Discovery of novel heat shock protein (Hsp90) inhibitors based on luminespib with potent antitumor activity

Article abstract of DOI:10.1016/j.bmcl.2020.127165

A series of isosteric surrogates of the 4-phenyl group in luminespib were investigated as new scaffolds of the Hsp90 inhibitor for the discovery of novel antitumor agents. Among the synthesized surrogates of isoxazole and pyrazole, compounds 4a, 5e and 12b exhibited potent Hsp90 inhibition in ATPase activity and Her2 degradation assays and significant antitumor activity in A2780 and HCT116 cell lines. Animal studies indicated that compared to luminespib, their activities were superior in A2780 or NCI-H1975 tumor xenograft models. A molecular modeling study demonstrated that compound 4a could fit nicely into the N-terminal ATP binding pocket.

Full text of DOI:10.1016/j.bmcl.2020.127165

Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel heat shock protein (Hsp90) inhibitors based on
luminespib with potent antitumor activity
Juyoung Junga,c, Jinsun Kwonb, Soojung Honga, An-Na Moona, Jinah Jeonga, Sungwook Kwona,
⁎
Jeong-ah Kima, Myoungjae Leea, Hongsub Leea, Jin Hee Leea, Jeewoo Leec,
a Research Laboratories, Ildong Pharmaceutical Co., Hwaseong-si, Gyeonggi-do 18449, South Korea
b AIMS BioScience Co., 2, Baumoe-ro 27-gil, Seocho-gu, Seoul 06752, South Korea
c Laboratory of Medicinal Chemistry, College of Pharmacy, Seoul National University, Seoul 08826, South Korea
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of isosteric surrogates of the 4-phenyl group in luminespib were investigated as new scaffolds of the
Hsp90 inhibitor for the discovery of novel antitumor agents. Among the synthesized surrogates of isoxazole and
pyrazole, compounds 4a, 5e and 12b exhibited potent Hsp90 inhibition in ATPase activity and Her2 degradation
assays and significant antitumor activity in A2780 and HCT116 cell lines. Animal studies indicated that com-
pared to luminespib, their activities were superior in A2780 or NCI-H1975 tumor xenograft models. A molecular
modeling study demonstrated that compound 4a could fit nicely into the N-terminal ATP binding pocket.
Heat shock protein 90
Luminespib
Antitumor agent
An ATP-dependent molecular chaperone, heat shock protein 90
(Hsp90), regulates the stability and activity of client proteins that play
critical roles in proliferation, survival, transformation and apoptosis.1–4
A significant number of these client proteins, including protein kinases
such as HER2, C-RAF, B-RAF, and AKT, have been implicated in on-
cogenesis. Simultaneous degradation of multiple oncogenic client pro-
teins is caused by inhibition of Hsp90 in the ubiquitin-proteasome
pathway.5–8 Due to its the selectivity in inhibiting the growth of cancer
cells over nontransformed cells, Hsp90 inhibition has emerged as a
promising mode of action for anticancer chemotherapy.9–10
In our program to discover Hsp90 inhibitors as novel antitumor
agents, we investigated novel scaffolds based on luminespib in which
the phenyl group was substituted with isoxazole or 2,3- or 3,4-pyrazole
moieties (Fig. 1). The synthesized compounds were tested for Hsp90
inhibition by measuring their ATPase activity and ability to degrade a
client protein and were evaluated for their in vitro cytotoxicity against
cancer cell lines. The selected compounds were further evaluated for
their antitumor activities in in vivo tumor xenograft models. The mo-
lecular modeling of representative inhibitors was performed to identify
the binding mode in the N-terminal ATP binding pocket.
Geldanamycin (GD) is a natural 1,4-benzoquinone ansamycin that
inhibits Hsp90 by binding to its N-terminal ATP-binding pocket11 and
has provided an important opportunity for understanding the role of
Hsp90 in tumor growth.12–14 Tanespimycin (17-AAG), a derivative of
geldanamycin, as a single or combination drug in clinical trials, has
shown broad activity and reduces drug resistance. However, 17-AAG has
limited applications due to its poor solubility, bioavailability, hepato-
toxicity and extensive metabolism by polymorphic enzymes.15–17
Therefore, novel small molecule Hsp90 inhibitors have been studied to
overcome this issue. Among them, resorcinol scaffolds have emerged as a
new class of Hsp90 inhibitor. This class of compounds, such as lumine-
spib (NVP-AUY922) and ganetespib (STA-9090), exhibit potent and se-
lective Hsp90 inhibition and display significant oral activities in cellular
and animal models of cancer.18,19 In addition, purine scaffold compounds
(e.g., PU-H71, BIIB021) have also been reported as Hsp90 inhibitors.20
The synthesis of R1-CH2 substituted isoxazole derivatives (4a–x) is
described in Scheme 1. The known iodide key intermediate 1 was
prepared18 and coupled with ethyl 5-(tributylstannyl)isoxazole-3-car-
boxylate21 in the presence of tetrakis(triphenylphosphine)palladium(0)
to provide the isoxazole compound 2a. Reduction of carboxylate 2a
afforded the alcohol intermediate 3a, which was subsequently con-
verted into the mesylate intermediate 3e. Alkylation of the alcohol
intermediate 3a with methylbromoacetate afforded the ethylcarba-
moylether compound 3b. Treatment of the ester 3b with 2 N LiOH or
7 N NH3 provided the acid 3c or amide 3d, respectively. Next, fluoro or
cyano substitution reactions from the mesylate intermediate 3e readily
afforded the fluoromethyl 3f and cyanomethyl 3g isoxazoles. In addi-
tion, aminomethyl 3i was obtained by thermal treatment of the po-
tassium phthalimide. The others 3h–x were obtained from the mesylate
intermediate 3e by a similar substitution reaction. Finally, removal of
⁎ Corresponding author.
E-mail address: jeewoo@snu.ac.kr (J. Lee).
https://doi.org/10.1016/j.bmcl.2020.127165
Received 4 March 2020; Received in revised form 27 March 2020; Accepted 1 April 2020
0960-894X/©2020ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:JuyoungJung,etal.,Bioorganic&MedicinalChemistryLetters,https://doi.org/10.1016/j.bmcl.2020.127165

J. Jung, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Fig. 1. Design of new scaffold Hsp90 inhibitors based on luminespib.
the dibenzyl protecting group in 3a–x using boron trichloride provided
the final compounds 4a–x.
The synthesis of R2-CO substituted isoxazole derivatives 5a–g is il-
lustrated in Scheme 2. Acid hydrolysis of the ethylcarboxylate 2a
furnished the acid 2b. Amination of the ester 2a with commercially
available various amines afforded the requisite amide derivatives 2c–g.
Final products 5a–g were obtained after the deprotection of the di-
benzyl group.
The synthesis of 1,3-disubstituted pyrazole derivatives is shown in
Scheme 3. The iodide intermediate 1 was converted into the alkyne 6
by ethnyltributyltin via the Stille reaction and as then converted into
the acetyl 7. The acetyl 7 was condensed with dimethylformamide di-
methylacetal to give the dimethylaminoacryloyl 8, which was quanti-
tatively cyclized into the unsubstituted C3-linked pyrazole 9a using
hydrazine hydrate. Alkyl substituted pyrazoles (9b–e) were obtained by
using appropriate alkylation reagents, followed by the deprotection of
benzyl groups to produce the desired product 10a–e.
The synthesis of 1,4-disubstituted pyrazole derivatives is presented
in Scheme 4. Stille coupling of the iodide 1 with trityl pyrazole tribu-
tylstannane using Pd(0) afforded the protected C4-linked pyrazole 11a
almost quantitatively, which was deprotected to furnish C4-linked 11b.
The 1-methyl, 1-ethyl and 1-isopropyl substituted pyrazoles 11c–e were
also obtained from the iodide 1 by direct coupling with the corre-
sponding substituted pyrazoles. The final deprotection readily provided
the C4-linked pyrazoles 12b–e.
Scheme 1. Synthesis of isoxazole derivatives. Reagents and conditions: (a)
ethyl 5-(tributylstannyl)isoxazole-3-carboxylate, Pd(PPh3)4, toluene, reflux,
2.5 h, 78%; (b) LAH, THF, 0 °C to r.t., 2 h, 61%; (c) BrCH2CO2Me, Cs2CO3,
CH3CN, r.t., 24 h, 61%; (d) 2 N LiOH, THF/water, 0 °C, 1 h, 63%; (e) 7 N NH3/
MeOH, KCN, 50 °C, 12 h, 89%; (f) MsCl, Et3N, 0 °C, 3 h, 61%; (g) KF, 18-crown-
6, r.t., 18 h, 78%; (h) KCN, 18-crown-6, r.t., 12 h, 98%; (i) phthalimide po-
tassium salt, CH3CN, reflux, 24 h, 95% and then MeNH2, EtOH, reflux, 6.5 h,
99%; (j)-(y) R1NH2 or R1NH, CH2Cl2, 8 h, 60–99%; (z) BCl3, CH2Cl2, 0 °C, 0.5 h,
45–99%.
All the synthesized compounds were tested for Hsp90 inhibition by
the measurement of the ATPase activity of Hsp90 as well as by the cell-
based Her2 degradation assay. For antitumor activity, their cytotoxi-
cities were evaluated against the A2780 (human ovarian cancer) and
HCT116 (human colon cancer) cell lines. The results are summarized in
Tables 1–3, together with the values of luminespib (NVP) for compar-
ison.
activity compared to that of the corresponding acyclic amines.
Next, we explored the isoxazole surrogates (5a–g) with a COR2
group at the C3 position (Table 2). Whereas the ethyl ester 5a and
carboxylic acid 5b showed weak activities, most amide analogues dis-
played reasonable inhibition toward Hsp90 and cancer cells. In parti-
cular, the ethylamide 5e showed significantly improved activity, in
which its Hsp90 inhibition and cytotoxicity in HCT116 were more po-
tent than those of NVP.
First, we investigated the isoxazole surrogates bearing a CH2R1
group (4a–x) at the C3 position (Table 1). The hydroxylmethyl iso-
xazole analogue 4a and the O-alkyl substituted analogues 4b–d, in
which R1 is OCH2CO2Me, OCH2CO2H, and OCH2CONH2, respectively,
were examined. Among them, 4a exhibited potent inhibitory activity
toward Hsp90 and cancer cells, which was slightly better than that of
NVP. The substitution of hydroxyl in 4a with OMs, fluoro and cyano
groups did not improve the activity. Mono- or di-substituted amino-
methyl isoxazole analogues 4h–s were also explored. Although some of
the amine analogues 4j, 4l, 4m, 4q, and 4r demonstrated promising
potency, none of compounds were found to be better than NVP for
inhibition of Hsp90 and tumors. Cyclicamino, such as morpholinyl,
piperidinyl, pyrrolidinyl and N-methylpiperazinyl, analogues 4t-x were
also examined. However, this modification resulted in diminished
Finally, we examined N-alkyl pyrazole surrogates in which the two
isomers, the 1,3-pyrazole (10a-d) and 1,4-pyrazole (12b-e) analogues,
were tested. Compared to NVP, most of the 1,3-pyrazole analogues have
similar potencies for Hsp90 inhibition and antitumor activity. The SAR
in cell-based assays clearly showed a preference for an alkyl substitu-
tion on the pyrazole moiety. The potency was enhanced when alkyl
groups, such as methyl, ethyl and isopropyl, were introduced (10b–c,
2

J. Jung, et al.
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Scheme 4. Synthesis of 4-substituted pyrazole derivatives. Reagents and
conditions: (a) 4-(tributylstannyl)-1-R4-1H-pyrazole, Pd(PPh3)4, toluene, reflux,
12 h, 60–99%; (b) CF3CO2H, CH2Cl2/MeOH, 75 °C, 3 h, 99%; (c) BCl3, CH2Cl2,
0 °C, 0.5 h, 34–87%.
Scheme 2. Synthesis of isoxazole-3-carboxylate derivatives. Reagents and
conditions: (a) ethyl 5-(tributylstannyl)isoxazole-3-carboxylate, Pd(PPh3)4, to-
luene, reflux, 2.5 h, 78%; (b) 2 N LiOH, THF/water, 0 °C, 1 h, 93%; (c) 7 N NH3/
MeOH, KCN, 50 °C, 12 h, 91%; (d) aq. 40% MeNH2, EtOH, reflux, 48 h, 90%; (e)
2 M EtNH2, EtOH, reflux, 1 h, 86%; (f) morpholine, EtOH, reflux, 24 h, 87%; (g)
thiomorpholine, EtOH, reflux, 48 h, 67%; (h) BCl3, CH2Cl2, 0 °C, 0.5 h, 56–99%.
Table 1
In vitro activity of the isoxaole-CH2R1 surrogates.
R1
IC50 (μM)
A2780
ATPase
Her2
HCT116
NVP
4a
4b
4c
4d
4e
4f
0.500
0.279
0.443
1.28
0.012
0.042
ND
0.006
0.005
ND
0.02
OH
0.013
0.262
ND
OCH2CO2Me
OCH2CO2H
OCH2CONH2
OMs
ND
ND
0.452
0.476
0.41
0.05
ND
ND
0.097
0.4
ND
F
0.027
ND
0.028
ND
0.063
0.079
0.084
0.164
0.045
0.117
0.109
0.121
0.167
0.078
0.111
0.092
0.038
0.183
0.028
0.093
0.051
0.34
4g
4h
4i
CN
0.47
NH2
0.674
0.436
0.475
0.465
0.521
0.435
0.5
0.171
ND
ND
NHMe
ND
4j
NHEt
0.023
0.024
0.018
0.067
0.071
0.05
ND
0.045
ND
4k
4l
NH-n-propyl
NH-isopropyl
NH-allyl
0.038
0.043
ND
4m
4n
4o
4p
4q
4r
NHCOMe
NHCOEt
NH(CH2)2OH
NMe2
0.382
0.975
0.49
ND
ND
0.033
0.052
0.117
0.05
0.062
0.038
ND
0.031
0.078
ND
NEt2
0.468
0.413
0.595
0.752
0.686
0.572
0.44
4s
N(n-propyl)2
morphonyl
piperidinyl
pyrrolidinyl
N-methylpiperazinyl
thiomorphonyl
4t
ND
4u
4v
4w
4x
0.062
0.055
ND
0.038
0.064
0.067
ND: not determined.
10e). However, the addition of an n-propyl group caused reduced ac-
tivity. Compared to NVP and the corresponding 1,3-pyrazoles, the 1,4-
pyrazole analogues showed similar potencies. Unsubstituted pyrazole
12b proved to be a potent Hsp90 inhibitor.
Scheme 3. Synthesis of 3-substituted pyrazole derivatives. Reagents and
conditions: (a) ethynyltributyltin, Pd(PPh3)4, toluene, reflux, 2 h, 75%; (b)
HCOOH, NaHCO3, 95 °C, 1 h, 45%; (c) N,N-dimethylformamide dimethylacetal,
EtOH, reflux, 4 h, 76%; (d) NH2NH2, EtOH, r.t., 48 h, 99%; (e)-(h) R3I, K2CO3,
CH3CN, 1 h, 78–87%; (i) BCl3, CH2Cl2, 0 °C, 0.5 h, 40–99%.
The eight compounds selected from the in vitro study were evaluated
for in vivo antitumor activity in a mouse A2780 tumor xenograft model.
3

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Table 2
Table 5
In vitro activity of the isoxaole-COR2 surrogates.
Antitumor activity of selected compounds against human tumor xenograft
models (NCI-H1975).a
Dose (mg/kg)
route
Frequency
TGI (%)b
mortality
NVP
4a
150
200
200
200
i.v.
i.v.
i.v.
i.v.
(1/week) × 3
(1/week) × 3
(1/week) × 3
(1/week) × 3
43.5
50.9
44.0
55.4
0/6
0/6
0/6
0/6
5e
12b
a
BALB/c-nu/nude mice (n = 6) transplanted with tumor cells were treated
with resorcinol compounds.
R2
IC50 (μM)
A2780
b
Tumor growth inhibition.
ATPase
Her2
HCT116
NVP
5a
5b
5c
0.500
0.447
1.87
0.012
ND
0.006
9
0.02
ND
OEt
OH
ND
20
ND
NH2
0.657
0.489
0.284
0.583
0.566
0.052
0.024
0.028
0.052
ND
0.015
0.1
0.079
0.124
0.014
0.060
ND
5d
5e
NHMe
NHEt
0.043
0.085
0.207
5f
morphonyl
thiomorphonyl
5g
ND: not determined.
Table 3
In vitro activity of the pyrazole surrogates.
R3
R4
IC50 (μM)
ATPase
Her2
A2780
HCT116
NVP
10a
10b
10c
10d
10e
12b
12c
12d
12e
–
–
0.500
0.524
0.522
0.553
0.584
0.543
0.469
0.482
0.532
0.542
0.012
0.045
0.016
0.012
0.024
0.014
0.013
0.019
0.013
0.019
0.006
0.033
0.012
0.013
0.027
0.045
0.038
0.02
0.02
H
–
0.036
0.016
0.025
0.049
0.019
0.043
0.041
0.026
0.021
Me
–
Et
–
n-pro
–
i-pro
–
–
–
–
–
H
Me
Et
i-pro
0.03
0.021
Table 4
Antitumor activity of selected compounds against human tumor xenograft
models (A2780).a
Dose (mg/kg)
route
Frequency
TGI (%)b
mortality
NVP
4a
50
50
50
50
50
50
50
50
50
i.v.
i.v.
i.v.
i.v.
i.v.
i.v.
i.v.
i.v.
i.v.
(3/week) × 3
(3/week) × 3
(3/week) × 2
(3/week) × 2
(3/week) × 2
(3/week) × 3
(3/week) × 2
(3/week) × 2
(3/week) × 2
48.0
47.0
40.3
53.0
12.8
52.0
46.4
49.8
47.6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
Fig. 2. Predicted binding mode. (A) The binding mode of 4a (orange) and co-
crystal ligand (green) in the ATP-binding site of Hsp90. Hydrogen bonds are
shown as yellow dashed lines. (B) Ligand interaction diagram. Hydrogen bonds
are depicted by magenta lines, and the corresponding π-cation interaction is
depicted by the red line.
4j
4m
4r
5e
10a
10e
12b
carboxymethyl cellulose (CMC) and were administered intravenously
(iv) at a dose of 50 mg/kg three times weekly for two or three weeks.
Luminespib (NVP) was used as a reference for comparison.
a
BALB/c-nu/nude mice (n = 6) transplanted with tumor cells were treated
with resorcinol compounds.
As shown in Table 4, all the tested compounds except 4r sig-
nificantly inhibited tumor growth with a range of TGI = 40.3–53.0%
without any death or body weight loss. In particular, compounds 4m,
5e and 10e showed higher inhibitions compared to that of NVP.
A further in vivo study was conducted in another tumor model (See
Table 5). The three compounds (4a, 5e, 12b) were administered i.v. at
200 mg/kg once weekly for three weeks to athymic mice bearing NCI-
b
Tumor growth inhibition: [1-(mean volume of treated tumors)/(mean vo-
lume of control tumors)] × 100%.
For the tumor model, A2780 human ovarian adenocarcinoma cells were
cultured and implanted into nude mice, and 5 or 6 days later, tumor
size and volume were measured. The compounds were suspended in 1%
4

J. Jung, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
H1975 human lung carcinoma xenografts. While compound 5e ex-
hibited comparable activity to NVP, compounds 4a and 12b displayed
higher antitumor growth inhibition than NVP without any death. This
dose schedule was well tolerated, and body weight loss was not ob-
served.
influence the work reported in this paper.
Acknowledgments
This work was supported by a grant (HA17C0053, 1720340) from
the Korea Health Technology R&D Project through the Korea Health
Industry Development Institute (KHIDI), funded by the Ministry of
Health & Welfare, Republic of Korea.
To understand the binding mode of 4a, we performed a docking
study of 4a with an Hsp90 X-ray crystal structure (2VCI.pdb) using
Glide SP.22 As shown in Figure 2, the binding mode of 4a was similar to
that of luminespib (green), which binds to the Hsp90 ATP binding site.
The resorcinol moiety of 4a was located deep in the pocket and had key
hydrogen bonding interactions with Asn51, Asp93 and water mole-
cules. Furthermore, the amide group of 4a formed hydrogen bonds with
Gly97 and Lys58. The isoxazole ring made a π-cation interaction with
Lys58, and its hydroxymethyl group showed hydrogen bonding with
Asp54, features that are not shown with luminespib.
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Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to
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