Ring-opening of five-membered heterocycles conjugated 4-isopropylresorcinol scaffold-based benzamides as HSP90 inhibitors suppressing tumor growth in vitro and in vivo

Article abstract of DOI:10.1016/j.ejmech.2021.113428

A series of ring-opened dihydroxybenzamides have been designed and synthesized as heat shock protein 90 inhibitors. One of derivatives, compound 6b ((N-ethyl-2,4-dihydroxy-5-isopropyl-N-(pyridin-3-yl)benzamide)) demonstrated remarkable antiproliferative activity against in human KRAS mutant A549 and EGFR T790 M mutant H1975 lung cancer cell lines with GI₅₀ values of 0.07 and 0.05 μM, respectively. It is also active against in other cancer cell lines, such as colorectal HCT116 (GI₅₀ = 0.09 μM), liver Hep3B (GI₅₀ = 0.20 μM) and breast MDA-MB-231 (GI₅₀ = 0.09 μM), and shows no evidence of toxicity in normal cell line. Compound 6b has an IC₅₀ of 110.18 nM in HSP90α inhibitory activity, slightly better than reference compound 1 (17-AAG, IC₅₀ = 141.62 nM) and achieves the degradation of multiple HSP90 client proteins in a dose- and time-dependent manner and downstream signaling of Akt in a concentration- and time-dependent manner in the human A549 lung cancer cell line. In the Boyden chamber assay, compound 6b can efficiently inhibit the migration of A549 cells when compared to the reference compound 1. It also induce significant activity through the apoptotic pathway. Treatment with 6b showed no vision toxicity (IC₅₀ > 10 μM) on 661w photoreceptor cells as compared to AUY922 (3a) with a 0.04 μM values of IC₅₀ and has no effect in hERG test. In a bidirectional Caco-2 permeability assay, compound 6b was classified as a highly permeable compound which is not a substrate of efflux transporters. In a pharmacokinetic study in rats, 6b showed an F = 17.8% of oral bioavailability. The effect of metabolic stability of compound 6b in human hepatocytes showed a T_1/2 of 67.59 min. Compound 6b (50 mg/kg, po, daily) exhibits antitumor activity with a 72% TGD (tumor growth delay) in human A549 lung xenograft. The combination of 6b and afatinib, orally administered, showed tumor growth suppression with 67.5% of TGI in lung H1975 xenograft model. Thus compound 6b is a lead compound for further development of potential agents to treat lung cancer.

Full text of DOI:10.1016/j.ejmech.2021.113428

European Journal of Medicinal Chemistry 219 (2021) 113428
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Ring-opening of five-membered heterocycles conjugated 4-
isopropylresorcinol scaffold-based benzamides as HSP90 inhibitors
suppressing tumor growth in vitro and in vivo
,
b
,
,
,
,
Yi-Min Liu a 1, Huang-Ju Tu c 1, Chueh-Heng Wu e 1, Mei-Jung Lai b d, Shu-Chieh Yu a,
Min-Wu Chao c, Yi-Wen Wu c, Che-Ming Teng e, Shiow-Lin Pan b
,
, c, d, **
Jing-Ping Liou a
, b, d, *
a School of Pharmacy, College of Pharmacy, Taipei Medical University, 250 Wuxing Street, Taipei, 11031, Taiwan
b Biomedical Commercialization Center, Taipei Medical University, Taipei, 11031, Taiwan
c Graduate Institute of Cancer Molecular Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, 11031,
Taiwan
d Ph.D. Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, Taipei, 11031, Taiwan
e Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of ring-opened dihydroxybenzamides have been designed and synthesized as heat shock protein
90 inhibitors. One of derivatives, compound 6b ((N-ethyl-2,4-dihydroxy-5-isopropyl-N-(pyridin-3-yl)
benzamide)) demonstrated remarkable antiproliferative activity against in human KRAS mutant A549
Received 13 November 2020
Received in revised form
12 March 2021
Accepted 29 March 2021
Available online 6 April 2021
and EGFR T790 M mutant H1975 lung cancer cell lines with GI50 values of 0.07 and 0.05
m
M, respectively.
It is also active against in other cancer cell lines, such as colorectal HCT116 (GI50 ¼ 0.09
mM), liver Hep3B
mM), and shows no evidence of toxicity in normal
(GI50 ¼ 0.20
m
M) and breast MDA-MB-231 (GI50 ¼ 0.09
cell line. Compound 6b has an IC50 of 110.18 nM in HSP90
a inhibitory activity, slightly better than
Keywords:
Ring-opening
Heat shock protein 90 inhibitors
Lung cancer
reference compound 1 (17-AAG, IC50 ¼ 141.62 nM) and achieves the degradation of multiple HSP90 client
proteins in a dose- and time-dependent manner and downstream signaling of Akt in a concentration-
and time-dependent manner in the human A549 lung cancer cell line. In the Boyden chamber assay,
compound 6b can efficiently inhibit the migration of A549 cells when compared to the reference
compound 1. It also induce significant activity through the apoptotic pathway. Treatment with 6b
showed no vision toxicity (IC50 > 10
mM) on 661w photoreceptor cells as compared to AUY922 (3a) with a
0.04 M values of IC50 and has no effect in hERG test. In a bidirectional Caco-2 permeability assay,
m
compound 6b was classified as a highly permeable compound which is not a substrate of efflux trans-
porters. In a pharmacokinetic study in rats, 6b showed an F ¼ 17.8% of oral bioavailability. The effect of
metabolic stability of compound 6b in human hepatocytes showed a T1/2 of 67.59 min. Compound 6b
(50 mg/kg, po, daily) exhibits antitumor activity with a 72% TGD (tumor growth delay) in human A549
lung xenograft. The combination of 6b and afatinib, orally administered, showed tumor growth sup-
pression with 67.5% of TGI in lung H1975 xenograft model. Thus compound 6b is a lead compound for
further development of potential agents to treat lung cancer.
© 2021 Published by Elsevier Masson SAS.
Abbreviations: DCM, dichloromethane; DMAP, 4-(Dimethylamino)pyridine; DMF, dimethylformamide; EDC．HCl, N-(3-Dimethylaminopropyl)-N0-ethylcarbodiimide
hydrochloride; HOBt, 1-Hydroxybenzotriazole hydrate; NMM, N-methylmorpholine; TFA, trifluoroacetic acid; THF, tetrahydrofuran.
* Corresponding author. School of Pharmacy, College of Pharmacy, Taipei Medical University, 250 Wuxing Street, Taipei, 11031, Taiwan.
** Corresponding author. Biomedical Commercialization Center, Taipei Medical University, Taipei, 11031, Taiwan.
E-mail addresses: slpan@tmu.edu.tw (S.-L. Pan), jpl@tmu.edu.tw (J.-P. Liou).
Authors contributed equally.
1
https://doi.org/10.1016/j.ejmech.2021.113428
0223-5234/© 2021 Published by Elsevier Masson SAS.

Y.-M. Liu, H.-J. Tu, C.-H. Wu et al.
European Journal of Medicinal Chemistry 219 (2021) 113428
1. Introduction
benzamide (5b) have also been reported [10] (Fig. 1).
Among HSP90 inhibitors in the clinical trials, the resorcinol
derivatives are commonly used alone or in combination with pro-
teasome inhibitors in the treatment of non-small cell lung cancer
(NSCLC) and can have significant outcomes [11e14]. With the
appearance of these related results, HSP90 has emerged as a
notable anticancer target.
Heat shock proteins (HSPs) are molecular chaperones which
play an essential role in the development of proteins. They are
required for basic functions like protein folding and participate in
many higher-order functions such as post-translational regulation
of signaling molecules and assembly or disassembly of transcrip-
tional complexes. Heat shock proteins are classified into different
families based on their molecular weight. HSPs, especially HSP90
which is associated with a number of signaling pathways [1,2], have
been widely investigated in recent decades.
HSP90 has a primary role in both normal and cancer cells in
activation of a wide range of proteins known as HSP90 client pro-
teins. These client proteins can be separated into three main clas-
ses: steroid hormone receptors, tyrosine and serine/threonine
kinases, and proteins with miscellaneous functions. Cancer cells
overexpress a number of HSP90 client proteins, such as hypoxia-
Though the use of an HSP90 inhibitor, the treatment of non-
small cell lung cancer (NSCLC) has enjoyed some success, but
vision-related disorders have been reported. In the Phase 2 study of
the AUY922 (3a) in previously treated patients with advanced
NSCLC for example, vision-related disorders were reported in 79.7%
of patients (most were grade 1/2). 22.9% of patients reported night
blindness and 22.2% of patients reported photopsia. Other vision-
related disorders have also been reported, such as vision blurred
(19.6%), visual impairment (19.6%), and visual acuity reduced
(17.0%) [15]. In another Phase 2 study of the activity of AUY922 (3a)
against NSCLC harboring EGFR exon 20 insertions, vision changes
were reported in 76% of patients (most were grade 1/2) [16].
Because of the significant treatment outcomes and the concern
of vision disorders, we investigated the concept of ring-opening in a
rational design process. Compounds 3a-3b (Fig. 1) possess a ring
system connected to a resorcinol and compound 3c has a ring
connected to a resorcinol through an amide linker. Attempts were
made to modify this ring system by introducing various aryl rings
connected by an amide linker, for example, compounds 6 (3-
aminopyridine), 7 (4-aminopyridine) and 8 (5-aminoindole). In
addition, an attempt was also made to utilize different-sized alkyl
groups in the substituents linked to resorcinol through the amide
linkage, to generate a series of potential HSP90 inhibitors (Fig. 2). In
this way, a number of designed target compounds (6e8) (Table 1)
were synthesized and their antiproliferative activity was evaluated
and discussed below.
inducible factor-1a (HIF-1a), mutant p53, AKT (protein kinase B)
and epidermal growth factor receptor (EGFR), which are all
involved in tumor proliferation and metastasis [3,4].
HSP90 plays a pivotal role in the hallmarks of cancer cells and its
inhibition and disruption can affect processes involved in the
initiation of cancer. Consequently, some compounds behaving as
HSP90 inhibitors have entered clinical trials. HSP90 inhibitors can
be divided into several classes based on distinct structures. Exam-
ples include ansamycin derivatives as 17-AAG (1, Tanespimycin,
Phase III) [5], purine derivatives as BIIB021 (2, Phase II) [6], resor-
cinol derivatives such as AUY922 (3a, Luminespib, Phase II) [7],
STA-9090 (3b, Ganetespib, Phase III) [8] and AT-13387 (3c, Ona-
lespib, Phase II) [9] (Fig. 1). Especially the resorcinol derivatives,
there are few compounds under investigation with different core
structures have shown significant activities and served as HSP90
inhibitors. For example, the aryl-triazolyl acetate (4a), tetraisoqui-
nolinecarboxamide (4b), amide-tethered quinoline (5a), N-benzyl
Fig. 1. Structures of HSP90 inhibitors in clinical trials and compounds under investigation.
2

Y.-M. Liu, H.-J. Tu, C.-H. Wu et al.
European Journal of Medicinal Chemistry 219 (2021) 113428
Table 1
Structures of designed and synthesized compounds (6e8).
2. Results and discussion
2.1. Chemistry
Scheme 1 shows the synthesis of compounds 6a-6d and 7a-7d.
Compound 9 was reacted with m- or p-aminopyridine in the
presence of a coupling reagent such as EDC.HCl to yield compounds
10e11. The benzyl group in compounds 6a and 7a was removed by
hydrogenolysis. The amide NH group of intermediates (10e11) was
deprotonated by strong base to generate the alkylated products,
then completed with the debenzylation by hydrogenolysis to afford
compounds 6b-6d and 7b-7d.
R
R1
R2
Scheme 2 depicts the synthesis of compounds 8. Compound 12
is commercially available and was reacted with Boc anhydride or
methyl iodide to attack the N-1 position of indole, followed by
reduction of the nitro group and an amide coupling reaction with
compound 9 to yield intermediates 13e14. The protecting groups in
compounds 8a and 8f were directly removed treatment with acid
or heterolytic H2 cleavage. Compounds with an alkyl group at the
amide linkage (8b-8e and 8g-8i) were used in similar synthetic
procedures.
6a
6b
6c
6d
7a
7b
7c
7d
H
8a
8b
8c
8d
8e
8f
8g
8h
8i
H
H
H
H
H
CH3
C2H5
C3H7
CH(CH3)2
H
CH3
C2H5
C3H7
C2H5
C3H7
CH(CH3)2
H
C2H5
C3H7
CH(CH3)2
H
CH3
CH3
CH3
CH3
Synthesis of the intermediate compound (9) began from com-
pound 15, which is commercially available, and proceeded through
five steps to yield the product (Scheme 3). First, the hydroxyl
groups of compound 15 were protected by benzyl groups (16). Next,
the acetyl group was converted to a tertiary alcohol (17) by a
Grignard reaction and then reacted with triethylsilane and TFA to
get compound 18. Finally, to undergo formylation with the Vils-
meier reaction (19) and then oxidation, compound 19 was
converted to the intermediate compound 9.
2.2. Biological evaluation
2.2.1. In vitro cell growth inhibitory activity
The synthetic compounds (6e8) were evaluated for their
Fig. 2. The rational design of compounds 6e8 with the ring-opening concept.
3

Y.-M. Liu, H.-J. Tu, C.-H. Wu et al.
European Journal of Medicinal Chemistry 219 (2021) 113428
Scheme 1. Synthetic route to compounds 6-7a. aReagents and conditions: (a) 3-or 4-aminopyridine, EDC.HCl, HOBt, NMM, DMF, r.t., 71e73%; (b) for 6a and 7a, 10% Pd/C, H2, MeOH,
r.t., 87e88%; for 6b-6d and 7b-7d, alkyl iodide, NaH, DMF, r.t. then 10% Pd/C, H2, MeOH, r.t., 60e70%.
Scheme 2. Synthetic route to designed compounds 8a. aReagents and conditions: (a) (i) for 13, Boc anhydride, DMAP, DCM, r.t.; for 14, methyl iodide, NaH, DMF, r.t.; (ii) Fe powder,
NH4Cl, IPA/H2O, reflux, (iii) 9, EDC.HCl, HOBt, NMM, DMF, r.t., 33e79% in three steps; (b) for 8a, (i) TFA, DCM, r.t. (ii) 10% Pd/C, H2, MeOH, r.t., 73% in two steps; for 8b-8e, (i) alkyl
iodide, NaH, DMF, r.t. (ii) TFA, DCM, r.t. (iii) 10% Pd/C, H2, MeOH, THF, 40 psi, r.t., 48e57% in three steps; for 8f, 10% Pd/C, H2, MeOH, r.t., 48%; for 8g-8i, (i) alkyl iodide, NaH, DMF, r.t.,
(ii) 10% Pd/C, H2, MeOH, r.t., 71e75% in two steps.
Scheme 3. Synthetic route to intermediate compound 9a. aReagents and conditions: (a) benzyl bromide, K2CO3, acetone, reflux, 93%; (b) methylmagnesium bromide, THF, 0 ꢀC to r.t.,
77%; (c) triethylsilane, TFA, DCM, ꢁ78 ꢀC to r.t., 92%; (d) POCl3, DMF, 0 ꢀCe80 ꢀC, 92%; (e) sulfamic acid, NaClO2, H2O, THF, dimethyl sufoxide, 0 ꢀC to r.t., 65%.
antiproliferative activity against human A549 lung cancer cell line
(Table 2). Based on the activity results, compounds such as 6a, 7a,
8a and 8f with a free amide linkage displayed loss of activity. The
varying carbon length of alkyl groups in the substituted amide
linkage exhibits a regular pattern in the growth inhibitory activity.
The inhibitory activity increases with a methyl group (8b, 8g) or an
4

Y.-M. Liu, H.-J. Tu, C.-H. Wu et al.
European Journal of Medicinal Chemistry 219 (2021) 113428
ethyl group (6b, 7b, 8c, 8h), but decreases with a propyl group (6c,
7c, 8d, 8i) or an isopropyl group (6d, 7d, 8e). Interestingly, com-
pounds with an ethyl-substituted amide linkage (6b, 7b, 8c, 8h)
show the best antiproliferative activity in every series. Amongst the
synthetic compounds (6e8), compound 6b shows significant
inhibitory activity against to A549 cancer cell line with GI50 value of
cancer cell line in a concentration- and time-dependent manner
(Fig. 4A and B). As well as in human H1975 lung cancer cell line,
which is a T790M-positive cell line harboring the EGFR L858R/
T790 M double mutation, the compound 6b was tested for inhibi-
tory activity and showed a GI50 value of 0.05 0.01 mM.
0.07 0.01
m
M.
2.2.5. Evaluation of compound 6b on inhibition of migration
Cancer metastasis is an important issue due to the migration or
invasion of neoplastic cells from the primary tumor to distant parts
of the body. Herein, we have examined the anti-migration effect of
compound 6b by a Boyden chamber assay (Fig. 5). The results
2.2.2. Antiproliferative activity and HSP90 enzyme inhibition
against normal and cancer cell lines
We sought to learn if these synthetic compounds also possess
antiproliferative activity and HSP90 enzyme inhibition in other
cancer cell lines and the results are shown in Table 3. We chose to
investigate compounds 6b, 7b, 8b, 8c and 8g, which possess good
antiproliferative activity against the A549 lung cancer cell line.
Compound 6b has exhibited similar activity in human HCT116
colorectal cancer cell line (0.09 0.01
cancer cell line (0.09 0.01 M) compared to that of the A549 cell
line and slightly decreased activity against the Hep3B hepatocel-
lular carcinoma cell line (0.20 0.01 M). In addition, the inhibitory
activity of compound 6b is also better than the reference com-
pounds 1 (17-AAG) and 2 (BIIB021). Moreover, compound 6b shows
no evidence of toxicity in normal cell line.
Compound 6b also possesses the best therapeutic index among
these compounds with a value of 12.4 and an IC50 value of
110.18 3.13 nM for the HSP90 enzyme inhibition which is similar
revealed that at 1
mM, compound 6b can efficiently inhibit the
migration of A549 cells as compared to the reference compound, 1
(17-AAG).
2.2.6. Evaluation of compound 6b in cell cycle progression and
apoptotic pathways
mM) and MDA-MB-231 breast
The effect of compound 6b on cell cycle distribution was
investigated by Western blotting. The data show that compound 6b
increases the expression of cyclin B1 and enhances the phosphor-
ylation of cdc2 at Thr161 while suppressing phosphorylation of
cdc2 at Tyr15 and cdc25c at Ser216 (Fig. 6A). Furthermore, the
mitotic markers p-MPM-2 and p-H3 (Ser10) are also increased by
compound 6b. These results indicated that compound 6b induces
M stage arrest in A549 cells. We also found that the levels of p53 are
markedly increased after cells are treated with compound 6b or
compound 1 (Fig. 6B). This overexpressed-p53 caused caspase-3,
caspase-6, caspase-7, caspase-9 cleavage and induced PARP acti-
vation. Fig. 6C shows that the accumulation of p53 started at 6 h,
while the activation of caspase and PARP started at 18 h. Ectopic
expression of WT-p53 partially reduced caspase-3 cleavage and
rescued the cell apoptosis which was caused by compound 6b
(Fig. 6D).
m
m
to that of reference compound 2 (BIIB021) (105.06
7.75 nM).
These data imply that compound 6b, with remarkable inhibitory
activity against several cancer cell lines has potential for further
development and it was thus selected for further investigations as
an HSP90 inhibitor.
2.2.3. Evaluation of compound 6b in degradation of HSP90 client
proteins and downstream signaling of Akt
2.2.7. Evaluation of compound 6b with respect to vision-related
toxicity
The in vitro western immunoblotting assay was used to confirm
the mechanism of action of compound 6b as an HSP90 inhibitor
(Fig. 3). The results reveal that compound 6b triggers the degra-
dation of multiple HSP90 client proteins such as FAK and Src along
with concomitant induction of HSP70 protein in a dose- and time-
dependent manner in the human A549 lung cancer cell line (Fig. 3A
and B). In addition, compound 6b can also trigger degradation of
downstream signaling of Akt in a concentration- and time-
dependent manner (Fig. 3C and D). These results are consistent
with the signature features of HSP90 inhibition.
As mentioned above, vision-related disorders of HSP90 in-
hibitors have been reported. In order to confirm whether com-
pound 6b induces vision-related toxicity, we have tested the
compound 6b on 661W photoreceptor cells (Table 4). Both com-
pound 3a (AUY922) and geldanamycin (an ansamycin-related
HSP90 inhibitor) have displayed significantly toxicity in 661W
cells with IC50 values of 0.04 and 1.65
compound 6b has an IC50 value over 10
no cytotoxicity to photoreceptor cells.
m
M, respectively. However,
mM, which indicates little or
2.2.4. Evaluation of compound 6b against mutant cancer lines
Based on recent reports of increasing resistance, we have
investigated if compound 6b can act against mutant cancer cell
lines (Fig. 4). Compound 6b can induce EGFR degradation and
strongly suppresses the MAPK pathways in K-ras mutant A549
2.2.8. Evaluation of compound 6b with a potassium channel hERG
test
hERG channels are involved in cardiac action potential repolar-
ization. Inhibition of the hERG current can cause QT interval pro-
longation resulting in potentially fatal ventricular tachyarrhythmia.
A number of drug candidates have been withdrawn from safety
studies due to these cardiotoxic effects, and it is important to
examine compounds for activity on hERG channels early in the lead
optimization process. In order to evaluate the effect of hERG test,
Table 2
Antiproliferative activity (GI50) of synthetic compounds 6e8.
Compound
A549 (GI50 SDa,
m
M)
Compound
A549 (GI50 SDa,
mM)
compound 6b (10 mM) was conducted by the Eurofin Cerep Panlabs.
The results showed the safety of compound 6b which failed to
reduce the hERG function (Table 5).
6a
6b
6c
6d
7a
7b
7c
7d
>10
8a
8b
8c
8d
8e
8f
8g
8h
8i
>10
0.07 0.01
0.39 0.01
0.26 0.02
>10
0.12 0.01
0.37 0.05
0.19 0.03
0.17 0.01
0.11 0.01
0.14 0.02
0.21 0.02
>10
0.28 0.02
0.08 0.01
0.30 0.01
2.2.9. Evaluation of compound 6b by CYP inhibition and
permeability assays
Cytochrome P450s (CYP) are a family of enzymes which play a
major role in the metabolism of drugs. Inhibition of CYPs by co-
administered drugs is one of the most common causes of drug-
drug interaction and leads to substantial increase of the parent
a
SD: standard deviation, all experiments were independently performed at least
three times.
5

Y.-M. Liu, H.-J. Tu, C.-H. Wu et al.
European Journal of Medicinal Chemistry 219 (2021) 113428
Table 3
In vitro efficacy of synthetic compounds 6b, 7b, 8b, 8c and 8g.
Compounds
Lung
A549
Colorectal HCT116
Liver
Hep3B
Breast MDA-MB-231
HUVEC
TIb
HSP90 IC50 (nM)
(GI50 SDa,
mM)
6b
0.07
0.01
0.12
0.01
0.17
0.01
0.11
0.01
0.28
0.02
0.08
0.01
0.26
0.03
0.09
0.01
0.64
0.41
1.00
0.49
0.37
0.05
0.70
0.06
0.34
0.06
0.25
0.02
0.20
0.03
0.10
0.01
0.18
0.04
0.11
0.02
0.57
0.32
0.08
0.01
0.24
0.02
0.09
0.01
0.08
0.01
0.07
0.01
0.08
0.01
0.10
0.02
0.27
0.01
0.24
0.01
0.87
0.01
<0.1
12.4
0.8
3.4
0.9
2.9
1.3
5.5
110.18
3.13
109.23
5.35
115.39
10.66
117.34
9.62
105.54
7.58
7b
8b
0.57
0.02
<0.1
8c
8g
0.82
0.03
<0.1
1 (17-AAG)
2 (BIIB021)
141.62
12.75
105.06
7.75
1.42
0.09
a
SD: standard deviation, all experiments were independently performed at least three times.
TI: Therapeutic Index. (HUVEC divided by the cancer cell line IC50).
b
Fig. 3. Compound 6b and compound 1 triggered degradation of multiple HSP90 client proteins and signaling downstream of Akt in a concentration- and time-dependent manner.
(A, C) A549 cells were treated with DMSO or 0.1e3 mM of drugs (compound 6b or compound 1) for 24 h. (B, D) A549 cells were treated with 0.3 mM of indicated drugs for indicated
times.
drug concentration. Compound 6b was tested for its CYP inhibitory
potential for CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4 (Table 6). In
direct CYP inhibition assays, compound 6b most potently inhibited
CYP3A4 with IC50 values of 1.15 and 3.23 M, using a substrate of
m
6

Y.-M. Liu, H.-J. Tu, C.-H. Wu et al.
European Journal of Medicinal Chemistry 219 (2021) 113428
Fig. 4. Compound 6b and compound 1 induced EGFR degradation and strongly suppressed MAPK pathways in K-ras mutant A549 cancer cell line in a concentration- and time-
dependent manner. (A) A549 cells were treated with DMSO or 0.1e3 mM of drugs (compound 6b or compound 1) for 24 h. (B) A549 cells were treated with 0.3 mM of indicated drugs
for indicated times.
Fig. 5. Evaluation of inhibition of migration by Boyden chamber assay. (A) A549 cells were seeded in the upper compartment of the Boyden chamber (1 ꢂ 105 cells), and incubated
with DMSO (CTL), 0.1e3 mM compound 6b or 1 mM 17-AAG for 6 h. 10% FBS was placed in the lower chamber as a chemo-attractant. Migrated cells in the lower compartment were
fixed, stained with crystal violet and viewed by microscopy. Two lanes of immunoblotting below were conducted to observe the inhibition of phosphorylation of Src or FAK. (B) Bar
graph represents the percentage of cell migration. *p < 0.05, **p < 0.01, and ***p < 0.001 as compared with the control group.
testosterone and midazolam. This was followed by CYP2C19
(7.80 M), CYP2C9 (14.97 M), and CYP2B6 (37.17 M). Compound
6b partially inhibited CYP1A2, 2C8 and 2D6 with IC50
values > 50 M.
2.2.10. Pharmacokinetics profile and metabolic stability of
compound 6b
m
m
m
The pharmacokinetic parameters of compound 6b after a sub-
sequent single intravenous (IV) administration of 2 mg/kg and oral
administration (PO) of 20 mg/kg to male Sprague Dawley rats are
shown in Table 8 and Figure S1. Following a single IV dosing
administration, 6b showed a t1/2 of 2.9 h. Systemic clearance was
4.39 L/h/kg, and Vss was 4.59 L/kg. Following a single administra-
tion of 6b PO, the median Tmax and t1/2 were 0.25 h and 16.4 h,
respectively. The oral bioavailability (F) was 17.8% based on the
AUC0-∞ ratio and was 15.1% based on the AUC0-t ratio.
m
A bidirectional Caco-2 permeability assay was used to examine
the in vitro permeability of compound 6b (Table 7). Verapamil and
atenolol, with high and low permeability respectively, served as
reference compounds. The Caco-2 cell permeability coefficients for
apical to basolateral (AP/BL) movement and basolateral to apical
(BL/AP) movement of compound 6b were 31.9 5.1 ꢂ 10ꢁ6cm/s
and 30.6 2.1 ꢂ 10ꢁ6cm/s and compound 6b can be classified as a
highly permeable compound (Papp,A/B > 10 ꢂ 10ꢁ6 cm/s) (Table 7).
It has an efflux ratio (ratio of BL/AP/AP/BL) of 0.96 and might not
be a substrate of efflux transporters.
To determine the in vitro metabolism among different species,
the metabolic stability of compound 6b was tested in mouse, rat,
dog and human hepatocytes (Fig. 7). The apparent half-life of
7

Y.-M. Liu, H.-J. Tu, C.-H. Wu et al.
European Journal of Medicinal Chemistry 219 (2021) 113428
Fig. 6. Effect of compound 6b on cell cycle progression and apoptotic pathways in human A549 lung cancer cell line. (A) Cells were treated with vehicle (DMSO) or 0.3 mM of
indicated drugs for indicated times. After treatment, whole cell lysates were collected and subjected to western blot analysis for the detection of cell cycle-related protein
expression. (B) Concentration-dependent and (C) time-dependent effects of compound 6b-induced p53 accumulation, PARP cleavage, and caspase activation. (D) A549 cells were
transfected with si-RNA specific to p53 or scrambled prior to treatment of compound 6b. After then, western blot was conducted for the detection of p53 and cleaved C3 protein
levels, and 48 h MTT assay was performed to measure A549 cell viability. *p < 0.05, **p < 0.01, and ***p < 0.001 as compared with the control group.
Table 4
The cytotoxic effects of compound 6b and HSP90 inhibitors in 661W by MTT assay.
Compounds
6b
1 (17-AAG)
>10
2 (BIIB021)
>10
3a (AUY922)
3b (STA-9090)
>10
Geldanamycin
1.65 0.6
IC50
(
m
M)a
>10
0.04 0.00
a
SD: standard deviation, all experiments were independently performed at least three times.
Table 5
compound 6b in mouse, rat, dog and human hepatocytes was 5.31,
11.22, 39.24, and 67.59 min, respectively (Table 9). The intrinsic
clearance (CLint,app) and predicted hepatic clearance (CLhep) of
compound 6b were 185.00 and 5.25 L/h/kg in mouse hepatocytes,
45.03 and 3.07 L/h/kg in rat hepatocytes, 7.15 and 1.50 L/h/kg in dog
hepatocytes, and 3.32 and 0.88 L/h/kg in human hepatocytes,
The potassium channel hERG test of compound 6b.
Compound
Concentration (
m
M)
Inhibition (%)a
6b
10
ꢁ18
a
Potassium channel hERG test was examined by the Eurofin Cerep Panlabs.
8

Y.-M. Liu, H.-J. Tu, C.-H. Wu et al.
European Journal of Medicinal Chemistry 219 (2021) 113428
Table 6
Table 8
Evaluation of pharmacokinetics parameters in rat.a.
Direct inhibitory effects (IC50 values) of compound 6b on seven human CYP450
enzymes in pooled human liver microsomes.
Parameter
IV (2 mg/kg)
PO (20 mg/kg)
F (%)
CYP enzyme
Substrate
IC50 (m
M)a
Tmax (h)b
0.03 (0.02e0.03)
1580 135
487 32
456
2.9
4.59
0.25 (0.25e6.0)
40.5 10.8
737 127
811 164
16.4 (14.0e17.4)
NA
NA
NA
CYP1A2
CYP2B6
CYP2C8
CYP2C9
CYP2C19
CYP2D6
CYP3A4
CYP3A4
Phenacetin
Bupropion
>50 (50.6%)
37.17
>50 (54.3%)
14.97
7.80
>50 (84.0%)
3.23
Cmax (ng/mL)
AUC(0-t) (ng mLꢁ1 h)
AUC0-∞ (ng mLꢁ1 h)
t1/2 (hr)
Vss (L/kg)
CL (L/L/kg)
15.1c
17.8d
NA
Amodiaquine
Diclofenac
S-Mephenytoin
Dextromethorphan
Midazolam
NA
NA
4.39
NA
a
The studies were performed by QPS Taiwan.
Median (range).
F(%) was calculated as (AUC0-t, PO/DosePO)/(AUC0-t, IV/DoseIV).
Testosterone
1.15
b
c
a
When IC50 was greater than 50
m
M, the percent inhibition value at 50
m
M was
d
included (in parentheses); The studies were performed by Development Center for
Biotechnology Taiwan.
F(%) was calculated as (AUC0-∞, PO/DosePO)/(AUC0-∞, IV/DoseIV); NA: Not
applicable.
respectively. In addition, compound 6b showed a high hepatic
extraction ratio in mouse (E ¼ 0.97) and rat (E ¼ 0.93) hepatocytes,
and moderate to high hepatic extraction ratio in dog (E ¼ 0.79) and
human (E ¼ 0.73) hepatocytes. This indicates that 6b is likely to be
cleared rapidly through the liver.
2.2.11. In vivo antitumor efficacy of compound 6b in human A549
lung cancer xenografts
Based on a few significant outcomes of HSP90 inhibitors used in
NSCLC treatment that have been reported recently, compound 6b
was evaluated for in vivo efficacy against tumor xenografts in hu-
man A549 lung cancer xenografts (n ¼ 7) (Fig. 8). Compound 6b
displayed antitumor activity at doses of 25 mg/kg and 50 mg/kg,
Fig. 7. Time course of stability in mouse, rat, dog and human hepatocytes.
suppressing the growth with 46.7% (*p
< 0.05) and 46.9%
(*p < 0.05) of TGI (tumor growth inhibition) after oral adminis-
tration, respectively (Fig. 8A). No significant differences in weight
loss were observed during all the treatments (Fig. 8B). Moreover,
compound 6b significant enhanced tumor growth delay (TGD, 72%)
compared to a vehicle treated group. (Fig. 8C) (Table 10).
weight loss (Fig. 9C and D). Compound 6b combined with afatinib
showed synergistic antitumor effect compared to the single treat-
ment. Compound 3b (150 mg/kg, iv, once weekly) [17] single or
combined with afatinib, did not exhibit antitumor activity
(p > 0.05) but was associated with treatment-related animal death
in monotherapy (4/9 dead) or combination therapy (8/9 dead).
Compared with reference compound 3b, compound 6b seems to be
a safer and more efficacious HSP90 inhibitor.
2.2.12. In vivo antitumor efficacy of compound 6b in human H1975
lung cancer xenografts
The antitumor efficacy of compound 6b in human H1975 lung
cancer xenograft was evaluated employing afatinib and STA-9090
(3b) as reference compounds (Fig. 9). Compound 6b has shown
antitumor activity and reduction in tumor volume at 50 mg/kg with
TGI as 34.3% (**p < 0.01) without any significant body weight loss
during the treatment period (Fig. 9A and B, n ¼ 8). The effect of
afatinib, STA-9090, and compound 6b in combination was exam-
ined as shown in Fig. 9C and D (n ¼ 9). The combination therapy led
to substantially higher reductions in the tumor volume. Mono-
therapy with 6b resulted in TGI of 25.5% (***p < 0.001) at 200 mg/
kg (po, q3d) and 27.2% (***p < 0.001) at 50 mg/kg (po, qd). While
compound 6b at doses of 200 mg/kg (po, q3d) and 50 mg/kg (po,
qd) in combination with afatinib (25 mg/kg, po, qd), resulted in
suppressing tumor growth by a TGI of 50.5% (***p < 0.001) and
67.5% (***p < 0.001), respectively, without considerable body
3. Conclusion
We have synthesized
ybenzamide compounds (6a-6d, 7a-7d, and 8a-8i). Among all these
synthetic compounds, compound 6b was observed to be a HSP90
a series of ring-opened dihydrox-
inhibitor with an HSP90
a inhibitory IC50 of 110.18 nM, which is
slightly better than reference compound 1. It can efficiently inhibit
the migration of A549 cells compared to the reference compound 1.
Compound 6b also has substantial in vitro antiproliferative activity
and in vivo antitumor activity. Our results show that 6b exhibits
significant inhibitory activity in human KRAS mutant A549, EGFR
T790 M mutant H1975, HCT116 and MDA-MB-231 with IC50 values
of 70, 50, 90 and 90 nM, respectively, and shows no evidence of
Table 7
Permeability and efflux ratio of compound 6b and reference compounds in both directions across Caco-2 cell monolayers.
Compound
6b
Verapamil
Atenolol
Rhodamine 123
0.53 0.68
Papp (A to B)a
31.86 5.12
14.39 1.11
0.45 0.06
(x 10ꢁ6 cm/s)
Papp (B to A)a
30.60 2.14
e
e
8.04 1.68
(x 10ꢁ6 cm/s)
Assay acceptance criteria (cm/sec)
Efflux ratio
Papp ꢃ 10 ꢂ 10ꢁ6
0.96
Papp ꢃ 10 ꢂ 10ꢁ6
e
Papp < 5 ꢂ 10ꢁ6
e
e
15.17
Classification
High permeable
High permeable
Low permeable
Substrate of efflux transporter
a
Permeability coefficient (Papp) for apical to basolateral (A to B); The studies were performed by Development Center for Biotechnology Taiwan.
9

Y.-M. Liu, H.-J. Tu, C.-H. Wu et al.
European Journal of Medicinal Chemistry 219 (2021) 113428
Table 9
Hepatocytes stability of compound 6 in mouse, rat, dog and human.a.
Compound
Species Hepatocytes
T1/2 (min)
CLint (L/h/kg)
CLhep (L/h/kg)
Extraction ratio
Clearance classification
6b
Mouse
Rat
Dog
5.31
185.00
45.03
7.15
5.25
3.07
1.50
0.88
0.97
0.93
0.79
0.73
High
High
Moderate to High
Moderate to High
11.22
39.24
67.59
Human
3.32
a
The studies were performed by QPS Taiwan.
Fig. 8. In vivo antitumor efficacy of compound 6b on human A549 lung xenograft. (A) 1 ꢂ 107 A549 cells were injected into 8 wk-old Balb/c nude mice. The mice were divided into
three groups (each group n ¼ 7) and treated with vehicle, compound 6b 25 mg/kg and 50 mg/kg by daily oral administration route. Tumor growth is tracked by the mean tumor
volume (mm3) SE and calculated as % tumor growth inhibition (%TGI). (B) Body weight change after drug treatment. (C) Tumor growth delay (TGD) induced by compound 6b in
A549 xenograft. *p < 0.05, **p < 0.01, and ***p < 0.001 as compared with the control group.
toxicity in normal cell line. Compound 6b inhibits tumor growth by
46.9% and has a tumor growth delay of 72% at 50 mg/kg (po, qd) in
human A549 lung xenografts. It has a TGI of 27.2% at 50 mg/kg (po,
qd) in a lung H1975 xenograft model. Combination therapy of 6b at
dose of 50 mg/kg (po, qd) and afatinib led to substantially higher
reductions in the tumor volume and 67.5% TGI. Combined with
afatinib, compound 6b showed a synergistic antitumor effect
compared to treatment with 6b alone. No significant differences in
weight loss were observed during all the treatments. Treatment
with 6b exhibited no vision toxicity on 661w photoreceptor and
without effect in hERG test. From the results of the pharmacoki-
netic study in rats, 6b shows an oral bioavailability (F) of 17.8%. In a
Caco-2 permeability assay, it was indicated that compound 6b
could be classified as a highly permeable compound and might not
be a substrate of efflux transporters. In the metabolic stability assay,
the half-life and the intrinsic clearance of compound 6b were
67.59 min and 3.32 L/h/kg in human hepatocytes. The CYP inhibi-
tory potential of 6b on CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4 in
pooled human liver microsomes was evaluated and CYP3A4 was
found to be the most sensitive to 6b. In conclusion, our study offers
evidence for the efficacy and safety of compound 6b, and reveals its
potential as an anti-lung cancer agent.
Table 10
Tumor growth delay by compound 6b in human A549 lung xenograft.
Treatment
Dose (mg/kg)
Median TTEa
T-Cb
TGD (%)c
Control
Compound 6b
Compound 6b
e
13.8
19.5
23.8
e
e
25
50
5.7
10
41
72
a
TTE: time to endpoint.
T-C: difference between median TTE of treated vs control group.
% TGD (Tumor growth delay): [(T-C)/C] x 100.
b
c
10

Y.-M. Liu, H.-J. Tu, C.-H. Wu et al.
European Journal of Medicinal Chemistry 219 (2021) 113428
Fig. 9. Anticancer activities of HSP90 inhibitors and afatinib in human lung cancer H1975 xenograft models. (A) 1 ꢂ 107 H1975 cells were injected into 8 wk-old Balb/c nude mice.
The mice were divided into four groups (each group n ¼ 9) and treated with vehicle, afatinib 25 mg/kg, compound 6b 25 mg/kg and 50 mg/kg by daily oral administration route.
Tumor growth is tracked by the mean tumor volume (mm3) SE and calculated as % tumor growth inhibition (%TGI). (B) Body weight change after treatment. (C) The effect of
compound 6b, afatinib and STA-9090 alone or in combination treatment. (each group n ¼ 9) (D) Body weight change after treatment. *p < 0.05, **p < 0.01, and ***p < 0.001 as
compared with the control group. TR, treatment related death.
4. Experimental section
4.1.1. Syntheses of compound 6-8
4.1.1.1. 2,4-Dihydroxy-5-isopropyl-N-(pyridin-3-yl)benzamide (6a).
A mixture of 10 (0.42 g, 0.93 mmol), 10% palladium on activated
carbon (0.04 g, 0.04 mmol) and MeOH (8 ml) was placed under
hydrogen gas. The reaction was stirred at room temperature for 2 h.
The 10% palladium on activated carbon was removed by a methyl
alcohol wash through celite packing. The organic layer was
concentrated in vacuo and purified by silica gel chromatography
(EtOAc:n-hexane ¼ 2: 1, Rf ¼ 0.3) to afford 6a (0.22 g, 88.0%) as a
4.1. Chemistry
Nuclear magnetic resonance (1H and 13CNMR) spectra were
obtained with a Bruker DRX-500 spectrometer operating at 500 or
125 MHz. Chemical shifts are reported in parts per million (ppm, d)
downfield from TMS as an internal standard. Low-resolution mass
spectra (LRMS) were measured with TSQ-700 (Finnigan, Germany).
High-resolution mass spectra (HRMS) were measured with a JEOL
(JMS-700) electron impact (EI) mass spectrometer. The purities of
the final compounds were determined using a Waters Acquity
UPLC/BSM with PhotoDiode Array detector using C-18 column
white solid. m.p. 269.1e270.3 ꢀC. 1H NMR (500 MHz, MeOD):
d 1.24
(d, J ¼ 7 Hz, 6H), 3.20 (Sep, J ¼ 7 Hz, 1H), 6.35 (s, 1H), 7.41e7.43 (m,
1H), 7.75 (s, 1H), 8.17e8.20 (m, 1H), 8.27e8.28 (m, 1H), 8.82 (d,
J ¼ 2.5 Hz,1H). 13C NMR (125 MHz, DMSO‑d6):
d 22.64, 26.15,102.59,
(Waters Acquity BEH-C18, 2.1 mm (ID) x 50 mm (L), 1.7
mm particle
107.23, 123.55, 126.58, 126.66, 128.50, 134.99, 142.82, 144.80,
159.03, 159.99, 167.54. hMS (ESI) for C15H15N2O3 [M ꢁ H]-: calcd,
271.1088; found, 271.1081.
size) with the solvent system consisting of water containing 0.1%
formic acid þ 2 mM NH4OAc (mobile phase A) and acetonitrile
(mobile phase B), and were found to be S 95%. Flash column
chromatography was accomplished on silica gel (Merck Kieselgel
60, No. 9385, 230e400 mesh ASTM). All reactions were carried out
under an atmosphere of dry nitrogen.
4.1.1.2. N-Ethyl-2,4-dihydroxy-5-isopropyl-N-(pyridin-3-yl)benza-
mide (6b). A mixture of 10 (0.35 g, 0.77 mmol), NaH (0.04 g,
1.0 mmol) and DMF (4 ml) was added to ethyl iodide (0.1 ml,
11

Y.-M. Liu, H.-J. Tu, C.-H. Wu et al.
European Journal of Medicinal Chemistry 219 (2021) 113428
1.24 mmol) at R.T. for 2 h. The reaction was quenched with water
and extracted with EtOAc (30 ml x 3). The organic layer was
collected and dried over anhydrous MgSO4 concentrated in vacuo
and purified by silica gel chromatography (EtOAc:n-hexane ¼ 2: 1,
Rf ¼ 0.4) to get the oily product, then the oily product was dissolved
in MeOH (8 ml) and added 10% palladium on activated carbon
(0.04 g, 0.04 mmol) under the hydrogen gas. The reaction was
stirred at R.T. for 2 h. The 10% palladium on activated carbon was
removed by methyl alcohol washed through celite packing. The
organic layer was concentrated in vacuo and purified by silica gel
chromatography (EtOAc:n-hexane ¼ 2: 1, Rf ¼ 0.3) to afford 6b
(0.16 g, 69.57%) as a yellow solid. m.p. 179.6e181.5 ꢀC. 1H NMR
4.1.1.7. 2,4-Dihydroxy-5-isopropyl-N-propyl-N-(pyridin-4-yl)benza-
mide (7c). The synthetic procedure for compound 7c is similar to
that used for compound 6b and gave a yield of 63.64% as a white
solid. m.p. 196.3e198.2 ꢀC. 1H NMR (500 MHz, MeOD):
d 0.86 (d,
J ¼ 7 Hz, 6H), 0.94 (t, J ¼ 7.5 Hz, 3H), 1.65 (Sex, J ¼ 7.5 Hz, 2H), 2.97
(Sep, J ¼ 7 Hz, 1H), 3.92 (t, J ¼ 7.5 Hz, 2H), 6.20 (s, 1H), 6.68 (s, 1H),
7.17e7.18 (m, 2H), 8.38e8.39 (m, 2H). 13C NMR (125 MHz, MeOD):
d
11.67, 22.36, 22.93, 27.06, 52.35, 103.55, 111.09, 122.95, 127.81,
129.51, 151.07, 154.35, 159.16, 160.36, 173.42. hMS (ESI) for
C
18H22N2NaO3 [MþNa]þ: calcd, 337.1523; found, 337.1514.
4.1.1.8. 2,4-Dihydroxy-N,5-diisopropyl-N-(pyridin-4-yl)benzamide
(7d). The synthetic procedure for compound 7d is similar to that
used for compound 6b and gave a yield of 60.25% as a white solid.
(500 MHz, MeOD):
d
0.90 (d, J ¼ 7 Hz, 6H), 1.20 (t, J ¼ 7 Hz 3H), 2.96
(Sep, J ¼ 7 Hz, 1H), 3.95 (q, J ¼ 7 Hz, 2H), 6.15 (s, 1H), 6.66 (s, 1H),
7.38e7.41 (m, 1H), 7.69e7.71 (m, 1H), 8.28 (d, J ¼ 2 Hz, 1H),
m.p. 165.4e166.6 ꢀC. 1H NMR (500 MHz, MeOD):
d
0.89 (d, J ¼ 7 Hz,
8.33e8.34 (m, 1H). 13C NMR (125 MHz, MeOD):
d
13.26, 23.08, 27.18,
6H), 1.27 (d, J ¼ 6.5 Hz, 6H), 2.96 (Sep, J ¼ 7 Hz, 1H), 4.89 (Sep,
46.48, 103.47, 112.03, 125.60, 127.68, 128.91, 137.08, 142.36, 147.93,
J ¼ 7 Hz, 1H), 6.15 (s, 1H), 6.63 (s, 1H), 7.21 (d, J ¼ 6 Hz, 2H),
149.64, 157.95, 159.54, 172.97. hMS (ESI) for
C
17H20N2NaO3
8.43e8.45 (m, 2H). 13C NMR (125 MHz, MeOD):
d 21.38, 23.02, 27.11,
[MþNa]þ: calcd, 323.1366; found, 323.1357.
51.03, 103.40, 112.91, 126.41, 127.54, 128.66, 150.98, 151.43, 157.95,
159.35, 173.09. hMS (ESI) for C18H22N2NaO3 [MþNa]þ: calcd,
337.1523; found, 337.1513.
4.1.1.3. 2,4-Dihydroxy-5-isopropyl-N-propyl-N-(pyridin-3-yl)benza-
mide (6c). The synthetic procedure for compound 6c is similar to
that used for compound 6b and gave a yield of 67.85% as a white
4.1.1.9. 2,4-Dihydroxy-N-(1H-indol-5-yl)-5-isopropylbenzamide
(8a). A mixture of 13 (0.5 g, 0.85 mmol) was dissolved in DCM
(5 ml) and added TFA (1 ml) at R.T. for 2 h. The reaction was
quenched with water and extracted with EtOAc (30 ml x 3). The
organic layer was collected and dried over anhydrous MgSO4
concentrated in vacuo and without further purification. The crude
product was dissolved in MeOH (8 ml) and added 10% palladium on
activated carbon (0.04 g, 0.04 mmol) under the hydrogen gas. The
reaction was stirred at R.T. for 2 h. The 10% palladium on activated
carbon was removed by a methyl alcohol wash through celite
packing. The organic layer was concentrated in vacuo and purified
by silica gel chromatography (EtOAc:n-hexane ¼ 1: 1, Rf ¼ 0.4) to
afford 8a (0.19 g, 73.07%) as a green solid. m.p. 221.1e223.7. 1H NMR
solid. m.p. 196.3e198.2 ꢀC. 1H NMR (500 MHz, MeOD):
d 0.92 (m,
9H), 1.63 (6, J ¼ 7.5 Hz, 2H), 2.97 (Sep, J ¼ 7 Hz, 1H), 3.87 (t,
J ¼ 7.5 Hz, 2H), 6.14 (s, 1H), 6.66 (s,1H), 7.37e7.41 (m,1H), 7.69e7.71
(m, 1H), 8.28e8.32 (m, 2H). 13C NMR (125 MHz, MeOD):
d 11.69,
22.14, 23.10, 27.21, 52.97, 103.47, 112.38, 125.56, 127.72, 128.82,
136.95, 142.60, 147.85, 149.54, 157.67, 159.46, 173.18. hMS (ESI) for
C
18H22N2NaO3 [MþNa]þ: calcd, 337.1523; found, 337.1516.
4.1.1.4. 2,4-Dihydroxy-N,5-diisopropyl-N-(pyridin-3-yl)benzamide
(6d). The synthetic procedure for compound 6d is similar to that
used for compound 6b and gave a yield of 60.25% as a yellow solid.
m.p. 172.4e174.3 ꢀC. 1H NMR (500 MHz, MeOD):
d
0.96 (d,
(500 MHz, MeOD):
d
1.22 (d, J ¼ 6.5 Hz, 6H), 3.19 (Sep, J ¼ 7 Hz, 1H),
J ¼ 6.5 Hz, 6H), 1.19 (d, J ¼ 7 Hz, 6H), 2.98 (Sep, J ¼ 7 Hz, 1H), 4.96
6.35 (s, 1H), 6.41 (d, J ¼ 7 Hz, 1H), 7.15 (d, J ¼ 2 Hz, 1H), 7.15e7.22 (m,
(Sep, J ¼ 7 Hz, 1H), 6.11 (s, 1H), 6.66 (s, 1H), 7.36e7.39 (m, 1H),
1H), 7.32 (d, J ¼ 8.5 Hz, 1H), 7.70e7.73 (m, 2H). 13C NMR (125 MHz,
7.67e7.69 (m, 1H), 8.29 (d, J ¼ 2.5 Hz, 1H), 8.36e8.37 (m, 1H). 13
C
MeOD): d 23.20, 28.11, 102.66, 103.79, 109.12, 112.22, 115.85, 118.99,
NMR (125 MHz, MeOD):
d
21.48, 23.18, 27.28, 49.84, 103.36, 114.37,
126.64, 127.37, 128.67, 129.63, 130.76, 135.63, 160.99, 161.33, 170.16.
hMS (ESI) for C18H18N2NaO3 [MþNa]þ: calcd, 333.1210; found,
333.1212.
125.15, 127.61, 127.99, 138.58, 139.93, 148.82, 151.68, 156.24, 158.64,
173.26. hMS (ESI) for C18H22N2NaO3 [MþNa]þ: calcd, 337.1523;
found, 337.1517.
4.1.1.10. 2,4-Dihydroxy-N-(1H-indol-5-yl)-5-isopropyl-N-methyl-
benzamide (8b). A mixture of 13 (0.8 g, 1.35 mmol), NaH (0.07 g,
1.75 mmol) and DMF (5 ml) was added to methyl iodide (0.2 ml,
3.22 mmol) at R.T. for 2 h. The reaction was quenched with water
and extracted with EtOAc (30 ml x 3). The organic layer was
collected and dried over anhydrous MgSO4 concentrated in vacuo
and purified by silica gel chromatography (EtOAc:n-hexane ¼ 1: 2,
Rf ¼ 0.6) to get the oily product. This was then dissolved in DCM
(5 ml) and added TFA (1 ml) at R.T. for 2 h. The reaction was
quenched with water and extracted with EtOAc (30 ml x 3). The
organic layer was collected and dried over anhydrous MgSO4
concentrated in vacuo and without more purification. The crude
product was dissolved in MeOH (8 ml), THF (8 ml) and added to 10%
palladium on activated carbon (0.04 g, 0.04 mmol) under hydrogen
gas and 40 psi at R.T. for overnight. The 10% palladium on activated
carbon was removed by a methyl alcohol wash through celite
packing. The organic layer was concentrated in vacuo and purified
by silica gel chromatography (EtOAc:n-hexane ¼ 1: 2, Rf ¼ 0.55) to
afford 8b (0.25 g, 56.82%)as a white solid. m.p. 217.7e219.5. 1H NMR
4.1.1.5. 2,4-Dihydroxy-5-isopropyl-N-(pyridin-4-yl)benzamide (7a).
The synthetic procedure for compound 7a is similar to that used for
compound 6a and gave a yield of 86.95% as a yellow solid. m.p.
235.9e238.3 ꢀC. 1H NMR (500 MHz, MeOD):
d
1.23 (d, J ¼ 7 Hz, 6H),
3.19 (Sep, J ¼ 7 Hz, 1H), 6.36 (s, 1H), 7.76e7.77 (m, 3H), 8.40 (d,
J ¼ 6.5 Hz, 2H). 13C NMR (125 MHz, MeOD):
d 23.14, 28.17, 103.84,
109.12, 116.18, 128.31, 129.24, 148.40, 150.73, 160.85, 162.36, 169.99.
hMS (ESI) for
273.1240.
C
15H17N2O3 [MþH]þ: calcd, 273.1234; found,
4.1.1.6. N-Ethyl-2,4-dihydroxy-5-isopropyl-N-(pyridin-4-yl)benza-
mide (7b). The synthetic procedure for compound 7b is similar to
that used for compound 6b and gave a yield of 63.64% as a white
solid. m.p. 185.2e186.1 ꢀC. 1H NMR (500 MHz, MeOD):
d 0.86 (d,
J ¼ 7 Hz, 6H), 1.21 (t, J ¼ 7 Hz, 3H), 2.97 (Sep, J ¼ 7 Hz, 1H) 4.01 (q,
J ¼ 7 Hz, 2H), 6.20 (s, 1H), 6.68 (s, 1H), 7.17e7.18 (m, 2H), 8.39e8.41
(m, 2H). 13C NMR (125 MHz, MeOD):
d 13.53, 22.93, 27.07, 45.98,
103.58, 110.91, 123.09, 127.81, 129.57, 151.14, 154.14, 159.34, 160.44,
173.25. hMS (ESI) for C17H20N2NaO3 [MþNa]þ: calcd, 323.1366;
found, 323.1360.
(500 MHz, MeOD):
d
0.49 (d, J ¼ 7 Hz, 6H), 2.74 (qui, J ¼ 7 Hz, 1H),
3.46 (s, 3H), 6.16 (s,1H), 6.41 (d, J ¼ 3 Hz,1H), 6.48 (s,1H), 6.93e6.95
(m, 1H), 7.26 (d, J ¼ 3 Hz, 1H), 7.33 (s, 1H), 7.37 (d, J ¼ 8.5 Hz, 1H). 13
C
12

Y.-M. Liu, H.-J. Tu, C.-H. Wu et al.
European Journal of Medicinal Chemistry 219 (2021) 113428
NMR (125 MHz, MeOD):
d
22.69, 26.73, 40.30, 102.95, 103.29,
4.1.1.16. N-Ethyl-2,4-dihydroxy-5-isopropyl-N-(1-methyl-1H-indol-
5-yl)benzamide (8h). The synthetic procedure for compound 8h is
similar to that used for compound 6b and gave a yield of 70.83% as a
113.34, 119.66, 121.33, 126.72, 127.40, 130.00, 130.22, 136.61, 138.85,
159.45, 173.50. hMS (ESI) for C19H20N2NaO3 [MþNa]þ: calcd,
347.1366; found, 347.1365.
white solid. m.p. 167.6e169.8 ꢀC. 1H NMR (500 MHz, MeOD):
d 0.47
(d, J ¼ 6.5 Hz, 6H), 1.19 (t, J ¼ 7 Hz, 3H), 2.73 (Sex, J ¼ 7 Hz, 1H), 3.74
(s, 3H), 3.93 (q, J ¼ 7 Hz, 2H), 6.17 (s, 1H), 6.37 (d, J ¼ 3 Hz, 1H), 6.44
(s, 1H), 6.94e6.96 (m, 1H), 7.15 (d, J ¼ 3 Hz, 1H), 7.30e7.33 (m 2H).
4.1.1.11. N-Ethyl-2, 4-dihydroxy-N-(1H-indol-5-yl)-5-
isopropylbenzamide (8c). The synthetic procedure for compound 8c
is similar to that used for compound 8b and gave a yield of 51.16% as
a white solid. m.p. 234.4e236.7 ꢀC. 1H NMR (500 MHz, MeOD):
13C NMR (125 MHz, MeOD):
d 13.04, 22.73, 26.72, 33.14, 47.59,
102.26, 103.28, 111.32, 121.03, 122.42, 126.65, 129.81, 130.74, 131.74,
d
0.46 (d, J ¼ 6.5 Hz, 6H), 1.21 (t, J ¼ 7 Hz, 3H), 2.72 (Sex, J ¼ 7 Hz,
136.96, 137.27, 159.30, 172.99. hMS (ESI) for C21H24N2NaO3
1H), 3.94 (q, J ¼ 7 Hz, 2H), 6.17 (s. 1H), 6.39 (d, J ¼ 3 Hz, 1H), 6.45 (s,
1H), 6.90e6.92 (m, 1H), 7.24 (d, J ¼ 3 Hz, 1H), 7.30 (d, J ¼ 2 Hz, 1H),
[MþNa]þ: calcd, 375.1679; found, 375.1679.
7.37 (d, J ¼ 8.5 Hz, 1H). 13C NMR (125 MHz, MeOD):
d
13.03, 22.70,
4.1.1.17. 2,4-Dihydroxy-5-isopropyl-N-(1-methyl-1H-indol-5-yl)-N-
propylbenzamide (8i). The synthetic procedure for compound 8i is
similar to that used for compound 6b and gave a yield of 75.0% as a
26.73, 47.67, 102.94, 103.27, 113.25, 120.69, 122.23, 126.65, 127.35,
129.94, 130.19, 136.68, 136.80, 159.33, 172.96. hMS (ESI) for
C
20H22N2NaO3 [MþNa]þ: calcd, 361.1523; found, 361.1524.
white solid. m.p. 179.9e182.4 ꢀC. 1H NMR (500 MHz, MeOD):
d 0.49
(d, J ¼ 6.5 Hz, 6H), 0.93 (t, J ¼ 7 Hz, 3H), 1.66 (Sex, J ¼ 7.5 Hz, 2H),
2.73 (Sex, J ¼ 7 Hz, 1H), 3.77 (s, 1H), 3.87 (t, J ¼ 7.5 Hz, 2H), 6.15 (s,
1H), 6.38 (d, J ¼ 2.5 Hz, 1H), 6.43 (s, 1H), 6.96e6.98 (m, 1H), 7.17 (d,
4.1.1.12. 2,4-Dihydroxy-N-(1H-indol-5-yl)-5-isopropyl-N-pro-
pylbenzamide (8d). The synthetic procedure for compound 8d is
similar to that used for compound 8b and gave a yield of 51.06% as a
J ¼ 3 Hz,1H), 7.32e7.35 (m, 2H). 13C NMR (125 MHz, MeOD):
d 11.81,
white solid. m.p. 206.0e207.8 ꢀC. 1H NMR (500 MHz, MeOD):
d
0.48
21.78, 22.72, 26.70, 33.11, 54.23, 102.23, 103.26, 111.25, 120.84,
122.29, 126.62, 129.69, 130.67, 131.70, 137.20, 159.19, 173.13. hMS
(ESI) for C22H26N2NaO3 [MþNa]þ: calcd, 389.1836; found, 389.1833.
(d, J ¼ 6.5 Hz, 6H), 0.93 (t, J ¼ 7.5 Hz, 3H), 1.66 (Sex, J ¼ 7.5 Hz, 2H),
2.73 (Sex, J ¼ 6.5 HZ, 1H), 3.86 (t, J ¼ 7.5 Hz, 2H), 6.16 (s, 1H), 6.40 (d,
J ¼ 3 Hz,1H), 6.46 (s,1H), 6.91 (s, 1H), 7.25 (d, J ¼ 3.5 Hz,1H), 7.31 (d,
J ¼ 1.5 Hz, 1H), 7.34 (d, J ¼ 8.5 Hz, 1H). 13C NMR (125 MHz, MeOD):
4.1.2. 2,4-bis(Benzyloxy)-5-isopropylbenzoic acid (9)
d
11.81, 21.77, 22.68, 26.71, 54.31, 102.91, 103.25, 113.18, 120.51,
A mixture of 19 (3 g, 8.32 mmol), sulfamic acid (6.5 g,
66.94 mmol), DMSO (2 ml), THF (20 ml) and water (20 ml) was
added to NaClO2 (6 g, 66.34 mmol) dissolved in water (20 ml) at
0 ꢀC. The reaction was allowed back to R.T. and stirred for 2 h. The
residue was extracted with EtOAc (40 ml x 3) and purified by silica
gel chromatography (EtOAc:n-hexane ¼ 1: 4, Rf ¼ 0.25) to afford 9
122.10, 126.62, 127.33, 129.82, 130.14, 136.62, 137.01, 159.23, 173.11.
hMS (ESI) for C21H24N2NaO3 [MþNa]þ: calcd, 375.1679; found,
375.1676.
4.1.1.13. 2,4-Dihydroxy-N-(1H-indol-5-yl)-N,5-diisopropylbenzamide
(8e). The synthetic procedure for compound 8e is similar to that
used for compound 8b and gave a yield of 47.91% as a white solid.
(2.02 g, 64.61%) as a yellow solid.1H NMR (500 MHz, CDCl3):
d 1.23
(d, J ¼ 7 Hz, 6H), 2.98 (s, 1H), 3.30 (Sep, J ¼ 7 Hz, 1H), 5.10 (s, 2H),
m.p. 226.1e227.9 ꢀC. 1H NMR (500 MHz, MeOD):
d 0.56 (d,
5.20 (s, 2H), 6.57 (s, 1H), 7.34e7.44 (m, 10H), 8.03 (s, 1H).
J ¼ 5.5 Hz, 6H), 1.19 (d, J ¼ 7 Hz, 6H), 2.76 (qui, J ¼ 7 Hz, 1H), 5.04 (t,
J ¼ 5.5 Hz, 1H), 6.13 (s, 1H), 6.42 (d, J ¼ 3 Hz, 1H), 6.49 (s, 1H),
6.85e6.88 (m, 1H), 7.24 (d, J ¼ 3 Hz, 1H), 7.31 (d, J ¼ 1.5 Hz, 1H), 7.33
4.1.3. 2,4-bis(Benzyloxy)-5-isopropyl-N-(pyridin-3-yl)benzamide
(10)
(d, J ¼ 9 Hz, 1H). 13C NMR (125 MHz, MeOD):
d
21.43, 22.77, 26.76,
A mixture of 9 (2 g, 5.31 mmol), EDC.HCl (1.5 g, 7.85 mmol),
HOBt (0.9 g, 6.67 mmol), NMM (1.4 ml,12.75 mmol) and DMF (8 ml)
was stirred for 10 min then 3-aminopyridine (0.6 g, 6.38 mmol) was
added at R.T. for overnight, the reaction was quenched with water
and extracted with EtOAc (30 ml x 3). The organic layer was
collected and dried over anhydrous MgSO4 concentrated in vacuo to
yield colorless oil product. The residue was purified by silica gel
chromatography (EtOAc:n-hexane ¼ 2: 1, Rf ¼ 0.4) to afford 10
102.93, 103.20, 112.46, 122.92, 124.60, 126.53, 127.11, 129.17, 129.73,
136.87, 158.58, 173.25. hMS (ESI) for C21H24N2NaO3 [MþNa]þ: calcd,
375.1679; found, 375.1681.
4.1.1.14. 2,4-Dihydroxy-5-isopropyl-N-(1-methyl-1H-indol-5-yl)ben-
zamide (8f). The synthetic procedure for compound 8f is similar to
that used for compound 6a and gave a yield of 47.91% as a pink
solid. m.p. 221.2e223.8 ꢀC. 1H NMR (500 MHz, MeOD):
d
1.25 (d,
(1.76 g, 73.33%) as a white solid. 1H NMR (500 MHz, CDCl3):
d 1.27
J ¼ 7 Hz, 6H), 3.21 (Sep, J ¼ 7 Hz, 1H), 3.75 (s, 1H), 6.35 (s, 1H), 6.39
(d, J ¼ 3 Hz, 1H), 7.11 (d, J ¼ 3 Hz, 1H), 7.26e7.28 (m, 1H), 7.31 (d,
J ¼ 8.5 Hz, 1H), 7.74 (d, J ¼ 1.5 Hz, 1H), 7.77 (s, 1H). 13C NMR
(d, J ¼ 7.0 Hz, 6H), 3.36 (Sep, J ¼ 7.0 Hz, 1H), 5.15 (s, 2H), 5.18 (s, 2H),
6.64 (s, 1H), 7.16e7.18 (m, 1H), 7.35e7.46 (m, 10H), 7.93e7.94 (m,
1H), 8.13e8.16 (m, 1H), 8.18 (s, 1H), 8.22e8.24 (m, 1H), 9.93 (s, 1H).
(125 MHz, MeOD):
d 23.22, 28.13, 33.08, 101.93, 103.82, 109.15,
110.29, 116.10, 118.95, 127.41, 128.68, 130.18, 131.04, 136.27, 161.03,
161.36, 170.13. hMS (ESI) for C19H20N2NaO3 [MþNa]þ: calcd,
347.1366; found, 347.1370.
4.1.4. 2,4-bis(Benzyloxy)-5-isopropyl-N-(pyridin-4-yl)benzamide
(11)
The synthetic procedure for compound 11 is similar to that used
for compound 10 and gave a yield of 70.83% as a white solid. 1H
4.1.1.15. 2,4-Dihydroxy-5-isopropyl-N-methyl-N-(1-methyl-1H-
indol-5-yl)benzamide (8g). The synthetic procedure for compound
8g is similar to that used for compound 6b and gave a yield of 75.0%
as a white solid. m.p. 157.2e158.4 ꢀC. 1H NMR (500 MHz, MeOD):
NMR (500 MHz, CDCl3):
1H), 5.14 (s, 2H), 5.18 (s, 2H), 6.64 (s, 1H), 7.07e7.08 (m, 2H),
7.35e7.50 (m, 10H), 8.16 (s, 1H), 8.33e8.34 (m, 2H), 10.01 (s, 1H).
d
1.27 (d, J ¼ 7 Hz, 6H), 3.35 (qui, J ¼ 7 Hz,
d
0.49 (d, J ¼ 6.5 Hz, 6H), 2.74 (qui, J ¼ 7 Hz, 1H), 3.46 (s, 3H), 3.78 (s,
4.1.5. tert-Butyl 5-(2,4-bis(benzyloxy)-5-isopropylbenzamido)-1H-
indole-1-carboxylate (13)
3H), 6.15 (s, 1H), 6.39 (d, J ¼ 8 Hz, 1H), 6.45 (s, 1H), 6.99e7.01 (m,
1H), 7.18 (d, J ¼ 3 Hz, 1H), 7.35 (d, J ¼ 5 Hz, 1H), 7.37 (s, 1H). 13C NMR
A mixture of 5-nitroindole (2 g, 12.33 mmol) was dissolved in
DCM (15 ml) and added DMAP (3 g, 24.56 mmol) and Boc anhydride
(4 g, 18.33 mmol) at R.T. for overnight. The reaction was quenched
with water and extracted with EtOAc (30 ml x 3). The organic layer
was collected and dried over anhydrous MgSO4 concentrated in
(125 MHz, MeOD):
d 22.73, 26.73, 33.13, 40.20, 102.28, 103.30,
111.41, 120.03, 121.50, 126.73, 129.88, 130.76, 131.79, 137.20, 139.00,
159.41, 173.53. hMS (ESI) for C20H22N2NaO3 [MþNa]þ: calcd,
361.1523; found, 361.1520.
13

Y.-M. Liu, H.-J. Tu, C.-H. Wu et al.
European Journal of Medicinal Chemistry 219 (2021) 113428
vacuo and without more purification. The crude product was dis-
solved in IPA (100 ml) and H2O (25 ml) and added iron powder
(3.5 g, 63.64 mmol) and NH4Cl (3 g, 55.56 mmol). The reaction was
stirred and reflux for 1h. The iron powder was removed by methyl
alcohol wash through celite packing. The organic layer was
concentrated in vacuo and without more purification. Then the
mixture of 9 (2 g, 5.31 mmol), EDC.HCl (1.5 g, 7.85 mmol), HOBt
(0.9 g, 6.67 mmol), NMM (1.4 ml, 12.75 mmol) and DMF (8 ml) was
stirred for 10 min then added crude product at R.T. for overnight,
the reaction was quenched with water and extracted with EtOAc
(30 ml x 3). The organic layer was collected and dried over anhy-
drous MgSO4 concentrated in vacuo to yield colorless oil product.
The residue was purified by silica gel chromatography (EtOAc:n-
hexane ¼ 1: 1, Rf ¼ 0.6) to afford 13 (2.46 g, 78.59%) as a white solid.
CDCl3):
d
1.53 (s, 3H), 1.59 (s, 3H), 3.98 (s, 1H), 5.04 (s, 2H), 5.10 (s,
2H), 6.53e6.55 (m, 1H), 6.67 (d, J ¼ 2.5 Hz, 1H), 7.24 (d, 1H),
7.33e7.42 (m, 10H).
4.1.9. (((4-Isopropyl-1,3-phenylene)bis(oxy))bis(methylene))
dibenzene (18)
To a mixture of 17 (16 g, 45.92 mmol) and CH2Cl2 (125 ml) was
added triethylsilane (9 ml, 56.35 mmol) and TFA (4.5 ml,
58.77 mmol) at ꢁ78 ꢀC. The reaction was allowed back to R.T. and
stirred for 4 h. The reaction was quenched with 1 N NaOH (aq.) and
extracted with CH2Cl2 (30 ml x 3). The organic layer was collected
and dried over anhydrous MgSO4 concentrated in vacuo to yield a
colorless oil product. The residue was purified by silica gel chro-
matography (EtOAc:n-hexane ¼ 1: 19, Rf ¼ 0.35) to afford 18
1H NMR (500 MHz, CDCl3):
d
1.28 (d, J ¼ 7.0 Hz, 6H),1.16 (s, 9H), 3.36
(14.06 g, 92.08%) as a white solid.1H NMR (500 MHz, CDCl3):
d 1.25
(qui, J ¼ 7.0 Hz,1H), 5.15 (s, 4H), 6.45 (s,1H), 6.46 (s,1H), 6.80 (s,1H),
(d, J ¼ 6.5 Hz, 6H), 3.37 (Sep, J ¼ 7.0 Hz,1H), 5.05 (s, 2H), 5.07 (s, 2H),
6.58e6.60 (m, 1H), 6.63 (d, J ¼ 2.5 Hz, 1H), 7.16 (d, J ¼ 8.5 Hz, 1H),
7.34e7.47 (m, 10H).
7.34e7.52 (m, 11H), 7.91 (s, 2H), 8.24 (s, 1H), 9.95 (s, 1H).
4.1.6. 2,4-bis(Benzyloxy)-5-isopropyl-N-(1-methyl-1H-indol-5-yl)
benzamide (14)
A mixture of 5-nitroindole (2 g, 12.33 mmol), NaH (0.6 g,
15 mmol) and DMF (10 ml) was added methyl iodide (1.5 ml,
24.09 mmol) at R.T. for 2 h. The reaction was quenched with water
and extracted with EtOAc (30 ml x 3). The organic layer was
collected and dried over anhydrous MgSO4 concentrated in vacuo
and purified by silica gel chromatography (EtOAc:n-hexane ¼ 1: 4,
Rf ¼ 0.2) to afford product as a yellow solid. The above product was
dissolved in IPA (68 ml) and H2O (17 ml) and added iron powder
(2.5 g, 45.45 mmol) and NH4Cl (2 g, 37.04 mmol). The reaction was
stirred and reflux for 1 h. The iron powder was removed by a
methyl alcohol wash through celite packing. The organic layer was
concentrated in vacuo and without more purification. Then the
mixture of 9 (2 g, 5.31 mmol), EDC.HCl (1.5 g, 7.85 mmol), HOBt
(0.9 g, 6.67 mmol), NMM (1.4 ml, 12.75 mmol) and DMF (8 ml) was
stirred for 10 min then added crude product at R.T. for overnight,
the reaction was quenched with water and extracted with EtOAc
(30 ml x 3). The organic layer was collected and dried over anhy-
drous MgSO4 and concentrated in vacuo to yield a colorless oil
product. The residue was purified by silica gel chromatography
(EtOAc:n-hexane ¼ 1: 2, Rf ¼ 0.6) to afford 14 (0.96 g, 32.85%) as a
4.1.10. 2,4-bis(Benzyloxy)-5-isopropylbenzaldehyde (19)
A mixture of POCl3 (3.3 ml, 35.40 mmol) and DMF (2.7 ml) was
stirred at 0 ꢀC for 10 min then added 18 (4 g, 12.03 mmol) dissolved
in DMF (3 ml). The reaction was heated at 80 ꢀC and stirred for 1.5 h.
The reaction was quenched with 6 N NaOH (aq.) and extracted with
EtOAc (30 ml x 3). The organic layer was collected and dried over
anhydrous MgSO4 concentrated in vacuo to yield a yellow product.
The residue was without more purification to afford 19 (4.01 g,
92.40%) as a yellow solid.1H NMR (500 MHz, CDCl3):
d 1.21 (d,
J ¼ 7.0 Hz, 6H), 3.28 (Sep, J ¼ 7.0 Hz, 1H), 5.10 (s, 2H), 5.12 (s, 2H),
6.50 (s, 1H), 7.34e7.42 (m, 10H), 7.74 (s, 1H), 10.39 (s, 1H).
4.2. Biology
4.2.1. Cell lines
The human non-small cell lung cancer cell line A549, human
breast adenocarcinoma cell line MDA-MB-231, human colorectal
carcinoma cell line HCT-116, human hepatoma cell line Hep3B and
human umbilical vein endothelial cells (HUVEC) were obtained
from Bioresource Collection and Research Center (Taiwan). Cells
were maintained in RPMI-1640 medium, L-15 medium, McCoy’s 5a
medium and MEM medium supplemented with 10% (v/v) fetal
bovine serum (Gibco, Carlsbad, CA, USA) and 1% of a mixture of
penicillin-streptomycin-amphotericin B (Kibbutz Beit Haemek,
Israel). Photoreceptor-derived 661W cells were the kind gift of Prof
Muayyad Al-Ubaidi, University of Houston. Cells were maintained
in DMEM high glucose medium supplemented with 10% (v/v) fetal
bovine serum and 1% of a mixture of penicillin-streptomycin-
amphotericin B.
white solid. 1H NMR (500 MHz, CDCl3):
d
1.28 (d, J ¼ 7.0 Hz, 6H),
3.36 (qui, J ¼ 7.0 Hz, 1H), 3.73 (s, 3H), 5.15 (s, 2H), 5.16 (s, 2H), 6.36
(d, J ¼ 3.0 Hz, 1H), 6.62 (s, 1H), 7.02e7.05 (m, 2H), 7.11 (d, J ¼ 8.5 Hz,
1H), 7.35 (t, J ¼ 7.0 Hz, 1H), 7.40e7.53 (m, 10H), 7.70 (d, J ¼ 1.5 Hz,
1H), 8.24 (s, 1H), 9.86 (d, 1H).
4.1.7. 1-(2,4-bis(Benzyloxy)phenyl)ethanone (16)
A
mixture of 1-(2,4-dihydroxyphenyl)ethanone (10 g,
65.77 mmol), benzyl bromide (18 ml, 151.33 mmol), potassium
carbonate (28 g, 202.60 mmol) and acetone (250 ml) was refluxed
overnight, the precipitate was filtered. The organic layer was
collected and concentrated in vacuo to yield an oily product, and the
oily product was added n-hexane and stirred overnight. The res-
idue was filtered by suction filtration to yield white product and
without more purification to afford 16 (20.43 g, 93.42%) as a white
4.2.2. Chemicals and antibodies
SRB (sulforodamine B) and MTT (3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide) were purchased from Sigma
Chemical Co (St. Louis, MO, USA). Antibodies against Hsp90, FAK, p-
solid. 1H NMR (500 MHz, CDCl3):
d
2.55 (s, 3H), 5.09 (s, 2H), 5.11 (s,
FAK, p-Src, p-Rb, Akt, p-Akt, p-GSK3b, p-mTOR, 4EBP-1, p-4EBP-1,
2H), 6.60e6.62 (m, 2H), 7.34e7.43 (m, 10H), 7.84 (d, J ¼ 9 Hz, 1H).
eIF-4E, p-p70S6K, EGFR, p-EGFR, MEK, p-MEK, ERK, p-ERK, p-cdc2
(Thr161), p-cdc2 (Tyr15), p-cdc2 (Ser216), caspase-9, caspase-3,
cleaved caspase-3, p-H2AX (Ser139) and p-ERBB2 were obtained
from Cell Signaling Technologies (Beverly, MA, USA). Antibodies
against PARP, cyckin B1 and cdc25c were purchased from Santa
Cruz Biotechnology (Santa Cruz, CA, USA). p-MPM2 and p-Histone 3
(Ser10) were purchased from Upstate Biotechnology Inc. (Teme-
cula, CA, USA). Src and p-p53 (Ser46) were purchased from Abcam
(Cambridge, MA, USA).
4.1.8. 2-(2,4-bis(Benzyloxy)phenyl)propan-2-ol (17)
To a mixture of 16 (20 g, 60.17 mmol) and THF (100 ml) was
added 3 M methylmagnesium bromide (33 ml) at 0 ꢀC. The reaction
was allowed back to R.T. and stirred for 4 h. The reaction was
quenched with water at 0 ꢀC.The residue was filtered by suction
filtration to yield white product and without more purification to
afford 17 (16.21 g, 77.30%) as a white solid.1H NMR (500 MHz,
14

Y.-M. Liu, H.-J. Tu, C.-H. Wu et al.
European Journal of Medicinal Chemistry 219 (2021) 113428
4.2.3. Cell toxicity and cell proliferation assays
Cremophor (1:4:5), ip, qwk, once weekly) and afatinib (dissolved in
1% carboxymethyl cellulose þ 0.5% Tween 80 in D5W, po, daily)
alone or a combination of both. Body weights and tumor sizes were
measured twice a week. All mouse tumors were allowed to reach an
endpoint volume of 2500 mm3. Tumors were then dissected and
subjected to further analysis.
Cell cytotoxicity was measured using a colorimetric MTT assay.
5 ꢂ 103 cells/well were seeded in a 96-well plate and then treated
with the indicated concentrations of test compounds for 48 h.
0.5 mg/ml MTT solution was then added to the 96-well plate in the
dark and the plate was incubated at 37 ꢀC for 1.5 h. MTT-containing
medium were removed and DMSO were added to each well to lyse
cells, the absorbance was spectrophotometrically recorded at
570 nm.
4.2.8. Statistical analysis
All data were expressed as mean values
S.E.M. and were
Cell proliferation was measured using the sulforhodamine B
(SRB) assay. 5 ꢂ 103 cells/well were incubated for 48 h with the
indicated concentrations of test compounds, fixed with 10% tri-
chloroacetic acid, stained for 30 min with SRB (0.4% in 1% acetic
acid), and washed repeatedly with 1% acetic acid. Protein-bound
dye was finally dissolved in 10 mM Tris base solution, and the
optical density at 515 nm was measured.
measured independently three times. The significance of differ-
ences between the experimental groups and controls was assessed
by Student’s t-test. P < 0.05 was considered statistically significant
(*p < 0.05; **p < 0.01; ***p < 0.001; compared with the respective
control group).
4.2.9. Inhibition effects on Cytochrome P450 in human liver
microsomes
4.2.4. Hsp90 enzyme activity assay
In direct CYP inhibition experiments, incubations were per-
formed in triplicate in 96-well plates containing test compound
and known positive control inhibitors. All incubations contained
0.1e1 mg/mL of pooled human liver microsomes (depending on
which enzyme was assessed), 1 mM NADPH, and 2.5 mM MgCl2 in
100 mM potassium phosphate buffer, pH 7.4. Eight concentrations
of test article and positive control inhibitors or control solvent
(DMSO) were added to the incubation. Final solvent concentration
was 0.1% (v/v). Incubations were commenced with the addition of
Enzyme inhibition assays were measured with a specific HSP90
enzyme activity assay (BPS Bioscience, San Diego, CA, USA). The
assay is based on the competition of fluorescently labeled gelda-
namycin for binding to purified recombinant HSP90a. Briefly, all
reagents were mixed per the instructions then added into wells and
incubated at R.T. for 1e2 h with slow shaking. Fluorescent polari-
zation of the sample in a microtiter-plate reader was read at exci-
tation wavelengths ranging from 475 to 495 nm and detection of
emitted light ranging from 518 to 538 nm.
probe substrates to a final incubation volume of 200
maintained at 37 ꢀC for the defined period. Incubations were
terminated by addition of methanol (200 L). Aliquots of 30 mL
mL and
4.2.5. Immunoblot and immunoprecipitation analyses
m
After treatment with the indicated conditions, cells were incu-
bated for 10 min at 4 ꢀC in lysis buffer (Cell Signaling Technology,
Danvers, MA, USA), then scraped from the culture surface, incu-
bated on ice for 10 min, and centrifuged for 30 min at 17,000 g and
terminated incubation mixtures were extracted by adding 3-fold
volume of deprotein solvent (0.1% formic acid in methanol or
acetonitrile) containing internal standard and centrifuged at
20000 g for 5 min. The supernatants were analyzed by LC-MS/MS.
4
ꢀC. Protein samples were then electrophoresed on sodium
dodecyl sulfate polyacrylamide gels and transferred onto nitrocel-
lulose membranes, which were blocked by 5% milk in tris-buffered
saline (TBS) for 1h at R.T.. Membranes were incubated with primary
antibodies in TBS overnight at 4 ꢀC, followed by incubation with hP-
conjugated secondary antibodies for 1 h at R.T.. To measure bound
antibodies, the membranes were treated with an enhanced
chemiluminescence reagent (Advansta Corp., Menlo Park, CA, USA)
and exposed to photographic film.
4.2.10. Bidirectional Caco-2 permeability assay
Caco-2 cells were seeded at a density of 8 ꢂ 104 cells/cm2 onto
cell culture inserts with polycarbonate membrane for 21 days. For
absorptive (AP / BL) permeability, transport was initiated by
adding 0.4 mL of drug solution (HBSS/MES, pH 6.5 containing 10 mM
test compound, 0.1% DMSO) to the apical chamber (donor chamber)
of inserts bathed with 0.6 mL of transport medium (HBSS/HEPES,
pH 7.4) in the basolateral chamber (receiver chamber). For secre-
tory (BL/AP) permeability, transport was initiated by adding
4.2.6. Cell migration assay
0.6 mL of drug solution (HBSS/HEPES, pH 7.4 containing 10 mM test
Cell migration was determined using a 24-well Boyden chamber
compound, 0.1% DMSO) to the basolateral chamber (donor cham-
ber) of wells with 0.4 mL of transport medium (HBSS/MES, pH 6.5)
with 12
mm pore size polycarbonate polyvinylpyrrolidone-free
Nucleopore filters (Millipore, MA, USA). The membrane was
coated with 0.5% gelatin for 4 h. First, 1 ꢂ 105 A549 cells were
seeded in upper chamber and added 10% FBS medium as chemo-
attractant in the lower chamber. Both wells were treated with
different drugs. After 6 h, the membranes were fixed with 4%
formalin for 10 min and then stained with 1% crystal violet for
10 min. The cell number was counted using a microscope. Finally,
the crystal violet on the membrane were dissolved with 0.1 M so-
dium citrate and detected with 550 nM wavelength.
in the apical chamber (receiver chamber). Samples (100 mL) were
withdrawn from the receiver chamber at 30, 60, 90 and 120 min
and from the donor chamber at 0 and 120 min. The volume with-
drawn was replaced with fresh transport medium. The concentra-
tions of test compound were analyzed by LC-MS/MS.
4.2.11. Pharmacokinetics study in male SD rats
Dose formulations were prepared on the dosing day prior to
dosing. Vehicle for IV formulation consisted of 5% v/v N,N-
dimethylacetamide (DMA) and 5% v/v Kolliphor® HS 15 (Solutol
HS) in D5W (dextrose 5% in water solution). The vehicle for PO
formulation consisted of 1% w/v carboxymethyl cellulose (CMC)
and 0.5% v/v Tween 80. Complete mixing was ensured by stirring.
The male Sprague Dawley rats were assigned to 2 dose groups of
three animals each. Compound 6b was administered to the study
animals either intravenously (IV; Group 1) or orally by gavage (PO;
Group 2) at the dose levels of 2 mg/kg (IV dosing) or 20 mg/kg (PO
dosing). Blood samples were obtained pre-dose and at 0.03, 0.08,
0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 h post-dose from the animals in
4.2.7. Tumor xenograft model
8-week-old male BALB/c nude mice were fed water ad libitum
and Pico-Lab Rodent Diet. All procedures were performed in
accordance with the NIH guidelines on laboratory animal welfare.
A549 or H1975 cells (1 ꢂ 107 cells) were subcutaneously injected
into the flanks of mice. When tumor sizes reached 100 mm3, mice
were randomized into different groups with an indicated dosage of
compound 6b (dissolved in 1% carboxymethyl cellulose þ 0.5%
Tween 80 in D5W, p.o), STA-9090 (dissolved in DMSO/ethanol/
15

Y.-M. Liu, H.-J. Tu, C.-H. Wu et al.
European Journal of Medicinal Chemistry 219 (2021) 113428
[4] H. Mellatyar, S. Talaei, Y. Pilehvar-Soltanahmadi, A. Barzegar, A. Akbarzadeh,
A. Shahabi, M. Barekati-Mowahed, N. Zarghami, Targeted cancer therapy
through 17-DMAG as an Hsp90 inhibitor: overview and current state of the
art, Biomed. Pharmacother. 102 (2018) 608e617.
[5] R.C. Schnur, M.L. Corman, R.J. Gallaschun, B.A. Cooper, M.F. Dee, J.L. Doty,
M.L. Muzzi, C.I. DiOrio, E.G. Barbacci, P.E. Miller, V.A. Pollack, D.M. Savage,
D.E. Sloan, L.R. Pustilnik, J.D. Moyer, M.P. Moyer, erbB-2 oncogene inhibition
by geldanamycin derivatives: synthesis, mechanism of action, and structure-
activity relationships, J. Med. Chem. 38 (1995) 3813e3820.
Group 1, and pre-dose and at 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 48 and
72 h post-dose from the animals in Group 2. Blood samples were
processed to plasma within 60 min after collection. Plasma samples
were analyzed at QPS Taiwan using an LC-MS/MS method. Indi-
vidual concentration-time data were used in the calculation of PK
parameters of compound 6b using Phoenix® WinNonlin® version
6.3.
[6] S.R. Kasibhatla, K. Hong, M.A. Biamonte, D.J. Busch, P.L. Karjian,
J.L. Sensintaffar, A. Kamal, R.E. Lough, J. Brekken, K. Lundgren, R. Grecko,
G.A. Timony, Y. Ran, R. Mansfield, L.C. Fritz, E. Ulm, F.J. Burrows, M.F. Boehm,
Rationally designed high-affinity 2-amino-6-halopurine heat shock protein 90
inhibitors that exhibit potent antitumor activity, J. Med. Chem. 50 (2007)
2767e2778.
[7] P.A. Brough, W. Aherne, X. Barril, J. Borgognoni, K. Boxall, J.E. Cansfield, K.-
M.J. Cheung, I. Collins, N.G.M. Davies, M.J. Drysdale, B. Dymock, S.A. Eccles,
H. Finch, A. Fink, A. Hayes, R. Howes, R.E. Hubbard, K. James, A.M. Jordan,
A. Lockie, V. Martins, A. Massey, T.P. Matthews, E. McDonald, C.J. Northfield,
L.H. Pearl, C. Prodromou, S. Ray, F.I. Raynaud, S.D. Roughley, S.Y. Sharp,
A. Surgenor, D.L. Walmsley, P. Webb, M. Wood, P. Workman, L. Wright, 4,5-
Diarylisoxazole HSP90 chaperone inhibitors: potential therapeutic agents for
the treatment of cancer, J. Med. Chem. 51 (2008) 196e218.
[8] T.-Y. Lin, M. Bear, Z. Du, K.P. Foley, W. Ying, J. Barsoum, C. London, The novel
HSP90 inhibitor STA-9090 exhibits activity against Kit-dependent and
eindependent malignant mast cell tumors, Exp. Hematol. 36 (2008)
1266e1277.
[9] A.J. Woodhead, H. Angove, M.G. Carr, G. Chessari, M. Congreve, J.E. Coyle,
J. Cosme, B. Graham, P.J. Day, R. Downham, L. Fazal, R. Feltell, E. Figueroa,
M. Frederickson, J. Lewis, R. McMenamin, C.W. Murray, M.A. O’Brien, L. Parra,
S. Patel, T. Phillips, D.C. Rees, S. Rich, D.M. Smith, G. Trewartha, M. Vinkovic,
B. Williams, A.J. Woolford, Discovery of (2,4-dihydroxy-5-isopropylphenyl)-
[5-(4-methylpiperazin-1-ylmethyl)-1,3-dihydroisoindol-2-yl]methanone
(AT13387), a novel inhibitor of the molecular chaperone Hsp90 by fragment
based drug design, J. Med. Chem. 53 (2010) 5956e5969.
4.2.12. In vitro hepatocytes stability
The metabolism of 6b was investigated in cryopreserved hepa-
tocytes from CD-1 mice, Sprague-Dawley rat, Beagle dog and hu-
man. Incubations of 6b (10 mM) and hepatocytes were conducted in
12-well plates containing approximately 0.5 ꢂ 106 cells/well in
triplicate for 4 h at 37 ꢀC with 90e120 rpm orbital shaking. Meta-
bolic reactions were stopped at 0.5, 1, 2 and 4 h by adding 1x vol-
ume of acetonitrile. The test article depletion and metabolite
identification were performed using a QTRAP 5500 LC-MS/MS
system (AB SCIEX). Multiple reaction monitoring, precursor ion,
neutral loss, and glucuronide neutral loss scans in positive ion
electrospray mode were used to identify compound 6b and its
metabolites.
4.2.13. The hERG human potassium channel assay
This assay measures binding of [3H] labeled Astemizole to po-
tassium channel hERG. Briefly, HEK-293 cells stably transfected
with a plasmid encoding the human potassium channel hERG are
used to prepare membranes in modified HEPES pH 7.4 buffer. A
[10] (a) S. Jung, N.G. Yoon, S. Yang, D. Kim, W.S. Lee, K.B. Hong, C. Lee, B.H. Kang,
J.H. Lee, S. Kang, Discovery of 2-((4-resorcinol)-5-aryl-1,2,3-triazol-1-yl)ace-
tates as potent Hsp90 inhibitors with selectivity over TRAP1, Bioorg. Med.
Chem. Lett 30 (2020) 126809;
10
m
g aliquot of membrane is incubated with 1.5 nM [3H]Astemizole
for 60 min at 25 ꢀC. Non-specific binding is estimated in the pres-
ence of 10 mM Astemizole. Membranes are filtered and washed 3
(b) C. Liang, X. Wu, Z. Li, J. Zhu, C. Lu, Y. Shen, Design, synthesis and phar-
macological evaluation of N-(5-chloro-2,4-dihydroxybenzoyl)-(R)-N-aryl-
methyl-1,2,3,4-tetrahydro-3-isoquinolinecarboxamides as potent Hsp90
inhibitors, Eur. J. Med. Chem. 143 (2018) 85e96;
times and the filters are counted to determine [3H]Astemizole
specifically bound.
(c) K. Nepali, M.H. Lin, M.W. Chao, S.J. Peng, K.C. Hsu, T. Eight Lin, M.C. Chen,
M.J. Lai, S.L. Pan, J.P. Liou, Amide-tethered quinoline-resorcinol conjugates as a
new class of HSP90 inhibitor suppressing the growth of prostate cancer cells,
Bioorg. Chem. 91 (2019) 103119;
(d) S.Y. Park, Y.J. Oh, Y. Lho, J.H. Jeong, K.H. Liu, J. Song, S.H. Kim, E. Ha,
Y.H. Seo, Design, synthesis, and biological evaluation of a series of resorcinol-
based N-benzyl benzamide derivatives as potent Hsp90 inhibitors, Eur. J. Med.
Chem. 143 (2018) 390e401.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
[11] (a) M. Wang, A. Shen, C. Zhang, Z. Song, J. Ai, H. Liu, L. Sun, J. Ding, M. Geng,
A. Zhang, Development of heat shock protein (Hsp90) inhibitors to combat
resistance to tyrosine kinase inhibitors through hsp90-kinase interactions,
J. Med. Chem. 59 (2016) 5563e5586;
Acknowledgment
This paper is dedicated to the memory of our dear supervisor
Professor Che-Ming Teng (Pharmacological Institute, College of
Medicine, National Taiwan University), who passed away recently.
Professor Teng took part in the study design and the provided
critical supervision for the experiments. We thank Prof. Muayyad
Al-Ubaidi from University of Houston for providing photoreceptor-
derived 661W cells. This research was supported by the Ministry of
Science and Technology, Taiwan (grant no. MOST 108-2113-M-038
-005 and MOST 109-2113-M-038 -005).
(b) A. Courtin, T. Smyth, K. Hearn, H.K. Saini, N.T. Thompson, J.F. Lyons,
N.G. Wallis, Emergence of resistance to tyrosine kinase inhibitors in non-
small-cell lung cancer can be delayed by an upfront combination with the
HSP90 inhibitor onalespib, Br. J. Canc. 115 (2016) 1069e1077.
[12] (a) J. Han, L.A. Goldstein, W. Hou, S. Chatterjee, T.F. Burns, H. Rabinowich,
HSP90 inhibition targets autophagy and induces a CASP9-dependent resis-
tance mechanism in NSCLC, Autophagy 14 (2018) 958e971;
ꢀ
(b) S. Cedres, E. Felip, C. Cruz, A. Martinez de Castro, N. Pardo, A. Navarro,
~
A. Martinez-Marti, J. Remon, J. Zeron-Medina, J. Balmana, A. Llop-Guevara,
J.M. Miquel, I. Sansano, P. Nuciforo, F. Mancuso, V. Serra, A. Vivancos, Activity
of HSP90 inhibiton in a metastatic lung cancer patient with a germline BRCA1
mutation, J. Natl. Cancer Inst. 110 (2018) 914e917.
[13] E. Kurihara, K. Shien, H. Torigoe, T. Takeda, Y. Takahashi, Y. Ogoshi,
T. Yoshioka, K. Namba, H. Sato, K. Suzawa, H. Yamamoto, J. Soh, M. Okazaki,
T. Shien, S. Tomida, S. Toyooka, Ganetespib in epidermal growth factor
receptor-tyrosine kinase inhibitor-resistant non-small cell lung cancer, Anti-
cancer Res. 39 (2019) 1767e1775.
[14] A. Akram, S. Khalil, S.A. Halim, H. Younas, S. Iqbal, S. Mehar, Therapeutic uses
of HSP90 inhibitors in non-small cell lung carcinoma (NSCLC), Curr. Drug
Metabol. 19 (2018) 335e341.
[15] E. Felip, F. Barlesi, B. Besse, Q. Chu, L. Gandhi, S.W. Kim, E. Carcereny,
L.V. Sequist, P. Brunsvig, C. Chouaid, E.F. Smit, H.J.M. Groen, D.W. Kim, K. Park,
E. Avsar, S. Szpakowski, M. Akimov, E.B. Garon, Phase 2 study of the HSP-90
inhibitor AUY922 in previously treated and molecularly defined patients
with advanced non-small cell lung cancer, J. Thorac. Oncol. 13 (2018)
576e584.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113428.
References
[1] L. Whitesell, S.L. Lindquist, HSP90 and the chaperoning of cancer, Nat. Rev.
Canc. 5 (2005) 761e772.
[2] S.D. Gupta, M.K. Bommaka, A. Banerjee, Inhibiting protein-protein interactions
of Hsp90 as a novel approach for targeting cancer, Eur. J. Med. Chem. 178
(2019) 48e63.
[16] Z. Piotrowska, D.B. Costa, G.R. Oxnard, M. Huberman, J.F. Gainor, I.T. Lennes,
A. Muzikansky, A.T. Shaw, C.G. Azzoli, R.S. Heist, L.V. Sequist, Activity of the
ꢀ
ꢀ
ꢁ ꢁ
ꢀ
[3] Z. Solarova, J. Mojzis, P. Solar, Hsp90 inhibitor as a sensitizer of cancer cells to
different therapies (review), Int. J. Oncol. 46 (2015) 907e926.
16

Y.-M. Liu, H.-J. Tu, C.-H. Wu et al.
European Journal of Medicinal Chemistry 219 (2021) 113428
Hsp90 inhibitor luminespib among non-small-cell lung cancers harboring
EGFR exon 20 insertions, Ann. Oncol. 29 (2018) 2092e2097.
[17] T. Shimamura, S.A. Perera, K.P. Foley, J. Sang, S.J. Rodig, T. Inoue, L. Chen, D. Li,
J. Carretero, Y.C. Li, P. Sinha, C.D. Carey, C.L. Borgman, J.P. Jimenez,
M. Meyerson, W. Ying, J. Barsoum, K.K. Wong, G.I. Shapiro, Ganetespib (STA-
9090), a nongeldanamycin HSP90 inhibitor, has potent antitumor activity in
in vitro and in vivo models of non-small cell lung cancer, Clin. Canc. Res. 18
(2012) 4973e4985.
17