Syntheses and studies of amamistatin B analogs reveals that anticancer activity is relatively independent of stereochemistry, ester or amide linkage and select replacement of one of the metal chelating groups

Article abstract of DOI:10.1016/j.bmcl.2011.01.084

A series of analogs of the amamistatin natural products was designed and synthesized to facilitate additional anticancer structure-activity relationships. The results indicate that the anticancer activity is relatively independent of stereochemistry, ester or amide linkage and replacement of the oxazoline/oxazole based iron-binding group with a catechol.

Full text of DOI:10.1016/j.bmcl.2011.01.084

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2611–2615
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Syntheses and studies of amamistatin B analogs reveals that anticancer
activity is relatively independent of stereochemistry, ester or amide linkage
and select replacement of one of the metal chelating groups
⇑
Chunrui Wu, Patricia A. Miller, Marvin J. Miller
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of analogs of the amamistatin natural products was designed and synthesized to facilitate addi-
tional anticancer structure–activity relationships. The results indicate that the anticancer activity is rel-
atively independent of stereochemistry, ester or amide linkage and replacement of the oxazoline/oxazole
based iron-binding group with a catechol.
Received 13 December 2010
Revised 18 January 2011
Accepted 19 January 2011
Available online 22 January 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Amamistatins
Anti-cancer
Structure–activity relationships (SAR)
Design
Syntheses
Studies
Iron binding natural products
Amamistatins A (1) and B (2) are natural products isolated from
the actinomycete Nocardia asteroides (Fig. 1).1–3 Amamistatin A (1)
exhibits anti-proliferative activity against a variety of cancer cell
lines, including MCF-7 breast, A549 lung, and MKN45 stomach can-
cer. Amamistatins A and B also show cytotoxicity against mouse
lymphocytic leukemia cells P388 (IC50 15 and 16 ng/mL, respec-
tively).1 In addition, these compounds structurally resemble myco-
bactins,4 analogs of which exhibit anti-tuberculosis activity and
have other therapeutic potential due to the chelation of iron
(III),4–7 and other more recently reported siderophores including
the nocardimicins,8,9 nocobactin NA,10,11 formobactin,12 and brasi-
libactin A.13–15 Iron chelation by these similar siderophores arises
from three coordination sites, a hydroxyphenyl oxazoline or oxa-
zole and two hydroxamic acids. The origin of the anticancer activ-
ity of amamistatins is not yet known, but because of they are
natural metal ligands, they are capable of chelating metals essen-
tial for enzymatic activities. It was previously proposed that the
anticancer activity of amamistatins was due to HDAC inhibition
by the N-formyl hydroxylamine ligand.5 However, recent studies
showed that the synthetic amamistatin B and an analog lacking
the N-hydroxy group on the cyclic hydroxamate displayed no
Herein, we wish to report the design and syntheses of new
analogs of amamistatins and their activity against MCF-7 and PC-
3 human cancer cell lines. The first series is represented by analogs
3–6, with variation of the oxazole moiety and the ester linkage as
well as controlled stereochemical modification. The second com-
pound type involves change of the hydroxyphenyl oxazole to a cat-
echol (7) since catechols are frequent ligands in other natural
siderophores16 (Fig. 1).
The retro-synthetic analysis of amamistatin B analogs is shown
in Figure 1. Disconnection of the ester reveals the constituent
mycobactic acid and cobactin and further simplification gives four
fragments: a hydroxyphenyl oxazole or its analog (A), a linear
hydroxamic acid (B), a b-hydroxy or b-amino acid (C), and a
lysine-based cyclic hydroxamic acid, N-hydroxycaprolactam (D).
Syntheses of fragments A (compounds 8–10), B (compound 11),
and D (compound 12) (Scheme 2) were carried out according to
published procedures.5–7 The oxazole, and oxazoline were pre-
pared from salicylic acid and the corresponding amino acid, serine
(R1 = H) or threonine (R1 = Me). Both the linear and cyclic hydroxa-
mic acids were prepared from Cbz-protected lysine.
The b-hydroxy carboxylic acid 16 (fragment C) was derived
from Meldrum’s acid (13) and octanoyl chloride using an acylation,
decarboxylation, and asymmetric reduction protocol (Scheme 1).
Thus, treatment of Meldrum’s acid with octanoyl chloride in the
presence of pyridine followed by refluxing in MeOH afforded
b-keto ester 14 in 77% yield.17 Asymmetric hydrogenation of 14
HDAC activity at the concentration of 100 l
M.6
⇑
Corresponding author.
E-mail address: mmiller1@nd.edu (M.J. Miller).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.084

2612
C. Wu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2611–2615
R
R1
O
N
O
O
O
O
O
O
H
H
N
N
N
N
N
O
N
N
H
YN
OH
OH
H
OH
OH
O
O
O
O
R1 = H, CH3
OH Y = O, NH
H
H
N
OH
O
1 Amamistatin A R = OMe
2 Amamistatin B R = H
R1
O
R1
O
N
X
N
O
O
O
O
O
H
N
H
N
N
N
OH
HY
N
H
YN
N
OH
OH
H
OH
OH
O
O
O
mycobactic acid
cobactin
H
N
H
OH
OH
O
3 X = O, oxazole, R1 = Me, Y = O
4 X = O, oxazole, R1 = Me, Y = NH
5 X = O, oxazole, R1 = H, Y = O
6 X = O, oxazoline, R1 = Me, Y = O
O
H2N
OH
N
R1
O
OH
N
N
O
H2N
OH
O
O
H
N
OH
O
O
HY
OH
N
H
HO
O
N
N
H
OH
OH
O
OH
O
O
hydroxyphenyl oxazole
or
hydroxyphenyl oxazoline
cyclic
hydroxamic
acid
β-hydroxy
or amino
acid
liner
hydroxamic
acid
H
OH
7
O
A
B
D
C
Figure 1. Amamistatins, analogs and their retro-synthetic analysis.
O
amamistatins) L-(S)-configuration of the linear lysine components
R1
OH
CbzHN
were first targeted for syntheses, because we had previously
shown that the diastereomer of amamistatin B with the (S) config-
uration of the linear lysine had anticancer activity similar to ama-
O
N
OH
N
H3BrN
OBn
OH
O
O
mistatin B, itself, which has a D-(R)-linear lysine component. Thus,
H
N
8
9
X = O, oxazole, R1 = Me
X = O, oxazole, R1 = H
additional changes of stereochemistry at other stereogenic centers
including the b-hydroxy amide were anticipated to help elaborate
structure–activity relationship (SAR) studies. A convergent ap-
proach was designed to couple oxazole A with lysine derivative B
to give the corresponding mycobactic acid and couple separate
enantiomers of b-hydroxy acids C (Y = O) with cyclic hydroxamate
D to generate diastereomeric cobactins. Final coupling of the
mycobactic acid with the individual diastereomers of the cobactins
would complete the syntheses.
OBn
O
11
10 X = O, oxazoline, R1 = H
12
Fragment B
Fragment D
Fragment A
1) H15C7COCl
O
O
O
Pyridine, rt
2) MeOH, reflux
O
OMe
77% 2 steps
O
O
14
13
As anticipated, DCC-mediated coupling of b-hydroxy carboxylic
acids 16R and 16S with hydroxamate 12 provided diastereomeri-
cally pure cobactins 17R and 17S in high yields (Scheme 2). Inter-
estingly, HOAt/EDC-mediated coupling of 16R and 12 was much
less effective and gave only a 34% yield of 17R. However, use of
HOAt/EDC was more effective than a DCC-mediated processes for
coupling of oxazole 8 and hydroxamic acid 18 to give protected
mycobactic acid 19, especially with addition of DMAP. LiOH-
mediated saponification of methyl ester 19 gave mycobactic acid
20 cleanly.
The direct coupling of mycobactic acid 20 and cobactins 17R
and 17S was anticipated to provide the dibenzyl-protected forms
of diastereomeric amamistatin analogs 3. However, all attempts
at esterification were unsuccessful, including use of EDC/4-PPY,
HOAt/EDC or Yamaguchi coupling conditions. Since we had previ-
ously found that coupling of simple protected forms of linear lysine
derivatives (fragment B) with other cobactins, including those
Ru*, H2
MeOH
OH
O
OH
O
LiOH
∗
∗
OMe
OH
16R 85%
16S 90%
(R)-BINAP
(S)-BINAP
15R 89% 98% ee
15S 90% 96% ee
Fragment C
Scheme 1.
with a catalyst generated in situ from (cod)Ru(2-methylallyl)2 and
(R)- or (S)-BINAP worked very efficiently under one atmosphere of
hydrogen to provide the (R)- or (S)-b-hydroxy ester 15R/15S in
excellent optical (ee) and chemical yields.18 Saponification of these
b-hydroxy esters with LiOH and subsequent acidification gave the
corresponding b-hydroxy carboxylic acids 16R/16S.
With all fragments in hand, analogs 3, with either the (R) or (S)
stereochemistry of the b-hydroxy amide and the unnatural (for

C. Wu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2611–2615
2613
1) NHS, DCC
2) NaHCO3
OH
O
OH
O
+
N
N
∗
∗
OBn
OBn
OH
BrH3N
N
H
O
O
12
17R 92%
17S 75%
16R
16S
O
O
O
O
N
O
O
N
H
H
BrH3N
N
N
OMe
OH
OH
N
O
N
N
LiOH
90%
OH
OH
O
O
OH
O
N
H
8
H
H
BnO
18
BnO
BnO
O
O
O
20
19
method A: HOBt, EDC, Et3N
method B: HOAt, EDC, DMAP
method C: NHS, DCC, NaHCO3
35%
70%
59%
Scheme 2.
containing a tertiary alcohol instead of the secondary alcohol of 18,
was effective,5 we attempted the same method on separate sub-
strates 17R/S. Again, to our surprise, these less hindered alcohols
(17R/S) did not react with the linear lysine component 11 under
the same conditions. A variety of other conditions, including
1-methylimidazole/TsCl mediated coupling,19,20 Mitsunobu
reaction,21 EDC/4-PPY all failed to generate 21. Fortunately, in this
case, Yamaguchi conditions provided the desired individual diaste-
reomers of 21 in good yields.22 HOAt/EDC-mediated coupling of
acid 8 and the individual diastereomers of dibenzyl-protected
amines 22R/S, derived from treatment of the Cbz-protected deriv-
atives 21R/S with HBr/HOAc provided both isomers (23R/S) of the
benzyl-protected amamistatin analogs in approximately 80% yield.
Separate hydrogenolysis of 23R and 23S generated analogs 3R and
3S, respectively, in about 70% yield each (Scheme 3).
or inhibition of Mycobacterium smegmatis and Mycobacterium tuber-
culosis only on the basis of their diastereomeric relationship,23 indi-
vidual amamistatin diastereomers 6, each with varied configuration
at the stereogenic center of the oxazoline and the b-hydroxy ester
components, were also prepared from 21R and 21S. The yields for
the four isomers of 6 and 25 are also summarized in Table 1.
We next turned our attention to analogs 4R and 4S in which the
normal ester was replaced by an amide. This required preparation
of the corresponding diastereomeric amines, 25 which was accom-
plished by reaction of the individual diastereomeric alcohols 17R
and 17S with diphenyl phosphoryl azide under Mitsunobu-like
conditions and subsequent reduction of the resulting azides
(Scheme 4). To complete the syntheses of the amides, we again
used a sequential process in which compounds 26R/S were synthe-
sized first before the final coupling to the oxazole. Diastereomers
26 were produced by coupling individual diastereomers of 25 with
the NHS activated ester of 11. Removal of the Cbz-protecting
groups followed by coupling with the NHS-activated ester 27 of
oxazole 8 provided the benzyl-protected analogs 28R/S. Hydrogen-
olytic deprotection of diastereomeric forms of 28 afforded the de-
sired amide-linked analogs 4 in excellent yields.
The des-methyl oxazole analogs 5R and 5S were also synthe-
sized using a similar strategy. Starting from individual diastereo-
mers of 21, removal of the Cbz-protecting groups with HBr/
HOAc, followed by EDC/HOAt-mediated couplings with oxazole
acid 9 afforded the protected analogs 24, which were debenzylated
by hydrogenolysis to give analogs 5 (Table 1).
Because of the different biological activities exhibited by myco-
bactins S and macobactin T which differ in their growth promotion
Alteration of iron-binding moieties of amamistatins was also
considered. Thus, in a second series of analogs, we replaced the
O
O
O
C7H15
O
CbzHN
OH
N
CbzHN
∗
O
N
OBn
N
2,4,6-trichlorobenzoyl chloride
DIPEA
DMAP
H
OH
O
O
+
N
∗
OBn
C7H15
N
H
O
110 oC, tol/THF, 16 h
H
N
H
OBn
OBn
O
11
17R
17S
21R 82%
21S 76%
O
O
O
C7H15
O
C7H
∗
15 O
H
HOAt, EDC•HCl
N
N
BrH3N
N
N
∗
HBr/HOAc
O
DMAP, ACN
OBn
O
N
N
H
N
H
OR
21R
21S
OH
O
O
O
O
O
N
H
N
H
O
OH
OBn
OH
RO
O
8
23R R = Bn
23S R = Bn
76%
22
Pd/C
H2
81%
Pd/C
H2
3S R = H
3R R = H
73%
71%
Scheme 3.

2614
C. Wu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2611–2615
Table 1
O
O
C7H
∗
O
O
15 O
C7H15 O
H
∗
N
N
CbzHN
N
1) HBr/HOAc
N
∗
O
N
O
N
N
H
OBn
OR
N
H
OH
O
O
O
2) Acid 10 or 11, EDC
HOAt, DMAP
H
H
RO
OBn
OH
O
O
25 R = Bn
(Oxazoline)
24 R = Bn
(Oxazole)
N
21R or 21S
O
Pd/C
H2
Pd/C
H2
OH
6 R = H
5 R = H
Oxazole
9
Oxazoline 10
Product
Acid
Compound 21
% Yield of 24 or 25
% Yield of 5 or 6
5R
5S
6SR
6SS
6RR
6RS
9
9
10S
10S
10R
10R
21R
21S
21R
21S
21R
21S
70
73
36
52
59
51
85
80
94
90
93
92
oxazoline/oxazole group with a catechol to form the separate dia-
stereomers 7R and 7S. The syntheses, summarized in Scheme 5, are
similar to those used to prepare 4 and 6.24
The analogs of amamistatin B were submitted for biological
studies using MCF-7 and PC-3 tumor cellular assays. All of the
analogs were found to have moderate to good activity. As shown
in Table 2, the anticancer activity varied only slightly among all
of the analogs, including diastereomers, with the catechol (7),
being only slightly less active overall.
OH
O
NH2
O
1) DPPA, PPh3, DIAD
2) PPh3, THF/H2O
N
N
OBn
OBn
N
H
C7H15
N
H
C7H15
O
O
25S 75%
25R 63%
17R
17S
O
O
C7H15
O
N
CbzHN
N
H
OBn
N
H
O
Conclusion: The syntheses of a series of amamistatin analogs
was accomplished and all of the analogs were found to be
1) DCC, NHS
11
2) 25S or 25R
H
N
NaHCO3
OBn
26S 41%
26R 51%
Table 2
Compound
MCF-7
PC-3
%Inhibition @
%Inhibition @
IC50
M)
IC50
M)
O
O
C7H
15 O
20
l
M
(
l
20
lM
(l
H
N
N
N
1) HBr/HOAc
N
H
3R
3S
4R
4S
5R
5S
6SR
6SS
6RR
6RS
7R
94
91
96
95
91
93
87
89
92
90
86
81
0.2
0.32
0.55
2
0.45
0.8
0.24
0.5
0.32
3
72
62
72
68
85
60
83
79
90
79
40
63
4
8
9
14
3.75
9
3
3
2
7.5
—
N
H
OR
OH
O
O
2) 27,DIPEA
N
H
RO
O
O
ONHS
28S R = Bn
28R R = Bn
N
Pd/C
H2
Pd/C
H2
OH
69%
63%
O
27
4S R = H
4R R = H
83%
98%
6
1
7S
7
Scheme 4.
O
C7H
∗
O
O
15 O
C7H15
O
H
N
N
CbzHN
N
1) HBr/HOAc
∗
O
O
N
N
OBn
RO
OR
N
H
H
O
O
O
OR
2) Acid 29, EDC
HOAt, DMAP
N
H
H
RO
COOH
OBn
OBn
O
30R R = Bn
30S R = Bn
58%
21R
21S
Pd/C
H2
39%
Pd/C
H2
OBn
29
7S R = H
7R R = H
97%
79%
Scheme 5.

C. Wu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2611–2615
3. Yokokawa, F.; Izumi, K.; Omata, J.; et al. Tetrahedron 200, 56, 3027.
2615
comparably active against MCF-7 and PC-3 human cancer cell lines
indicating that metal chelation might be more important than ste-
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Acknowledgment
We gratefully acknowledge support from the National Institutes
of Health (AI 054193).
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Supplementary data
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Supplementary data associated with this article can be found, in
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