Synthesis and antiviral activity of coumarin derivatives against infectious hematopoietic necrosis virus

Article abstract of DOI:10.1016/j.bmcl.2019.05.019

Infectious hematopoietic necrosis virus (IHNV)is a highly contagious disease of juvenile salmonid species. However, robust anti-IHNV drugs currently are extremely scarce. For the purpose of seeking out anti-IHNV drugs, here a total of 24 coumarin derivatives are designed, synthesized and evaluated for their anti-viral activities. By comparing the half maximal inhibitory concentrations (IC₅₀)of the 12 screened candidate drugs in epithelioma papulosum cyprini (EPC)cells infected with IHNV, the imidazole coumarin derivative C4 is selected for additional validation studies, with an IC₅₀ of 2.53 μM at 72 h on IHNV glycoprotein. Further experiments revealed that C4 could significantly inhibit apoptosis and cellular morphological damage induced by IHNV. On account of these findings, derivative C4 could be a viable way of controlling IHNV and considered as a promising lead compound for the development of commercial drugs.

Full text of DOI:10.1016/j.bmcl.2019.05.019

Accepted Manuscript
Synthesis and antiviral activity of coumarin derivatives against infectious hem-
atopoietic necrosis virus
Yang Hu, Weichao Chen, Yufeng Shen, Bin Zhu, Gao-Xue Wang
PII:
S0960-894X(19)30317-8
https://doi.org/10.1016/j.bmcl.2019.05.019
BMCL 26439
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
23 March 2019
9 May 2019
13 May 2019
Please cite this article as: Hu, Y., Chen, W., Shen, Y., Zhu, B., Wang, G-X., Synthesis and antiviral activity of
coumarin derivatives against infectious hematopoietic necrosis virus, Bioorganic & Medicinal Chemistry Letters
(2019), doi: https://doi.org/10.1016/j.bmcl.2019.05.019
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Synthesis and antiviral activity of coumarin derivatives against
infectious hematopoietic necrosis virus
Yang Hu, Weichao Chen, Yufeng Shen, Bin Zhu*, Gao-Xue Wang*
College of Animal Science and Technology, Northwest A&F University, Xinong Road 22nd, Yangling,
Shaanxi, 712100, China;
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* Author to whom correspondence should be addressed; E-mail address: zhubin1227@126.com (Bin
Zhu); wanggaoxue@126.com (Gao-Xue Wang).
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Tel./Fax: +86-029-8709-2102.
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Abstract
Infectious hematopoietic necrosis virus (IHNV) is a highly contagious disease of juvenile
salmonid species. However, robust anti-IHNV drugs currently are extremely scarce. For the purpose of
seeking out anti-IHNV drugs, here a total of 24 coumarin derivatives are designed, synthesized and
evaluated for their anti-viral activities. By comparing the half maximal inhibitory concentrations (IC50)
of the 12 screened candidate drugs in epithelioma papulosum cyprini (EPC) cells infected with IHNV,
the imidazole coumarin derivative C4 is selected for additional validation studies, with an IC50 of 2.53
μM at 72 h on IHNV glycoprotein. Further experiments revealed that C4 could significantly inhibit
apoptosis and cellular morphological damage induced by IHNV. On account of these findings,
derivative C4 could be a viable way of controlling IHNV and considered as a promising lead
compound for the development of commercial drugs.
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Keywords: coumarin derivatives, antiviral activity, IHNV
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As the causative agent of infectious hematopoietic necrosis (IHN), Infectious hematopoietic
necrosis virus (IHNV) is a rhabdovirus belonging to the genus Novirhabdovirus.1 Like other
rhabdoviruses, IHNV is a single-stranded, nonsegmented, RNA virus that encodes five structural
proteins: the RNA-dependent RNA polymerase, glycoprotein, matrix protein, phosphoprotein,
nucleoprotein, and a nonstructural protein nonvirion.2,3 IHNV is an important viral disease in salmonid
aquaculture and outbreaks of IHN usually cause 80%~100% mortality.4-6 At the present time, vaccines
are used as a preventive measure against these viral diseases; however, their efficacy is not complete.
Therefore, the establishment of effective antiviral agents against fish viral diseases is urgently needed.
Natural products have been used to treat disease for thousands of years and play an increasingly
important role in drug discovery and development. In fact, the majority of anti-infectious agents are of
natural origin.7 Coumarins are a class of compounds found widely in nature, and they show a broad
spectrum of activities including anticancer, antioxidant, anti-inflammatory and antiviral.8-15 Moreover,
coumarins can be considered as a privileged scaffold and an ideal framework for the design of
compounds that can interact with different targets as their inherent affinity for several biological
targets.16 Our previous study demonstrated two coumarin derivatives could reduce the replication of
spring viraemia of carp virus (SVCV), another rhabdovirus of fish.17 The results suggest that coumarin
derivatives be synthesized for developing the anti-IHNV agents.
Another promising group, imidazole, has emerged as one of the most important heterocycles in
current medicinal chemistry and its applications have also been extended to widespread diseases.18-23 In
recent years, a library of coumarin derivatives conjugated with imidazole were synthesized and proved
to possess different bioactivity.24,25 As far as anti-parasites activity was concerned, Liu et al. discovered
7-(6-benzimidazole-hexyloxy)coumarin derivative exhibiting anti-parasites activity against

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Dactylogyrus intermedius.26 These results promoted us to synthesize new derivatives of coumarins with
imidazole skeleton for the sake of finding new effectively antiviral agents.
In this study, our work is directed toward the coumarin antiviral agents and their structural
modification. According to the previous studies, we hypothesized that the chemical structures of
coumarin derivatives influenced the antiviral activity. Based on these things mentioned above, we
synthesized several series of novel coumarins derivatives owning nitrogen-containing heterocyclic with
different length of chemical bridges. We evaluated their antiviral activities against IHNV following by
secondary assays, including virus titration assay, cytopathic effects (CPE) reduction analysis, nucleus
damage observation, and microtubule structure. This study helped rationalize activities at the
investigated drugs chemical structures, pointing to potential strategies for designing more selective
compounds.

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Synthetization of coumarin derivatives
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The synthesis of 24 coumarin derivatives is shown in Scheme 1. Compounds A1-A6, which are
crucial to the synthesis of all heterocyclic coumarin derivatives, were synthesized from 7-
hydroxycoumarin by reacting with the corresponding α, ω-dibromoalkanes in anhydrous acetone at
reflux condition. Compounds B1-D6 were synthesized in 55-90% yield by treatment of A1-A6
compounds with corresponding amines and anhydrous potassium carbonate in acetonitrile at room
1
temperature. The structures of all synthesized compounds were confirmed by ESI-MS, H, and 13C
NMR.
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Scheme 1
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A screen of synthesized coumarins with anti-IHNV activity
From this preliminary screen, a set of 25 compounds was identified. A plot displays the

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percentage inhibition on glycoprotein cDNA copy by 100, 25, or 10 uM of 25 coumarin derivatives
(Fig. 1). The preliminary screen results showed that a total of 2 coumarins in 100 μM, 4 coumarins in
25 μM and 6 coumarins in 10 μM inhibited IHNV glycoprotein production by more than 50%. These
12 coumarins were further measured for their IC50 (Table 1) and C4 was the most effective agent with
EC50 of 2.53 µM and CC20 of 17.13 µM (Fig. 2).
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Fig. 1

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Table 1. The IC50 values of 12 compounds at the 95% confidence interval.
Compounds
Constitutional Formula
IC50 values (μM)
O
O
O
O
O
O
O
O
O
O
O
O
O
Br
A4
94.22 (84.64-110.90)
O
O
O
O
Br
A5
B3
B4
B5
B6
79.67 (73.81-87.92)
21.87 (20.71-23.41)
14.31 (12.46-16.72)
6.47 (5.65-7.46)
N
N
N
N
N
N
N
N
O
O
N
N
7.35 (5.90-9.64)
O
O
O
O
C4
C5
2.53 (2.20-2.91)
3.49 (2.38-5.71)
N
O
N
N
O
O
O
O
O
O
C6
D2
D5
D6
7.28 (5.99-9.18)
17.91 (15.82-20.96)
7.65 (6.61-9.47)
N
N
N
O
O
O
O
O
O
N
N
N
N
8.67 (7.58-11.09)
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Fig. 2
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To further confirm the antiviral activity of derivative C4 against IHNV, we further evaluated CPE
and the titer of after treatment with C4. As shown in Fig. 3B, CPE of IHNV-infected cells in presence
of C4 was decreased significantly at 72 h. Similarly, significant inhibition of IHNV was shown in C4-
treated EPC cells in the measurement of the viral titer. IHNV titers were 104.22 (48 h p.i.), 106.08 (72 h
p.i.) and 107.15 (96 h p.i.) 50% tissue culture infective dose/0.1 mL (TCID50/0.1 mL); whereas IHNV
titers were 102.92 (48 h p.i.), 105.32 (72 h p.i.) and 106.34 (96 h p.i.) TCID50/0.1 mL in C4-treated group.
The results above indicated that C4 could significantly inhibit IHNV replication in EPC cells.

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Fig. 3
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Effects of compound C4 on IHNV-induced apoptosis
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Typical apoptotic features including cell membrane rupture and nuclear fragmentation were
observed in the IHNV-infected cells (Fig. 4). However, C4 treated cells maintained a normal spindle

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shape, and apoptosis body was a tiny bit even after 72 h of infection (Fig. 4). These results suggested
that treatment with C4 can block the occurrence of apoptosis in IHNV-infected cells to some extent.
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Fig. 4
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Compound C4 had a morphologically protective effect on EPC cells
The EPC cell microstructure was reflected in Fig. 5. Fig. 5A shows that normal cells were intact,
round and plump, and retained spherical shape; while after 72 h viral infection, the cells were seriously
shrunk, and observed typical apoptotic features including cell shrinkage, volume reduction and cell
blebbing (Fig. 5C). Remarkably, treated with C4, the whole IHNV-infected cell morphology was
similar to the normal, with keeping spherical shapes with a clear edge (Fig. 5B and D).

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Fig. 5
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Among natural products, the coumarins exhibit a positive antiviral effect on the replication of
hepatitis C virus,17 influenza A virus (H1N1),27 and herpes simplex virus (HSV),28 highlighting the
importance of this natural origin. Through structure modification based on the leading compound 7-
hydroxycoumarin, coumarin derivatives show a range of biological activities. For example, derivatives
have displayed large potentiality for the treatment of cancer.29,30 The structural optimization of 7-
hydroxycoumarin has been demonstrated as a promising lead compound that exhibits excellent activity
against bacteria.25 In this study, We sought to characterize the structure-activity relationship (SAR) for
this class of compounds by introducing substituents on the aromatic ring of hydroxy and investigating
those compounds with different length of “C-linker”. We also considered the role of the imidazole
analogs in the other end of the linker.
In this paper, we identified a number of newly synthesized coumarins that exhibited anti-IHNV
activity. By analyzing the SAR, we considered that different types of imidazole derivatives (2-
methylimidazole) might play a key role in the anti-IHNV activity of the screened coumarins in the
same length of the linker group. Moreover, imidazole analogs had been reported for other antiviral
activities which were consistent with our result that it could increase the activity against IHNV.31-33
Interestingly, the linker lengths also distinctively affected the antiviral activity of coumarins, where six,
eight and ten carbons had a higher inhibitory effect on IHNV. Otherwise, the toxicity of the
synthesized compounds in EPC cells was increased with extended linker lengths, especially for eight or
ten carbon atoms. In addition, our previous studies demonstrated that the bioactivities were influenced
by the carbon linker, such as antibacterial and anti-parasites.25,34
In summary, a series of coumarin derivatives were designed, synthesized and evaluated for antiviral
efficacy against IHNV in EPC cells. Among the 24 derivatives screened, 12 derivatives were found to

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have a potential anti-IHNV effect. Moreover, six carbon atoms length of the linker and 2-
methylimidazole could significantly increase the antiviral activity. Importantly, C4 displays a robust
inhibitory effect on IHNV proliferation at a lower concentration, without overt cytotoxicity to host cells.
Taken together, derivative C4 was regarded as an anti-IHNV agent in aquaculture.
Acknowledgment
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The authors thank Prof. Ling-bing Zeng in Yangtze River Fisheries Research Institute for
providing EPC cells and Professor Tong-yan Lu in Heilongjiang River Fishery Research Institute
Chinese Academy of Fishery Sciences for providing IHNV strains. This work was supported by the
National Natural Science Foundation of China (No. 31772873) and the Excellent Young Talents
Program of Northwest A&F University (No. 2452018029).
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Conflict of interest
The authors declare no competing financial interest.
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Figure captions
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Fig. 1. The percent inhibition of 25 coumarin derivatives on the IHNV of G expression is shown. The
inhibitory percentage of the marked red compounds were examined more than 50%. Error bars indicate
the SD.
Fig. 2. Confirmatory six-point dose-response curves for compound C4 in EPC cells. The percent
inhibition of the compound in the IHNV assay is shown in red, and the percent cytotoxicity of the
compound on the host cell is shown in blue. Error bars indicate the SEM.
Fig. 3. (A) Workflow of the experimental design followed in B. (B) Morphologically protective effect
of compound C4 against IHNV in EPC cells. CPE was shown in red ovals. (C) Compound C4 reduced
the titers of IHNV in EPC cells. Data were shown as mean ± SD.
Fig. 4. Fluorescence microscopy images of nucleus damage in EPC cells. EPC cells in 12-well plate
were treated in the presence or absence of compound C4 with 1 × 103 TCID50 IHNV infections. The
cells were dyed after 72 h post infection and apoptosis body was detected as arrows indicating.
Fig. 5. Scanning electron microscopy images showed the morphology of EPC cells in normal (A), C4-
treated (B), the morphological changes of cells after IHNV infection (C), and protective effect of C4
(17 μM) in cell morphology after 72 h (D) treatment.
Scheme 1. Synthetic route of coumarin derivatives. Reagents and conditions: (a) alkyl dibromide,
K2CO3, dry acetone, reflux, 20-24 h; (b) imidazole / 2-methylimidazole / 4-methylimidazole, K2CO3,
CH3CN, r.t., 20-24 h.
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Graphical Abstract

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Highlights
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Coumarin-imidazoles with anti-IHNV properties were synthesized.
Derivative C4 effectively inhibited IHNV replication with an IC50 value of 2.53 μM.
C4 could significantly inhibit apoptosis and cellular morphological damage induced by IHNV in EPC
cells.