New branching inhibitors and their potential as strigolactone mimics in rice

Article abstract of DOI:10.1016/j.bmcl.2011.06.019

Strigolactones (SLs) are rhizosphere communication chemicals. Recent studies of highly branched mutants revealed that SL or its metabolites work as a phytohormone to inhibit shoot branching. When SLs are exogenously applied to the rice d10-1 mutant that has a highly branched phenotype caused by a defect in the SL biosynthesis gene (CCD8), they inhibit tiller bud outgrowth (branching in rice) of the mutant. We focused our attention on the SL function as a phytohormone and tried to find new chemicals mimicking the hormonal action of SL by screening chemicals that inhibit branching of rice d10-1 mutant. Fortunately, we found 5-(4-chlorophenoxy)-3-methylfuran-2(5H)-one (3a) as a new chemical possessing SL-like activity against the rice d10-1 mutant. Then, we prepared several derivatives of 3a (3b-3k) to examine their ability to inhibit shoot branching of rice d10-1. These derivatives were synthesized by a one-pot coupling reaction between phenols and halo butenolide to give 5-phenoxy 3-methylfuran-2(5H)-one (3) derivatives, which possess a common substructure with SLs. Some of the derivatives showed SL-like activity more potently than GR24, a typical SL derivative, in a rice assay. As SLs also show activity by inducing seed germination of root parasitic plants, the induction activity of these derivatives was also evaluated. Here we report the structure-activity relationships of these compounds.

Full text of DOI:10.1016/j.bmcl.2011.06.019

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4905–4908
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
New branching inhibitors and their potential as strigolactone mimics in rice
Kosuke Fukui a, Shinsaku Ito a, Kotomi Ueno a, Shinjiro Yamaguchi b, Junko Kyozuka a, Tadao Asami a,
⇑
a Graduate School of Agriculture and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan
b RIKEN Plant Science Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Strigolactones (SLs) are rhizosphere communication chemicals. Recent studies of highly branched
mutants revealed that SL or its metabolites work as a phytohormone to inhibit shoot branching. When
SLs are exogenously applied to the rice d10-1 mutant that has a highly branched phenotype caused by
a defect in the SL biosynthesis gene (CCD8), they inhibit tiller bud outgrowth (branching in rice) of the
mutant. We focused our attention on the SL function as a phytohormone and tried to find new chemicals
mimicking the hormonal action of SL by screening chemicals that inhibit branching of rice d10-1 mutant.
Fortunately, we found 5-(4-chlorophenoxy)-3-methylfuran-2(5H)-one (3a) as a new chemical possessing
SL-like activity against the rice d10-1 mutant. Then, we prepared several derivatives of 3a (3b–3k) to
examine their ability to inhibit shoot branching of rice d10-1. These derivatives were synthesized by a
one-pot coupling reaction between phenols and halo butenolide to give 5-phenoxy 3-methylfuran-
2(5H)-one (3) derivatives, which possess a common substructure with SLs. Some of the derivatives
showed SL-like activity more potently than GR24, a typical SL derivative, in a rice assay. As SLs also show
activity by inducing seed germination of root parasitic plants, the induction activity of these derivatives
was also evaluated. Here we report the structure–activity relationships of these compounds.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 13 April 2011
Revised 30 May 2011
Accepted 6 June 2011
Available online 22 June 2011
Keywords:
Chemical synthesis
Drug design
Strigolactone
Mimic
Branching
Root parasitic weed
Germination
Plant hormone
Strigol, a member of the strigolactones (SLs), was first discov-
ered from cotton root exudates as a seed germination stimulant
of root parasitic plants such as Striga.1 This compound was identi-
fied as a member of the terpenoids whose structural core consists
of tricyclic-lactone (ABC ring) and a butenolide group (D-ring) that
are connected via an enolether moiety (Fig. 1 and 1). A variety of
SLs, chemically substituted at A- and/or B-rings, has been isolated
from the root exudates of many different host plants as seed ger-
mination stimulants of several kinds of root parasitic plants.2 The
discovery of natural SLs led chemists to synthesize various artificial
analogues based on the structure of natural products for the appli-
cation of such compounds to the field of agriculture.3 Recent stud-
ies revealed that SLs stimulate not only seed germination of root
parasitic plants but also hyphal branching in Arbuscular mycorrhizal
fungi (AM fungi), which establish a symbiotic relationship with
plant roots.4 This relationship provides the fungus with carbohy-
drates produced by the plant, and the plant with water and phos-
phate taken up by the fungus. Therefore, it was thought that SLs
were originally released as symbiotic chemicals and abused by root
parasitic plants. More recently, two research groups reported that
these rhizosphere communication chemicals or their metabolites
also work as a novel class of plant hormones inhibiting shoot
branching.5
Until now, many kinds of artificial SL analogs were synthesized
and studied as stimulants of seed germination of root parasitic
plants, some of which also showed hypal branching activity.6
Structure–activity relationship studies of SL analogs as seed germi-
nation stimulants of root parasitic plants revealed that D-ring
lactone connected by an enolether-moiety is important for their
activity.7 GR24 (Fig. 1 and 2) is the first artificial SL analog synthe-
sized more easily than natural SLs and is therefore widely used
as a mimic of natural SLs in several biological situations.2a,3b,5,6 In
the first report GR24 was synthesized in a 6-step reaction from
trans-2-bromo-1-indanol. Later, an improved method which re-
quires a 4-step reaction from 1-indanone, was reported.8 This
method facilitated the formation of the ABC ring part and reduced
the number of steps, although GR24 diastereomer (epi-GR24) was
simultaneously prepared. Although the activity of epi-GR24 is the
same as that of GR24, however, in research GR24 should be evalu-
ated separately from epi-GR24. Subsequently, asymmetric synthe-
sis of GR24 was reported,9 but this report did not mention the
formation of the ABC ring. At present the formation of the ABC ring
of GR24 still required at least a one-step reaction followed by
formylation. Therefore efficient and short routes for the synthesis
of potent SL mimics are still required. To date, the activity of SL
analogs has been evaluated by measuring the activity of germina-
tion stimulation of root parasitic plants, although their activity as a
phytohormone has not been well investigated.
In this report, we focused our attention on chemicals that inhib-
ited shoot branching. This is because, ever since the discovery of
⇑
Corresponding author.
E-mail address: asami@pgr1.ch.a.u-tokyo.ac.jp (T. Asami).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.019

4906
K. Fukui et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4905–4908
(Table 1, entries 1–6 and 10) likely gave better yield than those
with electron-donating groups (Table 1, entries 7–9 and 11).
We then evaluated the inhibitory activity of the newly synthe-
sized compounds on the shoot branching of rice grown in a hydro-
ponic culture system as reported previously.11 In this test, we used
an SL-deficient d10-1 mutant in which a gene encoding the SL bio-
synthesis enzyme (CCD8) is mutated.12 This mutant, whose pheno-
type shows more tillering, reverts back to a phenotype almost
identical to that of wild type after the application of SLs. Second til-
ler bud outgrowth was observed in 2-week-old rice d10-1 seed-
lings without chemical treatment, but this characteristic
phenotype in mutant rice was not observed in the SL-treated
d10-1 mutant. We examined the inhibitory effect of the com-
pounds synthesized as described above in this assay system at
Figure 1. Structures of natural SL; (+)-strigol (1), synthetic SL GR24 (2), and a lead
compound CPMF (3a) used in this report.
SLs as a phytohormone, easily obtainable SL mimics have been in
demand by many plant biologists to investigate the functions of
SLs in crop production and plant physiology. Chemicals mimicking
the hormonal action of SLs can be useful for agriculture in the
future. Thus, we attempted to explore the compounds that can
mimic the action of SL as a phytohormone. Recently, Kondo et al.
reported that the imino analog of GR24 showed germination stim-
ulating activity in some root parasitic weeds.3e This finding indi-
cates that it is not always essential to have the Michael acceptor
1
1
l
l
M (Fig. 2). Treatment of GR24 (2), a synthetic SL analog, at
M inhibited the second tiller outgrowth of d10-1. All of the
compounds synthesized and listed in Table 1 are 4-substituted
phenoxyfuranone derivatives that show clear biological activity
depending on the substituent on the phenoxy ring. Halogenated
compounds, which contain fluorine (3b), bromine (3c) or iodine
(3d), are highly active; they also include a chlorine-substituted
compound (3a). Compounds substituted with an electron-
withdrawing group such as trifluoromethyl (3e) or nitrile (3f),
are also more active than GR24, although, interestingly, the com-
pound substituted with methoxycarbonyl (3j) as an electron-with-
drawing group was not so active. In contrast, the compound
substituted with an electron-donating group, such as methyl
(3g), was active. On the other hand, compounds substituted with
bulky groups, such as tert-butyl (3h), methoxy (3i) or butenolide
(3k), were likely not so active as compounds 3a–3g. When combin-
ing the results in Figure 2, compounds including a bulky substitu-
of the C–D ring junction moiety. We expected that the p-bond con-
jugated ether is important for SL activity. To examine this expecta-
tion, we synthesized new chemicals lacking an enolether moiety,
5-phenoxy-3-methylfuran-2(5H)-one (PMF) and 5-(4-chlorophen-
oxy)-3-methylfuran-2(5H)-one (3a, CPMF), and estimate their
activity in a shoot branching inhibition assay at 1 lM, as reported
for SL biosynthesis inhibitors.10 Fortunately, in this assay, CPMF
had distinct activity but PMF, which has no substituent on the phe-
nyl group, had almost no activity. We then initiated structure–
activity relationship studies of 3a. Here we report the chemical
synthesis of 3a and analogs of 3a and their efficacy in inhibiting
shoot branching in the rice d10-1 mutant.
Compounds 3a–3k in Table 1 were synthesized using modified
procedures reported in the literature.11 The first step was
bromination at the 5-position of 3-methylfuran-2(5H)-one using
N-bromo-succinimide, which was then coupled with a variety of
phenols under basic conditions to give a crude mixture of phenol
ethers in two steps. Purification with silica gel flash column
chromatography and re-crystallization from n-hexane and ethyl
acetate gave pure 3a–3k in 13–73% yield as shown in Table 1.
The compounds substituted with electron-withdrawing groups
ent were likely less active than those including
a smaller
substituent. This result obtained by structure–activity relationship
studies of the derivatives reported here could be useful for design-
ing new SL mimics in future research.
Next, we examined the concentration dependency of the inhib-
itory activity of each compound substituted with an electron-
withdrawing but not a bulky group (Fig. 3A). All of the compounds
tested here inhibited second tiller bud outgrowth at 0.1
effectively as GR24 at 1 M. Compounds 3c–3f were so potent in
our assay system even at 0.1 M that little or no second tiller
lM as
l
l
bud outgrowth was observed in the d10-1 mutant treated with
3c–3f at this concentration. To further estimate the activity of
3c–3f at a low concentration (Fig. 3B), we again treated the
Table 1
Percent yields of phenyl ether derivatives
d10-1 mutant with 3c–3f at 0.01 lM. The result showed that 3c
is likely the most active. There were statistically unclear points
in these data but every time 3c was the most active in the same
Entrya
R=
R1=
Yieldb (%)
1
2
3
4
5
6
7
8
9
Cl
F
Br
I
CF3
CN
Me
tBu
OMe
Cl
F
Br
I
CF3
CN
Me
tBu
OMe
3a
3b
3c
3d
3e
3f
3g
3h
3i
29
40
53
73
59
55
29
24
38
10
3j
50
11
OH
3k
13
a
Reaction conditions : The reaction was carried out in 4.0 ml of CH2Cl2 and
3.0 ml of H2O with 1.0 mmol of TBABr, 1.2 mmol of K2CO3, 1.0 mmol of phenol and
1.0 mmol of substrate at room temperature for over 8 h.
Figure 2. Effect of chemicals on 16-day-old rice seedlings. Length of second tiller of
rice seedlings treated with 1
samples.
lM of chemicals. The data are means SE of six
b
Isolated yield.

K. Fukui et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4905–4908
4907
Figure 4. Effect of 3c on rice tillering. A comparison of second tiller length in 16-
day-old wild type (WT) and d mutants. Concentration of 3c was 1 lM in hydroponic
culture.
Table 2
Effect of chemicals on inducing seed germination of Striga hermonthica
No.
Germination rate (%)
10
0.001
l
M
0.1
l
M
1
l
M
lM
100 lM
2
13.8 8.4
29.5 5.2
33.8 0.8
1.0 1.7
1.1 2.0
0.0
0.9 1.6
1.4 2.5
4.5 7.9
0.0
24.0 11.1
3.3 3.8
1.4 2.5
5.4 4.8
5.7 5.2
3.4 3.1
22.2 6.8
0.0
1.1 1.9
3.1 3.2
14.0 7.2
0.7 1.2
—
3a
3b
3c
3d
3e
3f
3g
3h
3i
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
2.1 1.8
4.8 4.4
19.9 1.6
8.3 14.4
11.1 2.9
21.7 2.9
0.0
2.1 3.6
4.5 4.8
11.9 7.7
4.4 5.3
Figure 3. Effect of chemical concentration on 16-day-old rice seedlings. (A) Length
of second tiller of rice seedlings treated with chemicals at the described concen-
tration. The data are means SE of six samples. (B) Length of second tiller of rice
0.0
1.1 1.9
3.7 3.5
0.6 1.0
3j
3k
seedlings treated with 0.01
samples.
lM of chemicals. The data are means SE of six
Each data value is mean germination percentage standard deviation from three
replicates.
assay. In this assay, our compounds showed a clear inhibitory ef-
fect on rice second tiller bud outgrowth in a dose-dependent man-
ner; however, even at a high concentration (10
lM, data not
clearly stimulated the germination of Striga seeds at 100
lM, but
shown) no morphological change was observed such as dwarfing
all of the debranones barely induced germination at 1 lM. On
or bleaching in rice treated with these chemicals.
the other hand, GR24 showed germination stimulant activity even
at 0.001 M. That is, GR24 was 10,000-times more active than
debranones in this biological test although GR24 was 10-times less
Next, we investigated the mechanism by which 3c exerts its
hormonal activity in rice. To examine the possibility that 3c inhib-
its tiller bud outgrowth through the SL signaling pathway, we trea-
ted 3c in SL-insensitive mutants, d3-1 and d14-1,5b,13 in which SL
signal transduction is blocked. Tiller bud outgrowth of d3-1 and
d14-1 is not affected by the application of GR24, while GR24 inhib-
ited tiller bud outgrowth of d10-1. As a result, a similar trend was
l
active than 3c in the rice branching test.
The important structural requirements of SLs and their syn-
thetic derivatives for hyphal branching in AM fungi and seed
germination of root parasitic plants have been studied, revealing
the existence of several similarities in the structural requirements
for both activities.6a,8 Specifically, the existence of proper substitu-
ents on the A- and/or B-ring and a bond between the C-ring and the
D-ring are important for exerting both activities. However, there is
a distinct difference in the structural requirements for AM fungi
and for parasitic plants. For example, carbon–carbon or carbon–
nitrogen double bonds next to an ether bond are required for the
induction activity of seed germination of root parasitic plants but
not for stimulating hyphal branching. In contrast, there has been
no structure–activity relationship study on the functions of SL
derivatives as a phytohormone because both SL-deficient mutants
and a relatively large amount of SLs are necessary for estimating
the hormonal activity of SLs making it difficult to evaluate the hor-
monal activity of SLs and their derivatives. Therefore, whether
structural requirements of SLs as a phytohormone were different
from those for stimulating root parasitic seed germination or
inducing hyphal branching or not is still unknown. In this report,
we demonstrate that our chemicals inhibit shoot branching in
rice maybe through the SL signaling pathway and therefore our
observed for the application of 1 lM of 3c to these three mutants
and wild type rice (Fig. 4). That is, 3c inhibited tiller bud outgrowth
of d10-1, but by contrast, did not affect tiller bud outgrowth of both
d3-1 and d14-1. These results suggest that our compounds should
inhibit tiller bud outgrowth by activating the SL signal transduc-
tion pathway, not by a side effect, and be good lead compounds
for branching inhibitors as SL mimics. Here we propose the name
of the chemicals possessing a 5-phenoxy-3-methylfuran-2(5H)-
one skeleton as debranones (furanones showing de-branching
activity).
SLs are not only shoot branching inhibitors but also seed germi-
nation stimulants of root parasitic plants Striga and Orobanche. To
confirm whether debranones also act as SL mimics in the induction
of seed germination of parasitic plants, we used a highly sensitive
germination assay using Striga hermonthica seeds. The assay meth-
od was the same as that used in a former report by Ito et al.10 We
compared the activity of synthesized debranones with that of GR24
(Table 2). As a result, some of the derivatives (3c, 3e, 3f, and 3j)

4908
K. Fukui et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4905–4908
2. (a) Hauck, C.; Müller, S.; Schildkenecht, H. J. Plant Physiol. 1992, 139, 474; (b)
Müller, S.; Hauck, C.; Schildkenecht, H. J. Plant Growth Regul. 1992, 11, 77; (c)
Yokota, T.; Sakai, H.; Okuno, K.; Yoneyama, K.; Takeuchi, Y. Phytochemistry
1998, 49, 1967; (d) Yoneyama, K.; Xie, X.; Yoneyama, K.; Takeuchi, Y. Pest
Manag. Sci. 2009, 65, 467; (e) Takikawa, H.; Jikumaru, S.; Sugimoto, Y.; Xie, X.;
Yoneyama, K.; Sasaki, M. Tetrahedron Lett. 2009, 50, 4549.
3. (a) Jhonson, A. W.; Roseberry, G.; Parker, C. Weed Res. 1976, 16, 223; (b)
Jhonson, A. W.; Gowda, G.; Hassanti, A.; Knox, J.; Monaco, S.; Razawi, Z.;
Roseberry, G. J. Chem. Soc., Perkin Trans. 1 1981, 1734; (c) Nefkens, G. H. L.;
Thuring, J. W. J. F.; Beenakkers, M. F. M.; Zwanenburg, B. J. Agric. Food Chem.
1997, 45, 2273; (d) Flematti, G. R.; Ghisalberti, E. L.; Dixon, K. W.; Trengove, R.
D. Tetrahedron Lett. 2005, 46, 5719; (e) Kondo, Y.; Tadokoro, E.; Matsuura, M.;
Iwasaki, K.; Sugimoto, Y.; Miyake, H.; Takikawa, H.; Sasaki, M. Biosci. Biotechnol.
Biochem. 2007, 71, 2781; (f) Mwakaboko, A. S.; Zwanenburg, B. Plant Cell
Physiol. 2011, 52, 699.
4. Akiyama, K.; Matsuzaki, K.; Hayashi, H. Nature 2005, 435, 824.
5. (a) Gomez-Roldan, V.; Fermas, S.; Brewer, P. B.; Puech-Pagès, V.; Dun, E. A.;
Pillot, J.-P.; Letisse, F.; Matusova, R.; Danoun, S.; Portais, J.-C.; Bouwmeester, H.;
Bécard, G.; Beveridge, C. A.; Rameau, C.; Rochange, S. F. Nature 2008, 455, 189;
(b) Umehara, M.; Hanada, A.; Yoshida, S.; Akiyama, K.; Arite, T.; Takeda-
Kamiya, N.; Magome, H.; Kamiya, Y.; Shirasu, K.; Yoneyama, K.; Kyozuka, J.;
Yamaguchi, S. Nature 2008, 455, 195.
6. (a) Zwanenburg, B.; Mwakaboko, A. S.; Reizelman, A.; Anilkumar, G.;
Sethumadhavan, D. Pest Manag. Sci. 2009, 65, 478; (b) Akiyama, K.;
Ogasawara, S.; Ito, S.; Hayashi, H. Plant Cell Physiol. 2010, 51, 1104.
7. (a) Mangnus, E. M.; Zwanenburg, B. J. Agric. Food Chem. 1992, 40, 1066; (b)
Wigchert, S. C. M.; Zwanenburg, B. J. Agric. Food Chem. 1999, 47, 1320.
8. Mangnus, E. M.; Dommerholt, F. J.; Jong, R. L. P. D.; Zwanenburg, B. J. Agric. Food
Chem. 1992, 40, 1230.
chemicals can be said to be SL mimics. However, the activity of
debranones in the branching inhibition test differed greatly from
that in the germination induction test. Therefore, it is possible to
say that debranones are the first selective SL mimics specialized
for plant hormone action and that 3c is the most potent and selec-
tive compound inducing the de-branching phenotype in rice. At
present, whether debranones function as agonists of SLs or not is
unclear, but further investigations on SL-related gene expression
and identification of SL receptor(s) will reveal the action mecha-
nism(s) of debranones.
In this report, we demonstrate that our compounds could be
used as selective SL mimics to inhibit the branching of plants.
Further structure–activity relationship studies on debranone deriv-
atives can provide selective SL mimics active only for the induction
of root parasitic seed germination. Such types of SL mimics would
be useful for reducing damage to crops by a parasitic plant attack
by stimulating suicidal germination of seeds of parasitic plants.
At present the synthesis of 3-methyl-2(5H)-furanone on a large-
scale is not easy,14 but further exploration of SL mimics may enable
to find chemicals that exert SL-like activity without the butenolide
moiety in our compounds.
9. Thuring, J. W. J. F.; Nefkens, G. H. L.; Zwanenburg, B. J. Agric. Food Chem. 1997,
45, 2278.
Acknowledgements
10. Ito, S.; Kitahata, N.; Umehara, M.; Hanada, A.; Kato, A.; Ueno, K.; Mashiguchi,
K.; Kyozuka, J.; Yoneyama, K.; Yamaguchi, S.; Asami, T. Plant Cell Physiol. 2010,
51, 1143.
11. Wolff, S.; Hoffmann, H. M. R. Synthesis 1987, 760.
12. Arite, T.; Iwata, H.; Oshima, K.; Maekawa, M.; Nakajima, M.; Kojima, M.;
Sakakibara, H.; Kyozuka, J. Plant J. 2007, 51, 1019.
We thank Professor A. G. T. Babiker for supplying Striga seeds.
This research was supported by PROBRAIN (grant to T.A.) and the
Japan Society for the Promotion of Science Fellows to S.I.
References and notes
13. Arite, T.; Umehara, M.; Ishikawa, S.; Hanada, A.; Maekawa, M.; Yamaguchi, S.;
Kyozuka, J. Plant Cell Physiol. 2009, 50, 1416.
14. Malik, H.; Rutjes, F. P. J. T.; Zwanenburg, B. Tetrahedron 2010, 66, 7198.
1. Cook, C. E.; Whichard, L. P.; Turner, B.; Wall, M. E. Science 1966, 154, 1189.