Potent antiviral activity of novel multi-substituted 4-anilinoquin(az)olines

Article abstract of DOI:10.1016/j.bmcl.2020.127284

Screening a series of 4-anilinoquinolines and 4-anilinoquinazolines enabled identification of potent novel inhibitors of dengue virus (DENV). Preparation of focused 4-anilinoquinoline/quinazoline scaffold arrays led to the identification of a series of high potency 6-substituted bromine and iodine derivatives. The most potent compound 6-iodo-4-((3,4,5-trimethoxyphenyl)amino)quinoline-3-carbonitrile (47) inhibited DENV infection with an EC₅₀ = 79 nM. Crucially, these compounds showed very limited toxicity with CC₅₀ values >10 μM in almost all cases. This new promising series provides an anchor point for further development to optimize compound properties.

Full text of DOI:10.1016/j.bmcl.2020.127284

Journal Pre-proofs
Potent antiviral activity of novel multi-substituted 4-anilinoquin(az)olines
Sirle Saul, Szu-Yuan Pu, William J. Zuercher, Shirit Einav, Christopher R.M.
Asquith
PII:
S0960-894X(20)30394-2
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127284
BMCL 127284
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
1 May 2020
19 May 2020
20 May 2020
Please cite this article as: Saul, S., Pu, S-Y., Zuercher, W.J., Einav, S., Asquith, C.R.M., Potent antiviral activity
of novel multi-substituted 4-anilinoquin(az)olines, Bioorganic & Medicinal Chemistry Letters (2020), doi:
https://doi.org/10.1016/j.bmcl.2020.127284
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Potent antiviral activity of novel multi-substituted
4-anilinoquin(az)olines Sirle Saul, Szu-Yuan Pu,
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Leave this area blank for abstract info.
OMe
MeO
OMe
MeO
MeO
N
MeO
NH
N
S
NH
N
+
O
O
N
N
OMe
OMe
NC
I
63
Analogs
N
O
Erlotinib
2
47
: DENV EC50 = 0.079 µM
: DENV EC50 = 6.5 µM
: DENV EC50 = 2.8 µM

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Potent antiviral activity of novel multi-substituted 4-anilinoquin(az)olines
Sirle Saula, Szu-Yuan Pua, William J. Zuercherb,c, Shirit Einava,*, Christopher R. M. Asquithb,d,*
aDepartment of Medicine, Division of Infectious Diseases and Geographic Medicine, and Department of Microbiology and Immunology, Stanford University
School of Medicine, Stanford, CA 94305, USA
bStructural Genomics Consortium, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
c Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
dDepartment of Pharmacology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Screening a series of 4-anilinoquinolines and 4-anilinoquinazolines enabled identification of
potent novel inhibitors of dengue virus (DENV). Preparation of focused 4-
anilinoquinoline/quinazoline scaffold arrays led to the identification of a series of high potency 6-
substituted bromine and iodine derivatives. The most potent compound 6-iodo-4-((3,4,5-
trimethoxyphenyl)amino)quinoline-3-carbonitrile (47) inhibited DENV infection with an EC50=
79 nM. Crucially, these compounds showed very limited toxicity with CC50 values greater than
10 µM in almost all cases. This new promising series provides an anchor point for further
development to optimize compound properties.
Keywords:
Dengue Virus
Flavivirus
2020 Elsevier Ltd. All rights reserved.
4-anilinoquinoline
4-anilinoquinazoline
Antiviral
In the past five decades there has been a dramatic increase in
the global burden of the mosquito-borne dengue virus (DENV)
infection. The geographical range of dengue infections, including
those in the developed world, has been expanding due to climate
 Corresponding authors: e-mail: chris.asquith@unc.edu (C. R. M. Asquith),
seinav@stanford.edu (S. Einav)
N
O
F3C
change and rapid urbanization.1 Consequently, ~400 million
people are estimated to get infected with one or more of the 4
distinct DENV serotypes annually in over 128 endemic countries.2-
H
N
O
N
H
CF3
N
N
F
N
Cl
H
3
The majority of symptomatic individuals experience an
O
O
N
N
uncomplicated dengue fever infection. However, 5-20% progress
to a life-threatening disease, known as severe dengue, particularly
upon a secondary infection with a heterologous DENV serotype.4-5
The development of an effective dengue vaccine has been
hampered by the necessity to generate simultaneous protection
against the 4 distinct DENV serotypes.6-7 Moreover, since there are
no approved antiviral therapies currently available, the
management of dengue infections remains focused on the
treatment of symptoms, rather than the underlying disease.8 This
results in continued morbidity and mortality.
In recent years, there has been a flurry of activity to identify
novel DENV inhibitors,9-24 but none of these compounds have yet
entered clinical trials. These compounds include a number of
kinase inhibitor scaffolds, such as the oxindole (sunitinib,
JMX0395),9-10 azaindole (1)11 and isothiazolo[4,3-b]pyridine (2-
3)12-13 (Fig. 1).
H
H
JMX0395
DENV (Huh7): EC50 = 0.038 µM
Sunitinib
DENV (Huh7): EC50 = 0.51 µM
R=
OMe
N
R
N
MeO
S
N
MeO
H2N
MeO
MeO
N
2
O
Figure
MeO
1.
of
A
N
N
selection
H
3
2
3
= DENV (Huh7): EC50 = 2.8 µM
= DENV (Huh7): EC50 = 0.87 µM
1
= DENV (Huh7): EC50 = 0.72 µM
previously reported inhibitors and associated activities on DENV (the data is
all for serotype 2 for consistency). Huh7 = human hepatoma cells.
looked to the chemically tractable 4-anilinoquin(az)oline scaffold.
Our team recently reported erlotinib as a promising starting point
with activity against DENV replication (Fig. 2).9 There are a
number of other quin(az)oline based inhibitors that have shown
To identify new chemical starting points to inhibit DENV, we

potent anti-DENV activity in the low nanomolar range, including
RYL-634, 4 and 5 among others (Fig. 2).9,22-26 These results
focused our attention on exploring the possible anti-DENV
activity of the quin(az)oline scaffold.
Scheme 1. General synthetic procedure
We tested the compounds for antiviral activity in human
hepatoma (Huh7) cells infected with DENV2.38-41 Their effect on
overall infection was measured at 48 hours post-infection via
luciferase assays and the half-maximal effective concentration and
the 90% effective concentrations (EC50 and EC90 values,
respectively) were calculated. In parallel, we tested the effect of
these compounds on cell viability via an AlamarBlue assay in the
DENV-infected Huh7 cells and measured the half-maximal
cytotoxic concentration (CC50) values.42
F
O
NH
N
F
N
O
O
N
H
N
OMe
OMe
O
Erlotinib
RYL-634
We first screened a series of trimethoxyanilinoquinazolines (6-
24) (Tab. 1).32 The unsubstituted trimethoxyanilinoquinazoline
(6) showed a 2-fold increase in potency over erlotinib. However,
further simple substitutions with a 6-methyl (7), 6-fluoro (8) or
6,7-difluoro (9) showed no activity or toxicity. Interestingly, the
switch from 6-fluoro (9) to 6-chloro (10) led to a >10-fold increase
in activity and an almost 7-fold increase with respect to erlotinib.
Similar activity values were observed for compounds possessing
6-bromo (11) and 6-iodo (12). However, when there was an
increase in size and electronegativity to 6-trifluoromethyl (13) the
compound was inactive. Switching the halogen to the 7-postion
(14-18) led to net decrease in activity. The 7-fluoro (14) was
inactive and the 7-chloro (15) was equivalent to erlotinib and 6-
fold less potent than the 6-chloro counterpart (10). The 7-bromo
(16) and 7-iodo (17) were only 2- and 3-fold less potent
respectively. In contrast, the 7-trifluoromethyl (18) was more
active than 13, its regioisomer at the 6-position, with equivalent
activity to erlotinib. The 7-cyano (19) saw an increase in
DENV: EC50 = 6.5 µM
DENV: EC50 = 0.0070 µM
Cl
NH2
N
O
S
N
N
H2N
N
N
N
H
4
5
DENV: EC50 = 0.070 µM
DENV: EC50 = 0.0028 µM
Figure 2. Structures of quinazolines with anti-DENV activity.
To further explore antiviral quin(az)oline activity, we probed
the structure activity relationships (SAR) of the
quinoline/quinazoline by profiling several focused arrays of
compounds. We developed a series of hybrid molecules
combining structural features of erlotinib and 2 to expand the SAR
in the current literature and assess tractability of the scaffolds (Fig.
Table 1. Screening results of trimethoxy quinazolines on the DENV
OMe
MeO
3).
OMe
MeO
MeO
MeO
NH
N
N
R1
R2
NH
N
S
N
O
O
N
N
OMe
OMe
N
O
DENV Inhibition
Erlotinib
DENV (Huh7): EC50 = 6.5 µM
2
Cmpd
R1
R2
DENV (Huh7): EC50 = 2.8 µM
EC50a (µM) CC50b (µM)
6
H
Me
F
H
H
H
F
2.8
>10
>10
>10
1.0
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
5.8
7
OMe
MeO
MeO
8
R
9
F
N
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Cl
H
H
H
H
F
X
R
R
Figure
3.
Depiction
Br
0.97
1.0
N
6
63
-
I
Hybrid Scaffold
of a hybrid series of trimethoxyanilinoquin(az)olines developed by combining
CF3
H
>10
>10
6.1
erlotinib and 2.
We hence synthesized a series of compounds (6-64) to follow up
on the initial results of erlotinib and 2 to explore the 4-
anilinoquinazoline and 4-anilinoquinoline scaffolds through
nucleophilic aromatic displacement of 4-chloroquin(az)olines
(Sch. 1).27-36 We were able to access products in good to excellent
yields (34-83 %) consistent with previous reports for literature and
H
Cl
Br
I
H
2.0
H
2.9
H
CF3
CN
H
5.1
>10
>10
>10
>10
>10
>10
>10
H
1.4
novel compounds.27-37
OMe
CN
SO2Me
OMe
OMe
H
3.7
OMe
MeO
MeO
H
>10
>10
6.6
MeO
MeO
R3
N
N
H
Cl
R3
N
H
X
X
OMe
OMe
R1
R1
R2
R2
N
Ethanol,
2.7
Reflux, 18 h
6
64
-
X=CH, N, C-CN

a= infectivity assay in Huh7 cells mean average n=2; b= cytotoxicity in Huh7
cells mean average n=2
a= infectivity assay in Huh7 cells mean average n=2; b= cytotoxicity in Huh7
cells mean average n=2
potency to almost 5-fold more than erlotinib with an EC50=1.4 µM.
The 6-cyano (20) was 2-fold weaker, while the 6-methylsulfone
(21) was inactive as was the corresponding 6-methoxy substitution
(22). The 6,7-dimethoxy analog (23) had equivalent potency to
erlotinib. Interestingly the 7-methoxy analog (24) showed a 2-fold
increase in activity relative to erlotinib and 23.
The incorporation of a slightly larger substituent, a 6-tert-butyl
(34), led to an activity down to that of erlotinib. Switching to the
6-methoxy analog (35) recovered the potency loss with an almost
8-fold increase over erlotinib. Surprisingly, the 6,7-dimethoxy
analog (36) showed no activity (n=4 biological replicates) and
neither did the 7-methoxy substitution (37). The other 7-position
analogs (38-43) all demonstrated weaker activity. Variation of the
halogen (38-42) resulted in no clear trend with the 7-iodo (41) the
only compound with a 2-fold improvement over erlotinib. The 7-
cyano (43) yielded equipotent activity to 41 with an EC50=3.3 µM
with an increased ligand efficiency.
To increase the plane angle to near orthogonality, we switched
to the 3-cyanoquinoline (44-54) (Tab. 3).32 The initial result was
disappointing with the unsubstituted analog (44) having no
observable activity up to the highest compound concentrations
employed. Switching to the 6-chloro (45) yielded an analog with
activity just above micromolar (EC50=1.3 µM). However, the 6-
bromo (46) and 6-iodo (47) had activities equivalent to 29 and with
a more than 80-fold increase over erlotinib and 120-fold over the
unsubstituted analog. Switching to the 6-methylsulfone (48)
removed all activity. Some recovery of activity to EC50=3.3 µM
was possible switching to the 6-methoxy (49). However, the 6,7-
dimethoxy (50) showed a slight reduction in potency and the 7-
methoxy (51) was inactive. Results were similar for the 7-chloro
(52) and 7-bromo (53). Interestingly, there was some recovery
with the 7-iodo (54) with an EC50= 4.7 µM.
Even small changes to the central plane angle can have radical
effects on the scaffold’s biological properties. Consequently, we
hypothesized that switching from the quinazoline to the quinoline
core would more closely mimic the central plane angle present in
1 and lead to more potent activity. The change from quinoline to
quinazoline was expected increase the plane angle from nearly
planar to around 50-60 degrees.32 Our hypothesis proved to be
valid as the unsubstituted trimethoxyanilinoquinoline (25) was 3-
fold more potent than the corresponding quinoline (6) and 8-fold
more potent than erlotinib (Tab. 2). The 6-fluoro analog (26) had
equivalent potency to 25. The 6,7-difluoro (27) was also
equivalent but showed toxicity at higher concentrations (CC50= 5.5
µM). The 6-chloro analog (28) was equivalent to the 6-fluoro (26),
and the 6-bromo (29) exhibited a substantial increase in potency
(EC50=80 nM), with a more than 80-fold increase over erlotinib
and 10-fold over the unsubstituted analog (25). The 6-bromo
analog (29) demonstrated a local activity minimum as further
substitution to increase the halogen size to 6-iodo (30) and 6-
trifluoromethyl (31), along with the corresponding 6-cyano (32)
and methylsulfone (33) all showed potency in the range 0.5-1 µM.
Table 2. Screening results of SAR of trimethoxy quinolines and anti-DENV
Table 3. Screening results of SAR of trimethoxy 3-cyanoquinolines and anti-
activity.
DENV activity.
OMe
OMe
MeO
MeO
MeO
NH
N
MeO
NH
N
R1
R2
NC
R1
R2
DENV Inhibition
DENV Inhibition
Cmpd
R1
R2
Cmpd
R1
R2
EC50a (µM) CC50b (µM)
EC50a (µM) CC50b (µM)
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
H
F
H
H
0.82
0.52
1.0
>10
>10
5.5
44
45
46
47
48
49
50
51
52
53
54
H
Cl
H
H
>10
1.3
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
F
F
Br
H
0.082
0.079
>10
3.1
Cl
H
0.52
0.080
0.82
0.59
0.76
0.84
7.4
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
I
H
Br
H
SO2Me
OMe
OMe
H
H
I
H
H
CF3
CN
SO2Me
tBu
OMe
OMe
H
H
OMe
OMe
Cl
Br
I
5.5
H
>10
>10
>10
4.7
H
H
H
H
H
0.75
>10
>10
4.1
H
OMe
OMe
F
a= infectivity assay in Huh7 cells mean average n=2; b= cytotoxicity in Huh7
cells mean average n=2
H
We sought to further understand the observed SAR between the
quinazoline scaffold and the DENV inhibition profiles. This was
achieved by synthesizing a series of derivatives with different
electronic and steric profiles (55-63) (Tab. 4).28,36 First, the direct
6-bromo analog (55) of 29 with a 2-methyl substitution was >20-
fold more potent than erlotinib but with an unexpected increase in
toxicity (CC50=2.5 µM) with only a 10-fold selectivity index
H
Cl
Br
I
>10
8.4
H
H
3.6
H
CF3
CN
6.8
H
3.3

(EC50/CC50). Switching to the 6-methoxy (56), 6,7-dimethoxy
(57), 7-methoxy (58) all yielded analogs with limited or no activity
in addition to no toxicity. The 7-trifluoromethyl (59) was also
inactive. However, moving the trifluoromethyl to the 6-position
(60) yielded a compound that was 3-fold less potent than the 6-
bromo (55), but with no toxicity and 7-fold more potency than
erlotinib. Moving the methyl group to block the amino bridge (61)
removed all activity. Switching the trifluoromethyl for a simple
fluorine (62) decreased potency by over 2-fold. The 7-methyl
group (63) was weakly active and the 8-methyl substitution (64)
was inactive. These two results taken together suggest that the
quinoline nitrogen is an important contributor to activity.
control. Shown are representative experiments from at least two conducted,
each with 5 biological replicates; shown are means ± SD.
This body of work provides several exciting starting points for
further optimization. The mechanism of the observed antiviral
activity has yet to be defined and could be a combination of factors
including kinases and other proteins. Some of these compounds
are known to act as kinase inhibitors having been originally
prepared as inhibitors targeting the ATP-binding site of human
kinases
including
cyclin-G-associated
kinase
(GAK),
serine/threonine-protein kinase 10 (STK10) and STE20-like
serine/threonine-protein kinase (SLK).32 It is possible that these
kinases may be involved, but their ATP-binding sites may not be
directly involved. Alternatively, other undefined proteins may
participate in the mechanism of antiviral action. Indeed,
compounds having structural features expected to impede
interaction with a kinase hinge region (Tab. 4) also demonstrated
antiviral activity. It is also possible that the observed phenotypes
may originate from modulation of other, non-kinase ATP or non-
ATP binding proteins.43 Lastly, we cannot exclude a possibility
that these compounds target a viral protein.
Erlotinib and 2 have both previously been reported to inhibit
DENV replication.9 The synthetic combination of these two
molecules to form a series of trimethoxyquin(az)olines has defined
a new series of DENV inhibitors. Several key results act as a
signpost for further chemical optimization. The 6,7-dimethoxy-N-
(3,4,5-trimethoxyphenyl)quinolin-4-amine analog 23 had
equivalent potency to erlotinib showing the glycol ether side
chains are less important for activity. The 6-bromo-N-(3,4,5-
trimethoxyphenyl)quinolin-4-amine analog 29 demonstrated a
local minimum in the SAR, highlighting a medium sized group
was optimal in this position. The 6-bromo (46) and 6-iodo (47) 3-
cyanoquinoline compounds supported this with activities
equivalent to 29 and a more than 80-fold increase over erlotinib
and 120-fold over the unsubstituted analog (44). This initial series
of results are the first step in defining a medicinal chemistry
trajectory towards a series of optimized antiviral compounds with
potential to treat DENV and possibly other viral agents.
These results delineate the SAR of anti-DENV activity and the
4-anilinoquinoline/quinazoline scaffold. The 6-position bromine
and iodine analogs on the quinoline were by far the most potent
series of analogs (29, 46, 47 and 55) (Fig. 4).
Table 4. Screening results of SAR of the trimethoxy quinoline core and anti-
DENV activity.
OMe
MeO
R4
MeO
N
N
R1
R2
R3
R5
DENV
Inhibition
EC50 CC50
Cmpd
R1
R2
R3
R4
R5
a
b
(µM) (µM)
55
56
57
58
59
60
61
62
63
64
Br
OMe
OMe OMe
H
H
CF3
CF3
F
H
H
H
H
Me
Me
Me
Me
Me
Me
H
Me
Me
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
0.28
10
10
>10
>10
0.91
>10
2.3
2.5
>10
>10
>10
>10
>10
>10
>10
>10
>10
OMe
CF3
H
H
H
Acknowledgments
H
H
8.0
>10
This work was supported by award number W81XWH-16-1-0691
from the Department of Defense (DoD), Congressionally Directed
Medical Research Programs (CDMRP) and HDTRA11810039
from the Defense Threat Reduction Agency (DTRA)/Fundamental
Research to Counter Weapons of Mass Destruction to SE. The
SGC is a registered charity (number 1097737) that receives funds
from AbbVie, Bayer Pharma AG, Boehringer Ingelheim, Canada
Foundation for Innovation, Eshelman Institute for Innovation,
Genome Canada, Innovative Medicines Initiative (EU/EFPIA)
[ULTRA-DD grant no. 115766], Janssen, Merck KGaA Darmstadt
Germany, MSD, Novartis Pharma AG, Ontario Ministry of
Economic Development and Innovation, Pfizer, São Paulo
Research Foundation-FAPESP, Takeda, and Wellcome
[106169/ZZ14/Z]. We are grateful Dr. Brandie Ehrmann and Miss
Diane E. Wallace for LC-MS/HRMS support provided by the
Mass Spectrometry Core Laboratory at the University of North
Carolina at Chapel Hill. The opinions, interpretations,
conclusions, and recommendations are those of the authors and are
not necessarily endorsed by the U.S. Army or the other funders.
Me
a= infectivity assay in Huh7 cells mean average n=2; b= cytotoxicity in Huh7
cells mean average n=2
References and notes
Figure 4. Compounds 29, 46, 47 and 55 suppress DENV infection. Dose
response of DENV infection (black) and cell viability (blue) to compounds 29
(A), 46 (B), 47 (C) and 55 (D) measured by luciferase and alamarBlue assays,
respectively, 48 hours after infection. Data are plotted relative to vehicle
1.
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37. General procedure for the synthesis of 4-anilinoquin(az)olines:
4-chloroquin(az)oline derivative (1.0 eq.), aniline derivative (1.1
eq.), were suspended in ethanol (10 mL) and refluxed for 18 h. The
crude mixture was purified by flash chromatography using
EtOAc:hexane followed by 1-5 % methanol in EtOAc; After
solvent removal under reduced pressure, the product was obtained
as a free following solid or recrystallized from ethanol/water.
Compounds 6-54 were synthesized as previous described32 and 60-
61 as previously reported.28
6-bromo-2-methyl-N-(3,4,5-trimethoxyphenyl)quinolin-4-
amine (55) was obtained as a light yellow solid (149 mg, 0.370
mmol, 63%). m.p. >250 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.87
– 10.61 (m, 1H), 9.06 (d, J = 2.0 Hz, 1H), 8.28 – 7.87 (m, 2H), 6.82,
(s, 1H), 6.78 (s, 2H), 3.80 (s, 6H), 3.72 (s, 3H), 2.63 (s, 3H). 13C
NMR (100 MHz, DMSO-d6) δ 155.1, 153.6, 153.3, 137.6, 136.4,
136.1, 132.7 (s, 2C), 125.8, 122.1, 119.1, 117.6, 103.0 (s, 2C),
101.0, 60.2, 56.1 (s, 2C), 19.2. HRMS m/z [M+H]+ calcd for
C19H20BrN2O3: 403.0657, found 403.0662, LC tR = 4.00 min, > 98%
Purity.
6-methoxy-2-methyl-N-(3,4,5-
trimethoxyphenyl)quinolin-4-amine (56) was obtained as
a
1
yellow solid (213 mg, 0.601 mmol, 83%). m.p. 135-137 °C; H
NMR (400 MHz, DMSO-d6) δ 10.68 (s, 1H), 8.22 (d, J = 2.6 Hz,
1H), 8.05 (d, J = 9.2 Hz, 1H), 7.59 (dd, J = 9.2, 2.5 Hz, 1H), 6.80
(s, 2H), 6.74 (s, 1H), 3.98 (s, 3H), ), 3.81 (s, 6H), 3.72 (s, 3H), 2.61
(s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 157.5, 153.5, 153.4,
152.2, 136.2, 133.5, 133.1 (s, 2C), 124.7, 121.3, 117.3, 103.2 (s,
2C), 103.0, 100.1, 60.2, 56.5, 56.1 (s, 2C), 19.5. HRMS m/z
[M+H]+ calcd for C20H23N2O4: 355.1658, found 355.1655, LC tR =
19. Saudi, M.; Zmurko, J.; Kaptein, S.; Rozenski, J.; Neyts, J.; Van
Aerschot, A. Eur J Med Chem. 2014, 87, 529.
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Shi, P. Y.; Smith, P. W. J Med Chem. 2016, 59, 3935.
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Lou, Z.; Rao, Y. J Med Chem. 2019, 62, 4056.
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Jaber, D.; Schul, W.; Gu, F.; Heudi, O.; Ma, N. L.; Poh, M. K.;
Phong, W. Y.; Keller, T. H.; Jacoby, E.; Vasudevan, S. G.
Antimicrob Agents Chemother. 2009, 53, 1823.
25. Chao, B.; Tong, X. K.; Tang, W.; Li, D. W.; He, P. L.; Garcia, J.
M.; Zeng, L. M.; Gao, A. H.; Yang, L.; Li, J.; Nan, F. J.; Jacobs,
M.; Altmeyer, R.; Zuo, J. P.; Hu, Y. H. J Med Chem. 2012, 55,
3135.
3.84 min,
> 98% Purity. 6,7-dimethoxy-2-methyl-N-(3,4,5-
trimethoxyphenyl)quinolin-4-amine (57) was obtained as a light
yellow solid (187 mg, 0.483 mmol, 72%). m.p. >250 °C; 1H NMR
(400 MHz, DMSO-d6) δ 10.49 (s, 1H), 8.11 (s, 1H), 7.48 (s, 1H),
6.77 (s, 2H), 6.65 (s, 1H), 3.98 (s, 3H), 3.94 (s, 3H), 3.81 (s, 6H),
3.72 (s, 3H), 2.57 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 154.2,
153.5, 152.9, 151.2, 148.8, 136.1, 135.3, 133.2 (s, 2C), 110.2, 103.2
(s, 2C), 102.7, 99.6, 99.4, 60.2, 56.7, 56.1 (s, 2C), 56.0, 19.4. HRMS
m/z [M+H]+ calcd for C21H25N2O5: 385.1763, found 385.1762, LC
tR = 3.82 min, > 98% Purity. 7-methoxy-2-methyl-N-(3,4,5-
trimethoxyphenyl)quinolin-4-amine (58) was obtained as
a
colourless solid (194 mg, 0.504 mmol, 80%). m.p. >350 °C; 1H
NMR (400 MHz, DMSO-d6) δ 10.63 (s, 1H), 8.67 (d, J = 9.3 Hz,
1H), 7.48 (d, J = 2.5 Hz, 1H), 7.35 (dd, J = 9.3, 2.5 Hz, 1H), 6.77
(s, 2H), 6.65 (s, 1H), 3.94 (s, 3H), 3.80 (s, 6H), 3.72 (s, 3H), 2.59
(s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 162.8, 154.1, 153.9,
153.5 (s, 2C), 140.8, 136.3, 133.0, 125.3, 117.2, 110.3, 103.3 (s,
2C), 99.8, 99.4, 60.2, 56.1 (s, 2C), 55.9, 19.6. HRMS m/z [M+H]+
calcd for C20H23N2O4: 355.1658, found 355.1639, LC tR = 3.18 min,
26. Opsenica, I.; Burnett, J. C.; Gussio, R.; Opsenica, D.; Todorović,
N.; Lanteri, C. A.; Sciotti, R. J.; Gettayacamin, M.; Basilico, N.;
Taramelli, D.; Nuss, J. E.; Wanner, L.; Panchal, R. G.; Solaja, B.
A.; Bavari, S. J Med Chem. 2011, 54, 1157.
>
98%
trimethoxyphenyl)quinolin-4-amine (59) was obtained as
yellow solid (165 mg, 0.421 mmol, 69%). m.p. >250 °C; 1H NMR
Purity.
2-methyl-7-(trifluoromethyl)-N-(3,4,5-
a

(400 MHz, DMSO-d6) δ 11.01 (s, 1H), 9.01 (d, J = 8.8 Hz, 1H),
8.53 (d, J = 1.9 Hz, 1H), 8.06 (dd, J = 9.0, 1.8 Hz, 1H), 6.89 (s, 1H),
6.81 (s, 2H), 3.81 (s, 6H), 3.73 (s, 3H), 2.67 (s, 3H). 13C NMR (100
MHz, DMSO-d6) δ 155.1 (d, J = 212.2 Hz), 153.6 (s, 2C), 138.2,
136.5, 132.6, 132.5 (q, J = 32.2 Hz), 132.0, 125.7, 124.6, 122.0 –
121.6 (m), 118.4, 117.5 – 116.9 (m), 103.1 (s, 2C), 101.7, 60.2, 56.1
(s, 2C), 19.9. HRMS m/z [M+H]+ calcd for C20H20F3N2O3:
393.1426, found 393.1422, LC tR = 4.07 min, > 98% Purity. 6-
fluoro-2-methyl-N-(3,4,5-trimethoxyphenyl)quinolin-4-amine
(62) was obtained as a light yellow solid (179 mg, 0.523 mmol,
Supplementary Material
Supplementary material
Declaration of interests
☒ The authors declare that they have no known
competing financial interests or personal
relationships that could have appeared to influence
1
68%). m.p. >250 °C; H NMR (400 MHz, DMSO-d6) δ 10.72 (s,
1H), 8.71 (dd, J = 10.5, 2.7 Hz, 1H), 8.21 (dd, J = 9.3, 5.1 Hz, 1H),
7.92 (ddd, J = 9.3, 8.0, 2.7 Hz, 1H), 6.80 (d, J = 6.2 Hz, 3H), 3.80
(s, 6H), 3.72 (s, 3H), 2.64 (s, 3H). 13C NMR (100 MHz, DMSO-d6)
δ
159.5
N/a
(d,
=
J
244.9
Hz),
154.5, 153.9 (d, J = 3.8 Hz), 153.6 (s, 2C), 136.4, 135.6, 132.8,
122.8 (d, J = 17.2 Hz), 122.6, 117.2 (d, J = 9.5 Hz), 108.3 (d, J =
25.1 Hz), 103.1 (s, 2C), 100.4, 60.2, 56.1 (s, 2C), 19.7. HRMS m/z
[M+H]+ calcd for C19H20FN2O3: 343.1458, found 343.1456, LC tR
the work reported in this paper.
☐The authors declare the following financial
interests/personal relationships which may be
considered as potential competing interests:
=
3.67
min,
>
98%
Purity.
2-methyl-N-(3,4,5-
trimethoxyphenyl)quinolin-4-amine (63) was obtained as
a
colourless solid (200 mg, 0.616 mmol, 73%). m.p. >250 °C; 1H
NMR (400 MHz, DMSO-d6) δ 10.76 (s, 1H), 8.77 (dd, J = 8.6, 1.3
Hz, 1H), 8.11 (dd, J = 8.5, 1.2 Hz, 1H), 7.97 (ddd, J = 8.4, 7.0, 1.1
Hz, 1H), 7.72 (ddd, J = 8.3, 7.0, 1.2 Hz, 1H), 6.80 (s, 2H), 6.77 (s,
1H), 3.81 (s, 6H), 3.73 (s, 3H), 2.64 (s, 3H). 13C NMR (100 MHz,
DMSO-d6) δ 154.6, 154.4, 153.6 (s, 2C), 138.5, 136.4, 133.5, 132.9,
126.4, 123.4, 119.8, 116.1, 103.2 (s, 2C), 100.3, 60.2, 56.1 (s, 2C),
19.8. HRMS m/z [M+H]+ calcd for C19H21N2O3: 325.1552, found
325.1551, LC tR = 3.59 min, > 98% Purity. 8-methyl-N-(3,4,5-
trimethoxyphenyl)quinolin-4-amine (64) was obtained as
a
1
mustard solid (93.1 mg, 0.287 mmol, 34%). m.p. 130-132 °C; H
NMR (400 MHz, DMSO-d6) δ 13.45 (s, 1H), 10.77 (s, 1H), 8.56 (d,
J = 8.5 Hz, 1H), 8.39 (d, J = 6.9 Hz, 1H), 7.86 (d, J = 7.1 Hz, 1H),
7.68 (dd, J = 8.5, 7.1 Hz, 1H), 6.90 (d, J = 6.8 Hz, 1H), 6.80 (s, 2H),
3.75 (d, J = 31.4 Hz, 9H), 2.68 (s, 3H). 13C NMR (100 MHz,
DMSO-d6) δ 155.4, 155.2, 153.7 (s, 2C), 136.5, 134.3, 134.2, 133.0,
126.6, 121.1, 117.2, 112.9, 103.2 (s, 2C), 100.6, 60.2, 56.2 (2, 2C),
17.8. HRMS m/z [M+H]+ calcd for C19H21N2O3: 325.1552, found
325.1550, LC tR = 3.60 min, > 98% Purity.
38. Virus construct. DENV2 (New Guinea C strain)44,45 Renilla
reporter plasmid used for in vitro assays was a gift from Pei-Yong
Shi (The University of Texas Medical Branch)
39. Cells. Huh7 (Apath LLC) cells were grown in DMEM
(Mediatech) supplemented with 10% FBS (Omega Scientific),
nonessential amino acids, 1% L-glutamine, and 1% penicillin-
streptomycin (ThermoFisher Scientific) and maintained in a
humidified incubator with 5% CO2 at 37 °C.
40. Virus Production. DENV2 RNA was transcribed in vitro using
mMessage/mMachine (Ambion) kits. DENV was produced by
electroporating RNA into BHK-21 cells, harvesting supernatants on
day 10 and titering via standard plaque assays on BHK-21 cells. In
parallel, on day
2 post-electroporation, DENV-containing
supernatant was used to inoculate C6/36 cells to amplify the virus.
41. Infection assays. Huh7 cells were infected with DENV in
replicates (n = 5) at a multiplicity of infection (MOI) of 0.05.
Overall infection was measured at 48 hours using
a Renilla luciferase substrate.
42. Viability assays. Viability was assessed using AlamarBlue®
reagent (Invitrogen) assay according to manufacturer’s protocol.
Fluorescence was detected at 560 nm on InfiniteM1000 plate
reader.
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