Triazole containing novobiocin and biphenyl amides as Hsp90 C-terminal inhibitors

Article abstract of DOI:10.1039/c4md00102h

Hsp90 C-terminal inhibitors are advantageous for the development of new cancer chemotherapeutics due to their ability to segregate client protein degradation from induction of the prosurvival heat shock response, which is a major detriment associated with

Full text of DOI:10.1039/c4md00102h

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CONCISE ARTICLE
Triazole containing novobiocin and biphenyl
amides as Hsp90 C-terminal inhibitors†
Cite this: Med. Chem. Commun., 2014,
5, 1317
Jinbo Zhao,a Huiping Zhao,a Jessica A. Hall,a Douglas Brown,d Eileen Brandes,b
Joseph Bazzill,c Patrick T. Grogan,be Chitra Subramanian,b George Vielhauer,d
a
Mark S. Cohenbce and Brian S. J. Blagg
*
Hsp90 C-terminal inhibitors are advantageous for the development of new cancer chemotherapeutics due
to their ability to segregate client protein degradation from induction of the prosurvival heat shock
response, which is a major detriment associated with Hsp90 N-terminal inhibitors under clinical
investigation. Based upon prior SAR trends, a 1,2,3-triazole side chain was placed in lieu of the aryl side
chain and attached to both the coumarin and biphenyl scaffold. Antiproliferative studies against SKBr3
and MCF-7 breast cancer cell lines demonstrated these triazole-containing compounds to exhibit
improved activity. These compounds were shown to manifest Hsp90 inhibitory activity through Western
blot analysis and represent a new scaffold upon which more potent inhibitors can be pursued.
Received 7th March 2014
Accepted 15th May 2014
DOI: 10.1039/c4md00102h
www.rsc.org/medchemcomm
proteins have been identied,8,9 many of which are directly
associated with the six hallmarks of cancer.10 Consequently,
Introduction
Hsp90 inhibition results in the simultaneous disruption of
multiple signaling pathways required for the viability of trans-
formed cells. 17-AAG is a semi-synthetic derivative of geldana-
mycin that targets the N-terminal ATP-binding site and exhibits
therapeutic benet at tolerable doses in the clinic. In addition
to 17-AAG, 16 other Hsp90 N-terminal inhibitors have been
investigated in the clinical setting, which highlights the
importance of Hsp90 as a cancer therapeutic target.11 Unfortu-
nately, these Hsp90 N-terminal inhibitors induce the pro-
survival heat shock response at the same concentration needed
to induce client protein degradation, resulting in primarily
cytostatic activity. Thus, the clinical effectiveness of Hsp90 N-
terminal inhibitors remains unclear.12 In contrast to N-terminal
inhibitors, studies have demonstrated that inhibitors of the
Hsp90 C-terminus13 and some allosteric modulators14,15 do not
induce the heat shock response, and consequently provide an
attractive paradigm for the development of Hsp90 inhibitors
that exhibit improved activities.16,17
Seminal work by Neckers and coworkers reported that the
DNA gyrase inhibitor, novobiocin (1), and related natural
products bind the Hsp90 C-terminus nucleotide binding pocket
with low affinity (IC50 ꢀ 700 mM).18 Subsequent modication to
novobiocin including alteration of coumarin scaffold, optimi-
zation of the benzamide side chain, and replacement of the
sugar moiety led to compounds that manifest increased inhib-
itory activity.19–24 Although no co-crystal structure of Hsp90
bound to C-terminal inhibitors has been solved, insights gained
from SAR studies on novobiocin has produced valuable infor-
mation on the Hsp90 C-terminal binding pocket. Among the key
elements on novobiocin identied, the secondary amide linker
Developments in molecular biology have led to identication of
new targets for the pursuit of cancer chemotherapies during
recent decades. The most successful demonstration thus far is
Gleevec, a small molecule BCR/ABL kinase inhibitor used for
the treatment of chronic myelogenous leukemia.1,2 Due to their
genetic instability and signaling redundancy, cancer cells
exhibit compensatory mechanisms and oen acquire resistance
to inhibition of a single protein.3 The most common approach
to overcome this obstacle relies upon combination therapy, in
which inhibitors of multiple oncogenic targets are adminis-
tered. In contrast, the development of anti-cancer agents that
target signaling nodes represent an alternative method to target
multiple signaling pathways simultaneously while minimizing
the potential for resistance.4,5 The 90 kDa heat shock protein
(Hsp90) represents
a paradigmatic target for the latter
approach, as molecular chaperones are responsible for the
conformational maturation of signaling proteins associated
with cancer cell growth and proliferation.6,7 In fact, recent
studies indicated that almost 400 Hsp90-depedent client
aDepartment of Medicinal Chemistry, The University of Kansas, 1251 Wescoe Hall
Drive, Malott 4070, Lawrence, Kansas 66045, USA. E-mail: bblagg@ku.edu; Fax: +1-
785-864-5326; Tel: +1-785-864-2288
bDepartments of Surgery, University of Michigan, Ann Arbor, MI, 48109, USA
cPharmaceutical Chemistry, University of Michigan, Ann Arbor, MI, 48109, USA
dDepartments of Urology, The University of Kansas Medical Center, 3901 Rainbow
Blvd., Mail Stop 1016, Kansas City, KS 66160, USA
eDepartments of Pharmacology, Toxicology and Therapeutics, The University of Kansas
Medical Center, 3901 Rainbow Blvd., Mail Stop 1016, Kansas City, KS 66160, USA
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4md00102h
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and an aryl side chain were shown to be particularly important replacement of the amide linker on novobiocin with a 1,2,3-
(Fig. 1A).9 Structural modication to these moieties has triazole resulted in decreased antiproliferative activity,25 indi-
produced analogues that exhibit improved activity, such as cating that the amide N–H may be acting as a hydrogen bond
compound 2, which contains a simplied coumarin core, a donor. Therefore, it was proposed to incorporate the 1,2,3-tri-
piperidine moiety, and a benzamide side chain (Fig. 1B). azole into the aryl side chain while retaining the secondary
Increasing evidence suggests that hydrogen bonding interac- amide linker. To test this hypothesis, compounds 4 and 5 were
tions on the side chain are also benecial,21 which is in accord designed to combine the features noted above, wherein the
with the improved activities observed for analogues possessing noviose sugar was replaced with a readily available piperidine
a 2-21 or 3-indole25 or a 4-methoxyphenyl23 side chain. Therefore, moiety (Fig. 1C).22 The results from these studies are presented
it was proposed that incorporation of additional hydrogen herein and describe the identication of compounds that
bonding interactions could further increase inhibitory activity. manifest mid-nanomolar activity, which is ꢀ1000 fold more
In addition, it was recently demonstrated that the coumarin efficacious than novobiocin.
core could be replaced with simplied surrogates,26 which is
imperative, due to the multi-step synthesis required for the
preparation of this ring system. Indeed, replacement of the
Results and discussion
coumarin ring (2) system with a biphenyl scaffold (3) resulted in
compounds that manifest increased antiproliferative activity
(Fig. 1B).27 Therefore, it was proposed that incorporation of a
hydrogen bonding ring system onto both the coumarin and
reaction between the corresponding azides and propiolic acid
biphenyl scaffold could provide compounds that exhibit
improved activity. Through exploratory methods, it was deter-
mined that the 1,2,3-triazole moiety represents an optimal side
propiolic acid and the corresponding azides.31 Aminocoumarin
chain surrogate for the benzamide side chain present in
novobiocin.
The copper catalyzed azido alkyne cycloaddition (CuAAC)
corresponding acid chlorides and coupled with aminocoumarin
reaction has found wide application to various elds of chem-
istry, especially biomedical and medicinal chemistry, due to the
Retrosynthetically, coumarin derivative 4 was envisioned for
assembly by an amide coupling reaction between aniline 6 and
triazolic acid 7, with the latter being accessed through a CuAAC
(Scheme 1). Synthesis of both the phenyl and benzyl derivatives
(4a and 4b) commenced by preparation of 7a and 7b from
6 was prepared via reported procedures.22 Upon treatment with
oxalyl chloride, triazolic acids 7a and 7b were converted to the
6 to provide 4a and 4b (Scheme 2).
The cellular activity manifested by 4a and 4b was evaluated
against a panel of cancer lines (see ESI† for details), including
mild conditions required for assembly and its broad functional
group tolerance.28,29 The product obtained from such reactions,
SKBr3 (estrogen receptor negative, Her2 over-expressing)
the 1,2,3-triazole, is an amide bioisostere that demonstrates
lymphatic metastatic MDA-MB-468LN (estrogen receptor nega-
enhanced hydrogen bonding interactions compared to the
tive, Ah receptor-positive) and MCF-7 (estrogen receptor posi-
amide. In addition, the triazole ring is present in various ther-
tive) breast cancer cell lines, head and neck squamous cell
apeutic classes due to its hydrogen bonding capabilities and
carcinoma (HNSCC) MDA1986 and JMAR cell lines, as well as
high dipole.29,30 Prior studies have shown that bioisosteric
prostate cancer cell lines PC3-MM2 and LNCaP. Both
Fig. 1 The design of novel Hsp90 C-terminal inhibitors.
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chloro analogues (4g) manifested similar potencies compared
to the unsubstituted analogue, 4b. However, the 4-NO2
substituent (4l) exhibited improved activity against all cancer
cell lines tested, manifesting an IC50 of 0.38 and 0.64 mM against
SKBr3 and MCF-7 cell lines, respectively. Sterically bulky groups
such as 4-tert-butyl (4k) were found favorable, although their
effects on the MCF-7 cell line were not clear. When a hydro-
phobic cyclohexylmethyl group was installed as the appendage
(4q), improved inhibitory activities against SKBr3, MDA1986
and JMAR cells were observed, indicating the existence of a
hydrophobic region in this area of the binding pocket. Although
current data is limited and warrants further investigation, the
observed inhibitory activities manifested by these triazole-con-
taining analogues indicate that hydrogen bonding interactions
on the side chain are favorable for Hsp90 C-terminal inhibition,
which might result from either direct hydrogen bonding inter-
actions or conformational rigidity on the side chain which
might direct hydrophobic substituent into the hydrophobic
pocket.
Increasing exibility between the triazole side chain and the
terminal phenyl ring was sought to determine whether
improved inhibitory activity could be achieved. Therefore,
compounds bearing two (4r) or three carbons (4s) were
synthesized by an amide coupling reaction between the corre-
sponding triazolylic acids (7r and 7s) and amine 6 (Scheme 2).
The antiproliferative activities manifested by these compounds
were then determined. A comparison of the data shown for 4a,b
and 4r,s (Table 1) illustrates that compound 4r, which contains
a two carbon linker, exhibited the most improved activity
amongst the four analogues evaluated, exhibiting IC50s of
Scheme 1 Retrosynthetic analysis of the triazole 4.
compounds manifested activity at low micromolar concentra-
tions against all cell lines tested. Encouraged by these prelim-
inary studies, substitutions on the phenyl and benzyl side
chains were explored to determine structure–activity relation-
ships for this series of compounds. Compounds 4c–q were
synthesized efficiently in a manner similar to 4a and 4b, from
the substituted triazolic acids 7c–q and aniline 6 (Scheme 2). It
was determined that both 4-chloro (4c) and 3-chloro (4f)
substitutions improved inhibitory activity against SKBr3 cell
lines. Due to comparable activities manifested by both the
phenyl and benzyl analogues, studies were directed towards
modication of the benzyl ring system. Electron-donating
groups such as 4-methyl (4i) and 4-methoxy (4j) as well as 4-
Scheme 2 Synthesis of triazole containing coumarin derivatives.
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Table 1 Antiproliferative activity of coumarin-based triazole derivatives 4a
R (4, IC50, mM)
SKBr3b
MCF-7b
MDA-MB-468LNc
MDA1986c
JMARc
PC3-MM2d
LNCaPd
Ph (4a)
Bn (4b)
0.94 Æ 0.02a
0.99 Æ 0.08
0.58 Æ 0.03
1.38 Æ 0.05
1.08 Æ 0.05
0.71 Æ 0.14
1.14 Æ 0.19
0.73 Æ 0.01
1.21 Æ 0.07
1.31 Æ 0.20
0.45 Æ 0.03
0.38 Æ 0.13
1.08 Æ 0.01
1.06 Æ 0.01
1.15 Æ 0.19
1.30 Æ 0.07
0.61 Æ 0.00
0.13 Æ 0.01
1.73 Æ 0.05
1.33 Æ 0.13a
1.08 Æ 0.00
2.04 Æ 0.25
5.07 Æ 1.86
1.38 Æ 0.07
1.14 Æ 0.18
1.44 Æ 0.31
1.36 Æ 0.08
1.50 Æ 0.19
1.42 Æ 0.14
1.22 Æ 0.07
0.64 Æ 0.01
1.34 Æ 0.19
1.68 Æ 0.01
1.61 Æ 0.06
0.99 Æ 0.08
1.29 Æ 0.16
0.55 Æ 0.01
1.65 Æ 0.03
1.10 Æ 0.21
2.40
0.81 Æ 0.15
2.80 Æ 0.50
4.00 Æ 1.92
4.60
2.20 Æ 0.30
2.10 Æ 0.60
2.40 Æ 0.25
3.40 Æ 1.27
0.74 Æ 0.22
0.81 Æ 0.23
2.60 Æ 6.12
1.14 Æ 1.17
2.69 Æ 1.33
2.10 Æ 0.52
0.99 Æ 0.24
0.99 Æ 0.10
NT
1.50 Æ 3.40
1.60 Æ 0.21
0.18 Æ 0.11
2.90 Æ 0.33
0.29
2.30 Æ 0.40
2.10 Æ 0.23
0.43 Æ 0.38
3.60 Æ 0.63
3.64
2.69
4.72 Æ 0.71
15.5 Æ 8.75
2.45 Æ 0.47
15.2 Æ 9.19
>100
13.3 Æ 11.9
4.65 Æ 0.71
4.96 Æ 0.67
7.24 Æ 1.02
>100
1.84 Æ 0.00
1.28 Æ 0.71
3.31 Æ 0.00
3.03 Æ 0.05
3.49 Æ 0.30
>100
1.20 Æ 0.06
2.36 Æ 0.53
NT
9.8 Æ 13.5
2.79 Æ 0.49
3.81 Æ 4.99
>100
4-ClC6H5 (4c)
4-BrC6H5 (4d)
4-MeC6H5 (4e)
3-ClC6H5 (4f)
4-ClBn (4g)
4-BrBn (4h)
4-MeBn (4i)
4-MeOBn (4j)
4-t-BuBn (4k)
4-NO2Bn (4l)
4-FBn (4m)
3-ClBn (4n)
3-MeOBn (4o)
2-ClBn (4p)
CH2C6H13 (4q)
Ph(CH2)2 (4r)
Ph(CH2)3 (4s)
6.40
6.40
3.90 Æ 5.11
3.67 Æ 1.38
3.80 Æ 1.42
7.32 Æ 5.44
7.85 Æ 0.42
2.37 Æ 0.00
2.49 Æ 0.32
8.82 Æ 1.14
6.00 Æ 1.13
7.20 Æ 0.15
6.66 Æ 5.44
3.13 Æ 0.14
1.40 Æ 0.04
NT
1.70 Æ 0.52
2.00 Æ 0.50
1.50 Æ 0.20
2.60 Æ 0.30
0.34 Æ 0.09
0.18 Æ 0.22
1.77 Æ 4.10
0.23 Æ 0.22
1.34 Æ 0.31
3.20 Æ 1.37
0.24 Æ 0.32
0.11 Æ 0.30
NT
1.70 Æ 0.20
1.60 Æ 0.31
1.80 Æ 0.95
6.70
0.58 Æ 0.14
0.26 Æ 0.34
2.38 Æ 5.38
0.19 Æ 0.10
1.49 Æ 0.31
3.70 Æ 0.85
0.30 Æ 0.26
0.74 Æ 0.86
NT
a
b
Values represent mean Æ standard deviation for at least two separate experiments performed in triplicate. Cellular activities were determined
c
d
with MTS/PMS cell viability assay. Cellular activities were determined with Promega CellTiter-Glo (CTG) luminescent assay. Cellular activities
were determined with sulforhodamine B assay.
130 nM against SKBr3 cell line and about 110 nM against 7a,b, 7g–q, 7r and 7t were converted to their acid chlorides upon
MDA1986 cell line.
treatment with oxalyl chloride, followed by coupling with
To conrm that the observed antiproliferative activities were aniline 8 to generate the biphenyl containing derivatives 5a–o
resulting from Hsp90 inhibition, western blot analyses of Hsp90 (Scheme 3).
client proteins in MCF-7 cell lysates were performed. Actin,
The antiproliferation activities manifested by these
whose maturation does not require the Hsp90 machinery, was compounds are reported in Table 2. In contrast to the
used as a control. As shown in Fig. 2, Hsp90 client proteins coumarin-based analogues 4a and 4b, which showed similar
Her2, Akt and Raf-1 were degraded upon exposure of 4f, 4o or 4q anti-proliferative activity, the phenyl and benzyl analogues 5a
at concentrations that mirror their anti-proliferative values, and 5b displayed very different activities, with the latter being
conrming that cell viability is directly linked to Hsp90 about 2.5–8 times more potent against all the cell lines evalu-
inhibition.
ated, manifesting IC50s of 170 and 500 nM against SKBr3 and
Attention was then shied to determine whether the anti- MCF-7 cell lines, respectively. A compound that contains the
proliferative activities of these derivatives were similar to those two carbon linker (5c) was found to exhibit activity similar to
of the recently identied biphenyl ring system. Biphenyl aniline those of the benzyl analogue, 5b. Subsequent studies were then
8 was synthesized according to a previously reported proce- aimed to modify the benzylic ring. As can be seen in Table 2,
dure.25 Similar to the coumarin derivatives, triazolylic acids halogens at the 4-position were in general detrimental to
inhibitory activity (5d,e); electron-donating groups such as 4-
methyl (5f) and 4-methoxy (5g) retained potencies against SKBr3
and MDA-MB-468LN cells, but manifested increased inhibitory
activities against the MCF-7 cell line (120 and 270 nM, respec-
tively) and prostate cancer cell lines (360 and 480 nM, respec-
tively). 3-Chloro (5k), 3-methoxy (5l), and 2-chloro (5m)
produced decreased activity against SKBr3 cells, but increased
activities against MCF-7 and MDA-MB-468LN cells. Combina-
tion of 2-chloro and 4-methyl substitution (5n) retained activity
against SKBr3 cells, but did not improve activity against MCF-7
cells compared to the 4-methyl substituted analogue, 5f.
However, this combination indeed improved activities against
MDA-MB-468LN, MDA1986 and LNCaP cell lines. Of particular
note, the 4-tert-butyl substituted analogue, 5h, manifested
decreased antiproliferative activity against all cell lines tested
Fig. 2 Western blot analyses of the Hsp90 client protein degradation
in MCF-7 breast cancer cells lysis after treatment of triazole analogues.
L represents a concentration 1/2 of the anti-proliferative IC50 value,
while H represents a concentration 5 times the antiproliferative IC50
value. Geldanamycin (G, 500 nM) represents a positive control, while compared to the 4-methyl analogue, 5f, which is in contrast to
DMSO (D), vehicle, serves as the negative control.
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Scheme 3 Synthesis of biphenyl triazole derivatives 5.
Table 2 Antiproliferative activity of biphenyl triazole analogues 5a
R (5, IC50, mM)
SKBr3b
MCF-7b
MDA-MB-468LNc
MDA1986c
JMARc
PC3-MM2d
LNCaPd
Ph (5a)
Bn (5b)
1.14 Æ 0.10a
0.17 Æ 0.02
0.19 Æ 0.02
0.32 Æ 0.10
0.49 Æ 0.03
0.17 Æ 0.03
0.16 Æ 0.02
3.72 Æ 0.68
0.45 Æ 0.04
0.72 Æ 0.18
0.39 Æ 0.01
0.32 Æ 0.02
0.34 Æ 0.04
0.16 Æ 0.01
0.20 Æ 0.02
1.44 Æ 0.18a
0.50 Æ 0.02
0.38 Æ 0.10
0.44 Æ 0.04
0.56 Æ 0.04
0.12 Æ 0.01
0.27 Æ 0.06
10.48 Æ 0.40
0.55 Æ 0.01
1.04 Æ 0.08
0.34 Æ 0.02
0.16 Æ 0.02
0.38 Æ 0.03
0.20 Æ 0.05
0.31 Æ 0.04
0.83 Æ 0.16
0.34 Æ 0.07
0.36 Æ 0.04
0.74 Æ 0.19
2.80 Æ 0.61
0.41 Æ 0.26
0.17
2.61 Æ 0.76
0.56 Æ 0.25
1.09 Æ 0.23
0.17 Æ 0.02
0.19 Æ 0.05
0.22 Æ 0.03
0.15 Æ 0.02
0.24 Æ 0.06
1.30 Æ 0.09
0.28 Æ 0.10
0.10 Æ 0.23
1.30 Æ 0.37
3.10 Æ 0.87
0.36 Æ 0.05
0.48 Æ 0.09
2.34 Æ 1.81
0.19 Æ 0.17
0.93 Æ 0.34
0.17 Æ 0.12
0.28 Æ 0.09
0.81 Æ 0.05
0.15 Æ 0.06
0.38 Æ 0.14
2.20 Æ 0.24
0.50 Æ 0.06
0.58 Æ 0.46
2.30 Æ 0.82
7.80
0.42 Æ 0.07
0.61 Æ 0.09
3.49 Æ 2.05
0.21 Æ 0.35
1.39 Æ 0.52
0.27 Æ 0.04
0.21 Æ 0.12
0.65
4.26 Æ 5.59
0.55 Æ 0.46
0.83 Æ 0.34
0.66 Æ 0.21
19.9 Æ 27.9
0.36 Æ 0.04
0.44 Æ 0.19
>100
1.03 Æ 0.16
2.81 Æ 1.82
0.35 Æ 0.14
0.55 Æ 0.37
1.04 Æ 0.51
0.35 Æ 0.12
5.12 Æ 6.54
3.89 Æ 1.09
0.65 Æ 0.54
0.43 Æ 0.22
1.59 Æ 0.78
0.54 Æ 0.12
0.20 Æ 0.04
0.24 Æ 0.07
1.01 Æ 1.39
0.60 Æ 0.34
1.64 Æ 0.97
0.12 Æ 0.10
0.16
CH2Bn (5c)
4-ClBn (5d)
4-BrBn (5e)
4-MeBn (5f)
4-MeOBn (5g)
4-t-BuBn (5h)
4-NO2 (5i)
4-FBn (5j)
3-ClBn (5k)
3-MeOBn (5l)
2-ClBn (5m)
2-Cl,4-MeBn (5n)
CH2C6H13 (5o)
0.61 Æ 0.34
0.05 Æ 0.04
0.48 Æ 0.68
0.43 Æ 0.25
0.26 Æ 0.45
a
b
Values represent mean Æ standard deviation for at least two separate experiments performed in triplicate. Cellular activities were determined
c
d
with MTS/PMS cell viability assay. Cellular activities were determined with Promega CellTiter-Glo (CTG) luminescent assay. Cellular activities
were determined with sulforhodamine B assay.
the coumarin derivatives 4i and 4k, indicating a smaller also degraded in a concentration-dependent manner upon
hydrophobic pocket exists when the biaryl scaffold is present. exposure to the most potent analogue 5f and the representative
Similar to the coumarin scaffold, replacement of the benzyl analogue 5b, against MCF-7 cells (Fig. 3B and C), while actin
group with
a cyclohexylmethyl substituent resulted in levels remain unchanged. Examination of Hsp90 expression
compounds that manifested good inhibitory activities. Differ- when exposed to these compounds have shown that while GDA,
ences in the structure–activity relationships for the two scaf- the positive control, induced Hsp90 upregulation due to
folds suggest the biphenyl ring system presents the side chain induced heat shock response, Hsp90 levels were constant or
through different binding interactions. In general, installation even decreased upon treatment of these compounds (Fig. 3),
of triazole side chain on the recently identied biphenyl side consistent with the observations manifested by many Hsp90 C-
chain greatly improves antiproliferative activity against a wide terminal inhibitors,20,22,24,27,32 indicating that these compounds
spectrum of cell lines, resulting in more potent analogues.
exhibit Hsp90 inhibitory activity through C-terminal inhibition.
The cellular activity manifested by these biphenyl analogues It is worthwhile to note that at a concentration as low as 1 mM, 5f
was shown to result from Hsp90 inhibition by performing was able to deplete Hsp90 client proteins as well as Geldana-
western blot analyses of MCF-7 cell lysates treated with such mycin at 500 nM, demonstrating the remarkable activity
compounds. Hsp90-dependent client proteins were decreased exhibited by this compound for Hsp90 inhibition.
upon exposure to of 5b, 5f and 5g at concentrations that mirror
Furthermore, no increase in heat shock proteins Hsp27,
their antiproliferative IC50s. Clear degradation of Hsp90 client Hsp70, or Hsp90 was observed with increasing concentrations
proteins Her2, Akt and Raf-1 was observed, while actin, which of 5f (Fig. 4A). Depletion of client proteins without increased
does not rely upon the Hsp90 chaperone machinery, remained levels of heat shock proteins is a hallmark of Hsp90 C-terminal
constant, indicating that the antiproliferative activities man- inhibition. To determine whether 5f binds the Hsp90 C-
ifested by these compounds resulted from Hsp90 inhibition terminus, proteolytic ngerprinting of Hsp90 from TnT rabbit
(Fig. 3A). These client proteins Her2, Akt, Raf-1 and CDK6 were reticulocyte in the presence of 5f was performed. Novobiocin
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Fig. 3 Western blot analyses of the Hsp90 client protein degradation in MCF-7 breast cancer cells lysis 24 h after treatment of biphenyl triazole
analogues 5b, 5f and 5g. (A) L represents a concentration 1/2 of the anti-proliferative IC50 value, while H represents a concentration 5 times the
antiproliferative IC50 value. (B) Concentrations (in mM) of 5f are indicated above each lane. (C) Concentrations of 5b (in mM) are indicated above
each lane. Geldanamycin (G, 500 nM) and DMSO (D) were employed respectively as positive and negative controls.
molecular weight from vehicle control. The C-terminal Hsp90
antibody AC88 detects the emergence of a 50 kDa band in the
presence of novobiocin and other C-terminal inhibitors. A 50
kDa band was detected with 5 mM novobiocin and 1 mM 5f that
is not detected for the vehicle control (Fig. 4B). Together, these
data further support that 5f binds and inhibits the Hsp90 C-
terminus, which leads to client protein degradation without
induction of the heat shock response.
A brief summary of observed structure–activity relationships
for these derivatives is depicted in Fig. 5. In support of the
existence a hydrophobic pocket, incorporation of either an
aromatic or saturated carbocycle was found favorable to
inhibitory activity. Two carbons were found optimal for the
coumarin derivatives, compared to one carbon for the biphenyl
derivatives; and suggests that projection of the side chain is
different for the biaryl scaffold. This observation is further
supported by the tolerance of bulky groups exhibited at the para
position of benzylic moiety (4k), but not for the biphenyl
derivative, 5h. These observations highlight the signicance of
Fig. 4 (A) Western blot analyses of the heat shock proteins Hsp27,
Hsp70 and Hsp90 in MCF-7 breast cancer cells lysates 24 h after
treatment with 5f. Concentrations (in mM) of 5f are indicated above
each lane. Geldanamycin (G, 500 nM) and DMSO (D) are positive and
negative controls. (B) Proteolysis of Hsp90 from TnT reticulocyte
lysate incubated under conditions of protein synthesis with vehicle (1%
DMSO) 5 mM novobiocin and 1 mM 5f. An antibody specific to the C-
terminus of Hsp90 was used to identify the Hsp90 fragments
produced in the presence of increasing amounts of trypsin.
locks Hsp90 in the “open conformation” when bound to the C-
terminus.33,34 In this conformation, amino acids Lys615 and
Arg620 are not solvent exposed and are “protected” from
Fig. 5 A summary of SARs for triazole containing coumarin and
cleavage by trypsin. This results in bands that differ in
biphenyl derivatives.
1322 | Med. Chem. Commun., 2014, 5, 1317–1323
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both hydrogen bonding and hydrophobic interactions at this 13 J. D. Eskew, T. Sadikot, P. Morales, A. Duren, I. Dunwiddie,
location of the Hsp90 C-terminal binding pocket.
M. Swink, X. Zhang, S. Hembruff, A. Donnelly,
R. A. Rajewski, B. S. J. Blagg, J. R. Manjarrez, R. L. Matts,
J. M. Holzbeierlein and G. A. Vielhauer, BMC Cancer, 2011,
11, 468.
Conclusion
In conclusion, a series of 1,2,3-triazole side chain containing
analogues that contain the coumarin or biphenyl backbone
were designed, synthesized and evaluated for antiproliferative
activities against a panel of breast, HNSCC, and prostate cancer
cell lines. Compounds manifesting nanomolar activity were
identied through preliminary SAR studies and their Hsp90
inhibitory activity conrmed by Western blot analysis. The
improved Hsp90 inhibitory activities exhibited by these
compounds might result from increased hydrogen bonding
properties on the amide side chain when connected to a
hydrophobic appendage, or from the conformational rigidity
due to the incorporation of 1,2,3-triazole which directs the
hydrophobic appendage into the hydrophobic pocket. These
observations are expected to aid in the development of more
potent Hsp90 C-terminal inhibitors, and such studies will be
reported in due course.
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Acknowledgements
We gratefully acknowledge nancial support of this project by
the NIH/NCI (CA120458 and CA167079) and University of
Michigan Comprehensive Cancer Center CCSG Development
Grant (MSC).
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