Identification of 4-anilino-6-aminoquinazoline derivatives as potential MERS-CoV inhibitors

Article abstract of DOI:10.1016/j.bmcl.2020.127472

New therapies for treating coronaviruses are urgently needed. A series of 4-anilino-6-aminoquinazoline derivatives were synthesized and evaluated to show high anti-MERS-CoV activities. N⁴-(3-Chloro-4-fluorophenyl)-N⁶-(3-methoxybenzyl

Full text of DOI:10.1016/j.bmcl.2020.127472

Journal Pre-proofs
Identification of 4-Anilino-6-aminoquinazoline Derivatives as Potential
MERS-CoV Inhibitors
Jun Young Lee, Young Sup Shin, Jihye Lee, Sunoh Kwon, Young-hee Jin,
Min Seong Jang, Seungtaek Kim, Jong Hwan Song, Hyoung Rae Kim, Chul
Min Park
PII:
S0960-894X(20)30583-7
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127472
BMCL 127472
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
10 April 2020
27 July 2020
4 August 2020
Please cite this article as: Young Lee, J., Sup Shin, Y., Lee, J., Kwon, S., Jin, Y-h., Seong Jang, M., Kim, S.,
Hwan Song, J., Rae Kim, H., Min Park, C., Identification of 4-Anilino-6-aminoquinazoline Derivatives as
Potential MERS-CoV Inhibitors, Bioorganic & Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/
j.bmcl.2020.127472
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover
page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version
will undergo additional copyediting, typesetting and review before it is published in its final form, but we are
providing this version to give early visibility of the article. Please note that, during the production process, errors
may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier Ltd.

Identification of 4-Anilino-6-aminoquinazoline Derivatives as Potential MERS-CoV
Inhibitors
Jun Young Lee,a Young Sup Shin,a Jihye Lee,b Sunoh Kwon,a,c Young-hee Jin,a,d Min Seong
Jang,a,e Seungtaek Kim,b Jong Hwan Song,a Hyoung Rae Kim,a Chul Min Parka,*
a
Center for Convergent Research of Emerging Virus Infection (CEVI), Korea Research
Institute of Chemical Technology, 141 Gajeong-ro, Yuseong-gu, Daejeon 34114, South Korea
b Zoonotic Virus Laboratory, Institut Pasteur Korea, Seongnam-si, Gyeonggi-do 13488, South
Korea
c
Herbal Medicine Research Division, Korea Institute of Oriental Medicine, Daejeon 34054,
South Korea
d KM Application Center, Korea Institute of Oriental Medicine, Dong-gu, Daegu 41062, South
Korea
e
Department of Non-Clinical Studies, Korea Institute of Toxicology, Yuseong-gu, Daejeon
34114, South Korea
Abstract : New therapies for treating coronaviruses are urgently needed. A series of 4-
anilino-6-aminoquinazoline derivatives were synthesized and evaluated to show high anti-
MERS-CoV activities. N4-(3-Chloro-4-fluorophenyl)-N6-(3-methoxybenzyl)quinazoline-4,6-
diamine (1) has been identified in a random screen as a hit compound for inhibiting MERS-
CoV infection. Throughout optimization process, compound 20 was found to exhibit high
inhibitory effect (IC50 = 0.157 μM, SI = 25) with no cytotoxicity and moderate in vivo PK
properties.

Coronaviruses (CoVs) are a group of positive-sense, single-stranded RNA viruses that cause
severe respiratory diseases in a broad range of animal species, including humans.1-3 In 2003,
one of the novel coronaviruses, severe acute respiratory syndrome CoV (SARS-CoV), caused
a total of 8,422 cases of SARS with 916 deaths.4 The other novel coronavirus, Middle East
respiratory syndrome CoV (MERS-CoV), has emerged in April 2012 and posed a serious threat
to public health. As of 4 April 2020, a total of 2,494 human MERS-CoV infections with 858
deaths had been reported from 27 countries.5 Although MERS-CoV can cause primary
infections from direct contact with animal reservoirs like camels,6 person-to-person
transmission of this virus has mainly occurred in health-care facilities and family clusters.7-9
Recently outbreak of COVID-19, which is caused by the severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2), emerged in China and has spread to several other countries.
Drug repositioning for an FDA-approved compound library found that numerous compounds
inhibited MERS-CoV infection. However, there are still no approved drugs for coronaviruses.10
Through high content screening (HCS) platform of Institut Pasteur Korea (IPK) using the
Korea Chemical Bank (KCB),11 we found several novel compounds that can inhibit MERS-
CoV
infection.12
We
found
that
N4-(3-chloro-4-fluorophenyl)-N6-(3-
methoxybenzyl)quinazoline-4,6-diamine 1 was effective for inhibiting MERS-CoV infection.
Quinazoline derivatives have previously been reported as potent inhibitors of the protein kinase
of epidermal growth factor receptor (EGFR).13 Most of those compounds were approved as
anticancer drugs, including Gefitinib,14 Lapatinib,15 Erlotinib,16 and Afatinib17. We thought
that quinazoline compounds can exhibit good bioavailability and be easily extended to
treatment of MERS-CoV infection. Here we report on the synthesis and biological effects of
4-anilino-6-aminoquinazoline derivatives.
The general synthetic route for 4-aminoquinazoline derivatives is described in Scheme 1. 2-
Amino-5-nitrobenzonitrile was reacted with dimethylformamide-dimethyl acetal (DMF-DMA)
in toluene to give (E)-N’-(2-cyano-4-nitrophenyl)-N,N’-dimethylformimidamide 2. N,N-
o
dimethylformamidine 2 was treated with acetic acid and anilines at 120 C to produce 6-
nitroquinazoline 3.18 Reduction of the nitro group of 3 was carried out using iron powder and
o
NH4Cl in isopropyl alcohol and water at 100 C to afford 6-aminoquinazoline 4. Aromatic
amine 4 was reductive alkylated with aldehydes using NaBH(OAc)3 and trifluoroacetic acid in
isopropyl acetate at 100 oC to give N-substituted quinazolines 5.

F
HN
N
Cl
H
N
MeO
N
1
IC50 : 5.6 M
CC50 : > 25 M
SI : 4.5
Figure 1. Hit against MERS-CoV from a KCB library screen
Y
HN
N
O2N
X
CN
N
O2N
X
CN
O2N
X
a
b
N
N
NH2
2
3
Y
Y
R2
N
HN
HN
c
d
H2N
X
N
R1
N
N
X
N
4
5
Scheme 1. Synthesis of 6-substituted 4-anilinoquinazoline derivatives. Reagents and
conditions: (a) DMF-DMA, toluene, 110 oC, 5 h; (b) ArNH2, AcOH, 120 oC, 7 h; (c) Fe, NH4Cl,
i-PrOH/H2O (10/1), 110 oC/4 h; (d) Aldehyde, NaBH(OAc)3, trifluoroacetic acid, i-PrOAc, 90
oC, 3 h; Acyl chloride, Et3N, dichloromethane, rt; or carboxylic acid, EDCI, i-Pr2NEt, DMF,
rt.
The antiviral activities of the synthesized compounds in Vero cells were determined by
immunofluorescence assay (IFA). Vero cells were infected with a korean clinical MERS-CoV
isolates and the inhibitory concentration (IC50) and cytotoxic concentration (CC50) values of
the compounds were calculated by nonlinear regression analysis.11 Molecules with higher
selective index (SI) were considered as active molecules.

Our preliminary structure activity relationship (SAR) studies surrounding 1 were carried out
by introducing halide groups on phenyl ring of 4-anilino group (Table 1). 4-Bromo substituent
(6) maintained the potency. 4-Fluoro (7), 3-chloro (8), 4-cyano (9), and 3-acetyl (10) reduced
potency compared to 1. Interestingly, 4-trifluoromethyl (11) can slightly improve the potency.
In the next phase optimization, we observed the effects of the substituents of 6 position of
quinazoline ring, having fixed with 3-chloro-4-fluoro and 4-trifluoromethyl substituents on
phenyl ring at 4 position (Table 2).
Table 1. Antiviral effect and toxicity of 4-anilino groups of hit compound 1
Y
HN
H
N
MeO
N
N
Compound
Y
3-Cl,4-F
4-Br
IC50(μM)a
5.6
CC50(μM)b
>25
SIc
4.5
3.4
1.1
1.0
1.0
1.0
1.0
1
6
7.4
>25
7
4-F
22.7
>25
>25
8
3-Cl
23.0
9
4-CN
3-COCH3
4-CF3
>25
>25
10
11
>25
>25
1.8
>25
a,b IC50 and CC50 were derived from the results of at least two dependent experiment in Vero
cells infected with MERS-CoV.
c SI(selective index) = CC50/IC50 for inhibiting MERS-CoV infection.
First, substituent effects at 6 position of 1 having 3-chloro-4-fluoro aniline at 4 position were
evaluated. Changing 3-methoxybenzyl amine to 2-methoxybenzyl amine group 12 showed
similar activity (IC50 = 3.8 μM). Introducing 2-hydroxybenzyl amine at 6 position of
quinazoline ring 13 was tolerated (IC50 = 3.6 μM). 3,4-Difluoro (14) and 4-fluoro (15)
compounds gave similar activities (IC50 = 4.6 μM). We next explored the effects of electron-
withdrawing groups. Compounds with nitro groups (16 to 18) showed similar inhibitory effects
(IC50 = 3.3, 6.1 and 2.7 μM, respectively). Compound with 2-cyanobenzyl amine at 6 position

(19) showed no inhibitory effect, whereas 4-cyanoebnzyl amine (21) functionality was
tolerated (IC50 = 4.8 μM). Gratifyingly, 3-cyanobenzyl amine analogue 20 resulted in
significant higher activity (IC50 = 0.157 μM). We tested other electron-withdrawing groups at
meta position of benzyl amine substituent. 3-Amidobenzyl amine analogue 22 was detrimental
for activity but trifluoromethyl substituent at 3 position 23 retained the inhibitory effect (IC50
= 2.3 μM). Then benzyl amine substituents at 6 position were changed to aliphatic amines and
amides (24–28). Aliphatic amine substituents, such as di-n-butyl (24) and cyclohexyl (25),
showed little inhibitory effects and amide groups (26–28) were detrimental. We introduced
several substituents at 7 position of 20. Methoxy at 7 position gave a little decreased activity
(29, IC50 = 5.8 μM) but hydroxy (30) showed no inhibitory effects.
The antiviral effects of derivatives at 6 position of 11 with 4-trifluoromethyl aniline group at
4 position were also examined. Most of substituents were well tolerated. 3-Cyanobenzyl (32),
4-cyanobenzyl (33), 3-nitrobenzyl (34) and 4-nitrobenzyl (35) showed similar activities (IC50
= 3.3 to 4.1 μM) to 11. However, 2-cyanobenzyl (31) substituent exhibited decreased potency.
Table 2. Structure-Activity Relation of Derivatives of 1 and 11
CF3
F
R2
N
R2
N
HN
N
HN
Cl
R1
N
R1
N
X
X
N
12-31
32-36
Compound
X
H
H
H
H
H
H
H
R1
R2
IC50(μM)a
5.6
CC50(μM)b
SIc
4.5
7
1
3-CH3O-PhCH2
2-CH3O-PhCH2
2-OH-PhCH2
3,4-F2-PhCH2
4-F-PhCH2
H
H
H
H
H
H
H
>25
>25
>25
23.0
14.4
>25
>25
12
13
14
15
16
17
3.8
3.6
7
4.6
5
4.6
3
2-NO2-PhCH2
3-NO2-PhCH2
3.3
8
6.1
4

18
19
20
H
H
H
4-NO2-PhCH2
2-CN-PhCH2
3-CN-PhCH2
H
H
H
2.7
>25
>25
8
1
>25
25
0.157±0.002
3.59±1.1
21
22
23
24
H
H
H
H
4-CN-PhCH2
3-H2NCO-PhCH2
3-CF3-PhCH2
n-butyl
H
H
4.8
>25
2.3
>25
>25
9.2
5
1
4
1
H
n-
butyl
H
>25
>25
25
26
27
28
29
30
11
31
32
33
34
35
H
H
cyclohexyl
CH3CO
15
>25
>25
>25
5.8
>25
>25
>25
>25
13.2
>25
>25
>25
>25
>25
>25
>25
1
1
1
1
2
1
1
1
7
6
7
8
H
H
3-CN-PhCO
H
H
3-CN-trans-cynnamoyl
3-CN-PhCH2
3-CN-PhCH2
3-CH3O-PhCH2
2-CN-PhCH2
3-CN-PhCH2
4-CN-PhCH2
3-NO2-PhCH2
4-NO2-PhCH2
H
OMe
OH
H
H
H
>25
1.8
H
H
H
>25
3.6
H
H
H
H
4.1
H
H
3.7
H
H
3.3
a,b IC50 and CC50 were derived from the results of at least two dependent experiment in Vero
cells infected with MERS-CoV.
c SI(selective index) = CC50/IC50 for inhibiting MERS-CoV infection.
Considering activity and cytotoxicity, 20 was thought to be the best compounds for anti-
MERS drug and evaluated for its hERG, metabolic stability, cytotoxicity (Table 3). Our
optimized lead compound 20 was found to show no hERG binding and have good microsomal
stability in both mouse and human. Compound 20 showed no cytotoxicity toward Vero (not
infected with MERS-CoV) as well as toward HFL-1, L929 NIH 3T3 and CHO-K1 cell lines,
which shows that potential interaction between compound 20 and viruses might affect the cell
viability. The pharmacokinetic parameters of 20 were evaluated in rats by intravenous (i.v.)

and oral (p.o.) routes at 2 and 5 mg/kg, respectively (Table 4). 20 showed reasonable oral
bioavailability (21 %)
Table 3. Result of hERG, Microsomal stability, cytotoxicity of 20
MSa
Cytotoxicity (μM)b
hERG
(μM)
>50
Compound
ma
ha
Veroc
>100
HFL-1
>100
L929
>100
NIH 3T3
>100
CHO-K1
>100
20
60.7 56.4
a % original compound remained after 30 min incubation
b Cell information. Vero: African green monkey kidney cell line, HFL-1: human embryonic
lung cell line, L929: NCTC clone 929, mouse fibroblast cell line, NIH 3T3 : mouse embryonic
fibroblast cell line, CHO-K1 : Chinese hamster ovary cell line.
c Vero cell was not infected with MERS-CoV.
Table 4. In vivo pharmacokinetic profiles in rat of 20
Parameters*
I.V., 2 mg/kg
NA**
P.O., 5 mg/kg
Tmax (h)
2.0
0.2
NA
Cmax (μg/h)
T1/2 (h)
5.9
5.5
1.11
0.57
AUC (μg·h/mL)
CL (L/h/kg)
Vss (L/Kg)
Ft (%)
1.73
6.3
NA
NA
21
NA
*All results are the mean of experiments using three rats.
**NA : not applicable
In conclusion, we have synthesized a series of 4-anilino-6-aminoquinazoline derivatives and
most of the compounds showed anti-MERS-CoV activity in Vero cell. The best compound
among the derivatives was 20, which had the form of quinazoline with 3-Chloro-4-

fluoroaniline at 4 position and 3-cyanobenzyl amine at 6 position. Compound 20 showed high
anti-MERS-CoV activity (IC50 = 0.157 μM, SI = 25) with no cytotoxicity and moderate in vivo
PK property. Further studies on additional SAR and pharmacological investigation of these
compounds are currently underway.
Acknowledgements
The chemical library used in this study was kindly provided by Korea Chemical Bank
(http://www.chembank.org/) of Korea Research Institute of Chemical Technology. This work
was supported by a grant of National Research Council of Science & Technology (NST) by
the Korean government (MSIP) (No. CRC-16-01-KRICT).
Reference
1.
2.
3.
Woo PC, Lau SK, Huang Y, et al. Exp Biol Med. 2009;234:1117.
Chan JF, Li KS, To KK, et al. J Infect. 2012;65:477.
Chan JF, Lau SK, Woo PC. J Formos Med Assoc. 2013;112:372.
World Health Organization (WHO). Summary table of SARS cases by
4.
country,
1
November
2002
-
7
August
2003.
https://www.who.int/csr/sars/country/2003_08_15/en/ [online].
5.
coronavirus
cov/2020_04_04/en/[online].
World Health Organization (WHO). Middle East respiratory syndrome
(MERS-CoV). https://www.who.int/emergencies/mers-
6.
Azhar EI, El-Kafrawy SA, Farraj SA, et al. N Eng J Med. 2014;370:2499.
Zumla A, Chan JF, Azhar EI, et al. Nat Rev Drug Discov. 2016;15:327.
Assiri A, McGeer A, Perl TM, et al. N Eng J Med. 2013;369:407.
Memish ZA, Zumla AI, Al-Hakeem RF, et al. N Eng J Med. 2013;368:2487.
Liang R, Wang L, Zhang N, et al. Viruses 2018;10:721.
7.
8.
9.
10.

11.
12.
Cruz DJ, Bonotto RM, Gomes RG, et al. PLoS Negl Trop Dis. 2013;7:2471.
(a) Yoon JH, Lee J, Lee JY, et al. Bull Korean Chem Soc. 2019;40:906 (b)
Yoon JH, Lee JY, Lee J, et al. Bioorg Med Chem Lett. 2019; 29:126727.
13.
14.
15.
16.
17.
18.
Li H-Q, Li D-D, Lu X, et al. Bioorg Med Chem Lett. 2012;20:317.
Lynch TJ, Bell DW, Sordella R, et al. N Engl J Med. 2004;350:2129.
Wood ER, Truesdale AT, McDonald OB, et al. Cancer Res. 2004;64:6652.
Raymond E, Faivre S, Armand JP. Drugs 2000;60:15.
Vavalà T. Clin Pharmacol. 2017;9:147.
Chandregowda V, Rao GV, Reddy GC. Org Proc Res Dev. 2007;11:813.
Declaration of interests
■ The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
Chul Min Park
19.
F
HN
N
Cl
H
N
MeO
N
1
IC50 : 5.6 M
CC50 : > 25 M
SI : 4.5

Figure 1. Hit against MERS-CoV from a KCB library screen

Y
HN
N
O2N
X
CN
N
O2N
X
CN
O2N
X
a
b
N
N
NH2
2
3
Y
Y
R2
N
HN
HN
c
d
H2N
X
N
R1
N
N
X
N
4
5
Scheme 1. Synthesis of 6-substituted 4-anilinoquinazoline derivatives. Reagents and
conditions: (a) DMF-DMA, toluene, 110 oC, 5 h; (b) ArNH2, AcOH, 120 oC, 7 h; (c) Fe, NH4Cl,
i-PrOH/H2O (10/1), 110 oC/4 h; (d) Aldehyde, NaBH(OAc)3, trifluoroacetic acid, i-PrOAc, 90
oC, 3 h; Acyl chloride, Et3N, dichloromethane, rt; or carboxylic acid, EDCI, i-Pr2NEt, DMF,
rt.
20.
F
HN
N
Cl
H
N
NC
N
20
IC50 = 0.157 M
SI = 25
21.
Table 1. Antiviral effect and toxicity of 4-anilino groups of hit compound 1
Y
HN
H
N
MeO
N
N
Compound
Y
IC50(μM)a
CC50(μM)b
SIc

1
6
3-Cl,4-F
4-Br
5.6
7.4
>25
>25
>25
23.0
>25
>25
>25
4.5
3.4
1.1
1.0
1.0
1.0
1.0
7
4-F
22.7
>25
>25
>25
1.8
8
3-Cl
9
4-CN
10
11
3-COCH3
4-CF3
a,b IC50 and CC50 were derived from the results of at least two dependent experiment in Vero
cells infected with MERS-CoV.
c SI(selective index) = CC50/IC50 for inhibiting MERS-CoV infection.
Table 2. Structure-Activity Relation of Derivatives of 1 and 11
CF3
F
R2
N
R2
N
HN
N
HN
Cl
R1
N
R1
N
X
X
N
12-31
32-36

Compound
X
H
H
H
H
H
H
H
H
H
H
R1
R2
H
H
H
H
H
H
H
H
H
H
IC50(μM)a
5.6
CC50(μM)b
>25
SIc
4.5
7
1
3-CH3O-PhCH2
2-CH3O-PhCH2
2-OH-PhCH2
3,4-F2-PhCH2
4-F-PhCH2
12
13
14
15
16
17
18
19
20
3.8
>25
3.6
>25
7
4.6
23.0
14.4
>25
5
4.6
3
2-NO2-PhCH2
3-NO2-PhCH2
4-NO2-PhCH2
2-CN-PhCH2
3-CN-PhCH2
3.3
8
6.1
>25
4
2.7
>25
8
>25
>25
1
25
0.157±0.002
3.59±1.1
21
22
23
24
H
H
H
H
4-CN-PhCH2
3-H2NCO-PhCH2
3-CF3-PhCH2
n-butyl
H
H
4.8
>25
2.3
>25
>25
9.2
5
1
4
1
H
n-
butyl
H
>25
>25
25
26
27
28
29
30
11
31
32
33
34
35
H
H
cyclohexyl
CH3CO
15
>25
>25
>25
5.8
>25
>25
>25
>25
13.2
>25
>25
>25
>25
>25
>25
>25
1
1
1
1
2
1
1
1
7
6
7
8
H
H
3-CN-PhCO
H
H
3-CN-trans-cynnamoyl
3-CN-PhCH2
3-CN-PhCH2
3-CH3O-PhCH2
2-CN-PhCH2
3-CN-PhCH2
4-CN-PhCH2
3-NO2-PhCH2
4-NO2-PhCH2
H
OMe
OH
H
H
H
>25
1.8
H
H
H
>25
3.6
H
H
H
H
4.1
H
H
3.7
H
H
3.3
a,b IC50 and CC50 were derived from the results of at least two dependent experiment in Vero
cells infected with MERS-CoV.
c SI(selective index) = CC50/IC50 for inhibiting MERS-CoV infection.

Table 3. Result of hERG, Microsomal stability, cytotoxicity of 20
MSa
Cytotoxicity (μM)b
hERG
(μM)
>50
Compound
ma
ha
Veroc
>100
HFL-1
>100
L929
>100
NIH 3T3
>100
CHO-K1
>100
20
60.7 56.4
a % original compound remained after 30 min incubation

b Cell information. Vero: African green monkey kidney cell line, HFL-1: human embryonic
lung cell line, L929: NCTC clone 929, mouse fibroblast cell line, NIH 3T3 : mouse embryonic
fibroblast cell line, CHO-K1 : Chinese hamster ovary cell line.
c Vero cell was not infected with MERS-CoV.
Table 4. In vivo pharmacokinetic profiles in rat of 20
Parameters*
I.V., 2 mg/kg
NA**
P.O., 5 mg/kg
Tmax (h)
2.0
0.2
NA
Cmax (μg/h)
T1/2 (h)
5.9
5.5

1.11
0.57
AUC (μg·h/mL)
CL (L/h/kg)
Vss (L/Kg)
Ft (%)
1.73
6.3
NA
NA
21
NA
*All results are the mean of experiments using three rats.
**NA : not applicable
22.