Strigolactone analogues and mimics derived from phthalimide, saccharine, p-tolylmalondialdehyde, benzoic and salicylic acid as scaffolds

Article abstract of DOI:10.1016/j.bmc.2011.10.057

A series of new strigolactone (SL) analogues is derived from simple and cheap starting materials. These SL analogues are designed using a working model. The first analogue is a modified Nijmegen-1, the second contains saccharin as substituent (bio-isoster

Full text of DOI:10.1016/j.bmc.2011.10.057

Bioorganic & Medicinal Chemistry 19 (2011) 7394–7400
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Bioorganic & Medicinal Chemistry
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Strigolactone analogues and mimics derived from phthalimide, saccharine,
p-tolylmalondialdehyde, benzoic and salicylic acid as scaffolds
Binne Zwanenburg , Alinanuswe S. Mwakaboko 1
⇑
Radboud University Nijmegen, Institute for Molecules and Materials, Department of Organic Chemistry, 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:
A series of new strigolactone (SL) analogues is derived from simple and cheap starting materials. These SL
analogues are designed using a working model. The first analogue is a modified Nijmegen-1, the second
contains saccharin as substituent (bio-isosteric replacement of a carbonyl in Nijmegen-1 by a sulfonyl
group) and the third one is derived from p-tolylmalondialdehyde. These new SL analogues are apprecia-
bly to highly active as germination stimulants of seeds of Striga hermonthica and Orobanche cernua. The SL
analogue derived from saccharin is the most active one.
A serendipitous and most rewarding finding is that the compound obtained by a direct coupling of sac-
charin with the chlorobutenolide exhibits a high germination activity especially towards O. cernua seeds.
Two other SL mimics are obtained from benzoic and salicylic aid by a direct coupling reaction with chlo-
robutenolide, both of them are very active germinating agents. These SL mimics represent a new type of
germination stimulants. A tentative molecular mechanism for the mode of action of these SL mimics has
been proposed.
Received 11 July 2011
Revised 17 October 2011
Accepted 18 October 2011
Available online 24 October 2011
Keywords:
Strigolactone
Parasitic weeds
Bioassay
Striga
Orobanche
Germination
Molecular mechanism
Alternative mode of action
New type stimulants
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
formation of a radicle from the seed and its subsequent attachment
to the host roots allowing the developing parasite to take nutrients,
Strigolactones (SLs) are currently in the focus of interest as they
are considered as a new type of plant hormones.1–6 Natural strigo-
lactones have been isolated from root exudates of various plants,
especially those that are parasitised by the noxious weeds Striga
and Orobanche spp. The first natural SL that has been isolated is
strigol (1)7 (Fig. 1). Full details of the structure of this germination
stimulant for seeds of the just mentioned weeds were described
about 20 years after its initial isolation.8 Up to now several natural
germination stimulants have been isolated and identified from the
root exudates of both host and nonhost plants.7,9–14 The structures
of SLs invariably contain four rings, the main structural differences
are encountered in the AB part, whereas all stimulants contain the
same D-ring.12–15 It is highly relevant to mention that these stimu-
lants occur only in minute amounts in root exudates. The production
of strigol per plant is estimated to be ca. 25–30 pg per day.16,17 As a
consequence the isolation and identification of natural SLs is extre-
mely difficult. The natural life cycle of the parasitic weeds Striga and
Orobanche spp. involves the germination of their seeds induced by a
SL exuded by the roots of the host plant. This is followed by the
hormones and water from the host plant which, as a consequence,
will suffer and not be able to grow normally.18 Infestation of cereal
crops (main staple food) by Striga results in considerable crop losses,
which is considered to be the major obstacle to food production in
Central and Southern Africa.19 The estimated loss of cereal crop
amounts to of million of tons each year.19,20 Weed pest control is
therefore of utmost importance.19,20 The concept of suicidal germi-
nation is an appealing approach to control these noxious
weeds.15,21–25 In this approach the soil is treated with stimulant in
absence of a host, hence the germinated seeds cannot develop and
will die. The natural stimulants cannot be used for this purpose as
their synthesis26 is far too laborious and expensive. Therefore, ana-
logues have been prepared in whichthe bioactivityis predominantly
retained. The first successful series of analogues are the GR com-
pounds,27 as is exemplified by GR 24 (3)28 (Fig. 1).
An extensive study of structure–activity relationship revealed
that the bioactiphore resides in the CD part of the molecule.29,30 In
addition, a molecular mechanism has been proposed for the initial
reaction of the stimulants with protein receptor in the seeds
(Scheme 1).29,30 In essence this is nucleophilic addition to the
a,
b-unsaturated unit followed by a retro reaction with the concurrent
elimination of the D-ring. Combining the information of the bioacti-
phore and the molecular mechanism leads to a working model for
designing new germination stimulants as shown in Figure 2.15
⇑
Corresponding author. Tel.: +31 24 3653159.
E-mail address: B.Zwanenburg@science.ru.nl (B. Zwanenburg).
Current address: Department of Chemistry, University of Dar es Salaam, PO Box
1
35061, Dar es Salaam, Tanzania.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.10.057

B. Zwanenburg, A. S. Mwakaboko / Bioorg. Med. Chem. 19 (2011) 7394–7400
7395
O
O
O
O
O
O
O
O
O
C
A
B
O
O
O
O
OH
OH
D
O
O
O
(+)-Strigol (1)
Orobanchol (2)
GR 24 (3)
O
N
O
OMe
O
O
O
O
O
O
O
O
O
O
O
Nijmegen-1 (4)
5
6
Figure 1. Chemical structures of some natural and synthetic SLs.
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 triggering of germination by SLs.
which leads to a saccharine derived analogue. The third one is de-
rived from a 1,3-dialdehyde leading to the enal derived SL
analogue.
The second part of the paper deals with a serendipitous finding
of a new type of stimulant which contains no a,b-unsaturated unit,
Stereochemistry
is very important
O
O
O
C
∗
∗
A
B
R
but the D-ring only. These new SL mimics are derived from saccha-
rine, as well as from benzoic and salicylic acid.
O
D
∗
O
A-ring modifications
hardly affect activity
2. Results and discussion
2.1. Synthesis of SL analogues and mimics
α, β-unsaturated system
and D-ring are essential
The synthesis of compound 11 was achieved by first treating tri-
mellitic anhydride 7 with glycine33 followed by subsequent esterifi-
cation of both carboxylic acid groups of 8 using dimethoxypropane
and methanol. Hydroxymethylidenation of thus obtained 9 with
methyl formate, using metallic sodium, gave the expected hydroxy-
methylene compound as its sodium enolate. This salt was treated
in situ with chlorobutenolide 10. Using this one-pot procedure the
desired product 11 was obtained in a modest yield (Scheme 2).
A saccharin SL analogue was obtained by first reacting of pota-
sium saccharinate 12b with chloroacetone to give acetonylsaccha-
rin 13 in 86% yield.34 Subsequent hydroxymethylidenation with
methyl formate using metallic sodium gave crystalline enol 14
and a colourless oily byproduct which could not be identified. Cou-
pling of 14 with chlorobutenolide 10 gave the desired saccharin
derived SL analogue 15 in 43% yield (Scheme 3).
Figure 2. Working model for designing SL analogues.
In the early stages of developing this model Nijmegen-1 was de-
signed as a potential stimulant.31 Gratifyingly, this compound
showed an appreciable stimulation activity towards seeds of both
parasitic weeds.31 Even more rewarding was the successful appli-
cation of Nijmegen-1 in the field in controlling Orobanche in tobac-
co crops.15 Recently, we reported the synthesis and bioactivity of a
series of ketone derived SL analogues.23,32 The best performing ke-
tone SL analogue is 5, which is readily prepared from 1-tetralone in
two steps. A second series of SL analogues was obtained from sim-
ple cyclic keto enols in a single step operation.24 The SL analogue 6
from dimedone was one of the most active stimulants. Both series
of SL analogues were designed on the basis of the model15 shown
in Figure 2.
The first SL analogue (17) having an enal as the a,b-unsaturated
In this paper, we report the design of three new SL analogues.
The first one is a structural modification of the phthalimide scaf-
fold of Nijmegen-1. The second one is an isosteric replacement of
one of the carbonyl groups in Nijmegen-1 by a sulfonyl group
unit was obtained from 2-p-tolyl-malondialdehyde 16 by a one-pot
procedure as shown in Scheme 4 in 52% yield.
For the sake of comparison and curiosity, the saccharin derivative
(18) in which the D-ring is coupled directly to saccharin was also

7396
B. Zwanenburg, A. S. Mwakaboko / Bioorg. Med. Chem. 19 (2011) 7394–7400
O
O
O
O
O
H
O
O
Glycine
Nitrobenzene
DMP
N
N
O
O
HO
HO
Methanol
O
O
O
O
O
O
7
9
8
O
O
O
O
Na, HCO2CH3,
N
O
O
DMF, (One-pot)
O
O
Cl
O
O
O
10
11
Scheme 2. Synthesis of modified Nijmegen-1.
O
O
O
O
O
ClCH2COCH3
DMF, Reflux
(i) HCO2CH3, Na
(ii) H+
NR
N
N
O
S
S
S
O
O
O
O
O
HO
12a
R=H
13
14
12b R=K
O
O
KOBut
N
DMF, 10
S
O
O
O
O
O
15
Scheme 3. Synthesis of a SL analogue from acetonyl saccharin.
O
O
O
H
KOBut
DMF, 10
H
O
O
O
R
O
ONa
O
DMF
16
HO
O
O
10
20a
19a
R=H
R=H
O
20b R=OH
19b R=OH
17
H
Scheme 6. Synthesis of SL mimics from benzoic and salicylic acid.
H
O
As it was expected that direct coupling was a new option for
preparing SL mimics (see Section 2.2) also benzoic and salicylic
acid were directly coupled with the D-ring. In this manner the SL
mimics 20a and 20b were obtained in a single step (Scheme 6).
The yields of these preparations of SL analogues and mimics
have not been optimised. At this stage of the work, the bioactivities
are most relevant.
Scheme 4. Synthesis of a SL analogue from p-tolylmalondialdehyde.
O
O
O
O
KOBut, DMF
N
NH
S
10
S
O
O
O
O
saccharin (12a)
2.2. Germination activity of the new SL analogues and mimics
18
Scheme 5. Synthesis of SL mimic from saccharin.
The stimulatory germination activities of SL analogues 11, 15
and 17 were assayed35 using seeds of Striga hermonthica and
Orobanche cernua. The germination results are collected in Tables
1 and 2. There is a considerable difference in the induction of ger-
mination for both seed types. It should be noted however, that the
positive control, that is, the activity of GR 24, is different for both
prepared. The coupling reaction took place in the presence of potas-
sium tert-butoxide and DMF as the solvent. The desired product 18
was obtained as a crystalline product in 29% yield (Scheme 5).

B. Zwanenburg, A. S. Mwakaboko / Bioorg. Med. Chem. 19 (2011) 7394–7400
7397
Table 1
Table 2
Germination stimulatory activitya of the new analogues and mimics towards the
Germination stimulatory activitya of the new analogues and mimics towards the
seeds of S. hermonthica
seeds of O. cernua
Entry Starting material
D-ring
derivative
% Germination SEb at a
concentration of
Entry Starting material
D-ring
derivative
% Germination SEb at a
concentration of
1 mg/L
nde
0.1 mg/L 0.01 mg/L
19.7 1.4 43.2 4.4
1 mg/L
nde
0.1 mg/L 0.01 mg/L
85.7 1.7 81.6 3.5
1
GR 24
(3)d,f
11
1
GR 24
(3)d,f
11
2
Methoxycarbonyl
phthalimidoacetic ester
Acetonyl saccharin
p-Tolylmalondialdehyde
Saccharin
17.6 3.6 35.4 7.3 6.4 1.4
2
Methoxycarbonyl
phthalimidoacetic ester
Acetonyl saccharin
Arylmalondialdehyde
Saccharin
74.5 4.4 68.7 13.0 6.9 0.5
15
17
18
20a
20b
64.2 6.9 68.4 1.7 55.9 4.6
0.0 0.0c 9.6 2.7 34.4 3.5
0.0 0.0c 18.5 2.3 11.1 2.1
2.7 1.1c 8.7 2.7 6.7 2.4
2.6 0.8c 6.9 1.1 21.3 3.5
3
4
5
6
7
15
17
18
20a
20b
84.9 3.2 79.0 3.5 54.5 3.8
63.4 5.1 89.0 3.5 87.1 5.0
70.2 2.9 31.0 5.3
87.3 1.7 77.7 5.9 20.6 4.2
51.9 10.6 81.7 8.3 72.4 4.0
4
5
6
7
3.7 1.0c
Sodium benzoate
Sodium salicylate
Sodium benzoate
Sodium salicylate
a
a
Activities are given as germination percentages obtained after treatment of the
Activities are given as germination percentages obtained after treatment of the
seeds with the stimulant solution. Germination percentages given are means SE of
one representative experiment.
seeds with the stimulant solution. Germination percentages given are means SE of
one representative experiment.
b
b
Values are the mean germination percentages SE obtained by treatment of the
Values are the mean germination percentages SE obtained by treatment of the
seeds with GR 24 (3) in the same bioassay.
seeds with GR 24 (3) in the same bioassay.
c
c
No activity. Values are not significantly different from germination percentages
No activity. Values are not significantly different from germination percentages
obtained in the aqueous control.
obtained in the aqueous control.
d
d
Equimolar mixture of two racemic diastereomers of GR 24 (3).
Not determined (nd).
Equimolar mixture of two racemic diastereomers of GR 24 (3).
Not determined (nd).
e
e
f
f
The germination percentage of GR 24 (3) at 0.001 mg/L was 64.3 7.5.
The germination percentage of GR 24 (3) at 0.001 mg/L was 55.9 5.1.
test runs, but the conclusions will be influenced only marginally by
this difference.
These three new stimulating agents show a similar activity pro-
file. The best performing stimulant for both seed types is saccharin
derived compound 15, as it is active over a wide concentration
range. Therefore, this SL analogue is an interesting candidate for
the use in the suicidal approach for reducing the devastating effect
of the parasitic weeds. A drawback of this compound 15 is that its
synthesis requires optimisation.
These results constitute another demonstration of the validity
of the model shown in Figure 2 for designing SL analogues. A series
of SL analogues with the enol ether unit conjugated with an es-
ter24,27,31,37, ketone23,24,32,38 or aldehyde38 are now available for
further evaluation. For all these analogues the molecular mecha-
nism shown in Scheme 1 is a conceivable mode of action. Recently
it has been shown23 that the same holds for SL imino analogues
[ArC(CN)@N–O–D-ring where Ar is Ph or 2-pyridyl],39 in spite of
an earlier contrasting opinion.39
For S. hermonthica the ester substituted Nijmegen-1 (11) is only
moderately active at low concentration, but at 0.1 mg/L it is appre-
ciably active when compared with GR24 at the same concentra-
tion. The saccharin derived SL analogue 15 shows a high activity.
The SL analogue 17 obtained from the 1,3-dialdehyde 16 is reason-
ably active at low concentration when compared with GR24. In
contrast, the Orobanche seeds respond much better to the newly
prepared stimulants. Analogue 11 is almost as active as GR24,
but SL analogue 15 derived from saccharin is the most active. Also
the enal derived SL analogue 17 is very active as well.
The compound 18 derived from saccharin by direct coupling
with the D-ring shows an unexpected high activity for O. cernua
seeds at the concentration of 1 mg/L. Also the compounds obtained
from benzoic acid and salicylic acid, 20a and 20b, respectively, are
surprisingly active. Because of the germinating activities of com-
pounds 18, 20a and 20b we call them SL mimics. The activity of
these SL mimics towards S. hermonthica seeds is much lower. By
taking into account that the GR24 response is rather low, the SL
analogues still seem appreciably active, especially for the saccharin
derived mimic 18.
The germinating activity of the saccharin derivative 18 is a great
surprise. The same holds for 20a and 20b. These compounds are
lacking the a,b-unsaturated unit which is considered to be a prere-
quisite for active germination stimulants (see model compound,
Fig. 2). The identification of these compounds as SL mimics is a
clear case of serendipity. The intriguing question is whether the
activity of these furanone derivatives can be explained by a modi-
fied molecular mechanism. As in the receptor site only nucleophilic
functionalities are available for initiating a reaction, it is suggested
that such a nucleophile adds to the furanone in a Michael fashion
as shown in Scheme 7. Subsequent internal proton shift, followed
by an elimination of the group at C-2, leads to a furanone with a
2.3. Discussion
The three newly SL analogues 11, 15 and 17 based on the model
compound shown in Figure 2, are all are active as germination
agents. This observation is another demonstration of the validity
of this model for the design of SL analogues. Modified Nijmegen-
b,c-olefinic bond which is in equilibrium with a 2-hydroxylfuran.
1 11 has an
analogues 15 has an
logue 17 obtained from malondialdehyde has an
a
,b unsaturated ester unit, the saccharin derived SL
,b unsaturated ketone unit and the SL ana-
,b-unsaturated
In essence, this tentative explanation is again an addition-elimina-
tion reaction. A condition for such a reaction to place is that the
substituent at the 2-postion of the SL mimic is a good leaving
group. In the case of the saccharin, benzoate and salicylate mimics
this condition is fulfilled. It important to note that presence of the
D-ring still is a prerequisite. Recently, germination was also ob-
served for a 2-aryloxyfuranone.40 Provided that this aryloxy group
is a good leaving group, which is the case for the p-bromoophenoxy
group, the same molecular mechanistic explanation as shown in
Scheme 7 is conceivable. It is relevant to note here that alkoxy-
2(5)-furanones are inactive as germination stimulants as has been
previously described in great detail.41 This is in agreement with the
a
a
aldehyde moiety. In fact, the latter compound is the first SL ana-
logue having an enal group connected to the D-ring. The saccharin
SL analogue is of interest for two reasons. Firstly, it shows that the
isosteric replacement of the carbonyl in the phthalomido scaffold
by a sulfonyl group is allowed. Secondly, it throws some doubt
on the claim that the phthalimido part of Nijmegen-1 is an essen-
tial molecular fragment for seed germination and development as
was suggested by McCourt et al. on the basis of a molecular simi-
larity study.36

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B. Zwanenburg, A. S. Mwakaboko / Bioorg. Med. Chem. 19 (2011) 7394–7400
fused-silica capillary column (DB-5, 30 m  0.25 mm) and helium
O
O
O
L
O
L
OH
L
O
was used as the carrier gas. GLC was conducted with a Hewlet-
Packard HP 5890 gas chromatograph using a capillary column
(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 visualised 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 at140 °C.
Column chromatography was performed on silica gel (Kieselgel,
Merck) using eluents indicated. All solvents were dried under
standard conditions.
Nu
H
Nu
Nu
L = saccharin anion
benzoate anion
salicylate anion
O
O
OH
O
p-bromophenoxy anion
L-
Nu
Nu
Scheme 7. Tentative molecular mechanism for the mode of action of SL mimics.
The chlorobutenolide 10 was prepared as previously
described.45
tentative mechanism shown in Scheme 7. Alkoxy groups cannot
serve as good leaving groups in this mode of action. Alternatively,
a simple accommodation of the SL mimic in the receptor through
dipole–dipole interactions can be envisaged, but then the differ-
ence between alkoxy and aryloxy furanones can not readily be
explained.
3.1.2. Nomenclature
The IUPAC nomenclature has been used for all compounds. The
systematic names were generated using the ACD/Name programme
provided by Advanced Chemistry Development Inc. (Toronto,
Canada).
Whether the SL analogues and SL mimics are interacting with
the same or with different receptors is a question that cannot be
answered in this stage. For the moment we hypothesise that both
stimulants interact with the same receptor but with different
modes of action. Both interaction modes are, to certain extend, re-
lated as both of them are similar addition–elimination reactions. It
is worth noting that the enol unit, for example, OCH@CH–C(@O)–
OMe, as present in the working model shown in Figure 2, may also
serve as leaving group L in Scheme 7.
3.1.2.1. Methyl 2-(2-methoxy-2-oxoethyl)-1,3-dioxo-5-isoindo-
linecarboxylate (9).
Carboxyphthalimidoacetic acid 833 (7.0 g,
28 mmol) dissolved in a mixture of methanol (70 mL) and 2,2-dime-
thoxypropane (80 mL) containing p-toluenesulfonic acid (500 mg)
was stirred at room temperature for 1 h. The temperature of the
reaction mixture was then elevated to 60 °C and set aside for 18 h.
The mixture was cooled to room temperature, concentrated in va-
cuo, and the resultant solid material was recrystallised from 2-pro-
panol to give 9 as a pure white solid, 2.1 g, (27%), mp 125–130 °C. The
spectroscopic data were in complete agreement with the assigned
structure. 1H NMR (CDCl3, 100 MHz): d 8.54–7.94 (m, 3H, ArH),
4.48 (s, 2H, NCH2CO), 4.00 (s, 3H, CH3O), 3.78 (s, 3H, OCH3).
The interesting outcome of this study is that appropriately 2-
substituted 2(5H)-furanones are new candidates for parasitic weed
control using the suicidal approach.
Currently SLs receive much attention in the literature because
of the newly discovered bioproperties. Natural SLs have been iden-
tified as the branching factor for arbusular mycorrhizal (AM) fun-
gi5,42 and as inhibitor of plant shoot branching.43,44 SLs are now
considered as a new class of plant hormones1,2 for which more
functions will be uncovered in the coming years. Interestingly,
the synthetic SL analogue GR24 is also active for both func-
tions.5,42–44 Therefore, searching for other SL analogues for the
new activities is another incentive for this study. Now there two
options, namely SL analogues and SL mimics. It has been shown
that some aryloxy-2-(5H)-furanones are active as branching inhib-
itors.40 Predictably, the SL mimics described in this paper offer
good prospects for being active as shoot branching inhibitors or
as hyphal branching factors for AM fungi.
3.1.2.2. Methyl 2-{(Z)-1-(methoxycarbonyl)-2-[(4-methyl-5-oxo-
2,5-dihydro-2-furanyl)oxy]-1-ethenyl}-1,3-dioxo-5-isoindoline-
carboxylate (11).
A stirred solution of the phthalimido ester 9
(600.0 mg, 2.17 mmol) in methyl formate (15 mL), whilst main-
tained under nitrogen, was treated with small pieces of metallic so-
dium (61.0 mg). The mixture was set aside for 24 h and then
concentrated in vacuo. The residue was dissolved in anhydrous
DMF (15 mL), cooled (À50 °C) and then treated dropwise with the
solution of chlorobutenolide 10 (378.8 mg, 2.86 mmol) in DMF
(5 mL). The mixture was allowed to warm to room temperature,
set aside for 5 days. The mixture was concentrated in vacuo, the res-
idue treated with a mixture of water (50 mL) and ethyl acetate
(30 mL), the separated aqueous layer was extracted with ethyl ace-
tate(2 Â 20 mL), andthe combinedorganiclayers werewashedwith
water, then dried (MgSO4), and concentrated in vacuo. The resultant
crude product gave compound 11 as a white foam (228.8 mg, 26.3%)
after column chromatography (hexane/ethyl acetate, 1:1, v/v). 1H
NMR (CDCl3, 400 MHz): d 8.53–7.93 (m, 4H, ArH + @CHO), 6.91 (s,
1H, CH@), 6.20 (s, 1H, OCHO), 4.00 (s, 3H, CH3OCO), 3.79 (s, 3H,
OCH3) 1.98 (s, 3H, CH3). 13C NMR, (CDCl3, 100 MHz): d 169.9,
165.2, 162.9, 153.7, 140.9, 135.7, 132.3, 124.9, 123.9, 106.0, 100.1,
60.4, 52.9, 52.4, 31.8, 22.6, 14.2, 14.1, 10.7; MS [EI, m/z, rel. intensity
(%)]: 402 ([M+1]+, 0.8); 401 ([M]+, 3.4); 370 ([M+À31], [C18H12NO8]+,
10.4), 304 ([M+À97], [C14H10NO7]+, 100), 97 ([M+À304], [C5H5O2]+,
3. Experimental section
3.1. Synthesis
3.1.1. 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 focusing 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
82.3); HRMS/EI: m/z calcd for
C19H15NO9: 401.0746. Found:
401.0745.
3.1.2.3. 2-(2-Oxopropyl)-2,3-dihydro-1H-1k6-benzo[d]isothiazole-
1,1,3-trione (13). stirred solution of saccharin (10.0 g,
A
54.64 mmol) in DMF (80 mL), whilst maintained under nitrogen, was
treated with potassium tert-butoxide (6.13 g, 54.64 mmol) followed
by chloroacetone (6.1 g, 65.6 mmol). The mixture was set aside for

B. Zwanenburg, A. S. Mwakaboko / Bioorg. Med. Chem. 19 (2011) 7394–7400
7399
15 min at room temperature and then heated at 125 °C for 2 h. The
cooled mixture was concentrated in vacuo, the residue was dissolved
in dichloromethane (80 mL) and washed with water (100 mL). The
dichloromethane layer was dried (MgSO4) and concentrated in vacuo.
The residue was crystallised from hot methanol to give 16 (11.2 g, 86%)
as a white solid, which was sufficiently pure for the subsequent step
(mp 143–144 °C).34 1H NMR (CDCl3, 100 MHz): d 8.14–7.86 (m, 4H,
ArH.), 4.48 (s, 2H, NCH2CO), 2.29 (s, 3H, COCH3).
chromatography (heptane/ethyl acetate, 1:1, v/v), 1H NMR (CDCl3,
400 MHz): d 9.48 (s, 1H, COH (aldehydic proton), 7.30–7.18 (m,
4H, ArH + CH@), 6.94 (s, 1H, CH@), 6.23 (s, 1H, OCHO), 2.35 (s, 3H,
Ar–CH3), 2.01 (s, 3H, CH3). 13C NMR (CDCl3, 100 MHz): d 190.4
(C@O), 170.1 (C@O), 161.4, 141.0, 138.1, 135.9, 129.2, 128.9,
126.2, 125.7, 100.5, 21.3, 10.7; MS [CI, m/z, rel. intensity (%)]: 259
([M+1]+, 77.4); 258 ([M]+, 15.5); 241 ([M++1]À18, [C15H12O3]+,
58.2), 161 ([M+À97], [C10H19O2]+, 18.6), 97 ([M+À161], [C5H5O2]+,
100); Anal. Calcd for C15H14O4: C, 69.76; H, 5.46. Found: C, 69.83;
H, 5.37.
3.1.2.4. 2-[(Z)-1-Acetyl-2-hydroxy-1-ethenyl]-2,3-dihydro-1H-1k6
-benzo[d]isothiazole-1,1,3-trione (14).
A
stirred, cooled
(À10 °C) solution of acetonyl saccharin 16 (5.0 g, 20.9 mmol) in
methyl formate (30 mL), whilst maintained under nitrogen was trea-
ted with small pieces of metallic sodium (481.2 mg, 20.9 mmol). The
mixture was allowed to attain room temperature, set aside for 14 h,
and then concentrated in vacuo. The residue was treated with a mix-
ture of glacial acetic acid (2 mL) and 1 M HCl (5 mL), and extracted
with ethyl acetate(50 mL), andthe separated aqueous layer extracted
with ethyl acetate (2 Â 50 mL). The combined organic layers were
washed with water, dried (MgSO4) and concentrated in vacuo. The
resultant crude material was purified by column chromatography
(hexane/ethyl acetate, 2:1, v/v) to give compound 14 (1.92 g in
34.3%) as a yellow solid and an unidentified compound (1.50 g,
26.9%) as oil. Recrystallisationof 14from di-isopropyl ether gave pure
material, mp 150–151 °C; 1H NMR (CDCl3, 100 MHz): d 14.93 (s, 1H,
OH (hydrogen bonding), 8.19–7.69 (m, 4H, ArH), 6.07 (br s, 1H, OH),
2.44 (s, 3H, CH3). MS [EI, m/z, rel. intensity (%)]: 267 ([M]+, 0.2); 239
([M+À28], [C10H9NO4S]+, 100); HRMS/EI: m/z calcd for C11H9NO5S:
267.0201. Found: 267.01993.
3.1.2.7. 2-(4-Methyl-5-oxo-2,5-dihydro-2-furanyl)-2,3-dihydro-1H-
1k6 -benzo[d]isothiazole-1,1,3-trione (18).
A stirred solution of
saccharin(250.0 mg, 1.34 mmol) inanhydrousDMF(10 mL) wastreated
with potassium tert-butoxide (153.3 mg, 1.34 mmol), followed by the
chlorobutenolide 10 (252.8 mg, 1.91 mmol), whilst being maintained
under nitrogen. The mixture was set-aside at room temperature for
ꢀ67 h and then processed in a manner similar to that described above.
The resultant crude material was triturated with a mixture of heptane/
ethyl acetate (1:1, v/v) to give pure 19 as a white solid, 111.5 mg
(29.3%), mp 173–176 °C; 1H NMR (CDCl3, 300 MHz): d 8.09–7.77 (m,
4H, ArH + @CH), 6.82 (m, 1H, OCHO), 2.09 (m, 3H, CH3); MS [EI m/z,
rel. intensity (%)]: 280 ([M+1]+, 2.8); 279 ([M]+, 16.1); 250 ([M+À29],
81.3); 183 ([M++1]À97, [C7H4 NO3 S]+, 32.9); 97 ([M++1]À183,
[C5H5O2]+, 100); HRMS/EI: m/z calcd for C12H9NO5 S: 279.0201. Found:
279.02006.
3.1.2.8. 4-Methyl-5-oxo-2,5-dihydro-2-furanyl benzoate (20a).
A stirred mixture of sodium benzoate 19a (429.2 mg, 3.0 mmol)
and the chlorobutenolide 10 (470.2 mg, 3.55 mmol) in an anhydrous
DMF (15 mL), whilst maintained under nitrogen, was set aside at
room temperature for 18 h. Then TLC analysis indicated the total
consumption of 10. The mixture was concentrated in vacuo, the res-
idue treated with a mixture of water (50 mL) and ethyl acetate
(30 mL), the separated aqueous layer was extracted with ethyl ace-
tate (2 Â 20 mL), and the combined organic layers were washed
with water, then dried (MgSO4), and concentrated in vacuo. The
resultant crude product was crystallised from di-isopropyl ether to
give 20a as pure white crystals, 387.8 mg (59.3%), mp 96–102 °C;
1H NMR (CDCl3, 400 MHz): d 8.06–7.47 (m, 5H, ArH), 7.13 (m, 1H,
CH@), 7.04 (m, 1H, OCHO), 2.04 (s, 3H, CH3). 13C NMR, (CDCl3,
400 MHz): d 171.0 (C@O), 164.7 (C@O), 142.1, 134.6, 134.0, 130.1,
128.6, 128.4, 93.0, 10.7; MS [EI m/z, rel. intensity (%)]: 219 ([M+1]+,
8.4); 218 ([M]+, 61.3); 189 [M+À29], 54.1, 105 [M+À113], 100, 97
([M+À121], [C5H5O2]+, 90.6); Anal. Calcd for C12H10O4: C, 66.05; H,
4.62. Found: C, 65.51; H, 4.59.
3.1.2.5. 2-{(Z)-1-Acetyl-2-[(4-methyl-5-oxo-2,5-dihydro-2-fura-
nyl)oxy]-1-ethenyl}-2,3-dihydro-1H-k6-benzo[d]isothiazole-1,1,
3-trione (15).
Compound 14 (300.0 mg, 1.12 mmol) was
added to a stirred, cooled (À40 °C) solution of potassium tert-
butoxide (126.1 mg, 1.12 mmol) in dry DMF (8 mL), whilst main-
tained under nitrogen. The mixture was stirred for 0.5 h at room
temperature, cooled to À60 °C and then treated dropwise with a
solution of the chlorobutenolide 10 (156.3 mg, 1.18 mmol) in
DMF (4 mL). The mixture was allowed to warm to room tempera-
ture and set aside for 24 h. The mixture was concentrated in vacuo,
the residue treated with a mixture of water (50 mL) and ethyl ace-
tate (30 mL), the separated aqueous layer was extracted with ethyl
acetate (3 Â 20 mL), and the combined organic layers were washed
with water, then dried (MgSO4), and concentrated in vacuo. The
resultant crude product 18 was obtained as
a yellow solid
(423.0 mg). Recrystallisation from di-isopropyl ether gave pure
product 15 as yellowish crystals (176.5 mg, 43.3%), mp 245–
253 °C; 1H NMR (CDCl3, 300 MHz): d 7.87–7.77 (m, 4H, ArH + @-
CHO), 6.88 (m, 1H, CH@), 6.58 (s, 1H, OCHO), 2.33 (s, 3H, COCH3),
1.93 (s, 3H, CH3). 13C NMR, (CDCl3, 100 MHz): d 194.1 (C@O),
174.7 (C@O), 171.4 (C@O), 165.6 (C@O), 140.0, 138.5, 135.9,
134.5, 133.9, 129.4, 128.2, 123.6, 89.9, 88.0, 22.4, 10.8; MS [EI, m/
z, rel. intensity (%)]: 335 ([M+1]+, 2.4); 335 ([M]+, 6.1); 335
([M+À18], 7.7), 211 ([M+À152], 3.2), 97 ([M+À266], 60.2), 98
([M+À265], 100); Anal. Calcd for C16H13NO7S: C, 52.89; H, 3.61;
N, 3.85; S, 8.85. Found: C, 53.45; H, 3.87; N, 4.20; S, 9.02. HRMS/
EI: m/z calcd for C15H13NO6S: 335.0464. Found: 335.04631.
3.1.2.9. 4-Methyl-5-oxo-2,5-dihydro-2-furanyl salicylate (20b).
A
stirred mixture of sodium salicylate 19b (600.0 mg,
3.75 mmol) and compound 10 (541.6 mg, 4.09 mmol) in anhydrous
DMF (15 mL), whilst maintained under nitrogen, was set-aside at
room temperature for ꢀ65 h. Then the mixture was processed in
the same manner as described above. The resultant solid was crys-
tallised from di-isopropyl ether to give 20b as pure white crystals,
365.4 mg (41.6%), mp 80–81 °C; 1H NMR (CDCl3, 400 MHz): d 10.28
[s, 1H, OH (hydrogen bonding)], 7.81–7.00 (m, 4H, ArH + @CH + O-
CHO), 2.04 (m, 3H, CH3); 13C NMR, (CDCl3, 100 MHz), d 170.8
(C@O), 168.4 (C@O), 162.2, 141.6, 136.9, 135.0, 130.1, 119.5,
117.9, 110.9, 92.8, 10.7; MS [EI m/z, rel. intensity (%)]: 235
([M+1]+, 11.2); 234 ([M]+, 73.9); 138 ([M++1]À97, [C7H5O3]+,
91.2); 97 ([M+À137, [C5H5O2]+, 100); HRMS/EI: m/z calcd for
3.1.2.6. E-3-[(4-Methyl-5-oxo-2,5-dihydro-2-furanyl)oxy]-2-(4-
methylphenyl)-2-propenal (17).
A stirred mixture of p-tolyl-
malondialdehyde 16 (200.0 mg, 1.23 mmol), DBU (206.5 mg,
1.36 mmol) and the chlorobutenolide 10 (163.4 mg, 1.23 mmol)
in dry dichloromethane (10 mL), whilst maintained under nitrogen,
was set aside at room temperature for 24 h, after which the mixture
was processed in the manner described above to give compound 17
as white crystals (165.0 mg, 51.8%), mp 119–121 °C, after column
C12H10 O5: 234.0528. Found: 234.05289.
3.2. Germination
The bioassays were carried out as described previously.23,24,35

7400
B. Zwanenburg, A. S. Mwakaboko / Bioorg. Med. Chem. 19 (2011) 7394–7400
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