Influence of pyranose and spacer arm structures on phloem mobility and insecticidal activity of new tralopyril derivatives

Article abstract of DOI:10.3390/molecules22071058

Six new conjugates were designed and synthesized by introducing glucose, methyl glucuronate or glucuronic acid moieties on tralopyril. Phytotoxicity and phloem mobility results demonstrated that the introduction of glucose, methyl glucuronate or glucuronic acid moieties can simultaneously solve the tough phytotoxicity problem and phloem mobility transformation of tralopyril. Conjugates 12 and 18 containing the glucuronic acid moiety exhibited higher phloem mobility than conjugates 9, 11, 15 and 17. Conjugates 15, 17 and 18 with methoxymethyl groups on the tralopyril pyrrole nitrogen atom showed activity against Plutella xylostella, while conjugates 9, 11 and 12 with a methene group on the pyrrole N showed no activity. Cabbage roots were incubated in a buffered solution containing conjugates 15, 17 and 18 at 4 mM for 72 h. Only 18 showed systemic insecticidal activity with 100% mortalityagainst P. xylostella, while 15 and 17 showed lower activity andchlorfenapyr showed no activity. The glucuronic acid promoiety imparted more phloem mobility to tralopyril than glucose and methyl glucuronate. The methoxymethyl group bond on the tralopyril skeleton was the key factor in determining the insecticidal activity of the conjugates. A promising systemic proinsecticide containing glucuronic acid and tralopyril moieties was proposed.

Full text of DOI:10.3390/molecules22071058

molecules
Article
Influence of Pyranose and Spacer Arm Structures on
Phloem Mobility and Insecticidal Activity of
New Tralopyril Derivatives
Yao Chen 1,2,3, Zhi Wei Lei 3, Ying Zhang 4, Wen Yang 3, Hui Fang Liu 3, Yu Feng Zhou 3 and
Mao Fa Yang 1,2,
*
1
Institute of Entomology, Guizhou University, Guiyang 550025, Guizhou, China; cheny0221@163.com
Guizhou Provincial Key Laboratory for Agricultural Pest Management of the Mountainous Region,
Guizhou University, Guiyang 550025, Guizhou, China
Guizhou Tea Research Institute, Guizhou Academy of Agricultural Sciences, Guiyang 550006, Guizhou,
China; leizhiwei816@163.com (Z.W.L.); yangwen3409@126.com (W.Y.); 18300865026@163.com (H.F.L.);
gzzhouyf@163.com (Y.F.Z.)
2
3
4
Guizhou Institute of Plant Protection, Guizhou Academy of Agricultural Sciences,
Guiyang 550006, Guizhou, China; zhangying201201@126.com
*
Correspondence: mfyang@gzu.edu.cn; Tel.: +86-851-8385-0050
Received: 22 May 2017; Accepted: 20 June 2017; Published: 25 June 2017
Abstract: Six new conjugates were designed and synthesized by introducing glucose, methyl
glucuronate or glucuronic acid moieties on tralopyril. Phytotoxicity and phloem mobility results
demonstrated that the introduction of glucose, methyl glucuronate or glucuronic acid moieties
can simultaneously solve the tough phytotoxicity problem and phloem mobility transformation of
tralopyril. Conjugates 12 and 18 containing the glucuronic acid moiety exhibited higher phloem
mobility than conjugates
the tralopyril pyrrole nitrogen atom showed activity against Plutella xylostella, while conjugates
11 and 12 with a methene group on the pyrrole N showed no activity. Cabbage roots were incubated
in a buffered solution containing conjugates 15 17 and 18 at 4 mM for 72 h. Only 18 showed systemic
9, 11, 15 and 17. Conjugates 15, 17 and 18 with methoxymethyl groups on
9,
,
insecticidal activity with 100% mortalityagainst P. xylostella, while 15 and 17 showed lower activity
andchlorfenapyr showed no activity. The glucuronic acid promoiety imparted more phloem mobility
to tralopyril than glucose and methyl glucuronate. The methoxymethyl group bond on the tralopyril
skeleton was the key factor in determining the insecticidal activity of the conjugates. A promising
systemic proinsecticide containing glucuronic acid and tralopyril moieties was proposed.
Keywords: glucuronic acid; glucose; tralopyril; chlorfenapyr; phloem mobility
1. Introduction
For various practical reasons insecticides with phloem mobility are preferred for pest species
hidden on unpredictable non-exposed plant parts, such as growing tips, roots, inside leaf deformations,
and galls. However, there are very few existing synthetic insecticides with phloem mobility. Currently,
only spirotetramat can be bidirectionally transported in plants [1]. Attempts have been made to achieve
phloem-mobile insecticides by introducing a carboxyl group [
2,3
], amino acid [ ], or sugar [ ] into
3
4,5
the parent compounds of existing non-phloem-mobile types. Three research groups have focused on
the addition of a sugar moiety [4–9].
In 1995, Hsu et al. described a phloem-mobile pronematicide to solve the inherent incompatibility
between phloem mobility transformation and activity loss by structural modification of pesticides [4].
Two conjugates, hydroxymethyloxamyl glucuronide (JR522) and its methyl ester (JM775), were
successfully synthesized. These conjugates showed more phloem-mobile attributes and greater
Molecules 2017, 22, 1058; doi:10.3390/molecules22071058
www.mdpi.com/journal/molecules

Molecules 2017, 22, 1058
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nematicidal activity than oxamyl alone. However, the applied strict reaction conditions limited
further development of hydroxymethyloxamyl glucuronides.
Since 2011, Xu and his team have made great efforts to improve the phloem mobility of pesticides
through the addition of a sugar group. Yang et al. described the phloem mobility of a conjugate
containing glucose and fipronil (GTF), and they reported that GTF can be reconverted into fipronil
in adult castor bean plants [
proved that the uptake and phloem transport of GTF involved a plant sugar carrier system [
5
]. Subsequent studies on the phloem transport mechanism of GTF
].
6
Yuan et al. synthesized 10 conjugates containing both fipronil and monosaccharide moieties through
an O-glycosidic linkage; phloem mobility test results indicated that eight conjugates show phloem
mobility [
7
]. Lei et al. confirmed that glucose conjugation at the C-2, C-3, C-4, and C-6 positions has a
]. Qin et al. synthesized six conjugates
significant influence on the phloem mobility of GTF conjugates [
8
containing both rotenone or dalpanol and monosaccharide moieties; their phloem systemicity tests
showed that only D-glucose conjugates exhibit phloem transport properties, and phloem mobility is
affected by the parent molecule [9]. The above mentioned studies compared the differences in the
phloem mobility among different monosaccharide moieties, and the effect of glucose positions on
phloem mobility. In 2016, Bonnemain and his team had demonstrated that glutamic acid imparts
more phloem mobility to fenpiclonil than glucose [10]. However, differences in the phloem mobility of
phloem-mobile derivatives among glucose, methyl glucuronate and glucuronic acid promoieties have
not yet been reported.
Chlorfenapyr (Figure 1) is the first commercial pesticide derived from a class of compounds
produced by bacteria and known as halogenated pyrroles [11]. This specific pesticide is a pro-insecticide
with an ethoxymethyl group on the pyrrole N of tralopyril (Figure 1), which is the primary
active compound [12,13]. Modification by ethoxymethyl group introduction is necessary to solve
the intolerable phytotoxicity of tralopyril to the applied plants. Tralopyril uncouples oxidative
phosphorylation in the mitochondria, resulting in the disruption of ATP production and loss of energy.
These processes can lead to cell dysfunction and subsequent death of the organism. A recent study on
phloem-mobile proinsecticides used tralopyril, which is a non-phloem-mobile candidate, to impart
phloem mobility by introducing theanine or glutamic acid moieties [14]. Insecticidal activity test results
indicated that amethoxymethyl group linker on thepyrrole N of the conjugates was dramatically more
favorable to activity than a methene group. However, this structure–activity phenomenon has not yet
been extended to other promoieties such as sugars.
The experimental scheme in the present study can be described as follows: (I) Six new tralopyril
conjugates containing glucose, methyl glucuronate or glucuronic acid moieties were synthesized.
(II) Their phytotoxicities to Fuding white tea seedlings were tested. (III) A phloem mobility test in
Ricinus communis L. seedlings was performed. Their phloem mobility mechanism was predicted via
the “Rule of Five” and a Kleier model. (IV) A poison fodder method was adopted to determine the
insecticidal activity of the conjugates against the third-instar larvae of Plutella xylostella. (V) Their
systemic mobility on cabbage and systemic insecticidal activity against the third-instar larvae of
P. xylostella were evaluated. This study was performed to investigate the influence of different
sugar promoieties on phloem mobility and insecticidal activity. Moreover, the influence of the two
linkage arms (a methene and a methoxymethyl group) between the sugar promoiety and tralopyril on
insecticidal activity was determined.
2. Results and Discussion
2.1. Synthesis and Characterization
Conjugates that share structural similarities should be designed and synthesized in order to
accurately compare the differences in phloem mobility impartedby glucose, methyl glucuronate
and glucuronic acid moieties to tralopyril. Therefore, azides
2 and 4 (Scheme 1), which showed
small structuraldifferences at the C-6 positions of pyranose, were selected and prepared according to

Molecules 2017, 22, 1058
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previously reported procedures [15,16]. A propargyl group was introduced into tralopyril to afford 7
(Scheme 2) and 13 (Scheme 4) as a coupling partner for the subsequent click reactions. Schemes 2–5
show that the terminalalkynes in
7 and 13 were reacted with azide intermediates 2 and 4, respectively,
to afford intermediates 8, 10, 14 and 16 with yields up to 90% via click reactions. The removal of
acetyl groups was performed by the Zemplén procedure. The resulting crude products were purified
by column chromatography to yield the deprotected conjugates 9, 11, 15 and 17 as white solids.
Conjugates 11 and 17 were de-esterified in the presence of KOH in MeOH to afford conjugates 12 and
18 as white solids.
OAc
OAc
O
AcO
AcO
O
N
3
AcO
AcO
OAc
AcO
2
AcO
1
MeO
O
MeO
O
O
O
AcO
AcO
N
3
AcO
AcO
OAc
AcO
AcO
4
3
Scheme 1. Synthesis of Azide Intermediates
2
and
4. Reagents and conditions: azidotrimethylsilane,
SnCl4, CH2Cl2, rt, 2 h.
NC
OAc
NC
Br
Br
O
(1)
Cl
AcO
+
Cl
+
N
3
AcO
Br
N
6
N
CF3
CF3
AcO
H
7
2
5
NC
NC
Br
Br
Cl
Cl
(3)
(2)
N
N
OH
CF3
OAc
CF3
O
O
HO
HO
AcO
AcO
N
N
N
N
N
N
HO
AcO
9
8
Scheme 2. Synthesis of glucose conjugate
9
with a space arm of methene group. Reagents and
5H2O (0.4 equiv), sodium ascorbate (0.8 equiv),
◦
conditions: (1) NaH,◦THF, 65 C, 24 h; (2) CuSO4
·
1:1 t-BuOH/H2O, 60 C, 3 h; (3) 0.05 M NaOMe/MeOH, rt, 0.5 h, Amberlite IR 120H+.
NC
Br
Cl
NC
MeO
O
N
O
Br
CF3
(1)
H CO
3
O
Cl
AcO
AcO
O
+
N
3
AcO
N
N
N
CF3
AcO
AcO
4
N
AcO
7
10
NC
NC
Br
CF3
Br
CF3
Cl
O
Cl
N
N
(3)
O
(2)
H CO
3
HOOC
HO
O
HO
HO
N
N
N
N
HO
N
N
12
11
HO
HO
Scheme 3. Synthesis of methyl glucuronate or glucuronic acid conjugate
methene group. Reagents and conditions: (1) CuSO4
s
with a space arm of
·
5H2O (0.4 equiv), sodium ascorbate (0.8 equiv),
1:1 t-BuOH/H2O, 60 C, 3 h; (2) 0.05 M NaOMe/MeOH, Room Temperature, 0.5 h, Amberlite IR 120H+;
◦
◦
(3) KOH, MeOH, 0 C, rt, overnight, Amberlite IR 120H+.

Molecules 2017, 22, 1058
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OAc
NC
NC
Br
Br
(2)
(1)
O
AcO
Cl
Cl
N
3
+
AcO
N
N
H
CF3
CF3
AcO
O
13
5
2
NC
NC
Br
Br
Cl
Cl
N
N
N
(3)
CF3
CF3
OH
HO
OAc
AcO
O
O
O
O
HO
HO
AcO
AcO
N
N
N
N
N
14
15
Scheme 4. Synthesis of glucose conjugate 15 with a space arm of methoxymethyl group. Reagents
and conditions: (1) THF, Bromochloromethane(10 equiv), NaH, Propargyl alcohol, 65 ◦C, 48 h;
(2) CuSO4
·
5H2O (0.4 equiv), sodium ascorbate (0.8 equiv), 1:1 t-BuOH/H2O, 60 ◦C, 3 h; (3) 0.05 M
NaOMe/MeOH, rt, 0.5 h, Amberlite IR 120H+.
1
The structures of conjugates
spectroscopy. The characteristic signals of the 1,2,3-triazole and acetyl groups on the sugar rings could
be observed in the 1H- and 13C-NMR spectra of conjugates 14 and 16. In the 1H-NMRspectra
10
8–12 and 14–
18 were confirmed by H-, 13C-NMR and ESI-MS
8,
,
of these compounds, single peaks corresponding to the 1,2,3-triazole and acetyl groups were found
at 8.25–8.31 and 1.76–2.03 ppm; in their 13C-NMRspectra, the characteristic peaks of acetyl groups
were found at 20 and 169–170 ppm. The removal of acetyl groups from conjugates
8, 10, 14 and 16 was
confirmed by the 1H-and 13C-NMR spectra of conjugates
9
,
11 12, 15 17 and 18. Thecharacteristic
,
,
peaks at 1.76–2.03 ppm in the 1H-NMR spectra, and the peaks at 20 and 169–170 ppm in the 13C-NMR
spectra disappeared after deprotection. The removal of the methyl group from conjugates 11 and 17
was confirmed by the 1H and 13C-NMR spectra of conjugates 12 and 18. The characteristic peaks at
3.66–3.74 ppm in 1H-NMR spectra and the peaks at 52–53 ppm in the 13C-NMR spectra disappeared
after deprotection. The
the 1H-NMR spectroscopic data, in which H-1 appeared as a doublet,
conjugates and 15 as well as 5.56–5.71 (J = 9.3 Hz) for conjugates 11
β-configuration of conjugates 9, 11, 12, 15, 17 and 18 was determined from
δ
5.45–5.50 (J = 9.2–9.4 Hz) for
17 and 18. The mass spectra
9
δ
,
12
,
of the products exhibited excellent correlation with the calculated molecular mass.
NC
Br
CF3
Cl
NC
N
O
H3CO
Br
(1)
O
O
O
H3CO
AcO
Cl
AcO
+
N
3
AcO
O
N
CF3
N
N
AcO
4
AcO
O
N
13
AcO
16
NC
NC
Br
Br
Cl
Cl
N
N
N
CF3
CF3
(3)
(2)
O
O
O
HO
O
H CO
3
O
O
HO
HO
HO
N
N
N
HO
N
N
HO
HO
18
17
Scheme 5. Synthesis of methyl glucuronate or glucuronic acid conjugate
s
with a space arm of
methoxymethyl group. Reagent◦s and conditions: (1) CuSO4
·
5H2O (0.4 equiv), sodium ascorbate
◦
(0.8 equiv), 1:1 t-BuOH/H2O, 60 C, 3 h; (2) 0.05 M NaOMe/MeOH, 45 C, 1 h, Amberlite IR 120H+;
◦
(3) KOH, MeOH, 0 C, rt, over-night, Amberlite IR 120H+.

Molecules 2017, 22, 1058
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2.2. Phytotoxicity to Tea Shoots
Chlorfenapyr is a pro-insecticide optimized from tralopyril which exhibits intolerable
phytotoxicity to the applied plants [17]. The tea shoots were selected for phytotoxicity assay, mainly
because chlorfenapyr could be used to control tea infection pests and it has a lower European Union
MRLs in tea [14]. As shown in Table 1, no phytotoxicity symptoms were observed up to 63 days after
smearing conjugates 9, 11, 12, 15, 17 and 18 (0.02, 0.2, and 2 mM, respectively; score: 0–5%; grade 1)
under pot experimental conditions. By contrast, tralopyril showed obvious phytotoxicity symptoms
at 0.2 mM (score: 61–70%; grade 7) and 2 mM (score: 71–80%; grade 8), but not at 0.02 mM (score:
0–5%; grade 1). These results indicated that the introduction of a sugar group may solve the difficult
phytotoxicity problem of tralopyril.
Table 1. Phytotoxicity of tralopyril, 9, 11, 12,15, 17 and 18 to tea shoots.
Phytotoxicity Symptom Scales a
Tralopyril
0.2 mM
20
9
11
12
15
17
18
Symptoms
Score (%)
0.02 mM
2 mM
20
2 mM
2 mM
2 mM
2 mM
2 mM
2 mM
Injury to leaf tips
Injury to leaf
surfaces
20
20
0
0
0
0
0
0
0
0
0
0
0
0
0
0
20
20
Leaf wilting
Necrosis
Vein clearing
Epinasty
20
10
10
10
10
0
0
0
0
0
0
1
20
10
0
0
0
70
7
20
10
10
0
0
80
0
0
0
0
0
0
1
0
0
0
0
0
0
1
0
0
0
0
0
0
1
0
0
0
0
0
0
1
0
0
0
0
0
0
1
0
0
0
0
0
0
1
Hyponasty
Total score
grade b
8
a
b
Phytotoxicity symptoms and their scores were determined according to references [18,19]. The phytoxicity effect
was graded as per the following scale: 0–10, 1; 11–20, 2; 21–30, 3; 31–40, 4; 41–50, 5; 51–60, 6; 61–70, 7; 71–80, 8; 81–90,
9; 91–100, 10.
2.3. Phloem Mobility
The castor bean system was selected for the phloem mobility assay, mainly because it is widely
employed to evaluate phloem mobility of nutrients as well as xenobiotics [
conjugates 11 12, 15 17 18 and chlorfenapyr was collected at 3 h for detection. When Ricinus
cotyledons were incubated with 200 M conjugates 11 12, 15 17 and 18 for 3 h, they were clearly
detected, and their concentrations in the phloem sap were about 4, 1.5, 40, 2.5, 1 and 50 M, respectively
5]. The phloem sap of
9,
,
,
,
µ
9,
,
,
µ
(Figure 2). When the cotyledons were incubated in chlorfenapyr solution at the same concentration,
it was not detected in the phloem sap (Figure 1).
NC
NC
Br
Br
Cl
Cl
N
O Et
chlorfenapyr
CF3
N
H
CF3
tralopyril, 5
Figure 1. Chemical structure of tralopyril and chlorfenapyr.
These results indicated that the introduction of the three different sugar promoieties could impart
phloem mobility to tralopyril. Based on the concentrations of conjugates
phloem sap, the concentration of conjugates 12 and 18 in the phloem sap was approximately 10-fold
and 20-fold greater higher than that of conjugates and 15, which showed that the glucuronic acid
9, 12, 15 and 18 for 3 h in the
9
moiety caused higher phloem mobility than the glucose moiety. The concentration of conjugates 12 and
18 was approximately 27-fold and 50-fold greater than that of conjugates 11 and 17. This result indicates
that the carboxyl group has a positive contribution to phloem mobility. Conjugates 9 and 15 shared

Molecules 2017, 22, 1058
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the same glucose and 1,2,3-triazole moieties with GTF, but their concentrations were approximately
0.1-fold greater than that of GTF at 3 h [
affected by the parent molecule.
5]. This result again demonstrates that phloem mobility was
Figure 2. Phloem mobility of
a buffered solution containing 200
9
,
11
,
12
,
15
9
,
,
17
11
,
,
18 and chlorfenapyr. The cotyledons were incubated in
12 15 17 18 and chlorfenapyr at pH 5.5. The sap was
µM
,
,
,
collected at the third hour of incubation. For box plots, n = 8.
Time-course experiments were conducted during a 5 h period to further elucidate the time-course
phloem mobility for conjugates 12 and 18. The results (Figures 3 and 4) indicated that the concentrations
of conjugates 12 and 18 in the phloem sap increased during the test period. Their concentration reached
55 µM and 56 µM at 5 h, which was approximately 9-fold and 7-fold higher than those at 1 h.
Figure 3. Time course of conjugate 12 concentration in phloem sap of R. communis. The cotyledons
were incubated in a buffered solution containing 200 µM 12 at pH 5.5. The hook was severed at time
1 h, and then the sap was collected every 1 h during 5 h. For box plots, n = 8.
The effectiveness of the addition of a carboxyl group, amino acid, or sugar to the parent compound
for converting a non-mobile crop protection product into a phloem-mobile type was proven. In the
first approach, uptake and translocation of conjugates may involve an ion-trapping effect. For example,

Molecules 2017, 22, 1058
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the phloem mobility of acidic derivatives of the fungicide fenpiclonil was caused by the ion trap
mechanism [ ]. In the second approach, uptake and translocation of conjugates may involve two
2
components of specific active carrier-mediated systems and passive transport. For example, 2,4D-Lys
uptake involves an active carrier-mediated system aside from diffusion [20]. In the last approach, uptake
and translocation of conjugates may involve a specific active carrier-mediated system. For example,
the uptake and phloem transport of GTF involve an active carrier-mediated mechanism [6].
Figure 4. Time course of conjugate 18 concentration in phloem sap of R. communis. The cotyledons
were incubated in a buffered solution containing 200 µM 18 at pH 5.5. The hook was severed at time
1 h, and then the sap was collected every 1 h during 5 h. For box plots, n = 8.
The “Rule of Five”, which is widely adopted in the pharmaceutical industry, can also be used
to predict the diffusion of endogenous molecules or xenobiotics through plant membranes [21,22].
According to this rule, poor absorption or permeation of small molecules is more probable when their
MW, log P, HBDs, and HBAs are more than 500D, 5, 5, and 10, respectively. Moreover, at least three in
four parameters should satisfy these values [23]. Compound classes that are substrates for biological
transporters are exceptions to this rule [23]. Considering that the two parameters (MW and HBAs) of
conjugates 9, 11, 12, 15, 17 and 18 in Table 2 are out of range, they were poorly diffused through the
membranes. Therefore, a carrier system is probably involved in the long-distance transport of these
conjugates. The Kleier model is also used to predict the phloem systemicity of a compound based
on the physicochemical properties (log Kow pKa and log Cf) of molecules [24–26]. According to this
model, large values of log Cf correspond to high predicted phloem mobility. As shown in Table 2,
the introduction of a sugar moiety to the parent compound resulted in the lowering of log Kow for
conjugates
pKa values. In terms of log Cf values, conjugates
area, whereas conjugates 12 and 18 were in the mobile molecule area [
partly explained why the phloem mobility of conjugates 12 and 18 were higher than that of conjugates
11 15 and 17. As stated by Hsu et al., glucuronidation is indeed a good method to confer phloem
9,
11,
12, 15
,
17 and 18, but only the glucuronidation products 12 and 18 possessed lower
11 15 and 17 were in the non-mobile molecule
25 26]. This observation
9
,
,
2,6, ,
9
,
,
mobility to immobile or poorly mobile pesticides. The effect of this process is exerted by a combined
reduction in log Kow (over two units) and impartation of desirable acidity to exploit the ion-trapping
effect [4]. Therefore, the uptake and translocation of conjugates 12 and 18 may involve two components
of carrier-mediated systems and ion trap mechanism. This inference should be confirmed by obtaining
direct evidence in further experiments.

Molecules 2017, 22, 1058
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Table 2. Predicted physicochemical descriptor MW, log D, pKa, HBD, HBA of tralopyril, chlorfenapyr,
9, 11, 12, 15, 17 and 18 a.
Feature
MW (D)
Log D (pH 5.0)
Log D (pH 8.0)
pKa
HBD HBA
Log P
Log Cf c
14 b
14
14
14
2.75
14
Tralopyril
chlorfenapyr
349.53
407.61
592.75
620.76
606.73
622.78
650.79
636.76
4.75
4.76
2.02
2.50
0.48
2.29
2.49
0.63
4.75
4.76
2.02
1
0
4
3
4
4
3
4
2
3
4.75
4.76
2.02
2.50
2.15
2.29
2.49
2.30
<−99
<−99
<−99
<−99
0.32
<−99
<−99
0.49
9
11
12
15
17
18
10
11
11
11
12
12
2.50
−1.84
2.29
2.49
−1.69
14
2.77
a
b
All parameters were computed using ACD/Lab sversion 12.01 software. Assumed nonacidic and assigned pKa
c
of 14. Log Cf values were calculated for a short plant [
6,26]. Plant parameters for these calculations are L = 0.15 m,
l = 0.05 m, r = 5
membrane was calculated using log P = 1.2 (log Kow) − 7.5.
×
10−6 m, v = 3.0
×
10−4 m s−1, pH (phloem) = 8, and pH (apoplast) = 6. The permeability of the
2.4. Insecticidal Activity Against P. xylostella
The poison fodder method was adopted to determine the insecticidal activity of conjugates 9,
11
when P. xylostella was exposed to 4 mM conjugates for 72 h
group on the pyrrole N of tralopyril moiety exhibited no activity, while conjugates 15
,
12, 15
,
17 and 18 against P. xylostella at concentrations of 1, 2 and 4 mM. As shown in Table 3,
conjugates 11 and 12 with a methylene
17 and 18 with
,
9,
,
a methoxymethyl group on the pyrrole N of tralopyril moiety showed 100% mortality. The results
again proved that the methoxymethyl group on the pyrrole N of tralopyril moiety was akey factor to
determine the insecticidal activity [14]. Among 15, 17 and 18, conjugate 15 with the glucose promoiety
showed the lowest insecticidal activity, while 17 with the methyl glucuronate promoiety showed
the best insecticidal activity. These observed differences indicated that the methyl glucuronate and
glucuronic acid promoieties were clearly more favorable to the insecticidal activity to tralopyril than
glucose. Therefore, further systemic insecticidal activity assessment of conjugates 15, 17 and 18 were
carried out.
Table 3. The fatality rates of tralopyril, 9, 11, 12, 15, 17 and 18 against P. xylostella.
48 h Fatality Rate (%)
2 mM
72 h Fatality Rate (%)
2 mM
1 mM
4 mM
1 mM
4 mM
Chlorfenapyr
100.00
0.00
100.00
0.00
100.00
0.00
100.00
0.00
100.00
0.00
100.00
0.00
9
11
12
15
17
18
0.00
0.00
0.00
100.00
0.00
0.00
0.00
10.00
100.00
33.33
0.00
0.00
100.00
100.00
93.33
0.00
0.00
10.00
100.00
0.00
0.00
0.00
30.00
100.00
96.66
0.00
0.00
100.00
100.00
100.00
2.5. Systemic Mobility on Cabbage and Systemic Insecticidal Activity
When 4 mM of conjugate 15 was root-applied, its detected concentrations after 48 h and 72 h were
about 252 g/g and 47 g/g, respectively (Table 4). When 4 mM of conjugate 17 was root-applied,
µ
µ
it was not detected, but conjugate 18 was detected (Table 4).This result is due to the fact that conjugate
17 was hydrolyzedin to conjugate 18 in aqueous solution. When 4 mM of conjugate 18 was root-applied,
it was clearly detected, and its concentrations at 48 h and 72 h were about 1905 µg/g and 1947 µg/g,
respectively (Table 4). By contrast, after root-application of 4 mM chlorfenapyr, it was not detected
(Table 4). These observed differences indicate that the introduction of sugar promoieties could impart
xylem mobility to tralopyril, and the glucuronic acid promoiety was clearly more favorable for the
xylem mobility of tralopyril than glucose.

Molecules 2017, 22, 1058
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Table 4. Detected concentration for the harvested leaves from the treatment of root-applied compound.
Concentration of Detected Compound (µg/g)
Root-Applied Compound
15
17
18
Chlorfenapyr
15
17
18
252.96
-
-
34.98
1905.27
-
-
-
-
-
-
-
ND a
48 h harvested
leaves
-
-
Chlorfenapyr
ND
15
17
18
47.68
-
-
68.79
1947.95
-
-
-
-
-
-
-
ND
-
-
72 h harvested
leaves
Chlorfenapyr
ND
a
ND = not detected.
The harvested leaves from the treatments of root-applied conjugates 15
,
17 and 18 were used
to determine the systemic insecticidal activity. Table 5 shows that when P. xylostella was exposed to
the 48 h and 72 h harvested leaves, the treatments of root-applied conjugates 15 17 and 18 exhibited
,
different insecticidal activities, especially conjugate 18 which showed excellent activity with a mortality
of 93.33% and 100% at 48 h and 72 h. However, the harvested leaves from the treatments of root-applied
chlorfenapyr showed no insecticidal activity. The results of the root-applied activity screening model
proved that glucuronic acid promoiety imparts more systemic insecticidal activity to tralopyril than
glucose and methyl glucuronate.
Previous studies showed that specific promoiety introduction that confer phloem mobility to
pesticides usually lower direct biological activity [
the present work. Hsu et al. described the strategy of phloem-mobile pro-pesticide to overcome this
inherent incompatibility [ ]. Conjugate 18 may meet this strategy as the excellent systemic mobility
3–5,14]. This phenomenon was also observed in
4
and systemic insecticidal activity. Although, conjugate 18 exhibited lower direct insecticidal activity
than chlorfenapyr, its excellent property of systemic insecticidal activity enables this conjugate to
maintain the insecticidal activity for the insects hidden on unpredictable non-exposed plant parts.
Table 5. The systemicinsecticidal activity of the conjugates against P. xylostella.
Root-Applied
Compound
48 h Mortalities Post
Feeding (%)
72 h Mortalities Post
Feeding (%)
15
17
18
0.00
0.00
20.00
0.00
0.00
36.66
10.00
93.33
0.00
48 h harvested leaves
72 h harvested leaves
Chlorfenapyr
CK a
0.00
15
17
18
0.00
0.00
53.33
0.00
0.00
0.00
46.66
100.00
0.00
Chlorfenapyr
CK
0.00
a
CK= control.
3. Materials and Methods
3.1. Synthesis
3.1.1. General Information
All reagents were purchased from a commercial company. Analytical thin-layer chromatography
(TLC) was performed on silica gel GF254 plates (Qingdao Marine Chemical Ltd., Qingdao, China).

Molecules 2017, 22, 1058
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Silica gel (100–200 and 200–300 mesh, Qingdao Marine Chemical Ltd., Qingdao, China) was used
for column chromatography. 1H-and 13C-NMR spectra were obtained on a WNMR-I 500 (Wuhan
Institute of Physics and Mathematics (WIPM) of Chinese Academy of Sciences, Wuhan, Hubei, China)
or INOVA 400 (Agilent Technologies Inc., Santa Clara, CA, USA,) instrument. Chemical shifts were
expressed in δ (ppm) values, with TMS as an internal standard. The mass spectra of new conjugates
were obtained by high-performance liquid chromatography (HPLC)-mass spectrometry (MS) (Thermo,
San Jose, CA, USA) using electrospray ionization (ESI) and are reported as m/z values.
3.1.2. Synthesis of Azide Intermediates 2 and 4
Azide intermediates were synthesized according to previously reported procedures [15
,16]. Briefly,
compounds or (2 mmol) were added to a solution of azidotrimethylsilane (2.2 mmol) in anhydrous
1
3
CH2Cl2 (10 mL), followed by dropwise addition of SnCl4 (2 mmol). The resulting mixture was stirred at
room temperature (rt) for 2 h, then extracted three times with aqueous sodium hydrogen carbonate and
washed with brine, dried over sodium sulfate, filtered, and evaporated in vacuo. The residues were
then purified byrecrystallization from ethanol to give the azide intermediates 2 or 4 as white solids.
3.1.3. Synthesis of N-Propargyl Tralopyril (7)
Synthesis of the title compound was accomplished according to a previously reported
procedure [14]. Briefly, NaH (60% in oil, 0.144 g, 3.6 mmol) was added to the solution of tralopyril
(1.05 g, 3 mmol) in THF (15 mL). The mixture was then stirred at room temperature for 2 h. Propargyl
bromide (3.3 mmol) in THF (15 mL) was dropwise added at room temperature. The mixture was then
stirred at 65 ◦C for 48 h. The reaction mixture was quenched by adding ice-water, and the resulting
mixture was extracted with ethyl acetate (15 mL
×
3). The combined organic layers were washed with
aqueous sodium hydrogen carbonate and brine, dried with sodium sulfate, filtered, and evaporated
in vacuo. The residue was purified by column chromatography (10 mL min−1) to afford the desired
product 7.
3.1.4. Synthesis of N-Propynoxymethyl Tralopyril (13)
The title compound was synthesized according to a previously reported procedure [14]. Briefly,
NaH (60% in oil, 0.288 g, 7.2 mmol) was added to the solution of tralopyril (1.05 g, 3 mmol) in mixture
solvent of THF (20 mL) and bromochloromethane (1.95 g, 15 mmol, 5 equiv). The mixture was then
stirred at room temperature for 2 h. Propargyl alcohol (3 mmol) in THF (10 mL) was dropwise added
◦
◦
at 65 C. The mixture was then stirred at 65 C for 48 h. The reaction mixture was quenched by adding
ice-water, and the resultant mixture was extracted with ethyl acetate (20 mL 3). The combined
×
organic layers were washed with aqueous sodium hydrogen carbonate and brine, dried with sodium
sulfate, filtered, and evaporated in vacuo. The residue was purified by column chromatography
(10 mL min−1) to give the desired product 13.
3.1.5. General Procedure for the Click Reaction
The conjugates
terminal alkyne derivatives (compounds
1 mmol) were dissolved in t-BuOH (3 mL). The reaction was initiated by the addition of CuSO4
(0.2 mmol) and sodium ascorbate (0.4 mmol) in distilled water (3 mL). The mixture was stirred at 60 C
until TLC indicated the disappearance of the starting materials. The mixture was poured into distilled
water (20 mL), and the product was extracted three times with CH2Cl2 (20 mL). The organic layer
was dried with Na2SO4 and filtered. The solvent was removed under reduced pressure. The residues
were purified by column chromatography (1:2 petroleum ether/ethyl acetate) to obtain the glycosyl
tralopyril conjugates 8, 10, 14 and 16.
8
,
10
,
14 and 16 were synthesized using the following procedure: tralopyril
7
or 13, 1 mmol) and an azidoglycose (compounds or 4,
2
·
5H2O
◦

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4-Bromo-2-(4-chlorophenyl)-1-((1-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl)-1H-1,2,3-triazol-4-yl)methyl)-
1
5-(trifluoromethyl)-1H-pyrrole-3-carbonitrile (
8
). H-NMR (500 MHz, DMSO-d6)
δ
8.31 (s, 1H), 7.66–7.59
(m, 4H), 6.31 (d, J = 9.2 Hz, 1H), 5.62 (t, J = 9.4 Hz, 1H), 5.53 (t, J = 9.5 Hz, 1H), 5.28 (s, 2H), 5.17
(t, J = 9.8 Hz, 1H), 4.34 (m, 1H), 4.16–4.05 (m, 2H), 2.02 (s, 3H), 2.00 (s, 3H), 1.96 (s, 3H), 1.77 (s, 3H).
13C-NMR (126 MHz, DMSO-d6)
δ 170.04, 169.59, 169.42, 168.42, 144.43, 142.72, 135.87, 131.80, 129.33,
125.50, 122.08, 113.69, 102.68, 97.92, 83.86, 73.24, 72.10, 70.13, 67.43, 61.59, 43.30, 20.52, 20.42, 20.26,
19.81. ESI-MS m/z: 761 [M + 1]+, 783 [M + Na]+.
2-(4-((3-Bromo-5-(4-chlorophenyl)-4-cyano-2-(trifluoromethyl)-1H-pyrrol-1-yl)methyl)-1H-1,2,3-triazol-1-yl)-
1
6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (10). H-NMR (400 MHz, DMSO-d6)
δ
8.31
(s, 1H), 7.62 (m, 4H), 6.30 (d, J = 9.1 Hz, 1H), 5.62 (t, J = 9.3 Hz, 1H), 5.52 (t, J = 9.5 Hz, 1H), 5.28 (s, 2H),
5.17 (t, J = 9.7 Hz, 1H), 4.33 (m, 1H), 3.65 (s, 3H), 2.02 (s, 3H), 2.00 (s, 3H), 1.96 (s, 3H). 13C-NMR
(101 MHz, DMSO-d6)
δ 170.07, 169.61, 169.44, 168.45, 144.44, 142.73, 135.89, 131.82, 129.34, 125.51,
122.10, 113.71, 83.87, 73.26, 72.12, 70.14, 67.44, 61.60, 52.10, 43.31, 20.55, 20.45, 20.29. ESI-MS m/z: 748
[M + 1]+, 770 [M + Na]+.
4-Bromo-2-(4-chlorophenyl)-5-(trifluoromethyl)-1-((1-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl-1H-1,2,3-
1
triazol-4-yl)methoxy)methyl)-1H-pyrrole-3-carbonitrile (14). H-NMR (400 MHz, DMSO-d6)
δ 8.26 (s, 1H),
7.67 (d, J = 1.1 Hz, 4H), 6.33 (d, J = 8.7 Hz, 1H), 5.66–5.51 (m, 2H), 5.36 (m, 2H), 5.19 (t, J = 9.5 Hz,
1H), 4.42 (s, 2H), 4.37 (m, 1H), 4.18–4.03 (m, 2H), 2.03 (s, 3H), 2.00 (s, 3H), 1.97 (s, 3H), 1.76 (s, 3H).
13C-NMR (101 MHz, DMSO-d6)
δ 170.08, 169.62, 169.44, 168.52, 144.53, 143.05, 135.90, 131.97, 129.35,
125.35, 123.29, 113.63, 98.01, 83.86, 75.44, 73.29, 72.12, 70.16, 67.53, 61.85, 61.16, 20.55, 20.44, 20.30, 19.90.
ESI-MS m/z: 792 [M + 1]+, 814 [M + Na]+.
2-(4-(((3-Bromo-5-(4-chlorophenyl)-4-cyano-2-(trifluoromethyl)-1H-pyrrol-1-yl)methoxy)methyl)-1H-1,2,3-
triazol-1-yl)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (16). 1H-NMR (500 MHz,
DMSO-d6)
(m, 2H), 5.19 (t, J = 9.6 Hz, 1H), 4.42 (s, 2H), 4.36 (m, 1H), 3.70 (s, 3H),2.03 (s, 3H), 2.00 (s, 3H), 1.97
(s, 3H).13C-NMR (126 MHz, DMSO-d6)
170.09, 169.63, 169.44, 168.53, 144.53, 143.06, 135.90, 131.97,
δ 8.25 (s, 1H), 7.71–7.63 (m, 4H), 6.33 (d, J = 8.8 Hz, 1H), 5.65–5.50 (m, 2H), 5.40–5.32
δ
129.35, 125.36, 123.29, 113.63, 103.29, 98.02, 83.86, 75.43, 73.29, 72.12, 70.16, 67.53, 61.84, 61.16, 52.11,
20.54, 20.43, 20.29. ESI-MS m/z: 778 [M + 1]+, 800 [M + Na]+.
3.1.6. General Procedure for the Removal of Acetyl Groups
The conjugates 8, 10, 14 or 16 (1 mmol) were added to a solution of sodium methoxide in
dry methanol (0.05 M, 15 mL). The resulting solution was stirred for 30 min at room temperature.
The mixture was neutralized with Amberlite IR120 (H+) resin and filtered. Subsequently, the filtrate
was evaporated. The residues were purified by column chromatography (9:1 CH2Cl2/MeOH) to afford
the glycosyl tralopyril conjugate 9, 11, 15 or 17.
4-Bromo-2-(4-chlorophenyl)-1-((1-(beta-D-glucopyranosyl)-1H-1,2,3-triazol-4-yl)methyl)-5-(trifluoromethyl)-
1H-pyrrole-3-carbonitrile (
9 δ 7.94 (s, 1H), 7.62 (m, 4H), 5.45
). 1H-NMR (400 MHz, DMSO-d6):
(d, J = 9.4 Hz, 1H), 5.27 (s, 2H), 3.90–3.88 (m, 2H), 3.70–3.65 (m, 3H), 3.50–3.48 (m, 1H).13C-NMR
(101 MHz, DMSO-d6)
δ 144.4, 141.6, 135.7, 131.9, 129.3, 127.1, 125.6, 122.3, 121.1, 113.7, 102.5, 97.9, 87.5,
80.0, 76.9, 72.0, 70.0, 60.7, 43.2. ESI-MS m/z: 594 [M + 1]+, 617 [M + Na]+.
Methyl6-(4-((3-bromo-5-(4-chlorophenyl)-4-cyano-2-(trifluoromethyl)-1H-pyrrol-1-yl)methyl)-1H-1,2,3-triazol-
1
1-yl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylate (11). H NMR (400 MHz, MeOD)
δ
7.89 (s, 1H),
7.48 (m, 4H), 5.59 (d, J = 9.3 Hz, 1H), 5.34 (s, 2H), 4.11 (d, J = 9.8 Hz, 1H), 3.89 (t, J = 9.2 Hz, 1H), 3.74
(s, 3H), 3.66 (t, J = 9.5 Hz, 1H), 3.53 (t, J = 9.1 Hz, 1H). 13C-NMR (101 MHz, MeOD)
δ
170.49, 146.03,
143.76, 138.11, 132.83, 130.51, 127.10, 123.53, 122.96, 121.11, 120.24, 114.26, 103.51, 101.12, 89.14, 79.07,
77.80, 73.30, 72.66, 53.02, 44.17. ESI-MS m/z: 622 [M + 1]+, 644 [M + Na]+.

Molecules 2017, 22, 1058
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4-Bromo-2-(4-chlorophenyl)-5-(trifluoromethyl)-1-((1-(beta-D-glucopyranosyl-1H-1,2,3-triazol-4-
yl)methoxy)methyl)-1H-pyrrole-3-carbonitrile (15). 1H-NMR (400 MHz, DMSO-d6)
δ 8.11 (s, 1H),
7.74–7.62 (m, 4H), 5.50 (d, J = 9.2 Hz, 1H), 5.40 (s, 2H), 5.38 (d, J = 5.9 Hz, 1H), 5.31 (d, J = 4.9 Hz,
1H), 5.18 (d, J = 5.5 Hz, 1H), 4.64 (t, J = 5.5 Hz, 1H), 4.39 (s, 2H), 3.69 (m, 2H). 13C-NMR (101 MHz,
DMSO-d6)
δ 144.55, 142.23, 135.97, 131.99, 129.45, 125.37, 123.54, 113.68, 98.01, 87.49, 80.01, 76.95, 75.58,
72.14, 69.59, 61.36, 60.80. ESI-MS m/z: 624 [M + 1]+, 646 [M + Na]+.
Methyl 6-(4-(((3-bromo-5-(4-chlorophenyl)-4-cyano-2-(trifluoromethyl)-1H-pyrrol-1-yl)methoxy)methyl)-1H-
1
1,2,3-triazol-1-yl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylate (17). H-NMR (400 MHz, DMSO-d6)
δ
8.18 (s, 1H), 7.70–7.57 (m, 4H), 5.71 (d, J = 9.3 Hz, 1H), 5.57 (m, 2H), 5.39 (s, 2H), 4.41 (s, 2H), 4.15
(
d, J = 9.6 Hz, 1H), 3.82 (m, 1H), 3.66 (s, 3H). 13C-NMR (101 MHz, DMSO-d6)
δ
168.78, 144.52, 142.39,
135.93, 131.91, 129.37, 125.31, 123.71, 113.66, 103.33, 87.07, 77.46, 76.10, 75.50, 71.61, 71.19, 61.25, 52.12.
ESI-MS m/z: 652 [M + 1]+, 674 [M + Na]+.
3.1.7. General Procedure for the Removal of Methyl Ester Groups
◦
KOH (1.3mmol) was added to a solution of 11 or 17 (1 mmol) in MeOH (8 mL) at 0 C. The reaction
mixture was stirred overnight at room temperature and acidified via treatment with Amberlite
IR 120H+. Filtration, evaporation under reduced pressure, and purification afforded 12 or 18 as
a white solid.
6-(4-((3-Bromo-5-(4-chlorophenyl)-4-cyano-2-(trifluoromethyl)-1H-pyrrol-1-yl)methyl)-1H-1,2,3-triazol-1-yl)-
1
3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (12). H-NMR (400 MHz, MeOD)
δ 7.91 (s, 1H,
Ar-H), 7.50 (q, J = 8.6 Hz, 4H, Ar-H), 5.60 (d, J = 9.3 Hz, 1H, H-1), 5.36 (s, 2H , NCH2), 4.07 (d, J = 9.7
Hz, 1H, H-5), 3.89 (t, J = 9.1 Hz, 1H, H-3), 3.67 (t, J = 9.4 Hz, 1H, H-4), 3.55 (t, J = 9.1 Hz, 1H, H-2).
13C-NMR (101 MHz, MeOD)
δ 171.75, 146.05, 143.80, 138.13, 132.83, 130.51, 130.25, 127.09, 123.48,
122.97, 121.14, 120.29, 114.26, 103.47, 100.14, 89.16, 78.99, 77.96, 73.38, 72.70, 44.21. ESI-MS m/z: 608
[M + 1]+, 630 [M + Na]+.
6-(4-(((3-Bromo-5-(4-chlorophenyl)-4-cyano-2-(trifluoromethyl)-1H-pyrrol-1-yl)methoxy)methyl)-1H-1,2,3-
1
triazol-1-yl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (18). H-NMR (500 MHz, DMSO-d6)
δ
8.17 (s, 1H), 7.71–7.60 (m, 4H), 5.65 (d, J = 9.3 Hz, 1H), 5.39 (s, 2H), 4.41 (m, 2H), 3.99 (d, J = 9.7 Hz, 1H),
3.79 (t, J = 9.1 Hz, 1H), 3.50 (t, J = 9.3 Hz, 1H), 3.43 (t, J = 8.9 Hz, 1H). 13C-NMR (126 MHz, DMSO-d6)
δ
169.70, 144.54, 142.35, 135.96, 131.91, 129.39, 125.32, 123.72, 113.66, 103.31, 97.97, 87.14, 77.80, 76.32,
75.49, 71.70, 71.21, 61.26. ESI-MS m/z: 638 [M + 1]+, 660 [M + Na]+.
3.2. Phytotoxicity Assay to Tea
Seedlings of Fuding white tea were grown as previously described [14]. At eight weeks after
sowing, average-sized seedlings were selected for the experiments. Four treatments were tested,
namely, three concentrations of three conjugates and tralopyril (0.02, 0.2 and 2 mM), along with the
untreated control (aqueous solution containing 0.1% Tween 80 and 10% methanol). A volume of 100 µL
was applied to the whole tea shoots (a bud with two leaves) for each tea seedling. The score method
used to assess the phytotoxic effect of the test substances was conducted according to previously
described procedures [18,19]. Weekly observations of the phytotoxic symptoms for seven weeks
in each plot and their corresponding scores were pooled and compared with the untreated control.
Finally, the phytotoxic effect of the test substances was graded according to the previously described
standard [18,19].
3.3. Sap Collection from Ricinus communis L. Seedlings and Analysis
Castor bean (R. communis L.) seeds were obtained from the Agricultural Science Academy of Zibo
(Shandong, China), and grown as previously described [2]. At 6 days after sowing, average-sized
seedlings were selected for the experiments. Phloem sap was collected from the upper part of the

Molecules 2017, 22, 1058
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hypocotyl according to previously described methods [
2,
20,
24]. The endosperm of seedlings was
carefully removed without bending or crushing the cotyledons. These latter organs were incubated in
buffer solution containing 20 mM MES (pH 5.5), 0.25 mM MgCl2, and 0.5 mM CaCl2 supplemented
with 200 µM 9, 11, 12, 15, 17 and 18 or 200 µM chlorfenapyr. After 1 h of incubation, the hypocotyl
was severed in the hook region for phloem exudation, and the collected sap was stored in ice until
analysis.The phloem sap was analyzed by HPLC (HP 1260 system, Agilent Technologies, Santa Clara,
CA, USA, equipped with a quaternary pump, autosampler, and UV–visible detector) after dilution with
pure water (phloem sap/pure water, 1/3, v/v). Separations were performed with a C18 reversed-phase
◦
column (5
µm, 150
×
4.6 mm i.d., Agilent Technologies) at 25 C. The mobile solvent system consisted
of acetonitrile and water containing 0.1% trifluoroacetic acid (40/60, v/v for
15
17, and 18) or water (80/20, v/v for chlorfenapyr) at a flow rate of 0.5 mL min−1. The detector
wave length was 210 nm for 11 12, 15 17 18 and chlorfenapyr. External calibration was used to
determine their concentrations. A series of standard solutions (0.5, 1, 5, 10, 25 and 50 M) for the
9, 11, and 12; 45/55, v/v for
,
9,
,
,
,
µ
calibration curves was prepared in methanol. Their linear equations of the different compounds are
shown in Table 6.
Table 6. The linear equations of the compounds.
Compound
Linear Equations
R2
Chlorfenapyr
y = 38.04x + 68.58
y = 25.96x + 8.59
y = 28.50x + 9.55
y = 29.91x + 9.69
y = 50.09x – 34.16
y = 33.77x – 10.19
y = 35.45x + 11.11
0.9974
0.9998
0.9999
0.9998
0.9987
0.9968
0.9995
9
11
12
15
17
18
3.4. Insecticidal Activity of Six Conjugates Against P. xylostella
Third-instar larvae of P. xylostella and its artificial fodder were obtained from the He Nan Ji Yuan
Bai Yun Industrial Co., Ltd. (Jiyuan, Henan, China). The bioactivities of 11 12 15 17 18 and
9,
,
,
,
,
chlorfenapyr on the third-instar larvae of P. xylostella were assessed by mixing them directly into the
fodder. Three concentrations (1, 2and 4 mM) of the six conjugates and chlorfenapyr were dissolved in
methanol and acetone (10/90, v/v). Each test solution (1 mL) and artificial fodder (1 g) were placed into
Petri dishes (9 cm in diameter). Artificial fodder (1 g) containing methanol and acetone (1 mL, 10/90,
v/v), but not the tested compound, was used as the control. At 8 h after the solvent was evaporated,
ten P. xylostella larvae were then introduced into each dish. The dishes were incubated at 26 ◦C and
70% relative humidity under a photoperiod of 16:8 h (light/dark). All treatments were repeated four
times. Mortalities were determined at 48 h and 72 h post treatment.
3.5. Systemic Mobility on Cabbage and Systemic Insecticidal Activity of Conjugates Against P. xylostella
Two-weeks-old seedlings of Brassica oleracea were fertilized with half-strength Hoagland’s solution
once a week. The growing plants were used in the tests for three weeks. The roots of the cabbage
seedlings were dipped in the conjugates (4 mM) and chlorfenapyr aqueous solution (50 mL) containing
Tween80 (0.1%) and methanol (5%). The aqueous solution containing Tween80 (0.1%) and methanol
(5%) was used as the control. All treatments were repeated three times. The leaves were harvested at
48 h and 72 h after the root-applied chemical and then were divided into two parts. One part of the
harvested samples was stored at
samples was used to the assessments of systemic insecticidal activity.
−
15 ◦C until they could be analyzed. Another part of the harvested

Molecules 2017, 22, 1058
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3.5.1. Analysis
Plant samples (1 g) were ground into powder, transferred to volumetric flasks, and ultrasonically
treated for 15 min with 10 mL of methanol. The extracts were filtered, and fil◦trate residues were
reextracted twice. The combined extracts were rotary-evaporated to dryness at 65 C, and the residues
were transferred in 2 mL of methanol to a solid-phase extraction (SPE) cartridge (AccuBond C18 SPE,
Agilent Technologies). The SPE columns were eluted in another 2 mL of methanol, and the collected
solution was evaporated to a volume of 1 mL. The resulting solutions were diluted with methanol
and used for HPLC analysis. The HPLC analyses were performed according to above mentioned
procedures. The recovery rate ranged from 85% to 96%, which was acceptable for the test.
3.5.2. Assessments of Systemic Insecticidal Activity
The harvested fresh leaves were placed into Petri dishes (9 cm in diameter). Ten P. xylostella
◦
larvae were then introduced into each dish. The Petri dishes were incubated at 26 C and 70% relative
humidity under a photoperiod of 16:8 h (light/dark). All treatments were repeated three times.
Mortalities were determined at 48 h and 72 h post treatment.
3.6. Physicochemical Properties
The physicochemical properties (molecular mass [MW], the octanol : water partition coefficient
[logD], acid dissociation constant polar [pKa], number of hydrogen bond donors [HBDs], and number
of hydrogen bond acceptors [HBAs]) of the six conjugates, tralopyril and chlorfenapyr were predicted
using ACD LogD suite version 12.01 software (Advanced Chemistry Development, Inc., Toronto, ON,
Canada).
4. Conclusions
In summary, six new conjugates containing tralopyril and pyranose moieties (glucose, methyl
glucuronate or glucuronic acid) were synthesized via click chemistry. The applied mild reaction
conditions provided a application case for the addition of a sugar moiety to pesticides. No phytotoxicity
symptoms were observed up to 63 days after smearing 2 mM of the six conjugates on seedlings, which
indicated that the introduction of a sugar group may solve the difficult phytotoxicity problem of
tralopyril. The phloem mobility tests on Ricinus system demonstrated that the presence of a glucuronic
acid core conferred more phloem mobility to tralopyril than glucose. The translocation of conjugates 12
and 18 with a glucuronic acid core may involve two components of carrier-mediated systems and
an ion trap mechanism. This inference requires further confirmation. Insecticidal activity results
indicated that the methoxymethyl group on the pyrrole N of tralopyril moiety was a determinant
factor in the insecticidal activity, and the pyranose structure also affects the insecticidal activity.
Their insecticidal mechanism of action should be confirmed by obtaining direct evidence in further
experiments. Based on the systemic mobility and systemic insecticidal activity test results, conjugate 18
with a glucuronic acid group and methoxymethyl group on the pyrrole N of tralopyril could be a
promising systemic pro-insecticide.
Acknowledgments: The authors would like to acknowledge and thank Jean-François Chollet for providing the
physicochemical property calculations. This reseach was supported by the National Natural Science Foundation
of China (Grant No. 31301704) and Independent Innovation Project of Guizhou academy of agricultural sciences
(Grant No. [2014]005 and [2014]009).
Author Contributions: Chen, Y.; Lei, Z.W. and Yang, W. conceived, designed and analyzed the data;
Liu, H.F.; Zhang, Y. and Chen, Y. performed the experiments; Zhou, Y.F. and Yang, M.F. contributed
reagents/materials/analysis tools; Chen, Y. and Yang, W. wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.

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References
1.
Brück, E.; Elbert, A.; Fischer, R.; Krueger, S.; Kühnhold, J.; Klueken, A.M.; Nauen, R.; Niebes, J.F.;
Reckmann, U.; Schnorbach, H.J.; et al. Movento , an innovative ambimobile insecticide for sucking
®
insect pest control in agriculture: Biological profile and field performance. Crop Prot. 2009, 28, 838–844.
[CrossRef]
2.
3.
4.
Chollet, J.; Rocher, F.; Jousse, C.; Delétage-Grandon, C.; Bashiardes, G.; Bonnemain, J.L. Synthesis and
phloem mobility of acidic derivatives of the fungicide fenpiclonil. Pest Manag. Sci. 2004, 60, 1063–1072.
[CrossRef] [PubMed]
Jiang, D.X.; Lu, X.L.; Hu, S.; Zhang, X.B.; Xu, H.H. A new derivative of fipronil: Effect of adding a glycinyl
group to the 5-amine of pyrazole on phloem mobility and insecticidal activity. Pestic. Biochem. Phys. 2009, 95,
126–130. [CrossRef]
Hsu, F.C.; Sun, K.; Kleier, D.A.; Fielding, M.J. Phloem mobility of xenobiotics VI. A phloem-mobile
pro-nematicide based on oxamyl exhibiting root-specific activation in transgenic tobacco. Pestic. Sci. 1995
44, 9–19. [CrossRef]
,
5.
6.
Yang, W.; Wu, H.X.; Xu, H.H.; Hu, A.L.; Lu, M.L. Synthesis of Glucose–Fipronil conjugate and its phloem
mobility. J. Agric. Food Chem. 2011, 59, 12534–12542. [CrossRef] [PubMed]
Wu, H.X.; Yang, W.; Zhang, Z.X.; Huang, T.; Yao, G.K.; Xu, H.H. Uptake and phloem transport of
glucose-fipronil conjugate in ricinus communis tnvolve a carrier-mediated mechanism. J. Agric. Food Chem.
2012, 60, 6088–6094. [CrossRef] [PubMed]
7.
8.
Yuan, J.G.; Wu, H.X.; Lu, M.L.; Song, G.P.; Xu, H.H. Synthesis of a series of monosaccharide–fipronil
conjugates and their phloem mobility. J. Agric. Food Chem. 2013, 61, 4236–4241. [CrossRef] [PubMed]
Lei, Z.W.; Wang, J.; Mao, G.L.; Wen, Y.J.; Tian, Y.Q.; Wu, H.X.; Li, Y.F.; Xu, H.H. Glucose positions affect
the phloem mobility of glucose-fipronil conjugates. J. Agric. Food Chem. 2014, 62, 6065–6071. [CrossRef]
[PubMed]
9.
Qin, P.W.; Wang, J.; Wang, H.; Wen, Y.J.; Lu, M.L.; Li, Y.F.; Xu, Y.S.; Xu, H.H. Synthesis of
rotenone-O-monosaccharide derivatives and their phloem mobility. J. Agric. Food Chem. 2014, 62, 4521–4527.
[CrossRef] [PubMed]
10. Wu, H.X.; Marhadour, S.; Lei, Z.W.; Yang, W.; Marivingt-Mounir, C.; Bonnemain, J.L.; Chollet, J.F.
Vectorization of agrochemicals: amino acid carriers are more efficient than sugar carriers to translocate
phenylpyrrole conjugates in the Ricinus system. Environ. Sci. Pollut. Res. 2016. [CrossRef] [PubMed]
11. Albers, P.H.; Klein, P.N.; Green, D.E.; Melancon, M.J.; Bradley, B.P.; Noguchi, G. Chlorfenapyr and mallard
ducks: Overview, study design, macroscopic effects, and analytical chemistry. Environ. Toxicol. Chem. 2006
25, 438–445. [CrossRef] [PubMed]
,
12. Liu, A.P.; Ming, T.; Yu, S.Y.; Liu, X.P.; Wang, X.G.; Hui, P.; Wu, D.X.; Li, H.; Ou, X.M.; Huang, M.Z.; et al.
Synthesis, insecticidal and acaricidal activities of novel 2-arylpyrroles. Sci. China Chem. 2013, 56, 117–123.
[CrossRef]
13. Rahman, M.M.; Choi, J.H.; El-Aty, A.M.A.; Park, J.H.; Park, J.Y.; Im, G.J.; Shim, J.H. Determination of
chlorfenapyr in leek grown under greenhouse conditions with GC-
µECD and confirmation by mass
spectrometry. Biomed. Chromatogr. 2012, 26, 172–177. [CrossRef] [PubMed]
14. Yang, W.; Chen, Y.; Zhang, Y.; Gao, X.B.; Zhou, Y.F. Effects of introducing theanine or glutamic acid core to
tralopyril on systemicity and insecticidal activity. Pestic. Biochem. Phys. 2016. [CrossRef]
15. Cagnoni, A.J.; Varela, O.; Gouin, S.G.; Kovensky, J.; Uhrig, M.L. Synthesis of multivalent glycoclusters from
1-thio-β-D-galactose and their inhibitory activity against the β-galactosidase from E. coli. J. Org. Chem. 2011,
76, 3064–3077. [CrossRef] [PubMed]
16. Wang, J.; Lei, Z.W.; Wen, Y.J.; Mao, G.L.; Wu, H.X.; Xu, H.H. A novel fluorescent conjugate applicable to
visualize the translocation of glucose–fipronil. J. Agric. Food Chem. 2014, 62, 8791–8798. [CrossRef] [PubMed]
17. Ma, Q.; Liu, Y.; Zhang, P.; Li, Y.; Xiong, L.; Wang, Q. Design, synthesis, and biological evaluation of various
α
-substituted benzylpyrroles based on the structures of insecticidal chlorfenapyr and natural pyrrolomycins.
J. Agric. Food Chem. 2014, 62, 6072–6081. [CrossRef] [PubMed]
18. Roy, S.; Mukhopadhyay, A.; Gurusubramanian, G. Field efficacy of a biopesticide prepared from Clerodendrum
viscosum Vent. (Verbenaceae) against two major tea pests in the sub Himalayan tea plantation of North
Bengal, India. J. Pest. Sci. 2010, 83, 371–377. [CrossRef]

Molecules 2017, 22, 1058
16 of 16
19. Roy, S.; Rahman, A.; Phukan, A.K.; Muraleedharan, N.N. Terminalia chebula Retz. (Combretaceae): Source
of a botanical acaricide against Oligonychus coffeae Nietner (Acarina: Tetranychidae). Int. J. Acarol. 2014, 40,
138–144. [CrossRef]
20. Delétagegrandon, C.; Chollet, J.F.; Faucher, M.; Rocher, F.; Komor, E.; Bonnemain, J.L. Carrier-mediated
uptake and phloem systemy of a 350-Dalton chlorinated xenobiotic with an
α-amino acid function.
Plant Physiol. 2001, 125, 1620–1632. [CrossRef]
21. Bhal, S.K.; Kassam, K.I.; Peirson, I.G.; Pearl, G.M. The rule of five revisited: applying log D in place of log P
in drug-likeness filters. Mol. Pharm. 2007, 4, 556–560. [CrossRef] [PubMed]
22. Rocher, F.; Chollet, J.F.; Legros, S.; Jousse, C.; Lemoine, R.; Faucher, M.; Bush, D.R.; Bonnemain, J.L. Salicylic
acid transport in Ricinus communis involves a pH-dependent carrier system in addition to diffusion.
Plant Physiol. 2009, 150, 2081–2091. [CrossRef] [PubMed]
23. Lipinski, C.A.; Lombardo, F.; Dominy, B.W.; Feeney, P.J. Experimental and computational approaches to
estimate solubility and permeability in drug discovery and development settings. Adv. Drug Deliv. Rev. 2012
,
64, 4–17. [CrossRef]
24. Kallarackal, J.; Orlich, G.; Schobert, C.; Komor, E. Sucrose transport into the phloem of Ricinus communis L.
seedlings as measured by the analysis of sieve-tube sap. Planta 1989, 177, 327–335. [CrossRef] [PubMed]
25. Kleier, D.A. Phloem mobility of xenobiotics: I. Mathematical model unifying the weak acid and intermediate
permeability theories. Plant Physiol. 1988, 86, 803–810. [CrossRef] [PubMed]
26. Kleier, D.A. Phloem mobility of xenobiotics. V. Structural requirements for phloem-systemic pesticides.
Pestic. Sci. 1994, 42, 1–11. [CrossRef]
Sample Availability: Samples of the compounds 2, 4 and 7–18 are available from the authors.
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