Discovering High Potent Hsp90 Inhibitors as Antinasopharyngeal Carcinoma Agents through Fragment Assembling Approach

Article abstract of DOI:10.1021/acs.jmedchem.0c01521

Hsp90 is a new promising target for cancer treatment. Many inhibitors have been discovered as therapeutic agents, and some have passed Phase I and II. However, no one is approved by FDA yet. Novel and druggable Hsp90 inhibitors are still demanding. Here,

Full text of DOI:10.1021/acs.jmedchem.0c01521

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Article
Discovering High Potent Hsp90 Inhibitors as Antinasopharyngeal
Carcinoma Agents through Fragment Assembling Approach
Mengyang Xu, Chao Zhao, Biying Zhu, Liangyue Wang, Huihao Zhou, Daoguang Yan,* Qiong Gu,*
and Jun Xu*
Cite This: J. Med. Chem. 2021, 64, 2010−2023
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ABSTRACT: Hsp90 is a new promising target for cancer treatment. Many inhibitors have been discovered as therapeutic agents,
and some have passed Phase I and II. However, no one is approved by FDA yet. Novel and druggable Hsp90 inhibitors are still
demanding. Here, we report a new way to discover high potent Hsp90 inhibitors as antinasopharyngeal carcinoma agents through
assembling fragments. With chemotyping analysis, we extract seven chemotypes from 3482 known Hsp90 inhibitors, screen 500
fragments that are compatible with the chemotypes, and confirm 15 anti-Hsp90 fragments. Click chemistry is employed to construct
172 molecules and synthesize 21 compounds among them. The best inhibitor 3d was further optimized and resulted in more potent
4f (IC50 = 0.16 μM). In vitro and in vivo experiments confirmed that 4f is a promising agent against nasopharyngeal carcinoma. This
study may provide a strategy in discovering new ligands against targets without well-understood structures.
smaller number of fragments26 rather than screening a large
INTRODUCTION
■
number of druglike compounds. There can be 1060−200 possible
Heat shock protein 90 (Hsp90, a molecular chaperone
druglike compounds, but only 107 possible fragments.27 FBDD
participating in the regulation of cell proliferation and
can significantly reduce chemical space for lead identification.28
apoptosis) is highly expressed in tumor cells. More than 200
However, optimizing fragments to leads (F2L) is still a
oncogenic proteins are clients of Hsp90, such as HER2, EGFR,
challenge for FBDD. Fragment growing, merging, and linking
AKT, p53, BCR-ABL, and Raf.1 Targeting Hsp90 is a new
are common techniques for F2L. These techniques require
promising strategy for anticancer drug development.2 Recently,
well-understood fragment−receptor binding models, which
Hsp90 was reported to play an important role in virus infection
have to be supported by structure biology experiments.29
such as Dengue virus and Epstein−Barr virus,3−8 and Hsp90
Moreover, F2L has to chemically combine fragments with the
inhibitors were used to treat aging-related disease.9,10
binding model restructions.30
Hsp90 consists of N-terminal domain for adenosine
To develop new approaches for combining fragments, we
triphosphate (ATP) binding, middle domain for client protein
reported a strategy that discovered liver X receptor-β (LXRβ)
binding, and C-terminal domain for dimerization. ATP binding
agonists by connecting LXRβ privileged fragments that
causes Hsp90 conformational change, which activates Hsp90
extracted from known LXRβ agonists.31 Later on, we reported
chaperone function. Thus, many ATP-competitive inhibitors
a promoted strategy called chemotype-based drug discovery
were developed to disrupt Hsp90 function for treating cancers.
(CBDD) and identified antiosteoporosis leads with the
These inhibitors belong to three major classes: (1)
chemotyping analysis approach and their assembly rules.32
geldanamycin analogues,11 such as 17-DMAG,12 17-AAG,13
These results indicate that compounds with same target or
and IPI-504;14 (2) radicicol derivatives like NVP-AUY922,15
STA-9090,16 and AT13387;17 and (3) purine-based Hsp90
inhibitors such as BIIB021.18,19 Over 20 Hsp90 N-terminal
Received: September 3, 2020
Published: February 5, 2021
inhibitors entered clinical trials; however, they were all
discontinued due to toxicity. Therefore, novel druggable
Hsp90 inhibitors are still demanding.
Fragment-based drug discovery (FBDD) has been employed
for years with significant success.20−25 FBDD starts with a
https://dx.doi.org/10.1021/acs.jmedchem.0c01521
© 2021 American Chemical Society
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Figure 1. (A) SSSR of molecules in Library P. (B) The SSSR of molecules in Library M. (C) The SSSR of molecules in Library N. (D) The
example of chemotypes assembly to known Hsp90 inhibitor ACO1. The pink color shows that the functional group does not include in chemotype-
assembled motif. SSSR = smallest set of smallest rings.
function constructed with chemotypes through certain rules.
This may help us to accelerate the F2L process and discovery
novel Hsp90 inhibitors.
In this study, we report a second-generation CBDD method
that utilizes both fragment-linking approach and the chemo-
typing analysis approach. And we apply this method in the
discovery of novel Hsp90 inhibitors.
M. The popularities of chemotypes C4−6 are evenly
distributed in all three libraries. Chemotype C7 is a rare
chemotype. Thus, we hypothesize that linking chemotypes C1
and C4 can generate more potent Hsp90 inhibitors. A potent
known Hsp90 inhibitor ACO1 is assembled by C1, C3, and
C4 (Figure 1D). The chemotypes C1 and C3 link through
merging six-membered aromatic rings, and then newly formed
motif link with C4 through overlapping the same five-
membered aromatic ring. This indicated that Hsp90 inhibitor
can be formed by linking anti-Hsp90 chemotypes.
Novel Hsp90 Inhibitors Design Based on Newly
Discovered Active Anti-Hsp90 Fragments. A library
containing 500 fragments were virtually screened based on
the PFs and chemotypes via substructure retrieval, resulting in
328 fragments, which were then acquired and assayed with
surface plasmon resonance (SPR)33 experiments. Thus, we
identified 28 hits (Figure 2A) that exhibited a response value
higher than 30. The hits are all compatible with the privileged
chemotypes of Hsp90 inhibitors. For instance, hits F01-04
belong to mono-ring PF1 and F14-16 belong to chemotype
C1. These results imply that they all can be used to generate
potential Hsp90 inhibitors.
Linking chemotypes C1, C4, and C7 to form triazole
derivatives can be efficiently executed with click chemistry
syntheses. This requires converting out hits (fragments) to
brominated or alkynyl products as starting materials for click
chemistry reactions. Thus, the hits with multisubstituted
fragments were simplified to mono-substituted fragments
(precursors of brominated or alkynyl products) with the
condition of commercial availability (Figure 2E). This resulted
in seven bromides and two alkynes as the precursors, and two
designed scaffolds for potential Hsp90 inhibitory molecules, as
shown in Figure 2B. For negative control, we purposely
assembled molecules that contained chemotypes that were not
considered as Hsp90 inhibitory fragments (such as 2-
RESULTS
■
Chemotypes Derived from Known Anti-Hsp90
Agents. We started with 3482 known Hsp90 inhibitors
derived from ChEMBL database. These inhibitors partitioned
into potent library (library P, 1384 inhibitors with pChEMBL
values greater than 6), moderate potent library (library M,
1233 inhibitors with pChEMBL values between 6 and 5), and
nonactive library (library N, 865 inhibitors with pChEMBL
values less than 5). The smallest set of smallest rings (SSSR)
analyses indicated that inhibitors with three rings were most
popular (Figure 1), and potent Hsp90 inhibitors had at least
two rings. Therefore, we hypothesize that Hsp90 inhibitors can
be generated by linking chemotypes that have same ring in
each fragment.
Using a de novo substructure generation algorithm (DSGA),
we derived 29 privileged fragments (PF) and 164 other
fragments. Many privileged fragments of Hsp90 inhibitors have
five- or six-membered rings. The privileged fragments were
summarized into four motifs (PF1−4, Table 1). PF1 and PF4
exhibited much higher popularity in library P than libraries M
and N. PF2 and PF3 showed that even frequency in all
libraries.
The chemotypes and their popularities are listed in Table 1,
which indicates that library P contains more chemotypes C1
and C2 than libraries M and N do. Thus, we hypothesize that
compounds containing C1 and C2 can be more potent Hsp90
inhibitors. Chemotype C3 is associated with mild Hsp90
inhibition, which is related to the compounds in libraries P and
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hydrogen bonds is through a water bridge). Also, a water-
mediated interaction net connects 3d’s triazole ring to Phe138
and Asn51, which makes the t-butyl benzene ring be docked
into a hydrophobic pocket formed with Ala111, Tyr139,
Leu107, Phe22, Trp162, and Phe138. This compound does
not interact with the key residue Asp93. To further enhance
the activity of 3d, the benzene ring needs to be modified to
establish the binding between 3d and Asp93 (Table 4).
Compared to NVP-HSP990 and NVP-AUY992, compound 3d
occupied a different hydrophobic pocket by the t-butyl benzyl
group (Figure 3B,C), and this is similar to TAS-116 (Figure
3D).
Table 1. Privileged Anti-Hsp90 Chemotypes and Their
Frequency in Different Libraries
ae
Anti-Hsp90 Lead Compound 4f Discovered. Hence, we
synthesized seven 3d derivatives (4a−4f) by introducing −OH
or −NH2 groups onto the scaffold of 3d. These derivatives
exhibited enhanced Hsp90α or Hsp90β inhibitory activities.
Specifically, the compounds with para-hydroxyl benzene have
significantly increased Hsp90 inhibitory activities. However,
the −NH2 group in ortho position suppresses the activity. The
intermediate compound 5a further revealed that ortho-NH2
decreases the Hsp90 inhibition effect. As we hypothesize
before, the hydroxy groups do increase the activity like 4f 16-
fold more potent than 5a. Among them, compound 4f showed
5-fold more potent Hsp90 inhibition effects than 17-AAG.
A molecular dynamics simulation indicates that more
binding interactions were formed between 4f and Hsp90α
(PDB ID: 4W7T) compared to compound 4a (Figure 4). As
expected, two hydroxy groups of 4f provide two more
hydrogen bonds with Asp93 and one more hydrogen bond
with Thr184 compared to 3d. Another hydroxy group forms a
water-mediated hydrogen bond with Met98. The α-carbon of
benzyl provides a hydrogen-bond network with Asn51, Gly135,
and Phe138 through water. A p−π conjugation is formed
between Leu107 and tert-butyl benzene ring. When Asp93 was
mutated to Gly93, the binding affinity between 4f and Hsp90α
was decreased in 1600-fold (the table in Figure 4). The
mutations at Leu71 and Thr184 also result in a significant
decrease of Hsp90 binding affinity. These data proved that the
binding model is reasonable.
a
A = nonhydrogen atom, usually is C, O, N, and S; Q = nonhydrogen
heteroatom; Y = any function group, such as C, N, O, S, CONH2,
CH2OH, etc. Dashed lines = any bonds (single or double bond).
b
Double dashed lines = aromatic bond. Library P = potent library.
c
d
Library M = moderate potent library. Library N = nonactive library.
PF = privileged fragments.
e
Compound 4f Prevents Nasopharyngeal Carcinoma
in vivo and in vitro through Inhibiting Hsp90. The
activity of these newly synthesized compounds was tested on
nasopharyngeal carcinoma (see the last column in Tables 4
and S3). Compound 4f exhibited the most potent activity in a
dose-dependent manner without cytotoxicity on normal cells
(Figures 5A and S2, Table S3). Protein kinase B (PKB, aka
AKT, an Hsp90 client protein) (Figures 5B and S3) was
degraded through the induction of compound 4f without
changing Hsp90 expression. Thus, PI3K/AKT signaling
pathway, which activates many cell survival-related proteins,
is regulated. Consequently, 4f causes the CNE2 cell apoptosis
through PI3K/AKT signaling pathway. The degradation of
protein B-cell lymphoma-extra large (Bcl-xL) indicates that
compound 4f may induce mitochondrial-mediated apoptosis in
CNE2 cells. Another key protein HER2 related to cell death
was also decreased at 500 nM. All of these data revealed that 4f
causes cell apoptosis through a multipathway, proteostasis
disruption.
ethynylthiophene). Twenty-one compounds (Table 2) were
synthesized with the scheme shown in Figure 2D.
Anti-Hsp90 and Nasopharyngeal Carcinoma Hit
Discovered. We validated the inhibitory activities of the
synthesized compounds with Hsp90α ATPase assay. Our
results indicated that most compounds containing the Hsp90
binding chemotypes (see 3a−3n in Table 2) exhibited Hsp90
inhibitory activities indeed. However, compounds (see 3o−3u
in Table 2) with other chemotypes (see Figure 2C)
significantly lost activities (lower than 10% inhibition at 10
μM). Compounds 3d and 3n also showed potent binding
affinity with Hsp90α and Hsp90β as well as their inhibition
effects (Table 3). Furthermore, the cytotoxicities of 3d and 3n
were tested on liver cancer, cervical carcinoma, and
nasopharyngeal carcinoma (Table 3). Compound 3d selec-
tively inhibits nasopharyngeal carcinoma with an IC50 value of
3.98 μM. Compound 3d provides a hit scaffold for further
study.
Figure 5C,D also demonstrates that 4f cannot induce the
CNE2 cell apoptosis after two different Hsp90 siRNAs were
treated and 4f cannot alter the amount of AKT and Bcl-xL
after knockdown of the Hsp90 gene. These data proved that 4f
does induce the CNE2 cell apoptosis through Hsp90
Binding Model of Hit Compound 3d with Hsp90.
Molecular dynamic34 simulations indicate that 3d binds to the
ATP binding pocket of Hsp90α (Figure 3, PDB ID: 4W7T)
through two hydrogen bonds with Thr184 (one of the
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Figure 2. Designing Hsp90 inhibitors through assembling privileged chemotypes. (A) Thirty fragments for building potential Hsp90 inhibitors.
5Har = five-membered heteroaromatic ring; 6Ar = six-membered aromatic ring; 6r = six-membered aliphatic ring. (B) Chemotypes assembling to
form potential anti-Hsp90 scaffolds. (C) Chemotype C1 or C4 assembles with a non-anti-Hsp90 chemotype to form potential non-anti-Hsp90
scaffolds. (D) Click chemistry synthetical scheme for assembling potential Hsp90 inhibitors from privileged fragments (chemotypes). (E)
Commercially available starting materials from active fragments.
inhibition. Another rescue assay further confirmed that 4f
cause CNE2 apoptosis through Hsp90 inhibition. As shown in
Figure 5E, the overexpression of Hsp90 can rescue the cell
compared to the mock group (not Hsp90 overexpression) after
4f treatment. However, without treating 4f, the cell apoptosis is
quite similar between the mock group and the Hsp90
overexpression group. The amounts of related proteins like
AKT and BCL2 were also increased by Hsp90 overexpression.
As shown in Figure 6A,B, in vivo studies demonstrated that
the tumor sizes were shrunken after 32-day treatment with 4f.
Figure 6C indicates that 4f can inhibit the nasopharyngeal
tumor growth in a time-dependent manner. Finally, a
pharmacokinetic (PK) study was applied to test the druggable
property. As shown in Figure 6D, t1/2 of 4f is 1.10 h and the
bioavailability (F value) is 12%.
(NFs, Table S1), and the last one is considered as a bad
fragment (BF, Table S1). Like PFs, the NFs also exhibit higher
frequency in library P than the other two libraries (Table S1).
However, their frequency in library P is much lower than PFs
(NF1 is 4.19% and NF2 is 2.82%). Different from PFs and
NFs, the frequency of BF1 in nonactive library is higher than
the other two libraries (4.28% in library N vs 3.68% in library P
and 2.11% in library M). Therefore, we consider it as BF
because it may decrease activities. The fragments that contain
BF1 were also filtered before SPR screening. According to the
PFs and BF, 328 hit fragments were extracted from 500 and
then confirmed by SPR. The chemotypes are defined as two
fragments from PFs and BFs assembled through the linker
between them. The linkers are discovered according to our
previous study.32 In this study, PFs and chemotypes are
applied for screening fragments to discover potential anti-
Hsp90 hit fragments. Since the PFs and chemotypes are
extracted from known compounds, they can be employed to
build specific fragment databases for given targets in future.
These databases may provide a higher success rate of hit
fragment identification.
DISCUSSION
■
Here, we reported a novel strategy of hit fragments discovering
and assembling. The most potent compound 4f was confirmed
as an Hsp90 inhibitor to treat nasopharyngeal carcinoma by in
vitro and in vivo studies. First, we discovered PFs and
chemotypes from known Hsp90. These PFs and chemotypes
were then applied for hit fragments discovery. Here, seven
main fragments were found, four of which were considered as
privileged fragments, two were considered as normal fragments
The process of optimizing the hit fragments to leads is
usually considered as the bottleneck of FBDD. In most cases, it
is difficult to understand fragment−protein interactions
(reasonable binding model of fragment−protein), which
plays a key role in the F2L process. Theoretically, chemotypes
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Table 2. Structures of 15 Newly Designed Compounds and Their Hsp90 Inhibitory Activities
a
The percent of Hsp90α ATPase activity inhibition at the 10 μM of testing compounds.
Table 3. Chemotype-Constructed Substructures and Their
Frequency
click chemistry for Hsp90 inhibitors and obtained a more
potent lead compared to NVP-AUY992.35 At the same time,
the triazole ring also matches the chemotypes C1, C2, C4, and
C7. All of these encourage us to perform click chemistry for
chemotypes assembling. According to the chemotypes and
click chemistry, several virtual leads were formed and
synthesized. A series in vitro studies were then applied, 3d
and 3n were selected due to their potent Hsp90 binding
affinity and inhibition effects. Compound 3d exhibited
selectivity on nasopharyngeal carcinoma against liver cancer
and cervical carcinoma compared to 3n and 17-AAG. A
possible reason is the isoform selectivity of 3d (Table S2).36
Compound 3d exhibited around 10-fold activity on Hsp90α,
Hsp90β, and TRAP1 over GRP94, while 17-AAG showed
potent activity on GRP94. Compound 3d was further
optimized due to its selectivity on nasopharyngeal carcinoma.
Compound 4f with the most potent activity (IC50 of 160 nM
on Hsp90 and 40 nM on nasopharyngeal carcinoma) was
obtained. Unusually, the cell apoptosis effect is stronger than
the ATPase inhibition effect. Hsp90 is a central chaperone that
directs the folding and conformational maturation of its clients.
The inhibition of Hsp90 will cause several proteins disfunction
or degradation like AKT and HER2. This will lead a
multipathway disturbed and therefore cause more potent
inhibition effects on cell death.
bioassays
3d
3n
17-AAG
a
Hsp90α Kd (μM)
Hsp90β Kd (μM)
4.8 0.5
4.8 1.0
5.6 0.3
5.6 1.1
>50
3.1 0.4
7.0 1.5
6.2 1.4
9.8 1.5
18.1 1.9
22.4 3.3
14.1 2.1
0.9 0.1
0.9 0.2
0.9 0.3
0.8 0.1
8.0 2.0
19.4 3.2
3.0 0.6
a
b
Hsp90α IC50 (μM)
Hsp90β IC50 (μM)
HepG2 IC50 (μM)
b
c
d
Hela IC50 (μM)
>50
4.0 0.5
e
CNE2 IC50 (μM)
a
b
Kd is tested by SPR. IC50 indicates ATPase inhibition effects.
c
d
HepG2 indicates liver cancer. Hela indicates cervical carcinoma.
CNE2 denotes nasopharyngeal carcinoma.
e
can assemble with each other to grow up through same ring
overlapping (including homo- and hetero-assembling, Figure
1D). This strategy provides a possibility for optimizing
fragments when the fragment−protein binding model is
difficult to obtain. Our study proved that chemotype assembly
can be used for the F2L process without understanding
fragment−protein interactions. The novelty of leads discovered
by this method is warranted. The combination of different
chemotypes provides various novel scaffolds. For example, only
two chemotypes assembling can provide more than 80 motifs
of scaffold (Markush structures).
The triazole ring formed by click chemistry is a good
bioisostere in drug discovery. Norrild et al. also applied the
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Figure 3. (A) Binding model of compound 3d with Hsp90α (PDB ID: 4W7T). Compound 3d binds to Thr184 through a water bridge (red ball).
(B) Overlay of compound 3d with NVP-HSP990 (blue color, PDB ID: 4W7T). (C) Overlay of compound 3d with NVP-AUY922 (red color, PDB
ID: 2VCI). (D) Overlay of compound 3d with TSA-116 (yellow color, PDB ID: 5ZR3).
phosphate-buffered saline (PBS) was purchased from Gibco (Gibco,
NY). Cell apoptosis was detected using Annexin V-FITC Apoptosis
Detection Kit (Yeasen, China). ATPase activity was detected using
CONCLUSIONS
■
In summary, with chemotyping analysis approach and click
chemistry, we have designed, synthesized, and validated new
Hsp90 inhibitors with a novel scaffold. In vitro and in vivo
experiments demonstrate that the new inhibitor can be a
promising lead for nasopharyngeal carcinoma treatment. Our
method might be applied in discovering new ligands against
the targets that structures of the binding models are not well
understood. Of cause, it will be even better if the binding
mechanisms are understood.
the ATP, nicotinamide adenine dinucleotide (NADH), phosphoe-
nolpyruvate (PEP), and pyruvate kinase/lactic dehydrogenasea. 3-
(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide
(MTT) and cell dissociation solution were obtained from Sigma-
Aldrich (Sigma-Aldrich, St Louis, MO).
1
NMR Spectroscopy and Mass Spectrometry. H NMR and
13C NMR spectra were collected on a BRUKER 400 MHz
spectrometer and were calibrated with tetramethylsilane. The NMR
data are displayed as follows: chemical shifts (δ) are recorded as ppm,
coupling constants (J) in hertz (Hz), integrity as the number of
protons, and multiplicity as s (singlet), d (doublet), t (triplet), q
(quartet), quintet, sextet, and m (multiplet). Mass spectra were
obtained on a Shimadzu LCMS-2010EV utilizing the electron-spray
ionization method (ESI-MS). Thin-layer chromatography (TLC) was
carried out using plates coated with silica gel GF254, purchased from
Merck. Column chromatography was performed using Merck silica
gel 60 (70−230 mesh).
EXPERIMENTAL SECTION
■
Materials. Chemicals were purchased from Energy Chemical Co.,
Sigma-Aldrich Co., or Tokyo Chemical Industry Co. and utilized
without further purification. Solvents were distilled prior to use. For in
vitro and in vivo assay, RPMI 1640 medium and Lipofectamine 2000
were purchased from Thermo Fisher (Thermo Fisher Scientific);
Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine
serum (FBS) were purchased from Gibco (Gibco); penicillin and
streptomycin were purchased from Hyclone (Hyclone); and
Purity. Purity of all final compounds was ≥95%, as determined by
high-performance liquid chromatography (HPLC, SHIMADZU LC-
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(208 mg, 1 mmol), NaN3 (65 mg, 1 mmol), CuI (19 mg, 0.1 mmol),
1-ethynyl-4-methoxybenzene (132 mg, 1 mmol), and DBU (304 mg,
2 mmol) to provide product 3b as a white solid (163 mg, 54%). H
Table 4. Compounds 4a−f and 5a,b with Their Binding
Affinities on Hsp90 Homologues and Inhibition Effects on
CNE2 Cells
1
NMR (400 MHz, CDCl3) δ 8.98 (s, 1H), 8.25 (d, J = 8.0 Hz, 1H),
7.98 (d, J = 8.0 Hz, 1H), 7.66 (s, 1H), 7.55 (s, 1H), 7.55 (d, J = 8.0
Hz, 2H), 7.42−7.40 (m, 2H), 7.05 (d, J = 8.0 Hz, 2H), 3.83 (s, 3H).
13C NMR (100 MHz, CDCl3) δ 160.6, 152.5, 148.0, 143.4, 136.6,
131.4, 130.2, 128.9, 127.4, 123.9, 122.7, 120.9, 114.8, 55.8. ESI-MS
+
m/z: 302.11 [M + H] .
1-(4-(tert-Butyl)benzyl)-4-(4-(tert-butyl)phenyl)-1H-1,2,3-triazole
(3c). The general procedure was applied with 1-bromomethyl-4-(t-
butyl)benzene (212 mg, 1 mmol), NaN3 (65 mg, 1 mmol), CuI (19
mg, 0.1 mmol), 4-t-butylphenylacetylene (158 mg, 1 mmol), and
DBU (304 mg, 2 mmol) to provide product 3c as a white solid (246
1
mg, 71%). H NMR (400 MHz, CDCl3) δ 7.66 (d, J = 8.0 Hz, 2H),
7.64 (s, 1H), 7.36−7.33 (m, 3H), 7.16 (d, J = 8.0 Hz, 2H), 5.45 (s,
2H), 1.25 (s, 9H), 1.24 (s, 9H). 13C NMR (100 MHz, CDCl3) δ
150.8, 150.2, 147.1, 130.7, 126.8, 126.7, 125.0, 124.7, 124.4, 118.2,
+
52.9, 33.6, 30.2, 30.2. ESI-MS m/z: 348.24 [M + H] .
1-(4-(tert-Butyl)benzyl)-4-(4-methoxyphenyl)-1H-1,2,3-triazole
(3d). The general procedure was applied with 1-bromomethyl-4-(t-
butyl)benzene (212 mg, 1 mmol), NaN3 (65 mg, 1 mmol), CuI (19
mg, 0.1 mmol), 1-ethynyl-4-methoxybenzene (132 mg, 1 mmol), and
DBU (304 mg, 2 mmol) to provide product 3d as a white solid (189
1
mg, 59%). H NMR (400 MHz, CDCl3) δ 7.66 (d, J = 8.0 Hz, 2H),
7.50 (s, 1H), 7.32 (d, J = 8.0 Hz, 2H), 7.17 (d, J = 8.0 Hz, 2H), 6.84
(d, J = 8.0 Hz, 2H), 5.44 (s, 2H), 3.74 (s, 3H), 1.21 (s, 9H). 13C
NMR (100 MHz, CDCl3) δ 158.5, 150.8, 147.0, 130.7, 126.8, 126.0,
125.0, 122.3, 117.7, 113.1, 113.1, 54.3, 52.9, 33.6, 30.2, 28.7. ESI-MS
m/z: 322.18 [M + H]+.
4-(4-(tert-Butyl)phenyl)-1-(4-phenoxyphenyl)-1H-1,2,3-triazole
(3e). The general procedure was applied with 1-bromo-4-phenox-
ybenzene (248 mg, 1 mmol), NaN3 (65 mg, 1 mmol), CuI (19 mg,
0.1 mmol), 4-t-butylphenylacetylene (158 mg, 1 mmol), and DBU
(304 mg, 2 mmol) to provide product 3e as a white solid (239 mg,
1
65%). H NMR (400 MHz, CDCl3) δ 8.04 (s, 1H), 7.76 (d, J = 8.0
20AT, Japan). Compounds were dissolved at a concentration of 0.5
mg/mL in methanol. Then, 150 μL of the sample was injected into an
Agilent XDB-C18 HPLC column (4.6 m × 250 mm; particle size, 5
μm) and chromatographed using a gradient of water/MeCN from
90:10 to 50:50 for 15 min at a flow rate of 250 μL/min. UV
absorption was detected from 100 to 950 nm using a diode array
detector.
Animals. We performed all procedures on mice and rats with
approval by the Sun Yat-sen University Institutional Animal Care and
Use Committee. The C57BL/6 mice and female Sprague-Dawley
(SD) rats were obtained from Guangdong Medical Lab Animal
Center. The mice and rats were maintained in pressurized ventilated
cages under conditions of repeated controlled illumination (12 h dark;
12 h light) with ad libitum access to sterilized water and food.
General Synthesis Procedure. Bromide-substituted compound
1, NaN3, and CuI were added into dimethyl sulfoxide (DMSO) and
H2O (5:1). The mixture was stirred at room temperature for 0.5 h.
Then, the alkene-substituted materials 2 and DBU were added into
the mixture and stirred at 70 °C for 8 h. The mixture was followed by
extraction with EtOAc. The extract was then washed with water brine,
dried over Na2SO4, and evaporated in vacuo. The residue was purified
by column chromatography to give products as a solid.
6-(4-(4-Methoxyphenyl)-1H-1,2,3-triazol-1-yl)isoquinoline (3a).
The general procedure was applied with 6-bromoisoquinoline (208
mg, 1 mmol), NaN3 (65 mg, 1 mmol), CuI (19 mg, 0.1 mmol), 4-t-
butylphenylacetylene (158 mg, 1 mmol), and DBU (304 mg, 2 mmol)
to provide product 3a as a white solid (259 mg, 79%). 1H NMR (400
MHz, CDCl3) δ 8.91 (s, 1H), 8.20 (d, J = 8.0 Hz, 1H), 8.07 (s, 1H),
7.89 (d, J = 8.0 Hz, 1H), 7.71 (d, J = 8.0 Hz, 2H), 7.62 (s, 1H), 7.43
(d, J = 8.0 Hz, 1H), 7.39 (d, J = 8.0 Hz, 1H), 7.38 (d, J = 8.0 Hz, 2H),
1.35 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 152.5, 151.3, 148.0,
143.4, 136.6, 131.4, 130.2, 128.9, 128.0, 127.4, 127.3, 125.5, 125.4,
123.9, 120.9, 34.2, 31.3. ESI-MS m/z: 329.16 [M + H]+.
Hz, 2H), 7.66 (d, J = 8.0 Hz, 2H), 7.43−7.41 (m, 2H), 7.34−7.30 (m,
2H), 7.13−7.06(m, 3H), 7.02−6.99 (m, 2H). 13C NMR (100 MHz,
CDCl3) δ 156.79, 155.35, 150.55, 147.36, 131.31, 128.99, 126.38,
124.82, 124.56, 123.09, 121.22, 118.37, 118.31, 116.39, 33.70, 30.27.
ESI-MS m/z: 370.18 [M + H]+.
4-(4-Methoxyphenyl)-1-(4-phenoxyphenyl)-1H-1,2,3-triazole
(3f). The general procedure was applied with 1-bromo-4-phenox-
ybenzene (248 mg, 1 mmol), NaN3 (65 mg, 1 mmol), CuI (19 mg,
0.1 mmol), 1-ethynyl-4-methoxybenzene (132 mg, 1 mmol), and
DBU (304 mg, 2 mmol) to provide product 3f as a white solid (209
mg, 61%). 1H NMR (400 MHz, CDCl3) δ 7.98 (s, 1H), 7.76 (d, J =
8.0 Hz, 2H), 7.66 (d, J = 8.0 Hz, 2H), 7.35−7.31 (m, 2H), 7.13−7.08
(m, 4H), 6.99−6.91 (m, 3H), 3.79 (s, 3H). 13C NMR (100 MHz,
CDCl3) δ 158.78, 156.79, 131.31, 128.99, 126.13, 123.10, 121.91,
121.21, 118.39, 118.30, 115.91, 113.31, 54.33. ESI-MS m/z: 344.13
[M + H]+.
4-(4-(tert-Butyl)phenyl)-1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-
1H-1,2,3-triazole (3g). The general procedure was applied with 6-
bromo-2,3-dihydrobenzo[b][1,4]dioxine (213 mg, 1 mmol), NaN3
(65 mg, 1 mmol), CuI (19 mg, 0.1 mmol), 4-t-butylphenylacetylene
(158 mg, 1 mmol), and DBU (304 mg, 2 mmol) to provide product
1
3g as a white solid (211 mg, 66%). H NMR (400 MHz, CDCl3) δ
8.07 (s, 1H), 7.84 (d, J = 8.0 Hz, 1H), 7.50 (d, J = 8.0 Hz, 2H), 7.34
(s, 1H), 7.26 (d, J = 8.0 Hz, 1H), 4.34 (s, 3H), 1.39 (s, 9H). 13C
NMR (100 MHz, CDCl3) δ 151.5, 148.2, 144.1, 144.0, 131.0, 127.5,
125.8, 125.6, 118.0, 117.4, 113.7, 110.2, 64.4, 64.4, 34.7, 31.3. ESI-MS
+
m/z: 336.16 [M + H] .
1-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-4-(4-methoxyphenyl)-
1H-1,2,3-triazole (3h). The general procedure was applied with 6-
bromo-2,3-dihydrobenzo[b][1,4]dioxine (213 mg, 1 mmol), NaN3
(65 mg, 1 mmol), CuI (19 mg, 0.1 mmol), 1-ethynyl-4-
methoxybenzene (132 mg, 1 mmol), and DBU (304 mg, 2 mmol)
to provide product 3h as a white solid (127 mg, 51%). 1H NMR (400
MHz, DMSO-d6) δ 7.78 (s, 1H), 7.27 (s, 1H), 7.18 (d, J = 8.0 Hz,
6-(4-(4-(tert-Butyl)phenyl)-1H-1,2,3-triazol-1-yl)isoquinoline
(3b). The general procedure was applied with 6-bromoisoquinoline
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Figure 4. Binding model of compound 4f with Hsp90α (PDB ID: 4W7T) and binding affinity of 4f with WT-Hsp90α or mutated Hsp90α. The H-
bonds are represented as yellow dashed lines. Waters are represented as the red ball.
1
2H), 6.95 (s, 1H), 6.89−6.77 (m, 3H), 4.30−4.29 (m, 4H), 3.82 (s,
3H). 13C NMR (100 MHz, DMSO-d6) δ 159.7, 147.5, 144.3, 144.0,
130.9, 127.1, 123.4, 119.1, 118.4, 114.9, 113.4, 109.6, 64.8, 64.6, 55.7.
3k as a white solid (183 mg, 51%). H NMR (400 MHz, CDCl3) δ
7.67 (d, J = 8.0 Hz, 2H), 7.60 (s, 1H), 7.56 (d, J = 8.0 Hz, 2H), 7.37
(d, J = 8.0 Hz, 2H), 7.34 (d, J = 8.0 Hz, 2H), 5.56 (s, 2H), 1.26 (s,
9H). 13C NMR (100 MHz, CDCl3) δ 150.50, 147.53, 137.75, 127.07,
126.39, 125.10, 125.07, 125.03, 124.76, 124.44, 118.28, 52.46, 33.65,
+
ESI-MS m/z: 310.11 [M + H] .
1-(Benzo[d][1,3]dioxol-5-yl)-4-(4-(tert-butyl)phenyl)-1H-1,2,3-tri-
azole (3i). The general procedure was applied with 5-bromobenzo-
[d][1,3]dioxole (200 mg, 1 mmol), NaN3 (65 mg, 1 mmol), CuI (19
mg, 0.1 mmol), 4-t-butylphenylacetylene (158 mg, 1 mmol), and
DBU (304 mg, 2 mmol) to provide product 3i as a white solid (179
+
30.24. ESI-MS m/z: 360.05 [M + H] .
4-(4-Methoxyphenyl)-1-(4-(trifluoromethyl)benzyl)-1H-1,2,3-tria-
zole (3l). The general procedure was applied with 1-bromomethyl-4-
(trifluoromethyl)benzene (225 mg, 1 mmol), NaN3 (65 mg, 1 mmol),
CuI (19 mg, 0.1 mmol), 1-ethynyl-4-methoxybenzene (132 mg, 1
mmol), and DBU (304 mg, 2 mmol) to provide product 3k as a white
solid (179 mg, 54%). 1H NMR (400 MHz, CDCl3) δ 7.66 (d, J = 8.0
Hz, 2H), 7.58−7.55 (m, 3H), 7.33 (d, J = 8.0 Hz, 2H), 6.87 (d, J =
8.0 Hz, 2H), 5.55 (s, 2H), 3.76 (s, 3H). 13C NMR (100 MHz,
CDCl3) δ 158.7, 147.4, 137.7, 133.0, 127.1, 126.0, 125.1, 125.1, 125.0,
121.9, 117.7, 113.2, 113.1, 54.3, 52.5. ESI-MS m/z: 334.10 [M + H] +.
1-(3,5-Bis(trifluoromethyl)benzyl)-4-(4-(tert-butyl)phenyl)-1H-
1,2,3-triazole (3m). The general procedure was applied with 1-
(bromomethyl)-3,5-bis(trifluoromethyl)benzene (305 mg, 1 mmol),
NaN3 (65 mg, 1 mmol), CuI (19 mg, 0.1 mmol), 4-t-
butylphenylacetylene (158 mg, 1 mmol), and DBU (304 mg, 2
1
mg, 56%). H NMR (400 MHz, CDCl3) δ 7.98 (s, 1H), 7.75 (d, J =
8.0 Hz, 2H), 7.41 (d, J = 8.0 Hz, 2H), 7.24 (d, J = 4.0 Hz, 1H), 7.13
(d, J = 8.0 Hz, 1H), 6.85 (d, J = 8.0 Hz, 1H), 6.01 (s, 2H), 1.29 (s,
9H). 13C NMR (100 MHz, CDCl3) δ 150.5, 147.6, 147.2, 146.9,
130.6, 126.4, 124.8, 124.5, 116.6, 113.1, 107.5, 101.7, 101.1, 33.7,
+
30.3, 28.7. ESI-MS m/z: 322.14 [M + H] .
1-(Benzo[d][1,3]dioxol-5-yl)-4-(4-methoxyphenyl)-1H-1,2,3-tria-
zole (3j). The general procedure was applied with 5-bromobenzo-
[d][1,3]dioxole (200 mg, 1 mmol), NaN3 (65 mg, 1 mmol), CuI (19
mg, 0.1 mmol), 1-ethynyl-4-methoxybenzene (132 mg, 1 mmol), and
DBU (304 mg, 2 mmol) to provide product 3j as a white solid (126
mg, 43%). 1H NMR (400 MHz, DMSO-d6) δ 8.01 (s, 1H), 7.65 (d, J
= 8.0 Hz, 2H), 7.10 (d, J = 8.0 Hz, 2H), 7.08 (d, J = 8.0 Hz, 1H), 6.89
(d, J = 8.0 Hz, 1H), 6.83 (s, 1H), 6.07(s, 2H), 3.84 (s, 3H). 13C NMR
(100 MHz, DMSO-d6) δ 160.6, 148.8, 148.0, 131.4, 128.5, 122.7,
121.8, 118.9, 114.8, 114.6, 109.3, 101.2, 55.8. ESI-MS m/z: 296.09
1
mmol) to provide product 3m as a white solid (277 mg, 65%). H
NMR (400 MHz, CDCl3) δ 7.80 (s, 1H), 7.69−7.66 (m, 5H), 7.37
(d, J = 8.0 Hz, 2H), 5.62 (s, 2H), 1.26 (s, 9H). 13C NMR (100 MHz,
CDCl3) δ 150.7, 147.8, 136.4, 131.8, 131.4, 126.9, 126.9, 126.2, 124.8,
124.5, 121.8, 121.8, 121.7, 118.3, 51.9, 33.7, 30.22. ESI-MS m/z:
+
[M + H] .
+
4-(4-(tert-Butyl)phenyl)-1-(4-(trifluoromethyl)benzyl)-1H-1,2,3-
triazole (3k). The general procedure was applied with 1-
bromomethyl-4-(trifluoromethyl)benzene (225 mg, 1 mmol), NaN3
(65 mg, 1 mmol), CuI (19 mg, 0.1 mmol), 4-t-butylphenylacetylene
(158 mg, 1 mmol), and DBU (304 mg, 2 mmol) to provide product
428.14 [M + H] .
1-(3,5-Bis(trifluoromethyl)benzyl)-4-(4-methoxyphenyl)-1H-
1,2,3-triazole (3n). The general procedure was applied with 1-
(bromomethyl)-3,5-bis(trifluoromethyl)benzene (305 mg, 1 mmol),
NaN3 (65 mg, 1 mmol), CuI (19 mg, 0.1 mmol), 1-ethynyl-4-
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Figure 5. In vitro and in vivo experiments prove 4f is a promising lead against nasopharyngeal carcinoma. (A) Quantification of the apoptotic rate in
control and different-dose 4f-treated Annexin V-FITC/PI stained CNE2 cells at 48 h. (B) Western blots analysis of the effects of 4f on Hsp90,
AKT, p-mTOR, and Bcl-xL. (C) Changes in cell viability upon Hsp90α knockdown with and without compound 4f at 70 nM. (D) Western blots
analysis of the effects of 4f on Hsp90, AKT, and Bcl-xL when Hsp90 is knockdown. (E) Cell apoptosis of CNE2 in the mock group and the Hsp90
overexpression group without and with 4f treatment. (F) AKT and BCL-2 expression in the mock group and the Hsp90 overexpression group
without and with 4f treatment. ***: P ≤ 0.001 compared to related groups; ns: nonsignificant difference compared to related groups.
methoxybenzene (132 mg, 1 mmol), and DBU (304 mg, 2 mmol) to
provide product 3n as a white solid (236 mg, 59%). H NMR (400
1-(4-Phenoxyphenyl)-4-(thiophen-2-yl)-1H-1,2,3-triazole (3q).
The general procedure was applied with 1-bromo-4-phenoxybenzene
(248 mg, 1 mmol), NaN3 (65 mg, 1 mmol), CuI (19 mg, 0.1 mmol),
2-ethynylthiophene (108 mg, 1 mmol), and DBU (304 mg, 2 mmol)
1
MHz, CDCl3) δ 7.81 (s, 1H), 7.65 (d, J = 8.0 Hz, 2H), 7.66 (s, 1H),
7.48 (s, 2H), 7.31 (d, J = 8.0 Hz, 2H), 5.50 (s, 2H), 3.84 (s, 3H). 13C
NMR (100 MHz, CDCl3) δ 158.7, 147.4, 137.7, 133.0, 127.1, 126.0,
125.1, 125.1, 125.0, 121.9, 117.7, 113.2, 113.1, 54.3, 52.5. ESI-MS m/
1
to provide product 3q as a yellow solid (178 mg, 56%). H NMR
(400 MHz, CDCl3) δ 7.98 (s, 1H), 7.66−7.62 (m, 2H), 7.41−7.40
(m, 1H), 7.35−7.31 (m, 2H), 7.30−7.27 (m, 1H), 7.11−6.99 (m,
6H). 13C NMR (100 MHz, CDCl3) δ 156.9, 129.0, 126.7, 124.4,
123.5, 123.2, 121.3, 118.4, 118.3, 116.2, 28.7. ESI-MS m/z: 320.07
+
z: 402.09 [M + H] .
6-(4-(Thiophen-2-yl)-1H-1,2,3-triazol-1-yl)isoquinoline (3o). The
general procedure was applied with 6-bromoisoquinoline (206 mg, 1
mmol), NaN3 (65 mg, 1 mmol), CuI (19 mg, 0.1 mmol), 2-
ethynylthiophene (108 mg, 1 mmol), and DBU (304 mg, 2 mmol) to
+
[M + H] .
1-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-4-(thiophen-2-yl)-1H-
1,2,3-triazole (3r). The general procedure was applied with 6-bromo-
2,3-dihydrobenzo[b][1,4]dioxine (213 mg, 1 mmol), NaN3 (65 mg, 1
mmol), CuI (19 mg, 0.1 mmol), 2-ethynylthiophene (108 mg, 1
mmol), and DBU (304 mg, 2 mmol) to provide product 3r as a white
1
provide product 3o as a yellow solid (239 mg, 86%). H NMR (400
MHz, CDCl3) δ 9.15 (s, 1H), 8.56 (d, J = 4.0 Hz, 1H), 7.91−8.86 (m,
3H), 7.39 (t, J = 4.0 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 151.7,
143.4, 133.9, 132.9, 128.6, 126.7, 126.4, 120.5, 118.3. ESI-MS m/z:
279.16 [M + H]+.
1
solid (131 mg, 46%). H NMR (400 MHz, CDCl3) δ 7.92(s, 0.5H),
8.82 (s, 0.5H), 7.27−7.12 (m, 2H), 7.04−6.90 (m, 3H), 4.24 (s, 2H).
13C NMR (100 MHz, CDCl3) δ 143.1, 131.4, 129.6, 126.7, 124.8,
124.3, 124.1, 123.5, 117.0, 116.3, 112.8, 109.3, 63.4. ESI-MS m/z:
1-(4-(tert-Butyl)benzyl)-4-(thiophen-2-yl)-1H-1,2,3-triazole (3p).
The general procedure was applied with 1-bromomethyl-4-(t-
butyl)benzene (212 mg, 1 mmol), NaN3 (65 mg, 1 mmol), CuI
(19 mg, 0.1 mmol), 2-ethynylthiophene (108 mg, 1 mmol), and DBU
(304 mg, 2 mmol) to provide product 3p as a white solid (216 mg,
73%). 1H NMR (400 MHz, DMSO-d6) δ 7.50−7.47 (m, 4H), 7.24−
7.22 (m, 3H), 5.48 (s, 2H), 2.21 (s, 9H). 13C NMR (100 MHz,
DMSO-d6) δ 202.6, 175.3, 169.4, 165.6, 158.2, 138.3, 138.1, 114.4,
+
286.05 [M + H] .
1-(Benzo[d][1,3]dioxol-5-yl)-4-(thiophen-2-yl)-1H-1,2,3-triazole
(3s). The general procedure was applied with 5-bromobenzo[d][1,3]-
dioxole (200 mg, 1 mmol), NaN3 (65 mg, 1 mmol), CuI (19 mg, 0.1
mmol), 2-ethynylthiophene (108 mg, 1 mmol), and DBU (304 mg, 2
1
mmol) to provide product 3s as a white solid (143 mg, 53%). H
+
113.9, 95.70, 91.2, 51.3, 30.7, 21.7. ESI-MS m/z: 298.41 [M + H] .
NMR (400 MHz, DMSO-d6) δ 9.08 (s, 1H), 7.59 (d, J = 4.0 Hz, 1H),
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Figure 6. (A) Images of tumors (n = 10) treated with 4f or vehicle after 32 days. (B) Tumor weight treated with 4f or vehicle after 32 days. (C)
Tumor volume in mice (n = 10) treated for the indicated time with 4f or vehicle. (D) Pharmacokinetic study of 4f in mice.
7.53 (d, J = 4.0 Hz, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.42 (d, J = 8.0 Hz,
1H), 7.19 (t, J = 4.0 Hz, 1H), 7.16 (d, J = 8.0 Hz, 1H). 13C NMR
(100 MHz, DMSO-d6) δ 148.7, 148.0, 142.9, 132.9, 131.4, 128.5,
126.3, 124.9, 119.6, 114.4, 109.1, 102.7, 102.5. ESI-MS m/z: 272.057
CDCl3) δ 142.9, 136.1, 131.9, 131.6, 131.3, 127.1, 126. 7, 124.5,
123.6, 121.9, 120.5, 118.0, 52.0. ESI-MS m/z: 378.04 [M + H] .
+
4-(1-(4-(tert-Butyl)benzyl)-1H-1,2,3-triazol-4-yl)phenol (4a).
Compound 3d (160 mg, 0.5 mmol) was added into hydrobromic
acid (48% in water, 10 mL) and stirred at 120 °C for 12 h. Then, the
solvent was removed in vacuo to give targeted compound 4a as a
white solid (291 mg, 95%). 1H NMR (400 MHz, CDCl3) δ 7.56 (d, J
= 8.0 Hz, 2H), 7.51 (s, 1H), 7.33 (d, J = 8.0 Hz, 2H), 7.11 (d, J = 8.0
Hz, 1H), 6.81 (d, J = 8.0 Hz, 1H), 5.45 (s, 2H), 1.24 (s, 9H). 13C
NMR (100 MHz, DMSO-d6) δ 157.7, 151.1, 147.4, 133.7, 128.1,
127.0, 125.9, 122.2, 120.5, 116.1, 53.1, 34.8, 31.5. ESI-MS m/z:
308.39 [M + H]+.
2-(tert-Butyl)-5-((4-(4-hydroxyphenyl)-1H-1,2,3-triazol-1-yl)-
methyl)phenol (4b). The general procedure was applied with 4-
(bromomethyl)-1-(tert-butyl)-2-methoxybenzene (256 mg, 1 mmol),
NaN3 (65 mg, 1 mmol), CuI (19 mg, 0.1 mmol), 4-t-
butylphenylacetylene (158 mg, 1 mmol), and DBU (304 mg, 2
mmol) to provide intermediate as a white solid. The intermediate
(176 mg, 0.5 mmol) was added into hydrobromic acid (48% in water,
10 mL) and stirred at 120 °C for 12 h. Then, the solvent was removed
in vacuum to give targeted compound 4b as a white solid (141 mg,
88%). 1H NMR (400 MHz, DMSO-d6) δ 9.57 (s, 1H), 9.44 (s, 1H),
8.39 (s, 1H), 7.65 (d, J = 8.0 Hz, 2H), 7.13 (d, J = 8.0 Hz, 1H), 6.83
(d, J = 8.0 Hz, 2H), 6.72−6.55 (m, 2H), 5.48 (s, 2H), 1.31 (s, 9H).
13C NMR (100 MHz, DMSO-d6) δ 157.7, 156.5, 147.4, 135.6, 135.2,
+
[M + H] .
4-(Thiophen-2-yl)-1-(4-(trifluoromethyl)benzyl)-1H-1,2,3-triazole
(3t). The general procedure was applied with 1-bromomethyl-4-
(trifluoromethyl)benzene (225 mg, 1 mmol), NaN3 (65 mg, 1 mmol),
CuI (19 mg, 0.1 mmol), 2-ethynylthiophene (108 mg, 1 mmol), and
DBU (304 mg, 2 mmol) to provide product 3t as a white solid (207
mg, 67%). 1H NMR (400 MHz, CDCl3) δ 7.57 (s, 1H), 7.55 (s, 2H),
7.32 (d, J = 8.0 Hz, 2H), 7.27 (d, J = 4.0 Hz, 1H), 7.21 (d, J = 4.0 Hz,
1H), 7.19 (s, 1H), 6.98 (t, J = 4.0 Hz, 1H), 5.54 (s, 2H). 13C NMR
(100 MHz, CDCl3) δ 142.58, 137.45, 131.52, 127.16, 126.62, 125.14,
125.07, 124.24, 123.33, 118.05, 52.53. ESI-MS m/z: 310.05 [M + H]
+
.
1-(3,5-Bis(trifluoromethyl)benzyl)-4-(thiophen-2-yl)-1H-1,2,3-tri-
azole (3u). The general procedure was applied with 1-(bromometh-
yl)-3,5-bis(trifluoromethyl)benzene (305 mg, 1 mmol), NaN3 (65
mg, 1 mmol), CuI (19 mg, 0.1 mmol), 2-ethynylthiophene (108 mg, 1
mmol), and DBU (304 mg, 2 mmol) to provide product 3u as a white
1
solid (297 mg, 79%). H NMR (400 MHz, CDCl3) δ 7.82 (s, 1H),
7.70 (s, 1H), 7.62 (s, 1H), 7.31 (d, J = 4.0 Hz, 1H), 7.24 (d, J = 4.0
Hz, 1H), 7.00 (t, J = 4.0 Hz, 1H), 5.61 (s, 2H). 13C NMR (100 MHz,
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127.1, 127.0, 120.6, 118.4, 116.1, 115.5, 53.0, 34.6, 29.7. ESI-MS m/z:
324.38 [M + H]+.
polyacrylamide gel electrophoresis (SDS-PAGE), and fractions
showing a single band corresponding to the expected molecular
weight were pooled, resulting in >95% pure protein samples.
2-(1-(4-(tert-Butyl)benzyl)-1H-1,2,3-triazol-4-yl)-5-methoxyani-
line (4c). The general procedure was applied with 1-bromomethyl-4-
(t-butyl)benzene (212 mg, 1 mmol), NaN3 (65 mg, 1 mmol), CuI (19
mg, 0.1 mmol), 1-ethynyl-4-methoxy-2-nitrobenzene (177 mg, 1
mmol), and DBU (304 mg, 2 mmol) to provide intermediate as a
white solid. The intermediate (188 mg, 0.5 mmol) was then added
into MeOH with Pd/C (50 mg). The air was replaced by H2. The
mixture was stirred at r.t. for 8 h and filtered. The filtrate was collected
and purified by falsh column to afford the desired compound 4c as a
Surface Plasmon Resonance (SPR) Assay. SPR technology-
based binding assays were performed on a Biacore T200 instrument
(GE Healthcare) at 25 °C with running buffer PBST (phosphate-
buffered saline, pH 7.4, containing 0.005% Tween-20). Protein was
dissolved in the coupling buffer (20 μg/mL in 10 mM sodium acetate
[pH 5.0]) and immobilized onto the CM5 chip. The chip was first
equilibrated with PBST overnight. Inhibitors with 10 μM were serially
diluted and injected at a flow rate of 30 μL/min for 120 s (contact
phase) followed by 120 s (dissociation phase). The binding data were
determined using Biacore T200 evaluation software (GE Healthcare).
ATPase Activity Assay. The ATPase activity assay is based on the
conversion of phosphoenolpyruvate (PEP) to pyruvate by pyruvate
kinase (PK) coupled to the conversion of pyruvate to lactate by
lactate dehydrogenase (LDH) at the expense of NADH.37 NADH has
an excitation wavelength of 340 nm, and the oxidation of NADH to
NAD+ produces a decrease of absorbance at 340 nm. The reaction
buffer contained 150 mM Tris-HCl, pH 7.6, 40 mM KCl, 10 mM
MgCl2, 1.2 mM ATP, 0.1 mM NADH, 8 mM PEP, and 10 μL of
pyruvate kinase/lactic dehydrogenase enzymes (Sigma). The reaction
was started by addition of 2 μM Hsp90 protein at 37 °C. The ATPase
activity was determined by tracking the decrease in the absorbance at
340 nm. The compounds were added to the reaction mixture to test
the inhibition of Hsp90 ATPase activity. 17-AAG as a positive control.
Cells Culture and Cytotoxicity Assay. The poorly differentiated
human nasopharyngeal carcinoma (CNE2), HeLa, and HepG2 cell
lines were obtained from the Cancer Center of Sun Yat-sen
University. CNE2 was cultured in RPMI 1640 medium with 10%
fetal bovine serum (v/v) and supplementary 1% (v/v) penicillin/
streptomycin. HeLa and HepG2 cell lines were cultured in DMEM
with 10% fetal bovine serum (v/v) and supplementary 1% (v/v)
penicillin/streptomycin. All cell lines were maintained at 37 °C in a
humidified atmosphere with 5% CO2. Exponentially growing cells
were seeded at 3 × 103 cells per well in 96-well culture plates for 24 h.
The cells were exposed to increasing concentrations (0−20 μM) of
inhibitors for 48 h. The equal volume of DMSO was used as the
solvent control. MTT solution (10 μL) was added to each well (0.5
mg/mL) and incubated for another 3 h. Then, the formazan crystals
were dissolved in 150 μL of DMSO and light absorbance of the
solution was measured at 570 nm, with a reference wavelength of 630
nm, on a multiscanner autoreader (M450; Bio-Rad). The IC50 values
were calculated using the PrismPad program.
1
white solid (145 mg, 86%). H NMR (400 MHz, DMSO-d6) δ 8.47
(s, 1H), 7.42−7.29 (m, 4H), 6.34−6.18 (m, 3H), 5.59 (s, 2H), 3.35
(s, 3H), 1.26 (s, 9H). 13C NMR (100 MHz, DMSO-d6) δ 160.2,
151.1, 148.2, 147.6, 133.6, 129.2, 128.2, 126.0, 120.8, 106.6, 103.2,
100.5, 55.2, 53.2, 34.7, 31.5. ESI-MS m/z: 337.43 [M + H]+.
4-(1-(4-(tert-Butyl)benzyl)-1H-1,2,3-triazol-4-yl)-3-nitrophenol
(4d). The general procedure was applied with 1-bromomethyl-4-(t-
butyl)benzene (212 mg, 1 mmol), NaN3 (65 mg, 1 mmol), CuI (19
mg, 0.1 mmol), 1-ethynyl-4-methoxy-2-nitrobenzene (177 mg, 1
mmol), and DBU (304 mg, 2 mmol) to provide intermediate as a
white solid. The intermediate (188 mg, 0.5 mmol) was added into
hydrobromic acid (48% in water, 10 mL) and stirred at 120 °C for 12
h. Then, the solvent was removed in vacuum to give targeted
1
compound 4d as a white solid (169 mg, 96%). H NMR (400 MHz,
DMSO-d6) δ 10.82 (brs, 1H), 8.40 (s, 1H), 7.61 (d, J = 8.0 Hz, 1H),
7.42 (d, J = 8.0 Hz, 2H), 7.28 (d, J = 8.0 Hz, 2H), 7.23 (d, J = 8.0 Hz,
1H), 7.44−7.11 (m, 1H), 5.60 (s, 2H), 1.27 (s, 9H). 13C NMR (100
MHz, DMSO-d6) δ 158.4, 149.4, 142.6, 133.5, 132.2, 128.2, 126.0,
123.2, 120.0, 114.8, 110.9, 53.1, 34.8, 31.5. ESI-MS m/z: 353.38 [M +
H]+.
3-Amino-4-(1-(4-(tert-butyl)benzyl)-1H-1,2,3-triazol-4-yl)phenol
(4e). Compound 4d (176 mg, 0.5 mmol) was then added into MeOH
with Pd/C (50 mg). The air was replaced by H2. The mixture was
stirred at r.t. for 8 h and filtered. The filtrate was collected and purified
by flash column to afford desired compound 4e as a white solid (148
mg, 92%). 1H NMR (400 MHz, DMSO-d6) δ 8.52 (s, 1H), 7.58 (d, J
= 8.0 Hz, 1H), 7.41 (d, J = 8.0 Hz, 2H), 7.26 (d, J = 8.0 Hz, 2H), 6.33
(s, 1H), 6.19 (d, J = 8.0 Hz, 1H), 5.58 (s, 2H). 13C NMR (100 MHz,
DMSO-d6) δ 159.4, 148.3, 145.9, 133.1, 130.3, 128.6, 126.1, 123.2,
120.1, 104.7, 101,2, 56.5, 34.7, 31.4. ESI-MS m/z: 323.41 [M + H]+.
5-(1-(4-(tert-Butyl)benzyl)-1H-1,2,3-triazol-4-yl)benzene-1,2,3-
triol (4f). The general procedure was applied with 1-bromomethyl-4-
(t-butyl)benzene (212 mg, 1 mmol), NaN3 (65 mg, 1 mmol), CuI (19
mg, 0.1 mmol), 5-ethynyl-1,2,3-trimethoxybenzene (192 mg, 1
mmol), and DBU (304 mg, 2 mmol) to provide intermediate as a
white solid. The intermediate 5a (190 mg, 0.5 mmol) was added into
hydrobromic acid (48% in water, 10 mL) and stirred at 120 °C for 12
h. Then, the solvent was removed in vacuum to give targeted
Apoptosis Assay. CNE2 cells were seeded in a 24-well plate (5 ×
104 cells/well). After 24 h, the cells were treated with compound
(DMSO 0.05%). After 12 h, the cells were washed twice with PBS and
collected from the 24-well plate into FACS tubes. The tubes were
centrifuged at 1500 rpm for 5 min and washed with 1 mL of PBS. The
cells were centrifuged again at 1500 rpm for 5 min and prepared in
mastermix (100 μL of 1 × binding buffer, 5 μL of Annexin V/FITC,
and 3 μL of PI in each sample). The resuspended cells in 100 μL of
mastermix were incubated for 15 min at room temperature in the dark
and tested by FACS to evaluate apoptotic cells. Results are
represented as mean and s.e.m. of at least three independent
experiments.
1
compound 4f as a white solid (160 mg, 94%). H NMR (400 MHz,
DMSO-d6) δ 8.91 (s, 1H), 8.32 (s, 1H), 8.20 (s, 1H), 7.40 (d, J = 8.0
Hz, 2H), 7.28 (d, J = 8.0 Hz, 2H), 6.72 (d, J = 8.0 Hz, 1H), 5.53 (s,
2H), 1.26 (s, 9H). 13C NMR (100 MHz, DMSO-d6) δ 151.0, 147.7,
146.8, 133.7, 133.6, 128.2, 125.9, 121.7, 120.6, 104.9, 53.1, 34.7, 31.5.
ESI-MS m/z: 340.38 [M + H]+. Purity: 97.3%.
Expression and Purification of Proteins. The Escherichia coli
strain BL21 was transformed with pET28a-Hsp90α, pET28a-
Hsp90β, and pET28a-Hsp90α-mutants; cultured in LB medium
containing 50 mg/mL kanamycin at 37 °C to an absorbance of 0.6 at
600 nm; and induced with 1 mM IPTG for 16 h at 16 °C before being
harvested by centrifugation. The cell pellets were suspended in lysis
buffer (10 mM Tris, pH 7.5, 200 mM NaCl, 1 mM DTT) and
disrupted by sonication. After centrifugation, the supernatant was
applied to a Ni-NTA column and proteins were eluted with elution
buffer (10 mM Tris, pH 7.5, 200 mM NaCl, 1 mM DTT, and 400
mM imidazole). The 6-His-tag was removed by digestion with
thrombin. The samples were exchanged and further purified with the
buffer using size exclusion chromatography (S200 Sephacryl column,
GE) in 50 mM HEPES, 200 mM NaCl, and 1 mM DTT. Fractions
containing the protein were analyzed using sodium dodecyl sulfate
Immunoblotting. CNE2 (15 × 106 cells/mL) cells were
incubated with inhibitors for 24 h. The cells were collected and
washed with ice-cold PBS three times. Then, the cells were lysed with
RIPA buffer for 30 min on ice. Total cellular protein from each sample
was loaded onto 8−12% sodium dodecyl sulfate (SDS) polyacryla-
mide gel electrophoresis (PAGE) gels. The proteins were transferred
to poly(vinylidene fluoride) (PVDF) membranes (Millipore), and the
membranes were blocked with 5% nonfat milk for 1 h. The
membranes were probed with primary antibodies overnight, washed
with TBST for 30 min, exposed to horseradish peroxidase (HRP)-
conjugated secondary antibodies for 1 h, and washed again in TBST
for 30 min. Immunoreactive material was detected using the
chemiluminescence method. Actin was used as the loading control.
2020
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Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Hsp90α Knockdown Assay. For transient transfection, the cells
were transfected with siHsp90-1 (5′-AAAGCGUUCAUGGAAG-
CUUUG-3′) or siHsp90-2 (5′-GCUUGACAGAUCCCAGUAATT-
3′) at a final concentration of 150 nM using Lipofectamine 2000. The
cells were then collected for cytotoxicity assay and immunoblotting
assay.
Xenograft Studies. Nude mice (nu/nu, male 6−8 weeks old)
were subcutaneously injected with 10 × 106 CNE2 cell on their right
flanks. For evaluation of inhibitor using xenograft mice, the molecule
was administered daily by i.p. oral with a dose of 1 mg/kg for 6 days
after subcutaneous injection of CNE2 cells on the right flank of each
mouse. Tumor growth was recorded by measurement of two
perpendicular diameters of the tumors over a 3-week period using
the formula 4π/3 × (width/2)2 × (length/2). The tumors were
harvested and weighed at the experimental endpoint, and the masses
of tumors (g) treated with vehicle control (DMSO) and 4f were
compared by a two-tailed unpaired Student’s t test.
PK Study. Nine male Kunming mice were treated on
pharmacokinetic study. All of the animals were 9−11 weeks old
with body weight of about 23 g. For two PK studies, four mice per
group received a single dose of 4f. For both intravenous injection and
oral administration PK studies, 2 mg/kg of 4f were given. Blood
samples were collected at 0 min, 10 min, 15 min, 30 min, 1 h, 2 h, 4 h,
and 6 h post dose. Preparation of samples from the pharmacokinetic
studies on the mice was performed using 40 μL of plasma sample,
then centrifugation supernatant was obtained. Acetonitrile was added
(200 μL) to precipitate plasma proteins, and then the sample was
centrifuged for 10 min at 12 000 rpm. The supernatant was
transferred to a new tube and frozen at −80 °C until analysis by
HPLC (WOOKING, K2025, China). Pharmacokinetic data were
analyzed by Pkweb.
Authors
Mengyang Xu − Research Center for Drug Discovery, School of
Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou
510006, China; Department of Biotechnology, College of Life
Science and Technology, Jinan University, Guangzhou
510632, China
Chao Zhao − Research Center for Drug Discovery, School of
Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou
510006, China; Shenzhen Cell Inspire Therapeutics Co.,
Ltd., Shenzhen 518101, China
Biying Zhu − Department of Biotechnology, College of Life
Science and Technology, Jinan University, Guangzhou
510632, China
Liangyue Wang − Research Center for Drug Discovery, School
of Pharmaceutical Sciences, Sun Yat-Sen University,
Guangzhou 510006, China
Huihao Zhou − Research Center for Drug Discovery, School of
Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou
510006, China; orcid.org/0000-0002-9675-5007
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01521
Author Contributions
M.X. and C.Z. contributed equally to this work. M.X., C.Z.,
Q.G., and J.X. designed the overall study. C.Z. performed the
in silico experiments and chemical experiments. M.X. and L.W.
performed in vitro biological experiments and SPR study. B.Z.
performed in vivo study. H.Z., D.Y., Q.G., and J.X. supervised
the overall study. C.Z., H.Z., D.Y., Q.G., and J.X. wrote and
edited the manuscript.
Statistics. Mean s.d. was calculated to express numerical data
and histograms. A two-tailed Student’s t-test was performed between
two groups and a difference was considered statistically significant
with P < 0.05.
Notes
The authors declare no competing financial interest.
ASSOCIATED CONTENT
■
ACKNOWLEDGMENTS
sı
* Supporting Information
■
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01521.
This work was funded in part by the National Natural Science
Foundation of China (8180131061, 81473138, 81573310,
81803361), National Key R&D Program of China
(2017YFB0203403), the Science and Technology Program of
Guangzhou (201604020109), the Guangdong Provincial Key
Lab. of New Drug Design and Evaluation (Grant
2011A060901014), and the China Postdoctoral Science
Foundation(2020M673089).
NMR spectra and HPLC traces of compound 4f; SPR
response of hit fragments with HSP90 (Figure S1);
nontoxicity of compound 4f on normal human hepatic
cells (Figure S2); gray values of AKT expression in
CNE2 cells after 4f treatment (Figure S3); anti-Hsp90
fragments and their frequencies (Table S1); selectivity of
17-AAG, 3d, and 4f among Hsp90 isoforms (Table S2);
and cytotoxicity of 4a−f on normal cell 293T (Table S3)
(PDF)
ABBREVIATIONS
■
FBDD, fragment-based drug design; HER2, receptor tyrosine-
protein kinase erbB-2; EGFR, epidermal growth factor
receptor; AKT, protein kinase B; BCR-ABL, breakpoint cluster
region-c-abl oncogene 1; F2L, fragment to lead; SPR, surface
plasmon resonance; NMR, nuclear magnetic resonance; TSA,
thermal shift assays; SSSR, smallest set of smallest rings; LXRβ,
liver X receptor-β; DSGA, de novo substructure generation
algorithm; SCA, scaffold-based classification approach; Hsp90,
heat-shock protein 90; SAR, structure−activity relationship;
ATP, adenosine triphosphate; PI3K, phosphoinositide 3-
kinase; Bcl-xL, B-cell lymphoma-extra large; POC, percent of
control
Molecular Formula Strings (CSV)
AUTHOR INFORMATION
■
Corresponding Authors
Jun Xu − Research Center for Drug Discovery, School of
Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou
510006, China; School of Biotechnology and Health Sciences,
Wuyi University, Jiangmen 529020, China; orcid.org/
0000-0002-1075-0337; Email: junxu@biochemomes.com
Qiong Gu − Research Center for Drug Discovery, School of
Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou
510006, China; orcid.org/0000-0001-6011-3697;
Email: guqiong@mail.sysu.edu.cn
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https://dx.doi.org/10.1021/acs.jmedchem.0c01521
J. Med. Chem. 2021, 64, 2010−2023