Modified biphenyl Hsp90 C-terminal inhibitors for the treatment of cancer

Article abstract of DOI:10.1016/j.bmcl.2017.07.030

Heat Shock Protein 90 (Hsp90) is a molecular chaperone under clinical investigation for the treatment of neurodegenerative diseases and cancer. Neuroprotective Hsp90 C-terminal inhibitors (novologues) contain a biaryl ring system, and include KU-596, which was modified and investigated for potential anti-cancer activity. Incorporation of a benzamide group onto the biaryl novologues in lieu of the acetamide yielded compounds that manifest anti-cancer activity. Further exploration of the central phenyl ring led to compounds with enhanced anti-proliferative activity. The design, synthesis, and evaluation of these new analogs against breast and prostate cancer cell lines is reported herein, where it was found that 8b and 10 manifest potent anti-proliferative activity and a robust degradation of Hsp90 client-dependent proteins.

Full text of DOI:10.1016/j.bmcl.2017.07.030

Accepted Manuscript
Modified Biphenyl Hsp90 C-terminal Inhibitors for the Treatment of Cancer
Leah K. Forsberg, Weiya Liu, Jeffrey Holzbeierlein, Brian S.J. Blagg
PII:
S0960-894X(17)30727-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.07.030
BMCL 25143
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
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30 June 2017
10 July 2017
Please cite this article as: Forsberg, L.K., Liu, W., Holzbeierlein, J., Blagg, B.S.J., Modified Biphenyl Hsp90 C-
terminal Inhibitors for the Treatment of Cancer, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://
dx.doi.org/10.1016/j.bmcl.2017.07.030
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Modified Biphenyl Hsp90 C-terminal Inhibitors for the Treatment of
Cancer
Leah K. Forsberg aWeiya Liub, Jeffrey Holzbeierleinb, and Brian S. J. Blagg*,a
aDepartment of Medicinal Chemistry, 1251 Wescoe Hall Drive, Malott 4070, The
University of Kansas, Lawrence, Kansas 66045-7563.
b Department of Urology, 3901 Rainbow Boulevard, Stop 3016, The University of Kansas
Medical Center, Kansas City, Kansas 66160.
*Author to whom correspondence should be addressed. Phone: (785) 864-2288. Fax:
(785) 864-5326. Email: bblagg@ku.edu.
TOC Graphic
Abstract
Heat shock protein 90 (Hsp90) is a chaperone under investigation for the
treatment of neurodegenerative diseases and cancer. Neuroprotective Hsp90 C-terminal
inhibitors (novologues) contain a biaryl ring system, and include KU-596, which was
modified and investigated for potential anti-cancer activity. Incorporation of a
benzamide group on the biaryl novologues in lieu of the acetamide yields compounds that
manifest anti-cancer activity against breast cancer cell lines. Further exploration on the
1

central phenyl ring led to compounds with enhanced anti-proliferation activity. The
design, synthesis, and evaluation of these new analogs against breast and prostate cancer
cell lines is reported herein, where it was found that 8b and 10 manifest potent anti-
proliferative activity and a robust degradation of Hsp90 client dependent proteins.
Keywords
Breast Cancer, Prostate Cancer, Heat Shock Protein 90, Hsp90 inhibitors, Novobiocin
Analogs
Heat Shock Protein 90 (Hsp90) is a molecular chaperone that is responsible for
the maturation of more than 300 nascent polypeptides.1-5 Hsp90-dependent client proteins
are associated with all 10 hallmarks of cancer, making it an excellent therapeutic target
for the treatment of cancer.6-8 As a result, 17 Hsp90 inhibitors have been investigated in
clinical trials for the treatment of cancer.9-13 Hsp90 exists as a homodimer and contains a
C-terminus, a middle domain, and an N-terminal ATP-binding site.3 In stressed cells,
Hsp90 exists as a heteroprotein complex bound to various co-chaperones and partner
proteins, and Hsp90 inhibitors exhibit 200-fold higher affinity for the heteroprotein
complex versus the homodimer that resides in non-stressed cells.14-20 Inhibition of Hsp90
by traditional inhibitors that bind the N-terminal ATP-binding site results in induction of
the heat shock response (HSR) and the overexpression of Hsp27, Hsp40, Hsp70,
Hsp90.21-23 Since N-terminal inhibitors induce this HSR, challenges associated with
dosing and scheduling have arisen in the clinic.9, 13, 24, 25 As a result, new inhibitors that
do not induce the heat shock response are actively sought.26, 27
The natural product, novobiocin, was the first Hsp90 C-terminal inhibitor
identified, 28, 29 and preliminary SAR studies resulted in the identification of KU-32, a C-
2

terminal inhibitor that contains an acetamide in lieu of the benzamide side chain and the
23, 30
acetamide on noviose sugar.
Interestingly, KU-32 induces the HSR, but at
significantly lower concentrations than that needed to induce the degradation of client
proteins.31, 32 Since KU-32 was able to induce the pro-survival HSR, it was investigated
for the treatment of neurodegenerative diseases. Studies in mice demonstrated that KU-32
was able to protect against neuronal glucotoxicity and reverse the clinical endpoints of
diabetic peripheral neuropathy.33 A second generation of noviosylated analogs contained
a biphenyl scaffold in lieu of the coumarin ring, and allowed the preparation of additional
analogs. KU-596, the lead compound resulting from this latter generation, was found to
exhibit comparable biological activity to KU-32.34, 35 Since the biphenyl scaffold resulted
in improved neuroprotection, compounds containing this new core were investigated for
the treatment of cancer. An initial study found that the biphenyl scaffold in KU-596
could be modified to append a biaryl side chain in lieu of the acetamide as well as the
replacement of the noviose sugar with N-methyl piperidine to produce compounds that
manifested anti-cancer activity without induction of the HSR.35 Since modifications on
the central biaryl ring have not been investigated, analogs were prepared to determine
whether such modifications could improve the anti-cancer activity of this class of
compounds, Figure 1.
3

Figure 1. Rationale for the development of new analogs.
Molecular modeling studies were performed with a homology model of the C-
terminal binding site to identify potential interactions with the biaryl core. Two
hydrogen-bonding interactions appeared attainable by inclusion of a phenol onto the
central biphenyl ring as suggested in Figure 2. Molecular modeling studies suggested that
compounds containing substituents pointing toward amino acids Glu537 and Thr540
could also form hydrogen-bonding interactions, and consequently may be more
efficacious. In silico studies suggested that the attachment of a methyl group onto the
biphenyl core could produce hydrophobic interactions within the binding site and
increase affinity. These interactions might be similar to previously studied Hsp90 C-
terminal inhibitors that contain a coumarin core. Additionally, incorporation of a methyl
4

group may reinstate interactions lost upon transitioning from the coumarin core to the
biaryl ring system.36-38 Consequently, analogs were sought to include such modifications.
Thr-540
Glu-537
OMe
OMe
OH
H
N
H
N
OMe
OMe
O
O
O
O
OH
10
O
O
MeO
9
MeO
HO
F
HO
F
OH
OH
Figure 2. Molecular modeling of compounds 9 and 10 docked into the Hsp90 C-terminal
homology model.39
Derivatives of the central ring were selected for initial investigation, as illustrated
in Scheme 1A. Synthesis of each derivative began via a different core. Synthesis of the
derivative containing a methyl substituent (19) began by reduction of 2,4-
dihydroxybenzaldehyde, 12, with sodium cyanoborohydride in acidic tetrahydrofuran to
yield 13 (Scheme 1B).40 Silver triflate and dichloro-methyl methyl ether were then used
to formylate 13, which produced aldehyde 14.41 The resulting methylbenzaldehyde, 14,
was then benzylated to give 15, which was then transformed unto the
5

trifluoromethanesulfonate derivative under microwave conditions.42 Subsequent Suzuki
coupling of 16 with 3-fluorophenyl boronic acid generated the biaryl core of 17. Henry
olefination conditions were used to homologate the aldehyde into the α,β-unsaturated
nitro compound 18, which was subsequently reduced to provide amine 19.
Scheme 1. A. Proposed analogs with substitutions off of the central ring. B. Synthesis of
free amine, 19. a). NaBH3CN, HCl, THF, 90%; b). AgOTf, Cl2CHOMe, DCM, 40%; c).
NaHCO3, BnBr, MeCN, 70%; d). N-PhTf2,K2CO3, THF, 120 oC, microwave, 95%; e).
FC6H4B(OH)2, Pd(dppf)Cl2, K2CO3, DMF, 75%; f). NH4OAc, CH3NO2, 90%; g). LAH,
THF, 60%.
The remaining analogs required alternative routes for their preparation, however
installment of the triflate and subsequent steps to yield the free amine were identical to
the conditions described for 19, as outlined in Scheme 1B. Synthesis of the methoxy and
phenol derivatives began by formylation of phloroglucinol, followed by selective benzyl
ether formation to give 20 (Scheme 2A and B). Aldehyde 20 was then treated with
dimethyl sulfide in water and acetone to yield 21 (Scheme 2A).43 Intermediate 21 was
6

subjected to conditions previously outlined in Scheme 1B to produce amine 22. In order
to obtain the phenol, derivative 20 was treated with chloromethyl methyl ether to produce
23 (Scheme 2B),44, 45 which was then subjected to conditions described in Scheme 1B to
attain amine 24. Synthesis of amine 27 began by selective benzylation of 2,3,4-
trihydroxybenzaldehyde, which was subsequently reacted with chloromethyl methyl ether
and diisopropylamine to yield aldehyde 26 (Scheme 2C). Compound 26 was then
subjected to the conditions described in scheme 1B to afford amine 27. Amine 30, was
attained via selective benzylation of 2,4-dihydroxy-3-methyl benzaldehyde to yield ether
29 (Scheme 2D), which was subjected to previously described methods to produce amine
30. Final derivative 33 was synthesized by subjecting 2-nitrobenzene-1,3-diphenol to
Duff reaction conditions to yield 31 (Scheme 2E). Aldehyde 31 then underwent selective
benzylation to produce the key intermediate, 32, which was subsequently modified to
give free amine 33.
7

Scheme 2. Synthesis of free amines 22, 24, 27, 30, and 33. Conditions: a). Me2SO4,
K2CO3, H2O/Acetone (1/1), 55%; b). MOMCl, DIPA, DCM, 65%; c). NaHCO3, BnBr,
MeCN, 45%; d). MOMCl, DIPEA, DCM, 70%; e). NaHCO3, BnBr, KI, MeCN, 75%.
Two different amide side chains were investigated with these analogs, the original
prenylated side chain present in novobiocin and the optimized biaryl side chain found in
4. Additionally, compounds were made to contain either the noviose sugar or the noviose
surrogate, N-methyl piperidine.46 Installation of the biaryl side chain began by an acid
chloride coupling of previously synthesized 3436 with the free amine of each central ring
derivative (Scheme 3A). Once the coupled product was obtained (35-40), hydrogenolysis
was employed to produce the free phenols, 41-46. The free phenol was then coupled
with the activated noviose sugar (47),47 using Schmidt conditions.48 The carbonate was
then removed under solvolysis conditions to procure the corresponding noviose products,
8

6-10.49 Simultaneously, free phenols 41-46 were subjected to Mitsunobu conditions with
1-methylpiperidin-4-ol (48) to yield the amine-containing products, 6a-11a (Scheme 3A).
Scheme 3. A. Synthesis of final products 6-10 and 6a-11a. B. Synthesis of final products
6b-9b. Conditions: a). DMAP, Pyridine, DCM, 60%; b). H2 (g), Pd/C, EtOAc, 70%; c).
BF3 OEt, DCM, 40%; d). Et3N, MeOH, 85%; e). Ph3P, DIAD, THF, 30%; f).DMAP,
Et3N, DCM, 35%; g). Pd(OAc), Et3Si, Et3N, DCM, 75%; h). TBAF, THF, 0 oC, 15 min,
60%; i). Ph3P, DIAD, THF, 30%; j). K2CO3, MeOH,60%.
Analogs that contained the prenylated side chain were synthesized by an acid
chloride coupling reaction between 5137 and the free amine of each analogue (Scheme
3B). Coupled products 52-55 were subjected to Pd(OAc)Cl2, Et3SiH, Et3N, to give the
9

silyl protected phenols. Upon cleavage of the silyl ether with TBAF at 0oC, 56-59 were
obtained in good yields (Scheme 3B). The resulting phenols, 56-59, were then subjected
to a Mitsunobu reaction with 48, to give the amino products, 6b-9b, respectively.
Upon preparation of these new analogs, an MTS50 anti-proliferation assay was
conducted against two breast cancer cell lines, MCF-7 (estrogen receptor positive cells)
and SkBr3 (estrogen receptor negative HER2 overexpressing), and two prostrate cancer
cell lines, LNCap-LN3 (androgen receptor sensitive prostate cancer cells) and PC3-MM2
(androgen receptor insensitive prostate cancer cells). These cell lines were selected
because the oncogenic drivers of these cancers are Hsp90-dependent client proteins:
estrogen receptor, HER-2, and the androgen receptor.50 All new analogs exhibited
moderate anti-proliferative activity against the 4 cancer cell lines as detailed in Table 1.
Compounds containing an N-methyl piperidine exhibited greater inhibitory activity than
those containing noviose. Additionally, compounds with a prenylated amide side chain
were found to be more efficacious than compounds that contain the biaryl amide side
chain.
Table 1. Anti-proliferative values of new analogs
SkBr3a
Compound
X
Y
Z
R R1
MCF7a
LNCap- PC3-
LN3
(µM)
>500
MM2
(µM)
>500
(µM)
(µM)
3
H
H
H
A
-
>500
>500
10

5
5a
6
H
H
H
H
H
H
A
A
A
A
B
A
A
B
A
A
B
A
A
B
A
A
A
A
B
A
B
B
A
B
B
A
B
B
A
B
B
A
B
B
>50
15.4 1.75 23.7 0.04
>50 43.9 4.4
>50
15.9
22.5
>100
20.8
>100
8.09
>100
>100
>50
>50
5.49
>100
13.9
>100
10.6
32.1
1.84
>50
CH3
CH3
CH3
H
H
H
6a
6b
7
H
H
9.14 2.72 7.81 1.1
35.6 3.03 15.2 1.67
44.7 4.15 49.4 2.03
8.71 1.20 5.72 0.89
7.86 0.20 8.16 0.29
23.8 0.47 23.5 0.44
12.2 0.89 15.7 1.5
5.89 0.67 7.59 0.58
13.6 2.01 16.7 0.93
11.3 1.62 9.29 1.07
1.45 0.87 3.25 1.2
8.99 0.28 43.5 1.03
17.8 1.48 21.3 2.82
H
H
OCH3
OCH3
OCH3
OH
OH
OH
H
H
7a
7b
8
H
H
H
H
H
H
8a
8b
9
H
H
44.8
22.8
>100
22.4
23.1
25.1
>50
>50
H
H
1.32
9.37
8.61
42.3
>50
H
OH
OH
OH
CH3
CH3
NH2
9a
9b
10
10a
11a
H
H
H
H
H
H
H
H
>50
H
H
11.3 1.3
15.3 2.3
22.4
18.9
a
Values represent mean standard deviation for at least three separate experiments performed in
triplicate.
Interestingly, the synthesized analogs followed the same trend in activity
independent of the sugar surrogate or amide side chain. The methyl derivatives were
more active than the parent compound; the methoxy derivatives were more active than
the methyl derivatives, and the phenol derivatives more active than both the methoxy and
the methyl derivatives. Analogs that contained the sugar surrogate, N-methyl piperidine,
were more active than the analogs containing the noviose sugar. Additionally, analogs
that contained the prenylated side chain were more active than analogs containing the
biaryl side chain. Analogue 6 was similarly active to the parent compound, 5. However
11

analogs 6a and 6b, which contained N-methyl piperidine in lieu of noviose, were more
active than 6. In addition, analogs containing moieties that could participate in hydrogen
bonding interactions manifested improved inhibitory activity as compared to 5.
After determination of IC50 values, compounds 8a, 9, and 10 were further
evaluated by western blot analysis of MCF7 cell lysates. Compound 10 exhibited
significant Hsp90-dependent client protein degradation at concentrations that mirrored its
anti-proliferative activity, clearly linking Hsp90 inhibition to cell viability, as illustrated
in Figure 3. In fact, Hsp90 dependent clients Her-2, Cyclin D1, and c-Raf were degraded
upon treatment with 10 for 24 hours. 8b and 9 also demonstrated client protein
degradation, but to a lesser extent than 10. Treatment with analog 8b resulted in HER2
degradation at the lowest concentrations, which may be due to mechanisms of cellular
51, 52
resistance.
Importantly, no increase in Hsp90 levels was observed with these C-
terminal inhibitors, which is an important attribute manifested by C-terminal inhibitors
that exhibit anti-cancer activity.
Figure 3. Western Blot analysis from MCF-7 cells. The high concentration is five times
the IC50 value and the low concentration is half the IC50 value. Concentrations for each
12

analog tested were as follows: 8b (high: 29.45 µM, low: 2.945 µM), 9 (high: 68 µM, low:
6.8 µM), and 10 (high: 44.95 µM, low: 4.49 µM). The positive control was GDA
(500nM) and DMSO was used as the negative control.
In summary, a series of Hsp90 C-terminal inhibitors with modifications to the
central ring of the biphenyl scaffold were designed, synthesized, and biologically
evaluated. Modifications to the central ring produced compounds that exhibited
increased biological activity. Structure-activity relationship studies revealed that
modifications that include a hydrogen bond donor was most beneficial. Finally, western
blot analysis confirmed these compounds to manifest their anti-proliferative activity via
Hsp90 inhibition, as client protein degradation was observed at concentrations that
mirrored anti-proliferative activity.
Acknowledgment
The authors gratefully acknowledge support of this project by the NIH/NCI (CA120458).
The NMR support for this project was provided by NSF (9512331). The authors are
greatly thankful to Frontier Scientific, Inc. for providing high quality boronic acid for this
project.
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homolytic cleavage of carbon-oxygen bonds in radical anions obtained by electrochemical
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985-990.
49.
N-(2-(5-(((2R,3R,4S,5R)-3,4-dihydroxy-5-methoxy-6,6-dimethyltetrahydro-
2H-pyran-2-yl)oxy)-3'-fluoro-6-methyl-[1,1'-biphenyl]-2-yl)ethyl)-3',6-dimethoxy-
[1,1'-biphenyl]-3-carboxamide (10): Activated noviose sugar 47 in anhydrous DCM
(0.5mL) was added to a mixture of 45 (0.035g, 0.066mmol) in anhydrous DCM (0.5mL) at
rt. Boron trifluoride diethyl ethrate (0.07mL) was then added drop wise. The reaction was
monitored via TLC. Upon completion of the reaction (~1 hour), triethylamine (0.1mL) was
added. The mixture was concentrated and purified via column chromatography (SiO2, 3:1,
Hexane: EtOAc- 20:1, DCM:MeOH). The product was obtained as a white solid in 30% yield
and used directly in the next step. The product (0.019g, 0.03mmol) was dissolved in MeOH
(12mL) and triethylamine (4mL) was added. The mixture was stirred over night. After 12
hours the reaction was concentrated and purified via column chromatography (SiO2, 20:1,
DCM: MeOH). The product was obtained in 70% yield to produce a white solid: 1H NMR
(500 MHz, Chloroform-d) δ 7.69 (dd, J = 8.6, 2.4 Hz, 1H), 7.59 (t, J = 2.5 Hz, 1H), 7.33 (t, J =
8.0 Hz, 2H), 7.17 (dd, J = 8.6, 1.1 Hz, 1H), 7.12 (s, 1H), 7.08 – 7.00 (m, 3H), 6.97 (d, J = 8.6 Hz,
1H), 6.95 – 6.86 (m, 2H), 5.94 (td, J = 5.7, 2.0 Hz, 1H), 5.53 (d, J = 2.2 Hz, 1H), 4.23 (dd, J =
9.3, 3.2 Hz, 1H), 4.19 (t, J = 2.8 Hz, 1H), 3.84 (d, J = 3.0 Hz, 6H), 3.60 (s, 3H), 3.53 (d, J = 1.3
Hz, 1H), 3.49 – 3.38 (m, 2H), 3.39 – 3.31 (m, 1H), 2.81 – 2.77 (m, 1H), 2.64 (d, J = 25.0 Hz,
2H), 1.82 (s, 3H), 1.39 (s, 3H), 1.21 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 166.83, 163.76,
159.25, 158.91, 153.55, 142.44, 141.67, 138.96, 130.38, 130.05, 129.49, 129.38, 129.11,
128.00, 127.49, 126.87, 125.27, 121.97, 116.42, 115.30, 114.15, 112.93, 112.84, 110.81,
97.83, 71.45, 68.75, 61.92, 55.78, 55.34, 40.73, 32.90, 29.25, 22.61, 13.83. HRMS (ESI+) m/z:
[M + Na+] calculated for C38H42FNO8Na 682.2792; found 682.2806, 2.1ppm.
50.
Cells were maintained in Advanced DMEM/F12 (Gibco) media and grown in a
humidified atmosphere (37° C, 5% CO2). The cells were grown to confluence, seeded
(3000cells/well, 100 μL) in a 96-well plate, and placed in the incubator for 24 hours.
Compound or vehicle at varying concentrations in DMSO (1% DMSO final concentration)
were administered and then incubated for 72 hours. At 72 h, the % of viable cells was
determined using the MTS/PMS cell proliferation kit (Promega) per the manufacturer’s
instructions. Cells incubated with vehicle were normalized to 100% proliferation, and
values were adjusted accordingly.
51.
Nam SK, Yun S, Koh J, et al. BRAF, PIK3CA, and HER2 Oncogenic Alterations
According to KRAS Mutation Status in Advanced Colorectal Cancers with Distant Metastasis.
PloS one. 2016;11(3): e0151865.
52.
Pagliarini R, Shao W, Sellers WR. Oncogene addiction: pathways of therapeutic
response, resistance, and road maps toward a cure. EMBO reports. 2015;16: 280-296.
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