Synthesis of Piperine Analogs Containing Isoxazoline/Pyrazoline Scaffold and Their Pesticidal Bioactivities

Article abstract of DOI:10.1021/acs.jafc.8b03690

In continuation of our program to discover new potential pesticidal agents, thirty-one piperine analogs containing isoxazoline/pyrazoline scaffold were prepared, and confirmed by infrared spectra, proton/carbon-13 nuclear magnetic resonance spectra, and high-resolution mass spectra. The structures of compounds VIIb and VIIIc were further determined by ¹H-¹H COSY spectra. Especially the configuration of compound VIIIc was unambiguously confirmed by single-crystal X-ray diffraction. Their pesticidal activities were evaluated against three serious and typically crop-threatening agricultural pests, Tetranychus cinnabarinus Boisduval (spider mite), Mythimna separata Walker (Oriental armyworm), and Plutella xylostella Linnaeus (diamondback moth). Compounds VIIIb and VIIIc exhibited greater than 40-fold more potent acaricidal activity than the lead compound piperine against T. cinnabarinus. Notably, compounds VIa-c exhibited more pronounced oral toxicity against P. xylostella than toosendanin; compounds VIb and VIc displayed more promising growth inhibitory activity against M. separata than toosendanin. It demonstrated that the methylenedioxy and isoxazoline scaffolds were important for the oral toxicity and growth inhibitory activity against P. xylostella and M. separata, respectively; the ethylenedioxy and isoxazoline scaffolds were vital for the acaricidal activity against T. cinnabarinus. Moreover, compounds VIb, VIIf, and VIIIc showed very low toxicity against NRK-52E cells.

Full text of DOI:10.1021/acs.jafc.8b03690

Subscriber access provided by UNIV TEXAS SW MEDICAL CENTER
Agricultural and Environmental Chemistry
Synthesis of Piperine Analogs Containing Isoxazoline/
Pyrazoline Scaffold and Their Pesticidal Bioactivities
ruige yang, Min Lv, and Hui Xu
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03690 • Publication Date (Web): 08 Oct 2018
Downloaded from http://pubs.acs.org on October 8, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 46
Journal of Agricultural and Food Chemistry
Synthesis of Piperine Analogs Containing Isoxazoline/Pyrazoline
Scaffold and Their Pesticidal Bioactivities
†
,‡
†,‡
,
†
Ruige Yang , Min Lv , and Hui Xu*
†
Research Institute of Pesticidal Design & Synthesis, College of Chemistry and
Pharmacy/Plant Protection, Northwest A&F University, Yangling 712100, Shaanxi Province,
China
‡
These authors contributed equally to this work.
*H. Xu, Tel: 8629ꢀ8709ꢀ1952. Fax: 8629ꢀ8709ꢀ1952. Eꢀmail: orgxuhui@nwsuaf.edu.cn.
1
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 2 of 46
1
Abstract
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
In continuation of our program to discover new potential pesticidal agents, thirtyꢀone piperine
analogs containing isoxazoline/pyrazoline scaffold were prepared, and confirmed by infrared
spectra, proton/carbonꢀ13 nuclear magnetic resonance spectra, and highꢀresolution mass
1
1
spectra. The structures of compounds VIIb and VIIIc were further determined by Hꢀ H
COSY spectra. Especially the configuration of compound VIIIc was unambiguously
confirmed by singleꢀcrystal Xꢀray diffraction. Their pesticidal activities were evaluated
against three serious and typically cropꢀthreatening agricultural pests, Tetranychus
cinnabarinus Boisduval (spider mite), Mythimna separata Walker (Oriental armyworm) and
Plutella xylostella Linnaeus (diamondback moth). Compounds VIIIb and VIIIc exhibited
greater than 40 folds more potent acaricidal activity than the lead compound piperine against
T. cinnabarinus. Notably, compounds VIa–c exhibited more pronounced oral toxicity against
P. xylostella than toosendanin; compounds VIb and VIc displayed more promising growth
inhibitory activity against M. separata than toosendanin. It demonstrated that the
methylenedioxy and isoxazoline scaffolds were important for the oral toxicity and growth
inhibitory activity against P. xylostella and M. separata, respectively; the ethylenedioxy and
isoxazoline scaffolds were vital for the acaricidal activity against T. cinnabarinus. Moreover,
compounds VIb, VIIf and VIIIc showed very low toxicity against NRKꢀ52E cells.
1
1
1
1
1
1
1
1
1
1
2
2
2
KEYWORDS: Piperine, Structural modification, Acaricidal activity, Oral toxicity, Growth
inhibitory activity
2
ACS Paragon Plus Environment

Page 3 of 46
Journal of Agricultural and Food Chemistry
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
INTRODUCTION
Tetranychus cinnabarinus Boisduval (spider mite), Mythimna separata Walker (Oriental
armyworm) and Plutella xylostella Linnaeus (diamondback moth) are three serious and
typical agricultureꢀthreatening pests, and their outbreaks can result in a significant loss of
1
ꢀ4
crops. For instance, due to the intermittent outbreaks of thirdꢀgeneration larvae of M.
5
separata occurring in 2012, nearly 4 million hectares of crops in China were entirely lost.
On the other hand, because of extensive and unreasonable application of synthetic
agrochemicals to deal with pests outbreaks, currently, resistances in pest populations, and
6
ꢀ12
negative impacts on human health and environment have been simultaneously developed.
Therefore, discovery of the potential alternatives to efficiently control pests for crop
protection is highly urgent. Natural products could play an important role for affording lead
13ꢀ26
compounds in the discovery of pesticide candidates.
Piperine (Figure 1) is isolated as a simple alkaloid from Piper nigrum Linn., and exhibits
lots of biological properties such as antiꢀinflammatory, antimicrobial, antitumor, and pesticidal
2
7,28
activities.
In addition, molecules containing isoxazoline (I) or pyrazoline (II, Figure 1)
fragment show antimicrobial, fungicidal, mosquitocidal, antiꢀAlzheimer, antiꢀcancer,
2
9ꢀ39
monoamine oxidase inhibitory, pesticidal, or antiꢀinflammatory activities.
Previously, we
studied isoxazolopodophyllic acidꢀbased esters (III, Figure 1), isoxazolopodophyllolꢀbased
esters (IV, Figure 1) and isoxazolopodophyllalꢀbased hydrazones (V, Figure 1) from
podophyllotoxin, and found some derivatives showed more promising insecticidal activity
4
0,41
than toosendanin, a commercial botanical insecticide isolated from Melia azedarach.
Based upon the above results, and in continuation of our program aimed at the development
42ꢀ44
of new potential pesticidal agents,
therefore, a series of piperine analogs containing
3
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 4 of 46
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
6
6
6
6
6
6
6
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
isoxazoline/pyrazoline scaffold (VI–IX, Figure 1) were prepared. Their insecticidal and
acaricidal activities were evaluated against M. separata, T. cinnabarinus and P. xylostella.
MATERIALS AND METHODS
Chemicals. All reagents and solvents were of reagent grade or purified according to standard
methods before use. All the different substituted acetophenone, hydroxylamine hydrochloride,
all the different substituted phenylhydrazine hydrochloride, piperidine, lithium aluminium
4 3 2
hydride (LiAlH ), aluminum chloride (AlCl ), manganese dioxide (MnO ), sodium hydride
(NaH) and trimethyl phosphonoacetate were purchased from Aladdin Chemistry Co., Ltd.
(Shanghai, China). N,NꢀDimethylformamide (DMF), ethyl acetate (EA), petroleum ether (PE),
dichloroethane (DCE), dichloromethane (DCM), absolute methanol (MeOH), and absolute
ethanol (EtOH) were analyticalꢀgrade and purchased from Bodi Chemical Co., Ltd. (Tianjin,
China). Malonic acid, pyridine, potassium carbonate (K
2 3 4
CO ), ammonium chloride (NH Cl),
anhydrous sodium sulfate (Na SO ), sodium chloride (NaCl), sodium hydroxide (NaOH) and
2
4
potassium hydroxide (KOH) were purchased from Kelong Chemical Reagent Co., Ltd.
(Chengdu, China). Hydrochloric acid (HCl) and sulfuric acid (H SO ) were purchased from
2
4
Luoyang Chemical Reagent Factory (Luoyang, China). Analytical thinꢀlayer chromatography
(TLC) was performed on silica gel plates using silica gel 60 GF254 (Qingdao Haiyang
Chemical Co., Ltd., Qingdao, China). Silica gel column chromatography was performed with
silica gel 200−300 mesh (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China). An
intermediate, (2E,4E)ꢀ5ꢀ(1,3ꢀbenzodioxolꢀ5ꢀyl)ꢀ2,4ꢀpentadienal (2, 37% yield for four steps
28
from piperine (1)) (Figure 2), was prepared as previously described.
Instruments. Melting point (mp) was determined using the XTꢀ4 digital melting point
apparatus (Beijing Tech Instrument Ltd., Beijing, China). Infrared (IR) spectra were measured
4
ACS Paragon Plus Environment

Page 5 of 46
Journal of Agricultural and Food Chemistry
6
7
7
7
7
7
7
7
7
7
7
9
0
1
2
3
4
5
6
7
8
9
by a TENSOR 27 spectrometer (Bruker, Ettlingen, Germany). Nuclear magnetic resonance
1
13
spectra ( H and C NMR) were carried out on an Avance III 500 MHz equipment (Bruker,
Karlsruhe, Germany). Highꢀresolution mass spectra (HRMS) were carried out on a LTQ FT
Ultra instrument (Thermo Fisher Scientific Inc., MA). Xꢀray crystallography was recorded on
a SMART APEX II equipment (Bruker, Karlsruhe, Germany).
General Procedure for Synthesis of Compounds 4a–c.
A
solution of
(2E,4E)ꢀ5ꢀ(1,3ꢀbenzodioxolꢀ5ꢀyl)ꢀ2,4ꢀpentadienal (2, 2.0 mmol), appropriate substituted
acetophenone (2.0 mmol) and KOH (0.4 mmol) in EtOH (10 mL) was stirred at room
temperature for 2–11 h. When the reaction was complete checked by TLC analysis, the
precipitate was collected by filtration, washed with water (2 mL×2) and EtOH (1 mL×2), and
dried to afford compounds 4a–c in 57–76% yields.
o
1
8
8
8
8
8
8
8
8
8
8
9
0
1
2
3
4
5
6
7
8
9
0
Data for Compound 4a: Yield: 57%, yellow solid. Mp: 132–134 C. H NMR (500 MHz,
CDCl ) δ: 7.96 (d, J = 7.0 Hz, 2H), 7.46–7.57 (m, 4H), 6.98–7.01 (m, 2H), 6.90 (dd, J = 1.0,
3
8.0 Hz, 1H), 6.67–6.79 (m, 4H), 6.52–6.57 (m, 1H), 5.98 (s, 2H). HRMS (ESI): Calcd for
+
C H O ([M+H] ), 305.1172; found, 305.1172.
20
17
3
o
1
Data for Compound 4b: Yield: 76%, yellow solid. Mp: 145–147 C. H NMR (500 MHz,
CDCl ) δ: 8.00 (dd, J = 5.5, 8.5 Hz, 2H), 7.55 (dd, J = 11.5, 14.5 Hz, 1H), 7.13–7.16 (m, 2H),
3
6.95–6.99 (m, 2H), 6.91 (d, J = 8.0 Hz, 1H), 6.68–6.79 (m, 4H), 6.56 (dd, J = 11.5, 14.0 Hz,
+
1H), 5.98 (s, 2H). HRMS (ESI): Calcd for C20
H
16
O
3
F ([M+H] ), 323.1078; found, 323.1077.
o
1
Data for Compound 4c: Yield: 69%, yellow solid. Mp: 140–142 C. H NMR (500 MHz,
CDCl ) δ: 7.91 (d, J = 8.0 Hz, 2H), 7.50–7.56 (m, 1H), 7.45 (d, J = 7.5 Hz, 2H), 6.89–6.99
3
(m, 3H), 6.68–6.83 (m, 4H), 6.50–6.57 (m, 1H), 5.98 (s, 2H). HRMS (ESI): Calcd for
5
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 6 of 46
+
9
9
9
9
9
9
9
9
9
1
2
3
4
5
6
7
8
9
C
20
H
16
O
3
Cl ([M+H] ), 339.0782; found, 339.0782.
Synthesis of 2,3-Dihydrobenzo[b][1,4]dioxine-6-carbaldehyde (6). A solution of
3,4ꢀdihydroxybenzaldehyde (5, 0.087 mol), DCE (0.174 mol) and K CO (0.174 mol) in
2
3
DMF (150 mL) was stirred at 105 °C. After 4 h, the mixture was cooled and filtered. The
filtered cake was washed with EA (100 mL). The filtrate was diluted with water (100 mL)
and extracted with EA (200 mL×3). The combined organic phase was washed by brine (200
mL×3), dried over anhydrous Na
2 4
SO , concentrated in vacuo, and purified by silica gel
column chromatography (PE:EA = 4:1–2:1, v/v) to afford compound 6 in 90% yield as a
o
o
45 1
white solid. Data for Compound 6: CAS: 29668ꢀ44ꢀ8. Mp: 49–51 C (lit., mp 51 C).
H
1
1
00
01
NMR (500 MHz, CDCl ) δ: 9.82 (s, 1H), 7.39–7.40 (m, 2H), 6.97–6.99 (m, 1H), 4.33–4.34
3
(m, 2H), 4.28–4.30 (m, 2H).
1
1
1
1
1
1
1
1
1
1
1
02
03
04
05
06
07
08
09
10
11
12
Synthesis of Methyl (E)-3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)acrylate (8). A solution
of compound 6 (0.047 mol), malonic acid (0.07 mol) and piperidine (1.4 mL) in pyridine (14
mL) was stirred at 85 °C. When the reaction was complete checked by TLC analysis after 6
h, the mixture was cooled to room temperature and poured into ice water (30 mL). Then the
pH value was adjusted to pH 5–6 by 1 M aq. HCl. The precipitate was collected by filtration
and washed with ice water to afford (E)ꢀ3ꢀ(2,3ꢀdihydrobenzo[b][1,4]dioxinꢀ6ꢀyl)acrylic acid
(7) as a white solid, which was used directly for the next step without further purification.
2 4
When a solution of compound 7 (0.036 mol) and conc. H SO (fourteen drops) in MeOH
(100 mL) was refluxed for 12 h, the mixture was cooled to room temperature and the white
precipitate (8) was collected by filtration. Data for Compound 8: Yield: 85% (for two steps
o
o
46 1
3
from compound 6 to 8). Mp: 66–68 C (lit., mp 66–68 C). H NMR (500 MHz, CDCl ) δ:
6
ACS Paragon Plus Environment

Page 7 of 46
Journal of Agricultural and Food Chemistry
1
1
13
14
7.59 (d, J = 16.0 Hz, 1H), 7.01–7.05 (m, 2H), 6.86 (d, J = 8.0 Hz, 1H), 6.29 (d, J = 16.0 Hz,
1H), 4.26–4.28 (m, 4H), 3.78 (s, 3H).
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
Synthesis of (E)-3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)acrylaldehyde (10). A solution of
compound 8 (0.035 mol) in THF (100 mL) was added dropwise to a wellꢀstirred suspension
of LiAlH (0.105 mol) and AlCl (0.035 mol) in dry THF (30 mL) at 0 °C. The mixture was
4
3
stirred at this temperature for 2 h and quenched by dropwise addition of ice water (2 mL).
The solid was removed by filtration and washed with DCM/MeOH (5:1, v/v). The filtrate was
2 4
dried over anhydrous Na SO , concentrated in vacuo, and purified by silica gel column
chromatography to afford compound 9 in 81% yield as a light yellow oil. Subsequently, a
solution of compound 9 (0.03 mol) and MnO (0.30 mol) in dry THF (100 mL) was refluxed
2
for 4 h. The solid was removed by filtration and washed with DCM. The filtrate was
concentrated in vacuo, and purified by silica gel column chromatography (PE:EA:DCM =
5:1:1, v/v/v) to give compound 10 in 52% yield as a white solid. Data for Compound 10:
o
47 1
CAS: 261913ꢀ24ꢀ0. Mp: 69–71 C (lit., not reported). H NMR (500 MHz, CDCl
(d, J = 7.5 Hz, 1H), 7.36 (d, J = 16.0 Hz, 1H), 7.06–7.10 (m, 2H), 6.91 (d, J = 8.5 Hz, 1H),
6.59 (dd, J = 7.5, 15.5 Hz, 1H) 4.30–4.31 (m, 2H), 4.27–4.29 (m, 2H).
3
) δ: 9.65
,
Synthesis of Methyl (2E,4E)-5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)penta-2,4-dienoate
(11). To a stirred solution of NaH (0.044 mol) (60% dispersion in mineral oil) in dry THF
(180 mL) was added trimethyl phosphonoacetate (0.026 mol) dropwise at 0 °C. After adding,
a solution of compound 10 (0.022 mol) in dry THF (50 mL) was added to the above mixture
at 0 °C. When the reaction was complete checked by TLC analysis after 1.5 h, it was
quenched with saturated aq. NH
4
Cl (15 mL). Water (50 mL) was added to the solution, which
7
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 8 of 46
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
was extracted with EA (100 mL×3). The combined organic phase was dried over anhydrous
Na
2 4
SO , concentrated in vacuo, and purified by silica gel column chromatography (PE:EA =
5:1–3:1, v/v) to afford compound 11 in 90% yield as a white solid. Data for Compound 11:
o
1
3
Mp: 91–93 C. H NMR (500 MHz, CDCl ) δ: 7.44 (dd, J = 10.5, 15.0 Hz, 1H), 6.94–6.98 (m,
2H), 6.84 (d, J = 8.5 Hz, 1H), 6.68–6.80 (m, 2H), 5.95 (d, J = 15.0 Hz, 1H), 4.25–4.28 (m,
4H), 3.76 (s, 3H).
Synthesis of (2E,4E)-5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)penta-2,4-dienal (13). A
solution of compound 11 (0.015 mol) in THF (80 mL) was added dropwise to a wellꢀstirred
4 3
suspension of LiAlH (0.045 mol) and AlCl (0.015 mol) in dry THF (20 mL) at 0°C. After
adding, the reaction mixture was stirred at this temperature for 2 h and quenched by dropwise
addition of ice water (2 mL). The solid was removed by filtration and washed with
DCM/MeOH (5/1, v/v). The filtrate was dried over anhydrous Na
vacuo to afford compound 12, which was used directly for the next step without further
purification. A solution of compound 12 (0.01 mol) and MnO (0.10 mol) in dry THF (60 mL)
2 4
SO , and concentrated in
2
was refluxed for 4 h. The solid was removed by filtration and washed with DCM. The filtrate
was concentrated in vacuo, and purified by silica gel column chromatography (PE:EA:MeOH
= 10:2:1, v/v/v) to afford compound 13 as a yellow solid. Data for Compound 13: Yield: 51%
o
1
3
(for two steps from compound 11 to 13). Mp: 114–115 C. H NMR (500 MHz, CDCl ) δ:
9.59 (d, J = 8.0 Hz, 1H), 7.20–7.25 (m, 1H), 6.99–7.02 (m, 2H), 6.81–6.91 (m, 3H), 6.24 (dd,
J = 8.0, 15.5 Hz, 1H), 4.26–4.29 (m, 4H).
General Procedure for Synthesis of Compounds 14a–c. A solution of compound 13 (2.0
mmol), appropriate substituted acetophenone (2.0 mmol) and KOH (0.4 mmol) in EtOH (10
mL) was stirred at room temperature for 8–12 h. When the reaction was complete checked by
8
ACS Paragon Plus Environment

Page 9 of 46
Journal of Agricultural and Food Chemistry
1
1
58
59
TLC analysis, the precipitate was collected, washed with water (2 mL×2) and EtOH (1
mL×2), and dried to afford compounds 14a–c in 65–72% yields.
o
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
Data for Compound 14a: Yield: 65%, yellow solid. Mp: 134–136 C. H NMR (500 MHz,
CDCl ) δ: 7.96 (d, J = 7.5 Hz, 2H), 7.46–7.57 (m, 4H), 6.94–7.00 (m, 3H), 6.74–6.84 (m, 3H),
6.67 (d, J = 14.5 Hz, 1H), 6.51–6.56 (m, 1H), 4.27 (s, 4H). HRMS (ESI): Calcd for C H O
3
2
1
19
3
+
([M+H] ), 319.1329; found, 319.1328.
Data for Compound 14b: Yield: 71%, yellow solid. Mp: 168–170 C. H NMR (500 MHz,
CDCl ) δ: 7.97–8.00 (m, 2H), 7.50–7.56 (m, 1H), 7.12–7.16 (m, 2H), 6.94–6.98 (m, 3H),
o
1
3
6.74–6.84 (m, 3H), 6.68 (d, J = 14.5 Hz, 1H), 6.55 (t, J = 12.5 Hz, 1H), 4.27 (s, 4H). HRMS
+
(ESI): Calcd for C H FO ([M+H] ), 337.1234; found, 337.1239.
2
1
18
3
o
1
Data for Compound 14c: Yield: 72%, yellow solid. Mp: 152–154 C. H NMR (500 MHz,
CDCl ) δ: 7.89–7.93 (m, 2H), 7.45–7.55 (m, 3H), 6.96–7.01 (m, 3H), 6.74–6.84 (m, 3H),
3
6.68 (d, J = 11.5 Hz, 1H), 6.50–6.55 (m, 1H), 4.27 (s, 4H). HRMS (ESI): Calcd for
+
C
21
H
18ClO
3
([M+H] ), 353.0939; found, 353.0937.
General Procedure for Synthesis of Compounds VIa–c and VIIIb,c. A mixture of
compounds 4a–c or 14b,c (0.2 mmol), hydroxylamine hydrochloride (0.5 mmol), and NaOH
(1.2 mmol) in absolute EtOH (2 mL) was stirred at room temperature for 3 h and then at
65 °C for 6–9 h. When the reaction was complete checked by TLC analysis, the mixture was
cooled and filtered. The filtered cake was washed with ice water (1 mL×2) and cold EtOH (1
mL×2), and then dried to afford compounds VIa–c and VIIIb,c in 5–45% yields. Exemplary
data for compounds VIa and VIIIb are as follows:
o
ꢀ1
Data for Compound VIa: Yield: 23%, pale yellow solid. Mp: 125–127 C. IR cm (KBr):
9
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 10 of 46
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
00
01
02
3024, 2921, 2851, 1502, 1488, 1445, 1252, 1041, 988, 759, 692; H NMR (500 MHz,
DMSOꢀd ) δ: 7.67–7.69 (m, 2H), 7.45–7.46 (m, 3H), 7.15 (s, 1H), 6.86–6.92 (m, 2H), 6.83
(dd, J = 10.5, 15.5 Hz, 1H, Hꢀ3’), 6.61 (d, J = 15.5 Hz, 1H, Hꢀ4’), 6.50 (dd, J = 11.0, 15.0 Hz,
1H, Hꢀ2’), 6.01 (s, 2H, OCH O), 5.91 (dd, J = 7.5, 15.0 Hz, 1H, Hꢀ1’), 5.20–5.25 (m, 1H,
Hꢀ5), 3.63 (dd, J = 10.5, 16.5 Hz, 1H, Hꢀ4), 3.25 (dd, J = 9.0, 17.0 Hz, 1H, Hꢀ4). C NMR
(125 MHz, DMSOꢀd ) δ: 157.2, 148.3, 147.6, 133.85, 133.80, 131.7, 131.2, 130.5, 129.9,
6
2
1
3
6
129.2, 127.0, 126.7, 122.1, 108.8, 105.8, 101.6, 81.9, 40.4. HRMS (ESI): Calcd for
+
C
26
H
18
O
3
N ([M+H] ), 320.1281; found, 320.1281.
o
ꢀ1
Data for Compound VIIIb: Yield: 45%, pale yellow solid. Mp: 198–200 C. IR cm (KBr):
1
3000, 2920, 2875, 1604, 1457, 1295, 1069, 988, 865, 668; H NMR (500 MHz, DMSOꢀd ) δ:
6
7.71–7.74 (m, 2H), 7.28–7.31 (m, 2H), 6.99 (s, 1H), 6.95 (d, J = 8.0 Hz, 1H), 6.75–6.82 (m,
2H), 6.57 (d, J = 15.5 Hz, 1H, Hꢀ4’), 6.44–6.49 (m, 1H, Hꢀ2’), 5.91 (dd, J = 7.5, 15.0 Hz, 1H,
Hꢀ1’), 5.19–5.24 (m, 1H, Hꢀ5), 4.23 (s, 4H, OCH
2
CH
2
O), 3.62 (dd, J = 10.5, 16.5 Hz, 1H,
) δ: 164.4 (J =
1
3
Hꢀ4), 3.24 (dd, J = 8.5, 17.0 Hz, 1H, Hꢀ4). C NMR (125 MHz, DMSOꢀd
6
246.2 Hz), 156.4, 143.96, 143.91, 133.8, 133.6, 131.0, 130.7, 129.4 (J = 8.7 Hz), 126.6, 126.5,
120.3, 117.7, 116.4 (J = 22.5 Hz), 115.1, 82.1, 64.6, 64.5, 40.4. HRMS (ESI): Calcd for
+
C
27
H
24
O
2
N
2
Br ([M+H] ), 487.1016; found, 487.1015.
General Procedure for Synthesis of VIIa–m and IXa–m. A mixture of compounds 4a–c or
14a–c (0.2 mmol), substituted phenylhydrazine hydrochlorides (15a–e, 0.5 mmol), and
NaOH (1.2 mmol) in absolute EtOH (2 mL) was stirred at room temperature for 3 h and then
at 65 °C for 6–9 h. When the reaction was complete checked by TLC analysis, the mixture
was cooled and filtered. The filtered cake was washed with ice water (1 mL×2) and cold
EtOH (1 mL×2), and then dried to afford compounds VIIa–m and IXa–m in 27–87% yields.
1
0
ACS Paragon Plus Environment

Page 11 of 46
Journal of Agricultural and Food Chemistry
2
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Exemplary data for compounds VIIa,b and IXa,b are as follows:
o
ꢀ1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
Data for Compound VIIa: Yield: 59%, pale yellow solid. Mp: 106–108 C. IR cm (KBr):
1
3023, 2895, 1595, 1492, 1251, 1038, 984, 749, 691; H NMR (500 MHz, DMSOꢀd
6
) δ:
7.73–7.75 (m, 2H), 7.45 (t, J = 7.0 Hz, 2H), 7.35–7.38 (m, 1H), 7.22–7.25 (m, 2H), 7.16 (d, J
= 8.0 Hz, 2H), 7.10 (d, J = 1.0 Hz, 1H), 6.83–6.88 (m, 2H), 6.72–6.78 (m, 2H), 6.53 (d, J =
15.5 Hz, 1H), 6.46 (dd, J = 10.5, 15.0 Hz, 1H), 5.99 (s, 2H), 5.84 (dd, J = 7.5, 15.0 Hz, 1H),
13
5.00–5.05 (m, 1H), 3.67 (dd, J = 11.5, 17.0 Hz, 1H), 3.14 (dd, J = 6.0, 17.0 Hz, 1H).
C
NMR (125 MHz, DMSOꢀd ) δ: 148.5, 148.3, 147.4, 145.2, 132.9, 132.6, 132.4, 131.8, 129.3,
6
129.1, 126.9, 126.2, 126.1, 126.0, 121.9, 119.2, 113.9, 108.8, 105.6, 101.5, 62.1, 40.3. HRMS
+
(ESI): Calcd for C H O N ([M+H] ), 395.1754; found, 395.1753.
2
6
23
2
2
o
ꢀ1
Data for Compound VIIb: Yield: 87%, pale yellow solid. Mp: 117–119 C. IR cm (KBr):
1
3018, 2888, 1506, 1446, 1254, 1044, 982, 820, 690; H NMR (500 MHz, DMSOꢀd
6
) δ: 7.74
(d, J = 7.5 Hz, 2H), 7.36–7.44 (m, 3H), 7.07–7.14 (m, 5H), 6.84–6.89 (m, 2H), 6.78 (dd, J =
10.5, 15.0 Hz, 1H), 6.53 (d, J = 15.5 Hz, 1H), 6.47 (dd, J = 10.5, 15.0 Hz, 1H), 6.00 (s, 2H),
5.83 (dd, J = 7.5, 15.0 Hz, 1H), 4.97–5.02 (m, 1H), 3.66 (dd, J = 11.5, 17.0 Hz, 1H), 3.14 (dd,
1
3
6
J = 6.0, 17.0 Hz, 1H). C NMR (125 MHz, DMSOꢀd ) δ: 157.4 (J = 233.7 Hz), 148.8, 148.2,
147.4, 142.1, 132.9, 132.8, 132.6, 132.5, 131.8, 129.2, 129.1, 126.9, 126.1, 121.9, 115.9 (J =
22.5 Hz), 115.2 (J = 7.5 Hz), 108.8, 105.7, 101.5, 62.8, 40.5. HRMS (ESI): Calcd for
+
C
26
H
22
O
2
N
2
F ([M+H] ), 413.1660; found, 413.1659.
o
ꢀ1
Data for Compound IXa: Yield: 72%, pale yellow solid. Mp: 170–172 C. IR cm (KBr):
1
3024, 2926, 1596, 1508, 1379, 1290, 1068, 988, 750, 692; H NMR (500 MHz, DMSOꢀd
6
) δ:
7.74 (d, J = 7.0 Hz, 2H), 7.36–7.42 (m, 3H), 7.14–7.22 (m, 4H), 6.89–6.92 (m, 2H), 6.78 (d, J
= 6.0 Hz, 2H), 6.72 (dd, J = 11.0, 15.0 Hz, 1H), 6.40–6.48 (m, 2H), 5.86 (dd, J = 7.0, 14.5 Hz,
11
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 12 of 46
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
1H), 5.00–5.02 (m, 1H), 4.21 (s, 4H), 3.67 (dd, J = 11.5, 16.5 Hz, 1H), 3.13 (dd, J = 5.5, 17.5
1
3
6
Hz, 1H). C NMR (125 MHz, DMSOꢀd ) δ: 148.5, 145.2, 143.9, 143.7, 132.9, 132.65,
132.62, 132.4, 130.8, 129.3, 129.1, 126.9, 126.1, 120.1, 119.2, 117.6, 115.0, 113.8, 64.6, 64.5,
+
62.1, 40.4. HRMS (ESI): Calcd for C27
H
25
O
2
N
2
([M+H] ), 409.1911; found, 409.1910.
o
ꢀ1
Data for Compound IXb: Yield: 33%, Yellow solid. Mp: 100–102 C. IR cm (KBr): 3013,
1
2922, 1579, 1507, 1446, 1384, 1288, 1068, 988, 822, 691; H NMR (500 MHz, DMSOꢀd
6
) δ:
7.74 (d, J = 7.5 Hz, 2H), 7.44 (t, J = 7.5 Hz, 2H), 7.35–7.38 (m, 1H), 7.05–7.19 (m, 5H), 6.95
(s, 1H), 6.92 (d, J = 8.5 Hz, 1H), 6.79 (d, J = 8.0 Hz, 1H), 6.75 (dd, J = 11.0, 15.5 Hz, 1H),
6.41–6.49 (m, 2H), 5.84 (dd, J = 7.5, 15.0 Hz, 1H), 4.96–5.01 (m, 1H), 4.21 (s, 4H), 3.66 (dd,
1
3
J = 11.5, 17.0 Hz, 1H), 3.14 (dd, J = 6.0, 17.0 Hz, 1H). C NMR (125 MHz, DMSOꢀd ) δ:
6
157.4 (J = 233.7 Hz), 148.7, 143.9, 143.7, 142.1, 132.8, 132.7, 132.6, 132.4, 130.8, 129.2,
129.1, 126.9, 126.1, 120.1, 117.6, 115.9 (J = 21.2 Hz), 115.2 (J = 7.5 Hz), 115.0, 64.6, 64.4,
+
62.8, 40.5. HRMS (ESI): Calcd for C27
H
24
O
2
N
2
F ([M+H] ), 427.1816; found, 427.1816.
Biological Assay.
Acaricidal Activity of Compounds 4a
–
c, 14a
The acaricidal activity of compounds 4a
m against the female adults of T. cinnabarinus was assessed by
slideꢀdipping method. Spirodiclofen (a commercial acaricidal agent) was used as a positive
control. The solutions of compounds 4a c, 14a c, VIa c, VIIa m, VIIIb,c, IXa m and
–
c, VIa
–
c, VIIa
–
m, VIIIb,c and IXa
–
m against
4
8,49
Tetranychus cinnabarinus.
VIIa m, VIIIb,c and IXa
–c, 14a
–
c, VIa c,
–
–
–
–
–
–
–
–
spirodiclofen were prepared in acetone/deionized water (v/v = 1/1) at 0.5 mg/mL. For each
compound, 90−120 healthy and sizeꢀconsistency female adults of spider mites (30−40 mites
per group) were selected. 30−40 spider mites were adfixed dorsally in two lines to a strip of
doubleꢀcoated masking tape on a microscope slide by using a small brush. Then the slides
1
2
ACS Paragon Plus Environment

Page 13 of 46
Journal of Agricultural and Food Chemistry
2
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
were dipped into the corresponding solution for 5 s, and taken out. Excess solutions on the
slides were removed by filter paper. The slides treated with acetone/deionized water (v/v =
1/1) alone were used as a blank control group (CK). The experiment was carried out at 26 ±
1°C and 60−80% relative humidity (RH), and on 14 h/10 h (light/dark) photoperiod. The
results were checked by binocular dissecting microscope. Their mortalities were recorded at
48 h and 72 h after treatment. Their corrected mortality rate values were calculated as follows:
corrected mortality rate (%) = (T − C) × 100/(100% − C); C is the mortality rate of CK, and T
is the mortality rate of the treated T. cinnabarinus.
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
Oral Toxicity of Compounds 4a
–
c, 14a
Plutella xylostella. Thirty 3rdꢀinstar larvae of P. xylostella were chosen as the tested insects
for each compound. The solutions of compounds 4a c, 14a c, VIa c, VIIa m, VIIIb,c,
IXa m and toosendanin (a positive control) were prepared in acetone at 20 mg/mL. The
–c, VIa–c, VIIa–m, VIIIb,c and IXa–m against
16
–
–
–
–
–
corresponding solution (1 µL) was added to a fresh Brassica oleracea leaf disc (0.5×0.5 cm),
and dried. A fresh Brassica oleracea leaf disc was treated by acetone alone as the blank control
group (CK). One piece of the above discs was offered to and consumed by each insect, which
was raised in each well of 12ꢀ or 24ꢀwell culture plates for 48 h (temperature: 25 ± 2 °C; RH:
65–80%; photoperiod: light/dark = 16 h/8 h). Their corrected mortality rate values were
calculated as follows: corrected mortality rate (%) = (T − C) × 100/(100% − C); C is the
mortality rate of CK, and T is the mortality rate of the treated P. xylostella.
Growth Inhibitory Activity of Compounds 4a
–
c, 14a
Thirty early 3rdꢀinstar larvae of M. separata were chosen as
the tested insects for each compound. The solutions of compounds 4a c, 14a c, VIa c,
VIIa m, VIIIb,c, IXa m and toosendanin (a positive control) were prepared in acetone at 1
–c, VIa–c, VIIa–m, VIIIb,c and IXa–m
50,51
against Mythimna separata.
–
–
–
–
–
1
3
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 14 of 46
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
mg/mL. After dipped into the corresponding solution for 3 s, wheat leaf discs (1×1 cm) were
taken out, and dried. Wheat leaf discs were treated by acetone alone as the blank control
group (CK). Several above discs were added to each culture dish (ten insects per dish). Once
the discs were consumed, additional ones were added. After 48 h, the rest of
compoundꢀsoaked discs was cleaned out, and the untreated ones were added till the end of
pupae (temperature: 25 ± 2 °C; RH: 65–80%; photoperiod: light/dark = 12 h/12 h). Their
corrected mortality rate values were calculated as follows: corrected mortality rate (%) = (T −
C) × 100/ (100% − C); C is the mortality rate of CK, and T is the mortality rate of the treated
M. separata.
Cytotoxic Assay.
Normal rat kidney tubular epithelial cells (NRKꢀ52E) were purchased from the Chinese
Academy of Sciences Cell Bank (Shanghai, China). NRKꢀ52E cells, maintained in a 37 °C
2
humidified incubator with 5% CO , were grown in high glucose Dulbecco's Modified Eagle's
Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL
5
2
penicillin and 100 ꢀg/mL streptomycin. The cell line was routinely cultured in 6ꢀwell plate
and trypsinized using trypsin/ethylenediaminetetraacetic acid (EDTA) when the cells reached
approximately 80% confluence. The cytotoxicity of compounds VIb, VIIf and VIIIc was
5
3,54
evaluated using the Cell Counting Kitꢀ8 (CCKꢀ8, Dojindo Laboratories, Japan) assay.
NRKꢀ52E cells were seeded at a density of 5000 cells per well of 96ꢀwell plate and incubated
at 37 °C in a atmosphere of 5% CO for 24 h. The DMEM was discarded, and replaced with
2
200 ꢀL DMEM containing candidate compounds at various concentrations. The DMEM
without NRKꢀ52E cells and compounds was as a blank group. After incubation for 24 h, 10
ꢀL CCKꢀ8 was added. After 1–2 h at 37 °C, the OD (optical density) value of each well was
1
4
ACS Paragon Plus Environment

Page 15 of 46
Journal of Agricultural and Food Chemistry
2
95
96
97
98
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
measured using an enzyme linked immunosorbent assay (ELISA) hybrid microplate reader at
a wavelength of 450 nm. Cell survival rate values were calculated as follows: cell survival
rate (%) = (ODtreated − ODblank)/(ODcontrol − ODblank) × 100
2
2
2
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
RESULTS AND DISCUSSION
Synthesis.
As
shown
in
Figure
2,
firstly,
an
intermediate,
(2E,4E)ꢀ5ꢀ(1,3ꢀbenzodioxolꢀ5ꢀyl)ꢀ2,4ꢀpentadienal (2) was obtained in 37% yield for four
28
steps from piperine (1) as described previously. Then, compounds 4a
–c were smoothly
prepared by reaction of compound 2 with appropriate substituted acetophenone in the
5
5
presence of KOH.
On the other hand, as described in Figure 3, reaction of
3,4ꢀdihydroxybenzaldehyde (5) with DCE in the presence of K CO3 gave
2
56
2,3ꢀdihydrobenzo[b][1,4]dioxineꢀ6ꢀcarbaldehyde (6). Compound 6 then reacted with
malonic acid in the presence of piperidine and pyridine to afford
57
(E)ꢀ3ꢀ(2,3ꢀdihydrobenzo[b][1,4]dioxinꢀ6ꢀyl)acrylic acid (7), which was esterified with
methanol catalyzed by conc. sulfuric acid to give methyl
(E)ꢀ3ꢀ(2,3ꢀdihydrobenzo[b][1,4]dioxinꢀ6ꢀyl)acrylate (8) in 85% yield for two steps from
2
8
compound 6 to 8. Next, compound 8 reacted with LiAlH
4
and AlCl
3
to produce compound
to afford
9
in 81% yield, which was further oxidized by MnO
2
2
8
(E)ꢀ3ꢀ(2,3ꢀdihydrobenzo[b][1,4]dioxinꢀ6ꢀyl)acrylaldehyde (10) in 52% yield. Subsequently,
reaction of compound 10 with trimethyl phosphonoacetate in the presence of NaH gave
methyl (2E,4E)ꢀ5ꢀ(2,3ꢀdihydrobenzo[b][1,4]dioxinꢀ6ꢀyl)pentaꢀ2,4ꢀdienoate (11) in 90%
5
8
yield. Compound 11 reacted with LiAlH
oxidized by MnO to afford (2E,4E)ꢀ5ꢀ(2,3ꢀdihydrobenzo[b][1,4]dioxinꢀ6ꢀyl)pentaꢀ2,4ꢀdienal
(13) in 51% yield for two steps from compound 11 to 13. Finally, compounds 14a
4 3
and AlCl to produce compound 12, which was
2
2
8
–c were
1
5
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 16 of 46
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
easily prepared by reaction of compound 13 with appropriate substituted acetophenone in the
5
5
presence of KOH. As described in Figures 4 and 5, piperine analogs VIa
–c and VIIIb,c
were obtained by reaction of compounds 4a–c or 14b,c with hydroxylamine hydrochloride in
5
5
the presence of NaOH; piperine analogs VIIa
–m and IXa–m were synthesized by reaction
of compounds 4a–c or 14a–c with different phenylhydrazine hydrochlorides (15a–e) in the
4
5
1
13
presence of NaOH. Their structures were determined by IR, H NMR, C NMR, and
HRMS.
The assignments of the chemical shifts for the protons were further determined by
1
1
1
1
Hꢀ H COSY spectra. As shown in Figure 6, the Hꢀ H COSY correlation of Hꢀ5 and Hꢀ1’ of
1
1
compound VIIb indicated that its configuration should be as A. Similarly, the Hꢀ H COSY
correlation of Hꢀ5 and Hꢀ1’ of compound VIIIc demonstrated that its configuration should be
as B (Figure S1). Especially threeꢀdimensional structure of compound VIIIc was determined
by Xꢀray crystallography (Figure 7). It clearly showed that the nitrogen atom of the
isoxazoline fragment was on the same side of the 4ꢀchlorophenyl. Crystallographic data
(excluding structure factors) of compound VIIIc was deposited at the CCDC (Cambridge
Crystallographic Data Centre) with deposition numbers of 1585248.
Pesticidal Activities. The acaricidal activity of compounds 4a–c, 14a–c, VIa–c, VIIa–m,
VIIIb,c and IXa m against the female adults of T. cinnabarinus was evaluated by
–
slideꢀdipping method at a concentration of 0.5 mg/mL. As shown in Table 1, compounds VIa,
VIb, VIIf, VIIg, VIIl, VIIIb, VIIIc, IXf, and IXl exhibited more potent acaricidal activity
when compared with piperine (1). The 72 h mortality rates (MRs) of compounds VIa, VIb,
VIIf, VIIg, VIIl, VIIIb, VIIIc, IXf, and IXl against T. cinnabarinus were 45.4%, 41.0%,
48.5%, 47.7%, 46.0%, 56.7%, 63.7%, 46.7% and 42.9%, respectively; whereas the 72 h MR
1
6
ACS Paragon Plus Environment

Page 17 of 46
Journal of Agricultural and Food Chemistry
3
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
of compound 1 against T. cinnabarinus was only 12.1%. Among them, compound VIIIc
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
showed the most promising acaricidal activity. Compared with andrographolideꢀrelated esters
5
0,59
and quinolinomatrine derivatives,
herein piperine analogs generally exhibited more potent
acaricidal activity. For piperine analogs containing isoxazoline scaffold (e.g., VIb,c and
VIIIb,c), the ethylenedioxy group was an important factor for increasing the acaricidal
activity. For example, the 72 h MRs of compounds VIb,c (containing the methylenedioxy
group) against T. cinnabarinus were 41.0% and 26.7%, respectively; whereas the 72 h MRs
of compounds VIIIb,c (containing the ethylenedioxy group) against T. cinnabarinus were
56.7% and 63.7%, respectively. However, for piperine analogs containing pyrazoline scaffold
(VIIa-m and IXa-m), their ethylenedioxy or methylenedioxy group to the acaricidal activity
was not very obvious. It was noteworthy that when introduction of two fluorine or chlorine
atoms on the two phenyl rings of compound VIIa or IXa, respectively, four promising
compounds VIIf,l and IXf,l were obtained; and the 72 h MRs of compounds VIIf,l and IXf,l
against T. cinnabarinus were 48.5%, 46.0%, 46.7% and 42.9%, respectively.
As described in Table 2, the LC50 values of nine potent compounds against T.
cinnabarinus were assessed. The LC50 values of VIa, VIb, VIIf, VIIg, VIIl, VIIIb, VIIIc,
IXf, and IXl were 0.67, 0.82, 0.60, 0.60, 0.63, 0.42, 0.38, 0.65, and 0.72 mg/mL, respectively.
Especially compounds VIIIb and VIIIc exhibited 41 or 45 folds more pronounced activity
than compound 1 (LC50 value: 17.3 mg /mL). The LC90 values of VIIIb and VIIIc were 1.22,
and 1.23 mg/mL, respectively (LC value of compound 1: 49.9 mg /mL).
9
0
As shown in Table 3, the oral toxicity of compounds 4a
VIIIb,c and IXa m against P. xylostella treated at 20 µg/larvae was described at 24 h and 48
h, respectively. Compounds VIa c, VIIb, VIIf, VIIg, and VIIIc showed the good activity
–c, 14a–c, VIa–c, VIIa–m,
–
–
1
7
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 18 of 46
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
against P. xylostella, and their corresponding 48 h MRs were 62.1%, 72.4%, 69.0%, 55.2%,
58.6%, 51.7%, and 51.7%, respectively; whereas the 48 h MR of compound 1 was only
27.6%. Among them, compounds VIa–c exhibited more promising oral toxicity than
toosendanin. Interestingly, for piperine analogs containing isoxazoline scaffold (e.g., VIb,c
and VIIIb,c), the methylenedioxy group was an vital factor for increasing the oral toxicity.
For example, the 48 h MRs of compounds VIIIb,c (containing the ethylenedioxy group)
against P. xylostella were 44.8% and 51.7%, respectively; whereas the 48 h MRs of
compounds VIb,c (containing the methylenedioxy group) against P. xylostella were 72.4%
and 69.0%, respectively. Similarly, for piperine analogs containing pyrazoline scaffold
(VIIa
–
m and IXa
–
m), the methylenedioxy group was also an important factor for the good
m (containing the methylenedioxy group) generally displayed
oral toxicity. Compounds VIIa
–
more promising activity than compounds IXa–m (containing the ethylenedioxy one).
2
Additionally, when the hydrogen atom (R ) of VIIa was substituted by a fluorine atom, the 48
h MR of the corresponding compound VIIb was increased from 31.1% to 55.2%; when two
1
2
hydrogen atoms (R , R ) of VIIa were substituted by two fluorine atoms or the fluorine and
chlorine atoms, respectively, the corresponding compounds VIIf (48 h MR: 58.6%) and VIIg
(48 h MR: 51.7%) also showed the activity better than compound VIIa (48 h MR: 31.1%).
As shown in Table 4, compounds 4a
tested for their growth inhibitory activity against M. separata at 1 mg/mL. Compounds VIa
VIIf h, and VIIIb,c showed the good activity with the final mortality rates (FMRs) greater
–
c, 14a–c, VIa
–
c, VIIa
–
m, VIIIb,c and IXa
–
m were
–c,
–
than 50%. Especially compounds VIb and VIc displayed the most pronounced activity. The
FMRs of VIb and VIc were 62.1%, and 65.5%, respectively; whereas the FMR of compound
1 was 41.4%. The symptoms for the treated M. separata during the larval, pupal and adult
1
8
ACS Paragon Plus Environment

Page 19 of 46
Journal of Agricultural and Food Chemistry
4
8,50,51
3
87
88
89
90
91
92
93
94
95
96
97
98
99
00
01
02
03
04
05
06
07
08
09
periods were observed as the same as previously described.
For instance, the dead
3
3
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
larvae with thin and wrinkled bodies (Figure S2), the malformed and dead pupae (Figure S3),
and the malformed moths (Figure S4) appeared at three different growth stages, respectively.
However, the percentages of FMRs of the treated M. separata at three growth stages were
different (Figure 8). The percentages of FMRs at the larval stage of compounds VIa
VIIf g; and VIIIb,c were greater than 45%; especially the percentage of FMR at the larval
stage of compound VIIh was greater than 78%. Whereas the percentages of FMRs at the
pupal stage of compounds VIa c; VIIf g; and VIIIb,c were at the range of 2.7% 26.7%.
–c;
–
–
–
–
These results were different to those of 2′(2′,6′)ꢀ(di)chloropicropodophyllotoxins derivatives,
2
and more than half of their FMRs were generally at the pupal stage.
Additionally, as described in Table 4, in general, piperine analogs containing isoxazoline
scaffold (VIa
–
c and VIIIb,c) showed more potent activity against M. separata than those
m and IXa m). For piperine analogs containing
containing pyrazoline scaffold (VIIa
–
–
isoxazoline scaffold, the methylenedioxy group was an important factor for the growth
inhibitory activity. The FMRs of compounds VIb,c (containing the methylenedioxy group)
against M. separata were 62.1% and 65.5%, respectively; whereas the FMRs of compounds
VIIIb,c (containing the ethylenedioxy one) against M. separata were 51.7% and 58.6%,
1
2
respectively. Among compounds VIIa–m, when two hydrogen atoms (R ,R ) of VIIa were
substituted by two fluorine atoms or the fluorine and chlorine/bromine atoms, respectively,
the corresponding compounds VIIf (FMR: 51.7%), VIIg (FMR: 51.7%) and VIIh (FMR:
55.2%) exhibited more pronounced activity than compound VIIa (FMR: 27.6%).
Finally, the toxicity of compounds VIb, VIIf and VIIIc was evaluated against NRKꢀ52E
cells. It is noteworthy that compounds VIb, VIIf and VIIIc showed very low toxicity to
1
9
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 20 of 46
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
NRKꢀ52E cells, and their CC50 values were 186.2, > 200, and 127.0 ꢀg/mL, respectively.
In conclusion, a series of piperine analogs containing isoxazoline (VIa
–c and
VIIIb,c)/pyrazoline (VIIa m and IXa m) scaffold were prepared, and their structures were
–
–
characterized by infrared spectra, nuclear magnetic resonance spectra, and highꢀresolution
mass spectra. Moreover, the configuration of compound VIIIc was further determined by
singleꢀcrystal Xꢀray diffraction. Their pesticidal activities were evaluated against three
serious and typically cropꢀthreatening agricultural pests, T. cinnabarinus, M. separata and P.
xylostella. Among them, compounds VIIIb and VIIIc showed greater than 40ꢀfold more
pronounced acaricidal activity than their precursor piperine against T. cinnabarinus.
Compounds VIa–c exhibited more potent oral toxicity than piperine and toosendanin against
P. xylostella. Compounds VIb and VIc displayed more promising growth inhibitory activity
than piperine and toosendanin against M. separata. In general, piperine analogs containing
isoxazoline scaffold (e.g., VIa
–
c and VIIIb,c) showed more potent pesticidal activities than
m and IXa m). The methylenedioxy and
those containing pyrazoline scaffold (e.g., VIIa
–
–
isoxazoline scaffolds were the important factors for piperine analogs exhibiting good oral
toxicity and growth inhibitory activity; on the contrary, the ethylenedioxy and isoxazoline
scaffolds were the vital factors for piperine analogs showing good acaricidal activity. This
will lay the foundation for further structural modifications and application of piperine analogs
as pesticidal agents for agriculture.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at
1
13
1
13
DOI: Spectra of H NMR and C NMR, and data on H NMR, C NMR, HRMS, IR, and
2
0
ACS Paragon Plus Environment

Page 21 of 46
Journal of Agricultural and Food Chemistry
4
33
34
35
36
37
38
39
40
41
42
melting points of target compounds.
4
4
4
4
4
4
4
4
4
AUTHOR INFORMATION
Corresponding Author
*(H.X.) Phone/fax: +86ꢀ29ꢀ87091952. Eꢀmail: orgxuhui@nwsuaf.edu.cn.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The present research was supported by National Natural Science Foundation of China
(Project No. 31872013), and Special Funds of Central Colleges Basic Scientific Research
Operating Expenses (Project No. 2452015096).
2
1
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 22 of 46
REFERENCES
(
1) Wingfield, M. J.; Roux, J.; Slippers, B.; Hurley, B. P.; Garnas, J.; Myburg, A. A.;
Wingfield, B. D. Established and new technologies reduce increasing pest and pathogen
threats to Eucalypt plantations. Forest Ecol. Manage. 2013, 301, 35–42.
(2) Wang, R.; Zhi, X. Y.; Li, J.; Xu, H. Synthesis of novel oxime sulfonate derivatives of
2′(2′,6′)ꢀ(di)chloropicropodophyllotoxins as insecticidal agents. J. Agric. Food Chem.
2015, 63, 6668–6674.
(
3) Chen, Y. J.; Dai, G. H. Effect of the extract and compound from Solanum nigrum Linn
on Tetranychus cinnabarinus. J. Appl. Entomol. 2017, 141, 458−469.
(
4) Shi, L.; Wei, P.; Wang, X. Z.; Shen, G. M.; Zhang, J. ; Xiao, W.; Xu, Z. F.; Xu, Q.; He, L.
Functional analysis of esterase TCE2 gene from Tetranychus cinnabarinus (Boisduval)
involved in acaricide resistance. Sci. Rep. 2016, 6, 18646.
(
5) Zeng, J.; Jiang Y.; Liu, J. Analysis of the armyworm outbreak in 2012 and suggestions of
monitoring and forecasting. Plant Prot. 2013, 39, 117−121.
(
6) Etebari, K.; Furlong, M. J.; Asgari, S. Genome wide discovery of long intergenic
nonꢀcoding RNAs in diamondback moth (Plutella xylostella) and their expression in
insecticide resistant strains. Sci. Rep. 2015, 5, 14642.
(
7) Sun, Z.; Lv, M.; Yu, X.; Xu, H. Application of sustainable natural bioresources in crop
protection: insight into a podophyllotoxinꢀderived botanical pesticide for regulating insect
vestigial wing of Mythimna separata Walker. ACS Sust. Chem. Eng. 2017, 5, 3945−3954.
8) Heckel, D. G. Insecticide resistance after silent spring. Science 2012, 337, 1612−1614.
9) Tabashnik, B. E.; Brevault, T.; Carriere, Y. Insect resistance to Bt crops: lessons from the
first billion acres. Nat. Biotechnol. 2013, 31, 510–521.
(
(
2
2
ACS Paragon Plus Environment

Page 23 of 46
Journal of Agricultural and Food Chemistry
(10) Topuz, E.; Erler, F.; Gumrukcu, E. Survey of indigenous entomopathogenic fungi and
evaluation
of
their
pathogenicity
against
the
carmine
spider
mite, Tetranychus cinnabarinus (Boisd.), and the whitefly, Bemisia tabaci (Genn.) biotype
B. Pest Manage. Sci. 2016, 72, 2273−2279.
(
11) Lu, F. P.; Liang, X.; Lu, H.; Li, Q.; Chen, Q.; Zhang, P.; Li, K. M.; Liu, G. H.; Yan,
W.; Song, J. M. Overproduction of superoxide dismutase and catalase confers
cassava resistance to Tetranychus cinnabarinus. Sci. Rep. 2017, 7, 40179.
(
12) Lu, W. C.; Wang, M. Y.; Xu, Z. F.; Shen, G. M.; Wei, P.; Li, M.; Reid, W.; He, L.
Adaptation of acaricide stress facilitates Tetranychus urticae expanding
against Tetranychus cinnabarinus in China. Ecol. Evol. 2017, 7, 1233−1249.
13) Zuo, Y.; Wu, Q. Y.; Su, S. W.; Niu, C. W.; Xi, Z.; Yang, G. F. Synthesis, herbicidal
activity, and QSAR of novel Nꢀbenzothiazolylꢀpyrimidineꢀ2,4ꢀdiones as
(
(
protoporphyrinogen oxidase inhibitors. J. Agric. Food Chem. 2016, 64, 552–562.
14) Wei, H.; Liu, J.; Zhan, Z.; Chen, Y.; Tian, H.; Lin, S.; Gu, X. The toxicity and
physiological effect of essential oil from Chenopodium ambrosioides against the
diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae). Crop Prot. 2015, 76,
68−74.
(
15) Prasifka, J. R.; Spring, O.; Conrad, J.; Cook, L. W.; Palmquist, D. E.; Foley, M. E.
Sesquiterpene lactone composition of wild and cultivated sunflowers and biological
activity against an insect pest. J. Agric. Food Chem. 2015, 63, 4042–4049.
(
16) Li, Q.; Huang, X.; Li, S.; Ma, J.; Lv, M.; Xu, H. Semisynthesis of esters of fraxinellone
C4/10ꢀoxime and their pesticidal activities. J. Agric. Food Chem. 2016, 64, 5472–5478.
17) Zhi, X. Y.; Zhang, Y. Y.; Huang, J. L.; Xu, H. Sevenꢀmembered lactam derivatives of
(
2
3
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 24 of 46
podophyllotoxins as new pesticidal agents. Sci. Rep. 2017, 7, 3917.
(
(
(
(
18) Yang, Z. B.; Hu, D. Y.; Zeng, S.; Song, B. A. Novel hydrazone derivatives containing
pyridine amide moiety: design, synthesis, and insecticidal activity. Bioorg. Med. Chem.
Lett. 2016, 26, 1161−1164.
19) Huang, X.; Zhang, B.; Xu, H. Synthesis of some monosaccharideꢀrelated ester
derivatives as insecticidal and acaricidal agents. Bioorg. Med. Chem. Lett. 2017, 27,
4336−4340.
20) Kaziem, A. E. ; Gao, Y. H.; He, S.; Li, J. H. Synthesis and insecticidal activity of
enzymeꢀtriggered functionalized hollow mesoporous silica for controlled release. J. Agric.
Food Chem. 2017, 65, 7854–7864.
21) Onozaki, Y.; Horikoshi, R.; Ohno, I. ; Kitsuda, S.; Durkin, K. A.; Suzuki, T.; Asahara,
C.; Hiroki, N.; Komabashiri, R.; Shimizu, R. Flupyrimin: a novel insecticide acting at the
nicotinic acetylcholine receptors. J. Agric. Food Chem. 2017, 65, 7865–7873.
22) Casida, J. E. Pesticide interactions: mechanisms, benefits, and risks. J. Agric. Food Chem.
(
2017, 65, 4553–4561.
(
23) Lin, L.; Mulholland, N.; Wu, Q. Y.; Beattie, D.; Huang, S. W.; Irwin, D.; Clough, J.; Gu,
Y. C.; Yang, G. F. Synthesis and antifungal activity of novel sclerotiorin analogues. J.
Agric. Food Chem. 2012, 60, 4480–4491.
(
24) Zhang, M. Z.; Chen, Q.; Mulholland, N.; Beattie, D.; Irwin, D.; Gu, Y. C.; Yang, G. F.;
Clough, J. Synthesis and fungicidal activity of novel pimprinine analogues. Eur. J. Med.
Chem. 2012, 53, 283–291.
(25) Bagwell, C. L.; Moloney, M. G.; Yaqoob, M. Oxazolomycins: natural product lead
structures for novel antibacterials by click fragment conjugation. Bioorg. Med. Chem.
2
4
ACS Paragon Plus Environment

Page 25 of 46
Journal of Agricultural and Food Chemistry
Lett. 2010, 20, 2090–2094.
(
26) Zhang, M. Z.; Chen, Q.; Xie, C. H.; Mulholland, N.; Turner, S.; Irwin, D.; Gu, Y. C.;
Yang, G. F.; Clough, J. Synthesis and antifungal activity of novel streptochlorin
analogues. Eur. J. Med. Chem. 2015, 92, 776–783.
(
27) Qu, H.; Lv, M.; Xu, H. Piperine: bioactivities and structural modifications. MiniꢁRev.
Med. Chem. 2015, 15, 145–156.
(
28) Qu, H.; Yu, X.; Zhi, X. Y.; Lv, M.; Xu, H. Naturalꢀproductꢀbased insecticidal agents 14.
semisynthesis and insecticidal activity of new piperineꢀbased hydrazone derivatives
against Mythimna separata Walker in vivo. Bioorg. Med. Chem. Lett. 2013, 23,
5
552–5557.
29) Yang, R.; Zhang, Y.; Xu, H. Synthesis of novel isoxazolineꢀcontaining
podophyllotoxin/2′(2′,6′)ꢀ(di)halogenopodophyllotoxin derivatives and their
insecticidal/acaricidal activities. Bioorg. Med. Chem. Lett. 2018, 28, 1410–1416.
30) Zaki, Y. H.; Sayed, A. R.; Elroby, S. A. Regioselectivity of 1,3ꢀdipolar cycloadditions and
antimicrobial activity of isoxazoline, pyrrolo[3,4ꢀd]isoxazoleꢀ4,6ꢀdiones,
pyrazolo[3,4ꢀd]pyridazines and pyrazolo[1,5ꢀa]pyrimidines. Chem. Centr. J. 2016, 10,
7.
(
(
1
(31) Suresh, G.; Nadh, R. V.; Srinivasu, N.; Kaushal, K. Novel coumarin isoxazoline
derivatives: synthesis and study of antibacterial activities. Synth. Commun. 2016, 46,
1
972–1980.
32) Ning, G. H.; Zhao, W. T.; Bian, Q.; Tang, X. Y. Synthesis and fungicidal activity of some
,5ꢀdisubstituted isoxazoline derivatives. Chin. J. Org. Chem. 2014, 34, 1800–1805.
33) Jiang, S.; Tsikolia, M.; Bernier, U. R.; Bloomquist, J. R. Mosquitocidal activity and mode
(
(
3
2
5
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 26 of 46
of action of the isoxazoline fluralaner. Inter. J. Environ. Res. Public Health 2017, 14,
154.
(
(
(
34) EvranosꢀAksoz, B.; YabanogluꢀCiftci, S.; Ucar, G.; Yelekci, K.; Ertan, R. Synthesis of
some novel hydrazone and 2ꢀpyrazoline derivatives: monoamine oxidase inhibitory
activities and docking studies. Bioorg. Med. Chem. Lett. 2014, 24, 3278−3284.
35) Desai, N. C.; Patel, B. Y.; Dave, B. P. Synthesis and antimicrobial activity of novel
quinoline derivatives bearing pyrazoline and pyridine analogues. Med. Chem. Res. 2017,
26, 109−119.
36) Sid, A.; Messai, A.; Parlak, C.; Kazanci, N.; Luneau, D.; Kesan, G.; Rhyman, L.;
Alswaidan, I. A.; Ramasami, P. 1ꢀFormylꢀ3ꢀphenylꢀ5ꢀ(4ꢀisopropylphenyl)ꢀ2ꢀpyrazoline:
synthesis, characterization, antimicrobial activity and DFT studies. J. Mol. Struct. 2016,
1121, 46−53.
(
(
(
37) Badavath, V. N.; Baysal, I.; Ucar, G.; Sinha, B. N.; Jayaprakash, V. Monoamine oxidase
inhibitory activity of novel pyrazoline analogues: curcumin based design and synthesis.
ACS Med. Chem. Lett. 2016, 7, 56−61.
38) Zhao, P. L.; Wang, F.; Zhang, M. Z.; Liu, Z. M.; Huang, W.; Yang, G. F. Synthesis,
fungicidal, and insecticidal activities of βꢀmethoxyacrylateꢀcontaining Nꢀacetyl
pyrazoline derivatives. J. Agric. Food Chem. 2008, 56, 10767–10773.
39) Zhao, P. L.; Wang, L.; Zhu, X. L.; Huang, X.; Zhan, C. G.; Wu, J. W.; Yang, G. F.
Subnanomolar inhibitor of cytochrome bc1 complex designed by optimizing interaction
with conformationally flexible residues. J. Am. Chem. Soc. 2010, 132, 185–194.
(40) Wang, Y.; Shao, Y. H.; Wang, Y. Y.; Fan, L. L.; Yu, X.; Zhi, X. Y.; Yang, C.; Qu, H.; Yao,
X. J.; Xu, H. Synthesis and quantitative structureꢀactivity relationship (QSAR) study of
2
6
ACS Paragon Plus Environment

Page 27 of 46
Journal of Agricultural and Food Chemistry
novel isoxazoline and oxime derivatives of podophyllotoxin as insecticidal agents. J.
Agric. Food Chem. 2012, 60, 8435–8443.
(
41) Wang, Y.; Yu, X.; Zhi, X. Y.; Xiao, X.; Yang, C.; Xu, H. Synthesis and insecticidal
activity of novel hydrazone compounds derived from a naturally occurring lignan
podophyllotoxin against Mythimna separata (Walker). Bioorg. Med. Chem. Lett. 2014,
24, 2621–2624.
(42) Yu, X.; Che, Z. P.; Xu, H. Recent advances in the chemistry and biology of
podophyllotoxins. Chem. −Eur. J. 2017, 23, 4467−4526.
(
43) Zhang, Y. Y.; Xu, H. Recent progress in the chemistry and biology of limonoids. RSC
Adv. 2017, 7, 35191−35220.
(
44) Yu, X.; Feng, G.; Huang, J. L.; Xu, H. Evaluation of some quinolineꢀbased hydrazone
derivatives as insecticidal agents. RSC Adv. 2016, 6, 30405−30411.
(
(
(
45) Khan, S. A.; Ahmad, B.; Alam, T. Synthesis and antihepatotoxic activity of some new
chalcones containing 1,4ꢀdioxane ring system. Pak. J. Pharm. Sci. 2006, 19, 290−294.
46) Weaver, D. F.; Campbell, A. J. Heterocyclic antiꢀepileptogenic agents and methods of use
thereof. USA 2003114441, 2003.
47) Osman, N. A.; Mahmoud, A. H.; Klein, C. D.; Allarà, M.; Marzo, V. D.; Abouzid, K. M.;
Abadi, A. H. Naphthalene and 2,3ꢀdihydrobenzo[b][1,4]dioxine derivatives with extended
side chains as new scaffolds of CB
571−1576.
2
ꢀselective ligands. Med. Chem. Commun. 2014, 5,
1
(
48) Yang, R.; Huang, X.; Che, Z.; Zhang, Y.; Xu, H. Application of sustainable natural
resources in crop protection: podophyllotoxinꢀbased botanical pesticides derived from
Podophyllum hexandrum for controlling cropꢀthreatening insect pests. Ind. Crops Prod.
2
7
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 28 of 46
2017, 107, 45−53.
(49) Chang, K. M.; Knowles, C. O. Formamidine acaricides. Toxicity and metabolism studies
with two spotted spider mites, Tetranychus urticae Koch. J. Agric. Food Chem. 1977, 25,
493−501.
(
50) Huang, J.; Lv, M.; Thapa, S.; Xu, H. Synthesis of novel quinolinomatrine derivatives and
their insecticidal/acaricidal activities. Bioorg. Med. Chem. Lett. 2018, 28, 1753−1757.
51) Yu, M.; Liu, G.; Zhang, Y.; Feng, T.; Xu, M.; Xu, H. Design, synthesis and evaluation of
novel isoxazolines/oxime sulfonates of 2’(2’,6’)ꢀ(di)chloropodophyllotoxins as
insecticidal agent. Sci. Rep. 2016, 6, 33062.
(
(
(
(
52) Zhang, Y. Q.; Li, H. H.; Zhu, J. J.; Wei, T. T.; Peng, Y. X.; Li, R.; Xu, R.; Li, M.; Xia, A.
Z. Role of artesunate in TGFꢀβ1ꢀinduced renal tubular epithelialꢀmesenchymal
transdifferentiation in NRKꢀ52E cells. Mol. Med. Rep. 2017, 16, 8891−8899.
53) Yao, Y. W.; Liao, C. Z.; Li, Z.; Wang, Z.; Sun, Q.; Liu, C. P.; Yang, Y.; Tu, Z. C.; Jiang, S.
Design, synthesis, and biological evaluation of 1,3ꢀdisubstitutedꢀpyrazole derivatives as
new class I and IIb histone deacetylase inhibitors. Eur. J. Med. Chem. 2014, 86, 639−652.
54) Hou, W.; Zhang, G. J.; Luo, Z.; Li, D.; Ruan, H. Q.; Ruan, B. H.; Sun, L.; Xu, H. T.
Identification of a diverse synthetic abietane diterpenoid library and insight into the
structureꢀactivity relationships for antibacterial activity. Bioorg. Med. Chem. Lett. 2017,
27, 5382−5386.
(
55) AbdelꢀHalim, M.; Diesel, B.; Kiemer, A. K.; Abadi, A. H.; Hartmann, R. W.; Engel, M.
Discovery and optimization of 1,3,5ꢀtrisubstituted pyrazolines as potent and highly
selective allosteric inhibitors of protein kinase Cꢀzeta. J. Med. Chem. 2014, 57, 6513−6530.
56) Qin, Y. J.; Li, Y. J.; Jiang, A. Q.; Yang, M. R.; Zhu, Q. Z.; Dong, H.; Zhu, H. L. Design,
(
2
8
ACS Paragon Plus Environment

Page 29 of 46
Journal of Agricultural and Food Chemistry
synthesis and biological evaluation of novel pyrazolineꢀcontaining derivatives as potential
tubulin assembling inhibitors. Eur. J. Med. Chem. 2015, 94, 447−457.
(
(
(
57) Wu, M.; Wang, Z. W; Meng, C. S.; Wang, K. L.; Hu, Y. N.; Wang, L. Z.; Wang, Q. M.
Discovery and SARs of transꢀ3ꢀaryl acrylic acids and their analogs as novel antiꢀtobacco
mosaic virus (TMV) agents. PLoS ONE 2013, 8, e56475.
58) Lu, K.; Luo, T. P.; Xiang, Z.; You, Z. J.; Fathi, R.; Chen, J. H.; Yang, Z. A concise and
diversityꢀoriented strategy for the synthesis of benzofurans and indoles via Ugi and
DielsꢀAlder reactions. J. Comb. Chem. 2005, 7, 958−967.
59) Huang, X.; Zhang, B.; Xu, H. Synthesis of andrographolideꢀrelated esters as insecticidal
and acaricidal agents. Bioorg. Med. Chem. Lett. 2018, 28, 360−364.
2
9
ACS Paragon Plus Environment