Novobiocin analogues with second-generation noviose surrogates

Article abstract of DOI:10.1016/j.bmcl.2012.11.022

Hsp90 is a promising therapeutic target for the treatment of cancer. Novobiocin is the first Hsp90 C-terminal inhibitor ever identified and recent structure-activity relationship studies on the noviose sugar identified several commercially available amines as suitable surrogates. In an effort to further understand this region of the molecule, analogues containing various N′-amino substituents were prepared and evaluated against two breast cancer cell lines for determination of their efficacy. Compound 37j manifested the most potent anti-proliferative activity from these studies and induced Hsp90-dependent client protein degradation at mid nano-molar concentrations.

Full text of DOI:10.1016/j.bmcl.2012.11.022

Accepted Manuscript
Novobiocin analogues with second-generation noviose surrogates
Huiping Zhao, Brian S.J. Blagg
PII:
S0960-894X(12)01465-5
DOI:
http://dx.doi.org/10.1016/j.bmcl.2012.11.022
BMCL 19786
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
17 September 2012
1 November 2012
7 November 2012
Please cite this article as: Zhao, H., Blagg, B.S.J., Novobiocin analogues with second-generation noviose surrogates,
Bioorganic & Medicinal Chemistry Letters (2012), doi: http://dx.doi.org/10.1016/j.bmcl.2012.11.022
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Novobiocin analogues with second-generation
noviose surrogates
Huiping Zhao and Brian S. J. Blagg*
Department of Medicinal Chemistry, 1251 Wescoe Hall Drive, Malott 4070
The University of Kansas, Lawrence, Kansas 66045-7563.
* Author to whom correspondence should be addressed. Phone: (785) 864-2288. Fax:
(785) 864-5326. Email: bblagg@ku.edu.
The University of Kansas
ABSTRACT Hsp90 is a promising therapeutic target for the treatment of cancer.
Novobiocin is the first Hsp90 C-terminal inhibitor ever identified and recent structure-
activity relationship studies on the noviose sugar identified several commercially
available amines as suitable surrogates. In an effort to further understand this region of
the molecule, analogues containing various N’-amino substituents were prepared and
evaluated against two breast cancer cell lines for determination of their efficacy.
Compound 37j manifested the most potent anti-proliferative activity from these studies
and induced Hsp90-dependent client protein degradation at mid nano-molar
concentrations.
KEYWORDS Heat shock protein 90, Hsp90 inhibitors, Novobiocin analogues, Breast
cancer
1

The 90 kDa heat shock protein is an ATP-dependent chaperone that belongs to the
GHKL superfamily.1 It is one of the most abundant molecular chaperones in the cytosol
and promotes the folding, activation and stabilization of more than 200 client proteins,
approximately 50 of which are directly associated with cell growth and/or signaling
pathways.2 Malignant or mutated oncogenic proteins, such as Her-2, Raf-1, Akt, CDK4,
Src, Flt-3, hTert, c-Met, etc, are distributed amongst the six hallmarks of cancer and are
highly dependent upon the Hsp90 protein folding machinery for their ability to promote
cell survival, proliferation, and adaptation.3 In contrast to the homodimeric chaperone
that is present in normal cells, Hsp90 exists as a heteroprotein super-chaperone complex
in cancer cells and is bound to oncogenic proteins that are sensitive to physiological
stress.4 In such scenarios, Hsp90 inhibition results in the simultaneous disruption of
multiple oncogenic pathways and eventually leads to cancer cell death, while largely
sparing normal cells. As a consequence, Hsp90 has emerged as a promising therapeutic
target for the treatment of cancer.
Hsp90 contains three highly conserved domains: the 25 kDa N-terminus is
responsible for ATPase activity, the 35 kDa middle domain is utilized for substrate
recognition, and the 12 kDa C-terminus elicits dimerization and co-chaperone binding.5
The N-terminal domain has been extensively studied and 16 small molecules targeting
this region have been evaluated in clinical trials.6,7 However, detriments such as heat
shock induction and cytostatic activity associated with N-terminal inhibition has limited
their potential use against cancer.8 However, some small molecules that bind to the C-
terminal domain do not induce the pro-survival heat shock response, and in some cases,
even cause Hsp70 and Hsp90 degradation.9-12 Because the Hsp90 C-terminus is
2

responsible for mediating interactions with co-chaperones such as HOP (Hsp70-Hsp90
organization protein) and the immunophilins (eg. FK506 binding protein) to facilitate
client protein maturation,13,14 small molecule modulation of this region exhibits activities
not observed with N-terminal inhibitors.11,15
Novobiocin was first identified as an Hsp90 C-terminal inhibitor in 2000 by
Neckers and coworkers.16 Subsequent modification to novobiocin has led to the
elucidation of structure-activity relationships and analogues that exhibit superior
inhibitory activity.17-21 Deletion of both the 4-hydroxy substituent on the coumarin ring
and the 3’-carbamoyl group on noviose resulted in DHN2, which transformed novobiocin
from a DNA gyrase inhibitor to a selective Hsp90 inhibitor (Figure 1).22 Subsequent
replacement of the synthetically complex noviose sugar with readily available amines led
to molecules represented by NA-1 (novobiocin analogue 1, Figure 1) and NA-2
(novobiocin analogue 2) that manifest increased anti-proliferative activity and
solubility.21 During the course of studies aimed at the modification of these amines,
Huang and coworkers developed a three-dimensional quantitative structure-activity
relationship (3D-QSAR) model that suggested modifications to the amine may improve
anti-proliferative activity.23 Consequently, second generation amino-analogues were
designed, synthesized and evaluated against two breast cancer cell lines.
3

Figure 1. Evolution of novobiocin analogues.
SAR generated from novobiocin analogues suggested that a three-carbon linker
between the 7-coumarin phenol and the amine was optimal.21 Although the anti-
proliferative activity of NA-1 and NA-2 suggested flexibility of the amine moiety may be
required, increasing the number of rotatable bonds is generally considered detrimental,
due to entropic penalties. Therefore, novobiocin analogues containing rigid heterocyclic
piperidine and pyrroridine derivatives were synthesized and evaluated. As illustrated in
Figure 2, the piperidine or pyrrolidine ring system was linked to the 7-phenolic oxygen
by 1−3 carbons.
Figure 2. Proposed cyclic amino derivatives of novobiocin
4

These amines were assembled with the coumarin core in modular fashion utilizing
Boc-protected secondary amines (1 and 3) or tertiary amines (2 and 4), which underwent
Mitsunobu esterification with phenol 5 to afford 6−9.21 Subsequent hydrogenolysis to
unmask the amine allowed for amide coupling with 4-(chlorocarbonyl)-2-(3-methylbut-2-
en-1-yl)phenyl acetate (10) in the presence of pyridine afforded novobiocin C-linked
heterocyclic analogues 11─14. Acid-catalyzed deprotection of 11 and 13 resulted in
secondary amines 15 and 16. Finally, hydrolysis of ester 12 and 14─16 generated phenols
17─20 (Scheme 1).
Scheme 1. Synthesis of C-linked heterocyclic derivatives of novobiocin
Along with the C-linked heterocyclic derivatives (12, 14, 15−20), N-linked
heterocycles (29−33) were also synthesized (Scheme 2). Compound 23 was regarded as a
versatile intermediate and was prepared in 3 steps. Benzyl (7-hydroxy-8-methyl-2-oxo-
2H-chromen-3-yl)carbamate (5) was treated with ten equivalents of 1,3-dibromopropane
5

(21) in the presence of potassium carbonate to afford alkyl bromide 22. Subsequent
hydrogenolysis followed by amide coupling with acid chloride 10 produced the
corresponding amide 23. Displacement of the bromide in 23 with amines 24─28 in N,N-
dimethylformamide and simultaneous hydrolysis of the phenolic ester gave amines
29─33.
Scheme 2. Synthesis of terminal cycloamine derivatives of novobiocin
Upon construction of the C- and N-linked heterocyclic novobiocin analogues,
these molecules were evaluated for anti-proliferative activity against SKBr3 (estrogen
receptor negative, Her2 over-expressing breast cancer cells) and MCF-7 (estrogen
receptor positive breast cancer cells) cell lines. As shown in Table 1, the anti-proliferative
activities manifested by C-linked heterocyclic analogues (12─20) were similar to NA-1,
suggesting that various ring structures are accommodated within the binding site.
Interestingly, the secondary amino analogues exhibit greater inhibitory activity than the
corresponding tertiary amines (16 vs 12, 19 vs 18), which is further exacerbated against
the MCF-7 cell line. Although piperizine analogues (29 and 30) maintained activity
comparable to NA-1, compounds 31 and 32 were inactive at the highest concentration
6

tested. However, shrinking the ring size to five atoms (33) restored activity, suggesting
that hydrophobicity is limited in this region and that the inclusion of heteroatoms is
necessary for further extension.
Table 1. Anti-proliferative activities NA-1 analogues.
R
R’
SKBr3 (μM)
0.76±0.17a
MCF-7 (μM)
1.09±0.10
H
KU-398
12
OAc
0.74±0.01
1.65±0.11
OAc
OAc
OAc
H
0.88±0.09
0.64±0.06
0.62±0.04
0.64±0.06
1.30±0.63
1.88±0.45
0.71±0.03
0.82±0.07
0.71±0.03
2.64±0.45
14
15
16
17
18
H
H
H
H
0.60±0.01
0.92±0.56
1.01±0.03
0.79±0.10
1.55±0.04
1.20±0.12
19
20
29
7

H
0.82±0.02
0.93±0.21
30
H
H
H
>50
>50
31
32
33
>50
>50
0.72±0.01
0.99±0.04
a
Values represent mean ± standard deviation for at least two separate experiments
performed in triplicate
Data from the first set of compounds suggest there is limited hydrophobic space,
however, further extension is possible, but requires an H-bond donor (e.g., 30), which
cannot be replaced with a hydrophobic moiety (32) or an H-bond acceptor (31). In
addition, Huang and co-workers suggested in their 3D-QSAR study that steric bulk N-
substitution of NA-2 could lead to increased anti-proliferative activity.23 Therefore,
analogues containing various linear and branched N-substitutions were pursued in an
effort to maintain both the hydrophobicity and the H-bond donor properties. As shown in
Scheme 3, compound 23 was treated with primary and secondary amines (34a─34o) to
produce the corresponding secondary and tertiary amine derivatives containing various
linear or branched chains (35a─35k). Similarly, treatment of 23 with amines 34l─34o
afforded compounds 35j−35o that contain various H-bond properties to further verify
whether H-bond donors or acceptors were favored.
8

Scheme 3. Synthesis of NA-2 analogues with linear and branched chains.
Anti-proliferative activity manifested by these analogues was evaluated against
SKBr3 and MCF-7 cell lines as previously described. As shown in Table 2, a bulky group
(up to 3 linear carbons, 35a, 35b, 35d, and 35f−35k except 35i) was generally well-
tolerated in the binding site. It appears as though this hydrophobic chain can be linear
(35a, 35b, and 35d) or branched (35f─35h, 35j and 35k). The diethylamine (35c) and
diisopropylamine (35i) analogues exhibited decreased anti-proliferative activity against
SKBr3 cells, while the diisopropylamine analogue manifested no activity (35e),
indicating a limited amount of space is available. Glycine ester analogue 35l and amide
analogue 35n exhibited decreased anti-proliferative activity against SKBr3 cells or MCF-
7 cells while alcohol 35o retained activity against both cancer cell lines, supporting
further extension into the binding site requires hydrogen bond donors. The significantly
9

decreased activity of glycine acid analogue 35m may result from limited cell membrane
penetration.
Table 2. Anti-proliferative activities of NA-2 analogues
R1
R2
SKBr3 (μM)
0.44±0.02a
MCF-7 (μM)
1.35±0.38
CH3
CH3
KU-407
CH2CH3
H
0.72±0.34
0.73±0.06
4.90±0.42
0.99±0.03
0.98±0.35
2.19±0.50
6.39±0.04
0.98±0.00
>50
35a
35b
35c
35d
35e
35f
CH2CH3
CH3
CH2CH3
CH2CH3
H
CH2CH2CH3
CH2CH2CH3
CH2CH(CH3)2
CH(CH3)2
CH(CH3)2
CH(CH3)2
C(CH3)3
CH2CH2CH3 >50
H
1.03±0.02
1.32±0.06
0.74±0.16
1.05±0.06
1.31±0.39
0.95±0.10
1.14±0.11
2.82±0.27
18.11±5.35
1.46±0.35
H
0.75±0.26
0.57±0.13
1.60±0.02
0.66±0.06
0.88±0.10
1.06±0.11
7.57±2.86
2.29±0.47
35g
35h
35i
CH3
CH(CH3)2
H
35j
C(CH3)3
CH3
H
35k
35l
CH2COOMe
CH2COOH
CH2CONH2
H
35m
35n
H
10

CH2CH2OH
H
0.61±0.06
1.56±0.09
35o
a
Values represent mean ± standard deviation for at least two separate experiments
performed in triplicate
Although none of the first two sets of compounds showed significantly improved
anti-proliferative activity, it is clear that steric bulk is tolerated in the binding site.
Therefore, a set of NA-2 derivatives containing various rings (37a−37j) were prepared in
a similar synthetic sequence, as described in Scheme 4.
Scheme 4. Synthesis of NA-2 derivatives with cyclic N-substitutions
The anti-proliferative data (Table 3) shows that cyclohexyl (37a and 37b), benzyl
(37e), substituted benzyl (37f and 37g), and bicyclic alkyls (37i and 37j) are tolerated in
the binding site. Aniline-containing analogues were inactive (37c and 37d), possibly due
to the altered hydrogen bonding property manifested by amine due to the incorporation of
the phenyl ring. Compound 37j, which contained an N-adamantyl substitution,
manifested the most potent anti-proliferative activity against both SKBr3 and MCF-7
breast cancer cell lines, indicating that significant space is available, but the molecule
must contain a free H-bond donor in the form of a secondary amine. In addition, 37j
11

exhibited lower activity against normal cells (MRC5, 5.42 μM; HMLE, 15.05 μM), thus
provide a considerable therapeutic window for the treatment of cancer.
Table 3. Anti-proliferative activity of NA-2 derivatives with cyclic N-substitutions
R1
R2
H
SKBr3 (μM)
0.66±0.12a
0.97±0.03
>50
MCF-7 (μM)
1.07±0.35
1.08±0.07
>50
Cyclohexyl
Cyclohexyl
Phenyl
37a
37b
37c
37d
37e
37f
37g
37h
37i
CH3
H
Phenyl
CH3
H
>50
>50
Benzyl
0.47±0.16
1.00±0.31
0.96±0.01
10.73±0.49
0.77±0.13
0.91±0.01
1.45±0.20
1.07±0.01
11.52±4.16
1.03±0.12
(R)-1-phenylethyl
Cumenyl
Benzyl
H
H
CH3
H
Adamantyl
H
0.31±0.04
0.32±0.03
37j
a
Values represent mean ± standard deviation for at least two separate experiments
performed in triplicate
To confirm these novobiocin analogues that contain amino groups in lieu of noviose
manifest inhibitory activity through Hsp90 inhibition, western blot analyses of cell
lysates following the administration of most active compound, 37j, were performed. As
12

shown in Figure 3, the Hsp90-dependent client proteins, Raf and Akt, were degraded in
MCF-7 cells in a concentration-dependent manner upon treatment with 37j. The non-
Hsp90-dependent protein, actin, was not degraded upon the administration of 37j,
indicating selective degradation of Hsp90-dependent proteins. In accordance with
previous studies, Hsp90 levels remained constant at all concentrations tested,
demonstrating that 37j does not induce the pro-survival heat shock response.
Figure 3. Western blot analyses of MCF-7 cell lysates for Hsp90 client protein
degradation after 24 h incubation. Concentrations (in μM) of 37j are indicated above each
lane. Geldanamycin (G, 500 nM) and DMSO (D) were employed respectively as positive
and negative controls.
In conclusion, a library of novobiocin analogues containing a second generation
of amino-appendages was designed, synthesized and evaluated. The results showed that a
variety of bulky N-alkyl groups are well tolerated and can lead to compounds with
efficacious activity against breast cancer cells. Compound 37j, a secondary amino
derivative containing an N-adamatyl group, manifested the most potent anti-proliferative
activity, which through western blot analyses confirmed this compound inhibits the
Hsp90 protein folding machinery.
Acknowledgement
13

The authors gratefully acknowledge support of this project by the NIH/NCI (CA120458)
and the DOD Prostate Cancer Research Program (QH815179).
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15

Novobiocin analogues with second-generation
noviose surrogates
Huiping Zhao and Brian S. J. Blagg*
Department of Medicinal Chemistry, 1251 Wescoe Hall Drive, Malott 4070
The University of Kansas, Lawrence, Kansas 66045-7563.
* Author to whom correspondence should be addressed. Phone: (785) 864-2288. Fax:
(785) 864-5326. Email: bblagg@ku.edu.
The University of Kansas
For Table of Contents Only
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