2-((1-Phenyl-1H-1,2,3-triazol-4-yl)methyl)-2-azabicyclo[3.2.1]octan-3-one derivatives: Simplification and modification of aconitine scaffold for the discovery of novel anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2020.112988

The molecular chaperone heat shock protein 90 (Hsp90) is a promising target for cancer therapy. Natural product aconitine is a potential Hsp90 inhibitor reported in our previous work. In this study, we designed and synthesized a series of 2-((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)-2-azabicyclo[3.2.1]octan-3-one derivatives as potent Hsp90 inhibitors by simplifying and modifying aconitine scaffold. Among these compounds, 14t exhibited an excellent antiproliferative activity against LoVo cells with an IC₅₀ value of 0.02 μM and a significant Hsp90α inhibitory activity with an IC₅₀ value of 0.71 nM. Molecular docking studies provided a rational binding model of 14t in complex with Hsp90α. The following cell cycle and apoptosis assays revealed that compound 14t could arrest cell cycle at G1/S phase and induce cell apoptosis via up-regulation of bax and cleaved-caspase 3 protein expressions while inhibiting the expressions of bcl-2. Moreover, 14t could inhibit cell migration in LoVo and SW620 cell lines. Consistent with in vitro results, 14t significantly repressed tumor growth in the SW620 xenograft mouse model.

Full text of DOI:10.1016/j.ejmech.2020.112988

Journal Pre-proof
2-((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)-2-azabicyclo[3.2.1]octan-3-one derivatives:
simplification and modification of aconitine scaffold for the discovery of novel
anticancer agents
Yi Zhang, Ting-jian Zhang, Xin-yang Li, Jing-wei Liang, Shun Tu, Hai-li Xu, Wen-han
Xue, Xin-hua Qian, Zhen-hao Zhang, Xu Zhang, Fan-hao Meng
PII:
S0223-5234(20)30960-0
DOI:
https://doi.org/10.1016/j.ejmech.2020.112988
EJMECH 112988
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 October 2020
Revised Date: 29 October 2020
Accepted Date: 1 November 2020
Please cite this article as: Y. Zhang, T.-j. Zhang, X.-y. Li, J.-w. Liang, S. Tu, H.-l. Xu, W.-h. Xue,
X.-h. Qian, Z.-h. Zhang, X. Zhang, F.-h. Meng, 2-((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)-2-
azabicyclo[3.2.1]octan-3-one derivatives: simplification and modification of aconitine scaffold for the
discovery of novel anticancer agents, European Journal of Medicinal Chemistry, https://doi.org/10.1016/
j.ejmech.2020.112988.
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2-((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)-2-azabicyclo[3.2.1]octan-3-
one derivatives: simplification and modification of aconitine scaffold
for the discovery of novel anticancer agents
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a
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Yi Zhang a, 1, Ting-jian Zhang a, 1, Xin-yang Li a, b, Jing-wei Liang , Shun Tu , Hai-li Xu ,
Wen-han Xue a, Xin-hua Qian a, Zhen-hao Zhang a, Xu Zhang a, Fan-hao Meng a, *
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a School of Pharmacy, China Medical University, 77 Puhe Road, North New Area, Shenyang,
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110122, China
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b Department of Pharmacy, Shengjing Hospital of China Medical University, Shenyang 110122,
China
1 These two authors contributed equally to this work
* Corresponding author: Professor Fan-hao Meng
E-mail address: fhmeng@cmu.edu.cn (F. H. Meng)
Fax: +86-24-31939448
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Abstract
The molecular chaperone heat shock protein 90 (Hsp90) is a promising target for cancer therapy.
Natural product aconitine is a potential Hsp90 inhibitor reported in our previous work. In this
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study,
we
designed
and
synthesized
a
series
of
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2-((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)-2-azabicyclo[3.2.1]octan-3-one derivatives as potent
Hsp90 inhibitors by simplifying and modifying aconitine scaffold. Among these compounds, 14t
exhibited an excellent antiproliferative activity against LoVo cells with an IC50 value of 0.02 µM
and a significant Hsp90α inhibitory activity with an IC50 value of 0.71 nM. Molecular docking
studies provided a rational binding model of 14t in complex with Hsp90α. The following cell
cycle and apoptosis assays revealed that compound 14t could arrest cell cycle at G1/S phase and
induce cell apoptosis via up-regulation of bax and cleaved-caspase 3 protein expressions while
inhibiting the expressions of bcl-2. Moreover, 14t could inhibit cell migration in LoVo and SW620
cell lines. Consistent with in vitro results, 14t significantly repressed tumor growth in the SW620
xenograft mouse model.
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Key words: Hsp90 inhibitors; Aconitine; Structural simplification; Antiproliferative activity
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1. Introduction
Heat shock protein 90 (Hsp90, 90 kDa) is a ubiquitous molecular chaperone that is
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responsible for the maturation, stabilization and activation of numerous client proteins related to
signal transduction in the cell [1, 2]. Hsp90 regulates more than 500 clients include many
oncogenic proteins (e.g., EGFR, c-Met, Src, Akt, c-Raf, Cdk4, MMP2, HIF-1α), which are
necessary for the development and progression of cancer [3-9]. Oncogenic mutations of client
proteins require increased Hsp90 activity. In fact, Hsp90 is overexpressed in cancer cells 2-10 fold
higher than normal cells and exists as activated multi-chaperone complexes in cancer cells [10].
Therefore, inhibition of the molecular chaperone Hsp90 represents a promising chemotherapeutic
strategy toward the treatment of various types of cancers [2, 11-13].
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In humans, Hsp90 exists as four isoforms: Hsp90α, Hsp90β, glucose-regulated protein 94
kDa (Grp94), and tumor necrosis receptor-associated protein 1 (Trap1) [14]. Hsp90α and Hsp90β
are localized to the cytoplasm, whereas Grp94 resides in the endoplasmic reticulum, and Trap1 is
localized within the mitochondria [15]. Most Hsp90 inhibitors are pan-inhibitors that target both
cytosolic isoforms (Hsp90α and Hsp90β) [16]. Hsp90 predominantly exists as a homodimer and is
composed of three principal domains: the N-terminal ATP-binding domain, the middle domain and
the C-terminal dimerization domain [9, 17]. Over the past decades, a substantial number of Hsp90
inhibitors have been developed, including N-terminal inhibitors and C-terminal inhibitors. Most
Hsp90 inhibitors, such as geldanamycin (1) [18], SNX-5422 (2) [19], radicicol (3) [20] and
17-allylamino-17-demethoxygeldanamycin (17-AAG, 4) [21] are known to interact with the
ATP-binding pocket in the N-terminal domain of Hsp90, whereas KU-174 (5) [22] and novobiocin
(6) [23], have shown effective inhibitory activity by interacting with the ATP-binding pocket in
the C-terminal domain of Hsp90 (Fig. 1). However, none of the Hsp90 inhibitors have been
approved by the U.S. Food and Drug Administration (FDA) for the treatment of cancer, challenges
such as concomitant induction of the pro-survival heat shock response, hepatotoxicity and
multidrug resistance, have impeded their clinical application [13, 24, 25]. Hence, the discovery of
novel Hsp90 inhibitors with better biological activities and fewer side effects is still a demanding
task.
Natural products (NPs) have been prominent in the development of new antitumor agents [11,
26]. However, NPs generally have structurally complex frameworks and high molecular weights,
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leading to synthetic difficulty, unfavorable pharmacokinetic profiles, and poor drug-likeness [26].
Therefore, simplifying complex structures of NPs, while retaining the desired biological activity,
is a valid and meaningful strategy in drug development process. This strategy has been
successfully used in the lead optimization of NPs and yielded a number of marketed drugs and
drug candidates [27-29]. A classic example of the structural simplification of NPs is the
development of simplified morphine-derived analgesics (e.g., butophanol, pentazocine, pethidine
and methadone), in which the complex pentacyclic system of morphine was simplified via the
removal of unnecessary fragments [30].
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Aconitine (7), a diterpenoid alkaloid isolated from the Chinese plants of Aconitum genera
(Ranunculaceae family), has been reported to show effective anticancer properties such as
interfering with the cell cycle, inducing apoptosis, and altering multidrug resistance [31, 32]. Our
previous investigation indicated the potential for aconitum alkaloids as Hsp90 inhibitor for cancer
treatment [33]. However, the structural complexity and natural scarcity hampered the possibility
of its clinical application [34, 35]. Thus, the research for more available aconitine analogs with
concise framework could be a meaningful work. As shown in Fig. 2, most aconitum alkaloids
share the general structures and ring numbering systems. Previous structure-activity relationship
(SAR) studies have indicated that A-E-F ring framework is essential to biological activity of
aconitine-type natural products [34]. Additionally, our team recently illustrated the
structure-toxicity relationship (STR) of aconitum alkaloids, and revealed that hydrophilic groups
substituted at C2 and C3 (ring A), C8 and C15 (ring C) may be related to neurological toxicities
[36]. Analogously, the ester substituents at C14 (ring B) and C8 (ring D) could cause cardio
problems according to Nyirimigabo’s reports [37]. To this end, our design strategy thus focuses on
retaining the E-F ring framework that simplified as a 2-azabicyclo[3.2.1]octane. In addition, the
C1 and C10 methyl group are highly conserved and carbonyl group at C19 plays crucial roles in
biological functions [38], which thus maintained in our design. Based on the deep understanding
of the crystallographic structure of Hsp90 (PDB ID: 2XJX), fragment-based drug design strategy
was carried out with 2-azabicyclo[3.2.1]octan-3-one as a core moiety. Using fragment growing
method, a lipophilic phenyl moiety was introduced into the molecule, which was connected to the
N18 atom of 2-azabicyclo[3.2.1]octan-3-one core through a 1,2,3-triazole. Continuing our
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previous work [39], we reported herein the design and synthesis of a series of
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2-((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)-2-azabicyclo[3.2.1]octan-3-one derivatives (14a-v) as
potent anticancer agents. In addition to the cellular and enzymatic activities evaluations, the
molecular docking study was carried out. Moreover, cell cycle arrest, apoptosis and cell migration
were investigated and we further biologically evaluated anticancer effects through a xenograft
mouse model.
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2. Results and discussion
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2.1. Chemistry
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The
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2-((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)-2-azabicyclo[3.2.1]octan-3-one derivatives (14a-v) is
shown in Scheme 1. Commercially available (1S)-(‒)-camphor (8) was treated with
hydroxylamine to give oxime 9 according to the synthetic protocol described by Kotsikorou [40].
In this procedure, compound 10, as a key intermediate, was produced from the Beckmann
rearrangement of (1S)-(‒)-camphor oxime (9). Briefly, (1S)-(‒)-camphor oxime (9) was activated
with methanesulfonyl chloride to produce sulfonate intermediate, then Beckmann rearrangement
proceeded via the departure of the sulfonate group and migration of the antiperiplanar carbon to
the nitrogen, and tautomerized to camphor lactam 10 [41]. Camphor lactam 10 shared the same
stereo configuration with the corresponding moiety of aconitine. Compound 11 was synthesized
from the camphor lactam 10 by the action of sodium hydride in dry THF followed by addition of
the propargyl bromide. The preparation of various substitute azido benzenes 13a-v was performed
via diazo-reaction and displacement reaction with sodium azide. The 1,3-dipolar cycloaddition of
compound 11 with various azido benzenes 13a-v was subsequently carried out in the presence of
copper (II) sulfate and ascorbic acid to give target compounds 14a-v.
2.2. Biological evaluation
2.2.1.
In vitro cytotoxicity assay and heat shock protein 90 inhibition
In this study, MTT assay was used as a preliminary screen for evaluation of in vitro
cytotoxicity. The antiproliferative effect of all target compounds (14a-v) was evaluated against six
cancer cell lines: A549, OVCAR-3, HepG2, LoVo, PANC-1 and SGC7901. The results were
expressed as IC50 values and summarized in Table 1, and 17-AAG (4), a typical Hsp90 N-terminal
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inhibitor, was used as a reference compound.
As shown in Table 1,
most
of
the
newly
synthesized
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2-((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)-2-azabicyclo[3.2.1]octan-3-one derivatives presented
excellent potency against all six cancer cell lines than 17-AAG, revealing that our designed
skeleton was greatly beneficial to inhibit tumor cell growth. In addition, all target compounds,
except compounds 14a-c, 14p and 14r, had better antiproliferative ability against LoVo cells
compared to other five cancer cell lines. Among these compounds, 14j, 14l, 14m, 14p, 14q, 14t
and 14v showed robust activity against A549, OVCAR-3, HepG2 and LoVo cells. Most
importantly, compound 14t showed potent activities against six cancer cell lines with IC50 values
from 0.02 µM to 0.12 µM, and displayed the best activity against LoVo cells with an IC50 value of
0.02 µM. Compound 14q also exhibited strong antiproliferative activity against A549, OVCAR-3,
HepG2 and LoVo cells, with IC50 values of 0.06 µM, 0.05 µM, 0.06 µM and 0.07 µM,
respectively.
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The structure-activity relationship (SAR) studies revealed that the halogen substituents were
advantageous to anticancer activities. From the results, it seemed that the meta-derivatives usually
possessed higher potency in LoVo cell line than its ortho- and para-derivatives when R group was
a halogen (e.g., 14q versus 14p, 14r; 14t versus 14s, 14u). For the meta-derivatives, when R = F
(14n), Cl (14q) or Br (14t), the antiproliferative activity of compounds was better than the other
groups (e.g., 14b, 14e, 14h and 14k). Particularly, the antiproliferative activity of the bromo
analogs (14s, 14t and 14u) were superior to the chloro analogs (14p, 14q and 14r) and the fluoro
analogs (14m, 14n and 14o) in LoVo cell line. Correspondingly, compound 14t with R group
substituted by a bromo at the meta-position presented excellent anticancer activities.
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On the basis of antiproliferative activity, inhibitory effects of compounds 14j, 14l, 14m, 14p,
14q, 14t and 14v were tested against six normal cell lines: MRC-5, IOSE80, LO2, NCM460,
HPDE6-c7 and GSE-1 to evaluate their toxicity and safety. The results were expressed as CC50
values and recorded in Table 2. Selectivity index (SI) of the drug represents a safe range for
evaluating and judging drug effects [42]. The SI value of the compound was obtained by dividing
CC50 value of the compound against the normal cell, by IC50 value of this compound against the
corresponding cancer cell line (Fig. 3). The larger the index is, the more extensive the safety
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ranges. It can be seen from the Fig. 3, seven candidate compounds showed high drug selectivity
toward liver and colon cancer cell lines. Moreover, compound 14t was the most selective, showing
SI values in the range of 82−527 toward the six cancer cell lines. Besides, in vitro human Hsp90α
inhibitory activity studies were performed on seven compounds and the results were summarized
in Table 3. From the results, compounds 14j, 14m, 14p, 14q and 14t show significant
improvement in Hsp90α inhibitory potency compared to 17-AAG. Especially, compound 14t
exhibited the best Hsp90α inhibition (IC50 = 0.71 nM) among the tested compounds. This result
was consistent with MTT assays.
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In summary, our results indicated that most of the target compounds had excellent
antiproliferative ability against LoVo cells compared to other five cancer cell lines, and the
introduction of halogens was superior to the other groups. Based on selectivity index, seven
candidate compounds displayed the highest drug selectivity for liver and colon cancer cell lines.
Among these compounds, 14t had stronger ability to inhibit both cell viability and Hsp90α
enzyme activity than 17-AAG and had more potential of medicinal research significance on the
discovery of novel Hsp90 inhibitors. With the above consideration, our study mainly focused on
the inhibitory effects of compound 14t on colon cancer cell lines.
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2.2.2.
Molecular docking study
In order to identify the interactions of compound 14t with the amino acid residues of Hsp90,
docking studies were performed using Molecular Operating Environment software (MOE, Version
2015.10). The target protein Hsp90α (PDB ID: 2XJX, Resolution 1.66 Å) was retrieved from
Protein Data Bank (PDB) and used for docking simulation. As illustrated in Fig. 4, compound 14t
occupied a deep ATP-binding pocket of Hsp90α, the O atom of lactam group accepted an H-bond
from the Asn106 and H-bond distance was 2.6 Å, and hydrogen bonding energy component
contributed −1.5 kcal/mol to the binding. The azabicyclo group linked to the amino acid residue
Asp102 via a water bridge. Moreover, the (1S,5S)-1,8,8-trimethyl-2-azabicyclo[3.2.1]octan-3-one
moiety formed hydrophobic interactions with residues Met98, Lys58 and Asp54. In addition,
docking study identified a key hydrophobic interaction between the 1,2,3-triazole moiety and
Asn51. Interestingly, bromine atom formed a crucial halogen bond with carboxyl group of Asp93,
which contributed −5.9 kcal/mol to the binding.
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2.2.3.
Cell cycle assay
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Colon cancer is one of the most common malignancies in the world, approximately 90% of
colon cancer deaths arise from cancer metastasis [43, 44]. To investigate the mechanism of
cellular cytotoxicity, high metastatic LoVo and SW620 cell lines were treated with different
concentrations of compound 14t (0, 20, 40, and 80 nM) for 24 h. The effects of compound 14t on
cell cycle distribution were determined by flow cytometry analysis using propidium iodide (PI)
staining (Fig. 5A). The analysis indicated that, as the concentration of the 14t increased, the cells
in the G0/G1 phase increased, and the cells in the G2/M phase decreased. Eventually, compound
14t induced cycle arrest of LoVo and SW620 cells at the G1/S phase (Fig. 5B), suggesting that
this might be one of the possible mechanisms for cellular cytotoxicity.
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2.2.4.
Cell apoptosis assay
Most chemotherapeutic drugs eventually induce apoptosis in cancer cells when they inhibit
cell cycle progression, thereby achieving antitumor effects [45]. Therefore, to determine whether
the cell cycle arrest caused by the compound 14t could ultimately induce apoptosis, LoVo and
SW620 cells were treated with different concentrations of compound 14t (0, 20, 40, and 80 nM)
for 24 h, then collected and stained with Annexin V-FITC and PI double staining. The apoptotic
effects of compound 14t were determined by flow cytometry analysis. As can be seen from the
Fig. 6A, the apoptotic rate of LoVo cells was 3.2%, 6.1%, 8.6%, and 32.3% for each concentration
respectively. With the increase in concentration, the apoptotic rate increased, of which the late
apoptosis rates were 1.6%, 3.1%, 5.4%, and 24.9%. However, the apoptotic rate of SW620 cells
was 3.0%, 13.6%, 23.2%, and 49.9% for each concentration respectively. With the increase in
concentration, the percentages of apoptotic cell were significantly increased from 3.0% to 49.9%
(Fig. 6B). The late apoptotic rates of SW620 cells were 1.3%, 8.5%, 15.1%, and 29.3%. These
results indicated that compound 14t could induce apoptosis in the two colon cancer cell lines, and
that SW620 cells were more prone to apoptosis than LoVo cells after 14t treatment.
2.2.5.
The effects on apoptosis-related proteins of 14t-treated LoVo and SW620 cells
To elucidate the underlying mechanism of apoptosis induced by 14t, western blot analysis
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was performed to study the expression levels of apoptosis-related proteins. It has been
demonstrated that bcl-2 family proteins play a crucial role in regulating the apoptotic pathway,
including pro-apoptotic bax and anti-apoptotic bcl-2 proteins [46]. Thus, proteins were treated
with 14t at different concentrations (0, 20, 40 and 80 nM), and the results showed that the levels
of bax were markedly increased in the presence of 14t, whereas levels of bcl-2 were decreased. To
further explore the mechanism of apoptosis, we detected the protein expression of
cleaved-casepase 3 and caspase 3. As shown in Fig. 7A, the activation of caspase-3 caused an
increase in cleaved-caspase 3 protein levels, eventually, induced apoptosis.
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2.2.6.
Cell migration and wound healing assays
Cell migration is a vital possession involved in the dissemination of cancerous cells mostly
during cancer metastasis [47]. Herein, we investigated the effects of 14t on the migration of LoVo
and SW620 cells in a transwell assay. From the results, it was clearly observed that 14t preventing
the migration of LoVo and SW620 cells in a dose dependent manner, and the inhibition of SW620
cells migration was more prominent (Fig. 8A and 8B). Wound healing assays also supported the
conclusion that 14t suppressed the migration of LoVo and SW620 cells. The results showed that,
comparison of inhibitory effects on the migration of LoVo and SW620 cells with control group,
the effects of compound 14t were significantly different (P < 0.001). In addition, the effect was
statistically significant after 24 h of incubation at the concentration of 80 nM, in comparison with
control cells, keeping the wound nearly intact in SW620 cell monolayers (Fig. 8B).
2.2.7.
In vivo antitumor effects of compound 14t
To evaluate the in vivo anti-colon cancer activity of compound 14t, BABL/c nude mice were
inoculated subcutaneously with colonic cancer SW620 cells. After the establishment of solid
tumor, two groups of the mice were intraperitoneally treated with 5 mg/kg of 17-AAG and 14t
daily for 21 consecutive days, respectively. There was no significant body weight loss and no
obvious adverse effects were observed during the study (Fig. 9A), indicating that 14t has low
toxicity in mice. Moreover, it could be seen from Fig. 9B and 9C that in comparison with the
vehicle-treated controls, treatment with 14t at 5 mg/kg significantly inhibited the growth of
implanted colon tumors. As depicted in Fig. 9D, compound 14t achieved significant tumor growth
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inhibition with tumor growth inhibition (TGI) value of 81.6%. In contrast, 17-AAG failed to exert
significant tumor growth inhibition (TGI = 29.6%) as compared with 14t.
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Immunohistochemical labeling of tumor tissue was performed using antibodies for cell
proliferation marker (Ki67) and apoptotic cell death (Cleaved-caspase 3). The result was
consistent with the above results that the expression of Ki67 in tumors was significantly decreased
by treatment with 14t. Simultaneously, the expression of cleaved-caspase 3 in tumors was
significantly increased, and 14t-treated group exhibited greater efficacy than the 17-AAG-treated
and control group (Fig. 9E).
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3. Conclusion
In this study, in order to find novel Hsp90 inhibitors,
a
series of
2-((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)-2-azabicyclo[3.2.1]octan-3-one derivatives consisting
of simplified and modified aconitine scaffold was designed, synthesized and tested for their
antiproliferative ability against six cancer cell lines. Among these compounds, 14t was identified
to be the most promising one with excellent antiproliferative effects against LoVo cells (IC50
=
0.02 µM) and significant Hsp90α inhibitory activities (IC50 = 0.71 nM). Molecular docking studies
were employed to support the experimental results and the key interactions were identified. Flow
cytometry analysis indicated that compound 14t could induce cell cycle arrest at the G1/S phase.
Additionally, Annexin V-FITC assay and western blot analysis revealed that 14t inhibited
proliferation of cancer cells by inducing apoptosis via up-regulating bax, cleaved-caspase 3, and
down-regulating bcl-2 protein expression levels. Consistent with the in vitro results, treatment
with 14t effectively suppressed the volumes and size of colon tumors in mice. Overall, our work
successfully identified a series of novel Hsp90 inhibitors derived from aconitine through structural
simplification and modification. Compound 14t, as a promising lead, is worthy for the further
investigation.
4. Experimental
4.1. Chemistry
All reagents and solvents were purchased from commercial sources and dried prior to use
unless noted otherwise. Reactions were monitored by thin-layer chromatography on 0.25 mm
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silica gel plates (GF254) and visualized under UV light. Melting points were determined with an
electro thermal melting point apparatus, are uncorrected. Optical rotations were measured by
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WZZ-3 polarimeter in the solvent indicated. H NMR and 13C NMR spectra were recorded at
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23 °C in CDCl3 on a Bruker spectrometer using TMS as the internal standard. Chemical shifts
were reported as δ (ppm) and signal splitting patterns were described as singlet (s), doublet (d),
triplet (t), quartet (q), quintet (quint), or multiplet (m), with coupling constants (J) in hertz.
High-resolution mass spectra (HRMS) were obtained on an electron spray injection (ESI) Bruker
micrOTOF-Q mass spectrometer. The purity of the target compounds was confirmed by HPLC
analysis performed on Waters BDS C18 (200 mm ×4.6 mm, 5 µm) eluting at 1.0 mL/min of
methanol and water (50:50 v/v).
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4.1.1.
Synthesis of (1S) -(‒)-camphor oxime (9)
A solution of (1S)-(‒)-camphor (8) (3.0 g, 19.7 mmol), hydroxylamine hydrochloride (2.1 g,
30.2 mmol) in water (11 mL) was heated at 80 °C under nitrogen. To the stirred suspension was
added MeOH (16 mL) followed by a solution of sodium acetate (3.9 g, 47.5 mmol) in water (8
mL). The reaction mixture was heated at reflux under nitrogen for 9 h, the methanol was removed
in vacuo, and the aqueous suspension was filtered. The filtered solid was washed with water,
recrystallized from 95% EtOH, and dried to get (1S)-(‒)-camphor oxime (9) as a white solid
1
(2.33g, 71% yield). Mp: 117-119 °C. H NMR (600 MHz, CDCl3) δ 8.13 (s, 1H), 2.56 (dt, J =
17.8, 3.8 Hz, 1H), 2.06 (d, J = 17.8 Hz, 1H), 1.93 (t, J = 4.4 Hz, 1H), 1.85 (ddd, J = 12.4, 8.1, 3.9
Hz, 1H), 1.71 (td, J = 12.2, 4.2 Hz, 1H), 1.49 – 1.44 (m, 1H), 1.27 – 1.21 (m, 1H), 1.01 (s, 3H),
0.93 (s, 3H), 0.81 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 169.96, 51.86, 48.35, 43.72, 33.11,
32.75, 27.73, 19.45, 18.64, 11.19.
4.1.2.
Synthesis of (1S,5S)-1,8,8-trimethyl-2-azabicyclo[3.2.1]octan-3-one (10)
To a stirred solution of (1S)-(‒)-camphor oxime (9) (5.38 g, 32.20 mmol) in pyridine (25 mL)
at −22 °C, then methanesulfonyl chloride (5.0 mL, 65.10 mmol) was added dropwise. The reaction
mixture was stirred at −22 °C for 3 h and then the temperature was raised. After 2 h of additional
stirring at room temperature, the mixture was poured onto iced water and extracted with DCM (3
× 50 mL). The combined organic extracts were washed with sodium bicarbonate and brine,
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concentrated under reduced pressure. The residue was purified by flash chromatography on silica
gel (chloroform-acetone, 3:1) to get camphor lactam 10 as a white solid (0.39 g, 7.2% yield). Mp:
1
3
196-197 °C. H NMR (600 MHz, CDCl3) δ 6.77 (s, 1H), 2.60 (ddd, J = 18.1, 4.7, 2.5 Hz, 1H),
4
2.17 (dd, J = 18.1, 1.6 Hz, 1H), 2.08 – 2.01 (m, 1H), 2.01 – 1.96 (m, 1H), 1.95 – 1.88 (m, 2H),
1.54 – 1.47 (m, 1H), 1.13 (s, 3H), 1.02 (s, 3H), 0.97 (s, 3H). 13C NMR (150 MHz, CDCl3) δ
172.69, 63.65, 43.44, 42.84, 40.11, 39.44, 27.77, 23.44, 18.43, 17.95.
5
6
7
8
4.1.3.
Synthesis of (1S,5S)-1,8,8-trimethyl-2-(prop-2-yn-1-yl)-2-azabicyclo[3.2.1]octan-3-one
9
(11)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
To a stirred solution of (1S,5S)-1,8,8-trimethyl-2-azabicyclo[3.2.1]octan-3-one (10) (0.65 g,
3.89 mmol) in dry THF (20 mL) at 0 °C was added NaH (0.11 g, 4.66 mmol). After being stirred
for 1 h, propargyl bromide (0.51 g, 4.27 mmol) was added, and then the solution was stirred for 4
h at room temperature. The mixture was quenched with saturated water and extracted with DCM
(3 × 20 mL). The combined organic extracts were washed with brine, dried over Na2SO4, filtered,
and concentrated to give the crude product. The crude product was purified by flash
chromatography on silica gel (petroleum ether-EtOAc, 3:1) to get compound 11 as a grey solid
1
(0.33 g, 41% yield). Mp: 183-187 °C. H NMR (600 MHz, CDCl3) δ 4.32 (dd, J = 17.7, 2.1 Hz,
1H), 4.01 (dd, J = 17.7, 2.1 Hz, 1H), 3.08 (t, J = 2.4 Hz, 1H), 2.56 – 2.51 (m, 1H), 2.16 – 2.05 (m,
2H), 1.97 (tdd, J = 11.2, 5.7, 3.2 Hz, 1H), 1.85 (t, J = 5.4 Hz, 1H), 1.80 (ddd, J = 13.5, 12.0, 5.2
Hz, 1H), 1.41 – 1.35 (m, 1H), 1.32 (s, 3H), 0.93 (s, 3H), 0.89 (s, 3H). 13C NMR (150 MHz, CDCl3)
δ 169.52, 82.14, 73.42, 69.11, 44.69, 42.46, 38.29, 30.42, 28.39, 25.01 (2C), 18.26, 17.02.
4.1.4.
Synthesis of 1-azido-2-hydroxybenzene (13a)
To a solution of 2-hydroxyaniline (12a) (1.0 g, 9.16 mmol) in DCM (15 mL) at 0 °C was
added concentrated hydrochloride (37%, 1.08 g, 11.0 mmol), followed by a solution of NaNO2
(0.76 g, 11.0 mmol) in 10mL H2O by consecutive dropwise addition over a period of 10 min with
stirring. After stirring for a further 1 h, a solution of NaN3 (0.77g, 11.91 mmol) in H2O (5 mL) was
added to the above reaction mixture with stirring. The reaction mixture was warm to room
temperature and stirred for 4 h. Then the solution was poured into H2O, extracted with DCM (3 ×
20 mL), dried over Na2SO4, filtered and concentrated in vacuo to give 13a as yellow oil (1.20 g,
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2
3
4
97% yield), which was used for the next step without further purification.
Compounds 13b-v were prepared with the same method and the appropriate substituted
aniline.
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6
4.1.5.
Synthesis of (1S,5S)-2-((1-(2-hydroxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-1,8,8-trimet
hyl-2-azabicyclo[3.2.1]octan-3-one (14a)
7
To a stirred solution of 1-azido-2-hydroxybenzene (13a) (180 mg, 1.65 mmol) in tBuOH (10
mL), the water solution (10 mL) of copper sulfate pentahydrate (37 mg, 0.15 mmol) and ascorbic
acid (79 mg, 0.45 mmol) was added, and then (1S,5S)-1,8,8-trimethyl-2-(prop-2-yn-1-yl)
-2-azabicyclo[3.2.1]octan-3-one (11) (287 mg, 1.5 mmol) was added. The reaction mixture was
stirred at 60 °C for 8 h and then was concentrated in vacuo, diluted with H2O and extracted with
EtOAc (3 × 15 mL). The combined organic phases were dried over anhydrous Na2SO4, filtered,
and concentrated under reduced pressure. The residue was purified by flash chromatography on
silica gel (petroleum ether-EtOAc, 1:1) to give the target compound 14a.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
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30
Target compounds 14b-v were prepared with the same method and the corresponding
substitute azide benzene (13b-v).
4.1.6.
(1S,5S)-2-((1-(2-hydroxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-1,8,8-trimethyl-2-azabicy
25
clo[3.2.1]octan-3-one (14a). A grey oil, yield: 72%. [α]D = + 23.4 (c = 1.0 in EtOH). 1H NMR
(600 MHz, CDCl3) δ 10.12 (s, 1H), 8.25 (s, 1H), 7.49 (dd, J = 8.1, 1.4 Hz, 1H), 7.28 (dd, J = 6.9,
1.3 Hz, 1H), 7.17 (dd, J = 8.2, 1.2 Hz, 1H), 7.00 – 6.96 (m, 1H), 4.81 (d, J = 15.3 Hz, 1H), 4.74 (d,
J = 15.3 Hz, 1H), 2.73 (ddd, J = 18.2, 4.8, 2.5 Hz, 1H), 2.27 (dd, J = 18.1, 1.3 Hz, 1H), 2.04 (dd, J
= 6.3, 3.8 Hz, 1H), 1.92 – 1.86 (m, 2H), 1.85 (dd, J = 11.6, 5.7 Hz, 1H), 1.45 (s, 3H), 1.43 (dd, J =
9.2, 5.7 Hz, 1H), 0.98 (s, 3H), 0.92 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 171.92, 149.18, 145.50,
129.55, 122.99, 122.05, 120.20, 120.13, 119.03, 69.94, 44.69, 42.37, 39.94, 38.77, 37.45, 28.31,
25.05, 18.19, 17.50. ESI-HRMS calcd for C19H25N4O2 [M + H] + 341.19775, found: 341.19712.
HPLC purity of 98.831% (retention time = 2.422).
4.1.7.
(1S,5S)-2-((1-(3-hydroxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-1,8,8-trimethyl-2-azabicy
clo[3.2.1]octan-3-one (14b). A brown oil, yield: 76%. [α]D25 = + 43.9 (c = 1.0 in EtOH). 1H NMR
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(600 MHz, CDCl3) δ 10.25 (s, 1H), 8.17 (s, 1H), 7.88 (t, J = 2.1 Hz, 1H), 7.34 (t, J = 8.1 Hz, 1H),
7.20 (dd, J = 7.9, 1.4 Hz, 1H), 6.97 (dd, J = 8.2, 1.8 Hz, 1H), 4.85 (d, J = 15.3 Hz, 1H), 4.73 (d, J
= 15.2 Hz, 1H), 2.76 (ddd, J = 18.2, 4.8, 2.4 Hz, 1H), 2.35 – 2.28 (m, 1H), 2.07 – 2.01 (m, 1H),
1.93 – 1.88 (m, 2H), 1.84 (ddd, J = 13.8, 12.0, 5.4 Hz, 1H), 1.47 (dd, J = 9.4, 4.0 Hz, 1H), 1.44 (s,
3H), 0.97 (s, 3H), 0.91 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 172.35, 158.64, 145.93, 137.62,
130.62, 121.78, 116.44, 110.50, 107.68, 70.15, 44.67, 42.27, 39.90, 38.71, 37.54, 28.31, 25.02,
18.16, 17.40. ESI-HRMS calcd for C19H25N4O2 [M + H] + 341.19775, found: 341.19748. HPLC
purity of 98.047% (retention time = 2.814).
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
4.1.8.
(1S,5S)-2-((1-(4-hydroxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-1,8,8-trimethyl-2-azabicy
25
1
clo[3.2.1]octan-3-one (14c). A grey oil, yield: 74%. [α]D = + 67.2 (c = 1.0 in EtOH). H NMR
(600 MHz, CDCl3) δ 9.89 (s, 1H), 7.93 (s, 1H), 7.46 (d, J = 8.8 Hz, 2H), 6.98 (d, J = 8.8 Hz, 2H),
4.76 (dd, J = 38.6, 15.2 Hz, 2H), 2.81 – 2.73 (m, 1H), 2.31 (d, J = 18.2 Hz, 1H), 2.05 (s, 1H), 1.95
(ddd, J = 16.4, 9.7, 4.8 Hz, 2H), 1.91 – 1.84 (m, 1H), 1.52 (s, 3H), 1.49 – 1.42 (m, 1H), 1.00 (s,
3H), 0.93 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 172.65, 158.13, 145.36, 129.36, 121.93, 121.90
(2C), 116.41 (2C), 70.32, 44.72, 42.23, 39.83, 38.92, 37.75, 28.39, 25.07, 18.19, 17.49.
ESI-HRMS calcd for C19H25N4O2 [M + H] + 341.19775, found: 341.19700. HPLC purity of 97.913%
(retention time = 2.325).
4.1.9.
(1S,5S)-1,8,8-trimethyl-1-((2-(o-tolyl)-1H-1,2,3-triazol-4-yl)methyl)-2-azabicyclo[3.2.1]
octan-3-one (14d). A grey oil, yield: 79%. [α]D25 = + 28.4 (c = 1.0 in EtOH). 1H NMR (600 MHz,
CDCl3) δ 7.79 (s, 1H), 7.41 – 7.37 (m, 1H), 7.35 (d, J = 7.4 Hz, 1H), 7.32 (dd, J = 3.9, 1.2 Hz, 2H),
4.85 (d, J = 15.3 Hz, 1H), 4.74 (d, J = 15.3 Hz, 1H), 2.72 (ddd, J = 18.1, 4.9, 2.5 Hz, 1H), 2.25 (dd,
J = 18.1, 1.4 Hz, 1H), 2.18 (s, 3H), 2.04 (ddd, J = 9.3, 4.5, 2.4 Hz, 1H), 1.91 (t, J = 5.3 Hz, 1H),
1.89 – 1.80 (m, 2H), 1.45 (s, 3H), 1.44 – 1.41 (m, 1H), 0.98 (s, 3H), 0.92 (s, 3H). 13C NMR (150
MHz, CDCl3) δ 171.40, 145.78, 136.56, 133.52, 131.38, 129.71, 126.77, 125.95, 124.95, 69.71,
44.64, 42.49, 40.06, 38.70, 37.55, 28.34, 25.05, 18.14, 17.84, 17.53. ESI-HRMS calcd for
C20H27N4O [M + H] + 339.21849, found: 339.21812. HPLC purity of 96.178% (retention time =
2.322).
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4.1.10. (1S,5S)-1,8,8-trimethyl-1-((2-(m-tolyl)-1H-1,2,3-triazol-4-yl)methyl)-2-azabicyclo[3.2.1
]octan-3-one (14e). A grey oil, yield: 81%. [α]D25 = + 30.9 (c = 1.0 in EtOH). 1H NMR (600 MHz,
CDCl3) δ 8.06 (s, 1H), 7.57 (s, 1H), 7.54 (d, J = 8.0 Hz, 1H), 7.37 (t, J = 7.8 Hz, 1H), 7.22 (d, J =
7.5 Hz, 1H), 4.76 (dd, J = 40.8, 15.2 Hz, 2H), 2.73 (dd, J = 18.1, 1.9 Hz, 1H), 2.43 (s, 3H), 2.26 (d,
J = 18.0 Hz, 1H), 2.04 (s, 1H), 1.92 – 1.80 (m, 3H), 1.49 – 1.41 (m, 4H), 0.97 (s, 3H), 0.92 (s, 3H).
13C NMR (150 MHz, CDCl3) δ 171.52, 146.39, 139.84, 136.99, 129.44, 129.27, 121.51, 120.89,
117.35, 69.72, 44.64, 42.46, 40.03, 38.70, 37.52, 28.31, 25.05, 21.37, 18.18, 17.51. ESI-HRMS
calcd for C20H27N4O [M + H] + 339.21849, found: 339.21807. HPLC purity of 98.729% (retention
time = 2.325).
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4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
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30
4.1.11. (1S,5S)-1,8,8-trimethyl-1-((2-(p-tolyl)-1H-1,2,3-triazol-4-yl)methyl)-2-azabicyclo[3.2.1]
octan-3-one (14f). A brown oil, yield: 89%. [α]D25 = + 32.4 (c = 1.0 in EtOH). 1H NMR (600 MHz,
CDCl3) δ 8.04 (s, 1H), 7.62 (d, J = 8.1 Hz, 2H), 7.29 (d, J = 7.9 Hz, 2H), 4.75 (dd, J = 42.2, 15.2
Hz, 2H), 2.73 (d, J = 18.0 Hz, 1H), 2.41 (s, 3H), 2.26 (d, J = 18.1 Hz, 1H), 2.02 (d, J = 4.4 Hz,
1H), 1.94 – 1.86 (m, 2H), 1.83 (dd, J = 12.1, 5.1 Hz, 1H), 1.48 – 1.39 (m, 4H), 0.97 (s, 3H), 0.92
(s, 3H). 13C NMR (150 MHz, CDCl3) δ 171.54, 146.34, 138.61, 134.79, 130.14 (2C), 121.45,
120.19 (2C), 69.73, 44.65, 42.46, 40.03, 38.71, 37.54, 28.32, 25.06, 21.07, 18.18, 17.52.
ESI-HRMS calcd for C20H27N4O [M + H] + 339.21849, found: 339.21826. HPLC purity of 98.181%
(retention time = 2.327).
4.1.12. (1S,5S)-2-((1-(2-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-1,8,8-trimethyl-2-azabic
yclo[3.2.1]octan-3-one (14g). A grey oil, yield: 84%. [α]D25 = + 23.8 (c = 1.0 in EtOH). 1H NMR
(600 MHz, CDCl3) δ 8.10 (s, 1H), 7.74 (dd, J = 7.9, 1.6 Hz, 1H), 7.43 – 7.39 (m, 1H), 7.10 – 7.04
(m, 2H), 4.85 (d, J = 15.3 Hz, 1H), 4.75 (d, J = 15.3 Hz, 1H), 3.85 (s, 3H), 2.73 (ddd, J = 18.1, 4.9,
2.5 Hz, 1H), 2.26 (dd, J = 18.1, 1.3 Hz, 1H), 2.04 (s, 1H), 1.92 – 1.88 (m, 2H), 1.81 (ddd, J = 13.8,
12.0, 5.4 Hz, 1H), 1.47 – 1.43 (m, 1H), 1.42 (s, 3H), 0.97 (s, 3H), 0.95 (s, 3H). 13C NMR (150
MHz, CDCl3) δ 171.34, 151.16, 145.32, 130.00, 126.38, 125.44 (2C), 121.05, 112.12, 69.65,
55.90, 44.66, 42.49, 40.08, 38.63, 37.60, 28.36, 25.07, 18.13, 17.49. ESI-HRMS calcd for
C20H27N4O2 [M + H] + 355.21340, found: 355.21283. HPLC purity of 95.873% (retention time =
2.345).
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4.1.13. (1S,5S)-2-((1-(3-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-1,8,8-trimethyl-2-azabic
yclo[3.2.1]octan-3-one (14h). A grey oil, yield: 86%. [α]D25 = + 47.1 (c = 1.0 in EtOH). 1H NMR
(600 MHz, CDCl3) δ 8.07 (s, 1H), 7.39 (t, J = 8.1 Hz, 1H), 7.35 (s, 1H), 7.29 (t, J = 8.8 Hz, 1H),
6.97 – 6.92 (m, 1H), 4.76 (dd, J = 43.5, 15.2 Hz, 2H), 3.87 (s, 3H), 2.73 (dd, J = 18.0, 1.9 Hz, 1H),
2.26 (d, J = 18.1 Hz, 1H), 2.06 – 2.00 (m, 1H), 1.92 – 1.80 (m, 3H), 1.47 – 1.41 (m, 4H), 0.98 (s,
3H), 0.93 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 171.48, 160.51, 146.46, 138.07, 130.42, 121.53,
114.58, 112.20, 105.87, 69.71, 55.62, 44.64, 42.45, 40.03, 38.70, 37.49, 28.31, 25.05, 18.18, 17.51.
ESI-HRMS calcd for C20H27N4O2 [M + H] + 355.21340, found: 355.21268. HPLC purity of 94.876%
(retention time = 2.362).
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5
6
7
8
9
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12
13
14
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17
18
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30
4.1.14. (1S,5S)-2-((1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-1,8,8-trimethyl-2-azabic
yclo[3.2.1]octan-3-one (14i). A grey oil, yield: 81%. [α]D25 = + 27.6 (c = 1.0 in EtOH). 1H NMR
(600 MHz, CDCl3) δ 7.99 (s, 1H), 7.64 (d, J = 9.0 Hz, 2H), 7.00 (d, J = 9.0 Hz, 2H), 4.78 (d, J =
15.2 Hz, 1H), 4.71 (d, J = 15.2 Hz, 1H), 3.86 (s, 3H), 2.73 (ddd, J = 18.1, 4.9, 2.5 Hz, 1H), 2.26
(dd, J = 18.1, 1.5 Hz, 1H), 2.05 (s, 1H), 1.92 – 1.86 (m, 2H), 1.82 (ddd, J = 13.8, 11.8, 5.4 Hz,
1H), 1.45 (s, 3H), 1.43 (dd, J = 9.4, 5.3 Hz, 1H), 0.98 (s, 3H), 0.93 (s, 3H). 13C NMR (150 MHz,
CDCl3) δ 171.49, 159.64, 146.33, 130.59, 121.90 (2C), 121.59, 114.67 (2C), 69.72, 55.61, 44.66,
42.48, 40.05, 38.71, 37.55, 28.32, 25.06, 18.19, 17.53. ESI-HRMS calcd for C20H27N4O2 [M + H]
+ 355.21340, found: 355.21291. HPLC purity of 98.480% (retention time = 2.422).
4.1.15. 2-(4-(((1S,5S)-1,8,8-trimethyl-3-oxo-2-azabicyclo[3.2.1]octan-2-yl)methyl)-1H-1,2,3-tri
azol-1-yl)benzonitrile (14j). A brown oil, yield: 64%. [α]D25 = + 32.9 (c = 1.0 in EtOH). 1H NMR
(600 MHz, CDCl3) δ 8.20 (s, 1H), 7.86 (d, J = 7.9 Hz, 1H), 7.80 (d, J = 2.9 Hz, 2H), 7.61 (ddd, J
= 8.5, 5.5, 3.2 Hz, 1H), 4.87 (d, J = 15.4 Hz, 1H), 4.74 (d, J = 15.4 Hz, 1H), 2.74 (ddd, J = 18.1,
4.6, 2.3 Hz, 1H), 2.27 (d, J = 18.1 Hz, 1H), 2.05 (s, 1H), 1.94 – 1.89 (m, 2H), 1.88 – 1.82 (m, 1H),
1.53 – 1.46 (m, 1H), 1.44 (s, 3H), 0.98 (d, J = 10.6 Hz, 6H). 13C NMR (150 MHz, CDCl3) δ
171.83, 146.77, 138.58, 134.26, 134.23, 129.59, 125.50, 124.00, 115.50, 107.19, 69.86, 44.69,
42.36, 39.95, 38.62, 37.56, 28.27, 25.04, 18.20, 17.46. ESI-HRMS calcd for C20H24N5O [M + H]+
350.19809, found: 350.19768. HPLC purity of 96.157% (retention time = 2.422).
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4.1.16. 3-(4-(((1S,5S)-1,8,8-trimethyl-3-oxo-2-azabicyclo[3.2.1]octan-2-yl)methyl)-1H-1,2,3-tri
azol-1-yl)benzonitrile (14k). A brown oil, yield: 70%. [α]D25 = + 23.7 (c = 1.0 in EtOH). 1H NMR
(600 MHz, CDCl3) δ 8.22 (s, 1H), 8.15 (s, 1H), 8.05 (d, J = 8.1 Hz, 1H), 7.71 (d, J = 7.7 Hz, 1H),
7.67 (t, J = 7.9 Hz, 1H), 4.80 (d, J = 15.2 Hz, 1H), 4.71 (d, J = 15.3 Hz, 1H), 2.73 (ddd, J = 18.1,
4.6, 2.3 Hz, 1H), 2.27 (d, J = 18.0 Hz, 1H), 2.05 (s, 1H), 1.92 (t, J = 5.2 Hz, 1H), 1.90 – 1.80 (m,
2H), 1.46 (s, 3H), 1.44 (dd, J = 9.3, 5.5 Hz, 1H), 0.99 (s, 3H), 0.93 (s, 3H). 13C NMR (150 MHz,
CDCl3) δ 171.86, 146.99, 137.58, 131.82, 130.81, 124.18, 123.49, 121.64, 117.49, 114.02, 69.91,
44.67, 42.39, 39.94, 38.77, 37.49, 28.30, 25.06, 18.19, 14.20. ESI-HRMS calcd for C20H24N5O [M
+ H] + 350.19809, found: 350.19763. HPLC purity of 97.223% (retention time = 2.542).
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6
7
8
9
10
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13
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30
4.1.17. 4-(4-(((1S,5S)-1,8,8-trimethyl-3-oxo-2-azabicyclo[3.2.1]octan-2-yl)methyl)-1H-1,2,3-tri
25
1
azol-1-yl)benzonitrile (14l). A brown oil, yield: 67%. [α]D = +27.8 (c = 1.0 in EtOH). H NMR
(600 MHz, CDCl3) δ 8.19 (s, 1H), 7.95 (d, J = 8.6 Hz, 2H), 7.84 (d, J = 8.6 Hz, 2H), 4.80 (d, J =
15.3 Hz, 1H), 4.71 (d, J = 15.3 Hz, 1H), 2.72 (ddd, J = 18.0, 4.6, 2.3 Hz, 1H), 2.26 (d, J = 17.9 Hz,
1H), 2.05 (s, 1H), 1.94 – 1.91 (m, 1H), 1.90 – 1.80 (m, 2H), 1.46 (s, 3H), 1.43 (dd, J = 9.2, 5.5 Hz,
1H), 0.99 (s, 3H), 0.93 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 171.56, 147.19, 139.84, 133.86
(2C), 121.45, 120.37 (2C), 117.79, 112.09, 69.78, 44.65, 42.41, 40.01, 38.72, 37.40, 28.29, 25.06,
18.18, 17.55. ESI-HRMS calcd for C20H24N5O [M + H] + 350.19809, found: 350.19793. HPLC
purity of 98.325% (retention time = 2.362).
4.1.18. (1S,5S)-2-((1-(2-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-1,8,8-trimethyl-2-azabicycl
o[3.2.1]octan-3-one (14m). A yellow oil, yield: 75%. [α]D25 = + 25.8 (c = 1.0 in EtOH). 1H NMR
(600 MHz, CDCl3) δ 8.11 (d, J = 2.7 Hz, 1H), 7.91 (td, J = 7.9, 1.1 Hz, 1H), 7.45 – 7.39 (m, 1H),
7.34 – 7.26 (m, 2H), 4.86 (d, J = 15.4 Hz, 1H), 4.73 (d, J = 15.3 Hz, 1H), 2.74 (ddd, J = 18.1, 4.6,
2.3 Hz, 1H), 2.27 (d, J = 18.0 Hz, 1H), 2.04 (s, 1H), 1.94 – 1.88 (m, 2H), 1.87 – 1.81 (m, 1H),
1.46 (dd, J = 9.5, 4.9 Hz, 1H), 1.43 (s, 3H), 0.98 (s, 3H), 0.94 (s, 3H). 13C NMR (150 MHz,
CDCl3) δ 171.54, 153.38 (d, J = 251.4 Hz), 146.19, 130.13 (d, J = 7.8 Hz), 125.29 (d, J = 10.3 Hz),
125.08 (d, J = 3.8 Hz), 124.84, 124.56 (d, J = 7.8 Hz), 116.96 (d, J = 20.0 Hz), 69.70, 44.62, 42.39,
39.95, 38.63, 37.46, 28.27, 25.02, 18.10, 17.46. ESI-HRMS calcd for C19H24FN4O [M + H] +
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343.19341, found: 343.19314. HPLC purity of 97.942% (retention time = 2.364).
3
4.1.19. (1S,5S)-2-((1-(3-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-1,8,8-trimethyl-2-azabicycl
25
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o[3.2.1]octan-3-one (14n). A grey oil, yield: 78%. [α]D = + 30.4 (c = 1.0 in EtOH). H NMR
(600 MHz, CDCl3) δ 8.15 (s, 1H), 7.61 – 7.54 (m, 2H), 7.48 (dt, J = 14.1, 7.2 Hz, 1H), 7.12 (td, J
= 8.2, 1.8 Hz, 1H), 4.80 (d, J = 15.3 Hz, 1H), 4.72 (d, J = 15.2 Hz, 1H), 2.73 (ddd, J = 18.0, 4.5,
2.2 Hz, 1H), 2.26 (d, J = 18.1 Hz, 1H), 2.04 (s, 1H), 1.93 – 1.87 (m, 2H), 1.86 – 1.81 (m, 1H),
1.46 (s, 3H), 1.43 (dd, J = 9.5, 5.3 Hz, 1H), 0.98 (s, 3H), 0.93 (s, 3H). 13C NMR (150 MHz, CDCl3)
δ 171.63, 162.95 (d, J = 248.2 Hz), 146.56, 138.13 (d, J = 10.2 Hz), 131.07 (d, J = 9.0 Hz), 121.53,
115.50 (d, J = 3.2 Hz), 115.32 (d, J = 21.1 Hz), 107.92 (d, J = 26.3 Hz), 69.75, 44.58, 42.34, 39.90,
38.65, 37.39, 28.23, 24.97, 18.10, 17.43. ESI-HRMS calcd for C19H24FN4O [M + H] + 343.19341,
found: 343.19286. HPLC purity of 96.725% (retention time = 2.538).
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4.1.20. (1S,5S)-2-((1-(4-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-1,8,8-trimethyl-2-azabicycl
25
1
o[3.2.1]octan-3-one (14o). A grey oil, yield: 75%. [α]D = + 35.1 (c = 1.0 in EtOH). H NMR
(600 MHz, CDCl3) δ 8.07 (s, 1H), 7.77 – 7.71 (m, 2H), 7.24 – 7.17 (m, 2H), 4.79 (d, J = 15.3 Hz,
1H), 4.71 (d, J = 15.3 Hz, 1H), 2.73 (ddd, J = 18.1, 4.8, 2.5 Hz, 1H), 2.26 (dd, J = 18.1, 1.3 Hz,
1H), 2.04 (s, 1H), 1.91 (d, J = 5.0 Hz, 1H), 1.90 – 1.82 (m, 2H), 1.46 (s, 3H), 1.43 (dd, J = 9.3, 5.2
13
Hz, 1H), 0.98 (s, 3H), 0.93 (s, 3H). C NMR (150 MHz, CDCl3) δ 171.54, 162.21 (d, J = 248.7
Hz), 146.52, 133.32 (d, J = 3.0 Hz), 122.19 (d, J = 8.5 Hz, 2C), 121.69, 116.54 (d, J = 23.2 Hz,
2C), 69.72, 44.60, 42.39, 39.96, 38.67, 37.43, 28.26, 25.00, 18.13, 17.47. ESI-HRMS calcd for
C19H24FN4O [M + H] + 343.19341, found: 343.19282. HPLC purity of 98.306% (retention time =
2.416).
4.1.21. (1S,5S)-2-((1-(2-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-1,8,8-trimethyl-2-azabicyc
lo[3.2.1]octan-3-one (14p). A yellow oil, yield: 72%. [α]D25 = + 17.7 (c = 1.0 in EtOH). 1H NMR
(600 MHz, CDCl3) δ 8.00 (s, 1H), 7.61 – 7.58 (m, 1H), 7.57 – 7.54 (m, 1H), 7.46 – 7.41 (m, 2H),
4.87 (d, J = 15.3 Hz, 1H), 4.74 (d, J = 15.3 Hz, 1H), 2.73 (ddd, J = 18.1, 4.8, 2.4 Hz, 1H), 2.26 (d,
J = 18.0 Hz, 1H), 2.08 – 2.02 (m, 1H), 1.93 – 1.88 (m, 2H), 1.84 (dd, J = 11.9, 5.4 Hz, 1H), 1.45
(dd, J = 9.4, 5.1 Hz, 1H), 1.43 (s, 3H), 0.98 (s, 3H), 0.94 (s, 3H). 13C NMR (150 MHz, CDCl3) δ
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171.39, 145.82, 134.96, 130.69, 130.64, 128.72, 127.82, 127.76, 125.39, 69.68, 44.62, 42.43,
40.04, 38.59, 37.53, 28.30, 25.03, 18.14, 17.48. ESI-HRMS calcd for C19H24ClN4O [M + H] +
359.16386, found: 359.16345. HPLC purity of 98.440% (retention time = 2.378).
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4.1.22. (1S,5S)-2-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-1,8,8-trimethyl-2-azabicyc
lo[3.2.1]octan-3-one (14q). A yellow oil, yield: 79%. [α]D25 = + 32.5 (c = 1.0 in EtOH). 1H NMR
(600 MHz, CDCl3) δ 8.11 (s, 1H), 7.82 (s, 1H), 7.66 (d, J = 8.0 Hz, 1H), 7.44 (t, J = 8.0 Hz, 1H),
7.39 (d, J = 8.0 Hz, 1H), 4.79 (d, J = 15.2 Hz, 1H), 4.71 (d, J = 15.1 Hz, 1H), 2.76 – 2.70 (m, 1H),
2.26 (d, J = 18.1 Hz, 1H), 2.05 (s, 1H), 1.93 – 1.82 (m, 3H), 1.45 (s, 3H), 1.43 (dd, J = 9.4, 5.5 Hz,
1H), 0.98 (s, 3H), 0.92 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 171.66, 146.68, 137.88, 135.47,
130.75, 128.57, 121.57, 120.53, 118.21, 69.80, 44.65, 42.43, 40.00, 38.73, 37.46, 28.30, 25.05,
18.18, 17.52. ESI-HRMS calcd for C19H24ClN4O [M + H] + 359.16386, found: 359.16311. HPLC
purity of 96.760% (retention time = 2.329).
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4.1.23. (1S,5S)-2-((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-1,8,8-trimethyl-2-azabicyc
lo[3.2.1]octan-3-one (14r). A yellow oil, yield: 77%. [α]D25 = + 24.6 (c = 1.0 in EtOH). 1H NMR
(600 MHz, CDCl3) δ 8.06 (s, 1H), 7.70 (d, J = 8.8 Hz, 2H), 7.48 (d, J = 8.8 Hz, 2H), 4.78 (d, J =
15.3 Hz, 1H), 4.70 (d, J = 15.1 Hz, 1H), 2.72 (ddd, J = 18.1, 4.8, 2.5 Hz, 1H), 2.25 (dd, J = 18.1,
1.2 Hz, 1H), 2.03 (dd, J = 6.9, 4.6 Hz, 1H), 1.92 – 1.82 (m, 3H), 1.45 (s, 3H), 1.43 (dd, J = 9.2,
5.3 Hz, 1H), 0.98 (s, 3H), 0.92 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 171.52, 146.76, 135.58,
134.25, 129.83 (2C), 121.49, 121.45 (2C), 69.75, 44.67, 42.47, 40.05, 38.73, 37.48, 28.32, 25.08,
18.20, 17.56. ESI-HRMS calcd for C19H24ClN4O [M + H] + 359.16386, found: 359.16348. HPLC
purity of 98.876% (retention time = 2.336).
4.1.24. (1S,5S)-2-((1-(2-bromophenyl)-1H-1,2,3-triazol-4-yl)methyl)-1,8,8-trimethyl-2-azabicyc
lo[3.2.1]octan-3-one (14s). A yellow oil, yield: 83%. [α]D25 = + 22.7 (c = 1.0 in EtOH). 1H NMR
(600 MHz, CDCl3) δ 7.96 (s, 1H), 7.73 (dd, J = 8.1, 1.2 Hz, 1H), 7.53 (dd, J = 7.9, 1.6 Hz, 1H),
7.47 (td, J = 7.7, 1.3 Hz, 1H), 7.38 (td, J = 7.8, 1.7 Hz, 1H), 4.87 (d, J = 15.4 Hz, 1H), 4.74 (d, J =
15.3 Hz, 1H), 2.73 (ddd, J = 18.1, 4.9, 2.5 Hz, 1H), 2.26 (dd, J = 18.1, 1.4 Hz, 1H), 2.04 (tdd, J =
7.4, 6.4, 3.7 Hz, 1H), 1.94 – 1.89 (m, 2H), 1.85 – 1.80 (m, 1H), 1.45 (dd, J = 9.3, 5.4 Hz, 1H),
19

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1.43 (s, 3H), 0.98 (s, 3H), 0.94 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 171.37, 145.85, 136.66,
133.80, 131.11, 128.42, 128.23, 125.46, 118.77, 69.71, 44.67, 42.48, 40.09, 38.61, 37.57, 28.35,
25.07, 18.25, 17.53. ESI-HRMS calcd for C19H24BrN4O [M + H] + 403.11335, found: 403.11224.
HPLC purity of 98.533% (retention time = 2.368).
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6
4.1.25. (1S,5S)-2-((1-(3-bromophenyl)-1H-1,2,3-triazol-4-yl)methyl)-1,8,8-trimethyl-2-azabicyc
lo[3.2.1]octan-3-one (14t). A yellow oil, yield: 77%. [α]D25 = + 23.0 (c = 1.0 in EtOH). 1H NMR
(600 MHz, CDCl3) δ 8.18 (s, 1H), 7.98 (s, 1H), 7.73 (d, J = 8.0 Hz, 1H), 7.52 (d, J = 8.0 Hz, 1H),
7.38 (t, J = 8.1 Hz, 1H), 4.79 (d, J = 15.3 Hz, 1H), 4.72 (d, J = 15.2 Hz, 1H), 2.73 (dd, J = 18.1,
2.2 Hz, 1H), 2.26 (d, J = 18.2 Hz, 1H), 2.04 (s, 1H), 1.90 (s, 1H), 1.89 – 1.80 (m, 2H), 1.45 (s, 3H),
1.42 (dd, J = 9.4, 5.3 Hz, 1H), 0.98 (s, 3H), 0.92 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 171.50,
146.52, 137.84, 131.33, 130.93, 123.11, 123.03, 121.47, 118.56, 69.66, 44.52, 42.29, 39.85, 38.61,
37.34, 28.19, 24.94, 18.07, 17.39. ESI-HRMS calcd for C19H24BrN4O [M + H] + 403.11335, found:
403.11306. HPLC purity of 98.269% (retention time = 2.377).
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4.1.26. (1S,5S)-2-((1-(4-bromophenyl)-1H-1,2,3-triazol-4-yl)methyl)-1,8,8-trimethyl-2-azabicyc
lo[3.2.1]octan-3-one (14u). A yellow oil, yield: 80%. [α]D25 = + 21.2 (c = 1.0 in EtOH). 1H NMR
(600 MHz, CDCl3) δ 8.07 (s, 1H), 7.68 – 7.60 (m, 4H), 4.78 (d, J = 15.2 Hz, 1H), 4.70 (d, J = 15.2
Hz, 1H), 2.72 (dd, J = 18.1, 1.9 Hz, 1H), 2.25 (d, J = 18.1 Hz, 1H), 2.07 – 2.01 (m, 1H), 1.90 (d, J
= 7.2 Hz, 1H), 1.88 – 1.79 (m, 2H), 1.45 (s, 3H), 1.43 (dd, J = 9.1, 5.4 Hz, 1H), 0.98 (s, 3H), 0.92
(s, 3H). 13C NMR (150 MHz, CDCl3) δ 171.57, 146.77, 136.05, 132.80 (2C), 122.13, 121.70 (2C),
121.44, 69.77, 44.67, 42.47, 40.04, 38.75, 37.49, 28.32, 25.08, 18.21, 17.56. ESI-HRMS calcd for
C19H24BrN4O [M + H] + 403.11335, found: 403.11206. HPLC purity of 98.628% (retention time =
2.328).
4.1.27. (1S,5S)-1,8,8-trimethyl-2-((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)-2-azabicyclo[3.2.1]o
ctan-3-one (14v). A brown oil, yield: 87%. [α]D25 = + 33.7 (c = 1.0 in EtOH). 1H NMR (600 MHz,
CDCl3) δ 8.13 (s, 1H), 7.76 (d, J = 8.1 Hz, 2H), 7.49 (t, J = 7.9 Hz, 2H), 7.39 (t, J = 7.4 Hz, 1H),
4.80 (d, J = 15.3 Hz, 1H), 4.73 (d, J = 15.3 Hz, 1H), 2.75 – 2.69 (m, 1H), 2.25 (d, J = 18.1 Hz,
1H), 2.03 (s, 1H), 1.91 – 1.85 (m, 2H), 1.84 – 1.79 (m, 1H), 1.45 (s, 3H), 1.44 – 1.39 (m, 1H),
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0.96 (s, 3H), 0.92 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 171.36, 146.32, 136.91, 129.53 (2C),
128.40, 121.34, 120.08 (2C), 69.58, 44.50, 42.30, 39.88, 38.56, 37.36, 28.19, 24.92, 18.05, 17.38.
ESI-HRMS calcd for C19H25N4O [M + H] + 325.20284, found: 325.20266. HPLC purity of 97.794%
(retention time = 2.585).
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6
4.2. Biology
7
4.2.1.
Cell culture
8
All cancer cell lines were obtained from the American Type Culture Collection (ATCC). All
healthy cell lines were obtained from the Shanghai Cell Resource Bank. Cultured cells using the
culture guidelines provided by the supplier and performed relevant mycoplasma tests once a
month.
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4.2.2.
MTT assay
The cultured cells were collected in the logarithmic growth phase, digested with 0.25%
trypsin, and diluted into a single cell suspension with phosphate-buffered saline (PBS). Then 100
µL of each cell type (4 × 104 cells/mL) was seeded into 96-well plate and incubated for 24 h.
Different concentrations of compounds were added into the 96-well plates. Respective
concentrations of DMSO were used as control. Then these plates were incubated for 24 h, and 20
µL MTT solution (5 mg/mL) (Wuhan Bost Biotechnology Co., Ltd., China) was added and
cultured for 4 h. The nutrient solution was discarded slowly and 100 µL of DMSO was added to
each well. The optical density (OD) of each well was detected at 490 nm using a microplate reader.
The IC50 value was determined as the inhibitor’s concentration, which produced 50% inhibition
calculated IC50 values for the compounds using probability units and weighted regression methods.
Determined the CC50 value as the concentration of the compound at which 50% of the healthy cell
died. Every assay was performed in triplicate. Data are presented as the mean ± SD (n = 3).
4.2.3.
Inhibition of heat shock protein 90
Fluorescence polarization assay was used to screen target compounds for their inhibition
effects for Hsp90α. Briefly, various concentrations of compounds and fluorescein
isothiocyanate-labeled geldanamycin were added to each 96-well plate. Recombinant Hsp90α
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protein (ProSpec-Tany Company, China) was diluted with the reaction buffer (20 mM HEPES pH
7.3, 50 mM KCl, 5 mM MgCl2, 20 mM Na2MoO4, 0.01% Triton X-100, 100 μg/mL bovine serum
albumin, and 2 mM dithiothreitol) and added to each well to get the final concentration of 22.2
nM. The enzyme reaction was initiated as soon as the Hsp90α protein was added. After incubation
for 60 min at 4 °C, the fluorescent polarization values were measured at an excitation wavelength
at 495 nm and an emission wavelength at 530 nm.
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4.2.4.
Molecular docking study
9
Molecular modeling studies were carried out with MOE (Molecular Operating Environment,
Version 2015.10) software [48]. The crystal structure of Hsp90α (PDB ID: 2XJX, Resolution 1.66
Å) downloaded from RCSB Protein Data Bank was adopted in docking calculations [2]. The
receptor was optimized by a Quickprep protocol with the following procedures of Structure
Preparation, Protonate 3D and Structure Refine (RMSD gradient = 0.1 kcal/mol, AMBER10: EHT
field) [49, 50]. The docking procedure was adopted the standard protocol implemented in MOE
and all parameters were maintained as the defaults. The binding interactions are illustrated by
PyMOL software [51].
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4.2.5.
Cell cycle analysis
LoVo and SW620 cells (2 × 105 cells) were incubated in 6 cm dishes for 24 h. Then 14t was
added to the dishes with different concentrations. After incubation for 24 h, the cells were
harvested and fixed in 80% ethanol at 4 °C overnight, and then incubated with the
DNA-interacting dye propidium iodide (PI) (Promega Corporation, USA) for 30 min at 37 °C.
Cell cycle analysis was performed using a flow cytometry (Becton Dickinson and Company,
USA).
4.2.6.
Annexin V/propidium iodide (PI) staining
The Annexin V-FITC/PI dual staining assay was used to determine the percentage of
apoptotic cells. Briefly, 1 mL of LoVo and SW620 cells (1 × 106 cells/mL) were plated in 6-well
plates and incubated for 24 h. Then 14t was added to the cells with indicated concentrations. After
another incubation of 24 h, the cells were harvested softly and washed twice with cold PBS. Then
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the cells were re-suspended in 150 µL of PBS, 10 µL of fluorescein isothiocyanate (FITC)-labeled
Annexin V (Annexin V-FITC) (Promega Corporation, USA) and 5 µL of PI were added. The
mixture was incubated at room temperature for 10 min in the dark and then analyzed by flow
cytometry (Becton Dickinson and Company, USA).
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4.2.7.
Western blot analysis
7
LoVo and SW620 cells (1 × 107 cells) were seeded into the flask (Becton Dickinson and
Company, USA), and these cells were incubated with different concentrations of 14t for 24 h.
Then the cells were lysed, and the proteins were extracted. Extracted proteins were separated from
each other using polyacrylamide electrophoresis on a 10% gel (Beyotime Biotechnology Company,
China) and transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad Company, USA),
followed by incubated with the primary specific antibodies (bax, bcl-2, cleaved-caspase 3, caspase
3 and β-actin). After binding of appropriate secondary antibodies, proteins were visualized with an
ECL detection system.
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4.2.8.
Cell migration and wound healing assays
Cell migratory ability was measured using transwell chambers (8-μm pore size; Corning
Costar, USA). For the transwell assay, LoVo and SW620 cells suspended in serum-free
RPMI-1640 medium containing different concentrations of 14t were seeded into the upper
chamber. The lower chamber contained RPMI-1640 medium supplemented with 20% serum. After
24 h incubation, the filters were fixed in methanol and stained with 0.1% crystal violet. The upper
faces of the filters were gently abraded, and the lower faces with cells migrated across the filters
were imaged and counted under the microscope. For wound healing assay, cells were placed into
6-well plates and cultured until 100% confluence. An artificial scratch was created using a 200 µL
pipette tip. Add serum-free RPMI-1640 medium containing different concentrations of 14t. At 24
h after culturing in serum-free medium, wound closure images were captured in the same field
under magnification. Cell healing rates were calculated by the fraction of cell coverage across the
line. These experiments were performed in triplicate and repeated three times.
4.2.9.
Evaluation of in vivo antitumor activity
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All animal experimental protocols were approved by the Laboratory Animal Welfare and
Ethical Committee of China Medical University. Five-week-old immunodeficient BABL/c female
nude mice, weighing 18 to 20 g, were purchased and maintained under specific pathogen-free
conditions. SW620 cells (1× 107 cells) were injected subcutaneously into the right flank of nude
mice. When the tumor reached a volume of about 50 mm3, the mice were randomized to
drug-treated or vehicle groups (five mice per group). Tumor growth was measured every 3 days
using a vernier caliper. For a total of 21 days, the mice were injected intraperitoneally with
compound 14t and 17-AAG (5 mg/kg) every day, after which the mice were euthanized and the
xenograft tumors were dissected. As controls, vehicle groups of five mice were treated with the
PBS. The tumor volume (V) was calculated using the formula: V (mm3) = (length × width2)/2. The
tumor growth inhibition (TGI) values were calculated by the following formula: TGI (%) = [1–
RTV (drug–treated)/RTV (control)] × 100%, The individual relative tumor volume: RTV = (tumor
volume on day 21)/(tumor volume on day 1).
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4.2.10. Immunohistochemistry
Sections derived from formalin-fixed and paraffin-embedded mouse tissues were
deparaffinized by incubation overnight at 65 °C followed by rehydration in sequential xylene and
ethanol rinses. After incubation with hydrogen peroxide, the slides were washed with PBS and
then incubated with 0.4% Triton X-100. The sections were incubated with blocking solution
(Dako Protein Block, Denmark) for 30 min at room temperature after washing with PBS. The
sections were further incubated with primary antibodies (Ki67 and cleaved-caspase 3) overnight at
4 °C, washed with PBS several times, incubated with the corresponding biotinylated secondary
antibodies, and then washed with PBS multiple times. After adding avidin-biotin complexes, the
sections were visualized using diaminobenzidine (DAB) detection reagent (Enzo Life Sciences,
USA) and mounted with a mounting solution (Vector Laboratories, USA).
Acknowledgements
This work was supported by the National Natural Science Foundation of China (Grant Nos.
81803355 and 81573687), the Scientific Research Fund of Liaoning Provincial Education
Department (LQNK201739), the Key Research and Development Project of Liaoning (2019 NO.
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26) and the Doctoral Research Funding (229181).
3
Appendix A. Supplementary data
4
Supplementary data to this article can be found online.
5
6
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