Study on chemical modification and analgesic activity of N-(4-tert-butylphenyl)-4-(3-chloropyridin-2-yl) piperazine-1-carboxamide

Article abstract of DOI:10.1016/j.ejmech.2020.112236

N-(4-Tert-butylphenyl)-4-(3-chloropyridin-2-yl) piperazine-1-carboxamide (BCTC) is a potent and extensively studied urea-based TRPV1 antagonist. Although BCTC was effective in alleviating chronic pain in rats, it showed obvious hyperthermia side-effect an

Full text of DOI:10.1016/j.ejmech.2020.112236

European Journal of Medicinal Chemistry 194 (2020) 112236
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Study on chemical modification and analgesic activity of
N-(4-tert-butylphenyl)-4-(3-chloropyridin-2-yl)
piperazine-1-carboxamide
,
,
Cunbin Nie a 1, Qifei Li b 1, Yue Qiao a, Jing Hu a, Mengkang Gao a, Yusui Wang a,
Zhenrui Qiao a, Qiang Wang c, Lin Yan a *, Hai Qian b
,
, **
a Institute for Innovative Drug Design and Evaluation, School of Pharmacy, Henan University, N. Jinming Ave., Kaifeng, Henan, 475004, China
b State Key Laboratory of Natural Medicines, Center of Drug Discovery, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, Jiangsu, 210009, China
c School of Pharmaceutical Sciences, South-Central University for Nationalities, 182 Minyuan Road, Wuhan, Hubei, 430074, China
a r t i c l e i n f o
a b s t r a c t
Article history:
N-(4-Tert-butylphenyl)-4-(3-chloropyridin-2-yl) piperazine-1-carboxamide (BCTC) is
a potent and
Received 9 February 2020
Received in revised form
11 March 2020
Accepted 11 March 2020
Available online xxx
extensively studied urea-based TRPV1 antagonist. Although BCTC was effective in alleviating chronic
pain in rats, it showed obvious hyperthermia side-effect and unsatisfactory pharmacokinetic profile,
therefore, it was not developed further. In order to enrich the structural types of urea-based TRPV1
antagonists, two series of novel analogs, in which the pyridine ring of BCTC was replaced with a mildly
basic pyrimidine ring or 1,2,3,4-tetrahydro-b-carboline scaffold, were designed and synthesized.
Advancing the structure-activity relationship of these two series led to the discovery of N-(4-
methoxyphenyl)-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole-2-carboxamide (7o), with an improved
pharmacological and tolerability profile compared with the lead compound BCTC.
© 2020 Elsevier Masson SAS. All rights reserved.
Keywords:
Transient receptor potential vanilloid type 1
BCTC
Pyrimidine
Tetrahydro-b-carboline
Hyperthermia
1. Introduction
antagonists have been developed so far, but owing to their hyper-
thermic effect in preclinical trials, none have advanced beyond the
Pain, especially chronic pain, is a significant problem in public
health management affecting one-fifth of the global population [1].
Among different targets involved in pain transmission [2,3], the
transient receptor potential vanilloid 1 (TRPV1) receptor has been
the focus of pain-related research since its discovery [4]. TRPV1, a
calcium permeable nonselective cation channel expressed on sen-
sory neurons, is selectively activated by multiple factors such as
protons (pH < 6.8), heat (>43 ꢀC), endogenous ligands, and natural
vanilloids including capsaicin and resiniferatoxin [5]. The expres-
sion of TRPV1 is upregulated under certain nerve injury and in-
flammatory disease conditions. Furthermore, TRPV1 antagonists
relieved pain of inflammation, osteoarthritis, and, even, cancer in
rodent models [4]. Therefore, the TRPV1 antagonists are considered
as an attractive analgesic [4,6]. Numerous potent TRPV1
clinical trial stage [7,8]. Novel TRPV1 antagonists with excellent
analgesic effect and no hyperthermic adverse-effect are yet to be
developed.
Piperazine ureas represent one of the major classes of TRPV1
antagonists. Among them, N-(4-(tert-butyl) phenyl)-4-(3-
chloropyridin-2-yl) piperazine-1-carboxamide (BCTC) is the most
representative antagonist of this family. It showed potent analgesic
activity in the animal models of inflammatory and neuropathic pain
[9]. However, BCTC inhibited TRPV1 activation by capsaicin (CAP),
protons, and, especially, protons, which led to obvious hyperther-
mia. Further optimization efforts undertaken during the past
decade led to the development of several series of BCTC analogs. As
far as we know, the structure of BCTC can be divided into A-, B- and
C-regions (Fig. 1). These structural modifications mainly included
the following: (ⅰ) To improve the pharmacokinetic profile of BCTC,
the central piperazine core was bioisosterically replaced with 2-
methylpiperazine [10] or tetrahydropyridine ring [11,12]. (ⅱ)
Several amide [13] or quinazoline [14] and 6,6-heterocycle [15]
analogs have been prepared by substituting the piperazine urea in
* Corresponding author.
** Corresponding author.
E-mail addresses: yanlin@henu.edu.cn (L. Yan), qianhai24@163.com (H. Qian).
These authors contributed equally to this work.
1
https://doi.org/10.1016/j.ejmech.2020.112236
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

2
C. Nie et al. / European Journal of Medicinal Chemistry 194 (2020) 112236
Scheme 2. Synthesis of the target compounds 7. Reagents and conditions: (a) tri-
phosgene/DMAP, CH2Cl2, one pot.
Evodiamine and rutaecarpine possess vanilloid activity and can be
considered as conformationally restricted tryptamine derivatives,
which are similar to N-arachidonoylserotonin (an endogenous
modulator of TRPV1, AA-5-HT, Fig. 2) [19] to some extent. More
recently, we investigated the rigidified derivatives, designed using
conformationally constrained analogs of tryptamine with a triazole
ring, which behaved as relatively potent TRPV1 antagonists [20].
Here, with the aim to develop novel TRPV1 antagonists with
improved pharmacological profile, we designed novel analogs
based on the hypothesis that the conformationally restricted analog
of tryptamine can be used to replace the pyridine moiety of BCTC.
Based on the modification of the pyridine unit of BCTC, two series of
novel TRPV1 antagonists were synthesized and evaluated in this
study (Fig. 1).
2. Results and discussion
2.1. Chemistry
Fig. 1. Design of TRPV1 antagonists by chemical modification of the pyridine unit of
BCTC.
The target compounds, 4 and 7, were prepared as shown in
Schemes 1 and 2, respectively. In order to obtain compounds 4a-4z,
the synthesis of intermediate 3 was critical. Using a nucleophilic
aromatic substitution reaction, piperazine (2) was mostly intro-
duced at position 4 of 2,4-dichloropyrimidine (1). This reaction
yielded two regioisomers, and 2-chloro-4-(piperazin-1-yl) pyrim-
idine (3) was yielded as the major product. The regiochemistry of
this reaction was verified by X-ray crystallographic analysis of
compound 4g (Fig. 3), which also confirmed the structure of 3. In
the presence of triphosgene, intermediate 3 was coupled with the
aromatic amines to obtain the desired compounds 4a-4ae with
acceptable yield. In addition, another series of derivatives, 7a-7ab,
were also successfully synthesized by treating commercially
BCTC. (ⅲ) By employing the conformational restriction of imidazole
[16] or pyrrolidinyl [17] as a bioisosteric alternate for the amide
bond of BCTC, two series of novel TRPV1 antagonists were devel-
oped. The structural analysis of BCTC and its analogs revealed that
the chemical modification of the pyridine unit of BCTC is rare, and
most of these studies focused on the structure-activity relationship
(SAR) of regions B and C. Therefore, to enhance the structural
novelty of analogs of BCTC, we tried to replace the pyridine group
with suitable moieties that allow a better interaction with the
TRPV1 receptor. It is known that the pyrimidine moiety is widely
used in the pharmaceutical industry. Compared with pyridine
structure, pyrimidine structure provides more versatility in the
available tetrahydro-b-carboline (5) with aromatic amines and
synthesis because of lower
p-electron density [18]. Thus, we
triphosgene in the presence of N,N-dimethylaminopyridine
(DMAP). The details of chemical synthesis are presented in the
supporting information.
initially chose bioisosteric pyrimidine to replace the pyridine of
BCTC in order to increase the interaction with the TRPV1 receptor.
Fig. 2. Structure of AA-5-HT, evodiamine, and rutaecarpine.
Scheme 1. Synthesis of the target compounds 4. Reagents and conditions: (a) CH2Cl2, 0 ꢀC-rt; (b) aromatic amines, triphosgene/DMAP, CH2Cl2, one pot.

C. Nie et al. / European Journal of Medicinal Chemistry 194 (2020) 112236
3
significantly. This result suggests that the type of region A is
important for the activity of such antagonists. Modifications of aryl
moiety of BCTC were performed. As shown in Table 1, a variety of
substituents such as alkyl, halogen group, bulky group (tert-butyl
and i-propyl), and, even, the multi-substituted benzene groups
were investigated. We found that the introduction of electron-
withdrawing groups (for example fluoro or trifluoromethyl) on
the phenyl ring resulted in higher potency. In addition, 2-CF3 analog
4h was the most effective compound in this series, but its activity
was still significantly lower than that of the lead compound BCTC.
Furthermore, the substitution of the phenyl group at region C with
other types of aryl rings, that is, pyridine (4ac and 4ad) and iso-
quinoline (4ae), resulted in a dramatic loss of activity. This
prompted us to further investigate novel scaffolds.
Fig. 3. X-ray crystal structure of 4g.
2.2. In vitro evaluation
For comparison with the 2-chloropyrimidin analogs, the tetra-
hydro-b-carboline scaffold that has been proved to have TRPV1
activity [19,20], was coupled with the aryl fragment to prepare
ureas 7 (Table 2). As shown in Table 2, it is interesting to note that
replacing the 2-chloropyrimidin with tetrahydro-b-carboline
resulted in a significant increase in the antagonist activity. For
instance, the first compound of this class, 7a, which contains the
p-tBu-phenyl fragment, displayed more potent antagonism than 4a
We initiated the exploration of the SAR of pyrimidine analogs
4a-4ae. Compounds 4a-4ae were compared with the prototype
antagonist BCTC for their blockade ability of CAP or protons-
induced activation of human TRPV1 channels. Unexpectedly,
when the pyridine ring of BCTC was replaced with 2-
chloropyrimidin (4a), the activity of the antagonist decreased
Table 1
In vitro ability of compounds 4a-4ae to inhibit the activation of hTRPV1 receptors.
Compounds
Ar
hTRPV1(CAP)a IC50
(
mM)
hTRPV1(pH)b % inhib @ 10
mM
or % inhib @ 10 mM
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4ꢀ
4p
4q
4r
4s
4t
4u
4v
4w
4x
p-tBu-phenyl
o-Br-phenyl
m-Br-phenyl
p-Br-phenyl
o-F-phenyl
m-F-phenyl
p-F-phenyl
o-CF3-phenyl
29 6 %
1.072 0.615
1.451 0.647
ND
0.787 0.034
0.952 0.147
1.141 0.318
0.136 0.042
0.543 0.178
0.874 0.229
1.248 0.341
7.531 0.875
ND
9.417 1.014
41 7 %
0.854 0.269
37 6 %
ND
ND
35 12 %
ND
14
55
46
ND
66
57
3
4
7
7
9
45 11
94
61
53
31 11
29
ND
23
ND
62
ND
ND
ND
23
ND
17
5
9
7
m-CF3-phenyl
p-CF3-phenyl
p-Cl-phenyl
o-iPr-phenyl
4
6
8
m-iPr-phenyl
o-CH3O-phenyl
p-CH3O-phenyl
o-NO2-phenyl
3,4-diCH3O-phenyl
2,5-diCH3-phenyl
2,4-diCH3-phenyl
2,4,6-triCH3-phenyl
2-CH3-5-C2H5-phenyl
3-Cl-4-CH3-phenyl
2,5-diCl-phenyl
3,4-diCl-phenyl
4-Cl-2-NO2-phenyl
4-CH3-2-NO2-phenyl
2-CH3O-4-NO2-phenyl
2,4,6-triCl-phenyl
4,6-dimethylpyridin-2-
2-chloropyridin-3-
isoquinolin-5-
5
3
45 10 %
ND
54 9 %
43 5 %
ND
42 11 %
68 12 %
ND
ND
27
24
15
17
7
9
4
6
7
4y
4z
4aa
4 ab
4ac
4ad
4ae
BCTC
37
ND
ND
ND
97
ND
22 9 %
0.031 0.034
2
Unless otherwise stated, all values are the mean (SEM of at least three separate experiments).
ND, not determined.
a
Human TRPV1 receptor activated by capsaicin.
Human TRPV1 receptor activated by low pH (5.0).
b

4
C. Nie et al. / European Journal of Medicinal Chemistry 194 (2020) 112236
Table 2
In vitro ability of compounds 7a-7 ab to inhibit the activation of hTRPV1 receptors.
Compounds
Ar
hTRPV1(CAP)a IC50
(
m
M)
hTRPV1(pH)b % inhib @ 10
mM
or % inhib @ 10 mM
7a
7b
7c
7d
7e
p-tBu-phenyl
o-Br-phenyl
m-Br-phenyl
p-Br-phenyl
o-F-phenyl
m-F-phenyl
p-F-phenyl
o-CF3-phenyl
0.125 0.084
0.387 0.056
0.424 0.115
0.212 0.065
0.365 0.115
0.463 0.081
0.317 0.128
0.336 0.079
0.569 0.212
0.302 0.124
0.284 0.082
0.138 0.079
0.179 0.087
0.217 0.097
0.091 0.057
0.347 0.146
ND
0.684 0.122
0.389 0.175
0.594 0.115
0.765 0.148
0.568 0.126
0.098 0.075
ND
57
62 16
59 10
74
65
52 11
7
5
4
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7r
67
37
33
45
61 11
69 14
23
33
43
35
15
34
47
28
19
28
93
6
7
6
9
m-CF3-phenyl
p-CF3-phenyl
p-Cl-phenyl
o-iPr-phenyl
m-iPr-phenyl
6
8
7
6
5
6
9
6
8
5
7
o-CH3O-phenyl
p-CH3O-phenyl
2-CH3-5-C2H5-phenyl
4-CH3-2-NO2-phenyl
2,5-diCH3-phenyl
2,4-diCH3-phenyl
2-CH3O-4-NO2-phenyl
2,5-diCl-phenyl
3,4-diCl-phenyl
2,4,6-triCl-phenyl
4-Cl-2-NO2-phenyl
3-Cl-4-CH3-phenyl
3,4-diCH3O-phenyl
2-chloropyridin-3-
isoquinolin-5-
7s
7t
7u
7v
7w
7x
7y
7z
7aa
7 ab
BCTC
51 12
55 13
36 11
21
15
98
0.297 0.126
0.481 0.134
ND
8
7
2
ND
0.024 0.046
Unless otherwise stated, all values are the mean (SEM of at least three separate experiments).
ND, not determined.
a
Human TRPV1 receptor activated by capsaicin.
Human TRPV1 receptor activated by low pH (5.0).
b
against CAP and proton (pH 5.0) activation. An interesting trend was
observed during the analysis of SAR for substituents of benzene
moiety, performed to gain a deeper insight. In the CAP test, the
antagonism activity of para-substituted analogs was slightly
stronger than that of ortho- or meta-substituted analogs. Typical
examples include phenyls substituted with bromine (7d vs. 7b, 7c),
fluorine (7g vs. 7e, 7f), and trifluoromethyl (7j vs. 7h, 7i) (Table 2).
Furthermore, the electron-donating group favored the antagonism
activity of the second structural class. For instance, p-tBu-phenyl
(7a) and p-CH3O-phenyl (7o) analogs exhibited good antagonism.
Thereafter, we introduced multiple substitutions on the benzene
ring and replaced the benzene ring with heteroaryl groups (such as
pyridine and isoquinoline). As illustrated in Table 2, during the
introduction of multiple substitutions on the phenyl ring, a modest
decrease in antagonism activity was observed. Unexpectedly, a
complete loss in antagonism activity was observed when the phenyl
ring was replaced with a 2-chloropyridin ring (7aa) and isoquino-
line ring (7ab). In the pH assay, almost all analogs exhibited a partial
2.3. In vivo evaluation
On the basis of the in vitro studies, the selected compounds (4h,
7a, 7l, 7o, and 7w) were screened for further studies in vivo. It is
worth noting that the efficacy of the same TRPV1 receptor antag-
onist is different in different models of hyperalgesia. To evaluate the
analgesic activity of each compound, we performed nociception
tests in three different pain models, with BCTC as the control
(Fig. 4). The rats were administered a single oral dose of 30 mg/kg
test compounds, because oral administration of BCTC at 30 mg/kg
dose has been reported to exhibit potent analgesic activity in
various in vivo nociceptive trials in rats [23]. In the capsaicin model,
all test compounds significantly reduced the total paw licking time
compared with the vehicle. Particularly, compound 7o exhibited
higher potency than the positive control BCTC (Fig. 4A). In the
proton-induced abdominal constriction model, BCTC, 4h, and 7w
considerably reduced the number of writhes, whereas compounds
7a, 7l, and 7o showed a certain inhibitory effect (Fig. 4B). This is
consistent with results of studies in vitro, that is, 7a, 7l, and 7o
incompletely blocked proton (pH 5.0) activation, with a modest
inhibition activity of 57%, 69%, and 43%, respectively. In the case of
the tail-flick model, all compounds could increase %MPE in com-
parison with the vehicle. Interestingly, the highest %MPE produced
by 7o was significantly higher than that generated by the other
blockade (<74%) of protons-inducted activation of TRPV1 at 10 mM
dose, except for 2,4,6-triCl-phenyl (7w, 93%) derivative and BCTC
(98%), which manifested a full blockade of acid activation. It is
noteworthy that several temperature-neutral analogs have strong
antagonistic effects on capsaicin-induced activation of TRPV1, but
only partially blocked acid-activated TRPV1 [4,21,22].

C. Nie et al. / European Journal of Medicinal Chemistry 194 (2020) 112236
5
Fig. 4. Analgesic activities of synthesized compounds at 30 mg/kg dose after oral administration. (A) The antinociceptive effect in the capsaicin test; (B) suppression of acetic acid-
induced writhing response; (C) inhibition of thermal nociception by synthesized compounds. Each bar represents the mean SEM (n ¼ 6). Statistical analysis was evaluated using a
one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test. *p < 0.05, **p < 0.01, and, ***p < 0.001 compared with the vehicle group.
drugs, for unknown reasons (Fig. 4C). It was observed that all the
test compounds had a certain degree of antinociceptive activity. As
shown in Fig. 4, 7o showed good antinociceptive potency that was
more potent than BCTC, in the models of capsaicin- and heat-
induced pain. Importantly, 7o showed a weak effect on low pH,
indicating that it was probably free of hyperthermia.
In order to confirm our hypothesis, we carried out the body
temperature investigation of test compounds using a rectal ther-
mometer, with BCTC as the positive control (Fig. 5). Rats were
administered a single oral dose of 30 mg/kg, and the rectal body
temperatures were recorded prior to dosing and at 30, 60, 90, 120,
150, and 180 min after dosing. As shown in Fig. 5, a transient in-
crease in body temperature was observed in rats following BCTC
dosing. The mean temperatures of the BCTC group significantly
differed from that of the vehicle-treated group (ANOVA/Dunnett’s)
at 30 and 60 min post dosing. Similar to the results of BCTC, com-
pounds 4h, 7a, 7l, and 7w also caused transient hyperthermia in the
test rats, with the mean temperatures at some evaluated time
points being significantly higher than those in the control group. As
expected, there was no significant difference between the com-
pound 7o-treated and vehicle-treated groups from before dosing to
180 min post dosing. In addition, the dose-dependency of body
temperature was evaluated. As shown in Fig. 6, at all time points
and doses of 7o, the patterns of drug-related effects on body
temperature were not obvious (no other significant differences
were observed when compared with the control groups).
Fig. 6. Effects of compound 7o at different doses on body temperature in rats. Data are
expressed as mean SEM (n ¼ 6). *p < 0.05, **p < 0.01, and, ***p < 0.001 by Dunnett’s
multiple comparison test compared with the vehicle-treated group.
rapid absorption (Tmax
(278.163 86.843 mg/mL), high AUC (622.907
¼
0.106
0.020 h), high Cmax
60.132 mg/
mL ꢁ h), and modest half-life (t1/2 ¼ 1.581 0.653 h). As shown in
Table 3 and Fig. 7, the pharmacokinetic property of compound 7o
was obviously better than 4h when administered orally. Upon oral
administration, compound 7o exhibited slower absorption
(Tmax ¼ 3.933 0.389 h), lower clearance (CL 0.136 0.025 mL/h/
kg), and a longer half-life (t1/2 ¼ 3.085 0.484 h), which resulted in
reasonable residence time (7.95 0.86 h after a 10 mg/kg dose).
2.4. Molecular modeling
Due to the excellent in vivo and in vitro activities of compound
7o, the pharmacokinetic properties of this compound was assessed
in Sprague-Dawley rats (Table 3 and Fig. 7; the pharmacokinetic
parameters of compound 4h was also included for comparison).
Compounds 4h and 7o were administered at 10 mg/kg dose by oral
gavage to three rats. As shown in Table 3, compound 4h exhibited
Furthermore, the docking study was carried out with a rat
TRPV1 (PDB ID: 5IS0) model [24]. The binding interaction between
antagonist 7o and the receptor was analyzed, and its binding mode
was compared with that of BCTC.
Table 3
Pharmacokinetic parameters of 4h and 7o following oral administrationa to ratsb.
Parameters
Unit
Compound
4h
7o
t1/2, ka
h
h
1/h
1/h
mL/kg
mL/h/kg
h
mg/mL
mg/mL*h
h
0.016 0.002
1.581 0.653
44.072 5.783
0.479 0.198
0.036 0.011
0.016 0.002
0.106 0.020
278.163 86.843
622.907 60.132
2.304 0.945
2.426 0.109
3.085 0.484
0.286 0.013
0.227 0.036
0.613 0.204
0.136 0.025
3.933 0.389
7.050 2.001
74.848 13.564
7.951 0.855
t
1/2, k10
ka
k10
V
CL
Tmax
Cmax
AUC
MRT
0-inf
Fig. 5. Effects of compounds at 30 mg/kg dose after oral administration on body
temperature in rats. Data are expressed as mean SEM (n ¼ 6). *p < 0.05, **p < 0.01,
and, ***p < 0.001 by Dunnett’s multiple comparison test compared with the vehicle-
treated group.
a
Compound was prepared in 0.5% sodium carboxymethyl cellulose and admin-
istered at 10 mg/kg dose.
b
n ¼ 3.

6
C. Nie et al. / European Journal of Medicinal Chemistry 194 (2020) 112236
Fig. 7. (A) Plasma concentrations of compound 4h at each time point after intragastric gavage (10 mg/kg) in rats. (B) Plasma concentrations of compound 7o at each time point after
intragastric gavage (10 mg/kg) in rats. Each point is the average concentration, and the bars are standard deviations of the mean (n ¼ 3).
Fig. 8. Molecular modeling of compound 7o and BCTC. (A) Three dimensional model of BCTC that interacts with the key amino acids of rTRPV1. Hydrogen bonds are shown as green
dashed lines. (B) Three dimensional model of compound 7o that interacts with the key amino acids of rTRPV1. Hydrogen bonds are shown as green dashed lines. (C) Three
dimensional model of compounds in the hydrophobic pocket of rTRPV1. Compound 7o is depicted by sticks colored by atom type (C green, O red, N blue, polar H white). BCTC is
depicted by sticks colored by atom type (C yellow, O red, Cl green, N blue, polar H white). (D) Three dimensional model of compounds that interacts with the hydrophobic
transmembrane region of rTRPV1. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

C. Nie et al. / European Journal of Medicinal Chemistry 194 (2020) 112236
7
As illustrated in Fig. 8A, when binding to rTRPV1, BCTC achieved
https://doi.org/10.1016/j.ejmech.2020.112236.
favorable contacts with diverse residues of amino acids. An oxygen
atom of the urea formed a hydrogen bond with Thr550. The
piperazine ring occupied the main hydrophobic receptor slot and
interacted tightly with Ala665, Ala546, Phe591, and Met547 via
hydrophobic interaction. Furthermore, we also observed hydro-
phobic interactions between the 4-tert-butylphenyl group and
Leu553, Leu669; the 3-chloropyridine group and Met547, Phe543,
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Crystallographic data (excluding structure factors) on the
structure of compounds have been deposited in the Cambridge
Crystallographic Data Centre as supplementary publication
numbers CCDC-1965573 and 1965572. Copies of the data can be
obtained, free of charge, from CCDC, 12 Union Road, Cambridge CB2
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uk].
Declaration of competing interest
The authors declare that they have no known competing
financial interest or personal relationships that could have
appeared to influence the work reported in this paper.
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This study was financially supported by the National Natural
Science Foundation of China (U1704185) and the Key Scientific and
Technological Project of Henan Province (202102310147). We also
thank Dr. Mingxue Li for X-ray analysis of compounds 4g and 7o.
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