Fluoro-substituted and 13C-labeled styrylbenzene derivatives for detecting brain amyloid plaques

Article abstract of DOI:10.1016/j.ejmech.2004.02.013

Two styrylbenzene derivatives, (E,E)-1-fluoro-2,5-bis-(3-hydroxycarbonyl-4- hydroxy)styrylbenzene (FSB) and (E,E)-1-bromo-2,5-bis(3-hydroxycarbonyl-4- hydroxy)styrylbenzene-α,α′-¹³C₂ ([ ¹³C]BSB), were synthesized for use as a histochemical stain to detect amyloid plaques of Alzheimer's disease (AD) brain sections. An analysis of fluorescence spectra demonstrated that FSB shows approximately twofold fluorescence intensity relative to the conventional styrylbenzene derivative, (E,E)-1-bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene (BSB). Moreover, FSB was found to stain amyloid plaques and neurofibrillary tangles of AD brains with greater fluorescence intensity and a lower level of background signals compared to BSB. These finding indicate that FSB can be an excellent fluorescent compound to label human amyloid lesions with high sensitivity and specificity. Because of the possession of a nuclide with a quantized angular momentum, both FSB and [¹³C]BSB are also potential contrast agents for magnetic resonance imaging to locate AD pathologies in vivo.

Full text of DOI:10.1016/j.ejmech.2004.02.013

European Journal of Medicinal Chemistry 39 (2004) 573–578
www.elsevier.com/locate/ejmech
Original article
Fluoro-substituted and 13C-labeled styrylbenzene derivatives
for detecting brain amyloid plaques
Kumi Sato a, Makoto Higuchi b, Nobuhisa Iwata b, Takaomi C. Saido b, Kazumi Sasamoto a,*
a Dojindo Laboratories, Tabaru 2025-5, Mashiki-machi, Kumamoto 861-2202, Japan
b Laboratory for Proteolytic Neuroscience, RIKEN Brain Science Institute, Wako-shi, Saitama 351-0198, Japan
Received 13 October 2003; received in revised form 19 February 2004; accepted 26 February 2004
Available online 28 May 2004
Abstract
Two styrylbenzene derivatives, (E,E)-1-fluoro-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene (FSB) and (E,E)-1-bromo-2,5-bis(3-
hydroxycarbonyl-4-hydroxy)styrylbenzene-a,a′-13C2 ([13C]BSB), were synthesized for use as a histochemical stain to detect amyloid plaques
of Alzheimer’s disease (AD) brain sections. An analysis of fluorescence spectra demonstrated that FSB shows approximately twofold
fluorescence intensity relative to the conventional styrylbenzene derivative, (E,E)-1-bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)
styrylbenzene (BSB). Moreover, FSB was found to stain amyloid plaques and neurofibrillary tangles of AD brains with greater fluorescence
intensity and a lower level of background signals compared to BSB. These finding indicate that FSB can be an excellent fluorescent compound
to label human amyloid lesions with high sensitivity and specificity. Because of the possession of a nuclide with a quantized angular
momentum, both FSB and [13C]BSB are also potential contrast agents for magnetic resonance imaging to locate AD pathologies in vivo.
© 2004 Elsevier SAS. All rights reserved.
Keywords: Alzheimer’s disease; Histochemical staining; Strylbenzenes; Magnetic resonance imaging
1. Introduction
vivo as a possible antemortem diagnostic tool for AD [2,3].
Lee and her co-workers [4,5] reported one of such ligands,
(E,E)-1-bromo-2,5-bis(3-hydroxycarbonyl-4-hydroxy)styryl-
benzene (BSB), with which they were able to detect amyloid
plaques in vivo in a mouse model of AD. BSB was also found
to be useful as a stain with bright fluorescence for detecting
amyloid deposits in systemic amyloidosis both in vitro and in
Alzheimer’s disease (AD) has now become one of the
most common diseases among the elderly, affecting more
than 4 million people in United States alone. The disease is
neuropathologically characterized by the formations of se-
nile plaques (SPs), which consist of mainly amyloid b (Ab)
peptide, and of filamentous tau lesions including neurofibril-
lary tangles (NFTs), neuropil threads (NPs) and plaque
neurites, and finally loss of neurons. The definitive diagnosis
of AD is based on histochemical and/or immunohistochemi-
cal staining of these neuropathological hallmarks in postmor-
tem brain sections. Congo red and thioflavin-S have been
most widely used as standard stains that detect brain amy-
loids by fluorescence [1]. Recently, however, much effort
have been directed toward derivatization of Congo red to
develop a small ligand molecule that binds brain amyloid in
vivo [6]
.
We were interested in exploiting an analogous compound
to BSB that contains a fluoro group, instead of bromo, be-
cause fluoro-substitution has several advantages that the
fluorescence intensity is normally greater than that of the
corresponding bromo-substituted compounds (known as
‘heavy atom effect’) and that the smaller molecular weight is
favorable for enhanced permeability of the blood-brain bar-
rier (BBB) as well as a rapid clearance from the body when
used as an in vivo probe molecule. In addition, fluoro-
substituted probes have a potential to be used as a contrast
agent in magnetic resonance imaging (MRI), by which detec-
tion of AD amyloid plaques has never been achieved to date.
Toward this goal, we synthesized fluoro-substituted and 13C-
labeled BSB analogs, (E,E)-1-fluoro-2,5-bis-(3-hydroxy-
carbonyl-4-hydroxy)styrylbenzene (FSB) and (E,E)-1-
bromo-2,5-bis(3-hydroxycarbonyl-4-hydroxy)styrylbenzene-
Abbreviations: Ab, amyloid b; AD, Alzheimer’s disease; NFTs, neurofi-
brillary tangles; NPs, neuropil threads; SPs, senile plaques.
* Corresponding author.
E-mail address: sasamoto@dojindo.co.jp (K. Sasamoto).
© 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2004.02.013

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K. Sato et al. / European Journal of Medicinal Chemistry 39 (2004) 573–578
a,a′-13C2 ([13C]BSB), and report herein our preliminary re-
sults obtained with these analogs in staining AD brain amy-
loid lesions.
the synthesis was done in an identical manner to BSB.
[13C]BSB showed very strong peaks in 13C-NMR spectrum
at 124.8 and 125.5 ppm, those corresponding to the vinylic
1
carbons; it also showed 13C–H couplings in its H-NMR
spectrum.
2. Results and discussion
The emission and excitation spectra of FSB and
[13C]BSB, in comparison with BSB, are shown in Fig. 1.
FSB fluoresces with maximum emission and excitation
wavelengths identical to those of BSB (kex = 390 nm;
kem = 520 nm) but with intensities being roughly twofold.
The increased fluorescence intensities can be attributed to the
heavy atom effect, which is well known particularly with
halide-substituted fluorophores whose fluorescence is
quenched in the order Cl < Br < I. These fluorescence prop-
erties seem to indicate that FSB is more sensitive as a stain
for histochemical localization of AD pathology than BSB,
although it is not clear whether and to what extent the fluo-
rescence is enhanced upon binding to amyloid fibrils.
Fig. 2compares FSB and [13C]BSB with BSB in staining
temporal cortex and hippocampus tissue sections from AD
brains, both of which were ethanol-fixed. The same protocol
was used for the FSB and BSB stains, and thus was not
optimized for FSB. FSB clearly stained SPs, NFTs, NPs and
plaque neurites with a greater intensity than BSB. The differ-
ence in the contrast of staining between FSB and BSB was
particularly pronounced in some regions of the temporal
cortex, where relatively immature, non-neuritic plaques were
abundant (Fig. 2A, C).As BSB was reported to label a variety
of amyloid forms such as SPs and NFTs, it is likely that FSB
The syntheses of FSB and [13C]BSB were carried out in a
similar manner to BSB [5], starting from 2,5-dimethylaniline
or p-xylene-a,a′-13C2, respectively, as outlined in Scheme 1.
Since initial attempts to fluorinate p-xylene using various
electrophilic fluorinating reagents failed due primarily to
difficulties in purification, we finally converted 2,5-dimethyl-
aniline to fluoride 3 via the corresponding diazonium tet-
rafluoroborate, which decomposed spontaneously to give the
desired product. The key step was the Wittig–Horner cou-
pling reaction between phosphate ester 5 and aldehyde 1 that
produced (E,E)-isomer 6 as a sole product as confirmed by
1H-NMR. Deprotection of 6 in two steps gave FSB in a
moderate yield, whose NMR spectra were very similar to
those of BSB except a strong peak at –118 ppm in 19F-NMR
spectrum. In the synthesis of [13C]BSB, p-xylene-a,a′-13C2,
in which the two methyl groups of p-xylene are 13C-labeled,
was used as a starting material because it is commercial
available and 13C-labeling of 1 at its aldehyde group turned
out to be of very low yield. To install a bromo group into this
compound, we employed a method reported by Kitagawa et
al. [7] that utilized a ferrocenium complex, affording bro-
mide 9 in 59% yield. From 9 to the final product [13C]BSB,
Scheme 1. Synthesis of FSB and [13C]BSB.

K. Sato et al. / European Journal of Medicinal Chemistry 39 (2004) 573–578
575
80
60
40
20
0
FSB
FSB
[13C]BSB
[13C]BSB
BSB
BSB
200
300
400
500
600
700
Wavelength (nm)
Fig. 1. Fluorescence spectra of FSB and BSB taken at 1.0 µM in DMSO.
Fig. 2. Temporal cortex (A–C) or hippocampal (D–F) sections from AD brains (ethanol-fixed) were stained with FSB (A and D), [13C]BSB (B and E) or BSB
(C and F).

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K. Sato et al. / European Journal of Medicinal Chemistry 39 (2004) 573–578
Fig. 3. Subadjacent sections of frontal (A and B) or temporal (C and D) cortex fromAD brains, fixed, respectively, in isotonic ethanol or NBF, were stained with
FSB (A and C) or [13C]BSB (B and D). The number indicates the same plaque in FSB- and [13C]BSB-stained sections.
also has similar affinities for these amyloid forms containing
the b-pleated sheet structure. [13C]BSB showed fluorescence
staining that was comparable to the staining with BSB in
both hippocampus and temporal cortex.
[4,5]. Although it will be required to clarify the kinetics FSB
in animals and humans, and both FSB and BSB exist possibly
as a monocarboxylate anion due to their similar pKa values of
the carboxylic groups, it is likely that FSB penetrates intact
BBB with a higher permeability than BSB because of its less
bulky structure. In addition, our results of histochemical
staining imply high specificity of FSB in labeling amyloid
plaques of living AD patients compared to BSB. It gives a
bluish green fluorescence under UV light, which allows the
plaques to be readily distinguished. FSB is also in contrast to
Congo red that fluoresces pink to orange-red, with which
small plaques are unlikely to be detected because of this
fluorescence. FSB might also be useful as a histochemical
stain for systemic amyloidosis [6]. In vitro binding studies as
well as in vivo studies for FSB and [13C]BSB are currently
underway and will be reported elsewhere.
Using subadjacent ethanol-fixed frontal and NBF-fixed
temporal cortex sections, staining of the same amyloid
plaque with FSB and [13C]BSB was compared (Fig. 3).
Again, FSB gave a better contrast than [13C]BSB with a
greater fluorescence intensity and a lower background level.
The observations of the present study clearly demonstrate
that both FSB and [13C]BSB effectively stain hallmark le-
sions of AD including SPs and NFTs. Those BSB analogs
were designed in the hope that they can be used as a sensitive
MRI probe for an antemortem diagnosis of AD. Since 19F
and 13C are NMR active, amyloid lesions can be located by
detecting MR signals from these nuclides. Notably, FSB was
found to label these AD pathologies with higher sensitivity
and specificity than BSB, suggesting its superiority in the use
for neuropathological examinations. Our observation
showed that [13C]BSB binds to amyloid plaques and NFTs
with an affinity similar to BSB. The biodistribution and
pharmacokinetics of [13C]BSB are supposed to be identical
to those of BSB, and thus it may be useful for imaging
amyloid plaques in living AD patients and animal models as
BSB has been demonstrated to visualize these lesions in vivo
3. Experimental section
p-Xylene-a,a′-13C2 was purchased from Isotec (Miamis-
burg, Ohio, USA) and used as purchased. 1H-Nuclear mag-
netic resonance (NMR) spectra were taken on a JEOL JNM
ECP-300 spectrometer operating at 300 MHz. Mass spectra
were recorded on a JEOL JMS-AX505W spectrometer. IR

K. Sato et al. / European Journal of Medicinal Chemistry 39 (2004) 573–578
577
spectra were measured on a Hitachi 270-30 spectrometer.
Fluorescence spectra were taken on a Hitachi F-4500 spec-
trometer. Fluorescent tissue sections were viewed using a
Zeiss Axiophot 2 fluorescence microscope (Göttingen, Ger-
many).
Hz, 2H), 3.17 (d, J = 20.6 Hz, 2H), 3.98–4.10 (m, 8H), 7.04
(d, J = 9.9 Hz, 1H), 7.27–7.34 (m, 2H); 19F-NMR (CDCl3):
–117.1 ppm; MS: m/z 397 (M + 1).
3.5. (E,E)-1-Fluoro-2,5-bis(3-methoxycarbonyl-4-methoxy)
styrylbenzene (6)
3.1. 3-Methoxycarbonyl-4-methoxybenzaldehyde (1)
Sodium methoxide in methanol (28 wt.%, 2 ml) was
added to an anhydrous methanol solution (5 ml) containing 1
(1.16 g, 6.0 mmol) and 5 (1.18 g, 3.0 mmol) at 0 °C, and the
solution was refluxed overnight. Precipitates formed were
collected and suspended in water (100 ml). The pH of this
suspension was adjusted to 3 with 1 M HCl, and the solution
was extracted with chloroform. The chloroform layer was
washed with water, dried (MgSO4) and concentrated to give a
crude coupling product, which was chromatographed on
SiO2 (10% EtOAc in CHCl3) affording 0.42 g (30%) of 6 as
5-Formylsalicylic acid (22.0 g, 132.4 mmol) was dis-
solved in acetone (1100 ml) with heating, to which was
added K2CO3 (36.5 g, 264.1 mmol) and methyl iodide. The
mixture was heated at 50 °C for 12 h, during which time a
total of 253.4 g of methyl iodide (1.78 mol) was added every
2 h. After the solvent was stripped off, water (200 ml) was
added to leave crude methylated product, which was crystal-
lized from ethanol to give 16.59 g (65%) of 1 as a white
crystalline solid. 1H-NMR (CDCl3): d 3.93 (s, 3H), 4.06 (s,
3H), 7.12 (d, J = 8.7 Hz, 1H), 8.03 (dd, J = 8.5, 2.2 Hz, 1H),
8.33 (d, J = 2.1 Hz, 1H), 9.92 (s, 1H).
1
yellow powder. H-NMR (CDCl3): d 3.93 (s, 6H), 3.94 (s,
6H), 6.94–7.26 (m, 8H), 7.55 (t, J = 7.9 Hz, 1H), 7.61 (dd,
J = 9.1, 2.2 Hz, 1H), 7.64 (dd, J = 9.3, 2.2 Hz, 1H), 7.97 (d,
J = 2.2 Hz, 1H), 7.98 (d, J = 2.2 Hz, 1H); 19F-NMR (CDCl3):
–118.1 ppm; MS: m/z 476 (M+).
3.2. 2-Fluoro-p-xylene (3)
Concentrated HCl (17 ml) was slowly added to a stirred
suspension of 2,5-dimethylaniline (10 g, 82.5 mmol) in water
(30 ml). An aqueous solution (10 ml) of NaNO2 (7.0 g,
101.4 mmol) was then added dropwise with stirring to this
suspension at 5 °C. After being stirred for 1 h at 5 °C, the
solution was filtered. Addition of NaBF4 (11.0 g,
100.2 mmol) to this filtrate caused precipitation of the corre-
sponding diazonium salt, which decomposed spontaneously
at room temperature to leave the crude fluorinated product.
Column chromatography on SiO2 (hexane) afforded 1.51 g
3.6. (E,E)-1-Fluoro-2,5-bis(3-methoxycarbonyl-4-hydroxy)
styrylbenzene (7)
To 6 (0.42 g, 0.87 mmol) in dichloromethane (50 ml) was
added 1.0 M BBr3 in dichloromethane (9.0 ml, 9.0 mmol) at
–78 °C, and the mixture was stirred overnight at room tem-
perature. Water (30 ml) and methanol (~5 ml) were added to
the reaction mixture, and the organic layer was separated.
The aqueous layer was extracted with dichloromethane; the
combined organic layer was washed with water, dried
(MgSO4) and concentrated to give 7 in almost quantitative
yield as yellow powder. 1H-NMR (DMSO-d6): d 3.93 (s, 6H),
7.03 (d, J = 8.7 Hz, 1H), 7.04 (d, J = 8.5 Hz, 1H), 7.15 (d,
J = 16.5 Hz, 1H), 7.16 (d, J = 16.5 Hz, 1H), 7.36 (d,
J = 16.5 Hz, 1H), 7.37 (d, J = 16.5 Hz, 1H), 7.43–7.52 (m,
2H), 7.75–7.89 (m, 3H), 7.98 (s, 2H), 10.59 (s, 2H); 19F-
NMR (DMSO-d6): –118.6 ppm.
1
(15%) of 3 as a colorless oil. H-NMR (CDCl3): d 2.22 (s,
3H), 2.30 (s, 3H), 6.79–6.83 (m, 2H), 7.03 (t, J = 8.0 Hz, 1H);
19F-NMR (CDCl3): –118.5 ppm.
3.3. 1-Fluoro-2,5-bis(bromomethyl)benzene (4)
A chloroform solution (100 ml) containing 3 (1.02 g,
8.2 mmol), N-bromosuccinimide (3.1 g, 17.4 mmol) and
2,2′-azobis(isobutyronitrile) (10 mg, 0.8 mol%) was refluxed
for 100 min. After being cooled, the chloroform solution was
washed with water, dried (MgSO4), and concentrated to leave
colored solids, which crystallized by addition of hexane to
give 0.99 g (43%) of 4 as off-white crystalline powder.
1H-NMR (CDCl3): d 4.43 (s, 2H), 4.49 (s, 2H), 7.08–7.41 (m,
3H); 19F-NMR (CDCl3): –115.9 ppm; MS: m/z 281 (M + 1).
3
3.7. (E,E)-1-Fluoro-2,5-bis(3-hydroxycarbonyl-4-hydroxy)
styrylbenzene (FSB)
A suspension of 7 (0.42 g, 0.94 mmol) in 0.06 M aqueous
KOH (150 ml) was refluxed for 4 h, and the pH of the
solution was adjusted to 2 by slow addition of 6 M HCl.After
being cooled, precipitates were collected to give 0.23 g (64%
from 6) of FSB as yellow powder. mp 294 °C (decomp.);
1H-NMR (DMSO-d6): d 6.95 (d, J = 8.5 Hz, 1H), 6.96 (d,
J = 8.5 Hz, 1H), 7.12 (d, J = 16.2 Hz, 1H), 7.13 (d,
J = 16.5 Hz, 1H), 7.33 (d, J = 16.5 Hz, 1H), 7.34 (d,
J = 16.2 Hz, 1H), 7.42–7.49 (m, 2H), 7.74–7.76 (m, 1H), 7.79
(d, J = 8.8 Hz, 1H), 7.80 (d, J = 8.8 Hz, 1H), 7.98 (d,
J = 2.2 Hz, 1H), 8.00 (d, J = 2.2 Hz, 1H); 19F-NMR (DMSO-
d6): –118.7 ppm (s); MS: m/z 419 (M – 1); IR (KBr): 3430
(OH), 3025 (CO2H), 1670 (C=O), 1210, 1190, 955 cm–1.
3.4. 2-Fluoro-1,4-bis(diethylphosphonato)xylene (5)
A mixture of 4 (0.99 g, 3.5 mmol) and triethyl phosphite
(1.16 g, 7.0 mmol) was heated at 160 °C for 4 h. The reaction
mixture was chromatographed on SiO2 (5% MeOH in
CH2Cl2) to afford 1.18 g (85%) of 5 as a colorless oil.
1H-NMR (CDCl3): d 1.23–1.29 (m, 12H), 3.11 (d, J = 20.3

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K. Sato et al. / European Journal of Medicinal Chemistry 39 (2004) 573–578
3.8. 2-Bromo-p-xylene-␣,␣′-13C2 (9)
3.13. (E,E)-1-Bromo-2,5-bis(3-hydroxycarbonyl-4-hydroxy)
styrylbenzene-␣,␣′-13C2 ([13C]BSB)
Bromination of p-xylene-a,a′-13C2 was carried out ac-
cording to a method reported by Kitagawa et al. [7]. Briefly,
bromine (1.65 g, 10.3 mmol) was added to a dichlo-
romethane solution (5 ml) containing ferrocenium
tetrakis[3,5-bis(trifluoromethyl)phenyl]borate [7] (0.49 g,
0.46 mmol) and ZnO (0.84 g, 10.3 mmol). The solution was
stirred at room temperature for 1 h, to which was then added
p-xylene-a,a′-13C2 (1.0 g, 9.2 mmol) in dichloromethane
(10 ml) at –50 °C. After being stirred for 2 h at 0 °C, the
reaction mixture was partitioned between ethyl acetate and
saturated Na2S2O3. Ethyl acetate layer was washed with
brine, dried (MgSO4) and concentrated in vacuo. Chroma-
tography on SiO2 (hexane) gave 1.03 g (59%) of 9 as a
colorless oil. 1H-NMR (CDCl3): d 2.28 (d, J = 127.0 Hz, 3H),
2.33 (d, J = 127.0 Hz, 3H), 6.99 (dd, J = 7.7, 4.1 Hz, 1H), 7.09
(dd, J = 7.7, 4.7 Hz, 1H), 7.34 (d, J = 4.4 Hz, 1H).
The compound was synthesized in the same manner as for
FSB in 50% yield as yellow-brown powder. mp 303 °C
(decomp.); 1H-NMR (DMSO-d6): d 6.86–7.55 (m, 6H),
7.61–7.68 (m, 1H), 7.81–7.89 (m, 4H), 8.00 (d, J = 2.2 Hz,
1H), 8.02 (d, J = 2.2 Hz, 1H); 13C-NMR (DMSO-d6): 124.8,
125.5 ppm; MS: m/z 483(M – 2); IR (KBr): 3450 (OH), 3025
(CO2H), 1670 (C=O), 1210, 1180, 950 cm–1.
3.14. Staining
Brain tissues from four AD patients obtained at autopsy
were used in this study. Tissues were fixed either in 70%
ethanol containing 150 mM NaCl or in 10% neutral buffered
formalin (NBF) overnight. Four-micrometre-thick sections
of parafin-embedded blocks were stained with FSB or
[13C]BSB according to a protocol for BSB [4]. Briefly, tis-
sues sections were deparaffinized and immersed in a 0.01%
FSB or [13C]BSB in 50% ethanol for 30 min. Sections were
then differentiated in saturated Li2CO3 and rinsed in 50%
ethanol to be examined with the fluorescence microscopy.
Upon the staining, autofluorescence from endogenous lipo-
fuscin was not quenched by using an oxidizing step as re-
ported by Barden [8].
3.9. 1-Bromo-2,5-bis(bromomethyl)benzene-␣,␣′-13C2 (10)
The compound was synthesized in the same manner as for
4 in 43% yield as a white crystalline solid. 1H-NMR
(CDCl3): d 4.41 (d, J = 154.0 Hz, 2H), 4.58 (d, J = 154.0 Hz,
2H), 7.32 (ddd, J = 7.7, 4.7, 1.7 Hz, 1H), 7.43 (dd, J = 7.7,
4.7 Hz, 1H), 7.61 (dd, J = 5.5, 1.7 Hz, 1H).
3.10. 2-Bromo-1,4-bis(diethylphosphonato)xylene-␣,␣′-13C2
References
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The compound was synthesized in the same manner as for
5 in 94% yield as yellow oil. H-NMR (CDCl3): d 1.25 (t,
J = 7.1 Hz, 12H), 3.08 (dd, J = 128.0, 21.0 Hz, 2H), 3.37 (dd,
J = 128.0, 21.0 Hz, 2H), 3.98–4.09 (m, 8H), 7.20–7.26 (m,
1H), 7.39–7.43 (m, 1H), 7.50–7.52 (m, 1H).
1
3.11. (E,E)-1-Bromo-2,5-bis(3-methoxycarbonyl-4-methoxy)
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styrylbenzene-␣,␣′-13C2 (12
)
The compound was synthesized in the same manner as for
6 in 50% yield as yellow powder. 1H-NMR (CDCl3): d 3.93
(s, 6H), 3.94 (s, 6H), 6.94 (dd, J = 154.0, 16.0 Hz, 1H),
6.98–7.14 (m, 4H), 7.43–7.45 (m, 1H), 7.60–7.72 (m, 5H),
7.97 (s, 1H), 7.98 (s, 1H).
3.12. (E,E)-1-Bromo-2,5-bis(3-methoxycarbonyl-4-hydroxy)
styrylbenzene-␣,␣′-13C2 (13
)
The compound was synthesized in the same manner as for
1
7 in 97% yield as yellow powder. H-NMR (DMSO-d6): d
3.93 (s, 6H), 6.85–7.54 (m, 6H), 7.62–7.68 (m, 1H), 7.82–
7.88 (m, 4H), 7.97 (d, J = 2.0 Hz, 1H), 7.99 (d, J = 2.1 Hz,
1H), 10.59 (s, 1H), 10.61 (s, 1H).
[8] H. Barden, The presence of ethylenic bonds and vic-glycol groups in
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