Synthesis and Biological Evaluation of Novobiocin Core Analogues as Hsp90 Inhibitors

Article abstract of DOI:10.1002/chem.201504955

Development of heat shock protein 90 (Hsp90) C-terminal inhibitors has emerged as an exciting strategy for the treatment of cancer. Previous efforts have focused on modifications to the natural products novobiocin and coumermycin. Moreover, variations in both the sugar and amide moieties have been extensively studied, whereas replacements for the coumarin core have received less attention. Herein, 24 cores were synthesized with varying distances and angles between the sugar and amide moieties. Compounds that exhibited good anti-proliferative activity against multiple cancer cell lines and Hsp90 inhibitory activity, were those that placed the sugar and amide moieties between 7.7 and 12.1 ? apart along with angles of 180°.

Full text of DOI:10.1002/chem.201504955

DOI: 10.1002/chem.201504955
Full Paper
&
Medicinal Chemistry |Hot Paper|
Synthesis and Biological Evaluation of Novobiocin Core Analogues
as Hsp90 Inhibitors
[
a]
[b]
[b]
[b]
Katherine M. Byrd, Chitra Subramanian, Jacqueline Sanchez, Hashim F. Motiwala,
[c]
[b]
[c]
[a]
Weiya Liu, Mark S. Cohen, Jeffrey Holzbeierlein, and Brian S. J. Blagg*
Abstract: Development of heat shock protein 90 (Hsp90) C-
terminal inhibitors has emerged as an exciting strategy for
the treatment of cancer. Previous efforts have focused on
modifications to the natural products novobiocin and cou-
mermycin. Moreover, variations in both the sugar and amide
moieties have been extensively studied, whereas replace-
ments for the coumarin core have received less attention.
Herein, 24 cores were synthesized with varying distances
and angles between the sugar and amide moieties. Com-
pounds that exhibited good anti-proliferative activity against
multiple cancer cell lines and Hsp90 inhibitory activity, were
those that placed the sugar and amide moieties between
7.7 and 12.1 Š apart along with angles of 1808.
Introduction
fact, Hsp90 C-terminal inhibitors have proven to exhibit dis-
[
13,14]
tinct properties when compared to N-terminal inhibitors.
Heat shock protein 90 (Hsp90) is a molecular chaperone that is
responsible for folding nascent polypeptides into their biologi-
cally active conformation. More than 200 client proteins
depend upon Hsp90 for their maturation/activation, and many
Therefore, the development of Hsp90 C-terminal inhibitors
could provide a clinically useful alternative for the treatment of
[
15–19]
cancer.
Studies have shown that natural product C-terminal inhibi-
[1–4]
[20–24]
are essential to cancer cell survival and proliferation.
Unlike
tors,
such as novobiocin and coumermycin A1, do not
induce the heat shock response. Unfortunately, these com-
pounds manifest IC values of 700 and 70 mm, respective-
drugs that target these individual client proteins, inhibition of
Hsp90 results in the degradation of more than 30 anticancer
targets simultaneously. Furthermore, Hsp90 is overexpressed
and can be selectively targeted in cancer, thus its inhibition
50
[
25,26]
ly.
Although no crystal structure of the Hsp90 C terminus
bound to inhibitors exists, homology models have been devel-
[5–7]
[27–29]
can diminish potential side effects.
oped to aid the design of new inhibitors.
Using these
Seventeen Hsp90 inhibitors have entered clinical trials for
the treatment of cancer. All of these inhibitors bind the N-ter-
models, structure–activity-relationship (SAR) studies on novo-
biocin and coumermycin have led to compounds that manifest
activity in the mid to low nanomolar range against a variety of
[8–10]
minal ATP binding site.
The major problem associated with
[
30]
these inhibitors is induction of the heat shock response (pro-
survival) at the same concentration that leads to protein degra-
dation. Due to increased Hsp90 levels observed upon adminis-
tration of N-terminal inhibitors, the scheduling and dosing of
cancers,
which supports the potential use of these com-
pounds as anticancer therapeutics.
To date, SAR studies on novobiocin have primarily focused
on finding replacements for the noviose sugar and the prenyl-
ated aryl side chain (Figure 1). In fact, more potent Hsp90 in-
hibitors can be accessed by replacement of the noviose sugar
with N-methylpiperidine (2) or by substitution of the prenylat-
[11]
patients is often difficult. Presently, no Hsp90 inhibitor is
FDA-approved for the treatment for cancer.
In contrast to N-terminal inhibitors, C-terminal inhibitors can
be prepared that do not induce the pro-survival heat shock re-
[
31]
ed aryl side chain with a biaryl side chain (3). Both of these
modifications not only lead to more efficacious analogues, but
also a simplified synthesis, as the noviose sugar requires more
than 10 synthetic transformations. In addition to the coumarin
core, biaryl cores (4 and 5) have been discovered that manifest
[
12]
sponse that is responsible for these dosing difficulties. In
[
a] K. M. Byrd, Prof. B. S. J. Blagg
Department of Medicinal Chemistry, The University of Kansas
Wescoe Hall Drive, Malott 4070, Lawrence, KS 66045-7563 (USA)
E-mail: bblagg@ku.edu
[
32]
superior anticancer activity.
Since compounds containing
a biaryl core exhibit improved inhibitory activity over the cou-
marin-based compounds, it suggests that some flexibility
within the C-terminal binding pocket may exist. Additionally,
orientation of the N-methylpiperidine and the biaryl side chain
may also be important for the increased efficacy. In fact, com-
pounds prepared to date project the sugar and amide side
chains linearly at approximately 1808. Therefore, the focus of
[
b] C. Subramanian, J. Sanchez, H. F. Motiwala, Prof. M. S. Cohen
Department of Surgery, University of Michigan, Ann Arbor, MI 48109 (USA)
[
c] W. Liu, Prof. J. Holzbeierlein
Department of Urology, The University of Kansas Medical Center
3
901 Rainbow Boulevard,Stop 3016, Kansas City, Kansas 66160 (USA)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201504955.
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Figure 1. Novobiocin and analogues previously evaluated.
this study was to determine the optimal distance and angle
between the N-methylpiperidine and the biaryl side chain.
Results and Discussion
Design and synthesis of novel scaffolds
In order to determine the optimal distance and angle between
the N-methylpiperidine and the biaryl side chain, cores were
designed to place these groups between 6.2 and 14.3 Š apart
as presented in Table 1.
From a design perspective, initial efforts were focused on
modification of the bicyclic core represented by the coumarin
ring in novobiocin. First, the biaryl side chain was placed at
the 1-position of both the tetrahydronaphthalene and naph-
thalene cores (6 and 7). Second, the biaryl side chain was
placed at the 2-position of the tetrahydronaphthalene core.
Compounds 6–8 would provide insight into both the position
and planarity of the biaryl side chain on the bicyclic ring
system.
Figure 2. Analogues with phenyl-naphthalene core.
ties between these compounds would help establish the role
played by the core.
Compounds 21 and 22 contain 4-ring analogues that were
designed for this investigation. Additionally, compound 23 rep-
resents a derivative of 5, where an alkyne was placed between
the phenyl rings to increase distance between the N-methylpi-
peridine and the biaryl side chain by two carbons, while main-
taining linearity. The alkyne in 23 could be reduced to both
the alkene (24 and 25) and alkane (26) to provide insights into
the flexibility and geometry of the two-carbon linker. Finally,
an additional series of alkyne-containing compounds was envi-
sioned (27–29) that extend from an aromatic core.
Various angles between the N-methylpiperidine and the
biaryl side chain were accessed by placement of these groups
at different positions within the same core structure. Com-
pounds 9, 11 and 13 are representative examples wherein this
strategy was utilized. Alternatively, different angles were ob-
served when the biaryl side chain was placed out of plane
with regards to the core (Figure 3).
Next, a series of compounds with a three-ring core was de-
signed. The first set of compounds contained a phenylnaphtha-
lene core, wherein the biaryl side chain or the N-methylpiperi-
dine was placed at all three positions on the benzene ring
(
Figure 2). A variety of distances and angles could be quickly
accessed with this scaffold, as advanced intermediates could
be used for the preparation of additional compounds.
The second set of compounds investigated were the 1-
phenyl-di(tetra)hydronaphthalene analogues (15–18). Initially,
the 1-phenyl-dihydronaphthalene scaffold was designed to
compare placement of the phenyl ring on the 1- versus 2-posi-
tion of the naphthalene ring. Later, 1-phenyl-tetrahydronaph-
thalene analogues could also be accessed by use of an inter-
mediate. The 1-phenyl-tetrahydronaphthalene scaffold would
provide data on planarity of the three-ring system and its
effect on anti-proliferative activity. The third set of compounds
contained two fused three-ring cores (19 and 20). Unlike the
previous sets of compounds, where one of the rings was able
to rotate freely, the fused ring system provides ridged counter-
parts for investigation. A comparison of anti-proliferative activi-
Synthesis of select analogues
Scheme 1 illustrates the synthetic protocol used to prepare 1-
amido and 2-amido analogues. Amines 30a/b, which were syn-
thesized from the corresponding 6-methoxy-tetralones (see
the Supporting Information), were subjected to EDCI coupling
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Table 1. List of cores with varying distance and angles.
Table 1. (Continued)
[
a,b]
[b]
[a,b]
[b]
Cmpd
Structure
Dist. [Š]
6.2
Angle [8]
Cmpd
Structure
Dist. [Š]
Angle [8]
6
73.4
1
7
8
9.2
108.3
104.3
7
6.3
120
1
9.8
8
9
7.8
7.7
160.9
115
19
9.1
9.4
124.6
154.5
2
2
0
1
1
0
7.9
115
9.2
168.3
180
11
10.4
139.3
2
2
2
3
14.1
1
2
10.5
141.6
12.1
180
1
1
3
4
11.8
11.8
168.9
172.1
2
2
4
5
9.1
120
11.8
170.7
1
1
5
6
7.8
9.1
79.4
2
2
6
7
11.9
8.7
166.6
163
118.8
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of commercially available 2-amino-6-bromonaphthalene (33)
with C to form amide 34 in good yield. Amide 34 was the
common intermediate used to prepare analogues wherein the
N-methylpiperidine was placed at all three positions on the
phenyl substituent. A Suzuki coupling between amide 34 and
boronic acids 35a–c produced the phenylnaphthalenes, 36a–
c, in moderate yield. Finally phenols 36a–c were coupled with
B through a Mitsunobu reaction to generate the final products
(10, 12, 14) in 53–60% yield.
Table 1. (Continued)
[
a,b]
[b]
Cmpd
Structure
Dist. [Š]
10.9
Angle [8]
2
8
9
172.2
2
14.3
160.5
[
a] Distance measured between the oxygen bound to N-methylpiperidine
and amide nitrogen of the biaryl side chain. [b] Both distance and angles
were measured in PyMOL after the 3D structures had been minimized
through molecular dynamic simulations.
Scheme 2. Synthesis of 2-phenyl-naphthalene analogue.
Figure 3. Graphic representation of the biaryl side chain that rests either in
or out-of-plane.
Synthesis of 1-phenyl-di(tetra)hydronaphthalene analogues
commenced by a Suzuki coupling between commercially avail-
[
34]
able boronic acids, 38a/b, and vinyl triflate 37 to yield the
corresponding nitro aromatics, 39a/b (Scheme 3). Following
demethylation of 39a/b, N-methyl-4-piperidinol was coupled
with phenols 40a/b under Mitsunobu conditions. At this point,
the synthesis diverged and the nitro group in 41a/b selectively
reduced to the corresponding aniline using iron-catalyzed con-
ditions (42a/b); or both the nitro group and the alkene were
reduced via a palladium-catalyzed hydrogenation (43a/b). The
resulting anilines were then coupled with acid chloride C to
form amides 15/18 or 16/17, respectively.
Preparation of the dibenzo[b,d]furan cores is shown in
[
35]
Scheme 4.
Synthesis of the dibenzofuran-containing ana-
logues began by a Suzuki coupling between boronic acid 44
and aryl bromides 45a/b to yield 46a/b in moderate yield. Pyr-
idine-HCl was used to cleave the methyl ethers of 46a/b and
provide resorcinols 47a/b. An intramolecular nucleophilic sub-
stitution of the resorcinol moiety (47a/b) generated dibenzo-
furanols 48a/b in good yields. N-Methyl-4-piperidinol was
added to 48a/b through a Mitsunobu reaction, which pro-
duced 49a/b. Following reduction of the nitro group present
in 49a/b, the resulting aniline was coupled with acid chloride
C to furnish 19 and 20.
Scheme 1. Synthesis of 1 and 2-amido novobiocin analogues.
with carboxylic acid A. After removal of the benzyl group by
hydrogenolysis, phenol 32a/b was coupled with N-methyl-4-pi-
[33]
peridinol (B) through Mitsunobu etherification.
The synthesis of an alkyne-containing compound is outlined
in Scheme 5. Commercially available 4-ethynylanisole (51) was
coupled with 1-bromo-4-nitrobezene (52) under Sonogashira
A representative synthesis of the phenylnaphthalene ana-
logues is shown in Scheme 2. Synthesis began by the coupling
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Scheme 3. Synthesis of 1-phenyl-di(tetra)hydronaphthalene analogues.
Scheme 5. Synthesis of 1,2-diphenyl-ethyne analogue.
Scheme 4. Synthesis of dibenzo[b,d]furan analogues.
Evaluation of anti-proliferative activity of analogues
conditions to yield alkyne 53 in 82% yield. Following cleavage
of the methyl ether present in 53, phenol 54 was treated with
N-methyl-4-piperidinol under Mitsunobu conditions to furnish
Upon preparation of the compounds listed Table 1, the anti-
proliferative activity manifested by these compounds was eval-
uated against MCF-7 (estrogen receptor positive breast cancer
cells), SKBr3 (estrogen receptor negative, HER2 overexpressing
breast cancer cells), PC3-MM2 (androgen-independent prostate
cancer cells) and MDA-MB-468Ln (human breast cancer triple
5
5 in moderate yield. The nitro group on 55 was subsequently
[
36]
reduced, and the resulting aniline was coupled with C to
produce the final product, 23.
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negative) cell lines (Table 2). In the bicyclic core series, the po-
sition of the amide was important for anti-proliferative activity.
When the amide was attached at the 1-position, no anti-prolif-
erative activity observed for the aromatic core (in-plane; 7),
whereas moderate activity was observed when the amide was
placed out-of-plane (6). In contrast, when the amide was
placed at the 2-position, an increase in anti-proliferative activi-
ty was observed when the amide was in-plane (57) versus out-
of-plane (8).
The anti-proliferative activity manifested by the 2-phenyl-
napthalene cores were slightly worse than the 1-phenyl-di(tet-
ra)hydronaphthalene cores and better than the dibenzo[b,d]-
furan cores in the three-ring series. Compounds containing the
amide side chain on the phenyl ring (9, 11 and 13) were more
active than compounds containing the N-methylpiperidine on
the phenyl ring (compounds 10, 12 and 14), with the para-
substituted amide analogue being most efficacious for the 2-
phenyl-naphthalene series (13). When the amide was attached
to the para-position of the 1-phenyl-di(tetra)hydronaphthalene
core, a marginal difference in activity for the di- versus tetrahy-
dronaphthalene core was observed (17 and 18). On the other
hand, when the amide was placed at the ortho-position, an in-
crease in activity against MDA-MB-468Ln and PC3-MM2 cells
was observed (15 and 16). Finally, there was an increase in ac-
tivity when the N-methylpiperidine was located at the 6-posi-
tion (19) versus the 7-position (20) of the dibenzo[b,d]furan
core. The trends observed for these three analogues indicate
that compounds are less potent when the rings are fused,
versus compounds that can rotate freely.
The incorporation of alkynes into the structure did not pro-
duce more potent compounds than their counterparts. For ex-
ample, 5 exhibits mid-nanomolar activity against all of the cell
lines, whereas 23 (which contains an alkyne between the two
phenyl rings) manifested a sixfold decrease in activity. A similar
trend was also observed between 13 and 29. Interestingly,
when the alkyne of 23 was reduced to the E-alkene (25), an in-
crease in activity was observed. In contrast, the Z-alkene (24)
exhibited a slight decrease in activity when compared to 23.
However, when 23 was fully saturated, a threefold decrease in
activity was observed against the MCF-7 cell line, whereas
a twofold increase in activity was observed against SKBr3 cells.
These observations highlight the importance of identifying the
optimal distance and angle between the N-methylpiperidine
and the biaryl side chain.
Figure 4. 3D graphs for the anti-proliferative activity versus distance versus
angles for MCF-7, SKBr3 and PC3-MM2 cell lines.
N-methylpiperidine and the biaryl side chain at a 1808 angle
and between 7.7 to 9.6 Š apart from one another.
Based on the data outlined in Table 2, three-dimensional
[
37]
graphs were generated to compare the anti-proliferative ac-
tivity versus the angle and distance between the N-methylpi-
peridine and the biaryl side chain for MCF-7, SKBr3 and PC3-
MM2 cell lines (Figure 4). As the angle approaches 1808, the
anti-proliferative activity increased in all cell lines. The optimal
distance for good anti-proliferative activity (<2 mm) was vari-
able in these cell lines. In the MCF-7 cell line, the optimal dis-
tance for activity was 7.5 to 10.5 Š, although a small pocket
was located from 11.5 to 12 Š. In the SKBr3 cell line, the opti-
mal distance was 7.5 to 14 Š. In the PC3-MM2 cell line, the op-
timal distance for good activity was 7.5 to 10 Š. In general, the
most potent compounds (<1 mm) were those that placed the
Evaluation of Hsp90 inhibitory activity of analogues
After measuring the anti-proliferative activity manifested by
these compounds, the molecules were further investigated in
the Hsp90-dependent luciferase-refolding assay to correlate
[
38]
Hsp90 inhibitory activity with cell viability. Table 3 lists the
compounds that exhibited Hsp90 inhibitory activity. The data
in Table 3 was then displayed in a three-dimensional graph,
which compares Hsp90 inhibitory activity versus the distance
and angle between the N-methylpiperidine and the biaryl side
chain (Figure 5).
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Table 2. Anti-proliferative activity manifested by novobiocin analogues.
[a]
[a]
[a]
[a,d]
Cmpd
Structure
Dist. [Š]
Angle [8]
MCF-7
SKBr3
PC3-MM2
MDA-MB-468Ln
6
6.2
73.4
5.4Æ0.80
5.4Æ0.00
8.7Æ0.37
7.0
7
8
6.3
120
>50
>50
>50
22.1
7.8
7.8
160.9
180
6.7Æ0.00
1.5Æ0.30
7.6Æ0.00
1.3Æ0.22
10.7Æ0.10
3.1Æ0.67
12.83
N.T.
[
b]
5
9
7
7.7
7.9
115
115
3.9Æ0.016
2.6Æ0.738
2.0Æ0.016
3.3Æ0.05
2.2Æ0.03
7.2Æ1.1
3.0
4.3
1
0
1
1
10.4
10.5
139.3
141.6
2.9Æ0.639
2.3Æ0.13
2.3Æ0.06
3.5Æ0.02
7.5Æ0.26
5.7Æ0.12
3.0
3.3
1
2
1
1
3
4
11.8
11.8
168.9
172.1
1.5Æ0.139
3.4Æ0.09
1.4Æ0.09
2.4Æ0.17
7.9Æ1.2
2.2
4.2
7.49Æ0.17
1
1
5
6
7.8
9.1
79.4
2.1Æ0.03
1.8Æ0.43
1.8Æ0.64
1.7Æ0.70
5.2Æ1.0
3.8
2.6
118.8
3.3Æ0.25
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Table 2. (Continued)
Cmpd Structure
[a]
[a]
[a]
[a,d]
Dist. [Š]
Angle [8]
MCF-7
SKBr3
PC3-MM2
MDA-MB-468Ln
1
7
8
9.2
108.3
2.6Æ0.63
2.5Æ0.40
3.2Æ0.90
2.2Æ0.06
5.4Æ1.1
1.9
1
9.8
104.3
5.6Æ0.27
3.5
5.5
1
2
9
0
9.1
9.4
124.6
154.5
2.9Æ0.58
4.2Æ0.80
3.3Æ0.35
6.4Æ0.21
10.3Æ0.35
12.0Æ0.21
>10
2
2
1
2
9.2
168.3
180
3.3Æ0.37
4.9Æ0.43
7.9Æ0.76
>50
5.2
14.1
20.9Æ2.54
11.8Æ0.94
>10
2
3
12.1
180
2.7Æ0.79
4.3Æ0.01
5.8Æ0.32
4.9
2
2
4
5
9.1
120
5.9Æ0.00
1.3Æ0.59
7.5Æ0.00
7.8Æ1.1
6.0
11.8
170.7
0.68Æ0.10
1.3Æ0.26
0.50
2
5
6
11.9
9.6
166.6
180
9.8Æ3.4
2.9Æ1.108
0.47Æ0.06
5.3
4.6
[c]
0.71Æ0.02
0.98
N.T.
2
2
7
8
8.7
163
8.7Æ1.9
14.3Æ1.20
2.2Æ0.14
>50
26.8
1.9
10.9
172.2
8.8
2.1Æ0.41
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Table 2. (Continued)
Cmpd Structure
[
a]
[a]
[a]
[a,d]
Dist. [Š]
14.3
Angle [8]
MCF-7
SKBr3
PC3-MM2
MDA-MB-468Ln
2
9
160.5
4.3Æ0.42
3.5Æ0.63
5.4Æ0.07
2.2
[
a] Values are in mm, which represent mean standard deviation for at least two separate experiments performed in triplicate. [b] Reference [31b]. [c] Refer-
ence [32a]. N.T.=not tested.
data, the Hsp90 inhibitory activity increased as the angle ap-
proached 1808, although exceptions were noted for com-
pounds with a distance of less than 8 Š. There were several
compounds that fit within the parameters for distance and
angle but did not exhibit Hsp90 inhibitory activity. This infor-
mation suggests that identification of Hsp90 inhibitors cannot
be limited simply to distance and angle. Interestingly, the
fused 3- and 4-ring systems (19–21) were generally more
potent Hsp90 inhibitors as compared to the 3-ring systems
(14, 15, 16, 18) that contained a freely rotatable ring, which
was opposite to the observations in the anti-proliferation stud-
ies. These data suggest that the Hsp90 inhibitory activity is
best for compounds that are flat and exhibit a distance greater
than 8 Š.
Table 3. Hsp90 inhibitory activity of analogues.
[a]
Cmpd
Dist. [Š]
Angle [8]
Hsp90 IC50 [mm]
6
8
9
6.2
7.8
7.7
73.4
160.9
115
54.7
118
102
391
61.7
967
491
292
61.2
149
115
15.8
40.9
179
64.9
85.1
266
821
1
5
1
1
1
1
2
2
5
2
2
2
2
2
2
4
7
5
6
8
9
0
1
11.8
7.8
7.8
9.1
9.8
9.1
9.4
9.2
9.6
12.1
9.1
11.8
11.9
8.7
172.1
180
79.4
118.8
104.3
124.6
154.5
168.3
180
180
120
170.7
166.6
163
3
4
5
6
7
8
In order to provide further evidence that the anti-prolifera-
tive activity was due to Hsp90 inhibition, Western blot analysis
was performed on 25, which exhibited the most potent anti-
proliferative activity and good Hsp90 inhibitory activity. As
shown in Figure 6, 25 induced degradation of Hsp90-depen-
dent clients Her2, Raf-1 and CDK6. Actin was used as a control,
since this protein is not an Hsp90-dependent substrate.
10.9
172.2
[
a] Values represent mean standard deviation for at least two separate ex-
periments performed in triplicate.
Figure 6. Western blot analyses of Hsp90 client protein degradation in PC3-
MM2 cells treated with 25. L represents a concentration 1/2”IC50 value
while H represents a concentration of 5”IC50 value. Geldanamycin (GDA
Figure 5. 3D graph for the Hsp90 inhibitory activity versus distance versus
angles.
500 nm) represents a positive control, while DMSO, represents the negative
control.
A distance of 6.2 and 12.1 Š between the N-methylpiperi-
dine and the biaryl side chain was optimal for Hsp90 inhibitory
activity. In general, the distance observed for the compounds
in Table 3 fit within the optimal distance identified for the
manifestation of good anti-proliferative activity, though there
were some exceptions (6, 23). Similar to the anti-proliferation
Conclusions
In this study, the distance and angle between the N-methylpi-
peridine and the biaryl side chain was analyzed in an effort to
develop more potent Hsp90 C-terminal inhibitors. The best
Chem. Eur. J. 2016, 22, 6921 – 6931
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anti-proliferative and Hsp90 inhibitory activities were obtained
when the distance was between 7.7 and 12.1 Š and the angle
was close to 1808 (Figure 7). As a result of this investigation,
a compound (25) was developed, which exhibited mid-nano-
molar activity against SKBr3 cells. This study also revealed
other structural aspects such as planarity and flexibility are im-
portant to achieve good anti-proliferative and Hsp90 inhibitory
activities. Based on the information gathered in this study, the
compounds that exhibited good anti-proliferative and Hsp90
inhibitory activities can be used as a template to develop
more novel and useful Hsp90 inhibitors.
[5] a) D. Picard, Cell. Mol. Life Sci. 2002, 59, 1640–1648; b) J. S. Isaacs, W.
Xu, L. Neckers, Cancer Cell 2003, 3, 213–217; c) W. B. Pratt, D. O. Toft,
Exp. Biol. Med. 2003, 228, 111 –133; d) L. Whitesell, S. L. Lindquist, Nat.
Rev. Cancer 2005, 5, 761–772.
[6] C. Moser, S. A. Lang, O. Stoeltzing, Anticancer Res. 2009, 29, 2031–2042.
[7] F. Koga, K. Kihara, L. Neckers, Anticancer Res. 2009, 29, 797–807.
[
8] J. Franke, S. Eichner, C. Zeilinger, A. Kirschning, Nat. Prod. Rep. 2013, 30,
299–1323.
1
[9] A. Khandelwal, V. Crowley, B. S. J. Blagg, Med. Res. Rev. 2015, 35, 92–
118.
[10] K. Jhaveri, S. Modi, Onco Targets Ther. 2015, 8, 1849–1858.
11] D. S. Hong, U. Banerji, B. Tavana, G. C. George, J. Aaron, R. Kurzock,
Cancer Treat. Rev. 2013, 39, 375–387.
12] a) S. N. Shelton, M. E. Shawgo, S. B. Matthews, Y. Lu, A. C. Donnelly, K.
Szabla, M. Tanol, G. A. Vielhauer, R. A. Rajewski, R. L. Matts, B. S. Blagg,
J. D. Robertson, Mol. Pharmacol. 2009, 76, 1314–1322; b) R. Conde, Z. R.
Belak, M. Nair, R. F. O’Carroll, N. Ovsenek, Biochem. Cell Biol. 2009, 87,
[
[
845–851.
[
[
13] A. Donnelly, B. S. J. Blagg, Curr. Med. Chem. 2008, 15, 2702–2717.
14] I. Gomez-Monterrey, M. Sala, S. Musella, P. Campiglia, Recent Pat. Anti-
Cancer Drug Discov. 2012, 7, 313–336.
[15] X. Song, Z. Zhao, X. Qi, S. Tang, Q. Wang, T. Zhu, Q. Gu, M. Liu, J. Li, On-
cotarget. 2015, 6, 5263–5274.
[
[
16] Y. Wang, S. R. McAlpine, Org. Biomol. Chem. 2015, 13, 4627–4631.
17] M. Strocchia, S. Terracciano, M. G. Chini, A. Vassallo, M. C. Vaccaro, F. Dal
Piaz, A. Leone, R. Riccio, I. Bruno, G. Bifulco, Chem. Commun. 2015, 51,
3850–3853.
Figure 7. Summary of optimal distance and angle between the N-methylpi-
peridine and the biaryl side chain.
[18] Y. Wang, S. R. McAlpine, Chem. Commun. 2015, 51, 1410–1413.
19] J. Gavenonis, N. E. Jonas, J. A. Kritzer, Bioorg. Med. Chem. 2014, 22,
989–3993.
[
3
Experimental Section
[20] J. Davenport, J. R. Manjarrez, L. Peterson, B. Krumm, B. S. J. Blagg, R. L.
Matts, J. Nat. Prod. 2011, 74, 1085–1092.
[21] A. Khandelwal, J. A. Hall, B. S. J. Blagg, J. Org. Chem. 2013, 78, 7859–
7884.
1
H NMR spectra were recorded at 400 or 500 MHz (Bruker DRX-
4
00 Bruker with a H/C/P/F QNP gradient probe) spectrometer and
C NMR spectra were recorded at 125 MHz (Bruker DRX 500 with
1
3
[22] Z. Yin, E. C. Henry, T. A. Gasiewicz, Biochemistry 2009, 48, 336–345.
[
[
[
[
23] M. K. Hadden, L. Galam, J. E. Gestwicki, R. L. Matts, B. S. J. Blagg, J. Nat.
Prod. 2007, 70, 2014–2018.
24] H. Zhao, G. E. Brandt, L. Galam, R. L. Matts, B. S. J. Blagg, Bioorg. Med.
Chem. Lett. 2011, 21, 2659–2664.
25] M. G. Marcu, A. Chadli, I. Bouhouche, M. Catelli, L. M. Neckers, J. Biol.
Chem. 2000, 275, 3718137186.
26] M. G. Marcu, T. W. Schulte, L. Neckers, J. Natl. Cancer Inst. 2000, 92,
242–248.
broadband, inverse triple resonance, and high resolution magic
angle spinning HR-MA probe spectrometer); chemical shifts are re-
ported in d (ppm) relative to the internal reference [D]CDCl3
(
CDCl , 7.27 ppm). FAB (HRMS) spectra were recorded with a LCT
3
Premier (Waters Corp., Milford, MA) spectrometer. The purity of all
compounds was determined to be >95% as determined by
1
13
H NMR and C NMR spectroscopy, unless otherwise noted. TLC
was performed on glass-backed silica gel plates (Uniplate) with
spots visualized by UV light. All solvents were reagent grade and,
when necessary, were purified and dried by standard methods.
Concentration of solutions after reactions and extractions involved
the use of a rotary evaporator operating at reduced pressure.
[27] R. L. Matts, A. Dixit, L. B. Peterson, L. Sun, S. Voruganti, P. Kalyanaraman,
S. D. Hartson, G. M. Verkhivker, B. S. J. Blagg, ACS Chem. Biol. 2011, 6,
8
00–807.
28] E. Moroni, H. Zhao, B. S. J. Blagg, G. Colombo, J. Chem. Inf. Model. 2014,
4, 195–208.
29] S. Sattin, J. Tao, G. Vettoretti, E. Moroni, M. Pennati, A. Lopergolo, L.
Morelli, A. Bugatti, A. Zuehlke, M. Moses, T. Prince, T. Kijima, K. Beebe,
M. Rusnati, L. Neckers, N. Zaffaroni, D. A. Agard, A. Bernardi, G. Colom-
bo, Chem. Eur. J. 2015, 21, 13598–15608.
[
[
5
Acknowledgements
[30] a) G. Garg, H. Zhao, B. S. J. Blagg, ACS Med. Chem. Lett. 2015, 6, 204–
209; b) J. A. Hall, L. K. Forsberg, B. S. J. Blagg, Future Med. Chem. 2014,
We would like to thank Yusuf Oluwasegun Adeshina and
Arnab Bandyopadhyay for producing the 3D graphs for this
study. Also, we gratefully acknowledge support of this project
by the National Institute of Health (CA120458).
6, 1587–1605; c) J. Zhao, H. Zhao, J. A. Hall, D. Brown, E. Brandes, J. Baz-
zill, P. T. Grogan, C. Subramanian, G. Vielhauer, M. S. Cohen, B. S. J.
Blagg, MedChemComm 2014, 5, 1317–1323; d) H. Zhao, E. Moroni, G.
Colombo, B. S. J. Blagg, ACS Med. Chem. Lett. 2014, 5, 84–88; e) H.
Zhao, B. Yan, L. B. Peterson, B. S. J. Blagg, ACS Med. Chem. Lett. 2012, 3,
327–331; f) B. R. Kusuma, L. B. Peterson, H. Zhao, G. Vielhauer, J. Holz-
beierlein, B. S. J. Blagg, J. Med. Chem. 2011, 54, 6234–6253; g) J. A. Burli-
son, L. Neckers, A. B. Smith, A. Maxwell, B. S. J. Blagg, J. Am. Chem. Soc.
2006, 128, 15529–15536; h) J. A. Burlison, B. S. J. Blagg, Org. Lett. 2006,
Keywords: antitumor agents · C-terminal inhibitors · Hsp90 ·
luciferase refolding · medicinal chemistry
8, 4855–4858; i) X. M. Yu, G. Shen, L. Neckers, H. Blake, J. Holzbeierlein,
B. Cronk, B. S. J. Blagg, J. Am. Chem. Soc. 2005, 127, 12778–12779.
[
[
1] J. Trepel, M. Mollapour, G. Giaccone, L. Neckers, Nat. Rev. Cancer 2010,
0, 537–549.
2] J. Travers, S. Sharp, P. Workman, Drug Discovery Today 2012, 17, 242–
52.
[31] a) H. Zhao, B. R. Kusuma, B. S. J. Blagg, ACS Med. Chem. Lett. 2010, 1,
311–315; b) H. Zhao, A. C. Donnelly, B. R. Kusuma, G. E. L. Brandt, D.
Brown, R. A. Rajewski, G. Vielhauer, J. Holzbeierlein, M. S. Cohen, B. S. J.
Blagg, J. Med. Chem. 2011, 54, 3839–3853.
1
2
[
[
3] Y. H. Seo, Arch. Pharmacal Res. 2015, 38, 1582–1590.
4] L. Neckers, J. Biosci. 2007, 32, 517–530.
[32] a) H. Zhao, G. Garg, J. Zhao, E. Moroni, A. Girgis, L. S. Franco, S. Singh,
G. Colombo, B. S. J. Blagg, Eur. J. Med. Chem. 2015, 89, 442–466; b) H.
Chem. Eur. J. 2016, 22, 6921 – 6931
www.chemeurj.org
6930
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
Zhao, M. Anyika, A. Girgis, B. S. J. Blagg, Bioorg. Med. Chem. Lett. 2014,
4, 3633–3637.
33] T. Tsunoda, Y. Yamamiya, Y. Kawamura, S. Itô, Tetrahedron Lett. 1995, 36,
529–2530.
34] E. Richmond, N. Duguet, A. M. Z. Slawin, T. LØbl, A. D. Smith, Org. Lett.
012, 14, 2762–2765.
35] T. H. Jepsen, M. Larsen, M. Jørgensen, M. B. Nielsen, Synthesis 2013, 45,
115–1120.
[38] a) L. Galam, K. M. Hadden, Z. Ma, Q. Z. Ye, B. G. Yun, B. S. J. Blagg, R. L.
Matts, Bioorg. Med. Chem. 2007, 15, 1939–1946; b) T. Sadikot, M. Swink,
J. D. Eskew, D. Brown, H. Zhao, B. R. Kusuma, R. A. Rajewski, B. S. J.
Blagg, R. L. Matts, J. M. Holzbeirlein, G. A. Vielhauer, Assay Drug Dev.
Technol. 2013, 11, 478–488.
2
[
[
[
[
[
2
2
1
36] U. Sharma, P. K. Verma, N. Kumar, V. Kumar, M. Bala, B. Singh, Chem. Eur.
J. 2011, 17, 5903–5907.
37] Wolfram Research, Inc., Mathematica, Version 10.3, Champaign, IL 2015.
Received: December 9, 2015
Revised: February 11, 2016
Published online on April 1, 2016
Chem. Eur. J. 2016, 22, 6921 – 6931
www.chemeurj.org
6931
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim