Design, synthesis and biological evaluation of novel 2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole triazole derivatives as potent TRPV1 antagonists

Article abstract of DOI:10.1016/j.ejmech.2019.06.007

Reported herein is the design, synthesis, and pharmacologic evaluation of a class of TRPV1 antagonists constructed on 2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole as A-region and triazole as B-region. The SAR analysis indicated that 2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole analogues displayed excellent antagonism of hTRPV1 activation by capsaicin and showed better potency compared to the corresponding dihydroindole analogues. Optimization of this design led to the eventual identification of 2-((1-(2-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)methyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (6g), a potent TRPV1 antagonist. In vitro, using cells expressing recombinant human TRPV1 channels, 6g displayed potent antagonism activated by capsaicin (IC₅₀ = 0.075 μM) and only partially blocked acid activation of TRPV1. In vivo, 6g exhibited good efficacy in capsaicin-induced and heat-induced pain models and had almost no hyperthermia side-effect. Furthermore, pharmacokinetic studies revealed that compound 6g had a superior oral exposure after oral administration in rats. To understand its binding interactions with the receptor, the docking study of 6g was performed in rTRPV1 model and showed an excellent fit to the binding site. On the basis of its superior profiles, 6g could be considered as the lead candidate for the further development of antinociceptive drugs.

Full text of DOI:10.1016/j.ejmech.2019.06.007

Accepted Manuscript
Design, synthesis and biological evaluation of novel 2,3,4,9-tetrahydro-1H-
pyrido[3,4-b]indole triazole derivatives as potent TRPV1 antagonists
Jinyu Li, Cunbin Nie, Yue Qiao, Jing Hu, Qifei Li, Qiang Wang, Xiaohui Pu, Lin Yan,
Hai Qian
PII:
S0223-5234(19)30524-0
DOI:
https://doi.org/10.1016/j.ejmech.2019.06.007
EJMECH 11410
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 March 2019
Revised Date: 3 June 2019
Accepted Date: 3 June 2019
Please cite this article as: J. Li, C. Nie, Y. Qiao, J. Hu, Q. Li, Q. Wang, X. Pu, L. Yan, H. Qian,
Design, synthesis and biological evaluation of novel 2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole triazole
derivatives as potent TRPV1 antagonists, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.06.007.
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ACCEPTED MANUSCRIPT
Design, synthesis and biological evaluation of novel
2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole
triazole derivatives as potent TRPV1 antagonists
Jinyu Lia, Cunbin Niea, Yue Qiaoa, Jing Hua, Qifei Lib, Qiang Wangc, Xiaohui Pua, Lin Yana*, Hai
Qianb*
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
Reported herein is the design, synthesis, and pharmacologic evaluation of a class of TRPV1
antagonists constructed on 2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole as A-region and triazole as
B-region.
H
N
N
N
N
N
R
Region C
Lipophilic
Region A
Region B
H-bond
H-bond acceptor/
aryl interaction
interactions sidechain
* Corresponding authors.
E-mail addresses: yanlin0378@163.com (L. Yan), qianhai24@163.com (H. Qian).

ACCEPTED MANUSCRIPT
Design, synthesis and biological evaluation of novel
2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole
triazole derivatives as potent TRPV1 antagonists
Jinyu Lia, Cunbin Niea, Yue Qiaoa, Jing Hua, Qifei Lib, Qiang Wangc, Xiaohui Pua, Lin Yana*, Hai
Qianb*
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
ABSTRACT
Reported herein is the design, synthesis, and pharmacologic evaluation of a class of TRPV1
antagonists constructed on 2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole as A-region and triazole as
B-region. The SAR analysis indicated that 2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole analogues
displayed excellent antagonism of hTRPV1 activation by capsaicin and showed better potency
compared to the corresponding dihydroindole analogues. Optimization of this design led to the
eventual
identification
of
2-((1-(2-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)methyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-
b]indole (6g), a potent TRPV1 antagonist. In vitro, using cells expressing recombinant human
TRPV1
channels,
6g
displayed potent antagonism activated by capsaicin (IC50 = 0.075 µM) and only partially blocked
acid activation of TRPV1. In vivo, 6g exhibited good efficacy in capsaicin-induced and
heat-induced pain models and had almost no hyperthermia side-effect. Furthermore,
pharmacokinetic studies revealed that compound 6g had a superior oral exposure after oral
administration in rats. To understand its binding interactions with the receptor, the docking study
of 6g was performed in rTRPV1 model and showed an excellent fit to the binding site. On the
basis of its superior profiles, 6g could be considered as the lead candidate for the further
development of antinociceptive drugs.
Keywords:
Analgesic,
Transient
receptor
potential
vanilloid
type
1,
2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole, 1,2,3-triazole, hyperthermia.
1. Introduction
TRPV1, the transient receptor potential vanilloid 1, is arguably the best-characterized
member of the transient receptor potential (TRP) family: it is a calcium permeable nonselective
ion channel gated by a wide range of stimuli such as exogenous ligands (e.g., capsaicin or
resiniferatoxin), heat (>43 °C), acid (pH <6.8), and endogenous substances (e.g., anandamide and
oxidative metabolites of linoleic acid), and is a central nociceptor on sensory afferent neurons[1-5].
* Corresponding authors.
E-mail addresses: yanlin0378@163.com (L. Yan), qianhai24@163.com (H. Qian).

ACCEPTED MANUSCRIPT
Activation of this channel has been implicated in the pathophysiology of many neuronal (chronic
inflammatory pain and peripheral neuropathy) and non-neuronal diseases, such as cystitis, asthma,
and hearing loss [6, 7]. Therefore, TRPV1 antagonists have garnered great attention for the
treatment of a variety of disease states, particularly in the management of pain physiology and
neurogenic inflammation [8, 9]. To date, various structural classes of TRPV1 antagonists have
been discovered, some of which have reached clinical trials, but none have advanced beyond due
to their undesirable side effects such as hyperthermia that led to the termination of several drug
development programs [10, 11]. Thus, pharmacological separation of analgesic and hyperthermic
effects became the key challenge in developing TRPV1 antagonists as therapeutic agents for pain
management. Recent reports have revealed that, complete blocking of all modes of TRPV1
activation (capsaicin, endogenous lipids, acidic pH, heat) in some models of chronic pain models
can elicit hyperthermia [6, 12]. In addition, the magnitude and duration of the side effects seems to
be different for different molecules. With distinct molecular domains for activation by capsaicin,
protons, and heat, current intense interest exists to design modality-specific TRPV1 antagonists
[13, 14]. Very recently, a number of diverse structures as modality-selective TRPV1 antagonists,
which selectively inhibit capsaicin-induced TRPV1 activation but only partially block TRPV1
activation by acid, are recognized as the potential hyperthermia-free agents [6, 14, 15]. Taken
together, these studies support the need for a different class of antagonists that can work in a
particular activation mode, mainly by selectively blocking capsaicin-induced responses to avoid
hyperthermia.
On the other hand, chemical modification of natural products or endogenous ligands, which
can interfere with a given receptor, is a widely used strategy in medicinal chemistry programs [16].
Evodiamine and rutaecarpine are the two major components of Evodia rutaecarpa, which have
been reported to possess multiple biological effects, such as antinociceptive, anti-inflammatory,
antineoplastic, antidiabetic, and thermoregulatory effects, of which some are related to TRPV1
activity [17]. Furthermore, voacangine, a conformationally restricted tryptamine-derived natural
product, is a stimulus-selective antagonist which competitively inhibits capsaicin binding to
TRPV1 and blocks capsaicin- and heat-induced activation of this thermoreceptor [18]. These
potent natural products share structural features (2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole group)
that have been assumed to constitute a basic pharmacophore for TRPV1 blocking activity (Fig. 1).
Therefore, we sought to design
a
series of new compounds containing
2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole group with improved modality-selective TRPV1
activation and pharmacodynamic profiles. Additionally, click chemistry, which commonly
employs the 1,3-dipolar cycloaddition reaction between azides and alkynes to yield triazoles, has
been widely applied in drug discovery as a rapid method to assemble compound libraries [19].
Further, triazoles are amphoteric in nature, acting as both acids and bases. Such properties make
them usually soluble in aqueous medium. In order to rapidly generate a large number of diverse
modality-selective TRPV1 antagonists with better physiochemical properties as potential drug
candidates, we employed 1,3-dipolar azide-alkyne cycloaddition reaction to yield 1,4-disubstituted
1,2,3-triazoles. Herein, we report the design, synthesis, in vitro screening, and SAR analysis of a
new family of TRPV1 antagonists built around a 2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole
scaffold based on said pharmacophore (Fig. 2).

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O
O
N
MeO
N
N
N
N
H
N
N
N
H
H
H3C
MeO2C
Evodiamine
Rutaecarpine
Voacangine
Fig. 1. Chemical structures of evodiamine, rutaecarpine, and voacangine used in this study.
H
N
N
N
N
N
R
Region C
Lipophilic
Region A
Region B
H-bond
H-bond acceptor/
aryl interaction
interactions sidechain
Fig. 2. Structure of the target TRPV1 antagonists.
2. Results and discussion
2.1. Chemistry
The work described in this paper stems from the observation that the pharmacophore of the
TRPV1 antagonistic template can be divided into three regions: A, B and C (Fig. 2) [2]. The
structure-activity relationship (SAR) of the template has been investigated in the most detail for
the A-region, in which various functional groups including mono- or bicyclic-aryl and heteroaryl
rings with a properly positioned hydrogen-bond receptor in this part of the molecule improving
both potency and drug-like properties. In this study, a series of new TRPV1 antagonists were
studied by using 2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole as the A-region of antagonistic
template. Furthermore, we intended to explore whether the B-region such as urea, thiourea, amide
could be isosterically replaced by the 1,4-disubstituted 1,2,3-triazole ring. For this purpose, the
target compounds 6 were prepared according to Scheme 1. The starting material
2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (1) was purchased directly. The synthetic protocol was
straightforward enough to afford the two precursors in three steps: nucleophilic substitution,
diazotation-azidation, and click reaction. Firstly, 2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (1)
was mixed with potassium carbonate in acetone, followed by addition of 3-bromo-1-propyne (2)
and catalytic amount of potassium iodide to afford intermediate (3) in high yield. Secondly, a
variety of aromatic amines (4) were diazotized by sodium nitrite to form diazonium salts, which
were subsequently converted into azides (5). Lastly, the click reactions were performed under mild
conditions. In general, compound 3 reacted with diverse azides (5) in the presence of copper
sulfate and sodium ascorbate which guided the regioselectivity to obtain 1, 4-disubstituted
1,2,3-triazoles (6).

ACCEPTED MANUSCRIPT
H
H
N
N
a
Br
(1)
(2)
NH
N
2
1
3
b
Ar N3
Ar NH2
4
5
H
N
c
3
5
+
N
(3)
N Ar
N
N
6
Scheme 1. Synthesis of the target compounds 6. Reagents and conditions: (a) K2CO3, KI, acetone, rt; (b) NaNO2,
HCl, H2O, 0-5 , 30 min; NaN3, 0-5 , 2-4 h; (c) sodium ascorbate, CuSO4, 75% CH3OH, 24-48 h.
To probe the effect of 2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole on TRPV1 antagonism, a
second series of products (10) were prepared starting from substituted indoles 7 in a similar way
(Scheme 2). The synthesis of the dihydroindole-1,2,3-triazole derivatives was readily achieved by
reduction of appropriately substituted indole using sodium cyanoborohydride, alkylation with
3-bromo-1-propyne (2) followed by a click reaction to afford compounds 10 according to the
procedure as shown in Scheme 2.
Br
2
a
R1
R1
R1
N
N
N
b
H
H
9
8
7
Ar N3
R1
5
N
R2
N
N
c
N
10
Scheme 2. Synthesis of the target compounds 10. Reagents and conditions: (a) NaCNBH3, acetic acid, rt; (b)
K2CO3, KI, acetone, rt; (c) sodium ascorbate, CuSO4, 75% CH3OH, 24-48 h.
2.2. In vitro evaluation
With the obtained building blocks in hand, we initiated exploration of the SAR of the
C-region aryl group (Table 1). The ability of these compounds to block capsaicin (CAP) or low
pH-induced activation of human TRPV1 channels was assessed. As shown in Table 1, the phenyl
6A showed potent competitive antagonism at recombinant human TRPV1 activated by capsaicin
(IC50 = 0.314 µM) and incomplete blockade of acid-evoked response (68% block at 50 µM).

ACCEPTED MANUSCRIPT
However, replacement of the phenyl group with two other types of aryl groups, isoquinoline (6B),
pyridine (6C and 6D) resulted in a significant loss of activity, indicating that C-region is important
for the activity of this class of antagonists. Compound 6A was selected for further optimization
due to its potent TRPV1 antagonist potency against capsaicin activation. It was also noteworthy
that compound 6A exhibited different effects on capsaicin and pH 5.0 activation (only partially
inhibited activation of the channel by protons).
Table 1
Effect of arene substitution on TRPV1 antagonism.
H
N
N
N Ar
N
N
6
Compounds
Ar
hTRPV1(CAP) IC50a (µM)
hTRPV1(pH) b % inhib @ 50 µM
*
0.314 ± 0.087
68 ± 2
6A
*
2.156 ± 0.323
59 ± 5
13 ± 4
72 ± 5
6B
6C
6D
N
*
N
ND
Cl
*
3.489 ± 0.278
N
a Human TRPV1 receptor activated by capsaicin.
b Human TRPV1 receptor activated by low pH (5.0).
Unless otherwise stated, all values are the mean (SEM of at least three separate experiments).
ND, not determined.
Table 2
In vitro ability of compounds to inhibit the activation of hTRPV1 receptors.
H
N
R
N
N
N
N
6

ACCEPTED MANUSCRIPT
Compounds
R
hTRPV1(CAP) IC50a(µM)
hTRPV1(pH) b % inhib @ 50 µM
2-Cl
3-Cl
0.243 ± 0.069
0.416 ± 0.094
0.495 ± 0.074
0.232 ± 0.069
0.385 ± 0.082
0.494 ± 0.032
0.075 ± 0.029
0.089 ± 0.057
0.204 ± 0.043
0.108 ± 0.071
2.531 ± 0.055
1.878 ± 0.034
1.217 ± 0.095
1.195 ± 0.038
0.121 ± 0.047
0.788 ± 0.049
1.674 ± 0.032
64 ± 5
71 ± 8
73 ± 2
57 ± 3
71 ± 8
74 ± 2
58 ± 3
62 ± 4
79 ± 5
85 ± 3
67 ± 2
64 ± 3
61 ± 7
55 ± 8
86 ± 5
78 ± 2
40 ± 4
71 ± 1
67 ± 7
48 ± 3
73 ± 3
67 ± 2
74 ± 6
79 ± 5
13 ± 7
98 ± 1
6a
6b
6c
4-Cl
2-F
6d
6e
3-F
4-F
6f
2-CF3
3-CF3
4-CF3
2-NO2
2-iPr
2-OCH3
4-tBu
4-OCH3
4-NO2
4-Br
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
3-iPr
2-NO2, 4-CH3
2-NO2, 4-Cl
3-Cl, 4-CH3
3, 4-di-Cl
ND
0.394 ± 0.069
ND
6s
6t
0.341 ± 0.052
1.543 ± 0.047
1.122 ± 0.305
0.112 ± 0.019
ND
6u
6v
6w
6x
6y
BCTC
2, 5-di-CH3
3, 4-di-OCH3
2, 5-di-Cl
2, 4, 6-tri-CH3
0.019 ± 0.053
a Human TRPV1 receptor activated by capsaicin.
b Human TRPV1 receptor activated by low pH (5.0).
Unless otherwise stated, all values are the mean (SEM of at least three separate experiments).
ND, not determined.
Next, as part of our continuing effort to optimize the C-region of our TRPV1 antagonistic
template, we investigated the SAR of the substituted phenyl analogues. The results are presented
in Table 2, together with the potency of the classical TRPV1 antagonist BCTC
(N-(4-(tert-butyl)phenyl)-4-(3-chloropyridin-2-yl)piperazine-1-carboxamide).
A
variety of
substituents such as the small lipophilic halogen group, alkyl, bulky group (i-propyl and
tert-butyl), and even the multi-substituted benzene rings were investigated. An interesting trend
has been observed through a deeper analysis of the SAR of the position of the substituent. In CAP
assay, it was found that the ortho-substitution displayed higher potency than the meta-substitution
or para-substitution. The representative examples include phenyls with chlorine (6a vs 6b, 6c),
fluorine (6d vs 6e, 6f), and trifluoromethyl (6g vs 6h, 6i) substitutions (Table 2). Importantly, SAR
studies demonstrated that the electron withdrawing groups such as fluorine, chlorine,
trifluoromethyl and nitro-group were optimal. As illustrated in Table 2, 2-CF3 analogue 6g was the

ACCEPTED MANUSCRIPT
most potent compound. Migrating the CF3 group to the meta or para positions (6h and 6i) reduced
activity. The electron donating group substituent (e.g., 6k) obviously decreased potency, as did a
2-OCH3 group (6l). Moreover, introduction of the multisubstitution on the phenyl ring (6r, 6t and
6y) was poorly tolerated. However, in pH assay, in marked contrast to BCTC (a full blocker of
acid activation), most of these analogues exhibited only partially blockade (<75%) of acid
activation of TRPV1 at 50 µM. Interestingly, 2-CF3 derivative 6g exhibited differentiated effects
on capsaicin (potent, competitive antagonism, IC50 = 0.075 µM) and pH 5.0 activation (incomplete
blockade of acid-evoked response, 58% block at 50 µM) of recombinant human TRPV1.
Table 3
In vitro ability of compounds to inhibit the activation of hTRPV1 receptors.
R1
N
R2
N
N
N
10
Compounds
R1
R2
hTRPV1(CAP)
IC50a(µM)
22.573 ± 1.386
19.416 ± 2.357
4.945 ± 0.793
ND
hTRPV1(pH) b % inhib @
50 µM
32 ± 7
35 ± 6
55 ± 3
17 ± 6
39 ± 7
42 ± 5
34 ± 8
24 ± 4
12 ± 4
H
H
4-tBu
3-F
10a
10b
10c
10d
10e
10f
10g
10h
10i
H
2-CF3
3-iPr
2-iPr
4-Cl
H
H
ND
H
12.572 ± 4.653
ND
5-Cl
5-Cl
5-F
4-Cl
4-tBu
4-Cl
27.071 ± 3.298
ND
a Human TRPV1 receptor activated by capsaicin.
b Human TRPV1 receptor activated by low pH (5.0). Unless otherwise stated, all values are the mean (SEM of at
least three separate experiments).
ND, not determined.
With the above result in hand, we further turned our investigation to the A-region of our
TRPV1 antagonistic template, in order to assess the size of the substituents at the A-region and the
effect of the 2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole group (Table 3). A second series of
compounds (10) were synthesized (Scheme 2). Among these compounds, dihydroindole, which
was
also
the
nitrogen-containing
heterocycle,
was
employed
instead
of
2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole. A range of substituents were introduced at the C-region
phenyl moiety, including electron-withdrawing (F, Cl and CF3) and electron-donating (i-propyl,
and tert-butyl) groups. Intriguingly, with the size decreasing from three-ring to double-ring moiety,
that is replacement of the 2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole group at A-region with
dihydroindole resulted in drastic loss of activity. The most potent compound 10f showed about a
65-fold loss of potency in CAP assay and slight loss of potency in pH assay compared to that of its
parent compound 6g. Furthermore, when the dihydroindole core was replaced by the substituted

ACCEPTED MANUSCRIPT
dihydroindole, whereas chlorine and tert-butyl existed as substituents on the C-region phenyl
moiety, it was found that this was not tolerated for in vitro potency. These findings implied that the
2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole group at the A-region was preferred.
2.3. In vivo evaluation
Based on the in vitro studies, five compounds (6g, 6h, 6j, 6o and 6x) were selected for further
studies in vivo. In detail, the analgesic activity in vivo of each compound was evaluated employing
three different models of pain (Fig. 3). A single oral dose of 30 mg/kg was administered to mice,
as most of compounds were found to be almost not efficacious at 1 and 10 mg/kg in comparison
with vehicle group. In the capsaicin test, the total time spent licking the paw was significantly
reduced by all test compounds compared to the vehicle (Fig. 3A). Especially, compound 6g
exhibited greater potency than the positive control BCTC. In the abdominal constriction test, all
compounds reduced the number of writhes in the proto-induced pain models and compounds 6j,
6o and 6x exhibited better potency than BCTC (Fig. 3B). What is particularly interesting is that
compound 6g, the most active compound in the capsaicin test, had much weaker effect compared
to other compounds, which was consistent with the activity observed in pH assay. In the tail-flick
test, all compounds could increase %MPE compared to the vehicle. Compound 6g exhibited a
highest %MPE compared to other compounds in treatment of heat-induced pain, though it
exhibited lowest analgesic activity in treatment of proton-induced pain (Fig. 3C). Overall, all the
test compounds had antinociceptive activity to a certain extent. Among them, the most potent
compound 6g exhibited selective inhibitory activity in which it showed good antinociceptive
potency in capsaicin- and heat-induced pain models, but displayed weak effect to low pH.
Previous report indicated that hyperthermia depended on the blocking of TRPV1 activation by
protons, suggesting that weak pH antagonism of 6g was probably free of hyperthermia.
Fig. 3. Analgesic activities of synthesized compounds in 30 mg/kg after oral administration. (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 = 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.
We next conducted body temperature study and compared compounds effects with positive
control BCTC (Fig. 4). A single oral dose of 30 mg/kg was administered to mice, and the core
body temperature was measured every 30 min up to 120 min using a rectal thermometer. As
shown in Fig. 4, BCTC displayed a significant increase in the core body temperature beginning 30
min after administration and lasting for at least 90 min. Similar to the effect of BCTC, compounds

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6j, 6o and 6x also produced significant increase in the body temperature over vehicle-treated mice
30 min after administration, with a maximum of △ temperature occurring at 60 min. However,
in contrast to BCTC, compounds 6g and 6h, which had the lipophilic substitution CF3 group on
C-region and exhibited weak effect in low pH-induced activation of human TRPV1 channel test,
did not exhibit significant effects relative to vehicle. Furthermore, as previously mentioned in the
three analgesic tests, 6g was the most effective in treatment of capsaicin-induced and heat-induced
pain, and not effective in proton-induced pain (Fig. 3). These results indicate that compound 6g
was the most promising compound and selected for further studies.
Fig. 4. The effects of compounds in 30 mg/kg after oral administration on body temperature in mice. Data are
expressed as mean ± SEM (n = 6). *p < 0.05, **p < 0.01; ***p < 0.001 by Dunnett’s multiple comparison test
compared with the vehicle-treated group.
Fig. 5. The effects of compound 6g at different doses on body temperature in mice. The changes of body
temperature after dose. Data are expressed as mean ± SEM (n = 6). *p < 0.05, **p < 0.01; ***p < 0.001 by
Dunnett’s multiple comparison test compared with the vehicle-treated group.
To further explore the compound 6g, the dose-dependency of increased core body
temperature study were evaluated. More in detail, a single oral dose of 6g (3, 10, 50, or 100 mg/kg)
or BCTC (10 mg/kg) or vehicle was administered, and the core body temperatures were obtained
at 30, 60, 90, and 120 min after dosing using a rectal thermometer. As illustrated in Fig. 5, 6g
slightly elevated the core body temperature above that of vehicle treated mice in a manner that
was not clearly dose-dependent. Conversely BCTC, a potent blocker of both capsaicin and acid
activation of TRPV1 in vitro, induced a robust increase in the core body temperature following a
10 mg/kg oral dose. In general, over all times and doses of 6g, the average change of the core

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body temperature was not significantly above vehicle and did not increase more than 0.53°C.
Following in vitro and in vivo studies, the most potent compound (6g) and its corresponding
analogue (6h) were further assessed for their metabolic stability. The in vivo rat pharmacokinetic
data which were analyzed using the one-compartment model for the compounds selected have
been summarized in Table 4 and Fig. 6. As can be seen from Table 4, compound 6g showed a
favorable PK profile after a single oral administration of 10 mg/kg. Low clearance (CL 8.9 ± 1.1
L/h/kg) associated with moderate half-life (t1/2 = 2.3 ± 0.7 h), high Cmax (184.8 ± 53.4 ng/mL) and
high AUC (1122 ± 229 ng/mL × h) was seen with 6g oral administration. Compared to 6g,
compound 6h has rapid absorption (Tmax = 1.3 ± 0.3 h), but lower Cmax (122.7 ± 35.5 ng/mL),
higher clearance (CL = 22.7 ± 2.8 L/h/kg) and lower AUC (441 ± 89 ng/mL × h). These results
indicate that compound 6g had a superior PK profile compared to compound 6h, consistent with
the activity observed in pH assay and the analgesic activity in vivo.
Table 4
Pharmacokinetic parameters of 6g and 6h following oral administrationa to ratsb.
Compound
Parameters
Unit
6g
6h
t1/2, ka
t1/2, k10
ka
h
h
0.9 ± 0.3
0.9 ± 0.4
2.3 ± 0.7
0.9 ± 0.3
1/h
0.75 ± 0.11
0.31 ± 0.06
29.1 ± 1.1
8.9 ± 1.1
0.77 ± 0.13
0.75 ± 0.11
30.4 ± 1.8
22.7 ± 2.8
1.3 ± 0.3
k10
1/h
V
L/kg
L/h/kg
h
CL
Tmax
Cmax
AUC 0-inf
MRT
2.0 ± 0.4
ng/mL
ng/mL*h
h
184.8 ± 53.4
1122 ± 229
4.61 ± 0.34
122.7 ± 35.5
441 ± 89
2.64 ± 0.19
a Compound was prepared in 0.5% sodium carboxymethyl cellulose and administered at 10 mg/kg.
b n = 3.
(A)
(B)
Fig. 6. (A) Plasma concentrations in each time point of compound 6g after intragastric gavage (10 mg/kg) in rats.
(B) Plasma concentrations in each time point of compound 6h after intragastric gavage (10 mg/kg) in rats. Each
point is the average concentration, and the bars are standard deviations of the mean (n = 3).
2.4. Molecular modeling

ACCEPTED MANUSCRIPT
In order to analyze the binding interactions of antagonist 6g with the receptor, we carried out
a docking study with rat TRPV1 (PDB ID: 5IS0) model [20] and compared its binding mode with
that of BCTC.
(A)
(B)
(C)
Fig. 7. Molecular modeling of compound 6g and BCTC. (A) Three dimensional model of BCTC that interacts with
the key amino acids of rTRPV1. (B) Three dimensional model of compound 6g that interacts with the key amino
acids of rTRPV1. (C) Three dimensional model of compounds in the hydrophobic pocket of rTRPV1. Compound
6g is depicted by sticks colored by atom type (C dark green, F gray, N blue, polar H white). BCTC is depicted by
sticks colored by atom type (C orange, O red, Cl green, N blue, polar H white).
As shown in Fig. 7A, an oxygen atom of the urea group of BCTC participated in hydrogen
bonding with Thr550. The 4-tert-butylphenyl group was involved in a hydrophobic interaction
with Leu553 and Leu669. Furthermore, the piperazine group made tight interactions with the
binding site residues via the hydrophobic interaction with Ala665, Ala546, Phe591 and Met547.
Additionally, the 3-chloropyridine group made an additional hydrophobic interaction with the
hydrophobic region composed of Met547, Phe543, Phe522 and Ile661. As we expected,
compound 6g showed an excellent fit to the binding site (Fig. 7B). The
2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (A-region) of compound 6g made tight interactions
with the binding site residues via the hydrogen bonding with Tyr511, electrostatic interaction with
Glu570, and hydrophobic interaction with Leu553, Ala566, Ile569 and Ile573. Furthermore, the
1,2,3-triazole group (B-region) made a hydrogen bond with Thr550 and also contributed to the

ACCEPTED MANUSCRIPT
hydrophobic interaction with Leu669. Moreover, the aromatic group in the C-region made an
additional hydrophobic interaction with Ala546 and Met547. The 3-trifluoromethyl group in the
C-region also formed a hydrophobic interaction with Met547, in addition to the hydrogen bonding
with the 1,2,3-triazole group (B-region). Finally, as shown in Fig. 7C, compound 6g fitted well
into the active hydrophobic pocket better than BCTC. That might explain why the activity of 6g
was excellent.
3. Conclusions
In conclusion, we selected 2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole as the A-region, and a
series of 1,2,3-triazole TRPV1 antagonists were newly designed and synthesized based on the
click chemistry. Close scrutiny of our pharmacology data revealed compound 6g was a potent
TRPV1 antagonist that exhibited excellent in vitro functional activity and good efficacy in
capsaicin-induced and heat-induced pain models. Consistent with its action in vitro being through
TRPV1, compound 6g blocked heat-induced and capsaicin-induced TRPV1 activation but did not
cause TRPV1-related hyperthermia in mice. In addition, compound 6g displayed promising
pharmacokinetic properties in rats following oral administration. The docking study with the
rTRPV1 model indicated that 6g showed an excellent fit to the binding site resulting in its high
potency. Taken together, this investigation has provided us with novel scaffolds for the further
studies of related TRPV1 antagonists.
4. Experimental section
4.1. Biological methods
The synthesized compounds were investigated for TRPV1 antagonistic in vitro, in vivo
analgesic activity and the effect on body temperature. The test compounds and the standard drugs
were administered in the form of a suspension (using 0.5% sodium carboxymethyl cellulose as a
vehicle) by intragastric administration. Separate groups of KM male mice (n = 6), weighing 18-22
g, were pretreated with compounds (30 mg/kg unless otherwise indicated) 30 min before the test.
The animals were procured from the Comparative Medicine Centre of Yangzhou University
(Jiangsu, China) and were maintained in colony cages at 25 ± 2 °C, relative humidity 45-55%,
under a 12 h light/dark cycle; they were fed standard animal feed. All the animals were
acclimatized for a week before use. The Institutional Animal Ethics committee has approved the
protocol adopted for the experimentation of animals.
4.1.1. Transient receptor potential vanilloid type1 antagonistic activity assays in vitro
Culture plates with Ca2+- and Mg2+-free phosphate-buffered saline supplemented with 5 mM
ethylenediaminetetra-acetic acid were used for the TRPV1 aequorin cells (Perkin Elmer, Waltham,
MA, USA) growth. The cells were pelleted for 2 min at 1000 g; resuspended in Dulbecco’s
minimum essentialmedium-F12 medium with 15 mM HEPES (pH 7.0) and 0.1% BSA (assay
buffer) at a density of 3 × 105 cells/mL and incubated for 4 h in the dark in the presence of 5 mM
Coelenterazine (Promega, Madison, WI, USA). After loading, cells were diluted with assay buffer
to a concentration of 5 × 106 cells/mL. Twenty microliter of cells was injected over 20 µL of the
sample solution plated on 384-well plates, respectively, unless otherwise indicated. The Digitonin,
ATP (Sigma-aldrich, St Louis, MO, USA), and assay buffer were added in the blank control wells
for reference, and final concentration of Digitonin and ATP was100 and 50 µM. The sample

ACCEPTED MANUSCRIPT
solution and the cells were incubated for 2.5 min before added agonists capsaicin (Tocris, England)
and HCl solution at pH 5 and then immediately detected. The light emission was recorded during
variable times using EnVision2014 Multilabel Reader (PerkinElmer) [21, 22].
4.1.2. Analgesic activity
4.1.2.1. Capsaicin test
As previously described, we evaluated analgesic activity in the capsaicin-induced pain model
[23]. Twenty microliter of solution of capsaicin (16 µg/20 mL) was injected s.c. under the skin of
the dorsal surface of the right hind paw. The mouse was then placed in an individual cage. The
amount of time spent licking the injected paw was measured and expressed as the cumulative
licking time for 5 min after the capsaicin injection.
4.1.2.2. Abdominal constriction test
Abdominal constriction test was performed as described previously to assess analgesia of
pain activated by acid [24]. We placed mice in individual glass cylinders for a 30 min
acclimatization period, injected with 0.6% acetic acid (0.1 mL/10 g/mouse i.p.), and immediately
placed inside transparent glass cylinders. The number of writhes was recorded for 15 min.
4.1.2.3. Tail-flick test
Tail-flick test was carried out according to previous performation [24]. Briefly, in a water
bath maintained at 52 °C, the distal one-third of the mouse tail was immersed. Latency times until
a tail-flick response were recorded before and after drug treatment. The antinociception response
was presented as percentmaximal possible effect (%MPE) as defined by %MPE = 100% × (drug
response time - basal responsetime)/(cut-off time - basal response time). A cut-off time of 12 s was
applied to avoid tissue damage.
4.1.3. Effect on body temperature
Mice were intragastric administered with synthesized compounds (30 mg/kg, i.g.), BCTC (30
mg/kg, i.g.), or an equal volume of vehicle. The body temperature of mice was monitored by the
electric probe thermometer (MT-1C/F, Ruidien, Shenzhen, China) at 0, 30, 60, 90, and 120 min
after dosing. The effect on body temperature was presented as temperature or △ temperature = the
temperature at the certain time after dosing – the temperature at 0 min after dosing.
4.1.4. Pharmacokinetic Study.
The animal studies were performed according to committee approved procedures.
Spragur-Dawley male rats, each weighing 220-250 g, were quarantined for 1 week before use. The
animals were surgically implanted with a jugularvein cannula 1 day before treatment and were
fasted overnight before treatment. The compound was given to the rats (n = 3) as oral (10 mg/kg)
dose prepared in a mixture of dosing vehicles. The volume of the dosing solution given was
adjusted according to the body weight recorded before the drug was administered. At specific time
points (0.25, 0.5, 1, 2, 4, 6, 8, and 12 h), about 250-µL of blood sample was collected from the
jugular vein and transferred into heparin-pretreated Eppendorf (EP) tubes. The blood samples
were centrifuged at 8000 g (4 °C) for 10 min and the plasma was transferred into EP tubes and
stored in -20 °C.

ACCEPTED MANUSCRIPT
A 50-µL aliquot of plasma sample was subjected to the deproteinization using 200-µL
acetonitrile. After 1-min vortex, the sample mixture was centrifuged at 12 000 g (4 °C) for 20 min.
The resulting supernatant was collected and dried using Eppendorf Concentrator Plus (Hamburg,
Germany). The dry residuals were re-dissolved in 100-µL of acetonitrile/water (50:50, v/v). After
centrifugation (12 000 g, 15 min), a 5-µL aliquot of the supernatant was injected into a
UPLC-QTOF/MS system (Waters, Milford, MA, USA). The plasma concentration data were
analyzed with a standard one-compartmental method.
4.1.5. Statistical analysis of the data
Statistical analyses were performed using specific software (GRAPHPAD INSTAT version
5.00; GraphPad software, San Diego, CA, USA). Comparisons were analyzed using a one-way
analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test, unless otherwise
stated. p < 0.05 is regarded as statistically significant.
4.2. Chemistry
4.2.1. General
All reagents were purchased from Shanghai Chemical Reagent Company. Column
chromatography was carried out on silica gel (200-300 mesh) and monitored by thin layer
chromatography performed on GF/UV 254 plates and were visualized by using UV light at 365
1
and 254 nm. H NMR spectra: BrukerAVANCE
apparatus at 400 MHz, in CDCl3 unless
otherwise indicated; δ in ppm rel. to Me4Si, J in Hz. 13C NMR spectra: BrukerAVANCE
apparatus at 100 MHz, in CDCl3 unless otherwise indicated; δ in ppm rel. to Me4Si. HRMS
(high-resolution mass spectra) were taken with a Thermo QE spectrometer, in m/z.
General
procedure
for
the
preparation
To solution
of
2-(prop-2-ynyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole
(3).
a
of
1,2,3,4-Tetrahydro-9H-pyrido[3,4-b]indole (1, 0.5g, 2.9 mmol) in acetone (30 mL) at room
temperature, anhydrous K2CO3 (0.8g, 5.8 mmol) was added and then stirred for 45 min. To the
above reaction mixture, the solution of bromopropyne (2, 0.34g, 2.9 mmol) in acetone (10 mL)
with a catalytic amount of KI was added dropwise and then stirred at room temperature for 12-24
h. The mixture was filtered and the filtrate was evaporated and the crude product was purified by
silica gel column chromatography to give the 3 (0.46 g, 75% yield) as a tan solid.
General procedure for the preparation of 5. Compound 4 (10 mmol) was dissolved in HCl
(6 mol/mL, 10 mL) at 0 . To the above reaction mixture, the solution of sodium nitrite (0.6 g, 8.5
mmol) in H2O (25 mL) was added dropwise at -5 to 0
within 30 min. The solution was
vigorously stirred at 0-5 for 30 min. Sodium azide (40 mmol) in H2O (50 mL) was added
dropwise into the reaction mixture at 0-5 . The resulting solution was stirred at room
temperature for 2-4 h followed by diluting with ice water (200 mL) and extracting with EtOAc (3
× 100 mL). The combined organic layer was washed with water (2 × 60 mL), saturated aqueous
NaHCO3 (2 × 60 mL) and brine (2 × 50 mL), dried over anhydrous Na2SO4, filtered and
concentrated in vacuo to afford compound 5. The residual crude product was used directly without
purification.

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General procedure for the preparation of 6. To the solution of compound 3 (1 mmol) and
5 (1 mmol) in 75% methanol (40 mL), sodium ascorbate (30 mg) and CuSO4 (10 mg) were added
successively. The reaction solution was stirred at room temperature for 24-48 h. After filtration,
the solvent was evaporated and the crude product was purified by silica gel column
chromatography to give the desire product with high purity.
2-((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole
1
(6A). C20H19N5, brown yellow solid (95.7% yield), mp = 223.2-223.8 °C; H NMR (DMSO,
400MHz): δ ppm 10.69 (s, 1H, NH), 8.79 (s, 1H, C=CH), 7.93 (d, 2H, J = 8.0 Hz, Ar-H), 7.59 (t,
2H, J = 10.0 Hz, Ar-H), 7.48 (t, 1H, J = 10.0 Hz, Ar-H), 7.36 (d, 1H, J = 8.0 Hz, Ar-H), 7.27 (d,
1H, J = 8 Hz, Ar-H), 7.03-6.91 (m, 2H, Ar-H), 3.94 (s, 2H, CH2), 3.70 (s, 2H, CH2), 2.88 (t, 2H, J
= 8.0 Hz, CH2), 2.72 (t, 2H, J = 8.0 Hz, CH2); 13C NMR (DMSO, 100MHz):
δ ppm 145.5, 137.2,
136.3, 133.1, 130.3, 128.9, 127.1, 122.4, 120.7, 120.4, 118.7, 117.7, 111.3, 106.7, 52.2, 50.8, 50.0,
21.5; HRMS (ESI) calcd. for C20H20N5 [M+H]+ 330.1713, found 330.1712.
2-((1-(isoquinolin-5-yl)-1H-1,2,3-triazol-4-yl)methyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b
1
]indole (6B). C23H20N6, brown yellow solid (92.7% yield), mp = 147.2-149.6 °C; H NMR
(CDCl3, 400MHz): δ ppm 9.33 (s, 1H, NH), 8.70 (s, 1H, C=CH), 8.54 (s, 1H, Ar-H), 8.09 (d, 1H, J
= 12.0 Hz, Ar-H), 7.91 (s, 1H, Ar-H), 7.73-7.63 (m, 2H, Ar-H), 7.54 (d, 1H, J = 8.0 Hz, Ar-H),
7.44 (d, 1H, J = 12.0 Hz, Ar-H), 7.24 (t, 1H, J = 8.0 Hz, Ar-H),7.10-7.01 (m, 2H, Ar-H), 4.05 (s,
2H, CH2), 3.79 (s, 2H, CH2), 3.02 (t, 2H, J = 6.0 Hz, CH2), 2.86 (t, 2H, J = 6.0 Hz, CH2); 13C
NMR (CDCl3, 100MHz):
δ ppm 152.6, 145.0, 144.7, 136.2, 131.4, 130.7, 129.8, 128.9, 127.1,
126.6, 125.2, 121.3, 119.2, 117.9, 115.2, 110.8, 107.8, 53.4, 52.2, 51.0, 50.0, 21.3; HRMS (ESI)
calcd. for C23H20N6 [M+H]+ 381.1822, found 381.1819.
2-((1-(4,6-dimethylpyridin-2-yl)-1H-1,2,3-triazol-4-yl)methyl)-2,3,4,9-tetrahydro-1H-pyr
1
ido[3,4-b]indole (6C). C21H22N6, brown yellow solid (73.0% yield), mp = 176.6-178.2 °C; H
NMR (CDCl3, 400MHz): δ ppm 11.63 (s, 1H, NH), 7.93 (s, 1H, C=CH), 7.39 (s, 1H, Ar-H), 7.36
(d, 1H, J = 12.0 Hz, Ar-H), 7.22 (d, 1H, J = 8.0 Hz, Ar-H), 7.03 (s, 1H, Ar-H), 6.94 (s, 1H, Ar-H),
6.91 (s, 1H, Ar-H), 3.67 (s, 2H, CH2), 3.62 (s, 2H, CH2), 2.83 (s, 2H, CH2), 2.73 (s, 2H, CH2) ,
2.49 (s, 3H, CH3) , 2.47 (s, 3H, CH3); 13C NMR (CDCl3, 100MHz):
δ ppm 158.4, 147.3, 140.0,
136.3, 131.4, 127.6, 126.7, 124.3, 121.6, 119.7, 117.8, 111.1, 106.0, 57.2, 55.8, 53.6, 24.3, 21.7,
20.7; HRMS (ESI) calcd. for C21H23N6 [M+H]+ 359.1979, found 359.1981.
2-((1-(2-chloropyridin-3-yl)-1H-1,2,3-triazol-4-yl)methyl)-2,3,4,9-tetrahydro-1H-pyrido[
3,4-b]indole (6D). C19H17ClN6, cyan solid (74.2% yield), mp = 155.4-157.3 °C; 1H NMR (CDCl3,
400MHz):
7.66-7.21 (m, 3H, Ar-H), 6.97 (s, 1H, Ar-H), 6.94 (s, 1H, Ar-H), 3.65 (s, 2H, CH2), 3.61 (s, 2H,
CH2), 2.83 (s, 2H, CH2), 2.71 (s, 2H, CH2); 13C NMR (CDCl3, 100MHz):
ppm 149.7, 140.6,
δ ppm 10.65 (s, 1H, NH), 8.38 (s, 1H, C=CH), 8.31 (s, 1H, Ar-H), 8.07 (s, 1H, Ar-H),
δ
139.2, 136.2, 132.8, 131.1, 127.3, 126.7, 122.3, 121.6, 119.8, 118.7, 118.1, 111.1, 107.0, 57.2,
55.4, 52.3, 20.7; HRMS (ESI) calcd. for C19H18ClN6 [M+H]+ 365.1276, found 365.1279.
2-((1-(2-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b
1
]indole (6a). C20H18ClN5, pale yellow solid (91.6% yield), mp = 143.6-145.0 °C; H NMR

ACCEPTED MANUSCRIPT
(DMSO, 400MHz):
δ
ppm 10.62 (s, 1H, NH), 8.42 (s, 1H, C=CH), 7.68 (d, 1H, J = 8.0 Hz, Ar-H),
7.62 (d, 1H, J = 12.0 Hz, Ar-H), 7.52 (t, 2H, J = 10.0 Hz, Ar-H), 7.27 (d, 1H, J = 8.0 Hz, Ar-H),
7.18 (t, 1H, J = 12.0 Hz, Ar-H), 6.94-6.82 (m, 2H, Ar-H), 3.87 (s, 2H, CH2), 3.60 (s, 2H, CH2),
2.79 (t, 2H, J = 8.0 Hz, CH2), 2.65 (t, 2H, J = 14.0 Hz, CH2); 13C NMR (DMSO, 100MHz):
δ ppm
143.7, 135.8, 134.6, 132.6, 131.5, 130.4, 128.5, 128.4, 128.3, 126.6, 126.0, 120.2, 118.2, 117.2,
110.8, 106.2, 51.6, 50.2, 49.3, 21.0; HRMS (ESI) calcd. for C20H19ClN5 [M+H]+ 364.1323, found
364.1319.
2-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b
1
]indole (6b). C20H18ClN5, brown yellow solid (87.4% yield), mp = 181.4-182.4 °C; H NMR
(DMSO, 400MHz): δ ppm 10.66 (s, 1H, NH), 8.85 (s, 1H, C=CH), 8.07 (s, 1H, Ar-H), 7.95 (d, 1H,
J = 12.0 Hz, Ar-H), 7.64-7.52 (m, 2H, Ar-H), 7.36 (d, 1H, J = 8.0 Hz, Ar-H), 7.27 (d, 1H, J = 8.0
Hz, Ar-H), 7.03-6.91 (m, 2H, Ar-H), 3.94 (s, 2H, CH2), 3.71 (s, 2H, CH2), 2.88 (t, 2H, J = 6.0 Hz,
CH2), 2.73 (t, 2H, J = 6.0 Hz, CH2); 13C NMR (DMSO, 100MHz): δ ppm 145.7, 138.2, 136.3,
134.6, 133.0, 132.0, 128.7, 127.1, 122.6, 120.7, 120.1, 118.9, 118.7, 117.7, 111.3, 106.7, 52.2,
50.7, 50.0, 21.5; HRMS (ESI) calcd. for C20H19ClN5 [M+H]+ 364.1323, found 364.1320.
2-((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b
1
]indole (6c). C20H18ClN5, pale yellow solid (97.2% yield), mp = 237.0-237.2 °C; H NMR
(DMSO, 400MHz): δ ppm 10.69 (s, 1H, NH), 8.82 (s, 1H, C=CH), 7.98 (d, 2H, J = 8.0 Hz, Ar-H),
7.66 (d, 2H, J = 8.0 Hz, Ar-H), 7.36 (d, 1H, J = 8.0 Hz, Ar-H), 7.27 (d, 1H, J = 8.0 Hz, Ar-H), 7.01
(t, 1H, J = 8.0 Hz, Ar-H), 6.94 (t, 1H, J = 8.0 Hz, Ar-H), 3.94 (s, 2H, CH2), 3.70 (s, 2H, CH2), 2.88
(t, 2H, J = 6.0 Hz, CH2), 2.72 (t, 2H, J = 6.0 Hz, CH2); 13C NMR (DMSO, 100MHz):
δ ppm 136.3,
136.0, 133.2, 130.2, 129.3, 127.1, 126.1, 123.0, 122.0, 120.7, 118.7, 117.7, 111.1, 106.7, 52.3,
50.8, 50.0, 47.0, 21.5; HRMS (ESI) calcd. for C20H19ClN5 [M+H]+ 364.1323, found 364.1320.
2-((1-(2-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]
1
indole (6d). C20H18FN5, brown yellow solid (79.0% yield), mp = 209.4-209.6 °C; H NMR
(DMSO, 400MHz): δ ppm 10.72 (s, 1H, NH), 8.55 (s, 1H, C=CH), 7.86 (t, 1H, J = 10.0 Hz, Ar-H),
7.64-7.52 (m, 2H, Ar-H), 7.44 (t, 1H, J = 10.0 Hz, Ar-H), 7.36 (d, 1H, J = 8.0 Hz, Ar-H), 7.28 (d,
1H, J = 12.0 Hz, Ar-H), 7.04-6.91 (m, 2H, Ar-H), 3.96 (s, 2H, CH2), 3.71 (s, 2H, CH2), 2.88 (t, 2H,
J = 8.0 Hz, CH2), 2.72 (t, 2H, J = 8.0 Hz, CH2); 13C NMR (DMSO, 100MHz):
δ ppm 144.9, 136.3,
133.0, 131.6, 127.1, 126.3, 126.0, 125.7, 125.4, 120.7, 118.7, 117.8, 117.7, 117.4, 111.3, 106.7,
52.0, 50.7, 49.9, 21.5; HRMS (ESI) calcd. for C20H19FN5 [M+H]+ 348.1619, found 348.1618.
2-((1-(3-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]
1
indole (6e). C20H18FN5, brown yellow solid (92.4% yield), mp = 196.1-198.2 °C; H NMR
(DMSO, 400MHz): δ ppm 10.68 (s, 1H, NH), 8.86 (s, 1H, C=CH), 7.89-7.86 (m, 2H, Ar-H), 7.64
(d, 1H, Ar-H), 7.38-7.27 (m, 3H, Ar-H), 7.03-6.92 (m, 2H, Ar-H), 3.95 (s, 2H, CH2), 3.71 (s, 2H,
CH2), 2.89 (s, 2H, CH2), 2.73 (s, 2H, CH2); 13C NMR (DMSO, 100MHz): δ ppm 164.1, 161.7,
145.7, 136.3, 133.0, 132.3, 127.1, 122.6, 120.7, 118.7, 117.7, 116.2, 115.7, 111.3, 107.9, 106.7,
52.2, 50.8, 50.0, 21.5; HRMS (ESI) calcd. for C20H19FN5 [M+H]+ 348.1619, found 348.1618.
2-((1-(4-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]

ACCEPTED MANUSCRIPT
indole (6f). C20H18FN5, pale yellow solid (83.2% yield), mp = 237.9-238.8 °C; 1H NMR (DMSO,
400MHz): δ ppm 10.67 (s, 1H, NH), 8.78 (s, 1H, C=CH), 8.00-7.79 (m, 2H, Ar-H), 7.45 (t, 2H, J
= 8.0 Hz, Ar-H), 7.36 (d, 1H, J = 8.0 Hz Ar-H), 7.27 (d, 1H, J = 8.0 Hz, Ar-H), 7.01 (t, 1H, J = 8.0
Hz, Ar-H), 6.94 (t, 1H, J = 8.0 Hz, Ar-H), 3.94 (s, 2H, CH2), 3.70 (s, 2H, CH2), 2.89 (t, 2H, J = 6.0
Hz, CH2), 2.73 (t, 2H, J = 6.0 Hz, CH2); 13C NMR (DMSO, 100MHz): δ ppm 163.2, 160.7, 145.6,
136.3, 133.7, 133.1, 127.1, 122.7, 120.7, 118.7, 117.7, 117.2, 117.0, 111.3, 106.7, 60.2, 52.3, 50.8,
50.0, 21.5; HRMS (ESI) calcd. for C20H19FN5 [M+H]+ 348.1619, found 348.1617.
2-((1-(2-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)methyl)-2,3,4,9-tetrahydro-1H-p
yrido[3,4-b]indole (6g). C21H18F3N5, brown yellow solid (80.0% yield), mp = 145.5-145.8 °C; 1H
NMR (CDCl3, 400MHz): δ ppm 8.37 (s, 1H, NH), 7.84 (d, 2H, J = 8.0Hz, C=CH, Ar-H),
7.72-7.63 (m, 3H, Ar-H), 7.52 (d, 1H, J = 8.0 Hz, Ar-H), 7.43 (d, 1H, J = 8.0 Hz Ar-H), 7.10-7.02
(m, 2H, Ar-H), 4.02 (s, 2H, CH2), 3.77 (s, 2H, CH2), 2.97 (s, 2H, CH2), 2.84 (s, 2H, CH2); 13C
NMR (CDCl3, 100MHz): δ ppm 136.1, 133.0, 130.4, 128.9, 127.3, 127.2, 127.1, 126.0, 124.0,
121.2, 119.2, 117.8, 110.8, 107.8, 60.4, 53.4, 31.4, 29.7, 22.6, 21.0, 14.1; HRMS (ESI) calcd. for
C21H19F3N5 [M+H]+ 398.1587, found 398.1582.
2-((1-(3-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)methyl)-2,3,4,9-tetrahydro-1H-p
1
yrido[3,4-b]indole (6h). C21H18F3N5, pale yellow solid (86.7% yield), mp = 190.2-190.5 °C; H
NMR (CDCl3, 400MHz): δ ppm 8.35 (s, 1H, NH), 8.00 (s, 2H, C=CH, Ar-H), 7.89 (d, 1H, J = 8.0
Hz, Ar-H), 7.70-7.61 (m, 2H, Ar-H), 7.45 (d, 1H, J = 8.0 Hz , Ar-H), 7.24 (s, 1H, Ar-H), 7.12-7.05
(m, 2H, Ar-H), 3.99 (s, 2H, CH2), 3.72 (s, 2H, CH2), 2.99 (t, 2H, J = 6.0Hz, CH2), 2.85 (t, 2H, J =
6.0Hz, CH2); 13C NMR(CDCl3, 100MHz): δ ppm 146.0, 137.3, 136.1, 131.4, 130.5, 127.1, 125.3,
123.4, 121.3, 121.0, 119.3, 117.9, 117.3, 110.8, 107.9, 52.3, 51.0, 50.0, 21.3; HRMS (ESI) calcd.
for C21H19F3N5 [M+H]+ 398.1587, found 398.1585.
2-((1-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)methyl)-2,3,4,9-tetrahydro-1H-p
1
yrido[3,4-b]indole (6i). C21H18F3N5, yellow solid (83.7% yield), mp = 191.0-193.5 °C; H NMR
(DMSO, 400MHz): δ ppm 10.66 (s, 1H, NH), 8.92 (s, 1H, C=CH), 8.19 (d, 2H, J = 8.0 Hz, Ar-H),
7.97 (d, 2H, J = 12.0 Hz, Ar-H), 7.36 (d, 1H, J = 12.0 Hz, Ar-H), 7.27 (d, 1H, J = 8.0 Hz, Ar-H),
7.03-6.91 (m, 2H, Ar-H), 3.96 (s, 2H, CH2), 3.71 (s, 2H, CH2), 2.89 (t, 2H, J = 8.0 Hz, CH2), 2.73
(t, 2H, J = 6.0 Hz, CH2); 13C NMR(DMSO, 100MHz): δ ppm 146.0, 139.9, 136.3, 133.0, 129.2,
128.8, 127.6, 127.1, 122.7, 120.8, 118.7, 117.7, 111.3, 106.7, 52.2, 50.7, 50.0, 21.5; HRMS (ESI)
calcd. for C21H19F3N5 [M+H]+ 398.1587, found 398.1585.
2-((1-(2-nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]
1
indole (6j). C20H18N6O2, brown yellow solid (87.3% yield), mp = 169.0-169.7 °C; H NMR
(DMSO, 400MHz): δ ppm 9.84 (s, 1H, NH), 7.77 (s, 1H, C=CH), 7.34 (d, 1H, J = 12.0 Hz, Ar-H),
7.10-6.93 (m, 3H, Ar-H), 6.48 (d, 1H, J = 8.0 Hz, Ar-H), 6.39 (d, 1H, J = 12.0 Hz , Ar-H),
6.15-6.03 (m, 2H, Ar-H), 3.08 (s, 2H, CH2), 2.81 (s, 2H, CH2), 1.99 (t, 2H, J = 12.0 Hz, CH2),
13
1.84 (t, 2H, J = 12.0 Hz, CH2); C NMR (DMSO, 100MHz): δ ppm 144.5, 144.0, 136.3, 134.8,
133.0, 131.4, 129.7, 127.9, 127.1, 125.9, 125.6, 120.7, 118.7, 117.7, 111.3, 106.7, 52.0, 50.6, 49.8,
21.57; HRMS (ESI) calcd. for C20H19N6O2 [M+H]+ 375.1564, found 375.1563.

ACCEPTED MANUSCRIPT
2-((1-(2-isopropylphenyl)-1H-1,2,3-triazol-4-yl)methyl)-2,3,4,9-tetrahydro-1H-pyrido[3,
1
4-b]indole (6k). C23H25N5, brown yellow solid (95.0% yield), mp = 186.2-187.4 °C; H NMR
(DMSO, 400MHz): δ ppm 10.73 (s, 1H, NH), 8.39 (s, 1H, C=CH), 8.33 (s, 2H, Ar-H), 7.58 (s, 2H,
Ar-H), 7.37 (d, 2H, J = 4.0 Hz, Ar-H), 7.29 (d, 1H, J = 8.0 Hz, Ar-H), 7.04-6.03 (m, 2H, Ar-H),
3.97 (s, 2H, CH2), 3.74 (s, 2H, CH2), 3.42 (s, 2H, CH2), 2.74 (s, 2H, CH2), 1.14 (d, 6H, J = 8.0 Hz,
CH3); 13C NMR (DMSO, 100MHz): δ ppm 144.6, 136.3, 135.6, 133.1, 130.8, 127.2, 127.0, 126.5,
120.7, 118.7, 117.8, 111.3, 106.7, 60.2, 52.2, 50.7, 49.9, 28.0, 23.9, 21.6, 21.2, 14.5; HRMS (ESI)
calcd. for C23H26N5 [M+H]+ 372.2183, found 372.2178.
2-((1-(2-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4
1
-b]indole(6l). C21H21N5O, pale yellow solid (86.6% yield), mp = 168.6-169.3 °C; H NMR
(DMSO, 400MHz): δ ppm 10.71 (s, 1H, NH), 8.38 (s, 1H, C=CH), 7.65 (d, 1H, J = 8.0 Hz, Ar-H),
7.52 (t, 1H, J = 12.0 Hz, Ar-H), 7.37 (d, 1H, J = 12.0 Hz, Ar-H), 7.29 (t, 2H, J = 10.0 Hz, Ar-H),
7.14 (t, 1H, J = 10.0 Hz, Ar-H), 7.04-6.92 (m, 2H, Ar-H), 3.94 (s, 2H, CH2), 3.85 (s, 3H, OCH3),
3.71 (s, 2H, CH2), 2.89 (t, 2H, J = 8.0 Hz, CH2), 2.72 (t, 2H, J = 8.0 Hz, CH2); 13C NMR (DMSO,
100MHz): δ ppm 152.0, 144.0, 136.3, 133.1, 131.0, 127.1, 126.3, 126.1, 126.0, 121.3, 120.7, 118.7,
117.8, 113.4, 111.3, 106.7, 56.5, 52.2, 50.8, 49.9, 21.5; HRMS (ESI) calcd. for C21H22N5O
[M+H]+ 360.1819, found 360.1814.
2-((1-(4-tert-butylphenyl)-1H-1,2,3-triazol-4-yl)methyl)-2,3,4,9-tetrahydro-1H-pyrido[3,
1
4-b]indole (6m). C24H27N5, brown yellow solid (83.5% yield), mp = 224.4-227.4 °C; H NMR
(DMSO, 400MHz): δ ppm 10.68 (s, 1H, NH), 8.73 (s, 1H, C=CH), 7.84 (d, 2H, J = 8.0 Hz, Ar-H),
7.59 (d, 2H, J = 8.0 Hz, Ar-H), 7.36 (d, 1H, J = 4.0 Hz, Ar-H), 7.28 (d, 1H, J = 8.0 Hz, Ar-H), 7.01
(t, 1H, J = 8.0 Hz, Ar-H), 6.94 (t, 1H, J = 6.0 Hz, Ar-H), 3.94 (s, 2H, CH2), 3.71 (s, 2H, CH2), 2.89
(t, 2H, J = 4.0 Hz, CH2), 2.73 (t, 2H, J = 6.0 Hz, CH2), 1.32 (t, 9H, CH3); 13C NMR (DMSO,
100MHz): δ ppm 151.5, 145.4, 136.3, 134.9, 133.1, 127.1, 127.0, 122.3, 120.7, 120.1, 118.7, 117.7,
111.3, 106.7, 52.3, 50.8, 50.0, 34.92, 31.46, 21.56; HRMS (ESI) calcd. for C24H28N5 [M+H]+
386.2339, found 386.2335.
2-((1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4
1
-b]indole (6n). C21H21N5O, brown yellow solid (90.3% yield), mp = 216.7-219.7 °C; H NMR
(DMSO, 400MHz): δ ppm 10.69 (s, 1H, NH), 8.69 (s, 1H, C=CH), 7.84 (d, 2H, J = 12.0 Hz,
Ar-H), 7.36 (d, 1H, J = 8.0 Hz, Ar-H), 7.27 (d, 1H, J = 12.0 Hz, Ar-H), 7.14 (d, 2H, J = 12.0 Hz,
Ar-H), 7.03-6.91 (m, 2H, Ar-H), 3.92 (s, 2H, CH2), 3.83 (s, 3H, OCH3), 3.69 (s, 2H, CH2), 2.88 (t,
13
2H, J = 8.0 Hz, CH2), 2.72 (t, 2H, J = 8.0 Hz, CH2); C NMR (DMSO, 100MHz): δ ppm 159.5,
145.2, 136.3, 133.0, 130.6, 127.1, 122.4, 122.0, 120.7, 118.7, 117.8, 115.3, 111.3, 106.7, 56.0,
52.3, 50.8, 50.0, 21.5; HRMS (ESI) calcd. for C21H22N5O [M+H]+ 360.1819, found 360.1817.
2-((1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]i
1
ndole (6o). C20H18N6O2, brown yellow solid (89.2% yield), mp = 201.4-202.5 °C; H NMR
(DMSO, 400MHz): δ ppm 10.65 (s, 1H, NH), 9.00 (s, 1H, C=CH), 8.44 (d, 2H, J = 12.0 Hz,
Ar-H), 8.26 (t, 2H, J = 6.0 Hz, Ar-H), 7.36 (d, 1H, J = 8.0 Hz Ar-H), 7.26 (d, 1H, J = 6.0 Hz,
Ar-H), 7.03-6.91 (m, 2H, Ar-H), 3.97 (s, 2H, CH2), 3.71 (s, 2H, CH2), 2.90 (t, 2H, J = 8.0 Hz,
CH2), 2.73 (t, 2H, J = 8.0 Hz, CH2),; 13C NMR (DMSO, 100MHz): δ ppm 170.7, 147.0, 146.2,

ACCEPTED MANUSCRIPT
141.4, 136.3, 133.0, 127.1, 125.9, 122.9, 120.9, 118.7, 117.7, 111.3, 106.7, 60.2, 52.1, 50.7, 50.0,
21.1, 14.5; HRMS (ESI) calcd. for C20H19N6O2 [M+H]+ 375.1564, found 375.1558.
2-((1-(4-bromophenyl)-1H-1,2,3-triazol-4-yl)methyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b
1
]indole (6p). C20H18BrN5, brown yellow solid (87.2% yield), mp = 245.3-246.9 °C; H NMR
(DMSO, 400MHz): δ ppm 10.67 (s, 1H, NH), 8.82 (s, 1H, C=CH), 7.90 (s, 2H, Ar-H), 7.80 (s, 2H,
Ar-H), 7.36 (d, 1H, J = 8.0 Hz, Ar-H), 7.28 (d, 1H, J = 8.0 Hz, Ar-H), 7.01 (t, 1H, J = 8.0 Hz,
Ar-H), 6.95 (s, 1H, Ar-H), 3.94 (s, 2H, CH2), 3.71 (s, 2H, CH2), 2.89 (s, 2H, CH2), 2.73 (s, 2H,
CH2); 13C NMR (DMSO, 100MHz):
δ ppm 145.6, 136.3, 133.2, 127.1, 122.5, 122.3, 121.5, 120.7,
118.7, 117.8, 111.3, 106.7, 52.5, 50.8, 50.0, 21.5; HRMS (ESI) calcd. for C20H19BrN5 [M+H]+
408.0818, found 408.0817.
2-((1-(3-isopropylphenyl)-1H-1,2,3-triazol-4-yl)methyl)-2,3,4,9-tetrahydro-1H-pyrido[3,
1
4-b]indole (6q). C23H25N5, brown yellow solid (73.7% yield), mp = 167.0-168.9 °C; H NMR
(CDCl3, 400MHz): δ ppm 8.57 (s, 1H, NH), 7.94 (s, 1H, C=CH), 7.56 (s, 1H, Ar-H), 7.45-7.36 (m,
3H, Ar-H), 7.26-7.21 (m, 1H, Ar-H), 7.05 (t, 2H, J = 12.0 Hz, Ar-H), 3.94 (s, 2H, CH2), 3.68 (s,
2H, CH2), 3.54 (s, 2H, CH2), 2.82 (s, 2H, CH2), 1.27 (d, 7H, J = 8.0 Hz, CH-CH3); 13C NMR
(CDCl3, 100MHz):
δ ppm 151.0, 145.3, 137.0, 136.2, 131.6, 129.6, 127.1, 126.9, 121.2, 119.1,
118.7, 117.9, 110.9, 107.8, 53.4, 52.4, 50.9, 50.0, 34.1, 23.8, 21.3; HRMS (ESI) calcd. for
C23H26N5 [M+H]+ 372.2183, found 372.2180.
2-((1-(4-methyl-2-nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl)-2,3,4,9-tetrahydro-1H-pyri
1
do[3,4-b]indole (6r). C21H20N6O2, brown yellow solid (92.0% yield), mp = 128.7-129.4 °C; H
NMR (DMSO, 400MHz): δ ppm 10.73 (s, 1H, NH), 8.60 (s, 1H, C=CH), 8.05 (d, 1H, J = 8.0 Hz,
Ar-H), 7.74 (s, 2H, Ar-H), 7.37 (d, 1H, J = 8.0 Hz, Ar-H), 7.28 (d, 1H, J = 8.0 Hz, Ar-H), 7.02 (t,
1H, J = 8.0 Hz, Ar-H), 6.95 (t, 1H, J = 8.0 Hz, Ar-H), 3.97 (s, 2H, CH2), 3.71 (s, 2H, CH2), 2.89 (t,
2H, J = 6.0 Hz, CH2), 2.74 (t, 2H, J = 6.0 Hz, CH2), 2.50 (s, 3H, CH3); 13C NMR (DMSO,
100MHz): δ ppm 144.9, 144.3, 142.1, 136.3, 135.0, 133.1, 127.6, 127.4, 127.1, 125.9, 125.6,
120.7, 118.7, 117.8, 111.3, 106.7, 52.1, 50.7, 49.9, 21.6, 20.8; HRMS (ESI) calcd. for C21H21N6O2
[M+H]+ 389.1721, found 389.1719.
2-((1-(4-chloro-2-nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl)-2,3,4,9-tetrahydro-1H-pyri
do[3,4-b]indole (6s). C20H17ClN6O2, brown yellow solid (88.0% yield), mp = 131.3-132.4 °C; 1H
NMR (CDCl3, 400MHz): δ ppm 8.40 (s, 1H, NH), 8.00 (s, 1H, C=CH), 7.81 (s, 1H, Ar-H), 7.68 (d,
1H, J = 12.0 Hz, Ar-H), 7.42 (d, 2H, J = 8.0 Hz, Ar-H), 7.23 (d, 1H, J = 8.0 Hz, Ar-H), 7.11-7.02
(m, 2H, Ar-H), 4.00 (s, 2H, CH2), 3.69 (s, 2H, CH2), 2.98 (s, 2H, CH2), 2.83 (s, 2H, CH2); 13C
NMR (CDCl3, 100MHz): δ ppm 144.4, 136.6, 136.1, 133.8, 128.8, 127.0, 125.7, 121.3, 119.2,
117.9, 110.8, 107.8, 60.4, 53.4, 52.1, 51.0, 49.7, 21.2, 21.0, 14.2; HRMS (ESI) calcd. for
C20H18ClN6O2 [M+H]+ 409.1174, found 409.1173.
2-((1-(3-chloro-4-methylphenyl)-1H-1,2,3-triazol-4-yl)methyl)-2,3,4,9-tetrahydro-1H-py
1
rido[3,4-b]indole (6t). C21H20ClN5, brown yellow solid (90.3% yield), mp = 229.6-231.4 °C; H
NMR (DMSO, 400MHz): δ ppm 10.80 (s, 1H, NH), 8.96 (s, 1H, C=CH), 8.16 (s, 1H, Ar-H), 7.96
(d, 1H, J = 8.0 Hz, Ar-H), 7.67 (d, 1H, J = 8.0 Hz Ar-H), 7.47 (d, 1H, J = 8.0 Hz, Ar-H), 7.38 (d,

ACCEPTED MANUSCRIPT
1H, J = 8.0 Hz , Ar-H), 7.12 (t, 1H, J = 8.0 Hz, Ar-H), 7.05 (t, 1H, J = 8.0 Hz, Ar-H), 4.05 (s, 2H,
CH2), 3.81 (s, 2H, CH2), 3.48 (s, 3H, CH3), 3.00 (s, 2H, CH2), 2.84 (s, 2H, CH3); 13C NMR
(DMSO, 100MHz): δ ppm 150.3, 141.0, 140.9, 140.8, 139.3, 137.4, 131.8, 125.5, 125.2, 123.6,
123.4, 122.5, 116.0, 111.4, 99.9, 57.0, 55.5, 54.7, 26.3, 24.4; HRMS (ESI) calcd. for C21H21ClN5
[M+H]+ 378.1480, found 378.1480.
2-((1-(3,4-dichlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-2,3,4,9-tetrahydro-1H-pyrido[3,
4-b]indole (6u). C20H17Cl2N5, brown yellow solid (84.0% yield), mp = 226.1-227.1 °C; 1H NMR
(DMSO, 400MHz): δ ppm 9.80 (s, 1H, NH), 8.02 (s, 1H, C=CH), 7.43 (d, 1H, J = 4.0 Hz, Ar-H),
7.12 (d, 1H, J = 12.0 Hz, Ar-H), 6.98 (d, 1H, J = 8.0 Hz, Ar-H), 6.48 (d, 1H, J = 8.0 Hz, Ar-H),
6.39 (d, 1H, J = 8.0 Hz, Ar-H), 6.13 (t, 1H, J = 8.0 Hz, Ar-H), 6.06 (t, 1H, J = 8.0 Hz, Ar-H), 3.07
(s, 2H, CH2), 2.82 (s, 2H, CH2), 2.01 (s, 2H, CH2), 1.85 (s, 2H, CH2); 13C NMR (DMSO,
100MHz):
δ ppm 150.6, 141.4, 141.0, 137.8, 137.5, 136.9, 135.9, 131.8, 127.4, 126.8, 125.5,
125.1, 123.4, 122.5, 116.0, 111.4, 57.0, 55.5, 54.7, 26.3; HRMS (ESI) calcd. for C20H18Cl2N5
[M+H]+ 398.0934, found 398.0932.
2-((1-(2,5-dimethylphenyl)-1H-1,2,3-triazol-4-yl)methyl)-2,3,4,9-tetrahydro-1H-pyrido[3
1
,4-b]indole (6v). C22H23N5, brown yellow solid (81.0% yield), mp = 208.5-210.1 °C; H NMR
(CDCl3, 400MHz):
7.22-7.17 (m, 3H, Ar-H), 7.14-7.01 (m, 3H, Ar-H), 3.98 (s, 2H, CH2), 3.74 (s, 2H, CH2), 2.96 (s,
2H, CH2), 2.83 (s, 2H, CH2), 2.34 (s, 3H, CH3), 2.13 (s, 3H, CH3); 13C NMR (CDCl3, 100MHz):
δ ppm 8.51 (s, 1H, NH), 7.69 (s, 1H, C=CH), 7.42 (d, 1H, J = 8.0 Hz, Ar-H),
δ
ppm 136.8, 136.2, 131.2, 130.5, 130.1, 127.1, 126.4, 124.5, 121.2, 120.3, 120.2, 119.1, 117.8,
110.8, 107.8, 53.4, 52.3, 50.8, 50.2, 21.3, 20.7, 17.4; HRMS (ESI) calcd. for C22H24N5 [M+H]+
358.2026, found 358.2025.
2-((1-(3,4-dimethoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-2,3,4,9-tetrahydro-1H-pyrido
1
[3,4-b]indole (6w). C22H23N5O2, brown yellow solid (87.1% yield), mp = 180.9-181.6 °C; H
NMR (DMSO, 400MHz): δ ppm 10.70 (s, 1H, NH), 8.75 (s, 1H, C=CH), 7.50-7.43 (m, 2H, Ar-H),
7.37 (d, 1H, J = 12.0 Hz, Ar-H), 7.27 (d, 1H, J = 8.0 Hz, Ar-H), 7.13 (d, 1H, J = 12.0 Hz, Ar-H),
7.04-6.91 (m, 2H, Ar-H), 3.94 (s, 2H, CH2), 3.87 (s, 3H, CH3), 3.82 (s, 3H, CH3), 3.71 (s, 2H,
CH2), 2.91 (t, 2H, J = 6.0 Hz, CH2), 2.74 (t, 2H, J = 6.0 Hz, CH2); 13C NMR (DMSO, 100MHz):
δ
ppm 149.7, 149.2, 145.1, 136.3, 132.9, 130.6, 127.1, 122.5, 120.8, 118.7, 117.8, 112.4, 111.3,
106.6, 104.9, 56.3, 56.2, 55.3, 52.3, 50.8, 50.0, 21.4; HRMS (ESI) calcd. for C22H24N5O2 [M+H]+
390.1925, found 390.1922.
2-((1-(2,5-dichlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-2,3,4,9-tetrahydro-1H-pyrido[3,
1
4-b]indole (6x). C20H17Cl2N5, pale yellow solid (88.0% yield), mp = 211.5-211.9 °C; H NMR
(DMSO, 400MHz): δ ppm 10.62 (s, 1H, NH), 8.45 (s, 1H, C=CH), 7.84 (s, 1H, Ar-H), 7.72 (d, 1H,
J = 8.0 Hz, Ar-H), 7.64 (d, 1H, J = 4.0 Hz, Ar-H), 7.28 (d, 1H, J = 8.0 Hz, Ar-H), 7.19 (d, 1H, J =
8.0 Hz, Ar-H), 6.93 (t, 1H, J = 6.0 Hz, Ar-H), 6.86 (t, 1H, J = 8.0 Hz, Ar-H), 3.89 (s, 2H, CH2),
3.63 (s, 2H, CH2), 2.81 (t, 2H, J = 6.0 Hz, CH2), 2.64 (t, 2H, J = 6.0 Hz, CH2); 13C NMR (DMSO,
100MHz):
δ ppm 144.3, 136.3, 136.0, 133.0, 132.9, 132.3, 131.8, 128.6, 128.0, 127.1, 126.5,
120.7, 118.7, 117.7, 111.3, 106.7, 52.6, 50.7, 49.8, 21.5; HRMS (ESI) calcd. for C20H18Cl2N5
[M+H]+ 398.0934, found 398.0931.

ACCEPTED MANUSCRIPT
2-((1-mesityl-1H-1,2,3-triazol-4-yl)methyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole
1
(6y). C23H25N5, pale yellow solid (79.0% yield), mp = 148.5-149.9 °C; H NMR (CDCl3,
400MHz): δ ppm 8.56 (s, 1H, NH), 7.56 (s, 1H, C=CH), 7.44 (d, 1H, J = 8.0 Hz, Ar-H), 7.23 (s,
1H, J = 12.0 Hz, Ar-H), 7.10-7.02 (m, 2H, Ar-H), 6.98 (s, 2H, Ar-H), 4.03 (s, 2H, Ar-H), 3.73 (s,
2H, Ar-H), 2.95 (t, 2H, J = 8.0 Hz, CH2), 2.84 (t, 2H, J = 8.0 Hz, CH2), 2.35 (s, 3H, CH3), 1.85 (s,
6H, CH3); 13C NMR (CDCl3, 100MHz):
δ ppm 144.1, 140.3, 136.2, 134.9, 133.5, 131.7, 129.1,
127.1, 124.9, 121.2, 119.1, 117.8, 110.8, 107.7, 60.4, 53.4, 52.3, 50.6, 49.8, 21.3, 21.1, 17.2, 14.2;
HRMS (ESI) calcd. for C23H26N5 [M+H]+ 372.2183, found 372.2177.
General procedure for the preparation of 8. To a solution of compound 7 (1 mmol) in
glacial acetic acid (10 mL) at room temperature, sodium cyanoborohydride (0.19 g, 3 mmol) was
added. Then, the reaction was stirred at room temperature, monitored by TLC. Basification of the
solution by NaHCO3 (satd) was accomplished until pH value was about 8. The solution was
extracted with CH2Cl2. The combined organic layers were dried by MgSO4 and concentrated in
vacuo to afford compound 8 for the next step without further purification.
General procedure for the preparation of 9. To a solution of compound 8 (2.9 mmol) in
acetone (30 mL) at room temperature, anhydrous K2CO3 (0.8g, 5.8 mmol) was added and then
stirred for 45 min. To the above reaction mixture, the solution of bromopropyne (2, 0.34g, 2.9
mmol) in acetone (10 mL) with a catalytic amount of KI was added dropwise and then stirred at
room temperature for 12-24 h. The mixture was filtered and the filtrate was concentrated under
vacuum to afford compound 9 for the next step without further purification.
General procedure for the preparation of 10. To the solution of compound 9 (1 mmol) and
5 (1 mmol) in 75% methanol (40 mL), sodium ascorbate (30 mg) and CuSO4 (10 mg) were added
successively. The reaction solution was stirred at room temperature for 24-48 h. After filtration,
the solvent was evaporated and the crude product was purified by silica gel column
chromatography to give the desire product with high purity.
1-((1-(4-tert-butylphenyl)-1H-1,2,3-triazol-4-yl)methyl)indoline (10a). C21H24N4, brown
yellow solid (85.9% yield), mp = 148.2-150.2 °C; 1H NMR (CDCl3, 400MHz): δ ppm 7.86 (s, 1H,
C=CH), 7.60 (d, 2H, J = 8.0 Hz, Ar-H), 7.49 (d, 2H, J = 8.0 Hz, Ar-H), 7.08 (t, 2H, J = 10.0 Hz,
Ar-H), 6.69 (t, 1H, J = 8.0 Hz, Ar-H), 6.60 (d, 1H, J = 4.0 Hz, Ar-H), 4.49 (s, 2H, CH2), 3.43 (t,
2H, J = 8.0 Hz, CH2), 2.96 (t, 2H, J = 8.0 Hz, CH2), 1.34 (s, 9H, CH3); 13C NMR (CDCl3,
100MHz): δ ppm 152.1, 151.6, 145.2, 134.5, 130.3, 127.3, 126.6, 126.3, 124.7, 120.2, 118.3,
115.0, 107.5, 53.5, 44.7, 34.8, 31.3, 28.6; HRMS (ESI) calcd. for C21H25N4 [M+H]+ 333.2074,
found 333.2070.
1-((1-(3-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)indoline (10b). C17H15FN4, brown
yellow solid (73.1% yield), mp = 143.8-146.2 °C; 1H NMR (CDCl3, 400MHz): δ ppm 7.86 (s, 1H,
C=CH), 7.52-7.43 (m, 3H, Ar-H), 7.12-7.09 (m, 3H, Ar-H), 6.70 (t, 1H, J = 6.0 Hz, Ar-H), 6.59 (d,
1H, J = 8.0 Hz, Ar-H), 4.51 (s, 2H, CH2), 3.45 (t, 2H, J = 8.0 Hz, CH2), 2.98 (t, 2H, J = 8.0 Hz,
CH2); 13C NMR (CDCl3, 100MHz): δ ppm 151.5, 145.8, 138.1, 131.1, 130.3, 127.3, 124.7, 120.0,

ACCEPTED MANUSCRIPT
118.3, 115.7, 108.3, 107.4, 44.7, 31.6, 28.5, 22.6, 14.1; HRMS (ESI) calcd. for C17H16FN4 [M+H]+
295.1354, found 295.1352.
1-((1-(2-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)methyl)indoline
(10c).
1
C18H15F3N4, oily liquid (73.5% yield); H NMR (CDCl3, 400MHz): δ ppm 7.83 (s, 1H, C=CH),
7.72-7.63 (m, 3H, Ar-H), 7.53 (d, 1H, J = 8.0 Hz, Ar-H), 7.10-7.05 (m, 2H, Ar-H), 6.68 (t, 1H, J =
8.0 Hz, Ar-H), 6.61 (d, 1H, J = 8.0 Hz, Ar-H), 4.52 (s, 2H, CH2), 3.41 (t, 2H, J = 8.0 Hz, CH2),
2.96 (t, 2H, J = 10.0 Hz, CH2); 13C NMR (CDCl3, 100MHz): δ ppm 151.6, 144.6, 134.8, 133.0,
131.3, 130.4, 130.3, 128.9, 127.3, 124.8, 124.6, 121.2, 118.3, 107.5, 53.4, 53.3, 44.6, 28.5; HRMS
(ESI) calcd. for C18H16F3N4 [M+H]+ 345.1322, found 345.1319.
1-((1-(3-isopropylphenyl)-1H-1,2,3-triazol-4-yl)methyl)indoline (10d). C20H22N4, oily
liquid (83.2% yield); 1H NMR (CDCl3, 400MHz): δ ppm 7.88 (s, 1H, C=CH), 7.60 (s, 1H, Ar-H),
7.47 (d, 1H, J = 8.0 Hz, Ar-H), 7.41 (t, 1H, J = 8.0 Hz, Ar-H), 7.29 (d, 1H, J = 8.0 Hz, Ar-H), 7.09
(t, 2H, J = 8.0 Hz, Ar-H), 6.70 (t, 1H, J = 8.0 Hz, Ar-H), 6.62 (d, 1H, J = 8.0 Hz, Ar-H), 4.51 (s,
2H, CH2), 3.46 (t, 2H, J = 8.0 Hz, CH2), 2.99 (t, 3H, J = 10.0 Hz, CH2, C-CH), 1.30 (d, 6H, J =
4.0 Hz , CH3); 13C NMR (CDCl3, 100MHz): δ ppm 151.6, 151.0, 145.4, 137.1, 130.3, 129.5, 127.3,
126.9, 124.6, 120.3, 118.8, 118.2, 117.9, 107.4, 53.5, 44.8, 34.1, 28.6, 23.8, 22.6; HRMS (ESI)
calcd. for C20H23N4 [M+H]+ 319.1917, found 319.1914.
1-((1-(2-isopropylphenyl)-1H-1,2,3-triazol-4-yl)methyl)indoline (10e). C20H22N4, oily
liquid (85.1% yield); 1H NMR (CDCl3, 400MHz): δ ppm 7.61 (s, 1H, C=CH), 7.48 (t, 2H, J = 8.0
Hz, Ar-H), 7.32-7.28 (m, 1H, Ar-H), 7.25 (d, 1H, J = 8.0 Hz, Ar-H), 7.11-7.05 (m, 2H, Ar-H), 6.70
(t, 1H, J = 8.0 Hz, Ar-H), 6.62 (d, 1H, J = 8.0 Hz, Ar-H), 4.56 (s, 2H, Ar-H), 3.47 (t, 2H, J = 8.0
Hz, CH2), 2.99 (t, 2H, J = 8.0 Hz, CH2), 2.74-2.63 (m, 1H, C-CH), 1.16 (d, 6H, J = 8.0 Hz , CH3);
13C NMR (CDCl3, 100MHz): δ ppm 151.6, 144.7, 144.4, 135.2, 130.4, 130.3, 127.3, 126.8, 126.5,
124.6, 124.3, 118.2, 107.6, 53.5, 44.7, 28.6, 27.9, 23.8, 22.2; HRMS (ESI) calcd. for C20H23N4
[M+H]+ 319.1917, found 319.1914.
1-((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)indoline (10f). C17H15ClN4, oily
liquid (89.6% yield); 1H NMR (CDCl3, 400MHz): δ ppm 7.86 (s, 1H, C=CH), 7.65 (d, 2H, J = 8.0
Hz, Ar-H), 7.46 (d, 2H, J = 8.0 Hz, Ar-H), 7.12-7.07 (m, 2H, Ar-H), 6.71 (t, 1H, J = 8.0 Hz, Ar-H),
6.60 (d, 1H, J = 8.0 Hz, Ar-H), 4.50 (s, 2H, CH2), 3.45 (t, 2H, J = 8.0 Hz, CH2), 2.98 (t, 2H, J =
8.0 Hz, CH2); 13C NMR (CDCl3, 100MHz): δ ppm 151.5, 145.7, 135.5, 134.4, 130.3, 129.8, 127.3,
124.7, 121.6, 120.0, 118.3, 107.4, 53.5, 44.7, 28.6; HRMS (ESI) calcd. for C17H16ClN4 [M+H]+
311.1058, found 311.1054.
5-chloro-1-((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)indoline (10g). C17H14Cl2N4,
pale yellow solid (87.4% yield), mp = 137.0-138.5 °C; 1H NMR (CDCl3, 400MHz): δ ppm 7.85 (s,
1H, C=CH), 7.65 (d, 2H, J = 8.0 Hz, Ar-H), 7.46 (d, 2H, J = 8.0 Hz, Ar-H), 7.00 (d, 2H, J = 8.0
Hz, Ar-H), 6.48 (d, 1H, J = 8.0 Hz, Ar-H), 4.45 (s, 2H, CH2), 3.46 (t, 2H, J = 8.0 Hz, CH2), 2.94 (t,
2H, J = 8.0 Hz, CH2); 13C NMR (CDCl3, 100MHz): δ ppm 150.2, 135.4, 134.4, 132.2, 129.8,
126.9, 124.8, 122.8, 121.5, 120.0, 107.9, 53.5, 44.4, 28.3, 22.6, 21.0, 14.2; HRMS (ESI) calcd. for
C17H15Cl2N4 [M+H]+ 345.0668, found 345.0665.

ACCEPTED MANUSCRIPT
1-((1-(4-tert-butylphenyl)-1H-1,2,3-triazol-4-yl)methyl)-5-chloroindoline
1
(10h) .C21H23ClN4, brown yellow solid (73.6% yield), mp = 152.7-154.5 °C; H NMR (CDCl3,
400MHz): δ ppm 7.81 (s, 1H, C=CH), 7.61 (d, 2H, J = 8.0 Hz, Ar-H), 7.51 (d, 2H, J = 12.0 Hz,
Ar-H), 7.02 (d, 2H, J = 8.0 Hz, Ar-H), 6.50 (d, 1H, J = 8.0 Hz, Ar-H), 4.47 (s, 2H, CH2), 3.46 (t,
2H, J = 8.0 Hz, CH2), 2.95 (t, 2H, J = 8.0 Hz, CH2), 1.35 (s, 9H, CH3); 13C NMR (CDCl3,
100MHz): δ ppm 152.1, 150.3, 144.7, 134.5, 132.2, 126.9, 126.6, 124.8, 122.7, 122.2, 120.4,
120.2, 108.0, 53.4, 44.5, 34.7, 31.2, 28.3; HRMS (ESI) calcd. for C21H24ClN4 [M+H]+ 367.1684,
found 367.1682.
1-((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-5-fluoroindoline (10i). C17H14ClFN4,
brown yellow solid (79.8% yield), mp = 119.2-122.1 °C; 1H NMR (CDCl3, 400MHz): δ ppm 7.86
(s, 1H, C=CH), 7.66 (d, 2H, J = 8.0 Hz, Ar-H), 7.47 (d, 2H, J = 8.0 Hz, Ar-H), 6.88-6.73 (m, 2H,
Ar-H), 6.50-6.47 (m, 1H, Ar-H), 4.44 (s, 2H, CH2), 3.43 (t, 2H, J = 8.0 Hz, CH2), 2.94 (t, 2H, J =
8.0 Hz, CH2); 13C NMR (CDCl3, 100MHz): δ ppm 157.9, 155.6, 147.8, 145.5, 135.4, 134.4, 132.0,
129.8, 121.5, 120.0, 113.1, 112.4, 107.6, 54.0, 45.3, 31.5, 28.6; HRMS (ESI) calcd. for
C17H15ClFN4 [M+H]+ 329.0964, found 329.0960.
4.3. Docking experiments
Receptor protein (PDB ID: 5IS0) was prepared and optimized using relevant module by
Sybyl-X 1.10 software. The 3D-interaction was plotted by Discovery Studio 4.1 Client.
Acknowledgment
This study was financially supported by the National Natural Science Foundation of China
(U1704185) and the Science and Technology Opening and Co-production Project of Henan
Province of China (182106000060).
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Highlights
• Design, synthesis, and pharmacologic evaluation of a class of TRPV1 antagonists which
constructed on 2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole as A-region and triazole as B-region.
•
Optimization of this design led to the eventual identification of
2-((1-(2-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)methyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-
b]indole (6g), a potent TRPV1 antagonist.
• 6g exhibited potent antagonism activated by capsaicin (IC50 = 0.075 µM) and only partially
blocked acid activation of TRPV1, and demonstrated good efficacy in different pain models and
did not elevate core body temperature.