Syntheses of amamistatin fragments and determination of their HDAC and antitumor activity

Article abstract of DOI:10.1021/ol070382e

Amamistatins A and B are natural products found to have anti-proliferative effects against MCF-7, A549, and MKN45 human tumor cell lines (IC₅₀ 0.24-0.56 μM). It was proposed that their activity was due to histone deacetylase (HDAC) inhibition m

Full text of DOI:10.1021/ol070382e

ORGANIC
LETTERS
2007
Vol. 9, No. 9
1683-1685
Syntheses of Amamistatin Fragments
and Determination of Their HDAC and
Antitumor Activity
Kelley A. Fennell† and Marvin J. Miller*,†,‡
Department of Chemistry and Biochemistry and Walther Cancer Center,
UniVersity of Notre Dame, Notre Dame, Indiana 46556, and Leibniz Institute for
Natural Product Research and Infection Biology, Hans-Kno¨ll-Institute,
Beutenbergstrasse 11a, D-07745 Jena, Germany
mmiller1@nd.edu
Received February 14, 2007
ABSTRACT
Amamistatins A and B are natural products found to have anti-proliferative effects against MCF-7, A549, and MKN45 human tumor cell lines
(IC50 0.24 0.56 M). It was proposed that their activity was due to histone deacetylase (HDAC) inhibition mediated by the N-formyl-N-hydroxy
−
µ
lysine moiety. Amamistatin B fragment analogs were synthesized and screened for biological activity. These compounds were modest HDAC
inhibitors and showed antitumor activity against MCF-7 and PC-3 human tumor cells.
Histone deacetylase (HDAC) enzymes work with histone
acetyltransferases (HATs) to modulate levels of histone
acetylation, which are directly related to gene expression.1,2
Aberrant recruitment of histone deacetylases can lead to gene
silencing and tumor growth, and small molecule HDAC
inhibitors cause differentiation, growth arrest, and apoptosis
in tumor cells.3 Many HDAC inhibitors such as Trichostatin
A (TSA)4 and SAHA5 (Figure 1) contain a hydroxamic acid
moiety, used to chelate the catalytic Zn2+ found in the
enzyme active site. The N-formyl hydroxylamine, or retro-
hydroxamate, is an alternative zinc-binding ligand that has
also been employed in the synthesis of HDAC inhibitors.6,7
Amamistatins A and B are natural products isolated from
the actinomycete Nocardia asteroides.8 Amamistatin A was
found to have anti-proliferative effects against MCF-7 breast,
A549 lung, and MKN45 stomach human tumor cell lines,
with IC50 values of 0.48, 0.56, and 0.24 µM, respectively.
The amamistatin structure, containing a lysine-derived N-
formyl hydroxylamine, is very similar to that of related
natural products formobactin,9 nocobactin,10 brasilibactin,11
and the mycobactins12 (Figure 2). We proposed that the anti-
(6) Wu, T. Y. H.; Hassig, C.; Wu, Y.; Ding, S.; Schultz, P. G. Bioorg.
Med. Chem. Lett. 2004, 14, 449-453.
(7) Nishino, N.; Yoshikawa, D.; Watanabe, L. A.; Kato, T.; Jose, B.;
Komatsu, Y.; Sumida, Y.; Yoshida, M. Bioorg. Med. Chem. Lett. 2004,
14, 2427-2431.
(8) (a) Suenaga, K.; Kokubo, S.; Shinohara, C.; Tsuji, T.; Uemura, D.
Tetrahedron Lett. 1999, 40, 1945-1948. (b) Kokubo, S.; Suenaga, K.;
Shinohara, C.; Tsuji, T.; Uemura, D. Tetrahedron 2000, 56, 6435-6400.
(9) Murakami, Y.; Kato, S.; Nakajima, M.; Matsuoka, M.; Kawai, H.;
Shin-Ya, K.; Seto, H. J. Antibiot. 1996, 49, 839-845.
† University of Notre Dame.
‡ Hans-Kno¨ll-Institute.
(1) Meinke, P. T.; Liberator, P. Curr. Med. Chem. 2001, 8, 211-235.
(2) Johnstone, R. W. Nat. ReV. Drug DiscoVery 2002, 1, 287-299.
(3) Miller, T. A.; Witter, D. J.; Belvedere, S. J. Med. Chem. 2003, 46,
5097-5116.
(4) Yoshida, M.; Kijima, M.; Akita, M.; Beppu, T. J. Biol. Chem. 1990,
265, 17174-17179.
(5) Richon, V. M.; Emiliani, S.; Verdin, E.; Webb, Y.; Breslow, R.;
Rifkind, R. A.; Marks, P. A. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 3003-
3007.
(10) Ratledge, C.; Snow, G. A. Biochem. J. 1974, 139, 407-413.
(11) Tsuda, M.; Yamakawa, M.; Oka, S.; Tanaka, Y.; Hoshino, Y.;
Mikami, Y.; Sato, A.; Fujiwara, H.; Ohizumi, Y.; Kobayashi, J. J. Nat.
Prod. 2005, 68, 462-464.
(12) Snow, G. A. Bacteriol. ReV. 1970, 34, 99-125.
10.1021/ol070382e CCC: $37.00
© 2007 American Chemical Society
Published on Web 03/31/2007

Scheme 1. Synthesis of Hydroxyphenyl Oxazole
tion and oxidation13 led to oxazole 4 without the need to
isolate the intermediate oxazoline. Acid 5 was revealed via
hydrogenolytic removal of both benzyl protecting groups.
The first step in the synthesis of the N-formyl hydroxyl-
amine fragment was methyl esterification of N-R-Cbz-D-
lysine 6 (Scheme 2). Oxidation of the terminal amino group
Figure 1. Hydroxamic acid and retrohydroxamate HDAC inhibi-
tors.
cancer activity exhibited by the amamistatins and related
compounds was due to HDAC inhibition mediated by the
N-formyl hydroxylamine ligand.
Scheme 2. Synthesis of Protected Lysine Retrohydroxamate
As an initial test of our hypothesis, we synthesized a set
of compounds containing the retrohydroxamate, which
corresponds with a portion of the amamistatin structure. With
the N-formyl hydroxylamine in place for Zn2+ coordination,
the hydroxyphenyl oxazole moiety should be in a position
to mimic the aromatic cap groups found in many HDAC
inhibitors.
Synthesis of the hydroxyphenyl oxazole fragment began
with salicylic acid and threonine protected as the benzyl ether
and benzyl ester, respectively (Scheme 1). EDC-mediated
coupling afforded R-hydroxy amide 3, and one-pot cycliza-
with dibenzoyl peroxide14 was followed by formylation.
Removal of the benzoyl group and reprotection as the benzyl
hydroxamate led to the more stable compound 9. Cbz
deprotection with HBr gave the hydrobromide salt 10 in
quantitative yield.
Oxazole 5 and retrohydroxamate 10 were joined via an
EDC-mediated coupling to give amide 11. Deprotection by
hydrogenolysis revealed the N-formyl hydroxylamine (12,
Scheme 3). A small set of related retrohydroxamates was
prepared using the same method starting from benzoic acid
and N-R-Cbz-L-lysine.
The amamistatin-based retrohydroxamates 12-16 were
screened in an enzymatic HDAC assay, as well as in cellular
(13) Phillips, A. J.; Uto, Y.; Wipf, P.; Reno, M. J.; Williams, D. R. Org.
Lett. 2000, 2, 1165-1168.
(14) Wang, Q. X.; King, J.; Phanstiel, O., IV. J. Org. Chem. 1997, 62,
8104-8108.
Figure 2. Amamistatins A and B and related natural products.
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Org. Lett., Vol. 9, No. 9, 2007

hydroxylamine 16 was inactive. This mirrored what has been
reported in the literature for retrohydroxamate HDAC
inhibitors.6,7 Interestingly, the level of HDAC inhibition for
these compounds did not correlate with growth inhibition in
either the MCF-7 or PC-3 cellular assays. This indicates that
although the amamistatin-based N-formyl hydroxylamines
may have an effect on tumor cell growth via HDAC
inhibition, alternate mechanisms of action must also be
involved.
Scheme 3. Synthesis of Amamistatin-Based HDAC Inhibitors
To follow up on this work, the syntheses of Amamistatin
B and analogs were completed. These compounds, also
containing the N-formyl hydroxylamine ligand, showed no
HDAC inhibition, even at a concentration of 100 µM. This
same lack of HDAC inhibition was also observed with
synthetic brasilibactin A, as reported by Shaw.15 Amamistatin
B and analogs were, however, extremely potent growth
inhibitors of MCF-7 cells, with IC50 values of 120-200 nM.
Full details regarding the synthesis and biological activity
of these compounds will be reported shortly.
assays against MCF-7 (breast cancer) and PC-3 (prostate
cancer) cells. The assay results are summarized in Table 1.
Table 1. HDAC Inhibition and Antitumor Activity of
Amamistatin-Based Retrohydroxamates
Acknowledgment. This research was supported by the
NIH (AI054193) and the U.S. Army Medical Research &
Materiel Command (DAMD17-03-1-0206). The authors
gratefully acknowledge Mrs. Patty Miller for performing
MCF-7 and PC-3 cellular assays and Mr. Jose Chaverri for
performing HDAC assays. K.A.F. received support through
a Clare Boothe Luce Graduate Fellowship and a J. Peter
Grace fellowship. M.J.M. acknowledges the generous hos-
pitality of the Hans-Kno¨ll-Institute.
inhibition (%)
HDAC
(10 µM)
MCF-7
(20 µM)
PC-3
(20 µM)
R1
R2
/
12
13
14
15
16
OH
H
OH
H
H
H
H
H
R
R
S
S
S
45
22
73
53
0
19
18
65
65
50
63
22
43
23
18
Supporting Information Available: Experimental pro-
cedures and NMR spectra for all compounds. This material
is available free of charge via the Internet at http://pubs.acs.org.
OH
Me
OL070382E
The compounds containing N-formyl hydroxylamine (12-
15) showed modest HDAC inhibition, whereas the N-acetyl
(15) Mitchell, J. M.; Shaw, J. T. Org. Lett. 2007, 9, 1679-1682.
Org. Lett., Vol. 9, No. 9, 2007
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