3339
Reprinted from Journal of Medicinal Chemistry, 1991, 34.
Copyright @ 1991 by the American Chemical Society and reprinted by permission of the copyright owner.
Ct~H2 b
o c •L!~ 'fioC~'U H HN3
12NH 2.
O~3 0 HOI ~c
14 tS~617 N 19 21NH ~ I d
• 16fi;-0 \~ Ii
c •
• 0 c
b be.
b
2
(halichondrin B)
(6) By high-resolution FAB, NMR (2D, 400 MHJ and comparison
with an authentic sample of polyether 2 provided by Prof. D.
Uemura.
(7) Uemura, D.; Takahashi, K.; Yamamoto, T.; Katayama, C.;
Tanaka, J.; Okumura, Y.; Hirata, Y. J. Arn. Chern. Soc. 1985,
107,4796.
(8) (a) Hirata, Y.; Uemura, D. Pure Appl. Chern. 1988,58, 701. (b)
Tasumasa, T.; T08hitaka, M.; Keizo, Y.; Hiroyuki, K. Fujisawa
Pharmaceutical Co., Ltd., Jpn Kokai Tokkyo Kohe JP
61,191,687 [86,191,687] (C1. C07D493/22) 1986, Appl. 85/
32,253 1985; Chern. Abstr. 1987, 106, 23261g.
1
(homohalichondrin B)
the polyether macrolides, homohalichondrin B (1,900 p.g,
4.1 X lO-s0/0 yield, PS T /C 285 at 150 p.g/kg) and hali-
chondrin B (2,400 p.g, 1.8 X 10-80/0 yield, PS T /C 238 at
25 p.g/kg), heretofore found in trace amounts in one dif-
ficultly accessible Japanese sponge.7,8 A new PS inhibitory
(ED50 0.21p.g/mL) peptide (3, 100 mg, 4.54 X 1~0/0 yield)
designated axinastatin 1 was also isolated, accompanied
by axinohydantoin3 (30 mg) and hymenialdisine3 (0.53 mg).
~:HO l;lol;l:l;loH 'HO 0 0 °\. p-('""f'u,! ° 0" 0, ,~o~n~H HHH, H
HO 0H: 9~ °
I V .... __ : oC' 0 '.
3
(axinastatin 1)
Axinastatin 1 (3) crystallized from methylene chloride:
mp 283-7 °C dec; [a]25n -161.6° (c 0.099, CH30H); TLC(R, 0.18 in 95:5 CH2CI2/MeOH); UV (CH30H) Amu 208 nm
(E 18000); IR (NaCI plate), "mu 3320, 2960, 1640, 1520,
1465,1430 cm-l ; high-resolution FAB MS 753.4293 [M +
H]+, theoretical mass for [M + H]+ of C38H56NsOs requires
(1) (a) Antineoplastic Agents series contribution 219. For part
218, refer to Bradshaw, T. D.; Gescher, A.; Pettit, G. R. Int. J.
Cancer 1991,47,929. (b) Developmental Therapeutics Pro-
gram, Division of Cancer Treatment, National Cancer Insti-
tute, Frederick Cancer Research Facility, Frederick, MD
21701. (c) Midwest Center for Mass Spectrometry, The
University of Nebraska, Lincoln, NE 68588-0362. (d) Northern
Territory Museum of Arts and Sciences, Bullock Point, Fannie
Bay, Darwin, N.T. 0801, Australia. (e) National Museum of
Natural History, Smithsonian Institution, Washington, D.C.
20560.
(2) (a) Kashman, Y.; Hirsh, S.; McConnell, O. J.; Ohtani, I.; Ku-
sumi, T.; Kakisawa, H. J. Arn. Chern. Soc. 1989,111,8925. (b)
Cimino, G.; Mattia, C. A.; Mazzarella, L.; Puliti, R; Scognam-
iglio, G.; Spinella, A.; Trivellone, E. Tetrahedron 1989, 45,
3863. (c) Kobayashi, M.; Kawazoe, K.; Kitagawa, I. Chern.
Pharrn. Bull. 1989, 37 (6), 1676. (d) Kubo, A.; Kitahara, Y.;
Nakahara, S. Chern. Pharrn. Bull. 1989, 37 (5), 1384. (e)
Burres, N. S.; Sazesh, S.; Gunawardana, G. P.; Clement, J. J.
Cancer Res. 1989,49,5267. (0 Kobayashi, J.; Murayama, T.;
Ohizumi, Y.; Sasaki, T.; Ohta, T.; Nozoe, S. Tetrahedron Lett.
1989,30,4833. (g) Kobayashi, M.; Kawazoe, K.; Kitagawa, I.
Tetrahedron Lett. 1989,30,4149. (h) Fedoreyev, S. A.; nyin,
S. G.; Utkina, N. K.; Maximov, O. B.; Reshetnyak, M. V.;
Antipin, M. Y.; Struchkov, T. P. Tetrahedron 1989,45,3487.
(i) Fusetani, N.; Yasumuro, K.; Matsunaga, S.; Hashimoto, K.
Tetrahedron Lett. 1989,30,2809. (j) Sakemi, S.; Sun, H. H.;
Jefford, C. W.; Bernardinelli, G. Tetrahedron Lett. 1989,30,
2517.
(3) For leading references, see: Pettit, G. R.; Herald, C. L.; Leet,
J. E.; Gupta, R; Schaufelberger, D. E.; Bates, R B.; Clewlow,
P. J.; Doubek, D. L.; Manfredi, K. P.; Rdtzler, K.; Schmidt, J.
M.; Tackett, L. P.; Ward, F. B.; Bruck, M.; Camou, F. Can. J.
Chern. 1990, 68, 1621.
(4) Pettit, G. R.; Clewlow, P. J.; Dufresne, C.; Doubek, D. L.;
Cerny, R. L.; Rdtzler, K. Can. J. Chern. 1990,68,708.
(5) U.S. National Cancer Institute's murine P388 lymphocytic
leukemia cell line.
Isolation and Structure of the Cell Growth
Inhibitory Constituents from the Western Pacific
Marine Sponge Axinella Sp.l.
While isolation of alkaloids from marine Porifera has
been accelerating,2 only a small number of antineoplastic3
or peptide3.4 constituents have been recovered from these
invertebrates. Our isolation and structural determination
of the P388lymphocytic leukemia (PS system)6 cell growth
inhibitory cyclooctapeptide hymenistatin 14 from a Palau
sponge in the genus Hymeniacidon represented the first
such combination of source, structural type, and biological
activity. We have also found an Axinella sp. (Demos-
pongiae class) collected (in 1979) in Palau (at -40 m) to
yield a methylene chloride-2-propanol extract that pro-
vided a 101% increase in life span (at 100 mg/kg) against
the PS leukemia6 with ED60 2.5 p.g/mL in the corre-
sponding cell line.
Ip 1985 the sponge was recollected (palau) and preserved
in 2-propanol. A 220-kg (wet weight) portion was extracted
with methylene chloride-methanol. By means of PS
guided bioassay and a series of detailed3.4 solvent partition,
gel permeation (and gel partition, Sephadex LH-20),
partition (silica gel including reversed phase), and ad-
sorption column chromatographic techniques, a series of
structurally diverse antineoplastic constituents were de-
tected in this very productive sponge. We now report that
the most potent in vivo components were established6 as
0022-2623/91/1834-3339$02.50/0 © 1991 American Chemical Society
3340
753.4299; amino acid analyses Asp, (or Asn), Phe, Pro, and
Val in the ratio 1:1:2:3. The molecular formula for axin-
astatin 1 (3) was deduced from high-field (400 MHz) lH
and l3C NMR studies (see Table I of the supplementary
material) in conjunction with the high-resolution FAB MS
peak matching experiments just noted. Combined lH, lH
COSY, lH, l3C COSY, lH, lH relayed COSY,9a HMBC,9b
and NOESY experiments confirmed the amino acid se-
quence and cyclic structure 3. The amino acid components
and sequence ofaxinastatin 1 were confirmed as cyclo-
(Asn-Pro-Phe-Val-Val-Pro-Val) by tandem (MS/MS) mass
spectrometry.10
Protonation upon FAB results in ring opening of the
cyclic peptide at an N-acyl bond to give a linear acylium
ion.10 The major fragmentation processes observed by
tandem mass spectrometry involve losses of amino acid
residues from the C terminus. Protonation is favored at
proline, and with axinastatin 1 there are two possibilities.
The FAB MS/MS spectrum of the [M + H) species con-
tains two series (A and B) of ions resulting from proton-
ation at the two proline units. All of the ions in both series
197 311 408 555 654
pro-:=t-=t~==t-~v~
A
245 344 443 540 639
pro-~q-q-~q-Asn
B
were observed. Additional supporting information for the
sequence was obtained by MS/MS experiments on
source-produced fragment ions to confirm the interrela-
tionship of the fragment ions and by exact mass mea-
surements on the fragment ions to verify elemental com-
position and correct assignment.
The absolute configuration of cycloheptapeptide 3 was
ascertained by analyzing the acid hydrolysate N-penta-
fluoropropionyl-isopropyl ester' derivatives using chiral
GC (Chirasil-Val III column). Each amino acid was found
to have the L configuration. The disproportionatly high
representation of L-Pro and L-Val in axinastatin 1 (3) and
other strongly antineoplastic peptides' we have discovered
in marine animals suggests that the presence of these am-
ino acids may be an important structural requirement for
controlling cell growth in peptide mediated systems.
The halichondrins proved to be remarkably potent
against all of the 60 cell lines in the U.S. NCl's human
tumor cell line in vitro screen,11 yet with sufficient dif-
ferences in relative sensitivity among the lines to yield a
distinctive mean graph12 profile. For halichondrin B and
homohalichondrin B, the log molar GIoo'S for each line
ranged from -8.10 to -9.70 and -8.05 to -10.08, respec-
(9) (a) Bax, A.; Drobny, G. J. Magn. Res. 1985,61,306. (b) Bax,
A.; Summers, M. A. J. Am. Chem. Soc. 1986, 108, 2094.
(10) Cerny, R. L.; Gross, M. L. Tandem Mass Spectrometry for
Determining the Amino Acid Sequences of Cyclic Peptides and
for Assessing Interactions of Peptides and Metal Ions. In Mass
Spectrometry of Peptides; Desiderio, D. M., Ed.; CRC Press:
Boca Raton, FL, 1990; pp 289-314.
(11) Boyd, M. R. Status of the NCI preclinical antitumor drug
discovery screen. In Principles and Practices of Oncology;
DeVita, V. T., Jr., Hellman, S.; Rosenberg, S. A., Eds.; Lip·
pincott: Philadelphia, 1989; pp 1-12.
(12) Boyd, M. R.; Paull, K. D.; Rubinstein, L. R. Data display and
analysis strategies from the NCI disease-oriented in vitro an-
titumor drug screen. In Antitumor Drug Discovery and De-
velopment; Valeriote, F. A., Corbett, T., Baker, L., Eds.; Klu-
wer Academic Press: Amsterdam, 1990, in press.
tively. The mean log molar GIoo's were -8.95 and -8.99
for halichondrin B and homohalichondrin B, respectively.
The characteristic mean graph "fingerprints" of the hali-
chondrins were very similar; an analysis by the COMPARE
pattern-recognition algorithm13 showed their mean graph
profiles to be most highly correlated to those produced by
structurally unrelated, tubulin-binding standard agentsll
such as vincristine and taxol.
Discovery of the halichondrins in an Axinella sp., a
sponge unrelated to their original source,' suggests that
these exceptionally activel' Porifera constituents may have
a microorganism source. Either by exogenous and/or en-
dogenous biosynthetic processes the marine porifera con-
tinue to be an especially fruitful source of potentially useful
antineoplastic substances of novel structure.
Acknowledgment. With pleasure we acknowledge the
very necessary financial assistance provided by Out-
standing Investigator Grant CA 44344-01Al and PHS
Grants CA-I6049-07-12 awarded by the Division of Cancer
Treatment, National Cancer Institute, DHHS, the Fannie
E. Rippel Foundation, the Arizona Disease Control Re-
search Commission, the Robert B. Dalton Endowment
Fund, Virginia Piper, Eleanor W. Libby, and by the U.S.
Army Medical Research and Development Command un-
der Grant No. DAMDI7-89-Z-9021. Other helpful assis-
tance was provided by the Government of Palau (Dr. T.
Paulis and K. B. Batcheller), Drs. D. Uemura (for an au-
thentic sample of halichondrin B), Y. Kamano, K. Erick-
son, A. M. Yates, and L. P. Tackett, D. N. Tackett, P. J.
Daschner, L. Williams, S. Taylor, the Smithsonian Insti-
tution Oceanographic Sorting Center, the U.S. National
Science Foundation (Grant CHE-8409644), and the NSF
Regional Instrumentation Facility in Nebraska (Grant
CHE-8620177.
Supplementary Material Available: NMR spectra ofaxi-
nastatin 1 and interpretation of tandem MS-MS spectra (28
pages). Ordering information is given on any current masthead
page.
(13) Paull, K. D.; Shoemaker, R. H.; Hodes, L.; Monks, A.; Scu-
diero, D. A.; Rubinstein, L.; Plowman, J.; Boyd, M. R.: Display
and analysis of patterns of differential activity of drugs against
human tumor cell lines: Development mean graph and COM·
PARE algorithm. J. Natl. Cancer Inst. 1989,81,1088-1092.
(14) Cooper, A. J.; Salomon, R. G. Tetrahedron Lett. 1990,31,3813.
George R. Pettit,· Cherry L. Herald, Michael R. Boyd'"
John E. Leet, Claude Dufresne, Dennis L. Doubek
Jean M. Schmidt, Ronald L. Cerny'a
John N. A. Hooper,'d Klaus C. Rutzler'·
Cancer Research Institute and Department of Chemistry
Arizona State University
Tempe, Arizona 85287-1604
Received August 5, 1991