Design, synthesis and in vitro anti-influenza A virus evaluation of novel quinazoline derivatives containing S-acetamide and NH-acetamide moieties at C-4

Article abstract of DOI:10.1016/j.ejmech.2020.112706

It is an urgent need to develop more effective anti-influenza agents due to the emergence of highly pathogenic and drug-resistant influenza viruses. Herein, a series of 2,4-disubstituted quinazoline derivatives were designed, synthesized and their antiviral activities against influenza A virus were evaluated. Nine compounds (10a2, 16a, 16e, 16i, 16j, 16n, 16o, 16p and 16r) showed potent activity against influenza A virus (IAV) with IC₅₀ at the low-micromole level (1.29–9.04 μM). Particularly, 16e and 16r possess good anti-IAV activity (IC₅₀: 1.29 μM and 3.43 μM, respectively) and acceptable cytotoxicity, and inhibit the transcription and replication of viral RNA. Together with reasonable PK profiles of 16e, these results suggest their promising potential as candidates for further investigation.

Full text of DOI:10.1016/j.ejmech.2020.112706

Journal Pre-proof
Design, synthesis and in vitro anti-influenza A virus evaluation of novel quinazoline
derivatives containing S-acetamide and NH-acetamide moieties at C-4
Guoning Zhang, Minghua Wang, Jianyuan Zhao, Yujia Wang, Mei Zhu, Juxian Wang,
Shan Cen, Yucheng Wang
PII:
S0223-5234(20)30678-4
DOI:
https://doi.org/10.1016/j.ejmech.2020.112706
EJMECH 112706
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 April 2020
Revised Date: 28 July 2020
Accepted Date: 28 July 2020
Please cite this article as: G. Zhang, M. Wang, J. Zhao, Y. Wang, M. Zhu, J. Wang, S. Cen, Y. Wang,
Design, synthesis and in vitro anti-influenza A virus evaluation of novel quinazoline derivatives
containing S-acetamide and NH-acetamide moieties at C-4, European Journal of Medicinal Chemistry,
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Design, synthesis and in vitro anti-influenza A virus evaluation
of novel quinazoline derivatives containing S-acetamide and
NH-acetamide moieties at C-4
1
1
1
Guoning Zhang , Minghua Wang , Jianyuan Zhao , Yujia Wang, Mei Zhu, Juxian
*
*
*
Wang , Shan Cen , Yucheng Wang
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union
Medical College, Beijing 100050, PR China
*
Corresponding author.
E-mail addresses: imbjxwang@163.com (J. Wang), shancen@imb.pumc.edu.cn (S. Cen),
wangyucheng@imb.pumc.edu.cn (Y. Wang).
1
These authors made equal contributions to the work.

Design, synthesis and in vitro anti-influenza A virus evaluation
of novel quinazoline derivatives containing S-acetamide and
NH-acetamide moieties at C-4
1
1
1
Guoning Zhang , Minghua Wang , Jianyuan Zhao , Yujia Wang, Mei Zhu, Juxian
*
*
*
Wang , Shan Cen , Yucheng Wang
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union
Medical College, Beijing 100050, PR China
*
Corresponding author.
E-mail addresses: imbjxwang@163.com (J. Wang), shancen@imb.pumc.edu.cn (S. Cen),
wangyucheng@imb.pumc.edu.cn (Y. Wang).
1
These authors made equal contributions to the work.
Abstract：It is an urgent need to develop more effective anti-influenza agents due to
the emergence of highly pathogenic and drug-resistant influenza viruses. Herein, a
series of 2,4-disubstituted quinazoline derivatives were designed, synthesized and
their antiviral activities against influenza A virus were evaluated. Nine compounds
(10a2, 16a, 16e, 16i, 16j, 16n, 16o, 16p and 16r) showed potent activity against
influenza A virus (IAV) with IC50 at the low-micromole level (1.29–9.04 μM).
Particularly, 16e and 16r possess good anti-IAV activity (IC : 1.29 μM and 3.43 μM,
5
0
respectively) and acceptable cytotoxicity, and inhibit the transcription and replication
of viral RNA. Together with reasonable PK profiles of 16e, these results suggest their
promising potential as candidates for further investigation.
Key words: Anti-influenza A virus activity, quinazoline derivatives, synthesis,
structure-activity relationships
1
. Introduction

Influenza, or “flu”, is a highly contagious respiratory infectious diseases caused by
the influenza virus and is responsible for approximately 3–5 million cases of severe
disease, many hospitalisations and about 290,000 to 650,000 respiratory deaths
worldwide every year [1–5]. Influenza virus can be divided into four types: influenza
A, B, C and D [6]. Among them, influenza A virus (IAV), a linear, negative-sense,
single-stranded RNA virus, possesses high pathogenicity and is the main cause of
seasonal epidemics and occasional pandemics of respiratory diseases worldwide,
which belongs to the Orthomyxoviridae family[7–11].
Fig. 1. Four classes of approved antiviral agents for the treatment of influenza virus infection
However, there are limited options in the clinic treatment of influenza infections.
Up to now, only two classes of anti-influenza drugs are approved by the FDA, namely,
M2 ion-channel blockers (amantadine and rimantadine) and neuraminidase inhibitors
(oseltamivir, zanamivir, peramivir and laninamivir octanoate)(Fig. 1)[12]. In addition,
as a RNA polymerase inhibitor, Favipiravir(Fig. 1) was approved by Japan against
influenza infection in 2014(Fig. 1)[13] and Baloxavir marboxil (BXM)(Fig. 1), an
orally available small molecule inhibitor of cap-dependent endonuclease (CEN), was
approved for the treatment of uncomplicated influenza in Japan and the United States
of America in 2018(Fig. 1)[14]. Unfortunately, the M2 ion-channel inhibitors are no
longer recommended for treatment of influenza and NA inhibitors have several
limitations in clinical practice due to their drug resistance and severe side effects
[
15–19]. Safe and effective therapy for IAV infection is still a high unmet medical

need. Therefore, we are constantly required to develop new antiviral agents with new
scaffold and novel mechanism of action.
Fig. 2. Design of new quinazoline derivatives
In our previous studies [20], a series of substituted indole derivatives were
synthesized and most of them displayed significant activity against IAV A/WSN/33
(H1N1) strain. Among them, two compounds A and B with indole and naphthalene
ring displayed more potential in vitro anti-IAV activity (IC : 4.18 μM and 5.22 μM,
5
0
respectively, Fig.2). We also reported that 3-substituted indole derivatives inhibited
IAV replication at the post-entry stage and might target viral RNA transcription and
replication [21].
Based on the above research results, and as part of our persistent efforts to develop
potential antiviral candidates, we intended to replace the indole and naphthalene
scaffold with quinazoline scaffold employing the strategies of bioisosterism and
scaffold alteration to explore the structure-activity relationship (SAR) (Fig. 2). Here,
we designed and synthesized a series of 2,4-substitued quinazoline derivatives
containing S-acetamide and NH-acetamide moieties and evaluated their anti-IAV
activity in this study, aiming at developing these compounds into a new class of
anti-IAV drug candidates.
2
. Results and Discussion
2
.1. Chemistry
The target compounds 7a–i and 10a–b were synthesized by following the pathway

described in Scheme 1. The quinazoline-2,4(1H,3H)-dione core was constructed by
reacting anthranilic acid 1 with urea at 180℃ . Subsequently, the two carbonyl groups
in compound 2 were subjected to chlorination with phosphoryl chloride (POCl ) to
3
produce the 2,4-dichloroquinazoline 3. Under basic conditions, the 4-position chlorine
of 3 was selectively substituted with ethyl thioglycolate to give the key intermediate 4.
To confirm the regioselectivity of this reaction, we did HMBC spectrum analysis of
compound 4 and found that there is a coupling between the newly introduced
methylene hydrogen and the 4-position carbon atom (Fig. 3). Nucleophilic
substitution of intermediate
4
with pyrrolidine in the presence of
N,N-diisopropylethylamine (DIPEA) yielded the corresponding compound 5, which
was hydrolyzed subsequently to give acid 6. In the presence of HATU and DIPEA,
intermediate 6 reacted with different amines to afford the target compounds 7a–7i. On
the other hand, the key intermediate 4 was hydrolyzed in the presence of LiOH to give
the corresponding acid 8. Amidation of acid 8 with aniline or benzylamine in the
presence of HATU and DIPEA afforded the amides 9a–9b. Finally, the chlorine at the
2
-position of 9a–b was substituted with available piperazine or piperidine derivatives
to provide the target compounds 10a–b.
Scheme 1. Synthesis of the target compounds 7 and 10

A
D
4
.17
1
.38
O
O
S
N
7
.93
4.33
7
.64
O
N
7
.92
Cl
8
.10
3
2.5
1
4.23
B
168.2
121.9S
1
27.7
62.2
O
1
23.9
35.0
1
72.8
N
1
55.6
1
1
28.1
49.7
N
Cl
1
4
.17
C
H H
O
S
O
1
72.8
N
N
Cl
Fig. 3. Structure and HMBC spectrum of compound 4 ( A: Chemical shift of hydrogen atom in
1
13
HNMR; B: Chemical shift of carbon atom in CNMR; C: Coupling between the newly
introduced methylene hydrogen and the 4-position carbon atom; D: Part of HMBC-spectrum of
compound 4)
The synthesis of quinazoline derivatives 16a–r are shown in Scheme 2.
Commercially available glycine 11 was protected by ditertbutyl dicarbonate to afford
the corresponding Boc-glycine 12. Amidation of 12 with different amines in the
presence of HATU and DIPEA afforded the amides 13. The subsequent deprotection
afforded the desired intermediate 14, which underwent a coupling reaction with
2
,4-dichloroquinazoline 3 to generate the key intermediate 15. Finally, compounds 15
were reacted with appropriate amines in the presence N,N-diisopropylethylamine
DIPEA) to obtain the desired products 16a–r in moderate to good yields.
(

Scheme 2. Synthesis of the target compounds 16
2
.2 Anti-influenza virus activity
The target compounds 7, 10, and 16 were initially screened for their inhibitory
effect against IAV A/WSN/33 (H1N1) at a concentration of 10 μM in HEK293T-Gluc
cells and the results were shown in Fig.1. Notably, most of the tested compounds
exhibited a moderate to high inhibition ratio to IAV infection. Among them,
compounds 16a, 16e and 16j showed the most potent inhibition ratio (95.84%,
9
9.84%, 98.21%, respectively), which was higher than compounds A and B (90.21%
and 78.04%, respectively). Overall, the potency of the quinazoline derivatives in this
study depends on both of the groups at C-2 and C-4 positions and the NH-acetamide
derivatives (16) are more active than the S-acetamides (7, 10).
Fig. 4. Inhibition rate of the target compounds on IAV.
An exploration on C-4 (R) of quinazoline core revealed a flat SAR (Fig.1). All the
modifications on R are less beneficial to anti-IAV activity, even decreased the
antiviral activity, except for 7e and 7f, suggesting that naphthalen-2-yl and

cyclopropyl are acceptable. Furthermore, insertion of a methylene between the
benzene ring and amide bond leads to decreased activity. For example, N-phenyl
acetamide derivatives provided better anti-IAV activity than N-benzyl acetamide
derivatives (7a vs 7b, 7c vs 7h). Additionally, replacement of the pyrrolidin-1-yl at
C-2 with other amino groups has a significant effect on the activity and diethylamino
group (10a2, 10b2) is optimal for the C-2 position.
In the next round of optimization, we replaced the sulfur atom with nitrogen atom
employing the strategy of bioisosterism with the aim of improving the antiviral
potency. To our delight, we observed the NH-acetamide derivatives (16) are much
more active than the S-acetamides (7, 10), indicating that nitrogen atom is preferred
over sulfur atom. The naphthalen-2-yl and phenyl at C-4 were identified to be crucial
for antiviral activity. Compound 16a and compound 16e were optimal for excellent
anti-IAV activity (IR = 99.84% and 95.84%, respectively). On the other hand,
replacing the pyrrolidin-1-yl by other secondary amines (from 16j to 16q)
significantly enhances the anti-IAV activity, except for 16k, 16l and 16m. The
anti-IAV activity of the quinazoline derivatives in this study depends on both of the
groups at the C-4 and C-2 positions and the optimal combination needs further
discussion in the future.
Thirteen compounds identified with high inhibition rates in the initial screening
were selected for the dose response assays (Table 1). The IC of ribavirin used as a
5
0
positive control was 15.36 μM. Among them, eight compounds (10a2, 16a, 16e, 16i,
6j, 16n, 16o and 16p) showed stronger anti-influenza A/WSN/33 (H1N1) activities
with IC at micromole level (1.29–9.04 μM). Compound 16e showed the strongest
1
5
0
inhibitory activity with IC value of 1.29 μM in this study. Subsequently, compound
50
1
6r
synthesized
by
replacing
the
pyrrolidin-1-yl
of
16e
with
3
,4-dihydroisoquinolin-2(1H)-yl of 16j remains excellent activity (IC : 3.43±0.54
5
0
μM) and exhibited lower cytotoxicity with CC values of up to and beyond 100 μM.
5
0

Tale 1 Anti-influenza virus activity and cytotoxicity of the selected compounds in HEK293T-Gluc
cells
Cpd.
IC50（μM）
36.10±7.88
38.24±7.40
7.18±1.89
19.15±0.73
1.88±0.10
39.43±0.93
1.29±0.01
9.04±0.57
3.88±0.47
6.84±0.68
3.83±0.15
5.00±1.37
11.47±0.54
3.43±0.54
4.18±0.12
5.22±0.34
15.36±0.93
CC50（μM）
>100
SI
7e
>2.77
>2.61
>13.93
>5.22
12.38
>2.54
46.46
1.75
7f
>100
1
0a2
0b2
>100
1
>100
16a
23.28±2.91
>100
16d
1
6e
6i
6j
59.94±3.04
15.86±0.58
36.64±2.24
29.43±0.95
>100
1
1
9.44
16n
16o
16p
16q
4.30
>26.11
>20.00
>8.72
>29.15
10.52
>19.15
>6.51
>100
>100
16r
>100
A
B
43.98±2.17
>100
ribavirin
>100
As shown in Table 1, the cytotoxicity of the fourteen compounds was also
investigated in HEK293T-Gluc cells using a cell counting kit-8 (CCK-8) assay. Most
compounds displayed low cytotoxic (CC > 100 μM), whereas the introduction of
5
0
nitrogen atom at C-4 resulted in increased cytotoxicity (16a, 16i and 16n).
Considering both of the activity and cytotoxicity, compound 16e and 16r, both of that
hold the higher selective index (SI >29.15) among these analogues, could be selected
as a lead compound for further modification.
Encouraged by their strong potency against influenza virus IAV A/WSN/33
(H1N1) strain, eight compounds were evaluated against influenza virus IAV

A/PR/8/1934 (H1N1) strain in HEK293T-Gluc cells. As shown in Table 2, all tested
compounds turned out to be potent inhibitors against influenza virus IAV
A/PR/8/1934 (H1N1) with IC50 values from 0.12 to 6.05 μM, suggesting their
promising potential antiviral activity. In addition, we assessed the cytotoxicity of these
compounds on A549 cells, a cell line that is widely used in IAV study. The results
showed that most of the compounds displayed low to medium toxicity on A549 cells
(Table 3), similar to what found in 293T cells. Despite of an increasing cytotoxicity of
1
6e, 16i, 16j and 16n on A549 compared with 293T, much higher values of CC than
5
0
IC indicate that their antiviral activities are not due to cytotoxicity. Furthermore, the
5
0
similar cytotoxicity profile of these compounds on both cell lines would help us to
improve their safety by further optimization in the future.
Table 2 Anti-influenza virus activity of the selected compounds in 293T-GLUC cells
Cpd.
0a2
IC (μM)
Cpd.
16n
16o
16p
A
IC (μM)
5
0
50
1
1.69±0.21
1.47±0.12
0.63±0.02
0.95±0.08
0.41±0.09
0.12±0.03
6.05±0.32
5.80±0.68
0.33±0.01
16a
16e
1
6i
6j
1
Table 3 The cytotoxicity of selected compounds on A549 cells
Cpd.
0a2
6a
6e
CC （μM）
Cpd.
16n
CC （μM）
50
50
1
＞100
12.84±0.03
1
19.04±1.34
4.77±0.13
16o
＞100
1
16p
A
＞100
16i
5.92±0.14
71.41±3.4
16j
10.72±0.18
2
.3 Primary Mechanism of compound 16e against IAV
Next, compound 16e was selected for further mechanisms study against the
influenza A virus. We initially carried out a time-of-addition experiment for 16e to

investigate the possible time-dependent inhibitory effects on influenza replication, and
the results were shown in Figure 4. When compound 16e was added before viral
infection (from -1 to 0 h), no reduction in viral yield was observed. However, 16e
displayed a significant inhibitory effect on influenza virus when added after viral
infection, particularly in the early stage (from 2 to 4 h), suggesting that 16e affects the
early steps of the replication cycle.
Fig.5. Times-of-addition of 16e after infected with IAV. A549 cells were inoculated with
influenza single-cycle A/WSN/33(H1N1) virus at a multiplicity of infection of 0.2 and compound
1
6e (2.5 μM) was added. Viral yields were determined at 11 h post infection by measuring Gluc
activity. In this experiment, DMSO was used as the negative control and ribavirin (30 μM) as the
positive control.
In order to further confirm the mode of action of these compounds against influenza
virus, we investigated the effect on IAV RNA transcription and replication using an
IAV mini-genome replicon system. As shown in Fig.5, compared with the negative
control, compound 16e could significantly inhibited the activity of luciferase in a
dose-dependent manner, and the activity of luciferase was inhibited by more than 80%
at 2.5 μM. These results suggest that these compounds may target viral RNA
transcription and replication.

Fig.6. Inhibition of compound 16e on influenza viral vRNP. HEK239T cells were transfected with
pCAGGS expression plasmids encoding PB2、PB1、PA、NP、pol-LUC and SV40-Relina in the
absence or presence of compound 16e (1.25 μM or 2.5 μM). Effect of vRNA transcription was
evaluated by measuring luciferase and relina at 24 h post-transfection. ** indicates p<0.01 as
compared to negative control.
2
.4 Lipinski’s rules and in vivo PK profiles
Lipinski’s rules are important guidelines for determining drug-likeness
compounds [22]. The related theoretical values of most ten potent compounds were
obtained using Molinspiration free online software (https://www.molinspiration.com).
As shown in Table 4, none violation of Lipinski’s rule-of-five was found among
compounds 10a2, 16a, 16n, 16o, 16p and 16q. The logP of compounds 16e, 16i, 16j
and 16r are out of the recommended range (0≤logP≤5), which indicates they have a
bad hydrophilicity ratio to be bioavailable compounds. However, they are still
incorporate with the Lipinski’s rule-of-five (violations ≤1). The calculated polar
surface area values were much lower than 140, predicting adequate intestinal
absorption.

Table 4 Parameters of Lipinski’s rules
Lipinski’s
violations
3
4
5
nON
nOHNH
MW
1
2
3
Cpd.
LogP (≤5)
TPSA
Vol (Å )
(≤10)
(≤5)
(≤500)
(≤1)
1
0a2
4.77
4.05
5.24
5.34
5.04
4.88
3.33
4.24
3.38
6.22
58.12
70.15
70.15
70.15
70.15
70.15
79.38
70.15
73.39
70.15
5
6
6
6
6
6
7
6
7
6
1
2
2
2
2
2
2
2
2
2
336.96
320.87
364.86
347.94
375.48
392.28
346.66
348.03
367.02
419.47
366.49
347.42
397.48
416.31
409.49
423.52
377.45
363.46
390.49
459.55
0
0
1
1
1
0
0
0
0
1
1
6a
6e
1
1
6i
6j
1
1
6n
6o
1
1
1
6p
6q
1
6r
1
2
3
4
5
LogP, logarithm of compound partition coefficient between n-octanol and water.
TPSA, topological polar surface area.
nON, number of hydrogen bond acceptors.
nOHNH, number of hydrogen bond donors.
MW, molecular weight.
In order to identify the oral bioavailability of these compounds, compound 16e
was further evaluated for in vivo PK profiles in male SD rats (n = 3), following a
single oral dose (25 mg/kg, n = 3) and an intravenous dose administrations (1.25
mg/kg, n = 3). As shown in Table 5, compound 16e shows reasonable oral exposure
-1
and bioavailability (AUC0-inf: 183 h·ng·mL , F: 22.5%), but a relative high Vss (26.6
L/kg) might lead to unexpected accumulation and potential toxicity risk for basic
molecule. The oral T1/2 of 25 mg/kg was approximate 2.25 h, suggesting a need for
further optimization to improve the exposure. Thus far, all the above results support
compound 16e to be worth of further investigation.

Table 5 Rat PK profiles of compound 16e
Dose (mg/kg)
Parameter
1.25 (iv)
25 (po)
0.32±0.07
2.25±0.5
0.33±0.14
78.6±16
-
-
1
Plasma
K (h )
1.03±0.12
0.68±0.07
-
el
T1/2 (h)
tmax (h)
Cmax (ng/mL)
58.0±16
80.8±27
34.9±8.9
39.2±9.4
18.0±4.2
30.6±6.4
557±155
0.79±0.08
26.6±9.16
-
C (ng/mL)
0
AUC (h·ng/mL)
157±62
183±55
313±111
553±85
-
0
-t
AUC0-inf (h·ng/mL)
AUMC (h·h·ng/mL)
0
-t
AUMC0-inf (h·h·ng/mL)
CL (mL/min/kg)
MRT (h)
3.18±0.99
-
Vss (L/kg)
F(%)
22.5±8.8
The PK study in male SD rats was carried out according to the standard procedures (iv, 1.25 mg/kg; po, 25 mg/kg).
Major parameters, including plasma clearance (CL), volume of distribution at steady state (Vss), T1/2, area under
the cure (AUC), and oral bioavailability (F), are reported.
3
. Conclusions
In summary, we reported the synthesis and characterization of a series novel
quinazoline derivatives containing S-acetamide and NH-acetamide moieties as new
anti-IAV agents. Most of them exhibit potent in vitro anti-IAV activity. The
subsequent SAR studies showed that nine compounds (10a2, 16a, 16e, 16i, 16j, 16n,
1
6o,16p and 16r) showed much stronger anti-influenza A activities with IC50 at
micromole level (1.29–9.04 μM). Particularly, 16e and 16r exhibited excellent
anti-IAV activity (IC :1.29 μM and 3.43 μM, respectively) and displayed acceptable
5
0
cytotoxicity. The preliminary mechanism studies imply that these quinazoline
derivatives exert their antiviral activity at the viral post-entry stage. In addition,
compound 16e with reasonable PK profiles has been selected as a potential candidate
for further investigation.
4
4
. Experimental section
.1. Chemistry

Melting points were obtained from an X4 micromelting point meter and the
1
13
temperature was uncorrected. H NMR and C NMR spectra were determined on a
on a Bruker AVANCE III 400 MHz, 500 MHz or 600 MHz spectrometer (Bruker Inc)
in DMSO-d , Methanol-d or CDCl using tetramethylsilane as an internal standard.
6
4
3
High resolution mass spectra were obtained on an Autospee Ultima-TOF spectrometer.
The reagents were all of analytical grade or chemically pure. TLC was performed on
silica gel plates (Merck, ART5554 60F254).
4
4
.2. Synthesis
.2.1. Ethyl 2-((2-chloroquinazolin-4-yl)thio)acetate 4
Quinazoline-2,4(1H,3H)-dione (2) and 2,4-diichloroquinazoline (3) were
synthesized according to previously reported methods[23-24].
To a stirred solution of 3 (2.6 g, 13 mmol) and DIPEA (3.4 g, 26 mmol) in THF
(25 mL), ethyl thioglycolate (1.56 g, 13 mmol) was added dropwise. The reaction
mixture was stirred at r.t. for 1.5 h. The reaction mixture was concentrated under
reduced pressure and purified by column chromatography (silica gel) eluting with
petroleum ether and ethyl acetate (15:1, v / v) to get the title compound 4 (2.9 g, 80%
1
from 3) as light yellow solid. H NMR (400 MHz, Chloroform-d) δ 8.13 – 8.08 (m,
1
4
1
H), 7.97 – 7.88 (m, 2H), 7.64 (ddd, J = 8.2, 6.2, 1.9 Hz, 1H), 4.32 (q, J = 7.1 Hz, 2H),
1
3
.17 (s, 2H), 1.38 (t, J = 7.1 Hz, 3H). C NMR (101 MHz, Chloroform-d) δ 172.83,
68.24, 155.59, 149.69, 135.01, 128.08, 127.75, 123.88, 121.88, 62.18, 32.53, 14.23.
4
.2.2. General procedure for the synthesis of compounds 7a–7i
Pyrrolidine (0.6 g, 8.4 mmol) was added to the suspension of 4 (1.2 g, 4.2 mmol)
and DIPEA (1.1 g, 8.4mmol) in THF (15 mL) and then the system was heated to 80℃
for 1 h. The solvent was removed by evaporation and the residue was purified by
column chromatography (silica gel) eluting with petroleum ether and ethyl acetate
(10:1, v / v) to get the title compound 5 (1.2 g, 90% from 4) as yellow solid. The solid
was then dissoved in THF (10 mL) and lithium hydroxide (2 eq.) in water (10 mL)
was added. The reaction mixture was stirred for 1.5 h at room temperature and
evaporated under vacuum. The residue in water (20 mL) was adjusted to pH 2 with 1

N HCl and the precipitated solid was filtered, washed with distilled water (20 mL) to
give compound 6 as an off-white solid.
A solution of compound 6 (99.8 mg, 0.34 mmol), 2-(7-azabenzotriazol-1-yl)-
N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (157.7 mg, 0.42 mmol)
in DMF (2 mL) was stirred at room temperature under the atmosphere of nitrogen for
5
min. The substituted amines (0.42 mmol) and N,N-diisopropylethylamine (DIPEA)
(0.70 mmol) was added to the mixture and stirred for 4 h at room temperature. The
mixture was diluted with water and extracted with ethyl acetate. The combined
extracts were washed by saturated brine, dried over Na SO . After filtration, the
2
4
solvent was removed under reduced pressure. The residue was purified by column
chromatography (silica gel) eluting with hexanes/EtOAc 1 : 1 to give the target
compounds 7a–7i.
4
.2.2.1. N-phenyl-2-((2-(pyrrolidin-1-yl)quinazolin-4-yl)thio)acetamide 7a. According
to the general procedure, employing 6 and aniline afforded compound 7a as a light
1
yellow solid, 67% yield, mp: 227–229℃ . H NMR (500 MHz, Chloroform-d) δ 9.10 (s,
1
7
3
1
1
H), 7.82 (d, J = 8.0 Hz, 1H), 7.63 (p, J = 9.0, 8.3 Hz, 2H), 7.42 (d, J = 8.0 Hz, 2H),
.32 – 7.23 (m, 2H), 7.16 (t, J = 7.5 Hz, 1H), 7.07 (t, J = 7.5 Hz, 1H), 4.08 (s, 2H),
1
3
.73 (d, J = 6.0 Hz, 4H), 2.00 (d, J = 6.0 Hz, 4H). C NMR (151 MHz, DMSO-d ) δ
6
69.95, 166.28, 156.17, 151.09, 139.57, 134.51, 129.24, 125.99, 124.18, 123.76,
22.13, 119.48, 117.69, 46.88, 34.65, 25.36. HRMS-ESI (m/z): Calcd. for
+
C H N OS(M+H) : 365.1431, Found: 365.1428.
20
21
4
4
.2.2.2. N-benzyl-2-((2-(pyrrolidin-1-yl)quinazolin-4-yl)thio)acetamide 7b. According
to the general procedure, employing 6 and benzylamine afforded compound 7b as a
1
yellow solid, 55% yield, mp: 190–192℃ . H NMR (500 MHz, DMSO-d ) δ 8.67 –
6
8
1
=
.57 (m, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.66 (t, J = 7.0 Hz, 1H), 7.45 (d, J = 8.0 Hz,
H), 7.31 – 7.09 (m, 5H), 4.29 (d, J = 6.0 Hz, 2H), 4.07 (d, J = 4.0 Hz, 2H), 3.52 (t, J
1
3
6.0 Hz, 4H), 1.87 (s, 4H). C NMR (151 MHz, DMSO-d ) δ 169.77, 167.49,
6
1
4
56.15, 139.66, 134.48, 128.63, 127.74, 127.24, 125.97, 124.26, 122.04, 117.80,
+
6.83, 43.13, 33.32, 25.43. HRMS-ESI (m/z): Calcd. for C H N OS (M+H) :
2
1
23
4

3
79.1587, Found: 379.1593.
4
.2.2.3. N-(2-ethoxyphenyl)-2-((2-(pyrrolidin-1-yl)quinazolin-4-yl)thio)acetamide 7c.
According to the general procedure, employing 6 and o-phenetidine afforded
1
compound 7c as a yellow solid, 57% yield, mp: 176–178℃ . H NMR (500 MHz,
DMSO-d ) δ 9.27 (s, 1H), 8.08 (d, J = 8.0 Hz, 1H), 7.85 (d, J = 8.0 Hz, 1H), 7.74 –
6
7
–
5
1
1
.62 (m, 1H), 7.47 (d, J = 8.5 Hz, 1H), 7.28 – 7.17 (m, 1H), 7.07 – 6.94 (m, 2H), 6.93
6.84 (m, 1H), 4.26 (s, 2H), 3.95 (d, J = 7.5 Hz, 2H), 3.57 (d, J = 6.5 Hz, 5H), 1.87 (s,
1
3
H), 1.12 – 1.03 (m, 3H). C NMR (151 MHz, DMSO-d ) δ 169.31, 166.58, 156.12,
6
51.30, 148.10, 134.76, 127.84, 126.05, 124.53, 124.18, 122.27, 120.81, 120.58,
17.66, 112.36, 64.27, 46.85, 34.09, 25.39, 14.58. HRMS-ESI (m/z): Calcd. for
+
C H N O S (M+H) : 409.1693, Found: 409.1702.
22
25
4
2
4
.2.2.4. N-(furan-2-ylmethyl)-2-((2-(pyrrolidin-1-yl)quinazolin-4-yl)thio)acetamide 7d.
According to the general procedure, employing 6 and furfurylamine afforded
1
compound 7d as a yellow solid, 60% yield, mp: 178–180℃. H NMR (500 MHz,
DMSO-d ) δ 8.61 (t, J = 6.0 Hz, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.65 (t, J = 8.0 Hz, 1H),
6
7
.56 (s, 1H), 7.44 (d, J = 8.5 Hz, 1H), 7.17 (t, J = 7.5 Hz, 1H), 6.38 (s, 1H), 6.21 (d, J
3.5 Hz, 1H), 4.29 (d, J = 5.5 Hz, 2H), 4.04 (s, 2H), 3.53 (d, J = 6.0 Hz, 4H), 1.91 (s,
=
1
3
4
1
2
3
H). C NMR (151 MHz, DMSO-d ) δ 169.80, 167.35, 156.10, 152.42, 151.12,
6
42.62, 134.46, 125.94, 124.19, 122.04, 117.73, 110.88, 107.52, 46.82, 36.47, 33.30,
+
5.47. HRMS-ESI (m/z): Calcd. for C H N O S (M+H) : 369.1380, Found:
1
9
21
4
2
69.1382.
4
.2.2.5. N-(naphthalen-2-yl)-2-((2-(pyrrolidin-1-yl)quinazolin-4-yl)thio)acetamide 7e.
According to the general procedure, employing 6 and 2-naphthylamine afforded
1
compound 7e as an off-white solid, 66% yield, mp: 236–238℃. H NMR (500 MHz,
Chloroform-d) δ 9.35 (s, 1H), 8.15 (s, 1H), 7.84 (d, J = 8.0 Hz, 1H), 7.78 – 7.72 (m,
3
H), 7.67 (s, 2H), 7.44 (t, J = 7.5 Hz, 1H), 7.38 (t, J = 7.5 Hz, 2H), 7.20 (s, 1H), 4.15
1
3
(
s, 2H), 3.76 (s, 4H), 1.98 (s, 4H). C NMR (151 MHz, DMSO-d ) δ 169.99, 166.57,
6

1
56.14, 151.06, 137.15, 134.55, 133.90, 130.23, 128.90, 127.94, 127.74, 126.93,
1
25.96, 125.10, 124.20, 122.18, 120.22, 117.68, 115.60, 46.88, 34.73, 25.32.
+
HRMS-ESI (m/z): Calcd. for C H N OS (M+H) : 415.1587, Found: 415.1619.
2
4
23
4
4
.2.2.6. N-cyclopropyl-2-((2-(pyrrolidin-1-yl)quinazolin-4-yl)thio)acetamide 7f.
According to the general procedure, employing 6 and cyclopropylamine afforded
1
compound 7f as an off-white solid, 72% yield, mp: 244–246℃ . H NMR (500 MHz,
Chloroform-d) δ 7.78 (d, J = 8.0 Hz, 1H), 7.60 (d, J = 8.0 Hz, 2H), 7.13 (t, J = 7.5 Hz,
1
2
H), 6.94 (s, 1H), 3.90 (s, 2H), 3.69 (d, J = 6.0 Hz, 4H), 2.65 (dq, J = 7.5, 3.9 Hz, 1H),
1
3
.03 (d, J = 6.0 Hz, 4H), 0.73 (d, J =7.0 Hz, 2H), 0.39 (d, J = 4.5 Hz, 2H). C NMR
(
151 MHz, DMSO-d ) δ 169.96, 168.31, 156.17, 151.11, 134.44, 125.98, 124.18,
6
1
22.06, 117.74, 46.86, 33.49, 25.44, 23.18, 6.01. HRMS-ESI (m/z): Calcd. for
+
C H N OS (M+H) : 329.1431, Found: 329.1420.
17
21
4
4
.2.2.7. N-(thiazol-2-yl)-2-((2-(pyrrolidin-1-yl)quinazolin-4-yl)thio)acetamide 7g.
According to the general procedure, employing 6 and 2-aminothiazole afforded
1
compound 7g as a brown solid, 63% yield, mp: 234–236℃. H NMR (500 MHz,
Chloroform-d) δ 11.02 – 10.71 (m, 1H), 7.80 (d, J = 8.0 Hz, 1H), 7.65 (d, J = 6.0 Hz,
2
H), 7.39 (d, J = 4.0 Hz, 1H), 7.16 (t, J = 7.5 Hz, 1H), 6.96 (d, J = 4.0 Hz, 1H), 4.15
(s, 2H), 3.76 (t, J = 6.0 Hz, 4H), 2.01 (d, J = 6.0 Hz, 4H). HRMS-ESI (m/z): Calcd.
+
for C H N OS (M+H) : 372.0948, Found: 372.0932.
1
7
18
5
2
4
.2.2.8. N-(2-ethoxybenzyl)-2-((2-(pyrrolidin-1-yl)quinazolin-4-yl)thio)acetamide 7h.
According to the general procedure, employing 6 and 2-ethoxybenzylamine afforded
1
compound 7h as a light yellow solid, 89% yield, mp: 169–171℃. H NMR (500 MHz,
DMSO-d ) δ 8.29 (s, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.66 (t, J = 8.0 Hz, 1H), 7.45 (d, J
6
=
8.5 Hz, 1H), 7.18 (t, J = 8.0 Hz, 2H), 7.08 (d, J = 7.5 Hz, 1H), 6.88 (d, J = 8.0 Hz,
1
2
H), 6.82 – 6.76 (m, 1H), 4.25 (d, J = 6.0 Hz, 2H), 4.06 (s, 2H), 3.90 (q, J = 7.0z Hz,
1
3
H), 3.49 (s, 4H), 1.94 – 1.77 (m, 4H), 1.22 (t, J = 7.0 Hz, 3H). C NMR (151 MHz,
DMSO-d ) δ 169.60, 167.46, 156.43, 156.09, 151.23, 134.50, 128.58, 128.43, 125.98,
6

1
24.23, 122.04, 120.28, 117.78, 111.76, 63.60, 46.77, 38.52, 33.23, 25.40, 15.05.
+
HRMS-ESI (m/z): Calcd. for C H N O S (M+H) : 423.1849, Found: 423.1866.
2
3
27
4
2
4
.2.2.9. N-(3,5-dichlorophenyl)-2-((2-(pyrrolidin-1-yl)quinazolin-4-yl)thio)acetamide
7
i. According to the general procedure, employing 6 and 3,5-dichloroaniline afforded
1
compound 7i as a pink solid, 65% yield, mp: 240–241℃. H NMR (500 MHz,
DMSO-d ) δ 10.67 (s, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.65 (s, 3H), 7.44 (d, J = 8.5 Hz,
6
1
4
1
H), 7.29 (s, 1H), 7.19 (t, J = 8.0 Hz, 1H), 4.23 (s, 2H), 3.52 – 3.43 (m, 4H), 1.82 (s,
1
3
H). C NMR (151 MHz, DMSO-d ) δ 169.72, 167.17, 156.15, 151.09, 141.81,
6
34.62 (d, J = 7.1 Hz), 126.02, 124.15, 123.04, 122.21, 117.63, 46.90, 34.64, 25.31.
+
HRMS-ESI (m/z): Calcd. for C H Cl N OS (M+H) : 433.0651, Found: 433.0675.
2
0
19
2
4
4
.2.3. General procedure for the synthesis of compounds 10a,10b
To a solution of 4 (1.3 g, 4.5 mmol) in THF (10 mL) was added lithium
hydroxide (0.38 g, 9.0 mmol) in 10 mL water and stirred for 1 h at room temperature.
The solvent was removed by evaporation, and then the residue was re-dissolved in
water (10 mL) and adjusted to pH 2 with 1 N HCl. The precipitated solid was filtered,
washed with distilled water (10 mL) to give compound 8 as a white solid .
A solution of compound 8 (0.46 g, 1.8 mmol), 2-(7-azabenzotriazol-1-yl)-
N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (0.81 g, 2.1 mmol) in
DCM (10 mL) was stirred at room temperature under the atmosphere of nitrogen for 5
min. Aniline or benzylamine (2.1 mmol) and N,N-diisopropylethylamine (DIPEA)
(3.6 mmol) was added to the mixture and stirred for 4 h at the same temperature. The
mixture was washed by water, saturated brine and dried over Na SO . After filtration,
2
4
the solvent was removed under reduced pressure and the residue was recrystallized
from ethyl acetate to obtain a yellow solid 9a and an off-white solid 9b.
Secondary amines (2 eq.) was added to the suspension of 9a or 9b ( 0.3 mmol) and
DIPEA (3 eq.) in THF (5 mL) and then the system was heated to 110℃for 8-12 h. The
mixture was concentrated under reduced pressure and then re-dissolved in
DCM/MeOH(10:1,v/v), washed by water, saturated brine and dried over Na SO .
2
4
After filtration, the solvent was removed under reduced pressure and the residue was

was recrystallized from ethyl acetate to give the target compounds 10a, 10b.
4
.2.3.1.
N-phenyl-2-((2-(3,4-dihydroisoquinolin-2(1H)-yl)quinazolin-4-yl)thio)acetamide
1
0a1.
According
to
the
general
procedure,
employing
9a
and
1
,2,3,4-tetrahydroisoquinoline afforded compound 10a1 as a yellow solid, 59% yield,
1
mp: 192–194℃ . H NMR (500 MHz, Chloroform-d) δ 8.79 (s, 1H), 7.85 (d, J = 8.0 Hz,
1
7
=
H), 7.66 (d, J = 7.0 Hz, 2H), 7.43 (d, J = 8.0 Hz, 2H), 7.25 – 7.19 (m, 4H), 7.18 –
.13 (m, 2H), 7.11 (d, J = 6.5 Hz, 1H), 7.06 (t, J = 7.5 Hz, 1H), 5.05 (s, 2H), 4.17 (t, J
1
3
6.0 Hz, 2H), 4.10 (s, 2H), 2.91 (t, J = 6.0 Hz, 2H). C NMR (151 MHz, DMSO-d )
6
δ 170.59, 166.13, 157.17, 150.71, 139.66, 135.29, 134.74, 134.49, 129.29, 128.79,
1
3
4
26.90, 126.64, 126.34, 126.22, 124.18, 123.79, 122.87, 119.46, 118.00, 46.27, 41.81,
+
4.74, 28.65. HRMS-ESI (m/z): Calcd. for C H N OS (M+H) : 427.1587, Found:
2
5
23
4
27.1600.
4
.2.3.2.
N-phenyl-2-((2-(diethylamino)quinazolin-4-yl)thio)acetamide
10a2.
According to the general procedure, employing 9a and diethylamine afforded
1
compound 10a2 as an off-white solid, 47% yield, mp: 180–181℃. H NMR (500 MHz,
Chloroform-d) δ 8.66 (s, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.63 (t, J = 7.5 Hz, 1H), 7.57
(s, 1H), 7.43 (d, J = 8.0 Hz, 2H), 7.28 (d, J = 8.0 Hz, 1H), 7.15 (t, J = 7.5 Hz, 1H),
7
.08 (t, J = 7.5 Hz, 1H), 4.08 (s, 2H), 3.74 (q, J = 7.0 Hz, 4H), 1.20 (t, J = 7.0 Hz, 6H).
1
3
C NMR (151 MHz, DMSO-d ) δ 169.94, 166.01, 156.78, 151.16, 139.66, 134.47,
6
1
29.21, 126.09, 124.08, 123.71, 122.16, 119.42, 117.64, 41.74, 34.40, 13.71.
+
HRMS-ESI (m/z): Calcd. for C H N OS (M+H) : 367.1587, Found: 367.1597.
2
0
23
4
4
.2.3.3. N-phenyl-2-((2-(4-methylpiperazin-1-yl)quinazolin-4-yl)thio)acetamide 10a3.
According to the general procedure, employing 9a and 1-methylpiperazine afforded
1
compound 10a3 as an off-white solid, 56% yield. mp: 228–230℃. H NMR (500 MHz,
Chloroform-d) δ 8.73 (s, 1H), 7.85 (d, J = 8.0 Hz, 1H), 7.66 (t, J = 8.0 Hz, 1H), 7.55
(d, J = 8.5 Hz, 1H), 7.42 (d, J = 8.0 Hz, 2H), 7.28 (d, J = 8.0 Hz, 2H), 7.21 (t, J = 7.5

Hz, 1H), 7.08 (t, J = 7.5 Hz, 1H), 4.04 (s, 2H), 3.98 (t, J = 5.0 Hz, 4H), 2.48 (t, J = 5.0
13
Hz, 4H), 2.32 (s, 3H). C NMR (151 MHz, DMSO-d ) δ 170.58, 166.07, 157.26,
6
1
5
3
50.63, 139.55, 134.73, 129.23, 126.21, 124.13, 123.78, 122.95, 119.41, 118.01,
+
4.79, 46.04, 43.84, 34.67. HRMS-ESI (m/z): Calcd. for C H N OS (M+H) :
2
1
24
5
94.1696, Found: 394.1704.
4
.2.3.4.
N-benzyl-2-((2-(3,4-dihydroisoquinolin-2(1H)-yl)quinazolin-4-yl)thio)acetamide 10b1.
According to the general procedure, employing 9b and 1,2,3,4-tetrahydroisoquinoline
1
afforded compound 10b1 as an off-white solid, 53% yield, mp: 230–232℃ . H NMR
(500 MHz, Chloroform-d) δ 7.82 (d, J = 8.0 Hz, 1H), 7.66 (s, 1H), 7.24 – 7.10 (m,
8
H), 7.07 (d, J = 6.5 Hz, 2H), 7.01 (s, 1H), 4.96 (s, 2H), 4.40 (d, J = 6.0 Hz, 2H), 4.08
(s, 2H), 4.02 (s, 2H), 2.88 (s, 2H), 1.56 (s, 2H). HRMS-ESI (m/z): Calcd. for
+
C H N OS (M+H) : 441.1744, Found: 441.1764.
26
25
4
4
.2.3.5.
N-benzyl-2-((2-(diethylamino)quinazolin-4-yl)thio)acetamide
10b2.
According to the general procedure, employing 9b and diethylamine afforded
1
compound 10b2 as an off-white solid, 50% yield, mp: 189–191℃ . H NMR (500 MHz,
Chloroform-d) δ 7.78 (d, J = 8.1 Hz, 1H), 7.58 (d, J = 8.0 Hz, 2H), 7.22 (d, J = 7.0 Hz,
3
H), 7.12 (d, J = 7.0 Hz, 3H), 6.97 (s, 1H), 4.43 (d, J = 6.0 Hz, 2H), 4.00 (s, 2H), 3.67
1
3
(
q, J = 7.0 Hz, 4H), 1.19 (t, J = 7.0 Hz, 6H). C NMR (151 MHz, DMSO-d ) δ
6
1
1
69.79, 167.34, 156.76, 151.26, 139.66, 134.42, 128.67, 127.74, 127.25, 126.09,
24.13, 122.08, 117.73, 43.13, 41.80, 33.16, 13.74. HRMS-ESI (m/z): Calcd. for
+
C H N OS (M+H) : 381.1744, Found: 381.1739.
21
25
4
4
.2.3.6. N-benzyl-2-((2-(4-methylpiperazin-1-yl)quinazolin-4-yl)thio)acetamide 10b3.
According to the general procedure, employing 9b and 1-methylpiperazine afforded
1
compound 10b3 as an off-white solid, 38% yield. mp: 180–181℃. H NMR (500 MHz,
Chloroform-d) δ 7.80 (d, J = 8.0 Hz, 1H), 7.63 (t, J = 7.5 Hz, 1H), 7.53 (d, J = 8.5 Hz,
1
H), 7.23 – 7.15 (m, 4H), 7.10 – 7.05 (m, 2H), 7.01 (d, J = 6.0 Hz, 1H), 4.42 (d, J =

1
3
6
.0 Hz, 2H), 3.97 (s, 2H), 3.92 (s, 4H), 2.44 (s, 4H), 2.32 (s, 3H). C NMR (151 MHz,
DMSO-d ) δ 170.44, 167.40, 157.21, 150.71, 139.66, 134.70, 128.69, 127.65, 127.25,
6
1
26.20, 124.19, 122.89, 118.09, 54.74, 46.01, 43.77, 43.06, 33.48. HRMS-ESI (m/z):
+
Calcd. for C H N OS (M+H) : 408.1853, Found: 408.1841.
2
2
26
5
4
.2.4. General procedure for the synthesis of compounds 16a–r
Glycine (30.84 g, 0.41 mol) was dissolved in methanol (200 mL) and
triethylamine (180 mL). Di-tert-butyl dicarbonate (179.11 g, 0.82 mol) was added and
the mixture was stirred at 70℃for 5 h. The solvent was evaporated and water (100 mL)
and EtOAc (150 mL) were added to the residue. The two-phase mixture was
transferred to a separatory funnel. The aqueous phase was extracted with EtOAc (2 X
8
0 mL). The organic phases were combined, dried over MgSO , filtered and the
4
solvent was removed by rotary evaporation to yield 12 as a light yellow solid.
HATU(1.2 eq.) was added to a solution of Boc-Gly-OH and DIPEA(2.0 eq.) in
DCM (20 mL), and various primary amines (1.2 eq.) followed by stirring for 3 hours
at room temperature. The mixture was washed with brine (two times for each), and
the organic layer was dried over sodium sulfate. After filtration, the filtrate was
concentrated under reduced pressure obtaining the crude amide 13a–i. The obtained
amide compound was deprotected of N-Boc by hydrogen chloride gas to give 14a–i.
To a solution of 3 (0.5 g, 2.5 mmol) and DIPEA (0.97 g, 7.5 mmol) dissolved in
THF (5 mL) was added 14a–i (1 eq.) and stirred for 3-5 h at room temperature. The
mixture was concentrated under reduced pressure and then dissolved in ethyl acetate
(
20 mL), washed by water, saturated brine and dried over Na SO . After filtration, the
2 4
solvent was removed under reduced pressure to obtain the target compounds 15a–i.
To a solution of 15a–i (1 e.q.) and DIPEA (3 e.q.) dissolved in DMF (4 mL) was
added secondary amines (2 e.q.) and stirred for 3-8 h at 100℃. After cooling to room
temperature, the mixture was diluted with ethyl acetate (20 mL), washed by water,
saturated brine and dried over Na SO , and concentrated under reduced pressure. The
2
4
residue was recrystallized from ethyl acetate to get the target compound 16a–r.

4
.2.4.1.
N-phenyl-2-((2-(pyrrolidin-1-yl)quinazolin-4-yl)amino)acetamide
16a.
According to the general procedure, employing 15a and pyrrolidine afforded
1
compound 16a as an off-white solid, 75% yield, mp: 250–252℃ . H NMR (500 MHz,
DMSO-d ) δ 10.24 – 10.20 (m, 1H), 8.42 (q, J = 5.5 Hz, 1H), 8.04 (d, J = 8.0 Hz, 1H),
6
7
.61 (d, J = 8.0 Hz, 2H), 7.48 (t, J = 8.0 Hz, 1H), 7.32 – 7.23 (m, 3H), 7.03 (q, J = 8.0
1
3
Hz, 2H), 4.19 (d, J = 5.5 Hz, 2H), 3.49 – 3.40 (m, 4H), 1.79 (s, 4H). C NMR (151
MHz, DMSO-d ) δ 169.05, 160.18, 157.79, 152.63, 139.69, 132.71, 129.09, 125.21,
6
1
23.52, 123.47, 120.19, 119.65, 110.85, 46.51, 45.33, 25.43. HRMS-ESI (m/z): Calcd.
+
for C H N O (M+H) : 348.1819, Found: 348.1846.
2
0
22
5
4
.2.4.2.
N-benzyl-2-((2-(pyrrolidin-1-yl)quinazolin-4-yl)amino)acetamide
16b.
According to the general procedure, employing 15b and pyrrolidine afforded
1
compound 16b as a light yellow solid, 70% yield, mp: 161–162℃ . H NMR (500 MHz,
DMSO-d ) δ 8.44 (t, J = 6.0 Hz, 1H), 8.37 – 8.29 (m, 1H), 8.00 (d, J = 8.0 Hz, 1H),
6
7
.48 (t, J = 8.0 Hz, 1H), 7.33 – 7.16 (m, 5H), 7.03 (t, J = 7.5 Hz, 1H), 4.29 (d, J = 6.0
1
3
Hz, 2H), 4.07 (d, J = 6.0 Hz, 2H), 3.47 (t, J = 6.0 Hz, 4H), 1.85 (s, 4H). C NMR
(
151 MHz, DMSO-d ) δ 170.11, 160.25, 157.84, 152.66, 140.13, 132.65, 128.50,
6
1
27.54, 127.05, 125.18, 123.62, 120.07, 111.00, 46.53, 44.73, 42.42, 25.53.
+
HRMS-ESI (m/z): Calcd. for C H N O (M+H) : 362.1976, Found: 362.1997.
2
1
24
5
4
.2.4.3. N-(2-ethoxyphenyl)-2-((2-(pyrrolidin-1-yl)quinazolin-4-yl)amino)acetamide
1
6c. According to the general procedure, employing 15c and pyrrolidine afforded
1
compound 16c as a white solid, 72% yield, mp: 147–148℃ . H NMR (500 MHz,
DMSO-d ) δ 9.03 (s, 1H), 8.59 (t, J = 6.0 Hz, 1H), 8.15 (d, J = 8.0 Hz, 1H), 8.02 (d, J
6
=
8.0 Hz, 1H), 7.56 – 7.47 (m, 1H), 7.31 (d, J = 8.5 Hz, 1H), 7.07 (t, J = 7.5 Hz, 1H),
.02 – 6.96 (m, 1H), 6.93 (d, J = 8.0 Hz, 1H), 6.88 (t, J = 7.5 Hz, 1H), 4.25 (d, J = 6.0
Hz, 2H), 3.88 (q, J = 7.0 Hz, 2H), 3.54 – 3.40 (m, 4H), 1.83 (d, J = 6.0 Hz, 4H), 0.95
7
1
3
(
t, J = 7.0 Hz, 3H). C NMR (151 MHz, DMSO-d ) δ 168.48, 160.05, 150.35, 148.01
6
(d, J = 18.3 Hz), 140.72, 133.53, 133.14, 127.75, 124.34, 124.17, 123.48, 120.34,

1
12.34, 110.41, 64.29, 46.80, 45.96, 25.36, 17.55, 14.48. HRMS-ESI (m/z): Calcd. for
+
C H N O (M+H) : 392.2081, Found: 392.2104.
22
26
5
2
4
.2.4.4. N-(furan-2-ylmethyl)-2-((2-(pyrrolidin-1-yl)quinazolin-4-yl)amino)acetamide
1
6d. According to the general procedure, employing 15d and pyrrolidine afforded
1
compound 16d as a light yellow solid, 80% yield, mp: 199–201℃ . H NMR (500 MHz,
DMSO-d ) δ 8.69 (s, 1H), 8.46 (t, J = 6.0 Hz, 1H), 8.06 (d, J = 8.0 Hz, 1H), 7.55 (d, J
6
=
7.0 Hz, 2H), 7.42 (d, J = 8.0 Hz, 1H), 7.12 (t, J = 8.0 Hz, 1H), 6.37 (s, 1H), 6.19 (d,
J = 3.0 Hz, 1H), 4.27 (d, J = 6.0 Hz, 2H), 4.05 (d, J = 6.0 Hz, 2H), 3.47 (s, 4H), 1.88
1
3
(
s, 4H). C NMR (151 MHz, DMSO-d ) δ 169.44, 160.05, 152.85, 142.46, 133.40,
6
1
23.86, 123.13, 121.36, 110.82, 110.65, 107.26, 46.89, 44.64, 35.92, 25.42.
+
HRMS-ESI (m/z): Calcd. for C H N O (M+H) : 352.1768, Found: 352.1795.
1
9
22
5
2
4
.2.4.5. N-(naphthalen-2-yl)-2-((2-(pyrrolidin-1-yl)quinazolin-4-yl)amino)acetamide
1
6e. According to the general procedure, employing 15e and pyrrolidine afforded
1
compound 16e as a white solid, 69% yield, mp: 169–171℃. H NMR (500 MHz,
DMSO-d ) δ 10.32 (s, 1H), 8.40 (t, J = 6.0 Hz, 1H), 8.28 (d, J = 2.0 Hz, 1H), 8.03 (d,
6
J = 8.0 Hz, 1H), 7.86 (d, J = 9.0 Hz, 1H), 7.82 (d, J = 8.0 Hz, 1H), 7.78 (d, J = 8.0 Hz,
1
7
5
1
1
2
H), 7.63 (dd, J = 9.0, 2.1 Hz, 1H), 7.50 (t, J = 8.0 Hz, 1H), 7.45 (t, J = 7.5 Hz, 1H),
.38 (t, J = 7.5 Hz, 1H), 7.29 (d, J = 8.0 Hz, 1H), 7.06 (t, J = 7.5 Hz, 1H), 4.25 (d, J =
13
.5 Hz, 2H), 3.50 – 3.40 (m, 4H), 1.76 (s, 4H). C NMR (151 MHz, DMSO-d ) δ
6
69.35, 160.23, 157.78, 152.66, 137.23, 133.92, 132.75, 130.16, 128.75, 127.92,
27.67, 126.85, 125.27, 124.96, 123.44, 120.52, 120.25, 115.67, 110.85, 46.51, 45.48,
+
5.40. HRMS-ESI (m/z): Calcd. for C H N O (M+H) : 398.1976, Found: 398.2012.
2
4
24
5
4
.2.4.6. N-cyclopropyl-2-((2-(pyrrolidin-1-yl)quinazolin-4-yl)amino)acetamide 16f.
According to the general procedure, employing 15f and pyrrolidine afforded
1
compound 16f as an off-white solid, 74% yield, mp: 210–212℃. H NMR (500 MHz,
DMSO-d ) δ 8.14 (t, J = 6.0 Hz, 1H), 8.00 (d, J = 4.0 Hz, 1H), 7.95 (d, J = 8.0 Hz,
6

1
H), 7.51 – 7.42 (m, 1H), 7.26 (d, J = 8.5 Hz, 1H), 7.02 (t, J = 7.5 Hz, 1H), 3.93 (d, J
6.0 Hz, 2H), 3.56 – 3.44 (m, 4H), 2.63 (m, 1H), 1.88 (d, J = 6.0 Hz, 4H), 0.60 (dt, J
=
=
1
3
7.0, 3.4 Hz, 2H), 0.45 – 0.31 (m, 2H). C NMR (151 MHz, DMSO-d ) δ 170.94,
6
1
2
3
60.13, 157.61, 152.23, 132.71, 124.98, 123.47, 120.25, 110.87, 46.57, 44.45, 25.48,
+
2.73, 6.03. HRMS-ESI (m/z): Calcd. for C H N O (M+H) : 312.1819, Found:
1
7
22
5
12.1828.
4
.2.4.7. N-(thiazol-2-yl)-2-((2-(pyrrolidin-1-yl)quinazolin-4-yl)amino)acetamide 16g.
According to the general procedure, employing 15g and pyrrolidine afforded
1
compound 16g as a light yellow solid, 65% yield, mp: 155–157℃ . H NMR (500 MHz,
DMSO-d ) δ 12.31 (s, 1H), 8.51 (q, J = 6.0 Hz, 1H), 8.03 (dd, J = 8.0, 3.5 Hz, 1H),
6
7
3
1
1
2
3
.49 (t, J = 8.0 Hz, 1H), 7.46 (d, J = 3.5 Hz, 1H), 7.27 (d, J = 8.0 Hz, 1H), 7.17 (d, J =
.5 Hz, 1H), 7.04 (t, J = 7.5 Hz, 1H), 4.28 (d, J = 5.5 Hz, 2H), 3.43 – 3.37 (m, 4H),
1
3
.77 (d, J = 6.5 Hz, 4H). C NMR (151 MHz, DMSO-d ) δ 169.30, 160.08, 158.58,
6
57.63, 152.61, 138.09, 132.81, 125.21, 123.55, 120.26, 113.69, 110.75, 46.44, 44.52,
+
5.40. HRMS-ESI (m/z): Calcd. for C H N OS (M+H) : 355.1336, Found:
1
7
19
6
55.1360.
4
.2.4.8. N-(2-ethoxybenzyl)-2-((2-(pyrrolidin-1-yl)quinazolin-4-yl)amino)acetamide
1
6h. According to the general procedure, employing 15h and pyrrolidine afforded
1
compound 16h as an off-white solid, 60% yield, mp: 170–172℃ . H NMR (500 MHz,
DMSO-d ) δ 8.32 (t, J = 6.0 Hz, 1H), 8.11 (t, J = 6.0 Hz, 1H), 8.03 – 7.93 (m, 1H),
6
7
.52 – 7.44 (m, 1H), 7.28 (d, J = 8.0 Hz, 1H), 7.20 – 7.15 (m, 1H), 7.14 – 7.11 (m,
H), 7.03 (t, J = 7.5 Hz, 1H), 6.89 (d, J = 8.0 Hz, 1H), 6.78 (t, J = 7.5 Hz, 1H), 4.24 (d,
1
J = 6.0 Hz, 2H), 4.07 (d, J = 6.0 Hz, 2H), 3.94 (q, J = 7.0 Hz, 2H), 3.45 (s, 4H), 1.84
1
3
(
s, 4H), 1.23 (t, J = 7.0 Hz, 3H). C NMR (151 MHz, DMSO-d ) δ 170.09, 160.22,
6
1
1
57.79, 156.32, 152.70, 132.71, 128.29, 128.01, 127.47, 125.22, 123.53, 120.18,
20.11, 111.61, 110.93, 63.58, 46.49, 44.86, 37.78, 25.50, 15.05. HRMS-ESI (m/z):
+
Calcd. for C H N O (M+H) : 406.2238, Found: 406.2249.
2
3
28
5
2

4
.2.4.9.
N-(3,5-dichlorophenyl)-2-((2-(pyrrolidin-1-yl)quinazolin-4-yl)amino)acetamide 16i.
According to the general procedure, employing 15i and pyrrolidine afforded
1
compound 16i as an off-white solid, 66% yield, mp: 185–187℃ . H NMR (500 MHz,
DMSO-d ) δ 10.44 (s, 1H), 8.44 (t, J = 5.5 Hz, 1H), 8.00 (d, J = 8.0 Hz, 1H), 7.68 (d,
6
J = 2.0 Hz, 2H), 7.50 (t, J = 8.0 Hz, 1H), 7.36 – 7.22 (m, 2H), 7.06 (t, J = 7.5 Hz,
1
3
1
H), 4.16 (d, J = 5.5 Hz, 2H), 3.41 (t, J = 6.0 Hz, 4H), 1.79 (s, 4H). C NMR (151
MHz, DMSO-d ) δ 169.90, 160.13, 157.52, 152.37, 141.92, 134.53, 132.87, 125.12,
6
1
23.44, 122.78, 120.40, 117.72, 110.73, 46.55, 45.59, 25.38. HRMS-ESI (m/z): Calcd.
+
for C H Cl N O (M+H) : 416.1040, Found: 416.1063.
2
0
19
2
5
4
.2.4.10.
N-phenyl-2-((2-(3,4-dihydroisoquinolin-2(1H)-yl)quinazolin-4-yl)amino)acetamide
1
6j.
According
to
the
general
procedure,
employing
15a
and
1
,2,3,4-tetrahydroisoquinoline afforded compound 16j as an off-white solid, 56%
1
yield, mp: 151–153℃ . H NMR (500 MHz, DMSO-d ) δ 10.26 (s, 1H), 8.53 (t, J = 5.4
6
Hz, 1H), 8.06 (d, J = 8.0 Hz, 1H), 7.67 (d, J = 8.0 Hz, 2H), 7.54 (t, J = 8.0 Hz, 1H),
7
.37 – 7.26 (m, 3H), 7.15 – 6.97 (m, 6H), 4.84 (s, 2H), 4.21 (d, J = 5.5 Hz, 2H), 3.96
1
3
(
t, J = 6.0 Hz, 2H), 2.69 (s, 2H). C NMR (151 MHz, DMSO-d ) δ 168.97, 160.46,
6
1
1
58.65, 152.24, 139.74, 135.43, 135.17, 132.96, 129.17, 128.88, 126.78, 126.39,
26.16, 125.48, 123.55, 123.43, 121.00, 119.59, 111.06, 46.24, 45.57, 41.51, 28.60.
+
HRMS-ESI (m/z): Calcd. for C H N O (M+H) : 410.1976, Found: 410.1984.
2
5
24
5
4
.2.4.11. N-phenyl-2-((2-morpholinoquinazolin-4-yl)amino)acetamide 16k. According
to the general procedure, employing 15a and morpholine afforded compound 16k as a
1
light yellow solid, 60% yield, mp: 228–230℃ . H NMR (500 MHz, DMSO-d ) δ
6
1
0.24 (t, J = 7.7 Hz, 1H), 8.56 (q, J = 5.5 Hz, 1H), 8.07 (dd, J = 8.0, 3.2 Hz, 1H), 7.60
d, J = 8.0 Hz, 2H), 7.53 (t, J = 8.0 Hz, 1H), 7.29 (t, J = 7.5 Hz, 3H), 7.12 (t, J = 7.5
Hz, 1H), 7.03 (t, J = 7.5 Hz, 1H), 4.16 (d, J = 5.5 Hz, 2H), 3.66 (t, J = 5.0 Hz, 4H),
(

1
3
3
.47 (t, J = 5.0 Hz, 4H). C NMR (151 MHz, DMSO-d ) δ 168.90, 160.46, 158.78,
6
1
52.09, 139.59, 132.98, 129.13, 125.54, 123.56, 123.49, 121.23, 119.61, 111.22,
+
6
6.57, 45.50, 44.55. HRMS-ESI (m/z): Calcd. for C H N O (M+H) : 364.1768,
2
0
22
5
2
Found: 364.1779.
4
.2.4.12.
N-phenyl-2-((2-(diethylamino)quinazolin-4-yl)amino)acetamide
16l.
According to the general procedure, employing 15a and diethylamine afforded
1
compound 16l as a light yellow solid, 49% yield, mp: 142–144℃ . H NMR (500 MHz,
DMSO-d ) δ 10.07 (s, 1H), 8.32 (t, J = 6.0 Hz, 1H), 8.06 – 7.92 (m, 1H), 7.61 (d, J =
6
8
.0 Hz, 2H), 7.53 – 7.44 (m, 1H), 7.27 (q, J = 8.0 Hz, 3H), 7.03 (dt, J = 12.5, 8.0 Hz,
1
3
2
H), 4.16 (d, J = 6.0 Hz, 2H), 3.54 (q, J = 7.0 Hz, 4H), 1.00 (s, 6H). C NMR (151
MHz, DMSO-d ) δ 168.91, 160.33, 158.20, 152.67, 139.75, 132.69, 129.09, 125.33,
6
1
23.45, 123.32, 120.21, 119.54, 110.72, 45.25, 41.32, 13.99. HRMS-ESI (m/z): Calcd.
+
for C H N O (M+H) : 350.1976, Found: 350.1999.
2
0
24
5
4
.2.4.13.
N-phenyl-2-((2-(4-methylpiperazin-1-yl)quinazolin-4-yl)amino)acetamide
1
6m. According to the general procedure, employing 15a and 1-methylpiperazine
1
afforded compound 16m as an off-white solid, 65% yield, mp: 189–191℃ . H NMR
(
500 MHz, DMSO-d ) δ 10.21 (d, J = 6.0 Hz, 1H), 8.50 (q, J = 5.0 Hz, 1H), 8.05 (d, J
6
=
8.0 Hz, 1H), 7.60 (d, J = 8.0 Hz, 2H), 7.52 (t, J = 8.0 Hz, 1H), 7.33 – 7.23 (m, 3H),
7
5
1
1
.09 (t, J = 7.5 Hz, 1H), 7.03 (t, J = 7.5 Hz, 1H), 4.16 (d, J = 5.5 Hz, 2H), 3.68 (t, J =
1
3
.0 Hz, 4H), 2.17 (t, J = 5.0 Hz, 4H), 2.09 (s, 3H). C NMR (151 MHz, DMSO-d ) δ
6
68.87, 160.43, 158.71, 152.17, 139.57, 132.94, 129.11, 125.48, 123.57, 123.39,
21.06, 119.65, 111.07, 54.94, 46.15, 45.48, 43.72. HRMS-ESI (m/z): Calcd. for
+
C H N O (M+H) : 377.2085, Found: 377.2090.
21
25
6
4
.2.4.14.
N-benzyl-2-((2-(3,4-dihydroisoquinolin-2(1H)-yl)quinazolin-4-yl)amino)acetamide
1
6n.
According
to
the
general
procedure,
employing
15b
and
1
,2,3,4-tetrahydroisoquinoline afforded compound 16n as a yellow solid, 58% yield,

1
mp: 102–104℃ . H NMR (500 MHz, DMSO-d ) δ 8.52 (t, J = 6.0 Hz, 1H), 8.42 (t, J =
6
6
7
4
.0 Hz, 1H), 8.02 (d, J = 8.0 Hz, 1H), 7.57 – 7.50 (m, 1H), 7.34 (d, J = 8.5 Hz, 1H),
.27 – 7.13 (m, 9H), 7.09 (t, J = 7.5 Hz, 1H), 4.90 (s, 2H), 4.31 (d, J = 6.0 Hz, 2H),
1
3
.11 (d, J = 6.0 Hz, 2H), 4.00 (t, J = 6.0 Hz, 2H), 2.81 (t, J = 6.0 Hz, 2H). C NMR
(
151 MHz, DMSO-d ) δ 170.07, 160.51, 158.64, 152.21, 140.02, 135.51, 135.22,
6
1
1
32.93, 129.04, 128.57, 127.53, 127.07, 126.87, 126.49, 126.32, 125.39, 123.57,
20.90, 111.20, 46.25, 44.86, 42.52, 41.52, 28.74. HRMS-ESI (m/z): Calcd. for
+
C H N O (M+H) : 424.2132, Found: 424.2129.
26
26
5
4
.2.4.15. N-benzyl-2-((2-morpholinoquinazolin-4-yl)amino)acetamide 16o. According
to the general procedure, employing 15b and morpholine afforded compound 1o as a
1
pink solid, 46% yield, mp: 224–225℃. H NMR (500 MHz, DMSO-d ) δ 8.47 (q, J =
6
6
.0 Hz, 2H), 8.03 (d, J = 8.0 Hz, 1H), 7.58 – 7.45 (m, 1H), 7.33 – 7.29 (m, 1H), 7.27
(d, J = 7.0 Hz, 2H), 7.24 – 7.19 (m, 3H), 7.11 (t, J = 7.5 Hz, 1H), 4.29 (d, J = 6.0 Hz,
13
2
H), 4.06 (d, J = 6.0 Hz, 2H), 3.69 (t, J = 5.0 Hz, 4H), 3.60 (t, J = 5.0 Hz, 4H). C
NMR (151 MHz, DMSO-d ) δ 169.95, 160.54, 158.89, 152.08, 140.08, 132.92,
6
1
28.61, 127.49, 127.09, 125.52, 123.60, 121.12, 111.38, 66.65, 44.82, 44.58, 42.42.
+
HRMS-ESI (m/z): Calcd. for C H N O (M+H) : 378.1925, Found: 378.1939.
2
1
24
5
2
4
.2.4.16.
N-benzyl-2-((2-(diethylamino)quinazolin-4-yl)amino)acetamide
16p.
According to the general procedure, employing 15b and diethylamine afforded
1
compound 16p as an off-white solid, 40% yield, mp: 222–224℃. H NMR (500 MHz,
DMSO-d ) δ 12.13 (s, 1H), 9.90 (s, 1H), 8.74 – 8.66 (m, 1H), 8.38 – 8.30 (m, 1H),
6
8
.04 (d, J = 8.0 Hz, 1H), 7.81 (t, J = 8.0 Hz, 1H), 7.44 (t, J = 8.0 Hz, 1H), 7.34 – 7.30
(
(
m, 2H), 7.29 – 7.24 (m, 3H), 4.32 (d, J = 5.5 Hz, 2H), 4.21 (d, J = 5.5 Hz, 2H), 3.70
1
3
s, 4H), 1.18 (s, 6H). C NMR (151 MHz, DMSO-d ) δ 168.34, 159.97, 139.77,
6
1
3
3
35.10, 128.66, 128.62, 127.75, 127.68, 127.26, 127.23, 124.56, 110.09, 44.89, 42.66,
+
8.16, 13.35. HRMS-ESI (m/z): Calcd. for C H N O (M+H) : 364.2132, Found:
2
1
26
5
64.2162.

4
.2.4.17.
N-benzyl-2-((2-(4-methylpiperazin-1-yl)quinazolin-4-yl)amino)acetamide
1
6q. According to the general procedure, employing 15b and 1-methylpiperazine
1
afforded compound 16q as a light yellow solid, 68% yield, mp: 129–131℃ . H NMR
(
500 MHz, DMSO-d ) δ 8.46 (t, J = 6.0 Hz, 1H), 8.38 (t, J = 6.0 Hz, 1H), 8.00 (d, J =
6
8
.0 Hz, 1H), 7.51 (t, J = 8.0 Hz, 1H), 7.27 (dd, J = 11.0, 8.0 Hz, 3H), 7.21 (d, J = 8.0
Hz, 3H), 7.08 (t, J = 7.5 Hz, 1H), 4.29 (d, J = 6.0 Hz, 2H), 4.05 (d, J = 6.0 Hz, 2H),
1
3
3
.73 (t, J = 5.0 Hz, 4H), 2.31 (t, J = 5.0 Hz, 4H), 2.20 (s, 3H). C NMR (151 MHz,
DMSO-d ) δ 170.03, 160.48, 158.82, 152.21, 140.04, 132.88, 128.62, 127.46, 127.09,
6
1
25.44, 123.53, 120.91, 111.20, 55.11, 46.36, 44.79, 43.81, 42.43. HRMS-ESI (m/z):
+
Calcd. for C H N O (M+H) : 391.2241, Found: 391.2229.
2
2
27
6
4
.2.4.17.
N-(naphthalen-2-yl)-2-((2-(3,4-dihydroisoquinolin-2(1H)-yl)quinazolin-4-yl)amino)ac
etamide 16r. According to the general procedure, employing 15e and
1
,2,3,4-tetrahydroisoquinoline afforded compound 16r as a light yellow solid, 62%
1
yield, mp: 237–239℃. H NMR (500 MHz, DMSO-d ) δ 10.72 (s, 1H), 8.68 (t, J = 5.7
6
Hz, 1H), 8.36 (s, 1H), 8.12 (d, J = 8.0 Hz, 1H), 7.85 (dd, J = 19.0, 8.5 Hz, 2H), 7.76 (t,
J = 7.0 Hz, 2H), 7.53 (t, J = 8.0 Hz, 1H), 7.45 (t, J = 7.5 Hz, 1H), 7.38 (t, J = 7.5 Hz,
1
6
2
1
1
4
4
H), 7.33 (d, J = 8.0 Hz, 1H), 7.11 (t, J = 7.5 Hz, 1H), 7.00 (t, J = 7.0 Hz, 1H), 6.97 –
.81 (m, 3H), 4.82 (s, 2H), 4.29 (d, J = 5.5 Hz, 2H), 3.95 (t, J = 6.0 Hz, 2H), 2.66 (s,
1
3
H). C NMR (151 MHz, DMSO-d ) δ 169.35, 160.47, 158.65, 152.16, 137.50,
6
35.39, 135.17, 133.96, 132.93, 130.16, 128.77, 128.70, 127.91, 127.64, 126.80,
26.78, 126.31, 126.02, 125.40, 124.87, 123.62, 120.97, 120.50, 115.52, 111.11,
+
6.22, 45.70, 41.50, 28.60. HRMS-ESI (m/z): Calcd. for C H N O (M+H) :
2
9
26
5
60.2132, Found: 460.2128.
4
.3 Viruses, cells and cytotoxicity assay
HEK293T-Gluc cells were generated by transfection of plasmid DNA
pLenti6-Gluc constitutively expressing the negative-strand RNA of Gaussia luciferase
Gluc) gene, that is converted into the positive strand upon IAV infection, and
(