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A steroidal alkaloid from Pachysandra axillaris
Phytockmisrry. Vol. 31. No. 7, pp. 2571 -2572. 1992 Printed in Great Britain.
A STEROIDAL
ALKALOID
0031~9422/92 ss.oo+o.cKl Pcrgamon Press Ltd
FROM
PACHYSANDRA
AXILLARIS
CHIU MINGHUA, NIE RUILIN* and ZHOU JUN Kunming Institute of Botany, Academia Sinica, Kunming, 650204,P.R. China (Received in
revisedform 22 October 1991)
Key Word Index Pachysandra axillaris; Buxaceae; steroidal alkaloid; axillaridine
A.
Abstract-The chemical structure of a novel steroidal alkaloid, axillaridine A, from Pachysandra axillaris has been elucidated as 20a-dimethylamino-3-benzoylamino-5a-pregn-2(3)-en-4-one.
INTRODUCIION Pachysandra
axillaris,
herb of the Buxaceae family, is distributed over southern China, and has been used in folk drugs for the treatment of rheumatism. In previous publications, we have reported the isolation of 25 steroida1 alkaloids and identification of pachyaximines A and B, iso-spiropachysine and spiropachysine from this plant [l-3]. This paper describes the isolation and structural elucidation of a novel steroidal alkaloid, axillaridine A, from the same species, the structure of which has been identified as 20x-dimethylamino-3-benzoylamino-5apregn-2(3)-en+one. a
RESULTSAND
DISCUSSION
The total alkaloids mixture was isolated from the concentrated 95% extracts of P. axillaris by extraction at different pH values. The fraction obtained from the chloroform phase was evaporated to afford a gum. The acetone-insoluble portion of this fraction afforded the alkaloid axillaridine A (1) in 0.016% yield. The IR spectrum of axillaridine A (1) exhibited absorptions at 3380 (NH), 1660 (a&unsaturated cyclohexenone), 1650 (benzamide C=O), 1640 (C=C), 1600, 1490, 1458 and 705 cm - ’ (aromatics). The UV spectrum showed &n*, at 232(.s 10701) and 272 (E 10465) nm which indicated the presence of a secondary benzamide and an a&unsaturated carbonyl group. The high resolution mass spectrum of axillaridine A included the [M] + at m/z 462.32011, in agreement with the molecular formula Cs,H,,N,O,. In its mass spectrum, the peak at m/z 447 resulted from the loss of a methyl group from the [M] +. The alkaloid showed a base peak at m/z 72, resulting from the cleavage of the nitrogen-containing side chain on ring D, a commonly encountered fragment in other related alkaloids [4]. The weak peak at m/z 391 is also a common fragment resulting from the loss of the nitrogen-containing side chain on ring D from the CM]‘. The peak at m/z 105 was due to the benzoyl ion. The ‘H NMR spectrum (CDCI,, 400 MHz) displayed two tertiary methyl groups at 60.648 and 0.901, while a
*Author to whom correspondence
should
secondary methyl group resonated as a doublet at 60.872 (J=6.4Hz), corresponding to 18-, 19- and 21-methyl groups, respectively. A 6H singlet at 62.173 was assigned to the protons of two methyl groups attached to the nitrogen. The NH proton of the secondary benzamide resonated at 68.646 as a singlet, the downfield chemical shift of this proton being due to the proximity of the a$unsaturated carbonyl group on ring A. The C-2 olefinic proton appeared at 67.805 as a double doublet (5=2.5, 6.8 Hz), the downfield chemical shift of this proton being due to the proximity of the carbonyl group of the benzamide. The splitting of a double doublet supported the fact that the carbonyl group of the a&unsaturated carbonyl moiety is at C-4 and not at C- 1. We assumed the presence of intramolecular hydrogen bonds between the NH hydrogen and the a&unsaturated carbonyl group as well as the olefinic hydrogen at C-2 and the carbonyl group of the benzamide. Thus, the C-2 olefinic and NH hydrogen signals appeared at very downfield positions (67.805 and 8.646, respectively). The aromatic protons appeared as three groups of 2H, 1H and 2H multiplets centred at 67.838(2H, br d,J=7.0 Hz), 7.517(1H, br dd,./ = 7.0, 7.0 Hz) and 7.448 (2H, br dd, J=7.0, 7.0 Hz), corresponding to H-2’, 6, H-4’ and H-3’, 5’, respectively. The ‘%NMR spectrum (CDCI,) of 1 exhibited three signals at 6 12.3, 13.3 and 10.0, which were assigned to C18, C-19 and C-21 methyl carbons, respectively. The 13CNMR data of rings C and D as well as of the side chain were similar to those. of pachyaximines A and B, spiropachysine and iso-spiropachysine [2,3]. The signals
1
be addressed. 2571
2572 Table
Short Reports 1. r3CNMR
data and DEPT
Carbon no.
Chemical shift (6)
1 2 3 4 5
39.1 126.4 131.4 196.1 54.9
6
20.5
CH,
7 8 9 10 II 12 13 I4 I5 16 17
30.5 34.1 54.0 39.9 20.8 39.4 41.6 56.2 23.9 21.6 54.9
CH, CH CH C CH, CH, C CH
DEPT CH,
CH C C CH
Carbon no. 18 19 20 21
of axillaridine Chemical shift (6) 12.3 13.3 61.0 10.0
N-Me,
39.9
Ph-CO 1’ 2 3’ 4 5’ 6
165.4 134.6 128.7 126.9 131.7 126.9 128.7
EXI’ERIMENTAL
A (1)
DEPT Me Me CH Me
MC C C CH CH CH CH CH
CH, CH, CH
of the benzoyl group moieties were easily assigned. The x,/?-unsaturated carbonyl carbon appeared at 6 196.1. The assignments to the various carbons were confirmed by DEPT and 13C-‘H HETCOR (Table 1). The t3C-‘H HETCOR spectrum fully agreed with the proposed structure 1 and assignments of the data of 13C and ‘H NMR spectra of axillaridine A. In the ‘H NMR spectrum, the C-2 olefinic proton signal was interlaced with that of aromatics. The shift correlation between the C-2 carbon at 6 126.4 and the C-2 proton at 6 7.805 (br dd) could be clearly observed from r3C-*H HETCOR and DEPT spectra. The chemical shift of the C-6 methylene carbon shifts upfield due to the shielding influence of the C-4 carbonyl group; its signal was assigned to fi20.8 and DEPT. The multiplicities by 13C-rH HETCOR of overlapping carbon and proton signals could undoubtedly be assigned from these spectra.
‘H NMR, 13CNMR, DEPT and “C-‘H HETCOR spectra were recorded in CDCI,; chemical shifts (6) are given in ppm with TMS as int. std. UV were measured in 95% EtOH, EIMS at
70 eV.
The 95% EtOH exts of the dried whole plant (ca 45 kg) of P. axillarrs Franch. were evapd to a gum. The crude alkaloids were obtamed by extn into 5% HOAc. Partial sepn of the alkaloids was achieved by extn into CHCI, at different pH values. The fr. obtained from the CHCI, phase was cvapd to a gum. The Me,CO insoluble fr. obtamed from the gum was washed several times wrth CHCI,. IO afford a colourless powder alkaloid 1 (7.2 g) in 0.016% yield. Axillaridine A (1). Mp 223-224‘. [z];’ +51.3’ (CHCI,; c 0.517). UV j.::” nm: 232 (E lO701), 272 (E 10465). 1R vz; cm-‘: 3380,2950,2860,2770, 1660.1650, 1640,1600.1580,1534, 1490. 145X, 1378, 1366, 1340, 705. HRMSmjz: [M]’ 462.32011. MS m;‘z(%):462 [Ml’ @.I),447 [M -Me]’ (0.3), 391 [M-side chain]*, 312. 226 (8). 187 (12). 122. 105 (II), 77 (8). 72 (100). ‘HNMR (CDCI,): 68.646 (IH. s, NH), 7.805 (IH, dd, J=2.5, 6.8Hz, H-2). 7.838 (2H, hrd, J=7.0Hz, H-2’. 6’) 7.517 (IH, br dd. J = 7.0.7.0 HI, H-4’). 7.448 (2H. hr dd, J = 7.0.7.0 Hz. H-3’. 5’). 2.173 (6H, s, NMe,). 0.901 (3H, s, Me-l9), 0.872 (3H, d, J =6.4 Hz, Me-21). 0.648 (3H. s. Me-18). lZCNMR and DEPT (CDCI,): see Table I. “C ‘H HETCOR spectrum in CDCI,. Acknowledgements .- We are grateful lo Assoc. Prof. Kasai (lnstitute of Pharmaceutical Sciences, Htroshima Universtty School of Medicme, Japan) and Dr Matsuura Wakunaga
Pharmaceutical
(Institute for OTC
Co., Ltd.,
Research,
Japan) for the HRMS
measurements.
REFERENCFS 1. Chiu (1990)
Minghua.
2. Chiu Minghua,
Acta Botanica 3. Chiu
Nie
Youji Huaxue
Mtnghua,
Ruilin,
Li Zhongrong
and Zhou
Jun
10, 41.
Nie Ruilin, Wang Xun and Zhou Jun (1989) Sinica 31, 535. Nie
(1990) Phyrochemisrry
Li Zhongrong 29. 3927.
Rutlin,
and Zhou
Jun
4. Ktkuchi, T., Uyeo, S., Nishinaga, T., Ibuka. T. and Kato, A. ( 1967) Yakugaku Zasshr 87. 63 I.
A STEROIDAL
ALKALOID
0031~9422/92 ss.oo+o.cKl Pcrgamon Press Ltd
FROM
PACHYSANDRA
AXILLARIS
CHIU MINGHUA, NIE RUILIN* and ZHOU JUN Kunming Institute of Botany, Academia Sinica, Kunming, 650204,P.R. China (Received in
revisedform 22 October 1991)
Key Word Index Pachysandra axillaris; Buxaceae; steroidal alkaloid; axillaridine
A.
Abstract-The chemical structure of a novel steroidal alkaloid, axillaridine A, from Pachysandra axillaris has been elucidated as 20a-dimethylamino-3-benzoylamino-5a-pregn-2(3)-en-4-one.
INTRODUCIION Pachysandra
axillaris,
herb of the Buxaceae family, is distributed over southern China, and has been used in folk drugs for the treatment of rheumatism. In previous publications, we have reported the isolation of 25 steroida1 alkaloids and identification of pachyaximines A and B, iso-spiropachysine and spiropachysine from this plant [l-3]. This paper describes the isolation and structural elucidation of a novel steroidal alkaloid, axillaridine A, from the same species, the structure of which has been identified as 20x-dimethylamino-3-benzoylamino-5apregn-2(3)-en+one. a
RESULTSAND
DISCUSSION
The total alkaloids mixture was isolated from the concentrated 95% extracts of P. axillaris by extraction at different pH values. The fraction obtained from the chloroform phase was evaporated to afford a gum. The acetone-insoluble portion of this fraction afforded the alkaloid axillaridine A (1) in 0.016% yield. The IR spectrum of axillaridine A (1) exhibited absorptions at 3380 (NH), 1660 (a&unsaturated cyclohexenone), 1650 (benzamide C=O), 1640 (C=C), 1600, 1490, 1458 and 705 cm - ’ (aromatics). The UV spectrum showed &n*, at 232(.s 10701) and 272 (E 10465) nm which indicated the presence of a secondary benzamide and an a&unsaturated carbonyl group. The high resolution mass spectrum of axillaridine A included the [M] + at m/z 462.32011, in agreement with the molecular formula Cs,H,,N,O,. In its mass spectrum, the peak at m/z 447 resulted from the loss of a methyl group from the [M] +. The alkaloid showed a base peak at m/z 72, resulting from the cleavage of the nitrogen-containing side chain on ring D, a commonly encountered fragment in other related alkaloids [4]. The weak peak at m/z 391 is also a common fragment resulting from the loss of the nitrogen-containing side chain on ring D from the CM]‘. The peak at m/z 105 was due to the benzoyl ion. The ‘H NMR spectrum (CDCI,, 400 MHz) displayed two tertiary methyl groups at 60.648 and 0.901, while a
*Author to whom correspondence
should
secondary methyl group resonated as a doublet at 60.872 (J=6.4Hz), corresponding to 18-, 19- and 21-methyl groups, respectively. A 6H singlet at 62.173 was assigned to the protons of two methyl groups attached to the nitrogen. The NH proton of the secondary benzamide resonated at 68.646 as a singlet, the downfield chemical shift of this proton being due to the proximity of the a$unsaturated carbonyl group on ring A. The C-2 olefinic proton appeared at 67.805 as a double doublet (5=2.5, 6.8 Hz), the downfield chemical shift of this proton being due to the proximity of the carbonyl group of the benzamide. The splitting of a double doublet supported the fact that the carbonyl group of the a&unsaturated carbonyl moiety is at C-4 and not at C- 1. We assumed the presence of intramolecular hydrogen bonds between the NH hydrogen and the a&unsaturated carbonyl group as well as the olefinic hydrogen at C-2 and the carbonyl group of the benzamide. Thus, the C-2 olefinic and NH hydrogen signals appeared at very downfield positions (67.805 and 8.646, respectively). The aromatic protons appeared as three groups of 2H, 1H and 2H multiplets centred at 67.838(2H, br d,J=7.0 Hz), 7.517(1H, br dd,./ = 7.0, 7.0 Hz) and 7.448 (2H, br dd, J=7.0, 7.0 Hz), corresponding to H-2’, 6, H-4’ and H-3’, 5’, respectively. The ‘%NMR spectrum (CDCI,) of 1 exhibited three signals at 6 12.3, 13.3 and 10.0, which were assigned to C18, C-19 and C-21 methyl carbons, respectively. The 13CNMR data of rings C and D as well as of the side chain were similar to those. of pachyaximines A and B, spiropachysine and iso-spiropachysine [2,3]. The signals
1
be addressed. 2571
2572 Table
Short Reports 1. r3CNMR
data and DEPT
Carbon no.
Chemical shift (6)
1 2 3 4 5
39.1 126.4 131.4 196.1 54.9
6
20.5
CH,
7 8 9 10 II 12 13 I4 I5 16 17
30.5 34.1 54.0 39.9 20.8 39.4 41.6 56.2 23.9 21.6 54.9
CH, CH CH C CH, CH, C CH
DEPT CH,
CH C C CH
Carbon no. 18 19 20 21
of axillaridine Chemical shift (6) 12.3 13.3 61.0 10.0
N-Me,
39.9
Ph-CO 1’ 2 3’ 4 5’ 6
165.4 134.6 128.7 126.9 131.7 126.9 128.7
EXI’ERIMENTAL
A (1)
DEPT Me Me CH Me
MC C C CH CH CH CH CH
CH, CH, CH
of the benzoyl group moieties were easily assigned. The x,/?-unsaturated carbonyl carbon appeared at 6 196.1. The assignments to the various carbons were confirmed by DEPT and 13C-‘H HETCOR (Table 1). The t3C-‘H HETCOR spectrum fully agreed with the proposed structure 1 and assignments of the data of 13C and ‘H NMR spectra of axillaridine A. In the ‘H NMR spectrum, the C-2 olefinic proton signal was interlaced with that of aromatics. The shift correlation between the C-2 carbon at 6 126.4 and the C-2 proton at 6 7.805 (br dd) could be clearly observed from r3C-*H HETCOR and DEPT spectra. The chemical shift of the C-6 methylene carbon shifts upfield due to the shielding influence of the C-4 carbonyl group; its signal was assigned to fi20.8 and DEPT. The multiplicities by 13C-rH HETCOR of overlapping carbon and proton signals could undoubtedly be assigned from these spectra.
‘H NMR, 13CNMR, DEPT and “C-‘H HETCOR spectra were recorded in CDCI,; chemical shifts (6) are given in ppm with TMS as int. std. UV were measured in 95% EtOH, EIMS at
70 eV.
The 95% EtOH exts of the dried whole plant (ca 45 kg) of P. axillarrs Franch. were evapd to a gum. The crude alkaloids were obtamed by extn into 5% HOAc. Partial sepn of the alkaloids was achieved by extn into CHCI, at different pH values. The fr. obtained from the CHCI, phase was cvapd to a gum. The Me,CO insoluble fr. obtamed from the gum was washed several times wrth CHCI,. IO afford a colourless powder alkaloid 1 (7.2 g) in 0.016% yield. Axillaridine A (1). Mp 223-224‘. [z];’ +51.3’ (CHCI,; c 0.517). UV j.::” nm: 232 (E lO701), 272 (E 10465). 1R vz; cm-‘: 3380,2950,2860,2770, 1660.1650, 1640,1600.1580,1534, 1490. 145X, 1378, 1366, 1340, 705. HRMSmjz: [M]’ 462.32011. MS m;‘z(%):462 [Ml’ @.I),447 [M -Me]’ (0.3), 391 [M-side chain]*, 312. 226 (8). 187 (12). 122. 105 (II), 77 (8). 72 (100). ‘HNMR (CDCI,): 68.646 (IH. s, NH), 7.805 (IH, dd, J=2.5, 6.8Hz, H-2). 7.838 (2H, hrd, J=7.0Hz, H-2’. 6’) 7.517 (IH, br dd. J = 7.0.7.0 HI, H-4’). 7.448 (2H. hr dd, J = 7.0.7.0 Hz. H-3’. 5’). 2.173 (6H, s, NMe,). 0.901 (3H, s, Me-l9), 0.872 (3H, d, J =6.4 Hz, Me-21). 0.648 (3H. s. Me-18). lZCNMR and DEPT (CDCI,): see Table I. “C ‘H HETCOR spectrum in CDCI,. Acknowledgements .- We are grateful lo Assoc. Prof. Kasai (lnstitute of Pharmaceutical Sciences, Htroshima Universtty School of Medicme, Japan) and Dr Matsuura Wakunaga
Pharmaceutical
(Institute for OTC
Co., Ltd.,
Research,
Japan) for the HRMS
measurements.
REFERENCFS 1. Chiu (1990)
Minghua.
2. Chiu Minghua,
Acta Botanica 3. Chiu
Nie
Youji Huaxue
Mtnghua,
Ruilin,
Li Zhongrong
and Zhou
Jun
10, 41.
Nie Ruilin, Wang Xun and Zhou Jun (1989) Sinica 31, 535. Nie
(1990) Phyrochemisrry
Li Zhongrong 29. 3927.
Rutlin,
and Zhou
Jun
4. Ktkuchi, T., Uyeo, S., Nishinaga, T., Ibuka. T. and Kato, A. ( 1967) Yakugaku Zasshr 87. 63 I.