Antineoplastic Agents. 522. Hernandia peltata (Malaysia) and Hernandia nymphaeifolia (Republic of Maldives)

Article abstract of DOI:10.1021/np030125s

Bioassay (P388 lymphocytic leukemia cell line and human tumor cell lines)-guided separation of the extracts prepared from the tropical and coastal trees Hernandia peltata (Malaysia) and Hernandia nymphaeifolia (Republic of Maldives) led to the isolation of a new lignan designated as hernanol (1) and 12 previously known lignans: (-)-deoxypodophyllotoxin (2), deoxypicropodophyllin (3), (+)-epiaschantin (4), (+)-epieudesmin (5), praderin (6), 5′-methoxyyatein (7), podorhizol (8), deoxypodorhizone (9), bursehernin (10), kusunokinol (11), clusin (12), and (-)-maculatin (13). The oxidative cyclization (with VOF₃) of lignans 8, 9, and 10 resulted in a new and unusual benzopyran (14), isostegane (15), and a new dibenzocyclooctadiene lactone (16), respectively. The structure and relative stereochemistry of hernanol (1) and lignans 3, 7, 8, 9, 10, 11, and 12 were determined by 1D and 2DNMR and HRMS analyses. The structures and absolute stereochemistry of structures 2, 4, 5, 6, 13, 14, 15, and 16 were unequivocally determined by single-crystal X-ray diffraction analyses. Evaluation against the murine P388 lymphocytic leukemia cell line and human tumor cell lines showed podophyllotoxin derivatives 2 and 3 to be strong cancer cell line growth inhibitors and substances 4, 5, 8, and 15 to have marginal cancer cell line inhibitory activities. Seven of the lignans and one of the synthetic modifications (14) inhibited growth of the pathogenic bacterium Neisseria gonorrhoeae.

Full text of DOI:10.1021/np030125s

2
14
J . Nat. Prod. 2004, 67, 214-220
An tin eop la stic Agen ts. 522. Her n a n d ia pelta ta (Ma la ysia ) a n d Her n a n d ia
n ym ph a eifolia (Rep u blic of Ma ld ives)⊥ ,1
George R. Pettit,* Yanhui Meng, R. Patrick Gearing, Delbert L. Herald, Robin K. Pettit, Dennis L. Doubek,
J ean-Charles Chapuis, and Larry P. Tackett
Cancer Research Institute and Department of Chemistry and Biochemistry, Arizona State University,
Tempe, Arizona 85287-2404
Received March 21, 2003
Bioassay (P388 lymphocytic leukemia cell line and human tumor cell lines)-guided separation of the
extracts prepared from the tropical and coastal trees Hernandia peltata (Malaysia) and Hernandia
nymphaeifolia (Republic of Maldives) led to the isolation of a new lignan designated as hernanol (1) and
1
2 previously known lignans: (-)-deoxypodophyllotoxin (2), deoxypicropodophyllin (3), (+)-epiaschantin
4), (+)-epieudesmin (5), praderin (6), 5′-methoxyyatein (7), podorhizol (8), deoxypodorhizone (9),
bursehernin (10), kusunokinol (11), clusin (12), and (-)-maculatin (13). The oxidative cyclization (with
VOF ) of lignans 8, 9, and 10 resulted in a new and unusual benzopyran (14), isostegane (15), and a new
dibenzocyclooctadiene lactone (16), respectively. The structure and relative stereochemistry of hernanol
1) and lignans 3, 7, 8, 9, 10, 11, and 12 were determined by 1D and 2DNMR and HRMS analyses. The
(
3
(
structures and absolute stereochemistry of structures 2, 4, 5, 6, 13, 14, 15, and 16 were unequivocally
determined by single-crystal X-ray diffraction analyses. Evaluation against the murine P388 lymphocytic
leukemia cell line and human tumor cell lines showed podophyllotoxin derivatives 2 and 3 to be strong
cancer cell line growth inhibitors and substances 4, 5, 8, and 15 to have marginal cancer cell line inhibitory
activities. Seven of the lignans and one of the synthetic modifications (14) inhibited growth of the
pathogenic bacterium Neisseria gonorrhoeae.
The tropical and relatively small (∼54 species) plant
family Hernandiaceae of trees, shrubs, and vines is divided
among three genera. The largest, Hernandia, has some 24
species. References to the possible use of H. ovigera and
H. sonora in traditional medicine for cancer treatment
first appeared in a European medicinal plant treatise of
2
a
1
831-1836. Subsequent chemical investigations of H.
2
b-g
2h
2i
ovigera
and H. sonora (believed to correspond to H.
nymphaeifolia2 ) have provided a good basis for these
historical observations. Both species have been found to
contain cancer cell growth inhibitory lignans, aporphine
j-n
2
h,n
alkaloids, and podophyllotoxins.
To further explore the
anticancer constituents of H. nymphaeifolia (Presl.) Ku-
bitzki, we collected (in 1989) this plant in a new location,
the Republic of the Maldives. For the same purpose, we
collected H. peltata Meissner, which also may correspond
to H. sonora, on the east coast of Malaysia in 1990.
Both H. nymphaeifolia and H. peltata are common
coastal trees that grow to 12-20 m in height and occur
widely in tropical countries of Southeast Asia and Indo-
pacific regions. H. ovigera and H. peltata have been used
widely, for example, in Western Samoa,2i as a traditional
medicine for boils, cough, diarrhea, abdominal pains,
anticonvulsive treatment, eye problems, and a number of
other indications that appear to include anticancer, anti-
viral, and antiparasite. In our present investigation we
isolated and characterized eight previously known lignans
3
4
5
6
7
8
9
10,11
(
2, 3, 4, 5, 6, 7, 8, and 10
) from H. peltata.
3
4
6
9
12
10,11
Similarly, nine known lignans (2, 3, 5, 8, 9, 10,
1
3
14
15
1
1, 12, and 13 ) were isolated from H. nymphaeifolia,
together with one new lignan, an epitraxillagenin desig-
nated hernanol (1). The X-ray crystal structure (and
absolute stereochemistry) of deoxypodophyllotoxin 2 (also
⊥
Dedicated to the late Dr. Monroe E. Wall and to Dr. Mansukh C. Wani
of Research Triangle Institute for their pioneering work on bioactive natural
products.
known as anthricin) has been reported16 and has served
as a template in the conformational analysis and confor-
*
To whom correspondence should be addressed. Tel: 480-965-3351.
Fax: 480-965-8558. E-mail: bpettit@asu.edu.
1
0.1021/np030125s CCC: $27.50
© 2004 American Chemical Society and American Society of Pharmacognosy
Published on Web 02/12/2004

Antineoplastic Agents. 522
J ournal of Natural Products, 2004, Vol. 67, No. 2 215
Ta ble 1. 1H and 13C NMR Assignments for Hernanol (1) in CHCl3a
δ1H
1
H - H COSY
1
δ13
C
HMBCb
position
1
129.7
133.4
110.9
106.3
146.6
153.3
144.5
136.9
114.5
153.3
121.3
106.3
38.3
H-5, H-7a, b, H-8(w)
H-7′, H-8′, H-2′(w), H-6′(w)
H-6, H-7
H-6′, H-7′
OCH3-3, OH-4, H-2, H-5
OCH3-3′, H-2′
OH-4, H-2, H-5, H-6
OCH3-4′, H-2′, H-6′
OH-4
OCH3-5′, H-6′
H-2, H-7a, b
1
2
2
3
3
4
4
5
5
6
6
7
′
′
′
′
′
′
6.44s (1H)
6.34s (1H)
6.82d (1H, J ) 7.5 Hz)
H-6
H-5
6.53d (1H, J ) 7.5 Hz)
6.34s (1H)
H-2′, H-7′
H-2, H-6, H-8, H-8′, H-9a, H-9b(w)
2.65dd (1H, J ) 13.5, 6.5 Hz, H-7a)
H-7b, H-8
H-7a, H-8
H-8′
H-7a,b, H-8′, H-9,
H-7′, H-8
2
.56dd (1H, J ) 13.5, 8.5 Hz, H-7b)
7
8
8
9
′
′
2.92m (1H)
35.2
41.1
46.5
71.3
H-2′, H-6′, H-8, H-8′
H-7ab, H-7′, H-9a, b(w)
H-7, H-7′, H-8, H-9b(w)
H-7, H-8
2.48m (1H)
2.60m (1H)
3.89t (1H, J ) 8.5 Hz, H-9a)
H-9b, H-8
H-9a, H-8
4
.19dd (1H, J ) 8.5, 7.5 Hz, H-9b)
9
′
178.6
55.8
56.1
H-7′, H-8′, H-9a,b
OCH3-3
OCH3-3′
OH-4
OCH3-4′
OCH3-5′
3.81s (3H)
3.81s (3H)
5.51s (1H)
3.82s (3H)
3.81s (3H)
60.9
56.1
a
Measured at 500 MHz. b w ) weak.
mational-biological activity relationships of other podo-
phyllotoxin analogues.1
6,17
The absolute structure of the
lignan 15 (known as isostegane and here produced by
semisynthesis as described below) has also been deter-
mined, on the basis of X-ray structural analysis of the (+)-
1
1
2-bromo derivative of isostegane.18 Some (3, 6, 11, 12, and
3) were not previously known to occur in these two
Hernandia species. Another objective of this research was
to employ several of the more abundant lignans for
synthetic conversion to cyclic biaryls with the prospect of
obtaining new cancer cell growth inhibitors related to
podophyllotoxin. For that purpose, oxidative cyclization of
1
9
lignans 8, 9, and 10 with VOF
3
yielded the unusual
benzopyran 14, isostegane (15),19 and a new dibenzocy-
clooctadiene lactone (16), respectively. Herein we report
the structural elucidation, as well as the anticancer and
antimicrobial evaluations, of these 16 Hernandia constitu-
ents and structural modifications.
Resu lts a n d Discu ssion
The initial CH
phaeifolia and H. peltata were separately partitioned
between CH OH-H O (9:1 f 1:1) and hexane f CH Cl
Bioassay-guided (P388 lymphocytic leukemia cell line)
separation of the CH Cl fraction from each of the Her-
2
Cl
2
3
-CH OH (1:1) extracts of H. nym-
3
2
2
2
.
2
2
nandia species solvent partitioning sequences by a series
of Sephadex LH-20 column chromatographic steps followed
by final purification employing HPLC and recrystallization
proton signals confirmed their origin from a trisubstituted
and a tetrasubstituted benzene ring. The 1H- H COSY
spectrum (Table 1) confirmed the connection of four sat-
urated carbon atoms in the butyrolactone ring and also con-
firmed the connection of C-7 to C-8 and C-7′ to C-8′. These
assignments were further defined by HMQC and HMBC
spectra (Table 1), confirming the presence of 3-methoxy-
4-hydroxylbenzyl and 3′,4′,5′-trimethoxybenzyl groups. In
addition, the bonding of the 3-methoxy-4-hydroxylbenzyl
group to lactone position C-7 and the 3,4,5-trimethoxyben-
zyl group to C-7′ was established by the strong correlations
observed from H-7a,b (δ 2.65, 2.56) to C-1 (129.7) and from
H-7′ (δ 2.92) to C-1′ (133.4). Thus, the structure of hernanol
(1) was determined unambiguously.
-
4
1
led to the new lignan hernanol (1, 6.5 × 10 % yield from
H. nymphaeifolia) and 12 known lignans (2-13).
The hernanol (1) molecular formula was assigned as
+
C
22
H
26
O
7
on the basis of high-resolution APCI mass
+
spectroscopy ([M + 1] at m/z 403). The infrared absorption
at 1769 cm (CdO) and the appearance of signals at δ
.48, 2.60 (2H, m, C8,8′-H), 2.56, 2.65, and 2.92 (4H, m
-
1
2
1
C7,7′-H), and 3.89, 4.19 (2H, each dd, C9-H) in the H NMR
spectrum (Table 1) suggested a 2,3-dibenzylbutyrolactone-
1
type lignan. The H NMR spectrum of hernanol (1) also
revealed five aromatic proton signals at δ 6.44 (1H, s), 6.53
(
1H, d, J ) 7.5 Hz), 6.82 (1H, d, J ) 7.5 Hz), and 6.34 (2H,
s). The splitting pattern and coupling constants of the five

2
16 J ournal of Natural Products, 2004, Vol. 67, No. 2
Pettit et al.
F igu r e 2. X-ray structure of isostegane 15 with 50% thermal
probability ellipsoids.
F igu r e 1. X-ray crystal structure of benzopyran 14 with 50%
probability thermal ellipsoids.
The relative stereochemistry of the two chiral carbons
in hernanol (1) was deduced as 8R*, 8′R* on the basis of
comparing their carbon chemical shifts with those of known
lignans 7, 9, and 10, whose relative stereochemistry has
already been determined as 8R*, 8′R*, and especially with
lignan 13, whose absolute stereochemistry has already
been decided by single-crystal X-ray analysis. The trans-
relationship of the C-8 and C-8′ protons of the known 2,3-
dibenzylbutyrolactone lignans 7, 8, 9, 10, and 13 isolated
from H. nymphaeifolia suggests that they might be derived
from analogous biosyntheses. Interestingly, when compar-
1
13
ing the H and C NMR chemical shifts of hernanol (1)
with those of traxillagenin2
0,21
(first isolated from the stems
2
1
of Trachelospermum asiaticum and its structure deduced
on the basis of 13C NMR and MS data), it was found that
the carbon chemical shifts of the two compounds are very
similar (less than 0.8 ppm difference). Because the stere-
ochemistry of hernanol (1) was established by HMBC, it is
likely that the traxillagenin structure needs to be reas-
signed to that of lignan 1. The structures and relative
stereochemistry of lignans 3, 7, 8, 9, 10, 11, and 12 were
determined by 1D and 2D NMR and HRMS analyses as
F igu r e 3. X-ray structure of deoxyisosteganone 16 with 50% thermal
probability ellipsoids.
well as by comparison with published data.4
,8-14
The
structures of compounds 2, 4-6, and 13-16 were deter-
mined by single-crystal X-ray diffraction techniques. For
a majority of these compounds, due to the high oxygen
content, anomalous dispersion effects were sufficiently
intense as to allow determination of the absolute stereo-
chemistry. Thus, refinement of the absolute structure
mounted on the tip of a glass fiber with Vaseline and frozen
into position with a cooled N
2
gas stream, and data
collection was initiated. Frames of data were collected such
as to survey a complete sphere of reflections with at least
>95% coverage of the total number of theoretical reflections
possible. Structure solution and refinement were performed
with the Bruker SHELXTL22 software package. In this
manner, unambiguous structure determinations were ob-
tained for the known compounds 2, 4-6, and 13. Absolute
stereochemical structures were also determined for the
reaction products 14-16 (Figures 1-3), obtained from the
(Flack parameter) in the cases of compounds 2, 4, 6, and
1
3-16 allowed conclusive assignment of their absolute
stereochemical structures.
X-ray structure determinations of compounds 2, 4-6, 13,
and 15 confirmed the structures of these known compounds
as deoxypodophyllotoxin, (+)-epiashantin, (+)-epieudesmin,
praderin, maculatin, and isostegane, respectively. Figures
and X-ray tables for these known compounds are sum-
marized in the Supporting Information. The absolute
stereochemical structures of the remaining substances 14-
1
9
corresponding reaction of 8-10 with VOF
3
,
as described
below.23
A series of oxidative coupling methods using heavy metal
oxidants1
9,24
has been used to synthesize stegane and
1
6 (Figures 1-3 respectively), which were isolated via
isostegane lignans from cis- or trans-3,4-dibenzylbutyro-
lactones. As a part of our continuing efforts to discover
useful anticancer agents, cyclization of podorhizol (8),
deoxypodorhizone (9), and bursehernin (10) was conducted
synthetic modifications, are presented below.
All of the X-ray data collections were performed at -150
C (123 K) on a Bruker SMART 6000 X-ray diffractometer
°
1
9
using Cu radiation and a graphite monochromator. Suit-
able crystals, obtained via crystallization methods, were
by treating each with VOF
3
.
As expected, isostegane
(15, 14% yield) and a new deoxy isosteganone (16, 20%

Antineoplastic Agents. 522
J ournal of Natural Products, 2004, Vol. 67, No. 2 217
Ta ble 2. Murine P388 Lymphocytic Leukemia Cell Line and
Chemicals AB. Semipreparative HPLC was performed with a
Gilson model 805 HPLC coupled with a Gilson model 117 UV
detector. Analytical HPLC was conducted with a Hewlett-
Packard model 1050 HPLC coupled with a Hewlett-Packard
diode-array detector. Melting points were measured on an
Olympus electrothermal melting point apparatus and are
uncorrected. The optical rotation measurements were recorded
with a Perkin-Elmer 241 polarimeter. UV spectra were col-
lected with a Perkin-Elmer Lambda 3B UV/vis spectrometer.
The IR spectra were recorded with a Thermo Nicolet Avatar
360 infrared spectrometer. NMR spectra were determined with
a Varian XL-300 or a Varian UNITY INOVA-500 and 400
spectrometer using tetramethylsilane (TMS) as an internal
reference. High-resolution mass spectra were obtained using
a J EOL LCMate magnetic sector instrument in either the FAB
mode with a glycerol matrix or by APCI with a poly(ethylene
glycol) reference.
Human Cancer Cell Line Inhibition Values (ED50 expressed in
a
µg/mL) for Lignans 2, 3, 4, 5, 8, and 15
cancer cell lineb
2
3
4
5
8
15
P388
0.0023 0.030
1.5 1.7
>1.0 6.1
3.8 4.7
6.6 4.3
BXPC-3
MCF-7
SF268
NCI-H460
KM20L2
DU-145
0.0029 0.061 >10
0.0021 0.026 >10
0.0033 0.041 >10
0.0027 0.067 >10
0.0028 0.054 >10
0.0028 0.051 >10
3.4
3.5
3.8 >10
3.8 >10
3.0 >10
3.1 >10
4.9
5.8
4.8
5.3
a
In DMSO. b Cancer cell type (P388, lymphocytic leukemia;
BXPC-3, pancreas adenocarcinoma; MCF-7, breast adenocarci-
noma; SF268, CNS glioblastoma; NCI-H460, lung large cell;
KM20L2, colon adenocarcinoma; DU-145, prostate carcinoma).
yield) were synthesized from deoxypodorhizone (9) and
bursehernin (10), respectively. However, the cyclization of
podorhizol (8) resulted in an unusual benzopyran (14, 42%
yield), instead of the expected isostegane-type product. In
this respect, an O-benzoquinone was formed following
Her n a n d ia n ym ph a eifolia . On March 28, 1989, this tree
(∼15 m) was collected on the small island of Guradu in the
South Male Atoll, Republic of Maldives, and preserved in
absolute ethanol. Later in 1989, four 55 gal drums of the tree
(bark, stems, and leaves) were collected and 40 L of absolute
cleavage of its methylenedioxy group when isostegane was
treated with thallium(III) oxide in TGA.24 Each product
ethanol was added to each drum. Our procedure avoided the
problems (e.g., fungal overgrowth) associated with large-scale
plant drying in remote tropical areas and long shipment times.
The taxonomic identification was undertaken (by Drs. D. H.
Lorence and L. R. Landrum), and a voucher specimen is
maintained in our institute.
Her n a n d ia pelta ta . The initial collection of this tree (∼1
kg sample) was performed on September 9, 1990, at Pulau
Redang, on the east coast of Malaysia. The collected material
was preserved in MeOH and identified, and a voucher speci-
men was stored as noted above for H. nymphaeifolia.
Extr a ction a n d In itia l Sep a r a tion of H. n ym ph a eifolia
a n d H. pelta ta Con stitu en ts. Approximately 1000 kg of H.
was purified by a flash chromatography, HPLC, and
recrystallization sequence. Colorless crystals of each were
obtained, and their structures and absolute stereochemis-
try were determined by X-ray diffraction analyses.
The X-ray structure of the new, unusual benzopyran
product 14 is shown in Figure 1. Both inter- and intramo-
lecular H-bonding were observed between the hydroxyl
hydrogen and the carbonyl oxygen in the cyclohexadienone
ring in this compound. In contrast to the rather complex
and unique ring formation exemplified by 14, the cycliza-
tion of lactones 9 and 10 took a more normal course of
closure, forming the eight-membered rings of 15 and 16,
respectively, as shown in Figures 2 and 3.
nymphaeifolia was extracted (2×; 10, 5 days) using 1:1 CH
Cl -CH OH. After each extraction, 30% by volume of H O was
added to separate a CH Cl fraction, yielding 1329 and 316 g,
respectively. A 610 g aliquot of the first CH Cl phase was
2
-
2
3
2
2
2
All thirteen lignans (1-13) isolated from H. nymphaei-
folia and H. peltata and the three synthetic modifications
2
2
partitioned between CH OH-H O (9:1 f 1:1) and hexane (for
3
2
(14-16) were examined using the murine P388 lympho-
the 9:1 mix) followed by CH
2
Cl
2
(for the 1:1), which provided
Cl fraction. The
cytic leukemia cell line and a selection of human cancer
cell lines. Six lignans (2-5, 8, and 15) exhibited cancer cell
growth inhibitory activities ranging from potent to mar-
282 g of a (P388 ED50 0.034 µg/mL) CH
2
2
solvent partitioning sequence was a modification of the original
procedure of Bligh and Dyer.30 By applying the same partition-
ing procedure, 39.6 g of a CH
was partitioned between CH
2
Cl
OH-H
Cl
fraction (P388 ED50 0.022 µg/
2
-CH
O (9:1 f 3:2) and hexane
(for the 3:2), affording
3
OH extract of H. peltata
ginal (Table 2). Among the six lignans, deoxypodophyllo-
toxin (2), which is known as an anticancer agent,2
5-27
shows
3
2
(
for the 9:1 mix) followed by CH
.3 g of a P388 active CH Cl
mL).
2
2
the strongest activity in both P388 and human cancer lines,
while (+)-epiaschantin (4) shows activity only in the P388
leukemia cell line and podorhizol (8) is active in only two
types of human cell lines. The other 10 lignans were
inactive in both the P388 and human tumor cell lines.
Antimicrobial susceptibility testing was performed by the
7
2
2
Sep a r a tion of th e Meth ylen e Ch lor id e F r a ction fr om
H. n ym ph a eifolia . The CH Cl fraction was passed through
2
2
a Sephadex LH-20 column with CH
active fractions were obtained, and each was chromatographed
on a column of Sephadex LH-20 using CH OH-CH Cl (2:3)
3
OH as eluent. Three P388
reference broth microdilution assay.2
1
8,29
Lignans 1-3, 8,
3
2
2
as eluent. Again, three P388 active fractions (a, b, and c) were
obtained. Fractions a and b were combined and subjected to
separation on a Sephadex LH-20 column, using hexane-
1-13, and synthetic modification 14 inhibited growth of
the Gram-negative pathogen Neisseria gonorrhoeae (mini-
mum inhibitory concentrations 32-64 µg/mL). Lignan 9
was not available for antimicrobial evaluation.
toluene-CH
3
OH (3:1:1) as eluent, leading to three distinct
compounds and three P388 active fractions (d, e, and f). The
In conclusion, this detailed bioassay-directed investiga-
tion of H. nymphaeifolia and H. peltata has resulted in a
more definitive understanding of the lignan-type anticancer
constituents. In parallel, the structures of five lignans and
three oxidative cyclization products were established un-
equivocally by X-ray crystal structure determination. These
X-ray crystal structures will now provide a sound reference
point for structure determinations of related compounds
by spectral methods.
three nearly pure products were recrystallized (2×) from CH
3
-
OH. Three pure lignans, 2 (colorless needles, 1.98 g), 3
(colorless needles, 0.86 g), and 5 (colorless needles, 0.90 g),
were obtained. Fraction d was first separated on a Sephadex
LH-20 column, using 2-propanol-toluene-hexane (1:1:3) as
eluent, and then further separated on a silica gel column, using
2 2
CH Cl -EtOAc-hexane (3:1:1) as eluent. A pure colorless
semisolid (10) (0.39 g) and two other pure white solids were
obtained. Recrystallization (2×) of the two white solids from
3
CH OH resulted in 13 (colorless planar crystals, 14.5 mg) and
9
(colorless needles, 0.285 g). Fraction e was first separated
Exp er im en ta l Section
on a column of Sephadex LH-20, using 2-propanol-toluene-
hexane (1:1:3) as eluent, followed by separation on a silica gel
column, using EtOAc-hexane (1:1) as eluent. One pure lignan
(8) (colorless semisolid, 0.10 g) and another nearly pure
Gen er a l Exp er im en ta l Meth od s. All chromatographic
solvents were redistilled. Sephadex LH-20 used for partition
column chromatography was obtained from Pharmacia Fine

2
18 J ournal of Natural Products, 2004, Vol. 67, No. 2
Pettit et al.
fraction were obtained. The latter fraction was purified by
semipreparative HPLC using a SphereClone ODS(2) column
(1H, dd, J ) 8.4, 6.4 Hz, H-9b), 5.67 (1H, s, H-7′), 5.97 (1H, s,
H-5), 6.39 (1H, s, OH-3), 6.67 (1H, s, H-2′), 7.05 (1H, s, H-2),
and a gradient mobile phase (30% CH
3
CN in H
2
O for 60 min
3.88 (3H, s, OCH
3
-3′), 3.84 (3H, s, OCH
3 3
-4′), 3.78 (3H, s, OCH -
at a flow rate of 2.8 mL/min) to afford 4.0 mg of hernanol (1)
5′); 13C NMR (400 MHz in CDCl
3
) δ (ppm) 189.1 (C-4), 174.9
2
4
(
colorless semisolid): [R]
D
-36.2° (c 0.26, CH
3
OH); UV λmax
(C-6), 173.5 (C-9′), 153.6 (C-3′), 152.2 (C-5′), 143.6 (C-4′), 142.9
(C-3), 133.4 (C-1′), 120.1 (C-6′), 115.8 (C-2), 104.6 (C-5), 104.1
OH) (log ꢀ) 203 (5.0), 225 (4.5), 278 (3.8) nm; H and 13C
1
(CH
3
NMR (Table 1); HRAPCI MS (positive-ion mode) m/z 403.175
(C-2′), 75.0 (C-7′), 70.3 (C-9), 61.6 (OCH
3 3
-5′), 60.9 (OCH -4′),
+
[
M + H] (calcd for C22
H
26
O
7
, 402.4376).
56.2 (OCH -3′), 49.1 (C-8′), 45.5 (C-1), 40.3 (C-7), 39.3 (C-8);
3
The purification of fraction f, first on a Sephadex LH-20
HRAPCIMS (positive-ion mode) calcd for C21
20 8
H O 400.38787,
+
column, using 2-propanol-toluene-hexane (1:1:3) as eluent,
and then by analytical HPLC using a Zorbax C18 column with
found, m/z 401.1236 [M + 1] .
Cr ysta l Str u ctu r e of Ben zop yr a n 14. A large, colorless
block-shaped crystal, obtained via slow evaporation of a
methanol solution, with approximate dimensions of 0.96 × 0.64
× 0.48 mm, was mounted on the tip of a glass fiber. An initial
set of cell constants was calculated from reflections harvested
from three sets of 60 frames at 123(1) K on a Bruker 6000
diffractometer at -150 °C. Cell parameters indicated an
orthorhombic space group. A subsequent data collection, using
5 s scans/frame and 0.396° steps in ω, was conducted in such
a manner as to completely survey a complete sphere of
reflections. This resulted in >97.9% coverage of the total
reflections possible to a resolution of 0.87 Å. A total of 13 520
reflections were harvested from the total data collection, and
final cell constants were calculated from a set of 6377 reflec-
tions from these data. Subsequent statistical analysis of the
an isocratic mobile phase (41% CH
3 2
CN in H O at a flow rate
of 1 mL/min), yielded 11 (small colorless needles, 2.8 mg).
Fraction c was separated on a silica gel column, using CH
Cl
2
-
-
2
-EtOAc-hexane (3:1:1) as eluent, and one nearly pure
fraction was obtained which was recrystallized (2×) from CH
3
OH to provide 12 (small colorless needles, 10.8 mg).
Sep a r a tion of th e Meth ylen e Ch lor id e F r a ction fr om
H. pelta ta . A portion of the P388 active CH Cl fraction (0.14
g) was chromatographed on a column of Sephadex LH-20,
using CH OH as eluent. Again, three P388 active fractions (a,
b, and c) were selected for further separation. Fractions a and
b were both recrystallized (3×) from CH OH, to provide lignans
(colorless needles, 25 mg) and 5 (colorless needles, 9.8 mg).
Separation of the mother liquid from recrystallization of lignan
was first conducted on a Sephadex LH-20 column, using
toluene-CH OH-acetone-n-hexane (8:1:2:2) as eluent, and
then by semipreparative HPLC using a LUNA 5 µm C18
column with an isocratic mobile phase (36% CH CN in H
2
2
3
3
4
2
2
4
complete reflection data set using the XPREP program
20 8
indicated the space group was P2 . Crystal data: C21H O ,
3
1
2
1
2
1
a ) 7.3608(1) Å, b ) 11.0299(1) Å, c ) 22.7788(3) Å, V )
3
2
O
1849.39(4) Å, λ ) (Cu KR) ) 1.54178 Å, µ(Cu KR) ) 0.937
-
1
-3
at 2.8 mL/min), to give a pure compound and the nearly pure
fraction d. The pure compound was recrystallized from MeOH
mm , F
c
) 1.438 g cm for Z ) 4 and M ) 400.37, F(000) )
r
840. After data reduction, merging of equivalent reflections,
and rejection of systematic absences, 3365 unique reflections
remained (Rint ) 0.0191), of which 3328 were considered
(
2×) to afford colorless prism crystals of lignan 2 (51 mg).
Fraction d was further separated by semipreparative HPLC
again, using a LUNA 5 µm C18 column and an isocratic mobile
observed [I
o
o
> 2σ(I )] and were used in the subsequent
phase (37% CH
0 as a colorless semisolid (28 mg). The mother liquid from
lignan 5 was further separated on a Sephadex LH-20 column
using toluene-CH OH-hexane (5:1:1) as eluent and finally
by semipreparative HPLC using a SYNERGI 4 µm RP 80 A
column and an isocratic mobile phase (36% CH CN in H O at
3
CN in H
2
O at 2.8 mL/min), leading to lignan
structure solution and refinement. An absorption correction
was applied to the data with SADABS.31 Direct methods
structure determination and refinement were accomplished
with the SHELXTL NT ver. V6.1222 suite of programs. All non-
hydrogen atom coordinates for lactone 14 were located using
the default settings of that program. The remaining hydrogen
atom coordinates were calculated at optimum positions using
the program SHELXL.22 These latter atoms were assigned
thermal parameters equal to either 1.2 or 1.5 (depending upon
chemical type) of the Uiso value of the atom to which they were
attached, and then both coordinates and thermal values were
forced to ride that atom during the final cycles of refinement.
All non-hydrogen atoms were refined anisotropically in a full-
matrix least-squares refinement process. Both inter- and
intramolecular hydrogen bonding (O-9 hydroxyl hydrogen and
the O-10 carbonyl oxygen) was observed in the unit cell. The
absolute structure parameter (Flack x parameter) ) 0.0259
with an esd of 0.1130, indicating that the absolute structure
shown in Figure 1 represents the correct enantiomer. The final
1
3
3
2
a flow rate of 2.8 mL/min): the result was lignan 6 (colorless
needles, 3.1 mg). Fraction c was passed through a Sephadex
LH-20 column in CH OH and eluted in CH OH. A P388 active
3 3
fraction was obtained. Separation of this fraction by semi-
preparative HPLC using a LUNA 5 µm C18 column and an
isocratic mobile phase (40% CH CN in H O at a flow rate of
3 2
2
.8 mL/min) afforded three P388 active fractions (e, f, and g).
Fraction e was further separated by analytical HPLC using a
SYNERGI 4 µm RP 80 A column and isocratic mobile phase
3 2
(34% CH CN in H O at 1 mL/min). Pure lignan 8 (colorless
semisolid, 1.5 mg) was obtained. Fraction f was separated by
semipreparative HPLC (2×) (first on a SYNERGI 4 µm RP 80
3
A column with an isocratic mobile phase of 35% CH CN in
H
2
O at 2.8 mL/min and second on a Zorbax C18 column with
standard residual R
was 0.0268 (for observed data) and 0.0270 (for all data). The
corresponding Sheldrick R values were wR of 0.0697 and
1
value for the model shown in Figure 1
an isocratic mobile phase of 34% CH CN in H O at 4 mL/min).
3
2
Pure lignan 7 (colorless semisolid, 2.1 mg) was obtained. The
purification of fraction g by semipreparative HPLC using a
SYNERGI 4 µm RP 80 A column and an isocratic mobile phase
of 34% CH CN in H O at a flow rate of 2.8 mL/min afforded
3 2
lignan 3 (colorless needles, 5.2 mg).
2
0.0699, respectively. The goodness-of-fit factor was 1.052. The
difference Fourier map showed insignificant residual electron
density, the largest difference peak and hole being +0.209 and
3
-0.234 e/Å , respectively. The final bond distances and angles
Syn th esis of Ben zop yr a n 14. A solution of lignan 8 (0.103
g, 0.247 mmol in DCM 5 mL) was added dropwise to a solution
for the structural model, as shown in Figure 1, were all within
acceptable limits.
of VOF
3
(91.8 mg, 0.741 mmol) in 2:1 TFA-DCM (3 mL) and
Syn th esis of Isostega n e (15). The preceding reaction was
repeated using lignan 9 (0.28 g, 0.69 mmol in DCM, 15 mL)
TFA anhydride (0.1 mL) at -15 °C under argon. The reaction
mixture was stirred at -15 °C for 4 h and the reaction
and VOF
(15 mL) and TFA anhydride (0.4 mL)). Isolation of the product
(15) was performed using saturated NaHCO (35 mL) and
DCM (35 mL) followed by flash chromatography, using EtOAc-
CH Cl
3
(0.25 g, 1.97 mmol in a 2:1 mixture of TFA-DCM
3
terminated by addition of saturated NaHCO (12 mL) and
DCM (12 mL). The organic layer was separated, solvent
removed in vacuo, and the residue subjected to flash chroma-
tography, using EtOAc-CH
provide pyran 14 (43 mg, 42%). Recrystallization from CH
3
2
Cl
2
-hexane (1:1:3) as eluent, to
2
2
-hexane (1:1:3) as noted above (14) to give 40 mg of
3
-
isostegane (14% yield). Recrystallization (2×) from CH
3
OH
1
13
OH (2×) gave pure colorless planar crystals of pyran 14: mp
afforded colorless planar crystals: mp 169-171 °C; H and
C
2
4
1
32
2
18 °C (dec); [R]
D
+38.6° (c 0.22, CH
3
OH); H NMR (400 MHz
NMR data were the same as already published; EIMS calcd
+
in CDCl
3
) δ (ppm) 2.13 (1H, t, J ) 12.8 Hz, H-7a), 2.24 (1H,
22 7
for C22H O , 398.41, found m/z 398 [M] .
dd, J ) 12.8, 4.8 Hz, H-7b), 2.45 (1H, d, J ) 14.4 Hz, H-8′),
Cr yst a l St r u ct u r e of Isost ega n e 15. A large, colorless
plate, obtained via slow evaporation of a methanol solution,
2
.97 (1H, m, H-8), 3.82 (1H, dd, J ) 11.2, 8.4 Hz, H-9a), 4.36

Antineoplastic Agents. 522
J ournal of Natural Products, 2004, Vol. 67, No. 2 219
indicated the space group was P2 . Crystal data: C21 , a
with approximate dimensions of 0.6 × 0.64 × 0.16 mm, was
mounted on the tip of a glass fiber. Data collection, using 5 s
scans/frame and 0.396° steps in ω, was conducted at 123(1) K
on a Bruker 6000 diffractometer at -150 °C. Cell parameters
indicated a monoclinic space group. A total of 7003 reflections
were harvested from the total data collection, and final cell
constants were calculated from a set of 6682 reflections from
these data. Subsequent statistical analysis of the complete
1
20 8
H O
) 11.1061(2) Å, b ) 16.1220(2) Å, c ) 11.3073(2) Å, V )
3
1824.64(5) Å , λ ) (Cu KR) ) 1.54178 Å, µ(Cu KR) ) 0.860
-
1
-3
mm , F
c
) 1.399 g cm for Z ) 4 and M ) 384.39, F(000) )
r
812. After data reduction, merging of equivalent reflections,
and rejection of systematic absences, 6178 unique reflections
remained (Rint ) 0.0284), of which 5903 were considered
observed [I
o
o
> 2σ(I )] and were used in the subsequent
2
2
reflection data set using the XPREP program indicated that
the space group was P2
structure solution and refinement. An absorption correction
was applied to the data with SADABS.31 Direct methods
structure determination and refinement were accomplished
1
. Crystal data:
C
22
H
22
O
7
, a
)
1
1.6304(2) Å, b ) 6.85900(10) Å, c ) 12.5499(2) Å, V ) 954.68-
-
1
22
(
1
3) Å, λ ) (Cu KR) ) 1.54178 Å, µ(Cu KR) ) 0.863 mm , F
c
)
with the SHELXTL NT ver. V6.12 suite of programs. All non-
-
3
.386 g cm for Z ) 2 and M
r
) 398.40, F(000) ) 420. After
hydrogen atom coordinates for lactone 16 were located using
the default settings of that program. The remaining hydrogen
atom coordinates were calculated at optimum positions using
the program SHELXL.22 These latter atoms were assigned
thermal parameters equal to either 1.2 or 1.5 (depending upon
chemical type) of the Uiso value of the atom to which they were
attached, and then both coordinates and thermal values were
forced to ride that atom during the final cycles of refinement.
All non-hydrogen atoms were refined anisotropically in a full-
matrix least-squares refinement process. The final standard
data reduction, 2815 reflections were considered observed [I
2σ(I )] and were used in the subsequent structure solution
o
>
o
and refinement. An absorption correction was applied to the
data with SADABS.31 Direct methods structure determination
and refinement were accomplished with the SHELXTL NT ver.
2
2
V6.12 suite of programs. All non-hydrogen atom coordinates
for isostegane (15) were located using the default settings of
that program. The remaining hydrogen atom coordinates were
calculated at optimum positions using the program SHELXL.22
These latter atoms were assigned thermal parameters equal
to either 1.2 or 1.5 (depending upon chemical type) of the Uiso
value of the atom to which they were attached, then both
coordinates and thermal values were forced to ride that atom
during the final cycles of refinement. All non-hydrogen atoms
were refined anisotropically in a full-matrix least-squares
refinement process. The absolute structure parameter (Flack
x parameter) was also refined to a value of 0.0900 with an esd
of 0.1600, indicating that the absolute structure shown in
Figure 2 represents the correct enantiomer. The final standard
residual R
(for observed data) and 0.0477 (for all data). The corresponding
Sheldrick R values were wR of 0.1335 and 0.1350, respec-
1
value for the model shown in Figure 3 was 0.0466
2
tively, and the goodness-of-fit factor was 1.052. The absolute
structure parameter (Flack x parameter) ) 0.1407 with esd
of 0.1360. The difference Fourier map again showed insignifi-
cant residual electron density, the largest difference peak and
3
hole being +0.286 and -0.554 e/Å , respectively. The final bond
distances and angles for the structural model, as shown in
Figure 3, were all within acceptable limits.
residual R
for observed data) and 0.0355 (for all data). The corresponding
Sheldrick R values were wR of 0.0941 and 0.0943, respec-
1
value for the model shown in Figure 2 was 0.0353
Ca n cer Cell Lin e Bioa ssa y P r oced u r es. Inhibition of
human cancer cell growth was assessed using the National
Cancer Institute’s standard sulforhodamine B assay as previ-
(
2
tively. The goodness-of-fit factor was 1.043. The difference
Fourier map showed insignificant residual electron density,
the largest difference peak and hole being +0.179 and -0.240
33
ously described. Briefly, cells in a 5% fetal bovine serum/
RPMI1640 medium solution were inoculated in 96-well plates
and incubated for 24 h. Serial dilutions of the compounds were
then added. After 48 h, the plates were fixed with trichloro-
acetic acid, stained with sulforhodamine B, and read with an
automated microplate reader. A growth inhibition of 50% (GI50
or the drug concentration causing a 50% reduction in the net
protein increase) was calculated from optical density data with
Immunosoft software. Mouse leukemia P388 cells were incu-
bated in a 10% horse serum/Fisher medium solution for 24 h
followed by a 48 h incubation with serial dilutions of the
3
e/Å , respectively. The final bond distances and angles for the
structural model, as shown in Figure 2, were all within
acceptable limits.
Syn th esis of Deoxyisosta ga n on e 16. When the above (14,
1
5) oxidative cyclization reaction was applied to lignan 10 (0.39
g, 1.06 mmol in DCM, 20 mL) and VOF (0.40 g, 3.19 mmol in
:1 TFA-DCM (20 mL) and TFA anhydride (0.5 mL)), followed
by the preceding (15) isolation procedure, 82 mg (20%) of
lactone 16 was obtained. Two recrystallizations from CH OH
yielded colorless planar crystals: mp 155-157 °C; [R]
3
2
3
compounds. Cell growth inhibition (ED50) was then calculated
using a Z1 Beckman/Coulter particle counter.
2
4
D
1
+
3 3
160.3° (c 0.26, CH OH); H NMR (300 MHz in CDCl ) δ (ppm)
An tim icr obia l Su scep tibility Testin g. Lignans were
evaluated against the bacteria Stenotrophomonas maltophilia
ATCC 13637, Micrococcus luteus Presque Isle 456, Staphylo-
coccus aureus ATCC 29213, Escherichia coli ATCC 25922,
Enterobacter cloacae ATCC 13047, Enterococcus faecalis ATCC
4
8
2
.36 (1H, dd, J ) 8.1, 6.0 Hz, H-9a), 3.77 (1H, dd, J ) 11.0,
.1 Hz, H-9b), 2.20 (1H, m, H-8), 2.38 (1H, d, J ) 8.7 Hz, H-7a),
.60 (1H, d, J ) 13.2 Hz, H-7b), 2.15 (1H, m, H-8′), 3.14 (1H,
d, J ) 13.2 Hz, H-7′a), 2.28 (1H, d, J ) 8.7 Hz, H-7′b), 5.96
2H, d, J ) 4.5 Hz, OCH O), 6.77 (1H, s H-2′), 6.66 (1H, s,
H-2), 6.65 (1H, s, H-5), 6.64 (1H, s, H-5′), 3.84 (1H, s, OCH
(
2
2
9212, Streptococcus pneumoniae ATCC 6303, and Neisseria
3
-
gonorrhoeae ATCC 49226, and the fungi Candida albicans
ATCC 90028 and Cryptococcus neoformans ATCC 90112,
1
3
4
3 3
′), 3.90 (1H, s, OCH -3′); C NMR (400 MHz in CDCl ) δ
ppm) 176.6 (C-9′), 148.9 (C-3′), 147.5 (C-4), 147.2 (C-3), 146.1
C-4′), 132.1 (C-1′), 132.3 (C-6′), 132.4 (C-6), 133.5 (C-1), 113.9
O),
-4′), 50.0 (C-8′), 47.0 (C-
), 34.3 (C-7), 32.1 (C-7′); HRAPCI MS (positive-ion mode) calcd
368.3799, found m/z 369.
(
(
(
7
8
following established broth microdilution susceptibility as-
28,29
says.
The minimum inhibitory concentration was defined
C-5′), 111.7 (C-2′), 111.1 (C-5), 108.8 (C-2), 101.3 (OCH
0.1 (C-9), 56.1 (OCH -3′), 56.1 (OCH
3 3
2
as the lowest concentration of compound that inhibited all
visible growth of the test organism (optically clear). Assays
were repeated on separate days.
for C21
20 6
H O
Cr ysta l Str u ctu r e of Deoxyisostega n on e 16. A colorless,
block-shaped crystal, obtained via slow evaporation of a
methanol solution, with approximate dimensions of 0.48 × 0.48
Ack n ow led gm en t. The very necessary financial assis-
tance was provided by Outstanding Investigator Grant CA-
4344-01A1-12, and Grant ROI CA-16049-01-2 from the
4
×
0.32 mm, was mounted on the tip of a glass fiber. Data
Division of Cancer Treatment and Diagnosis, NCI, DHHS; the
Arizona Disease Control Research Commission; the Fannie E.
Rippel Foundation; Gary L. and Diane Tooker; the Caitlin
Robb Foundation; Polly J . Trautman; Dr. J ohn C. Budzinski;
Sally Schloegel; the Eagles Art Ehrmann Cancer Fund; the
Ladies Auxiliary to the Veterans of Foreign Wars; and the
Robert B. Dalton Endowment Fund. Very helpful technical
assistance was contributed by Drs. J . C. Knight, L. R. Lan-
drum, D. H. Lorence, B. Orr, and J . M. Schmidt, as well as by
collection at -150 °C [123(1) K], using 5 s scans/frame and
0
.396° steps in ω, was performed such that a complete sphere
of reflections was surveyed. This resulted in >96.6% coverage
of the total reflections possible to a resolution of 0.87 Å. Cell
parameters indicated a monoclinic space group. A total of
1
3 248 reflections were harvested from the total data collection,
and final cell constants were calculated from a set of 10 422
reflections from these data. Subsequent statistical analysis of
2
2
the complete reflection data set using the XPREP program

2
20 J ournal of Natural Products, 2004, Vol. 67, No. 2
Pettit et al.
D. M. Carnell, F. Craciunescu, M. Dodson, L. C. Garner, C. R.
Reimus, D. Nielsen Tackett, C. Weber, and L. Williams. We
also wish to thank the Governments of the Republic of
Maldives (M. H. Maniku, A. Naseer, and M. Shiham) and
Malaysia (S. Bin Haji Abdul Majid, M. Khan Bin Momin Khan,
and R. Mohamad Noordin Raja Omar) for other helpful
assistance.
(15) Mikaya, G. A.; Turabelidze, D. G.; Kemertelidze, E. P.; Zaikin, V. G.;
Vul’fson, N. S. Bioorg. Khim. 1980, 6, 1415-1419.
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(
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M. Chem. Pharm. Bull. 1988, 36, 3239-3247.
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(
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303-1312.
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1
(
(
(
Su p p or tin g In for m a tion Ava ila ble: Tables of X-ray crystal-
lographic data for compounds 2, 4-6, and 13-16. This material is
available free of charge via the Internet at http://pubs.acs.org.
2
078-2082.
21) Nishibe, S.; Fujimoto, T.; Nose, M.; Takada, T.; Ogihara, Y.; Xu, G.
Phytochemistry 1993, 32, 1579-1581.
22) SHELXTL-NT-Version 6.12, an integrated suite of programs for the
determination of crystal structures from diffraction data; Bruker AXS,
Inc.: Madison, WI, 2001. This package includes, among others,
XPREP (an automatic space group determination program), SHELXS
Refer en ces a n d Notes
(
1) For the preceding contribution, see: Pettit, R. K.; Fakoury B. R.;
Knight, J . C.; Pettit, G. R.; Pon, S. Antimicrob. Agents Chemother.,
submitted.
(
a structure solution program via Patterson or direct methods), and
SHELXL (structure refinement software).
(2) (a) Hartwell, J . L. Plants Used Against Cancer; Quarterman Publica-
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(
23) Tables of X-ray crystallographic data (98 pages) for compounds 2,
4
-6, and 13-16 are available free of charge via the Internet at http://
1
942, 63, 1540-1544. (c) Yamaguchi, H.; Nakajima, S.; Arimoto, M.;
pubs.acs.org. This crystallographic data has also been deposited
with the Cambridge Data Centre. Copies of the data can be obtained,
free of charge, on application to the Director, CCDC, 12 Union
Rd., Cambridge CB2 1EZ, UK [fax: +44(0)1223-336033 or e-mail:
deposit@ccdc.cam.ac.uk].
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