Development of Phenyl Cyclohexylcarboxamides as a Novel Class of Hsp90 C-terminal Inhibitors

Article abstract of DOI:10.1002/chem.201703206

Inhibition of the heat shock protein 90 (Hsp90) C-terminus represents a promising therapeutic strategy for the treatment of cancer. Novobiocin, a coumarin antibiotic, was the first Hsp90 C-terminal inhibitor identified, however, it manifested poor anti-proliferative activity (SKBr3, IC₅₀≈700 μm). Subsequent structure–activity relationship (SAR) studies on novobiocin led to development of several analogues that exhibited improved anti-proliferative activity against several cancer cell lines. Recent studies demonstrate that the biphenyl core could be used in lieu of the coumarin ring system, which resulted in more efficacious analogues. In continuation of previous efforts, the work described herein has identified the phenyl cyclohexyl core as a novel scaffold for Hsp90 C-terminal inhibition. Structure–activity relationship (SAR) studies on this scaffold led to the development of compounds that manifest mid-nanomolar activity against SKBr3 and MCF-7 breast cancer cell lines through Hsp90 inhibition.

Full text of DOI:10.1002/chem.201703206

A Journal of
Accepted Article
Title: Development of Phenyl Cyclohexylcarboxamides as a Novel
Class of Hsp90 C-terminal Inhibitors
Authors: leah forsberg, gaurav garg, huiping zhao, and Brian Blagg
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10.1002/chem.201703206
Chemistry - A European Journal
Development of Phenyl Cyclohexylcarboxamides as a Novel Class
of Hsp90 C-terminal Inhibitors
Gaurav Garg,1‡ Leah K. Forsberg,1‡ Huiping Zhao,1 and Brian S. J. Blagg1*
1. Department of Medicinal Chemistry, 1251 Wescoe Hall Drive, Malott 4070,
The University of Kansas, Lawrence, Kansas 66045-7563, USA
* Author to whom correspondence should be addressed. Phone: (785) 864-
2288. Fax: (785) 864-5326. Email: bblagg@ku.edu.
‡ Authors contributed equally
Author(s), Corresponding Author(s)*
Page No. – Page No.
Title
Backbone optimization: The central core was explored to identify an optimized
scaffold for Hsp90 C-terminal inhibition. Structural investigations led to the
development of phenyl cyclohexyl carboxamides as a novel class of Hsp90 C-
terminal inhibitors. This new scaffold exhibits improved biological activity and
provides a new template for future Hsp90 inhibitors.
Abstract
Inhibition of the heat shock protein 90 (Hsp90) C-terminus represents a promising
therapeutic strategy for the treatment of cancer. Novobiocin, a coumarin antibiotic, was
the first Hsp90 C-terminal inhibitor identified, however, it manifested poor anti-
proliferative activity (SKBr3, IC50 ~ 700 µM). Subsequent SAR studies on novobiocin
led to development of several analogues that exhibited improved anti-proliferative
activity against several cancer cell lines. Recently, we demonstrated that the biphenyl
core could be used in lieu of the coumarin ring system, which resulted in more
efficacious analogues. In continuation of previous efforts, the work described herein
has identified the phenyl cyclohexyl core as a novel scaffold for Hsp90 C-terminal
inhibition. SAR studies on this scaffold led to the development of compounds that
manifest mid-nanomolar activity against SKBr3 and MCF-7 breast cancer cell lines
through Hsp90 inhibition.
Keywords: Heat shock protein 90; Hsp90 C-terminal inhibitors; Phenyl
Cyclohexylcarboxamides; Structure-activity relationship; Breast cancer.
1
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1. Introduction
Heat shock protein 90 (Hsp90) is an evolutionarily conserved molecular
chaperone that plays a critical role in the maintenance of protein homeostasis as well
as adaptive response to cell stress.[1] Hsp90 is a core component of the protein folding
machinery that regulates the folding, stability, function, and proteolytic turnover of
more than 300 client proteins, including protein kinases, transcription factors, and
signal transducers.[2] These client proteins control a wide range of cellular functions,
such as cell signaling, protein trafficking, chromatin remodeling, cell proliferation and
survival.[2-3] However, in cancer, these clients are frequently mutated and/or over-
expressed to drive oncogenic processes, such as dysregulated proliferation,
metastasis, and angiogenesis. In fact, ~25% of the Hsp90-dependent clients (eg. Her2,
CDK6, Raf1, Akt, survivin, telomerase) represent oncoproteins that are directly
associated with all ten hallmarks of cancer.[3a, 4] Moreover, Hsp90 is overexpressed in
cancer cells to stabilize these oncoproteins against proteotoxic stresses and to fold
mutants that enable the growth and/or survival of tumorigenic cells.[5] Consequently,
Hsp90 inhibition provides an opportunity to simultaneously disrupt multiple oncogenic
pathways that are required for cancer cell survival. As a result, Hsp90 has emerged as
a promising therapeutic target for the development of cancer chemotherapeutics.[6] 17
Small molecules that target the Hsp90 N-terminus have entered clinical trials for the
treatment of cancer, demonstrating proof-of-concept for Hsp90 inhibitors as potential
anticancer agents.[7] Although these molecules have shown some promising clinical
responses, several concerns have arisen such as concomitant induction of the pro-
survival heat shock response (HSR), hepatotoxicity, and cardiotoxicity amongst others,
which represent additional risks that must be overcome during the development of new
inhibitors.[3b] Therefore, small molecules that modulate Hsp90 via alternative
mechanisms represent a novel approach towards the evolution of Hsp90 inhibitors.[3b, 8]
In 2000, Neckers and co-workers discovered that novobiocin, a clinically used
antibiotic and DNA gyrase inhibitor, binds the Hsp90 C-terminus, and allosterically
inhibits Hsp90 function.[9] Importantly, novobiocin and related natural products do not
induce the HSR and therefore, can avoid the clinical limitations associated with Hsp90
N-terminal inhibitors. Unfortunately, novobiocin exhibits low cellular activity (SKBr3 IC50
~ 700 µM) and thus, has limited therapeutic potential. Preliminary studies with
novobiocin identified key structural features required for Hsp90 inhibitory activity,
which led to analogues with improved inhibitory activity.[10] Subsequent studies
2
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revealed that the benzamide side chain of novobiocin is important for anti-proliferative
activity and modification of this side chain produced several promising compounds,
such as KU174 (Figure 1).[11] Additional studies demonstrated that the
stereochemically complex sugar moiety could be replaced with ionizable amines,
which produced analogues that manifest mid-nanomolar to low micromolar activity
against multiple cancer cell lines.[12] In contrast to these side chains, limited structural
investigations have been performed on the coumarin core of novobiocin.
Recent investigation of the novobiocin scaffold demonstrated that an aromatic
or heteroaromatic ring system could be used in lieu of the coumarin core without
compromising activity, suggesting that the central core may serve as a backbone to
orient both the benzamide and sugar/side chains within the binding pocket.[13]
Consequently, it was proposed that the coumarin core could be replaced with other
scaffolds to identify compounds upon which more potent inhibitors could be realized.
Moreover, the development of a structurally diverse set of compounds is necessary to
better understand the mechanism by which the C-terminal domain regulates the Hsp90
chaperone cycle. Towards this objective, we discovered that the coumarin core of
novobiocin could be replaced with a biphenyl scaffold, which led to analogues that
exhibit potent anti-proliferative activity.[14] More recently, we demonstrated that a
stilbene core (Figure 1) could be used in lieu of the coumarin core as well.[15] In
continuation of these studies, the current work identifies a novel core, which has led to
the development of more efficacious Hsp90 inhibitors.
Benzamide
OH
OMe
OH
O
H
N
H
N
Improtant for
hydrophobic
interactions
OMe
Noviose Sugar
O
O
O
O
O
O
O
OMe
O
O
MeO
Coumarin
Improtant for MeO
interactions
with Hsp90
KU174 (2)
(SkBr3, IC50 ~14 µM)
O
O
HO
OH
OH
Novobiocin (1)
(SkBr3, IC50 ~700 µM)
NH2
OMe
OMe
H
N
OMe
H
N
OMe
O
N
O
N
O
4
O
KU820(3)
(SkBr3, IC50 ~0.5 µM)
(SkBr3, IC50 ~0.7 µM)
Figure 1. HSP90 C-terminal inhibitors.
3
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2. Results and Discussion
2.1 Design, synthesis and evaluation of biphenyl mimics-
Prior SAR studies on novobiocin have revealed the benzamide side chain and
sugar are critical for Hsp90 inhibitory activity and that modification to these moieties
has produced several promising compounds, such as 5, which manifests improved
activity against several cancer cell lines.[11-12, 16] Recently, it was demonstrated that a
biphenyl scaffold could be used in lieu of the coumarin core, which also led to potent
inhibitors, such as KU820 (Figure 2), presumably due to the favorable conformation
adopted by the relatively flexible biphenyl core within the binding pocket.[14a, 17] In
addition, it was determined that the planarity and flexibility of the central core are
important for Hsp90 inhibitory activity.[15] Therefore, it was hypothesized that the
central core could be modified to optimize orientation of the side chains for increased
inhibitory activity. Since biphenyl analogues manifest superior activity, this scaffold
was chosen as the starting point for the discovery of more efficacious Hsp90 inhibitors.
Increasing evidence suggests that molecules with aromatic scaffolds offer
limited spatial diversity, while the incorporation of a saturated ring system can improve
drug-like properties, such as ligand/receptor interactions as well as solubility.[18] As a
flexible structure, the saturated ring system has the ability to adopt multiple
conformations and to project substituents into optimal orientations within the binding
pocket. Therefore, optimization of the core began by replacement of the A- and/or B-
ring of the biphenyl core with a saturated ring system. The previously optimized biaryl
amide side chain and N-methylpiperidine present in 3 were appended to these new
cores for evaluation of preliminary structure-activity relationships.
4
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Benzamide
Coumarin
OH
OH
H
N
Noviose Sugar
O
O
O
O
O
MeO
Novobiocin (1)
(SkBr3, IC50 ~700 µM)
O
O
OH
NH2
OMe
H
N
OMe
N
O
O
O
O
5
(SKBr3 ~ 1.0 µM)
OMe
H
N
OMe
B
O
N
A
O
KU820 (3)
(SKBr3 ~ 0.5 µM)
OMe
H
N
OMe
O
N
O
Figure 2. Rationale for proposed molecules.
As shown in Scheme 1, the preparation of analogues that contain a saturated
B-ring (15a and 15b) commenced via benzyl-protection of phenol 6 to afford ketone 7,
which was then stereoselectively reduced with either L-selectride or sodium
borohydride to give the syn (8a) or anti (8b) diastereomers in >90% de, respectively.
Inversion of stereochemistry was made possible by mesylation of the alcohols (8a,
8b), followed by SN2 substitution with sodium azide to yield 10a and 10b. Reduction of
the azides with palladium on carbon under a hydrogen atmosphere gave amines 11a
and 11b, which were then coupled with biaryl acid 12 using standard coupling
conditions to afford the corresponding amides, 13a and 13b. Mitsunobu etherification
of the resulting amides with 1-methyl-4-hydroxypiperidine (14a) gave the desired
products, 15a and 15b, in moderate yields.
5
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O
OMs
O
OH
b
c
a
BnO
N3
BnO
HO
BnO
9a = Syn
9b = Anti
8a = Syn
8b = Anti
7
6
NH2
OMe
f
d
e
HO
O
OMe
+
BnO
HO
10a = Anti
10b = Syn
11a = Anti
11b = Syn
12
OMe
OMe
H
N
H
N
OMe
OMe
g
O
O
N
15a = Anti
15b = Syn
13a = Anti
13b = Syn
O
HO
Scheme 1. Synthesis of phenylcyclohexyl carboxamides. Reagents and conditions: a BnBr, K2CO3, acetone, 0 oC to rt,
12 h, 95%; b L-selectride, THF, -40 oC to rt, 12 h, 92% or NaBH4, MeOH, 0 oC to rt, 12 h, 90%; c MsCl, Et3N, DCM, 0
o
oC to rt, 4 h, 92 − 94%; d NaN3, DMF, 100 C, 12 h, 80 − 85%; e 10% Pd(OH)2, H2, MeOH/THF, rt, 12 h, ~100%; f
EDCI•HCl, Et3N, DCM, 0 oC to rt, 12 h, 45 − 50%; g 1-Methylpiperdin-4-ol (14a), DIAD, PPh3, THF, 0 oC to rt, 12 h, 36 −
40%.
In addition, an analogue that contains a piperidine ring (21) was designed to
determine whether orientation of the cyclohexyl ring is important for activity. Synthesis
of 21 was achieved via a Buchwald coupling between aryl bromide 16 and piperidine
17 to produce 18, which then underwent hydrogenolysis with palladium hydroxide
under a hydrogen atmosphere to give the free phenol, 19 (Scheme 2). Mitsunobu
etherification of 19 with 1-methyl-4-hydroxypiperdine (14a) yielded the N-Boc-
protected amine 20, which was then treated with trifluoroacetic acid to remove the
Boc- protecting group before the resulting amine was coupled with biaryl acid 12 to
afford the desired product, 21.
H
H
H
N
N
I
N
Boc
Boc
Boc
b
c
a
N
N
+
HN
BnO
19
17
BnO
18
16
HO
OMe
H
N
H
Boc
N
OMe
d
N
N
N
O
N
O
21
O
20
t
Scheme 2. Synthesis of phenylpiperdin-4-yl carboxamide. Reagents and conditions: a Pd2(dba)3, X-Phos, BuONa,
toluene, 130 oC, 2 h, m.w., 37%; b 10% Pd(OH)2, H2, MeOH/THF, rt, 12 h, ~100%; c 14a, DIAD, PPh3, THF, 0 oC to rt,
12 h, 30%; d i. 30% TFA/DCM, 0 oC to rt, 4 h, ~100%; ii. EDCI•HCl, Et3N, DCM, 0 oC to rt, 12 h, 55%.
In parallel, an analogue containing a saturated A-ring (31) was prepared as
illustrated in Scheme 3. Synthesis of compound 31 was initiated by selective
6
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benzylation of cyclohexane-1,4-diol to give 23,[19] which was then oxidized with
pyridinium chlorochromate to yield ketone 24.[20] The ketone was then converted to the
vinyl triflate (25), before Suzuki coupling with boronic acid 26 to give the cyclohexyl
phenyl core, 27. Acid-catalyzed hydrolysis of the Boc-protecting group on 27 yielded
aniline 28, which underwent an amide coupling reaction with acid chloride 12 to afford
29. Hydrogenolysis of 29 with palladium on carbon under a hydrogen atmosphere
gave the free alcohol, 30, which underwent an SN2 substitution reaction with 14b to
afford 31 in moderate yield. Following a similar protocol as standardized for 15a and
15b, compound 37 was prepared to contain two cyclohexyl rings as shown in Scheme
4.
OH
NHBoc
OTf
OH
O
a
c
d
b
HO
(HO)2B
BnO
BnO
BnO
22
23
24
26
25
OMe
NHBoc
H
N
NH2
OMe
OMe
e
f
g
O
BnO
N
29
BnO
27
28
OMe
BnO
H
N
H
N
OMe
OMe
h
O
O
N
OTs
30
14b
31
O
HO
Scheme 3. Synthesis of a cyclohexylphenylamide. Reagents and conditions: a BnBr, NaH, DMF, 0 oC to rt, 12 h, 70%;
o
b PCC, DCM, rt, 12 h, 50%; c N-Ph2Tf, LDA, THF, 55%; d Pd(dppf)Cl2, Cs2CO3, DMF, 100 C, 12 h,30%; e 30% TFA,
o
DCM, rt, 12 h, ~100%; f 12, EDCI•HCl, Et3N, DCM, 0 C to rt, 12 h, 80%; g Pd(OH)2, H2, MeOH, 12 h, 40%; h K2CO3,
DMF, 90 oC, 48 h, 25%.
N3
OH
OMs
NH2
c
d
b
a
HO
HO
N
HO
33
35
HO
32
34
OMe
OMe
H
H
N
OMe
N
OMe
e
O
O
N
OTs
14b
O
37
36
HO
Scheme 4. Synthesis of a cyclohexyl derivative. Reagents and conditions: a MsCl, Et3N, DCM, 0 oC to rt, 12 h, 90%; b
NaN3, DMF, 100 oC, 12 h, 30%; c Pd/C, H2, MeOH, 12 h, ~100%; d 12a, DIPEA, DCM, 0 oC to rt, 12 h, 60%; e K2CO3,
DMF, 90 oC, 48 h, 25%.
Upon construction, analogues containing saturated A- and/or B-rings were
evaluated for their anti-proliferative activity against two cancer cell lines, SKBr3
7
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(estrogen receptor negative, Her2 overexpressing breast cancer cells) and MCF-7
(estrogen receptor positive breast cancer cells). As shown in Table 1, compound 15a
(anti) exhibited 4-fold greater anti-proliferative activity than the lead compound 3, and
10-15 fold better activity than 15b and 21, indicating the anti-stereochemistry is
important for anti-proliferative activity. Incorporation of a saturated ring into the A-
position was detrimental, as compounds 31 and 37 were both inactive up to 50µM,
suggesting that planarity of the A-ring is important for anti-proliferative activity.
Table 1. Anti-proliferative activity of analogues with the modified A- and/or B-rings.
OMe
H
N
OMe
B
O
N
A
O
SKBr3
(IC50, µM)
MCF-7
(IC50, µM)
Entry
3
Compound
0.47 ± 0.06 0.71 ± 0.02
0.17 ± 0.02 a 0.22 ± 0.01
-
OMe
OMe
OMe
OMe
OMe
H
N
OMe
15a
15b
21
O
N
O
H
N
OMe
OMe
OMe
OMe
2.57 ± 0.08 2.43 ± 0.05
O
N
O
H
N
2.87 ± 0.13
3.37 ± 0.14
N
O
O
O
N
O
H
N
31
>50
>50
>50
>50
N
O
H
N
37
N
O
aValues represent mean ± standard deviation for at least two separate experiments
performed in triplicate.
Preliminary SARs indicate that the phenyl A-ring is necessary for maintaining
anti-proliferative activity, while modifications to the B-ring could improve activity. Since
8
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no co-crystal structure of Hsp90 bound to C-terminal inhibitors has been solved, there
is limited knowledge about the size and nature of this binding pocket. Therefore, a
library of analogues containing the phenyl A-ring attached to various appendages in
lieu of the cyclohexyl B-ring was pursued to identify an optimal scaffold that could
orient both side chains and maximize interactions for increased inhibitory activity.
Recent studies have suggested that an optimal distance between 7.7 and 12.1 Å from
the amine to the amide is important for Hsp90 inhibitory activity.[15] Therefore,
analogues containing five-, six- and seven-membered rings with varying distances and
orientations of both the amine and amide were pursued.
The phenylpiperidine analogue (43), which represents a shorter distance
between the amine and amide, was prepared as described in Scheme 5. Synthesis of
the phenylpiperidine-1-yl core commenced via an amide coupling between piperidine
38 and biaryl acid 12 to give amide 39, which was converted to the triflate before
Suzuki coupling with boronic acid 41 to give 42. Hydrogenolysis of 42, followed by
Mitsunobu etherification of the resulting phenol with 1-methyl-4-hdroxypiperdine (14a)
gave 43.
OMe
OMe
OMe
OMe
OH
OH
b
a
N.HBr
+ BnO
B
TfO
N
O
N
O
O
O
38
39
41
OMe
40
OMe
OMe
OMe
N
c
d, e
O
N
BnO
N
O
O
42
43
Scheme 5. Synthesis of phenylpiperidin-1-yl carboxamide. Reagents and conditions: a 12, EDCI•HCl, Et3N, DCM, 0 oC
o
to rt, 12 h, 75%; b N-Ph2Tf, LDA, THF, -78 OC to rt, 12 h, 55%; c Pd(PPh3)4, K2CO3, toluene/EtOH/H2O, 110 C, 12 h,
81%; d 1,4-cyclohexadiene, MeOH, 70 oC, 48 h, 85%; e 14a, TMAD, PBu3, benzene, 90 oC, 12 h, 35%.
Preparation of analogues (53) that contain a five-membered saturated ring is
illustrated in Scheme 6. Synthesis began by a Heck coupling between aryl bromide 44
and cyclopent-2-enone to yield 45, which was subsequently reduced via palladium on
carbon and hydrogen gas to afford cyclopentanone 46.[21] Reduction of 46 with sodium
borohydride gave an inseparable mixture of anti and syn diastereomers, 47, in a 7:3
ratio, respectively. The mixture of 47 was converted to the methanesulfonate ester, 48,
before nucleophilic substitution with sodium azide to produce 49. Following reduction
of the azide, the resulting amine was coupled with biaryl acid 12 to form the
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corresponding amide 51. Removal of the methoxymethyl protecting group present in
51 provided the free phenol, 52. Mitsunobu etherification of the resulting phenol with 1-
methyl-4-hdroxypiperdine (14a) finally furnished the desired product 53 in moderate
yield.
O
O
OH
Br
c
d
a
b
MOMO
MOMO
44
MOMO
MOMO
45
46
47
OMe
OMe
NH2
NH2
OMs
f
e
g
HN
O
MOMO
MOMO
MOMO
51
49
48
50
MOMO
HN
OMe
OMe
OMe
OMe
i
h
HN
O
O
N
52
53
HO
O
Scheme 6. Synthesis of phenylcyclopentyl carboxamides. Reagents and conditions: a Cyclopent-2-en-1-one,
o
o
Pd(OAc)2, triethanolamine, toluene, 110 C, 12 h, 75%; b Pd/C, H2, EtOAc, rt, 12 h, ~100%; c NaBH4, MeOH, 0 C to
rt, 1 h, 90%; d MsCl, Et3N, THF, 0 oC to rt, 1 h, 90%; e. NaN3, DMF, 100 oC, 12 h, 85%; f. 10% Pd/C, H2, EtOAc, rt, 12
o
o
h, ~100%; g 12, EDCI•HCl, HOBt, DIPEA, DCM, 0 C to rt, 12 h, 80%; h 6N HCl, MeOH/THF, 0 C to rt, 12 h, 60%; i
14a, TMAD, PBu3, benzene, 90 oC, 12 h, 20%.
As shown in Scheme 7, synthesis of analogues that contain a seven-
membered ring began via a Grignard coupling reaction of commercially available ester
54 with allyl magnesium bromide to yield 55.[22] Ring-closing metastasis using Grubbs
first-generation catalyst, followed by removal of the hydroxyl group using triethylsilane
provided the desired phenylcycloheptyl core, 57. Oxidation of alkene 57 with meta-
chloroperoxybenzoic acid resulted in an inseparable mixture of epoxides 58a and 58b,
in a 1:1 ratio. Reduction of these epoxides with lithium aluminum hydride resulted in
alcohol 59, which upon acid-catalyzed cleavage of the methyl ether, provided 60. Next,
60 was converted to methanesulfonate ester 61 before nucleophilic substitution with
sodium azide and then subsequently reduced to afford amine 62. Amide coupling of 62
with acid chloride 12a produced 63, which after base-catalyzed deprotection of the
methanesulfonate group gave the free phenol, 64. Finally, Mitsunobu etherification of
the resulting phenol with 1-methyl-4-hdroxypiperdine (14a) furnished 65.
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O
O
OH
OH
OH
b
a
c
O
O
O
O
55
54
56
57
OH
O
O
d
e
f
+
O
O
58a
58b
HO
O
60
OMe
59
OMs
NH2
OMe
g
h, i
j
H
N
O
MsO
MsO
61
62
63
MsO
OMe
OMe
OMe
OMe
H
H
N
N
k
l
O
O
N
64
O
65
HO
Scheme 7. Synthesis of phenylcycloheptyl carboxamides. Reagents and conditions: a AllylMgBr, THF, 0 oC to rt, 12 h,
84%; b Grubbs I, DCM, 40 oC, 12 h, 54%; c Et3SiH, TFA, DCM, 48 h, 50%; d mCPBA, NaHCO3, DCM, 0 oC, 12 h, 89%;
e LAH, AlCl3, THF, 0 oC to rt, 12 h, 60%; f BBr3, DCM, -78 oC to rt, 2 h, 46%; g MsCl, Et3N, THF, 0 oC to rt, 1 h, 90%; h
o
o
NaN3, DMF, 100 C, 12 h, 40%; i Pd/C, H2, EtOAc, rt, 12 h, 90%; j 12a, DIPEA, DCM, 0 C to rt, 12 h, 90%; k 3.2 N
KOH, EtOH, 90 oC, 3 h, 60%; l 14a, TMAD, PBu3, benzene, 90 oC, 12 h, 32%.
Compounds (70 and 78) that contain a methylene linker were envisioned to
impart additional flexibility and explore potential binding interactions as a consequence
of the flexible benzamide side chain. As shown in Scheme 8, construction of the
phenylcyclopentyl methyl derivative (70) was accomplished by employing the
methanesulfonate ester 48 as a key intermediate. Briefly, 48 was first converted to
cyanide 66 via a nucleophilic substitution reaction with tetra-butylammonium cyanide
and then reduced with lithium aluminum hydride to afford the free amine, 67.[23] Amide
coupling of the resulting amine with acid chloride 12a gave the corresponding amide
68. Acid-catalyzed deprotection of the methoxymethyl-protecting group generated the
free phenol, 69, which underwent Mitsunobu etherification to yield an inseparable
mixture of anti and syn diastereomers of 70 in a 6:4 ratio respectively.
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OMs
CN
NH
NH2
OMe
O
b
a
c
OMe
MOMO
MOMO
MOMO
MOMO
68
48
66
67
NH
NH
O
OMe
OMe
O
f
d
OMe
OMe
N
HO
O
69
70
Scheme 8. Synthesis of phenylcyclopentyl methyl carboxamide. Reagents and conditions: a (CH3CH2CH2CH2)4N(CN),
o
o
o
DMF, 85 C, 12 h, 55%; b LAH, THF, 0 C to rt, 3 h, 88%; c 12, EDCI•HCl, HOBt, DIPEA, DCM, 0 C to rt, 80%; d p-
TSA, MeOH, rt, 12 h, 60%; e 14a, TMAD, PBu3, benzene, 90 oC, 12 h, 25%.
As outlined in Scheme 9, the phenylcyclohexyl methyl derivatives (78a and
78b) were assembled following the general strategy described earlier.
O
OH
OMs
O
b
a
c
73a=Syn
73b=Anti
HO
72a=Syn
72b=Anti
MOMO
NH2
MOMO
O
MOMO
71
6
CN
N
H
OMe
e
f
d
OMe
MOMO
75a=Anti
75b=Syn
74a=Anti
74b=Syn
MOMO
76a=Anti
76b=Syn
MOMO
O
O
N
H
OMe
g
h
N
H
OMe
N
OMe
OMe
77a=Anti
77b=Syn
78a=Anti
78b=Syn
O
HO
Scheme 9. Synthesis of phenylcyclohexyl methyl carboxamides. Reagents and condtions: a MOMCl, DIPEA, DCM, 0
oC to rt, 12 h, 65%; b L-selectride, THF, -40 oC to rt, 12 h, 70% or NABH4, MeOH, rt, 30 min, 85%; c MsCl, Et3N, DCM,
0 oC to rt, 12 h, 85 − 90%%; d (CH3CH2CH2CH2)4N(CN), DMF, 85 oC, 12 h, 40 − 49%; e Pd/C, H2, EtOAc, rt, 12 h, 65 −
o
o
70%%; f 12a, DIPEA, DCM, 0 C to rt, 12 h, 70 − 80%; g 6N HCl, MeOH, 0 C to rt, 12 h, 66 − 72%; h 14a, TMAD,
PBu3, benzene, 90 oC, 12 h, 31 − 39%.
Upon construction of the modified B-ring analogues, the compounds were
evaluated for their anti-proliferative activity against both breast cancer cell lines as
summarized in Table 2. In general, compounds containing other ring systems were
less active than the lead compound 15a, suggesting the 1,4-anti-cyclohexyl core is
optimal. Incorporation of either a five- or seven-membered ring system resulted in less
potent analogues as evidenced by 53 and 65, which were 25-40 fold less active than
15a, highlighting the orientation of the side chains for increased anti-proliferative
activity. Inclusion of a methylene linker (70, 78a and 78b vs 15a) did not improve
activity, suggesting that flexibility is not necessary for maximal activity. Surprisingly,
12
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10.1002/chem.201703206
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the stereochemistry of analogues 78a and 78b was not important for anti-proliferative
activity, as both the syn and anti-diastereomers manifested similar activities.
Overall, these studies suggest that the 1,4-anti-cyclohexyl core is optimal for
orientation of the appendages in a conformation that leads to increased anti-
proliferative activity. Therefore, molecules containing the phenyl cyclohexyl core were
further investigated.
Table 2. Anti-proliferative activity of analogues containing various central core.
OMe
H
N
OMe
O
N
O
SKBr3
(IC50, µM) (IC50, µM)
MCF-7
Entry
15a
Compound
0.17 ± 0.02 0.22 ± 0.01
N
43
55
>10
>10
5.46 ± 0.94 9.14 ± 0.69
3.51 ± 0.14 4.17 ± 0.01
65
70
4.11 ± 0.61 6.62 ± 0.15
78a
78b
2.98 ± 0.43 3.16 ± 0.31
4.13 ± 0.74 5.0 ± 0.29
2.2 Exploration of the amine moiety. Prior studies on novobiocin have demonstrated
that the noviose sugar or a surrogate is necessary for Hsp90 inhibitory activity and that
modifications to this moiety can improve activity. Therefore, a concise library of
13
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phenylcyclohexyl analogues containing various amines or the noviose sugar was
designed to probe linker length, hydrogen-bonding interactions, and steric bulk. As
shown in Scheme 10, analogues (80-82) containing various linker lengths were
synthesized by Mitsunobu etherification of phenol 13a with the corresponding amino
alcohols. Synthesis of the propylene-tethered amines (83-87) was accomplished by
O-alkylation of 13a with 1,3-dibromopropane to give ether 79, which was then treated
with primary or secondary amines to yield the desired products in moderate yield.
Compound 89 was prepared by coupling phenol 13a with the trichloroacetimidate of
noviose carbonate (88) in the presence of boron trifluroide etherate, followed by
solvolysis of the cyclic carbonate.
OMe
OMe
H
H
N
N
OMe
OMe
a
O
O
R2R1N
OMe
O
HO
n
13a
80-82
n = 0, 1, 2
OMe
OMe
H
H
N
N
OMe
b
c
13a
13a
O
O
R2R1N
O
Br
O
83-87
79
OMe
H
N
d, e
NH
OMe
CCl3
O
O
O
MeO
O
88
89
O
O
O
MeO
O
HO
OH
Scheme 10. Synthesis of phenylcyclohexyl carboxamides containing various amines. Reagents and conditions: a
o
alcohol, TMAD, PBu3, benzene, 90 OC, 12 h, 38 - 60%; b 1,3-dibromopropane, K2CO3, DMF, 0 C to rt, 12 h, 68%; c
amine, K2CO3, DMF, rt, 12 h, 48 − 68%; d 88, BF3OEt2, DCM, rt, 12 h, 55%; e 2%Et3N/MeOH, rt, 12 h, 68%.
Upon construction of the library, the individual compounds were evaluated
against SKBr3 and MCF-7 cancer cell lines as summarized in Table 3. Acyclic amines
(80, 81) exhibited comparable anti-proliferative activity as the cyclic amine, 15a, while
homologation of the linker resulted in decreased activity (82 vs 15a), indicating the 3-
carbon chain is optimal for anti-proliferative activity. The incorporation of bulky
substituents (83, 86, 87) maintained activity, indicating that various substituents could
be accommodated within the binding pocket. Interestingly, the inclusion of a second
hydrogen bond donor/acceptor (84, 85) produced activity comparable to 83. In
addition, secondary amines (86, 87) manifested similar activity as the tertiary amines.
14
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Chemistry - A European Journal
Surprisingly, compound 89 exhibited 5-20 fold decreased activity compared to 15a,
indicating the phenyl cyclohexyl core may bind differently than its coumarin
counterpart.
Table 3. Anti-proliferative activity of phenylcyclohexyl carboxamides with modified side
chain.
OMe
H
N
OMe
O
RO
SKBr3
(IC50, µM)
MCF-7
(IC50, µM)
S. No.
15a
R
N
0.17 ± 0.02 0.22 ± 0.01
0.25 ± 0.05 0.34 ± 0.02
N
80
81
82
83
84
85
86
87
N
0.27 ± 0.05 0.36 ± 0.01
0.82 ± 0.05 1.28 ± 0.71
N
N
0.28 ± 0.09 0.34 ± 0.01
0.40 ± 0.04 0.41 ± 0.08
0.32 ± 0.02 0.46 ± 0.01
0.33 ± 0.04 0.36 ± 0.01
0.40 ± 0.06 0.57 ± 0.01
N
HN
N
N
N
H
N
H
O
O
MeO
89
4.52 ± 0.25 1.20 ± 0.09
HO
OH
15
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2.3 Exploration of the benzamide side chain using optimal core and amines.
Exploration of the benzamide side chain was also pursued with the optimized
core and amine. Earlier structural investigations on the benzamide identified several
side chains that contained either a prenyl, indole, triazole or biaryl moiety, all of which
manifested mid-nanomolar to low micromolar activity against several cancer cell lines
while attached to the coumarin scaffold. Therefore, a focused library of phenyl
cyclohexyl derivatives was designed to contain these previously identified side chains
in an effort to gain additional interactions with the binding pocket. In addition, structural
investigation of the biaryl side chain was sought to identify locations to which
modifications can be made. As shown in Scheme 11, synthesis of the phenylcyclohexyl
analogues began by reduction of commercially available ketone 6, with L-selectride to
yield alcohol 90, which was then converted to the methanesulfonate ester 91 before
stereochemical inversion with sodium azide to afford 92.[24] Base-catalyzed
deprotection of the phenol, followed by Mitsunobu etherification with 1-methyl-4-
hydroxypiperidine (14a) produced azide 94. The azide was reduced via palladium on
carbon under a hydrogen atmosphere to give amine 95, which was then coupled with
acid chlorides 12g-12p to give amides 96-105, in moderate to low yields.
OMs
N3
O
OH
c
b
a
MsO
MsO
HO
HO
6
91
92
NH2
90
N3
N3
d
g
e
f
N
N
HO
O
O
93
95
94
H
N
Ar
O
N
O
96, 98 - 105
97
h
Scheme 11. Synthesis of phenylcyclohexyl derivatives that contain modified benzamide side chains. Reagents and
o
o
conditions: a L-selectride, THF, -78 C to rt, 12 h, 90%; b MsCl, Et3N, DCM, 0 C to rt, 12 h, 90%; c NaN3, DMF, 100
oC, 12 h, 65%; d 3.2 N KOH, ethanol, 90 oC, 12 h, 65%; e 14a, PPh3, DIAD, THF, 0 oC to rt, 12 h, 60%; f 10% Pd/C, H2,
MeOH/EtOAc, rt, 12 h, ~100%; g Ar-COCl, Et3N, DCM, 0 oC to rt, 12 h, 65 − 80%; h K2CO3, MeOH, rt, 1 h, 78%.
Upon completion, the library of phenylcyclohexyl analogues that contain
modified side chains was evaluated for antiproliferative activity against both breast
cancer cell lines. As shown in Table 4, the incorporation of prenylated, triazole or
indole (96-99 Vs 15a) side chains onto the phenylcyclohexyl core resulted in
significantly decreased activity compared to the biaryl ring system, suggesting such
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modifications are not well tolerated. These results suggest that the phenyl cyclohexyl
core projects the amine and benzamide side chains into a different orientation as
compared to the biphenyl and coumarin cores. The results also suggest that the 4-
methoxy is important for activity as compound 100 showed similar activity, while
compounds 101 and 102 exhibited 5-6 fold less activity compared to 15a. Removal of
the second phenyl ring resulted in a 20-40 fold loss in activity (103 vs 15a),
emphasizing a requirement for both aryl rings. In addition, the results suggest that a
hydrogen-bond donor at the para-position is beneficial, as replacement of the methoxy
group with either hydroxyl (104) or acetoxy (105) resulted in decreased activity.
Table 4. Anti-proliferative activity of cyclohexylphenylamides with modified side chain.
H
N
Ar
O
N
O
SKBr3
(IC50, µM)
MCF-7
(IC50, µM)
Entry
Ar
OMe
OMe
0.17 ± 0.02 0.22 ± 0.01
3.21 ± 0.19 2.61 ± 0.21
15a
OAc
96
97
OH
1.54 ± 0.04 2.34 ± 1.41
>10
>10
98
99
N
H
OMe
1.16 ± 0.36 1.66 ± 0.31
N
N
N
OMe
0.39 ± 0.02 0.66 ± 0.01
1.03 ± 0.02 1.13 ± 0.15
100
101
OMe
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1.22 ± 0.12 0.72 ± 0.08
7.39 ± 0.58 5.83 ± 0.31
102
103
OMe
OH
OMe
3.61 ± 0.14 1.81 ± 0.05
2.09 ± 0.05 1.75 ± 0.09
104
105
OAc
OMe
Validation of Hsp90 inhibition via Western Blot Analysis.
Western blot analyses were performed on MCF-7 cell lysates treated with five
representative compounds (15a, 80, 81, 55 and 100) to confirm these analogues
manifest anti-proliferative activity via Hsp90 inhibition. As shown in Figure 4A,
incubation with compound 15a caused degradation of Hsp90-dependent clients EGFR
and ERα, while treatment with compounds 80 and 81 led to the degradation of Her2,
EGFR, ERα, and Akt, clearly linking cell viability to Hsp90 inhibition. In addition,
treatment of MCF-7 cells with 55 and 100 induced the degradation of the Hsp90-
dependent client substrates ERα, and Akt (Figure 4B). The Hsp90-independent
protein, actin remained unchanged, suggesting selective degradation of Hsp90-
dependent client proteins. In addition, Hsp90 levels remained constant, which is a
hallmark shared by Hsp90 C-terminal inhibitors.
A.
18
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10.1002/chem.201703206
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B.
Figure 4 Western blot analyses of compounds after incubation with MCF-7 breast
cancer cells for 24 h. (A) Western blot analyses of Hsp90-dependent client proteins
(Her2, EGFR, ERα and Akt) degradation after treatment with 15a, 55 and 80. (B)
Western blot analyses of Hsp90-dependent client proteins (ERα and Akt) degradation
after treatment with 55 and 100. L represents a concentration 1/2 of the anti-
proliferative IC50 value, while H represents a concentration 5-fold of the anti-
proliferative IC50 value. Geldanamycin (G, 0.5 µM) and dimethylsulfoxide (D, 100%)
were employed as positive and negative controls, respectively. Protein levels were
measured compared to the level of Actin.
Conclusions:
In summary, the biphenyl core was explored to identify an optimized scaffold
for Hsp90 C-terminal inhibition. These studies led to the development of phenyl
cyclohexyl carboxamides that manifest sub-micromolar to mid-nanomolar anti-
proliferative activity against multiple breast cancer cell lines. Structural investigations
suggest that the central core is important for projection of the amine and amide side
chains as evidenced by increased anti-proliferative activity. Furthermore, SAR studies
on these side chains indicate the phenyl cyclohexyl derivatives exhibit a different
binding mode as compared to other scaffolds. Discovery of the phenyl cyclohexyl core
provides a new platform on which the development of more efficacious Hsp90 C-
terminal inhibitors can be explored.
3. Experimental section
3.1 Anti-proliferation assays. Cells were maintained in a 1:1 mixture of Advanced
DMEM/F12 (Gibco) supplemented with non-essential amino acids, L-glutamine (2
mM), streptomycin (500 µg/mL), penicillin (100 units/mL), and 10% FBS. Cells were
grown to confluence in a humidified atmosphere (37° C, 5% CO2), seeded (2000/well,
100 µL) in 96-well plates, and allowed to attach overnight. Compound or GDA at
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varying concentrations in DMSO (1% DMSO final concentration) was added, and cells
were returned to the incubator for 72 h. At 72 h, the number of viable cells was
determined using an MTS/PMS cell proliferation kit (Promega) per the manufacturer’s
instructions. Cells incubated in 1% DMSO were used at 100% proliferation, and values
were adjusted accordingly. IC50 values were calculated from separate experiments
performed in triplicate using GraphPad Prism.
3.2 Western blot Analyses. MCF-7 cells were cultured as described above and
treated with various concentrations of drug, GDA in DMSO (1% DMSO final
concentration), or vehicle (DMSO) for 24 h. Cells were harvested in cold PBS and
lysed in RIPA lysis buffer containing 1 mM PMSF, 2 mM sodium orthovanadate, and
protease inhibitors on ice for 1 h. Lysates were clarified at 14000g for 10 min at 4° C.
Protein concentrations were determined using the Pierce BCA protein assay kit per the
manufacturer’s instructions. Equal amounts of protein (20 µg) were electrophoresed
under reducing conditions, transferred to
a
nitrocellulose membrane, and
immunoblotted with the corresponding specific antibodies. Membranes were incubated
with an appropriate horseradish peroxidase-labeled secondary antibody, developed
with a chemiluminescent substrate, and visualized.
Acknowledgement
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
acids for this project.
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