A pregnane steroid from Aglaia lawii and structure confirmation of cabraleadiol monoacetate by X-ray crystallography

Article abstract of DOI:10.1016/S0031-9422(00)00463-5

The pregnane steroid, (E)-aglawone, along with four known triterpenes, and a known sterol mixture were isolated from the bark of Aglaia lawii (Wight) Saldanha ex Ramamoorty (Meliaceae). The structural determination/identification was accomplished by a combination of 1D- and 2D-NMR spectroscopic techniques. The relative stereochemistry of the known triterpene, 20S,24S-epoxydammarane-3α, 25-diol acetate, was also unequivocally determined for the first time by X-ray crystallography. The isolates were not active against various human cancer cell lines.

Full text of DOI:10.1016/S0031-9422(00)00463-5

Phytochemistry 56 (2001) 775±780
www.elsevier.com/locate/phytochem
A pregnane steroid from Aglaia lawii and structure con®rmation of
cabraleadiol monoacetate by X-ray crystallography
Sheng-Xiang Qiu a,1, Nguyen van Hung b, Le T hi Xuanb, Jian-Qiao Gu a, Emil Lobkovsky c,
Tran Cong Khanh d, Djaja D. Soejarto a, Jon Clardy c, John M. Pezzuto a, Yumi Dong a,
Mai Van Tri b, Le Mai Huong b, Harry H.S. Fong a,*
aProgram for the Collaborative Research in the Pharmaceutical Sciences, Department of Medicinal Chemistry and Pharmacognosy,
College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, IL 60612, USA
bInstitute of Chemistry, National Center for Science and Technology, Hanoi, Viet Nam
cBaker Laboratory, Chemistry Department, Cornell University, Ithaca, NY 14853, USA
dHanoi College of Pharmacy, Hanoi, Viet Nam
Received 24 January 2000; received in revised form 4 August 2000
Abstract
The pregnane steroid, (E)-aglawone, along with four known triterpenes, and a known sterol mixture were isolated from the bark
of Aglaia lawii (Wight) Saldanha ex Ramamoorty (Meliaceae). The structural determination/identi®cation was accomplished by a
combination of 1D- and 2D-NMR spectroscopic techniques. The relative stereochemistry of the known triterpene, 20S,24S-
epoxydammarane-3a,25-diol acetate, was also unequivocally determined for the ®rst time by X-ray crystallography. The isolates
were not active against various human cancer cell lines. # 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Aglaia lawii; Meliaceae; (E)-Aglawone; Pregnane steroid; Triterpenes; Phytosterols; 1D- and 2D-NMR Spectroscopic techniques; X-Ray
crystallography
1. Introduction
(3) and a mixture of the phytosterols, stigmasterol and
poriferasterol. Structures of the isolates were characterized
by means of spectroscopy, including 1D and 2D NMR
spectroscopic techniques. The relative stereochemistry
of 20S, 24S-epoxydammarane-25-ol-3a-yl acetate was
unequivocally determined by X-ray crystallography.
Aglaia lawii (Wight) Saldanha ex Ramamoorty is a tra-
ditional medicinal plant indigenous to Vietnam, having
been used for the treatment of bacterial infection, liver
and tumor diseases (Vo, 1997). In our continuing search
for biologically active constituents from Vietnamese higher
plants, the CHCl3 fraction of a MeOH extract of the stem
bark derived from A. lawii was found to exhibit cytotoxi-
city against several human cell lines (Le et al., 2000).
Fractionation of CHCl3-soluble components resulted in
the isolation of a new pregnane steroid, namely (E)-
aglawone (1), as well as the known triterpenes, b-amyr-
one and b-amyrin, 20S, 24S-epoxydammarane-25-ol-3a-
yl acetate (cabraleadiol monoacetate) (2), cabraleadiol
2. Results and discussion
(E)-Aglawone (1), obtained as white needles, exhibited
a molecular formula C21H32O2 (Mr=316) as evidenced by
a quasimolecular ion [M+H]+ at m/z 317, determined by
electrospray mass spectroscopy, and DEPTexperiments,
in which three methyls, nine methylenes, six methines
and four quaternary carbons were evident. Inspection of
its UV, IR, and 1H and 13C NMR spectral data revealed
that it was an a, b-unsaturated pregnane steroid closely
related in structure to (E)-volkendousin (Rogers et al.,
1998). The existence of the a,b-unsaturated cyclopenta-
none fragment was further con®rmed by the strong
* Corresponding author. Tel.: +1-312-413-5894; fax: +1-312-996-
3272.
E-mail address: hfong@uic.edu (H.H.S. Fong).
1
Current address: Monsanto Company, T4L, 800 N. Lindbergh
Blvd, St Louis, MO 63167, USA.
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(00)00463-5

776
S.-X. Qiu et al. / Phytochemistry 56 (2001) 775±780
absorption at 1718 and 1640 cmÀ1 in the IR spectrum, and
an UV absorption maximum at 242 nm (Nakano et al.,
1996). In addition to con®rming the tetracyclic frame-
work of 1 with the D-ring being an a,b-unsaturated
cyclopentanone, the interpretation of the NMR spec-
troscopic data from DQF-COSY, HOHAHA, ROESY,
HMQC, and HMBC also established the placement of
the hydroxyl group at the C-3 position (Fig. 1). The
small coupling constants of the carbinol proton H-3 at ꢀ
4.06 (t, J=2.6) was indicative of the C-3 hydroxyl being
axial from an examination of the Dreiding model.
strong 1H±13C correlations with the 13C signals of C-12,
C-13, C-14 and C-17 as expected. Similarly, the H sig-
1
nal at ꢀ 0.83, which showed correlations with the 13C
signals assignable to those of C-1, C-5, C-9 and C-10,
was assigned to CH3-19. The geometric stereochemistry
1
of 1 was assigned to E by a comparison of its H NMR
spectrum with those of (E)- and (Z)-volkendousin
(retrieved directly from the deposited supplemental
documents of cited reference via the ACS website)
1
(Rogers et al., 1998). It has been reported that the H
pro®les of E- and Z-isomers can be regarded as being
characteristic of the geometric stereochemistry of the
trisubstituted exocyclic double bond, since the chemical
shifts of the vinyl protons and the methyl groups of the
two epimers could be di€erentiated by the anisotropic
e€ect of the carbonyl group at C-16 (Rogers et al.,
1998). The assignment of an E geometric stereo-
chemistry to the C17±C20 double bond was justi®ed by
It has been reported that the angular methyl groups of
1
pregnane steroids usually show signi®cant H±13C cor-
relations in the long-range hetereonuclear coupling
(HMBC) spectrum, and therein provided the basis for
the assignments of both angular methyls and backbone
skeleton carbon signals (Qiu et al., 1997). In the HMBC
spectrum of 1, the CH3-18 signal at ꢀ 1.01 showed

S.-X. Qiu et al. / Phytochemistry 56 (2001) 775±780
777
Fig. 1. Selective ROESY correlations for compound 1.
1
the H-NMR spectrum of 1, in which the vinyl proton
resonated at a relatively higher frequency at ꢀ 6.49 (ꢀ
6.45 and 5.88 were observed for E- and Z-volkendousin,
respectively). This resonance is indicative of a syn con-
®guration to the carbonyl oxygen, and would conse-
quently experience a deshielding e€ect, leading the
doublet methyl signal to be detected at relatively lower
frequency (ꢀ 1.85). Indeed, Z- and E-volkendousin were
observed at ꢀ 2.05 and 1.85, respectively (Rogers et al.,
1998). Furthermore, the ROE cross peak observed
between the signals of 18-methyl at ꢀ 1.01 and 21-
methyl at ꢀ 1.85 in the ROESY spectrum provides solid
evidence for the E geometric stereochemistry of the C17±
Table 1
1H and 13C NMR spectral assignment for (E)-aglawone (1)a
No.
ꢀC (mult.)
ꢀ
H [mult., coupling (Hz)]
HMBC(H)
19
1
31.92 (t)
28.92 (t)
66.45 (d)
36.33 (t)
39.03 (d)
28.27 (t)
31.87 (t)
34.19 (d)
54.03 (d)
36.25 (s)
20.52 (t)
35.79 (t)
43.39 (s)
50.07 (d)
37.93 (t)
1.45 (m); 0.98 (m)
1.23 (m); 1.09 (m)
4.06 (t, 2.6)
2
3
4
1.25 (m); 1.05 (m)
1.65 (m)
5
19
6
1.21 (m); 1.04 (m)
1.44 (obsc); 1.02 (m)
1.56 (m)
7
8
9
0.91 (m)
19
19
10
11
12
13
14
15
1.70 (m); 1.45 (m)
1.40 (obsc); 1.25 (m)
18
18, 20
18
C
21 double bond. Therefore, the structure of 1 was deter-
mined as (E)-aglawone. An unambiguous NMR assign-
ment of 1 was achieved by means of a combination of 1D
and 2D NMR experiments (Table 1). The protonated
carbon signals were assigned to the corresponding pro-
ton signals by means of HMQC experiment, which also
allowed the construction of spin-systems by combina-
tion with DQF-COSY experiment. Finally, the HMBC
experiment enable the assignment of the quaternary
carbons.
Compound 2, which was assigned to a molecular for-
mula of C32H54O4 by HREIMS at m/z 502.4071 (requires
502.4022), exhibited eight tertiary methyl singlets at
ꢀ0.84, 0.87, 0.89, 0.93, 0.98, 1.11, 1.15 and 1.19, a car-
binol methine proton at ꢀ 4.47 (br. s), an oxymethine
proton at ꢀ3.50 (dd, J=5.7, 9.0 Hz), an acetyl methyl at
ꢀ 2.10 (s) and some heavily-overlapping unresolved
1.42 (m)
1.96 (dd, 14.5; 17.0)
2.20 (dd, 7.4; 17.0)
16
17
18
19
20
21
206.75 (s)
148.02 (s)
17.67 (q)
11.17 (q)
128.97 (d)
13.17 (q)
20
18, 20, 21
1.01 (s)
0.83 (s)
6.49 (q,7.5)
1.85 (d, 7.5)
21
20
a
Recorded in CDCl3, chemical shift values were reported as ꢀ
values (ppm) from internal TMS at 300 MHz, signal multiplicity and
coupling constants (Hz) are shown in parentheses: m=multiplex signals;
obsc.=obscured signals.
monoacetate) or ocotillone-II, which are epimeric at C-
24 (Nagai et al., 1973; Hisham et al., 1996), leaving the
stereochemistry at positions C-20 and C-24 to be
assigned.
1
multiplets extending from ꢀ 1.05 to 1.88 in its H NMR
spectrum, characteristic for dammarane-type triterpe-
noids (Hisham et al., 1996). The IR spectrum of 2
showed the presence of a hydroxyl absorption at 3622
(secondary OH) and an ester carbonyl absorption at
1730 and 1245 cmÀ1. T he13C NMR spectrum of 2
showed 32 13C signals which were resolved by a DEPT
experiment into nine methyls, ten methylenes, six
methines and six quaternary carbons, respectively. The
overall spectroscopic pro®les of 2 were found to be very
similar to those of 20S, 24S-epoxydammarane-25-ol-3a-
yl acetate (betulafolianediol 3-acetate; cabraleadiol
There has been some controversy regarding the
assignment of the con®guration of the C-24 chiral center
of 20, 24-trisubstituted epoxides. Nagai et al. (1973)
initially assigned the (R) con®guration to C-24 for oco-
tillone-II, based on a series of chemical conversions and
an X-ray crystallographic study, but Rao et al. (1975)
claimed 24(S) stereochemistry and suggested 24(R) ste-
reochemistry for cabraleone and its cogeners. A few
years later, the C-24 stereochemistry of ocotillone and
cabraleone was reassigned as R and S, respectively, by

778
S.-X. Qiu et al. / Phytochemistry 56 (2001) 775±780
Tanaka and Yahara (1978) and by Betancor et al.
(1983). There was no controversy in regard to the C-20
con®guration in both compounds.
by a comparison of its NMR pro®le with that of the
literature values (Hisham et al., 1996). The deacetylated
derivative prepared from 2 by treatment of K2CO3-
MeOH was found to be identical and possessing the
same stereochemistry as that of 2 (20S, 24S) by a direct
In order to resolve the geometric stereochemical assign-
ment at the chiral center at C-24 and validate the use of 1H
and 13C NMR chemical shifts as proposed by Hisham et
al. (1996) for predicting the C-20, 24 stereochemistry of
these compounds, an X-ray crystallographic study was
performed to conclusively determine the stereochemistry
of compound 2. In addition, the NMR spectroscopic
resonances were unequivocally assigned by use of 1D-
and 2D NMR spectroscopy.
A suitable crystal that formed in the monoclinic space
group C2 was selected for the study. Accurate lattice
constants were a=30.2897(6) A, b=7.1071(2) A,
c=13.8023(4) A. Four molecules of the composition
C32H54O4 formed the asymmetric unit. All unique dif-
fraction maxima with 2y 449ꢀ were collected using 2y:y
scans and graphite monochromated MoKa radiation. A
total of 7181 unique re¯ections were collected. A com-
puter-generated drawing of the ®nal X-ray model of
20S, 24S-epoxydammarane-25-ol-3a-yl acetate is given
in Fig. 2. All the 1H and 13C NMR chemical shifts were
unambiguously assigned by means of 2D NMR techni-
ques and were found to be consistent with those made by
Hisham et al. (1996). Thus, the stereochemistry of 20S,
24S-epoxydammarane-25-ol-3a-yl acetate was con-
clusively determined, and the published NMR spectral
assignments were con®rmed.
1
comparison of their mp, the H and 13C NMR spectral
data. Therefore, compound 3 was unambiguously iden-
ti®ed as 20S, 24S-epoxydammarane-3a, 25-diol (betula-
folianediol 3-acetate; cabraleadiol).
b-Amyrone (Seo et al., 1975; Kikuchi et al., 1980), b-
amyrin (Seo et al., 1975; Kikuchi et al., 1980), were
identi®ed by direct comparison of their physical and
NMR spectral data with those from the literature.
A mixture of two phytosterols was also isolated.
There are two very close but well-resolved sets of signals
1
of equal intensity observed in both H and 13C NMR
spectra taken in CDCl3, which suggested that the two
existed in equal population. The 13C NMR spectral data
were fully superimposable on those of stigmasterol and
poriferasterol (Matsumoto et al., 1984; Horibe et al.,
1989), resulting in the identi®cation of this mixture as
equimolecular epimeric mixture of stigmasterol and
poriferasterol.
None of the isolates were shown to be cytotoxic
against several human cancer cell lines (LNCaP, Col 2
or KB), using established protocols (Likhitwitayawuid
et al., 1993). Further bioassay-guided of the CHCl3
soluble fraction with an aim to isolate and identify the
bioactives responsible for the cytotoxcity observed for
the extract is in progress.
Compound 3 was readily discerned to be 20S, 24S-
epoxydammarane-3a, 25-diol, a deacetyl derivative of 2,
3. Experimental section
3.1. General experimental procedures
Melting points were determined on a Ko¯er hot-stage
apparatus and are uncorrected. IR spectra were recorded
with a MIDAC collegian FT±IR spectrometer. Optical
rotations were measured with a Perkin-Elmer 241
polarimeter. UV spectra were obtained with a Beckman
1
DU-7 spectrometer. The H and 13C NMR, DEPT,
DQF-COSY, HOHAHA, HMQC, HMBC and ROESY
spectra were recorded with TMS as internal standard,
using a Bruker Advance DPX-300 instrument equipped
with a Bruker standard program in CDCl3 solution.
Electrospray MS/MS was performed on a Macro Mass
Quattro II electrospray triple quadrupole mass spectro-
meter. Electron impact mass spectra (70 eV) were
recorded with a Varian MAT-112S mass spectrometer.
TLC was performed on precoated Kieselgel 60 F254
plates (Merck) and the visualization was achieved by
spraying with either 10% H2SO4 followed by heating or
phosphomobydbic acid spray reagent.
The X-ray di€raction study of compound 2 was con-
ducted on a R3M/V four-circled di€ractometer (Siemens)
Fig. 2. Computer-generated perspective drawing of the ®nal X-ray
model of 20S, 24S-epoxydammarane-25-ol-3a-yl acetate (2).

S.-X. Qiu et al. / Phytochemistry 56 (2001) 775±780
779
connected to a MicroVax II computer (Digital) using
CuKa or MoKa radiations.
(3H, s, Me-19), 0.88 (3H, s, Me-29), 0.93 (3H, s, Me-30),
0.98 (3H, s, Me-18), 1.11 (3H, s, Me-17), 1.15 (3H, s,
Me-21), 1.19 (3H, s, Me-26), 2.09 (3H, s, Ac-Me), 3.65
(1H, dd, J=9.5, 5.7 Hz, H-24), 4.62 (1H, br.s, H-3b).
13C NMR (ppm from solvent CDCl3 signal at 77.0) ꢀ :
33.9 (t, C-1), 22.5 (t, C-2), 77.8 (d, C-3), 36.3 (s, C-4),
50.3 (d, C-5), 17.7 (t, C-6), 34.7 (t, C-7), 40.1 (s, C-8),
50.2 (d, C-9), 36.7 (s, C-10), 21.2 (t, C-11), 26.8 (t, C-12),
42.4 (d, C-13), 49.7 (s, C-14), 31.1 (t, C-15), 25.5 (t, C-
16), 49.4 (d, C-17), 15,1 (q, C-18), 15.6 (q, C-19), 86.0 (s,
C-20), 26.6 (q, C-21), 34.4 (t, C-22), 26.0 (t, C-23), 85.9
(d, C-24), 69.8 (s, C-25), 27.2 (q, C-26), 23.9 (q, C-27),
27.5 (q, C-28), 21.4 (q, C-29), 16.2 (q, C-30), 20.9 (q, Ac-
Me).
3.2. Plant material
Stem bark of A. lawii was collected in Cuc Phuong
National Park, Vietnam, and identi®ed by one of the
authors (DDS). Voucher specimens (No. MAF-1414B)
have been deposited in the Herbaria of the Cuc Phuong
National Park, Vietnam, and the Field Museum of
Natural History, Chicago, IL, USA.
3.3. Extraction and isolation
The milled, air-dried stem bark of A. lawii (4 kg) was
extracted with MeOH (9 1Â3) by maceration. The
resulting pooled extract was concentrated to dryness in
vacuo and defatted with n-hexane to a€ord a n-hexane-
soluble fraction (25 g). The defatted residue was then
suspended in H2O and partitioned with CHCl3 to give,
on drying under reduced pressure, 98 g of a CHCl3-
soluble fraction. This fraction demonstrated cytotoxic
activity against the LNCaP (hormone-dependent pros-
tate human cancer), KB (human oral epidermoid carci-
noma), and Col 2 (human colon cancer) cell lines with
ED50 values of 8.2, 11.9, and 9.0 mg/ml, respectively.
Fractionation of the CHCl3-soluble fraction was
initiated by column chromatography over silica gel (450
g) using a gradient mixture of petroleum and EtOAc (0±
100%) as eluent. Sixteen fractions were pooled on the
basis of their similar TLC patterns. Work-up of fraction
2 a€orded compound b-amyrone (1.0 g) after recrys-
tallization from a mixture of petroleum ether and
CHCl3 (1:1). Work-up of fractions 4 and 5, followed by
recrystallization from CHCl3, resulted in the isolation of
b-amyrin (520 mg) and 2 (5.2 g). The epimeric mixture
of stigmasterol plus poriferasterol (400 mg) was isolated
from the work-up of fraction 6 and recrystallization from
CHCl3. Fraction 11 was further chromatographed on a
silica gel column (50 g) and eluted with a mixture of pet-
roleum/CHCl3/MeOH (20:20:1, v/v/v) followed by pre-
parative TLC (silica gel, solvent system: petroleum:
EtOAc=4:1) to a€ord compounds 1 (3.5 mg) and 3 (7
mg).
Crystal data for 2: C32H54O4, MW=502.75, monoclinic,
C2, a=30.2897(6) A, b=7.1071(2) A, c=13.8023(4) A,
ꢁ=90.0000 (2)ꢀ, ꢂ=95.3225(12)ꢀ, ꢃ=90.0000(10)ꢀ, V=
2958.44 (13) A, Z=4, crystal size: 0.5Â0.3Â0.23 mm,
R1=0.0507, WR2=0.1127. All unique di€raction
maxima with 2ꢄ449ꢀ were collected using 2ꢄ:ꢄ scans
and graphite monochromated MoKa radiation. A total
of 7181 unique re¯ections were collected. The structure
was solved by direct methods (SIR92) re®ned by full-
matrix least-squares techniques.
Additional crystallographic details (atomic coordi-
nates and equivalent isotropic displacement coecients,
lists of structure factors, interatomic distances, angles,
anisotropic displacement coecients for non-hydrogen
atoms, hydrogen atom coordinates and their isotropic
displacement coecients) have been deposited at the
Cambridge Crystallographic Data Centre. The coordi-
nates can be obtained, upon request, from Dr. Olga
Kennord, University Chemical Laboratory, 12 Union
Road, Cambridge CB2 1EZ, UK.
Acknowledgements
This study was supported in part by grants (No. 94-
26147 and No. 96-44652) from the John D. and Catherine
T. MacArthur Foundation, Chicago, IL, USA.
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(E)-Aglawone (1), white powder; mp 125±126ꢀC; [a]D
À0.1ꢀ (c, 0.2, MeOH); UV (MeOH) nm (log E) lmax
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