Stilbenoids from Bletilla striata

003l-9422/93$6.00+ 0.00 0 1993PergamonPressLtd

1993 Phytochemutry,Vol.33,No. 6, PP. 1481-1483, Pnnted in Great Britam.

STILBENOIDS FROM BLETILLA

STRIATA*

LI BAI, TOMOKO KATO, KEIKO INOUE, MASAE YAMAKIt and SHUZO TAKAGI Faculty of Pharmaceutical Sciences,Mukogawa Women’s University, 1l-68 Koshien Kyuban-cho, Nishinomiya Hyogo 663, Japan (Received 5 January 1993)

Key Word Index-Bletilla drophenanthrene.

striata;

Orchidaceae; bibenzyl; dibenzyldihydrophenanthrene;

benzoyldihy-

Abstract-Five new stilbenoids, three bibenzyls, one dibenzyldihydrophenanthrene and one benzoyldihydrophenanthrene, were isolated from tubers of Bletilla striata. The new structures were elucidated as 3,3’-dihydroxy-4-@ hydroxybenzyl)-S-methoxybibenzyl, 3,3’-dihydroxy-2-(p-hydroxybenzyl)-5-methoxybibenzyl, 3’,5-dihydroxy-2-(p2,7-dihydroxy-l,3-bi(p-hydroxybenzyl)-4-methoxy-9,1O-dihydrophenanthrene hydroxybenzyl)-3-methoxybibenzyl, and 2,7-dihydroxy-l-(phydroxybenzoyl)-4-methoxy-9,1O-dihydrophenanthrene, by their spectroscopic data.

INTRODUCTION In the course of our investigations on the constituents of Bletilla striata, we have isolated and characterized bibenzyls [l, 21, phenanthrenes [l-3, 6, 73, biphenanthrenes [2, 31, bis(phenanthrene)ethers [4, 51, dihydrophenanthropyrans [S], phenanthrene glucosides [9] and phenanthrenylspirolactone [lo] from this source. We now report on the structural elucidation of three bibenzyls (l-3), one dibenzyldihydrophenanthrene (4) and one benzoyldihydrophenanthrene (5).

OH 2

R,=OH

f+O%

3

R,=OCHJ

R&H

RESULTSAND DISCUSSION Compound 1 was obtained as needles, mp 171-173”. Its UV spectrum showed absorption maxima at 278 and 283 nm suggesting 1 to be a bibenzyl Cl]. The IR spectrum exhibited absorptions at 315O(OH), 1590 and 1450 cm - i (benzenoids). The mass spectrum exhibited a [M]’ at m/z 350 (C,,H,,O,) and significant peaks at m/z 243, resulting from the loss of a hydroxybenzyl group (C,H,O) from the [Ml’, and at m/z 107 ([C,H,O]‘). Acetylation of 1 afforded a triacetate (CM]’ m/z 478) indicating the presence of three hydroxyl groups. The ‘HNMR spectrum showed two doublets at 66.59 (2H) and 7.01 (2H) due to an A,B, system characteristic of a p-substituted aromatic ring, and one singlet at 6 3.79 due to a benzylic methylene, suggesting the presence of a p-hydroxybenzyl group. Additionally, the ‘H NMR spectrum exhibited signals of a singlet at 63.69 due to one methoxyl group and multiplets at 62.78 for four protons of an ethylene linkage of bibenzyl derivatives [ 1,7], along with signals for six protons on two benzene rings of

*Part 11 in the series on the constituents of Bletilla striata. For part 10 see ref. [lo]. t Author to whom correspondence should be addressed.

bibenzyl (Table 1).Two of the six protons appeared as a pair of doublets at 66.25 and 6.30 due to H-2 and H-6 in one aromatic ring; the remaining four protons resonating at 66.58,6.61,6.64 and 7.04 were assignable to H-4’, H-2’, H6’ and H-5’ in the other aromatic ring from their chemical shifts and splitting patterns. The signal assignments and the location of the functional groups were confirmed by NOE enhancement and acetylation shifts. Irradiation of the ethylene protons enhanced the signals for H-2 (ll%), H-6 (14%) and H-2’ (11%), H-6’ (8%). On irradiation of the methoxyl group no other signals except H-6 (11%) were enhanced. In turn, irradiation of the benzylic methylene caused enhancement of H-2” and H-6” (10%). These results show that the methoxyl group is attached to C-5, the p-hydroxybenzyl group to C-4, and hence two hydroxyl groups to C-3 and C-3’. Thii substituent pattern was consistent with the observations that the signals due to H-2, H-2, H-4’ and benzylic methylene were shifted largely to lower fields in the ‘HNMR spectrum of the

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LI BAI et al.

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Table 1. ‘H NMR data of 1-3 and their acetates 1

1 acetate

2

2 acetate

3

3 acetate

6.25 d (1.3)*

6.52 d (1.3) 6.56 d (1.3) 6.92 mt

-6.30 d (2.6) 6.24 d (2.6) 6.54 t (2.3)

6.65 d (2.7) 6.54 d (2.7) 6.78 t (1.9)

6.33 d (2.1) 6.28 d (2.1) 6.54 mt

_. 6.58 d (2.1) 6.55 d (2.1) 6.80 t (2.3)

6.92 mt 7.27 d (7 7) 7.02 d (7.7) 7.15 6.91 d (8.6) (8 6) 2.90 m 3.85 s 3.73 s 2.20 s, 2.26 s 2.28 s

6.56dd (7.9. 2.3) 7.03 t (7.9) 6.56 dd (7.9, 2.3) d 6.93 6.64 d (8.6) 2.55 m, 2.72 m 3.87 s 3.70 s -

6.9Odd (7.9, 1.9) 7.24 t (7.9) 6.90 dd (7.9. 1.9) 7.05 d 6.95 d (8.6) 2.71 m, 2.81 m 3.83 s 3.76 s 2.14 s, 2.26 s 2.28 s

2 4 6 2 4

6.30 d (1.3) 6.61 t (2.0) 6.58 dd (7.5, 2.0)

5’ 6

7.04 t (7.7, 7.5) 6.64 d (7 7)

2” 6” 3”:5”

-CH,-CH,benzylic-CH,OMe OCOMe

6.59 d (8.6) 7.01 2.78 m 3.79 s 3.69 s -

6.54mt 7.03 t (8.2) 6.57 dt (8.2, 1.7) 6.86 d 6.63 d (8.6) 2.56 m. 2.71 m 3.84 s 3.74 s

6.90 dd (7.8, 2.3) 7.24 t (7.8) 6.90 dd (7 8, 2.3) 7.07 d 6.93 d (8.6) 2.10 m, 2.83 m 3.99 s 3 16 s 2.26 s. 2.28 s 2.29 s

*Coupling constant (J in Hz) are given in parentheses. p Unresolved.

triacetate. On the basis of the above spectral evidences, the structure of 1 is established as 3,3’-dihydroxy-4-(phydroxybenzyl)-Smethoxybibenzyl. Compound 2 was obtained as needles, mp 160-161”. The UV spectrum showed absorption maxima at 227sh and 282 nm, and the IR spectrum showed the presence of hydroxyl groups and benzenoids. The mass spectrum of 2 exhibited the same [M]’ and two intense peaks as in 1, and suggested that 2 might be a p-hydroxybenzylbibenzyl in which the substitution pattern of the functional groups was different from that of 1. On comparing the ‘H NMR spectrum of 2 with that of 1, the most striking difference between them is the presence of a pair of split multiplets at 62.55 (2H) and 2.72 (2H) assignable to two methylenes of bibenzyl (&CH,-CH,-$). It was presumed that any substituent was located at either C-2 or C-6. This assumption was supported by WOE enhancements. In the NOE experiments, irradiation of the methoxyl group at 63.70 enhanced the signals from H-4 (8%) and H-6 (8%) indicating that the methoxyl group was at C-S. On the other hand, on irradiation of the benzylic methylene protons at 63.87, NOES were observed upon two signals, i.e. the H-2” and 6” (14%), methylene groups at 62.55 (3%) and 2.72 (7%), confirming that the p-hydroxybenzyl group is located at C-2. From the above spectral evidences, the structure of 2 was concluded to be 3,3’dihydroxy-2-(p-hydroxybenzyl)-Smethoxybibenzyl. Compound 3 was obtained as a powder and showed a [M]’ at m/z 350 (C,,H,,O,) in the mass spectrum. The spectral data of 3 (UV, IR, ‘H NMR) were very similar to those of 2, and indicated that 3 might be a structural isomer of 2. NOE enhancement was used to determine the positions of the functional groups. Irradiation of the methoxyl group at 63.74 enhanced the signals from both H-4 (21%) and H-2”, H-6” (2%). This finding indicated that the p-hydroxybenzyl group is at C-2 and the methoxyl group at C-3. Thus, the structure of 3 was assigned to be 3’,5-dihydroxy-2-(p-hydroxybenzyl)-3-methoxybibenzyl.

Compound 4 showed UV maxima at 204 and 282 nm resembling those of dihydrophenanthrene derivatives [3, 61. The IR spectrum had absorptions at 3250 (OH), 1590 and 1480 cm-’ (benzenoids). The mass spectrum exhibited a CM]’ at m/z 454 (CZ9HZ605) and two significant peaks at m/z 359 and 254 formed by sequential loss of one molecule of a hydroxyphenyl group and one molecule of a hydroxybenzyl group, respectively. Acetylation of 4 afforded a tetraacetate ([M] ’ m/z 622) indicating the presence of four hydroxyl groups. The ‘H NMR spectrum showed four doublets at 66.71 (2H), 6.75 (2H), 6.99 (2H) and 7.14 (2H) due to two pairs of an A,B, system characteristic of a p-substituted aromatic ring, and two singlets due to benzylic methylenes, supporting the presence of two p-hydroxybenzyl groups. Furthermore, the ‘H NMR spectrum showed signals for a doublet at 68.21 due to H-5, a double of doublets at 66.70 due to H-6, and a doublet at 66.73 due to H-8, together with one ethylene group due to H-9, 10 of a dihydrophenanthrene group and one methoxyl group. Irradiation of the methoxyl group at 63.46 enhanced the signals of H-5 (7%), H-2”, H-6” (3%) and the benzylmethylene at 64.07 (2%), while irradiation of the benzylic methylene at 63.98 caused enhancement of H-2’, H-6’ (lo%), and H-10 (9%). These results showed that the methoxyl group was placed at C-4 and that the p-hydroxybenzyl groups were located at C-l and 3. Thus, 4 is 2,7-dihydroxy-1,3-bi(phydroxybenzyl)-4-methoxy-9,1O-dihydrophenanthrene. Compound 5 showed UV maxima at 210, 279 and 295sh nm, and IR absorptions at 3250 (OH), 1690 (carbony]), 1590 and 1480cm-’ (benzenoids). The mass spectrum exhibited a [M]’ at m/z 362 (C,,H,,OS) and major fragment ions at m/z 268, due to removal of a hydroxyphenyl group from [Ml’, and at 121 due to a hydroxybenzoyl moiety. Acetylation of 5 afforded a triacetate ([M] + m/z 488). The ‘H NMR spectrum showed signals due to one methoxyl group, one p-substituted phenyl group and signals for four aromatic protons, a doublet at 68.01 due to H-5, a double of doublets at 66.63

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Stilbenoids from BIetilla striata

due to H-6, a doublet at 66.59 due to H-8, and a singlet at 66.52 due to H-3, along with two multiplets at 62.52 and 2.40 assignable to H-9, 10 of a dihydrophenanthrene group. On the p-substituted phenyl group, H-2’ and 6 (67.72, 2H) showed a striking down-field shift of 0.6-0.7 ppm from their usual values [1, 3, 61, supporting the presence of a hydroxybenzoyl group. The position of the methoxyl group is based on the observation of the NOE enhancements. Thus, irradiation of the methoxyl group at 63.89 gave enhancements of the signals of H-3 (22%) and H-S (3%), confirming that the methoxyl group is located at C-4. The structure of 5 was also supported by the 13CNMR spectrum. From the above spectral evidences, 5 is assigned to be 2,7-dihydroxy-l-(phydroxybenzoyl)-4-methoxy-9,1O-dihydrophenanthrene. EXPERIMENTAL

Mps: uncorr.; IR: KBr; UV: MeOH; ‘HNMR and 13CNMR: 500 and 125 MHz, respectively, MeOH-d, with TMS. MS; EIMS, 70 eV. CC and TLC were performed using Merck silica gel. Plant materials. See ref. [2]. Extraction and isolation. See ref. [2]. Fr. 4 and 5 were rechromatographed over silica gel to give 1 (7 mg), 2 (11 mg), 3 (113 mg), 4 (41 mg) and 5 (17 mg). Compound 1. Colourless needles from CHCl,-MeOH, mp 171-173”. IR v,,% cm -I: 3150, 1590, 1450, 1420; UV I,, nm (log E): 278 (3.83), 283 (3.83); ‘H NMR: Table 1; MS m/z (rel. int.): 350 (lOO), 243 (79), 225 (26), 107 (39). Triacetate: oil. ‘H NMR: Table 1; MS m/z (rel. int.): 476 (5), 434 (82), 392 (lOO), 350 (39), 107 (50). Compound 2. Colourless needles from CHCl,-MeOH, mp 16&161”. IR v,,, cm -I: 3100, 1590, 1495, 1420, UV A,,,,, nm (log E):227sh (4.38), 282 (3.94); ‘H NMR: Table 1; MS m/z (rel. int.): 350 (lOO),256 (16), 243 (48), 229 (56), 107 (50). Triacetate: oil. ‘H NMR: Table 1; MS m/z (rel. int.): 476 (5), 434 (lOO), 392 (84), 350 (25). Compound 3. Powder. IR v,,, cm- l: 3200, 1590, 1495; UV A,,,,, nm (log E): 230sh (4.36), 283 (3.94); ‘H NMR: Table 1; MS m/z (rel. int.): 350 (lOO),256 (25), 243 (40), 107 (50). Triacetate: oil. ‘H NMR: Table 1; MS m/z (rel. int.): 476 (46), 434 (lOO), 392 (87), 350 (28), 243 (25), 107 (35). Compound 4. Powder. IR v,,, cm-‘: 3250,1590,1480; UV 1,, nm (log E): 204 (4.80), 282 (4.51); ‘HNMR: 62.642.69 (4H, m, -CH,-CH,-), 3.46 (3H, s, OMe), 3.98 (2H, s, benzylic-CH,-), 4.07 (2H, s, benzylic-CH,-), 6.70 (lH,dd,J=8.6,2.3 Hz, H-6), 6.71 (2H,d, J=8.5 Hz, H-3’, 5’),6.73(1H,d,J=2.3Hz,H-8),6.75(2H,d,J=8.5Hz,H3”, 5’3, 6.99 (2H, d, .I= 8.5 Hz, H-2’, 6’), 7.14 (2H, d, J=8.5 Hz,H-2”,6”),8.21 (lH,d,J=8.6 Hz,H-5);MSm/z (rel. int.): 454 (lOO), 359 (28), 343 (20), 239 (3), 107 (20). Tetraacetate: powder. MS m/z (rel. int.): 622 (18), 580

(lOO), 538 (60), 496 (lo), 454 (l), 107 (14); ‘HNMR (CDCI,): 62.11,2.25,2.26,2.29 (3H, s, OAc x 4), 2.59-2.68 (4H, -CH,-CH,-), 3.36 (3H, s, OMe), 3.90 (4H, s, 2 benzylic-CH,-), 6.93695 (2H, m, H-6, 8)*, 6.96 (2H, d, J =8.6 Hz, H-3’, 5’), 6.98 (2H, d, J=8.6 Hz, H-3”, 5”), 7.06 (2H, d, .J=8.6 Hz, H-2’, 6’), 7.22 (2H, d, J=8.6 Hz, H-2”, 6”), 8.30 (lH, d, J=8.1 Hz, H-5). Compound 5. Yellow powder. IR v,,cm-‘: 3200, 1690, 1560, 1480; UV A,,,,, nm (log E): 210 (4.64), 279 (4.55), 295sh (4.53); ‘H NMR: 62.40 (2H, m, -CH,-), 2.52 (2H, m, -CH,-), 3.89 (3H, s, OMe), 6.52 (lH, s, H-3), 6.59 (lH, d, J =2.7 Hz, H-8), 6.63 (lH, dd, J=8.6, 2.7 Hz, H-6), 6.81 (2H, d, 5=8.8 Hz, H-3’, S), 7.72 (2H, d, 5=8.8 Hz, H-2’, 6’), 8.01 (lH, d, J = 8.6 Hz, H-5); “C NMR: 628.7 (t, C-9 or C-lo), 30.6 (t, C-9 or C-lo), 56.1 (q, OMe), 99.3 (d, C-3), 113.8 (d, C-6), 114.8 (d, C-8), 116.3 (d, C-3’, 53, 117.5 (s, C4a), 120.4(s,C-5a), 120.5(s,C-1), 130.5(d,C-5), 131.6(s,Cl’), 133.6 (d, C-2’, 6’), 139.1 (s, C-la), 140.4 (s, C-8a), 155.1 (s, C-4), 156.6 (s, C-4’), 159.7 (s, C-7), 164.4 (s, C-2), 199.7 (s, CO); MS m/z (rel. int.): 362 (lOO),268 (47), 240 (3), 121(27). Triacetate: powder. ‘HNMR: 61.93, 2.29, 2.31 (3H, s, OAc x 3), 2.56 (2H, m, -CH,-), 2.66 (2H, m, -CH,-), 3.92 (3H, s, OMe), 6.76 (lH, s, H-3), 6.94 (lH, d, J=2.6 Hz, H8), 7.00 (lH, dd, J=8.6, 2.6Hz, H-6), 7.17 (2H, d, J =8.8 Hz, H-3’, 5’), 7.85 (2H, d, J=8.8 Hz, H-2’, 6’), 8.26 (lH, d, J=8.6 Hz, H-5); MS m/z (rel. int.): 488.{30), 446 (95), 404 (lOO), 362 (12), 345 (32), 268 (18). *Peaks are overlapped and not resolvea. REFERENCES

1. Takagi, S., Yamaki, M. and Inoue, K. (1983) Phytochemistry 22, 1011. 2. Yamaki, M., Bai, L., Inoue, K. and Takagi, S. (1989) Phytochemistry 28, 3503. 3. Bai, L., Kate, T., Yamaki, M., Inoue, K. and Takagi, S. (1991) Phytochemistry 30, 2733. 4. Bai, L., Yamaki, M., Inoue, K. and Takagi, S. (1990) Phytochemistry 29, 1259. 5. Yamaki, M., Bai, L., Kate, T., Inoue, K., Takagi, S., Yamagata, Y. and Tomita, K. (1992) Phytochemistry 31, 3985. 6. Yamaki, M., Bai, L., Inoue, K. and Takagi, S. (1990) Phytochemistry 29, 2285. 7. Yamaki, M., Kate, T., Bai, L., Inoue, K. and Takagi, S. (1991) Phytochemistry 30, 2759. 8. Yamaki, M., Bai, L., Kato, T., Inoue, K. and Takagi, S. (1993) Phytochemistry 32, 427. 9. Yamaki, M., Kate, T., Bai, L., Inoue, K. and Takagi, S. (1993) Phytochemistry (in press).

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