Syntheses and biological activity of amamistatin B and analogs

Article abstract of DOI:10.1021/jo7020532

(Chemical Equation Presented) Amamistatins A and B, natural products isolated from a strain of Nocardia, showed growth inhibition against three human tumor cell lines (IC₅₀ 0.24-0.56 μM). Structurally related mycobactins affect the growth of bo

Full text of DOI:10.1021/jo7020532

Syntheses and Biological Activity of Amamistatin B and Analogs
Kelley A. Fennell,† Ute Mo¨llmann,‡ 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 e.V. Hans Kno¨ll Institute, Beutenbergstrasse 11a, 07745 Jena, Germany
mmiller1@nd.edu
ReceiVed September 19, 2007
Amamistatins A and B, natural products isolated from a strain of Nocardia, showed growth inhibition
against three human tumor cell lines (IC50 0.24-0.56 µM). Structurally related mycobactins affect the
growth of both mycobacterial and human cells through interference with iron chelation. To further probe
the biological activity of this class of compounds, the total syntheses of amamistatin B and two analogs
were completed, and the synthetic samples were screened for tumor cell growth inhibition, HDAC
inhibition, and Mycobacterium tuberculosis growth inhibition. Amamistatin B (15) and diastereomer 18
were both active against MCF-7 cells (IC50 0.12-0.20 µM), and less so against PC-3 cells (IC50 8-13
µM). Amamistatin B only moderately inhibited the growth of M. tuberculosis (MIC 47 µM) but showed
growth promotion of Mycobacterium smegmatis and other bacteria.
Introduction
from strains of Nocardia, with brasilibactin showing similar anti-
cancer activity.
Amamistatins A and B are a pair of natural products that
were isolated from the actinomycete Nocardia asteroides.1,2 As
shown below, their structures were determined and, similar to
mycobacterial siderophores, were found to contain a hydrox-
yphenyloxazole and both linear and cyclic forms of ꢀ-N-
hydroxylysine. Amamistatin A was reported to have anti-
proliferative effects against human tumor cell lines MCF-7
breast, A549 lung, and MKN45 stomach, with IC50 values of
0.48, 0.56, and 0.24 µM, respectively. Amamistatins A and B
both showed cytotoxicity against mouse lymphocytic leukemia
cells P388 (IC50 15 and 16 ng/mL).2 Related natural products
formobactin,3 nocobactin,4 and brasilibactin5 were also isolated
As indicated, the amamistatin structure is also related to that
of mycobactins, intracellular siderophores produced by myco-
bacteria6 for use in the acquisition and transport of iron.7
Mycobactins are known to bind Fe3+ in a 1:1 ratio using three
bidentate ligands: a hydroxyphenyl oxazoline and two hydrox-
amic acids. The addition of exogenous mycobactins can perturb
the growth of mycobacteria,6,8 and synthetic mycobactins have
been investigated as anti-tuberculosis compounds based on their
ability to interfere with the mycobacterial iron transport
process.9,10 Exochelin 772SM, an extracellular mycobacterial
siderophore, has also shown selective cytotoxicity toward breast
cancer cells versus normal cells due to effects of iron chelation.11
(5) 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.
† University of Notre Dame.
‡ Hans Kno¨ll Institute.
(1) Suenaga, K.; Kokubo, S.; Shinohara, C.; Tsuji, T.; Uemura, D.
(6) Snow, G. A. Bacteriol. ReV. 1970, 34, 99-125.
(7) Ratledge, C. Tuberculosis 2004, 84, 110-130.
Tetrahedron Lett. 1999, 40, 1945-1948.
(2) Kokubo, S.; Suenaga, K.; Shinohara, C.; Tsuji, T.; Uemura, D.
Tetrahedron 2000, 56, 6435-6440.
(8) Bosne-David, S.; Bricard, L.; Ramiandrasoa, F.; De´ Roussent, A.;
Kunesch, G.; Andremont, A. Antimicrob. Agents Chemother. 1997, 41,
1837-1839.
(9) Hu, J.; Miller, M. J. J. Am. Chem. Soc. 1997, 119, 3462-3468.
(10) Xu, Y.; Miller, M. J. J. Org. Chem. 1998, 63, 4314-4322.
(3) Murakami, Y.; Kato, S.; Nakajima, M.; Matsuoka, M.; Kawai, H.;
Shin-Ya, K.; Seto, H. J. Antibiot. 1996, 49, 839-845.
(4) Ratledge, C.; Snow, G. A. Biochem. J. 1974, 139, 407-413.
10.1021/jo7020532 CCC: $40.75 © 2008 American Chemical Society
Published on Web 01/04/2008
1018
J. Org. Chem. 2008, 73, 1018-1024

Amamistatin B and Analogs
along with two amamistatin analogs. All three compounds were
screened for growth inhibition against MCF-7 and PC-3 human
tumor cell lines as well as Mycobacterium tuberculosis H37Rv
and other mycobacteria.
Results and Discussion
The synthesis of amamistatin B began with the preparation
of its four primary components, a strategy also used for the
syntheses of amamistatin A22 and several mycobactin-type
compounds.9,10 The fragments obtained from retrosynthetic
disconnection of amamistatin B include a hydroxyphenyl
oxazole, linear and cyclic hydroxamic acids derived from lysine,
and a â-hydroxy acid. The fragments were then coupled in a
stepwise fashion to produce amamistatin B. Using this strategy,
alternate fragment structures can easily be included for the
synthesis of amamistatin analogs.
The growth inhibition observed in breast cancer and other12,13
cell lines upon treatment with exochelin 772SM is most likely
caused by inhibition of ribonucleotide reductase,14 an iron-
containing enzyme that serves as the rate-limiting step in DNA
synthesis.
The anti-tumor activity of the amamistatins may be explained
by a mode of action based on iron chelation, similar to that
employed by mycobacterial compounds. The authors also
proposed that the amamistatins could act as histone deacetylase
(HDAC) inhibitors via their N-formyl hydroxylamine, or ret-
rohydroxamate, moiety.15 Inhibition of the HDAC enzyme leads
to tumor growth suppression,16-19 and the retrohydroxamate
ligand has been previously employed in small molecule HDAC
inhibitors.20,21 In order to further probe the biological activities
of this class of compounds, amamistatin B was synthesized
Syntheses of hydroxyphenyl oxazole fragments 1 and 2 were
carried out according to a procedure described previously.23 The
synthesis of protected linear hydroxamate 3 from Cbz-D-lysine
has also been reported.23 â-Hydroxy acid 4 was synthesized
using the Mukaiyama aldol method24 reported during the
synthesis of amamistatin A.22
(11) Pahl, P. M. B.; Horwitz, M. A.; Horwitz, K. B.; Horwitz, L. D.
Breast Cancer Res. Treat. 2001, 69, 69-79.
(12) Pahl, P. M. B.; Yan, X.-D.; Hodges, Y. K.; Rosenthal, E. A.;
Horwitz, M. A.; Horwitz, L. D. J. Biol. Chem. 2000, 275, 17821-17826.
(13) Chong, T. W.; Horwitz, L. D.; Moore, J. W.; Sowter, H. M.; Harris,
A. L. Cancer Res. 2002, 62, 6924-6927.
(14) Hodges, Y. K.; Antholine, W. E.; Horwitz, L. D. Biochem. Biophys.
Res. Commun. 2004, 315, 595-598.
(15) Fennell, K. A.; Miller, M. J. Abstracts of Papers, 39th National
Organic Chemistry Symposium, Salt Lake City, UT, June 12-16, 2005;
American Chemical Society: Washington, DC, 2005; A80. Fennell, K. A.;
Miller, M. J. Abstracts of Papers, 231st National Meeting of the American
Chemical Society, Atlanta, GA, March 26, 2006; American Chemical
Society: Washington, DC, 2006; ORGN 642.
(16) Meinke, P. T.; Liberator, P. Curr. Med. Chem. 2001, 8, 211-235.
(17) Johnstone, R. W. Nat. ReV. Drug DiscoVery 2002, 1, 287-299.
(18) Grozinger, C. M.; Schreiber, S. L. Chem. Biol. 2002, 9, 3-16.
(19) Miller, T. A.; Witter, D. J.; Belvedere, S. J. Med. Chem. 2003, 46,
5097-5116.
(20) Wu, T. Y. H.; Hassig, C.; Wu, Y.; Ding, S.; Schultz, P. G. Bioorg.
Med. Chem. Lett. 2004, 14, 449-453.
(21) 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.
The synthetic route used for cyclic hydroxamate 9 was based
on earlier work on the synthesis of serine-derived â-lactams,
where it was found that â-mesylated benzyl hydroxamates could
be cyclized upon treatment with tBuOK at low temperature.25
This same idea was applied to the larger ring system of 9 with
(22) Yokokawa, F.; Izumi, K.; Omata, J.; Shioiri, T. Tetrahedron 2000,
56, 3027-3034.
(23) Fennell, K. A.; Miller, M. J. Org. Lett. 2007, 9, 1683-1685.
(24) Kiyooka, S.; Kaneko, Y.; Komura, M.; Matsuo, H.; Nakano, M. J.
Org. Chem. 1991, 56, 2276-2278.
J. Org. Chem, Vol. 73, No. 3, 2008 1019

Fennell et al.
SCHEME 1. Synthesis of Cyclic Hydroxamate Fragment
SCHEME 2. Synthesis of Amamistatin B and a
Diastereomer
moderate success. For the first step, Cbz-lysine was converted
to Cbz-hydroxynorleucine 6a using nitroferricyanide, according
to the procedure published by Baldwin26 (Scheme 1). This
reaction also resulted in the corresponding dehydration product
6b, and the two products were used as a mixture for the
following step. EDC-mediated coupling with O-benzyl hydroxy-
lamine gave benzyl hydroxamates 7a and 7b, which were easily
separated by chromatography. Conversion of 7a to mesylate 8
proceeded cleanly in near quantitative yield. Upon treatment
with tBuOK in DMF, mesylate 8 gave desired benzyl hydrox-
amate 9a along with the O-cyclized hydroximate isomer 9b in
∼3:1 ratio and 80% combined yield. Cyclization with K2CO3
in refluxing acetone gave 9a and 9b in similar ratio and total
yield, and the reaction was more consistent on a larger scale.
Deprotection of the Cbz group of 9a with HBr resulted in amine
10 as the hydrobromide salt.
SCHEME 3. Synthesis of Amamistatin Analog 24
The synthesis of amamistatin B from its fragments began with
EDC-mediated coupling between â-hydroxy acid 4 and cyclic
hydroxamate 10 (Scheme 2). Ester formation between the
resulting â-hydroxy amide 11 and linear hydroxamate 3 was
carried out using DCC and catalytic 4-pyrrolidinopyridine.27
Other esterification conditions, including acid chloride, acid
fluoride, mixed anhydride, and active ester formation, as well
as Mitsunobu conditions, were tried with no success. Global
deprotection of ester 12 was accomplished by hydrogenolysis
to give amine 13 with two free hydroxamic acids. Hydroxyphe-
nyl oxazole 1 was converted to NHS-ester 14 and purified prior
to use. Coupling of 14 with 13 led directly to amamistatin B
15. This same synthetic sequence was repeated using the
opposite enantiomer of linear hydroxamate 3 to give 18, a
diastereomer of amamistatin B.
â-hydroxy acid 4, and the resulting â-hydroxy amide 20 was
again esterified with S-3 using DCC and catalytic 4-pyrrolidi-
nopyridine. Cbz-deprotection of 21 was accomplished with 33
wt % HBr/AcOH. Coupling of amine 22 to phenyl oxazole 2
and deprotection of the benzyl hydroxamate with hydrogenolysis
afforded amamistatin analog 24.
A similar method, shown in Scheme 3, was used for the
synthesis of 24, an amamistatin analog lacking two of the metal-
binding sites present in the parent compound. Commercially
available L-(-)-R-amino-ꢀ-caprolactam‚HCl 19 was coupled to
The total synthesis of natural product amamistatin B (15) was
completed as described above. The syntheses of amamistatin B
diastereomer 18 and structural analog 24 were also carried out
using similar methodology. The synthesis of amamistatin B
(25) Krook, M. A.; Miller, M. J. J. Org. Chem. 1985, 50, 1126-1128.
(26) Baldwin, J. E.; Killin, S. J.; Adlington, R. M.; Spiegel, U.
Tetrahedron 1988, 44, 2633-2636.
(27) Hassner, A.; Alexanian, V. Tetrahedron Lett. 1978, 46, 4475-4478.
1020 J. Org. Chem., Vol. 73, No. 3, 2008

Amamistatin B and Analogs
TABLE 1. Cellular Growth Inhibition by Amamistatin B and
Analogs
TABLE 2. Bacterial Growth Promotion by Amamistatin B
diameter of growth zone (mm)
IC50 (µM)
M. smegmatis B. subtilis S. aureus P. aeruginosa
compound
MCF-7
PC-3
VERO
compound
mc2 155a
ATCC 6633 SG 511 K199 PAO1b
15 (amamistatin B)
18
24
0.12
0.20
14
8
13
16
24
26
40
amamistatin B 15
Fe-mycobactin J
(control)
Fe-ferricrocin
(control)
24
19
37
35
9 (inhib)
34
28
45
20
a Siderophore biosynthesis and transport deletion mutants exhibit growth
zones of 20-24 mm. b Mutants deficient for siderophore biosynthesis and
uptake exhibit growth zones of 24 and 27 mm, respectively.
required approximately the same number of synthetic steps as
were used in Shioiri’s synthesis of amamistatin A,22 and a
similar stepwise fragment coupling strategy was employed. In
order to facilitate the characterization of synthetic compounds,
spectral data for amamistatin B (15) and analogs 18 and 24
were compared with data reported for amamistatins A and B2
and formobactin3 upon isolation (Supporting Information).
Synthetic amamistatin B (15) and analogs (18, 24) were tested
for growth inhibitory activity against MCF-7 (breast) and PC-3
(prostate) human tumor cells and for inhibition of HDAC in
vitro. All three compounds possess an N-formyl hydroxylamine
ligand but differ in their ability to bind iron with a 1:1
stoichiometry. None of the compounds displayed HDAC activity
when tested at 100 µM. This mirrors the result obtained with a
synthetic sample of brasilibactin A,28 even though smaller
amamistatin fragments containing the same retrohydroxamate
ligand previously showed modest HDAC inhibition.23 Presum-
ably, the complete amamistatin and brasilibactin structures adopt
a conformation that prevents the retrohydroxamate moiety from
accessing the enzyme active site, thus resulting in the observed
lack of HDAC inhibition.
All three amamistatin-like compounds demonstrated signifi-
cant growth-inhibitory activity in the MCF-7 and PC-3 tumor
cell assays (Table 1). Amamistatin B (15) and its diastereomer
(18) both reached the nanomolar range of inhibition against
MCF-7 cells, which are often more sensitive than other cell
lines. In both assays, 15 showed the highest level of growth
inhibition, followed closely by 18. Analog 24, lacking two of
the bidentate metal ligands, was significantly less active. These
values are comparable to the 0.48 µM IC50 value reported for
amamistatin A against MCF-7 cells at the time of its isolation.1
Growth inhibition of VERO (monkey kidney) cells was used
as a measure of general mammalian cytotoxicity. Although all
three synthetic compounds exhibited a concerning level of
cytotoxicity, the amount needed for activity against MCF-7 cells
may be low enough to provide an acceptable therapeutic window
for compounds 15 and 18. The difference in activity between
analog 24 and stronger iron chelators 15 and 18 reinforces the
idea that growth inhibition (of both tumor cells and normal cells)
can be affected by changes in iron availability due to chelation.
These results further support the belief that amamistatins and
related natural products could serve as the source or inspiration
for effective therapeutic agents for the treatment of cancer.
On the basis of the results of earlier studies using natural6,8
and synthetic9,10 mycobactins, it was proposed that amamistatins
could have a similar inhibitory effect on the growth of M.
tuberculosis. Of the three synthetic compounds studied here (15,
18, 24), only amamistatin B (15) exhibited observable activity
against M. tuberculosis H37Rv in iron-sufficient (GAS) media
(MIC 46.7 µM). In iron-deficient media, 15 showed 84% growth
inhibition at 128 µM, but the activity quickly dropped off at
lower concentrations.
Further studies showed that amamistatin B (15) is a potent
growth promoter for a different mycobacteria, Mycobacterium
smegmatis mc2 155, as well as other Gram-positive and Gram-
negative bacteria (Table 2). Growth of wild-type M. smegmatis
mc2 15529 was promoted by 15 at a level similar to that of the
native siderophore, Fe-loaded mycobactin J. Growth of M.
smegmatis mutants30 deficient for siderophore biosynthesis and
transport was also promoted to the same extent, indicating that
the activity of amamistatin B (15) is not dependent on the
presence or uptake of native siderophores. The M. smegmatis
mutants used for this study included mc2155-B1 (exochelin
biosynthesis deleted), mc2155-M24 (mycobactin biosynthesis
deleted), mc2155-M24-B3 (exochelin and mycobactin biosyn-
thesis deleted), and mc2155-M24-U3 (mycobactin biosynthesis
and exochelin permease deleted).
Growth of wild-type Gram-positive bacteria Bacillus subtilis
ATCC 6633 was also promoted by 15, while Staphylococcus
aureus SG 511 was inhibited relative to the control (Fe-loaded
ferricrocin). Amamistatin B was also able to promote the growth
of Gram-negative Pseudomonas aeruginosa K199 PAO1 (wild-
type) as well as mutants deficient for pyoverdin and pyochelin
biosynthesis (K648) and the pyoverdin receptor (K690). Sal-
monella typhimurium (enb-7, enterobactin biosynthesis mutant)
and Eschericia coli (wild-type AB 2847 and iron transport
mutants BR 158, H1876, HK9/7, MS 172, and 41/2 lacking
siderophore receptors TonB, FepA, Cir, Fiu, FhuA, and FhuE
alone or in combination) failed to recognize or utilize 15, and
no growth promotion was observed (data not shown). Growth
promotion by amamistatin B can be attributed to either direct
uptake of the iron-loaded compound or transfer of solubilized
Fe3+ to another chelator recognized by the organism. For the
organisms in which no growth promotion is observed, it appears
that iron sequestered by 15 cannot be accessed by the organism
through direct uptake or ligand transfer, and growth in the iron-
depleted media is impaired.
The iron-binding ability of amamistatin B was confirmed
through the use of the chrome azurol S (CAS) assay.31 The
orange halo resulting from iron binding by a 5-µL sample of
15 had a diameter of 9 mm, a value typical for moderately
lipophilic iron chelators. Mycobactins, despite their strong iron-
binding ability, often display a negative result in the CAS assay,
since their increased hydrophobicity prevents diffusion through
the media. Although these studies show that amamistatin B (15)
can bind iron and promote the growth of a variety of bacterial
species under iron-deficient conditions, it is not yet clear whether
(29) Snapper, S. B.; Melton, R. E.; Mustafa, S.; Kieser, T.; Jacobs, W.
R. Mol. Microbiol. 1990, 4, 1911-1919.
(30) Schumann, G.; Mo¨llmann, U. Antimicrob. Agents Chemother. 2001,
45, 1317-1322.
(28) Mitchell, J. M.; Shaw, J. T. Org. Lett. 2007, 9, 1679-1681.
(31) Schwynn, B.; Neilands, J. B. Anal. Biochem. 1987, 160, 47-56.
J. Org. Chem, Vol. 73, No. 3, 2008 1021

Fennell et al.
169.1, 156.3, 135.9, 135.0, 129.3, 128.9, 128.6, 128.4, 128.1, 78.3,
69.5, 67.3, 52.2, 37.4, 31.5, 28.5, 21.4; HRMS (FAB) m/z calcd
for C22H29N2O7S [M + H]+ 465.1690, found 465.1693.
the compound is recognized as a siderophore and taken into
the cell or simply serves as a source of solubilized iron that is
then available for ligand exchange with the native siderophores
produced by the organism.
(S)-1-(Benzyloxy)-3-(benzyloxycarbonylamino)azepan-2-
one 9a. To a solution of 8 (1.94 g, 4.18 mmol) in acetone (100
mL) was added K2CO3 (1.73 g, 12.5 mmol), and the reaction was
heated at reflux in a 75 °C oil bath for 16 h. The opaque white
solution was cooled to room temperature, diluted with EtOAc (400
mL), washed with H2O (2 × 250 mL) and brine (250 mL), dried
over Na2SO4, filtered, and concentrated to a light yellow oil (1.57
g). The crude material was purified by flash chromatography (SiO2,
98:2-90:10 CH2Cl2/EtOAc) to give 9a as a light colored oil that
solidified to a waxy off-white solid upon standing (962 mg, 2.61
mmol, 63%): mp 58-61 °C; 1H NMR (500 MHz, CDCl3) δ 7.43-
7.31 (m, 10H), 6.15 (d, 1H, J ) 6.0 Hz), 5.11 (s, 2H), 4.93 (2d,
2H, J ) 10.5 Hz), 4.29-4.23 (m, 1H), 3.59-3.54 (m, 1H), 3.49-
3.43 (m, 1H), 2.03-1.99 (m, 1H), 1.94-1.83 (m, 1H), 1.73-1.61
(m, 2H), 1.58-1.43 (m, 1H), 1.41-1.34 (m, 1H); 13C NMR (125
MHz, CDCl3) δ 170.0, 155.4, 136.4, 135.0, 129.6, 128.8, 128.5,
128.4, 128.0, 127.9, 76.7, 66.6, 53.2, 52.6, 31.8, 27.5, 26.1; HRMS
(FAB) m/z calcd for C21H25N2O4 [M + H]+ 369.1809, found
369.1834.
The total syntheses of natural product amamistatin B (15)
and two analogs (18, 24) were carried out in order to further
study the biological activity of this class of compounds.
Amamistatin B and its analogs showed significant inhibitory
activity against MCF-7 cells (15 IC50 120 nM, 18 IC50 200 nM,
24 IC50 14 µM) but were less potent in the PC-3 assay (IC50
8-16 µM). All three compounds were inactive in the HDAC
enzyme assay, and only 15 showed modest activity against M.
tuberculosis (MIC ∼46 µM). Amamistatin B was also found
to be a growth promoter for a variety of Gram-positive and
Gram-negative bacteria. Insight into the structure-activity
relationships of amamistatin analogs gained from these studies
will help direct the future synthesis of compounds of this class
targeted toward tuberculosis or tumor growth inhibition.
Experimental Section
(S)-3-Amino-1-(benzyloxy)azepan-2-one Hydrobromide 10.
To a solution of 9a (817 mg, 2.22 mmol) in CH2Cl2 (20 mL) was
added 33 wt % HBr in AcOH (20 mL), and the reaction was stirred
for 30 min. Volatiles were removed under reduced pressure. The
orange residue was left under vacuum for 5 h to give 10 as an
off-white solid (695 mg, 2.20 mmol, 99%): mp 217-219 °C (d);
1H NMR (500 MHz, CD3OD) δ 7.48-7.45 (m, 2H), 7.40-7.36
(m, 3H), 4.97 (2d, 2H, J ) 10.5 Hz), 4.25-4.23 (m, 1H), 3.84-
3.78 (m, 1H), 3.67-3.62 (m, 1H), 2.01-1.97 (m, 2H), 1.80-1.64
(m, 3H), 1.42-1.37 (m, 1H); 13C NMR (125 MHz, CD3OD) δ
169.0, 136.6, 131.0, 130.1, 129.7, 77.8, 53.8, 53.5, 29.9, 28.0, 27.4;
HRMS (FAB) m/z calcd for C13H19N2O2 [M + H]+ 235.1441, found
235.1459.
(S)-N-(Benzyloxy)-2-(benzyloxycarbonylamino)-6-hydroxy-
hexanamide 7a. A solution of Cbz-Lys-OH 5 (3.0 g, 10.7 mmol)
in H2O (45 mL) at 60 °C (internal temp) was adjusted to pH 9.5
with 2 M NaOH. Na2[Fe(CN)5NO]‚2H2O (3.71 g, 12.5 mmol) was
added in portions over 30 min with vigorous stirring, and the pH
was adjusted to 9.5 after each addition. The muddy brown slurry
was stirred for 6 h at 60 °C and pH 9.5. The solution was cooled
and filtered through Celite to remove the red-brown precipitate.
The yellow-brown solution was acidified to pH 1 with 1 N HCl,
and the resulting bright yellow solution was extracted with EtOAc
(3 × 125 mL). The combined organic layers were washed with
brine, dried over Na2SO4, filtered, and concentrated to give a
mixture of (S)-2-(benzyloxycarbonylamino)-6-hydroxyhexanoic acid
6a and (S)-2-(benzyloxycarbonylamino)hex-5-enoic acid 6b as a
crude orange oil (3.07 g). To a solution of 6a and 6b (2.91 g) in
THF (95 mL) and H2O (75 mL) was added NH2OBn‚HCl (2.48 g,
15.5 mmol), and the pH was adjusted to 4.5 with 4 N NaOH. EDC
(11.2 g, 58.4 mmol) was added in portions, and the pH was adjusted
to 4.5 with 1 N HCl after each addition. When the pH stopped
rising (∼1.5 h), the solution was extracted with EtOAc (3 × 150
mL). The combined organic layers were washed with brine, dried
over Na2SO4, filtered, and concentrated. The off-white solid (3.73
g) was purified by flash chromatography (SiO2, 4:1 CHCl3/EtOAc)
to give 7a as a white solid (1.99 g, 5.16 mmol, 48% from 5): mp
115-116 °C; 1H NMR (500 MHz, CDCl3) δ 10.13 (s, 1H), 7.39-
7.31 (m, 4H), 7.30-7.24 (m, 6H), 5.90 (d, 1H, J ) 9.0 Hz), 4.97
(2d, 2H, J ) 12 Hz), 4.84 (d, 2H, J ) 3.5 Hz), 4.05 (q, 1H, J )
7.5 Hz), 3.52-3.43 (m, 2H), 2.96 (br s, 1H), 1.74-1.67 (m, 1H),
1.60-1.53 (m, 1H), 1.48-1.40 (m, 2H), 1.40-1.24 (m, 2H); 13C
NMR (125 MHz, CDCl3) δ 169.5, 156.4, 136.0, 135.1, 129.3, 128.7,
128.6, 128.5, 128.3, 128.0, 78.2, 67.1, 62.0, 52.4, 32.3, 31.8, 21.8;
HRMS (FAB) m/z calcd for C21H27N2O5 [M + H]+ 387.1914, found
387.1917.
(S)-N-((S)-1-(Benzyloxy)-2-oxoazepan-3-yl)-3-hydroxy-2,2-
dimethyl-decanamide 11. To a suspension of (3S)-3-hydroxy-2,2-
dimethyldecanoic acid 4 (456 mg, 2.11 mmol) and 10 (654 mg,
2.08 mmol) in CH3CN (15 mL) were added Et3N (0.293 mL, 2.09
mmol), HOBt (279 mg, 2.07 mmol), and EDC (435 mg, 2.27 mmol)
sequentially. The off-white suspension was stirred at room tem-
perature for 2 days. The reaction mixture was diluted with EtOAc
(150 mL), washed with H2O (75 mL), saturated NaHCO3 (2 × 75
mL), H2O (50 mL), 5% citric acid (2 × 75 mL), and brine (75
mL), dried over Na2O4, filtered, and concentrated to a white solid.
Purification by flash chromatography (SiO2, 5:1 CH2Cl2/EtOAc)
gave 11 as a white solid (637 mg, 1.47 mmol, 71%): mp 153.5-
154.5 °C; 1H NMR (500 MHz, CDCl3) δ 7.41-7.38 (m, 2H), 7.37-
7.30 (m, 3H), 7.29 (d, 1H, J ) 6.5 Hz), 4.92 (2d, 2H, J ) 10.5
Hz), 4.42-4.39 (m, 1H), 3.64-3.57 (m, 1H), 3.54 (br s, 1H), 3.51-
3.42 (m, 2H), 1.96-1.84 (m, 2H), 1.73-1.52 (m, 2H), 1.48-1.40
(m, 2H), 1.35-1.20 (m, s, 15H), 1.16 (s, 3H), 0.86-0.82 (m, 3H);
13C NMR (125 MHz, CDCl3) δ 177.6, 170.8, 135.1, 129.6, 128.9,
128.6, 77.4, 76.8, 52.8, 51.9, 46.1, 31.9, 31.7, 31.0, 29.6, 29.3, 27.6,
26.7, 26.3, 23.6, 22.7, 20.7, 14.1; HRMS (FAB) m/z calcd for
C25H41N2O4 [M + H]+ 433.3061, found 433.3050; LC/MS (5%-
80% CH3CN/10 mM ammonium acetate) tR 9.97, m/z for [M +
(S)-6-(Benzyloxyamino)-5-(benzyloxycarbonylamino)-6-oxo-
hexyl methane-sulfonate 8. To a solution of 7a (3.026 g, 7.83
mmol) in anhydrous pyridine (55 mL) in a flame-dried flask at
0 °C was added MsCl (0.73 mL, 9.43 mmol) slowly. The reaction
was stirred for 25 min at 0 °C and for 2 h 20 min at room
temperature. The solution was diluted with EtOAc (500 mL),
washed with 1 N HCl (2 × 300 mL) and brine (300 mL), dried
over Na2SO4, filtered, and concentrated to leave 8 as a white solid
(3.49 g, 7.51 mmol, 96%) with no need for purification: mp 110-
H]+ 433.4; [R]20 +13.7 (c 7.94, CHCl3).
D
((S)-1-((S)-1-(Benzyloxy)-2-oxoazepan-3-ylamino)-2,2-dimethyl-
1-oxodecan-3-yl) (2R)-2-(Benzyloxycarbonylamino)-6-(N-(ben-
zyloxy)formamido)hexanoate 12. A solution of (2R)-2-(benzy-
loxycarbonylamino)-6-(N-(benzyloxy)formamido) hexanoic acid
R-3 (176 mg, 0.43 mmol) and 11 (150 mg, 0.35 mmol) in anhydrous
CH2Cl2 (3 mL) in a flame-dried flask was stirred over 4 Å MS for
15 min. A solution of dried DCC (113 mg, 0.55 mmol) in CH2Cl2
(1 mL) was added, followed by a solution of dried 4-pyrrolidi-
nopyridine (51 mg, 0.34 mmol) in CH2Cl2 (1 mL). The reaction
was stirred overnight at room temperature. The solution was filtered
to remove sieves and white precipitate. The filtrate was diluted with
1
112 °C; H NMR (500 MHz, CDCl3) δ 9.30 (s, 1H), 7.39-7.30
(m, 10H), 5.51 (d, 1H, J ) 8.5 Hz), 5.08-4.96 (m, 2H), 4.87 (s,
2H), 4.19-4.11 (m, 2H), 4.06-3.99 (m, 1H), 2.95 (s, 3H), 1.81-
1.58 (m, 4H), 1.44-1.36 (m, 2H); 13C NMR (125 MHz, CDCl3) δ
1022 J. Org. Chem., Vol. 73, No. 3, 2008

Amamistatin B and Analogs
EtOAc (30 mL), washed with H2O (15 mL) and brine (20 mL),
dried over Na2SO4, filtered, and concentrated to a white residue
(305 mg). Purification by flash chromatography (SiO2, 9:1 CH2-
Cl2/EtOAc) gave 12 as a colorless oil (61 mg, 0.074 mmol, 21%)
along with recovered 11 (73 mg, 49%): 1H NMR (500 MHz,
CDCl3) δ 8.15 (s, 1H), 7.55-7.51 (m, 1H), 7.46-7.25 (m, 15H),
6.28-6.21 (m, 1H), 5.17-5.00 (m, 2H), 4.88-4.79 (m, 2H), 4.78-
4.75 (m, 2H), 4.71-4.69 (m, 1H), 4.41-4.31 (m, 2H), 3.62-3.46
(m, 4H), 2.06-1.35 (m, 12H), 1.33-1.14 (m, 14H), 1.17 (s, 3H),
0.86-0.81 (m, 3H); 13C NMR (125 MHz, CDCl) δ 174.2, 171.7,
170.3, 163.1, 156.4, 136.5, 135.0, 134.3, 129.6, 129.5, 129.2, 129.0,
128.8, 128.6, 128.5, 128.2, 128.1, 79.7, 77.7, 66.8, 54.6, 52.9, 52.1,
46.2, 46.1, 43.9, 31.9, 31.7, 31.0, 30.2, 29.3, 29.1, 27.6, 26.3, 26.2,
25.9, 24.9, 23.5, 23.0, 14.1; HRMS (FAB) m/z calcd for C47H65N4O9
[M + H]+ 829.4746, found 829.4745; [R]20D +0.9 (c 2.95, CHCl3).
(FAB) m/z calcd for C18H35N2O5 [M + H]+ 327.2642, found
327.2621. [R]20 +9.6 (c 0.70, CHCl3).
D
((S)-2,2-Dimethyl-1-oxo-1-((S)-2-oxoazepan-3-ylamino)decan-
3-yl) (2S)-2-(Benzyloxycarbonylamino)-6-(N-(benzyloxy)forma-
mido)hexanoate 21. To a solution of S-3 (90 mg, 0.22 mmol) and
20 (60 mg, 0.18 mmol) in dry CH2Cl2 (2 mL) was added DCC (61
mg, 0.29 mmol) followed by 4-pyrrolidinopyridine (5 mg, 0.04
mmol). The reaction was stirred at room temperature for 2 days.
The solution was diluted with CH2Cl2 (5 mL) and filtered to remove
the white solid. The filtrate was diluted with CH2Cl2 (50 mL),
washed with H2O and brine, dried over Na2SO4, filtered, and
concentrated. The crude material was purified by flash chroma-
tography (SiO2, 2:1 EtOAc/CH2Cl2) to give 21 as a colorless oil
(73 mg, 0.10 mmol, 55%): 1H NMR (300 MHz, CDCl3) δ 8.16 (s,
1H), 7.56-7.51 (m, 1H), 7.37-7.28 (m, 10H), 6.47-6.41 (m, 1H),
5.98 (d, 1H, J ) 8.7 Hz), 5.11-4.97 (m, 2H), 4.80 (s, 2H), 4.51-
4.45 (m, 1H), 4.39-4.31 (m, 1H), 3.57-3.40 (m, 1H), 3.25-3.07
(m, 4H), 2.09-1.40 (m, 12H), 1.31-1.09 (m, 12H), 1.22 (s, 3H),
1.15 (s, 3H), 0.90-0.81 (m, 3H); HRMS (FAB) m/z calcd for
C40H59N4O8 [M + H]+ 723.4327, found 723.4348.
((S)-1-((S)-1-(Hydroxy)-2-oxoazepan-3-ylamino)-2,2-dimethyl-
1-oxodecan-3-yl) 6-(N-(Hydroxy)formamido)-(2R)-2-(2-(2-hy-
droxyphenyl)-5-methyloxazole-4-carboxamido)hexanoate 15
(amamistatin B). A solution of 12 (20.5 mg, 0.025 mmol) in MeOH
(3 mL) was purged with Ar for 5 min, 10 wt % Pd on carbon (7.8
mg) was added, and the solution was purged again for 3 min. The
reaction was bubbled with H2 for 30 s and stirred under 1 atm H2
for 1.25 h. The catalyst was removed by gravity filtration and rinsed
with MeOH (15 mL). The solution was concentrated to give 13 as
a colorless residue (13.2 mg). 2,5-Dioxopyrrolidin-1-yl 2-(2-
hydroxyphenyl)-5-methyl oxazole-4-carboxylate 14 (9.6 mg, 0.030
mmol), 13, and NaHCO3 (17.1 mg, 0.204 mmol) were dissolved
in H2O (0.6 mL) and THF (0.6 mL). More NaHCO3 (5.5 mg) was
added to bring the pH to 8. The reaction was stirred overnight at
room temperature and then concentrated under reduced pressure
to a clumpy white residue. The crude material was dissolved in
CH3CN/H2O and purified by reversed-phase prep TLC (25:75 H2O/
CH3CN, 1.5 plates used) to give 15 as a white residue (4.5 mg,
0.006 mmol, 25%): (many peaks doubled due to formamide
((S)-2,2-Dimethyl-1-oxo-1-((S)-2-oxoazepan-3-ylamino)decan-
3-yl) 6-(N-(Benzyloxy)formamido)-(2S)-2-(5-methyl-2-phenylox-
azole-4-carboxamido)hexanoate 23. To a solution of 21 (67 mg,
0.093 mmol) in CH2Cl2 (0.7 mL) was added 33 wt % HBr in acetic
acid (0.7 mL), and the reaction was stirred for 30 min at room
temperature. Volatiles were removed under reduced pressure. The
resulting orange oil was dissolved in saturated NaHCO3 (10 mL)
and extracted with CH2Cl2 (3 × 7 mL). The organic solution was
dried over Na2SO4, filtered, and concentrated under reduced pressure
to a colorless residue (45 mg, 0.77 mmol, 83%). The residue was
dissolved in dry CH2Cl2 (1.0 mL) along with 5-methyl-2-pheny-
loxazole-4-carboxylic acid 2 (17 mg, 0.084 mmol). HOBt (10 mg,
0.076 mmol) was added, and the solution was cooled in an ice
bath. EDC (18 mg, 0.092 mmol) was added, and the reaction
mixture was stirred in an ice bath for 20 min and at room
temperature for 40 h. The solution was diluted with EtOAc (15
mL), washed with H2O (10 mL), saturated NaHCO3, 5% citric acid,
and brine, dried over Na2SO4, filtered, and concentrated under
reduced pressure. The crude material was purified by flash
chromatography (SiO2, 5:1 EtOAc/CH2Cl2) to give 23 as a colorless
oil (12 mg, 0.016 mmol, 20%): 1H NMR (600 MHz, CDCl3) δ
8.16 (s, 1H), 8.01 (s, 1H), 7.46-7.44 (m, 4H), 7.38-7.29 (m, 6H),
6.28-6.21 (m, 1H), 4.80 (s, 2H), 4.51-4.49 (m, 1H), 4.44-4.40
(m, 1H), 3.58-3.52 (m, 1H), 3.33-3.12 (m, 4H), 2.68 (s, 3H),
2.13-1.94 (m, 4H), 1.84-1.71 (m, 4H), 1.51-1.38 (m, 4H), 1.36-
1
rotamers) H NMR (600 MHz, DMSO) δ 10.29 (s, 1H), 9.88 (s,
0.5H), 9.71 (s, 1H), 9.44 (s, 0.5H), 8.88-8.81 (m, 1H), 8.20 (s,
0.5H), 7.87 (s, 0.5H), 7.78 (dd, 1H, J ) 1.8 Hz, 6 Hz), 7.46-7.43
(m, 1H), 7.41-7.38 (m, 1H), 7.04 (d, 1H, J ) 8.4 Hz), 6.99-6.96
(m, 1H), 5.08-5.06 (m, 1H), 4.49-4.42 (m, 1H), 4.41-4.36 (m,
1H), 3.87-3.82 (m, 1H), 3.46-3.36 (m, 3H), 2.63 (s, 3H), 1.87-
1.84 (m, 3H), 1.76-1.67 (m, 1H), 1.62-1.46 (m, 5H), 1.41-1.05
(m, 15H), 1.04 (s, 3H), 1.03 (s, 3H), 0.81-0.75 (m, 3H); 13C NMR
(150 MHz, DMSO) δ 173.3, 171.7, 168.6, 160.8, 157.2, 157.0,
155.9, 152.4, 150.6, 137.0(imp.), 134.3(imp.), 132.6, 128.5, 126.8-
(imp.), 126.5, 123.7(imp.), 119.7, 117.2, 110.4, 78.0, 52.3, 52.0,
50.9, 45.6, 34.3(imp.), 31.2, 31.0, 30.8, 30.0, 29.7, 28.9, 28.6, 28.4,
27.7, 27.0, 25.6, 25.5, 24.4, 22.8, 22.0, 15.8, 13.8, 11.3; HRMS
(FAB) m/z calcd for C36H54N5O10 [M + H]+ 716.3865, found
716.3866; LC/MS (5-80% CH3CN/10 mM ammonium acetate) tR
10.63, m/z for [M + H]+ 716.5, 769.5 (+Fe).
1.11 (m, 12H), 1.18 (s, 3H), 1.16 (s, 3H), 0.86-0.80 (m, 3H); 13
C
NMR (150 MHz, CDCl3) δ 177.9, 175.6, 174.4, 171.8, 163.1, 162.1,
153.6, 130.8, 130.1, 129.7, 129.3, 129.0, 128.8, 128.6, 127.0, 126.6,
78.0, 77.7, 52.5, 52.4, 46.4, 46.1, 42.4, 32.4, 32.0, 31.5, 31.3, 29.8,
29.5, 29.3, 29.1, 28.1, 26.9, 26.4, 23.9, 22.8, 14.3, 12.0; HRMS
(FAB) m/z calcd for C43H60N5O8 [M + H]+ 774.4436, found
774.4426; LC/MS (5%-80% CH3CN/10 mM ammonium acetate)
tR 11.55, m/z for [M + H]+ 774.5.
(S)-3-Hydroxy-2,2-dimethyl-N-((S)-2-oxoazepan-3-yl)decana-
mide 20. To a solution of 4 (150 mg, 0.69 mmol) and (S)-3-
aminoazepan-2-one hydrochloride 19 (113 mg, 0.69 mmol) in
CH3CN (4.5 mL) were added Et3N (97 µL, 0.69 mmol), HOBt (93
mg, 0.69 mmol) and EDC (145 mg, 0.76 mmol) sequentially. The
solution was stirred at room temperature overnight. The reaction
mixture was diluted with EtOAc (75 mL), washed with H2O (30
mL), saturated NaHCO3 (2 × 40 mL), H2O (30 mL), 5% citric
acid (2 × 40 mL), and brine (40 mL), dried over Na2O4, filtered,
and concentrated to a colorless oil. Purification by flash chroma-
tography (SiO2, 1:1-1:2 hexanes/EtOAc) gave 20 as a colorless
oil (126 mg, 0.39 mmol, 56%): 1H NMR (300 MHz, CDCl3) δ
7.27 (d, 1H, J ) 6.9 Hz), 6.73 (t, 1H, J ) 6.0 Hz), 4.53-4.43 (m,
1H), 3.62 (br s, 1H), 3.48-3.41 (m, 1H), 3.27-3.21 (m, 2H), 2.04-
1.96 (m, 2H), 1.86-1.69 (m, 2H), 1.58-1.37 (m, 2H), 1.34-1.21
(m, 12H), 1.20 (s, 3H), 1.14 (s, 3H), 0.86-0.81 (m, 3H); 13C NMR
(75 MHz, CDCl3) δ 177.6, 175.9, 77.4, 52.2, 46.0, 42.1, 31.9, 31.6,
31.0, 29.6, 29.3, 28.9, 28.0, 26.7, 23.5, 22.7, 20.4, 14.1; HRMS
((S)-2,2-Dimethyl-1-oxo-1-((S)-2-oxoazepan-3-ylamino)decan-
3-yl) 6-(N-(Hydroxy)formamido)-(2S)-2-(5-methyl-2-phenylox-
azole-4-carboxamido)hexanoate 24. A solution of 23 (6.8 mg,
0.009 mmol) in MeOH (1 mL) was purged with Ar for 5 min, 10
wt % Pd on carbon (1.5 mg) was added, and the solution was purged
again for 2 min. The reaction was bubbled with H2 for 30 s, stirred
under 1 atm H2 for 5.5 h, and bubbled with H2 again for 15 min.
The catalyst was removed by gravity filtration and rinsed well with
MeOH. LCMS showed starting material remaining in the filtrate.
The solution was concentrated under reduced pressure to a volume
of 1 mL and purged with Ar. Pd on carbon (1 mg, 10 wt %) was
added, and the solution was purged again. The reaction was stirred
under 1 atm H2 for 2.5 h, filtered, and concentrated. Purification
by reversed phase prep TLC (75% CH3CN/H2O, 1/2 plate) to
remove residual starting material gave 24 as a white residue (2
mg, 0.003 mmol, 33%): (many peaks doubled due to formamide
J. Org. Chem, Vol. 73, No. 3, 2008 1023

Fennell et al.
1
rotamers) H NMR (600 MHz, DMSO) δ 9.91 (s, 0.5H), 9.46 (s,
knowledge Mrs. Patty Miller for performing MCF-7 and PC-3
cellular assays and Mr. Jose Chaverri for performing HDAC
assays at Notre Dame. Professor Scott Franzblau and Baojie
Wan at the University of Chicago’s Institute for Tuberculosis
Research kindly provided M. tuberculosis inhibition data. The
excellent technical assistance of Irmgard Heinemann and Uta
Wohfield with microbial assays at the HKI is greatly appreci-
ated. K.A.F. received support through a Clare Boothe Luce
Graduate Fellowship and a J. Peter Grace fellowship.
0.5H), 8.24 (s, 1H), 8.02 -8.01 (m, 2H), 7.96-7.90 (m, 1H), 7.58-
7.54 (m, 3H), 7.48-7.36 (m, 2H), 5.10-5.08 (m, 1H), 5.02-4.98
(m, 1H), 4.48-4.42 (m, 1H), 4.36-4.28 (m, 1H), 3.44-3.38 (m,
1H), 3.19-3.11 (m, 1H), 3.08-3.02 (m, 1H), 2.64 (s, 3H), 1.93-
1.80 (m, 3H), 1.79-1.69 (m, 1H), 1.66-1.53 (m, 5H), 1.42-1.11
(m, 15H), 1.08-1.03 (m, 6H), 0.87-0.79 (m, 3H); 13C NMR (150
MHz, DMSO) δ 161.3, 150.6, 137.0, 134.4, 130.9, 129.7, 129.1,
128.5, 126.8, 126.2, 126.0, 123.7, 52.2, 51.5, 45.4, 34.4, 31.1, 30.8,
29.7, 29.5, 28.9, 28.6, 28.3, 27.7, 25.3, 22.4, 22.0, 18.8, 15.8, 13.8,
11.4; HRMS (FAB) m/z calcd for C36H54N5O8 [M + H]+ 684.3967,
found 684.3961; LC/MS (5%-80% CH3CN/10 mM ammonium
acetate) tR 9.63, m/z for [M + H]+ 684.5.
Supporting Information Available: Additional experimental
procedures, 1H and 13C NMR spectra for synthetic compounds, and
NMR data comparison tables for 15, 18, and 24 with natural
products. This material is available free of charge via the Internet
at http://pubs.acs.org.
Acknowledgment. This research was supported by the NIH
(AI054193) and the U.S. Army Medical Research & Material
Command (DAMD17-03-1-0206). The authors gratefully ac-
JO7020532
1024 J. Org. Chem., Vol. 73, No. 3, 2008