Synthesis of 3-(4-bromobenzyl)-5-(aryl methylene)-5H-furan-2-ones and their activity as inhibitors of the photosynthetic electron transport chain

Article abstract of DOI:10.1021/jf072120x

A series of 12 3-(4-bromobenzyl)-5-(arylmethylene)-5H-furan-2-one lactones, designed using the naturally occurring toxin nostoclides as a lead structure, were synthesized and screened as potential inhibitors of photosynthetic electron transport. The structures were confirmed by ¹H and ¹³C NMR, MS, and IR analyses. Their biological activity was evaluated both in vitro, as the ability to interfere with light-driven reduction of ferricyanide by isolated spinach chloroplasts, and in vivo, as the capability to inhibite the oxygen production by intact Chlorella cells. Some of the compounds exhibited inhibitory properties in the micromolar range against basal and phosphorylating electron flow from water to K₃[Fe(CN)₆], with no effect on uncoupled electron flow. Thus, they seem to behave as energy-transfer inhibitors. Although poor solubility in water may limit their effectiveness, the active derivatives could present structures to be exploited for the design of new substances endowed with herbicidal activity.

Full text of DOI:10.1021/jf072120x

8562 J. Agric. Food Chem. 2007, 55, 8562–8569
Synthesis of 3-(4-Bromobenzyl)-5-(aryl
methylene)-5H-furan-2-ones and Their Activity as
Inhibitors of the Photosynthetic Electron
Transport Chain
LUIZ C. A. BARBOSA,*,† MARCELO E. ROCHA,† RÓBSON R. TEIXEIRA,†,‡
§
CÉLIA R.A. MALTHA,† AND GIUSEPPE FORLANI
Chemistry Department, Federal University of Viçosa, Avenida P.H. Rolfs, CEP 36570-000, Viçosa,
MG, Brazil, Chemistry Department, Federal University of Minas Gerais, Avenida Antônio Carlos,
6627, CEP 31270-9001, Belo Horizonte, MG, Brazil, and Department of Biology & Evolution,
University of Ferrara, via L. Borsari 46, I-44100 Ferrara, Italy
A series of 12 3-(4-bromobenzyl)-5-(arylmethylene)-5H-furan-2-one lactones, designed using the
naturally occurring toxin nostoclides as a lead structure, were synthesized and screened as potential
1
inhibitors of photosynthetic electron transport. The structures were confirmed by H and 13C NMR,
MS, and IR analyses. Their biological activity was evaluated both in vitro, as the ability to interfere
with light-driven reduction of ferricyanide by isolated spinach chloroplasts, and in vivo, as the capability
to inhibite the oxygen production by intact Chlorella cells. Some of the compounds exhibited inhibitory
properties in the micromolar range against basal and phosphorylating electron flow from water to
K3[Fe(CN)6], with no effect on uncoupled electron flow. Thus, they seem to behave as energy-transfer
inhibitors. Although poor solubility in water may limit their effectiveness, the active derivatives could
present structures to be exploited for the design of new substances endowed with herbicidal activity.
KEYWORDS: Herbicides; nostoclide analogues; photosynthetic electron transport inhibitors; solubility
INTRODUCTION
mineralized by the soil microflora. In this context, natural
products may provide novel lead compounds that may be
optimized using well-established chemical strategies (5–9).
Improved agronomical techniques, rapid mechanization,
artificial fertilizers, and high-yielding crop varieties have been
responsible for the impressive increase of farm productivity over
the years. Among these factors, the availability of chemical
agents for crop protection has played a pivotal role, helping
farmers to control a variety of pests and diseases, among which
are weeds (1). Herbicide application has become the most
reliable and least expensive method of weed control (2, 3).
However, repeated use of the same active ingredients has led
to herbicide resistance. Starting from the 1960s, hundreds of
weed biotypes were reported to survive herbicide application
(4). As a consequence, new herbicides must be continuously
developed to overcome weed resistance. Modern pesticides
should have a favorable combination of properties, including
high levels of herbicidal activity, low application rates, crop
tolerance, and low toxicity to mammals. Moreover, increasing
public concern for the environmental pollution deriving from
agricultural practice strictly demands that phytochemicals be
endowed with low recalcitrancy and thus be able to be rapidly
Among the numerous phytotoxic products of microbial origin
is cyanobacterin (1), a natural lactone isolated from the blue-
green alga Scytonema hofmanni (10) (Figure 1). At a concen-
tration as low as 5 µM, compound 1 is toxic to most
cyanobacteria, and also inhibits the growth of eukaryotic algae
and several mono- and dicotyledones (11, 12). It acts mainly
by inhibiting the photosynthetic electron transport, with a
mechanism of action similar to that of the herbicide 3-(3,4-
diclorophenhyl)-1,1-dimethylurea (13). In isolated Euglena
gracilis chloroplasts, such interference leads to a cascade of
events that results in the disruption of the tylakoid membrane
(14). Two lactones similar to compound 1, namely, nostoclides
I and II (Figure 1, compounds 2a and 2b), were isolated from
the culture of a symbiotic cyanobacterium, Nostoc sp., in
Peltigera canina, a common lichen (15). Contrary to compound
1, for which several studies have been reported, the mode of
action of nostoclides has not yet been fully investigated.
Nostoclides has shown moderate cytotoxicity against two mouse
neuroblastoma cell lines (15). However, because of the high
structural similarity between nostoclides and cyanobacterin (they
share a 3-benzyl-5-benzylidene-4-isopropyl-dihydro-furan-2-one
ring), and the fact that P. canina cultures are usually not
* Corresponding author. Fax: (55) 31 3899 3065. E-mail: lcab@
ufv.br.
† Federal University of Viçosa.
‡ Federal University of Minas Gerais.
§ University of Ferrara.
10.1021/jf072120x CCC: $37.00
2007 American Chemical Society
Published on Web 10/17/2007

Photosynthesis-Inhibiting Nostoclide Analogues
J. Agric. Food Chem., Vol. 55, No. 21, 2007 8563
stoppered and shaken for 1–2 min. The hydrochloride was filtered by
suction and washed immediately with diethyl ether (6 mL) and pentane
(12 mL). The bottle was tightly stoppered and the filtrate was allowed
to stand in the refrigerator (+5 oC) overnight. The clear brown ethereal
phase was decanted from the lower phase, and the solvent was
evaporated. Diethyl ether (10 mL) was added to the residue. The flask
mixture was cooled in an ice–salt bath, and diethylamine (18.0 mL)
was added to the chilled solution over 1 h, utilizing a dropping funnel
connected to a calcium chloride tube. After the addition, the temperature
of the reaction mixture was raised to 35 oC and stirring was continued
for 20 h. The hydrochloride formed was removed by suction filtration
and washed with diethyl ether (3 × 20 mL). The organic phase was
concentrated under reduced pressure, affording a brown oil. This crude
material was purified by column chromatography eluted with a mixture
of hexane:ethyl acetate of increasing polarity (hexane:EtOAc ) 3:1,
2:1, 1:1 v/v). The required compound was obtained as a yellow oil in
Figure 1. Structure of cyanobacterin (1) and nostoclides (2).
contaminated with microorganisms, it has been suggested that
these chlorinated compounds may play a role as allelopathic
agents.
51% yield (3.35 g, 12.2 mmol). IR (film, NaCl, cm-1) νmax 3145; 3117,
j
2974; 2935; 2876; 1605; 1516; 1466; 1248; 1213; 1199; 1171; 1102;
1066; 1029; 962; 847; 792. 1H NMR (400 MHz, CDCl3): δ 1.11 (t,
12H, J ) 7.0 Hz; CH3); 3.06–3.19 (m, 8H, NCH2); 5.68 (brs, 1H; H3);
6.27 (brs, 1H, H4); 6.94 (brs, 1H; H5). 13C NMR (100 MHz, CDCl3):
Within the frame of research programs aimed at the utilization
of natural products as lead compounds to develop new
herbicides (16–19), we previously assessed the potential phy-
totoxicity of several nostoclide analogues (20). In this paper,
we describe the preparation of 12 3-(4-bromobenzyl)-5-(aryl-
methylene)-5H-furan-2-one lactones analogues to nostoclides.
Their ability to interfere with the photosynthetic electron
transport chain in isolated spinach chloroplasts and in intact
Chlorella cells was also investigated.
2
3
δ 13.71 (s, CH3); 39.76 (d, JC-P ) 4.5 Hz; NCH2); 88.78 (d, JC-P
)
3.2 Hz; C3); 111.13 (s, C4); 134.02 (s, C5); 152.58 (d, 2JC-P ) 3.3 Hz;
C2). MS, m/z (%): 274 (M+, C12H23N2O3P, 3); 191 (100); 163 (8); 83
(8); 72 (51); 44 (29).
3-(4-Bromobenzyl)-5H-furan-2-one (7). Previously titrated n-butyl-
lithium hexane solution (1.1 mol L-1, 7.5 mL, 8.25 mmol) was added
over 10 min to a solution of compound 5 (1.0 g; 3.65 mmol) in THF
(15 mL) and HMPA (5 mL), chilled to -78 °C. The addition was
carried out so that the temperature reached -55 °C but did not exceed
this level. The resulting mixture was chilled to -78 °C for 20 min;
then 4-bromobenzyl bromide (3.8 g, 15.0 mmol) dissolved in THF (15
mL) was added with a syringe over 15 min so that the temperature did
not rise above -55 °C. After the addition, the temperature was raised
to room temperature. Water (20 mL) and ethyl acetate (80 mL) were
added, the phases were separated, and the dark inorganic phase was
extracted with ethyl acetate (2 × 80 mL). The combined organic phases
were washed with brine (40 mL) and dried over magnesium sulfate.
The solvent was removed under reduced pressure to yield the substituted
furan 6, which need not be purified for the next reaction. To this crude
material formic acid (5 mL) was added in a 50 mL round bottom flask
on a water bath at room temperature, and the resulting mixture was
stirred until bubbling had ceased (45 minutes). Benzene (12 mL) was
added and most of the excess of formic acid was removed under reduced
pressure. To the residue, ethyl acetate (40 mL) and sodium chloride-
sodium carbonate solution (12 mL) were added. The organic phase
was washed twice with the latter solution (2 × 12 mL). The combined
inorganic phase was extracted with ethyl acetate (2 × 40 mL). The
combined organic phases were dried over magnesium sulfate, and the
solvent was removed under reduced pressure. The resulting material
was purified by column chromatography on silica gel eluted with
hexane:diethyl ether (2:1 v/v) to generate compound 7 as a yellow oil
MATERIALS AND METHODS
Chemicals. Dichloromethane, tetrahydrofuran (THF), hexamethyl-
phosphoramide (HMPA), diethyl ether, and amines were purified
according to standard methods (21). Analytical grade tert-butyldi-
methylsilyltrifluoromethanesulfonate (TBDMSOTf), piperonal, ben-
zaldehyde, 2,4,6-trimethoxybenzaldehyde, 3-methylbenzaldehyde, 4-
fluorobenzaldehyde, 4-bromobenzaldehyde, 4-chlorobenzaldehyde, 3-nitro-
benzaldehyde, 4-trifluoromethylbenzaldehyde, 4-cyanobenzaldehyde,
2-chloro-4-dimethylaminobenzaldehyde, 4-bromobenzyl bromide, 8-diaza-
byciclo[5.4.0]undec-7-ene (DBU), and phosphoryl chloride (POCl3) were
procured from Aldrich (Milwaukee, WI) and utilized without further
purification.
Synthesis. All reactions were carried out under a protective
atmosphere of dry nitrogen or utilizing a calcium chloride tube. The
aldehyde 2,5-dimethoxybenzaldehyde was prepared from the corre-
sponding benzylic alcohol by Swern oxidation (22). Lactone 3 was
synthesized employing a previously described methodology (23).
Commercially available n-butyllithium hexane solutions (1.6 mol L-1
)
were titrated prior to use (24). 1H and 13C NMR spectra were recorded
on a Bruker AVANCE DRX 400 spectrometer at 400 and 100 MHz,
respectively, using CDCl3 as solvent and TMS as internal standard.
Mass spectra were obtained on a SHIMADZU GCMS-QP5050A
instrument by direct injection using the following temperature program:
40 °C min-1 until temperature reaches 60 °C, and then 80 °C min-1
until temperature reaches 300 °C; the detector temperature was 280
°C. Infrared spectra were recorded on a Perkin Elmer Paragon 1000
FTIR spectrophotometer, using potassium bromide (1% w/w) disks,
scanning from 625 to 4000 cm-1. Melting points are uncorrected and
were obtained with a MQAPF-301 melting point apparatus (Micro-
quimica, Brazil). Analytical thin layer chromatography was carried out
on TLC plates recovered with 60GF254 silica gel. Column chromatog-
raphy was performed over silica gel (60–230 mesh).
in 76% yield (704 mg, 2.78 mmol). IR (KBr, cm-1) νmax 3087; 2929;
j
2868; 1748; 1651; 1591; 1488; 1349; 1069; 803. 1H NMR (400 MHz,
CDCl3): δ 3.56 (s, 2H, H6); 4.76 (d, 2H, J ) 1.8 Hz; H5); 6.95 (d, 1H,
J ) 1.8 Hz; H4); 7.11 (d, 2H, J ) 8.5 Hz; H2′/H6′); 7.44 (d, 2H, J )
8.5 Hz; H3′/H5′). 13C NMR (100 MHz, CDCl3): δ 31.52 (C6); 70.52
(C5); 121.02 (C4′); 130.89 (C2′/C6′); 132.10 (C3′/C5′); 133.98 (C3);
136.53 (C1′); 145.94 (C4); 173.95 (C2). MS, m/z (%): 252 (M+,
C11H9BrO2, 8); 254 (M + 2, 7); 209 (4); 191 (2); 173 (13); 144 (4);
128 (100); 115 (36); 89 (20); 63 (22); 57 (49); 51 (65).
N,N,N′,N′-Tetraethyldiamidophosphate (5). A 25 mL flask, protected
from moisture by a calcium chloride tube, was charged with 5H-furan-
2-one (3) (2.00 g, 24 mmol), dichloromethane (5.0 mL), and POCl3
(2.2 mL, 24 mmol). A solution of triethylamine (4.8 mL, 36 mmol) in
dichloromethane (4.0 mL) was added dropwise over 1 h. The resulting
mixture was stirred overnight (18 hours), after which triethylamine (0.8
mL, 6.0 mmol) was added in one portion, and stirring was continued
for 24 h. The solvent was removed on a rotatory evaporator and diethyl
ether (12 mL) was added carefully to the dark residue, followed by
pentane (6 mL), to precipitate the amine hydrochloride. The flask was
(5Z)-3(4-Bromobenzyl)-5-(1,3-dioxalanebenzylidene)-5H-furan-2-
one (8a). To a two-neck round bottom flask, under nitrogen
atmosphere, were added 3-(4-bromobenzyl)-5H-furan-2-one (7) (100
mg, 0.39 mmol), dichloromethane (3 mL), TBDMSOTf (108 µL,
0.47 mmol), diisopropyl ethyl amine (DIPEA) (200 µL, 1.2 mmol),
and piperonal (120 mg, 0.47 mmol). The resulting mixture was
stirred at room temperature for 1 h. After DBU (116 µL, 0.78 mmol)
was added, the reaction mixture was refluxed for an additional 3 h
before addition of dichloromethane (70 mL). The resulting organic
layer was washed with 3 mol L-1 HCl aqueous solution (2 × 25

8564 J. Agric. Food Chem., Vol. 55, No. 21, 2007
Barbosa et al.
mL) and brine (25 mL). After separation, the organic layer was dried
over MgSO4, filtered, and concentrated under reduced pressure. The
resulting material was purified by column chromatography on silica
gel eluted with hexane:dichloromethane (1:1 v/v). The procedure
described afforded compound 8a (65 mg, 0.15 mmol) as a yellow
solid in 59% yield. The product was recrystalized from a mixture
of hexane:dichloromethane (∼1:1 v/v).
Data for (5Z)-3(4-Bromobenzyl)-5-(4-fluorobenzylidene)-5H-furan-
o
2-one (8e). White solid; m.p. 138.2–138.9 C. IV (KBr, cm-1): νmax
j
3088; 1758; 1727; 1595; 1507; 1485; 1237; 1044; 822; 800. 1H NMR
(400 MHz, CDCl3): δ 3,68 (s, 2H, H7); 5.85 (s, 1H, H6); 6.96 (t, 1H,
J ) 1.3 Hz, H4); 7.06 (t, 2H, J ) 8.8 Hz, H3′′/H5′′); 7.13 (d, 2H, J )
8.3 Hz, H2′/H6′); 7.46 (d, 2H, J ) 8.3 Hz, H3′/H5′); 7.72 (dd, 2H, J
) 8.8, 5.5 Hz, H2′′/H6′′). 13C NMR (100 MHz, CDCl3): δ 31.14 (s,
C7); 111.75 (s, C6); 115.98 (d, 1JC-F ) 21.5 Hz, C3′′/C5′′); 120.96 (s,
C4′); 129.33 (s, C1′′); 130.63 (s, C2′/C6′); 131.72 (s, C3); 131.99 (s,
C3′/C5′); 132.37 (d, 2JC-F ) 8.3 Hz, C2′′/C6′′); 136.11 (s, C1′); 139.63
(s, C4); 146.92 (s, C5); 162.92 (d, JC-F ) 250; C4′′); 170.08 (s, C2).
MS, m/z (%): 358 (M+, C18H12BrFO2, 64); 360 (M + 2, 62); 340 (4);
313 (10); 279 (15); 261 (28); 235 (18); 233 (61); 220 (19); 203 (7);
183 (5); 169 (10); 143 (23); 136 (39); 115 (66); 108 (100); 89 (33); 75
(12); 63 (23); 51 (13).
Compounds 8b–8l were prepared employing a procedure similar to
that described for compound 8a, and yields are presented in the Results.
The synthesized compounds were fully characterized by IR, NMR (1H
and 13C), and MS spectrometry. HMBC and HSQC bidimensional
experiments helped in the 13C assignments. Structures of lactones 8a–8l
were supported by the following spectroscopic data.
Data for (5Z)-3(4-Bromobenzyl)-5-(1,3-dioxalanebenzylidene)-5H-
furan-2-one (8a). Yellow solid; m.p. 175.6–176.0 °C. IV (KBr, cm-1):
j
Data for (5Z)-3(4-Bromobenzyl)-5-(4-bromobenzylidene)-5H-furan-
νmax 3102; 2891; 1729; 1650; 1599; 1499; 1488; 1267; 1045; 1034;
2-one (8f). White solid; m.p. 164.3–165.2 oC. IV (KBr, cm-1): νmax
940. 1H NMR (400 MHz, CDCl3): δ 3.66 (s, 2H, H7); 5.81 (s, 1H,
H6); 5.99 (s, 2H, OCH2O); 6.80 (d, 1H, J ) 8.2 Hz, H5′′); 6.93 (s,
1H, H4); 7.11 (dd, 1H, J ) 8.2, 1.6 Hz, H6′′); 7.13 (d, 2H, J ) 8.3
Hz, H2′/H6′); 7.42 (d, 1H, J ) 1.6 Hz, H2′′); 7.46 (d, 2H, J ) 8.3 Hz,
H3′/H5′). 13C NMR (100 MHz, CDCl3): δ 31.11 (C7); 101.50
(OCH2O); 108.58 (C5′′); 109.98 (C2′′); 113.07 (C6); 120.87 (C4′);
125.87 (C6′′); 127.39 (C1′′); 130.63 (C2′/C6′); 130.75 (C3); 131.95
(C3′/C5′); 136.33 (C1′); 139.69 (C4); 146.06 (C5); 148.28 (C3′′); 148.53
(C4′′); 170.21 (C2). MS, m/z (%): 384 (M+, C19H13BrO4, 82); 386 (M
+ 2, 80); 339 (6); 304 (8); 276 (7); 259 (10); 219 (11); 189 (17); 169
(15); 162 (70); 134 (78); 115 (47); 104 (39); 89 (29); 76 (100); 63
(25); 50 (55).
j
3111; 1769; 1756; 1643; 1577; 1487; 1405; 1311; 1279; 1144; 1033;
813. 1H NMR (400 MHz, CDCl3): δ 3.67 (s, 2H, H7); 5.82 (s, 1H,
H6); 6.95 (s, 1H, H4); 7.13 (d, 2H, J ) 8.3 Hz, H2′/H6′); 7.47 (d, 2H,
J ) 8.3 Hz, H3′/H5′); 7.49 (d, 2H, J ) 8.6 Hz, H3′′/H5′′); 7.59 (d,
2H, J ) 8.6 Hz, H2′′/H6′′). 13C NMR (100 MHz, CDCl3): δ 31.19
(C7); 111.65 (C6); 121.01 (C4′); 123.33 (C4′′); 130.63 (C2′/C6′); 131.79
(C2′′/C6′′); 131.93 (C3); 132.02, 132.04 (C3′/C5’and C3′′/C5′′); 132.28
(C1′′); 135.98 (C1′); 139.55 (C4); 147.65 (C5); 169.87 (C2). MS, m/z
(%): 418 (M+, C18H12Br2O2, 29); 420 (M + 2, 60); 422 (M+4, 26);
375 (3); 339 (2); 323 (8); 311 (1); 295 (4); 260 (9); 232 (10); 215
(24); 203 (11); 169 (9); 143 (11); 115 (32); 101 (16); 89 (100); 75 (6);
63 (27); 50 (8).
Data for (5E)-3(4-Bromobenzyl)-5-(2,4,6-trimethoxybenzylidene)-5H-
furan-2-one (8b). Yellow solid; m.p. 155.8–156.5 °C. IV (KBr, cm-1):
Data for (5Z)-3(4-Bromobenzyl)-5-(4-chlorobenzylidene)-5H-furan-
2-one (8g). White solid; m.p. 148.9–150.5 °C. IV (KBr, cm-1) νmax
j
j
νmax 3142; 2990; 2946; 2840; 1733; 1598; 1573; 1462; 1337; 1218;
1160; 1060; 821. 1H NMR (400 MHz, CDCl3): δ 3.64 (s, 2H, H7);
3.74 (s, 6H, 2′′/6′′-OCH3); 3.83 (s, 3H, 4′′-OCH3); 6.12 (s, 2H, H3′′/
H5′′); 6.56 (s, 1H, H6); 7.07 (s, 1H, H4); 7.11 (d, 2H, J ) 8.3 Hz,
H2′/H6′); 7.43 (d, 2H, J ) 8.3 Hz, H3′/H5′). 13C NMR (100 MHz,
CDCl3): δ 31.25 (C7); 55.44 (4′′-OCH3); 55.64 (2′′/6′′-OCH3); 90.81
(C3′′/C5′′); 103.73 (C1′′); 106.12 (C6); 120.64 (C4′); 130.66 (C2′/C6′);
131.77 (C3′/C5′); 132.09 (C3); 136.76 (C1′); 137.90 (C4); 148.53 (C5);
158.72 (C2′′/C6′′); 162.00 (C4′′); 170.05 (C2). MS, m/z (%): 430 (M+,
C21H19BrO5, 98); 432 (M + 2, 100); 387 (2); 361 (6); 265 (3); 233 (6);
208 (15); 205 (9); 193 (22); 181 (30); 166 (54); 165 (24); 151 (23);
138 (52); 115 (39); 109 (13); 69 (23); 63 (19); 53 (15).
3110; 2928; 1770; 1758; 1645; 1584; 1488; 1407; 1091; 1034; 1012.
1H NMR (400 MHz, CDCl3): δ 3.68 (s, 2H, H7); 5.84 (s, 1H, H6);
6.95 (s, 1H, H4); 7.13 (d, 2H, J ) 8.2 Hz, H2′/H6′); 7.34 (d, 2H, J )
8.4 Hz, H3′′/H5′′); 7.47 (d, 2H, J ) 8.2 Hz, H3′/H5′); 7.66 (d, 2H, J
) 8.4 Hz, H2′′/H6′′). 13C NMR (100 MHz, CDCl3): δ 31.18 (C7);
111.60 (C6); 121.01 (C4′); 129.08 (C3′′/C5′′); 130.64 (C2′/C6′); 131.53
(C3); 131.60 (C2′′/C6′′); 132.02 (C3′/C5′); 132.19 (C4′′); 134.96 (C1′);
136.02 (C1′′); 139.55 (C4); 147.55 (C5); 169.90 (C2). MS, m/z (%):
374 (M+, C18H12BrClO2, 43); 376 (M + 2, 56); 378 (M + 4, 14); 321
(9); 311 (1); 295 (9); 277 (11); 259 (5); 232 (13); 215 (31); 203 (15);
193 (4); 169 (7); 152 (22); 143 (15); 124 (33); 115 (5); 101 (26); 89
(100); 63 (40); 49 (21); 44 (91); 32 (36).
Data for (5Z)-3(4-Bromobenzyl)-5-(benzylidene)-5H-furan-2-one
(8c). White solid; m.p. 127.3– 27.9 °C. IV (KBr, cm-1): νmax 3102;
j
Data for (5Z)-3(4-Bromobenzyl)-5-(2,5-dimetoxybenzylidene)-5H-
furan-2-one (8h). Fluorescent yellow solid; m.p. 84.0–85.3 °C. IV (KBr,
3058; 3022; 1764; 1649; 1609; 1486; 1362; 1026; 930; 759. 1H NMR
(400 MHz, CDCl3): δ 3.68 (brs, 2H, H7); 5.89 (s, 1H, H6); 6.96 (t,
1H, J ) 1.4 Hz, H4); 7.14 (d, 2H, J ) 8.4 Hz, H2′/H6′); 7.30 (t, 1H,
J ) 7.2 Hz, H4′′); 7.39 (t, 2H, J ) 7.2 Hz, H3′′/H5′′); 7.46 (d, 2H, J
) 8.4 Hz, H3′/H5′); 7.73 (d, 2H, J ) 7.2 Hz, H2′′/H6′′). 13C NMR
(100 MHz, CDCl3): δ 31.14 (C7); 113.06 (C6); 120.93 (C4′); 128.81
(C3′′/C5′′); 129.03 (C4′′); 130.49 (C2′′/C6′′); 130.62 (C2′/C6′); 131.75
(C3); 131.97 (C3′/C5′); 133.02 (C1′′); 136.16 (C1′); 139.74 (C4); 147.29
(C5); 170.18 (C2). EM, m/z (%): 340 (M+, C18H13BrO2, 41); 342 (M
+ 2, 38); 322 (3); 295 (5); 261 (9); 243 (23); 233 (13); 217 (16); 216
(30); 215 (41); 202 (18); 189 (3); 171 (8); 156 (3.1); 143 (13); 115
(56); 101 (13); 91 (15); 90 (100); 77 (9); 63 (27); 51 (15).
cm-1): νmax 2954; 2835; 1758; 1645; 1607; 1581; 1498; 1236; 1023;
j
1
884. H NMR (400 MHz, CDCl3): δ 3.67 (s, 2H, H7); 3.81, 3.80 (s,
6H, 3′′-OCH3 and 6′′-OCH3); 6.41 (s,1H, H6); 6.80 (d, 1H, J ) 9.0
Hz, H3′′); 6.85 (dd, 1H, J ) 9.0, J ) 2.9 Hz, H4′′); 6.98 (s, 1H, H4);
7.14 (d, 2H, J ) 8.3 Hz, H2′/H6′); 7.46 (d, 2H, J ) 8.3 Hz, H3′/H5′);
7.72 (d, 1H, J ) 2.9 Hz, H6′′). 13C NMR (100 MHz, CDCl3): δ 31.13
(C7); 55.92, 56.25 (2′′-OCH3 and 5′′-OCH3); 106.85 (C6); 111.80 (C3′′);
115.74 (C6′′); 116.65 (C4′′), 120.87 (C4′); 122.60 (C1′′); 130.65 (C2′/
C6′); 131.15 (C3); 131.94 (C3′/C5′); 136.33 (C1′); 140.13 (C4); 147.28
(C5); 152.16 (C2′′); 153.77 (C5′′); 170.26 (C2). MS, m/z (%): 400 (M+,
C20H17BrO4, 100); 402 (M + 2, 98); 357 (7); 329 (7); 306 (4); 278
(23); 261 (7); 250 (14); 234 (9); 178 (10); 163 (39); 153 (8); 136 (38);
123 (15); 115 (31); 92 (20); 77 (33); 63 (21); 51 (17); 44 (75); 32
(33).
Data for 5(Z)-3(4-Bromobenzyl)-5-(3-methylbenzylidene)-5H-furan-
2-one (8d). White solid; m.p. 111.2–111.9 °C. IV (KBr, cm-1): νmax
j
1
3108; 1747; 1648; 1488; 1419; 1341; 1072; 1034; 914; 899; 793. H
NMR (400 MHz, CDCl3): δ 2.37 (s, 3H, 3′′-CH3); 3.68 (brs, 2H, H7);
5.86 (s, 1H, H6); 6.95 (t, 1H, J ) 1.3 Hz, H4); 7.12 (d, 1H, J ) 7.6
Hz, H4′′); 7.14 (d, 2H, J ) 8.3 Hz, H2′/H6′); 7.26 (t, 1H, J ) 7.6 Hz,
H5′′); 7.47 (d, 2H, J ) 8.3 Hz, H3′/H5′); 7.54 (d, 1H, J ) 7.6 Hz,
H6′′); 7.55 (s, 1H, H2′′). 13C NMR (100 MHz, CDCl3): δ 21.41 (3′′-
CH3); 31.13 (C7); 113.32 (C6); 120.92 (C4′); 127.77 (C6′′); 128.71
(C5′′); 129.97 (C4′′); 130.65 (C2′/C6′); 131.01 (C2′′); 131.56 (C3);
131.97 (C3′/C5′); 132.94 (C1′′); 136.22 (C1′); 138.48 (C3′′); 139.82
(C4); 147.16 (C5); 170.30 (C2). MS, m/z (%): 354 (M+, C19H15BrO2,
95); 356 (M + 2, 93); 336 (7); 311 (9); 275 (11); 257 (35)247 (21);
230 (44); 229 (45); 215 (35); 204 (18); 169 (16); 143 (26); 132 (50);
115 (89); 104 (69); 89 (47); 78 (100); 63 (34); 51 (31).
Data for (5Z)-3(4-Bromobenzyl)-5-(3-nitrobenzylidene)-5H-furan-
o
2-one (8i). Pale yellow solid; m.p. 122.8–123.9 C. IV (KBr, cm-1):
j
νmax 3097; 2936; 2892; 2838; 1754; 1653; 1609; 1570; 1530; 1352;
1
1034; 907. H NMR (400 MHz, CDCl3): δ 3.71 (s, 1H, H7); 5.93 (s,
1H, H6); 7.01 (s, 1H, H4); 7.15 (d, 2H, J ) 8.3 Hz, H2′/H6′); 7.48 (d,
2H, J ) 8.3 Hz, H3′/H5′); 7.56 (t, 1H, J ) 8.1 Hz, H5′′); 8.13 (d, 1H,
J ) 8.0 Hz, H4′′); 8.16 (d, 1H, J ) 8.0 Hz, H6′′); 8.42 (s, 1H, H2′′).
13C NMR (100 MHz, CDCl3): δ 31.26 (C7); 109.82 (C6); 121.16 (C4′);
123.17 (C4′′); 124.74 (C2′′); 129.83 (C5′′); 130.65 (C2′/C6′); 132.10
(C3′/C5′); 133.65 (C1′); 134.65 (C1′′); 135.62 (C6′′); 135.67 (C3);
139.22 (C4); 148.59 (C3′′); 148.98 (C5); 169.32 (C2). MS, m/z (%):
485 (M+, C18H12BrNO4, 45); 487 (M + 2, 45); 370 (43); 368 (44);

Photosynthesis-Inhibiting Nostoclide Analogues
J. Agric. Food Chem., Vol. 55, No. 21, 2007 8565
350 (6); 306 (11); 288 (6); 278 (13); 259 (7); 231 (15); 216 (16); 202
(34); 193 (8); 169 (15); 143 (17); 115 (99); 89 (100); 63 (74); 51 (19);
44 (67); 39 (49); 30 (9).
initiated by exposure to saturating light (800 µmol m-2 s-1), and the
rate of ferricyanide reduction was measured at 30 s intervals for 10
min against an exact blank at 420 nm. Activity was calculated over
the linear portion of the curve from a molar extinction coefficient of
1,000 M-1 cm-1. Nostoclide analogues were dissolved in DMSO so
as to obtain 25 or 50 mM solutions that were then diluted with water,
as appropriate. Their effect upon the Hill reaction was evaluated in
parallel assays in which the compounds were added to the reaction
mixture to concentrations of 5 µM or 10 µM. Each dose was carried
out at least in triplicate, and results were expressed as percentage of
untreated controls. Phosphorylating electron flow was assessed under
the same conditions, but in the presence of 0.5 mM ADP and 2 mM
K2HPO4. Unless otherwise indicated, uncoupled activity was measured
following the addition of 2 mM NH4Cl to the basal reaction mixture
containing aliquots of membrane preparations corresponding to 10 µg
of chlorophyll.
In ViVo Inhibition of Photosynthetic ActiVity Rate. The ability of the
most active compounds to interfere with the photosynthetic process in
vivo was evaluated on eukaryotic algal cells, as described in the
literature (26). Chlorella protothecoides Kruger, ATCC 30411 strain,
was grown at 24 ( 1 °C in 250 mL Erlenmeyer flasks containing 50
mL of Proteose Peptone medium under continuous light (500 µmol
m-2 s-1). Mid-log-grown cells (10 ( 2.5 mg L-1 chlorophyll) were
harvested by centrifugation for 1 min at 14000 g, and pellets were
resuspended in a proper volume of Bg11 medium to a cellular density
of 8.0 ( 0.2 mg L-1 Chl. The rate of oxygen evolution under saturating
light (>2 mmol m-2 s-1 photosynthetic active radiation) was measured
polarographically using a Hansatech (King’s Lynn, Norfolk, England)
system consisting of a DW2/2 electrode unit, an A1 stirrer, an LS2
light source with A8 fiber optic, and a CB1-D control box. All assays
were performed at 24 °C. Reaction was started by switching the light
on, and the oxygen concentration (arbitrary units) in the 1.0 mL cell
was recorded every 15 s up to 8 min. The light was then switched off,
and oxygen consumption in the dark was followed for a further 5 min,
providing a measure of the cell respiratory activity. The rate of
photosynthetic oxygen production was calculated as the sum of oxygen
evolution in the light and oxygen consumption in the dark. The
inhibitory activity of the nostoclide analogues was evaluated by adding
100 µL of freshly prepared 1.1 mM water solutions to the stirred
electrode cell (final concentration 0.1 mM). The resulting rate was
expressed as percent of that of controls to which only water was added.
At least three independent measurements were performed, and means
( SE are presented. To improve the solubility of the compounds and
increase the rate of translocation to the chloroplast, assays were also
carried out in the presence of 0.001% (v/v) Triton X-100; at this level,
well below the critical micelle concentration (0.2 mM, corresponding
to about 0.013%), the detergent is thought to be unable to interfere
with membrane functionality and was found to be ineffective on algal
photosynthetic activity (data not shown).
Data for (5Z)-3(4-Bromobenzyl)-5-(4-trifluoromethylbenzylidene)-
5H-furan-2-one (8j). White solid; m.p. 110.7–111.4 oC. IV (KBr,
cm-1)” νmax 3110; 2938; 1766; 1647; 1615; 1488; 1417; 1323; 1163;
j
1
1133; 1069; 1014. H NMR (400 MHz, CDCl3): δ 3.70 (s, 2H, H7);
5.91 (s, 1H, H6); 6.98 (s, 1H, H4); 7.14 (d, 2H, J ) 8.3 Hz, H2′/H6′);
7.48 (d, 2H, J ) 8.3 Hz, H3′/H5′); 7.61 (d, 2H, J ) 8.2 Hz, H3′′/
H5′′); 7.83 (d, 2H, J ) 8.2 Hz, H2′′/H6′′). 13C NMR (100 MHz,
CDCl3): δ 31.23 (s, C7); 110.98 (s, C6); 121.10 (s, C4′); 125.66 (d,
3JC-F ) 3.9 Hz; C3′′/C5′′); 130.44 (s, C2′′/C6′′); 130.65 (s, C2′/C6′);
132.07 (s, C3′/C5′); 133.18 (s, C3); 135.80 (s, C1′); 136.38 (s, C1′′);
139.45 (s, C4); 148.66 (s, C5); 169.65 (s, C2). MS, m/z (%): 408 (M+,
C19H12BrF3O2, 59); 410 ( M + 2, 61); 391 (6); 363 (9); 329 (23); 311
(26); 285 (16); 283 (4); 265 (7); 233 (10); 215 (32); 204 (17); 186
(14); 158 (56); 143 (21); 126 (11); 115 (100); 90 (20); 89 (50); 63
(39); 49 (34); 39 (30).
Data for (5Z)-3(4-Bromobenzyl)-5-(4-cyanobenzylidene)-5H-furan-
2-one (8k). White solid; m.p. 178.0–179.0 °C. IV (KBr, cm-1): νmax
j
3098; 3062; 2229; 1767; 1650; 1609; 1505; 1487; 1027; 937; 899; 796.
1H NMR (400 MHz, CDCl3): δ 3.70 (s, 2H, H7); 5.88 (s, 1H, H6);
6.99 (s, 1H, H4); 7.14 (d, 2H, J ) 8.3 Hz; H2′/H6′); 7.48 (d, 2H, J )
8.3 Hz, H3′/H5′); 7.64 (d, 2H, J ) 8.4 Hz, H3′′/H5′′); 7.81 (d, 2H, J
) 8.4 Hz, H2′′/ H6′′). 13C NMR (100 MHz, CDCl3): δ 31.24 (C7);
110.40 (C6); 111.84 (C4′′); 118.57 (4′′-CN); 121.14 (C4′); 130.56,
130.61 (C2′/C6′ and C2′′/C6′′); 132.07 (C3′/C5′); 132.40 (C3′′/C5′′);
133.73 (C3); 135.60 (C1′); 137.35 (C1′′); 139.27 (C4); 149.27 (C5);
169.32 (C2). MS, m/z (%): 365 (M+, C19H12BrNO2, 25); 367 (M + 2,
24); 347 (2); 320 (5); 286 (14); 268 (16); 240 (26); 230 (12); 215 (5);
196 (5); 169 (7); 143 (33); 128 (8); 115 (100); 114 (32); 101 (16); 89
(24); 75 (8); 63 (23); 51 (14).
Data for (5Z)-3(4-Bromobenzyl)-5-(2-chloro-4-dimethylaminoben-
zylidene)-5H-furan-2-one (8l). Orange solid; m.p. 155.2–156.0 °C. IV
(KBr, cm-1): νmax 3094; 2990; 2906; 2888; 2820; 1777; 1751; 1613;
j
1594; 1516; 1485; 1370; 1295; 1155; 1030; 833. 1H NMR (400 MHz,
CDCl3): δ 3.02 (s, 6H, 4′′-N(CH3)2); 3.66 (brs, 2H, H7); 6.33 (s, 1H,
H6); 6.65 (dd, 1H, J ) 9.1, J ) 2.7 Hz; H5′′); 6.71 (d, 1H, J ) 2.7
Hz, H3′′); 6.99 (t, 1H, J ) 1.3 Hz, H4); 7.14 (d, 2H, J ) 8.4 Hz,
H2′/H6′); 7.45 (d, 2H, J ) 8.4 Hz, H3′/H5′); 8.17 (d, 1H, J ) 9.1 Hz,
H6′′). 13C NMR (100 MHz, CDCl3): δ 31.09 (C7); 40.31 (4′′-(CH3)2);
109.26 (C6); 111.38 (C5′′); 112.52 (C2′′); 119.10 (C3′′); 120.80 (C4′);
129.51 (C3); 130.65 (C2′/C6′); 131.91 (C3′/C5′); 132.77 (C6′′); 136.05
(C1′′); 136.56 (C1′); 140.09 (C4); 145.71 (C5); 150.62 (C4′′); 170.60
(C2). MS, m/z (%): 417 (M+, C20H17BrClNO2, 81); 419 (M + 2, 100);
421 (M + 4, 25); 382 (2); 338 (5); 303 (2); 275 (4); 231 (4); 209 (4);
195 (48); 192 (23); 167 (39); 152 (20); 140 (9); 132 (43); 115 (40);
101 (12); 89 (45); 75 (12); 63 (18); 51 (14).
Biological Tests. EValuation of Lactones 8a–8l as Inhibitors of the
Photosynthetic Electron Transport by the Hill Reaction. The ability of
the compounds to interfere in vitro with the electron transport chain
was evaluated as previously described (25). Briefly, photosynthetically
active thylakoid membranes were isolated from market spinach
(Spinacea oleracea L.) leaves. Deveined plant material was resuspended
in 5 mL g-1 of ice-cold 20 mM N-tris(hydroxymethyl)methylglycine
(Tricine)-NaOH buffer (pH 8.0) containing 10 mM NaCl, 5 mM MgCl2
and 0.4 M sucrose and homogenized for 30 s in a blender at maximal
speed. The homogenate was filtered through surgical gauze, and the
filtrate was centrifuged at 4 °C for 1 min at 500 g; the supernatant was
further centrifuged for 10 min at 1500 g. Pelleted chloroplasts were
osmotically swollen by resuspension in sucrose-lacking buffer. The
suspension was immediately diluted 1:1 with sucrose-containing buffer,
kept on ice in the dark and used within a few hours from the preparation.
Following proper dilution with 80% (v/v) acetone, the absorbance of
each sample was determined at 645 and 663 nm, and the chlorophyll
content was calculated on the basis of Arnon’s formula. The basal rate
of photosynthetic electron transport was measured following light-driven
ferricyanide reduction. Aliquots of membrane preparations correspond-
ing to 20 µg of chlorophyll were incubated at 24 °C in 1 mL cuvettes
containing 20 mM Tricine-NaOH buffer (pH 8.0), 10 mM NaCl, 5
mM MgCl2, 0.2 M sucrose, and 1 mM K3Fe(CN)6. The assay was
RESULTS AND DISCUSSION
Synthesis of Lactones 8a–8l. For the preparation of com-
pounds 8a–8l, the synthesis of furyl 5 was required. This was
accomplished, with a fair yield, employing a slightly modified
procedure described in the literature (23) and having lactone 3
as a starting material. The treatment of lactone 3 with POCl3 in
the presence of triethylamine afforded compound 4, which was
not isolated. Subsequent reaction of 4 with diethylamine
furnished 5 in 51% yield (Figure 2). Treatment of compound
5 with n-butyllithium, followed by the addition of 4-bromoben-
zyl bromide, resulted in the formation of the substituted furan
6 (Figure 3). The crude product was treated with formic acid
(27) to provide lactone 7, with an overall yield of 76%.
The vinylogous aldol reaction (28–31) between the silyloxy
diene furan synthon and the relevant aldehydes was then
employed to achieve the synthesis of compounds 8a–8l. The
reaction of lactone 7 with the corresponding aldehydes in the
presence of tert-butyl-dimethylsilyltrifluoromethanesulfonate and

8566 J. Agric. Food Chem., Vol. 55, No. 21, 2007
Barbosa et al.
Figure 2. Synthesis of N,N,N′,N′-tetraethyldiamidophosphate (compound 5).
Table 1. In vitro Effects of Nostoclide Analogues on Ferricyanide
Reduction by Functionally Intact Chloroplasts Isolated from Spinacia
oleracea Leavesa
compd
arylidene group
5 µM
10 µM
8a
8b
8c
8d
8e
8f
8g
8h
8i
1,3-dioxalanebenzylidene
2,4,6-trimethoxybenzylidene
benzylidene
3-methylbenzylidene
4-fluorobenzylidene
4-bromobenzylidene
4-chlorobenzylidene
2,5-dimethoxybenzylidene
3-nitrobenzylidene
4-trifluoromethylbenzylidene
4-cyanobenzylidene
91.1 ( 2.5 91.0 ( 1.3
84.2 ( 1.2 79.9 ( 3.6
54.1 ( 2.4 42.9 ( 2.2
72.8 ( 0.9 69.7 ( 3.7
74.0 ( 1.5 68.5 ( 4.9
95.4 ( 1.1 92.0 ( 0.6
94.1 ( 0.9 89.4 ( 0.1
94.1 ( 4.5 71.0 ( 0.5
72.6 ( 2.2 66.9 ( 0.2
71.7 ( 1.3 48.8 ( 0.9
100.4 ( 0.9 101.4 ( 2.6
8j
8k
8l
2-chloro-4-dimethylamino benzylidene 94.7 ( 8.2 91.0 ( 2.0
6.8 ( 0.5 4.9 ( 0.7
diuron
a Basal activity was measured as described in Materials and Methods in either
the absence or presence of nostoclide analogues at a concentration of 5 or 10
µM. Each sample was carried out in triplicate, and values were expressed as
percentage ( SD of untreated controls.
Biological Activity. Because of their structural similarity
to other nostoclide analogues, some of which were shown to
inhibit the photosynthetic electron transport chain (20), the
synthesized compounds were suspected to be endowed with
biological activity against photosynthetic organisms. To
verify such a hypothesis, we evaluated their ability to lower
the rate of ferricyanide reduction by isolated spinach chlo-
roplasts. Results, summarized in Table 1, showed that most
compounds are indeed able to interfere with the light-driven
electron transport chain. With the exception only of com-
pound 8k, in all cases, the rate of the electron transfer from
water to ferricyanide was significantly (P < 0.05) reduced
at 10 µM. The effectiveness even of the most active
compounds was strikingly lower than that of the commercial
herbicide diuron, taken as a comparison term. However,
several of them were more effective than two nostoclide
analogues previously characterized as Hill reaction inhibitors
(20), and equipotent to some pyrazole derivatives recently
described as a new class of potential inhibitors of oxygenic
photosynthesis (25, 26). From a qualitative point of view,
their effect varies with the substituent of the benzylidene ring,
but at this initial stage of the research, a structure–activity
relationship analysis would be limited by the low number of
derivatives available. The two most active compounds, 8c
and 8j, bear no substituent and a trifluoromethyl moiety at
position 4 of the benzylidenic ring, respectively. When the
concentration of these nostoclide analogues was raised above
10 µM, in all cases, the resulting inhibitory effect rapidly
reached a plateau and did not increase further (Figure 4).
The lack of a dose-effect relationship for the active com-
pounds above a certain threshold may depend upon their low
solubility in water. Millimolar solutions of nostoclides were
obtainable only with organic solvents, such as DMSO.
However, those solutions had to be diluted with water, in
order to avoid any interference of the solvent with the
integrity (and thus the functionality) of the thylakoid
membranes. A certain degree of lipophilicity is usually
required for a photosynthetic inhibitor, given that in order
Figure 3. Synthetic methodology employed to prepare nostoclide ana-
o
logues 8a–8l. (i) n-BuLi, THF/HMPA (5:1), –78 C; p-C6H4BrCH2Br (ii)
HCOOH (76% yield for steps i and ii); (iii) ArylCHO, TBDMSOTf, DIPEA,
CH2Cl2, r.t., 1 h; DBU, reflux, 1–3 h.
Hunig’s base, followed by treatment of the silyl ether generated
in situ with DBU, afforded the lactones 8a–8l, with yields
ranging from 23 to 98% (Figure 3). Reaction times were not
optimized, but most reactions were complete within 3 h or less.
The direct ꢀ-elimination of the silyl ether intermediates, under
the reaction conditions, led to the formation of the corresponding
Z stereoisomers. However, in the case of compound 8b, the
formation of the E isomer was observed. This fact is probably
due to a destabilizing steric repulsion between the lactone
oxygen lone pair and the methoxy groups at both C2′′ and C6′′.
In all cases, the geometry of the double bond was confirmed
by bidimensional nuclear Overhauser enhancement spectroscopy
(NOESY).

Photosynthesis-Inhibiting Nostoclide Analogues
J. Agric. Food Chem., Vol. 55, No. 21, 2007 8567
Figure 5. Reversal of the inhibition brought about by compound 8c at
increasing ammonium concentration. Basal electron flow was enhanced
by the progressive addition of NH4Cl, which acts as an uncoupler. As a
consequence, the inhibitory effect exerted by the nostoclide at 10 µM
was progressively reduced. Data are expressed as percent of the activity
in untreated samples containing the same amount of uncoupler. Results
are mean ( SE over at least six replications from two independent
experiments.
Figure 4. Effect of increasing concentrations of the most active compounds
upon ferricyanide reduction by functionally intact chloroplasts isolated from
spinach leaves.
Table 2. Effects of the Most Active Nostoclide Analogues on Basal,
Phosphorylating, and Uncoupled Electron Flow from water to Ferricyanide
in Spinach Chloroplasts.a
compd 8c
compd 8j
activityb
Table 3. In vivo Effects of the Most Active Nostoclide Analogues on the
Photosynthetic Oxygen Evolution Rate by Intact Cells of Chlorella
protothecoidesa
basal electron flow (µM)
0
10
0
0
10
0
0
10
0
0
0
10
0
0
10
0
100 ( 3.1
44.2 ( 2.5
47.7 ( 3.8
100 ( 3.8
47.9 ( 3.0
52.1 ( 2.8
100 ( 4.1
95.6 ( 3.1
79.8 ( 3.0
phosphorylating electron flow (µM)
uncoupled electron flow (µM)
compd 8c
compd 8j
diuron
activityb
in the absence of
0
0
0
100
0
0
0
0
100
0
0
0
0
0
100
0
0
0
0
100
0
10
100
0
0
0
10
100
0
100 ( 5.9
49.9 ( 3.5***
38.5 ( 4.8***
82.6 ( 5.7NS
94.5 ( 0.7NS
100 ( 5.1
detergent (µM)
0
10
a Basal activity was measured as described in Materials and Methods.
Phosphorylating electron flow was assessed under the same conditions in the
presence of 0.5 mM ADP and 2 mM K2HPO4. Activity was uncoupled by adding
2 mM NH4Cl to the reaction mixture. b Activity was expressed as percent of untreated
samples. Values are mean ( SE over at least six replications in two independent
experiments. Control value rates were 50.0 ( 1.6, 92.9 ( 3.5, and 141.9 ( 5.8
nmol of ferricyanide reduced s-1 (mg of chlorophyll)-1 under basal, phosphorylating,
and uncoupled conditions, respectively.
+0.001% (v/v) Triton
X-100 (µM)
32.2 ( 3.3***
20.7 ( 4.9***
74.2 ( 6.9*
89.5 ( 2.1NS
0
a Activity was evaluated as detailed in Materials and Methods on cells harvested
during the mid-log phase of growth. The compounds were added to a concentration
of 0.1 mM, in either the presence or absence of detergent. Values are expressed
as percent of the rate measured in untreated controls and are means ( SE over
three replications (six for untreated controls). b Results were subjected to Anova:
*, P < 0.10; **, P < 0.05; ***, P < 0.01; NS, not significant.
to exert its effect, it needs to diffuse through both the
plasmalemma and the chloroplast envelopes. Moreover, it
has to penetrate the thylakoid membrane to reach its
molecular target, and in several cases, the presence of a
hydrocarbon side chain has been reported to greatly impact
the activity of a given scaffold, as it provides the required
lipophilicity (26, 32). Consistently, some of the most effective
commercial herbicides targeting photosynthesis show a
relatively low solubility in water, such as lenacil. Nothwith-
standing, the opposite is also true, because sufficient hydro-
philicity is needed to allow herbicides to be translocated
within the plants and reach their target inside the chloroplast.
The recorded effect may thus be ascribed to the small
concentration of a given nostoclide analogue that is able to
dissolve in the buffer in which chloroplasts are resuspended.
To gain more information about their mode of action, we
also assessed the inhibitory properties of the two most active
compounds (8c and 8j) under phosphorylating or uncoupled
conditions. Results are shown in Table 2. In the presence of
substrates of ATP synthase, both compounds retained their
ability to inhibit ferricyanide reduction, and the residual
activity did not differ significantly from that evaluated under
basal conditions. On the contrary, the uncoupled electron flow
was found to be almost insensitive for the presence of the
compounds at a concentration at which the basal rate was
inhibited by 50%. Because the actual concentration of the
inhibitors could not be raised above such a level because of
their poor solubility (Figure 4), the effect of the same fixed
concentration was investigated at increasing levels of am-
monium, which acts as the uncoupler. Results (Figure 5)
confirmed that the inhibition brought about by compound 8c
is progressively reverted when increasing NH4Cl concentra-
tions dissipate the proton gradient across the tylakoid
membrane. This suggests that these nostoclide analogues may
inhibit phosphorylation in chloroplasts, acting as energy-
transfer inhibitors through an interaction with the coupling
factor, CF0-CF1. A similar mode of action has been reported,
for instance, in the case of euparin, a phytotoxic substance
isolated from Helianthella quinquenerVis (33), and chalepensin,
a coumarin isolated from Stauranthus perforatus (34), which
also inhibit basal and phosphorylating electron flow without
affecting the uncoupled rate.
Finally, the actual ability of compounds 8c and 8j to
interfere with the photosynthetic activity was evaluated in
vivo by measuring the light-driven oxygen production in
intact Chlorella cells. The results are outlined in Table 3.
Even though a higher dose was used (100 µM), one at which
the maximal level of compound that can dissolve in water is
most likely present, the effect of nostoclide analogues was

8568 J. Agric. Food Chem., Vol. 55, No. 21, 2007
Barbosa et al.
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quite poor. In the absence of detergents, the mild reduction
of the rate of oxygen evolution with respect to controls was
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the efficacy previously shown in vitro by the two compounds
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Conclusions. Several 3-(4-bromobenzyl)-5-(arylmethyl-
ene)-5H-furan-2-one lactones, analogues to the naturally-
occurring toxins nostoclides, were synthesized and purified.
Their potential as inhibitors of the photosynthetic process in
the eukaryotic cell was investigated. Several of them were
able to interfere with the light-driven ferricyanide reduction
by isolated chloroplasts, most likely at the level of the
coupling factor. However, their poor solubility in aqueous
environments severely limited their efficacy, as significant
rates of the electron transport chain were still present at
saturating concentrations. Because of the higher concentration
needed for Hill reaction inhibition, these compounds seem,
therefore, not to be satisfactory for use as agrochemicals.
However, their inhibitory potential is higher than that of other
nostoclide analogues previously characterized in our labo-
ratories (20). Thus, the most active derivatives could represent
structures to be further exploited for the design of new
substances endowed with herbicidal activity. Work is cur-
rently under way to achieve the synthesis of new nostoclide
analogues showing better photosynthesis-inhibiting properties.
ACKNOWLEDGMENT
The authors thank Mr. Andrea Duzzi and Lauro Gherardini
for technical assistance, and Kimberly Epps from University
of Florida for revising the manuscript.
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Received for review July 16, 2007. Revised manuscript received August
15, 2007. Accepted August 23, 2007. We are grateful to the following
Brazilian agencies: Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq) for research fellowships (LCAB, RRT, MER) and
financial support, and Fundação de Amparo à Pesquisa de Minas Gerais
(FAPEMIG) and FINEP for financial support. Partial support from the
University of Ferrara (Fondo d’Ateneo 2006 & 2007) is also gratefully
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