Synthesis of novel 3/5(3,5)-(di)nitropaeonol hydrazone derivatives as nematicidal agents

Article abstract of DOI:10.1080/10286020.2021.1874941

Eighteen novel 3/5(3,5)-(di)nitropaeonol hydrazone derivatives were prepared, and their structures well characterized by ¹H NMR, HRMS, and mp. Due to the steric hindrance, the substituents on the C = N double bond of all hydrazine compounds (ex

Full text of DOI:10.1080/10286020.2021.1874941

Journal of Asian Natural Products Research
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ganp20
Synthesis of novel 3/5(3,5)-(di)nitropaeonol
hydrazone derivatives as nematicidal agents
Gen-Qiang Chen, Li-Na Zhu, Jin-Ming Yang, Song Zhang, Yuan-Hao Li, Xiao-
Long Guo, Di Sun, Jia-Xuan He, Yue-E Tian, Sheng-Ming Liu, Jia Jiang, Xiao-Bo
Huang & Zhi-Ping Che
To cite this article: Gen-Qiang Chen, Li-Na Zhu, Jin-Ming Yang, Song Zhang, Yuan-Hao Li, Xiao-
Long Guo, Di Sun, Jia-Xuan He, Yue-E Tian, Sheng-Ming Liu, Jia Jiang, Xiao-Bo Huang & Zhi-
Ping Che (2021): Synthesis of novel 3/5(3,5)-(di)nitropaeonol hydrazone derivatives as nematicidal
agents, Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2021.1874941
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JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
https://doi.org/10.1080/10286020.2021.1874941
Synthesis of novel 3/5(3,5)-(di)nitropaeonol hydrazone
derivatives as nematicidal agents
Gen-Qiang Chen, Li-Na Zhu, Jin-Ming Yang, Song Zhang, Yuan-Hao Li, Xiao-
Long Guo, Di Sun, Jia-Xuan He, Yue-E Tian, Sheng-Ming Liu, Jia Jiang, Xiao-Bo
Huang and Zhi-Ping Che
Laboratory of Pesticidal Design & Synthesis, Department of Plant Protection, College of Forestry,
Henan University of Science and Technology, Luoyang 471023, China
ABSTRACT
ARTICLE HISTORY
Received 28 September 2020
Accepted 7 January 2021
Eighteen novel 3/5(3,5)-(di)nitropaeonol hydrazone derivatives
were prepared, and their structures well characterized by H NMR,
1
HRMS, and mp. Due to the steric hindrance, the substituents on
the C ¼ N double bond of all hydrazine compounds (except E/
Z ¼ 4/1 for IV-1g, IV-1l, IV-2b, and E/Z ¼ 3/2 for IV-1n, IV-3a)
adopted E configuration. Among all compounds, four compounds
2, 4, IV-1j, and IV-1n exhibited potent nematicidal activity than
their precursor paeonol, especially 5-nitropaeonol (2) and 3,5-dini-
tropaeonol (4) displayed the most potent nematicidal activity
Heterodera glycines in vivo with LC50 values of 32.3307 and
36.7074 mg/L, respectively.
KEYWORDS
Paeonol; structural
modification; hydrazine;
nematicidal activity;
botanical nematicides
1. Introduction
Soybean cyst nematode (SCN, Heterodera glycines Ichinohe), is the most damaging
nematode pest, which has been affecting soybean crop worldwide [1]. At present
there are only a few commercial nematicides left in use, and their repeated applica-
tions over the years have led to the development of resistance [2–5]. Developing the
high-efficiency and low-risk small-molecule green-nematocide is the key of effective
control of the nematodes [6]. Our previous research has shown that paeonol, a sec-
ondary metabolite of peony, had significant nematicidal activity.
CONTACT Zhi-Ping Che
zhipingche@163.com
Laboratory of Pesticidal Design & Synthesis, Department of
Plant Protection, College of Forestry, Henan University of Science and Technology, Luoyang 471023, China
ß 2021 Informa UK Limited, trading as Taylor & Francis Group

2
G.-Q. CHEN ET AL.
Figure 1. The chemical structures of paeonol (1), sulfonate derivatives of paeonol (I) and paeonol-
based phenylsulfonylhydrazone derivatives (II).
Paeonol (1, Figure 1), 2-hydroxyl-4-methoxyacetophenone, is a naturally occurring
phenolic compound, isolated from the root bark of Paeonia suffruticosa and the
whole plant of Cynanchum paniculatum [7–10]. Due to its crucial phenolic ketone
skeleton, modern biological science research has indicated that 1 has a wide range of
biological activities. Some 1 derivatives show good pharmacological activities, such as
anti-inflammatory [11], antimicrobial [12], antioxidant [13], antidiabetic [14], anti-
cancer [15], antipyretic analgesic [16], and so on. Some exhibit good agricultural
activities in terms of insecticidal [17], anti-oomycete [18], antifungal [19], antibacter-
ial [20], antiviral [21], refreshing [22], and other agricultural effects. More recently,
sulfonate esterification of hydroxyl of paeonol has been investigated in our group,
and some sulfonate derivatives of paeonol (I, Figure 1) exhibited more pronounced
anti-oomycete activity [18, 23]. In addition, paeonol-based phenylsulfonylhydrazone
derivatives (II, Figure 1) displayed potential insecticidal activity [17]. Meanwhile, we
have found that some N-arylsulfonyl-3-acylindole arylcarbonyl hydrazine derivatives
showed significant nematicidal activity [24]. Overall, to the best of our knowledge,
research on structure optimizations of paeonol as nematicidal agents against H. gly-
cines has not yet been reported. Therefore, we herein prepared three series, 18 novel
3/5(3,5)-(di)nitropaeonol hydrazone derivatives (IV-1a–n, IV-2a,b, and IV-3a,b,
Scheme 1) by introduction of the hydrazone fragments on the 3/5(3,5)-(di)nitropaeo-
nol skeleton. Moreover, their nematicidal activity was evaluated against H. glycines.
2. Results and discussion
2.1. Chemistry
Paeonol (1) reacting with concentrated nitric acid (HNO3) was investigated. As
described in Table 1, when 1 mmol of 1 reacted with 1 mmol of HNO3 at ꢀ30 ꢁC for
0.5 h, besides 5-nitropaeonol (2, 29% yield), another mononitrification product, 3-
nitropaeonol (3), was also obtained (13% yield), and no dinitrification product 3,5-
dinitropaeonol (4) was obtained (entries 1). Interestingly, when the molar ratio of
HNO3 and 1 was 3/1 at ꢀ30 ꢁC for 5 h, dinitrification product (4) was smoothly pro-
duced in a 74% yield. Therefore, the present dinitrification reaction depended on the
amount of HNO3.
As described in Scheme 1, 3/5(3,5)-(di)nitropaeonol hydrazone derivatives (IV-
1a–n, IV-2a,b, and IV-3a,b) were smoothly prepared by the reaction of three

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
Scheme 1. Synthetic route for the preparation of IV-1a–n, IV-2a,b, and IV-3a,b.
Table 1. The preparation of compounds 2–4 from 1a.
Amount of (mmol)
Isolated yield (%)
3
Entry
1
HNO3
t (h)
2
4
1
2
1
1
1
3
0.5
5
29
0
13
0
0
74
aTo a solution of 1 (1 mmol) in 5 ml of H2SO4 (18.4 mol/L) at -30 ꢁC, a certain amount of HNO3 (16 mol/L) was added
dropwise for 10 min.
intermediates (2, 3, and 4) with the corresponding hydrazine hydrochlorides. The
1
structures of all compounds were well characterized by H NMR, HRMS, MS, and
mp. Due to the steric hindrance, the substituents on the C ¼ N double bond of all
hydrazine compounds (except E/Z ¼ 4/1 for IV-1g, IV-1l, IV-2b, and E/Z ¼ 3/2 for
IV-1n, IV-3a) adopted E configuration.
2.2. Nematicidal activity
The nematicidal activity of 1, 2–3, and novel 3/5(3,5)-(di)nitropaeonol hydrazone
derivatives (IV-1a–n, IV-2a,b, and IV-3a,b) against H. glycines is described in Table
2. Avermectin, a commercial nematicide, was used as the positive control. Firstly,
three intermediates (2–4) were evaluated for their nematicidal activity against H. gly-
cines, and compounds 2 and 4 exhibited the promising nematicidal activity. It is note-
worthy that introduction of a nitro group at the C5 position of 1 could result in
more potent compound compared to their precursor paeonol (e.g. 2 vs. 1).
Interestingly, when a nitro group was introduced at the C3 position of 1, the nemati-
cidal activity of the corresponding compound was decreased sharply (e.g. 2 vs. 3). In
addition, the nematicidal activity of mononitrification products (e.g. 2 and 3) was

4
G.-Q. CHEN ET AL.
Table 2. Nematicidal activity of compounds IV-1a–n, IV-2a,b, and IV-3a,b against H. glycines.
Compounds
R1
LC50 (mg/L)
1
2
3
4
—
—
—
—
50.8042
32.3307
64.3901
36.7074
113.5217
88.1402
86.5749
86.5412
93.7017
81.1502
62.4183
79.4028
87.2408
48.4560
59.9017
85.8187
88.2901
45.1476
111.8550
54.7011
147.4196
97.7275
7.3537
IV-1a
IV-1b
IV-1c
IV-1d
IV-1e
IV-1f
IV-1g
IV-1h
IV-1i
IV-1j
IV-1k
IV-1l
IV-1m
IV-1n
IV-2a
IV-2b
IV-3a
IV-3b
Avermectin
H
3-Me
4-Me
2-F
3-F
4-F
3,4-F
2-Cl
3-Cl
2-Br
3-Br
2-NO2
3-NO2
4-NO2
H
3-NO2
H
3-NO2
—
compared with that of the corresponding dinitrification product (e.g. 4). For example,
the LC50 value of 1 was 50.8042 mg/L, whereas the values of 2, 3, and 4 were
32.3307, 64.3901, and 36.7074 mg/L, respectively. This demonstrated that a nitro
group introduced at the C5 position of 1 was important for obtaining the
potent compound.
Our research group has found that some N-arylsulfonyl-3-acylindole arylcarbonyl
hydrazine derivatives showed significant nematicidal activity [24]. On the other hand,
dinitrification product, 3,5-dinitropaeonol (4) exhibited the promising nematicidal
activity against H. glycines. Given that, fourteen 3,5-dinitropaeonol carbonyl hydra-
zone derivatives (IV-1a–n) were preferentially prepared by introduction of the hydra-
zone fragments on the 3,5-dinitropaeonol skeleton and evaluated for their nematicidal
activity against H. glycines. To further compare the nematicidal activity, four 5/3-
nitropaeonol hydrazone derivatives (IV-2a,b and IV-3a,b) were also prepared.
Meanwhile, some interesting results of the structure–activity relationships (SAR) for
1
IV-1a–n, IV-2a,b, and IV-3a,b were observed: (1) When R ¼ Me, F, Cl, Br or NO2,
1
introduction of R at different position of the benzene ring could lead to derivatives
with different nematicidal activity. For example, the LC50 values of IV-1b/IV-1c, IV-
1d/IV-1e/IV-1f, IV-1h/IV-1i, IV-1j/IV-1k, and IV-1l/IV-1m/IV-1n were 88.1402/
86.5749, 86.5412/93.7017/81.1502, 79.4028/87.2408, 48.4560/59.9017, and 85.8187/
1
88.2901/45.1476 mg/L, respectively. In addition, when R was introduced at the ortho-
position (e.g. IV-1d and IV-1l) on the phenyl ring, the nematicidal activity was more
potent than the meta-position (e.g. IV-1e and IV-1m), but they were not as high
as that of the para-position (e.g. IV-1f and IV-1n). For example, the LC50 values of
IV-1f/IV-1n and IV-1d/IV-1l were 81.1502/45.1476 and 86.5412/85.8187 mg/L,
respectively; however, the LC50 values of IV-1e/IV-1m were 93.7017/88.2901 mg/L,
respectively. (2) Interestingly, when the different halogen (e.g. fluorine, chlorine, or

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
bromine) group was introduced at the same position on the phenyl ring, the nemati-
cidal activity was significantly different, and bromine atom was more potent than
chlorine and fluorine atoms (IV-1j/IV-1k vs. IV-1h/IV-1i and IV-1d/IV-1e).
Moreover, it is noteworthy that the introduction of 3,4-difluorophenylhydrazinyl as a
two-electron-withdrawing substituent (such as 3-F and 4-F) could result in more
potent compound IV-1g relative to those containing phenylhydrazinyl as a one-elec-
1
1
tron-withdrawing substituent (e.g. R ¼ 3-F for IV-1e and R ¼ 4-F for IV-1f). For
example, the LC50 value of IV-1g was 62.4183 mg/L, whereas the values of IV-1e and
IV-1f were 93.7017 and 81.1502 mg/L, respectively. (3) When a nitro group was
introduced at the C5 position of 1, the nematicidal activities of the corresponding
derivatives were more than that of the corresponding derivatives of a nitro group
introduced at the C3 position of 1. For example, the LC50 values of IV-1a/IV-1b and
IV-2a/IV-2b were 113.5217/88.1402 and 111.8550/54.7011 mg/L, respectively, whereas
the values of IV-3a/IV-3b were 147.4196/97.7275 mg/L, respectively. It is generally
implied that a nitro group introduced at C5 position of 1 was important for obtaining
the potent compound.
3. Experimental
3.1. General experimental procedures
Paeonol (2-hydroxyl-4-methoxyacetophenone) and hydrazine hydrochloride
.
(R1PhNHNH2 HCl) were ordered from Shanghai Macklin Biochemical Co., Ltd.
(Shanghai, China). Concentrated nitric acid (HNO3, 16 mol/L) and concentrated sul-
furic acid (H2SO4, 18.4 mol/L) were purchased from Luoyang Haohua Chemical
Reagent Co. Ltd. (Henan, China). Anhydrous alcohol, ethyl acetate, petroleum ether
and dichloromethane were analytical grade, and ordered from Beichen Fangzheng
Reagent Factory (Tianjin, China). Thin layer chromatography (TLC) was performed
with silica gel 60 GF254 (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China).
Silica gel column chromatography (CC) was performed with silica gel 200–300 mesh
(Qingdao Haiyang Chemical Co., Ltd.). Proton nuclear magnetic resonance (1H
NMR) spectra were carried out with a Bruker Avance III 400 MHz instrument in deu-
terated chloroform (CDCl3) or dimethyl sulfoxide (DMSO) using tetramethylsilane
(TMS) as the internal standard. High resolution mass spectrometry (HRMS) was
determined on an IonSpec 4.7 T FTMS instrument. Melting points (m.p.) of all com-
pounds were detected on a digital melting point apparatus, and are uncorrected.
3.2. Synthesis of 5-nitropaeonol (2) and 3-nitropaeonol (3)
To a solution of 1 (1 mmol) in 5 ml of H2SO4 (18.4 mol/L) at ꢀ30 ꢁC, was added
dropwise concentrated nitric acid (HNO3, 1 mmol) over 10 min. After adding, the
reaction mixture was stirred at ꢀ30 ꢁC for 0.5 h. The reaction mixture was poured
into 50 ml ice water, and the precipitated product was collected, which was washed
by water until neutral, and purified by silica gel column chromatography to afford
the key intermediate compounds 2 and 3 in 29% and 13% yields, respectively [25].

6
G.-Q. CHEN ET AL.
3.2.1. Data for 2
Yield ¼ 29%, Pale yellow solid, m.p. 125–126 ꢁC.
3.2.2. Data for 3
Yield ¼ 13%, Pale yellow solid, m.p. 132–133 ꢁC.
3.3. Synthesis of 3,5-dinitropaeonol (4)
To a solution of 1 (1 mmol) in 5 ml of H2SO4 (18.4 mol/L) at ꢀ30 ꢁC, was added
dropwise concentrated nitric acid (HNO3, 3 mmol) over 10 min. After adding, the
reaction mixture was stirred at ꢀ30 ꢁC for 5 h. The reaction mixture was poured into
50 ml ice water, and the precipitated product was collected, which was washed by
water until neutral, and recrystallized from ethyl acetate to obtain the key intermedi-
ate compound 4 at a yield of 74% [25].
3.3.1. Data for 4
Yield ¼ 74%, Pale yellow solid, m.p. 129–130 ꢁC.
3.4. General procedure for preparation of 3/5(3,5)-(di)nitropaeonol hydrazone
derivatives (IV-1a–n, IV-2a,b and IV-3a,b)
Compounds 2, 3, or 4 (0.5 mmol) and the corresponding hydrazine hydrochloride
.
(R1PhNHNH2 HCl, 0.5 mmol) in molar ratio 1:1 were dissolved in 5 ml anhydrous
alcohol, and two drops of HOAc were added to the solution at ambient temperature.
After finishing dropping, the above mixture was refluxed for 2–12 h until the reaction
was completed by TLC analysis. Then the mixture was allowed to stand for 10 min or
until a larger amount of solid accumulated in the bottom of the flask. The gained
solid was collected after filtration, and then washed with a certain amount of cool
anhydrous alcohol until it was pure, monitored by TLC. Subsequently, the pure com-
pounds were dried in vacuum drying chamber to afford the desired compounds IV-
1a–n, IV-2a,b, and IV-3a,b in 49–93% yields [24].
3.4.1. Data for IV-1a
1
Yield ¼ 69%, Yellow solid. H NMR (400 MHz, DMSO) d: 15.80 (s, 1H), 10.04 (s,
1H), 8.38 (s, 1H), 7.36 (t, J ¼ 8.0 Hz, 2H), 7.07 (d, J ¼ 8.0 Hz, 2H), 6.94 (t, J ¼ 7.2 Hz,
1H), 3.94 (s, 3H), 2.48 (s, 3H). HRESIMS: m/z 347.0990 [M þ H]þ (calcd for
C15H15N4O6, 347.0986).
3.4.2. Data for IV-1b
1
Yield ¼ 57%, Yellow solid. H NMR (400 MHz, DMSO) d: 15.72 (s, 1H), 9.99 (s,
1H), 8.36 (s, 1H), 7.23 (t, J ¼ 8.0 Hz, 1H), 6.84–6.89 (m, 2H), 6.75 (d, J ¼ 7.2 Hz, 1H),
3.94 (s, 3H), 2.46 (s, 3H), 2.28 (s, 3H). HRESIMS: m/z 361.1145 [M þ H]þ (calcd for
C16H17N4O6, 361.1143).

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
3.4.3. Data for IV-1c
1
Yield ¼ 78%, Yellow solid. H NMR (400 MHz, DMSO) d: 15.87 (s, 1H), 9.95 (s,
1H), 8.35 (s, 1H), 7.15 (d, J ¼ 8.4 Hz, 2H), 6.97 (d, J ¼ 8.4 Hz, 2H), 3.94 (s, 3H), 2.45
(s, 3H), 2.23 (s, 3H). HRESIMS: m/z 361.1148 [M þ H]þ (calcd for
C16H17N4O6, 361.1143).
3.4.4. Data for IV-1d
1
Yield ¼ 82%, Yellow solid. H NMR (400 MHz, DMSO) d: 9.92 (s, 1H), 8.41 (s, 1H),
7.17-7.29 (m, 3H), 6.96–7.02 (m, 1H), 3.94 (s, 3H), 2.49 (s, 3H). HRESIMS: m/z
365.0896 [M þ H]þ (calcd for C15H14FN4O6, 365.0892).
3.4.5. Data for IV-1e
1
Yield ¼ 86%, Yellow solid. H NMR (400 MHz, DMSO) d: 10.19 (s, 1H), 8.38 (s,
1H), 7.33–7.39 (m, 1H), 6.87-6.89 (m, 1H), 6.79–6.83 (m, 1H), 6.69–6.74 (m, 1H),
3.94 (s, 3H), 2.46 (s, 3H). HRESIMS: m/z 365.0893 [M þ H]þ (calcd for
C15H14FN4O6, 365.0892).
3.4.6. Data for IV-1f
1
Yield ¼ 91%, Yellow solid. H NMR (400 MHz, DMSO) d: 15.57 (s, 1H), 10.01 (s,
1H), 8.37 (s, 1H), 7.16–7.21 (m, 2H), 7.03–7.09 (m, 2H), 3.94 (s, 3H), 2.46 (s, 3H).
HRESIMS: m/z 365.0899 [M þ H]þ (calcd for C15H14FN4O6, 365.0892).
3.4.7. Data for IV-1g (E/Z ¼ 4/1)
1
Yield ¼ 77%, Yellow solid. H NMR (400 MHz, DMSO) d: 10.24 (s, 0.8H), 9.85 (s,
0.2H), 8.35 (s, 0.8H), 8.15 (s, 0.2H), 7.31–7.43 (m, 1H), 7.05–7.11 (m, 0.8H),
6.96–7.02 (m, 0.2H), 6.86–6.90 (m, 0.8H), 6.81–6.83 (m, 0.2H), 3.93 (s, 2.4H), 3.06 (s,
0.6H), 2.45 (s, 2.4H), 2.36 (s, 0.6H).
3.4.8. Data for IV-1h
1
Yield ¼ 85%, Yellow solid. H NMR (400 MHz, DMSO) d: 9.09 (s, 1H), 8.46 (s, 1H),
7.50 (dd, J ¼ 8.0 Hz, 1.6 Hz, 1H), 7.36-7.41 (m, 1H), 7.23 (dd, J ¼ 8.0 Hz, 1.6 Hz, 1H),
7.00–7.04 (m, 1H), 3.95 (s, 3H), 2.54 (s, 3H). HRESIMS: m/z 381.0599 [M þ H]þ
(calcd for C15H14ClN4O6, 381.0596).
3.4.9. Data for IV-1i
1
Yield ¼ 93%, Yellow solid. H NMR (400 MHz, DMSO) d: 10.15 (s, 1H), 8.39 (s,
1H), 7.37 (t, J ¼ 8.0 Hz, 1H), 7.08 (t, J ¼ 2.0 Hz, 1H), 6.99–7.02 (m, 1H), 6.93–6.96
(m, 1H), 3.94 (s, 3H), 2.46 (s, 3H). HRESIMS: m/z 381.0601 [M þ H]þ (calcd for
C15H14ClN4O6, 381.0596).
3.4.10. Data for IV-1j
1
Yield ¼ 49%, Yellow solid. H NMR (400 MHz, DMSO) d: 8.89 (s, 1H), 8.46 (s, 1H),
7.65 (dd, J ¼ 8.0 Hz, 1.6 Hz, 1H), 7.40–7.44 (m, 1H), 7.21 (dd, J ¼ 8.0 Hz, 1.6 Hz, 1H),
6.94–6.98 (m, 1H), 3.95 (s, 3H), 2.53 (s, 3H). HRESIMS: m/z 425.0095 [M þ H]þ
(calcd for C15H14BrN4O6, 425.0091).

8
G.-Q. CHEN ET AL.
3.4.11. Data for IV-1k
1
Yield ¼ 88%, Yellow solid. H NMR (400 MHz, DMSO) d: 10.13 (s, 1H), 8.38 (s,
1H), 7.31 (t, J ¼ 8.0 Hz, 1H), 7.23 (t, J ¼ 2.0 Hz, 1H), 7.02–7.08 (m, 2H), 3.94 (s, 3H),
2.46 (s, 3H). HRESIMS: m/z 425.0093 [M þ H]þ (calcd for C15H14BrN4O6, 425.0091).
3.4.12. Data for IV-1l (E/Z ¼ 4/1)
1
Yield ¼ 56%, Yellow solid. H NMR (400 MHz, DMSO) d: 10.76 (s, 0.8H), 10.72 (s,
0.2H), 8.37 (s, 0.8H), 8.20 (dd, J ¼ 8.8 Hz, 1.2 Hz, 0.8H), 8.05 (s, 0.2H), 7.85 (dd,
J ¼ 8.8 Hz, 1.2 Hz, 0.2H), 7.77 (dd, J ¼ 8.8 Hz, 6.8 Hz, 1H), 7.64–7.68 (m, 0.8H),
7.58–7.61 (m, 0.2H), 7.00–7.04 (m, 0.8H), 6.80–6.84 (m, 0.2H), 3.90 (s, 2.4H), 3.06 (s,
0.6H), 2.45 (s, 2.4H), 2.29 (s, 0.6H).
3.4.13. Data for IV-1m
1
Yield ¼ 79%, Yellow solid. H NMR (400 MHz, DMSO) d: 10.39 (s, 1H), 8.40 (s,
1H), 7.90 (t, J ¼ 2.4 Hz, 1H), 7.71-7.73 (m, 1H), 7.63 (t, J ¼ 8.4 Hz, 1H), 7.43-7.46 (m,
1H), 3.95 (s, 3H), 2.48 (s, 3H). HRESIMS: m/z 392.0841 [M þ H]þ (calcd for
C15H14N5O8, 392.0837).
3.4.14. Data for IV-1n (E/Z ¼ 3/2)
1
Yield ¼ 87%, Yellow solid. H NMR (400 MHz, DMSO) d: 10.87 (s, 0.6H), 10.64 (s,
0.4H), 8.32 (s, 0.6H), 8.15–8.20 (m, 2H), 8.13 (s, 0.4H), 7.30 (d, J ¼ 8.8 Hz, 1.2H),
7.20 (d, J ¼ 8.8 Hz, 0.8H), 3.90 (s, 1.8H), 3.06 (s, 1.2H), 2.48 (s, 1.8H), 2.41 (s, 1.2H).
3.4.15. Data for IV-2a
1
Yield ¼ 77%, Yellow solid. H NMR (400 MHz, DMSO) d: 14.03 (s, 1H), 9.65 (s,
1H), 8.15 (s, 1H), 7.32 (t, J ¼ 8.8 Hz, 2H), 7.06 (d, J ¼ 8.8 Hz, 2H), 6.88 (t, J ¼ 7.2 Hz,
1H), 6.75 (s, 1H), 3.93 (s, 3H), 2.39 (s, 3H). HRESIMS: m/z 302.1142 [M þ H]þ (calcd
for C15H16N3O4, 302.1135).
3.4.16. Data for IV-2b (E/Z ¼ 4/1)
1
Yield ¼ 70%, Yellow solid. H NMR (400 MHz, DMSO) d: 13.37 (s, 0.8H), 12.79 (s,
0.2H), 10.35 (s, 0.2H), 10.11 (s, 0.8H), 8.51 (s, 0.2H), 8.19 (s, 0.8H), 7.91 (t,
J ¼ 2.4 Hz, 0.2H), 7.88 (t, J ¼ 2.4 Hz, 0.8H), 7.73 (dd, J ¼ 7.6 Hz, 2.4 Hz, 0.2H), 7.68
(dd, J ¼ 7.6 Hz, 2.4 Hz, 0.8H), 7.59 (d, J ¼ 8.4 Hz, 0.2H), 7.57 (d, J ¼ 8.4 Hz, 0.8H),
7.46 (dd, J ¼ 7.6 Hz, 2.4 Hz, 0.8H), 7.42 (dd, J ¼ 7.6 Hz, 2.4 Hz, 0.2H), 6.82 (s, 0.8H),
6.81 (s, 0.2H), 3.94 (s, 2.4H), 3.76 (s, 0.6H), 2.41 (s, 2.4H), 2.38 (s, 0.6H).
3.4.17. Data for IV-3a (E/Z ¼ 3/2)
1
Yield ¼ 84%, Yellow solid. H NMR (400 MHz, DMSO) d: 14.47 (s, 0.6H), 13.05 (s,
0.4H), 10.27 (s, 0.6H), 9.72 (s, 0.4H), 8.14–8.18 (m, 0.4H), 7.69–7.73 (m, 0.6H),
7.26–7.32 (m, 2H), 6.77–7.06 (m, 4H), 3.98 (s, 1.2H), 3.89 (s, 1.8H), 2.64 (s, 1.2H),
2.41 (s, 1.8H).

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
9
3.4.18. Data for IV-3b
1
Yield ¼ 72%, Yellow solid. H NMR (400 MHz, DMSO) d: 13.88 (s, 1H), 10.11 (s,
1H), 7.85 (t, J ¼ 2.4 Hz, 1H), 7.77 (d, J ¼ 9.2 Hz, 1H), 7.69 (dd, J ¼ 8.0 Hz, 1.6 Hz, 1H),
7.60 (t, J ¼ 8.0 Hz, 1H), 7.41 (dd, J ¼ 8.0 Hz, 1.6 Hz, 1H), 6.84 (d, J ¼ 8.8 Hz, 1H), 3.91
(s, 3H), 2.44 (s, 3H). HRESIMS: m/z 347.0991 [M þ H]þ (calcd for
C15H15N4O6, 347.0986).
3.5. Bioassay method
Eighteen 3/5(3,5)-(di)nitropaeonol hydrazone derivatives (IV-1a–n, IV-2a,b, and IV-
3a,b) were screened in vivo for their nematicidal activity against H. glycines. The
ranges of compounds 1, 2-4, IV-1a–n, IV-2a,b, and IV-3a,b and avermectin (used as
a positive control) concentrations for the assays were defined in preliminary experi-
ments. Dimethyl sulfoxide (DMSO) solutions of compounds 1, 2–4, IV-1a–n, IV-
2a,b, and IV-3a,b and avermectin, and the final concentrations of active ingredients
(a.i.) in medium were the following: for compounds 1, 2-4, IV-1a–n, IV-2a,b, IV-
3a,b 0.02, 0.04, 0.08, 0.16, and 0.32 mg/ml, for avermectin 0.005, 0.006, 0.007, 0.008,
0.009, and 0.010 mg/ml. Then fifty sterilized nematodes (second-stage juveniles (J2) of
H. glycines) were transferred to a 60-mm-diameter watch-glass with 2 ml of the above
solutions, and placed the watch-glass in Petri dishes and kept in an incubator at
25 ꢁC for 12 h. The blank control group was prepared in the same way but lacked the
tested compound. Three replicates in each trial were made and the independent
experiment was repeated three times. The activities of five concentrations of the
tested compounds were monitored under a microscope by recording the death rate of
the tested nematodes. Nematodes that did not move when prodded with a needle
were considered to be dead. The method for determining nematicidal activity was as
described in our previous paper [23, 24]. The median lethal concentration (LC50) val-
ues of compounds 1, 2-4, IV-1a–n, IV-2a,b, and IV-3a,b and emamectin benzoate
were calculated using the probit method.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was financially supported by Key Science and Technology Program of Henan
Province (Grant No. 202102110068), Young Teacher Funding Program of the Henan Higher
School (Grant No. 2020GGJS080), National Natural Science Foundation of China (Grant
No. 31901863).
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