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Neo-clerodane diterpenoids from ajuga macrosperma and ajuga pantantha
Phytvchemistry, Vol. 34,No. 4, pp. 109-1094, hnted in GreatBritain.
NEO-CLERODANE
003l--9422/93 $6.00 + 0.00 Q 1993PergamonPressLtd
1993
DITERPENOIDS FROM AJUGA AJUGA PANTANTHA
XIAOYU SHEN,*~ AKIRA ISOGAI,* KAZUO FURIHATA,* HANDING
MACROSPERMA
SUN-~
AND
and AKINORI SUZUKI*$
*Department of Agricultural Chemistry, The University, Tokyo 113, Japan; tKunming Institute of Botany, Academia Sin@ Kunming 650204,China (Received2 February 1993)
Key Word Index-Ajuga macrosperma, A. pantantha; Labiatae; neo-clerodane diterpenoids; ajugamacrins C, D and E, ajugapantin A.
Abstract-Five neo-clerodane diterpenoids, named ajugamacrins C, D, E and ajugapantin A, as well as a known compound ajugacumbin B, were isolated from dried whole herb of Ajuga macrosperma and A. pantantha. Their structures were determined by the NMR spectral analysis.
INTRODUCI’ION
In our previous paper, we reported on the isolation and the structural determination of two neo-clerodane diterpenoids, ajugamacrins A (5) and B (6) [ 11. All the protons of ajugamacrin A (5) were unambiguously assigned by NMR experiments, in particular, by HMBC and Time Sharing Gated Decoupling difference spectra. At the same time the positions of the substituted groups in 5 were confirmed by the HMBC method. As part of our ongoing study of the diterpenoids in plants belonging to the genus Ajuga, we have examined extracts of A. macrosperma Wall and A. pantantha Hand-M=, both of which are used in traditional Chinese folk medicine to alleviate fever and remove phlegm [2]. The present paper deals with the isolation and the structure elucidation of four neo-clerodane diterpenoids (l-4). RESULTS AND DISCUSSION
The EtOAc-soluble fractions of the methanol extracts of both plants were subjected to extensive chromatography and HPLC to yield ajugamacrins C (l), D (2) and E (3) from A. macrosperma and, ajugapantin A (4) and ajugacumbin B [3] from A. pantantha. Direct comparison of the NMR spectra of 1-4 with those of ajugamacrins A (5) and B (6) suggested that l-4 were neo-clerodane diterpenoids and were almost identical to 5 and 6, except for the positions and structures of the hydroxyl substituent groups. The 500 MHz ‘H NMR spectra (Table 1) of l-4 readily confirmed that the substituted ester groups located at C-l, C-6, C-12 and C19 were. the same as those of 5 and 6, since the chemical shifts and the coupling constants of the oxymethine protons in l-4 were in agreement with those of 5 and 6. The comparison of the mass and NMR spectra of I with those of 5, suggested that 1 had two acetates and two $Author to whom correspondence should be addressed
RI
1 COCHMep
2 COCHMe2 3 4 5 6
COCHMeEt AC AC AC
R2
COCHMes COCHMeEt COCHMe2 AC COCHMe2 COCHMeEt
isobutyrates, while 5 had three acetates and one isobutyrate, those acyl groups being at the above mentioned four positions. Similarly, both 2 and 3 should have two acetates, one isobutyrate and one 2-methylbutyrate. The acetoxy signals which were simultaneously observed in the NMR spectra of l-3 were assigned to the substituents at C-6 and C-19. The acetoxy signal at C-l in 5 and 6,6, 169.4 or 169.3 and 21.8, was not observed in the spectra of l-3 (Table 2). Moreover, the assignment was proved out by the HMBC spectra of 2 and 3 (see below). The positions of the two isobutyrate groups in 1 were verified to be at C-l and C-12, because the ‘HNMR resonances at 6, 5.54 (ddd, 11.0, 11.0, 4.8 Hz, H-l) and 5.76 (dd, 9.0, 3.0 Hz, H-12) were the same as those in 5. The ‘H signals, 6, 2.57 (quintet, d, 7.0 Hz), 1.25 and 1.23 (each 3H, d, 7.0 Hz), of 1 were assigned to an isobutyrate group at C-12 on the basis of their common identity to those observed in the ‘H NMR spectrum of 5. The other signals of the isobutyrate group, 6n2.44 (quintet, 7.0 Hz), 1.20 and 1.17 (each 3H, d, 7.0 HZ), which were absent in the spectrum of 5, must be at the C-l position.
1091
1092
XIAOYU SHEN et al.
Ajugamacrins D (2) and E (3) were first obtained as a mixture and only separated by careful HPLC (2 eluted a little earlier than 3). They constituted a pair of positional isomers, molecular formula C34H48011. In the HMBC spectrum of the mixture of 2 and 3, the ‘H signals at 6, 1.94 and 2.12 (s, each 6H, COMe) showed significant long-range correlations with the 13C signals at 6, 169.7 and 170.3, which in turn were correlated with the signals of the H-6 protons (6u4.66) and the H-19 protons (bH4.40 and 4.95), respectively. These data confirmed that the two acetate radicals were attached to the C-6 and C-19 positions. The 500 MHz ‘H NMR spectrum of 2 (Table 1) was almost identical with that of3; the only difference was the interchange of the C-l isobutyrate and C-12 2methylbutyrate groups. Precise analysis of the signals around C-l and C-12 in the NMR spectra suggested that the 2-methylbutyrate in 2 and the isobutyrate in 3 was located at C-12, because their chemical shifts and coupling patterns were in complete agreement with those in 6 and 5, respectively. Furthermore, comparison of the ‘H signals of the isobutyrate of 2 with those in 1 not only provided additional evidence for assignment of ‘H signals Table 1. ‘H
of both isobutyrates in 1, but also supported the deduction of the suggested structures of 2 and 3. Ajugapantin A (4), (12S)-lfi,6a,12,19-tetraacetoxy-4, 1%epoxy-neo-clerod-13(14)-en-l&16-olide, was first isolated as a natural product from A. pantantha, although it was proved to be identical with the diacetyl derivative of ajugamarin Cl [4]. The assignment of the 12s structure to 1-3, and the 2s configuration to the 2-methylbutyrate moiety in 2 and 3 was based on the comparison of the NMR data of the H12 protons as well as biogenetic considerations. Thus, the structures of ajugamacrins C, D and E were confirmed as (12S)-6a,l9-diacetoxy-l~,l2-diisobutyraloxy-4,18-epoxyneo-clerod-13(14)-en-15,16-olide (I), (12S)-6a,l9-diacetoxy-lP-isobutyrate-12-[(2S)-2-methylbutanoyloxy]-4, 18-epoxy-neoklerod- 13(14)-en- l&16-olide (2) and (12S)6a,19-diacetoxy-l~-[(2~)-2-methylbutanoyloxy]-l2-isobutyloxy-4,18-epoxy-neo-clerod-13(14)-en-l5,l6-olide (3). respectively. It is interesting that the ‘H NMR chemical shifts of the acetate, isobutyrate and (2S)-methylbutyrate groups involved a systematic exchange of the substituted groups
NMR data for compounds 1-6 (500 MHz., CDCI,, CHCl, as int. standard. J: Hz)
H
1
2
3
1 2 2
5.54 ddd 11.0, 11.0, 4.8 2.18 m 1.47 m 2.36 m 1.12 m 4.65 br dd 11.0,4.5 1.68* 1.53* 1.65* 2.14 d 11.0 1.58* 2.73 dd 16.0, 9.0 5.76 dd 9.0, 3.0 5.90 dd 3.0, 2.9 4.72 dd 17.5, 2.0 4.83 br dd 17.5, 2.0 0.84 d 7.0 2.97 dd 4.0, 2.0 2.24 d 4.0 4.39 br d 12.5 4.95 d 12.5 0.77 s 1.94 9 211 s 2.44 quintet 7.0 1.20 and 1.17 d 7.0 2.57 quintet 7.0 1.25 and 1.23 d 7.0
5.56 ddd 11.0, 11.0, 4.8 2.20 m 1.478 2.36 m 1.12* 4.65 br dd 11.0, 4.5 1.68* 1.53* 1.71* 2.15 d 11.0 1.56* 2.74 dd 16.0, 9.0 5.73 dd 9.0, 3.0 5.91 dd 3.0, 20 4.72 dd 17.5, 2.0 4.86 br dd 17.5, 2.0 0.84 d 7.0 2.97 dd 4.0, 2.0 2.23 d 4.0 4.40 br d 13.0 4.95 d 13.0 0.78 s 1.94 s 2.12 s 2.44 quintet 7.0 1.19 and 1.17 d 7.0 -
5.56 ddd 11.0, 11.0, 4.8 2.20 m 1.47* 2.36 m 1.12* 4.66 br dd 11.0, 4.5 1.68* 1.53* 1.71* 2.14 d 11.0 l-56* 2.74 dd 16.0, 9.0 5.76 dd 9.0, 3.0 5.90 dd 3.0, 2.0 4.72 dd 17.5, 2.0 4.84 br dd 17.5, 2.0 0.85 d 7.0 2.97 dd 4.0, 2.0 2.23 d 4.0 4.40 br d 13.0 4.95 d 13.0 0.78 s 1.94 s 2.12 s
-
0.93 t 7.5 1.19 d 7.0 2.40 qt 7.5, 7.0 1.55 and 1.70 m
3 6 7 8 10 11 11 12 14 16 16 Me-17 18 18 19 19 Me-20 AC-6 AC-19 COCHMe,-1 COCHMe,-12 COCHMeC,H,-1
COCHMeC,H,-12
*Ambiguous due to signal overlapping.
-
257 quintet 7.0 1.25 and 1.22 d 7.0 0.89 t 7.5 1.13 d 7.0 2.27 qt 7.5, 7.5 1.55 and 1.70 m
(Continued overleaf)
Diterpenoids from Ajuga
1093
Table 1. (Con&) H
4
5
6
1 2 2 3 3 6 1 7 8 10 11 11 12 14 16 16 Me-17 18 18 19 19 Me-20 AC-~ AC-6 AC-12 AC-19 COCHMe,-12
5.51 ddd 11.0, 11.0, 4.8 2.20 ddd 13.0, 5.5, 4.8 1.45 dddd 13.0, 14.0, 11.0, 5.5 2.37 m 1.14 m 4.67 br dd 11.0, 4.5 1.68* 1.54* 1.65* 2.16 d 11.0 1.60* 2.52 dd 16.0, 9.0 5.85 dd 9.0, 3.0 5.95 dd 3.0, 2.0 4.75 dd 17.5, 2.0 4.85 dd 17.5, 2.0 0.83 d 7.0 3.01 dd 4.0, 2.0 2.29 d 4.0 4.38 br d 12.5 4.95 d 12.5 0.78 s 2.07 s 1.94 s 2.13 s 2.11 s -
5.51 ddd 11.0, 11.0, 4.8 2.20 ddd 13.0, 5.5,4.8, 2.0 1.46 dddd 13.0, 14.0, 11.0, 5.5 2.36 dddd 14.0, 14.0, 5.5, 2.0 1.12 ddd 14.0, 5.5, 2.0 4.66 br dd 11.0, 4.5 1.682 1.52br dd 9.5,4.5 1.68’ 2.14 d 11.0 1.59 dd 16.0, 3.0 2.58 dd 16.0, 9.0 5.76 dd 9.0, 3.0 5.91 dd 3.0, 2.0
5.51 ddd 12.0, 11.0, 5.0 2.20 ddd 13.0, 5.5, 4.8, 2.5 1.47 dddd 13.0, 14.0, 11.0, 5.5 2.35 dddd 14.0, 14.0, 5.5, 2.0 1.12 ddd 14.0, 5.5, 2.5 4.65 br dd 10.5, 4.5 1.68*
COCHMeC2H,-12
-
4.72 dd 17.5,2.0 4.84 ddd 17.5,2.0, 0.5 0.84 d 7.0 2.98 dd 4.0, 2.0 2.25 d 4.0 4.36 br d 12.5 4.93 d 12.5 0.77 s 2.06 s 1.93 s -
1.53brdd 9.5, 4.5 1.66; 2.16 d 11.0 1.59dd 16.0, 3.0 2.58 dd 16.0, 9.0 5.73 dd 9.0, 3.0 5.92 dd 3.0, 2.0 4.73 dd 17.5, 2.0 4.86 br dd 17.5, 2.0 0.83 d 6.5 2.98 dd 4.0, 2.0 2.25 d 4.0 4.36 br d 13.0 4.93 d 13.0 0.77 s 2.06 s 1.93 s -
2.10 s 2.56 quintet
2.10 s
-
0.92 t 7.5 1.18 d 7.0 2.38 qt 7.5, 7.5 1.73 and 1.53 qd 7.5, 7.0
7.0 1.21 and 1.24d 7.0
*Ambiguous due to signal overlapping. attached at different positions in l-6. The proton resonances of the acetates at C-l, C-6 and C-19 were at S, 2.06-2.07,1.93-1.94 and 2.11-2.12. When the isobutyrate was attached at C-12, the signal of the methine appeared at 6,, 2.56-2.57 and those of two methyls at 6” 1.21-1.25. By contrast these were observed at 6,2.44,1.20 and 1.17, when the isobutyrate was attached at C-l. The ‘H signals at 6,0.92-0.93,2.39-2.40 and 1.18-1.19, contributed by the 2-methylbutyrate, were assigned at C-12; the ‘H signals of the same substituent at C-3 in 3 resonated at 6, 0.89, 2.27 and 1.13. These differences reflect their structural differences, although a few compounds were examined. EXPERIMENTAL
Mps: uncorr.; ‘H and 13CNMR: JEOL JNM GX-500, FABMS:JEOL SX-102. Plant material. Voucher specimens of A. macrosperma Wall collected in March 1990 near Xishuangbanna Botanic Garden, and A. pantantka Hand-Mazz collected in June 1991 at Qujing, Yunnan, China, are deposited in the Kunming Institute of Botany, Academia Sinica. Extraction and isolation. Dried and finely powdered whole herb of A. mucrosperma (3.0 kg) was extracted with
MeOH (3 x 3 1)at room temp. for 10 days. Filtration and evapn of the solvent yielded a residue which was treated with activated charcoal (2 x 50 g) in MeOH (2 1). The residue was partitioned between EtOAc and H,O, and the EtOAc solvent evapd in uacuo to yield 18 g of yellow gum which was subjected to CC over silica gel (360 g). The column was eluted successively with nhexane-EtOAc (5: 1, 5:3, 1: 1, 1:2), EtOAc and Me,CO. In the n-hexane-EtOAc (5:3) eluate, fr. 7 (300 ml) gave a crude crystal from Me&O. The crystal was applied to HPLC (OD&s; MeOH-H,O, 3: 1) to give crude 1 and a mixt. of 2 and 3 which were further purified by HPLC (silica gel; n-hexane-2-propanol, 24: 1) to yield l(3.5 mg), 2 (0.15 mg) and 3 (0.4 mg), respectively, by crystallization from Me&O. Dried and finely powdered whole herb of A. pantantha (3.9 kg) was extracted according to the procedure mentioned above to give 30 g of yellow gum, which was subjected to CC over silica gel (1 kg). The column was eluted successively with n-hexane, n-hexane-EtOAc (5 : 1, 5 : 3,1: 1) and Me&O. The n-hexane-EtOAc (5: 3) eluate, frs 11-24 and 25-45 (each 200 ml), gave ajugamarin Cl (0.9 g). From the residue (1.86 g) of the mother liquor of frs 25-45,4 (235 mg) was purified by CC over RP-8 (75 g) eluted with MeOH-CHCl, (19: 1).
1094
XIAoYu SHEN et al.
Table 2. ‘%NMR C
data for compounds l-6
1
2
70.4 31.9 30.3 64.0 46.0 71.3 32.5 35.1 39.2 50.8 41.3 66.4 169.2 116.0 172.4 70.5 15.6 48.6 61.8 17.0
70.5 31.7 30.4 64.1 46.0 71.3 32.6 35.1 39.2 50.8 41.1 66.6 169.1 115.8 172.4 70.6 15.4 48.7 61.6 17.1 -
70.5 31.9 30.4 64.1 46.0 71.3 32.6 35.3 39.2 50.8 41.1 66.6 169.1 115.7 172.4 70.6 15.4 48.8 61.6 17.1
AC-6
169.6 21.0 -
169.7 21.0 -
169.7 21.0
170.4 21.0 175.9 34.5 19.2 18.3 175.8 34.5 19.2 18.3
170.3 21.0 175.9 34.6 19.3 18.3 -
170.3 21.0
AC-19 COCHMe,-1
COCHMe,-12
COCHMe
Et-l
-
COCHMe
Et-12
175.4 40.9 27.0 15.7 11.4
4
3
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 AC-1
AC-12
(125 MHz., CDCI,, CHCI, as int. standard)
175.8 34.6 19.3 18.3 175.4 41.5 26.9 16.1 11.5
Ajugumacrin C (1). Needles from Me&O, mp 206-207”, CJzH,,O1,; IR vg; cm-i: 3035, 2940, 2880, 1780,1775,1730(br), 1635, 1455, 1380, 13651260, 1230, 1190,1150,1085,1040; ‘H and 13C NMR: Tables 1 and 2; FABMS: m/z 607 [MI-Q+, 547, 519, 518, 371, 311, 281, 201, 173, 121, 43. Ajugamucrin D (2). Needles from Me&O, mp. 273-274”, C3.+H4s01 i; ‘H and i3C NMR: Tables 1 and 2 FABMS: m/z 621 [MH]+. ’ Ajugumacrin E (3). Needles from Me&O, C,,H,,O, 1; ‘H and 13CNMR: Tables 1 and 2; FABMS: m/z 621 [MH]+. Ajugapuntin A (4). Needles from Me,CO, mp 204-205”, C,,H,,O, i; IR 4;; cm-l: 2970,2940,2870, 1780, 1760,
70.6 31.7 30.4 64.1 45.9 71.3 32.7 35.4 39.0 50.4 41.7 66.4 168.4 116.2 172.3 70.6 15.6 48.7 61.5 16.9 169.3 21.8 169.7 21.0 169.4 21.0 170.3 21.0 __
5
6
70.4 31.7 30.4 64.0 71.2 32.6 35.2 39.5 50.8 41.7 66.3 169.3 115.9 172.4 70.4 15.5 48.6 61.9 17.0 169.4 21.8 169.6 21.0 -_ 170.3 21.0 --
70.2 31.6 30.3 64.0 45.9 71.2 32.6 35.3 39.1 50.7 41.5 66.4 169.0 115.9 172.4 70.6 15.6 48.7 61.9 17.0 169.3 21.8 169.7 21.0
170.3 21.0
175.8 34.2 19.5 18.4 -
-
-
175.4 40.9 26.9 15.7 11.4
174O(br), 1640,1380,1250,1080,1040; ‘Hand “CNMR: Tables 1 and 2. FABMS: m/z 551 [MH]+, 491,431,371, 311, 281,201, 171,43.
REFERENCES
1. Shen, X., Isogai, A., Furihata, K., Sun, H. and Suzuki, A. (1993) Phytochamistry (in press). 2. Yunnan Inst. of Bot. (1977) Flora Yunnanica Tomus 1, Science Press, Peking. 3. Shen, X., Zhang, H. and Sun, H. (1993) Acta Bob. Yunnan. 15 (in press). 4. Shimomura, H., Sashida, Y. and Ogawa, K. (1989) Chem. Pharm. Bull. 37, 354.
NEO-CLERODANE
003l--9422/93 $6.00 + 0.00 Q 1993PergamonPressLtd
1993
DITERPENOIDS FROM AJUGA AJUGA PANTANTHA
XIAOYU SHEN,*~ AKIRA ISOGAI,* KAZUO FURIHATA,* HANDING
MACROSPERMA
SUN-~
AND
and AKINORI SUZUKI*$
*Department of Agricultural Chemistry, The University, Tokyo 113, Japan; tKunming Institute of Botany, Academia Sin@ Kunming 650204,China (Received2 February 1993)
Key Word Index-Ajuga macrosperma, A. pantantha; Labiatae; neo-clerodane diterpenoids; ajugamacrins C, D and E, ajugapantin A.
Abstract-Five neo-clerodane diterpenoids, named ajugamacrins C, D, E and ajugapantin A, as well as a known compound ajugacumbin B, were isolated from dried whole herb of Ajuga macrosperma and A. pantantha. Their structures were determined by the NMR spectral analysis.
INTRODUCI’ION
In our previous paper, we reported on the isolation and the structural determination of two neo-clerodane diterpenoids, ajugamacrins A (5) and B (6) [ 11. All the protons of ajugamacrin A (5) were unambiguously assigned by NMR experiments, in particular, by HMBC and Time Sharing Gated Decoupling difference spectra. At the same time the positions of the substituted groups in 5 were confirmed by the HMBC method. As part of our ongoing study of the diterpenoids in plants belonging to the genus Ajuga, we have examined extracts of A. macrosperma Wall and A. pantantha Hand-M=, both of which are used in traditional Chinese folk medicine to alleviate fever and remove phlegm [2]. The present paper deals with the isolation and the structure elucidation of four neo-clerodane diterpenoids (l-4). RESULTS AND DISCUSSION
The EtOAc-soluble fractions of the methanol extracts of both plants were subjected to extensive chromatography and HPLC to yield ajugamacrins C (l), D (2) and E (3) from A. macrosperma and, ajugapantin A (4) and ajugacumbin B [3] from A. pantantha. Direct comparison of the NMR spectra of 1-4 with those of ajugamacrins A (5) and B (6) suggested that l-4 were neo-clerodane diterpenoids and were almost identical to 5 and 6, except for the positions and structures of the hydroxyl substituent groups. The 500 MHz ‘H NMR spectra (Table 1) of l-4 readily confirmed that the substituted ester groups located at C-l, C-6, C-12 and C19 were. the same as those of 5 and 6, since the chemical shifts and the coupling constants of the oxymethine protons in l-4 were in agreement with those of 5 and 6. The comparison of the mass and NMR spectra of I with those of 5, suggested that 1 had two acetates and two $Author to whom correspondence should be addressed
RI
1 COCHMep
2 COCHMe2 3 4 5 6
COCHMeEt AC AC AC
R2
COCHMes COCHMeEt COCHMe2 AC COCHMe2 COCHMeEt
isobutyrates, while 5 had three acetates and one isobutyrate, those acyl groups being at the above mentioned four positions. Similarly, both 2 and 3 should have two acetates, one isobutyrate and one 2-methylbutyrate. The acetoxy signals which were simultaneously observed in the NMR spectra of l-3 were assigned to the substituents at C-6 and C-19. The acetoxy signal at C-l in 5 and 6,6, 169.4 or 169.3 and 21.8, was not observed in the spectra of l-3 (Table 2). Moreover, the assignment was proved out by the HMBC spectra of 2 and 3 (see below). The positions of the two isobutyrate groups in 1 were verified to be at C-l and C-12, because the ‘HNMR resonances at 6, 5.54 (ddd, 11.0, 11.0, 4.8 Hz, H-l) and 5.76 (dd, 9.0, 3.0 Hz, H-12) were the same as those in 5. The ‘H signals, 6, 2.57 (quintet, d, 7.0 Hz), 1.25 and 1.23 (each 3H, d, 7.0 Hz), of 1 were assigned to an isobutyrate group at C-12 on the basis of their common identity to those observed in the ‘H NMR spectrum of 5. The other signals of the isobutyrate group, 6n2.44 (quintet, 7.0 Hz), 1.20 and 1.17 (each 3H, d, 7.0 HZ), which were absent in the spectrum of 5, must be at the C-l position.
1091
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XIAOYU SHEN et al.
Ajugamacrins D (2) and E (3) were first obtained as a mixture and only separated by careful HPLC (2 eluted a little earlier than 3). They constituted a pair of positional isomers, molecular formula C34H48011. In the HMBC spectrum of the mixture of 2 and 3, the ‘H signals at 6, 1.94 and 2.12 (s, each 6H, COMe) showed significant long-range correlations with the 13C signals at 6, 169.7 and 170.3, which in turn were correlated with the signals of the H-6 protons (6u4.66) and the H-19 protons (bH4.40 and 4.95), respectively. These data confirmed that the two acetate radicals were attached to the C-6 and C-19 positions. The 500 MHz ‘H NMR spectrum of 2 (Table 1) was almost identical with that of3; the only difference was the interchange of the C-l isobutyrate and C-12 2methylbutyrate groups. Precise analysis of the signals around C-l and C-12 in the NMR spectra suggested that the 2-methylbutyrate in 2 and the isobutyrate in 3 was located at C-12, because their chemical shifts and coupling patterns were in complete agreement with those in 6 and 5, respectively. Furthermore, comparison of the ‘H signals of the isobutyrate of 2 with those in 1 not only provided additional evidence for assignment of ‘H signals Table 1. ‘H
of both isobutyrates in 1, but also supported the deduction of the suggested structures of 2 and 3. Ajugapantin A (4), (12S)-lfi,6a,12,19-tetraacetoxy-4, 1%epoxy-neo-clerod-13(14)-en-l&16-olide, was first isolated as a natural product from A. pantantha, although it was proved to be identical with the diacetyl derivative of ajugamarin Cl [4]. The assignment of the 12s structure to 1-3, and the 2s configuration to the 2-methylbutyrate moiety in 2 and 3 was based on the comparison of the NMR data of the H12 protons as well as biogenetic considerations. Thus, the structures of ajugamacrins C, D and E were confirmed as (12S)-6a,l9-diacetoxy-l~,l2-diisobutyraloxy-4,18-epoxyneo-clerod-13(14)-en-15,16-olide (I), (12S)-6a,l9-diacetoxy-lP-isobutyrate-12-[(2S)-2-methylbutanoyloxy]-4, 18-epoxy-neoklerod- 13(14)-en- l&16-olide (2) and (12S)6a,19-diacetoxy-l~-[(2~)-2-methylbutanoyloxy]-l2-isobutyloxy-4,18-epoxy-neo-clerod-13(14)-en-l5,l6-olide (3). respectively. It is interesting that the ‘H NMR chemical shifts of the acetate, isobutyrate and (2S)-methylbutyrate groups involved a systematic exchange of the substituted groups
NMR data for compounds 1-6 (500 MHz., CDCI,, CHCl, as int. standard. J: Hz)
H
1
2
3
1 2 2
5.54 ddd 11.0, 11.0, 4.8 2.18 m 1.47 m 2.36 m 1.12 m 4.65 br dd 11.0,4.5 1.68* 1.53* 1.65* 2.14 d 11.0 1.58* 2.73 dd 16.0, 9.0 5.76 dd 9.0, 3.0 5.90 dd 3.0, 2.9 4.72 dd 17.5, 2.0 4.83 br dd 17.5, 2.0 0.84 d 7.0 2.97 dd 4.0, 2.0 2.24 d 4.0 4.39 br d 12.5 4.95 d 12.5 0.77 s 1.94 9 211 s 2.44 quintet 7.0 1.20 and 1.17 d 7.0 2.57 quintet 7.0 1.25 and 1.23 d 7.0
5.56 ddd 11.0, 11.0, 4.8 2.20 m 1.478 2.36 m 1.12* 4.65 br dd 11.0, 4.5 1.68* 1.53* 1.71* 2.15 d 11.0 1.56* 2.74 dd 16.0, 9.0 5.73 dd 9.0, 3.0 5.91 dd 3.0, 20 4.72 dd 17.5, 2.0 4.86 br dd 17.5, 2.0 0.84 d 7.0 2.97 dd 4.0, 2.0 2.23 d 4.0 4.40 br d 13.0 4.95 d 13.0 0.78 s 1.94 s 2.12 s 2.44 quintet 7.0 1.19 and 1.17 d 7.0 -
5.56 ddd 11.0, 11.0, 4.8 2.20 m 1.47* 2.36 m 1.12* 4.66 br dd 11.0, 4.5 1.68* 1.53* 1.71* 2.14 d 11.0 l-56* 2.74 dd 16.0, 9.0 5.76 dd 9.0, 3.0 5.90 dd 3.0, 2.0 4.72 dd 17.5, 2.0 4.84 br dd 17.5, 2.0 0.85 d 7.0 2.97 dd 4.0, 2.0 2.23 d 4.0 4.40 br d 13.0 4.95 d 13.0 0.78 s 1.94 s 2.12 s
-
0.93 t 7.5 1.19 d 7.0 2.40 qt 7.5, 7.0 1.55 and 1.70 m
3 6 7 8 10 11 11 12 14 16 16 Me-17 18 18 19 19 Me-20 AC-6 AC-19 COCHMe,-1 COCHMe,-12 COCHMeC,H,-1
COCHMeC,H,-12
*Ambiguous due to signal overlapping.
-
257 quintet 7.0 1.25 and 1.22 d 7.0 0.89 t 7.5 1.13 d 7.0 2.27 qt 7.5, 7.5 1.55 and 1.70 m
(Continued overleaf)
Diterpenoids from Ajuga
1093
Table 1. (Con&) H
4
5
6
1 2 2 3 3 6 1 7 8 10 11 11 12 14 16 16 Me-17 18 18 19 19 Me-20 AC-~ AC-6 AC-12 AC-19 COCHMe,-12
5.51 ddd 11.0, 11.0, 4.8 2.20 ddd 13.0, 5.5, 4.8 1.45 dddd 13.0, 14.0, 11.0, 5.5 2.37 m 1.14 m 4.67 br dd 11.0, 4.5 1.68* 1.54* 1.65* 2.16 d 11.0 1.60* 2.52 dd 16.0, 9.0 5.85 dd 9.0, 3.0 5.95 dd 3.0, 2.0 4.75 dd 17.5, 2.0 4.85 dd 17.5, 2.0 0.83 d 7.0 3.01 dd 4.0, 2.0 2.29 d 4.0 4.38 br d 12.5 4.95 d 12.5 0.78 s 2.07 s 1.94 s 2.13 s 2.11 s -
5.51 ddd 11.0, 11.0, 4.8 2.20 ddd 13.0, 5.5,4.8, 2.0 1.46 dddd 13.0, 14.0, 11.0, 5.5 2.36 dddd 14.0, 14.0, 5.5, 2.0 1.12 ddd 14.0, 5.5, 2.0 4.66 br dd 11.0, 4.5 1.682 1.52br dd 9.5,4.5 1.68’ 2.14 d 11.0 1.59 dd 16.0, 3.0 2.58 dd 16.0, 9.0 5.76 dd 9.0, 3.0 5.91 dd 3.0, 2.0
5.51 ddd 12.0, 11.0, 5.0 2.20 ddd 13.0, 5.5, 4.8, 2.5 1.47 dddd 13.0, 14.0, 11.0, 5.5 2.35 dddd 14.0, 14.0, 5.5, 2.0 1.12 ddd 14.0, 5.5, 2.5 4.65 br dd 10.5, 4.5 1.68*
COCHMeC2H,-12
-
4.72 dd 17.5,2.0 4.84 ddd 17.5,2.0, 0.5 0.84 d 7.0 2.98 dd 4.0, 2.0 2.25 d 4.0 4.36 br d 12.5 4.93 d 12.5 0.77 s 2.06 s 1.93 s -
1.53brdd 9.5, 4.5 1.66; 2.16 d 11.0 1.59dd 16.0, 3.0 2.58 dd 16.0, 9.0 5.73 dd 9.0, 3.0 5.92 dd 3.0, 2.0 4.73 dd 17.5, 2.0 4.86 br dd 17.5, 2.0 0.83 d 6.5 2.98 dd 4.0, 2.0 2.25 d 4.0 4.36 br d 13.0 4.93 d 13.0 0.77 s 2.06 s 1.93 s -
2.10 s 2.56 quintet
2.10 s
-
0.92 t 7.5 1.18 d 7.0 2.38 qt 7.5, 7.5 1.73 and 1.53 qd 7.5, 7.0
7.0 1.21 and 1.24d 7.0
*Ambiguous due to signal overlapping. attached at different positions in l-6. The proton resonances of the acetates at C-l, C-6 and C-19 were at S, 2.06-2.07,1.93-1.94 and 2.11-2.12. When the isobutyrate was attached at C-12, the signal of the methine appeared at 6,, 2.56-2.57 and those of two methyls at 6” 1.21-1.25. By contrast these were observed at 6,2.44,1.20 and 1.17, when the isobutyrate was attached at C-l. The ‘H signals at 6,0.92-0.93,2.39-2.40 and 1.18-1.19, contributed by the 2-methylbutyrate, were assigned at C-12; the ‘H signals of the same substituent at C-3 in 3 resonated at 6, 0.89, 2.27 and 1.13. These differences reflect their structural differences, although a few compounds were examined. EXPERIMENTAL
Mps: uncorr.; ‘H and 13CNMR: JEOL JNM GX-500, FABMS:JEOL SX-102. Plant material. Voucher specimens of A. macrosperma Wall collected in March 1990 near Xishuangbanna Botanic Garden, and A. pantantka Hand-Mazz collected in June 1991 at Qujing, Yunnan, China, are deposited in the Kunming Institute of Botany, Academia Sinica. Extraction and isolation. Dried and finely powdered whole herb of A. mucrosperma (3.0 kg) was extracted with
MeOH (3 x 3 1)at room temp. for 10 days. Filtration and evapn of the solvent yielded a residue which was treated with activated charcoal (2 x 50 g) in MeOH (2 1). The residue was partitioned between EtOAc and H,O, and the EtOAc solvent evapd in uacuo to yield 18 g of yellow gum which was subjected to CC over silica gel (360 g). The column was eluted successively with nhexane-EtOAc (5: 1, 5:3, 1: 1, 1:2), EtOAc and Me,CO. In the n-hexane-EtOAc (5:3) eluate, fr. 7 (300 ml) gave a crude crystal from Me&O. The crystal was applied to HPLC (OD&s; MeOH-H,O, 3: 1) to give crude 1 and a mixt. of 2 and 3 which were further purified by HPLC (silica gel; n-hexane-2-propanol, 24: 1) to yield l(3.5 mg), 2 (0.15 mg) and 3 (0.4 mg), respectively, by crystallization from Me&O. Dried and finely powdered whole herb of A. pantantha (3.9 kg) was extracted according to the procedure mentioned above to give 30 g of yellow gum, which was subjected to CC over silica gel (1 kg). The column was eluted successively with n-hexane, n-hexane-EtOAc (5 : 1, 5 : 3,1: 1) and Me&O. The n-hexane-EtOAc (5: 3) eluate, frs 11-24 and 25-45 (each 200 ml), gave ajugamarin Cl (0.9 g). From the residue (1.86 g) of the mother liquor of frs 25-45,4 (235 mg) was purified by CC over RP-8 (75 g) eluted with MeOH-CHCl, (19: 1).
1094
XIAoYu SHEN et al.
Table 2. ‘%NMR C
data for compounds l-6
1
2
70.4 31.9 30.3 64.0 46.0 71.3 32.5 35.1 39.2 50.8 41.3 66.4 169.2 116.0 172.4 70.5 15.6 48.6 61.8 17.0
70.5 31.7 30.4 64.1 46.0 71.3 32.6 35.1 39.2 50.8 41.1 66.6 169.1 115.8 172.4 70.6 15.4 48.7 61.6 17.1 -
70.5 31.9 30.4 64.1 46.0 71.3 32.6 35.3 39.2 50.8 41.1 66.6 169.1 115.7 172.4 70.6 15.4 48.8 61.6 17.1
AC-6
169.6 21.0 -
169.7 21.0 -
169.7 21.0
170.4 21.0 175.9 34.5 19.2 18.3 175.8 34.5 19.2 18.3
170.3 21.0 175.9 34.6 19.3 18.3 -
170.3 21.0
AC-19 COCHMe,-1
COCHMe,-12
COCHMe
Et-l
-
COCHMe
Et-12
175.4 40.9 27.0 15.7 11.4
4
3
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 AC-1
AC-12
(125 MHz., CDCI,, CHCI, as int. standard)
175.8 34.6 19.3 18.3 175.4 41.5 26.9 16.1 11.5
Ajugumacrin C (1). Needles from Me&O, mp 206-207”, CJzH,,O1,; IR vg; cm-i: 3035, 2940, 2880, 1780,1775,1730(br), 1635, 1455, 1380, 13651260, 1230, 1190,1150,1085,1040; ‘H and 13C NMR: Tables 1 and 2; FABMS: m/z 607 [MI-Q+, 547, 519, 518, 371, 311, 281, 201, 173, 121, 43. Ajugamucrin D (2). Needles from Me&O, mp. 273-274”, C3.+H4s01 i; ‘H and i3C NMR: Tables 1 and 2 FABMS: m/z 621 [MH]+. ’ Ajugumacrin E (3). Needles from Me&O, C,,H,,O, 1; ‘H and 13CNMR: Tables 1 and 2; FABMS: m/z 621 [MH]+. Ajugapuntin A (4). Needles from Me,CO, mp 204-205”, C,,H,,O, i; IR 4;; cm-l: 2970,2940,2870, 1780, 1760,
70.6 31.7 30.4 64.1 45.9 71.3 32.7 35.4 39.0 50.4 41.7 66.4 168.4 116.2 172.3 70.6 15.6 48.7 61.5 16.9 169.3 21.8 169.7 21.0 169.4 21.0 170.3 21.0 __
5
6
70.4 31.7 30.4 64.0 71.2 32.6 35.2 39.5 50.8 41.7 66.3 169.3 115.9 172.4 70.4 15.5 48.6 61.9 17.0 169.4 21.8 169.6 21.0 -_ 170.3 21.0 --
70.2 31.6 30.3 64.0 45.9 71.2 32.6 35.3 39.1 50.7 41.5 66.4 169.0 115.9 172.4 70.6 15.6 48.7 61.9 17.0 169.3 21.8 169.7 21.0
170.3 21.0
175.8 34.2 19.5 18.4 -
-
-
175.4 40.9 26.9 15.7 11.4
174O(br), 1640,1380,1250,1080,1040; ‘Hand “CNMR: Tables 1 and 2. FABMS: m/z 551 [MH]+, 491,431,371, 311, 281,201, 171,43.
REFERENCES
1. Shen, X., Isogai, A., Furihata, K., Sun, H. and Suzuki, A. (1993) Phytochamistry (in press). 2. Yunnan Inst. of Bot. (1977) Flora Yunnanica Tomus 1, Science Press, Peking. 3. Shen, X., Zhang, H. and Sun, H. (1993) Acta Bob. Yunnan. 15 (in press). 4. Shimomura, H., Sashida, Y. and Ogawa, K. (1989) Chem. Pharm. Bull. 37, 354.