Synthesis and antiviral evaluation of novel 1,3,4-oxadiazole/thiadiazole-chalcone conjugates

Article abstract of DOI:10.1016/j.bmcl.2017.08.038

A series of novel 1,3,4-oxadiazole/thiadiazole–chalcone conjugates were synthesized and their in vitro and in vivo antiviral activities were evaluated via microscale thermophoresis method and half-leaf method, respectively. The in vitro results indicated that compounds 7g, 7l, 8h, and 8l displayed good antiviral activity against TMV, with the binding constant values of 5.93, 6.15, 6.02, and 5.04 μM, respectively, which were comparable to that of Ninnanmycin (6.78 μM) and even better than that of Ribavirin (99.25 μM). The in vivo results demonstrated that compounds 7g, 7l, 8h, and 8l exhibited remarkable anti-TMV activity with the EC₅₀ values of 33.66, 33.97, 33.87 and 30.57 μg/mL, respectively, which were comparable to that of Ningnanmycin (36.85 μg/mL) and superior to that of Ribavirin (88.52 μg/mL). Interestingly, the trend of antiviral activity in vivo was consistent with the in vitro results.

Full text of DOI:10.1016/j.bmcl.2017.08.038

Accepted Manuscript
Synthesis and antiviral evaluation of novel 1,3,4-oxadiazole/thiadiazole-chal-
cone conjugates
Xiuhai Gan, Deyu Hu, Zhuo Chen, Yanjiao Wang, Baoan Song
PII:
S0960-894X(17)30839-9
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.08.038
BMCL 25232
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
21 February 2017
21 June 2017
17 August 2017
Please cite this article as: Gan, X., Hu, D., Chen, Z., Wang, Y., Song, B., Synthesis and antiviral evaluation of novel
1
,3,4-oxadiazole/thiadiazole-chalcone conjugates, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://
dx.doi.org/10.1016/j.bmcl.2017.08.038
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Synthesis and antiviral evaluation of novel 1,3,4-oxadiazole/
thiadiazole-chalcone conjugates
a, b
Xiuhai Gan , Deyu Hu *, Zhuo Chen , Yanjiao Wang , Baoan Song *
a,
a
a
a,
*
Author to whom correspondence should be addressed. Tel.: +86-(851)-8829-2170; Fax:
+86-(851)-8829-2170. E-mail: dyhu@gzu.edu.cn; songbaoan22@yahoo.com
a
State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key
Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Research,
and Development Center for Fine Chemicals, Guizhou University, Guiyang 550025, China;
b
College of Chemistry and Life Science, Guizhou Education University, Guiyang 550018, China
1

ABSTRACT: A series of novel 1,3,4-oxadiazole/thiadiazole–chalcone conjugates were
synthesized and their in vitro and in vivo antiviral activities were evaluated via microscale
thermophoresis method and half-leaf method, respectively. The in vitro results indicated that
compounds 7g, 7l, 8h, and 8l displayed good antiviral activity against TMV, with the binding
constant values of 5.93, 6.15, 6.02, and 5.04 µM, respectively, which were comparable to that of
Ninnanmycin (6.78 µM) and even better than that of Ribavirin (99.25 µM). The in vivo results
demonstrated that compounds 7g, 7l, 8h, and 8l exhibited remarkable anti-TMV activity with the
EC50 values of 33.66, 33.97, 33.87 and 30.57 µg/mL, respectively, which were comparable to that
of Ningnanmycin (36.85 µg/mL) and superior to that of Ribavirin (88.52 µg/mL). Interestingly,
the trend of antiviral activity in vivo was consistent with the in vitro results.
Keywords: Chalcone, 1,3,4-Oxadiazole/thiadiazole, Tobacco mosaic virus coat protein, Antiviral
activity, Microscale thermophoresis
___________
Abbreviations used
1
1
13
13
TMV, tobacco mosaic virus; H NMR, H nuclear magnetic resonance; C NMR, C nuclear
magnetic resonance; HRMS, High-resolution mass spectrometer; EC50, half-maximal effective
concentration; MST, microscale thermophoresis; SAR, structure–activity relationship; TMV CP,
tobacco mosaic virus coat protein.
2

Tobacco mosaic virus (TMV), a well-known plant virus can infect at least 400 individual
1
species such as tobacco, cucumber, tomato, and many ornamental flowers. The loss caused by
,2
3
TMV is up to＄100 million worldwide each year. Unfortunately, few effective antiviral agents
for completely controlling TMV infection are available. Therefore, the search of novel, highly
effective, friendly to environmentally friendly antiviral agents with unique mode of action still
remains a challenging task in pesticide research.
TMV is composed of RNA and coat protein (CP) subunits; viral RNA and CP self-assemble
4
readily in vitro to virion. TMV CP can assemble into four main alpha-helices which are joined by
a prominent loop proximal to the axis of the virion. This alpha-helices can ensure that the virion
5
remains stable and play a positive role in regulating production of subgenomic mRNAs,
,6
translation of mRNAs, transcription and translation pathways of TMV RNA and production of one
7
or more viral proteins which are consistent with multi-functionality of CP molecules.
Furthermore, we recently reported the crystal structure of a four-layer aggregate of engineered
8
,9
d
TMV CP. The binding constant (K ) between compound and TMV CP, which is an important
parameter for drug discovery, was investigated by microscale thermophoresis (MST). The results
1
0–12
revealed that TMV CP was the key functional protein for virus infecting the host,
be a potential action target for development and screening of anti-TMV drugs.
and might
1
3,14
Chalcones, a kind of important natural products in plants,
have been used in drug and
1
5–17
pesticide design in consequence of their various biological activities, such as anticancer,
1
8,19
20,21
22
23
24
antibacterial,
anti-fungal,
anti-inflammatory,
anti-plasmodial,
antiparasitic,
and
1
4,25
antiviral
effects. In our previous work, we have reported a series of chalcone derivatives (A
1
1,26
and B, Fig. 1), with remarkable bioactivities against TMV.
The 1,3,4-oxadiazole/thiadiazole
3

scaffold represented a key motif in heterocyclic chemistry, possessing a variety of biological
2
7–29
30
anticancer, antitubercular, antiviral and other effects. In
31
32
activities, including antibacterial,
our previous work, we found that several 1,3,4-oxadiazole/thiadiazole derivatives (C and D, Fig. 1)
displayed good antiviral activities against TMV and the structure–activity relationship (SAR)
3
3,34
revealed that 5-phenyl-1,3,4-oxadiazole derivatives showed excellent antiviral action.
These
findings demonstrated that chalcone and 1,3,4-oxadiazole/thiadiazole derivatives exhibited good
curative and protective effects against TMV, but it was still lack of compounds with high
inactivating activity.
Fig. 1. Structures of the compounds in our previous studies.
As part of our ongoing drug discovery program in developing potential antiviral agents on the
basis of natural product chalcone scaffold, we combined 5-phenyl-1,3,4-oxadiazole/thiadiazole
and
chalcone
with
1,2-dibromoethane
to
generate
a
series
of
novel
5-phenyl-1,3,4-oxadiazole/thiadiazole–chalcone conjugates (Fig. 2). In this letter, we report their
synthesis, in vitro and in vivo antiviral activity, and preliminary structure–activity relationship
analyses.
4

Fig. 2. Design of novel 1,3,4-oxadiazole/thiadiazole–chalcone conjugates.
The synthetic route is depicted in Scheme 1. The 5-phenyl-1,3,4-oxadiazole/thiadiazole-2-thiols
2
9
3/4 were prepared by literature methods.
Chalcones 5a–5m were synthesized by
Claisen–Schmidt condensation reaction of p-hydroxy-benzaldehyde and substituted
2
acetophenones. The key intermediates 6a–6m were obtained according to previous report. The
6
11
5-phenyl-1,3,4-oxadiazole/thiadiazole–chalcone
conjugates
were
prepared
from
3
4
5-phenyl-1,3,4-oxadiazole/thiadiazole-2-thiols 3/4 and chalcones 6a–6m via thioetherification.
1
13
The target compounds were characterized by H NMR, C NMR and HRMS, the experimental
1
details and data are presented in the supplementary data file. The main characteristic of the H
NMR spectra of 7a as a representative compound was the appearance of two triplet peaks at δ
H
4
.45 and 3.69 ppm, which is the O–CH
2
– and S–CH
2
– proton absorption peak, respectively. The
low-frequency downfield doublets at δ
H
8.00–6.96 ppm indicated =C–H and phenyl protons, of
which, the two doublets at δ
H
H
7.76 (d, 1H, J = 15.5 Hz) and δ 7.37 (d, 1H, J = 15.5 Hz) ppm
demonstrated that the configuration of the double bond of chalcone is “E”. Typical chemical shifts
1
3
of C NMR spectra at around δ
C
166.16 and 163.90 ppm indicated the presence of the
1
,3,4-oxadiazole moiety, while peaks at around δ
C
188.87, 66.06, and 31.60 ppm are confirmed
+
–, respectively. HRMS found [M+H] mass to be
the presence of C=O, –OCH
2
–, and –SCH
2
+
ClS [M+H] 463.08777.
463.08820, which accord with the calculated value for C25
H
20
O
3
N
2
5

Scheme 1. Synthetic route of novel 1,3,4-oxadiazole/thiadiazole–chalcone conjugates.
In this study, the in vitro anti-TMV activity of the target compounds was performed via the
MST method. The commercially available agents Ribavirin and Ningnanmycin were used as
positive control and the results are listed in Table 1. According to Table 1, most of the compounds
displayed good antiviral activity against TMV due to the interactions between the target
6 4 6 3
compounds and TMV CP. Compounds 7f (Ar = 4-F-C H ), 7g (Ar = 2,4-diCl-C H ), 7l (Ar =
thiophene), 8h (Ar = Ph), 8l (Ar = thiophene), and 8m (Ar = furan) exhibited strong combining
capacity to TMV CP, with the K values of 8.78, 5.93, 6.15, 6.02, 5.04, and 8.89 µM, respectively,
d
which were better than that of Ribavirin (99.25 µM), lead compounds B (66.58 µM) and D (19.36
µM), but similar to that of Ningnanmycin (6.78 µM). SAR analyses indicated that
electron-withdrawing groups were favorable for combining capacity of compound, these findings
were evidenced by the activity order of 7f (Ar = 4-F-C
6
H
4
3 6 4
, 8.78 µM) > 7j (Ar = 4-OCH -C H ,
119.10 µM) and 8f (Ar = 4-F-C
6
H
4
3 6 4
, 75.62 µM) > 8j (4-OCH -C H , 88.58 µM). Bulky groups of
the aromatic ring were unfavorable for combining capacity, for example 7l (Ar = thiophene, 6.15
µM) > 7h (Ar = Ph, 263.10 µM) and 8l (Ar = thiophene, 5.04 µM) > 8h (Ar = Ph, 6.02 µM).
6

Table 1
a
Antiviral activity of the compounds against TMV in vitro and in vivo
In vivo effect
Inhibition ration at EC50
In vitro effect
b
Compounds
X
Ar
d
K (µM)
5
00 µg/mL (%)
87.65±1.78
89.25±2.12
90.21±2.83
88.26±3.15
91.65±2.10
90.21±1.12
95.12±1.53
70.07±1.22
94.12±1.11
83.65±3.73
66.72±1.10
93.25±2.83
84.09±1.72
91.24±2.94
84.45±2.33
92.05±0.65
89.68±2.31
88.15±2.70
82.26±3.35
93.81±1.71
93.06±2.72
93.18±0.25
85.28±2.51
81.90±3.22
93.75±2.06
92.02±2.71
73.89±2.55
91.38±0.98
87.65±1.45
90.58±1.12
(µg/mL)
64.34±4.10
69.69±2.11
83.71±2.37
42.64±2.36
44.55±1.25
38.03±2.11
33.66±3.75
175.59±2.18
48.91±4.52
124.76±2.15
169.83±1.07
33.97±1.58
81.79±5.13
55.47±2.45
65.19±2.63
41.05±2.99
51.23±1.56
57.55±3.58
73.90±2.90
46.07±0.88
33.87±3.15
50.49±2.18
76.09±2.02
84.15±2.36
30.57±3.11
35.43±1.14
88.52±2.56
36.85±1.58
68.24±2.27
50.36±1.85
7
7
a
O
4-Cl-C
6
H
4
64.70±2.12
78.90±5.09
99.80±5.40
22.80±1.48
30.40±2.26
8.78±1.12
b
O
O
O
O
O
O
O
O
O
O
O
O
S
3,4-diCl-C
6
H
3
7
c
2-Cl-C
6
H
4
7d
6 4
2-F-C H
7
e
6 4
3-Cl-C H
7
f
6 4
4-F-C H
7
7
g
2,4-diCl-C
6
H
3
5.93±0.56
h
Ph
263.10±12.81
52.70±3.62
119.10±9.70
251.35±2.56
6.15±0.19
7
i
3 6 4
4-CH -C H
7
j
4-OCH
3-OCH
3
3
-C
6
6
H
4
7k
-C
H
4
7
l
thiophene
furan
7
m
83.40±2.75
59.56±4.11
70.90±1.83
18.15±1.24
55.60±1.95
60.01±2.18
75.62±2.98
38.50±0.31
6.02 ±0.85
52.95±4.27
88.58±6.38
93.19±8.28
5.04±0.17
8
a
4-Cl-C
3,4-diCl-C
6 4
2-Cl-C H
6 4
H
8b
S
6 3
H
8
c
S
8d
S
6 4
2-F-C H
8
e
S
6 4
3-Cl-C H
8
f
S
6 4
4-F-C H
8
8
g
S
2,4-diCl-C
6 3
H
h
S
Ph
8
i
S
3 6 4
4-CH -C H
8
j
S
4-OCH
3-OCH
3
3
-C
6
6
H
4
8k
S
-C
H
4
8
l
S
thiophene
furan
8
m
S
8.89±0.69
c
Ribavirin
99.25±7.95
6.78±0.91
c
Ningnanmycin
d
B
68.58±5.09
19.36±1.56
d
D
a
b
c
d
Average of three replicates (n=3); half-maximal effective concentration; positive control; the compound of
chalcone and 1,3,4-oxadiazole derivatives with the best antiviral activity as positive control.
7

In order to confirm the MST results, the anti-TMV activity of the title compounds at 500 µg/mL
was tested in vivo, and the results are shown in Table 1. The results revealed that most of the
compounds exhibited good anti-TMV activity in vivo. Among them, compounds 7e (Ar =
3
-Cl-C
6
6
4 6 3 3 6 4
H ), 7g (Ar = 2,4-diCl-C H ), 7i (Ar = 4-CH -C H ), 7l (Ar = thiophene), 8c (Ar =
2-Cl-C
4 6 3 3 6 4
H ), 8g (Ar = 2,4-diCl-C H ), 8h (Ar = Ph), 8i (Ar = 4-CH -C H ), 8l (Ar = thiophene),
and 8m (Ar = furan) exerted remarkable antiviral activity, with the value of 91.65%, 95.12%,
94.12%, 93.25%, 92.05%, 93.81%, 93.06%, 93.18%, 93.75%, and 92.02%, respectively, which
were better than that of Ribavirin (73.89%), lead compounds B (87.65 %) and D (90.58%), but
similar to that of Ningnanmycin (91.38%). The EC50 values of the test compounds were also
investigated. Compounds 7g, 7l, 8h, 8l, and 8m exhibited good antiviral activity, with EC50 values
of 33.66, 33.97, 33.87, 30.57, and 35.43 µg/mL, respectively, which were better than that of
Ribavirin (88.52 µg/mL), lead compound B (68.24 µg/mL) and D (50.36 µg/mL), and
comparable to that of Ningnanmycin (36.85 µg/mL). Similarly to the in vitro results,
electron-withdrawing groups and smaller aromatic rings enhanced the antiviral activity, such as
6 4 3 6 4
the activity order of 7f (Ar = 4-F-C H , 38.03 µg/mL) > 7j (Ar = 4-OCH -C H , 124.76 µg/mL),
8
3
3
6 4 3 6 4
f (Ar = 4-F-C H , 73.90 µg/mL) > 8j (4-OCH -C H , 76.09 µg/mL) and 7l (Ar = thiophene,
3.97 µg/mL) > 7h (Ar = Ph, 175.59 µg/mL), 8l (Ar = thiophene, 30.57 µg/mL) > 8h (Ar = Ph,
3.87 µg/mL). In addition, the bioassay results indicated that most of 1,3,4-thiadiazole-chalcone
conjugates showed better antiviral activities than that of 1,3,4-oxadiazole-chalcone conjugates, for
example, 8a (55.47 µg/mL) > 7a (64.34 µg/mL), 8c (41.05 µg/mL) > 7c (83.71 µg/mL), 8h
(
33.87 µg/mL) > 7h (175.59 µg/mL), 8l (30.57 µg/mL) > 7l (33.97 µg/mL), 8m (35.43 µg/mL)
>
7m (81.79 µg/mL), and so on. Interestingly, the trend of antiviral activity against TMV in vivo
8

was consistent with the in vitro results, and the statistical analyses showed that the EC50 values
d
were significantly correlated with K values (R = 0.887, P < 0.01).
In summary, twenty-six novel 5-phenyl-1,3,4-oxadiazole/thiadiazole–chalcone conjugates were
designed and synthesized. Compounds 7g, 7l, 8h, and 8l exhibited excellent anti-TMV activity in
vitro and in vivo. SAR analyses revealed that smaller aromatic ring and electron-withdrawing
groups were favorable for antiviral effect, it will pave the way for further design and chemical
modification of chalcones. This work demonstrates that the method of MST based on TMV CP
can
be
used
to
evaluate
antiviral
activity
in
vitro
and
5-phenyl-1,3,4-oxadiazole/thiadiazole–chalcone conjugates can be utilized as lead molecules for
the development of potential antiviral agents.
Acknowledgements
The authors gratefully acknowledge the financial support from National Natural Science
Foundation of China (Nos. 21562013 and 21362004) and the Innovative Talents Project of
Guizhou Province in China (No. 201503) and the Provincial University Cooperation Plan of
Guizhou Province in China (No. 20147001).
Supplementary data
Supplementary data (containing information on the synthesis, characterization, and bioactivity
test methods of the title compounds) associated with this article can be found in the online version,
at http://dx.doi.org/10.1016/j.bmcl.
9

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11

Highlights
1. Novel 1,3,4-oxadiazole/thiadiazole–chalcone conjugates were synthesized
2. The method of MST was established and used to evaluate antiviral activity.
3. Compound 8l possessed remarkable binding affinity to TMV CP.
4. Compound 8l exhibited excellent anti-TMV activity.
12

35.
13