Syntheses of photolabile novobiocin analogues

Article abstract of DOI:10.1016/j.bmcl.2004.09.017

Novobiocin was recently shown to inhibit Hsp90 through a previously unrecognized C-terminal ATP binding site. Although the N-terminal region of Hsp90 has been solved by X-ray crystallography, the C-terminal region has not. In an effort to elucidate the C-

Full text of DOI:10.1016/j.bmcl.2004.09.017

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5903–5906
Syntheses of photolabile novobiocin analogues
Gang Shen, Xiao ming Yu and Brian S. J. Blagg*
Department of Medicinal Chemistry and The Center for Protein Structure and Function, The University of Kansas,
1251 Wescoe Hall Drive, Malott Hall 4070, Lawrence, KS 66045-7562, United States
Received 19 July 2004; revised 8 September 2004; accepted 8 September 2004
Available online 30 September 2004
Abstract—Novobiocin was recently shown to inhibit Hsp90 through a previously unrecognized C-terminal ATP binding site.
Although the N-terminal region of Hsp90 has been solved by X-ray crystallography, the C-terminal region hasnot. In an effort
to elucidate the C-terminal binding site of Hsp90, four photolabile analogues of novobiocin were prepared.
Ó 2004 Elsevier Ltd. All rights reserved.
The coumarin antibioticsnovobiocin, clorobiocin, and
coumermycin A1 (Fig. 1) have been isolated from several
streptomyces strains and exhibit potent activity against
Gram-positive bacteria.1–4 These compounds bind to
type II topoisomerases including DNA gyrase and inhibit
the enzyme catalyzed hydrolysis of ATP.5–8 Asa result,
novobiocin analogueshave garnered the attention of
numerousresearchersasan attractive agent for the treat-
ment of bacterial infection. Unlike norfloxacin, pipe-
midic acid, and nalidixic acid, which are clinically used
DNA gyrase inhibitors, novobiocin produces cytotoxic-
ity in a number of cancer cell lines.9–12
nucleotide-binding region. Subsequent studies have
shown that inhibition of Hsp90 by novobiocin leads to
a decrease in Hsp90 client proteins in various cancer cell
lines,18 an effect that issimilar to N-terminal inhibitors,
geldanamycin, and radicicol.19–26
Hsp90 is a molecular chaperone responsible for the con-
formational maturation of nascent polypeptides into
biological active, three-dimensional structures.16,27–36
Hsp90 dependent client proteins represented in the six
37
hallmarksof cancer include Her-2,
Src family kin-
ases,38 Raf,39 PLK,40 RIP,41 AKT,42 telomerase,43 and
MET.44 Inhibition of the Hsp90 protein folding machin-
ery results in the simultaneous disruption of multiple
oncogenic pathways and consequently, Hsp90 has
emerged asa promiisng target for the development of
cancer chemotherapeutics.45,46
The crystal structure of topoisomerase II has not been
solved, but the co-crystal structure of DNA gyrase
13–15
bound to novobiocin wasreported in 1991.
There
were little similarities between novobiocin and adenine
triphosphate, but the structure did indicate that ATP
is bound in an unusual, bent conformation, as opposed
to the typical extended form. Like DNA gyrase, the N-
terminal region of the 90kDa heat shock proteins
(Hsp90) also bind ATP in a very similar bent conforma-
tion.16 Because novobiocin binds to a similarly shaped
ATP binding site in DNA gyrase and has cytotoxicity
in cancer cell lines, it was proposed that this molecule
may be binding to the analogousATP binding pocket
in Hsp90. Elegant studies by Neckers and co-workers
demonstrated that novobiocin did bind to Hsp90, but
not to the N-terminal region.17 Instead, novobiocin
wasfound to bind a previously unrecognized C-terminal
Critical to the discovery of new Hsp90 inhibitors is elu-
cidation of the C-terminal ATP binding site. In an
effort to elucidate the location of thisnucleotide-binding
pocket, four photolabile novobiocin analogueswere pre-
pared by convergent syntheses.
The syntheses of photolabile novobiocin analogues be-
gan by the preparation of known compounds4,7-dihyd-
roxy-8-methyl-2H-1-benzopyran-2-one47 (4, Scheme 1)
and diazonium salt 5.48 Upon treatment of 4 with 5, a
yellow precipitate was immediately observed and subse-
quent characterization confirmed thismaterial wasthe
desired diaza coumarin derivative 6. Previoussynthetic
effortsaimed at the preparation of novobiocin analogues
have shown that a sulfone analogue of the coumarin ring
could be coupled directly with trichloracetoamidate 7 in
the presence of a catalytic amount of boron trifluoride
Keywords: Novobiocin; Hsp90; DNA gyrase.
*
Corresponding author. Tel.: +1 785 864 2288; fax: +1 785 864
5326; e-mail: bblagg@ku.edu
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.09.017

5904
G. Shen et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5903–5906
OH
OH
OH
O
H
N
OH
O
H
N
O
O
O
O
O
O
Cl
O
MeO
O
MeO
H2N
O
H
N
O
OH
Novobiocin 1
Clorobiocin 2
OH
O
O
H
N
O
H
N
O
O
O
O
NH
OH
O
HO
O
O
O
MeO
O
N
H
O
O
OMe
O
H
N
OH
Coumermycin A1 3
HO
O
Figure 1. Coumarin antibiotics.
OH
MeO
OH
OH
O
NH
CCl3
R
O
N
Ph
O
O
O
N
Cl
O
N2
7
O
O
5
HO
O
O
HO
O
O
89%
O
BF3OEt2 MeO
87%
4
6
O
O
O
8, R = NNPh
9, R = NH2
H2, Pd/C
Scheme 1. Synthesis of aminocoumarin.
N3
OH
O
H
N
O
O
O
N
3
N3
11, 42%
N
3
O
MeO
OH
H
N
O
HO
OH
H
OH
NH3/MeOH
N
O
H N
2
O
9
O
O
10
O
O
DCC, 63%
O
O
O
O
O
MeO
12, 16%
O
MeO
O
O
HO
O
O
O
NH
N
3
2
OH
H
N
O
O
O
O
O
14, 50%
N3
N3
O
MeO
OH
H
N
N3
O
HO
OH
H
OH
NH3/MeOH
O
N
H N
2
O
9
O
DCC, 76%
O
O
13
O
O
O
O
O
MeO
15, 21%
O
O
MeO
O
HO
O
O
O
NH
2
Scheme 2. Syntheses of photolabile novobiocin analogues.

G. Shen et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5903–5906
5905
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excess coumarin, the amount of dinoviosylated product
was drastically reduced and the desired 7-noviose prod-
uct, 8, wasprovided in good yield. The diaza-bridge con-
necting the phenyl and coumarin ringsesrved asa
masked amine, which could be cleaved under reducing
conditionsto provide the 3-amino coumarin 9. However,
9 turned out to be unstable under a variety of conditions,
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In this letter, we described the syntheses of four photo-
labile analoguesof novobiocin, an inhibitor of not
only type II topoisomerases, but also Hsp90. These
photolabile analoguesare currently under biological
investigation for their ability to covalently modify the
C-terminal ATP binding site of Hsp90. The outcome
of these studies will be reported in due course.
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Acknowledgements
The authorsgratefully acknowledge support of thispro-
ject by the NIH COBRE RR017708 and The University
of Kansas Center for Research. G.S. is the recipient of a
Susan G. Komen Breast Cancer Foundation Disserta-
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25
1
51. For 11: ½aꢀD ꢁ45.3 (c 0.23, 50% MeOH in CH2Cl2); H
NMR (CD2Cl2/CD3OD, 400MHz): 7.83 (d, J = 8.9Hz,
1H), 7.76 (d, J = 7.6Hz, 1H), 7.68 (s, 1H), 7.54 (t,
J = 7.8Hz, 1H), 7.29 (d, J = 7.9Hz, 1H), 7.23 (d,
J = 8.9Hz, 1H), 5.59 (s, 1H), 5.36 (d, J = 9.9Hz, 1H),
4.27 (s, 1H), 3.57 (d, J = 9.9Hz, 1H), 3.55 (s, 3H), 2.32 (s,
3H), 1.36 (s, 3H), 1.16 (s, 3H); 13C NMR (CD2Cl2/
CD3OD, 125MHz): d 167.5, 162.5, 158.0, 157.8, 150.8,
141.4, 134.7 (2C), 130.5, 124.2, 123.1, 122.5, 118.8, 114.2,
111.7, 110.8, 102.1, 98.9, 81.7, 79.2, 72.1, 69.9, 61.3, 28.4,
22.5, 8.2; IR (film) mmax 2115, 1736, 1718, 1701, 1684, 1652,
1607, 1580, 1540, 1523, 1509, 1369, 1323, 1270, 1118,
44. Sato, S.; Fujita, N.; Tsuruo, T. Proc. Natl. Acad. Sci.
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1093cmꢁ1
;
C26H27N5O10 requires570.1836).
HRMS 570.1887 (FAB+) m/z (M+H+,
25
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52. For 14: ½aꢀD ꢁ36.6 (c 0.06, 50% MeOH in CH2Cl2);
1H NMR (CD2Cl2/CD3OD, 400MHz): 8.04 (m, 2H), 7.84
(d, J = 8.9Hz, 1H), 7.22 (m, 3H), 5.59 (d, J = 2.2Hz, 1H),
5.36 (dd, J = 3.1, 9.8Hz, 1H), 4.20 (dd, J = 2.2, 3.1Hz,
1H), 3.58 (d, J = 9.8Hz, 1H), 3.56 (s, 3H), 2.33 (s, 3H),
1.37 (s, 3H), 1.17 (s, 3H); 13C NMR (CD2Cl2/CD3OD,
125MHz): d 167.6, 162.7, 162.3, 157.9 (2C), 150.9, 144.9,
141.0, 129.9 (2C), 129.3, 122.5, 119.3 (2C), 114.1, 110.7,
102.0, 98.9, 81.7, 79.1, 72.1, 69.9, 61.2, 28.3, 22.5, 8.1; IR
(film) mmax 2119, 1735, 1690, 1629, 1600, 1496, 1371, 1280,
1251, 1193, 1097, 937cmꢁ1; HRMS 570.1841 (FAB+) m/z
(M+H+, C26H27N5O10 requires570.1836).
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