Efficient synthesis of lactonic and thionolactoniclignans and evaluation of their anti-oxidant, anti-inflammatory and cytotoxic activities

Article abstract of DOI:10.1007/s00044-016-1709-5

A short and versatile synthesis of α,β-dibenzyl-γ-butyrolactones has been developed starting from inexpensive materials by applying Stobbe condensation and following the novel reductive cyclisation with sodiumborohydride. Natural products Suchilactone (5a

Full text of DOI:10.1007/s00044-016-1709-5

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1709-5
ORIGINAL RESEARCH
Efficient synthesis of lactonic and thionolactoniclignans and evaluation
of their anti-oxidant, anti-inflammatory and cytotoxic activities
1
Hari Krishna Kancharla1 Bharani Meka1
●
●
●
Trimurtulu Golakoti
Y. L. N. Murthy2
Received: 29 May 2015 / Accepted: 8 July 2016
© Springer Science+Business Media New York 2016
Abstract A short and versatile synthesis of α,β-dibenzyl-γ-
butyrolactones has been developed starting from inexpen-
sive materials by applying Stobbe condensation and
following the novel reductive cyclisation with sodiumbor-
ohydride. Natural products Suchilactone (5a), Kaerophyllin
(5b), savinin (5d) and anhydropodorhizol (5f) were pre-
pared in racemic form. New thionolactones (6a–6e) were
prepared by treating these lignans with Lawesson’s reagent.
For all the prepared compounds the antioxidant, anti-
inflammatory (5-lipoxygenase inhibition) and cytotoxic
(Brine shrimp lethality test) activities were tested. The
compounds 5a, 5b, 5c, 5d, 5e and 5f showed good
5-lipoxygenase inhibition activity. The thiono lignan 6d
showed impressive cytotoxic activity.
of medicinal properties. Many lignan containing plants have
been used for centuries, particularly in Asian communities,
as cures and remedies for various ailments. (Ayres and
Loike, 1990; Muhammad et al., 2005). Numerous synthetic
strategies have been reported for the chemical synthesis
of many lignan compounds (Robert, 2003; Kumada et al.,
1980; Feringa et al., 1994). Natural lignans belonging to the
γ-butyrolactone series have been interesting targets to syn-
thetic chemists, due to their broad and potent biological
properties, including antitumoral activity and cytotoxicity.
For example podophyllotoxin, steganacin, 5-methox-
ypodophyllotoxin, (-)-pluviatolide, (-)-hinokinin (Middel
et al., 1995), and burseran (Tomioka et al., 1985) are all
well known as antitumor compounds. As a result, many
synthetic routes for the preparation of important lignans
have been reported (Moritani et al., 1996; Bella et al., 1999;
Xia et al., 2009, 2010). Lignans exhibit interesting modes of
action. Podophyllotoxin for example exerts antitumor
actions by targeting microtubules. Microtubules are highly
dynamic assemblies of heterodimers of α and β-tubulins and
they are important in the execution of key cellular events
during cell reproduction. Podophyllotoxin inhibits the
assembly of tubulin into microtubule and disrupts the
dynamic equilibrium between microtubules and tubulin
leading to the destruction of the cytoskeletal framework and
arrest of cell division at the mitotic stage of the cell cycle by
destroying the spindle fibers and preventing the duplicated
chromosomes from separating. Another γ-butyrolactone
containing lignan, savinin (5d) was reported to exhibit anti-
inflammatory activity through potentially inhibiting TNF-α,
an important pro-inflammatory cytokine produced by
monocytes and macrophages (Jae et al. 2001). The
γ-butyrolactone containing lignans may explain some of the
biological activities attributed to the plants containing these
compounds. The γ-butyrolactone ring was conceived to be a
●
Keywords α,β-dibenzyl-γ-butyrolactones Thionolactones
●
●
●
Lawesson’s reagent Anti-oxidant 5-Lipoxygenase
●
inhibition activity Brine shrimp lethality test
Introduction
Lignans are an extremely large class of natural products
with very high structural diversity and extraordinary range
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-016-1709-5) contains supplementary material,
which is available to authorized users.
* Trimurtulu Golakoti
research@lailanutra.in
1
Laila Impex R&D Centre, Unit-I, Phase-III, Jawaharautonagar,
Vijayawada, Andhra Pradesh 520007, India
2
Andhra University, Visakhapatnam, Andhra Pradesh 530003,
India

Med Chem Res
key pharmacophore. Herein we report simple and con-
venient methods for the synthesis of lactonic and thiono-
lactonic lignans and also evaluation of their biological
[antioxidant, 5-lipoxygenase inhibitory activity (5-LOX) and
brine shrimp lethality test (BSLT)] properties. The lignans of
the current investigation are expected to show anti-inflam-
matory and cytotoxic activity due to their close resemblance
with the structures of savinin and podophyllotoxin.
of the aromatic aldehydes (1a–c) with dimethylsuccinate in
presence of NaOMe and MeOH gave the intermediate
compounds 3-methoxycarbonyl-4-(substitutedphenyl)but-3-
enoic acids 2a–c. The compounds 2a–c on esterification
with MeOH in H2SO4 yielded diester compounds 3a–c.
These intermediates 3a–c again on Stobbe condensation
with aromatic aldehydes produced the compounds (2E,3E)-
3-(methoxycarbonyl)-2-(substitutedbenzyli-dene)-4-(substi-
tutedphenyl)but-3-enoic acids (4a–f) (Das et al., 1996).
These precursor compounds 4a–f on reductive cyclisation
with sodium-borohydride gave the target α,β-dibenzyl-γ-
butyrolactones 5a–f in one step (Table 1). In this reaction
with sodiumborohydride we were able to reduce the double
bond and also the ester group to obtain the alcohol in one
step, which on acidic work up gave the cyclized product.
These α,β-dibenzyl-γ-butyrolactones 5a–f on treatment
with Lawesson’s reagent (A) (Ozturk et al., 2007) in toluene
at 100 °C furnished the novel thiono α,β-dibenzyl-γ-butyr-
olactones 6a–e (Table 1).
Results and discussion
Chemistry
Synthesis of compounds
We explored an alternative novel strategy to synthesize
the lignans having α,β-dibenzyl-γ-butyrolactones structure
(Scheme 1). Stobbe condensation (Jhonson and Daub, 1951)
Scheme 1 The substitutions of all compounds is given in Table 1. a (CH2COOCH3)2, NaOMe, MeOH, 80–85 %; b H2SO4, MeOH, 90–95 %; c
aldehyde, NaOMe, MeOH, 80–85 %; d NaBH4, THF/MeOH, 90 °C, 60–65 %; e Lawesson’s reagent, toluene, 100 °C, 85–90 %

Med Chem Res
Biological activity
micro plate spectropho-tometer (BIO-RAD) (Pater Rajesh
and Patel Natver, 2011). The cytotoxic activity of the
prepared compounds was tested by brine shrimp lethality
test (Meyer, 1982) and the anti-inflammatory activity was
checked by 5-lipoxygenase inhibition assay by micro plate
spectrophotometer (Chung, 2009). The ED50/IC50 values
are summarized in Table 2. The molarity unit values of the
biolog-ical activities were summarized in Table 3.
All the prepared compounds were tested for superoxide
scavenging activity (antioxidant activity) using X—mark
Table 1 Structure of compounds
Structure R1
R2
R3
R4
R5
R6
number
1a
1b
1c
2a
2b
2c
3a
3b
3c
4a
4b
4c
4d
4e
4f
–OCH2O–
H
–
–
–
–OCH3
–OCH3
–OCH3
H
–
–
–
Anti-inflammatory activity
–OCH3
–OCH3
–
–
–
–OCH2O–
–OCH3
H
–
–
–
–OCH3
–OCH3
H
–
–
–
The compounds 5a (ED50 8.95 μg/mL), 5b (12.18), 5c
(25.12), 5d (15.96), 5e (22.42) and 5f (22.96) showed good
–OCH3
–OCH3
–
–
–
–OCH2O–
–OCH3
H
–
–
–
–OCH3
–OCH3
H
–
–
–
Table 3 Biological activity reports of the prepared compounds (5a–5f
and 6a–6e) (micromolar)
–OCH3
–OCH3
–
–
–
–OCH2O–
–OCH3
H
–OCH3
–OCH2O–
–OCH3
–OCH2O–
–OCH3
–OCH2O–
–OCH3
–OCH2O–
–OCH3
–OCH2O–
–OCH3
–OCH2O–
–OCH3
–OCH2O–
–OCH3
–OCH3
–OCH2O–
–OCH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Structure number BSLTb molarity unit
5-LOXc molarity unit
(μm)
–OCH3
–OCH3
H
–OCH3
H
–OCH3
–OCH3
–OCH3
–OCH3
–OCH3
–OCH3
(μm)
–OCH2O–
–OCH3
H
5a
105.6793
120.8152
193.8802
292.4148
173.6715
136.1307
153.6458
2411.1
24.3207
33.0978
65.4167
45.3409
54.1546
57.6884
›260.4167
92.5521
›250.0
–OCH3
–OCH3
–OCH3
–OCH3
H
–OCH3
5b
5a
5b
5c
5d
5e
5f
–OCH2O–
–OCH3
5c
–OCH3
–OCH3
H
5d
–OCH3
H
5e
–OCH2O–
–OCH3
H
–OCH3
–OCH3
–OCH3
–OCH3
H
5f
–OCH3
6a
6a
6b
6c
6d
6e
–OCH2O–
–OCH3
6b
–OCH3
–OCH3
–OCH3
–OCH3
H
6c
272.8
–OCH3
H
–OCH3
–OCH3
–OCH3
–OCH3
–OCH3
6d
9.814
232.5581
–OCH3
6e
165.2657
8.5349
241.5459
Podophyllotoxina
Curcumina
24.9185
Table 2 Biological activity reports of the prepared compounds 5a–5f
and 6a–6e (ED50, IC50 values of BSLT, 5-LOX and NBT activities)
Reference compounds used as positive controls
a
Structure
number
BSLTa ED50
(μg/mL)
5-LOXb ED50
(μg/mL)
NBTc IC50
(μg/mL)
All the compounds were of the purity >98 % by HPLC
b
Brine shrimp lethality test
c
5a
38.89
44.46
74.45
102.93
71.90
54.18
59.00
925.86
109.12
4.22
8.95
›100
›100
›100
›100
›100
›100
›100
›100
›100
›100
›100
5-lipoxygenase inhibitory activity
5b
12.18
25.12
15.96
22.42
22.96
›100
5c
5d
5e
5f
6a
6b
35.54
6c
›100
›100
›100
6d
6e
68.42
3.67
Podophyllotoxind
Curcumind
Gallic acidd
9.17
1.40
All the compounds were of the purity >98 % by HPLC
a
brine shrimp lethality test
b
5-lipoxygenase inhibitory activity
c
nitro blue tetrazolium chloride assay
d
Reference compounds used as positive controls

Med Chem Res
5-lipoxygenase inhibition activity. Presence of a methyle-
nedioxy substitution on one aryl group and two electron-
donar methoxy substitutions on second aryl group in α,
β-dibenzyl-γ-butyrolactones (5a and 5b) seems to induce
superior 5-lipoxygenase inhibition than the corresponding
lignans with four methoxyl groups (5c) or five-methoxyl
groups (5e). The compounds 6a–6e obtained by replacing
ketonic oxygen by sulphur in α,β-dibenzyl-γ-butyrolactone
moiety either lost or showed very poor 5-LOX inhibition
when compared to their corresponding oxygen containing
cousins 5a, 5b, 5c, 5e and 5f respectively. In addition, the
observed data for compounds 6a–6e is difficult to rationa-
lize as the trend does not match with that observed for the
compounds 5a–5f.
retained the BSLT activity though at a diminished level
when compared to 5a–5f, but compound 6d is an exception.
It showed brine shrimp lethality closely equal to the positive
control podophyllotoxin.
Cytotoxic activity
The thionolactone compounds (6a–6e) exhibited good
BSLT activity, but it varied significantly from 5a–5c, 5e
and 5f respectively. The compounds 6a and 6e corre-
sponding to the lactones 5a and 5f retained the activity but
at slightly diminished level. The thionolactones 6b and 6c
significantly lost the activity when compared to the corre-
sponding lactones 5b and 5c. Interestingly, the thiono-
lactone 6d showed highly potent brine shrimp lethality with
an ED50 value of 4.22 µg/mL, which is comparable to the
ED50 value 3.67 µg/mL exhibited by the positive control
podophyllotoxin. The overall trend of compounds 6a–6e
does not match with that observed for compounds 5a–5f.
Structure activity relationship
The α,β-dibenzyl-γ-butyrolactones (5a–5f) showed potent
5-LOX inhibitory activity and three of them 5a, 5b and 5d
closely matched the efficacy of curcumin, which is com-
monly considered as a natural bench mark for anti-inflam-
matory activity. The compounds (5a and 5b) with a
methylenedioxy substitution on one aryl group and two
electron-donor methoxy substitutions on second aryl group
in α,β-dibenzyl-γ-butyrolactones showed better BSLT
activity and 5-lipoxygenase inhibition than savinin (5d), a
natural metabolite with methylenedioxy group on both the
aryl groups. The thionolactone compounds (6a–6e)
obtained by replacing lactone oxygen with sulfur, surpris-
ingly either lost the 5-LOX inhibition or showed highly
diminished activity when compared to the corresponding
lactone analogs including savinin. Podophyllotoxin is a
cyclized version related to the compounds 5a–5f and it is
highly potent compared to α,β-dibenzyl-γ-butyrolactones
(5a–5f). Annulation of the lactone ring with the aromatic
ring as observed in podophyllotoxin may impart superior
biological properties, when compared to the corresponding
uncyclized analogs (5a–5f). The thionolactonic analogs
Conclusion
In conclusion, we have described a flexible and novel route
for the preparation of 4-(subtitu-tedbenzyl)-3-(sub-
titutedbenzylidene)-dihydro-furan-2(3H)-one lignans start-
ing from simple aromatic aldehydes through a four-step
synthetic sequence, which involves a novel one step
reductive cyclisation with sodium-borohydride in the final
step. In addition some new (E)-4-(substituted benzyl)-3-
(substituted benzylidene)dihydrofuran-2(3H)-thionolignans
were synthesized from 4-(substituted benzyl)-3-(substituted
benzylidene)dihydrofuran-2(3H)-one lignans by treating
with Lawesson’s reagent. The biological (antioxidant,
5-LOX, and BSLT) activities of these compounds were
tested. α,β-dibenzyl-γ-butyrolactones with the methylene-
dioxy substitution on one aryl group and methoxy sub-
stitutions on the 2nd aryl group (5a and 5b) exhibited good
5-lipoxygenase inhibition activity. The compounds with
more number of methoxy substitutions on the aryl groups of
thiono lignans showed good cytotoxic activity.

Med Chem Res
Experimental
General procedure for the preparation of (2E,3E)-
3-(methoxycarbonyl)-2-(substituted-benzylidene)-
4-(substitutedphenyl)but-3-enoic acid 4(a–f)
General considerations
All the reagents used in this work were laboratory reagent
grade. Melting points were determined on MEL-TEMP
capillary melting point apparatus and are uncorrected. H
A mixture of (E)-dimethyl-2-(substitutedbenzylidene)suc-
cinate 3(a–c, 7.2 mmol) and aldehyde (7.6 mmol) in
methanol (50 mL) was added to a solution of NaOMe
(15 mmol) in methanol (100 mL). The mixture was heated
under reflux for 2 h, and then the reaction mixture was
cooled and acidified with HCl (2 N). The mixture was then
extracted with Et2O (3 × 100 mL). The Et2O fraction was
then re-extracted with aq. NaHCO3 (3 × 75 mL). Acid-
ification of the aq. NaHCO3 extract with con. HCl provided
an oily layer. This was then extracted with CH2Cl2 (3 × 100
mL), the combined CH2Cl2 layer was washed with water
(3 × 30 mL), dried on anhydrous Na2SO4 and evaporated to
yield compound as a yellow solid. This was recrystallized
from methanol to get pure compound 4 (a–f).
1
NMR spectra were recorded on Bruker Avance-400 MHz
instrument. 13C NMR spectra were obtained at 100 MHz.
Chemical shifts were expressed in ppm downfield relative
to tetramethylsilane, and the coupling constants (J) are
reported in Hertz. The LCMS spectra were obtained on
Agilent-1100 spectrometer. Silica gel (100–200 mesh) was
used for the column chromatography. High Resolution
Mass Spectra were recorded on UPLC-Acquity-XEVO-G2-
Q Tof. Purity of the compounds was determined on waters
alliance 2695 HPLC system.
General procedure for the preparation of
3-methoxycarbonyl)-4-(substitutedphenyl)but-3-enoic
acid (2a–c)
General procedure for the preparation of
4-(substitutedbenzyl)-3-(substitutedbenzylide-ne)
dihydrofuran-2(3H)-one (5a–f)
A mixture of aldehyde 1(a–c), 66.6 mmol) and dimethyl
succinate (79.6 mmol) in methanol was added to a solution
of NaOMe (100 mmol, prepared by dissolving Na metal in
dry methanol and followed by removing the excess
methanol) in methanol (100 mL). The mixture was heated
under reflux for 2 h, cooled and acidified with HCl (2 N).
This mixture was then extracted with Et2O (3 × 100 mL).
The Et2O fraction was then back-extracted with aq.
NaHCO3 (3 × 75 mL). Acidification of the aq. NaHCO3
extract with con. HCl provided an oily residue. This residue
was then extracted with CH2Cl2 (3 × 100 mL) and the
extract was washed with water (2 × 30 mL), dried over
NaSO4 and evaporated to yield a yellow solid. This was
recrystallized from methanol to get the intermediate com-
pound 2(a–c).
The suitable 3-(methoxycarbonyl)-2-(substitutedbenzylide-
ne)-4-(substitutedphenyl)but-3-enoic acid 4(a–f) (1.2 mmol)
in THF (30 mL) and NaBH4 (2.5 mmol) was heated at 70 °C
for 0.5 h. Then methanol (5 mL) was added at the same
temperature for 2 h. Stir the reaction mixture at reflux for
6 h. The reaction mixture was poured into ice cold water,
acidified with 2 N HCl, stir for 10 min. This mixture then
was extracted with EtOAc (3 × 30 mL). The combined
EtOAc layer was washed with water, brine and dried over
anhydrous Na2SO4. The crude obtained after evaporation of
EtOAc layer was purified by silica gel column chromato-
graphy to get compound 5(a–f).
General procedure for the preparation of (E)-4-
(substitutedbenzyl)-3-(substitutedbenzylide-ne)
dihydrofuran-2(3H)-thione (6a–e)
General procedure for the preparation of (E)-dimethyl-2-
(substitutedbenzylidene)succinate 3(a–c)
A
mixture of lignan 4-(substitutedbenzyl)-3-(sub-
stitutedbenzylidene)dihydrofuran-2(3H)-one (5a, 5b, 5c, 5e
and 5f, 1 mmol), toluene and Lawesson’s reagent (1.5
mmol) was stirred at 90–100 °C for 2 h. The reaction mix-
ture was quenched with ice cold water and the mixture was
extracted with EtOAc (2 × 25 mL). The combined ethyl
acetate layer was washed with water, brine and dried over
anhydrous Na2SO4. The crude product obtained after the
evaporation of the organic layer was purified by silica gel
column chromatography to yield pure thiono lignan analogs
(6a–e).
A mixture of 3-methoxycarbonyl)-4-(substitutedphenyl)but-
3-enoic acid 2(a–c) (50 mmol) in dry methanol (50 mL) and
con. H2SO4 (2.5 mL) was refluxed for 3 h and then the
methanol was distilled off. The residue thus obtained was
chilled and extracted with Et2O (3 × 50 mL). The combined
ether extract was washed with aqueous sodium
carbonate solution (3 × 20 mL) followed by water, dried
over Na2SO4 and evaporated to give the diester (3a–c) as
yellow solid.

Med Chem Res
(E)-3-methoxycarbonyl)-4-(benzo[d][1,3]dioxol-6-yl)but-3-
(E)-dimethyl-2-(3,4-dimethoxybenzelidene)-succinate (3b)
enoic acid (2a)
Compound 3b, yield—92 %, pale yellow oil, 1H NMR
(400 MHz, CDCl3) δ: 3.60 (2H, s, 2-CH2), 3.73 (3H, s, –
COOCH3), 3.83 (3H, s, –OCH3), 3.88 (3H, s, –OCH3), 3.90
(3H, s, –COOCH3), 6.89 (1H, d, J = 8.4 Hz, arom), 6.95–
6.98 (2H, m, arom), 7.85 (1H, s, 4-H); LCMS: m/z 317 (M
+Na)+.
Compound 2a, yield—85 %, pale yellow solid, m.p. 118–
120 °C; 1H NMR (400 MHz, CDCl3) δ: 3.59 (2H, s, –CH2),
3.84 (3H, s, –COOCH3), 6.01 (2H, s, –OCH2O–), 6.90–
6.98 (3H, m, arom), 7.82 (1H, s, 4-H); LCMS: m/z 287
(M+Na)+.
(E)-dimethyl-2-(3,4,5-trimethoxybenzylidene)-succinate
(E)-3-(methoxycarbonyl)-4-(3,4-dimethoxyphe-nyl)but-3-
(3c)
enoic acid (2b)
Compound 3c, yield—90 %, pale yellow oil, 1H NMR (400
MHz, CDCl3) δ: 3.59 (2H, s, 2-CH2), 3.73 (3H, s, –
COOCH3), 3.83 (3H, s, –COOCH3), 3.85 (6H, s, 2× –
OCH3), 3.87 (3H, s, –OCH3), 6.62 (2H, s, arom), 7.84 (1H,
s, 4-H); 13C NMR (100 MHz, CDCl3) δ: 33.7 (CH2, C-3),
52.2 (CH3, –COOCH3), 52.3 (CH3, –COOCH3), 56.1 (CH3,
2× –OCH3), 60.9 (CH3, –OCH3), 106.4 (CH, C-2′, 6′),
125.2 (C, C-1′), 130.3 (C, C-2), 138.8 (CH, ØCH), 142.4
(C, C-4′), 153.2 (C, C-3′, 5′), 167.8 (C=O, C-1), 171.7
(C=O, C-4); LCMS: m/z 325 (M+H)+.
Compound 2b, yield—80 %, pale brown solid, m.p. 140–
142 °C; 1H NMR (400 MHz, CDCl3) δ: 3.63 (2H, s, –CH2),
3.86 (3H, s, –OCH3), 3.89 (3H, s, –OCH3), 3.91 (3H, s, –
COOCH3), 6.91 (1H, d, J = 12 Hz, arom), 7.00–7.02 (2H,
m, arom), 7.86 (1H, s, 4-H); 13C NMR (100 MHz, CDCl3)
δ: 33.9 (CH2, C-2), 52.4 (CH3, –COOCH3), 55.9 (CH3, –
OCH3), 56.0 (CH3, –OCH3), 111.2 (CH, C-2′), 112.3 (CH,
C-5′), 122.8 (CH, C-6′), 122.9 (C, C-1′), 127.3 (C, C-3),
142.8 (CH, C-4), 149.0 (C, C-4′), 150.1 (C, C-3′), 168.6
(C=O, –COOCH3), 176.0 (C=O, C-1); LCMS: m/z 303
(M+Na)+.
(2E,3E)-3-(methoxycarbonyl)-2-((benzo[d][1,3]-dioxol-6-
yl)methylene)-4-(3,4-dimethoxy-phenyl)but-3-enoic acid
(4a)
(E)-3-(methoxycarbonyl)-4-(3,4,5-trimethoxy-phenyl)but-3-
enoic acid (2c)
Compound 4a, yield—83 %, pale yellow solid, m.p. 120–
122 °C; ,1H NMR (400 MHz, CDCl3) δ: 3.73 (3H, s, –
OCH3), 3.74 (3H, s, –OCH3), 3.85 (3H, s, –COOCH3), 5.94
(2H, d, J = 4 Hz, –OCH2O–), 6.74 (1H, d, J = 8.0 Hz, 5′-H),
6.79 (1H, d, J = 8.8 Hz, 5″-H), 7.04 (1H, d, J = 8.4 Hz, 6′-
H), 7.07 (1H, s, 2′-H), 7.11–7.13 (2H, m, 6″, 2″-H), 7.88
(1H, s, –CHC6H5), 7.91 (1H, s, 4-H); LCMS: m/z 411
(M–H)−.
Compound 2c, yield—83 %, pale brown oil; 1H NMR (400
MHz, CDCl3) δ: 3.62 (2H, s, –CH2), 3.86 (9H, s, 3× –
OCH3), 3.88 (3H, s, –COOCH3), 6.67 (2H, s, arom), 7.85
(1H, s, 4-H); 13C NMR (100 MHz, CDCl3) δ: 34.1 (CH2, C-
2), 52.6 (CH3, –COOCH3), 56.2 (CH3, 2× –OCH3), 56.5
(CH3, –OCH3), 106.6 (CH, C-2′), 106.7 (CH, C-6′), 124.2
(C, C-1′), 129.9 (C, C-3), 139.1 (CH, C-4), 142.0 (C, C-4′),
153.3 (C, C-3′, C-5′), 168.5 (C=O, –COOCH3), 175.4
(C=O, C-1); LCMS: m/z 333 (M+Na)+.
(2E,3E)-3-(methoxycarbonyl)-2-(3,4-dimethoxy-
benzylidene)-4-(benzo[d][1,3]dioxol-5-yl)but-3-enoic acid
(4b)
(E)-Dimethyl-2-((benzo[d][1,3]dioxol-6-yl)methylene)
succinate (3a)
Compound 4b, yield—85 %, pale brown solid, m.p. 148–
152 °C; 1H NMR (400 MHz, CDCl3) δ: 3.71 (3H, s, –
OCH3), 3.75 (3H, s, –OCH3), 3.86 (3H, s, –COOCH3), 5.94
(2H, d, J = 4 Hz, –OCH2O–), 6.74 (1H, d, J = 8 Hz, 5′-H),
6.80 (1H, d, J = 8.0 Hz, 5″-H), 7.03 (1H, d, J = 8.0 Hz, 6′-
H), 7.06 (1H, s, 2′-H), 7.13–7.15 (2H, m, 2″ and 6″-H),
7.87 (1H, s, –CHC6H5), 7.93 (1H, s, 4-H); 13C NMR (100
MHz, CDCl3) δ: 55.6 (CH3, –COOCH3), 55.7 (CH3, –
OCH3), 55.8 (CH3, –OCH3), 101.5 (CH2, –OCH2O–),
108.7 (CH, C-5″), 108.8 (CH, C-2″), 110.9 (CH, C-2′),
112.2 (CH, C-5′), 123.08 (CH, C-6′), 124.3 (CH, C-6″),
126.2 (C, C-1′), 126.4 (C, C-1″), 127.2 (C, C-2), 128.7 (C,
C-3), 142.5 (CH, C-4), 144.4 (CH, ØCH), 148.1 (C, C-4″),
Compound 3a, yield—95 %, pale yellow solid, m.p. 70–72
1
°C; H NMR (400 MHz, CDCl3) δ: 3.55 (2H, s, 2-CH2),
3.74 (3H, s, –COOCH3), 3.81 (3H, s, –COOCH3), 6.00
(2H, s, –OCH2O–), 6.82–6.88 (3H, m, arom), 7.80 (1H, s,
4-H); 13C NMR (100 MHz, CDCl3) δ: 33.5 (CH2, C-3),
52.3 (CH3, 2× –COOCH3), 101.4 (CH2, –CH2O–), 108.6
(CH, C-2′), 109.1 (CH, C-5′), 124.0 (CH, C-6′), 124.4 (C,
C-1′), 128.8 (C, C-2), 141.9 (CH, ØCH–), 148.0 (C, C-3′),
(148.4 (C, C-4′), 167.9 (C=O, C-1), 171.6 (C=O, C-4);
LCMS: m/z 301 (M+Na)+.

Med Chem Res
148.8 (C, C-3″), 149.2 (C, C-4′), 150.9 (C, C-3′), 167.6
(C=O, –COOCH3), 171.1 (C=O, C-1) LCMS: m/z 411
(M−H)−.
7.04 (1H, s, 2′-H), 7.87 (1H, s, –CHC6H5), 7.89 (1H, s, 4-
H); 13C NMR (100 MHz, CDCl3) δ: 52.6 (CH3, –
COOCH3), 56.0 (CH3, 2× –OCH3), 60.9 (CH3, –OCH3),
101.5 (CH2, –OCH2O–), 107.6 (CH, C-2′), 107.9 (CH, C-
6′), 108.6 (CH, C-5″), 108.7 (CH, C-2″), 124.2 (CH, C-6″),
124.8 (C, C-1″), 126.3 (C, C-2), 128.6 (C, C-1′), 129.5 (C,
C-3), 139.9 (CH, Ø″CH), 142.6 (C, C-4′), 144.4 (CH, Ø′
CH), 148.1 (C, C-4″), 149.2 (C, C-3″), 153.0 (C, C-3′,
C-5′), 167.5 (C=0, C-4), 171.6 (C=O, C-1); LCMS: m/z
441 (M–H)−.
(2E,3E)-3-(methoxycarbonyl)-2-(3,4-dimetho-
xybenzylidene)-4-(3,4-dimethoxyphenyl)but-3-enoic acid
(4c)
Compound 4c, yield—80 %, pale yellow solid, m.p. 160–
164 °C; 1H NMR (400 MHz, CDCl3) δ: 3.72 (3H, s, –
OCH3), 3.74 (6H, s, 2× –OCH3), 3.85 (3H, s, –OCH3), 3.86
(3H, s, –COOCH3), 6.79 (2H, d, J = 8.4 Hz, 5′ and 5″-H),
7.12–7.16 (4H, m, 2′, 2″, 6′ and 6″-H), 7.91 (1H, s, –
CHC6H5), 7.95 (1H, s, 4-H); LCMS: m/z 427 (M–H)−.
(E)-4-(3,4-dimethoxybenzyl)-3-((benzo[d][1,3]-dioxol-6-yl)
methylene)dihydrofuran-2(3H)-one (5a)
(2E,3E)-3-(methoxycarbonyl)-4-(benzo[d][1,3]-dioxol-5-
yl)-2-((benzo[d][1,3]dioxol-6-yl)-methylene)but-3-enoic
acid (4d)
Compound 5a, yield—65 %, pale yellow solid, m.p. 94–
96 °C, H NMR (400 MHz, CDCl3) δ: 2.64 (1H, t, J =
1
10.4 Hz, C6H5CH2–H–), 3.01 (1H, dd, 3.2, 14 Hz,
C6H5CH2–H–), 3.62–3.76 (1H, m, 4-H), 3.85 (3H, s,
–OCH3), 3.87 (3H, s, –OCH3), 4.26–4.34 (2H, m, 5-H),
6.04 (2H, s, –OCH2O–), 6.70 (1H, s, 2″-H), 6.76–6.81
(2H, m, 6″ and 5″-H), 6.87 (1H, d, J = 8.0 Hz, 5′-H),
Compound 4d, yield—84 %, pale yellow solid, m.p. 170–
172 °C; 1H NMR (400 MHz, CDCl3) δ: 3.73 (3H, s, –
COOCH3), 5.94 (4H, s, 2× –OCH2O–), 6.73 (1H, d, J = 7.6
Hz, 5′-H), 6.74 (1H, d, J = 8.0 Hz, 5″-H), 7.00–7.05 (4H,
m, 2′, 2″, 6′ and 6″-H), 7.84 (1H, s, –CHC6H5), 7.88 (1H, s,
4-H); 13C NMR (100 MHz, CDCl3) δ: 52.5 (CH3, –
COOCH3), 101.5 (CH2, 2× –OCH2O–), 108.5 (CH, C-5′),
108.6 (CH, C-5″), 108.8 (CH, C-2′), 108.9 (CH, C-2″),
123.6 (CH, C-6″), 123.8 (CH, C-6′), 126.4 (C, C-1′), 126.6
(C, C-1″), 128.5 (C, C-2), 128.6 (C, C-3), 142.8 (CH, C-4),
144.3 (CH, ØCH), 148.0 (C, C-4′), 148.1 (C, C-4″), 149.1
(C, C-3′), 149.4 (C, C-3″), 167.4 (C=O, –COOCH3), 172.2
(C=O, C-1); LCMS: m/z 395 (M–H)−.
7.07–7.09 (2H, m, 2′ and 6′-H), 7.50 (1H, s, ØCH–); 13
C
NMR (100 MHz, CDCl3) δ: 37.7 (CH2, –CH2C6H5), 40.0
(CH, C-4), 55.9 (CH3, –OCH3), 55.9 (CH3, –OCH3), 69.7
(CH2, C-5), 101.7 (CH2, –OCH2O–), 108.5 (CH, C-2′),
108.7 (CH, C-2″), 111.7 (CH, C-5′), 112.3 (CH, C-5″),
120.9 (CH, C-6′), 126.1 (CH, C-6″), 126.2 (C, C-3),
128.3 (C, C-1′), 130.5 (C, C-1″), 137.1 (CH, C6H5CH),
148.2 (C, C-4″), 148.4 (C, C-4′), 149.2 (C, C-3′), 149.3
(C, C-3″), 172.4 (C=O, C-2); LCMS: m/z 391 (M+Na)+.
(2E,3E)-3-(methoxycarbonyl)-2-(3,4,5-trimetho-
xybenzylidene)-4-(3,4-dimethoxyphenyl)but-3-enoic acid
(4e)
(E)-3-(3,4-dimethoxybenzylidene)-4-((benzo[d]-[1,3]
dioxol-5-yl)methyl)dihydrofuran-2(3H)-one (5b)
Compound 5b, yield—60 %, pale yellow solid, m.p. 146–
148 °C; 1H NMR (400 MHz, CDCl3) δ: 2.61 (1H, m,
C6H5CH2–H–), 3.04 (1H, dd, J = 4.0, 14.4 Hz,
C6H5CH2–H–), 3.76–3.82 (1H, m, 4-H), 3.91 (3H, s,
–OCH3), 3.94 (3H, s, –OCH3), 4.24–4.28 (2H, m, 5-H),
5.93 (2H, s, –OCH2O–), 6.63 (1H, d, 7.6 Hz, 5′-H), 6.66
(1H, s, 2′-H), 6.73 (1H, d, J = 7.6 Hz, 6′-H), 6.94 (1H, d,
J = 8.8 Hz, 6″-H), 7.05 (1H, s, 2″-H), 7.22 (1H, d, J = 8.4
Hz, 5″-H), 7.54 (1H, s, ØCH–); 13C NMR (100 MHz,
CDCl3) δ: 37.4 (CH2, ØCH), 41.1 (CH, C-4), 55.9 (CH3,
–OCH3), 56.0 (CH3, –OCH3), 69.5 (CH2, C-5), 101.1
(CH2, –OCH2O–), 108.5 (CH, C-5″), 109.0 (CH, 2″),
109.1 (C, C-2′), 111.4 (CH, C-5′), 122.1 (CH, C-6″),
122.2 (CH, C-6′), 125.7 (CH, C-3), 126.9 (CH, C-1′),
131.5 (C, C-1″), 137.5 (CH, ØCH), 146.6 (C, C-4″),
148.0 (C, C-3″), 149.1 (C, 4′), 150.8 (C, 3′), 172.6 (C=O,
C-2); LCMS: m/z 391 (M+Na)+.
Compound 4e, yield—85 %, pale yellow oil, 1H NMR (400
MHz, CDCl3) δ: 3.72 (3H, s, –OCH3), 3.75 (6H, s, 2× –
OCH3), 3.76 (3H, s, –OCH3), 3.84 (3H, s, –OCH3), 3.86
(3H, s, –COOCH3), 6.79–6.81 (3H, m, arom), 7.10 (1H, d,
J = 1.6 Hz, arom), 7.13 (1H, s, arom), 7.91 (2H, s,
–CHC6H5 and 4-H); LCMS: m/z 457 (M–H)−.
(2E,3E)-3-(methoxycarbonyl)-2-(3,4,5-trimetho-
xybenzylidene)-4-(benzo[d][1,3]dioxol-5-yl)but-3-enoic
acid (4f)
Compound 4f, yield—80 %, off white solid, m.p. 86–88 °
1
C; H NMR (400 MHz, CDCl3) δ: 3.73 (3H, s, –OCH3),
3.75 (6H, s, 2× –OCH3), 3.84 (3H, s, –COOCH3), 5.95
(2H, d, J = 2.4 Hz, –OCH2O–), 6.76 (1H, d, J = 8.0 Hz, 5′-
H), 6.80 (2H, s, 2″, 6″-H), 7.02 (1H, d, J = 8.4 Hz, 6′-H),

Med Chem Res
(E)-4-(3,4-dimethoxybenzyl)-3-(3,4-dimethox-ybenzylidene)
(E)-3-(3,4,5-trimethoxybenzylidene)-4-((benzo-[d][1,3]
dihydrofuran-2(3H)-one (5c)
dioxol-5-yl)methyl)dihydrofuran-2(3H)-one (5f)
Compound 5c, yield—60 %, pale yellow solid, m.p. 128–
130 °C; H NMR (400 MHz, CDCl3) δ: 2.66 (1H, q, 4.0,
Compound 5f, yield—60 %, off-white solid, m.p. 98–100 °
C; 1H NMR (400 MHz, CDCl3) δ: 2.66 (1H, q, 9.6, 4.4 Hz,
C6H5CH2–H–), 3.03 (1H, dd, J = 4.8, 14.4 Hz, C6H5CH2–
H–), 3.82–3.86 (1H, m, 4-H), 3.88 (6H, s, 2× –OCH3), 3.90
(3H, s, –OCH3), 4.24–4.32 (2H, m, 5-H), 5.92 (2H, d, J =
3.2 Hz, –OCH2O–), 6.59–6.61 (2H, m, 2″ & 6″-H), 6.70
(1H, d, J = 7.6 Hz, 5″-H), 6.77 (2H, s, 2′ and 6′-H), 7.51
(1H, s, ØCH–); LCMS: m/z 399 (M+H)+.
1
10.4 Hz, C6H5CH2–H–), 3.09 (1H, dd, J = 4.0, 14.4 Hz,
C6H5CH2–H–), 3.68–3.78 (1H, m, 4-H), 3.86 (6H, s, 2× –
OCH3), 3.90 (3H, s, –OCH3), 3.94 (3H, s, –OCH3), 4.24–
4.29 (2H, m, 5-H), 6.67 (1H, s, 2″-H), 6.74–6.82 (2H, m, 5″
and 6″-H), 6.93 (1H, d, J = 8.4 Hz, 5′-H), 7.09 (1H, s, 2′-
H), 7.22 (1H, d, J = 8.0 Hz, 6′-H), 7.54 (1H, s, ØCH–); 13
C
NMR (100 MHz, CDCl3) δ: 37.3 (CH2, ØCH2), 39.6 (CH,
C-4), 55.9 (CH3, –OCH3), 56.0 (CH3, 3× –OCH3), 69.7
(CH2, C-5), 111.3 (CH, C-2′), 111.6 (CH, C-5′), 112.4 (CH,
C-5″), 112.9 (CH, C-2″), 120.7 (CH, C-6′), 123.9 (CH, C-
6″), 125.9 (C, C-3), 127.0 (C, C-1′), 130.4 (C, C-1″), 137.3
(CH, ØCH), 148.1 (C, C-4″), 149.1 (C, C-4′), 149.2 (C, C-
3′), 150.8 (C, C-3″), 172.6 (C=O, C-2); LCMS: m/z 407
(M+Na)+.
(E)-4-(3,4-dimethoxybenzyl)-3-((benzo[d][1,3]-dioxol-6-yl)
methylene)dihydrofuran-2(3H)-thione (6a)
Compound 6a, yield—85 %, pale yellow oil, 1H NMR (400
MHz, CDCl3) δ: 2.60 (1H, q, J = 4,0, 10.4 Hz, C6H5CH2–
H–), 3.00 (1H, dd, J = 4.0, 14.8 Hz, C6H5CH2–H–), 3.86
(1H, m, 4-H), 3.86 (3H, s, –OCH3), 3.88 (3H, s, –OCH3),
4.47 (1H, q, J = 2.8, 6.8 Hz, 5-H), 6.05 (2H, s, –OCH2O–),
6.70 (1H, d, J = 1.6 Hz, 2″-H), 6.75 (1H, dd, J = 1.6, 8.0
Hz, 6″-H), 6.81 (1H, d, J = 8.0 Hz, 5″-H), 6.90 (1H, d, J =
8.0 Hz, 5′-H), 7.14 (1H, d, J = 1.2 Hz, 2′-H), 7.18 (1H, dd,
J = 2.0, 8.4 Hz, 6′-H), 7.87 (1H, s, ØCH–); LCMS: m/z 385
(M+H)+; 13C NMR (100 MHz, CDCl3) δ: 29.7 (CH2, –
CH2Ø), 37.5 (CH, C-4), 41.3 (CH2, C-5), 55.9 (CH3, –
OCH3), 56.0 (CH3, –OCH3), 101.9 (CH2, –OCH2O–),
108.5 (CH, C-2′), 109.0 (CH, C-2″), 111.6 (CH, C-5′),
112.3 (CH, C-5″), 121.0 (CH, C-6′), 127.3 (CH, C-6″),
128.7 (CH, ØCH), 130.4 (C, C-1′), 138.0 (C, C-3), 140.2
(C, C-1″), 148.2 (C, C-4″), 148.5 (C, C-4′), 149.3 (C, C-3′),
149.5 (C, C-3″), 213.5 (C=S, C-2); HRMS m/z calculated
for C21H21O5S: 385.1110 (M+H)+; found-385.1168; HPLC
purity: 99.76 %.
(E)-4-((benzo[d][1,3]dioxol-6-yl)methyl)-3-((benzo[d][1,3]
dioxol-6-yl)methylene)dihy-drofuran-2(3H)-one (5d)
Compound 5d, yield—62 %, pale yellow solid, m.p. 145–
47 °C; 1H NMR (400 MHz, CDCl3) δ: 2.58 (1H, m,
C6H5CH2–H–), 3.00 (1H, dd, J = 4.8, 14.0 Hz, C6H5CH2–
H–), 3.73 (1H, m, 4-H), 4.23–4.27 (2H, m, 5-H), 5.93 (2H,
d, J = 1.6 Hz, –OCH2O–), 6.05 (2H, s, –OCH2O–), 6.64
(1H, d, J = 7.6 Hz, 6″-H), 6.66 (1H, s, 2″-H), 6.74 (1H, d, J
= 8.0 Hz, 5″-H), 6.88 (1H, d, J = 8.0 Hz, 5′-H), 7.05 (1H, s,
2′-H), 7.08 (1H, d, J = 8.0 Hz, 6′-H), 7.50 (1H, s, ØCH–);
13C NMR (100 MHz, CDCl3) δ: 37.5 (CH2, –CH2C6H5),
39.9 (CH, C-4) 69.5 (CH2, C-5) 101.0 (CH2, –OCH2O–),
101.7 (CH2, –OCH2O–), 108.5 (CH, C-2′), 108.6 (CH, C-
2″), 108.8 (CH, C-5′), 109.1 (CH, C-5″), 122.0 (CH, C-6′),
122.1 (CH, C-6″), 125.8 (C, C-3), 126.1 (C, C-1′) 128.2 (C,
C-1″), 131.5 (CH, ØCH), 146.6 (C, C-4″), 148.0 (C, C-4′),
148.4 (C, C-3′), 149.2 (C, C-3″), 172.5 (C=O, C-2); LCMS:
m/z 353 (M+H)+.
(E)-3-(3,4-dimethoxybenzylidene)-4-((benzo-[d][1,3]
dioxol-5-yl)methyl)dihydrofuran-2(3H)-thione (6b)
Compound 6b, yield—85 %, pale yellow oil, 1H NMR
(400 MHz, CDCl3) δ: 2.52 (1H, q, J = 3.6, 10.8 Hz,
C6H5CH2–H–), 2.95 (1H, dd, J = 3.6, 14.8 Hz, C6H5CH2–
H–), 3.81 (1H, m, 4-H), 3.85 (3H, s, –OCH3), 3.89 (3H, s, –
OCH3), 4.40–4.49 (2H, m, 5-H), 5.87 (2H, s, –OCH2O–),
6.57 (1H, d, J = 7.6 Hz, 6″-H), 6.60 (1H, s, 2″-H), 6.67 (1H,
d, J = 8.0 Hz, 5″-H), 6.89 (1H, d, J = 8.4 Hz, 5′-H), 7.03
(1H, d, J = 1.6 Hz, 2′-H), 7.24 (1H, dd, J = 1.6, 8.0 Hz, 6′-
H), 7.83 (1H, s, ØCH); LCMS: m/z 385 (M+H)+; 13C NMR
(100 MHz, CDCl3) δ: 29.6 (CH2, –CH2Ø), 37.3 (CH, C-4),
40.9 (CH2, C-5), 56.0 (CH3, –OCH3), 56.0 (CH3, –OCH3),
101.1 (CH2, –OCH2O–), 108.5 (CH, C-2′), 109.0 (CH, C-
2′), 111.4 (CH, C-5′), 113.6 (CH, C-5″),, 121.9 (CH, C-6′),
123.9 (CH, C-6″), 127.3 (CH, ØCH), 131.5 (C, C-1′), 137.7
(C, C-3), 140.6 (C, C-1″), 146.6 (C, C-4″), 148.0 (C, C-4′),
(E)-3-(3,4,5-trimethoxybenzylidene)-4-(3,4-
dimethoxybenzyl)dihydrofuran-2(3H)-one (5e)
Compound 5e, yield—62 %, pale yellow oil, 1H NMR (400
MHz, CDCl3) δ: 2.65 (1H, m, C6H5CH2–H–), 3.10 (1H, dd,
J = 4.0, 14.4 Hz, C6H5CH2–H–), 3.81–3.83 (1H, m, 4-H),
3.84 (3H, s, –OCH3), 3.85 (3H, s, –OCH3), 3.88 (6H, s, 2×
–OCH3), 3.90 (3H, s, –OCH3), 4.26–4.30 (2H, m, 5-H),
6.65 (1H, d, J = 2.0 Hz, 2″-H), 6.72 (1H, d, J = 8.0 Hz, 6″-
H), 6.82–6.88 (3H, m, 2′ and 6′-H), 7.52 (1H, s, ØCH–);
LCMS: m/z 437 (M+Na)+.

Med Chem Res
149.3 (C, C-3′), 151.1 (C, C-3″), 213.4 (C=S, C-2); HRMS
m/z calculated for C21H21O5S: 385.1110 (M+H)+; found-
385.1128.
s, –OCH3), 4.50–4.55 (2H, m, 5-H), 5.93 (2H, d, J = 2.0
Hz, –OCH2O–), 6.60–6.62 (2H, m, arom), 6.72 (1H, d, J =
7.6 Hz, 5″-H), 6.84 (2H, s, 2′ & 6′-H), 7.87 (1H, s, ØCH–);
LCMS: m/z 415 (M+H)+; 13C NMR (100 MHz, CDCl3) δ:
29.7 (CH2, –CH2Ø), 37.7 (CH, C-4), 40.7 (CH2, C-5), 56.4
(CH3, –OCH3), 61.0 (CH3, –OCH3), 101.1 (CH2, –
OCH2O–), 107.9 (CH, C-2′ and 6′), 108.5 (CH, C-2″),
109.0 (CH, C-5″), 121.8 (CH, C-6″), 129.7 (CH, ØCH–),
131.3 (C, C-1′), 138.8 (C, C-1″), 140.4 (C, C-3), 140.6 (C,
C-4′), 146.7 (C, C-4″), 148.1 (C, C-3″), 153.5 (C, C-3′ and
5′), 213.2 (C=S, C-2); HRMS m/z calculated for
C23H23O6S : 415.1215 (M+H)+; found-415.1305.
(E)-4-(3,4-dimethoxybenzyl)-3-(3,4-dimethoxy-benzylidene)
dihydrofuran-2(3H)-thione (6c)
Compound 6c, yield—88 %, pale yellow oil, 1H NMR (400
MHz, CDCl3) δ: 2.64 (1H, q, J = 4.0, 10.4 Hz, C6H5CH2–
H–), 3.06 (1H, dd, J = 4.0, 14.8 Hz, C6H5CH2–H–), 3.84
(1H, m, 4-H), 3.86 (3H, s, –OCH3), 3.87 (3H, s, –OCH3),
3.91 (3H, s, –OCH3), 3.95 (3H, s, –OCH3), 4.48–4.57 (2H,
m, 5-H), 6.68 (1H, s, 2″-H), 6.75 (1H, d, J = 8.0 Hz, 6″-H),
6.81 (1H, d, 8.4 Hz, 5″-H), 6.95 (1H, d, 8.4 Hz, 5′-H), 7.14
(1H, s, 2′-H), 7.31 (1H, d, J = 8.4 Hz, 6′-H), 7.91 (1H, s,
ØCH–); 13C NMR (100 MHz, CDCl3) δ: 29.7 (CH2,
ØCH2), 39.8 (CH, C-4), 41.5 (CH2, C-5), 55.8 (CH3, –
OCH3), 56.1 (CH3, 3× –OCH3), 111.3 (CH, C-2′), 111.7
(CH, C-5′), 112.3 (CH, C-5″), 112.8 (CH, C-2″), 121.0
(CH, C-6′), 123.7 (CH, C-6″), 126.9 (C, C-3), 128.0 (C, C-
1′), 133.4 (C, C-1″), 140.3 (CH, ØCH), 148.3 (C, C-4″),
149.3 (C, C-4′), 149.6 (C, C-3′), 151.8 (C, C-3″), 191.6
(C=S, C-2); LCMS: m/z 401 (M+H)+; HRMS m/z calcu-
lated for C22H25O5S : 401.1423 (M+H)+; found-401.1438.
Biological activity assay
All the reagents used in this work were laboratory reagent
grade. All the prepared compounds were tested for super-
oxide scavenging activity by spectrophotometric method
(Pater Rajesh and Patel Natver, 2011) using the instrument
X-mark microplate spectro-photometer (BIO-RAD).
Antioxidant assay
Different concentrations of test items (5a–5f and 6a–6e) in
30 μL, 30 μL of EDTA (ethylenediaminetetraacetic acid, 60
mM), 30 μL of NBT (nitro blue tetrazolium chloride, 500
μM), 30 μL of riboflavin (20 μM) and 180 μL of phosphate
butter (96.67 μM) were added to a total volume of 300 μL in
96 well plate and the plate was illuminated for 15 min
and thereafter the absorbance was measured at 560 nm. The
solution prepared without the addition of the test substance
was used as control sample. Gallic acid was used as positive
control.
(E)-3-(3,4,5-trimethoxybenzylidene)-4-(3,4-di-
methoxybenzyl)dihydrofuran-2(3H)-thione (6d)
Compound 6d, yield—90 %, pale yellow oil, 1H NMR
(400 MHz, CDCl3) δ: 2.66 (1H, q, J = 4.0, 10.4 Hz,
C6H5CH2–H–), 3.07 (1H, dd, J = 4.4, 14.4 Hz, C6H5CH2–
H–), 3.81 (1H, m, 4-H), 3.85 (3H, s, –OCH3), 3.86 (3H, s, –
OCH3), 3.90 (6H, s, 2× –OCH3), 3.92 (3H, s, –OCH3),
4.49–4.57 (2H, m, 5-H), 6.65 (1H, d, J = 1.6 Hz, 2″-H),
6.72 (1H, d, J = 8.0 Hz, 6″-H), 6.80 (1H, d, J = 8.4 Hz, 5″-
H), 6.88 (2H, s, 2′ and 6′-H), 7.87 (1H, s, ØCH–); LCMS:
m/z 431 (M+H)+; 13C NMR (100 MHz, CDCl3) δ: 29.7
(CH2, –CH2Ø), 37.5 (CH, C-4), 40.7 (CH2, C-5), 56.0
(CH3, –OCH3), 56.0 (CH3, –OCH3), 56.5 (CH3, –OCH3),
61.0 (CH3, –OCH3), 108.1 (CH, C-2′ and 6′), 111.7 (CH,
C-2″), 112.4 (CH, C-5″), 120.7 (CH, C-6″), 129.8 (CH,
ØCH), 130.1 (C, C-1′), 139.1 (C, C-1″), 140.4 (C, C-3),
148.3 (C, C-4′), 149.3 (C, C-4″), 149.7 (C, C-3″), 153.6 (C,
C-3′ and 5′), 213.3 (C=S, C-2); HRMS m/z calculated for
C23H27O6S: 431.1528 (M+H)+; found-431.1521.
Percentage inhibition (%) of superoxide scavenging
activity was calculated using the formula:
½ðA ꢀ BÞ=Aꢁ ´ 100:
A is the difference in absorbance of control sample
between samples with and without riboflavin, B the differ-
ence in absorbance of test sample between samples with and
without riboflavin.
The IC50 values were obtained from a plot drawn
between concentrations (µM) versus the percentage inhibi-
tion. The super-oxide radical inhibitions exhibited by test
compounds are summarized in Table 2.
Cytotoxicity assay
(E)-3-(3,4,5-trimethoxybenzylidene)-4-((benzo-[d][1,3]
dioxol-5-yl)methyl)dihydrofuran-2(3H)-thione (6e)
Cytotoxic activity was evaluated by brine shrimp lethality
test (Meyer, 1982). A 4.9 mL of sterile brine solution was
pipetted into each test tube. 100 μL solution containing
different concentrations of each of the test substances
(5a–5f and 6a–6e) were added to separate test tubes with
control in triplicates and the solution was vortexed
Compound 6e, yield—88 %, pale yellow oil, 1H NMR
(400 MHz, CDCl3) δ: 2.64 (1H, q, 4.1, 10.0 Hz, C6H5CH2–
H–), 2.99 (1H, dd, J = 4.4, 14.4 Hz, C6H5CH2–H–),
3.81–3.86 (1H, m, 4-H), 3.90 (6H, s, 2× –OCH3), 3.92 (3H,

Med Chem Res
Compliance with ethical standards
thoroughly. Ten shirmps were added to each test tube and
the surviving larvae of test and control test tubes observed
after 24 h. Replicas were maintained for accurate results.
Percentage lethality was determined by comparing the
mean surviving larvae of test and control tubes. The ED50
values were determined using Finney probit analysis
software at 95 % confidence limits from observed data.
The data is summarized Table 2.
Conflict of interest The authors declare that they have no conflict of
interests.
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Percentage inhibition of 5 ꢀ lipoxygenese ¼
f½ðabsorbance of control ꢀ absorbance of back groundÞ
ꢀ ðAbsorbanceofsample ꢀ absorbanceofbackgroundÞꢁ=
ðAbsorbanceofcontrol ꢀ AbsbackgroundÞg ´ 100
An IC50 value will be determined as the concentration
that elicits the half maximal response. The data is sum-
marized in Table 2.
Gallic acid, podophyllotoxin and curcumin were used as
positive standards for comparing superoxide scavenging
activity, brine shrimp lethality and 5-lipoxygenase inhibi-
tion activity respectively. The assay mixture without the test
substance was used as blank (negative control).
Acknowledgments We express our sincere thanks to Gokaraju
Gangaraju (Chairman), Gokaraju Ramaraju (Managing Director) and
KiranBhupatiraju (CEO) for their continuous support for our work.
Xia YM, Zhang YY, Wang W, Ding Y, He R (2010) Synthesis and
bioactivity of erythro-nordihydro guaiaretic acid, threo-(-)-saur-
unin and their analogues. J Serb Chem Soc 75:1325