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Indole alkaloids from leaves and stems of Leuconotis eugenifolius
fhytochemisrry, Vol. 35, No. I, pp. 169-171, t994 Printed in Great Britain.
INDOLE ALKALOIDS
0
003 I -9422/w 36.00 + o.al 1993 Pcrgamon PressLtd
FROM LEAVES AND STEMS OF ~~UCO~~~~S EU~E~~F~~~~~ FUMIKO ABE and TATSUO YAMAUCHI
Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1, Nanakuma, Jonan-ku, Fukuoka 81441, Japan (Received 16
Key Word Index-leuconotis ~en~o~~~; rhazinaline N,oxide; di~spiroleu~no1~.
April 1993)
Apocynaceae; indole alkaloid; leuconolam;
yohimbine;
Abstract-From leaves and stems of Leuconotis eugenifolius, six indole alkaloids were isolated and five were identified as yohimbine, /?-yohimbine, leuconolam, 21-O-methyl-leuconolam and rhazinaline N,-oxide. The new alkaloid, having a pentacyclic diazaspiro system, was determined to be a diazaspiroleuconolam and named leuconoxine. Other constituents identified were 3@hydroxy-Sa,6a_epoxy+ionone, epigallocatechin, loganic acid, 2-methyl-3-buten-2-01 rutinoside and four flavonol glycosides.
INTRODUCIlON Leuconotis eugenifolius DC. is indigenous to Malaysia and Indonesia, and its latex was once used for treating yaws. Four plumeran-type alkaloids with C-2-C-7 bond cleavage were isolated from stems collected in Malaysia [l]. Since Apocynaceae plants in the tropicai districts occasionally show wide diversity in alkaloid content [Z, 31, we identified the alkaloids from leaves and stems of L. eugenifolius collected in Sumatra in order to compare the results with plants from Malaysia [l].
RESULIX
AND DISCUSSION
Six alkaloids (l-6) were isolated by successive column chromatography from the methanolic extract of the airdried leaves. Compounds 1 and 2 afforded the same molecular formula, Cz,Hz6N,0,, based on the high resolution mass spectra. The presence of one hydroxyl, one carbomethoxyl and five methylene groups was sugspectra, and the gested from the ‘H and ‘%NMR structures were confirmed as yohimbine (1) and &yohimbine (2), by comparison of their NMR data with those of authentic samples. Compound 3 has two carbonyl groups, one of which is conjugated with an olefinic bond. Signals in the ‘H and 1%I NMR spectra were in good agreement with those of leuconolam, reported previously from this species [l] and also from Alstonia scholaris collected in Java [3]. Since 4 showed similar features to those of 3 except for a 3H signal due to a methoxyl group at 63.17 in the ‘H NMR spec&um and aiTorded a molecular peak at m/z 340.1786, 14 mass units more than 3, its structure was determined as 21-O-methylleuconolam Cl]. In 5, the presence of a carbomethoxyl and a formyl (C1’7)groups linking to a quaternary carbon (C-16) and an
6,R-H 7:R-U
ethylidene group, respectively, was suggested in the NMR spectra and by COLOC measurement. The carbon signal at 6180.7 was assigned to C-2 and 5 was suggested to retain an indolenine framework which caused an upfield shift of a carbomethoxyl group (63.21). In the 2DNOESY spectrum, H-18 showed cross-peaks to H-15 and H-17. Chemical shifts and coupling patterns were the same as those of rhazinaline [3] except for downfield shifts of the proton and carbon signals assignable to H-3, H-5, H-21 and C-3, C-5, C-21. Based on the molecular formula, C21H12N104r one of the oxygens must be present as an N-oxide and 5 is characterized as rhazinaline N,oxide. Based on the presence of two carbonyl groups, 6 was considered to be an alkaioid related to 3. In the FAB-mass spectrum, 6 afforded a [M +H]+ peak at m/z 311.1761, suggesting a molecular formula of C,9H22NZ02. Since the unsaturation number was 10 and 169
F. ABE and T. YAMAUCHI
170 Table
1. ‘H and
“CNMR
spectral
data of compounds CDCI,]
5*
6
Atom 2 3
172.8 36.8
5
170.7
6
37.5
7 8 9 10 11 12 13 14 15
41.9 135.1 123.8 125.5 127.9 120.1 142.0 20.1 26.7
16
29.4
17
27.0
18 19 20 21
7.3 27.3 38.1 92.5
180.7 79.2
3.95 hr d (13) 2.79 hr r (13)
70.0 28.7
2.86 dd (17. 7) 2.67 d (17) 3.81 d (7) 7.16 7.12 7.25 7.77
5 and 6 [a (ppm) in
dd td rd dd
(7, (7, (7, (7,
1) 1) 1) 1)
1.6 m 1.97 hr f (12) 1.7 m 2.48 ddd (19, 6, 1) 2.75 m 1.84 ddd (14, 6, 1) 1.65 m 0.92 r (6) 1.38 qui (6)
56.2 145.3 127.5 128.7 130.9 123.2 157.1 29.6 32.9
3.42 m 3.0 m 2.10 m 3.0 m
7.53 7.25 7.39 7.60
br br br br
d (8) t (8) r (8) d (8)
3.0 m 4.13 br s
73.4 197.1
9.93 s
16.2 127.4 131.3 72.3
1.85 dd (7, 1) 5.82 br q (7)
169.5 53.8
others
4.94 dd (4, 2)
4.47 br d (18) 4.10 d (18) 3.21 s (-CO,Me)
(-CO,Me) *Dissolved
in CD,OD.
no olefinic carbon signals were observed in the “C NMR spectrum except for those due to a benzene ring, 6 was determined to be a pentacyclic compound. Of the two quaternary carbons at 638.1 and 92.5. the former seemed to be C-20 by comparison with that of 3, and the latter was assigned to C-21 based on cross-peaks from H-6 and H-17 in the COLOC spectrum. Although no hydroxyl group was present, 6 showed a similar chemical shift for C-21 as observed in 3, suggesting C-21 in 6 to be a diazaspirocarbon. Configuration of H-7 was determined to be fi, based on the 2D NOESY between H-7/H- 17 and H-7/H-19. In an earlier paper on 3 by Goh et al. [1], a chlorinated pentacyclic compound, 6_chlorodiazaspiroleuconolam (7) was formed from 3 with concentrated hydrochloric acid. In comparison of the “C NMR spectrum of 6 with that of 7, signals were in good agreement with each other except for C-5, C-6, C-7, C-8 and C-19. Compound 6 was, therefore, characterized as a diazaspiroleuconolam and named leuconoxine. Compounds 1-4 and 6 were also obtained from the stems. Compound 1 was the major alkaloid in the stems, and 3 in the leaves. In comparison with the alkaloid pattern of plants from Malaysia, it is apparent that 3 is the only common alkaloid present.
Resides alkaloids, 3/%hydroxy-Sq6a-epoxy-/?-ionone, epigallocatechin, loganic acid, fi-D-galactosides and 2-0fi-D-xylosyl-/?-D-galactosides of quercetin and kaempferol, and 2-methyl-3-buten-2-01 rutinoside were isolated from leaves. EXPERIMENTAL General. Mps: uncorr. iH NMR: 400 and “C NMR: 100 MHq CDCI, and/or CD,OD; TMS as int. standard. Optical rotations were measured in MeOH. For TLC and silica gel CC (normal phase), the following solvent systems were used, 1: C,H,-Me,CO (10: 1-3: 1). 2: CHCI,-MeOH (20: l-5: l), 3: CHCl,-MeOH-H,O (7 : 1: 2-7: 3 : 1, bottom layer). For reversed-phase columns (MCI-gel and Fuji-gel), MeOH-H,O or MeCN-H,O was used. Spray reagents: Dragendorlf reagent or Ehrlich reagent. Plant material. Leaves and stems of Leuconotis eugenifolius DC. were collected in Sumatra, Indonesia, in August 1990 (Herbarium number 4039 AT. F., stored at Osaka University). Extraction and isolation from the leaves. Air-dried and powdered leaves (1.25 kg) were percolated with MeOH.
lndole alkaloids
from Leuconotis eugenijolius
The MeOH soln was coned, diluted with eq. vol. of H,O, and then extracted with CHCl, after filtration of ppt. The extract was chromatographed on silica gel columns with solvents 1 and 2 to afford 1 (13 mg), 2 (12 mg), 3 (2 10 mg), 4 (21 mg) and 6 (17 mg). The MeOH soln was coned further and extracted with n-BuOH. The extract was passed through a polystyrene column (MCI-gel) with H,O-MeOH. The eluate with MeOH was coned and chromatographed on silica gel columns with solvents 2 and 3, and a Fuji-gel column with MeCN-Hz0 to give 5 (11 mg). Known alkaloids: yohimbine (1, mp 228-233” (dec), [ali +57.8” (c 0.90), negative FAB-MS m/z: 353.1869. CzlH,,N,O,-H), /?-yohimbine (2, mp 222-230” (de@, [a];’ - 19.1” (c 0.35), negative FAB-MS m/z 353.1867, C,,H,,N,O,-H), leuconolam (3, mp 262-267”. [a];s -515.8” (c 0.25); EIMS m/z: 326.1638, C,,H,,N,O,), 21-0-methylleuconolam (4, mp 140-597.8” (c 0.28), EIMS m/z: 340.1789, 150”, [ali CzoHz,NzG,). Neutral constituents. From the BuOH fraction, 3/?hydroxy-Sa&-epoxy+ionone (20 mg), epigallocatechin (60 mg), loganic acid (110 mg), quercetin 3-0-fl-D-galactoside (200 mg). quercetin 2-0-@-xylosyl-@-galactoside (1.9 g), kaempferol 3-0-B-D-galactoside (180 mg), kaempferol 2-O-/?-D-xylosyl-B-D-galactoside (540 mg) and 2-methyl-3-buten-2-ol-rutinoside (30 mg) were obtained. All compounds were identified from NMR and mass spectral considerations. Extraction and isolationfrom the stems. Air-dried stems (420 g) were treated as described above and the following compounds were obtained: 1(32 mg), 2 (17 mg), 3 (18 mg), 4 (3 mg) and 6 (2 mg). Rhazinaline &-oxide (5). A solid, [a];’ - 87.3” (c 0.70), FAB-MS m/z 367.1651, C2,HzzN,0d+H requires
*In ref. Cl], the [alo of lcuconolam from Leuconotisgrifithii is given as - 28.3”in CHCl,.
171
367.1658. ZD-NOESY cross-peaks: H-15/H-17, H-18, H-16/H-18, H-19/H-21a. COLOC cross-peaks: C-2/H-3, H-14, C-3/H-15, C-7/H-l!, C-8/H-6, C-10/H-12, C-l l/ H-9, C-16/H-14, H-17, C-19/H-18, H-21a, C-20/H-15, -CO,Me/ -CO,&. Leuconoxine (6). Prisms, mp 238-242” (CHCl,M&H), Cali’ -88.0” (c 1.2), FAB-MS m/z: 311.1761, CI,H,,N,O, +H requires 311.1759. ZD-NOESY crosspeaks: H-7/H-9, H-17/3, H-19, H-9/H-6a, H-15a/H-30; H18/H-15fi. COLOC cross-peaks: C-2/H-16, C-17, C-5/H3, H-7, C-8/H-6, H-7, *H-10, C-9/H-11, C-10/H-12, C-11/I-l-9, C-12/H-14 C-13/H-11, C-15/H-3, C-20/H-14, H-16, H-17, H-18, C-20/H-3/?, H-6a, H-17.
Acknowledgements-We thank Prof. I. Kitagawa of Osaka University and Prof. H. Shibuya of Fukuyama university for supplying the plant materials which were collected under the aid of Monbusho International Scientific Research Program (02041054). Our thanks are also due to MS Y. Iwase, J. Hbnda, and Mr Hanazono for NMR and mass spectral measurements.
REFERENCES
1. Goh, S. H., Ali, A. R. M. and Won& W. H. (1989) Tetrahedron 45, 7899. 2. Yamauchi, T., Abe, F., Padolina, W. G. and Dayrit, F. M. (1990) Phytochemistry 29, 3321.
3. Yamauchi, T., Abe, F., Chen, R.-F., Nom&a, G.-l., Santisuk, T. and Padolina, W. G. (1990) Phytochemistry 29, 3547.
INDOLE ALKALOIDS
0
003 I -9422/w 36.00 + o.al 1993 Pcrgamon PressLtd
FROM LEAVES AND STEMS OF ~~UCO~~~~S EU~E~~F~~~~~ FUMIKO ABE and TATSUO YAMAUCHI
Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1, Nanakuma, Jonan-ku, Fukuoka 81441, Japan (Received 16
Key Word Index-leuconotis ~en~o~~~; rhazinaline N,oxide; di~spiroleu~no1~.
April 1993)
Apocynaceae; indole alkaloid; leuconolam;
yohimbine;
Abstract-From leaves and stems of Leuconotis eugenifolius, six indole alkaloids were isolated and five were identified as yohimbine, /?-yohimbine, leuconolam, 21-O-methyl-leuconolam and rhazinaline N,-oxide. The new alkaloid, having a pentacyclic diazaspiro system, was determined to be a diazaspiroleuconolam and named leuconoxine. Other constituents identified were 3@hydroxy-Sa,6a_epoxy+ionone, epigallocatechin, loganic acid, 2-methyl-3-buten-2-01 rutinoside and four flavonol glycosides.
INTRODUCIlON Leuconotis eugenifolius DC. is indigenous to Malaysia and Indonesia, and its latex was once used for treating yaws. Four plumeran-type alkaloids with C-2-C-7 bond cleavage were isolated from stems collected in Malaysia [l]. Since Apocynaceae plants in the tropicai districts occasionally show wide diversity in alkaloid content [Z, 31, we identified the alkaloids from leaves and stems of L. eugenifolius collected in Sumatra in order to compare the results with plants from Malaysia [l].
RESULIX
AND DISCUSSION
Six alkaloids (l-6) were isolated by successive column chromatography from the methanolic extract of the airdried leaves. Compounds 1 and 2 afforded the same molecular formula, Cz,Hz6N,0,, based on the high resolution mass spectra. The presence of one hydroxyl, one carbomethoxyl and five methylene groups was sugspectra, and the gested from the ‘H and ‘%NMR structures were confirmed as yohimbine (1) and &yohimbine (2), by comparison of their NMR data with those of authentic samples. Compound 3 has two carbonyl groups, one of which is conjugated with an olefinic bond. Signals in the ‘H and 1%I NMR spectra were in good agreement with those of leuconolam, reported previously from this species [l] and also from Alstonia scholaris collected in Java [3]. Since 4 showed similar features to those of 3 except for a 3H signal due to a methoxyl group at 63.17 in the ‘H NMR spec&um and aiTorded a molecular peak at m/z 340.1786, 14 mass units more than 3, its structure was determined as 21-O-methylleuconolam Cl]. In 5, the presence of a carbomethoxyl and a formyl (C1’7)groups linking to a quaternary carbon (C-16) and an
6,R-H 7:R-U
ethylidene group, respectively, was suggested in the NMR spectra and by COLOC measurement. The carbon signal at 6180.7 was assigned to C-2 and 5 was suggested to retain an indolenine framework which caused an upfield shift of a carbomethoxyl group (63.21). In the 2DNOESY spectrum, H-18 showed cross-peaks to H-15 and H-17. Chemical shifts and coupling patterns were the same as those of rhazinaline [3] except for downfield shifts of the proton and carbon signals assignable to H-3, H-5, H-21 and C-3, C-5, C-21. Based on the molecular formula, C21H12N104r one of the oxygens must be present as an N-oxide and 5 is characterized as rhazinaline N,oxide. Based on the presence of two carbonyl groups, 6 was considered to be an alkaioid related to 3. In the FAB-mass spectrum, 6 afforded a [M +H]+ peak at m/z 311.1761, suggesting a molecular formula of C,9H22NZ02. Since the unsaturation number was 10 and 169
F. ABE and T. YAMAUCHI
170 Table
1. ‘H and
“CNMR
spectral
data of compounds CDCI,]
5*
6
Atom 2 3
172.8 36.8
5
170.7
6
37.5
7 8 9 10 11 12 13 14 15
41.9 135.1 123.8 125.5 127.9 120.1 142.0 20.1 26.7
16
29.4
17
27.0
18 19 20 21
7.3 27.3 38.1 92.5
180.7 79.2
3.95 hr d (13) 2.79 hr r (13)
70.0 28.7
2.86 dd (17. 7) 2.67 d (17) 3.81 d (7) 7.16 7.12 7.25 7.77
5 and 6 [a (ppm) in
dd td rd dd
(7, (7, (7, (7,
1) 1) 1) 1)
1.6 m 1.97 hr f (12) 1.7 m 2.48 ddd (19, 6, 1) 2.75 m 1.84 ddd (14, 6, 1) 1.65 m 0.92 r (6) 1.38 qui (6)
56.2 145.3 127.5 128.7 130.9 123.2 157.1 29.6 32.9
3.42 m 3.0 m 2.10 m 3.0 m
7.53 7.25 7.39 7.60
br br br br
d (8) t (8) r (8) d (8)
3.0 m 4.13 br s
73.4 197.1
9.93 s
16.2 127.4 131.3 72.3
1.85 dd (7, 1) 5.82 br q (7)
169.5 53.8
others
4.94 dd (4, 2)
4.47 br d (18) 4.10 d (18) 3.21 s (-CO,Me)
(-CO,Me) *Dissolved
in CD,OD.
no olefinic carbon signals were observed in the “C NMR spectrum except for those due to a benzene ring, 6 was determined to be a pentacyclic compound. Of the two quaternary carbons at 638.1 and 92.5. the former seemed to be C-20 by comparison with that of 3, and the latter was assigned to C-21 based on cross-peaks from H-6 and H-17 in the COLOC spectrum. Although no hydroxyl group was present, 6 showed a similar chemical shift for C-21 as observed in 3, suggesting C-21 in 6 to be a diazaspirocarbon. Configuration of H-7 was determined to be fi, based on the 2D NOESY between H-7/H- 17 and H-7/H-19. In an earlier paper on 3 by Goh et al. [1], a chlorinated pentacyclic compound, 6_chlorodiazaspiroleuconolam (7) was formed from 3 with concentrated hydrochloric acid. In comparison of the “C NMR spectrum of 6 with that of 7, signals were in good agreement with each other except for C-5, C-6, C-7, C-8 and C-19. Compound 6 was, therefore, characterized as a diazaspiroleuconolam and named leuconoxine. Compounds 1-4 and 6 were also obtained from the stems. Compound 1 was the major alkaloid in the stems, and 3 in the leaves. In comparison with the alkaloid pattern of plants from Malaysia, it is apparent that 3 is the only common alkaloid present.
Resides alkaloids, 3/%hydroxy-Sq6a-epoxy-/?-ionone, epigallocatechin, loganic acid, fi-D-galactosides and 2-0fi-D-xylosyl-/?-D-galactosides of quercetin and kaempferol, and 2-methyl-3-buten-2-01 rutinoside were isolated from leaves. EXPERIMENTAL General. Mps: uncorr. iH NMR: 400 and “C NMR: 100 MHq CDCI, and/or CD,OD; TMS as int. standard. Optical rotations were measured in MeOH. For TLC and silica gel CC (normal phase), the following solvent systems were used, 1: C,H,-Me,CO (10: 1-3: 1). 2: CHCI,-MeOH (20: l-5: l), 3: CHCl,-MeOH-H,O (7 : 1: 2-7: 3 : 1, bottom layer). For reversed-phase columns (MCI-gel and Fuji-gel), MeOH-H,O or MeCN-H,O was used. Spray reagents: Dragendorlf reagent or Ehrlich reagent. Plant material. Leaves and stems of Leuconotis eugenifolius DC. were collected in Sumatra, Indonesia, in August 1990 (Herbarium number 4039 AT. F., stored at Osaka University). Extraction and isolation from the leaves. Air-dried and powdered leaves (1.25 kg) were percolated with MeOH.
lndole alkaloids
from Leuconotis eugenijolius
The MeOH soln was coned, diluted with eq. vol. of H,O, and then extracted with CHCl, after filtration of ppt. The extract was chromatographed on silica gel columns with solvents 1 and 2 to afford 1 (13 mg), 2 (12 mg), 3 (2 10 mg), 4 (21 mg) and 6 (17 mg). The MeOH soln was coned further and extracted with n-BuOH. The extract was passed through a polystyrene column (MCI-gel) with H,O-MeOH. The eluate with MeOH was coned and chromatographed on silica gel columns with solvents 2 and 3, and a Fuji-gel column with MeCN-Hz0 to give 5 (11 mg). Known alkaloids: yohimbine (1, mp 228-233” (dec), [ali +57.8” (c 0.90), negative FAB-MS m/z: 353.1869. CzlH,,N,O,-H), /?-yohimbine (2, mp 222-230” (de@, [a];’ - 19.1” (c 0.35), negative FAB-MS m/z 353.1867, C,,H,,N,O,-H), leuconolam (3, mp 262-267”. [a];s -515.8” (c 0.25); EIMS m/z: 326.1638, C,,H,,N,O,), 21-0-methylleuconolam (4, mp 140-597.8” (c 0.28), EIMS m/z: 340.1789, 150”, [ali CzoHz,NzG,). Neutral constituents. From the BuOH fraction, 3/?hydroxy-Sa&-epoxy+ionone (20 mg), epigallocatechin (60 mg), loganic acid (110 mg), quercetin 3-0-fl-D-galactoside (200 mg). quercetin 2-0-@-xylosyl-@-galactoside (1.9 g), kaempferol 3-0-B-D-galactoside (180 mg), kaempferol 2-O-/?-D-xylosyl-B-D-galactoside (540 mg) and 2-methyl-3-buten-2-ol-rutinoside (30 mg) were obtained. All compounds were identified from NMR and mass spectral considerations. Extraction and isolationfrom the stems. Air-dried stems (420 g) were treated as described above and the following compounds were obtained: 1(32 mg), 2 (17 mg), 3 (18 mg), 4 (3 mg) and 6 (2 mg). Rhazinaline &-oxide (5). A solid, [a];’ - 87.3” (c 0.70), FAB-MS m/z 367.1651, C2,HzzN,0d+H requires
*In ref. Cl], the [alo of lcuconolam from Leuconotisgrifithii is given as - 28.3”in CHCl,.
171
367.1658. ZD-NOESY cross-peaks: H-15/H-17, H-18, H-16/H-18, H-19/H-21a. COLOC cross-peaks: C-2/H-3, H-14, C-3/H-15, C-7/H-l!, C-8/H-6, C-10/H-12, C-l l/ H-9, C-16/H-14, H-17, C-19/H-18, H-21a, C-20/H-15, -CO,Me/ -CO,&. Leuconoxine (6). Prisms, mp 238-242” (CHCl,M&H), Cali’ -88.0” (c 1.2), FAB-MS m/z: 311.1761, CI,H,,N,O, +H requires 311.1759. ZD-NOESY crosspeaks: H-7/H-9, H-17/3, H-19, H-9/H-6a, H-15a/H-30; H18/H-15fi. COLOC cross-peaks: C-2/H-16, C-17, C-5/H3, H-7, C-8/H-6, H-7, *H-10, C-9/H-11, C-10/H-12, C-11/I-l-9, C-12/H-14 C-13/H-11, C-15/H-3, C-20/H-14, H-16, H-17, H-18, C-20/H-3/?, H-6a, H-17.
Acknowledgements-We thank Prof. I. Kitagawa of Osaka University and Prof. H. Shibuya of Fukuyama university for supplying the plant materials which were collected under the aid of Monbusho International Scientific Research Program (02041054). Our thanks are also due to MS Y. Iwase, J. Hbnda, and Mr Hanazono for NMR and mass spectral measurements.
REFERENCES
1. Goh, S. H., Ali, A. R. M. and Won& W. H. (1989) Tetrahedron 45, 7899. 2. Yamauchi, T., Abe, F., Padolina, W. G. and Dayrit, F. M. (1990) Phytochemistry 29, 3321.
3. Yamauchi, T., Abe, F., Chen, R.-F., Nom&a, G.-l., Santisuk, T. and Padolina, W. G. (1990) Phytochemistry 29, 3547.