Synthesis and biological evaluation of quinolinone compounds as SARS CoV 3CLpro inhibitors

Article abstract of DOI:10.1002/cjoc.201300392

SARS CoV 3CL^pro is known to be a promising target for development of therapeutic agents against the severe acute respiratory syndrome (SARS). A quinolinone compound 1 was selected via virtual screening, and it was synthetized and tested for enz

Full text of DOI:10.1002/cjoc.201300392

FULL PAPER
DOI: 10.1002/cjoc.201300392
Synthesis and Biological Evaluation of Quinolinone
Compounds as SARS CoV 3CLpro Inhibitors
Yuanpei Sun,b Ning Zhang,d Jian Wang,c Yu Guo,b
Bo Sun,d Wei Liu,*,a Honggang Zhou,b and Cheng Yangb,d
a College of Sciences, Tianjin University of Science and Technology, Tianjin 300457, China
b College of Pharmacy, Nankai University, Tianjin 300071, China
c College of Life Sciences, Nankai University, Tianjin 300071, China
d High Throughput Molecular Drug Discovery Center, Tianjin International Joint Academy of Biotechnology and
Medicine, TEDA, Tianjin 300457, China
SARS CoV 3CLpro is known to be a promising target for development of therapeutic agents against the severe
acute respiratory syndrome (SARS). A quinolinone compound 1 was selected via virtual screening, and it was syn-
thetized and tested for enzymatic inhibition in vitro. Compound 1 showed potent inhibitory activity (IC50＝0.44
μmol/L) toward SARS CoV 3CLpro. Further work on a series of quinolinone derivatives resulted in the discovery of
the most potent compound 23, inhibiting SARS CoV 3CLpro with an IC50 of 36.86 nmol/L. The structure-activity
relationships were also discussed.
Keywords SARS, SARS CoV 3CLpro, inhibitors, quinolinone
Introduction
ketone group with effective inhibitory activity. Zhou
et al.[11] have reported 1-alkylisatin-5-carboxamide (IC50
＝0.37 μmol/L, Figure 1) and its analogues to be highly
potent SARS CoV 3CLpro inhibitors.
Severe acute respiratory syndrome (SARS), caused
by a novel coronavirus (SARS CoV), is a highly infec-
tive upper respiratory tract disease.[1-4] Due to the high
mortality rate (up to 10%) SARS quickly became a
global threat in 2003.[5,6] Although about a year later
from its first discovery the initial outbreak of the virus
was stymied, the reemergence of SARS is quite possible
and the development of new anti-SARS drugs is par-
ticularly important.[7,8]
O
O
H2N
O
N
The SARS CoV is a positive-sense single-stranded
RNA virus. Its genome is comprised of about 29700
nucleotides, most of them (over 21000 nucleotides) be-
longs to the replicase gene, which encodes two overlap-
ping polyproteins, pp1a (486 kDa) and pp1ab (790 kDa).
The SARS CoV 3CLpro, a ~33 kDa cysteine protease,
releases functional polypeptides from pp1a and pp1ab.
Without these polypeptides the viral replication and
transcription will be disabled. As the integral role of
SARS CoV 3CLpro in SARS CoV lifecycle, it is an ideal
target for drug discovery.[9-12]
1-Alkylisatin-5-carboxamide
O
NH
O
O
O
H
N
H
O
N
N
H
H
O
O
TG-0205221
Figure 1 1-Alkylisatin-5-carboxamide and TG-0205221.
Previously, a large number of SARS CoV 3CLpro in-
hibitors have been reported and their scaffolds are di-
verse. Yang et al.[6] developed a potent SARS CoV 3CL
protease inhibitor (TG-0205221, Ki＝50 nmol/L, Figure
1). Sydnes et al.[9] synthetized a series of glutamic acid
and glutamine peptides possessing a trifluoromethyl
We identified novel SARS CoV 3CLpro inhibitors
among a database containing more than 600000 com-
pounds by virtual screening. Compound 1 (Figure 2)
was selected, synthetized and tested against SARS CoV
3CLpro in vitro. 1 showed potent inhibitory activity (IC50
*
E-mail: liuwei2006@tust.edu.cn; Tel.: 0086-022-60600812
Received May 10, 2013; accepted June 9, 2013; published online July 19, 2013.
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cjoc.201300392 or from the author.
Chin. J. Chem. 2013, 31, 1199—1206
© 2013 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Sun et al.
FULL PAPER
＝0.44 μmol/L) toward SARS CoV 3CLpro.
Results and Discussion
23 compounds were synthesized and tested against
SARS CoV 3CLpro in vitro. Biological evaluation results
of the title compounds are listed in Table 1.
O
S
O
As shown in Table 1, replacement of the thiophene
moiety (compound 1) with benzene ring (compound 2)
leads to the decrease of the inhibitory activity, espe-
cially when the benzene ring was substituted. It is worth
N
O
H
Figure 2 Structure of compound 1.
Table 1 Inhibition activities of quinolinone compounds against SARS CoV 3CLpro
O
O
R2
O
N
R1
No. R1
R2
Inhibition or IC50
No. R1
R2
Inhibition or IC50
68.06%
S
H
H
H
0.437±0.03 μmol/L
H
H
H
1
2
3
13
14
15
O
0.66±0.10 μmol/L
52.20%
39.83%
11.19%
H
H
H
H
H
H
H
62.63%
38.15%
H
H
43.62%
39.37%
4
5
16
17
18
19
20
21
22
O
31.03%
H
50.97%
6
O
44.47%
H
CH2CH3
32.22±4.65 μM
41.70±10.35 μmol/L
58.49±11.60 μmol/L
158.7±4.95 nmol/L
Chin. J. Chem. 2013, 31, 1199—1206
7
Cl
83.70%
H
CH2CH2COOCH3
8
N
3.29±0.49 μmol/L
54.20%
H
OCH2CH(CH3)2
9
Cl
O
O
CH3
10
1200
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© 2013 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Synthesis and Biological Evaluation of Quinolinone Compounds as SARS CoV 3CLpro Inhibitors
Continued
Inhibition or IC50
No. R1
R2
Inhibition or IC50
No. R1
R2
O
S
H
H
1.41±0.12 μmol/L
CH3
36.86±1.50 nmol/L
11
12
23
O
4.45±0.76 μmol/L
noting that the introduction of substituent at meta-posi-
tion or ortho-position of benzene ester carbonyl shows
more inhibition potency than para-position. For in-
stance, the IC50 of compounds 11 and 12 are 1.41 and
4.45 μmol/L respectively, significantly more powerful
than compound 13 (68.06% inhibition at 1 mmol/L).
Meanwhile, to extend the distance between the benzene
ring and the ester carbonyl carbon, compounds 14－18
were prepared. Biological evaluation results showed
these compounds were mostly inactive against SARS
CoV 3CLpro compared with compound 2. Compounds
19－21 with a carbon chain instead of benzene ring
were partly kept the inhibitory potency, got an IC50 from
32.22 to 58.49 μmol/L. Dramatically, compound 23
(IC50＝36.86 nmol/L), methylation derivatives of the
N－H group of the quinolinone of compound 1, exhib-
ited approximately 12-fold higher potency relative to
compound 1 (IC50＝0.44 μmol/L). As well as compound
22, it was about 9-fold higher potency than compound
11.
ester carbonyl oxygen forms a hydrogen bond with the
NH of Gly 143 (2.26 Å). To compound 23, the hydro-
Figure 3 The docking model of compound 1 (grey) and 23
(blue) in the active site of SARS CoV 3CLpro
.
The nitrogen, oxygen, and sulfur atoms are colored
blue, red, and orange, respectively. Hydrogen bonds are
displayed as blue dashed lines. Figure 3 showed dock-
ing model of compounds 1 and 23, from which we can
conclude that compound 1 binds competitively and
strongly to the active site, and the quinolinone portion
was recognized by S1 pocket. Moreover, the thiophene
moiety extends into S1’ site with hydrophobic interac-
tions. The S1’ pocket is not quite large, this may explain
why compounds 14 and 15 , with long carbon chain
between the aromatic ring and ester carbonyl, show
lower potency than compound 2. Unlike compound 1,
methylation of the N-H of the quinolinone brings a dif-
ferent binding interaction, the thiophene moiety of
compound 23 occupies S2 pocket, and the methyl group
fills into the crack of S1 pocket closely. As S1’ pocket
is much nearer the surface of the protein than S2 pocket,
the new binding model makes compound 23 bind more
tightly than compound 1 with the protein. This may
partly explain why compound 23 showed more potent
inhibitory effect compared with compound 1. Molecular
insight into the two different binding modes of com-
pound 1 and 23 was shown in Figure 4, the oxygen at
C-2 of quinolinone moiety of compound 1 makes a
H-bond with the NH of His 163 (1.78 Å), and the NH of
quinolinone forms a H-bond with Glu 166 (2.26 Å). The
Figure 4 Simulated binding models of compound 1 (A) and 23
(B) in SARS CoV 3CLpro
.
Chin. J. Chem. 2013, 31, 1199—1206
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Sun et al.
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gen bond of the amide carbonyl oxygen with the NH of
His 163 (2.36 Å) was also conserved as well. But there
is no hydrogen bond with Glu 166, because of the meth-
ylation. The ester carbonyl oxygen of compound 23
makes hydrogen bonds with the active site residues, His
41 (3.36 Å) and Cys 145 (2.23 Å) . This is the most dif-
ference between the two binding modes. The active site
of SARS CoV 3CLpro contains a catalytic dyad consti-
tuted by Cys 145 and His 41, which functions as the
common nucleophile in the proteolytic process.[11] That
means that although the methylation disturbed the hy-
drogen bond of N－H of quinolinone, the distance be-
tween ester carbonyl oxygen and the S－H group of Cys
145 and N－H group of His 41 might be changed fa-
vorably resulting in dramatic inhibitory potency. In ad-
dition, the carbonyl of compound 23 may potentially
react with the Cys 145 residue. This suppose remains to
be further studied.
Scheme 1 General procedure for the synthesis of target com-
pounds 1－23
O
O
OH
O
R2
O
R2
Cl
CH2Cl2
Pyridine
N
R1
O
N
R1
1 － 23
4-hydroxy-1-methyl-2-quinolinone (1 mmol) in 25 mL
of anhydrous CH2Cl2, pyridine (1.2 mmol) was added,
then the mixture was stirred at room temperature. Cor-
responding acyl chloride (1.2 mmol) dissolved in 10 mL
of anhydrous CH2Cl2 was added dropwise over 25 min.
The reaction mixture was stirred for a further 5－8 h,
then washed several times with diluted hydrochloric
acid in a separatory funnel. The organic phase was dried
over anhydrous MgSO4 and the solvent removed by
evaporation. Product was purified by recrystallization
with CH2Cl2 and ethyl acetate.
Conclusions
In conclusion, twenty-three quinolinone compounds
were synthesized and tested in vitro as inhibitors against
SARS CoV 3CLpro. Compound 1, selected via virtual
screening, was proved to have a potent inhibitory activ-
ity (IC50 ＝0.44 μmol/L). Following optimization of
4-quinolinone ester derivatives resulted in the discovery
of compound 23 with the most powerful potency (IC50
＝36.86 nmol/L). The present work demonstrated that
4-quinolinone ester analogs could be used as new leads
for future Anti-SARS drugs discovery. Further struc-
tural optimization and in vivo activities about quinoli-
none compounds are well under way.
4-(Thiophene-2-carbonyl)oxy-quinol-2-one
(1)
Compound 1 was prepared according to general proce-
dure by using 4-hydroxy-2-quinolinone and 2-thio-
phenecarbonyl chloride, obtained a white solid in 93%
yield. m.p. 234 － 235 ℃ . 1H NMR (400 MHz,
DMSO-d6) δ: 6.62 (s, 1H), 7.23 (t, J＝8.0 Hz, 1H), 7.41
－7.36 (m, 2H), 7.64－7.58 (m, 2H), 8.20－8.16 (m,
2H), 11.97 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ:
113.0, 115.1, 116.1, 122.5, 122.7, 129.5, 131.1, 132.1,
136.7, 136.9, 139.4, 155.9, 159.1, 162.6; IR (KBr) ν:
3436, 1737, 1670, 1256, 1103, 733 cm−1; ESI-MS m/z:
272.02 ([M＋H＋]). Anal. calcd for C14H9NO3S: C 61.98,
H 3.34, N 5.16; found C 62.10, H 3.35, N 5.43.
Experimental
4-Benzoyloxy-quinol-2-one (2) Compound 2 was
prepared according to general procedure by using
4-hydroxy-2-quinolinone and benzoyl chloride, ob-
tained a white solid in 94% yield. m.p. 214－215 ℃.
1H NMR (400 MHz, DMSO-d6) δ: 6.61 (s, 1H), 7.22 (t,
J＝7.6 Hz, 1H), 7.41 (d, J＝8.4 Hz, 1H), 7.69－7.58 (m,
4H), 7.82 (t, J＝7.6 Hz, 1H), 8.23 (d, J＝8.4 Hz, 2H),
11.97 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ: 113.1,
115.3, 116.1, 122.7, 128.4, 129.7, 130.6, 132.0, 135.1,
139.4, 156.5, 162.7, 163.8; IR (KBr) ν: 3462, 1740,
1671, 1251, 1093, 749 cm−1; ESI-MS m/z: 266.09 ([M
＋H＋]). Anal. calcd for C16H11NO3: C 72.45, H 4.18, N
5.28; found C 72.74, H 4.21, N 5.31.
Materials and instruments
Unless otherwise noted, all chemicals and solvents
were commercially available and treated with standard
methods before use. Melting points were determined
using an SGW-X4B digital melting point apparatus and
uncorrected. SHANGHAI SANPONT GF254 plates
were used as analytical TLC. H and 13C NMR spectra
1
were recorded on a 400 MHz Bruker Avance DPX
spectrometers and using DMSO as solvent. All 13C
NMR spectra were recorded proton-decoupled. Chemi-
1
cal shifts for H and 13C spectra are quoted in ppm
downfield from TMS. Coupling constants are referred to
as J values in hertz. ESI mass spectra were acquired
using a Bruker ESQUIRELCTM ESI ion trap spec-
trometer. FT-IR spectra were recorded at room tem-
perature in the region of 4000―400 cm−1 with a Perkin-
Elmer spectrum 65 FT-IR spectrometer using KBr pel-
lets. Elemental analyses of carbon, hydrgen and nitro-
gen were obtained with an Elementar Vario MICRO
cube Elemental Analyser.
4-(4-Methylbenzoyl)oxy-quinol-2-one (3) Com-
pound 3 was prepared according to general procedure
by using 4-hydroxy-2-quinolinone and 4-methylbenzoyl
chloride, obtained a white solid in 90% yield. m.p. 239
1
－241 ℃. H NMR (400 MHz, DMSO-d6) δ: 2.46 (s,
3H), 6.59 (s, 1H), 7.21 (t, J＝7.6 Hz, 1H), 7.40 (d, J＝
8.0 Hz, 1H), 7.47 (d, J＝8.0 Hz, 2H), 7.59 (t, J＝7.6 Hz,
2H), 8.12 (d, J＝8.0 Hz, 2H), 11.95 (s, 1H); 13C NMR
(100 MHz, DMSO-d6) δ: 21.8, 113.0, 115.3, 116.1,
122.6, 122.7, 125.6, 130.2, 130.7, 132.0, 139.4, 139.8,
General procedure
To a suspension of 4-hydroxy-2-quinolinone or
1202
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Synthesis and Biological Evaluation of Quinolinone Compounds as SARS CoV 3CLpro Inhibitors
145.8, 156.6, 162.7, 163.7; IR (KBr) ν: 3436, 1740,
1672, 1257, 1092, 752 cm−1; ESI-MS m/z: 280.01 ([M
＋H＋]). Anal. calcd for C17H13NO3: C 73.11, H 4.69, N
5.02; found C 73.02, H 4.53, N 4.98.
([M＋H＋]). Anal. calcd for C17H12ClNO3: C 65.08, H
3.86, N 4.46; found C 65.34, H 3.78, N 4.31.
4-(2-Naphthoyl)oxy-quinol-2-one (8) Compound
8 was prepared according to general procedure by using
4-hydroxy-2-quinolinone and 2-naphthoyl chloride, ob-
tained a white solid in 95% yield. m.p. 259－260 ℃.
1H NMR (400 MHz, DMSO-d6) δ: 6.66 (s, 1H), 7.22 (t,
J＝8.0 Hz, 1H), 7.42 (d, J＝8.0 Hz, 1H), 7.61 (t, J＝8
Hz, 1H), 7.71－7.67 (m, 2H), 7.76 (t, J＝8.0 Hz, 1H),
8.10 (d, J＝8.0 Hz, 1H), 8.20－8.15 (m, 2H), 8.26 (d,
J＝8.0 Hz, 1H), 8.97 (s, 1H), 11.99 (s, 1H); 13C NMR
(100 MHz, DMSO-d6) δ: 113.2, 115.3, 116.1, 122.7,
122.8, 125.6, 125.7, 127.8, 128.3, 129.3, 129.8, 130.2,
132.1, 132.6, 132.6, 136.1, 139.5, 156.7, 162.7, 164.0;
IR (KBr) ν: 3436, 1744, 1675, 1276, 1191, 750 cm−1;
ESI-MS m/z: 315.99 ([M ＋ H ＋ ]). Anal. calcd for
C20H13NO3: C 76.18, H 4.16, N 4.44; found C 76.49, H
4.34, N 4.23.
4-(2-Methylbenzoyl)oxy-quinol-2-one (4) Com-
pound 4 was prepared according to general procedure
by using 4-hydroxy-2-quinolinone and 2-methylbenzoyl
chloride, obtained a white solid in 91% yield. m.p. 220
1
－221 ℃. H NMR (400 MHz, DMSO-d6) δ: 2.61 (s,
3H), 6.60 (s, 1H), 7.22 (t, J＝7.6 Hz, 1H), 7.41 (d, J＝
8.0 Hz, 1H), 7.49－7.45 (m, 2H), 7.67－7.58 (m, 3H),
8.24 (d, J＝7.6 Hz, 1H), 11.95 (s, 1H); 13C NMR (100
MHz, DMSO-d6) δ: 56.5, 112.9, 113.4, 115.4, 116.1,
118.0, 120.9, 122.7, 122.8, 132.0, 132.4, 135.8, 139.5,
156.6, 159.7, 162.7, 163.1; IR (KBr) ν: 3430, 1727,
1675, 1492, 1300, 1231, 745 cm−1; ESI-MS m/z: 280.33
([M＋H＋]). Anal. calcd for C17H13NO3: C 73.11, H 4.69,
N 5.02; found C 73.38, H 4.71, N 5.47.
4-(4-Ethylbenzoyl)oxy-quinol-2-one (5)
Com-
4-(6-Chloronicotinoyl)oxy-quinol-2-one
(9)
pound 5 was prepared according to general procedure
by using 4-hydroxy-2-quinolinone and 4-ethylbenzoyl
chloride, obtained a white solid in 96% yield. m.p. 233
Compound 9 was prepared according to general proce-
dure by using 4-hydroxy-2-quinolinone and 6-chloro-
nicotinoyl chlorid, obtained a white solid in 97% yield.
1
1
－234 ℃. H NMR (400 MHz, DMSO-d6) δ: 1.24 (t,
m.p. 250－251 ℃. H NMR (400 MHz, DMSO-d6) δ:
J＝7.6 Hz, 3H), 2.75 (q, J＝7.6 Hz, 2H), 6.59 (s, 1H),
7.21 (t, J＝8.0 Hz, 1H), 7.41 (d, J＝8.0 Hz, 1H), 7.50 (d,
J＝8.0 Hz, 2H), 7.59 (t, J＝8.0 Hz, 2H), 8.14 (d, J＝8.0
Hz, 2H), 11.95 (s, 1H); 13C NMR (100 MHz, DMSO-d6)
δ: 15.7, 28.8, 113.0, 115.3, 116.1, 122.7, 125.9, 129.1,
130.8, 132.0, 139.4, 151.8, 156.6, 162.7, 163.7; IR (KBr)
ν: 3435, 1756, 1655, 1243, 1087, 750 cm−1; ESI-MS m/z:
293.99 ([M＋H＋]). Anal. calcd for C18H15NO3: C 73.71,
H 5.15, N 4.78; found C 73.98, H 5.28, N 4.89.
6.65 (s, 1H), 7.21 (t, J＝8.0 Hz, 1H), 7.41 (d, J＝8.0 Hz,
1H), 7.60 (t, J＝8.0 Hz, 1H), 7.74 (d, J＝8.0 Hz, 1H),
7.82 (d, J＝8.4 Hz, 1H), 8.58 (dd, J＝8.4, 2.4 Hz, 1H),
9.18 (d, J＝2.4 Hz, 1H), 11.99 (s, 1H); 13C NMR (100
MHz, DMSO) δ: 113.1, 115.0, 116.0, 122.7, 123.0,
124.6, 125.3, 132.1, 139.4, 141.6, 152.0, 155.8, 156.2,
162.1, 162.6; IR (KBr) ν: 3436, 1753, 1683, 1276, 1112,
752 cm−1; ESI-MS m/z: 301.14 ([M＋H＋]). Anal. calcd
for C15H9ClN2O3: C 59.91, H 3.02, N 9.32; found C
60.12, H 3.11, N 9.41.
4-(4-tert-Butylbenzoyl)oxy-quinol-2-one (6) Com-
pound 6 was prepared according to general procedure
by using 4-hydroxy-2-quinolinone and 4-tert-butyl-
benzoyl chloride, obtained a white solid in 95% yield.
4-(2-Ethoxybenzoyl)oxy-quinol-2-one
(10)
Compound 10 was prepared according to general pro-
cedure by using 4-hydroxy-2-quinolinone and 2-ethoxy-
benzoyl chloride, obtained a white solid in 90% yield.
1
m.p. 263－264 ℃. H NMR (400 MHz, DMSO-d6) δ:
1
1.35 (s, 9H), 6.58 (s, 1H), 7.21 (t, J＝7.6 Hz, 1H), 7.41
(d, J＝7.6 Hz, 1H), 7.60 (t, J＝7.6 Hz, 2H), 7.68 (d, J＝
8.4 Hz, 2H), 8.15 (d, J＝8.4 Hz, 2H), 11.96 (s, 1H); 13C
NMR (100 MHz, DMSO-d6) δ: 31.2, 35.5, 113.0, 115.3,
116.1, 122.7, 125.7, 126.5, 130.6, 132.0, 139.4, 156.6,
158.4, 162.7, 163.7; IR (KBr) ν: 3467, 1758, 1661, 1254,
1108, 766 cm−1; ESI-MS m/z: 322.12 ([M＋H＋]). Anal.
calcd for C18H15NO3: C 74.75, H 5.96, N 4.36; found C
74.52, H 5.88, N 4.41.
m.p. 178－179 ℃. H NMR (400 MHz, DMSO-d6) δ:
1.37 (t, J＝6.8 Hz, 3H), 4.21 (q, J＝6.8 Hz, 2H), 6.53 (s,
1H), 7.13 (t, J＝7.6 Hz, 1H), 7.28－7.22 (m, 2H), 7.41
(d, J＝8.0 Hz, 1H), 7.60 (td, J＝8.0, 1.2 Hz, 1H), 7.67
(td, J＝8.0, 1.6 Hz, 1H), 7.75 (d, J＝8.0 Hz, 1H), 7.95
(dd, J＝7.6, 1.6 Hz, 1H), 11.95 (s, 1H); 13C NMR (100
MHz, DMSO-d6) δ: 15.0, 64.8, 112.9, 114.2, 115.3,
116.1, 118.5, 120.8, 122.5, 123.0, 132.0, 132.2, 135.5,
139.5, 156.8, 158.8, 162.7, 163.6; IR (KBr) ν: 3422,
1717, 1672, 1308, 1233, 1109, 744 cm−1; ESI-MS m/z:
210.09 ([M＋H＋]). Anal. calcd for C18H15NO4: C 69.89,
H 4.89, N 4.53; found C 69.52, H 4.88, N 4.61.
4-(4-Chloromethylbenzoyl)oxy-quinol-2-one (7)
Compound 7 was prepared according to general proce-
dure by using 4-hydroxy-2-quinolinone and 4-(chloro-
methyl)benzoyl chloride, obtained a white solid in 97%
4-(3-Methoxybenzoyl)oxy-quinol-2-one
(11)
1
yield. m.p. 234－236 ℃. H NMR (400 MHz, DMSO-
Compound 11 was prepared according to general pro-
cedure by using 4-hydroxy-2-quinolinone and 3-meth-
oxy-benzoyl chloride, obtained a white solid in 94%
yield. m.p. 199 － 201 ℃ . 1H NMR (400 MHz,
DMSO-d6) δ: 3.88 (s, 3H), 6.60 (s, 1H), 7.22 (t, J＝8.0
Hz, 1H), 7.42－7.37 (m, 2H), 7.62－7.56 (m, 3H), 7.67
(t, J＝2.0 Hz, 1H), 7.82 (d, J＝8.0 Hz, 1H), 11.97 (s,
1H); 13C NMR (100 MHz, DMSO-d6) δ: 56.0, 113.1,
d6) δ: 4.92 (s, 2H), 6.62 (s, 1H), 7.21 (t, J＝8.4 Hz, 1H),
7.41 (d, J＝8.4 Hz, 1H), 7.65－7.58 (m, 1H), 7.72 (d,
J＝8.4 Hz, 2H), 8.23 (d, J＝8.4 Hz, 2H), 11.97 (s, 1H);
13C NMR (100 MHz, DMSO-d6) δ: 45.6, 113.1, 115.2,
116.1, 122.69, 122.72, 128.2, 129.9, 131.0, 132.1, 139.4,
144.7, 156.5, 162.7, 163.4; IR (KBr) ν: 3436, 1741,
1675, 1264, 1097, 762, 711 cm−1; ESI-MS m/z: 314.02
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115.0, 115.2, 116.1, 121.2, 122.7, 122.7, 122.9, 129.7,
130.9, 132.1, 139.4, 156.5, 160.0, 162.7, 163.6; IR (KBr)
ν: 3435, 1752, 1668, 1277, 1196, 1102, 742 cm−1;
ESI-MS m/z: 296.11 ([M ＋ H ＋ ]). Anal. calcd for
C17H13NO4: C 69.15, H 4.44, N 4.74; found C 68.75, H
4.28, N 4.81.
131.9, 139.4, 140.5, 156.5, 162.6, 170.5; IR (KBr) ν:
3436, 1760, 1671, 1125, 750 cm−1; ESI-MS m/z: 294.10
([M＋H＋]). Anal. calcd for C18H15NO3: C 73.71, H 5.15,
N 4.78; found C 73.24, H 5.03, N 4.96.
4-(Cinnamoyl)oxy-quinol-2-one (16) Compound
16 was prepared according to general procedure by us-
ing 4-hydroxy-2-quinolinone and cinnamoyl chloride,
obtained a white solid in 95% yield. m.p. 207－208 ℃.
1H NMR (400 MHz, DMSO-d6) δ: 6.53 (s, 1H), 7.03 (d,
J＝16.0 Hz, 1H), 7.23 (t, J＝8.0 Hz, 1H), 7.39 (d, J＝
8.0 Hz, 1H), 7.51－7.48 (m, 3H), 7.59 (t, J＝8.0 Hz,
1H), 7.66 (d, J＝8.0 Hz, 1H), 7.88－7.86 (m, 2H), 7.99
(d, J＝16.0 Hz, 1H), 11.93 (s, 1H); 13C NMR (100 MHz,
DMSO-d6) δ: 112.7, 115.3, 116.0, 116.7, 122.6, 122.8,
129.4, 129.5, 131.7, 132.0, 134.2, 139.4, 148.3, 156.5,
162.7, 164.1; IR (KBr) ν: 3436, 1755, 1682, 1631, 1118,
750 cm−1; ESI-MS m/z: 291.93 ([M＋H＋]). Anal. calcd
for C18H13NO3: C 74.22, H 4.50, N 4.81; found C 74.02,
H 4.86, N 4.98.
2-Oxo-1,2-dihydroquinolin-4-yl phenyl carbonate
(17) Compound 17 was prepared according to general
procedure by using 4-hydroxy-2-quinolinone and
phenyl chloroformate, obtained a white solid in 91%
yield. m.p. 191 － 192 ℃ . 1H NMR (400 MHz,
DMSO-d6) δ: 6.74 (s, 1H), 7.28 (t, J＝7.6 Hz, 1H), 7.41
－7.34 (m, 1H), 7.53－7.45 (m, 4H), 7.62 (t, J＝7.6 Hz,
1H), 7.79 (d, J＝7.6 Hz, 1H), 12.00 (s, 1H); 13C NMR
(100 MHz, DMSO-d6) δ: 112.3, 114.6, 116.1, 121.7,
122.5, 122.8, 127.3, 130.3, 132.3, 139.4, 150.4, 151.0,
156.0, 162.6; IR (KBr) ν: 3444, 1784, 1672, 1243, 765
cm−1; ESI-MS m/z: 282.00 ([M＋H＋]). Anal. calcd for
C16H11NO4: C 68.32, H 3.94, N 4.98; found C 68.56, H
4.02, N 5.11.
4-(2-Methoxybenzoyl)oxy-quinol-2-one
(12)
Compound 12 was prepared according to general pro-
cedure by using 4-hydroxy-2-quinolinone and oanisoyl
chloride, obtained a white solid in 91% yield. m.p. 208
1
－209 ℃. H NMR (400 MHz, DMSO-d6) δ: 3.93 (s,
3H), 6.55 (s, 1H), 7.15 (t, J＝7.6 Hz, 1H), 7.25 (t, J＝
7.6 Hz, 1H), 7.30 (d, J＝8.4 Hz, 1H), 7.40 (d, J＝8.4 Hz,
1H), 7.60 (t, J＝7.6 Hz, 1H), 7.71 (dd, J＝13.2, 7.6 Hz,
2H), 8.01 (d, J＝7.2 Hz, 1H), 11.94 (s, 1H); 13C NMR
(100 MHz, DMSO-d6) δ: 21.8, 113.2, 115.4, 116.1,
122.69, 122.72, 126.9, 127.6, 131.6, 131.8, 132.0, 132.6,
134.1, 139.5, 141.4, 156.7, 162.7, 164.2; IR (KBr) ν:
3468, 1750, 1679, 1236, 1195, 1136, 728 cm−1; ESI-MS
m/z: 296.06 ([M＋H＋]). Anal. calcd for C17H13NO4: C
69.15, H 4.44, N 4.74; found C 69.32, H 4.72, N 5.06.
4-(4-Methoxybenzoyl)oxy-quinol-2-one
(13)
Compound 13 was prepared according to general pro-
cedure by using 4-hydroxy-2-quinolinone and 4-meth-
oxybenzol chloride, obtained a white solid in 95% yield.
1
m.p. 232－233 ℃. H NMR (400 MHz, DMSO-d6) δ:
3.91 (s, 3H), 6.57 (s, 1H), 7.18 (d, J＝8.8 Hz, 2H), 7.22
(d, J＝7.6 Hz, 1H), 7.40 (d, J＝8.8 Hz, 1H), 7.59 (t, J＝
7.6 Hz, 2H), 8.18 (d, J＝8.8 Hz, 2H), 11.94 (s, 1H); 13C
NMR (100 MHz, DMSO-d6) δ: 56.2, 113.0, 115.0,
115.4, 116.1, 120.4, 122.7, 132.0, 132.9, 139.4, 156.6,
162.7, 163.4, 164.7; IR (KBr) ν: 3436, 1743, 1655, 1605,
1252, 1093, 761 cm−1; ESI-MS: m/z 295.78 ([M＋H＋]).
Anal. calcd for C17H13NO4: C 69.15, H 4.44, N 4.74;
found C 69.01, H 4.52, N 4.33.
Benzyl 2-oxo-1,2-dihydroquinolin-4-yl carbonate
(18) Compound 18 was prepared according to general
procedure by using 4-hydroxy-2-quinolinone and benzyl
chloroformate, obtained a white solid in 92% yield. m.p.
4-(2-Phenylacetyl)oxy-quinol-2-one (14)
Com-
pound 14 was prepared according to general procedure
by using 4-hydroxy-2-quinolinone and phenylacetyl
chloride, obtained a white solid in 92% yield. m.p. 174
1
169－170 ℃. H NMR (400 MHz, DMSO-d6) δ: 5.35
(s, 2H), 6.58 (s, 1H), 7.22 (t, J＝8.0 Hz, 1H), 7.38 (d,
J＝8.0 Hz, 1H), 7.46－7.41 (m, 3H), 7.50 (d, J＝6.8 Hz,
2H), 7.59 (t, J＝6.8 Hz, 2H), 11.95 (s, 1H); 13C NMR
(100 MHz, DMSO-d6) δ: 112.7, 115.2, 116.0, 122.5,
122.7, 127.7, 129.0, 130.2, 132.0, 134.0, 139.4, 156.4,
162.6, 169.4; IR (KBr) ν: 3435, 1762, 1654, 1226, 1195,
770 cm−1; ESI-MS m/z: 295.89 ([M＋H＋]). Anal. calcd
for C17H13NO4: C 69.15, H 4.44, N 4.74; found C 69.54,
H 4.71, N 4.58.
1
－175 ℃. H NMR (400 MHz, DMSO-d6) ν: 4.16 (s,
2H), 6.41 (s, 1H), 7.16 (t, J＝8.0 Hz, 1H), 7.46－7.31
(m, 6H), 7.49 (d, J＝8 Hz, 1H), 7.56 (t, J＝8.0 Hz, 1H),
11.92 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ: 71.1,
112.0, 114.7, 116.1, 122.4, 122.7, 129.0, 129.1, 129.2,
132.1, 135.1, 139.4, 151.7, 156.1, 162.6; IR (KBr) ν:
3436, 1756, 1668, 1123, 755 cm−1; ESI-MS m/z: 280.35
([M＋H＋]). Anal. calcd for C17H13NO3: C 73.11, H 4.69,
N 5.02; found C 72.98, H 4.46, N 4.91.
4-(Propionyl)oxy-quinol-2-one (19)
Compound
4-(3-Phenylpropanoyl)oxy-quinol-2-one
(15)
19 was prepared according to general procedure by us-
ing 4-hydroxy-2-quinolinone and propionyl chloride,
obtained a white solid in 92% yield. m.p. 179－180 ℃.
1H NMR (400 MHz, DMSO-d6) δ: 1.18 (t, J＝7.6 Hz,
3H), 2.79 (q, J＝7.6 Hz, 2H), 6.40 (s, 1H), 7.21 (t, J＝
8.0 Hz, 1H), 7.36 (d, J＝8.0 Hz, 1H), 7.57 (t, J＝8.0 Hz,
1H), 7.63 (d, J＝8.0 Hz, 1H), 11.88 (s, 1H); 13C NMR
(100 MHz, DMSO-d6) δ: 9.2, 27.3, 112.7, 115.3, 116.0,
122.5, 122.9, 131.9, 139.4, 156.5, 162.7, 171.9; IR (KBr)
ν: 3436, 1761, 1674, 1134, 752 cm−1; ESI-MS m/z:
Compound 15 was prepared according to general pro-
cedure by using 4-hydroxy-2-quinolinone and hydro-
cinnamoyl chloride, obtained a white solid in 93% yield.
1
m.p. 167－168 ℃. H NMR (400 MHz, DMSO-d6) δ:
3.02 (t, J＝7.2 Hz, 2H), 3.12 (t, J＝7.2 Hz, 2H), 6.30 (d,
J＝1.2 Hz, 1H), 7.12 (t, J＝8.0 Hz, 1H), 7.29－7.23 (m,
1H), 7.35－7.33 (m, 6H), 7.55 (t, J＝8.0 Hz, 1H), 11.88
(s, 1H); 13C NMR (100 MHz, DMSO-d6) δ: 30.5, 35.4,
112.7, 115.2, 115.9, 122.4, 122.8, 126.8, 128.8, 128.9,
1204
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© 2013 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2013, 31, 1199—1206

Synthesis and Biological Evaluation of Quinolinone Compounds as SARS CoV 3CLpro Inhibitors
218.02 ([M＋H＋]). Anal. calcd for C12H11NO3: C 66.35,
H 5.10, N 6.45; found C 66.54, H 5.32, N 6.74.
285.95 ([M＋H＋]). Anal. calcd for C15H11NO3S: C
63.14, H 3.89, N 4.91; found C 63.53, H 3.57, N 4.65.
Methyl 2-oxo-1,2-dihydroquinolin-4-yl succinate
(20) Compound 20 was prepared according to general
procedure by using 4-hydroxy-2-quinolinone and
methyl 4-chloro-4-oxobutanoate, obtained a white solid
Biology enzyme assays
The sequence of SARS-CoV 3CLpro cloned into the
pGEX-6p-1 vector was transformed into E. coli BL21
(DE3) cells. The recombinant protein with GST-tag was
purified by GST-glutathione affinity chromatography
and the ion exchange column. Eventually purified pro-
tein was of high purity (＞95%) as judged by SDS-
PAGE analysis and the concentration is 0.5 μmol/L, and
the buffer is 50 mmol/L Tris-HCl, pH 7.3, 1 mmol/L
EDTA. The substrate synthesized in Shanghai Biologi-
cal Engineering Company is dissolved in DMSO, with
0.8 mmol/L liquid storage for used.
1
in 90% yield. m.p. 170－171 ℃. H NMR (400 MHz,
DMSO-d6) δ: 2.75 (t, J＝6.8 Hz, 2H), 3.02 (t, J＝6.8 Hz,
2H), 3.65 (s, 3H), 6.35 (s, 1H), 7.22 (t, J＝8.0 Hz, 1H),
7.37 (d, J＝8.0 Hz, 1H), 7.58 (t, J＝8.0 Hz, 1H), 7.66 (d,
J＝8.0 Hz, 1H), 11.91 (s, 1H); 13C NMR (100 MHz,
DMSO-d6) δ: 28.8, 29.3, 52.1, 112.6, 115.2, 116.0,
122.5, 122.9, 132.0, 139.4, 156.5, 162.6, 170.3, 172.8;
IR (KBr) ν: 3439, 1772, 1733, 1651, 1329, 1117, 773
cm−1; ESI-MS m/z: 276.04 ([M＋H＋]). Anal. calcd for
C14H13NO5: C 61.09, H 4.76, N 5.09; found C 61.21, H
4.34, N 4.96.
The SARS-CoV 3CLpro inhibition assays were con-
ducted via fluorescence resonance energy transfer
(FRET). The natural substrate amino acid sequence
(AVLQ. SGFRKK) of SARS-CoV 3CLpro ends with
MCA fluorescent group and Dnp fluorescence quench-
ing group. The system of screening is as follows (Table
2): The settled concentrations of proteins, compounds
and substrate were preheated at 37 ℃ and oscillated.
Excitation/emission light is 320/405 nm, test was car-
ried out every 3 s for 60 times. Drawing curves, the
maximum value of the negative control curve slope is
V0, and the largest compound curve slope is V1. The
inhibition ratio can be defined (1−V1/V0). And the IC50
value was calculated by the following equation:
Isobutyl 2-oxo-1,2-dihydroquinolin-4-yl carbon-
ate (21) Compound 21 was prepared according to
general procedure by using 4-hydroxy-2-quinolinone
and isobutyl chloroformate, obtained a white solid in
1
94% yield. m.p. 126－127 ℃. H NMR (400 MHz,
DMSO-d6) δ: 0.95 (d, J＝6.8 Hz, 6H), 2.07－1.97 (m,
1H), 4.08 (d, J＝6.8 Hz, 2H), 6.55 (s, 1H), 7.24 (t, J＝
7.6 Hz, 1H), 7.38 (d, J＝8.0 Hz, 1H), 7.59 (t, J＝7.6 Hz,
2H), 11.95 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ:
19.0, 27.7, 75.4, 112.1, 114.8, 116.1, 122.3, 122.7,
132.1, 139.4, 151.9, 156.2, 162.7; IR (KBr) ν: 3436,
1774, 1655, 1231, 1196, 759 cm−1; ESI-MS m/z: 262.33
([M＋H＋]). Anal. calcd for C14H15NO4: C 64.36, H 5.79,
N 5.36; found C 64.12, H 5.89, N 5.08.
V /V
0
1
[I]/IC
＝ ＋
50
V0 shows the initial rate of the reaction without inhibitor,
V means the initial rate of the reaction with the inhibitor
at various concentrations, [I] indicates the concentration
of the inhibitor.
1-Methyl-4-(3-methoxybenzoyl)oxy-quinol-2-one
(22) Compound 22 was prepared according to general
procedure by using4-hydroxy-1-methyl-2-quinolinone
and 3-methoxybenzol chloride, obtained a white solid in
1
96% yield. m.p. 160－162 ℃. H NMR (400 MHz,
Table 2 SARS CoV 3CLpro compound screening system
DMSO-d6) δ: 3.67 (s, 3H), 3.88 (s, 3H), 6.74 (s, 1H),
7.32 (t, J＝8.0 Hz, 1H), 7.39 (dd, J＝8.0, 2.0 Hz, 1H),
7.59 (t, J＝8.0 Hz, 1H), 7.75－7.65 (m, 4H), 7.84 (d,
J＝8.0 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ:
29.7, 56.0, 112.5, 115.1, 115.7, 116.2, 121.3, 122.88,
122.91, 123.2, 129.7, 130.9, 132.5, 140.3, 155.4, 160.0,
161.9, 163.6; IR (KBr) ν: 1742, 1664, 1275, 1102, 1023,
740 cm−1; ESI-MS m/z: 310.19 ([M＋H＋]). Anal. calcd
for C18H15NO4: C 69.89, H 4.89, N 4.53; found C 70.36,
H 5.02, N 4.86.
System
SARS-Cov 3CLpro
Substrate
Volume
97 µL
2 µL
Final concentration
0.5 µmol/L
16 µmol/L
Compound
Total
1 µL
1 mmol/L
100 µL
Docking study
The crystal structure of SARS-CoV 3CLpro (code:
3SN8), obtained from The Protein Data Bank as a com-
plex bound with inhibitors, was used in this docking
study. And the docking studies were carried out in the
Discovery Studio 3.0. To prepare the protein for dock-
ing, water molecules and inhibitors were removed and
hydrogen atoms were added by using Prepare Protein
Structure module, from the protein.
1-Methyl-4-(thiophene-2-carbonyl)oxy-quinol-2-
one (23) Compound 23 was prepared according to
general procedure by using 4-hydroxy-1-methyl-2-qui-
nolinone and 2-thiophenecarbonyl chloride, obtained a
1
white solid in 91% yield. m.p. 148－149 ℃. H NMR
(400 MHz, DMSO-d6) δ: 3.65 (s, 3H), 6.75 (s, 1H), 7.32
(t, J＝8.0 Hz, 1H), 7.37 (t, J＝4.4 Hz, 1H), 7.63 (d, J＝
8.0 Hz, 1H), 7.73－7.70 (m, 2H), 8.20－8.16 (m, 2H);
13C NMR (100 MHz, DMSO-d6) δ: 29.7, 112.3, 115.7,
116.1, 122.9, 123.0, 129.5, 131.1, 132.5, 136.7, 136.9,
140.2, 154.7, 159.1, 161.8; IR (KBr) ν: 3092, 1733,
1663, 1413, 1354, 1249, 1105, 739 cm−1; ESI-MS m/z:
Acknowledgement
This work was supported by Tianjin SME Technol-
ogy Innovation Fund (No. 11ZXCXSY03500) and Na-
Chin. J. Chem. 2013, 31, 1199—1206
© 2013 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.cjc.wiley-vch.de
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Sun et al.
FULL PAPER
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Chin. J. Chem. 2013, 31, 1199—1206