A scaffold merging approach to Hsp90 C-terminal inhibition: synthesis and evaluation of a chimeric library

Article abstract of DOI:10.1039/c6md00377j

Inhibition of the Hsp90 C-terminus is an attractive therapeutic paradigm for the treatment of cancer, however the developmental space of C-terminal inhibitors is limited. It was hypothesized that the combination of two previously identified scaffolds into a single structure could provide a platform for which to probe the three-dimensional space within the Hsp90 C-terminal binding pocket. The resulting chimeric compounds displayed anti-proliferative activity at low micromolar concentrations and manifested inhibitory activity in an Hsp90-dependent rematuration assay. Initial structure-activity relationships suggest that this new scaffold binds Hsp90 in a conformation different from that of the parent compounds, and consequently, provides a new opportunity to develop more efficacious inhibitors of the Hsp90 C-terminal binding pocket.

Full text of DOI:10.1039/c6md00377j

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A Scaffold Merging Approach to Hsp90 C-terminal Inhibition:
Synthesis and Evaluation of a Chimeric Library†
Rachel E. Davis, Zheng Zhang, and Brian S. J. Blagg*
Department of Medicinal Chemistry, The University of Kansas, 1251 Wescoe Hall Drive, 4070
Malott Hall, Lawrence, Kansas 66045, United States
*Author to whom correspondence should be addressed. Phone: (785) 864ꢀ2288. Fax: (785) 864ꢀ
5326. Email: bblagg@ku.edu.
†The authors declare no competing interests.
TOC Entry
Two previously identified Hsp90 Cꢀterminal inhibitors were merged into a single scaffold that
manifested improved Hsp90 inhibitory activity.
Abstract
Inhibition of the Hsp90 Cꢀterminus is an attractive therapeutic paradigm for the treatment of
cancer, however the developmental space of Cꢀterminal inhibitors is limited. It was hypothesized
that the combination of two previously identified scaffolds into a single structure could provide a
platform for which to probe the threeꢀdimensional space within the Hsp90 Cꢀterminal binding
pocket. The resulting chimeric compounds displayed antiꢀproliferative activity at low
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micromolar concentrations and manifested inhibitory activity in an Hsp90ꢀdependent
rematuration assay. Initial structureꢀactivity relationships suggest that this new scaffold binds
Hsp90 in a conformation different from that of the parent compounds, and consequently,
provides a new opportunity to develop more efficacious inhibitors of the Hsp90 Cꢀterminal
binding pocket.
Main Text
The 90 kDa heat shock protein (Hsp90) is a highly conserved and widely expressed
molecular chaperone that plays a role in the maturation and stabilization of more than 200
protein substrates (clients).[1] Hsp90 is upregulated in cancer to meet the demands of rapidly
growing cells that reside in the harsh microenvironment of a tumor.[2] Additionally, many of the
Hsp90ꢀdependent client proteins (e.g. Akt, Her2, Rafꢀ1, etc.) are associated with cellular growth
and signaling pathways that become dysregulated in cancer and represent wellꢀvalidated
therapeutic targets.[3ꢀ4] Therefore, inhibition of the Hsp90 folding machinery provides a unique
opportunity to simultaneously disrupt multiple oncogenic pathways, and consequently,
represents an attractive target to combat the many pathologies exhibited by cancer.[5]
Within the cell, Hsp90 exists as a homodimer with each monomer possessing both an Nꢀ
terminal ATPase domain, as well as a Cꢀterminal dimerization domain that contains a second
nucleotideꢀbinding pocket.[6] In the past decade, 17 inhibitors that target the Nꢀterminus of
Hsp90 have undergone clinical evaluation.[7] However, Nꢀterminal inhibitors induce both the
proꢀapoptotic degradation of client proteins and the proꢀsurvival heat shock response at similar
concentrations.[8] The combination of these effects most often results in cytostatic activity and
limits the potential utility of Nꢀterminal inhibitors in the clinic.[9] In contrast, inhibitors of the
Hsp90 Cꢀterminus can segregate these effects: Cytotoxic Cꢀterminal inhibitors promote client
2

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protein degradation without induction of the heat shock response,[10ꢀ11] whereas cytoprotective Cꢀ
terminal inhibitors induce the heat shock response at concentrations well below that which is
needed to induce the degradation of client proteins.[12ꢀ13] Despite the potential advantages offered
by Hsp90 Cꢀterminal inhibitors, they have yet to undergo clinical investigation. The development
of more efficacious analogs has been hampered by the lack of a coꢀcrystal structure between the
Cꢀterminal nucleotideꢀbinding pocket and an inhibitor.
In 2000, Neckers and coworkers discovered the antibiotic novobiocin (Figure 1) as the
first inhibitor of the Hsp90 Cꢀterminal binding region.[14ꢀ15] Subsequent SAR studies transformed
novobiocin from a DNA gyrase inhibitor into a selective inhibitor of Hsp90 (1).[16ꢀ17] However,
the coumarin core of novobiocin is not amenable to divergent modification and as a result,
considerable effort has been placed on the identification of new scaffolds that inhibit the Hsp90
Cꢀterminus.[18ꢀ21] Prior work has led to the discovery of 2,[11] which features a biaryl moiety and a
flexible linker attached to the amide side chain, and 3,[22] which contains a biphenyl core that
serves as a coumarin surrogate. In an attempt to improve potency and expand the chemical space
associated with Hsp90 Cꢀterminal inhibition, it was envisioned that these two scaffolds could be
combined into a single compound, which would exhibit the interactions manifested by both 2
and 3. In this communication, we report the design, synthesis, and evaluation of such chimeric
analogs.
Figure 1. Hsp90 Cꢀterminal inhibitors.
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Retrosynthetic analysis of triphenyl scaffold 4 suggested 5 as a common intermediate that
was amenable to lateꢀstage diversification (Scheme 1). Mitsunobu etherification of alcohol 6
with phenol 5 would install the piperidine ring and subsequent coupling of the aniline with acid
chloride 7 would produce the requisite amide side chain. The triphenyl core of 5 could be rapidly
assembled through sequential Suzuki coupling reactions between bromoresorcinol 8 and
phenylboronic acids, 9 and 10.
Scheme 1. Retrosynthetic analysis of triphenyl analogs.
Synthesis of the proposed analogs commenced upon preparation of 1ꢀbenzyloxyꢀ4ꢀ
bromoresorcinol (8, Scheme 2). Formation of the benzyl ether from 4ꢀbromoresorcinol (11) and
benzyl bromide gave the undesired regioisomer 12 as the major product in low yield. However,
treatment of 11 with pꢀtoluenesulfonyl chloride resulted in chemoselective formation of the para
sulfonate ester.[23] Subsequent masking of the ortho phenol with chloromethyl methyl ether gave
bromoresorcinol 13, which upon baseꢀcatalyzed hydrolysis of the sulfonate ester and installation
of the benzyl ether gave 14. Ultimately, the bisꢀprotected bromoresorcinol 14 was subjected to
acidꢀcatalyzed hydrolysis of the methoxymethyl acetal to furnish 8 in 81% yield over 4 steps.
Suzuki coupling of 8 with 3ꢀ or 4ꢀ(tertꢀbutoxycarbonylamino)phenylboronic acid (9) gave
carbamateꢀprotected anilines, 15a–b. Microwaveꢀassisted conversion of the hindered phenol into
a trifluoromethanesulfonate ester and subsequent Suzuki coupling[24] with metaꢀsubstituted
phenylboronic acids 10 yielded the triphenyl compounds, 16a–d. Hydrogenolysis of the benzyl
ethers followed by Mitsunobu etherification with 1ꢀmethylꢀ4ꢀhydroxypiperidine yielded 17a–d.
4

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Cleavage of the carbamate with trifluoroacetic acid yielded common intermediates 18a–d, which
were subsequently coupled with acid chlorides (7a–f) to afford the final products, 4a–x.
Scheme 2. Synthesis of triphenyl analogs 4a–x. Reagents and conditions: a) BnBr, NaHCO3, CH3CN, 80 °C, 31%;
b) TsCl, K2CO3, acetone, 65 °C, then MOMCl, 98%; c) 4 M KOH, EtOH, 60 °C, 99%; d) BnBr, K2CO3, acetone, 65
°C, 92%; e) HCl (conc.), THF/MeOH, rt, 91%.; f) 3ꢀ or 4ꢀ(tert-butoxycarbonylamino)phenylboronic acid,
Pd(dpppf)Cl2, K2CO3, dioxane, 85 °C, 70–90%; g) NꢀPhenylꢀbis(trifluoromethanesulfonimide), K2CO3, THF, mw
120 °C; h) 3ꢀ(trifluoromethyl)phenylboronic acid or 3ꢀfluorophenylboronic acid, SPhos, Pd2(dba)3, K3PO4, dioxane,
85 °C, 70–95% over two steps; i) H2, Pd/C, THF, rt; j) 1ꢀmethylꢀ4ꢀhydroxypiperidine, PPh3, DIAD, THF, rt, 35–
65% over two steps; k) TFA, DCM, rt, quant.; l) Et3N, DCM, rt, 10–90%.
Upon construction of the library, the chimeric compounds were evaluated for their antiꢀ
proliferative activity against MCF7 and SKBR3 breast cancer cell lines. As shown in Table 1,
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the new chimeric scaffold was efficacious against both cell lines, and manifested EC50 values in
the low micromolar range. In general, compounds with trifluoromethyl and fluorine substitutions
exhibited similar efficacies (e.g. 4a vs 4b), which correlated with previously reported SAR
trends for 2.[11] In contrast to SAR generated for the parent scaffolds,[11, 22, 25ꢀ26] neither the
location of the amide side chain (meta vs para) nor its substitutions were drivers for potency (e.g.
4q vs 4r and 4j vs 4n, respectively), which suggests the new scaffold may not interact with
Hsp90 in the same orientation as the lead compounds.
Table 1. Antiꢀproliferative activities of 4a–x.
MCF7a
(EC50, µM)
SKBR3a
(EC50, µM)
Cmpd NHCOR
R
R1
ꢀ
ꢀ
ꢀ
2.7 ± 0.40
1.6 ± 0.15
2.3 ± 0.75
4.2 ± 0.21
2.2 ± 0.96
2.1 ± 0.35
3.0 ± 0.58
4.4 ± 0.10
2.6 ± 0.96
3.3 ± 0.96
3.9 ± 0.69
5.6 ± 0.48
3.4 ± 0.41
5.1 ± 0.13
2.8 ± 0.22
5.2 ± 0.25
3.0 ± 0.10
4.2 ± 0.10
1.7 ± 0.87
2.9 ± 0.99
2.0 ± 0.74
1.0 ± 0.21
4 ± 1.2
3.5 ± 0.47
1.5 ± 0.15
1.9 ± 0.66
4.1 ± 0.14
1.8 ± 0.80
4.2 ± 0.12
2.9 ± 0.80
3.3 ± 0.98
2.3 ± 0.95
3.2 ± 0.99
3.0 ± 0.43
5.9 ± 0.97
3.5 ± 0.59
5.2 ± 0.21
1.8 ± 0.54
6.2 ± 0.31
2.4 ± 0.51
4.2 ± 0.11
2.2 ± 0.70
2.2 ± 0.95
1.9 ± 0.72
0.8 ± 0.36
4 ± 1.1
2
3
ꢀ
ꢀ
ꢀ
meta
meta
para
para
meta
meta
para
para
meta
meta
para
para
meta
meta
para
para
meta
para
meta
para
meta
para
A
CF3
F
4a
4b
4c
4d
4e
4f
A
A
CF3
F
A
B
CF3
F
B
B
CF3
F
4g
4h
4i
B
C1
C1
C1
C1
C2
C2
C2
C2
D1
D1
D2
D2
D3
D3
CF3
F
4j
CF3
F
4k
4l
CF3
F
4m
4n
4o
4p
4q
4r
4s
4t
CF3
F
CF3
CF3
CF3
CF3
CF3
CF3
4u
4v
0.9 ± 0.18
0.5 ± 0.14
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meta
para
D4
D4
CF3
CF3
3 ± 1.2
5 ± 1.3
3 ± 1.4
4w
4x
4.4 ± 0.87
aValues represent the mean ± SD of at least three separate experiments performed in triplicate.
In light of the relatively flat SAR trends observed for this chimeric scaffold in the antiꢀ
proliferation assay, the compounds were evaluated in an Hsp90ꢀdependent luciferase
rematuration assay. Briefly, PC3ꢀMM2 cells expressing firefly luciferase were heated to denature
luciferase before incubation with either DMSO or a proposed Hsp90 inhibitor.[27] After
incubation, luciferase activity was measured to determine whether the compounds inhibited the
refolding of luciferase, a process dependent upon Hsp90.[28] As shown in Figure 2A, analogs
containing the pꢀbromo substitution were the most efficacious of the benzamideꢀcontaining
compounds, regardless of whether the amide was in the meta or para position (4i–l). In contrast
to the benzamideꢀcontaining compounds, the triazolamideꢀcontaining compounds (4q–x, Figure
2B) exhibited a preference for metaꢀsubstitution. Additionally, with the exception of tꢀbutylꢀ
substituted analogs, 4w–x, the triazolamideꢀcontaining compounds were more efficacious than
the benzamideꢀcontaining compounds. Together, these data suggest the triazole ring may
participate in hydrogen bonding interactions that require substitution at the meta position.
Interestingly, there were little trends between the antiꢀproliferative and luciferase
rematuration assays. Indeed, despite the entire library manifesting good antiꢀproliferative
activity, only a subset inhibited the Hsp90 proteinꢀfolding machinery. Regardless, pꢀbromo
benzamides 4i–l and metaꢀsubstituted triazolamides 4q, 4s, and 4u displayed increased inhibition
of luciferase rematuration compared to parent compounds 2 and 3, validating the potential use of
this scaffold as a starting point for the development of more efficacious inhibitors.
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Figure 2. Inhibition of heatꢀdenatured luciferase refolding. Values are RLU relative to DMSO and represent the
mean ± SD of at least two separate experiments performed in triplicate. (A) Benzamideꢀcontaining analogs 4a–p.
(B) Parents 2 and 3 and triazolamideꢀcontaining analogs 4q–r.
To further characterize the most efficacious compounds, the levels of Hsp90ꢀdependent
client proteins from MCFꢀ7 cells treated with compounds 4q, 4i, and 4k were examined by
western blot analyses. As shown in Figure 3, the level of Hsp90ꢀdependent client proteins
(HER2, CDK4, ERꢀα, and Cyclin D1) decreased in a concentrationꢀdependent manner, while the
levels of actin, which is not dependent upon Hsp90, remained constant. The levels of Hsp90 and
Hsp70 also remained constant, indicating that these compounds did not induce the heat shock
response, a hallmark manifested by Hsp90 Cꢀterminal inhibitors that exhibit antiꢀcancer activity.
Compounds 4i and 4k appear to be more efficacious than 4q at inducing client protein
degradation, despite exhibiting a similar potency in the luciferase rematuration assay.
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Figure 3. Western blot of Hsp90ꢀdependent client proteins from MCF7 cell lysate upon treatment with
triphenyl compounds. L represents a concentration equal to 0.5ꢀfold of the antiꢀproliferative EC50. H represents a
concentration equal to 10ꢀfold of the antiꢀproliferative EC50.
In summary, a library of chimeric small molecules was designed, synthesized, and
biologically evaluated in Hsp90ꢀdependent cancer cell lines. The antiꢀproliferative activities
manifested by the triphenyl analogs against both MCF7 and SKBR3 cells were similar to the
parent compounds. The ability of select analogs to inhibit the refolding of denatured luciferase in
PC3ꢀMM2 cells confirm these molecules inhibit the Hsp90 protein folding machinery. In
addition, the degradation of Hsp90ꢀdependent client proteins treated with 4q, 4i, and 4k occurred
without induction of the heat shock response, which is consistent with Hsp90 Cꢀterminal
inhibiton. The ease with which this new triphenyl scaffold can be rapidly constructed and
divergently modified provides an excellent opportunity to explore the Cꢀterminal binding pocket
and to develop more efficacious inhibitors. Such studies are currently underway and will be
reported in due course.
Acknowledgements
The authors gratefully acknowledge the support of this work by NIH grant CA120458.
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