Heterocyclic chalcone activators of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) with improved in vivo efficacy

Article abstract of DOI:10.1016/j.bmc.2015.07.056

Nrf2 activators represent a good drug target for designing agents to treat diseases associated with oxidative stress. Building upon previous work, we designed and prepared a series of heterocyclic chalcone-based Nrf2 activators with reduced lipophilicity

Full text of DOI:10.1016/j.bmc.2015.07.056

Accepted Manuscript
Heterocyclic chlacone activators of nuclear factor (erythroid-derived 2)-like 2
(
Nrf2) with improved in vivo efficacy
Nicole Lounsbury, George Mateo, Brielle Jones, Srinivas Papaiahgari, Rajash
K. Thimmulappa, Christiana Teijaro, John Gordon, Kenneth Korzekwa, Min
Ye, Graham Allaway, Magid Abou-Gharbia, Shyam Biswal, Wayne Childers
Jr.
PII:
S0968-0896(15)00640-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.07.056
BMC 12485
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
27 May 2015
18 July 2015
26 July 2015
Please cite this article as: Lounsbury, N., Mateo, G., Jones, B., Papaiahgari, S., Thimmulappa, R.K., Teijaro, C.,
Gordon, J., Korzekwa, K., Ye, M., Allaway, G., Abou-Gharbia, M., Biswal, S., Childers, W. Jr., Heterocyclic
chlacone activators of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) with improved in vivo efficacy, Bioorganic
&
Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.07.056
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Heterocyclic chalcone activators of nuclear
factor (erythroid-derived 2)-like 2 (Nrf2)
with improved in vivo efficacy
Leave this area blank for abstract info.
a
a
b
Nicole Lounsbury , George Mateo , Brielle Jones , Srinivas
b
c
d
Papaiahgari , Rajesh K. Thimmulappa , Christiana Teijaro ,
a
a
a
b
John Gordon , Kenneth Korzekwa , Min Ye , Graham Allaway ,
Magid Abou-Gharbia , Shyam Biswal and Wayne E. Childers, Jr.
a
c*
a*
a
Moulder Center for Drug Discovery Research, Temple University School of Pharmacy, 3307 N. Broad Street, Philadelphia, PA, USA
Cureveda, LLC, 1450 South Rolling Road, Halethorpe, MD 21227, USA
b
c
Depertmant of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe Street, Baltimore, MD 21205, USA
d
Department of Chemistry, Temple University, Philadelphia, PA 19122, USA

Bioorganic & Medicinal Chemistry
Heterocyclic chlacone activators of nuclear factor (erythroid-derived 2)-like 2 (Nrf2)
with improved in vivo efficacy
a
a
b
b
c
Nicole Lounsbury , George Mateo , Brielle Jones , Srinivas Papaiahgari , Rajash K. Thimmulappa ,
d
a
a
a
b
Christiana Teijaro , John Gordon , Kenneth Korzekwa , Min Ye , Graham Allaway , Magid Abou-
a
c*
a*
Gharbia , Shyam Biswal and Wayne Childers, Jr
a
Moulder Center for Drug Discovery Research, Temple University School of Pharmacy, 3307 N. Broad Street, Philadelphia, PA, USA
Cureveda, LLC, 1450 South Rolling Road, Halethorpe, MD 21227, USA
b
c
Depertmant of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe Street, Baltimore, MD 21205, USA
d
Department of Chemistry, Temple University, Philadelphia, PA 19122, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Nrf2 activators represent a good drug target for designing agents to treat diseases associated with
oxidative stress. Building upon previous work, we designed and prepared a series of
heterocyclic chalcone-based Nrf2 activators with reduced lipophilicity and, in some cases,
greater in vitro potency compared to the respective carbocylic scaffold. These changes resulted
in enhanced oral bioavailability and a superior pharmacodynamic effect in vivo.
Available online
2
009 Elsevier Ltd. All rights reserved.
Keywords:
Chalcone
Nrf2
Keap1
Bioavailability
Solubility
1. Introduction
4
(
ROS/RNS) sensor that is anchored in the cytoplasm. When
Oxidative stress reflects an imbalance between reactive
bound to Keap1, Nrf2 is ubiquitinated through a Cul3-based E3
4,5
oxygen species (ROS) and reactive nitrogen species (RNS) and a
cell’s ability to detoxify these reactive species and repair the
resulting damage. While some of these reactive species can play
a beneficial role (such as in the immune system’s response to
pathogens), growing evidence implicates oxidative stress in a
number of disease states, including cancer, chronic
neurodegenerative diseases, cardiovascular disease, diabetes-
induced tissue damage and various respiratory diseases such as
ligase pathway and subsequently degraded.
Exposure to
oxidative stress disrupts Keap1-Cul3 ligase activity resulting in
stabilization of Nrf2 protein. Higher levels of Nrf2 result in its
increased translocation to the nucleus where it binds to the ARE
of target genes along with other binding partners, leading to their
6-10
transcriptional induction.
The Keap1-Nrf2 system has come
to be recognized as a major regulatory pathway of cytoprotective
gene expression against oxidative stress and, as such, represents a
1
11,12
COPD.
good drug target for oxidative stress-related diseases.
The cell contains a number of defense systems to combat
intrinsically and extrinsically induced stressors. For many years,
research into oxidative stress focused on superoxide dismutase
The multiple mechanisms through which Keap1 senses
oxidative stress are still not completely understood. However,
data from site-directed mutagenesis studies (recently summarized
2
12
and the glutathione scavenging system. In 1994, nuclear factor
by Suzuki et al.) suggests that one of the sensor mechanisms is
erythroid 2 p45-related factor 2 (Nrf2) was discovered and later
shown to be a positive regulator of nearly all cellular antioxidant
enzymes through binding to the regulatory element, Antioxidant
Response Element (ARE). Nrf2 levels are controlled by binding
to the Kelch-like ECH-associated protein 1 (Keap1), a stress
a series of cysteine residues that react with ROS/RNS or
chemical modifiers to reduce Keap1’s ability to target Nrf2 for
ubiquitination. In support of this hypothesis, many known Nrf2
activators contain electrophilic groups that are able to react with
soft nucleophiles like thiols. Mass spectroscopy and X-ray
crystallography have been employed to confirm modification of
3
-
-----------
13
Keap1 by a number of these compounds.
Representative
Corresponding Authors. Tel +1 215 707 1079. Fax +1 215 707 5620 (W. C.);
tel +1 410 502 1944. Fax +1 410 955 0116 (S. B.)
14
electrophilic Nrf2 activators are shown in Figure 1. Some of
Email
Addresses:
wayne.childers@temple.edu
(W.
Childers),
these compounds have advanced to clinical trials for various
sbilwal@jhsph.edu (S. Biswal).

were synthesized using the previously described modification of
1
8,19
the Claisen-Schmidt reaction (Scheme 1).
trifluoromethyl)pyridine carboxaldehydes 6a-d (Table 1) with
’-methoxyacetophenone in the presence of lithium hydroxide at
Condensation of
(
2
room temperature overnight gave chalcones 8a-d (Table 2) in
varying yield following purification on silica gel. A similar
procedure was used to synthesize chalcones 8e-h (Table 1)
starting with 2-(trifouoromethyl)benzaldehyde and the
appropriate methoxy pyridyl-ethanones 7a–d (Table 1). The
final products were obtained as viscous oils that hardened upon
standing to give waxy solids. Two of the required intermediates
(6c and 6d) were purchased from commercial vendors. Others
were not available commercially or their high cost precluded
their purchase. We elected to synthesize those intermediates
using variations of previously published procedures. The
commercially available starting materials and synthetic chemistry
references for 6a-d and 7a-d are shown in Table 1.
Table 1
Source of Intermediates 6a-d and 7a-d
Cd.
Structure
Starting
Material
Reference
20
Figure 1. Structures of some known Nrf2 activators.
indications. Among these are sulforaphane (1, the anti-cancer
6a
15
agent discovered in broccoli), CDDO-Me (4, which advanced to
clinical trials under the generic name bardoxolone methyl but
16
was terminated in Phase III due to adverse events) and dimethyl
6
b
21
fumarate (2), which recently received approval for the treatment
®
17
of multiple sclerosis under the trade name Tecfidera .
Some of the authors of the present manuscript recently
reported a series of non-cytotoxic chalcone-derived Nrf2
activators, exemplified by compound 5 (Figure 1), that increased
the expression of Nrf2-regulated antioxidant genes in human lung
epithelial cells and in an in vivo mouse model. As a group,
chalcones tend to demonstrate low-to-moderate aqueous
solubility and modest oral bioavailability due to their lipophilic
nature. We sought to enhance the bioavailability of our chalcone
series by reducing the lipophilicity through the incorporation of
heteroatoms into the aromatic rings. In this manuscript we
present the results of that effort.
Commercially
Available
6c
NA
NA
18
Commercially
Available
6d
7
a
2
2
2
2
. Results and Discussion
7
b
23
.1 Chemistry.
For the present study, we chose to hold the 2-trifluoromethyl
and 2-methoxy groups of 5 constant and introduce pyridine
nitrogen atoms into various positions of the two aromatic rings in
order to lower lipophilicity. The required heteroaryl chalcones
7
7
c
22
24
d
2.2 Biology.
To examine the ability of the pyridyl chalcones to activat
Nrf2, we measured their effect on the expression of the
antioxidant gene, heme oxygenase (HO-1), a well-characterized
2
5,26
ARE target of Nrf2.
Beas-2B) were treated with test compounds for 6 hours and HO-
gene expression levels were determined by quantitative
polymerase chain reaction (QPCR) analysis as previously
Normal human bronchial epithelial cells
(
1
2
7
described. Results for the pyridyl chalcones were compared to
Scheme 1. Synthesis of heteroarylchalcone Nrf2 actovators. Reagents and
conditions: (a) LiOH, MeOH, rt, overnight (35 – 83% yield).
those obtained for compound 5. Compound 4, the known Nrf2

TABLE 2.
Compounds 5 and 8a – 8d. Calculated and Measured Physicochemical Properties.
b
-6
f
Maximum Solubility, uM
Caco-2 Papp, 10 cm/sec
a
Compd
Structure
logP
4.61
3.78
3.39
3.39
3.63
3.78
3.39
3.39
3.99
TPSA
26.3
39.2
39.2
39.2
39.2
39.2
39.2
39.2
39.2
c
d
e
PB
SIF
SGF
A - B
B - A
14.6
5
13.5
50.8
172
134
90.8
170
23.6
53.8
12.8
49.8
166
11.1
52.3
197
176
137
174
184
184
34.6
17.4
g
8
8
a
nd
nd
nd
b
nd
nd
8
c
157
nd
8
d
98.6
172
17.3
nd
15.0
nd
8
8
e
f
22.5
48.2
33.6
nd
nd
8
8
g
nd
nd
h
40.5
nd
nd
a
e
b
c
d
Topological polar surface area; Maximum kinetic solubility; Phosphate buffer, pH 7.4; Simulated intestinal fluid;
f
g
Simulated gastric fluid; Permeability coefficient, bi-directional permeability in Caco-2 monolayers. nd = not determined.
activator clinical candidate CDDO-Me, was included as a
positive control and comparator at a concentration of 10 uM.
Data are presented in Figure 2.
and 8f) were superior to 5. In particular, compound 8d enhanced
HO1 expression at a concentration of 5 uM, nearly 3-fold higher
than the non-heterocyclic chalcone 5.
At a concentration of 5 uM, five of the eight pyridyl chalcone
analogs were more effective at activating the expression of HO-1
Several factors were considered while trying to understand the
structure-activity and structure-property relationships of this
series. In general, the pyridyl chalcones were more soluble in
aqueous media than the carboxylic analog 5 (Table 2). However,
since the cellular assay was run at a concentration of 5 uM, which
is less than the maximum aqueous solubility of both 5 and 8a-h,
it is unlikely that solubility limitations played a significant role in
the potency differences seen in the Beas-2B assay. Likewise,
crystal packing properties were not a contributing factor since all
of the test drugs were first dissolved in DMSO prior to use.
Compounds 8a-h displayed somewhat lower logP values
compared to 5, which could have resulted in different
permeability properties. But the data from a bi-directional Caco-
in Beas-2B cells than the known Nrf2 activator 4 (Figure 2 and
Table 3). Three of the five active pyridyl chalcones (8b, 8c and
8g) were essentially equipotent with the previously reported
chalcone 5, while two of the five active pyridyl chalcones (8d
2
assay suggested that this lower lipophilicity had little effect on
permeability (at least for one comparison), and there was no
correlation between HO1 levels and logP for the chalcones tested
(Table 2). Beas-2B cells can be stimulated to express the efflux
transporter p-glycoprotein, although basal expression levels are
28
low. However, literature precedence indicates that chalcones
which do interact with p-glycoprotein usually act as inhibitors
29-31
rather than substrates,
and the lack of efflux seen in the Caco-
2
assay (Table 2) suggests that efflux transport is not playing a
significant role in the differences in biological data seen for
compounds 5 and 8a-h.
Another factor that could influence the potency of these
chalcones is the reactivity of the electrophilic enone moiety. The
lower the LUMO energy, the easier it is for nucleophiles such as
thiols to share electrons with the enone, thereby resulting in
chemical reaction. This type of analysis is most accurate when
Figure 2. Activation of HO-1 expression in BEAS2-2B cells.

the LUMO energies are calculated using the conformation of the
enone-containing molecule that is attacked by the thiol on Keap1,
that is, the binding conformation.
Since the binding
conformation, if any, of these molecules with Keap1 is not
known, we used the global minimum energy of each chalcone for
our analysis. The results are presented in Table 3.
The calculated LUMO energy of the global minimum energy
conformation of 5 was -23.43 kcal/mole. The LUMO energies of
the global minimum energy conformations of some of the pyridyl
chalcones were indeed lower than that of compound 5. In
particular, the calculated LUMO for the potent analog 8d was
-28.03 kcal/mol. However, there was not a clear correlation
between the differences in activity shown in Figure 2 and the
differences in LUMO energy. For example, the calculated
LUMO of the global minimum energy conformation of 8b was
Table 3.
LUMO energies of the global minimum energy conformations
of compounds 5 and 8a-h.
LUMO
kcal/mol)
LUMO
(kcal/mol)
Compound
Compound
(
5
-23.42
-26.14
-30.09
-27.08
-28.03
8
8
8
8
a
b
c
8e
8f
-21.04
-23.19
-24.07
-22.44
8g
8h
d
-30.09 kcal/mol yet the compound was less potent than
Figure 3. Plasma exposure of 5 and 8d at 50, 200 and 400 mg/kg po. N = 3
compound 5 in the HO1 expression assay. It is likely that while,
overall, LUMO energies for the enone moiety may be playing a
role in the greater activity for at least some of the pyridyl
chalcones, other factors such as binding interactions with Keap1
may also be contributing to the differences in potency.
animals per time point. (iv data provided in supplementary data)
Table 4.
Pharmacokinetic properties for 5 and 8d.
b
c
We then compared the in vivo plasma exposure of the
carbocyclic chalcone 5 and the pyridyl chalcone 8d. The study
was supported with in vitro data from mouse liver microsomal
assays and plasma protein binding assays. Both compounds were
given iv at 2 mg/kg and doses orally via gavage at doses of 50,
%F
Microsomal Stability
Protein
Binding
a
AUC
e
c
Cd.
Clint
t1/2
(
ug/mL•hr)
(ul/min/mg)
minutes
(% free)
5
> 690
< 2
0.1 %
f
2
mg/kg
0.38
0.47
7.75
42.7
200 and 400 mg/kg. Plasma levels were quantified by
g
g
g
5
0 mg/kg
5%
20%
56%
LC/MS/MS at various time points. The results are summarized
in Figure 3 and Table 4 (iv data are provided in the
supplementary data).
2
4
8
00 mg/kg
00 mg/kg
d
> 690
< 2
3.8 %
Both 5 and 8d were metabolized rapidly in mouse liver
microsomes, with clearance values > 690 uL/min/mg. However,
it appears that both compounds were more stable in vivo than
predicted by the microsomal stability data, likely due to the high
protein binding. Overall, 8d demonstrated a higher AUC at all
three doses than did 5, resulting in higher bioavailabilities.
Based on the Caco-2 data (Table 1), permeability and transporter-
mediated efflux do not play an appreciable role in the difference
in bioavailability seen between 5 and 8d. Half-lives for the two
compounds following iv administration were similar (5 – 1.31 hr;
f
2
mg/kg
0.302
0.77
g
g
g
5
0 mg/kg
10%
38%
83%
200 mg/kg
11.49
50.4
400 mg/kg
a
b
Area under the curve; Bioavailability, based on AUC;
c
e
f
d
Mouse liver microsomes; Clint – Free intrinsic clearance
Plasma protein binding,
administered i.v.; administered p.o.
% free, unbound fraction;
g
8
d – 1.25 hr. See supplementary data). Accurate half-lives could
Pharmacodynamic studies were also performed on mice
treated orally with either 200 mg/kg or 400 mg/kg of compounds
not be obtained but from Figure 3 it appears that the half-live for
compound 8d is somewhat higher than that seen with compound
5
and 8d. Lung tissue from drug-treated and vehicle-treated mice
5, at least at the higher doses. The exposure for both compounds
was removed and analyzed for HO1 gene expression HO1 protein
levels. Results are shown in Figures 4 and 5. Higher levels of
HO1 gene expression (Figure 4) and HO1 protein expression
appear to be linear across the doses tested. One likely
contributing factor to the enhanced bioavailability seen with 8d is
its improved aqueous solubility compared to 5 (Table 2).
Solubility and dissolution rate are well known to have a
significant impact on drug absorption.
contributing factor is the longer half-life at the higher doses.
(Figure 5) were observed in the lungs of mice 6 hours post-
32
treatment with 8d compared to untreated mice, indicating Nrf2
activation. A greater effect on both HO1 gene and protein
Another likely

expression was seen in mice treated with 8d compared to those
treated with equivalent doses of 5. These data correlate with the
higher in vitro potency (Figure 2), higher oral bioavailability
otherwise, reactions were run under normal atmospheric
1 13
conditions. H-NMR (400 mHz) and C-NMR (100 MHz) data
were collected on a Bruker Avance III spectrometer at ambient
temperature in the identified solvent. Peak positions are given in
parts per million downfield from tetramethylsilane as the internal
standard for 1H-NMR spectra. Deuterated chloroform was used
(Figure 3, Table 4) and greater free, unbound fraction in plasma
(Table 4) demonstrated by compound 8d compared to that seen
with compound 5.
1
3
as the internal reference for
C-NMR Normal phase
chromatographic separations were performed on silica gel using a
Teledyne ISCO Combiflash Rf system. Purity of all final
compounds was determined to be ≥95% by reversed phase HPLC
on a C-18 column using a gradient of 0–100% acetonitrile in
water with 0.1% formic acid as a modifier and UV detection at
both 220 nM and 254 nM. High resolution MS was obtained on
an Agilent 6520 LC/Q-TOF MS. Compound 5 was synthesized
18
as previously described.
4
1
.1.1 General Method for Producing Pyridyl Chalcones. (E)-
-(2-Methoxyphenyl)-3-(2-(trifluoromethyl)pyridine-3-yl)-
prop-2-en-1-one (8a).
A solution of lithium hydroxide (0.8 mg, 0.03 mmol)) and 2’-
methoxyacetophenone (26 mg, 0.157 mmol) in absolute
methanol (1.5 mL) was stirred at room temperature for 15
minutes. To the resulting mixture was added a solution of 2-
Figure 4. HO1 gene expression in mouse lung relative to vehicle, 6 hours
post-treatment (* P ≤ 0.05).
(trifluoromethyl)-3-pyridinecarboxaldehyde (6a, 28 mg, 0.16
mmol) in absolute methanol (15 mL). The reaction was stirred
overnight at room temperature (approx. 18 hours). The reaction
was then concentrated on a rotary evaporator and the resulting
oily residue purified by chromatography on silica gel using a
gradient of 0 – 100% ethyl acetate in hexane to provide the
1
desired product (17 mg, 35%) as a light yellow waxy solid. H-
NMR (CDCl
1
1
3
):  8.71 (d, J = 4.9 Hz, 1H), 8.12 (d, J = 8.1 Hz,
H), 7.93 (dd, J = 15.8 Hz, 2.0 Hz, 1H), 7.68 (dd, J = 7.6, 1.8 Hz,
H), 7.56 (dd, J = 8.0, 4.6 Hz, 1H), 7.55 (d, J = 7.4 Hz, 1H), 7.54
(
7
dt, J = 7.4 , 1.9 Hz, 1H), 7.36 (d, J = 15,8 Hz, 1H), 7.09 (t, J =
13
.6, 1H), 7.03 (d, J = 8.2 Hz, 1H), 3.93 (s, 3H).
)  190.8, 157.3, 148.2, 147.1, 145.2, 136.5, 135.1, 134.4,
32.6, 131.6, 129.7, 129.6, 127.2, 125.5, 120.0, 110.6, 54.7.
NO + H = 308.0898, found
C-NMR
(CDCl
3
1
HRMS (FAB): calc’d C16
H
12
F
3
2
Figure 5. HO1 protein levels in mouse lung relative to vehicle, 6 hours post-
treatment (* P ≤ 0.05; ** P ≤ 0.01).
308.0889.
4
.1.2
(E)-1-(2-Methoxyphenyl)-3-(3-(trifluoromethyl)-
3. Conclusion
pyridin-4-yl)prop-2-en-1-one (8b).
Prepared using the general method from lithium hydroxide
hydrate (0.6 mg, 0.02 mmol), 2’-methoxyacetophenone (14 mg,
A series of pyridinyl-chalcones was synthesized and evaluated
for the ability to activate HO1, a known ARE target gene for
Nrf2. Two of the compounds, 8d and 8f, were superior to the
clinical candidate 4 and to the previously described carbocyclic
chalcone Nrf2 activator 5. The inclusion of the nitrogen atom
into the structure of these new analogs enhanced their aqueous
solubility compared to 5. This increased solubility translated to
enhanced oral bioavailability in the case of 8d, a result that was
not influenced by changes in permeability or efflux transport.
Lung tissue from mice treated orally with 8d showed higher
levels of both HO1 gene expression and protein expression
compared to untreated mice or mice treated with equivalent doses
of the carbocyclic chalcone derivative 5. These results in
agreement with the higher in vitro potency, greater oral
bioavailability and higher free, unbound fraction in plasma seen
with 8d and indicate that the compound functions as a Nrf2
activator in vivo.
0
.112 mmol) and 2-(trifluoromethyl)-4-pyridinecarboxaldehyde
(6b, 20 mg, 0.114 mmol) in absolute methanol (final reaction
volume = 2 mL). The reaction mixture was purified by
chromatography on silica gel (gradient of 10 – 100% ethyl
acetate in hexane) to give the desired product as a yellow oil (13
mg, 39%). H-NMR (CDCl
1H), 7.77 (dd, J = 15.8, 2.0 Hz, 1H), 7.62 (dd, J = 7.6, 1.8 Hz,
1H), 7.54 (d, J = 5.2 Hz, 1H), 7.46 (dt, J = 7.6, 1.8 Hz, 1H),7.42
(d, 15.8 Hz, 1H), 7.00 (t, J = 7.6 Hz, 1H), 6.94 (d, J = 8.3 Hz,
1H), 3.85 (s, 3H).
144.6, 140.9, 140.0, 134.6, 132.2, 129.6, 128.4, 126.3, 126.0,
1
3
),  8.87 (s, 1H), 8.76 (d, J = 5.2 Hz,
13
3
C-NMR (CDCl )  190.2, 156.9, 156.0,
12 3 2
122.3, 119.0, 113.0, 56.1. HRMS (FAB): calc’d C16H F NO +
Na = 330.0718, found 330.0719.
4.1.3
(E)-1-(2-Methoxyphenyl)-3-(4-(trifluoromethyl)-
pyridin-4-yl)prop-2-en-1-one (8c).
Prepared using the general method from lithium hydroxide (3
mg, 0.125 mmol)), 2’-methoxyacetophenone (94 mg, 0.625
mmol)) and 4-trifluoromethyl-3-pyridinecarboxaldehyde (6c, 111
mg, 0.635 mmol) in absolute methanol (final reaction volume = 4
mL). The reaction mixture was purified by chromatography on
silica gel (gradient of 0 – 100% ethyl acetate in hexane) to give
4
4
. Experimental
.1 Chemistry
Reagents were purchased from commercial suppliers and used
without further purification. Intermediate 6c was purchased from
Frontier Scientific (Logan, UT) and intermediate 6d was
purchased from Combi-Blocks (San Diego, CA). Unless stated
1
the desired product as a yellow oil (104 mg, 54%). H-NMR

(
1
CDCl
5.8, 2.0 Hz, 1H), 7.63 (dd, J = 7.6, 1.8 Hz, 1H), 7.51 (d, J = 5.5
Hz, 1H), 7.46 (dt, J = 8.4, 1.8 Hz, 1H), 7.42 (d, J = 15.8 Hz, 1H),
3
)  8.99 (s, 1H), 8.70 (d, J = 5.5 Hz, 1H), 7.81 (dd, J =
0.36 mmol) in absolute methanol (final reaction volume = 3 mL).
The reaction mixture was purified by chromatography on silica
gel (5% methanol in ethyl acetate) to give the desired product as
1
7
.00 (t, J = 7.6 Hz, 1H), 6.94 (d, J = 8.4 Hz, 1H), 3.94 (s, 3H).
a yellow solid (67 mg, 57%). H-NMR (CDCl
3
)  8.50 (s, 1H),
13
C-NMR (CDCl
3
)  191.2, 158.5, 151.4, 150.7, 149.4, 136.1,
8.40 (d, J = 4.3 Hz, 1H), 7.89 (dd, J = 15.8, 1.9 Hz, 1H), 7.78 (d,
J – 7.8 Hz, 1H), 7.72 (d, J = 7.8 Hz, 1H), 7.61 (t, J = 7.5 Hz, 1H),
7.52 (t, J = 7.5 Hz, 1H), 7.40 (d, J = 4.4 Hz, 1H), 7.16 (d, J = 15.8
1
5
3
34.2, 133.9, 132.4, 130.9, 129.1, 128.2, 121.7, 119.5, 111.6,
12 3 2
5.6. HRMS (FAB): calc’d C16H F NO + H = 308.0898, found
08.0900.
1
3
Hz, 1H), 3.98 (s, 3H). C-NMR (CDCl
1
1
3
)  191.8, 152.5, 143.0,
40.9, 135.1, 134.6, 133.4, 132.2, 130.0, 129.8, 129.4, 129.1,
2
26.4, 125.2, 122.6, 55.4. HRMS (FAB): calc’d C16 NO +
4
.1.4
(E)-1-(2-Methoxyphenyl)-3-(3-(trifluoromethyl)-
12
H F
3
pyridin-2-yl)prop-2-en-1-one (8d).
H = 308.0898, found 308.0899.
Prepared using the general method from lithium hydroxide
4.1.8 (E)-1-(2-Methoxypyridin-3-yl)-3-(2-(trifluoromethyl)-
(1.2 mg, 0.01 mmol), 2’-methoxyacetophenone (72 mg, 0.48
phenyl)prop-2-en-1-one (8h).
mmol) and 3-trifluoromethyl-2-pyridinecarboxaldehyde (6d, 85
mg, 0.49 mmol) in absolute methanol (final reaction volume = 4
mL). The reaction mixture was purified by chromatography on
silica gel (gradient of 0 – 100% ethyl acetate in hexane) to give
Prepared using the general method from lithium hydroxide (1
mg, 0.04 mmol), 2-(trifluoromethyl)benzaldehyde (34 mg, 0.2
mmol) and 1-(2-methoxypyridin-3-yl)ethanone (7d, 29 mg, 0.19
mmol) in absolute methanol (final reaction volume = 1.5 mL).
The reaction mixture was purified by chromatography on silica
the desired product as a yellow oil that hardened upon standing
1
(77 mg, 54%). H-NMR (CDCl
3
)  8.82 (d, J = 4.3 Hz 1H), 8.12
(
1
d, J = 15.0 Hz, 1H), 8.01 (dd, J = 8.0, 1.6 Hz, 1H), 7.93 (dd, J =
5.0, 1.9 Hz, 1H), 7.70 (dd, J = 7.6, 1.8 Hz, 1H), 7.51 (t, J = 7.4
Hz, 1H), 7.41 (dd, J = 7.5, 4.6 Hz, 1H), 7.07 (t, J = 7.5 Hz, 1H),
gel (40% ethyl acetate in hexane) to give the desired product as a
1
yellow solid (29 mg, 49%). H-NMR (CDCl
3
)  8.36 (dd, J =
4.8, 2.0 Hz, 1H), 8.08 (dd, J = 7.4, 2.0 Hz, 1H), 8.05 (dd, J =
15.6, 2.2 Hz, 1H), 7.83 (d, J = 7.8 Hz, 1H), 7.75 (d, J = 7.8 Hz,
1H), 7.63 (t, J = 7.6 Hz, 1H), 7.53 (d, J = 7.5 Hz, 1H), 7.47 (d, J
1
3
7
1
1
.02 (d, J = 8.1 Hz, 1H), 3.93 (s, 3H).
3
C-NMR (CDCl ) 
92.4, 158.6, 152.3, 151.6, 135.4, 134.1, 133.9, 133.5, 130.6,
13
28.8, 125.8, 125.1, 123.2, 120.8, 111.6, 55.7. HRMS (FAB):
= 15.7 Hz, 1H), 7.05 (dd, J = 7.4, 4.8 Hz, 1H), 4.09 (s, 3H). C-
NMR (CDCl )  189.3, 160.6, 149.5, 138.9, 137.9, 135.9, 131.0,
30.1, 129.8, 128.6, 126.9, 125.2, 124.5, 121.2, 116.1, 52.8.
HRMS (FAB): calc’d C16 NO + H = 308.0898, found
calc’d C16
12 3
H F NO
2
+ H = 308.0898, found 308.0897.
3
1
4
.1.5 (E)-1-(3-Methoxypyridin-2-yl)-3-(2-(trifluoromethyl)-
H
12
F
3
2
phenyl)prop-2-en-1-one (8e).
3
08.0874.
.2 Calculated logP and TPSA.
Predicted octanol/water partition coefficients (logP) and
Prepared using the general method from lithium hydroxide (2
mg, 0.05 mmol), 2-(trifluoromethyl)benzaldehyde (47 mg, 0.27
mmol) and 1-(3-methoxypyridin-2-yl)ethanone (7a, 40 mg, 0.26
mmol) in absolute methanol (final reaction volume = 6 mL). The
reaction mixture was purified by chromatography on silica gel
4
topological polar surface areas (TPSA) were calculated using the
Marvin calculator plugin available in MarvinSketch from
ChemAxon (Budapest, Hungary). The logP calculator employs a
(gradient of o – 100% ethyl acetate in hexane) to give the desired
3
3
product as yellow oil that hardened upon standing (43 mg, 54%).
variation on the atomic fragment method originally described
1
34
H-NMR (CDCl
3
)  8.33 (dd, J = 4.4, 1.3 Hz, 1H), 8.09 (dd, J =
by Viswanadhan et al. The TPSA calculator uses the fragment-
3
5
1
1
=
1
1
1
5.9, 2.1 Hz, 1H), 7.90 (d, J = 7.7 Hz, 1H), 7.73 ((d, J = 7.7 Hz,
H), 7.71 (d, J = 15.9 Hz, 1H), 7.60 (t, J = 7.6 Hz, 1H), 7.50 (t, J
based summation method described by Ertl et al.
.3 Molecular modeling.
Modeling and LUMO energy calculations were performed
4
7.6 Hz, 1H), 7.47 (dd, J = 8.6, 4.4 Hz, 1H), 7.41 (dd, J = 8.6,
13
.3 Hz, 1H), 3.96 (s, 3H). C-NMR (CDCl
44.3, 140.4, 139.7, 134.1, 132.0, 129.6, 129.3, 129.0, 128.4,
28.1, 127.1, 126.1, 119.8, 55.9. HRMS (FAB): calc’d
NO + H = 308.0898, found 308.0899.
.1.6 (E)-1-(4-Methoxypyridin-3-yl)-3-(trifluoromethyl)-
3
)  190.0, 155.6,
36
using the Molecular Operating Environment (MOE), the
37
38
MMFF94 potential and the MOPAC interface of MOE.
4
.4 Solubility assays.
C
H F
16 12 3
2
Solubility assays were performed using Millipore
4
MultiScreen®HTS-PFC Filter Plates designed for solubility
assays (EMD Millipore, Billerica, MA). The 96-well plates
consist of two chambers separated by a filter. Liquid handling
was performed using JANUS® Verispan and MTD workstations
phenyl)prop-2-en-1-one (8f).
Prepared using the general method from lithium hydroxide
(0.8 mg, 0.032 mmol), 2-(trifluoromethyl)benzaldehyde (28 mg,
0
0
.16 mmol) and 1-(4-methoxypyridin-3-yl)ethanone (7b, 24 mg,
.15 mmol) in absolute methanol (final reaction volume = 1.5
(Perkin Elmer, Waltham, MA). 4 uL of drug solutions (10 mM
in DMSO) are added to 196 uL of the appropriate medium in top
chamber to give a final DMSO concentration of 2% and a
theoretical drug concentration of 200 uM. Plates are gently
shaken for 90 minutes and then subjected to vacuum. Insoluble
drug is captured on the filter. 160 uL of the filtrate is transferred
to 96-well Griener UV Star® analysis plates (Sigma-Aldrich, St.
Louis, MO) containing 40 uL of acetonitrile. The drug
concentration in the filtrate is measured by UV absorbance on a
mL). The reaction was purified by chromatography on silica gel
5% methanol in ethyl acetate) to give the desired product as a
(
1
yellow solid (38 mg, 83%). H-NMR (CDCl
Hz, 1H), 7.97 (dd, J = 15.8, 2.0 Hz, 1H), 7.80 (d, J = 7.7 Hz, 1H),
.75 (d, J = 7.7 Hz, 1H), 7.62 (t, J = 7.5 Hz, 1H), 7.53 (t, J = 7.5
Hz, 1H), 7.35 (d, J = 15.8 Hz, 1H), 6.66 (d, J = 5.9 Hz, 1H), 3.98
3
)  8.64 (d, J = 5.9
7
1
3
(s, 3H). C-NMR (CDCl
3
)  190.7, 163.9, 154.0, 151.3, 139.9,
1
1
33.8, 132.1, 130.3, 129.8, 128.0, 126.3, 126.2, 125.3, 122.5,
®
Spectromax Plus microplate reader (Molecular Devices,
06.9, 55.8. HRMS (FAB): calc’d C16
H
12
F
3
NO
2
+ H = 308.0898,
NO + H =
Sunnyvale, CA) using Softmax Pro software v. 5.4.5.
Absorbances at 5 wavelengths (280, 300, 320, 340, and 360 nM)
were summed to generate the UV signal. Assays were performed
in triplicate. Standard curves were generated by adding 4 uL of
50x of five concentrations of test compounds in DMSO to 40 uL
of acetonitrile in UV Star plates followed by 156 uL of the
appropriate solubility medium. Analysis and statistics were
found 308.0899. HRMS (FAB): calc’d C16
3
H
12
F
3
2
08.0898, found 308.0904.
4
.1.7 (E)-1-(3-Methoxypyridin-4-yl)-3-(2-(trifluoromethyl)-
phenyl)prop-2-en-1-one (8g).
Prepared using the general method from lithium hydroxide
(1.75 mg, 0.073 mmol), 2-(trifluoromethyl)benzaldehyde (51 mg.
®
performed using GraphPad Prism v. 5.04. Data are reported as
0.37 mmol) and 1-(3-methoxypyridin-4-yl)ethanone (7c, 55 mg.

the maximum concentration observed in the filtrate. The
following solubility media were used in the assays.
plasma mixture (pH 7.4) and buffer (Dulbecco’s phosphate-
buffered saline 1X without calcium and magnesium (Mediatech,
Inc., Herndon, VA)) were placed in their respective sides, wells
were capped and the plate was then placed in the rotator and
allowed to dialyze for 22 hours. Following dialysis, 25 uL of
buffer and plasma mixture were removed and mixed with 25 uL
of the opposite matrix in 96-well deep plates. Concentrations of
analytes from each side of the dialysis plate were determined by
LC/MS/MS using a standard curve constructed in the same
matrix. The fraction unbound (fu) was calculated by dividing the
drug concentration in the buffer side of the dialysis plate by the
drug concentration in the plasma side.
Phosphate buffer (pH 7.4): Millipore-defined universal buffer –
45 mM potassium phosphate, 45 mM sodium acetate, 45 mM
ethanolamine, pH = 7.4.
Simulated Intestinal Fluid (SIF): United States Pharmacopeia-
defined SIF – 50 mM potassium phosphate, pH 6.8, without
pancreatin.
Simulated Gastric Fluid (SGF): United States Pharmacopeia-
defined SGF – 0.2% sodium chloride, 84 mM HCl, pH 1.2,
without pepsin.
4.4 Caco-2 assay.
4.7 Protocol for quantitative PCR (QPCR). HO1 expression
Bi-directional permeability was assessed in Caco-2
3
9
in Beas-2B cells.
monolayers. Caco-2 cells (clone C2BBe1) were obtained from
American Type Culture Collection (Manassas, VA). Cell
monolayers were grown to confluence on collagen-coated
microporous, polycarbonate membranes in 12-well plates. The
permeability assay buffer was Hanks Balanced Salt Solution
containing 10 mM HEPES and 15 mM glucose at a pH of 7.4.
The buffer in the receiver chamber also contained 1% bovine
serum albumin. The dosing solution concentration was 5 uM for
each test compound in the assay buffer. Cell monolayers were
The ability of the chalcones to upregulate expression of HO1
was examined in Beas-2B cells using a previously described RT-
25
PCR method.
Data are reported as fold-increase in RFC.
Normal human bronchial epithelial cells (Beas2B) obtained from
the American Type Culture Collection (Manassas, VA) were
cultured under recommended conditions. Cells were seeded onto
24-well plates for 16 hours before incubation with test
compounds. The cells were treated with indicated concentrations
of test compound for 6 hours and total RNA was extracted using
a Qiagen RNeasy kit (Qiagen Corporation, Valencia, CA). QPCR
of human HO-1 and endogenous control GAPDH was performed
by using Assay-on Demand primers and probe sets from Applied
Biosystems (Applied Biosystems, Foster City, CA).
dosed on the apical side (A-to-B) or basolateral side (B-to-A) and
o
incubated at 37 C with 5% CO
2
. Samples were taken from the
donor and receiver chambers at 120 minutes. Each determination
was performed in duplicate. The flux of co-doses of Lucifer
yellow was also measured for each monolayer to insure the
integrity of the monolayer.
LC/MS/MS. The apparent permeability constant, Papp (x 10
Samples were assayed by
4.8 In Vivo exposure in mice.
-6
The in vivo exposure of compounds 5 and 8d were measured
over a 24 hour period in 6 – 8 week old female C57BL/6 mice
(Harlan Labs, Indianapolis, IN; n = 3 animals per time point)
following a 2 mg/kg iv dose and doses of 50, 200 and 400 mg/kg
dose given orally via gavage. Animals were acclimatized for 72
hours prior to any experimental procedures. They were housed
40
cm/second), was calculated using established equations.
.5 Microsomal stability assay.
The clearance of test compounds in mouse liver microsomes
4
o
was determined at 37 C. Assays were conducted in 96-deep well
polypropylene plates. Test compounds (1 uM) were incubated in
3
-5 per cage on a 12 hour light/12 hour dark cycle and were
0.5 mL of 100 mM potassium phosphate buffer (pH 7.4) with 0.5
given free access to food and water. Animals were observed at
least once daily for any abnormalities or distress and for the
development of infectious disease. Treatment of animals was in
accordance with the regulations outlined in the USDA Animal
Welfare Act (9 CFR Parts 1, 2 and 3) and the conditions
specified in the Guide for the Care and Use of Laboratory
Animals (ILAR publication, NEC, 2011, The National
Academies Press). Test compounds were administered in a
vehicle consisting of 10% Ethanol-90% Sesame Oil/Cremophor
EL (55:35). The dosing volume was 0.2 mL/mouse. Animals
were observed hourly for signs of distress or poor health. Time
points (following drug administration) examined were 0, 30
minutes, 1 hour, 3 hours, 6 hours, 12 hours and 24 hours. Blood
mg/mL pooled liver microsomes from male CD-1 mice (Life
Technologies, Grand Island, NY), 2 mM tetra-sodium NADPH
and 3 mM magnesium chloride for 60 minutes at 37oC with
gentle shaking. At five time points, 75 uL of reaction mixture
was transferred to 96-shallow well stop plates on ice containing
225 mL of acetonitrile with 0.1 uM propafenone. Control
reactions (lacking NADPH) were performed in a similar manner
to demonstrate NADPH dependency of compound loss. Standard
curves for test compounds were generated using 5 concentrations
in duplicate that were processed as above but with zero
incubation time. Stop plates were centrifuged at 2,000 xg for 10
minutes and then 170 uL of the supernatants were transferred to a
Waters Aquity® UPLC 700 uL 96-well sample plate with cap
2
was drawn under CO anesthesia by cardiac puncture and
mat (Waters, Milford, MA).
The amount of compound
collected in tubes containing EDTA. Plasma was isolated by
remaining in the supernatant was quantified by LC/MS/MS using
a Waters TQ MS (electrospray positive mode) coupled to a
Waters Aquity® UPLC (BEH column, C18 1.7 uM, 2.1 x 50 cm,
gradient of acetonitrile/water/0.1% formic acid). Propafenone
was used as the internal standard. GraphPad® Prism v 5.04 was
used for nonlinear fitting of time course data to generate Clint
values.
o
centrifugation at 12,000 rpm for 7 minutes and stored at -20 C
until analysis. At the end of the 24 hour study all animals were
euthanized by CO inhalation. Drug concentrations in plasma
2
were quantified by LC/MS/MS. Statistics were analyzed using
WinNonLin.
4.9 Pharmacodynamic analysis. Effect on HO1 gene and
protein expression in vivo.
4
.6 Plasma protein binding.
For pharmacodynamics anslysis, mice were dosed orally as
described above with 200 or 400 mg/kg of compounds 5 and 8d,
or vehicle alone. Mice were sacrificed 6 hours post-treatment
and lungs were excised for gene and protein analysis. Half of the
lung’s left lobe was homogenized and RNA was extracted using
the RNeasy Mini Kit (Qiagen, Valencia, CA). HO1 gene
expression was determined by QPCR analysis as described
above. The remaining lung tissue was homogenized in o.5 mM
41
Equilibrium dialysis was performed as previously described
using 96-well equilibrium dialyzer plates with MW cutoff of 5K
Harvard Apparatus, Holliston, MA) and a dual-plate rotator set
to maximum speed (Harvard Aparatus, Holliston, MA) located in
(
o
2
a 37 C incubator with a 10% CO atmospheric environment.
Test compounds (2.5 uM) were added to plasma in DMSO (2 uL,
final DMSO concentration 0.4%) to give 10 uM. 200 uL of

sodium vanadate, 1 mM PMSF in Cell Lysis Buffer (Cell
Signaling, Canvers, MA) supplemented with 1 tablet/10mL
Complete Mini Protease Inhibitor (Roche, Mannheim, Germany).
HO1 protein levels were determined by ImmunoSet HO1 ELISA
Builarte, T. R.; Yamamoto, M.; Sporn, M. B.; Kensler, T. W.
Pharmacodynamic characterization of chemopreventative triterpenoids as
exceptionally potent inducers of Nrf2-regulated genes. Mol. Cancer Ther.
2
007, 6, 154-162.
17) Center for Drug Evaluation and Research. New Drug Application #
(
(
Enzo Life Science, Famingdale, NY) and normalized by the total
protein in homogenates found using the BCA Protein Assay
Thermo Scientific, Rockford, IL).
204063Orig1s000. Clinical Pharmacology and Biopharmaceutics Review.
Reference ID: 3123618. US Food and Drug Administration. Rockville, MD.
Dated
4/29/2012.
Available
from:
(
http://www.accessdata.fda.gov/drugsatfda_docs/nda/2013/204063Orig1s000T
OC.cfm. Last accessed 09/03/2014.
Acknowledgements
(
18) Kumar, V.; Kumar, S.; Hassan, M.; Wu, H.; Thimmulappa, R. K.;
Caco-2 data were provided by Absorption Systems (Exton, PA).
The in-life portion of the in vivo exposure and
pharmacodynamics studies and plasma collection were provided
by Novel Life Sciences, Inc. (Gaithersburg, MD). This work was
funded by Curveda, LLC (Halethorpe, MD).
Kumar, A.; Sharma, S. K.; Parmar, V. S.; Biswal, S.; Malhotra, S. V. Novel
chalcone derivatives as potent Nrf2 activators in mice and human lung
epithelial cells. J. Med. Chem. 2011, 54, 4147-4159.
(19) Bhagat, S.; Sharma, R.; Sawant, D. M.; Sharma, L.; Chakraborty, A.
2
K. LiOH·H O as a dual activation catalyst for highly efficient and easy
synthesis of 1,3-diaryl-2-propenones by Claisen-Schmidt concensation under
mild conditions. J. Mol. Catal. A: Chem. 2006, 244, 20-24.
Supplementary data
Synthetic procedures for producing intermediates 6a, 6b and 7a-
d, H-NMR and C-NMR spectroscopic data for compounds 8a-
(20) Kao, B.-C.; Doshi, H.; Reyes-Rivera, H.; Titus, D. D.; Yin, M.;
Dalton, D. R. 3-Substituted 2-pyridinecarbaldoximes. J. Heterocyclic Chem.
1
13
1
991, 28, 1315-1324.
21) Ashimori, A.; Ono, T.; Uchida, T.; Ohtaki, Y.; Fukaya, C.; Watanabe,
8
8
h and PK data following iv administration for compounds 5 and
d can be found in the online version at:
(
M.; Yokoyama, K. Novel 1,4-dihydropyridine calcium antagonists. I.:
Synthesis and hypotensive activity of 4-(substituted pyridyl)-1,4-
dihydropyridine derivatives. Chem. Pharm. Bull. 1990, 38, 2446-2458.
References and notes
(1) Hybertson, B. M.; Gao, B.; Bose, S. K.; McCord, J. M. Oxidative
(
22) LaMattina, J. L. The synthesis of 2-amino-4-(4-imidazolyl)pyridines.
J. Heterocycl. Chem. 1983, 20, 533-538.
23) Busto, E.; Gotor-Fernandez, V.; Gotor, V. Enantioselective synthesis
stress in health and disease: The therapeutic potential of Nrf2 activation.
Mol. Aspects Med. 2011, 32, 234-246.
(
(2) Moi, P.; Chan, K.; Asunis, I.; Cao, A.; Kan, Y. W. Isolation of NF-E2-
of 4-(dimethylamino)pyridines through a chemical oxi
related factor 2 (Nrf2), a Nf-E2-like basic leucine zipper transcriptional
activator that binds to the tandem Nf-E2/AP1 repeat of the beta-globulin
locus control region. Proc. Natl. Acad. Sci. USA 1994, 91, 9926-9930.
dation-enzymatic reduction sequence. Application in asymmetric catalysis.
Adv. Synth. Catal. 2006, 348, 2626-2632.
(24) Trecourt, F.; Marsais, F.; Gungor, T.; Queguiner, G. Improved
synthesis of 2,3-disubstituted pyridines by metallation of
(3) Venugopal, R.; Jaiswal, A. K. Nrf1 and Nrf2 positively and c-Fos and
Fra1 negatively regulate the human antioxidant response element-mediated
expression of NAD(P)H:quinone oxidoreductase21 gene. Proc. Natl. Acad.
Sci. USA 1996, 93, 14960-14965.
2-chloropyridine: A convenient route to fused poly heterocycles. J. Chem.
Soc., Perkin 1 1990, 2409-2415.
(25) Chen, X.-L.; Kunsch, C. Induction of cytoprotective genes through
Nrf2/antioxidajnt response element pathway: A new therapeutic approach for
the treatment of inflammatory diseases. Curr. Pharm. Design 2004, 10, 879-
891.
(26) Alam, J.; Stewart, D.; Touchard, C.; Bonapally, S.; Choi, A. M. K.;
Cook, J. L. Nrf2, a cap’n’collar transcription factor, regulates induction of
the heme oxygenase-1 gene. J. Biol. Chem. 1999, 274, 26071-36078.
(27) Kumar, S.; Kingh, B. K.; Prasad, A. K.; Parmer, V. S.; B iswal, S.;
Ghosh, B. Ethyl 3’,4’,5’-trimethoxythionocinnamate modulates NF-dB and
Nrf2 transcription factors. Eur. J. Pharmacol. 2013, 700, 32-41.
(28) Kuzuya, Y.; Adachi, T.; Hara, H.; Anan, A.; Izuhara, K.; Nagai, H.
Induction of drug-metabolizing enzymes and transporters iun human
bronchial epithelial cells by beclomethasone dipropionate. IUBMB Life 2004,
56, 355-359.
(4) Itoh, K.; Wakabayashi, N.; Katoh, Y.; Ishii, T.; Igarashi, K.; Engel, J.
D.; Yamamoto, M. Keap1 represses nuclear activation of antioxidant
responsive elements by Nrf2 through binding to the amino-terminal Neh2
domain. Genes Dev. 1999, 13, 76-86.
(
5) Niture, S. K.; Khatri, R. Jaiswal, A. K. Regulation of Nrf2 – an update.
Free Rad. Biol. and Med. 2014, 66, 36-44.
6) Kobayashi, A.; Kang, M.-I.; Okawa, H.; Ohtsuji, M.; Zenke, Y.;
(
Chiba, T.; Kazuhiko, I.; Yamamoto, M. Oxidative stress sensor Keap1
functions as an adaptor for Cul1-based E3 ligase to regulate proteasomal
degradation of Nrf2. Mol. Cell. Biol. 2004, 24, 7130-7139.
(7) Sussan, T. E.; Rangasamy, T.; Blake, D. J.; Malhotra, D.; El-Haddad,
H.; Bedja, D.; Yates, M. S.; Kombairaju, P.; Yamamoto, M.; Liby, K. T.;
Sporn, M. b.; Gabrielson, K. L.; Champoin, H. C.; Tuder, R. M.; Knesler, T.
W.; Biswal, S. Targeting Nrf2 with the triterpenoid CDDO-imidazole
attenuates cigarette smoke-induced emphysema and cardiac dysfunction in
mice. Proc. Natl. Acad. Sci. USA 2009, 106, 250-255.
(29) Bois, F.; Beney, C.; Boumendjel, A.; Mariotte, A.-M.; Conseil, G.;
DiPietro, A. Halogenated chalcones with high-affinity binding to p-
glycoprotein: potential modulators of multidrug resistance. J. Med. Chem.
1998, 41, 4161-4164.
(8) Kensler, T. W.; Wakabayashi, N.; Biswal, S. Cell survival responses
to environmental stresses via the Keap1-Nrf2-ARE pathway. Annual Rev.
Pharmacol. Toxicol. 2007, 47, 89-116.
(30) Liu, X.-L.; Tee, H.-W.; Go, M.-L. Functionalized chalcones as
selective inhibitors of p-glycoprotein and breast cancer resistance protein.
Bioorg. Med. Chem. 2008, 16, 171-180.
(9) Rangasamy, T.; Guo, J.; Mitzner, W. A.; Roman, J.; Singh, A.; Fryer,
A. D.; Yamamoto, M.; Kensler, T. W.; Tuder, R.M.; Georas, S. N.; Biswal, S.
Disruption of Nrf2 enhances susceptibility to severe airway inflammation and
asthma in mice. J. Exp. Med. 2005, 202, 47-59.
(31) Parveen, Z.; Brunhofer, G.; Jabeen, I.; Erker, T.; Chiba, P.; Ecker, G.
F. Synthesis, biological evaluation and 3D-QSAR studies of new chalcone
derivatives as inhibitors of human p-glycoprotein. Bioorg. Med. Chem. 2014,
22, 2311-2319.
(10) Thimmulappa, R. K.; Mai, K. H.; Srisuma, S.; Kensler, T. W.;
Yamamoto, M.; biswal, S. Identification of Nrf2-related genes induced by
the chemopreventative agent sulforaphane by oligonucleotide microarray.
Cancer Res. 2002, 62, 5196-5203.
(32) Amidon, G. L.; Lennernas, H.; Shah, V. P.; Crison, J. R.
A
theoretical basis for a biopharmaceutic drug classification: the correlation of
in vitro drug product dissolution and in vivo bioavailability. Pharm. Res.
1995, 12, 413-420.
(
11) Copple, Ii. M.; the Keap1-Nrf2 cell defense pathway – a promising
therapeutic target? Adv. Pharmacol. 2012, 63, 43-79.
12) Suzuki, T.; Motohashi, H.; Yamamoto, M. Toward clinical
application of the Keap1-Nrf2 pathway. Trends Pharmacol. Sci 2013, 34,
40-346.
13) Sekhar, K. R.; Rschakonda, G.; Freeman, M. L. Cysteine-based
regulation of the CUL3 adaptor protein Keap1. Toxicol. Appl. Pharmacol.
010, 244, 21-26.
14) Hur, W.; Gray, N. S. Small molecule modulators of antioxidant
response pathway. Curr. Opin. Chem. Biol. 2011, 15, 162-173.
15) Kensler, T. W.; Egner, P. A.; Agyeman, A. S.; Visvanathan, K.;
(33) Szegezdi, J.P. Scizmadia, F. Prediction of distribution coefficient
th
(
using microconstants. Poster presented at the 227 ACS National Meeting,
March 28 – April 1, 2004, Anaheim, CA. Available on the world wide web
at:
http://www.chemaxon.com/conf/Prediction_of_distribution_coefficient_using
_microconstants.pdf. Last accessed 09/16/2014.
(34) Viswanadhan, V. N.; Ghose, A. K.; Revankar, G. R.; Robins, R. K.
Atomic physicochemical parameters for three dimensional structure directed
quantitative structure-activity relationships. 4. Additional parameters for
hydrophobic and dispersive interactions and their application for an
automated superposition of certain naturally occurring nucleoside antibiotics.
J. Chem Inf. Comput. Sci. 1989, 3, 163-172.
3
(
2
(
(
Groopman, J. D.; Chen, J.-G., Chen, T.-Y., Fahey, J. W.; Talalay, P. Keap1-
Nrf2 signaling: A target for cancer prevention by sulphoraphane. Top. Curr.
Chem. 2013, 329, 163-178.
(35) Ertl, P.; Rohde, B.; Selzer, P. Fast calculation of molecular polar
surface area as a sum of fragment-based contributions and its application to
(16) Yates, M. S.; Tauchi, M.; Katsouda, F.; Flanders, K. C.; Liby, K. T.;
Honda, T.; Gribble, G. W.; Johnson, D. A.; Johnson, J. A.; Burton, N. C.;

the prediction of drug transport properties. J. Med. Chem. 2000, 43, 3714-
717.
36) MOE Version 2013.08, Chemical Computing Group (Montreal, CA),
available at: http://www.chemcomp.com/MOE-
Molecular_Operating_Environment.htm. Last accessed 10/02/2014.
37) Halgren, T. A. Merck molecular force field. I. basis, form, scope,
parameterization and performance of MMFF94. J. Comput. Chem. 1996, 17,
90-519.
38) Stewart, J. J. P. Optimization of parameters for semiempirical
methods I. method. J. Comput. Chem. 1989, 10, 209-22.
39) Hidalgo, I. J.; Raub, T. J.; Borchart, R. T. Characterization of the
3
(
(
4
(
(
human colon carcinoma cell line (Caco-2) as a model system for intestinal
epithelial permeability. Gastroenterology 1989, 96, 736-749.
(40) Hubatsch, I.; Ragnarsson, E. G. E.; Artursson, P. Determination of
drug permeability and prediction of drug absorption in Caco-2 monolayers.
Nat. Protocols 2007, 2, 2111-2119.
(41) Kochansky, C. J.; McMasters, D. R.; Lu, P.; Koeplinger, K. A.; Kerr,
H. H.; Shou, M.; Korzekwa, K. R. Impact of pH on plasma protein binding in
equilibrium dialysis. Mol. Pharm. 2008, 5, 438-448.