Syntheses, antiviral activities and induced resistance mechanisms of novel quinazoline derivatives containing a dithioacetal moiety

Article abstract of DOI:10.1016/j.bioorg.2018.06.026

A series of novel quinazoline derivatives containing a dithioacetal moiety were designed and synthesized, and their structures were characterized by ¹H nuclear magnetic resonance, ¹³C nuclear magnetic resonance, and high-resolution mass spectrometry. Bioassay results indicated that compound 4b exhibited remarkable protective activity against cucumber mosaic virus (CMV, EC₅₀ = 248.6 μg/mL) and curative activity against potato virus Y (EC₅₀ = 350.5 μg/mL), which were better than those of ningnanmycin (357.7 μg/mL and 493.7 μg/mL, respectively). Moreover, compound 4b could increase the chlorophyll content in plants, improve photosynthesis, and effectively induce tobacco anti-CMV activity.

Full text of DOI:10.1016/j.bioorg.2018.06.026

Accepted Manuscript
Syntheses, antiviral activities and induced resistance mechanisms of novel qui-
nazoline derivatives containing a dithioacetal moiety
Dandan Xie, Jing Shi, Awei Zhang, Zhiwei Lei, Guangcheng Zu, Yun Fu, Xiuhai
Gan, Limin Yin, Baoan Song, Deyu Hu
PII:
S0045-2068(18)30292-X
https://doi.org/10.1016/j.bioorg.2018.06.026
YBIOO 2406
DOI:
Reference:
Bioorganic Chemistry
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Revised Date:
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26 March 2018
9 June 2018
20 June 2018
Please cite this article as: D. Xie, J. Shi, A. Zhang, Z. Lei, G. Zu, Y. Fu, X. Gan, L. Yin, B. Song, D. Hu, Syntheses,
antiviral activities and induced resistance mechanisms of novel quinazoline derivatives containing a dithioacetal
moiety, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.06.026
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Syntheses, antiviral activities and induced resistance mechanisms of novel
quinazoline derivatives containing a dithioacetal moiety
Dandan Xie, Jing Shi, Awei Zhang, Zhiwei Lei, Guangcheng Zu, Yun Fu, Xiuhai Gan, Limin Yin,
Baoan Song*, Deyu Hu*
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
*Author to whom correspondence should be addressed; E-mail: dyhu@gzu.edu.cn;
songbaoan22@yahoo.com

Abstract
A series of novel quinazoline derivatives containing a dithioacetal moiety were designed and
1
synthesized, and their structures were characterized by H nuclear magnetic resonance, 13C nuclear
magnetic resonance, and high-resolution mass spectrometry. Bioassay results indicated that compound
4b exhibited remarkable protective activity against cucumber mosaic virus (CMV, EC50 = 248.6 μg/mL)
and curative activity against potato virus Y (EC50 = 350.5 μg/mL), which were better than those of
ningnanmycin (357.7 μg/mL and 493.7 μg/mL, respectively). Moreover, compound 4b could increase
the chlorophyll content in plants, improve photosynthesis, and effectively induce tobacco anti-CMV
activity.
Keywords: Quinazoline derivatives, Dithioacetal moiety, Antiviral activity, Cucumber mosaic virus,
Induced resistance mechanism
Abbreviations used
1
1H NMR, H nuclear magnetic resonance; 13C NMR, 13C nuclear magnetic resonance; HRMS, High
resolution mass spectrometry; CMV, cucumber mosaic virus; PVY, potato virus Y; DMSO, dimethyl
sulfoxide; CC, cellular components; MF, molecular functions; BP, biological processes; COS, chitosan
oligosaccharide; DEPs, differential proteomics analysis; GO, gene ontology; KEGG, Kyoto
Encyclopedia of Genes and Genomes; PS I, photosystem I; PS II, photosystem II; ATP, adenosine
triphosphate; NPR1, nonexpressor of pathogenesis related genes 1; CAT, catalase 1; PR1, related
protein 1; ICS1, isochorismate synthase 1; SOD, superoxide dismutase; PAL, phenylalanine
ammonia-lyase.
1. Introduction
Cucumber mosaic virus (CMV) and potato virus Y (PVY) are two well-known agricultural viruses,
which can infect many plants and cause serious economic losses in annual agricultural production
worldwide [1,2]. However, the control effects of existing anti-plant virus reagents against these plant
viruses are generally unsatisfactory. For instance, ningnanmycin, one of the successfully registered and
widely used as plant virus inhibitor, has only 30%–60% control effect against these plant viruses [3,4].
Thus, the development of potent novel molecules for controlling plant virus disease is still required.

Quinazoline is one important heterocyclic structure found in many synthetic and naturally occurring
products [5]. As a natural alkaloid, quinazoline has been the focus of considerable attention because of
its potential biological activities in agrochemicals, such as antibacterial [6], antifungal [7], and antiviral
activities [8]. In our previous work, several series of quinazoline derivatives were synthesized and
exhibited good antiviral activities [9–12]. Among these quinazoline derivatives, when the derivatization
site is at position 4 of the quinazoline ring, the compounds showed good activities against tobacco
mosaic virus (TMV) and CMV in vivo. For example, a series of 1,4-pentadien-3-one derivatives
containing the 4-thioquinazoline moiety were synthesized, and these derivatives exhibited good
antiviral
activity
against
CMV.
Moreover,
we
also
detected
(1E,4E)-1-aryl-5-(2-(quinazolin-4-yloxy)phenyl)-1,4-pentadien-3-one derivatives with excellent
protective activities against TMV in vivo.
Dithioacetal is the sulfur analog of acetal. Recent research on dithioacetal derivatives have mainly
focused on synthetic methodology [13–15]. In our previous study, we observed for the first time that
the dithioacetal derivatives exhibited remarkable antiviral activities against CMV and PVY, and the
compound 2,2′-((4-(chlorobenzyl)oxy)-3-methoxyphenyl)methylene-bis(2-hydroxyethyl) [16] (6f, Fig.
1) is being further developed as a potential antiviral agent. On the basis of these facts, we sought to
incorporate a dithioacetal substructural unit into the backbone of quinazoline to obtain novel
quinazoline derivatives with good antiviral activities. To continue our work on developing novel and
promising antiviral agents, a series of novel quinazoline derivatives containing a dithioacetal moiety
were designed and synthesized. The antiviral activities of all of the synthesized compounds against
CMV and PVY were assessed. Compound 4b showed excellent antiviral activities, which is related to
that compound 4b can increase the chlorophyll content in plants and improve photosynthesis, and then
effectively induce tobacco antiviral activity.
2. Results and discussions
2.1 Chemistry
The synthetic route of compounds 4a–4x is depicted in Scheme 1. Intermediate 2 was synthesized
according to the previously presented method in the literature [9]. Intermediate 3 was prepared
according to the method previously presented in the literature [8]. Target compounds 4a–4x were
synthesized by condensation of 1 equivalent of aldehyde and 2 equivalent of thiol, with NaHSO4·SiO2
as the catalyst. Notably, in the syntheses of these types of compounds, we previously used ZrCl4 as

catalyst and obtained a good yield (usually greater than 95%). However, in the syntheses of 4a–4x, the
yield when using ZrCl4 as catalyst is usually only approximately 50%. When ZrCl4 was replaced with
NaHSO4·SiO2 as the catalyst, the yield considerably increased (isolation yield was usually greater than
80%). In addition, compared with ZrCl4, NaHSO4·SiO2 has many advantages as catalyst in the reaction,
such as water insensitivity, inexpensive, and could be used repeatedly. The structures of all compounds
were confirmed by 1H NMR, 13C NMR, and HRMS.
2.2 Biological activity
2.2.1 Antiviral activities of compounds 4a–4x
In this study, the antiviral activities of target compounds 4a–4x against CMV and PVY were evaluated
using previously presented methods [16–18], and the commercial agent Ningnanmycin and compound
6f were used as post control. The results of the bioassay against CMV and PVY were shown in Table 1
and indicate that most of the title compounds exhibited good to excellent curative activities in vivo.
Compounds 4b, 4i, 4o, and 4p showed excellent curative activities against CMV at 500 μg/mL with
inhibition rates of 58.3%, 56.5%, 58.2%, and 56.0%, respectively, which were better than those of
Ningnanmycin (50.0%) and 6f (54.2%). Compound 4b showed excellent protective activities against
CMV with inhibition rate of 61.1%, which was better than those of Ningnanmycin and 6f. Moreover,
compounds 4b, 4h, 4i, and 4o exhibited similar or even higher curative activities (i.e., 53.5%, 51.7%,
53.7% and 51.9%, respectively) against PVY than that of 6f (52.0%) and better than that of
Ningnanmycin (46.5%). Compounds 4b, 4h, 4i, 4q, and 4s exhibited comparative protective activities
(i.e., 52.0%, 52.2%, 54.3%, 52.3%, and 51.3%, respectively) against PVY to that of 6f (54.5%) and
better than that of Ningnanmycin (47.3%).
On the basis of a preliminary biological evaluation, the EC50 values of the curative and protective
activities against CMV and PVY of some target compounds were evaluated. As shown in Table 1,
compound 4b showed excellent protective activity against CMV with EC50 value of 248.6 μg/mL,
which was better than that of Ningnanmycin (357.7 μg/mL) and 6f (298.5 μg/mL). Compounds 4a and
4o exhibited good curative activities against CMV, with EC50 values of 333.6 μg/mL and 359.4 μg/mL,
which were similar to that of Ningnanmycin but weaker than that of 6f. Meanwhile, compounds 4b, 4i,
and 4o exhibited excellent curative activity against PVY, with EC50 values of 350.5, 357.5, and 376.6
μg/mL, which were better than that of Ningnanmycin (493.7 μg/mL) and 6f (381.7 μg/mL).
2.2.2 Effect of compound 4b on the chlorophyll content in plants

As the most important and basic physiological process of plants, photosynthesis can provide energy
and organics for plant growth. Chlorophyll is the core pigment in plant chloroplasts, and its function is
to capture light energy and drive electrons to the reaction center; thus, it plays an important role in
plant growth [19]. In this study, we investigated the change of the chlorophyll content in tobacco that
was infected with CMV at different times, with chitosan oligosaccharide (COS) as a comparison. As
shown in Fig. 2, the chlorophyll content in tobacco plant decreased gradually with CMV inoculation
(Fig. 2A–2C). However, in CMV-inoculated tobacco leaves treated with compound 4b, the chlorophyll
content increased from day 1 to day 5 and reached the highest value at day 5 and the variation in trends
surpassed that of COS treatment. On the seventh day, the chlorophyll content in compound 4b-treated
tobacco decreased gradually. When the chlorophyll content was measured, similar variation tendencies
were observed in Ct and Ca/Cb ratios of all treatments (Fig. 2D). Therefore, compound 4b could
increase the chlorophyll content in plants, improve photosynthesis, and effectively induce tobacco
anti-CMV activity.
2.2.3 Effect of compound 4b on defense gene expression of plants
The difference of defense gene expression of 4b, COS, and CK treatment of tobacco on the third day
was investigated, and the results are shown in Fig. 3. The results indicated that the gene expression
levels of nonexpressor of pathogenesis related genes 1 (NPR1), isochorismate synthase 1 (ICS1), and
copper/zine superoxide dismutase (SOD) on tobacco after compound 4b treatment were significantly
higher than those of the CK and COS treatment groups. The NPR1 gene expression level of 4b+CMV
was even 45 and 68 times higher than those of CK+CMV and COS+CMV, respectively. The related
protein 1 (PR1) gene expression level of compound 4b treatment was significantly higher that of
CK+CMV but slightly lower than that of COS+CMV. In addition, the catalase 1 (CAT1) and
phenylalanine ammonia lyase 4 (PAL4) gene expression levels of 4b+CMV were slightly higher than
that of CK+CMV but significantly higher that of COS+CMV.
2.2.4 Results of proteomics analysis
To investigate the effect of proteins on CMV-infected tobacco after compound 4b treatment, we
extracted and identified the total proteins of samples on the third day through label-free LC-MS/MS.
The results showed significant changes in proteins in the treatment and control groups. As shown in Fig.
4, 1160 and 2026 proteins were detected in the compound 4b and CK treatment groups, respectively
(Supporting Information Tables S1 and S2). A total 984 proteins (44.7%) were common in both groups.

A total 176 and 1042 proteins exhibited different expression levels in the compound 4b and CK
treatment groups, respectively.
2.2.5 Functional classification by gene ontology (GO)
Considerable efforts on GO analysis were conducted to assign functional information to differential
proteomics analysis (DEPs) between 4b-treated and CK-treated tobacco. The hierarchical relationships
for the representation of information on cellular components (CC), molecular functions (MF), and
biological processes (BP) are illustrated in Fig. 5. The enrichment analysis of DEPs indicated that the
main CC involved the cytoplasm, membrane, nucleus, p, chloroplast, mitochondrion, cytosol, plasma
membrane, extracellular region, and cell wall. MF involved RNA binding, DNA binding, nuclease
activity, kinase activity, enzyme regulator activity, protein binding, helicase activity, signal transporter
activity, and ATPase activity. BP involved the generation of precursor metabolites and energy,
photosynthesis, transport, response to oxidative stress, lipid metabolism, and response to stress. We
investigated the hierarchical relationships for the representation of information on CC, MF, and BP. We
determined that a large number of proteins involved in photosynthesis, including photosystem II (PSII)
D2 protein, PSII CP43 reaction center protein, photosystem I (PSI) P700 chlorophyll a apoprotein A2,
and PSI P700 chlorophyll a apoprotein A1, were changed. The results indicated that compound 4b had
a significant effect on the photosynthesis of tobacco.
2.2.6 Discussion of proteins related to photoreaction system
In plants, different genes coordinate their biological functions. Pathway-based analysis was conducted
to analyze the biological functions of genes further. The Kyoto Encyclopedia of Genes and Genomes
(KEGG) was the main public database for pathways and used to identify potential biological pathways
of DEPs between CK+CMV and 4b+CMV treatment groups. Table 2 shows a total of 16 DEPs, which
include 8 up-regulated and 8 down-regulated expressed proteins. The results indicated that
photosynthesis (pathway ID: Ko00195) was enriched as one of the most important pathways for DEPs.
Photosynthesis is an important metabolic process of plants and algae, including PSI PSII, cytochrome
b6/f complex, and adenosine triphosphate (ATP) synthase. Among them, PSI uses light energy to
produce the high-energy carriers ATP and nicotinamide adenine dinucleotide phosphate, and thus has
an important role [20]. The PSI reaction center is a pigment–protein complex, which is composed of
two core polypeptide structures, namely, PsaA and PsaB [21]. PsaA and PsaB exist as heterodimers,
and nearly all chlorophyll a in PSI bind to the PsaA/B heterodimer [22]. In the compound 4b treatment

group, the upregulation of PsaA and PsaB may indirectly led to an increase in the quantity of
chlorophyll a (Fig. 6). Moreover, PsaN is a 10 kD protein, which played an important role in the
docking of plastocyanin to the PSI complex. In the absence of PsaN, the second-order rate constant for
the reduction of P700+ by plastocyanin was decreased by approximately 40% [22–24]. PsaN is related
to the transfer rate of electrons in PSI. In short, the results showed that compound 4b could induce the
expression of proteins during photosynthesis, thereby improving the quality and efficiency of
photosynthesis in plants and making plants more resistant to viral infections.
3. Conclusion
In summary, a series of novel quinazoline derivatives containing dithioacetal moiety were
synthesized. The structures of the title compounds were verified by 1H NMR, 13C NMR, and HRMS,
and their antiviral activities against CMV and PVY were evaluated. The results showed that some
compounds exhibited remarkable antiviral activities. Compound 4b exhibited excellent protective
activity against CMV and curative activity against PVY with EC50 values of 248.6 μg/mL and 350.5
μg/mL, respectively, which were obviously superior to those of ningnanmycin (357.7 μg/mL and 493.7
μg/mL, respectively). Moreover, Compound 4b can induce resistance of tobacco, and then inhibit virus
infection and replication. Further studies of the structural optimization and mechanisms of antivirals
are currently underway.
4. Experimental sections
4.1 Chemistry
All starting materials and reagents were commercially available and used without further purification,
1
except as indicated. The H NMR and 13C NMR spectra were recorded on a JEOL-ECX500 MHz
(JEOL, Tokyo, Japan) or a Bruker DPX 400 MHz (Bruker BioSpin GmbH, Rheinstetten, Germany)
NMR spectrometer with CDCl3 or dimethyl sulfoxide (DMSO) as the solvent. The following
abbreviations were used to explain the multiplicities: s, singlet; d, doublet; t, triplet; m, multiplet; and
br, broadened. The melting points were determined on a WRX-4 microscope melting point apparatus
(YiCe Apparatus & Equipment co., LTD, Shanghai, China). High-resolution mass spectrometry
(HRMS) was conducted using a Thermo Scientific Q Exactive (Thermo Fisher Scientific,
Massachusetts, America).
4.1.1 General procedure for the syntheses of intermediate 1
A mixture of 2-aminobenzoic acid (0.1 mol) and formamide (20 mL) was refluxed for 10 h. After the

mixture was cooled to room temperature, water (200 mL) was added and the precipitate was filtered off.
The crude product was recrystallized from ethanol (100 mL) to the desired intermediate 1 as a white
solid.
4.1.2 General procedure for the syntheses of intermediate 2
A mixture of 4-hydroxyquinazoline (0.02 mol) in SOCl2 (20 mL) containing DMF (2 drops) was
refluxed for 5 h. SOCl2 was removed under reduced pressure, and the residue was dissolved in
dichloromethane (DCM). The solution was washed with NaHCO3 solution and brine, dried over
anhydrous Na2SO4, and concentrated under reduced pressure to obtain the desired compound as a
yellow solid.
4.1.3 General procedure for the syntheses of intermediate 3
Hydroxyl aldehyde dissolved in acetonitrile (ACN; 5.0 mL) was added to a solution of intermediate 2
(1.0 mmol) and potassium carbonate (1.2 mmol) in ACN (25.0 mL) by dripping funnel (1.0 mmol),
refluxed for 8 h, filtered, and concentrated under vacuum. The crude compound was purified by flash
chromatography on silica gel with n-hexane/EtOAc (3:1, v/v) to obtain the desired intermediate 3 as a
white solid.
4.1.4 General procedure for the syntheses of compounds 4a–4x
NaHSO4·SiO2 (0.01 mmol) was added to the solution of intermediate 3 (0.1 mmol) and thiol (0.2 mmol)
in 30 mL DCM. The reaction mixture was stirred for 6 h at room temperature. The mixture was filtered,
and the solvent was removed in vacuo. The residue was purified by flash chromatography on silica gel
with n-hexane/EtOAc (3:1, v/v) to obtain the pure product. The NMR and HRMS data of compounds
4a–4x are listed as follows.
4-(4-(bis(ethylthio)methyl)phenoxy)quinazoline (4a): white solid; m.p. 55.3 °C–56.8 °C; yield, 81.2%;
1H NMR (400 MHz, CDCl3) δ 8.78 (s, 1H, quinazoline-H), 8.38 (d, J = 8.3 Hz, 1H, quinazoline-H),
8.02 (d, J = 8.4 Hz, 1H, quinazoline-H), 7.93 (t, J = 8.4 Hz, 1H, quinazoline-H), 7.78 (d, J = 8.6 Hz,
1H, quinazoline-H), 7.68 (t, J = 8.2 Hz, 2H, Ph-H), 7.26 (t, J = 8.2 Hz, 2H, Ph-H), 4.99 (s, 1H, SCHS),
2.75–2.51 (m, 4H, 2CH2CH3), 1.27 (t, J = 7.4 Hz, 6H, 2CH2CH3). 13C NMR (100 MHz, CDCl3) δ
166.8, 154.2, 151.7, 138.2, 134.3, 132.4, 129.2, 129.2, 127.8, 123.7, 121.8, 121.8, 116.5, 116.1, 52.0,
26.3, 26.3, 14.3, 14.3. HRMS (ESI): calculated for C19H21N2OS2 [M+H]+: 357.1089, found: 357.1084.
4-(4-(bis(ethylthio)methyl)-2-methoxyphenoxy)quinazoline (4b): white solid; m.p. 79.0 °C–81.9 °C;
1
yield, 86.5%; H NMR (500 MHz, CDCl3) δ 8.74 (s, 1H, quinazoline-H), 8.40 (d, J = 8.2 Hz, 1H,

quinazoline-H), 8.01 (d, J = 8.5 Hz, 1H, quinazoline-H), 7.92 (t, J = 7.7 Hz, 1H, quinazoline-H), 7.67 (t,
J = 7.6 Hz, 1H, quinazoline-H), 7.23 (d, J = 1.7 Hz, 1H, Ph-H), 7.17 (d, J = 8.1 Hz, 1H, Ph-H), 7.10
(dd, J = 8.1, 1.9 Hz, 1H, Ph-H), 4.97 (s, 1H, SCHS), 3.77 (s, 3H, OCH3), 2.72–2.56 (m, 4H, 2CH2CH3),
1.28 (t, J = 7.4 Hz, 6H, 2CH2CH3). 13C NMR (125 MHz, CDCl3) δ 166.8, 154.4, 151.7, 151.6, 140.8,
139.5, 134.1, 127.9, 127.6, 124.0, 122.6, 120.4, 116.3, 112.2, 56.0, 52.4, 26.5, 26.5, 14.4, 14.4. HRMS
(ESI): calculated for C20H23N2O2S2 [M+H]+: 387.1196, found: 387.1188.
4-(4-(bis(propylthio)methyl)-2-methoxyphenoxy)quinazoline (4c): white solid; m.p. 38.6 °C–39.6 °C;
1
yield, 81.4%; H NMR (500 MHz, CDCl3) δ 8.74 (s, 1H, quinazoline-H), 8.40 (d, J = 8.3 Hz, 1H,
quinazoline-H), 8.01 (d, J = 8.4 Hz, 1H, quinazoline-H), 7.91 (t, J = 8.4 Hz, 1H, quinazoline-H), 7.66 (t,
J = 8.1 Hz, 1H, quinazoline-H), 7.23 (d, J = 2.0 Hz, 1H, Ph-H), 7.16 (s, 1H, Ph-H), 7.10 (d, J = 2.0 Hz,
1H, Ph-H), 4.92 (s, 1H, SCHS), 3.76 (s, 3H, OCH3), 2.60 (m, 2.67–2.54, 4H, 2CH2CH3), 1.67–1.59 (m,
4H, 2SCH2), 1.00 (t, J = 7.3 Hz, 6H, 2CH3). 13C NMR (125 MHz, CDCl3) δ 166.7, 154.3, 151.6, 151.5,
140.7, 139.6, 134.0, 127.8, 127.4, 123.9, 122.5, 120.3, 116.2, 112.1, 55.9, 53.0, 34.4, 34.4, 22.6, 22.6,
13.6, 13.6. HRMS (ESI): calculated for C22H27N2O2S2 [M+H]+: 415.1508, found: 415.1502.
4-(4-(bis(isopropylthio)methyl)-2-methoxyphenoxy)quinazoline (4d): white solid; m.p. 61.8 °C–63.0 °C;
1
yield, 78.6%; H NMR (500 MHz, CDCl3) δ 8.74 (s, 1H, quinazoline-H), 8.40 (d, J = 8.2 Hz, 1H,
quinazoline-H), 8.01 (d, J = 8.5 Hz, 1H, quinazoline-H), 7.91 (dd, J = 11.5, 3.9 Hz, 1H, quinazoline-H),
7.67 (t, J = 7.6 Hz, 1H, quinazoline-H), 7.16 (d, J = 8.0 Hz, 1H, Ph-H), 7.10 (dd, J = 8.1, 1.6 Hz, 1H,
Ph-H), 4.99 (s, 1H, SCHS), 3.77 (s, 3H, OCH3), 3.09–2.98 (m, 2H, 2CHS), 1.29 (t, J = 5.0 Hz, 12H,
4CH3). 13C NMR (125 MHz, CDCl3) δ 166.8, 154.5, 151.7, 140.8, 134.0, 134.2, 127.9, 127.6, 126.5,
124.0, 122.6, 120.4, 116.3, 112.3, 56.1, 50.6, 36.0, 36.0, 23.3, 23.3, 23.2, 23.2. HRMS (ESI): calculated
for C22H27N2O2S2 [M+H]+: 415.1508, found: 415.1502.
4-(4-(bis(ethylthio)methyl)phenoxy)-7-chloroquinazoline (4e): white solid; m.p. 59.8 °C–60.7 °C; yield,
1
86.9%; H NMR (500 MHz, CDCl3) δ 8.76 (s, 1H, quinazoline-H), 8.30 (d, J = 8.7 Hz, 1H,
quinazoline-H), 8.01 (d, J = 2.0 Hz, 1H, quinazoline-H), 7.62 (dd, J = 8.8, 2.0 Hz, 1H, quinazoline-H),
7.58 (d, J = 8.6 Hz, 2H, Ph-H), 7.24 (d, J = 8.5 Hz, 2H, Ph-H), 4.99 (s, 1H, SCHS), 2.72–2.52 (m, 4H,
2CH2S), 1.27 (t, J = 7.4 Hz, 6H, 2CH3). 13C NMR (125 MHz, CDCl3) δ 166.6, 155.3, 152.4, 151.5,
140.5, 138.4, 129.2, 128.7, 128.7, 127.2, 125.1, 121.7, 121.7, 114.8, 51.9, 26.3, 26.3, 14.3, 14.3.
HRMS (ESI): calculated for C19H20N2OClS2 [M+H]+: 391.0700, found: 391.0704.
4-(4-(bis(ethylthio)methyl)-2-methoxyphenoxy)-7-chloroquinazoline
(4f).
white
solid;
m.p.

56.0–58.0 °C; yield, 87.2%; 1H NMR (500 MHz, CDCl3) δ 8.72 (s, 1H, quinazoline-H), 8.33 (d, J = 8.7
Hz, 1H, quinazoline-H), 8.00 (d, J = 1.9 Hz, 1H, quinazoline-H), 7.61 (dd, J = 8.7, 2.0 Hz, 1H,
quinazoline-H), 7.22 (d, J = 1.9 Hz, 1H, Ph-H), 7.15 (s, 1H, Ph-H), 7.09 (dd, J = 8.1, 2.0 Hz, 1H,
Ph-H), 4.96 (s, 1H, SCHS), 3.76 (s, 3H, OCH3), 2.70–2.58 (m, 4H, 2CH2S), 1.28 (t, J = 7.4 Hz, 6H,
2CH3). 13C NMR (125 MHz, CDCl3) δ 166.7, 155.6, 152.4, 151.5, 140.6, 140.4, 139.7, 128.6, 127.2,
125.4, 122.5, 120.4, 114.7, 112.2, 56.0, 52.5, 26.5, 26.5, 14.4, 14.4. HRMS (ESI): Calculated for
C20H22N2O2ClS2 [M+H]+: 421.0806, found: 421.0798.
4-(4-(bis(propylthio)methyl)-2-methoxyphenoxy)-7-chloroquinazoline (4g). white solid; m.p.
62.6–63.4 °C; yield, 80.5%; 1H NMR (400 MHz, CDCl3) δ 8.73 (s, 1H, quinazoline-H), 8.33 (d, J = 8.8
Hz, 1H, quinazoline-H), 8.00 (d, J = 2.0 Hz, 1H, quinazoline-H), 7.61 (dd, J = 8.8, 2.0 Hz, 1H,
quinazoline-H), 7.22 (d, J = 1.9 Hz, 1H, Ph-H), 7.16 (d, J = 8.1 Hz, 1H, Ph-H), 7.08 (dd, J = 8.1, 2.0
Hz, 1H, Ph-H), 4.91 (s, 1H, SCHS), 3.76 (s, 3H, OCH3), 2.69–2.51 (m, 4H, 2SCH2CH2CH3), 1.70–1.56
(m, 4H, 2SCH2CH2CH3), 1.00 (t, J = 7.3 Hz, 6H, 2SCH2CH2CH3). 13C NMR (100 MHz, CDCl3) δ
166.6, 155.5, 152.4, 151.4, 140.5, 140.3, 139.8, 128.5, 127.1, 125.4, 122.4, 120.3, 114.6, 112.1, 55.9,
53.0, 34.4, 34.4, 22.6, 22.6, 13.6, 13.6. HRMS (ESI): Calculated for C22H26N2O2ClS2 [M+H]+:
449.1119, found: 449.1119.
4-(4-(bis(isopropylthio)methyl)-2-methoxyphenoxy)-7-chloroquinazoline (4h). white solid; m.p.
109–111 °C; yield, 80.9%; 1H NMR (400 MHz, CDCl3) δ 8.72 (s, 1H, quinazoline-H), 8.33 (d, J = 8.8
Hz, 1H, quinazoline-H), 8.00 (d, J = 1.9 Hz, 1H, quinazoline-H), 7.61 (dd, J = 8.8, 2.0 Hz, 1H,
quinazoline-H), 7.26 (s, 1H, Ph-H), 7.15 (d, J = 8.1 Hz, 1H, Ph-H), 7.10 (dd, J = 8.1, 1.9 Hz, 1H,
Ph-H), 4.99 (s, 1H, SCHS), 3.76 (s, 3H, OCH3), 3.03 (dt, J = 13.4, 6.7 Hz, 2H, 2CH(CH3)2), 1.29 (dd, J
= 6.7, 3.9 Hz, 12H, 2CH(CH3)2). 13C NMR (100 MHz, CDCl3) δ 166.6, 155.5, 152.4, 151.5, 140.5,
140.3, 140.1, 128.5, 127.1, 125.4, 122.4, 120.3, 114.6, 112.2, 56.0, 50.5, 35.9, 35.9, 23.2, 23.2, 23.2,
23.2. HRMS (ESI): Calculated for C22H26N2O2ClS2 [M+H]+: 449.1119, found: 449.1118.
4-(4-(bis(ethylthio)methyl)-2-methoxyphenoxy)-6-chloroquinazoline
(4i).
white
solid;
m.p.
61.5–63.0 °C; yield, 86.5%; 1H NMR (500 MHz, CDCl3) δ 8.72 (s, 1H, quinazoline-H), 8.37 (d, J = 2.3
Hz, 1H, quinazoline-H), 7.94 (s, 1H, quinazoline-H), 7.84 (dd, J = 8.9, 2.4 Hz, 1H, quinazoline-H),
7.22 (d, J = 2.0 Hz, 1H, Ph-H), 7.15 (d, J = 8.1 Hz, 1H, Ph-H), 7.09 (dd, J = 8.1, 2.0 Hz, 1H, Ph-H),
4.97 (s, 1H, SCHS), 3.76 (s, 3H, OCH3), 2.72–2.57 (m, 4H, 2CH2S), 1.28 (t, J = 7.4 Hz, 6H, 2CH3).
13C NMR (125 MHz, CDCl3) δ 166.0, 154.6, 151.5, 150.2, 140.6, 139.8, 135.0, 133.2, 129.6, 123.1,

122.5, 120.4, 117.0, 112.2, 56.0, 52.5, 26.5, 26.5, 14.4, 14.4. HRMS (ESI): Calculated for
C20H22N2O2ClS2 [M+H]+: 421.0806, found: 421.0806.
4-(4-(bis(propylthio)methyl)-2-methoxyphenoxy)-6-chloroquinazoline (4j). white solid; m.p.
56.4–57.9 °C; yield, 83.9%; 1H NMR (500 MHz, CDCl3) δ 8.72 (s, 1H, quinazoline-H), 8.37 (d, J = 2.3
Hz, 1H, quinazoline-H), 7.95 (d, J = 9.0 Hz, 1H, quinazoline-H), 7.84 (dd, J = 8.9, 2.3 Hz, 1H,
quinazoline-H), 7.23 (d, J = 2.0 Hz, 1H, Ph-H), 7.14 (s, 1H, Ph-H), 7.09 (dd, J = 8.2, 2.0 Hz, 1H,
Ph-H), 4.92 (s, 1H, SCHS), 3.76 (s, 3H, OCH3), 2.67–2.52 (m, 4H, 2SCH2), 1.67–1.59 (m, 4H, 2CH2),
1.00 (t, J = 7.4 Hz, 6H, 2CH3). 13C NMR (125 MHz, CDCl3) δ 165.9, 154.5, 151.4, 150.1, 140.5, 139.9,
134.9, 133.1, 129.6, 123.0, 122.4, 120.3, 116.9, 112.2, 55.9, 53.1, 34.5, 34.5, 22.6, 22.6, 13.6, 13.6.
HRMS (ESI): Calculated for C22H26N2O2ClS2 [M+H]+: 449.1119, found: 449.1121.
4-(4-(bis(isopropylthio)methyl)-2-methoxyphenoxy)-6-chloroquinazoline (4k). white solid; m.p.
54.5–56.0 °C; yield, 80.3%; 1H NMR (400 MHz, CDCl3) δ 8.72 (s, 1H, quinazoline-H), 8.37 (d, J = 2.3
Hz, 1H, quinazoline-H), 7.95 (d, J = 8.9 Hz, 1H, quinazoline-H), 7.84 (dd, J = 9.0, 2.4 Hz, 1H,
quinazoline-H), 7.26 (s, 1H, Ph-H), 7.15 (d, J = 8.1 Hz, 1H, Ph-H), 7.10 (dd, J = 8.2, 1.9 Hz, 1H,
Ph-H), 4.99 (s, 1H, SCHS), 3.77 (s, 3H, OCH3), 3.04 (dt, J = 13.5, 6.7 Hz, 2H, 2CH(CH3)2), 1.29 (dd, J
= 6.7, 3.9 Hz, 12H, 2CH(CH3)2). 13C NMR (100 MHz, CDCl3) δ 165.9, 154.6, 151.4, 150.1, 140.4,
140.1, 134.9, 133.1, 129.5, 123.0, 122.4, 120.3, 116.9, 112.2, 56.0, 50.5, 35.9, 35.9, 23.2, 23.2, 23.1,
23.1. HRMS (ESI): Calculated for C22H26N2O2ClS2 [M+H]+: 449.1119, found: 449.1123.
4-(4-(bis(ethylthio)methyl)-2-methoxyphenoxy)-6-fluoroquinazoline
(4l).
white
solid;
m.p.
73.1–74.7 °C; yield, 87.0%; 1H NMR (400 MHz, CDCl3) δ 8.71 (s, 1H, quinazoline-H), 8.00 (ddd, J =
9.7, 8.8, 3.9 Hz, 2H, quinazoline-H), 7.66 (ddd, J = 9.1, 8.4, 2.9 Hz, 1H, quinazoline-H), 7.22 (d, J =
1.9 Hz, 1H, Ph-H), 7.16 (d, J = 8.1 Hz, 1H, Ph-H), 7.10 (dd, J = 8.2, 2.0 Hz, 1H, Ph-H), 4.97 (s, 1H,
SCHS), 3.76 (s, 3H, OCH3), 2.73–2.55 (m, 4H, 2CH2CH3), 1.28 (t, J = 7.4 Hz, 6H, 2CH2CH3). 13C
NMR (100 MHz, CDCl3) δ 166.4 (d, 4JFC = 5.1 Hz), 160.7 (d, 1JFC = 250.2 Hz), 153.7 (d, 4JFC = 2.5 Hz),
3
2
151.5, 148.8, 140.6, 139.6, 130.5 (d, JFC = 8.6 Hz), 124.0 (d, JFC = 25.3 Hz), 122.5, 120.3, 116.9 (d,
2
3JFC = 9.7 Hz), 112.2, 107.8 (d, JFC = 23.3 Hz), 56.0, 52.5, 26.4, 26.4, 14.3, 14.3. HRMS (ESI):
Calculated for C20H22N2O2FS2 [M+H]+: 405.1101, found: 405.1096.
4-(4-(bis(propylthio)methyl)-2-methoxyphenoxy)-6-fluoroquinazoline (4m). white solid; m.p.
1
60.3–60.7 °C; yield, 85.0%; H NMR (400 MHz, CDCl3) δ 8.71 (s, 1H, quinazoline-H), 8.02 (dd, J =
7.9, 3.7 Hz, 1H, quinazoline-H), 8.00–7.97 (m, 1H, quinazoline-H), 7.67 (td, J = 8.7, 2.9 Hz, 1H,

quinazoline-H), 7.23 (d, J = 2.0 Hz, 1H, Ph-H), 7.16 (d, J = 8.1 Hz, 1H, Ph-H), 7.09 (dd, J = 8.1, 2.0
Hz, 1H, Ph-H), 4.92 (s, 1H, SCHS), 3.76 (s, 3H, OCH3), 2.69–2.52 (m, 4H, 2CH2CH2CH3), 1.70–1.57
(m, 4H, 2CH2CH2CH3), 1.00 (t, J = 7.3 Hz, 1H, 2CH2CH2CH3). 13C NMR (100 MHz, CDCl3) δ 166.4
4
1
4
(d, JFC = 5.2 Hz), 160.6 (d, JFC = 250.1 Hz), 153.7 (d, JFC = 2.5 Hz), 151.4, 148.7, 140.5, 139.9,
2
3
130.5 (d, 3JFC = 8.6 Hz), 124.0 (d, JFC = 25.3 Hz), 122.4, 120.3, 116.8 (d, JFC = 9.7 Hz), 112.1, 107.8
2
(d, JFC = 23.3 Hz), 55.9, 53.0, 34.4, 34.4, 22.6, 22.6, 13.6, 13.6. HRMS (ESI): Calculated for
C22H26N2O2FS2 [M+H]+: 433.1414, found: 433.1412.
4-(4-(bis(isopropylthio)methyl)-2-methoxyphenoxy)-6-fluoroquinazoline (4n). white solid; m.p.
70.1–72.1 °C; yield, 82.4%; 1H NMR (400 MHz, CDCl3) δ 8.71 (s, 1H, quinazoline-H), 8.00 (ddd, J =
10.0, 8.8, 3.9 Hz, 1H, quinazoline-H), 7.66 (ddd, J = 9.1, 8.4, 2.9 Hz, 1H, quinazoline-H), 7.27 (s, 1H,
Ph-H), 7.16 (d, J = 8.1 Hz, 1H, Ph-H), 7.10 (dd, J = 8.1, 2.0 Hz, 1H, Ph-H), 5.00 (s, 1H, SCHS), 3.76
(s, 3H, OCH3), 3.04 (dt, J = 13.5, 6.7 Hz, 2H, 2CH(CH3)2), 1.29 (dd, J = 6.7, 3.9 Hz, 12H, 2CH(CH3)2).
13C NMR (100 MHz, CDCl3) δ 166.38 (d, 4JFC = 5.2 Hz), 160.65 (d, 1JFC = 250.1 Hz), 153.7 (d, 4JFC
=
3
2
2.5 Hz), 151.5, 148.8, 140.6, 140.1, 130.5 (d, JFC = 8.6 Hz), 123.9 (d, JFC = 25.3 Hz), 122.5, 120.3,
3
116.9 (d, JFC = 9.6 Hz), 112.3, 107.8 (d, 2JFC = 23.3 Hz), 56.0, 50.6, 35.9, 35.9, 23.2, 23.2, 23.2, 23.2.
HRMS (ESI): Calculated for C22H26N2O2FS2 [M+H]+: 433.1414, found: 433.1417.
4-(4-(bis(ethylthio)methyl)-2-methoxyphenoxy)-8-methylquinazoline
(4o).
white solid;
m.p.
80.0–82.0 °C; yield, 83.7%; 1H NMR (400 MHz, CDCl3) δ 8.78 (s, 1H, quinazoline-H), 8.24 (d, J = 8.2
Hz, 1H, quinazoline-H), 7.74 (d, J = 7.1 Hz, 1H, quinazoline-H), 7.54 (t, J = 7.6 Hz, 1H,
quinazoline-H), 7.22 (d, J = 1.9 Hz, 1H, Ph-H), 7.17 (d, J = 8.1 Hz, 1H, Ph-H), 7.09 (dd, J = 8.1, 2.0
Hz, 1H, Ph-H), 4.97 (s, 1H, SCHS), 3.75 (s, 3H, OCH3), 2.77 (s, 3H, quinazoline-CH3), 2.74–2.53 (m,
4H, 2CH2CH3), 1.27 (t, J = 7.4 Hz, 6H, 2CH2CH3). 13C NMR (100 MHz, CDCl3) δ 167.0, 153.3, 151.6,
150.8, 141.0, 139.3, 136.2, 134.0, 126.9, 122.6, 121.5, 120.3, 116.1, 112.3, 56.0, 52.5, 26.4, 26.4, 17.7,
14.3, 14.3. HRMS (ESI): Calculated for C21H25N2O2S2 [M+H]+: 401.1352, found: 401.1345.
4-(4-(bis(propylthio)methyl)-2-methoxyphenoxy)-8-methylquinazoline (4p). white solid; m.p.
1
71.6–72.3 °C; yield, 82.0%; H NMR (400 MHz, CDCl3) δ 8.79 (s, 1H, quinazoline-H), 8.25 (dd, J =
8.2, 0.7 Hz, 1H, quinazoline-H), 7.74 (dd, J = 6.5, 0.6 Hz, 1H, quinazoline-H), 7.54 (dd, J = 8.0, 7.4 Hz,
1H, quinazoline-H)., 7.23 (d, J = 2.0 Hz, 1H, Ph-H), 7.17 (d, J = 8.1 Hz, 1H, Ph-H), 7.08 (dd, J = 8.2,
2.0 Hz, 1H, Ph-H), 4.92 (s, 1H, SCHS), 3.76 (s, 3H, OCH3), 2.77 (s, 3H, CH3), 2.64–2.54 (m, 4H,
2CH2CH2CH3), 1.69–1.58 (m, 4H, 2CH2CH2CH3), 1.00 (t, J = 7.3 Hz, 6H, 2CH2CH2CH3). 13C NMR

(100 MHz, CDCl3) δ 166.9, 153.3, 151.5, 150.8, 140.9, 139.5, 136.1, 134.0, 126.9, 122.5, 121.5, 120.3,
116.1, 112.2, 55.9, 53.0, 34.4, 34.4, 22.6, 22.6, 17.7, 13.6, 13.6. HRMS (ESI): Calculated for
C23H29N2O2S2 [M+H]+: 429.1665, found: 429.1667.
4-(4-(bis(isopropylthio)methyl)-2-methoxyphenoxy)-8-methylquinazoline (4q). white solid; m.p.
1
82.6–83.8 °C; yield, 79.1%; H NMR (400 MHz, CDCl3) δ 8.78 (s, 1H, quinazoline-H), 8.24 (dd, J =
8.2, 0.6 Hz, 1H, quinazoline-H), 7.74 (d, J = 7.1 Hz, 1H, quinazoline-H), 7.59–7.48 (m, 1H,
quinazoline-H), 7.26 (s, 1H, Ph-H), 7.16 (d, J = 8.1 Hz, 1H, Ph-H), 7.09 (dd, J = 8.1, 2.0 Hz, 1H,
Ph-H), 4.99 (s, 1H, SCHS), 3.76 (s, 3H, OCH3), 3.03 (dt, J = 13.5, 6.7 Hz, 2H, 2CH(CH3)2), 2.77 (s,
3H, Ph-CH3), 1.29 (dd, J = 6.7, 4.0 Hz, 12H, 2CH(CH3)2). 13C NMR (100 MHz, CDCl3) δ 166.9, 153.4,
151.6, 150.8, 141.0, 139.8, 136.2, 134.0, 126.9, 122.6, 121.5, 120.3, 116.1, 112.4, 56.0, 50.6, 35.9, 35.9,
23.2, 23.2, 23.2, 23.2, 17.7. HRMS (ESI): Calculated for C23H29N2O2S2 [M+H]+: 429.1665, found:
429.1668.
2,2'-(((3-methoxy-4-((8-methylquinazolin-4-yl)oxy)phenyl)methylene)bis(sulfanediyl))bis(ethan-1-ol)
1
(4r). white solid; m.p. 67.3–68.0 °C; yield, 75.2%; H NMR (500 MHz, DMSO-d6) δ 8.74 (s, 1H,
quinazoline-H), 8.20 (d, J = 8.1 Hz, 1H, quinazoline-H), 7.90 (d, J = 7.1 Hz, 1H, quinazoline-H), 7.66
(t, J = 7.7 Hz, 1H, quinazoline-H), 7.29 (d, J = 8.1 Hz, 1H, Ph-H), 7.26 (s, 1H, Ph-H), 7.13 (d, J = 6.7
Hz, 1H, Ph-H), 5.33 (s, 1H, SCHS), 4.89 (t, J = 5.4 Hz, 2H, 2OH ), 3.69 (s, 3H, OCH3), 3.58 (dd, J =
12.5, 6.3 Hz, 4H, 2CH2S), 2.74 (dt, J = 13.5, 6.8 Hz, 2H, CH2OH), 2.69 (s, 3H, CH3), 2.63 (dt, J = 13.4,
6.9 Hz, 2H, CH2OH). 13C NMR (125 MHz, DMSO-d6) δ 166.8, 153.7, 151.3, 150.6, 140.6, 136.3,
134.9, 128.1, 123.5, 123.5, 121.5, 120.4, 115.6, 112.6, 61.2, 61.2, 56.3, 52.7, 35.2, 35.2, 17.7. HRMS
(ESI): Calculated for C21H25N2O4S2 [M+H]+: 433.1250, found: 433.1243.
4-(4-(bis(ethylthio)methyl)-2-methoxyphenoxy)-6,7-dimethoxyquinazoline (4s). white solid; m.p.
125.2–126.7 °C; yield, 86.2%; 1H NMR (400 MHz, CDCl3) δ 8.60 (s, 1H, quinazoline-H), 7.58 (s, 1H,
quinazoline-H), 7.32 (s, 1H, quinazoline-H), 7.22 (d, J = 1.9 Hz, 1H, Ph-H), 7.17 (d, J = 8.1 Hz, 1H,
Ph-H), 7.09 (dd, J = 8.1, 2.0 Hz, 1H, Ph-H), 4.97 (s, 1H, SCHS), 4.07 (s, 3H, Ph-OCH3), 4.06 (s, 3H,
quinazoline-OCH3), 3.77 (s, 3H, quinazoline-CH3), 2.70–2.57 (m, 4H, 2CH2), 1.28 (t, J = 7.4 Hz, 6H,
2CH3). 13C NMR (100 MHz, CDCl3) δ 165.2, 155.8, 153.0, 151.6, 150.0, 149.3, 140.9, 139.2, 122.6,
120.4, 112.2, 110.5, 106.8, 101.3, 56.4, 56.3, 56.0, 52.4, 26.4, 26.4, 14.3, 14.3. HRMS (ESI):
Calculated for C22H27N2O4S2 [M+H]+: 447.1407, found: 447.1399.
4-(4-(bis(propylthio)methyl)-2-methoxyphenoxy)-6,7-dimethoxyquinazoline (4t). white solid; m.p.

131.2–131.6 °C; yield, 83.0%; 1H NMR (400 MHz, CDCl3) δ 8.60 (s, 1H, quinazoline-H), 7.58 (s, 1H,
quinazoline-H), 7.33 (s, 1H, quinazoline-H), 7.22 (s, 1H, Ph-H), 7.16 (d, J = 8.1 Hz, 1H, Ph-H), 7.08
(dd, J = 8.1, 1.6 Hz, 1H, Ph-H), 4.92 (s, 1H, SCHS), 4.06 (s, 6H, 2OCH3), 3.77 (s, 3H, 2OCH3),
2.72–2.45 (m, 4H, 2SCH2), 1.60–1.67 (m, 4H, 2CH2CH3), 1.00 (t, J = 7.3 Hz, 6H, 2CH2CH3). 13C
NMR (100 MHz, CDCl3) δ 165.2, 155.8, 153.0, 151.6, 150.1, 149.3, 140.9, 139.4, 122.6, 120.4, 112.2,
110.5, 106.8, 101.3, 56.4, 56.3, 56.0, 53.0, 34.4, 34.4, 22.6, 22.6, 13.6, 13.6. HRMS (ESI): Calculated
for C24H31N2O4S2 [M+H]+: 475.1720, found: 475.1723.
4-(4-(bis(isopropylthio)methyl)-2-methoxyphenoxy)-6,7-dimethoxyquinazoline (4u). white solid; m.p.
1
159.8–160.3 °C; yield, 80.5%; H NMR (500 MHz, DMSO-d6) δ 8.52 (s, 1H, quinazoline-H), 7.53 (s,
1H, quinazoline-H), 7.38 (s, 1H, quinazoline-H), 7.31 (s, 1H, Ph-H), 7.24 (d, J = 8.1 Hz, 1H, Ph-H),
7.16 (d, J = 8.2 Hz, 1H, Ph-H), 5.23 (s, 1H, SCHS), 3.99 (s, 3H, , quinazoline-OCH3), 3.97 (s, 3H,
quinazoline-OCH3), 3.69 (s, 3H, , Ph-OCH3), 2.99–2.93 (m, 2H, 2CH(CH3)2), 1.24 (dd, J = 16.9, 6.7
Hz, 12H, 2CH(CH3)2). 13C NMR (125 MHz, DMSO-d6) δ 164.3, 155.6, 152.2, 150.8, 149.9, 148.6,
140.1, 134.0, 122.9, 119.8, 112.0, 109.2, 106.6, 100.6, 56.0, 55.9, 55.6, 49.6, 35.6, 35.6, 23.0, 23.0,
23.0, 23.0. HRMS (ESI): Calculated for C24H31N2O4S2 [M+H]+: 475.1720, found: 475.1715.
4-(4-(bis(ethylthio)methyl)-2-methoxyphenoxy)-6-chloro-8-methylquinazoline (4v). white solid; m.p.
1
76.3–77.3 °C; yield, 86.3%; H NMR (500 MHz, CDCl3) δ 8.76 (s, 1H, quinazoline-H), 8.22 (s, 1H,
quinazoline-H), 7.70 (s, 1H, quinazoline-H), 7.22 (s, 1H, Ph-H), 7.15 (d, J = 8.1 Hz, 1H, Ph-H), 7.09
(dd, J = 8.1, 2.0 Hz, 1H, Ph-H), 4.97 (s, 1H, SCHS), 3.76 (s, 3H, OCH3), 2.75 (s, 3H, quinazoline-CH3),
2.70–2.57 (m, 4H, 2CH2), 1.28 (t, J = 7.4 Hz, 6H, 2CH3). 13C NMR (125 MHz, CDCl3) δ 166.1, 153.4,
151.4, 149.4, 140.6, 139.5, 138.6, 134.6, 132.5, 122.4, 120.5, 120.3, 116.7, 112.1, 55.9, 52.3, 26.4, 26.4,
17.5, 14.3, 14.3. HRMS (ESI): Calculated for C21H24N2O2S2Cl [M+H]+: 435.0962, found: 435.0962.
4-(4-(bis(propylthio)methyl)-2-methoxyphenoxy)-6-chloro-8-methylquinazoline (4w). white solid; m.p.
71.7–72.6 °C; yield, 83.2%; 1H NMR (400 MHz, CDCl3) δ 8.76 (s, 1H, quinazoline-H), 8.22 (d, J = 2.3
Hz, 1H, quinazoline-H), 7.69 (d, J = 2.2 Hz, 1H, quinazoline-H), 7.22 (d, J = 1.9 Hz, 1H, Ph-H), 7.15
(d, J = 8.1 Hz, 1H, Ph-H), 7.08 (dd, J = 8.1, 1.9 Hz, 1H, Ph-H), 4.91 (s, 1H, SCHS), 3.76 (s, 3H,
OCH3), 2.75 (s, 3H, Ph-CH3), 2.68–2.51 (m, 4H, 2CH2CH2CH3), 1.69–1.58 (m, 4H, 2CH2CH2CH3),
1.00 (t, J = 7.3 Hz, 6H, 2CH2CH2CH3). 13C NMR (100 MHz, CDCl3) δ 166.1, 153.5, 151.4, 149.4,
140.6, 139.7, 138.6, 134.6, 132.5, 122.4, 120.5, 120.3, 116.7, 112.1, 55.9, 53.0, 34.4, 34.4, 22.6, 22.6,
17.5, 13.6, 13.6. HRMS (ESI): Calculated for C23H28N2O2S2Cl [M+H]+: 463.1275, found: 463.1276.

4-(4-(bis(isopropylthio)methyl)-2-methoxyphenoxy)-6-chloro-8-methylquinazoline (4x). white solid;
m.p. 97.1–97.7 °C; yield, 80.2%; 1H NMR (400 MHz, CDCl3) δ 8.75 (s, 1H, quinazoline-H), 8.21 (dd,
J = 2.4, 0.6 Hz, 1H, quinazoline-H), 7.69 (dd, J = 2.3, 1.0 Hz, 1H, quinazoline-H), 7.26 (d, J = 1.9 Hz,
1H, Ph-H), 7.14 (d, J = 8.1 Hz, 1H, Ph-H), 7.09 (dd, J = 8.2, 1.9 Hz, 1H, Ph-H), 4.99 (s, 1H, SCHS),
3.76 (s, 3H, OCH3), 3.03 (dt, J = 13.5, 6.7 Hz, 2H, 2CH(CH3)2), 2.74 (s, 3H, quinazoline-CH3), 1.29
(dd, J = 6.7, 4.0 Hz, 12H, 2CH(CH3)2). 13C NMR (100 MHz, CDCl3) δ 166.1, 153.5, 151.5, 149.4,
140.6, 140.0, 138.6, 134.6, 132.5, 122.4, 120.5, 120.3, 116.7, 112.2, 56.0, 50.5, 35.9, 35.9, 23.2, 23.2,
23.2, 23.2, 17.5. HRMS (ESI): Calculated for C23H28N2O2S2Cl [M+H]+: 463.1275, found: 463.1270.
4.2 Biological method
4.2.1 In vivo antiviral activities assay
Nicotiana tabacum cv. K326 and Chenopodium amaranticolor plants were cultivated in a greenhouse.
N. tabacum cv. K326 was used to determine systemic CMV or PVY infection, and C. amaranticolor
was used as local lesion host when plants grew to the 5−6-leaf stages. CMV and PVY were purified by
the reported method [16]. In vivo measures of compounds were determined through a previously
reported technique, and in vivo modes include curative and protective activities. Positive controls
included commercial antiviral agent ningnanmycin and lead compound 6f [16]. Measurements were
performed in triplicate.
4.2.2 Chlorophyll content
We used the methods reported in the literature [25] to measure the chlorophyll content in plants on 1, 3,
5, and 7 days after virus inoculation. Test samples in triplicate of each treatment were sliced into small
uniform pieces by a hole puncher while avoiding the midrib. The samples weighed 100 mg and were
placed in 10 mL of a cold solution of a 1:1 mixture of 85% acetone and 85% ethanol (v/v). The samples
were homogenized, clogged for 0.5 h at 35 °C, and centrifuged for 15 min at 6500 rpm. Absorbance
spectra were recorded at 663 nm and 645 nm for chlorophyll a (Ca) and chlorophyll b (Cb), respectively,
against a reference solution. Ca, Cb, and total chlorophyll content (Ct) were calculated as follows:
Ca (mg/L) = 9.784OD663 − 0.990OD645
Cb (mg/L) = 21.426OD645 − 4.650OD645
Ct (mg/L) = Ca + Cb = 5.134OD663 + 20.643OD645
Fresh weight (FW, mg/g) was determined using the following equation:
FW = CV/1000W

where C is the concentration (mg/L), V is the volume of the solvent (mL), and W refers to the sample
FW (g).
4.2.3 Gene expression analysis
Total RNA was extracted using the Trizol reagent kit (TaKaRa, Beijing, China). The
reverse-transcribed reagents of RNA were processed using the cDNA kit (TaKaRa, Beijing, China)
according to the manufacturer’s instructions, and a PCR instrument (Bio-Rad, USA) was used to
complete the operations. The primer sequence information is listed in Table 3. Gene expression was
normalized using β-actin as internal control, and the gene relative copy numbers were calculated using
a previously presented method [26].
4.2.4 DEPs analysis
Protein extraction: We used the methods reported in the literature for protein extraction [27,28]. The
sample (1.0 g) was ground to powder in liquid nitrogen, homogenized, and suspended in 5 mL of
ice-cold extraction buffer containing 0.7 M sucrose, 0.5 M Tris–HCl, 0.1 M KCl, 50 mM
ethylenediaminetetraacetic acid, and 40 mM dithiothreitol at room temperature for 10 min. An equal
volume of phenol saturated with Tris–HCl pH 7.5 was added. The mixture was shaken at 4 °C for 30
min and centrifuged at 5000 rpm for 10 min. The supernatant was collected and diluted with five times
volume of 100 mM ammonium acetate in methanol. After overnight incubation at −20 °C, the sample
was centrifuged at 5000 rpm for 20 min at 4 °C. The precipitate was washed with ice-cold 80% acetone
and air-dried. The precipitate was then solubilized with 150 μL solution containing 8 M urea, 0.1 M
Tris, and 10 mM DTT. The protein concentration was determined through the Bradford method. The
solutions were mixed with equal volume of 55 mM iodoacetamide and stored in the dark at 25 °C for
30 min. Then, the mixtures were centrifuged for 20 min at 12000 rpm and 4 °C with 3 kDa Millipore.
After washing with diluent rehydration, the mixtures were dissolved with 100 μL Milli-Q water. The
samples were mixed with 12.5 μg of trypsin (Sigma, USA), stored at 4 °C for 30 min, and incubated
overnight at 37 °C. After centrifugation for 20 min at 12000 rpm and 4 °C, the supernatant was
collected and air-dried. The final samples were solubilized and analyzed through HPLC.
LC-MS/MS analysis: The peptides were analyzed by the Nano-LC-1DTM system (Eksigent, Dublin,
CA, USA) through a TripleTOF 5600 mass spectrometer (Foster City, CA, USA). The samples (8 μL)
were injected using full-loop injection and desalted on a ChromXP Trap column (Nano LC TRAP
Column; 3 μm C18–CL, 120 Å, 350 μm × 0.5 mm; Foster City, CA, USA) by equilibration of 1% ACN

and 0.1% FA in H2O. After washing for 10 min at 300 nL min−1 flow rate, the samples were eluted by
reverse-phase column (3C18–CL, 75 μm × 15 cm; Foster City, CA, USA). The mobile phases A (5%
ACN and 0.1% FA) and B (95% ACN and 0.1% FA) were processed for 120 min at a flow rate of 300
nL/min. TripleTOF 5600 MS was used in the data-dependent mode to switch automatically between
TOF–MS and product ion acquisition through Analyst® software (TF1.6; AB SCIEX, Foster City, CA,
USA). β-Galactosidase digest was used to calibrate two samples by 10 min of elution and 30 min of
identification.
Proteomics data analysis: On the basis of the tobacco proteome downloaded from UniProt, we used
MaxQuant version 1.5.2.8 by the Andromeda search engine to process the raw data [29]. The initial
search at a precursor mass tolerance of 20 ppm was used for mass recalibration. The search also
included variable modifications of methionine oxidation, N-terminal acetylation, and cysteine
carbamidomethylation. In the main Andromeda search, the precursor and fragment masses presented
the initial mass tolerances of 6 ppm and 20 ppm, respectively. The false discovery rate was set to 0.01,
and the peptide length ranged from two to seven amino acid miscleavages. The protein intensity and
abundance of DEPs were determined through label-free quantification with a minimum of two ratio
counts and iBAQ algorithm, respectively [29,30]. The common contaminants were identified and
eliminated, and the reverse database was used to filter the protein tables. Differentially accumulated
proteins between treatment and control groups were identified through unpaired t-test of iBAQ data of
two samples.
4.2.5 Bioinformatics and annotations
Classification of DEPs was conducted with GO annotation on KEGG by the UniProt software
(http://www.uniprot.org/).Initially, GO items lacking the corresponding annotations were initially
removed from the protein list. Subsequently, the IDs of the listed proteins were plotted at the levels of
BP, CC, and MF [31]. DEPs were mapped to the GO database (http://www.geneontology.org/), and the
number of proteins at each GO term was computed. The results obtained from label-free proteomics
were used as the target list. The background list was generated by downloading the GO database.
5. Acknowledgments
This work was supported by the National Natural Science Foundation of China (No, 21562013) and
Science and Technology Plan Project of Guizhou Province in China (No. 20154001).
Conflict of interest

The authors confirmed that this article content has no conflict of interest.
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Figures and Scheme
Fig. 1. Design of the title compounds 4a–4x.

Fig. 2. Effects of compound 4b on Ca (A), Cb (B), Ct (C), and chlorophyll a/b (D) in tobacco
leaves. Vertical bars refer to mean ± SD (n = 3).

Fig. 3. Effect of compound 4b on defense gene expression of plant

Fig. 4. Venn diagram for proteins identified in 4b treatment and solvent (CK) treatment groups

Fig. 5. Cellular components, molecular functions, and biological processes involving DEPs in
4b+CMV versus solvent (CK) + CMV

Fig. 6. KEGG map of photosynthetic pathway of DEPs in 4b+CMV versus CK+CMV. The red
frames indicate the corresponding DEPs were up-regulated in 4b+CMV groups and the green
frames indicate that corresponding DEPs were down-regulated in 4b+CMV groups.

Scheme 1 Synthetic route of the title compounds 4a–4x.

Table 1
Antiviral activity of the target compounds against CMV and PVY in vivoa
Anti-CMV
Anti-PVY
Curative Protective
Compd.
Curative
activityb
(%)
Protective
activityb
(%)
EC50 of Protective
EC50 of Curative
activityb
activityb
activity (μg/mL)
activity (μg/mL)
(%)
(%)
29.9±5.0
58.3±4.6
40.7±3.7
40.5±4.2
48.1±1.2
28.0±3.1
40.3±2.4
38.2±2.5
56.5±6.1
43.5±1.3
43.7±0.8
52.1±1.2
43.5±2.5
40.6±2.1
58.2±5.4
56.0±1.4
50.3±2.0
37.8±3.4
46.0±2.7
40.0±1.6
35.1±6.3
35.1±1.6
33.3±1.3
25.2±2.3
50.0±3.4
54.2±4.7
52.0±5.2
61.1±3.5
44.1±2.3
37.8±2.3
40.9±4.8
28.5±3.1
34.8±1.4
26.5±4.2
48.7±1.2
48.6±2.5
31.7±4.8
51.2±8.2
26.0±3.4
23.7±2.0
51.4±3.7
48.8±3.0
42.9±2.1
25.1±5.0
26.0±1.2
30.5±5.7
40.2±5.0
32.4±1.7
30.8±3.3
24.1±2.0
51.8±3.5
55.2±1.2
333.6±4.5
47.5±2.5
53.5±4.1
44.9±3.2
40.2±2.0
43.8±2.1
30.7±1.2
34.3±0.9
51.7±2.6
53.7±3.9
31.3±3.1
37.0±2.8
48.6±3.5
34.0±4.0
28.3±2.8
51.9±2.4
45.5±2.0
49.2±3.0
38.3±0.9
49.4±2.5
37.1±2.5
39.4±2.3
35.6±3.8
27.0±2.8
32.1±2.5
46.5±2.2
52.0±2.1
45.4±4.0
52.0±2.4
45.0±3.4
44.22.1±
46.3±4.0
37.5±1.2
46.1±3.0
52.2±2.0
54.3±3.2
35.0±2.5
49.9±3.8
49.5±4.0
40.5±3.1
44.5±2.7
49.3±3.6
44.9±2.1
52.3±1.8
41.6±2.1
51.3±3.6
46.3±1.3
43.0±5.4
36.5±2.0
30.3±2.2
42.6±5.1
47.3±3.0
54.5±2.5
514.9±5.0
4a
248.6±2.9
350.5±3.6
4b
526.3±5.3
-
4c
582.3±2.6
-
4d
-
686.5±4.8
4e
-
-
4f
783.8±5.1
-
4g
-
489.5±5.0
4h
538.9±5.6
357.5±3.5
4i
552.3±2.9
-
4j
599.5±3.5
-
4k
395.4±5.2
-
4l
-
-
4m
-
-
4n
359.4±3.2
376.6±4.7
4o
510.4±2.5
621.7±4.1
4p
-
507.7±6.2
4q
678.5±8.4
-
4r
-
499.2±4.3
4s
624.4±3.5
-
4t
518.2±6.2
-
4u
-
-
4v
-
-
4w
-
-
4x
Ningnanmycinc
6fc
357.7±3.5
493.7±5.4
298.5±1.6
381.7±2.9
a Average of three replicates. b Concentration of compound is 500 μg/mL. c Used as the control.

Table 2
Major DEPs involved in photoreaction system
Sig/
Protein ID
Protein names
length
LogRatio
p Value
regulated
up
Specific
A0A140G1
Q8_TOBAC
A0A1S3XR
N0_TOBAC
A0A1L2BN
N8_NICOT
A0A1S3ZH
H0_TOBAC
I0B7K0_NI
CBE
Photosystem II CP43
reaction center protein
Photosystem II reaction
center Psb28 protein
Photosystem II CP47
reaction center protein
461
180
505
238
228
136
274
3.629444526 0.000302626
1
1
2.082291
1.176552
-
0.014975
0.004781
-
up
1
up
psbP-like protein 1
Chloroplast PsbQ2
-1
-1
-1
1
down
down
down
up
-1.82163
-2.13776
-
6.02E-05
0.004509
-
A0A1S4CA
S4_TOBAC
A0A1S4B6I
9_TOBAC
photosystem II 10 kDa
polypeptide
photosystem II 22 kDa
protein, chloroplastic-like
photosystem II repair protein
PSB27-H1,
A0A1S4DN
09_TOBAC
171
-3.18785
0.002091
-1
down
chloroplastic-like
A0A1S4AF
D5_TOBAC
A0A1L2BN
L5_NICOT
A0A1L2BN
Photosystem II reaction
center Psb28 protein
Photosystem I P700
chlorophyll a apoprotein A1
Photosystem I P700
180
750
734
-
-
-
-
1
1
1
up
up
up
0.004868634
-
M0_NICOT chlorophyll a apoprotein A2
photosystem I reaction
A0A1S4C5L
center subunit IV B,
2_TOBAC
143
1.501874982 0.039447547
1
up
chloroplastic
A0A1S3XV
cytochrome b6
T6_TOBAC
296
228
133
498
-1.188189171 0.001083161
-2.246939357 0.000162916
-1
-1
-1
-1
down
down
down
down
A0A1S4B83
2_TOBAC
Cytochrome b6-f complex
iron-sulfur subunit
A0A1L2BN ATP synthase epsilon chain,
-2.82819
-0.66562
4.53E-05
N3_NICOT
A0A140G1S
2_TOBAC
chloroplastic
ATP synthase subunit beta
0.006169623