Strigolactone analogues derived from dihydroflavonoids as potent seed germinators for the broomrapes

Article abstract of DOI:10.1021/acs.jafc.9b08044

The broomrapes (Orobanche and Phelipanche spp.) and witchweeds (Striga spp.) are a class of parasitic weeds, which are distributed widely in the tropical, subtropical, and temperate areas of the globe. Since they have completely consistent lifecycles with the host plants, it is difficult to control them selectively through using the conventional herbicides. Inducing suicidal germination of these weed seeds by small molecular signaling agents proved to be a promising strategy for the management of parasitic weeds. As a class of naturally occurring terpenoid metabolites, strigolactones (SLs) show significant biological activities including stimulation germination of weed seeds, inhibition of shoot-branching, and so on. However, the widespread application of these natural SLs is greatly limited by their extremely low natural abundance and complex molecular structures. Design and synthesis of the simplified analogues as natural SLs alternatives provide a viable avenue for the efficient control of these parasitic weeds. We herein disclose the development of a novel class of SLs analogues derived from dihydroflavonoids as potent seed germinators of parasitic weeds. It was shown that one of them displayed a higher potential toward the seed germination of the broomrapes than the positive control GR24. The structure-activity relationship of these SLs analogues was further validated on the basis of the binding affinity experiment to strigolactone receptor protein HTL7 by using a YLG fluorescent probe method.

Full text of DOI:10.1021/acs.jafc.9b08044

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Article
Strigolactone Analogues Derived from Dihydroflavonoids as Potent
Seed Germinators for the Broomrapes
Yunyao Kang, Zhili Pang, Niuniu Xu, Fangjie Chen, Zhong Jin,* and Xiaohua Xu*
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ABSTRACT: The broomrapes (Orobanche and Phelipanche spp.) and witchweeds (Striga spp.) are a class of parasitic weeds, which
are distributed widely in the tropical, subtropical, and temperate areas of the globe. Since they have completely consistent lifecycles
with the host plants, it is difficult to control them selectively through using the conventional herbicides. Inducing suicidal
germination of these weed seeds by small molecular signaling agents proved to be a promising strategy for the management of
parasitic weeds. As a class of naturally occurring terpenoid metabolites, strigolactones (SLs) show significant biological activities
including stimulation germination of weed seeds, inhibition of shoot-branching, and so on. However, the widespread application of
these natural SLs is greatly limited by their extremely low natural abundance and complex molecular structures. Design and synthesis
of the simplified analogues as natural SLs alternatives provide a viable avenue for the efficient control of these parasitic weeds. We
herein disclose the development of a novel class of SLs analogues derived from dihydroflavonoids as potent seed germinators of
parasitic weeds. It was shown that one of them displayed a higher potential toward the seed germination of the broomrapes than the
positive control GR24. The structure−activity relationship of these SLs analogues was further validated on the basis of the binding
affinity experiment to strigolactone receptor protein HTL7 by using a YLG fluorescent probe method.
KEYWORDS: strigolactones, parasitic weeds, seed germination, dihydroflavonoids, Orobanche
harm to the crops, it was demonstrated to be hard to control
them by common herbicides given that the lifecycles of these
parasitic weeds are almost in accord with those of the host
plants.9 It was reported by the USDA (United States
Department of Agriculture) staff that Striga spp. could be
successfully controlled through using ethylene gas to induce
the suicidal germination of parasite seeds.10 However, it is still
impractical in the developing countries considering the high
expense of this methodology. Moreover, this method was also
shown to be less effective to some types of the broomrapes.6
As mentioned above, one of the promising strategies to
control Orobanche and Striga is to induce the suicidal
germination of their seeds. The seed germination of the
parasitic weeds could be efficiently stimulated by a class of
specifically natural secondary metabolites such as strigol,
orobanchol, solanacol, etc., namely, strigolactones (SLs)
(Scheme 1a).11 Since strigol was first isolated from cotton
roots in 1966, up to 20 strigolactones have been isolated and
identified to date. As a class of naturally occurring plant
signaling molecules, these compounds generally show extensive
biological activities, including seed germination, shoot-
branching inhibition, and root-colonization by symbiotic
arbuscular mycorrhizal fungi.12,13 Structurally, these natural
INTRODUCTION
■
The broomrapes (Orobanche and Phelipanche spp.) and
witchweeds (Striga spp.) belong to a class of parasitic
angiosperms.1,2 They can directly invade to the roots of the
host plants, such as corn, sorghum, millet, rice, and sugar cane,
thereby competing for the nutritional uptake with the host
plants. Commonly, Striga species own the functional
chloroplasts and belong to a type of the hemiparasites. The
low photosynthesis efficiency of Striga spp. makes them
difficult to survive without host plants,2 whereas holoparasites,
i.e., Orobanche spp., depend completely on the supply of
nutrient from their host plants.3 Striga spp. are widely
distributed in the continent of Africa. According to the FAO
(Food and Agriculture Organization of the United Nations)
report from 2006, about six million hectacres (ha) of maize
growing in eastern, western, and southern Africa were infested
with Striga spp.4 In western Kenya, approximate 76% of lands
that grow maize and sorghum are infested with S. hermonthica,
leading to the serious loss of crop yields every year.5 Because
the requisite temperature for seed germination of the
broomrapes is normally not higher than that of Striga spp.
seeds, the broomrapes can survive in the most subtropical and
temperate zones including Asia, Australia, Mediterranean, and
eastern Europe, besides Africa.6,7 As far as we know, there are
11 species of Orobanche distributed in China, and O. cumana
and O. aegyptiaca were found to be among the most harmful
species toward the crops.7,8 In the Xinjiang Uygur Autono-
mous Region of China, the infestation of O. aegyptiaca has
caused 20−70% yield loss of melon and watermelon.8
Although Orobanche spp. and Striga spp. have caused great
Received: December 18, 2019
Revised: August 19, 2020
Accepted: September 14, 2020
Published: September 14, 2020
https://dx.doi.org/10.1021/acs.jafc.9b08044
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A

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Scheme 1. Natural Strigolactones and Related Synthetic Analogues
lactones contain a fused three-ring (ABC ring) skeleton,
connected with a butenolide ring (D ring) by an enol ether
unit.11 Because of the extremely low natural abundance and
complicated molecular structures, the large-scale syntheses and
application of these SLs as herbicides have yet to be
fulfilled.14,15 The structure−activity relationship (SAR) of
SLs on seed germination was first deduced by Zwanenburg et
al.,16 and they demonstrated that the syn-fused lactone C/D
ring moiety in their scaffolds is essential for their seed
germination activity. To date, a wide range of SL analogues
incorporating this active pharmacophore core, as GR24, GR7,
and Nijmegen-1 (Scheme 1b), has been exploited as potent
herbicides against the parasitic weeds.17,18 Regrettably, most of
synthetic analogues exert germination activity toward parasitic
weeds no better than natural SLs. In addition, it is also noted
that not all seeds of parasitic weeds respond to GR24 and its
analogues, e.g., O. crenata, O. fetida, O. hederae, and O.
densiflora.19 As a consequence, the development of novel SL
analogues against those irresponsive parasitic weeds still
remains highly desirable.
di-C-glycosylflavone isolated from the root extract and root
exudate of Desmodium uncinatum Jacq.,24 could effectively
prevent parasitism of Striga hermonthica (Del.) Benth.
(witchweed) through an allelopathic mechanism (Scheme
1c). As our ongoing research toward the development of novel
herbicides against the parasitic weeds, we envisioned that the
hybrid compounds derived from flavonoids and active
pharmacophore core of SLs might share both biological
modes of action, serving as a new class of herbicides against the
parasitic broomrapes and witchweeds. The incorporation of
α,β-unsaturated enol ether in the 3-position of dihydroflavone
scaffold would improve the stability of active pharmacophore
core due to the presence of conjugated arene. Meanwhile, 2-
substituted group in dihydroflavone might also adjust the
hydrolytic rate of enol ether and hemiacetal of D ring. Herein,
we report synthesis and seed germination assay toward the
parasitic weeds of this type of new SL analogues derived from
dihydroflavones. Of particular note, seed germination assay
showed that some of synthetic SL analogues revealed seed
germination activity toward the broomrapes 10 times higher
than that of the positive control GR24. Moreover, the binding
affinity of these dihydroflavonoid-derived SL analogues to the
strigolactone receptor HTL7 was also studied by using a YLG
fluorescent probe method.
To the best of our knowledge, in this class of compounds,
α,β-unsaturated enol ether connected with a lactone D ring is
the indispensable active pharmacophore core for seed
germination activity.19 The strigolactone signaling mechanism
study on the α,β hydrolase (At)D14 revealed that the
generation of the active form of strigolactone involved a
MATERIALS AND METHODS
■
D14-triggering open-to-closed transition state pathway to
release the active lactone D ring.20 Consequently, the stability
of α,β-unsaturated enol ether and hydrolytic rate of the lactone
D ring highly correlated with the biological activity of SL
analogues. Besides alkaloids and terpenes, flavones and the
related derivatives belong to an important class of natural
products that generally exhibits diverse biological activities
including anti-oxidant, anti-inflammatory, anti-microbial, and
anti-tumor activities.21−23 It was shown that isoschaftoside, a
Materials and Instruments. The seeds of O. cumana and O.
aegyptiaca were kindly provided by Professor Yongqing Ma (North-
west Agriculture & Forest University, Yangling, China). Prior to the
use, the seeds were sterilized by 1% (w/w) sodium hypochlorite
(NaClO) for 3 min and then soaked in 75% (v/v) ethanol for another
3 min. After the surface sterilization, all seeds were thoroughly rinsed
with sterile water and finally dried in air above a clean bench.
Both the expression and purification of HTL7 protein and synthesis
of Yoshimulactone Green (YLG) were performed according to the
B
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previously reported methods.25,26 Water for HTL7 inhibition assay
was distilled twice prior to use. Fluorescence measurements were
carried out on a Varian Cary Eclipse spectrofluorometer (Varian Co.).
1H and 13C NMR and HRMS spectra of all synthesized compounds
were recorded on a Bruker AvanceII400 MHz spectrometer and
Agilent 6520 Q-TOF LC/MS, respectively. The starting dihydro-
flavone derivatives 1a−17a were prepared from the corresponding
aromatic aldehydes according to the previously reported methods.27,28
The dihydroflavone derivatives 18a−24a and the corresponding enol
intermediates were synthesized according to the previously described
methods.29−31 The enol intermediate 25b,31 5-bromo-3-methyl-
2(5H)-furanone,32 and rac-GR2433,34 were prepared according to
the previously reported procedures.
Synthesis of Strigolactone Analogues. To a solution of N,N-
dimethylformamide dimethyl acetal (DMFDMA) (21 mmol) in
toluene (15 mL) at room temperature was added the corresponding
dihydroflavone (7 mmol). The resulting mixture was then warmed to
reflux overnight and monitored by TLC. After complete consumption
of the starting materials, toluene was removed under reduced pressure
and the residue was purified by silica gel column chromatography
(eluent: petroleum ether/ethyl acetate = 2:1, v/v) to give the desired
enamine intermediates 1b−17b.35
The enamine (2 mmol) thus obtained was dissolved in a mixture of
THF (5 mL), acetic acid (5 mL), and H2O (5 mL) at room
temperature. The mixture was then stirred vigorously and monitored
by TLC. After the completion of hydrolysis, the solution was
extracted with ethyl acetate exhaustively. The combined organic
phases were washed with saturated NaHCO3 aqueous solution, and
then, the solvent was evaporated under reduced pressure. The crude
enol obtained was subjected to the next reaction without further
purification.
To a solution of the crude enol intermediate in THF (15 mL) was
added t-BuOK (2.2 mmol) at 0 °C. The mixture was stirred for 15
min at 0 °C, and a solution of 5-bromo-3-methyl-2(5H)-furanone (3
mmol) in THF (5 mL) was added dropwise. The reaction mixture
was stirred overnight, and the solvent was then evaporated under
reduced pressure. The resulting residue was finally purified by silica
gel column chromatography (eluent: petroleum ether/ethyl acetate/
dichloromethane = 7:1:2, v/v/v) to give the desired SL analogues 1−
25.
Compound 3. Rf = 0.22 (petroleum ether/ethyl acetate/dichloro-
methane = 7:1:2, v/v/v). White solid, 260 mg, 45% yield from the
intermediate enamine 3b (470 mg). 1H NMR (400 MHz, CDCl3): δ
7.94−7.97 (1H, m, H-5), 7.73 (1H, s, H-1″), 7.36−7.41 (2H, m, H-7,
3′), 7.19−7.27 (2H, m, H-4′, 6′), 7.11 (1H, t, J = 8.0 Hz, H-5′), 7.00
(1H, t, J = 8.0 Hz, H-6), 6.73−6.87 (3H, m, H-8, 2″, 3″), 6.12 (1H, s,
H-2), 1.98 (3H, m, 4″-CH3). 13C NMR (100 MHz, CDCl3): δ
181.57, 181.51, 170.28, 170.19, 159.46, 159.42, 151.22, 140.97,
140.84, 136.49, 136.24, 136.19, 135.88, 135.82, 134.63, 134.51,
130.20, 130.15, 130.01, 128.98, 128.87, 127.10, 127.04, 126.96,
126.87, 121.89, 121.80, 121.64, 121.46, 118.64, 115.56, 115.45,
100.50, 73.22, 73.04, 10.82, 10.78. HR-ESI-MS: m/z ([M + H]+)
calcd for C21H15ClO5383.0686, found 383.0684.
Compound 4. Rf = 0.31 (petroleum ether/ethyl acetate/dichloro-
methane = 5:1:2, v/v/v). Yellow solid, 270 mg, 46% yield from the
intermediate enamine 4b (490 mg). 1H NMR (400 MHz, CDCl3): δ
7.98 (1H, dd, J = 4.0, 8.0 Hz, H-5), 7.77 (1H, s, H-1″), 7.58−7.63
(1H, m, H-7), 7.43 (1H, t, J = 8.0 Hz, H-3′), 7.15−7.29 (3H, m, H-4′,
5′, 6′), 7.01−7.05 (1H, m, H-6), 6.79−6.91 (2H, m, H-8, 3″), 6.70−
6.73 (1H, m, H-2″), 6.14 (1H, s, H-2), 2.01 (3H, m, 4″-CH3). 13C
NMR (100 MHz, CDCl3): δ 181.57, 181.49, 170.29, 170.20, 159.42,
159.35, 151.29, 140.98, 140.84, 138.13, 137.88, 136.24, 136.19,
135.86, 135.78, 133.55, 133.32, 130.43, 130.38, 129.16, 129.03,
127.62, 127.52, 127.11, 127.05, 124.71, 124.59, 121.92, 121.83,
121.65, 121.49, 118.68, 118.66, 115.70, 115.58, 100.47, 75.41, 75.19,
10.83, 10.78. HR-ESI-MS: m/z ([M + H]+) calcd for C21H15BrO5
427.0181, 429.0161, found 427.0171, 429.0155.
Compound 5. Rf = 0.35 (petroleum ether/ethyl acetate/dichloro-
methane = 5:1:2, v/v/v). Yellow solid, 210 mg, 30% yield from the
intermediate enamine 5b (590 mg). 1H NMR (400 MHz, CDCl3): δ
7.95 (1H, dd, J = 1.6, 8.0 Hz, H-5), 7.84−7.89 (1H, m, H-1″), 7.73−
7.74 (1H, m, H-7), 7.38−7.42 (1H, m, H-3′), 7.19 (2H, m, H-5′, 6′),
6.93−7.02 (2H, m, H-6, 4′), 6.86−6.89 (1H, m, H-3″), 6.76−6.83
(1H, m, H-8), 6.52 (1H, m, H-2″), 6.11 (1H, m, H-2), 1.98 (3H, m,
4″-CH3). 13C NMR (100 MHz, CDCl3): δ 181.55, 181.48, 170.31,
170.20, 159.31, 159.24, 151.35, 141.16, 141.02, 140.92, 140.85,
140.38, 140.13, 136.24, 136.19, 135.85, 135.78, 130.57, 130.53,
128.72, 128.57, 128.47, 128.38, 127.12, 127.06, 121.96, 121.87,
121.70, 121.54, 118.72, 118.69, 115.96, 115.85, 100.46, 100.43,
100.12, 100.02, 99.92, 79.40, 79.14, 10.84, 10.80. HR-ESI-MS: m/z
([M + H]+) calcd for C21H15IO5 475.0043, found 475.0031.
Compound 1. Rf = 0.35 (petroleum ether/ethyl acetate/dichloro-
methane = 5:1:2, v/v/v). Yellow oil, 290 mg, 34% yield from the
intermediate enamine 1b (680 mg). 1H NMR (400 MHz, CDCl3): δ
7.86−7.89 (1H, m, H-5), 7.77−7.81 (1H, m, H-1″), 7.43 (1H, t, J =
8.0 Hz, H-7), 7.22−7.33 (5H, m, H-2′, 3′, 4′, 5′, 6′), 6.89−7.00 (3H,
m, H-6, 8, 3″), 6.47−6.51 (1H, m, H-2″), 6.24 (1H, s, H-2), 2.01
(3H, s, 4″-CH3). 13C NMR (100 MHz, CDCl3): δ 181.35, 181.30,
170.31, 170.27, 159.59, 159.55, 151.00, 150.95, 141.10, 141.02,
138.69, 138.32, 136.14, 136.08, 135.86, 135.82, 128.59, 128.27,
128.22, 127.12, 127.06, 126.64, 126.60, 122.00, 121.78, 121.70,
121.63, 118.61, 116.57, 100.76, 74.91, 74.80, 10.71. HR-ESI-MS: m/z
([M + H]+) calcd for C21H16O5 349.1076, found 349.1070.
Compound 2. Rf = 0.30 (petroleum ether/ethyl acetate/dichloro-
methane = 5:1:2, v/v/v). White solid, 230 mg, 28% yield from the
intermediate enamine 2b (670 mg). 1H NMR (400 MHz, CDCl3): δ
7.93−7.96 (1H, m, H-5), 7.71−7.72 (1H, m, H-1″), 7.38−7.43 (1H,
m, H-7), 7.22−7.29 (1H, m, H-3′), 7.13−7.18 (1H, m, H-4′), 6.95−
7.07 (3H, m, H-6, 5′, 6′), 6.81−6.88 (2H, m, H-8, 3″), 6.69 (1H, s,
H-2″), 6.15−6.16 (1H, m, H-2), 1.99 (3H, m, 4″-CH3). 13C NMR
(100 MHz, CDCl3): δ 181.32, 181.29, 170.31, 170.22, 160.99 (d, JC−F
= 249 Hz), 160.92 (d, JC−F = 248 Hz), 159.50, 159.45, 151.09, 150.91,
141.01, 140.92, 136.24, 136.19, 135.92, 135.89, 130.71 (d, JC−F = 9
Hz), 130.68 (d, JC−F = 8 Hz), 128.76 (d, JC−F = 4 Hz), 128.71 (d, JC−F
Compound 6. Rf = 0.22 (petroleum ether/ethyl acetate/dichloro-
methane = 5:1:2, v/v/v). Yellow oil, 520 mg, 39% yield from the
1
intermediate enamine 6b (1.1 g). H NMR (400 MHz, CDCl3): δ
7.85−7.89 (1H, m, H-5), 7.53−7.55 (1H, m, H-1″), 7.28 (1H, t, J =
8.0 Hz, H-7), 7.12−7.19 (1H, m, H-6′), 7.02−7.08 (1H, m, H-6),
6.87 (1H, t, J = 8.0 Hz, H-5′), 6.65−6.79 (4H, m, H-8, 3′, 4′, 3″),
6.56−6.60(1H, m, H-2″), 6.00−6.04 (1H, m, H-2), 3.68 (3H, m, 2′-
OMe), 1.88 (3H, m, 4″-CH3). 13C NMR (100 MHz, CDCl3): δ
181.77, 181.70, 170.36, 170.24, 159.97, 159.94, 157.80, 157.74,
150.16, 149.78, 141.15, 141.08, 135.79, 135.70, 135.56, 130.19,
130.16, 128.85, 128.77, 127.63, 127.30, 126.76, 126.67, 121.45,
121.27, 121.15, 121.04, 120.27, 120.24, 118.27, 118.21, 116.35,
116.23, 111.29, 111.22, 100.60, 100.44, 72.32, 72.15, 55.57, 55.53,
10.70. HR-ESI-MS: m/z ([M + H]+) calcd for C22H18O6 379.1182,
found 379.1178.
Compound 7. Rf = 0.27 (petroleum ether/ethyl acetate/dichloro-
methane = 5:1:2, v/v/v). Yellow oil, 330 mg, 42% yield from the
intermediate enamine 7b (650 mg). 1H NMR (400 MHz, CDCl3): δ
7.85−7.88 (1H, m, H-5), 7.76−7.81 (1H, m, H-1″), 7.43 (1H, t, J =
8.0 Hz, H-7), 7.15−7.20 (1H, m, H-6′), 6.88−7.02 (5H, m, H-6, 8, 2′,
5′, 3″), 6.76 (1H, t, J = 8.0 Hz, H-4′), 6.44−6.48 (1H, m, H-2″), 6.24
(1H, s, H-2), 3.73 (3H, m, 3′-OMe), 2.00 (3H, s, 4″-CH3). 13C NMR
(100 MHz, CDCl3): δ 181.32, 181.26, 170.33, 170.25, 159.88, 159.86,
159.62, 150.94, 141.06, 140.95, 140.31, 139.94, 136.19, 136.14,
136.10, 136.04, 129.69, 127.29, 127.22, 122.11, 121.91, 121.84,
121.75, 119.00, 118.89, 118.64, 116.74, 116.69, 113.81, 113.42,
112.83, 112.29, 100.84, 100.76, 74.79, 74.71, 55.30, 10.86. HR-ESI-
MS: m/z ([M + H]+) calcd for C22H18O6 379.1182, found 379.1184.
= 4 Hz), 127.07, 127.03, 126.35 (d, JC−F = 14 Hz), 126.06 (d, JC−F
=
14 Hz), 124.13, 124.09, 124.05, 121.83, 121.75, 121.56, 121.37,
118.52, 118.49, 115.96 (d, JC−F = 22 Hz), 115.83 (d, JC−F = 22 Hz),
115.23, 115.19, 100.63, 100.55, 70.48, 70.45, 70.41, 70.38, 10.83,
10.80. HR-ESI-MS: m/z ([M + H]+) calcd for C21H15FO5 367.0982,
found 367.0976.
C
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Compound 8. Rf = 0.22 (petroleum ether/ethyl acetate/dichloro-
methane = 5:1:2, v/v/v). Yellow oil, 190 mg, 24% yield from the
intermediate enamine 8b (660 mg). 1H NMR (400 MHz, CDCl3): δ
8.22 (1H, m, H-2′), 8.10 (1H, t, J = 7.2 Hz, H-4′), 7.88 (2H, m, H-5,
6′), 7.58−7.66 (1H, m, H-1″), 7.44−7.51 (2H, m, H-7, 5′), 6.97−
7.07 (3H, m, H-6, 8, 3″), 6.54 (1H, s, H-2″), 6.28−6.32 (1H, m, H-
2), 2.03 (3H, s, 4″-CH3). 13C NMR (100 MHz, CDCl3): δ 180.54,
180.51, 170.21, 170.08, 159.08, 159.03, 152.02, 151.62, 148.56,
144.17, 141.23, 140.85, 140.77, 136.62, 136.58, 136.43, 136.29,
132.46, 132.39, 129.88, 129.83, 127.43, 127.40, 123.36, 123.35,
122.44, 122.37, 121.92, 121.89, 121.85, 121.70, 118.66, 118.61,
115.79, 115.70, 100.98, 100.80, 73.98, 10.90. HR-ESI-MS: m/z ([M +
H]+) calcd for C21H15NO7 394.0927, found 394.0916.
(1H, d, J = 6.4 Hz, H-2″), 6.25−6.28 (1H, m, H-2), 2.01 (3H, m, 4″-
CH3). 13C NMR (100 MHz, CDCl3): δ 180.93, 180.88, 170.19,
170.13, 159.34, 159.30, 151.43, 142.74, 142.40, 140.86, 140.77,
136.44, 136.37, 136.26, 127.40, 127.33, 127.09, 127.05, 125.73,
125.70, 125.67, 122.20, 122.12, 122.01, 121.80, 118.61, 116.12,
116.10, 100.87, 100.79, 74.42, 74.35, 10.86. HR-ESI-MS: m/z ([M +
H]+) calcd for C22H15F3O5 417.0950, found 417.0943.
Compound 14. Rf = 0.30 (petroleum ether/ethyl acetate/
dichloromethane = 5:1:2, v/v/v). Yellow oil, 210 mg, 40% yield
1
from the intermediate enamine 14b (430 mg). H NMR (400 MHz,
CDCl3): δ 7.91−7.94 (1H, m, H-5), 7.72−7.74 (1H, m, H-1″), 7.33−
7.37 (1H, m, H-7), 6.93−7.01 (3H, m, H-6, 6′, 3″), 6.77−6.85 (3H,
m, H-3′, 5′, 8), 6.55 (1H, s, H-2″), 6.15 (1H, m, H-2), 2.54 (3H, s,
2′-CH3), 2.24 (3H, m, 4′-CH3), 1.98 (3H, m, 4″-CH3). 13C NMR
(100 MHz, CDCl3): δ 182.19, 170.38, 170.05, 159.38, 159.36, 150.80,
150.14, 141.10, 140.90, 138.81, 137.85, 137.72, 136.16, 136.08,
136.03, 135.82, 133.71, 133.41, 131.98, 131.94, 127.48, 127.33,
127.10, 127.06, 126.40, 126.32, 121.95, 121.77, 121.62, 121.54,
118.53, 118.49, 116.38, 116.30, 100.76, 100.42, 73.51, 73.25, 21.13,
19.79, 19.76, 10.84. HR-ESI-MS: m/z ([M + H]+) calcd for C23H20O5
377.1389, found 377.1390.
Compound 9. Rf = 0.20 (petroleum ether/ethyl acetate/dichloro-
methane = 7:1:2, v/v/v). Yellow oil, 140 mg, 23% yield from the
intermediate enamine 9b (510 mg). 1H NMR (400 MHz, CDCl3): δ
7.86−7.89 (1H, m, H-5), 7.75−7.80 (1H, m, H-1″), 7.41 (1H, t, J =
8.0 Hz, H-7), 7.23−7.26 (2H, m, H-2′, 6′), 6.89−6.98 (3H, m, H-6,
3′, 5′), 6.77−6.80 (2H, m, H-8, 3″), 6.42−6.45 (1H, m, H-2″), 6.23
(1H, s, H-2), 3.73 (3H, m, 4′-OMe), 2.00 (3H, m, 4″-CH3). 13C
NMR (100 MHz, CDCl3): δ 181.58, 181.53, 170.36, 170.33, 159.61,
159.56, 159.54, 159.50, 150.76, 150.74, 141.10, 140.98, 136.16,
136.10, 136.03, 135.97, 130.74, 130.31, 128.21, 128.15, 127.19,
127.12, 122.10, 121.87, 121.71, 121.62, 118.72, 116.74, 113.99,
113.96, 100.77, 100.73, 74.70, 74.62, 55.32, 55.31, 10.85. HR-ESI-
MS: m/z ([M + H]+) calcd for C22H18O6 379.1182, found 379.1181.
Compound 10. Rf = 0.20 (petroleum ether/ethyl acetate/
dichloromethane = 7:1:2, v/v/v). Brown oil, 250 mg, 35% yield
Compound 15. Rf = 0.25 (petroleum ether/ethyl acetate/
dichloromethane = 4:1:2, v/v/v). Yellow oil, 180 mg, 46% yield
1
from the intermediate enamine 15b (320 mg). H NMR (400 MHz,
CDCl3): δ 7.86−7.89 (1H, m, H-5), 7.74−7.77(1H, m, H-1″), 7.41−
7.45 (1H, m, H-7), 6.90−6.99 (3H, m, H-6, 8, 6′), 6.84 (1H, s, H-3″),
6.72−6.77 (1H, m, H-2′), 6.66−6.68 (1H, m, H-5′), 6.36−6.39 (1H,
m, H-2″), 6.23 (1H, m, H-2), 5.90 (2H, m, −OCH2O−), 2.01 (3H,
m, 4″-CH3). 13C NMR (100 MHz, CDCl3): δ 181.41, 181.37, 170.31,
170.24, 159.44, 159.39, 150.92, 150.76, 148.14, 148.12, 147.75,
141.03, 140.92, 136.22, 136.15, 136.08, 132.59, 132.21, 127.26,
127.20, 122.10, 121.88, 121.84, 121.76, 120.54, 120.49, 118.73,
116.70, 108.21, 108.18, 107.51, 107.46, 101.33, 100.76, 100.74, 74.78,
74.74, 10.88. HR-ESI-MS: m/z ([M + H]+) calcd for C22H16O7
393.0974, found 393.0969.
1
from the intermediate enamine 10b (590 mg). H NMR (400 MHz,
CDCl3): δ 7.87−7.89 (1H, m, H-5), 7.76−7.81 (1H, m, H-1″), 7.44
(1H, t, J = 8.0 Hz, H-7), 7.26−7.31 (2H, m, H-3′, 5′), 6.90−7.00
(5H, m, H-6, 8, 2′, 6′, 3″), 6.43−6.46 (1H, m, H-2″), 6.24 (1H, s, H-
2), 2.02 (3H, m, 4″-CH3). 13C NMR (100 MHz, CDCl3): δ 181.23,
181.19, 170.24, 170.20, 162.68 (d, JC−F = 246 Hz), 159.39, 159.36,
150.96, 140.94, 140.85, 136.29, 136.21 (d, JC−F = 4 Hz), 128.64 (d,
JC−F = 8 Hz), 128.59 (d, JC−F = 8 Hz), 127.30, 127.23, 121.97, 121.89,
Compound 16. Rf = 0.27 (petroleum ether/ethyl acetate/
118.67, 116.55, 115.63 (d, JC−F = 22 Hz), 115.61 (d, JC−F = 22 Hz),
100.81, 100.74, 74.44, 74.38, 10.87. HR-ESI-MS: m/z ([M + H]+)
calcd for C21H15FO5 367.0982, found 367.0975.
dichloromethane = 5:1:2, v/v/v). Yellow oil, 230 mg, 40% yield
1
from the intermediate enamine 16b (460 mg). H NMR (400 MHz,
CDCl3): δ 7.89−7.92 (1H, m, H-5), 7.74−7.77 (1H, m, H-1″), 7.42−
7.46 (1H, m, H-7), 7.20−7.22 (1H, m, H-3′), 6.84−7.02 (5H, m, H-
6, 8, 1′, 2″, 3″), 6.63 (1H, d, J = 8.0 Hz, H-2′), 6.24 (1H, s, H-2), 2.02
(3H, m, 4″-CH3). 13C NMR (100 MHz, CDCl3): δ 180.97, 180.92,
170.27, 170.21, 159.06, 159.04, 150.95, 150.75, 142.42, 142.20,
141.00, 140.90, 136.25, 136.16, 136.11, 127.24, 127.19, 126.84,
126.73, 126.65, 126.53, 126.48, 122.16, 122.12, 122.08, 121.96,
118.92, 116.68, 116.63, 100.79, 100.76, 71.74, 71.60, 10.90, 10.88.
HR-ESI-MS: m/z ([M + H]+) calcd for C19H14O5S 355.0640, found
355.0630.
Compound 11. Rf = 0.25 (petroleum ether/ethyl acetate/
dichloromethane = 5:1:2, v/v/v). Brown oil, 210 mg, 33% yield
from the intermediate enamine 11b (530 mg). H NMR (400 MHz,
1
CDCl3): δ 7.88 (1H, d, J = 8.0 Hz, H-5), 7.76−7.82 (1H, m, H-1″),
7.43−7.46 (1H, m, H-7), 7.25 (4H, s, H-2′, 3′, 5′, 6′), 6.90−7.01
(3H, m, H-6, 8, 3″), 6.42−6.46 (1H, m, H-2″), 6.24 (1H, s, H-2),
2.02 (3H, s, 4″-CH3). 13C NMR (100 MHz, CDCl3): δ 181.13,
181.09, 170.23, 170.18, 159.35, 159.33, 151.10, 140.91, 140.80,
137.26, 136.88, 136.34, 136.27, 136.23, 134.34, 134.31, 128.91,
128.89, 128.24, 128.19, 127.35, 127.28, 122.07, 121.98, 121.85,
118.66, 116.36, 116.33, 100.80, 100.75, 74.39, 74.34, 10.90. HR-ESI-
MS: m/z ([M + H]+) calcd for C21H15ClO5 383.0686, found
383.0682.
Compound 17. Rf = 0.30 (petroleum ether/ethyl acetate/
dichloromethane = 5:1:2, v/v/v). Yellow oil, 80 mg, 13% yield
1
from the intermediate enamine 17b (500 mg). H NMR (400 MHz,
CDCl3): δ 7.93 (1H, d, J = 8.0 Hz, H-5), 7.69−7.71 (1H, m, H-1″),
7.40−7.45 (1H, m, H-7), 7.34 (1H, s, H-3′), 7.01 (1H, t, J = 8.0 Hz,
H-6), 6.90−6.92 (2H, m, H-8, 3″), 6.41 (1H, d, J = 4.0 Hz, H-2″),
6.19−6.23 (3H, m, H-2, 1′, 2′), 2.01 (3H, m, 4″-CH3). 13C NMR
(100 MHz, CDCl3): δ 181.11, 170.27, 159.15, 159.08, 151.44, 151.22,
151.10, 150.94, 143.55, 143.50, 141.01, 140.91, 136.23, 136.12,
136.07, 127.11, 127.08, 122.00, 121.93, 121.76, 118.54, 114.57,
114.53, 110.42, 110.35, 109.98, 109.65, 100.76, 100.67, 69.13, 69.06,
10.88. HR-ESI-MS: m/z ([M + H]+) calcd for C19H14O6 339.0869,
found 339.0862.
Compound 12. Rf = 0.20 (petroleum ether/ethyl acetate/
dichloromethane = 5:1:2, v/v/v). Yellow oil, 160 mg, 37% yield
from the intermediate enamine 12b (360 mg). H NMR (400 MHz,
1
CDCl3): δ 8.11−8.14 (2H, m, H-3′, 5′), 7.82−7.89 (2H, m, H-5, 1″),
7.46−7.50 (3H, m, H-7, 2′, 6′), 7.00−7.05 (2H, m, H-6, 3″), 6.93−
6.96 (1H, m, H-8), 6.54 (1H, d, J = 4.0 Hz, H-2″), 6.27−6.30 (1H, m,
H-2), 2.03 (3H, m, 4″-CH3). 13C NMR (100 MHz, CDCl3): δ
180.55, 180.52, 170.08, 170.02, 159.12, 159.10, 151.66, 151.55,
147.89, 146.00, 145.67, 140.73, 140.66, 136.57, 136.51, 136.48,
136.42, 127.63, 127.49, 127.43, 123.98, 123.96, 122.46, 122.39,
121.98, 121.77, 118.58, 118.56, 115.87, 115.85, 100.92, 100.79, 74.15,
75.11, 10.91. HR-ESI-MS: m/z ([M + H]+) calcd for C21H15NO7
394.0927, found 394.0922.
Compound 18. Rf = 0.20 (petroleum ether/ethyl acetate/
dichloromethane = 7:1:2, v/v/v). White solid, mp 123−124 °C,
1
100 mg, 31% yield from the intermediate enol (210 mg). H NMR
(400 MHz, CDCl3): δ 7.93 (1H, dd, J = 8.0, 1.2 Hz, H-5), 7.57 (1H,
s, H-1″), 7.45 (1H, t, J = 8.0 Hz, H-7), 7.02 (1H, t, J = 8.0 Hz, H-6),
6.95 (1H, s, H-3″), 6.94 (1H, d, J = 8.0 Hz, H-8), 6.21 (1H, s, H-2″),
5.03 (2H, d, J = 1.2 Hz, H-2a, 2b), 2.02 (3H, s, 4″-CH3). 13C NMR
(100 MHz, CDCl3): δ 181.81, 170.36, 161.44, 149.62, 141.08, 135.97,
Compound 13. Rf = 0.25 (petroleum ether/ethyl acetate/
dichloromethane = 5:1:2, v/v/v). Yellow oil, 220 mg, 56% yield
1
from the intermediate enamine 13b (330 mg). H NMR (400 MHz,
CDCl3): δ 7.82−7.88 (2H, m, H-5, 1″), 7.43−7.54 (5H, m, H-7, 2′,
3′, 5′, 6′), 6.98−7.03 (2H, m, H-6, 3″), 6.90−6.94 (1H, m, H-8), 6.50
D
https://dx.doi.org/10.1021/acs.jafc.9b08044
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
pubs.acs.org/JAFC
Article
a
Table 1. Seed Germination Activity towards O. aegyptiaca
test concentration (mg/L)
2.0
compound number
0.2
20
EC50 (M)
1
2
3
4
5
6
7
8
74.8 8.6%
36.9 8.0%
36.1 9.6%
26.4 9.5%
18.9 7.6%
52.0 12.4%
41.6 9.6%
24.7 9.4%
59.3 10.4%
36.2 12.6%
28.4 6.0%
26.9 10.6%
67.0 10.0%
41.7 12.8%
38.3 13.6%
57.8 8.3%
17.0 7.2%
75.2 4.8%
79.4 5.9%
70.9 10.3%
72.9 9.1%
75.0 8.6%
72.3 9.6%
83.8 6.5%
31.9 7.3%
82.9 4.6%
91.2 5.1%
85.1 6.0%
71.1 8.3%
41.9 9.0%
46.2 13.2%
83.0 5.2%
78.7 10.1%
49.4 11.4%
86.4 2.7%
67.1 8.6%
60.5 6.9%
66.4 11.9%
55.4 12.0%
75.8 6.7%
82.8 9.1%
83.8 8.6%
41.9 7.6%
79.4 6.6%
87.0 5.4%
75.2 5.9%
80.6 7.7%
79.0 5.8%
82.6 5.9%
92.5 4.9%
53.7 7.2%
95.5 3.6%
91.0 5.0%
92.1 4.6%
85.4 5.1%
76.2 7.9%
82.9 6.7%
91.0 4.0%
91.8 6.3%
73.2 6.9%
90.5 5.7%
86.8 4.1%
81.9 5.9%
81.0 8.4%
74.2 11.7%
88.2 4.8%
91.1 5.0%
86.6 5.1%
74.6 9.6%
85.0 5.5%
83.6 4.2%
82.8 4.7%
80.4 6.7%
79.3 9.7%
80.2 4.9%
94.8 5.6%
66.4 7.8%
93.1 7.5%
2.12 × 10−7
n.d.
n.d
n.d
n.d
6.71 × 10−7
n.d
n.d
9
4.84 × 10−7
n.d
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
GR24
n.d
n.d
n.d
n.d
n.d
6.69 × 10−7
n.d
3.31 × 10−8
3.49 × 10−9
2.33 × 10−8
3.50 × 10−8
5.18 × 10−8
2.92 × 10−7
8.99 × 10−8
n.d
5.70 × 10−8
a
b
The seed germination rate for negative control (distilled water) was lower than 5%. n.d: not determined.
135.80, 127.43, 122.12, 121.84, 118.06, 114.12, 100.76, 64.32, 10.84.
HR-ESI-MS: m/z ([M + H]+) calcd for 273.0763, found 273.0761.
Compound 19. Rf = 0.20 (petroleum ether/ethyl acetate/
dichloromethane = 7:1:2, v/v/v). Yellow oil, 40 mg, 9% yield from
the intermediate enol (290 mg). 1H NMR (400 MHz, CDCl3): δ 7.92
(1H, d, J = 8.0 Hz, H-5), 7.51−7.55 (1H, m, H-1″), 7.46 (1H, t, J =
8.0 Hz, H-7), 6.98−7.03 (1H, m, H-6), 6.95−6.96 (1H, m, H-3″),
6.90−6.93 (1H, m, H-8), 6.17−6.22 (1H, m, H-2″), 5.51−5.57 (1H,
m, H-2), 2.03 (3H, s, 4″-CH3), 1.41−1.44 (3H, m, 2-CH3). 13C NMR
(100 MHz, CDCl3): δ 181.42, 181.33, 170.42, 170.37, 159.45, 159.41,
149.36, 149.20, 143.10, 141.08, 136.12, 136.07, 136.00, 127.20,
127.16, 121.50, 121.43, 118.86, 118.72, 100.90, 100.74, 71.07, 70.99,
20.63, 20.60, 10.90. HR-ESI-MS: m/z ([M + H]+) calcd for 287.0919,
found 287.0917.
Compound 20. Rf = 0.25 (petroleum ether/ethyl acetate/
dichloromethane = 7:1:2, v/v/v). Yellow oil, 100 mg, 10% yield
from the intermediate enol (660 mg). 1H NMR (400 MHz, CDCl3):
δ 7.91 (1H, dt, J = 8.0, 1.2 Hz, H-5), 7.55−7.59 (1H, m, H-1″), 7.45
(1H, t, J = 8.0 Hz, H-7), 7.00 (1H, td, J = 8.0, 2.0 Hz, H-6), 6.94−
6.96 (1H, m, H-3″), 6.91−6.94 (1H, m, H-8), 6.18−6.22 (1H, m, H-
2″), 5.27−5.30 (1H, m, H-2), 2.03 (3H, s, 4″-CH3), 1.82−1.91 (1H,
m, 2-CH2−), 1.56−1.61 (1H, m, 2-CH2−), 0.94 (3H, m, 2-CH2CH3).
13C NMR (100 MHz, CDCl3): δ 181.65, 181.56, 170.42, 170.37,
159.56, 159.50, 149.85, 149.68, 143.10, 141.11, 141.08, 136.10,
136.08, 136.03, 135.97, 127.20, 127.15, 121.62, 121.44, 121.36,
118.60, 117.96, 117.94, 100.94, 100.72, 96.94, 75.94, 75.86, 27.62,
10.90, 10.80, 9.95, 9.92. HR-ESI-MS: m/z ([M + H]+) calcd for
301.1076, found 301.1072.
(1H, m, H-2), 2.03 (3H, s, 4″-CH3), 1.80−1.87 (1H, m, 2-CH2−),
1.36−1.55 (3H, m, 2-CH2CH2−), 0.89 (3H, q, J = 8.0 Hz, 2-
(CH2)2CH3). 13C NMR (100 MHz, CDCl3): δ 181.66, 181.57,
170.41, 170.35, 159.57, 159.52, 149.58, 149.41, 141.09, 141.07,
136.13, 136.08, 136.03, 135.98, 127.20, 127.15, 121.63, 121.46,
121.42, 121.35, 118.62, 118.19, 100.91, 100.68, 74.43, 74.35, 36.46,
36.41, 18.64, 13.79, 13.73, 10.89. HR-ESI-MS: m/z ([M + H]+) calcd
for 315.1232, found 315.1228.
Compound 22. Rf = 0.20 (petroleum ether/ethyl acetate/
dichloromethane = 7:1:2, v/v/v). Yellow oil, 350 mg, 28% yield
from the intermediate enol (890 mg). 1H NMR (400 MHz, CDCl3):
δ 7.90 (1H, dt, J = 8.0, 1.6 Hz, H-5), 7.52−7.56 (1H, m, H-1″), 7.42−
7.47 (1H, m, H-7), 6.97−7.01 (1H, m, H-6), 6.93−6.95 (1H, m, H-
3″), 6.91 (1H, d, J = 8.0 Hz, H-8), 6.17−6.22 (1H, m, H-2″), 5.34−
5.38 (1H, m, H-2), 2.01−2.03 (3H, m, 4″-CH3), 1.78−1.87 (1H, m,
2-CH2−), 1.49−1.56 (1H, m, 2-CH2−), 1.23−1.41 (4H, m, 2-
CH2CH2CH2−), 0.81−0.87 (3H, m, 2-(CH2)3CH3). 13C NMR (100
MHz, CDCl3): δ 181.65, 181.56, 170.41, 170.37, 159.51, 159.46,
149.64, 149.46, 141.12, 141.08, 136.06, 136.05, 136.01, 135.89,
127.16, 127.11, 121.56, 121.40, 121.32, 118.60, 118.59, 118.15,
118.12, 100.88, 100.68, 74.58, 74.51, 34.01, 27.43, 22.31, 22.27, 14.06,
14.02, 10.88, 10.86. HR-ESI-MS: m/z ([M + H]+) calcd for 329.1389,
found 329.1386.
Compound 23. Rf = 0.20 (petroleum ether/ethyl acetate/
dichloromethane = 7:1:2, v/v/v). Yellow oil, 290 mg, 20% yield
from the intermediate enol (1.07 g). 1H NMR (400 MHz, CDCl3): δ
7.89 (1H, d, J = 8.0 Hz, H-5), 7.52−7.56 (1H, m, H-1″), 7.44 (1H, t,
J = 8.0 Hz, H-7), 6.89−7.00 (3H, m, H-6, 8, 3″), 6.16−6.22 (1H, m,
H-2″), 5.33−5.37 (1H, m, H-2), 2.01 (3H, s, 4″-CH3), 1.83 (1H, m,
2-CH2−), 1.51 (1H, m, 2-CH2−), 1.36 (2H, brs, 2-CH2CH2−), 1.23
(4H, brs, 2-(CH2)2−CH2CH2−), 0.83 (3H, m, 2-(CH2)4CH3). 13C
NMR (100 MHz, CDCl3): δ 181.60, 181.51, 170.39, 170.36, 159.48,
159.44, 149.69, 149.48, 142.38, 141.14, 141.10, 136.02, 135.97,
135.80, 127.10, 127.06, 121.54, 121.39, 121.35, 121.29, 118.57,
Compound 21. Rf = 0.20 (petroleum ether/ethyl acetate/
dichloromethane = 7:1:2, v/v/v). Yellow oil, 320 mg, 29% yield
from the intermediate enol (780 mg). 1H NMR (400 MHz, CDCl3):
δ 7.91 (1H, d, J = 8.0 Hz, H-5), 7.53−7.57 (1H, m, H-1″), 7.45 (1H,
t, J = 8.0 Hz, H-7), 6.98−7.02 (1H, m, H-6), 6.94−6.95 (1H, m, H-
3″), 6.91 (1H, d, J = 8.0 Hz, H-8), 6.18−6.22 (1H, m, H-2″), 5.39
E
https://dx.doi.org/10.1021/acs.jafc.9b08044
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
pubs.acs.org/JAFC
Article
118.11, 118.05, 100.88, 100.69, 98.39, 74.56, 74.51, 34.22, 31.32,
31.27, 24.95, 22.57, 22.55, 14.05, 14.02, 10.82. HR-ESI-MS: m/z ([M
+ H]+) calcd for 343.1545, found 343.1540.
Compound 24. Rf = 0.25 (petroleum ether/ethyl acetate/
dichloromethane = 7:1:2, v/v/v). Brown oil, 290 mg, 17% yield
1
from the intermediate enol (1.16g). H NMR (400 MHz, CDCl3): δ
7.89 (1H, d, J = 8.0 Hz, H-5), 7.60 (1H, s, H-1″), 7.45 (1H, t, J = 8.0
Hz, H-7), 6.99 (1H, t, J = 8.0 Hz, H-6), 6.95 (1H, s, H-3″), 6.88 (1H,
d, J = 8.0 Hz, H-8), 6.17 (1H, s, H-2″), 2.03 (3H, s, 4″-CH3), 1.64
(3H, s, 2-CH3), 1.62 (3H, s, 2-CH3). 13C NMR (100 MHz, CDCl3):
δ 182.48, 170.40, 159.67, 150.70, 140.99, 136.05, 136.00, 127.19,
121.34, 121.29, 120.67, 118.57, 101.20, 79.82, 27.71, 10.91. HR-ESI-
MS: m/z ([M + H]+) calcd for C17H16O5 301.1076, found 301.1071.
Compound 25. Rf = 0.55 (dichloromethane/ethyl acetate = 50:1,
v/v). White solid, mp 104−105 °C, 30 mg, 2.5% yield from the
1
intermediate enol (700 mg). H NMR (400 MHz, CDCl3): δ 7.43
(1H, d, J = 4.0 Hz, H-1″), 6.91 (1H, s, H-3″), 6.13 (1H, s, H-2″),
4.52 (2H, s, H-2), 3.97 (2H, t, J = 4.0 Hz, H-6), 2.52 (2H, td, J = 8.0,
2.4 Hz, H-5), 2.01 (3H, s, 4″-CH3). 13C NMR (100 MHz, CDCl3): δ
195.58, 170.39, 149.41, 141.07, 135.97, 116.97, 100.68, 65.27, 64.71,
39.38, 10.87. HR-ESI-MS: m/z ([M + H]+) calcd for 225.0763, found
225.0764.
Figure 1. Seed germination activity toward O. Cumana. The seed
germination rate for negative control (distilled water) was lower than
10%.
Seed Germination Assay. The pretreated seeds of O. aegyptiaca
were evenly spread on each sterile water-wetted filter paper disk (6
mm diameter, approximately 25−65 seeds per disk) in Petri dishes.
The sealed Petri dishes were stored in the dark at room temperature
for 3−7 days. Then, a piece of the glass fiber filter paper disk was
placed in Petri dish and a solution of tested compound in acetone (25
μL) was added. After complete evaporation of acetone at room
temperature, the filter paper disk with preincubated seeds was coated
on it. Subsequently, another part of sterile water (25 μL) was added
on each disk and a piece of sterile water-wetted filter paper was placed
on the center of Petri dishes to preserve moisture. Finally, the sealed
Petri dishes were stored in the dark and incubated at room
temperature for 9−10 days. After the incubation, the percentage
ratios of germination were calculated with the assistance of a
microscope. In each concentration of tested compound, four disks of
pretreated seeds were used and each assay was carried out three times.
rac-GR24 was used as the positive control, and the disks that were
added with 25 μL of sterile water was used as negative control. The
EC50 values of the tested compounds were calculated with SPSS 19.0.
The filter paper disks used in seed germination assay were sterilized
prior to the use.
The germination activity assay of O. cumana seeds was carried out
by Prof. Yongqing Ma at the Forestry Institute of Northwest
Agriculture & Forest University, and the test method was slightly
modified on the basis of aforementioned method for O. aegyptiaca
seeds. The pretreated O. cumana seeds were evenly spread to sterile
water-wetted glass fiber filter paper disks (approximately 25−65 seeds
per disk) in Petri dishes and then preincubated in the dark at room
temperature for 3−7 days. After the incubation, the glass fiber filter
paper disks with preincubated O. cumana seeds were placed in Petri
dishes and a solution (20 μL) of tested compound (first dissolved in
acetone and then diluted with water to the content of acetone lower
than 5% (v/v)) was added directly. A piece of wet filter paper was
placed on the center of Petri dish to preserve moisture. Finally, the
sealed Petri dishes were stored in the dark and incubated at room
temperature for 9−10 days, and the percentage of germination was
determined by using a microscope. For the seed germination data of
all new compounds, see Table 1 and Figure 1.
successively added to the wells of a 96-well plate in turn. For the
blank test, water (5 μL) was used instead of the solution of the tested
compound. The fluorescent intensity was measured by spectrofluor-
ometer (excitation wavelength, 480 nm; emission wavelength, 520
nm, the excitation and emission slit widths were set as 10 and 20 nm,
respectively). The fluorescence intensity change rate was calculated
with Origin, where the lg[c(μM)]-(k′/k)% plot (k′ and k were the
fluorescence intensity change rates of the test compound and blank
control, respectively) was achieved by the Growth/Sigmoidal-
DoseResp curve fitting method of Origin. Finally, the IC50 value
and R2 were obtained from the above plot.
Docking Experiment. The molecular modeling computational
study was performed using Autodock vina 1.1.2 software. The crystal
structure of rice DWARF14 (PDB: 5DJ5) was used for the docking
study. The grid box was set as a 20 × 20 × 20 Å3 cube, and the center
of it was set at the position of original ligand GR24 (x = −30.80, y =
14.65, z = −21.05). The docking results were visualized by Pymol and
Discovery studio.
Stability Test. The stability of the strigolactone analogues is an
important factor affecting their seeds germination activity toward the
parasitic weeds. The hydrolytic stability of synthesized analogues was
tested under pH 5 and 8 conditions, respectively. Compound 24 was
selected as the representative test sample. First, it was dissolved in
methanol to give a stock solution (100 mM). This stock solution (10
μL) was then added to a mixture solution (1 mL) of phosphate buffer
(pH 8)/methanol (1:1) and phosphate citrate buffer (pH 5)/
methanol (1:1), respectively. The stability of compound 24 was
measured by HPLC/UV (SHIMADZU 228-45041-91 reservoir tray,
C18 column, 5u, 250 mm × 4.6 mm, eluent, 50% methanol in water,
wavelength, 254 nm) after 1, 2, 3, 4, 5, 6, 7, and 8 days. The stability
test of the control GR24 was performed in a similar manner.
RESULTS AND DISCUSSION
■
Synthesis of Strigolactone Analogues. First, 2-aryl-4-
chromanones 1a−17a were synthesized from 2-hydroxyaceto-
phenone and corresponding aromatic aldehyde via a Mannich
reaction by using I2/aniline catalytic system, originally
developed by Yao et al., and a sequential Aldol reaction.27,28
Condensation of these dihydroflavonoids 1a−17a with an
excess of N,N-dimethylformamide dimethyl acetal
(DMFDMA) gave the corresponding enamine intermediates
1b−17b in modest to good yield.35 All of these enamines were
isolated as the single E-isomers based on the analysis of their
NOESY spectra. Subsequently, the hydrolysis of the enamine
intermediates in the presence of acetic acid successfully
HTL7 Inhibition Assay. The competition assay of tested
compounds were performed according to the previously reported
method.26 Prior to the test, YLG stock solution (5 mM in DMSO)
was diluted with water to 50 μM, while the stock solution of GR24
(10 mM in DMSO) and tested compounds (1 mM in DMSO) were
diluted with water to 1000, 333.33, 111.11, 37.037, 12.346, 4.115,
1.372, and 0.457 μM, respectively.
The solutions of YLG (50 μM, 4 μL), tested compound (5 μL),
HEPES buffer (100 mM, 150 mM NaCl, pH 7, 88 μL), and HTL7
protein solution (1.8 mg/mL in HEPES buffer, 3 μL) were
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Scheme 2. Synthesis of SL Analogues 1−25
Chart 1. Structures of SLs 1−25
provided the precursor enols, which were found not to be
stable enough on silica gel column chromatography and thus
were directly subjected to condensation with 5-bromo-3-
methyl-2(5H)-furanone without further purification. Finally,
the target SL analogues 1−17 were obtained in good yields
(Scheme 2a). These new SL analogues were found to be stable
when kept in −20 °C for months.
For the synthesis of 2-alkyl-substituted 4-chromanone
derivatives 18a−24a, pyrrolidine-catalyzed cyclization of 2-
hydroxyacetophenone with aliphatic aldehydes or ketones
readily afforded the desired products via a known Mannich
procedure.29 The coupling of compounds 18a−24a with ethyl
formate using potassium tert-butanoate (tBuOK) as the base
successfully yielded the enol intermediates, which were
subsequently converted into the target SL analogues 18−24
upon condensation with 5-bromo-3-methyl-2(5H)-furanone in
the presence of t-BuOK (Scheme 2b). The E- configuration
was determined to be major isomers for these SLs based on the
analysis of NOESY spectra. The control compound 25 was also
prepared from commercially available dihydro-2H-pyran-
4(3H)-one 25a in a similar manner (Scheme 2c). For the
structures and synthetic routines of these novel SL analogues,
see Scheme 2 and Chart 1. Their spectroscopic character-
ization data are listed in the Materials and Methods section.
Seed Germination Assay. The synthetic SL analogues
derived from 2-aryl and 2-alkyl dihydroflavone derivatives were
first evaluated for their seed germination activity of O.
aegyptiaca. As shown in Table 1, for all 2-(hetero)aryl
substituted SL analogues, except compounds 4 (o-bromo), 8
(m-nitro), 13 (p-trifluoromethyl), and 17 (2-furyl), most
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compounds induced more than 80% germination rates at a
concentration of 20 mg/L, whereas compounds 1 (phenyl), 2
(o-fluoro), 6 (o-methoxy), and 16 (2-thienyl) still afforded
more than 80% germination rates at lower concentration of 2.0
mg/L. These compounds showed an EC50 value arranged from
2.12−6.71 × 10−7 M (compounds 1, 6, 9, and 16, Table 1).
Even at a concentration of as low as 0.2 mg/L, compound 1
achieved up to 74% germination rate and an EC50 value of 2.12
× 10−7 M.
Table 2. HTL7 Inhibition Activity of Compounds 1, 6, 9,
and 16 (first batch)
compound number
R
IC50 (μmol/L)
R2
1
6
9
16
phenyl
5.11274
5.44412
5.26619
6.69811
0.84217
0.9981
2-OMe-C6H4
4-OMe-C6H4
2-thienyl
0.99503
0.99252
0.9987
GR24
0.99876
In comparison to 2-aryl substituted SL analogues, the SL
analogues 18−24 derived from 2-mono- and 2-dialkyl
substituted dihydroflavonoids generally displayed remarkably
higher seed germination activity toward O. aegyptiaca. It was
shown that, at a lower dosage of 0.2 mg/L, all these 2-alkyl
substituted SL analogues revealed germination rates of more
than 70% toward O. aegyptiaca seeds. The steric effect of 2-
alkyl substituent was found to play an important role on seed
germination activity. The larger the alkyl group, the higher
EC50 values were observed (from 2.33 × 10−8 to 2.92 × 10−7
M). Of particular note, compound 19 (2-methyl) showed up
to 10 times higher seed germination activity than GR24 under
the same assay conditions, which showed the lowest EC50 value
of 3.49 × 10−9 M toward O. aegyptiaca seeds. As far as we
know, related to other known SL analogues derived from 3-
methyl-2(5H)-furanone, i.e., gibberellic acid hybrid strigolac-
tone mimics,36 this compound displays a higher seed
germination activity toward O. aegyptiaca. It is noteworthy
that compound 24 bearing two methyl groups at the C2
position also reveals a remarkable EC50 value of 8.99 × 10−8 M.
While for the control compound 25 without a conjugated
benzene ring, modest germination rates were provided,
suggesting that the aryl ring in the dihydroflavonoids is
indispensable for their seed germination potential toward
parasitic weeds.
Compounds 1 and 24 were selected to be further evaluated
for their seed germination activity against O. cumana. As
illustrated in Figure 1, these new SL analogues displayed
remarkable germination activity in comparison to the control
rac-GR24 at every tested concentration. At a concentration of
20 mg/L, both test samples showed a more than 60%
germination rate. Even at a lower concentration of 0.2 mg/L,
compound 24 still displays more than 50% germination rate
toward O. cumana seeds.
HTL7 Inhibition Assay. Yoshimulactone Green (YLG), a
small-molecule fluorescence turn-on probe, can activate
strigolactone signaling and illuminate signal perception by
the strigolactone receptors of the parasitic plant Striga
hermonthica, ShHTLs, thereby elucidating the regulatory
dynamics for strigolactone signal transduction in Striga.25,26
Among all subgroups of the strigolactone receptor ShHTLs,
ShHTL6 and ShHTL7 showed indiscriminately high affinity to
all of the tested SLs, making them ideal targets to detect
binding affinity of structurally diverse strigolactone analogues.
In particular, HTL7 was found to be sensitive to the
structurally diverse molecules at the levels of picomolar when
heterologously expressed in Arabidopsis.37 As a consequence,
the inhibition assay of these novel strigolactones toward HTL7
receptor were performed using YLG fluorescent probe method.
As shown in Table 2, for 2-aryl substituted dihydroflavo-
noid-derived analogues 1, 6, 9, and 16, the substituent pattern
in the aryl ring showed no prominent effect on the binding
affinity toward HTL7 proteins. Meanwhile, from all tested 2-
alkyl substituted dihydroflavonoid-derived analogues (com-
pounds 18−24, Table 3), compound 24 was found to show the
highest binding activity toward HTL7 protein receptor (IC50
=
Table 3. HTL7 Inhibition Activity of Compounds 18-24
(second batch)
compound number
R
IC50 (μmol/L)
R2
18
19
20
21
22
23
24
GR24
H
Me
Et
n-Pr
n-Bu
n-Pent
Me, Me
1.79543
2.19524
2.88826
2.63495
5.5582
7.76506
1.06094
1.98125
0.99456
0.99844
0.99272
0.99127
0.99124
0.99619
0.9918
0.99472
1.06 μmol/L), while the IC50 value for the control GR24 was
1.98 μmol/L. As seen in Table 3, the obvious decline in
binding affinity toward HTL7 proteins was observed with the
bulkiness of the 2-alkyl group in the dihydroflavonoids. Related
to the 2-aryl group, the 2-alkyl group on the C2 position of
dihydroflavonoid provided lower IC50 values.
Molecular Docking Experiment. The interaction of SLs
with a protein receptor is an essential step for stimulating the
seed germination of parasitic weeds. There is distinct evidence
that the D14 protein is involved in this process.11 The result of
this interaction is the detachment of the D-OH ring, which
leads to the change of the enzyme pocket conformation and
then subsequent cascade reactions, finally giving rise to the
germination of parasitic weed seeds.19 In order to rationalize
the seed germination activity of these dihydroflavonoid-derived
strigolactone analogues, the molecular docking of compounds
18, 19, and 20 into the catalytic site of OsD14 hydrolase
(PDB: 5DJ5) was thus performed (Figure 2).
The formed polar contact between carbonyl group in D ring
and Ser 97 is required for the successful hydrolysis of the
strigolactone analogues.38 As shown in Figure 2A,B, there was
no hydrogen bond formed between compound 19 and Ser 97,
but the orientation of the D ring in compound 19 was the
same as that of GR24. Moreover the electrostatic interaction
was formed between compound 19 and His 247, which
belongs to the catalytic triad of OsD14. The π−π interaction
and covalent bonding were also formed between the aromatic
ring and Tyr 159 and Trp155, respectively (Figure 2D). The
above interactions make compound 19 bound firmly in the
catalytic site of OsD14 with the right orientation. Additionally,
the molecular docking of compound 18 in OsD14 protein was
also performed. As shown in Figure 2C, the orientation of the
D ring in compound 18 was not same as that of compound 19.
This result indicates that the C2 substituent group in the
dihydroflavonoid scaffold has the potential to adjust the
orientation of D ring. Moreover, the docking result of
compound 20 was also similar to that of compound 19,
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Figure 2. Docking modes of compounds 18 and 19 with OsD14. (A) Close-up view of compound 19 as blue sticks bound in the catalytic site of
OsD14. (B) View from the top of the catalytic pocket with compound 19 shown as blue sticks. (C) Close-up view of compounds 18 (yellow sticks)
and 19 (blue sticks) bound in the catalytic site of OsD14. (D) 2D view of compound 19 in the catalytic site of OsD14. Note: The green sticks
shown in A and B are GR24, which is the original ligand of OsD14.
which might account for better germination activity of
compounds 19 and 20 in comparison with compound 18.
Hydrolytic Stability. Due to the presence of the labile
groups including enol ether, hemiacetal, and α,β-unsaturated
lactones, the stability of both natural and synthetic
strigolactone analogues plays a vital role in stimulating the
potential suicidal germination of parasitic plants. Generally,
regardless of the presence of additional stability-enhancing
agents and microorganisms, the pH values of the soil have an
important impact on the hydrolysis rate of SLs. As illustrated in
Figure S53, these dihydroflavonoid-derived SLs were found to
be stable at acidic conditions. For instance, about 45% of
compound 24 was hydrolyzed in an acidic (pH 5) solution of
water/methanol (1/1, v/v) after 8 days (see Figure S54 of the
Supporting Information). Comparatively, the control GR24
was only hydrolyzed no more than 10%. On the contrary,
complete hydrolysis within 1 day was observed for both
compound 24 and GR24 at alkaline conditions (water/
methanol, 1/1, v/v, pH 8). By comparison with the
resorcinol-type mimics,37 these compounds are shown to be
prone to hydrolysis to release the active D ring. These results
suggest that this class of dihydroflavonoid-derived SLs might
completely decompose in the soil and could be used as the
eco-friendly herbicides or promising lead compounds against
parasitic weeds.
In summary, to explore novel herbicides against parasitic
weeds, such as broomrapes (Orobanche and Phelipanche spp.)
and witchweeds (Striga spp.), a series of strigolactone
analogues derived from dihydroflavonoids were synthesized.
Their suicide-inducing seed germination potential toward the
broomrapes was evaluated, and the preliminary structure−
activity relationship was also discussed. The 2-alkyl group in
the dihydroflavone skeleton was found to play a key role in
their seed germination activity. From these, compound 19
bearing a methyl group displayed 10 times higher germination
activity than that of the control GR24. This result was further
demonstrated by the strigolactone receptor HTL7 inhibition
assay. In light of their hydrolytic stability, the SL analogues
described herein were shown to be promising candidates for
further study as suicidal germinators against parasitic weeds.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jafc.9b08044.
I
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traditional Chinese medicinal herbs. Sci. China: Life Sci. 2012, 55,
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Figures of NMR spectra, NOSEY spectra, stability test,
and seed germination activity (PDF)
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Peng, G. Harm of parasitic weed Orobanche and its prevention in
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AUTHOR INFORMATION
Corresponding Authors
■
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Zhong Jin − State Key Laboratory of Elemento-organic
Chemistry, College of Chemistry, Nankai University, Tianjin
300071, China; orcid.org/0000-0001-5722-7175;
Email: zjin@nankai.edu.cn
Xiaohua Xu − State Key Laboratory of Elemento-organic
Chemistry, College of Chemistry, Nankai University, Tianjin
300071, China; Email: xiaohuaxu@nankai.edu.cn
Authors
Yunyao Kang − State Key Laboratory of Elemento-organic
Chemistry, College of Chemistry, Nankai University, Tianjin
300071, China
Zhili Pang − State Key Laboratory of Elemento-organic
Chemistry, College of Chemistry, Nankai University, Tianjin
300071, China
Niuniu Xu − State Key Laboratory of Elemento-organic
Chemistry, College of Chemistry, Nankai University, Tianjin
300071, China
Fangjie Chen − College of Forestry, Northwest Agriculture &
Forest University, Xianyang 712100, Shanxi Province, China
(16) Zwanenburg, B.; Mwakaboko, A. S.; Reizelman, A.; Anilkumar,
G.; Sethumadhavan, D. Structure and function of natural and
synthetic signaling molecules in parasitic weed germination. Pest
Manage. Sci. 2009, 65, 478−491.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jafc.9b08044
Funding
(17) Zwanenburg, B.; Mwakaboko, A. S.; Kannan, C. Suicidal
germination for parasitic weed control. Pest Manage. Sci. 2016, 72,
2016−2025.
Financial support from the National Natural Science
Fundation of China (No. 21672116) and National Key
Research and Development Program of China
(2016YFD020050) is gratefully acknowledged.
(18) Samejima, H.; Babiker, A. G.; Takikawa, H.; Sasaki, M.;
Sugimoto, Y. Practicality of the suicidal germination approach for
controlling Striga hermonthica. Pest Manage. Sci. 2016, 72, 2035−
2042.
Notes
The authors declare no competing financial interest.
(19) Zwanenburg, B.; Pospisil, T.; Zeljkovic, S. C. Strigolactones:
new plant hormones in action. Planta 2016, 243, 1311−1326.
(20) Yao, R. F.; Ming, Z. H.; Yan, L. M.; Li, S. H.; Wang, F.; Ma, S.;
Yu, C. T.; Yang, M.; Chen, L.; Chen, L. H.; Li, Y. W.; Yan, C.; Miao,
D.; Sun, Z. Y.; Yan, J. B.; Sun, Y. N.; Wang, L.; Chu, J. F.; Fan, S. L.;
He, W.; Deng, H. T.; Nan, F. J.; Li, J. Y.; Rao, Z. H.; Lou, Z. Y.; Xie,
D. X. DWARF14 is a non-canonical hormone receptor for
strigolactone. Nature 2016, 536, 469−473.
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flavonoids. Int. J. Antimicrob. Agents 2005, 26, 343−356.
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ACKNOWLEDGMENTS
■
The authors would like to thank Professor Yongqing Ma
(Northwest Agriculture & Forest University, Xianyang, China)
for guidance on the seed germination experiment and kindly
providing the seeds of O. cumana and O. aegyptiaca.
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