Synthesis and antiviral bioactivity of novel (1E, 4E)-1-aryl-5-(2- (quinazolin-4-yloxy)phenyl)-1,4-pentadien-3-one derivatives

Article abstract of DOI:10.1016/j.ejmech.2013.02.035

A series of novel (1E, 4E)-1-aryl-5-[2-(quinazolin-4-yloxy)phenyl]-1,4- pentadien-3-one derivatives were designed and synthesized by reacting substituent aldehyde with intermediates 4a-f. Antiviral bioassays indicated that most of the compounds exhibited promising ex vivo antiviral bioactivities against tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV) at 500 μg/mL. The relationship between structure and antiviral activity was also discussed. Compounds 5a, 6e, and 6g could possess appreciable protective bioactivities on TMV ex vivo by approximately 50% (EC₅₀) at 257.7, 320.7 and 243.3 μg/mL. This study is the first to demonstrate that (1E, 4E)-1-aryl-5-(2-(quinazolin-4-yloxy)phenyl)-1,4-pentadien-3-one can be used to develop potential virucides for plants.

Full text of DOI:10.1016/j.ejmech.2013.02.035

European Journal of Medicinal Chemistry 63 (2013) 662e669
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antiviral bioactivity of novel (1E, 4E)-1-aryl-5-
(2-(quinazolin-4-yloxy)phenyl)-1,4-pentadien-3-one derivatives
*
*
Hui Luo, Jiaju Liu, Linhong Jin, Deyu Hu , Zhen Chen, Song Yang, Jian Wu, Baoan Song
State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key Laboratory of Green Pesticide and Agricultural Bioengineering,
Ministry of Education, Guizhou University, Huaxi District, Guiyang 550025, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel (1E, 4E)-1-aryl-5-[2-(quinazolin-4-yloxy)phenyl]-1,4-pentadien-3-one derivatives were
designed and synthesized by reacting substituent aldehyde with intermediates 4aef. Antiviral bioassays
indicated that most of the compounds exhibited promising ex vivo antiviral bioactivities against tobacco
Received 21 December 2012
Received in revised form
20 February 2013
Accepted 23 February 2013
Available online 14 March 2013
mosaic virus (TMV) and cucumber mosaic virus (CMV) at 500
and antiviral activity was also discussed. Compounds 5a, 6e, and 6g could possess appreciable protective
bioactivities on TMV ex vivo by approximately 50% (EC50) at 257.7, 320.7 and 243.3 g/mL. This study is
mg/mL. The relationship between structure
m
the first to demonstrate that (1E, 4E)-1-aryl-5-(2-(quinazolin-4-yloxy)phenyl)-1,4-pentadien-3-one can
be used to develop potential virucides for plants.
Keywords:
(1E, 4E)-1
Ó 2013 Elsevier Masson SAS. All rights reserved.
4-Pentadien-3-one derivatives
Quinazoline moiety
Tobacco mosaic virus
Cucumber mosaic virus
Antiviral activity
1. Introduction
rates and high control costs. Therefore, the search for new antiviral
agents remains a great task in pesticide science [2].
Viruses are microorganisms that can act as intracellular para-
sites, posing the risk of host genome integration. Thus, controlling
virus diseases is extremely difficult thus far. In recent years, prog-
ress has been made in screening antiviral agents with higher ac-
tivities via computer-aided drug design, discovery and application
of new targets of plant resistance diseases and viral replication, and
molecular discovery of new structures with high antiviral activities
from natural products in China. Several anti-plant viral agents were
used to prevent agricultural diseases in China, a successful anti-
plant viral agent was Ningnanmycin, which was isolated from
Strepcomces noursei var. Xichangensis and commercialized by the
Chengdu Institute of Biology, Chinese Academy of Sciences, and was
found to be more effective in the treatment of plants virus [1].
Although the agent is useful in treating plants affected by tobacco
mosaic virus (TMV) and cucumber mosaic virus (CMV), their use in
field trials is largely limited because of their unsatisfactory curative
Curcumin is the principal curcuminoid of the popular Indian
spice turmeric, which is a member of the ginger family. Curcumin
and its derivatives have extensive bioactivities, such as antiviral
[3,4], antibacterial [5], antioxidative [6,7,8], anti-inflammatory [9],
anticancer [10,11], anti-HIV [12], and anti-hyperglycemic actions
[13]. The health-promoting effects of food have recently attracted
the attention of researchers because food has the potential to
prevent disease and promote health in ways not anticipated by
traditional nutrition science. Eto et al. reported a new system that
allows the simultaneous estimation of the multiple health-
promoting effects of curcumin food constituents using infor-
matics [14]. This system was able to simultaneously estimate
health-promoting effects with reasonable precision from the same
expression data of marker proteins. This novel system should prove
to be an interesting platform for the evaluation of the health-
promoting effects of food [14]. Despite its broad effects on biolog-
ical functions, the potential use of curcumin as a therapeutic agent
is severely affected by its low water solubility, poor ex vivo
bioavailability, and rapid metabolism. In recent years, the structure
of curcumin has been widely modified, focusing mainly on omitting
the active methylene group and one carbonyl group, thereby
yielding 1, 4-pentadiene-3-ones with new structures that still
exhibit bioactivities. Our group reported several newly synthesized
Abrreviations: TMV, tobacco mosaic virus; CMV, cumber mosaic virus;
C. amaranticolor, Chenopodium amaranticolor; EC50, 50% effective concentration; 1H
NMR, 1H nuclear magnetic resonance; 13C NMR, 13C nuclear magnetic resonance.
* Corresponding authors. Tel.: þ86 851 362 0521; fax: þ86 851 362 2211.
E-mail addresses: fcc.dyhu@gzu.edu.cn (D. Hu), songbaoan22@yahoo.com
(B. Song).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.02.035

H. Luo et al. / European Journal of Medicinal Chemistry 63 (2013) 662e669
663
curcumin analogs, including 1, 4-pentadiene-3-ones containing a
phenoxy-substituted group with improved potential to inhibit PC3,
BGC-823, and Bcap-37 cells ex vivo [15]. Among the synthesized
the presence of a high-frequency downfield singlet d H 8.45 for Ne
CeH protons. NeCH3 absorption peaks showed a singlet at
3.75 ppm. The typical low-intensity carbon resonance frequencies
at dC 188.61 and dC 14.00e36.24 in the 13C NMR spectra of the
compounds also confirmed the presence of C]O and CH3,
respectively.
ethers,
pentadieneyl]-phenoxy} ethyl acetate exhibits substantial anti-
proliferative activity with IC50 values ranging from 7.1 mol/L to
7.3 mol/L against various human cancer cell lines. Preliminary
(1E,
4E)-4-{[5-(2,
3-dichlorophenyl)-3-one-1,4-
m
m
mechanism of antitumor action by DNA Ladder, Annexin V fluo-
rescein isothiocyanate/propidium iodide double staining, and
related studies indicated growth inhibition of PC3, BGC-823, and
Bcap-37 cells by induction of tumor cell apoptosis. Nevertheless, to
the best of our knowledge, no report exists on the inhibitory effects
of (1E, 4E)-1, 5-diarylpentadien-3-one derivatives bearing a qui-
nazolin pharmacophore on TMV and CMV.
To aid the development of highly active and readily available
virus inhibitors, in current study, a series of new (1E, 4E)-1-aryl-5-
(2-(quinazolin-4-yloxy)phenyl)-1,4-pentadien-3-one derivatives
were developed and evaluated for their antiviral activities (Fig. 1).
The results of bioassay revealed that the title compounds displayed
2.2. Antiviral activity
2.2.1. Ex vivo antiviral activities of 5aez, 6aeh against TMV
The inhibitory effects of the title compounds on TMV were
studied. The results of the preliminary bioassays were compared
with those of Ningnamycin virucide and listed in Table 1. From the
table, it can be seen that compounds 5aez, 6aeh indicated weak to
good antiviral activities against the tested virus. The title com-
pounds 5aez, 6aeh exhibited curative, inactivation, and protective
rates ranging from 1.2% to 62.7%, 3.4% to 75.3%, and 5.3% to 72.0%,
respectively. Compounds 5a, 5b, 5n, 5q, 5s, 6e, and 6h exhibited
significant protective effects against TMV, showing higher inhibi-
tory activities (72.0%, 64.2%, 64.8%, 65.8%, 68.2%, 71.9%, and 74.2%,
different antiviral activities at 500
6g could inhibit TMV ex vivo by approximately 50% (EC50 for pro-
tective effect) at 257.7, 320.7, and 243.3 g/mL, respectively.
mg/mL. Compounds 5a, 6e, and
respectively) at 500 mg/mL than Ningnamycin (64.0%) at 500 mg/mL.
m
Compounds 5a, 5b, 5n, 5q, 5s, 6e, and 6g were shown to inactivate
efficacy at 70.7%, 75.3%, 73.8%, 69.3%, 73.9%, 68.3%, and 68.2%,
respectively, and they exhibited slightly lower inactivation activ-
Compared with the other compounds, 5r and 5j showed more
potent antiviral activities. Their antiviral activities against CMV
were found similar to that of Ningnanmycin (EC50 ¼ 399.0
mg/mL).
ities than Ningnanmycin (87.2%) at 500 mg/mL. Compounds 5a, 5b,
The present work demonstrates that (1E, 4E)-1-aryl-5-(2-(quina-
zolin-4-yloxy)phenyl)-1, 4-pentadien-3-one can be used to develop
potential agrochemicals. To the best of our knowledge, this study is
the first to report the antiviral activities of (1E, 4E)-1-aryl-5-(2-
(quinazolin-4-yloxy) phenyl)-1, 4-pentadien-3-one derivatives.
5j, 5n, 6c, 6e, and 6g exhibited good curative activities on TMV at
40.7%, 45.4%, 51.9%, 54.7%, 45.2%, 62.7%, and 54.7%, respectively, and
they showed the same curative activity level as the standard
reference Ningnanmycin at 500
mg/mL (55.4%). The variation
among the different substitutes on R1 and R2 greatly affected the
antiviral activities of the compounds against TMV. For example,
when R1 was 6-CH3 and R2 was 4-nitrophenyl, an apparent increase
in antiviral activity was found. By contrast, when R2 was 4-
nitrophenyl and R1 was introduced to 8-CH3 and H, the corre-
2. Results and discussion
2.1. Chemistry
sponding compounds 5p and 5c at 500 mg/mL inhibited TMV at
Target compounds 5aez, 6aeh were synthesized by treatment
of the substituted aldehyde with intermediates 4aef in the present
of base (NaOH in water) in acetone at room temperature, as shown
in Fig. 2. The key intermediates 4aef were synthesized by reacting
(E)-4-(2-hydroxyphenyl)-3-butene-2-ones 3 with substituted 4-
chloroquiazoline in the present of K2CO3 in CH3CN at 30 ꢀCe
50 ꢀC for 6 h, followed by dropwise addition of water. The result-
ing precipitate was filtered, washed with water, and then dried. The
solid was recrystallized using ethanol to yield intermediates 4aef.
The structures of all the compounds were confirmed by IR, 1H
NMR, and 13C NMR spectral analyzes and elemental analysis. The IR
spectra of the title product 5e exhibited an absorption wavelength
at 3445 cmꢁ1, which indicates the presence of amidic NeCHeN. The
stretching frequency at 1653 cmꢁ1 was assigned to C]O vibrations,
and the characteristic absorptions at 1491 cmꢁ1 to 1620 cmꢁ1 and
1219 cmꢁ1 to 1316 cmꢁ1 were attributed to the presence of C]C,
NeC, and CeOeC groups, respectively. As indicated by 1H NMR, all
phenyl protons showed multiplets at 7.50 ppm to 6.71 ppm. The
main characteristic of the 1H NMR spectra for the compound was
20.3% and 45.4%, respectively. Further toxicity bioassays revealed
that 5a, 5j, 5q, 5s, 6e, and 6g showed remarkable protective effects
against TMV, with EC50 values of 257.7, 346.7, 349.8, 333.9, 320.7
and 243.3
protective activities against the tested virus and were even superior
g/mL). This
mg/mL, respectively. These compounds possessed good
to the commercial agent Ningnamycin (EC50 ¼ 370.8
m
finding suggests that 5a, 6e, and 6g may be promising lead struc-
tures for the discovery of new antiviral agents.
2.2.2. Ex vivo antiviral activities of the title compounds against
CMV
Ex vivo antiviral tests of 5aez, 6aeh against CMV were con-
ducted, and the results are summarized in Table 2. The title com-
pounds 5b, 5c, 5e, 5g, 5r, 5n, 6c, 6e and 6g showed good protective
effects against CMV, with efficacy rates of 56.1%, 55.6%, 55.2%, 57.2%,
58.5%, 62.8%, 55.1%, 54.0%, and 55.2%, respectively, which were
close to that of Ningnamycin (60.1%). The protective rates of the
other compounds ranged from 10.9% to 48.9%, which were lower
than that of the positive control Ningnamycin (60.1%). The curative
Fig. 1. Design of the target compounds.

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H. Luo et al. / European Journal of Medicinal Chemistry 63 (2013) 662e669
Fig. 2. Synthetic route of 5aez and 6aeh.
activity levels of compounds 5b, 5n, 6c, and 6g (47.2%, 47.3%, 49.2%,
and 47.8%, respectively) at 500 g/mL were similar to that of
Ningnanmycin at 500 g/mL (55.1%). Compounds 5b, 5g, 5r, 5n, 6c,
and 6e showed moderate inactivation activity levels of 60.7%, 61.6%,
58.9%, 58.9%, 60.1% and 56.5%, respectively, which were lower than
Second, compared with the same phenyl substituents for R2, the
monosubstituents had higher activities than the polysubstituents.
m
m
For example, the EC50 value of 6g (R2 ¼ 4-fluorophenyl) at 500
mL against TMV was 243.3 g/mL. By contrast, the inhibitory rates
of 6a (R2 ¼ 2, 4-dichloropheny) and 6b (R2 ¼ 2, 3-dichlorophenyl),
g/mL were 40.2%,
mg/
m
that of Ningnanmycin (89.2%) at 500
mg/mL.
and 6c (R2 ¼ 3-nitro-4-chlorophenyl) at 500
m
23.0% and 4.4%. Third and last, compared with the same sub-
stituents on quiazoline, the corresponding molecules containing a
6-methyl group always had higher inhibitory rates for TMV than
the compound containing a 8-methyl group. For example, the EC50
values of 6e (R1¼6-methyl, R2 ¼ 4-nitrophenyl) and 6g (R1 ¼ 6-
2.2.3. Structureeactivity relationship (SAR) analysis of antiviral
activities against TMV and CMV
As summarized in Table 3, some of the compounds showed
promising potency against TMV. To examine SAR, different sub-
stituent groups were introduced into R1 and R2 in the quiazoline
ring. When R2 was 4-flurophenyl-fixed and R1 was 8-methyl,
compound 5m showed weak activity. When R1 was H or 6-methyl,
the corresponding target compounds had excellent activities.
Several title compounds showed comparably good activities
compared with Ningnamycin. Based on the activity values indi-
cated in Table 3, the relationships of the antiviral activities with
different R1 and R2 (type, position, and number of substituents)
were deduced. Three main conclusions were drawn. First, at the
same substituted position of the phenyl for R2, the F group was
superior to the NO2 and Cl groups in terms of antiviral activity. For
example, the inhibitory rate of 6g (R2 ¼ 4-fluorophenyl) on TMV
methyl, R2
243.3
¼
4-fluorophenyl) against TMV were 320.7 and
g/mL, respectively. By contrast, the inhibitory rates of 6p
m
(R1 ¼8-methyl, R2 ¼ 4-nitrophenyl) and 6m (R1 ¼8-methyl, R2 ¼ 4-
fluorophenyl) at 500
mg/mL against TMV were 23.8% and 8.4%,
respectively.
The results of ex vivo bioassay against CMV are shown in Table 4.
Ningnanmycin was used as the reference antiviral agent. The re-
sults listed in Table 4 revealed that the compounds 5d, 5j, 5r, and 6e
displayed good anti-CMV activities. The protective effects of com-
pounds 5d, 5j, 5r, and 6e (420.3, 412.5, 402.0, and 448.2 mg/mL,
respectively) against CMV were significant. Compared with the
other compounds, 5j and 5r exhibited more potent antiviral ac-
tivities against CMV. Their antiviral activities against CMV were
was 74.2% at 500
m
g/mL, whereas those of 6e (R2 ¼ 4-nitrophenyl)
and 6h (R2 ¼ 4-chlorophenyl) were 71.9% and 49.4% at the same
conditions. In terms of protective effects against TMV, the EC50
values of 6g (R2 ¼ 4-fluorophenyl) and 6e (R2 ¼ 4-nitrophenyl)
found similar to that of Ningnanmycin (EC50 ¼ 399.0
mg/mL).
3. Conclusion
were 243.3 and 320.7
mg/mL, respectively. Upon modifying the
substituents at the 3- or 4-positions in the phenyl group, the
inhibitory rate of 6f (R2 ¼ 3-nitrophenyl) at 500 g/mL was 5.3%.
In summary, a series of new (1E, 4E)-1-aryl-5-(2-(quinazolin-4-
yloxy)phenyl)-1,4-pentadien-3-one derivatives were designed and
m

H. Luo et al. / European Journal of Medicinal Chemistry 63 (2013) 662e669
665
Table 1
Inhibition effect and toxicity of the testing compounds (500
m
g/mL) against tobacco mosaic virus ex vivo.
Curative effect
Compd.
R1
R2
Inactivation
effect (%)
Protection
effect (%)
activity (%)
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
H
H
H
H
H
H
H
H
4-FePh
3-NO2ePh
4-NO2ePh
40.7*
45.4*
42.8*
41.6*
45.2*
12.0
37.2
31.5
28.6
51.9 *
1.2
70.7**
75.3**
67.6**
65.4**
59.5**
17.6
53.2*
42.9*
42.8*
61.5*
3.4
14.7
10.5
73.8**
43.6*
20.4
72.0**
64.2**
63.1**
64.3**
62.2**
13.9
49.5*
43.8*
39.9
Furan-2-yl
5-Chloro-1,3-dimethyl-1H-pyrazol-4-yl
2-Cl-5-NO2ePh
4-Cl-3-NO2ePh
3,4-di-OCH3ePh
3,4-di-ClePh
4-Cl-2-NO2ePh
Furan-2-yl
5-Chloro-1,3-dimethyl-1H-pyrazol-4-yl
4-FePh
2-ClePh
2,3-di-ClePh
4-NO2ePh
2-OCH3ePh
3,4-di-ClePh
2-FePh
Furan-2-yl
H
H
63.8*
5.3
22.4
8-CH3
8-CH3
8-CH3
8-CH3
8-CH3
8-CH3
8-CH3
8-CH3
H
10.8
2.3
8.4
54.7*
31.3
20.3
15.7
21.5
34.9
31.8
8.4
64.8**
47.1*
23.8
5q
5r
5s
5t
32.8
26.3
53.4*
45.0*
49.8*
5.5
46.3*
42.1*
43.3*
16.3
H
H
5u
4-NO2ePh
5v
5w
5x
5y
5z
6a
6b
6c
6d
6e
6f
6g
H
H
H
H
4-Cl-3-NO2ePh
2,3-di-ClePh
3-CF3ePh
15.8
16.5
27.8
21.8
39.1
20.3
18.1
45.2*
1.9
62.7**
2.6
54.7*
31.5
55.4**
69.3*
33.2
73.9**
36.7
55.3*
34.1
17.5
63.1**
1.5
68.3**
9.2
68.2**
43.8*
89.2**
65.8*
28.0
68.2**
33.3
47.2*
40.2*
23.0
54.5*
4.4
71.9**
5.3
74.2**
49.4*
64.0**
Styrene
H
2-Cl-4-NO2ePh
2,4-di-ClePh
2,3-di-ClePh
Furan-2-yl
4-Cl-3-NO2ePh
4-NO2ePh
3-NO2ePh
4-FePh
4-ClePh
6-CH3
6-CH3
6-CH3
6-CH3
6-CH3
6-CH3
6-CH3
6-CH3
6h
Ningnamycin
n ¼ 3 for all groups; *P < 0.05, **P < 0.01.
synthesized. The title compounds 5a, 5j, 5v, 5x, 6e, and 6g exhibited
good protective activities against TMV ex vivo compared with the
commercial Ningnamycin. The antiviral tests showed that when R1
was 6-methyl and R2 was 4-F and 4-NO2Ph, the corresponding
compounds presented good anti-TMV activities. The antiviral test
demonstrated that the control effect of compound 6g was better
than that of the commercial virucide Ningnamycin. Compared with
the other compounds, 5r and 5j had more potent antiviral activities.
Their antiviral activities were similar to that of Ningnanmycin
(400 mesh). Column chromatographic operations were performed
on silica gel (100e200 or 200e300 mesh, Qingdao Haiyang Chemical
Co.). A SephadexLH-20 column chromatographic instrument (Beijing
Huideyi Tech Instrument Co.) and an XAD-7HP macroporous resin
(Rohm and Haas Co., USA) were used for the extraction and purifi-
cation of the chemical compounds. Tobacco seeds (Yun 85) were
provided by the Guizhou Institute of Tobacco. Chenopodium amar-
anticolor seeds were provided by Northwest Agriculture and Forestry
University. Fertilized soil (Baltisches substrat, HAWITA BALTICÒ) was
purchased from Hawita Gruppe GmbH for plant cultivation.
(EC50 ¼ 399.0
mg/mL) against CMV. To the best of our knowledge,
this study is the first to demonstrate the antiviral activities of (1E,
4E)-1-aryl-5-(2-(quinazolin-4-yloxy)phenyl)-1,4-pentadien-3-one
derivatives against TMV on tobacco plants and CMV on cumber
plants. Further antiviral studies on the biological efficacies, crop
safety, and toxicities of these compounds as virucide candidates
need to be conducted.
4.1.2. Synthetic procedures
Quinazolin-4(3H)-one, 6-methyl-quinazolin-4(3H)-one, 8-
methyl-quinazolin-4(3H)-one, 6-methyl-4-chloroquiazoline, 8-
methyl-4-chloroquiazoline, and 4-chloroquiazoline were prepared
according to a previously described method [16]. 6-Fluoro-4-
chloroquiazoline was prepared according to
a
previously
4. Experimental
described method [17]. Intermediates 4-chloroquiazoline, 4, 6-
dichloroquiazoline, 4, 6, 8-trichloro-quiazoline, and 4-chloro-6, 7,
8-trimethoxyquiazoline were prepared following standard syn-
thetic protocols [18,19]. (E)-4-(2-Hydroxyphenyl)-3-butylene-2-
one was prepared according to a previously reported [20]. In-
termediates 4aef were prepared, and their characterization data are
provided in the Supporting information.
4.1. Chemistry
4.1.1. General
1H and 13C nuclear magnetic resonance (NMR) (solvent CDCl3 or
acetone-d6 or DMSO-d6) spectral analyzes were performed on a
JEOL-ECX 500 NMR spectrometer at room temperature using tetra-
methylsilane (TMS) as an internal standard. The infrared (IR) spectra
were obtained using a SHIMADZU-IR Prestige-21 spectrometer in
KBr pellets. The melting points were determined using an XT-4
digital microscope (Beijing Tech Instrument Co.). Analytical thin-
layer chromatography (TLC) was performed on silica gel GF254
4.1.3. General synthetic procedures for title compounds
Compounds5aez and 6aehweresynthesized (Fig. 2). Thestarting
materials were substituted 2-aminobenzoic acid, formamide, salicy-
laldehyde, and acetone. Substituted O-aminobenzoicacidwas reacted
with formamide for 4.5 h at 140e145 ꢀC to obtain the intermediate 4-

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H. Luo et al. / European Journal of Medicinal Chemistry 63 (2013) 662e669
Table 2
Inhibition effect and toxicity of the testing compounds (500 mg/mL) against cumber mosaic virus ex vivo.
Compd.
R1
R2
Curative
effect (%)
Inactivation
effect (%)
Protection
effect (%)
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
5t
5u
5v
5w
5x
5y
5z
6a
6b
6c
6d
6e
6f
H
H
H
H
H
H
H
H
4-FePh
3-NO2ePh
4-NO2ePh
Furan-2-yl
44.0*
47.2*
45.8*
21.5
47.1*
24.2
46.7*
39.6
29.9
39.4
4.5
42.4*
60.7**
57.6**
40.9*
54.0*
29.4
61.6**
47.7*
38.1
53.6*
8.3
20.1
16.4
52.8*
38.5
24.4
32.5
58.9**
36.7
38.2
15.8
58.9**
28.6
44.1*
27.8
33.5
44.8*
56.1**
55.6**
34.3
55.2*
26.1
57.2*
48.1*
36.5
46.8*
10.9
25.8
14.3
48.9*
40.4*
26.8
28.4
58.5**
35.7
36.1
24.4
62.8**
27.2
48.7*
26.6
31.2
29.0
5-Chloro-1,3-dimethyl-1H-pyrazol-4-yl
2-Cl-5-NO2ePh
4-Cl-3-NO2ePh
3,4-di-OCH3ePh
3,4-di-ClePh
4-Cl-2-NO2ePh
Furan-2-yl
5-Chloro-1,3-dimethyl-1H-pyrazol-4-yl
4-FePh
2-ClePh
2,3-di-ClePh
4-NO2ePh
2-OCH3ePh
3,4-di-ClePh
2-FePh
Furan-2-yl
H
H
8-CH3
8-CH3
8-CH3
8-CH3
8-CH3
8-CH3
8-CH3
8-CH3
H
H
H
H
H
16.7
6.6
44.1*
31.6
24.4
20.9
41.9*
33.1
31.9
18.7
47.3*
23.2
35.7
22.0
28.6
21.3
24.6
49.2*
7.1
4-NO2ePh
4-Cl-3-NO2ePh
2,3-di-ClePh
3-CF3ePh
H
H
H
Styrene
2-Cl-4-NO2ePh
2,4-di-ClePh
2,3-di-ClePh
Furan-2-yl
4-Cl-3-NO2ePh
4-NO2ePh
3-NO2ePh
4-FePh
4-ClePh
6-CH3
6-CH3
6-CH3
6-CH3
6-CH3
6-CH3
6-CH3
6-CH3
26.9
24.1
60.1**
6.2
56.5*
19.5
51.0*
31.3
87.3**
28.0
55.1*
11.3
54.0*
16.5
55.2*
32.2
38.5
13.3
47.8*
28.2
55.1**
6g
6h
Ningnamycin
60.1**
quinazolinone 1. Substituted 4-chloroquinazoline 2 was synthesized
via chlorination of SOCl2. Intermediates 4aef were synthesized
starting from the (E)-4-(2-hydroxyphenyl)-3-butene-2-ones with
K2CO3 in acetonitrile for 3.5 h at 40e50 ꢀC. Finally, the target (1E, 4E)-
1, 4-pentadien-3-ones (5aez, 6aeh) was obtained via condensation
of intermediate 4 and substituted aldehydes. The physical character-
istics, IR, 1H NMR, 13C NMR, and elemental analysis data for all the
synthesized compounds are reported in the experimental protocols.
The data for 5a are shown below, whereas those for the others can be
found in the Supporting information.
filtrate was centrifuged at 10,000g, treated twice with PEG, and
then centrifuged again. The entire experiment was conducted at
4
ꢀC. Absorbance values were estimated at 260 nm using an ul-
traviolet spectrophotometer.
nm
Virus concentration ¼ ðA260 ꢂ dilution ratioÞ=E0:1%; 260
1
cm
4.2.2. Protective effects of the compounds against TMV ex vivo
The compound solution was smeared on the left side, whereas
the solvent served as the control on the right side of growing
N. tabacum L. leaves of the same age. The leaves were inoculated
with the virus after 12 h. A brush was dipped in 6 ꢂ 10ꢁ3 mg/mL
TMV to inoculate the leaves, which were previously scattered with
silicon carbide. Subsequently, the leaves were washed with water
and rubbed softly along the nervature once or twice. The number of
local lesions appearing 3e4 d after inoculation was counted [22].
Three repetitions were conducted for each compound.
4.1.4. (1E, 4E)-1-(2-Fluorophenyl)-5-(2-(quinazolin-4-yloxy)
phenyl)penta-1,4-dien-3-one (5a)
Yield 51.2%; yellow solid; m.p. 162e164 ꢀC; 1H NMR (500 MHz,
CDCl3)
d
: 8.75 (s,1H, Qu-2-H), 8.47 (d, J ¼ 8.0 Hz,1H, Qu-8-H). 8.04 (d,
J ¼ 8.6 Hz, 1H, Qu-5-H), 7.96 (t, J ¼ 6.7 Hz, J ¼ 6.8 Hz, 1H, Qu-7-H),
7.80e7.85 (m, 2H, AreCH], FeAreCH]), 7.72 (t, J ¼ 8.2 Hz,1H, Qu-6-
H), 7.49e7.52 (m, 2H, FeAr-2,6-H), 7.39e7.52 (m, 3H, Ar-3,5-H, FeAre
C]CH), 7.30 (d, J ¼ 12.7 Hz, 1H, AreC]CH), 7.01e7.11 (m, 3H, FeAr-
3,5-H, Ar-2-H), 6.76 (d, J ¼ 16.5 Hz, 1H, Ar-6-H); 13C NMR (125 MHz,
4.2.3. Inactivation effects of the compounds against TMV ex vivo
The virus was inhibited by mixing with the compound solution
at the same volume for 30 min. The mixture was inoculated on the
left side of N. tabacum L. leaves, whereas the right side of the leaves
was inoculated with the mixture of solvent and the virus to serve as
the control. The number of local lesion numbers appearing 3e4 d
after inoculation was counted [22]. Three repetitions were con-
ducted for each compound.
CDCl3, ppm) d: 188.56, 166.89, 165.12, 163.11, 154.23, 151.92, 151.57,
142.28, 136.68, 134.53, 131.70, 129.31, 130.24, 128.32, 128.23, 128.11,
127.27, 126.75, 123.57, 123.54, 116.27; Anal. Calcd for C25H17FN2O2
(397): C, 75.75; H, 4.32; N, 7.07. Found: C, 75.93; H, 4.64; N, 6.86.
4.2. Antiviral bioassay against TMV
4.2.1. Purification of TMV
4.2.4. Curative effects of the compounds against TMV ex vivo
Growing N. tabacum L. leaves of the same age were selected.
TMV at a concentration of 6 ꢂ 10ꢁ3 mg/mL was dipped and inoc-
ulated on the whole leaves. Subsequently, the leaves were washed
Using Gooding’s method [21], the upper leaves of Nicotiana
tabacum L. inoculated with TMV were selected, ground in phos-
phate buffer, and then filtered through a double-layer pledget. The

H. Luo et al. / European Journal of Medicinal Chemistry 63 (2013) 662e669
667
Table 3
Results of EC50 values of 5aez, and 6aeh against tobacco mosaic virus.
Compd.
R1
R2
EC50 for curative
EC50 for inactivation
EC50 for protection
effect (
m
g/ml)
effect (
mg/ml)
effect (mg/ml)
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
H
H
H
H
H
H
H
H
4-FePh
3-NO2ePh
4-NO2ePh
Furan-2-yl
499.2
532.4
672.2
437.5
636.1
e
381.3
396.4
424.7
383.5
455.5
e
257.7
435.8
378.5
376.9
441.8
e
5-Chloro-1,3-dimethyl-1H-pyrazol-4-yl
2-Cl-5-NO2ePh
4-Cl-3-NO2ePh
3,4-di-OCH3ePh
3,4-di-ClePh
4-Cl-2-NO2ePh
Furan-2-yl
5-Chloro-1,3-dimethyl-1H-pyrazol-4-yl
4-FePh
2-ClePh
2,3-di-ClePh
4-NO2ePh
2-OCH3ePh
3,4-di-ClePh
2-FePh
Furan-2-yl
618.7
e
456.1
629.8
596.1
400.9
e
479.7
549.2
e
H
H
e
461.9
e
346.7
e
8-CH3
8-CH3
8-CH3
8-CH3
8-CH3
8-CH3
8-CH3
8-CH3
H
e
e
e
e
e
e
475.3
e
418.3
602.6
e
449.8
551.4
e
e
5q
5r
5s
5t
e
e
e
566.7
e
451.6
560.8
496.4
e
470.8
574.1
553.7
e
H
H
e
5u
4-NO2ePh
e
5v
5w
5x
5y
H
H
H
H
4-Cl-3-NO2ePh
2,3-di-ClePh
3-CF3ePh
352.0
e
349.0
e
349.8
e
571.4
e
328.1
e
333.9
e
Styrene
5z
6a
6b
6c
6d
6e
6f
6g
H
2-Cl-4-NO2ePh
2,4-di-ClePh
2,3-di-ClePh
Furan-2-yl
4-Cl-3-NO2ePh
4-NO2ePh
3-NO2ePh
4-FePh
4-ClePh
e
478.9
e
514.8
6-CH3
6-CH3
6-CH3
6-CH3
6-CH3
6-CH3
6-CH3
6-CH3
e
e
e
e
580.2
e
418.7
e
471.0
e
455.8
e
352.7
e
320.7
e
441.3
e
364.6
555.9
86.5
243.3
504.7
370.8
6h
Ningnamycin
437.6
with water and dried. The compound solution was smeared on the
left side, and the solvent was smeared on the right side to serve as
the control. The number of local lesion appearing 3e4 d after
inoculation was counted [22]. Three repetitions were conducted for
each compound. The inhibitory rate of the compound was calcu-
lated according to the following formula (“av” means average):
N,N-dimethylformamide and then dissolved with distilled water
containing 1% Tween 20 at 500 g/mL concentration [23].
m
4.3.3. Curative effects of the compounds against CMV ex vivo
Growing C. amaranticolor leaves of the same age were selected.
Crude extracts of CMV were dipped and inoculated using a brush on
avlocallesionnumbers of controlðnottreatedwithcompoundÞꢁavlocallesionnumberssmeared with drugs
avlocallesionnumbersof controlðnottreatedwith compoundÞ
ꢂ100%
Inhibitoryrateð%Þ ¼
4.3. Antiviral bioassay against CMV
the whole leaves, which were previously scattered with silicon
carbide. The leaves were washed with water after inoculation for
0.5 h and then dried. The compound solution was smeared on the
left side of the leaves, and the solvent was smeared on the right side
to serve as the control. All plants were cultivated in an incubator at a
temperature of 28 ꢃ 1 ꢀC and an illumination of 10,000 Lux. The
number of local lesions appearing 6e7 d after inoculation was
counted [24]. Three repetitions were conducted for each compound.
4.3.1. Extraction of CMV
N. tabacum L. leaves inoculated with CMV were selected, ground
in phosphate butter, and then filtered through a double-layer
pledget. The filtrate was centrifuged at 8000g, and the superna-
tant liquid was the crude extract of the virus. The entire experiment
was performed at 4 ꢀC.
4.3.2. Preparation of antiviral control
The test compounds and 2% Ningnanmycin aqua (reference
antiviral agent) were initially dissolved with a small amount of
4.3.4. Inactivation effects of the compounds against CMV ex vivo
The virus was inhibited by mixing with the compound solution
at the same volume for 30 min. The mixture was inoculated on the

668
H. Luo et al. / European Journal of Medicinal Chemistry 63 (2013) 662e669
Table 4
Results of EC50 values of 5aez, and 6aeh against cumber mosaic virus.
Compd.
R1
R2
EC50 for curative
EC50 for inactivation
EC50 for protection
effect (
m
g/ml)
effect (
mg/ml)
effect (mg/ml)
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
H
H
H
H
H
H
H
H
4-FePh
3-NO2ePh
4-NO2ePh
Furan-2-yl
529.1
583.1
600.5
498.1
584.3
e
547.1
454.5
479.7
405.7
497.8
e
447.9
501.1
505.4
420.3
470.4
e
5-Chloro-1,3-dimethyl-1H-pyrazol-4-yl
2-Cl-5-NO2ePh
4-Cl-3-NO2ePh
3,4-di-OCH3ePh
3,4-di-ClePh
4-Cl-2-NO2ePh
Furan-2-yl
5-Chloro-1,3-dimethyl-1H-pyrazol-4-yl
4-FePh
2-ClePh
2,3-di-ClePh
4-NO2ePh
2-OCH3ePh
3,4-di-ClePh
2-FePh
550.1
e
438.2
534.8
e
492.0
540.0
e
H
H
e
605.6
e
398.4
e
412.5
e
8-CH3
8-CH3
8-CH3
8-CH3
8-CH3
8-CH3
8-CH3
8-CH3
H
e
e
e
e
e
e
588.2
e
489.3
e
509.9
597.4
e
e
e
5q
5r
5s
e
e
e
525.2
e
399.7
e
402.0
e
5t
H
Furan-2-yl
e
e
e
5u
H
4-NO2ePh
e
e
e
5v
5w
5x
5y
H
H
H
H
4-Cl-3-NO2ePh
2,3-di-ClePh
3-CF3ePh
530.8
e
566.2
e
476.7
e
592.7
e
438.1
e
460.1
e
Styrene
5z
6a
6b
6c
6d
6e
6f
6g
H
2-Cl-4-NO2ePh
2,4-di-ClePh
2,3-di-ClePh
Furan-2-yl
4-Cl-3-NO2ePh
4-NO2ePh
3-NO2ePh
4-FePh
4-ClePh
e
e
e
6-CH3
6-CH3
6-CH3
6-CH3
6-CH3
6-CH3
6-CH3
6-CH3
e
e
e
e
e
e
500.7
e
406.6
e
456.7
e
513.3
e
473.5
e
448.2
e
533.6
e
490.7
e
471.6
e
6h
Ningnamycin
443.3
98.7
399.0
left side of C. amaranticolor leaves, whereas the right side of the
leaves was inoculated with the mixture of solvent and the virus to
serve as the control. All leaves were previously scattered with sil-
4.3.6. Calculation of inhibitory rate
The inhibitory rate of the compounds was calculated according
to the following formula (“av” denotes average):
Inhibitory rateð%Þ
av local lesion no: of right leaveðnot treated with compoundÞ ꢁ av local lesion no: of left leaveðsmeared with compoundÞ
av local lesion no: of right leaveðnot treated with compoundÞ
¼
ꢂ 100%
icon carbide. All plants were cultivated using an incubator at a
SPSS 17.0 was used for the statistical analysis of inhibition data.
ANOVA (least significant difference method) was performed to
analyze the differences among the treatment groups.
temperature of 28 ꢃ 1 ꢀC and an illumination of 10,000 Lux. The
number of local lesions appearing 6e7 d after inoculation was
counted [24]. Three repetitions were conducted for each
compound.
Acknowledgments
4.3.5. Protective effects of the compounds against CMV ex vivo
The compound solution was smeared on the left side, whereas
the solvent was smeared on the right side of growing
C. amaranticolor leaves of the same age to serve as the control. The
leaves were inoculated with CMV after 1 d with a brush. The leaves
were washed with water after inoculation for 2 h. All plants were
cultivated in an incubator at a temperature of 28 ꢃ 1 ꢀC and an
illumination of 10,000 Lux. The number of local lesions appearing
6e7 d after inoculation was counted [24]. Three repetitions were
conducted for each compound.
The authors gratefully acknowledge the financial support of the
National Key Program for Basic Research (No. 2010CB 126105), the
Key Technologies R&D Program (No. 2011BAE06B05-6), and the
National Natural Science Foundation of China (Nos. 21132003,
21172048).
Appendix A. Supporting information
Supporting information related to this article can be found at
http://dx.doi.org/10.1016/j.ejmech.2013.02.035. These data include

H. Luo et al. / European Journal of Medicinal Chemistry 63 (2013) 662e669
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described in this article.
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