Click chemistry to probe Hsp90: Synthesis and evaluation of a series of triazole-containing novobiocin analogues

Article abstract of DOI:10.1016/j.bmcl.2010.04.140

A series of triazole-containing novobiocin analogues has been designed, synthesized and their inhibitory activity determined. These compounds contain a triazole ring in lieu of the amide moiety present in the natural product. The anti-proliferative effects of these compounds were evaluated against two breast cancer cell lines (SKBr-3 and MCF-7), and manifested activities similar to their amide-containing counterparts. In addition, Hsp90-dependent client protein degradation was observed via Western blot analyses, supporting a common mode of Hsp90 inhibition for both structural classes.

Full text of DOI:10.1016/j.bmcl.2010.04.140

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3957–3960
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Click chemistry to probe Hsp90: Synthesis and evaluation of a series
of triazole-containing novobiocin analogues
*
Laura B. Peterson, Brian S. J. Blagg
Department of Medicinal Chemistry, The University of Kansas, 1251 Wescoe Hall Drive, Malott 4070, Lawrence, KS 66045-7582, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of triazole-containing novobiocin analogues has been designed, synthesized and their inhibitory
activity determined. These compounds contain a triazole ring in lieu of the amide moiety present in the
natural product. The anti-proliferative effects of these compounds were evaluated against two breast
cancer cell lines (SKBr-3 and MCF-7), and manifested activities similar to their amide-containing coun-
terparts. In addition, Hsp90-dependent client protein degradation was observed via Western blot analy-
ses, supporting a common mode of Hsp90 inhibition for both structural classes.
Received 7 April 2010
Revised 28 April 2010
Accepted 30 April 2010
Available online 6 May 2010
Keywords:
Hsp90
Ó 2010 Elsevier Ltd. All rights reserved.
Novobiocin
Antiproliferation
Triazole
Current cancer therapy strategies utilize the administration of
multiple drug regimens that aim to halt multiple malignant pro-
cesses simultaneously. Heat shock protein 90 (Hsp90) represents
an exciting target for the treatment of cancer, as inhibition of this
chaperone can affect multiple proteins that are directly associated
with all six hallmarks of cancer.1–4 Hsp90 is a 90 kDa molecular
chaperone and is intimately involved in the post-translational
conformational maturation of nascent polypeptides as well as
the re-folding of denatured proteins and the re-solubilization of
protein aggregates.5 Pharmacological inhibition of Hsp90 effec-
tively inhibits protein substrates dependent upon Hsp90 for con-
formational maturation, resulting in destabilization of the Hsp90-
client protein heteroprotein complex, which leads to degradation
of substrates via the ubiquitin-proteasome pathway.4,6,7 Many
proteins associated with malignant progression; including steroid
hormone receptors, transcription factors and protein kinases, rely
upon Hsp90 to reach their biologically active, three-dimensional
conformation. As such, Hsp90 has emerged as a promising anti-
cancer target, with more than 20 clinical trials currently in pro-
gress with small molecules that bind the N-terminal ATP binding
site.8
icity and/or complicated dosing schedules. In contrast, the devel-
opment of Hsp90 inhibitors that target other small molecule
binding regions, such as that contained in the C-terminus remains
minimally investigated.10 For example, novobiocin was shown to
bind the C-terminus of Hsp90 in 2000 and provided the first exam-
ple of a small molecule binding site outside of the N-terminus
(Fig. 1).11,12 However, novobiocin manifests only modest inhibitory
activity (ꢀ500
lM). Since 2000, other inhibitors of the C-terminus
have also been identified, such as epigallocatechin gallate (EGCG)
(Fig. 1), but the development of such compounds has not been
thoroughly sought after.10
Since the discovery of the Hsp90 C-terminal binding site, ana-
logues of novobiocin have been synthesized and evaluated, with
many of the compounds manifesting micromolar anti-proliferative
activities.13–17 Modifications to both the coumarin core and benz-
amide side chain have been pursued, resulting in the production
of preliminary structure–activity relationships (SARs). The hydro-
gen bonding capabilities and the geometry of the amide bond ap-
pear important for novobiocin binding, however, modifications to
this moiety have not been fully realized. It was proposed that
inclusion of 1,2,3-triazoles as a bioisosteric replacement for the
amide moiety could facilitate SAR analysis for the aryl side chain
by utilizing ‘click’ chemistry. The triazole serves as a bioisostere
due to similarities in both electronic and spacial characteristics
to the amide bond. In addition, it is metabolically stable to hydro-
lysis and easily incorporated into small molecules.18,19 In contrast,
triazoles exhibit different hydrogen bonding capabilities and an al-
tered geometry as compared to their amide counterparts, which
aids in further elucidation of SAR. For these reasons, a series of
1,2,3-triazole-containing novobiocin analogues was prepared. The
The Hsp90 protein folding machinery requires co-chaperones
and partner proteins to aid in the topological reorientation of poly-
peptide substrates.7 This protein folding process is ATP-dependent,
with hydrolysis occurring at the N-terminal nucleotide binding site
of the Hsp90 homodimer.9 Although promising data has emerged
from these trials, many of these compounds exhibit undesired tox-
* Corresponding author. Tel.: +1 785 864 2288; fax: +1 785 864 5326.
E-mail address: bblagg@ku.edu (B.S.J. Blagg).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.140

3958
L. B. Peterson, B. S. J. Blagg / Bioorg. Med. Chem. Lett. 20 (2010) 3957–3960
Figure 1. Hsp90 C-terminal inhibitors.
Scheme 1. Reagents: (a) (i) POCl3, DMF, MeCN; (ii) H2O (55%); (b) N-acetyl glycine, NaOAc, Ac2O; (c) HCl, EtOH; (d) NaNO2, HCl, EtOH, H2O, then NaN3 (52% three steps); (e)
Ac2O, pyridine, CH2Cl2 (>95%).
design, syntheses, and biological evaluation of these compounds
are described herein.
rin 4 to the azide, which was required for the copper-catalyzed
Huisgen 1,3-dipolar cycloaddition, was accomplished by in situ
generation of the 3-diazonium salt upon treatment with sodium
nitrite in aqueous acid, followed by the addition of sodium azide
to afford 3-azido-coumarin, 5a.20 Acetylation of coumarin 5a was
accomplished with acetic anhydride in pyridine to afford 5b.
Upon the generation of compounds 5a and 5b, the copper-cata-
lyzed Huisgen 1,3-dipolar cycloaddition with the corresponding
alkynes was set to generate compounds 6–14a and 6–14b
(Scheme 2). Standard conditions were used to effect this transfor-
mation, and a combination of DMSO and H2O were found most
suitable for optimal product formation. Alkynes 6–10 were chosen
Synthesis of the 8-methyl coumarin core, as found in novobio-
cin, was commenced with commercially available 2-methyl resor-
cinol, 1 (Scheme 1). Compound 1 was formylated under Vilsmeier–
Haack conditions enlisting POCl3 and DMF, followed by hydrolysis
to afford formyl-resorcinol 2. Similar to the procedure of Sivaku-
mar and co-workers, condensation of 2 with N-acetyl glycine in
the presence of acetic anhydride, produced the bis-acylated cou-
marin, 3.20 Deacetylation of both the phenol and amine was
accomplished upon heating with HCl and EtOH to afford 3-ami-
no-7-hydroxy-8-methyl-coumarin, 4. Conversion of amino-couma-
Scheme 2. ‘Click’ procedure with various alkenes used. (a) The TMS protected alkynes were used in these reactions with the addition of 1 equiv TBAF.

L. B. Peterson, B. S. J. Blagg / Bioorg. Med. Chem. Lett. 20 (2010) 3957–3960
3959
Scheme 3. Noviosylation of triazole appended coumarins.
to investigate the electronic nature of the aryl side chain, including
neutral (Me), electron-withdrawing (F) and electron-donating
(OMe, NMe2) properties as originally identified during classical
novobiocin SAR studies.13 Alkynes 11–14 were chosen based on
prior observations that the analogous amide derivatives mani-
fested good anti-proliferative activities against multiple cell
lines,13 and thus provide direct comparison between amide and
triazole analogues.
Completion of the triazole-containing novobiocin analogues
was achieved by incorporation of the noviose sugar into 6–14a
(Scheme 3). The phenols of compounds 6–14a were noviosylated
with the trichloroacetimidate of noviose carbonate (15) in the
presence of boron trifluoride etherate.21 Solvolysis of the cyclic car-
bonate with methanolic triethylamine afforded novobiocin ana-
logues 6–14c.
These results provide evidence that compounds 14a–14c, and 11c
are the most potent analogues prepared in this series. The struc-
ture–activity relationships suggest that analogues bearing sterically
demanding side chains exhibit greater potency, as compounds with
biaryl, indole, or homologated aryl groups (11–14) were more
efficacious than substituted aryl compounds (6–10). In addition,
comparison of compounds 6–10 suggests that para-substitution of
the aryl ring with Me, OMe, NMe2 or F is not well tolerated, as these
compounds displayed minimal anti-proliferative activity. These
results suggest that the C-terminal binding pocket has a large hydro-
phobic pocket that can easily accommodate substituents as in com-
pounds 11–14.
Additionally, comparison between the amide-containing ana-
logues and triazole-containing analogues provides further SAR
(Table 2). As evident from the data presented in Table 2, the tria-
zole moiety has little effect on anti-proliferative activity. Both
the triazole and amide analogues containing the biaryl and 3-in-
dole side chains indicate the activities for both sets of compounds
Upon preparation of compounds 6–14a, b and c, their growth
inhibitory activities against MCF-7 (ER+) and SKBr-3 (ER-, HER2
overexpressing) breast cancer cell lines were determined (Table 1).
Table 1
Anti-proliferative activities of triazole-containing novobiocin analoguesa
Novobiocin
MCF-7b (IC50
481.3 1.5
,
lM)
SKBr-3b (IC50
474.7 13.9
, lM)
a
b
c
a
b
c
6
7
8
25.16 4.52
23.15 0.82
33.47 1.64
>100
40.83 8.39
>100
>100
64.44 5.79
>100
>100
39.02 3.11
>100
34.18 4.12
>100
76.37 2.31
90.82 9.19
>100
>100
>100
>100
>100
9
>100
>100
>100
10
11
12
13
14
>100
>100
>100
>100
>100
50.81 0.71
65.12 12.70
50.39 1.77
6.52 0.76
>100
28.00 0.60
33.68 3.22
38.30 5.95
8.17 0.02
42.37 4.96
28.46 2.20
76.13 8.37
7.45 0.69
13.16 3.85
14.62 0.44
NT
44.91 3.35
33.92 6.72
42.53 0.88
13.30 2.88
21.22 5.99
51.91 2.98
NT
18.33 4.67
8.17 0.11
NT = not tested.
a
Values represent mean standard error for at least two separate experiments performed in triplicate.
IC50 is defined as the concentration of compound necessary to inhibit cellular growth by 50%.
b
Table 2
Comparison of anti-proliferative activities: amide-containing versus triazole-containing novobiocin analogues
H
N
N
N
MeO
HO
N
R
R
O
MeO
HO
O
O
O
O
O
O
O
O
OH
OH
R = Biaryl
R = Biaryl (11c)
IC50 (MCF-7)
IC50 (SKBr-3)
18.7 1.8a
7.5 1.0a
13.16 3.85
21.22 5.99
R = 3-Indole
5.3 0.3a
R = 3-Indole (14c)
18.33 4.67
8.17 0.11
IC50 (MCF-7)
IC50 (SKBr-3)
12.2 1.5a
a
Biological data taken from Ref. 13.

3960
L. B. Peterson, B. S. J. Blagg / Bioorg. Med. Chem. Lett. 20 (2010) 3957–3960
lines. Western blot analysis affirmed Hsp90 inhibition by this class
of compounds. The compounds described herein exhibit compara-
ble activities to the corresponding amide-containing analogues.
These results indicate that the amide moiety can be replaced by
the triazole functionality, however, in some cases the loss of the
hydrogen bond donor appears detrimental, but can be overcome
by the inclusion of steric bulk in the triazole substituent.
Acknowledgements
Figure 2. Western blot analysis for compound 14b.
The authors gratefully acknowledge the support of this project
by NIH CA120458 and the NIH Training Grant (T32 GM008545)
on Dynamic Aspects in Chemical Biology (L.B.P.).
are comparable against the two cell lines tested. One discrepancy
observed between the amide and triazole analogues is that in
which simple aryl side chains (6–10) manifest IC50 values in the
M range for the amide-containing molecules while the tri-
azole compounds 6 and 9 display IC50 values above 50
Supplementary data
10–20
l
l
M.13 These
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.04.140.
results suggest that the triazole moiety affects biological activity in
two ways: the availability of a hydrogen bond donor in the amide
linkage and the steric bulk of the side chain. This hydrogen bond
contact is lost upon introduction of the triazole moiety, but can
be overcome by the introduction of steric bulk in the side chain.
A preference for steric bulk has been observed in several series of
novobiocin analogues, and appears to be a contributing factor to
biological activity.
After analysis of the triazole-containing analogues anti-prolifer-
ative activity, Western blot analysis was preformed for the most
active compound, 14b, to confirm anti-proliferative activity results
from Hsp90 inhibition (Fig. 2). Western blot analysis of MCF-7 cells
treated with increasing concentrations of 14b, induce Hsp90-
dependent client protein degradation in a dose-dependent manner,
with an apparent IC50 value that correlates directly to the anti-pro-
liferative IC50 value. Her2 and c-Raf are client proteins of Hsp90,
and pharmacological inhibition of Hsp90 leads to their degradation
via ubiquitinylation and proteome-mediated hydrolysis. Addition-
ally, compound 14b appears to have little effect on the heat shock
response, as unaltered levels of Hsp90 were observed, consistent
with inhibition of the Hsp90 C-terminus. In contrast, client protein
degradation and heat shock induction was not observed for the
inactive compound 10b (data not shown). Western blot analysis
indicates that these compounds are interfering with the Hsp90-
mediated protein folding process, and that the anti-proliferative
activities for these compounds are directly related to Hsp90
inhibition.
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In conclusion, a series of triazole-containing novobiocin ana-
logues was prepared and evaluated against two breast cancer cell