Synthesis and evaluation of a ring-constrained Hsp90 C-terminal inhibitor that exhibits neuroprotective activity

Article abstract of DOI:10.1016/j.bmcl.2018.03.071

KU-596 is a second-generation C-terminal heat shock protein 90 KDa (Hsp90) modulator based on the natural product, novobiocin. KU-596 has been shown to induce Hsp70 levels and manifest neuroprotective activity through induction of the heat shock response. A ring-constrained analog of KU-596 was designed and synthesized to probe its binding orientation and ability to induce Hsp70 levels. Compound 2 was found to exhibit comparable or increased activity compared to KU-596, which is under clinical investigation for the treatment of neuropathy.

Full text of DOI:10.1016/j.bmcl.2018.03.071

Accepted Manuscript
Synthesis and evaluation of a ring-constrained Hsp90 C-terminal inhibitor that
exhibits neuroprotective activity
Zheng Zhang, Zhenyuan You, Rick T. Dobrowsky, Brian S.J. Blagg
PII:
S0960-894X(18)30274-9
https://doi.org/10.1016/j.bmcl.2018.03.071
BMCL 25725
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
21 February 2018
22 March 2018
24 March 2018
Please cite this article as: Zhang, Z., You, Z., Dobrowsky, R.T., Blagg, B.S.J., Synthesis and evaluation of a ring-
constrained Hsp90 C-terminal inhibitor that exhibits neuroprotective activity, Bioorganic & Medicinal Chemistry
Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.03.071
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphical Abstract
To create your abstract, type over the instructions in the template box below.
Fonts or abstract dimensions should not be changed or altered.
Synthesis and evaluation of a ring-
constrained Hsp90 C-terminal inhibitor that
exhibits neuroprotective activity
Leave this area blank for abstract info.
Zheng Zhang†, Zhenyuan You‡, Rick T. Dobrowsky‡, and Brian S. J. Blagg*†

Bioorganic & Medicinal Chemistry Letters
Synthesis and evaluation of a ring-constrained Hsp90 C-terminal inhibitor that
exhibits neuroprotective activity
a
b
b
Zheng Zhang , Zhenyuan You , Rick T. Dobrowsky , and Brian S. J.
a
Blagg*
a Department of Chemistry and Biochemistry, The University of Notre Dame, 251 Nieuwland Science Hall, Notre Dame, Indiana 46556, United States
b Department of Pharmacology and Toxicology Department, The University of Kansas, Lawrence, Kansas 66045, United States
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
KU-596 is a second-generation C-terminal heat shock protein 90 KDa (Hsp90) modulator based
on the natural product, novobiocin. KU-596 has been shown to induce Hsp70 levels and
manifest neuroprotective activity through induction of the heat shock response. A ring-
constrained analog of KU-596 was designed and synthesized to probe its binding orientation and
ability to induce Hsp70 levels. Compound 2 was found to exhibit comparable or increased
activity compared to KU-596, which is under clinical investigation for the treatment of
neuropathy.
Keywords:
Hsp90 C-terminal inhibitor
Novologue
Ring-constrained
Neuroprotective
2
009 Elsevier Ltd. All rights reserved.
novel probe to investigate the potential neuroprotective
activity of such compounds.
New analogs of KU-32 have since been developed and
structure-activity relationships of these inhibitors have clearly
The 90 KDa heat shock protein (Hsp90) is a molecular
chaperone that is responsible for the folding and maturation of
more than 300 client proteins and consequently, represents a
promising target for the development of both cancer and
defined attributes required for neuroprotective versus anti-
24,25
cancer activity.
For example, the acetamide moiety of KU-
1
-7
neurodegenerative agents. Six candidates that target the
Hsp90 N-terminus are currently under clinical investigation to
3
2 is necessary to induce the HSR, but replacing this group
with a benzamide results in client protein degradation without
induction of the HSR. Although no co-crystal structure has
8
,9
assess their efficacy for the treatment of cancer. Despite
these efforts, Hsp90 N-terminal inhibitors induce an
undesired, pro-survival heat shock response (HSR), which has
impeded their clinical potential for cancer, as increased Hsp90
26-28
been solved for inhibitors bound to the Hsp90 C-terminus,
models have been developed that support the binding of KU-
3
2 at this location. One model suggests an unexplored pocket
1
0
levels result from the administration of such inhibitors. This
same HSR is also reponsible for the upregulation of other heat
shock proteins, including Hsp70, which exhibit pro-survival
activity and can refold protein aggregates that accumulate
during the pathology of several neurodegenerative
to reside in close proximity to KU-32, which provides an
opportunity to design compounds that exhibit increased
activity. Accordingly, a second generation of novobiocin
analogs was developed to contain a biaryl core, which led to
small molecules that manifest enhanced neuroprotective
1
1,12
diseases.
24
activity. Among the novologues prepared, KU-596 was
In contrast to N-terminal inhibitors, Hsp90 C-terminal
inhibitors can segregate the HSR from the degredation of
client proteins, allowing the opportunity to develop small
molecules that manifest selective activity towards cancer or
found to be most active in both luciferase reporter and
24, 25
mitochondria bioenergetic assays (Figure 1).
When KU-
5
96 was docked into the Hsp90 C-terminal model, the noviose
sugar was found to project into a subpocket that contains
amino acid side chains that contribute hydrogen bonding
interactions. In addition, the fluorine atom on KU-596
appeared to form a hydrogen bond with Lys560 (Figure 3A),
which is supported by prior structure-activity relationship
1
3-21
neurodegeneration.
KU-32 is derived from novobiocin, the
first Hsp90 C-terminal inhibitor identified (Figure 1), and was
shown to exhibit cytoprotective activity at concentrations that
2
2,23
induce the HSR.
00-fold lower concentrations than that required to promote
client protein degradation. Consequently, KU-32 represents a
In fact, KU-32 can induce the HSR at ≥
5
25
studies.
Since it is well-known that conformationally constrained
molecules often exhibit lower entropic penalties as well as

improved affinity upon binding, an analog of KU-596 was
pursued. The inclusion of a lactam to constrain the biaryl ring
system also represented an opportunity to introduce hydrogen
bonding interactions with the peptide backbone while
rigidifying the biaryl ring system. Prior studies with KU-32
analogs supported substitution at this position, as replacement
of the 8-methyl group with other functionalities led to
B
2
0, 29
improved activity (Figure 2).
Such data supports the
existence of unoccupied space in the binding site and
correlates well with the proposed model.
(
(
A) KU-596 docked into the Hsp90 C-terminal binding site.
B) 2 docked into the Hsp90 C-terminal binding site.
The dash line indicates the distance between the fluorine atom and hydrogen
from the amino residue
Figure 3. Molecular modeling for KU596 and compound 2
Figure 1. C-terminal Hsp90 inhibitors
In addition, the lactam carbonyl participated in a
hydrogen bonding network with Gln488. As expected, the
sugar moiety maintained a similar orientaion inside the pocket
as was predicted for KU-596 (Figure 3A). Similarly, the 3-
amido side chain projected into the same hydrophobic region
as observed with KU-596.
Compound 2 was pursued via the synthetic route
described in Scheme 1. Commercially available 3-nitro-4-
hydroxy benzaldehyde was converted to benzyl ether 3 via
benzyl bromide, followed by iron-mediated reduction of the
nitro substituent to form the corresponding aniline, 4.
Subsequent amide coupling was performed via conversion of
Figure 2. Design of ring-constrained novologue
2
-bromo-6-fluoro benzoic acid to the acid chloride, followed
by reaction with aniline 4 to ultimately yield 5. The aldehyde
of 5 was then converted to acetal 6 to mask its reactive nature
during subsequent steps. The intramolecular Heck coupling
was known to be catalyzed by palladium in the presence of
The phenanthridone containing compound, 2, was
designed and then docked into the Hsp90 C-terminal binding
site to further investigate the mode of binding (Figure 3B).
Interestingly, the fluorine atom on 2 pointed towards Gln488
rather than the aforementioned Lys560 (Figure 3B), which
can still form hydrogen bonding interactions via the amide
NH.
30
silver carbonate. However, attempts to perform an
intramolecular Heck coupling without protection of amide 6
was unsuccessful. Therefore, the amide was methylated to
give 7 before cyclization was successfully achieved to
31
produce 8 under modified conditions. Compound 8 was then
converted to 9 via Henry olefination. Compound 11 was then
synthesized from 9 via a two-step reduction before acetylation
with acetyl chloride. Finally, the activated noviose sugar 13
was prepared and appended to phenol 12 upon removal of
benzyl group to yield the final product, 2.
A
3
2
To evaluate the ability of 2 to promote Hsp70
induction compared to 1, western blot analyses were
performed in immortalized dorsal root ganglia sensory
neuronal 50B11 cells. As depicted in Figure 4, compound 2 is
more effective than 1 at the induction of Hsp70 levels. The
slight decrease of Hsp70 at the highest concentration of 2 is a
common attribute ascribed to this class of modulators, which
typically give a U-shape curve upon dosing with Hsp90 C-
2
4
terminal inhibitors.

Scheme 1: Synthesis of 2
Reaction conditions: (a) BnBr, K
chloride, Et N, CH Cl , rt, overnight, 83%; (d) trimethyl orthoformate, TsOH, MeOH, 50 C, 2h, 87%; (e) NaH, MeI, THF, 0 C to rt, overnight; (f) Pd(PPh
Ag CO , dioxane, 90 C, overnight; (g) TsOH monohydrate, acetone, rt, 2h, 76% over two steps; (h) CH
Dioxane/MeOH, 0 C to rt, 2h, 83%; (j) Zn, 1N HCl, EtOH/CH
r.t., overnight, 75%; (m) 13, BF ·OEt , CH Cl , rt, 3h; (n) Et N, MeOH, rt, overnight, 30% over two steps.
2
CO , Acetonitrile, reflux, overnight, 94%; (b) Fe, NH Cl, THF/EtOH/H O, reflux, 2h, 75%; (c) 2-bromo-6-fluoro benzoyl
3
4
2
o
o
3
2
2
o
3 4
) ,
o
2
3
3
NO
Cl
2
, NH
4
OAc, 80 C, overnight, 86%; (i) NaBH
4
,
o
Cl
2 2
, rt, 4h; (k) Acetyl chloride, Et
3
N, CH
2
2
, rt, 3h, 60% over two steps; (l) Pd/C, H
2
, EtOAc,
3
2
2
2
3
it can significantly increase cellular maximal respiratory
capacity (MRC), starting at concentrations of ~100 nM
(
Figure 5C). The improvement of mitochondrial bioenergetics
in sensory neurons may be one of the key mechanisms that
contributes to the neuroprotective efficacy of both 1 and 2 as
well as related molecules.
A .
1
5
0
5
0
N o Treatm ent
H2 O 2
1
0nM K U 596
1
Figure 4. Western Blot analysis of Hsp70 induction for 2. 50B11 cells
were seeded at 2 x 10 cells per well and cultured for 24hr at 37 °C
prior to treatment with DMSO or the indicated concentrations of
KU-596 or 3 for an additional 24 hr. The cells were harvested to
determine Hsp70 protein expression level by immunoblotting with
and Hsp70/Hsp72 biotin-conjugated monoclonal antibody.
30nM K U 596
100nM K U 596
300nM K U 596
5
1M K U 596
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
T im e (m in u te s)
Excessive oxidative stress can also cause
mitochondria dysfunction in diabetic sensory neurons, which
is a key contributor to the pathogenesis of diabetic peripheral
3
3
neuropathy (DPN). Thus, oxidative stress can be induced by
treating 50B11 cells with 0.5mM hydrogen peroxide to
decrease mitochondrial bioenergetics (mtBE) as measured by
the Seahorse assay and as indicated by a decreased response
to the protonophore, FCCP. Both 1 (Figure 5A) and 2 (Figure
5
B) can effectively improve mtBE in a dose-dependent
manner. However, drug 2 may be more effective than 1, since

3
.
Zhao, H.; Mary, L. M.; Blagg, B. S. J. Advances in Pharmacology
012, 64, 1-25.
4. Karagoz, G. E.; Rudiger, S. G.; Trends Biochem Sci 2015, 40, 117.
B .
2
1
5
0
5
0
N o Treatm ent
5
.
Yoshihiko, M.; Hitoshi, N.; Len, N. Current Pharmaceutical
Design 2013, 19, 347.
H2 O 2
1
1M 247
6. Pratt, W. B.; Morishma, Y.; Gestwicki, J. E.; Lieberman, A. P.;
Osawa, Y. Experimental biology and medicine 2014, 239, 1405.
Trepel, J.; Mollapour, M.; Giaccone, G.; Neckers, L. Nature Rev.
Cancer 2010, 10, 537-549
Neckers, L.; Trepel, J. B. Clinical cancer research: an official
journal of the American Association for Cancer Research 2014,
3
1
3
1
00nM 247
00nM 247
0nM 247
0nM 247
7
8
.
.
2
39, 1405.
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
9. Neckers, L.; Workman, P. Clinical cancer research: an official
T im e (m in u te s)
journal of the American Association for Cancer Research 2012,
1
8, 64.
1
0. Hong, Banerji, U.; Tavana, B.; George, G. C.; Aaron, J.; Kurzock,
R. Cancer Treatment Reviews 2013, 39, 375-387
C .
1
1
1. Mayer, M. P.; Bukau, B. Cell. Mol. Life Sci. 2005, 62, 670-684.
2. Peterson, L. B.; Blagg, B. S. J. Future Med. Chem. 2009, 1, 2670-
283.
3. Shelton, S. N.; Shawgo, M. E.; Comer, S. B.; Lu, Y.; Donnelly, A.
C.; Szabla, K.; Tanol, M.; Vielhauer, G. A.; Rajewski, R. A.;
Matts, R. L.; Blagg, B. S. J.; Robertson, J. D. Mol. Pharmacol.
1
2
0
8
6
4
2
0
*
*
*
*
1
1
*
2
009, 76, 1314−1322
1
1
1
1
4. Conde, R.; Belak, Z. R.; Nair, M.; O’Carroll, R. F.; Ovsenek, N.
Biochem. Cell Biol. 2009, 87, 845−851
5. Zhao, H.; Blagg, B. S. J. In inhibitors of Molecular Chaperones as
Therapeutic Agents.
6. Cohen, M. S.; Mukerji, R.; Samadi, A. K.; Zhang, X.; Zhao, H.;
Blagg, B. S. J. Ann. Surg. Oncol. 2012, 19, S483-490.
7. Burlison, J. A.; Avila, C.; Vielhauer, G.; Lubbers, D. J.;
Holzbeierlein, J.; Blagg, B. S. J. J. Org. Chem. 2008, 73, 2130-
2
137.
H2 02
-
+
+
+
+
+
+
+
0
+
0
+
0
+
+
0
1
1
8. Burlison, J. A.; Blagg, B. S. J. Org. Lett. 2006, 8, 4855-4858.
9. Burlison, J. A.; Neckers, L.; Smith, A. B.; Maxwell, A.; Blagg, B.
S. J. J. Am. Chem. Soc. 2006, 128, 15529-15536.
1
2
,
,
 M
 M
0
0
.0 1 .0 3 0 .1
0 .3 1 .0
0
0
0
0
0
0
0
0
.0 1 .0 3
0 .1 0 .3 1 .0
2
2
0. Donnelly, A.; Mays, J. R.; Burlison, J. A.; Nelson, J. T.;
Vielhauer, G.; Holzbeierlein, J.; Blagg, B. S. J. J. Org, Chem.
Figure 5. Improvement in mitochondrial bioenergetics by 2. The 50B11
cells were seeded into a 96 well plate and after 12 hrs the cells were
changed into high glucose DMEM medium with 1% FBS, containing
2
008, 73, 8901-8920.
1. Yu, X. M.; Shen, G.; Neckers, L.; Blake, H.; Holzbeierlein, J.;
o
Cronk, B.; Blagg, B. S. J. J. Am. Chem. Soc. 2005, 127, 12778-
the indicated concentrations of 1 (A) or 2 (B) for 24 hr at 37 C. The
1
2779.
cells were stressed with 0.5mM H O for 2 hr, the medium changed
2
2
22. 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. Bioorg Med Chem Lett 2007, 17, 1984.
to serum free unbuffered DMEM with 5.5 mM glucose and 1mM
o
pyruvate and the cells incubated at 37 C for 1 hr prior to assessing
oxygen consumption rate (OCR) with an XF96 Extracellular Flux
Analyzer. (C) Maximal respiratory capacity (MRC) was calculated
for each treatment. Results are from one experiment using 5 wells of
cells per treatment. *, p < 0.05 vs H O alone.
23. Lu, Y.; Ansar, S.; Michaelis, M. L.; Blagg, B. S. J. Bioorganic &
medicinal chemistry 2009, 17, 1709.
2
4. Kusuma, B. R.; Zhang, L.; Sundstrom, T.; Peterson, L. B.;
Dobrowsky, R. T.; Blagg, B. S. J. J. Med. Chem. 2012, 55, 5797-
2
2
5
812.
2
5. Anyika, M.; McMullen, M.; Forsberg, L. K.; Dobrowsky, R. T.;
Blagg, B. S. J. ACS Med. Chem. Lett. 2015, 7, 67-71.
In conclusion, a ring-constrained novologue was
synthesized and then evaluated by western blot analyses and
mitochondrial bioenergetic assays. Compound 2 exhibited
increased activity for mitochondrial bioenergetics against
oxidative stress as compared to 1 at all concentrations except
a slightly better activity was observed at 100 nM. This result
is consistent with the proposed docking studies and supports a
similar binding pose for both 1 and 2.
26. Matts, R. L.; Dixit, A.; Peterson, L. B.; Sun, L.; Voruganti, S.;
Kalyanaraman, P.; Hartson, S. D.; Verkhivker, G. M.; Blagg, B. S.
J. ACS Chem. Biol. 2011, 6, 800-807.
7. Moroni, E.; Zhao, H.; Blagg, B. S. J.; Colombo, G. J. Chem. Inf.
Model 2014, 54, 195-208.
28. Sattin, S.; Tao, J.; Vettoretti, G.; Moroni, E.; Pennati, M.;
Lopergolo, A.; Morelli, L.; Bugatti, A.; Zuehlke, A.; Moses, M.;
Prince, T.; Kijima, T.; Beebe, K.; Rusnati, M.; Neckers, L.;
Zaffaroni, N.; Agard, D. A.; Bernardi, A.; Colombo, G. Chem.
Eur. J. 2015, 21, 13598-15608.
2
2
9. Zhao, H.; Donnelly, A. C.; Kusuma, B. R.; Brandt, G. E.; Brown,
D.; Rajewski, R. A.; Vielhauer, G.; Holzbeierlein, J.; Cohen, M.
S.; Blagg, B. S. J. J. Med. Chem. 2011, 54, 3839-3853.
Acknowledgments
3
3
0. Ames, D. E.; Opalko, A. Tetrahedron 1984, 40, 1919.
1. Calder, E. D. D.; McGonagle, F. I.; Harkiss, A. H.; McGonagle,
G. A.; Sutherland, A. J. Org. Chem. 2014, 79, 7633-7648.
This work was supported by grants from The National
Institutes of Health to B.S.J.B [CA120458], R.T.D.
32. Burlison, J. A.; Avila, C.; Vielhauer, G.; Lubbers, D. J.;
Holzbeierlein, J.; Blagg, B. S. J. J. Org. Chem. 2008, 73, 2130-
[
DK095911], and B.S.J.B and R.T.D. [NS075311].
2
137
3. Farmer, K.L.; Li, C.; Dobrowsky, R.T. Pharmacol. Rev., 2012, 64,
80-900.
3
8
References and notes
1
2
.
.
Khandelwal, A.; Crowley, V. M.; Blagg, B. S. J. Med. Res. Rev.
016, 36, 92.
Hall, J. A.; Forsberg, L. K.; Blagg, B. S. J. Future Med. Chem.
014, 6, 1587.
2
2

Supplementary Material
Procedure for the docking study, synthesis of 3-12, western
blot analysis, Seahorse assay, characterization and NMR spectra
of 3-12.