Engineering an antibiotic to fight cancer: Optimization of the novobiocin scaffold to produce anti-proliferative agents

Article abstract of DOI:10.1021/jm200148p

Development of the DNA gyrase inhibitor, novobiocin, into a selective Hsp90 inhibitor was accomplished through structural modifications to the amide side chain, coumarin ring, and sugar moiety. These species exhibit ?700- fold improved anti-proliferative activity versus the natural product as evaluated by cellular efficacies against breast, colon, prostate, lung, and other cancer cell lines. Utilization of structure- activity relationships established for three novobiocin synthons produced optimized scaffolds, which manifest midnanomolar activity against a panel of cancer cell lines and serve as lead compounds that manifest their activities through Hsp90 inhibition.

Full text of DOI:10.1021/jm200148p

ARTICLE
pubs.acs.org/jmc
Engineering an Antibiotic to Fight Cancer: Optimization of the
Novobiocin Scaffold to Produce Anti-proliferative Agents
Huiping Zhao,†,^ Alison C. Donnelly,†,^ Bhaskar R. Kusuma,†,^ Gary E. L. Brandt,† Douglas Brown,‡
Roger A. Rajewski,§^ George Vielhauer,|| Jeffrey Holzbeierlein,|| Mark S. Cohen,|| and Brian S. J. Blagg*,†
†Department of Medicinal Chemistry, The University of Kansas, 1251 Wescoe Hall Drive, Malott 4070, Lawrence, Kansas 66045-7563,
United States
‡Biotechnology Innovation & Optimization Center, The University of Kansas, 2099 Constant Avenue, Lawrence, Kansas 66047-2535,
United States
§^Department of Pharmaceutical Chemistry, The University of Kansas, 2095 Constant Avenue, Lawrence, Kansas 66047-2535,
United States
Department of Urology, The University of Kansas Medical Center, 3901 Rainbow Boulevard, Mail Stop 3016, Kansas City,
Kansas 66160, United States
S Supporting Information
b
ABSTRACT: Development of the DNA gyrase inhibitor,
novobiocin, into a selective Hsp90 inhibitor was accomplished
through structural modifications to the amide side chain,
coumarin ring, and sugar moiety. These species exhibit ∼700-
fold improved anti-proliferative activity versus the natural product
as evaluated by cellular efficacies against breast, colon, prostate,
lung, and other cancer cell lines. Utilization of structureꢀ
activity relationships established for three novobiocin synthons
produced optimized scaffolds, which manifest midnanomolar
activity against a panel of cancer cell lines and serve as lead
compounds that manifest their activities through Hsp90
inhibition.
’ INTRODUCTION
The Hsp90 molecular chaperone has emerged as a promising
target for the treatment of cancer.1 While most current therapies
are directed at disrupting a single molecular function, Hsp90 is
distinctive as it serves to modulate multiple oncogenic pathways.
Moreover, Hsp90 is overexpressed in human malignancies and
its inhibition provides a unique opportunity to develop small
molecules that exhibit high differential selectivity.1ꢀ4 There are
more than 150 client proteins dependent upon Hsp90 for their
folding and conformational maintenance, many of which con-
tribute to cancer cell proliferation and growth.5ꢀ12 Small mole-
cule Hsp90 inhibitors transform the normal protein folding
machinery into a catalyst for protein degradation via utilization
of the ubiquitinꢀproteasome pathway.13,14 As such, Hsp90
inhibition is similar to a combinatorial attack, in which multiple
signaling pathways that contribute to malignancy are simulta-
neously disrupted.1,2,15ꢀ17
Novobiocin (Figure 1), a member of the coumermycin family
of antibiotics, manifests potent antimicrobial activity through
inhibition of DNA gyrase-mediated ATP-hydrolysis.18ꢀ22 Co-
crystal structures of DNA gyrase B bound to novobiocin and
ADP revealed both molecules to bind in an atypical, bent
conformation.23ꢀ25 It was this unique binding conformation
Figure 1. Structure of novobiocin.
that led Neckers and co-workers to hypothesize that novobiocin
exhibits its antitumor activity (∼700 μM) against SKBr3 breast
cancer cells through Hsp90 inhibition. Through subsequent
Western blot analyses, Neckers and co-workers confirmed that
novobiocin induces degradation of Hsp90-dependent clients
in a concentration-dependent manner, a hallmark of Hsp90
Received: February 9, 2011
Published: May 09, 2011
r
2011 American Chemical Society
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inhibition. In contrast to other Hsp90 inhibitors, it was shown
that novobiocin bound to the C-terminus, revealing a previously
unrecognized binding site, and perhaps, a new opportunity to
modulate the Hsp90 protein folding machinery.26
1000ꢀ10000-fold lower than that required for client protein
degradation, a phenomenon that had not been previously ob-
served. Because of its activity and nontoxic nature, 1 was evaluated
as a neuroprotective agent and produced an EC50 of 6 nM in a
culture model for Alzheimer’s disease.28
To confirm SAR trends observed by Yu and co-workers, two
natural product analogues, 2 and 3 (Figure 2), were designed and
subsequently evaluated by Burlison and co-workers. This study
aimed at elucidating functionalities on novobiocin that are
responsible for DNA gyrase inhibition and to deconvolute
Hsp90-selective inhibitory compounds. Studies with these com-
pounds confirmed that the 4-hydroxyl and the 30-carbamate are
detrimental to Hsp90 inhibitory activity but critical for DNA
gyrase inhibition.29 This study confirmed the trends observed
from a library based on compound 1, and produced the first
selective inhibitors of the Hsp90 C-terminus.
Although no cocrystal structure of Hsp90 bound to C-terminal
inhibitors has been reported, the structure and function of the
Hsp90 C-terminal nucleotide binding site are under intense
investigation. Compounds exhibiting higher affinity for Hsp90
than novobiocin are likely to provide the tools necessary for
elucidation of the Hsp90 C-terminal nucleotide-binding pocket
and provide new Hsp90 modulators that manifest chemother-
apeutic potential.27 The poor Hsp90 inhibitory activity of
novobiocin26 has been greatly improved upon by the preparation
of analogues. Yu and co-workers produced a library of com-
pounds that established the first structureꢀactivity relationships
for novobiocin as an Hsp90 inhibitor. This focused library
explored essential features of the benzamide side chain, coumarin
core, and noviose sugar, highlighting that attachment of noviose
to the 7-position and an amide linker at the 3-position of the
coumarin ring are critical for Hsp90 inhibitory activity. More-
over, modifications to the sugar identified the diol as most potent
versus the cyclic carbonate and corresponding carbamates as
found in novobiocin.28
Figure 3. Structures of 5 and coumermycin A1.
On the basis of the cytotoxicity of coumermycin A1 (Figure 3),
another member of the coumermycin family of antibiotics, it
appeared that dimerization could increase Hsp90 inhibitory
activity. Burlison and co-workers dimerized 1 in an attempt to
transform a nontoxic agent into a more potent analogue. The
resulting compound, 5, the most potent identified, exhibited low
micromolar anti-proliferative activity but did not induce the heat
shock response. These results suggested that modification of the
amide side chain could convert a nontoxic molecule into an anti-
proliferative agent.31 Consequently, a series of monomeric species
based on 5 was synthesized and evaluated in cell proliferation
studies.32 Monomeric analogues were designed to include biaryl
and heterocyclic amide derivatives that explored hydrogen-bond-
ing interactions within the putative binding pocket that typically
accommodates the prenylated benzamide of novobiocin. This
library identified key functionalities and established the first set of
SAR for the amide side chain (Figure 4).
The coumarin core of novobiocin was recently explored
and SAR established. Derivatives of 1 with variations to the
coumarin ring were designed to probe the importance of inter-
actions manifested by the natural substrate purine ring as well the
size and nature of the novobiocin binding pocket. The 6- and
8-positions of the coumarin scaffold proved tolerable to sub-
stitution, and incorporation of alkoxy groups at these locations
led to improved inhibitory activity. Moreover, the lactone moiety
of the coumarin ring was found to be dispensable (Figure 5).33
Figure 2. Structures of 1, 2, 3, and 4.28ꢀ30
Compound 1 (Figure 2) was the most notable compound
identified from this library. 1, with a shortened N-acyl side chain, a
4-deshydroxy substituent and no carbamoyl group on noviose,
induced degradation of Hsp90-dependent client proteins at ∼70-
fold lower concentration than novobiocin.28 However, in contrast
to N-terminal inhibitors, 1 induced Hsp90 at concentrations
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Sugar mimics were originally attached to the 7-position of
coumarin containing an 8-methyl substituent and a biaryl
benzamide. The most potent coumarin scaffolds obtained
from the coumarin studies were coupled with the optimal
biaryl side chain to yield a scaffold upon which sugar surro-
gates were appended.32,33 On the basis of the inhibitory
activity manifested by the biaryl analogues, the most promis-
ing sugar surrogates were coupled with coumarins containing
the 2-indole side chain.32 The simplified sugars and related
azasugars were found to conserve structural units present in
noviose but lacked the complexity of multi-step synthesis.
These sugar surrogates were designed to probe potential
hydrogen bonds as well as to determine the dimensions of
the pocket. In addition to simplified sugars, several surrogates
containing heteroatoms and various appendages were also
explored. These diverse sugar and nonsugar analogues sim-
plify the preparation of novobiocin analogues while providing
a handle to improve both solubility and efficacy. The design,
synthesis, and biological evaluation of these optimized com-
pounds are reported herein.
’ RESULTS AND DISCUSSION
Design, Synthesis, and Evaluation of Modified Sugar
Analogues of Novobiocin. Numerous biologically active nat-
ural products contain carbohydrates appended to their scaffolds
that serve to increase solubility and provide interactions with
their cognate receptor. In many cases, removal of the carbohy-
drate moiety renders the aglycon inactive, whereas alteration of
the sugar ring size can alter affinity of the compounds toward
specific targets.37ꢀ39 Modifications to the noviose pyranose ring
were proposed to elucidate functionalities required for inhibitory
activity as well as to determine whether different sized sugars can
be utilized as replacements for noviose. Thus, five-, six-, and
seven-membered sugars were synthesized and coupled to the
aforementioned scaffolds to determine optimal interactions.
When available, the R- and β-anomers were also evaluated. A
set of protected mono-, di- and trihydroxylated furanoses,
pyranoses, and oxepanose sugars previously synthesized by Yu
and co-workers40 (EꢀL, Figure 6) was chosen based on these
considerations.
Figure 4. Benzamide SAR.32
Figure 5. Coumarin SAR.33
Renoir and co-workers examined the role of noviose in Hsp90
inhibition. Their structureꢀactivity relationship studies demon-
strated that Hsp90 inhibition was observed by analogues that lack
the noviose moiety if a tosyl substituent is attached to the C-4 or
C-7 position of the coumarin. These analogues manifested mid-
micromolar IC50 values.34 In a subsequent paper, the same group
suggested that Hsp90 inhibition can be enhanced by removal of
C7/C8 substituents in denoviose analogues bearing a tosyl group
at the 4-position. These studies produced inhibitors with simpli-
fied coumarins that also exhibited mid-micromolar IC50 values.35
The compounds produced in these two publications represented
the first novobiocin analogues that lacked the noviose sugar while
manifesting Hsp90 inhibition.
The noviose sugar found in novobiocin is complex and is prepared
via laborious efforts. Even the most efficient syntheses of noviose
require more than 10 steps and the overall yields are less than
optimal.36 In an effort to gain further insight into this region of
novobiocin, simplified noviose derivatives were attached to the
novobiocin core. This study, which resulted in compounds that
exhibit a 700-fold improvement in activity over novobiocin, indicated
that while the sugar moiety plays a critical role in maintaining binding
interactions with Hsp90, not all functionalities are necessary.
Figure 6. Five-, six-, and seven-membered noviose alternatives.
The analogues were assembled in modular fashion, allowing
sequential coupling of sugars and the biaryl acid side chain with
the desired scaffold. As shown in the retrosynthetic analysis
(Scheme 1), the biaryl acid side chain was assembled through a
Suzuki coupling reaction, as described previously.33 Next, the
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Scheme 1. Retrosynthesis of Novobiocin Biaryl Analogues
with Sugars EꢀL
biaryl acid was converted to its corresponding acid chloride and
then coupled with the protected coumarin, 9. Finally, Mitsunobu
etherification between coumarin phenols 6aꢀc and sugars EꢀL
yielded the desired analogues in good yields.
Synthesis of these analogues, as described in Scheme 2, began
via protection of phenol 732,33 as the corresponding ester, 8.
Next, hydrogenolysis was employed to liberate vinylogous amide
9, which was subsequently coupled with biaryl acyl chloride, 11.
The biaryl acid chloride was generated from the corresponding
biaryl acid 10, as described previously.33 Finally, solvolysis of
ester 12 afforded phenol 6aꢀc in good yield.
Coumarin phenols 6aꢀc and protected sugar F were coupled
via a Mitsunobu etherification to yield an inseparable mixture of
diastereomers, 13, in a 3:2 ratio, respectively (Scheme 3). The
cyclic carbonates of the resulting mixture were solvolyzed to
afford a mixture of diols 14 and 15, which were separated by silica
chromatography. The stereochemistry at the anomeric carbon
was assigned by 2D-NOESY spectroscopy.
An anomeric mixture of compound 13 was synthesized via
Mitsunobu etherification between sugar F and coumarin 6.
Scheme 2. Synthesis of Phenol 6
Scheme 3. Synthesis of Pyranose-Containing Biaryl Analogues
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Scheme 4. Synthesis of Furanose- and Oxepanose-Containing Biaryl Analogues
Scheme 5. Synthesis of Cyclohexyl-Containing Biaryl Analogues
Likewise, the Mitsunobu conditions were used to append sugar
G to coumarin 6 and furnish the anomeric mixture of compound
16 and to attach sugar H to coumarin 6 to yield anomeric mixture
19. Solvolysis of the cyclic carbonate present in 13 furnished a
mixture of separable diols, 14 and 15. Moreover, benzoyl (Bz)
deprotection of 16 yielded analogues 17 and 18 in a 3:2 ratio,5
while TIPS removal of 19 furnished analogues 20 and 21 in a 4:3
ratio. Not surprisingly, a single diastereomer of 22 was exclusively
formed when coumarin 6 and protected sugar E were subjected
to Mitsunobu conditions. Subsequent deprotection of the acet-
onide and benzyl protecting groups afforded triol 23 in good
yield (Scheme 3).
As shown in Scheme 4, protected furanose sugars JꢀL were
coupled with coumarin 6 by enlisting Mitsunobu etherification to
afford the corresponding analogues in good yields. Mitsunobu
coupling of protected sugar J with coumarin 6 yielded exclusively
diastereomer 24, which was subsequently hydrolyzed to afford
diol 25. Mitsunobu etherification was used to append sugar K to
yield analogue 26, which, after silyl removal, furnished separable
compounds 27 and 28. Likewise, the same conditions were
applied to append sugar L to 29, which, after silyl cleavage,
yielded analogues 30 and 31. Protected oxepanose I was coupled
to coumarin 6 using Mitsunobu conditions to afford 32 as the
major product, which was hydrolyzed to give 33.
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Table 1. Anti-proliferative Activity of Various Sugar Analogues
compd (IC50, μM)
R
R1
R2
R3
anomer
X
Y
SKBr3
MCF-7
LNCAP-LN3
PC3-MM2
14a
15a
14b
15b
14c
15c
17a
18a41
20a
21a41
20b
20c
21c
23a
23b
23c
25a
25b
25c
27a
28a
30a
31a
33a
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
B
B
B
B
B
B
B
C
OH
OH
OH
OH
OH
OH
OH
OH
H
OH
OH
OH
OH
OH
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
β
R
β
R
β
R
β
R
β
R
β
β
R
β
β
β
β
β
β
β
R
β
R
β
H
H
H
H
CH3
CH3
OCH3
OCH3
CH3
CH3
CH3
CH3
CH3
CH3
OCH3
CH3
CH3
CH3
OCH3
CH3
CH3
OCH3
CH3
CH3
CH3
CH3
CH3
CH3
6.23 ( 0.52a,c
6.71 ( 0.80
1.46 ( 0.46
11.10 ( 0.43
7.18 ( 0.20
12.52 ( 0.54
>100
2.56 ( 0.08a
42.56 ( 2.48
17.46 ( 1.22
10.85 ( 0.18
9.50 ( 0.16
64.51 ( 3.52
>100
3.59 ( 3.40b
1.20 ( 0.83
2.29 ( 1.25
NT
NTb
12.88 ( 9.30
10.72 ( 10.85
NT
OCH3
OCH3
H
12.76 ( 5.12
14.93 ( 9.24
3.75 ( 0.01d
7.34 ( 11.82
3.11 ( 2.58
8.56 ( 9.00
11.50 ( 5.66
1.49 ( 0.51
3.27 ( 0.09d
3.23 ( 3.76
6.78 ( 2.25
4.66 ( 1.48
3.65 ( 2.83
2.60 ( 1.51
4.72 ( 1.48
NT
24.03 ( 17.14
25.37 ( 7.26
10.05 ( 0.01d
NT
H
H
>100
>100
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
H
>100
>100
>100
H
H
9.28 ( 0.04
8.75 ( 0.49
>100
11.20 ( 0.49
95.77 ( 3.21
>100
NT
H
H
NT
1.24d
H
OCH3
OCH3
H
H
1.37 ( 0.14
9.59 ( 0.42
11.76 ( 0.06
9.45 ( 0.19
12.46 ( 0.52
7.57 ( 1.05
9.45 ( 0.19
>100
>100
2.35 ( 1.11
4.58 ( 2.78
17.94 ( 10.33
5.90 ( 2.58
NT
OH
OH
OH
OH
OH
OH
OH
OH
H
CH2OH
CH2OH
CH2OH
H
11.07 ( 0.47
14.34 ( 0.16
3.37 ( 0.06
37.17 ( 1.68
11.73 ( 0.78
3.37 ( 0.07
>100
H
OCH3
H
H
H
10.64 ( 20.83
9.56 ( 2.58
NT
H
OCH3
H
H
H
>100
>100
5.34 ( 4.76
3.72d
NT
OH
OH
OH
H
35.24 ( 1.76
>100
>100
10.05 ( 6.75d
26.79 ( 58.24
NT
H
H
>100
4.96 ( 10.58
>100
OH
H
2.38 ( 0.06
>100
a Values represent mean ( standard deviation for at least two separate experiments performed in triplicate. b Values represent mean ( standard deviation
c
from dose response curves for at least two separate experiments performed in duplicate. All error values listed represent 95% confidence intervals
throughout the manuscript except where indicated. d Error values listed as standard deviations.
Table 2. Anti-proliferative Activity of Cyclohexyl Analogues
compd (IC50, μM)
R1
R2
X
Y
SKBr3
>100a
MCF-7
>100a
LNCAP-LN3
PC3-MM2
43a
43b
43c
44a
44b
44c
4541
45b
45c
CH3
CH3
CH3
H
CH3
CH3
CH3
CH3
CH3
CH3
H
H
H
CH3
1.24 ( 0.17b,c
NT
2.49 ( 1.70b
NT
OCH3
CH3
3.45 ( 1.73
>100
1.56 ( 0.03
>100
OCH3
H
NT
NT
CH3
>100
>100
1.14 ( 0.67
NT
4.09 ( 1.63
NT
H
H
OCH3
CH3
6.38 ( 0.71
>100
>100a
8.52 ( 0.36
>100
H
OCH3
H
NT
NT
4.04 ( 0.38c
H
CH3
>100
1.58 ( 0.75
NT
H
H
H
OCH3
7.44 ( 0.36
8.18 ( 0.79
5.46 ( 0.36
10.13 ( 1.04
NT
H
H
OCH3
CH3
NT
NT
a Values represent mean ( standard deviation for at least two separate experiments performed in triplicate. b Values represent mean ( standard deviation
from dose response curves for at least two separate experiments performed in duplicate. c Error values listed as standard deviations.
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In addition to the variable-sized sugar-containing analogues,
cyclohexyl analogues were designed to examine if a sugar was
necessary. Additionally, to probe the tolerance of steric bulk,
analogues with and without alkyl substituents were pursued.
Many of the simplified cyclohexyl sugar mimics were accessible
using common procedures (Scheme 5) such as reduction with
lithium aluminum hydride to produce compounds 37 and 38,
or Luche reduction to yield 39. Compounds containing
a double bond were designed to be coupled with the scaff-
old and subsequently oxidized to the corresponding diols
(e.g., 43ꢀ45).
contained a single hydroxyl at the 20-position proved inactive,
while most analogues with a 30-hydroxyl exhibited modest
activity. These data indicate that the 30-position hydroxy group
is essential for anti-proliferative activity. Interestingly, the natural
substrate for the C-terminal pocket of Hsp90, ATP/ADP, also
contains a 30-hydroxyl. Although the β epimer exhibited better
activity than its R counterpart (14aꢀc vs 15aꢀc) for most
analogues, this was not a general trend observed throughout the
series (20aꢀc vs 21aꢀc). In addition, the majority of the
compounds from this series demonstrated modest activity
against the prostate cancer cell lines. Notably, however, some
compounds (17a) that were completely inactive against both
breast cancer cells lines were efficacious against prostate cancer.
Analogues containing a cyclohexyl moiety in lieu of the sugar
demonstrated a range of activities. While analogues containing an
8-methyl group (43a, 44a, 45a) were inactive against breast
cancer, those with an 8-methoxy group (43b, 44b, 45b) exhibited
an IC50 value of ∼10 μM, which is comparable to its noviosylated
counterpart (Table 2).33 Interestingly, the gem-dimethyl group is
well tolerated in the case of the 8-methoxy coumarin (43b) but
detrimental in the case of the 6-methoxy coumarin (43c). This
finding suggests a mutually exclusive orientation between the
gem-dimethyl group and substituents on the coumarin, which
may preclude proper binding orientation.
Upon construction of the modified sugar analogues of novo-
biocin, the compounds were evaluated for anti-proliferative
activity against SKBr3 (estrogen receptor negative, HER2 over-
expressing breast cancer cells), MCF-7 (estrogen receptor posi-
tive breast cancer cells), LNCaP-LN3 (androgen-dependent
human prostate cancer cells), and PC3-MM2 (androgen-indepen-
dent prostate cancer cells) cell lines. As shown in Table 1,
analogues containing six-membered pyranose moieties (scaffold A)
were found to be the most active compounds against the two
breast cancer cell lines. Analogues containing dihydroxyl groups
consistently exhibited good to modest anti-proliferative activities
against both breast cancer cell lines. In contrast, analogues that
Scheme 6. Retrosynthesis of Azasugar-Containing Biaryl
Analogues
Scheme 8. Synthesis of 1,4-Azasugar Biaryl Analogues
Scheme 7. Synthesis of 1,3-Azasugar-Containing Biaryl Analogues
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Table 3. Anti-proliferative Activity of Azasugar Analogues
compd (IC50, μM)
R
X
Y
R0
SKBr3
MCF-7
1.73a
LNCAP-LN3
PC3-MM2
48a
48b
48c
50a
51a
51b
51c
52a
52b
52c
53a
54a
55a
55b
55c
57a
57b
57c
59a
59b
59c
60a
60b
60c
61a41
61b
61c
A
A
A
A
A
A
A
B
H
H
CH3
H
1.61 ( 0.05a
3.07 ( 0.78
1.21 ( 0.07
>50
4.27 ( 0.05b,c
6.45 ( 2.70c
5.40 ( 9.99
0.90 ( 0.59
1.22c
4.40 ( 0.06b,c
4.88 ( 2.20
3.57 ( 0.71c
3.73 ( 0.67
1.73 ( 1.80
0.98 ( 0.40
3.87 ( 0.03c
6.33 ( 4.12
14.43 ( 7.39
6.84 ( 2.80
3.42 ( 1.24
2.22 ( 1.04
2.99 ( 1.27
3.52 ( 1.43
4.17 ( 0.16c
2.57 ( 1.13
10.43 ( 2.15
2.59 ( 13.91
4.12 ( 1.29
8.47 ( 4.28
4.02 ( 2.13
4.23 ( 1.68
2.16 ( 1.28
6.86 ( 2.92c
3.13 ( 0.67
3.71 ( 1.27
2.84 ( 1.88
OCH3
CH3
H
1.43 ( 0.37
1.68 ( 0.04
>50
OCH3
H
H
CH3
Ac
H
CH3
CH3
CH3
CH3
H
2.92 ( 1.33
3.42 ( 0.45
1.96 ( 0.48
2.91 ( 0.90
8.98 ( 0.44
3.65 ( 0.22
>50
5.29 ( 0.23
1.65 ( 0.28
5.27 ( 0.22
2.07 ( 0.86
10.17 ( 0.02
3.34
H
OCH3
CH3
0.36 ( 0.14
4.49 ( 0.17c
4.90 ( 9.16
12.19 ( 6.30
9.45 ( 5.90
1.23 ( 0.67
0.71 ( 0.54
0.59 ( 0.54
1.50 ( 0.62
4.69 ( 0.57c
3.02 ( 0.97c
12.72 ( 3.25
3.66c
OCH3
H
CH3
B
H
OCH3
CH3
H
B
OCH3
H
H
C
B
CH3
Ac
>50
H
CH3
Ac
10.79 ( 0.08
3.92 ( 0.32
6.64 ( 0.54
2.77 ( 0.90
1.16 ( 0.16
2.61 ( 0.37
2.27 ( 0.61
1.19 ( 0.06
1.79
9.18 ( 0.60
1.85 ( 0.02
11.02 ( 1.12
2.01 ( 0.46
1.63 ( 0.28
3.29 ( 0.42
2.90 ( 0.67
1.47 ( 0.02
3.23 ( 0.20
2.82 ( 1.10
5.04 ( 0.02
1.38 ( 0.14
1.41 ( 0.13
1.51 ( 0.24
1.39 ( 0.28
1.79 ( 0.09
B
H
CH3
CH3
CH3
CH3
H
B
H
OCH3
CH3
B
OCH3
H
C
C
C
D
D
D
C
C
C
D
D
D
CH3
H
OCH3
CH3
H
OCH3
H
H
CH3
H
3.38 ( 1.25
10.85 ( 5.92
5.27 ( 22.18
1.22 ( 0.17c
1.78 ( 0.80
10.48 ( 5.46c
4.12 ( 0.16c
4.75 ( 2.03
4.17 ( 0.15c
H
OCH3
CH3
H
OCH3
H
H
1.20 ( 0.08
2.06 ( 0.57
1.40 ( 0.14
1.49 ( 0.06
1.34 ( 0.18
0.97 ( 0.07
1.19 ( 0.17
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
OCH3
CH3
OCH3
H
CH3
H
OCH3
CH3
OCH3
a Values represent mean ( standard deviation for at least two separate experiments performed in triplicate. b Values represent mean ( standard deviation
from dose response curves for at least two separate experiments performed in duplicate. c Error values listed as standard deviations.
Scheme 9. Synthesis of Alkyl Sugar Biaryl Analogues
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Journal of Medicinal Chemistry
ARTICLE
Overall, this library containing sugar surrogates and cyclohexyl
groups confirmed the 30-hydroxy as the most important func-
tional group. Moreover, the 40-methoxy, 50-gem-dimethyl and
anomeric oxygen found in noviose were identified as dispensable
moieties. Several analogues (15b, 24c, and 37a) exhibited low
micromolar anti-proliferative activity against SKBr3 cells, which
makes them ∼500-fold more active than novobiocin.
interactions with the binding pocket as well as to improve
solubility.
In modular fashion, the analogues were assembled similarly
to those described previously, allowing sequential coupling of
various sugar surrogates and the biaryl side chain. As shown in the
retrosynthetic analysis depicted in Scheme 6, the biaryl acid
chloride, prepared from the corresponding biaryl acid, was
coupled with coumarin 9. Following deprotection, coumarin
phenol 6 was etherified using Mitsunobu conditions to yield the
desired analogues.
To avoid synthetic complications, only a single hydroxyl group
was installed on the piperidine ring prior to Mitsunobu coupling
and the amine was masked as the carbamate, 46. With compound
6 and 46 in hand, compound 47 was synthesized via Mitsunobu
etherification, followed by deprotection to yield amine 48 in
quantitative yield. Acetylation of 48 gave amide 50, while
treatment with methyl iodide furnished the tertiary amine, 51,
which underwent dihydroxylation with OsO4/NMO to give 55.
Dihydroxylation of 47 afforded compound 49, which, upon
removal of Boc, afforded 52. Compounds 53 and 54 were
synthesized from compound 50 via reduction or dihydroxylation
conditions, respectively (Scheme 7).
Design, Synthesis, and Biological Evaluation of Azasugar
Analogues of Novobiocin. After evaluation of the initial library,
it was proposed that other six-membered heteroatom-containing
ring systems may serve as replacements for noviose. N-Hetero-
cycles are found in a wide variety of biologically active com-
pounds, imparting solubility to otherwise insoluble aglycons.
Several heterocycles were designed to probe hydrogen-bonding
Scheme 10. Synthesis of Non-Sugar Biaryl Analogues
In addition, compounds lacking the diol functionality and
containing a transposed nitrogen within the heterocycle were
selected for investigation. Boc-protected 1,3- or 1,4-azasugars
were coupled with phenol 6 to afford compounds 56 and 58,
which were subsequently deprotected to yield amines 57 and
59. Similarly, N-methylated analogues 60 and 61 were synthe-
sized via Mitsunobu etherification with commercially available
piperidines in good yields (Scheme 8).
Upon construction of the azasugar-containing analogues, they
were evaluated for anti-proliferative activity against two breast
and two prostate cancer cell lines. As shown in Table 3, the
activity against breast cancer cells exhibited by the majority of
Table 4. Anti-proliferative Activity of Novobiocin Analogues with Aliphatic Sugar Replacement
compd (IC50, μM)
R
X
Y
R0
SKBr3
MCF-7
LNCAP-LN3
PC3-MM2
64a
64b
64c
65a
65b
65c
66a
66b
66c
68a
68b
68c
A
A
A
A
A
A
B
B
B
C
C
C
H
H
CH3
H
5.36 ( 0.08a
3.60 ( 0.28
3.15 ( 0.54
1.02 ( 0.13
1.77 ( 0.12
1.42 ( 0.21
0.60 ( 0.01
0.91 ( 0.14
0.49 ( 0.20
>50
9.80 ( 0.11a
4.32 ( 0.32
6.23 ( 0.14
1.46 ( 0.08
3.08 ( 0.08
1.29 ( 0.17
0.50 ( 0.03
1.53 ( 0.14
0.70 ( 0.15
>50
>100b
14.01 ( 0.28b,c
9.12 ( 5.92
11.77 ( 5.94
4.17 ( 19.64
6.20 ( 2.22
4.87 ( 2.91
12.85 ( 11.42
4.08 ( 1.83
5.40 ( 3.15
0.19 ( 0.33
1.66 ( 6.05
NT
OCH3
CH3
H
15.24 ( 7.96
>100
OCH3
H
H
CH3
CH3
CH3
CH3
6.65 ( 12.43
5.22 ( 3.25
6.28 ( 2.22c
37.51 ( 54.24
8.03 ( 7.93
4.71 ( 0.21c
NT
H
OCH3
CH3
OCH3
H
CH3
H
OCH3
CH3
OCH3
H
CH3
H
OCH3
CH3
>50
>50
0.96 ( 1.20
NT
OCH3
19.36 ( 2.45
20.47 ( 5.54
a Values represent mean ( standard deviation for at least two separate experiments performed in triplicate. b Values represent mean ( standard deviation
from dose response curves for at least two separate experiments performed in duplicate. c Error values listed as standard deviations.
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Table 5. Anti-proliferative Activity of Non-Sugar Analogues
compd (IC50, μM)
X
Y
R
SKBr3
MCF-7
LNCAP-LN3
PC3-MM2
6a41
12a41
8532
73a41
74a41
69a
70a41
71a
72a
6b
H
H
H
H
H
H
H
H
H
CH3
H
8.88 ( 0.48a
0.98 ( 0.02
7.50 ( 0.80
7.33 ( 0.96
>100
6.93 ( 0.48a
1.40
3.21 ( 1.78b
1.50 ( 1.00
NT
5.36 ( 2.71b
2.85 ( 1.66
NT
CH3
COCH3
Noviose
Ms
CH3
18.70 ( 1.44
8.85 ( 0.88
>100
CH3
19.96 ( 42.67
0.58 ( 1.34
2.61 ( 1.09
3.75 ( 0.76
5.40 ( 6.58
3.65 ( 5.73
15.79 ( 32.60
1.05 ( 0.59
26.57 ( 67.44
>100
>100
CH3
Ts
>100
CH3
CONH2
CONHMe
CONMe2
PO(OMe)2
H
3.02 ( 0.56
2.40 ( 0.16
39.85 ( 0.48
>100
1.16 ( 0.08
1.72 ( 0.16
74.35 ( 3.92
>100
6.37 ( 4.31
5.22 ( 2.16
>100
CH3
CH3
CH3
>100
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
CH3
3.23 ( 1.20
5.72 ( 0.02
58.80 ( 1.04
>100
11.70 ( 1.92
1.50 ( 0.24
>100
11.23 ( 30.70
1.69 ( 2.05
>100
12b
8633
73b
74b
69b
70b
71b
72b
6c
CH3
COCH3
Noviose
Ms
CH3
CH3
>100
NT
CH3
Ts
>100
>100
NT
>100
CH3
CONH2
CONHMe
CONMe2
PO(OMe)2
H
6.83 ( 0.24
1.53 ( 0.16
6.17 ( 1.68
26.48 ( 0.80
>100
6.29 ( 0.88
7.78 ( 1.68
34.4 ( 7.20
24.22 ( 2.40
5.32 ( 0.08
8.62 ( 2.00
9.00 ( 4.32
>100
3.55 ( 3.44
4.77 ( 8.37
12.75 ( 0.32c
1.36 ( 2.80
6.18 ( 7.43
14.62 ( 23.63
0.11 ( 0.02
0.87 ( 1.32
0.11 ( 4.68
4.83 ( 3.87
2.37 ( 1.44
15.73 ( 14.09
6.58 ( 4.14
2.62 ( 2.04
3.13 ( 2.34
4.04 ( 16.68
31.32 ( 54.87
>100
CH3
CH3
CH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
12c
8733
73c
74c
69c
70c
71c
72c
H
COCH3
Noviose
Ms
20.50 ( 1.28
13.90 ( 0.96
>100
19.33 ( 50.90
1.44 ( 0.32
NT
H
H
H
Ts
>100
>100
NT
H
CONH2
CONHMe
CONMe2
PO(OMe)2
76.83 ( 1.84
>100
5.56 ( 0.24
6.54 ( 1.84
>100
>100
H
1.68 ( 1.89
11.57 ( 10.04
11.46 ( 6.54
H
>100
H
18.07 ( 2.88
>100
a Values represent mean ( standard deviation for at least two separate experiments performed in triplicate. b Values represent mean ( standard deviation
from dose response curves for at least two separate experiments performed in duplicate. c Error values listed as standard deviations.
these secondary and tertiary amines varied between 1 and 3 μM,
making them ∼700-fold more active than novobiocin. Although
dihydroxylation of the piperidine ring is well-tolerated when
appended to the 8-methyl scaffold (52a), this is not the case for
the other two scaffolds (52b and 52c). These data suggest that
dihydroxylation of the piperidine ring is not essential for anti-
proliferative activity. Inclusion of unsaturation within the piper-
idine ring is also well tolerated (scaffold A vs C and D, Table 3),
while methylation of the amine within this unsaturated ring
decreases activity (48 vs 51). It was observed that acetylation of
the amine functionality (50a, 53a, 54a) severely decreased
solubility and produced inactive compounds. Although insolu-
bility can be overcome through dihydroxylation of the piperidine
ring (54a), the resulting compound is ∼5-fold less active than
secondary or tertiary amines. There is no significant effect when
the nitrogen is transposed within the piperidine ring, as all of
the analogues exhibited activity between ∼1ꢀ3 μM. In addi-
tion, piperidine compounds exhibited comparable activity to
Scheme 11. Retrosynthesis of Azasugar Indole Novobiocin
Analogues
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Journal of Medicinal Chemistry
ARTICLE
methylated piperidine compounds (57 vs 60, 59 vs 61). Overall,
these azasugar analogues consistently exhibited low micromolar
anti-proliferative activity, representing an improved sugar mimic.
Design, Synthesis, and Biological Evaluation of Novobio-
cin Analogues with Acyclic Sugar Replacements. The pro-
mising results obtained when azasugars were appended to these
scaffolds encouraged the synthesis of acyclic nitrogen-containing
replacements. To probe the importance of a constrained ring
system, a library of ring-opened, amine-containing sugar surro-
gates was designed. A series of heteroatom-containing aliphatic
chains were synthesized to impart flexibility and explore the
potential of additional interactions with the binding pocket.
Aliphatic amines, either commercially available or prepared over
a modest number of steps, were appended to coumarin core 6
through standard Mitsunobu coupling to yield compounds
64ꢀ66. The dihydroxylated aliphatic chain was attached via
treatment with allyl bromide followed by dihydroxylation to
afford compound 68 (Scheme 9).
Scheme 12. Synthesis of Optimized Compounds
In addition to the aliphatic amine analogues synthesized,
several simplified nonsugar molecules were appended in lieu of
the noviose sugar. These nonsugars represent simplified func-
tionalities manifested by compound 4,30 a recently described
inhibitor of the Hsp90 C-terminus (Figure 2).30 Substitutions
were selected to probe hydrogen-bonding interactions, dimen-
sions, and improve solubility. Carbamates (69ꢀ71) with variable
substitution were prepared through standard conditions. Phos-
phate ester 71 was introduced through an esterification reaction
to increase the hydrophilicity of the inhibitor, and finally, methyl
and toluene sulfonic esters (73, 74) were incorporated using the
corresponding sulfonic chlorides to explore both the hydrogen
bonding network and dimensions of the pocket similarly to that
reported by Renoir and co-workers34 (Scheme 10).
Upon construction of these alkyl and nonsugar compounds,
they were evaluated for anti-proliferative activity against
various cancer cell lines. The anti-proliferative data are sum-
marized in Tables 4 and 5. All of the tertiary amine analogues
demonstrated notable anti-proliferative activity of ∼1 μM,
while the secondary amine analogues maintained activity of
∼3ꢀ5 μM against both breast cancer cell lines. The tertiary
amine analogues homologated by two methylene groups
(65aꢀc) exhibited activity of 1ꢀ3 μM, which is comparable
to the piperidine analogues. Moreover, homologation by three
methylene groups (66aꢀc) resulted in compounds that man-
ifested anti-proliferative activity in the mid-nanomolar range,
a finding that is consistent with the piperidine ring analogues.
In contrast, the diol-containing analogues, when appended to
the 8-methyl (68a) or 8-methoxy (68b) coumarin scaffolds
were inactive but resulted in modest activity when appended
to the 6-methoxy coumarin (68c). Furthermore, the aliphatic
amine and corresponding dihydroxylated analogues mani-
fested modest activities against the prostate cancer cell lines
as well.
The anti-proliferative data for the nonsugar analogues revealed
several interesting trends. The free phenol of each scaffold was
nearly equal in activity to its noviosylated counterpart. Moreover,
the acetylated phenols, in the case of the 8-methyl and 6-methoxy
coumarins, displayed activities of ∼1 μM against breast cancer
cells. Similarly, the carbamate and methylated carbamate were
well tolerated when appended to 8-methyl and 6-methoxy
coumarins, resulting in low micromolar analogues. Addition of
another methyl group to the carbamate severely compromised
activity. The methyl sulfonic ester was only tolerated when
appended to the 8-methyl scaffold, while the toluene sulfonic
ester was not tolerated by any analogue. Likewise, addition of the
phosphate ester decreased activity of the analogues. Overall,
hydrogen bonding and limited hydrophobic bulk are essential
features for optimal binding.
Synthesis of Optimized Scaffolds Using Most Promising
Noviose Replacements. On the basis of the biological evalua-
tion discussed thus far, we identified the N-methyl-4-hydroxy-
piperidine and 3-(dimethylamino)propan-1-ol as optimal noviose
replacements. Therefore, these surrogates were appended to three
previously identified and optimized coumarin scaffolds, containing
either the 2-indole or prenylated benzamide side chain in lieu of
the biaryl system.32,42 As detailed earlier, the analogues were
assembled in a modular fashion, allowing sequential coupling of
sugar mimics and the indole acid chloride or benzoic acid chloride
withthe desired scaffold (Scheme 11). Unlikepreviouslydiscussed
scaffolds, the Mitsunobu ether coupling was performed prior to
amide coupling due to solubility issues arising from the indole-
containing free phenol.
As seen in Scheme 12, phenol 732,33 was coupled with the
azasugar using Mitsunobu conditions to yield protected scaffold
75. The vinylogous amide was liberated via hydrogenolysis and
coupled with indole acyl chloride 76, which was generated from
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Table 6. Anti-proliferative Activity of Optimized Analogues
compd (IC50, μM)
R
X
Y
R0
R00
SKBr3
MCF-7
LNCAP-LN3
PC3-MM2
78a
78b
78c
79a
79b
79c
80a
80b
80c
82a
82b
82c
83a
83b
83c
84a
84b
84c
A
A
A
A
A
A
A
A
A
B
B
B
B
B
B
B
B
B
H
H
CH3
C
C
C
D
D
D
D
D
D
C
C
C
D
D
D
D
D
D
0.48 ( 0.09a
2.58 ( 0.28
0.11 ( 0.01
0.58 ( 0.04
1.07 ( 0.14
0.42 ( 0.01
0.76 ( 0.14
0.92 ( 0.01
0.42 ( 0.01
1.13 ( 0.01
1.50 ( 0.13
0.57 ( 0.09
0.46 ( 0.15
0.78 ( 0.17
0.36 ( 0.03
0.44 ( 0.02
0.77 ( 0.08
0.39 ( 0.06
0.57 ( 0.03a
1.86 ( 0.08
0.52 ( 0.04
1.18 ( 0.16
1.64 ( 0.24
0.58 ( 0.02
1.09 ( 0.08
1.54 ( 0.21
0.54 ( 0.02
5.23 ( 0.22
1.41 ( 0.09
0.56
11.83 ( 0.54b
11.40 ( 5.25b
7.93 ( 4.18
0.87 ( 0.46
2.12 ( 3.32
3.98 ( 0.06c
1.41 ( 0.04c
1.37 ( 1.42
3.53 ( 0.01c
2.26 ( 1.43
13.69 ( 0.18c
8.95 ( 6.11
2.58 ( 4.47
1.42 ( 0.05c
4.59 ( 4.23
1.46 ( 0.03c
1.81 ( 1.22
9.24 ( 17.79
1.38 ( 0.02c
OCH3
CH3
12.64 ( 0.32c
OCH3
H
1.47 ( 0.53
NT
CH3
Ac
Ac
Ac
H
H
OCH3
CH3
NT
OCH3
H
NT
CH3
NT
H
OCH3
CH3
H
NT
OCH3
H
H
NT
CH3
NT
4.71 ( 1.23c
H
OCH3
CH3
OCH3
H
NT
CH3
Ac
Ac
Ac
H
1.18 ( 0.02
2.14 ( 0.22
0.70 ( 0.03
1.35 ( 0.30
3.26 ( 0.26
0.80 ( 0.07
NT
H
OCH3
CH3
NT
OCH3
H
NT
CH3
NT
H
OCH3
CH3
H
NT
OCH3
H
NT
a Values represent mean ( standard deviation for at least two separate experiments performed in triplicate. b Values represent mean ( standard deviation
from dose response curves for at least two separate experiments performed in duplicate. c Error values listed as standard deviations.
commercially available indole acid, to yield 78.32 The analogues
containing a prenylated benzamide side chain (77) were as-
sembled via the same sequence (Scheme 12), to yield alkyl amine
analogues 79 and 80. The prenylated side chain, 77, was
assembled as described previously by Burlison and co-workers.29
These compounds were evaluated for their anti-proliferative
activity. The piperidine-containing analogues with an indole side
chain (78aꢀc) exhibited increased anti-proliferative activity when
attached to the 8-methyl and 6-methoxy coumarin (78a and 78c)
(Table 6). 78c was notably 10-fold more active against SKBr3
cells when compared to its biaryl counterpart 61c. In contrast,
attachment of this sugar surrogate to the 8-methoxy coumarin
(78b) compromised its activity versus the biaryl-containing
analogue 61b. In the case of the prenylated benzamide side
chain (79aꢀc and 80aꢀc), attachment of the azasugar increased
activity against both cell lines, with the 6-methoxy coumarin
manifesting superior activity (79c and 80c). In addition, the
intermediate acetylated phenols 79aꢀc demonstrated compar-
able activity to the hydrolyzed phenol products 80aꢀc. On the
basis of the results obtained with the biaryl containing analogues,
it was proposed that appendage of the aliphatic amine to the
coumarins with an indole side chain would result in increased
activity (see Table 4). However, the anti-proliferative activity of
these indole analogues (82aꢀc) was maintained or slightly
decreased in nearly every case. When these alkyl sugars were
attached to the coumarins containing a prenylated benzamide side
chain, the compounds manifested anti-proliferative activity in the
mid-nanomolar range against SKBr3 cells. Moreover, compounds
built upon the 6-methoxy coumarin scaffold (83c and 84c)
exhibited the best activity against both cell lines.
Finally, several analogues from each of the libraries were selected
for testing against a panel of melanoma and head and neck
squamous cell carcinoma cell lines. In preliminary studies, C-
terminal Hsp90 inhibitors have demonstrated modest activity
against melanoma cells and have shown promise in treating head
and neck cancers as well. Unpublished in vivo data produced by the
Cohen lab has demonstrated tumor regression upon sustained
treatment with a C-terminal Hsp90 inhibitor previously synth-
esized.33 Analogues were selected that exhibited diverse structural
features to probe the efficacy of these analogues against these
cancers. B16F10 (murine melanoma), SKMEL28 (human mel-
anoma), MDA1986 (human head and neck squamous cell carcino-
ma (HNSCC)), and JMAR (human oral squamous cell carcinoma)
were selected for this panel of testing. The majority of the analogues
tested manifested mid to low micromolar activity against these cell
lines. While some of the compounds exhibited better activity against
melanoma than head and neck cancers (61b), others were potent in
a specific melanoma (57b) or a specific head and neck (17a) cell
line (Table 7). The low micromolar activity of 78c and 82c against
all of the cell lines warrant further investigation.
Validation of Hsp90 Inhibition via Western Blot Analysis.
To confirm that the anti-proliferative activities exhibited by the
optimized sugar analogues result from Hsp90 inhibition, analo-
gues 80c and 83c were evaluated by Western blot analyses
(Figure 7). Notably, compounds 61a, 12b, 70a, and 87 have
already been shown to exert their anti-proliferative activities
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Table 7. Anti-proliferative Activity of Selected Analogues against Melanoma and HNSCC
compd (IC50, μM)
B16F10
SKMEL28
MDA1986
JMAR
6b
5.77 ( 0.96 a
2.30 ( 0.32
37.50 ( 6.56
8.56 ( 2.16
1.01 ( 0.48
10.40 ( 1.20
9.60 ( 1.12
4.55 ( 1.20
1.30 ( 0.16
42.70 ( 3.36
12.70 ( 1.44
9.37 ( 1.04
>50
12.40 ( 0.64 a
2.14 ( 0.08
14.90 ( 2.88
15.80 ( 0.48
1.47 ( 0.24
19.60 ( 2.48
10.65 ( 0.40
5.74 ( 2.08
6.41 ( 0.56
18.40 ( 2.96
21.40 ( 3.28
12.40 ( 2.48
18.50 ( 5.76
23.80 ( 2.88
20.60 ( 2.16
18.60 ( 2.00
3.47 ( 1.20
11.41 ( 1.28
2.94 ( 1.20
5.41 ( 0.08
5.49 ( 1.12
5.41 ( 0.16
27.60 ( 1.20
10.79 ( 0.64
1.48 ( 0.48
31.50 ( 1.84
6.14 ( 1.28
11.40 ( 2.56
13.80 ( 2.56
20.9 ( 2.56
29.40 ( 3.36
5.90 ( 1.68
5.73 ( 0.32
2.79 ( 0.96
14.67 ( 0.40
7.19 ( 0.24
5.19 ( 0.16
16.45 ( 1.36
5.75 ( 0.24
6.14 ( 0.56
7.14 ( 0.40
5.86 ( 1.20
6.14 ( 0.32
3.74 ( 0.24
25.60 ( 2.96
14.70 ( 1.92
1.47 ( 0.08
1.35 ( 0.08
6.70 ( 1.20 a
1.67 ( 0.16
13.40 ( 4.24
3.70 ( 0.64
0.81 ( 0.40
18.80 ( 3.68
5.70 ( 0.56
1.94 ( 0.40
5.73 ( 0.96
21.40 ( 3.12
6.90 ( 2.08
18.80 ( 3.76
22.90 ( 4.96
24.40 ( 2.00
21.80 ( 4.32
22.40 ( 3.36
18.80 ( 0.64
6.74 ( 2.08
17.90 ( 1.44
6.54 ( 0.56
5.97 ( 0.40
5.90 ( 0.40
25.10 ( 2.08
7.70 ( 0.56
2.36 ( 0.08
16.40 ( 1.68
5.60 ( 1.04
21.70 ( 1.12
13.80 ( 3.44
22.40 ( 1.36
36.40 ( 1.20
5.90 ( 0.48
12.80 ( 1.28
23.60 ( 2.88
12.80 ( 1.92
6.40 ( 0.96
2.74 ( 0.08
10.90 ( 0.64
14.60 ( 1.20
4.50 ( 0.08
11.40 ( 2.56
13.20 ( 0.72
13.10 ( 1.68
1.54 ( 0.08
21.90 ( 2.56
17.50 ( 3.28
1.42 ( 0.16
1.81 ( 0.32
24.60 ( 1.92a
12.60 ( 0.40
2.79 ( 0.64
6.14 ( 1.28
16.10 ( 2.16
3.60 ( 0.40
5.80 ( 1.04
12.64 ( 1.28
2.71 ( 0.32
14.90 ( 3.68
24.10 ( 3.28
6.19 ( 2.48
7.40 ( 1.68
6.70 ( 2.32
24.90 ( 2.80
7.50 ( 1.84
8.50 ( 1.68
2.73 ( 0.32
13.90 ( 2.00
3.40 ( 0.16
6.71 ( 0.24
11.60 ( 0.32
35.10 ( 1.20
11.40 ( 1.20
1.94 ( 0.16
18.40 ( 2.56
5.89 ( 0.48
11.60 ( 1.92
11.50 ( 0.72
15.14 ( 1.84
32.80 ( 3.36
6.70 ( 0.24
24.50 ( 0.72
>50
12a
14a
15a
17a
18a
23a
25a
25b
27a
30a
31a
33a
40a
41a
42a
43a
44a
45a
48a
48b
48c
50c
51a
51b
51c
52a
52b
52c
53a
53c
55b
55c
57a
57b
57c
59a
59b
59c
60a
60b
60c
61b
61c
64a
65a
78c
82c
>50
>50
>50
15.90 ( 2.16
2.50 ( 0.48
14.10 ( 2.08
2.94 ( 0.08
3.41 ( 0.32
2.86 ( 0.24
21.40 ( 1.28
6.40 ( 1.92
6.47 ( 2.80
6.14 ( 0.64
2.80 ( 0.40
11.75 ( 0.40
5.04 ( 0.32
12.40 ( 2.00
23.90 ( 2.88
6.58 ( 0.48
2.81 ( 0.32
1.90 ( 0.24
0.64 ( 0.08
1.84 ( 0.08
2.76 ( 0.32
2.14 ( 0.08
1.84 ( 0.16
2.60 ( 0.32
1.51 ( 0.24
2.67 ( 0.32
0.41 ( 0.08
36.73 ( 0.64
37.40 ( 2.16
15.70 ( 4.56
1.19 ( 0.32
2.03 ( 0.08
12.70 ( 1.44
5.70 ( 0.32
5.91 ( 0.40
12.60 ( 0.32
10.90 ( 0.48
12.60 ( 0.32
7.50 ( 0.64
19.10 ( 2.56
13.10 ( 0.64
1.84 ( 0.08
35.10 ( 2.16
18.40 ( 2.00
1.24 ( 0.24
2.65 ( 0.24
a Values represent mean ( standard deviation for at least two separate experiments performed in triplicate.
through an Hsp90-dependent mechanism, as confirmed through
previously executed Western blot analyses.30,33,41 It was essential
that these optimized derivatives demonstrate selective Hsp90-
dependent client protein degradation, versus a loading control, to
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ARTICLE
’ ASSOCIATED CONTENT
S
Supporting Information. Experimental procedures and
b
characterization for all compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: (785) 864-2288. Fax: (785) 864-5326. E-mail: bblagg@
ku.edu.
Author Contributions
^These authors contributed equally to this work
’ ACKNOWLEDGMENT
We gratefully acknowledge support of this project by the
NIH/NCI (CA120458) and the ACS Division of Organic
Chemistry Fellowship (A.D.).
Figure 7. Western blot analyses of Hsp90 client protein degradation
assays against MCF-7 cells. Concentrations (in μM) of 80c (top) and
83c (bottom) are indicated above each lane. G (geldanamycin, 500 nM)
and D (DMSO) were respectively employed as positive and negative
controls.
’ ABBREVIATIONS USED
Hsp90, 90 kDa heat shock protein; ATP, adenosine tripho-
sphate; DNA, deoxyribonucleic acid; SAR, structureꢀactivity
relationships; Akt, protein kinase B; Her2, human epidermal
growth factor receptor 2
confirm the compounds reported herein also manifest Hsp90
inhibition. Figure 7 shows that in MCF-7 cells, the Hsp90-dependent
client proteins Akt, Raf-1, and Her2 were degraded in a concentra-
tion-dependent manner upon treatment with the compounds.
Hsp90 client protein degradation occurred at concentrations that
paralleled the observed anti-proliferative IC50 values of 0.54 (
0.03 μM for 80c and 0.70 ( 0.04 μM for 83c, confirming that
Hsp90-dependent client protein degradation is causative for inhibi-
tion of cell growth. Furthermore, Hsp90 protein levels remained
constant at all concentrations tested, which is consistent with C-
terminal Hsp90 inhibition. Because the non-Hsp90-dependent
protein, actin, was not affected by these analogues, it was concluded
that selective degradation of Hsp90-dependent proteins occurred.
’ REFERENCES
(1) Powers, M. V.; Workman, P. Targeting of multiple signalling
pathways by heat shock protein 90 molecular chaperone inhibitors.
Endocr.-Relat. Cancer 2006, 13, S125–S135.
(2) Maloney, A.; Workman, P. HSP90 as a new therapeutic target for
cancer therapy: the story unfolds. Expert Opin. Biol. Ther. 2002, 2, 3–24.
(3) Sreedhar, A. S.; Kalmar, E.; Csermely, P.; Shen, Y. F. Hsp90
isoforms: functions, expression and clinical importance. FEBS Lett.
2004, 562, 11–15.
(4) Brandt, G. E. L.; Blagg, B. S. J. Alternative strategies of Hsp90
modulation for the treatment of cancer and other diseases. Curr. Med.
Chem. 2009, 9, 1447–1461.
(5) Pratt, W. B.; Toft, D. O. Regulation of signaling protein function
and trafficking by the hsp90/hsp70-based chaperone machinery. Exp.
Biol. Med. 2003, 228, 111–133.
’ CONCLUSION
(6) Terasawa, K.; Minami, M.; Minami, Y. Constantly updated
knowledge of Hsp90. J. Biochem. 2005, 137, 443–447.
(7) Buchner, J. Hsp90 & Co.—a holding for folding. Trends Biochem.
Sci. 1999, 24, 136–141.
(8) Picard, D. Heat-shock protein 90, a chaperone for folding and
regulation. Cell. Mol. Life Sci. 2002, 59, 1640–1648.
(9) Yonehara, M.; Minami, Y.; Kawata, Y.; Nagai, J.; Yahara, I. Heat-
induced chaperone activity of Hsp90. J. Biol. Chem. 1996, 271,
2641–2645.
(10) Xiao, L.; Lu, X.; Ruden, D. M. Effectiveness of Hsp90 inhibitors
as anti-cancer drugs. Mini-Rev. Med. Chem. 2006, 6, 1137–1143.
(11) Zhao, R.; Houry, W. A. Hsp90: a chaperone for protein folding
and gene regulation. Biochem. Cell Biol. 2005, 83, 703.
(12) Issacs, J. S.; Xu, W.; Neckers, L. Heat shock protein 90 as a
molecular target for cancer therapeutics. Cancer Cell 2003, 3,
213–217.
Several libraries of novobiocin analogues with various struc-
tural features were designed, synthesized, and evaluated. From
the library of sugar mimics, we identified the pyranose as optimal
and that a 20-hydroxy group is indispensable. Several low micro-
molar analogues containing modified sugars were also identified.
From the series of azasugar-containing analogues, we concluded
that replacement of noviose with a piperidine ring resulted in
consistent low micromolar anti-proliferative activities. The piper-
idine sugars confirmed that interactions made between noviose
and the binding pocket could also be maintained with azasugars.
In addition, the acyclic library produced several aliphatic sugar
surrogates that manifested mid-nanomolar activity. This library
confirmed that flexibility is tolerated and that noviose can be
replaced with a simple acyclic moiety to maximize the potency of
novobiocin analogues. Finally, the synthesis of optimized scaf-
folds with various cytotoxic benzamide side chains identified
several novobiocin analogues that exhibited low nanomolar anti-
proliferative activity. These analogues confirmed that results
obtained from earlier studies could be applied to these appen-
dages and produce compounds that warrant further study.
(13) 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.
(14) Peterson, L. B.; Blagg, B. S. J. To fold or not to fold: modulation
and consequences of Hsp90 inhibition. Future Med. Chem. 2009,
1, 267–283.
3852
dx.doi.org/10.1021/jm200148p |J. Med. Chem. 2011, 54, 3839–3853

Journal of Medicinal Chemistry
ARTICLE
(15) Workman, P. Combinatorial attack on multistep oncogenesis
by inhibiting the Hsp90 molecular chaperone. Cancer Lett. 2004, 206,
149–157.
(16) Donnelly, A.; Blagg, B. S. Novobiocin and additional inhibitors
of the Hsp90 C-terminal nucleotide-binding pocket. Curr. Med. Chem.
2008, 15, 2702–2717.
(17) 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.
(18) Lewis, R. J.; Tsai, F. T.; Wigley, D. B. Molecular mechanisms of
drug inhibition of DNA gyrase. BioEssays 1996, 18, 661–671.
(19) Reece, R. J.; Maxwell, A. DNA gyrase: structure and function.
Crit. Rev. Biochem. Mol. Biol. 1991, 26, 335–375.
(20) Laurin, P.; Ferroud, D.; Schio, L.; Klich, M.; Dupuis-Hamelin,
C.; Mauvais, P.; Lassaigne, P.; Bonnefoy, A.; Musicki, B. Structure-
activity relationship in two series of aminoalkyl substituted coumarin
inhibitors of gyrase B. Bioorg. Med. Chem. Lett. 1999, 9, 2875–2880.
(21) Ali, J. A.; Jackson, A. P.; Howells, A. J.; Maxwell, A. The 43-
kilodalton N-terminal fragment of the DNA gyrase B protein hydrolyzes
ATP and binds coumarin drugs. Biochemistry 1993, 32.
(22) Amolins, M. W.; Blagg, B. S. J. Natural product inhibitors of
Hsp90: potential leads for drug discovery. Mini-Rev. Med. Chem. 2009, 9,
140–152.
antiproliferative agents in breast cancer cells and potential inhibitors of
heat shock protein 90. J. Med. Chem. 2007, 50, 6189–6200.
(35) Radanyi, C.; LeBras, G.;Messaoudi, S.; Bouclier, C.; Peyrat, J.-F.;
Brion, J.-D.; Marsaud, V.; Renoir, J.-M.; Alami, M. Synthesisand biological
activity of simplified denoviose-coumarins related to novobiocin as potent
inhibitorsofheat-shockprotein 90 (hsp90). Bioorg. Med. Chem. Lett. 2008,
18, 2495–2498.
(36) 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.
(37) Hosmane, R. S.; Hong, M. How important is the N-3 sugar
moiety in the tight-binding interaction of coformycin with adenosine
deaminase? Biochim. Biophys. Res. Commun. 1997 236, 88–93.
(38) Eis, C.; Nidetzky, B. Substrate-binding recognition and speci-
ficity of trehalose phosphorylase from Schizophyllum commune exam-
ined in steady-state kinetic studies with deoxy and deoxyfluoro substrate
analogues and inhibitors. Biochem. J. 2002, 363, 335–340.
(39) Werz, D. B.; Seeberger, P. H. Carbohydrates as the next frontier
in pharmaceutical research. Chemistry 2005, 11, 3194–3206.
(40) Yu, Y. M.; Han, H.; Blagg, B. S. J. Synthesis of mono- and
dihydroxylated furanoses, pyranoses, and an oxepananose for the
preparation of natural product analogue libraries. J. Org. Chem. 2005,
70, 5599.
(23) Holdgate, G. A.; Tunnicliffe, A.; Ward, W. H. J.; Weston, S. A.;
Rosenbrock, G.; Barth, P. T.; Taylor, I. W. F.; Paupit, R. A.; Timms, D.
The entropic penalty of ordered water accounts for weaker binding of the
antibiotic novobiocin to a resistant mutant of DNA gyrase: a thermo-
dynamic and crystallographic study. Biochemistry 1997, 36, 9663–9673.
(24) Lewis, R. J.; Singh, O. M. P.; Smith, C. V.; Skarzyknski, T.;
Maxwell, A.; Wonacott, A. J.; Wigley, D. B. The nature of inhibition of
DNA gyrase by the coumarins and the cyclothialidines revealed by X-ray
crystallography. EMBO J. 1996, 15, 1412–1420.
(41) Donnelly, A.; Zhao, H.; Kusuma, B. R.; Blagg, B. S. J. Cytotoxic
sugar analogues of an optimized novobiocin scaffold. MedChemComm
2010, 1, 165–170.
(42) Zhao, H.; Kusuma, B. R.; Blagg, B. S. J. Synthesis and evaluation
of noviose replacements on novobiocin that manifest antiproliferative
activity. ACS Med. Chem. Lett. 2010, 1, 311–315.
(25) Tsai, F. T. F.; Singh, O. M. P.; Skarzynski, T.; Wonacott, A. J.;
Weston, S.; Tucker, A.; Pauptit, R. A.; Breeze, A. L.; Poyser, J. P.;
O’Brien, R.; Ladbury, J. E.; Wigley, D. B. The high-resolution crystal
structure of a 24 kDa gyrase B fragment from E. coli complexed with one
of the most potent coumarin inhibitors, clorobiocin. Proteins 1997,
28, 41–52.
(26) Marcu, M. G.; Schulte, T. W.; Neckers, L. Novobiocin and
related coumarins and depletion of heat shock protein 90-dependent
signaling proteins. J. Natl. Cancer Inst. 2000, 92, 242–248.
(27) Allan, R. K.; Mok, D.; Ward, B. K.; Ratajczak, T. Modulation of
chaperone function and cochaperone interaction by novobiocin in the
C-terminal domain of Hsp90. J. Biol. Chem. 2006, 281, 7161–7171.
(28) 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.
(29) 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.
(30) Shelton, S. N.; Shawgo, M. E.; Comer, S. B.; Lu, Y.; Donnelly,
A. C.; Szabla, K.; Tanol, M.; Vielhauer, G. A.; Rajewski, R. A.; Matts,
R. L.; Blagg, B. S.; Robertson, J. D. KU135, a novel novobiocin-derived
C-terminal inhibitor of Hsp90, exerts potent antiproliferative effects in
human leukemic cells. Mol. Pharmacol. 2009, 76, 1314–1322.
(31) 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.
(32) 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.
(33) 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 activity. J. Org. Chem. 2008, 73, 8901–8920.
(34) Le Bras, G.; Radanyi, C.; Peyrat, J.-F.; Brio, J.-D.; Alami, M.;
Marsaud, V.; Stella, B.; Renoir, J.-M. New novobiocin analogues as
3853
dx.doi.org/10.1021/jm200148p |J. Med. Chem. 2011, 54, 3839–3853