Bioactivity-guided mixed synthesis and evaluation of α-alkenyl-γ and δ-lactone derivatives as potential fungicidal agents

Article abstract of DOI:10.1039/c7ra12471f

In view of the great antifungal activities of sesquiterpene lactones and natural product Tulipalin A, 52 derivatives derived from α-methylene-γ-butyrolactone substructures were synthesized to study antifungal activities. In vitro and in vivo antifungal activity results revealed that compounds 2-25, which contain a γ-butyrolactone scaffold and cinnamic aldehyde moiety, have greater potent fungicidal activity than other compounds. The preliminary structure-activity relationships (SARs) demonstrated that compounds with electron-withdrawing groups and small steric hindrance would have more desirable potency. Meanwhile, the quantitative structure-activity relationship (QSAR) model (R² = 0.947, F = 65.77, and S² = 0.0028) revealed a convincing correlation of antifungal activity against B. cinerea with molecular structures of title compounds. The present study provided a more detailed insight into the antifungal activity of the α-methylene-γ-butyrolactone substructure, which provided a potential expectation for the exploration of α-alkenyl-γ-butyrolactone structures in agriculture.

Full text of DOI:10.1039/c7ra12471f

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PAPER
Bioactivity-guided mixed synthesis and evaluation
of a-alkenyl-g and d-lactone derivatives as
potential fungicidal agents†
Cite this: RSC Adv., 2017, 7, 56496
Yong-Ling Wu,a Yan-Qing Gao,ab De-Long Wang,a Chen-Quan Zhong,a
ab
and Xing Zhangab
*
Jun-Tao Feng
In view of the great antifungal activities of sesquiterpene lactones and natural product Tulipalin A, 52
derivatives derived from a-methylene-g-butyrolactone substructures were synthesized to study
antifungal activities. In vitro and in vivo antifungal activity results revealed that compounds 2-25, which
contain a g-butyrolactone scaffold and cinnamic aldehyde moiety, have greater potent fungicidal activity
than other compounds. The preliminary structure–activity relationships (SARs) demonstrated that
compounds with electron-withdrawing groups and small steric hindrance would have more desirable
potency. Meanwhile, the quantitative structure–activity relationship (QSAR) model (R2 ¼ 0.947, F ¼ 65.77,
and S2 ¼ 0.0028) revealed a convincing correlation of antifungal activity against B. cinerea with
molecular structures of title compounds. The present study provided a more detailed insight into the
Received 15th November 2017
Accepted 29th November 2017
DOI: 10.1039/c7ra12471f
antifungal activity of the a-methylene-g-butyrolactone substructure, which provided
expectation for the exploration of a-alkenyl-g-butyrolactone structures in agriculture.
a potential
rsc.li/rsc-advances
structures reacting with the sulydryl moieties of enzymes or
functional proteins via the Michael type addition among
numerous natural products are the important chemical scaffolds
of STLs.11,12 In our previous work, carabrone and its alcohol
analogue carabrol, two known sesquiterpene lactones isolated
from the fruits of Carpesium macrocephalum, were found to possess
potent antifungal activity in a protective manner which blocked
the early penetrative infection process of pathogenic spores.13 The
study of the structure–activity relationships (SARs) of carabrone
derivates also revealed that the a-methylene-g-butyrolactone core
boosts their antifungal potency.14,15 At the same time, Tulipalin A
and B, two natural pharmacophores, both of which contain
a methylene group, exerted great antimicrobial activities.16 There-
fore, as shown in Fig. 1, the bioactivity of these compounds guided
us to continue to seek new analogous a-methylene-g-lactone
bearing templates with potent fungicidal efficacy.
a-Alkenyl-g-butyrolactone derivates with a chain-ring have
been demonstrated to reduce their cytotoxicity.17 Moreover, little
is known about the effect of the lactone ring conformation and
constraints on the bio-activity of a-methylene-g-lactone. Thus
this prompted us to design and synthesize a series of a-alkenyl-g-
butyrolactone and d-valerolactone derivates. Since the in vitro
antifungal activity of fungicides does not always match the in vivo
activity, i.e. in compounds with high in vitro antifungal activity,
the in vivo activities were also tested. At the same time, SAR and
constructed QSAR models were built to elaborate the relationship
between structural features of the activity and mechanism
actions.18,19 The objective of this article was to seek an efficient
candidate based on bioactivity-guided mixed synthesis.
Introduction
In recent years, fungicide resistance and food security problems
have becoming increasingly serious.1,2 Since it is difficult to nd
appropriate fungal-specic targets of fungal cells, the discovery of
a new mode of action or novel fungicidal agents based on novel
scaffolds from natural products has become important to over-
come the chemical resistance problem. In fact, natural products
with structural modication have been broadly used for discov-
ering and developing pesticides in the market today, due to the
structural diversities, environmental compatibilities, easy
biodegradation, and lower environmental and mammalian
toxicity characteristics.3–6 In addition, discovering and screening
candidates with antifungal activity from thousands of natural
products is virtually and economically impossible, while employ-
ing a bioactivity-guided mixed synthesis method would reduce the
problems with cost and time requirements for screening.
A wide variety of structures of sesquiterpene lactones (STLs)
have a diversity of biological activities, such as cytotoxic, anti-
cancer, antifungal, anti-inammatory, and antimicrobial proper-
ties.7–10 In fact, a-methylene-g-lactone and cyclopentenone
aResearch and Development Center of Biorational Pesticide, Northwest A&F University,
Yangling 712100, Shaanxi, China. E-mail: fengjt@nwsuaf.edu.cn; Fax: +86-29-
87092122; Tel: +86-29-87092122
bResearch Center of Biopesticide Technology
& Engineering, Yangling 712100,
Shaanxi, China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c7ra12471f
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Fig. 1 The design strategy to find promising fungicidal candidates.
ꢀ
Data for compound 2-2. White crystal; mp: 162.3–163.5 C;
Experimental
Chemicals and instruments
52% yield; 1H NMR (500 MHz, CDCl3) d 7.68 (t, J ¼ 2.9 Hz, 1H),
7.38 (dd, J ¼ 7.6, 1.6 Hz, 1H), 7.27 (dt, J ¼ 6.2, 3.1 Hz, 1H), 7.23–
Chemicals and reagents used in this research were of analytical 7.16 (m, 2H), 4.32–4.20 (m, 2H), 3.06–2.94 (m, 2H). 13C NMR
grade (purchased from Aladdin Industrial Inc., Shanghai, China); (126 MHz, CDCl3): d 172.10 135.20, 132.41, 131.68, 130.80,
all solvents were dried and redistilled before use. Analytical thin- 129.98, 129.36, 127.23, 126.63, 68.01, 26.62. HR-MS (ESI): m/z
layer chromatography (TLC) was performed on silica gel GF254
Column chromatographic (CC) purication was carried out using
.
calcd for C11H9ClO2 ([M + Na]+) 208.0291, found 231.0183.
ꢀ
Data for compound 2-3. White crystal; mp: 163.5–164.2 C;
silica gel (200–300 mesh), which was obtained from Qingdao 51% yield; 1H NMR (500 MHz, CDCl3) d 7.68 (t, J ¼ 2.9 Hz, 1H),
Haiyang Chemical Co., Ltd. (Qingdao, China). The melting points 7.38 (dd, J ¼ 7.6, 1.6 Hz, 1H), 7.27 (dt, J ¼ 6.2, 3.1 Hz, 1H), 7.22–
of these synthetic derivatives were determined on an X-5 appa- 7.15 (m, 2H), 4.31–4.23 (m, 2H), 2.99 (ddd, J ¼ 15.8, 10.0, 5.0 Hz,
ratus and uncorrected, and the apparatus was purchased from 2H). 13C NMR (126 MHz, CDCl3): 172.01, 135.20, 132.41, 131.68,
Beijing Tech. Nuclear magnetic resonance spectra (NMR) were 130.80, 129.98, 129.36, 127.26, 126.63, 69.22, 26.62. HR-MS
obtained on a Bruker Avance 500 MHz instrument. HR-MS (ESI) (ESI): m/z calcd for C11H9ClO2 ([M + Na]+) 208.0291, found
was observed using a Bruker Apex-Ultra 7.0 T spectrometer. The 231.0183.
ꢀ
reaction progress was monitored by thin-layer chromatography
on silica gel GF-254 with detection by UV light.
Data for compound 2-4. White crystal; mp: 159.3–159.9 C;
55% yield; 1H NMR (500 MHz, CDCl3) d 7.53 (t, J ¼ 2.9 Hz, 1H),
7.48–7.42 (m, 4H), 4.50 (t, J ¼ 7.2 Hz, 2H), 3.25 (td, J ¼ 7.3,
3.0 Hz, 2H). 13C NMR (126 MHz, CDCl3): 172.25, 135.85, 135.22,
133.14, 131.15, 129.25, 124.24, 65.44, 27.59. HR-MS (ESI): m/z
calcd for C11H9ClO2 ([M + Na]+) 208.0291, found 231.0183.
Synthesis of compound a-alkenyl-g-butyrolactone
As shown in Scheme 1, to a cooled (0 ꢀC) solution of g-butyr-
olactone (5.0 g, 58 mmol) and benzaldehyde (5.85 g, 55.1 mmol) in
dry benzene (75 mL) was added KOtBu (7.82 g, 69.7 mmol) por-
tionwise.20,21 Aer the addition, the thick orange solution was
stirred at room temp for 6 h. The mixture was acidied with dilute
H2SO4 (aq) and extracted with Et2O (3 ꢁ 30 mL). The organic layer
was dried (MgSO4) and evaporated under reduced pressure. The
residue was puried using ash column chromatography (silica
gel, EtOAc/petroleum ether ¼ 1 : 5) to yield 2 (5.4 g, 56%) as
a white solid. The data of compound 2 is shown as follows.
1
Data for compound 2-5. White oil; 57% yield; H NMR (500
MHz, CDCl3) d 7.57 (t, J ¼ 2.9 Hz, 1H), 7.43 (dd, J ¼ 8.1, 1.0 Hz,
1H), 7.33 (dd, J ¼ 7.9, 1.2 Hz, 1H), 7.23 (dd, J ¼ 11.3, 3.9 Hz, 1H),
7.06 (td, J ¼ 7.9, 1.5 Hz, 1H), 4.20 (dt, J ¼ 12.1, 5.8 Hz, 2H), 2.99–
2.90 (m, 2H). 13C NMR (126 MHz, CDCl3): d 171.89, 134.11,
133.25, 130.92, 129.52, 127.66, 127.58, 126.81, 125.68, 68.01,
29.41. HR-MS (ESI): m/z calcd for C11H9BrO2 ([M + Na]+)
251.9786, found 274.9678.
ꢀ
Data for compound 2-6. White crystal; mp: 161.5–162.2 C;
44% yield; 1H NMR (500 MHz, CDCl3) d 7.57 (t, J ¼ 2.9 Hz, 1H),
7.43 (dd, J ¼ 8.1, 1.0 Hz, 1H), 7.33 (dd, J ¼ 7.9, 1.2 Hz, 1H), 7.23
(dd, J ¼ 11.3, 3.9 Hz, 1H), 7.06 (td, J ¼ 7.9, 1.5 Hz, 1H), 4.27 (dt,
J ¼ 12.1, 5.8 Hz, 2H), 2.96 (m, 2H). 13C NMR (126 MHz, CDCl3):
d 172.89, 133.11, 133.25, 130.92, 129.52, 128.66, 127.58, 125.81,
124.68, 66.01, 28.41. HR-MS (ESI): m/z calcd for C11H9BrO2 ([M +
Na]+) 251.9786, found 274.9678.
ꢀ
Data for compound 2-1. White crystal; mp: 166.3–167.5 C;
55% yield; 1H NMR (500 MHz, CDCl3) d 7.39 (t, J ¼ 3.0 Hz, 1H),
7.38–7.35 (m, 2H), 7.34–7.31 (m, 2H), 7.30–7.28 (m, 1H), 4.28 (s,
1H), 4.25 (s, 1H), 3.04 (td, J ¼ 7.3, 2.9 Hz, 2H). 13C NMR (126
MHz, CDCl3): 172.37, 136.02, 134.62, 130.13, 129.72, 128.71,
124.02, 65.47, 27.27. HR-MS (ESI): m/z calcd for C11H10O2 ([M +
H]+) 174.0861, found 175.0754.
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Scheme 1 Synthesis route of the target compounds 2-1–26 and 4-1–26.
ꢀ
7.15 (t, J ¼ 7.6 Hz, 1H), 7.08 (d, J ¼ 7.7 Hz, 1H), 7.05 (s, 1H), 7.02
(d, J ¼ 7.5 Hz, 1H), 4.23–4.08 (m, 2H), 2.92 (dd, J ¼ 9.6, 4.8 Hz,
2H), 2.18 (d, J ¼ 15.2 Hz, 3H). 13C NMR (126 MHz, CDCl3):
d 172.31, 138.50, 135.79, 134.51, 130.75, 130.49, 128.55, 126.77,
123.71, 67.38, 29.13, 23.18. HR-MS (ESI): m/z calcd for C12H12O2
([M + Na]+) 188.0837, found 211.0730.
Data for compound 2-7. White crystal; mp: 166.2–167.2 C;
57% yield; 1H NMR (500 MHz, CDCl3) d 7.52 (t, J ¼ 2.9 Hz, 1H),
7.35 (dd, J ¼ 8.1, 1.0 Hz, 1H), 7.24 (dd, J ¼ 7.9, 1.2 Hz, 1H), 7.21
(dd, J ¼ 11.3, 3.9 Hz, 1H), 7.06 (td, J ¼ 7.9, 1.5 Hz, 1H), 4.22 (dt,
J ¼ 12.1, 5.8 Hz, 2H), 3.03 (m, 2H). 13C NMR (126 MHz, CDCl3):
d 172.54, 135.13, 133.25, 131.63, 128.32, 127.63, 126.52, 125.82,
123.68, 65.03, 28.41. HR-MS (ESI): m/z calcd for C11H9BrO2 ([M +
Na]+) 251.9786, found 274.9678.
Data for compound 2-13. White crystal; mp: 159.5–160.2 ꢀC;
61% yield; 1H NMR (500 MHz, CDCl3) d 7.55 (t, J ¼ 2.9 Hz, 1H),
7.42 (d, J ¼ 8.1 Hz, 2H), 7.27 (d, J ¼ 8.0 Hz, 2H), 4.47 (t, J ¼
7.3 Hz, 2H), 3.24 (td, J ¼ 7.3, 2.9 Hz, 2H), 2.42 (s, 3H). 13C NMR
(126 MHz, CDCl3): d 172.74, 140.37, 136.60, 131.93, 130.07,
129.72, 122.42, 65.45, 27.46, 21.51. HR-MS (ESI): m/z calcd for
ꢀ
Data for compound 2-8. White crystal; mp: 161.1–162.4 C;
56% yield; 1H NMR (500 MHz, CDCl3) d 7.47 (s, 1H), 7.24 (t, J ¼
9.6 Hz, 1H), 7.21 (dd, J ¼ 11.3, 3.9 Hz, 1H), 7.06 (td, J ¼ 7.9,
1.5 Hz, 1H), 6.97 (dd, J ¼ 11.4, 5.5 Hz, 1H), 4.29 (t, J ¼ 7.1 Hz,
C
12H12O2 ([M + Na]+) 188.0837, found 211.0730.
2H), 3.04 (dd, J ¼ 8.9, 4.6 Hz, 2H). 13C NMR (126 MHz, CDCl3):
d 172.35, 134.31, 133.31, 132.70, 131.93, 131.89, 123.54, 115.98,
112.81, 66.26, 28.38. HR-MS (ESI): m/z calcd for C11H9FO2 ([M +
Na]+) 192.0587, found 215.0478.
Data for compound 2-14. White crystal; mp: 161.5–162.2 ꢀC;
57% yield; 1H NMR (500 MHz, CDCl3) d 7.75 (t, J ¼ 2.9 Hz, 1H),
7.24 (d, J ¼ 7.7 Hz, 1H), 7.19 (t, J ¼ 7.9 Hz, 1H), 6.82 (dd, J ¼ 14.6,
7.1 Hz, 1H), 6.76 (t, J ¼ 7.6 Hz, 1H), 4.18 (t, J ¼ 7.3 Hz, 2H), 3.62
(d, J ¼ 20.1 Hz, 3H), 2.92 (td, J ¼ 7.3, 2.9 Hz, 2H). 13C NMR (126
MHz, CDCl3): d 171.68, 160.74, 131.69, 130.57, 128.84, 123.36,
123.29, 120.45, 112.14, 69.22, 55.22, 27.29. HR-MS (ESI): m/z
calcd for C12H12O3 ([M + H]+) 204.0786, found 205.0859.
Data for compound 2-15. White crystal; mp: 165.2–166.4 ꢀC;
44% yield; 1H NMR (500 MHz, CDCl3) d 7.21–7.06 (m, 2H), 6.82
(dd, J ¼ 12.0, 7.7 Hz, 1H), 6.79–6.66 (m, 2H), 4.22–4.09 (m, 2H),
3.68–3.45 (m, 3H), 2.89 (dd, J ¼ 9.1, 6.1 Hz, 2H). 13C NMR (126
MHz, CDCl3): d 172.23, 159.61, 138.21, 135.53, 130.99, 124.29,
122.19, 115.18, 65.43, 54.98, 27.32. HR-MS (ESI): m/z calcd for
ꢀ
Data for compound 2-9. White crystal; mp: 158.5–159.2 C;
46% yield; 1H NMR (500 MHz, CDCl3) d 7.45 (s, 1H), 7.30 (s, 1H),
7.27 (t, J ¼ 9.6 Hz, 1H), 6.97 (dd, J ¼ 11.4, 5.5 Hz, 2H), 4.27 (t, J ¼
7.1 Hz, 2H), 3.24 (dd, J ¼ 8.9, 4.6 Hz, 2H). 13C NMR (126 MHz,
CDCl3): d 172.35, 135.64, 135.33, 134.72, 131.26, 131.61, 121.53,
115.98, 112.31, 66.29, 26.35. HR-MS (ESI): m/z calcd for
C
11H9FO2 ([M + Na]+) 192.0587, found 215.0478.
Data for compound 2-10. White crystal; mp: 161.3–162.6 ꢀC;
53% yield; 1H NMR (500 MHz, CDCl3) d 7.47–7.30 (m, 2H), 7.27
(t, J ¼ 9.6 Hz, 1H), 6.97 (dd, J ¼ 11.4, 5.5 Hz, 2H), 4.29 (t, J ¼
7.1 Hz, 2H), 3.04 (dd, J ¼ 8.9, 4.6 Hz, 2H). 13C NMR (126 MHz,
CDCl3): d 172.35, 134.70, 131.96, 131.91, 123.54, 115.98, 115.81,
66.29, 27.35. HR-MS (ESI): m/z calcd for C11H9FO2 ([M + Na]+)
192.0587, found 215.0478.
C
12H12O3 ([M + H]+) 204.0786, found 205.0859.
Data for compound 2-16. White crystal; mp: 161.5–162.2 ꢀC;
54% yield; 1H NMR (500 MHz, CDCl3) d 7.22 (d, J ¼ 8.7 Hz, 2H),
7.18 (s, 1H), 6.73 (d, J ¼ 8.8 Hz, 2H), 4.18 (t, J ¼ 7.3 Hz, 2H), 3.57
(d, J ¼ 31.6 Hz, 3H), 2.93 (td, J ¼ 7.3, 2.5 Hz, 2H). 13C NMR (126
MHz, CDCl3): d 172.60, 160.67, 135.03, 132.83, 126.85, 120.97,
115.62, 68.40, 55.05, 27.06. HR-MS (ESI): m/z calcd for C12H12O3
([M + H]+) 204.0786, found 205.0859.
Data for compound 2-11. White crystal; mp: 167.2–168.4 ꢀC;
53% yield; 1H NMR (500 MHz, CDCl3) d 7.38 (s, 1H), 7.13 (dd, J ¼
4.9, 3.9 Hz, 1H), 7.03–6.94 (m, 2H), 6.94–6.87 (m, 1H), 4.12–3.85
(m, 2H), 2.83–2.56 (m, 2H), 2.03 (d, J ¼ 4.3 Hz, 3H). 13C NMR
(126 MHz, CDCl3): d 171.15, 138.19, 133.63, 132.61, 130.85,
129.36, 128.21, 126.45, 125.06, 67.38, 27.00, 21.90. HR-MS (ESI):
m/z calcd for C12H12O2 ([M + Na]+) 188.0837, found 211.0730.
Data for compound 2-12. White crystal; mp: 155.5–156.4 ꢀC;
56% yield; 1H NMR (500 MHz, CDCl3) d 7.22 (d, J ¼ 1.2 Hz, 1H),
Data for compound 2-17. White crystal; mp: 162.5–163.1 ꢀC;
45% yield; 1H NMR (500 MHz, CDCl3) d 7.61 (d, J ¼ 8.0 Hz, 2H),
7.58–7.52 (m, 2H), 7.45 (s, 1H), 4.41 (t, J ¼ 7.1 Hz, 2H), 3.23 (dd,
J ¼ 26.6, 10.9 Hz, 2H). 13C NMR (126 MHz, CDCl3): d 172.19,
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Data for compound 2-25. White crystal; mp: 161.2–162.4 ꢀC;
55% yield; 1H NMR (500 MHz, CDCl3) d 7.51 (dd, J ¼ 8.9, 7.5 Hz,
2H), 7.42–7.37 (m, 2H), 7.35 (dt, J ¼ 5.1, 2.0 Hz, 1H), 7.26 (dt, J ¼
11.2, 2.8 Hz, 1H), 6.94 (d, J ¼ 15.6 Hz, 1H), 6.90–6.80 (m, 1H),
4.61–4.23 (m, 2H), 3.08 (td, J ¼ 7.4, 2.6 Hz, 2H). 13C NMR (126
MHz, CDCl3): d 171.97, 141.46, 135.99, 135.85, 129.35, 129.07,
127.35, 124.53, 123.84, 65.54, 25.70. HR-MS (ESI): m/z calcd for
137.98, 134.45, 130.14, 126.93, 125.94, 125.78, 67.66, 46.85,
27.43. HR-MS (ESI): m/z calcd for C12H9F3O2 ([M + Na]+)
242.0555, found 265.0447.
Data for compound 2-18. White crystal; mp: 167.3–168.3 ꢀC;
42% yield; 1H NMR (500 MHz, CDCl3) d 7.96 (d, J ¼ 6.2 Hz, 1H),
7.91–7.85 (m, 3H), 7.71 (t, J ¼ 2.8 Hz, 1H), 7.59 (dd, J ¼ 8.6,
1.5 Hz, 1H), 7.57–7.52 (m, 2H), 4.52–4.43 (m, 2H), 3.33 (td, J ¼
7.3, 2.9 Hz, 2H). 13C NMR (126 MHz, CDCl3): d 173.28, 136.68,
133.62, 133.18, 132.18, 130.87, 128.62, 128.60, 127.72, 127.49,
126.81, 126.39, 123.76, 65.40, 27.60. HR-MS (ESI): m/z calcd for
C
13H12O2 ([M + H]+) 200.0837, found 201.0910.
Data for compound 2-26. White crystal; mp: 159.2–160.2 ꢀC;
43% yield; 1H NMR (500 MHz, CDCl3) d 7.71 (dd, J ¼ 8.9, 7.5 Hz,
2H), 7.56–7.47 (m, 2H), 7.36 (dt, J ¼ 5.1, 2.0 Hz, 1H), 7.16 (dt, J ¼
11.2, 2.8 Hz, 1H), 6.85 (d, J ¼ 15.6 Hz, 1H), 4.65 (m, 2H), 3.08 (td,
J ¼ 7.4, 2.6 Hz, 2H). 13C NMR (126 MHz, CDCl3): d 172.94,
143.45, 136.93, 135.84, 128.35, 128.07, 127.34, 124.73, 122.84,
65.64, 26.71. HR-MS (ESI): m/z calcd for C13H12O2 ([M + H]+)
277.9942, found 279.0015.
C
15H12O2 ([M + H]+) 224.0837, found 225.0910.
Data for compound 2-19. White crystal; mp: 160.3–161.4 ꢀC;
43% yield; 1H NMR (500 MHz, CDCl3) d 8.46 (s, 1H), 8.43 (s, 1H),
8.06–8.00 (m, 2H), 7.97 (t, J ¼ 7.4 Hz, 2H), 7.58–7.48 (m, 4H),
4.48–4.23 (m, 2H), 2.57 (td, J ¼ 7.1, 3.2 Hz, 2H). 13C NMR (126
MHz, CDCl3): d 171.06, 132.98, 131.27, 131.01, 129.16, 128.67,
128.62, 128.25, 126.57, 125.53, 125.17, 65.43, 26.95. HR-MS
(ESI): m/z calcd for C19H14O2 ([M + H]+) 274.0994, found
275.1067.
Synthesis of compound a-alkenyl-d-valerolactone
Data for compound 2-20. White crystal; mp: 164.2–165.3 ꢀC; A solution of aromatic aldehyde (5 mmol), d-valerolactone (5
45% yield; 1H NMR (500 MHz, CDCl3) d 10.14 (d, J ¼ 1.4 Hz, 1H), mmol) in anhydrous methyl alcohol was added dropwise to
8.04 (dd, J ¼ 8.2, 1.4 Hz, 2H), 7.88 (dd, J ¼ 18.9, 12.1 Hz, 2H), a methyl alcohol solution of NaOEt (10 mmol) under ice-
3.80–3.66 (m, 2H), 2.09 (d, J ¼ 1.6 Hz, 2H). 13C NMR (126 MHz, cooling. The mixture was stirred at 0–10 ꢀC for 3 hours. TLC
CDCl3): d 190.56, 139.24, 132.92, 132.25, 130.11, 129.90, 117.94, was used to monitor whether the reaction was completed.
52.29, 30.60. HR-MS (ESI): m/z calcd for C12H9NO2 ([M + H]+) Thereaer, the mixture was diluted with EtOAc, and acidied
199.0633, found 200.0706.
with dilute H2SO4 (aq). The organic layer was then washed with
Data for compound 2-21. White crystal; mp: 155.5–155.9 ꢀC; NaHCO3 solution and water, dried with MgSO4 and removed
47% yield; 1H NMR (500 MHz, CDCl3) d 7.53 (t, J ¼ 2.9 Hz, 1H), under reduced pressure to give the crude product which was
7.19 (t, J ¼ 7.9 Hz, 1H), 6.86 (dd, J ¼ 14.6, 7.1 Hz, 1H), 6.56 (t, J ¼ puried by column chromatography with petroleum ether–
7.6 Hz, 1H), 4.12 (t, J ¼ 7.3 Hz, 2H), 2.82 (td, J ¼ 7.3, 2.9 Hz, 2H). EtOAc (10 : 1) to afford compound 4. The structures of all
13C NMR (126 MHz, CDCl3): d 172.64, 162.74, 134.69, 133.52, compounds were conrmed by 1H and 13C NMR and HR-MS
124.83, 123.36, 121.25, 120.43, 113.14, 68.52, 56.24, 27.23. HR- analyses. The synthesis route is shown in Scheme 1 and the
MS (ESI): m/z calcd for C11H8Cl2O2 ([M + H]+) 241.9901, found data of compound 4 is shown as follows.
ꢀ
242.9974.
Data for compound 4-1. White crystal; mp: 167.4–168.3 C;
Data for compound 2-22. White crystal; mp: 157.5–158.4 ꢀC; 45% yield; 1H NMR (500 MHz, CDCl3) d 7.66 (s, 1H), 7.46 (s, 1H),
46% yield; 1H NMR (500 MHz, CDCl3) d 7.48 (s, 1H), 7.16 (t, J ¼ 7.28 (s, 1H), 7.17 (m, 3H), 4.22 (m, 2H), 2.71 (m, 2H), 1.76 (m,
2.6 Hz, 1H), 6.56 (d, J ¼ 3.5 Hz, 1H), 6.43 (dd, J ¼ 3.4, 1.7 Hz, 2H). 13C NMR (126 MHz, CDCl3): d 168.23, 138.34, 136.66,
1H), 4.32 (t, J ¼ 7.4 Hz, 2H), 3.12 (td, J ¼ 7.3, 2.6 Hz, 2H). 13C 136.21, 129.21, 128.98, 128.67, 128.54, 128.59, 74.05, 27.20,
NMR (126 MHz, CDCl3): d 172.17, 151.19, 145.25, 122.48, 24.10. HR-MS (ESI): m/z calcd for C12H12O2 ([M + H]+) 188.0837,
121.28, 115.73, 112.49, 65.63, 27.21. HR-MS (ESI): m/z calcd for found 189.0910.
C9H8O3 ([M + H]+) 164.0473, found 165.0546.
Data for compound 4-2. White crystal; mp: 162.3–163.2 C;
ꢀ
Data for compound 2-23. White crystal; mp: 166.5–167.2 ꢀC; 44% yield; 1H NMR (500 MHz, CDCl3) d 7.59 (s, 1H), 7.16 (s, 1H),
43% yield; 1H NMR (500 MHz, CDCl3) d 7.28 (t, J ¼ 2.9 Hz, 1H), 7.11–7.09 (m, 3H), 4.29–4.09 (m, 2H), 2.72–2.55 (m, 2H), 1.76–
7.21 (d, J ¼ 8.3 Hz, 2H), 7.07 (dd, J ¼ 13.9, 6.1 Hz, 2H), 4.17 (t, J ¼ 1.72 (m, 2H). 13C NMR (126 MHz, CDCl3): d 169.63, 138.38,
7.3 Hz, 2H), 2.93 (td, J ¼ 7.3, 2.9 Hz, 2H), 2.54–2.41 (m, 2H), 1.09 135.56, 135.41, 129.71, 129.18, 128.82, 128.13, 127.55, 72.65,
(t, J ¼ 7.7 Hz, 3H). 13C NMR (126 MHz, CDCl3): d 174.83, 146.25, 27.23, 23.12. HR-MS (ESI): m/z calcd for C12H11ClO2 ([M + H]+)
135.78, 131.95, 130.68, 128.41, 122.82, 68.58, 28.49, 27.22, 222.0448, found 223.0521.
15.13. HR-MS (ESI): m/z calcd for C13H14O2 ([M + H]+) 202.0994,
Data for compound 4-3. White crystal; mp: 156.5–157.7 C;
47% yield; 1H NMR (500 MHz, CDCl3) d 7.56 (s, 1H), 7.18 (s, 1H),
ꢀ
found 225.0886.
Data for compound 2-24. White crystal; mp: 154.2–155.3 ꢀC; 7.14–7.09 (m, 3H), 4.29–4.09 (m, 2H), 2.73–2.56 (m, 2H), 1.78–
43% yield; 1H NMR (500 MHz, CDCl3) d 7.25 (d, J ¼ 2.3 Hz, 1H), 1.70 (m, 2H). 13C NMR (126 MHz, CDCl3): d 167.23, 139.35,
7.23–7.13 (m, 2H), 7.12–6.96 (m, 2H), 4.13 (dd, J ¼ 13.5, 6.4 Hz, 136.52, 134.51, 129.81, 129.58, 128.82, 128.24, 127.59, 72.05,
2H), 3.00–2.85 (m, 2H), 1.79 (t, J ¼ 4.7 Hz, 1H), 1.08–1.01 (m, 28.20, 24.10. HR-MS (ESI): m/z calcd for C12H11ClO2 ([M + H]+)
6H). 13C NMR (126 MHz, CDCl3): d 171.35, 150.69, 135.62, 222.0448, found 223.0521.
ꢀ
132.10, 130.63, 128.51, 122.92, 65.23, 35.36, 26.95, 25.02. HR-MS
Data for compound 4-4. White crystal; mp: 158.8–159.2 C;
(ESI): m/z calcd for C14H16O2 ([M + H]+) 216.1150, found 43% yield; 1H NMR (500 MHz, CDCl3) d 7.55 (d, J ¼ 2.1 Hz, 1H),
217.1223.
7.16–7.12 (m, 5H), 4.23–4.06 (m, 2H), 2.59 (dd, J ¼ 8.7, 4.4 Hz,
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2H), 1.77–1.68 (m, 2H). 13C NMR (126 MHz, CDCl3): d 168.60, 7.23 (t, J ¼ 7.5 Hz, 1H), 7.16 (t, J ¼ 7.8 Hz, 2H), 7.10 (d, J ¼ 7.4 Hz,
141.05, 133.37, 131.68, 130.69, 128.91, 126.60, 68.76, 26.81, 1H), 4.42–4.20 (m, 2H), 2.79 (td, J ¼ 6.6, 2.3 Hz, 2H), 2.29 (d, J ¼
24.10. HR-MS (ESI): m/z calcd for C12H11ClO2 ([M + H]+) 8.8 Hz, 3H), 1.87 (td, J ¼ 11.2, 6.4 Hz, 2H). 13C NMR (126 MHz,
222.0448, found 223.0521.
CDCl3): d 166.91, 141.44, 138.51, 134.85, 130.81, 130.41, 128.74,
ꢀ
Data for compound 4-5. White crystal; mp: 162.3–163.4 C; 127.80, 124.96, 70.78, 25.89, 23.46, 22.25. HR-MS (ESI): m/z
46% yield; 1H NMR (500 MHz, CDCl3) d 7.56–7.46 (m, 1H), 7.42– calcd for C13H14O2 ([M + H]+) 202.0994, found 203.0167.
7.35 (m, 1H), 7.27–7.16 (m, 2H), 7.03 (td, J ¼ 7.2, 3.7 Hz, 1H),
Data for compound 4-13. White crystal; mp: 157.5–158.2 ꢀC;
4.31–4.11 (m, 2H), 2.60–2.44 (m, 2H), 1.82–1.70 (m, 2H). 13C 47% yield; 1H NMR (500 MHz, CDCl3) d 7.78 (s, 1H), 7.31 (dd, J ¼
NMR (126 MHz, CDCl3): d 172.32, 133.17, 132.47, 130.54, 50.5, 26.7 Hz, 2H), 6.88 (d, J ¼ 8.5 Hz, 2H), 4.45–4.23 (m, 2H),
128.77, 127.53, 124.52, 120.97, 70.82, 45.35, 26.58, 22.84. HR-MS 3.90–3.71 (m, 3H), 2.79 (s, 2H), 1.92 (dd, J ¼ 17.7, 12.1 Hz, 2H).
(ESI): m/z calcd for C12H11BrO2 ([M + Na]+) 265.9942, found 13C NMR (126 MHz, CDCl3): d 167.31, 159.71, 141.16, 134.10,
288.9834.
Data for compound 4-6. White crystal; mp: 160.5–161.2 C; m/z calcd for C13H14O2 ([M + H]+) 202.0994, found 203.0167.
42% yield; 1H NMR (500 MHz, CDCl3) d 7.50–7.46 (m, 1H), 7.40 Data for compound 4-14. White crystal; mp: 165.3–166.2 ꢀC;
130.35, 123.26, 116.89, 68.48, 57.67, 26.28, 24.73. HR-MS (ESI):
ꢀ
(d, J ¼ 8.1 Hz, 1H), 7.34–7.30 (m, 1H), 7.29 (t, J ¼ 3.0 Hz, 1H), 46% yield; 1H NMR (500 MHz, CDCl3) d 7.76 (d, J ¼ 1.9 Hz, 1H),
7.22 (t, J ¼ 7.8 Hz, 1H), 4.42–4.26 (m, 2H), 3.10 (tt, J ¼ 28.8, 7.31 (m, 1H), 6.98 (d, J ¼ 7.3 Hz, 1H), 6.88 (s, 1H), 6.81 (dd, J ¼
14.4 Hz, 2H), 2.19–2.10 (m, 2H). 13C NMR (126 MHz, CDCl3): 4.4, 3.8 Hz, 1H), 4.26 (dd, J ¼ 10.2, 5.1 Hz, 2H), 3.72 (t, J ¼
d 172.12, 136.62, 134.46, 132.63, 132.38, 130.97, 128.57, 125.46, 12.5 Hz, 3H), 2.71 (d, J ¼ 3.8 Hz, 2H), 1.91 (m, 2H). 13C NMR (126
123.28, 69.39, 67.38, 28.20. HR-MS (ESI): m/z calcd for MHz, CDCl3): d 165.67, 158.15, 142.52, 135.45, 128.44, 126.54,
C
12H11BrO2 ([M + Na]+) 265.9942, found 288.9834.
Data for compound 4-7. White crystal; mp: 166.1–166.8 C; m/z calcd for C13H14O3 ([M + H]+) 218.0943, found 219.1016.
123.53, 114.67, 112.53, 68.63, 55.21, 26.86, 23.90. HR-MS (ESI):
ꢀ
42% yield; 1H NMR (500 MHz, CDCl3) d 7.62–7.56 (m, 2H), 7.51
Data for compound 4-15. White crystal; mp: 161.2–162.4 ꢀC;
(dd, J ¼ 6.1, 3.2 Hz, 1H), 7.38 (d, J ¼ 8.5 Hz, 2H), 4.50 (t, J ¼ 43% yield; 1H NMR (500 MHz, CDCl3) d 7.77 (d, J ¼ 1.9 Hz, 1H),
7.2 Hz, 2H), 3.24 (td, J ¼ 7.3, 2.9 Hz, 2H), 2.43–1.91 (m, 0H). 13
C
7.32–7.10 (m, 1H), 6.94 (d, J ¼ 7.3 Hz, 1H), 6.87 (s, 1H), 6.83 (dd,
NMR (126 MHz, CDCl3): d 172.24, 135.30, 133.56, 132.22, J ¼ 4.4, 3.8 Hz, 1H), 4.29 (dd, J ¼ 10.2, 5.1 Hz, 2H), 3.75 (t, J ¼
131.33, 124.43, 124.23, 65.45, 27.61. HR-MS (ESI): m/z calcd for 12.5 Hz, 3H), 2.78 (d, J ¼ 3.8 Hz, 2H), 1.93 (m, 2H). 13C NMR (126
C
12H11BrO2 ([M + Na]+) 265.9942, found 288.9834.
Data for compound 4-8. White crystal; mp: 167.2–168.5 C; 122.54, 115.67, 114.64, 68.67, 55.22, 25.86, 22.90. HR-MS (ESI):
MHz, CDCl3): d 166.76, 159.18, 141.12, 136.15, 129.40, 126.24,
ꢀ
43% yield; 1H NMR (500 MHz, CDCl3) d 7.67 (d, J ¼ 1.9 Hz, 1H), m/z calcd for C13H14O3 ([M + H]+) 218.0943, found 219.1016.
7.43 (m, 1H), 7.28 (s, 1H), 7.13 (m, 2H), 4.57–4.34 (m, 2H), 2.98–
Data for compound 4-16. White crystal; mp: 152.5–153.2 ꢀC;
2.72 (m, 2H), 1.96 (m, 2H). 13C NMR (126 MHz, CDCl3): d 168.41, 42% yield; 1H NMR (500 MHz, CDCl3) d 7.78 (s, 1H), 7.34 (t, J ¼
142.34, 140.36, 132.24, 131.12, 125.56, 123.43, 115.83, 115.53, 16.1 Hz, 2H), 6.88 (d, J ¼ 8.5 Hz, 2H), 4.42–4.12 (m, 2H), 3.95–
67.26, 27.61, 21.89. HR-MS (ESI): m/z calcd for C12H11FO2 ([M + 3.60 (m, 3H), 2.79 (s, 2H), 1.92 (dd, J ¼ 17.7, 12.1 Hz, 2H). 13C
Na]+) 206.0743, found 229.0635.
NMR (126 MHz, CDCl3): d 167.31, 160.37, 141.16, 133.82,
ꢀ
Data for compound 4-9. White crystal; mp: 152.5–153.3 C; 129.07, 123.26, 115.97, 68.48, 56.40, 25.19, 23.26. HR-MS (ESI):
42% yield; 1H NMR (500 MHz, CDCl3) d 7.85 (d, J ¼ 1.9 Hz, 1H), m/z calcd for C13H14O3 ([M + H]+) 218.0943, found 219.1016.
7.43–7.32 (m, 2H), 7.28 (s, 1H), 7.13 (m, 2H), 4.45 (m, 2H), 2.96
Data for compound 4-17. White crystal; mp: 158.1–159.5 ꢀC;
(m, 2H), 1.96 (m, 2H). 13C NMR (126 MHz, CDCl3): d 166.73, 45% yield; 1H NMR (500 MHz, CDCl3) d 7.70 (d, J ¼ 7.0 Hz, 1H),
142.34, 140.21, 132.24, 132.18, 125.56, 125.21, 115.63, 112.73, 7.53–7.45 (m, 2H), 7.38 (d, J ¼ 7.5 Hz, 2H), 4.21 (dd, J ¼ 26.4,
68.61, 27.53, 22.43. HR-MS (ESI): m/z calcd for C12H11FO2 ([M + 16.2 Hz, 2H), 2.81–2.59 (m, 2H), 1.85–1.74 (m, 2H). 13C NMR
Na]+) 206.0743, found 229.0635.
(126 MHz, CDCl3): d 166.36, 139.19, 138.29, 130.53, 128.37,
Data for compound 4-10. White crystal; mp: 158.3–159.2 ꢀC; 125.07, 125.07, 68.73, 27.21, 23.18. HR-MS (ESI): m/z calcd for
44% yield; 1H NMR (500 MHz, CDCl3) d 7.77 (d, J ¼ 1.9 Hz, 1H),
7.45–7.32 (m, 2H), 7.10–6.97 (m, 2H), 4.47–4.24 (m, 2H), 2.94–
C
13H11F3O2 ([M + H]+) 256.0711, found 257.0784.
Data for compound 4-18. White crystal; mp: 157.7–158.5 ꢀC;
2.70 (m, 2H), 1.93–1.82 (m, 2H). 13C NMR (126 MHz, CDCl3): 42% yield; H NMR (500 MHz, CDCl3) d 8.05 (s, 1H), 7.85–7.79
d 167.71, 141.33, 132.24, 132.18, 125.56, 116.03, 115.53, 68.66, (m, 4H), 7.53–7.47 (m, 4H), 4.45–4.34 (m, 2H), 2.89 (dd, J ¼ 8.4,
27.57, 22.83. HR-MS (ESI): m/z calcd for C12H11FO2 ([M + Na]+) 4.4 Hz, 2H), 1.93–1.85 (m, 2H). 13C NMR (126 MHz, CDCl3):
1
206.0743, found 229.0635.
d 167.14, 141.35, 133.24, 133.02, 132.45, 130.45, 128.55, 128.18,
Data for compound 4-11. White crystal; mp: 157.5–157.9 ꢀC; 127.66, 127.25, 127.19, 126.62, 126.02, 68.76, 26.01, 24.38. HR-
1
46% yield; H NMR (500 MHz, CDCl3) d 8.02 (s, 1H), 7.28–7.20 MS (ESI): m/z calcd for C16H14O2 ([M + H]+) 238.0994, found
(m, 4H), 4.56–4.31 (m, 2H), 2.72 (td, J ¼ 6.7, 2.2 Hz, 2H), 2.34– 239.1067.
2.20 (m, 3H), 1.93 (td, J ¼ 11.4, 6.1 Hz, 2H). 13C NMR (126 MHz,
Data for compound 4-19. White crystal; mp: 161.2–161.7 ꢀC;
CDCl3): d 166.65, 140.58, 137.61, 134.00, 130.36, 128.87, 128.58, 43% yield; 1H NMR (500 MHz, CDCl3) d 8.41 (s, 1H), 8.40 (s, 1H),
126.65, 125.55, 69.96, 26.04, 23.43, 20.43. HR-MS (ESI): m/z 8.06–8.00 (m, 2H), 7.93 (t, J ¼ 7.4 Hz, 2H), 7.52–7.48 (m, 4H),
calcd for C13H14O2 ([M + H]+) 202.0994, found 203.0167.
4.48–4.23 (m, 2H), 2.55 (td, J ¼ 7.1, 3.2 Hz, 2H), 1.85–1.74 (m,
Data for compound 4-12. White crystal; mp: 161.5–162.2 ꢀC; 2H). 13C NMR (126 MHz, CDCl3): d 172.36, 133.92, 131.57,
42% yield; 1H NMR (500 MHz, CDCl3) d 7.79 (t, J ¼ 2.2 Hz, 1H), 131.01, 129.86, 128.43, 128.54, 128.25, 126.57, 125.21, 125.11,
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66.44, 26.65. HR-MS (ESI): m/z calcd for C20H16O2 ([M + H]+)
288.1150, found 289.1223.
Biological assays
Fungi. The pathogenic fungi Botrytis cinerea, Colletotrichum
lagenarium, Fusarium graminearum, Gaeumannomyces graminis
var. tritici, and Sclerotinia sclerotiorum were provided by the
Center of Pesticide Research, Northwest A&F University, China.
These fungi were grown on potato dextrose agar (PDA) plates at
Data for compound 4-20. White crystal; mp: 159.6–160.4 ꢀC;
43% yield; 1H NMR (500 MHz, CDCl3) d 10.03 (d, J ¼ 1.4 Hz, 1H),
8.21 (dd, J ¼ 8.2, 1.4 Hz, 2H), 7.86 (dd, J ¼ 18.9, 12.1 Hz, 2H),
3.82–3.66 (m, 2H), 2.53 (d, J ¼ 1.6 Hz, 2H), 1.82 (m, 2H). 13C
NMR (126 MHz, CDCl3): d 193.33, 138.21, 132.73, 132.22,
130.41, 129.90, 118.54, 54.24, 32.63. HR-MS (ESI): m/z calcd for
ꢀ
25 C and maintained at 4 ꢀC with periodic subculturing.
C
13H11NO2 ([M + H]+) 213.0790, found 214.0863.
Data for compound 4-21. White crystal; mp: 158.5–159.5 ꢀC;
In vitro antifungal assay
42% yield; 1H NMR (500 MHz, CDCl3) d 7.47 (t, J ¼ 10.5 Hz, 1H),
7.30 (t, J ¼ 2.9 Hz, 1H), 7.20–7.12 (m, 4H), 6.81 (dd, J ¼ 19.0,
7.8 Hz, 1H), 4.23 (ddd, J ¼ 15.9, 10.1, 5.4 Hz, 3H), 3.70 (dd, J ¼
7.2, 1.4 Hz, 2H), 1.96–1.84 (m, 2H). 13C NMR (126 MHz, CDCl3):
d 157.71, 136.60, 130.96, 130.69, 129.81, 129.53, 126.94, 121.28,
111.86, 56.68, 44.27, 25.90. HR-MS (ESI): m/z calcd for
According to the mycelium linear growth rate method reported
previously,22–24 the in vitro antifungal activities of compounds
against ve strains of plant pathogenic fungi were tested.
The compounds with higher initial activities against B. cin-
erea and G. graminis var. tritici were selected to assay 50%
inhibition concentration values (IC50). In order to get a series of
stock solutions, the test compound (2 mM) was diluted with 5%
DMSO aqueous solution by a double-fold dilution method. Each
stock solution (10 mL) was mixed with the autoclaved PDA
medium (190 mL) to provide a set of mediums with different
concentrations of the test compound (3.125, 6.25, 12.5, 25, 50
and 100 mM). The culture medium containing only 0.25%
DMSO was used as a blank control, and Tulipalin A, carabrone
and carbendazim were used as positive controls. Each test was
performed in triplicate. Antifungal toxicity regression equations
and IC50 values were established according to the method
previously reported.14
C
12H10Cl2O2 ([M + H]+) 256.0058, found 257.0131.
Data for compound 4-22. White crystal; mp: 161.5–162.4 ꢀC;
43% yield; 1H NMR (500 MHz, CDCl3) d 7.43 (s, 1H), 7.21 (t, J ¼
2.6 Hz, 1H), 6.52 (d, J ¼ 3.5 Hz, 1H), 6.41 (dd, J ¼ 3.4, 1.7 Hz,
1H), 4.32 (t, J ¼ 7.4 Hz, 2H), 3.32 (td, J ¼ 7.3, 2.6 Hz, 2H), 2.06–
1.96 (m, 2H). 13C NMR (126 MHz, CDCl3): d 171.87, 152.23,
142.21, 123.55, 121.18, 116.32, 112.49, 66.83, 46.63, 21.23. HR-
MS (ESI): m/z calcd for C10H10O3 ([M + H]+) 178.0630, found
179.0703.
Data for compound 4-23. White crystal; mp: 161.6–162.3 ꢀC;
41% yield; 1H NMR (500 MHz, CDCl3) d 7.69 (d, J ¼ 81.8 Hz, 1H),
7.40–7.21 (m, 2H), 7.14 (d, J ¼ 8.1 Hz, 2H), 4.30–4.20 (m, 2H),
2.76 (dd, J ¼ 6.1, 3.9 Hz, 2H), 2.64–2.53 (m, 2H), 1.88–1.79 (m,
2H), 1.15 (td, J ¼ 7.5, 3.8 Hz, 3H). 13C NMR (126 MHz, CDCl3):
d 166.94, 145.64, 141.25, 132.61, 132.26, 127.89, 126.59, 68.52,
In vivo antifungal assay
Fungi strains and fungicides. The fungal pathogens B. cin-
23.46, 16.23. HR-MS (ESI): m/z calcd for C14H16O2 ([M + H]+) erea, G. graminis and B. graminis were provided by the Agricul-
216.1150, found 217.1223.
tural Culture Collection of China (Yangling, Shaanxi, China). B.
Data for compound 4-24. White crystal; mp: 152.2–153.5 ꢀC; cinerea and G. graminis were cultured for 2 weeks at 25 C on
41% yield; 1H NMR (500 MHz, CDCl3) d 7.27 (d, J ¼ 2.3 Hz, 1H), potato dextrose agar (PDA) aer being retrieved from the
7.21–7.10 (m, 2H), 7.10 (m, 2H), 4.11 (dd, J ¼ 13.5, 6.4 Hz, 2H), storage tube.
ꢀ
3.03–2.82 (m, 2H), 2.11–1.96 (m, 2H). 1.77 (t, J ¼ 4.7 Hz, 1H),
The stock solution of the test compound was diluted with
1.03–1.02 (m, 6H). 13C NMR (126 MHz, CDCl3): d 172.35, 153.54, water containing 0.1% tween-80 to 200 mg mLꢂ1
137.65, 132.10, 131.32, 128.51, 121.92, 66.23, 35.36, 27.76, Against B. cinerea. The protective activity of test samples
24.03. HR-MS (ESI): m/z calcd for C15H18O2 ([M + H]+) 230.1307, against B. cinerea on tomato fruits was determined with the
found 231.1380. following methods. Tomato fruits were surface disinfected in
.
Data for compound 4-25. White crystal; mp: 157.7–158.4 ꢀC; sodium hypochlorite (7%, w/v) solution for 5 min and washed
44% yield; 1H NMR (500 MHz, CDCl3) d 7.56 (dd, J ¼ 8.9, 7.5 Hz, thoroughly with sterile water, then samples were spray tested on
2H), 7.44–7.32 (m, 2H), 7.32 (dt, J ¼ 5.1, 2.0 Hz, 1H), 7.28 (dt, J ¼ the tomato fruits until liquid owed on the surface at 24 h before
11.2, 2.8 Hz, 1H), 6.94 (d, J ¼ 15.6 Hz, 1H), 6.91–6.80 (m, 1H), inoculation. Inoculated fruits were placed in plastic boxes at
4.63–4.23 (m, 2H), 3.18 (td, J ¼ 7.4, 2.6 Hz, 2H), 1.96–1.95 (m, 2H). 25 ꢀC with a 16 h photoperiod and 80% relative humidity for
13C NMR (126 MHz, CDCl3): d 172.67, 142.54, 136.91, 132.55, disease development, and carbendazim was used as the positive
128.35, 129.07, 126.35, 122.53, 121.84, 66.54, 24.72. HR-MS (ESI): control. Aer 3 days, the average lesion diameter was determined
m/z calcd for C14H14O2 ([M + H]+) 214.0994, found 215.1067.
by measuring each lesion in two perpendicular directions. The
Data for compound 4-26. White crystal; mp: 157.3–158.8 ꢀC; lengths of the long and short axes were averaged and disease
43% yield; 1H NMR (500 MHz, CDCl3) d 7.72 (dd, J ¼ 8.9, 7.5 Hz, control efficacy was calculated as follows: disease control
2H), 7.54–7.41 (m, 2H), 7.32 (dt, J ¼ 5.1, 2.0 Hz, 1H), 7.26 (dt, J ¼ efficacy ¼ (lesion diameter in the water control ꢂ lesion diameter
11.2, 2.8 Hz, 1H), 6.86 (d, J ¼ 15.6 Hz, 1H), 4.63 (m, 2H), 3.18 (td, in the treatment)/lesion diameter in the water control ꢁ 100.
J ¼ 7.4, 2.6 Hz, 2H), 2.13–1.65 (m, 2H). 13C NMR (126 MHz,
Against G. graminis.25 G. graminis agar plugs (4 mm diam-
CDCl3): d 173.94, 147.43, 135.93, 135.24, 129.15, 126.17, 125.14, eter) were taken from the growing edges of 7 day old PDA
122.73, 121.84, 65.64, 25.71. HR-MS (ESI): m/z calcd for cultures of each isolate. The wheat seeds were surface dis-
C
14H12BrO2 ([M + H]+) 292.0099, found 293.0172.
infected in sodium hypochlorite (0.5%, v/v) for 4 min, and aer
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that the seeds were placed in tested samples for 30 min and put Fisher signicance ratio (F) were used to clarify the standards of
on top of the agar plug with each isolate culture, then covered statics. Moreover, the tested IC50 values were converted into the
with 2 cm thick sand in 9 cm diameter sterile plates. Tri- corresponding plog IC50 values and used as dependent vari-
adimefon was used as the control. There were three replicates of ables to get better linear regression. The quality of the nal
each isolate and each replicate consisted of nine wheat seed- model was ensured using internal validation and the “leave-
lings. The seedlings were kept moist and at room temperature one-out” cross-validation methods.30,31
at 21 ꢀC. The percentage of the affected root area was calculated
aer 21–25 days inoculation. Disease severity was recorded on
Results and discussion
Chemistry
a 0–4 visual scale of the rhizomes and roots, in which 0 ¼
rhizomes and roots with no symptoms, 1 ¼ lesions on <25%,
2 ¼ lesions on 25–50%, 3 ¼ lesions on 50–75%, and 4 ¼ lesions
The synthetic routes of the title compounds are outlined in
Scheme 1. To evaluate the essentiality of the substituent on the
exocyclic carbon–carbon double bond of g-lactone and d-
lactone, we synthesized a series of a-methylene-g-lactone and a-
methylene-d-lactone compounds. g-Butyrolactone and d-valer-
olactone compounds were reacted with different substituent
on 75–100%. The disease index and biocontrol effect meet the
following equation.
Against B. graminis.26 Wheat seeds were surface disinfected
in sodium hypochlorite (7%, w/v) solution for 5 min, washed
thoroughly with sterile water, then germinated in 9 cm pots.
Aer 14 days, the plants reached the desired leaf stage, and the
aldehydes, carried out in an ice-cooling bath under KOtBu or
tested samples were uniformly sprayed on the leaves 24 h before
NaOEt catalysts. With these concise and stereospecic
methods, the stereochemistry of the exocyclic double bond was
exclusively E in moderate yields. It is worth noting that the yield
of the g-butyrolactone derivates (44–61%) was obviously higher
than that of the d-valerolactone derivates (41–47%), which may
be due to the ring tension of the former lactone compounds
being lower than that of the latter, and results in the g-butyr-
olactone derivates tending to be more stable.
shaking the spores. Aer inoculation, the plants were incubated
at 20 ꢀC/15 ꢀC in a growth chamber with cycles of 16 h light and
8 h darkness. Two controls were included in each experiment:
untreated leaves and leaves treated with 15% triadimefon.
Assessment of disease was made 7 days aer inoculation by
counting the number of colonies on 2.5 cm of the middle part of
the treated leaf area. In most cases, leaves with colonies were
assessed visually and classied according to a scale from 0 to 4,
in which 0 ¼ leaves with no symptoms, 1 ¼ lesions on <25%, 2 ¼
Antifungal activity and SARs
lesions on 25–50%, 3 ¼ lesions on 50–75%, and 4 ¼ lesions on
75–100%. The disease index and biocontrol effect meet the
The in vitro screening results of the title compounds for
preliminary antifungal activities against ve pathogenic fungi
following equation.
X
ðgrade of disease severity ꢁ diseased plants of this gradeÞ
Disease indexð%Þ ¼
ꢁ 100
total plants that were assessed ꢁ the highest grade of disease severity
disease index of pathogen control ꢂ disease index of bacteria treatment
Biocontrol effectð%Þ ¼
ꢁ 100
disease index of pathogen control
(Botrytis cinerea, Colletotrichum lagenarium, Phytophthora cap-
sici, Gaeumannomyces graminis var. tritici, and Sclerotinia scle-
rotiorum, Fusarium graminearum) at 100 mg mLꢂ1 are listed in
Table 1 and Fig. 2. The results showed that most of the
compounds exhibited certain to great inhibition activity against
each of the fungi at 100 mg mLꢂ1 (inhibition rate ¼ 46.0–95.8%).
Particularly, the antifungal activity of compounds 2-3–4, 2-7–10,
2-17, 2-17–19, 2-21, 2-25–26, 4-8–10, 4-17, 4-21, and 4-25–26 was
more potent than that of Tulipalin A and equal or higher than
that of the natural product carabrone. Furthermore, from the
results we can see that all the derivatives showed higher activity
against B. cinerea and G. graminis (inhibition rate > 55%)
compared to the other three pathogenic fungi.
Building and validation of the QSAR model
Building and validation of the QSAR model was carried out with
a common procedure. Briey, using the Gaussian 03W package
of programs (Gaussian Inc.), the optimal conformers of the title
compounds with the lowest energy were computed at the DFT/6-
31G (d) level.27 Meanwhile, the most stable congurations of the
compounds were generated and the corresponding “.log” and
“.chk” les were gained. Aerwards, the calculated results were
transformed into a form compatible with CODESSA 2.7.15 using
Ampac 9.1.3.28,29 At last, all of the molecular descriptors
involved in these compounds were calculated by CODESSA
2.7.15, and the heuristic analysis method was used to build the
QSAR model, which determines the most signicant structural
features for antifungal activity against B. cinerea. During the
model development process, the squared correction coefficient
(R2), the squared standard error of the estimates (S2), and the
According to these preliminary results, the half maximal
inhibitory concentration (IC50) of all the derivatives against G.
graminis and B. cinerea was tested by the mycelial growth
inhibitory rate method. It is notable that most of the g-
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Table 1 Initial antifungal activity of compounds at 100 mg mLa
Compound
No.
Average inhibition rate (%) (100 mg mLꢂ1; 72 h)
R
B. c.
C. l.
G. g.
S. s.
F. g.
1
2
3
4
5
6
7
8
2-1
2-2
2-3
2-4
2-5
2-6
2-7
2-8
Ph
2-PhCl
3-PhCl
4-PhCl
2-PhBr
3-PhBr
4-PhBr
2-PhF
81.4 ꢃ 0.7
81.4 ꢃ 0.5
82.7 ꢃ 0.8
85.6 ꢃ 0.8
78.5 ꢃ 0.2
79.1 ꢃ 0.7
82.6 ꢃ 0.5
86.3 ꢃ 0.1
92.6 ꢃ 0.3
95.8 ꢃ 0.4
73.3 ꢃ 0.6
75.6 ꢃ 0.3
77.6 ꢃ 0.8
69.7 ꢃ 0.8
72.6 ꢃ 0.5
73.4 ꢃ 0.2
96.1 ꢃ 0.7
85.2 ꢃ 0.3
87.1 ꢃ 0.8
75.2 ꢃ 0.8
88.2 ꢃ 0.7
81.6 ꢃ 0.4
72.4 ꢃ 0.6
69.7 ꢃ 0.7
92.2 ꢃ 0.5
96.0 ꢃ 0.9
79.2 ꢃ 0.4
78.4 ꢃ 0.8
79.5 ꢃ 0.8
80.6 ꢃ 0.4
76.7 ꢃ 0.9
77.2 ꢃ 0.4
80.6 ꢃ 0.5
83.1 ꢃ 0.3
86.4 ꢃ 0.8
89.5 ꢃ 0.6
69.9 ꢃ 0.3
71.2 ꢃ 0.3
74.1 ꢃ 0.6
65.7 ꢃ 0.6
68.2 ꢃ 0.3
69.2 ꢃ 0.5
91.4 ꢃ 0.7
80.2 ꢃ 0.8
81.6 ꢃ 0.9
69.5 ꢃ 0.5
83.4 ꢃ 0.5
76.5 ꢃ 0.8
70.8 ꢃ 0.2
68.2 ꢃ 0.6
85.0 ꢃ 0.3
86.6 ꢃ 0.2
82.2 ꢃ 0.7
64.9 ꢃ 0.7
76.7 ꢃ 0.3
77.9 ꢃ 0.1
77.6 ꢃ 0.5
80.1 ꢃ 0.6
72.3 ꢃ 0.3
74.9 ꢃ 0.8
78.6 ꢃ 0.3
80.5 ꢃ 0.5
87.5 ꢃ 0.5
90.4 ꢃ 0.5
68.3 ꢃ 0.2
70.8 ꢃ 0.5
81.2 ꢃ 0.1
64.5 ꢃ 0.3
68.1 ꢃ 0.4
69.8 ꢃ 0.2
91.3 ꢃ 0.6
80.5 ꢃ 0.7
81.3 ꢃ 0.7
71.3 ꢃ 0.1
82.6 ꢃ 0.5
75.3 ꢃ 0.3
65.2 ꢃ 0.8
63.4 ꢃ 0.1
87.2 ꢃ 0.6
88.3 ꢃ 0.9
73.4 ꢃ 0.8
74.3 ꢃ 0.2
74.4 ꢃ 0.8
75.4 ꢃ 0.7
70.7 ꢃ 0.8
72.5 ꢃ 0.3
74.1 ꢃ 0.3
78.2 ꢃ 0.1
81.5 ꢃ 0.3
83.4 ꢃ 0.8
63.1 ꢃ 0.2
66.5 ꢃ 0.7
68.3 ꢃ 0.7
64.6 ꢃ 0.4
66.8 ꢃ 0.2
63.7 ꢃ 0.4
87.9 ꢃ 0.6
74.1 ꢃ 0.8
78.5 ꢃ 0.3
61.8 ꢃ 0.5
78.9 ꢃ 0.6
72.5 ꢃ 0.3
65.4 ꢃ 0.4
62.2 ꢃ 0.6
80.4 ꢃ 0.2
83.1 ꢃ 0.2
75.6 ꢃ 0.7
57.3 ꢃ 0.7
81.2 ꢃ 0.8
83.6 ꢃ 0.4
84.1 ꢃ 0.2
86.8 ꢃ 0.2
81.6 ꢃ 0.7
80.2 ꢃ 0.4
83.2 ꢃ 0.5
91.7 ꢃ 0.3
93.3 ꢃ 0.5
95.1 ꢃ 0.2
75.5 ꢃ 0.3
73.6 ꢃ 0.4
77.6 ꢃ 0.4
71.2 ꢃ 0.1
76.6 ꢃ 0.6
74.4 ꢃ 0.3
93.9 ꢃ 0.5
84.4 ꢃ 0.3
84.8 ꢃ 0.6
76.2 ꢃ 0.7
88.4 ꢃ 0.3
73.6 ꢃ 0.6
71.2 ꢃ 0.3
71.2 ꢃ 0.3
92.3 ꢃ 0.7
92.8 ꢃ 0.6
77.7 ꢃ 0.3
75.5 ꢃ 0.4
74.2 ꢃ 0.3
82.1 ꢃ 0.5
76.8 ꢃ 0.9
77.4 ꢃ 0.5
75.3 ꢃ 0.2
85.2 ꢃ 0.6
87.1 ꢃ 0.5
89.5 ꢃ 0.4
68.7 ꢃ 0.3
83.6 ꢃ 0.2
72.3 ꢃ 0.4
55.2 ꢃ 0.5
66.5 ꢃ 0.9
62.0 ꢃ 0.6
87.3 ꢃ 0.1
78.4 ꢃ 0.2
77.5 ꢃ 0.1
78.7 ꢃ 0.2
83.6 ꢃ 0.9
65.6 ꢃ 0.2
63.2 ꢃ 0.4
55.3 ꢃ 0.5
87.6 ꢃ 0.5
86.2 ꢃ 0.4
86.9 ꢃ 0.7
68.3 ꢃ 0.7
63.2 ꢃ 0.4
68.7 ꢃ 0.3
69.1 ꢃ 0.2
72.3 ꢃ 0.4
63.6 ꢃ 0.5
65.4 ꢃ 0.7
66.3 ꢃ 0.2
72.4 ꢃ 0.5
75.5 ꢃ 0.7
76.2 ꢃ 0.1
58.6 ꢃ 0.3
56.2 ꢃ 0.2
58.6 ꢃ 0.3
53.4 ꢃ 0.3
52.4 ꢃ 0.7
55.3 ꢃ 0.9
79.8 ꢃ 0.7
71.3 ꢃ 0.6
72.2 ꢃ 0.3
65.1 ꢃ 0.3
74.5 ꢃ 0.7
69.3 ꢃ 0.8
57.2 ꢃ 0.4
55.6 ꢃ 0.2
75.6 ꢃ 0.3
78.2 ꢃ 0.7
56.2 ꢃ 0.1
61.8 ꢃ 0.4
61.3 ꢃ 0.1
68.2 ꢃ 0.5
58.3 ꢃ 0.2
61.8 ꢃ 0.7
61.4 ꢃ 0.5
68.3 ꢃ 0.2
70.1 ꢃ 0.4
72.6 ꢃ 0.8
53.5 ꢃ 0.3
51.1 ꢃ 0.3
52.7 ꢃ 0.6
48.8 ꢃ 0.3
46.0 ꢃ 0.9
50.4 ꢃ 0.3
74.2 ꢃ 0.1
66.6 ꢃ 0.7
65.4 ꢃ 0.3
61.2 ꢃ 0.1
69.6 ꢃ 0.5
63.4 ꢃ 0.8
52.5 ꢃ 0.4
50.6 ꢃ 0.9
71.1 ꢃ 0.4
72.6 ꢃ 0.3
71.3 ꢃ 0.7
59.2 ꢃ 0.7
72.5 ꢃ 0.2
75.6 ꢃ 0.4
76.3 ꢃ 0.5
76.0 ꢃ 0.1
68.5 ꢃ 0.3
70.9 ꢃ 0.5
71.2 ꢃ 0.5
77.3 ꢃ 0.4
80.8 ꢃ 0.6
82.2 ꢃ 0.1
63.6 ꢃ 0.8
61.4 ꢃ 0.3
64.5 ꢃ 0.2
60.6 ꢃ 0.5
56.6 ꢃ 0.7
61.5 ꢃ 0.4
83.1 ꢃ 0.5
75.5 ꢃ 0.2
68.9 ꢃ 0.4
62.3 ꢃ 0.8
78.2 ꢃ 0.7
73.5 ꢃ 0.3
63.2 ꢃ 0.4
60.0 ꢃ 0.1
82.9 ꢃ 0.4
83.1 ꢃ 0.7
62.2 ꢃ 0.8
66.6 ꢃ 0.3
67.3 ꢃ 0.2
74.8 ꢃ 0.9
64.2 ꢃ 0.5
66.3 ꢃ 0.8
68.2 ꢃ 0.7
80.8 ꢃ 0.3
76.3 ꢃ 0.1
77.6 ꢃ 0.2
59.2 ꢃ 0.4
57.3 ꢃ 0.7
58.8 ꢃ 0.4
54.1 ꢃ 0.6
48.4 ꢃ 0.3
51.6 ꢃ 0.7
75.2 ꢃ 0.6
71.1 ꢃ 0.2
70.3 ꢃ 0.8
63.6 ꢃ 0.6
75.4 ꢃ 0.9
69.2 ꢃ 0.6
56.5 ꢃ 0.8
53.6 ꢃ 0.3
77.3 ꢃ 0.5
80.1 ꢃ 0.7
68.2 ꢃ 0.7
56.5 ꢃ 0.7
9
2-9
3-PhF
4-PhF
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
2-10
2-11
2-12
2-13
2-14
2-15
2-16
2-17
2-18
2-19
2-20
2-21
2-22
2-23
2-24
2-25
2-26
4-1
4-2
4-3
4-4
4-5
4-6
4-7
4-8
4-9
4-10
4-11
4-12
4-13
4-14
4-15
4-16
4-17
4-18
4-19
4-20
4-21
4-22
4-23
4-24
4-25
4-26
Carabrone
Tulipalin A
2-PhCH3
3-PhCH3
4-PhCH3
2-PhCH3O
3-PhCH3O
4-PhCH3O
4-PhCF3
2-C10H7
9-C14H9
4-PhCN
2,4-PhCl2
2-Furfural
4-PhCH2CH3
4-PhCH(CH3)2
Cinnamyl
a-Br-cinnamyl
Ph
2-PhCl
3-PhCl
4-PhCl
2-PhBr
3-PhBr
4-PhBr
2-PhF
3-PhF
4-PhF
2-PhCH3
3-PhCH3
4-PhCH3
2-PhCH3O
3-PhCH3O
4-PhCH3O
4-PhCF3
2-C10H7
9-C14H9
4-PhCN
2,4-PhCl2
2-Furfural
4-PhCH2CH3
4-PhCH(CH3)2
Cinnamyl
a-Br-cinnamyl
a
B. c., Botrytis cinerea; C. l., Colletotrichum lagenarium; G. g., Gaeumannomyces graminis var. tritici; S. s., Sclerotinia sclerotiorum; F. g., Fusarium
graminearum.
butyrolactone derivates (IC50 ¼ 14.54–134.56 mM) showed plausibly ascribed to the intrinsic biological feature of the
a superior activity against G. graminis compared to the d-valer- lactone ring and the fact that the g-butyrolactone derivates bear
olactone derivates (IC50 ¼ 24.45–154.88 mM), which might be conformation core size for receptor binding sites; on the
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Fig. 2 In vitro antifungal activity of compounds against Gaeumannomyces graminis var. tritici, Colletotrichum lagenarium and Fusarium gra-
minearum at 100 mg mLꢂ1
.
contrary, the d-valerolactone derivates disrupt the bioactive 21.54 mM) and 2-7 (4-PhBr, IC50 ¼ 33.64 mM). Meanwhile, with
conformation. In order to investigate the SARs of the substitu- the bulky aryl groups, the derivatives (2-18–19 and 4-18–19)
ents on the lactone ring, different kinds and positions of bearing a 20-naphthyl or a 90-anthryl group have no obvious
substituent were introduced, and G. graminis was selected for increase in activity (IC50 ¼ 47.85, 50.54, 75.25, 70.74 mM).
SAR research for all compounds. According to the data in Moreover, isopropyl with a bulky substituent was unfavorable to
Table 2, we can construct SARs from the preliminary conclu- the activity. Taken together, these results could demonstrate the
sions. The compounds 2-2–10 and 4-2–10 containing a halogen importance of substituents with a proper size, the para-position
atom (-PhF, -PhCl, and -PhBr) exhibited obviously potent anti- and the electron-withdrawing characteristic. Compared with
fungal activity compared to the -PhCH3O and -PhCH3 deriva- aromatic substituent compounds 2-1 and 4-1 (IC50 ¼ 88.21,
tives, which maybe due to the former derivates containing the 102.41 mM), a small decrease in activity was observed for
electron-withdrawing group being benecial to antifungal compounds 2-22 and 4-22 (IC50 ¼ 98.74, 114.36 mM) with the 2-
activity. In general, the activity of the compounds follows: F > Cl furfural substituent. Unexpectedly, the cinnamic aldehyde
> Br > CH3 > CH3O. Remarkably, 2-17 displayed an IC50 value of compounds 2-25–26 and 4-25–26 (IC50 ¼ 14.54, 16.44, 24.45,
14.78 mM, an involved the -PhCF3 electron-withdrawing and 27.02 mM) showed higher antifungal activity toward G.
substituent, the value was higher than that of the isopropyl (2- graminis than other compounds and the natural product car-
24, IC50 ¼ 123.75 mM) and ethyl substituent (2-23, IC50 ¼ 104.21 abrone (IC50 ¼ 28.54 mM), and derivatives 2-25 also displayed
mM). In contrast with 2-17, disubstitution of the benzylidene approximately 2-fold more activity than carbendazim (IC50
¼
ring with a 2,4-PhCl2 substituent did not improve activity, as 7.84 mM). This phenomenon may be due to the cinnamic alde-
seen from compound 2-21 (IC50 ¼ 33.85 mM). A plausible hyde structure have some antifungal capability.
explanation on such a decrease assumes that the steric effect
interferes with receptor binding when a Cl atom is on the ortho-
position.
As we can see, the steric effect also played an essential role
for the antifungal activity. For the title compounds, substitution
at the para-position was strongly favored over the correspond-
tomato fruits and against G. graminis and B. graminis on wheat
ing meta- and/or ortho-positions, which was veried between the
derivatives 2-2–16 and 4-2–16, and the derivatives 2-2 (2-PhCl,
In vivo fungicidal bioactivity
In order to further conrm the antifungal activity of these title
compounds, the in vivo fungicidal activities (protective effect) of
the 14 promising derivatives were tested against B. cinerea on
seedlings.
As can be seen in Table 3 and Fig. 3, the inhibitory rates of
IC50 ¼ 45.45 mM) and 2-5 (2-PhBr, IC50 ¼ 46.65 mM) were less
compounds 2-10 and 2-25 exceed 80% against all three fungi at
potent than the corresponding derivatives 2-4 (4-PhCl, IC50
¼
200 mg mLꢂ1, meanwhile, compound 2-25 is equivalent with
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Table 2 In vitro fungicidal activity of compounds against B. cinerea and G. graminisa
G. graminis
G. graminis
B. cinerea
B. cinerea
c
c
R
Compd
IC50, mM
IC50, mM
pIC50
Compd
IC50, mM
IC50, mM
pIC50
Ph
2-PhCl
3-PhCl
4-PhCl
2-PhBr
3-PhBr
4-PhBr
2-PhF
2-1
2-2
2-3
2-4
2-5
2-6
2-7
2-8
88.21
45.45
34.87
21.54
46.65
41.76
33.64
45.47
28.52
20.15
122.13
98.47
78.77
134.54
115.64
104.12
14.78
47.85
50.54
87.76
33.85
98.74
104.21
123.75
14.54
16.44
58.27
7.84
94.96
63.50
50.14
46.39
66.80
58.54
50.92
49.31
39.26
28.33
136.06
127.45
107.72
145.29
138.43
131.62
21.65
57.88
39.84
106.80
42.77
113.69
145.63
152.10
23.73
21.12
67.86
8.34
ꢂ1.98
ꢂ1.80
ꢂ1.70
ꢂ1.67
ꢂ1.83
ꢂ1.77
ꢂ1.71
ꢂ1.69
ꢂ1.59
ꢂ1.45
ꢂ2.13
ꢂ2.11
ꢂ2.03
ꢂ2.16
ꢂ2.14
ꢂ2.12
ꢂ1.34
ꢂ1.76
ꢂ1.60
ꢂ2.03
ꢂ1.63
ꢂ2.06
ꢂ2.16
ꢂ2.18
ꢂ1.38
ꢂ1.32
—
4-1
4-2
4-3
4-4
4-5
4-6
4-7
4-8
102.41
64.98
51.20
48.58
66.73
64.54
51.41
62.35
48.55
40.05
124.38
103.47
96.24
154.88
126.37
123.24
31.38
75.25
70.74
98.54
55.98
114.36
148.47
142.55
24.45
27.02
28.54
—
119.26
82.11
79.00
66.44
83.58
78.47
70.12
79.83
74.35
66.78
165.43
147.27
130.25
175.22
173.16
153.30
56.00
89.55
60.88
135.50
65.04
133.45
163.54
176.65
38.95
46.17
25.74
—
ꢂ2.08
ꢂ1.91
ꢂ1.90
ꢂ1.82
ꢂ1.92
ꢂ1.89
ꢂ1.85
ꢂ1.90
ꢂ1.87
ꢂ1.82
ꢂ2.22
ꢂ2.17
ꢂ2.11
ꢂ2.24
ꢂ2.24
ꢂ2.19
ꢂ1.75
ꢂ1.95
ꢂ1.78
ꢂ2.13
ꢂ1.81
ꢂ2.13
ꢂ2.21
ꢂ2.25
ꢂ1.84
ꢂ1.66
—
3-PhF
4-PhF
2-9
4-9
2-10
2-11
2-12
2-13
2-14
2-15
2-16
2-17
2-18
2-19
2-20
2-21
2-22
2-23
2-24
2-25
2-26
4-10
4-11
4-12
4-13
4-14
4-15
4-16
4-17
4-18
4-19
4-20
4-21
4-22
4-23
4-24
4-25
4-26
Carabrone
—
2-PhCH3
3-PhCH3
4-PhCH3
2-PhCH3O
3-PhCH3O
4-PhCH3O
4-PhCF3
2-C10H7
9-C14H9
4-PhCN
2,4-PhCl2
2-Furfural
4-PhCH2CH3
4-PhCH(CH3)2
Cinnamyl
a-Br-cinnamyl
Tulipalin A
Carbendazimb
—
—
a
All half maximal inhibitory concentration (IC50) values are presented as the mean ꢃ SD (n ¼ 3), mM. b Commercial fungicide, carbendazim was
used as the positive control. c pIC50, the tested IC50 values were converted into the corresponding plog IC50 values.
Table 3 In vivo fungicidal activity (protective effect) of compounds
against B. cinerea, G. graminis and B. graminisa
commercial fungicide (carbendazim or triadimefon).
Compounds 2-17 and 4-7 also showed great activity, and their
protective inhibitory rates were higher than 70%. The above
results indicated that the in vivo fungicidal activities in
this research were aligned with in vitro activities; moreover,
the in vivo fungicidal activities were better than the in vitro
activity.
Compounds
Against
Against
Against
No. (200 mg mLꢂ1
)
B. cinerea (%) G. graminis (%) B. graminis (%)
1
2
3
4
5
6
7
8
2-2
2-10
2-13
2-17
2-19
2-21
2-23
2-25
4-1
4-7
4-15
4-18
4-20
4-25
Tulipalin A
Carabrone
Triadimefon
52.21d
82.43ab
61.23cd
78.65b
56.54d
55.65d
66.51c
86.61a
56.42d
77.85b
56.24d
68.57c
66.33c
80.47ab
54.21d
70.36bc
—
57.65d
83.59a
59.65d
81.54ab
41.68e
66.58c
48.68e
85.67a
52.24d
70.36b
62.87c
66.34c
62.24c
76.39b
58.36d
80.24ab
87.29a
—
56.36d
81.67a
52.34d
78.65ab
42.52e
64.38c
51.25d
80.24a
46.75e
73.51b
46.27
59.68d
57.46d
79.38ab
42.94e
73.58b
82.39a
—
QSAR study and antifungal activity against B. cinerea
Conformer optimization and minimum energy calculations are
the essential procedures in the construction of a QSAR model.
In this paper, 52 g-butyrolactone and d-valerolactone
compounds were used as samples and 5 groups of descriptors
were obtained. Heuristic regression was chosen to acquire
a QSAR model with satisfactory values of R2, F, and S2, which
were used to establish a relationship between antifungal activity
and molecular descriptors.
The number of descriptors was achieved by the “breaking
point” rule, and according to the t values in the test, the
descriptors were obtained. Meanwhile, the numbers of samples
and descriptors also meet the equation 3D # S ꢂ 3 (S means the
number of samples; D means the number of descriptors). At
last, the nal 5-descriptor model was generated. In the ESI,† the
9
10
11
12
13
14
15
16
17
18
Carbendazim 89.54a
a
Values are the means of three replicates; letters a–d represent
signicant difference at 0.05; commercial fungicide,
a
p
¼
triadimefon and carbendazim were used as the positive control.
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Fig. 3 In vivo antifungal activity of compounds against Botrytis cinerea, Gaeumannomyces graminis var. tritici, and Blumeria graminis at 200 mg
mLꢂ1 aCommercial fungicide; carbendazim was used as the B. cinerea positive control and triadimefon was used as the G. graminis and B.
.
graminis positive control.
ve signicant descriptors, the “breaking point” rule gure and model.32 Internal validation results were presented in the ESI.†
2
2
their values are listed.
The RTraining and RTest were within 5% for all three sets, and
The fabricated QSAR model equation has ve descriptors the average values of RTraining2 and RTest2 approached the overall
presented in a grading down way based on the importance of R2 value. So, the obtained QSAR model indicated the predictive
the statistical analysis, as shown in Table 4. According to this power of 3-fold cross-validation. In the “leave-one-out” method,
optimized model, the comparison chart of the predictive and every fourth compound 1, 5, 9, etc., was an external test set, and
practical activity of the 52 compounds was exhibited in Fig. 4. the others were the training set. The R2 values of the training set
Compared with experimental pIC50 (negative log IC50), we could and test set were close, and the QSAR model acquired in this
conclude that the generated model was reliable. The nal QSAR study was available.
model with 5 descriptors can be described as
By explaining the descriptors of this model, we could gain
some insight into the structural features and antifungal activity.
The 1st and 5th descriptors obtained in this study were the max.
net atomic charge for an O atom and the max. net atomic
pIC50 ¼ ꢂ4.6536 + 5.4325 ꢁ qOmax. ꢂ 3.1128 ꢁ no + 2.5461
ꢁ MAOEP + 0.0665 ꢁ mc ꢂ 3.5452 ꢁ qmax.
,
N ¼ 52, R2 ¼ 0.947, F ¼ 65.77, S2 ¼ 0.0028
Internal validation and “leave-one-out” cross-validation
methods were carried out to validate the established QSAR
Table 4 The best five-descriptor model
Descriptor no.
X
ꢃDX
t-Text
Descriptor
0
1
2
3
4
5
ꢂ4.6536
5.4325
3.1128
2.5461
3.2135 ꢁ 10ꢂ1 6.6525
Intercept
a
3.4576 ꢁ 10ꢂ1 ꢂ2.7665 qmO ax.
b
5.7687
1.0893
ꢂ1.6532 no
2.6675
MAOEPc
mc
6.6523 ꢁ 10ꢂ2 3.2254 ꢁ 10ꢂ2 1.9878
d
2.2153 ꢁ 10ꢂ1 2.6673
qmax.
e
3.5452
a
b
Max. net atomic charge for an O atom. Number of occupied
c
electronic levels of atoms. Max. atomic orbital electronic population.
d
e
Total point-charge composition of the molecular dipole. Max. net
atomic charge.
Fig. 4 Graphic of pEC50 values versus respective errors.
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charge. These two descriptors belonged to electrostatic
descriptors and they reect the charge distribution of the
molecules as shown in the contour maps indicating the occu-
pied electronic levels of compounds 2-4 and 4-17 (Fig. 5), which
represent the geometric mean of atomic electronegativities.33,34
According to the frontier molecular orbital theory (FMO) of
chemical reactivity, the formation of a transition state is due to
an interaction between the frontier orbitals (HOMO and LUMO)
of the reacting species. So the electrostatic properties of an O
atom and max. net atomic charge were important elements for
the antifungal activity of the title compounds.
The second and third most important descriptors obtained
in this model were the maximum atomic orbital electronic
population and the number of occupied electronic levels of
atoms. The former is an important descriptor to reect the
nucleophilicity of the molecule, which is directly in contact with
the molecular nucleophilic capacity and describes the suscep-
tibility of the molecule to electrophilic attack.35 The number of
occupied electronic levels of atoms belongs to quantum-
chemical descriptors, which therefore act directly on the
quantum chemically calculated charge distribution in the
molecules and describes the molecular polar interactions.
Meanwhile, as the result of the of electron activity difference
between the atoms, the optimized geometries and permanent
polarization are shown in the molecular electrostatic potential
map simultaneously (Fig. 5), and we can see that the exocyclic
carbon–carbon double bond exhibits greater negative electro-
static potential which easily occurred in the nucleophilic reac-
tion.36,37 Actually, a-methylene-g-butyrolactone derivatives with
Fig. 6 Optimized geometries and charge distribution of compounds
2-4, 2-9, 2-13, 4-17, 4-21, and 4-24.
an electrophilic a,b-unsaturated carbonyl system (Michael
acceptor), which had higher electron deciency, could react
with biological nucleophiles.38,39
The h important descriptor was the total point-charge
component of the molecular dipole (mc), which measured the
hydrophilic/lipophilic property of the compounds.40–42 The
appropriate mc value illustrated the penetration ability of the
molecules to the cell as well as interaction with the action target
to a large extent. As shown in Fig. 6, the optimized geometries
and charge distribution on the atoms of compounds 2-4, 2-9, 2-
13, 4-17, 4-21 and 4-24 were demonstrated simultaneously. It
was illustrated that, aer lactone ring modication with
aromatics, the hydrophilic and lipophilic property of the
compounds was regulated, therefore the mc value was an
essential factor in adjusting the antifungal activity.
Conclusion
Inspired by the bioactivity of sesquiterpene lactones and the
natural product Tulipalin A, a series of a-methylene-g-lactone
and a-methylene-d-lactone compounds were prepared through
structural modication. The antifungal activity of all
compounds against G. graminis and B. cinerea were evaluated,
and the g-lactone derivatives exhibited higher antifungal
activity than the d-lactone derivatives. Particularly, compound
2-25 connected with a cinnamic aldehyde structure showed
superior in vitro and in vivo fungicide activities. The SAR indi-
cated that electron-withdrawing groups and steric effects played
important roles simultaneously. Moreover, the QSAR model
(R2 ¼ 0.947, F ¼ 65.77, and S2 ¼ 0.0028) indicated that the net
Fig.
5 Molecular electrostatic potential and contour maps of
compounds 2-4 and 4-17. The green parts represent positive
molecular orbitals, and the red parts represent negative molecular
orbitals.
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atomic charges, nucleophilicity and hydrophilic/lipophilic 19 C. Hansch and R. P. Verma, Eur. J. Med. Chem., 2009, 44,
property of the compounds are the most important in this 260–273.
research. In view of these results, we know more about the 20 A. K. Mahalingam, A. Linda, K. E. Jenny, W. Johan, K. Jacob,
antifungal activity of a-methylene-g-lactone substructures,
especially that compound 2-25 contains a g-butyrolactone
T. Unge, W. Hans, M. Larhed and H. Anders, J. Med. Chem.,
2010, 53, 607–615.
scaffold and cinnamic aldehyde moiety and has the potential to 21 Y. Ru Meng and Y. D. Guan, Chin. Chem. Lett., 2002, 13,
be a fungicide candidate.
1039–1042.
22 S. Brase, A. Encinas, J. Keck and C. F. Nising, Chem. Rev.,
2009, 109, 3903–3990.
23 M. Wang, Q. Zhang, Q. Ren, X. Kong, L. Wang, H. Wang,
J. Xu and Y. Guo, J. Agric. Food Chem., 2014, 62, 10945–10953.
24 L. Fang, M. Wang, S. Gou, X. Liu, H. Zhang and F. Cao, J.
Med. Chem., 2014, 57, 1116–1120.
Conflicts of interest
The authors have no conicts of interest to declare.
Acknowledgements
´
25 R. P. John, R. D. Tyagi, D. Prevost, S. K. Brar, S. Pouleur and
We greatly appreciate the funding support for this research
provided by the National Natural Science Foundation of China
(No. 31471800). We also thank the Kunming Institute of Botany,
Chinese Academy of Science for the HR-MS spectral data.
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