Novologues containing a benzamide side chain manifest anti-proliferative activity against two breast cancer cell lines

Article abstract of DOI:10.1016/j.bmcl.2014.05.020

Hsp90 represents a promising target for the development of both anti-cancer and neuroprotective agents. Structure-activity relationship studies on novobiocin and novobiocin analogues, led to the development of KU-32 and recently, KU-596, as lead compounds for the potential treatment of neurodegenerative diseases. Similar to KU-32, we have demonstrated that upon replacement of the acetamide side chain present in KU-32 with a benzamide, this neuroprotective agent was transformed into a scaffold that manifests anti-proliferative activity. To assess structure-activity relationships for this new scaffold, a library of benzamide-containing novologues was prepared and evaluated against two breast cancer cell lines. Compound 14a manifested the most potent anti-proliferative activity from these studies and induced Hsp90-dependent client protein degradation in a concentration-dependent manner.

Full text of DOI:10.1016/j.bmcl.2014.05.020

Accepted Manuscript
Novologues Containing a Benzamide Side Chain Manifest Anti-proliferative
Activity Against Two Breast Cancer Cell Lines
Huiping Zhao, Mercy Anyika, Antwan Girgis, Brian S.J. Blagg
PII:
S0960-894X(14)00507-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.05.020
BMCL 21633
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
2 April 2014
30 April 2014
5 May 2014
Please cite this article as: Zhao, H., Anyika, M., Girgis, A., Blagg, B.S.J., Novologues Containing a Benzamide
Side Chain Manifest Anti-proliferative Activity Against Two Breast Cancer Cell Lines, Bioorganic & Medicinal
Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.05.020
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Novologues Containing a Benzamide Side Chain Manifest Anti-
proliferative Activity Against Two Breast Cancer Cell Lines
Huiping Zhao, Mercy Anyika, Antwan Girgis 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 heat shock protein 90 (Hsp90) folding machinery is essential for the maturation
of nascent polypeptides into their biologically active three-dimensional-structures and for the
rematuration/clearance of misfolded proteins that form under cellular stress.1-3 As a pro-
survival chaperone, Hsp90 overexpression is commonly observed in transformed cells, which
is required to sustain the hostile tumor micro-environment associated with nutrient
deprivation and hypoxia. Pharmacological inhibition of Hsp90 has been shown to induce the
degradation of oncogenic proteins associated with all six hallmarks of cancer that rely upon
Hsp90.4-8 Consequently, Hsp90 represents a highly sought after target for the treatment of
cancer. In fact, 17 small molecules that bind competitively to the N-terminal ATP-binding
pocket are under clinical evaluation against various cancers.9,10 However, heat shock factor 1
(HSF-1), the master regulator of the pro-survival heat shock response also binds Hsp90.
Ultimately, Hsp90 N-terminal inhibition results in HSF-1 release, and upon phosphorylation,
trimerizes and translocates to the nucleus wherein it binds the heat shock elements to activate
the pro-survival, heat shock response (HSR). The HSR serves to expand the cellular buffering
capacity and to assist in the maturation of mutated and oncogenic substrates.11 This
concomitant heat shock response is detrimental to the treatment of cancer and may lead to
1

drug resistance and tumor metastasis.12 Recent studies have demonstrated that allosteric
modulation of the Hsp90 C-terminus can separate the pro-survival heat shock response from
pro-apoptotic, client protein degradation.13-20 Two classes of small molecules derived from
novobiocin 1, (Figure 1) the first identified Hsp90 C-terminal inhibitor, were discovered via
the structure-activity relationship studies. KU-32 (2), which lacks a 4-hydroxyl, the 3’-
carbamate, and contains an acetamide in lieu of a prenylated benzamide, represents a lead
compound that induces the heat shock response at concentrations much lower than that
needed for client protein degradation.2,21 Consequently, this class of analogues has been
evaluated as neuroprotective agents to refold protein aggregates.22-24 In contrast, KU-174 (3)
contains a biarylamide side chain in lieu of the acetamide, and induces Hsp90 client protein
degradation without induction of the heat shock response.25-26 Therefore, this class of
novobiocin analogues manifests optimal properties for the treatment of cancer, as no HSR is
observed with such compounds.
Recently, a second generation of novologues that contains a scaffold that mimics KU-
32 was identified, and biological evaluation against primary sensory neurons showed these
molecules to possess enhanced neuroprotective properties.27 Novologue 4 contains a 3-
trifluoromethylphenyl ring in lieu of the coumarin lactone present in novobiocin and
incorporates the acetamide onto the flexible ethylene linker to maintain hydrogen-bonding
interactions with the Hsp90 C-terminal binding pocket. As shown during the discovery and
development of novobiocin-based Hsp90 C-terminal inhibitors, restoration of the benzamide
side chain onto KU-32 transforms the molecule from manifesting neuroprotective activity
into one that serves as an anti-cancer agent. Therefore, we hypothesized that replacement of
the acetamide present in novologue 4 with a biarylamide should also transform this
novologue into an anti-proliferative agent (5a).
2

Figure 1. Hypothesis for the design of cytotoxic novologues.
As shown in Scheme 1, retrosynthesis of compound 5a (and related analogues) was
envisioned for construction via noviosylation of phenol 7 by activated noviose carbonate, 6.
Intermediate 7 could be assembled via an amide coupling reaction between amine 8 and acid
chloride 9, followed by hydrogenolysis. As reported previously,27 the synthesis of amine 8
could be achieved from 2,4-dihydroxybenaldehyde through phenol protection, Suzuki
coupling, a Henry reaction, and reduction of the corresponding α,β-unsaturated nitro styrene.
Scheme 1. Retrosynthesis of cytotoxic novologues.
Synthesis of these novologues commenced via selective phenol protection of 2,4-
dihydroxybenaldehyde (10) with benzyl bromide. The resulting benzyl ether was converted
to trifluoromethanesulfonate 11, in the presence of trifluoromethanesulfonic anhydride and
3

triethylamine. Subsequent Suzuki coupling with commercially available aryl boronic acids
(phenyl, m-CF3, m-Cl, m-F and m-OMe) was employed to generate the respective biaryl ring
systems, 12a-e. These boronic acids were selected based on prior studies that suggested the
inclusion of electronegative substituents at the meta-position of the B-ring exhibit favorable
interactions with Lys539 in the Hsp90 C-terminal binding pocket.27
Benzaldehydes 12a-e were subjected to a Henry reaction to afford the corresponding
nitrostyrenes, 13a-e. Subsequent lithium aluminum hydride reduction of the nitrostyrenes to
the corresponding amines, followed by acylation with benzoyl chlorides 9a-e gave the benzyl
ether containing benzamides, which upon hydrogenolysis produced phenols 7a-i.
Scheme 2. Synthesis of phenol 7a-e.
Preparation of the novologues required noviosylation of phenols 7a-e, which occurred
upon treatment with the trichloroacetimidate of noviose carbonate 6 in the presence of
catalytic boron trifluoride etherate, followed by solvolysis of the cyclic carbonate with
methanolic ammonia to generate the desired products, 5a-e, in good yields.
In addition, novologues 14a-i were synthesized to investigate prior observations that
suggested the replacement of noviose with amines could further increase inhibitory activity
4

and solubility.28-31 Therefore, Mitsunobu etherification of phenols 7a-i with 1-methyl-4-
hydroxypiperidine was performed to yield the amino containing novologues, 14a-i.
Scheme 3. Synthesis of cytotoxic novologues.
Upon construction of the library, these novologues were evaluated for anti-
proliferative activity against two breast cancer cell lines: SKBr3 (estrogen receptor negative,
HER2 over-expressing breast cancer cells) and MCF-7 (estrogen receptor positive breast
cancer cells). As shown in Table 1, the novologues manifested anti-proliferative activity
against both breast cancer cell lines. Novologues containing a piperidine ring were shown to
exhibit greater activity than their noviose containing counterparts (14a-e vs 5a-e), which is
consistent with prior studies on novobiocin.28 Although substitutions on the benzamide side
chain were beneficial to anti-proliferative activity, 4-Cl (5e) and 4-methoxy (5c) were better
than 4-t-butyl (5d) for the compounds containing noviose. However, compounds containing
the piperidine ring manifested better activity when 4-t-butyl (15c) or 4-Cl (15d) were present,
compared to the 4-methoxy derivative (15b). Substitutions on the B ring did not produce
significant differences in anti-proliferative activity, however, as predicted, the inclusion of an
5

electronegative atom (F, Cl and CF3) at the meta-position of the B-ring was most active. As
hypothesized, all the novologues were more active than the parent, acetamide-containing,
neuroprotective compound 4.
Table 1. Anti-proliferative activity manifested by novologues containing a benzamide side
chain
Entry
4
5a
R
-
R1
-
R2
R3
SKBr3
>500
MCF-7
>500
-
-
A
A
A
A
A
B
B
B
B
B
B
B
B
B
OMe
H
m-methoxyphenyl
H
-
13.98 2.04 18.40 2.11
41.90 7.69a 44.44 1.81
14.02 0.64 12.10 0.37
21.78 0.62 23.69 0.91
14.48 2.34 11.69 0.84
-
5b
5c
OMe
t-Butyl
Cl
H
-
H
-
5d
5e
H
m-methoxyphenyl
-
CF3
OMe
OMe
OMe
OMe
OMe
H
1.52 0.05
5.63 1.45
2.82 0.93
1.62 0.15
6.87 1.05
3.19 0.15
14a
14b
14c
14d
14e
15a
15b
15c
15d
m-methoxyphenyl
m-methoxyphenyl
Cl
F
OMe
m-methoxyphenyl
m-methoxyphenyl
11.54 0.75 12.36 0.86
6.77 1.35 8.12 0.35
13.22 1.50 15.07 1.41
H
-
H
H
H
H
OMe
t-Butyl
Cl
-
2.71 0.62
1.16 0.10
1.87 0.64
4.82 0.11
1.48 0.26
2.11 0.04
-
-
Values in µM unless otherwise indicated. aValues represent mean standard deviation for at
least two separate experiments performed in triplicate.
In order to gain further insight into this class of compounds, a comparison between
the anti-proliferative activities manifested by novobiocin-derived inhibitors and these
novologues was performed. Although the benzamide side chain is attached to a freely
rotating ethylene linker in novologues 5a and 14a, they manifested similar activity to
novobiocin derivatives 16 and 17, suggesting that this new scaffold can serve as a surrogate
for the coumarin ring system. More importantly, the entropic penalty introduced by
incorporation of the ethylene linker appears to be compensated by interactions provided by
incorporation of the trifluorophenyl ring. Alternatively, the rigid coumarin ring system may
not be optimal for binding interactions.
6

Table 2. Comparison of anti-proliferative activities: benzamide-containing novologue versus
novobiocin derivatives
Novologues
Novobiocin derivatives
7.50 0.80
1.38 0.18
R
A
B
A
B
13.98 2.04
SKBr3
MCF-7
5a
15a
5a
16
17
16
17
1.30 0.19
18.40 2.11
1.62 0.15
18.70 1.44
1.51 0.24
15a
To confirm that Hsp90 inhibition was responsible for the observed anti-proliferative
activities manifested by these novologues, Western blot analyses of several Hsp90 dependent
client protein levels were examined in MCF-7 cell lysates treated with 14a.32 As shown in
Figure 2, Her2, Raf-1 and p-Akt, all of which are well-validated Hsp90-dependent client
proteins, were degraded in a concentration-dependent manner, while non-Hsp90-dependent
actin levels remained constant. In addition, Hsp90 levels remained unchanged, indicating
that these novologues bind the Hsp90 C-terminus.33-36
Figure 2. Western blot analysis of Hsp90-dependent client proteins from MCF-7 breast
cancer cell lysate upon treatment with 14a. Concentrations (in µM) were indicated above
each line. Geldanamycin (G, 0.5 µM) and dimethylsulfoxide (D, 100%) were employed as
positive and negative controls.
7

In conclusion, a library of benzamide-containing novologues was designed,
synthesized and evaluated against two breast cancer cell lines. Initial structure-activity
relationships for the amide appendage were elucidated and novologue 14a was shown to
exhibit lead-like activity. Western blot analyses demonstrated these compounds to manifest
anti-proliferative activity through Hsp90 inhibition. Replacement of the acetamide side chain
on the novologue scaffold with a benzamide side chain successfully transformed this class of
neuroprotective agents into molecules that exhibit anti-proliferative activity. Further
structure-activity relationship studies on this novologue scaffold are currently underway and
will be reported in due course.
8

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10

OCH3
H
N
H
N
OCH3
O
O
O
O
N
O
MeO
HO
F
OH
CF3
KU-596
14a
Neuroprotective property
Anti-proliferative activity

Elsevier Editorial System(tm) for Bioorganic & Medicinal Chemistry Letters
Manuscript Draft
Manuscript Number: BMCL-D-14-00546R1
Title: Novologues Containing a Benzamide Side Chain Manifest Anti-proliferative Activity Against
Breast Cancer Cell Lines
Article Type: Full Length Article
Keywords: Heat shock protein 90, Hsp90 inhibitors, Novobiocin analogues, Breast cancer
Corresponding Author: Dr. Brian S. J. Blagg, Ph.D.
Corresponding Author's Institution: The University of Kansas
First Author: huiping zhao
Order of Authors: huiping zhao; mercy anyika; antwan girgis; Brian S. J. Blagg, Ph.D.
Abstract: Hsp90 represents a promising target for the development of both anti-cancer and
neuroprotective agents. Structure-activity relationship studies on novobiocin and novobiocin
analogues, led to the development of KU-32 and recently, KU-596, as lead compounds for the potential
treatment of neurodegenerative diseases. Similar to KU-32, we have demonstrated that upon
replacement of the acetamide side chain present in KU-32 with a benzamide, this neuroprotective
agent was transformed into a scaffold that manifests anti-proliferative activity. To assess structure-
activity relationships for this new scaffold, a library of benzamide-containing novologues was prepared
and evaluated against two breast cancer cell lines. Compound 14a manifested the most potent anti-
proliferative activity from these studies and induced Hsp90-dependent client protein degradation in a
concentration-dependent manner.