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Home > Synthesis and Biological Evaluation of Substituted Indole and Its Analogs as Influenza A Virus Inhibitors
Synthesis and Biological Evaluation of Substituted Indole and Its Analogs as Influenza A Virus Inhibitors
Xuandi Zhang,a Guo-Ning Zhang,b Yujia Wang,b Mei Zhu,b Juxian Wang,b Ziqiang Li,a Donghui Li,*a Shan Cen,*b and Yucheng Wang*b
School of Pharmacy, Jinzhou Medical University, Jinzhou 121001, P. R. China, e-mail: lidonghuilx@sina.com
Institute of Medicinal Biotechnology, Chinese Academy of Medical Science and Peking Union Medical College, Beijing 100050, P. R. China, e-mail: shancen@imb.pumc.edu.cn; wangyucheng@imb.pumc.edu.cn
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 signifi- cant 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 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.
The N/O-containing heterocycles, such as coumar- ins 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 functionaliza- tion of aza-heterocycles under metal[20] and metal- free[21] conditions. 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-naph- thol, β-naphthol, 4-hydroxycoumarin, 6-hydroxycou- marin and 7-hydroxycoumarin to investigate the structure– activity relationship of indoles, respectively. The sulfur atom in the chemicals was 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).
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-bromo- indole core structure exhibit a moderate to high inhibition ratio to IAV infection, and compound D9 showed 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 showed equivalent or decreased inhibition ratio to IAV infection, and com- pounds 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 com- pounds D7 and D8. All of the α-naphthol, β-naphthol, 4-hydroxycoumarin and 6-hydroxycoumarin series showed anti-IAV effects comparable to the 5-bromo- indole series.
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- and 3-F-aniline moiety, 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.
In summary, six series of anti-IAV target products were designed and synthesized based on the sub- stitution 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, analogs with α-naphthol, β-naphthol, 4- hydroxycoumarin and 6-hydroxycoumarin replace- ments 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.
Conclusions
In this work, six 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 four series of analogs were designed and synthesized. Among these, three series show similar anti-IAV activity when compared with the 5-bromoindole series, except for the 6-hydroxycou- marin 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
The products D1–9 were synthesized according to the route depicted in Scheme 1. Ethyl bromoacetate (59.88 mmol, 1 equiv.), Na2S2O3 · 5H2O (71.86 mmol,
1.2 equiv.), 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 °C for 2 h. After completion of the reaction, the solvent was evaporated, and the residue was sonicated in 100 ml EtOH for 5 min and filtered under vacuum. The filtrate was evaporated to dryness to obtain the crude product A.
To a solution of A (22.53 mmol, 1.2 equiv.) in 30 ml of DMSO was added 5-bromoindole (18.78 mmol, 1 equiv.) and I2 (1.88 mmol, 0.1 equiv.), and the solution was stirred for 2 h at 60 °C. After the completion of the reaction, monitored by TLC, the solution was diluted with AcOEt, extracted with saturated Na2S2O3 and brine, and dried on anhydrous sodium sulfate. After removing the solvent, crude product B was afforded as yellow solid.
NaOH (17.98 mmol, 2 equiv.), EtOH (30 ml) and H2O (7.5 ml) were added to B (8.99 mmol, 1 equiv.) and stirred for about 30 min at room temperature. After the reaction was completed, 6 N HCl was added to adjust pH 3, most of the solvent was distilled off, the mixture was extracted with AcOEt, and the solvent was evaporated to give C.
To a solution of C (0.70 mmol, 1 equiv.), aniline (0.63 mmol, 0.9 equiv.) and HATU (0.77 mmol, 1.1 equiv.) in 10 ml of CH2Cl2, DIEA (1.68 mmol, 2.4 equiv.) was added. The solution was stirred for 3 – 5 h at room temperature until the completion of the reaction, monitored by TLC. The solution was diluted with CH2Cl2 and washed with H2O and brine, dried over anhydrous sodium sulfate, and purified by column chromatography to afford products D1–9.
The products G1–30 were synthesized according to the route depicted in Scheme 2. Naphthol or coumarin (10 mmol, 1 equiv.) was added to the reaction flask, ethyl bromoacetate (12 mmol, 1.2 equiv.), K2CO3 (40 mmol, 4 equiv.) 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 were added to the reaction solution and extracted with AcOEt. The solution was concen- trated to afford E1–2 (After replacing coumarin as the raw material, solids precipitated when H2O was added, and E3–5 was obtained through filtration).
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 the control. After 48 h incubation, 10 μL of CCK-8 solution were 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 determined as described previously by Tan- nous.[24] Briefly, coelenterazine h (16.7 μM in PBS) was equilibrated for 30 min in the dark at room temper- ature. 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
This 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 (2018ZX09711003-002-02), the Fundamental Research Funds for Jinzhou Medical University (JYTQN201731) and the 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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