Phenylquinoline transient receptor potential vanilloid 1 antagonists for the treatment of pain: Discovery of 1-(2-phenylquinoline-4-carbonyl)-N-(4-(trifluoromethyl)phenyl)pyrrolidine-3-carboxamide

Article abstract of DOI:10.1016/j.bmc.2017.12.048

Reported herein is the design, synthesis, and pharmacologic characterization of a class of TRPV1 antagonists constructed on a phenylquinoline platform that evolved from Cinchophen lead. This design composes three sections: a phenylquinoline headgroup attached to an aliphatic carboxamides, which is tethered at a phenyl tail group. Optimization of this design led to the identification of 37, comprising a pyrrolidine linker and a trifluoromethyl–phenyl tail. In the TRPV1 functional assay, using cells expressed hTRPV1, 37 antagonized capsaicin-induced Ca²⁺ influx, with an IC₅₀ value of 10.2 nM. In the complete mice analgesic model, 37 exhibited better antinociceptive activity than the positive control BCTC in diverse pain models. All of these results suggested that 37 could be considered as a lead candidate for the further development of antinociceptive drugs.

Full text of DOI:10.1016/j.bmc.2017.12.048

Accepted Manuscript
Phenylquinoline Transient Receptor Potential Vanilloid 1 Antagonists for the
Treatment of Pain: Discovery of 1-(2-phenylquinoline-4-carbonyl)-N-(4- (tri-
fluoromethyl)phenyl)pyrrolidine-3-carboxamide
Chen Liao, Yan Liu, Chunxia Liu, Jiaqi Zhou, Huilan Li, Nasi Wang, Jieming
Li, Taiyu Liu, Hesham Ghaleb, Wenlong Huang, Hai Qian
PII:
S0968-0896(17)31996-X
https://doi.org/10.1016/j.bmc.2017.12.048
BMC 14153
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
13 October 2017
13 December 2017
29 December 2017
Please cite this article as: Liao, C., Liu, Y., Liu, C., Zhou, J., Li, H., Wang, N., Li, J., Liu, T., Ghaleb, H., Huang,
W., Qian, H., Phenylquinoline Transient Receptor Potential Vanilloid 1 Antagonists for the Treatment of Pain:
Discovery of 1-(2-phenylquinoline-4-carbonyl)-N-(4- (trifluoromethyl)phenyl)pyrrolidine-3-carboxamide,
Bioorganic & Medicinal Chemistry (2017), doi: https://doi.org/10.1016/j.bmc.2017.12.048
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Phenylquinoline Transient Receptor Potential Vanilloid 1 Antagonists for the
Treatment of Pain: Discovery of 1-(2-phenylquinoline-4-carbonyl)-N-(4-
(trifluoromethyl)phenyl)pyrrolidine-3-carboxamide
Chen Liaoa, Yan Liub,*, Chunxia Liua, Jiaqi Zhoua, Huilan Lia, Nasi Wanga, Jieming Lia,
Taiyu Liua, Hesham Ghaleba, Wenlong Huanga,c,*, Hai Qiana,c,*
a Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical
University, 24 Tongjiaxiang, Nanjing 210009, PR China
b School of Pharmacy, Chongqing Medical University, Chongqing 400016, PR China
c
Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease, China Pharmaceutical
University, 24 Tongjiaxiang, Nanjing 210009, PR China
* Corresponding authors. Tel./fax: +86 25 83271051.
E-mail addresses: drliuyan@126.com (Y. Liu), ydhuangwenlong@126.com (W. Huang),
qianhai24@163.com (H. Qian)
ABSTRACT: Reported herein is the design, synthesis, and pharmacologic
characterization of a class of TRPV1 antagonists constructed on a Phenylquinoline
platform that evolved from Cinchophen lead. This design composes three sections: a
phenylquinoline headgroup attached to an aliphatic carboxamides, which is tethered at
a phenyl tail group. Optimization of this design led to the identification of 37,
−phenyl tail. In
comprising a pyrrolidine linker and a trifluoromethyl
the TRPV1
functional assay, using cells expressed hTRPV1, 37 antagonized capsaicin-induced
Ca2+ influx, with an IC50 value of 10.2 nM. In the complete mice analgesic model, 37
exhibited better antinociceptive activity than the positive control BCTC in diverse pain
models. All of these results suggested that 37 could be considered as a lead candidate
for the further development of antinociceptive drugs.
Keywords: TRPV1; analgesia; Phenylquinoline.

1. Introduction
Transient receptor potential vanilloid subfamily type 1 (TRPV1) is a
thermosensitive, nonselective cation channel and a member of the TRP channel
superfamily.1,2 Structural features include six transmembrane domains, with
intracellular N- and C-termini. The channel is selectively activated by thermal stimuli
(>43 ), acidic pH (<6.8), and endogenous lipidic mediators (e.g., anandamide and
oxidative metabolites of linoleic acid). In humans, disease-related changes in TRPV1
expression have been demonstrated in patients suffering from painful diabetic
neuropathy, osteoarthritis, and rheumatoid arthritis as well as from a variety of visceral
disorders, including inflammatory bowel disease, inflammatory bowel syndrome,
detrusor hyperreflexia, and rectal hypersensitivity.3-6 Together, these results suggest an
important role for TRPV1 in the development and maintenance of chronic
inflammatory and neuropathic conditions.
During the past decade, various structural classes of TRPV1 antagonists have been
reported in the literature.7 Piperazinecarboxamides analogues such as BCTC are potent
TRPV1 antagonists, and diverse BCTC analogues have been synthesized to improve
the pharmaceutical and pharmacological profile (Fig.1). Lead compounds from this
series were highly potent and efficacious across a range of preclinical pain models.8
Despite this, development of lead compounds from this series was hindered due to a
variety of issues including poor aqueous solubility and oral bioavailability. We,
therefore, initiated our follow-up SAR effort with the aim of identifying novel potent
TRPV1 antagonists with improved pharmacokinetic properties, overall pharmaceutical
profile, and aqueous solubility. 6,9 Our strategy to achieve this goal was based on the
design, optimization, and pharmacologic characterization of TRPV1 antagonists
constructed on quinoline that evolved from GCR-6211, V349, A-1165442 and
Cinchophen -- several effective analgesics.10 (i) Modification of the ring C and A: we
investigate the influence of different substitutes of lipophillic sidechain and
pheylquinoline on TRPV1 analgesic activity; (ii) Modification of the ring B: we
introduce different linkers to improve aqueous solubility and pharmacological profile.
This SAR study effort has culminated with the identification of 1, a potent TRPV1
antagonist that is currently being evaluated in treatment of pain.

Figure 1. Graphical abstract approach based on piperazine carboxamides
2. Result and discussion
2.1 Chemistry
−3
The synthetic route employed to prepare analogues 9 4 is shown in Scheme 1.
Commercially available indoline-2,3-dione (2) reacted with acetophenone (3) in the
presence of KOH in ethanol to produce the 2-phenylquinoline-3-carboxylic acid (4a).
Commercially
available
substituted
anilines
(5)
reacted
with
N,N'-Carbonyldiimidazole (CDI) in Dimethyl Sulfoxide (DMSO) to produce
isocyanatobenzene (6). Intermediate 6 then gave the corresponding 1-Boc-piperazine in
79% yield. The Boc protecting group was removed by treatment with 4 N HCl in
dichloromethane, leading to a hydrochloride salt in 95% yield. Washing with alkaline
water and organic solvent respectively produce N-phenylpiperazine-1-carboxamide (8).
Chlorination of 4 with thionyl chloride and displacement of the chloride with 8 gave the
targets 9-34.
The synthetic route employed to prepare analogues 35−38 is shown in Scheme 2.
Chlorination of 4a reacted with thionyl chloride in dichloromethane to produce the acyl
chloride intermediate 39. Subsequent acyl chloride 39 reacted with γ-aminobutanoic
acid,
piperidine-4-carboxylic
acid
acid,
pyrrolidine-2-carboxylic
in the
acid,
and
of
pyrrolidine-3-carboxylic
respectively
presence
N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDCI) and
4-dimethylaminopyridine (DMAP) to give the corresponding targets 35-38 in moderate
yields.
Scheme1. Synthesis of A-ring and C-ring Modified Analogues 9-34

4f: R1 = 8-Cl, R2 = 4’-CH3
4g: R1 = H, R2 = 4’-CH3
4h: R1 = H, R2 = 4’-tBu
4a: R1 = H, R2 = H (Cinchophen)
4b: R1 = 8-CH3, R2 = H
4c: R1 = 8-Cl, R2 = H
4d: R1 = 8-F, R2 = H
4e: R1 = 8-CF3, R2 = H
Reagents and conditions: (a) KOH, EtOH, 80 ; (b) N,N'-Carbonyldiimidazole, DMSO; (c)
1-Boc-piperazine, DCM; (d) 4 M HCl in EtOAc, NaHCO3; (e) SOCl2, Et3N, DCM → rt. Total
yield: 20.76 %-59.29%.
Scheme2. Synthesis of Linker Modified Analogues 35-38
N
HO
O
4 a (Cinchophen)
Reagents and conditions: (a) SOCl2, Et3N, DCM; (b) substituted piperidine-4-carboxylic acid,
substituted pyrrolidine-2-carboxylic acid, substituted pyrrolidine-3-carboxylic acid, or substituted
→ rt.
4-aminobutanoic acid; (c) EDCI, DMAP, DCM
Total yield: 29.64 %-31.25%.
2.2 Biological studies

2.2.1 Evaluation of TRPV1 antagonism activity and SAR study of target compounds
In vitro TRPV1 assays designed to assess the functional inhibitory activity of test
compounds utilized highthroughput screening model, all the compounds inhibited Ca2+
inflow. The hTRPV1 (CAP) IC50 values obtained from eight-point
concentration−response studies are shown in Tables 1−3.
The data show that almost all of the compounds, except 9, 16, 17 and 21, emerged
as preferable inhibition of TRPV1. Especially, 22 (IC50=12.7 nM), 34 (IC50=12.2 nM),
37 (IC50=10.2 nM) and 38 (IC50=10.6 nM) exerted potency similarity with the positive
control BCTC (IC50=9.6 nM). SAR studies demonstrated that the three fluoromethyl is
optimal (vide infra). The initial optimization focused on the tail phenyl substitution
(Table 1), ortho-posion and para-position increased potency, as did a 4-methyl group
(10) and 2-methyl group (11), but meta-substituents (21) substantially decreased
potency. When the substituent is the para halogen atom (14), the activity increased
slightly, and the ortho halogen atom had not this feature (e.g., 16). In addition, the
activity of tert-butyl group substituded compound (19) was greatly improved, the
potency of 22 suggests that its 4-CF3 group is a suitable isostere for a tert-butyl group
(19), it exhibited stronger antagonism activity in vitro.
Besides, the results of aqueous solubility experiment show that the solubility of
9-38 is better than that of the positive control BCTC, except a few compounds (e.g., 11,
31). The ortho-posion was weaker than para-position on the tail phenyl substitution,
and Phenylquinoline R2 Substitution reduced solubility but R1 wouldn’t. The change
of linker L had little effect on solubility.
The results indicated that the varying TRPV1 antagonistic potencies of the
derivatives were attributed to the diverse substitution groups of the benzene ring and
the linker. To develop antinociceptive candidates in vivo, we test all the compounds for
further antinociceptive tests.
2.2.2 Evaluation of antinociceptive activity in vivo and SAR study of target compounds
To attest the preferable compounds, three different models of pain were selected
for antinociceptive activity in vivo. Antinociceptive activities results are shown in
−4
Figures 2
.
Firstly, we chose compounds 9-26 to be assayed in antinociceptive tests (Figure 2).
In the capsaicin test, 9-20 and 22-26 seemed to be as effective as compound 4, while 21
showed weak effects. Compounds 10, 12, 14, 17, 22, 23 etc. significantly reduced the

number of writhes in protoninduced pain models, especially 12, 22, 23 was superior to
positive control BCTC and compound 4, while 16 showed nearly no effects. In
treatment of heat-induced pain 12, 18, 22, 23 and 25 showed similar %MPE as BCTC
in the tail-flick test. All the test compounds had antinociceptive activity to a certain
extend. The initial optimization focused on the tail phenyl group with the
phenylquinoline moiety held constant, 22 exhibited good antinociceptive potency in
three different models of pain. It shows that when the -CF3 on phenyl group, compound
is favorable in vivo potency.
We next exaimed the effect of inducing different groups on phenylquinoline of the
lead compound 22 (Figure 3), but unfortunate all the compunds 27-34 is similar to 22
in vivo potency. Therefore, we speculate the changes on phenylquinoline have no effect
on TRPV1 antagonism activity. Then we focused our attention on fine-tuning the linker
(Figures 4). Result shows that the propylamine 37 is better than 22, the proton-induced
time spent licking shorter (37/22: 11.64±1.68/21.91±2.33) and heat-induced MPE％
more (37/22: 13.63±0.83/9.47±0.62), having significant difference with 22 in pain
models. All the results of biological assays in vitro and in vivo proved that 37 may be
considered as a promising candidate for development of potent agents for treatment of
pain.
Table 1. In Vitro hTRPV1 Functional Activity and Solubility of Tail Phenyl R3 Substitution
Solubility
hTRPV1(CAP)IC50
a(nM)
compd
R3
(mg/ml)
0.87
-H
9
136.2±7.6
43.5±2.2
4-CH3
0.86
10

2-CH3
4-OCH3
2-OCH3
4-Cl
0.39
2.63
2.20
0.92
0.92
0.91
0.88
0.62
0.85
11
12
13
14
15
16
17
18
19
40.7±1.8
76.0±2.7
45.2±1.3
80.3±3.5
37.0±0.9
250.3±9.4
125.2±4.2
16.4±1.1
36.4±1.7
3-Cl
2-Cl
2-F
4-nBu
4-tBu
2-Et
3-isopropyl
4-CF3
0.36
0.33
1.40
20
21
22
46.8±2.3
NDb
12.7±0.5
2-CF3
4-OCF3
4-NO2
1.15
2.68
0.88
0.73
0.62
23
24
47.1±1.4
46.0±3.2
39.6±1.9
81.9±2.6
9.2±1.2
25
26
BCTC

a Human TRPV1 receptor activated by capsaicin. Unless otherwise stated, all values are the mean
(SEM of at least three separate experiments).
b ND = not determined
Table 2. In Vitro hTRPV1 Functional Activity and Solubility of Phenylquinoline R1 and R2
Substitution
F
R2
F
F
O
N
N
N
H
N
R1
O
hTRPV1(CAP)IC
a(nM)
Solubility
(mg/ml)
compd
R1
R2
50
H
8-CH3
8-Cl
8-F
H
-H
1.40
0.77
0.82
0.65
0.89
0.46
0.83
0.71
0.98
22
27
28
29
30
31
32
33
34
12.7±0.5
40.5±1.6
70.3±2.3
14.0±1.2
37.6±1.9
25.7±2.0
12.8±1.1
12.3±0.8
12.2±1.5
-H
-H
8-CF3
8-Cl
-H
-H
4’-CH3
4’-CH3
4’-tBu
4’-OH
-H
-H

BCTC
0.62
9.2±1.2
a Human TRPV1 receptor activated by capsaicin. Unless otherwise stated, all values are the mean
(SEM of at least three separate experiments).
Table 3. In Vitro hTRPV1 Functional Activity and Solubility of Linker L
Solubility
hTRPV1(CAP)IC50
compd
L
a(nM)
12.7±0.5
22±1.3
(mg/ml)
1.40
22
35
0.95
0.90
36
37
14±0.7
0.93
10.2±1.9
0.89
0.62
38
10.6±1.1
9.6±1.2
BCTC
a Human TRPV1 receptor activated by capsaicin. Unless otherwise stated, all values are the mean
(SEM of at least three separate experiments).

Figure 2. Antinociceptive activities of Tail Phenyl R3 Substitution
(A) The antinociceptive effects 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 = 8). 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; ***p
<0.001 compared with the vehicle group.

Figure 3. Antinociceptive activities of Phenylquinoline R1 and R2 Substitution
(A) The antinociceptive effects 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 = 8). 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; ***p
<0.001 compared with the vehicle group. #p <0.05; ##p <0.01; ###p <0.001 compared with the
compoud 22.
Figure 4. Antinociceptive activities of Linker L
(A) The antinociceptive effects 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 = 8). 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; ***p
<0.001 compared with the vehicle group. #p <0.05; ##p <0.01; ###p <0.001 compared with the
compoud 22.
3. Conclusion
To sum up, we have synthesized 30 carbamide derivatives of phenylquinoline
TRPV1 antagonists and conducted SAR studies with the goal of identifying the novel
developed analgesic candidates. This was achieved through bioisosteres replacements
for the original piperazine and combined with phenyl quinoline compounds.
Preliminary biological evaluation revealed that several compounds presented TRPV1
antagonistic activity, antinociceptive activity and aqeous solubility. Especially, 37
showed superior potency in proton-induced and heat-induced pain models. Therefore,
37 may be considered as a good candidate which and held the most promising potential
for further development.
4. Experimental
4.1 Chemistry
All reagents and solvents were obtained from commercial sources and used
without further purification. Melting points were determined on RY-1 melting point
apparatus, and were not corrected. Mass spectra (MS) were acquired using a Waters
UPLC/MS (ACQUITY UPLC/TQD (Waters UPLC with the ACQUITY TQD [Waters
Corporation, Milford, MA, USA])) operating in electron spray ionization mode (ESI+)
and was used to confirm the purity of each compound. TLCs and preparative TLCs
were performed on silica gel GF/UV 254. Purifications by column chromatography
were carried out over silica gel (200–300 mesh) and visualized under UV light at 254
nm and 365 nm. Proton nuclear magnetic resonance (1H NMR) spectra were recorded
on Bruker ACF-300/500 MHz instruments. Chemical shifts were reported as δ values
(ppm) downfield from internal tetramethylsilane of the indicated organic solution. Peak
multiplicities were expressed as follows: s, singlet; d, doublet; t, triplet; q, quartet; dd,
doublet of doublet; br, broad singlet; m, multiplet. Coupling constants (J values) were

given in hertz (Hz).
4.1.1 General procedure for the preparation of 4
Isatin (0.5g, 3.34mmol) was added into a stirred solution of KOH (0.56g,
10.02mmol) in ethanol 10 ml at 80 . The reaction mixture was stirred at 0 for 24 h.
The mixture was added to 50 g ice water. The pH was adjusted by the solution of 2 N
HCl to 4-5. The mixture was cooled overnight and filtered. The residue was
recrystallized by the mixture of petroleum/ethyl acetate (5:1). Compound 4 (0.8 g) was
obtained and the yield was 96.87%.
4.1.2 General procedure for the preparation of 8
Aniline (1 g, 10 mmol) was dissolved in DMSO (10ml), adding CDI (2g, 12mmol)
into stirred solution. The reaction mixture was stirred at room temperature for 2h. The
reaction mixture was monitored by TLC and concentrated in vacuo. The resulting oil
was dissolved in methylene chloride (20ml), adding 1-Boc-piperazine (1.67g, 9mmol),
The reaction mixture was stirred at room temperature for 2h. The residue was purified
by flash column chromatography on silica gel using ethyl petroleum/ethyl acetate (1:1)
as eluent to obtain 8. Compound 4 (1.2 g) was obtained and the yield was 62.69%.
4.1.3 General procedure for the preparation of 9-38
2- phenyl quinoline -3- formic acid (0.5g, 2.01mmol) reacted with SOCl2 (0.4ml,
6.02mmol) and DMF (2-3 drops) in dichloromethane (10ml). The reaction mixture was
stirred at 40 for 2h, The reaction mixture was monitored by TLC and concentrated in
vacuo. N- phenyl piperazine -1- formamide (0.41g, 2.01mmol) dissolved in
dichloromethane (10ml), adding Et3N (0.56ml, 4.02mmol) into the above mixture
solution. The reaction mixture was stirred at room temperature for 12h. The residue was
purified by flash column chromatography on silica gel using ethyl petroleum/ethyl (1:6)
as eluent to obtain 9-34.
4.1.4 General procedure for the preparation of 35-38
2- phenyl quinoline -3- formic acid (0.2g, 0.8mmol) reacted with SOCl2 (0.3ml,
4mmol) and DMF (2-3 drops) in dichloromethane (10ml). The reaction mixture was
stirred at 40 for 2h, The reaction mixture was monitored by TLC and concentrated in
vacuo. The product was dissolved in dichloromethane (10ml) at 0 , and dropping Et3N
until white smoke disappeared. The gamma aminobutyric acid (0.08g, 0.8mmol) was
added into the mixture solution and stirring at room temperature for 12h, then adding

EDCI (0.17g, 0.88mmol) and a catalytic amount of DMAP, stirring for 2h, The reaction
mixture was monitored by TLC and concentrated in vacuo. The residue was purified by
flash column chromatography on silica gel using ethyl petroleum/ethyl (1:1) as eluent
to obtain 35-38.
N-phenyl-4-(2-phenylquinoline-4-carbonyl)piperazine-1-carboxamide (9)
Yield 64.0%, white solid, m.p. 143-145°C; 1H NMR (300 MHz, DMSO-d6) δppm: 8.64
(s, 1H, NH), 8.35 (d, J = 8.0 Hz, 2H, Ar-H), 8.27 - 8.07 (m, 2H, Ar-H), 7.87 (t, J = 7.8
Hz, 2H, Ar-H), 7.76 - 7.36 (m, 5H, Ar-H), 7.24 (t, J = 7.9 Hz, 2H, Ar-H), 6.95 (t, J = 7.3
Hz, 1H, Ar-H), 4.07 - 3.63 (m, 4H, piperazine), 3.32 (d, J = 18.2 Hz, 2H); 13C NMR (75
MHz, DMSO-d6) δppm: 166.47 , 156.32 , 155.48 , 148.13 , 143.75 , 140.76 , 138.54 ,
131.00 , 130.29 , 129.35 , 128.78 , 127.86 , 125.23 , 123.35 , 122.34 , 120.10 , 116.05 ,
46.93 , 44.46 , 44.06 ; ESI-MS m/z: 437.21 ([M + H]+). Anal. Calcd for C27H24N4O2: C,
74.29; H, 5.54; N, 12.84. Found: C, 74.31; H, 5.57; N, 12.85.
4-(2-phenylquinoline-4-carbonyl)-N-(p-tolyl)piperazine-1-carboxamide (10)
1
Yield 55.2%, yellow solid, m.p. 105-109°C; H NMR (300 MHz, DMSO-d6) δppm:
8.53 (s, 1H, NH), 8.34 (d, J = 6.5 Hz, 2H, Ar-H), 8.27 - 8.09 (m, 2H, Ar-H), 7.86 (t, J =
7.3 Hz, 2H, Ar-H), 7.76 - 7.46 (m, 4H, Ar-H), 7.31 (d, J = 8.4 Hz, 2H, Ar-H), 7.04 (d, J
= 8.3 Hz, 2H, Ar-H), 3.97 - 3.60(m, 4H, piperazine), 3.53 - 3.12 (m, 4H, piperazine),
2.22 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6) δppm: 166.45, 156.32, 155.56,
148.12, 143.76, 138.52, 138.14, 131.17, 131.01, 130.43, 130.17, 129.36, 129.21,
127.96, 127.78, 125.24, 123.35, 120.29, 119.24, 116.05, 46.95, 44.43, 44.03, 41.73,
20.80; ESI-MS m/z: 451.5 ([M + H]+). Anal. Calcd for C28H26N4O2: C, 74.65; H, 5.82;
N, 12.44. Found: C, 74.66; H, 5.83; N, 12.46.
4-(2-phenylquinoline-4-carbonyl)-N-(o-tolyl)piperazine-1-carboxamide (11)
1
Yield 43.7%, yellow solid, m.p. 143-145°C; H NMR (300 MHz, DMSO-d6) δppm:
8.36 (d, J = 6.5 Hz, 2H, Ar-H), 8.20 (d, J = 8.9 Hz, 2H, Ar-H), 8.03 - 7.80 (m, 2H, Ar-H),
7.69 (t, J = 7.1 Hz, 1H, Ar-H), 7.67 - 7.49 (m, 3H, Ar-H), 7.47 - 6.99 (m, 4H, Ar-H),
4.15 - 3.64 (m, 4H, piperazine), 3.62 - 2.94 (m, 4H, piperazine), 2.17 (s, 3H, CH3); 13C
NMR (75 MHz, DMSO-d6) δppm: 166.48 , 156.65, 156.36 , 156.18, 148.14 , 143.78 ,
138.56, 138.19, 133.60, 130.99, 130.68, 129.71 , 129.34, 127.86 , 126.32 , 125.17,
123.36, 116.05, 46.96, 44.64, 44.12, 41.74, 18.40; ESI-MS m/z: 467.2 ([M + H]+). Anal.
Calcd for C28H26N4O2: C, 74.65; H, 5.82; N, 12.44. Found: C, 74.67; H, 5.83; N, 12.46.
N-(4-methoxyphenyl)-4-(2-phenylquinoline-4-carbonyl)piperazine-1-carboxamide
(12)

1
Yield 58.8%, yellow solid, m.p. 101-103°C; H NMR (300 MHz, DMSO-d6) δppm:
8.67 - 8.32 (m, 1H, Ar-H), 8.18 (d, J = 6.8 Hz, 2H, Ar-H), 8.09 - 7.82 (m, 3H, Ar-H),
7.77 - 7.49 (m, 4H, Ar-H), 7.23 (d, J = 8.7 Hz, 2H, Ar-H), 6.81 (d, J = 8.6 Hz, 2H, Ar-H),
6.64 (s, 1H, NH), 4.20 - 3.85 (m, 2H, piperazine), 3.77 (s, 3H, OCH3), 3.73 - 3.54 (m,
2H, piperazine), 3.53 - 3.18 (m, 4H, piperazine); 13C NMR (75 MHz, DMSO-d6) δppm:
166.50, 156.36, 155.77, 155.07, 148.17, 143.80, 138.58, 133.73, 131.00, 130.32,
129.37, 127.88, 125.26, 123.39, 122.12, 116.07, 114.03, 55.58, 46.95, 44.44, 44.02,
41.76; ESI-MS m/z: 467.6 ([M + H]+). Anal. Calcd for C28H26N4O3: C, 72.09; H, 5.62;
N, 12.01. Found: C, 72.10; H, 5.65; N, 12.01.
N-(2-methoxyphenyl)-4-(2-phenylquinoline-4-carbonyl)piperazine-1-carboxamide
(13)
Yield 60.6%, pale yellow solid, m.p. 23-124°C; 1H NMR (300 MHz, DMSO-d6) δppm:
8.68 - 8.44 (m, 1H, Ar-H), 8.22 (d, J = 6.3 Hz, 2H, Ar-H), 8.03 - 7.78 (m, 3H), 7.78 -
7.48 (m, 4H, Ar-H), 7.12 (s, 1H, NH), 7.08 - 6.91 (m, 2H, Ar-H), 6.90 - 6.82 (m, 1H,
Ar-H), 4.24 - 3.92 (m, 2H, piperazine), 3.87 (s, 3H, OCH3), 3.83 - 3.69 (m, 2H,
piperazine), 3.61 - 3.18 (m, 4H, piperazine); 13C NMR (75 MHz, DMSO-d6) δppm:
166.54, 156.41, 155.46, 150.72, 148.20, 143.79, 138.60, 131.01, 130.42, 130.22,
129.37, 128.80, 127.97, 127.82, 125.24, 124.09, 123.39, 122.97, 120.67, 116.10,
111.37, 56.08, 46.85, 44.49, 43.93, 41.71; ESI-MS m/z: 467.2([M + H]+). Anal. Calcd
for C28H26N4O3: C, 72.09; H, 5.62; N, 12.01. Found: C, 72.11; H, 5.65; N, 12.03.
N-(4-chlorophenyl)-4-(2-phenylquinoline-4-carbonyl)piperazine-1-carboxamide (14)
Yield 56.3%, white solid, m.p. 126-128°C; 1H NMR (300 MHz, DMSO-d6) δppm: 8.77
(s, 1H, NH), 8.34 (d, J = 7.1 Hz, 2H, Ar-H), 8.27 - 8.05 (m, 2H, Ar-H), 7.96 - 7.71 (m,
2H, Ar-H), 7.67 (t, J = 7.5 Hz, 1H, Ar-H), 7.63 - 7.38 (m, 5H), 7.29 (d, J = 8.8 Hz, 2H),
4.00 - 3.60 (m, 4H, pyrrolidine), 3.59 - 3.02 (m, 4H, pyrrolidine); 13C NMR (75 MHz,
DMSO-d6) δppm: 166.53, 156.36, 155.28, 148.16, 143.74, 139.84, 138.56, 131.01,
130.31, 129.37, 128.68, 127.88, 125.96, 125.24, 123.37, 121.52, 116.08, 46.90, 44.44,
44.03, 41.73; ESI-MS m/z: 471.1 ([M + H]+). Anal. Calcd for C27H23ClN4O2: C, 68.86;
H, 4.92; N, 11.90. Found: C, 68.88; H, 4.93; N, 11.92.
N-(3-chlorophenyl)-4-(2-phenylquinoline-4-carbonyl)piperazine-1-carboxamide (15)
1
Yield 64.4%, yellow solid, m.p. 111-113°C; H NMR (300 MHz, DMSO-d6) δppm:
8.83 (s, 1H, NH), 8.35 (d, J = 6.6 Hz, 2H, Ar-H), 8.28 - 8.04 (m, 2H, Ar-H), 7.87 (t, J =
8.8 Hz, 2H, Ar-H), 7.79 - 7.47 (m, 5H, Ar-H), 7.40 (d, J = 8.3 Hz, 1H, Ar-H), 7.26 (t, J
= 8.1 Hz, 1H, Ar-H), 6.99 (d, J = 6.9 Hz, 1H, Ar-H), 4.00 - 3.63 (m, 4H, pyrrolidine),
3.62 - 3.09 (m, 4H, pyrrolidine); 13C NMR (75 MHz, DMSO-d6) δppm: 166.55, 156.37,
155.15, 148.18, 143.73, 142.46, 138.58, 133.26, 131.00, 130.32, 129.36, 127.87,
125.23, 123.37, 121.90, 119.31, 118.19, 116.09, 46.88, 44.46, 44.06, 41.72; ESI-MS

m/z: 471.2 ([M + H]+). Anal. Calcd for C27H23ClN4O2: C, 68.86; H, 4.92; N, 11.90.
Found: C, 68.87; H, 4.93; N, 11.92.
N-(2-chlorophenyl)-4-(2-phenylquinoline-4-carbonyl)piperazine-1-carboxamide (16)
1
Yield 67.4%, yellow solid, m.p. 109-110°C; H NMR (300 MHz, DMSO-d6) δppm:
8.25 (d, J = 8.4 Hz, 1H, Ar-H), 8.14 (d, J = 6.7 Hz, 2H, Ar-H), 7.76 (t, J = 9.6 Hz, 2H,
Ar-H), 7.69 - 7.45 (m, 4H, Ar-H), 7.27 (d, J = 5.5 Hz, 1H, Ar-H), 7.18 (d, J = 8.6 Hz, 2H,
Ar-H), 7.12 - 6.96 (m, 1H, Ar-H), 6.83-6.62 (m, 1H, Ar-H), 4.07 - 3.52 (m, 4H,
piperazine), 3.37 - 3.03 (m, 4H, piperazine); 13C NMR (75 MHz, DMSO-d6) δppm:
166.49, 156.34, 155.49, 148.13, 143.74, 138.55, 136.91, 130.99, 130.17, 129.73,
129.35, 128.77, 127.95, 127.78, 127.70, 127.50, 126.23, 125.23, 123.25, 116.07, 46.89,
44.61, 44.10, 41.70; ESI-MS m/z: 471.1([M + H]+). Anal. Calcd for C27H23ClN4O2: C,
68.86; H, 4.92; N, 11.90. Found: C, 68.88; H, 4.94; N, 11.93.
N-(2-fluorophenyl)-4-(2-phenylquinoline-4-carbonyl)piperazine-1-carboxamide (17)
1
Yield 56.1%, yellow solid, m.p. 103-105°C; H NMR (300 MHz, DMSO-d6) δppm:
8.20 (d, J = 7.0 Hz, 2H, Ar-H), 8.14 - 7.98 (m, 2H, Ar-H), 7.98 - 7.77 (m, 3H, Ar-H),
7.77 - 7.44 (m, 4H, Ar-H), 7.06 - 6.83 (m, 3H, Ar-H), 6.62 (s, 1H, NH), 4.25 - 3.71 (m,
4H, piperazine), 3.61 - 3.26 (m, 4H, piperazine); 13C NMR (75 MHz, DMSO-d6) δppm:
166.46, 157.40, 156.32, 155.55, 154.15, 148.12, 143.75, 138.53, 131.00, 130.42,
130.16, 129.71, 129.35, 129.00, 127.95, 127.78, 126.54, 125.24, 124.46, 123.35,
116.05, 115.80, 114.99, 46.89, 44.54, 44.11, 41.69; ESI-MS m/z: 455.2([M + H]+).
Anal. Calcd for C27H23FN4O2: C, 71.35; H, 5.10; N, 12.33. Found: C, 71.37; H, 5.11; N,
12.36.
N-(4-butylphenyl)-4-(2-phenylquinoline-4-carbonyl)piperazine-1-carboxamide (18)
Yield 77.4%, pale yellow solid, m.p. 109-110°C; 1H NMR (300 MHz, DMSO-d6) δppm:
9.06 (s, 1H, NH), 8.26 (s, 2H, Ar-H), 7.90 (d, J = 6.9 Hz, 3H, Ar-H), 7.77 (d, J = 6.7 Hz,
1H, Ar-H), 7.60 (s, 3H, Ar-H), 7.24 (s, 2H, Ar-H), 7.07 (d, J = 7.9 Hz, 2H, Ar-H), 6.73
13
(s, 1H, Ar-H), 4.19 - 3.63 (m, 4H, piperazine), 3.63 - 3.09 (m, 4H, piperazine); C
NMR (75 MHz, DMSO-d6) δppm: 168.89, 157.87, 148.71, 147.13 ,146.86, 132.05,
128.15, 126.24, 125.76, 123.03, 120.42, 112.49, 111.83, 111.30, 109.89, 56.73, 55.37,
47.00, 38.13, 34.55, 28.03; ESI-MS m/z: 493.2 ([M + H]+). Anal. Calcd for C31H32N4O2:
C, 75.58; H, 6.55; N, 11.37. Found: C, 75.61; H, 6.57; N, 11.38.
N-(4-(tert-butyl)phenyl)-4-(2-phenylquinoline-4-carbonyl)piperazine-1-carboxamide
(19)
Yield 62.5%, white solid, m.p. 115-117°C; 1H NMR (300 MHz, DMSO-d6) δppm: 8.55
(s, 1H, NH), 8.34 (d, J = 6.6 Hz, 2H, Ar-H), 8.24 - 8.02 (m, 2H, Ar-H), 7.86 (t, J = 7.6
Hz, 2H, Ar-H), 7.75 - 7.44 (m, 4H, Ar-H), 7.34 (d, J = 8.7 Hz, 2H, Ar-H), 7.24 (d, J =

8.6 Hz, 2H, Ar-H), 3.67 - 3.56 (m, 4H, piperazine), 3.55 - 3.02 (m, 4H, piperazine), 1.24
13
(s, 9H, tBu); C NMR (75 MHz, DMSO-d6) δppm: 165.99, 155.84, 155.14, 147.65,
144.15, 143.27, 138.05, 137.63, 130.50, 129.92, 129.69, 128.86, 127.45, 124.91,
124.75, 122.87, 119.51, 115.56; ESI-MS m/z: 493.3 ([M + H]+). Anal. Calcd for
C31H32N4O2: C, 75.58; H, 6.55; N, 11.37. Found: C, 75.60; H, 6.57; N, 11.38.
N-(2-ethylphenyl)-4-(2-phenylquinoline-4-carbonyl)piperazine-1-carboxamide (20)
Yield 79.1%, white solid, m.p. 111-113°C; 1H NMR (300 MHz, DMSO-d6) δppm: 8.39
- 8.11 (m, 3H, Ar-H), 8.11 - 7.71 (m, 3H, Ar-H), 7.71 - 7.44 (m, 4H, Ar-H), 7.25 - 7.03
(m, 3H, Ar-H), 6.21 (s, 1H, Ar-H), 4.13-3.87 (m, 4H, piperazine), 3.57 - 3.12 (m, 4H,
13
piperazine), 2.59 (q, J = 7.6 Hz, 2H), 1.23 (t, J = 7.6 Hz, 3H); C NMR (75 MHz,
DMSO-d6) δppm: 166.48, 156.40, 148.14, 143.78, 139.77, 138.56, 137.49, 130.99,
130.29, 129.35, 128.57, 127.87, 127.49, 126.19, 125.76, 125.23, 123.36, 116.06, 46.99,
44.64, 44.11, 41.76, 24.18, 14.43; ESI-MS m/z: 465.2 ([M + H]+). Anal. Calcd for
C29H28N4O2: C, 74.98; H, 6.08; N, 12.06. Found: C, 74.99; H, 6.07; N, 12.08.
N-(3-isopropylphenyl)-4-(2-phenylquinoline-4-carbonyl)piperazine-1-carboxamide
(21)
Yield 71.1%, yellow solid, m.p. 115-117°C; 1H NMR (300 MHz, DMSO-d6) δppm:
8.57 (s, 1H, NH), 8.34 (d, J = 6.6 Hz, 2H, Ar-H), 8.30 - 8.09 (m, 2H, Ar-H), 7.96 - 7.75
(m, 2H, Ar-H), 7.71 - 7.54 (m, 4H, Ar-H), 7.30 (d, J = 8.8 Hz, 2H, Ar-H), 7.14 (t, J = 7.7
Hz, 1H, Ar-H), 6.82 (d, J = 7.6 Hz, 1H, Ar-H), 3.99-3.50 (m, 4H, piperazine), 3.34 -
3.03 (m, 4H, piperazine), 2.93 - 2.77 (m, J = 13.7, 6.9 Hz, 1H, isoproyl), 1.17 (d, J = 6.9
Hz, 6H, isoproyl); 13C NMR (75 MHz, DMSO-d6) δppm: 166.46, 156.31, 155.47,
148.95, 148.13, 143.77, 140.71, 138.53, 130.99, 130.41, 130.17, 129.35, 128.65,
127.94, 127.77, 125.25, 123.36, 120.47, 118.03, 117.72, 116.04, 46.94, 44.45, 44.08,
41.74, 33.95, 24.35; ESI-MS m/z: 479.2 ([M + H]+). Anal. Calcd for C30H30N4O2: C,
75.29; H, 6.32; N, 11.71. Found: C, 75.29; H, 6.34; N, 11.72.
4-(2-phenylquinoline-4-carbonyl)-N-(4-(trifluoromethyl)phenyl)piperazine-1-carboxa
mide (22)
Yield 82.5%, pale yellow solid, m.p. 101-103°C; 1H NMR (300 MHz, DMSO-d6) δppm:
9.05 (s, 1H, NH), 8.34 (d, J = 6.8 Hz, 2H, Ar-H), 8.28 - 8.08 (m, 2H, Ar-H), 7.97 - 7.80
(m, 2H, Ar-H), 7.78 - 7.43 (m, 8H, Ar-H), 4.04-3.53 (m, 4H, piperazine), 3.52 - 3.10 (m,
4H, piperazine); 13C NMR (75 MHz, DMSO-d6) δppm: 165.99, 155.81, 154.54, 147.65,
144.23, 143.22, 138.05, 130.51, 129.93, 129.68, 128.87, 127.45, 127.29, 125.61,
124.76, 122.87, 118.87, 116.14, 115.58, 46.38, 43.95, 43.58, 41.19, 39.45; ESI-MS m/z:
505.2([M + H]+). Anal. Calcd for C28H23F3N4O2: C, 66.66; H, 4.60; N, 11.11. Found: C,
66.67; H, 4.62; N, 11.12.

4-(2-phenylquinoline-4-carbonyl)-N-(2-(trifluoromethyl)phenyl)piperazine-1-carboxa
mide (23)
Yield 35.4%, yellow solid, m.p. 94 ~ 96°C; 1H NMR (300 MHz, DMSO-d6) δppm: 8.93
(s, 1H, NH), 8.06 (d, J = 6.7 Hz, 2H, Ar-H), 7.73 - 7.52 (m, 2H, Ar-H), 7.47 - 7.32 (m,
2H, Ar-H), 7.24 - 7.05 (m, 8H, Ar-H), 3.88-3.37 (m, 4H, piperazine), 3.06 - 2.73 (m, 4H,
piperazine)；13C NMR (75 MHz, DMSO-d6) δ ppm: 160.38, 147.22, 147.06, 145.38,
143.33, 139.72, 131.23, 129.13, 127.92, 127.64, 127.10, 126.29, 125.83, 122.99,
116.05, 111.88, 111.42, 109.97, 56.72, 55.50, 54.85, 50.07, 47.02, 40.10, 38.98, 38.67,
28.01; ESI-MS m/z: 505.2 ([M + H]+). Anal. Calcd for C28H23F3N4O2: C, 66.66; H,
4.60; N, 11.11. Found: C, 66.67; H, 4.63; N, 11.12.
4-(2-phenylquinoline-4-carbonyl)-N-(4-(trifluoromethoxy)phenyl)piperazine-1-carbo
xamide (24)
Yield 33.5%, pale yellow solid, m.p. 95~97°C; 1H NMR (300 MHz, DMSO-d6) δppm:
8.84 (s, 1H, NH), 8.34 (d, J = 6.6 Hz, 2H, Ar-H), 8.30 - 8.12 (m, 2H, Ar-H), 7.86 (t, J =
7.9 Hz, 2H, Ar-H), 7.82 - 7.43 (m, 7H, Ar-H), 7.24 (d, J = 8.6 Hz, 2H, Ar-H), 4.23 -
3.40 (m, 4H, piperazine), 3.31 - 2.83 (m, 4H, piperazine); 13C NMR (75 MHz,
DMSO-d6) δppm: 166.46, 156.31, 155.29, 148.13, 143.72, 140.14, 138.53, 130.99,
130.29, 129.35, 127.85, 125.24, 123.35, 121.72, 121.07, 116.05, 46.89, 44.44, 41.70；
ESI-MS m/z: 521.4 ([M + H]+). Anal. Calcd for C28H23F3N4O3: C, 64.61; H, 4.45; N,
10.76. Found: C, 64.63; H, 4.43; N, 10.75.
N-(4-nitrophenyl)-4-(2-phenylquinoline-4-carbonyl)piperazine-1-carboxamide (25)
Yield 47.5%, pale yellow solid, m.p. 84~86°C; 1H NMR (300 MHz, DMSO-d6) δppm:
9.36 (s, 1H, NH), 8.34 (d, J = 6.6 Hz, 3H, Ar-H), 8.30 - 8.07 (m, 4H, Ar-H), 7.86 (t, J =
7.8 Hz, 2H, Ar-H), 7.81 - 7.63 (m, 3H, Ar-H), 7.57 (d, J = 7.3 Hz, 2H, Ar-H), 3.96 -
3.46 (m, 4H, piperazine), 3.31 - 2.90 (m, 4H, piperazine); 13C NMR (75 MHz,
DMSO-d6) δppm: 166.88, 156.74, 155.63, 144.67, 144.46, 143.99, 138.73, 133.94,
131.05, 130.32, 129.43, 128.04, 127.93, 126.06, 124.53, 123.66, 122.43, 119.70,
116.96, 46.86, 44.46, 44.13, 41.72; ESI-MS m/z: 482.1([M + H]+). Anal. Calcd for
C27H23N5O4: C, 67.35; H, 4.81; N, 14.54. Found: C, 67.37; H, 4.82; N, 14.56.
N-(4-(diethylamino)phenyl)-4-(2-phenylquinoline-4-carbonyl)piperazine-1-carboxami
de (26)
Yield 35.9%, yellow solid, m.p. 92~94°C; 1H NMR (300 MHz, DMSO-d6) δppm: 8.89

- 8.12 (m, 5H, Ar-H), 7.87 (d, J = 7.6 Hz, 2H, Ar-H), 7.77-7.39 (m, 4H, Ar-H), 7.18 (d,
J = 7.7 Hz, 2H, Ar-H), 6.60 (s, 1H, Ar-H), 4.01-3.56 (m, 4H, piperazine), 3.26 - 3.01 (m,
4H, piperazine), 2.50 (q, J = 6.3 Hz, 4H, diethylin) 1.02 (t, J = 6.3 Hz, 6H, diethylin);
13C NMR (75 MHz, DMSO-d6) δppm: 166.43, 156.31, 155.99, 148.12, 143.79, 138.53,
130.99, 130.16, 129.35, 128.90, 127.94, 127.77, 127.45, 125.24, 123.35, 122.86,
116.03, 112.65, 46.97, 44.43, 41.74, 12.84; ESI-MS m/z: 508.6 ([M + H]+). Anal. Calcd
for C31H33N5O2: C, 73.35; H, 6.55; N, 13.80. Found: C, 73.38; H, 6.57; N, 13.83.
4-(8-methyl-2-phenylquinoline-4-carbonyl)-N-(4-(trifluoromethyl)phenyl)piperazine-
1-carboxamide (27)
Yield 32.3%, yellow solid, m.p. 92~94°C; 1H NMR (300 MHz, DMSO-d6) δppm: 9.04
(s, 1H, NH), 8.39 (d, J = 6.8 Hz, 2H, Ar-H), 8.20 (s, 1H, Ar-H), 7.84 - 7.51 (m, 10H,
Ar-H), 3.99 - 3.66 (m, 4H, piperazine), 3.64 - 3.08 (m, 4H, piperazine), 2.86 (s, 3H,
CH3); 13C NMR (75 MHz, DMSO-d6) δppm: 166.75, 154.99, 146.93 (s), 144.70,
144.00, 138.86, 137.69, 130.86, 130.31, 129.35, 127.66, 126.11, 123.12, 119.40,
115.63, 46.86, 44.47, 44.09, 41.66, 18.18; ESI-MS m/z: 519.4 ([M + H]+). Anal. Calcd
for C29H25F3N4O2: C, 67.17; H, 4.86; N, 10.80. Found: C, 67.18; H, 4.88; N, 10.83.
4-(8-chloro-2-phenylquinoline-4-carbonyl)-N-(4-(trifluoromethyl)phenyl)piperazine-1
-carboxamide (28)
1
Yield 23.5%, yellow solid, m.p.133~135°C; H NMR (300 MHz, DMSO-d6) δppm:
9.05 (s, 1H, NH), 8.47 - 8.39 (m, 2H, Ar-H), 8.35 (s, 1H, Ar-H), 8.05 (d, J = 7.5 Hz, 1H,
Ar-H), 7.87 (d, J = 8.3 Hz, 1H, Ar-H), 7.74 - 7.74 (m, 2H, Ar-H), 7.65 - 7.53 (m, 5H,
Ar-H), 4.04 - 3.67 (m, 4H, piperazine), 3.58 - 3.14 (m, 4H, piperazine); 13C NMR (75
MHz, DMSO-d6) δppm: 166.08, 156.74, 155.03, 144.67, 144.40, 143.99, 138.13,
133.59, 131.02, 130.83, 129.43, 128.02, 127.94, 126.06, 124.88, 124.66, 122.41,
119.40, 116.90, 46.86, 44.47, 44.03, 41.76; ESI-MS m/z: 539.5 ([M + H]+). Anal. Calcd
for C28H22ClF3N4O2: C, 62.40; H, 4.11; N, 10.40. Found: C, 62.42; H, 4.12; N, 10.43.
4-(8-fluoro-2-phenylquinoline-4-carbonyl)-N-(4-(trifluoromethyl)phenyl)piperazine-1
-carboxamide (29)
1
Yield 28.9%, yellow solid, m.p. 98~100°C; H NMR (300 MHz, DMSO-d6) δppm:
9.04 (s, 1H, NH), 8.58 (d, 2H, J = 7.3 Hz, Ar-H), 8.32 (s, 1H, Ar-H), 8.06 - 7.45 (m,
10H, Ar-H), 4.05 - 3.59 (m, 4H, piperazine), 3.58 - 3.10 (m, 4H, piperazine); 13C NMR
(75 MHz, DMSO-d6) δppm: 166.08, 162.07, 156.58, 155.03, 144.71, 143.74, 138.19,
130.75, 129.42, 127.90, 126.12, 121.27, 119.40, 117.11, 115.13, 46.85, 44.27, 41.73;
ESI-MS m/z: 523.5([M + H]+). Anal. Calcd for C28H22F4N4O2: C, 64.36; H, 4.24; N,
10.72. Found: C, 64.37; H, 4.25; N, 10.74.

4-(2-phenyl-8-(trifluoromethyl)quinoline-4-carbonyl)-N-(4-(trifluoromethyl)phenyl)pi
perazine-1-carboxamide (30)
Yield 33.5%, pale yellow solid, m.p. 99~102°C; 1H NMR (300 MHz, DMSO-d6) δppm:
9.05 (s, 1H, NH), 8.58 - 8.35 (m, 3H, Ar-H), 8.29 (d, J = 7.1 Hz, 1H, Ar-H), 8.18 (d, J
= 8.0 Hz, 1H, Ar-H), 7.80 (t, J =7.7 Hz, 1H, Ar-H), 7.75 - 7.51 (m, 8H, Ar-H), 3.93 -
3.72 (m, 4H, piperazine), 3.52 - 3.03 (m, 4H, piperazine); 13C NMR (75 MHz,
DMSO-d6) δppm: 166.04, 156.65, 155.74, 155.04, 144.57, 143.77, 138.12, 133.61,
130.36, 129.41 , 127.08, 124.98, 124.41, 122.39, 116.37, 113.09, 55.61, 46.87, 44.40,
43.74; ESI-MS m/z: 573.5([M + H]+). Anal. Calcd for C29H22F6N4O2: C, 60.84; H, 3.87;
N, 9.79. Found: C, 60.86; H, 3.89; N, 9.80.
4-(8-methoxy-2-phenylquinoline-4-carbonyl)-N-(4-(trifluoromethyl)phenyl)piperazin
e-1-carboxamide (31)
Yield 38.9%, pale yellow solid, m.p. 99~102°C; 1H NMR (300 MHz, DMSO-d6) δ ppm:
8.62 - 8.16 (m, 4H, Ar-H), 8.06 (d, J = 6.5 Hz, 1H, Ar-H), 7.86 (d, J = 7.4 Hz, 1H, Ar-H),
7.80 - 7.48 (m, 5H, Ar-H), 7.34 (d, J = 9.0 Hz, 1H, Ar-H), 6.84 (d, J = 9.0 Hz, 1H, Ar-H),
4.13 - 3.76 (m, 4H, piperazine), 3.71 (s, 3H, CH3), 3.55 - 3.06 (m, 4H, piperazine); 13C
NMR (75 MHz, DMSO-d6) δppm: 166.04, 156.73, 155.74, 155.02, 144.47, 143.97,
138.11, 133.61, 130.94, 129.44 , 127.99, 124.78, 122.09, 116.87, 113.99, 55.55, 46.90,
44.40, 43.93, 41.79; ESI-MS m/z: 501.5 ([M + H]+). Anal. Calcd for C29H24ClF3N4O2:
C, 65.16; H, 4.71; N, 10.48. Found: C, 65.17; H, 4.73; N,10.50.
4-(2-(p-tolyl)quinoline-4-carbonyl)-N-(4-(trifluoromethyl)phenyl)piperazine-1-carbox
amide (32)
1
Yield 37.5%, pale yellow solid, m.p.105~107°C; H NMR (300 MHz, DMSO-d6) δ
ppm: 8.19 (d, J = 8.4 Hz, 1H, NH), 8.02 (d, J = 8.2 Hz, 2H, Ar-H), 7.71 (m, 3H, Ar-H),
7.60 - 7.08 (m, 8H, Ar-H), 4.02 - 3.46 (m, 4H, piperazine), 3.46 - 3.02 (m, 4H,
piperazine), 2.45 (s, 3H, CH3); 13C NMR (75 MHz, CDCl3) δppm: 167.63, 157.11,
154.31, 148.49, 142.00, 140.24, 135.90, 130.50, 129.77, 127.37, 126.18, 123.94,
122.77, 119.36, 115.71, 46.63, 44.25, 41.51, 21.35; ESI-MS m/z: 519.5([M + H]+).
Anal. Calcd for C29H25F3N4O2: C, 67.17; H, 4.86; N, 10.80. Found: C, 67.17; H, 4.84;
N,10.79.
4-(2-(4-(tert-butyl)phenyl)quinoline-4-carbonyl)-N-(4-(trifluoromethyl)phenyl)pipera
zine-1-carboxamide (33)
Yield 45.3%, pale yellow solid, m.p. 99~102°C; 1H NMR (300 MHz, DMSO-d6) δ ppm:

9.03 (s, 1H, NH), 8.26 (d, J = 8.2 Hz, 2H, Ar-H), 8.15 (d, J = 7.3 Hz, 2H, Ar-H), 7.85 (t,
J = 8.6 Hz, 2H, Ar-H), 7.63 - 7.46 (m, 7H, Ar-H), 4.00 - 3.53 (m, 4H, piperazine), 3.52
13
- 2.96 (m, 4H, piperazine), 1.33 (s, 9H, tBu); C NMR (75 MHz, DMSO-d6) δppm:
166.56 , 156.31, 155.04, 153.15, 148.20, 144.73, 143.56, 135.88, 135.51, 135.24,
130.92, 130.14, 127.66, 126.13, 125.24, 123.29, 119.39, 115.96, 46.88, 44.49, 44.12,
41.70, 34.97, 31.47; ESI-MS m/z: 561.7([M + H]+). Anal. Calcd for C32H31F3N4O2: C,
68.56; H, 5.57; N, 9.99. Found: C, 68.57; H, 5.58; N,10.02.
4-(2-(4-hydroxyphenyl)quinoline-4-carbonyl)-N-(4-(trifluoromethyl)phenyl)piperazin
e-1-carboxamide (34)
Yield 39.6% , pale yellow solid, m.p. 96~98°C; 1H NMR (300 MHz, DMSO-d6) δ ppm:
9.07 (s, 1H, NH), 8.37 (d, J = 6.8 Hz, 2H, Ar-H), 8.33 - 8.28 (m, 2H, Ar-H), 8.07 - 7.89
(m, 2H, Ar-H), 7.85 - 7.56 (m, 8H, Ar-H), 5.77 (s, 1H, OH), 4.24-3.59 (m, 4H,
13
piperazine), 3.57 - 3.19 (m, 4H, piperazine); C NMR (75 MHz, DMSO-d6) δ ppm:
156.80, 147.74, 147.16, 146.86, 145.32, 143.14, 127.06, 126.25, 125.78, 122.94,
118.99, 116.11, 114.31, 111.86, 111.48, 109.88, 56.74, 55.45, 54.84, 50.08, 46.99,
38.65, 27.99; ESI-MS m/z: 525.6([M + H]+). Anal. Calcd for C28H23F3N4O3: C, 64.61;
H, 4.45; N, 10.76. Found: C, 64.62; H, 4.48; N,10.78.
1-(2-phenylquinoline-4-carbonyl)-N-(4-(trifluoromethyl)phenyl)piperidine-4-carboxa
mide (35)
Yield 39.6%, yellow solid, m.p. 107~109°C;1H NMR (300 MHz, DMSO-d6) δ ppm:
10.35 (s, 1H, NH), 9.94 - 8.02 (m, 5H, Ar-H), 8.01 - 6.69 (m, 9H, Ar-H), 3.28 - 2.60 (m,
4H, piperidine), 2.06 (m, 1H, piperidine), 1.96 - 0.65 (m, 4H, piperidine); 13C NMR (75
MHz, DMSO-d6) δppm: 173.81, 166.14, 156.34, 148.16, 144.17, 143.25, 138.57,
130.99, 130.37, 130.20, 129.34, 127.81, 126.47, 125.14, 123.44, 119.47, 115.73, 46.66,
43.09, 42.96, 29.42, 28.91; ESI-MS m/z:504.5([M + H]+). Anal. Calcd for
C29H24F3N3O2: C, 69.18; H, 4.80;N, 8.35. Found: C, 69.20; H, 4.82; N,8.37.
1-(2-phenylquinoline-4-carbonyl)-N-(4-(trifluoromethyl)phenyl)pyrrolidine-2-carbox
amide (36)
Yield 45.3%, pale yellow solid, m.p. 99~102°C; 1H NMR (300 MHz, DMSO-d6) δ ppm:
10.56 (s, 1H, NH), 8.60 - 8.02 (m, 5H), 7.84 - 8.73 (m, 3H, Ar-H), 7.82- 7.66 (m, 6H,
Ar-H), 4.65 (t, 1H, J = 6.1 Hz, pyrrolidine), 3.92 - 3.69 (m, 2H, pyrrolidine), 2.47 - 2.19
(m, 2H, pyrrolidine), 2.08 - 1.93 (m, 2H, pyrrolidine); 13 C NMR (75 MHz, DMSO-d6)
δppm: 170.86, 167.13, 157.06, 148.48, 143.74, 141.66, 138.81, 131.14, 129.67, 128.94,
127.42, 125.82, 124.76, 122.67, 120.38, 118.79, 115.83, 61.67, 58.34, 49.34, 47.85;
ESI-MS m/z:490.5([M + H]+). Anal. Calcd for C28H22F3N3O2: C, 68.70; H, 4.53; N,
8.58. Found: C, 68.73; H, 4.55; N,8.60.

1-(2-phenylquinoline-4-carbonyl)-N-(4-(trifluoromethyl)phenyl)pyrrolidine-3-carbox
amide (37)
Yield 39.8%, pale yellow solid, m.p. 89~91°C ; 1H NMR (300 MHz, DMSO-d6) δ ppm:
10.45 (s, 1H, NH), 10.05 - 8.00 (m, 4H, Ar-H), 8.00 - 6.49 (m, 9H, Ar-H), 4.31 - 3.80
(m, 2H, pyrrolidine), 3.80 - 3.53 (m, 2H, pyrrolidine), 2.51 (m, 1H, pyrrolidine), 2.45 -
1.95 (m, 2H, pyrrolidine); 13C NMR (75 MHz, DMSO-d6) δppm: 172.18, 171.50,
165.93, 156.43, 148.27, 144.76, 143.70, 138.60, 130.89, 130.12, 129.34, 129.07,
127.78, 127.74, 126.56, 125.30, 122.95, 119.66, 119.54, 115.93, 48.37, 47.93, 43.60,
30.12; ESI-MS m/z: 490.3 ([M + H]+). Anal. Calcd for C28H22F3N3O2: C, 68.70; H,
4.53; N, 8.58. Found: C, 68.72; H, 4.56; N,8.60.
N-(4-oxo-4-((4-(trifluoromethyl)phenyl)amino)butyl)-2-phenylquinoline-4-carboxami
de (38)
1
Yield 45.3%, pale yellow solid, m.p.101~103°C ; H NMR (300 MHz, DMSO-d6) δ
ppm: 10.36 (s, 1H, NH), 8.93 (s, 1H, Ar-H), 8.32 (d, J = 6.4 Hz, 2H, Ar-H), 8.27 - 7.91
(m, 3H, Ar-H), 7.83 (d, J = 7.5 Hz, 2H, Ar-H), 7.75 - 7.15 (m, 5H, Ar-H), 3.46 (t, J = 5.7
Hz, 2H, CH2), 2.61 - 2.47 (m, 2H, CH2), 1.99 (t, J = 6.7 Hz, 2H, CH2); 13C NMR (75
MHz, DMSO-d6) δppm: 172.03, 167.14, 156.23, 148.37, 143.61, 143.28, 138.68,
130.91, 130.73, 130.46, 129.96, 129.34, 127.65, 126.44, 125.85, 123.85, 119.31,
117.10, 114.99, 39.26, 34.38, 25.1; ESI-MS m/z: 478.3 ([M + H]+). Anal. Calcd for
C27H22F3N3O2: C, 67.92; H, 4.64; N, 8.80. Found: C, 67.94; H, 4.66; N,8.81.
4.2 Biological studies
4.2.1 Evaluation of TRPV1 antagonism activity
The TRPV1 aequorin cells (PerkinElmer, USA) were collected from culture plates
with Ca2+ and Mg2+-free phosphatebuffered saline supplemented with 5 mM
ethylenediaminetetra-acetic acid; pelleted for 2 min at 1000g; resuspended in
Dulbecco’s minimum essential medium–F12 medium with 15 mM HEPES (pH 7.0)
and 0.1% BSA (assay buffer) at a density of 3×105 cells/ml; incubated for 4 h in the
dark in the presence of 5 mM coelenterazine h (Promega, USA). After loading, cells
were diluted with assay buffer to a concentration of 5×106 cells/ml. Twenty microliters
of cells were injected over 20 mL of the sample solution plated on 384-well plates,
respectively, unless otherwise indicated. The digitonin, ATP (Sigma–Aldrich, USA)
assay buffer was added in the blank control wells for reference, and final concentrations
of digitonin and ATP were 100 mM and 50 mM. The sample solution and the cells were
incubated for 2.5 min before adding agonist capsaicin (Tocris, England), then
immediately detected. The light emission was recorded during variable times using

EnVision 2014 Multilabel Reader (PerkinElmer, USA). 11,12 Cells were exposed to test
compounds at varying concentrations, and intracellular Ca2+ was measured for 5 min.
All data points are the average value obtained from quadruplicate wells in the same
assay plate. IC50 values were obtained by eight-point nonlinear regression of sigmoidal
concentration-response modeled by GraphPad Prism 6.02 (Graph Pad Software Inc.,
CA, USA).
4.2.2 Evaluation of antinociceptive activity in vivo
Healthy male mice weighting 18-20 g and were purchased from laboratory animal
center of Nanjing Qinglongshan. Animal license number: SYXK(Su)2017-0001.
Separate groups of male mice (n = 6) were pretreated with compounds (30 mg/kg
unless otherwise indicated) 30 min before the test. Mice were kept at 24±1 °C and 55±
10% humidity, with 12 h of light (artificial illumination; 08:00–20:00) for 1 week.
Capsaicin test: The capsaicin test was assessed as previously described.13
Following an acclimation period of 30 min, 20 µl of solution of capsaicin (0.8 µg/ml)
was injected subcutaneously into the dorsal aspect of the right hind paw. The mouse
was then placed in an individual cage. Mice were observed for a continuous period of 5
min. The amount of time spent licking the injected paw was measured and expressed as
the cumulative licking time during the 5 min observation period.
Abdominal constriction test: Abdominal constriction test was performed as
described previously. Mice were placed in individual glass cylinders for a 30 min
acclimatization period. The writhes were induced by injection with 0.6% acetic acid
(0.1 mL/10 g/mouse i.p.), and immediately placed inside transparent glass cylinders.14
The number of muscular contractions was counted for 15 min after acetic acid injection.
Tail-flick test: Tail-flick test was performed as described previously.15 In the
experiment evaluating the time course of antinociception, we elected to use the warm
water tail withdrawal test in order to minimize damage to the tail because of the
repeated testing. In brief, the distal one-third of the tail was immersed into 52.0 water
and latency to withdrawal the tail was recorded before and after drug
treatment. The antinociception response was presented as percent maximal possible
effect (%MPE) as defined by percent maximal possible effect=100%×(drug response
time - basal response time)/(cut-off time - basal response time). To avoid tissue damage,
the cut-off time was suitable for 12 s.
All animal experimental protosols were approved by the ethical committee at
China Pharmaceutical University and conducted according to the Laboratory Animal

Management Regulations in China and adhered to the Guide for the Care and Use of
Laboratory Animals published by the National Institutes of Health (NIH Publication
NO. 85-23, revised 2011).
Statistical analysis of the data
Statistical analyses were performed using specific software (GraphPadInStat
version 5.00; GraphPad Software, San Diego, CA, USA). Unpaired comparisons were
analyzed using the two-tailed Student t-test, unless otherwise stated.
Acknowledgments
This study was supported by National Natural Science Foundation of China (No.
81673299) and Scientific and Technological Research Program of Chongqing
Municipal Education Commission (No.KJ1702012).
References and notes
[1] William H. P.; Raul R. C.; Wing C.; Lee Y.; Sharmila P.; Liu, J.; Mark A. Y.; Scott L.
D.; Dennis S.; Qin N.; Tasha H.; Mary L. L.; Zhang S.; Michael F.; Liu Y.; Michael R.
B.; Christopher M. F.; Mark R. P. J. Med. Chem. 2015, 58, 3859-3874.
[2] Brederson, J. D.; Kym, P. R.; Szallasi, A. Eur. J. Pharmacol. 2013, 716, 61-67.
[3] Mojgan A. T.; Pier G. B.; Stefania B.; Stefania G.; Stefania M.; Pier A. B. Medicinal
Research Reviews 2016, 0, 1-47.
[4] Liao M.; Cao E.; David J.; Cheng Y. Nature 2013, 504(7478): 107-112.
[5] Szallasi A.; Sheta M. Expert Opinion on Investigational Drugs 2012, 21,
1351-1369.
[6] Laykea T.; Toshiyuki K.; Noriyuki K.; Yu J.; Toshiyuki A.; Wu G.; Yuka I.;
Yoshitaka Y.; Ni C.; John E.; Naoki T.; Aniket P.; Zhou X.; Takuya S.; Kevin B.;
Tsuyoshi H.; Manjunath S.; Yasuyoshi I.; Akira K.; Donald J. K. J. Med. Chem. 2014,
57, 6781-6794.
[7] (a) Kyle, D. J.; Tafesse, L. Expert Opin. Ther. Pat. 2006, 16, 977-996 (b) Voight, E.
A.; Kort, M. E. Expert Opin. Ther. Pat. 2010, 20, 1107-1122.
[8] (a) Tafesse, L.; Sun, Q.; Schmid, L. A.; Valenzano, K. J.; Rotshteyn, Y.; Su, S.; Kyle,
D. J. Med. Chem. Lett. 2004, 14, 5513-5519. (b) Sun, Q.; Tafesse, L.; Islam, K.; Zhou,

X.; Victory, S. F.; Zhang, C.; Hachicha, M.; Schmid, L. A.; Patel, A.; Rotshteyn, Y.;
Valenzano, K. J.; Kyle, D. J. Bioorg. Med. Chem. Lett. 2003, 13, 3611-3616.
[9] Reilly, R. M.; McDonald, H. A.; Puttfarcken, P. S.; Joshi, S. K.; Lewis, L.; Pai, M.;
Franklin, P. H.; Segreti, J. A.; Neelands, T. R.; Han, P.; Chen, J.; Mantyh, P. W.;
Ghilardi, J. R.; Turner, T. M.; Voight, E. A.; Daanen, J. F.; Schmidt, R. G.; Gomtsyan,
A.; Kort, M. E.; Faltynek, C. R.; Kym, P. R. J. Pharmacol. Exp. Ther. 2012, 342, 416.
[10] Kean W.F., Buchanan W.W. Inflammopharmacology 2005, 13, 343-370.
[11] Le Poul, E.; Hisada, S.; Mizuguchi, Y.; Dupriez, V. J.; Burgeon, E.; Detheux, M. J.
Biomol. Screening 2002, 7, 57-65.
[12] Lehto, S. G.; Tamir, R.; Deng, H.; Klionsky, L.; Kuang, R.; Le, A.; Lee, D.; Louis,
J. C.; Magal, E.; Manning, B. H.; Rubino, J.; Surapaneni, S.; Tamayo, N.; Wang, T.;
Wang, J.; Wang, J.; Wang, W.; Youngblood, B.; Zhang, M.; Zhu, D.; Norman, M. H.;
Gavva, N. R. J. Pharmacol. Exp. Ther. 2008, 326, 218-229.
[13] Wang, J.; Dai, D.; Qiu, Q.; Deng, X.; Lin, H.; Qian, H.; Huang, W. Chem. Biol.
Drug Des. 2015, 85, 623.
[14] Tang, L.; Chen, Y.; Chen, Z.; Blumberg, P. M.; Kozikowski, A. P.; Wang, Z. J. J.
Pharmacol. Exp. Ther. 2007, 321, 791.
[15] Ge L.; Zhang X.; Zhang H.; Qian H.; Huang W.; Zhou J. Lett. Drug Des. Discov.
2011, 8, 76-81.

Amining to develop pharmacological and improve aqueous solubility, we have identified series of
TRPV1 antagonists bearing phenylquinoline moiety, exemplified by the antinoceptive potent and
solubility antagonist 37.