Synthesis and Biological Evaluation of Substituted Indole and Its Analogs as Influenza A Virus Inhibitors

Article abstract of DOI:10.1002/cbdv.201800577

Influenza A virus (IAV), a highly pathogenic virus to human beings, is most susceptible to mutation and thus causes rapid, severe global pandemics resulting in millions of fatalities worldwide. Since resistance to the existing anti-influenza drugs is developing, innovative inhibitors with a different mode of action are urgently needed. The lead compound 6092B-E5 has proven to be an effective antiviral reagent in our previous work. Using the principles of substitution and bioisosterism of the indole ring, six series of novel anti-IAV target products were designed, synthesized and evaluated for their antiviral effect in this work. Compounds D₁, D₃, D₉, G₁, G₃, G₁₂ and G₂₃ were identified as promising anti-IAV candidates with excellent anti-IAV efficacy (IC₅₀ values of 3.06–5.77 μm) and low cytotoxicity (CC₅₀ values up to and beyond 100 μm). This work represents a successful application of the substitution and bioisosteric replacement strategy for the discovery of novel antiviral molecules that can be used for further structural optimization.

Full text of DOI:10.1002/cbdv.201800577

Title: Synthesis and biological evaluation of substituted indole and its
analogs as influenza A virus inhibitors
Authors: Xuandi Zhang, Guo-Ning Zhang, Yujia Wang, Mei Zhu, Juxian
Wang, Ziqiang Li, Donghui Li, Shan Cen, and Yucheng Wang
This manuscript has been accepted after peer review and appears as an
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content of this Accepted Article.
To be cited as: Chem. Biodiversity 10.1002/cbdv.201800577
Link to VoR: http://dx.doi.org/10.1002/cbdv.201800577

10.1002/cbdv.201800577
Chemistry & Biodiversity
Chem. Biodiversity
Synthesis and biological evaluation of substituted indole and its analogs as
influenza A virus inhibitors
Xuandi Zhanga, Guo-Ning Zhangb, Yujia Wangb, Mei Zhub, Juxian Wangb,Ziqiang Lia, Donghui Lia*,a, Shan
Cen*,b, Yucheng Wang*,b
a: School of Pharmacy, Jinzhou Medical University, Jinzhou, Liaoning 121001, China；
b: Institute of Medicinal Biotechnology, Chinese Academy of Medical Science and Peking Union Medical College, Beijing 100050, China；
*. Corresponding author: lidonghuilx@sina.com (to D. L.); shancen@imb.pumc.edu.cn (to S. C.); wangyucheng@imb.pumc.edu.cn. (to Y. W.)
Abstract Influenza A virus (IAV), a highly pathogenic virus to human beings, is most susceptible to mutation and thus causes rapid, severe global
pandemics resulting in millions of fatalities worldwide. Since resistance to the existing anti-influenza drugs is developing, innovative inhibitors with a
different mode of action are urgently needed. The lead compound 6092B-E5 has proven to be an effective antiviral reagent in our previous work. Using
the principles of substitution and bioisosterism of the indole ring, six series of novel anti-IAV target products were designed, synthesized and evaluated
for their antiviral effect in this work. Compounds D1, D3, D9, G1, G3, G12 and G23 were identified as promising anti-IAV candidates with excellent anti-IAV
efficacy (IC50 values of 3.06-5.77 μM) and low cytotoxicity (CC50 values up to and beyond 100 μM). This work represents a successful application of the
substitution and bioisosteric replacement strategy for the discovery of novel antiviral molecules that can be used for further structural optimization.
Keywords: Influenza A virus •substitution and bioisosteric replacement • indole• naphthol • coumarin
Introduction
The flu is an acute respiratory disease that was discovered in the 16th century and spreads quickly to the community during epidemics.[1] According to
statistics, there are 0.3-0.6 million respiratory deaths related to seasonal influenza annually, including approximately ten thousand flu-related respiratory
deaths in children under 5 years of age.[2-11] As major respiratory disease pathogens, influenza A viruses (IAV) cause sporadic infections or spread
worldwide in a pandemic when novel strains emerge in the human population after transmission from an animal host. All of the famous influenza
outbreaks in history were caused by the influenza A virus (IAV), including the Spanish flu in 1918, the Asian flu in 1957, the Hong Kong flu in 1968, the
Russian flu in 1977, the H5N1 avian flu in 2003, the H1N1 influenza in 2009 and the H7N9 avian flu in 2013.
Four classes of drugs are currently available for treatment. The first is the amantadine derivative M2 blocker, to which all currently circulating seasonal
influenza viruses are resistant.[12-14] The second class includes neuraminidase inhibitors (NAls): oseltamivir (high side effects), zanamivir and peramivir (for
severe cases). Although neuraminidase inhibitors are widely used, their effectiveness has been the subject of much debate.[15-17] Resistance to
neuraminidase inhibitors can occur through a variety of mechanisms.[18] The third and fourth types, membrane fusion inhibitors and RNA polymerase
inhibitors, both lead to significant side effects.[19] Controversial therapeutic data and potential resistance are the reasons driving the search for new
compounds.
In our previous study, the lead compound 6092B-E5 (Figure 1) and its derivatives were validated as good syncytial virus inhibitors (data not reported). As
the RSV and IAV are both negative-stranded RNA viruses, these RSV inhibitors were also tested for their anti-influenza A activity. Luckily, most of the
compounds derived from compound 6092B-E5 in Figure 1 exhibited moderate to excellent activity. This result inspired us to systematically further
research the anti-influenza A effect of these compounds.
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Figure 1. Structure of compound 6092B-E5
The N/O-containing heterocycles, such as coumarins and indoles, occur in both bioactive natural products and privileged structure in the synthetic
pharmaceutical chemistry frequently. Many successful efforts were made for the regioselective functionalization of aza-heterocycles under metal[20] and
metal-free[21] conditions. And the synthesis and bioactivities of fused[22] and conjuncted[23] aza-heteroaromatics were also well reported. Thus, according
to the principles of electron isosterism and skeletal transition, the core structure was replaced with 5-bromoindole, α-naphthol, β-naphthol, 4-
hydroxycoumarin, 6-hydroxycoumarin and 7-hydroxycoumarin to investigate the structure-activity relationship of indoles, respectively. The sulfur in the
chemicals were also replaced with its electron isostere, oxygen. Then, different anilines were selected to study the effect of halogen substitution on the
structure-activity relationship of aniline (Figure 2).
Figure 2. Six series of novel anti-IAV compounds designed
Results and Discussion
All of the target compounds were tested for their inhibitory effect on IAV at 10 μM, and the results are shown in Table 1. All compounds bearing a 5-
bromoindole core structure exhibit a moderate to high inhibition ratio to IAV infection, and compound D9 shows the most potent inhibition ratio of
92.48%. Compounds G1-30 are generated according to the principles of electron isosterism and skeletal transition. Most of these compounds shown
equivalent or decreased inhibition ratio to IAV infection, and compounds G1, G11, G23 and G24 were the most potent ones with an inhibition ratio above
80.00%. When the fragment of 5-bromoindole was substituted with 7-hydroxycoumarin, most of these compounds (G25, G26, G28 and G29) lost their anti-
IAV activity. When the core structure of compounds D3 and D9 was replaced with α-naphthol, β-naphthol, 4-hydroxycoumarin and 6-hydroxycoumarin,
their activity decreased slightly. However, a contrary result was observed for compounds D7 and D8. All of the α-naphthol, β-naphthol, 4-
hydroxycoumarin and 6-hydroxycoumarin series show anti-IAV effects comparable to the 5-bromoindole series.
Table 1. Inhibitory effect of the target compounds on IAV
Compound
R
IAV Inhibition Rate a(%)
R1=2-Me
R1=4-Me
R1=2-OMe
R1=4-OMe
D1
D2
D3
D4
85.82±4.09
48.01±7.14
80.27±7.11
79.99±4.75
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R1=3-Br
D5
D6
D7
51.77±2.47
73.40±10.75
55.17±8.51
67.87±12.15
92.48±5.75
84.44±7.64
62.35±8.27
75.80±3.23
56.40±5.83
70.34±8.88
61.00±5.63
64.99±4.11
68.20±9.04
74.53±8.81
65.00±2.67
87.98±9.69
77.91±7.38
19.29±4.14
75.39±13.54
R1=4-Br
R1=2-Cl
R1=4-F
D8
D9
G1
R1=4-OEt
R2=α-Naph R3=4-OEt R4=H
R2=α-Naph R3=2-Cl R4=H
G2
G3
R2=α-Naph R3=4-F R4=H
R2=α-Naph R3=2-OMe R4=H
R2=α-Naph R3=4-F R4=3-Cl
R2=α-Naph R3=4-Cl R4=H
G4
G5
G6
G7
R2=α-Naph R3=4-Br R4=H
R2=β-Naph R3=2-OMe R4=H
R2=β-Naph R3=2-Cl R4=H
G8
G9
G10
G11
G12
G13
G14
R2=β-Naph R3=4-OEt R4=H
R2=β-Naph R3=4-F R4=H
R2=β-Naph R3=4-F R4=3-Cl
R2=β-Naph R3=4-Cl R4=H
R2=4-hydroxycoumarin R3=4-F R4=H
R2=4-hydroxycoumarin R3=2-OMe R4=H
R2=4-hydroxycoumarin R3=4-OEt R4=H
R2=4-hydroxycoumarin R3=4-Cl R4=H
R2=4-hydroxycoumarin R3=4-Br R4=H
R2=4-hydroxycoumarin R3=4-F R4=3-Cl
R2=6-hydroxycoumarin R3=4-OEt R4=H
R2=6-hydroxycoumarin R3=4-F R4=H
R2=6-hydroxycoumarin R3=2-OMe R4=H
R2=6-hydroxycoumarin R3=4-F R4=3-Cl
R2=6-hydroxycoumarin R3=4-Cl R4=H
R2=7-hydroxycoumarin R3=4-F R4=H
R2=7-hydroxycoumarin R3=4-OEt R4=H
R2=7-hydroxycoumarin R3=2-Cl R4=H
R2=7-hydroxycoumarin R3=2-OMe R4=H
R2=7-hydroxycoumarin R3=4-F R4=3-Cl
R2=7-hydroxycoumarin R3=4-Cl R4=H
NA*
G15
G16
63.39±3.39
39.58±7.90
71.39±4.43
31.29±4.82
52.55±14.98
79.00±11.37
17.12±13.02
69.36±7.46
88.96±6.10
89.08±10.68
10.15±6.12
34.94±4.89
60.91±17.87
30.44±23.90
37.61±11.09
69.90±18.74
99.61±0.06
G17
G18
G19
G20
G21
G22
G23
G24
G25
G26
G27
G28
G29
G30
ribavirin
a: IAV inhibition rate is the average of three independent sets of data. NA*: Not Applicable
Among these molecules, 11 compounds that demonstrated a high inhibition rate (>60%) in the preliminary experiments were selected for dose-response
analysis. As shown in Figure. 3, all of the compounds exhibited a dose-dependent inhibition effect. Compounds D1, D3, D9, G1, G3, G12 and G23 exhibit
excellent anti-IAV effects with IC50 values of 3.06-5.77 μM (See Table 2). These compounds share a common 3-ethoxyaniline, 3-F aniline, 3-Cl aniline or 2-
Cl, 3-F aniline substituent group, which revealed that an electron-donating group at position 3 on the aniline served a major purpose for the IAV inhibitory
effect. Most of these compounds exhibit low cytotoxicity with CC50 values of up to and beyond 100 μM. Thus, compounds D1, D3, D9, G1, G3, G12 and G23
were identified as promising anti-IAV candidates.
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Figure 3. IAV inhibition effect of selected compounds at different concentrations
Table 2. IAV inhibition effect and cytotoxicity of selected compounds
Compound
IC50 (μM)
CC50 (μM)
Safety Index= CC50/IC50
4.41
3.98
3.06
5.77
53
12.02
10.35
D1
D3
41.2
35
11.44
NT*
D9
NT*
>100
>100
>100
NT*
>100
>100
>100
G1
4.49
12.11
4.76
>22.27
>8.26
>21.01
>NT*
>8.94
>20.88
>9.50
G3
G11
G12
G14
13.49
11.18
4.79
G17
G23
10.53
G24
NT*: not tested
In summary, 6 series of anti-IAV target products were designed and synthesized based on the substitution and bioisosteric replacement of the indole ring.
All of the 5-bromoindole derivatives demonstrate moderate to excellent anti-IAV activities, and the most potent compounds in this work, D3 and D9,
originated from this series. When the indole is replaced with its bioisosteres, analogues with α-naphthol, β-naphthol, 4-hydroxycoumarin and 6-
hydroxycoumarin replacements exhibit similar anti-IAV activities compared with the 5-bromoindole derivatives. This result suggests that our bioisosteric
replacement of the indole ring is a successful strategy for the chemotype enrichment of the antivirus hits that can be used for further structure
optimization. Compounds D1, D3, D9, G1, G3, G12 and G23 were identified as promising anti-IAV candidates.
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Conclusions
In this work, 6 series of novel anti-IAV products were designed and synthesized based on the substitution and bioisosterism of the indole ring. Most of
these compounds exhibit an anti-IAV inhibition ratio of 50% or more at 10 μM. All of the compounds in the first series bear a 5-bromoindole core structure
and exhibit good to excellent anti-IAV activities. Using the bioisosteric strategy, another 4 series of analogs were designed and synthesized. Among these,
3 series show similar anti-IAV activity when compared with the 5-bromoindole series, except for the 6-hydroxycoumarin series, which indicates that the
bioisosteric replacement of the indole ring is a successful strategy for the discovery of novel antiviral entities.
Experimental Section
Chemistry
Ethyl bromoacetate (59.88 mmol, 1equi), Na2S2O3·5H2O (71.86 mmol, 1.2equi), 30 ml of H2O and 90 ml of MeOH were added to a reaction flask with an
electromagnetic stirring bar, and the mixture was refluxed at 65 oC for 2 h. After completion of the reaction solvent is evaporated and the residue is
sonicated in 100 ml EtOH for 5 min and filtered under vacuum. The filtrate is evaporated to dryness to obtain the crude product A.
To a solution of A (22.53 mmol, 1.2 equi) in 30 ml of DMSO, was added 5-bromoindole (18.78 mmol, 1equi) and I2 (1.88 mmol, 0.1 equi), and the solvent
was stirred for 2h at 60 oC. After the completion of the reaction monitored by TLC, the solvent was diluted with EA, extracted with saturated Na2S2O3 and
saturated brine, and dried on anhydrous sodium sulfate. After removing the solvent, crude product B was afforded as a yellow solid.
NaOH (17.98 mmol, 2equi), EtOH 30 ml, H2O 7.5 ml were added to B (8.99 mmol, 1 equi) and stirred for about 30 min at room temperature. After the
reaction was completed, 6N HCl was added to adjust the pH≈3, most of the solvent was distilled off, the mixture was extracted with EA, and the solvent
was evaporated to give C.
To a solution of C (0.70 mmol, 1 equi), aniline (0.63 mmol, 0.9 equi) and HATU (0.77 mmol, 1.1 equi) in 10 ml of DCM, DIEA (1.68 mmol, 2.4 equi) was
added. The solvent was stirred for 3-5 h at room temperature until the completion of the reaction monitored by TLC.the solvent was diluted with DCM
and wahed with H2O and saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography to afford products D1-9
.
Scheme 1. Synthesis of Thioether Derivatives. Reagents and conditions: (a) MeOH, H2O, Na2S2O3, 65 oC, 2h. (b)5-Bromo indole, I2, DMSO, 60 oC, 1h. (c) EtOH: 10% sodium
hydroxide solution=4:1. (d) HATU, DIEA, aniline, RT, 2-3h.
Naphthol or coumarin (10 mmol, 1equi) was added to the reaction flask, ethyl bromoacetate (12 mmol, 1.2 equi), K2CO3 (40 mmol, 4 equi) and DMF were
added, and the reaction was performed at 65° C for about 2 h. TLC detection was used. After the reaction was completed, 100 ml of H2O was added to the
reaction solution and extracted with EA. The solvent was concentrate to afford E1-2(After replacing coumarin as the raw material, solids precipitated when
H2O was added, and E3-5 was obtained through filtration.).
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E1-5 was dissolved in a mixture of 12ml of EtOH and 3ml of 10% sodium hydroxide solution,and stirred at room temperature for about 30 min, after the
end of the reaction, 6N HCl was added to adjust the PH ≈ 3, most of the solvent was distilled off, and the remnant was extracted with ethyl acetate. The
organic layer was evaporated to dryness to afford F1-2(The reaction to replace the coumarin as a raw material is over. After adjusting the pH and remove
the solvent, it is directly filtered and washed with a small amount of EA to obtain F3-5).Add F1-5 (1 equi), aniline (1 equi), HATU (1.1 equi), DIEA (2.4 equi),
DCM (10ml) to the reaction flask and monitor at room temperature for 3-5 h. TLC monitoring. After the end of the reaction, spin down the solvent.
Chromatography to obtain the final product G1-13 (After the reaction of the substituted coumarin as a raw material, there are a lot of solids precipitated,
direct filtration can be washed with EA to get pure product G14-30).
Scheme 2. synthesis of compounds G1-30. Reagents and conditions: (a) K2CO3, DMF, 65 oC, 2h. (b) EtOH: 10% sodium hydroxyl=4:1 (v/v), RT, 30 min. (c) HATU, DIEA, anline,
DCM, RT, 3-5h.
2-((5-bromo-1H-indol-3-yl)thio)-N-(o-tolyl)acetamide:C(0.7mmol, 1equi), HATU (0.77mmol, 1.1equi), DIEA (1.68mmol, 2.4equi), 2-Methyl-1-
aminobenzene (0.63mmol, 0.9equi) were reacted at room temperature for 3-5 hours and the reaction was completed. After treatment with saturated
saline, column chromatography(P/E = 5:1) gave the title compound as D1 (48% yield). 1H NMR (600 MHz, Methanol-d4) δ 7.86 (d, J = 1.9 Hz, 1H), 7.49 (s,
1H), 7.32 (d, J = 8.5 Hz, 1H), 7.26 (dd, J = 8.6, 1.9 Hz, 1H), 7.18 (dd, J = 7.8, 1.5 Hz, 1H), 7.16 – 7.06 (m, 3H), 3.49 (s, 2H), 2.00 (s, 3H).13C NMR (151 MHz,
Methanol-d4) δ 171.4, 136.8, 136.6, 133.8, 133.7, 132.5, 131.5, 127.3, 127.2, 126.6, 126.1, 122.5, 114.6, 114.6, 103.9, 41.9, 17.8. HRMS (ESI): calcd for
C17H15BrN2OS (M-H+): 373.0016, found: 373.0010.
2-((5-bromo-1H-indol-3-yl)thio)-N-(p-tolyl)acetamide: The synthesis method is the same as D1. Column chromatography (P/E = 4:1) to give D2 (60% yield).
1H NMR (600 MHz, Methanol-d4) δ 7.77 (d, J = 1.8 Hz, 1H), 7.41 (s, 1H), 7.27 – 7.15 (m, 4H), 7.05 – 6.99 (m, 2H), 3.34 (s, 2H), 2.23 (s, 3H). 13C NMR (151 MHz,
Methanol-d4) δ 170.9, 136.8, 136.7, 135.1, 133.8, 132.6, 130.1, 126.0, 122.5, 121.5, 114.6, 114.5, 103.9, 42.6, 20.9. HRMS (ESI): calcd for C17H15BrN2OS (M-
H+): 373.0016, found: 373.0007.
2-((5-bromo-1H-indol-3-yl)thio)-N-(2-methoxyphenyl)acetamide: The synthesis method is the same as D1. Column chromatography (P/E = 5:1) to give D3
(54% yield). 1H NMR (600 MHz, DMSO-d6) δ 11.63 (s, 1H), 9.20 (s, 1H), 7.94 (d, J = 7.8 Hz, 1H), 7.77 (s, 1H), 7.66 – 7.57 (m, 1H), 7.39 (d, J = 8.5 Hz, 1H), 7.29 –
7.21 (m, 1H), 7.05 (dt, J = 16.2, 7.8 Hz, 2H), 6.89 (t, J = 7.5 Hz, 1H), 3.82 (s, 3H), 3.59 (s, 2H).13C NMR (151 MHz, DMSO-d6) δ 168.0, 149.5, 135.5, 133.1, 131.3,
127.5, 124.8, 124.7, 121.4, 121.3, 120.7, 114.6, 113.1, 111.5, 102.6, 56.2, 41.5. HRMS (ESI): calcd for C17H15BrN2O2S (M-H+): 388.9965, found: 388.9972.
2-((5-bromo-1H-indol-3-yl)thio)-N-(4-methoxyphenyl)acetamide: The synthesis method is the same as D1. Column chromatography (P/E = 2:1) to give D4
(38% yield). 1H NMR (600 MHz, Methanol-d4) δ 7.81 (d, J = 1.8 Hz, 1H), 7.46 (s, 1H), 7.31 – 7.20 (m, 4H), 6.81 (d, J = 9.0 Hz, 2H), 3.75 (s, 3H), 3.37 (s, 2H), 2.81
(s, 1H).13C NMR (151 MHz, Methanol-d4) δ 170.8, 158.0, 136.7, 133.8, 132.6, 132.4, 126.0, 123.2, 122.5, 114.9, 114.6, 114.5, 103.9, 55.8, 42.6. HRMS(ESI):
calcd for C17H15BrN2O2S (M-H+): 388.9965, found: 388.9979.
2-((5-bromo-1H-indol-3-yl)thio)-N-(3-bromophenyl)acetamide: The synthesis method is the same as D1. Column chromatography (P/E = 3:1) to give D5
(45% yield). 1H NMR (600 MHz, Methanol-d4) δ 7.76 (d, J = 1.9 Hz, 1H), 7.66 (t, J = 2.0 Hz, 1H), 7.45 (s, 1H), 7.30 – 7.25 (m, 2H), 7.20 (dt, J = 8.0, 1.7 Hz, 2H),
7.14 (t, J = 8.0 Hz, 1H), 3.37 (s, 2H).13C NMR (151 MHz, Methanol-d4) δ 171.2, 141.0, 136.7, 133.8, 132.6, 131.2, 128.0, 126.1, 123.9, 123.2, 122.5, 119.6, 114.6,
114.5, 103.7, 42.7. HRMS (ESI): calcd for C16H12Br2N2OS (M-H+): 436.8964, found: 436.8905
2-((5-bromo-1H-indol-3-yl)thio)-N-(4-bromophenyl)acetamide: The synthesis method is the same as D1. Column chromatography (P/E = 3:1) to give D6
(60% yield). 1H NMR (600 MHz, Methanol-d4) δ 7.76 (d, J = 1.9 Hz, 1H), 7.45 (s, 1H), 7.40 – 7.35 (m, 2H), 7.33 – 7.26 (m, 3H), 7.20 (dd, J = 8.6, 1.9 Hz, 1H), 3.37
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(s, 2H).13C NMR (151 MHz, Methanol-d4) δ 171.1, 138.8, 136.7, 133.8, 132.7, 126.0, 122.9, 122.5, 117.6, 114.6, 114.5, 103.7, 42.7. HRMS (ESI): calcd for
C16H12Br2N2OS (M-H+): 436.8964, found: 436.8907.
2-((5-bromo-1H-indol-3-yl)thio)-N-(2-chlorophenyl)acetamide: The synthesis method is the same as D1. Column chromatography (P/E = 4:1) to give D7
(8% yield). 1H NMR (600 MHz, Methanol-d4) δ 7.83 (d, J = 1.9 Hz, 1H), 7.76 (dd, J = 8.1, 1.6 Hz, 1H), 7.50 (s, 1H), 7.39 (dd, J = 8.0, 1.5 Hz, 1H), 7.34 – 7.21 (m,
3H), 7.13 (td, J = 7.8, 1.6 Hz, 1H), 3.53 (s, 2H).13C NMR (151 MHz, Methanol-d4) δ 171.1, 136.8, 135.6, 133.7, 132.3, 130.5, 128.4, 127.3, 127.2, 126.1, 125.8,
122.4, 114.6, 103.6, 42.3. HRMS (ESI): calcd for C16H12BrClN2OS (M-H+): 392.9469, found: 392.9480.
2-((5-bromo-1H-indol-3-yl)thio)-N-(4-fluorophenyl)acetamide: The synthesis method is the same as D1. Column chromatography (P/E = 4:1) to give D8
(72% yield).1H NMR (600 MHz, Methanol-d4) δ 7.81 – 7.73 (m, 1H), 7.46 (s, 1H), 7.37 – 7.33 (m, 2H), 7.29 (dd, J = 8.6, 0.5 Hz, 1H), 7.21 (dd, J = 8.6, 1.9 Hz, 1H),
7.07 – 6.93 (m, 2H), 3.38 (s, 2H). 13C NMR (151 MHz, Methanol-d4) δ 171.0, 160.8 (J = 243.11 Hz), 136.7, 135.7, 133.8, 132.7, 126.0, 123.3, 123.2, 122.5, 116.2,
116.1, 114.6, 114.5, 103.8, 42.6. HRMS (ESI): calcd for C16H12BrFN2OS (M-H+): 376.9765, found: 376.9757.
5-bromo-3-((2-((4-ethoxyphenyl)amino)-2-oxoethyl)thio)indol-1-ide: The synthesis method is the same as D1. Column chromatography (P/E = 2:1) to give
D9 (80% yield). 1H NMR (600 MHz, DMSO-d6) δ 11.74 – 11.50 (m, 1H), 9.76 (s, 1H), 7.79 – 7.73 (m, 1H), 7.59 (d, J = 2.8 Hz, 1H), 7.42 – 7.34 (m, 3H), 7.24 (dd, J
= 8.6, 2.2 Hz, 1H), 6.85 – 6.80 (m, 2H), 3.96 (q, J = 6.9 Hz, 2H), 1.29 (t, J = 7.0 Hz, 3H). 13C NMR (151 MHz, DMSO-d6) δ 167.1, 154.5, 135.0, 132.7, 131.9, 131.0,
124.3, 120.9, 120.8, 114.3, 114.0, 112.6, 102.4, 63.0, 41.2, 38.2, 14.7. HRMS (ESI): calcd for C18H17BrN2O2S (M-H+): 403.0121, found: 403.0113.
N-(4-ethoxyphenyl)-2-(naphthalen-1-yloxy)acetamide: The reaction flask was added with F1 (1mmol, 1equi), p-ethoxyaniline (1mmol, 1equi), HATU
(1.1mmol, 1.1equi), DIEA (2.4mmol, 2.4equi), and DCM 10ml and stirred for 3-5 h at room temperature. Then the solvent was removed under vacuum and
the crude product was purified by column chromatography (P/E = 10:1) to obtain G1 (82% yield) as a dark yellow solid. 1H NMR (400 MHz, Chloroform-d) δ
8.30 – 8.20 (m, 2H), 7.89 – 7.84 (m, 1H), 7.60 – 7.53 (m, 3H), 7.51 – 7.45 (m, 2H), 7.41 (t, J = 8.0 Hz, 1H), 6.92 – 6.86 (m, 3H), 4.81 (s, 2H), 4.02 (q, J = 7.0 Hz,
2H), 1.41 (t, J = 7.0 Hz, 3H). 13C NMR (151 MHz, Chloroform-d) δ 166.2, 156.4, 152.9, 134.8, 129.8, 128.1, 126.9, 126.0, 125.9, 125.3, 122.3, 122.1, 121.3, 115.0,
106.2, 68.2, 63.8, 15.0. HRMS (ESI): calcd for C20H19NO3 (M-H+): 320.1292, found: 320.1302.
N-(2-chlorophenyl)-2-(naphthalen-1-yloxy)acetamide: The synthesis method is the same as G1. Column chromatography (P/E = 10:1) to give G2 (78%
yield). 1H NMR (600 MHz, Chloroform-d) δ 9.19 (s, 1H), 8.54 (dd, J = 8.3, 1.6 Hz, 1H), 8.43 – 8.37 (m, 1H), 7.89 – 7.84 (m, 1H), 7.59 – 7.53 (m, 3H), 7.44 – 7.39
(m, 2H), 7.36 – 7.31 (m, 1H), 7.10 (ddd, J = 8.0, 7.4, 1.6 Hz, 1H), 6.89 (dd, J = 7.7, 0.9 Hz, 1H), 4.85 (s, 2H). 13C NMR (151 MHz, Chloroform-d) δ 166.3, 152.7,
134.8, 134.0, 129.3, 128.0, 128.0, 127.0, 126.1, 125.8, 125.3, 123.1, 122.3, 121.5, 121.4, 105.8, 67.9. HRMS (ESI): calcd for C18H14ClNO2 (M-H+): 310.0640,
found: 310.0657.
N-(4-fluorophenyl)-2-(naphthalen-1-yloxy)acetamide: The synthesis method is the same as G1. Column chromatography (P/E = 10:1) to give G3 (92%
yield). 1H NMR (600 MHz, Chloroform-d) δ 8.34 (s, 1H), 8.26 (dd, J = 8.1, 1.7 Hz, 1H), 7.90 – 7.84 (m, 1H), 7.62 – 7.53 (m, 5H), 7.40 (t, J = 8.0 Hz, 1H), 7.09 –
7.01 (m, 2H), 6.87 (d, J = 7.6 Hz, 1H), 4.79 (d, J = 1.8 Hz, 2H). 13C NMR (151 MHz, Chloroform-d) δ 166.44, 159.85 (J = 244.62 Hz) , δ 166.4, 159.9 (J = 244.62
Hz) , 152.8, 134.8, 132.9, 128.1, 127.0, 126.1, 125.9, 125.3, 122.4, 122.2, 122.1, 121.2, 116.0, 115.9, 106.3, 68.2. HRMS (ESI): calcd for C18H14FNO2 (M-H+):
294.0936, found: 294.0941.
N-(2-methoxyphenyl)-2-(naphthalen-1-yloxy)acetamide: The synthesis method is the same as G1. Column chromatography (P/E = 10:1) to give G4 (90%
yield). 1H NMR (600 MHz, Chloroform-d) δ 9.34 (s, 1H), 8.47 (dd, J = 8.0, 1.6 Hz, 1H), 8.42 – 8.38 (m, 1H), 7.90 – 7.83 (m, 1H), 7.62 – 7.51 (m, 3H), 7.41 (t, J =
7.9 Hz, 1H), 7.10 (td, J = 7.8, 1.6 Hz, 1H), 7.02 (td, J = 7.8, 1.3 Hz, 1H), 6.92 (dd, J = 8.1, 1.3 Hz, 1H), 6.87 (dd, J = 7.6, 0.9 Hz, 1H), 4.80 (s, 2H), 3.90 (s, 3H). 13C
NMR (151 MHz, Chloroform-d) δ 165.9, 152.8, 148.2, 134.8, 128.0, 127.1, 126.8, 125.9, 125.8, 125.3, 124.4, 122.0, 121.4, 121.3, 119.7, 110.2, 105.7, 67.7, 55.9
HRMS (ESI): calcd for C19H17NO3 (M-H+): 306.1136, found: 306.1124.
N-(3-chloro-4-fluorophenyl)-2-(naphthalen-1-yloxy)acetamide: The synthesis method is the same as G1. Column chromatography (P/E = 10:1) to give G5
(75% yield). 1H NMR (400 MHz, Chloroform-d) δ 8.34 – 8.21 (m, 2H), 7.91 – 7.85 (m, 1H), 7.79 (dd, J = 6.5, 2.7 Hz, 1H), 7.62 – 7.54 (m, 3H), 7.44 – 7.38 (m, 2H),
7
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Chem. Biodiversity
7.12 (t, J = 8.7 Hz, 1H), 6.88 (dd, J = 7.7, 0.9 Hz, 1H), 4.82 (s, 2H). 13C NMR (151 MHz, Chloroform-d) δ 166.6, 155.3 (J = 247.64 Hz)，152.8, 134.8, 133.5, 128.2,
127.0, 126.2, 125.9, 125.3, 122.6, 122.5, 121.1, 120.0, 120.0, 116.9 (J = 21.14 Hz), 106.3, 68.2. HRMS (ESI): calcd for C18H13ClFNO2 (M-H+): 328.0546, found:
308.0542.
N-(4-chlorophenyl)-2-(naphthalen-1-yloxy)acetamide: The synthesis method is the same as G1. Column chromatography (P/E = 10:1) to give G6 (87%
1
yield). H NMR (600 MHz, Chloroform-d) δ 8.35 (s, 1H), 8.28 – 8.22 (m, 1H), 7.87 (dd, J = 7.8, 1.7 Hz, 1H), 7.63 – 7.52 (m, 5H), 7.40 (t, J = 7.9 Hz, 1H), 7.35 –
7.28 (m, 2H), 6.86 (d, J = 7.7 Hz, 1H), 4.78 (s, 2H). 13C NMR (151 MHz, Chloroform-d) δ 166.5, 152.8, 135.5, 134.8, 130.1, 129.3, 128.1, 126.97, 126.1, 125.9,
125.2, 122.5, 121.5, 121.2, 106.3, 68.2. HRMS(ESI): calcd for C18H14ClNO2 (M-H+): 310.0640, found: 310.0660.
N-(4-bromophenyl)-2-(naphthalen-1-yloxy)acetamide: The synthesis method is the same as G1. Column chromatography (P/E = 10:1) to give G7 (72%
yield). 1H NMR (600 MHz, Chloroform-d) δ 8.34 (s, 1H), 8.28 – 8.22 (m, 1H), 7.93 – 7.82 (m, 1H), 7.62 – 7.54 (m, 3H), 7.52 – 7.45 (m, 4H), 7.40 (t, J = 8.0 Hz,
1H), 6.87 (d, J = 7.6 Hz, 1H), 4.80 (s, 2H). 13C NMR (151 MHz, Chloroform-d) δ 166.5, 152.8, 136.0, 134.8, 132.2, 128.1, 127.0, 126.2, 125.9, 125.3, 122.5, 121.8,
121.2, 117.7, 106.3, 68.3. HRMS (ESI): calcd for C18H14BrNO2 (M-H+): 354.0135, found: 354.0148.
N-(2-methoxyphenyl)-2-(naphthalen-2-yloxy)acetamide: The reaction flask was added with F2 (1mmol, 1equi), o-anisidine (1mmol, 1equi), HATU
(1.1mmol, 1.1equi), DIEA (2.4mmol, 2.4equi), and DCM 10ml at room temperature for 3-5 h. After spin-drying, solvent column chromatography (P/E =
10:1) was used to obtain G8 (78% yield). 1H NMR (600 MHz, Chloroform-d) δ 9.04 (s, 1H), 8.43 (dd, J = 8.0, 1.6 Hz, 1H), 7.85 – 7.75 (m, 3H), 7.48 (ddd, J = 8.2,
6.8, 1.3 Hz, 1H), 7.40 (ddd, J = 8.1, 6.8, 1.2 Hz, 1H), 7.28 (dd, J = 8.9, 2.6 Hz, 1H), 7.23 (d, J = 2.5 Hz, 1H), 7.09 (td, J = 7.8, 1.6 Hz, 1H), 7.00 (td, J = 7.8, 1.3 Hz,
1H), 6.89 (dd, J = 8.2, 1.4 Hz, 1H), 4.76 (s, 2H), 3.87 (s, 3H). 13C NMR (151 MHz, Chloroform-d) δ 166.1, 155.1, 148.4, 134.4, 130.1, 129.7, 127.8, 127.2, 126.9,
124.6, 124.5, 121.2, 120.1, 118.3, 110.3, 108.0, 68.0, 56.0. HRMS (ESI): calcd for C19H17NO3 (M-H+): 306.1136, found: 306.1123.
N-(2-chlorophenyl)-2-(naphthalen-2-yloxy)acetamide: The synthesis method is the same as G8. Column chromatography (P/E = 10:1) to give G9 (86%
yield). 1H NMR (600 MHz, Chloroform-d) δ 9.09 (s, 1H), 8.49 (dd, J = 8.2, 1.5 Hz, 1H), 7.86 – 7.75 (m, 3H), 7.49 (ddd, J = 8.2, 6.8, 1.3 Hz, 1H), 7.40 (td, J = 7.9,
1.3 Hz, 2H), 7.33 – 7.27 (m, 2H), 7.23 (d, J = 2.5 Hz, 1H), 7.08 (td, J = 7.7, 1.5 Hz, 1H), 4.78 (s, 2H). 13C NMR (151 MHz, Chloroform-d) 13C NMR (151 MHz,
Chloroform-d) δ 166.3, 154.9, 134.4, 133.97, 130.2, 129.8, 129.3, 128.0, 127.9, 127.2, 126.98, 125.2, 124.7, 123.3, 121.5, 118.2, 107.9, 67.8. HRMS (ESI): calcd
for C18H14ClNO2 (M-H+): 310.0640, found: 310.0657.
N-(4-ethoxyphenyl)-2-(naphthalen-2-yloxy)acetamide: The synthesis method is the same as G8. Column chromatography (P/E = 10:1) to give G10 (88%
yield). 1H NMR (400 MHz, Chloroform-d) δ 8.22 (s, 1H), 7.85 – 7.74 (m, 3H), 7.52 – 7.45 (m, 3H), 7.40 (ddd, J = 8.1, 6.8, 1.3 Hz, 1H), 7.29 – 7.20 (m, 3H), 6.91 –
6.85 (m, 2H), 4.73 (s, 2H), 4.02 (q, J = 7.0 Hz, 2H), 1.41 (t, J = 7.0 Hz, 3H). 13C NMR (151 MHz, Chloroform-d) δ 166.1, 156.4, 155.0, 134.4, 130.2, 129.9, 129.7,
127.8, 127.2, 127.0, 124.7, 122.1, 118.2, 115.0, 108.1, 67.8, 63.8, 15.0. HRMS (ESI): calcd for C20H19NO3 (M-H+): 320.1292, found: 320.1299.
N-(4-fluorophenyl)-2-(naphthalen-2-yloxy)acetamide: The synthesis method is the same as G8. Column chromatography (P/E = 10:1) to give G11 (86%
yield). 1H NMR (400 MHz, Chloroform-d) δ 8.30 (s, 1H), 7.85 – 7.75 (m, 3H), 7.60 – 7.55 (m, 2H), 7.49 (ddd, J = 8.2, 6.8, 1.3 Hz, 1H), 7.41 (ddd, J = 8.1, 6.9, 1.3
Hz, 1H), 7.28 – 7.21 (m, 3H), 7.09 – 7.02 (m, 2H), 4.74 (s, 2H). 13C NMR (151 MHz, Chloroform-d) δ 166.3 ,159.5(J = 244.62 Hz), 154.9, 134.4, 132.9, 130.3,
129.8, 127.9, 127.2, 127.1, 124.8, 122.2, 122.1, 118.1, 116.0, 115.9, 108.1, 67.7. HRMS (ESI): calcd for C18H14FNO2 (M-H+): 294.0936, found: 294.0936.
N-(3-chloro-4-fluorophenyl)-2-(naphthalen-2-yloxy)acetamide: The synthesis method is the same as G8. Column chromatography (P/E = 10:1) to give G12
(80% yield). 1H NMR (400 MHz, Chloroform-d) δ 8.31 (s, 1H), 7.86 – 7.74 (m, 4H), 7.53 – 7.38 (m, 3H), 7.28 – 7.20 (m, 3H), 7.12 (t, J = 8.7 Hz, 1H), 4.74 (s, 2H).
13C NMR (151 MHz, Chloroform-d) δ 166.3, 155.3 (J = 247.6 Hz), 154.8, 134.3, 133.6, 133.5, 130.3, 129.8, 127.9, 127.2, 127.1, 124.8, 122.5, 121.5, 121.4, 120.0,
119.9, 118.0, 117.0, 116.8, 108.1, 67.7. HRMS (ESI): calcd for C18H13ClFNO2 (M-H+): 328.0546, found: 328.0542.
N-(4-chlorophenyl)-2-(naphthalen-2-yloxy)acetamide: The synthesis method is the same as G8. Column chromatography (P/E = 10:1)to give G13 (85%
yield). 1H NMR (400 MHz, Chloroform-d) δ 8.33 (s, 1H), 7.86 – 7.73 (m, 3H), 7.59 – 7.55 (m, 2H), 7.49 (d, J = 1.3 Hz, 1H), 7.44 – 7.38 (m, 1H), 7.35 – 7.30 (m, 2H),
8
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Chemistry & Biodiversity
Chem. Biodiversity
7.28 – 7.24 (m, 1H), 7.21 (d, J = 2.6 Hz, 1H), 4.73 (s, 2H). 13C NMR (151 MHz, Chloroform-d) 13C NMR (151 MHz, Chloroform-d) δ 166.2, 154.7, 135.4, 134.2,
130.2, 130.0, 129.7, 129.2, 127.7, 127.1, 126.9, 124.7, 121.4, 117.9, 108.0, 67.6. HRMS (ESI): calcd for C18H14ClNO2 (M-H+): 310.0640, found: 310.0654.
N-(4-fluorophenyl)-2-((2-oxo-2H-chromen-4-yl)oxy)acetamide: The reaction flask was added with F3 (1mmol, 1equi), Fluoroaniline (1mmol, 1equi), HATU
(1.1mmol, 1.1equi), DIEA (2.4mmol, 2.4equi), and DCM 10ml at room temperature for 3-5 h. After spin-drying, solvent column chromatography (P/E =
10:1) was used to obtain G14 (75% yield). 1H NMR (600 MHz, DMSO-d6) δ 10.32 (s, 1H), 7.96 (dd, J = 7.9, 1.6 Hz, 1H), 7.69 (ddd, J = 8.3, 7.3, 1.6 Hz, 1H), 7.66
– 7.61 (m, 2H), 7.52 – 7.34 (m, 2H), 7.27 – 7.11 (m, 2H), 5.90 (s, 1H), 5.02 (s, 2H). 13C NMR (151 MHz, DMSO-d6) δ 164.6, 164.4, 161.4, 158.3 (J = 240.10 Hz)，
152.7, 134.5, 132.9, 124.2, 123.3, 121.6, 121.5, 116.4, 115.5, 115.3, 115.0, 91.3, 67.6. HRMS (ESI): calcd for C17H12FNO4 (M-H+): 312.0678, found: 312.0666.
N-(2-methoxyphenyl)-2-((2-oxo-2H-chromen-4-yl)oxy)acetamide: The synthesis method is the same as G14. Column chromatography (P/E = 10:1) to give
G15 (80% yield). 1H NMR (600 MHz, DMSO-d6) δ 9.47 (s, 1H), 8.01 (d, J = 7.9 Hz, 1H), 7.95 (d, J = 7.8 Hz, 1H), 7.74 – 7.67 (m, 1H), 7.46 (t, J = 7.4 Hz, 2H), 7.17 –
7.07 (m, 2H), 6.95 (td, J = 7.6, 1.6 Hz, 1H), 5.94 (s, 1H), 5.07 (s, 2H), 3.88 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 164.5, 164.1, 161.4, 152.8, 149.4, 133.0,
126.3, 125.0, 124.3, 122.8, 121.3, 120.4, 116.5, 115.0, 111.3, 91.5, 67.7, 55.8. HRMS (ESI): calcd for C18H15NO5 (M-H+): 324.0877, found: 324.0877.
N-(4-ethoxyphenyl)-2-((2-oxo-2H-chromen-4-yl)oxy)acetamide: The synthesis method is the same as G14. Column chromatography (P/E = 10:1) to give
G16 (85% yield). 1H NMR (600 MHz, DMSO-d6) δ 10.12 (s, 1H), 7.97 (dd, J = 7.9, 1.5 Hz, 1H), 7.69 (ddd, J = 8.7, 7.3, 1.6 Hz, 1H), 7.54 – 7.48 (m, 2H), 7.45 – 7.37
(m, 2H), 6.93 – 6.86 (m, 2H), 5.89 (s, 1H), 4.98 (s, 2H), 3.99 (q, J = 7.0 Hz, 2H), 1.31 (t, J = 7.0 Hz, 3H). 13CNMR(151MHz,DMSO-d6) δ 164.6, 164.0, 161.4,
154.9, 152.7, 132.9, 131.1, 124.2, 123.3, 121.3, 116.4,115.0, 114.4, 91.2, 67.7, 63.1, 14.6. HRMS (ESI): calcd for C19H17NO5 (M-H+): 338.1034, found: 338.1030.
N-(4-chlorophenyl)-2-((2-oxo-2H-chromen-4-yl)oxy)acetamide: The synthesis method is the same as G14. Column chromatography (P/E = 10:1) to give G17
(74% yield). 1H NMR (600 MHz, DMSO-d6) δ 10.40 (s, 1H), 7.95 (dd, J = 8.0, 1.7 Hz, 1H), 7.72 – 7.63 (m, 3H), 7.46 – 7.37 (m, 4H), 5.90 (s, 1H), 5.03 (s, 2H). 13
C
NMR (151 MHz, DMSO-d6) 13C NMR (151 MHz, DMSO-d6) δ 164.7, 164.5, 161.4, 152.7, 137.1, 132.9, 128.7, 127.5, 124.2, 123.3, 121.2, 116.4, 115.0, 91.3, 67.6.
HRMS (ESI): calcd for C17H12ClNO4 (M-H+): 328.0382, found: 328.0374.
N-(4-bromophenyl)-2-((2-oxo-2H-chromen-4-yl)oxy)acetamide: The synthesis method is the same as G14. Column chromatography (P/E = 10:1) to give
G18 (69% yield). 1H NMR (600 MHz, DMSO-d6) δ 10.40 (s, 1H), 7.95 (dd, J = 7.9, 1.6 Hz, 1H), 7.70 (ddd, J = 8.4, 7.3, 1.6 Hz, 1H), 7.63 – 7.57 (m, 2H), 7.55 – 7.51
(m, 2H), 7.46 – 7.39 (m, 2H), 5.90 (s, 1H), 5.03 (s, 2H). 13C NMR (151 MHz, DMSO-d6) δ 164.7, 164.5, 161.4, 152.7, 137.6, 132.9, 131.7, 124.2, 123.3, 121.6,
116.4, 115.5, 115.0, 91.3, 67.6. HRMS (ESI): calcd for C17H12BrNO4 (M-H+): 371.9877, found: 371.9862.
N-(3-chloro-4-fluorophenyl)-2-((2-oxo-2H-chromen-4-yl)oxy)acetamide: The synthesis method is the same as G14. Column chromatography (P/E = 10:1)
to give G19 (84% yield). 1H NMR (600 MHz, DMSO-d6) δ 10.45 (s, 1H), 8.00 – 7.94 (m, 1H), 7.92 (dd, J = 6.9, 2.7 Hz, 1H), 7.76 – 7.63 (m, 1H), 7.58 – 7.48 (m,
1H), 7.47 – 7.35 (m, 3H), 5.91 (s, 1H), 5.03 (s, 2H). 13C NMR (151 MHz, DMSO-d6) 13C NMR (151 MHz, DMSO-d6) δ 164.8, 164.5, 161.4, 153.4 (J = 244.6 Hz),
152.7, 135.4, 135.4, 132.9, 124.2, 123.3, 121.2, 120.1, 119.2, 117.0, 116.4, 115.0, 91.3, 67.5. HRMS (ESI): calcd for C17H11ClFNO4 (M-H+): 346.0288, found:
346.0277.
N-(4-ethoxyphenyl)-2-((2-oxo-2H-chromen-6-yl)oxy)acetamide: The reaction flask was added with F4 (1mmol, 1equi), P-ethoxyaniline (1mmol, 1equi),
HATU (1.1mmol, 1.1equi), DIEA (2.4mmol, 2.4equi), and DCM 10ml at room temperature for 3-5 h. After spin-drying, solvent column chromatography
(P/E = 10:1) was used to obtain G20 (80% yield). 1H NMR (600 MHz, DMSO-d6) δ 9.95 (s, 1H), 8.05 – 7.99 (m, 1H), 7.55 – 7.49 (m, 2H), 7.39 (d, J = 8.9 Hz, 1H),
7.35 – 7.27 (m, 2H), 6.91 – 6.84 (m, 2H), 6.50 (d, J = 9.6 Hz, 1H), 4.72 (s, 2H), 3.98 (q, J = 7.0 Hz, 2H), 1.30 (t, J = 7.0 Hz, 3H). 13C NMR (151 MHz, DMSO-d6) 13
C
NMR (151 MHz, DMSO-d6) δ 165.7, 160.0，154.8, 154.2, 148.2, 144.0, 131.2, 121.4, 119.9, 119.1, 117.4, 116.7, 114.3, 112.0, 67.6, 63.1, 14.7. HRMS (ESI):
calcd for C19H17NO5 (M-H+): 338.1034, found: 338.1046.
N-(4-fluorophenyl)-2-((2-oxo-2H-chromen-6-yl)oxy)acetamide: The synthesis method is the same as G20. Column chromatography (P/E = 10:1) to give G21
(79% yield). 1H NMR (600 MHz, DMSO-d6) δ 10.16 (s, 1H), 8.02 (d, J = 9.6 Hz, 1H), 7.70 – 7.62 (m, 2H), 7.38 (d, J = 8.8 Hz, 1H), 7.35 – 7.28 (m, 2H), 7.17 (t, J =
9
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10.1002/cbdv.201800577
Chemistry & Biodiversity
Chem. Biodiversity
8.9 Hz, 2H), 6.49 (d, J = 9.5 Hz, 1H), 4.76 (s, 2H). 13C NMR (151 MHz, DMSO-d6) δ 166.2, 160.0, 158.3 (J = 240.10 Hz), 154.1, 148.2, 144.0, 134.7, 134.7，
121.6, 121.6, 119.9, 119.1, 117.4, 116.7, 115.3, 115.2, 112.0, 67.5 HRMS (ESI): calcd for C17H12FNO4 (M-H+): 312.0678, found: 312.0681.
N-(2-methoxyphenyl)-2-((2-oxo-2H-chromen-6-yl)oxy)acetamide: The synthesis method is the same as G20. Column chromatography (P/E = 10:1) to give
G22 (76% yield). 1H NMR (400 MHz, DMSO-d6) δ 9.24 (s, 1H), 8.04 (dd, J = 10.8, 8.8 Hz, 2H), 7.44 – 7.29 (m, 3H), 7.15 – 7.04 (m, 2H), 6.93 (ddd, J = 8.7, 7.1,
1.8 Hz, 1H), 6.51 (d, J = 9.6 Hz, 1H), 4.82 (s, 2H), 3.86 (s, 3H). 13C NMR (151 MHz, Chloroform-d) δ 165.9, 160.0, 153.8, 149.1, 148.3, 144.0, 126.4, 124.7, 120.9,
120.4, 119.9, 119.2, 117.5, 116.8, 112.2, 111.1, 67.6， 55.8. HRMS (ESI): calcd for C18H15NO5 (M-H+): 324.0877, found: 324.0876.
N-(3-chloro-4-fluorophenyl)-2-((2-oxo-2H-chromen-6-yl)oxy)acetamide: The synthesis method is the same as G20. Column chromatography (P/E = 10:1)
to give G23 (86% yield). 1H NMR (600 MHz, DMSO-d6) δ 10.32 (s, 1H), 8.03 (d, J = 9.6 Hz, 1H), 7.96 (dd, J = 6.9, 2.8 Hz, 1H), 7.58 (ddd, J = 9.1, 4.4, 2.6 Hz, 1H),
7.40 (t, J = 8.9 Hz, 2H), 7.36 – 7.25 (m, 2H), 6.50 (d, J = 9.6 Hz, 1H), 4.77 (s, 2H). 13C NMR (151 MHz, DMSO-d6) 13C NMR (151 MHz, DMSO-d6) δ 166.6, 160.0,
153.3(J = 243.1 Hz), 154.0, 148.2, 144.0, 135.5, 121.2, 120.1, 119.9, 119.1, 119.0, 117.4, 117.0, 116.9, 116.7, 112.0, 67.5. HRMS (ESI): calcd for C17H11ClFNO4
(M-H+): 346.0288, found: 346.0274.
N-(4-chlorophenyl)-2-((2-oxo-2H-chromen-6-yl)oxy)acetamide: The synthesis method is the same as G20. Column chromatography (P/E = 10:1) to give G24
(75% yield). 1H NMR (600 MHz, DMSO-d6) δ 10.25 (s, 1H), 8.03 (d, J = 9.5 Hz, 1H), 7.70 (d, J = 2.1 Hz, 1H), 7.69 (d, J = 2.2 Hz, 1H), 7.39 (dd, J = 8.7, 1.3 Hz, 3H),
7.35 – 7.29 (m, 2H), 6.50 (d, J = 9.5 Hz, 1H), 4.78 (s, 2H). 13C NMR (151 MHz, DMSO-d6) δ 166.4, 160.0, 154.1, 148.2, 143.9, 137.2, 128.6, 127.3, 121.3, 119.9,
119.1, 117.3, 116.6, 112.0, 67.6. HRMS (ESI): calcd for C17H12ClNO4 (M-H+): 328.0382, found: 328.0380.
N-(4-fluorophenyl)-2-((2-oxo-2H-chromen-7-yl)oxy)acetamide: The reaction flask was added with F5 (1mmol, 1equi), Fluoroaniline (1mmol, 1equi), HATU
(1.1mmol, 1.1equi), DIEA (2.4mmol, 2.4equi), and DCM 10ml at room temperature for 3-5 h. After spin-drying, solvent column chromatography (P/E =
10:1) was used to obtain G25 (84% yield). 1H NMR (600 MHz, DMSO-d6) δ 10.25 (s, 1H), 8.00 (d, J = 9.5 Hz, 1H), 7.71 – 7.62 (m, 3H), 7.21 – 7.13 (m, 2H), 7.04
(d, J = 7.6 Hz, 2H), 6.31 (d, J = 9.5 Hz, 1H), 4.84 (s, 2H). 13C NMR (151 MHz, DMSO-d6) δ 165.7, 160.9, 160.2, 158.3(J = 240.1 Hz), 155.1, 144.2, 134.7, 129.5,
121.5, 115.3, 112.9, 112.7, 101.7, 67.2. HRMS (ESI): calcd for C17H12FNO4 (M-H+): 312.0678, found: 312.0678.
N-(4-ethoxyphenyl)-2-((2-oxo-2H-chromen-7-yl)oxy)acetamide: The synthesis method is the same as G25. Column chromatography (P/E = 10:1) to give
1
G26 (88% yield). H NMR (600 MHz, DMSO-d6) δ 10.01 (s, 1H), 8.00 (d, J = 9.5 Hz, 1H), 7.66 (d, J = 8.3 Hz, 1H), 7.56 – 7.48 (m, 2H), 7.04 (d, J = 8.3 Hz, 2H),
6.92 – 6.84 (m, 2H), 6.31 (d, J = 9.5 Hz, 1H), 4.80 (s, 2H), 3.98 (q, J = 6.9 Hz, 2H), 1.30 (t, J = 6.9 Hz, 3H). 13C NMR (151 MHz, DMSO) δ 165.3, 160.9, 160.2,
155.1, 154.8, 144.2, 131.2, 129.5, 121.3, 114.3, 112.8, 112.8, 112.7, 101.6, 67.3, 63.1, 14.6. HRMS (ESI): calcd for C19H17NO5 (M-H+): 338.1034, found: 338.1018.
N-(2-chlorophenyl)-2-((2-oxo-2H-chromen-7-yl)oxy)acetamide: The synthesis method is the same as G25. Column chromatography (P/E = 10:1) to give G27
(83% yield). 1H NMR (600 MHz, DMSO-d6) δ 9.76 (s, 1H), 8.01 (d, J = 9.5 Hz, 1H), 7.79 (dd, J = 8.1, 1.6 Hz, 1H), 7.72 – 7.64 (m, 1H), 7.53 (dd, J = 8.0, 1.5 Hz,
1H), 7.36 (td, J = 7.8, 1.5 Hz, 1H), 7.23 (td, J = 7.7, 1.6 Hz, 1H), 7.06 (d, J = 7.5 Hz, 2H), 6.32 (d, J = 9.5 Hz, 1H), 4.93 (s, 2H). 13C NMR (151 MHz, DMSO) δ 166.2,
160.6, 160.1, 155.1, 144.2, 144.2, 134.0, 129.6, 129.5, 129.5, 127.6, 126.7, 126.7, 125.8, 125.8, 112.9, 112.7, 101.7, 101.7, 67.1. HRMS (ESI): calcd for
C17H12ClNO4 (M-H+); 328.0382, found: 328.0377.
N-(2-methoxyphenyl)-2-((2-oxo-2H-chromen-7-yl)oxy)acetamide: The synthesis method is the same as G25. Column chromatography (P/E = 10:1) to give
G28 (77% yield). 1H NMR (600 MHz, DMSO-d6) δ 9.32 (s, 1H), 8.00 (dd, J = 9.0, 4.0 Hz, 2H), 7.67 (d, J = 8.4 Hz, 1H), 7.19 – 7.01 (m, 4H), 6.96 – 6.85 (m, 1H),
6.32 (d, J = 9.4 Hz, 1H), 4.91 (s, 2H), 3.86 (s, 3H). 13C NMR (151 MHz, DMSO) δ 165.7, 160.6, 160.1, 155.2, 149.3, 144.2, 129.6, 126.4, 124.8, 121.2, 120.4,
112.9, 112.7, 111.2, 101.7, 67.3, 55.8, 39.9, 39.8, 39.6, 39.5, 39.4, 39.2, 39.1. HRMS (ESI): calcd for C18H15NO5(M-H+): 324.0877, found: 324.0870.
N-(3-chloro-4-fluorophenyl)-2-((2-oxo-2H-chromen-7-yl)oxy)acetamide: The synthesis method is the same as G25. Column chromatography (P/E = 10:1) to
give G29 (92% yield). 1H NMR (600 MHz, DMSO-d6) δ 10.36 (s, 1H), 8.00 (d, J = 9.5 Hz, 1H), 7.94 (dd, J = 6.8, 2.6 Hz, 1H), 7.70 – 7.63 (m, 1H), 7.56 (ddd, J =
9.0, 4.3, 2.6 Hz, 1H), 7.40 (t, J = 9.1 Hz, 1H), 7.04 (dq, J = 3.8, 2.4 Hz, 2H), 6.32 (d, J = 9.5 Hz, 1H), 4.86 (s, 2H). 13C NMR (151 MHz, DMSO) δ 166.2, 160.8,
10
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10.1002/cbdv.201800577
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160.1, 155.1, 154.4 (J = 243.1 Hz), 144.2, 135.5, 135.5, 129.5, 121.1, 120.1, 120.0, 119.2, 119.1, 117.0, 116.9, 112.9, 112.9, 112.7, 101.7, 67.2. HRMS (ESI): calcd
for C17H11ClFNO4(M-H+): 346.0288, found: 346.0281.
N-(4-chlorophenyl)-2-((2-oxo-2H-chromen-7-yl)oxy)acetamide: The synthesis method is the same as G25. Column chromatography (P/E = 10:1) to give G30
(86% yield). 1H NMR (600 MHz, DMSO-d6) δ 10.29 (s, 1H), 8.00 (d, J = 9.5 Hz, 1H), 7.67 (dt, J = 9.6, 3.4 Hz, 3H), 7.43 – 7.35 (m, 2H), 7.08 – 6.95 (m, 2H), 6.31
(d, J = 9.5 Hz, 1H), 4.85 (s, 2H). 13C NMR (151 MHz, DMSO) δ 166.0, 160.8, 160.1, 155.1, 144.2, 137.2, 129.5, 128.7, 127.3, 121.2, 112.9, 112.9, 112.7, 101.6,
67.2. HRMS (ESI): calcd for C17H12ClNO4(M-H+): 328.0382, found: 328.0396.
Cytotoxicity assay
All of these compounds were individually subjected to the cell counting kit-8 (CCK-8) cytotoxicity assay. Briefly, HEK293T-Gluc cells were cultured in a 96-
well plate and incubated with various concentrations of each compound. Cells cultured in the medium with DMSO alone were utilized as a control. After
48 h incubation, 10 μL CCK-8 solution was added to each well and incubated for an additional 1 h at 37°C. The optical density (OD) of each well at 450 nm
was recorded on a microplate reader (Thermo, Varioskan Flash).
IAV replication assay
HEK293T-Gluc cells were incubated with IAV for 1 h at room temperature and then cultured for 24 h at 37°C in fresh DMEM. Gluc activity in the culture
medium was measured as described previously by Tannous.[24] Briefly, coelenterazine-h (16.7 μM in PBS) was equilibrated for 30 min in the dark at room
temperature. Then, cell culture supernatants were added to the wells in white and opaque 96-well plates, followed by automated injection of 60 μL of
coelenterazine-h per well. The photon counts over 0.5 s were acquired using a Centro XS3 LB 960 microplate luminometer (Berthold Technologies).
Acknowledgements
The research was supported by the National Natural Science Foundation of China (81473098, 81473099 and 81703366), the Fundamental Research Funds
for the Central Universities (332017078), the National Mega-project for Innovative Drugs (2018ZX09711003-002-002), Fundamental Research Funds for
Jinzhou Medical University (JYTQN201731) and CAMS innovation fund for Medical Sciences (2016-I2M-3-014, 2016-I2M-1-011, 2017-I2M-3-019, and 2018-
I2M-3-004).
Author Contribution Statement
Shan Cen, Donghui Li, Juxian Wang and Yucheng Wang conceived and planned the experiments. Xuandi Zhang, Mei Zhu ,Ziqiang Li and Guoning Zhang
designed and synthesized the compounds. Yujia Wang tested the anti-IAV activity of the compounds. Xuandi Zhang wrote the manuscript with support
from Guoning Zhang and Shan Cen. Yucheng Wang supervised the project.
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