Insight into reduction of obacunone, and their ester derivatives as insecticidal agents against Mythimna separata Walker

Article abstract of DOI:10.1016/j.bmcl.2014.11.027

Here we have prepared a series of ester compounds of obacunone, a naturally occurring limonoid, isolated from plants such as Citrus and Dictamnus angustifolius. Their insecticidal activity was evaluated at 1 mg/mL against the pre-third-instar larvae of or

Full text of DOI:10.1016/j.bmcl.2014.11.027

Bioorganic & Medicinal Chemistry Letters 25 (2015) 25–29
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Insight into reduction of obacunone, and their ester derivatives as
insecticidal agents against Mythimna separata Walker
Xiang Yu a, Guodong Ding a, Xiaoyan Zhi a, Hui Xu a,b,
⇑
a Research Institute of Pesticidal Design & Synthesis, College of Sciences, Northwest A&F University, Yangling 712100, Shaanxi Province, People’s Republic of China
b State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest A&F University, Yangling 712100, Shaanxi Province, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Here we have prepared a series of ester compounds of obacunone, a naturally occurring limonoid, iso-
lated from plants such as Citrus and Dictamnus angustifolius. Their insecticidal activity was evaluated at
1 mg/mL against the pre-third-instar larvae of oriental armyworm (Mythimna separata Walker), a typical
lepidopteran pest. When obacunone reacted with NaBH4, the ratio of two reduction products,
Received 18 August 2014
Revised 15 October 2014
Accepted 8 November 2014
Available online 15 November 2014
C7
C7
a
a
-hydroxyobacunone (2) and C7b-hydroxyobacunone (3), was related to the reaction mixing solvents.
-Propionyloxybacunone (4b) and C7b-(n)heptanoyloxybacunone (5g) exhibited the more promising
Keywords:
Obacunone
Ester
insecticidal activity than their precursor obacunone and toosendanin.
Ó 2014 Elsevier Ltd. All rights reserved.
Insecticidal activity
Natural-product-based insecticide
Structural modification
Oriental armyworm (Mythimna separata
Walker)
Recently, more eco-friendly pesticides for use in integrated pest
management (IPM) is required. Nowadays, the discovery of new
pesticidal agents from plant secondary metabolites, or by using
them as the lead compounds for further structural modification,
has received much research attention.1–9 Obacunone (1, Fig. 1), is
isolated as an naturally occurring limonoid from many species of
plants such as Citrus and its hybrids,10 and Dictamnus angustifo-
lius.11 Besides exhibiting some medicinal activities such as antipro-
liferative,12 anticancer,13 antimalarial,14 and antioxidant
activities,15 compound 1 also showed insecticidal activities such
as the moult inhibiting activity against mosquito Culex quinquefas-
ciatus,16 and the antifeedant activity against Spodoptera fru-
giperda.17 As part of an ongoing search for new potential natural-
product-based insecticidal agents to control the lepidopteran
pests,18–21 herein we synthesized a series of ester compounds of
obacunone by modification of its B ring. Their insecticidal activity
was evaluated against the oriental armyworm (Mythimna separata
Walker), an important and typical lepidopteran pest.
solvents. For example, when a solution of NaBH4 (1.0 mmol) in CH3-
OH was added dropwise to a solution of 1 (0.5 mmol) in CH3OH at
ꢀ5 °C, after reaction for 1 h, the ratio of 2 and 3 was 1/3.8 (entry
1); when a solution of NaBH4 (1.0 mmol) in THF, CH3OH, CH3CN or
CH2Cl2 was added dropwise to a solution of 1 (0.5 mmol) in CH3OH
or CH3CN at ꢀ5 °C, respectively, after reaction for 1–2 h, the ratio of
2 and 3 was between 1/2.87 and 1/2.63 (entries 2–6). When a
solution of NaBH4 (1.0 mmol) in CH3OH was added dropwise to a
solution of 1 (0.5 mmol) in CH2Cl2 at ꢀ5 °C, after reaction for 2 h,
23
O
22
18 20
21
12
17O
11
1
13 D
19
C28
16
2
14
As described in Table 1, first, reduction of 1 with NaBH4 in the
presence of different solvents such as CH3OH, THF, CH2Cl2, and CH3-
CN, was investigated. Interestingly, we found that the ratio
of two reduction isomers, C7a-hydroxyobacunone (2) and
C7b-hydroxyobacunone (3), was related with the reaction mixing
9
O
8
15
O
10 B
O
A
4
3
O
5
7
O
6
29
30
1
⇑
Corresponding author. Tel./fax: +86 (0) 29 87091952.
E-mail address: orgxuhui@nwsuaf.edu.cn (H. Xu).
Figure 1. Chemical structure of obacunone (1).
http://dx.doi.org/10.1016/j.bmcl.2014.11.027
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

26
X. Yu et al. / Bioorg. Med. Chem. Lett. 25 (2015) 25–29
Table 1
Investigation of reduction of 1 with NaBH4 in the presence of different solvents
O
NaBH4 (1 mmol) in
B solvent (1.6 mL)
O
O
O
+
1 (0.5 mmol) in A
solvent (10 mL)
O
O
O
O
O
O
O
O
-5 oC
OH
OH
2
3
Yield (%)
Entry
Reaction time (h)
Ratio of 2/3
2
3
1
2
3
4
5
6
7
8
9
CH3OH
CH3OH
THFa
CH3OH
CH3CN
CH2Cl2
THF
CH3OH
CH3OH
THF
CH3CN
CH2Cl2
THF
1
1
1.5
1
1
2
2
1
2.5
2
2
20
23
25
19
22
24
34
36
47
43
46
0
76
66
71
51
58
63
60
37
26
32
44
0
1:3.8
CH3OH
CH3CN
CH3OH
CH3OH
CH3CN
CH2Cl2
THF
THF
THF
THF
CH2Cl2
1:2.87
1:2.84
1:2.68
1:2.64
1:2.63
1:1.76
1:1.03
1.81:1
1.34:1
1.05:1
/
10
11
12
10
a
THF: Tetrahydrofuran.
Figure 3. X-ray crystal structure of C7b-hydroxyobacunone (3).
Figure 2. X-ray crystal structure of C7a-hydroxyobacunone (2).
the ratio of 2 and 3 was 1/1.76 (entry 7). However, when a solution
of NaBH4 (1.0 mmol) in CH3OH or CH2Cl2 was added dropwise to a
solution of 1 (0.5 mmol) in THF at ꢀ5 °C, after reaction for 1 or
2 h, the ratio of 2 and 3 was nearly 1 (entries 8 and 11). If a solution
of NaBH4 (1.0 mmol) in CH3CN was added dropwise to a solution of
1 (0.5 mmol) in THF at ꢀ5 °C, after reaction for 2 h, the ratio of 2 and
3 was 1.34/1 (entry 9). Moreover, when a solution of NaBH4
(1.0 mmol) in THF was added dropwise to a solution of 1 (0.5 mmol)
in THF at ꢀ5 °C, after reaction for 2.5 h, the ratio of 2 and 3 was
increased to 1.81/1 (entry 10). But when a solution of NaBH4
(1.0 mmol) in THF was added dropwise to a solution of 1 (0.5 mmol)
in CH2Cl2 at ꢀ5 °C, even if the reaction time was prolonged to 10 h, 2
and 3 were not obtained (entry 12). The steric structures of 2 and 3
were further determined by X-ray crystallography as shown in
Figures 2 and 3.22 It demonstrated that the C7-hydroxy groups of
2 and 3 were present in and b configuration, respectively.
Second, 2 or 3 reacted with different carboxylic acids in the
presence of DCC and DMAP. It is noteworthy that the reaction
activity of 2 or 3 with different carboxylic acids in the presence
of DCC and DMAP was associated with the stereo configuration
of their hydroxy groups at the C-7 position. For example, as shown
a
in Schemes 1 and 2 containing 7a-hydroxy group did not reacted
with carboxylic acids such as acetic acid, propanoic acid, benzoic
acid, and 4-formylbenzoic acid in the presence of DCC and DMAP,
even if the reaction time was prolonged to 72 h. Moreover, when 2
reacted with benzoyl chloride or chloroacetyl chloride for 48 h, no
target compounds were obtained. Only when 2 reacted with acetic
or propionic anhydride under reflux, the corresponding esters 4a

X. Yu et al. / Bioorg. Med. Chem. Lett. 25 (2015) 25–29
27
O
R3CO2H
DCC/DMAP
O
O
CH2Cl2, r.t./72 h
O
O
O
X
O
O
R3
R3= CH3, CH3CH2, Ph, 4-formylphenyl
O
O
O
R2COCl
O
C
D
O
O
O
O
X
Et3N/CH2Cl2
0 oC-r.t./48 h
O
O
15
A
O
B
O
O
O
R2
O
OH
7
R2 = ClCH2, Ph
2
(R1CO)2O
reflux/2 or 4 h
O
O
O
O
O
O
O
R1
4a and 4b
R1 = CH3
(73%)
4a
4b
R1= CH2CH3
(86%)
Scheme 1. Investigation of synthesis of C7
a-ester derivatives of obacunone.
O
O
O
O
R3CO2H
O
O
O
O
O
DCC/DMAP
CH2Cl2, r.t./6-24 h
40-97%
O
O
O
O
OH
O
R3
3
5a-e'
R3
a'
q
v
a
b
CH3
j
(CH2)9CH3
Cl
CHO
CH2Cl
k
l
(CH2)8CH CH2
(CH2)10CH3
Br
c
w
CH2CH3
b'
c'
r
NO2
NO2
m
d
e
(CH2)2CH3
(CH2)3CH3
(CH2)14CH3
(CH2)16CH3
x
n
s
Br
O2N
f
(CH2)4CH3
(CH2)5CH3
Y
Z
g
o
d'
N
t
h
(CH2)6CH3
(CH2)8CH3
F
i
p
Cl
e'
u
N
CH3
Br
Scheme 2. Synthesis of C7b-ester derivatives of obacunone (5a–e0).

28
X. Yu et al. / Bioorg. Med. Chem. Lett. 25 (2015) 25–29
and 4b were afford, respectively. It may be due to the steric hin-
drance between the hydrogen atom at the C-15 position and
the hydroxy group at the C-7 position of 2. On the contrary, when
the final mortality rate of 3 (containing C7b-hydroxy) was 46.4%.
(2) To C7 -alkylacyloxy derivatives 4a and 4b, introduction of
the propionyloxy group at the C-7 position of 1 could lead to
a
a
a
a
3 reacted with different carboxylic acids in the presence of DCC
and DMAP, the title products 5a–e0 were smoothly obtained in
40–97% yields (Scheme 2). The structures of all target compounds
were well characterized by 1H NMR, HRMS, optical rotation, IR, and
mp (see Supporting information). The steric configuration of 5q
was further confirmed by X-ray crystallography (Fig. 4).22 It
suggested that the (4-formyl)benzoyloxy group at the C-7 position
of 5q adopted b configuration.
the more promising compound 4b (e.g., the final mortality rates:
57.1% for 4b versus 39.3% for 4a). (3) To C7b-acyloxy series
(5a–e0), in general, the insecticidal activity of C7b-alkylacyloxy
derivatives (5a–n) was more potent than that of C7b-arylacyloxy
ones (5o–e0). (4) To C7b-alkylacyloxy series 5a–n, the proper
length of their side chain at the C-7b position to the insecticidal
activity was very necessary. Especially introduction of the (n)hep-
tanoyloxy or (n)dodecanoyloxy group at the C-7b position of 1
resulted in the more potential compounds 5g and 5l than toosend-
anin. However, to C7b-arylacyloxy series 5o–e0, whether introduc-
tion of the electron-withdrawing or electron-donating groups on
the phenyl ring, or heterocyclic rings at the C-7b position of 1
did not lead to the potential compounds, and their final mortality
rates were even less than their precursors (e.g., 1 and 3).
In conclusion, we have prepared a series of ester compounds of
obacunone modified in the B ring and tested their insecticidal
activity against the pre-third-instar larvae of M. separata in vivo.
When obacunone was reduced by NaBH4, the ratio of products 2
and 3 was related to the reaction mixing solvents. The key steric
structures of three compounds 2, 3, and 5q were determined by
single-crystal X-ray diffraction. Especially 4b and 5g exhibited
the more promising insecticidal activity than their precursor
obacunone and toosendanin. It will pave the way for further design
As shown in Table 2, the insecticidal activity of compounds 1–3,
4a, 4b, and 5a–e0 against the pre-third-instar larvae of M. separata
in vivo was tested by the leaf-dipping method at a concentration of
1 mg/mL.23 Toosendanin,
a commercial botanical insecticide
isolated from Melia azedarach, was used as the positive control at
1 mg/mL. Leaves treated with acetone alone were used as a blank
control group. It was found that the obacunone derivatives
exhibited the delayed insecticidal activity against M. separata. For
example, the corrected mortality rates of 4b against M. separata
after 10 and 20 days were 3.3% and 13.3%, respectively; whereas
after 35 days it was sharply increased to 57.1%, which was more
than 17-fold of that after 10 days. In addition, the symptoms of
M. separata treated by the obacunone derivatives were observed
in the same way as our previous reports.18–21 For instance, in the
treated groups, many larvae generally died with the slim and wrin-
kled bodies during the stage of larva (Fig. S1, see Supporting infor-
mation); a majority of larvae molted to malformed pupae or died
during the pupation period (Fig. S2, see Supporting information);
some malformed moths were with imperfect wings during the
stage of adult emergence (Fig. S3, see Supporting information).
Among all derivatives, compounds 4b, 5g and 5l showed the more
potential insecticidal activity than their precursor 1 and toosenda-
nin (a positive control). For example, the final mortality rates of 4b,
5g and 5l were 57.1%, 57.1% and 50%, respectively. Meanwhile, the
preliminary relationship between the structures of the obacunone
derivatives and their insecticidal activity was also analysed: (1)
The configuration of the hydroxy group at the C-7 position of 1
was important for the insecticidal activity. For instance, the final
Table 2
Insecticidal activity of ester compounds of obacunone (4a, 4b, and 5a–e0) against M.
separata on leaves treated with a concentration of 1 mg/mL
Compd
Corrected mortality rate (%)
20 days
10 days
6.7 6.7
35 days
1
2
3
4a
4b
5a
5b
20.0 5.8
16.7 3.3
30.0 5.8
13.3 3.3
13.3 3.3
26.7 3.3
23.3 6.7
42.9 3.3
32.1 3.3
46.4 5.8
39.3 3.3
0
0
10.0 5.8
6.7 6.7
3.3 3.3
57.1
0
20.0
0
0
0
46.4 5.8
42.9 3.3
39.3 3.3
42.9 3.3
39.3 3.3
21.4 3.3
mortality rate of 2 (containing C7a-hydroxy) was 32.1%, whereas
5c
5d
5e
5f
13.3 3.3
16.7 6.7
10.0 5.8
10.0 5.8
20.0
0
23.3 6.7
13.3 6.7
16.7 3.3
5g
0
0
30.0
0
57.1
0
5h
5i
3.3 3.3
13.3 3.3
16.7 3.3
39.3 6.7
17.9 3.3
10.0
0
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
5t
5u
5v
5w
5x
5y
6.7 6.7
20.0
26.7 3.3
20.0
33.3 6.7
20.0
16.7 3.3
6.7 3.3
13.3 3.3
20.0 5.8
16.7 3.3
0
25.0
35.7
50.0 3.3
46.4 5.8
35.7
25.0
28.6 3.3
28.6 3.3
25.0 5.8
28.6 3.3
28.6 3.3
28.6 3.3
28.6 3.3
39.3 3.3
21.4 3.3
28.6 3.3
28.6 3.3
39.3 3.3
17.9 3.3
0
0
0
0
3.3 3.3
0
0
0
0
0
0
0
0
13.3 3.3
0
0
6.7 6.7
0
0
10.0 5.8
6.7 6.7
13.3 6.7
10.0 5.8
10.0
0
23.3 3.3
13.3 6.7
13.3 3.3
10.0
0
0
0
10.0
0
6.7 3.3
6.7 3.3
3.3 3.3
6.7 6.7
13.3 6.7
13.3 6.7
26.7 3.3
10.0 5.8
10.0 5.8
10.0 5.8
20.0 5.8
23.3 3.3
5z
5a0
5b0
5c0
0
0
25.0
0
5d0
3.3 3.3
6.7 3.3
3.3 3.3
17.9 3.3
32.1 6.7
46.4 5.8
6.7 3.3
5e0
Toosendanin
Blank control
0
0
0
0
Figure 4. X-ray crystal structure of C7b-(4-formyl)benzoyloxybacunone (5q).

X. Yu et al. / Bioorg. Med. Chem. Lett. 25 (2015) 25–29
29
13. Miller, E. G.; Porter, J. L.; Binnie, W. H.; Guo, I. Y.; Hasegawa, S. J. Agric. Food
Chem. 2004, 52, 4908.
14. Lee, S. E.; Kim, M. R.; Kim, J. H.; Takeoka, G. R.; Kim, T. W.; Park, B. S.
Phytomedicine 2008, 15, 533.
and chemical modifications of obacunone as botanical insecticidal
agents.
15. Mandadi, K. K.; Jayaprakasha, G. K.; Bhat, N. G.; Patil, B. S. Z. Naturforsch. 2007,
62C, 179.
Acknowledgments
16. Jayaprakasha, G. K.; Singh, R. P.; Pereira, J.; Sakariah, K. K. Phytochemistry 1997,
44, 843.
17. Ruberto, G.; Renda, A.; Tringali, C.; Napoli, E. M.; Simmonds, M. S. J. J. Agric.
Food Chem. 2002, 50, 6766.
18. Qu, H.; Yu, X.; Zhi, X. Y.; Lv, M.; Xu, H. Bioorg. Med. Chem. Lett. 2013, 23, 5552.
19. Yang, C.; Shao, Y. H.; Zhi, X. Y.; Qu, H.; Yu, X.; Yao, X. J.; Xu, H. Bioorg. Med.
Chem. Lett. 2013, 23, 4806.
The present research was supported by National Natural Sci-
ence Foundation of China (no. 31171896), and Special Funds of
Central Colleges Basic Scientific Research Operating Expenses
(YQ2013008), Northwest A&F University.
20. Wang, Y.; Shao, Y.; Wang, Y.; Fan, L.; Yu, X.; Zhi, X.; Yang, C.; Qu, H.; Yao, X.; Xu,
H. J. Agric. Food Chem. 2012, 60, 8435.
Supplementary data
21. Xu, H.; Wang, Q. T.; Guo, Y. Chem. Eur. J. 2011, 17, 8299.
22. Crystallographic data (excluding structure factors) for the structures of 2, 3,
and 5q have been deposited with the Cambridge Crystallographic Data Centre
as supplementary publication number CCDC 965981, 970433, and 965982,
respectively. Copies of the data can be obtained, free of charge, on application
to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK. Fax: +44 (0) 1223 336033 or
e-mail: deposit@ccdc.cam.ac.uk.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.11.
027.
23. Biological assay: The insecticidal activity of compounds 4a, 4b, and 5a–e0
against the pre-third-instar larvae of Mythimna separata was assessed by leaf-
dipping method. Toosendanin, a commercial insecticide isolated from Melia
References and notes
1. Lin, L.; Mulholland, N.; Wu, Q.; Beattie, D.; Huang, S.; Irwin, D.; Clough, J.; Gu,
Y.; Yang, G. F. J. Agric. Food Chem. 2012, 60, 4480.
azedarach, was used as
a positive control and supplied by Research &
2. Chen, J. T.; Su, H. J.; Huang, J. W. J. Agric. Food Chem. 2012, 60, 7341.
3. Ando, D.; Fukushima, M.; Fujisawa, T.; Katagi, T. J. Agric. Food Chem. 2012, 60,
2607.
4. Li, Y. Q.; Zhang, P. X.; Ma, Q. Q.; Song, H. B.; Liu, Y. X.; Wang, Q. M. Bioorg. Med.
Chem. Lett. 2012, 22, 6858.
5. Che, Z. P.; Yu, X.; Fan, L. L.; Xu, H. Bioorg. Med. Chem. Lett. 2013, 23, 5592.
6. Yadav, P. A.; Suresh, G.; Rao, M. S. A.; Shankaraiah, G.; Rani, P. U.; Babu, K. S.
Bioorg. Med. Chem. Lett. 2014, 24, 888.
7. Qiu, S.; Sun, H.; Zhang, A. H.; Xu, H. Y.; Yan, G. L.; Han, Y.; Wang, X. J. Chin. J. Nat.
Med. 2014, 12, 401.
Development Center of Biorational Pesticide, Northwest A&F University,
Shaanxi province, China. For each compound, 30 larvae (10 larvae per group)
were used. Acetone solutions of all the above tested compounds, and
toosendanin were prepared at the concentration of 1 mg/mL. Fresh wheat
leaves were dipped into the corresponding solution for 3 s, then taken out, and
dried in a room. Leaves treated with acetone alone were used as a blank control
group. Several treated leaves were kept in each dish, where every 10 larvae
were raised. If the treated leaves were consumed, additional treated leaves
were added to the dish. After 48 h, untreated fresh leaves were added to all
dishes until adult emergence. The experiment was carried out at 25 2 °C and
on 12 h/12 h (light/dark) photoperiod. The insecticidal activity of the tested
compounds against the pre-third-instar larvae of M. separata was calculated by
the following formula:
8. Sreelatha, T.; Hymavathi, A.; Rama Subba Rao, V.; Devanand, P.; Usha Rani, P.;
Madhusudana Rao, J.; Suresh Babu, K. Bioorg. Med. Chem. Lett. 2010, 20, 549.
9. Cheng, S. S.; Lin, C. Y.; Chen, Y. J.; Chung, M. J.; Chang, S. T. Pest Manage. Sci.
2014, 70, 1215.
corrected mortality rate ð%Þ ¼ ðT ꢀ CÞ ꢁ 100=ð100% ꢀ CÞ
10. Roussef, G.; Nagy, S. Phytochemistry 1982, 21, 85.
11. Jung, H. J.; Sok, D. E.; Kim, Y. H.; Min, B. S.; Lee, J. P.; Bae, K. H. Planta Med. 2000,
66, 74.
where T is the mortality rate in the treated group expressed as a percentage and
C is the mortality rate in the untreated group expressed as a percentage.
12. Kim, J.; Jayaprakasha, G. K.; Patil, B. S. Food Funct. 2013, 4, 258.