Single step synthesis of strigolactone analogues from cyclic keto enols, germination stimulants for seeds of parasitic weeds

Article abstract of DOI:10.1016/j.bmc.2011.06.057

The single step synthesis of a newly designed series of strigolactones (SLs) from cyclic keto enols is described. The germinating activity of these SL analogues towards seeds of the parasitic weeds Striga and Orobanche spp. is reported. The first of these

Full text of DOI:10.1016/j.bmc.2011.06.057

Bioorganic & Medicinal Chemistry 19 (2011) 5006–5011
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Single step synthesis of strigolactone analogues from cyclic keto enols,
germination stimulants for seeds of parasitic weeds
⇑
Alinanuswe S. Mwakaboko , Binne Zwanenburg
Department of Organic Chemistry, Institute for Molecules and Materials, Radboud University Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
The single step synthesis of a newly designed series of strigolactones (SLs) from cyclic keto enols is
Received 10 May 2011
Revised 17 June 2011
Accepted 18 June 2011
Available online 25 June 2011
described. The germinating activity of these SL analogues towards seeds of the parasitic weeds Striga
and Orobanche spp. is reported. The first of these SL analogues are derived from the hydroxyl
c-pyrones
kojic acid and maltol, the second type from hydroxyl -pyrones, namely, 4-hydroxy-6-methyl-2H-pyran-
a
2-one and 4-hydroxy-coumarin and the third type from 1,3-diketones, namely, 1,3-cyclohexane-dione
(dimedone) and tricyclic 1,3-dione. All keto enols are coupled in a single step with the appropriate D-ring
precursor in the presence of a base to give the desired SL analogues. All SL analogues are acceptably
biologically active in inducing the germination of seeds of Striga hermonthica and Orobanche cernua. Most
interesting are the analogues derived from 4-hydroxy coumarin and dimedone, as they have a remark-
ably high biological activity towards the seeds of parasitic weeds at relatively low concentrations, com-
parable with that of the general standard stimulant GR24.
Keywords:
Strigolactone
Parasitic weeds
Bioassay
Striga
Orobanche
Germination
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Natural stimulants can only be obtained from plant roots in
minute amounts. Their structures are too complicated for a gram
Important food crops such as maize, sorghum and millet and
rice in third world countries are enormously damaged by the par-
asitic weeds, Striga and Orobanche spp.1,2 The seeds of these weeds
germinate by a chemical signal which is present in the root exu-
dates of the host plants. The concentration of these stimulants in
root exudates is extremely low, which hampers their isolation
and characterization.3–9 These compounds which are collectively
named as strigolactones (SLs), are structurally similar, all having
four rings, as shown in the formula of the first isolated stimulant,
strigol (1)3 (Fig. 1).
Extensive structure–activity studies revealed that the bioacti-
phore for germination resides in the C–D part of these mole-
cules.10–18 A tentative molecular mechanism12 for understanding
the interaction of the stimulant molecule in the receptor site in-
volves an addition–elimination reaction initiated by a Michael
addition of a nucleophilic group as is depicted in Scheme 1.
The working model for the design of stimulant molecules
shown in the Figure 2, is constructed by combining the structural
information of the bioactiphore and the features of the molecular
mechanism.13,18
scale synthesis.19 Therefore, there has been a continuous search
for structurally simplified strigolactones that can be synthesized
relatively easy. The first series developed was the GR family,10,11
typically represented by GR 24 (2) and GR 7 (3). SL analogues lack-
ing the B-ring16 or having a modified C-ring16 have been described.
Also analogues with an imino bioisosteric unit20 replacing the enol
ether and with a heterocyclic ABC moiety21 have been reported.
Nijmegen-122 which has an appreciable stimulant activity has been
designed on the basis of the working model. This molecule con-
tains all essential structural elements for activity, namely, the D-
ring which is invariably present in all natural strigolactones, and
the enone moiety necessary for the addition–elimination reaction
to take place. It has been argued that the molecular mechanism
shown in Scheme 1 is not appropriate for imino bioisosters.20 How-
ever, recently is was shown that these compounds can also be fit-
ted into this mechanism.23 In a previous paper,23 we designed a
series of cyclic ketone derived SL analogues with considerable
stimulant activity. The analogue 5 obtained from 1-tetralone has
an activity comparable to that of GR 24, the world wide used stan-
dard stimulant.10,24
This paper deals with the design of a new series of SL analogues
derived from readily available starting materials, all being cyclic
keto enols. The enol is then coupled with the D-ring in a single
step, thereby giving structures containing the structural elements
required for activity, namely the enone, the vinyl ether and the
D-ring unit. The prime objective of this work is to check the
⇑
Corresponding author. Tel.: +31 24 3653159.
E-mail address: B.Zwanenburg@science.ru.nl (B. Zwanenburg).
Present address: Department of Chemistry, University of Dar es Salaam, PO Box
35061, Dar es Salaam, Tanzania.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.057

A. S. Mwakaboko, B. Zwanenburg / Bioorg. Med. Chem. 19 (2011) 5006–5011
5007
O
O
O
O
O
O
O
O
C
A
B
O
O
O
O
D
OH
O
O
O
3
GR 7 ( )
(+)-Strigol (1)
GR 24 (2)
O
O
O
OMe
O
O
N
O
O
O
O
O
5
4)
Nijmegen-1 (
Figure 1. Some natural and synthetic strigolactones.
O
O
O
O
O
+
O
Nu
Nu
O
Nu
O
O
O
O
O
O
O
O
O
O
O
Scheme 1. Molecular mechanism for the initiation of germination.12
SL analogue 9 in 37% yield. Structural analysis revealed that cou-
pling had taken place at the enolic hydroxyl group (Scheme 2).
Stereochemistry
is very important
The second hydroxy c-pyrone chosen was maltol 10. This start-
O
O
O
C
ing material is naturally occurring and can be isolated from pine
needles and larch bark. The coupling of maltol (10) with bromo
butenolide 7 using potassium carbonate as the base resulted in
the desired crystalline compound 11 in 65% yield (Scheme 3).
A
B
R
O
D
O
Two hydroxy a-pyrones, both having the required enolone unit,
A-ring modifications
hardly affect activity
were included in this study, namely the commercially available, 4-
hydroxy-6-methyl-2H-pyran-2-one (12) and 4-hydroxy-coumarin
(14). The coupling with halo butenolide in the presence of potas-
sium carbonate afforded in a single step the desired SL analogues
13 and 15, respectively, in good yields (Schemes 4 and 5). The
structures of these products were ascertained by spectroscopic
analysis.
-unsaturated system
and D-ring are essential
Figure 2. The working model for designing stimulant molecules.18
The third type of enol ketones constitutes the 1,3-diketones 16
and 18 (dimedone) and the rather unusual tricyclic 1,3-dione 20.
All three diketones are predominantly present in the enol form.
Coupling with the D-ring precursor in a single step proceeded
smoothly in the same manner described above to give the desired
SLs shown in the Schemes 6 and 7.
validity of the working model for designing new synthetically
readily accessible stimulant molecules. When sufficiently active
the new stimulants may be candidates for the use in parasitic weed
control applying the concept of suicidal germination,18,25–27 that is
treatment of seeds of the parasite in the absence of a suitable host.
Due to lack of nutrients and water, the germinated seeds will die
after a few days. We already demonstrated that Nijmegen-1 can
successfully be used for this purpose.18
The yields for the coupling reactions have not been optimized.
At this stage of the work, the bioactivity is the most relevant issue.
O
O
O
2. Results and discussion
OH
O
O
K2CO3, DMF
O
2.1. Synthesis of new SL analogues
HO
HO
O
O
Br
O
6
9
The first keto enol that was selected for this project is the hy-
droxyl
c-pyrone 6 (Kojic acid) which is commercially available
7
for a reasonable price. Coupling of 6 with bromo butenolide 7
was achieved by using potassium carbonate as the base, giving
Scheme 2. Conversion of Kojic acid into SL analogue 9.

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A. S. Mwakaboko, B. Zwanenburg / Bioorg. Med. Chem. 19 (2011) 5006–5011
2.2. Germination activity of the newly prepared SL analogues
O
O
O
OH
O
O
K2CO3, DMF
O
Seeds of Striga hermonthica (from Sorghum bicolor (L.) Moench)
were harvested in Sudan in 1987. Orobanche cernua seeds were
harvested in Spain in 1994 and were stored in the dark at room
temperature until required. The bioassay experiments were carried
out according to the standardized procedure.28 An aqueous solu-
tion of acetone (0.1% v/v) was used as a negative control and the
diastereomeric mixture of GR 24 (2) as a positive control. The re-
sults of the bioassays with S. hermonthica are shown as bar graph
representations in Figure 3. The germination activities towards O.
cernua seeds are presented in Figure 4.
The bioassays of the newly prepared SL analogues 9, 11, 13 and
15 reveal that all these compounds have an appreciable activity in
stimulating the germination of Striga seeds. Analogues 13 and 15
give the best performance. The analogue 19 derived from dime-
done has a very good activity, in fact comparable with that of GR
24. Remarkably, the analogue 21 from tricyclic dione is also very
active. The response of the newly prepared stimulants towards
Orobanche seed shows a very similar profile. In general, the
dose–response curve of the best performing newly prepared stim-
ulants may have an optimum at a higher concentration than GR 24.
The most promising stimulants for Striga and Orobanche seeds that
came out of this study are the SL analogues 13 derived from 4-
hydroxy-6-methyl-2H-pyran-2-one, 15 obtained from 4-hydroxy-
coumarin and 19 prepared from dimedone, the latter being the
best. Especially, SL analogue 19 from dimedone is a potential can-
didate for parasitic weed control using the concept of suicidal
germination.
O
7
10
11
Scheme 3. Synthesis of SL analogue 11 from maltol.
O
O
O
O
O
O
K2CO3, DMF
7
OH
12
13
O
Scheme 4. Synthesis of SL analogue 13 from 4-hydroxy-6-methyl-2H-pyran-2-one.
O
O
O
O
O
O
K2CO3, DMF
O
Cl
O
OH
14
8
15
O
Scheme 5. Single step conversion of hydroxy coumarin into SL analogue 15.
2.3. Discussion
O
O
O
All new strigolactone analogues derived from cyclic keto enols
were prepared in a single step by coupling the selected keto enols
with the halo butenolide in the presence of a base. All these com-
pounds have appreciable germination activity towards seeds of
Striga and Orobanche, hence the working model18 for designing
new stimulants (Fig. 2) is proven to be valid. The analogues 9
and 11 are the least active ones, particularly for Orobanche seeds.
This may be attributed to the fact that these structures are in fact
cross-conjugates which necessitates the addition–elimination
mechanism (see Scheme 1) to take a slightly deviant course. This
implies that 9 and 11 are not ideally suited for the molecular mod-
el. Among these newly prepared stimulants, the most promising
ones are those prepared from hydroxy coumarin and dimedone:
SL analogues 15 and 19, respectively. It should be noted that espe-
cially the activity towards Orobanche seeds is remarkably high. The
best performing stimulants are synthetically readily accessible in a
single step synthetic operation. The application protocol for the
suicidal germination approach in combating plagues of parasitic
K2CO3,DMF
O
8
R
O
R
O
R
R
16
17
R = Me 19
R = H
R = Me 18
R = H
Scheme 6. SL analogues derived from cyclohexane-1,3-diones.
O
O
K2CO3, DMF
8
O
OH
O
O
20
21
Scheme 7. An SL analogue derived from a tricyclic 1,3-dione.
Figure 3. Bar graph representation of percentages of the germinated seeds of S. hermonthica after exposure to various concentrations of analogues 9, 11, 13, 15 17, 19 and 21
with GR 24 as a positive control.

A. S. Mwakaboko, B. Zwanenburg / Bioorg. Med. Chem. 19 (2011) 5006–5011
5009
Figure 4. Bar representation of the percentages of the germinated seeds of O. cernua after exposure to various concentrations of analogues 9, 11, 13, 15 17, 19 and 21 with GR
24 as a positive control.
weeds involves formulation of the stimulants as an emulsion in or-
der to avoid untimely decomposition in the soil and slowing down
of the leaching of the stimulants to lower soil layers where it will
umn (25 m) HP-1 with nitrogen (2 mL/min, 0.5 atm) as the carrier
gas. Thin layer chromatography (TLC) was carried out on Merck
pre-coated silica gel 60 F254 plates (0.25 mm) using the eluents
indicated. Spots were visualized using a UV lamp, or with a potas-
sium dichromate spray (prepared from 7.5 g of K2Cr2O7 in 250 mL
H2O containing 12.5 mL 1 M H2SO4) followed by heating at 140 °C.
Column chromatography was performed on silica gel (Kieselgel,
Merck) using eluents indicated. All solvents were dried under stan-
dard conditions. Solvents like DMF, DME and THF were distilled
using standard purification procedures.
not be effective. In addition, factors such as bioavailability18,29
,
optimum concentration18,30 of stimulant and optimum stimulation
time18,29 have to be taken into account. The idea of suicidal germi-
nation was coined for the first time as early as 197625 and applied
in soil boxes using aqueous solution of the GR 7, but discontinued
due to untimely decomposition of the stimulant. Several discour-
aging reports31–33 about the soil stability of GR 24 and GR 7 ap-
peared and therefore, the suicidal approach was not considered
as vey realistic.26,27 Recently, this approach was reconsidered using
formulated Nijmegen-1 for the reduction of seed banks of Oroban-
che ramosa in tobacco.18 The results are very encouraging. Hence,
this is a stimulus for the design and synthesis of new SL analogues
with high germinating activity. This paper constitutes a contribu-
tion to this objective.
The halo butenolides 7 and 8 were prepared as previously
described.37,38
Nomenclature. The IUPAC nomenclature has been used for all
compounds. The systematic names were generated using the
ACD/Name programme provided by Advanced Chemistry Develop-
ment Inc. (Toronto, Canada).
It is relevant to note that SLs recently received much attention
in the literature because of the newly discovered bioproperties. SLs
have been identified as the branching factor for arbusular mycor-
rhizal (AM) fungi34 and as inhibitor of plant shoot branching.35 It
has been suggested that SLs constitute a new class of plant hor-
mones36 for which more functions will be uncovered in the coming
years. It was found that GR 24 is also active for both functions.
Therefore, searching for other SL analogues for the new activities
is another incentive for this study. It remains to be seen whether
the some model compound as shown in Figure 2 is applicable for
these new activities.
3.1.1. 2-(Hydroxymethyl)-5-[(4-methyl-5-oxo-2,5-dihydro-2-
furanyl)oxy]-4H-pyran-4-one (9)
A stirred mixture of 5-hydroxy-2-(hydroxymethyl)-4H-pyran-
4-one (6) (400.0 mg, 2.82 mmol), anhydrous potassium carbonate
(428.2 mg, 3.10 mmol) and bromo butenolide
7 (548.3 mg,
3.10 mmol) in dry DMF (4 mL), while maintained under nitrogen,
was set aside at room temperature for 18 h until TLC indicated
the total consumption of 7. The mixture was treated with a mix-
ture of water (50 mL) and dichloromethane (25 mL), the separated
aqueous layer was extracted with dichloromethane (2 Â 15 mL),
the combined organic layers were washed with brine, dried
(Na2SO4), filtered and then concentrated in vacuo. The resultant
crude product was purified by column chromatography (hexane/
ethyl acetate, 1:1, v/v) to give 9, 245.7 mg (36.6%) as a white solid.
An analytical sample was obtained by recrystallization from di-iso-
propyl ether, mp 91–94 °C; 1H NMR (CDCl3, 300 MHz): d 7.93 (s,
1H, OCH@CO), 7.09 (s, 1H, @CH), 6.58 (s, 1H, @CH), 6.37 (s, 1H,
OCHO), 4.51 (s, 2H, CH2), 3.10 (s, 1H, OH), 1.97 (s, 3H, CH3). 13C
NMR, (CDCl3, 100 MHz): d 174.6 (C@O), 171.2 (C@O), 167.8,
147.5, 144.0, 142.4, 134.6, 113.3, 99.3, 60.7, 10.6; MS [EI m/z, rel.
intensity (%)]: 239 ([M+1]+, 1.1); 238 ([M]+, 6.6); 97 ([M+À141],
[C5H5O2]+, 100); Anal. Calcd for C11H10O6: C, 55.47; H, 4.23. Found:
C, 55.35; H, 4.17.
3. Experimental section
3.1. Synthesis
General remarks. IR spectra were recorded using a Perkin-Elmer
298 infrared spectrophotometer and a Bio-Rad FTS-25 instrument.
1H NMR spectra were recorded on a Bruker AC 100 spectrometer
(100 MHz), AC 300 (300 MHz), and a Bruker AM-400 (400 MHz),
respectively, using Me4Si (TMS) as the internal standard. 13C
NMR spectra were recorded on a Bruker AC 300 (operating at
75 MHz) and AM 400 (operating at 100 MHz) spectrometers with
CDCl3 (77.0 ppm), acetone-d6 (29.206 and 206 ppm) as standards.
Melting points were determined with a Reichert Thermopan
microscope and are uncorrected. Elemental analyses were con-
ducted on a Carlo-Erba instruments CHNSO EA 1108 elemental
analyzer. Mass spectra were recorded using a double focussing
VG 7070E mass spectrometer in the mode indicated, or a Varian
Saturn 2 GC–MS ion-trap system. GC–MS separations were carried
out on a fused-silica capillary column (DB-5, 30 m  0.25 mm) and
helium was used as the carrier gas. GLC was conducted with a
Hewlet–Packard HP 5890 gas chromatograph using a capillary col-
3.1.2. 2-Methyl-3-[(4-methyl-5-oxo-2,5-dihydro-2-furanyl)oxy]-
4H-pyran-4-one (11)
A stirred mixture of 3-hydroxy-2-methyl-4H-pyran-4-one (10)
(200.0 mg, 1.59 mmol), anhydrous potassium carbonate (241.3
mg, 1.75 mmol) and bromo butenolide 7 (309.0 mg, 2.33 mmol)
in dry DMF (2 mL) was maintained under nitrogen at room temper-
ature for 5 h and then processed in the manner described above.
The crude product was purified by column chromatography (hex-
ane/ethyl acetate, 1:1, v/v) (306.7 mg (65%) and then

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A. S. Mwakaboko, B. Zwanenburg / Bioorg. Med. Chem. 19 (2011) 5006–5011
recrystallized (di-isopropyl ether) to give 11 as white crystals,
207.8 mg, mp 104 °C; 1H NMR (CDCl3, 300 MHz): d 7.68 (d, 1H,
J = 5.7 Hz, CH@), 7.14 (m, 1H, CH@), 6.48 (m, 1H, OCHO), 6.41 (d,
1H, J = 5.7 Hz, @CH), 2.35 (s, 3H, CH3), 1.99 (s, 3H, CH3). 13C NMR
(CDCl3, 100 MHz): d 173.9 (C@O), 171.5 (C@O), 161.3, 154.1,
143.0, 141.8, 134.1, 117.3, 100.3, 15.2, 10.6; MS [EI m/z, rel. inten-
sity (%)]: 222 ([M]+), 8.9; 223 ([M]++1), 0.1; 126 ([M]+À97,
[C6H5O3]+), 44.5; 97 ([M+À125], [C5H5O2]+), 100); Anal. Calcd for
was purified by column chromatography (hexane/ethyl acetate,
1:1, v/v) to give compound 19 as white crystals, 78.5 mg (14.5%).
An analytical sample was obtained by recrystallization from di-
isopropyl ether, mp 96–99 °C; 1H NMR (CDCl3, 400 MHz): d 6.94
(s, 1H, CH@), 6.24 (s, 1H, OCHO), 5.73 (s, 1H, @CH), 2.30 (s, 2H,
CH2), 2.26 (s, 2H, CH2), 2.02 (s, 3H, CH3), 1.09 (s, 6H, 2 Â CH3).
13C NMR (CDCl3, 100 MHz): d 199.0 (C@O), 172.4 (C@O), 170.5,
141.2, 134.9, 105.1, 95.9, 50.6, 42.1, 32.5, 28.5, 27.9, 10.6; MS [EI
m/z, rel. intensity (%)]: 237 ([M+1]+, 1.0); 236 ([M]+, 8.9); 97
([M+À139], [C5H5O2]+, 100); Anal. Calcd for C13H16O4: C, 66.09; H,
6.83. Found: C, 66.11; H, 6.71%.
Method B. A stirred mixture of 5,5-dimethyl-1,3-cyclohexanedi-
one (18) (300.0 mg, 2.14 mmol), DBU (358.8 mg, 2.36 mmol) and
chloro butenolide 8 (283.9 mg, 2.14 mmol) in dichloromethane
(15 mL) while maintained under nitrogen was set aside at room
temperature for 5 h, until TLC indicated the total consumption of
the chloro butenolide 8. The mixture was processed in the manner
described above. Column chromatography (hexane/ethyl acetate,
2:1, v/v) gave compound 19 as white crystals, 73.3 mg (14%), mp
98–100 °C; 1H NMR (CDCl3, 400 MHz): d 6.94 (s, 1H, CH@), 6.24
(s, 1H, OCHO), 5.73 (s, 1H, @CH), 2.30 (s, 2H, CH2), 2.26 (s, 2H,
CH2), 2.02 (s, 3H, CH3), 1.09 (s, 6H, 2 Â CH3). 13C NMR (CDCl3,
100 MHz): d 199.0 (C@O), 172.4 (C@O), 170.5, 141.2, 134.9,
105.1, 95.9, 50.6, 42.1, 32.5, 28.5, 27.9, 10.6; MS [EI m/z, rel. inten-
sity (%)]: 237 ([M+1]+, 1.0); 236 ([M]+, 8.9); 97 ([M+À139],
[C5H5O2]+, 100); Anal. see above.
C11H10O5: C, 59.46; H, 4.54. Found: C, 59.22; H, 4.41.
3.1.3. 6-Methyl-4-[(4-methyl-5-oxo-2,5-dihydro-2-furanyl)oxy]-
2H-pyran-2-one (13)
Treatment of 4-hydroxy-6-methyl-2H-pyran-2-one (12)
(200.0 mg, 1.59 mmol) with anhydrous potassium carbonate
(241.3 mg, 1.75 mmol) and bromo butenolide
7 (309.0 mg,
2.33 mmol) in dry DMF (4 mL) in the manner described above, fol-
lowed by column chromatography (hexane/ethyl acetate, 2:1, v/v)
gave 13 (121.4 mg, 58.8%). Recrystallization (di-isopropyl ether)
gave an analytically pure sample (107.9 mg) as white crystals,
mp 125–127 °C; 1H NMR (CDCl3, 400 MHz): d 6.97 (s, 1H, CH@),
6.31 (s, 1H, OCHO), 5.84 (s, 1H, CH@CO), 5.77 (s, 1H, CH@C), 2.24
(s, 3H, CH3), 2.02 (s, 3H, CH3). 13C NMR (CDCl3, 100 MHz): d
170.1 (C@O), 167.7 (C@O), 163.8, 163.4, 140.8, 135.3, 99.4, 96.2,
91.5, 20.0, 10.6; MS [EI m/z, rel. intensity (%)]: 222 ([M]+, 7.6);
193 ([M+À29], 2.8, [C10H10O4]+); 97 ([M+À125], [C5H5O2]+, 100);
Anal. Calcd for C11H10O5: C, 59.46; H, 4.54. Found: C, 59.42; H, 4.29.
3.1.4. 4-[(4-Methyl-5-oxo-2,5-dihydro-2-furanyl)oxy]-2H-
chromen-2-one (15)
3.1.7. 3-Methyl-5-[(5-oxotricyclo[5.2.1.02,6]deca-3,8-dien-3-
yl)oxy]-2(5H)-furanone (21)
The reaction of 4-hydroxy-coumarin (14) (400.0 mg,
2.47 mmol) with anhydrous potassium carbonate (412.5 mg,
2.71 mmol) and chloro butenolide 8 (359.6 mg, 2.71 mmol) in
dry DMF (4 mL) was conducted in the manner described above, fol-
lowed by recrystallization from di-isopropyl ether to give 15,
315.4 mg (49.5%) as pure yellowish crystals, mp 177–182 °C; 1H
NMR (CDCl3, 400 MHz): d 7.74–7.27 (m, 4H, ArH protons), 7.11
(m, 1H, CH@), 6.49 (s, 1H, @CHCO), 6.11 (s, 1H, OCHO), 2.09 (s,
3H, CH3). 13C NMR, (CDCl3, 100 MHz): d 170.0 (C@O), 163.0
(C@O), 161.7, 153.4, 140.7, 135.6, 132.9, 124.1, 122.8, 116.9,
114.7, 96.6, 94.2, 10.6; MS [EI m/z, rel. intensity (%)]: 258 ([M]+),
12.9; 259 ([M]++1), 1.9; 97 ([M+À161], [C5H5O2]+, 100); Anal. Calcd
for C14H10O5: C, 65.11; H, 3.90. Found: C, 64.74; H, 3.83.
The tricyclic keto-enol 20 (400.0 mg, 2.47 mmol) was treated
with potassium carbonate (375.4 mg, 2.72 mmol) and chloro bute-
nolide 8 (359.9 mg, 2.72 mmol) in dry DMF (4 mL) at room temper-
ature for 18 h. When TLC indicated total consumption of chloro
butenolide 8, the mixture was treated with ice water (50 mL)
and dichloromethane (20 mL), the separated aqueous layer was ex-
tracted with dichloromethane (2 Â 15 mL), the combined organic
layers washed with brine, dried (Na2SO4) and concentrated in va-
cuo. Column chromatography (hexane/ethyl acetate, 1:1, v/v) of
the resultant material, followed by recrystallization from di-iso-
propyl ether gave 21 (112.6 mg, 18%) as white crystals, mp 168–
171 °C; 1H NMR (CDCl3, 400 MHz): d 6.93 (s, 1H, @CH), 6.13 (s,
1H, OCHO), 6.03 (s, 1H), 5.89 (s, 1H), 5.36 (s, 1H), 3.27 (m, 1H),
3.22 (br s, 1H), 3.02 (br s, 1H), 2.97 (m, 1H), 2.03 (s, 3H, CH3),
1.75 (d, J = 8.5 Hz, 1H), 1.5 (d, J = 8.5 Hz, 1H). 13C NMR (CDCl3,
100 MHz): d 205.4 (C@O), 186.1 (C@O), 170.4, 140.8, 135.2,
133.8, 131.9, 110.3, 97.7, 52.0, 51.3, 46.2, 44.1, 43.6, 10.6; MS [EI
m/z, rel. intensity (%)]: 259 ([M+1]+, 2.0); 258 ([M]+, 13.3); 230
([M+À28], [C14H14O3]+, 3.9); 161 ([M+À97], [C10H9O2]+, 63.3); 97
([M+À161], [C5H5O2]+, 100); Anal. Calcd for C15H14O4: C, 69.76; H,
5.46. Found: C, 69.87; H, 5.28.
3.1.5. 3-Methyl-5-[(3-oxo-1-cyclohexenyl)oxy]-2(5H)-furanone
(17)
Treatment of 1,3-cyclohexanedione (16) (300.0 mg, 2.68 mmol)
with potassium carbonate (407.2 mg, 2.95 mmol) and chloro bute-
nolide 8 (390.3 mg, 2.95 mmol) in dry DMF (3 mL) was conducted
in the manner as described above, to give the desired compound 17
as white crystals, 90.9 mg (16.3%) after column chromatography
(hexane/ethyl acetate, 1:1, v/v). An analytical sample was obtained
by recrystallization from di-isopropyl ether, mp 120–124 °C; 1H
NMR (CDCl3, 400 MHz): d 6.94 (s, 1H, CH@), 6.24 (s, 1H, OCHO),
5.73 (s, 1H, @CH), 2.46–2.28 (m, 6H, 2 Â CH2), 2.01 (s, 3H, CH3).
13C NMR (CDCl3, 100 MHz) d 199.0 (C@O), 174.1 (C@O), 170.5,
141.2, 134.9, 106.3, 95.8, 36.6, 28.2, 20.9, 10.6; MS [EI m/z, rel.
intensity (%)]: 209 ([M+1]+, 2.9); 208 ([M]+, 21.7); 97 ([M+À111],
[C5H5O2]+, 100); Anal. Calcd for C11H12O4: C, 63.45; H, 5.81. Found:
C, 63.26; H, 5.65.
3.2. Bioassays28
3.2.1. Seeds
Seeds of S. hermonthica (from Sorghum bicolor (L.) Moench) were
harvested in the Sudan in 1987. O. cernua seeds were harvested in
Spain in 1994. Seeds were stored in the dark at room temperature
until required.
3.2.2. Preparation of test solutions
3.1.6. 5-[(5,5-Dimethyl-3-oxo-1-cyclohexenyl)oxy]-3-methyl-
2(5H)-furanone (19)
Method A. 5,5-Dimethyl-1,3-cyclohexanedione (18) (300.0 mg,
2.14 mmol) was treated with potassium carbonate (325.8 mg,
2.36 mmol) and chloro butenolide 8 (312.3 mg, 2.36 mmol) in
dry DMF (3 mL) in the manner described above. The crude product
The compounds to be tested were accurately weighed (ca. 1 or
ca. 2 mg) using a 5 decimal balance, dissolved in analytical grade
acetone (1 mL) and then diluted with distilled water to a volume
of 50 mL. Aliquots of these analytical grade solutions were further
diluted with distilled water to obtain test solutions containing 1,
0.1 and 0.01 mg/L test compound and 0.01% (v/v) acetone,

A. S. Mwakaboko, B. Zwanenburg / Bioorg. Med. Chem. 19 (2011) 5006–5011
5011
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Lefevre, J., Musselman, L. J., Parker, C., Peckering, A., Eds.; ICSU Press: Paris,
1984; pp 125–132.
respectively. Distilled water was boiled for a minimum of 30 min
and then cooled to room temperature before use.
12. Mangnus, E. M.; Zwanenburg, B. J. Agric. Food Chem. 1992, 40, 1066.
13. B. Zwanenburg, E.M. Mangnus, J.W.J.F. Thuring, Strigol Analogues: Design
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Food Chem. 1992, 40, 1222.
3.2.3. Bio-assays
For surface sterilization, seeds of S. hermonthica and O. cernua
were exposed to an aqueous solution of sodium hypochlorite (2%
active chlorine) for 5 min with agitation. The seeds were then
rinsed thoroughly with demineralised water and dried on a blot-
ting paper for ca. 30 min. For conditioning, the sterilized seeds
were spread on glass fiber filter paper disks (8 mm diameter;
approximately 30–70 seeds per disk) in Petri dishes, wetted with
demineralised water, stored in the dark for 14 days at 20 °C for
Orobanche seeds, at 30 °C for Striga seeds. The conditioning water
17. Thuring, J. W. J. F.; Nefkens, G. H. L.; Zwanenburg, B. J. Agric. Food Chem 1997,
45, 1409.
18. Zwanenburg, B.; Mwakaboko, A. S.; Reizelman, A.; Anilkumar, G.;
Sethumadhavan, D. Pest Manag. Sci 2009, 65, 478.
was then removed and replaced by 100 lL of test solution per disk.
19. For some total syntheses of natural SLs, see: (a) MacAlpine, G. A.; Raphael, R. A.;
Shaw, A.; Taylor, A.; Wild, H. J. J. Chem. Soc., Chem. Perkin Trans 1976, 410; (b)
Sugimoto, Y.; Wigchert, S. C. M.; Thuring, J. W. J. F.; Zwanenburg, B. J. Org. Chem
1998, 63, 1259; (c) Matsui, J.; Yokota, T.; Bando, M.; Takeuchi, Y.; Mori, K. Eur. J.
Org. Chem. 1999, 2201; (d) Reizelman, A.; Scheren, M.; Nefkens, G. H. L.;
Zwanenburg, B. Synthesis 2000, 1944.
20. Kondo, Y.; Tadokoro, E.; Matsuura, M.; Iwasaki, K.; Sugimoto, Y.; Miyake, H.;
Takikawa, H.; Sasaki, M. Biosci., Biotechnol., Biochem. 2007, 71, 2781.
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Jong, R. L. P.; Zwanenburg, B. J. Agric. Food Chem. 1992, 40, 1230; (b) Malik, H.;
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After incubation for 24 h (Striga), and 5 days (Orobanche) in the
dark at the temperatures indicated, the germination percentage
was determined using a microscope. Seeds were considered to
have germinated if the radical protruded through the seed coat.
In each test series, aqueous solutions containing 0.01% (v/v) ace-
tone were used as negative controls. Test solutions of the stimulant
GR 24 (2) (concentrations of 0.1 and 0.01 and 0.001 mg/L) were
used as positive controls. In each test, the germination percentages
were determined on 9 disks per treatment. All bio-assays were
compared with that of GR 24 in the same run, thus giving a reliable
relative activity with respect to this positive control. All assays
were carried out in triplicate; no statistical analyses were per-
formed as for that more concentrations of the stimulants should
have been investigated.
27. Parker, C.; Riches, C. R. Parasitic Weeds of the World, Biology and Control; CAB
International: Wallingford, Oxon, UK, 1993.
Acknowledgements
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We thank H. Amatdjais, P. van Galen, and A. Swolfs for conduct-
ing elemental analysis, mass, and 400 MHz 1H NMR measure-
ments, respectively, Henk Regeling for his experimental advice
and Dr. G.J.F. Chittenden for helpful discussions.
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Supplementary data
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