Modified approach for preparing (E)-Stilbenes related to resveratrol, and evaluation of their potential immunobiological effects

Article abstract of DOI:10.1135/cccc2009531

Resveratrol and closely related stilbenoids belong to the most intensively studied biologically active compounds. This interest evoked several attempts to prepare such compounds in a convenient synthetic way. Our approach allowed obtaining largely methoxystilbenes, formed as E-isomers only (using Wittig-Horner synthesis as the key step), which were further demethylated by boron tribromide. The hydroxymethoxystilbenes (e.g. pterostilbene) were prepared using isopropyl protection, later selectively deprotected by boron trichloride. The method is suitable for preparing such compounds in a large amount. Effects of the obtained stilbene derivatives on immunobiological responses triggered by lipopolysacharide and interferon-a were tested under in vitro conditions. Namely production of nitric oxide (NO) was investigated, and relation between the molecular structure and immunobiological activity was assessed.

Full text of DOI:10.1135/cccc2009531

Modified Approach for Preparing (E)-Stilbenes
175
MODIFIED APPROACH FOR PREPARING (E)-STILBENES
RELATED TO RESVERATROL, AND EVALUATION OF
THEIR POTENTIAL IMMUNOBIOLOGICAL EFFECTS
b
Jan ŠMIDRKALa1, Juraj HARMATHAb1,*, Miloš BUDĚŠÍNSKÝb2, Karel VOKÁČ ,
c1
c
a
a
Zdeněk ZÍDEK , Eva KMONÍČKOVÁ , Roman MERKL and Vladimír FILIP
a
Department of Dairy and Fat Technology, Faculty of Food and Biochemical Technology,
Institute of Chemical Technology, Prague, 166 28 Prague 6, Czech Republic;
1
e-mail: jan.smidrkal@vscht.cz
b Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i.,
1
2
166 10 Prague 6, Czech Republic; e-mail: harmatha@uochb.cas.cz, budesinsky@uochb.cas.cz
Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, v.v.i.
142 20 Prague 4, Czech Republic; e-mail: zidekz@biomed.cas.cz
c
1
Received October 23, 2009
Accepted February 12, 2010
Published online February 20, 2010
Resveratrol and closely related stilbenoids belong to the most intensively studied biologi-
cally active compounds. This interest evoked several attempts to prepare such compounds in
a convenient synthetic way. Our approach allowed obtaining largely methoxystilbenes,
formed as E-isomers only (using Wittig–Horner synthesis as the key step), which were fur-
ther demethylated by boron tribromide. The hydroxymethoxystilbenes (e.g. pterostilbene)
were prepared using isopropyl protection, later selectively deprotected by boron trichloride.
The method is suitable for preparing such compounds in a large amount. Effects of the ob-
tained stilbene derivatives on immunobiological responses triggered by lipopolysacharide
and interferon-γ were tested under in vitro conditions. Namely production of nitric oxide
(NO) was investigated, and relation between the molecular structure and immunobiological
activity was assessed.
Keywords: Stilbene; Resveratrol; Pinosylvin; Pterostilbene; Polyphenols; Antioxidants;
Stilbenes; Nitric oxide (NO) bioassay; Immunobiological in vitro activity.
Resveratrol (3,4′,5-trihydroxystilbene (1)) is a phytoalexin found in various
plant species, but mainly in grapevine (Vitis vinifera L.), as well as in wines
prepared from resveratrol-containing grapevines1,2. Resveratrol belongs
now to the most intensively studied biologically active compounds3,4. This
interest evoked several attempts to prepare it in a synthetic way, too.
Wittig–Horner synthesis is the key step of total synthesis for resveratrol and
for other structurally related stilbenes. Our approach allowed stereoselective
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© 2010 Institute of Organic Chemistry and Biochemistry
doi:10.1135/cccc2009531

176
Šmidrkal et al.:
preparation of differently substituted hydroxy- and methoxystilbenes solely
as E-isomers.
Resveratrol 1 was at the first time isolated and identified from the roots
of Veratrum grandiflorum A. Gray (Liliaceae)3,5. Soon after, Späth and co-
workers6–9 synthesized a series of hydroxylated and methoxylated stilbenes,
e.g. 4′-hydroxy-3,5-dimethoxystilbene (2), known as pterostilbene, identi-
fied as a phytoalexin together with resveratrol in leaves of Vitis vinifera,
too10. They prepared also pterostilbene methyl ether, confirming the struc-
tures of both pterostilbene 2 and resveratrol 1. Späth also directly synthe-
sized resveratrol by condensation of sodium 4-hydroxyphenylacetate and
3,5-dihydroxybenzaldehyde followed by decarboxylation of the resulting
carboxylic acid8. Pinosylvin 3 was prepared by Späth using the same syn-
thetic strategy9. Later, also the 4′-methylresveratrol (4) was prepared11.
However, the previously published synthetic methods5–9, as well as the
reinvestigated and upgraded methods published later11–15 are convenient
only for preparations low quantity of these compounds, or only as mixtures
of isomers in various E/Z ratio16,17. Our current study of chemoecological18
and pharmacological19,20 properties of selected grapevine stilbenoids de-
rived from the indicated basic stilbenes (1–4), evoked the necessity of access
to their various synthetic analogues in a reasonable high quantity. This was
available in our case by upgrading the method by a new more convenient
approach21.
A remarkable interest in biological effects of resveratrol has mainly
emerged after the discovering its relation to the so called French para-
dox1,22. The initial interest to explain the role of resveratrol and its deriva-
tives in wine, or in other natural sources (e.g. vegetables) was due to their
antioxidative effect against reactive oxygen species, which may cause oxi-
dative stress damages to biological units involved in lipid metabolism, in
aging, inflammation and neurodegenerative disorders3,23. Resveratrol has
been identified also as a potent anticancer agent24. Studies on anticarcino-
genic mechanisms of action revealed multiple intracellular targets of
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Modified Approach for Preparing (E)-Stilbenes
177
resveratrol25. Wine stilbenes are efficient also in cancer prevention26, espe-
cially in chemoprevention of skin cancer27. Beneficial effect of resveratrol
and related phenolics has been observed in brain health prevention28 with
therapeutic potential in neurodegenerative processes including Alzheimer’s
disease29, Parkinson’s desease, and stroke28,29. Resveratrol has been reported
to be estrogenic (however with indistinct properties), as well as exhibiting
antiviral and antiinflamatory effects30. In our study, functional groups
modification of the resveratrol family stilbenes resulted in differentiation of
their antioxidative properties, targeting mainly extracellular reactive oxy-
gen species, which are responsible for tissue damage during chronic inflam-
mation19. Our interest is focused on various types of plant secondary
metabolites31–33, including resveratrol and other structurally related stil-
benes as modulators of inducible NO production20 associated with the
function of immune defense system34.
RESULTS AND DISCUSSION
In our approach, the readily available starting 3,5-dihydroxybenzoic acid (5)
was methylated to 3,5-dimethoxybenzoic acid (6), esterified to a methyl-
ester 7, reduced to alcohol 8, and transformed by thionyl chloride to the
3,5-dimethoxybenzylchloride (9) with a favorable high yield, and compara-
ble quality (m.p. 42–44 °C, see e.g. ref.35) . Chloride 9 was subsequently
converted into diethylphosphonate 10 by Arbuzov reaction (Scheme 1).
Afterwards, the phosphonate 10 was used in Wittig–Horner reaction
together with 4-methoxybenzaldehyde to provide (E)-3,4′,5-trimethoxy-
stilbene (11). Final demethylation by boron tribromide provided trans-
resveratrol 1 in overall yield 32%. Dimethylphosphonate 10 was in the
same way reacted with 4-isopropyloxybenzaldehyde to provide (E)-3,5-
dimethoxy-4′-isopropoxystilbene (12), which was selectively dealkylated by
boron trichloride to provide (E)-4′-hydroxy-3,5-dimethoxystilbene, i.e.
pterostilben 2 in overall yield 27%. The same synthetic procedure was used
to prepare and (E)-3,5-dihydroxystilbene (13) which, after demethylation,
provided pinosylvin 3 in a total yield 14%.
A slightly modified way (Scheme 2) was used for the synthesis of
(E)-3,5-dihydroxy-4′-methoxystilbene (4), however, obtained only in over-
all yield 4%.
All tested compounds (1–4, 11) have been found to suppress the immune-
stimulated production of NO, with only marginal differences in the corre-
sponding IC50 values estimates (Fig. 1a, Table I). The NO-inhibitory effects
of resveratrol and related hydroxystilbenes are well recognized36–38. It
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Šmidrkal et al.:
should be mentioned that in opposite to the inhibition of NO production,
which depends on the activity of inducible NO synthase (iNOS), resveratrol
in nanomolar concentration can activate endothelial NO synthase (eNOS),
and thus, enhance production of NO by endothelial cells39. Our data are in
agreement with the findings40,41 showing that pterostilbene 2 is equally
potent to reduce the iNOS-mediated NO production as resveratrol. To the
SCHEME 1
(i) (CH3O)2SO2, NaOH, H2O, 96%; (ii) CH3OH, H2SO4, 98%; (iii) LiAlH4, THF, 96%; (iv)
SOCl2, 82%; (v) (C2H5O)3P; (vi) 4-methoxybenzaldehyde, NaH, 76% both steps; (vii) BBr3,
CH2Cl2, 56%; (viii) 4-isopropoxybenzaldehyde, NaH, 92% both steps; (ix) BCl3, CH2Cl2,
39%; (x) benzaldehyde, NaH, 61% both steps; (xi) BBr3, CH2Cl2, 32%
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Modified Approach for Preparing (E)-Stilbenes
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SCHEME 2
(i) CH3OH, H2SO4, 89%; (ii) i-C3H7Br, KI, K2CO3, DMF, 82%; (iii) LiAlH4, THF, 67%; (iv)
SOCl2, 32%; (v) (C2H5O)3P; (vi) 4-methoxybenzaldehyde, NaH, 92% both steps; (vii) BCl3,
CH2Cl2, 28%
TABLE I
IC50 estimate values (in µM) of compounds 1–4 and 11 for their NO-inhibiting and cell
viability (cytocidal) effects. The data are mean 95% limits of confidence (l.c.)
NO
Viability
Compound
IC50
95% l.c.
IC50
95% l.c.
Resveratrol 1
33.7
27.1
29.9
35.2
44.4
30.3–37.6
22.5–32.7
24.3–36.7
28.9–42.9
38.9–50.7
100.1
>200
85.0–117.8
Pterostilbene 2
Pinosylvin 3
115.8
67.2
102.4–131.1
50.3–89.7
4′-Methylresveratrol 4
3,4′,5-Trimethoxystilbene 11
112.9
102.3–124.5
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Šmidrkal et al.:
best of our knowledge, reports on the interaction of pinosylin 3 and other
compounds with biosynthesis of NO are missing.
The NO-inhibitory activity is obviously not due to the cytocidal effects
(Fig. 1b) because, as determined by the estimated IC50 values, the com-
pounds are several-fold less potent to kill the cells (Table I). The dissocia-
tion between the NO-inhibitory and cytotoxic activities is most clearly
expressed by pterostilbene 2. It inhibits NO with IC50 of 27 µM, while the
a
Concentration, µM
b
Concentration, µM
FIG. 1
Effects of compounds 1–4 and 11 on immunobiological responses triggered by lipopoly-
sacharide (LPS) and interferon-γ (IFN-γ) under in vitro conditions using murine resident
peritoneal macrophages. a Inhibitory effects of compounds on LPS + INF-γ induced nitric oxide
(NO) production. b Effects of the compounds on viability of macrophages (cytocidal activity).
The data are mean SEM and represent one of two identical experiments
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IC50 for suppression of cell viability is >200 µM. Applied at the 50-µM con-
centration, pterostilbene 2 reduced the NO production by approximately
75%, while this concentration remained without any effect on the viability
of cells.
The present data support the view that pterostilbene 2 is a promising can-
didate for further preclinical studies. It has been shown that its pharmaco-
kinetic properties are more favorable than those of resveratrol41,42, which
has proved to possess antiinflammatory19, hepatoprotective, cardiovascular
protective, and cancer chemopreventive properties43.
The synthetic trans-stilbenes (E-isomers) 1–4, and their activities, served
us for further photochemical and biochemical transformations performed
for preparing various structural analogues (Z-isomers, cyclic isosters)20, and
other derivatives (as e.g. oxidative coupled oligomers)4,44, targeted for as-
sessing their immunobiological activities33.
EXPERIMENTAL
The melting points were determined on a Boetius microblock and are uncorrected. The ana-
lytical samples were dried over phosphorus pentoxide at 14 Pa and room temperature or
60 °C for 6 h. Conversion of the compounds in each synthetic step and purity of the prod-
ucts during crystallizations were checked by TLC on silica gel plates (GF254 Merck,
0.25 mm). Plates were developed in solvent mixture of hexane–dichloromethane–
propan-2-ol 1:1:0.1 (v/v/v) and detected by UV (254 nm). Purity of final products and of the
compounds determined for biological assays were analyzed by reversed-phase HPLC using
4 × 250 mm column, packed with 5 mm size S5 ODS2 C-18 (Phase Separation). Elution was
performed in gradient mode, with solvent system acetonitrile–water (2.5–70%)/60 min,
0.6 ml/mim, detected by UV at 220 nm.
1H and 13C NMR spectra were measured on Bruker AVANCE-600 instrument (1H at 600.13
and 13C at 150.9 MHz) in d6-DMSO at 300 K. Homonuclear 2D-H,H-COSY and heteronuclear
2D-H,C-HSQC and 2D-H,C-HMBC spectra were used for the structural assignment of proton
and carbon-13 signals. Chemical shifts are given in ppm (δ-scale), coupling constants (J) are
given in Hz.
Mass spectra, including HR-MS, were recorded on a Thermo Scientific LCQ Fleet ion trap
mass spectrometer (in negative ESI-MS mode, excepting compound 11).
(E)-3,4′,5-Trihydroxystilbene (trans-Resveratrol; 1)
A solution of 3,4′,5-trimethoxystilbene (11; 8.1 g, 30 mmol) in absolute dichloromethane
(200 ml) was under continuous stirring cooled down to –5 °C, and a solution of boron
tribromide (13 ml, 135 mmol) dissolved in absolute dichloromethane (130 ml) was stepwise
added during 30 min. The reaction mixture under continuous stirring was let to warm-up to
room temperature and stirred further 24 h. The obtained red solution was poured into
a mixture of ice in water (500 ml), and after evaporating the dichloromethane, a grey sus-
pension was obtained. The precipitate was filtered, washed with water, dried and, after crys-
tallization from a benzene–ethanol mixture, pale-grey needles of resveratrol 1 (3.8 g, 56%)
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Šmidrkal et al.:
were obtained, with m.p. 265–268 °C (according to ref.8 m.p. 260–261 °C after sublimation).
TLC detection at RF 0.08, and RP-HPLC analysis indication at tR 31.8 (both under conditions
indicated above).
1H NMR (600.13 MHz; d6-DMSO): 9.57 (bs, 1 H, 4′-OH); 9.22 (bs, 2 H, 3-OH and 5-OH);
7.39 (m, 2 H, H-2′ and H-6′); 6.92 (dt, 1 H, J(7,8) = 16.4, J(7,2) = J(7,6) = 0.5, H-7); 6.81 (bd,
1 H, J(8,7) = 16.4, H-8); 6.75 (m, 2 H, H-3′ and H-5′); 6.38 (dd, 2 H, J(2,4) = J(6,4) = 2.2,
J(2,7) = J(6,7) = 0.5, H-2 and H-6); 6.11 (t, 1 H, J(4,2) = J(4,6) = 2.2, H-4). 13C NMR
(150.9 MHz; d6-DMSO): 158.72 (C-3 and C-5); 157.43 (C-4′); 139.48 (C-1); 128.27 (C-1′);
128.08 (C-8, C-2′ and C-6′); 125.84 (C-7); 115.68 (C-3′ and C-5′); 104.49 (C-2 and C-6);
101.95 (C-4).
Negative ESI-MS (in MeOH), m/z (%): 227.0 [M – H]– (100), 185 (95), 183 (42), 159 (29),
157 (27), 143 (14). HR ESI-MS, m/z: 227.0705 [M – H]– for C14H11O3 required 227.0703.
(E)-4′-Hydroxy-3,5-dimethoxystilbene (Pterostilbene; 2)
A solution of 4′-isopropoxy-3,5-dimethoxystilbene (12; 17.9 g, 60 mmol) in absolute
dichloromethane (200 ml) was stirred, and a solution of 1 M boron trichloride (36 ml,
36 mmol) was added. Reaction was monitored by TLC, and after 8 h the reaction mixture
did not contain any starting compound. The reaction mixture was poured in water (250 ml),
the organic phase was separated and washed with water (2 × 200 ml). The organic phase was
extracted with a solution of 2% NaOH (3 × 200 ml), and the combined alkaline extract was
washed with dichloromethane (200 ml) and acidified by a solution of 5 M HCl (125 ml). The
separated oil was extracted with diethylether (2 × 200 ml), and the combined extract was
washed with water (2 × 200 ml), dried with anhydrous sodium sulfate and evaporated. The
residue (16 g) was recrystallized (2×) from xylene, and the resulting pterostilbene 2 was
obtained as pale yellow needles (6.0 g, 39%), with m.p. 86–88 °C (according to ref.7 m.p.
87–88 °C from petroleum ether). TLC detection at RF 0.38, and RP-HPLC analysis indication
at tR 49.6 (both under conditions indicated above).
1H NMR (600.13 MHz; d6-DMSO): 9.63 (bs, 1 H, OH); 7.41 (m, 2 H, H-2′ and H-6′); 7.16
(bd, 1 H, J(8,7) = 16.4, H-8); 6.94 (dt, 1 H, J(7,8) = 16.4, J(7,2) = J(7,6) = 0.4, H-7); 6.76 (m,
2 H, H-3′ and H-5′); 6.71 (dd, 2 H, J(2,4) = J(6,4) = 2.3, J(2,7) = J(6,7) = 0.4, H-2 and H-6);
6.37 (t, 1 H, J(4,2) = J(4,6) = 2.3, H-4); 3.76 (s, 6 H, 2 × OCH3). 13C NMR (150.9 MHz;
d6-DMSO): 160.84 (C-3 and C-5); 157.61 (C-4′); 139.82 (C-1); 129.17 (C-8); 128.14 (C-1′, C-2′
and C-6′); 125.34 (C-7); 115.78 (C-3′ and C-5′); 104.24 (C-2 and C-6); 99.48 (C-4); 55.38 (2 ×
OCH3).
Negative ESI-MS (in MeOH), m/z (%): 255.0 [M – H]– (25), 240 (100), 239 (8). HR ESI-MS,
m/z: 255.1018 [M – H]–, for C16H15O3 required 255.1016.
(E)-3,5-Dihydroxystilbene (Pinosylvin; 3)
A solution of 3,5-dimethoxystilbene (13; 10.6 g, 50 mmol) in absolute dichloromethane
(100 ml) was stirred and cooled to 5 °C, and a solution of boron tribromide (15.4 ml, 16 mmol)
in absolute dichloromethane (500 ml) was added during 30 min. The reaction mixture was
allowed to warm-up to room temperature and stirred for 24 h. Next day, the reaction mix-
ture was refluxed for another 4 h. The red reaction mixture was cooled (with water and ice),
water (250 ml) was added, and allowed to warm-up to room temperature. The organic phase
was washed with a solution of 4% NaCl (4 × 250 ml), solution of 4% sodium hydrogen car-
bonate (250 ml) and once more with a solution of NaCl (250 ml), then dried with anhy-
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Modified Approach for Preparing (E)-Stilbenes
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drous sodium sulfate, and evaporated. The residue was crystallized from benzene as colorless
needles (3.3 g, 32%), with m.p. 156–157 °C (according to ref.10 m.p. 155–155.5 °C from ben-
zene). TLC detection at RF 0.19, and RP-HPLC analysis indication at tR 41.6 (both under con-
ditions indicated above).
1H NMR (600.13 MHz; d6-DMSO): 9.29 (bs, 2 H, 3-OH and 5-OH); 7.57 (m, 2 H, H-2′ and
H-6′); 7.35 (m, 2 H, H-3′ and H-5′); 7.25 (m, 1 H, H-4′); 7.06 (d, 1 H, J(8,7) = 16.4, H-8); 7.03
(d, 1 H, J(7,8) = 16.4, H-7); 6.45 (d, 2 H, J(2,4) = J(6,4) = 2.2, H-2 and H-6); 6.16 (t, 1 H,
J(4,2) = J(4,6) = 2.2, H-4). 13C NMR (150.9 MHz; d6-DMSO): 158.77 (C-3 and C-5); 138.95
(C-1); 137.23 (C-1′); 129.15 (C-7); 128.89 (C-3′ and C-5′); 128.05 (C-8); 127.76 (C-4′); 126.70
(C-2′ and C-6′); 104.91 (C-2 and C-6); 102.56 (C-4).
Negative ESI-MS (in MeOH), m/z (%): 211.1 [M – H]– (23), 169 (100), 167 (67), 152 (7).
HR ESI-MS, m/z: 211.0758 [M – H]–, for C14H11O2 required 211.0754.
(E)-3,5-Dihydroxy-4′-methoxystilbene (Resveratrol-4′-methyl Ether; 4)
Resveratrol-4′-methyl ether 4 was prepared from 3,5-diisopropoxy-4′-methoxystilbene (15)
by identical method, differing only in using twice a higher amount of boron trichloride so-
lution. Compound 4 was obtained as colorless needles (360 mg, 28%), with m.p. 172–175 °C
(crystallized from ethyl acetate–benzene mixture). TLC detection at RF 0.18, and RP-HPLC
analysis indication at tR 42.4 (both under conditions indicated above).
1H NMR (600.13 MHz; d6-DMSO): 9.25 (bs, 2 H, 3-OH and 5-OH); 7.51 (m, 2 H, H-2′ and
H-6′); 6.98 (bd, 1 H, J(8,7) = 16.4, H-8); 6.92 (m, 2 H, H-3′ and H-5′); 6.89 (dt, 1 H, J(7,8) =
16.4, J(7,2) = J(7,6) = 0.5, H-7); 6.41 (dd, 2 H, J(2,4) = J(6,4) = 2.2, J(2,7) = J(6,7) = 0.5, H-2
and H-6); 6.13 (t, 1 H, J(4,2) = J(4,6) = 2.2, H-4); 3.76 (s, 3 H, OCH3). 13C NMR (150.9 MHz;
d6-DMSO): 159.10 (C-4′); 158.74 (C-3 and C-5); 139.32 (C-1); 129.87 (C-1′); 128.00 (C-2′ and
C-6′); 127.70 (C-7); 126.86 (C-8); 114.34 (C-3′ and C-5′); 104.62 (C-2 and C-6); 102.14 (C-4);
55.35 (OCH3).
Negative ESI-MS (in MeOH), m/z (%): 242 (16), 241.0 [M – H]– (100), 227 (3), 197 (1).
HR ESI-MS, m/z: 241.0863 [M – H]–, for C15H13O3 required 241.0859.
(E)-3,4′,5-Trimethoxystilbene (11)
A mixture of 3,5-dimethoxybenzylchloride (9; 9.3 g, 50 mmol) and triethyl phosphite (8.3 g,
50 mmol) was stirred and heated under argon at 160 °C for 2 h. During the first hour,
evolution of gas (ethyl chloride) was observed. After the reaction has been finished, the ob-
tained crude 3,5-dimethoxybenzyl diethyl phosphonate (10) was cooled to room tempera-
ture. To a stirred absolute 1,2-dimethoxyethane (under argon), sodium hydride (80%
dispersion in mineral oil, 2.1 g, 70 mmol) was added, then stepwise 4-methoxybenzaldehyde
(6.8 g, 50 mmol), and finally the whole amount of the above acquired phosphonate 10. The
mixture was stirred and slowly heated up to 85 °C. Already at 70 °C, appearing evolution of
gas (hydrogen) started and a semisolid material deposited. The reaction mixture was
refluxed another 30 min, and then a half amount of the 1,2-dimethoxyethane solvent was
distilled off. The residue was poured into water (500 ml) and the oily phase was extracted
with diethyl ether (2 × 200 ml). The combined ether extract was dried with anhydrous cal-
cium chloride, partly evaporated, and the residue was crystallized from ethanol. The final
stilbene 11 was obtained in a form of colorless crystals (10.3 g, 76%), with m.p. 54–56 °C
(according to ref.7 m.p. 56–57 °C). TLC detection at RF 0.7, and RP-HPLC analysis indication
at tR 60.1 (both under conditions indicated above).
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1H NMR (600.13 MHz; d6-DMSO): 7.53 (m, 2 H, H-2′ and H-6′); 7.21 (bd, 1 H, J(8,7) =
16.4, H-8); 7.02 (dt, 1 H, J(7,8) = 16.4, J(7.2) = J(7,6) = 0.5, H-7); 6.94 (m, 2 H, H-3′ and
H-5′); 6.74 (dd, 2 H, J(2,4) = J(6,4) = 2.2, J(2,7) = J(6,7) = 0.5, H-2 and H-6); 6.38 (t, 1 H,
J(4,2) = J(4,6) = 2.2, H-4); 3.77 (s, 9 H, 3-, 5- and 4′-OMe). 13C NMR (150.9 MHz; d6-DMSO):
160.87 (C-3 and C-5); 159.26 (C-4′); 139.67 (C-1); 129.77 (C-1′); 128.80 (C-8); 128.09 (C-2′
and C-6′); 126.37 (C-7); 114.41 (C-3′ and C-5′); 104.38 (C-2 and C-6); 99.70 (C-4); 55.41
(3- and 5-OMe); 55.37 (4′-OMe).
Positive HR ESI-MS, m/z: 271.1325 [M + H]+, for C17H19O3 required 271.1329.
(E)-4-Isopropoxy-3,5-dimethoxystilbene (12)
Compound 12 was prepared from 3,5-dimethoxybenzylchloride (9) and 4-isopropyloxybenz-
aldehyde by identical method as for (E)-3,4′,5-trimethoxystilbene (11) as a glassy solid
(92%), which crystallized afterwards reaching m.p. 35–38 °C. TLC detection at RF 0.7 (under
conditions indicated above).
1H NMR (600.13 MHz; CDCl3): 7.42 (m, 2 H, H-2′ and H-6′); 7.02 (d, 1 H, J(7,8) = 16.3,
H-7); 6,89 (d, 1 H, J(8,7) = 16.3, H-8); 6.87 (m, 2 H, H-3′ and H-5′); 6.64 (d, 2 H, J(2,4) =
J(6,4) = 2.3, H-2 and H-6); 6.37 (t, 1 H, J(4,2) = J(4,6) = 2.3, H-4); 4.55 (h, 1 H, J = 6.1,
O-CH< (iPr)); 3.81 (s, 6 H, 3- and 5-OMe); 1.34 (d, 6 H, J = 6.1, 2 × CH3 (iPr)). 13C NMR
(150.9 MHz; CDCl3): 160.88 (C-3 and C-5); 157.66 (C-4′); 139.68 (C-1); 129.58 (C-1′); 128.74
(C-7); 127.77 (C-2′ and C-6′); 126.33 (C-8); 115.91 (C-3′ and C-5′); 104.22 (C-2 and C-6);
99.48 (C-4); 69.82 (O-CH< (iPr)); 55.28 (3- and 5-OMe); 21.99 (2 × CH3 (iPr)).
Positive HR ESI-MS, m/z: 299.1641 [M + H]+, for C19H23O3 required 299.1642.
(E)-3,5-Dimethoxystilbene (13)
Compound 13 was prepared from 3,5-dimethoxybenzylchloride (9) and benzaldehyde by
identical method as (E)-3,4′,5-trimethoxystilbene (11) as colorless needles (61%), with m.p.
55–56 °C (crystallized from propan-2-ol). TLC detection at RF 0.7 (under conditions indi-
cated above).
1H NMR (600.13 MHz; CDCl3): 7.50 (m, 2 H, H-2′ and H-6′); 7.35 (m, 2 H, H-3′ and H-5′);
7.26 (m, 1 H, H-4′); 7.08 (bd, 1 H, J(8,7) = 16.5, H-8); 7.03 (bd, 1 H, J(7,8) = 16.5, H-7); 6.67
(dd, 2 H, J(2,4) = J(6,4) = 2.3, J(2,7) = J(6,7) = 0.6, H-2 and H-6); 6.40 (t, 1 H, J(4,2) = J(4,6) =
2.3, H-4); 3.82 (s, 6 H, 3- and 5-OMe). 13C NMR (150.9 MHz; CDCl3): 160.92 (C-3 and C-5);
139.30 (C-1); 137.06 (C-1′); 129.15 (C-8); 128.65 (C-3′ and C-5′); 128.61 (C-7); 127.70 (C-4′);
126.54 (C-2′ and C-6′); 104.51 (C-2 and C-6); 99.92 (C-4); 55.32 (3- and 5-OMe).
Positive HR ESI-MS, m/z: 241.1222 [M + H]+, for C16H17O2 required 241.1223.
(E)-3,5-Diisopropoxy-4′-methoxystilbene (15)
Compound 15 was prepared from 3,5-diisopropoxybenzylchloride (14; Scheme 2) and
4-methoxybenzaldehyde by identical method as (E)-3,4′,5-trimethoxystilbene (11) as a color-
less liquid (92%). TLC detection at RF 0.7 (under conditions indicated above).
1H NMR (500.13 MHz; CDCl3): 7.43 (m, 2 H, H-2′ and H-6′); 7.00 (bd, 1 H, J(8,7) = 16.2,
H-8); 6.89 (m, 2 H, H-3′ and H-5′); 6.87 (bd, 1 H, J(7,8) = 16.2, H-7); 6.61 (d, 2 H, J(2,4) =
J(6,4) = 2.3, H-2 and H-6); 6.35 (t, 1 H, J(4,2) = J(4,6) = 2.3, H-4); 4.56 (h, 2 H, J = 6.1,
2 × O-CH< (iPr)); 3.82 (s, 3 H, 4′-OMe); 1.34 (d, 12 H, J = 6.1, 4 × CH3 (iPr)).
HR EI-MS, m/z: 326.1897 [M]+, for C21H26O3 required 326.1882.
Collect. Czech. Chem. Commun. 2010, Vol. 75, No. 2, pp. 175–186

Modified Approach for Preparing (E)-Stilbenes
185
Nitric Oxide (NO) Bioassay
Isolation and cultivation of the murine peritoneal macrophages for the bioassay were per-
formed in a routine way32,33. Macrophages (2 × 106/ml) were cultured 24 h in presence of
test compounds, applied either alone or in the presence of NO-priming immune stimuli, i.e.
murine recombinant interferon-γ (IFN-γ), and lipopolysaccharide (LPS). The concentration of
nitrites in supernatants of cells was taken as a measure of NO production. It was detected in
individual cell-free samples (50 µl) incubated 5 min at ambient temperature with an aliquot
of a Griess reagent. The absorbance at 540 nm was recorded using a microplate spectrophoto-
meter.
Cytotoxicity Assay
Viability of cells was determined using a colorimetric assay based on the cleavage of the
tetrazolium salt WST-1 by mitochondrial dehydrogenases in viable cells (Roche Diagnostics,
Mannheim, Germany). The cells (1 × 106/ml) were cultured as described above. After the
24-h culture, the WST-1 was added and the cells were kept in the Heraeus incubator at 37 °C
for additional 3 h. Optical density at 450–690 nm was evaluated.
The test compounds were dissolved in dimethylsulfoxide (DMSO). The final concentra-
tion of DMSO was lower than 0.02%. This amount was devoid of any effects on production
of NO and on the cell viability.
The research was supported by the Czech Science Foundation (grant No. 203/07/1227) and in part
from the research project AV0Z 40550506. We thank Ms K. Kertisová for recording mass spectra,
Ms V. Krejčová and Mgr. J. Křížková for technical assistance.
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