Chemical synthesis, crystal structure, versatile evaluation of their biological activities and molecular simulations of novel pyrithiobac derivatives

Article abstract of DOI:10.1016/j.ejmech.2019.02.002

Since pyrithiobac (PTB) is a successful commercial herbicide with very low toxicity against mammals, it is worth exploring its derivatives for an extensive study. Herein, a total of 35 novel compounds were chemically synthesized and single crystal of 6–6

Full text of DOI:10.1016/j.ejmech.2019.02.002

Accepted Manuscript
Chemical synthesis, crystal structure, versatile evaluation of their biological activities
and molecular simulations of novel pyrithiobac derivatives
Ren-Jun Wu, Kai-Xuan Zhou, Haijin Yang, Guo-Qing Song, Yong-Hong Li, Jia-Xin
Fu, Xiao Zhang, Shu-Jing Yu, Li-Zhong Wang, Li-Xia Xiong, Cong-Wei Niu, Fu-Hang
Song, Haitao Yang, Jian-Guo Wang
PII:
S0223-5234(19)30112-6
DOI:
https://doi.org/10.1016/j.ejmech.2019.02.002
EJMECH 11094
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 December 2018
Revised Date: 18 January 2019
Accepted Date: 1 February 2019
Please cite this article as: R.-J. Wu, K.-X. Zhou, H. Yang, G.-Q. Song, Y.-H. Li, J.-X. Fu, X. Zhang,
S.-J. Yu, L.-Z. Wang, L.-X. Xiong, C.-W. Niu, F.-H. Song, H. Yang, J.-G. Wang, Chemical synthesis,
crystal structure, versatile evaluation of their biological activities and molecular simulations of novel
pyrithiobac derivatives, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.02.002.
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ACCEPTED MANUSCRIPT
Chemical synthesis, crystal structure, versatile evaluation of their biological activities
and molecular simulations of novel pyrithiobac derivatives
Ren-Jun Wua,1 Kai-Xuan Zhoub,c,1 Haijin Yangd,e, 1 Guo-Qing Songa, Yong-Hong Lia，Jia-Xin Fua, Xiao Zhanga, Shu-Jing Yua, Li-Zhong Wanga,
Li-Xia Xionga, Cong-Wei Niua, Fu-Hang Songd,**, Haitao Yangc,***, Jian-Guo Wanga,*

ACCEPTED MANUSCRIPT
Chemical synthesis, crystal structure, versatile evaluation of their
biological activities and molecular simulations of novel pyrithiobac
derivatives
Ren-Jun Wua,1 Kai-Xuan Zhoub,c,1 Haijin Yangd,e,1 Guo-Qing Songa, Yong-Hong Lia，
Jia-Xin Fua, Xiao Zhanga, Shu-Jing Yua, Li-Zhong Wanga, Li-Xia Xionga, Cong-Wei
Niua, Fu-Hang Songd,**, Haitao Yangc,***, Jian-Guo Wanga,*
a. State-Key Laboratory and Research Institute of Elemento-Organic Chemistry,
National Pesticide Engineering Research Center (Tianjin), College of Chemistry,
Nankai University, Tianjin 300071, China
b. State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin
300071, China
c. Tianjin International Joint Academy of Biotechnology and Medicine, Tianjin
300457, China.
d. State Key Laboratory of Microbial Resources and CAS Key Laboratory of
Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese
Academy of Sciences, Beijing, 100101, China.
e. School of Ocean Sciences, China University of Geosciences, Beijing 100083,
China.
1 These three authors contributed equally to this work.
*corresponding author. E-mail address: nkwjg@nankai.edu.cn (J.-G. Wang)
**corresponding author. E-mail address: songfuhang@163.com (F.-H. Song)
*** corresponding author. E-mail address: yanght@tju.edu.cn (H. Yang)
Abstract: Since pyrithiobac (PTB) is a successful commercial herbicide with very low
toxicity against mammals, it is worth exploring its derivatives for an extensive study.
Herein, a total of 35 novel compounds were chemically synthesized and single crystal
of 6-6 was obtained to confirm the molecular structure of this family of compounds.
The novel PTB derivatives were fully evaluated against various biological platforms.
From the bioassay results, the best AHAS inhibitor 6-22 displayed weaker herbicidal
activity but stronger anti-Candida activity than PTB did. For plant pathogenic fungi,
6-26 showed excellent activity at 50 mg/L dosage. Preliminary insecticidal activity
and antiviral activity were also observed for some title compounds. Strikingly, 6-5
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exhibited a promising inhibitory activity against SARS-CoV Mpro with IC50 of 4.471
µM and a low cellular cytotoxicity against mammalian 293T cells. Based on the
results of molecular modeling, HOMO-1 was considered to be a factor that affects
AHAS inhibition and a possible binding mode of 6-5 with SARS-CoV Mpro was
predicted. This is the first time that PTB derivatives have been studied as biological
agents other than herbicides. The present research hence has suggested that more
attentions should be paid to compounds belonging to this family to develop novel
agrochemicals or medicines.
Keywords: pyrithiobac derivative, acetohydroxyacid synthase, SARS-CoV Mpro,
crystal structure, biological activity.
1. Introduction
The thioether (or sulfoether) compounds are of great interest because they are either
useful intermediates for organic synthesis or meaningful agents with biological
activities [1]. For example, dimethyl sulfide and phenyl sulfide are common industrial
chemicals for the synthesis of pesticides, medicines and dyes. Diallyl sulfide, a
naturally occurring component of garlic, exhibits anticancer activity [2]. Methionine
was reported in the 1950s to have therapeutic effect against infective hepatitis [3].
Song et al. synthesized a series of 1,3,4-oxadiazole thioether derivatives (example-a)
and found them to display strong antibacterial activity [4]. Yang et al prepared some
novel thioether-substituted flavonoids (example-b) and some of them showed
satisfactory antiproliferative activity [5]. Chu et al. designed some nucleoside
conjugates of thioether phospholipids (example-c) and favorable anti-HIV activity
was observed [6].
Pyrithiobac (PTB) is a particular thioether compound in that the sulfur atom is
connected by a phenyl ring and a pyrimidine ring, which in fact is a powerful
herbicide used in cotton fields with excellent selectivity [7]. The target of this
herbicide is acetehydroxyacid synthase (AHAS, EC 2.2.1.6), a special biological
enzyme that exists only in plants and microbes [8]. A recent crystallography study
showed that PTB binds with plant AHAS in a similar cavity provided by two
neighboring subunits [9]. Nevertheless, PTB derivatives have not been fully explored
till now. As is known, compounds containing heterocycle ring such as pyrimidine or
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triazine often show diverse agricultural or medicinal activities. For instances, pyramat
is a successful insecticide with a pyrimidine ring developed in the 1960s and anilazine
is a traditional fungicide with a triazine ring that have been used for over 60 years [10,
11]. Recently, some aromatic disulfides with pyrimidine ring (example-d) were
discovered to be potent inhibitors of main protease of severe acute respiratory
syndrome coronavirus (SARS-CoV Mpro) [12]. The chemical structures of dimethyl
,
sulfide, phenyl sulfide, diallyl sulfide methionine, example-a, example-b, example-c,
pyrithiobac, pyramat, anilazine and example-d mentioned above are shown in Fig. 1.
In view of these backgrounds, herein we have designed and synthesized a series of 35
novel PTB derivatives and investigated their biological activities upon various
bioassay platforms. To our delight, the title compounds exhibited multiple
bioactivities, against agricultural weeds, insects, pathogenic plant fungi or plant virus,
and medicinal infections caused by fungus or SARS-associated coronavirus. For
compounds that showed remarkable activity against SARS-CoV Mpro, very low
cellular cytotoxicity were observed against mammalian 293T cells. In addition, the
chemical structure of
was elucidated clearly from single crystal X-ray diffraction,
6-6
which evidently indicated that these PTB derivatives were chemically prepared
successfully. Molecular simulations were performed and it was found that HOMO-1
might contribute to AHAS inhibition from DFT calculation and a plausible binding
mode of inhibitor
with SARS-CoV Mpro was generated via molecular docking.
6-5
2. Results and discussion
2.1 Synthesis of the PTB derivatives
The chemistry of intermediates and is illustrated in Scheme 1, and the synthetic
2
3
route for the target compound is presented in Scheme 2. Although and were
6
2
3
commercially available, they were chemically synthesized in our laboratory. Various
mercaptobenzonitriles were prepared from corresponding fluorobenzonitriles via a
2
1
nucleophilic substitution attacked by a sulfhydryl group provided by sodium sulfide
nonahydrate to give desirable outputs. were further hydrolyzed under alkaline
2
condition from mercaptobenzonitriles to mercaptobenzoic acids with satisfactory
3
yields. Some intermediates were hard to be purified and were used directly in the
following reaction. According to the literature [13], hydrolysis of occurs easily
2
under the condition of 15% sodium hydroxide at 150 °C, however, this reaction was
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not observed until the condition was changed to 30% sodium hydroxide at 175 °C
with 0.9 equivalent of sodium sulfide nonahydrate added to the solution in an
autoclave reactor. It should be noted that when there is a trifluoromethyl group in the
ortho or para position of the hydrosulphonyl group of , the trifluoromethyl group
2
itself will be hydrolyzed at the same time when the cyanogroup is hydrolyzed,
however when the trifluoromethyl group is at the meta position of the hydrosulphonyl
group, the hydrolysis of trifluoromethyl group does not happen, which has been
confirmed by the final compounds. This is in agreement with an early report by Jones
in 1947 [14]. The synthesis and characterizations of the intermediates and are
2
3
detailed in the supplementary material.
For the target compounds, in total there are four different synthesis routes. Compound
to
6-1 6-13
were prepared from and 2-chloro-4,6-dimethoxy-1,3,5-triazine using
2
4
triethylamine as a base in acetonitrile [15]. Compound
to
6-14 6-21
were synthesized
from and , but a more potent base sodium methoxide was used in methanol.
3
4
Compound
and
were also started from , with substituted phenyl as the
2'
6-34
6-35
4
nucleophile, potassium hydroxide as the base in tetrahydrofuran [16]. In all these
three routes, the chlorine atom in the triazine ring is attacked and substituted by the
hydrosulphonyl or hydroxyl group in , and . For the synthesis of compound
2 2'
3
6-22
to
, intermediate or reacted with 4,6-dimethoxy-2-(methylsulfonyl)pyrimidine
6-33
2
3
in ethanol to give the final product, which is also a nucleophilic substitution but in
5
this case the leaving group is a methanesulfonyl group in the pyrimidine ring and the
corresponding base is sodium bicarbonate [17]. The yields for the target compounds
were in the range of 10%-58%, with a majority of 20%-30%, indicating that further
optimization of the reaction conditions is required.
The chemical structures of 6-1 to 6-35 were fully characterized by means of 1H NMR,
13C NMR and HRMS. The melting points of all the target compounds were also
determined. To further confirm the molecular structure and chemical bonding of the
target compounds, 6-6 was successfully recrystallized from ethyl acetate/petroleum
ether to give colorless crystals suitable for single crystal X-ray diffraction. As shown
in Fig. 2, the two aromatic rings are connected by a sulfur atom and the whole
molecule adopts a bent shape, which is highly similar to the conformation of PTB in
complex with plant AHAS [9].
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2.2 Biological activity
2.2.1 Inhibition against fungal AHAS and Candida albicans
As can be seen in Table 1, most PTB derivatives displayed preferable inhibitory
activities against C. albicans AHAS at 100 µM concentration, and a few of them still
showed >50% inhibition at 10 µM. In contrast, PTB itself was a very weak inhibitor
of this enzyme, the inhibition of which was only 40% at 100 µM. This is close to the
reported data by Garcia et al [18]. The other control ethoxysulfuron, one of the best
AHAS inhibitors to date, had 96.8% inhibition even at 10 µM concentration. Among
the target compounds, 6-1, 6-3, 6-5, 6-9, 6-14, 6-17, 6-18, 6-22 and 6-27 had some
antifungal activities from the YNB media cell based assay. For the anti-Candida
activity, there is some difference between the results from RPMI 1640 media and that
from YNB media, and from the latter the activity is higher. It is known that, YNB
media does not contain the BCAAs while RPMI media contains these amino acids,
therefore there will be a shift of the minimum inhibitory concentration (MIC) values
between the data from the two medias if AHAS inhibition is the cause of the
antifungal activity. The results here indicated that the observed anti-Candida activity
was due to this reason, which is also in accordance with our previous report [19].
When the observation time changed to 48h instead of 24h, MIC data increased to
different extent (data now shown), which means that the anti-Candida activity here
was an inhibitory effect, not a fungicidal effect. There exists a strong correlation
between the fungal AHAS inhibition data and the antifungal activity for 6-9, 6-22 and
6-27. No antifungal activity was found for any compound with very weak AHAS
inhibition. For comparison, PTB did not have any inhibitory activity in the cell based
assay at all the conditions, however, ethoxysulfuron was a very potent inhibitor in the
YNB media, with MIC values of 0.39 mg/L against all the three fungal isolates. These
experimental results were similar to the reported potencies of the two commercial
AHAS inhibitors [18, 19].
2.2.2 Inhibition against plant AHAS and herbicidal activity
The in vitro inhibition of Arabidopsis thiliana AHAS and herbicidal activity of the
target compounds are listed in Table 2. Generally, most compounds demonstrated
excellent inhibition of plant AHAS at 100 µM concentration. Compound
,
,
6-13 6-22
,
,
,
and
displayed complete inhibition of AHAS, as well as
6-23 6-24 6-25 6-26 6-27
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PTB. At 10 µM concentration, compound
,
and
still contributed >60%
6-24
6-21 6-22
AHAS inhibition. For the in vivo herbicidal activity, compound
,
,
,
6-22 6-24 6-25 6-34
and
displayed >80% rape root growth inhibitory activity at 100 mg/L dosage,
6-35
while compound
,
,
,
,
,
,
,
,
and
6-4 6-5 6-9 6-10 6-16 6-18 6-23 6-26 6-28
6-29
possessed >50% herbicidal activity. At 10 mg/L, compound and
,
still
6-22 6-24
6-35
maintained >70% rape root inhibition. As can be seen, PTB was not only a very
strong inhibitor of plant AHAS, but also an extremely potent herbicide. Taken
together the plant AHAS inhibition data and herbicidal activity,
and
were
6-24
6-22
subjected to greenhouse pot assay for further evaluation, which is illustrated in Table
S1. At 300 g/ha dosage, these two compounds displayed potent herbicidal activities,
Brassica
in both pre-emergence and post-emergence assays for dicot weed species
campestris and Amaranthus retroflexu. Interestingly also showed 72.3%
, 6-24
inhibition against monocot Echinochloacrus-galli from the post-emergence assay. For
comparison, the herbicidal activity of PTB was much stronger even at the dosage of
30 g/ha, with the only exception of the post-emergence activity against
Echinochloacrus-galli.
PTB is the best commercial inhibitors of plant AHAS in this family but it is a rather
weak inhibitor of fungal AHAS. Meanwhile, the commercial sulfonylurea herbicide
ethoxysulfuron is also a potent a strong anti-Candida agent. This implied that the two
family of herbicidal AHAS inhibitors are different when subjected to fungal inhibition.
Nevertheless, here the synthesized new derivatives demonstrated some interesting
difference with PTB. For instance,
was more potent regarding the fungal AHAS
6-22
and anti-Candida activity than PTB was, while the herbicidal activity of
was
6-22
significantly weaker than that of PTB. In both fungal AHAS and plant AHAS
inhibition tests, was one of the best inhibitor. For , there is a fluorine atom at
6-22
6-22
the para position of the sulfur atom, while for PTB, a chlorine atom locates at the
meta position of the sulfur atom. This minor structural difference has led to significant
difference in their biological activities. A compound that combines the feature of PTB
and
has been designed, which has fluorine atoms or chlorine atoms at both para
6-22
and meta positions of the sulfur atom in the molecule. This new compound is
expected to have improved AHAS inhibition.
2.2.3 Antifungal activity against plant fungi disease
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Most of the target compounds together with PTB were also subjected to the
bioactivity against six common plant pathogenic fungi, and the corresponding results
are summarized in Table 3. The commercial fungicide chlorothalonil was used as a
positive control. At 50 mg/L concentration, the tested compounds showed moderate to
excellent antifungal activity against the plant fungi. For Sclerotinia sclerotiorum, 6-19,
6-20, 6-25, 6-26 and 6-31 exhibited >70% inhibition. For Phytophthora capsici, the
best compound was 6-28 with inhibitory activity of 66.7%. For Physalospora piricola,
6-3, 6-5, 6-6, 6-7 and 6-26 displayed >70% biological activity. For Rhizoctonia
cerealis, 6-11, 6-23, 6-25, 6-26, 6-27, 6-35 and PTB had >85% inhibitory rates, while
6-24, 6-28 and 6-31 had >70% potency. For Watermelon anthracnose and Rice
bakanae, none of the tested compounds showed powerful inhibition. The
concentrations for 50% of the maximal effect (EC50) of 6-11, 6-23, 6-25, 6-26, 6-27,
6-35 and PTB against R. cerealis were determined subsequently and the
corresponding EC50 data were 12.4 mg/L, 14.1 mg/L, 11.8 mg/L, 9.2 mg/L, 11.4 mg/L,
10.9 mg/L and 15.4 mg/L, respectively. The control chlorothalonil was active against
all the selected fungi with inhibition data >75%, and EC50 against R. cerealis was 5.3
mg/L. Compound 6-26 was the best inhibitor among all the compounds, showing >75%
inhibition against three fungi and the best inhibition of 60.6% against R. bakanae,
which might be considered as a potential fungicide for further study. It should be
pointed out that the media for this assay contains the amino acids and we failed to try
these assays under YNB media, therefore antifungal activity determined here does not
necessarily have direct relationship with AHAS inhibition, or AHAS inhibition might
not be the sole factor. Although 6-22 was not subjected to the plant fungi assay, there
were some overlaps between the anti-Candida activity and antifungal activities
against plant fungi disease, such as 6-3, 6-5 and 6-27. Obviously, C. albicans and the
plant pathogenic fungi are different species. PTB did not exhibit any anti-Candida
activity, but it was an inhibitor of R. cerealis.
2.2.4 Insecticidal activity against armyworm
Table 4 presents the larvicidal activity of the target compounds against Mythimna
separata at 600 mg/L concentration. 6-3, 6-14 and 6-20 exhibited >75% insecticidal
activity, while 6-6 and 6-9 showed no activity for this test. In contrast, the commercial
pesticide rynaxypyr displayed 100% insecticidal activity at a much lower
concentration of 10 mg/L, much stronger than any of the tested target compounds.
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This is a valuable attempt despite that these compounds possess very weak
insecticidal activity, and it implies that some PTB derivatives are might be useful for
the research of novel insecticides.
2.2.5 Antiviral activity against tobacco mosaic virus (TMV)
Most of the title compounds were bioassayed their passivation efficacies against TMV,
and the determined inhibitory values are listed in Table 5. Ten of the tested
compounds, comprising
,
,
,
,
,
,
,
,
and
,
6-35
6-4 6-5 6-10 6-19 6-20 6-22 6-25 6-28 6-34
were found to have anti-TMV activity at 500 mg/L concentration. Among these
compounds, and showed >40% antiviral activity, better than that of
,
6-20 6-22
6-25
commercial antiviral ribaviron (37.6%) but weaker than another commercial antiviral
ningnanmycin (56.4%). Therefore these compounds were subjected to further
evaluation of the biological activity at 100 mg/L concentration, and the determined
passivation efficacies were 6.2% for
, 11.4% for
and 6.8% for
,
6-25
6-20
6-22
respectively. At this condition, ribavirin had 11.9% antiviral activity and
ningnanmycin had 27.8% efficiency. These data suggests that the ningnanmycin is
superior to the PTB derivatives, and
conditions.
is comparable to ribaviron at the tested
6-22
2.2.6 Inhibitory activity against SARS-CoV Mpro
The biological activities against SARS-CoV Mpro of the target compounds were also
evaluated in this study and the inhibitory results that were tested at 20 µM are listed in
Table 6. Compound
,
and
exhibited 100% inhibition, while , , ,
6-1 6-2 6-3
6-4 6-5
and displayed >75% inhibitions. At the same condition, PTB
6-30
6-7
,
,
6-6 6-14 6-17
only showed 12.2% inhibition towards SARS-CoV Mpro.
,
and
were
6-7
6-4 6-5
additionally measured their half maximal inhibitory concentration (IC50) values for
SARS-CoV Mpro, together with their half cytotoxic concentration (CC50) data for
293T cells. The determined IC for
was 3.30 µM and its CC was 30.5 µM, the
50
6-4
50
corresponding data for
were 4.47 µM (IC ) and >200 µM (CC ), and for
6-5
6-7
has a surprising selectivity
50
50
were 4.54 µM (IC ) and 53.5 µM (CC ). Therefore
6-5
index of 44.5, showing that it is a promising hit for further investigation. The
50
50
inhibition curve of
against SARS-CoV Mpro is drawn in Fig. 3.
6-5
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For a comparison,
and
both showed anti-TMV activity and inhibition against
6-5
6-4
SARS-CoV Mpro, while other compounds are not within the overlap. There exist big
difference between TMV and SARS-CoV, and it is not likely that major overlap exists
between them.
Although different compounds showed different biological activities.
displayed
6-5
desirable activities from all the bioassays and it has a low cellular cytotoxicity against
293T cells. Therefore it is an excellent starting point for further molecular design. Of
course, other normal cell lines treated
need to be accessed for better estimation of
6-5
the biological safety of potent compound and this is anticipated to be done in the near
future.
2.3 Molecular simulation
2.3.1 DFT calculation of typical compounds for AHAS inhibition
It is generally accepted that frontier molecular orbital contributes significantly to the
inhibitor-enzyme interaction [20-23]. Among the title compounds, 6-22 showed
potent inhibitions for both fungal AHAS and plant AHAS, as well as its desirable
anti-Candida activity and herbicidal activity. Meanwhile, 6-8 and 6-12 were very
weak inhibitors towards both AHASs, and no in vivo activities were observed. These
compounds were then subjected to theoretical calculations by means of DFT/B3LYP
method. From the crystal structure of PTB bound with A. thaliana AHAS, it is clear
that the pyrimidine ring forms a π-π interaction with a neighboring residue Trp574 [9],
which means that the pyrimidine ring or the triazine ring in the newly synthesized
compounds might be involved in this interaction. The HOMO-1, HOMO, LUMO and
LUMO+1 of these compounds were drawn Fig 4 or Fig S1. Comparisons of these
maps revealed that here HOMO-1 might be an important factor in AHAS inhibition.
For 6-22, HOMO-1 covers not only the pyrimidine ring, but also the phenyl ring,
whereas for 6-8 and 6-12, the HOMO-1s only distribute in the heterocycle ring (Fig.
4). This suggests that 6-22 is possible to have more interactions with the enzyme than
6-8 and 6-12 do. However for HOMO, LUMO and LUMO+1 maps (seen in Fig S1),
there are either no obvious differences (HOMO and LUMO+1), or the difference is in
conflict with the known π-π interaction with W574 (LUMO). Based on these thoughts,
it is concluded that HOMO-1 is a non-negligible element for the further molecular
design of AHAS-inhibiting PTB derivatives.
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2.3.2 Molecular docking for SARS-CoV Mpro inhibitor
Because neither PTB nor its derivative has previously been reported to be an inhibitor
of SARS-CoV Mpro, little is known about the binding mode. Now that we have
identified that a lot of the target compounds are good inhibitors of this enzyme and
6-5 even showed very low cellular cytotoxicity, it is interesting to dock the inhibitor
to the binding site to investigate the probable mode of action. FlexX is a fast, flexible
docking method that uses an incremental construction algorithm to place ligands into
an active site [24], which has been used previously to successfully predict the binding
modes of several families of inhibitors [12, 19, 25]. This program was utilized to
divine the binding mode in this study. Fig. 5a is a two-dimensional illustration of the
interactions between 6-5 and the surrounding residues of SARS-CoV Mpro drawn by
LIGPLOT [26]. As can be seen, multiple hydrophobic interactions were formed by
6-5 with Thr26, Leu27, Phe140, Leu141, Gly143, His163, Glu166 and His172, while
four intermolecular hydrogen bonds could be seen between 6-5 and Asn142, Ser144
and Cys145. Fig. 5b shows the three-dimensional molecular surface of the cavity
generated by PyMOL (DeLano Scientific, South San Francisco, CA), and 6-5 adopted
a relaxed conformation to inhabit in the cavity. The predicted binding mode has
provided meaningful information for design more potent SARS-CoV Mpro inhibitors.
3. Conclusion
Previous studies on pyrithiobac and its derivatives mostly focused on their herbicidal
use. Since thioether compounds are always associated with considerable biological
activities, it is necessary to investigate different bioactivities to widen their
agricultural or medicinal use. As PTB itself is a highly safe compound towards
mammals, compounds with similar structures are expected to have biologically safe
property. In this research, a total of 35 PTB derivatives were synthesized and
characterized, and this is the first time that such derivatives have been evaluated
comprehensively against different bioassays. The target compounds were found to
have anti-Candida activity, herbicidal activity, antifungal activity against plant fungi,
insecticidal activity, anti-TMV activity and inhibitions against SARS-CoV Mpro. As a
promising AHAS inhibitor, 6-22 exhibited desirable C. albicans inhibition and potent
herbicidal activity. Interestingly, 6-26 displayed potent antifungal activity against
plant pathogenic diseases. 6-22 also showed desirable passivation efficacies against
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TMV. Furthermore, some compound such as 6-5 was identified to be potent inhibitor
of SARS-CoV Mpro. From a DFT calculation, HOMO-1 might have impact on AHAS
inhibitory activity. A feasible binding mode was constructed to explain the interaction
of 6-5 with SARS-CoV Mpro. Overall, because 6-5 displayed activities from all the
bioassays and its cell cytotoxicity data was very low, it is regarded as a lead
compound for further molecular design.
4. Experimental section
4.1. General synthesis and instruments
The starting materials were procured from Bejing Ouhe, Shanghai Bide, Shanghai
Energy Chemical, Qingdao Haiyang Chemical, Alfa-Aesar, Sigma-Aldrich, TCI and
some local chemical suppliers. All solvents and liquid reagents were dried in advance
using standard methods and distilled before use. Sodium sulfide nonahydrate was
dried and stored in a drying column before use. Melting points were determined using
1
an RT-2 melting apparatus and were uncorrected. H NMR spectra and 13C NMR
were obtained using a Bruker Avance 400 MHz spectrometer. The chemical shift
values (d) for the NMR spectra were reported as parts per million (ppm), using
deuterated chloroform (CDCl3) or dimethyl sulfoxide (DMSO-d6) as the solvent and
tetramethylsilane (TMS) as an internal reference standard. Mass spectra were
recorded on an Ionspect FT-MS 7.0T LC/mass detector instrument. Single-crystal
X-ray diffraction analyses were performed on a Bruker Smart 1000 CCD
diffractometer. The synthesis of intermediate 2 and 3 is depicted in Scheme 1 and the
experimental data are detailed in the supplementary material. The synthesis for target
PTB derivatives is shown in Scheme 2 and the experimental details are stated below.
4.2. Synthesis of the target PTB derivatives
For the synthesis of 6-1 to 6-13, intermediate 4 (4.5 mmol) was added to the solution
of intermediate 2 (3.0 mmol) in 40 mL acetonitrile under stirring, subsequently the
reaction proceeded overnight at room temperature in the presence triethylamine (4.5
mmol). The mixture was then vacuumed and the remaining solid was further extracted
by using a combination of ethyl acetate and water and the organic layer was kept for
further process. The final product was purified by flash column chromatography using
petroleum ether/ethyl acetate (80:1) and the yields were in the range of 15%-28%.
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For the synthesis of 6-14 to 6-21, intermediate 4 (2.4 mmol), intermediate 3 (1.6
mmol) and sodium methoxide (4.8 mmol) were added to 40 mL methanol. The
reaction proceeded for 5 h under reflux and the mixture was then vacuumed to get rid
of the solvent. The crude product was further extracted three times by using sodium
hydroxide solution and ethyl acetate. The aqueous layer was then adjusted by
hydrochloric acid to a pH value of 3-4. The product was extracted by ethyl acetate and
finally purified by flash column chromatography using dichloromethane/methanol
(80:1) and the yields were in the range of 10%-28%.
For the synthesis of 6-22 to 6-33, intermediate 5 (4.0 mmol), intermediate 2 or 3 (2.66
mmol) and tetrabutyl ammonium bromide (0.3 mmol) were added to 50 mL 90%
ethanol. After that the reaction continued for 15 h under reflux and every two hours
the pH value of the mixture was adjusted to >7 by adding saturated sodium
bicarbonate solution. The crude product was further extracted three times by using
sodium hydroxide solution and ethyl acetate. The aqueous layer was then adjusted by
hydrochloric acid to a pH value of 3-4. The product was extracted by ethyl acetate and
finally purified by flash column chromatography using dichloromethane/methanol
(80:1) and the yields were in the range of 14%-35%.
For the synthesis of 6-34 and 6-35, intermediate 4 (5.0 mmol), intermediate 2’ (5.0
mmol) and potassium hydroxide (5.0 mmol) was added to 40 mL tetrahydrofuran,
after that the reaction proceeded overnight at room temperature. The mixture was then
vacuumed and the remaining solid was further extracted by using a combination of
ethyl acetate and water and the organic layer was kept. The final product was purified
by recrystallization from ethanol and the yields were 47% and 58%.
4.2.1. 2-((4,6-dimethoxy-1,3,5-triazin-2-yl)thio)-6-fluorobenzonitrile (6-1)
1
Yield 22%; m. p.: 122.8-123.4 °C; white solid; HNMR (400 MHz, DMSO-d6) δ
13
7.97-7.87 (m, 1H, ArH),7.82-7.70 (m, 2H, ArH), 3.87 (s, 6H, OCH3); CNMR (100
MHz, DMSO-d6) δ 182.2, 171.5, 136.6, 136.4, 133.9, 132.4, 119.1, 118.9, 113.0, 55.6;
HRMS (ESI)m/z: [M + H]+ calcd for C12H9FN4O2S: 293.0503, found 293.0506.
4.2.2. 2-chloro-6-((4,6-dimethoxy-1,3,5-triazin-2-yl)thio)benzonitrile (6-2)
1
Yield 25%; m. p.: 140.8-142.2 °C; white solid; HNMR (400 MHz, DMSO-d6) δ 7.94
(t, J = 19.2 Hz, 1H, ArH), 7.89 (d, J = 7.2 Hz, 1H, ArH), 7.85 (d, J =7.6 Hz, 1H, ArH),
12

ACCEPTED MANUSCRIPT
3.86 (s, 6H, OCH3); 13CNMR (100 MHz, DMSO-d6) δ 182.0, 171.3, 137.3, 136.3,
135.4, 133.2, 132.1, 119.3, 114.9, 55.6; HRMS (ESI) m/z[M + H]+ calcd for
C12H9ClN4O2S: 309.0208, found 309.0201.
4.2.3. 2-bromo-6-((4,6-dimethoxy-1,3,5-triazin-2-yl)thio)benzonitrile (6-3)
1
Yield 28%; m. p.: 153.3-155.4 °C; white solid; HNMR (400 MHz, DMSO-d6) δ 8.06
(d, J = 8.4 Hz, 1H, ArH), 7.92(d, J = 8.0 Hz, 1H, ArH), 7.78 (t, J = 16.0 Hz,1H, ArH),
3.86 (s, 6H, OCH3); 13CNMR (100 MHz, DMSO-d6) δ 182.1, 171.4, 137.0, 135.5,
135.2, 133.4, 126.4, 121.6, 116.3, 55.6; HRMS (ESI) m/z[M + H]+ calcd for
C12H9BrN4O2S: 352.9702, found 352.9709.
4.2.4. 2-((4,6-dimethoxy-1,3,5-triazin-2-yl)thio)-6-iodobenzonitrile (6-4)
1
Yield 22%; m. p.: 170.4-172.8 °C; white solid; HNMR (400 MHz, DMSO-d6) δ 8.20
(d, J = 8.8 Hz, 1H, ArH), 7.89 (d, J = 8.0 Hz, 1H, ArH), 7.54 (t, J = 16.0 Hz, 1H, ArH),
3.87 (s, 6H, OCH3); 13CNMR (100 MHz, DMSO-d6) δ 182.3, 171.3, 141.3, 136.8,
135.1, 132.7, 126.2, 118.6, 102.3, 55.8; HRMS (ESI,) m/z[M + H]+ calcd for:
C12H9IN4O2S: 400.9564, found 400.9569.
4.2.5. 2-((4,6-dimethoxy-1,3,5-triazin-2-yl)thio)-5-fluorobenzonitrile (6-5)
Yield 22%; m. p.: 128.8-130.8 °C; white solid; 1HNMR (400 MHz, DMSO-d6) δ 8.31 (s,
1H, ArH),7.92 (d, J = 8.0Hz, 2H, ArH), 3.87 (s, 6H, OCH3); 13CNMR (100 MHz,
DMSO-d6) δ 172.2, 166.2, 139.1, 136.3, 134.5, 134.2, 129.8, 120.8, 116.4, 55.8. HRMS
(ESI) m/z [M + H]+ calcd for C12H9FN4O2S: 293.0503, found 293.0504.
4.2.6. 5-chloro-2-((4,6-dimethoxy-1,3,5-triazin-2-yl)thio)benzonitrile (6-6)
1
Yield 20%; m. p.: 158.9-160.4 °C; white solid; HNMR (400 MHz, DMSO-d6) δ 8.42
(d, J = 2.0Hz, 1H, ArH), 8.04 (dd, J =10.4 Hz, 1H, ArH), 7.84 (d, J =8.4 Hz, 1H, ArH),
3.87 (s, 6H, OCH3); 13CNMR (100 MHz, DMSO-d6) δ 182.1, 171.3, 139.1, 137.5,
137.0, 130.2, 124.5, 120.8, 116.2, 55.8; HRMS (ESI) m/z [M + H]+ calcd for
C12H9ClN4O2S: 309.0208, found 309.0210.
4.2.7. 5-bromo-2-((4,6-dimethoxy-1,3,5-triazin-2-yl)thio)benzonitrile (6-7)
1
Yield 15%; m. p.: 131.2-132.4 °C; white solid; HNMR (400 MHz, DMSO-d6) δ 7.76
(d, J = 7.6 Hz, 1H, ArH), 7.54 (d, J = 7.2 Hz, 1H, ArH), 7.38 (s, 1H, ArH), 3.87 (s, 6H,
OCH3); 13CNMR (100 MHz, DMSO-d6) δ 182.5, 171.3, 139.2, 127.1, 121.8, 121.3,
121.0, 120.8, 116.8, 55.5; HRMS (ESI)m/z [M + H]+ calcd for C12H9BrN4O2S:
352.9702, found 352.9698.
4.2.8. 2-((4,6-dimethoxy-1,3,5-triazin-2-yl)thio)-5-(trifluoromethyl)benzonitrile (6-8)
Yield 20%; m. p.: 91.3-94.1 ºC; white solid; 1HNMR (400 MHz, CDCl3) δ 8.02 (d, 1H,
13

ACCEPTED MANUSCRIPT
ArH), 7.90(s, 1H, ArH), 7.88(d, 1H, ArH), 3.92(s, 3H, OCH3); 13CNMR (100 MHz,
CDCl3) δ 187.7, 171.3, 137.4, 135.9, 132.8, 132.5 130.5, 129.6, 120.6, 115.7, 77.0, 55.5;
HRMS (ESI)m/z [M + H]+ calcd for C13H9F3N4O2S: 343.0471, found 343.0473.
4.2.9. 4-((4,6-dimethoxy-1,3,5-triazin-2-yl)thio)-3-fluorobenzonitrile (6-9)
1
Yield 18%; m. p.:124.4-126.4 °C; white solid; HNMR (400 MHz, DMSO-d6) δ 8.10
(d, J = 8.8 Hz, 1H, ArH), 7.94 (t, J = 14.4 Hz, 1H, ArH), 7.85 (d, J = 8.0 Hz, 1H, ArH),
3.85 (s, 6H, OCH3); 13CNMR (100 MHz, DMSO-d6) δ 182.0, 171.3, 137.8, 128.3,
122.1, 119.7, 119.4, 117.0, 115.7, 55.5; HRMS (ESI) m/z [M + H]+ calcd for
C12H9FN4O2S: 293.0503, found:293.0508.
4.2.10. 3-chloro-4-((4,6-dimethoxy-1,3,5-triazin-2-yl)thio)benzonitrile (6-10)
Yield 20%; m. p.: 145.3-146.4 °C; white solid; 1HNMR (400 MHz, DMSO-d6) δ 8.31 (t,
J = 8.6 Hz, 1H, ArH), 8.06 (d, J = 8.0 Hz, 1H, ArH), 7.96 (d, J =6.8 Hz, 1H, ArH), 3.85
(s, 6H, OCH3); 13CNMR (100 MHz, DMSO-d6) δ 181.3, 170.9, 138.8, 138.4, 133.2,
132.8, 131.4, 117.0, 114.5, 55.3; HRMS (ESI) m/z [M + H]+ calcd for C12H9ClN4O2S:
309.0208, found 309.0255.
4.2.11. 3-bromo-4-((4,6-dimethoxy-1,3,5-triazin-2-yl)thio)benzonitrile (6-11)
1
Yield 24%; m. p.: 148.3-149.8 °C; white solid; HNMR (400 MHz, DMSO-d6) δ 7.73
(s, 1H, ArH), 7.53 (d, J = 8.4 Hz, 1H, ArH), 7.49 (d, J = 6.8 Hz, 1H, ArH), 3.92 (s, 6H,
OCH3); 13CNMR (100 MHz, DMSO-d6) δ 142.0, 138.0, 136.4, 135.9, 131.6, 131.0,
126.4, 120.9, 111.9, 55.5; HRMS (ESI) m/z [M + H]+ calcd for C12H9BrN4O2S:
352.9702, found 352.9698.
4.2.12.
(6-12)
4-((4,6-dimethoxy-1,3,5-triazin-2-yl)thio)-3-(trifluoromethyl)benzonitrile
1
Yield 18%; m. p.: 146.3-149.9 ºC; white solid; HNMR (400 MHz, CDCl3) δ 8.05(d,
1H, ArH), 7.90(s, 1H, ArH), 7.87(d, 1H, ArH) ,3.88 (s, 3H, OCH3); 13CNMR (100
MHz, CDCl3) δ 182.7, 171.2, 140.5, 134.6, 132.6, 130.5, 130.4, 123.5, 116.8, 114.2,
55.4; HRMS (ESI) m/z [M + H]+ calcd for C13H9F3N4O2S: 343.0471, found 343.0473.
4.2.13. 4-chloro-2-((4,6-dimethoxy-1,3,5-triazin-2-yl)thio)benzonitrile (6-13)
Yield 25%; m. p.: 146.8-147.5 °C; white solid; 1HNMR (400 MHz, DMSO-d6) δ 7.76
(s, 1H, ArH), 7.63 (d, J = 8.0 Hz,1H, ArH), 7.56 (d, J = 8.0 Hz 1H, ArH), 3.89 (s, 6H,
13
OCH3); CNMR (100 MHz, DMSO-d6) δ 181.6, 170.3, 135.9, 134.7, 134.5, 133.3,
132.8, 132.2, 114.4, 54.5; HRMS (ESI) m/z [M + H]+ calcd for C12H9ClN4O2S:
309.0208, found 309.0211.
4.2.14. 2-((4,6-dimethoxy-1,3,5-triazin-2-yl)thio)-5-fluorobenzoic acid (6-14)
14

ACCEPTED MANUSCRIPT
Yield 18%; m. p.: 198.4-200.3 °C; white solid; 1HNMR (400 MHz, DMSO-d6) δ 13.97
(s, 1H, COOH) 7.74 (d, J = 7.2 Hz, 2H, ArH), 7.49 ( d, J = 8.4 Hz, 1H, ArH), 3.89 (s,
6H, OCH3); 13CNMR (100 MHz, DMSO-d6) δ 173.6, 171.7, 167.2, 134.8, 129.1,
128.7, 121.1, 117.9, 167.2, 55.7; HRMS (ESI) m/z [M + H]+ calcd for C12H10FN3O4S:
312.0449, found 312.0447.
4.2.15. 5-chloro-2-((4,6-dimethoxy-1,3,5-triazin-2-yl)thio)benzoic acid (6-15)
Yield 10%; m. p.: 170.2-172.0 °C; white solid; 1HNMR (400 MHz, DMSO-d6) δ 12.44
(s, 1H, COOH), 7.99 (s, 1H, ArH), 7.65 (d, J = 9.6 Hz, 1H, ArH), 3.89 (s, 6H, OCH3);
13CNMR (100 MHz, DMSO-d6) δ 169.8, 166.5, 137.6, 133.1, 130.8, 129.6, 127.1,
126.4, 123.5, 55.7; HRMS (ESI) m/z [M + H]+ calcd for C12H10ClN3O4S: 328.0153,
found: 328.0151.
4.2.16. 5-bromo-2-((4,6-dimethoxy-1,3,5-triazin-2-yl)thio)benzoic acid (6-16)
Yield 28%; m. p.: 186.8-189.2 °C; white solid; 1HNMR (400 MHz, DMSO-d6) δ 13.62
(s, 1H, COOH), 8.04 (d, J = 9.6 Hz, 1H, ArH), 7.65 (d, J = 9.6 Hz,1H, ArH), 3.89 (s,
6H, OCH3); 13CNMR (100 MHz, DMSO-d6) δ 183.2, 171.2, 166.6, 139.0, 136.5,
134.8, 133.1, 127.1, 123.4, 55.6; HRMS (ESI) m/z [M - H]- calcd for C12H10BrN3O4S:
369.9503, found 369.9498.
4.2.17. 4-((4,6-dimethoxy-1,3,5-triazin-2-yl)thio)-3-fluorobenzoic acid (6-17)
Yield 27%; m. p.: 177.2-179.4 °C; white solid; 1HNMR (400 MHz, DMSO-d6) δ 13.19
(s, 1H, COOH) 7.65 (s, 1H, ArH), 7.56-7.62 (m, 2H, ArH), 3.92 (s, 6H, OCH3);
13CNMR (100 MHz, DMSO-d6) δ 173.6, 171.5, 166.1, 133.4, 131.5, 127.9, 127.4,
126.7, 116.9, 55.5; HRMS (ESI) m/z [M + H]+ calcd for C12H10FN3O4S: 312.0449,
found 312.0447.
4.2.18. 3-chloro-4-((4,6-dimethoxy-1,3,5-triazin-2-yl)thio)benzoic acid (6-18)
1
Yield 28%; m. p.: 195.0-196.4 °C; white solid; HNMR (400 MHz, DMSO-d6) δ 13.62
(s, 1H, COOH) 8.09 (s, 1H, ArH), 7.86-8.01 (s, 2H, ArH), 3.84 (s, 6H, OCH3); 13CNMR
(100 MHz, DMSO-d6) δ 182.1, 171.3, 166.1, 138.9, 138.6, 134.6, 131.8, 130.6, 128.7,
55.7; HRMS (ESI) m/z [M - H]- calcd for C12H10ClN3O4S: 326.0008 found: 326.0009.
4.2.19. 3-bromo-4-((4,6-dimethoxy-1,3,5-triazin-2-yl)thio)benzoic acid (6-19)
1
Yield 26%; m. p.: 252.4-252.8 °C; white solid; HNMR (400 MHz, DMSO-d6) δ 13.35
(s, 1H, COOH) 8.13 (s, 1H, ArH), 7.95 (d, J = 7.6 Hz, 1H,ArH), 7.58 (d, J = 7.2 Hz, 1H,
ArH) 3.92 (s, 6H, OCH3); 13CNMR (400 MHz, DMSO-d6) δ 173.2, 165.4, 149.7, 139.9,
133.4, 131.2, 129.5, 126.4, 119.9, 55.0; HRMS (ESI) m/z [M - H]- calcd for
C12H10BrN3O4S: 369.9503, found 369.9497.
15

ACCEPTED MANUSCRIPT
4.2.20.
(6-20)
4-((4,6-dimethoxy-1,3,5-triazin-2-yl)thio)-2-(trifluoromethyl)benzoic
acid
1
Yield 22%; m. p.: 127.5-129.8 °C; white solid; HNMR (400 MHz, DMSO-d6) δ 13.43
(s, 1H, COOH), 8.28 (s, 1H, ArH), 8.11 (d, J = 7.6 Hz, 1H, ArH), 7.93 (d, J = 8.0 Hz,
13
1H, ArH), 3.85 (s, 6H, OCH3); CNMR (400 MHz, DMSO-d6) δ 182.7, 172.4, 165.6,
137.1, 135.5, 132.6, 132.4, 131.0, 129.2, 55.6; HRMS (ESI) m/z [M + H]+ calcd for
C14H12F3N2O4S: 362.0417, found 362.0414.
4.2.21. 4-((4,6-dimethoxypyrimidin-2-yl)thio)isophthalic acid (6-21).
1
Yield 26%; m. p.: 121.4-123.9 °C; yellow solid; HNMR (400 MHz, DMSO-d6) δ
13.40(s, 1H, ArH), 8.38 (s, J = 7.6 Hz, 1H, ArH), 8.09 (s, 1H, ArH), 7.97 (s, 1H, ArH),
6.03 (s, 1H, ArH), 3.75 (s, 6H, OCH3); 13CNMR (100 MHz, DMSO-d6) δ 171.4, 167.6,
166.7, 136.6, 136.6, 136.1, 132.8, 131.1, 128.7, 118.0, 87.0, 54.7; HRMS (ESI) m/z [M
+ H]+ calcd for C14H12F3N2O4S: 338.0441, found 338.0442.
4.2.22. 2-((4,6-dimethoxypyrimidin-2-yl)thio)-5-fluorobenzoic acid (6-22)
1
Yield 18%, m. p.: 140.5-142.7 °C; white solid; HNMR (400 MHz, DMSO-d6) δ 13.43
(s, 1H, COOH), 7.83 (d, J = 7.0 Hz, 1H, ArH), 7.65 (d, J = 8.8 Hz, 1H, ArH), 7.47 (s,
13
1H, ArH), 3.71 (s, 6H, OCH3); CNMR (100 MHz, DMSO-d6) δ 170.0, 168.4, 168.0,
138.4, 124.5, 118.5, 118.2, 117.5, 117.3, 85.8, 53.3; HRMS (ESI) m/z [M + H]+ calcd
for C13H11FN2O4S: 311.0496, found 311.0497.
4.2.23. 5-chloro-2-((4,6-dimethoxypyrimidin-2-yl)thio)benzoic acid (6-23)
1
Yield 14%; m. p.: 170.2-172.4 °C; white solid; HNMR (400 MHz, DMSO-d6) δ 13.61
(s, 1H, COOH), 8.08 (s, 1H, ArH), 7.89 -7.96 (m, 2H, ArH), 6.04 (s, 1H, ArH), 3.70 (s,
13
6H, OCH3); CNMR (100MHz, DMSO-d6) δ 171.4, 166.2, 140.1, 138.6, 138.2, 133.9,
133.8, 130.5, 128.6, 87.0, 54.7; HRMS (ESI) m/z [M - H]- calcd for C13H11ClN2O4S:
325.0055, found 325.0060.
4.2.24. 5-bromo-2-((4,6-dimethoxypyrimidin-2-yl)thio)benzoic acid (6-24)
1
Yield 21%; m. p.: 182.4-185.6 °C; white solid; HNMR (400 MHz, DMSO-d6) δ 13.43
(s, 1H, COOH), 7.99 (s, 1H, ArH), 7.79 (d, J = 8.4 Hz, 1H, ArH), 7.72 (d, J = 8.4 Hz,
1H, ArH), 5.98 (s, 1H, ArH), 3.74 (s, 6H, OCH3); 13CNMR (100 MHz, DMSO-d6) δ
171.1, 169.2, 166.9, 138.5, 137.9, 134.5, 132.7, 129.3, 122.5, 86.7, 54.6; HRMS (ESI)
m/z [M - H]- calcd for C13H11BrN2O4S: 368.9550, found: 368.9543.
4.2.25. 4-((4,6-dimethoxypyrimidin-2-yl)thio)-3-fluorobenzoic acid (6-25)
Yield 27%; m. p.: 187.6-189.2 °C; white solid; 1HNMR (400 MHz, DMSO-d6) δ 13.68
(s, 1H, COOH), 7.83 (s, 2H, ArH), 7.79 (s, 1H, ArH), 6.02 (s, 1H, ArH) 3.70 (s, 6H,
16

ACCEPTED MANUSCRIPT
OCH3); 13CNMR (100 MHz, DMSO-d6) δ 171.3, 167.8, 166.6, 137.6, 125.9, 121.5,
120.7, 116.6, 116.4, 86.6, 54.6; HRMS (ESI) m/z [M + H]+ calcd for C13H11FN2O4S:
311.0496, found 311.0502.
4.2.26. 3-chloro-4-((4,6-dimethoxypyrimidin-2-yl)thio)benzoic acid (6-26)
Yield 31%; m. p.: 179.6-180.2 °C; white solid; 1HNMR (400 MHz, DMSO-d6) δ 13.80
(s, 1H, COOH), 8.08 (s, 1H, ArH), 7.87-8.03 (2H, ArH), 6.02 (s, 1H, ArH) 3.70 (s, 6H,
OCH3); 13CNMR (100 MHz, DMSO-d6) δ 171.3, 168.1, 166.5, 154.8, 138.6, 138.0,
133.1, 130.5, 128.6, 86.7, 54.6; HRMS (ESI) m/z [M - H]- calcd for C13H11ClN2O4S:
325.0055, found 325.0062.
4.2.27. 3-bromo-4-((4,6-dimethoxypyrimidin-2-yl)thio)benzoic acid (6-27)
1
Yield 24%; m. p.: 172.8-174.6 °C; white solid; HNMR (400 MHz, DMSO-d6) δ
13.45 (s, 1H, COOH), 8.23 (s, 1H, ArH), 7.92-8.01 (2H, ArH), 6.03 (s, 1H, ArH), 3.70
13
(s, 6H, OCH3); CNMR (100 MHz, DMSO-d6) δ 170.8, 167.5, 165.5, 137.5, 135.4,
133.2, 130.0, 129.6, 128.6, 86.2, 54.1; HRMS (ESI)m/z [ M - H]- calcd for
C13H11BrN2O4S: 368.9550, found 368.9549.
4.2.28. 2-((4,6-dimethoxypyrimidin-2-yl)thio)-4-(trifluoromethyl)benzonitrile (6-28)
Yield 28%; m. p.: 105.5-107.6 °C; white solid; 1HNMR (400 MHz, DMSO-d6) δ 8.12
(s, 1H, ArH), 7.88 (d, J = 8.0Hz,1H, ArH), 7.56 (d, J = 8.0 Hz, 1H, ArH), 3.89 (s, 6H,
13
OCH3); CNMR (100 MHz, DMSO-d6) δ 172.8, 171.0, 167.1, 135.7, 133.8, 128.9,
126.1, 122.8, 121.4, 116.1, 87.5, 54.1; HRMS (ESI) m/z [M + H]+calcd for
C14H10F3N3O2S: 342.0519, found 342.0524.
4.2.29. 2-((4,6-dimethoxypyrimidin-2-yl)thio)-4-(trifluoromethyl)benzoic acid (6-29).
1
Yield 24%; m. p.: 127.4-129.3 °C; yellow solid; H NMR (400 MHz, DMSO-d6) δ
13.71 (s, 1H, COOH), 8.28-8.12 (m, 1H,ArH), 8.03 (d, J = 8.3 Hz, 1H, ArH), 7.89 (d, J
= 8.1 Hz, 1H, ArH), 7.74 (d, J = 8.1 Hz, 1H, ArH), 6.07 (d, J = 40.3 Hz, 1H, Het-H),
3.73 (s, 6H, OCH3); 13CNMR (100 MHz, DMSO-d6) δ 171.1, 168.7, 167.4, 139.7,
133.0, 132.6, 131.4, 131.0, 125.9, 123.2, 87.1, 54.5; HRMS (ESI) m/z [M + H]+ calcd
for C14H12F3N2O4S: 361.0464, found 361.0469.
4.2.30. 4-((4,6-dimethoxypyrimidin-2-yl)thio)-2-(trifluoromethyl)benzonitrile (6-30).
1
Yield 17%; m. p.: 83.3-85.4 °C; yellow solid; HNMR (400 MHz, DMSO-d6) δ 8.07
(s, 1H, ArH), 7.83 (d, J =8.0Hz 1H, ArH), 7.71 (d, J =8.0Hz 1H, ArH), 5.73 (s, 1H,
ArH), 3.67 (s, 6H, OCH3), 13CNMR (100 MHz, DMSO-d6) δ 171.1, 167.3, 166.6,
148.4, 137.1, 135.5, 133.8, 132.2, 131.9, 131.3, 86.7, 53.4; HRMS (ESI) m/z [M +
H]+ calcd for C14H10F3N3O2S: 342.0519, found 342.0522.
17

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4.2.31. 4-((4,6-dimethoxypyrimidin-2-yl)thio)-2-(trifluoromethyl)benzoic acid (6-31).
Yield 35%; m. p.: 131.4-132.9 °C; yellow solid; 1HNMR (400 MHz, DMSO-d6) δ 13.65
(s, 1H, COOH), 8.14 (s,1H, ArH), 8.01 (d, J = 7.6 Hz, 1H, ArH), 7.91 (d, J = 8.4Hz,
13
1H, ArH), 6.05 (s, 1H, ArH), 3.74 (s, 6H, OCH3); CNMR (100 MHz, DMSO-d6) δ
193.6, 171.3, 168.4, 167.9, 138.4, 132.7, 131.6, 130.8, 130.5, 124.9, 86.9, 54.6; HRMS
(ESI) m/z [M + H]+ calcd for C14H12F3N2O4S: 361.0464, found 361.0463.
4.2.32. 2-((4,6-dimethoxypyrimidin-2-yl)thio)-5-(trifluoromethyl)benzonitrile (6-32).
1
Yield 21%; m. p.: 85.6-87.4 °C; yellow solid; HNMR (400 MHz, DMSO-d6) δ 8.31
(s, 1H, ArH), 8.06 (d, J = 7.6 Hz, 1H, ArH), 8.00 (d, J = 7.6 Hz, 1H, ArH), 5.68 (s,
1H, ArH), 3.90 (s, 6H, OCH3); 13CNMR (400 MHz, DMSO-d6) δ 172.7, 171.0, 164.4,
139.7, 137.4, 134.9, 128.8, 125.1, 124.7, 121.4, 83.2, 54.1; HRMS (ESI) m/z [M +
H]+ calcd for C14H10F3N3O2S: 342.0519, found 342.0521.
4.2.33. 4-((4,6-dimethoxypyrimidin-2-yl)thio)isophthalic acid (6-33)
1
Yield 26%; m. p.: 121.4-123.9 °C; yellow solid; HNMR (400 MHz, DMSO-d6) δ
13.40(s, 1H, ArH), 8.38 (s, J = 7.6Hz, 1H, ArH), 8.09 (s, 1H, ArH), 7.97 (s, 1H, ArH),
6.03 (s, 1H, ArH), 3.75 (s, 6H, OCH3); 13CNMR (100 MHz, DMSO-d6) δ 171.4, 167.6,
166.7, 136.6, 136.6, 136.1, 132.8, 131.1, 128.7, 118.0, 87.0, 54.7; HRMS (ESI) m/z [M
+ H]+ calcd for C14H12F3N2O4S: 337.0489, found 337.0490.
4.2.34. 2-(3-bromophenoxy)-4,6-dimethoxy-1,3,5-triazine (6-34)
1
Yield 47%; m. p.: 83.5-85.2 °C; white solid; HNMR (400 MHz, CDCl3) δ 7.63
(s, 1H), 7.31 (s, 1H), 7.24 (s, 1H), 3.81 (s, 7H); 13CNMR (100 MHz, DMSO-d6)
155.6, 129.7, 128.3, 124.1, 122.8, 121.7, 117.8, 113.3, 55.5; HRMS (ESI) m/z [M +
H]+ calcd for C11H10BrN3O3: 311.9978, found: 311.9984.
4.2.35. 2-(3-iodophenoxy)-4,6-dimethoxy-1,3,5-triazine (6-35)
1
Yield 58%; m. p.: 80.2-81.4 °C; white solid; HNMR (400 MHz, DMSO-d6) δ
7.63-7.73 (m, 2H, ArH), 7.23-7.31 (m, 2H, ArH), 3.91 (s, 6H, OCH3); 13CNMR (100
MHz, DMSO-d6) δ 173.7, 173.0, 152.4, 135.2, 131.8, 130.8, 121.8, 94.8, 55.8; HRMS
(ESI) m/z [M + H]+ calcd for C11H10BrN3O3:359.9840, found 359.9833.
4.3. Evaluation of biological activities
4.3.1. Fungal AHAS inhibition and plant AHAS inhibition
The C. albican AHAS catalytic subunit and A. thaliana AHAS catalytic subunit were
expressed and purified as describe previously [19, 27]. AHAS activity was measured
using the colorimetric assay in 50 mM potassium phosphate (pH 7.0) containing 50
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mM pyruvate, 1 mM thiamine diphosphate, 10 mM MgCl2, and 10 µM FAD. After
incubation for 30 min at 37 °C (for fungal AHAS at 30 °C), acetolactate was
estimated [28].
4.3.2. Antifungal activity against C. albicans
The Candica albicans strains used in this study include standard strain SC5314, two
clinical isolates g5 and 17# and the recipe and procedure for the measurement of
antifungal activity is similar with the published method [19, 29]. Yeast nitrogen base
(YNB) medium and RPMI 1640 medium were both used for the determination
whether AHAS is a probable cause of the antifungal data. The microdilution plates
were incubated at 35 °C over a period of 72 h and visual readings of the cultures were
taken at 24h. MICs were defined as the lowest concentrations of the compounds that
can inhibit visible fungal growth.
4.3.3. Herbicidal activity
The rape (Brassica campestris L.) root growth inhibition assay was detailed before
[27]. Rape seeds were soaked in distilled water for 4 h before they were placed on a
filter paper in a 6-cm Petri plate, to which 2 mL of inhibitor solution had been added
in advance. Usually, 15 seeds were used on each plate. The plate was placed in a dark
room and allowed to germinate for 48 h at 28 °C. The lengths of 10 rape roots
selected from each plate were measured and the means were calculated.
The pre-emergence and post-emergence greenhouse pot herbicidal activities were
evaluated against monocotyledonous weeds (Echinochloacrus-galli and Digitaria
adscendens) and dicotyledonous weeds (Brassica campestris and Amaranthus
retroflexus) using methods reported previously [30].
4.3.4. Antifungal activities against plant pathogenic fungus
The fungicidal activities of the target compounds were tested against S. sclerotiorum,
P. capsici, P. piricola, R. cerealis, W. anthracnose and R. bakanae, the method of
which was described previously [31]. The relative inhibitory ratio (%) was determined
by the mycelium growth rate method at 50 mg/L concentration. Chlorothalonil and
PTB were used as controls. The EC50 values of selected compounds against R.
cerealis were calculated by equation (1) [27].
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Probit=m(log10[I]-log10[EC50])+5
(1)
4.3.5. Larvicidal activity against Mythimna separata
The insecticidal activity of title compounds and rynaxypyr against oriental armyworm
(Mythimna separata) was tested according to the leaf-dip method using the reported
procedure at 600 mg/L dose [32].
4.3.6. Passivation efficacies against TMV
TMV was inhibited by mixing with the compound solution at the same volume for 30
min. The mixture was then inoculated on the left side of the leaves of N. tabacum L.,
whereas the right side of the leaves was inoculated with the mixture of solvent and the
virus for control. The local lesion numbers were recorded 3−4 days after inoculation,
as reported previously [33]. The inactivation effect was evaluated at 500 mg/L or 100
mg/L.
4.3.7. SARS-CoV Mpro inhibition assay
SARS-CoV Mpro was expressed and purified previously [34], and the inhibition assay
was detailed before by us [12]. Briefly, the assays were conducted by fluorescence
resonance energy transfer. The settled concentrations of proteins, compounds and
substrate were preheated at 37 °C and oscillated. The excitation/emission light was
320/405 nm, and the test was carried out every 5 s for 200 times. IC50 values for
selected compounds were calculated by equation (2).
V0/V=1+[I]/IC50
(2)
4.3.8. Cytotoxicity assay for the determination of CC50
MTT assay was employed to test the cytotoxic effect of selected compounds, which
is similar to a published procedure [35]. 293T Cells in logarithmic phase were
collected and moved to 96-microwell plates (12000ꢀcells/well) overnight. After
treatment of 48ꢀh with various concentrations of compounds, 15ꢀµL MTT dye was
added to each well, followed by incubating for an additional 4ꢀh. The suspension was
discarded and 100ꢀµL DMSO was added to each well, after that OD570 was read. CC50
value was defined as the concentration of the compound reducing cell viabilityby
50%.
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4.4. In silico molecular modeling
Sybyl 7.3 (Tripos Inc., St Louis, MO) was used to construct the chemical structures
for the molecular simulations. The crystal structure of 6-6 was regarded as the starting
conformation of the investigated compounds. The molecules were assigned
Gasteiger-Hückel charges and minimized by the Tripos force field when convergence
reached 0.001 kcal/mol/Å.
4.4.1. DFT calculation
Representative compounds 6-6, 6-12 and 6-22 were chosen for DFT geometry
optimization by the SCF method using the B3LYP function with a basis set of
6-31G(d, p) to describe their molecular properties. Gaussian03 was used to perform
the calculations [36]. The optimized molecular structures by Sybyl were used as the
initial conformations. All computations were conducted for the ground states of these
molecules as singlet states. All of the convergent precisions used were the system’s
default values.
4.4.2. Molecular docking using FlexX
The crystal structure of SARS-CoV Mpro in complex with inhibitor (pdb code 2AMD)
was retrieved from the protein databank. All water molecules were removed, and
hydrogen atoms were added in the standard geometry. Any amino acid residue within
6.5 Å of the location of the original inhibitor was considered to be in the binding
pocket. Cscore calculation was enabled and set to serial mode.
Acknowledgment
This work was financially supported by the Natural Science Foundation of China (No.
21672114). This work was also dedicated to 100th anniversary of Nankai University.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.doi.org/XXXXXX.
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Fig. 1 Examples of industrial or bioactive thioethers and bioactive compounds
containing pyrimidine or triazine ring.
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Fig. 2 Crystal structure of compound 6-6.
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100
80
60
40
20
0
2.5
3.0
3.5
4.0
log[6-5]
Fig. 3 Inhibition curve of compound 6-5 against SARS-CoV Mpro (Concentration was
expressed in nM).
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