Design, synthesis and biological evaluation of N1-(isoquinolin-5-yl)-N2-phenylpyrrolidine-1,2-dicarboxamide derivatives as potent TRPV1 antagonists

Article abstract of DOI:10.1016/j.bioorg.2018.09.033

Reported herein is the design, synthesis, and pharmacologic evaluation of a class of TRPV1 antagonists constructed on a N¹-(isoquinolin-5-yl)-N²-phenylpyrrolidine-1,2-dicarboxamide platform that evolved from a 5-aminoisoquinoline urea lead. Advancing the SAR of this series led to the eventual identification of 3b, comprising a p-Br substituted phenyl. In a TRPV1 functional assay, using cells expressing recombinant human TRPV1 channels, 3b displayed potent antagonism activated by capsaicin (IC₅₀ = 0.084 μM) and protons (IC₅₀ = 0.313 μM). In the preliminary analgesic and body temperature tests, 3b exhibited good efficacy in capsaicin-induced and heat-induced pain models and without hyperthermia side-effect. On the basis of its superior profiles, 3b could be considered as the lead candidate for the further development of antinociceptive drugs.

Full text of DOI:10.1016/j.bioorg.2018.09.033

Accepted Manuscript
Design, synthesis and biological evaluation of N 1-(isoquinolin-5-yl)-N 2-phe-
nylpyrrolidine-1,2-dicarboxamide derivatives as potent TRPV1 antagonists
Mingxiang Gao, Cunbin Nie, Jinyu Li, Beibei Songm, Xinru Chengm, Erying
Sun, Lin Yan, Hai Qian
PII:
S0045-2068(18)30664-3
DOI:
https://doi.org/10.1016/j.bioorg.2018.09.033
YBIOO 2530
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
5 July 2018
22 September 2018
24 September 2018
Please cite this article as: M. Gao, C. Nie, J. Li, B. Songm, X. Chengm, E. Sun, L. Yan, H. Qian, Design, synthesis
and biological evaluation of N 1-(isoquinolin-5-yl)-N 2-phenylpyrrolidine-1,2-dicarboxamide derivatives as potent
TRPV1 antagonists, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.09.033
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Design, synthesis and biological evaluation of
N1-(isoquinolin-5-yl)-N2-phenylpyrrolidine-1,2-dicarboxamide derivatives as potent TRPV1
antagonists
Mingxiang Gaoa, Cunbin Niea, Jinyu Lia, Beibei Songa, Xinru Chenga, Erying Suna, 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
ABSTRACT
Reported herein is the design, synthesis, and pharmacologic evaluation of a class of TRPV1
antagonists constructed on
a
N1-(isoquinolin-5-yl)-N2-phenylpyrrolidine-1,2-dicarboxamide
platform that evolved from a 5-aminoisoquinoline urea lead. Advancing the SAR of this series led
to the eventual identification of 3b, comprising a p-Br substituted phenyl. In a TRPV1 functional
assay, using cells expressing recombinant human TRPV1 channels, 3b displayed potent
antagonism activated by capsaicin (IC50 = 0.084 μM) and protons (IC50 = 0.313 μM). In the
preliminary analgesic and body temperature tests, 3b exhibited good efficacy in capsaicin-induced
and heat-induced pain models and without hyperthermia side-effect. On the basis of its superior
profiles, 3b could be considered as the lead candidate for the further development of
antinociceptive drugs.
Keywords: analgesic, transient receptor potential vanilloid type 1, 5-aminoisoquinoline urea,
hyperthermia.
1. Introduction
Transient receptor potential vanilloid 1 (TRPV1) is an ion channel expressed on sensory
neurons triggering an influx of cations, which is selectively activated 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) [1-4].
Activation of this channel is associated to chronic inflammatory pain and peripheral neuropathy
[5]. Therefore, inhibition of TRPV1 function represents a strategy for the treatment of a variety of
disease states, particularly in the management of chronic intractable pain [6, 7]. Over the past
decade, a number of potent and selective small molecule TRPV1 antagonists has confirmed that
pharmacological blockade of this receptor provided analgesic efficacy in several models of
inflammatory and neuropathic pain [8-10]. However, the tendency of some TRPV1 antagonists to
induce hyperthermia side-effect in preclinical models turned out to be a hurdle and led to its
withdrawal from clinical development [11]. As a result, pharmacological separation of analgesic
and hyperthermic effects became the key challenge in developing TRPV1 antagonists as
therapeutic agents for pain management. Recently, efforts to eliminate hyperthermia led to the
identification of the relative responses of TRPV1 to various stimulatory modulators, where
*
Corresponding authors.
E-mail addresses: yanlin0378@163.com (L. Yan), qianhai24@163.com (H. Qian).

whether TRPV1 antagonists block responses both to capsaicin and low pH or whether they
selectively antagonize capsaicin only [12, 13]. Reported herein is the design, synthesis and
evaluate for their activity of TRPV1 antagonists constructed on a 5-aminoisoquinoline urea
fragment (Fig. 1). The early lead compound 1 which belonged to the “first-generation” TRPV1
antagonists exhibited good potency at the target, modest efficacy in animal pain models, and less
than desirable pharmacokinetic profile [14, 15]. It bears emphasizing, that most of the
“first-generation” antagonists block, in a dose-dependent manner, all modes of TRPV1 activation
(capsaicin, endogenous lipids, acidic pH, heat) can elicit hyperthermia [16]. More recently, the
isoquinoline urea derivative 2 which including chromane moiety and with R configuration was
found to be a modality-differentiated second-generation TRPV1 antagonist with good analgesic
efficacy and a temperature-neutral profile [9]. In contrast to first-generation antagonists that
inhibit all modes of TRPV1 activation and can elicit hyperthermia, compound 2 fully block
TRPV1 activation by capsaicin but only partially block TRPV1 activation by acid and devoid of
hyperthermic effects at high dose. These data encouraged a more focused investigation of SAR to
optimization of the isoquinoline urea series TRPV1 antagonists. In this study, to look for another
class of potent TRPV1 antagonists and further explore the isoquinoline urea, the pyrrolidine ring
was designed to maintain the distance and constrained nature of the phenyl ring, whereas the
chromane ring was cleaved, leading to compound 3b, a novel TRPV1 antagonist with good
analgesic efficacy and a temperature-neutral profile (Fig. 1).
F
F
O
O
O
O
O
N
HN
N
R
HN
N
H
HN
N
H
H
CF3
Cl
N
N
N
2
3
1
Figure 1. Structural modifications of TRPV1 antagonists.
2. Results and discussion
2.1. Chemistry
The synthesis of the target compounds 3 were accomplished by urea coupling between the
two corresponding amines (6, 10) as described in Scheme 1. Intermediate 6 was prepared via
nitration of commercially available isoquinoline (4) and subsequent reduction using Pd/C as a
catalyst. The synthesis of another intermediate (10) was accomplished starting from
N-Boc-L-proline (7). Reaction of compound 7 with substituted anilines produced pyrrolidine
carboxamides 9a-w. The Boc protecting group was removed under acidic conditions (HCl),
leading to 10a-w in high yield for coupling. The coupling between 6 and 10a-w in the presence of
triphosgene provided the target compounds 3a-w. The structures of all target compounds are
presented in Table 1.

NO2
NH2
a
b
d
N
N
N
6
4
5
NH2
O
O
O
c
+
R
N
HN
N
Boc
N
Boc
HN
OH
H
.HCl
R
R
7
8a - w
9a - w
10a - w
R
O
O
NH
e
+
6
10a - w
HN
N
N
3a - w
Scheme 1. Synthesis of the target compounds 3. Reagents and conditions: (a) HNO3, H2SO4, -15°C; (b) Pd/C,
CH3OH, rt; (c) EDCI/DMAP; (d) HCl/EtOAc; (e) triphosgene/DMAP, CH2Cl2, one pot.
2.2. Structure–activity relationship (SAR) analysis
Firstly, all of the new compounds were evaluated for their ability to block capsaicin (CAP) or
low pH-induced activation of human TRPV1 channels. The results are presented in Table 1,
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, bulky tert-butyl group, even the
multisubstituted benzene rings were basically well tolerated. The effects of changing the position
of the substituent were also investigated. In CAP assay, the para-substitution was superior to
meta-substitution or ortho-substitution. The representative examples include phenyls with bromine
(3b vs 3c, 3d), chlorine (3g vs 3e, 3f), methoxyl (3i vs 3h), and methyl (3j vs 3k) substitutions
(Table 1). However, in pH assay, these para-substitution compounds showed dramatic decrease in
potency than substitutions at meta- and ortho-positions, while that of 3i, resulted in a minor
increase in activity (compare 3i IC50 = 0.049 μM vs 3h IC50 = 0.054 μM).
Table 1
In vitro ability of compounds to inhibit the activation of hTRPV1 receptors
hTRPV1(CAP)
IC50a (μM)
hTRPV1(pH)
IC50b (μM)
Compounds
R
H
0.481 ± 0.072
0.084 ± 0.019
0.239 ± 0.087
0.216 ± 0.102
0.038 ± 0.015
0.313 ± 0.023
0.059 ± 0.042
0.064 ± 0.017
3a
3b
3c
3d
4-Br
3-Br
2-Br

2-Cl
3-Cl
ND
0.041 ± 0.036
0.056 ± 0.049
0.128 ± 0.092
0.054 ± 0.043
0.049 ± 0.047
0.307 ± 0.124
0.046 ± 0.075
0.035 ± 0.014
0.135 ± 0.062
0.171 ± 0.078
0.214 ± 0.056
ND
3e
3f
3.1 ± 0.1
4-Cl
0.416 ± 0.064
0.451 ± 0.087
0.195 ± 0.041
0.314 ± 0.094
0.382 ± 0.029
ND
3g
2-OCH3
3h
3i
4-OCH3
4-CH3
3j
3-CH3
3k
3l
3-iPr
4-tBu
0.211 ± 0.031
0.199 ± 0.053
0.208 ± 0.047
ND
3m
3n
3o
4-F
2-CH3, 4-OCH3
2-Cl, 4-Br
3-Cl, 4-CH3
2, 6-di-CH3
2, 4-di-CH3
3-Cl, 4-OCH3
3, 4-di-Cl
3, 5-di-OCH3
2, 4, 6-tri-CH3
3p
3q
3r
0.234 ± 0.071
ND
0.011 ± 0.025
0.0067 ± 0.0097
ND
0.497 ± 0.068
2.8 ± 0.2
3s
ND
3t
0.204 ± 0.062
1.9 ± 0.3
0.0056 ± 0.0042
ND
3u
3v
0.412 ± 0.082
0.017 ± 0.045
0.0089 ± 0.0059
0.0032 ± 0.0098
3w
BCTC
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, to develop more efficient analgesic candidates with minimized side-effects, all of the
new compounds were advanced into our preliminary in vivo analgesic studies and side-effects tests.
Initially, when administered orally in mice, most of compounds were found to be nearly not
efficacious at 1 and 10 mg/kg, in comparison with vehicle group (data not shown). We
subsequently examined the analgesic activities using the dose of 30 mg/kg. The analgesic activity
in vivo of each compounds were evaluated by three different models of pain (Fig. 2). In the
capsaicin test, the total time spent licking the paw was significantly reduced by all test compounds
compared to the vehicle (Fig. 2A). In addition, most of the test compounds were superior to
positive control BCTC, especially 3b, 3i, 3n, 3o and 3u exhibited greater potency than BCTC.
Once more, the para-substitution was the favorable site for higher potency. For instance, p-Br
substituted 3b was more potent than m-Br substituted 3c or o-Br substituted 3d. In the abdominal
constriction test, all compounds reduced the number of writhes in proto-induced pain models and
many test compounds exhibited better potency than BCTC (Fig. 2B). Of particular interest was the
compound 3b which was the most active compound in the capsaicin test, had much weaker effect
compared to other compounds, but consistent with the activity observed in pH assay. In the
tail-flick test, a variety of substituents are well tolerated (Fig. 2C). Evidently, better %MPE in
treatment of heat-induced pain were obtained from para-substitution compounds such as 3b, 3g,
3j and 3n. Overall, all the test compounds had antinociceptive activity to a certain extend.
Basically all of the compounds were efficacious in capsaicin-induced pain and in the tail-flick test,

but some of test compounds such as 3b, 3j and 3n lacked efficacy in the abdominal constriction
test. To our delight, recent reports have indicated that the hyperthermic effect of a TRPV1
antagonist is related to the extent to which it causes blockade in proton mode. Indeed, some
TRPV1 antagonists which only minimally blocking acid activation of TRPV1, have been
identified do not elevate core body temperature in preclinical models. 9
Figure 2. 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 = 8).

Statistical analysis was evaluated using a one-way analysis of variance (ANOVA) followed by Dunnett’s multiple
comparison test. *p <0.05; **p <0.01; ***p <0.001 compared with the vehicle group.
In a follow-up experiment, potent compounds (i.e., those exhibiting acceptable in vivo and in
vitro potency) were further assessed for their body temperature and compared their effects with
positive control BCTC (Fig. 3). A single oral dose of 30 mg/kg was administered to mice, and core
body temperature was evaluated over 2 h using a rectal thermometer. In our tests, there were
significant increases by almost all test compounds on body temperature beginning 30 min after
administration and lasting for at least 90 min, and with a maximum of △temperature occurring at
60 min. In contrast, compound 3b which exhibited very 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, 3b was effective in treatment of
capsaicin-induced and heat-induced pain, and not effective in proton-induced pain. Compared to
3b, compounds 3q and 3u exhibited good analgesic potencies in acid-induced, capsaicin-induced
pain and caused hyperthermia in mice. Of the compounds evaluated in vitro TRPV1 antagonistic
activity, in vivo model of pain and body temperature, 3b emerged as a preferred compound and
exhibited superior pharmacodynamic properties. Therefore, it was evaluated in more detail.
Figure 3. The effects of compounds in 30 mg/kg after oral administration on body temperature in mice. The
changes of body temperature after dose. Data are expressed as mean ± SEM (n = 8). *p < 0.05, **p < 0.01; ***p <
0.001 by Dunnett’s multiple comparison test compared with the vehicle-treated group.
Focusing in particular on dose-response analyses to better determine the efficacy of 3b, some
additional experiments had been carried out. First, analgesic activities of compound 3b at different
doses were assessed (Fig. 4). In the capsaicin-induced pain model study, 3b (i.g., 30 min before
capsaicin) dose-dependently reduced the total time spent licking the paw (Figure 4A). In addition,
the oral administration of 10 mg/kg and 60 mg/kg of 3b were similar to an oral dose of 30 mg/kg
of 3b, which exhibited greater potency than BCTC. In the abdominal constriction test, though
pretreatment with 3b (i.g., 30 min before the injection of acetic acid) dose-dependently reduced
the number of writhes the effect was not significant and much weaker than BCTC (30 mg/kg i.g.)
did (Figure 4B). Similarly, in the tail-flick test compound 3b-produced antinociception was
dose-dependent (Figure 4C). Compound 3b (1 mg/kg) had a similar %MPE to the vehicle group,
while 3b (30 mg/kg) and 3b (60 mg/kg) showed higher %MPE than BCTC (30 mg/kg). From the
above analgesic activity tests, the compound 3b exhibited good analgesic potency in

capsaicin-induced and heat-induced pain models and the antinociception effect was
dose-dependent. However, the analgesic activity tests also indicated that the compound 3b
exhibited differentiated effects on capsaicin-induced and acetic acid-induced pain model.
Figure 4. Analgesic activities of compound 3b at different doses. (A) The antinociceptive effects in the capsaicin
test; (B) Suppression of acetic acid-induced writhing response; (C) Inhibition of thermal nociception. Each bar
represents the mean ± SEM (n = 8). Statistical analysis was evaluated using a one-way analysis of variance
(ANOVA) followed by Dunnett’s multiple comparison test. *p < 0.05; **p < 0.01; ***p < 0.001 compared with
the vehicle group; #p < 0.05; ##p < 0.01; ###p < 0.001 compared with the 3b (30 mg/kg) group.
To explore this further with the compound 3b, the dose-response studies were administered
to mice, and core body temperature was evaluated over 2 h using a rectal thermometer. As
illustrated in Fig. 5, no significant changes in core body temperature were observed in all the
doses of 3b versus vehicle when monitored over a period of 2 h. While a 30 mg/kg po dose of
BCTC significantly increased temperature relative to vehicle, beginning 30 min after
administration and lasting for at least 2 h. In general, the analgesic activity of 3b was in a
dose-dependent manner and over all times and doses of 3b, the average change of core body
temperature was not significantly above vehicle.
Figure 5. The effects of compound 3b at different doses on body temperature in mice. The changes of body
temperature after dose. Data are expressed as mean ± SEM (n = 8). *p < 0.05, **p < 0.01; ***p < 0.001 by
Dunnett’s multiple comparison test compared with the vehicle-treated group.
On the basis of its potent in vitro activity at hTRPV1, good analgesic activity in
capsaicin-induced and heat-induced pain models and without hyperthermia side-effect, compound
3b was selected for further study. The results of both the in vitro TRPV1 activities in rat and the
pharmacokinetic profiles were reported in Table 6. Results from in vitro rTRPV1 antagonistic

activities showed that compound 3b was also potent antagonist against rat TRPV1 (rTRPV1).
Consistent with its in vitro hTRPV1 antagonistic activities, 3b exhibited poor antagonism toward
pH. In addition, high aqueous solubility is widely recognized as a requirement for good in vivo
bioavailability. According to the Log P values (the calculated octanol-water partition coefficient),
compound 3b was less lipophilic (logP 2.56) than compound 1 (logP 3.16) and 2 (logP 3.46). Of
course, one concern is that predicted Log P values have significant uncertainty. We subsequently
identified compound 3b showed satisfactory aqueous solubility (﹥100 μM). The pharmacokinetic
parameters of 3b administered in oral were also summarized in Table 6. As shown in Table 6,
compound 3b exhibited moderate Cmax (119.3 ± 35.1 ng/mL), and modest half-life (1.4 ± 0.3 h)
as well as relatively moderate bioavailability (F = 23%) in rats after oral administration of 30
mg/kg.
Table 6. In vitro TRPV1 activities and the pharmacokinetic profiled of compound 3b
characteristic
3b
rTRPV1(CAP) IC50a (μM)
rTRPV1(pH) IC50b (μM)
Log Pc
0.1543 ± 0.058
0.6984 ± 0.187
2.56
Solubility pH 6.8 (μM)
﹥100
t1/2 (h)
1.4 ± 0.3
119.3 ± 35.1
23
Cmax (ng/mL)
bioavailability, Foral (%)
a Rat TRPV1 receptor activated by capsaicin.
b Rat TRPV1 receptor activated by low pH (5.0). Unless otherwise stated, all values are the mean (SEM of at least
three separate experiments).
cLog P values calculated by ChemDraw.
dPharmacokinetic analysis determined in rat (three animals per group each oral) following administration of 30
mg/kg.
3. Conclusions
In conclusion, this work summarizes the transformation of an early 5-aminoisoquinoline urea
motif into a N1-(isoquinolin-5-yl)-N2-phenylpyrrolidine-1,2-dicarboxamide-based design and its
optimization to produce 3b. Close scrutiny of our pharmacology data revealed 3b was a potent
TRPV1 antagonist that exhibited excellent in vitro functional activity and good efficacy in
capsaicin-induced and heat-induced pain models. Further experiments showed that the
antinociception effects produced by 3b were dose-dependent and the analgesic mechanism of 3b
may be mainly by blocking heat-induced and capsaicin-induced TRPV1 activation without
hyperthermia. In addition, compound 3b displayed promising pharmacokinetic properties in rats
following oral administration. Taken together, this investigation has provided us with novel
scaffolds for the further study of related TRPV1 antagonists.
4. Experimental
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 = 8), 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
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) [17, 18].
4.1.2. Analgesic activity
4.1.2.1. Capsaicin test
As previously described, we evaluated analgesic activity in the capsaicin-induced pain model
[19]. 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 [20]. 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.20 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 = the temperature at
the certain time after dosing – the temperature at 0 min after dosing.
4.1.4. Aqueous solubility
Small volumes of the DMSO solutions of test compounds were diluted to 130 μM by adding
second liquid for a disintegration test of Japanese Pharmacopoeia (JP2, pH=6.8). After incubation
at 25 °C for 20 h, precipitates were separated by filtration. The solubility was determined by
HPLC analysis of each filtrate.
4.1.5. Pharmacokinetic Study.
The animal studies were performed according to committee approved procedures. Male rats,
each weighing 330−380 g (9−10 weeks old), were quarantined for 1 week before use. The animals
were surgically implanted with a jugularvein cannula 1 day before treatment and were fasted
before treatment. The compound was given to the rats (n = 3) as oral (30 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 0 (immediately before dosing), 1,
2, 4, 6, 8, and 24 h after dosing, a blood sample (∼150 mL) was taken from each animal via the
jugular-vein cannula and stored in ice (0−4 °C). The processing of the plasma and analysis by high
performance liquid chromatography−tandem mass spectrometry (HPLC−MS/MS) was carried out
as described. The plasma concentration data were analyzed with a standard noncompartmental
method.
4.1.6. 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 Ⅲ apparatus at 300 MHz, in CDCl3 unless
otherwise indicated; δ in ppm rel. to Me4Si, J in Hz. 13C NMR spectra: BrukerAVANCE Ⅲ
apparatus at 75 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.
4.2.2. General procedure for the preparation of isoquinolin-5-amine (6)
To a solution of isoquinoline (3.0 g, 23.2 mmol) in 40 mL H2SO4 at -15°C was added solid
KNO3 (2.8 g, 27.8 mmol) in four successive equal portions in 30 min. The reaction mixture was
warmed to room temperature and stirred for 3 h, then poured into ice-water (100 mL). The pH of
the mixture was adjusted to 8-10 with a few drops of ammonia. The precipitated solids were
collected by filtration, washed with methyl-tert-butyl ether (100 ml × 2) and dried to provide 5
(4.0 g, 100% yield) as a yellow solid. To compound 5 (2.5 g, 14.4 mmol) in MeOH (50 mL) was
added 10% Pd/C (300 mg), and the mixture was stirred under an atmosphere of hydrogen at room
temperature for 3 h. The mixture was filtered and the filtrate was concentrated under vacuum to
give the 6 (0.85 g, 41% yield) as a tan solid.
4.2.3. General procedure for the preparation of 10a-w
The solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 1.78
g, 9.30 mmol) in CH2Cl2 (50 mL) with N, N-dimethylaminopyridine (DMAP, cat) was added
dropwise to a stirred solution of Boc-protected L- proline 7 (1 g, 4.64 mmol) in CH2Cl2 (20 mL) at
room temperature. The reaction mixture was stirred at room temperature for 0.5 h. After this
period, a solution of substituted phenylamines 8a-w (4.64 mmol) in CH2Cl2 (20 mL) was added
dropwise at 0°C. The resulting mixture was then heated to room temperature and stirred,
monitored by TLC. Then the reaction mixture was washed with 1N hydrochloric acid (60 mL × 2),
aqueous brine (60 mL), saturated NaHCO3 aqueous solution (60 mL × 2), brine (60 mL), dried
over Na2SO4, and concentrated in vacuo to provide 9a-w as yellow solid. The resulting solid of
9a-w was dissolved in saturated HCl ethyl acetate solution (15 mL) and the reaction mixture was
stirred at room temperature, monitored by TLC. Then the resulting solid was filtered and dried
under reduced pressure to provide 10a-w as an HCl salt.
4.2.4. General procedure for the preparation of 3a-w
The solution of 6 (300 mg, 2.08 mmol) in CH2Cl2 (20 mL) was added dropwise to a stirred
solution of triphosgene (415 mg, 1.39 mmol) in CH2Cl2 (20 mL) under nitrogen. Then DMAP
(750 mg, 6.15 mmol) was added to the mixture. The reaction mixture was stirred for 30 min,
followed by added solution of 10a-w (2.08 mmol) in CH2Cl2 (20 mL) and stirred for an additional
12 h at room temperature. The reaction mixture was monitored by TLC and washed with water to
remove DMAP. The organic layer was dried over Na2SO4 and concentrated by rotary evaporation
to give the red-brown crude product. The residue was purified by flash column chromatography
eluting with CH2Cl2/CH3OH (20:1) to obtain 3a-w.
4.2.4.1. (R)-N1-(isoquinolin-5-yl)-N2-phenylpyrrolidine-1,2-dicarboxamide (3a)
Yield 50.5%, red-brown solid, mp = 232.0-234.0℃; 1H NMR (DMSO, 300 MHz) δ ppm 10.00 (s,
1H, NH), 9.29 (s, 1H, NH), 8.55 (s, 1H, isoquinoline), 8.46 (d, 1H, J = 3.0 Hz, isoquinoline),
7.91-7.86 (m, 2H, isoquinoline), 7.74 (t, 1H, J = 6.0 Hz, isoquinoline), 7.63 (t, 3H, J = 4.5 Hz,
isoquinoline and Ar-H), 7.29 (t, 2H, J = 6.0 Hz, Ar-H), 7.03 (t, 1H, J = 6.0 Hz, Ar-H), 4.54 (d, 1H,
J = 6.0 Hz, CH), 3.81-3.66 (m, 2H, NCH2), 2.26-2.22 (m, 1H, NCH2CH2), 2.02-1.86 (m, 3H,
CHCH2 and NCH2CH2); 13C NMR (DMSO, 75 MHz) δ ppm 171.8, 155.2, 152.7, 142.5, 139.6,

135.0, 131.8, 130.9, 129.0, 127.5, 126.4, 124.5, 123.5, 119.6, 117.2, 61.0, 47.2, 30.5, 24.7;
HRMS(ESI) Calcd for C21H20N4O2[M+H]+ 361.1659, found: 361.1652.
4.2.4.2. (R)-N2-(4-bromophenyl)-N1-(isoquinolin-5-yl)pyrrolidine-1,2-dicarboxamide (3b)
Yield 80.5%, red-brown solid, mp = 108.3-110.1℃; 1H NMR (CDCl3, 400MHz) δ ppm 9.75 (s, 1H,
NH), 9.11 (s, 1H, NH), 8.36 (s, 1H, isoquinoline), 7.76 (d, 1H, J = 9.0 Hz, isoquinoline), 7.68 (d,
1H, J = 9.0 Hz, isoquinoline), 7.50-7.42 (m, 2H, isoquinoline), 7.20 (t, 4H, J = 9.0 Hz, Ar-H), 7.01
(s, 1H, isoquinoline), 4.61 (d, 1H, J = 6.0 Hz, CH), 3.62-3.32 (m, 2H, NCH2), 2.32-2.13 (m, 2H,
NCH2CH2), 2.02-1.86 (m, 2H, CHCH2); 13C NMR (CDCl3, 75 MHz) δ ppm 170.1, 155.9, 152.8,
142.8, 137.3, 132.5, 131.6, 130.9, 129.0, 127.2, 125.6, 125.2, 121.1, 116.3, 114.8, 61.0, 46.9, 27.8,
25.2; HRMS(ESI) Calcd for C21H19BrN4O2 [M+H]+439.0764, found: 439.0761.
4.2.4.3. (R)-N2-(3-bromophenyl)-N1-(isoquinolin-5-yl)pyrrolidine-1,2-dicarboxamide (3c)
1
Yield 25.8%, red-brown solid, mp 112.1-114.0℃; H NMR(CDCl3, 300MHz) δ ppm 9.80 (s, 1H,
NH), 9.22 (s, 1H, NH), 8.48 (s, 1H, isoquinoline), 7.90 (d, 1H, J = 9.0 Hz, isoquinoline), 7.80 (d,
1H, J = 3.0 Hz, isoquinoline), 7.60-7.57 (m, 2H, isoquinoline), 7.47 (d, 1H, J = 6.0 Hz, Ar-H),
7.32 (t, 1H, J = 7.5 Hz, Ar-H), 7.15 (d, 1H, J = 9.0 Hz, Ar-H), 7.07 (t, 1H, J = 7.5 Hz, Ar-H), 6.90
(s, 1H, isoquinoline), 4.72 (d, 1H, J = 6.0 Hz, CH), 3.80-3.54 (m. 2H, NCH2), 2.57-2.51 (m, 1H,
NCH2CH2), 2.36-2.11 (m, 3H, NCH2CH2 and CHCH2); 13C NMR (CDCl3, 75MHz) δ ppm 169.8,
156.1, 152.9, 143.0, 139.5, 132.4, 131.1, 130.2, 130.0, 128.8, 127.2, 126.8, 125.5, 125.2, 124.3,
122.5, 118.1, 61.0, 46.9, 27.7, 25.3; HRMS(ESI) Calcd for C21H19BrN4O2 [M+H]+ 439.0764,
found: 439.0760.
4.2.4.4. (R)-N2-(2-bromophenyl)-N1-(isoquinolin-5-yl)pyrrolidine-1,2-dicarboxamide (3d)
1
Yield 18.9%, red-brown solid, mp = 115.9-117.1℃; H NMR (CDCl3, 300 MHz) δ ppm 9.48(s,
1H, NH), 9.14 (d, 1H, J = 6.0 Hz, isoquinoline), 9.06 (s, 1H, NH), 8.43-8.40 (m, 1H, isoquinoline),
8.23 (d, 1H, J = 3.0 Hz, isoquinoline), 7.90 (t, 1H, J = 6.0 Hz, isoquinoline), 7.70 (t, 1H, J = 7.5
Hz, isoquinoline), 7.57 (d, 1H, J = 3.0 Hz, Ar-H), 7.52-7.46 (m, 1H, Ar-H), 7.22 (t, 1H, J = 7.5 Hz,
Ar-H), 6.97 (d, 1H, J = 3.0 Hz, isoquinoline), 6.95-6.91 (m, 1H, Ar-H), 4.73 (d, 1H, J = 6.0 Hz,
CH), 3.73-3.53 (m, 2H, NCH2), 2.52-2.44 (m, 1H, NCH2CH2), 2.18-2.07 (m, 3H, NCH2CH2 and
CHCH2); 13C NMR (CDCl3, 75 MHz) δ ppm 170.2, 155.8, 152.8, 142.9, 135.8, 133.1, 132.4,
130.9, 129.7, 129.0, 128.2, 127.2, 124.9, 124.6, 122.3, 121.7, 114.4, 61.2, 46.8, 28.6, 25.2;
HRMS(ESI) Calcd for C21H19BrN4O2 [M+H]+ 439.0764, found: 439.0756.
4.2.4.5. (R)-N2-(2-chlorophenyl)-N1-(isoquinolin-5-yl)pyrrolidine-1,2-dicarboxamide (3e)
Yield 38.9%, red-brown solid, mp = 96.3-98.1℃; 1H NMR (CDCl3, 300 MHz) δ ppm 9.33 (s, 1H,
NH), 9.14 (s, 1H, NH), 8.42 (d, 1H, J = 6.0 Hz, isoquinoline), 8.29 (d, 1H, J = 9.0 Hz,
isoquinoline), 7.83 (d, 1H, J = 6.0 Hz, isoquinoline), 7.68 (d, 1H, J = 9.0 Hz, isoquinoline),
7.60-7.44 (m, 2H, isoquinoline and Ar-H ), 7.35-7.20 (m, 3H, Ar-H), 7.03 (t, 1H, J = 7.5 Hz,
isoquinoline), 4.70 (d, 1H, J = 6.0 Hz, CH), 3.64-3.47 (m, 2H, NCH2), 2.48-2.46 (m, 1H,
NCH2CH2), 2.04-1.98 (m, 3H, NCH2CH2 andCHCH2); 13C NMR(CDCl3, 75 MHz) δ ppm170.0,
155.7, 152.7, 142.8, 134.7, 132.5, 130.7, 129.1, 128.9, 127.4, 127.1, 125.6, 124.9, 124.8, 123.5,

122.0, 114.7, 61.2, 46.8, 28.1, 25.1; HRMS (ESI) Calcd for C22H19ClN4O2 [M+H]+ 395.1269,
found: 395.1267.
4.2.4.6. (R)-N2-(3-chlorophenyl)-N1-(isoquinolin-5-yl)pyrrolidine-1,2-dicarboxamide (3f)
1
Yield 19.7%, red-brown solid, mp = 209.9-210.4℃; H NMR (CDCl3, 300 MHz) δ ppm 9.88 (s,
1H, NH), 9.16 (s, 1H, NH), 8.43-8.41 (s, 1H, isoquinoline), 7.80 (d, J = 9.0 Hz, 1H, isoquinoline),
7.73 (d, J = 9.0 Hz, 1H, isoquinoline), 7.61-7.47 (m, 3H, isoquinoline and Ar-H), 7.21 (d, J = 9.0
Hz, 1H, Ar-H), 7.07 (t, J = 9.0 Hz, 2H, isoquinoline and Ar-H), 6.94 (d, J = 6.0 Hz, 2H, Ar-H),
4.67 (d, J = 6.0 Hz, 1H, CH), 3.66-3.42 (m, 2H, NCH2), 2.40 (s, 1H, NCH2CH2), 2.19-1.92 (m, 3H,
NCH2CH2 and CHCH2); 13C NMR (CDCl3, 75 MHz) δ ppm 170.2, 156.0, 152.8, 142.8, 139.4,
134.3, 132.5, 131.0, 129.7, 129.0, 127.2, 125.8, 125.2, 123.9, 119.6, 117.6, 114.9, 61.0, 46.9, 27.7,
25.2; HRMS (ESI) Calcd for C22H19ClN4O2 [M+H]+ 395.1269, found: 395.1267.
4.2.4.7. (R)-N2-(4-chlorophenyl)-N1-(isoquinolin-5-yl)pyrrolidine-1,2-dicarboxamide (3g)
1
Yield 17.9%, red-brown solid, mp = 104.6-105.5℃; H NMR (CDCl3, 300 MHz) δ ppm 9.78(s,
1H, NH), 9.18 (d, 1H, J = 9.0 Hz, NH), 8.42 (t, 1H, J = 7.5 Hz, isoquinoline), 7.83 (t, 1H, J = 9.0
Hz, isoquinoline), 7.74 (t, 1H, J = 9.0 Hz, isoquinoline), 7.58 (d, 1H, J = 6.0 Hz, isoquinoline),
7.50 (t, 1H, J = 9.0 Hz, isoquinoline), 7.42-7.37 (m, 2H, Ar-H), 7.15-7.02 (m, 3H, isoquinoline
and Ar-H), 4.69 (d, 1H, J = 9.0 Hz, CH), 3.70-3.49 (m, 2H, NCH2), 2.43 (d, 1H, J = 15.0 Hz,
NCH2CH2), 2.21-1.95 (m, 3H, NCH2CH2 and CHCH2); 13C NMR (CDCl3, 75 MHz) δ ppm 170.1,
156.0, 152.8, 142.8, 136.9, 132.6, 131.0, 129.1, 128.7, 127.2, 125.6, 125.1, 120.8, 114.8, 114.7,
61.0, 46.9, 29.6, 25.2; HRMS (ESI) Calcd for C22H19ClN4O2 [M+H]+ 395.1269, found: 395.1266.
4.2.4.8. (R)-N1-(isoquinolin-5-yl)-N2-(2-methoxyphenyl)pyrrolidine-1,2-dicarboxamide (3h)
1
Yield 42.3%, red-brown solid, mp = 105.5-107.1℃; H NMR (CDCl3, 300 MHz) δ ppm 9.17 (s,
1H, NH), 9.12 (s, 1H, NH), 8.43 (d, 1H, J = 9.0 Hz, isoquinoline), 8.31 (d, 1H, J = 6.0 Hz,
isoquinoline), 7.97 (d, 1H, J = 6.0 Hz, isoquinoline), 7.71 (d, 1H, J = 6.0 Hz, isoquinoline),
7.60-7.49 (m, 2H, isoquinoline and Ar-H ), 7.02 (t, 2H, J = 9.0 Hz, Ar-H), 6.77 (d, 2H, J = 6.0 Hz,
Ar-H), 6.93 (d, 1H, J = 9.0 Hz, isoquinoline), 6.83 (d, 1H, J = 9.0 Hz, Ar-H), 4.70 (s, 1H, CH),
3.77-3.60 (m, 5H, NCH2 and OCH3), 2.47-2.37 (m, 2H, NCH2CH2), 2.19-2.12 (m, 2H, CHCH2);
13C NMR(CDCl3, 75 MHz) δ ppm 170.0, 155.3, 152.8, 148.3, 142.9, 132.7, 130.4, 129.0, 127.4,
127.2, 124.8, 124.5, 124.1, 120.9, 120.0, 114.4, 110.1, 61.5, 55.7, 46.9, 28.8, 24.9; HRMS (ESI)
Calcd for C22H22N4O3 [M+H]+ 391.1765, found: 391.1760.
4.2.4.9. (R)-N1-(isoquinolin-5-yl)-N2-(4-methoxyphenyl)pyrrolidine-1,2-dicarboxamide (3i)
1
Yield 42.3%, red-brown solid, mp = 114.1-116.0℃; H NMR (CDCl3, 300 MHz) δ ppm 9.44 (s,
1H, NH), 9.18 (s, 1H, NH), 8.44 (d, 1H, J = 3.0 Hz, isoquinoline), 7.89 (d, 1H, J = 6.0 Hz,
isoquinoline), 7.75 (d, 1H, J = 6.0 Hz, isoquinoline), 7.60-7.51 (m, 2H, isoquinoline), 7.41 (d, 2H,
J = 9.0 Hz, Ar-H), 7.00 (s, 1H, isoquinoline), 6.77 (d, 2H, J = 6.0 Hz, Ar-H), 4.71 (d, 1H, J = 9.0
Hz, CH), 3.74 (s, 3H, OCH3), 3.69-3.53 (m, 2H, NCH2), 2.50-2.22 (m, 2H, NCH2CH2), 2.11-1.95

(m, 2H, CHCH2); 13C NMR (CDCl3, 75 MHz) δ ppm 169.4, 156.1, 152.9, 143.0, 136.8, 132.4,
131.4, 130.7, 129.1, 127.2, 125.2, 125.0, 121.4, 114.5, 114.0, 61.0, 55.4, 46.9, 27.6, 25.3; HRMS
(ESI) Calcd for C22H22N4O3 [M+H]+ 391.1765, found: 391.1759.
4.2.4.10. (R)-N1-(isoquinolin-5-yl)-N2-p-tolylpyrrolidine-1,2-dicarboxamide (3j)
1
Yield 59.3%, red-brown solid, mp = 205.3-207.1℃; H NMR (CDCl3, 300 MHz) δ ppm 9.62 (s,
1H, NH), 9.04 (s, 1H, NH), 8.30 (d, 1H, J = 9.0 Hz, isoquinoline), 7.66 (d, 1H, J = 6.0 Hz,
isoquinoline), 7.59-7.51 (m, 3H, isoquinoline), 7.39-7.23 (m, 3H, Ar-H ), 7.06 (t, 1H, J = 7.5 Hz,
isoquinoline), 6.82 (d, 1H, J = 9.0 Hz, Ar-H), 4.58 (d, 1H, J = 6.0 Hz, CH), 3.56-3.41 (m, 2H,
NCH2), 2.30 (d, 1H, J = 9.0 Hz, NCH2CH2), 2.21 (s, 3H, CH3), 2,04 (t, 1H, J = 7.5 Hz, NCH2CH2),
1.88 (s, 2H, CHCH2) ; 13C NMR (CDCl3, 75 MHz) δ ppm 170.3, 156.1, 152.4, 142.3, 138.6, 138.0,
132.9, 131.2, 128.9, 128.5, 127.1, 126.1, 124.9, 124.8, 120.4, 116.9, 115.5, 61.0, 46.8, 28.2, 24.9,
21.4; HRMS (ESI) Calcd for C22H22N4O2 [M+H]+ 375.1816, found: 375.1812.
4.2.4.11. (R)-N1-(isoquinolin-5-yl)-N2-m-tolylpyrrolidine-1,2-dicarboxamide (3k)
1
Yield 85.0%, red-brown solid, mp = 223.6-224.0℃; H NMR (CDCl3, 300 MHz) δ ppm 9.50 (s,
1H, NH), 9.09 (s, 1H, NH), 8.35 (d, 1H, J = 6.0 Hz, isoquinoline), 7.72 (d, 1H, J = 9.0 Hz,
isoquinoline), 7.63 (d, 1H, J = 9.0 Hz, isoquinoline), 7.50 (d, 1H, J = 6.0 Hz, isoquinoline), 7.40 (t,
1H, J = 7.5 Hz, isoquinoline), 7.31 (d, 3H, J = 6.0 Hz, Ar-H), 6.98 (d, 2H, J = 9.0 Hz, Ar-H and
isoquinoline), 4.59 (d, 1H, J = 9.0 Hz, CH), 3.57-3.39 (m, 2H, NCH2), 2.38-2.06 (m, 5H,
NCH2CH2 and CH3), 1.90 (t, 2H, J = 16.5 Hz, CHCH2); 13C NMR (CDCl3, 75 MHz) δ ppm 169.9,
156.0, 152.6, 142.6, 135.5, 133.6, 132.7, 131.0, 129.3, 129.0, 127.1, 125.7, 124.9, 119.8, 115.0,
60.9, 46.8, 27.8, 25.1, 20.8; HRMS (ESI) Calcd for C22H22N4O2[M+H]+ 375.1816, found:
375.1812.
4.2.4.12. (R)-N2-(3-isopropylphenyl)-N1-(isoquinolin-5-yl)pyrrolidine-1,2-dicarboxamide (3l)
Yield 16.6%, red-brown solid, mp = 98.4-101.1℃; 1H NMR (CDCl3, 300 MHz) δ ppm 9.53 (s, 1H,
NH), 9.15 (s, 1H, NH), 8.41 (d, 1H, J = 6.0 Hz, isoquinoline), 7.82 (d, 1H, J = 6.0 Hz,
isoquinoline), 7.71 (d, 1H, J = 9.0 Hz, isoquinoline), 7.57 (d, 1H, J = 6.0 Hz, isoquinoline), 7.49 (t,
1H, J = 9.0 Hz, isoquinoline), 7.41 (d, 1H, J = 6.0 Hz, Ar-H), 7.28 (t, 1H, J = 7.5 Hz, Ar-H), 7.16
(t, 1H, J = 7.5 Hz, Ar-H), 7.10 (s, 1H, isoquinoline), 6.93 (d, 1H, J = 9.0 Hz, Ar-H), 4.68 (d, J =
6.0 Hz, 1H, CH), 3.66-3.47 (m, 2H, NCH2), 2.87-2.78 (m, 1H, NCH2CH2), 2.53-2.06 (m, 3H,
NCH2CH2 and CHCH2), 1.20-1.18 (m, 6H, C(CH3)2); 13C NMR (CDCl3, 75 MHz) δ ppm 169.8,
156.0, 152.8, 149.8, 142.8, 138.1, 132.6, 130.9, 129.1, 128.7, 127.2, 125.6, 125.0, 122.2, 118.0,
117.3, 114.8, 61.1, 46.9, 34.1, 27.7, 25.2, 23.9; HRMS (ESI) Calcd for C24H26N4O2 [M+H]+
403.2129, found: 403.2121.
4.2.4.13. (R)-N2-(4-tert-butylphenyl)-N1-(isoquinolin-5-yl)pyrrolidine-1,2-dicarboxamide (3m)
1
Yield 23.9%, red-brown solid, mp = 114.0-115.5℃; H NMR (CDCl3, 300 MHz) δ ppm 9.62 (s,
1H, NH), 9.05 (s, 1H, NH), 8.31 (d, J = 6.0 Hz, 1H, isoquinoline), 7.60 (d, J = 3.0 Hz, 1H,

isoquinoline), 7.55 (d, J = 9.0 Hz, 1H, isoquinoline), 7.49 (s, 2H, isoquinoline), 7.38-7.29 (m, 3H,
isoquinoline and Ar-H), 7.19 (d, J = 9.0 Hz, 2H, Ar-H), 4.52 (d, J = 6.0 Hz, 1H, CH), 3.47-3.35 (m,
2H, NCH2), 2.26 (d, J = 9.0 Hz, 1H, NCH2CH2), 2.00 (t, J = 7.5 Hz, 1H, NCH2CH2), 1.79 (s, 2H,
CHCH2), 1.23 (s, 9H, CH3); 13C NMR (CDCl3, 75 MHz) δ ppm 170.0, 156.0, 152.6, 146.9, 142.5,
135.6, 132.9, 131.1, 128.9, 127.0, 126.1, 125.9, 125.5, 125.4, 124.9, 119.4, 115.4, 60.9, 46.8, 34.3,
31.3, 31.2, 24.9; HRMS (ESI) Calcd for C25H28N4O2 [M+H]+ 417.2285, found: 417.2280.
4.2.4.14. (R)-N2-(4-fluorophenyl)-N1-(isoquinolin-5-yl)pyrrolidine-1,2-dicarboxamide (3n)
1
Yield 20.6%, red-brown solid, mp = 112.9-114.9℃; H NMR (CDCl3, 300 MHz) δ ppm 9.70 (d,
1H, J = 3.0 Hz, NH), 9.08 (d, 1H, J = 3.0 Hz, NH), 8.34-8.28 (m, 1H, isoquinoline), 7.72-7.53 (m,
3H, isoquinoline), 7.47-7.33 (m, 4H, isoquinoline and Ar-H), 6.82-6.74 (m, 2H, isoquinoline and
Ar-H), 4.60 (d, 1H, J = 6.0 Hz, CH), 3.66-3.44 (m, 2H, NCH2), 2.28-2.91 (m, 4H, NCH2CH2 and
CHCH2); 13C NMR(CDCl3, 75 MHz) δ ppm 170.5, 160.1, 157.7, 156.0, 152.5, 142.5, 134.3, 132.9,
131.2, 129.0, 127.1, 125.9, 124.9, 121.4, 115.1, 61.0, 45.9, 28.5, 25.0; HRMS (ESI) Calcd for
C21H19FN4O2 [M+H]+379.1565, found: 379.1561.
4.2.4.15. (R)-N1-(isoquinolin-5-yl)-N2-(4-methoxy-2-methylphenyl)pyrrolidine-1,2-dicarboxamide
(3o)
1
Yield 38.7%, red-brown solid, mp = 109.8-111.1℃; H NMR (CDCl3, 300 MHz) δ ppm 9.06 (s,
1H, NH), 8.91 (s, 1H, NH), 8.31 (d, 1H, J = 6.0 Hz, isoquinoline), 7.68 (d, 1H, J = 6.0 Hz,
isoquinoline), 7.61 (d, 1H, J = 9.0 Hz, isoquinoline), 7.49 (d, 1H, J = 6.0 Hz, isoquinoline),
7.44-7.36 (m, 3H, isoquinoline and Ar-H ), 6.55 (d, 2H, J = 9.0 Hz, isoquinoline and Ar-H), 4.58
(d, 1H, J = 9.0 Hz, CH), 3.66 (s, 3H, OCH3), 3.49-3.33 (m, 2H, NCH2), 2,32 (d, 1H, J = 9.0 Hz,
NCH2CH2), 2.99 (d, 4H, J = 12.0 Hz, Ar-CH3 and NCH2CH2), 1.93-1.86 (m, 2H, CHCH2) ; 13C
NMR (CDCl3, 75 MHz) δ ppm 170.4, 156.8, 156.0, 152.6, 142.6, 132.9, 132.0, 131.0, 129.0,
128.7, 127.1, 125.7, 124.8, 124.7, 115.7, 115.1, 111.1, 60.7, 55.3, 46.8, 28.0, 25.0, 18.1; HRMS
(ESI) Calcd for C23H24N4O3 [M+H]+ 405.1921, found: 405.1916.
4.2.4.16.
(R)-N2-(4-bromo-2-chlorophenyl)-N1-(isoquinolin-5-yl)pyrrolidine-1,2-dicarboxamide
(3p)
1
Yield 23.4%, red-brown solid, mp = 115.5-113.0℃; H NMR (CDCl3, 300 MHz) δ ppm 9.46 (s,
1H, NH), 9.17 (s, 1H, NH), 8.45 (d, 1H, J = 3.0 Hz, isoquinoline), 8.20 (d, 1H, J = 9.0 Hz,
isoquinoline), 7.91 (d, 1H, J = 6.0 Hz, isoquinoline), 7.73 (d, 1H, J = 6.0 Hz, isoquinoline), 7.59 (d,
1H, J = 3.0 Hz, isoquinoline), 7.52 (t, 1H, J = 6.0 Hz, Ar-H), 7.46 (d, 1H, J = 3.0 Hz, Ar-H),
7.33-7.30 (m, 1H, Ar-H), 7.11 (s, 1H, isoquinoline), 4.74 (d, 1H, J = 6.0 Hz, CH), 3.72-3.53 (m,
2H, NCH2), 2.55-2.50 (m, 1H, NCH2CH2), 2.15-1.96 (m, 3H, NCH2CH2 and CHCH2); 13C NMR
(CDCl3, 75 MHz) δ ppm 170.1, 155.8, 152.9, 143.0, 134.2, 132.4, 131.6, 130.6, 130.4, 129.0,
127.2, 125.3, 124.9, 124.3, 123.0, 116.4, 114.4, 61.2, 46.9, 27.7, 25.2; HRMS (ESI) Calcd for
C21H18BrClN4O2 [M+H]+ 473.0374, found: 473.0371.
4.2.4.17. (R)-N2-(3-chloro-4-methylphenyl)-N1-(isoquinolin-5-yl)pyrrolidine-1,2-dicarboxamide

(3q)
1
Yield 31.2%, red-brown solid, mp = 116.9-118.5℃; H NMR (CDCl3, 300 MHz) δ ppm 9.68 (s,
1H, NH), 9.10 (s, 1H, NH), 8.37 (d, 1H, J = 6.0 Hz, isoquinoline), 7.75 (d, 1H, J = 6.0 Hz,
isoquinoline), 7.66 (d, 1H, J = 9.0 Hz, isoquinoline), 7.56 (d, 2H, J = 3.0 Hz, isoquinoline), 7.44 (t,
1H, J = 6.0 Hz, Ar-H), 7.33-7.27 (m, 1H, Ar-H), 7.09 (d, 1H, J = 3.0 Hz, isoquinoline), 6.94 (d, 1H,
J = 6.0 Hz, Ar-H), 4.62 (d, 1H, J = 6.0 Hz, CH), 3.66-3.47 (m, 2H, NCH2), 2.33-2.29 (m, 1H,
NCH2CH2), 2.22 (s, 3H, CH3), 2.15-2.11 (m, 1H, NCH2CH2), 2.00-1.92 (m, 2H, CHCH2); 13C
NMR (CDCl3, 75 MHz) δ ppm 170.3, 155.9, 152.6, 142.6, 136.9, 134.0, 132.7, 131.2, 131.1,
130.6, 129.0, 127.1, 125.8, 125.0, 120.2, 118.0, 115.1, 61.0, 46.9, 28.2, 25.1, 19.3; HRMS (ESI)
Calcd for C22H21ClN4O2[M+H]+ 409.1426, found: 409.1421.
4.2.4.18. (R)-N2-(2,6-dimethylphenyl)-N1-(isoquinolin-5-yl)pyrrolidine-1,2-dicarboxamide (3r)
1
Yield 15.7%, red-brown solid, mp = 109.4-111.6℃; H NMR (CDCl3, 300 MHz) δ ppm 9.04 (s,
1H, NH), 8.60 (s, 1H, NH), 8.29 (d, 1H, J = 3.0 Hz, isoquinoline), 7.59 (d, 2H, J = 6.0 Hz,
isoquinoline), 7.48 (d, 2H, J = 24.0 Hz, isoquinoline), 7.35 (t, 1H, J = 7.5 Hz, Ar-H), 6.94-6.83 (m,
3H, isoquinoline and Ar-H), 4.51 (s, 1H, CH), 3.37-3.24 (m, 2H, NCH2), 2.10 (s, 1H, NCH2CH2),
1.95 (s, 6H, CH3), 1.81 (s, 3H, NCH2CH2 and CHCH2); 13C NMR (CDCl3, 75 MHz) δ ppm 171.1,
155.9, 152.5, 142.4, 135.1, 133.7, 133.1, 131.1, 128.9, 127.9, 127.0, 125.9, 124.7, 115.4, 60.5,
46.7, 28.7, 24.9, 18.2; HRMS (ESI) Calcd for C23H24N4O2 [M+H]+ 389.1972, found: 389.1967.
4.2.4.19. (R)-N2-(2,4-dimethylphenyl)-N1-(isoquinolin-5-yl)pyrrolidine-1,2-dicarboxamide (3s)
1
Yield 21.3%, red-brown solid, mp = 102.2-104.2℃; H NMR (CDCl3, 300 MHz) δ ppm 9.20 (d,
1H, J = 12.0 Hz, NH), 9.03 (s, 1H, NH), 8.49-8.43 (m, 1H, isoquinoline), 7.98-7.89 (m, 1H,
isoquinoline), 7.80-7.68 (m, 2H, isoquinoline), 7.61-7.51 (m, 2H, isoquinoline and Ar-H),
7.06-6.92 (m, 3H, isoquinoline and Ar-H), 4.76 (t, 1H, J = 9.0 Hz, CH), 3.73-3.51 (m, 2H, NCH2),
2.59 (s, 1H, NCH2CH2), 2.26 (d, 3H, J = 3.0 Hz, CH3), 2.15 (d, 3H, J = 6.0 Hz, CH3), 2.06-1.97
(m, 3H, NCH2CH2 and CHCH2); 13C NMR (CDCl3, 75 MHz) δ ppm 170.1, 156.0, 152.7, 142.7,
134.5, 133.3, 132.7, 131.0, 130.8, 129.3, 129.0, 127.2, 126.9, 125.5, 124.8, 122.6, 114.8, 60.8,
46.9, 27.8, 25.2, 20.8, 17.8; HRMS (ESI) Calcd for C23H24N4O2 [M+H]+ 389.1972, found:
389.1968.
4.2.4.20. (R)-N2-(3-chloro-4-methoxyphenyl)-N1-(isoquinolin-5-yl)pyrrolidine-1,2-dicarboxamide
(3t)
1
Yield 42.7%, red-brown solid, mp = 105.3-107.1℃; H NMR (CDCl3, 300 MHz) δ ppm 9.63 (s,
1H, NH), 9.19 (s, 1H, NH), 8.47 (d, 1H, J = 3.0 Hz, isoquinoline), 7.87 (d, 1H, J = 9.0 Hz,
isoquinoline), 7.76 (d, 1H, J = 9.0 Hz, isoquinoline), 7.61-7.51 (m, 3H, Ar-H and isoquinoline),
7.19 (d, 1H, J = 9.0 Hz, Ar-H), 6.95 (s, 1H, isoquinoline), 6.70 (d, 1H, J = 9.0 Hz, Ar-H), 4.68 (d,
1H, J = 6.0 Hz, CH), 3.80 (s, 3H, OCH3), 3.69-3.44 (m, 2H, NCH2), 2.45-2.21 (m, 2H, NCH2CH2),
2.11-1.95 (m, 2H, CHCH2); 13C NMR (CDCl3, 75 MHz) δ ppm 169.7, 155.9, 152.9, 151.4, 143.0,
132.5, 131.9, 130.8, 129.1, 127.2, 125.4, 125.1, 122.1, 121.9, 119.1, 114.6, 111.9, 60.9, 56.3, 46.9,

27.7, 25.2; HRMS (ESI) Calcd for C22H21ClN4O3 [M+H]+ 425.1375, found: 425.1370.
4.2.4.21. (R)-N2-(3,4-dichlorophenyl)-N1-(isoquinolin-5-yl)pyrrolidine-1,2-dicarboxamide (3u)
1
Yield 27.1%, red-brown solid, mp = 105.7-107.6℃; H NMR (CDCl3, 300 MHz) δ ppm 9.89 (s,
1H, NH), 9.16 (s, 1H, NH), 8.44 (d, 1H, J = 6.0 Hz, isoquinoline), 7.83 (d, 1H, J = 9.0 Hz,
isoquinoline), 7.75 (d, 1H, J = 9.0 Hz, isoquinoline), 7.66 (s, 1H, isoquinoline), 7.60 (d, 1H, J =
6.0 Hz, isoquinoline), 7.52 (t, 1H, J = 9.0 Hz, Ar-H), 7.13 (s, 2H, Ar-H), 7.05 (s, 1H, isoquinoline),
4.68 (d, 1H, J = 9.0 Hz, CH), 3.76-3.52 (m, 2H, NCH2), 2.51 (s, 1H, NCH2CH2), 2.38-1.91 (m, 3H,
NCH2CH2 and CHCH2); 13C NMR (CDCl3, 75 MHz) δ ppm 170.4, 155.9, 152.8, 142.8, 137.7,
132.5, 132.3, 131.1, 130.0, 129.1, 127.2, 126.7, 125.8, 125.3, 121.1, 118.8, 114.9, 61.1, 45.9, 28.0,
25.2; HRMS (ESI) Calcd for C21H18Cl2N4O2 [M+H]+ 429.0880, found: 429.0879.
4.2.4.22. (R)-N2-(3,5-dimethoxyphenyl)-N1-(isoquinolin-5-yl)pyrrolidine-1,2-dicarboxamide (3v)
Yield 82.7%, red-brown solid, mp = 99.9-101.1℃; 1H NMR (CDCl3, 300 MHz) δ ppm 9.67 (s, 1H,
NH), 9.16 (s, 1H, NH), 8.43 (s, 1H, isoquinoline), 7.80 (d, 1H, J = 9.0 Hz, isoquinoline), 7.71 (d,
1H, J = 6.0 Hz, isoquinoline), 7.55-7.46 (m, 2H, isoquinoline), 7.04 (s, 1H, isoquinoline), 6.75 (d,
2H, J = 9.0 Hz, Ar-H), 6.14 (s, 1H, Ar-H), 4.64 (d, 1H, J = 6.0 Hz, CH), 3.74-3.60 (m, 7H, 2CH3
and NCH2), 3.48 (t, 1H, J = 9.0 Hz, NCH2), 2.45 (s, 1H, NCH2CH2), 2.16-1.09 (m, 3H, NCH2CH2
and CHCH2); 13C NMR (CDCl3, 75 MHz) δ ppm 170.1, 160.8, 156.0, 152.8, 142.8, 140.0, 132.6,
130.9, 129.0, 127.1, 125.6, 125.0, 114.9, 97.8, 96.5, 61.1, 55.3, 46.8, 27.7, 25.2; HRMS (ESI)
Calcd for C23H24N4O4 [M+H]+ 421.1870, found: 421.1865.
4.2.4.23. (R)-N1-(isoquinolin-5-yl)-N2-mesitylpyrrolidine-1,2-dicarboxamide (3w)
Yield 21.3%, red-brown solid, mp = 96.8-98.8℃; 1H NMR (CDCl3, 300 MHz) δ ppm 9.14 (d, 1H,
J = 12.0 Hz, NH), 8.50-8.38 (m, 2H, isoquinoline and NH), 7.88-7.78 (m, 1H, isoquinoline), 7.70
(t, 1H, J = 11.5 Hz, isoquinoline), 7.61 (d, 1H, J = 3.0 Hz, isoquinoline), 7.53-7.32 (m, 1H,
isoquinoline and Ar-H), 6.76 (d, 2H, J = 6.0 Hz, isoquinoline and Ar-H), 4.68 (d, 1H, J = 12.0 Hz,
CH), 3.62-3.47 (m, 2H, NCH2), 3.13-2.68 (m, 2H, NCH2CH2), 2.19 (d, 2H, J = 6.0 Hz, CHCH2),
2.06 (d, 9H, J = 9.0 Hz, CH3); 13C NMR (CDCl3, 75 MHz) δ ppm 171.0, 155.8, 152.7, 142.6,
136.6, 134.9, 133.0, 131.1, 130.9, 129.0, 128.7, 127.1, 125.4, 124.6, 115.0, 60.5, 46.9, 45.8, 29.6,
25.1, 20.8, 18.1; HRMS (ESI) Calcd for C24H26N4O2 [M+H]+ 403.2129, found: 403.2124.
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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Graphical abstract
Design, synthesis and biological evaluation of
N1-(isoquinolin-5-yl)-N2-phenylpyrrolidine-1,2-dicarboxamide derivatives as potent TRPV1
antagonists
Mingxiang Gaoa, Cunbin Niea, Jinyu Lia, Beibei Songa, Xinru Chenga, Erying Suna, 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
Reported herein is the design, synthesis, and pharmacologic evaluation of a class of TRPV1
antagonists constructed on
platform that evolved from a 5-aminoisoquinoline urea lead.
a
N1-(isoquinolin-5-yl)-N2-phenylpyrrolidine-1,2-dicarboxamide
F
F
O
O
O
O
O
N
HN
N
R
HN
N
H
HN
N
H
H
CF3
Cl
N
N
N
2
3
1
†
Corresponding authors.
E-mail addresses: yanlin0378@163.com (L. Yan), qianhai24@163.com (H. Qian).

Highlights
Highlights
1. Design, synthesis, and pharmacologic evaluation of a class of TRPV1 antagonists.
2. 3b was orally bioavailable and exhibited potent antagonism of both human and rat TRPV1.
3. 3b exhibited good efficacy in different pain models and did not elevate core body temperature.