Synthesis and evaluation of noviose replacements on novobiocin that manifest antiproliferative activity

Article abstract of DOI:10.1021/ml100070r

Structural modifications to the coumarin core and benzamide side chain of novobiocin have successfully transformed the natural product from a selective DNA gyrase inhibitor into a potent inhibitor of the Hsp90 C-terminus. However, no structure-activity relationship studies have been conducted on the noviose appendage, which represents the rate-limiting synthon in the preparation of analogues. Therefore, a series of sugar mimics and nonsugar derivatives were synthesized and evaluated to identify simplified compounds that exhibit Hsp90 inhibition. Evaluation against two breast cancer cell lines demonstrated that replacement of the stereochemical complex noviose with simplified alkyl amines increased antiproliferative activity, resulting in novobiocin analogues that manifest IC₅₀ values in the midnanomolar range.

Full text of DOI:10.1021/ml100070r

pubs.acs.org/acsmedchemlett
Synthesis and Evaluation of Noviose Replacements on
Novobiocin That Manifest Antiproliferative Activity
Huiping Zhao, Bhaskar Reddy Kusuma, and Brian S. J. Blagg*
Department of Medicinal Chemistry, 1251 Wescoe Hall Drive, Malott 4070, The University of Kansas, Lawrence,
Kansas 66045-7563
ABSTRACT Structural modifications to the coumarin core and benzamide side
chain of novobiocin have successfully transformed the natural product from a
selective DNA gyrase inhibitor into a potent inhibitor of the Hsp90 C-terminus.
However, no structure-activity relationship studies have been conducted on the
noviose appendage, which represents the rate-limiting synthon in the preparation
of analogues. Therefore, a series of sugar mimics and nonsugar derivatives were
synthesized and evaluated to identify simplified compounds that exhibit Hsp90
inhibition. Evaluation against two breast cancer cell lines demonstrated that
replacement of the stereochemical complex noviose with simplified alkyl amines
increased antiproliferative activity, resulting in novobiocin analogues that manifest
IC50 values in the midnanomolar range.
KEYWORDS Heat shock protein 90, Hsp90 inhibitors, novobiocin, stucture-activity
relationships, breast cancer
he 90 kDa heat shock proteins (Hsp90) are responsible
for the conformational maturation of more than 200
T
Hsp90-dependent client proteins,1,2 of which Her2,
Src family kinases, Raf, PLK, RIP, AKT, telomerase, and Met
are directly associated with the six hallmarks of cancer.3,4
Consequently, inhibition of the Hsp90 protein folding machi-
nery simultaneously disrupts multiple oncogenic pathways,
leading to cell death.5,6 Since the first proof-of-concept drug,
17-AAG (a synthetic analogue of geldanamycin), entered
clinical trials and demonstrated therapeutic benefit at toler-
able doses,7 extensive research has led to more than 20 sub-
sequent clinical trials,8 highlighting Hsp90 as a promising
therapeutic target for the development of anticancer agents.9-11
Novobiocin, a natural product comprised of a noviose sugar,
a coumarin core, and a prenylated benzamide side chain, is
isolated from Streptomyces strains12 and is known to exhibit
antimicrobial activity by binding to the DNA gyrase ATP-
binding pocket,13 a unique nucleotide-binding motif shared
only by members of the GHKL superfamily.14 Because of the
similar bent conformation exhibited by ADP bound to both
DNA gyrase and the Hsp90 N-terminal domain, Neckers and
co-workers hypothesized that novobiocin may manifest anti-
cancer activity through Hsp90 inhibition.15 Their pioneering
studies revealed novobiocin to bind Hsp90, but instead of
binding to the well-recognized N-terminal domain, it bound
to a previously unrecognized C-terminal region, albeit with low
affinity (∼700 μM in SKBr3 cells).15 Since this study, structural
modifications of novobiocin have been pursued to identify
molecules that exhibit increased inhibitory activity.16-22
The first library of such compounds was designed and
synthesized by Yu and co-workers to identify functionalities
Figure 1. SAR generated from previous investigations.
necessary for Hsp90 inhibition on the coumarin ring and
benzamide side chain of novobiocin.17 Their studies revealed
that attachment of the noviose appendage to the 7-position of
the coumarin ring and an amide linker at the 3-position is
critical, while the 4-hydroxy substituent and the 30-carbamoyl
are detrimental. The most efficacious compound identified
Received Date: April 9, 2010
Accepted Date: July 7, 2010
Published on Web Date: July 13, 2010
r
2010 American Chemical Society
311
DOI: 10.1021/ml100070r ACS Med. Chem. Lett. 2010, 1, 311–315
|

pubs.acs.org/acsmedchemlett
Scheme 1. Preparation of Coumarin Phenol 10
Subsequent structural modifications and SAR studies ex-
plored the coumarin ring and benzamide side chain, and
several lead-like compounds were identified and remain under
investigation.19,21,22 As shown in Figure 1, the analogues
prepared thus far retain the noviose appendage. However, the
synthesis of noviose is laborious and hinders analogue deve-
lopment, as it requires more than 10 steps to prepare and
activate for subsequent coupling with the coumarin phenol.25,26
Acknowledging the limited SAR for this moiety and its cumber-
some preparation, simplified analogues were pursued in an
effort to increase activity while simultaneously increasing solu-
bility. In this article, we provide the first biologically active
substitutions for the noviose appendage on novobiocin and
the first nonsugar mimics that exhibit increased antiprolifera-
tive activity.
It is well understood that sugar moieties in natural pro-
ducts play a key role in solubility, activity, and bioavailability
for these compounds. Furthermore, the ring size can impart
significant affinity toward their cognate protein. With these
considerations in mind, a series of mono- and dihydroxy-
lated furanose and pyranose sugars (1-5, Scheme 2) were
synthesized according to previously disclosed procedures26
for incorporation onto the novobiocin scaffold, 10. The
preparation of 10 is described in Scheme 1. The coumarin
phenol 6 was converted to the methoxymethyl (MOM) ether
using MOM chloride and Hunig's base in dimethylform-
amide. The free aniline, liberated through hydrogenolysis
with 10% Pd/C and hydrogen in tetrahydrofuran from 7,
was coupled with acid chloride 8 to give benzamide 9.
from this library was compound A4, which induced degrada-
tion of Hsp90-dependent client proteins at ∼70-fold lower
concentration than novobiocin. Intriguingly, compound A4
induced the heat shock response at concentrations ∼1000-fold
lower than that required for client protein degradation.23,24
To confirm whether structure-activity relationship (SAR)
trends observed by Yu and co-workers conformed to the
natural product, DHN2 was synthesized to delineate function-
alities responsible for DNA gyrase versus Hsp90 inhibition.18
This novobiocin analogue confirmed that the 4-hydroxyl and
the 30-carbamate are detrimental to Hsp90 inhibitory activity
but critical for DNA gyrase inhibition, thus confirming the SAR
trends observed for A4.
Scheme 2. Synthesis of Novobiocin Analogues Containing Mono- and Dihydroxylated Furanoses and Pyranoses
r
2010 American Chemical Society
312
DOI: 10.1021/ml100070r ACS Med. Chem. Lett. 2010, 1, 311–315
|

pubs.acs.org/acsmedchemlett
Subsequent cleavage of the MOM ether with 4 N hydrochlor-
ide in dioxane provided phenol 10 in high yield.
Table 1. Anti-proliferative Activities of Sugar-Derived Novobiocin
Analogues
Once prepared, the phenol of 10 was coupled with sugars
1-5 under Mitsunobu conditions to give an inseparable
diastereomeric mixture of 11-13 and 15 (Scheme 2). In
the case of compound 14, a single diastereomer was formed.
Subsequent hydrolysis of the cyclic carbonates and acetyl
esters of 11a,b and 14 with lithium hydroxide in THF/MeOH/
H2O (3:2:2, v/v) afforded a diastereomeric mixture of 16a,b
and 19, respectively. At this stage, diastereomers 16a and
16b were separated by silica chromatography. The assign-
ment of stereochemistry at the anomeric center was establi-
shed through two-dimensional NMR studies utilizing nuclear
Overhauser effect spectroscopy. Hydrolysis of the benzoyl
and acetyl ester of 12 with basic methanol yielded 17a and
17b, which could be separated by silica chromatography.
The tri-isopropylsilyl of 13 and tert-butyldimethylsilyl groups
of 15 were removed by the addition of tetrabutylammonium
fluoride to give separable 18a and 18b, and 20a and 20b,
respectively.
Upon construction of the noviose surrogates, compounds
were subjected to evaluation of anti-proliferative activity
against SKBr3 (estrogen receptor negative, Her2 overexpres-
sing breast cancer cells) and MCF-7 (estrogen receptor
positive breast cancer cells) cell lines. As shown in Table 1,
the six-membered sugar mimics (16a-18a and 16b-18b)
were found to be more potent than their five-membered
counterparts (19 and 20a,b). Compound 18b displayed an
IC50 value of 3.11 ( 0.03 and 1.56 ( 0.20 μM against SKBr3
and MCF-7 cell lines, respectively, which is ∼3-8-fold more
active than DHN2 (Table 1) and ∼200 times greater than the
activity manifested by novobiocin. Surprisingly, the stereo-
chemistry of these sugar mimics was not critical for the
observed increase in anti-proliferative activity, as both the R-
(16b) and the β-anomers (17a) produced similar activities.
Placement of the hydroxyl group (30-OH or 40-OH) on the
etheral ring also did not impart preferential activity.
Although simplified sugar mimics were found to increase
anti-proliferative activity as compared to DHN2 and novo-
biocin, more simplified analogues exhibiting enhanced solu-
bility and activity were desired. N-Heterocycles are found in
a variety of biologically active compounds and, in contrast to
carbohydrates, are generally ionized at physiological pH.27
Upon review of the first set of studies, we proposed that the
noviose appendage played a siginficant role in solublizing
the relatively hydrophobic coumarin core and benzamide
side chain. Thus, commercially available amines, 21-27
(Scheme 3, secondary amines were protected with Boc),
were selected as potential replacements for the noviose
moiety. These alkylamines and heterocyclic analogues con-
tain an ionizable amine located at various positions within
the structure to afford potential hydrogen-bonding interac-
tions while simultaneously enhancing solubility through
their ionized counterparts.
compound
n
R2
R1
SKBr3 (μM)
MCF7 (μM)
16a
16b
17a
17b
18a
18b
19
2
2
2
2
2
2
1
1
1
OH
OH
H
OH
OH
OH
OH
H
10.68 ( 0.05a
6.96 ( 0.06
7.87 ( 0.04
29.98 ( 2.07
5.07 ( 0.26
3.11 ( 0.03
14.37 ( 0.52
22.16 ( 0.94
21.46 ( 2.28
10.86 ( 0.47
10.04 ( 0.03
13.30 ( 0.21
6.45 ( 0.13
10.24 ( 0.21
1.34 ( 0.18
1.56 ( 0.20
14.31 ( 0.40
>100
H
OH
OH
OH
OH
OH
H
OH
H
20a
20b
DHN2
H
22.50 ( 0.40
11.29 ( 0.41
a Values represent mean ( standard deviation for at least two
separate experiments performed in triplicate.
issue, the amine was first coupled with the coumarin ring
and subsequently with the benzamide side chain to afford
the desired analogues. The detailed synthesis is described as
follows: Tertiary amines or Boc-masked secondary amines
were reacted with Cbz-protected coumarin 6 in the presence
of 2 equiv of triphenylphosphine and diisopropylazodicarboxy-
late in tetrahydrofuran to give amine-derived coumarins, 28a-g.
The Cbz-protecting group was removed by hydrogenolysis
to give the free amines, which were then coupled with acid
chloride 8 to give compounds 29a-g in good yield. Removal of
the Boc protecting group with trifluoroacetic acid in methylene
chloride afforded the secondary amine analogues 30b, 30d,
and 30g. Hydrolysis of the phenolic ester with methanolic
triethyl amine gave analogues, 31a-g, in good to excellent yields.
The anti-proliferative activity manifested by these analogues
was assessed against SKBr3 and MCF-7 cell lines. As shown in
Table 2, the IC50 values for the secondary and tertiary amines
varied between 0.4 and 1.5 μM, making them 1500-fold more
potent than novobiocin. Generally, the ester series exhibited
comparable anti-proliferative activity to their phenol counter-
parts, suggesting that the ester analogues may rapidly hydro-
lyze in cells due to esterases. With regard to the piperidine
analogues, 4-substituted analogues exhibited greater potency
than the 3-substituted analogues against both cell lines. For
example, against the SKBr3 cell line, compound 29a is ∼3-fold
more active than compound 29c and compounds 30b and 31b
are ∼2 times more active than compounds 30d and 31d,
respectively. The same trend was observed for the noncyclic
amino analogues as well (29f vs 29e and 31f vs 31e). These
results indicate that the location of the amine is important for
binding/manifesting inhibitory activity. A surprising finding
from this work was that analogues containing noncyclic amines
exhibit equivalent potencies to their piperidine counterparts,
Originally, coupling of these amines with phenol 10 was
expected to easily afford the desired products. However, the
acetyl ester on the benzamide side chain was hydrolyzed
under these conditions and resulted in an inseparable mix-
ture of mono- or dialkylated products. To circumvent this
r
2010 American Chemical Society
313
DOI: 10.1021/ml100070r ACS Med. Chem. Lett. 2010, 1, 311–315
|

pubs.acs.org/acsmedchemlett
Scheme 3. Synthesis of Amine Analogues
Table 2. Anti-proliferative Activities of Amine Analogues
compound
R
R0
R00
SKBr3 (μM)
MCF-7 (μM)
29a
29c
29e
29f
A
B
C
D
A
B
C
A
A
B
B
C
D
C
Ac
Ac
Ac
Ac
Ac
Ac
Ac
H
Me
Me
Me
0.58 ( 0.05a
1.42 ( 0.02
1.32 ( 0.23
0.46 ( 0.19
0.56 ( 0.05
1.23 ( 0.00
0.91 ( 0.21
0.76 ( 0.17
0.47 ( 0.10
4.69 ( 0.16
0.79 ( 0.11
9.45 ( 0.22
0.44 ( 0.02
0.75 ( 0.12
1.18 ( 0.20
1.57 ( 0.05
4.76 ( 0.52
1.18 ( 0.03
1.53 ( 0.14
1.54 ( 0.41
2.08 ( 0.13
1.09 ( 0.10
0.85 ( 0.09
10.12 ( 0.17
1.68 ( 0.25
13.48 ( 0.38
1.35 ( 0.38
1.33 ( 0.01
30b
30d
30g
31a
31b
31c
31d
31e
31f
H
H
H
Me
H
H
Figure 2. Western blot analyses of Hsp90-depedent client pro-
teins from MCF-7 breast cancer cell lysates upon treatment with
18b (top) or 31b (bottom). Concentrations (in μM) were indicated
above each line, and geldanamycin (G, 0.5 μM) and dimethyl
sulfoxide (D) were employed as positive and negative controls.
H
Me
H
H
H
Me
H
dependent manner upon treatment with 18b or 31b. The
non-Hsp90-dependent protein, actin, was not altered upon
administration of 18b or 31b, indicating that selective de-
gradation of Hsp90-dependent proteins takes place in the
presence of 18b or 31b. In addition, neither of these two
compounds induced the heat shock response, which is a
characteristic shared by benzamide-containing novobiocin
analogues that bind the Hsp90 C-terminus.28,29
In a conclusion, sugar mimics and amino analogues of the
noviose appendage on the coumarin ring of novobiocin that
exhibit improved solubility and anti-proliferative activity have
been produced. The cyclic and acyclic amino surrogates can be
synthesized expeditiously and will enable rapid identification of
31g
H
H
a Values represent mean ( standard deviation for at least two
separate experiments performed in triplicate.
specifically, 29a and 29f and 31a and 31f, indicating that
inclusion of a constrained ring is not required.
To confirm that replacement of noviose with a sugar or
amino surrogate did not alter inhibitory activity against
Hsp90, Western blot analyses of cell lysates following the
administration of 18b or 31b were performed. As shown in
Figure 2, the Hsp90-dependent client proteins, Her2, Raf,
and Akt, were degraded in MCF-7 cells in a concentration-
r
2010 American Chemical Society
314
DOI: 10.1021/ml100070r ACS Med. Chem. Lett. 2010, 1, 311–315
|

pubs.acs.org/acsmedchemlett
novobiocin analogues that may provide clinical opportunities for
the treatment of cancer. The development of improved com-
pounds that exhibit such activity is underway, and the results
from those studies will be reported in due course.
(15) Marcu, M. G.; Chadli, A.; Bouhouche, I.; Catelli, M.; Neckers,
L. M. The Heat Shock Protein 90 Antagonist Novobiocin
Interacts with a Previously Unrecognized ATP-binding Do-
main in the Carboxyl Terminus of the Chaperone. J. Biol.
Chem. 2000, 275, 37181–37186.
(16) Shen, G.; Yu, X. M.; Blagg, B. S. J. Syntheses of Photolabile
Novobiocin Analogues. Bioorg. Med. Chem. Lett. 2004, 14,
5903–5906.
(17) Yu, X. M.; Shen, G.; Neckers, L.; Blake, H.; Holzbeierlein, J.;
Cronk, B.; Blagg, B. S. J. Hsp90 Inhibitors Identified from a
Library of Novobiocin Analogues. J. Am. Chem. Soc. 2005,
127, 12778–12779.
(18) Burlison, J. A.; Neckers, L.; Smith, A. B.; Maxwell, A.; Blagg,
B. S. J. Novobiocin: Redesigning a DNA Gyrase Inhibitor for
Selective Inhibition of Hsp90. J. Am. Chem. Soc. 2006, 128,
15529–15536.
SUPPORTING INFORMATION AVAILABLE Experimental
procedures for the synthesis and characterization of new com-
pounds (1H and 13C NMR and HR-MS). This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author: *To whom correspondence should be
addressed. Tel: 785-864-2288. Fax: 785-864-5326. E-mail: bblagg@
ku.edu.
(19) Burlison, J. A.; Blagg, B. S. J. Synthesis and Evaluation of
Coumermycin A1 Analogues that Inhibit the Hsp90 Protein
Folding Machinery. Org. Lett. 2006, 8, 4855–4858.
(20) Huang, Y. T.; Blagg, B. S. J. A Library of Noviosylated Coumarin
Analogues. J. Org. Chem. 2007, 72, 3609–3613.
Funding Sources: We gratefully acknowledge support of this
project by the NIH/NCI (CA120458).
(21) Burlison, J. A.; Avila, C.; Vielhauer, G.; Lubbers, D. J.;
Holzbeierlein, J.; Blagg, B. S. J. Development of Novobiocin
Analogues That Manifest Anti-proliferative Activity against
Several Cancer Cell Lines. J. Org. Chem. 2008, 73, 2130–2137.
(22) Donnelly, A. C.; Mays, J. R.; Burlison, J. A.; Nelson, J. T.;
Vielhauer, G.; Holzbeierlein, J.; Blagg, B. S. J. The Design,
Synthesis, and Evaluation of Coumarin Ring Derivatives of
the Novobiocin Scaffold that Exhibit Antiproliferative Activ-
ity. J. Org. Chem. 2008, 73, 8901–8920.
(23) Ansar, S.; Burlison, J. A.; Hadden, M. K.; Yu, X. M.; Desino,
K. E.; Bean, J.; Neckers, L.; Audus, K. L.; Michaelis, M. L.;
Blagg, B. S. J. A non-toxic Hsp90 Inhibitor Protects Neurons
from Abeta-Induced Toxicity. Bioorg. Med. Chem. Lett. 2007,
17, 1984–1990.
(24) Lu, Y.; Ansar, S.; Michaelis, M. L.; Blagg, B. S. J. Neuroprotec-
tive Activity and Evaluation of Hsp90 Inhibitors in an Im-
mortalized Neuronal Cell Line. Bioorg. Med. Chem. 2009, 17,
1709–1715.
(25) Yu, X. M.; Shen, G.; Blagg, B. S. J. Synthesis of (-)-Noviose
from 2,3-O-Isopropylidene-D-erythronolactol. J. Org. Chem.
2004, 69, 7375–7378.
(26) Yu, X. M.; Han, H.; Blagg, B. S. J. Synthesis of Mono- and
Dihydroxylated Furanoses, Pyranoses, and an Oxepanose for
the Preparation of Natural Product Analogue Libraries. J. Org.
Chem. 2005, 70, 5599–5605.
(27) Brown, E. G. Ring Nitrogen and Key Biomolecules: The Bio-
chemistry of N-Heterocycles; Kluwer Academic: Boston, 1998.
(28) Donnelly, A.; Blagg, B. S. J. Novobiocin and Additional
Inhibitors of the Hsp90 C-terminal Nucleotide Binding Pocket.
Curr. Med. Chem. 2008, 15, 2702–2717.
(29) Shelton, S. N.; Shawgo, M. E.; Matthews, S. B.; Lu, Y.;
Donnelly, A. C.; Szabla, K.; Tanol, M.; Vielhauer, G. A.;
Rajewski, R. A.; Matts, R. L.; Blagg, B. S. J.; Robertson, J. D.
KU135, a Novel Novobiocin-Derived C-Terminal Inhibitor of
the 90-kDa Heat Shock Protein, Exerts Potent Antiprolifera-
tive Effects in Human Leukemic Cells. Mol. Pharmacol. 2009,
76, 1314–1322.
REFERENCES
(1)
(2)
(3)
Pearl, L. H.; Prodromou, C.; Workman, P. The Hsp90 Mole-
cular Chaperone: An Open and Shut Case for Treatment.
Biochem. J. 2008, 410, 439–453.
Blagg, B. S. J.; Kerr, T. D. Hsp90 Inhibitors: Small Molecules that
Transform the Hsp90 Protein Folding Machinery into A Catalyst
for Protein Degradation. Med. Res. Rev. 2006, 26, 310–338.
Xu, W.; Neckers, L. Targeting the Molecular Chaperone Heat
Shock Protein 90 Provides a Multifaceted Effect on Diverse
Cell Signaling Pathways of Cancer Cells. Clin. Cancer Res.
2007, 13, 1625–1629.
(4)
(5)
Hanahan, D.; Weinberg, R. A. The Hallmarks of Cancer. Cell
2000, 100, 57–70.
Zhang, H.; Burrows, F. J. Targeting Multiple Signal Transduc-
tion Pathways Through Inhibition of Hsp90. Mol. Med. 2004,
82, 488–499.
(6)
(7)
Bishop, S. C.; Burlison, J. A.; Blagg, B. S. J. Hsp90: A Novel
Target for the Disruption of Multiple Signaling Cascades. Curr.
Cancer Drug Targets 2007, 7, 369–388.
Sausville, E. A.; Tomaszewski, J. E.; Ivy, P. Clinical Develop-
ment of 17-Allylamino, 17-Demethoxygeldanamycin. Curr.
Cancer Drug Targets 2003, 3, 377–383.
(8)
(9)
Pacey, S.; Banerji, U.; Judson, I.; Workman, P. Hsp90 Inhibi-
tors in the Clinic. Handb. Exp. Pharmacol. 2006, 331–358.
Chaudhury, S.; Welch, T. R.; Blagg, B. S. J. Hsp90 as ATarget for
Drug Development. ChemMedChem 2006, 1, 1331–1340.
(10) Workman, P.; Burrows, F.; Neckers, L.; Rosen, N. Drugging the
Cancer Chaperone HSP90: Combinatorial Therapeutic Expo-
litation on Oncogene Addiction and Tumor Stress. Ann. N.Y.
Acad. Sci. 2007, 1113, 202–216.
(11) Neckers, L.; Neckers, K. Heat-shock Protein 90 Inhibitors as
Novel Cancer Chemotherapeutics;An Update. Expert Opin.
Emerging Drugs 2005, 10, 137–149.
(12) Hoeksema, H.; Johnson, J. L.; Hinman, J. W. Structural Studies
on Streptonivicin, A New Antibiotic. J. Am. Chem. Soc. 1955,
77, 6710–11 .
(13) Hooper,D.C.;Wolfson,J.S.;McHugh,G.L.;Winters,M.B.;Swartz,
M. N. Effects of Novobiocin, Coumermycin A1, Clorobiocin, and
Their Analogs on Escherichia Coli DNA Gyrase and Bacterial
Growth. Antimicrob. Agents Chemother. 1982, 22, 662–671.
(14) Dutta, R.; Inouye, M. GHKL, An Emergent ATPase/kinase
Superfamily. Trends Biochem. Sci. 2000, 25, 24–8.
r
2010 American Chemical Society
315
DOI: 10.1021/ml100070r ACS Med. Chem. Lett. 2010, 1, 311–315
|