumlnm Alkaloid* (log E t ) (H) 2 16(4.30) 256(4.33) 299(3.55) 2 19(4.22) 261 (4.43) 336(3.52) 320w)ii (1) Excitationlemission wavelengthlnm ( I $ ) h$lnm 260,308,3331437 104 (71.90, 100) 260,308,33314398 1068 (71-93, l00)T 263,3441468 124 (44, loo) * Both alkaloids were in the form of their chlorides. I E = Molar absorptivity (dm’ mol-’ cm-I).$ 1 = Intensity ratio of the fluorescence bands in %. 9 6 = Difference between the emission and excitation wavelengths. f i Medium: 0.1 mol dm-3 HCIO., in methanol. 11 sh = Shoulder. wavelengths of the excitation and emission maxima indicate monosubstitution. Hence it can be deduced that this molecule has a substituent in position 8. The stability of the emission spectrum with respect to the medium reflects the similarity between the chromophoric groups of the molecule isolated from the cultured cells and balfourodinium [Fig.2(b) and ( h ) ] . For the alkaloid ( I ) , isolated from leaf stems, the excitation spectrum has two narrow bands, which are not affected by HCIO,; this indicates substitution in positions 7 and 8. However, the difference between the wavelengths of the excitation and emission maxima is less than that expected for two substituents, and this suggests substitution with a dioxolo group which is the auxochromic group giving an almost identical wavelength difference for hydroxyluninium [Fig. 2(e) and (i)]. The RF values of these two alkaloids in the solvent system ethyl acetate-formic acid-water (10 + 1 + 1) are 0.39 for (H) and 0.41 for (1). These higher RF values indicate that these alkaloids have a methyl-l-ethenyl group. Therefore, compound (H) is thought to be 2,3-dihydro-4,8- dimethoxy-9-methyl-2-( l-methylethenyl)furo[2,3-b]quino- linium and compound (I) to be 7,8-dihydro-8-( l-methyl- ethenyl)-6-methoxy- 10-methyl-1,3-dioxolo[4,.5-h]furo[2,3-b]- quinolinium. After isolation, these two assignments were confirmed by *H NMR spectroscopy and MS.”.” The alkaloids were designated as ptelecultinium (H) and ptelefolidonium ( I ) .Conclusion The results presented here show that it is possible to elucidate the structures of dihydrofuro[2,3-b]quinolinium alkaloids by studying their fluorescence spectra and chromatographic behaviour. The specificity and sensitivity of the fluorescence allows the identification of a molecule contained in the eluate from a chromatographic layer, with only a few picomoles of the alkaloid.At this level, purification is not sufficient to identify a molecule from its electronic absorption spectrum and by MS, and the available amounts preclude any possibility of determining the structure by NMR spectroscopy. In a few instances, thin-layer chromatography cannot separate alkaloids sufficiently. The use of overpressured thin-layer chromatography might lead to some improve- ment,z3 but it should also be noted that it is possible to differentiate the fluorescence spectrum of each component in a mixture by synchronous fluorescence spectrometry. A recent example was provided by a study of the alkaloidic composition of in vitro cultures of Ruta graveolens.’4 In conclusion, the method described here provides a simple means of characterization, and is faster than classical methods of identification.1 2 3 4 5 6 7 8 9 10 References Mester. I . . in Chemistry and Chemical Taxonomy of the Rutales. eds. Waterman. P. G., and Grundon. M. F., Academic Press, New York, 1983. pp. 31-96. Sangster. A. W., and Stuart, K. L., Chem. Rev., 1965, 65. 69. Rapoport. H.. and Holden, K. G., J . Am. Clrem. Soc., 1959.81. 3738. Szendrei. K., Minker. E.. Koltai, M., Reisch. J . . Novak. I . . and Buzas. G., Pliarmazie. 1968, 23, 519. Boyd, D. R., and Grundon. M. F., J . Clrem. SOC. C , 1970,556. Reisch, J . , Mirhom, Y. W,, Korosi. J., Szendrei. K., and Novak, I . , Phytochemistry, 1973, 12, 2552. Montagu. M., Levillain, P., Ridcau, M., and Chenicux. J . C.. Talunra, 1981, 28, 709. Montagu, M., Levillain, P . , ChCnieux, J .C., and Ridcau. M., J . Chromatogr.. 1986, 351, 144. Tr6mouilloux-Guiller, J., Kodja. H., Andreu. F., Creche, J.. Chknieux. J . C.. and Rideau. M., Plant Cell Rep.. 1988, 7,456. Rideau, M.. Verchere, C., Hibon, P., ChCnieux, J. C., Maupas, P., and Viel. C., Pliytocliemisrry, 1979, 18, 155.ANALYST, JANUARY 1992, VOL. 117 81 1 1 12 13 14 1s 16 17 18 I9 Platt. J . R., J . Cliem. Phys., 1949, 17, 484. Jaffc. H . H., and Orchin, M.. Theory and Applications of Ultraviolet Spectroscopy. Wiley, New York, 5th edn., 1970. Zanker, V.. Z . Phys. Chem.. 1954, 2, 52. Mitschcr, L. A.. Bathala, M. S.. Clark, G. W.. and Bcal, J . L., Lloydia. 1975.38, 109. Szcndrci, K . , Rcisch, J . . Novak, I . , Simon, L.. Rozsa, Zs.. Minkcr. E . , and Koltai, M., Herhu Hung.. 1971, 10. 131. Bccker, R . S . . Theory and Interpretation of Fluorescence und PI1 osphorcwen ce . W i 1 c y- I n t c rscic ncc . New Y ork . 1 969, Bourdon. R., in Mises au Point de Chimie Analytique, Organ- ique, Pliurmuceutique et Bromatologique. eds. Gautier, J . A.. and Malangcau. P., 15rh Series. Masson, Paris, 1967, pp. 1-41. Udcnfricnd. S . , Fluorescence Assay in Biology and Medicine, Acadcmic Press. New York. 3rd edn., 1962, pp. 125-190. Montagu. M., Thesis. Univcrsite dc Tours, 1983. pp. 1-193. pp. 345-383. pp. 155-189. 20 21 22 23 24 Montagu, M.. Lcvillain, P., ChCnieux. J. C.. and Rideau, M., J. Chromatogr.. 1985, 331, 437. Petit-Paly, G . . Montagu, M., Viel, C., Rideau, M., and Chknieux, J . C., Plant Cell Rep., 1987, 6, 309. Petit-Paly, G., Montagu. M., Merienne, C . , Ambrosc. J . D., Viel, C., Rideau, M., and Chknieux, J . C.. Planta Med., 1989. 55. 209. Pothier, J . . Pctit-Paly. G., Montagu, M.. Galand, N.. ChCni- eux, J. C., Rideau, M.. and Viel, C., J . Planar Ckromatogr.. 1990, 3, 356. Montagu, M., Pctit-Paly, G.. Levillain, P., Baumcrt, A.. Groger, D., ChCnicux, J. C., and Rideau, M., Pharmazie, 1989, 44.342. Puper 11024786 Received May 28, 1991 Accepted August 7. 1991