Design, synthesis and bioactivities of phenithionate analogues or derivatives for anti-schistosomiasis

Article abstract of DOI:10.1039/c7md00590c

A novel series of phenithionate analogues or derivatives were designed and synthesized using phenithionate as the lead compound, and their bioactivities were studied. Their structures were confirmed by ¹H NMR, ¹³C NMR, HR-ESI-MS, and elemental analysis, respectively. The results of in vitro inhibitory activity measurement proved that compounds 5a, 5c, 5g, 5i, 5m and 5o had a better inhibitory effect on larva and imago schistosoma. Among them, the inhibitory activity of compound 5i for larva schistosoma was IC₅₀ = 5.21 ± 0.04 μg mL^-1, and for imago schistosoma it was IC₅₀ = 6.35 ± 0.08 μg mL^-1. Moreover, the experimental results of in vivo anti-schistosomiasis activity measurement showed that they had good anti-schistosomiasis activity. Therefore, these compounds had better drugability.

Full text of DOI:10.1039/c7md00590c

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Design, synthesis and bioactivities of phenithionate analogues or
derivatives for anti-Schistosomiasis
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Shiyang Zhou1,2, Gangliang Huang*,1,2
1. College of Chemistry, Chongqing Normal University, Chongqing 401331,China;
2. Engineering Research Center of Ministry of Education for Biotechnology of Active
Substances in Chongqing Normal University,Chongqing 401331,China
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Eꢀmail: huangdoctor226@163.com
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Abstract: A series novel of phenithionate analogues or derivatives were designed and synthesized using
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phenithionate as the lead compound, and their bioactivities were studied. Their structures were confirmed by H
NMR, 13C NMR, HRꢀESIꢀMS, and elemental analysis, respectively. The results of inhibitory activity in vitro
proved that compounds 5a, 5c, 5g, 5i, 5m and 5o had better inhibitory effect on larva and imago schistosoma.
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Among them, the inhibitory activity of compound 5i to larva schistosoma was IC50=5.21 0.04ꢁg/mL, and to
±
imago schistosoma was IC50=6.35±0.08ꢁg/mL. Moreover, the experimental results of antiꢀSchistosomiasis activity
in vivo showed that they had good antiꢀSchistosomiasis activity. Therefore, these compounds had better
drugability.
Keyword: Phenithionate, design, synthesis, bioactivities
19 1. Introduction
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Parasitic diseases spread widely around the world. They are a common disease, especially in tropical and
subtropical developing countries. Some parasitic diseases can develop into epidemics in a region1ꢀ5. When parasitic
diseases are prevalent, they have a serious impact on the society and economy of the region. Schistosomiasis is the
most prevalent parasitic disease in the world, which is the most harmful to people's health. According to the World
Health Organization (WHO), schistosomiasis is endemic in 76 countries and regions, and there are about 200
million schistosomiasis patients, and 500ꢀ600 million people are threatened by the infection. Schistosomiasis is a
parasitic disease caused by Schistosoma mansoni parasitic in human veins6ꢀ10. There are three species of
Schistosoma mansoni parasitic on human body, including Schistosoma haematobium (S. haematobium),
Schistosoma mansoni (S. mansoni) and Schistosoma japonicum (S. japonicum)11ꢀ14. Among the three species,
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Schistosoma japonicum is the most widely distributed, mainly distributed in China, Japan, Malaysia, Indonesia and
other countries15ꢀ17. Schistosoma japonicum not only can cause acute or chronic enteritis, cirrhosis, severe diarrhea,
anemia and emaciation, but also can cause great harm to livestock. Chemical treatment of Schistosoma japonicum
happened in 1918, antimony potassium tartrate injection was used for the treatment of Schistosoma japonicum, and
achieved good treatment effect, but it had high toxicity. There were adverse reactions to lifeꢀthreatening by clinical
findings if people had the longer course of treatment with this medicine18ꢀ21. The treatment of Schistosoma
japonicum with this kind of antimony medicine is very toxic and must be intravenously injected15ꢀ17. It is rarely
used in clinic, and even some countries have banned it. So looking for Schistosoma japonicum agent drugs in the
treatment of nonꢀantimony is necessary, the first nonꢀantimony drug nithiocyanamine and derivative nitroscanate
were shown in Fig. 1. In 1975, nithiocyanamine was designed and synthesized, and it was a broadꢀspectrum
anthelminthics. Nithiocyanamine had a significant role in the killing of Schistosoma japonicum. The mechanism
was that the body tricarboxylic acid cycle metabolism was disturbed, so, lack of energy supply happened, and this
ultimately led to cell death22ꢀ25. For the treatment of various types of Schistosoma japonicum clinically, subsequent
clinical manifestations of slow metabolism can cause accumulation of poisoning, about 4%ꢀ8% patients showed
jaundice, transaminases and other side effects, so it was not used clinically. Nithiocyanamine derivative
nitroscanate also has obvious antiꢀSchistosoma japonicum effect and toxicity is slightly lower than nithiocyamine26.
The study on the structure and activity relationship shows that the isothiocyanate is not only a pharmacophore, but
also a toxic group. Therefore, it is also possible to resist the effect of Schistosoma japonicum by transforming the
cyano group into amino carbamate groups. The modified derivative of diphenyl ester reduced the toxicity greatly.
In this research, we modified the structure by replacing the linked nitrogen atom with oxygen atom and sulfur atom
with the principle of bioisosteres. The nitro phenithionate benzene ring also was replaced with trifluoromethyl and
trichloromethyl groups. The end of benzene ring was modified with hydroxyl and acetylamino groups, while its
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pharmacophore thioamide was retained (Fig.
2 and Scheme 1). The modified compounds had good
antiꢀSchistosoma japonicum effect, and some of them had better insect resistance than phenyl nitrate. According to
the structureꢀactivity relationships (SAR) of the compounds, we have retained the basic framework. A new group
has been introduced on the basis of phenithionate to change the logP and pKa, which affects the activity of drug.
When ꢀCCl3 and ꢀOH were introduced to benzene ring, the activity was very high. In addition, the connecting
atoms between benzene rings also have certain effects on the activity of drug. When the connecting atoms are N
and O, their activity is relatively high, because these atoms can form hydrogen bond with the receptor
macromolecule, which can enhance the activity of drug.
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Fig. 1. The structures of antiꢀSchistosomiasis drugs.
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Fig. 2. Design of phenithionate analogues or derivatives.
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Scheme 1. The synthesis of phenithionate analogues or derivatives.
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2. Results and discussion
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2.1. Design and synthesis of phenithionate analogues or derivatives
Based on phenithionate esters as a lead compound, a series of novel antiꢀSchistosomiasis drugs were designed,
and their structures were modified by the principle of biological electron exclusion (Fig. 2). In terms of drug
structure, these compounds have the same spatial structure as phenithionate esters, so the possibility of drug
formation is relatively large. In the structural modification, the basic framework of the compound was retained,
and the amino group with the same antiꢀSchistosomiasis efficacy was retained. At the same time, the substitution
of phenyl ester for N atom between benzene rings and substituent on benzene ring have been modified. In the
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process of structural design, N, O, S atoms were selected, and the substituent R1 in the benzene ring increased ꢀCF3
and ꢀCCl3, and R2 selected ꢀOH and ꢀNHCOCH3. Such structural design, mainly taking into account the
substituents on the benzene rings and connecting atoms will have an impact on the logP, pKa and the interaction
between drugs and receptors, so as to achieve the purpose of affecting the efficacy. In the synthesis process, a total
of two design schemes (Fig. 3). The target product was obtained by two steps of condensation and addition
(Scheme 2). Although the steps of the two schemes are different in sequence, the total yield of target and the
difficulty of operation are different. If the synthesis route is first condensed and then added, this route will have
many side reactions in the condensation process, the intermediates are difficult to handle, and in addition, the yield
is low due to the larger steric hindrance. The specific operation route scheme can be seen (Scheme 1), the addition
reaction with THF as solvent, AlCl3 as catalyst in refluxing for 8 hours to complete the first step reaction;
condensation reaction with toluene as solvent, anhydrous sodium carbonate as acid binding agent and anti reflux
should be 12 h can reach the synthesis of the target product superiority is a simple operation, mild reaction
conditions, the target product yield high yield characteristics, 80.5%ꢀ93.4%.
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HO
R2
H
N
N=C=S
O
2a-2b
S
THF AlCl3 reflux 8 h
Scheme 1
X
X
R2
3a-3f
1a-1c
Scheme 2
Toluene Na2CO3
reflux 12 h
R1
Cl
Toluene Na2CO3
reflux 16 h
R1
Cl
4a-4c
4a-4c
HO
R2
H
R1
N=C=S
R1
N
O
2a-2b
S
X
X
R2
THF AlCl3 reflux 12 h
6a-6l
5a-5r
R1:-NO2,-CF3,-CCl3
R2:-OH,-NHCOCH3
X:NH,O,S
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Fig. 3. The synthetic route of target compounds.
2.2. The biological activities
2.2.1. The inhibitory activity to anti-Schistosomiasis in vitro
Before studying the inhibitory activity of target compounds in vitro, we first studied the physical and
chemical properties of phenithionate analogues or derivatives, namely the lipid/water partition coefficient (logP)
and dissociation constant (pKa), which might affect their activity (Table 1). As could be seen from Tabel 1, these
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compounds had good fatꢀsoluble,and the logP was from 4.32 to 7.43. The higher the partition coefficient of lipid
and water was, the better the absorption of drugs was,and the activity was increased.In addition,from the
experimental results,it could also be seen that the compounds could be digested and absorbed by the small
intestine,and the pKa was from 8.99 to 9.24. The difference in the pKa of drug affected the effective time because
the compounds needed to be digested and absorbed by the small intestine,and the effective time would be slightly
delayed.After the completion of logP and pKa studies, we focused on the antiꢀSchistosomiasis in vitro. In the
experiment, Larva and Imago Schistosomiasis were used as the inhibitory targets, and the inhibitory activity was
measured by semi inhibitory concentration (IC50). Finally, the anti Imago Schistosoma haematobium (S.
haematobium), Schistosoma mansoni (S. mansoni) and Schistosoma japonicum (S. japonicum) in vitro (Fig. 4).
The results showed that the compounds 5a, 5c, 5g, 5i, 5m and 5o had high antiꢀSchistosomiasis activity, especially
for Schistosoma japonicum (S. japonicum).
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Table 1. The inhibitory activity in vitro and some physicoꢀchemical properties.
IC50±SD（ꢁg/mL）
Compounds
R1
R2
X
logP
pKa
Larva
Imago
ꢀNO2
ꢀNO2
ꢀNO2
ꢀNO2
ꢀNO2
ꢀNO2
ꢀCF3
ꢀCF3
ꢀCF3
ꢀCF3
ꢀCF3
ꢀCF3
ꢀCCl3
ꢀCCl3
ꢀCCl3
ꢀOH
NH
NH
O
4.54
4.32
5.01
4.81
5.25
5.06
6.04
5.32
6.11
5.41
6.66
5.96
6.79
6.07
6.84
9.22
9.04
9.23
9.04
9.21
8.99
9.23
9.05
9.23
9.03
9.22
9.02
9.23
9.01
9.24
6.42±0.12
8.22±0.48
6.38±0.09
8.24±0.33
8.05±0.15
15.22±0.37
8.02±0.12
14.11±0.51
5a
5b
5c
5d
5e
5f
ꢀNHCOCH3
ꢀOH
ꢀNHCOCH3
ꢀOH
O
S
15.67±0.58 30.54±0.39
16.66±0.54 31.20±0.38
ꢀNHCOCH3
ꢀOH
S
NH
NH
O
5.43±0.11
11.20±1.02 20.02±1.28
5.21+0.04 6.35±0.08
6.68±0.09
5g
5h
5i
ꢀNHCOCH3
ꢀOH
5j
ꢀNHCOCH3
ꢀOH
O
10.37±0.89 17.29±1.10
16.56±0.83 31.86±0.98
19.01±0.82 33.69±0.96
S
5k
5l
ꢀNHCOCH3
ꢀOH
S
NH
NH
O
7.35±0.26
11.60±1.20 20.11±1.34
7.98±0.41 13.55±0.46
12.39±0.35
5m
5n
5o
ꢀNHCOCH3
ꢀOH
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ꢀCCl3
ꢀCCl3
ꢀCCl3
ꢀNHCOCH3
O
S
S
6.16
9.02
9.22
9.01
——
10.19±1.01 18.29±1.21
21.09±1.15 37.28±1.19
23.54±1.22 40.39±1.31
5p
5q
ꢀOH
7.43
6.71
——
ꢀNHCOCH3
——
5r
Blank control
（DMSO）
Phenithionate
Praziquantel
/
/
6.53±0.13
6.33±0.11
8.21±0.21
7.98±0.18
——
——
——
——
——
——
1
100
90
80
70
60
50
40
30
20
10
0
S.haematobium
S.mansoni
S.japonicum
5a
5c
5g
5i
5m
5o
2
3
Fig. 4. The inhibition rate.
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2.2.2. The anti-Schistosomiasis in vivo
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In the course of the bioactivity study,compounds 5a, 5c, 5g, 5i, 5m and 5o with high inhibitory activity and low
acute toxicity and tested in vivo (Tables 2 and 3). The oral dose were 25mg.kgꢀ1.dꢀ1 and 50mg.kgꢀ1.dꢀ1, and the
antiꢀSchistosomiasis was observed in different times. Phenithionate and praziquantel were used as the positive
reference substance. The inhibitory activity experimental results showed that these two compounds had fast
effective time and good antiꢀSchistosomiasis activity. And acute toxicity ecperimental results showed that these
four compounds had low acute toxicity. In Table 3, for the common antiꢀSchistosomiasis drugs, phenithionate was
LD50=310.5±4.1mg/kg and praziquantel was LD50>500mg/kg. Compared with commonly used
antiꢀSchistosomiasis drugs, compounds 5a and 5c, LD50 was smaller (280.1±3.2mg/kg and 275.3±2.6mg/kg), and
the acute toxicity was higher. For other compounds 5g, 5i, 5m and 5o, LD50 was greater than 500mg/kg, this
compounds belonged to low toxicity or nonꢀtoxicity substances.
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Table 2. The inhibition activity in vivo.
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Time
Compounds
Dosage of drugs(mg.kgꢀ1.dꢀ1)
1d
+++ +++ +++ ++
+++ ++ ++
+++ +++ +++ ++
2d
3d
4d
5d
++
6d
7d
8d
9d
25
50
25
50
25
50
25
50
25
50
25
50
25
+
—
+
+
—
+
—
—
+
+
—
—
5a
++
—
—
+++ ++
+++ +++ ++
+++ ++
+++ +++ ++
+++ ++
++
+
+
+
—
—
—
—
—
—
5c
5g
5i
++
—
++
—
—
—
+
—
+
—
—
—
—
—
—
—
—
+
—
—
+
—
—
—
+++
—
+++
+++
++
++
++
++
+
+
+
5m
5o
+++ +++ ++
++
+
+
—
—
+
—
+++
—
+++
+++
+
+
+++ +++ ++
++
+
—
—
—
Blank control
（DMSO）
Phenithionate
Praziquantel
25
25
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++ +++ +++ ++
+++ +++ +++ ++
++
++
+
+
—
—
—
—
—
—
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2
Table 3. The median lethal dose (LD50).
Compounds
LD50±SD（mg/kg）
280.1±3.2
275.3±2.6
>500
5a
5c
5g
>500
5i
>500
5m
>500
5o
Phenithionate
Praziquantel
3
310.5±4.1
>500
4
5
3. Conclusion
In general, we reported the design and synthesis of a class of antiꢀSchistosomiasis compounds, which was simple,
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efficient, and had high yield. All the newly synthesized compounds had good inhibitory activity and low acute
toxicity. Among them, it showed by the antiꢀSchistosomiasis experiments in vitro that compounds 5a, 5c, 5g, 5i,
5m and 5o had good antiꢀSchistosomiasis activity, especially for Schistosoma japonicum (S. japonicum). It also
showed by the experiments in vivo that compounds 5a, 5c, 5g, 5i, 5m and 5o had good antiꢀSchistosomiasis
activity and low acute toxicity, especially the compounds 5g and 5i.
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4. Experimental
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4.1. Synthesis of compounds 3a-3f
The 4ꢀisothiocyanatoaniline 1a (0.10 mol) and hydroquinone 2a (0.10 mol) were place in a 250 mL round
bottom flask.100mL of tetrahydrofuran (THF) was added as a solvent.Under constant pressure conditions,0.01mol
of aluminium trichloride was addtiion as a reaction catalyst.After completion of addition, the reaction was refluxed
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for
8 h. After completion of the reflux,the filtrate was filtered while it was hot.The filtrate was
collected,cooled,allowed to stand for 12 h, and then filtered and dried in vacuo to give the crude product of
compound 3a. The crude 3a product was recrystallized in toluene, filtered and dried in vacuo to give pure product
as a white crystal. General experimental method was used for the synthesis of compounds 3b-3f.
4.2. A general method for all titled analogues or derivatives 5a-5r
0.10 mol of compound 3a, 0.06 mol of anhydrous sodium carbonate,and 0.10 mol of compound 4a were placed
in a 500 mL round bottom flask.200 mL toluene was added as solvent. Under magnetic stirring, the reaction was
heated and refluxed for 12 h. When the feflux was completed, the filtrate was filtered while it was hot. The filtrate
was collected, cooled, allowed to stand for 24 h, and then filtered and dried in vacuo to give the crude product of
compound 5a. The crude 5a product was recrystallized in toluene, filtered and dried in vacuo to give pure product
as a white crystal. General experimental method was used for the synthesis of compounds 5b-5r.
Oꢀ(4ꢀhydroxyphenyl) (4ꢀ((4ꢀnitrophenyl)amino)phenyl)carbamothioate（5a）：yield 90.1%；m.p. 163ꢀ165℃；1H
NMR（300MHz，DMSO）δ：9.46（s,1H,ꢀOH）,8.39(s,1H,ꢀNHꢀ),8.03(m,2H,PhꢀH),7.44(m,2H,PhꢀH),7.37
(m,2H,PhꢀH),7.25(m,2H,PhꢀH),6.94(m,2H,PhꢀH),6.70(m,2H,PhꢀH);
13C
NMR (75MHz,
m/z:
DMSO)
calcd
δ:
151.1,148.5,148.1,145.0,138.4,137.4,127.4,126.6,124.8,122.0,120.6,117.3;HRꢀESIꢀMS
for
C19H15N3O4S｛［M+H］+｝381.0785,found 381.4060;Anal.calcd forC19H15N3O4S:C, 59.83; H, 3.96; N, 11.02; O,
16.78; S, 8.41;found:C, 59.84; H, 3.96; N, 11.01; O, 16.79; S, 8.40%.
Oꢀ(4ꢀacetamidophenyl) (4ꢀ((4ꢀnitrophenyl)amino)phenyl)carbamothioate (5b): yield 89.3%；m.p. 171ꢀ173℃；1H
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NMR（300MHz，DMSO）δ：9.86(s,1H,ꢀꢀNHꢀ);8.39(s,1H,ꢀꢀNHꢀ);8.03(m,2H,PhꢀH),7.44(m,2H,PhꢀH),7.43
(m,2H,PhꢀH),7.37(m,2H,PhꢀH),7.25(m,2H,PhꢀH),6.86(m,2H,PhꢀH),2.06(s,3H,ꢀCH3);13C NMR（75MHz，DMSO）
δ ： 168.9,151.1,148.5,145.0,138.4,137.4,131.1,127.4,126.6,124.8,123.0,122.0,120.6,116.1,24.0;HRꢀESIꢀMS m/z:
calcd for C21H18N4O4S｛［M+H］+｝422.1048 ,found 422.4591;Anal.calcd forC21H18N4O4S:C, 59.71; H, 4.29; N,
13.26; O, 15.15; S, 7.59;found:C, 59.70; H, 4.30; N, 13.26; O, 15.16; S, 7.58%.
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Oꢀ(4ꢀhydroxyphenyl) (4ꢀ(4ꢀnitrophenoxy)phenyl)carbamothioate (5c): yield 92.6%；m.p. 166ꢀ168℃；1H NMR
（300MHz，DMSO）δ：9.46（s,1H,ꢀOH）,8.27(m,2H,PhꢀH),7.40(m,2H,PhꢀH),7.21(m,2H,PhꢀH),6.96
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(m,2H,PhꢀH),6.94(m,2H,PhꢀH),6.70(m,2H,PhꢀH);
13C
NMR
（
75MHz
，
DMSO
）
δ
：
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163.1,151.1,150.2.148.1,145.0,141.0,130.2,126.2,124.6,117.3,116.2,115.7;HRꢀESIꢀMS
m/z:
calcd
for
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14
15
16
17
18
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21
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23
24
25
26
27
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C19H14N2O5S｛［M+H］+｝382.0622,found 382.3902;Anal.calcd forC19H14N2O5S:C, 59.68; H, 3.69; N, 7.33; O,
20.92; S, 8.38;found:C, 59.67; H, 3.69; N, 7.34; O, 20.93; S, 8.37%.
Oꢀ(4ꢀacetamidophenyl) (4ꢀ(4ꢀnitrophenoxy)phenyl)carbamothioate (5d): yield 89.9%；m.p. 176ꢀ178℃；1H NMR
（300MHz，DMSO）δ：9.86(s,1H,ꢀꢀNHꢀ);8.27(m,2H,PhꢀH),7.43(m,2H,PhꢀH),7.40(m,2H,PhꢀH),7.21
(m,2H,PhꢀH),6.96(m,2H,PhꢀH),6.86(m,2H,PhꢀH),2.06(s,3H,ꢀCH3);13C NMR （ 75MHz ， DMSO ） δ ：
168.9,163.1,151.1,150.2,145.0,141.0,131.1,130.2,126.2,124.6,123.0,116.2,116.1,115.7,24.0;HRꢀESIꢀMS
m/z:
+
calcd for C21H17N3O5S｛［M+H］｝423.0891,found 423.4431;Anal.calcd forC21H17N3O5S:C, 59.57; H, 4.05; N, 9.92;
O, 18.89; S, 7.57;found:C, 59.58; H, 4.05; N, 9.91; O, 18.90; S, 7.56%.
Oꢀ(4ꢀhydroxyphenyl) (4ꢀ((4ꢀnitrophenyl)thio)phenyl)carbamothioat(5e): yield 87.3%；m.p. 170ꢀ172℃；1H NMR
（300MHz，DMSO）δ：9.46（s,1H,ꢀOH）,7.92(m,2H,PhꢀH),7.75(m,2H,PhꢀH),7.67m,2H,PhꢀH),7.29
(m,2H,PhꢀH),6.94(m,2H,PhꢀH),6.70(m,2H,PhꢀH);13C
NMR
（
75MHz
，
DMSO
）
δ
：
151.1,148.1,146.4,145.0,141.8,135.6,131.7,131.0,128.3,125.7,123.4,117.3;HRꢀESIꢀMS
m/z:
calcd
for
C19H14N2O4S2｛［M+H］+｝398.0397,found 398.4511;Anal.calcd forC19H14N2O4S2: C, 57.27; H, 3.54; N, 7.03; O,
16.06; S, 16.09;found: C, 57.26; H, 3.54; N, 7.04; O, 16.07; S, 16.08%.
Oꢀ(4ꢀacetamidophenyl) (4ꢀ((4ꢀnitrophenyl)thio)phenyl)carbamothioate (5f): yield 85.6%；m.p. 184ꢀ186℃；1H
NMR（300MHz，DMSO）δ：9.86(s,1H,ꢀꢀNHꢀ);7.92(m,2H,PhꢀH),7.75(m,2H,PhꢀH),7.67(m,2H,PhꢀH),7.43
(m,2H,PhꢀH),7.29(m,2H,PhꢀH),6.86(m,2H,PhꢀH),2.06(s,3H,ꢀCH3);13C NMR （ 75MHz ， DMSO ） δ ：
168.9,151.1,146.4,145.0,141.8,135.6,131.7,131.1,131.0,128.3,125.7,123.4,123.0,116.1,24.0;HRꢀESIꢀMS
m/z:
calcd for C21H17N3O4S2｛［M+H］+｝439.0660,found 439.5042;Anal.calcd for C21H17N3O4S2: C, 57.39; H, 3.90;
N, 9.56; O, 14.56; S, 14.59;found: C, 57.37; H, 3.91; N, 9.57; O, 14.57; S, 14.58%.
Oꢀ(4ꢀhydroxyphenyl) (4ꢀ((4ꢀ(trifluoromethyl)phenyl)amino)phenyl)carbamothioate (5g): yield 92.1% ； m.p.
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152ꢀ154℃；H NMR（300MHz，DMSO）δ：9.46（s,1H,ꢀOH）,7.79(s,1H,ꢀNHꢀ)7.43(m,2H,PhꢀH),7.37(m,2H,PhꢀH),
7.31(m,2H,PhꢀH),7.25(m,2H,PhꢀH),6.94(m,2H,PhꢀH),6.70(m,2H,PhꢀH);13C NMR （ 75MHz ， DMSO ） δ ：
3
151.1,148.1,145.7,145.0,138.4,127.4,126.6,126.0,124.1,123.5,120.6,117.3;HRꢀESIꢀMS
m/z:
calcd
for
+
4
C20H15F3N2O2S｛［M+H］｝404.0802,found 404.4071;Anal.calcd forC20H15F3N2O2S: C, 59.40; H, 3.74; F, 14.09;
N, 6.93; O, 7.91; S, 7.93;found: C, 59.41; H, 3.74; F, 14.08; N, 6.93; O, 7.92; S, 7.92%.
5
6
Oꢀ(4ꢀacetamidophenyl) (4ꢀ((4ꢀ(trifluoromethyl)phenyl)amino)phenyl)carbamothioate (5h): yield 90.4%； m.p.
157ꢀ159℃；1H NMR（300MHz，DMSO）δ：9.86(s,1H,ꢀꢀNHꢀ);7.79(s,1H,ꢀꢀNHꢀ);7.43(m,4H,PhꢀH),7.37
(m,2H,PhꢀH),7.31(m,2H,PhꢀH),7.25(m,2H,PhꢀH),6.86(m,2H,PhꢀH),2.06(s,3H,ꢀCH3);13C NMR（75MHz，DMSO）
δ ： 168.9,151.1,145.7,145.0,138.4,131.1,127.4,126.6,126.0,124.1,123.5,123.0,120.6,116.1,24.0;HRꢀESIꢀMS m/z:
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
calcd for
C22H18F3N3O2S｛［M+H］+｝445.1073,found 445.4601;Anal.calcd forC22H18F3N3O2S:C, 59.32; H,
4.07; F, 12.79; N, 9.43; O, 7.18; S, 7.20;found:C, 59.33; H, 4.06; F, 12.77; N, 9.43; O, 7.19; S, 7.21%.
Oꢀ(4ꢀhydroxyphenyl) (4ꢀ(4ꢀ(trifluoromethyl)phenoxy)phenyl)carbamothioate (5i): yield 93.4%；m.p. 170ꢀ172℃；
1H NMR（300MHz，DMSO）δ：9.46（s,1H,ꢀOH）,7.43(m,2H,PhꢀH),7.40(m,2H,PhꢀH),7.26(m,2H,PhꢀH),
6.96(m,2H,PhꢀH),6.94(m,2H,PhꢀH),6.70(m,2H,PhꢀH);13C
NMR
（
75MHz
，
DMSO
m/z:
）
δ
：
160.3,151.1,150.2,148.1,145.0,130.2,126.9,126.2,124.122.0,117.3,115.7;HRꢀESIꢀMS
calcd
for
C20H14F3NO3S｛［M+H］+｝405.0645,found 405.3913;Anal.calcd forC20H14F3NO3S:C, 59.26; H, 3.48; F, 14.06;
N, 3.46; O, 11.84; S, 7.91;found:C, 59.27; H, 3.49; F, 14.05; N, 3.46; O, 11.83; S, 7.90%.
Oꢀ(4ꢀacetamidophenyl) (4ꢀ(4ꢀ(trifluoromethyl)phenoxy)phenyl)carbamothioate(5j): yield 90.2%；m.p. 172ꢀ174℃；
1H NMR（300MHz，DMSO）δ：9.86(s,1H,ꢀꢀNHꢀ);7.43(m,4H,PhꢀH),7.40(m,2H,PhꢀH),7.26(m,2H,PhꢀH),
6.96(m,2H,PhꢀH),6.86(m,2H,PhꢀH),2.06(s,3H,ꢀCH3);13C
168.9,160.3,151.1,150.2,145.0,131.1,130.2,126.9,126.2,124.1,123.0,122.0,116.1,115.7,24.0;HRꢀESIꢀMS
calcd for
NMR
（
75MHz
，
DMSO
）
δ
：
m/z:
C22H17F3N2O3S｛［M+H］+｝446.0913,found 446.4443;Anal.calcd forC22H17F3N2O3S:C, 59.19; H,
3.84; F, 12.77; N, 6.27; O, 10.75; S, 7.18;found:C, 59.18; H, 3.85; F, 12.77; N, 6.26; O, 10.75; S, 7.19%.
Oꢀ(4ꢀhydroxyphenyl) (4ꢀ((4ꢀ(trifluoromethyl)phenyl)thio)phenyl)carbamothioate(5k): yield 90.2% ； m.p.
173ꢀ175℃；1H NMR（300MHz，DMSO）δ：9.46（s,1H,ꢀOH）,7.75(m,2H,PhꢀH),7.45(m,2H,PhꢀH),7.29
(m,2H,PhꢀH),7.19(m,2H,PhꢀH),6.94(m,2H,PhꢀH),6.70(m,2H,PhꢀH);13C NMR （ 75MHz ， DMSO ） δ ：
151.1,148.1,145.0,139.0,135.6,131.7,131.5,129.5,128.3,125.7,124.1,117.3;HRꢀESIꢀMS
m/z:
calcd
for
+
C20H14F3NO2S｛［M+H］｝421.0417,found
421.4523;Anal.calcd forC20H14F3NO2S2:C, 57.00; H, 3.35; F, 13.52;
2
N, 3.32; O, 7.59; S, 15.21;found:C, 57.01; H, 3.36; F, 13.50; N, 3.32; O, 7.58; S, 15.22%.
Oꢀ(4ꢀacetamidophenyl) (4ꢀ((4ꢀ(trifluoromethyl)phenyl)thio)phenyl)carbamothioate (5l): yield 90.2% ； m.p.
10

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173ꢀ175℃；1H NMR（300MHz，DMSO）δ：9.86(s,1H,ꢀꢀNHꢀ);7.75(m,2H,PhꢀH),7.45(m,2H,PhꢀH),7.43
(m,2H,PhꢀH),7.29(m,2H,PhꢀH),7.19(m,2H,PhꢀH),6.86(m,2H,PhꢀH),2.06(s,3H,ꢀCH3);13C NMR（75MHz，DMSO）
δ ： 168.9,151.1,145.0,139.0,135.6,131.7,131.5,131.1,129.5,128.3,125.7,124.1,123.0,116.124.0;HRꢀESIꢀMS m/z:
3
4
calcd for C22H17F3N2O2S2｛［M+H］+｝ 462.0685,found
462.5053;Anal.calcd forC22H17F3N2O2S2:C, 57.13; H,
5
3.71; F, 12.32; N, 6.06; O, 6.92; S, 13.86;found:C, 57.12; H, 3.72; F, 12.32; N, 6.06; O, 6.91; S, 13.87%.
Oꢀ(4ꢀhydroxyphenyl) (4ꢀ((4ꢀ(trichloromethyl)phenyl)amino)phenyl)carbamothioate (5m): yield 91.3% ； m.p.
165ꢀ167℃；1H NMR（300MHz，DMSO）δ：9.46(s,1H,ꢀꢀOH);7.79(s,1H,ꢀꢀNHꢀ);7.78(m,2H,PhꢀH),7.37
(m,2H,PhꢀH),7.31(m,2H,PhꢀH),7.25(m,2H,PhꢀH),6.94(m,2H,PhꢀH),6.70m,2H,PhꢀH);13C NMR（75MHz，DMSO）
6
7
8
9
δ ： 151.1,148.1,145.0,144.2,138.4,133.5,127.5,127.4,126.6,123.1,120.6,117.3,97.8;HRꢀESIꢀMS m/z: calcd for
+
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
C20H15Cl3N2O2S｛［M+H］｝ 451.9921,found
453.7623;Anal.calcd for C20H15Cl3N2O2S: C, 52.94; H, 3.33; Cl,
23.44; N, 6.17; O, 7.05; S, 7.07;found: C, 52.93; H, 3.32; Cl, 23.43; N, 6.17; O, 7.05; S, 7.08%.
Oꢀ(4ꢀacetamidophenyl) (4ꢀ((4ꢀ(trichloromethyl)phenyl)amino)phenyl)carbamothioate (5n): yield 91.3%； m.p.
165ꢀ167℃；1H NMR（300MHz，DMSO）δ：9.86(s,1H,ꢀꢀNHꢀ),7.79(s,1H,ꢀꢀNHꢀ),7.78(m,2H,PhꢀH),7.43
(m,2H,PhꢀH),7.37(m,2H,PhꢀH),7.31(m,2H,PhꢀH),7.25(m,2H,PhꢀH),6.86(m,2H,PhꢀH),2.06(s,3H,ꢀCH3);13C NMR
（75MHz，DMSO）δ：168.9,151.1,145.0,144.2,138.4,133.5,131.1,127.5,127.4,126.6,123.1,123.0,120.6,116.1,
97.8,24.0;HRꢀESIꢀMS m/z: calcd for C22H18Cl3N3O2S｛［M+H］+｝ 493.0184,found
494.8152;Anal.calcd for
C22H18Cl3N3O2S:C, 53.40; H, 3.67; Cl, 21.49; N, 8.49; O, 6.47; S, 6.48;found:C, 53.41; H, 3.66; Cl, 21.49; N, 8.49;
O, 6.48; S, 6.47%.
Oꢀ(4ꢀhydroxyphenyl) (4ꢀ((4ꢀ(trichloromethyl)phenoxy)phenyl)carbamothioate (5o): yield 92.3%；m.p. 177ꢀ179℃；
1H NMR（300MHz，DMSO）δ：9.46(s,1H,ꢀꢀOH);7.78(m,2H,PhꢀH),7.40(m,2H,PhꢀH),7.26(m,2H,PhꢀH),
6.96(m,2H,PhꢀH),6.94(m,2H,PhꢀH),6.70m,2H,PhꢀH);13C
NMR
（
75MHz,
DMSO
）
δ
：
158.8,151.1,150.2,148.1,145.0,137.1,130.2,126.3,126.2,121.6,117.3,115.7,97.8;HRꢀESIꢀMS m/z: calcd for
+
C20H14Cl3NO3S｛［M+H］｝ 452.9762,found
454.7463;Anal.calcd for C20H14Cl3NO3S:C, 52.83; H, 3.10; Cl,
23.39; N, 3.08; O, 10.55; S, 7.05;found:C, 52.82; H, 3.10; Cl, 23.38; N, 3.09; O, 10.55; S, 7.06%.
Oꢀ(4ꢀacetamidophenyl) (4ꢀ(4ꢀ(trichloromethyl)phenoxy)phenyl)carbamothioate(5p):yield 92.3%；m.p. 181ꢀ183
1H NMR（300MHz，DMSO）δ：9.86(s,1H,ꢀꢀNHꢀ),7.78(m,2H,PhꢀH),7.43(m,2H,PhꢀH),7.40(m,2H,PhꢀH),
℃；
7.26(m,2H,PhꢀH),6.96(m,2H,PhꢀH),6.86(m,2H,PhꢀH),2.06(s,3H,ꢀCH3);13C NMR （ 75MHz ， DMSO ） δ ：
168.9,158.8,151.1,150.2,145.0,137.1,131.1,130.2,126.3,126.2,123.0,121.6,116.1,115.7,97.8,24.0;HRꢀESIꢀMS m/z:
calcd for C22H17Cl3N2O3S｛［M+H］+｝ 494.0026,found
495.7992;Anal.calcd for C22H17Cl3N2O3S:C,
53.30; H, 3.46; Cl, 21.45; N, 5.65; O, 9.68; S, 6.47;found:C, 53.31; H, 3.45; Cl, 21.45; N, 5.64; O, 9.68; S, 6.48%.
11

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Oꢀ(4ꢀhydroxyphenyl) (4ꢀ((4ꢀ(trichloromethyl)phenyl)thio)phenyl)carbamothioate (5q): yield 84.1% ； m.p.
175ꢀ177
；1H NMR（300MHz，DMSO）δ：9.46(s,1H,ꢀꢀOH);7.75(m,2H,PhꢀH),7.63(m,2H,PhꢀH),7.45
℃
3
(m,2H,PhꢀH),7.29(m,2H,PhꢀH),6.94(m,2H,PhꢀH),6.70m,2H,PhꢀH);13C NMR （ 75MHz ， DMSO ） δ ：
4
151.1,148.1,145.0,142.5,137.5,135.6,131.7,131.1,128.3,127.2,125.6,117.3,97.8;HRꢀESIꢀMS m/z: calcd for
+
5
C20H14Cl3NO2S2｛［M+H］｝ 468.9533,found
470.8072;Anal.calcd for C20H14Cl3NO2S2: C, 51.02; H, 3.00;
6
Cl, 22.59; N, 2.98; O, 6.80; S, 13.62;found: C, 51.03; H, 3.00; Cl, 22.59; N, 2.97; O, 6.81; S, 13.61%.
7
Oꢀ(4ꢀacetamidophenyl) (4ꢀ((4ꢀ(trichloromethyl)phenyl)thio)phenyl)carbamothioate (5r): yield 80.1% ； m.p.
8
189ꢀ191℃
；1H NMR（300MHz，DMSO）δ：9.86(s,1H,ꢀꢀNHꢀ),7.75(m,2H,PhꢀH),7.63(m,2H,PhꢀH),7.45
9
(m,2H,PhꢀH),7.43(m,2H,PhꢀH),7.25(m,2H,PhꢀH),6.86(m,2H,PhꢀH),2.06(s,3H,ꢀCH3);13C NMR（75MHz，DMSO）
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
δ ： 168.9,151.1,145.0,142.5,137.5,135.6,131.7,131.1,128.3,127.2,125.7,123.0,116.1,97.8,24.0;HRꢀESIꢀMS m/z:
+
calcd for C22H17Cl3N2O2S｛［M+H］｝509.9798,found
511.8605;Anal.calcd for C22H17Cl3N2O2S2:C, 51.62;
2
H, 3.35; Cl, 20.78; N, 5.47; O, 6.25; S, 12.53;found:C, 51.63; H, 3.36; Cl, 20.78; N, 5.47; O, 6.24; S, 12.52%.
4.3 Biological activities
4.3.1.Screening of biological activity of compounds in vitro
A total of 100 healthy male mus musculus weighing 20ꢀ22g were selected. Each mus musculus was divided
into two parts and 50 mice after abdominal shaving. One of them was infected with abdominal skin with 260ꢀ280
cercariae of Schistosoma japonicum. The mus musculus were killed after 15 days of infection. Low temperature
Heinz balanced salt solution containing heparin (HBSS solution) was injected through the thoracic aorta,
mesenteric vein and intrahepatic collection of Schistosoma japonicum, and washing with HBSS solution of
toxoplasma gondii 3ꢀ4 after in vitro culture experiments for larva. In addition a mouse with 120ꢀ140 cercariae
infect mus musculus abdominal skin infection, 35 days after the mus musculus were killed, with the same HBSS
solution, and the worms collected for imago culture experiments in vitro. 10% calf serum ꢀRPMI 1640 culture
medium containing penicillin sodium salt (150 IU/mL), streptomycin (150 IU/mL) and amphotericin B (1 ꢁg/mL)
was used to culture Schistosoma japonicum larva and imago worms in vitro. The larva and imago were cultured in
6 hole culture plate, each placed 5 male and female worms, incubated in a constant temperature incubator at 37
℃
with 5% CO2 for 4 h, and then added different concentrations of compounds, and continued to culture for 72 h. The
viability of the insect in 72 h was observed and recorded. When the survival rate was 50%, it was the semi
inhibitory concentration (IC50) of the compound. Using DMSO as blank control experiment, phenithionate and
praziquantel were used as positive control experiment.
4.3.2.Biological activity of compounds anti-Schistosomiasis in vivo
12

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A total of 50 healthy male mus musculus weighing 20ꢀ22g were injected with 120ꢀ140 cercariae of
Schistosoma japonicum to infect the abdominal skin of mus musculus. After 35 days of infection, oral dose of 25
mg.kgꢀ1.dꢀ1 and 50mg.kgꢀ1.dꢀ1 compounds and were administered orally for 5 days. After 5 h of oral administration,
the mus musculus were collected for venous blood, the blood was coagulated and the serum was centrifuged for 15
min, then the serum was drawn out for 3500 r/min. The obtained serum was detected by triple dot ELISA, and the
round spot color was observed. When the round spot showed brown strong positive (+ + +), yellow was positive (+
+), showing slight yellow was weak positive (+), colorless was negative (ꢀ). Using DMSO as blank control
experiment, phenithionate and praziquantel were used as positive control experiment.
3
4
5
6
7
8
9
10
11
12
13
14
15
LIVE SUBJECT STATEMENT:
The reported experiments were in accordance with the standards set forth in the 8th Edition of Guide for the
Care and Use of Laboratory Animals published by the National Academy of Sciences, The National Academies
Press, Washington DC, United States of America. The academic committee of Chongqing Normal University has
approved the experiment.
16 Acknowledgements
17
18
19
20
21
22
23
24
25
26
27
28
29
The project was sponsored by Innovation Team Project of Universities in Chongqing (No. CXTDX201601018).
The Project was also Sponsored by Chongqing Scientific and Technological Innovation Special Project of Social
Undertakings and people’s Livelihood Guarantee(No. cstc2015shmszx80060), Open Foundation Project of
Engineering Research Center of Ministry of Education for Biotechnology of Active Substances(No. 20150408;
AS201605; AS201605; AS201610; AS201608; AS201607), Chongqing University Students’ Training Project of
Innovation and Undertaking (201510637085), Doctoral Program of Chongqing Normal University (No.
12XLB006), and Outstanding Achievements Transformation Project in Chongqing Normal University
(No.15XZH08), China.
At the same time, the project was sponsored by the Scientific Research Foundation for the Returned Overseas
Chinese Scholars, State Education Ministry. The work was also supported by Chongqing Key Research Project of
Basic Science & Frontier Technology (No. cstc2017jcyjBX0012), Scientific and Technological Research Program
of Chongqing Municipal Education Commission (Grant No. KJ1400523), Foundation Project of Chongqing
Normal University (No. 14XYY020), Chongqing General Research Program of Basic Research and Frontier
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Technology (No. cstc2015jcyjA10054), Chongqing Normal University Postgraduate's Research and Innovation
Project (No. YKC17004), Open Foundation Project of Engineering Research Center for Bioactive Substances (No.
3
AS201614), and Chongqing University Students' Training Project of Innovation & Undertaking (No.
4
201610637076), China.
Conflict of Interest
5
6
The authors declare no competing interests.
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