Triterpene lactones from the stem bark of Abies firma

Phytochemistry. Vol. 30. No. 6, pp. 1983 1987, 1991 Printed in Grrat Britain.

TRITERPENE

LACTONES REIKO

c

003 l-9422/91 $3.00 + 0.00 1991 Pergamon Press plc

FROM THE STEM BARK OF ABZES FIRMA TANAKA and

SHUNYO

MATSUNAGA*

Osaka University of Pharmaceutical Sciences, 2-10-65 Kawai, Matsubara City, Osaka 580, Japan (Received

25

September

1990)

Key Word Index--Abiesjirma; Pinaceae; stem bark; triterpenoids; 3/?-hydroxy-9/?-lanosta-7,24-dien-26,23R-olide; 27-hydroxy-3-oxo-9P-lanosta-7.24-dien-26.23R-olide.

Abstract-Two

new tetracyclic triterpene lactones were isolated from the stem bark of Abiesjirma and their structures determined as 3~-hydroxy-9~-lanosta-7,24-dien-26,23R-olide and 27-hydroxy-3-oxo-9/?-lanosta-7,24-dien-26,23Rolide on the basis of spectral evidence.

INTRODUCTIOIV

Several Abies species have been reported to contain highly oxygenated 9fi-lanostanes [l-7], lanostane [8] and cycloartane [9, IO] types of triterpenoids and their migrated analogues [I l-133 in barks, needles and seeds. Recently, we have reported the isolation of 3-0x09/3-lanosta-7,24dien-26,23R-olide (3-oxo-3-demethoxyabieslactone) (1) from the neutral ether extract of the stem bark of Abiesjirma Sieb. et Zucc. together with campesterol, sitosterol, a mixture of five n-alkyl trans-ferulates involving Czz-C,, alkyl moieties [6]. Chromatography of the bark extract led to the isolation of two new triterpene lactones, 2 and 3, and this paper describes the structure elucidation of these compounds. RESULTS AND DISCUSSION

Compound 2 gave the molecular formula ofC,,H,,O, in the high resolution mass spectrum. It indicated positive colour with the Liebermann-Burchard reagent and showed the presence of a hydroxyl group (v,,, 3520 and 1058 cm - ’ ) and an Q-unsaturated y&tone (v,. 1760 and 174Ocn-‘) in the IR spectrum. In the ‘H and “C NMR spectra (see Tables 1 and 2), it exhibited signals for five tertiary methyl groups, one secondary methyl group and one vinyhc methyl group, one secondary hydroxyl group which could be placed at the C-3/3 position (6 3.20, 1H, dd) as in those of the usual triterpene alcohols, one methine proton attached to an oxygen function (64.97, lH, ddd), one lactone carbonyl (6 174.43) and two trisubstituted double bonds cS5.56, lH, dt and 6.99, lH, t; 6121.70 (=CH-) and 148.59 (=C<), and 129.51 (=C<) and 149.66 (=CH-)], one of which was in the lactone ring. Acetylation of compound 2 gave an acetate (2a), in which the carbinolic methine proton signal was shifted to 64.46 (1H, dd). In the El mass spectrum (Scheme l), compound 2 gave three characteristic fragment ion peaks due to the tetracyclic triterpene skeleton at m/z 315 (ion g), the Cs side chain moiety involving the lactone ring in the terminal position by the cleavage of the C-17/C-20 bond [6,7] at m/z 139 (ion n) and the y-lactone portion at m/z

97 (ion o), together with the peaks arising from the retroDiels-Alder cleavage of the ring B at m/z 314 (ion h) and 299 (ion i) and the cleavage of the ring C at m/z 237 (ion k), 189 (ion I) and 187 (ion m). Further, the fragment ion peaks arising from the cleavage of the C-2O,K-22 bond followed by the loss of a methyl group and the cleavage of the ring D were observed at m/z 327 (ion f) and 273 (ion j), respectively. In the CD spectrum, it showed a negative Cotton effect curve similar to that of compound 1 [6]. All the above data indicated that compound 2 must be 3/G hydroxy derivative of 1. This assumption was proved by the following experiment. Reduction of compound 1 with sodium borohydride furnished 3/?-hydroxy-9/Glanosta7,24-dien-26,23R-olide, which was identical in all respects with compound 2. Compound 3 was assigned the molecular formula CJoH,,O, (HRMS). It also gave positive colour with the Liebermann-Burchard reagent and showed IR absorption bands for a hydroxyl group, an a&unsaturated ylactone, and a saturated six-membered ring ketone. In the ‘H and ‘%NMR spectra (Tables 1 and 2), it displayed signals for five tertiary methyl groups, one secondary and one vinylic methyl groups, one methylene group vicinal to the keto-group (62.50, 2H, dd), one hydroxymethyl group (64.45,2H, s; 657.20). one methine proton attached to an oxygen function [S5.09, lH, ddd; SSO.Ol), and two trisubstituted double bonds cS5.65, lH, dt and 7.26, lH, d; 6121.73 (=CH-), 148.45 (=C<), 133.07 (=C<), 150.14

*Author to whom correspondence should be addressed. 1983

1 2

R1 = R’ = 0, R3= ,, R’ = R’ = H. R’ = OH

2a

R’ =

R3 =

H. R’ = OAc

3 4

R’ =

R’ =

0. R’ =

R’=

OMe.R’=

R’=

OH H

1984

R. T.ANAKA~~~ S. MATSUNA~~A

Table

I. ‘H NMR chemical

shifts of compounds

1, 2, 2s and 3 (in CDCI,,

TMS=O)*

H

It

2

2a

3

Me-18 Me-19 Me-21 Me-27 Me-28 Me-29 Me-30

0.81 1.00 1.01 1.91 1.10 1.09 I .03 2.49 (8.5,

0.93 0.99 1.00 d (6.5) 1.91 d (1.7) 0.87 I .02 1.02

0.92’ 1.01 I .OO d (6.5) 1.92 d (1.7) 0.94* 0.90 1.01

0.8 1 1.00 I .02 d (6.5)

3.20 dd (10.2, 5.5) 5.56 dr (7.5. 3.0) 4.91 ddd (9.5, 4.1, 1.8) 6.99 r (1.3)

4.46 dd (10.2, 5.5) 5.56 dr (7.5, 3.0) 4.98 ddd (9.5, 4.1, 1.8) 7.00 I(l.3)

2&B

d (6.5) d (1.7)

dd 6.5)

32 7 23 24 21 OAc

5.53 (7.5, 4.98 (9.5, 7.01

1.10 1.09 1.01 2.50 dd (8.6, 6.5)

dr 3.0) ddd 4.1, 1.8) I(l.3)

5.65 (7.5, 5.09 (9.5, 7.26 4.45

dt 3.0) ddd 4.1, 1.8) d (1.8) s

2.05

*Assignments were range ‘H-‘%Z COSY tData taken from ‘Assignments may Coupling constants

made on the basis of 2D ‘H-‘H experiments. ref. [6]. be reversible vertically. (in Hz) in parentheses.

COSY, 2D ‘H-‘%I

COSY and 2D long

2 m/z436 (7) b 2 3

2 m/z 273(5)[CL~Hz90]* 3 m/z '271(lI)[C19HI,0]t

?I 2 m/z 315(3) [CzlH~lO]+

m/2439(5) m/z453 (24)

3 m/z 313 (5)[CllHJsO]+

a

2

m/z42l(IoO) c

2

R' = o-OH,R'=

3

m/z454(37)[C30H~603]+ R’ = 0. R’ = OH

f 2 m/z 327(3) [C1,H,,O]' 3 m/z 325(54) [CIIHJ,O]+

H

m/z 468(3)[C~oH~~O~]*

2 m/s314(3)[C11H,,0,]t

m/z 299(12)

i

n

2 m/z139(14)[C,H~,0,]+

0 2 m/z 97(46)(CIHsOIj+

m/z 189(9)

I Scheme

I. Mass spectral

fragmentation

of compounds

2 and 3.

m/z 187(25)

m

Triterpene

Table

C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 OAc OAc

2. 13C NMR chemical shifts of compounds (in CDCl,, TMS=O, 74.5 MHz)*

1t

2

34.20 34.28 218.92 47.0 I 52.35 23.01 121.64 148.48 45.67 35.80 20.88 34.39 44.15 51.93 33.01 28.21 53.47 22.51 23.12 33.49 18.38 40.46 78.96 149.68 129.48 177.44 10.65 21.30 28.00 27.41

35.52 27.94 79.33 38.83 48.62 23.07 121.70 148.59 48.33 35.90 22.93 35.26 43.74 52.76 33.32 28.55 53.96 23.66 24.49 33.47 18.41 40.46 79.01 149.66 129.51 174.43 10.65 16.36 28.89 30.45

--.

1,2,2a and 3

t 35.21 24.33 81.12 37.71 48.61 22.9 I 121.42 148.68 48.34 35.74 22.9 I 35.21 43.70 52.77 33.27 28.53 53.96 23.69 24.50 33.45 18.35 40.45 79.00 149.70 129.46 174.47 10.65 17.48 28.77 30.49 21.33 171.06

lactones

3 33.06 34.23 219.01 47.04 52.39 23.04 121.73 148.45 45.51 35.82 20.90 34.38 44.22 51.96 29.73 28.27 53.50 22.53 23.16 33.60 18.35 40.29 80.01 150.14 133.07 172.80 57.20 21.33 27.99 27.41

*Assignments were made on the basis of 2D ‘H-‘H 2D ‘H_13C COSY and 2D long range ‘H-13C experiments. tData taken from ref. [6].

COSY, COSY

(=CH-)], one of which was in the lactone ring. The lJCNMR signals due to one lactone carbonyl and one ketone were observed at 6 172.80 and 219.01, respectively. Except for the fact that compound 3 showed ‘H and 13CNMR signals for one hydroxymethyl and one sixmembered ring ketone instead of the absence of those for one vinyl methyl and the C-3fl hydroxyl which were in compound 2, all the chemical shift values of the other signals of both compounds were closely similar to each other, suggesting compound 3 to be the 27-hydroxymethyl derivative of 1. The CD and EI mass spectra gave conclusive evidence for this assumption. Although three fragment ion peaks observed at m/z 325, 313 and 271 in the EI mass spectrum of compound 3 (Schemes 1 and 2) were compatible with ions f, g and j in compounds 1 [6] and 2 indicating their carbon skeletons, all other peaks useful for structure analysis appeared between mass numbers 90 and 400 and were considerably different from those assigned for both 1 and 2. In the high mass number region, compound 3 showed three characteristic fragment ion peaks due to [M -H,O]+ (ion b’), [M-Me

from Abies firma

1985

-H,O]+ (base peak, ion c’) and [M-Me-CH,O]’ (ion d) and m/z 450, 435 and 423, respectively, in which, the relative intensity of the ion peak b’ was 3.3 times more predominant than that of ion b in 2 under the same ionization conditions. The fragment ion d was also typical for 3. The mode of formation of ion b’ from compound 3 is fundamentally different from that of ion b from compound 2. As shown in Scheme 2, it is evident that the dehydration involving the 27-hydroxymethyl and H-23 in the lactone ring for the formation of the ion b’ takes precedence over almost all the other fragmentations in 3. Ion b’ undergoes further fragmentation via the same route to that described for compound 2 (Scheme 1) to give the ions h’, i’and k’-o’ at m/z 312,297,231,187, 185, 137 and 95, respectively, all including an cx-methylene-P,yunsaturated y-lactone ring produced by dehydration of the original cx-hydroxymethyl-cz$-unsaturated y-lactone moiety in the terminal position. Ion p was characteristic

for compound 3, which could be derived by allylic cleavage of the C-20/C-22 bond from ion b’. In the CD spectrum, compound 3 exhibited a similar negative Cotton effect curve that seen for compounds 1 and 2 in the lower wave length region, indicating the lactone ring to have the same 23R-configuration. All the above results clearly proved the structure of compound 3 to be 27hydroxy-3-oxo-9/l-lanosta-7,24-dien-26,23R-olide. This is the first report of the isolation of 2 and 3 from natural sources. It is of chemotaxonomic interest that the stem bark of A. firma contains compound 2 next in abundance to compound 1 and compound 3 is one of the minor constituent, whereas no abieslactone (4), the most abundant triterpenoid in the barks of A. muriesii and A. ueitchii, has been detected from this plant. EXPERIMENTAL General. Mps: uncorr. Optical rotations: CHCI,. IR: KBr discs. ‘H NMR (300 MHz) and 13CNMR (74.5 MHz): CDCI, with TMS as internal standard. EIMS (probe.): 70eV. CD: dioxane. CC: silica gel 60 (70-230 mesh, Merck). TLC: silica gel and PF,,, (Merck). HF,,, Extraction and isolation ojcompowds. Collection of the plant material, extraction, and separation of the crude crystalline solid (5.238 g) deposited from the cont. Et,0 extract of the chopped and air-dried stem bark of A. firma (2.9 kg) on the silica gel CC has already been described [6]. After 3-oxo-9@nosta-7,24dien-26,23R-olide (1) (4.161 g) was obtained from the frs eluted with C,H,-CHCl, (1: l), the above CC was continued using CHCI, and CHCI,-EtOAc (2O:l) to give 2 (76mg) and 3 (14 mg), respectively, as needles. Compound 2. Mp 239-241” (MeOH), [a];’ - 58.9” (c 0.21). R, 0.42 (plate: 0.25 mm thick, C,H,CHCl,-EtOAc 1: 1: 1); IR v,,, cm-‘: 3520 (OH), 3065 (=CH-), 2938, 2870. 1760 and 1740 (a&unsaturated y-lactone), 1658 (GXJ, 1462, 1443, 1378, 1362, 1215, 1106, 1058, 1022,980,%2,878 and 805 (-HW<); ‘H and “CNMR: see Tables 1 and 2; EIMS m/z (rel. int.): 454.3449 (34) [M] + (talc. for &H,,O,: 454.3447). 439 (5) [M -Me]+ (ion a), 436 (7) [M-H,O]+ (b), 421 (100) [M-Me -H,O]+ (c), 327 (3) (f), 315 (3) (g), 314 (3) (b), 299 (12) (i), 273 (5) (j), 237 (4) (k), I89 (9) (I), 187 (25) (m), 139 (14) (a) and 97 (46) (0). CD: [0]2,0 -26000” [01z15 -29OW (trough). [B],,, - 19ooo” and [f&,, -4000”. Acetylarion ofcompound 2. Compound 2 (21 mg) was acetylated (Ac,O-pyridine, 1: 1, 1 ml) at room temp. for 24 hr. Usual work-up afforded a residue, which was subjected to prep. TLC (2 mm thick, 20 x 20 cm; solvent: CHCl,-EtOAc 20: 1) to give a

R. TANAKA and S. MATS~JNAW

1986

m/z 34I(5)

[Cz4Hl,0]’

n’ m/z 137(15) (CaHP02]’

0

m/z 297 (IO) .I I m/2185(12)

- HCOIH -

-co, -

+

m/z 187 ( 231 I’

m’

0' m/z 95 (40) [CIH,OZ]’

m/z 231(12)IC1~H~901] Scheme

2. Characteristic

mass spectral

solid. Recrystallization of the solid from MeOHCHCI, furnished 3/?-acetoxy-9~-lanosta-7,24-dien-26,23R-olide (2a) (20 mg) as needles, mp 28l-283.5’, [z];’ -43.5” (c 0.34); IR Y,,,_ cm-‘: 3035, 2930, 2865, 1750sh. 1735. 1705, 1655. l650sh. 1460, 1430, 1382, 1373, 1240, 1093, 1052. 1018. 968, 937. 888,872,827, 780 and 755; ‘H and *jCNMR: see Tables I and 2. EIMS m/z (rel. mt.): 496 (11) [Ml’, 481 (13) [M-Me](ion a). 436 (9) [M -HOAc]~(b),421(100)[M-Me-HOAc]f(c),315(5)(j),299 (10) (i). 233 (4) (k), I89 (7)(l), I87 (16)(m), 139 (3) (n) and 97(S) (0). Reduction ojcompound I wth NaBH,. A soln of NaBH, (6 mg) in MeOH (IO ml) was gradually added to a soln of compound I (I8 mg) in MeOH (35 ml) under stirring and the mixt. was kept at room temp. for 18 hr. After one drop of HOAc was added to destroy excess NaBH,. the mixt. was evapd in wcuo to give a residue, which was dissolved in Et,0 (30 ml) and the Et,0 soln was washed with H,O, dried with Na,SO, and filtered. Removal of the solvent afforded a solid (I8 mg), which was purified by prep. TLC (plate: 2 mm thick, 20 x 2Ocm; solvent: CHCI,-EtOAc, 5: I) to give 3/I-hydroxy-9/I-lanosta-7,24-dien26,23R-olide, mp 239-241’ (MeOH), as prisms (I2 mg). It was identified by direct comparison (mmp, co-TLC. IR, ‘H and 13C NMR and EIMS) with compound 2. Compound 3. Mp 216-218, (MeOH), [z]g +27.O’ (c 0.27), R, 0.3 I (plate 0.25 mm thick, C,H,-CHCI,-EtOAc, 1 : I : I); IR v,,,_ cm - ’ : 3445 (OH), 3048, 2952, 2925, 2877, 1740 (q!Iunsaturated y-lactone), 1692 (6-membered ring CO), 1640 and 1625 (>C=C<), 1467, 1382, 1198. 1052. 1037,940 and 812; ‘H and ‘-‘C NMR, see Tables I and 2; EIMS m.‘; (rcl. int.): 468.3243

+ fragmentation

of compound

3.

(3) [M] + (talc. for C30H4404: 468.3239). 453 (24) [M-Me] + (Iona). 450(23)(b’),435 (lOO)(c’), 423 (12)(d), 341 (S)(e), 325 (54) (I), 313 (S)(g). 312(S)(h’). 297(lO)(i’), 271 (II)(j),231 (12)(k’). I87 (23) [C,,H,,J* (I’), I85 (12) [C,,H,,]’ (m’), 137 (15) [C,,H,O,](n’). IO9 (28) [C,H,O,]+ (p) and 95 (40) [C,H,02J(0’). CD: [Olros -8000’. [O],,, -2SCOO’(trough), and [Olz,,, -5SW. [Ul*,, -2looo’ Acknowled~emenf -The authors are grateful to Mrs M. Nabae and Miss M. Kurlmoto. ex-members of this University, for NMR and MS measurements.

REFERENCES I. Uyeo, S.. Okada. Tetrahedron 2. Allen, 3. 4. 5.

6. 7.

J.. Matsunaga,

S. and Rowe, J. W. (1968)

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F. A.. Isaacs, N. W., Kennard, 0. and Motherwell, W. D. S. (1973) J. Chem. Sot.. Perkin Trans I 3463. Roshchln, V. 1.. Raldugin, V. A.. Baranova, R. A. and Pentegova. V. A. (1986) Khim. Prir. Soedin. 648. Raldugm, V. A.. Gatilov, Yu. V., Rybalova, T. V. and Rashkes, Ya. V. (1986) Khim. Prir. Soedin. 688. Hasegawa, S., Kaneko. N. and Hirose, Y. (1987) Phytochrmistry 26, 1095. Tanaka, R.. lnosturi. A., Yoneda, M., Ishida. T., Numada, A. and Matsunaga. S. (I 990) Phyrochetmstrg 29, 3263. Tanaka. R. and Matsunaga. S. (1990) Phytochemistry 29, 3267.

Tritcrpene lactones from Abies firma 8. Muller, J.-C. and Our&on, G. (1974) Phytochemistry 13, 1615. 9. Kutney, J. P., Grierson, D. S., Knowles, G. D., Westcott, N. D. and Rogers, I. H. (1973) Tetrahedron 29, 13. 10. Steglich, W., Klaar, M., Zechlin, L. and Hecht, H. J. (1979) Angew. chenl. 91, 751. 11. Hasegawa, S., Miura, T., Hirose., Y. and Iitaka, Y. (1985)

1987

Chemistry Letters 1589. 12. Hasegawa, S., Miura, T., Kaneko, N., Hirose, Y. and Iitaka, Y. (1987) Tetrahedron 43. 1775. 13. Raldugin, V. A., Shevtsov, S. A., Yaroshenko, N. I., Gatilov, Yu. V., Bagryanskaya, 1. Yu.. Demenkova, V. A. and Pentegova, V. A. (1987) Khim. Prir. Swdin. 824.