Sesquiterpenoids from the Liverwort Porella canariensis

Sesquiterpenoids from the Liverwort Porella canariensis* Frank Cullmann and Hans Becker Pharm akognosie und A nalytische P hytochem ie, U nversität des Saarlandes, D66041 Saarbrücken, G erm an y Z. N aturforsch. 54c, 1 5 1 -1 5 5 (1999); received N ovem ber 23/D ecem ber 29, 1998 Porella, Liverw ort, Pinguisane, D rim ane, G erm acran e, A rom ad en d ran e From the liverw ort Porella canariensis, collected in M adeira, Portugal, nine pinguisanes, four drim anes, including the pun g en t polygodial, two germ acranes, and two aro m ad en d ran es w ere isolated. The m ajo r com pounds w ere the aro m ad en d ran e ent-cyclocolorenone and the nor-pinguisane norpinguisone. T h ree hydro carb o n s w ere isolated by low tem p eratu re colum n chrom atography. O ne g erm acran e and one pinguisane proved to be new natural products.

Introduction Liverworts of the genus Porella exhibit a great variety of sesquiterpenoids. Besides arom aden dranes and drimanes, which are also found in higher plants, they produce pinguisanes and re lated structures, rearranged terpenoid skeletons, restricted to liverworts (Asakawa, 1995). Some Porella species produce the very pungent drimane dialdehyde polygodial up to 10-30 per cent in their crude extracts. This am ount is much bigger than in higher plants like e. g. Polygonum hy dropiper, a spice for grilled fish in Japan (A sa kawa, 1990). In a recent study of P. canariensis from the Ca nary Islands, ent-cyclocolorenone, three drimanes: cinnamolide, cis-didehydrocinnamolide, and isodrimeninol and one pinguisane derivative, an arte fact from the methanol extraction, could be iso lated (Nagashima et al., 1996). The availability of larger amounts from M adeira led us to reinvesti gate the species. Result and Discussion A combination of vacuum phy, column chromatography, chlorom ethane extract of P. six pinguisanes: a-pinguisene

liquid chrom atogra and HPLC of the dicanariensis afforded (1) (Asakawa et al.,

R eprint requests to Prof. D r. H an s B ecker. Fax: +49-681-302-2476 E-mail: pbl3hb@ rz.uni-sb.de * Publication No. 132 in th e series „A rb eitsk reis Chem ie und Biologie d er M o o se“. For publication No. 131 see C ullm ann and B ecker. 0939-5075/99/0300-0151 $ 06.00

1978), pinguisenol (2) (Asakawa et al., 1976), 7keto-pinguisenol-12-oic acid methyl ester (3) (Toy ota et al., 1991), deoxopinguisone (4) (Krutov et al., 1973), the new deoxopinguisone-12-oic acid methyl ester (5), and deoxopinguisone-15-oic acid methyl ester (6) (Asakawa and Aratani, 1976), two norpinguisanes: norpinguisone (7) (ibid.) and norpinguisone methyl ester (8) (Fukuyama et al., 1988) and the spiropinguisane ptychanolide (9) (Takeda et al., 1983). From the drimane series isodrimeninol (10) (Asakawa et al., 1979), drimenin (11) (He and Wu, 1988), cinnamolide (12) (Kioy et al., 1990) and the pungent polygodial (13) (ibid.) could be obtained. Additional sesquiterpenes were germacrene D (14) (Yoshihara et al., 1969), a mimeticum of the sex pheromone of the American cockroach, Periplaneta americana (Tahara et al., 1975), the new l(10),5,ll-germ acratriene-7a-ol (15) and finally the two arom adendranes a-gurjunene (16) (Asakawa et al., 1978) and ent-cycloco lorenone (17) (Wu and Chen, 1992). Norpingui sone (7) and ent-cyclocolorenone (17) were the major compounds of this liverwort, with 1.5 g iso lated each. Compound 5 showed in its 'H (Table I) and 13C NMR (Table II) data a close similarity to deoxo pinguisone (4) except for the presence of a carboxylic carbon atom (6C 174.8, s) and a methyl ester (6C 51.0, q) and consequently the loss of one of the four methyl carbon atoms. Since both doublet methyl signals of H-13 and H-15 were still present, the carbomethoxy group must be located either on C-8 or C-9 of the pinguisane skeleton. From the 2D NMR data, the structure of the deoxopinguisone-12-oic acid methyl ester could be deduced

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F. C ullm ann and H. B ecker

152 15 I

10

|

14

1

4 Ri = R 2 = H 5 R i = COOMe R 2 = H 6

S esquiterpenoids from Porella canariensis

7 R=H 8

R = COOMe

R i = H R 2 = COOMe CHO CHO

10 R i = a-OH, H R 2 = H, H 11 R i = 0 R 2 = H, H 12 R i =H , H R 2 = 0

15

"h 16 R = H, H 17 R = O

18

unambiguously. This is the first report of this structure, but related compounds, 12-acetoxy-deoxopinguisone and deoxopinguisone-12-al had already

19

been described for Dicranolejeunea yoshinagana (Toyota et a l, 1995).


153

F. C ullm ann and H. B ecker • S esquiterpenoids from Porella canariensis Table I. ’H NMR spectral d ata and coupling constants (in Hz in parenthesis) for com pounds 5 and 15 (CDC13).

Table II. 13C NMR spectral data for com pounds 5 and

Proton

Carbon

1

a ß 3a ß 4 5 2

6

7a ß 8 a ß 9a ß

C om pound 5

C om pound 15

m 1.58 m 1.98 m 1.69 m

-

2 .1 2

2 .0 2

2.53 m 2.70 d (18.0) 3.20 dd (18.0, 2.4)

1 1

6.16 d (1.9) 7.22 d (1.9)

1 2

-

1 0

13 14 15 16

d ( 6 .8 ) 0.73 s 1 . 1 2 d (7.1) 3.66 s

0 .8 6

1 2

2.27 m 1.47 1.54 2.77 5.16 5.34

15 (CDCI3).

m m ddd (10.2, 10.2, 3.2) dd (16.0, 9.7) d (16.0)

3 4 5 6

7 8

9 1 0 11 1 2

1.63 m 1.67 m 2.05 m 2 .2 0 m 4.90 brd (11.3)

13 14 15 16

C om pound 5

C om pound 15

38.5 29.8 34.8 34.5 117.2 147.1 24.2 62.1 48.3 108.9 140.9 174.8 15.1 16.0 14.4 51.0

131.8 41.1 27.4 53.3 130.5 139.7 73.0 40.8 24.7 129.5 149.0 108.6

d t t d s s t s s d d s q q q q

s t t d d d s t t d s t 2 1 .2 q 23.5 q 16.6 q -

-

4.64 1.67 1.33 1.50 -

brs s s s

The mass spectrometry of compound 15 estab lished a molecular formula of Q 5 H 24O, supported by 13C NMR (Table II). The 13C NM R spectrum of 15 also established that three d efin e bonds (one disubstituted, one trisubstituted, and one exo methylene) were present, and that there was a ter tiary alcohol (a singlet carbon at 6 C 73.0). Con sideration of the unsaturation present in the molecule formula gave a monocyclic compound. The similarity of the !H NM R data of compound 15 and germacrene D (14) led to the assumption of a germacrane-type sesquiterpene, which was confirmed by the 2D NM R data. From these data, the plain structure of a l(10),5,ll-germ acratriene7-ol was derived. The interpretation of the NOESY spectrum, shown in Fig. 1, established the relative configuration of the new l(10),5,ll-germacratrien-7a-ol. A similar compound, the l(10),5-germacradien7a-ol (18) had been reported from L e m n a lia a fricana (Izac et al., 1982) and the stereochemistry was established by means of an E u(fod ) 3 experi ment. The data presented herein were the basis for the reports of this compound from P e u c e d a n u m p a lu s tre (Schmaus et al., 1989) and from the liver-

worts M a r c h a n tia p o ly m o r p h a , C o n o c e p h a lu m c o n ic u m , and P o rella sw a r tz ia n a (Asakawa, 1995). However, the NMR spectral data of this com pound were not in accordance with our data, espe cially the chemical shift of C-14 was significantly different ( 6 C 21.2, q for 15 compared to 6 C 30.8 in the literature). By carefully checking the litera ture, we learned that San Feliciano et al. (1995) and Kitagawa et al. (1987) described a germacradienol from J u n ip e r u s c o m m u n is subsp. h e m is p h a e r ic a and N e p h th e a sp., respectively, with data identical to those of Izac et al. (1982), but their structure elucidation by means of modern 2D NM R methods established the structure of 1(10),5germacradiene-7ß-ol (19). Moreover, Bohlmann et al. (1977, 1984) reported both epimers, but pub lished merely identical data (being identical to Izac et a l.). Thus, to our opinion, all the above reported germacradienols should be l(10),5-germacradiene-


154

F. C ullm ann and H. B ecker • S esquiterpenoids from Porella canariensis

7ß-ol (19). Furtherm ore, this finding is in accor dance with the typical range of the 13C NMR shift of a methyl group geminal to an alcohol function as published in literature (öc 30.3-32.9 for equatorial, öc 22.5-26.1 for axial position, respectively) (Joseph-Nathan et a l, 1984). Consequently, the structures of the liverwort germacranes derived from the data of Izac should be revised to l(10),5-germacradien-7ß-ol, thus having the same stereochemistry as the similar l(10),5-germ acradien-7ß,ll-diol reported from Bryopteris filicina (Nagashima et a l, 1994) and Ptychanthus striatus (Asakawa, 1995). Once more, a Porella species proved to be a valu able source of sesquiterpenoids, especially the high contents of norpinguisone (7) and ent-cyclocolorenone (17) are worth mentioning. The occurrence of polygodial (13) for P. canariensis is reported for the first time. Thus, unlike stated in literature (Müller, 1954), P. canariensis belongs to the pun gent type Porella species. The differences in the chemical constituents of P. canariensis from the Ca nary Islands and Madeira suggests the existence of different chemical races of this liverwort in Europe.

perature ( - 2 0 °) column chromatography on silica gel (hexane) to yield 1 (373 mg), 4 (255 mg), 14 (105 mg) and 16 (50.5 mg). Fraction II was pre purified by HPLC (Si, CH 2Cl2/hexane 25/75) and gave rise to 5 (112 mg), 7 (240 mg) and a subfrac tion whose separation by HPLC (Si, EtOAc/hexane 0.5/99.5) resulted in 54.5 mg of compound 2 and 161 mg of compound 6 . Fraction III was al most pure and by recrystallisation from hexane another 1216 mg of compound 7 could be ob tained. Fraction IV was separated by HPLC (RP18, M eO H /H zO 75/25) and gave compounds 8 (59.5 mg), 9 (51.5 mg), and 11 (33.1 mg). Due to its high amount, Fraction V was chromatographed by CC, (Si, CHC13) to yield compound 17 (993 mg) and a mixture, from which after separation by HPLC (Si, EtOAc/hexane 12/88) another 455 mg of compound 17 could be obtained together with compounds 12 (160 mg) and 15 (11.8 mg). Fraction VI was separated by HPLC (Si, EtOAc/hexane 12/ 8 8 ) into compound 13 (399 mg) and a fraction con taining sterols, which were not investigated fur ther. A fter separation by HPLC, Fraction VII, the last fraction, yielded (Si, EtOAc/hexane 20/80) compounds 3 (144 mg) and 10 (246 mg).

Experim ental Plant material

Spectroscopic m ethods

Porella canariensis (F. Web.) Bryhn.: a) Madeira, M ontado dos Pecegueros. Coll. et det. S. Sä Fontinha, 04.07.1996, Herbarium SAAR 5451. b) Madeira, Faja da Nogueira. Coll. et det. S. Sä Fontinha, 31.07.1996, Herbarium SAAR 5452

NMR-spectroscopy: BRU K ER, CDCI 3 , ambient tem perature, 400 MHz ('H ), 100 MHz ( 13 C) for one-dimensional, 500 MHz and 125 MHz for twodimensional techniques, respectively; chemical shifts are given in b values (ppm) from TMS; mass spectroscopy: VARIA N MAT 311, 70 eV; GC-MS was perform ed on a HP-1 capillary column with a G 1800A GCD system (HP).

Extraction and isolation

General: All HPLC separations were performed isocratic. The composition of the mobile phase is given as v/v (in parenthesis). Since the liverworts of both collecting sites ex hibited identical TLC and HPLC chromatograms, they were combined and the resulting 575 g of air dried gametophytes of P. canariensis were ground and successively extracted with a total of 2 0 1 C H 2C12 to yield 18.2 g of lipophilic extract. This extract was subjected to VLC on silicagel in a hexane-ethyl acetate gradient (0 - 1 0 0 %) to yield seven main fractions (I - VII). Fraction I, contain ing the hydrocarbons, was separated by low tem

Spectroscopic data

Compound 5: yellowish oil, [a ]^ = -50.1° (c = 0.696); ’H NMR: see Table I; 13C NMR: see Ta ble II; EIMS m /z (rel. int.) = 262 (3) [M]+, 201 (2), 145 (3), 108 (100), 91 (5), 79 (7) Compound 15: colourless oil, [a]o = -20.0° (c = 0.280); 'H NMR: see Table I; 13C NMR: see Ta ble II; EIMS m /z (rel. int.) = 220 (1) [M]+, 205 (20), 202 (14), 187 (22), 159 (21), 145 (22), 134 (42), 119 (47), 107 (100), 91 (51), 79 (62), 67 (38), 55(34).


F. C ullm ann and H. B ecker • S esquiterpenoids from Porella canariensis

Acknowledgem ents

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