Pan 2010

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J. Nat. Prod.

2010, 73, 1873–1878 1873

Isolation and Characterization of Minor Analogues of Silvestrol and Other Constituents from a
Large-Scale Re-collection of Aglaia foWeolata

Li Pan,† Leonardus B. S. Kardono,‡ Soedarsono Riswan,§ Heebyung Chai,† Esperanza J. Carcache de Blanco,†,⊥
Caroline M. Pannell,| Djaja Doel Soejarto,3 Thomas G. McCloud,o David J. Newman,# and A. Douglas Kinghorn*,†
DiVision of Medicinal Chemistry, College of Pharmacy, The Ohio State UniVersity, Columbus, Ohio 43210, United States, Research Center for
Chemistry, Indonesian Institute of Science, Tangerang 15310, Indonesia, Herbarium Bogoriense, Research Center for Biology, Indonesian
Institute of Science, Bogor 16122, Indonesia, DiVision of Pharmacy Practice and Administration, College of Pharmacy, The Ohio State
UniVersity, Columbus, Ohio 43210, United States, Department of Plant Sciences, UniVersity of Oxford, South Parks Road, Oxford OX1 3RA, U.K.,
Program for CollaboratiVe Research in the Pharmaceutical Science and Department of Medicinal Chemistry and Pharmacognosy, College of
Pharmacy, UniVersity of Illinois at Chicago, Chicago, Illinois 60612, United States, SAIC-Frederick, Inc., P.O. Box B, Frederick,
Maryland 21702, United States, and National Cancer Institute, NCI-Frederick, FairView Center, P.O. Box B, Frederick,
Maryland 21702, United States

ReceiVed July 23, 2010

Two new minor silvestrol analogues [2′′′-episilvestrol (1) and 2′′′,5′′′-diepisilvestrol (2)], together with a new 21-
norbaccharane-type triterpene (3), two new 3,4-secodammarane triterpenes (4 and 5), and a new eudesmane sesquiterpene
(6), as well as nine known compounds, were isolated from a large-scale re-collection of the CHCl3-soluble extract of
the stem bark of Aglaia foVeolata obtained in Kalimantan, Indonesia. The structures of the new compounds were
established by interpretation of their spectroscopic data. All of the isolates were tested for cytotoxicity against HT-29
cells. The new silvestrol analogues, 1 and 2, were considerably less active as cytotoxic agents than silvestrol (7) and
episilvestrol (5′′′-episilvestrol) (8) against this cell line, showing the importance of the configuration at C-2′′′ in mediating
such activity within this compound class. Several of the compounds isolated were also evaluated in a NF-κB (p65)
inhibition assay.

Silvestrol, a rocaglate derivative possessing a dioxanyloxy unit investigation has indicated that silvestrol causes increased apoptosis,
affixed to a cyclopenta[b]benzofuran skeleton, and its 5′′′S epimer, decreased proliferation, and inhibition of angiogenesis, and it
episilvestrol, were isolated from the tropical tree Aglaia foVeolata inhibits the translation of malignancy-related mRNA by regulating
Pannell (Meliaceae) and fully characterized by Hwang et al. in 2004. the activity of initiation factor elF4A.6,7 The total synthesis of
The structure and absolute configuration of silvestrol were con- silvestrol has been accomplished independently by two different
firmed by single-crystal X-ray crystallography.1 Silvestrol was found groups.8-10
to possess comparable cytotoxic potencies for a small panel of The presence of the substituted 1,4-dioxanyloxy moiety in the
human cancer cell lines to those of the well-known anticancer silvestrol structure appears to be unique in nature and has been
compounds paclitaxel (Taxol) and camptothecin and was further found essential for the exhibition of potent biological activity when
demonstrated as being active in the in vivo hollow fiber assay. This compared with rocaglate derivatives lacking this functionality.1,11
compound also showed activity in the P-388 lymphocytic leukemia
Thus far, only silvestrol and episilvestrol (5′′′-epi-silvestrol) have
test system in vivo, when administered both ip and iv.1 In addition,
been isolated with this structural feature among the cyclopent-
silvestrol has been documented as an antineoplastic constituent of
a[b]benzofuran derivatives from species in the genus Aglaia.1,2,11
Aglaia leptantha, using human PC-3 prostate cancer cells in a
murine xenograft experiment, but with only the planar structure of Previous phytochemical work on the different plant parts of A.
the dioxanyl ring moiety reported.2 foVeolata, collected in Kalimantan, Indonesia, has demonstrated that
In preliminary mechanistic studies, Swanson and associates silvestrol occurs in the fruits, leaves, stem bark, and twigs of this
demonstrated that silvestrol produces a p53-independent cell-cycle species, with the highest yield (0.02% w/w) occurring in the stem
blockage at the G2/M checkpoint using LNCaP human prostate bark.1,12 In the present study, a large-scale re-collection of the stem
cells.3 In a follow-up investigation, silvestrol was shown to induce bark of A. foVeolata from Kalimanatan, Indonesia, was conducted
apoptosis in LNCaP cells via the involvement of caspases 2, 9, to afford the scale-up isolation of silvestrol at the gram level, in
and 10, but not caspases 3 and 7.4 More recently, silvestrol was order for more extensive biological testing to be performed. While
found to exhibit B-cell selectivity in both chronic lymphocytic this reisolation work was underway, an opportunity was taken to
leukemia and acute lymphocytic leukemia models.5 Silvestrol was search for the presence of minor new analogues of silvestrol.
observed to cause an early reduction in Mcl-1 expression in chronic Cytotoxicity assay-guided fractionation of the CHCl3-soluble
lymphocytic leukemia cells from patients.5 Additional mechanistic extract of the stem bark of A. foVeolata led to the isolation of two
new minor silvestrol analogues (1 and 2), three new triterpenoids
* To whom correspondence should be addressed. Tel: +1-614-247-8094. (3-5), and a new sesquiterpene (6), as well as nine known
Fax: +1-614-247-8081. E-mail: kinghorn.4@osu.edu. compounds. The structures of compounds 1-6 were established

Division of Medicinal Chemistry and Pharmacognosy, College of by spectroscopic data interpretation. Besides silvestrol and episil-
Pharmacy, The Ohio State University. vestrol, the other known compounds were identified as 17,24-epoxy-

Research Center for Chemistry, Indonesian Institute of Science.
§
Research Center for Biology, Indonesian Institute of Science. 25-hydroxybaccharan-3-one,1 17,24-epoxy-25-hydroxy-21-methoxy-

Division of Pharmacy Practice and Administration, College of Phar- 3,4-secobaccharane,12 eichlerianic acid,13 cabraleone,13 foveolin
macy, The Ohio State University. A,13 methyl foveolate A (dymalol),14 (-)-dehydrodiconiferyl
|
University of Oxford.
3
University of Illinois at Chicago.
alcohol,15 and 3-oxo-15-hydroxy-T-muurolol,16 by comparison of
o
SAIC-Frederick, Inc. their spectroscopic data with published values. All of the isolates
#
National Cancer Institute. were tested for cytotoxicity against the human colon cancer cell

10.1021/np100503q  2010 American Chemical Society and American Society of Pharmacognosy


Published on Web 10/12/2010
1874 Journal of Natural Products, 2010, Vol. 73, No. 11 Pan et al.

line (HT-29). The new compounds 3-6, together with silvestrol Table 1. 1H and 13
C NMR Spectroscopic Data of Compounds 1
and episilvestrol, were also evaluated in a NF-κB (p65) inhibition and 2a
assay. 1 2
position δH, (J in Hz) δC δH, (J in Hz) δC
1 5.04, d (7.2) 79.7 5.04, d (7.2) 79.7
2 3.90, dd (14.4, 6.6) 50.4 3.90, dd (14.4, 6.6) 50.2
3 4.27, d (14.4) 55.0 4.28, d (14.4) 55.0
3a 101.9 101.9
4a 160.4 160.6
5 6.48, d (1.8) 92.7 6.48, d (1.8) 92.9
6 160.0 160.0
7 6.40, d (1.8) 95.2 6.40, d (1.8) 95.4
8 157.1 157.1
8a 109.6 109.6
8b 93.3 93.4
1′ 126.6 126.6
2′,6′ 7.09, d (9.0) 129.0 7.09, d (9.0) 129.0
3′,5′ 6.69, d (9.0) 112.8 6.69, d (9.0) 112.8
4′ 158.8 158.8
1′′ 136.7 136.7
2′′,6′′ 6.84, m 127.8 6.85, m 127.8
3′′,5′′ 7.05, m 127.8 7.05, m 127.8
4′′ 7.05, m 126.6 7.06, m 126.6
1′′′ 5.40, brs 93.5 5.36, brs 92.7
2′′′ 4.65, d (1.2) 98.8 4.63, d (1.2) 98.9
3′′′R 4.04, brd (12.0, 3.0) 66.5 4.24, dd (11.4, 1.8) 67.2
3′′′β 4.01, t (11.0) 3.82, t (11.4)
4′′′ 4.18, brd (10.2) 68.3 4.05, ddd (9.6, 7.8, 1.8) 67.4
5′′′ 3.70, brs 70.2 3.58, dd (10.8, 6.0) 71.2
6′′′ 3.66, d (10.8) 63.7 3.59-3.61, m 62.7
3.68, d (10.8) 3.74, brd (10.8)
Results and Discussion COOCH3-2 170.6 170.6
3.65, s 52.1 3.65, s 52.1
Compound 1 was obtained as a colorless gum with a negative OCH3-8 3.87, s 56.0 3.87, s 56.0
specific rotation ([R]20D -33.5, c 0.05, MeOH) and afforded a OCH3-4′ 3.72, s 55.1 3.72, s 55.1
sodiated molecular ion peak at m/z 677.2233 [M + Na]+ in the OCH3-2′′′ 3.63, s 57.4 3.63, s 57.3
HRESIMS, corresponding to a molecular formula of C34H38O13, a1
H NMR spectrum measured at 600 MHz, 13C NMR spectrum
the same as silvestrol. The 1H and 13C NMR spectra of compound measured at 150 MHz; obtained in CDCl3 with TMS as internal
1 were observed to be very similar to those of silvestrol and standard. Assignments supported with HSQC and HMBC NMR spectra.
episilvestrol.1 In the 1H NMR spectrum, 11 proton signals were
detected in the low-field range from δH 6.30 to 7.20 ppm and were spectrum (Table 1). All of this information suggested that compound
recognized as belonging to three aromatic rings, including two meta- 1 is an isomer of silvestrol. By comparison of the 1H NMR data of
coupled aromatic signals at δH 6.40 (1H, d, J ) 1.8 Hz, H-7) and these two compounds, the major differences were evident in the
6.48 (1H, d, J ) 1.8 Hz, H-5), four AA′BB′-coupled proton signals 1,4-dioxanyloxy ring. A downfield shift of the methoxy group at
of a 1,4-disubstituted phenyl group at δH 6.69 (2H, d, J ) 9.0 Hz, C-2′′′ from δH 3.48 to δH 3.63 was clearly discernible for 1.
H-3′ and H-5′) and 7.07 (2H, d, J ) 9.0 Hz, H-2′ and H-6′), and Moreover, downfield shifts of 0.18 ppm for H-1′′′, 0.09 ppm for
proton signals of a monosubstituted phenyl ring at δH 6.84 (2H, H-2′′′, and 0.51 ppm for H-3R′′′, as well as an upfield shift of 0.10
m, H-2′′ and H-6′′) and 7.07 (3H, m, H-3′′, 4′′ and H-5′′). The ppm for H-3β′′′, were also observed. For the rocaglate moiety of
chemical shifts at δH 5.04 (1H, d, J ) 7.2 Hz, H-1), 3.90 (1H, dd, 1, most proton signals were found to be almost identical to those
J ) 14.4, 6.6 Hz, H-2), and 4.27 (1H, d, J ) 14.4 Hz, H-3) are of silvestrol, except for H-7 and H-5, two aromatic protons spatially
typical signals of H-1, H-2, and H-3 of the five-membered close to the 1,4-dioxanyloxy ring, for which slight downfield shifts
carbocyclic ring of a cyclopenta[b]benzofuran unit (Table 1).1 The of 0.13 and 0.08 ppm, respectively, were observed. These subtle
above analysis of the 1H NMR spectrum suggested the presence of differences allowed the inference to be made that the methoxy group
a rocaglate unit in the molecule of 1.1 In the 13C NMR spectrum at C-2′′′ on the 1,4-dioxanyloxy ring in 1 adopts an R-equatorial
of this compound, which was sorted using its DEPT and HSQC orientation rather than a β-axial orientation as in silvestrol. This
spectra, seven quaternary carbons at δC 160.4 (C-4a), 160.0 (C-6), presumption was consistent with the downfield shift of ap-
157.1 (C-8), 109.6 (C-8a), 126.6 (C-1′), 158.8 (C-4′), and 136.7 proximately 7.5 ppm for the carbon signal of C-3′′′ (δC 66.5) in
(C-1′′) were found to be consistent with the occurrence of the three the 13C NMR spectrum, due to the absence of the cis-γ substitution
substituted benzene rings indicated above. The observed resonances effect of the methoxy group on C-2′′′ to H-3β′′′. Furthermore, the
of two oxygenated quaternary carbons at δC 101.9 (C-3a) and 93.4 R position of the methoxy group on C-2′′′ was confirmed by the
(C-8b), two oxymethine groups at δC 79.7 (C-1) and 50.2 (C-2), key NOE effects between H-2′′′ and H-3β′′′. Additional perusal of
and an alkyl methine group at δC 55.0 (C-3) are characteristic for the 1H NMR spectrum indicated that the proton signals of H-3R′′′
a cyclopenta[b]benzofuran moiety.1 Besides the rocaglate feature, (δH 4.04, 1H, dd, J ) 12.0, 3.0 Hz), H-3β′′′ (δH 4.01, 1H, t, J )
the presence in 1 of an unusual [6-(1,2-dihydroxyethyl)-3-methoxy- 11.0 Hz), H-4′′′ (δH 4.18, brd, J ) 10.2 Hz), and H-5′′′ (3.70, brs)
1,4-dioxan-2-yl]oxy feature was also recognized, on the basis of adopted comparable splitting patterns and exhibited similar coupling
the observation of oxygenated proton signals at δH 5.40 (1H, brs, constants to those for silvestrol (7), suggesting the configurations
H-1′′′), 4.65 (1H, d, J ) 1.2 Hz, H-2′′′), 4.04 (1H, dd, J ) 12.6, of C-4′′′ and C-5′′′ to be the same for both substances. This
3.0, H-3R′′′), 4.01 (1H, t, J ) 11.0, H-3β′′′), 4.18 (1H, brd, J ) deduction was consistent with the analysis of the NOESY spectrum
10.2 Hz, H-4′′′), 3.70 (1H, brs, H-5′′′), 3.66 (1H, d, J ) 10.8 Hz, (Figure 1). Thus, the structure of compound 1 was determined to
Ha-6′′′), and 3.68 (1H, d, J ) 10.8 Hz, Hb-6′′′) in the 1H NMR be 2′′′-episilvestrol.
spectrum, as well as signals of four oxymethine carbons at δC 93.5 The HRESIMS of compound 2 showed a sodiated molecular ion
(C-1′′′), 98.8 (C-2′′′), 68.3 (C-4′′′), and 70.2 (C-5′′′) and two peak at m/z 677.2212, corresponding to a molecular formula of
oxymethylenes at δC 66.5 (C-3′′′) and 63.7 (C-6′′′) in the 13C NMR C34H38O13Na, the same as that of compound 1. The NMR spectrum
Minor Analogues of SilVestrol from Aglaia foVeolata Journal of Natural Products, 2010, Vol. 73, No. 11 1875

Table 2. 1H NMR Chemical Shifts of Compounds 3-6a


position 3 4 5 6
1 1.48b 1.62b 1.63b 3.88, m
1.95, m 1.82b 1.82b
2 2.46b 2.65, td 2.67, td ΗR, 2.72,
(14.0, 4.6) (14.0, 4.5) dd (6.1, 17.3)
2.50, ddd 2.52, ddd Ηβ, 2.66,
(14.2, 5.7, 3.5) (14.2, 5.7, 3.5) dd (12.1, 17.3)
b b
5 1.35 1.78 1.80b
Figure 1. Selected NOESY (T) correlations observed for 1. 6 1.50 b
1.43 b
1.44b 6.46, s
1.77b 1.77b
of compound 2 exhibited a very close resemblance to that of 7 1.42b 1.30b 1.31b
compound 1 and could be recognized as a second new silvestrol 8 2.30, m
isomer (Table 1). The same R-orientation of the methoxy group 9 1.43b 1.42b 1.42b ΗR, 1.43,
ddd (6.0, 5.7, 12.5)
on the 1,4-dioxane ring of 2 was deduced by the analysis of its 1D Ηβ, 2.16,
and 2D NMR spectroscopic data and by comparison with those of ddd (2.0, 5.0, 12.8)
compound 1. The major differences between compounds 1 and 2 11 1.35b 1.55b 1.55b 2.46, hep (6.6)
1.55b 1.26b 1.26b
were evident in the ethane 1,2-diol functionality on the 1,4- 12 1.19b 1.10b 1.12, d (6.8)
dioxanyloxy moiety. In the 1H NMR spectrum of compound 2, the 2.03, 1.86b 1.12, d (6.8)
proton signals of H-3R′′′ (δH 4.24, 1H, dd, J ) 11.4, 1.8 Hz) and brd (10.0)
13 2.07, td 1.75b 1.70b
H-3β′′′ (δH 3.82, 1H, t, J ) 11.4 Hz) adopted a similar coupling (12.0, 3.0)
pattern to that in compound 1 and silvestrol, while the splitting 14 1.09, s
patterns of H-4′′′ (δH 4.05, ddd, J ) 9.6, 7.8, 1.8) and H-5′′′ (δH 15 1.58b 1.10b 1.10b 4.45, brs
1.20b 1.45b 1.45b
3.58, 1H, dd, J ) 10.8, 6.0 Hz) were comparable with those of the 16 1.60b 1.30b 1.30b
known 5′′′-epimer of silvestrol (episilvestrol).1 Thus, compound 2 1.75b 1.78b 1.78b
was determined to be 2′′′,5′′′-diepisilvestrol. This structural proposal 17 3.48, d (11.4) 1.79b 1.91b
18 1.07, s 1.02, s 1.05, s
was confirmed by analysis of its HSQC, HMBC, and NOESY 2D- 19 0.96, s 1.08, s 1.10, s
NMR spectra. 21 1.15, s 1.17, s
Compound 3 was obtained as a white, amorphous powder with 22 HR1.48b 1.71b 1.71b
Hβ 2.03, td
a molecular formula of C29H48O4, as determined from the sodiated (13.2, 4.8)
molecular ion peak at m/z 483.3431 [M + Na]+ in the HRESIMS. 23 Hβ1.56b 1.80b 1.88b
The 1H NMR spectrum of 3 exhibited signals for seven tertiary HR1.73b 1.82b
24 3.26, d (12.0) 3.77, 3.65, dd
methyl groups at δH 0.96 (3H, s, H-19), 0.98 (3H, s, H-30), 1.03 t (7.2) (9.9, 5.6)
(3H, s, H-29), 1.07 (3H, s, H-18), 1.08 (3H, s, H-28), 1.16 (3H, s, 26 1.16, s 1.20, s 1.21, s
H-26), and 1.20 (3H, s, H-27), two typical methine protons at δH 27 1.20, s 1.10, s 1.13, s
28 1.08, s 1.48, s 1.50, s
3.26 (1H, d, J ) 12.0 Hz) and 3.48 (1H, d, J ) 11.4 Hz), and a 29 1.03, s 1.40, s 1.42, s
group of highly overlapped alkyl protons in the high field that 30 0.98, s 0.86, s 0.88, s
occurred in the range from δH 1.20 to 2.60 (Table 2). Altogether, a
Measured at 600 MHz and obtained in CDCl3 with TMS as internal
29 carbon signals in the 13C NMR spectrum were sorted by DEPT standard; J values (Hz) are given in parentheses. Assignments supported
and HSQC into seven methyls, 10 methylenes, three methines, four with 1H-1H COSY, HSQC, and HMBC spectra. b Multiplicity patterns
quaternary carbons, two oxygenated methines (δC 73.8, C-17 and unclear due to signal overlapping.
81.9, C-24), two quaternary oxygenated carbons (δC 69.2, C-20
and 71.9, C-25), and a ketone group (δC 217.7, C-3) (Table 3). was found around 289 nm, suggesting that this compound adopts
These characteristic signals as observed in the 1H NMR and 13C the same absolute configuration as found in structurally closely
NMR spectra were comparable with those of 17,24-epoxy-25- related 3-oxotriterpenoids.17,18 Accordingly, the structure of com-
hydroxybaccharan-3-one, which is a baccharane-type triterpene first pound 3 was elucidated as 17,24-epoxy-20R,25-dihydroxy-21-
isolated from A. foVeolata in an earlier study, and structurally norbaccharan-3-one.
confirmed by X-ray crystallographic analysis.1 Comparison of the Compound 4 was obtained as a white, amorphous resin. The
1
H NMR and 13C NMR spectroscopic data of 3 with those of this molecular formula was determined to be C30H50O4, on the basis of
known compound revealed that the only difference was the absence the sodiated molecular ion peak at m/z 497.3651 [M + Na]+ in the
of a tertiary methyl group (C-21) at C-20 and the presence of an HRESIMS. Its 1H NMR spectrum exhibited signals for eight tertiary
oxygenated quaternary carbon (δC 69.2) instead of an alkyl methyl groups at δH 0.86 (3H, s, H-30), 1.02 (3H, s, H-18), 1.08
quaternary carbon. The above observations, together with the (3H, s, H-19), 1.10 (3H, s, H-27), 1.15 (3H, s, H-21), 1.20 (3H, s,
molecular formula discerned from the HRESIMS, suggested that H-26), 1.40 (3H, s, H-29), and 1.48 (3H, s, H-28), an oxygen-
compound 3 is a baccharane-type nor-triterpene, with the C-21 bearing methine group at δH 3.77 (1H, t, J ) 6.9 Hz, H-24), a
methyl group having been replaced by a hydroxy group. In order methylene vicinal to a carbonyl group at δH 2.65 (1H, td, J ) 14.0,
to obtain further information on the free hydroxy groups attached 4.6, H-2R) and 2.50 (1H, ddd, J ) 14.2, 5.7, 3.5, H-2β), and a
at the quaternary carbons, CDCl3 and DMSO-d6 were both used as number of alkyl protons in the high-field region at δH 1.0-2.2 ppm
NMR solvent. In the 1H NMR spectrum with DMSO-d6 as solvent, (Table 2). The 30 carbon signals in the 13C NMR spectrum were
the hydroxy proton signals at C-20 and C-24 appeared at δH 4.17 classified by DEPT and HSQC NMR experiments into eight
and 3.97, respectively. HMBC correlations were observed between methyls, 10 methylenes, four methines, three quaternary carbons,
the signal for OH-20 and C-20, C-17, C-16, and C-22 and between four-oxygen bearing carbons (including one secondary and three
OH-24 and C-25, C-26, and C-27 and confirmed the locations of tertiary), and a carbonyl group. The NMR data of compound 4 were
these two hydroxy groups (Figure 2). In the NOESY spectrum, characteristic of the resonances of a 3,4-secodammarane derivative
enhancements of OH-20 with H-17, H-22R, and H-23R suggested with a tetrahydrofuran (20,24-epoxy) ring formed by the closure
an R-orientation of OH-20. In addition, NOE effects were also of a side chain attached to the D ring.11,17 Compounds based on
observed between CH3-19 and CH3-18, CH3-18 and H-13, H-17 this triterpenoid skeleton have been obtained previously from certain
and CH3-30, and H-24 and H-22β, which were consistent with the species belonging to the genera Aglaia and Cabralea of the plant
relative configuration of the known baccharane-type triterpenes family Meliaceae.11,17 On comparing the NMR data of 4 with those
(Figure 2).1 In the CD spectrum of 3, a positive n-π* Cotton effect of the structurally closely related known compound foveolin B, a
1876 Journal of Natural Products, 2010, Vol. 73, No. 11 Pan et al.

Table 3. 13
C NMR Chemical Shifts of Compounds 3-6a
position 3 4 5 6
1 39.7 40.7 40.3 71.8
2 34.0 32.8 32.4 42.4
3 217.7 175.5 175.0 198.7
4 47.3 86.4 86.0 129.4
5 54.9 53.5 53.1 157.9
6 19.6 23.9 23.6 116.9
7 33.0 34.6 34.2 160.3
8 42.4 40.5 40.2 23.7
9 50.0 51.7 51.4 32.0 Figure 3. Selected HMBC (f) and NOESY (T) correlations
10 36.9 39.7 39.3 38.7 observed for 4 and 5.
11 21.2 23.1 23.0 36.4
12 23.7 27.3 27.2 21.4
13 35.2 43.5 43.1 20.8 correlations between CH3-21 and both H-17 and H-24, respectively.
14 40.8 50.2 49.9 56.2 Additional NOE cross-peaks of H-5 and H-9, H-9 and CH3-30,
15 27.5 31.4 30.9 15.0 CH3-18 and CH3-19, H-13 and CH3-19, and H-17 and CH3-30 were
16 33.8 26.1 25.7 used to establish the relative configuration of the remaining
17 81.9 50.3 49.6
18 15.6 15.3 14.9 stereocenters of compound 4 (Figure 3), which were identical to
19 16.2 18.8 18.4 those of known derivatives.13,19 Thus, the structure of compound
20 69.2 86.8 86.5 4 was determined to be 20R,24S-epoxy-25-hydroxy-A-homo-4-
21 22.1 27.2 oxadammaran-3-one.
22 29.1 37.8 34.8
23 20.1 26.2 26.3
The molecular formula of compound 5 was determined to be
24 73.8 84.9 86.4 C30H50O4, the same as that of compound 4, from the sodiated
25 71.9 71.5 70.2 molecular ion peak at m/z 497.3639 in the HRESIMS. The 1H and
26 24.0 27.6 27.8 13
C NMR spectra of 5 were almost identical to those of 4 except
27 26.3 24.4 24.0
28 26.8 31.3 30.9
for some slight but distinctive differences in the epoxide ring signals,
29 21.0 26.7 26.9 which suggested that compound 5 is a stereoisomer of 4 (Tables 2
30 14.4 16.2 16.1 and 3). The 13C NMR resonances of C-20, C-21, C-22, C-23, C-24,
a
Measured at 150 MHz and obtained in CDCl3 with TMS as internal and C-25 were assigned with chemical shift values of δC 86.5, 27.2,
standard. Assignments supported with HSQC and HMBC NMR spectra. 34.8, 26.3, and 86.4, respectively, and the proton signal for H-24
appeared as a double doublet with coupling constants of 9.9 and
5.6 Hz (Tables 2 and 3). The typical NMR parameters of the epoxy
ring corresponded to the 20S, 24S configurations, as described in
the literature.13 In the NOESY spectrum, correlations between CH3-
21 and H-24 as well as H-17 as in compound 4 were absent, while
an enhancement between CH3-21 and H-13 was observed, consistent
with a configurational change (Figure 3). Other important NOE
effects observed for 5 were similar to those observed for 4.
Consequently, compound 5 was designated as 20S,24S-epoxy-25-
hydroxy-A-homo-4-oxadammaran-3-one, the 20S epimer of com-
pound 4.
Compound 6 was obtained as a colorless resin. The HRESIMS
Figure 2. Selected HMBC (f) and NOESY (T) correlations
observed for 3. of 6 afforded a sodiated molecular ion peak at m/z 273.1441,
corresponding to an elemental formula of C15H22O3Na. In the 1H
known constituent of A. foVeolata, it was revealed that the major NMR spectrum, the resonance at δH 6.46 (1H, s, H-6) was ascribed
differences were evident in the A ring.11 In the 13C NMR spectrum, as a signal from an endocyclic double bond, and the proton signals
the signals of the methylene groups at C-1 and C-2 were shifted at δH 3.88 (1H, m, H-2) and 4.45 (2H, brs, H-15) suggested the
downfield by 5.8 and 5.0 ppm, respectively, while the C-3 carbonyl presence of an oxymethine and an oxymethylene, respectively.
group resonance was shifted upfield by 4.2 ppm. In turn, the signal Besides a tertiary methyl group signal at δH 1.09 (3H, s, H-14), an
of the quaternary oxygenated C-4 carbon was also shifted downfield isopropyl group could be recognized on the basis of the proton
by nearly 10 ppm. These differences, together with the molecular signals of two secondary methyl groups at δH 1.09 (2 × 3H, d, J
formula of compound 4, suggested the possibility of an ester bond ) 6.8 Hz) and a heptet at δH 1.09 (1H, J ) 6.6 Hz). The 15 carbon
between the carbonyl group of C-3 and the quaternary oxygenated signals observed in the 13C NMR spectrum were sorted by DEPT
carbon of C-4, resulting in the closure of the 3,4-seco ring to form and HSQC into three methyls, three methylenes, a methine, a
a seven-membered lactone. In the HMBC spectrum, correlations quaternary carbon, two oxygenated carbons (including one primary
were observed from H-1, H-2, CH3-28, and CH3-29 to the carbonyl and one secondary), a conjugated ketone group, a trisubstituted
group (δC 175.5, C-3), from CH3-28 and CH3-29 to C-4 and C-5, double bond, and a tetrasubstituted double bond. These NMR
and from CH3-19 to C-1, C-10, and C-9 and provided confirmatory spectroscopic observations suggested that compound 6 is a ses-
evidence for this proposal (Figure 3). According to the previous quiterpenoid. In its 1H-1H COSY spectrum, the H-1 signal coupled
literature, 13C NMR chemical shifts of the carbons of the epoxide with the geminal protons of H-2R (δH 2.72, 1H, dd, J ) 6.1, 17.3)
ring, as well as the coupling pattern of H-24, have been used to and H-2β (δH 2.16, 1H, J ) 12.1, 17.3), which were located next
determine the absolute configuration of the 20,14-epoxy group of to the ketone group (δC 198.7, C-3). The geminal protons of H-9R
3,4-secodammarane derivatives.13 In the 13C NMR spectrum of 4, (δH 1.43, 1H, ddd, J ) 5.7, 6.0, 12.5) and H-9β (δH 2.16, 1H, J )
the signals of C-20, C-21, C-22, C-23, and C-24 appeared at δC 2.0, 5.0, 12.5) showed coupling to the allylic methylene protons at
86.8, 22.1, 37.8, 26.2, and 84.9, respectively (Table 3). In turn, in δH 2.30 (2H, m, H-8). The partial structures were further connected
the 1H NMR spectrum, the H-24 signal was exhibited as a triplet by HMBC correlations from CH3-14 to C-5, C-2, and C-9, from
with a J value of 6.8 Hz. This information was supportive of a H-13 and H-12 to C-7, from H-6 to C-4 and C-5, and from H-15
20R, 24S configuration of the epoxy group, as described previ- to C-5, C-4, and C-3 (Figure 4). Compound 6 was therefore
ously.13 These assignments were supported by the key NOESY elucidated as a 4,6-diene eudesmane derivative, with C-3 substituted
Minor Analogues of SilVestrol from Aglaia foVeolata Journal of Natural Products, 2010, Vol. 73, No. 11 1877

Sunfire PrepC18 column (Waters, Milford, MA), along with a Waters


system equipped with a 600 controller, a 717 Plus autosampler, and a
2487 dual wavelength absorbance detector.
Plant Material. A re-collection of A. foVeolata stem bark was done
in the autumn of 2007, in Kalimantan, Indonesia, by S.R. (Herbarium
Bogoriense, Bogor, Indonesia), through the cooperation of L.B.S.K
(LIPI, Tangerang, Indonesia). A voucher specimen (AA6126) has been
deposited at the Herbarium of the Field Museum of Natural History,
Figure 4. Selected HMBC (f) and NOESY (T) correlations Chicago, IL.
observed for 6. Extraction and Isolation. The dried stem bark of A. foVeolata
(40-45 kg) was ground at the University of Illinois Pharmacognosy
Table 4. Cytotoxicity of Compounds Isolated from the Stem Field Station, Downers Grove, IL, where it was also bulk extracted
Bark of Aglaia foVeolataa with MeOH. The MeOH extract was reduced in volume, and 20% (4
L) was dispatched for isolation work at The Ohio State University.
compound HT-29b This MeOH extract (4 L) of stem bark of A. foVeolata was concentrated
5′′′-episilvestrol (1) 2.29 under reduced pressure to yield 2 kg of thick, dark brown syrup. A
2′′′,5′′′-diepisilvestrol (2) 1.07 part of the extract (500 g) was partitioned sequentially with hexane (3
silvestrol (7) 0.0007 × 1 L) and CHCl3 (3 × 1 L). The CHCl3 partition was washed with
2′′′-episilvestrol (8) 0.001
1% saline solution to yield 200 g of a partially detannified CHCl3-
paclitaxelc 0.0006
camptothecinc 0.06 soluble extract, which was found to be active against the HT-29 cell
a
line (ED50 0.4 µg/mL). Accordingly, part of this fraction (180 g) was
Compounds 3-6 and all other known compounds obtained in this subjected to separation over a silica gel column (11 × 100 cm,
investigation were inactive against HT-29 cells (ED50 >10 µM). CH2Cl2-acetone, 20:1 to 100% acetone) to yield eight fractions
b
Results are expressed as ED50 values (µM). c Used as a positive
(F01-F08). Fraction F07 (HT-29 cell line, ED50 < 0.16 µg/mL, 7 g)
control substance.
was chromatographed over a LH-20 gel column (5 × 50 cm; eluted
with 100% MeOH), to furnish three pooled subfractions (F701-F703).
by a ketone group, and hydroxy groups present at C-1 and C-15, Subfraction F703 was demonstrated by TLC to be a silvestrol-rich
respectively. The β-orientation of the hydroxy group located at C-1 subfraction and was subjected to separation over a preparative RP-18
was deduced by the observed NOE effects of H-1 with H-2R and column (150 mm × 19 mm i.d.), using MeOH-H2O (55:45, 8 mL/
H-9R (Figure 4). A positive Cotton effect was observed in the range min) as solvent, to afford compounds 1 (0.8 mg; tR ) 20.5 min), 2
250 to 375 nm in the CD spectrum of 6, consistent with that of (0.9 mg; tR ) 26.0 min), silvestrol (7, 100 mg; tR ) 36.5 min), and
β-cyperone, a known 4,6-dien-3-one eudesmane.20 Accordingly, episilvestrol (8, 4.0 mg; tR ) 42.5 min). Subfraction F702 was
the absolute configuration of 6 was established as 1R, 10R, as chromatographed on an open ODS column (2 × 15 cm) with a
shown. Therefore, the structure of compound 6 was determined to MeOH-H2O gradient solvent system (50:50 to 90:10), to yield
compounds 3 (5.0 mg) and 17,24-epoxy-25-hydroxybaccharan-3-one
be 4,6-diene-1,15-dihydroxyeudesma-3-one.
(4.0 mg).
All pure compounds obtained in the present investigation were The bulk of the MeOH extract prepared at the University of Illinois
evaluated for their cytotoxic activity against the HT-29 human colon at Chigaco (ca. 80%) was transferred to SAIC-Frederick, Inc. Thus,
cancer cell line (Table 4). Among these compounds, the highly an 11 kg MeOH extract of A. foVeolata was subjected to separation on
active agents silvestrol (7) and 5′′′-episilvestrol (8) exhibited potent a silica gel column to yield 154 fractions, from which nine silvestrol-
cytotoxicity, with ED50 values 0.0007 and 0.001 µM, respectively. enriched subfractions were retained for the purification of gram
The minor new compounds 1 and 2, the C-2′′′ epimers of silvestrol quantities of silvestrol. Follow-up isolation work on the remaining 145
and 5′′′-episilvestrol, were found to be much less active, with ED50 side-cut fractions was conducted at The Ohio State University. The
values of 2.3 and 1.1 µM, respectively. Accordingly, it is noted active fraction F112 (HT-29 cell line, ED50 1.1 µg/mL, 110 g) was
that when the axial methoxy group on the 1,4-dioxanyloxy ring at chromatographed on a silica gel column, using CH2Cl2-acetone
C-2′′′ in silvestrol and 5′′′-episilvestrol was changed to an equatorial mixtures for elution, to yield eight subfractions (F11201-F11208). The
presence of silvestrol and episilvestrol was detected by HPLC analysis
orientation, as in compounds 1 and 2, the resultant cytotoxicity
in subfractions F11206 and F11207, respectively [C18 column, 150 mm
decreased dramatically. This investigation has thus demonstrated × 4.6 mm i.d.; MeOH-H2O 60:40; flow rate 1.5 mL/min; tR (silvestrol)
that the configuration of the chiral carbon C-2′′′ in the 1,4- ) 6.0 min; tR (episilvestrol) ) 6.7 min]. Subfraction F11203 was
dioxanyloxy unit of silvestrol derivatives plays an important role chromatographed on a silica gel column, using CHCl3-acetone mixtures
in mediating biological activity among these plant secondary for elution, to give 17,24-epoxy-25-hydroxy-21-methoxy-3,4-secobac-
metabolites. An enzyme-based ELISA NF-κB assay was also charane (50 mg), eichlerianic acid (20 mg), and cabraleone (3.5 mg).
employed to test the p65 (RelA) inhibitory activity of the new In an attempt to find additional new compounds, two side-cut
compounds 3-6, in addition to silvestrol and 5′′′-episilvestrol. All subfractions, F115-38-21 and F115-38-26 (1.0 g), were also investi-
of these substances exhibited IC50 values of >20 µM and were gated. These were prepared at SAIC-Frederick, Inc., from a silvestrol-
considered inactive. Compounds 1 and 2 were not tested in the containing fraction, F115, by passage over a RP-8 column using a
NF-κB assay due to the very small amounts isolated. CH3CN-H2O gradient eluent. Subfraction F115-38-21 (500 mg) was
chromatographed on a semipreparative RP-18 column by HPLC, using
MeOH-H2O (50:50, 5 mL/min) as solvent system, to give (-)-
Experimental Section dehydrodiconiferyl alcohol (3.5 mg, tR ) 12.5 min), 6 (1.2 mg, tR )
General Experimental Procedures. Optical rotations were obtained 15.5 min), and 3-oxo-15-hydroxy-T-muurolol (1.0 mg, tR ) 19.4 min).
on a Perkin-Elmer 343 automatic polarimeter. UV spectra were recorded Subfraction F115-38-26 (1.0 g) was subjected repeatedly to silica gel
with a Perkin-Elmer Lambda 10 UV/vis spectrometer. CD spectra were columns, using CH2Cl2-acetone gradient solvent systems (20:1 to 1:1)
run on a JASCO J-810 spectrometer. NMR spectroscopic data were for elution, to afford compounds 4 (4.0 mg), 5 (3.8 mg), foveolin A
obtained on a Bruker Avance DRX-400 or -600 MHz spectrometer. (15 mg), and methyl foveolate A (dymalol, 6 mg).
IR spectra were measured on a Thermo Scientific Nicolet 6700 FT-IR 2′′′-Episilvestrol (1): colorless gum; [R]20D -33.5 (c 0.05, MeOH);
spectrometer. Column chromatography was performed with 65-250 UV (MeOH) λmax (log ε) 211 (4.54), 233 (4.07), 278 (3.19) nm; CD (c
or 230-400 mesh silica gel (Sorbent Technologies, Atlanta, GA). 3.06 × 10-5 M, MeOH) λmax (∆ε) 217 (-6.05), 250 (+1.86), 294
Analytical thin-layer chromatography was conducted on precoated, 250 (+2.40) nm; IR (film) νmax 3460, 2919, 2850, 1733, 1717, 1683, 1616,
µm thick silica gel plates (UV254, glass backed, Sorbent Technologies, 1558, 1540, 1507, 1457, 1251, 1217, 1119, 1057, 1031, 753 cm-1; 1H
Atlanta, GA). Analytical HPLC was conducted on a 150 mm × 4.6 NMR (600 MHz, CDCl3) and 13C NMR (150 MHz, CDCl3) data, see
mm i.d. Sunfire PrepC18 column (Waters, Milford, MA), and semi- Table 1; HRESIMS m/z 677.2233 [M + H]+ (calcd for C34H38O13Na,
preparative HPLC was conducted on a 150 mm × 19 mm i.d., 5 µm 677.2210).
1878 Journal of Natural Products, 2010, Vol. 73, No. 11 Pan et al.

2′′′,5′′′-Diepisilvestrol (2): colorless gum; [R]20D -53.0 (c 0.05, We are grateful to Dr. J. Orjala and Mr. M. Totura of the University
MeOH); UV (MeOH) λmax (log ε) 211 (4.51), 232 (4.04), 278 (3.23) of Illinois at Chicago for performing an initial large-scale extraction
nm; CD (c 3.06 × 10-5 M, MeOH) λmax (∆ε) 215 (-7.85), 251 (+1.20), of the plant material. Mr. M. Harris, SAIC-Frederick, Inc., is acknowl-
292 (+1.72) nm; IR (film) νmax 3461, 2926, 2850, 1734, 1717, 1683, edged for assistance with plant fractionation studies. We thank Mr. J.
1653, 1616, 1558, 1540, 1507, 1457, 1250, 1118, 1043, 753 cm-1; 1H Fowble, College of Pharmacy, The Ohio State University, and Dr. C.-
NMR (600 MHz, CDCl3) and 13C NMR (150 MHz, CDCl3) data, see H. Yuan, OSU Campus Chemical Instrument Center, for facilitating
Table 1; HRESIMS m/z 677.2212 [M + H]+ (calcd for C34H38O13Na, the acquisition of the 400 and 600 MHz NMR spectra. We acknowledge
677.2210). Dr. M. Apsega, Campus Chemical Instrument Center, The Ohio State
17,24-Epoxy-20r,25-dihydroxy-21-norbaccharan-3-one (3): color- University, for the mass spectrometric data.
less gum; [R]20D +77.0 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 208
(3.10) nm; CD (c 1.09 × 10-4 M, MeOH) λmax (∆ε) 289 (+1.31) nm; Supporting Information Available: Comparison of 1H NMR
IR (film) νmax 3395, 2948, 2868, 1701, 1456, 1384, 1161, 1067, 991, spectra of four silvestrol derivatives (1, 2, 7, and 8); 1H and 13C NMR
946, 753 cm-1; 1H NMR (600 MHz, CDCl3) and 13C NMR (150 MHz, and selected 2D NMR spectra of new compounds 1-6. This material
CDCl3) data, see Tables 2 and 3; HRESIMS m/z 483.3431 [M + Na]+
is available free of charge via the Internet at http://pubs.acs.org.
(calcd for C29H48O4Na, 483.3450).
20S,24S-Epoxy-25-hydroxy-A-homo-4-oxadammaran-3-one (4):
colorless gum; [R]20D +77.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) References and Notes
206 (3.19) nm; CD (c 2.09 × 10-3 M, MeOH) λmax (∆ε) 210 (+0.52),
(1) Hwang, B. Y.; Su, B. N.; Chai, H.-B.; Mi, Q.; Kardono, L. B. S.;
283 (+0.03) nm; IR (film) νmax 3420, 2939, 2870, 1717, 1457, 1387, Afriastini, J. J.; Riswan, S.; Santarsiero, B. D.; Mesecar, A. D.; Wild,
1374, 1286, 1139, 1111, 1027, 755 cm-1; 1H NMR (600 MHz, CDCl3) R.; Fairchild, C. R.; Vite, G. D.; Rose, W. C.; Farnsworth, N. R.;
and 13C NMR (150 MHz, CDCl3) data, see Tables 2 and 3; HRESIMS Cordell, G. A.; Pezzuto, J. M.; Swanson, S. M.; Kinghorn, A. D. J.
m/z 497.3651 [M + Na]+ (calcd for C30H50O4Na, 497.3607). Org. Chem. 2004, 69, 3350–3358; 6156.
20S,24S-Epoxy-25-hydroxy-A-homo-4-oxadammaran-3-one (5): (2) Meurer-Grimes, B. M.; Yu, J.; Vairo, G. L. U.S patent 6710075 B2,
colorless gum; [R]20D +111.0 (c 0.04, MeOH); UV (MeOH) λmax (log 2004.
ε) 205 (3.16) nm; CD (c 2.09 × 10-4 M, MeOH) λmax (∆ε) 214 (+0.28), (3) Mi, Q.; Kim, S.; Hwang, B. Y.; Su, B.-N.; Chai, H.-B.; Arbieva, Z. H.;
288 (+0.12) nm; IR (film) νmax 3423, 2967, 2933, 2865, 1718, 1457, Kinghorn, A. D.; Swanson, S. M. Anticancer Res. 2006, 26, 3349–
1387, 1374, 1288, 1143, 1111, 1058, 755 cm-1; 1H NMR (600 MHz, 3356.
CDCl3) and 13C NMR (150 MHz, CDCl3) data, see Tables 2 and 3; (4) Kim, S.; Hwang, B. Y.; Su, B.-N.; Chai, H.-B.; Mi, Q.; Kinghorn, A. D.;
Wild, R.; Swanson, S. M. Anticancer Res. 2007, 27, 2175–2183.
HRESIMS m/z 497.3639 [M + Na]+ (calcd for C30H50O4Na, 497.3607).
(5) Lucas, D. M.; Edwards, R. B.; Lozanski, G.; West, D. A.; Shin, J. D.;
4,6-Diene-1β,15-dihydroxyeudesma-3-one (6): colorless gum; Vargo, M. A.; Davis, M. E.; Rozewski, D. M.; Johnson, A. J.; Su,
[R]20D +217.0 (c 0.04, MeOH); UV (MeOH) λmax (log ε) 204 (3.65), B. N.; Goettl, V. M.; Heerema, N. A.; Lin, T. S.; Lehman, A.; Zhang,
302 (4.42) nm; CD (c 1.00 × 10-4 M, MeOH) λmax (∆ε) 295 (+4.3) X. L.; Jarjoura, D.; Newman, D. J.; Byrd, J. C.; Kinghorn, A. D.;
nm; IR (film) νmax 3406, 2962, 2917, 2873, 1645, 1613, 1569, 1465, Grever, M. R. Blood 2009, 113, 4656–4666.
1423, 1362, 1328, 1295, 1220, 1035, 997, 755 cm-1; 1H NMR (600 (6) Bordeleau, M. E.; Robert, F.; Gerard, B.; Lindqvist, L.; Chen, S. M. H.;
MHz, CDCl3) and 13C NMR (150 MHz, CDCl3) data, see Tables 2 Wendel, H. G.; Brem, B.; Greger, H.; Lowe, S. W.; Porco, J. A., Jr.;
and 3; HRESIMS m/z 273.1441 [M + Na]+ (calcd for C15H22O3Na, Pelletier, J. J. Clin. InVest. 2008, 118, 1–11.
273.1467). (7) Cencic, R.; Carrier, M.; Galicia-Vázquez, G.; Bordeleau, M.-E.;
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to a published protocol.22,23 Rocaglamide was used as a positive control, Chai, H.-B.; Sugiarso, S.; Kardono, L. B. S.; Fong, H. H. S.; Pezzuto,
with an ED50 value of 0.08 µM in this assay. J. M.; Swanson, S. M.; Carcache-Blanco, E. J.; Kinghorn, A. D. J.
Nat. Prod. 2009, 72, 1165–1169.
Acknowledgment. This study was supported, in part, by grants U19
CA52956 and P01 CA125066 (awarded to A.D.K.) from NCI, NIH. NP100503Q

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