New strigolactone mimics: Structure-activity relationship and mode of action as germinating stimulants for parasitic weeds

Article abstract of DOI:10.1016/j.bmcl.2013.07.004

Strigolactones (SLs) are new plant hormones with varies important bio-functions. This Letter deals with germination of seeds of parasitic weeds. Natural SLs have a too complex structure for synthesis. Therefore, there is an active search for SL analogues and mimics with a simpler structure with retention of activity. SL analogues all contain the D-ring connected with an enone moiety through an enol ether unit. A new mechanism for the hydrolysis SL analogues involving bidentate bound water and an α,β-hydrolase with a Ser-His-Asp catalytic triad has been proposed. Newly discovered SL mimics only have the D-ring with an appropriate leaving group at C-5. A mode of action for SL mimics was proposed for which now supporting evidence is provided. As predicted an extra methyl group at C-4 of the D-ring blocks the germination of seeds of parasitic weeds.

Full text of DOI:10.1016/j.bmcl.2013.07.004

Bioorganic & Medicinal Chemistry Letters 23 (2013) 5182–5186
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
New strigolactone mimics: Structure–activity relationship and mode
of action as germinating stimulants for parasitic weeds
a,
Binne Zwanenburg a, , Sandip K. Nayak , Tatsiana V. Charnikhova b, Harro J. Bouwmeester b
⇑
a Radboud University Nijmegen, Institute for Molecules and Materials, Cluster of Organic Chemistry, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands
b Wageningen University, Laboratory of Plant Physiology, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
Strigolactones (SLs) are new plant hormones with varies important bio-functions. This Letter deals with
germination of seeds of parasitic weeds. Natural SLs have a too complex structure for synthesis. There-
fore, there is an active search for SL analogues and mimics with a simpler structure with retention of
activity. SL analogues all contain the D-ring connected with an enone moiety through an enol ether unit.
Received 10 May 2013
Revised 2 July 2013
Accepted 4 July 2013
Available online 13 July 2013
A new mechanism for the hydrolysis SL analogues involving bidentate bound water and an a,b-hydrolase
with a Ser-His-Asp catalytic triad has been proposed. Newly discovered SL mimics only have the D-ring
with an appropriate leaving group at C-5. A mode of action for SL mimics was proposed for which now
supporting evidence is provided. As predicted an extra methyl group at C-4 of the D-ring blocks the ger-
mination of seeds of parasitic weeds.
Keywords:
Strigolactones
Strigolactone analogues
Germination parasitic weeds
Structure–activity relationship
Mode of action
Ó 2013 Elsevier Ltd. All rights reserved.
New plant hormones
Design and synthesis
Strigolactones (SLs) are new plant hormones that are currently
much in focus.1–10 The predominant activities of these SLs are
stimulation of germination of seeds of parasitic weeds,1–10 branch-
ing factor of AM fungi,4,11,12 and inhibition of shoot branching and
bud outgrowth.13–15 This Letter deals primarily with the activity of
SLs as germination stimulants.
Naturally occurring SLs are present in the root exudates of
many plants, especially host plants for the parasitic weeds. They
invariably contain three annulated rings, the ABC scaffold,
connected with a butenolide ring (D-ring) via an enol ether
unit.1,2,7–10 Typical examples are (+)-strigol (1)16 and (ꢀ)-ent-20-
epi-orobanchol (2)17–19 (Fig. 1). For practical application these
natural SLs have a too complex structure, and therefore, SL
analogues have been developed with a much simpler structure
but with retention of the essential bioactivity.9,10 The most well
known analogue is GR24 (3)9,10,20,21 (Fig. 1).
for the bioactivity.9,10,22 Illustrative examples of such SL analogues
designed on the basis of the modelcompound shown in Figure 2 are
Nijmegen-1 (4)23 and analogues derived from tetralone (5),24,25 hy-
droxy coumarin (6)26 and saccharin (7)27 (Fig. 1).
Although the full details of the germination process are not
known yet, it is assumed that on a molecular level germination is
triggered by an initial reaction of water with the enol ether moiety
in a Michael fashion whereby the water molecule is bound in a
bidentate manner.10 In a subsequent retro-Michael reaction a
cleavage process gives the hydroxy butenolide and the formylated
ABC scaffold.9,10,22 There is some evidence that this hydrolytic
cleavage is catalyzed by an
a/b hydrolase having a Ser-His-Asp
canonical catalytic triad at the active site that is capable of accom-
modating an SL molecule.28 This newly proposed mechanism of
hydrolytic cleavage reaction is depicted in Figure 3, whereby histi-
dine serves as the base to initiate the Michael addition of water.
Recently, we reported our serendipitous finding that com-
pounds lacking the enol ether unit, such as the saccharine D-ring
derivative 8 and the aroyloxy substituted butenolides 9 (Fig. 4)
which are not in accordance with the model shown in Fig. 2, are ac-
tive as germinating agents for parasitic weeds.27 These compounds
are named as SL mimics.
For germination stimulants a model compound was used for the
design and preparations of new bioactive analogues. This model
(Fig. 2) was based on a structure–activity analysis combined with
a tentative mode of action for germination.9,10,22 It was shown that
the bioactiphore of SLs resides in the CD part of the SL molecules,
implying that the
a,b-unsaturated carbonyl moiety connected via
an enol ether unit with the D-ring is predominantly responsible
A structurally related SL mimic is butenolide 10, which shows a
moderate germination activity at a relative high concentration.29
In order to rationalize the activity of the SL mimics a mode of
action shown in Figure 5 was tentatively proposed.27 An essential
feature of this proposal is a proton shift prior to the elimination
⇑
Corresponding author. Tel.: +31 24 3653159.
E-mail address: B.Zwanenburg@science.ru.nl (B. Zwanenburg).
On leave of Bhabha Atomic Centre, Trombay, Mumbai, India.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.07.004

B. Zwanenburg et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5182–5186
5183
O
O
O
O
O
O
O
O
C
S
R
A
B
S
OH
O
O
OH
O
O
D
R
O
O
O
(+)-(3a ,5 ,8b ,2' )-strigol (1)
R
S
S
R
(-)-3aR,4R,8bR,2'R-orobanchol (2)
3aR*,8aS*,2'R*-GR 24 (3)
O
O
O
O
O
O
O
O
N
N
S
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
derived from
tetralone (5)
derived from
hydroxycoumarin (6)
derived from
saccharin (7)
Nijmegen-1 (4)
Figure 1. Structures of some natural SLs, analogue GR24 and newly designed SL analogues.
Stereochemistry
is (very) important
of the leaving group L. In the SL mimics a benzoate and a saccha-
ride ion serve as L.
In this Letter we provide supporting evidence for this mode of
action. By replacing the hydrogen atom at C-4 by a methyl group
a modified SL mimic is obtained in which the essential proton
transfer cannot take place anymore and accordingly these modified
SL mimics are predicted to be inactive as germinating agents.
The synthesis of such C-4 methyl containing SL mimics is
straightforward. A set of 4 SL mimics with a C-4 hydrogen and 3
mimics with a C-4 methyl was prepared as shown in Figure 6.
The SL mimics 9 were readily obtained by a coupling of bromo-but-
enolide with an appropriate sodium carboxylate.30 Coupling of a
O
O
O
C
*
*
B
R
A
O
D
*
O
A-ring modifications
hardly affect activity
Me
α,β-unsaturated system
and D-ring are essential
Figure 2. Model for the design of new bioactive SL analogues with germinating
suitable acid chloridewith 3,4-dimethyl hydroxy butenolide in
activity.
32
the presence of pyridine resulted in the SL mimics 11.
There
O
O
O
O
O
.
O
.
.
.
Asp217
O
.
.
HO
O
O
O
O
H
H
O
.
.
His246
Ser96
O
.
O
.
elimination
.
+
.
.
H
addition
.
.
.
OH
Formyl-ABC fragment
O
O
HO
O
O
D-ring fragment
Water is bound in a bidentate fashion. His246 base abstracts a proton and induces a Michael
additon of water to the enol ether bond, followed by elimination of the D-ring
Figure 3. Proposal for the hydrolysis mechanism of SLs in the catalytic triad of Ser96-His246-Asp217
.
O
O
O
O
O
O
O
NC
O
O
5
N
4
X
S
5
O
O
O
saccharin-butenolide (8)
5-benzoyloxy-butenolides (9)
a: X = H; b: X = o-OH
5-p-cyanophenoxy-butenolide (10)
Figure 4. SL mimics.

5184
B. Zwanenburg et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5182–5186
enolide ring, is the only conceivable reactive functionality that can
O
O
O
O
L
O
L
OH
L
O
Nu
serve as bioactiphore, whereby substituents and functional groups
are decisive for the bioactivity. This is exemplified by the SL mim-
ics describedin this Letter.
In this preliminary study only two types of seeds have been bio-
assayed. It is well known that the response of seeds of different
parasitic weeds to SL analogues can be quite different.18,39 That
possibly may also be the case for SL mimics.
4
H
Nu
Nu
L = saccharin anion
benzoate anion
p-cyanophenoxy anion
O
OH
O
L-
The new SL mimics 9 are of interest for the reduction of seeds
banks of parasitic weeds using the concept of suicidal germina-
tion.9,10 It is well documented that parasitic weeds are causing se-
vere damage to important food crops especially in African
countries.40 Reduction of seeds banks of these weeds is an option
to eradicate these noxious parasites.9,10,40,41,42
The results described above demonstrate that small changes in
the molecular structure of a SL mimic may have an enormous im-
pact on the bioactivity.
Nu
Nu
Figure 5. Mode of action proposed for SL mimics.
are a good reasons to believe that the SL mimics 9 and 11 are suf-
ficiently stable in aqueous solution. It was shown previously that
enantiopure 4-methyl-5-oxo-2,5-dihydro-2-furanyl acetate (SL
mimic 9a with an acetate instead of a benzoate) retains its optical
activity in phosphate buffers of pH 6.7 and 8.34 In addition, aque-
ous 30 lM solution of 9c and 11d were monitored with time using
Acknowledgments
HPLC. No change was observed after 5 days; hence, untimely
hydrolysis of these SL mimics does not occur. The mimics 9d and
11c slowly hydrolyze with an estimated half life of 3 days.35
These seven compounds were assayed as germinating agents
for seeds of Striga hermonthica, Phelipanche ramosa (France) and
P. ramosa (Italy), using GR 24 as positive and water as negative
control.36, 37 The results of these bioassays are collected in Figure 7.
The SL mimics 9a, b, c and d show a good response for Striga seeds
although the required concentrations are higher than needed for
germination of both P. ramosa’s. The activity of 9d is different for
thetwo types of ramosa’s for an unknown reason. The data clearly
demonstrate that the 3,4-dimethyl butenolides 11a, 11b and 11c
are practically inactive at concentrations at which the SL mimics
with one methyl group at C-3 only are highly active. This observa-
tion is in full accordance with the prediction on the basis of the
mode of action shown in Figure 5. The SL mimic29 10 also fits in
this picture. It should be noted that mimics in which the cyano
group in 10 is replaced by a weaker electron-withdrawing group
are inactive towards S. hermonthica.29
There is a considerable differencebetween SL analogues having
an enol ether unit and SL mimics that lack such a unit. When an ex-
tra methyl group is introduced in the butenolide ring in analogue
Nijmegen-1 (4) the germination activity is hardly affected,38
whereas an extra methyl in mimics 9 results in a dramatic loss
of activity. This difference in behavior strongly suggests that ana-
logues and mimics have different receptor sites as is reflected by
their different modes of action. The SL mimics cannot undergo a
hydrolytic process as described above for SL analogues. Conse-
quently, the bioactiphore for SL analogues and SL mimics must
be different. For SL mimics the unsaturated lactone, that is, the but-
S.K.N. is grateful for the special leave enabling him to take part
in this project. We thank Yanxia Zhang and Xie Cheng (Wagenin-
gen Univ.) for skillfully performing the bioassays.
References and notes
1. Tsuchiya, Y.; McCourt, P. Curr. Opin. Plant Biol. 2009, 12, 556.
2. Chen, C.; Zou, J.; Zhang, S.; Zaitlin, D.; Zhu, L. Sci. China, Ser. C 2009, 52, 693.
3. Bouwmeester, H. J.; Matsusova, R.; Zhongkui, S.; Beale, M. H. Curr. Opin. Plant
Biol. 2003, 6, 358.
4. Bouwmeester, H. J.; Roux, R.; Lopez-Raez, J. A.; Bécard, G. Trends Plant Sci. 2007,
12, 224.
5. Vurro, M.; Yoneyama, K. Pest Manag. Sci. 2012, 68, 664.
6. Yoneyama, K.; Xie, X.; Yoneyama, K.; Takeuchi, Y. Pest Manag. Sci. 2009, 65, 467.
and references therein.
7. Xie, X.; Yoneyama, K.; Yoneyama, K. Annu. Rev. Phytopathol. 2010, 48, 93.
8. Yoneyama, K.; Awad, A. A.; Xie, X.; Yoneyama, K.; Takeuchi, Y. Plant Cell Physiol.
2010, 51, 1095.
9. Zwanenburg, B.; Mwakaboko, A. S.; Reizelman, A.; Anilkumar, G.;
Sethumadhavan, D. Pest Manag. Sci. 2009, 65, 478.
10. Zwanenburg, B.; Pospišil, T. Mol. Plant 2013, 6, 38.
11. Akiyama, K.; Matsuzaki, K.; Hayashi, H. Nature 2005, 435, 824.
12. Akiyama, K.; Ogasawara, S.; Ito, S.; Hayashi, H. Plant Cell Physiol. 2010, 51, 1104.
13. 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.
14. 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.-Ch.; Bouwmeester, H. J.;
Bécard, G.; Beveridge, C. A.; Rameau, C.; Rochange, S. F. Nature 2008, 455, 189.
15. Boyer, F.-D.; de Saint Germain, A.; Pillot, J.-P.; Pouvreau, J.-B.; Chen, V. X.;
Ramos, S.; Stévenin, A.; Simier, P.; Delavault, P.; Beau, J.-M.; Rameau, C. Plant
Physiol. 2012, 159, 1524.
16. (a) Cook, C. E.; Whichard, L. P.; Turner, B.; Wall, M. E.; Egley, G. H. Science 1966,
154, 1189; (b) Cook, C. E.; Whichard, L. P.; Wall, M. E.; Egley, G. H.; Coggon, P.;
Luhan, P. A.; McPhail, A. T. J. Am. Chem. Soc. 1972, 94, 6189.
O
O
O
O
DMF
O
O
Br
O
ONa
+
+
X
X
O
5
4
X
H
X
9 a: X=H; b: X=o-OH; c: X=
X
p-OMe; d: X=p-NO2
O
O
O
O
pyridine
CH2Cl2
HO
O
Cl
O
5
4
X
X
X
11 a: X=H; b: p-OMe; c: X=p-NO2
Figure 6. Synthesis of SL mimics.

B. Zwanenburg et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5182–5186
5185
60%
50%
40%
30%
20%
10%
0%
3x10-3
3x10-2
3x10-1
3.0
30
9a
9b
9c
9d
11a
11b
11c
GR24 water
-10%
90
80
70
60
50
40
30
20
10
0
3x10-7
3x10-6
3x10-5
3x10-4
3x10-3
3x10-2
3x10-1
3.0
9a
9b
9c
9d
11a
11b
11c
GR24
water
-10
80.0
70.0
60.0
50.0
40.0
30.0
20.0
10.0
0.0
3x10-7
3x10-6
3x10-5
3x10-4
3x10-3
3x10-2
3x10-1
3.0
9a
9b
9c
9d
11a
11b
11c
GR24
water
-10.0
Figure 7. Bioassays of Striga hermonthica and Phelipanche ramosa collected in France and in Italy. Seed germination induced by SL mimics 9a, 9b, 9c, 9d, 11a, 11b and 11c in
the concentration range 3 ꢁ 10ꢀ7, 3 ꢁ 10ꢀ6, 3 ꢁ 10ꢀ5, 3 ꢁ 10ꢀ4, 3 ꢁ 10ꢀ3, 3 ꢁ 10ꢀ2, 3 ꢁ 10ꢀ1, 3.0 and 30
positive and negative control, respectively (n = 6). Bars represent means s.e.
lM. The SL analogue GR24 and dematerialized water were used as
17. This is the revised structure18 of this SL in root exudate of Red Clover. It is a
stereoisomer of the originally proposed (3aS,4S,8bS,20R)-orobanchol.19
18. Ueno, K.; Nomura, S.; Muranaka, S.; Mizutani, M.; Takikawa, H.; Sugimoto, Y. J.
Agric. Food Chem. 2011, 59, 10485.
Takeuchi, Y. Tetrahedron Lett. 1999, 40, 943; (c) Matsui, J.; Yokota, T.; Bando,
M.; Takeuchi, Y.; Mori, K. Eur. J. Org. Chem. 1999, 2201; (d) See also Ref. 10.
20. Johnson, A. W.; Gowda, G.; Hassanali, A.; Knox, J.; Monaco, S.; Razavi, Z.;
Roseberry, G. J. Chem. Soc., Perkin Trans. 1 1981, 1734.
19. (a) Yokota, T.; Sakai, H.; Okuno, K.; Yoneyama, K.; Takeuchi, Y. Phytochemistry
1998, 49, 1967; (b) Mori, K.; Matsui, J.; Yokota, T.; Sakai, H.; Bando, M.;
21. For improved syntheses of GR 24, see: (a) Mangnus, E. M.; Dommerholt, F. J.; de
Jong, R. L. P.; Zwanenburg, B. J. Agric. Food Chem. 1992, 40, 1230; (b) Malik, H.;
Rutjes, F. P. J. T.; Zwanenburg, B. Tetrahedron 2010, 66, 7198.

5186
B. Zwanenburg et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5182–5186
22. Mangnus, E. M.; Zwanenburg, B. J. Agric. Food Chem. 1992, 40, 1066.
23. Nefkens, G. H. L.; Thuring, J. W. J. F.; Beenakkers, M. F. M.; Zwanenburg, B. J.
Agric. Food Chem. 1997, 45, 2273.
24. Mwakaboko, A. S.; Zwanenburg, B. Plant Cell Physiol. 2011, 52, 699.
25. For another type of ketone derived SL analogue, see: Bhattacharya, C.;
Bonfante, P.; Deagostino, A.; Kapulnik, Y.; Larini, P.; Occhiato, E. G.; Prandi,
C.; Venturello, P. Org. Biomol. Chem. 2009, 7, 3413.
0.648 g, 82%, colorless tiny crystals, mp 141.8–142.1. 1H NMR (CDCl3,
400 MHz): d 8.38 (d, 2H, J = 8.4 Hz), 8.23 (d, 2H, J = 8.4 Hz), 7.13 (s, 1H), 7.06
(s, 1H), 2.06 (s, 3H); 13C NMR (CDCl3, 100 MHz): d 170.7, 163.0, 151.1, 141.5,
135.1, 133.7, 131.2, 123.7, 93.3, 10.7. m/z 263.04395, calc. for C12H9NO6:
263.04432.
31. Malik, H.; Kohlen, W.; Jamil, M.; Rutjes, F. P. J. T.; Zwanenburg, B. Org. Biomol.
Chem. 2011, 9, 2286.
26. Mwakaboko, A. S.; Zwanenburg, B. Bioorg. Med. Chem. 2011, 19, 5006.
27. Zwanenburg, B.; Mwakaboko, A. S. Bioorg. Med. Chem. 2011, 19, 7394.
28. Hamiaux, C.; Drummond, R. S. M.; Janssen, B. J.; Ledger, S. E.; Cooney, J. M.;
Newcomb, R. D.; Snowden, K. C. Curr. Biol. 2012, 22, 2032.
29. Fukui, K.; Ito, S.; Ueno, K.; Yamaguchi, S.; Kyozuka, J.; Asami, T. Bioorg. Med.
Chem. Lett. 2011, 21, 4905.
32. 3,4-Dimethyl-5-oxo-2,5-dihydro-2-furanyl-benzoate (11a). To a solution of 5-
hydroxy-3,4-dimethylbutenolide33 (0.385 g, 3.0 mmol) and benzoyl chloride
(0.492 g, 3.5 mmol) in dry dichloromethane (10 ml), was added dry pyridine
(0.5 ml) and the mixture was stirred at ambient temperature until all the
butenolide had reacted (3 h, monitored by TLC, heptane/ethyl acetate 7:3). The
mixture was quenched with water, acidified with cold aqueous 1 N HCl and
extracted with ethyl acetate (2 ꢁ 20 ml). The organic layer was washed with
water, brine, dried (Na2SO4) and concentrated in vacuo. The resultant crude
product was crystallized from ethyl acetate/heptane to yield pure 11a as a
colorless solid (0.512 g, 73%), mp 86.7–86.8 °C; 1H NMR (CDCl3, 400 MHz): d
8.06 (m, 2H), 7.49 (m, 1H), 7.45 (m, 2H), 7.00 (s, 1H), 2.04 (s, 3H), 1.92 (s, 3H);
13C NMR (CDCl3, 100 MHz): d 171.6, 165.0, 153.6, 134.0, 130.0, 128.6, 128.4,
127.2, 94.2, 11.4, 8.6. m/z 232.07394, calcd for C13H12O4: 232.07356. 3,4-
Dimethyl-5-oxo-2,5-dihydro-2-furanyl-40-methoxybenzoate (11b). Prepared as
described for 11a starting from hydroxybutenolide (0.385 g, 3.0 mmol) and 4-
methoxybenzoyl chloride (0.546 g, 3.2 mmol). Yield 0.632 g, 80%, colorless tiny
crystals, mp 141.1–141.4. 1H NMR (CDCl3, 400 MHz): d 8.01 (d, 2H, J = 7.2 Hz),
30. Compounds 9a and 9b were also prepared previously.27 3-Methyl-bromo-
2(5H)-furanone. To a solution of 3-methyl-2(5H)-furanone (9.81 g, 0.10 mol) in
dry 1,2-dichloroethane (150 ml) was added N-bromosuccinimide (22.25 g,
0.125 mol) and dibenzoyl peroxide (0.1 g) and the mixture was refluxed under
Dean–Stark conditions. The turbid mixture slowly became clear and turned
brown when the reaction progressed. After completion of the reaction (3 h,
monitored by TLC), the mixture was cooled to ambient temperature and
diluted with heptane (150 ml) whereby succinimide precipitated. The obtained
suspension was passed through Celite and the Celite bed was washed with
heptane/1,2-dichloroethane (50 ml). Removal of solvents in vacuo afforded a
thick yellow oil which was immediately purified by flash chromatography
(silica gel; heptane/ethyl acetate 19:1) to give pure product (12.1 g, yield 68%).
1H NMR (CDCl3, 400 MHz): d 7.22 (s, 1H), 6.86 (s, 1H), 2.03 (s, 3H); 13C NMR
6.98 (s, 1H), 6.93 (d, 2H, J = 7.2 Hz); 73.87 (s, 3H), 2.03 (s, 3H), 1.91 (s, 3H); 13
NMR (CDCl3, 100 MHz): d 171.7, 164.6, 164.2, 153.7, 132.2, 127.0, 120.6, 113.8,
94.0, 55.5, 11.4, 8.5. m/z 262.08439, calcd for 14H14O5: 262.08412. 3,4-
C
(CDCl3, 100 MHz):
d
170.7, 147.6, 130.7, 74.89, 10.5. For an alternative
C
synthesis, see: Ref. 31. 4-Methyl-5-oxo-2,5-dihydro-2-furanyl benzoate (9a). To
a stirred solution of bromobutenolide (0.48 g, 2.71 mmol) in anhydrous DMF
(4 ml) was added solid sodium benzoate (5) (0.325 g, 2.26 mmol) under argon
and the mixture was set aside at room temperature for 18 h. Sodium benzoate
slowly dissolved when the reaction progressed and ultimately a clear solution
was obtained. TLC indicated complete consumption of brombutenolide. The
mixture was concentrated in vacuo, the residue was treated with a mixture of
water (20 ml) and ethyl acetate (2 ꢁ 20 ml), and the combined organic layers
were washed with water, brine, dried (Na2SO4) and concentrated in vacuo. The
resultant crude product was crystallized from ethyl acetate/heptane to afford
pure 9a as colorless tiny crystals (0.395 g, 81%). For physical and spectral data,
see Ref. 27. 4-Methyl-5-oxo-2,5-dihydro-2-furanyl salicylate (9b). Prepared as
described for 9a starting from sodium salicylate (0.448 g, 2.8 mmol). The crude
product was purified by chromatography (silica gel, heptane/ethyl acetate
19:1) to give pure 9b (0.493 g, 70%). For physical and spectral data, see Ref. 27.
Dimethyl-5-oxo-2,5-dihydro-2-furanyl-40-nitrobenzoate (11c). Prepared as
described for 11a starting from hydroxybutenolide (0.385 g, 3.0 mmol) and
4-nitrobenzoyl chloride (0.576 g, 3.1 mmol). Yield 5.34 g, 64%), slightly yellow
crystals, mp 145.1–145.3. 1H NMR (CDCl3, 400 MHz): d 8.33 (d, 2H, J = 7.2 Hz),
8.23 (d, 2H, J = 7.2 Hz), 7.00 (s, 1H), 2.06 (s, 3H), 1.94 (s, 3H); 13C NMR (CDCl3,
100 MHz): d 171.2, 163.3, 152.9, 133.8, 131.7, 131.2, 127.8, 124.1, 123.7, 94.5,
11.5, 8.6. m/z 277.06022, calcd for C13H11NO6: 277.05864.
33. Harrison, T.; Myers, P. L.; Pattenden, G. Tetrahedron 1989, 45, 5247.
34. Wigchert, S. C. M.; Zwanenburg, B. J. Agric. Food Chem. 1999, 47, 1320.
35. It is important to note that the effective contact time of seeds with stimulant
solution is much shorter than the half life of the stimulant in water. The
stimulant, after the diffusion controlled penetration of into the seed, initiates
the germination process ultimately leading to extrusion of a radical. This
relatively slow process takes a few days. The germinated seeds are counted.
36. Solutions of mimics 9 and 11 were obtained by dissolving ca 5 mg (0.02 mmol)
of stimulant in 25 ml of analytical grade acetone. Of this stock solution 1 ml
was diluted with demineralized water in a measuring flask of 25 ml. This
4-Methyl-5-oxo-2,5-dihydro-2-furanyl-40-methoxybenzoate (9c). To
a cooled
(ꢀ50 °C) and stirred suspension of potassium tert-butoxide (0.393 g,
3.5 mmol) in dry DMF (8 ml) a solution of 4-methoxybenzoic acid (0.487 g,
3.2 mmol) in dry DMF(8 ml) was gradually added under argon. After stirring
for 30 min. at room temperature, the mixture was cooled (ꢀ50 °C) and a
solution of bromobutenolide (0.531 g, 3.0 mmol) in DMF (8 ml) was gradually
added. Then the mixture was allow warmed to ambient temperature and
stirred until all bromobutenolide had reacted (3 h, monitored by TLC, heptane/
ethyl acetate 6:4), whereon acetic acid (0.5 ml) was added and the mixture was
concentrated in vacuo. The residue was quenched with water (20 ml) and
extracted with ethyl acetate (3 ꢁ 15 ml). The combined organic layers were
washed with saturated NaHCO3, water, brine, dried (Na2SO4) and then
concentrated in vacuo. The resultant crude product was purified by flash
chromatography (silica gel, heptane/ethyl acetate 19:1) to give pure 9c
(0.512 g, 69 % yield), mp 103.2–103.6. 1H NMR (CDCl3, 400 MHz): d 8.06 (d,
2H, J = 8.4 Hz), 7.11 (m, 1H), 7.02 (m, 1H), 6.93 (m, 2H), 3.87 (s, 3H), 2.03 (s,
3H); 13C NMR (CDCl3, 100 MHz): d 171.2, 164.4, 164.2, 142.4, 134.4,132.2,
120.6, 113.9, 92.9, 55.5, 10.7. m/z 248.06833, calc. for C13H12O5: 248.06847. 4-
solution of 30 lM was further diluted to the indicated test concentrations
(Fig. 7). Bioassays were performed using standard protocols.37 Germinated
Striga seeds were counted after 48 h of incubation at 30 °C. Pelipanche seeds
were incubated for 5 days at 25 °C and then germinated seeds were counted.
The batch of seeds of P. ramosa (Italy) was probably not homogeneous,
although no anomalies were observed during visual inspection. At lower
concentrations the response was too low for 9a and 9b.
37. (a) Mangnus, E. M.; Stommen, P. L. A.; Zwanenburg, B. J. Plant Growth Regul.
1992, 11, 91; (b) Ref.24; (c) Matusova, R.; Rani, K.; Verstappen, F. W. A.;
Franssen, M. C. R.; Beale, M. H.; Bouwmeester, H. J. Plant Physiol. 2005, 139, 920.
38. Thuring, J. W. J. F.; Bitter, H. H.; de Kok, M.; Nefkens, G. H. L.; van Riel, A. M. D.
A.; Zwanenburg, B. J. Agric. Food Chem. 1997, 45, 2284.
39. Fernández-Aparicio, M.; Yoneyama, K.; Rubiales, D. Seed Sci. Res. 2011, 21, 55.
40. (a) Parker, C. Weed Sci. 2012, 60, 269; (b) Parker, C. Pest Manag. Sci. 2009, 65,
453.
41. Johnson, A. W.; Roseberry, G.; Parker, C. Weed Res. 1976, 16, 223.
42. Rubiales, D.; Fernández-Aparicio, M.; Wegmann, K.; Joel, D. Weed Res. 2009, 49,
23 (Special issue).
Methyl-5-oxo-2,5-dihydro-2-furanyl-40-nitrobenzoate
described for 9c starting from p-nitrobenzoic acid (0.535 g, 3.2 mmol). Yield
(9d).
Prepared
as