The design and synthesis of transient receptor potential vanilloid 3 inhibitors with novel skeleton

Article abstract of DOI:10.1016/j.bioorg.2021.105115

Transient receptor potential vanilloid 3 (TRPV3) channel as a member of thermo TRPV subfamily is primarily expressed in the keratinocytes of the skin and sensory neurons, and plays critical roles in various physiological and pathological processes such as

Full text of DOI:10.1016/j.bioorg.2021.105115

Bioorganic Chemistry 114 (2021) 105115
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
The design and synthesis of transient receptor potential vanilloid 3
inhibitors with novel skeleton
,
,
,
Mengqi Lva 1, Han Wub 1, Yaxuan Qub 1, Siyi Wua, Junxia Wanga, Congcong Wanga,
Yepeng Luana, Zhongyin Zhanga
,
*
a Department of Medicinal Chemistry, School of Pharmacy, Qingdao University Medical College, Qingdao University, Qingdao, Shandong, China
b Department of Pharmacology, School of Pharmacy, Qingdao University Medical College, Qingdao University, Qingdao, Shandong, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
TRPV3
Transient receptor potential vanilloid 3 (TRPV3) channel as a member of thermo TRPV subfamily is primarily
expressed in the keratinocytes of the skin and sensory neurons, and plays critical roles in various physiological
and pathological processes such as inflammation, pain sensation and skin disorders. However, TRPV3 studies
have been challenging, in part due to a lack of research tools such as selective antagonists. Recently, we syn-
thesized a series of cinnamate ester derivatives and evaluated their inhibitory activities on human TRPV3
channels expressed in HEK293 cells using whole-cell patch clamp recordings. And, we identified two potent
Small molecular
Inhibitor
Selectivity
TRPV3 antagonists 7c and 8c which IC50 values were 1.05
μM and 86 nM, respectively. It also showed good
selectivity to other subfamily members of TRPV, such as TRPV1 and TRPV4.
1. Introduction
necrosis factor (TNF)-α and IL-6 [11–16]. After TRPV3, which is
expressed in skin keratinocytes, is activated, the released factors can
cause skin itching and dermatitis. Recently, several studies have re-
ported that patients with Olmsted syndrome (OS) [17–19] caused by
TRPV3 gene mutation also have obvious skin pruritus, and studies have
shown that the expression of TRPV3 in patients with allergic dermatitis
is significantly increased. These indicated that TRPV3 is a key molecule
of the skin signaling pathway, and inhibiting TRPV3 overexpression may
exert a beneficial influence on skin health.
TRPV3, a member of the TRPV subfamily, is a non-selective cation
channel widely distributed over the peripheral and central nervous
system with high calcium permeability and ligand-gated property [1,2].
As a temperature sensor, TRPV3, in addition to responding to temper-
ature stimulation (>33 ◦C), can also be activated by some chemical
agonists and is also regulated by a variety of physiological factors of the
body [3–6]. Although its physiological functions have yet to be fully
elucidated, unique attributes of TRPV3 have distinguished it from other
vanilloid family members. Unlike other vanilloid TRPs, TRPV3 has
sensitization characteristics, that is, the number of stimulation channels
increases in a certain range, and the channel current also gradually in-
creases [7].
As so far, only a few compounds that can inhibit TRPV3 are reported,
such as osthole, forsythoside B and ruthenium red [20–22]. Moreover,
Example #64, Example #82 and FTP-THQ developed by Hydra Biosci-
ence (Fig. 1), GRC15133, GRC17173 and GRC15300 (unpublished
structures) of Glenmark pharmaceutical company [23] and 74a reported
by AbbVie company [24] showed a good inhibitory activity on TRPV3.
In terms of endogenous inhibitors, isoprene pyrophosphate [25] and 17
(R)-resolvin D1 have been studied [26]. However, there is still a lack of
specific antagonists.
Although many natural products can activate TRPV3, they have little
selectivity, such as carvacrol, camphor, menthol, thymol, etc [8]. The
most commonly used agonist in current research is the synthetic com-
pound 2-aminoethoxybiphenyl borate (2-APB) [9,10]. Studies have
shown that chemical activation of TRPV3 increases the release of many
proinflammatory and inflammatory factors, such as ATP, nitric oxide,
prostaglandin E2, nerve growth factor, interleukin (IL)-1α, tumor
* Corresponding author at: Department of Medicinal Chemistry, School of Pharmacy, Qingdao University Medical College, Qingdao University, Qingdao, Shan-
dong, China.
E-mail address: zyzhang@qdu.edu.cn (Z. Zhang).
1
Co-first author.
https://doi.org/10.1016/j.bioorg.2021.105115
Received 13 March 2021; Received in revised form 28 May 2021; Accepted 18 June 2021
Available online 21 June 2021
0045-2068/© 2021 Published by Elsevier Inc.

M. Lv et al.
Bioorganic Chemistry 114 (2021) 105115
2. Results and discussion
Previous work showed that forsythoside B has a certain inhibitory
activity against TRPV3 (IC50 = 6.7 μM) [21]. We proposed that cinna-
mate ester was the crucial fragment, and a series compounds were
synthesized. In the design of the target compound, the basic structure
skeleton was divided into three parts (Part A, B, C) (Fig. 2). H, hydroxyl,
acetyl, trifluoromethoxy substituents were introduced into the benzene
ring of Part A, and various substituents are introduced into the benzene
Fig. 2. Modification of cinnamate ester.
ring of Part B. Part C was consisted of
α,β-unsaturated ester and
carbonyl. Through a series of modification, a total of 23 target com-
pounds were synthesized.
in the para- position of Part A, the activities of compounds 4 and 5 were
higher than those of compounds 1 and 2. The inhibitory activities of
compounds 1 and 4 were similar, but the inhibitory activities of benzyl
ester 5 were much higher than that of benzyl ester 2.
2.1. Chemistry
Further, acetoxy group were introduced to the para- substitution of
Part A in 6a-6f. However, the activity of compound 6a decreased
significantly when p-methylbenzyl ester was retained. At the same time,
compounds 6b, 6c and 6f with higher inhibition rate than positive
control were determined against TRPV3. The inhibitory activity of 6b
was better than that of 6c, which indicated that fluorine substituted sites
had a certain effect on the inhibitory activity. It was also showed that
substitution of fluorine (compound 6b) was better than that of tri-
fluoromethyl (compound 6e) and two halogen (compound 6d). The
difluoro substitution of ring B (compound 6f) made the compound
exhibiting good inhibitory activity of TRPV3, and its inhibitory rate was
much higher than that of other compounds.
The synthetic route of 17 target compounds is shown in Scheme 1. By
esterification reaction of acids and alcohol/phenol with EDCI, DMAP,
triethylamine in CH2Cl2 as solvent overnight gave compounds 1–5, 6a-
6f in yields of 80–87%. Then, the target compounds 7a-7f were obtained
by dissolving 6a-6f in the solution of methanol and THF added with
potassium carbonate for 2 h, and the yields were about 60–76%.
In addition, the α,β-unsaturated carbonyl compounds were obtained
through the aldol condensation reaction. Under the condition of 10%
sodium hydroxide, compound 8a-8f was synthesized overnight in
anhydrous ethanol, and the yields were about 82–89%. Then, the pro-
tecting group MOM removed with 3 N HCl condition in methanol so-
lution for 3 h to obtain target compounds 9a and 9b in 72–76% yields
(Scheme 2).
Still in Table 1, compounds 7a-7f were showed to compare the ac-
tivity with A-ring acetoxy substituents. It can be seen from the table that
compound 7a showed lower inhibition rate than positive control, which
similar to compound 6a. The difluoro substituted compound 6f showed
the best inhibition in these acetoxy substituted compounds, while the 7f
was not. Compound 7c was identified as the best inhibitor in this series
with inhibition rate value of 85.45%.
2.2. Biological assays
2.2.1. Preliminary screening of TRPV3 inhibitory activity
The activities of 23 compounds were determined by patch clamp
According to Table 2, these compounds were featured with
technique in 50
μM concentration, and forsythoside B was used as
α
,β-unsaturated carbonyl as linker in Part C. When compound 9a
positive control. The results of inhibiting TRPV3 activity are shown in
the following table. In Table 1, 17 ester compounds were modified and
obtained. Compounds 1–5 showed lower TRPV3 inhibitory activity than
the positive control. When the trifluoromethoxy group was substituted
retained the hydroxyl of ring A and the fluorine of ring B, it displayed
high inhibition. But when the substituent of ring B was changed by p-
methyl (compound 9b), it resulted in almost disappearance of the
Fig. 1. The chemical structure of TRPV3 antagonists.
2

M. Lv et al.
Bioorganic Chemistry 114 (2021) 105115
Scheme 1. The synthetic route of 17 target compounds. Reagents and conditions: (a) phenol or benzyl alcohol, EDCI, DMAP, Et3N, CH2Cl2, 0 ◦C to rt, overnight,
yields 80–87%; (b) K2CO3, CH3OH, THF, rt, 2 h, yields 60–76%
Scheme 2. The synthetic route of 6 target compounds. Reagents and conditions: (c) 10% NaOH, EtOH, rt, overnight; (d) 3 N HCl, CH3OH, rt, 3 h, yields 72–76%
activity. Then, the fluorine substitution of ring B was retained, and ring
A was modified to obtain 8c-8f compounds. These compounds with halo
substituted in ring B exhibited poor inhibitory activities. While the tri-
fluoromethoxy substituted compound 8c showed the excellent inhibi-
tion (77.17%).
2.2.2. IC50 values and TRPV selectivity
All compounds were tested on TRPV3 to evaluate their TRPV3
inhibitory activities in 50
μМ concentration. Compound 7c and 8c
performed well in this test, then the IC50 values were evaluated. In Fig. 3,
The IC50 values were 1.05 М and 86 nM, respectively. While the IC50
μ
3

M. Lv et al.
Bioorganic Chemistry 114 (2021) 105115
the isoform selectivity profile, 7c and 8c were selected as the candidate
to test its inhibitory activities against TRPV1 and TRPV4. We can see
from the Fig. 4. The results of selectivity experiments showed that 7c
and 8c hardly inhibited TRPV1 and TRPV4.
Table 1
The
α,β-unsaturated ester target compounds of inhibitory activity in vitro
3. Conclusion
Compound
R1
R5
R6
R7
n
50 μМ Inhibition rate (%)
In summary, we reported that a novel series of cinnamate ester de-
rivatives were designed, synthesized and evaluated against TRPV3. The
target compounds were synthesized by the corresponding reaction based
on bioisosterism. The results showed that the target compound 7c and
1
H
H
CN
H
H
CN
H
H
F
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
Cl
H
H
–
0
1
0
0
1
1
0
0
0
0
0
1
0
0
0
0
0
–
19.26
32.6
2
H
CH3
CH3
H
3
H
28.58
29.12
60.39
22.65
75.59
66.9
4
OCF3
OCF3
OAc
OAc
OAc
OAc
OAc
OAc
OH
OH
OH
OH
OH
OH
–
5
CH3
CH3
H
8c have high TRPV3 inhibitory activity, with IC50 values of 1.05 μM and
6a
86 nM, respectively. Furthermore, 7c and 8c were tested have a good
selectivity to TRPV1 and TRPV4. At the same time, the results of SAR
research and analysis indicated that these target compounds have the
potential of further structural optimization.
6b
6c
F
H
H
H
F
6d
F
56.54
58.8
6e
CF3
F
6f
81.87
47.24
63.99
85.45
79.56
55.25
51.26
61.07
7a
CH3
H
H
F
4. Experimental section
7b
7c
F
H
H
H
F
4.1. Chemistry
7d
F
7e
CF3
F
7f
4.1.1. A general method for synthesis of compounds 1–5, 6a-6f and 7a-7f
To a stirred solution of cinnamic acid (1.5 mmol) in 8 mL dichloro-
methane solution was added alcohol or phenol (1 mmol), EDCI (2 mmol)
and DMAP (0.05 mmol) at 0 ◦C. The triethylamine (2 mmol) was added
to the previous liquor and the reaction was warmed to room temperature
and stirred mechanically overnight. After that, the reaction mixture was
quenched with saturated aqueous NaHCO3 solution (20 mL) and
extracted with CH2Cl2 (3 × 20 mL). The combined organic phases were
washed with saturated aqueous NaCl solution (2 × 20 mL) and dried
over anhydrous Na2SO4, then filtrated and evaporated the solvent under
vacuum. The residue was subjected to silica gel column chromatography
for purification using EtOAc/petroleum ether (1:6) as eluent to give
compound 1–5 and 6a-6f as white foam (80–87% yield). To a stirred 6a-
6f (0.4 mmol) and K2CO3 (0.12 mmol) were put into 2.5 mL methanol
and 2.5 mL THF for 2 h. The reaction was acidified with 2 N HCl to pH 1.
The resultant mixture was extracted with EtOAc (3 × 20 mL). The
combined organic phases were washed with saturated aqueous NaCl
solution (2 × 20 mL) and dried over anhydrous Na2SO4, then filtrated
and evaporated the solvent under vacuum. The residue was subjected to
silica gel column chromatography for purification using EtOAc/petro-
leum ether (1:3) as eluent to give compound 7a-7f as white foam
(60–76% yield).
Forsythoside B*
–
–
* positive control.
Table 2
The
α
,β-unsaturated carbonyl target compounds of inhibitory activity in vitro
.
Compound
R2
R3
R4
50 μМ Inhibition rate (%)
9a
OH
OH
H
H
F
73.58
2.586
77.17
7.837
21.28
41.03
61.07
9b
H
CH3
F
8c
OCF3
F
8d
H
F
8e
H
Cl
Br
–
F
8f
H
F
Forsythoside B*
–
–
* positive control.
4.1.2. 3-cyanophenyl cinnamate (1)
1H NMR (500 MHz, CDCl3) δ 7.90 (d, J = 16.0 Hz, 1H), 7.60 (dd, J =
7.1, 2.3 Hz, 2H), 7.55 – 7.50 (m, 3H), 7.46 – 7.44 (m, 4H), 6.62 (d, J =
16.0 Hz, 1H). 13C NMR (126 MHz, CDCl3) δ 164.69, 150.92, 147.78,
133.82, 131.12, 130.39, 129.49, 129.09, 128.45, 126.70, 125.47,
117.93, 116.25, 113.45. HRMS (ESI): m/z 250.0863 [M+H]+; calcd for
C16H12NO2, 250.0868.
4.1.3. 4-methylbenzyl cinnamate (2)
1H NMR (500 MHz, CDCl3) δ 7.74 (d, J = 16.0 Hz, 1H), 7.56 – 7.50
(m, 2H), 7.39 (dd, J = 3.8, 2.5 Hz, 3H), 7.33 (d, J = 8.0 Hz, 2H), 7.21 (d,
J = 7.8 Hz, 2H), 6.49 (d, J = 16.0 Hz, 1H), 5.23 (s, 2H), 2.38 (s, 3H). 13
C
NMR (126 MHz, CDCl3) δ 166.85, 145.06, 138.14, 134.39, 133.04,
130.32, 129.29, 128.89, 128.49, 128.10, 126.80, 118.00, 66.35, 21.25.
HRMS (ESI): m/z 275.1043 [M+Na]+; calcd for C17H16NaO2, 275.1048.
4.1.4. p-tolyl cinnamate (3)
Fig. 3. IC50 values of Compound 7c and 8c.
1H NMR (500 MHz, CDCl3) δ 7.86 (d, J = 16.0 Hz, 1H), 7.59 (dd, J =
6.2, 3.2 Hz, 2H), 7.46 – 7.40 (m, 3H), 7.20 (d, J = 8.2 Hz, 2H), 7.05 (d, J
= 8.4 Hz, 2H), 6.63 (d, J = 16.0 Hz, 1H), 2.36 (s, 3H). 13C NMR (126
MHz, CDCl3) δ 165.63, 148.54, 146.40, 135.43, 134.22, 130.65, 129.97,
128.99, 128.29, 121.31, 117.41, 20.92. HRMS (ESI): m/z 239.1067
value of Forsythoside B was tested as 6.71
μМ. As an important branch of
TRPV family, TRPV3 has been paid more and more attentions due to the
lack of specific inhibitors. Accordingly, with the aim of characterizing
4

M. Lv et al.
Bioorganic Chemistry 114 (2021) 105115
Fig. 4. Selectivity of compound 7c and 8c to other subfamily members of TRPV.
[M+H]+; calcd for C16H15O2, 239.1072.
4.1.10. (E)-2-chloro-4-fluorophenyl 3-(4-acetoxyphenyl)acrylate (6d)
1H NMR (400 MHz, CDCl3) δ 7.89 (d, J = 16.0 Hz, 1H), 7.62 (d, J =
8.6 Hz, 2H), 7.24 – 7.16 (m, 4H), 7.08 – 6.98 (m, 1H), 6.61 (d, J = 16.0
Hz, 1H), 2.33 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 169.07, 164.32,
161.21, 158.75, 152.64, 146.47, 143.36, 131.67, 129.62, 127.91,
127.80, 124.60, 124.51, 122.32, 117.67, 117.41, 116.27, 114.85,
114.62, 21.16. 19F NMR (376 MHz, CDCl3) δ ꢀ 114.39.
4.1.5. (E)-3-cyanophenyl 3-(4-(trifluoromethoxy)phenyl)acrylate (4)
1H NMR (400 MHz, CDCl3) δ 7.87 (d, J = 16.0 Hz, 1H), 7.64 (d, J =
8.7 Hz, 2H), 7.58 – 7.50 (m, 3H), 7.47 – 7.43 (m, 1H), 7.28 (d, J = 8.4
Hz, 2H), 6.59 (d, J = 16.0 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 164.38,
150.99, 150.97, 150.84, 145.92, 132.39, 130.44, 129.94, 129.60,
126.63, 125.42, 121.65, 121.27, 119.08, 117.88, 117.25, 113.53. 19F
NMR (376 MHz, CDCl3) δ ꢀ 57.70. HRMS (ESI): m/z 356.0505
[M+Na]+; calcd for C17H10F3NNaO3, 356.0510.
4.1.11. (E)-4-(trifluoromethyl)phenyl 3-(4-acetoxyphenyl)acrylate (6e)
1H NMR (500 MHz, CDCl3) δ 7.87 (d, J = 16.0 Hz, 1H), 7.68 (d, J =
8.5 Hz, 2H), 7.62 (d, J = 8.6 Hz, 2H), 7.31 (d, J = 8.6 Hz, 2H), 7.18 (d, J
= 8.5 Hz, 2H), 6.59 (d, J = 16.0 Hz, 1H), 2.33 (s, 3H). 13C NMR (101
MHz, CDCl3) δ 169.09, 164.71, 153.30, 152.65, 146.26, 131.66, 129.59,
128.22, 127.90, 126.88, 126.84, 126.80, 126.76, 125.27, 122.57,
122.36, 122.15, 116.78, 21.14. HRMS (ESI): m/z 373.0658 [M+Na]+;
calcd for C18H13F3NaO4, 373.0664. 19F NMR (376 MHz, CDCl3) δ
ꢀ 62.20.
4.1.6. (E)-4-methylbenzyl 3-(4-(trifluoromethoxy)phenyl)acrylate (5)
1H NMR (400 MHz, CDCl3) δ 7.68 (d, J = 16.0 Hz, 1H), 7.53 (t, J =
5.6 Hz, 2H), 7.31 (d, J = 8.0 Hz, 2H), 7.21 (t, J = 8.7 Hz, 4H), 6.44 (d, J
= 16.0 Hz, 1H), 5.21 (s, 2H), 2.37 (s, 3H). 13C NMR (101 MHz, CDCl3) δ
166.52, 150.44, 143.27, 138.26, 133.01, 132.91, 129.52, 129.33,
128.54, 121.67, 121.16, 121.16, 119.10, 118.99, 77.38, 77.06, 76.74,
66.52, 21.23. 19F NMR (376 MHz, CDCl3) δ ꢀ 57.74. HRMS (ESI): m/z
359.0866 [M+Na]+; calcd for C18H15F3NaO3, 359.0871.
4.1.12. (E)-3,4-difluorophenyl 3-(4-acetoxyphenyl)acrylate (6f)
1H NMR (500 MHz, CDCl3) δ 7.84 (d, J = 16.0 Hz, 1H), 7.61 (d, J =
8.5 Hz, 2H), 7.19 (dd, J = 18.6, 8.8 Hz, 3H), 7.10 – 7.02 (m, 1H), 6.95 –
6.89 (m, 1H), 6.55 (d, J = 16.0 Hz, 1H), 2.33 (s, 3H). 13C NMR (101
MHz, CDCl3) δ 169.09, 164.85, 152.63, 151.43, 151.29, 149.56, 149.44,
148.95, 148.81, 147.12, 146.99, 146.42, 146.39, 146.34, 146.30,
146.20, 131.64, 129.57, 122.35, 117.69, 117.65, 117.62, 117.59,
117.39, 117.20, 116.67, 111.81, 111.61, 21.14. 19F NMR (376 MHz,
CDCl3) δ ꢀ 134.65 (d, J = 21.4 Hz), ꢀ 140.99 (d, J = 21.4 Hz). HRMS
(ESI): m/z 341.0596 [M+Na]+; calcd for C17H12F2NaO4, 341.0601.
4.1.7. (E)-4-methylbenzyl 3-(4-acetoxyphenyl)acrylate (6a)
1H NMR (400 MHz, CDCl3) δ 7.69 (d, J = 16.0 Hz, 1H), 7.53 (d, J =
8.6 Hz, 2H), 7.32 (d, J = 8.0 Hz, 2H), 7.20 (d, J = 7.8 Hz, 2H), 7.12 (d, J
= 8.6 Hz, 2H), 6.43 (d, J = 16.0 Hz, 1H), 5.21 (s, 2H), 2.37 (s, 3H), 2.31
(s, 3H). 13C NMR (101 MHz, CDCl3) δ 169.16, 166.76, 152.13, 143.94,
138.18, 132.99, 132.14, 129.97, 129.31, 129.24, 128.51, 128.44,
122.15, 118.18, 115.91, 66.42, 21.24, 21.14. HRMS (ESI): m/z
333.1097 [M+Na]+; calcd for C19H18NaO4, 333.1103.
4.1.8. (E)-3-fluorophenyl 3-(4-acetoxyphenyl)acrylate (6b)
4.1.13. (E)-4-methylbenzyl 3-(4-hydroxyphenyl)acrylate (7a)
1H NMR (500 MHz, CDCl3) δ 7.85 (d, J = 16.0 Hz, 1H), 7.61 (d, J =
8.1 Hz, 2H), 7.36 (dd, J = 15.2, 7.6 Hz, 1H), 7.17 (d, J = 8.0 Hz, 2H),
6.97 (dd, J = 15.8, 8.9 Hz, 3H), 6.57 (d, J = 16.0 Hz, 1H), 2.33 (s, 3H).
13C NMR (101 MHz, CDCl3) δ 169.11, 164.83, 164.16, 161.70, 152.56,
151.69, 151.58, 145.94, 131.74, 130.25, 130.16, 129.55, 122.32,
117.47, 117.43, 117.00, 112.97, 112.76, 109.90, 109.66, 21.15. 19F
NMR (376 MHz, CDCl3) δ ꢀ 111.06. HRMS (ESI): m/z 323.0690
[M+Na]+; calcd for C17H13FNaO4, 323.0696.
1H NMR (400 MHz, CDCl3) δ 7.66 (d, J = 16.0 Hz, 1H), 7.40 (d, J =
8.5 Hz, 2H), 7.31 (d, J = 8.0 Hz, 2H), 7.19 (d, J = 7.9 Hz, 2H), 6.84 (d, J
= 8.6 Hz, 2H), 6.33 (d, J = 16.0 Hz, 1H), 5.21 (s, 2H), 2.36 (s, 3H). 13
C
NMR (101 MHz, CDCl3) δ 167.74, 158.28, 158.13, 145.32, 145.17,
138.18, 132.98, 130.07, 129.31, 128.45, 126.96, 126.86, 115.98,
115.96, 115.10, 114.98, 66.48, 66.43, 21.24. HRMS (ESI): m/z
291.0992 [M+Na]+; calcd for C17H16NaO3, 291.0997.
4.1.14. (E)-3-fluorophenyl 3-(4-hydroxyphenyl)acrylate (7b)
4.1.9. (E)-4-fluorophenyl 3-(4-acetoxyphenyl)acrylate (6c)
1H NMR (500 MHz, CDCl3) δ 7.81 (d, J = 15.9 Hz, 1H), 7.50 (d, J =
8.3 Hz, 2H), 7.36 (dd, J = 14.8, 7.9 Hz, 1H), 7.00 – 6.91 (m, 3H), 6.87 (d,
J = 8.4 Hz, 2H), 6.47 (d, J = 15.9 Hz, 1H). 13C NMR (101 MHz, DMSO) δ
165.44, 163.91, 161.48, 160.97, 152.15, 152.04, 147.65, 131.29,
131.21, 131.12, 125.30, 118.70, 118.67, 116.40, 113.19, 113.13,
112.98, 110.50, 110.26. 19F NMR (376 MHz, CD3OD) δ ꢀ 113.69. HRMS
(ESI): m/z 257.0619 [M - H]+; calcd for C15H10FO3, 257.0614.
1H NMR (400 MHz, CDCl3) δ 7.84 (d, J = 16.0 Hz, 1H), 7.64 – 7.56
(m, 2H), 7.19 – 7.06 (m, 6H), 6.57 (d, J = 16.0 Hz, 1H), 2.33 (s, 3H). 13
C
NMR (101 MHz, CDCl3) δ 169.12, 165.31, 161.46, 159.04, 152.51,
146.61, 146.58, 145.71, 131.80, 129.51, 123.07, 122.98, 122.31,
117.14, 116.22, 115.99, 21.15. 19F NMR (376 MHz, CDCl3) δ ꢀ 117.04.
HRMS (ESI): m/z 323.0690 [M+Na]+; calcd for C17H13FNaO4,
323.0696.
5

M. Lv et al.
Bioorganic Chemistry 114 (2021) 105115
4.1.15. (E)-4-fluorophenyl 3-(4-hydroxyphenyl)acrylate (7c)
1H NMR (400 MHz, CDCl3) δ 7.80 (t, J = 13.7 Hz, 1H), 7.48 (d, J =
8.6 Hz, 2H), 7.16 – 7.03 (m, 4H), 6.85 (d, J = 8.6 Hz, 2H), 6.47 (d, J =
15.9 Hz, 1H), 5.57 (s, 1H). 13C NMR (101 MHz, CDCl3) δ 166.03, 161.44,
159.01, 158.19, 146.68, 133.06, 130.34, 126.92, 123.11, 123.03,
116.20, 116.03, 115.97, 115.10, 114.26. 19F NMR (376 MHz, CDCl3) δ
ꢀ 117.13. HRMS (ESI): m/z 257.0619 [M - H]+; calcd for C15H10FO3,
257.0614.
(dd, J = 7.9, 1.2 Hz, 1H). 13C NMR (101 MHz, DMSO) δ 188.23, 166.76,
164.26, 158.20, 144.92, 136.37, 134.75, 134.72, 132.04, 131.94,
130.35, 122.15, 120.41, 118.36, 116.37, 116.16, 115.80. HRMS (ESI):
m/z 243.0816 [M+H]+; calcd for C15H12FO2, 243.0821.
4.1.18.3. (E)-3-(3-hydroxyphenyl)-1-(p-tolyl)prop-2-en-1-one (9b). 1H
NMR (400 MHz, CD3OD) δ 7.99 (dd, J = 8.3, 1.9 Hz, 2H), 7.75 – 7.63 (m,
2H), 7.42 – 7.33 (m, 2H), 7.28 – 7.21 (m, 2H), 7.15 (s, 1H), 6.92 – 6.85
(m, 1H), 2.45 (s, 3H). 13C NMR (101 MHz, CD3OD) δ 190.63, 157.74,
144.75, 144.02, 136.18, 135.37, 129.68, 129.09, 128.96, 128.42,
121.49, 119.85, 117.53, 114.40, 20.26. HRMS (ESI): m/z 237.0921 [M -
H]+; calcd for C16H13O2, 237.0916.
4.1.16. (E)-2-chloro-4-fluorophenyl 3-(4-hydroxyphenyl)acrylate (7d)
1H NMR (400 MHz, CD3OD) δ 7.82 (t, J = 17.0 Hz, 1H), 7.55 (d, J =
8.6 Hz, 2H), 7.34 (dd, J = 8.3, 2.9 Hz, 1H), 7.26 (dd, J = 9.0, 5.2 Hz,
1H), 7.17 – 7.08 (m, 1H), 6.84 (d, J = 8.6 Hz, 2H), 6.55 (d, J = 15.9 Hz,
1H). 13C NMR (101 MHz, CD3OD) δ 169.06, 165.18, 164.49, 162.73,
151.79, 147.67, 136.96, 134.24, 131.68, 131.57, 129.39, 128.85,
128.76, 120.86, 120.59, 119.53, 118.50, 118.27, 115.67. 19F NMR (376
MHz, CD3OD) δ ꢀ 116.68. HRMS (ESI): m/z 315.0195 [M+Na]+; calcd
for C15H10ClFNaO3, 315.0200.
4.1.18.4. (E)-1-(4-fluorophenyl)-3-(4-(trifluoromethoxy)phenyl)prop-2-
en-1-one (8c). 1H NMR (400 MHz, CDCl3) δ 8.08 – 7.98 (m, 2H), 7.76 (d,
J = 15.7 Hz, 1H), 7.65 (d, J = 8.7 Hz, 2H), 7.45 (d, J = 15.7 Hz, 1H),
7.24 (d, J = 7.8 Hz, 2H), 7.18 – 7.14 (m, 2H). 13C NMR (101 MHz,
CDCl3) δ 188.49, 167.00, 164.47, 150.64, 143.19, 134.33, 134.30,
133.37, 131.18, 131.09, 129.90, 122.35, 121.66, 121.23, 119.10,
115.96, 115.75. 19F NMR (376 MHz, CDCl3) δ ꢀ 61.65, ꢀ 109.09. HRMS
(ESI): m/z 311.0690 [M+H]+; calcd for C16H11F4O2, 311.0695.
4.1.17. (E)-4-(trifluoromethyl)phenyl 3-(4-hydroxyphenyl)acrylate (7e)
1H NMR (400 MHz, DMSO) δ 7.73 – 7.79 (m, 3H), 7.66 (d, J = 8.7 Hz,
2H), 7.45 (d, J = 8.4 Hz, 2H), 6.84 (d, J = 8.6 Hz, 2H), 6.65 (d, J = 15.9
Hz, 1H). 13C NMR (101 MHz, DMSO) δ 165.35, 161.00, 154.07, 147.92,
131.34, 127.35, 127.31, 127.27, 127.24, 126.93, 126.61, 125.88,
125.30, 123.40, 123.17, 116.39, 112.97. 19F NMR (376 MHz, CD3OD) δ
ꢀ 63.48. HRMS (ESI): m/z 307.0588 [M - H]+; calcd for C16H10F3O3,
307.0582.
4.1.18.5. (E)-1,3-bis(4-fluorophenyl)prop-2-en-1-one (8d). 1H NMR
(400 MHz, CDCl3) δ 8.08 – 8.02 (m, 2H), 7.78 (d, J = 15.7 Hz, 1H), 7.67
– 7.61 (m, 2H), 7.43 (d, J = 15.6 Hz, 1H), 7.21 – 7.15 (m, 2H), 7.15 –
7.08 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 188.60, 166.92, 165.39,
164.39, 162.88, 143.73, 134.47, 134.44, 131.14, 131.04, 130.45,
130.36, 121.27, 121.25, 116.30, 116.08, 115.90, 115.69. 19F NMR (376
4.1.18. (E)-3,4-difluorophenyl 3-(4-hydroxyphenyl)acrylate (7f)
1H NMR (400 MHz, DMSO) δ 10.16 (s, 1H), 7.78 (d, J = 15.9 Hz, 1H),
7.65 (d, J = 8.6 Hz, 2H), 7.56 – 7.40 (m, 2H), 7.13 – 7.05 (m, 1H), 6.83
(d, J = 8.6 Hz, 2H), 6.61 (d, J = 15.9 Hz, 1H). 13C NMR (101 MHz,
DMSO) δ 165.49, 160.90, 150.94, 150.80, 149.04, 148.92, 148.49,
148.35, 147.76, 147.13, 147.11, 147.04, 147.01, 146.63, 146.50,
131.32, 125.33, 119.30, 118.16, 117.97, 116.37, 112.98, 112.73,
112.54. 19F NMR (376 MHz, CD3OD) δ ꢀ 138.05 (d, J = 20.8 Hz),
ꢀ 144.31 (d, J = 20.8 Hz). HRMS (ESI): m/z 275.0525 [M - H]+; calcd for
C15H9F2O3, 275.0520.
MHz, CDCl3)
δ ꢀ 105.42, ꢀ 108.82. HRMS (ESI): m/z 267.0592
[M+Na]+; calcd for C15H10F2NaO, 267.0597.
4.1.18.6. (E)-3-(4-chlorophenyl)-1-(4-fluorophenyl)prop-2-en-1-one
(8e). 1H NMR (400 MHz, CDCl3) δ 8.08 – 8.03 (m, 2H), 7.76 (d, J =
15.7 Hz, 1H), 7.60 – 7.56 (m, 2H), 7.48 (d, J = 15.7 Hz, 1H), 7.42 – 7.38
(m, 2H), 7.22 – 7.15 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 188.55,
166.97, 164.44, 143.55, 136.59, 134.39, 134.36, 133.26, 131.17,
131.08, 129.63, 129.31, 121.97, 115.95, 115.73. 19F NMR (376 MHz,
CDCl3) δ ꢀ 109.18. HRMS (ESI): m/z 283.0296 [M+Na]+; calcd for
C15H10ClFNaO, 283.0302.
4.1.18.1. A general method for synthesis of compounds 9a, 9b and 8c-8f.
The acetophenone (1.3 mmol) and 10% NaOH (3 mmol) were dissolved
in anhydrous ethanol (5 mL) under stirring for 10 min. Then the benz-
aldehyde (1.2 mmol) was added to the previous liquor and the reaction
mixture was stirred overnight at room temperature. After that, the
resultant mixture was extracted with EtOAc (3 × 20 mL). The combined
organic phases were washed with saturated aqueous NaCl solution (2 ×
20 mL) and dried over anhydrous Na2SO4, then filtrated and evaporated
the solvent under vacuum. The residue was subjected to silica gel col-
umn chromatography for purification using EtOAc/petroleum ether
(1:15) as eluent to give compounds 8a-8b as oil and 8c-8f as white foam.
To a stirred solution of 8a-8b (1 mmol) in methanol solution (5 mL) was
added 3 N HCl (3 mmol). The reaction mixture was stirred at room
temperature for 3 h and then quenched with saturated aq.NaHCO3 (20
mL). The resultant mixture was extracted with EtOAc (3 × 20 mL). The
combined organic phases were washed with saturated aqueous NaCl
solution (2 × 20 mL) and dried over anhydrous Na2SO4, then filtrated
and evaporated the solvent under vacuum. The residue was subjected to
silica gel column chromatography for purification using EtOAc/petro-
leum ether (1:10) as eluent to give compound 9a, 9b as light yellow
foam (72–76% yield).
4.1.18.7. (E)-3-(4-bromophenyl)-1-(4-fluorophenyl)prop-2-en-1-one
(8f). 1H NMR (400 MHz, CDCl3) δ 8.09 – 8.01 (m, 2H), 7.75 (d, J = 15.7
Hz, 1H), 7.58 – 7.54 (m, 2H), 7.53 – 7.47 (m, 3H), 7.22 – 7.15 (m, 2H).
13C NMR (101 MHz, CDCl3) δ 188.53, 166.98, 164.44, 143.61, 134.37,
134.34, 133.69, 132.27, 131.17, 131.08, 129.83, 124.96, 122.06,
115.95, 115.74. 19F NMR (376 MHz, CDCl3) δ ꢀ 109.14. HRMS (ESI): m/
z 326.9791 [M+Na]+; calcd for C15H10BrFNaO, 326.9797.
4.2. Screening of TRPV3 inhibitory activity
HEK-293 cells were seeded into a small dish. On the next day, HEK-
293 cells were transiently transfected with hTRPV3 plasmid. After 4 h,
the medium was changed and incubated overnight in a 5% CO2 incu-
bator at 37 ◦C. On the third day, the whole cell current was recorded by
patch clamp amplification system. The extracellular fluid containing 2-
APB (50
μ
M) was perfused first, and then the extracellular fluid con-
taining different concentrations of inhibitor and 2-APB (50
μ
M) was
perfused. The maximum outward current mediated by mTRPV3 channel
induced by 2-APB and the outward current mediated by hTRPV3
channel under the action of 2-APB were recorded. The half inhibitory
concentration (IC50) was calculated according to the inhibition rate of
outward current mediated by hTRPV3 channel [21].
4.1.18.2. (E)-1-(4-fluorophenyl)-3-(3-hydroxyphenyl)prop-2-en-1-one
(9a). 1H NMR (400 MHz, DMSO) δ 9.64 (d, J = 2.5 Hz, 1H), 8.24 (dd, J
= 8.4, 5.7 Hz, 2H), 7.84 (d, J = 15.6 Hz, 1H), 7.65 (d, J = 15.6 Hz, 1H),
7.42 – 7.35 (m, 2H), 7.31 (d, J = 7.6 Hz, 1H), 7.28 – 7.22 (m, 2H), 6.88
When we evaluated the channel selectivity of the compounds, we
selected known potent agonists for each channel to control, 1 µM
capsaicin for TRPV1 and 0.1 µM GSK101 for TRPV4 [21].
6

M. Lv et al.
Bioorganic Chemistry 114 (2021) 105115
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The authors declare that they have no known competing financial
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Acknowledgements
We acknowledge financial support of this work from the National
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