Synthesis and biological evaluation of 3,5-disubstituted-4-alkynylisoxozales as a novel class of HSP90 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2015.06.009

Abstract A series of 3,5-disubstitute-4-alkynylisoxazole derivatives were designed and synthesized through palladium(II)-copper(I) catalyzed Sonogashira cross-coupling reaction of an alkynyl moiety and an isoxazole scaffold as novel HSP90 inhibitors. The resultant compounds displayed moderate to potent binding affinity to HSP90 proteins, and also demonstrated good cell growth inhibitory activity against various cancer cell lines (A549, K562, MCF-7, DU145 and Hela). Some compounds (18d, 18e, 19a, 19d, 20c and 20q) show similar or better binding affinity towards HSP90α and HSP90β comparing to NVP-AUY922. In addition, compounds 18e, 19a and 20q exhibited potent inhibitory activity against various human cancer cell lines.

Full text of DOI:10.1016/j.bmcl.2015.06.009

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of 3,5-disubstituted-4-
alkynylisoxozales as a novel class of HSP90 inhibitors
Jian Sun a, , Cai Lin b, , Xiaochu Qin b, Xiaoping Dong a, Zhengchao Tu b, Fei Tang b, Chaonan Chen b,
,
⇑
Jiancun Zhang b,c,
⇑
a Chengdu University of TCM, 37 Shierqiao Road, Chengdu 610075, China
b Guangzhou Institutes of Biomedicine and Heath, Chinese Academy of Science, 190 Kaiyuan Road, Guangzhou 510530, China
c State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedical and Heath, Chinese Academy Of Science, 190 Kaiyuan Road, Guangzhou 510530, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 3,5-disubstitute-4-alkynylisoxazole derivatives were designed and synthesized through
palladium(II)–copper(I) catalyzed Sonogashira cross-coupling reaction of an alkynyl moiety and an
isoxazole scaffold as novel HSP90 inhibitors. The resultant compounds displayed moderate to potent
binding affinity to HSP90 proteins, and also demonstrated good cell growth inhibitory activity against
various cancer cell lines (A549, K562, MCF-7, DU145 and Hela). Some compounds (18d, 18e, 19a, 19d,
Received 31 March 2015
Revised 21 May 2015
Accepted 1 June 2015
Available online xxxx
20c and 20q) show similar or better binding affinity towards HSP90
NVP-AUY922. In addition, compounds 18e, 19a and 20q exhibited potent inhibitory activity against
various human cancer cell lines.
a and HSP90b comparing to
Keywords:
HSP90
3,5-Disubstitute-4-alkynylisoxazole
HSP90 inhibitor
Ó 2015 Elsevier Ltd. All rights reserved.
Heat shock proteins (HSPs) are a family of stress proteins that
act as protective factors against misfolding of various essential
proteins involved in maintaining cell functionality. Among the
HSPs, HSP90 is a ubiquitous ATP-dependent molecular chaperone
that represents about 1–2% of total proteins in normal cells.
However, it may increase up to 4–6% under stress conditions, such
for homodimerization and binding of client proteins, co-chaper-
ones and other accessory proteins operating in a chaperone cycle.10
Inhibition of HSP90 triggers disruption of folding cycle, leading to
proteasomal degradation of client proteins, which causes loss of
function and, thus, inhibition of cell growth.11–13
HSP90 has higher ATPase activity with higher binding affinity to
HSP90 inhibitors in tumor cells than in normal cells.14 Although a
variety of HSP90 inhibitors have been identified since the
clinical evaluation of natural-product-based HSP90 inhibitor,
Geldanamycin analogs 17-AAG and 17-DMAG (Fig. 1). Many of
these compounds have been reported to have unwanted side
effects.15–24 It is therefore desirable to develop new and potent
inhibitors with improved specificity for HSP90.
as in tumors.1–4 There are two subtypes of HSP90, HSP90
a and
HSP90b. Functionally, both subtypes are similar, having both tissue
and substrate specificity.5 It is well established that HSP90 plays a
vital role in maintaining the stability, conformation and function of
a variety of signaling proteins that are involved in pathways asso-
ciated with cell cycle progression, cellular proliferation, invasion
and metastasis. Furthermore, many of the client proteins of
HSP90 including AKT, vascular endothelial growth factor (VEGF),
human epidermal growth factor receptor 2 (Her2), MET, and ALK,
are involved in signal transduction, cell cycle regulation and apop-
tosis. HSP90 is associated with a variety of co-chaperones and ATP
to form a mature complex. When the competitive inhibitors
occupy the ATP-binding site of HSP90, the complex is disrupted
and the client proteins are degraded. Some client proteins of
HSP90 are key oncogenic proteins, which make it an attractive
therapeutic target for cancer therapy.6–9 HSP90 is also essential
NVP-AUY922, a 4,5-diarylisoxazole-based HSP90 chaperone
inhibitor developed by Vernalis, entered clinical trials in 2007.25
Various analogues of NVP-AUY922 were also evaluated for their
binding affinities to HSP90a protein and their cell growth inhibi-
tory activities against various tumor cell lines, such as HCT-116,
NCI-H460 or A549. Several of the analogues displayed potent
HSP90 binding affinity and cell growth inhibitory activity
(Fig. 1).26–29 Based on these results, we designed and synthesized
a series of new alkynes-linked isoxazole scaffolds to investigate
their inhibitory activities for human HSP90a and HSP90b proteins,
and further to determine their anti-proliferative activity against
tumor cell lines. While maintaining the interactions of the resorci-
nol OH groups and C-3 amide, we speculated that the introduction
⇑
Corresponding authors. Tel.: +86 020 32015323.
E-mail addresses: chen_chaonan@gibh.ac.cn (C. Chen), zhang_jiancun@gibh.ac.
cn (J. Zhang).
These authors contributed equally.
http://dx.doi.org/10.1016/j.bmcl.2015.06.009
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Sun, J.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.06.009

2
J. Sun et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
OH
O
O
O
HO
X
O
O
O
HO
O
N
H
O
N
N
NH2
NH
O
N
N
OH
HO
N
O
O
N
O
HN
O
O
N
O
O
O
OCONH2
17-AAG
17-DMAG
X=CH2CHCH2NH2
X=Me2NCH2CH2NH
KW-2478
XL888
AT13387
O
O
Cl
N
CF3
N
N
N
N
N
N
N
N
H2N
HO
NH2
O
HO
N
O
O
N
H
NH2
N
NH
OH
N
O
O
N
H
OH
O
BIIB0221
SNX-5422
STA-9090
NVP-AUY922
O
N
O
O
O
N
N
Cl
N
HO
O
N
O
HO
N
O
HO
O
N
HO
O
OH
NH
N
O
N
H
N
N
N
N
N
H
OH
N
N
O
O
O
H
OH
O
OH
4
1
2
3
Figure 1. HSP90 inhibitors currently in clinical trials and reported NVP/AUY-922 analogues.
Figure 3. Docking of compound 19a in the active site of HSP90. Color scheme is
atom type: grey: 19a; cyan: protein residues involved in non-polar interaction;
green: protein residues involved in polar interactions. H-Bonds are shown as dotted
line.
Compound 11, a key intermediate, was synthesized through sev-
eral conventional transformations starting from 4-dihydroxyphenyl-
methylketone (Scheme 1). 2,4-Dihydroxyphenylmethylkeone was
first protectedwith benzylgrouptogive5, followedbyaWittingreac-
tion to give alkene 6.30 Hydrogenation of compound 6 gave resorcinol
7 in a good yield (85%), which was first protected by benzyl group to
give 8, and followed by a Friedel–Crafts acylation giving compound
9.25 The subsequent reaction of an intermediate sodium enolate with
diethyl oxalate yielded keto-enol eater 10. The isoxazole heterocyclic
compound 11 was obtained by treating 10 with hydroxylamine
hydrochloride in ethanol.
Figure 2. Docking pose of compound 19a (green) and NVP-AUY922 (cyan) in the
active site of HSP90.
of an alkynes group at C-4, as shown for compound 19a (Figs. 2 and
3), could provide good cation–
due of HSP90 protein. Thus, we designed a series of analogs with
alkynes at C-4 position of the isoxazole ring, and herein we report
the results on the syntheses, binding affinities for both HSP90 and
p bond interaction with Lys58 resi-
a
a
HSP90b proteins, and anti-proliferative activities of a series of 3,5-
disubstitute-4-alkynylisoxazole molecules.
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J. Sun et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
O
HO
BnO
BnO
HO
HO
a
b
c
d
OH
O
OBn O
OBn
OH
OH
5
6
8
7
BnO
BnO
BnO
e
f
g
O
O
O
O
O
11
N
OBn O
OBn O
10
OH
OBn
9
Scheme 1. Reagents and conditions: (a) BnBr, K2CO3, MeCN, reflux, overnight, 92%; (b) MePPh3I, t-BuOK, THF, 0 °C–rt, overnight; (c) H2 (10 atm), 10% Pd/C, AcOH, EtOH, rt,
2 d, 85%; (d) AcOH, BF3ÁEt2O, 85% (e) BnBr, K2CO3, MeCN, rt, overnight, 90%; (f) (CO2Et)2, Na, EtOH, rt, 4 h, 82%; (g) hydroxylamine hydrochloride, EtOH, rt, 89%.
R1
R1
BnO
BnO
I
HO
O
O
a
b
c, e
or d, e
O
11
O
O
R2
O
N
O N
OBn
12
OBn
13a-b
O
N
OH
14a-b
15a
13a R1
=
=
14a R1
=
R2 = NH2
O
N
O
N
H
N
13b R1
14b R1
=
=
R2
R2
=
=
N
O
H
N
1
15a
R
Scheme 2. Reagents and conditions: (a) NIS, CAN, MeCN, 80 °C, overnight, 72%; (b) terminal alkyne, Pd(PPh3)2Cl2, CuI, Et3N or diisopropylamine, DMF, 55 °C, overnight, 65%;
(c) LiOHÁH2O, THF–MeOH–H2O, rt, overnight, followed by (CH2)2CHNH2, EDCI, HOBt, DCM, rt, overnight; (d) NH3ÁMeOH, 60 °C, overnight; (e) BBr3, DCM, 0 °C–rt, 1 h.
R
HO
O
N
O
N
H
OH
OH
18
R
BnO
BnO
I
HO
O
c,d
b
a
O
O
11
N
H
N
H
N
H
O
N
O
N
OBn
OBn
O
N
16
17
19
R
HO
O
N
H
O
N
OH
20
Scheme 3. Reagents and conditions: (a) EtNH2, EtOH, reflux, 5 h, 87%; (b) NIS, CAN, MeCN, 80 °C, overnight, 70%; (c) terminal alkyne, Pd(PPh3)2Cl2, CuI, Et3N or
diisopropylamine, DMF, 55 °C, overnight, 65%; (d) BBr3, DCM, 0-rt, 1 h.
A moderate yield of 12 was obtained by mixing a catalytic
amount of ceric ammonium nitrate (CAN) with 11 as shown in
Table 1
Docking Scores calculated by Glide and Binding free energies calculated by the
Scheme 2. Conversion of the iodide 12 to 13a–b was achieved via
a Sonogashira cross-coupling with corresponding substituted alky-
nyl compounds.31,32 14a–b and 15a were obtained by ester saponi-
fication, subsequent coupling reaction with corresponding amine,
and finally removal of the benzyl group with boron tribromide.
The 3-N-ethylformamide-4-alkynylisoxazoles was synthesized
as shown in Scheme 3. Under azeo-tropic conditions, compound
MMGBSA methoda,b
XP docking Score
DGbind
NVP-AUY922
19a
À9.603
À9.057
À126.053
À111.774
a
All energies are in kcal/mol and calculated by Prime.
b
D
Gbind
=
D
Gbind,vauum
+
D
Gsolv,complex
À
D
Gsolv,ligand
À
DGsolv,receptor.
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4
J. Sun et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Table 2
Binding (FP assay) and cell growth inhibition data
R2
HO
O
R1
O
N
OH
Entry Compound
R1
R2
—
HSP90 (IC50
a
:
l
M) HSP90b (IC50
:
l
M) A549
K562
MCF-7
(IC50: lM)
DU145 Hela
1
2
NVP-AUY922
14a
—
0.059
0.066
0.085
0.636
0.0082 0.0087
0.0064
0.047
0.0047
0.047
0.0077
N
N
O
O
NH2
0.060
0.283
0.051
2.210
0.014
0.168
0.054
0.205
0.043
1.500
0.016
0.144
0.056
0.201
0.052
3.880
0.044
0.228
3
4
5
6
7
14b
15a
18a
18b
18c
NHCH(CH2)2
NHCH(CH2)2
NHCH2CH3
NHCH2CH3
NHCH2CH3
0.108
0.045
0.347
0.125
0.152
0.182
0.133
0.736
0.144
0.141
0.196
0.038
2.220
0.016
0.123
0.165
0.048
4.140
0.043
0.741
8
18d
18e
19a
NHCH2CH3
NHCH2CH3
NHCH2CH3
0.054
0.033
0.066
0.099
0.035
0.083
0.110
0.042
0.021
0.093
0.035
0.020
0.092
0.033
0.019
0.048
0.021
0.057
0.124
0.041
0.039
9
N
N
O
O
10
N
11
12
19b
19c
NHCH2CH3
NHCH2CH3
0.048
0.091
0.142
0.135
0.161
0.093
0.166
0.087
0.146
0.072
0.281
0.274
0.319
0.295
O
N
O
N
N
S
S
13
14
19d
19e
NHCH2CH3
NHCH2CH3
0.028
0.114
0.087
0.114
0.164
0.575
0.133
0.220
0.093
0.189
0.350
0.947
0.334
0.573
O
N
N
NH
O
15
16
19f
NHCH2CH3
NHCH2CH3
0.144
0.045
0.087
0.160
0.433
0.035
0.333
0.037
0.472
0.036
0.286
0.042
0.460
0.050
CF3
N
N
O
19g
CF3
N
11 was converted into 16 with 70% aqueous ethylamine in ethanol.
In the presence of a catalytic amount of CAN, compound 16 was
iodinated to afford 17 with N-iodosuccinimide. Compound 17
underwent a subsequent Sonogashira coupling reaction with cor-
responding alkynes, followed by reactions with boron tribromide
to give compounds 18, 19 and 20.
The synthesized compounds were tested for their HSP90 inhibi-
tory properties (Tables 2 and 3). A fluorescence polarization (FP)
assay was used for measurement of binding affinity. The resorcinol
moiety, considered the privilege structural moiety to interact with
HSP90 protein, was retained.25 Different amides were synthesized
at C-3 position of the isoxazole ring. Compounds 14a, 14b, and 19a
(Table 2) showed that the ethyl amide at C-3 demonstrates slightly
Propargylamine derivatives (i.e., 19a–g) showed good inhibitory
activities to HSP90 in the range of 28–144 nM, and to HSP90b
a
in the range of 83–160 nM. The results of 19f and 19g suggested
that larger groups of C-4 alkynyl moiety may be allowed for
HSP90 binding unlike the linear conformations of 18a–18e.
Amide groups (19h–k), which were introduced to the propargyl
position, showed binding activity in the nanomolar to low
micromolar ranges.
Taking into account that the adjacent CH2 moiety of a propargy-
lamine may be susceptible to oxidation during metabolic process
when sterically unhindered, we also synthesized dimethyl-substi-
tuted propargyl derivatives (20a–20k) shown in Table 3. Biological
evaluation of these derivatives showed generally decreased bind-
ing affinity in the range of 40–546 nM except for compound 20k.
20c–f with aliphatic amides showed similar inhibitory activities.
Various heterocyclic amides 20l–q showed similar activities with
electron-donating and electron-withdrawing groups. However, a
fluorine substituted pyridinylcarboxyamide (20q) showed better
better binding affinity to HSP90a and HSP90b proteins while also
showing potent cytotoxic activity as compared to the cyclopropyl
amide or simple amide. The prediction calculation by Glide and
the MMGBSA method in general matched with the test results
(Tables 1 and 2). For compounds directly linked with an alkynyl
group (Table 2), unsubstituted arylalkynes had better inhibitory
activity than substituted arylalkynes (i.e., 18a and 18b). The
smaller the saturated ring attached to the alkynyl, the better the
affinity is (18c–8e). The affinity with saturated rings generally
demonstrated better activity than those with aromatic rings.
inhibitory activities against HSP90a and HSP90b proteins.
All compounds were further evaluated for their anti-prolifera-
tive activities against five cancer cell lines including: A549,
adenocarcinomic human alveolar basal epithelial cell; K562,
human immortalised myelogenous leukemia line; MCF-7, breast
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J. Sun et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
5
Table 3
Binding (FP assay) and cell growth inhibition data
R2
R2
HO
HO
O
O
R1
R1
O
N
OH
O
N
OH
19
20
Entry
Compound
R1
R2
—
HSP90
a
(IC50
:
lM)
HSP90b (IC50
:
l
M)
A549
K562
MCF-7
(IC50: lM)
DU145
Hela
1
2
NVP-AUY922
19h
—
0.059
0.207
0.085
0.149
0.0082
1.970
0.0087
1.520
0.0064
0.0047
2.480
0.0077
1.860
O
CF3
NHCH2CH3
1.260
NH
O
CF3
3
4
19i
19j
NHCH2CH3
NHCH2CH3
0.066
0.546
0.146
1.100
0.820
0.834
0.965
0.714
0.592
0.572
0.655
0.977
0.730
1.250
NH
O
N
N
NH
O
5
6
19k
20a
NHCH2CH3
NHCH2CH3
1.660
0.120
0.167
0.101
0.882
0.259
0.672
0.166
0.459
0.112
0.997
0.166
0.976
0.221
NH
O
S
O
O
S
H
N
H
N
7
8
20b
20c
NHCH2CH3
NHCH2CH3
0.078
0.050
0.237
0.076
0.711
0.229
0.695
0.257
0.618
0.199
0.677
0.329
1.300
0.527
O
H
N
O
O
H
N
9
20d
20e
NHCH2CH3
NHCH2CH3
0.127
0.183
0.108
0.116
0.210
0.626
0.230
0.596
0.157
0.344
0.170
0.472
0.347
1.070
H
N
10
O
11
12
13
20f
20g
20h
NHCH2CH3
NHCH2CH3
NHCH2CH3
0.099
0.307
0.471
0.384
0.068
0.126
1.090
0.337
1.350
1.070
0.359
1.190
0.630
0.245
0.604
0.778
0.242
0.785
2.110
0.477
1.530
H
N
O
N
O
H
N
O
N
O
H
N
O
H
N
14
20i
NHCH2CH3
0.108
0.111
0.258
0.190
0.123
0.165
0.294
O
HN
O
15
16
20j
NHCH2CH3
NHCH2CH3
0.546
1.660
0.446
1.770
0.651
5.350
0.664
6.190
0.514
4.180
0.845
7.460
1.040
4.610
CF3
CF3
20k
NH
HN
17
18
19
20l
NHCH2CH3
NHCH2CH3
NHCH2CH3
0.040
0.295
0.069
0.151
0.143
0.120
0.416
0.810
0.348
0.313
0.402
0.216
0.292
0.400
0.282
0.343
0.549
0.221
0.789
0.857
0.448
N
N
O
HN
20m
20n
O
O
N
NH
(continued on next page)
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J. Sun et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Table 3 (continued)
Entry
Compound
R1
R2
HSP90
a
(IC50
:
lM)
HSP90b (IC50
:
lM)
A549
K562
MCF-7
(IC50: lM)
DU145
Hela
HN
O
20
21
22
20o
20p
20q
NHCH2CH3
NHCH2CH3
NHCH2CH3
0.057
0.121
0.066
0.393
0.389
0.073
0.729
0.557
0.051
0.748
0.567
0.047
0.720
0.550
0.038
0.659
0.507
0.030
1.390
0.929
0.075
N
N
N
O
HN
CF3
F
O
HN
O
cancer cell line; DU145, prostate cancer cell line, and Hela cell line
(Tables 2 and 3). Results showed that MCF-7 was the most sensi-
tive cell line as the IC50 of ten compounds was in low nanomolar
range. For A549 cell line, eight compounds showed good cytotoxic
activity in the low nanomolar range with compound 18b showing
an IC50 value of 14 nM. In the case of K562 cell line, nine com-
pounds demonstrated good cell growth inhibitory properties. For
DU145 cell line, 18e displayed the highest potency with an IC50
of 21 nM. Finally, seven compounds showed cytotoxic activity in
the nanomolar range (39–75 nM) against the Hela cell line.
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In summary, a series of novel alkynylisoxazoles were designed
and synthesized. Some compounds (i.e., 15a, 18d, 18e, 19b, 19d,
19g, 20c, 20i and 20o) exhibited improved binding affinity to
HSP90a and HSP90b comparing to NVP-AUY922. Some compounds
also displayed good anti-proliferative activity against different
cancer cell lines in vitro with IC50 values in the low nanomolar
range (18e, 19a and 20q). Compounds 14a, 15a and 19g demon-
strated good binding affinity to HSP90
a and tumor cell growth
inhibitory activity notwithstanding a weak binding activity to
HSP90b. Compounds 19d and 20c, despite possessing a good bind-
ing affinity to HSP90, displayed poor cell growth inhibitory proper-
ties likely due to poor cellular membrane penetrations. The results
of this novel class of alkynylisoxazoles may provide further
insights to design new HSP90 inhibitors in the future.
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The research was supported in part by Foshan Municipal Funds
for
Scientific
and
Technological
Innovation
Projects
(2013HK100012), Guangzhou Science and Technology Project
(2014J4100222), National Natural Science Foundation of China
Grant (21402205). The authors thank Yang Zhou and Dr. Yong Xu
for their help in the modeling calculations.
29. Meng, T.; Zhang, D. D.; Xie, Z. Q.; Yu, T.; Wu, S. C.; Huang, M.; Geng, M. Y.; Shen,
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Supplementary data
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Supplementary data (experimental procedure of biological
assay, molecular modeling, spectral characterizations of the syn-
thesized compounds) associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.06.009.
Please cite this article in press as: Sun, J.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.06.009