Biological activity of the tryprostatins and their diastereomers on human carcinoma cell lines

Article abstract of DOI:10.1021/jm0155953

Tryprostatin A 1 and B 2 are indole alkaloid-based fungal products that act in the G2/M phase of the cell cycle. Tryprostatin A and B as well as their two enantiomers and four diastereomers have been synthesized via a common strategy. As a measure of cyto

Full text of DOI:10.1021/jm0155953

J . Med. Chem. 2002, 45, 1559-1562
1559
Letters
Biologica l Activity of th e Tr yp r osta tin s
a n d Th eir Dia ster eom er s on Hu m a n
Ca r cin om a Cell Lin es
Shuo Zhao,† Kirsten S. Smith,‡ Amy Morin Deveau,‡
Christine M. Dieckhaus,‡ Michael A. J ohnson,‡
Timothy L. Macdonald,‡ and J ames M. Cook*,†
Department of Chemistry, University of
WisconsinsMilwaukee, Milwaukee, Wisconsin 53201, and
Department of Chemistry, University of Virginia,
McCormick Road, Charlottesville, Virginia 22904
Received December 19, 2001
Abstr a ct: Tryprostatin A 1 and B 2 are indole alkaloid-based
fungal products that act in the G2/M phase of the cell cycle.
Tryprostatin A and B as well as their two enantiomers and
four diastereomers have been synthesized via a common
strategy. As a measure of cytotoxicity, these eight stereo-
isomers were assayed for their growth inhibitory properties
in human breast, prostate, and lung cancer cell lines. The
ability of the tryprostatins and the tryprostatin stereoisomers
to induce topoisomerase II-mediated DNA relaxation or to
inhibit tubulin polymerization was also examined. Although
none of the stereoisomers were significantly active in topo-
isomerase II- or tubulin-based assays, ds2-try B 11 was found
to exihibit a cytotoxicity profile more potent than etoposide 3
in the human cancer cell lines examined. In addition, ds2-try
B 11 is comprised of an L-tryptophan derivative coupled to a
D-proline moiety, the latter stereochemistry of which may
enhance the activity of 11 and potential analogues in vivo.
F igu r e 1.
Tryprostatins A 1 and B 2 (Figure 1) have been
isolated as secondary metabolites from the fermentation
broth of a marine fungal strain of Aspergillus fumigatus
BM939. It was found that tryprostatins A 1 and B 2
completely inhibited cell cycle progression of tsFT210
cells in the G2/M phase at a final concentration of 50
µg/mL of 1 and 12.5 µg/mL of 2, respectively.1-3 Trypro-
statins A 1 and B 2 contain a 2-isoprenyltryptophan
moiety and a proline residue, the latter of which is fused
to the diketopiperazine unit.
The biological activity of these alkaloids has stimu-
lated research on their total synthesis.4-10 The interest
in tryprostatin A and B in the present work stems from
the desire to determine whether these alkaloids inhibit
topoisomerase II or tubulin polymerization. The struc-
tures of the tryprostatins resemble an indole-based class
of topo II inhibitors that includes azatoxin 4,11 a dual
topo II/tubulin inhibitor designed as a structure-based
hybrid of etoposide 3 and ellipticine 5 by Macdonald et
al.12 Etoposide 3, a topoisomerase II inhibitor, is one of
the most commonly used agents in cancer chemo-
therapy13 and has improved significantly the treatment
F igu r e 2.
of leukemia, lymphomas, and many solid tumors, in-
cluding testicular and ovarian cancers. Ellipticine 5, a
linear tetracyclic azasubstituted aromatic compound, is
the anticancer active indole alkaloid which intercalates
strongly with DNA.14 The similarities in the structures
of the tryprostatins and azatoxin 4 led us to investigate
topoisomerase II inhibition and tubulin inhibitory activ-
ity of the tryprostatins. A synthetic route to tryprostatin
A 1 and B 2 as well as their enantiomers (6 and 7)7 was
extended to the mismatched pairs 8-11 in order to
assess the effect of the stereochemistry of the diketo-
piperazine structure on the biological activity (Figure
2). The eight diastereomers were evaluated for their
ability to inhibit topoisomerase II (G2 phase) or tubulin
binding protein (M phase). The ultimate goals of this
research are to shed light on the mechanism of action
of the tryprostatins and employ this knowledge to design
agents active against human carcinoma cell lines.
Ch em istr y. The synthesis of ds2-try B 11 is described
here and serves as an example of the preparation of
* To whom correspondence should be addressed. Tel: 414-229-5856.
Fax: 414-229-5530. E-mail: capncook@uwm.edu.
† University of WisconsinsMilwaukee.
‡ University of Virginia.
10.1021/jm0155953 CCC: $22.00 © 2002 American Chemical Society
Published on Web 03/15/2002

1560 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 8
Letters
Sch em e 1
Ta ble 1. Cell Growth Inhibition of Tryprostatins and Their Enantiomers and Diastereomers (at 10, 100 µM) on Human Lung,
Breast, and Prostate Cancer Cell Lines
percent cell survivala
H520
MCF7
PC-3
compound
10 µM
100 µM
10 µM
100 µM
10 µM
100 µM
try A 1
en-try A 6
try B 2
en-try B 7
ds1-try A 8
ds2-try A 10
ds1-try B 9
ds2-try B 11
80.1 ( 4.1
81.7 ( 3.9
77.6 ( 3.6
>100
79.4 ( 4.2
75.2 ( 3.5
60.5 ( 3.5
99.8 ( 1.6
>100
98.5 ( 3.1
76.5 ( 11.2
0.1 ( 0.1
>100
95.0 ( 4.7
>100
99.2 ( 4.2
>100
95.5 ( 2.8
95.8 ( 1.3
>100
>100
97.3 ( 5.9
59.3 ( 3.9
95.6 ( 5.0
83.7 ( 4.2
68.9 ( 6.6
78.9 ( 2.1
>100
>100
88.2 ( 5.8
>100
66.7 ( 5.3
>100
>100
>100
>100
99.3 ( 1.8
>100
88.3 ( 8.4
>100
99.0 ( 4.6
>100
>100
>100
68.5 ( 3.4
0.2 ( 0.0
73.6 ( 5.3
0.0 ( 0.0
a
CellTiter 96 AQueous nonradioactive cell proliferation assay (Promega) was used to determine growth inhibition. Percent inhibition
values were calculated versus control wells and were done in quadruplicate. Control wells contained 0.2% DMSO and the positive control
was either etoposide or m-AMSA. Values are reported ( the standard deviation of the mean.
other analogues 8-10 in the series. In brief, the
protected 3-methylindole 1215 was stirred with N-
bromosuccinimide (NBS) in the presence of 2,2-azo-
bisisobutyronitrile (AIBN) at reflux to provide the
3-(bromomethyl)indole 13, as illustrated in Scheme 1.
When bromide 13 was coupled with the anion of the
Scho¨llkopf chiral auxiliary 14 (derived from D-valine),
the desired trans diastereomer 15 was obtained with
100% trans diastereoselectivity. To introduce the iso-
prenyl group at the indole C(2) position of 15 and
decrease the number of steps earlier reported by Gan
et al.,5 lithium diisopropylamide (LDA) was employed
to form the anion at C(2)7 (Scheme 1). The indole 15
was stirred with LDA at -78 °C, and this was followed
by addition of dry, pure isoprenyl bromide 16 to furnish
2-isoprenyl-pyrazine 17 (82% yield). Since the Scho¨llkopf
chiral auxiliary can tolerate strongly alkaline conditions,
it served as an excellent protecting group for the amino
acid functionality to prevent racemization. The pyrazine
moiety was removed from 17 under acidic conditions
(aqueous HCl, tetrahydrofuran) in greater than 92%
yield to provide the 2-isoprenyltryptophan 18 and
D-valine ethyl ester which could be recycled. With the
key 2-isoprenyltryptophan derivative in hand, the di-
ketopiperazine unit was now constructed. As illustrated
in Scheme 1, 2-isoprenyl-tryptophan (represented by 18)
was stirred with N-Fmoc-D-prolyl chloride 1916 in the
presence of triethylamine (in CHCl3) at room temper-
ature, and this was followed by removal of the solvent
(CHCl3). The Fmoc protecting group was then cleaved
by addition of diethylamine (DEA) in acetonitrile. The
acetonitrile and excess diethylamine were then removed
under reduced pressure. Formation of the diketopiper-
azine unit in 11 as well as removal of the BOC
protecting group from the indole N(H) function were
achieved after each diastereomer was heated individu-
ally in refluxing xylenes (high dilution). Therefore, a
stereospecific, enantiospecific total synthesis of the
diastereomer of tryprostatin B (11), for example, was
accomplished from 12 via alkylation of the correspond-
ing 2-lithio-indole derivative.
Resu lts a n d Discu ssion . The growth inhibition
properties of all eight diastereomers were studied on
three human cancer cell linessMCF7 (breast), PC3
(prostate), and H520 (lung). Data from the assay results
are illustrated in Table 1, and GI50 values are depicted
in Figure 3. Outlined in Table 2 are the results obtained
from the National Cancer Institute (NCI) on the same
cancer cell lines which are in complete agreement with
the present work.

Letters
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 8 1561
sembly assay was prepared as described in the litera-
ture20) indicated that only tryprostatin A 1 was active
against tubulin polymerization (∼250 µM). This was in
agreement with Osada et al.21 who recently reported
that tryprostatin A 1 was a novel inhibitor of MAP-
dependent (MAP ) microtubule-associated protein)
microtubule assembly.
Interest in 11 and the development of new analogues
based on the structure of ds2-try B 11 arises from
examination of results of cell growth inhibition studies.
In brief, ds2-try B 11 was more potent than etoposide 3
in the assay on the three human cancer cell lines
described in this work.
Examination of the data in Tables 1 and 2 indicated
the GI50 of ds2-try B 11 was 17.0 µM whereas the GI50
of etoposide 3 on MCF-7 cells was 55.6 µM. This clearly
demonstrated that ds2-try B 11 was more potent at
MCF-7 cells than etoposide 3. Although the GI50 of ds2-
try B 11 on H520 (GI50 ) 11.9 µM) and PC-3 (GI50
)
12.3 µM) cell lines was similar to that of etoposide 3,
ds2-try B 11 was much more potent than etoposide 3
at higher concentrations (see Figure 3).
Further research is underway to determine the mech-
anism of action of the cytotoxic activity of ds2-try B 11
and the scope of its activity against other cancer cell
lines (NCI data). In addition, results of this work
provide the nature of the stereochemistry (L-Try-D-Pro)
required for the synthesis of active analogues in the
series related to 11.
Refer en ces
(1) Cui, C.; Kakeya, H.; Okada, G.; Onose, R.; Ubukata, M.;
Takahashi, I.; Isono, K.; Osada, H. Tryprostatins A and B, Novel
Mammalian Cell Cycle Inhibitors Produced by Aspergillus
Fumigatus. J . Antibiot. 1995, 48, 1382-1384.
(2) Cui, C.; Kakeya, H.; Osada, H. Novel Mammalian Cell Cycle
Inhibitors, Tryprostatins A, B and Other Diketopiperazines
Produced by Aspergillus Fumigatus II. Physico-Chemical Prop-
erties and Structures. J . Antibiot. 1996, 49, 534-540.
(3) Cui, C.; Kakeya, H.; Okada, G.; Onose, R.; Osada, H. Novel
Mammalian Cell Cycle Inhibitors, Tryprostatins A, B and Other
Diketopiperazines Produced by Aspergillus Fumigatus I. Tax-
onomy, Fermentation, Isolation and Biological Properties. J .
Antibiot. 1996, 49, 527-533.
(4) Depew, K. M.; Danishefsky, S. J .; Rosen, N.; Sepp-Lorenzino,
L. Total Synthesis of Tryprostatin B: Generation of a Nucleo-
philic Prenylating Species from a Prenylstannane. J . Am. Chem.
Soc. 1996, 118, 12463-12464.
(5) Gan, T.; Cook, J . M. Enantiospecific Total Synthesis of Trypro-
statin A. Tetrahedron Lett. 1997, 38, 1301-1304.
F igu r e 3. Comparison of GI50 determinations for ds2-try B
11 and etoposide 3 on selected human cancer cell lines.
Concentrations were tested in quadruplicate or greater and
are shown as ( the standard deviation of the mean.
Ta ble 2. Growth Inhibition Data for ds2-try B 11 for
NCI-H522 (Lung Cancer), MCF-7 (Breast Cancer), and PC-3
(Prostate Cancer) Cell Linesa
NCI-H522
15.8
MCF-7
15.9
PC-3
11.9
GI50 (µM)
a
Data were obtained from NCI.
(6) Gan, T.; Liu, R.; Yu, P.; Zhao, S.; Cook, J . M. Enantiospecific
Synthesis of Optically Active 6-Methoxytryptophan Derivatives
and Total Synthesis of Tryprostatin A. J . Org. Chem. 1997, 62,
9298-9304.
(7) Zhao, S.; Gan, T.; Yu, P.; Cook, J . M. Total Synthesis of
Tryprostatin A and B As Well As Their Enantiomers. Tetra-
hedron Lett. 1998, 39, 7009-7012.
(8) Wang, H.; Usui, T.; Osada, H.; Ganesan, A. Synthesis and
Evaluation of Tryprostatin B and Demethoxyfumitremorgin C
Analogues. J . Med. Chem. 2000, 43, 1577-1585.
(9) van Loevezijn, A.; van Maarseveen, J . H.; Koomen, G.-J . Solid-
Phase Synthesis of Fumitremorgin, Verruculogen and Trypro-
statin Analogues based on a Cyclization/Cleavage Strategy.
Tetrahedron Lett. 1998, 39, 4737-4740.
One of the diastereomers of the tryprostatins, ds2-
try B 11, exhibited potent cytotoxic activity (GI50 < 20
µM, see Table 2) against all three cancer cell lines. This
indicated that (1) the L-Try unit was required since none
of the other tryprostatins (6, 7, 8, and 9) which
contained the D-Try unit exhibited activity; (2) the
presence of the 6-methoxy group on ds2-try A 10 nearly
eliminated the activity; (3) the stereochemistry of ds2-
try B 11 is novel for the unnatural proline residue, may
retard metabolism in vivo, and may provide an agent
with a very long half-life. All eight of the diastereomers
were examined for their ability to inhibit topoisomerase
II-mediated DNA relaxation using an established pro-
tocol.17,18 Analysis of the results for all eight diastereo-
mers indicated there was no activity against topo II
(data19 not shown) for all eight compounds.
(10) Cardoso, A. S.; Lobo, A. M.; Prabhakar, S. Studies in the Aza-
Cope Reaction: a Formal Highly Enantioselective Synthesis of
Tryprostatin A. Tetrahedron Lett. 2000, 41, 3611-3613.
(11) Cline, S. D.; Macdonald, T. L.; Osheroff, N. Azatoxin is
a
Mechanistic Hybrid of the Topoisomerase II-Targeted Anticancer
Drugs Etoposide and Ellipticine. Biochemistry 1997, 36 (42),
13095-13101.
(12) Solary, E.; Leteurtre, F.; Paull, K. D.; Scudiero, D.; Hamel, E.;
Pommier, Y. Dual Inhibition of Topoisomerase II and Tubulin
Polymerization by Azatoxin, a Novel Cytotoxic Agent. Biochem.
Pharmacol. 1993, 45 (12), 2449-2456.
Analysis of the data for inhibition of tubulin poly-
merization of all eight compounds (microtubule as-

1562 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 8
Letters
(13) Marchetti, F.; Bishop, J . B.; Lowe, X.; Generoso, W. M.; Hozier,
J .; Wyrobek, A. J . Etoposide Induces Heritable Chromosomal
Aberrations and Aneuploidy During Male Meiosis in the Mouse.
Proc. Natl. Acad. Sci. U.S.A. 2001, 98 (7), 3952-3957.
(14) Behravan, G.; Leijon, M.; Sehlstedt, U.; Norden, B.; Vallberg,
H.; Bergman, J .; Graslund, A. The Interaction of Ellipticine
Derivatives with Nucleic Acids Studied by Optical and 1H NMR
Spectroscopy: Effect of Size of the Heterocyclic Ring System.
Biopolymers 1994, 34 (5), 599-609.
(18) Muller, M. T.; Spitzner, J . R.; Didonato, J . A.; Metha, V. B.;
Tsutsui, K. Single-strand DNA Cleavages by Eukaryotic Topo-
isomerase II. Biochemistry 1988, 27, 8369-8379.
(19) Zhao, S. Part I. The Enantiospecific Total Synthesis of Trypro-
statin A and B As Well As Their Enantiomers and Mismatched
Pairs. Part II. The Enantiospecific Total Synthesis of the Ring-A
Oxygenated Sarpagine Indole Alkaloids (+)-Majvinine, (+)-10-
Methoxyaffinisine and (+)-Na-Methylsarpagine As Well As
the First Total Synthesis of the Alstonia Bisindole Alkaloid
Macralstonidine. Ph.D. Thesis, University of Wisconsins
Milwaukee, Milwaukee, WI, 2001.
(15) Liu, R.; Zhang, P.; Gan, T.; Cook, J . M. Regiospecific Bromination
of 3-Methylindoles with NBS and Its Application to the Concise
Synthesis of Optically Active Unusual Tryptophans Present in
Marine Cyclic Peptides. J . Org. Chem. 1997, 62 (21), 7447-7456.
(16) Beyermann, M.; Bienert, M.; Niedrich, H. Rapid Continuous
Peptide Synthesis via FMOC Amino Acid Chloride Coupling and
4-(Aminomethyl)piperidine Deblocking. J . Org. Chem. 1990, 55,
721-728.
(20) Bulman, A. L. Kinases and Drugs Targeting the Microtuble
System. Ph.D. Thesis, University of Virginia, Charlottesville, VA,
1998.
(21) Usui, T.; Kondoh, M.; Cui, C.; Mayumi, T.; Osada, H. Trypro-
statin A, a Specific and Novel Inhibitor of Microtubule Assembly.
Biochemistry J . 1998, 333, 543-548.
(17) Spitzner, J . R.; Muller, M. T. A Consensus Sequence for Cleavage
by Vertebrate DNA Topoisomerase II. Nucleic Acid Res. 1988,
16, 5533-5556.
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