Synthesis, antiviral bioactivity of novel 4-thioquinazoline derivatives containing chalcone moiety

Article abstract of DOI:10.3390/molecules200711861

A series of novel 4-thioquinazoline derivatives containing chalcone moiety were designed, synthesized and systematically evaluated for their antiviral activity against TMV. The bioassay results showed that most of these compounds exhibited moderate to good anti-TMV activity. In particular, compounds M₂ and M₆ possessed appreciable protection activities against TMV in vivo, with 50% effective concentration (EC₅₀) values of 138.1 and 154.8 μg/mL, respectively, which were superior to that of Ribavirin (436.0 μg/mL). The results indicated that chalcone derivatives containing 4-thioquinazoline moiety could effectively control TMV. Meanwhile, the structure-activity relationship (SAR) of the target compounds, studied using the three-dimensional quantitative structure-activity relationship (3D-QSAR) method of comparative molecular field analysis (CoMFA) based on the protection activities against TMV, demonstrated that the CoMFA model exhibited good predictive ability with the cross-validated q² and non-cross-validated r² values of 0.674 and 0.993, respectively. Meanwhile, the microscale thermophoresis (MST) experimental showed that the compound M₆ may interaction with the tobacco mosaic virus coat protein (TMV CP).

Full text of DOI:10.3390/molecules200711861

Molecules 2015, 20, 11861-11874; doi:10.3390/molecules200711861
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis, Antiviral Bioactivity of Novel 4-Thioquinazoline
Derivatives Containing Chalcone Moiety
Zhihua Wan, Deyu Hu *, Pei Li, Dandan Xie and Xiuhai Gan
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, Guizhou Province, China;
E-Mails: wzhuacool@163.com (Z.W.); pl19890627@126.com (P.L.); xddxed@163.com (D.X.);
gxh200719@163.com (X.G.)
* Author to whom correspondence should be addressed; E-Mail: fcc.dyhu@gzu.edu.cn;
Tel./Fax: +86-851-829-2170.
Academic Editor: Derek J. McPhee
Received: 21 April 2015 / Accepted: 16 June 2015 / Published: 29 June 2015
Abstract: A series of novel 4-thioquinazoline derivatives containing chalcone moiety were
designed, synthesized and systematically evaluated for their antiviral activity against TMV.
The bioassay results showed that most of these compounds exhibited moderate to good
anti-TMV activity. In particular, compounds M2 and M6 possessed appreciable protection
activities against TMV in vivo, with 50% effective concentration (EC50) values of 138.1
and 154.8 μg/mL, respectively, which were superior to that of Ribavirin (436.0 μg/mL). The
results indicated that chalcone derivatives containing 4-thioquinazoline moiety could
effectively control TMV. Meanwhile, the structure-activity relationship (SAR) of the target
compounds, studied using the three-dimensional quantitative structure-activity relationship
(3D-QSAR) method of comparative molecular field analysis (CoMFA) based on the protection
activities against TMV, demonstrated that the CoMFA model exhibited good predictive
ability with the cross-validated q2 and non-cross-validated r2 values of 0.674 and 0.993,
respectively. Meanwhile, the microscale thermophoresis (MST) experimental showed that
the compound M6 may interaction with the tobacco mosaic virus coat protein (TMV CP).
Keywords: 4-thioquinazoline derivatives; chalcone moiety; synthesis; antiviral activity;
TMV; 3D-QASR; MST

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1. Introduction
Tobacco mosaic virus (TMV) is a positive-sense single stranded RNA virus that infects members of
nine plant families and at least 125 species, including tobacco, tomato, pepper, cucumbers, and a number
of ornamental flowers. Once infected with TMV, leaves tend to become mosaic, yellow, necrosis, and
ruguse, which would affect crop production and quality, and caused $100 million in economic losses in
worldwide [1]. However, there are no effective antiviral agents for controlling TMV. Therefore, it is a
challenge to the development of novel, potent, and structurally concise antiviral agents.
4-Thioquinazoline and their derivatives, an important class of heterocyclic compounds, have a wide
range of biological properties [2], including antibacterial [3,4], antifungal [5–7], and anticancer [8]
activities. Over the past few years, the synthesis and study of bioactivity of 4-thioquinazoline derivatives
have attracted considerable attention. In our previous study, we have designed and synthesized a series
of S-substituted 6-fluoro-4-alkyl(aryl)thioquinazoline derivatives and 6-bromo-4-alkylthioquinazoline
derivatives which exhibited good antifungal activities [6,7].
Chalcones, belonging to the flavonoid family and obtained from Carthamus tinctorius first, possessed
a broad spectrum of biological activities, including antibacterial [9–12], antifungal [13], anti-Alzheimer’s
disease [14], anticancer [15], antitrichomonal [16], and anti-trypanosomacruzi [17] activities. It is
reported that Verma et al., had reported a series of chalcones derivatives, which were used to control
plant viruses [18]. Meanwhile, some studies also demonstrated that chalcone derivatives could better
control TMV [19], PVX [20], and ToRSV [21,22], respectively. Moreover, in our previous study, we
have reported a series of chalcone derivatives with better bioactivities against TMV and CMV [23].
Tobacco mosaic virus coat protein (TMV CP) assembly systems include helix and four-layer aggregate
disk systems. The helix forms of TMV CP mainly exist in the presence of TMV RNA. In these helix
forms, TMV CP has an important role in the self-assembly of TMV through an initial RNA recognition
reaction that triggers the assembly, which is believed to be necessary for virus assembly initiation and
elongation. The four-layer aggregate disk forms consisting of 34 subunit aggregates of TMV CP are
crystallized as a dimer of bilayer disks with 17 subunits per layer in the absence of TMV RNA [24].
In this work, a series of novel 4-thioquinazoline derivatives containing chalcone moiety were
designed, synthesized and systematically evaluated their antiviral activities against TMV. Biological
results showed that some of the title compounds displayed moderate to good antiviral activity. Among
the title compounds, compounds M2 and M6 showed appreciable protection activities against TMV
in vivo, which were better than that of the commercial agricultural antiviral agent Ribavirin. In addition,
the structure-activity relationship (SAR) of the compounds was also discussed using comparative
molecular field analysis (CoMFA) of the three-dimensional quantitative structure-activity relationship
(3D-QSAR) method based on the protection activities against TMV. The microscale thermophoresis
(MST) experimental showed that the compound M6 may interactions with the TMV CP. To the best of
our knowledge, this is the first report on the synthesis, antiviral activity, 3D-QSAR, and interaction
study of 4-thioquinazoline derivatives containing chalcone moiety.

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2. Results and Discussion
2.1. Chemistry
The summary of the synthetic route designed for the 4-thioquinazoline derivatives containing chalcone
moiety were shown in Scheme 1. Substituted 2-aminobenzoicacid, using as the starting materials, was
reacted with formamide for 4.5 h at 140 to 145 °C to obtain the intermediate 4(3H)-quinazolinone (1).
Substituted 4-chloroquinazoline (2) was prepared by chlorination reaction with SOCl2 [8]. Then, using
ethanol as the solvent, nucleophilic substitution reaction of 2 and carbamimidothioic acid, transformed
from thiourea via keto-enol tautomerism equilibrium, was reacted 8 h at 87 °C to obtain the intermediate
quinazolin-4-yl carbamimidothioate, then quinazolin-4-yl carbamimidothioate was reacted with potassium
hydrate, and adjusted pH value to 7 by acetic acid to get the key intermediate quinazoline-4-thiol
(3) [25,26]. Intermediates 4 was obtained via condensation of the 1-(4-hydroxyphenyl)ethanone with
substituted aldehydes. Then, intermediates Z1–Z21 were obtained via nucleophilic substitution reaction
of 4 and 1,2-dibromoethane in DMF for 8 h at 80 °C. Finally, the target compounds M1–M21 were
synthesized by the etherification reaction of intermediates 3 and Z with KOH in DMF at 40 °C for
8 to 10 h.
Scheme 1. Synthetic route of the target compounds.
1
The structures of all the title compounds confirmed through IR, H-NMR, 13C-NMR spectral
and elemental analyses. The IR spectra of the title compounds M1–M21 exhibited characteristic
absorption bands at 1690–1640 cm−1, which indicated the presence of C=O. The stretching frequency at
1
1620–1600 cm−1 was assigned to C=N vibrations. H-NMR indicated that all of the phenyl protons
1
showed multiplets at 8.27–6.93 ppm. The main characteristic of the H-NMR spectra for the target
compounds was the presence of a high-frequency downfield singlet δH 9.00–9.02 for Qu-2-H. And
–O–CH2– and –S–CH2– showed a triplet at 4.40–4.45 ppm and 3.80–3.84 ppm, respectively. Moreover,

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13
the chemical shifts shown at nearly 188.40–189.40, 66.50–66.60, and 28.22–28.33 ppm in C-NMR
were also confirmed the presence of C=O, –OCH2–, and –SCH2–, respectively.
2.2. Antiviral Activity Screening of Title Compounds against TMV in Vivo
The antiviral activities of the target compounds M1–M21 against TMV were evaluated by the half-leaf
method [27] and the results were summarized in Table 1. Most of the title compounds generally
exhibited good antiviral activity against TMV in vivo. The results of the preliminary bioassays indicated
that compounds M1, M2, and M6 exhibited better curative activities against TMV at 500 μg/mL, with
the values of 52.5% ± 5.2%, 47.7% ± 4.3%, and 48.3% ± 3.3%, respectively. Which were better than
that of Ribavirin (37.9% ± 1.9%). Compounds M2 and M6 exhibited significant protection activities
against TMV at 500 μg/mL, with the values of 68.6% ± 7.7% and 72.3% ± 5.2%, respectively, which
were even better than that of Ribavirin (51.8% ± 2.3%). The inactivation activities of compounds M2,
M6, M8, M10, and M14 at 500 μg/mL were 82.1% ± 4.1%, 83.2% ± 4.4%, 85.6% ± 2.6%, and 85.1% ±
3.6%, respectively, which were better than that of Ribavirin (72.9% ± 2.4%).
Table 1. Antiviral activities of the test compounds against tobacco mosaic virus (TMV)
in vivo at 500 μg/mL.
Compd. Curative Activity (%) a Protection Activity (%) a Inactivation Activity (%) a
M1
M2
52.5 ± 5.2
47.7 ± 4.3
42.1 ± 6.1
45.3 ± 2.3
29.8 ± 4.2
48.3 ± 3.3
43.2 ± 6.7
45.1 ± 6.6
43.2 ± 3.5
46.5 ± 5.6
42.4 ± 4.3
38.4 ± 4.5
46.2 ± 5.8
46.5 ± 3.7
38.7 ± 6.2
38.5 ± 4.3
46.3 ± 4.3
45.7 ± 3.8
41.2 ± 6.2
41.2 ± 3.2
45.2 ± 6.2
37.9 ± 1.9
60.7 ± 4.3
68.6 ± 7.7
65.4 ± 2.9
82.1 ± 4.1
63.4 ± 6.8
83.2 ± 4.4
63.9 ± 6.1
79.6 ± 3.4
66.1 ± 5.0
85.6 ± 2.6
75.2 ± 3.8
85.1 ± 3.6
71.5 ± 5.1
66.0 ± 2.8
70.8 ± 2.7
83.6 ± 2.3
62.9 ± 6.0
57.3 ± 3.5
61.5 ± 3.2
66.4 ± 4.2
65.6 ± 4.7
63.6 ± 4.3
60.3 ± 5.9
72.9 ± 2.4
M3
57.4 ± 2.4
M4
50.2 ± 2.2
M5
59.3 ± 5.9
M6
72.3 ± 5.2
M7
56.3 ± 4.6
M8
51.1 ± 4.2
M9
49.4 ± 5.2
M10
M11
M12
M13
M14
M15
M16
M17
M18
M19
M20
M21
Ribavirin
62.2 ± 2.1
57.3 ± 3.6
57.6 ± 1.2
51.8 ± 5.5
62.3 ± 6.2
52.8 ± 4.7
48.3 ± 5.2
57.4 ± 5.1
61.3 ± 4.5
60.3 ± 3.5
47.3 ± 4.1
55.6 ± 6.1
51.8 ± 2.3
a: Average of three replicates.
Based on the previous bioassays, the 50% effective concentration (EC50) values of protection
activities against TMV of the title compounds were tested and presented in Table 2. As indicated in

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Table 2, most of the target compounds showed good anti-TMV activities. Compounds M1, M2, M4,
M6, M10, M11, M12, M14, M15, M18, and M19 exhibited higher protection activity than Ribavirin
(436.0 ± 4.3 μg/mL), with the EC50 values range from 138.1 ± 3.4 to 274.3 ± 6.2 μg/mL. Especially,
compounds M2 and M6 exhibited the best protection activity against TMV, with the EC50 values of
156.4 ± 4.1 and 138.1 ± 3.4 μg/mL, respectively, which were better than that of Ribavirin
(436.0 ± 4.3 μg/mL). Compounds M5, M7, M13, M16, and M21 exhibited moderate protection activity
against TMV, with EC50 values of 355.9 ± 3.5, 406.9 ± 5.2, 424.0 ± 1.9, 345.1 ± 3.6, and 342.5 ± 4.3 μg/mL.
This finding suggests that these compounds may be potential lead structures for the discovery of new
antiviral agents.
Table 2. Actual and predicted protection activities against TMV.
Compd.
M1
EC50 (μg/mL) a Actual pEC50 (μM) b Predicted pEC50 (μM) b Residual
255.2 ± 3.2
156.4 ± 4.1
444.4 ± 2.6
208.1 ± 4.2
355.9 ± 3.5
138.1 ± 3.4
406.9 ± 5.2
442.1 ± 4.3
486.3 ± 6.3
204.6 ± 3.6
218.1 ± 5.2
314.6 ± 2.6
424.0 ± 1.9
319.5 ± 3.7
274.3 ± 6.2
345.1 ± 3.6
198.2 ± 5.2
246.6 ± 3.2
211.2 ± 4.2
488.2 ± 3.5
342.5 ± 4.3
436.0 ± 4.3
3.239
3.487
3.020
3.356
3.114
3.534
3.012
3.037
2.988
3.371
3.291
3.148
3.055
3.111
3.212
3.153
3.363
3.299
3.358
2.945
3.099
/
3.249
3.494
3.015
3.351
3.066
3.531
3.069
3.042
2.994
3.373
3.286
3.194
3.051
3.083
3.257
3.057
3.349
3.268
3.295
2.942
3.110
/
−0.010
−0.007
0.005
0.005
0.048
0.003
−0.057
−0.005
−0.006
−0.002
0.005
−0.046
0.004
0.028
−0.045
0.096
0.014
0.031
0.063
0.003
−0.011
/
M2
M3
M4
M5
M6
* M7
M8
M9
M10
M11
* M12
M13
M14
M15
M16
M17
M18
* M19
M20
M21
Ribavirin
a: Average of three replicates; b: pEC50 = −lg (EC50); *: Samples of the testing set.
2.3. Antiviral Activity and Structure Activity Relationship against TMV
As an extension of this approach, the structure-activity relationships were deduced on the basis of
activity values in Tables 1 and 2. Some of the compounds showed potency against TMV. When the R were
2-CH3-Ph (M1), 4-CF3-Ph (M2), 3,4-di-OCH3-Ph (M6), and 2-CF3-Ph (M10) groups, the corresponding
target compounds exhibited good curative activity. When R group was substituted with fused ring,
it was disfavored for the anti-TMV following the order of M5 (Naphthalene-2-yl) < M7 (Thiophene-2-yl).
And, when the R were 4-CF3-Ph (M2) and 3,4-di-OCH3-Ph (M6) groups, the corresponding compounds
exhibited good protection activities against TMV which was surpassed that of Ribavirin. However,

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compared with M3 (2-Cl-6-F-Ph), M8 (2,6-di-Cl-Ph), M15 (4-Cl-Ph), and M19 (3,4-di-Cl-Ph), we found
that the one who has dichlorophenyl substituent groups could increase the activity against TMV (M8 and
M15 > M3 and M13). Furthermore, the presence of 4-(trifluoromethyl)phenyl (M2), 3,4-dimethoxypheny
(M6), and 3,4-dichlorophenyl (M19) groups in a compound effectively improved the antiviral activity of
the compound more than that of other groups. Moreover, compared with M2 (4-CF3-Ph), M10 (2-CF3-Ph),
M3 (2-Cl-6-F-Ph), M20 (2-F-Ph), and M21 (4-F-Ph), we found that the one who has trifluoromethyl
substituent groups increases in the activity against TMV (M2 and M10 > M3, M20 and M21).
2.4. 3D-QSAR Study
In order to analyze the SAR base on the protection activity against TMV, the CoMFA of 3D-QSAR
model [28] with a total of twenty-one target compounds were developed by using Sybyl 7.3 software [29]
from Tripos Inc. (St. Louis, MO, USA). Predicted pEC50 [30] values of compounds in both the training
and testing sets were presented together with their actual pEC50 values in Table 2, and correlations
between predicted and actual pEC50 in CoMFA model were presented in Figure 1. Overall, predicted
EC50 values were very close to the corresponding actual values for compounds in both the training and
testing sets. The mostly linear correlations in Figure 1 demonstrated high predictive power of the
CoMFA model. Meanwhile, as shown in Table 3, the non-cross-validated PLS analysis was repeated
with the optimum number of components, as determined by the cross-validated analysis. To obtain
statistical confidence limits, the non-cross-validated analysis was repeated, which yielded an r2 value
of 0.993, and the q2 value of highly predictive CoMFA was 0.674 with 8 ONC, which suggested that
the model has good predictive ability (r2 > 0.9, q2 > 0.5). Meanwhile, as shown in Table 3, the SEE
was 0.020 and the F value was 161.503, respectively. The relative contributions to bioactivity from
steric and electrostatic fields in the CoMFA model were 0.478 and 0.522, respectively, suggesting that
bioactivity was mainly determined by electrostatic interactions.
Figure 1. Plot of actual predicted activities for training set and test set based on the
comparative molecular field analysis (CoMFA) model.

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Table 3. Statistical parameters of the CoMFA model.
Statistical Parameter
CoMFA
0.674
8
q2 a
ONC b
r2 c
SEE d
F e
Steric f
0.993
0.020
161.503
0.478
0.522
Electrostatic g
b
c
a: Cross-validated correlation; : Optimum number of components; : Non-cross-validated correlation;
d: Standard error of estimate; e: F value; f: Stericfield contribution; g: Electrostatic field electrostatic.
The CoMFA contour map of the steric was shown in Figure 2A. Green contours in the CoMFA steric
field indicated regions where bulky groups would increase activity, whereas yellow contours indicated
regions where bulky groups would decrease activity. As shown in Figure 2A, a green contour around
3- and 4-positions of aromatic ring suggested that anti-TMV activity increases with bulky substituents
in the order of M6 (3,4-di-OCH3-Ph, 1.898 Å3) > M9 (3,4-di-Cl-Ph, 1.707 Å3), and M2 (4-CF3-Ph,
1.616 Å3) > M12 (4-OCH3-Ph, 1.586 Å3). A larger yellow contour around 2- and 3-positions of aromatic
ring showed that compounds with bulky groups at this position exhibited lower anti-TMV activity in
the order of M13 (2,3-di-Cl-Ph, 1.725 Å3) > M9 (2,3-di-OCH3-Ph, 1.494 Å3).
Figure 2. CoMFA contour map for the steric (A) and electrostatic (B) component.
The CoMFA contour map of the electrostatic was shown in Figure 2B. Blue contours in the CoMFA
electrostatic field indicated regions where electron withdrawing groups would increase activity and red
contours indicated regions where electron-donating groups would increase activity. As shown in Figure 2B,
a red contour and a blue contour around 3- and 4-position of aromatic ring, which indicated that
suitable groups in the region could increase the activities follow the activity order of M15 (4-Cl-Ph) >
M12 (4-OCH3-Ph) > M21 (4-F-Ph). Meanwhile, a blue contour around 2-position of aromatic ring
showed that the electron withdrawing groups were favored at improvement of activity in the order of
M10 (2-CF3-Ph) > M1 (2-OCH3-Ph).
3D-QASR results indicated that introduction of small and electron withdrawing groups at 2-position
of aromatic ring could largely improve the activity. Bulky groups at 3- and 4-position of the aromatic
ring played a favorable role in improving the activity.

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2.5. Binding Sites of M6, M15 and Ribavirin to TMV CP
In order to study the interactions between target compounds and TMV CP, the MST analysis method
was used. The MST results indicated that the compounds M6, M15, and Ribavirin binding to TMV CP
protein yielded Kd values of 31.1 ± 1.83, 346 ± 23.9, and 511 ± 33.1 μM, respectively (Figure 3).
As predicted in MST, M6 shares, indeed, moderate affinity, in the contrary to M15 and Ribavirin, which
share weak affinity. The results showed that the combining capacity in the order of M6 > M15 > Ribavirin
is consistent with the trend of antiviral activity screening. The experimental results showed that the
compound M6 may interactions with the TMV CP. As shown in the Figure 3 and Table 4, bulky groups
at 3- and 4-position of aromatic ring played a favorable role in improving the activity. The bioactivity
was mainly determined by electrostatic interactions. As shown in Figure 3 and Table 4, when bulky
group at 3- and 4-position of aromatic ring (3,4-di-OCH3-Ph), the corresponding compound (M6) played
a stronger combining capacity, with the Kd values of 31.1 ± 1.83 μM, compared with that of compound
M15 (346 ± 23.9 μM), which was substituted with smaller group at 4-position of aromatic ring (4-Cl-Ph).
Figure 3. Microscale thermophoresis (MST) of M6 (A); M15 (B); and Ribavirin (C).
Table 4. The dissociation constant of M6, M15, and Ribavirin with TMV- coat protein (CP).
Compd.
M6
Kd (μM)
31.1 ± 1.83
346 ± 23.9
511 ± 33.1
M15
Ribavirin
3. Experimental Section
3.1. Instruments
1H-NMR and 13C-NMR spectra were obtained at 500 MHz using a JEOL-ECX500 NMR
spectrometer at room temperature using tetramethylsilane as an internal standard (solvent CDCl3).
Elemental analysis was performed on an Elementar Vario-III CHN analyzer. The melting points of the
products were determined under an XT-4 binocular microscope (Beijing Tech. Instrument Co., Beijing,
China) and left untouched. Analytical thin-layer chromatography (TLC) was conducted on a silica gel
GF254 (400 mesh). Column chromatographic operations were performed on silica gel (200–300 mesh).
Tobacco seeds were provided by the Guizhou Institute of Tobacco.

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3.2. Chemistry
3.2.1. General Procedure for Preparation of Intermediates 3
Quinazolin-4(3H)-one (1) and4-chloroquiazoline (2) were prepared according to a previously
described method [8]. 4-thioquinazoline (3) was gained by 4-chloroquiazoline with thiourea at reflux
temperature for 8 h. The data for intermediates quinazolin-4(3H)-one (1), 4-chloroquiazoline (2), and
4-thioquinazoline (3) can be found in the previously references [8,25].
3.2.2. General Procedure for Preparation of Intermediates Z
–
1 21
A mixture of 4 (4.95 mmol) and K2CO3 (9.99 mmol) in DMF (10 mL) was stirred at 80 °C for 1 h.
Then, 1,2-dibromoethane (14.84 mmol) was added dropwise. The mixture was stirred until the TLC
showed the reaction finished. The reaction mixture was poured into water (20 mL). The mixture was
extracted with EtOAc (3 × 10 mL) and the combined organic layers were dried over anhydrous Na2SO4
and concentrated under reduced pressure. The crude product was purified by column chromatography
over silicagel by using petroleum ether and ethyl acetate (v/v = 2:1) as eluent to give Z1 21 as a solid.
–
3.2.3. General Procedure for Preparation of Title Compounds (M1–M21)
The target compounds M1–M21 were synthesized as schematized in Scheme 1. A 25 mL
round-bottomed flask equipped with a magnetic stirrer was charged with intermediates 3 (0.74 mmol),
KOH (0.89 mmol), DMF (5 mL). The flask was stirred at 40 °C for 1 h, and then Z (0.59 mmol) in
DMF (2 mL) was dropwise into the flask. The resulting mixture was stirred at 40 °C for 8 to 10 h. TLC
monitored the progress of the reaction. Upon completion of the reaction (as indicated by TLC), the
reaction mixture was poured into saturated brine, the solid was filtered off and then dissolved with
dichloromethane and washed by 10% KOH. The organic fraction was evaporated under reduced pressure.
The solid was recrystallized from ethyl acetate/petroleum ether (3:1, v/v) to obtain the title compounds
1
13
M1–M21 with the yields from 56% to 78%. The physical characteristics, IR, H-NMR, C-NMR, and
elemental analysis data, for all the synthesized compounds were reported in Supplementary data and
the representative data of M6 were shown below.
(E)-3-(3,4-Dimethoxyphenyl)-1-(4-(2-(quinazolin-4-ylthio)ethoxy)phenyl)prop-2-en-1-one (M6). Yellow
solid; m.p. 151.2–153.1 °C; yield, 72.5%; IR (KBr, cm−1) ν: 3023.5–3068.8 (C-H of benzene), 2832.5
(–OCH3), 1654.0 (C=N), 1604.9 (C=O), 1490.0–1562.4 (C=C and benzene and Qu-ring), 1328.0 (C–N),
1
1260.5 (C–O); 1172.7 (–O–CH3); H-NMR (500 MHz, CDCl3) δ: 9.01 (s, 1H, Qu-2-H), 8.10 (d,
J = 7.45 Hz, 1H, Qu-8-H), 8.04 (d, J = 9.15 Hz, 2H, CO–Ph-2,6-H), 7.98 (d, J = 8.55 Hz, 1H, Qu-5-H),
7.86 (t, J1 = 8.60 Hz, J2 = 6.85 Hz, 1H, Qu-7-H), 7.77 (d, 1H, J = 15.45 Hz, Ar–CH), 7.60 (t, J1 = 7.40 Hz,
J2 = 6.90 Hz, 1H, Qu-6-H), 7.41 (d, J = 15.45 Hz, 1H, Ph–CO=CH), 7.23 (dd, J1 = 8.50 Hz,
J2 = 1.70 Hz, 1H, Ar-2-H), 7.15 (d, J = 1.75 Hz, 1H, Ar-6-H), 7.07 (d, J = 8.60 Hz, 2H, CO–Ph-3,5-H),
6.90 (d, J = 8.50 Hz, 1H, Ar-5-H), 4.42 (t, J1 = 6.60 Hz, J2 = 7.40 Hz, 2H, –OCH2–), 3.95 (s, 3H,
13
–CH3), 3.92 (s, 3H, –CH3), 3.81 (t, J1 = 7.35 Hz, J2 = 6.30 Hz, 2H, –SCH2–); C-NMR (125 MHz,
CDCl3) δ: 188.71, 170.25, 162.13, 153.41, 151.26, 149.20, 148.09, 144.24, 133.92, 131.62, 130.76,

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128.90, 128.01, 127.54, 123.89, 123.77, 122.98, 119.75, 114.41, 111.10, 110.07, 66.46, 55.99, 55.97,
28.22; Anal. Calcd for C27H22N2O4: C, 68.62; H, 5.12; N, 5.93; Found: C, 68.30; H, 5.11; N, 5.91.
3.3. Antiviral Biological Assay
3.3.1. Purification of TMV
Using Gooding’s method [21], the upper leaves of N. tabacum cv. K326 inoculated with TMV were
selected, ground in phosphate buffer, and then filtered through a double-layer pledget. The filtrate was
centrifuged at 10,000 g, 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 ultraviolet spectrophotometer.
virus concn = (ꢀꢁꢂꢃ × diluton ratio)/ꢄꢅꢃ.ꢆꢅꢇ%,ꢁꢂꢃ ꢈꢇ
(1)
3.3.2. Curative Effects of the Target Compounds against TMV in Vivo
Growing 5–6-leaf stage Nicotiana tabacum L. tobaccos were selected. TMV (concentration of
6 × 10−3 mg/mL) was dipped and inoculated using a brush on the whole leaves, which were previously
then dried. The compound solution was smeared on the left side of the leaves, scattered with silicon
carbide. The leaves were then washed with water after inoculation for 0.5 h and the solvent was smeared
on the right side for control. All plants were cultivated in an incubator at a temperature of 23 ± 1 °C
and an illumination of 10,000 Lux. The number of local lesions was counted and recorded 3 to 4 days
after inoculation. Three repetitions were conducted for each compound [27].
3.3.3. Protection Effects of the Target Compounds against TMV in Vivo
The compound solution was smeared on the left side, whereas the solvent was smeared on the right
side of Nicotiana tabacum L. leaves of the same age to serve as the control. The leaves were inoculated
with the virus after 12 h. A brush was dipped in 6 × 10−3 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. All plants were cultivated in an incubator at a temperature
of 23 ± 1 °C and an illumination of 10,000 Lux. The number of local lesions was counted and recorded
3 to 4 days after inoculation. Three repetitions were conducted for each compound [27].
3.3.4. Inactivation Effects of the Target Compounds against TMV in Vivo
The virus was inhibited by mixing with the compound solution at the same volume for 30 min.
The mixture was then inoculated on Nicotiana tabacum L. leaves, and the right side of the leaves was
inoculated with solvent and virus mixture for control. All of the leaves were previously scattered with
silicon carbide. All plants were cultivated in an incubator at a temperature of 23 ± 1 °C and an
illumination of 10,000 Lux. The number of local lesions was counted and recorded 3 to 4 days after
inoculation. Three repetitions were conducted for each compound [27].
The inhibitory rate of the compounds was calculated according to the following formula
(“av” denotes average):

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Inhibition rate (%) = [(av local lesion No. of control (not treated with compd.) − av local lesion
No. smeared with drugs)/av local lesion No. of control (not treated with compd.)] × 100%
(2)
3.4. 3D-QSAR Study
The protection activity used in study was expressed as pEC50 listed in Table 2, 18 molecules of total
compounds were randomly chosen as the training set for CoMFA and the other three compounds
(asterisk labeled) were used as the testing set.
3.4.1. Molecular Modeling and Alignment
Molecular modeling, CoMFA analysis was performed using Sybyl 7.3 (Tripos Inc., St. Louis, MO,
USA) software. The 3D structures of all molecules were built using the “Sketch Molecule” function in
Sybyl. Initial optimization of the structures were carried out using the Gasteiger-Hückel charge, Tripos
force field, and Powell conjugate gradient algorithm with a convergence criterion of 0.005 kcal/mol·Å [28].
The 3D structures of the 21 molecules were aligned on a common template molecule with M6 for the
CoMFA modeling study.
3.4.2. Partial Least-Squares Analysis
The partial least squares (PLS) analysis was used to derive the 3D-QSAR models. In which,
molecules were placed in a rectangular grid, the steric and electrostatic fields were calculated using a
volume-dependent lattice with a 2.0 Å grid spacing [29], and the CoMFA descriptors was used as the
independent variables, and the experimental pEC50 values were presented as the dependent variables.
Then, 3D-QSAR analysis was carried out using the PLS technique. The cross-validation and the ONC were
used to evaluate the performance of the models, ONC was determined with the highest cross-validated
q2 [30,31]. Then, the non-cross-validated correlation coefficient r2 value, standard error of estimate (SEE),
and F value and standard error were calculated according to the definitions in Sybyl 7.3 package, and
as factors for estimating. The contour maps and standard deviations values of CoMFA was generated
by the PLS coefficients.
3.5. MST Studies
TMV CP was purified according to a previously described method [24]. A range of concentrations
of the required compounds (range from 0.1 to 2 mM) were incubated with 0.1 mM of purified
recombinant TMV CP for 5 min with the Monolith NT Protein Labeling Kit Red (Nano Temper
Technologies, München, Germany) in assay buffer (10 mM Tris/HCl and 100 mM sodium chloride,
pH 7.4). The sample was loaded into the NanoTemper glass capillaries and microthermophoresis
carried out using 50% LED power and 40% MST. The Kd values were calculated from the duplicate
reads of three separate experiments using the mass action equation in the NanoTemper software [32].

Molecules 2015, 20
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4. Conclusions
In summary, a series of 4-thioquinazoline derivatives containing chalcone moiety were prepared
and evaluated for their antiviral activities against TMV using half-leaf method in vivo. Bioassay results
indicated that compounds M2 and M6 possessed appreciable protection activities against TMV in vivo,
with the EC50 values of 156.4 and 138.1 μg/mL, respectively, which were superior to that of Ribavirin
(436.0 μg/mL). Meanwhile, the CoMFA model was generated base on the protection activities against
TMV and exhibited good predictive abilities with the cross-validated q2 and non-cross-validated r2 values
of 0.674 and 0.993, respectively. The MST experimental showed that the compound M6 may interaction
with the TMV CP. The model provided a practical tool for the modification and optimization of
4-thioquinazoline derivatives containing chalcone moiety to further improve the antiviral activity.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/20/07/11861/s1.
Acknowledgments
The authors gratefully acknowledge the financial support of the National Natural Science
Foundation of China (No. 21362004) and Collaborative Innovation Center for Natural Products and
Biological Drugs of Yunnan.
Author Contributions
D.H. conceived and designed the experiments. Z.W. and P.L. performed the experiments and
analyzed the data; X.G. and D.X. tested all the antiviral activities of the compounds; D.H. analyzed the
data and wrote the paper, D.H. and P.L. revised the paper. All authors contributed to this study, read
and approved the final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds M1–M21 are available from the authors.
© 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/4.0/).