Development of novobiocin analogues that manifest anti-proliferative activity against several cancer cell lines

Article abstract of DOI:10.1021/jo702191a

(Chemical Equation Presented) Recent studies have shown that the DNA gyrase inhibitor, novobiocin, binds to a previously unrecognized ATP-binding site located at the C-terminus of Hsp90 and induces degradation of Hsp90-dependent client proteins at ～700 μM. As a result of these studies, several analogues of the coumarin family of antibiotics have been reported and shown to exhibit increased Hsp90 inhibitory activity; however, the monomeric species lacked the ability to manifest anti-proliferative activity against cancer cell lines at concentrations tested. In an effort to develop more efficacious compounds that produce growth inhibitory activity against cancer cell lines, structure - activity relationships were investigated surrounding the prenylated benzamide side chain of the natural product. Results obtained from these studies have produced the first novobiocin analogues that manifest anti-proliferative activity against several cancer cell lines.

Full text of DOI:10.1021/jo702191a

Development of Novobiocin Analogues That Manifest
Anti-proliferative Activity against Several Cancer Cell Lines
Joseph A. Burlison,† Christopher Avila,‡ George Vielhauer,‡ Donna J. Lubbers,†
Jeffrey Holzbeierlein,‡ and Brian S. J. Blagg*,†
Department of Medicinal Chemistry, 1251 Wescoe Hall DriVe, Malott 4070, The UniVersity of Kansas,
Lawrence, Kansas 66045-7563, and Department of Urology, The UniVersity of Kansas Medical Center,
3901 Rainbow BlVd., Mail Stop 3016, Kansas City, Kansas 66160
bblagg@ku.edu.
ReceiVed October 17, 2007
Recent studies have shown that the DNA gyrase inhibitor, novobiocin, binds to a previously unrecognized
ATP-binding site located at the C-terminus of Hsp90 and induces degradation of Hsp90-dependent client
proteins at ∼700 µM. As a result of these studies, several analogues of the coumarin family of antibiotics
have been reported and shown to exhibit increased Hsp90 inhibitory activity; however, the monomeric
species lacked the ability to manifest anti-proliferative activity against cancer cell lines at concentrations
tested. In an effort to develop more efficacious compounds that produce growth inhibitory activity against
cancer cell lines, structure-activity relationships were investigated surrounding the prenylated benzamide
side chain of the natural product. Results obtained from these studies have produced the first novobiocin
analogues that manifest anti-proliferative activity against several cancer cell lines.
Introduction
molecular chaperone is responsible for the conformational
maturation of nascent polypeptides associated with cell signal-
ing, steroid hormone receptors, and telomerase.3 Interestingly,
it is these proteins/pathways that are most commonly mutated
or hijacked in transformed cells to produce immortality.4 Not
only does Hsp90 fold these wild-type proteins, but also many
of the corresponding mutants that contribute to oncogenic
phenotypes.5,6 In fact, proteins represented in all six hallmarks
of cancer have been shown to be dependent upon Hsp90 for
conformational maturation.7-9 Consequently, Hsp90 has emerged
as a promising target for the development of cancer chemothera-
peutics.10-26
Although transcription and translation are vital processes, the
overall transformation of linear genetic information into three-
dimensional proteins that manifest biological activity represents
the most remarkable feature of the genetic code.1 Proteins
responsible for the conformational maturation of linear polypep-
tides into biologically active, three-dimensional enzymes are
molecular chaperones. Many chaperones are relatively promis-
cuous and bind to partially or completely unfolded peptides like
those excreted by the ribosome upon translation. However,
higher order chaperones appear to be much more selective and
fold proteins associated with specific processes, an example of
which is the 90 kDa heat shock proteins, Hsp90.2 The Hsp90
(3) Blagg, B. S. J.; Kerr, T. D. Med. Res. ReV. 2006, 26, 310-338.
(4) Zhang, H.; Burrows, F. J. Mol. Med. 2004, 82, 488-499.
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Targets 2007, 7, 369-388.
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* Author to whom correspondence should be addressed. Phone: (785) 864-
2288. Fax: (785) 864-5326.
† The University of Kansas.
‡ The University of Kansas Medical Center.
(1) Frydman, J. Annu. ReV. Biochem. 2001, 70, 603-647.
(2) Wegele, H.; Mueller, L.; Buchner, J. ReV. Physiol. Biochem.
Pharmacol. 2004, 151, 1-44.
10.1021/jo702191a CCC: $40.75 © 2008 American Chemical Society
Published on Web 02/23/2008
2130
J. Org. Chem. 2008, 73, 2130-2137

DeVelopment of NoVobiocin Analogues
The Hsp90-mediated protein folding process is dependent
upon the hydrolysis of ATP by the N-terminal ATP-binding
pocket to provide the requisite source of energy.27-28 This
nucleotide-binding domain is unique when compared to typical
ATP-binding proteins because the nucleotide is bound in a bent
conformation as opposed to the extended conformation utilized
by most proteins.29-32 In fact, only four proteins are known to
bind ATP in this manner and they include DNA Gyrase, Hsp90,
histidine Kinase, and MutL, all of whom compose the GHKL
superfamily.33 Due to similarities between the ATP-binding sites
of DNA gyrase, a well-validated target for bacterial infection
and Hsp90, Neckers proposed that a small molecule inhibitor
of DNA gyrase may also inhibit Hsp90 function and serve as a
lead compound for the development of more efficacious
chemotherapeutics.34-37 Upon completion of such an experi-
ment, Neckers and colleagues determined that the DNA gyrase
inhibitor, novobiocin,38 was indeed an Hsp90 inhibitor, but
instead of binding to the well-documented N-terminal nucleotide
binding site, it bound to a previously unrecognized C-terminal
ATP-binding motif.39-41 Although small molecule inhibitors of
the N-terminus (geldanamycin and radicicol) manifest IC50
values in the low nanomolar range,42 novobiocin exhibited an
IC50 value of ∼700 µM, which is significantly less potent than
N-terminal inhibitors. However, novobiocin remained the only
known inhibitor of the Hsp90 C-terminal nucleotide binding
pocket for several years.
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FIGURE 1. Previously reported inhibitors of the Hsp90 C-terminal
ATP-binding pocket.
In 2005, the first attempt to improve the inhibitory activity
of novobiocin against Hsp90 was reported.43 A4 was identified
as an Hsp90 C-terminal inhibitor that induced degradation of
Hsp90-dependent client proteins at ∼70-fold lower concentration
than novobiocin, Figure 1. In contrast to N-terminal inhibitors
that induce Hsp70 and 90 levels at the same concentration that
leads to degradation of client proteins, the C-terminal inhibitor,
A4, induced Hsp90 levels at concentrations 1000-10000-fold
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J. Org. Chem, Vol. 73, No. 6, 2008 2131

Burlison et al.
lower than that required for client protein degradation. On the
basis of this observation, A4 was evaluated for its neuropro-
tective activity in an experiment in which neurons were exposed
to lethal levels of Aâ, the precursor to amyloid formation in
Alzheimer’s disease.44 In these studies, A4 manifested neuro-
protective activity at the same concentration (EC50 ) 6 nM)
required to induce heat shock proteins, indicating that increased
chaperone levels were required to refold and clear protein
aggregates that typically lead to neuronal cell death. In addition
to these outstanding neuroprotective properties, A4 exhibited
no toxicity, which is in sharp contrast to Hsp90 N-terminal
inhibitors. To determine whether the structural features of A4
correlated directly with novobiocin, modifications to the natural
product were undertaken as represented by DHN2, which was
found to be more effective than A4 at inducing client protein
degradation and also remained nontoxic.45
acids were chosen that contained various functionalities in an
effort to probe for steric and electronic interactions with the
putative hydrophobic pocket that is believed to bind to this
region of novobiocin. Structure-activity relationships elucidated
from this first library could then be utilized to develop more
effective inhibitors.
SCHEME 1. Retrosynthesis of Novobiocin Analogues
Since Hsp90 exists as a homodimer and the putative C-
terminal ATP binding pocket on each monomeric species is
proximal to the dimerization domain, dimers of A4, the nontoxic
inhibitor, were prepared.46 In contrast to the monomeric species,
the dimeric molecule (A4-Dimer) was found to manifest anti-
proliferative activity. The conversion of a nontoxic molecule
(A4) into a potent anti-proliferative agent (A4-Dimer) occurred
through modification of the amide side chain, suggesting that
further modification of A4 in a similar manner may produce
monomeric species that also exhibit antitumor activity. The
identification of such a compound would clearly demonstrate
that C-terminal inhibitors function via a mechanism distinct from
that of N-terminal inhibitors and thus provide a unique op-
portunity to produce molecules that can be developed specifi-
cally for antitumor or neuroprotective activity via modulation
of the same biological target.
Novobiocin analogues were prepared by the condensation of
N,N-dimethylformamide dimethyl acetal (2) with Cbz-protected
glycine (1) to produce the vinylagous carbmate, 3 (Scheme 2).49
SCHEME 2. Synthesis of Novobiocin Analogues with
Monosubstituted Benzamides
On the basis of these previous studies we proposed to
determine whether monomeric compounds based on the A4
scaffold and the natural product novobiocin could exhibit anti-
proliferative activity against various cancer cell lines. In this
article we report the rationale utilized toward the successful
development of novobiocin analogues that possess potent
antitumor activity.
Results and Discussion
Synthesis and Biological Evaluation of Monosubstituted
Benzamide Novobiocin Derivatives. For determination of
structure-activity relationships for the benzamide side chain
of novobiocin, we envisioned the construction of novobiocin
analogues with highly substituted benzamides. As shown in
Scheme 1, the derivatives were assembled from three compo-
nents: noviose carbonate,47 8-methylcoumarin,48 and a series
of substituted benzoic acids. Previously, we demonstrated that
the trichloroacetimidate of noviose carbonate couples directly
to the coumarin phenol to afford the R-anomer in excellent
yield.47 Since no cocrystal structure of Hsp90 bound to
C-terminal inhibitors exists, commercially available benzoic
The 8-methylcoumarin 5 was prepared by a modified Pechmann
condensation of 2-methylresorcinol (4) with 3.48 The resulting
phenol was noviosylated with the trichloroacetimidate of noviose
carbonate (6)50 in the presence of catalytic boron trifluoride
etherate to give 7 in good yield.47 The benzyl carbonate was
removed via hydrogenolysis to produce aminocoumarin 7, which
proved to be a versatile intermediate throughout this project.
The amine was readily coupled to a preselected library of
benzoic acids in the presence of N-(3-(dimethylamino)propyl)-
N′-ethylcarbodiimide hydrochloride (EDCI) and 4-DMAP. Dur-
ing the course of our investigation, it was determined that
utilization of 4-DMAP led to bisacylation, which proved difficult
(44) 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.
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Chem. 1999, 36, 225-235.
2132 J. Org. Chem., Vol. 73, No. 6, 2008

DeVelopment of NoVobiocin Analogues
TABLE 1. Anti-proliferation Activities of Novobiocin Analogues (in µM) (n ) 3)
entry (IC50)
SkBr3
MCF-7
HCT-116
PL45
LNCaP
PC-3
8
9
21.5 ( 1.4
>100
20.6 ( 0.4
5.3 ( 1.3
5.6 ( 2.5
10.3 ( 0.9
18.9 ( 7.0
18.0 ( 3.8
8.1 ( 6.0
28.7 ( 4.8
>100
13.0 ( 2.1
>100
3.4 ( 0.6
35.8 ( 3.4
2.8 ( 0.8
5.9 ( 2.1
14.4 ( 2.4
1.6 ( 0.2
1.6 ( 0.2
20.1 ( 4.7
>100
72.0 ( 4.0
N/T
67.6 ( 9.7
9.1 ( 0.5
N/T
17.1 ( 4.3
65.3 ( 5.6
11.6 ( 1.4
19.3 ( 5.1
N/T
>100
>100
57.9 ( 10.1
14.7 ( 2.6
65.2 ( 2.8
10
11
12
13
14
15
16
17
18
19
20
>100
1.9 ( 0.6
15.9 ( 1.9
32.7 ( 1.6
12.8 ( 2.3
3.6 ( 2.0
44.3 ( 4.8
>100
21.7 ( 2.0
7.3 ( 0.9
17.3 ( 5.2
1.6 ( 0.5
44.9 ( 31.6
16.8 ( 0.8
N/T
69.3 ( 4.1
12.7 ( 0.8
24.8 ( 9.7
26.4 ( 15.8
15.6 ( 4.2
39.1 ( 4.1
13.0 ( 1.4
16.3 ( 1.6
21.8 ( 0.8
>100
>100
>100
>100
>100
17.7 ( 1.4
61.4 ( 4.7
33.1 ( 1.1
17.2 ( 3.3
30.1 ( 3.4
14.0 ( 0.5
14.4 ( 0.8
21.2 ( 2.2
24.4 ( 5.8
6.2 ( 1.3
12.5 ( 0.9
8.4 ( 0.3
TABLE 2. Anti-proliferation Activities of Novobiocin Analogues (in µM) (n ) 3)
entry (IC50)
SkBr3
MCF-7
HCT-116
PL45
LNCaP
PC-3
21
22
23
24
25
>100
>100
>100
>100
>100
>100
21.4 ( 2.2
10.2 ( 2.3
17.8 ( 2.4
2.6 ( 0.6
16.4 ( 0.4
6.9 ( 0.3
12.1 ( 0.1
4.0 ( 0.3
13.2 ( 0.6
5.4 ( 0.6
13.0 ( 0.2
3.2 ( 0.5
8.5 ( 1.7
9.8 ( 0.2
11.4 ( 0.6
3.0 ( 1.0
10.4 ( 0.2
92.8 ( 0.9
N/T
43.3 ( 13.4
22.0 ( 5.8
N/T
4.5 ( 0.7
3.9 ( 0.05
to separate from the monoacylated product. Therefore pyridine
was employed as the base and provided exclusively monoacy-
lated products. With the desired benzamides in hand, the cyclic
carbonates underwent solvolysis with triethylamine in methanol
to give the diol in excellent yield. To complete our small library
of inhibitors, aryl nitro compounds (15-17) were subjected to
hydrogenation to afford the corresponding anilines.
Simultaneous with the investigation of aryl substitutes,
modification of the amide and tether functionalities was also
explored (Scheme 3). Sulfonamide 21 was assembled by
positive breast cancer cells), HCT-116 (colon cancer cells
characterized by wild-type p53), PL45 (pancreatic cancer cells),
LNCaP (androgen sensitive prostate cancer cells), and PC-3
(androgen independent prostate cancer cells) cell lines. As shown
in Table 1, the simplified benzamide 8 manifested anti-
proliferative activity. This is in stark contrast to A4 (Figure 1),
which does not.44 The monosubstituted benzamide variants
improved activity. In fact, the most potent anti-proliferative
agents identified were the methoxy (9-11) and phenyl (12-
14) derivatives that produced activities against most cell lines.
Thus, our data suggest that a p-hydrogen bond acceptor and an
m-aryl side chain on the benzamide were most effective. In the
nitro (15-17) and aniline (18-20) series, the ortho-derivatives
in each respective series were more active than the correspond-
ing regioisomers, which indicates hydrogen bond interactions
may be important in this region of the molecule. To our surprise,
activity was abolished upon replacement of the amide with a
sulfonamide (21, Table 2). This suggests that key hydrogen-
bonding interactions also exist between the amide and the protein
target that are critical for manifesting anti-proliferative activity.
Concerning the spatial requirements of the hydrophobic pocket,
we found that a two-carbon spacer between the amide and
phenyl ring (23) results in compounds that are more potent than
those that contain a methylene linker (22), a benzyl carbamate
(24), or a simplified benzamide (8). Furthermore, as seen with
trans-cinnamide 25, increasing the rigidity in the ethyl linker
provided an additional ∼3-fold increase in inhibitory activity
versus the saturated derivative (23). These results suggest that
the hydrophobic pocket into which the benzamide projects may
accommodate larger aromatic systems that exhibit increased
affinity.
SCHEME 3. Preparation of Novobiocin Derivatives with
Various Linkers
sulfonylation of amine 7 with benzenesulfonyl chloride, the
carbonate of which was subjected to solvolysis to provide the
resulting diol. The Cbz-containing product 24 was obtained by
direct solvolysis of 5. Amides 22, 23, and 25 were prepared by
coupling the appropriate acid with amine 7 in the presence of
EDCI and pyridine, followed by solvolysis of the cyclic
carbonate.
Upon completion of this initial library, compounds were
evaluated for anti-proliferative activity against SkBr3 (Her2
overexpressing breast cancer cells), MCF-7 (estrogen receptor
Overall, we were pleased to have identified novobiocin
analogues that manifest anti-proliferative activity against mul-
tiple transformed cells, in particular, the drug resistant pancreatic
ductal adenocarcinoma (PL45), a very aggressive cancer that
is associated with high mortality rates in patients.51
(51) Greenlee, R. T.; Hill-Harmon, M. B.; Murray, T.; Thun, M. Ca-
Cancer J. Clin. 2001, 51, 15-36.
(52) Greenfield, A. A.; Butera, J. A.; Caufield, C. E. Tetrahedron Lett.
2003, 44, 2729-2732.
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Burlison et al.
PC-3
TABLE 3. Anti-proliferation Activities of Novobiocin Biaryl Analogues (in µM) (n ) 3)
entry (IC50)
SkBr3
MCF-7
HCT-116
PL45
LNCaP
28
29
30
31
32
33
34
35
36
37
38
16.5 ( 4.7
32.4 ( 5.1
20.4 ( 3.5
25.4 ( 1.7
7.1 ( 0.6
7.5 ( 1.0
7.8 ( 0.5
1.5 ( 0.1
2.9 ( 1.2
1.6 ( 0.2
>100
19.5 ( 2.3
18.9 ( 2.7
20.4 ( 0.8
16.7 ( 6.3
15.6 ( 6.3
18.7 ( 1.8
37.9 ( 2.3
1.5 ( 0.1
5.3 ( 1.5
2.3 ( 0.8
>100
11.4 ( 0.0
37.5 ( 3.4
26.4 ( 0.6
2.4 ( 1.0
5.2 ( 2.1
5.1 ( 1.1
24.0 ( 0.3
4.7 ( 1.4
4.5 ( 1.2
1.4 ( 0.1
>100
2.8 ( 0.4
6.8 ( 1.5
4.2 ( 1.0
1.4 ( 0.1
3.8 ( 1.7
2.0 ( 0.6
2.6 ( 0.5
1.4 ( 0.2
1.1 ( 0.0
1.9 ( 0.8
1.9 ( 0.2
22.6 ( 3.3
4.2 ( 1.2
3.7 ( 0.9
6.0 ( 1.0
2.1 ( 0.3
3.3 ( 0.6
2.6 ( 0.6
2.5 ( 1.2
2.6 ( 0.5
8.8 ( 0.6
25.4 ( 5.2
64.7 ( 17.1
50.7 ( 4.3
31.9 ( 10.3
52.3 ( 34.7
53.3 ( 4.5
44.0 ( 20.3
16.6 ( 4.4
14.7 ( 2.4
22.3 ( 3.6
1.0 ( 0.1
>100
SCHEME 5. Preparation of the Biaryl Novobiocin
Derivatives
In an effort to incorporate the initial structure-activity
relationships into more efficacious inhibitors, two small libraries
of novobiocin derivatives were prepared. The first library
explored optimization of the benzamide that contained a
p-methoxy and a m-phenyl substituent. It was proposed that such
a compound may exhibit synergistic or additive activity and
lead to more effective compounds. The second library focused
on the incorporation of heterocycles into the benzamide region
in order to investigate hydrogen bond donor/acceptor interactions
and the effects of rigidity as suggested by our initial findings.
It was believed that this set of compounds could be expeditiously
prepared by the coupling of a 3-iodo-4-methoxy benzoic acid
with 7, to produce an intermediate (27) upon which the
incorporation of additional phenyl substituents could be pursued
for elucidation of structure-activity relationships (Scheme 4).
SCHEME 4. Retrosynthetic Analysis of Biaryl Novobiocin
Analogues
pared to 11 and 13). Likewise, the tolyl derivatives (29-31)
also manifested lower growth inhibition than biaryl 28, and
complete activity was lost against most cell lines upon incor-
poration of dihydrothianthrene, 38. Dihydrodibenzofuran 39
lacked reasonable solubility in DMSO and was therefore not
evaluated in our studies. However, as seen in the methoxy series
(32-34) and the phenol series (35-37), activity increased as
polarity and hydrogen bond donor/acceptor properties of the
inhibitor increased. For example, introduction of an o-OMe (32)
improved inhibition ∼2-fold when compared to 28, but the
corresponding phenol (35) was ∼8 times more effective.
Key structure-function relationships observed for the
biaryl benzamide novobiocin derivatives are summarized in
Figure 2.
Synthesis and Biological Evaluation of the Biaryl Novo-
biocin Library. The Suzuki precursor 27 was prepared by
coupling aminocoumarin 7 with benzoic acid 26 in the presence
of EDCI and pyridine (Scheme 5). The biaryl substituents also
included various hydrogen bond acceptors and donors to further
probe key binding interactions with Hsp90. After an extensive
survey of experimental conditions, it was found that dichloro-
[1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichlo-
romethane [Pd(dppf)Cl2] in the presence of substituted phenyl
boronic acids and 2 M potassium carbonate in dioxane at 50
°C provided the most reproducible cross-coupling conditions.52
To complete the synthesis, the carbonates were removed upon
solvolysis with methanolic triethylamine.
Synthesis and Biological Evaluation of Heterocyclic No-
vobiocin Derivatives. With the initial structure-function
relationships for novobiocin in hand, we wished to introduce
Upon completion of the biaryl derivatives, the compounds
were evaluated for anti-proliferative activity against the same
cell lines described above. Unfortunately, as presented in Table
3, combinations of the p-methoxy and m-phenyl substituents
on the benzamide (28) did not produce compounds that inhibit
cell growth more effectively as originally proposed (28 com-
FIGURE 2. SAR for the biaryl novobiocin derivatives and Hsp90.
2134 J. Org. Chem., Vol. 73, No. 6, 2008

DeVelopment of NoVobiocin Analogues
TABLE 4. Anti-proliferation Activities of Novobiocin Heterocyclic Analogues (in µM) (n ) 3)
entry (IC50)
SkBr3
MCF-7
HCT-116
PL45
LNCaP
PC-3
40
41
42
43
44
45
46
47
28.4 ( 2.0
65.9 ( 15.8
>100
20.3 ( 4.1
47.8 ( 4.2
>100
28.6 ( 4.1
96.3 ( 3.7
>100
23.3 ( 5.9
37.3 ( 1.0
>100
9.6 ( 0.3
78.9 ( 3.5
>100
7.9 ( 0.7
13.2 ( 1.0
19.2 ( 2.5
3.8 ( 0.4
33.2 ( 0.7
5.2 ( 1.1
0.47 ( 0.34
1.8 ( 0.3
44.9 ( 31.1
50.1 ( 3.3
34.8 ( 16.0
7.9 ( 1.1
>100
9.3 ( 0.6
12.2 ( 0.0
2.3 ( 0.1
15.1 ( 2.1
67.0 ( 20.2
N/T
18.8 ( 4.4
7.4 ( 2.1
10.9 ( 2.3
22.3 ( 10.1
4.8 ( 2.4
N/T
N/T
>100
0.37 ( 0.06
12.2 ( 1.5
0.57 ( 0.07
5.3 ( 0.3
0.17 ( 0.01
3.5 ( 0.5
heterocycles into the benzamide region of novobiocin to further
increase both solubility and polarity. We proposed that hetero-
cycles may increase desired pharmacological properties and
provide additional opportunities for hydrogen-bonding interac-
tions within the binding pocket, as was observed in the nitro
(15-17) and aniline (18-20) series reported in Table 1. To
this end, heterocyclic novobiocin derivatives were prepared as
described above by coupling commercially available carboxylic
acids with aminocoumarin 7 via treatment with EDCI and
pyridine, and the carbonates of the resulting molecules were
then solvolyzed with methanolic triethylamine to afford the
requisite diols, 40-47 (Scheme 6).
effective than compound 46. A summary of the observed trends
for 46 is provided in Figure 3.
SCHEME 6. Preparation of Heterocyclic Novobiocin
Derivatives
FIGURE 3. Structure-activity relationships observed for 46 and
novobiocin.
Finally, to provide additional evidence that the growth
inhibitory activity manifested by 46 resulted from Hsp90
inhibition, 46 was evaluated by its ability to induce degradation
of Hsp90-dependent client proteins. As seen in Figure 4, Hsp90
client proteins such as Her2, Raf, and Akt were degraded in a
concentration-dependent manner while Hsp90 was induced in
the presence of 46. Since non-Hsp90-dependent substrates such
as actin remained unchanged, the inhibition of cell growth was
directly correlated to the degradation of Hsp90-dependent client
proteins.
The inhibitory values were obtained by evaluation against
our panel of cancer cell lines. In the nicotinic (40-42) series,
the ortho-analogue was found to represent the most active
regioisomer, consistent with the trend observed for the nitro
(15-17) and aniline (18-20) derivatives (Table 4). Likewise,
we examined benzofuran 43 and benzothiophene 44. We
postulated that because these structures contain a hydrogen bond
acceptor in the same proximity as the other ortho derivatives
they would therefore introduce a rigid two-carbon spacer
between the amide and the phenyl ring. Unfortunately, benzo-
furan (43) was less active than the o-OMe variant (9), and the
bioisoelectronic benzothiophene (44) was even worse. The
2-indole was included for its potential to provide a hydrogen
bond donor as was observed for o-anilines. Anti-proliferation-
activity was significantly increased against the majority of our
cell lines indicating that the indole does indeed properly account
for the activities observed for a trans two-carbon tether and an
o-aniline. Substitution of the benzamide with a 2-indoleamide
increased the activity >500-fold against SkBr3 cells when
compared to the natural product, novobiocin. Interestingly, the
position of the nitrogen on the indole is critical for activity, as
the 3-indoleamide (47) was approximately 3-10 times less
FIGURE 4. Western blot analyses of Hsp90 client protein degradation
assays against MCF-7 breast cancer cells. Concentration of 46 (in µM)
is denoted above each lane. GDA (geldanamycin) and DMSO were
used as positive and negative controls, respectively.
Conclusions
In conclusion, we have identified key structure-activity
relationships for novobiocin analogues. Two small molecule
libraries were prepared and evaluated for anti-proliferative
activity against several cancer cell lines. The 2-indoleamide (46)
was identified as the most potent inhibitor resulting from these
studies, which resulted from rational development through
J. Org. Chem, Vol. 73, No. 6, 2008 2135

Burlison et al.
159.6, 158.1, 155.3, 153.6, 149.5, 134.3, 132.6, 126.4, 124.4, 123.5,
121.9, 121.3, 115.5, 115.1, 112.0, 111.5, 94.8, 83.3, 78.4, 77.4,
77.1, 61.0, 56.6, 27.9, 22.6, 8.8; IR (film) νmax 3308, 3055, 2986,
2939, 2930, 1817, 1807, 1707, 1655, 1603, 1533, 1481, 1466, 1367,
1263, 1232 cm-1; HRMS (ESI+) m/z 526.1691 (M + H+, C27H28-
NO10 requires m/z 526.1713).
observed structure-activity trends. Studies are currently un-
derway with this and other novobiocin analogues to confirm
their Hsp90 inhibitory activity and to identify the location of
the C-terminal nucleotide-binding region. The results from such
studies will be reported in due course along with optimized
derivatives of 46.
N-(7-((2R,3R,4S,5R)-3,4-Dihydroxy-5-methoxy-6,6-dimeth-
yltetrahydro-2H-pyran-2-yloxy)-8-methyl-2-oxo-2H-chromen-3-
yl)-2-methoxybenzamide (9). General procedure for solvolysis
of the cyclic carbonate: Et3N (10% total volume) was added
dropwise to a solution of cyclic carbonate in methanol. The resulting
mixture was stirred for 14 h, and then concentrated. The residue
was purified via preparative TLC or column chromtography (SiO2,
4:1 CH2Cl2:acetone) to yield a colorless, amorphous solid (74%):
[R]25D -16.1 (c 0.16, 20% MeOH in CH2Cl2); 1H NMR (400 MHz,
20% CD3OD in CD2Cl2) δ 8.79 (s, 1H), 8.15 (d, J ) 7.9 Hz, 1H),
7.57 (t, J ) 7.8 Hz, 1H), 7.38 (d, J ) 8.7 Hz, 1H), 7.20 (d, J ) 8.7
Hz, 1H), 7.16 - 7.10 (m, 2H), 5.55 (s, 1H), 4.20-4.14 (m, 2H),
4.11 (s, 3H), 3.58 (s, 3H), 3.35 (d, J ) 8.0 Hz, 1H), 2.28 (s, 3H),
1.34 (s, 3H), 1.11 (s, 3H); 13C NMR (100 MHz, 20% CD3OD in
CD2Cl2) δ 164.9, 160.2, 158.4, 156.7, 149.8, 134.6, 132.4, 126.3,
125.3, 123.0, 122.9, 121.9, 121.2, 114.7, 112.5, 111.7, 99.1, 84.8,
79.2, 71.9, 69.0, 62.1, 56.8, 29.1, 22.9, 8.5; IR (film) νmax 3373,
Experimental Section
Benzyl 7-((3aR,4R,7R,7aR)-7-Methoxy-6,6-dimethyl-2-oxotet-
rahydrdo-3aH-[1.3]dioxolo[4,5-c]pyran-4-yloxy)-8-methyl-2-oxo-
2H-chromen-3-ylcarbamate (7a). Boron trifluoride etherate (61
µL, 69 mg, 0.49 mmol, 30 mol %) was added dropwise to a solution
of (3aR,4S,7R,7aR)-7-methoxy-6,6-dimethyl-2-oxo-tetrahydro-3aH-
[1.3]dioxolo[4,5-c]pyran-4-yl 2,2,2-trichloroacetimidate (6, 588 mg,
1.62 mmol) and benzyl 7-hydroxy-8-methyl-2-oxo-2H-chromen-
3-yl carbamate (5, 527 mg, 1.62 mmol) in CH2Cl2 (16 mL) at rt.
The mixture was stirred for 14 h, before Et3N (150 µL) was added
and the mixture concentrated. The residue was purified by chro-
matography (SiO2, DCMf100:1 CH2Cl2:acetone) to afford 7a (670
mg, 81%) as a yellow foam: [R]22D -19.7 (c 1.54, 20% MeOH in
CH2Cl2); 1H NMR (CDCl3, 400 MHz) δ 8.27 (s, 1H), 7.85 (s, 1H),
7.55-7.35 (m, 5H), 7.29 (d, J ) 2.9 Hz, 1H), 7.11 (d, J ) 8.7 Hz,
1H), 5.77 (d, J ) 1.9 Hz, 1H), 5.23 (s, 2H), 5.05 (d, J ) 1.9 Hz,
1H), 4.95 (t, J ) 7.7 Hz, 1H), 3.59 (s, 3H), 3.30 (d, J ) 7.6 Hz,
1H), 2.27 (s, 3H), 1.34 (s, 3H), 1.19 (s, 3H); 13C NMR (CDCl3,
100 MHz) δ 159.0, 155.2, 153.6, 153.6, 149.2, 136.0, 129.1 (2C),
129.0, 128.7 (2C), 125.8, 122.6, 122.1, 115.2, 115.1, 111.6, 94.8,
83.3, 78.4, 77.6, 77.0, 67.9, 61.0, 27.9, 22.6, 8.8; IR (film) νmax
3402, 3319, 3063, 3034, 2984, 2939, 2839, 1817, 1709, 1634, 1609,
1587, 1522, 1456, 1383, 1366, 1331, 1296, 1263, 1229, 1205, 1175
cm-1; HRMS (ESI+) m/z 526.1688 (M + H+, C27H28NO10 requires
m/z 526.1713).
3-Amino-7-((3aR,4R,7R,7aR)-7-methoxy-6,6-dimethyl-2-oxo-
oxotetrahydrdo-3aH-[1.3]dioxolo[4,5-c]pyran-4-yloxy)-8-methyl-
2H-chromen-2-one (7). Palladium on carbon (10%, 67 mg) was
added to a solution of carbamate 5 (670 mg, 1.31 mmol) in
THF (13 mL). The suspension was stirred for 6 h under a hydrogen
atmosphere before it was filtered through a plug of SiO2 and eluted
with THF (20 mL). The eluent was concentrated and the residue
purified by chromatography (SiO2, 100:1f50:1 CH2Cl2:acetone)
to afford 7 (425 mg, 83%) as a yellow foam: [R]23D -26.4 (c 0.780,
20% MeOH in CH2Cl2); 1H NMR (CDCl3, 400 MHz) δ 7.10 (d, J
) 8.6 Hz, 1H), 7.05 (d, J ) 8.6 Hz, 1H), 6.68 (s, 1H), 5.73 (d, J
) 2.0 Hz, 1H), 5.04 (dd, J ) 2.0, 7.9 Hz, 1H), 4.95 (t, J ) 7.7 Hz,
1H), 4.11 (s, 2H), 3.54 (s, 3H), 3.29 (d, J ) 7.6 Hz, 1H), 2.28 (s,
3H), 1.34 (s, 3H), 1.21 (s, 3H); 13C NMR (CDCl3, 200 MHz) δ
159.6, 153.3, 153.0, 148.1, 130.2, 122.7, 116.1, 114.8, 111.9, 111.0,
94.5, 83.0, 78.0, 77.3, 76.4, 60.6, 27.5, 22.2, 8.6; IR (film) νmax
3462, 3362, 2984, 2937, 2839, 1807, 1707, 1636, 1595, 1497, 1387,
1371, 1331, 1263, 1169, 1109, 1078, 1036 cm-1; HRMS (ESI+)
m/z 392.1357 (M + H+, C19H22NO8 requires m/z 392.1346).
2947, 2835, 2525, 1641, 1630, 1610, 1448, 1412, 1398 cm-1
;
HRMS (ESI+) m/z 500.1893 (M + H+, C26H30NO9 requires m/z
500.1921).
N-(7-((2R,3R,4S,5R)-3,4-Dihydroxy-5-methoxy-6,6-dimeth-
yltetrahydro-2H-pyran-2-yloxy)-8-methyl-2-oxo-2H-chromen-3-
yl)-6-methoxybiphenyl-3-carboxamide (28). General procedure
for Suzuki coupling and solvolysis of the cyclic carbonate: Aryl
iodide 27 (1.0 equiv), 2 M K2CO3(aq) (3.0 equiv), and the aryl
boronic acid were dissolved in dioxane before PdCl2(dppf)‚CHCl3
(3 mol %) was added to the solution at rt. The resulting solution
was stirred at rt for 30 min and then warmed to 55 °C for 3-16 h,
after which the mixture was concentrated, filtered through a pad
of silica gel (eluted with 40:1 CH2Cl2:acetone), and purified via
preparative TLC (SiO2, 40:1 CH2Cl2:acetone). The resulting product
was dissolved in methanol containing 10% Et3N and stirred for 14
h before being concentrated. The residue was purified by preparative
TLC (4:1 CH2Cl2:acetone) to afford the corresponding diol as a
colorless, amorphous solid (46%, 2 steps): [R]25 -8.7 (c 0.23,
D
1
CH2Cl2); H NMR (800 MHz, CD2Cl2) δ 8.78 (s, 1H), 8.69 (s,
1H), 7.93 (dd, J ) 4.4, 8.0 Hz, 1H), 7.87 (d, J ) 2.4 Hz, 1H), 7.55
(d, J ) 8.0 Hz, 2H), 7.45 (t, J ) 8.0 Hz, 2H), 7.93 (dd, J ) 4.4,
8.0 Hz, 2H), 7.21 (d, J ) 8.8 Hz, 1H), 7.10 (d, J ) 8.8 Hz, 2H),
5.60 (s, 1H), 4.27-4.20 (m, 2H), 3.90 (s, 3H), 3.59 (s, 3H), 3.36
(d, J ) 9.6 Hz, 1H), 2.73 (s, 2H), 2.28 (s, 3H), 1.36 (s, 3H), 1.13
(s, 3H); 13C NMR (200 MHz, CD2Cl2) δ 167.2, 161.7, 161.2, 157.9,
151.0, 139.4, 133.0, 131.8, 131.5 (3C), 130.0 (2C), 129.4, 128.1,
127.6, 125.8, 124.0, 116.1, 116.0, 113.0 (2C), 99.7, 86.1, 80.4, 73.1,
70.5, 63.7, 57.8, 30.8, 24.2, 10.1; IR (film) νmax 3402, 3086, 3055,
3028, 2974, 2934, 2849, 2837, 1709, 1670, 1607, 1526, 1504, 1489,
1367, 1265, 1231, 1095 cm-1; HRMS (ESI+) m/z 576.2231 (M +
H+, C32H34NO9 requires m/z 576.2234).
N-(7-((2R,3R,4S,5R)-3,4-Dihydroxy-5-methoxy-6,6-dimeth-
yltetrahydro-2H-pyran-2-yloxy)-8-methyl-2-oxo-2H-chromen-3-
yl)-1H-indole-3-carboxamide (47). General EDCI Coupling
Procedure B: N-(3-(Dimethylamino)propyl)-N′-ethylcarbodiimide
hydrochloride (2.5 equiv) was added to a solution of aminocoumarin
7 (1.0 equiv) and carboxylic acid (2.0 equiv) in CH2Cl2 containing
30% pyridine at rt. The solution was stirred for 14 h, then
concentrated, and the residue was purified via preparative TLC
(SiO2, 40:1 CH2Cl2:acetone) to afford the amide. The resulting
product was dissolved in methanol containing 10% Et3N and stirred
for 14 h at rt. The mixture was concentrated and the residue was
purified by preparative TLC (10:1 CH2Cl2:methanol or 4:1 CH2-
Cl2:actone) to afford the corresponding diol as a colorless,
amorphous solid (19%, 2 steps): [R]26D -11.4 (c 0.18, 20% MeOH
2-Methoxy-N-(7-((3aR,4R,7R,7aR)-7-methoxy-6,6-dimethyl-2-
oxotetrahydro-3aH-[1.3]dioxolo[4,5-c]pyran-4-yloxy)-8-methyl-
2-oxo-2H-chromen-3-yl)benzamide (9a). General EDCI coupling
procedure A: N-(3-(Dimethylamino)propyl)-N′-ethylcarbodiimide
hydrochloride (3 equiv) was added to a solution of aminocoumarin
7 (1 equiv), benzoic acid (3 equiv), and 4-DMAP (2.0 equiv) in
CH2Cl2 at rt. The solution was stirred for 14 h then concentrated,
and the residue was purified via preparative TLC or column
chromatography (SiO2, 40:1 CH2Cl2:acetone) to afford the benza-
mide as a colorless, amorphous solid (66%): [R]24D -29.6 (c 0.61,
1
20% MeOH in CH2Cl2); H NMR (400 MHz, CDCl3) δ 8.77 (s,
1H), 8.18 (d, J ) 7.8 Hz, 1H), 7.46 (t, J ) 7.8 Hz, 1H), 7.27 (d,
J ) 8.3 Hz, 1H), 7.09-7.02 (m, 2H), 6.99 (d, J ) 8.3 Hz, 1H),
5.71 (s, 1H), 4.99 (d, J ) 7.9 Hz, 1H), 4.91 (t, J ) 7.9 Hz, 1H),
4.05 (s, 3H), 3.53 (s, 3H), 3.24 (d, J ) 7.9 Hz, 1H), 2.23 (s, 3H),
1.29 (s, 3H), 1.14 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 164.5,
2136 J. Org. Chem., Vol. 73, No. 6, 2008

DeVelopment of NoVobiocin Analogues
1
in CH2Cl2); H NMR (500 MHz, 20% CD3OD in CD2Cl2) δ 8.75
(ESI+) m/z 509.1924 (M + H+, C27H29N2O8 requires m/z 509.
(s, 1H), 8.12 (dd, J ) 2.0, 6.5 Hz, 1H), 7.97 (s, 1H), 7.49 (dd, J )
2.0, 6.5 Hz, 1H), 7.38 (d, J ) 8.5 Hz, 1H), 7.31-7.24 (m, 2H),
7.20 (d, J ) 8.5 Hz, 1H), 5.55 (d, J ) 2.5 Hz, 1H), 4.16 (dd, J )
4.5, 9.5 Hz, 1H), 4.12 (t, J ) 4.5 Hz, 1H), 3.57 (s, 3H), 3.33-3.31
(m, 1H), 2.28 (s, 3H), 1.33 (s, 3H), 1.10 (s, 3H); 13C NMR
(125MHz, 20% CD3OD in CD2Cl2) δ 165.7, 161.3, 157.6, 150.6,
138.4, 131.0, 127.1, 126.4, 125.1, 124.5, 123.8, 123.5, 121.4, 115.8
(2C), 113.9, 112.8, 112.7, 100.0, 85.9, 80.1, 72.9, 70.1, 63.2, 30.2,
23.9, 9.6; IR (film) νmax 3439, 3418, 3394, 2957, 2924, 2853, 1636,
1529, 1437, 1379, 1261, 1180, 1128, 1082, 1020 cm-1; HRMS
1924).
Acknowledgment. The authors gratefully acknowledge
support of this project by the NIH/NCI (CA120458) and the
DOD Prostate Cancer Research Program (QH815179).
Supporting Information Available: Full experimental proce-
dures and characterization for all compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
JO702191A
J. Org. Chem, Vol. 73, No. 6, 2008 2137