Potential anticancer activity of naturally occurring and semisynthetic derivatives of aculeatins A and B from Amomum aculeatum

Article abstract of DOI:10.1021/np070584j

Activity-guided fractionation of hexanes- and CHCl₃-soluble extracts of Amomum aculeatum leaves, collected in Indonesia, led to the isolation of three new dioxadispiroketal-type (3-5) and two new oxaspiroketal-type (6 and 7) derivatives. Nine semisynthetic derivatives (1a-1h and 2a) of the parent compounds, aculeatins A (1) and B (2), were prepared. All isolates and semisynthetic compounds were tested against a small panel of human cell lines. Of these, aculeatin A (1; ED₅₀ 0.2-1.0 μM) was found to be among the most cytotoxic of the compounds tested and was further evaluated in an in vivo hollow fiber assay; it was found to be active against MCF-7 (human breast cancer) cells implanted intraperitoneally at doses of 6.25, 12.5, 25, and 50 mg/kg. However, when 1 was tested using P388 lymphocytic leukemia and human A2780 ovarian carcinoma in vivo models, it was deemed to be inactive at the doses used.

Full text of DOI:10.1021/np070584j

390
J. Nat. Prod. 2008, 71, 390–395
Potential Anticancer Activity of Naturally Occurring and Semisynthetic Derivatives of
Aculeatins A and B from Amomum aculeatum#
Young-Won Chin,† Angela A. Salim,† Bao-Ning Su,† Qiuwen Mi,‡ Hee-Byung Chai,† Soedarsono Riswan,§
Leonardus B. S. Kardono, Agus Ruskandi,§ Norman R. Farnsworth,‡ Steven M. Swanson,‡ and A. Douglas Kinghorn*,†
DiVision of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State UniVersity, Columbus, Ohio 43210, Program for
CollaboratiVe Research in the Pharmaceutical Sciences and Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy,
UniVersity of Illinois at Chicago, Chicago, Illinois 60612, Herbarium Bogoriense, Research Center for Biology, Indonesian Institute of Science,
Bogor 16122, Indonesia, and Research Center for Chemistry, Indonesian Institute of Science, Tangerang 15310, Indonesia
ReceiVed October 17, 2007
Activity-guided fractionation of hexanes- and CHCl3-soluble extracts of Amomum aculeatum leaves, collected in Indonesia,
led to the isolation of three new dioxadispiroketal-type (3–5) and two new oxaspiroketal-type (6 and 7) derivatives.
Nine semisynthetic derivatives (1a-1h and 2a) of the parent compounds, aculeatins A (1) and B (2), were prepared.
All isolates and semisynthetic compounds were tested against a small panel of human cell lines. Of these, aculeatin A
(1; ED50 0.2–1.0 µM) was found to be among the most cytotoxic of the compounds tested and was further evaluated in
an in vivo hollow fiber assay; it was found to be active against MCF-7 (human breast cancer) cells implanted
intraperitoneally at doses of 6.25, 12.5, 25, and 50 mg/kg. However, when 1 was tested using P388 lymphocytic leukemia
and human A2780 ovarian carcinoma in vivo models, it was deemed to be inactive at the doses used.
Amomum aculeatum Roxb. (Zingiberaceae) is a herbaceous plant,
distributed in Indonesia, Malaysia, and Papua New Guinea, and
has been used as a folk medicine for the treatment of fever and
malaria.1 In initial phytochemical work on this species, the 1,7-diox-
adispiro[5.1.5.2]pentadecane-type compounds, aculeatins A-D,
were characterized and found to exhibit antibacterial, antiprotozoal,
and cytotoxic activities.2,3 Of these compounds, aculeatins A, B,
and D have been chemically synthesized due to their unusual
structures and interesting bioactivities, and through enantioselective
synthetic methods, the absolute configurations of these three
substances were resolved definitively.4–9
In a preliminary short report, we have described the isolation,
structural characterization, and cytotoxicity testing of aculeatols
A-D, four C-9-hydroxylated analogues, from the leaves of A.
aculeatum.10 Aculeatols A-D are of structural and spectroscopic
interest since they contain five stereocenters, as opposed to only
three in the parent compounds, aculeatins A (1) and B (2), which
By bioactivity-guided fractionation of the hexanes- and chloroform-
were also present in the plant samples investigated.10
Results and Discussion
soluble extract of A. aculeatum leaves, using MCF-7 (human breast
cancer)cellsasamonitor,threenew1,7-dioxadispiro[5.1.5.2]pentadecane-
type compounds (3-5) and two new 1-oxaspiro[4,5]decane-type
compounds (6 and 7) were isolated and characterized.
In the present study, as part of a collaborative approach to the
discovery of new anticancer agents of plant origin,11 we have delved
more fully into the potential as cancer chemotherapeutic agents of
compounds based on the unusual 1,7-dioxadispiro[5.1.5.2]pentadecane
Compound 3 was isolated as a yellow oil and displayed a sodiated
chemotype. Five new constituents (3-7) of A. aculeatum have been
isolated and characterized and are reported herein. Since aculeatins
A (1) and B (2) were isolated in reasonably high yields, these
compounds have been derivatized to produce compounds 1a-1h
and 2a, respectively, in an attempt to generate biologically potent
analogues. Isolates (3-7) and derivatives (1a-1h and 2a) were
then evaluated against a small panel of human cancer cell lines. In
addition, follow-up testing has been performed on aculeatin A (1)
in an in vivo hollow fiber assay and also in P388 lymphocytic
murine leukemia and human A2780 ovarian carcinoma in vivo
models.
molecular ion peak at m/z 413.2668 in the HRESIMS, corresponding
to an elemental formula of C24H38O4Na (calcd for C24H38O4Na,
413.2668). The 1H NMR spectrum of 3 was closely comparable to
that of aculeatin B (2),2,10 suggesting the presence of a 1,7-
dioxadispiro[5.1.5.2]pentadeca-9,12-dien-11-one unit from signals
in the range δH 1.28 to 6.99 and a long-chain aliphatic group from
resonances between δH 0.88 and 1.47 (Table 1). However, the
signals of 3 at δH 3.85 (1H, tt, J ) 11.2, 4.4 Hz, H-4) and 3.36
(1H, m, H-2) appeared in a more upfield region when compared
with analogous signals of aculeatin B [H-4 (δH 4.35) and H-2 (δH
3.86)]. These differences suggested that compound 3 has a different
configuration at the C-4 position from aculeatin B (2). The coupling
constants of 11.2 and 4.4 Hz for H-4 implied an axial position of
this proton in the cyclohexane ring system, as opposed to the
equatorial orientation in 2 (3.0 Hz, H-4).10 From the 2D NOESY
data of 3, it was found that H-4 exhibited NOE correlations with
H-2 and H-15b (Figure 1), which showed the same orientation for
both H-2 and H-4, and was used to establish the R and S
configurations of C-2 and C-4, respectively. The configuration of
# Dedicated to Dr. G. Robert Pettit for his pioneering work on bioactive
natural products.
* To whom correspondence should be addressed. Tel: +1 614 247 8094.
Fax: +1 614 247 8081. E-mail: kinghorn.4@osu.edu.
† The Ohio State University.
‡ University of Illinois at Chicago.
§ Herbarium Bogoriense.
Research Center for Chemistry.
10.1021/np070584j CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 02/09/2008

DeriVatiVes of Aculeatins A and B from Amomum aculeatum
Journal of Natural Products, 2008, Vol. 71, No. 3 391
aculeatol E (5)
Table 1. 1H and 13C NMR Spectroscopic Data for Compounds 3 and 5
aculeatin E (3)
position
δC, mult.a
δH (J in Hz)
3.36, m
1.96, m
δC, mult.a
δH (J in Hz)
3.91, m
1.61, m
2
3
71.6, CH
40.8, CH2
69.7, CH
37.8, CH2
1.23, m
1.51, m
4
5
66.8, CH
43.5, CH2
3.85, tt (11.2, 4.4)
2.12, m
1.78, m
65.3, CH
40.3, CH2
4.38, m
2.03, dd (13.9, 3.1)
1.86, m
6
8
109.2, qC
78.0, qC
106.7, qC
81.9, qC
9
148.9, CH
127.1, CH
185.5, qC
127.3, CH
151.6, CH
34.9, CH2
6.79, dd (10.0, 2.9)
6.13, dd (10.0, 1.8)
69.7, CH
44.2, CH2
198.6, qC
129.6, CH
148.0, CH
31.7, CH2
3.93, dd (8.7, 5.4)
2.73, m
10
11
12
13
14
6.14, dd (10.4, 1.8)
6.99, dd (10.4, 2.9)
2.28, m
5.99, d (10.0)
6.61, d (10.0)
2.52, dd (11.7, 8.2)
1.86, m
2.06, m
15
33.3, CH2
2.39, m
1.83, m
34.6, CH2
2.65, dd (12.2, 8.2)
1.87, m
16
17
35.7, CH2
25.8, CH2
1.62, m, 1.52, m
1.47, m
35.7, CH2
25.5, CH2
1.57, m 1.44, m
1.44, m
1.31, m
1.30, m
18–25
26
27
29.3–29.6, CH2
31.9, CH2
22.6, CH2
1.26–1.30, m
1.28, m
1.28, m
29.4–29.7, CH2
31.9, CH2
22.7, CH2
1.23–1.30, m
1.23–1.30, m
1.26, m
28
14.1, CH3
0.88, t (6.6)
14.1, CH3
0.89, t (7.0)
a Multiplicity was deduced from the DEPT and HSQC spectroscopic data.
Figure 1. Key NOESY correlations of 3.
the C-6 position was confirmed as S by the observation of a NOE
correlation between H-2 and H-15a. When all of the spectroscopic
data were taken into account, the structure of compound 3 was
determined as 2R*,4S*,6S*-4-hydroxy-2-undecyl-1,7-dioxadis-
piro[5.1.5.2]pentadeca-9,12-dien-11-one, and this isolate has been
named aculeatin E.
The molecular formula of compound 4 was assigned as C24H38O4
and was inferred from a sodiated molecular ion peak at m/z
413.2672 (calcd for C24H38O4Na, 413.2668) in the HRESIMS. The
1H NMR spectrum of 4 disclosed resonances at δH 6.86 (1H, dd, J
) 10.0, 2.9 Hz, H-13), 6.77 (1H, dd, J ) 9.9, 2.9 Hz, H-9), 6.15
(1H, dd, J ) 10.0, 1.8 Hz, H-12), 6.11 (1H, dd, J ) 9.9, 1.8 Hz,
H-10), 4.13 (2H, m, H-2 and H-4), 2.37 (1H, m, H-14a), 2.24 (1H,
m, H-15a), 2.00 (3H, m, H-5a, H-14b, and H-15b), 1.94 (1H, m,
H-5b), 1.80 (1H, brd, J ) 13.1 Hz, H-3a), 1.50 (2H, m, H-16),
1.43 (1H, m, H-17a), and 1.41 (1H, dd, J ) 13.1, 2.6 Hz, H-3b),
assignable to the same dioxadispiroketal-type skeleton as aculeatin
2
A (1). Comparison of the H and 13C NMR chemical shifts of 4
with those of aculeatin A indicated that both compounds have
identical carbon skeletons. From detailed NMR assignments, the
relative configurations at the chiral carbons in 4 were confirmed as
being the same as aculeatin A (1) by the observation of correlations
in several 2D NMR (1H-1H COSY, HSQC, HMBC, and NOESY)
experiments. The only difference was observed in the respective
molecular formula of these substances. As shown by the MS data,
compound 4 has two less methylene units in the aliphatic side chain
when compared with aculeatin A (C26H42O4). Thus, the structure
of 4 (aculeatin F) was determined as 2R*,4R*,6R*-4-hydroxy-2-
undecyl-1,7-dioxadispiro[5.1.5.2]pentadec-9,12-dien-11-one.
1
The 1H NMR spectroscopic data (Table 1) of compound 5
exhibited three oxymethine signals at δH 4.38 (1H, m, H-4), 3.93

392 Journal of Natural Products, 2008, Vol. 71, No. 3
Chin et al.
Table 2. 1H and 13C NMR Spectroscopic Data for Compounds 6 and 7
amomol A (6)
amomol B (7)
a
position
δC, mult.a
δH (J in Hz)
δC
δH (J in Hz)
2
3
4
111.6, qC
36.6, CH2
34.9, CH2
111.5
36.6
35.1
2.34, m 2.15, m
2.39, m
2.07, m
2.24, m
2.43, m
2.07, m
5
79.1, qC
78.7
6
7
8
9
10
11
148.4, CH
127.1, CH
185.2, qC
127.7, CH
150.6, CH
40.7, CH2
6.78, d (10.0, 2.7)
6.13, d (10.0)
148.2
6.81, d (10.0)
6.14, d (10.0)
.
6.17, d (10.5)
6.85, d (10.5)
1.96, m
127.4b
185.3
127.5b
150.6
40.1
6.17, d (10.0)
6.85, d (10.0, 2.7)
2.13, m
1.70, brd (14.7)
3.84, m
1.54, m
1.96, m
3.79, m
1.53, m
12
13
69.1, CH
37.8, CH2
68.2
37.8
1.43, m
1.43, m
14
25.4, CH2
1.43, m,
25.5
1.43, m,
1.33, m
1.33, m
15–24
25
29.6–29.7, CH2
31.9, CH2
1.26–1.33, m
1.26, m
29.6–29.7
31.9
1.26–1.33, m
1.26, m
26
22.7, CH2
1.30, m
22.7
1.30, m
27
OCH3
14.1, CH3
48.9, CH3
0.88, t (6.8)
3.35, s
14.1
48.5
0.89, t (6.7)
3.35, s
a Multiplicity was deduced from the DEPT and HSQC spectroscopic data. b Assignments are interchangeable.
(1H, dd, J ) 8.7, 5.4 Hz, H-9), and 3.91 (1H, m, H-2), two olefinic
proton signals at δH 6.61 (1H, d, J ) 10.0 Hz, H-13) and 5.99
(1H, d, J ) 10.0 Hz, H-12), and one methylene signal at δH 2.73
(2H, m, H-10), attributable to an aculeatol-type structure.10 In
addition, it was found that the 13C NMR spectroscopic data of 5
were generally comparable to those of aculeatol D.10 The 13C NMR
chemical shifts at δC 69.7 (C-2) and 34.6 (C-15) were supportive
of the configuration of C-6 as being S.10 The coupling constants of
H-9 (8.7 and 5.4 Hz) and H-5a (13.9 and 3.1 Hz), and NOE
correlations between H-2/H-15a, H-9/H-14a, and H-13/H-14b in
the 2D NOESY spectrum of 5, suggested that the relative
configurations at the other chiral centers in this structure are the
same as those of aculeatol D.10 The distinction between compound
5 and aculeatol D is in the length of the alkyl side chain at C-2, as
shown in the molecular formula of C26H44O5 for 5 compared with
C24H40O5 for aculeatol D. Therefore, compound 5 (aculeatol E) was
elucidated as 2R*,4R*,6S*,8R*,9S*-4,9-dihydroxy-2-tridecyl-1,7-
dioxadispiro[5.1.5.2]pentadec-12-en-11-one.
The HRESIMS of 6 provided a sodiated molecular ion peak at
m/z 457.3294 [M + Na]+, suggesting C27H46O4 to be its molecular
formula. The 1H NMR spectrum of compound 6 exhibited
resonances at δH 6.85 (1H, dd, J ) 10.0, 2.7 Hz, H-10), 6.78 (1H,
dd, J ) 10.0, 2.7 Hz, H-6), 6.17 (1H, d, J ) 10.0 Hz, H-9), 6.13
(1H, d, J ) 10.0 Hz, H-7), 2.39 (1H, m, H-4a), 2.34 (1H, m, H-3a),
2.15 (1H, m, H-3b), and 2.07 (1H, m, H-4b), due to the presence
of an oxaspiro[4,5]deca-6,9-dien-8-one unit, which has not been
described in the literature to date (Table 2). From the remaining
proton signals, only one oxymethine proton at δH 3.84 was observed
when compared with two oxymethines present in aculeatin A (1),
suggesting that the cyclohexane ring of a dioxadispiroketal-type
compound has been cleaved in compound 6. This assumption was
supported by the observed HMBC correlation between the methoxy
protons at δH 3.32 and the spiro carbon at δC 111.6 (C-2), instead
of a correlation between H-14 and C-2 as exhibited by aculeatin-
type compounds.2,10 The proton resonance signals at δH 2.39 and
2.07 (H-4) exhibited three-bond correlations with C-6 and C-10,
respectively, which enabled the H-3 and H-4 signals to be assigned
unambiguously. The preparation of Mosher ester derivatives of 6
was used to provide the absolute configuration of C-12 as R, based
on the differences ∆δR-S +0.081 (H11a), +0.049 (H-11b), and
-0.014 (H-13a) between the (R)-MTPA and (S)-MTPA esters of
6.12–15 Although the NOESY correlations of δH 6.82 (H-10) to 2.36
(H-4a) and 3.32 (OMe) and δH 6.75 (H-6) to 2.04 (H-4b) and 1.67
(H-11b) were observed, those correlations were not sufficient to
demonstrate the relative configuration at the C-2 position. Thus,
compound 6 (amomol A) was determined structurally as 2ꢀ,12R-
2-(2-hydroxyheptadecyl)-2-methoxy-1-oxaspiro[4.5]deca-6,9-dien-
8-one.
Compound 7 gave the same molecular formula (C27H46O4) as
compound 6, and these compounds showed a close resemblance in
their 1H and 13C NMR spectroscopic data (Table 2). Furthermore,
by analysis of the 2D NMR (1H-1H COSY, HSQC, HMBC, and
NOESY) spectroscopic data, it was evident that the relative
configuration of the 2-methoxy-1-oxaspiro[4.5]deca-6,9-dien-8-one
unit of compound 7 was identical to that of 6. On preparing (R)-
and (S)-MTPA esters of compound 7, the absolute configuration
of C-12 in the alkyl side chain was established as R, the same
configuration as in compound 6. Since these two similar compounds
were able to be separated on a nonchiral HPLC column and their
optical rotations are of the opposite signs, the configuration of C-2
in compounds 6 and 7 was inferred as being opposite, suggesting
that these compounds are isomers. Therefore, the structure of 7
(amomol B) was also designated as 2ꢀ,12R-2-(2-hydroxyheptade-
cyl)-2-methoxy-1-oxaspiro[4.5]deca-6,9-dien-8-one.
All isolated compounds were evaluated for their cytotoxic activity
using a small panel of human cancer cell lines, and their cytotoxic
activities are summarized in Table 3.16,17 It was found that the five
new compounds 3–7 were cytotoxic against the human cancer cell
lines in which they were evaluated and comparable in potency to
the parent compounds, aculeatins A (1) and B (2). Since aculeatin
A (1) was isolated in a large quantity in the present study and
exhibited potent cytotoxic activity, modifications in the structure
of this parent compound were performed in an attempt to develop
more active analogues. Eight semisynthetic compounds from
aculeatin A, 1a-1h, and aculeatin B acetate, 2a, were synthesized.
Modification of the free hydroxy group at the C-4 position in
aculeatin A (1) resulted in decreased cytotoxic activities for these
compounds, as shown in Table 3. Likewise, the same cytotoxic
trend was evident for aculeatin B (2) even though only one analogue
(2a) was tested in the present study. Thus, a free hydroxy group at
the C-4 position in aculeatins A (1) and B (2) seems to be necessary
for the elicitation of potent cytotoxic activity within this compound
group.
Aculeatin A (1) was chosen for follow-up evaluation in an in
vivo hollow fiber assay18–21 due to its cytotoxicity in the human
cancer cell panel and its availability in a reasonably large quantity.

DeriVatiVes of Aculeatins A and B from Amomum aculeatum
Journal of Natural Products, 2008, Vol. 71, No. 3 393
Table 3. Cytotoxicity Data of Compounds 1, 1a-1h, 2, 2a,
and 3-7
only be investigated further as potential anticancer agents as low-
priority leads. However, owing to the cytotoxic potency of aculeatin
A (1) against the MCF-7 cell line and its efficacy against MCF-7
cells in hollow fibers, it may be worth performing a xenograft study
on 1 with this same model.
cell linea,b
compound
Lu1
LNCaP
MCF-7
1
1.0
2.6
3.4
0.5
1.1
1.9
3.0
17.9
5.1
5.8
0.8
22.1
1.2
2.2
1.8
0.7
4.4
0.7
0.9
0. 2
1.5
1.3
3.2
14.6
4.7
4.8
0.7
7.5
1.9
3.5
0.5
0.5
3.9
0.9
0.7
1a
1b
1c
1d
1e
1f
1g
1h
2
2a
3
4
5
6
Experimental Section
General Experimental Procedures. Optical rotations were mea-
sured using a Perkin-Elmer 241 automatic polarimeter. UV spectra were
obtained with a Perkin-Elmer UV/vis lambda 10 spectrometer. IR
spectra were run on a Nicolet Protégé 460 FT-IR spectrophotometer.
NMR spectroscopic data were recorded at room temperature using a
Bruker DRX-400 spectrometer. Chemical shifts were reported as ppm
with reference to the residual solvent or tetramethylsilane (TMS).
Electrospray ionization (ESI) mass spectrometric analyses were obtained
on a 3-T Finnigan FTMS-2000 Fourier transform mass spectrometer.
A SunFire PrepC18OBD column (5 µm, 150 × 19 mm i.d., Waters,
Milford, MA) and a SunFire PrepC18 guard column (5 µm, 10 × 19
mm i.d., Waters) were used for preparative HPLC, along with two
Waters 515 HPLC pumps and a Waters 2487 dual λ absorbance detector
(Waters). Column chromatography was carried out with Purasil
(230–400 mesh, Whatman, Clifton, NJ) and Sephadex LH-20 (Sigma,
St. Louis, MO). Analytical thin-layer chromatography (TLC) was
performed on precoated 250 µm thickness Partisil K6F (Whatman) glass
plates, while preparative thin-layer chromatography was conducted on
precoated 20 × 20 cm, 500 µm Partisil K6F (Whatman) glass plates.
Semisynthetic reagents were purchased from Sigma (St. Louis, MO).
All solvents used for chromatographic separations were purchased from
Fisher Scientific (Fair Lawn, NJ).
4.0
22.2
10.3
12.8
1.3
30.5
3.1
5.7
1.8
1.0
1.8
0.9
0.5
7
a Results are expressed as ED50 values (µM), and values < 10 µM
are considered to be active. b Key to cell lines used: Lu1 (human lung
carcinoma); LNCaP (hormone-dependent human prostate carcinoma);
MCF-7 (human breast carcinoma).
Plant Material. The leaves of A. aculeatum Roxb. were collected
at Gunung Kancana, West Java, Indonesia, in August 2003. The plant
was identified by S.R. and A.R. A voucher specimen (SR-CJR 8) has
been deposited at the Herbarium Bogoriense, Bogor, Indonesia.
Extraction and Isolation. The dried and milled leaves (803 g) of
A. aculeatum were extracted with MeOH (3 × 1.5 L) overnight at room
temperature. The solvent was evaporated in vacuo to afford a
concentrated MeOH extract, which was then diluted with H2O (0.9 L)
to give an aqueous MeOH extract (1.0 L). This aqueous extract was
partitioned in turn with hexanes (3 × 1.0 L), CHCl3 (3 × 1.0 L), and
EtOAc (3 × 1.0 L), to afford dried hexanes- (10 g), CHCl3- (2.1 g),
EtOAc- (6.6 g), and H2O-soluble (21 g) residues. The hexanes- and
CHCl3-soluble extracts exhibited significant cytotoxicity against a
human breast cancer cell line (MCF-7). Accordingly, the hexanes (ED50
) 1.0 µg/mL) and CHCl3 (ED50 ) 1.2 µg/mL) extracts were combined
and subjected to silica gel column chromatography (10 × 40 cm,
230–400 mesh), eluted with pure CHCl3 initially, then with a gradient
mixture of CHCl3-MeOH (100:1 to 1:1), to give eight fractions
(F01-F08). These fractions were evaluated in the MCF-7 cell line,
and the ED50 (µg/mL) values were >20, 0.3, 0.1, 0.9, 1.2, 5.0, 8.7,
and >20, respectively. Fraction F02 (3.5 g) was chromatographed on
a silica gel column (4 × 45 cm) eluted with hexanes-EtOAc-MeOH
(50:10:1) to give aculeatin A10 (1, 300 mg) and aculeatin B10 (2, 12
mg). During purification of a portion (40 mg) of crude aculeatin A
(300 mg) using HPLC separation (MeCN-H2O ) 90:10, 8 mL/min),
aculeatin F (4, 10 mg, tR 20 min) was isolated. Fraction F03 (1.25 g)
was also subjected to silica gel chromatography (2.5 × 45 cm) eluted
with hexane-EtOAc-MeOH (30:15:1 to 30:15:2) to give nine subfrac-
tions (F0301-0309). The constituents of subfraction F0306 were further
purified by HPLC using a reversed-phase C18 column (H2O-MeOH
85%-100% in 60 min) to furnish 3 (2.1 mg, tR 24.6 min), 5 (1.3 mg,
tR 35.4 min), and a mixture of 6 and 7 (10.0 mg, tR 57.5 min). Repetitive
HPLC separation of this mixture using the same conditions afforded
compounds 6 (4.6 mg, tR 56.8 min) and 7 (3.2 mg, tR 57.7 min).
Aculeatin E (3): yellow oil; [R]22D +5.1 (c 0.1, CHCl3); UV (MeOH)
λmax (log ꢀ) 230 (4.18) nm; IR (film) νmax 3440, 2923, 1671, 1634,
1080, 1014 cm-1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3,
100 MHz), see Table 1; HRESIMS m/z 413.2668 [M + Na], (calcd
for C24H38O4Na, 413.2668).
Figure 2. Effect of aculeatin A (1) on the growth of MCF-7 cells
in the hollow fiber assay. Hollow fibers containing either Lu1,
LNCaP, or MCF-7 cells were propagated subcutaneously (SC) or
within the peritoneum (IP) of immunodeficient mice. The animals
were treated with vehicle or aculeatin A (1) once daily by
intraperitoneal injection (6.25, 12.5, 25, 50 mg/kg) from days 3–6
after implantation.
This procedure is used as a secondary bioassay in our program on
the discovery of natural product anticancer agents, since it is
predictive of efficacy in follow-up in vivo assays and does not
consume much test compound.20,21 Aculeatin A (1) inhibited the
growth of the MCF-7 cell line (10–60%), propagated in hollow
fibers via the intraperitoneal site, in the dose range 6.25–50 mg/kg
(Figure 2). However, this compound was inactive when evaluated
against the Lu1 (human lung cancer) and LNCaP (human prostate
cancer) cells implanted at the intraperitoneal site at the same dose
range (Figure S1, Supporting Information). Moreover, compound
1 was not active against any of the three cell lines used when
implanted subcutaneously (dose range 6.25–50 mg/kg). When
aculeatin A (1) was assessed in the in vivo P388 lymphocytic
leukemia model,22,23 by intraperitoneal injection (15 mg/kg/
injection), it was found to be inactive (T/C ) 122%) in this model.
Furthermore, compound 1 was inactive in a human A2780S ovarian
carcinoma xenograft mouse model24,25 and gave 0.4 log cell kill
at 3 mg/kg/injection. Accordingly, it seems that compounds of the
1,7-dioxadispiro[5.1.5.2]pentadec-12-en-11-one structural type should
Aculeatin F (4): yellow oil; [R]22D -5.0 (c 0.4, CHCl3); UV (MeOH)
λmax (log ꢀ) 230 (4.27) nm; IR (film) νmax 3442, 2921, 1671, 1634,
1
1080, 1014 cm-1; H NMR (CDCl3, 400 MHz) δ 6.86 (1H, dd, J )
10.0, 2.9 Hz, H-13), 6.77 (1H, dd, J ) 9.9, 2.9 Hz, H-9), 6.15 (1H, dd,
J ) 10.0, 1.8 Hz, H-12), 6.11 (1H, dd, J ) 9.9, 1.8 Hz, H-10), 4.13
(2H, m. H-2 and H-4), 2.37 (1H, m, H-14a), 2.24 (1H, m, H-15a),

394 Journal of Natural Products, 2008, Vol. 71, No. 3
Chin et al.
2.00 (3H, m, H-5a, H-14b, and H-15b), 1.94 (1H, m, H-5b), 1.80 (1H,
brd, J ) 13.1 Hz, H-3a), 1.50 (2H, m, H-16), 1.43 (1H, m, H-17a),
1.41 (1H, dd, J ) 13.1, 2.6 Hz, H-3b), 1.30 (1H, m, H-17b), 1.23–1.27
(16H, m, H-18 to H-25), 0.88 (3H, t, J ) 6.7 Hz, H-26); 13C NMR
(CDCl3, 100 MHz) δ 185.3 (C, C-11), 150.9 (CH, C-13), 148.7 (CH,
C-9), 127.3 (CH, C-12), 127.0 (CH, C-10), 109.1 (C, C-6), 79.7 (C,
C-8), 65.3* (CH, C-2), 64.8* (CH, C-4), 39.1 (CH2, C-5 and C-15),
37.9 (CH2, C-3), 35.9 (CH2, C-16), 34.1 (CH2, C-14), 31.9 (CH2, C-24),
29.6–29.7 (CH2, C-18 to C-22), 29.3 (CH2, C-23), 25.6 (CH2, C-17),
22.7 (CH2, C-25), 14.1 (CH3, C-26) (*assignments are interchangeable);
HRESIMS m/z 413.2672 [M + Na] (calcd for C24H38O4Na, 413.2672).
Aculeatol E (5): yellow oil; [R]22D +8.4 (c 0.1, CHCl3); UV (MeOH)
λmax (log ꢀ) 221 (4.15) nm; IR (film) νmax 3441, 2921, 1670, 1637,
1080, 1014 cm-1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3,
100 MHz), see Table 1; HRESIMS m/z 459.3092 [M + Na] (calcd for
C26H44O5Na, 459.3086).
(1H), 4.17 (4H), 2.35 (1H), 2.20 (1H), 2.20 (1H), 1.99 (1H), 1.99 (1H),
1.97 (1H), 1.80 (1H), 1.53 (1H), 1.48 (2H), 1.48 (1H), 1.34 (1H),
1.33–1.23 (20H), 0.88 (3H); 13C NMR (CDCl3, 100 MHz) δ 185.9
(C), 172.5 (C), 170.5 (C), 151.9 (CH), 149.5 (CH), 127.0 (CH), 126.8
(CH), 107.2 (C), 79.3 (C), 68.8 (CH), 68.8 (CH2) 65.5 (CH), 39.4 (CH2),
35.8 (CH2), 35.7 (CH2), 34.6 (CH2), 34.0 (CH2), 31.9 (CH2), 29.7–29.6
(CH2), 29.4 (CH2), 25.6 (CH2), 22.7 (CH2), 14.1 (CH3); HRESIMS
m/z 557.3109 [M + Na]+ (calcd for C30H46O8Na, 557.3085).
Preparation of 1c. Compound 1 (10 mg) was treated with
(S)-campanoyl chloride (10 mg) and 4-(dimethylamino)pyridine (10
mg) in pyridine (1.0 mL) at room temperature for 48 h. The reaction
product was purified by preparative TLC (hexanes-EtOAc ) 2:1) to
give compound 1c (8 mg, Rf 0.5): 1H NMR (CDCl3, 400 MHz) δ 6.86
(1H), 6.77 (1H), 6.15 (1H), 6.11 (1H), 5.23 (1H), 4.13 (1H), 2.39 (1H),
2.23 (1H), 2.18 (1H), 2.01 (1H), 1.99 (1H), 1.98 (1H),1.84 (1H), 1.57
(1H), 1.47 (2H), 1.47 (1H), 1.33 (1H), 1.33–1.23 (20H), 1.06 (3H),
0.97 (3H), 0.92 (3H), 0.88 (3H); 13C NMR (CDCl3, 100 MHz) δ 185.4
(C), 178.1 (C), 167.0 (C), 151.7 (CH), 149.3 (CH), 126.9 (CH), 126.5
(CH), 107.0 (C), 91.3 (C), 79.1 (C), 69.0 (CH), 65.4 (CH), 54.8 (C),
53.8 (C), 39.2, (CH2), 35.9 (CH2), 35.7 (CH2), 34.6 (CH2), 33.9 (CH2),
31.9 (CH2), 30.4 (CH2), 29.6–29.5 (CH2), 29.3 (CH2), 28.7 (CH2), 25.5
(CH2), 22.6 (CH2), 16.9 (CH3), 16.8 (CH3), 14.1 (CH3), 9.6 (CH3);
HRESIMS m/z 621.3774 [M + Na]+ (calcd for C36H54O7Na, 621.3762).
Preparation of 1d. Compound 1 (10 mg) was treated with 2,6-
dichloro-5-fluoropyridinecarbonyl chloride (15 mg) and 4-(dimethy-
lamino)pyridine (10 mg) in pyridine at room temperature for 48 h. The
reaction product was purified by preparative TLC (hexanes-EtOAc )
2:1) to give compound 1d (9 mg, Rf 0.6): 1H NMR (CDCl3, 400 MHz)
δ 8.17 (1H), 6.90 (1H), 6.67 (1H), 6.19 (1H), 6.07 (1H), 5.40 (1H),
4.18 (1H), 2.43 (1H), 2.28 (1H), 2.18 (1H), 2.07 (1H), 2.03 (1H), 2.03
(1H), 1.91 (1H), 1.62 (1H), 1.50 (1H), 1.50 (1H), 1.34 (1H), 1.33–1.23
(20H), 0.88 (3H); 13C NMR (CDCl3, 100 MHz) δ 185.4 (C), 161.5
(C), 154.9 (C), 152.3 (C), 150.9 (CH), 148.4 (CH), 127.4 (CH), 127.1
(CH), 107.6 (C), 79.2 (C), 69.4 (CH), 65.7 (CH), 39.3 (CH2), 36.1
(CH2), 35.8 (CH2), 34.4 (CH2), 33.9 (CH2), 31.9 (CH2), 29.7–29.6
(CH2), 29.4 (CH2), 25.6 (CH2), 22.7 (CH2), 14.1 (CH3); HRESIMS
m/z 632.2312 [M + Na]+ (calcd for C32H42Cl2FNO5Na, 632.2316).
Preparation of 1e. Compound 1 (10 mg) was treated with
tetrabromophthalic anhydride (15 mg) and 4-(dimethylamino)pyridine
(10 mg) in pyridine (1.0 mL) at room temperature for 48 h. The reaction
product was purified by preparative TLC (hexanes-EtOAc ) 1:1) to
give compound 1e (9 mg, Rf 0.4): 1H NMR (CDCl3, 400 MHz) δ 6.80
(1H), 6.74 (1H), 6.04 (1H), 6.02 (1H), 5.18 (1H, m), 4.08 (1H), 2.32
(1H), 2.15 (2H), 2.00 (4H), 1.43–1.26 (26H), 0.878 (3H); 13C NMR
(CDCl3, 100 MHz) δ 185.6 (C), 169.7 (C), 166.6 (C), 151.9 (CH),
149.8 (CH), 138.0 (C), 135.3 (C), 133.4 (C), 130.6 (C), 129.9 (C),
128.5 (C), 126.9 (CH), 126.6 (CH), 106.7 (C), 79.5 (C), 71.4 (CH),
65.2 (CH), 39.4 (CH2), 35.8 (CH2), 34.3 (CH2), 34.2 (CH2), 31.9 (CH2),
29.8–29.7 (CH2), 29.4 (CH2), 25.6 (CH2), 22.7 (CH2), 14.1 (CH3);
HRESIMS m/z 904.9512 [M + Na]+ (calcd for C34H42Br4O7Na,
904.9520).
Preparation of 1f. Compound 1 (10 mg) was treated with tetra-
chlorophthalic anhydride (15 mg) and 4-(dimethylamino)pyridine (10
mg) in pyridine (1.0 mL) at room temperature for 24 h. The reaction
product was purified by preparative TLC (hexanes-EtOAc ) 1:1) to
give compound 1f (7 mg, Rf 0.4): 1H NMR (CDCl3, 400 MHz) δ 6.81
(1H), 6.78 (1H), 6.06 (1H), 6.04 (1H), 5.24 (1H), 4.09 (1H), 2.33 (1H),
2.20 (2H), 2.08 (4H), 1.42–1.25 (26H), 0.88 (3H); 13C NMR (CDCl3,
100 MHz) δ 185.6 (C), 169.6 (C), 166.6 (C), 152.0 (CH), 149.9 (CH),
134.3 (C), 132.0 (C), 130.7 (C), 126.9 (CH), 126.6 (CH), 122.2 (C),
121.7 (C), 106.8 (C), 79.6 (C), 71.3 (CH), 65.3 (CH), 39.5 (CH2), 35.8
(CH2), 35.7 (CH2), 34.3 (CH2), 31.9 (CH2), 29.8–29.7 (CH2), 29.4
(CH2), 25.6 (CH2), 22.7 (CH2), 14.1 (CH3); HRESIMS m/z 727.1562
[M + Na]+ (calcd for C34H42Cl4O7Na, 727.1553).
Preparation of 1g. Compound 1 (20 mg) was treated with
p-bromobenzoyl chloride (40 mg) and 4-(dimethylamino)pyridine (20
mg) in pyridine (1.0 mL) at room temperature for 24 h. The reaction
mixture was purified using silica gel column chromatography
(hexanes-EtOAc ) 3:1) to afford 18 mg of 1g: 1H NMR (CDCl3, 400
MHz) δ 7.76 (2H), 7.47 (2H), 6.94 (1H), 6.67 (1H), 6.16 (1H), 6.04
(1H), 5.39 (1H), 4.23 (1H), 2.38 (1H), 2.26 (1H), 2.05 (2H), 2.04 (1H),
2.04 (1H), 1.91 (1H), 1.58 (1H), 1.47 (2H), 1.47 (1H), 1.41 (1H),
1.33–1.23 (20H), 0.88 (3H); 13C NMR (CDCl3, 100 MHz) δ 186.1
(C), 165.9 (C), 152.2 (CH), 149.9 (CH), 132.0 (CH), 131.6 (CH), 130.1
(C), 128.4 (C), 127.3 (CH), 127.0 (CH), 107.7 (C), 79.5 (C), 68.1 (CH),
Amomol A (6): amorphous, white powder; [R]22 -11.4 (c 0.4,
D
CHCl3); UV (MeOH) λmax (log ꢀ) 230 (4.16) nm; IR (film) νmax 3442,
2921, 2851, 1671, 1634, 1080, 1014 cm-1; 1H NMR (CDCl3, 400 MHz)
and 13C NMR (CDCl3, 100 MHz), see Table 2; HRESIMS m/z 457.3294
[M + Na]+ (calcd for C27H46O4Na, 457.3294).
Amomol B (7): amorphous, white powder; [R]22 +8.8 (c 0.3,
D
CHCl3); UV (MeOH) λmax (log ꢀ) 230 (3.98) nm; IR (film) νmax 3442,
2917, 2850, 1673, 1634, 1081, 1017 cm-1; 1H NMR (CDCl3, 400 MHz)
and 13C NMR (CDCl3, 100 MHz), see Table 2; HRESIMS m/z 457.3294
[M + Na]+ (calcd for C27H46O4Na, 457.3294).
Preparation of Mosher Ester Derivatives of 6 and 7. The (R)-
MTPA and (S)-MTPA esters of 6 and 7 were prepared by a convenient
Mosher ester method described in the literature.15 Compound 6a [12-
(R)-MTPA ester of 6]: 1H NMR (pyridine-d5, 400 MHz) δ 6.958 (1H,
H-10), 6.931 (1H, H-6), 6.310 (1H, H-9), 6.281 (1H, H-7), 5.533 (1H,
m, H-12), 3.389 (3H, OCH3), 2.613 (1H, H-11a), 2.191 (1H, H-11b),
1.818 (2H, H-13), 1.393–1.191 (CH2), 0.874 (3H, H-27); HRESIMS
m/z 650.3794 [M + Na]+ (calcd for C37H53F3O6, 650.3794). Compound
6b [12-(S)-MTPA ester of 6]: 1H NMR (pyridine-d5, 400 MHz) δ 6.945
(1H, H-10), 6.878 (1H, H-6), 6.292 (1H, H-9), 6.273 (1H, H-7), 5.531
(1H, H-12), 3.284 (3H, OCH3), 2.532 (1H, H-11a), 2.142 (1H, H-11b),
1.832 (2H, H-13), 1.391–1.182 (CH2), 0.875 (3H, H-27); HRESIMS
m/z 650.3794 [M + Na]+ (calcd for C37H53F3O6, 650.3794). Compound
7a [12-(R)-MTPA ester of 7]: 1H NMR (pyridine-d5, 400 MHz) δ 6.964
(1H, H-10), 6.916 (1H, H-6), 6.311 (1H, H-9), 6.251 (1H, H-7), 5.549
(1H, H-12), 3.332 (3H, OCH3), 2.358 (2H, H-11), 1.780 (2H, H-13),
1.486–1.152 (CH2), 0.875 (3H, H-27); HRESIMS m/z 650.3794 [M +
Na]+ (calcd for C37H53F3O6, 650.3794). Compound 7b [12-(S)-MTPA
ester of 7]: 1H NMR (pyridine-d5, 400 MHz) δ 6.963 (1H, H-10), 6.899
(1H, H-6), 6.304 (1H, H-9), 6.252 (1H, H-7), 5.487 (1H, H-12), 3.280
(3H, OCH3), 2.355 (2H, H-11), 1.861 (2H, H-13), 1.481–1.198 (CH2),
0.874 (3H, H-27); HRESIMS m/z 650.3794 [M + Na]+ (calcd for
C37H53F3O6, 650.3794).
Preparation of 1a. Aculeatin A (1) (10 mg) was acetylated with
acetic anhydride (0.5 mL) and pyridine (0.5 mL) at room temperature
for 24 h. The reaction product was purified by preparative TLC using
hexanes-acetone (2:1) to give aculeatin A 4-acetate (1a, 7.0 mg, Rf
1
0.7): H NMR (CDCl3, 400 MHz) δ 6.89 (1H, dd, J ) 10.1, 3.0 Hz,
H-13), 6.74 (1H, dd, J ) 10.1, 3.0 Hz, H-9), 6.12 (1H, dd, J ) 10.1,
2.0 Hz, H-12), 6.08 (1H, dd, J ) 10.1, 2.0 Hz, H-10), 5.05 (1H, pentet,
J ) 2.8 Hz, H-4eq), 4.13 m (1H, H-2ax), 2.35 (1H, brt, J ) 7.1 Hz,
H-14a), 2.19 (1H, dd, J ) 9.4, 7.1 Hz, H-15a), 2.14 (1H, td, J ) 15.2,
2.3 Hz, H-5a), 2.00 (3H, s, COCH3), 1.96 (1H, m, H-14b), 1.96 (1H,
m, H-15b), 1.92 (1H, dd, J ) 14.9, 3.8 Hz, H-5b), 1.76 (1H, brd, J )
14.2 Hz, H-3a), 1.47 (3H, m, H-16 and H-17a), 1.32 (1H, m, H-17b),
1.30 (1H, m, H-3b), 1.33–1.23 (22H, m, H-18 to H-27), 0.88 (3H, t, J
) 6.8 Hz, H-28); 13C NMR (CDCl3, 100 MHz) δ 185.8 (C, C-11),
171.2 (C, COO), 152.2 (CH, C-13), 149.8 (CH, C-9), 126.8 (CH, C-12),
126.6 (CH, C-10), 107.4 (C, C-6), 79.3 (C, C-8), 67.2 (CH, C-4), 65.5
(CH, C-2), 39.5 (CH2, C-15), 35.9 (CH2, C-5), 35.9 (CH2, C-16), 34.8
(CH2, C-3), 34.1 (CH2, C-14), 31.9 (CH2, C-26), 29.7–29.6 (CH2, C-18
to C-24), 25.6 (CH2, C-17), 29.4 (CH2, C-25), 22.7 (CH2, C-27), 21.5
(CH3, OCOCH3), 14.1 (CH3, C-28); HRESIMS m/z 483.3083 [M +
Na]+ (calcd for C28H44O5Na, 483.3080).
Preparation of 1b. Compound 1 (10 mg) was treated with diglycolic
anhydride (10 mg) and pyridine (0.5 mL) at room temperature for 24 h.
The reaction product was purified by preparative TLC (hexanes-EtOAc
1
) 2:1) to give compound 1b (7 mg, Rf 0.3): H NMR (CDCl3, 400
MHz) δ 6.86 (1H), 6.74 (1H), 6.13 (1H), 6.09 (1H), 5.17 (1H), 4.10

DeriVatiVes of Aculeatins A and B from Amomum aculeatum
Journal of Natural Products, 2008, Vol. 71, No. 3 395
66.1 (CH), 39.8 (CH2), 37.0 (CH2), 36.3 (CH2), 35.1 (CH2), 34.5 (CH2),
32.3 (CH2), 30.2–30.0 (CH2), 29.7 (CH2), 26.1 (CH2), 23.1 (CH2), 14.4
(CH3); HRESIMS m/z 625.2335 [M + Na]+ (calcd for C33H45BrO5Na,
625.2328).
Supporting Information Available: Data for in vivo hollow fiber
evaluation of aculeatin A (1) against Lu1 (human lung cancer) and
LNCaP (human prostate cancer) cells and spectroscopic data for
compounds 3-7. This information is provided free of charge via the
Internet at http://pubs.acs.org.
Preparation of 1h. Compound 1 (22 mg) was oxidized with
pyridinium chlorochromate (40 mg) in CH2Cl2-pyridine (1:1, 2 mL)
at room temperature for 24 h. The reaction mixture was purified using
silica gel column chromatography (dicholoromethane) to yield 20 mg
of 1h: 1H NMR (CDCl3, 400 MHz) δ 6.79 (1H), 6.75 (1H), 6.13 (1H),
6.11 (1H), 4.13 (1H), 2.77 (1H), 2.55 (1H), 2.42 (1H), 2.41 (1H), 2.40
(1H), 2.23 (1H), 2.11 (1H), 2.10 (1H), 1.66 (1H), 1.55 (1H), 1.38 (2H),
1.23–1.35 (20H), 0.88 (3H); 13C NMR (CDCl3, 100 MHz) δ 204.5
(C), 185.2 (C), 50.3 (CH), 148.5 (CH), 127.5 (CH), 127.1 (CH), 109.5
(C), 79.7 (C), 69.6 (CH), 50.2 (CH2), 47.2 (CH2), 38.6 (CH2), 36.1
(CH2), 34.8 (CH2), 31.8 (CH2), 29.5–29.6 (CH2), 29.3 (CH2), 25.4
(CH2), 22.6 (CH2), 14.1 (CH3); HRESIMS m/z 439.2828 [M + Na]+
(calcd for C26H40O4Na, 439.2819).
Preparation of 2a. Aculeatin B (2) (5.0 mg) was acetylated
according to the same method described for 1a to give aculeatin B
4-acetate (2b, 2.5 mg): 1H NMR (CDCl3, 400 MHz) δ 6.98 (1H, dd, J
) 10.1, 3.0 Hz, H-13), 6.75 (1H, dd, J ) 10.1, 3.0 Hz, H-9), 6.15 (1H,
dd, J ) 10.1, 2.0 Hz, H-12), 6.11 (1H, dd, J ) 10.1, 2.0 Hz, H-10),
5.26 (1H, pentet, J ) 3.0 Hz, H-4eq), 3.74 (1H, brt, J ) 7.3 Hz, H-2ax),
2.63 (1H, dd, J ) 12.9, 6.8 Hz, H-14a), 2.32 (1H, dt, J ) 12.6, 7.6
Hz, H-15a), 2.09 (3H, s, COCH3), 2.09 (1H, m, H-5a), 2.06 (1H, m,
H-15b), 1.99 (1H, m, H-5b), 1.84 (1H, dd, J ) 12.9, 8.3 Hz, H-14b),
1.73 (1H, brd, J ) 14.1 Hz, H-3a), 1.61 (1H, m, H-16a), 1.54 (1H, m,
H-3b), 1.48 (1H, m, H-17a), 1.47 (1H, m, H-16b), 1.32 (1H, m, H-17b),
1.33–1.23 (20H, m, H-18 to H-27), 0.88 (3H, t, J ) 6.8 Hz, H-28);
13C NMR (CDCl3, 100 MHz) δ 185.5 (C, C-11), 170.0 (C, COO), 151.9
(CH, C-13), 148.8 (CH, C-9), 127.2 (CH, C-10), 127.2 (CH, C-12),
108.2 (C, C-6), 77.8 (C, C-8), 70.1 (CH, C-2), 68.4 (CH, C-4), 37.5
(CH2, C-5), 35.7 (CH2, C-16), 35.2 (CH2, C-15), 34.8 (CH2, C-14),
34.7 (CH2, C-3), 31.9 (CH2, C-26), 29.7–29.6 (CH2, C-18 to C-24),
29.4 (CH2, C-25), 25.8 (CH2, C-17), 22.7 (CH2, C-27), 21.4 (CH3,
OCOCH3), 14.1 (CH3, C-28); HRESIMS m/z 483.3083 [M+ Na]+
(calcd for C28H44O5Na, 483.3080).
References and Notes
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Acknowledgment. This work was supported by grants U19 CA52956
and P01CA125066-01A1 funded by the National Cancer Institute, NIH,
Bethesda, MD. We are grateful to Drs. W. C. Rose and R. Wild,
Pharmaceutical Research Institute, Bristol-Myers Squibb, Princeton, NJ,
for the in vivo evaluation of aculeatin A (1). We thank the College of
Pharmacy, The Ohio State University, for the provision of NMR
spectroscopic equipment used in this investigation. We are grateful to
Dr. C. M. Hadad, Department of Chemistry, and Mr. P. Eichenseer of
the Campus Chemical Instrument Center, The Ohio State University,
for the mass spectrometric data.
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