In the course of our investigation for bioactive compounds from Annonaceae natural resources,1—4)we have studied the stems of Goniothalamus amuyon (BLANCO) MERR., which is
not only indigenous to southern Taiwan near the coastal re-gion but also the only species of Goniothalamus in Taiwan. The seeds were reported to be useful in the treatment of edema and rheumatism.5) Literature surveys indicated that
two major types of bioactive compounds, styryllactones and acetogenins, have been found from Goniothalamus species. In this study, we report herein the isolation and structure elu-cidation of two new compounds, goniothalesacetate (1) and goniothaesdiol A (2), together with eight known compounds, include three styryllactones, goniodiol-7-monoacetate,4) go-niodiol-8-monoacetate,4) leiocarpin C,6) and five alkaloids, liriodenine,3) griffithazanone A,7)
4-methyl-2,9,10-(2H)-1-azaanthracencetrione,7)velutinam8)and aristolactam BII.8)
Compound 1 was isolated as yellow oil. The presence of hydroxyl and methoxyl groups were suggested in EI-MS by two fragment peaks at m/z 321 [MOH] and 307 [MOCH3]. Its molecular formula, C17H22O7, was obtained by HR-FAB-MS (Found m/z: 339.1441, Calcd: 339.1444). The UV spectrum of 1 revealed the maximum absorption at 210 nm. The IR absorptions at 3433 and 1743 cm1were in accordance with hydroxyl and carbonyl groups.
The 1H-NMR signals at d 7.30—7.50 (5H) represented a mono-substituted phenyl moiety. Five oxygen-bearing me-thine carbons were suggested by the 1H-NMR (d 4.00, 4.05,
4.28, 4.72, 5.10) and 13C-NMR (d 78.1, 81.1, 82.8, 83.2,
87.9). One methylene group, two methoxy groups, and one acetyl group were also indicated at d 2.55 (2H), d 3.51 (3H), d 3.68 (3H), and d 1.97 (3H) in the 1H-NMR spectrum,
re-spectively. These groups were also confirmed in the 13
C-NMR spectrum.
Further spectral evidence was required to confirm the structure of 1. The COSY spectrum showed coupling corre-lations through the sequence of H-2 to H-7 (Fig. 2). The HBMC spectrum (Fig. 2) showed cross peaks between the aromatic signals (H-2, 6) and 2, and between H-2 and
C-1, which indicated the aromatic ring was connected to C-2. In addition, the HMBC cross peaks between dH 4.72 (H-2)
and dC83.2 (C-3), dH5.10 (H-4) and dC87.9 (C-2), dC83.2
(C-3) and dC82.8 (C-5) were also observed. The HMBC cor-relations from dH 4.00 (H-6) to dC 59.3 (OCH3-6) and dH
3.51 (OCH3) to dC 78.1 (C-6) showed that the methoxyl
group was linked to C-6. In addition, a cross peak between dH5.10 (H-4) and dC171.7 (C-9) indicated the acetyl group was located on C-4. Accordingly, an ether linkage and a free hydroxyl should be assigned on positions 2, 3, or 5. The 1 H-NMR spectrum of the acetate derivative (3) confirmed that the hydroxyl group was located on C-3 according to the obvi-ous down-field shift of H-3 (from d 4.05 to 5.04) after acety-lation of 1. Therefore, a 2,5-ether linkage can be confirmed. The plane structure of 1 was determined as methyl 3-(3-ace-toxy-4-hydroxy-5-phenyloxolan-2-yl)-3-methoxypropanoate.
The stereochemistry of 1 was established by the NOESY spectrum. The cross peaks of H-2/H-5 were in agreement with cis-relationship for the tetrahydrofuranal moiety. The presence of a correlation between H-2 and H-4 and without correlation between H-3 and H-5 indicated that H-4 has the same orientation with H-2 and H-5. These correlations showed the relative configuration of H-2/H-3, H-3/H-4, and H-4/H-5 as erythero, threo, and threo. The H-5 and H-6 were also determined to be in the threo configuration as the result of J5/6 (7.2 Hz) value.
9)
In order to determine of the absolute stereochemistry of 1, (R)- and (S)-methoxy fluoromethyl phenylacetic acid (MTPA) esters of 1 (1r, 1s) were prepared. The 1H-NMR data (see Table 2) of 1r and 1s indicated the absolute configuration of C-3 to be R. Thus, the chiral cen-ters of 1 were evidenced as 2R,3R,4S,5S,6R. From the fore-going spectral analyses, the structure 1 was established and named goniothalesacetate.
Compound 2 was isolated as white powder. Its molecular formula, C14H18O5, was obtained by HR-FAB-MS (Found m/z: 267.1234, Calcd: 267.1232). The UV spectrum of 2
re-1040 Vol. 54, No. 7
New Constituents from Stems of Goniothalamus amuyon
Yu-Hsuan LAN,aFang-Rong CHANG,aYu-Liang YANG,band Yang-Chang WU*,a
aGraduate Institute of Natural Products, Kaohsiung Medical University; Kaohsiung, 807 Taiwan: and bInstitute of Biological Chemistry, Academia Sinica; Taipei, 115 Taiwan. Received January 24, 2006; accepted April 21, 2006
Two new compounds, goniothalesacetate (1) and goniothalesdiol A (2) together with goniodiol-7-monoac-etate, goniodiol-8-monoacgoniodiol-7-monoac-etate, leiocarpin C, liriodenine, griffithazanone A, 4-methyl-2,9,10-(2H)-1-azaanthra-cencetrione, velutinam and aristolactam BII were isolated and characterized from the stems of Goniothalamus amuyon. Structures of new compounds were determined by spectral analysis.
Key words Goniothalamus amuyon; styryllactone; goniothalesacetate; goniothalesdiol A
Notes Chem. Pharm. Bull. 54(7) 1040—1043 (2006)
© 2006 Pharmaceutical Society of Japan ∗ To whom correspondence should be addressed. e-mail: [email protected]
Fig. 1. Structure of Goniothalesacetate (1) and Goniothalesdiol A (2)
Fig. 2. Key NOESY Correlations and COSY and Key HMBC Correla-tions for Goniothalesacetate (1)
vealed the maximum absorption at 208 nm. The IR absorp-tions at 3409 and 1732 cm1 indicated the presence of hy-droxyl and carbonyl groups.
The existence of a mono-substituted phenyl moiety was in-dicated by the proton resonances at d 7.29—7.45 (5H). Four oxygen-bearing methines were suggested by the 1H-NMR (d 3.68, 4.38, 4.47, 4.87) and 13C-NMR (d 72.8, 73.4, 74.4, 85.7). The NMR spectra showed the existence of two methyl-ene groups at dH 1.88 and 2.34/dC 39.8 and dH 2.74 and 2.83/dC 40.3. A singlet at d 3.71 (3H) and a carbon
reso-nance at d 51.8 also indicated the presence of a methoxyl group.
The coupling correlations through a sequence from H-2 to H-6 were found in the COSY spectrum. The HBMC cross peaks between aromatic signals (H-2, H-6) and C-2 as well as between H-2 and C-1 indicated that the aromatic ring was connected to C-2. The HMBC correlations between dH3.71 and dC172.3 (C-8) as well as between d 2.74/2.84 (H-7) and
d 172.3 (C-8) suggested that the acetic acid methyl ester tail was linked to C-6 as in 1. However, the arrangements of the sequence from H-2 to H-6 are different in NMR spectra with those of 1. On the basis of aforementioned spectral features, tetrahydropyranal central skeleton was proposed.
The stereochemistry of 2 was assigned by analysis of 1
H-NMR coupling constant and circular dichroism. The J2/3 value (8.0 Hz) indicated an axial–axial position of H-2 and H-3.10)The observed J3/4 value was 3.6 Hz, which indicated
that H-3 and H-4 should be an axial–equitorial position, and the J5/6(8.4, 4.8 Hz) determined the conformation of H-6 as axial. Hence, the relative conformations H-2/H-3 and H-3/H-4 are assigned to be erythero and erythero. In addition, the
[a]Dvalues and circular dichroism spectrum was measured; however, the data can not lead to the decision of the absolute configuration of 2. The [a]Dvalues of glycopyranosylarenes
had been reported in either positive and negative values. Therefore, the negative [a]Dvalue of 2 is an useless data in stereochemistry. Moreover, in comparison with the previous CD data of glycopyranosylarenes,11) the ignorable negative cotton effect (De 0.5) of 2 at the 250 nm region is also am-biguous to the structural elucidation. The Mosher’s ester re-action also failed due to the insufficient amount of the sam-ple. Thus, we predicted the absolute stereochemistry should follow the ratiocination in biosynthesis and assign as 2R*,3S*,4S*, and 6R*, respectively, which was named gonio-thalesdiol A. It is a new-type styryllactone derivative from the genus Goniothalamus.
According to the previous literatures,12)the possible partial
biosynthesis pathway of styryllactones was published. Due to the unusual structural features of 1 and 2, we supposed their possible biogenetic pathway (Fig. 3). By key hydroxylation procedures (steps a, b) passing through different oxidative cyclization, possible metabolites, including the new com-pounds 1 and 2, deoxygoniopypyrone, and goniofupyrone, can be generated. In addition to our and previous supposi-tion,12)the biogenetic originals of most styryllactone
deriva-tives from Goniothalamus species can be completed. All pro-posed approaches were designed on the basis of refs. 12 and 13.
In the biological assay, compound 1 did not show signifi-cant inhibition against several cancer cell lines, including Hep2G (human hepatocellular carcinoma), Hep3B (human hepatoma cells), MDA-MB-231 (human breast carcinoma)
July 2006 1041
Table 2. 1H-NMR Spectral Data for 1r and 1s (in d) in CD 3OD
H-2 H-3 H-4 H-5 H-6 H-7
1s 4.940 5.345 5.284 4.242 4.016 2.529
1r 4.799 5.345 5.345 4.234 4.029 2.553
DdH(S–R) 0.141 0.000 0.061 0.01 0.01 0.04
Table 1. 1H- and 13C-NMR Spectral Data for 1 and 2 in CD
3OD and (CD3)2CO a) 1 2 H C H Cb) 2 4.72 (1H, d, J4.4 Hz) 87.9 4.87 (1H, d, J8.0 Hz) 73.4 3 4.05 (1H, dd, J4.4, 2.4 Hz) 83.2 3.68 (1H, dd, J8.0, 3.6 Hz) 85.7 4 5.10 (1H, dd, J4.2, 2.4 Hz) 81.1 4.47 (1H, ddd, J5.6, 3.6, 2.4 Hz) 72.8 5 4.28 (1H, dd, J7.2, 4.2 Hz) 82.8 1.77 (1H, ddd, J13.6, 4.8, 2.4 Hz) 39.8 2.35 (1H, ddd, J13.6, 8.4, 5.6 Hz) 6 4.00 (1H, td, J7.2, 4.4 Hz) 78.1 4.38 (1H, dddd, J8.4, 7.2, 6.4, 4.8 Hz) 74.4 7 2.60 (1H, dd, J15.4, 4.4 Hz) 37.4 2.61 (1H, dd, J15.2, 6.4 Hz) 40.3 2.55 (1H, dd, J15.4, 7.2 Hz) 2.75 (1H, dd, J15.2, 7.2 Hz) 8 173.2 172.3 6-OCH3 3.47 (3H, s) 59.3 8-OCH3 3.67 (3H, s) 52.3 51.8 4-OCOCH3 1.97 (3H, s) 171.7 20.7 1 141.7 147.7 2, 6 7.50 (2H, d, J7.2 Hz) 126.8 7.40 (2H, m) 126.2 3, 5 7.37 (2H, t, J7.2 Hz) 128.9 7.31 (2H, m) 128.4 4 7.30 (1H, d, J7.2 Hz) 128.2 7.28 (1H, m) 127.8
a) Chemical shift values are given in ppm, and J values in parenthese are given in Hz. Assignments were confirmed by 1H–1H COSY, HMQC, and HMBC experiments.
b)13C-NMR 100 MHz, in CDCl 3.
and MCF-7 (human breast carcinoma) cells. The significant antiplatelet aggregation and cytotoxic activities of other known compounds had been reported in the previous studies.4,8)
Experimental
Melting points were measured on a Yanagimoto micro-melting point ap-paratus and were uncorrected. The UV spectra were obtained on a Hitachi 200-20 spectrophotometer in CH3OH solution. The IR spectra were recorded
on a Mattson Genesis II spectrophotometer. 1H-NMR (400 MHz) and 13
C-NMR (100 MHz) spectra were recorded with Varian C-NMR spectrometers. LR-EI-MS were collected on a JEOL JMS-SX/SX 102A mass spectrometer. HR-FAB-MS were collected on a Finnigan/Thermo Quest MAT 95XL mass spectrometer. TLC analysis was carried out on Si gel GF254pre-coated plates
with detection using 50% H2SO4followed by heating on a hot plate. Plant Material Fresh stems of G. amuyon (BLANCO) MERR. were
col-lected in Hengchun, Pingtung Hsien, Taiwan in September, 2001. The voucher specimen (Goniothalamus 1) is deposited in the Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung, Taiwan.
Extraction and Isolation The procedures employed for extraction and partition were described previously.4)The CHCl
3(Fr. C-1) residue was
sub-jected to silica gel (2.5 kg, 1153 cm) column chromatography, gradient eluting with n-hexane/CHCl3/CH3OH. The collected fractions were
com-bined on the basis of their TLC characteristics to give 15 fractions after re-moval of solvents. Fraction 6, eluted with pure CHCl3, was further separated
by CC over silica gel with CHCl3to give velutinam (1.6 mg), liriodenine
(6.2 mg), griffithazanone A (2.3 mg), 4-methyl-2,9,10,-(2H)-l-azaanthra-cencetrione (1.9 mg) and aristolactam BII (3.5 mg). Fraction 8 eluted with CHCl3 and purified by preparative TLC to give goniodiol-7-monoacetate
(1.1 mg) and goniodiol-8-monoacetate (0.9 mg). Fraction 9 was rechro-matographed on silica gel eluting with CHCl3to afford 1 (45 mg).
The CHCl3layer (25 g) (Fr. C-2) was subjected to silica gel column
chro-matography (630 g, 727 cm) and eluted with gradient mixtures of CHCl3/CH3OH. The eluates were combined into 10 fractions on the basis of
TLC monitoring. Fraction 9 was further purified by RP-HPLC (Hypersil ODS column, i.d. 21.2250 mm, CH3CN–water, 20 : 80, flow rate 3.5
ml/min; UV detector set at 210 nm) to give 2 (1.2 mg) and leiocarpin C (1.4 mg) (Hypersil ODS column, i.d. 21.2250 mm, CH3OH–CH3CN–
water, 10 : 10 : 80, flow rate 3.5 ml/min; UV detector set at 210 nm).
Goniothalesacetate (1): Yellow oil. [a]D1.03° (c0.39, CH3OH). 1
H-NMR (CD3OD, 400 MHz) and 13C-NMR (CD3OD, 100 MHz) see Table 1.
IR nmaxcm1: 3433, 2927, 1743, 1437, 1372, 1235, 1203, 1092, 1048. UV
lmaxnm: 210 (loge 3.87). FAB-MS m/z (rel. int. %): 339 ([MH], 75).
EI-MS (70 eV) (rel. int. %): m/z339 [MH](4), 321 (2), 307 (3), 246 (18), 229 (23), 133 (27), 91 (70). HR-FAB-MS: Calcd for C17H22O7m/z [MH]
339.1444, Found 339.1441.
Goniothalesdiacetate (3): 1 (5.2 mg) was dissolved in acetic an-hydride/pyridine and the reaction mixture left at room temperature for 12 h. The mixture on concentration and chromatograpgy afforded 3 (4.5 mg, 86%). 1H-NMR (CD 3OD, 400 MHz) d: 4.90 (1H, d, J4.0 Hz, H-2), 5.04 (1H, dd, J4.0, 2.0 Hz, H-3), 5.28 (1H, dd, J4.0, 2.0 Hz, H-4), 4.30 (1H, dd, J7.6, 4.0 Hz, H-5), 4.05 (1H, td, J7.6, 4.0 Hz, H-6), 2.61 (1H, dd, J16.0, 4.0 Hz, H-7b), 2.54 (1H, dd, J16.0, 7.6 Hz, H-7b), 7.29—7.37 (5H, m, ph). 13C-NMR (CD 3OD, 100 MHz) d: 173.2, 171.4, 171.3, 140.5, 129.3, 129.0, 127.3, 86.2, 84.1, 83.9, 78.2, 78.1, 59.5, 52.3, 37.5, 20.7, 20.6. EI-MS (70 eV) (rel. int. %): m/z349 [MOCH3].
Goniothalesdiol A (2): White powder. 1H-NMR ((CD
3)2CO, 400 MHz)
and 13C-NMR (CDCl
3, 100 MHz) see Table 1. IR nmaxcm1: 3409, 2915,
2841, 2361, 1732, 1439, 1200, 1167, 1063. UV lmaxnm: 208. HR-FAB-MS:
Calcd for C14H18O5 m/z [MH] 267.1232, Found 267.1234. CD (c
3.75103, CH3OH): De2500.50.
Preparation of (R)- and (S)-MTPA Esters of 1 Compound 1 (4.5 mg) was dried completely under vaccum. Deuterated pyridine (0.5 ml) and (R)-()-a-methoxy-a-(trifluoromethyl) phenylacetyl chloride (15 mg) were added under a N2gas stream. The mixture was stirred 2 h at room
tempera-ture and purified by HPLC with CH3CN/H2O to give (R)-MTPA ester 1r
(3.2 mg). By the same procedure, the (S)-MTPA ester 1s (2.5 mg) was pre-pared. 1H-NMR data of 1r and 1s, see Table 2.
References
1) Wu Y. C., Duh C. Y., Chang F. R., Chang G. Y., J. Nat. Prod., 54, 1077—1081 (1991).
2) Wu Y. C., Chang F. R., Duh C. Y., Wang S. K., Wu T. S.,
Phytochem-istry, 31, 2851—2853 (1992).
3) Lan Y. H., Chang F. R., Yu J. H., Yang Y. L., Chang Y. L., Lee S. J., Wu Y. C., J. Nat. Prod., 66, 487—480 (2003).
4) Lan Y. H., Chang F. R., Liaw C. C., Wu C. C., Chiang M. Y., Wu Y. C., Planta Med., 71, 153—159 (2005).
5) Kan W. S., “Pharmaceutical Botany,” National Research Institute of
1042 Vol. 54, No. 7
Chinese Medicine, Taipei, 1979, p. 247.
6) Mu Q., Tang W., Li C., Lu Y., Sun H., Zheng H., Hao X., Zheng Q., Wu N., Lou L., Xu B., Heterocycles, 51, 2969—2976 (1999). 7) Zhang Y. J., Kong M., Chen R. Y., Yu D. Q., J. Nat. Prod., 62, 1050—
1052 (1999).
8) Chia Y. C., Chang F. R., Teng C. M., Wu Y. C., J. Nat. Prod., 63, 1160—1163 (2000).
9) Cao S. H., Wu X. H., Sim K. Y., Tan B. K. H., Pereira J. T., Goh S. H.,
Tetrahedron, 54, 2143—2148 (1998).
10) Dondoni A., Catozzi N., Marra A., J. Org. Chem., 69, 5023—5035 (2004).
11) Bellosta V., Chassagnard C., Carbohydr. Res., 219, 1—7 (1991). 12) Blázquez M. A., Bermejo A., Zafra-Polo M. C., Cortes D., Phytochem.
Anal., 10, 161—170 (1999).
13) Dewick P. M., “Medicinal Natural Products: A Biosynthetic Ap-proach,” John Willey & Sons, England, 2002.
