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Longirabdolides A and B, 6,7-seco-ent-kaurenoids from Rabdosia longituba
Phytochemistry, Vol.
0031-9422(93)E011- 3
Pergamon
LONGIRABDOLIDES
35, No. 5, pp. 1275 1278, 1994 Copyright 9 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0031-9422/94 $6.00 + 0.00
A A N D B, 6,7-SECO-ENT-KAURENOIDS F R O M
RABDOSIA LONGITUBA YOSHIO TAKEDA,* TAKASHI MATSUMOTOand HIDEAKI OTSUKA*'~ Faculty of Integrated Arts and Sciences,The University of Tokushima, Tokushima 770, Japan; tInstitute of Pharmaceutical Sciences, Hiroshima University School of Medicine, Minami-ku, Hiroshima 734, Japan
(Received 23 August 1993) Key Word Index--Rabdosia longituba; Labiatae, longirabdolides A and B; 6,7-seco-ent-kaurenoid.
Abstract--Two new diterpenoids, longirabdolides A and B, were isolated from the aerial parts of Rabdosia longituba, together with the known compounds, isolongirabdiol, effusanin E, oridonin and nodosin. The structures of the new compounds were elucidated mainly by spectroscopic methods.
O
12
INTRODUCTION
R 2 " I ' ~
Rabdosia longituba (Miq.) Hara El.1 contains many kinds of ditcrpcnoids having ent-kaurene and 6,7-seco-entkaurenc skeletons [2-9-1, many of which show cytotoxic and antibacterial activities 1-10.1.During the course of our studies on the biologically active substances from Labiatac plants, we examined the constituents of the aerial parts of the title plant collected in Hiroshima Prefecture, Japan and isolated two new diterpcnoids, which we have named longirabdolide A (1) and longirabdolidc B (5), together with the known compounds, isolongirabdiol (4) r8.1, cffusanin E (8) 1.11.1,oridonin (9) 1.12.1and nodosin (10) 1.13.1. This paper describes the characterization of the new compounds.
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t~-
1
6
(5) RI=Ra--H ; Fr~---CI-~ (6) RI=R3=H ; FI==cx-CHa,~H (7) Rl=R3'---Ac; R2=C1"12
0
o II _OH
I ,.
I[
I"~
RESULTS AND DISCUSSION
Compound 1, [~]v+ 39-1~ (MeOH) was obtained as an amorphous powder and the molecular formula was assigned as C2oH2aO6 (HRMS). It contains an exo-methylene group conjugated with a carbonyl group on a fivemembered ring t.v-l-llV-max~MeOH 229 nm (e 5674); IK'~Fma xKBr 1740 and 1640 cm-1; 1H NMR (CsDsN): c55.35 and 5.98 (each 1H, br s, H2-17); 13CNMR (CsDsN): &116.4 (t), 151.4 (s) and 202.7 (s)], a 6-1actone group [Vm,x 1710cm-1; 6c172.2 (s)] a tertiary methyl group [tSn0.95 (3H, s); 6c 18.2 (q)], three primary carbinyl groups of which one is involved in the formation of a 6-1actone moiety [6.4.99 (2H, br s); 6c 70.8 (t)] and two in hydroxyl groups [6.4.00 ( 1H, dd, J = 5.5 and 11.9 Hz) and 4.10 (1H, dd, J = 2.9 and 11.9 Hz), and 3.38 and 3.83 (each 1H, d, J=10.8 Hz); 6c58.6 (t) and 71.5 (t)], and a secondary carbinyl group [6,.15.06 (1H, m); &c65.2 (d)] as partial structures. The ~3C
*Authors to whom correspondence should be addressed.
"
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OH
OH
(8) RlffiOH; R2=H (9) RI=H ; R2--OH
(10)
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o p 0
(11)
:"
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(12)
NMR spectrum (Table 1) of I further shows the presence of five methylene groups, three methine groups and three quaternary carbon atoms in addition to the aforementioned signals. These spectral data, coupled with the
1275
1276
Y. TAKEDAet al. Table 1. 13CNMR data* of 1 and 5
C
1
5
1 2 3 4 5 6 7 8 9 10 I1 12 13 14 15 16 17 18 19 20
30.3"t 18.2 37.0 39.2 45.4 58.6 172.2 58.1 48.5 44.3 65.2 42.5 35.2 30.4~" 202.7 151.4 116.4 71.5 18.2 70.8
81.0 25.3 40.0 34.8 51.1 60.7 172.2 55.6 43.7 46.3 65.6 42.3 35.3 34.6 199.6 151.4 117.5 34.0 22.7 60.5
*The spectra were measured in CsDsN and the chemical shifts are given in ppm (6) relative to the internal TMS. tThe assignments may be interchanged. consideration on the structures of closely related compounds, suggested that 1 has an ent-spiro-seco-kaurene skeleton (11) as a basic skeleton and has a structure in which three hydroxyl groups are introduced into 11. Compound ! gave the triacetate 2 [6u 1.99, 2.09 and 2.10 (each 3H, s)-I on acetylation with Ac20 and pyridine. Catalytic hydrogenation of 1 over Pd-C gave the dihydro-compound 3 [3,1.12 (3H, d, J = 6 . 6 Hz)] which showed a negative Cotton effect [Ae3o5 -0.70 (MeOH)] in the CD spectrum, proving the supposed absolute stereochemistry [14, 15]. A secondary hydroxyl group was assigned to C-11 by following the cross-peaks starting from 6,3.38 (H-9) to 5.06 (the signal due to a proton on a carbon having a hydroxyl group, H-11), 1.86 (HI2//), 2.43 (H-12ct) and 3.15 (H-13), successively, in the t H COSY spectrum. The stereochemistry of the hydroxyl group was assigned as ~ based on the fact that the signal assigned to H-14~t [6,3.68 (d, J = 11.6 Hz)] suffered an abnormal downfield shift and the signal at 6 5.06 crossed peaks with the signal at 6 2.89 (1H, m) assigned to H-I//in the tH NOESY spectrum (Fig. 1). Judged from the coupling patterns, two of the primary carbinyl groups should be located adjacent to a methine group and a quaternary carbon atom, respectively. The possible positions are at C-6 and at either C-18 or C-19. The positions were finally determined as being at C-6 and C-18 based on the results of tH NOESY experiments (Fig. 1). Namely, the signal at 6 4.99 (H2-20) crossed peaks with the signals at 6n0.95 (H3-19) and 4.00 (H~-6). Thus, the structure of longirabdolide A was elucidated as 1. Compound 5, [ct]D -52.5 ~(MeOH) was obtained as an amorphous powder and has the same molecular formula,
. OH
H
F"\
2
11
~O
Fig. 1. Summary of the results of 1HNOESY spectrum for longirabdolide A (1).
C2oH2sO6, as that of 1. This compound contains an exomethylene group conjugated with a carbonyl group on a five-membered ring (see Experimental), two tertiary methyl groups [6nI.28 and 1.35 (each 3H, s); 6c22.7 and 34.0 (each q)], a 6-1actone [Vma~ 1700; 6n5.71 (1H, dd, J =4.9 and 12.2 Hz); 6c81.0 (d) and 172.2 (s)], two methylene groups having a hydroxyl group [6.4.30 and 4.34 (each 1H, d, J=11.8 Hz), and 4.33 (1H, dd, J = 5 . 2 and 12.2 Hz) and 4.50 (1H, dd, J = 2.1 and 12.2 Hz); 6 c 60.5 and 60.7 (each t)] and a methine group having a hydroxyl group [3,5,19 (1H, m); 6c65.6 (d)]. The 13CNMR spectrum of 5 (Table 1) showed, in addition to the signals mentioned above, the presence of four methylene groups, three methine groups and three quaternary carbon atoms. The spectral data suggested that 5 has an ent-6, 7seco-kaurene skeleton (12) as a basic skeleton and has a structure in which the three hydroxyl groups, i.e. two primary and a secondary hydroxyl groups, are introduced into a basic skeleton (12). Catalytic hydrogenation of 5 over Pd-C gave the dihydro-compound 6 which showed a negative Cotton effect 1-A~3o9.6--0.20 (MeOH)] in the CD spectrum, proving the absolute stereochemistry [14, 15]. Acetylation of 5 gave the diacetate 7 [6 a 2.03 and 2.09 (each 3H, s)'l. In the 1H NMR spectrum of 7, the signal due to a proton on the carbon having a secondary hydroxyl group did not show any downfield shift. This fact suggested that a hydroxyl group is located on a sterically hindered position and is presumed to be located at C-11fl, because it is known that this position in this carbon skeleton is hardly acetylated on treatment with Ac20 and pyridine [-10]. The suggestion was confirmed by 1H COSY and 1H NOESY experiments. Starting from the signal at 6 2.92 (H-9), the cross-peaks can be followed to 6 5.19 (H-11), 1.75 (H-12~), 2.46 (H-12//) and 3.12 (HI3), successively, in the 1HCOSY spectrum, and the signal of the H-11 crossed peaks with that of H-5 [32.63 (1H, dd, J=2.1 and 5.2 Hz)] in the IH NOESY spectrum (Fig. 2). One of the two primary hydroxyl groups should be located at C-6 judged from its coupling pattern. Another primary hydroxyl group was indicated to be located at C-20 based on the observations that the proton signals at 6,4.30 and 4.34 crossed peaks with that at 6c46.3 (C-10) in the ~H-13C long range COSY (d = 10 Hz) spectrum. The suggestion was confirmed based on the results of the ~H NOESY experiment shown in Fig.
Longirabdolides A and B from Rabdosia longituba
1277
(200 ml), CHC13-MeOH 97: 3 (200 ml), CHCI3-MeOH 24: 1 (100 ml), CHC13-MeOH 19 : 1 (1 1), CHC13-MeOH 47 : 3 (500 ml) and CHC13-MeOH 93 : 7 (500 ml), 8-ml frs H H/ were collected. Frs 81-83 gave 9 (42 mg). Frs 94-105 gave a residue (51 rag) which was purified by prep. TLC (CHC13-MeOH 19:1, developed twice and then CHC13-MeOH 9 : 1) to give 1 (37 rag). Compounds 4, 8, 9 and 10 were identified by direct comparison with authentic samples. Longirabdolide A (1). Amorphous powder, [~]~2 19OH ~ ~'OH + 39.1 ~ (MeOH; c 1.05); ~ITV nm (e): 229 (5674); IR - - JMeOH "'max KBr Fig. 2. Summary of the results of ~HNOESY spectrum for Vmax c m - 1. 3400 (br.), 1740, 1710, 1640, 1550, 1460, 1400, 1360, 1305, 1270, 1240, 1120, 1040, 1025 and 930; longirabdolide B (5). 1H NMR (CsDsN, 400 MHz): 6 0.95 (3H, s, H3-19 ), 1.37 (1H, br s, J = 13.1 Hz, H-3~), 1.52 (1H, dr, J = 13.8 and 2. Thus, the structure of longirabdolide B was elucidated 3.4 Hz, H-2fl), 1.74 (1H, m, H-2al), 1.86 (1H, dd, J = 4 . 7 and 14.5 Hz, H-12/~), 1.99 (1H, ddd, J = 13.1, 13.1 and as 5. Among the diterpenoids isolated so far from R. long- 4.0 Hz, H-3fl), 2.24 (1H, br d, J = 14.3 Hz, H-I~), 2.43 (1H, ituba, many were oxidized at C-19. On the other hand, the dd, J = 9 . 3 and 14.5 Hz, H-12~), 2.65 (1H, dd, J = 11.6 and compounds oxidized at C-18 are few, including 1 [-8, 9, 3.8 Hz, H- 14/~), 2.88 (1H, dd, J = 5.5 and 2.9 Hz, H-5), 2.89 (1H, m, H-l/3), 3.15 (1H, dd, J = 3.8 and 9.3 Hz, H-13), 3.38 161. (1H, br d, J=3.1 Hz, H-9), 3.38 (1H, d, J = 10.8 Hz, H118), 3.68 (1H, d, J = l l . 6 H z , H-14~t), 3.83 (1H, d, d EXPERIMENTAL = 10.8 Hz, H1-18), 4.00 (1H, dd, J = 5 . 5 and 11.9 Hz, H 1General. NMR: 1H (200 or 400 MHz) and x3C 6), 4.10 (1H, dd, J = 2 . 9 and 11.9 Hz, H1-6 ), 4.99 (2H, br s, (100 MHz); ELMS: 70 eV; CC: silica gel 60 (Merck); prep. H2-20), 5.06 (1H, m, H-11), 5.35 and 5.98 (each 1H, br s, TLC: silica gel 60 F254. H2-17), and 6.34 and 6.75 (each 1H, m, OH• Plant material. Plant material was collected in Geihoku 13CNMR: Table 1; ElMS m/z: 364.1862 I-M] + Town, Hiroshima Prefecture, Japan in late September, C2oH2sO 6 requires 364.1886. 1987 and identified as Rabdosia lonoituba (Miq.) Hara by Longirabdolide B (5). Amorphous powder, [ct] 22 one (H. O.) of the authors. A voucher specimen [RL-87- - 52.5 ~ (MeOH; c 0.88); UV 2um]~ nm (~): 229 (7064); IR Geihoku] was kept in the laboratory of H. O. Vmax~rcm-1.. 3400 (br), 1740, 1700, 1640, 1550, 1460, 1370, Isolation. Dried aerial parts (146 g) of R. lonoituba were 1305, 1280, 1190, 1150, 1060 and 940; 1H NMR (CsDsN, extracted with MeOH for 1 month at room temp. The 400 MHz): 61.28 (3H, s, H3-18), 1.35 (3H, s, H3-19 ), 1.46 extract was concd in vacuo and the residue was dissolved (1H, dt, J = 13.4 and 3.8 Hz, H-3ct), 1.53 (1H, ddd, J = 13.4, in 90% MeOH (330ml). The 90% MeOH soln was 13.4 and 3.4 Hz, H-3/~), 1.75 (1H, dd, J = 14.7 and 4.6 Hz, washed with n-hexane (300 ml x 3) and the 90% MeOH H-12~t), 1.87 (1H, m, H-2/3), 2.10 (1H, m, H-2~t), 2.26 (1H, br layer concd in vacuo. The residue was suspended in H 2 0 dd, J = l l . 3 and 4.6 Hz, H-I), 2.46 (1H, dd, J = 9 . 2 and (300 ml) and the suspension was extracted with EtOAc 14.7 Hz, H-12fl), 2.63 (1H, dd, J=2.1 and 5.2 Hz, H-5), (300 ml x 3). After being washed with H 2 0 and dried, the 2.92 (1H, d, J = 2 . 8 Hz, H-9), 3.12 (1H, dd, J = 4 . 6 and EtOAc extract was concd in vacuo to give a residue 8.9 Hz, H-13) 3.88 (1H, d, J = 11.3 Hz, H-14fl), 4.30 (1H, d, (3.99 g). An aliquot (3.80 g) of the residue was chromato- J = 11.8 Hz, H1-20), 4.33 (1H, dd, J = 5.2 and 12.2 Hz, H 1graphed over silica gel (200 g) with a mixt. of CHCI3 and 6), 4.34 (1H, d, J = 11.8 Hz, H1-20), 4.50 (1H, dd, J=2.1 Me2CO as eluant with increasing Me2CO content: and 12.2 Hz, H1-6 ), 5.19 (1H, m, H-11), 5.34 (1H, br s, H 1CHC13 (1.3 1), CHC13-Me2CO 19:1 (1 1), 17), 5.71 (1H, dd, J = 4 . 9 and 12.2 Hz, H-l), 5.91 (1H, m, CHC13-Me2CO 9:1 (1 1), CHC13-Me2CO 17:3 (11), OH), 6.00 (1H, br s, H1-17) 6.69 (1H, m, OH) and 7.09 (1H, CHC13-Me2CO 4:1 (1 1), CHCI3-Me2CO 7:3 (1 1), d, J = 3 . 7 H z , OH); 13CNMR: Table 1; EIMS m/z CHCla-Me2CO 3:2 (250 ml) and Me2CO (11). 364.1916 [M] +. C2oH2sO 6 requires 364.1886. Frs (75 ml) were collected. Longirabdolide A triacetate 2. Compound 1 (10 rag) Frs 57-59 gave a residue (352 mg), an aliquot (118 mg) was dissolved in a mixt. of pyridine (0.5 ml) and Ac20 of which was sepd by prep. TLC (Et20) to give 10, (0.5 ml) and the soln was left overnight at room temp. (19 mg). Excess MeOH was added to the soln and the solvent was Frs 64-78 gave a residue (149 mg) which was purified removed in vacuo to give a residue which was purified by by prep. TLC (Et20, developed 3 x ) to give 4 (92 mg): prep. TLC (CHCI3-Me2CO 17 : 3) to give 2 (6.5 mg) as an "cncl~ cm- 1.. 1745, 1645, 1240, Frs 79-84 gave a residue (121 rag) which sepd by prep. amorphous powder. IR Vm,x TLC (Et20, developed 4 x ). The faster moving zone gave 1220 and 1130; IH NMR (CDC13): 6 0.96 (3H, s, H3-19), 8 (15 mg) and the slower moving one 5 (60 rag). 1.99, 2.09 and 2.10 (each 3H, s, 3 • OAc), 2.62 (1H, d, J The eluate (366 mg) from 100% Me2CO was subjected = 3.5 Hz, H-9), 2.71 (1H, d, J = 11.2 Hz), 3.97-4.07 (2H), to CC (signal, 50 g) with mixts of CHC13 and MeOH 4.18 (1H, dd, J = 3 . 9 and 12.7 Hz, H1-6), 4.29 and 4.60 containing increasing amounts of MeOH, CHC13 (each 1H, d, J = 11.5 Hz), 5.53 (1H, br t, J = 3 . 5 Hz, H-11), OI4 \
1278
Y. TAKEDAet al.
and 5.58 and 6.09 (each IH, br s, H2-17); ElMS m/z: 490.2167 [M] +. Calcd for C26H3409:490.2203. Dihydrolongirabdolide A (3). Five percent Pd-C (10 mg) was added to the soln of 1 (10 mg) in MeOH (5 ml) and the mixt. was stirred in an arm. of H2 for 2 hr. After removing the catalyst by filtration, the solvent was removed in vacuo to give 3 (9.6 mg) as an amorphous powder. IR Vma, KBr cm-1:3400 (br), 1745, 1710, 1240 and 1030; 1H NMR (CsDsN): 60.97 (3H, s, H3-19), 1.12 (3H, d, J=6.6 Hz, H3-17 ), 2.93 (1H, t, J=4.4 Hz, H-5), 3.18 (IH, d, J=3.9 Hz, H-9), 3.42 (1H, d, J = 10.8 Hz, H1-18), 3.66 (IH, d, J = 10.8 Hz, H-14~t), 3.82 (1H, d, J = 10.8 Hz, HI-18), 4.09 (2H, m, H2-6), 4.95 (2H, br s, H2-20), 6.39 (2H, m, OH x2) and 6.75 (1H, m, OH); CD (MeOH, 5.246mM): Aeaos-0.70; EIMS m/z: 366.2055 [M] +. Calcd for C2oH3oO6: 366. 2042. Dihydrolongirabdolide B (6). Five per cent Pd-C (20 mg) was added to the soln of 5 (19 mg) in MeOH (5 ml) and the mixt. was stirred under an atm. of H 2 for 1.5 hr at room temp. After removing the catalyst by filtration, the filtrate was concd in vacuo to give a residue which was purified by prep. TLC (solvent: CHCIa-Me2CO 7 : 3) to KBr give 6 (10.8 mg) as an amorphous powder. IR Vmax cm- 1: 3400 (br), 1755, 1705, 1280 and 1075; tH NMR (CsDsN): 61.04 (3H, d, J = 6.6 Hz, Ha-17), 1.30 and 1.36 (each 3H, s, tert. Me x 2), 2.63 (IH, d, J=3.2 Hz, H-9), 2.78 (1H, dd, J =5.5 and 2.5Hz, H-5), 4.01 (1H, d, J = 11.2 Hz, H1-20), 4.29-4.47 (3H, H2-6 and H1-20), 5.08 (1H, m, H-11), 5.66 (1H, dd, J = 12.1 and 5.0 Hz, H-l), 5.87 and 6.63 (each IH, m, OH x2) and 7.08 (1H, br d, J=2.5 Hz, OH); CD (MeOH, 5.902 mM): Aealo-0.20; EIMS m/z: 366.2021 [M] +. Calcd for C2oH3oO6;366.2042. Longirabdolide B diacetate (7). Compound 5 (18.1 mg) was dissolved in a mixt. of pyridine (0.5 ml) and Ac20 anhydride (0.5 ml) and the soln was left for 35 hr at room temp. Work-up as before gave a residue which was purified by prep. TLC (solvent: CHC13-Me2CO 9: 1) to give the diacetate 7 (13.7 mg) as an amorphous powder. CHCI 3 IR vmax cm-l: 3450(br), 1750, 1710, 1645, 1370, 1280and 1240 (br); 1H NMR (CDCI3): 60.96 and 0.99 (each 3H, s, tert. Me • 2), 2.03 and 2.09 (each 3H, s, 2 x OAc), 3.21 (1H, dd, J =4.5 and 8.7 Hz, H-13), 3.36 (1H, d, J = 11.6 Hz, H-14fl), 4.06-4.30 (3H, H1-20 and H2-6), 4.39 (1H, m, H11), 4.41 (1H, d, J = 13.0 Hz, H~-20), 5.16 (1H, dd, J=7.1
and 9.8 Hz, H-l), and 5.54 and 6.08 (each 1H, br s, H2-17); EIMS m/z: 448.2114 [M] +. Calcd for C24H32Os: 448.2097. Acknowledgements--The authors thank the staff of the Analytical Centre of the Faculty of the Pharmaceutical Sciences, The University of Tokushima for measurements of NMR and mass spectra. REFERENCES
1. Hara, H. (1972) J. Jpn Botany 47, 193. 2. Isobe, T., Kamikawa, T. and Kubota, T. (1972) Nippon Kagaku Kaishi 2143. 3. Takeda, Y., Fujita, T. and Shingu, T. (1988) J. Chem. Soc. Perkin Trans 1 379. 4. Fujita, T., Takeda, Y. and Shingu, T. (1981) Heterocycles 16, 227. 5. Takeda, Y. and Fujita, T. (1988) Planta Med. 54, 327. 6. Ichihara, T., Takeda, Y. and Otsuka, H. (1988) Phytochemistry 27, 2261. 7. Takeda, Y., Ichihara, T. and Otsuka, H. (1988) Chem. Pharm. Bull. 36, 2079. 8. Takeda, Y., Ichihara, T. and Otsuka, H. (1990) J. Nat. Prod. 53, 138. 9. Takeda, Y, Ikawa, A., Matsumoto, T., Terao, H. and Otsuka, H. (1992) Phytochemistry 31, 1687. 10. Fujita, E. and Node, M. (1984) in Progress in the Chemistry of Organic Natural Products (Herz, W., Griesebach, H., Kirby, G. W. and Tamm, Ch., eds), Vol. 46, p. 77. Springer, Vienna. 11. Fujita, T., Takeda, Y. and Ueno, A. (1980) Chem. Letters 1635. 12 Fujita, E., Fujita, T., Katayama, H., Shibuya, M. and Shingu, T. (1970) J. Chem. Soc. (C) 1674. 13. Fujita, E., Fujita, T. and Shibuya, M. (1968) Chem. Pharm. Bull. 16, 509. 14. MacMillan, J. and Walker, E. R. H. (1972) J. Chem. Soc. Perkin Trans. I 986. 15. Kido, M., Ichihara, T., Otsuka, H. and Takeda, Y. (1992) Chem. Pharm. Bull. 40, 3324. 16. Takeda, Y., Futatsuishi, Y., Ichihara, T., Matsumoto, T., Terao, H., Terada, H. and Otsuka, H. (1993) Chem. Pharm. Bull. 41, 685.
0031-9422(93)E011- 3
Pergamon
LONGIRABDOLIDES
35, No. 5, pp. 1275 1278, 1994 Copyright 9 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0031-9422/94 $6.00 + 0.00
A A N D B, 6,7-SECO-ENT-KAURENOIDS F R O M
RABDOSIA LONGITUBA YOSHIO TAKEDA,* TAKASHI MATSUMOTOand HIDEAKI OTSUKA*'~ Faculty of Integrated Arts and Sciences,The University of Tokushima, Tokushima 770, Japan; tInstitute of Pharmaceutical Sciences, Hiroshima University School of Medicine, Minami-ku, Hiroshima 734, Japan
(Received 23 August 1993) Key Word Index--Rabdosia longituba; Labiatae, longirabdolides A and B; 6,7-seco-ent-kaurenoid.
Abstract--Two new diterpenoids, longirabdolides A and B, were isolated from the aerial parts of Rabdosia longituba, together with the known compounds, isolongirabdiol, effusanin E, oridonin and nodosin. The structures of the new compounds were elucidated mainly by spectroscopic methods.
O
12
INTRODUCTION
R 2 " I ' ~
Rabdosia longituba (Miq.) Hara El.1 contains many kinds of ditcrpcnoids having ent-kaurene and 6,7-seco-entkaurenc skeletons [2-9-1, many of which show cytotoxic and antibacterial activities 1-10.1.During the course of our studies on the biologically active substances from Labiatac plants, we examined the constituents of the aerial parts of the title plant collected in Hiroshima Prefecture, Japan and isolated two new diterpcnoids, which we have named longirabdolide A (1) and longirabdolidc B (5), together with the known compounds, isolongirabdiol (4) r8.1, cffusanin E (8) 1.11.1,oridonin (9) 1.12.1and nodosin (10) 1.13.1. This paper describes the characterization of the new compounds.
R217
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16 17
.14.
?_~.,3
%
+
R+O.--/~ (I) (2) (3) (4)
-
6
-
3
RI=R4=H ; R2---OH; R3fCH2 R~=R4-~c; R=---O/r : R3=CH= RI=R4=H ; R2=OH ; R3=ot-H, ~CH 3 RI--R2=R4=H ; R3--'CI'12
""
t~-
1
6
(5) RI=Ra--H ; Fr~---CI-~ (6) RI=R3=H ; FI==cx-CHa,~H (7) Rl=R3'---Ac; R2=C1"12
0
o II _OH
I ,.
I[
I"~
RESULTS AND DISCUSSION
Compound 1, [~]v+ 39-1~ (MeOH) was obtained as an amorphous powder and the molecular formula was assigned as C2oH2aO6 (HRMS). It contains an exo-methylene group conjugated with a carbonyl group on a fivemembered ring t.v-l-llV-max~MeOH 229 nm (e 5674); IK'~Fma xKBr 1740 and 1640 cm-1; 1H NMR (CsDsN): c55.35 and 5.98 (each 1H, br s, H2-17); 13CNMR (CsDsN): &116.4 (t), 151.4 (s) and 202.7 (s)], a 6-1actone group [Vm,x 1710cm-1; 6c172.2 (s)] a tertiary methyl group [tSn0.95 (3H, s); 6c 18.2 (q)], three primary carbinyl groups of which one is involved in the formation of a 6-1actone moiety [6.4.99 (2H, br s); 6c 70.8 (t)] and two in hydroxyl groups [6.4.00 ( 1H, dd, J = 5.5 and 11.9 Hz) and 4.10 (1H, dd, J = 2.9 and 11.9 Hz), and 3.38 and 3.83 (each 1H, d, J=10.8 Hz); 6c58.6 (t) and 71.5 (t)], and a secondary carbinyl group [6,.15.06 (1H, m); &c65.2 (d)] as partial structures. The ~3C
*Authors to whom correspondence should be addressed.
"
"
OH
OH
(8) RlffiOH; R2=H (9) RI=H ; R2--OH
(10)
O
o p 0
(11)
:"
II '"
(12)
NMR spectrum (Table 1) of I further shows the presence of five methylene groups, three methine groups and three quaternary carbon atoms in addition to the aforementioned signals. These spectral data, coupled with the
1275
1276
Y. TAKEDAet al. Table 1. 13CNMR data* of 1 and 5
C
1
5
1 2 3 4 5 6 7 8 9 10 I1 12 13 14 15 16 17 18 19 20
30.3"t 18.2 37.0 39.2 45.4 58.6 172.2 58.1 48.5 44.3 65.2 42.5 35.2 30.4~" 202.7 151.4 116.4 71.5 18.2 70.8
81.0 25.3 40.0 34.8 51.1 60.7 172.2 55.6 43.7 46.3 65.6 42.3 35.3 34.6 199.6 151.4 117.5 34.0 22.7 60.5
*The spectra were measured in CsDsN and the chemical shifts are given in ppm (6) relative to the internal TMS. tThe assignments may be interchanged. consideration on the structures of closely related compounds, suggested that 1 has an ent-spiro-seco-kaurene skeleton (11) as a basic skeleton and has a structure in which three hydroxyl groups are introduced into 11. Compound ! gave the triacetate 2 [6u 1.99, 2.09 and 2.10 (each 3H, s)-I on acetylation with Ac20 and pyridine. Catalytic hydrogenation of 1 over Pd-C gave the dihydro-compound 3 [3,1.12 (3H, d, J = 6 . 6 Hz)] which showed a negative Cotton effect [Ae3o5 -0.70 (MeOH)] in the CD spectrum, proving the supposed absolute stereochemistry [14, 15]. A secondary hydroxyl group was assigned to C-11 by following the cross-peaks starting from 6,3.38 (H-9) to 5.06 (the signal due to a proton on a carbon having a hydroxyl group, H-11), 1.86 (HI2//), 2.43 (H-12ct) and 3.15 (H-13), successively, in the t H COSY spectrum. The stereochemistry of the hydroxyl group was assigned as ~ based on the fact that the signal assigned to H-14~t [6,3.68 (d, J = 11.6 Hz)] suffered an abnormal downfield shift and the signal at 6 5.06 crossed peaks with the signal at 6 2.89 (1H, m) assigned to H-I//in the tH NOESY spectrum (Fig. 1). Judged from the coupling patterns, two of the primary carbinyl groups should be located adjacent to a methine group and a quaternary carbon atom, respectively. The possible positions are at C-6 and at either C-18 or C-19. The positions were finally determined as being at C-6 and C-18 based on the results of tH NOESY experiments (Fig. 1). Namely, the signal at 6 4.99 (H2-20) crossed peaks with the signals at 6n0.95 (H3-19) and 4.00 (H~-6). Thus, the structure of longirabdolide A was elucidated as 1. Compound 5, [ct]D -52.5 ~(MeOH) was obtained as an amorphous powder and has the same molecular formula,
. OH
H
F"\
2
11
~O
Fig. 1. Summary of the results of 1HNOESY spectrum for longirabdolide A (1).
C2oH2sO6, as that of 1. This compound contains an exomethylene group conjugated with a carbonyl group on a five-membered ring (see Experimental), two tertiary methyl groups [6nI.28 and 1.35 (each 3H, s); 6c22.7 and 34.0 (each q)], a 6-1actone [Vma~ 1700; 6n5.71 (1H, dd, J =4.9 and 12.2 Hz); 6c81.0 (d) and 172.2 (s)], two methylene groups having a hydroxyl group [6.4.30 and 4.34 (each 1H, d, J=11.8 Hz), and 4.33 (1H, dd, J = 5 . 2 and 12.2 Hz) and 4.50 (1H, dd, J = 2.1 and 12.2 Hz); 6 c 60.5 and 60.7 (each t)] and a methine group having a hydroxyl group [3,5,19 (1H, m); 6c65.6 (d)]. The 13CNMR spectrum of 5 (Table 1) showed, in addition to the signals mentioned above, the presence of four methylene groups, three methine groups and three quaternary carbon atoms. The spectral data suggested that 5 has an ent-6, 7seco-kaurene skeleton (12) as a basic skeleton and has a structure in which the three hydroxyl groups, i.e. two primary and a secondary hydroxyl groups, are introduced into a basic skeleton (12). Catalytic hydrogenation of 5 over Pd-C gave the dihydro-compound 6 which showed a negative Cotton effect 1-A~3o9.6--0.20 (MeOH)] in the CD spectrum, proving the absolute stereochemistry [14, 15]. Acetylation of 5 gave the diacetate 7 [6 a 2.03 and 2.09 (each 3H, s)'l. In the 1H NMR spectrum of 7, the signal due to a proton on the carbon having a secondary hydroxyl group did not show any downfield shift. This fact suggested that a hydroxyl group is located on a sterically hindered position and is presumed to be located at C-11fl, because it is known that this position in this carbon skeleton is hardly acetylated on treatment with Ac20 and pyridine [-10]. The suggestion was confirmed by 1H COSY and 1H NOESY experiments. Starting from the signal at 6 2.92 (H-9), the cross-peaks can be followed to 6 5.19 (H-11), 1.75 (H-12~), 2.46 (H-12//) and 3.12 (HI3), successively, in the 1HCOSY spectrum, and the signal of the H-11 crossed peaks with that of H-5 [32.63 (1H, dd, J=2.1 and 5.2 Hz)] in the IH NOESY spectrum (Fig. 2). One of the two primary hydroxyl groups should be located at C-6 judged from its coupling pattern. Another primary hydroxyl group was indicated to be located at C-20 based on the observations that the proton signals at 6,4.30 and 4.34 crossed peaks with that at 6c46.3 (C-10) in the ~H-13C long range COSY (d = 10 Hz) spectrum. The suggestion was confirmed based on the results of the ~H NOESY experiment shown in Fig.
Longirabdolides A and B from Rabdosia longituba
1277
(200 ml), CHC13-MeOH 97: 3 (200 ml), CHCI3-MeOH 24: 1 (100 ml), CHC13-MeOH 19 : 1 (1 1), CHC13-MeOH 47 : 3 (500 ml) and CHC13-MeOH 93 : 7 (500 ml), 8-ml frs H H/ were collected. Frs 81-83 gave 9 (42 mg). Frs 94-105 gave a residue (51 rag) which was purified by prep. TLC (CHC13-MeOH 19:1, developed twice and then CHC13-MeOH 9 : 1) to give 1 (37 rag). Compounds 4, 8, 9 and 10 were identified by direct comparison with authentic samples. Longirabdolide A (1). Amorphous powder, [~]~2 19OH ~ ~'OH + 39.1 ~ (MeOH; c 1.05); ~ITV nm (e): 229 (5674); IR - - JMeOH "'max KBr Fig. 2. Summary of the results of ~HNOESY spectrum for Vmax c m - 1. 3400 (br.), 1740, 1710, 1640, 1550, 1460, 1400, 1360, 1305, 1270, 1240, 1120, 1040, 1025 and 930; longirabdolide B (5). 1H NMR (CsDsN, 400 MHz): 6 0.95 (3H, s, H3-19 ), 1.37 (1H, br s, J = 13.1 Hz, H-3~), 1.52 (1H, dr, J = 13.8 and 2. Thus, the structure of longirabdolide B was elucidated 3.4 Hz, H-2fl), 1.74 (1H, m, H-2al), 1.86 (1H, dd, J = 4 . 7 and 14.5 Hz, H-12/~), 1.99 (1H, ddd, J = 13.1, 13.1 and as 5. Among the diterpenoids isolated so far from R. long- 4.0 Hz, H-3fl), 2.24 (1H, br d, J = 14.3 Hz, H-I~), 2.43 (1H, ituba, many were oxidized at C-19. On the other hand, the dd, J = 9 . 3 and 14.5 Hz, H-12~), 2.65 (1H, dd, J = 11.6 and compounds oxidized at C-18 are few, including 1 [-8, 9, 3.8 Hz, H- 14/~), 2.88 (1H, dd, J = 5.5 and 2.9 Hz, H-5), 2.89 (1H, m, H-l/3), 3.15 (1H, dd, J = 3.8 and 9.3 Hz, H-13), 3.38 161. (1H, br d, J=3.1 Hz, H-9), 3.38 (1H, d, J = 10.8 Hz, H118), 3.68 (1H, d, J = l l . 6 H z , H-14~t), 3.83 (1H, d, d EXPERIMENTAL = 10.8 Hz, H1-18), 4.00 (1H, dd, J = 5 . 5 and 11.9 Hz, H 1General. NMR: 1H (200 or 400 MHz) and x3C 6), 4.10 (1H, dd, J = 2 . 9 and 11.9 Hz, H1-6 ), 4.99 (2H, br s, (100 MHz); ELMS: 70 eV; CC: silica gel 60 (Merck); prep. H2-20), 5.06 (1H, m, H-11), 5.35 and 5.98 (each 1H, br s, TLC: silica gel 60 F254. H2-17), and 6.34 and 6.75 (each 1H, m, OH• Plant material. Plant material was collected in Geihoku 13CNMR: Table 1; ElMS m/z: 364.1862 I-M] + Town, Hiroshima Prefecture, Japan in late September, C2oH2sO 6 requires 364.1886. 1987 and identified as Rabdosia lonoituba (Miq.) Hara by Longirabdolide B (5). Amorphous powder, [ct] 22 one (H. O.) of the authors. A voucher specimen [RL-87- - 52.5 ~ (MeOH; c 0.88); UV 2um]~ nm (~): 229 (7064); IR Geihoku] was kept in the laboratory of H. O. Vmax~rcm-1.. 3400 (br), 1740, 1700, 1640, 1550, 1460, 1370, Isolation. Dried aerial parts (146 g) of R. lonoituba were 1305, 1280, 1190, 1150, 1060 and 940; 1H NMR (CsDsN, extracted with MeOH for 1 month at room temp. The 400 MHz): 61.28 (3H, s, H3-18), 1.35 (3H, s, H3-19 ), 1.46 extract was concd in vacuo and the residue was dissolved (1H, dt, J = 13.4 and 3.8 Hz, H-3ct), 1.53 (1H, ddd, J = 13.4, in 90% MeOH (330ml). The 90% MeOH soln was 13.4 and 3.4 Hz, H-3/~), 1.75 (1H, dd, J = 14.7 and 4.6 Hz, washed with n-hexane (300 ml x 3) and the 90% MeOH H-12~t), 1.87 (1H, m, H-2/3), 2.10 (1H, m, H-2~t), 2.26 (1H, br layer concd in vacuo. The residue was suspended in H 2 0 dd, J = l l . 3 and 4.6 Hz, H-I), 2.46 (1H, dd, J = 9 . 2 and (300 ml) and the suspension was extracted with EtOAc 14.7 Hz, H-12fl), 2.63 (1H, dd, J=2.1 and 5.2 Hz, H-5), (300 ml x 3). After being washed with H 2 0 and dried, the 2.92 (1H, d, J = 2 . 8 Hz, H-9), 3.12 (1H, dd, J = 4 . 6 and EtOAc extract was concd in vacuo to give a residue 8.9 Hz, H-13) 3.88 (1H, d, J = 11.3 Hz, H-14fl), 4.30 (1H, d, (3.99 g). An aliquot (3.80 g) of the residue was chromato- J = 11.8 Hz, H1-20), 4.33 (1H, dd, J = 5.2 and 12.2 Hz, H 1graphed over silica gel (200 g) with a mixt. of CHCI3 and 6), 4.34 (1H, d, J = 11.8 Hz, H1-20), 4.50 (1H, dd, J=2.1 Me2CO as eluant with increasing Me2CO content: and 12.2 Hz, H1-6 ), 5.19 (1H, m, H-11), 5.34 (1H, br s, H 1CHC13 (1.3 1), CHC13-Me2CO 19:1 (1 1), 17), 5.71 (1H, dd, J = 4 . 9 and 12.2 Hz, H-l), 5.91 (1H, m, CHC13-Me2CO 9:1 (1 1), CHC13-Me2CO 17:3 (11), OH), 6.00 (1H, br s, H1-17) 6.69 (1H, m, OH) and 7.09 (1H, CHC13-Me2CO 4:1 (1 1), CHCI3-Me2CO 7:3 (1 1), d, J = 3 . 7 H z , OH); 13CNMR: Table 1; EIMS m/z CHCla-Me2CO 3:2 (250 ml) and Me2CO (11). 364.1916 [M] +. C2oH2sO 6 requires 364.1886. Frs (75 ml) were collected. Longirabdolide A triacetate 2. Compound 1 (10 rag) Frs 57-59 gave a residue (352 mg), an aliquot (118 mg) was dissolved in a mixt. of pyridine (0.5 ml) and Ac20 of which was sepd by prep. TLC (Et20) to give 10, (0.5 ml) and the soln was left overnight at room temp. (19 mg). Excess MeOH was added to the soln and the solvent was Frs 64-78 gave a residue (149 mg) which was purified removed in vacuo to give a residue which was purified by by prep. TLC (Et20, developed 3 x ) to give 4 (92 mg): prep. TLC (CHCI3-Me2CO 17 : 3) to give 2 (6.5 mg) as an "cncl~ cm- 1.. 1745, 1645, 1240, Frs 79-84 gave a residue (121 rag) which sepd by prep. amorphous powder. IR Vm,x TLC (Et20, developed 4 x ). The faster moving zone gave 1220 and 1130; IH NMR (CDC13): 6 0.96 (3H, s, H3-19), 8 (15 mg) and the slower moving one 5 (60 rag). 1.99, 2.09 and 2.10 (each 3H, s, 3 • OAc), 2.62 (1H, d, J The eluate (366 mg) from 100% Me2CO was subjected = 3.5 Hz, H-9), 2.71 (1H, d, J = 11.2 Hz), 3.97-4.07 (2H), to CC (signal, 50 g) with mixts of CHC13 and MeOH 4.18 (1H, dd, J = 3 . 9 and 12.7 Hz, H1-6), 4.29 and 4.60 containing increasing amounts of MeOH, CHC13 (each 1H, d, J = 11.5 Hz), 5.53 (1H, br t, J = 3 . 5 Hz, H-11), OI4 \
1278
Y. TAKEDAet al.
and 5.58 and 6.09 (each IH, br s, H2-17); ElMS m/z: 490.2167 [M] +. Calcd for C26H3409:490.2203. Dihydrolongirabdolide A (3). Five percent Pd-C (10 mg) was added to the soln of 1 (10 mg) in MeOH (5 ml) and the mixt. was stirred in an arm. of H2 for 2 hr. After removing the catalyst by filtration, the solvent was removed in vacuo to give 3 (9.6 mg) as an amorphous powder. IR Vma, KBr cm-1:3400 (br), 1745, 1710, 1240 and 1030; 1H NMR (CsDsN): 60.97 (3H, s, H3-19), 1.12 (3H, d, J=6.6 Hz, H3-17 ), 2.93 (1H, t, J=4.4 Hz, H-5), 3.18 (IH, d, J=3.9 Hz, H-9), 3.42 (1H, d, J = 10.8 Hz, H1-18), 3.66 (IH, d, J = 10.8 Hz, H-14~t), 3.82 (1H, d, J = 10.8 Hz, HI-18), 4.09 (2H, m, H2-6), 4.95 (2H, br s, H2-20), 6.39 (2H, m, OH x2) and 6.75 (1H, m, OH); CD (MeOH, 5.246mM): Aeaos-0.70; EIMS m/z: 366.2055 [M] +. Calcd for C2oH3oO6: 366. 2042. Dihydrolongirabdolide B (6). Five per cent Pd-C (20 mg) was added to the soln of 5 (19 mg) in MeOH (5 ml) and the mixt. was stirred under an atm. of H 2 for 1.5 hr at room temp. After removing the catalyst by filtration, the filtrate was concd in vacuo to give a residue which was purified by prep. TLC (solvent: CHCIa-Me2CO 7 : 3) to KBr give 6 (10.8 mg) as an amorphous powder. IR Vmax cm- 1: 3400 (br), 1755, 1705, 1280 and 1075; tH NMR (CsDsN): 61.04 (3H, d, J = 6.6 Hz, Ha-17), 1.30 and 1.36 (each 3H, s, tert. Me x 2), 2.63 (IH, d, J=3.2 Hz, H-9), 2.78 (1H, dd, J =5.5 and 2.5Hz, H-5), 4.01 (1H, d, J = 11.2 Hz, H1-20), 4.29-4.47 (3H, H2-6 and H1-20), 5.08 (1H, m, H-11), 5.66 (1H, dd, J = 12.1 and 5.0 Hz, H-l), 5.87 and 6.63 (each IH, m, OH x2) and 7.08 (1H, br d, J=2.5 Hz, OH); CD (MeOH, 5.902 mM): Aealo-0.20; EIMS m/z: 366.2021 [M] +. Calcd for C2oH3oO6;366.2042. Longirabdolide B diacetate (7). Compound 5 (18.1 mg) was dissolved in a mixt. of pyridine (0.5 ml) and Ac20 anhydride (0.5 ml) and the soln was left for 35 hr at room temp. Work-up as before gave a residue which was purified by prep. TLC (solvent: CHC13-Me2CO 9: 1) to give the diacetate 7 (13.7 mg) as an amorphous powder. CHCI 3 IR vmax cm-l: 3450(br), 1750, 1710, 1645, 1370, 1280and 1240 (br); 1H NMR (CDCI3): 60.96 and 0.99 (each 3H, s, tert. Me • 2), 2.03 and 2.09 (each 3H, s, 2 x OAc), 3.21 (1H, dd, J =4.5 and 8.7 Hz, H-13), 3.36 (1H, d, J = 11.6 Hz, H-14fl), 4.06-4.30 (3H, H1-20 and H2-6), 4.39 (1H, m, H11), 4.41 (1H, d, J = 13.0 Hz, H~-20), 5.16 (1H, dd, J=7.1
and 9.8 Hz, H-l), and 5.54 and 6.08 (each 1H, br s, H2-17); EIMS m/z: 448.2114 [M] +. Calcd for C24H32Os: 448.2097. Acknowledgements--The authors thank the staff of the Analytical Centre of the Faculty of the Pharmaceutical Sciences, The University of Tokushima for measurements of NMR and mass spectra. REFERENCES
1. Hara, H. (1972) J. Jpn Botany 47, 193. 2. Isobe, T., Kamikawa, T. and Kubota, T. (1972) Nippon Kagaku Kaishi 2143. 3. Takeda, Y., Fujita, T. and Shingu, T. (1988) J. Chem. Soc. Perkin Trans 1 379. 4. Fujita, T., Takeda, Y. and Shingu, T. (1981) Heterocycles 16, 227. 5. Takeda, Y. and Fujita, T. (1988) Planta Med. 54, 327. 6. Ichihara, T., Takeda, Y. and Otsuka, H. (1988) Phytochemistry 27, 2261. 7. Takeda, Y., Ichihara, T. and Otsuka, H. (1988) Chem. Pharm. Bull. 36, 2079. 8. Takeda, Y., Ichihara, T. and Otsuka, H. (1990) J. Nat. Prod. 53, 138. 9. Takeda, Y, Ikawa, A., Matsumoto, T., Terao, H. and Otsuka, H. (1992) Phytochemistry 31, 1687. 10. Fujita, E. and Node, M. (1984) in Progress in the Chemistry of Organic Natural Products (Herz, W., Griesebach, H., Kirby, G. W. and Tamm, Ch., eds), Vol. 46, p. 77. Springer, Vienna. 11. Fujita, T., Takeda, Y. and Ueno, A. (1980) Chem. Letters 1635. 12 Fujita, E., Fujita, T., Katayama, H., Shibuya, M. and Shingu, T. (1970) J. Chem. Soc. (C) 1674. 13. Fujita, E., Fujita, T. and Shibuya, M. (1968) Chem. Pharm. Bull. 16, 509. 14. MacMillan, J. and Walker, E. R. H. (1972) J. Chem. Soc. Perkin Trans. I 986. 15. Kido, M., Ichihara, T., Otsuka, H. and Takeda, Y. (1992) Chem. Pharm. Bull. 40, 3324. 16. Takeda, Y., Futatsuishi, Y., Ichihara, T., Matsumoto, T., Terao, H., Terada, H. and Otsuka, H. (1993) Chem. Pharm. Bull. 41, 685.