Discovery of Quinoline Analogues as Potent Antivirals against Enterovirus D68 (EV-D68)

Article abstract of DOI:10.1021/acs.jmedchem.9b00115

Enterovirus D68 (EV-D68) is an atypical nonpolio enterovirus that mainly infects the respiratory system of humans, leading to moderate-to-severe respiratory diseases. In rare cases, EV-D68 can spread to the central nervous system and cause paralysis in infected patients, especially young children and immunocompromised individuals. There is currently no approved vaccine or antiviral available for the prevention and treatment of EV-D68. In this study, we aimed to improve the antiviral potency and selectivity of a previously reported EV-D68 inhibitor, dibucaine, through structure-activity relationship studies. In total, 60 compounds were synthesized and tested against EV-D68 using the viral cytopathic effect assay. Three compounds 10a, 12a, and 12c were identified to have significantly improved potency (EC₅₀ < 1 μM) and a high selectivity index (>180) compared with dibucaine against five different strains of EV-D68 viruses. These compounds also showed potent antiviral activity in neuronal cells, such as A172 and SH-SY5Y cells, suggesting they might be further developed for the treatment of both respiratory infection as well as neuronal infection.

Full text of DOI:10.1021/acs.jmedchem.9b00115

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Article
Discovery of quinoline analogs as potent
antivirals against enterovirus D68 (EV-D68)
Rami Musharrafieh, Jiantao Zhang, Peter Tuohy, Naoya Kitamura,
Shreya Bellampalli, Yanmei Hu, Rajesh Khanna, and Jun Wang
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 26 Mar 2019
Downloaded from http://pubs.acs.org on March 26, 2019
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Discovery of quinoline analogs as potent antivirals against enterovirus
D68 (EV-D68)
†
,§
†
†
†
‡
Rami Musharrafieh, Jiantao Zhang, Peter Tuohy, Naoya Kitamura, Shreya Sai Bellampalli,
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Yanmei Hu, Rajesh Khanna, Jun Wang*
†Department of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona,
Tucson, Arizona 85721, United States
§Department of Chemistry and Biochemistry, The University of Arizona, Tucson, Arizona 85721,
United States
‡Department of Pharmacology, College of Medicine, The University of Arizona, Tucson, Arizona,
8
5721, United States
*Corresponding author:
Jun Wang, Tel: 520-626-1366, Fax: 520-626-0749, email: junwang@pharmacy.arizona.edu
KEYWORDS. Enterovirus D68, EV-D68, antiviral, quinoline, 2C protein.
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ABSTRACT
Enterovirus D68 (EV-D68) is an atypical non-polio enterovirus that mainly infected the respiratory
system of humans, leading to moderate to severe respiratory diseases. In rare cases, EV-D68 can
spread to the central nervous system and causes paralysis in infected patients, especially young
children and immunocompromised individuals. There is currently no approved vaccine or antiviral
available for the prevention and treatment of EV-D68. In this study, we aimed to improve the
antiviral potency and selectivity of a previously reported EV-D68 inhibitor, dibucaine, through
structure-activity relationship studies. In total, sixty compounds were synthesized and tested against
EV-D68 using the viral cytopathic effect (CPE) assay. Three compounds 10a, 12a, and 12c were
identified to have significantly improved potency (EC < 1 µM) and a high selectivity index (SI >
0
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80) compared to dibucaine against five different strains of EV-D68 viruses. These compounds also
showed potent antiviral activity in neuronal cells such as A172 and SH-SY5Y cells, suggesting they
might be further developed for the treatment of both respiratory infection as well as neuronal
infection.
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INTRODUCTION
The human enteroviruses (EVs) are a group of small non-enveloped single-stranded positive-
sense RNA viruses that belong to the piconaviridae family.1, 2 Up to date, more than 110 serotypes
of enteroviruses have been identified, among which the most notable ones are polioviruses,
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coxsackieviruses, EV-D68, EV-A71, and rhinoviruses. Although poliovirus was nearly eradicated
4
by immunization, no vaccines are available for the non-polio EVs except EV-A71. The non-polio
enteroviruses such as EV-D68 are of particular concern as they have been shown to be causative
5
-8
pathogens of numerous epidemics of moderate to severe respiratory illness. Although infections in
most cases are mild and self-limiting, EV-D68 infections can lead to serious and life-threatening
neurological complications such as acute flaccid myelitis (AFM), most often in infants, young
9
children, and immunocompromised individuals. Cases of EV-D68 infection were rarely reported
1
0
before 2005, however, recent years have seen an increasing surge of EV-D68 infections around
the world, especially in the United States.6, 11 EV-D68 attracted public attention when an outbreak
spread to 49 states in the United States in 2014 that led to more than 1,100 reports of severe
6
respiratory disease and more than 100 cases of acute flaccid myelitis. Since then, EV-D68 infection
has been continuously reported.6
EV-D68 is an atypical enterovirus and behaves more like a rhinovirus. For example, unlike
o
other enteroviruses that replicate in the gastrointestinal tract at 37 C, EV-D68 is cold adapted and
o
12
replicates more efficiently at 33 C. Individuals with pre-existing respiratory condition such as
asthma or chronic obstructive pulmonary disease (COPD) are more likely to develop a severe
7
infection. More alarmingly, there has been evidence from both animal studies and human patients
that EV-D68 can spread to central nervous systems (CNS) such as spinal cord and cerebrospinal
fluid through viremia, causing neurological complications such as AFM.13-18
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Due to its global medical and socioeconomic impact, EV-D68 is classified as a priority
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pathogen by NIAID. However, despite decades of research efforts, there is currently no vaccine
2
nor small molecule antiviral exist for EV-D68 infection, and treatment is primarily limited to
supportive care. To reduce the morbidity and mortality associated with EV-D68 virus infection,
there is an urgent need to develop potent and selective small molecule antivirals. Towards this end,
we are interested in developing EV-D68 antivirals by targeting the viral 2C protein.
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The non-structural protein EV-D68 2C is a multi-functional protein. Genetic studies of 2C have
revealed that it has functional roles in viral uncoating, RNA binding and replication, membrane
rearrangement, encapsidation of the viral genome, and progeny viral assembly, all of which are
essential for viral replication.1, 2 Therefore, EV-D68 2C appears to be an attractive target for anti-
enterovirus drug development. Previous phenotypic screenings identified several small molecules
such as pirlindole mesylate, fluoxetine, formoterol, dibucaine, and guanidine as EV-D68
antivirals.20-23 Resistance selection through serial viral passage experiments led to the identification
of mutations in viral 2C protein that confer resistance to these compounds, suggesting 2C might be
the potential drug target. In this study, we first independently verified the antiviral activity and
cellular cytotoxicity of these reported 2C protein inhibitors. It was found that all these molecules
had only moderate antiviral activity against EV-D68 with a low selectivity index (Table 1),
consistent with previously reported results.21, 22 Next, we chose dibucaine as a starting hit compound
for structure-activity relationship (SAR) studies due to its modular synthetic accessibility. The aim
of this study is therefore to optimize the potency and selectivity index of dibucaine through SAR
studies. In total sixty quinoline analogues have been synthesized and tested against EV-D68
(US/KY/14-18953) virus in the primary viral cytopathic effect (CPE) assay. Three compounds (10a,
1
2a, and 12c) were identified to have significantly improved antiviral activity with EC values in
50
the submicromolar range and a selectivity index over 180. We further profiled the broad range
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antiviral activity of these three compounds against additional four contemporary human EV-D68
viruses. It was found that these three compounds all had potent antiviral activity and a high
selectivity index. These three lead compounds also potently inhibited EV-D68 infection in neuronal
cells such as A172 and SH-SY5Y cells, suggesting they can be further developed to treat both
respiratory and neuronal infections. The mechanism of action of these three compounds was studied
by RT-qPCR, western blot, and immunofluorescence imaging. These compounds were found to
inhibit viral RNA and protein synthesis, which agrees with the inhibition of viral 2C protein.
Table 1. Literature reported enterovirus 2C protein inhibitors.
1
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0
EV-D68
RD cells
Structure
(US/KY/14)
SI
b
CC (µM)
a
50
EC (µM)
5
0
Pirlindole mesylate
N
8.4 ± 1.1
19.0 ± 1.6
11.9 ± 4.9
2.3
O
NH
OH
S
O
Fluoxetine
F
F
F
1
.0 ± 0.1
11.9
5.9
N
O
H
Formoterol
O
NH
2
4.7 ± 1.1
146.3 ± 27.8
HO
O
N
H
OH
Dibucaine
H
O
N
N
5
.3 ± 1.2
56.6 ± 28.7
>300.0
10.7
N
O
Guanidine
NH
2
19.4 ± 15.0
> 1.4
H N
2
NH2
aAntiviral efficacy was determined in the CPE assay with EV-D68 US/KY/14-18953 virus and RD
b
cells. Cytotoxicity was determined using the neutral red method. The results are the mean ±
standard deviation of three repeats. SI = selectivity index (CC /EC ).
5
0
50
RESULTS AND DISCOSSION
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CHEMISTRY
Three positions of dibucaine were varied in the SAR study: the 2-position substituent was
substituted with either an alkoxy (O-R ) or an aromatic group (R ); the 4-position was substituted
1
2
with different amides; and the 6-position was substituted with either F, Cl, or methoxy group. We
adapted a stepwise iterative optimization strategy. First, the 4-position substitution was fixed while
the 2-position substitution was optimized. Second, the 2-position substitution was fixed while the 4-
position substitution was optimized. Third, optimal substitutions from both the 2- and 4-positions
were merged. Finally, the 6-position was diversified with the most potent analogs.
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amide
H
F, Cl, OCH3
O
N
N
4
6
H
2
O
N
alkoxy, aromatic
Dibucaine (1)
Figure 1. Chemical structure of dibucaine. Three positions, 2, 4, and 6, were examined in the
current SAR studies and were circled.
The synthesis of quinoline analogs was designed to allow for expeditious late-stage
diversification. For example, for varying the 2-position substitution, intermediate 4 was first
synthesized by amide coupling. Next, two methods were developed to install alkoxy substitution at
the 2-position (Scheme 1, synthesis route 1). Method 1 used sodium metal and excess alcohol,
which served as both the reagent and the solvent. This worked for alcohols with a low boiling point
such that they can be easily removed during the work up procedure. For alcohols with a high boiling
point, method 2 was developed which used DMF as the solvent and two equivalents of sodium
hydride and the alcohol. To introduce aromatic substitutions at the 2-position, Suzuki-Miyaura cross
coupling was employed using microwave heating (Scheme 1, synthesis route 2). Once the optimal
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substations at the 2-position were identified, the next step was to optimize the 4-position amide
substitution. For this, quinoline 4-carboxylic acids 7, 9, and 11 with optimal 2-position substitutions
(isopropoxyl, phenyl, and thienyl) were synthesized. Next, amide coupling was applied to introduce
different amines (Scheme 1, synthesis routes 3 & 4). Finally, for analogs with substitutions at the 6-
position of quinoline, two synthesis strategies were developed (Scheme 1, synthesis routes 5).24
The first strategy involved Pfitzinger reaction with 5-chloroisatin or 5-methoxylisatin 13 and
acetophenone using potassium hydroxide as a base. Coupling of 14 with different amines gave the
final product 15. The second strategy involved the reaction of 5-fluoroisatin with malonic acid
under refluxing condition in acetic acid to give intermediate 17. Next, a one pot chlorination and
amide coupling was achieved by first forming the 2-chloroquinoline-4-carbonyl chloride
intermediate through reacting intermediate 17 with thionyl chloride in the presence of catalytic
amount of DMF, then different amines were added to convert the acid chloride intermediate to the
intermediate 18. Finally, Suzuki-Miyaura cross-coupling was applied to install the 2-position
substitution to give the final product 19.
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Scheme 1. Synthesis routes of dibucaine analogs.
Synthesis Route 1:
Na, R1-OH
method 1
H
O
OH
O
N
H
N
N
O
HATU. DIEA
DMF
N
N
+
H2N
NaH, R1-OH
DMF
N
Cl
N
Cl
R1
N
O
2
3
0
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0
4
method 2
5
Synthesis Route 2:
H
H
O
N
O
N
N
HO
Pd(PPh3)4, K2CO3
N
B
R2
+
OH
Dioxane/H2O
microwave 140 oC 30 mins
N
Cl
N
R2
4
6
Synthesis Route 3:
R3
O
OH
O
OH
O
N
R4
Na
HATU. DIEA
DMF
R4
+
+
HN
R3
HO
N
Cl
N
O
N
O
2
7
8
Synthesis Route 4:
R6
N
R6
N
O
OH
O
O
OH
O
R5
R5
HATU. DIEA
DMF
HATU. DIEA
DMF
R6
R6
+
HN
R5
+
HN
R5
N
N
N
N
S
S
10
12
9
11
Synthesis Route 5:
R3
N
O
OH
O
R4
O
O
KOH
EtOH/H2O
R7
HATU. DIEA
DMF
R7
R7
R4
+
+
HN
O
microwave 125 o
C
R3
N
N
N
H
R7 = Cl, OCH3
1
1
4a R7 = Cl
13
14
15
4b R7 = OCH3
R3
R3
N
O
O
O
OH
O
N
O
1. SOCl2, DMF,
DCM reflux
HO
O
R4
Cl
3 4 2 3
Pd(PPh ) , K CO
R4
F
HO
OH
B
F
F
OH
F
O
+
R4
Dioxane/H2O
microwave 140 oC 30 mins
N
acetic acid, reflux
2. HN
THF
H
N
OH
N
N
R3
16
17
18
1
9
STRUCTURE-ACTIVITY RELATIONSHIP STUDIES
As an initial screening, all compounds were tested for antiviral activity and cellular cytotoxicity
in standard viral CPE assay and neutral red cell viability assay, respectively, with the human
rhabdomyosarcoma (RD) cells. The CPE assay involved infecting RD cells with the EV-D68 virus
(US/KY/14-18953) and monitoring cell viability after 3 days of incubation with and without
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compound treatment. Dibucaine was included as a positive control. For compound 5 series with
different alkoxy substitutions at the 2-position (Table 2), compound 5e with 2-isopropoxy
substitution was found to have the highest potency and selectivity (EC = 2.5 ± 0.5 µM, CC =
5
0
50
1
11.2 ± 15.4 µM, SI = 44.5). Compound 5k also had improved potency and selectivity compared to
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
3
4
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4
4
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5
5
5
5
5
5
6
0
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2
3
4
5
6
7
8
9
0
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2
3
4
5
6
7
8
9
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0
dibucaine. Other compounds (5a, 5b, 5c, 5d, 5f, 5g, 5h, 5i, and 5j) either had reduced antiviral
activity (5a, 5b, 5g, 5h, 5i, 5j) or reduced selectivity index (5c, 5d, 5f) compared to dibucaine.
From these results, it can be concluded that isopropoxy is one of the optimal substitutions at the 2-
position of quinoline. Therefore, compound 5e was chosen as a reference compound for following
optimization.
Table 2. SAR study of the 2-position R substituent: alkoxy
1
H
O
N
N
R1
N
O
5
Compound ID
R
1
EC50 (µM)a CC50 (µM)b
SI
5
a
52.5 ± 7.1
5.9 ± 3.2
3.5 ± 0.4
4.9 ± 2.0
> 250.0
>4.8
5
b
164.9 ± 57.6 27.9
5
c
CF3
33.1 ± 1.8
9.5
9.5
5
d
46.7 ± 14.7
5
e
2.5 ± 0.5
5.3 ± 1.2
2.8 ± 1.3
7.1 ± 5.0
111.2 ± 15.4 44.5
Dibucaine 1
56.6 ± 28.7
15.7 ± 1.1
38.5 ± 16.8
10.7
5.6
5
f
5
g
5.4
N.A.c
5
h
> 10
9.5 ± 1.1
5
i
6.8 ± 0.5
11.7 ± 1.2
1.7
O
5
j
12.2 ± 1.1 143.7 ± 40.5 11.8
1.1 ± 0.1 13.5 ± 2.9 12.3
5k
aAntiviral efficacy was determined in the CPE assay with EV-D68 US/KY/14-18953 virus and RD
b
c
cells. Cytotoxicity was determined using the neutral red method. N.A.= not applicable. The results
are the mean ± standard deviation of three repeats. SI = selectivity index (CC /EC ).
5
0
50
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1
1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
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3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
For compound 6 series with aromatic substitutions at the 2-position (Table 3), compounds 6d,
6f, 6g, and 6j had improved antiviral activity and selectivity index compared to compound 5e.
Compounds 6e, 6l, 6m, 6r, and 6s had improved antiviral activity compared to 5e, however they
were compromised by a lower selectivity index. Other compounds (6a, 6b, 6c, 6h, 6i, 6k, 6n, 6o,
6p, 6q, 6t, and 6u) in this series were less active than 5e and were not further explored. These
results suggested unsubstituted thienyl, furanyl, and benzyl are optimal substitutions at the 2-
position of quinoline.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3. SAR study of the 2-position R substituent: Aromatic Groups
2
H
O
N
N
N
R2
6
a
CC50 (µM)b
Compound ID
R
2
EC50 (µM)
SI
111.2 ± 15.4 44.5
5
e
2.5 ± 0.5
O
6
a
19.4 ± 1.2 132.3 ± 53.3
6.8
HN
N
N
N
250.7 ± 28.3 N.A.c
6
b
>100.0
6
c
36.3 ± 1.6 132.6 ± 28.7
3.7
N
N
6
d
0.6 ± 0.5
0.8 ± 0.6
37.4 ± 6.3
6.1 ± 2.2
46.9 ± 5.2
62.3
S
S
6
e
7.6
Cl
6f
1.0 ± 0.4
1.5 ± 0.9
46.9
S
6
g
122.5 ± 30.8 81.7
128.6 ± 67.2 N.A.c
O
6
h
O
N
>50.0
6
i
6.6 ± 3.3
0.8 ± 0.3
13.9 ± 8.1
173.5 ± 55.6 26.3
N
6j
45.1 ± 13.7
75.9 ± 6.4
56.4
5.5
6
k
O
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6l
1.6 ± 0.8
36.5 ± 3.1
22.8
13.2
O
6
m
2.4 ± 0.2
31.6 ± 4.2
46.6 ± 2.4
79.2 ± 8.0
O
6
n
17.4 ± 8.9
2.7
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
N.A.c
6
o
>80.0
N
N
6
6
p
q
24.6 ± 2.2
74.2 ± 7.0
3.0
N
N
N
O
N
87.6 ± 3.1 173.5 ± 20.3
2.0
14.4
18.4
5.5
6
r
0.8 ± 0.2
1.6 ± 0.1
9.0 ± 1.3
11.5 ± 6.7
29.5 ± 1.6
49.9 ± 13.8
6
s
O
O
6t
O
>10.0
9.8 ± 0.5
N.A.c
6
u
aAntiviral efficacy was determined in the CPE assay with EV-D68 US/KY/14-18953 virus and RD
b
c
cells. Cytotoxicity was determined using the neutral red method. N.A.= not applicable. The results
are the mean ± standard deviation of three repeats. SI = selectivity index (CC /EC ).
5
0
50
Once the 2-position substitution was optimized, the next step was to optimize the 4-position
amide. For compound 8 series with 2-position substitution being isopropoxyl (Table 4), compounds
8
a and 8e had improved antiviral activity compared with 5e, however they had a lower selectivity
index. All other compounds (8b, 8c, 8d, 8f, 8g, and 8h) had reduced antiviral activity. For
compound 10 and 12 series with 2-position being benzyl and thienyl, respectively (Table 5),
compound 10a had significantly improved antiviral activity and selectivity index (EC = 0.4 ± 0.2
5
0
µM, CC = 73.7 ± 19.1 µM, SI = 184.3). Similarly, compounds 12a and 12c were also found to be
5
0
highly potent EV-D68 antivirals (EC ≤ 0.1 µM) with a high selectivity index (SI > 600).
5
0
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 4. SAR study of the 4-position amide substituent
R3
O
N
R4
N
O
8
R3
N
EC50 (µM)a CC50 (µM)b
SI
111.2 ± 15.4 44.5
38.5 ± 1.5 22.6
148.8 ± 15.3 42.5
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Compound ID
R4
N
H
N
5
e
2.5 ± 0.5
H
N
8
a
1.7 ± 1.2
3.5 ± 0.3
N
H
N
8
b
NH2
N
H
8
c
N
11.4 ± 0.7 121.7 ± 39.2 10.7
H
N
8
d
N
17.5 ± 10.2
1.7 ± 1.2
25.9 ± 2.1
17.8 ± 1.1
> 100.0
> 250.0
36.9 ± 20.1
88.8 ± 21.5
100.7 ± 8.8
> 300.0
>14.3
21.7
3.4
O
H
N
8
e
N
H
N
8
f
N
N
H
N
8g
5.7
N
N
8
h
N.A.c
N
aAntiviral efficacy was determined in the CPE assay with EV-D68 US/KY/14-18953 virus and RD
b
c
cells. Cytotoxicity was determined using the neutral red method. N.A.= not applicable. The results
are the mean ± standard deviation of three repeats. SI = selectivity index (CC /EC ).
5
0
50
Table 5. SAR study of the 4-position amide substituent
R6
O
N
R5
N
R4 10 & 12
R6
N
Compound ID
R
4
EC50 (µM) CC50 (µM)
SI
56.4
73.7 ± 19.1 184.3
45.4 ± 14.0 4.7
R5
N
H
N
6j
0.8 ± 0.3
0.4 ± 0.2
9.6 ± 6.7
45.1 ± 13.7
H
N
1
0a
N
H
10b
N
N
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H
N
1
0c
3.5 ± 0.3
46.5 ± 14.7
13.3
N
H
1
0d
N
100.1 ± 8.5
34.7 ± 1.4
> 300.0
>250
>3
NH2
H
N
NH2
NH
10e
10f
10g
>7.2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H
N
>100.0
>350.0
>3.5
N
N
H
N
N
0.4 ± 0.1
3.5 ± 0.6
46.7 ± 8.3
116.8
12.8
H
N
1
0h
2a
2b
2c
2d
2e
44.9 ± 17.7
H
N
1
0.04 ± 0.01 42.3 ± 14.3 1057.5
0.7 ± 0.3 40.5 ± 14.0 57.9
0.06 ± 0.03 40.0 ± 13.1 666.7
N
S
S
H
1
N
N
H
N
1
N
S
N
H
1
N
8.9 ± 5.8
55.7 ± 3.5
>100.0
6.3
N
S
S
H
1
N
11.9 ± 1.3
> 8.4
NH2
aAntiviral efficacy was determined in the CPE assay with EV-D68 US/KY/14-18953 virus and RD
b
cells. Cytotoxicity was determined using the neutral red method. The results are the mean ±
standard deviation of three repeats. SI = selectivity index (CC /EC ).
5
0
50
For compounds 19 and 15 with substitutions at the 6-position (Table 6), none of them had
improved potency and selectivity index compared to compound 10a, suggesting the 6-position of
quinoline preferred to be unsubstituted.
Table 6. SAR study of the R substituent
7
R3
O
N
R2
R4
R7
N
1
5&19
R3
N
Compound ID
R
4
R
7
EC50 (µM)
CC50 (µM)
SI
R2
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
H
N
H
1
1
0a
9a
0.4 ± 0.2
73.7 ± 19.1
38.8 ± 19.0
24.9 ± 1.5
14.3 ± 0.8
13.5 ± 8.1
31.8 ± 1.3
27.5 ± 7.3
184.3
64.7
19.2
11.9
7.1
N
N
H
N
F
F
0.6 ± 0.3
1.3 ± 0.3
1.2 ± 0.2
1.9 ± 0.1
8.1 ± 0.6
12.3 ± 1.1
H
N
1
9b
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H
N
Cl
Cl
15a
5b
N
H
N
1
N
H
N
O
15c
5d
3.9
N
H
N
O
1
N
2.2
Overall our SAR study suggest that the optimal substitutions at the 2-position were
isopropoxyl, thienyl, furanyl, and benzyl (Fig. 2); the optimal substitutions at the 4-position were
N-(2-(diethylamino)ethyl)amide, N-(2-(dimethylamino)ethyl)amide, and N-(2-(pyrrolidin-1-
yl)ethyl)amide; the 6-position preferred to be un-substituted.
H
H
N
H
O
N
O
O
N
N
N
N
preferred
H
H preferred
H
O
N
N
4
6
2
O
N
O
preferred
S
Dibucaine (1)
S
O
Figure 2. Summary of SAR studies of dibucaine.
BROAD RANGE ANTIVIRAL ACTIVITY
Given the potent antiviral activity and the high selectivity index of lead compounds 10a, 12a,
and 12c in inhibiting the EV-D68 US/KY/14-18953 strain, we were interested in exploring their
broad range antiviral activity against other contemporary EV-D68 strains. For this, these three
compounds were tested against four additional human EV-D68 strains (Table 7). It was found that
compounds 10a, 12a, and 12c had similarly potent antiviral activity and high selectivity index
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against all five EV-D68 strains tested, corroborating that 2C protein is a conserved and high-profile
antiviral drug target. The three lead compounds 10a, 12a, and 12c were also well tolerated in two
additional cell lines A549 and HeLa (Table 7). Furthermore, as EV-D68 also infects CNS such as
spinal cord and brain, we were intrigued to figure out whether these three lead compounds could
also inhibit EV-D68 virus replication in the neuronal cells. For this purpose, we infected the human
brain glioblastoma A172 cells or the neuroblastoma cell line SH-SY5Y with the EV-D68 virus and
quantified viral replication in CPE assay with and without compound treatment (Table 8).
Encouragingly, all three compounds had potent antiviral activity against EV-D68 virus replication
in neuronal cell lines (EC = 0.01 to 0.4 µM). Notably, they were less cytotoxic to A172 and SH-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
SY5Y cells than the RD cells, resulting in a higher selectivity index. In conclusion, the three lead
compounds identified from the SAR studies, 10a, 12a, and 12c inhibits multiple EV-D68 strains
with high potency and selectivity index in both muscle cells (RD) and neuronal cells (A172 and SH-
SY5Y).
Table 7. Antiviral activity of potent leads against different serotypes of EV-D68 in RD cells
and cytotoxicity of lead compounds in different cell lines
Compound ID
Antiviral activity and cytotoxicity in RD cells
Cellular cytotoxicity
US/KY/14-
US/MO/14-
18947
US/MO/14-
18949
US/IL/14-
18956
(µM)
UA/IL/14-
18952
(µM)
A549
(µM)
HeLa
(µM)
1
8953
µM)
(
(µM)
(µM)
H
O
N
EC50 = 3.5 ± 2.3
CC50 = 56.6 ±
EC50 = 2.9 ± 1.8
CC50 = 56.6 ±
28.7
EC50 = 1.8
CC50 = 56.6 ±
28.7
EC50 = 2.0 ± 0.7
CC50 = 56.6 ±
28.7
N
EC50 = 5.3 ± 1.2
CC50 = 56.6 ±
CC50 = 37.5 ±
4.7
CC50 = 36.0 ±
4.0
2
8.7
2
8.7
SI = 16.2
SI = 19.5
SI = 31.4
SI = 28.3
N
O
SI = 10.7
Dibucaine (1)
H
O
N
EC50 = 0.2 ±
N
EC50 = 0.4 ± 0.2
CC50 = 73.7 ±
EC50 = 0.3 ± 0.09
CC50 = 73.7 ±
19.1
EC50 = 0.6 ± 0.1
CC50 = 73.7 ±
19.1
EC50 = 0.6 ± 0.1
CC50 = 73.7 ±
19.1
0
.07
CC50 = 73.7 ±
9.1
CC50 = 121.1 ±
6.2
CC50 = 68.7 ±
3.8
1
9.1
1
N
SI = 184.3
SI = 245.7
SI = 122.8
SI = 122.8
SI = 368.5
1
0a
H
N
EC50 = 0.06 ±
O
EC50 = 0.04 ±
EC50 = 0.05 ±
0.03
CC50 = 42.3 ±
14.3
EC50 = 0.06 ±
0.04
CC50 = 42.3 ±
14.3
EC50 = 0.05 ±
0.03
CC50 = 42.3 ±
14.3
N
0
.03
CC50 = 42.3 ±
4.3
SI = 705
0
.01
CC50 = 42.3 ±
4.3
SI = 1057.5
CC50 = 98.8 ±
3.5
CC50 = 51.2 ±
2.0
1
1
N
SI = 846
SI = 705
SI = 846
S
1
2a
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
H
O
N
EC50 = 0.06 ±
.03
CC50 = 40.0 ±
13.1
EC50 = 0.06 ±
EC50 = 0.05 ±
0.03
CC50 = 40.0 ±
13.1
EC50 = 0.07 ±
0.03
CC50 = 40.0 ±
13.1
N
EC50 = 0.05
CC50 = 40.0 ±
0
0.02
CC50 = 40.0 ±
13.1
CC50 = 83.2 ±
4.1
CC50 = 41.7 ±
4.3
1
3.1
N
SI = 800
SI = 666.7
SI = 666.7
SI = 800
SI = 571.4
S
12c
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 8. Antiviral activity of potent leads against different serotypes of EV-D68 in neuronal
cells
Compound ID
Antiviral activity and cytotoxicity in neuronal cells
US/KY/14-18953 US/KY/14-18953
US/MO/14-18947
US/MO/14-18947
in A172 cells
µM)
in SH-SY5Y cells
(µM)
in A172 cells (µM) in SH-SY5Y cells (µM)
(
H
O
N
N
EC50 = 3.7 ± 1.5
CC50 > 30.0
SI > 8.1
EC50 = 1.7 ± 0.8
CC50 > 30.0
SI > 17.6
EC50 = 1.9 ± 0.1
CC50 > 30.0
SI > 15.8
EC50 = 1.9 ± 0.3
CC50 > 30.0
SI > 15.8
N
O
Dibucaine (1)
H
O
N
N
EC50 = 0.4 ± 0.1
CC50 = 88.4 ± 31.6
SI = 221.0
EC50 = 0.4 ± 0.1
CC50 > 300.0
SI > 750.0
EC50 = 0.3 ± 0.02
CC50 = 88.4 ± 31.6
SI = 294.7
EC50 = 0.2 ± 0.07
CC50 > 300.0
SI > 1500.0
N
10a
H
N
O
N
EC50 = 0.02 ± 0.01
CC50 = 73.8 ± 41.0
SI = 3690.0
EC50 = 0.1 ± 0.05
CC50 = 136.3 ± 69.3
SI = 1363.0
EC50 = 0.1 ± 0.01
CC50 = 73.8 ± 41.0
SI = 738.0
EC50 = 0.01 ± 0.008
CC50 =136.3 ± 69.3
SI = 13630.0
N
S
1
2a
H
O
N
N
EC50 = 0.05 ± 0.00
CC50 = 65.4 ± 22.2
SI = 1308.0
EC50 = 0.1 ± 0.08
CC50 = 93.3 ± 30.7
SI = 933.0
EC50 = 0.2 ± 0.02
CC50 = 65.4 ± 22.2
SI = 327.0
EC50 = 0.02
CC50 = 93.3 ± 30.7
SI = 4665.0
N
S
1
2c
MECHANISM OF ACTION
It was reported that dibucaine inhibits EV-D68 through the inhibition of genome replication by
21
targeting the 2C protein. The EV-D68 2C protein is a highly conserved viral protein that plays
multiple functions in genome replication. It has been implicated in RNA replication, membrane
1
rearrangements, encapsidation, and uncoating. Inhibition of viral 2C protein is expected to inhibit
viral RNA and protein synthesis. To test this hypothesis and explore the cellular antiviral
mechanism of dibucaine analogs 10a, 12a and 12c, we detected viral capsid protein VP1 expression
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level by immunofluorescence imaging and western blot, and viral RNA levels by RT-qPCR (Fig.
3). Compared to DMSO control, 3 µM of dibucaine significantly reduced the fluorescent signal of
VP1 at 9 hours post infection (hpi) (Fig. 3A). The reduction of VP1 protein level was also
independently confirmed in western blot experiment (Fig. 3B), whereas 3 µM of dibucaine nearly
completely inhibited VP1 expression (Fig. 3B) while 1 µM dibucaine had no effect. In contrast,
when tested at 1 µM, dibucaine analogs, 10a, 12a and 12c, completely blocked the VP1 protein
expression (Fig. 3A & 3B) as shown by immunofluorescence imaging and western blot. Similarly,
RT-qPCR quantification of viral RNAs showed that 1 µM of dibucaine analogs, 10a, 12a and 12c,
completely inhibited the viral RNA synthesis (> 99%), whereas 3 µM of dibucaine reduced viral
RNA level by ~80% (Fig. 3C). Taken together, our results confirmed that dibucaine and its analogs
share similar mechanism of action, and the analogs had more potent antiviral activity than
dibucaine.
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Figure 3. Dibucaine and its analogs 10a, 12a, and 12c reduced the EV-D68 viral protein and RNA
levels. (A) Dibucaine and its analogs inhibited EV-D68 replication. RD cells infected with EV-D68
strain US/KY/14-18953 (MOI = 1) were fixed at 9 hpi and stained with anti-VP1 and DAPI for
detecting VP1 and nucleus, respectively. (B and C) Dibucaine and its analogs reduced the EV-D68
viral protein and RNA levels. RD cells were infected with EV-D68 strain US/KY/14-18953 (MOI =
0
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) with treatment of indicated compound. At 9 hpi, cells were harvested for viral protein
quantification by western or viral RNA quantification by RT-qPCR. Asterisks indicate statistically
significant difference in comparison with the DMSO control (one-way ANOVA analysis by Prism
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, ***P < 0.001). The value is the mean of two independent experiments ± standard deviation.
POTENTIAL SIDE EFFECTS
Dibucaine is a FDA-approved topical anesthetic drug. Its anesthetic activity is due to binding
and inhibiting sodium channels within neuronal cell membranes. For the purpose of antiviral
development, inhibition of the sodium channel might be an unwanted side effect. As the three lead
compounds 10a, 12a, and 12c were derived from dibucaine, we were concerned that they might
have anesthetic side effect by inhibiting the sodium channel. To ease this doubt, we chose one
representative compound 10a and tested its sodium channel inhibition using the standard sodium
flux assay. Dibucaine was included as a positive control. In this assay, rat dorsal root ganglia (DRG)
neurons were incubated with various concentrations of testing compounds. Sodium channels were
then activated by the sodium channel agonist veratridine. The resulting sodium flux was measured
indirectly by quantifying the sodium channel triggered calcium flux, which was measured by a
calcium-specific fluorescence dye (Fig. 4). It was found that dibucaine potently inhibited sodium
current conductance at both 20 µM and 50 µM, while compounds 10a had minimal to no effect
from 2 µM to 50 µM, nearly 100-fold higher than its antiviral EC value. These results confirmed
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that the quinoline lead compounds identified from this study such as 10a are promising candidates
with no potential anesthetic side effects, suggesting they are promising for further development.
Dibucaine (1)
Compound 10a
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Figure 4. Inhibition of sodium channels by dibucaine and lead compound 10a.
To address the concern that the optimized lead compounds might be promiscuous antivirals, we
tested the three lead compounds 10a, 12a, and 12c against two influenza viruses, the influenza A
virus A/California/07/2009 (H1N1), and the influenza B virus B/Brisbane/60/2008 (Victoria), in
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plaque assay. It was found that dibucaine and the three lead compounds had no inhibition against
these two strains of influenza viruses when tested at 10 µM (Fig. 5), indicating the antiviral activity
of the lead compounds 10a, 12a, and 12c, against EV-D68 was not due to non-specific effect of
these compounds.
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Figure 5. Dibucaine and the optimized lead compounds did not inhibit influenza A and B viruses.
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Plaque assay was performed using the condition described previously. Dibucaine, 10a, 12a, and
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2c were tested at 10 µM. Oseltamivir carboxylate was included as a positive control and was tested
at 200 nM.
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CONCLUSIONS
The magnitude and severity of EV-D68 outbreak in recent years underscores a need for
effective antivirals, not only to be used for prophylaxis, but also for the treatment of EV-D68
infection. In this study, we chose a previously reported hit compound, dibucaine, as a starting point
for the structure-activity relationship studies. Dibucaine was identified as an EV-D68 antiviral from
a drug repurposing phenotypic screening. However, the moderate antiviral activity and a low
selectivity index of dibucaine impeded its clinical use as an antiviral. As such, we aim to optimize
the potency and selectivity index of dibucaine through structure-activity relationship studies. This
effort led to the identification of three lead compounds 10a, 12a, and 12c, with significantly
improved antiviral efficacy and selectivity index (EC < 1 µM, SI > 180). The antiviral mechanism
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of action of these three compounds were investigated by immunofluorescence staining, western
blot, and RT-qPCR assays. Collectively, all three compounds inhibited viral protein and RNA
synthesis, consistent with the proposed mechanism of 2C protein inhibition. Nevertheless, to
confirm the mechanism of action, serial viral passage experiments need to be designed to select
resistant mutant in viral 2C protein. Alternatively, direct binding needs to be measured between lead
compounds and viral 2C protein. The lead compounds might either affect the function of viral 2C
protein directly or disrupt essential interactions between viral 2C protein and host factors. Such
experiments are ongoing and will be reported when available. To our knowledge, these three
compounds represent the most potent and select EV-D68 2C inhibitors reported so far. Of particular
highlights are the easy synthesis accessibility of these compounds which can be made in two steps
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from commercial available materials, broad range antiviral activity against all five contemporary
EV-D68 strains, and consistent antiviral activity across different cell lines including muscle cells
(RD) and neuronal cells (A172 and SH-SY5Y). It has been shown that recent circulating EV-D68
strains such as the ones tested in this study acquired the fitness of replication in neuronal cells.
Therefore it is important for an antiviral drug candidate to show consistent antiviral activity in
different cell lines. It should be pointed out that although EV-D68 infection has a direct link with
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AFM, it is not clear whether AFM is a result of viral replication in the CNS (spinal cord and
brain) or due to inflammation caused by viral infection. Therefore it is desired to develop both
blood-brain-barrier (BBB) permeable and impermeable EV-D68 antivirals and test them in mouse
model studies to dissect whether BBB permeability is essential for AFM treatment. The compounds
reported herein represent not only lead candidates for further translational development into EV-
D68 antivirals, but also valuable chemical probes to help understand the structure, function, and
mechanism of EV-D68 2C protein. Accordingly, future efforts will focus on optimizing the in vitro
and in vivo pharmacokinetic properties of this series of compounds and evaluating their in vivo
antiviral efficacy in mouse model studies. On another hand, the structure of the EV-D68 2C protein
is currently unknown. The inhibitors discovered herein might be able to stabilize the 2C protein and
help facilitate its structure determination by either NMR or X-ray crystallography. A high-
resolution crystal structure of EV-D68 2C is highly desired as it will be invaluable in guiding the
rational design of next-generation of 2C inhibitors.
EXPERIMENTAL SECTION
Chemistry. Chemicals were ordered from commercial sources and were used without further
purification. Synthesis procedures for reactions described in Scheme 1 were shown below. All final
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compounds were purified by flash column chromatography. H and C NMR spectra were recorded
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on a Bruker-400 NMR spectrometer. Chemical shifts are reported in parts per million referenced
with respect to residual solvent (CD OD) 3.31 ppm, (DMSO-d ) 2.50 ppm, and (CDCl ) 7.24 ppm
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or from internal standard tetramethylsilane (TMS) 0.00 ppm. The following abbreviations were
used in reporting spectra: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; dd, doublet of
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doublets; ddd, doublet of doublet of doublets. All reactions were carried out under N atmosphere
2
unless otherwise stated. HPLC-grade solvents were used for all reactions. Flash column
chromatography was performed using silica gel (230–400 mesh, Merck). High-resolution mass
spectra were obtained using the positive ESI method for all the compounds, obtained in an Ion
Cyclotron Resonance (ICR) spectrometer. Low-resolution mass spectra were obtained using an ESI
technique on a 3200 Q Trap LC/MS/MS system (Applied Biosystems). The purity was assessed by
using a Shimadzu LC-MS with a Waters XTerra MS C-18 column (part no. 186000538), 50 mm ×
2.1 mm, at a flow rate of 0.3 mL/min; λ = 250 and 220 nm; mobile phase A, 0.1% formic acid in
H O, and mobile phase B′, 0.1% formic in 60% 2-propanol, 30% CH CN, and 9.9% H O. All
2
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compounds submitted for antiviral CPE assay, cytotoxicity assay, and mechanistic studies were
confirmed to be >95.0% purity by LC-MS traces.
Synthesis procedures.
General procedure of amide coupling. To a DMF solution of carboxylic acid (1 mmol) was added
HATU (1 mmol) and DIEA (1.2 mmol). After stirring for 2 minutes, amine (1 mmol) was added.
The resulting solution was stirred overnight at room temperature. The reaction was diluted with
dichloromethane and extracted with aqueous NaHCO solution, followed by brine. The organic
3
layer was dried over MgSO , filtered, and concentrated under reduced pressure. The crude product
4
was purified by flash column chromatography (1-10% CH OH/CH Cl ) to give the final product.
3
2
2
General procedure of Suzuki-Miyaura cross coupling. To a solution of 2-chloroquinoline (1
mmol) and boronic acid (1mmol) in 1,4-dioxane in a microwave reaction vial was added an aqueous
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solution of K CO (2 mmol). The resulting solution was purged with N for 5 minutes. The catalyst,
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Pd(PPh ) (0.1 mmol), was added in one portion. The vial was capped and heated to 140 C for 30
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minutes with microwave irradiation. After cooling down to room temperature, the reaction solution
was diluted with CH Cl and extracted with aqueous NaHCO solution, followed by brine. The
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organic layer was dried over MgSO , filtered, and concentrated under reduced pressure. The crude
4
product was purified by flash column chromatography (1-10% CH OH/CH Cl ) to give the final
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product.
Compounds 4, 8, 10, 12, and 15 were synthesized using the general procedure of amide coupling.
Compounds 6 and 19 were synthesized using the general procedure of Suzuki-Miyaura cross
coupling.
Procedure for the synthesis of compound 5 through method 1. Sodium metal (10 mmol) was cut
into small pieces and was added to the alcohol solution. The solution was stirred at room
o
temperature under N until all the sodium metal was dissolved (heating to 60 C when necessary).
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2
‐chloro‐N‐[2‐(diethylamino)ethyl]quinoline‐4‐carboxamide (4) (1 mmol) was added in one portion
and the resulting solution was heated to reflux overnight. Reaction was quenched with H O and
2
excess of alcohol was removed under reduced pressure. The crude product was diluted with CH Cl
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2
and extracted with aqueous NaHCO solution, followed by brine. The organic layer was dried over
3
MgSO , filtered, and concentrated under reduced pressure. The crude product was purified by flash
4
column chromatography (1-10% CH OH/CH Cl ) to give the final product.
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2
Procedure for the synthesis of compound 5 through method 2. To a solution of alcohol (2 mmol)
in DMF was added NaH (3 mmol), and the mixture was stirred at room temperature for 1 hour. A
solution of 2‐chloro‐N‐[2‐(diethylamino)ethyl]quinoline‐4‐carboxamide (4) (1 mmol) in DMF was
o
added dropwise, and the resulting solution was stirred at 80 C for 4 hours. The reaction mixture
was quenched with aqueous NaHCO solution, and extracted with CH Cl . The organic layer was
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dried over MgSO , filtered, and concentrated under reduced pressure. The crude product was
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purified by flash column chromatography (1-10% CH OH/CH Cl ) to give the final product.
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Procedure for the synthesis of compound 14. To a solution of isatin (1 mmol) and acetophenone
(1 mmol) in ethanol was added the aqueous solution of KOH (10 mmol). The reaction mixture was
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heated to 125 C under microwave irradiation for 20 minutes. After cooling down to room
temperature, the reaction was quenched with HCl. The precipitate was collected by filtration,
washed sequentially with water, ethyl acetate, and dichloromethane. The crude product was purified
by flash column chromatography (10-20% CH OH/CH Cl ) to give the final product.
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Procedure for the synthesis of compound 17. A solution of 5-fluoroisatin (1 mmol) and malonic
acid (3 mmol) in acetic acid was refluxed for 16 hours. Acetic acid was removed under reduced
pressure, and the residue was diluted with water. Insoluble solid was collected by filtration and was
suspended in saturated NaHCO aqueous solution. The mixture was filtered and the filtrate was
3
acidified to pH 1 using concentrated HCl (12 N). Precipitate formed was filtered and washed with
water. The resulting solid was further purified by flash column chromatography (10-20%
CH OH/CH Cl ) to give the final product.
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General procedure for the synthesis of compound 18. To a solution of 6-fluoro-2-
hydroxyquinoline-4-carboxylic acid (17) (1 mmol) in CH Cl was added a few drops of DMF and
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thionyl chloride (4 mmol). The mixture was refluxed for 3 hours. Solvent was removed under
reduced pressure and the resulting acid chloride was dissolved in THF. Amine (3 mmol) was added
and the reaction mixture was stirred at room temperature overnight. Solvent was removed under
reduced pressure and the resulting residue was dissolved in CH Cl . The solution was extracted with
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aqueous NaHCO solution, followed by brine. The organic layer was dried over MgSO , filtered,
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and concentrated under reduced pressure. The crude product was purified by flash column
chromatography (1-10% CH OH/CH Cl ) to give the final product.
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‐chloro‐N‐[2‐(diethylamino)ethyl]quinoline‐4‐carboxamide (4). Yield: 82%. H NMR (400 MHz,
CD OD-d ) δ 8.18 (d, J = 8.9 Hz, 1H), 7.94 (d, J = 8.4 Hz, 1H), 7.79 (t, J = 7.7 Hz, 1H), 7.63 (t, J =
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.3 Hz, 1H), 7.58 (s, 1H), 3.62 (t, J = 6.9 Hz, 2H), 2.89 (t, J = 6.9 Hz, 2H), 2.78 (q, J = 7.2 Hz, 4H),
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.14 (t, J = 7.2 Hz, 6H). C NMR (101 MHz, CD OD) δ 168.19, 151.20, 149.28, 146.61, 132.35,
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129.29, 128.99, 126.69, 124.63, 121.29, 52.30, 48.20, 37.90, 11.18. C H ClN O, EI-MS: m/z
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(M+H ): 306.8 (calculated), 306.8 (found).
1
N‐[2‐(diethylamino)ethyl]‐2‐methoxyquinoline‐4‐carboxamide (5a). Yield: 72%. H NMR (400
MHz, CD OD-d ) δ 8.06 (dd, J = 8.4, 1.4 Hz, 1H), 7.85 (dd, J = 8.5, 1.5 Hz, 1H), 7.66 (ddd, J = 8.4,
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.9, 1.5 Hz, 1H), 7.43 (ddd, J = 8.3, 6.9, 1.3 Hz, 1H), 7.02 (s, 1H), 4.07 (s, 3H), 3.66 – 3.56 (m,
1
3
H), 2.90 (t, J = 7.0 Hz, 2H), 2.81 (q, J = 7.2 Hz, 4H), 1.16 (t, J = 7.2 Hz, 6H). C NMR (101
MHz, CD OD) δ 169.71, 163.13, 148.38, 146.29, 131.18, 128.50, 126.32, 125.79, 122.73, 112.16,
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+
5
4.12, 52.35, 48.32, 37.75, 11.13. C H N O , HRMS (ESI): m/z (M+H ): 302.1869 (calculated),
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02.1863 (found).
1
N‐[2‐(diethylamino)ethyl]‐2‐ethoxyquinoline‐4‐carboxamide (5b). Yield: 84%. H NMR (400 MHz,
DMSO-d ) δ 8.72 – 8.61 (m, 1H), 8.08 (dd, J = 8.3, 1.4 Hz, 1H), 7.87 – 7.74 (m, 1H), 7.68 (ddd, J =
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.3, 6.8, 1.5 Hz, 1H), 7.44 (ddd, J = 8.3, 6.8, 1.3 Hz, 1H), 6.99 (d, J = 11.4 Hz, 1H), 4.49 (q, J = 7.0
Hz, 2H), 3.46 – 3.32 (m, 2H), 2.75 – 2.52 (m, 6H), 1.39 (t, J = 7.0 Hz, 3H), 1.01 (t, J = 7.1 Hz, 6H).
13C NMR (101 MHz, DMSO) δ 166.03, 160.91, 146.40, 145.71, 130.05, 130.00, 127.08, 125.61,
1
25.58, 124.51, 124.41, 121.43, 110.87, 61.50, 53.37, 51.29, 46.57, 14.39, 11.62. C H N O ,
18 25 3 2
+
HRMS (ESI): m/z (M+H ): 316.2025 (calculated), 316.2020 (found).
1
N‐[2‐(diethylamino)ethyl]‐2‐(2,2,2‐trifluoroethoxy)quinoline‐4‐carboxamide (5c). Yield: 71%. H
NMR (400 MHz, DMSO-d ) δ 8.81 – 8.67 (m, 1H), 8.14 (ddd, J = 8.3, 1.5, 0.6 Hz, 1H), 7.86 (ddd,
6
J = 8.5, 1.4, 0.7 Hz, 1H), 7.75 (ddd, J = 8.4, 6.9, 1.5 Hz, 1H), 7.52 (ddd, J = 8.3, 6.9, 1.3 Hz, 1H),
7
.17 (s, 1H), 5.20 (q, J = 9.1 Hz, 2H), 3.53 – 3.33 (m, 2H), 2.76 – 2.52 (m, 6H), 1.01 (t, J = 7.1 Hz,
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H). C NMR (101 MHz, DMSO): δ 165.68, 158.94, 146.77, 145.60, 130.55, 127.19, 125.76,
125.41, 125.37, 122.65, 122.10, 110.08, 61.91, 61.56, 61.22, 60.87, 51.30, 46.57, 37.17, 11.62.
+
C H F N O , HRMS (ESI): m/z (M+H ): 370.1742 (calculated), 370.1737 (found).
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8
22
3
3
2
1
N‐[2‐(diethylamino)ethyl]‐2‐propoxyquinoline‐4‐carboxamide (5d). Yield: 87%. H NMR (400
0
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2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
MHz, DMSO-d ) δ 8.80 – 8.64 (m, 1H), 8.08 (dd, J = 8.3, 1.4 Hz, 1H), 7.79 (dd, J = 8.4, 1.2 Hz,
6
1
H), 7.68 (ddd, J = 8.4, 6.9, 1.5 Hz, 1H), 7.44 (ddd, J = 8.2, 6.8, 1.3 Hz, 1H), 7.02 (s, 1H), 4.39 (t, J
6.6 Hz, 2H), 3.51 – 3.35 (m, 2H), 2.90 – 2.52 (m, 6H), 1.79 (p, J = 7.1 Hz, 2H), 1.14 – 0.91 (m,
=
1
3
9H). C NMR (101 MHz, DMSO) δ 166.08, 161.08, 146.40, 145.54, 130.00, 127.08, 125.57,
1
24.42, 121.42, 110.94, 67.22, 51.08, 48.57, 46.56, 21.75, 11.30, 10.43. C H N O , HRMS (ESI):
1
9
27
3
2
+
m/z (M+H ): 330.2182 (calculated), 330.2176 (found).
N‐[2‐(diethylamino)ethyl]‐2‐(propan‐2‐yloxy)quinoline‐4‐carboxamide (5e). Yield: 80%. H NMR
1
(
400 MHz, CD OD-d ) δ 8.04 (dd, J = 8.4, 1.4 Hz, 1H), 7.81 (dd, J = 8.6, 1.2 Hz, 1H), 7.65 (ddd, J
3 4
=
8.4, 6.9, 1.4 Hz, 1H), 7.41 (ddd, J = 8.3, 6.9, 1.3 Hz, 1H), 6.96 (s, 1H), 5.55 (p, J = 6.2 Hz, 1H),
3
.59 (dd, J = 7.6, 6.4 Hz, 2H), 2.86 (dd, J = 7.7, 6.3 Hz, 2H), 2.77 (q, J = 7.2 Hz, 4H), 1.41 (d, J =
1
3
6.2 Hz, 6H), 1.15 (t, J = 7.2 Hz, 6H). C NMR (101 MHz, MeOD) δ 169.79, 162.31, 148.50,
1
46.28, 131.06, 128.49, 126.24, 125.57, 122.54, 112.77, 69.59, 52.39, 48.26, 37.88, 22.30, 11.28.
+
C H N O , HRMS (ESI): m/z (M+H ): 330.2182 (calculated), 330.2176 (found).
1
9
27
3
2
1
2‐(butan‐2‐yloxy)‐N‐[2‐(diethylamino)ethyl]quinoline‐4‐carboxamide (5f). Yield: 72%. H NMR
(
400 MHz, DMSO-d ) δ 8.85 – 8.67 (m, 1H), 8.06 (dd, J = 8.3, 1.4 Hz, 1H), 7.77 (dd, J = 8.5, 1.2
6
Hz, 1H), 7.67 (ddd, J = 8.4, 6.8, 1.5 Hz, 1H), 7.43 (ddd, J = 8.3, 6.9, 1.3 Hz, 1H), 6.99 (s, 1H), 5.35
(q, J = 6.2 Hz, 1H), 3.57 – 3.36 (m, 2H), 2.98 – 2.52 (m, 6H), 1.81 – 1.61 (m, 2H), 1.33 (d, J = 6.2
13
Hz, 3H), 1.06 (d, J = 7.5 Hz, 6H), 0.95 (t, J = 7.4 Hz, 3H). C NMR (101 MHz, DMSO) δ 166.16,
1
60.74, 146.42, 129.98, 127.06, 125.50, 124.33, 121.26, 111.41, 72.37, 46.54, 28.38, 19.17, 9.62.
+
C H N O , HRMS (ESI): m/z (M+H ): 344.2338 (calculated), 344.2333 (found).
20 29
3
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‐(cyclopropylmethoxy)‐N‐[2‐(diethylamino)ethyl]quinoline‐4‐carboxamide (5g). Yield: 71%. H
NMR (400 MHz, DMSO-d ) δ 8.79 – 8.64 (m, 1H), 8.08 (dd, J = 8.3, 1.4 Hz, 1H), 7.78 (dd, J = 8.4,
6
1
4
.2 Hz, 1H), 7.67 (ddd, J = 8.4, 6.8, 1.5 Hz, 1H), 7.44 (ddd, J = 8.3, 6.8, 1.3 Hz, 1H), 7.03 (s, 1H),
.28 (d, J = 7.2 Hz, 2H), 3.53 – 3.37 (m, 2H), 2.88 – 2.57 (m, 6H), 1.38 – 1.25 (m, 1H), 1.04 (t, J =
1
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2
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2
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2
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5
5
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5
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0
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2
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9
0
1
2
3
4
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7
8
9
0
1
2
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7
8
9
0
1
2
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3
7.1 Hz, 6H), 0.66 – 0.51 (m, 2H), 0.45 – 0.32 (m, 2H). C NMR (101 MHz, DMSO) δ 166.10,
1
9
61.00, 146.36, 130.02, 127.05, 125.56, 124.43, 121.40, 110.99, 70.30, 54.90, 51.03, 46.57, 11.17,
+
.96, 3.19. C H N O , HRMS (ESI): m/z (M+H ): 342.2182 (calculated), 342.2176 (found).
2
0
27
3
2
1
N‐[2‐(diethylamino)ethyl]‐2‐(pentan‐3‐yloxy)quinoline‐4‐carboxamide (5h). Yield: 77%. H NMR
(
400 MHz, DMSO-d ) δ 8.74 – 8.62 (m, 1H), 8.06 (dd, J = 8.3, 1.4 Hz, 1H), 7.75 (dd, J = 8.5, 1.2
6
Hz, 1H), 7.66 (ddd, J = 8.4, 6.8, 1.5 Hz, 1H), 7.42 (ddd, J = 8.2, 6.8, 1.3 Hz, 1H), 6.97 (s, 1H), 5.30
p, J = 6.0 Hz, 1H), 3.47 – 3.32 (m, 2H), 2.72 – 2.51 (m, 6H), 1.82 – 1.61 (m, 4H), 1.01 (t, J = 7.1
(
1
3
Hz, 6H), 0.92 (t, J = 7.4 Hz, 6H). C NMR (101 MHz, DMSO) δ 166.08, 162.24, 161.18, 146.36,
1
45.74, 129.93, 127.03, 125.51, 124.25, 121.33, 111.19, 76.67, 51.23, 46.57, 37.00, 35.74, 30.73,
+
2
5.67, 11.52, 9.46. C H N O , HRMS (ESI): m/z (M+H ): 358.2495 (calculated), 358.2489
21 31
3
2
(found).
1
2
‐(cyclopentyloxy)‐N‐[2‐(diethylamino)ethyl]quinoline‐4‐carboxamide (5i). Yield: 82%. H NMR
(
400 MHz, DMSO-d ) δ 8.75 – 8.60 (m, 1H), 8.08 (d, J = 8.2 Hz, 1H), 7.78 (d, J = 8.4 Hz, 1H),
6
7.67 (t, J = 7.7 Hz, 1H), 7.42 (t, J = 7.9 Hz, 1H), 6.94 (s, 1H), 5.65 – 5.54 (m, 1H), 3.49 – 3.31 (m,
2
7
H), 2.80 – 2.53 (m, 6H), 2.09 – 1.95 (m, 2H), 1.83 – 1.68 (m, 4H), 1.68 – 1.54 (m, 2H), 1.02 (t, J =
13
.2 Hz, 6H). C NMR (101 MHz, DMSO) δ 166.07, 160.68, 146.48, 145.48, 129.93, 127.16,
125.54, 124.32, 121.29, 111.30, 77.53, 54.89, 51.17, 46.57, 36.90, 32.36, 23.48, 11.43. C H N O ,
2
1
29
3
2
+
HRMS (ESI): m/z (M+H ): 356.2338 (calculated), 356.2333 (found).
N‐[2‐(diethylamino)ethyl]‐2‐(2‐ethoxyethoxy)quinoline‐4‐carboxamide (5j). Yield: 62%. H NMR
1
(
400 MHz, DMSO-d ) δ 8.78 – 8.64 (m, 1H), 8.09 (ddd, J = 7.4, 5.8, 1.4 Hz, 1H), 7.79 (dd, J = 8.4,
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.4 Hz, 1H), 7.68 (dddd, J = 8.4, 6.9, 3.2, 1.5 Hz, 1H), 7.44 (dddd, J = 8.3, 6.9, 4.0, 1.3 Hz, 1H),
7.02 (d, J = 18.9 Hz, 1H), 4.63 – 4.41 (m, 2H), 3.83 – 3.70 (m, 2H), 3.51 (q, J = 7.0 Hz, 2H), 3.47 –
13
3
.31 (m, 2H), 2.83 – 2.54 (m, 6H), 1.13 (t, J = 7.0 Hz, 3H), 1.03 (t, J = 7.1 Hz, 6H). C NMR (101
MHz, DMSO) δ 166.02, 160.86, 146.27, 145.66, 130.06, 127.09, 125.60, 124.53, 121.50, 110.92,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
68.03, 65.61, 65.13, 61.51, 51.13, 46.57, 15.09, 14.39. C H N O , HRMS (ESI): m/z (M+H ):
2
0
29
3
3
3
60.2287 (calculated), 360.2282 (found).
‐(benzyloxy)‐N‐[2‐(diethylamino)ethyl]quinoline‐4‐carboxamide (5k). Yield: 84%. H NMR (400
1
2
MHz, CD OD-d ) δ 8.07 (dd, J = 8.4, 1.4 Hz, 1H), 7.86 (dd, J = 8.5, 1.3 Hz, 1H), 7.66 (ddd, J = 8.4,
3
4
6
1
4
.9, 1.4 Hz, 1H), 7.56 – 7.47 (m, 2H), 7.47 – 7.41 (m, 1H), 7.41 – 7.33 (m, 2H), 7.33 – 7.24 (m,
H), 7.08 (s, 1H), 5.54 (s, 2H), 3.60 (t, J = 7.0 Hz, 2H), 2.90 (t, J = 7.0 Hz, 2H), 2.80 (q, J = 7.2 Hz,
1
3
H), 1.15 (t, J = 7.2 Hz, 6H). C NMR (101 MHz, MeOD) δ 169.71, 162.46, 148.30, 146.41,
138.44, 131.23, 129.43, 129.25, 128.95, 128.58, 126.31, 125.89, 122.84, 112.31, 68.92, 52.32,
+
4
8.36, 37.71, 11.07. C H N O , HRMS (ESI): m/z (M+H ): 378.2182 (calculated), 378.2176
2
3
27
3
2
(found).
1
N‐[2‐(diethylamino)ethyl]‐2‐(1H‐pyrazol‐5‐yl)quinoline‐4‐carboxamide (6a). Yield: 71%. H NMR
(400 MHz, CD OD-d ) δ 8.25 – 8.02 (m, 3H), 7.84 – 7.72 (m, 2H), 7.59 (ddd, J = 8.3, 6.9, 1.2 Hz,
3 4
1
H), 7.12 (d, J = 2.3 Hz, 1H), 3.60 (dd, J = 8.0, 6.3 Hz, 2H), 2.79 (dd, J = 7.9, 6.4 Hz, 2H), 2.68 (q,
1
3
J = 7.2 Hz, 4H), 1.12 (t, J = 7.2 Hz, 6H). C NMR (101 MHz, MeOD) δ 169.88, 149.49, 144.51,
1
31.44, 130.09, 128.27, 126.43, 125.09, 117.66, 105.41, 52.51, 48.14, 38.33, 11.68. C H N O,
1
9
23
5
+
HRMS (ESI): m/z (M+H ): 338.1981 (calculated), 338.1975 (found).
N‐[2‐(diethylamino)ethyl]‐2‐(1‐methyl‐1H‐pyrazol‐4‐yl)quinoline‐4‐carboxamide (6b). Yield: 78%.
1
H NMR (400 MHz, DMSO-d ) δ 8.81 – 8.71 (m, 1H), 8.52 (s, 1H), 8.20 (s, 1H), 8.14 (ddd, J = 8.4,
6
1
1
.5, 0.6 Hz, 1H), 7.97 (ddd, J = 8.5, 1.3, 0.6 Hz, 1H), 7.86 (s, 1H), 7.74 (ddd, J = 8.4, 6.8, 1.4 Hz,
H), 7.53 (ddd, J = 8.2, 6.8, 1.3 Hz, 1H), 3.94 (s, 3H), 3.52 – 3.42 (m, 2H), 2.82 – 2.70 (m, 2H),
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.70 – 2.53 (m, 4H), 1.04 (t, J = 7.1 Hz, 6H). C NMR (101 MHz, DMSO) δ 166.66, 151.51,
147.97, 143.17, 137.82, 130.62, 129.93, 128.64, 125.95, 125.50, 122.83, 122.68, 116.48, 51.29,
+
4
3
6.60, 38.83, 36.97, 11.45. C H N O, HRMS (ESI): m/z (M+H ): 352.2137 (calculated),
2
0
25
5
52.2132 (found).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
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2
2
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5
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0
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9
0
N‐[2‐(diethylamino)ethyl]‐2‐(1‐methyl‐1H‐pyrazol‐5‐yl)quinoline‐4‐carboxamide (6c). Yield: 65%.
1
H NMR (400 MHz, DMSO-d ) δ 8.87 – 8.79 (m, 1H), 8.24 (ddd, J = 8.4, 1.5, 0.6 Hz, 1H), 8.11
6
(
ddd, J = 8.4, 1.3, 0.6 Hz, 1H), 7.97 (s, 1H), 7.83 (ddd, J = 8.4, 6.9, 1.5 Hz, 1H), 7.66 (ddd, J = 8.3,
6.9, 1.3 Hz, 1H), 7.58 (d, J = 2.0 Hz, 1H), 7.10 (d, J = 2.0 Hz, 1H), 4.35 (s, 3H), 3.53 – 3.43 (m,
13
2
H), 2.80 – 2.70 (m, 2H), 2.70 – 2.54 (m, 4H), 1.04 (t, J = 7.1 Hz, 6H). C NMR (101 MHz,
DMSO) δ 166.24, 148.85, 147.21, 143.44, 139.95, 137.91, 130.39, 129.31, 127.42, 125.53, 122.95,
+
1
18.36, 108.21, 51.23, 46.60, 39.91, 37.00, 11.37. C H N O, HRMS (ESI): m/z (M+H ):
20 25
5
352.2137 (calculated), 352.2132 (found).
1
N‐[2‐(diethylamino)ethyl]‐2‐(thiophen‐2‐yl)quinoline‐4‐carboxamide (6d). Yield: 82%. H NMR
(
400 MHz, DMSO-d ) δ 9.00 – 8.88 (br s, 1H), 8.26 – 8.15 (m, 2H), 8.09 (d, J = 3.7 Hz, 1H), 8.06 –
6
7.94 (m, 1H), 7.87 – 7.72 (m, 2H), 7.62 – 7.55 (m, 1H), 7.27 – 7.21 (m, 1H), 3.70 – 3.46 (m, 2H),
13
3
1
.04 – 2.57 (m, 6H), 1.11 (t, J = 7.1 Hz, 6H). C NMR (101 MHz, DMSO) δ 166.51, 151.61,
47.64, 144.25, 143.00, 130.33, 130.02, 128.77, 128.59, 127.65, 126.72, 125.56, 123.31, 115.64,
+
50.81, 46.55, 36.25, 10.60. C H N OS, HRMS (ESI): m/z (M+H ): 354.1640 (calculated),
2
0
23
3
3
54.1629 (found).
1
2
‐(5‐chlorothiophen‐2‐yl)‐N‐[2‐(diethylamino)ethyl]quinoline‐4‐carboxamide (6e). Yield: 85%. H
NMR (400 MHz, CD OD-d ) δ 8.15 (ddd, J = 8.4, 1.4, 0.7 Hz, 1H), 8.09 – 7.95 (m, 2H), 7.75 (ddd,
3
4
J = 8.4, 6.9, 1.5 Hz, 1H), 7.70 (d, J = 4.0 Hz, 1H), 7.56 (ddd, J = 8.3, 6.9, 1.3 Hz, 1H), 7.05 (d, J =
.0 Hz, 1H), 3.70 (dd, J = 7.4, 6.4 Hz, 2H), 3.04 (t, J = 6.9 Hz, 2H), 2.93 (q, J = 7.2 Hz, 4H), 1.22
4
1
3
(
t, J = 7.2 Hz, 6H). C NMR (101 MHz, CD OD) δ 169.95, 152.46, 149.54, 144.76, 143.89,
3
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