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Bitter kaurane-type diterpene glucosides from the liverwort Jungermannia infusca
Phytochemistry,Vol. 29, No. 5, pp. 1619-1623,1990. Printed in Great Britain.
BITTER KAURANE-TYPE LIVERWORT
003l-9422/90 $3.00+ 0.00 0 1990Pergamon Press plc
DITERPENE GLUCOSIDES JUNGERMANNIA INFUSCA
FROM THE
FUMIHIRO NAGASHIMA, MASAO TOYOTA and YOSHINORI ASAKAWA* Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho 770, Tokushima, Japan (Receioed
Key Word Index--Jungermannia glucosides; bitter principles.
9 August 1989)
infusca; Jungermanniales;
Hepaticae;
infuscaside
A-E; kaurene-type diterpene
Abstract-Five new kaurene-type bitter diterpene glucosides, infuscasides A-E, have been isolated from the liverwort Jungermannia infisca together with the previously known ent-1%hydroxykaurene and stigmasteryl glucoside and their structures elucidated by chemical data and the extensive 2D-COSY NMR spectroscopy. The new diterpenoids have a very bitter taste.
INTRODUCTION Jungermannia species (Hepaticae) are rich sources of diterpenoids Cl]. Fresh J. infusca tissue is intensely bittertasting. Recently we isolated clerod-3,13(16)-15trien-l7oic acid, which inhibits the release of superoxide from J. infisca [2]. In this paper we wish to report the isolation of five novel kaurene-type glucosides which comprise the characteristic bitterness of J. infusca.
RESULTS AND DISCUSSION A combination of column chromatography on silica gel, Sephadex LH-20 and preparative HPLC of the methanol extract of J. infusca resulted in the isolation of five new diterpene glucosides named infuscasides A-E (1, 5, 6-8) possessing intense bitterness, together with ent-1%hydroxykaurene (4) [3] and stigmasteryl glucoside [4]. The molecular formula of infuscaside A (1) was determined to be C,,H,,O, by high resolution mass spectroscopy. The IR and NMR spectral data (Tables 1 and 2) exhibited the presence of a hydroxyl group (3350 cm- ‘), an acetoxyl group Cl737 cm-‘; 6ul.96 (3H, s); 6,170.6], two tertiary methyl groups [S, 1.08,1.66 (each 3H, s)] and an exomethylene group [S, 5.14, 5.52 (each lH, s); 6, 103.7, 160.11. The i3CNMR spectrum of 1 further contained the signals of seven methylenes, three methines, three quaternary carbons, two methylenes bearing an oxygen atom and seven methine groups bearing an oxygen atom, one of which might be an acetal carbon (6, 105.3) [S]. Acetylation of 1 gave a hexaacetate (2), [Su 1.98, 2.02, 2.03, 2.07, 2.09 and 2.17 (each 3H, s)], indicating that 1 contained five hydroxyl groups. The mass spectrum of 2 indicated the characteristic fragment ions of tetraacetyl glucoside at m/z 331, 169 and 109 [6, 71, respectively. Treatment of 1 with cellulase for three months afforded an aglycone (3) C,,H,,O,, m/z 320.2352, which contained two secondary hydroxyl groups [S, 4.20, 4.64 (each lH, br s); 6, 78.5 (C-6) and 83.2 (C-15)] and one primary
hydroxyl group [S, 3.94, 4.33 (each lH, d, J= 11.7); 6, 62.1 (t) C-201, showing that one acetoxyl group in 1 was located at C-6’ IS, 4.95 m; ~3~64.7(t)]. The position of the acetoxyl group in 1 was further confirmed by the analysis of the cross peaks which appeared in the lower field of ‘H-‘H and ‘H-13C and rH-13C long range ZD-COSY NMR spectra. The ‘HNMR spectrum of the aglycone 3, had the signals of two tertiary methyl groups and an exomethylene group and the characteristic signals at 6u2.81 (lH, br s, H-13) and 3.60 (lH, d, J= 11.7, H-14) for kaurene, indicating that the aglycone was a kaurene-type diterpene triol. In fact, the ‘H NMR signal pattern of the aglycone part of 1 was very similar to that of ent-15cc-hydroxykaurene (4) [3], which occurs in the same liverwort. Furthermore, the position of each hydroxyl group in 3 was elucidated by the analysis of 2D COSY (‘H-‘H and ‘H-13C) NMR spectra and the linkage of glucose of 1 at C-6 was established by the absence of the lower chemical shift of H-6 [S, 4.64 (lH, br s)]. The stereochemistry of 3 was determined by the NOESY experiment. The cross peaks were observed between (i) H-14 and H-15, (ii) H-7 and H-15, (iii) Me-19 and H-20 and (iv) H-6 and Me-18. The axial linkage of the glucose was confirmed by the resistance of hydrolysis of 1 by cellulase. The fi-configuration of the glucose at C-6 of 1 was also confirmed by the coupling constant (J = 7.6 Hz) of an anomeric proton (H-l’). On the basis of the above data, and by the consideration of co-occurrence with 4, the structure of infuscaside A was determined to be 6/I-(6’-acetoxy)+glucopyranosyl-15x,20-dihydroxy-ent-kaur-16-ene (1). Infuscaside B (S), C2sH4209 ([Ml’ 522.2829), showed the presence of a carbonyl group (1745 cm-‘; 6, 211.2). The ‘H and 13C NMR spectra (Tables 1 and 2) closely resembled those of 1, suggesting that 5 was another kaurene-type diterpene glucoside. This assumption was confirmed by the formation of a pentaacetate (6) whose mass spectrum showed the characteristic fragment ions for the tetraacetyl glucose as previously described. The location of an acetoxyl group at C-2’ in the glucose
1619
Diterpene
I
R’=Ac,
2
R1=R2=Ac
9
R1=R2=R
glucosides
from Jungermannia
R2=H
5
R1=Ac,
6
R1=R2=Ac
6
R1=R2=H
injiisca
1621
R2=H
HO.
3
R1=R2=OH
4
Rl=H,
R2=OH
Table 2. 13C NMR data of compounds TMS) C 1 2 3 4 5 6 7 8 9 10 11 12 13 14
1* 39.0 20.0 44.7 34.4 58.1 78.5 43.6 44.7 47.4 44.3 19.0 33.3 41.3 38.0
St 35.4 19.2 42.7 32.4 64.3 211.2 53.7 49.6 47.2 47.3 18.5 33.1 40.5 37.2
7t 35.8 19.3 42.2 33.3 56.4 20.3 39.4 42.3 47.2 46.3 18.6 33.4 41.2 38.0
*Assignments were confirmed iment. tINEPT experiment, tentative
7
c
1, 5 and 7 (pyridine-d,,
1*
15 83.2 16 160.1 17 103.7 18 34.3 19 24.1 20 62.1 1’ 105.3 2’ 75.0 3’ 79.0 4 71.6 5 75.4 6 64.7 OAc 20.8 170.6 by the ‘H-‘%Z
St 82.1 158.8 104.9 33.3 22.5 68.8 102.8 76.2 75.1 71.8 78.6 62.5 21.1 169.8 COSY
7t 82.7 160.2 103.8 35.3 22.7 69.4 103.1 76.2 75.3 72.2 78.7 62.9 21.2 169.9 exper-
assignments.
moiety and the carbonyl group at C-6 was established by the analysis of the cross peaks in the ‘H-‘HNMR spectrum of 5. The linkage of glucose was also confirmed to be at C-20 by the lower shifts of all the protons on carbon bearing hydroxyl groups, except for the chemical shifts of 6, 4.41 and 4.02 (each d, J = 11.7) of H-20. The stereochemistry of 5 was elucidated by NOE difference spectrum of 5. The NOE was observed between the axial
methyl group at C-4 and H-20. The ,%configuration of glucose was determined by the coupling constant (J = 7.8 Hz) of the anomeric proton. Thus the structure of infuscaside B was established as 20-(2’-acetoxy)-flglucopyranosyl-6-keto-lScr-hydroxy-enr-kaur-16-ene (5). Infuscaside C (7), C2sH4409 ([M+Na]+ 531) indicated the presence of a hydroxyl (3420 cm- ‘) and an acetoxyl group (1735 cm- ‘). The ‘H and 13C NMR spectra of 7 were similar to those of 5, except for the absence of ketone group, indicating that 7 might be the deoxo compound of 5. The position of the acetyl group at C-2’in glucose and the linkage of the glucose at C-20 were also determined by the comparison of the ‘H and 13C NMR spectra with those of 1 and 5. The stereochemistry of 7 was determined by NOESY spectrum in which the cross peak was observed between C- 19 Me and H-20. Infuscaside C was thus 15a-hydroxy-20-(2’-acetoxy)glucopyranosyl-ent-kaur16-ene (7). The IR spectrum of infuscaside D (8) showed the presence of a hydroxyl (3400 cm _ ‘) and a carbonyl groups (1692 cm-t). The signal patterns of the ‘H and 13C NMR spectra of 8 were almost identical to those of infuscaside B (5), except for the presence of one acetoxyl group. Acetylation of 8 in acetic anhydride gave a pentaacetate whose spectral data were identical to those of pentaacetate (6) derived from infuscaside B (5). Thus, structure 8 was given to infuscaside D. The spectral data of the last compound, infuscaside E (9), were very close to those of infuscaside A (I), except for the absence of an acetoxyl group, suggesting that 9 might be 68-glucopyranosyl-15u,20-dihydroxy-ent-kaur-16ene. This assumption was further confirmed by the following chemical and spectral data. Acetylation of 9 with
Diterpene
glucosides
from Jungermannia infusca
work was supported in part by a Grant-in-Aid for Cancer Research from Ministry of Health and Welfare (Y.A.). Supplementary material available. Copies of the ‘H-‘H, ‘H-r3C 2D COSY and ‘H-‘%I long range 2D COSY NMR spectra of 1 and ‘H-‘H 2D COSY spectra of 4, 68 can be obtained on request from the authors.
REFERENCES 1. Asakawa, Y. (1982) in Progress in the Chemistry ofOrganic Natural Products Vol. 42 (Herz, W., Grisebach, H. and Kirby, G. W., eds), p. 1. Springer, Wien.
1623
2. Toyota, M., Nagashima, F. and Asakawa, Y. (1989) Phytochemistry 28, 2507. 3. Matsuo, A., Kodama, J., Nakayama, N. and Hayashi, S. (1977) Phytochemistry 16, 489. 4. Asakawa, Y., Toyota, M. and Harrison, L. J. (1985) Phytochemistry 24, 1505. 5. Hashimoto, T., Tori, M. and Asakawa, Y. (1987) Phytochemistry 26, 3323. 6. Budzikiewicz, H., Djerassi, C. and Williams, D. H. (1964) in Structure Elucidation of Natural Products by Mass Spectrometry Vol. 11, p. 207. Holden-Day, San Francisco. 7. Biemann, K., Dejough, D. C. and Schnoes, H. K. (1963) J. Am. Chem. Sot. 85, 1763.
BITTER KAURANE-TYPE LIVERWORT
003l-9422/90 $3.00+ 0.00 0 1990Pergamon Press plc
DITERPENE GLUCOSIDES JUNGERMANNIA INFUSCA
FROM THE
FUMIHIRO NAGASHIMA, MASAO TOYOTA and YOSHINORI ASAKAWA* Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho 770, Tokushima, Japan (Receioed
Key Word Index--Jungermannia glucosides; bitter principles.
9 August 1989)
infusca; Jungermanniales;
Hepaticae;
infuscaside
A-E; kaurene-type diterpene
Abstract-Five new kaurene-type bitter diterpene glucosides, infuscasides A-E, have been isolated from the liverwort Jungermannia infisca together with the previously known ent-1%hydroxykaurene and stigmasteryl glucoside and their structures elucidated by chemical data and the extensive 2D-COSY NMR spectroscopy. The new diterpenoids have a very bitter taste.
INTRODUCTION Jungermannia species (Hepaticae) are rich sources of diterpenoids Cl]. Fresh J. infusca tissue is intensely bittertasting. Recently we isolated clerod-3,13(16)-15trien-l7oic acid, which inhibits the release of superoxide from J. infisca [2]. In this paper we wish to report the isolation of five novel kaurene-type glucosides which comprise the characteristic bitterness of J. infusca.
RESULTS AND DISCUSSION A combination of column chromatography on silica gel, Sephadex LH-20 and preparative HPLC of the methanol extract of J. infusca resulted in the isolation of five new diterpene glucosides named infuscasides A-E (1, 5, 6-8) possessing intense bitterness, together with ent-1%hydroxykaurene (4) [3] and stigmasteryl glucoside [4]. The molecular formula of infuscaside A (1) was determined to be C,,H,,O, by high resolution mass spectroscopy. The IR and NMR spectral data (Tables 1 and 2) exhibited the presence of a hydroxyl group (3350 cm- ‘), an acetoxyl group Cl737 cm-‘; 6ul.96 (3H, s); 6,170.6], two tertiary methyl groups [S, 1.08,1.66 (each 3H, s)] and an exomethylene group [S, 5.14, 5.52 (each lH, s); 6, 103.7, 160.11. The i3CNMR spectrum of 1 further contained the signals of seven methylenes, three methines, three quaternary carbons, two methylenes bearing an oxygen atom and seven methine groups bearing an oxygen atom, one of which might be an acetal carbon (6, 105.3) [S]. Acetylation of 1 gave a hexaacetate (2), [Su 1.98, 2.02, 2.03, 2.07, 2.09 and 2.17 (each 3H, s)], indicating that 1 contained five hydroxyl groups. The mass spectrum of 2 indicated the characteristic fragment ions of tetraacetyl glucoside at m/z 331, 169 and 109 [6, 71, respectively. Treatment of 1 with cellulase for three months afforded an aglycone (3) C,,H,,O,, m/z 320.2352, which contained two secondary hydroxyl groups [S, 4.20, 4.64 (each lH, br s); 6, 78.5 (C-6) and 83.2 (C-15)] and one primary
hydroxyl group [S, 3.94, 4.33 (each lH, d, J= 11.7); 6, 62.1 (t) C-201, showing that one acetoxyl group in 1 was located at C-6’ IS, 4.95 m; ~3~64.7(t)]. The position of the acetoxyl group in 1 was further confirmed by the analysis of the cross peaks which appeared in the lower field of ‘H-‘H and ‘H-13C and rH-13C long range ZD-COSY NMR spectra. The ‘HNMR spectrum of the aglycone 3, had the signals of two tertiary methyl groups and an exomethylene group and the characteristic signals at 6u2.81 (lH, br s, H-13) and 3.60 (lH, d, J= 11.7, H-14) for kaurene, indicating that the aglycone was a kaurene-type diterpene triol. In fact, the ‘H NMR signal pattern of the aglycone part of 1 was very similar to that of ent-15cc-hydroxykaurene (4) [3], which occurs in the same liverwort. Furthermore, the position of each hydroxyl group in 3 was elucidated by the analysis of 2D COSY (‘H-‘H and ‘H-13C) NMR spectra and the linkage of glucose of 1 at C-6 was established by the absence of the lower chemical shift of H-6 [S, 4.64 (lH, br s)]. The stereochemistry of 3 was determined by the NOESY experiment. The cross peaks were observed between (i) H-14 and H-15, (ii) H-7 and H-15, (iii) Me-19 and H-20 and (iv) H-6 and Me-18. The axial linkage of the glucose was confirmed by the resistance of hydrolysis of 1 by cellulase. The fi-configuration of the glucose at C-6 of 1 was also confirmed by the coupling constant (J = 7.6 Hz) of an anomeric proton (H-l’). On the basis of the above data, and by the consideration of co-occurrence with 4, the structure of infuscaside A was determined to be 6/I-(6’-acetoxy)+glucopyranosyl-15x,20-dihydroxy-ent-kaur-16-ene (1). Infuscaside B (S), C2sH4209 ([Ml’ 522.2829), showed the presence of a carbonyl group (1745 cm-‘; 6, 211.2). The ‘H and 13C NMR spectra (Tables 1 and 2) closely resembled those of 1, suggesting that 5 was another kaurene-type diterpene glucoside. This assumption was confirmed by the formation of a pentaacetate (6) whose mass spectrum showed the characteristic fragment ions for the tetraacetyl glucose as previously described. The location of an acetoxyl group at C-2’ in the glucose
1619
Diterpene
I
R’=Ac,
2
R1=R2=Ac
9
R1=R2=R
glucosides
from Jungermannia
R2=H
5
R1=Ac,
6
R1=R2=Ac
6
R1=R2=H
injiisca
1621
R2=H
HO.
3
R1=R2=OH
4
Rl=H,
R2=OH
Table 2. 13C NMR data of compounds TMS) C 1 2 3 4 5 6 7 8 9 10 11 12 13 14
1* 39.0 20.0 44.7 34.4 58.1 78.5 43.6 44.7 47.4 44.3 19.0 33.3 41.3 38.0
St 35.4 19.2 42.7 32.4 64.3 211.2 53.7 49.6 47.2 47.3 18.5 33.1 40.5 37.2
7t 35.8 19.3 42.2 33.3 56.4 20.3 39.4 42.3 47.2 46.3 18.6 33.4 41.2 38.0
*Assignments were confirmed iment. tINEPT experiment, tentative
7
c
1, 5 and 7 (pyridine-d,,
1*
15 83.2 16 160.1 17 103.7 18 34.3 19 24.1 20 62.1 1’ 105.3 2’ 75.0 3’ 79.0 4 71.6 5 75.4 6 64.7 OAc 20.8 170.6 by the ‘H-‘%Z
St 82.1 158.8 104.9 33.3 22.5 68.8 102.8 76.2 75.1 71.8 78.6 62.5 21.1 169.8 COSY
7t 82.7 160.2 103.8 35.3 22.7 69.4 103.1 76.2 75.3 72.2 78.7 62.9 21.2 169.9 exper-
assignments.
moiety and the carbonyl group at C-6 was established by the analysis of the cross peaks in the ‘H-‘HNMR spectrum of 5. The linkage of glucose was also confirmed to be at C-20 by the lower shifts of all the protons on carbon bearing hydroxyl groups, except for the chemical shifts of 6, 4.41 and 4.02 (each d, J = 11.7) of H-20. The stereochemistry of 5 was elucidated by NOE difference spectrum of 5. The NOE was observed between the axial
methyl group at C-4 and H-20. The ,%configuration of glucose was determined by the coupling constant (J = 7.8 Hz) of the anomeric proton. Thus the structure of infuscaside B was established as 20-(2’-acetoxy)-flglucopyranosyl-6-keto-lScr-hydroxy-enr-kaur-16-ene (5). Infuscaside C (7), C2sH4409 ([M+Na]+ 531) indicated the presence of a hydroxyl (3420 cm- ‘) and an acetoxyl group (1735 cm- ‘). The ‘H and 13C NMR spectra of 7 were similar to those of 5, except for the absence of ketone group, indicating that 7 might be the deoxo compound of 5. The position of the acetyl group at C-2’in glucose and the linkage of the glucose at C-20 were also determined by the comparison of the ‘H and 13C NMR spectra with those of 1 and 5. The stereochemistry of 7 was determined by NOESY spectrum in which the cross peak was observed between C- 19 Me and H-20. Infuscaside C was thus 15a-hydroxy-20-(2’-acetoxy)glucopyranosyl-ent-kaur16-ene (7). The IR spectrum of infuscaside D (8) showed the presence of a hydroxyl (3400 cm _ ‘) and a carbonyl groups (1692 cm-t). The signal patterns of the ‘H and 13C NMR spectra of 8 were almost identical to those of infuscaside B (5), except for the presence of one acetoxyl group. Acetylation of 8 in acetic anhydride gave a pentaacetate whose spectral data were identical to those of pentaacetate (6) derived from infuscaside B (5). Thus, structure 8 was given to infuscaside D. The spectral data of the last compound, infuscaside E (9), were very close to those of infuscaside A (I), except for the absence of an acetoxyl group, suggesting that 9 might be 68-glucopyranosyl-15u,20-dihydroxy-ent-kaur-16ene. This assumption was further confirmed by the following chemical and spectral data. Acetylation of 9 with
Diterpene
glucosides
from Jungermannia infusca
work was supported in part by a Grant-in-Aid for Cancer Research from Ministry of Health and Welfare (Y.A.). Supplementary material available. Copies of the ‘H-‘H, ‘H-r3C 2D COSY and ‘H-‘%I long range 2D COSY NMR spectra of 1 and ‘H-‘H 2D COSY spectra of 4, 68 can be obtained on request from the authors.
REFERENCES 1. Asakawa, Y. (1982) in Progress in the Chemistry ofOrganic Natural Products Vol. 42 (Herz, W., Grisebach, H. and Kirby, G. W., eds), p. 1. Springer, Wien.
1623
2. Toyota, M., Nagashima, F. and Asakawa, Y. (1989) Phytochemistry 28, 2507. 3. Matsuo, A., Kodama, J., Nakayama, N. and Hayashi, S. (1977) Phytochemistry 16, 489. 4. Asakawa, Y., Toyota, M. and Harrison, L. J. (1985) Phytochemistry 24, 1505. 5. Hashimoto, T., Tori, M. and Asakawa, Y. (1987) Phytochemistry 26, 3323. 6. Budzikiewicz, H., Djerassi, C. and Williams, D. H. (1964) in Structure Elucidation of Natural Products by Mass Spectrometry Vol. 11, p. 207. Holden-Day, San Francisco. 7. Biemann, K., Dejough, D. C. and Schnoes, H. K. (1963) J. Am. Chem. Sot. 85, 1763.