Antineoplastic Agents. 380. Isolation and X-ray Crystal Structure Determination of Isoaaptamine from the Republic of Singapore Hymeniacidon sp. and Conversion to the Phosphate Prodrug Hystatin 1

Article abstract of DOI:10.1021/np0204592

By use of bioassay (murine P388 lymphocytic leukemia cell line) guided isolation procedures, extracts of the Republic of Singapore marine sponge Hymeniacidon sp. were found to contain demethyl-oxyaaptamine (1) and aaptamine (3) as prominent cancer cell growth inhibitory constituents accompanied by the trace, albeit more active, component isoaaptamine (4). The isolation, X-ray structure elucidation, and antineoplastic and antimicrobial activities of isoaaptamine (4) have been summarized. Because of instability, isoaaptamine (4) was converted to a stable sodium phosphate prodrug designated hystatin 1 (7).

Full text of DOI:10.1021/np0204592

506
J . Nat. Prod. 2004, 67, 506-509
An tin eop la stic Agen ts. 380. Isola tion a n d X-r a y Cr ysta l Str u ctu r e Deter m in a tion
of Isoa a p ta m in e fr om th e Rep u blic of Sin ga p or e Hym en ia cid on sp . a n d
Con ver sion to th e P h osp h a te P r od r u g Hysta tin 11
George R. Pettit,*,† Holger Hoffmann,† J ames McNulty,† Kerianne C. Higgs,† Alison Murphy,† David J . Molloy,†
Delbert L. Herald,† Michael D. Williams,† Robin K. Pettit,† Dennis L. Doubek,† J ohn N. A. Hooper,‡
Leslie Albright,§ J ean M. Schmidt,† J ean-Charles Chapuis,† and Larry P. Tackett†
Cancer Research Institute and Department of Chemistry and Biochemistry, Arizona State University,
Tempe, Arizona 85287-2404, Sessile Marine Invertebrates, Queensland Museum, South Brisbane, Queensland 4101, Australia,
and Department of Biology, Western Oregon State University, Monmouth, Oregon 97361
Received October 2, 2002
By use of bioassay (murine P388 lymphocytic leukemia cell line) guided isolation procedures, extracts of
the Republic of Singapore marine sponge Hymeniacidon sp. were found to contain demethyl-
oxyaaptamine (1) and aaptamine (3) as prominent cancer cell growth inhibitory constituents accompanied
by the trace, albeit more active, component isoaaptamine (4). The isolation, X-ray structure elucidation,
and antineoplastic and antimicrobial activities of isoaaptamine (4) have been summarized. Because of
instability, isoaaptamine (4) was converted to a stable sodium phosphate prodrug designated hystatin 1
(7).
Marine Porifera continue to be a rapidly expanding
source of new drug candidates2,3 with unprecedented
structures4-6 and very important biological activities.7,8
From the onset6 of our investigations directed at exploring
marine organisms as sources of new anticancer drug
candidates, we have continued to evaluate marine sponge
constituents.6-8 In 1992, during our marine invertebrate
survey in the Republic of Singapore, we located a previously
unknown species of sponge belonging to the genus Hyme-
niacidon (order Halichondrida) that yielded methanol-
dichloromethane extracts active against the murine P388
lymphocytic leukemia cell line. A 500 kg (wet wt) re-
collection of the organism was employed to isolate the
cancer cell line inhibitory components utilizing the P388
cell line to guide the separations.
The sponge methanol-water and methanol-dichlo-
romethane extracts (see Experimental Section) were par-
titioned between water and dichloromethane. The dichlo-
romethane phase was partitioned between hexane and
methanol-water (9:1) to give polar fraction A. The initial
aqueous phase was next extracted with ethyl acetate
followed by 1-butanol to yield alcohol-soluble fraction B
(1.08 kg). A series of Sephadex LH-20 gel permeation and
partition chromatographic steps were used to separate
fraction A and led to the previously known demethy-
loxyaaptamine (1)9 and the dimethyl ketal 2, likely an
artifact of the isolation process.9 Demethyloxyaaptamine
(1) was isolated in gram quantities and exhibited a P388
ED50 of 0.31 µg/mL, while the much less abundant dimethyl
ketal 2 was inactive (P388 ED50 59 µg/mL).
Fraction B was separated, and aaptamine (3)10a was also
isolated in gram quantities along with the minor (10-5
%
yields) constituent isoaaptamine (4),11 each as the hydro-
chloride salt. The cancer cell growth inhibitory activities
of compounds 1, 3, and 4 are summarized in Table 1.
The structures of compounds 1, 2, and 3 were readily
deduced from spectral properties and literature compari-
sons.9,10a However, the structure of compound 4 proved to
be troublesome. Analysis of the H and 13C NMR spectra
1
* To whom correspondence should be addressed. Tel: 480-965-3351.
Fax: 480-965-8558.
indicated that it was nearly identical to isoaaptamine (4),
which was first reported by Fedoreev.11a Subsequently, it
was again isolated by Kashman11b and Shen.11c In our
experiments, all of the proton and carbon signals were
shifted downfield slightly owing to its isolation as the
† CRI. We dedicate this contribution to the memory of Dr. Harry B. Wood
(J uly 30, 1919-August 7, 2002), an outstanding pioneer of anticancer drug
development.
‡ Queensland Museum.
§ Western Oregon State University.
10.1021/np0204592 CCC: $27.50
© 2004 American Chemical Society and American Society of Pharmacognosy
Published on Web 02/28/2004

Notes
J ournal of Natural Products, 2004, Vol. 67, No. 3 507
Ta ble 1. Comparative Cancer Cell Line Results (ED50 µg/mL) for Demethyloxyaaptamine (1), Aaptamine (3), Isoaaptamine (4), and
Hystatin 1 (7)
cell type
cell line
demethyloxyaaptamine (1)
aaptamine (3)
isoaaptamine (4)
hystatin 1 (7)
leukemia
ovary
CNS
renal
lung
murine P388
OVCAR-3
SF-295
0.31
0.39
2.80
4.20
2.30
2.10
1.00
3.60
4.90
4.10
3.20
3.20
3.60
4.30
0.28
1.20
2.60
2.20
2.40
2.30
1.60
3.0
ND
ND
ND
>10
>10
ND
A498
NCI-H460
KM20L2
SK-MEL-5
colon
melanoma
hydrochloride salt. The shift of one of the methyl carbon
resonances from δ 60.7 in aaptamine (3) to 45.9 in
isoaaptamine (4) suggested the likelihood that an O-methyl
had shifted to an N-methyl position. Since isoquinoline
structures 5 and 6 also seemed consistent with the NMR
data, it was apparent that structure 4 could not initially
be assigned with certainty. But scrutiny of the NMR
NOESY and ROESY spectra did point to the likelihood of
structure 4. As an example, a cross-peak in the ROESY
spectrum between H-7 (δ 7.15) and the pyridine-like
hydrogen H-6 (δ 6.80) was consistent with structure 4.
NOESY cross-peaks between the proposed methoxy on C-8
(δ 4.03) and both H-7 and the phenolic hydroxy (δ 9.4) were
commensurate with structure 4 or 5 and allowed us to rule
out the methoxy analogue 6. On the basis of the NMR data,
we were unable to rule out the possibility of structure 5
unambiguously. Because of possible long-range correlations
that can occur through azaheterocycles, and in consider-
ation of the important cancer cell line and antimicrobial
activity of isoaaptamine (4), approaches to an unequivocal
structure elucidation were undertaken based on X-ray
crystal structure determination.
Since compound 4 was isolated as a yellow amorphous
hydrochloride salt that resisted crystallization, other salt
derivatives were explored. Changing the anion to perchlo-
rate, for example, gave beautiful yellow-red needles. Un-
fortunately, these proved to be too thin for X-ray analysis.
Other anions that were evaluated were even less promis-
ing. In view of the electron-rich structure of 4, a picric acid
derivative was considered, since it might crystallize as both
an acid-base and π-π complex. This proved to be the case.
Single crystals of the picrate were formed by slow crystal-
lization of a methanol-toluene solution of picric acid and
isoaaptamine (4). The structure of the colorful π-π picrate
complex verifies that our compound was indeed identical
with structure 4. More recently, isoaaptamine (4) has been
synthesized by Walz and Sundberg.12
One noteworthy feature of the crystal structure of the
picrate was the very unique behavior exhibited by the
toluene solvate. The molecule lies on the cell edges and is
centered on an inversion center at 0.00, 0.00, 0.500. As a
result, the toluene molecule was found to exist in two
conformations, each of 1/2 occupancy and overlapping,
inverted images of one another. The methyl substituent of
one toluene molecule (e.g., CS4A of the molecule composed
of ring atoms CS1, CS2, CS3, CS4, CS5, and CS2A) and
the carbon atom at the para-ring position of the other
toluene molecule (e.g., the ring consisting of CS1A, CS2A,
CS3A, CS4A, CS5A, and CS2) both occupy the same site
(within the resolution of the data), and as a consequence,
both of these atoms were assigned to the same site.
For a variety of reasons, the isolation of isoaaptamine
(4) as the most interesting cancer cell growth inhibitor of
the Singapore sponge Hymeniacidon sp. is noteworthy.
Aaptamine (3), as the parent alkaloid, was first isolated
from the marine sponge Aaptos aaptos.10a Later, it was
isolated from several other species of sponges.10b The
Ta ble 2. Comparative Antimicrobial Activities of Isoaaptamine
(4) and Hystatin 1 (7)
range of minimum inhibitory
concentration (ug/mL)
microorganism
4
7
Cryptococcus neoformans
Candida albicans
Staphylococcus aureus
Streptococcus pneumoniae
Enterococcus faecalis
Micrococcus luteus
64
n.i.
n.i.
64
16-32
n.i.
n.i.
n.i.
n.i.
n.i.a
16-32
8-32
32-64
32-64
n.i.
n.i.
n.i.
<0.5
Escherichia coli
Enterobacter cloacae
Stenotrophomonas maltophilia
Neisseria gonorrhoeae
<0.5
a
n.i.: no inhibition at 64 µg/mL.
discovery of the aaptamines in Hymeniacidon sp. suggests
that unusual isoquinolines may enjoy a broader species and
geographic distribution. Also, the large quantities of
aaptamine (3) contained in Hymeniacidon sp. would sug-
gest a relatively important biological role. Consequently,
we have been proceeding with an extended study of the
aaptamines, especially isoaaptamine (4).
When newly isolated, isoaaptamine (4) appears as a
yellow-colored powder that rapidly changes to dark green
and finally brown or black, presumably due to air oxidation.
Because of this instability and resultant degradation,
attempts at preparation of a variety of simple derivatives
generally proved to be elusive. Hence, attention was
directed at preparation of a stable prodrug that would
retain the biological activity. For that purpose, isoaaptamine
(4) was first phosphorylated13 with dibenzyl phosphite.
Cleavage of the benzyl ester by means of trimethylsilyl
bromide14 and reaction of the resulting phosphoric acid
with sodium methoxide afforded the relatively more stable
disodium phosphate prodrug designated hystatin 1 (7).
Biology. Evaluation of aaptamines 1, 3, 4, and 7 against
our Institute’s human tumor cell line panel (Table 1) and
the NCI 60-cell line human tumor screen15-18 was under-
taken. Isoaaptamine (4) had antifungal and antibacterial
activities, and the prodrug hystatin 1 (7) retained some of
the antibacterial activity (Table 2).
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Uncorrected melting
points were determined on a digital Electrothermal apparatus.
The NMR experiments were performed with a Varian VXR-
500 instrument in the indicated solvent. Mass spectra were
obtained using a Kratos MS-50 spectrometer (70 eV). The
X-ray crystallographic experiments were conducted with an
Enraf-Nonius CAD-4 diffractometer. Solvents used for chro-
matographic procedures were redistilled. Sephadex LH-20
(25-100 µM) employed for gel permeation and partition
chromatography was obtained from Pharmacia Fine Chemicals
AB, Uppsala, Sweden. Gilson FC-220 and FC-202 fraction
collectors were used for chromatographic fractionation experi-

508 J ournal of Natural Products, 2004, Vol. 67, No. 3
Notes
ments. Silica gel GF Uniplates were employed for monitoring
the chromatographic separations. All TLC plates were visual-
ized under UV light (254 nm) and developed with phospho-
molybdic acid spray reagent.
Hymen ia cidon sp. (Halich on dr ida:Hym en iacidon idae).
The initial specimen of this bright orange encrusting-lobular
sponge, a previously undescribed species, was collected on the
north side of Terumbu Pemalang Besar Reef, Republic of
Singapore, at a depth of 5-10 m in 1989. During 1992, 500
kg (wet wt) of the sponge was re-collected in this general area.
An im a l E xt r a ct ion a n d Solven t P a r t it ion in g. The
methanol shipping solution was removed and extract concen-
trated to 100 L of an aqueous suspension, which was succes-
sively extracted with dichloromethane (6 × 50 L), ethyl acetate
(6 × 50 L), and 1-butanol (4 × 50 L). The 1-butanol fraction
was concentrated to dryness and partially redissolved in 2 L
of methanol.
correction (based on a series of psi-scans).20 Structure deter-
mination was readily accomplished with the direct-methods
program SIR92.21 All non-hydrogen atom coordinates (exclud-
ing toluene solvent atoms) were located in a structure solution
run using the RANDOM option and allowing NREF to have
the maximum value (499) for that program. The non-hydrogen
solvent and the hydrogen atom positions on isoaaptamine (4)
and picric acid were determined from difference Fourier maps
using the program SHELXL-93.22 The hydrogen atoms were
assigned thermal parameters equal to 1.5 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 final
cycles of refinement. All non-hydrogen atoms were refined
anisotropically in a full-matrix least-squares refinement pro-
cess with the SHELXL-93 software package. The assignment
of nitrogen atom positions in the aromatic ring of the hetero-
cycle could not be made with any certainty based upon bond
distance information alone. Consequently, N versus C oc-
cupancy refinements were made on the individual models
produced by alternating the N atom over all possible suspected
positions which might contain nitrogen. The N and C character
of the various positions investigated was calculated to be the
following: 1, 77% N, 23% C; 2, 31% N, 69% C; 3, 15% N, 85%
C; 3A, 0% N, 100% C; 4, 97% N, 3% C; 5, 15% N, 85% C; 6,
38% N, 62% C. As a result, the two nitrogen atoms were
assigned to the 1 and 4 positions, respectively.23 The final
standard residual R value for the solvated complex model was
0.0769 for observed data (2353 reflections) and 0.0985 for all
data (3141 reflections). The corresponding Sheldrick R values
were wR2 of 0.2094 and 0.2269, respectively. A final difference
Fourier map showed insignificant residual electron density,
the largest difference peak and hole being 0.432 and -0.326
e/Å3, respectively. Final bond distances and angles were all
within acceptable limits.
Isola tion of th e Aa p ta m in es. The methanol-soluble por-
tion (1084 g) of the 1-butanol fraction was fractionated by a
gel permeation/partition sequence. The major component
isolated was aaptamine (3, 8.8 g, 1.7 × 10-3%), having spectral
properties in complete accord with the literature values. The
minor but more active isoaaptamine (4, 0.22 g, 4.4 × 10-5%)
was obtained as an amorphous yellow powder: mp 200-205
1
°C (dec); H NMR (DMSO) δ 3.96 (3H, s, OMe), 4.03 (3H, s,
NMe), 6.25 (1H, d, J ) 7.2, H-3), 6.80 (1H, d, J ) 7.3, H-6),
7.15 (1H, s, H-7), 7.25 (1H, d, J ) 7.3, H-5), 7.73 (1H, d, J )
7.2, H-2), 9.4 (1H, br s, OH), 12.7 (1H, br s, NH); 13C NMR
(DMSO) δ 45.9 (NMe), 56.5 (OMe), 97.3 (C-3), 101.5 (C-7),
113.1 (C-6), 118.1 (C-9b), 127.7 (C-5), 129.3 (C-6a and C-9a),
132.2 (C-9), 148.9 (C-2), 149.2 (C-3a), 153.6 (C-8); MS m/z 228
(100), 213 (94), 185 (60), 170 (47), 155 (10), 142 (22), 127 (7),
115 (12), 84 (7); HREIMS m/z 228.0903 (calcd for C13H12N2O2,
228.0899).
Isoa a p ta m in e P er ch lor a te. A sample of isoaaptamine (4)
(1.0 mg) was dissolved in methanol (1.0 mL) in a small
crystallization tube and two drops of perchloric acid (70%)
allowed to run down the side of the tube. Thin red needles
were slowly deposited after standing at room temperature for
several hours. The crystals proved to be too thin for X-ray
analysis and were not further characterized due to decomposi-
tion on standing.
Isoa a p ta m in e P icr a te. Picric acid was washed with water,
and the damp acid was dissolved in methanol at a concentra-
tion of about 1 M. To a 1.0 mg sample of isoaaptamine (4)
hydrochloride in a few drops of methanol was added 0.25 mL
of the picric acid solution. The orange solution was heated to
about 60 °C and diluted to 1.0 mL with toluene. The solution
was filtered hot, allowed to cool to room temperature, and then
placed in a freezer (approximately -20 °C), where red-colored
single crystals (dp 95-97 °C) slowly formed over 72 h.
X-r a y Cr yst a l St r u ct u r e Det er m in a t ion of Isoa a p t -
a m in e (4). A few, marginal quality, red, rod-shaped crystals
of the complex were obtained from toluene-methanol solution.
The best specimen, 0.48 × 0.10 × 0.04 mm, was mounted on
the tip of a glass fiber with Superglue. Data collection was
performed at 24 ( 1° for a monoclinic system. All reflections
corresponding to a complete quadrant, with 2θ e 130°, were
measured using the ω/2θ scan technique. Subsequent statisti-
cal analysis of the complete reflection data set using the
XPREP19 program indicated the space group was P21/c, the
asymmetric unit of the cell containing a single molecule each
of the parent heterocyclic compound and picric acid, along with
1/2 a molecule of toluene. Crystal data: C13H12N2O2‚C6H3N3H7‚
1/2C7H8, monoclinic space group P21/c, with a ) 7.983(1) Å, b
) 12.839(3) Å, c ) 21.522(5) Å, â ) 92.920(1)°, V ) 2203.0(8)
Å3, λ(Cu KR) ) 1.54184 Å, Fc ) 1.518 g cm-3 for Z ) 4 and fw
) 503.43, F(000) ) 1044. After Lorentz and polarization
corrections, merging of equivalent reflections, and rejection of
systematic absences, 3141 unique reflections (R(int) ) 0.0505)
remained, of which 2353 were considered observed (Io > 2σ-
(Io)) and were used in the subsequent structure determination
and refinement. Linear and anisotropic decay corrections were
applied to the intensity data as well as an empirical absorption
Syn t h esis of H yst a t in
1 (Disod iu m Isoa a p t a m in e
9-O-P h osp h a te) (7). To a dry flask equipped with a septum,
magnetic stirrer, thermometer, and Ar inlet containing dry
acetonitrile (20 mL) was added isoaaptamine hydrochloride
(4, 0.26 g, 0.97 mmol). Upon cooling to -10 °C, tetrachlo-
romethane (0.47 mL, 4.85 mmol) was added followed by
diisopropylethylamine (0.51 mL, 2.91 mmol) and a catalytic
amount of (dimethylamino)pyridine. After 2 min, dropwise
addition of dibenzyl phosphite (0.30 mL, 1.36 mmol) was begun
while maintaining a temperature of -10 °C. The resultant
yellow solution was stirred for 2 h, and 0.5 M aqueous KH2-
PO4 (30 mL) was added. The water layer was extracted with
CH2Cl2 (4 × 50 mL), and the combined organic extract was
washed with brine (100 mL). The organic layer was dried, and
the solvent was removed in vacuo. Purification by column
chromatography (neutral alumina, CH2Cl2-CH3OH, 20:1, as
eluent) gave the dibenzyl phosphate as a yellow oil (0.27 g),
which was immediately dissolved in dichloromethane. Bro-
motrimethylsilane (0.2 mL) was added at room temperature.
After stirring for 2 h, the solvent was removed and the residue
was dissolved in water (20 mL). The water layer was washed
with dichloromethane (3 × 10 mL), and the water was
removed. The yellow solid was dissolved in methanol, a 30%
solution of sodium methoxide (0.22 mL, 1.16 mmol) was added,
and the mixture was stirred for 12 h. The reaction mixture
was concentrated to a yellow solid that was recrystallized from
acetone-water. The isoaaptamine prodrug (7) was obtained
as a yellow-green powder (0.15 g, 45% yield): mp 166-168 °C
(dec); UV (CH3OH) λmax (log ꢀ) 209 nm (4.22), 241 (4.17), 260
(4.28), 316 (3.49), 326 (3.39), 389 (3.78); IR (KBr) ν 3406 (s),
3227 (m), 1649 (s), 1604 (s), 1525 (w), 1465 (m), 1429 (w), 1342
(m), 1298 (m), 1240 (w), 1111 (s), 977 (s), 848 (m), 723 (m),
630 (m), 563 (m); 1H NMR (D2O and a few drops of CD3OD) δ
3.77 (s, 3H, NMe), 3.87 (s, 3H, OMe), 5.84 (d, J ) 7.5 Hz, 1H,
H-3), 6.47 (d, J ) 7.5 Hz, 1H, H-6), 6.62 (d, J ) 7.5 Hz, 1H,
H-5), 6.65 (s, 1H, H-7), 7.43 (d, J ) 7.5 Hz, 1H, H-2); 13C NMR
(D2O) δ 45.81, 56.32, 98.05, 101.49, 112.90, 117.34, 128.96,
132.80, 134.30, 147.76, 148.32, 158.62; 31P NMR (162 MHz,
D2O) δ 1.58; EIMS m/z 228 (100) [M+ - (NaO)2P(O)H], 213

Notes
J ournal of Natural Products, 2004, Vol. 67, No. 3 509
(8) (a) Pettit, G. R.; Cichacz, Z. A.; Tan, R.; Hoard, M. S.; Melody, N.;
Pettit, R. K. J . Nat. Prod. 1998, 61, 13-16. (b) Pettit, G. R.; McNulty,
J .; Herald, D. L.; Doubek, D. L.; Chapuis, J .-C.; Schmidt, J . M.;
Tackett, L. P.; Boyd, M. R. J . Nat. Prod. 1997, 60, 180-183. (c) Pettit,
G. R.; Tan, R.; Gao, F.; Williams, M. D.; Doubek, D. L.; Boyd, M. R.;
Schmidt, J . M.; Chapuis, J .-C.; Hamel, E.; Bai, R.; Hooper, J . N. A.;
Tackett, L. P. J . Org. Chem. 1993, 58, 2538-2543.
(58), 185 (39), 170 (24), 142 (13), 114 (12), 28 (31); HRFABMS
([M - Na2O3P + H] m/z 229.09392 (cald for C13H13N2O2,
229.09771).
An tim icr obia l Su scep tibility Testin g. Compounds were
screened against the bacteria Staphylococcus aureus, Strep-
tococcus pneumoniae, Enterococcus faecalis, Micrococcus lu-
teus, Escherichia coli, Enterobacter cloacae, Stenotrophomonas
maltophilia, and Neisseria gonorrhoeae and the fungi Candida
albicans and Cryptococcus neoformans, according to estab-
lished broth microdilution susceptibility assays.24,25 The mini-
mum inhibitory concentration was defined as the lowest
concentration of compound that inhibited all visible growth
of the test organism (optically clear). Assays were repeated
on separate days.
(9) Nakamura, H.; Kobayashi, J .; Ohizumi, Y.; Hirata, Y. J . Chem. Soc.,
Perkin Trans. 1 1987, 173-176.
(10) (a) Aaptamine was first isolated from Aaptos aaptos: Nakamura, H.;
Kobayashi, J .; Ohizumi, Y. Tetrahedron Lett. 1982, 23, 5555-5558.
Subsequently, it has been isolated from certain other sponge spe-
cies: (b) Berquist, P. R.; Combie, R. C.; Kernan, M. R. Biochem. Syst.
Ecol. 1991, 19, 289-290. (c) Mohamed, R.; Lee, L. J .; Omar, S. Proc.
16th Malays. Biochem. Soc. Conf. 1991, 37-42. (d) Park, S. K.; Kim,
S. S.; Park, J . D.; Hong, J . S.; Kim, I. K. J . Korean Chem. Soc. 1995,
39, 559-563.
(11) (a) Fedoreev, S. A.; Prokofeva, N. G.; Denisenko, V. A.; Rebachuk, N.
M. Khim.-Farm. Zh. 1988, 22, 943-946. (b) Kashman, Y.; Rudi, A.;
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Chem. 1990, 14, 729-740. (c) Shen, Y.-C.; Chein, C.-C.; Hsieh, P.-
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(12) Walz, A. J .; Sundberg, R. J . J . Org. Chem. 2000, 65, 8001-8010.
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(14) (a) Pettit, G. R.; Rhodes, M. R.; Herald, D. L.; Chaplin, D. J .; Stratford,
M.; Hamel, E.; Pettit, R. K.; Chapuis, J .-C.; Oliva, D. Anti-Cancer
Drug Des. 1998, 13, 981-993. (b) Lazar, S.; Guillaumet, G. Synth.
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Ack n ow led gm en t. The research reported herein was
made possible by the following financial assistance: Outstand-
ing Investigator Grant CA44344-01A1-12 and Grant RO1
CA90441-01 awarded by the Division of Cancer Treatment,
National Cancer Institute, DHHS, the Fannie E. Rippel
Foundation, the Arizona Disease Control Research Commis-
sion, the Robert B. Dalton Endowment Fund, Virginia Piper,
Diane Cummings (The Nathan Cummings Foundation, Inc.),
Gary L. and Diane Tooker, Polly J . Trautman, and the Eagles
Art Ehrmann Cancer Fund. For other very helpful assistance
we thank the National University of Singapore (and especially
C. L. Ming and T. J . Lam) and M. Suffness, C. L. Herald, J .
C. Knight, Z. A. Cichacz, F. Hogan, D. Nielsen-Tackett, L.
Williams, and M. Filiatrault.
(15) Boyd, M. R. In CANCER: Principles and Practices of Oncology
Updates; DeVita, V. T., J r., Hellman, S., Rosenberg, S. A., Eds.;
Lippincott: Philadelphia, 1989; Vol. 10, No. 3, pp 1-12.
(16) Boyd, M. R. In Current Therapy in Oncology; Niederhuber, J . E., Ed.;
B. C. Decker, Inc.: Philadelphia, 1993; pp 11-22.
(17) Boyd, M. R.; Paull, K. D.; Rubinstein, L. R. In Cytotoxic Anticancer
Drugs: Models and Concepts for Drug Discovery and Development;
Valeriote, F. A., Corbett, T., Baker, L., Eds.; Kluwer Academic
Publishers: Amsterdam, 1992; pp 11-34.
(18) Boyd, M. R.; Paull, K. D. Drug Dev. Res. 1995, 34, 91-109.
(19) XPREP-The automatic space group determination program in the
SHELXTL PC program package.
(20) North, A. C.; Phillips, D. C.; Mathews, F. S. Acta Crystallogr. 1968,
A24, 351-359.
(21) Altomare, A.; Cascarano, G.; Giacovazzo, C.; Guagliardi, A.; Burla,
M.; Polidori, G.; Camalli, M. SIR92-A Program for Automatic
Solution of Crystal Structures by Direct Methods; Dipartimento
Geomineralogico, University of Bari, Italy, 1992.
(22) Sheldrick, G. M. SHELXL93. Program for the Refinement of Crystal
Structures; University of Gu¨ttingen: Germany, 1993.
(23) Preparation of Figures S1A and S1B in the Supporting Information
was done with: SHELXTL-PC Version 5.03, an integrated software
system for the determination of crystal structures from diffraction
data; Siemens Industrial Automation, Inc., Analytical Instrumenta-
tion: Madison, WI, 1994.
Su p p or tin g In for m a tion Ava ila ble: Illustrations of the crystal
structure of isoaaptamine (4) picrate-toluene complex and of the
overlappng toluene conformers are given, together with tables of X-ray
coordinates, bond lengths and angles, and thermal parameters. This
material is available free of charge via the Internet at http://pubs.ac-
s.org.
Refer en ces a n d Notes
(1) For part 379 of this series see: Pettit, G. R.; Toki, B.; Boyd, M. R.;
Hamel, E.; Pettit, R. K. J . Med. Chem. 1998, 41, 1688-1695.
(2) Kerr, R. G.; Kerr, S. S. Expert Opin. Ther. Pat. 1999, 9, 1207-1222.
(3) Bongiorni, L.; Pietra, F. Chem. Ind. 1996, 54-58.
(4) (a) Faulkner, D. J . Nat. Prod. Rep. 2001, 18, 1-49. (b) Faulkner, D.
J . Nat. Prod. Rep. 2000, 17, 7-55. (c) Davidson, B. S. Chem. Rev.
1993, 93, 1771-1791.
(5) Pettit, G. R.; Hogan-Pierson, F.; Herald, C. L. Anticancer Drugs from
Animals, Plants, and Microorganisms; Wiley-Interscience: New York,
1993.
(24) National Committee for Clinical Laboratory Standards. Methods for
Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow
Aerobically. Approved Standard M7-A4. Wayne, PA: NCCLS, 1997.
(25) National Committee for Clinical Laboratory Standards. Reference
Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts.
Approved Standard M27-A. Wayne, PA: NCCLS, 1997.
(6) (a) Pettit, G. R.; Day, J . F.; Hartwell, J . L.; Wood, H. B. Nature 1970,
227, 962-963. (b) Pettit, G. R. J . Nat. Prod. 1996, 59, 812-821. (c)
Pettit, G. R. Evolutionary Biosynthesis of Anticancer Drugs. In
Anticancer Agents: Frontiers in Cancer Chemotherapy; Ojima, I., Vite,
G. D., Altmann, K.-H., Eds.; American Chemical Society: Washing-
ton, DC, 2001; pp 16-42.
(26) Crystallographic data for the structure reported in this paper have
been deposited with the Cambridge Crystallographic Data Centre.
Copies of the data can be obtained, free of charge, on application to
the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [Fax:
+44 (0) 1223-336033 or e-mail: deposit@ccdc.cam.ac.uk].
(7) (a) Pettit, G. R.; Knight, J . C.; Collins, J . C.; Herald, D. L.; Pettit, R.
K.; Boyd, M. R.; Young, V. G. J . Nat. Prod. 2000, 63, 793-798. (b)
Pettit, G. R.; Cichacz, Z. A.; Gao, F.; Herald, C. L.; Boyd, M. R.;
Schmidt, J . M.; Hooper, J . N. A. J . Org. Chem. 1993, 58, 1302-1304.
(c) Pettit, G. R.; Cichacz, Z. A.; Herald, C. L.; Gao, F.; Boyd, M. R.;
Schmidt, J . M.; Hamel, E.; Bai, R. J . Chem. Soc., Chem. Commun.
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