Synthesis of a bromotyrosine-derived natural product inhibitor of mycothiol-S-conjugate amidase

Article abstract of DOI:10.1016/j.bmcl.2004.04.095

Recently we described the structures of two new bromotyrosine-derived alkaloids that inhibit the detoxification enzyme mycothiol-S-conjugate amidase (MCA) from Mycobacterium tuberculosis. Here we describe a concise total synthesis of bromotyrosine oxime 1

Full text of DOI:10.1016/j.bmcl.2004.04.095

Bioorganic & Medicinal Chemistry Letters 14 (2004) 3785–3788
Synthesis of a bromotyrosine-derived natural product inhibitor
of mycothiol-S-conjugate amidaseq
Brandon Fetterolf and Carole A. Bewley*
Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases,
National Institutes of Health, Bethesda, MD 20892-0820, USA
Received 26 March 2004; revised 27 April 2004; accepted 28 April 2004
Available online 28 May 2004
Abstract—Recently we described the structures of two new bromotyrosine-derived alkaloids that inhibit the detoxification enzyme
mycothiol-S-conjugate amidase (MCA) from Mycobacterium tuberculosis. Here we describe a concise total synthesis of bromoty-
rosine oxime 1. The six-step synthesis of 1 utilized a trifluoromethyloxazole intermediate, whose hydrolysis product underwent
alkylation and coupling to agmatine to give the inhibitor in ꢀ40% overall yield. Oxime 1 inhibited MCA and its homolog AcGI
deacetylase with IC50 values of 30 and 150 lM, respectively.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Mycothiol (MSH) is the major low molecular weight
thiol limited to actinomycetes, which include mycobac-
teria. Analogous to glutathione in Gram negative bac-
teria and eukaryotes, MSH plays a major role in
detoxification and maintenance of a reductive intracel-
lular environment.1 Owing to the continuing need for
new classes of antibiotics effective against Myco-
bacterium tuberculosis,2 studies of enzymes involved in
mycothiol biosynthesis and mycothiol-dependent
detoxification, as well as those of inhibitors to these
enzymes, are rapidly increasing in number. Recently we
described a series of marine natural product inhibitors
of MCA, some of which are lethal to M. smegmatis.3;4
Of those structures studied, kinetics experiments estab-
lished that the bromotyrosine-derived series of alka-
loids, such as 1, are among the most active inhibitors of
the group and are also competitive inhibitors of the
M. tuberculosis detoxification enzyme MCA. Kinetics,
NMR and modeling experiments suggest that this class
of compounds presumably acts by chelation of a metal
cation in the active site via the oxime moiety.4;5 Sub-
sequent structure–activity relationship studies using a
synthetic library of compounds6 inspired by the natural
product disulfide psammaplin A7 (5) yielded additional
information concerning structural features that con-
tribute to, or abrogate, inhibitory activity toward
MCA.8 Together, these studies established that phenolic
oximes exclusive of disulfides or mixed disulfides present
in the synthetic library represented the best inhibitors of
MCA reported to date. Moreover, inhibitors lacking the
disulfides may be more desirable in terms of stability and
specificity. Motivated by these findings, we report a
concise total synthesis of inhibitor 1.
Many marine natural products that appear to be bio-
genetically derived from a bromotyrosine precursor
have been described previously (several examples are
shown in Fig. 1). Owing to the variety of interesting
biological activities exhibited by members of this class of
compounds, a number of syntheses have also been
completed. Wasserman et al. employed a cyano ylid
coupling for chain elongation, followed by formation of
an oxime to obtain the natural products verongamine 3,
hemibastadin 2, and aerothionin 4.9 Hoshino et al.
synthesized the bromotyrosine disulfide psammaplin A 5
and its mixed disulfide derivatives by opening an oxa-
zole to generate an alpha keto acid intermediate fol-
lowed by formation of the oxime.10 During the course of
our synthetic studies, Kende et al. completed the first
total synthesis of 1 from two complementary schemes
starting with 4-hydroxyphenylpyruvic acid, and dibro-
mination with NBS was carried out prior to their
Keywords: Mycobacteria; Marine natural products; Deacetylase;
Enzyme.
q Supplementary data associated with this article can be found, in the
online version, at doi:10.1016/j.bmcl.2004.04.095
* Corresponding author. Tel.: +1-301-594-5187; fax: +1-301-402-0008;
e-mail: cb194k@nih.gov
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.04.095

3786
B. Fetterolf, C. A. Bewley / Bioorg. Med. Chem. Lett. 14 (2004) 3785–3788
H
OH
NH2
O
H2N
O
N
NH2
NH
1 R =
2 R =
Br
Br
Br
Br
Br
Br
O
Br
NHBoc
7
OH
N
+
OH
N
NH
R
N
H
NH2
N
H
H
N
N
NHBoc
NBoc
N
NH2
NH
O
N
H
HO
O
N
H
R1
6
1
O
R2
MeO
OH
OH
N
Br
Br
Br
Br
N
Br
MeO
H
NH
NHBoc
NBoc
N
OBn
N
R1 = H, R2
=
H2N
3 Verongamine
+
NH2
N
8
NH2
OH
Br
OMe
O
O
OH
R1 = Br, R2
=
4 Hemibastidin-2
10
9
Scheme 1. Retrosynthetic analysis.
OH
Br
OH
Br
unstable trifluoromethyloxazolone intermediate 11.12
Oxazole 11 was dissolved in 70% aq trifluoroacetic acid
(TFA) and allowed to stand overnight, precipitating the
alpha keto acid 12 as a white solid in 91% yield over the
two steps. Treatment of ketone 12 with O-benzyl
hydroxylamine in ethanol provided the alpha protected
oxime 9 in 87% yield. For coupling N-Boc-protected
agmatine13 8 to intermediate 9,14 several different cou-
pling reagents were tested in an attempt to improve
yields. These included the carbonyl activators (1,10)-
carbonyldiimidazole (CDI),15 dicyclohexylcarbodiimide
(DCC), and 1-(3-dimethylaminopropyl)-3-ethyl-carbo-
diimide hydrochloride (EDCI). Of the three, we found
that treating 9 with EDCI and N-hydroxylsuccinimide
gave slightly higher yields (63%) of intermediate 6
compared to yields of 53% and 56% for CDI and DCC,
respectively.
O
O
S
S
N
N
N
N
HO
OH
5 Psammaplin A
Figure 1. Examples of marine natural product oximes derived from
bromotyrosine.
coupling reactions.11 For the synthesis of 1, we felt
Hoshino’s approach to the synthesis of psammaplin A
would provide a general route to the oxime, starting
with commercially available 3,5-dibromotyrosine. A
retrosynthetic analysis (Scheme 1) indicated that 1 could
be obtained by O-alkylation to connect phenyl alcohol 6
and bromide 7, and compound 6 could be obtained by
coupling protected agmatine 8 with alpha-oximino acid
9.
As illustrated in Scheme 2, commercially available 3,5-
dibromotyrosine 10 was treated with trifluoroacetic
anhydride (TFAA) at 80 ꢁC for 18 h to generate the
In the final steps of the synthesis, alkylation of 6 with
bromide 7 under basic conditions provided 13 in 86%
yield.16 Initial attempts at hydrogenation of 13 using
O
OH
OH
F3C
Br
O
Br
Br
Br
Br
Br
1) N-hydroxylsuccinimide,
EDC, dioxane
70% aq. TFA
(CF3CO)2O, ∆
2) 8, Et3N, MeOH:dioxane
NH2
OH
R
N
O
CF3
O
O
OH
O
NH2OBn
EtOH
12 R=O
9 R=NOBn
10
11
BocNH
H2N
O
O
OH
Br
Br
Br
Br
Br
Br
20% TFA:CH2Cl2
7, K2CO3, DMF
OH
OBn
N
OR
N
N
H
H
H
N
N
N
NHBoc
NBoc
NH2
NH
NHBoc
NBoc
O
N
O
N
H
O
N
H
H
6
Pd-black
AcOH:dioxane
13 R=OBn
14 R=OH
1
Scheme 2. Reactions and conditions.

B. Fetterolf, C. A. Bewley / Bioorg. Med. Chem. Lett. 14 (2004) 3785–3788
3787
6. Nicolaou, K. C.; Hughes, R.; Pfefferkorn, J. A.; Barlu-
enga, S.; Roecker, A. J. Chem. Eur. J. 2001, 7, 4280.
palladium on carbon resulted in deprotection of the
benzyl group and undesirably, reduction of the oxime to
an amine as the major product. We next attempted
hydrogenation of 13 using palladium black in equal
amounts of acetic acid and 1,4-dioxane,17 which pro-
vided 14 in 89% yield with a minimal amount of amine
formation.18 Cleavage of the three N-tert-butyl carba-
mate protecting groups using 20% TFA in dichloro-
methane completed the synthesis of 1 in 38% overall
yield starting from the 3,5-dibromotyrosine.19
~ ꢀ
7. (a) Quinoa, E.; Crews, P. Tetrahedron Lett. 1987, 28, 3229;
(b) Arabshahi, L.; Schmitz, F. J. Org. Chem. 1987, 52,
3584.
8. Nicholas, G. M.; Eckman, L. L.; Ray, S.; Hughes, R. O.;
Pfefferkorn, J. A.; Barluenga, S.; Nicolaou, K. C.; Bewley,
C. A. Bioorg. Med. Chem. Lett. 2002, 12, 2487.
9. Wasserman, H. H.; Wang, J. J. Org. Chem. 1998, 63, 5581,
and references cited therein.
10. Hoshino, O.; Maruakata, M.; Yamada, K. Bioorg. Med.
Chem. Lett. 1992, 12, 1561.
11. Kende, A. S.; Lan, J.; Fan, J. Tetrahedron Lett. 2004, 45,
133.
12. Wilson, M. L.; Coscia, C. J. J. Org. Chem. 1979, 44, 301.
13. Agmatine 8 was synthesized as described by: Exposito, A.;
Fernandez-Suarez, M.; Iglesias, T.; Munoz, L.; Riguera,
R. J. Org. Chem. 2001, 66, 4206.
Spectroscopic data for synthetic 1 were recorded and
compared to those of natural 1. Consistent with data
from the natural product, HR-FABMS showed an iso-
topic cluster of 1:2:1 at m=z 521, 523, 525 in the
FABMS, and HR-FABMS showed a molecular ion at
523.0336 (calcd 523.0667); and proton and carbon
spectra (provided in Supporting Information) were
identical to those of the natural product. Last, synthetic
1 was tested for inhibitory activity against recombinant
M. tuberculosis MCA and the biosynthetic enzyme
AcGI deacetylase in an identical manner as described
previously.4;20 As an internal reference in the MCA
assays, synthetic 1 was tested in parallel to the natural
products psammaplysin21 and oceanapaside.22 Multiple
independent assays carried out in duplicate confirmed
levels of MCA inhibition for synthetic 1 and the natural
products equivalent to those reported previously, and
yielded an IC50 value of approximately 150 lM for
inhibition of AcGI deacetylase. In summary, a short and
highly efficient synthesis of natural product 1 has been
accomplished, and with minor modifications, can be
envisaged to supply a plethora of different synthetic
derivatives. Related synthetic and biological studies are
underway.
14. Experimental procedure for coupling product 6: to a
solution of acid 9 (0.45 mmol) in 4.5 mL of 1,4-dioxane at
room temperature was added N-hydroxylsuccinimide
(0.86 mmol) followed by addition of EDCI (0.77 mmol).
The reaction was stirred for 2 h at which time all starting
material had been consumed. The yellow solution was
concentrated in vacuo and the crude yellow oil diluted
with EtOAc (50 mL). The mixture was washed with satd
aq NaHCO3 (2 · 25 mL), 1 N HCl (2 · 25 mL), and brine
(25 mL). The clear organic layer was dried over Na2SO4
and concentrated to a white solid. The crude succinimide
ester was dissolved in 1,4-dioxane (4.5 mL) and to this
solution was added a second solution containing amine 8
(0.9 mmol) and triethylamine (0.9 mmol) in anhydrous
MeOH (4.5 mL). The yellow solution was stirred for 18 h
at ambient temperature and concentrated in vacuo. The
crude foam was dissolved in EtOAc and purified by flash
chromatography (35:65:1 EtOAc–hexane–Et3N) to yield
amide 6. 1H NMR (CDCl3, 300 MHz) d 1.49 (s, 18H), 1.59
(m, 4H), 3.33 (m, 2H), 3.44 (br d, J ¼ 5:1 Hz, 2H), 3.80 (s,
2H), 5.21 (s, 2H), 6.76 (t, J ¼ 6:3 Hz, 1H), 7.30–7.42 (m,
7H), 8.36 (br s, 1H). 13C NMR (CDCl3, 75 MHz) 14.3,
21.1, 26.7, 26.9, 28.2, 28.4, 28.6, 39.2, 40.6, 60.5, 77.7, 79.5,
83.3, 109.9, 126.4, 128.5, 128.8, 130.7, 133.2, 136.4, 148.3,
151.7, 153.4, 156.2, 162.3, 163.5. FTIR (film): m 2977, 1717,
1615 cmꢁ1. FABMS (pos) 888.2 (M+Cs).
Acknowledgements
We thank Lisa Eckman for preparation of recombinant
mycothiol-S-conjugate amidase, Noel Whittaker, and
Victor Livengood for MS data and Belhu Metaferia for
helpful discussions. This work was supported in part by
the Intramural AIDS Targeted Antiviral Program of the
Office of the Director (to C.A.B.).
15. Paul, R.; Anderson, G. W. J. Am. Chem. Soc. 1960, 82,
4596.
16. Spectroscopic data for compound 13: white solid. 1H
NMR (CDCl3, 300 MHz) d 1.45 (s, 9H), 1.50 (s, 18H), 1.6
(m, 4H), 1.97–2.07 (m, 2H), 3.34 (m, 2H), 3.42–3.46 (m,
4H), 3.82 (s, 2H), 4.01 (t, J ¼ 6 Hz, 2H), 4.96 (br s, 1H),
5.22 (s, 2H), 6.77 (t, J ¼ 6:3 Hz, 1H), 7.29–7.43 (m, 7H),
8.34 (br s, 1H), 11.50 (br s, 1H). 13C NMR (CDCl3,
75 MHz) d 26.7, 27.0, 28.2, 28.4, 28.5, 28.6, 28.8, 29.8,
30.1, 38.2, 39.2, 40.5, 71.3, 77.7, 79.4, 83.3, 118.0, 128.4,
128.5, 128.8, 133.7, 135.0, 136.4, 151.3, 151.6, 153.4, 156.2,
References and notes
156.3, 162.2, 163.6. FTIR m 1715, 1637, 1131 cmꢁ1
.
1. Reviewed by Newton, G. L.; Fahey, R. C. Arch. Micro-
biol. 2002, 178, 388.
2. (a) Dye, C.; Williams, B. G.; Espinal, M. A.; Raviglione,
M. C. Science 2002, 295, 2042; (b) Pozniak, A. Ann. N.Y.
Acad. Sci. 2001, 953, 192; (c) Grange, J. M.; Zumla, A.
Lancet 1999, 353, 996.
3. Nicholas, G. M.; Newton, G. M.; Fahey, R. C.; Bewley, C.
A. Org. Lett. 2001, 3, 1543.
4. Nicholas, G. M.; Eckman, L. L.; Newton, G. L.; Fahey,
R. C.; Ray, S.; Bewley, C. A. Bioorg. Med. Chem. 2003,
11, 601.
FABMS (pos) 1045.1 (M+Cs).
17. Masatoshi, M.; Tamura, M.; Hoshino, O. J. Org. Chem.
1997, 62, 4428.
18. Spectroscopic data for compound 14: clear oil/foam: 1H
NMR (CDCl3, 300 MHz) d 1.44 (s, 9H), 1.49 (s, 18H), 1.56
(m, 4H), 2.00 (m, 2H), 3.27–3.33 (m, 2H), 3.40 (m, 4H),
3.85 (s, 2H), 3.89 (t, J ¼ 5:1 Hz, 1H), 3.99 (t, J ¼ 5:7 Hz,
2H), 4.99 (br s, 1H), 6.77 (t, J ¼ 6:3 Hz, 1H), 7.48 (s, 2H),
11.47 (br s, 1H). 13C NMR (CDCl3, 75 MHz) d 15.4, 26.7,
28.1, 28.2, 28.4, 28.6, 30.1, 38.3, 39.1, 40.97, 66.1, 71.3,
79.5, 80.4, 83.9, 118.0, 133.7, 135.7, 151.2, 151.4, 153.3,
156.1, 156.5, 163.3. FTIR (film) m 2977, 1717, 1615,
1132 cmꢁ1. FABMS (pos) 823.1 (M+H).
5. Mahadevan, J.; Nicholas, G. M.; Bewley, C. A. J. Org.
Chem. 2003, 68, 3380.

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B. Fetterolf, C. A. Bewley / Bioorg. Med. Chem. Lett. 14 (2004) 3785–3788
1
19. Spectroscopic data for synthetic 1: white solid. H NMR
(CD3OD, 300 MHz) d 1.56 (m, 4H), 2.12–2.21 (m, 3H),
3.16 (m, 2H), 3.24–3.30 (m, 4H), 3.83 (s, 2H), 4.07 (t,
J ¼ 5:7 Hz, 2H), 7.49 (s, 2H). 13C NMR (CD3OD,
75 MHz) d 24.3, 27.3, 27.8, 29.0, 29.1, 39.0, 39.8, 42.2,
71.7, 118.7, 134.7, 138.0, 152.2, 152.4, 165.7. FTIR (film)
3333, 2972, 2930, 1715, 1637, 1131 cmꢁ1. FABMS (pos)
522.23 (M+H).
20. Nicholas, G. M.; Eckman, L. E.; Kovac, P.; Otero-Quin-
tero, S.; Bewley, C. A. Bioorg. Med. Chem. 2003, 11, 2641.
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Shoolery, J. N.; Matsumoto, G. K.; Van Duyne, G. D.;
Clardy, J. C. J. Am. Chem. Soc. 1985, 107, 2916.
22. Nicholas, G. M.; Hong, T. W.; Molinski, T. F.; Lerch, M.
L.; Cancilla, M. T.; Lebrilla, C. B. J. Nat. Prod. 1999, 62,
1678.