Synthesis and biological evaluation of novel Ani9 derivatives as potent and selective ANO1 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2018.10.002

Anoctamin 1 (ANO1), a calcium-activated chloride channel, is highly expressed and amplified in a number of carcinomas including breast, pancreatic and prostate cancers. Downregulation of ANO1 expression and function significantly inhibits cell proliferation, migration, and invasion of various cancer cell lines. Development of potent and selective ANO1 inhibitors is currently desirable, which may provide a new strategy for cancer treatment. Our previous study revealed a new class of ANO1 inhibitor, (E)-2-(4-chloro-2-methylphenoxy)-N'-(2-methoxybenzylidene)acetohydrazide (Ani9) and structural optimization via chemical modification of Ani9 basic skeleton was undertaken for the development of more potent and specific inhibitors of ANO1. Structure-activity relationship studies with newly synthesized derivatives revealed a number of potent ANO1 inhibitors, among which 5f is the most potent inhibitor with an IC₅₀ value of 22 nM. The selectivity analyses showed that 5f has excellent selectivity to ANO1 (>1000-fold over ANO2). In cellular assays, 5f significantly inhibited cell proliferation of PC3, MCF7, and BxPC3 cells expressing high levels of ANO1. In addition, 5f strongly reduced the protein levels of ANO1 in PC3 cells. This study will be useful in the development of ANO1 inhibitors for treatment of cancer and other ANO1-related diseases.

Full text of DOI:10.1016/j.ejmech.2018.10.002

Accepted Manuscript
Synthesis and biological evaluation of novel Ani9 derivatives as potent and selective
ANO1 inhibitors
Yohan Seo, Jinhwang Kim, Jiwon Chang, Seong Soon Kim, Wan Namkung, Ikyon
Kim
PII:
S0223-5234(18)30864-X
10.1016/j.ejmech.2018.10.002
EJMECH 10788
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 August 2018
Revised Date: 18 September 2018
Accepted Date: 1 October 2018
Please cite this article as: Y. Seo, J. Kim, J. Chang, S.S. Kim, W. Namkung, I. Kim, Synthesis and
biological evaluation of novel Ani9 derivatives as potent and selective ANO1 inhibitors, European
Journal of Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.ejmech.2018.10.002.
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ACCEPTED MANUSCRIPT
variation
(4) substitution
(2)
(3)
Structural
Optimization
O
O
O
O
N
N
N
N
H
H
OCF3
OMe
Cl
Cl
(1) ring formation
Ani9
5f
- potent ANO1 inhibitor with an IC50 value of 22 nM
- excellent selectivity to ANO1 over ANO2 (>1000)
- inhibition of cell proliferation of PC-3, MCF7, and BxPC3 cells
expressing high levels of ANO1
- reduction of the level of ANO1 protein expression in PC-3 cells

ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of novel Ani9 derivatives as potent and
selective ANO1 inhibitors
Yohan Seoa,b,‡, Jinhwang Kima,‡, Jiwon Changa, Seong Soon Kimc, Wan Namkunga,b,*, and
Ikyon Kima,*
a College of Pharmacy and Yonsei Institute of Pharmaceutical Sciences, Yonsei University, 85
Songdogwahak-ro, Yeonsu-gu, Incheon, 21983, Republic of Korea
b Interdisciplinary Program of Integrated OMICS for Biomedical Science Graduate School,
Yonsei University, Seoul, 03722, Republic of Korea
c Bio & Drug Discovery Division, Korea Research Institute of Chemical Technology, Daejeon,
34114, Republic of Korea
* Corresponding authors.
Tel.: +82 32 749 4519; fax: +82 32 749 4105; e-mail: wnamkung@yonsei.ac.kr
Tel.: +82 32 749 4515; fax: +82 32 749 4105; e-mail: ikyonkim@yonsei.ac.kr
‡ These authors contributed equally.
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Table of Contents
Keywords: Structure-activity relationship; Structural modification; Anoctamin 1 (ANO1);
Ani9; Anticancer agent.
Abstract
Anoctamin 1 (ANO1), a calcium-activated chloride channel, is highly expressed and
amplified in a number of carcinomas including breast, pancreatic and prostate cancers.
Downregulation of ANO1 expression and function significantly inhibits cell proliferation,
migration, and invasion of various cancer cell lines. Development of potent and selective
ANO1 inhibitors is currently desirable, which may provide a new strategy for cancer
treatment. Our previous study revealed a new class of ANO1 inhibitor, (E)-2-(4-chloro-2-
methylphenoxy)-N'-(2-methoxybenzylidene)acetohydrazide
(Ani9)
and
structural
optimization via chemical modification of Ani9 basic skeleton was undertaken for the
development of more potent and specific inhibitors of ANO1. Structure-activity relationship
studies with newly synthesized derivatives revealed a number of potent ANO1 inhibitors,
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among which 5f is the most potent inhibitor with an IC50 value of 22 nM. The selectivity
analyses showed that 5f has excellent selectivity to ANO1 (>1000-fold over ANO2). In
cellular assays, 5f significantly inhibited cell proliferation of PC3, MCF7, and BxPC3 cells
expressing high levels of ANO1. In addition, 5f strongly reduced the protein levels of ANO1
in PC3 cells. This study will be useful in the development of ANO1 inhibitors for treatment
of cancer and other ANO1-related diseases.
Introduction
ANO1, transmembrane member 16A (TMEM16A), is a calcium-activated chloride channel
(CaCC) and plays important roles in a wide range of biological processes in various cell types
including epithelial cells, smooth muscle cells, intestinal pacemaker cells and sensory
neurons [1-4]. In addition, ANO1 is highly expressed and amplified in a variety of
carcinomas including breast cancer, pancreatic cancer, prostate cancer, glioblastoma, head-
and-neck squamous cell carcinoma (HNSCC) and gastrointestinal stromal tumor (GIST), and
play a pivotal role in the regulation of cancer cell proliferation, tumorigenesis and cancer
progression [5-11]. Recent evidence suggests that ANO1 inhibitors may have potential utility
in treatment of ANO1-related diseases such as inflammatory airway diseases, hypertension
and pain [12, 13]. For example, ANO1 inhibition may have a beneficial effect on
inflammatory airway diseases via inhibition of excessive mucus secretion in airway
epithelium and airway smooth muscle contraction [12]. Inhibition of ANO1 significantly
reduced blood pressure in spontaneously hypertensive rats [14, 15]. In an animal model of
thermal pain, nocifensive behaviors are significantly decreased by inhibition of ANO1, and
capsaicin-mediated pain-related behaviors are also reduced by ANO1 inhibition [16, 17]. In
addition, recent studies showed that inhibition of ANO1 by small-molecule inhibitors
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significantly decreased cell proliferation and migration in a wide variety of cancer cell lines
including breast, pancreatic and prostate cancer cells expressing high levels of ANO1 [18-21].
ANO1 and ANO2 are verified as CaCCs and share the highest sequence homology among
ANO family members [22-24]. ANO2 is highly expressed in olfactory sensory neurons and
plays an important role in the regulation of olfactory transduction. Interestingly, recent
evidence suggests that ANO2 is involved in reductions of spike generation in thalamocortical
and CA1 hippocampal neurons [25, 26], and ANO2 knockout mice show deficits in executive
functions and severe impairment in motor learning [27, 28]. Thus, development of selective
inhibitors of ANO1 over ANO2 may be a more appropriate therapeutic approach to ANO1-
related disease. To date, several small-molecule inhibitors of ANO1 have been identified,
including CaCCinh-A01, T16Ainh-A01, MONNA, Ani9, and AACT (10bm) (Figure 1) [12,
29-32], and Ani9 is the most selective ANO1 inhibitor among the ANO1 inhibitors.
Figure 1. Chemical structures of reported ANO1 inhibitors
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With the basic knowledge of Ani9 in hand, we decided to conduct systematic structural
optimization via chemical modification of Ani9 to find the more potent ANO1 inhibitor with
better selectivity over ANO2, which is the topic of this paper. As shown in Scheme 1, we
hoped to synthesize Ani9 derivatives by following the four principles: (1) formation of a ring
(2) substitution of 2-methoxyphenyl by different (hetero)aryl moieties (purple region) (3)
replacement of the acylhydrazone with different pharmacophores (green region) (4)
introduction of substituent(s) at the alpha position of the carbonyl group.
Scheme 1. Synthetic Plan
Results and discussion
Our synthetic plans for Ani9 derivatization is outlined in Scheme 2. Reduction of the
hydrazone unit in Ani9 with NaBH4 afforded acylhydrazine 1. As conformational restriction
of initial hit compounds via additional ring introduction may result in more potent biological
activities, benzofuran analogue 3 was synthesized (Scheme 2). To evaluate the substituent
effect of the phenyl ring in Ani9, acylhydrazone derivatives 5 were prepared. Acylhydrazide
derived from ethyl 4-chloro-2-methylphenoxyacetate was allowed to react with various
(hetero)arylaldehydes to afford 5. The corresponding oxadiazoles 6 were obtained by
oxidative ring closure of acylhydrazones 5 to see if biological activity could be retained with
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oxadiazole unit. Furthermore, derivatives 7 having an amide instead of an acylhydrazone
were synthesized by EDC coupling of the commercially available 4-chloro-2-
methylphenoxyacetic acid with several amines.
Scheme 2. Syntheses of Ani9 Derivatives 1, 3-7
ANO1 and ANO2 inhibitory activities of the compounds were measured using YFP
fluorescence quenching assay in FRT-ANO1 and –ANO2 cells (Figure 2). Hydrazone moiety
of Ani9 seemed to be crucial as the corresponding hydrazine 1 exhibited the decreased
activity. A conformationally restricted benzofuran analogue 3 lost ANO1 inhibitory activity.
Overall, either oxadiazole (6a-k) or amide derivatives (7a-f) displayed decreased ANO1
inhibition compared with acylhydrazones (5). With respect to the substituent effects of the
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phenyl ring, substitution at the meta-position did not increase the activity. Interestingly,
electron-withdrawing groups such as fluorine and trifluoromethyl at the ortho-position helped
to inhibit ANO1 although the activities are less than that of Ani9. Finally, acylhydrazone (5f)
bearing a trifluoromethoxy group at the ortho-position showed more potent ANO1 inhibitory
activity than Ani9 while 5g with a trifluoromethoxy group at the meta-position did not inhibit
ANO1, indicating the importance of the orientation of this functional moiety. The
corresponding ketone analogue (5h) displayed rather weaker activity than 5f.
Figure 2. ANO1 and ANO2 inhibition by Ani9 analoguesa
IC50 (µM) of IC50 (µM) of
Compound
Structure
ANO1
ANO2
0.097 ± 0.01
>100
Ani9
10.7 ± 0.14
>100
>100
>100
>100
1
3
4
>100
19.4 ± 0.01
12.4 ± 0.05
6a
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>100
>100
NAb
>100
NA
5b
6b
5c
6c
5d
6d
5e
6e
0.89 ± 0.02
21.2 ± 0.17
>100
>100
NA
>100
>100
NA
0.21 ± 0.04
>100
>100
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0.021 ± 0.005
NA
NA
NA
NA
5f
6f
>100
>100
>100
5g
6g
0.3 ± 0.02
15.7 ± 0.01
25.7 ± 0.01
>100
59.4 ± 0.45
NA
5h
5i
5j
63.8 ± 0.02
>100
6j
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31.3 ± 0.57
>100
NA
5k
6k
N
O
>100
N
O
N
Cl
>100
>100
>100
NA
5l
5m
>100
NA
13.1 ± 0.07
>100
5n
7a
7b
20.6 ± 0.02
53.7 ± 0.05
14.2 ± 0.07
NA
7c
>100
>100
7d
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>100
NA
7e
7f
14.8 ± 0.11
>100
a
IC50 values were determined using YFP fluorescence quenching assay in FRT cells
expressing ANO1 and ANO2 (mean ± S.E., n = 3). b NA when the inhibition rate is less than
20%.
With these results in hand, more analogues based on 5f were prepared, which contain methyl,
dimethyl, and difluoro groups at the α position of the carbonyl to see the substituent effects
(Scheme 3). O-alkylation of 4-chloro-2-methylphenol with three different α-bromoesters
followed by sequential reactions with hydrazine and 2-trifluoromethoxybenzaldehyde
furnished 8a-c, respectively. Unfortunately, these substituents did not lead to increased
activity (Figure 3).
Scheme 3. Syntheses of Ani9 Derivatives 8a-c
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Figure 3. ANO1 and ANO2 inhibition by Ani9 analogues 8a-ca
IC50 (µM) of
IC50 (µM) of
Compound
Structure
ANO1
ANO2
>100
NA
NAb
>100
>100
8a
8b
8c
>100
a
IC50 values were determined using YFP fluorescence quenching assay in FRT cells
expressing ANO1 and ANO2 (mean ± S.E., n = 3). b NA when the inhibition rate is less than
20%.
Taken together, the SAR studies led to a series of new Ani9 ((E)-2-(4-chloro-2-
methylphenoxy)-N'-(2-methoxybenzylidene)acetohydrazide) derivatives as ANO1 inhibitors,
and 5f was the most potent and selective compound.
Electrophysiological studies on inhibitory effect of 5f on chloride channels
We further determined the inhibitory effect of 5f on ANO1, ANO2 and cystic fibrosis
transmembrane conductance regulator (CFTR) using Ussing chamber study. As shown in
Figure 4A and 4B, apical membrane current measurement in FRT cells expressing ANO1 and
ANO2 revealed that 5f potently inhibited ATP-induced ANO1 chloride current in a dose
dependent manner with an IC50 value of 22 nM (Figure 4C), but ANO2 was very poorly
inhibited by 5f. Only 17.1 ± 3.5% of ANO2 chloride current was blocked by 100 µM of 5f.
To investigate the effect of 5f on CFTR, a cAMP-regulated apical chloride channel, CFTR
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was activated by forskolin and inhibited with CFTRinh-172, a selective CFTR inhibitor
(Figure 4D). CFTR chloride current was not significantly altered by high concentrations (30
and 100 µM) of 5f. These results revealed that 5f is at least 1,000-fold more selective for
ANO1 than for ANO2 and CFTR.
Figure 4. Selective inhibition of ANO1 over ANO2 by 5f
A-B) Representative apical membrane current measurement in FRT cells expressing ANO1
and ANO2. The indicated concentrations of 5f were applied 20 minutes before ANO1 and
ANO2 activation by 100 µM ATP. C) Summary of dose responses (mean ± S.E., n = 3–6). D)
Apical membrane current measured in FRT cells expressing human WT-CFTR cells. CFTR
was activated by 20 µM forskolin and indicated concentrations of 5f were applied. CFTR was
completely inhibited by 10 µM CFTRinh-172.
Effect of 5f on cell viability in PC3, MCF7 and BxPC3 cells expressing high levels of
ANO1
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Downregulation of ANO1 significantly reduces cell proliferation and migration in prostate,
breast and pancreatic cancer cell lines expressing high levels of ANO1 (PC3, MCF7 and
BxPC3, respectively) [11, 18, 19]. To investigate the effect of ANO1 inhibitors on
proliferation of PC3, MCF7 and BxPC3 cells, the cells were treated with Ani9, 5f or 5g (a
nonfunctional analogue of 5f) for 48 hours and cell viability was estimated with MTS assay.
Figure 5. Effect of 5f, 5g and Ani9 on cell proliferation in PC3, MCF7 and BxPC3 cells
A-C) PC3, MCF7 and BxPC3 cells expressing high levels of ANO1 were treated with Ani9,
5f and 5g at the indicated concentration for 48 hours. Cell viability was determined by MTS
assay (mean ± S.E., n = 3).
As shown in Figure 5, high concentrations of Ani9 did not strongly inhibit the cell viability in
PC3, MCF7 and BxPC3 cells. However, 5f strongly inhibited cell viability of PC3, MCF7
and BxPC3 cells in a dose dependent manner, and the nonfunctional compound 5g showed
only small decrease in cell viability at high concentration as expected. In this experiment, 5f
much more potently inhibited the viability of prostate, breast and pancreatic cancer cells
compared with Ani9, but electrophysiological studies showed that IC50 values of Ani9 and 5f
are 77 and 22 nM, respectively [31].
To investigate the low activity of Ani9 on the inhibition of cell viability, we observed plasma
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stability of Ani9 and 5f and the effect of these compounds on the protein levels of ANO1 in
PC3 cells expressing high levels of ANO1. As shown in Figure 6A, Ani9 showed low plasma
stability but 5f displayed longer stability in rat plasma compared to Ani9. The half-life of
Ani9 and 5f in rat plasma was 32 and 104 min, respectively. Of interest, 5f strongly decreased
protein levels of ANO1 at 3 and 10 µM, but Ani9 weakly reduced the protein levels of ANO1
only at 10 µM (Figure 6B). These results showed 5f is a more stable, potent and selective
inhibitor of ANO1 compared with Ani9.
Figure 6. Plasma stability of Ani9 and 5f, and effect of the compounds on protein levels
of ANO1 in PC3 cells
A) Stability profiles of Ani9 and 5f in rat plasma. Ani9 (5 µM) and 5f (5 µM) were incubated
in rat plasma at 37℃for different incubation time of 0, 30, 60, 120 and 240 min. (mean ± S.E.,
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n = 3). B) Western blot analysis of ANO1 in PC3 cells expressing high levels of ANO1. Cells
were incubated with indicated concentration of Ani9 and 5f for 24 hours. (right) Summary of
band-intensity. The ANO1 band intensity was normalized to β-actin (mean ± S.E., n = 3). *P
< 0.05 **P < 0.01, ***P < 0.001.
Conclusion
We have evaluated the inhibitory effect of a series of Ani9 derivatives against ANO1. The
SAR analysis led to the identification of new potent ANO1 inhibitors including 5f (with an
IC50 value of 22 nM). The selectivity analyses revealed that 5f is highly selective for ANO1,
showing >1,000-fold selectivity over ANO2. These results show that 5f is currently the most
potent and selective ANO1 inhibitor. In vitro studies showed that 5f potently inhibited the
viability of PC3, MCF7 and BxPC3 cells in a dose-dependent manner. In addition, 5f showed
>3 times longer plasma stability and strong reduction of ANO1 protein levels compared to
Ani9. This study can be helpful in further efforts to identify potent and selective ANO1
inhibitors as a pharmacological tool in studies on ANO1, as well as a promising drug
candidate for treating ANO1-related diseases.
Experimental Section
General Methods
Unless specified, all reagents and starting materials were purchased from commercial sources
and used as received without purification. “Concentrated” refers to the removal of volatile
solvents via distillation using a rotary evaporator. “Dried” refers to pouring onto, or passing
through, anhydrous magnesium sulfate followed by filtration. Flash chromatography was
performed using silica gel (230−400 mesh) with hexanes, ethyl acetate, and dichloromethane
as the eluent. All reactions were monitored by thin-layer chromatography on 0.25 mm silica
plates (F-254) visualized with UV light. Melting points were measured using a capillary
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1
melting point apparatus. H and 13C NMR spectra were recorded on a 400 MHz NMR
spectrometer and were described as chemical shifts, multiplicity (s, singlet; d, doublet; t,
triplet; q, quartet; m, multiplet), coupling constant in hertz (Hz), and number of protons.
HRMS were measured with an electrospray ionization (ESI) and Q-TOF mass analyzer.
Synthesis of Ani9: A mixture of 2-(4-chloro-2-methylphenoxy)acetohydrazide (100 mg, 0.47
mmol) and 2-methoxybenzaldehyde (76 mg, 1.2 equiv) in EtOH (2.5 mL) was stirred at
80 °C for 16 h. The precipitated product was collected by filtration and dried to give Ani9 as
a white solid.
(E)-2-(4-Chloro-2-methylphenoxy)-N'-(2-
methoxybenzylidene)acetohydrazide (Ani9). White
solid, mp: 160.8-162.9 °C; 1H NMR (400 MHz, DMSO-
d6) δ 11.57 (s, 1H, isomer a), 11.56 (s, 1H, isomer b),
8.62 (s, 1H, isomer b), 8.34 (s, 1H, isomer a), 7.83 (d, J = 7.6 Hz, 1H, isomer a), 7.80 (d, J =
7.6 Hz, 1H, isomer b), 7.44-7.37 (m, 1H, isomer a, b), 7.26-7.13 (m, 1H, isomer a, b), 7.09 (d,
J = 8.4 Hz, 1H, isomer a, b), 7.03-6.96 (m, 1H, isomer a ,b), 6.88 (d, J = 8.8 Hz, 1H, isomer
b), 6.84 (d, J = 8.8 Hz, 1H, isomer a), 5.16 (s, 2H, isomer a), 4.66 (s, 2H, isomer b), 3.84 (s,
3H isomer a, b), 2.23 (s, 3H, isomer b), 2.20 (s, 3H, isomer a); 13C NMR (100 MHz, DMSO-
d6) δ 168.8, 164.0, 157.6, 155.3, 143.3, 139.5, 131.5, 130.1, 130.1, 129.9, 128.4, 126.4, 126.3,
125.6, 123.9, 121.9, 120.7, 113.1, 111.8, 66.6, 65.3, 55.7, 15.9; HRMS (ESI-QTOF) m/z
[M+H]+ calcd for C17H18ClN2O3 333.1000, found 333.1005.
Synthesis of 1: To a solution of Ani9 (20 mg, 0.06 mmol) in TFA (1 mL) was added Et3SiH
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(19.2 µL, 2 equiv) at 0 °C. After being stirred at 0 °C for 1 h, the reaction mixture was diluted
with 15% aq. HCl (1 mL) and washed with hexanes (1 mL). Then, the aqueous layer was
carefully basified with KOH pellet and extracted with CH2Cl2 (1 mL × 2). The organic layer
was dried over MgSO4 and concentrated under reduced pressure to give the crude residue,
which was purified by silica gel column chromatography (hexanes:ethyl
acetate:dichloromethane = 10:1:2) to give 1 as a white solid.
2-(4-Chloro-2-methylphenoxy)-N'-(2-
methoxybenzyl)acetohydrazide (1). White solid, mp:
98.2-100.6 °C; 1H NMR (400 MHz, CDCl3) δ 7.73 (br s,
1H), 7.29 (t, J = 7.0 Hz, 1H), 7.18 (dd, J = 1.2, 7.6 Hz,
1H), 7.14-7.09 (m, 2H), 6.93-6.87 (m, 2H), 6.56 (d, J = 8.4 Hz, 1H), 5.04 (s, 1H), 4.50 (s,
13
2H), 4.04 (s, 2H), 3.86 (s, 3H), 2.11 (s, 3H); C NMR (100 MHz, CDCl3) δ 166.8, 158.1,
154.0, 131.0, 130.9, 129.5, 128.5, 126.9, 126.8, 125.1, 120.6, 112.5, 110.6, 67.5, 55.6, 51.7,
16.3; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C17H20ClN2O3 335.1157, found 335.1154.
Synthesis of 2: A mixture of 5-chloro-2-hydroxybenzaldehyde (300 mg, 1.91 mmol), ethyl
bromoacetate (0.25 mL, 1.2 equiv), and K2CO3 (795 mg, 3 equiv) in CH3CN (6.5 mL) was
stirred at 130 °C for 6 h. After being concentrated under reduced pressure, the reaction
mixture was diluted with CH2Cl2 (5 mL) and water. The water layer was extracted with
CH2Cl2 (5 mL) one more time. The organic layer was dried over MgSO4 and concentrated in
vacuo to give the crude residue, which was purified with silica gel column chromatography
(hexanes:ethyl acetate:dichloromethane = 50:1:2) to afford 2 as a white solid.
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Ethyl 5-chlorobenzofuran-2-carboxylate (2). White solid, mp:
49.1-51.3 °C; 1H NMR (400 MHz, CDCl3) δ 7.63 (d, J = 2.4 Hz,
1H), 7.50 (d, J = 9.2 Hz, 1H) 7.44 (s, 1H), 7.38 (dd, J = 2.0, 8.8
13
Hz, 1H), 4.44 (q, J = 7.2 Hz, 2H), 1.42 (t, J = 6.7 Hz, 3H); C NMR (100 MHz, CDCl3) δ
159.3, 154.0, 147.1, 129.5, 128.3, 128.0, 122.3, 113.5, 113.1, 61.9, 14.4; HRMS (ESI-QTOF)
m/z [M+H]+ calcd for C11H10ClO3 225.0313, found 225.0315.
Synthesis of 3: A mixture of 2 (50 mg, 0.23 mmol) and hydrazine (36 µL, 5 equiv) in EtOH
(1 mL) was stirred at 80 °C for 3 h. After being concentrated under reduced pressure, the
crude residue (acylhydrazide) was redissolved in EtOH (1 mL) and 2-methoxybenzaldehyde
(36.5 mg, 1.2 equiv) was added at rt. After being stirred at 80 °C for 16 h, the resulting
precipitated product was collected by filtration and dried to give 3 as a white solid.
(E)-5-Chloro-N'-(2-methoxybenzylidene)benzofuran-
2-carbohydrazide (3). White solid, mp: 193.0-196.0 °C;
1H NMR (400 MHz, DMSO-d6) δ 12.22 (s, 1H), 8.87 (s,
1H), 7.93 (s, 1H), 7.88 (d, J = 7.6 Hz, 1H), 7.77-7.70 (m,
2H), 7.51 (dd, J = 2.0, 8.8 Hz, 1H), 7.44 (t, J = 7.8 Hz, 1H), 7.11 (d, J = 8.4 Hz, 1H), 7.03 (t,
J = 7.6 Hz, 1H), 3.88 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 157.9, 154.2, 152.9, 149.3,
144.3, 131.9, 128.6, 128.2, 127.1, 125.6, 122.3, 122.1, 120.8, 113.6, 111.9, 110.1, 55.7;
HRMS (ESI-QTOF) m/z [M+H]+ calcd for C17H14ClN2O3 329.0687, found 329.0688.
Synthesis of 4: To a solution of 3 (30 mg, 0.091 mmol) in CH2Cl2 (1 mL) was added
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iodobenzene diacetate (32 mg, 1.1 equiv) at rt. After being stirred at rt for 6 h, the reaction
mixture was concentrated in vacuo to furnish the crude residue, which was purified by silica
gel column chromatography (hexanes:ethyl acetate:dichloromethane = 10:1:2 to 7:1:2) to
give 4 as a light yellow solid.
2-(5-Chlorobenzofuran-2-yl)-5-(2-methoxyphenyl)-
1,3,4-oxadiazole (4). Light yellow solid, mp: 156.4-
159.0 °C; 1H NMR (400 MHz, CDCl3) δ 8.04 (d, J = 7.6
Hz, 1H), 7.68 (d, J = 1.6 Hz, 1H), 7.60-7.49 (m, 3H),
7.40 (dd, J = 2.0, 8.8 Hz, 1H), 7.15-7.07 (m, 2H), 4.02 (s, 3H); 13C NMR (100 MHz, CDCl3)
δ 163.7, 158.2, 157.3, 154.1, 142.4, 133.7, 130.8, 129.8, 128.8, 127.4, 121.8, 121.0, 113.3,
112.4, 112.1, 109.3, 56.2; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C17H12ClN2O3
327.0531, found 327.0532.
2-((4-Chloro-2-methylphenoxy)methyl)-5-(2-
methoxyphenyl)-1,3,4-oxadiazole (6a). White solid,
1
mp: 104.8-105.9 °C; H NMR (400 MHz, CDCl3) δ
7.93 (d, J = 8.0 Hz, 1H), 7.52 (t, J = 8.0 Hz, 1H), 7.16-
7.04 (m, 4H), 6.95 (d, J = 8.8 Hz, 1H), 5.231 (s, 2H),
3.95 (s, 3H), 2.23 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 164.7, 162.0, 158.1, 154.6, 133.6,
131.0, 129.5, 126.8, 126.7, 120.9, 113.2, 112.7, 112.1, 60.7, 56.1, 16.3; HRMS (ESI-QTOF)
m/z [M+Na]+ calcd for C17H15ClN2NaO3 355.0820, found 355.0822.
20

ACCEPTED MANUSCRIPT
Compounds 5b-5n and 6b-6k were prepared by following the similar procedures as those for
the syntheses of 3 and 4.
(E)-2-(4-Chloro-2-methylphenoxy)-N'-(3-
methoxybenzylidene)acetohydrazide (5b). White
1
solid, mp: 188.2-190.5 °C; H NMR (400 MHz,
DMSO-d6) δ 11.64 (s, 1H, isomer a), 11.55 (s, 1H,
isomer b), 8.26 (s, 1H, isomer b), 7.97 (s, 1H, isomer a), 7.45-7.1 (m, 4H, isomer a, b), 7.0 (s,
1H, isomer a, b), 6.95-6.75 (m, 1H, isomer a, b), 5.18 (s, 2H, isomer a) 4.69 (s, 2H, isomer b),
3.78 (s, 3H, isomer a, b), 2.30-2.13 (m, 3H, isomer a, b); 13C NMR (100 MHz, DMSO-d6) δ
169.0, 164.2, 159.5, 155.3, 154.9, 147.8, 143.7, 135.5, 135.4, 130.1, 130.0, 129.9, 128.8,
128.4, 126.4, 126.3, 124.5, 123.9, 120.1, 119.6, 116.4, 115.9, 113.2, 113.0, 111.5, 111.3, 66.9,
65.3, 55.2, 15.9; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C17H18ClN2O3 333.1000, found
333.1003.
2-((4-Chloro-2-methylphenoxy)methyl)-5-(3-
methoxyphenyl)-1,3,4-oxadiazole (6b). Light yellow
solid, mp: 86.0-86.1 °C; 1H NMR (400 MHz, CDCl3) δ
7.64 (d, J = 7.6 Hz, 1H), 7.59 (s, 1H) 7.42 (t, J = 8.0 Hz,
1H), 7.17-7.07 (m, 3H), 6.94 (d, J = 9.2 Hz, 1H), 5.31
(s, 2H), 3.88 (s, 3H), 2.24 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 165.9, 162.3, 160.1, 154.5,
131, 130.4, 129.5, 126.9, 126.7, 124.6, 119.6, 118.7, 113.1, 111.8, 60.6, 55.7, 16.2; HRMS
(ESI-QTOF) m/z [M+H]+ calcd for C17H16ClN2O3 331.0844, found 331.0845.
21

ACCEPTED MANUSCRIPT
(E)-2-(4-Chloro-2-methylphenoxy)-N'-(2-
fluorobenzylidene)acetohydrazide (5c). White solid,
1
mp: 177.3-178.7 °C; H NMR (400 MHz, DMSO-d6) δ
11.8-11.63 (m, 1H, isomer a, b), 8.53 (s, 1H, isomer b),
8.21 (s, 1H, isomer a), 7.93 (s, 1H, isomer a, b), 7.6-7.05 (m, 5H, isomer a, b), 6.87 (s, 1H,
isomer a, b), 5.19 (s, 2H, isomer a), 4.70 (s, 2H, isomer b), 2.21 (s, 3H, isomer a, b); 13C
NMR (100 MHz, DMSO-d6) δ 169.0, 164.3, 161.9, 159.4, 155.2, 154.9, 136.6, 132.0, 131.9,
129.9, 128.4, 126.5, 126.4, 126.3, 124.9, 123.0, 121.6, 16.1, 115.9, 113.2, 113.1, 67.0, 65.3,
15.9; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C16H15ClFN2O2 321.0801, found 321.0799.
2-((4-Chloro-2-methylphenoxy)methyl)-5-(2-
fluorophenyl)-1,3,4-oxadiazole (6c). White solid, mp:
96.8-98.7 °C; 1H NMR (400 MHz, CDCl3) δ 8.08 (t, J
= 7.6 Hz, 1H), 7.60-7.53 (m, 1H), 7.31 (t, J = 7.6 Hz,
1H), 7.26 (t, J = 9.2 Hz, 1H), 7.16-7.11 (m, 2H), 6.94
13
(d, J = 9.2 Hz, 1H), 5.34 (s, 2H), 2.24 (s, 3H); C NMR (100 MHz, CDCl3) 162.7, 161.5,
158.9, 154.5, 134.1, 134.0, 131.1, 130.0, 129.99, 129.6, 127.0, 126.7, 124.9, 124.86, 117.3,
117.1, 113.1, 60.5, 16.2; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C16H13ClFN2O2
319.0644, found 319.0641.
(E)-2-(4-Chloro-2-methylphenoxy)-N'-(3-
fluorobenzylidene)acetohydrazide (5d). White solid,
22

ACCEPTED MANUSCRIPT
mp: 200.3-201.8 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.7 (s, 1H, isomer a, b), 8.29 (s, 1H,
isomer b) 8.0 (s, 1H, isomer a), 7.61-7.43 (m, 3H, isomer a, b), 7.32-7.12 (m, 3H, isomer a, b),
6.91-6.85 (m, 1H, isomer a, b) 5.20 (s, 2H, isomer a), 4.70 (s, 2H, isomer b), 2.23 (s, 3H,
isomer b), 2.2 (s, 3H, isomer a); 13C NMR (100 MHz, DMSO-d6) δ 169.1, 164.3, 163.6,
161.2, 155.3, 154.9, 142.4, 136.6, 130.9, 130.8, 130.1, 129.9, 128.4, 126.4, 126.3, 123.9,
123.6, 116.8, 113.1, 112.9, 112.7, 66.9, 65.3, 15.9; HRMS (ESI-QTOF) m/z [M+H]+ calcd for
C16H15ClFN2O2 321.0801, found 321.0806.
2-((4-Chloro-2-methylphenoxy)methyl)-5-(3-
fluorophenyl)-1,3,4-oxadiazole (6d). White solid, mp:
1
85.2-87.8 °C; H NMR (400 MHz, CDCl3) δ 7.87 (d, J
= 8.0 Hz, 1H), 7.77 (d, J = 9.2 Hz, 1H), 7.55-7.47 (m,
1H), 7.3-7.23 (m, 1H), 7.17-7.12 (m, 2H), 6.93 (d, J =
8.4 Hz, 1H), 5.32 (s, 2H), 2.24 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 165.0, 164.96, 164.2,
162.6, 161.7, 154.5, 131.2, 131.1, 131.1, 129.5, 127.0, 126.7, 125.5, 125.4, 123.0, 122.98,
119.5, 119.3, 114.4, 114.2, 113.0, 60.5, 16.2; HRMS (ESI-QTOF) m/z [M+H]+ calcd for
C16H13ClFN2O2 319.0644, found 319.0643.
(E)-2-(4-Chloro-2-methylphenoxy)-N'-(2-
(trifluoromethyl)benzylidene)acetohydrazide (5e).
1
White solid, mp: 158.2-160.0 °C; H NMR (400 MHz,
DMSO-d6) δ 11.95 (s, 1H, isomer b), 11.87 (s, 1H,
isomer a), 8.66 (s, 1H, isomer b), 8.36 (s, 1H, isomer a), 8.22 (d, J = 8 Hz, 1H, isomer a),
23

ACCEPTED MANUSCRIPT
8.17 (d, J = 8 Hz, 1H, isomer b), 7.83-7.7 (m, 3H, isomer a), 7.67-7.58 (m, 3H, isomer b),
7.27-7.12 (m, 3H, isomer a), 6.93-6.86 (m, 3H, isomer b), 5.23 (s, 2H, isomer a), 4.72 (s, 2H,
13
isomer b), 2.24 (s, 3H, isomer b), 2.21 (s, 3H, isomer a); C NMR (100 MHz, DMSO-d6) δ
169.2, 164.6, 155.2, 155.0, 139.0, 132.8, 131.8, 130.1, 130.0, 129.9, 128.4, 127.1, 126.9,
126.4, 126.2, 125.9, 124.0, 113.3, 113.1, 67.0, 65.3, 15.9; HRMS (ESI-QTOF) m/z [M+H]+
calcd for C17H15ClF3N2O2 371.0769, found 371.0765.
2-((4-Chloro-2-methylphenoxy)methyl)-5-(2-
(trifluoromethyl)phenyl)-1,3,4-oxadiazole (6e). White
1
solid, mp: 70.8-72.0 °C; H NMR (400 MHz, CDCl3) δ
8.09-8.05 (m, 1H), 7.89-7.85 (m, 1H), 7.74-7.70 (m, 2H),
7.17-7.11 (m, 2H), 6.91 (d, J = 8.4 Hz, 1H), 5.34 (s, 2H),
2,23 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 164.4, 163.5, 154.4, 132.4, 132.4, 132.1, 132.1,
132.1, 131.1, 129.6, 126.7, 112.8, 60.4, 16.1; HRMS (ESI-QTOF) m/z [M+H]+ calcd for
C17H13ClF3N2O2 369.0612, found 369.0613.
(E)-2-(4-Chloro-2-methylphenoxy)-N'-(2-
(trifluoromethoxy)benzylidene)acetohydrazide (5f).
1
White solid, mp: 157.2-158.0 °C; H NMR (400 MHz,
DMSO-d6) δ 11.83 (s, 1H, isomer b), 11.76 (s, 1H,
isomer a), 8.57 (s, 1H, isomer b), 8.27 (s, 1H, isomer a), 8.06 (d, J = 7.6 Hz, 1H, isomer a),
8.02 (d, J = 8.0 Hz, 1H, isomer b), 7.62-6.54 (m, 3H, isomer b), 7.52-7.43 (m, 3H, isomer a),
7.28-7.12 (m, 3H, isomer a), 6.92-6.84 (m, 3H, isomer b), 5.2 (s, 2H, isomer a), 4.71 (s, 2H,
24

ACCEPTED MANUSCRIPT
13
isomer b), 2.24 (s, 3H, isomer b), 2.21 (s, 3H, isomer a); C NMR (100 MHz, DMSO-d6) δ
169.0, 155.2, 140.8, 137.2, 131.6, 130.1, 129.9, 128.4, 128.1, 128.0, 127.0, 126.9, 126.6,
126.4, 126.2, 124.0, 121.8, 113.1, 67.0, 65.3, 15.9; HRMS (ESI-QTOF) m/z [M+H]+ calcd
for C17H15ClF3N2O3 387.0718, found 387.0719.
2-((4-Chloro-2-methylphenoxy)methyl)-5-(2-
(trifluoromethoxy)phenyl)-1,3,4-oxadiazole (6f). Light
1
yellow solid, mp: 85.6-86.6 °C; H NMR (400 MHz,
CDCl3) δ 8.18 (d, J = 7.2 Hz, 1H), 7.62 (t, J = 7.2 Hz,
1H), 7.52-7.41 (m, 2H), 7.18-7.09 (m, 2H), 6.92 (d, J =
13
8.0 Hz, 1H), 5.34 (s, 2H), 2.23 (s, 3H); C NMR (100 MHz, CDCl3) δ 163.2, 163.0, 154.4,
146.6, 133.5, 131.0, 130.9, 129.5, 127.6, 126.9, 126.6, 122.5, 121.8, 119.2, 117.8, 112.8, 60.4,
16.0; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C17H13ClF3N2O3 385.0561, found 385.0563.
(E)-2-(4-Chloro-2-methylphenoxy)-N'-(3-
(trifluoromethoxy)benzylidene)acetohydrazi
1
de (5g). White solid, mp: 176.4-179.2 °C; H
NMR (400 MHz, DMSO-d6) δ 11.76 (s, 1H,
isomer a), 11.70 (s, 1H, isomer b), 8.33 (s, 1H, isomer b), 8.03 (s, 1H, isomer a), 7.76-7.65 (m,
3H, isomer a), 7.62-7.53 (m, 3H, isomer b), 7.42 (t, J = 8.4 Hz, 1H, isomer a, b), 7.28-7.12
(m, 3H, isomer a), 6.92-6.83 (m, 3H, isomer b), 5.20 (s, 2H, isomer a), 4.71 (s, 2H, isomer b),
2.24 (s, 3H, isomer b), 2.20 (s, 3H, isomer a); 13C NMR (100 MHz, DMSO-d6) δ 169.2,
164.4, 155.2, 154.9, 148.8, 146.1, 142.1, 136.6, 136.5, 131.0, 130.8, 130.1, 129.9, 128.7,
25

ACCEPTED MANUSCRIPT
128.4, 126.4, 126.4, 126.2, 124.5, 123.9, 122.5, 122.1, 121.3, 118.8, 118.7, 113.2, 113.1, 67.0,
65.3, 15.9; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C17H15ClF3N2O3 387.0718, found
387.0715.
2-((4-Chloro-2-methylphenoxy)methyl)-5-(3-
(trifluoromethoxy)phenyl)-1,3,4-oxadiazole
(6g).
1
White solid, mp: 80.3-80.8 °C; H NMR (400 MHz,
CDCl3) δ 8.02 (d, J = 8.0 Hz, 1H), 7.93 (s, 1H), 7.58 (t,
J = 8.0 Hz, 1H), 7.42 (d, J = 8.4, 1H), 7.17-7.10 (m,
2H), 6.94 (d, J = 8.0 Hz, 1H), 5.33 (s, 2H), 2.24 (s, 3H); 13C NMR (100 MHz, CDCl3) δ
164.7, 162.7, 154.4, 149.8, 131.1, 131.0, 129.5, 127.1, 126.7, 125.5, 125.4, 124.6, 121.8,
119.7, 113.0, 60.5, 16.2; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C17H13ClF3N2O3
385.0561, found 385.0568.
(E)-2-(4-Chloro-2-methylphenoxy)-N'-(1-(2-
(trifluoromethoxy)phenyl)ethylidene)acetohydraz
ide (5h). White solid, mp: 119.2-121.0 °C; 1H NMR
(400 MHz, CDCl3) δ 9.56 (s, 1H, isomer a), 9.35 (s,
1H, isomer b), 7.67 (d, J = 7.6 Hz, 1H, isomer a), 7.48 (d, J = 7.6 Hz, 1H, isomer b), 7.52-7.4
(m, 3H, isomer b), 7.37-7.28 (m, 3H, isomer a), 7.23-7.15 (m, 2H, isomer a), 7.12 (s, 1H,
isomer b), 7.05 (dd, J = 2.0, 8.6 Hz, 1H, isomer b), 5.09 (s, 2H, isomer b), 4.69 (s, 2H, isomer
13
a), 2.34 (s, 3H, isomer a), 2.32-2.26 (m, 3H, isomer a, b), 2.21 (s, 3H, isomer b); C NMR
(100 MHz, CDCl3) δ 170.4, 163.7, 155.1, 153.5, 152.4, 147.8, 146.9, 146.7, 131.0, 130.8,
26

ACCEPTED MANUSCRIPT
130.8, 130.6, 130.5, 130.0, 127.1, 127.0, 127.96, 126.2, 1120.5, 112.6, 112.4, 67.4, 66.1, 16.4,
16.3, 16.2, 16.16; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C18H17ClFN2O3 401.0874,
found 401.0873.
Ethyl
(E)-2-(2-((2-(2-(4-chloro-2-
methylphenoxy)acetyl)hydrazono)methyl)
phenoxy)-2-fluoroacetate (5i). White solid,
1
mp: 100.3-101.5 °C; H NMR (400 MHz,
CDCl3) δ 9.50 (s, 1H, isomer a), 9.03 (s, 1H,
isomer b), 8.51 (s, 1H, isomer a), 8.20-8.15 (m, 1H, isomer a, b), 7.91 (d, J = 7.2 Hz, isomer
b), 7.47-7.40 (m, 1H, isomer a, b), 7.25-7.12 (m, 4H for isomer a, 3H for isomer b), 7.07 (dd,
J = 8.4 Hz, 1H, isomer b), 6.77 (d, J = 8.4 Hz, 1H, isomer a), 6.71 (d, J = 8.8 Hz, 1H, isomer
b), 6.05-5.86 (m, 1H, isomer a, b), 5.13 (s, 2H, isomer b), 4.66 (s, 2H, isomer a), 4.41-4.32
(m, 2H, isomer a, b), 2.33 (s, 3H, isomer a), 2.30 (s, 3H, isomer b), 1.40-1.32 (m, 3H, isomer
a, b); 13C NMR (100 MHz, CDCl3) δ 169.4, 164.1, 163.8, 155.1, 154.4, 153.9, 144.0, 139.5,
132.3, 132.0, 131.0, 130.7, 127.6, 127.0, 126.2, 125.0, 116.9, 116.7, 113.1, 112.7, 103.9,
103.8, 101.6, 101.56, 67.8, 66.2, 62.9, 62.8, 16.3, 16.2, 14.0, 13.99; HRMS (ESI-QTOF) m/z
[M+Na]+ calcd for C20H20ClFN2NaO5 445.0937, found 445.0936.
(E)-2-(4-Chloro-2-methylphenoxy)-N'-(furan-2-
ylmethylene)acetohydrazide (5j). White solid, mp:
1
140.5-143.7 °C; H NMR (400 MHz, DMSO-d6) δ
27

ACCEPTED MANUSCRIPT
11.57 (s, 1H, isomer a), 11.48 (s, 1H, isomer b), 8.18 (s, 1H, isomer b), 7.89 (s, 1H, isomer a),
7.86-7.81 (m, 1H, isomer a, b), 7.27-7.11 (m, 2H, isomer a, b), 6.93-6.90 (m, 1H, isomer a, b),
6.88 (d, J = 8.8 Hz, 1H, isomer b), 6.8 (d, J = 8.8 Hz, 1H, isomer a), 6.65-6.61 (m, 1H,
isomer a, b), 5.09 (s, 2H, isomer a), 4.67 (s, 2H, isomer b), 2.23 (s, 3H, isomer b), 2.20 (s, 3H,
isomer a); 13C NMR (100 MHz, DMSO-d6) δ 168.6, 164.1, 155.2, 154.9, 149.1, 149.0, 145.4,
145.1, 137.7, 134.0, 130.1, 129.9, 128.8, 128.4, 126.4, 126.3, 124.6, 124.0, 114.0, 113.8,
113.2, 113.0, 112.2, 112.19, 67.0, 65.0, 15.9; HRMS (ESI-QTOF) m/z [M+H]+ calcd for
C14H14ClN2O3 293.0687, found 293.0687.
2-((4-Chloro-2-methylphenoxy)methyl)-5-(furan-2-yl)-
1
1,3,4-oxadiazole (6j). White solid, mp: 110.6-112.3 °C; H
NMR (400 MHz, CDCl3) 7.66 (s, 1H), 7.21 (d, J = 3.6 Hz,
1H), 7.16-7.10 (m, 2H), 6.92 (d, J = 8.0 Hz, 1H), 6.62 (dd,
13
J = 3.2 Hz, 1H), 5.30 (s, 2H), 2.23 (s, 3H); C NMR (100
MHz, CDCl3) δ 161.6, 158.7, 154.4, 146.2, 139.1, 131.1, 129.5, 127.0, 126.7, 115.0, 113.0,
112.4, 60.3, 16.2; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C14H12ClN3O2 291.0531, found
291.0530.
(E)-2-(4-Chloro-2-methylphenoxy)-N'-(pyridin-2-
ylmethylene)acetohydrazide (5k). White solid, mp:
1
179.8-180.2 °C; H NMR (400 MHz, DMSO-d6) δ
11.81 (s, 1H, isomer a, b), 8.61 (s, 1H, isomer a, b),
8.31 (s, 1H, isomer b), 8.04 (s, 1H, isomer a), 8.02-7.80 (m, 2H, isomer a, b), 7.41 (s, 1H,
28