Novel benzodiazepines derivatives as analgesic modulating for Transient receptor potential vanilloid 1

Article abstract of DOI:10.1016/j.bmc.2018.08.002

A new series of derivatives of 3-(7-chloro-5-(2-fluorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)propanoic acid were designed and synthesized as analgesic modulating for Transient receptor potential vanilloid 1. They were investigated for TRPV1 antagonistic activity in vitro, analgesic activity and sedative activity in vivo and aqueous solubility. Preliminary studies identified 3-(7-chloro-5-(2-fluorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)-N,N-dimethylpropanamide(Compound 11), as a potent analgesic modulating for TRPV1 with potent activity and good aqueous solubility.

Full text of DOI:10.1016/j.bmc.2018.08.002

Accepted Manuscript
Novel Benzodiazepines Derivatives as analgesic Modulating for Transient re-
ceptor potential vanilloid 1
Yan Liu, Chen Liao, Jiaqi Zhou, Chunxia Liu, Qifei Li, Yue Jiang, Hai Qian
PII:
S0968-0896(18)31210-0
https://doi.org/10.1016/j.bmc.2018.08.002
BMC 14489
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 July 2018
28 July 2018
1 August 2018
Please cite this article as: Liu, Y., Liao, C., Zhou, J., Liu, C., Li, Q., Jiang, Y., Qian, H., Novel Benzodiazepines
Derivatives as analgesic Modulating for Transient receptor potential vanilloid 1, Bioorganic & Medicinal
Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.08.002
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Novel Benzodiazepines Derivatives as analgesic Modulating for Transient
receptor potential vanilloid 1
Yan Liua,* , Chen Liaob, Jiaqi Zhoub, Chunxia Liub, Qifei Lib, Yue Jianga, Hai Qianb,c,*
a School of Pharmacy, Chongqing Medical University, 1 Yixueyuan Road, Chongqing 400016, PR
China
b
Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical
University, 24 Tongjiaxiang, Nanjing 210009, 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: y.liu@cqmu.edu.cn (Y. Liu), qianhai24@163.com (H. Qian)
Abstract
A
new
series
of
derivatives
of
3-(7-chloro-5-(2-fluorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)propanoic
acid were designed and synthesized as analgesic modulating for Transient receptor potential
vanilloid 1. They were investigated for TRPV1 antagonistic activity in vitro, analgesic
activity and sedative activity in vivo and aqueous solubility. Preliminary studies identified
3-(7-chloro-5-(2-fluorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)-N,N-dimet
hylpropanamide(Compound 11), as a potent analgesic modulating for TRPV1 with potent
activity and good aqueous solubility.
Keywords: benzodiazepine, transient receptor potential vanilloid 1, analgesic, sedation

1. Introduction
Pain is an unpleasant feeling and also a global health challenge, which has strong clinical
demand. In the past two decades, advancements have come in the form of quality
improvement initiatives, the introduction of novel analgesics, the advent of innovative
techniques and improved understanding of pain signaling[1]. Unfortunately, most analgesic
drugs on the market today is saddled by some side-effects[2]. Transient receptor potential
vanilloid type 1 (TRPV1) is a non-selective cation channel and found on unmyelinated C
fibers in the periphery, for which many have been developed and entered clinical trials for the
treatment of pain [3, 4]. TRPV1 antagonists are usually made up of three parts, including: aryl
interaction head, H-bond interaction linker and lipophilic sidechain tail [5]. For example, the
TRPV1 antagonist BCTC (1), containing the three parts described above, possesses the potent
analgesic activity.
Moreover, benzodiazepines drugs, which act on γ-aminobutyric acid type A (GABAA),
have a strong sedative anesthesia effect and are widely used in postoperative analgesia[6]
(Fig.1), Diazepam (2) is usually used to be as sedative-hypnotic medicine. And Midazolam (3)
was optimized from Diazepam, which possesses potent sedative and anesthetic activity[7].
Furthermore, optimization of adding the side chain of methyl propionate make Remimazolam
(4) become safer and more effective, for its metabolite having very little activity, so that
Remimazolam exerts and loses efficacy quickly[8]. However, a variety of side effects
including dependence, unwanted sedation and amnesia, complicating their long-term
use[9-12]. Thus, there is a great need for novel, potent analgesic drugs with improved safety
and tolerability.
In this context, in order to keep analgesia potency and reduce the side effects, we regard
aromatic area of benzodiazepine derivatives as aryl interaction head, and γ-aminobutyric as
linker, and connect them to different lipophilic sidechain by using amide bond, resulting in
novel benzodiazepine derivatives as analgesic modulated for Transient receptor potential
vanilloid 1.

Figure 1. Structure of BCTC and benzodiazepine sedative drugs
Figure 2. Modified benzodiazepines structure by amidation according to the common
structure of TRPV1 antagonists
2. Result and Discussion
2.1 Chemistry
The synthetic route employed to prepare benzodiazepine derivatives 11-23 is shown in
Scheme 1. Commercially available 2-Amino-5-chloro-2'-fluorobenzophenone reacted with
Fmoc-L-Glu(OMe) under the catalysis of dicyclohexylcarbodiimide (DCC) in
dichloromethane at room temperature for 4 h to produce intermediate 7. The Fmoc protecting
group of intermediate 7 then was removed by dissolving the intermediate in the mixed
solvents of trimethylamine-dichloromethane, and the seven-membered ring was synthesized
by dissolving in the mixed solvents of acetic acid-ethanol, producing methyl

4-amino-5-((4-chloro-2-(2-fluorobenzoyl)phenyl)amino)-5-oxopentanoate.
Hydrolyzing
methyl ester in THF-MeOH-H2O with LiOH produced intermediate 10. Compound 10 reacted
with differently substituted primary amines or amino heteroaromatic compounds to produce
corresponding targets 11-23 in moderate yields. Total yield range is 40 to 65%.
Scheme 1. Reagents and conditions: (i) DCC, DCM; (ii) 50% morpholine/DCM; (iii) 10%
acetic acid/EtOH, 85°C; (iv) LiOH, THF/MeOH/H2O=1:1:1; (v) HOBt, DIC, DCM→rt.
2.2 Biological studies
2.2.1 Transient receptor potential vanilloid type 1 antagonistic activity assays in vitro
In order to test the antagonistic activity of the compounds, we used TRPV1
over-expressed cells HEK-293 for vitro activity test (Table 1). Compound 11, 19, 20 and 23
has relatively good antagonistic activity against capsaicin-activated TRPV1. But the inhibition
rate is lower than that of positive control BCTC, and the remaining compounds have weaker
antagonistic activity. The more carbon atoms of the aliphatic chain connected at the terminal
nitrogen atom, the weaker its antagonistic activity is; compound with six-membered
nitrogen-containing heterocycle is more potent than one with five-membered
nitrogen-containing heterocycle, and the inhibition rate significantly increases when the
benzyl group is connected to the terminal nitrogen. However, as the steric hindrance between
the aromatic ring and the three-carbon linker increases, the inhibition rate decreases.
Table 1. Structures and inhibitory activity against TRPV1 of target compounds 10-23

hTRPV1(CAP)
IC50a (µM)
hTRPV1(CAP)
IC50a (µM)
Comp
R
Comp
R
ND
BCTC
10
0.016
ND
17
18
19
20
21
22
23
-N(CH2)5
ND
OH
morpholinyl
Thiomorpholiyl
benzyl
0.033
0.025
0.458
0.461
0.020
11
-NMe2
-NEt2
0.046
0.297
0.352
0.684
0.621
ND
12
13
-NPr2
O
14
-NiPr2
-NnBu2
-N(CH2)4
N
N
15
16
a Human TRPV1 receptor activated by capsaicin. Unless otherwise stated, all values are the
mean (SEM of at least three separate experiments). ND, not determined.
2.2.2 Analgesic activity in vivo
The TRPV1 antagonist BCTC with better analgesic activity was used as a positive
control. In the capsaicin test, results showed that all the compounds can reduce the time of the
mice licking paw to varying degrees, so all the compound are able to reduce the pain induced
by capsaicin. Among them, compound 12, 13, 14, 15 and 22 have best analgesic activities.
And their analgesic activity was better than that of positive control BCTC; In abdominal
constriction test, compound 11, 12, 17, 19 and 23 can significantly reduce the number of
abdominal constriction. Among them 11, 12, 17 and 23 have better analgesic activity than
positive control BCTC; In the tail-flick test, compared with the blank group, except 12, 13, 17
and 21, the remaining compounds can increase the mice MPE %, Compounds 11, 15, 16, 18,
19 and 20 have a significant effect of reducing the pain induced by the hot. 16, 18 and 20 have
better analgesic activity than BCTC.

Figure 3. Antinociceptive activities of compound 11~23. (A) The results of capsaicin test; (B)
The results of abdominal constriction test; (C)The results of tail-flick test. Each bar represents
the mean SEM (n = 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;
***p <0.001 compared with the vehicle group.
2.2.3 Sedative activities screened in vivo
Table 2. Sedative activities of target compounds 11-23 in mice
Comp
Pharmacodynamics
5 min after the administration, the mice stopped running. They walked slowly and stayed
motionless within a short time after 10 min, and after 20 min, they recover to normal
gradually.
11
12
13
5 min after the administration, the mice stopped running and shook. After 8 min, they stayed
still, but minor activities such as finishing hair began to appear, then they moved once every 2
min; After 15 min, they began to recover to normal.
After administration, the mice appeared some phenomenon such as dizziness and unstable
behavior gradually, after about 19 min they recovered to normal and they did not enter a state
of complete sedation.
After administration, the mice appeared some phenomenon such as dizziness and unstable
behavior gradually, they did not enter a state of complete sedation.
After administration, the mice appeared some phenomenon such as dizziness and unstable
behavior gradually, they did not enter a state of complete sedation.
6 min after administration, the mice stayed still, then they crawled once every four min. About
after 12 min they recovered to normal.
14
15
16
17
18
Within 2 min after administration, the mice began to stay still, they completely recovered to
normal after about 13 min
Within 5 min after administration, the mice stayed still, they crawled once every 4.5 min, after
about 12 min they recovered to normal.
Within 5 min after administration, the mice began to stay still. The mice appeared some minor
movements and then stayed still again, after about 10 min they began to run and jump and
recovered to normal basically.
19
20
21
No significant effect.
No significant effect.

22
23
No significant effect.
5 min after the administration, the mice stopped running. They walked slowly and stayed
motionless within a short time after 8 min, and after 18 min, they recovered to normal
gradually.
Within 10 s after administration, it entered a state of sedation with weakness and disability.
After 8 min, there was an activity phenomenon. Minor activities such as finishing hairs began
to appear, but they could not move around. After 44 min, they basically recovered and
appeared normal behaviors such as running and jumping.
triazolam
2.2. 4 Aqueous solubility test
Data in the Table 3 displays that expect for compound 16, 19 and 22, aqueous solubility
of other compounds has improved to varying degrees compared to BCTC.
Table 3 Aqueous solubility of compound 11-23
Comp
11
Solubility(mg/mL)
Comp
17
Solubility(mg/mL)
1.78
1.16
2.93
2.74
6.35
0.20
0.60
0.66
0.63
0.20
1.70
1.89
0.30
1.64
12
18
13
19
14
20
15
21
16
22
BCTC
23
2.3 Docking results
Docking experiments have been conducted to study structure-activity relationships of
target compounds (Figure 4). Results show that both BCTC and Compound 11 occupied the
cavity of the TRPV1 ion channel. BCTC molecular has aromatic H-bond interactions with the
Key residue ALA 566 and THR 550. Compound 11 with designed novel structures has
H-bond interaction with residue TYR 511, elucidating that designed structure change is of
significance. Comparing compound 15 with 11, compound with longer carbon chain may
show better affinity. Actually compound with heterocycle attached to terminal nitrogen
(Compound 18) may bind relatively weakly with the receptor.
B
A

C
D
Figure 4 (A) Structure docking results with TRPV1 for BCTC;(B) Structure docking results
with TRPV1 for 11;(C) Structure docking results with TRPV1 for 15;(D) Structure docking
results with TRPV1 for 18
3. Conclusions
Designed and synthesized benzodiazepines derivatives as novel TRPV1 antagonists were
studied on TRPV1 antagonistic activity in vitro, analgesic activity in vivo and sedative
activity in vivo. Their antagonistic activity against TRPV1 is basically consistent with their
analgesic activity in vivo. The longer the carbon chain on the terminal nitrogen is, the stronger
the analgesic effect of the compounds is in capsaicin-induced Tail-flick test (15 > 14 > 13 >
11). When the nitrogen-containing heterocycle and aromatic ring are attached to the terminal
nitrogen, the analgesic activity in this model would be reduced to some extent. However, such
law was not found in abdominal constriction test and capsaicin test. Compound 11 and 19
have significant activity in all three analgesia models in vivo. When a saturated aliphatic
chain and a five-membered or six-membered nitrogen-containing heterocycle is connected to
the terminal nitrogen, the compounds have a certain degree of sedation. But compounds with
unsaturated aromatic group connected to the terminal nitrogen have little sedative activity. It
is speculated that the steric hindrance of the terminal aromatic ring hinders the combination of
the compound and the benzodiazepine receptor and then the sedative effect disappears. In
addition, compounds 11-15 with saturated aliphatic chains linked to the terminal nitrogen
atom have good aqueous solubility. Based on the results above, compound 11 was found
possessing strong analgesic, certain sedative activity and good aqueous solubility, providing
the potential development prospects.
4. Experimental
4.1 General

All reagents were purchased from Shanghai Chemical Reagent Company. Column
chromatography (CC): silica gel 60 (100-200 mesh). Thin-layer chromatography (TLC): silica
gel 60 F254 plates (250 mm; Qingdao Ocean Chemical Company, Qingdao, China). M.p.:
1
capillary tube; uncorrected. IR spectra: Shimadzu FTIR-8400S spectrophotometer; in cm-1. H
NMR spectra: Bruker ACF-300Q apparatus at 300 MHz, in DMSO or CDCl3 unless otherwise
indicated; δ in ppm rel. to Me4Si, J in Hz. 13C NMR spectra: Bruker ACF-300Q apparatus at
75 MHz; δ in ppm rel. to Me4Si, J in Hz. Mass spectrometry (MS): Waters UPLC/MS/MS:
ACQUITY UPLC/TQD, in m/z; Elemental analyses: CHN-O-Rapid instrument; %Purity of
the target compounds (> 97%) were determined by HPLC analysis (UV detector, wavelength:
254 nm).
4.1.1
Synthesis
of
3-(7-chloro-5-(2-fluorophenyl)-2-oxo-2,3-dihydro-1H-benzo
[e][1,4]diazepin-3-yl)propanoic acid (9)
4.1.1.1 The preparation of 7
(2-amino-5-chlorophenyl)(2-fluorophenyl)methanone(4.0 mmol)and Fmoc-L-Glu(OMe)
(4.8
mmol)
were
dissolved
into
10
mL
dichloromethane,
adding
1,3-dicyclohexylcarbodiimide (4.8mmol) into stirred solution. The reaction mixture was
stirred at room temperature for 2 h. The mixture was filtered through celite and washed with
ethyl acetate. The combined organic layers were washed with water and brine, then dried over
anhydrous Na2SO4 and concentrated. The residue was recrystallized by the mixture of
1
petroleum/ethyl acetate (4:1). Compound 7 was obtained and the yield was 56.87%. H NMR
(300 MHz, DMSO-d6) δppm: 9.66 (s, 1H, NH), 8.02-7.58 (m, 9H, Ar-H), 7.41-7.29 (m, 6H,
Ar-H), 4.76 (d, J = 6.7 Hz, 2H, CH2), 4.57- 4.49 (m, 2H, CH), 3.78 (s, 3H, CH3), 2.45-2.27 (m,
4H, CH2).
4.1.1.2 The preparation of 9
Compound 7 was dissolved into the mixed solution of morpholine and dichloromethane
(1:1). The reaction mixture was stirred at room temperature for 4 h. The reaction mixture was
monitored by TLC and concentrated in vacuo. Then the concentrated product was dissolved
into ethanol with 10% acetic acid. The reaction mixture was stirred at 85°C for 2 h. The
reaction mixture was monitored by TLC and concentrated in vacuo. The residue was

recrystallized by the mixture of petroleum/ethyl acetate (1:1). Compound 9 was obtained and
the yield was 60.32%. 1H NMR (300 MHz, DMSO-d6) δppm: 9.80 (s, 1H, NH), 8.14-7.71 (m,
4H, Ar-H), 7.59-7.33 (m, 3H, Ar-H), 4.12 (t, J = 8.0 Hz, 1H, CH), 3.68 (s, 3H, CH3),
2.53-2.30 (m, 4H, CH2).
4.1.1.3 The preparation of 10
Compound 9 (0.5 mmol, 1 eq) was dissolved into the mixed solution of THF/MeOH/H2O
(2:1:1). Adding LiOH (1.5 mmol, 3 eq). The reaction mixture was stirred at room temperature
for 2 h. The mixture was added to ice water. The pH was adjusted by the solution of 2 N HCl
to 4-5. The mixture was cooled overnight and filtered. Compound 10 was obtained and the
1
yield was 93.58%. H NMR (300 MHz, DMSO-d6) δppm: 10.80 (s, 1H, OH), 9.83 (s, 1H,
NH), 8.16-7.88 (m, 4H, Ar-H), 7.71-7.43 (m, 3H, Ar-H), 4.16 (t, J = 8.1 Hz, 1H, CH),
2.58-2.34 (m, 4H, CH2).
4.1.2 Preparation of derivatives 11-23
Compound 10 (0.8 mmol, 1 eq) was dissolved into dichloromethane (5 mL) , then HOBt
(1.20 mmol, 1.5 eq) , DIC (1.20 mmol, 1.5 eq) and nitrogen substituent (0.8 mmol, 1 eq) were
added into the solution in turn, the mixture was stirred at room temperature for 2 h and
monitored by TLC. The reaction mixture was washed with saturated brine(15 mL) and
extracted with ethyl acetate (15 mL x 2). The organic layers were combined and dried by
anhydrous sodium sulfate. The organic layer was concentrated in vacuo. The residue was
purified by flash column -chromatography on silica gel using ethyl petroleum/ethyl (2:1) as
eluent to obtain 11-23.
4.1.2.1 3-(7-chloro-5-(2-fluorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)-
N,N-dimethylpropanamide (11)
1
Yield 40.59%, white solid, m.p.104-106°C; H NMR (300 MHz, DMSO-d6) δppm: 10.69 (s,
1H, NH), 7.91 – 7.49 (m, 3H, Ar-H), 7.49 – 7.18 (m, 3H, Ar-H), 7.13 (s, 1H, Ar-H), 5.53 (d, J
= 5.9 Hz, 1H, CH), 3.48 (d, J = 84.0 Hz, 2H, CH2), 2.98 (s, 3H, CH3), 2.80 (s, 3H, CH3), 2.25
(d, J = 5.4 Hz, 2H, CH2); 13C NMR (75 MHz, DMSO-d6) δppm: 172.33, 170.37, 164.00,
137.65, 132.06, 129.51, 128.73, 127.32, 125.09, 123.61, 116.62, 62.97, 37.13, 33.80, 27.12,

24.90; ESI-MS m/z: 386.21 ([M+H]-). Anal. Calcd for C20H19ClFN3O2: C, 61.94; H, 4.94; N,
10.83. Found: C, 61.95; H, 4.96; N,10.84.
4.1.2.2 3-(7-chloro-5-(2-fluorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)
-N,N-diethylpropanamide (12)
1
Yield 65.26%, white solid, m.p.107-108°C; H NMR (300 MHz, DMSO-d6) δppm: 10.70 (s,
1H, NH), 7.70-7.48 (m, 3H, Ar-H), 7.48-7.19 (m, 3H, Ar-H), 7.13 (s, 1H, Ar-H), 5.54 (s, 1H,
CH), 3.47 (m, 4H, 2CH2), 2.26 (s, 2H, CH2), 1.83-1.57 (m, 2H, CH2), 1.11 (s, 3H, CH3), 0.98
(s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6) δppm: 171.41, 170.38, 137.69, 132.89, 132.06,
128.71, 127.32, 123.60, 116.35, 92.24, 62.96, 33.82, 28.88, 27.49, 24.90, 14.72, 13.52;
ESI-MS m/z: 414.24 ([M+H]-). Anal. Calcd for C22H23ClFN3O2: C, 63.54; H, 5.57; N, 10.10.
Found: C, 63.55; H, 5.56; N,10.11.
4.1.2.3 3-(7-chloro-5-(2-fluorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)
-N,N-dipropylpropanamide (13)
Yield 45.16%, white solid, m.p.108-110°C; 1H NMR (300 MHz, CDCl3-d6) δppm: 9.90 (s, 1H,
NH), 7.60-7.33 (m, 3H, Ar-H), 7.30-6.96 (m, 4H, Ar-H), 4.64 (d, J = 5.6 Hz, 1H, CH), 3.27 (s,
4H, CH2), 2.91-2.36 (m, 4H, CH2), 1.79-1.37 (m, 4H, CH2), 0.88 (m, 6H, CH3); 13C NMR (75
MHz, DMSO-d6) δppm: 172.13, 170.40, 164.20, 157.53, 137.46, 133.03, 132.09, 129.49,
128.59, 127.46, 127.12, 125.10, 123.61, 62.70, 49.24, 40.04, 28.80, 23.65, 22.28, 20.92, 11.53,
11.37; ESI-MS m/z: 422.31 ([M+H]-). Anal. Calcd for C24H27ClFN3O2: C, 64.93; H, 6.13; N,
9.47. Found: C, 64.94; H, 6.16; N,9.48.
4.1.2.4 3-(7-chloro-5-(2-fluorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-3
-yl)-N,N-diisopropylpropanamide (14)
Yield 50.06%，white solid, m.p.109-112°C; 1H NMR (300 MHz, DMSO-d6) δppm: 9.80 (m,
1H, NH), 7.74 (m, 2H, Ar-H), 7.48-7.30 (m, 4H, Ar-H), 7.10 – 6.80 (m, 1H, Ar-H), 4.15 (s,
1H, CH), 3.92-3.36 (m, 2H, CH2), 2.61 (d, J = 6.6 Hz, 2H, CH2), 2.55-2.29 (m, 2H, CH), 1.57
-1.12 (m, 12H, CH3); 13C NMR (75 MHz, DMSO-d6) δppm: 172.50, 171.22, 136.17, 135.16,

132.07, 131.62, 129.20, 126.55, 125.84, 124.32, 122.83, 122.52, 117.51, 116.18, 110.94,
62.54, 45.89, 42.42, 31.34, 29.70, 29.36, 23.30, 20.92, 20.55; ESI-MS m/z: 442.27 ([M+H]-).
Anal. Calcd for C24H27ClFN3O2: C, 64.93; H, 6.13; N, 9.47. Found: C, 64.93; H, 6.15; N,9.48.
4.1.2.5 N,N-dibutyl-3-(7-chloro-5-(2-fluorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]
diazepin-3-yl)propanamide (15)
Yield 45.76%，white solid, m.p.112-113°C; 1H NMR (300 MHz, DMSO-d6) δppm: δ 9.12 (s,
1H, NH), 7.45 (m, 3H, Ar-H), 7.13 (m, 4H, Ar-H), 6.34 (s, 1H, CH), 4.34 (s, 4H, CH2), 3.29 (s,
4H, CH2), 2.68-2.45 (m, 4H, CH2), 1.36-1.23 (m, 10H, CH2, CH3).13C NMR (75 MHz,
CDCl3-d6) δppm:172.21, 157.16, 151.80, 131.59, 129.53, 122.65, 109.96, 59.22, 52.38, 42.07,
35.22, 29.01, 28.92, 25.68, 24.84, 23.49, 20.17, 13.88; ESI-MS m/z: 470.34 ([M+H]-). Anal.
Calcd for C26H31ClFN3O2: C, 66.16; H, 6.62; N, 8.90. Found: C, 66.17; H, 6.62; N,8.91.
4.1.2.6 7-chloro-5-(2-fluorophenyl)-3-(3-oxo-3-(pyrrolidin-1-yl)propyl)-1,3-dihydro-2H
-benzo[e][1,4]diazepin-2-one (16)
Yield 56.26%，white solid, m.p.107-109°C; 1H NMR (300 MHz, DMSO-d6) δppm: 10.69 (s,
1H, NH), 7.66-7.48 (m, 3H, Ar-H), 7.47-7.18 (m, 3H, Ar-H), 7.13 (s, 1H, Ar-H), 4.01 (s, 1H,
CH), 3.51 (m, 4H, 2CH2), 2.46 (d, J = 7.9 Hz, 2H, CH2), 2.26 (d, J = 6.3 Hz, 2H, CH2),
1.86-1.70 (m, 4H, 2CH2); 13C NMR (75 MHz, DMSO-d6) δppm: 174.59, 170.64, 166.79,
157.05, 138.64, 138.07, 132.34, 132.09, 132.02, 130.23, 129.21, 128.50, 127.88, 127.20,
123.55, 62.83, 55.35, 50.51, 30.41, 26.10, 25.88, 24.42; ESI-MS m/z: 428.20 ([M+H]-). Anal.
Calcd for C22H21ClFN3O2:C, 63.85; H, 5.11; N, 10.15. Found: C, 63.85; H, 5.12; N,10.16.
4.1.2.7 7-chloro-5-(2-fluorophenyl)-3-(3-oxo-3-(piperidin-1-yl)propyl)-1,3-dihydro-2H
-benzo[e][1,4]diazepin-2-one (17)
1
Yield 65.20%, white solid, m.p.101-103°C； H NMR (300 MHz, DMSO-d6) δppm: 10.69 (s,
1H, NH), 7.85-7.47 (m, 3H, Ar-H), 7.31 (dd, J = 23.8, 8.1 Hz, 3H, Ar-H), 7.13 (s, 1H, Ar-H),
5.53 (d, J = 7.1 Hz, 1H, CH), 3.42 (s, 4H, 2CH2), 3.19 (d, J = 4.3 Hz, 2H, CH2), 2.24 (d, J =
6.9 Hz, 2H, CH2), 1.67-1.44 (m, 6H, 3CH2); 13C NMR (75 MHz, DMSO-d6) δppm: 170.71,

170.58, 170.40, 164.04, 161.88, 158.57, 137.59, 132.96, 132.10, 129.50, 128.71, 127.33,
125.16, 123.61, 116.64, 116.36, 62.89, 46.34, 45.67, 30.38, 29.09, 26.08, 25.79, 24.41;
ESI-MS m/z: 426.25 ([M+H]-). Anal. Calcd for C23H23ClFN3O2: C, 64.56; H, 5.42; N, 9.82.
Found: C, 64.57; H, 5.44; N,9.82.
4.1.2.8 7-chloro-5-(2-fluorophenyl)-3-(3-morpholino-3-oxopropyl)-1,3-dihydro-2H-
benzo[e][1,4]diazepin-2-one (18)
1
Yield 55.74%, white solid, m.p.101-102°C; H NMR (300 MHz, DMSO-d6) δppm: 10.69 (s,
1H, NH), 7.59 (s, 4H, Ar-H), 7.29 (dt, J = 21.0, 9.7 Hz, 3H, Ar-H), 7.11 (s, 1H, Ar-H), 5.55 (d,
J = 7.1 Hz, 1H, CH), 3.60 (s, 8H, 4CH2), 2.52 (s, 2H, CH2), 2.25 (d, J = 6.7 Hz, 2H, CH2); 13C
NMR (75 MHz, DMSO-d6) δppm: 171.36, 170.38, 164.13, 137.48, 132.14, 129.53, 128.69,
127.40, 125.17, 123.63, 66.55, 62.85, 45.85, 41.90, 28.90, 27.09; ESI-MS m/z: 428.20
([M+H]-). Anal. Calcd for C22H21ClFN3O3: C, 61.47; H, 4.92; N, 9.78. Found: C, 61.48; H,
4.94; N,9.80.
4.1.2.9 7-chloro-5-(2-fluorophenyl)-3-(3-oxo-3-thiomorpholinopropyl)-1,3-dihydro-2H
-benzo[e][1,4]diazepin-2-one (19)
1
Yield 60.15%, white solid, m.p.103-105°C; H NMR (300 MHz, DMSO-d6) δppm: 10.70 (s,
1H, NH), 8.30-7.48 (m, 4H, Ar-H), 7.30 (dt, J = 21.0, 9.8 Hz, 3H, Ar-H), 7.13 (s, 1H, Ar-H),
5.53 (d, J = 7.0 Hz, 1H, CH), 3.72 (s, 4H, 2CH2), 3.54 (m, 4H, 2CH2), 2.59 (s, 2H, CH2), 2.26
(d, J = 7.0 Hz, 2H, CH2); 13C NMR (75 MHz, DMSO-d6) δppm: 171.31, 170.35, 157.18,
137.59, 132.90, 132.15, 129.52, 128.70, 127.35, 125.12, 123.63, 116.49, 66.58, 62.93, 48.02,
33.79, 28.94, 25.79, 24.90; ESI-MS m/z: 444.18 ([M+H]-). Anal. Calcd for C22H21ClFN3O2S:
C, 59.26; H, 4.75; N, 9.42. Found: C, 59.28; H, 4.76; N,9.42.
4.1.2.10 3-(7-chloro-5-(2-fluorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)
-N-phenylpropanamide (20)
1
Yield 49.85%, white solid, m.p.105-107°C; H NMR (300 MHz, DMSO-d6) δppm: 9.80 (s,
1H, NH), 7.78 (s, 1H, Ar-H), 7.34 (dt, J = 17.4, 16.4 Hz, 4H, Ar-H), 7.12-6.60 (m, 5H, Ar-H),
6.59-6.08 (m, 3H, Ar-H), 4.48 (d, J = 7.5 Hz, 1H, CH), 2.17 (d, J = 14.4 Hz, 2H, CH2), 1.97

(d, J = 14.0 Hz, 2H, CH2); 13C NMR (75 MHz, DMSO-d6) δppm: 173.42, 169.12, 161.60,
158.34, 144.76, 137.81, 132.15, 131.51, 131.08, 129.99, 128.56, 128.29, 125.69, 124.23,
120.20, 119.47, 116.63, 116.33, 78.17, 30.91, 24.91; ESI-MS m/z: 434.24 ([M+H]-). Anal.
Calcd for C24H19ClFN3O2: C, 66.13; H, 4.39; N, 9.64. Found: C, 66.12; H, 4.39; N,9.63.
4.1.2.11 7-chloro-5-(2-fluorophenyl)-3-(3-oxo-3-(4-phenylpiperazin-1-yl)propyl)-1,3
-dihydro-2H-benzo[e][1,4]diazepin-2-one (21)
Yield 56.65%, white solid, m.p.110-112°C;1H NMR (300 MHz, DMSO-d6) δppm: 10.71 (s,
1H, NH), 7.58 (d, J = 7.6 Hz, 3H, Ar-H), 7.49-7.07 (m, 7H, Ar-H), 6.88 (m, 4H, Ar-H), 4.03 (s,
1H, CH), 3.62 (s, 4H, piperazine), 3.14 (s, 4H, piperazine), 2.60 (s, 2H, CH2), 2.29 (d, J = 6.3
Hz, 2H, CH2); 13C NMR (75 MHz, DMSO-d6) δppm: 171.04, 170.39, 170.06, 164.07, 157.05,
151.29, 137.60, 133.00, 132.20, 129.45, 128.74, 127.34, 125.16, 123.63, 119.78, 116.32,
62.90, 55.38, 49.20, 48.83, 45.18, 29.07, 27.25; ESI-MS m/z: 503.32 ([M+H]-). Anal. Calcd
for C28H26ClFN4O2: C, 66.60; H, 5.19; N, 11.09. Found: C, 66.61; H, 5.19; N,11.10.
4.1.2.12 3-(3-(4-benzoylpiperazin-1-yl)-3-oxopropyl)-7-chloro-5-(2-fluorophenyl)-1,3
-dihydro-2H-benzo[e][1,4]diazepin-2-one (22)
Yield 40.36%, white solid, m.p.112-113°C;1H NMR (300 MHz, DMSO-d6) δppm: 10.69 (s,
1H, NH), 7.57 (d, J = 8.6 Hz, 4H, Ar-H), 7.43 (d, J = 11.3 Hz, 6H, Ar-H), 7.27 (d, J = 7.9 Hz,
3H, Ar-H), 7.11 (s, 1H, Ar-H), 4.03 (d, J = 5.9 Hz, 1H, CH), 3.61 (s, 2H, CH2), 3.53 (d, J =
35.9 Hz, piperazine), 2.26 (d, J = 6.4 Hz, 2H, CH2); 13C NMR (75 MHz, DMSO-d6) δppm:
171.51, 170.34, 169.87, 164.13, 158.56, 137.53, 136.11, 132.95, 132.10, 130.10, 129.54,
128.79, 127.39, 125.15, 123.64, 116.31, 62.93, 29.16, 27.14; ESI-MS m/z: 531.35 ([M+H]-).
Anal. Calcd for C29H26ClFN4O3: C, 65.35; H, 4.92; N, 10.51; O, 9.01. Found: C, 65.36; H,
4.93; N,10.52.
4.1.2.13 N-(4-(tert-butyl)phenyl)-4-(7-chloro-5-(2-fluorophenyl)-2-oxo-2,3-dihydro
-1H-benzo[e][1,4]diazepin-3-yl)butanamide (23)
1
Yield 40.22%, white solid, m.p.108-111°C; H NMR (300 MHz, DMSO-d6) δppm: 10.45 (s,
1H, NH), 7.67 (d, J = 8.6 Hz, 4H, Ar-H), 7.43 (d, J = 7.9 Hz, 3H, Ar-H), 7.27-7.39 (m, 6H,

Ar-H), 4.14 (d, J = 5.9 Hz, 1H, CH), 2.37 (m, 2H, CH2), 2.10 (t, J = 6.4 Hz, 2H, CH2), 1.33(s,
9 H,3CH3); 13C NMR (75 MHz, DMSO-d6) δppm: 179.77, 172.05, 168.64, 163.25, 146.85,
136.36, 135.54, 132.58, 131.24, 130.02, 129.50, 128.54, 128.16, 127.88, 127.87, 125.26,
124.48, 121.23, 121.14, 115.58, 64.88, 37.88, 37.62, 34.18, 31.34, 20.03. ESI-MS m/z: 491.18
([M+H]-). Anal. Calcd for C28H27ClFN3O2: C, 68.36; H, 5.53; N, 8.54; O, 9.01. Found: C,
68.36; H, 5.52; N, 8.52.
4.2 Pharmacology
4.2.1 Transient receptor potential vanilloid type 1 antagonistic activity assays in vitro
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)[13].
4.2.2 Analgesic activity in vivo
Capsaicin test: The capsaicin test was assessed as previously described. License number
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[14]. 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[15]. 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. 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. 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[15]. In
brief, the distal one-third of the tail was immersed into 52.0°C 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.
License number for test animals: NO.201811021.All animal experimental protocols 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).
4.2.3 Sedative activities screened in vivo
Healthy male BALB/c mice weighting 16-20 g and were purchased from laboratory
animal center of Nanjing Qinglongshan. 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. The test compound was
administered intravenously to animals through tail veins, while those animals were placed in
plastic restrainers[16]. When onset of loss of righting reflex was recorded, the animals were
immediately removed from the restrainers. The loss of ability to right itself from supine
positions was defined as LRR. Once an animal was able to right itself for the first time, it was
placed on its back once again. If they recovered itself consecutively two more times back to
normal, the animals actions were labeled as recovered from LRR. Each compound, including
positive controls, was prepared in a 0.9% wt/vol saline solution. A dose of 9.0 mg/kg b.w. was
administered by rapid bolus via the lateral tail vein to six mice.
4.2.4 Aqueous solubility test

HPLC chromatographic conditions: Column: 4.6×150mm, Inertsil ODS-SP; Mobile
phase: CH3CN/H2O; Detection wavelength: 254 nm; Flow rate: 1 mL/min; Detector:
Photo-Diode Array; Column temperature: 40°C; Injection volume: 10 µL ; Run time：20 min.
Establishment of a standard curve: Weigh compounds accurately, dissolve the compound
in the 100 mL volumetric flask and add distilled water to the scale. Respectively take 0.5 mL,
1 mL, 1.5 mL, 2 mL and 2.5 mL from solution prepared above into 10 mL centrifuge tubes
and add distilled water to the scale. Each solution was measured in parallel for three times by
HPLC. Calculate linear regression equation.
Preparation of compounds’ saturated solution: Weigh excess compounds and dissolve in
distilled water, shake on the shaker for 72 h in 37°C. Centrifuge then take the supernatant.
Analyze the supernatant by HPLC and then calculate the solubility according to linear
regression equation of standard curve.
4.3 Docking experiments
Receptor protein (PDB ID:5IS0) was prepared and optimized using relevant module by
Schrodinger software. Receptor Grid Generation Module was used to generate docking area.
Structure of ligands were drawn by ChemOffice2014 software and prepared using LigPrep
module. In the end, molecular docking between ligands and receptor was conducted with
extra precision.
Acknowledgments
This study was supported by Scientific and Technological Research Program of
Chongqing and Municipal Education Commission (No. KJ1702012) and National Natural
Science Foundation of China (No.81673299).
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Novel Benzodiazepines Derivatives as analgesic Modulating for Transient
receptor potential vanilloid 1
Yan Liua,* , Chen Liaob, Jiaqi Zhoub, Chunxia Liub, Qifei Lib, Yue Jianga, Hai Qianb,c,*
a School of Pharmacy, Chongqing Medical University, 1 Yixueyuan Road, Chongqing
400016, PR China
b
Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical
University, 24 Tongjiaxiang, Nanjing 210009, 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: y.liu@cqmu.edu.cn (Y. Liu), qianhai24@163.com (H. Qian)
∗ Co-corresponding authors: Yan Liu and Hai Qian, Centre of Drug Discovery, State Key Laboratory of Natural
Medicines, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China.
Tel: +86-25-83271051; Fax: +86-25-83271051.
E-mail: y.liu@cqmu.edu.cn (Y. Liu), qianhai24@163.com (H. Qian)