Activation of anti-oxidant Nrf2 signaling by substituted trans stilbenes

Article abstract of DOI:10.1016/j.bmc.2017.01.005

Nrf2, which is a member of the cap'n’ collar family of transcription factors, is a major regulator of phase II detoxification and anti-oxidant genes as well as anti-inflammatory and neuroprotective genes. The importance of inflammation and oxidative stress in many chronic diseases supports the concept that activation of anti-oxidant Nrf2 signaling may have therapeutic potential. A number of Nrf2 activators have entered into clinical trials. Nrf2 exists in the cytosol in complex with its binding partner Keap1, which is a thiol-rich redox-sensing protein. In response to oxidative and electrophilic stress, select cysteine residues of Keap1 are modified, which locks Keap1 in the Nrf2-Keap1 complex and allows newly synthesized Nrf2 to enter the nucleus. Numerous Nrf2-activating chemicals, including a number of natural products, are electrophiles that modify Keap1, often by Michael addition, leading to activation of Nrf2. One concern with the design of Nrf2 activators that are electrophilic covalent modifiers of Keap1 is the issue of selectivity. In the present study, substituted trans stilbenes were identified as activators of Nrf2. These activators of Nrf2 are not highly electrophilic and therefore are unlikely to activate Nrf2 through covalent modification of Keap1. Dose-response studies demonstrated that a range of substituents on either ring of the trans stilbenes, especially fluorine and methoxy substituents, influenced not only the sensitivity to activation, reflected in EC₅₀values, but also the extent of activation, which suggests that multiple mechanisms are involved in the activation of Nrf2. The stilbene backbone appears to be a privileged scaffold for development of a new class of Nrf2 activators.

Full text of DOI:10.1016/j.bmc.2017.01.005

Accepted Manuscript
Activation of Anti-oxidant Nrf2 Signaling by Substituted trans Stilbenes
Lorraine M. Deck, Lisa J. Whalen, Lucy A. Hunsaker, Robert E. Royer, David
L. Vander Jagt
PII:
S0968-0896(16)31010-0
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.01.005
BMC 13484
Reference:
Bioorganic & Medicinal Chemistry
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Accepted Date:
19 October 2016
2 January 2017
4 January 2017
Please cite this article as: Deck, L.M., Whalen, L.J., Hunsaker, L.A., Royer, R.E., Vander Jagt, D.L., Activation of
Anti-oxidant Nrf2 Signaling by Substituted trans Stilbenes, Bioorganic & Medicinal Chemistry (2017), doi: http://
dx.doi.org/10.1016/j.bmc.2017.01.005
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Activation of Anti-oxidant Nrf2 Signaling by Substituted trans Stilbenes
Lorraine M. Deck,a Lisa J. Whalen,a Lucy A. Hunsaker,b Robert E. Royer,b David L.
Vander Jagt,b,*
a Department of Chemistry and Chemical Biology, University of New Mexico,
Albuquerque, NM 87131
b Department of Biochemistry and Molecular Biology, University of New Mexico School of
Medicine, Albuquerque, NM 87131
* Corresponding author: DL Vander Jagt, dlvanderjagt@salud.unm.edu, 505-272-5788

Abstract- Nrf2, which is a member of the cap’n’collar family of transcription factors, is a major
regulator of phase II detoxification and anti-oxidant genes as well as anti-inflammatory and
neuroprotective genes. The importance of inflammation and oxidative stress in many chronic
diseases supports the concept that activation of anti-oxidant Nrf2 signaling may have therapeutic
potential. A number of Nrf2 activators have entered into clinical trials. Nrf2 exists in the cytosol
in complex with its binding partner Keap1, which is a thiol-rich redox-sensing protein. In
response to oxidative and electrophilic stress, select cysteine residues of Keap1 are modified,
which locks Keap1 in the Nrf2-Keap1 complex and allows newly synthesized Nrf2 to enter the
nucleus. Numerous Nrf2-activating chemicals, including a number of natural products, are
electrophiles that modify Keap1, often by Michael addition, leading to activation of Nrf2. One
concern with the design of Nrf2 activators that are electrophilic covalent modifiers of Keap1 is
the issue of selectivity. In the present study, substituted trans stilbenes were identified as
activators of Nrf2. These activators of Nrf2 are not highly electrophilic and therefore are unlikely
to activate Nrf2 through covalent modification of Keap1. Dose-response studies demonstrated
that a range of substituents on either ring of the trans stilbenes, especially fluorine and methoxy
substituents, influenced not only the sensitivity to activation, reflected in EC50 values, but also
the extent of activation, which suggests that multiple mechanisms are involved in the activation
of Nrf2. The stilbene backbone appears to be a privileged scaffold for development of a new
class of Nrf2 activators.

1. Introduction
Inflammation and oxidative stress are associated with most chronic diseases, including
neurodegenerative and neuropsychiatric diseases. In Alzheimer’s disease (AD), for example,
focal activation of microglia, the resident brain macrophage, which is a source of pro-
inflammatory signals and oxidative stress, is seen consistently in the vicinity of plaques, both in
pathological samples from AD patients and in animal models.1-5 A normal response to oxidative
stress is activation of a number of anti-oxidant genes to counteract the development of oxidative
stress. The promoters for these anti-oxidant genes commonly have an anti-oxidant response
element (ARE) where transcription factor Nuclear factor erythroid 2 related factor 2 (Nrf2) can
bind to induce gene expression.
Nrf2, which is a member of the cap’n’collar family of transcription factors, is a major regulator
of phase II detoxification and anti-oxidant genes as well as anti-inflammatory and
neuroprotective genes.6-12 In unstressed cells, Nrf2 is retained in the cytosol through formation
of a complex between Nrf2, Kelch-like ECH associated protein 1 (Keap1), and Cul3, which is an
adaptor to link Nrf2 to an E3 ubiquitin ligase complex. Nrf2 is continuously synthesized and then
degraded by ubiquitination and proteosomal degradation, which limits the cytosolic
concentration of Nrf2. In response to oxidative and other stress, select cysteine residues of Keap1
are modified. This strengthens the interaction between Nrf2 and Keap1, which locks Keap1 in
the complex.13,14 This allows newly synthesized Nrf2 to accumulate and translocate to the
nucleus where it interacts with small Maf proteins and subsequently binds to promoters with
ARE sequences. Nrf2 can also be locked to Keap1 by covalent modification of Keap1 by
electrophiles.
Nrf2 is widespread in the CNS and is recognized as an important regulator of brain
inflammation. In animal models with Nrf2 deficiency, neurodegeneration is observed, especially
in areas with high oxidative metabolism.7,15 In animal models of stroke and traumatic brain
injury, Nrf2 deficiency leads to more intense inflammation and more severe injury.16,17 In
humans, Nrf2 declines with age and may contribute to age as a risk factor for neurological
disease. Activation of Nrf2 in astrocytes can protect neurons from oxidative stress.18 The
impaired translocation of Nrf2 to the nucleus in neurons from AD subjects19 suggests that defects
in Nrf2 activation may play a major role in the development of oxidative damage in AD brains.
Recently we reported that the natural product resveratrol, which is a trans stilbene, and a
synthetic trans stilbene ((E)-2-fluoro-4’-methoxystilbene, designated LD55) were effective at
reducing both brain inflammation and plaque accumulation in a transgenic animal model of
AD.20 LD55 was selected based upon its ability to inhibit activation of the pro-inflammatory
transcription factor NF-κB in a reporter cell-based assay and on its ability to inhibit expression
of pro-inflammatory signals in a microglia cell line in response to LPS.21 Resveratrol also
inhibits the activation of NF-κB.22 In the present study, we demonstrate that LD55 also shares
with resveratrol the ability to activate Nrf2, and we describe the activities of a series of trans
stilbenes that were compared to resveratrol and LD55 for their abilities to promote activation of

Nrf2 in reporter assays. The natural product isothiocyanate sulforaphane, which is a well-
established activator of Nrf2,23 was used as a positive control.
2. Materials and method
2.1. Synthesis of substituted trans stilbenes
Initial screening for activators of Nrf2 utilized a library of substituted trans stilbenes available
from our earlier studies of inhibitors of NF-κB.21 This initial screen identified LD55 and several
other fluorine-substituted or methoxy-substituted trans stilbenes. Therefore synthesis and testing
of substituted trans stilbenes to develop SAR was focused on these substituents. The synthesis of
56 substituted trans (E)-stilbenes (Table 1) was accomplished using Horner-Wadsworth-
Emmons (HWE) olefination chemistry.24 The starred compounds in Table 1 are new to the
literature, and their synthesis is described in Scheme 1.25 Synthesis of the other compounds in
Table 1 was accomplished using a method reported previously.21 HWE chemistry was used to
avoid formation of a mixture of E and Z isomers and formation of triphenylphosphine oxide,
which complicates the purification process.
The required phosphonate ester starting materials were prepared in high yields by the classical
solvent free Michaelis-Arbuzov reaction of substituted benzyl chlorides or bromides with
triethylphosphite at 130oC.26 Removal of excess triethylphosphite and chloroethane or
bromoethane product was carried out by vacuum distillation. Further purification using a short
silica gel column and eluting with ethyl acetate/hexane provided pure phosphonate esters as
oils.27
Reaction of the appropriately substituted diethyl benzylphosphonate ester with a corresponding
freshly distilled substituted benzaldehyde in dry tetrahydrofuran using sodium hydride as the
base (Scheme 1) afforded the 20 new substituted stilbenes (Table 1) exclusively in the E
conformation. There was no detectable Z isomer, and the trans geometry of the substituted
stilbenes was confirmed by the coupling constants of approximately 16.5 Hz for the olefinic
protons in the proton NMR spectra. The reaction conditions allowed for a straightforward
workup since the diethylphosphoric acid byproducts are water-soluble and were easily removed
by extraction. Purification of the benzaldehydes and phosphonate esters was found to be critical
for obtaining good yields and pure products.
O
CHO
R2
CH2P(OEt)2
a
R1
R2
Aldehyde
R1
+
Stilbene
Phosphate ester
Scheme 1 Reagents and conditions for synthesis of the starred compounds in Table 1: (a) R1
o
substituted phosphonate ester (1.5 equiv), NaH (2 equiv), dry THF, 0 C 30 min, R2 substituted
aldehyde (1.0 equiv), 0 oC to rt, 20 h, then H2O/HCl.

Table 1
a
Structures of (E)-Stilbenes
Product
1
R1 R2
4-OCH3 2-F
Referenceb Product R1
R2
Referenceb
21
21
21
21
21
21
28
28
28
-
29
30
31
32
33
-
-
-
29
34
35
-
-
-
36
-
29*
30*
31
32
33
34*
35*
36*
37*
38
39
40*
41
42
43
44
45
46
47
48*
49*
50*
51
52
53
54*
55*
56
2-F
3-F
2-F
3-F
3-F
3-F
3-F
2-F
4-CH3
4-F
4-F
4-F
-
2,5-diF
3,4-diF
2,6-diF
3-F
-
-
35
31
36
-
2
3
4
5
6
7
8
9
10*
11
12
13
14
15
16*
17*
18*
19
20
21
22*
23*
24*
25
26*
27
28
-
-
-
2-F
3-F
4-F
4-OCH3 3-F
4-OCH3 4-F
4-F
2,5-diF
2,4-diF
2,4-diF
2,4-diF
2,4-diF
3,4-diF
2,5-diF
2,3-diOCH3
2-OCH3
2,4-diOCH3
2-Cl
4-CH3
4-CH3
4-CH3
2-F
4-F
3-F
2-F
2-OCH3
-
-
-
39
39
-
21
21
21
21
34
40
31
-
3-OCH3 2-F
3-OCH3 4-F
3-OCH3 3-F
3-F
4-F
3-F
4-F
3,4-diOCH3
-
3,4-diOCH3
2,3-diOCH3
2,3-diOCH3
2,5-diOCH3
2-F
4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-CH3
2,3-OCH3
3-OCH3
3-OCH3
4-OCH3
-
3-OH
4-F
3-OCH3, 4-OH
3-CF3
4-CH3
2-OCH3
2,5-diOCH3
3-NO2
4-Cl
4-OH
-
2-F
2,6-diF
-
4-CH3 3,4-diF
4-OCH3 2,5-diF
-
41
42
43
-
2-F
2-F
3,4-diF
3-F
3-NO2
3-NO2
4-CH3
2,3-diOCH3 4-CF3
4-OCH3 3,4-diF
2-F
4-F
2-F
2-F
37
38
2,3-diOCH3 4-CH(CH3)2
2,4-diOCH3 2,5-diOCH3
-
44
a data for starred compounds is reported in the experimental section
b melting points and NMR data of known compounds are in agreement with the literature data
3. Results and discussion
3.1. Activation of Nrf2 by resveratrol, LD55 and sulforaphane
Resveratrol, LD55 and sulforaphane were compared in an Nrf2-ARE reporter-HepG2 stable cell
line, which was designed for use as a luciferase reporter-based assay for activators of Nrf2.
Sulforaphane was used as a positive control. The EC50 value obtained for sulforaphane was
identical to the value reported by the manufacturer of the assay kit. The concentrations that
produced 50% activation (EC50) were obtained from dose-response curves, in which at least 6
concentrations were used and measurements were made at least in triplicate. In addition, cells
were washed extensively to remove the activating stilbene, followed by further dilution when the

cells were lysed. Additional dilution of any remaining activator occurred when a lysed sample
was assayed for luciferase activity. Low concentrations that might remain were shown to have no
direct effect on luciferase activity, either as inhibitor of activator. The ability of both resveratrol
and LD55 to activate Nrf2 signaling extends the known activities of these two trans stilbenes to
include both anti-inflammatory activities as inhibitors of the pro-inflammatory NF-κB signaling
pathway as well as anti-oxidative stress activities as activators of Nrf2. It is noteworthy that the
extent of activation of Nrf2 differs for these three activators (Table 2). LD55 was 5-fold better
than resveratrol in the extent of Nrf2 activation.. Resveratrol and LD55 show low EC50 values
(5.4µM), comparable to sulforaphane (1.2µM) (Table 2). The fact that these activators of Nrf2
can vary both in their EC50 values and in the extent of activation requires that both of these
factors be considered in evaluation of the activities of Nrf2 activators.
3.2. Activation of Nrf2 by monofluoro trans stilbenes
A library of substituted trans stilbenes was screened to identify activators of Nrf2. With few
exceptions, such as resveratrol, only compounds with fluorine or methoxy substituents showed
significant activity. From this screen and synthesis of additional compounds, a group of 56
substituted trans stilbenes was identified as activators of Nrf2. A series of monofluoro trans
stilbenes was compared with resveratrol and LD55. The series was screened at 15µM
concentrations of the compounds, a concentration that did not produce any solubility problems
and generally was sufficiently higher than the EC50 values to allow a comparison of the differing
extents of activation, obtained from dose-response studies (Table 2). All of these monofluoro
trans stilbenes were activators of Nrf2, although the extent of activation varied almost 20-fold.
Many of the compounds were better Nrf2 activators than sulforaphane and were much better than
resveratrol. The EC50 values for these monofluoro trans stilbenes ranged from 0.7µM (trans
stilbene 2) to 12.4 µM (trans stilbene 4). Comparison of trans stilbenes 2, 3 and 4, all of which
contain a single fluorine substituent on one of the aromatic rings and no substituent on the other
ring, suggests that fluorine substitution in the ortho position, as in LD55, is preferred. However,
this is not consistently observed. Comparison of LD55 with 5 and 6, all of which contain a para
methoxy substituent, shows little effect of altering the position of the fluorine on EC50 values
which ranged from 4.2 to 8.9 µM (Table 2). Likewise, altering the positions of both the fluorine
and methoxy substituents as in LD55, 5, 6, 10-13, all of which are isomers, has a modest effect
with EC50 values ranging from 2.3 to 8.9 µM. The extent of activation, however, differs
markedly. For example, isomer 10 is activated almost 10-fold more than isomer 6 (Table 2).
Replacing the methoxy substituent with a methyl substituent (7, 8 and 9) lowers EC50 to low or
even sub-micromolar values.

Table 2. Activation of Nrf2 by monofluoro trans stilbenes
Fold
activation
Number
Structure
EC50 (µM)
1.2 ± 0.08
O
S
S
C
N
Sulforaphane
12.4 ± 0.3
OH
Resveratrol
5.4 ± 0.5
3.2 ± 0.2
OH
HO
F
1 (LD55)
5.4 ± 0.3
0.7 ± 0.1
3.1 ± 0.4
12.4 ± 0.5
14.9 ± 1.2
10.7 ± 0.6
4.9 ± 2.3
4.8 ± 0.3
H3CO
F
2
3
4
F
F
F
5
8.9 ± 0.9
3.6 ± 0.2
H3CO
F
6
7
4.2 ± 0.4
1.3 ± 0.2
1.7 ± 0.1
1.1 ± 0.2
H3CO
H3C
F
F
8
0.8 ± 0.08
4.7 ± 0.03
H3C
9
1.4 ± 0.2
3.1 ± 0.2
3.8 ± 0.2
10.1 ± 0.5
18.4 ± 0.6
14.4 ± 0.4
F
H3C
F
10
11
OCH3
F
H3CO
F
12
6.0 ± 0.5
4.4 ± 0.8
OCH3
H3CO
13
14
2.3 ± 0.5
9.5 ± 0.7
15.1 ± 0.8
5.9 ± 0.7
F
OCH3
OCH3
F
OCH3
OCH3
15
16
2.6 ± 0.5
1.9 ± 0.1
1.6 ± 0.02
19.4 ± 0.5
F
OCH3
H3CO
H3CO
F
OCH3
17
2.9 ± 0.1
16.9 ± 0.5
F
H3CO
18
19
20
5.1 ± 0.5
1.6 ± 0.2
11.4 ± 1.6
10.7 ± 0.5
5.3 ± 0.05
13.4 ± 1.3
OCH3
F
F
Cl
F
HO

3.3. Activation of Nrf2 by polyfluoro trans stilbenes
A series of polyfluoro trans stilbenes was compared with resveratrol and LD55. EC50 values
ranged from 0.3 to >15 µM (Table 3) and extent of activation varied about 5-fold, with all of
these stilbenes better than resveratrol with respect to fold activation. For several compounds (24-
26, 39), solubility problems precluded obtaining maximum activations and, therefore, EC50
values from dose-response studies. For these trans stilbenes, fold activation is reported at the 15
µM concentrations used in the initial screening. As with the monofluoro trans stilbenes, the
location of the fluorine substituents markedly affected EC50 values and fold activations, but not
in readily predictable patterns. For example, EC50 for the difluoro trans stilbene 33 is sub-
micromolar (0.65 µM) while EC50 for its isomer 25 is >15 µM. Likewise, the EC50 for the
trifluoro trans stilbene 34 is sub-micromolar (0.3 µM) while EC50 for its isomer 24 is >15 µM.
Multiple trifluoro isomers (34, 35, 38) exhibited sub-micromolar EC50 values. Isomer 34 was the
most effective activator when expressed as fold activation divided by EC50. The conclusion from
the data in Tables 2 and 3 is that all of these fluorine substituted trans stilbenes demonstrate
measurable activity as activators of Nrf2.

Table 3. Activation of Nrf2 by polyfluoro trans stilbenes
Fold
activation
Number
21
Structure
EC50 (µM)
6.9 ± 0.1
2.6 ± 0.18
3.2 ± 0.5
>15
F
11.4 ± 0.4
F
F
F
22
10.2 ± 0.2
17.0 ± 0.9
11.5 ± 2.0a
H3C
F
23
F
H3CO
F
F
F
24
F
F
25
26
>15
>15
8.5 ± 0.2a
4.6 ± 0.6a
F
F
H3CO
F
F
27
28
29
4.3 ± 0.4
3.6 ± 0.3
5.9 ± 0.2
13.9 ± 0.9
12.7 ± 0.4
22.3 ± 0.5
F
F
F
F
F
F
F
F
30
31
32
5.0 ± 0.1
1.4 ± 0.5
1.0 ± 0.12
12.2 ± 0.8
21.2 ± 2.0
7.0 ± 0.7
F
F
F
F
F
F
F
33
34
35
0.65 ± 0.12
0.3 ± 0.02
0.45 ± 0.01
4.5 ± 0.4
10.7 ± 1.0
8.2 ± 0.2
F
F
F
F
F
F
F
F
F
36
37
38
9.6 ± 0.14
2.0 ± 0.07
5.0 ± 0.8
13.5 ± 0.2
16.8 ± 1.8
F
F
H3C
F
F
0.9 ± 0.08
>15
F
F
F
39
40
7.0 ± 0.5a
11.2 ± 0.1
F
F
F
2.9 ± 0.59
F
a. Fold activation at 15 µM

3.4. Activation of Nrf2 by nonfluoro trans stilbenes
A series of substituted trans stilbenes, most of which contained methoxy groups at one or more
positions, demonstrated activity as activators of Nrf2, as shown in Table 4. EC50 values ranged
from 0.8 µM (trans stilbene 56) to >15 µM. All of these trans stilbenes were as good as or better
than sulforaphane with respect to fold activation and were much better than resveratrol.
Compounds 51 and 54 demonstrated the highest fold activations of all of the trans stilbenes, 69
and 65.5 fold, respectively. The results from this study support the conclusion that the stilbene
scaffold is a useful structure upon which to explore chemical space to develop activators of Nrf2,
especially those that include fluorine and methoxy functional groups as in LD55.
The importance of inflammation and oxidative stress in many chronic diseases supports the
concept that activation of anti-oxidant Nrf2 signaling may have therapeutic potential. Cells have
developed complex adaptive responses to oxidative stress to maintain redox homeostasis and to
reduce oxidative stress.45 Stress activation of Nrf2 may be the result of exposure to xenobiotics,
including many natural products.46 Many of these Nrf2 activators are electrophiles. One of the
major mechanisms of Nrf2 activation is through covalent modification of select cysteine
sulfhydryl residues in Keap1. Numerous Nrf2-activating chemicals, including natural products
such as sulforaphane, are electrophiles that modify Keap1, often by Michael addition.47-49
Cysteine residues 273, 288 and 151 appear to be especially important targets, as determined by
site-directed mutagenesis. Resveratrol and other natural product phenols activate Nrf2 after
oxidation to electrophilic quinones that contain Michael acceptor functionalities.49 Moreover,
some natural product phenols including resveratrol can activate Nrf2 by additional mechanism,
such as increasing the level of Nrf2 mRNA.49
A number of Nrf2 activators have entered into clinical trials. Bardoxolone methyl, an oleanolic
acid-derived synthetic triterpene, is a potent Nrf2 activator that was evaluated for its ability to
slow progression to end-stage renal disease in patients with type 2 diabetes and stage 4 chronic
kidney disease. Bardoxolone methyl is able to modify Keap1 as a Michael acceptor. The clinical
trial was terminated in phase III owing to an increase in heart failure.50 However, bardoxalone
methyl is currently in phase III studies for pulmonary hypertension (ClinicalTrials.gov).
Dimethyl fumarate, a simple derivative of the metabolic intermediate fumaric acid, has recently
been FDA-approved for the treatment of relapsing-remitting multiple sclerosis.51 Dimethyl
fumarate is active as the monomethyl derivative formed by the action of non-specific esterases.
Monomethyl fumarate modifies Keap1 through electrophilic addition. For both bardoxolone
methyl and dimethyl fumarate, however, mechanisms in addition to modification of Keap1 may
play a role in activation of Nrf2.

Table 4. Activation of Nrf2 by nonfluoro trans stilbenes
Fold
activation
Number
41
Structure
EC50 (µM)
4.7 ± 0.7
OCH3
H3CO
25.4 ± 0.1
H3CO
42
43
4.4 ± 0.1
4.6 0.4
23.1 ± 0.6
15.6 ± 2.0
H3CO
OCH3
H3CO
Cl
44
45
3.1 ± 0.2
>15
17.8 ± 0.7
28.1 ± 0.1a
H3CO
H3CO
OH
OCH3
OH
46
47
3.7 ± 0.5
1.5 ± 0.5
27.7 ± 1.1
19.2 ± 1.1
H3CO
CF3
H3C
CH3
OCH3
H3CO
48
49
50
1.1 ± 0.2
4.4 ± 0.2
>15
16.1 ± 2.3
29.2 ± 1.2
15.7 ± 3.2a
H3CO
H3CO
H3CO
H3CO
OCH3
NO2
51
52
53
3.3 ± 0.4
3.0 ± 0.4
3.7 ± 0.2
69.0 ± 1.6
18.6 ± 0.5
11.9 ± 2.1
H3CO
NO2
NO2
H3C
CF3
OCH3
H3CO
54
55
2.2 ± 0.1
2.7 ± 0.3
65.5 ± 2.5
33.9 ± 0.7
CH(CH3)2
OCH3
H3CO
OCH3
OCH3
56
0.8 ± 0.03
12.9 ± 1.2
OCH3
H3CO
a. Fold activation at 15 µM
One concern with the design of Nrf2 activators that are electrophilic covalent modifiers of Keap1
is the issue of selectivity. Bardoxolone methyl, for example, has been shown to react with
multiple targets.52 To address this concern, a number of recent studies have focused on
development of non-electrophilic activators of Nrf2,53 aided by the availability of the crystal
structures of the BTB domain of Keap1, which contains residue cysteine151 that is the target for
covalent modification by bardoxolone methyl,54 the Kelch domain of Keap155 and the Keap1-

Nrf2 interface.56 Peptide inhibitors of the Keap1-Nrf2 protein-protein interaction have been
described57,58 as well as a variety of small molecules that inhibit the Keap1-Nrf2 interaction.59-62
There are additional mechanisms for activation of Nrf2. Epigenetic modifications of CpG
methylation status of Nrf2 by the anti-cancer drug 3,3’-diindolylmethane resulted in enhanced
expression of Nrf2 and of Nrf2-target genes in cell-based studies.63 This was suggested as an
explanation for the chemopreventive activity of this drug. The promoter of the gene encoding
Nrf2 contains XRE sequences, which can recruit the aromatic hydrocarbon receptor AhR; this
allows the Nrf2 gene to be activated by polycyclic aromatic hydrocarbons. 64 The promoter of the
Nrf2 gene also contains ARE-like sequences, which allows Nrf2 to regulate its own expression.65
The Nrf2 promoter also contains an NF-κB binding site, which allows activation of Nrf2 by pro-
inflammatory stimuli.66
The autophagy cargo receptor and signaling adaptor protein p62 contains a binding site for
Keap1. The attraction of Keap1 to this site is further regulated by phosphorylation by mTORC1,
which leads to autophagosome–mediated destruction of Keap1 and activation of Nrf2.67,68 This is
one example of proteins other than Nrf2 that interact specifically with Keap1, which indicates a
broad role for Keap1 and a complex set of mechanisms that impact Nrf2 levels.69 There are,
therefore, numerous targets for potential activators of Nrf2.
There are also mechanisms for activation of Nrf2 that do not appear to involve Keap1.70-72 Nrf2
activity is inhibited by the serine/threonine kinase GSK-3. GSK-3 constitutive activity is
inhibited by PKB/Akt phosphorylation. It is conceivable that trans stilbenes might activate Nrf2
independently of Keap1 through inhibition of GSK-3 or activation of kinases such as Akt.
In the present study, we identified numerous substituted trans stilbenes as activators of Nrf2,
especially trans stilbenes with fluorine and/or methoxy ring substituents. The double bond in the
trans stilbene scaffold is generally a low-reactivity center which would be unlikely to modify
Keap1 by electrophilic addition. This was tested by treating trans stilbene with N-acetylcysteine
under a variety of conditions involving different pH, solvent composition and concentrations and
examining the reaction mixture by NMR. There was no evidence of thiol addition to the trans
stilbene double bond under these conditions. It remains to be determined which site(s) is the
target for activation of Nrf2 by substituted trans stilbenes. Likewise, it remains to be determined
how different trans stilbenes produce markedly different fold activations, which likely involves
multiple mechanisms. There is always concern about toxicity, which includes concern about
potential estrogenic activity of stilbenes, especially hydroxylated stilbenes.73 However, the
natural product resveratrol, which has been studied extensively including numerous clinical
trials, is a hydroxylated stilbene with little evidence of toxicity. It is also noteworthy that our
initial study of LD55 in a transgenic animal model of AD involved feeding of LD55 at 100ppm
in the diet for one year with no apparent toxicity.
4. Conclusions
Substituted trans stilbenes represent promising compounds for the development of potential
drugs that target oxidative stress in chronic diseases, through activation of the anti-oxidant Nrf2
signaling pathway.

5. Experimental
5.1. Reporter Assay:
A Nrf2-ARE reporter-HepG2 stable cell line (BPS Bioscience, San Diego, CA) is grown in a
humidified atmosphere at 37oC in 5% CO2/95% air. The cells are maintained in MEM medium
with Earles balanced salts and L-glutamine supplemented with 10% fetal bovine serum (FBS), 1
mM sodium pyruvate, 1% non-essential amino acids, 100 units/ml penicillin, 100 µg/ml
streptomycin, and 400 µg/ml Geneticin. One day prior to treatment, the Nrf2-ARE cells are
plated into 24-well cell culture plates at approximately 30% confluency in the above media
without Geneticin. The following day, fresh media with or without substituted trans stilbene or
sulforaphane is applied to the cells. DMSO concentrations are kept at 0.1%. The cells are again
placed in a humidified atmosphere at 37oC in 5% CO2/95% air for 5 hours. Plate wells are
gently washed with phosphate buffered saline (PBS) pH 7.4 and lysed with 1x passive lysis
buffer (Promega, Madison, WI, USA). The subsequent lysates are analyzed with the Luciferase
Assay System (Promega) utilizing a GloMax 20/20 luminometer (Promega, Sunnyvale, CA,
USA). The firefly luciferase relative light units are normalized to protein (mg/ml) with BCATM
Protein Assay Kit protein (Pierce, Rockford, IL, USA)
5.2. Synthesis:
Reagents were purchased from commercial sources (Aldrich, Acros). Tetrahydrofuran was
distilled from lithium aluminum hydride. Thin layer chromatography was carried out on silica
13
gel 60F254 plates. All compounds were shown to be > 98% pure by 1H NMR and/or C NMR
unless otherwise noted. Column chromatographic separations were performed by using EM type
60 silica gel (230-400 mesh). Melting points were taken on a Thomas-Hoover Uni-Melt capillary
1
melting point apparatus and reported uncorrected. Unless otherwise noted, H spectra were
recorded by using CDCl3 solutions at 300 MHz; 13C NMR spectra were recorded in CDCl3 at 75
19
MHz; F were recorded in CDCl3 at 282 MHz. Chemical shifts are reported in ppm relative to
1
CDCl3 at 7.24 ppm for H NMR and 77.0 ppm for 13C NMR and the external standard
hexafluorobenzene for 19F NMR at -164.9 ppm. Peak assignments were made with the aid of
DEPT spectra. 1H NMR data are reported as follows: chemical shift, multiplicity (s = singlet, d =
doublet, t = triplet, q = quartet, dd = doublet of doublets, td = triplet of doublets, m = multiplet),
coupling constant (J in Hz) and integration. High resolution mass spectra (HRMS) were obtained
at the UNM Mass Spectrometry Facility, Albuquerque, New Mexico.
General Procedure for Synthesis of Phosphonate Esters
Benzyl chloride or benzyl bromide derivatives (1eq) were added to triethylphosphite (1.5 eq) and
heated to 130oC for 20 h. After cooling, the resulting crude product was distilled in vacuo to
remove excess triethylphosphite and ethyl chloride or ethyl bromide. Purification by filtration
through a pad of silica gel (70% ethyl acetate/30% hexanes) gave the phosphonate ester products
as colorless oils.

General Procedure for Synthesis of Stilbenes
The appropriately substituted phosphonate ester (10 mmol) was dissolved in dry tetrahydrofuran
(20 ml) and stirred at 0-5oC. Sodium hydride (25 mmol) was added to the solution slowly and
after thirty minutes the appropriate freshly distilled aldehyde (10 mmol) in tetrahydrofuran (30
ml) was added dropwise. The mixture was allowed to stir at room temperature overnight. In
order to increase the yield, compounds 35, 37 and 40 were heated under reflux for 3-4 hours.
The mixture was cooled and quenched with ice water (10 ml) and poured onto ice. Dilute
hydrochloric acid (1M) was added until acidic and the solution was extracted with ethyl acetate
(4 x 50 ml). The combined organic layers were washed with saturated salt and dried over
magnesium sulfate. Filtration and evaporation of the ethyl acetate afforded crude stilbene
products as oils or solids. The solids were crystallized from 95% ethanol to afford crystalline
stilbenes. The oils were chromatographed on silica gel using methylene chloride to give pure
products.
o
1
(E)-1-Fluoro-2-(2-methoxystyryl)benzene 10: 98% yield, white crystals; mp 34-35 C; H NMR
(CDCl3, 300 MHz): δ 7.66 (dt, J = 7.8, 1.6 Hz, 1H), 7.62 (dd, J = 7.6, 1.5 Hz, 1H), 7.54 (d, J =
16.6 Hz, 1H), 7.27 (d, J = 16.6 Hz, 1H), 7.22 (m, 2H), 7.13 (dt, J = 7.5, 1.3 Hz, 1H), 7.05 (dt, J =
13
8.0, 1.3 Hz, 1H), 6.98 (t, J = 7.4 Hz, 1H), 6.91 (d, J = 8.3 Hz, 1H), 3.88 (s, 3H). C NMR
(CDCl3, 75 MHz): δ 160.5 (d, J = 247.7 Hz), 157.2 (s), 129.2 (s), 128.6 (d, J = 8.4 Hz), 127.0 (d,
J = 3.4 Hz), 126.7 (s), 126.5 (s), 126.0 (d, J = 12.1 Hz), 125.7 (d, J = 3.6 Hz), 124.2 (d, J = 3.1
Hz), 121.1 (d, J = 4.0 Hz), 120.9 (s), 115.5 (d, J = 22.2 Hz), 110.8 (s), 55.7 (s). 19F (CDCl3, 282
MHz): δ -116.9 (s, 1F). HRMS (EI) calcd for C15H13FO [M]+: 228.0950; found, 228.0950.
(E)-1-(3-Fluorostyryl)-2,3-dimethoxybenzene 16: 98% yield, oil; 1H NMR (CDCl3, 300 MHz): δ
7.45 (d, J = 16.5 Hz, 1H), 7.27 (m, 3H), 7.23 (dd, J = 7.9, 1.3 Hz, 1H), 7.08 (d, J = 16.5 Hz, 1H),
7.06 (t, J = 8.1Hz, 1H), 6.96 (m, 1H), 6.86 (dd, J = 8.1, 1.4 Hz, 1H), 3.88 (s, 3H), 3.87 (s, 3H).
13C NMR (CDCl3, 75 MHz): δ 163.2 (d, J = 245.1 Hz), 153.3 (s), 147.3 (s), 140.1 (d, J = 7.6
Hz), 131.0 (s), 130.1 (d, J = 8.4 Hz), 128.7 (s), 124.3 (s), 124.2 (s), 122.6 (s), 117.9 (s), 114.4 (d,
J = 21.5 Hz), 112.9 (d, J = 21.8 Hz), 111.8 (s), 61.2 (s), 56.0 (s). 19F (CDCl3, 282 MHz): δ -111.9
(s, 1F). HRMS (EI) calcd for C16H15FO2 [M]+: 258.1056; found, 258.1058.
o
1
(E)-1-(4-Fluorostyryl)-2,3-dimethoxybenzene 17: 87% yield, white crystals; mp 50-51 C; H
NMR (CDCl3, 300 MHz): δ 7.50 (dd, J = 8.4, 5.6 Hz, 2H), 7.36 (d, J = 16.5 Hz, 1H), 7.21 (d, J =
7.8 Hz, 1H), 7.07 (d, J = 16.4 Hz, 1H), 7.04 (m, 3H), 6.83 (d, J = 7.6 Hz, 1H), 3.87 (s, 3H), 3.85
(s, 3H). 13C NMR (CDCl3, 75 MHz): δ 162.4 (d, J = 247.1 Hz), 153.2 (s), 147.0 (s), 133.9 (d, 2.9
Hz), 131.4 (s), 128.7 (s), 128.2 (d, J = 7.9 Hz), 124.2 (s), 122.8 (s), 117.8 (s), 115.6 (d, J = 21.6
19
Hz), 111.5 (s), 61.1 (s), 55.8 (s). F (CDCl3, 282 MHz): δ -112.7 (s, 1F). HRMS (EI) calcd for
C16H15FO2 [M]+: 258.1056; found, 258.1060.
o
1
(E)-2-(4-Fluorostyryl)-1,4-dimethoxybenzene 18: 74% yield, white crystals; mp 58-59 C; H
NMR (CDCl3, 300 MHz): δ 7.49 (dd, J = 8.7, 5.5 Hz, 2H), 7.37 (d, J = 16.4 Hz, 1H), 7.13 (d, J =
2.6 Hz, 1H), 7.05 (d, J = 16.4 Hz, 1H), 7.03 (t, J = 8.7 Hz, 2H), 6.80 (m, 2H), 3.83 (s, 3H), 3.81

(s, 3H). 13C NMR (CDCl3, 75 MHz): δ 162.3 (d, J = 247.0 Hz), 153.8 (s), 151.4 (s), 134.0 (d, 3.1
Hz), 128.1 (s), 128.0 (d, J = 7.7 Hz), 123.1 (s), 115.6 (d, J = 21.6 Hz), 113.7 (s), 112.2 (s), 111.7
19
(s), 56.2 (s), 55.7 (s). F (CDCl3, 282 MHz): δ -112.9 (s, 1F). HRMS (EI) calcd for C16H15FO2
[M]+: 258.1056; found, 258.1057.
o
1
(E)-1,2-Difluoro-4-(4-methylstyryl)benzene 22: 97% yield, white crystals; mp 90-92 C; H
NMR (CDCl3, 300 MHz): δ 7.38 (d, J = 8.1 Hz, 2H), 7.29 (m, 1H), 7.16 (d, J = 8.1 Hz, 2H), 7.13
(m, 2H), 6.99 (d, J = 16.4 Hz, 1H), 6.92 (d, J = 16.4 Hz, 1H), 2.36 (s, 3H). 13C NMR (CDCl3, 75
MHz): δ 151.7 (dd, J = 69.7, 13.3 Hz), 148.5 (dd, J = 70.7, 13.3 Hz), 138.0 (s), 134.9 (t, J = 5.0
Hz), 133.9 (s), 129.7 (s), 129.5 (s), 126.5 (s), 125.5 (s), 122.6 (dd, J = 6.1, 2.9 Hz), 117.3 (d, J =
17.5 Hz), 114.5 (d, J = 17.6 Hz), 21.3 (s). 19F (CDCl3, 282 MHz): δ -136.3 (d, J = 20.9 Hz, 1F), -
137.7 (d, J = 20.9 Hz, 1F). HRMS (EI) calcd for C15H12F2 [M]+: 230.0907; found, 230.0911.
(E)-1,4-Difluoro-2-(4-methoxystyryl)benzene 23: 60% yield, pale yellow crystals; mp 109-110
oC; 1H NMR (CDCl3, 300 MHz): δ 7.46 (d, J = 8.6 Hz, 2H), 7.25 (m, 1H), 7.06 (s, 2H), 6.98 (m,
13
1H), 6.89 (d, J = 8.6 Hz, 2H), 6.86 (m, 1H), 3.81 (s, 3H). C NMR (CDCl3, 75 MHz): δ 159.8
(s), 159.2 (d, J = 200.2 Hz), 156.0 (d, J = 201.6 Hz), 131.5 (d, J = 3.9 Hz), 129.5 (s), 128.1 (s),
127.0 (dd, J = 14.5, 8.2 Hz), 117.6 (s), 116.7 (dd, J = 25.4, 8.7 Hz), 114.5 (dd, J = 24.5, 9.0 Hz),
114.2 (s), 112.4 (dd, J = 24.6, 3.8 Hz), 55.3 (s). 19F (CDCl3, 282 MHz): δ -117.7 (d, J = 17.2 Hz,
1F), -122.9 (d, J = 17.2 Hz, 1F). HRMS (EI) calcd for C15H12F2O [M]+: 246.0856; found,
246.0852.
(E)-1,2-Difluoro-4-(2-fluorostyryl)benzene 24: 82% yield, white crystals; mp 85-86 oC; 1H NMR
(CDCl3, 300 MHz): δ 7.56 (dt, J = 7.7, 1.6 Hz, 1H), 7.33 (dt, J = 7.7, 1.8 Hz, 1H), 7.23 (m, 2H),
7.17 (d, J = 16.4 Hz, 1H), 7.10 (m, 3H), 7.05 (d, J = 16.3 Hz, 1H). 13C NMR (CDCl3, 75 MHz):
δ 160.7 (d, J = 247.9 Hz), 152.1 (dd, J = 44.2, 12.0 Hz), 148.8 (dd, J = 46.6, 12.0 Hz), 134.7 (t, J
= 6.2 Hz), 129.4 (d, J = 8.4 Hz), 128.9 (s), 127.3 (d, J = 3.2 Hz), 124.7 (d, J = 11.9 Hz), 124.4 (d,
J = 2.7 Hz), 123.1 (dd, J = 5.3, 3.0 Hz), 122.3 (s), 117.6 (d, J = 17.5 Hz), 116.1 (d, J = 22.1 Hz),
19
115.0 (d, J = 17.6 Hz). F (CDCl3, 282 MHz): δ -116.0 (s, 1F), -136.0 (d, J = 20.8 Hz, 1F), -
136.8 (d, J = 21.1 Hz, 1F). HRMS (EI) calcd for C14H9F3 [M]+: 234.0656; found, 234.0652.
o
1
(E)-1,2-Difluoro-4-(4-methoxystyryl)benzene 26: 66% yield, white crystals; mp 73-74 C; H
NMR (CDCl3, 300 MHz): δ 7.41 (d, J = 8.6 Hz, 2H), 7.27 (m, 1H), 7.11 (m, 2H), 6.94 (d, J =
16.5 Hz, 1H), 6.88 (d, J = 8.7 Hz, 2H), 6.83 (d, J = 16.5 Hz, 1H), 3.81 (s, 3H). 13C NMR (CDCl3,
75 MHz): δ 159.8 (s), 151.8 (dd, J = 79.6, 12.7 Hz), 148.6 (dd, J = 81.0, 13.2 Hz), 135.2 (t, J =
4.9 Hz), 129.6 (d, J = 13.0 Hz), 129.5 (s), 128.0 (s), 124.5 (s), 122.5 (dd, J = 5.4, 2.8 Hz), 117.5
(d, J = 17.4 Hz), 114.5 (d, J = 16.0 Hz), 114.4 (s), 55.5 (s). 19F (CDCl3, 282 MHz): δ -136.4 (d,
J = 20.9 Hz, 1F), -138.1 (d, J = 20.8 Hz, 1F). HRMS (EI) calcd for C15H12F2O [M]+: 246.0856;
found, 246.0860.
(E)-1,4-Difluoro-2-(2-fluorostyryl)benzene 29: 88% yield, white crystals; mp 79-81 oC; 1H NMR
(CDCl3, 300 MHz): δ 7.60 (dt, J = 7.7, 1.6 Hz, 1H), 7.27 (s, 2H), 7.26 (m, 2H), 7.13 (dd, J = 7.6,
1.1 Hz, 1H), 7.08 (m, 1H), 7.00 (m, 1H), 6.90 (m, 1H). 13C NMR (CDCl3, 75 MHz): δ 160.6 (d,
J = 250.4 Hz), 159.3 (d, J = 185.2 Hz), 156.1 (d, J = 189.0 Hz), 129.6 (d, J = 8.5 Hz), 127.2 (d, J

= 2.9 Hz), 126.5 (dd, J = 14.4, 7.9 Hz), 124.6 (d, J = 12.0 Hz), 124.3 (d, J = 3.3 Hz), 124.1 (s),
122.0 (s), 116.8 (dd, J = 25.3, 8.9 Hz), 115.9 (d, J = 22.2 Hz), 115.5 (dd, J = 24.5, 8.8 Hz), 112.8
(dd, J = 24.7, 3.5). 19F (CDCl3, 282 MHz): δ -115.9 (s, 1F), -117.3 (d, J = 17.2 Hz, 1F), -122.4
(d, J = 17.2 Hz, 1F). HRMS (EI) calcd for C14H9F3 [M]+: 234.0656; found, 234.0654.
(E)-1,2-Difluoro-4-(3-fluorostyryl)benzene 30: 95% yield, white crystals; mp 48-49 oC; 1H NMR
(CDCl3, 300 MHz): δ 7.30 (m, 2H), 7.16 (m, 4H), 6.98 (m, 1H), 6.96 (d, J = 16.5 Hz, 1H), 6.90
(d, J = 16.5 Hz, 1H). 13C NMR (CDCl3, 75 MHz): δ 163.2 (d, J = 245.6 Hz), 152.0 (dd, J = 42.1,
11.1 Hz), 148.7 (dd, J = 44.7, 11.1 Hz), 139.1, (d, J = 7.8 Hz), 134.2 (t, J = 5.9 Hz), 130.2 (d, J =
8.4 Hz), 128.5 (s), 127.8 (s), 122.9 (dd, J = 6.2, 3.6 Hz), 122.6 (d, J = 2.3 Hz), 117.5 (d, J = 17.5
Hz), 114.9 (d, J = 5.3 Hz), 114.7 (s), 112.8 (d, J = 21.9 Hz). 19F (CDCl3, 282 MHz): δ -111.4 (s,
1F), -135.8 (d, J = 20.9 Hz, 1F), -136.5 (d, J = 20.8 Hz, 1F). HRMS (EI) calcd for C14H9F3
[M]+: 234.0656; found, 234.0660.
(E)-1,4-Difluoro-2-(3-fluorostyryl)benzene 34: 89% yield, white crystals; mp 73-74 oC; 1H NMR
(CDCl3, 300 MHz): δ 7.27 (m, 4H), 7.18 (d, J = 16.5 Hz, 1H), 7.06 (d, J = 16.6 Hz, 1H), 7.01
13
(m, 2H), 6.91 (m, 1H). C NMR (CDCl3, 75 MHz): δ 163.2 (d, J = 245.7 Hz), 159.3 (d, J =
179.4 Hz), 156.0 (d, J = 183.7 Hz), 139.0 (d, J = 7.7 Hz), 130.8 (s), 130.2 (d, J = 8.3 Hz), 126.1
(m), 122.8 (d, J = 2.0 Hz), 121.2 (s), 116.9 (dd, J = 25.3, 8.8 Hz), 115.5 (dd, J = 24.5, 8.7 Hz),
19
115.1 (d, J = 21.6 Hz), 113.1 (d, J = 21.9 Hz), 112.8 (dd, J = 24.6, 3.8 Hz). F (CDCl3, 282
MHz): δ -111.5 (s, 1F), -117.3 (d, J = 17.2 Hz, 1F), -122.0 (d, J = 17.2 Hz, 1F). HRMS (EI)
calcd for C14H9F3 [M]+: 234.0656; found, 234.0659.
(E)-2,4-Difluoro-1-(3-fluorostyryl)benzene 35: 67% yield, white crystals; mp 60-61 oC; 1H NMR
(CDCl3, 300 MHz): δ 7.50 (dd, J = 15.0, 8.3 Hz, 1H), 7.23 (m, 3H), 7.16 (d, J = 16.5 Hz, 1H),
7.04 (d, J = 16.5 Hz, 1H), 6.88 (m, 3H). 13C NMR (CDCl3, 75 MHz): δ 163.2 (d, J = 245.5 Hz),
163.1 (dd, J = 143.9, 12.3 Hz), 159.8 (dd, J = 146.1, 12.2), 139.5 (d, J = 7.6 Hz), 130.2 (d, J =
8.4 Hz), 129.4 (s), 128.0 (dd, J = 9.4, 5.0 Hz), 122.5 (d, J = 2.2 Hz), 121.3 (s), 121.1 (d, J = 3.5
Hz), 114.8 (d, J = 21.4 Hz), 112.9 (d, J = 21.9 Hz), 11.7 (dd, J = 22.0, 3.7 Hz), 104.2 (t, J = 25.8
19
Hz). F (CDCl3, 282 MHz): δ -108.6 (d, J = 6.1 Hz, 1F), -111.6 (s, 1F), -111.7 (d, J = 6.3 Hz,
1F). HRMS (EI) calcd for C14H9F3 [M]+: 234.0656; found, 234.0658.
(E)-2,4-Difluoro-1-(2-fluorostyryl)benzene 36: 70% yield, white crystals; mp 84-85 oC; 1H NMR
(CDCl3, 300 MHz): δ 7.60 (t, J = 8.8 Hz, 1H), 7.58 (t, J = 8.7 Hz, 1H), 7.25 (s, 2H), 7.22 (m,
1H), 7.13 (dt, J = 7.6, 1.3 Hz, 1H), 7.06 (dt, J = 9.5, 1.3 Hz, 1H), 6.83 (m, 2H). 13C NMR
(CDCl3, 75 MHz): δ 162.6 (dd, J = 250.5, 13.9 Hz), 162.3 (t, J = 5.1 Hz), 158.9 (t, J = 10.2 Hz),
129.3 (d, J = 8.4 Hz), 128.1 (dd, J = 9.5, 5.2 Hz), 127.2 (d, J = 3.1 Hz), 125.1 (d, J = 11.9 Hz),
124.4 (d, J = 3.2 Hz), 122.9 (s) 122.2 (s), 121.7 (dd, J = 11.7, 3.6 Hz), 116.0 (d, J = 22.2 Hz),
19
111.8 (dd, J = 21.6, 3.5 Hz), 104.3 (t, J = 25.7 Hz). F (CDCl3, 282 MHz): δ -108.8 (s, 1F), -
112.2 (d, J = 5.6 Hz, 1F), -116.4 (s, 1F). HRMS (EI) calcd for C14H9F3 [M]+: 234.0656; found,
234.0658.
o
(E)-2,4-Difluoro-1-(4-methylstyryl)benzene 37: 32% yield, white crystals; mp 72-73 C; H
1
NMR (CDCl3, 300 MHz): δ 7.55 (dd, J = 15.0, 8.4 Hz, 1H), 7.89 (d, J = 7.9 Hz, 2H), 7.47 (d, J =

7.5 Hz, 2H), 7.16 (d, J = 16.5 Hz, 1H), 7.07 (d, J = 16.5 Hz, 1H), 6.85 (m, 2H), 2.35 (s, 3H). 13C
NMR (CDCl3, 75 MHz): δ 162.9 (dd, J = 131.0, 12.1 Hz), 159.5 (dd, J = 133.5, 12.0 Hz), 137.9
(s), 134.3 (s), 130.5 (s), 129.5 (s), 127.7 (dd, J = 9.2, 5.4 Hz), 126.5 (s), 121.8 (dd, J = 12.5, 3.8
19
Hz), 118.9 (s), 111.5 (dd, J = 21.5, 2.8 Hz), 104.1 (t, J = 25.7 Hz), 21.4 (s). F (CDCl3, 282
MHz): δ -109.7 (d, J = 5.3 Hz, 1F), -112.4 (d, J = 4.4 Hz, 1F). HRMS (EI) calcd for C15H12F2
[M]+: 230.0907; found, 230.0912.
o
(E)-1,4-Difluoro-2-(4-fluorostyryl)benzene 40: 50% yield, buff crystals; mp 76-77 C; H NMR
1
(CDCl3, 300 MHz): δ 7.48 (dd, J = 8.5, 5.5 Hz, 2H), 7.25 (m, 1H), 7.09 (s, 2H), 7.00 (m, 3H),
13
6.90 (m, 1H). C NMR (CDCl3, 75 MHz): δ 162.8 (d, J = 248.3 Hz), 159.2 (d, J = 190.6 Hz),
156.0 (d, J = 192.8 Hz), 132.9 (d, J = 3.0 Hz), 130.7 (d, J = 4.3 Hz), 128.3 (d, J = 8.0 Hz), 126.5
(m), 119.7 (s), 116.8 (dd, J = 25.3, 8.9 Hz), 115.7 (d, J = 21.8 Hz), 115.1 (dd, J = 24.6, 8.9 Hz),
112.7 (dd, J = 24.7, 3.0 Hz). 19F (CDCl3, 282 MHz): δ -111.5 (s, 1F), -117.4 (d, J = 17.0 Hz, 1F),
-122.5 (d, J = 16.9 Hz, 1F). HRMS (EI) calcd for C14H9F3 [M]+: 234.0656; found, 234.0659.
o
1
(E)-1,2-Dimethoxy-3-(4-methylstyryl)benzene 48: 92% yield, white crystals; mp 38-40 C; H
NMR (CDCl3, 300 MHz): δ 7.43 (d, J = 7.7 Hz, 2H), 7.41 (d, J = 16.7 Hz, 1H), 7.22 (d, J = 7.8
Hz, 1H), 7.14 (d, J = 7.9 Hz, 2H), 7.08 (d, J = 16.6 Hz, 1H), 7.02 (t, J = 8.2 Hz, 1H), 6.79 (d, J =
7.3 Hz, 1H), 3.83 (s, 6H), 2.34 (s, 3H). 13C NMR (CDCl3, 75 MHz): δ 153.1, 146.8, 137.5,
134.9, 131.7, 129.8, 129.3, 126.6, 124.1, 121.9, 117.8, 111.1, 61.0, 55.7, 21.2. HRMS (EI) calcd
for C17H18O3 [M]+: 270.1256; found, 270.1258.
(E)-1-Methoxy-2-(3-methoxystyryl)benzene 49: 86% yield, white crystals; mp 49-50 oC; 1H NMR
(CDCl3, 300 MHz): δ 7.55 (d, J = 7.6 Hz, 1H), 7.48 (d, J = 16.5 Hz, 1H), 7.23 (t, J = 7.7 Hz,
1H), 7.18 (t, J = 7.4 Hz, 1H), 7.09 (d, J = 8.9 Hz, 1H), 7.06 (d, J = 16.6 Hz, 1H), 7.05 (s, 1H),
6.92 (t, J = 7.4 Hz, 1H), 6.83 (d, J = 8.2 Hz, 1H), 6.76 (dd, J = 8.0, 1.9 Hz, 1H), 3.80 (s, 3H),
3.76 (s, 3H). 13C NMR (CDCl3, 75 MHz): δ 159.8, 156.9, 139.4, 129.5, 128.9, 128.7, 126.4,
126.2, 123.8, 120.7, 119.3, 113.0, 111.7, 110.9, 55.4, 55.1. HRMS (EI) calcd for C16H16O2 [M]+:
240.1150; found, 240.1156.
o
1
(E)-1,4-Dimethoxy-2-(3-methoxystyryl)benzene 50: 96% yield, white crystals; mp 48-49 C; H
NMR (CDCl3, 300 MHz): δ 7.44 (d, J = 16.4 Hz, 1H), 7.24 (t, J = 7.9 Hz, 1H), 7.10 (m, 3H),
7.05 (d, J = 16.4 Hz, 1H), 6.78 (m, 3H), 3.81 (s, 3H), 3.80 (s, 3H), 3.78 (s, 3H). 13C NMR
(CDCl3, 75 MHz): δ 159.9, 153.8, 151.5, 139.3, 129.5, 129.2, 127.1, 123.6, 119.4, 113.8, 113.2,
112.3, 111.8, 111.7, 56.2, 55.7, 55.2. HRMS (EI) calcd for C17H18O3 [M]+: 270.1256; found,
270.1258.
(E)-1,2-Dimethoxy-3-(4-(trifluoromethyl)styryl)benzene 54: 84% yield, white crystals; mp 67-69
oC; 1H NMR (CDCl3, 300 MHz): δ 7.62 (d, J = 9.3 Hz, 2H), 7.59 (d, J = 9.7 Hz, 2H), 7.54 (d, J =
16.7 Hz, 1H), 7.24 (d, J = 7.8 Hz, 1H), 7.13 (d, J = 16.6 Hz, 1H), 7.07 (t, J = 8.0 Hz, 1H), 6.87
(d, J = 8.0 Hz, 1H), 3.88 (s, 3H), 3.87 (s, 3H). 13C NMR (CDCl3, 75 MHz): δ 153.1, 147.3,
141.2, 130.9, 128.3, 126.7, 125.6, 125.5, 124.2, 118.0, 112.0, 61.3, 56.0. 19F (CDCl3, 282 MHz):
δ -60.9 (s, 3F). HRMS (EI) calcd for C17H15 F3O2 [M]+: 308.3002; found, 308.3000.

(E)-1-(4-Isopropylstyryl)-2,3-dimethoxybenzene 55: 96% yield, oil; 1H NMR (CDCl3, 300 MHz):
δ 7.35 (d, J = 8.2 Hz, 2H), 7.26 (d, J = 16.5 Hz, 1H), 7.24 (dd, J = 6.3, 1.6 Hz, 1H), 7.17 (d, J =
8.1 Hz, 2H), 7.14 (d, J = 16.5 Hz, 1H), 7.07 (t, J = 8.0 Hz, 1H), 6.84 (dd, J = 8.1, 1.2 Hz, 1H),
3.89 (s, 3H), 3.87 (s, 3H), 2.94 (sept, J = 6.9 Hz, 1H), 1.30 (d, J = 6.9 Hz, 6H). 13C NMR
(CDCl3, 75 MHz): δ 153.1, 148.5, 146.8, 135.3, 131.8, 129.8, 126.7, 126.6, 124.1, 122.0, 117.8,
111.1, 61.0, 55.7, 33.9, 23.9. HRMS (EI) calcd for C19H22O2 [M]+: 282.1620; found, 282.1618.
Acknowledgments
This work was supported in part by grant BC043125 from the U.S. Army/DOD Breast Cancer
Program.
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