Synthesis and evaluation of novel α-aminoamides containing benzoheterocyclic moiety for the treatment of pain

Article abstract of DOI:10.3390/molecules26061716

Novel α-aminoamide derivatives containing different benzoheterocyclics moiety were synthesized and evaluated as voltage-gated sodium ion channels blocks the treatment of pain. Compounds 6a, 6e, and 6f containing the benzofuran group displayed more potent in vivo analgesic activity than ralfinamide in both the formalin test and the writhing assay. Interestingly, they also exhibited potent in vitro anti-Na_v 1.7 and anti-Na_v 1.8 activity in the patch-clamp electrophysiology assay. Therefore, compounds 6a, 6e, and 6f, which have inhibitory potency for two pain-related Na_v targets, could serve as new leads for the development of analgesic medicines.

Full text of DOI:10.3390/molecules26061716

molecules
Article
Synthesis and Evaluation of Novel α-Aminoamides Containing
Benzoheterocyclic Moiety for the Treatment of Pain
Kun Tong 1,†, Ruotian Zhang 2,†, Fengzhi Ren 3, Tao Zhang 1, Junlin He 1, Jingchao Cheng 1, Zixing Yu 1,
Fengxia Ren 1, Yatong Zhang 2,* and Weiguo Shi 1,
*
1
State Key Laboratory of Toxicology and Medical Countermeasures,
Beijing Institute of Pharmacology & Toxicology, 27 Tai-Ping Road, Beijing 100850, China;
tongkq@126.com (K.T.); believe890521@163.com (T.Z.); hejunlin@bmi.ac.cn (J.H.);
chengjingchao3017@126.com (J.C.); yuyuzx1990@163.com (Z.Y.); ren2019victory@126.com (F.R.)
Hebei University of Science & Technology, 26 Yuxiang Street, Shijiazhuang City 050018, China;
zrtian18@163.com
2
3
New Drug Research & Development Co., Ltd., North China Pharmaceutical Group Corporation,
Shijiazhuang City 050015, China; rfzhhj@163.com
*
†
Correspondence: zhangyatong68@126.com (Y.Z.); shiweiguo@bmi.ac.cn (W.S.); Tel.: +86-10-669-316-39 (W.S.)
These authors contributed equally to this work.
Abstract: Novel
synthesized and evaluated as voltage-gated sodium ion channels blocks the treatment of pain.
Compounds 6a 6e, and 6f containing the benzofuran group displayed more potent in vivo analgesic
activity than ralfinamide in both the formalin test and the writhing assay. Interestingly, they also
exhibited potent in vitro anti-Nav1.7 and anti-Nav1.8 activity in the patch-clamp electrophysiology
α-aminoamide derivatives containing different benzoheterocyclics moiety were
,
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Tong, K.; Zhang, R.; Ren, F.;
Zhang, T.; He, J.; Cheng, J.; Yu, Z.;
Ren, F.; Zhang, Y.; Shi, W. Synthesis
and Evaluation of Novel
assay. Therefore, compounds 6a, 6e, and 6f, which have inhibitory potency for two pain-related Nav
targets, could serve as new leads for the development of analgesic medicines.
Keywords: α-aminoamides; sodium channel blocker; Nav1.7; Nav1.8; dual channel inhibitors
α-Aminoamides Containing
Benzoheterocyclic Moiety for the
Treatment of Pain. Molecules 2021, 26,
1716. https://doi.org/10.3390/
molecules26061716
1. Introduction
Chronic pain syndrome, such as neuropathic pain, severely affects the quality of life
of patients. However, specific analgesics for managing chronic pain are an unmet clinic
need [1–5]. Voltage-gated sodium ion channels subtype 1.7 (Nav1.7) and 1.8 (Nav1.8) have
Academic Editors: Mariana Spetea
and Massimo Bertinaria
been proven as promising targets for the discovery of new drugs to treat chronic pain and
numerous small-molecule inhibitors targeting Nav1.7 and Nav1.8 that have been developed
in preclinical or clinical studies in recent years [6–12].
Received: 5 February 2021
Accepted: 18 March 2021
Published: 19 March 2021
In our previous study [13], we synthesized a series of novel
containing an indole ring group based on modified ralfinamide, a Nav1.7-selective in-
hibitor for the treatment of neuropathic pain [14 17]. We found that the new compounds
showed robust in vivo potency but lower Nav1.7 inhibitory activity in vitro compared to
ralfinamide, indicating that further modifications and structure–activity relationship (SAR)
investigations are necessary.
α-aminoamide analogues
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iations.
–
We hypothesized that the H-acceptor properties (virtually missing in indole) for
H-bonding will likely be key to afford a lower Nav1.7 selectivity. Here, different benzo-
heterocyclic moiety including benzimidazole, benzofuran, and quinoline groups were
introduced to replace the indolyl group, and novel compounds were synthesized and
evaluated as sodium ion channel inhibitors. In addition, the in vivo potencies of the
compounds as painkillers were assessed in an animal model. Interestingly, the novel
compounds containing the benzofuran group exhibited potent activity in the formalin
assay and state-dependent inhibition of both Nav1.7 and Nav1.8, suggesting that the novel
compounds might possess dual channel activity. Their exact mechanisms of action needed
to be further investigated.
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Molecules 2021, 26, 1716. https://doi.org/10.3390/molecules26061716
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 1716
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2. Results and Discussion
2.1. Synthesis
The synthetic route is shown in Scheme 1. The target compounds were synthesized
from isophthalaldehyde (1a) or terephthalaldehyde (1b), followed by the reduction of one
formyl group with sodium borohydride to obtain 3-(hydroxymethyl)benzaldehyde (2a
and 4-(hydroxymethyl)benzaldehyde (2b), respectively. Then, compounds 2a and 2b were
converted to the intermediates 3-(bromomethyl)benzaldehyde (3a) and 4-(bromomethyl)
benzaldehyde (3b) by bromination, respectively. Compounds 3a and 3b were etherified
with a different hydroxylbenzoaromatic ring (benzimidazole, benzofuran, or quinoline)
by a Williamson reaction to obtain compounds 4a–h. Finally, compounds 4a–4h were
)
converted to the target α-aminoamide derivatives 5a–d, 6a–f, and 7a–h with L-alaninamide
hydrochloride by reductive amination.
Scheme 1. Synthesis of compounds 5a–d, 6a–f, and 7a–h.
2.2. Analgesic Effect of Synthesized Compounds in the Formalin Test
The analgesic potency of the new compounds was first tested with the formalin
assay, as described previously [18]. As shown in Table 1, generally, the tested compounds
containing the benzofuran group displayed more potent in vivo analgesic potency in phase
2 than others containing the benzimidazole and quinoline ones. The percentage of analgesia
of compounds 6a, 6e, and 6f was nearly 1.5–2 times higher than that of ralfinamide at a
dosage of 10 mg/kg. The most potent compound 6a showed a percentage of analgesia of
84%, compared to 42.4% for ralfinamide. The linked positions of the benzofuran ring
(A ring) and the benzene ring (B ring) affected the potency of the compounds. The
presence of linkages at positions 4 and 6 of the benzofuranyl group seemed to increase the
analgesic activity.

Molecules 2021, 26, 1716
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Table 1. The analgesic activity of the synthesized compounds in the formalin test.
Substituted Position of the
Substituted Position of the
Pharmacophore in the
% Analgesia a
(10 mg/kg, i.p.)
Compound
Ether Bond in the A Ring
B Ring
0
51.0 ± 21.6 $
27.4 ± 19.2
5a
5b
5c
5d
6a
6b
6c
6d
6e
6f
7a
7b
7c
7d
7e
7f
4
4
5
5
4
4
5
5
6
6
5
5
6
6
7
7
8
8
—
3
4
3
4
3
4
3
4
3
4
3
4
3
4
3
4
3
4
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
48.8 ± 15.5 $
60.8 ± 32.2 $
84.0 ± 16.2 $ *
48.4 ± 26.4 $
30.2 ± 19.8
42.2 ± 16.3
71.7 ± 22.0 $ @
67.8 ± 22.7 $ @
48.0 ± 27.8 $
37.2 ± 25.3
81.5 ± 14.8 $ *
20.9 ± 18.7 $
30.6 ± 14.7
15.9 ± 17.6
7g
7h
35.6 ± 26.1
57.2 ± 30.1 $
42.4 ± 23.3 #
Ralfinamide
—
a
$
#
Data are presented as the mean
vehicle. p < 0.001 compared to ralfinamide. p < 0.05 compared to ralfinamide.
±
standard deviation. Two-tailed Student’s t test. p < 0.001 compared to vehicle. p < 0.05 compared to
*
@
Further studies illustrated that compound 6a was more efficient than ralfinamide
in both the dose-effect relationship assays and the oral administration assays (Figure 1).
Compound 6a showed a dose-dependent activity at a dosage of 2.5 mg/kg, 5 mg/kg,
and 10 mg/kg by intraperitoneal injection. The 5 mg/kg dose of compound 6a yielded
a higher (69.6%) analgesic response than 20 mg/kg ralfinamide (59.7%). The results of
intragastric administration of compound 6a by mouth also showed a higher analgesic
activity than ralfinamide.
2.3. Analgesic Effect of Synthesized Compounds in the Acetic Acid-Induced Writhing Test
The analgesic effect of the novel compounds containing the benzofuran group was
further evaluated in a mouse writhing assay, which is a method used to assess pain induced
by a chemical, such as acetic acid. As shown in Table 2, compounds 6a, 6b, 6e, and 6f
displayed higher activities than ralfinamide, while compounds 6c and 6d showed a lower
potency. These compounds exhibited very similar analgesic effects in the two assays.

Molecules 2021, 26, 1716
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(a)
(b)
(c)
(d)
Figure 1. The analgesic potency of compound 6a and ralfinamide (n = 8). (a). The results of the dose-effect relationship
assay of compound 6a. (b). The results of the oral administration assay of compound 6a. (c). The results of the dose-effect
relationship assay of ralfinamide. (d). The results of the oral administration assay of ralfinamide.
Table 2. The analgesic activity of the synthesized compounds in the acetic acid-induced writhing test.
Substituted Position of the
Pharmacophore in the B
Ring
Substituted Position of the
Ether Bond in the A Ring
% Analgesia a
(10 mg/kg, i.p.)
Compound
0
49.7 ± 11.3 $
48.0 ± 5.8 $
11.2 ± 7.6
6a
6b
6c
6d
6e
6f
4
4
5
5
6
3
4
3
4
3
4
0
0
0
28.9 ± 8.5 #
0
45.7 ± 17.2 $
63.2 ± 13.3 $ @
44.4 ± 15.5 $
0
6
—
Ralfinamide
—
a
$
#
Data are presented as the mean
vehicle. p < 0.05 compared to ralfinamide.
±
standard deviation. Two-tailed Student’s t test. p < 0.001 compared to vehicle. p < 0.05 compared to
@
2.4. Inhibitory Activity of the Compounds and Their Effects on Tetrodotoxin (TTX)–Sensitive
Inactivated Nav1.7 Current
The compounds were also evaluated as sodium channel inhibitors. Their effects
on TTX-sensitive activated (TP
screened at a single dosage (10
−
1) and inactivated (TP
M), based on the IC50 value (7.10
−
2) states of Nav1.7 current were
1.41 M) of ralfinamide
µ
±
µ
(Table 3). The compounds with the benzimidazole group showed no activity against
Nav1.7, while compounds containing the quinoline group showed modest state-dependent
inhibitory potency on Nav1.7, which is consistent with the result of indole compounds
described in the previous study [13]. The compounds containing the benzofuran group
exhibited potent inhibitory activities against Nav1.7. Compounds 6e and 6f had IC50 values
close to that of ralfinamide, while compound 6a was the most effective inhibitor with an
IC50 value three times lower than that of ralfinamide (Table 4). This is consistent with the
in vivo effects in the mouse formalin and writhing assays.

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Table 3. Inhibitory activity of the compounds against Nav1.7.
Substituted Position
of the Ether Bond in
the A Ring
Substituted Position
of the Pharmacophore
in the B Ring
Inhibitory Effect against Nav1.7 (%, 10 µM)
Compound
Activated State
Inactivated State
(TP−1)
(TP−2)
0
5a
5b
5c
5d
6a
6b
6c
6d
6e
6f
7a
7b
7c
7d
7e
7f
4
4
5
5
4
4
5
5
6
6
5
5
6
6
7
7
8
8
—
3
4
3
4
3
4
3
4
3
4
3
4
3
4
3
4
5.1 ± 1.2
3.7 ± 2.0
1.7 ± 0.3
0.7 ± 0.8
15.6 ± 0.8
11.8 ± 0.3
12.2 ± 0.7
10.9 ± 1.1
11.4 ± 1.6
12.4 ± 2.9
6.4 ± 4.9
14.7 ± 5.4
8.6 ± 1.2
3.7 ± 0.5
3.6 ± 3.3
−1.8 ± 3.2
14.7 ± 0.2
9.1 ± 4.3
8.1 ± 3.8
10.7 ± 0.4
0
1.4 ± 0.6
0
2.6 ± 0.8
0
1.5 ± 0.9
0
61.8 ± 0.2 $
55.5 ± 2.5 $
48.1 ± 4.2 $
31.8 ± 0.3 #
58.5 ± 1.8 $
50.4 ± 3.6 #
23.8 ± 3.6
0
0
0
0
0
0
0
29.8 ± 0.6 #
30.0 ± 2.9 #
13.5 ± 0.4
0
0
0
47.7 ± 2.7 $
31.6 ± 3.4 #
46.0 ± 0.1 $
38.6 ± 4.7 #
57.7 ± 3.4 $
0
0
7g
7h
3
4
0
Ralfinamide
—
a
#
Data are presented as the mean
±
standard deviation. Two-tailed Student’s t test, $ p < 0.001 compared to vehicle. p < 0.05 compared
to vehicle.
Table 4. In vitro inhibitory activity of novel compounds.
Substituted Position
Substituted Position
of the Ether Bond in
the A Ring
of the
Pharmacophore in
the B Ring
Compound
IC50 to Nav1.7 (µM)
0
6a
6e
6f
4
6
6
3
3
4
2.3 ± 0.2
9.4 ± 2.7
8.1 ± 1.1
6.6 ± 1.6
0
0
Ralfinamide
—
—
2.5. Inhibitory Activity of the Compounds against Nav1.8 and Nav1.5
For the active compounds 6a 6e, and 6f, further evaluation of their inhibitory effects
,
on Nav1.8 and Nav1.5 was conducted. Much higher inhibition for Nav1.8 inactivated
state Na+ currents was observed for these compounds, when compared to ralfinamide.
Compound 6a showed nearly nine-fold more potent against Nav1.8 than ralfinamide
(Table 5). These findings suggest that the benzofuran moiety could play a critical role in
the inhibitory activity. Meanwhile, 6a, 6e, and 6f displayed moderate inhibitory activity
against Nav1.5, similarly to ralfinamide, indicating a low selectivity over the cardiac sodium
channel (Table 6).
Table 5. Inhibitory activity of the compounds against Nav1.8.
Substituted Position
Substituted Position
of the
Compound
of the Ether bond in
the A ring
IC50 to Nav1.8 (µM)
Pharmacophore in
the B ring
0
6a
6e
6f
4
6
6
3
3
4
4.3 ± 0.5
10.1 ± 1.9
4.8 ± 0.7
36.3 ± 8.9
0
0
Ralfinamide
—
—

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Table 6. Inhibitory activity of the compounds against Nav1.5.
Substituted Position
Substituted Position
of the Ether Bond in
the A Ring
of the
Pharmacophore in
the B Ring
Inhibitory Effect to
Nav1.5 (%, 10 µM)
Compound
0
6a
6e
6f
4
6
6
3
3
4
66.8 ± 1.5
70.1 ± 2.2
48.0 ± 0.1
64.6 ± 3.2
0
0
Ralfinamide
—
—
2.6. Discussion
The voltage-gated sodium channel isoforms Nav1.7 and Nav1.8 are attractive drug
targets for novel analgesics. However, achieving selectivity for specific subtypes with
small-molecule inhibitors has been challenging [19
as ralfinamide were previously developed as potential drug candidates for the specific
treatment of neuropathic pain [21 22]. Studying the relationship between target selectivity
and potency for novel -aminoamide derivatives is critically needed. Thus, we synthesized
a set of novel -aminoamide derivatives to further investigate the SARs in our previous
work [13]. We confirmed that the chemical moiety of the A ring affected the selectivity
to Nav channels and the structure needed to be further modified. Novel -aminoamide
,20]. α-Aminoamide derivatives such
,
α
α
α
derivatives containing different benzo-heterocyclics moiety were synthesized in this work
and evaluated as voltage-gated sodium ion channels block the treatment of pain.
Our previous studies have demonstrated that some novel compounds exhibit very
high in vivo potency in the mouse formalin assay compared to ralfinamide, but lower or
little in vitro inhibitory activity against Nav1.7. Therefore, in the present study, we first
assayed the activities of the novel compounds in vivo to confirm their potency as analgesics.
The results of the formalin test and writhing assay revealed the most potent compounds
6a, 6e, and 6f containing the benzofuran group. Consistent with our previous study, these
compounds showed no analgesic effect in a mouse hotplate assay and a tail flick assay,
even at a high dose of 100 mg/kg (data not shown).
Next, we assayed the anti-Nav1.7 in vitro activity of the new compounds by whole-cell
patch-clamp electrophysiology experiments. Compounds 6a, 6e, and 6f also showed a
highly potent inhibitory activity against TTX-sensitive inactivated Nav1.7 current, which
differs from the result of the previous compounds containing an indole group, demonstrat-
ing that the benzofuran moiety is a key effective functional group for Nav1.7 inhibition.
Moreover, compounds 6a, 6e, and 6f also exhibited potent anti-Nav1.8 activity, which is an-
other valid drug target for treating pain. To date, no drug candidates targeting only Nav1.7
or Nav1.8 are licensed in the market. Therefore, inhibitors with a broader selectivity for
both Nav1.7 and Nav1.8 ion channels might be potential drug candidates for the treatment
of pain.
Replacing the benzofuran group of compounds 6a, 6e, and 6f with benzimidazole
or a quinoline ring decreased the in vivo potency in the formalin assay, and the in vitro
inhibitory activity against Nav1.7. The SAR results suggested that the benzofuran group
was an essential factor for maintaining the analgesic effect in vivo as well as making it a
dual target inhibitor of Nav1.7 and Nav1.8 channels in vitro.
The novel compounds 6a, 6e, and 6f could serve as new leads for the further de-
velopment of candidates for treating pain. Further work evaluating the potency of new
compounds for treating neuropathic pain, and the inhibitory activity against other Nav
isoforms concerned with pain including Nav1.3, Nav1.6, and Nav1.9, is underway in
our laboratory.

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3. Materials and Methods
3.1. General Information
Commercially available reagents from Labter Pharmatech (Beijing, China), Innochem
(Beijing, China), Energy (Shanghai, China), Ark Pharm (Arlington Heights, IL, USA),
Fluorochem (Hadfield, UK), and Acros Organics (Geel, Belgium) were used without fur-
ther purification. Nuclear magnetic resonance (NMR) spectra (1H-NMR, 400 MHz) were
recorded on a JNM-ECA
(IR) data were recorded using a Nicolet 6700 spectrophotometer (Thermo Fisher Scientific
Co., Ltd., Waltham, MA, USA). Mass spectra were obtained on an API 150 mass spectrom-
−
400 spectrometer (JEOL Co. Ltd., Tokyo, Japan). Infrared spectra
−
eter (ABI Inc., Foster City, CA, USA). Thin-layer chromatography (TLC) plates and silica
gel (200–300 mesh) were purchased from Qingdao Haiyang Chemical Co. Ltd., (Shandong,
China). Sample purification was conducted using Combiflash Companion Preparative
Chromatography (Teledyne Isco Inc., Lincoln, NE, USA).
3.2. Chemistry
3.2.1. Synthesis of 2a and 2b
Synthesis of 3-(hydroxymethyl)benzaldehyde (2a) and 4-(hydroxymethyl)benzaldehyde
(2b): Isophthalaldehyde (1a) or terephthalaldehyde (1b) (4.0 equivalent 10 g, 74.55 mmol)
was dissolved in a mixture of ethanol (50 mL) and tetrahydrofuran (80 mL). After cooling
in an ice bath, sodium borohydride (1 equivalent 0.85 g, 18.60 mmol) was added to the
solution. The reaction mixture was stirred at 0 ◦C for at least 6 h until the disappearance
of 1a or 1b. Then the reaction was quenched with 3 M HCl (to pH 4–5). The solution was
filtered and evaporated to dryness under a vacuum. The residue was mixed with water
and extracted with EtOAc (2
×
50 mL). The combined organic layers were washed with
brine and dried over anhydrous sodium sulfate. After filtration, the solvent was evaporated
to obtain a crude product. The crude product was purified by flash column chromatog-
raphy (petroleum ether: EtOAc = 3:1) to afford 3-(hydroxymethyl)benzaldehyde (2a) or
4-(hydroxymethyl)benzaldehyde (2b) as a colorless and transparent liquid.
3.2.2. Synthesis of 3a and 3b
Synthesis of 3-(bromomethyl)benzaldehyde (3a) or 4-(bromomethyl)benzaldehyde
(
3b): Compound 2a or 2b (2.50 g, 18.36 mmol) was dissolved in CH2Cl2 (40 mL), and
n-bromosuccinimide (4.90 g, 27.53 mmol) was added to the solution. After cooling in an
ice bath, PPh3 (9.63 g, 36.72 mmol) was added to the solution. The mixture was stirred at
room temperature for at least 3 h. The reaction mixture was filtered, and water (40 mL) was
added to the filtrate. The solution was extracted with CH2Cl2 (2
×
20 mL). The organic
layers were dried over anhydrous sodium sulfate, and the solvent was evaporated under
a reduced pressure to obtain a residue. The residue was purified by silica gel column
chromatography (petroleum ether: EtOAc = 10:1) to afford 3-(bromomethyl)benzaldehyde
(3a) or 4-(bromomethyl)benzaldehyde (3b) as a white porous solid.
3.2.3. Synthesis of 4a–h
A mixture of 4-hydroxybenzenfuran, 5-hydroxybenzenfuran, or 6-hydroxybenzenfuran
or other hydroxylbenzoaromatics (0.52 g, 3.88 mmol) and Cs2CO3 (1.31 g, 4.03 mmol) in
ethanol (30 mL) was stirred at room temperature for 1 h. Then, compound 3a or 3b (0.92 g,
4.66 mmol) and kalium iodidum (0.10 g, 0.58 mmol) were added to the solution, and the
mixture was refluxed for at least 4 h, until the reaction was completed. The mixture was
evaporated under a reduced pressure to obtain a pale yellow solid. Then it was mixed with
water and extracted with EtOAc (2
×
15 mL). The organic layers were washed with water
and brine and dried over anhydrous sodium sulfate. The solvent was evaporated under a
reduced pressure. The residue was purified by flash column chromatography (petroleum
ether:EtOAc = 10:1) to obtain compound 4a–h as a pale yellow oil.

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3.2.4. Synthesis of 5a–d, 6a−6f, 7a−7h
A mixture of L-alaninamide hydrochloride (0.53 g, 4.28 mmol) and Et3N (0.87 g,
8.57 mmol) in absolute methanol (30 mL) was stirred at room temperature for 1 h. To
the solution, compound 4a–h (0.72 g, 2.86 mmol) was added. After stirring for another
2 h, potassium borohydride (0.92 g, 17.14 mmol) was added to the solution, and the
solution was refluxed for 3 h. The solvent was evaporated under a reduced pressure to
obtain a pale yellow solid. The residue was purified by silica gel column chromatography
(MeOH/CH2Cl2, 0-5%) to afford compound 5a–d, 6a–f, 7a–h as a white solid.
(S)-2-((3-(((1H-Benzo[d]imidazol-4-yl)oxy)methyl)benzyl)amino)propanamide (5a)
Yield: 74%. IR (KBr, cm−1
)
ν
3369, 3143, 2953, 2806, 2580, 1687 (C=O), 1622 (C=N),
1
1552, 1514. H-NMR (400 MHz, DMSO-d6, ppm) δH 12.75 (s, 1H, NH, imidazole), 8.11 (s,
1H, HAr), 7.50 (br s, 1H, CONH2), 7.30–7.38 (m, 4H, HAr), 7.04–7.17 (m, 3H, HAr, CONH2),
6.81 (br s, 1H, HAr), 5.30 (s, 2H, OCH2), 3.71 (d, J = 13.6 Hz, 1H, Ar-CH2), 3.58 (d, J = 13.6
Hz, 1H, Ar-CH2), 3.04 (q, J = 7.0 Hz, 1H, CH
-CH3), 1.14 (d, J = 7.0 Hz, 3H, CH3). 13C-NMR
(100 MHz, DMSO-d6, ppm) δC 170.3 (C=O), 146.3, 139.8, 136.7, 132.5, 132.0, 129.8, 129.2,
128.7, 127.8, 127.0, 121.5, 107.7, and 106.7 (13C, Ar-C), 69.7 (CH2), 54.6 (CH), 48.3 (CH2),
16.0 (CH3). HR-ESI MS m/z 325.1659 [M + H]+.
(S)-2-((4-(((1H-Benzo[d]imidazol-4-yl)oxy)methyl)benzyl)amino)propanamide (5b)
Yield: 88%. IR (KBr, cm−1
(C=N), 1512, 1496. H-NMR (400 MHz, DMSO-d6, ppm) δH 12.74 (s, 1H, NH, imidazole),
)
ν
3402, 3115, 3086, 2973, 2774, 2566, 1721 (C=O), 1621
1
8.10 (s, 1H, HAr), 7.46 (m, 2H, HAr, CONH2), 7.35-7.37 (m, 3H, HAr), 7.04–7.16 (m, 3H, HAr
,
CONH2), 6.79 (br s, 1H, HAr), 5.29 (s, 2H, OCH2), 3.70 (d, J = 13.5 Hz, 1H, Ar-CH2), 3.56 (d,
J = 13.5 Hz, 1H, Ar-CH2), 3.02 (q, J = 7.0 Hz, 1H, CH-CH3), 1.13 (d, J = 7.0 Hz, 3H, CH3).
13C-NMR (100 MHz, DMSO-d6, ppm) δC 170.4 (C=O), 146.3, 139.9, 136.9, 132.4, 131.5, 130.4,
127.7, 127.0, 121.5, 107.7, and 106.7 (13C, Ar-C), 69.6 (CH2), 54.6 (CH), 48.1 (CH2), 16.0
(CH3). HR-ESI MS m/z 325.1659 [M + H]+.
(S)-2-((3-(((1H-Benzo[d]imidazol-5-yl)oxy)methyl)benzyl)amino)propanamide (5c)
Yield: 54%. IR (KBr, cm−1
)
ν
3385, 3254, 3084, 2997, 2771, 2552, 1688 (C=O), 1635
1
(C=N), 1530, 1448. H-NMR (400 MHz, DMSO-d6, ppm) δH 12.30 (br s, 1H, NH, imidazole),
8.09 (s, 1H, HAr), 7.05–7.45 (m, 8H, HAr, CONH2), 6.89 (d, J = 8.4 Hz, 1H, HAr), 5.11 (s,
2H, OCH2), 3.71 (d, J = 13.5 Hz, 1H, Ar-CH2), 3.57 (d, J = 13.5 Hz, 1H, Ar-CH2), 3.04 (q,
J = 7.0 Hz, 1H, CH-CH3), 1.14 (d, J = 7.0 Hz, 3H, CH3). 13C-NMR (100 MHz, DMSO-d6,
ppm) δC 170.4 (C=O), 156.9, 139.6, 137.0, 132.2, 131.6, 129.9, 129.6, 128.9, 128.3, 125.1, 116.7,
115.4, and 97.7 (13C, Ar-C), 69.8 (CH2), 54.6 (CH), 48.4 (CH2), 16.0 (CH3). HR-ESI MS m/z
325.1659 [M + H]+.
(S)-2-((4-(((1H-Benzo[d]imidazol-5-yl)oxy)methyl)benzyl)amino)propanamide (5d)
−1
Yield: 58%. IR (KBr, cm
) ν 3326, 3258, 3145, 2929, 2763, 2659, 2557, 1706 (C=O), 1636
1
(C=N), 1513, 1493. H-NMR (400 MHz, DMSO-d6, ppm) δH 12.25 (irregular d, 1H, NH,
imidazole), 8.06 (irregular d, 1H, HAr), 7.24–7.53 (m, 7H), 6.86–7.07 (m, 2H, HAr, CONH2),
5.10 (s, 2H, OCH2), 3.68 (d, J = 13.6 Hz, 1H, Ar-CH2), 3.54 (d, J = 13.6 Hz, 1H, Ar-CH2), 3.00
(q, J = 7.0 Hz, 1H, CH-CH3), 1.13 (d, J = 7.0 Hz, 3H, CH3). 13C-NMR (100 MHz, DMSO-d6,
ppm) δC 170.4 (C=O), 156.9, 139.7, 137.4, 131.6, 131.5, 130.4, 127.9, 125.1, 116.7, 115.4 and
97.7 (13C, Ar-C), 69.5 (CH2), 54.4 (CH), 48.1 (CH2), 16.0 (CH3). HR-ESI MS m/z 325.1658
[M + H]+.
(S)-2-((3-((Benzofuran-4-yloxy)methyl)benzyl)amino)propanamide (6a)
−1
Yield: 91%. IR (KBr, cm
) ν 3314, 3175, 2973, 2754, 1697 (C=O), 1631, 1604, 1545, 1493,
1
1436, 1368. H-NMR (400 MHz, DMSO-d6, ppm) δH 7.91 (d, J = 2.0 Hz, 1H, HAr), 7.48 (br s,
1H, CONH2), 7.30–7.42 (m, 4H, HAr), 7.19–7.26 (m, 2H, HAr), 7.03 (br s, 1H, CONH2), 6.98
(dd, J = 2.2, 0.8 Hz, 1H, HAr), 6.89 (dd, J = 7.3, 1.1 Hz, 1H, HAr), 5.24 (s, 2H, OCH2), 3.72 (d, J

Molecules 2021, 26, 1716
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= 13.5 Hz, 1H, Ar-CH2), 3.58 (d, J = 13.5 Hz, 1H, Ar-CH2), 3.03 (q, J = 6.7 Hz, 1H, CH-CH3),
1.14 (d, J = 6.7 Hz, 3H, CH3). 13C-NMR (100 MHz, DMSO-d6, ppm) δC 170.5 (C=O), 155.7,
152.2, 144.7, 137.4, 132.2, 129.8, 129.3, 128.8, 128.1, 125.3, 117.4, 105.2, 104.7 and 104.1 (14C,
Ar-C), 69.3 (CH2), 54.7 (CH), 48.4 (CH2), 16.0 (CH3). HR-ESI MS m/z 325.1547 [M + H]+.
(S)-2-((4-((Benzofuran-4-yloxy)methyl)benzyl)amino)propanamide (6b)
Yield: 56%. IR (KBr, cm−1
) ν 3315, 3184, 2971, 2797, 1693 (C=O), 1606, 1544, 1494.
1H-NMR (400 MHz, DMSO-d6, ppm) δH 7.90 (d, J = 2.2 Hz, 1H, HAr), 7.35–7.46 (m, 5H,
HAr, CONH2), 7.19–7.25 (m, 2H, HAr), 7.04 (br s, 1H, CONH2), 6.96 (dd, J = 2.2, 0.8 Hz,
1H, HAr), 6.88 (dd, J = 7.3, 1.3 Hz, 1H, HAr), 5.23 (s, 2H, OCH2), 3.70 (d, J = 13.2 Hz, 1H,
Ar-CH2), 3.57 (d, J = 13.2 Hz, 1H, Ar-CH2), 3.03 (m, 1H, CH-CH3), 1.14 (d, J = 7.0 Hz, 3H,
CH3). 13C-NMR (100 MHz, DMSO-d6, ppm) δC 170.4 (C=O), 155.7, 152.1, 144.8, 137.8, 131.4,
130.4, 127.7, 125.2, 117.4, 105.2, 104.7, and 104.0 (14C, Ar-C), 69.1 (CH2), 54.5 (CH), 48.2
(CH2), 16.0 (CH3). HR-ESI MS m/z 325.1546 [M + H]+.
(S)-2-((3-((Benzofuran-5-yloxy)methyl)benzyl)amino)propanamide (6c)
−1
Yield: 87%. IR (KBr, cm
) ν 3364, 3313, 3150, 2973, 2746, 1697 (C=O), 1613, 1570, 1462.
1H-NMR (400 MHz, DMSO-d6, ppm) δH 7.95 (d, J = 2.2 Hz, 1H, HAr), 7.50 (d, J = 9.2 Hz,
1H, HAr), 7.44 (br s, 1H, CONH2), 7.29–7.35 (m, 4H, HAr), 7.25 (d, J = 2.5 Hz, 1H, HAr), 7.02
(br s, 1H, CONH2), 6.98 (dd, J = 9.0, 2.5 Hz, 1H, HAr), 6.88 (dd, J = 2.2, 0.8 Hz, 1H, HAr),
5.10 (s, 2H, OCH2), 3.70 (d, J = 13.6 Hz, 1H, Ar-CH2), 3.56 (d, J = 13.6 Hz, 1H, Ar-CH2), 3.03
(q, J = 6.8 Hz, 1H, CH-CH3), 1.14 (d, J = 6.8 Hz, 3H, CH3). 13C-NMR (100 MHz, DMSO-d6,
ppm) δC 170.4 (C=O), 154.6, 149.4, 146.8, 137.7, 132.1, 129.6, 129.4, 128.7, 128.2, 127.9, 113.6,
111.8, 106.9, and 105.1 (14C, Ar-C), 69.7 (CH2), 54.6 (CH), 48.4 (CH2), 16.0 (CH3). HR-ESI
MS m/z 325.1547 [M + H]+.
(S)-2-((4-((Benzofuran-5-yloxy)methyl)benzyl)amino)propanamide (6d)
Yield: 70%. IR (KBr, cm−1
)
ν
3307, 3265, 3167, 2967, 2798, 1690 (C = O), 1614, 1546,
1
1473. H-NMR (400 MHz, DMSO-d6, ppm) δH 7.94 (d, J = 2.2 Hz, 1H, HAr), 7.49 (d, J = 9.0
Hz, 1H, HAr), 7.33–7.42 (m, 5H, HAr, CONH2), 7.24 (d, J = 2.5 Hz, 1H, HAr), 7.01 (br s, 1H,
CONH2), 6.97 (dd, J = 9.0, 2.5 Hz, 1H, HAr), 6.88 (dd, J = 2.2, 0.9 Hz, 1H, HAr), 5.10 (s, 2H,
OCH2), 3.69 (d, J = 13.4 Hz, 1H, Ar-CH2), 3.55 (d, J = 13.4 Hz, 1H, Ar-CH2), 3.01 (q, J = 7.0
Hz, 1H, CH
-CH3), 1.13 (d, J = 7.0 Hz, 3H, CH3). 13C-NMR (100 MHz, DMSO-d6, ppm) δC
170.4 (C=O), 154.5, 149.3, 146.8, 138.1, 131.2, 130.3, 127.8, 127.7, 113.6, 111.8, 106.9, and 105.0
(14C, Ar-C), 69.4 (CH2), 54.5 (CH), 48.1 (CH2), 16.0 (CH3). HR-ESI MS m/z 325.1547 [M +
H]+.
(S)-2-((3-((Benzofuran-6-yloxy)methyl)benzyl)amino)propanamide (6e)
Yield: 79%. IR (KBr, cm−1
) ν 3316, 3150, 2952, 2746, 1686 (C=O), 1621, 1565, 1489.
1H-NMR (400 MHz, DMSO-d6, ppm) δH 7.87 (d, J = 2.0 Hz, 1H, HAr), 7.52 (d, J = 9.0 Hz,
1H, HAr), 7.45 (br s, 1H, CONH2), 7.30–7.34 (m, 5H, HAr), 7.01 (br s, 1H, CONH2), 6.96
(dd, J = 8.7, 2.2 Hz, 1H, HAr), 6.87 (dd, J = 2.2, 0.8 Hz, 1H, HAr), 5.13 (s, 2H, OCH2), 3.70
(
d, J = 13.6 Hz, 1H, Ar-CH2), 3.56 (d, J = 13.6 Hz, 1H, Ar-CH2), 3.02 (q, J = 7.0 Hz, 1H,
CH-CH3), 1.13 (d, J = 7.0 Hz, 3H, CH3). 13C-NMR (100 MHz, DMSO-d6, ppm) δC 170.4
(C=O), 156.5, 155.2, 145.1, 137.4, 132.1, 129.7, 129.4, 128.7, 128.3, 121.4, 120.6, 112.5, 106.5,
and 97.2 (14C, Ar-C), 69.6 (CH2), 54.7 (CH), 48.4 (CH2), 16.0 (CH3). HR-ESI MS m/z 325.1546
[M + H]+.
(S)-2-((4-((Benzofuran-6-yloxy)methyl)benzyl)amino)propanamide (6f)
−1
Yield: 82%. IR (KBr, cm
) ν 3388, 3262, 3167, 2967, 2770, 1693 (C=O), 1621, 1544, 1489.
1H-NMR (400 MHz, DMSO-d6, ppm) δH 7.86 (d, J = 2.2 Hz, 1H, HAr), 7.52 (d, J = 8.4 Hz,
1H, HAr), 7.34–7.42 (m, 5H, HAr, CONH2), 7.29 (d, J = 1.7 Hz, 1H, HAr), 7.01 (br s, 1H,
CONH2), 6.94 (dd, J = 8.7, 2.2 Hz, 1H, HAr), 6.86 (dd, J = 2.2, 0.8 Hz, 1H, HAr), 5.12 (s, 2H,
OCH2), 3.69 (d, J = 13.6 Hz, 1H, Ar-CH2), 3.54 (d, J = 13.6 Hz, 1H, Ar-CH2), 3.00 (q, J =

Molecules 2021, 26, 1716
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6.7 Hz, 1H, CH-CH3), 1.13 (d, J = 6.7 Hz, 3H, CH3). 13C-NMR (100 MHz, DMSO-d6, ppm)
δC 170.4 (C=O), 156.4, 155.2, 145.0, 137.8, 131.3, 130.3, 127.8, 121.4, 120.6, 112.6, 106.5, and
97.2 (14C, Ar-C), 69.2 (CH2), 54.5 (CH), 48.1 (CH2), 15.9 (CH3). HR-ESI MS m/z 325.1546
[M + H]+.
(S)-2-((3-((Quinolin-5-yloxy)methyl)benzyl)amino)propanamide (7a)
Yield: 80%. IR (KBr, cm−1
1558. 1H-NMR (400 MHz, DMSO-d6, ppm) δH 8.91 (dd, J = 4.1, 1.6 Hz, 1H, HAr), 8.58
d, J = 8.4 Hz, 1H, HAr), 7.68 (m, 1H, HAr), 7.62 (d, J = 8.4 Hz, 1H, HAr), 7.51–7.54 (m, 2H,
) ν 3320, 3157, 2659, 2050, 1690 (C=O), 1639 (C=N), 1595,
(
HAr, CONH2), 7.32–7.44 (m, 4H, HAr), 7.18 (d, J = 7.6 Hz, 1H, HAr), 7.04 (s, 1H, CONH2),
5.32 (s, 2H, OCH2), 3.74 (d, J = 13.6 Hz, 1H, Ar-CH2), 3.59 (d, J = 13.6 Hz, 1H, Ar-CH2), 3.04
(q, J = 7.0 Hz, 1H, CH-CH3), 1.14 (d, J = 7.0 Hz, 3H, CH3). 13C-NMR (100 MHz, DMSO-d6
,
ppm) δC 170.4 (C=O), 154.1, 145.5, 140.2, 139.4, 136.4, 135.1, 132.3, 130.1, 129.4, 128.8, 128.1,
121.3, 121.1, 113.3, and 109.0 (15C, Ar-C), 70.2 (CH2), 54.6 (CH), 48.4 (CH2), 16.0 (CH3).
HR-ESI MS 336.1707 [M + H]+.
(S)-2-((4-((Quinolin-5-yloxy)methyl)benzyl)amino)propanamide (7b)
Yield: 66%. IR (KBr, cm−1
1558. 1H-NMR (400 MHz, DMSO-d6, ppm) δH 8.90 (dd, J = 3.9, 1.4 Hz, 1H, HAr), 8.56
d, J = 8.4 Hz, 1H, HAr), 7.67 (m, 1H, HAr), 7.60 (d, J = 8.4 Hz, 1H, HAr), 7.49–7.53 (m, 3H,
) ν 3384, 3156, 2659, 2058, 1689 (C=O), 1639 (C=N), 1595,
(
HAr, CONH2), 7.34–7.39 (m, 3H, HAr), 7.17 (d, J = 7.6 Hz, 1H, HAr), 7.02 (br s, 1H, CONH2),
5.31 (s, 2H, OCH2), 3.70 (d, J = 13.6 Hz, 1H, Ar-CH2), 3.56 (d, J = 13.6 Hz, 1H, Ar-CH2), 3.02
(q, J = 7.0 Hz, 1H, CH-CH3), 1.14 (d, J = 7.0 Hz, 3H, CH3). 13C-NMR (100 MHz, DMSO-d6
,
ppm) δC 170.4 (C=O), 154.0, 145.8, 139.9, 139.4, 136.8, 134.8, 131.8, 130.5, 127.9, 121.3, 121.1,
113.6, and 108.9 (15C, Ar-C), 70.0 (CH2), 54.6 (CH), 48.1 (CH2), 16.0 (CH3). HR-ESI MS m/z
336.1707 [M + H]+.
(S)-2-((3-((Quinolin-6-yloxy)methyl)benzyl)amino)propanamide (7c)
Yield: 85%. IR (KBr, cm−1
)
ν
3290, 3158, 2913, 2660, 2062, 1964, 1696 (C=O), 1617
1
(C=N), 1617, 1600. H-NMR (400 MHz, DMSO-d6, ppm) δH 8.75 (dd, J = 4.2, 1.7 Hz, 1H,
HAr), 8.25 (m, 1H, HAr), 7.94 (dd, J = 9.5, 0.8 Hz, 1H, HAr), 7.47–7.50 (m, 4H, HAr, CONH2),
7.31–7.40 (m, 4H, HAr), 7.02 (br s, 1H, CONH2), 5.23 (s, 2H, OCH2), 3.72 (d, J = 13.6 Hz, 1H,
Ar-CH2), 3.58 (d, J = 13.6 Hz, 1H, Ar-CH2), 3.03 (q, J = 7.0 Hz, 1H, CH-CH3), 1.14 (d, J = 7.0
Hz, 3H, CH3). 13C-NMR (100 MHz, DMSO-d6 ppm) δC 170.4 (C=O), 157.9, 143.7, 142.4,
,
136.3, 134.6, 132.2, 130.3, 130.1, 130.0, 128.9, 128.5, 126.9, 123.1, 122.4, and 107.9 (15C, Ar-C),
69.9 (CH2), 54.6 (CH), 48.3 (CH2), 16.0 (CH3). HR-ESI MS m/z 336.1708 [M + H]+.
(S)-2-((4-((Quinolin-6-yloxy)methyl)benzyl)amino)propanamide (7d)
Yield: 82%. IR (KBr, cm−1
)
ν
3357, 3175, 3076, 2969, 2661, 1689 (C=O), 1616 (C=N),
1
1600, 1493. H-NMR (400 MHz, DMSO-d6, ppm) δH 8.74 (dd, J = 4.2, 1.7 Hz, 1H, HAr), 8.24
(dd, J = 8.4, 1.1 Hz, 1H, HAr), 7.93 (d, J = 8.9 Hz, 1H, HAr), 7.45–7.49 (m, 5H, HAr, CONH2),
7.36–7.38 (m, 3H, HAr), 7.01 (br s, 1H, CONH2), 5.21 (s, 2H, OCH2), 3.70 (d, J = 13.6 Hz, 1H,
Ar-CH2), 3.55 (d, J = 13.6 Hz, 1H, Ar-CH2), 3.01 (q, J = 7.0 Hz, 1H, CH-CH3), 1.13 (d, J = 7.0
Hz, 3H, CH3). 13C-NMR (100 MHz, DMSO-d6 ppm) δC 170.6 (C=O), 158.2, 143.8, 143.2,
,
137.2, 135.1, 131.7, 130.7, 130.5, 128.6, 127.2, 123.8, 122.7, 108.1 (15C, Ar-C), 69.9 (CH2), 54.7
(CH), 48.3 (CH2), 16.1 (CH3). HR-ESI MS m/z 336.1708 [M + H]+.
(S)-2-((3-((Quinolin-7-yloxy)methyl)benzyl)amino)propanamide (7e)
Yield: 81%. IR (KBr, cm−1
) ν 3300, 3137, 2743, 1694 (C=O), 1643 (C=N), 1643, 1609.
1H-NMR (400 MHz, DMSO-d6, ppm) δH 8.81 (dd, J = 4.5, 2.0 Hz, 1H, HAr), 8.28 (dd, J = 8.1,
1.7 Hz, 1H, HAr), 7.90 (d, J = 9.0 Hz, 1H, HAr), 7.48–7.50 (m, 2H, HAr, CONH2), 7.30–7.39
(m, 6H, HAr), 7.02 (br s, 1H, CONH2), 5.27 (s, 2H, OCH2), 3.71 (d, J = 13.6 Hz, 1H, Ar-CH2),
3.57 (d, J = 13.6 Hz, 1H, Ar-CH2), 3.03 (q, J = 7.0 Hz, 1H, CH-CH3), 1.14 (d, J = 7.0 Hz, 3H,
CH3). 13C-NMR (100 MHz, DMSO-d6, ppm) δC 170.4 (C=O), 162.2, 145.0, 144.5, 140.7, 135.8,

Molecules 2021, 26, 1716
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132.2, 130.9, 130.3, 130.0, 129.0, 128.7, 124.3, 122.4, 119.6, and 101.3 (15C, Ar-C), 70.1 (CH2),
54.6 (CH), 48.3 (CH2), 16.0 (CH3). HR-ESI MS m/z 336.1705 [M + H]+.
(S)-2-((4-((Quinolin-7-yloxy)methyl)benzyl)amino)propanamide (7f)
Yield: 78%. IR (KBr, cm−1
1637 (C = N), 1604. H-NMR (400 MHz, DMSO-d6, ppm) δH 8.81 (dd, J = 4.2, 1.7 Hz, 1H,
HAr), 8.28 (dd, J = 8.2, 1.4 Hz, 1H, HAr), 7.90 (d, J = 9.0 Hz, 1H, HAr), 7.45–7.48 (m, 3H,
HAr, CONH2), 7.31–7.39 (m, 5H, HAr), 7.00 (br s, 1H, CONH2), 5.26 (s, 2H, OCH2), 3.69
)
ν
3365, 3177, 3020, 2719, 2661, 2396, 2085, 1689 (C=O),
1
(
d, J = 13.6 Hz, 1H, Ar-CH2), 3.55 (d, J = 13.6 Hz, 1H, Ar-CH2), 3.00 (q, J = 7.0 Hz, 1H,
CH-CH3), 1.13 (d, J = 7.0 Hz, 3H, CH3). 13C-NMR (100 MHz, DMSO-d6 ppm) δC 170.8
,
(C=O), 163.0, 145.9, 145.1, 140.9, 137.0, 132.0, 131.5, 130.9, 128.9, 124.8, 123.0, 120.1, and
101.5 (15C, Ar-C), 70.4 (CH2), 54.9 (CH), 48.6 (CH2), 16.2 (CH3). HR-ESI MS m/z 336.1706
[M + H]+.
(S)-2-((3-((Quinolin-8-yloxy)methyl)benzyl)amino)propanamide (7g)
Yield: 96%. IR (KBr, cm−1
)
ν
3338, 3177, 2971, 2788, 1689 (C=O), 1631 (C=N), 1595,
1
1546. H-NMR (400 MHz, DMSO-d6, ppm) δH 8.86 (dd, J = 4.2, 1.7 Hz, 1H, HAr), 8.32 (dd,
J = 8.1, 1.7 Hz, 1H, HAr), 7.49–7.57 (m, 4H, HAr, CONH2), 7.28–7.43 (m, 5H, HAr), 7.01 (br
s, 1H, CONH2), 5.29 (s, 2H, OCH2), 3.72 (d, J = 13.6 Hz, 1H, Ar-CH2), 3.58 (d, J = 13.6 Hz,
1H, Ar-CH2), 3.03 (q, J = 7.0 Hz, 1H, CH-CH3), 1.13 (d, J = 7.0 Hz, 3H, CH3). 13C-NMR
(100 MHz, DMSO-d6 ppm) δC 170.3 (C=O), 149.3, 145.9, 144.9, 136.2, 132.0, 130.0, 129.8,
,
129.6, 129.6, 128.7, 128.2, 122.8, 120.5, 115.5, and 113.8 (15C, Ar-C), 70.5 (CH2), 54.6 (CH),
48.4 (CH2), 16.0 (CH3). HR-ESI MS m/z 336.1708 [M + H]+.
(S)-2-((4-((Quinolin-8-yloxy)methyl)benzyl)amino)propanamide (7h)
Yield: 85%. IR (KBr, cm−1
) ν 3323, 2958, 2746, 1689 (C=O), 1629 (C=N) 1597, 1547.
1H-NMR (400 MHz, DMSO-d6, ppm) δH 8.85 (dd, J = 4.0, 1.7 Hz, 1H, HAr), 8.32 (dd, J = 8.4
,
1.7 Hz, 1H, HAr), 7.51 (dd, J = 8.4, 4.0 Hz, 1H, HAr), 7.44–7.48 (m, 4H, HAr, CONH2),
7.36–7.38 (m, 3H, HAr), 7.26–7.31 (m, 1H, HAr), 7.01 (br s, 1H, CONH2), 5.28 (s, 2H, OCH2),
3.71 (d, J = 13.6 Hz, 1H, Ar-CH2), 3.55 (d, J = 13.6 Hz, 1H, Ar-CH2), 3.02 (q, J = 7.0 Hz, 1H,
CH-CH3), 1.13 (d, J = 7.0 Hz, 3H, CH3). 13C-NMR (100 MHz, DMSO-d6 ppm) δC 170.4
,
(C=O), 148.9, 145.6, 145.4, 136.5, 131.7, 130.4, 129.7, 129.6 128.1, 122.8, 120.4, 115.5, and
114.0 (15C, Ar-C), 70.2 (CH2), 54.5 (CH), 48.1 (CH2), 16.0 (CH3). HR-ESI MS m/z 336.1707
[M + H]+.
Due to the influence of the surrounding groups, the hydrogen atom of the -NH- group
in these compounds was unobserved in the 1H-NMR spectra.
3.3. Formalin Test
The formalin test was carried out using the method described previously [19]. During
the formalin test, Institute of Cancer Research (ICR; CD 1) male mice (weight: 22–25 g)
−
were acclimated to the environment for two days before the experiment with food and
water available freely. The mice were housed under a 12 h/12 h light/dark cycle. The
temperature and humidity of the room were kept at 25
±
2 ◦C and 50–60%, respectively.
The mice were divided into a vehicle group, a ralfinamide group, and test compound
groups randomly, with eight mice in each group. All compounds were dissolved in saline
and administered by intraperitoneal injection (n = 8) or intragastric administration by
mouth (n = 8) at 10 mg/kg. The vehicle group was given the same volume of saline.
After administration, the mice were placed into polyvinyl chloride (PVC) observation
chambers. Thirty minutes later, the mice were injected subcutaneously with 20 µL of
2.7% formalin solution into the surface of their right hind paw and were returned to their
PVC chambers immediately to observe their behavior. The cumulative time each mouse
licked its right hind paw during phase II (15–30 min) was recorded as its painful time in
phase II. The analgesic effects of the compounds were presented as the mean
deviation. The data were analyzed by SPSS using one-way analysis of variance followed
±
standard

Molecules 2021, 26, 1716
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by Dunnett’s test. The analgesic activity of the test compounds was calculated according to
the following formula.
Average Time (Vehicle) − Average Time (Drug)
%Analgesia =
× 100%
Average Time (Vehicle)
3.4. Acetic Acid-Induced Writhing Test
The writhing test was performed according to a previously reported method [20].
The ICR (CD-1) male mice (weight: 22–25 g) were treated as for the formalin test in the
same environment. The mice were divided into a vehicle group, a ralfinamide group,
and test compound groups randomly, with six mice in each group. All compounds were
dissolved in saline and administered by intraperitoneal injection at 10 mg/kg. The vehicle
group was given the same volume of saline. After administration, the mice were placed
into PVC chambers. Thirty minutes later, the mice were injected with 1% acetic acid
solution at 10 mL/kg by intraperitoneal injection and placed into the observation chambers
immediately to record the number of writhes each mouse had in 20 min. The results were
presented as the mean
for the formalin test, and the analgesic activity of each compound was calculated by the
following formula.
±
standard deviation. The analytical method was the same as that
Average Number (Vehicle) − Average Number (Drug)
%Analgesia =
× 100%
Average Number (Vehicle)
3.5. Assay for hNav1.7 Inhibition
The compounds were tested as hNav1.7 peak current inhibitors at room temperature
using human embryonic kidney 293 cells stably expressing hNav1.7. The holding voltage
was depolarized to 0 mV from
that, the membrane potential was recovered to
to 0 mV for another 20 ms. Finally, the voltage was renewed to
potential was applied every 20 s. The compounds were administered when the voltage of
hNav1.7 recorded by the whole-cell patch clamp was stable. Every compound concentration
was tested for 5 min, and all compounds were tested at several concentrations and with
multiple cells.
−
120 mV for 20 ms and restored to
120 mV for 20 ms and then depolarized
120 mV for 30 ms. The
−
75 mV for 8 s. After
−
−
3.6. Assay for hNav1.8 Inhibition
The whole-cell patch clamp assay assessing the hNav1.8 current was carried out
at room temperature with Chinese hamster ovary cells stably expressing hNav1.8. The
holding voltage of the membrane was transferred to 0 mV from
then restored to 50 mV for 8 s. After that, the membrane potential was renewed to
120 mV for 20 ms, which was followed by a depolarization to 0 mV for 50 ms. Finally,
the potential was recovered to the holding voltage of 120 mV for 30 ms. This process was
−
120 mV for 50 ms and
−
−
−
performed every 20 s. The compounds were administered when the voltage of hNav1.8
was stable. Every compound concentration was tested for 5 min, and all compounds were
tested at several concentrations and with multiple cells.
3.7. Assay for hNav1.5 Inhibition
The inhibition of the compounds to hNav1.5 peak current was performed at room
temperature using HEK293 cells that could express hNav1.5 stably. The holding voltage
of the membrane was transferred to 0 mV from
50 mV for 8 s. After that, the membrane potential was renewed to
followed by a depolarization to 0 mV for 50 ms. At last, the potential was recovered
to the holding voltage 120 mV for 30 ms. This progress was performed every 20 s.
−
120 mV for 50 ms and then restored to
−
−
120 mV for 20 ms,
−
The compounds were administrated when the voltage of hNav1.5 was stable. Every
concentration was tested for 5 min and all compounds were tested at several concentrations
and with multiple cells.

Molecules 2021, 26, 1716
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4. Conclusions
In conclusion, novel
α
-aminoamide derivatives containing benzo-aromatic hetero-
cyclic groups were synthesized and evaluated as sodium channel isoform blocks for treating
pain. Compounds 6a 6e, and 6f displayed a greater in vivo analgesic potency than ralfi-
,
namide in both the formalin test and the writhing assay. Interestingly, they exhibited potent
both anti-Nav1.7 and anti-Nav1.8 activity in the patch-clamp electrophysiology assay. The
new compounds could serve as new leads for the development of analgesic drugs.
Author Contributions: W.S. and Y.Z.: study design, director of the study, revision and final approval
of the version to be published. K.T. and R.Z.: compounds synthesis, biological activity assay, data
collection, data analysis, and manuscript writing. K.T. and F.R. (Fengxia Ren): data analysis, data
interpretation. T.Z., J.H., J.C., and Z.Y.: director of the compounds’ synthesis and data analysis. J.H.,
F.R. (Fengzhi Ren), and W.S.: manuscript revision. All authors have read and agreed to the published
version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: All the procedures adopted were approved and agreed upon
by the Institutional Animal Ethical Committee of Beijing Institute of Pharmacology & Toxicology.
The research was conducted in accordance with the ethical standards. All care and use guidelines for
laboratory animals were followed.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available in this article.
Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role
in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the
manuscript, or in the decision to publish the results. The authors agreed to submit the manuscript to
the journal unanimously.
Sample Availability: Samples of the compounds are available from the authors.
References
1.
2.
3.
Jang, J.Y.; Lee, S.H.; Kim, M.Y.; Ryu, J.S. Characteristics of neuropathic pain in patients with spinal cord injury. Ann. Rehabil. Med.
2014, 38, 327–334. [CrossRef] [PubMed]
Colloca, L.; Ludman, T.; Bouhassira, D.; Baron, R.; Dickenson, A.H.; Yarnitsky, D.; Freeman, R.; Truini, A.; Attal, N.; Finnerup,
N.B.; et al. Neuropathic pain. Nat. Rev. Dis. Primers. 2017, 3, 17002. [CrossRef] [PubMed]
Snedecor, S.J.; Sudharshan, L.; Cappelleri, J.C.; Sadosky, A.; Desai, P.; Jalundhwala, Y.J.; Botteman, M. Systematic review and
comparison of pharmacologic therapies for neuropathic pain associated with spinal cord injury. J. Pain Res. 2013, 6, 539–547.
[CrossRef] [PubMed]
4.
5.
6.
7.
8.
9.
Van Hecke, O.; Austin, S.K.; Khan, R.A.; Smith, B.H.; Torrance, N. Neuropathic pain in the general population: A systematic
review of epidemiological studies. Pain 2014, 155, 654–662. [CrossRef] [PubMed]
Kiguchi, N.; Kobayashi, D.; Saika, F.; Matsuzaki, S.; Kishioka, S. Pharmacological regulation of neuropathic pain driven by
inflammatory macrophages. Int. J. Mol. Sci. 2017, 18, 2296. [CrossRef] [PubMed]
Mulcahy, J.V.; Pajouhesh, H.; Beckley, J.T.; Delwig, A.; Du Bois, J.; Hunter, J.C. Challenges and opportunities for therapeutics
targeting the voltage-gated sodium channel isoform Nav1.7. J. Med. Chem. 2019, 62, 8695–8710. [CrossRef] [PubMed]
Wulff, H.; Christophersen, P.; Colussi, P.L.; Chandy, K.G.; Yarov-Yarovoy, V. Antibodies and venom peptides: New modalities for
ion channels. Nat. Rev. Drug Discov. 2019, 18, 339–357. [CrossRef]
Dib-Hajj, S.D.; Waxman, S.G. Sodium channels in human pain disorders: Genetics and pharmacogenomics. Annu. Rev. Neurosci.
2019, 42, 87–106. [CrossRef] [PubMed]
Catterall, W.A.; Lenaeus, M.J.; Gamal El-Din, T.M. Structure and pharmacology of voltage-gated sodium and calcium channels.
Annu. Rev. Pharmacol. Toxicol. 2020, 60, 133–154. [CrossRef] [PubMed]
10. Chew, L.A.; Bellampalli, S.S.; Dustrude, E.T.; Khanna, R. Mining the Nav 1.7 interactome: Opportunities for chronic pain
therapeutics. Biochem. Pharmacol. 2019, 163, 9–20. [CrossRef] [PubMed]
11. Verma, P.; Kienle, A.; Flockerzi, D.; Ramkrishna, D. Using bifurcation theory for exploring pain. Ind. Eng. Chem. Res. 2020, 59,
2524–2535. [CrossRef]
12. Zhou, Y.; Cai, S.; Moutal, A.; Yu, J.; Gomez, K.; Madura, C.L.; Shan, Z.; Pham, N.Y.N.; Serafini, M.J.; Dorame, A.; et al. The Natural
Flavonoid Naringenin Elicits Analgesia through Inhibition of NaV1.8 Voltage-Gated Sodium Channels. ACS Chem. Neurosci.
2019, 10, 4834–4846. [CrossRef]

Molecules 2021, 26, 1716
14 of 14
13. Li, H.; Fan, S.; Cheng, J.; Zhang, P.; Zhong, B.; Shi, W. Synthesis and evaluation of novel
α
-aminoamides containing an indole
moiety for the treatment of neuropathic pain. Molecules 2016, 21, 793. [CrossRef] [PubMed]
14. Yamane, H.; de Groat, W.C.; Sculptoreanu, A. Effects of ralfinamide, a Na+ channel blocker, on firing properties of nociceptive
dorsal root ganglion neurons of adult rats. Exp. Neurol. 2007, 208, 63–72. [CrossRef] [PubMed]
15. Di Stefano, A.F.D.; Radicioni, M.M.; Rusca, A. Pressor response to oral tyramine and monoamine oxidase inhibition during
treatment with ralfinamide (NW-1029). Neurotox. Res. 2013, 23, 315–326. [CrossRef] [PubMed]
16. Wang, Y.Y.; Wilson, S.M.; Brittain, J.M.; Ripsch, M.S.; Salomé, C.; Park, K.D.; White, F.A.; Khanna, R.; Kohn, H. Merging structural
motifs of functionalized amino acids and α-aminoamides results in novel anticonvulsant compounds with significant effects on
slow and fast inactivation of voltage-gated sodium channels and in the treatment of neuropathic pain. ACS Chem. Neurosci. 2011
2, 317–332. [CrossRef]
,
17. Liang, X.; Yu, G.; Su, R. Effects of ralfinamide in models of nerve injury and chemotherapy-induced neuropathic pain. Eur. J.
Pharmacol. 2018, 823, 27–34. [CrossRef]
18. Ma, M.; Sun, J.; Li, M.; Yu, Z.; Cheng, J.; Zhong, B.; Shi, W. Synthesis and evaluation of novel biased µ-opioid-receptor (µOR)
agonists. Molecules 2019, 24, 259. [CrossRef] [PubMed]
19. Ma, R.S.Y.; Kayani, K.; Whyte-Oshodi, D.; Whyte-Oshodi, A.; Nachiappan, N.; Gnanarajah, S.; Mohammed, R. Voltage gated
sodium channels as therapeutic targets for chronic pain. J. Pain Res. 2019, 12, 2709–2722. [CrossRef]
20. Kushnarev, M.; Pirvulescu, L.P.; Candido, K.D.; Knezevic, N.N. Neuropathic pain: Preclinical and early clinical progress with
voltage-gated sodium channel blockers. Expert Opin. Inv. Drug 2020, 29, 259–271. [CrossRef]
21. Zhang, S.H.; Blech-Hermoni, Y.; Faravelli, L.; Seltzer, Z. Ralfinamide administered orally before hindpaw neurectomy or
postoperatively provided long-lasting suppression of spontaneous neuropathic pain-related behavior in the rat. Pain 2009, 139,
293–305. [CrossRef] [PubMed]
22. Bauer, M.; Bliesath, H.; Leuratti, C.; Lackner, E.; Dieterle, W.; Müller, M.; Brunner, M. Disposition and metabolism of ralfinamide,
a novel Na-channel blocker, in healthy male volunteers. Pharmacology 2010, 86, 297–305. [CrossRef] [PubMed]