Importance of the proximity and orientation of ligand-linkage to the design of cinnamate-GW9662 hybrid compounds as covalent PPAR agonists

Article abstract of DOI:10.3390/molecules24102019

Covalent agonists of PPAR cause unique receptor conformational changes and behave as selective PPAR modulators, whereas there are few covalent agonists other than endogenous unsaturated fatty acids metabolites. Previously, we established a cell-based stra

Full text of DOI:10.3390/molecules24102019

molecules
Article
Importance of the Proximity and Orientation of
Ligand-Linkage to the Design of Cinnamate-GW9662
Hybrid Compounds as Covalent PPARγ Agonists
Yuki Utsugi 1,2, Hirona Kobuchi 3, Yukio Kawamura 3, Ahmed Salahelden Aboelhamd Atito 2,
Masaya Nagao 4, Hiroko Isoda 2,5,6 and Yusaku Miyamae 2,4,6,
*
1
College of Agro-Biological Resources Sciences, University of Tsukuba, Ibaraki 305-8572, Japan;
utsugi.yuki.sp@alumni.tsukuba.ac.jp
Master’s/Doctoral Program in Life Science Innovation, School of Integrative and Global Majors, University
of Tsukuba, Ibaraki 305-8572, Japan; fopcu91@gmail.com (A.S.A.A.); isoda.hiroko.ga@u.tsukuba.ac.jp (H.I.)
Department of Food and Nutrition, Faculty of Home Economics, Kyoto Women’s University, Kyoto 605-8501,
Japan; kobuchi34@gmail.com (H.K.); kawamury@kyoto-wu.ac.jp (Y.K.)
Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan; mnagao@kais.kyoto-u.ac.jp
Alliance for Research on the Mediterranean and North Africa, University of Tsukuba, Ibaraki 305-8572, Japan
Faculty of Life and Environmental Sciences, University of Tsukuba, Ibaraki 305-8572, Japan
Correspondence: miyamae.yusaku.fw@u.tsukuba.ac.jp; Tel.: +81-29-853-7442
2
3
4
5
6
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 16 March 2019; Accepted: 24 May 2019; Published: 27 May 2019
Abstract: Covalent agonists of PPAR
as selective PPAR modulators, whereas there are few covalent agonists other than endogenous
unsaturated fatty acids metabolites. Previously, we established a cell-based strategy to identify new
PPAR ligands and synthesized a new-type of covalent agonist that possesses the hybrid structure of
γ cause unique receptor conformational changes and behave
γ
γ
a plant-derived cinnamic acid derivative and GW9662, a covalent antagonist. Herein, we report six
analogues that differ in how the two fragments are linked together. Compounds with a simplified
linker showed potent agonistic activity with improved EC50 values (less than 5 nM), indicating that
close proximity between the two fragments improves binding affinity. When the position of cinnamic
acid moiety was placed at 40 carbon of aniline ring, PPAR
Docking studies suggested that the activation profile likely depends on interaction with the cavity
around helix 3, -sheet, and -loop region in the ligand-binding domain. Furthermore, a cell-based
assay revealed that agonist-type compounds activate PPAR transcription in a manner dependent
γ agonist activity was completely abolished.
β
Ω
γ
on covalent linkage with the Cys285 residue leading to prolonged transactivation. This activation
feature reflects pharmacological benefits of covalent drugs, suggesting that these hybrid compounds
may serve as potential leads for a new-class of covalent PPARγ ligands.
Keywords: PPARγ; covalent agonist; ligand-linkage; structure-activity relationship
1. Introduction
Peroxisome proliferator-activated receptor (PPAR)
superfamily and is found in adipose tissue and macrophages [
with retinoid X receptors (RXRs), which triggers the transcriptional switch of target genes in a
ligand-dependent manner [ ]. Ligand binding to PPAR stabilizes their conformation and subsequently
modulates cofactor recruitment, resulting in transcriptional activation of downstream genes [
related to glucose metabolism, adipogenesis, and macrophage function [ ]. These functions make
PPAR a therapeutic target for type 2 diabetes and other metabolic diseases such as obesity and
γ
is a member of the nuclear receptor
1
]. PPAR forms obligate heterodimers
γ
2
γ
3,4]
5–7
γ
atherosclerosis. Thiazolidinedione (TZD), a known full PPAR
γ
agonist, has been used for the treatment
Molecules 2019, 24, 2019; doi:10.3390/molecules24102019
www.mdpi.com/journal/molecules

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of type 2 diabetes, however its use has been limited because of adverse effects such as body weight
gain and edema. These side effects are closely related with the undesirable expression of a subset of
genes as a result of the complete activation of PPAR
agonists can selectively modulate the transcription of the downstream gene set, resulting in fewer
side effects while retaining beneficial pharmacological effects [10 11]. This is related to the distinct
γ transcription by full agonists [8,9]. Partial PPARγ
,
conformational changes caused by the partial agonists, leading to different coactivator recruitment and
activation of discrete sets of the downstream genes [12,13].
Recently, our group reported a synthetic covalent PPAR
The first step involved the identification of a combination of compounds that cooperatively activated
the PPAR , followed by the design and synthesis of a hybrid structure. This strategy is based on the
unique character of the PPAR ligand binding pocket (LBP) that can be simultaneously occupied by
multiple ligands because of its large cavity coupled with the synergistic activation of the transcriptional
switch of PPAR 15]. Using this approach, we successfully identified the combination of a synthetic
irreversible antagonist, called GW9662 ( ) [16], and a cinnamic acid derivative ( ). We synthesized a
single molecule containing a hybrid structure of the two ( ) and determined its subtype-selective partial
γ agonist by using a two-step strategy [14].
γ
γ
γ
[
1
2
3
agonistic activity (EC50 value = 14 nM) that was dependent on its covalent linkage with the Cys285
residue. A recent study demonstrated that some types of oxidized fatty acids and their metabolites
form covalent bonds with Cys285, effectively stabilizing the conformation of the ligand-binding
domain (LBD) and activating the transcriptional switch [17]. Another group demonstrated that a
metabolite of oleic acids, which has a nitro moiety, forms a covalent bond with Cys285 and behaves as
a selective PPAR
gain in diabetic model mice [18]. These studies indicate that covalent PPAR
significance and pharmacological potential. However, there are few covalent agonists known other
γ
modulator, causing the reduction of the blood glucose level without body weight
γ
ligands have biological
than endogenous ligands like fatty acids metabolites. Our synthesized ligand is a rare covalent PPAR
agonist, although we have not yet clarified its structure-activity relationship.
γ
The key features of the structural designing of the identified compound were the site and the
linker moiety connecting the two fragments. Previously, we selected the hydroxyethyl linker to connect
the two units based on the computational analysis. A docking study predicted that the length of the
linker should be less than two carbon atoms because linkers with more than three carbons occupy
most of the space in the binding subpocket, causing the cinnamate unit to protrude from the space.
This prompted us to hypothesize that the proximity between the two fragments is a key factor in
determining that the fused hybrid molecule could fit into the available space. As reported herein,
we attempted to create a combination where the two fragments were connected with a diphenyl ether
moiety instead of an alkyl chain linker (Figure 1). The simplified compounds were synthesized using
an improved synthetic route, allowing access to the desired compounds with fewer reaction steps.
The improvement of the synthetic protocol also provided us several types of analogues with different
orientations of the cinnamate moiety. Using these chemical derivatives, the results indicated that
the position of the cinnamate moiety is critical for activation of PPAR
γ transcription because of the
possible interactions with the -sheet and -loop region. These results provide us with the principles
β
Ω
for structural designing by ligand-linking using two identified fragments. Finally, the pharmacological
characterization of the newly synthesized covalent agonist was assessed.

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Figure 1. Structures of ligands synthesized in previous and current studies.
2. Results and Discussion
2.1. Retrosynthetic Analyses
The simplified analogue 4a was designed by replacing the hydroxyethyl linker of the previous
compound to diphenyl ether. The principle of the synthetic route to 4a 4c, and 4d is depicted in
3
,
Scheme 1. The 2-chloro-5-nitrobenzoyl group was planed to be coupled in the last step because of its
high reactivity. The amine compound can be obtained by the reduction of nitrobenzene anchored with
cinnamic acid ethyl ester. The α,β-unsaturated carbonyl of the cinnamate moiety can be produced
by the Horner-Wadsworth-Emmons reaction. The central diphenyl ether was a key intermediate of
this study. This can be easily synthesized through one step by using a standard etherification reaction,
enabling the number of steps to be reduced. Several types of aromatic ring compounds with different
positions of the reactive groups are commercially available, and were used as starting materials to
synthesize three types of intermediates, 6a
, 6b, and 6c. It allowed us to obtain several analogues with
different orientations of the cinnamate moiety.

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Scheme 1. Retrosynthetic analysis.
2.2. Synthesis
The syntheses of compounds 4a
were linked via an ether bond at one of three positions in the nitro-benzene. The aldehyde groups of
the resultant diphenyl ethers were converted to the -unsaturated carbonyl compounds 7a via
the Horner-Wadsworth-Emmons reaction. After reduction of the nitro groups of 7a , amines 8a
were bound to 2-chloro-5-nitrobenzoyl chloride or 3-nitrobenzoyl chloride to produce 4a 4e was
obtained by the same amide bond formation using , which was produced by catalytic hydrogenation
–e are outlined in Scheme 2. Nitro-benzene and benzaldehyde
α
,β
–c
–
c
–c
–d.
9
of 8a. Compound 16 was synthesized using 10 as a starting material, according to the procedure
previously reported by our group [14] (Scheme 3). The amino group of 10 was protected with a Boc
group using a reported protocol [19]. The alcohol moiety of 11 was tosylated with p-toluensulfonyl
chloride. The sulfonate compound was connected with p-hydroxybenzaldehyde through an ether bond.
The aldehyde group of diphenyl ether was converted to the α,β-unsaturated carbonyl compound 14
via the Horner-Wadsworth-Emmons reaction. After removal of the Boc group, amine 15 was bound to
2-chloro-5-nitrobenzoyl chloride to produce 16.

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Scheme 2. Reagents and conditions: (a) K2CO3, DMF, 80 ◦C, 18 h. (b) (EtO)2P(O)CH2COOEt, LiCl,
DBU, CH3CN, rt, 18 h. (c) Zn, NH4Cl, MeOH, reflux, 1 h. (d) Et3N, CH2Cl2, rt, 18 h. (e) Pd/C, H2,
EtOAc, rt 6 h.
Scheme 3. Reagents and condition: (a) ref. [19]. (b) p-TsCl, CH2Cl2, DMAP, Et3N, rt, 24 h. (c) K2CO3,
CH3CN, p-hydroxybenzaldehyde, reflux, 18 h. (d) (EtO)2P(O)CH2COOEt, LiCl, DBU, CH3CN, rt, 18 h.
(e) HCl, EtOAc, rt, 18 h. (f) Et3N, CH2Cl2, rt, 18 h.
2.3. PPARγ Agonist Activity
PPAR
LBD chimera protein using the plasmid pGAL4- PPAR
pUAS-tk-Luc containing the target sequence of GAL4 [20]. The test results of all the synthesized
compounds are shown in Figure 2 and Table 1. Compound 4a (EC50 = 5.13 0.45 nM) and 4c
(EC50 = 4.11 ± 0.13 nM) exhibited potent PPAR agonist activity, which was about 3-fold more potent
than a previously synthesized compound . This supports our hypothesis that close proximity between
the two fragments contributes to the improvement of binding affinity. Compound 4e activated PPAR
γ
agonist activity was assessed by expression of a GAL4 DNA-binding domain/PPAR
γ
γ
LBD and the luciferase reporter plasmid
±
γ
3
γ

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at a low concentration (EC50 = 2.54
±
1.30 nM) and to a similar extent as 4a and 4c, suggesting that the
double bond of the
changes of the agonist induction activity of these compounds were almost half that of troglitazone,
indicating that these compounds act as partial PPAR agonists. Compound 4b, in which the chlorine
α,β-unsaturated carbonyl moiety is not necessary for the agonist activity. The fold
γ
atom of 4a was substituted with hydrogen, had no agonist activity, indicating that the agonist activity
of 4a requires the covalent bond formation with the Cys285 residue via nucleophilic substitution.
Interestingly, when the position of the cinnamic acid moiety was placed at the 40 carbon of the aniline
ring (4d), the PPARγ agonist activity was completely abolished. The disappearance of activity was
also observed for compound 16, in which the cinnamic acid moiety was connected with an ethyl linker
at the 40 position of the aniline ring. These results indicate that the connecting position of cinnamic
acid is critical for agonist activity.
Figure 2. Transactivation potentials of PPAR
γ
by 4a
,
b
,
c,
d,
e, and 16. The luciferase reporter
assay was performed in HepG2 cells transiently cotransfected with pGal4-humanPPAR
γ
LBD plasmid,
pUAS-tk-Luc reporter, and pGL4.74[hRluc/TK] (Promega). Firefly luciferase activities were normalized
to Renilla luciferase activities. Vehicle control data are denoted as 1. HepG2 cells were treated with
the vehicle (0.1% DMSO, shown as –), troglitazone (10
µM, shown as Tro), 4a, b, c, d, e, or 16 for 6 h.
All the results are presented as the mean
compared with vehicle control.
±
SD (n = 3) of three independent experiments. * p < 0.001,

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Table 1. EC50 values of tested compounds.
Compounds
EC50 (nM)
4a
4b
4c
4d
4e
16
5.13 ± 0.45
N.D.
4.11 ± 0.13
N.D.
2.54 ± 1.30
N.D.
2.4. Validation of Covalent Bond Formation of the Synthesized Compounds with the Cys285 Residue
To assess the interactions between the receptor and the synthesized compounds, we initially
validated the formation of a covalent bond with Cys285 by performing a rhodamine-maleimide
assay [21]. This assay can detect the presence of a covalent modification of Cys285 in the PPAR
because it has only one cysteine residue in its sequences. As shown in Figure 3A, the fluorescence of
the rhodamine-labeled PPAR LBD decreased after treatment with GW9662 alone. The decrease of
labeled PPAR LBD was also found when it was incubated with agonist-type compounds 4a and 4c
indicating that these ligands can form a covalent bond with the Cys285 residue in the PPAR LBD.
Interestingly, non-agonist type ligands 4d and 16 also showed the decrease of fluorescence, indicating
that these two compounds are able to bind to PPAR LBD through the formation of covalent bond
γ LBD
γ
γ
,
γ
γ
with Cys285 but do not exhibit the binding manners which cause the receptor activation. In contrast,
decrease of fluorescence was not observed by the treatment of compound 4b, which lacks the ability
of nucleophilic addition to the Cys285 residue because of the replacement of chlorine to hydrogen.
To further confirm the covalent bonding of the synthesized compounds, we conducted proteolytic
mapping studies via mass spectroscopy using 4a as a representative compound and 4b as a negative
control. The complexes between recombinant PPAR
and subjected to MALDI-TOF MS. Covalent binding of 4a (mass of 466.1 Da) to the PPAR
assumed to result in a mass change of 431.1 Da for the modified tryptic peptide. By comparison of
the tryptic digests of PPAR , with and without 4a, a candidate peptide containing the attachment site
γ
LBD and the test compounds were trypsinized
γ
LBD is
γ
for 4a (IFQGCQFR, m/z 499.7492 Da) was detected by its mass shift to m/z 714.8080 Da (detected as
double charged; Figure 3B). The binding site of 4a was identified as the Cys285 residue using MS/MS
analysis to detect the mass difference between the y3- and y4-ions (m/z 533.1 Da), as shown in Figure 3B.
In contrast, these mass shifts were not exhibited by the non-covalent analogue 4b. Taken together,
these experiments indicated that compounds that possess a 2-chloro-5-nitrobenzoyl moiety form a
covalent bond with the Cys285 residue in the PPARγ LBD.

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Figure 3. Assessment of the abilities of the synthesized compounds to form a covalent bond with the
Cys285 residue in the PPAR LBD. ( ) PPAR LBD recombinant protein was incubated with the vehicle
(0.1% DMSO, shown as –), troglitazone (shown as Tro),
residue was detected by the rhodamine-maleimide assay. (
to Cys285 in the PPAR
γ
A
γ
1,
3,
4a,
4b
, 4c, 4d, or 16, and the free cysteine
B), (C) Detection of covalent binding of 4a
γ
LBD by ESI-MS/MS analysis. Fragmentation pattern of peptide IFQGCQFR
was obtained from the trypsin digest. The calculated y- and b-ions of this peptide with observed ions
underlined and the native or modified peptides detected in chromatogram are shown. The spectrum in
(B) shows the adduct of 4a with Cys285 residue in the PPARγ LBD; in contrast, in panel (C), no adduct
of 4b is observed.
2.5. Docking Study
To gain further insight into the interaction between the synthesized covalent ligands and the
PPAR LBD, a molecular docking simulation was carried out. The PPAR LBD comprises 13 helices
and a small four-stranded -sheet. Full agonists such as TZD occupy the binding site around helix 3
and 12, and cause a drastic conformational change of the AF-2 region because of the hydrogen bond
with the Tyr473 residue [ ]. Partial agonists are shown to bind to the cavity around helix 3, the -sheets,
and -loop region (termed the alternate binding site) [22 24] rather than the AF-2 sites. Compound
γ
γ
β
3
β
Ω
–
1
(GW9662) covalently reacted with the Cys285 residue through nucleophilic substitution [16] irreversibly
blocking other agonists such as TZDs from entering the AF-2 sites, whereas there was an open space
around the alternate binding site. We, and others, revealed that another small ligand, such as
2,
could access the alternate binding site in the GW9662-bound PPAR LBP [14 24]. To determine the
γ
,

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possible binding manner of the newly synthesized ligands, we docked the compounds into the crystal
structure of the human PPAR LBD bound to (PDB code 3B0R). Taking into account the covalent bond
γ
1
formation, the fifth carbon of the nitrobenzene ring in the ligand was constrained to the sulfur atom of
the thiol group in Cys285 and then the constrained ligand was docked. Because agonist-type ligands
synthesized in this study act as partial agonists, we defined the potential binding site for docking as the
cavity near helix3, the
H12. To validate the docking protocol, we first performed cross-docking simulations by using several
crystal structures (5DV3, 5DV6, and 5DV8) of the complexes between PPAR LBD and different types
β-sheet, and Ω-loop region in the PPARγ LBD rather than the AF-2 pocket near
γ
of covalent ligands. We observed that the reported binding manner could be reproduced when the
ligands were docked into the original protein structure (Supplementary Figure S1A). Similar binding
poses were obtained when the ligands were docked in the crystal structure other than originally
reported structural data (Supplementary Figure S1B). The root mean square deviation (RMSD) values
of small-size compounds like GW9662, SB1404, and SB1405 were less than 1.2 while that of SB1451,
which has huge lipophilic moiety in the anilin ring of GW9662 unit, was less than 1.9. Encouraged
by these data, the binding poses of newly synthesized compounds were analyzed by the validated
protocol. The binding manner of
Compound was located behind helix3 and formed a covalent bond to Cys285. The docking simulation
predicted that the GW9662 units of docked compounds 4a 4c, and 4d interact with Phe282, Gln284, and
Arg288 in the helix3 as well as Leu469 and Tyr473 in the helix12. The binding poses of GW9662 units
of docked compounds aligned with the binding position of in the original crystal structure, while the
1 in the original crystal structure is displayed in green (Figure 4A–C).
1
,
1
binding sites of the cinnamic acid moiety are different among the agonist- and non-agonist-type ligands.
In the binding mode of the agonist-type ligands 4a and 4c, the cinnamate moiety is able to interact
with Arg280, Ile281, and Gly284 in the helix3, and Ile341 and Ser342 in the
β-sheet, and Lys263 in the
Ω
-loop (Figure 4A,B), implying that the region near helix 3 and the -loop may be robustly stabilized
Ω
by binding with these ligands. In contrast, non-agonist type ligand 4d exhibited a different binding
pose in which the whole structure was positioned orthogonal to the helix 3 axis and the cinnamic acid
moiety interact with Pro227, Leu228, and Lys232 in the helix1 as well as Glu343 in the
β-sheet, located
outside the cavity (Figure 4C). The Mol Dock Scores of these ligands were
−
175.697 (4a), 184.234 (4c),
−
and −157.507 (4d).
These binding poses of both the agonist and non-agonist types of compounds were similar to
those of endogenous unsaturated fatty acids. Previous studies reported that oxidized fatty acids
occupy the PPAR
(15-oxo-ETE) and 4-oxo-docoxahexanoiec acid (4-oxo-DHA) effectively stabilize the
through the formation of a covalent bond with Cys285, leading to the transcriptional activation [17
The configuration of 15-oxo-ETE is parallel with the helix 3 axis and interacts with the -sheet, helix 5
and helix 3, while 4-oxo-DHA binds to the LBP in a U-shaped conformation, wrapping around near
the Cys285 residue with the interactions with the helix3 and the -sheet. 8-Oxo-ETE does not activate
the PPAR , although it forms a covalent bond with Cys285 [25]. The configuration of 8-oxo-ETE is
γ
LBP by covalently modifying the Cys285 residue. 15-Oxo-eicosatetraenoic acid
-loop region
25].
Ω
,
β
β
γ
orthogonal to the helix 3 axis similar to the binding pose of 4d. Indeed, the binding poses of the
synthesized compounds showed good alignment with those of endogenous ligands (Figure 4D,E).
These observations suggested that our synthesized ligands may mimic the binding mode of endogenous
fatty acids that covalently bind to the PPARγ LBD.

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Figure 4. Characterization of the putative binding modes of the synthesized ligands. (
structure of the complex between the PPAR LBD and GW9662 (green) (PDB code 3B0R), and docking
poses of 4a (blue, ), 4c (cyan, ), and 4d (yellow, ). Yellow spheres indicate hydrophobic contacts.
) Superposition of 15-oxo-ETE (gray, PDB code 2ZK4), 4-oxo-DHA (magenta, PDB code 2VV3), and
A–C) Co-crystal
γ
A
B
C
(
D
the docking poses of 4a and 4c. (
docking pose of 4d.
E) Superposition of 8-oxo-ETE (orange, PDB code 2ZK3) and the
2.6. Characterization of Agonist-Type Covalent Ligands
Finally, we assessed the pharmacological properties of the agonist-type covalent ligands. We first
examined the relationship between covalent binding to the Cys285 residue and the agonist activity
by a luciferase reporter gene assay using PPARγ mutants where the cysteine was substituted to an
alanine (C285A). The mutation did not influence the transcriptional response to troglitazone treatment,
indicating that the mutated LBD retained its agonist-inducible properties. In the C285A mutant, 4a and
4c did not show the transactivation (Figure 5A), indicating that the covalent modification of Cys285
is indispensable for the agonist activity of these compounds. Next, we investigated the potential
advantages of covalent binding for pharmacological applications. One of the features of covalent
ligands is that the compounds can be anchored to the receptor, mimicking a situation where the drug
captures the protein [26]. Based on this consideration, we assumed that the transcription level is
related to transient exposure to the ligands and is sustained after ligand elimination. To examine
this idea, we monitored the levels of luciferase activity upon withdrawal of the synthesized agonists.
In cells treated with troglitazone, a non-covalent agonist, luciferase transcriptional levels decreased
upon withdrawal of troglitazone, with luciferase activity approaching background levels after 24 h.
In contrast, the treatment of 4a and 4c resulted in sustained transcription activity 24 h after withdrawal
of the ligands, as expected.

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Figure 5. Characterization of the synthesized agonists 4a and 4c. (
abolishes the PPAR agonist activities of 4a and 4c. The luciferase reporter assay was performed using
wild-type and the C285A mutant of the pGal4-humanPPAR LBD plasmid. The experimental procedure
was described in the caption for Figure 2. HepG2 cells were treated with the vehicle (0.1% DMSO, shown
as –), troglitazone (10 M, shown as Tro), (100 nM), 4a (indicated concentrations), or 4c (indicated
concentrations) for 6 h. * p < 0.005, compared with vehicle control (WT). # p < 0.005, compared with
vehicle control (C285A). ( ) Monitoring the agonistic activities after ligand withdrawal. The luciferase
reporter assay was performed using the pGal4-humanPPAR LBD plasmid. The experimental procedure
was described in the caption for Figure 2. The transfected HepG2 cells were treated with troglitazone
(10 M, shown as Tro), 4a (100 nM), or 4c (100 nM) for 6 h and then washed with media to remove the
treated compounds. Cells were further cultured for the indicated times in the absence of ligands, after
which the luciferase activities were measured. All the results are presented as the mean SD (n = 3) of
A) Mutation of the cysteine residue
γ
γ
µ
3
B
γ
µ
±
†
three independent experiments. p < 0.01, compared with 0 h after ligand withdrawal.
3. Materials and Methods
3.1. Chemistry
3.1.1. General Remarks
All reagents and solvents were purchased and used without further purification. Flash
chromatography was performed using Wako gel C-200 (Fujifilm Wako Pure Chemical Corporation,
Osaka, Japan) and Parallel FR-360 (Yamazen Corporation, Osaka, Japan). The following spectroscopic
and analytical instruments were used: 1H and 13C NMR, Avance III 400 (reference TMS, Bruker,
Germany), Avance 500 (reference TMS, Bruker, Germany), JNM-ECS 400 (reference TMS, JOEL, Tokyo,
Japan), HR-ESI-TOF-MS, Waters Xevo G2-S QTof (Waters, Tokyo, Japan).
3.1.2. 4-(3-Nitrophenoxy)benzaldehyde (6a)
K2CO3 (621 mg, 4.5 mmol) was added to a solution of 3-nitrophenol 5a (417 mg, 3 mmol) in DMF
(12 mL). The reaction mixture was stirred at room temperature for 30 min. Then, p-fluorobenzaldehyde
(501 mg, 3.6 mmol) was added to the reaction mixture, and stirred at 80 ◦C for 18 h. The solution
was extracted with Hexane/EtOAc (4:1). The organic layer was washed with brine and dried over
MgSO4, filtered, and the solvents were evaporated in vacuo. The residue was purified by silica gel
chromatography (
(CDCl3, 400 MHz):
7.91 (1H, m), 7.93 (2H, d, J = 8.8 Hz), 8.07 (1H, ddd, J = 0.9, 2.1, 8.2 Hz), 9.98 (1H, s) ppm. 13C NMR
ϕ
20
×
150 mm; Hexane/EtOAc, 80:20) to afford 6a (691 mg, 2.8 mmol, 95%). 1H NMR
δ
7.15 (2H, d, J = 8.7 Hz), 7.42 (1H, ddd, J = 0.9, 2.4, 8.2 Hz), 7.59 (1H, t, J = 8.2 Hz),
(CDCl3, 100 MHz):
δ 114.8, 118.7, 118.7, 119.3, 125.8, 130.8, 132.2, 132.2, 132.6, 149.4, 156.4, 161.3,
190.6 ppm. HR ESI-MS (negative ion) m/z: 242.0434 (M − H)− (Calcd for C13H8NO4: 242.0453).

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3.1.3. General Procedure of Preparation for Compounds 6b and c
K2CO3 (850 or 1270 mg, 1.5 eq) was added to a solution of 4-hydroxybenzaldehyde 5b (500 or
750 mg, 1 eq) in DMF (15 mL). The reaction mixture was stirred at room temperature for 30 min.
Then, 4-fluoro-nitrobenzen (690 mg, 1.2 eq) or 1-fluoro-2-nitrobenzen (1040 mg, 1.2 eq) was added to
the reaction mixture, and stirred at 80 ◦C for 18 h. The solution was extracted with Hexane/EtOAc
(2:1). The organic layer was washed with brine and dried over MgSO4, filtered, and the solvents
were evaporated in vacuo. The residue was purified by silica gel chromatography (
Hexane/EtOAc, 80:20) to yield 6b or c.
ϕ
20
×
150 mm;
3.1.4. 4-(2-Nitrophenoxy)benzaldehyde (6b)
Colorless liquid, yield 93%. 1H NMR (CDCl3, 400 MHz):
dd, J = 1.2, 8.2 Hz), 7.38 (1H, ddd, J = 1.2, 7.6, 8.1 Hz), 7.65 (1H, ddd, J = 1.6, 7.5, 8.2 Hz), 7.90 (2H, d,
J = 8.6 Hz), 8.04 (1H, dd, J = 1.6, 8.2 Hz), 9.96 (1H, s) ppm. 13C NMR (CDCl3, 100 MHz):
117.9, 117.9,
δ 7.10 (2H, d, J = 8.6 Hz), 7.21 (1H,
δ
123.3, 125.6, 126.3, 132.2, 132.2, 132.4, 134.9, 142.5, 148.5, 161.9, 190.8 ppm. HR ESI-MS (negative ion)
m/z: 242.0434 (M − H)− (Calcd for C13H8NO4: 242.0453).
3.1.5. 4-(4-Nitrophenoxy)benzaldehyde (6c)
Colorless liquid, yield 93%. 1H NMR (CDCl3, 400 MHz):
d, J = 8.6 Hz), 7.96 (2H, d, J = 8.7 Hz), 8.28 (2H, d, J = 9.2 Hz), 10.0 (1H, s) ppm. 13C NMR (CDCl3,
δ 7.14 (2H, d, J = 9.3 Hz), 7.20 (2H,
100 MHz):
δ 118.8, 118.8, 119.7, 119.7, 126.1, 126.1, 132.1, 132.1, 133, 143.8, 160.3, 161.3, 190.5 ppm.
HR ESI-MS (negative ion) m/z: 242.0434 (M − H)− (Calcd for C13H8NO4: 242.0453).
3.1.6. General Procedure of Preparation for Compounds 7a–c
(EtO)2P(O)CH2COOEt (615 µl, 1.1 eq) was added to LiCl (130 mg, 1.1 eq) and DBU (830 µL) in
dry CH3CN (10 mL), and stirred at room temperature for 1 h. Then corresponding aldehyde (500 mg,
1 eq) in dry CH3CN (1 mL) was added to the solution and stirred at room temperature for 18 h.
After addition of water, the solution was extracted with EtOAc. The organic layer was washed with
brine and dried over MgSO4, filtered, and the solvents were evaporated in vacuo. The residue was
purified by silica gel chromatography (ϕ20 × 150 mm; Hexane/EtOAc, 90:10) to yield 7a–c.
3.1.7. (E)-Ethyl 3-[4-(3-nitrophenoxy)phenyl]acrylate (7a)
1
Colorless liquid, yield 77%. H NMR (CDCl3, 400 MHz):
δ 1.35 (3H, t, J = 7.1 Hz), 4.28 (2H, q,
J = 7.1 Hz), 6.40 (1H, d, J = 16 Hz), 7.06 (2H, d, J = 8.7 Hz), 7.37 (1H, ddd, J = 0.9, 2.3, 8.2 Hz), 7.53 (1H,
t, J = 8.2 Hz), 7.57 (2H, d, J = 8.7 Hz), 7.68 (1H, d, J = 16 Hz), 7.85 (1H, t, J = 2.3 Hz), 8.00 (1H, ddd,
J = 0.9, 2.2, 8.2 Hz) ppm. 13C NMR (CDCl3, 100 MHz):
δ 14.3, 60.5, 113.6, 118, 118.4, 119.5, 119.5, 124.8,
130, 130, 130.5, 130.9, 143.3, 149.3, 157.4, 157.5, 166.9 ppm. HR ESI-MS (negative ion) m/z: 312.0869
(M − H)− (Calcd for C17H14NO5: 312.0872).
3.1.8. (E)-Ethyl 3-[4-(2-nitrophenoxy)phenyl]acrylate (7b)
1
Colorless liquid, yield 94%. H NMR (CDCl3, 400 MHz):
δ 1.34 (3H, t, J = 7.1 Hz), 4.27 (2H, q,
J = 7.1 Hz), 6.37 (1H, d, J = 16 Hz), 7.02 (2H, d, J = 8.7 Hz), 7.11 (1H, dd, J = 0.9, 8.1 Hz), 7.28 (1H, td,
J = 0.8, 8.2 Hz), 7.53 (2H, d, J = 8.8 Hz), 7.56 (1H, td, J = 1.4, 8.1 Hz), 7.66 (1H, d, J = 16 Hz), 7.99 (1H, dd,
J = 1.5, 8.2 Hz) ppm. 13C NMR (CDCl3, 100 MHz):
δ 14.5, 60.7, 118, 118.9, 118.9, 121.9, 124.4, 126, 130,
130, 130.7, 134.5, 142, 143.5, 149.7, 158, 167.1 ppm. HR ESI-MS (negative ion) m/z: 312.0869 (M − H)−
(Calcd for C17H14NO5: 312.0872).
3.1.9. (E)-Ethyl 3-[4-(4-nitrophenoxy)phenyl]acrylate (7c)
Colorless liquid, yield 90%. 1H NMR (CDCl3, 400 MHz):
q, J = 7.1 Hz), 6.41 (1H, d, J = 16 Hz), 7.07 (2H, d, J = 9.3 Hz), 7.10 (2H, d, J = 8.6 Hz), 7.58 (2H, d,
δ 1.35 (3H, t, J = 7.1 Hz), 4.28 (2H,

Molecules 2019, 24, 2019
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J = 8.6 Hz), 7.68 (1H, d, J = 16 Hz), 8.23 (2H, d, J = 9.3 Hz) ppm. 13C NMR (CDCl3, 100 MHz):
δ 14.5,
60.8, 118, 118, 118.6, 120.7, 120.7, 126.2, 126.2, 130.2, 130.2, 131.8, 132.4, 143.4, 156.7, 162.6, 167 ppm. HR
ESI-MS (negative ion) m/z: 312.0869 (M − H)− (Calcd for C17H14NO5: 312.0872).
3.1.10. General Procedure of Preparation for Compounds 8a–c
The corresponding nitrobenzene cinnamic acid derivatives (7a: 300 mg, 7b: 500 mg, 7c: 250 mg,
1 eq) were dissolved in hot MeOH (15 mL). Zinc (522–1044 mg, 10 eq) and NH4Cl (641–1282 mg,
20 eq) were added to the solution. The reaction mixture was refluxed for 1 h. Then, the solution was
filtered and evaporated. After addition of water, the solution was extracted with EtOAc. The organic
layer was washed with brine and dried over MgSO4, filtered, and the solvents were evaporated in
vacuo. The residue was purified by silica gel chromatography (ϕ20 × 150 mm; Hexane/EtOAc, 75:25)
to yield 8a–c.
3.1.11. (E)-Ethyl 3-[4-(3-aminophenoxy)phenyl]acrylate (8a)
1
Colorless liquid, yield 92%. H NMR (CDCl3, 400 MHz):
δ 1.33 (3H, t, J = 7.3 Hz), 3.73 (2H, s),
4.26 (2H, q, J = 7.3 Hz), 6.34 (1H, d, J = 16.0 Hz), 6.36 (1H, t, J = 2.3 Hz), 6.42 (1H, ddd, J = 1.0, 2.3,
8.2 Hz), 6.47 (1H, ddd, J = 1.0, 2.3, 8.2 Hz), 6.99 (2H, d, J = 8.7 Hz), 7.12 (1H, t, J = 8.2 Hz), 7.47 (2H, d,
J = 8.6 Hz), 7.65 (1H, d, J = 16 Hz) ppm. 13C NMR (CDCl3, 125 MHz):
δ 14.5, 60.5, 106.4, 109.7, 111,
117.1, 118.7, 118.7, 129.4, 129.8, 129.8, 130.7, 144, 148.4, 157.5, 159.6, 167.3 ppm. HR ESI-MS (positive
ion) m/z: 284.1282 (M + H)+ (Calcd for C17H18NO3: 284.1287).
3.1.12. (E)-Ethyl 3-[4-(2-aminophenoxy)phenyl]acrylate (8b)
1
Colorless liquid, yield 97%. H NMR (CDCl3, 400 MHz):
δ 1.33 (3H, t, J = 7.1 Hz), 3.77 (2H, s),
4.26 (2H, q, J = 7.1 Hz), 6.33 (1H, d, J = 16.0 Hz), 6.75 (1H, td, J = 1.5, 8.0 Hz), 6.84 (1H, dd, J = 1.5,
7.9 Hz), 6.92 (1H, dd, J = 1.4, 8.0 Hz), 6.95 (2H, d, J = 8.7 Hz), 7.03 (1H, td, J = 1.5, 8.0 Hz), 7.47 (2H, d,
J = 8.6 Hz), 7.64 (1H, d, J = 16.0 Hz) ppm. 13C NMR (CDCl3, 100 MHz):
δ 14.5, 60.6, 116.9, 116.9, 117.1,
117.1, 119.1, 121.2, 125.9, 129.1, 130, 130, 139.1, 142.3, 144.1, 159.7, 167.4 ppm. HR ESI-MS (positive ion)
m/z: 284.1282 (M + H)+ (Calcd for C17H18NO3: 284.1287).
3.1.13. (E)-Ethyl 3-[4-(4-aminophenoxy)phenyl]acrylate (8c)
1
Colorless liquid, yield 82%. H NMR (CDCl3, 400 MHz):
δ 1.33 (3H, t, J = 7.1 Hz), 3.64 (2H, s),
4.25 (2H, q, J = 7.1 Hz), 6.31 (1H, d, J = 16 Hz), 6.69 (2H, d, J = 8.8 Hz), 6.88 (2H, d, J = 8.8 Hz), 6.90 (2H,
d, J = 8.7 Hz), 7.44 (2H, d, J = 8.7 Hz), 7.63 (1H, d, J = 16 Hz) ppm. 13C NMR (CDCl3, 100 MHz):
δ
14.5,
60.6, 116.4, 116.5, 116.5, 117.2, 117.2, 121.8, 121.8, 128.5, 129.8, 129.8, 143.5, 144.2, 147.7, 161.2, 167.4 ppm.
HR ESI-MS (positive ion) m/z: 284.1282 (M + H)+ (Calcd for C17H18NO3: 284.1287).
3.1.14. Ethyl 3-[4-(3-aminophenoxy)phenyl]propanoate (9)
8a (150 mg, 0.48 mmol) was diolved in EtOAc (5 mL). Following to addition of Pd/C (50 mg),
the reaction mixture was stirred at room temperature under a H2 atmosphere. After 6 h, Pd/C was
removed with celite and the filtrate was evaporated in vacuo. The residue was purified by silica gel
1
chromatography (
(CDCl3, 400 MHz):
ϕ
10
δ
×
300 mm; Hexane/EtOAc, 80:20) to afford
9
(127 mg, 0.45 mmol, 93%). H NMR
1.24 (3H, t, J = 7.1 Hz), 2.61 (2H, t, J = 7.5 Hz), 2.92 (2H, t, J = 7.5 Hz), 3.65 (2H, s),
4.13 (2H, q, J = 7.1 Hz), 6.30 (1H, t, J = 2.2 Hz), 6.37 (1H, ddd, J = 0.9, 2.3, 8.1 Hz), 6.40 (1H, ddd, J = 0.8,
2.2, 8.0 Hz), 6.93 (2H, d, J = 8.6 Hz), 7.07 (1H, t, J = 8.0 Hz), 7.15 (2H, d, J = 8.6 Hz) ppm. 13C NMR
(CDCl3, 100 MHz):
δ 14.4, 30.4, 36.3, 60.6, 105.5, 108.8, 110.1, 119.4, 119.4, 129.6, 129.6, 130.5, 135.6,
148.2, 155.6, 158.8, 173.1 ppm. HR ESI-MS (positive ion) m/z: 286.1450 (M + H)+ (Calcd for C17H20NO3:
286.1443).

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3.1.15. General Procedure of Preparation for Compounds 4a–e
Et3N (19–25
µL) in dry CH2Cl2 (5–8 mL) was added to the solution of corresponding amine (8a
:
100 or 40 mg, 8b: 100 mg, 8c: 75 mg,
9: 50 mg, 1 eq), and stirred at room temperature until dissolved.
Then, 2-chloro-5-nitrobenzoyl chloride (42–85 mg, 1.1 eq) or 3-nitrobenzoyl chloride (24 mg, 1.1 eq) was
added to the solution and stirred at room temperature for 18 h. After addition of water, the solution
was extracted with CHCl3. The organic layer was washed with brine and dried over MgSO4, filtered,
and the solvents were evaporated in vacuo. The residue was purified by silica gel chromatography
(ϕ10 × 300 mm; Hexane/EtOAc, 75:25) to yield 4a–e.
3.1.16. (E)-Ethyl 3-{4-[3-(2-chloro-5-nitrobenzamido)phenoxy]phenyl}acrylate (4a)
1
Yellow powder, yield 88%. H NMR (CDCl3, 400 MHz):
δ 1.32 (3H, t, J = 7.1 Hz), 4.23 (2H, q,
J = 7.1 Hz), 6.32 (1H, d, J = 16.0 Hz), 6.88 (1H, ddd, J = 1.6, 2.2, 7.7 Hz), 7.02 (2H, d, J = 8.6 Hz), 7.37 (1H,
t, J = 7.8 Hz), 7.41 (2H, m), 7.48 (2H, d, J = 8.7 Hz), 7.61 (1H, d, J = 16 Hz), 7.62 (1H, d, J = 8.9 Hz), 8.20
(1H, m), 8.22 (1H, dd, J = 2.7, 8.8 Hz), 8.53 (1H, d, J = 2.7 Hz) ppm. 13C NMR (CDCl3, 100 MHz): δ 14.3,
60.5, 111.5, 115.6, 116.2, 117.1, 118.8, 118.8, 125.1, 126, 129.6, 129.8, 129.8, 130.4, 131.6, 136.3, 137.6, 138.5,
143.7, 146.5, 157, 158.8, 162.3, 167.2 ppm. HR ESI-MS (negative ion) m/z: 465.0863 (M − H)− (Calcd for
C24H18N2O6Cl: 465.0853).
3.1.17. (E)-Ethyl 3-{4-[3-(3-nitrobenzamido)phenoxy]phenyl}acrylate (4b)
1
Colorless powder, yield 55%. H NMR (CDCl3, 400 MHz):
δ 1.32 (3H, t, J = 7.1 Hz), 4.23 (2H,
q, J = 7.1 Hz), 6.29 (1H, d, J = 16 Hz), 6.85 (1H, ddd, J = 0.7, 2.2, 8.2 Hz), 6.98 (2H, d, J = 8.7 Hz), 7.32
(1H, t, J = 8.1 Hz), 7.43 (2H, d, J = 8.7 Hz), 7.45 (2H, m), 7.58 (1H, d, J = 16 Hz), 7.64 (1H, t, J = 8.1 Hz)
8.24 (1H, dd, J = 1.1, 7.9 Hz), 8.34 (1H, ddd, J = 1.0, 2.2, 8.2 Hz), 8.68 (2H, m) ppm. 13C NMR (CDCl3,
100 MHz): δ 14.5, 60.8, 111.9, 116.2, 116.2, 117.2, 119, 119, 122.2, 126.6, 129.7, 130, 130, 130.2, 130.6,
133.8, 136.5, 139.2, 144.1, 148.3, 157.1, 159.1, 163.8, 167.5 ppm. HR ESI-MS (negative ion) m/z: 431.1250
(M − H)− (Calcd for C24H19N2O6: 431.1243).
,
3.1.18. (E)-Ethyl 3-{4-[2-(2-chloro-5-nitrobenzamido)phenoxy]phenyl}acrylate (4c)
1
Colorless powder, yield 99%. H NMR (CDCl3, 400 MHz):
δ 1.33 (3H, t, J = 7.1 Hz), 4.25 (2H,
q, J = 7.1 Hz), 6.35 (1H, d, J = 15.9 Hz), 6.99 (1H, m), 7.02 (2H, d, J = 8.6 Hz), 7.17 (1H, td, J = 1.6, 8.1
Hz), 7.27 (1H, m), 7.51 (2H, d, J = 8.6 Hz), 7.59 (1H, d, J = 8.8 Hz), 7.64 (1H, d, J = 16 Hz), 8.23 (1H,
dd, J = 2.7, 8.8 Hz), 8.57 (1H, dd, J = 1.4, 8.1 Hz), 8.58 (1H, s), 8.61 (1H, d, J = 2.7 Hz) ppm. 13C NMR
(CDCl3, 100 MHz):
δ 14.3, 60.5, 117.7, 118.3, 118.3, 118.9, 121.7, 125, 125.4, 125.9, 126.1, 129.3, 129.9,
129.9, 130.3, 131.7, 136, 137.3, 143.3, 145.2, 146.7, 158.2, 161.8, 166.9 ppm. HR ESI-MS (negative ion) m/z:
465.0863 (M − H)− (Calcd for C24H18N2O6Cl: 465.0853).
3.1.19. (E)-Ethyl 3-{4-[4-(2-chloro-5-nitrobenzamido)phenoxy]phenyl}acrylate (4d)
1
Colorless powder, yield 79%. H NMR (CDCl3, 400 MHz):
δ 1.33 (3H, t, J = 7.1 Hz), 4.24 (2H, q,
J = 7.1 Hz), 6.32 (1H, d, J = 16.0 Hz), 6.99 (2H, d, J = 8.6 Hz), 7.08 (2H, d, J = 8.9 Hz), 7.48 (2H, d, J = 8.8
Hz), 7.62 (1H, d, J = 16.0 Hz), 7.64 (1H, d, J = 8.8 Hz), 7.65 (2H, d, J = 8.9 Hz), 8.19 (1H, s), 8.24 (1H,
dd, J = 2.7, 8.8 Hz), 8.55 (1H, J = 2.7 Hz) ppm. 13C NMR (CDCl3, 100 MHz):
δ
14.5, 60.7, 117.2, 118.5,
118.5, 120.6, 120.6, 122.4, 122.4, 125.4, 126.1, 129.6, 130, 130, 131.8, 133.3, 136.7, 137.8, 144.0, 146.8, 153.6,
159.6, 162.5, 167.4 ppm. HR ESI-MS (negative ion) m/z: 465.0863 (M
465.0853).
−
H)− (Calcd for C24H18N2O6Cl:
3.1.20. Ethyl 3-{4-[3-(2-chloro-5-nitrobenzamido)phenoxy]phenyl}propanoate (4e)
1
Colorless powder, yield 86%. H NMR (CDCl3, 400 MHz):
δ 1.23 (3H, t, J = 7.1 Hz), 2.59 (2H, t,
J = 7.8 Hz), 2.89 (2H, t, J = 7.7 Hz), 4.09 (2H, q, J = 7.1 Hz), 6.78 (1H, ddd, J = 1.0, 2.2, 8.0 Hz), 6.94 (2H,
d, J = 8.6 Hz), 7.14 (2H, d, J = 8.6 Hz), 7.24 (1H, t, J = 2.1 Hz), 7.28 (1H, t, J = 8.0 Hz), 7.33 (1H, m), 7.57

Molecules 2019, 24, 2019
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(1H, d, J = 8.8 Hz), 8.16 (1H, dd, J = 2.7, 8.8 Hz), 8.39 (1H, s), 8.40 (1H, d, J = 2.7 Hz) ppm. 13C NMR
(CDCl3, 100 MHz): 14.4, 30.4, 36.1, 60.7, 110.7, 114.9, 115.3, 119.5, 119.5, 125, 126, 129.8, 129.8, 130.3,
δ
131.6, 136.1, 136.7, 137.9, 138.5, 146.5, 155.1, 158.3, 162.7, 173.2 ppm. HR ESI-MS (negative ion) m/z:
467.1017 (M − H)− (Calcd for C24H20N2O6Cl: 467.1010).
3.1.21. tert-Butyl N-(4-{2-[(4-methylbenzenesulfonyl)oxy]ethyl}phenyl)carbamate (12)
The solution of 11 (209 mg, 1 mmol) in dry CH2Cl2 (5 mL) was added to p-toluensulfonyl chloride
(210 mg, 1.1 mmol) with 4-dimethylaminopyridine (12 mg, 0.1 mmol) and Et3N (279 µL, 2 mmol) in
dry CH2Cl2 (7 mL) at 0 ◦C. Then, the reaction mixture was stirred at room temperature for 24 h. After
addition of water, the solution was extracted with CH2Cl2. The organic layer was washed with brine
and dried over MgSO4, filtered, and the solvents were evaporated in vacuo. The residue was purified
by silica gel chromatography (
81%). 1H NMR (CDCl3, 400 MHz):
J = 7.0 Hz), 6.64 (1H, s), 6.99 (2H, d, J = 8.5 Hz), 7.24 (2H, d, J = 8.5 Hz), 7.27 (2H, d, J = 8.5 Hz), 7.67
(2H, d, J = 8.5 Hz) ppm. 13C NMR (CDCl3, 100 MHz):
21.6, 28.3, 28.3, 28.3, 34.7, 70.7, 80.5, 118.7,
ϕ
20
×
150 mm; Hexane/EtOAc, 90:10) to afford 12 (293 mg, 0.81 mmol,
δ
1.51 (9H, s), 2.42 (3H, s), 2.87 (2H, t, J = 7.0 Hz), 4.16 (2H, t,
δ
118.7, 127.8, 127.8, 129.4, 129.4, 129.8, 129.8, 130.7, 132.9, 137.2, 144.6, 152.7 ppm. HR ESI-MS (positive
ion) m/z: 430.1073 (M + K)+ (Calcd for C20H25NO5SK: 430.1091).
3.1.22. tert-Butyl N-{4-[2-(4-formylphenoxy)ethyl]phenyl}carbamate (13)
K2CO3 (142 mg, 1 mmol) was added to a solution of 12 (250 mg, 0.69 mmol) in dry CH3CN
(7 mL) at room temperature. The reaction mixture was stirred at room temperature for 20 min.
p-hydroxybenzaldehyde (101 mg, 0.83 mmol) was added to the reaction mixture at room temperature.
The reaction mixture was refluxed for 18 h and cooled to room temperature. The solution was
added H2O, and extracted with CHCl3. The organic layer was washed with brine and dried over
MgSO4, filtered, and the solvents were evaporated in vacuo. The residue was purified by silica gel
chromatography (
1H NMR (CDCl3, 400 MHz):
6.98 (2H, d, J = 8.7 Hz), 7.20 (2H, d, J = 8.5 Hz), 7.32 (2H, d, J = 8.4 Hz), 7.81 (2H, d, J = 8.8 Hz),
9.87 (1H, s) ppm. 13C NMR (CDCl3, 100 MHz):
28.5, 28.5, 28.5, 35.1, 69.5, 81, 115.5, 115.5, 119.6, 119.6,
ϕ
20
×
150 mm; Hexane/EtOAc, 90:10) to afford 13 (220 mg, 0.69 mmol, quant.).
δ
1.51 (9H, s), 3.07 (2H, t, J = 7.0 Hz), 4.21 (2H, t, J = 7.0 Hz), 6.65 (1H, s),
δ
130.3, 130.3, 130.7, 132.8, 132.8, 133.1, 137.9, 153.8, 164.9, 192 ppm. HR ESI-MS (negative ion) m/z:
340.1522 (M − H)− (Calcd for C20H22NO4: 340.1549).
3.1.23. (E)-Ethyl 3-{4-[2-(4-{[(tert-butoxy)carbonyl]amino}phenyl)ethoxy]phenyl}acrylate (14)
(EtO)2P(O)CH2COOEt (144
µL, 0.72 mmol) was added to LiCl (31 mg, 0.72 mmol) and
1,8-diazabicyclo [5.4.0]undec-7-ene (200
µL) in dry CH3CN (4 mL), and stirred at room temperature for
1 h. Then 13 (150 mg, 0.48 mmol) in dry CH3CN (1 mL) was added to the solution and stirred at room
temperature for 18 h. After addition of water, the solution was extracted with EtOAc. The organic layer
was washed with brine and dried over MgSO4, filtered, and the solvents were evaporated in vacuo.
The residue was purified by silica gel chromatography (
14 (179 mg, 0.44 mmol, 91%). H NMR (CDCl3, 400 MHz):
ϕ
20
×
150 mm; Hexane/EtOAc, 90:10) to afford
1.32 (3H, t, J = 7.1 Hz), 1.51 (9H, s), 3.04
1
δ
(2H, t, J = 7.0 Hz), 4.14 (2H, t, J = 7.0 Hz), 4.25 (2H, q, J = 7.1 Hz), 6.30 (1H, d, J = 15.9 Hz), 6.54 (1H, s),
6.87 (2H, d, J = 8.8 Hz), 7.19 (2H, d, J = 8.5 Hz), 7.31 (2H, d, J = 8.4 Hz), 7.44 (2H, d, J = 8.7 Hz), 7.63
(1H, d, J = 15.9 Hz) ppm. 13C NMR (CDCl3, 100 MHz):
δ 14.3, 28.7, 28.7, 28.7, 34.9, 60.2, 68.8, 80.4,
114.8, 114.8, 115.7, 118.8, 118.8, 127.2, 129.4, 129.4, 129.6, 129.6, 132.5, 136.9, 144.2, 152.8, 160.5, 167.3
ppm. HR ESI-MS (positive ion) m/z: 450.1684 (M + K)+ (Calcd for C24H29NO5K: 450.1683).
3.1.24. (E)-Ethyl 3-{4-[2-(4-aminophenyl)ethoxy]phenyl}acrylate (15)
4N HCl/EtOAc (2 mL) was added to a solution of 14 (107 mg, 0.27 mmol) in EtOAc (1 mL) at
room temperature. The reaction mixture was stirred at room temperature for 18 h. Then, the solvents
1
were evaporated in vacuo to afford 15 (83 mg, 0.27 mmol, quant.). H NMR (DMSO-d6, 400 MHz):

Molecules 2019, 24, 2019
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δ
1.25 (3H, t, J = 7.1 Hz), 3.07 (2H, t, J = 6.6 Hz), 4.17 (2H, q, J = 7.2 Hz), 4.25 (2H, t, J = 6.6 Hz), 6.47
(1H, d, J = 16 Hz), 6.97 (2H, d, J = 8.8 Hz), 7.31 (2H, d, J = 8.4 Hz), 7.42 (2H, d, J = 8.3 Hz), 7.60 (1H, d,
J = 16 Hz), 7.65 (2H, d, J = 8.8 Hz), 10.2 (2H, s) ppm. 13C NMR (DMSO-d6, 100 MHz):
14.2, 34.2, 59.8,
δ
68, 114.9, 114.9, 114.9, 114.9, 115.5, 122.8, 126.7, 130.1, 130.1, 130.1, 130.1, 137.8, 144.1, 160.2, 166.4 ppm.
HR ESI-MS (positive ion) m/z: 312.1585 (M + H)+ (Calcd for C19H22NO3: 312.1600).
3.1.25. (E)-Ethyl 3-(4-{2-[4-(2-chloro-5-nitrobenzamido)phenyl]ethoxy}phenyl)acrylate (16)
Et3N (50 µL) in dry CH2Cl2 (2 mL) was added to the solution of 15 (40 mg, 0.12 mmol), and stirred
at room temperature until dissolved. Then, 2-chloro-5-nitrobenzoyl chloride (28 mg, 0.13 mmol) was
added to the solution and stirred at room temperature for 18 h. After addition of water, the solution
was extracted with CHCl3. The organic layer was washed with brine and dried over MgSO4, filtered,
and the solvents were evaporated in vacuo. The residue was purified by silica gel chromatography
1
(
δ
ϕ
10
×
300 mm; Hexane/EtOAc, 75:25) to afford 16 (38 mg, 77
µ
mol, 67%). H NMR (CDCl3, 400 MHz):
1.32 (3H, t, J = 7.1 Hz), 3.10 (2H, t, J = 6.8 Hz), 4.20 (2H, t, J = 6.8 Hz), 4.23 (2H, q, J = 7.1 Hz), 6.27
(1H, d, J = 16 Hz), 6.90 (2H, d, J = 8.7 Hz), 7.29 (2H, d, J = 8.4 Hz), 7.44 (2H, d, J = 8.7 Hz), 7.59 (1H, d,
J = 8.8 Hz), 7.60 (1H, d, J = 15.9 Hz), 7.61 (2H, d, J = 8.4 Hz), 8.19 (1H, dd, J = 2.7, 8.8 Hz), 8.41 (1H, s),
8.52 (1H, d, J = 2.7 Hz) ppm. 13C NMR (CDCl3, 100 MHz):
δ
14.3, 35.1, 60.3, 68.6, 114.8, 114.8, 115.7,
120.5, 120.5, 125, 125.7, 127.2, 129.7, 129.7, 129.7, 129.7, 131.4, 135.2, 135.6, 136.7, 137.7, 144.2, 146.4,
160.5, 162.3, 167.3 ppm. HR ESI-MS (negative ion) m/z: 493.1168 (M
493.1166).
−
H)− (Calcd for C26H22N2O6Cl:
3.2. Cell Line and Cell Culture
The human hepatocyte carcinoma cell line, HepG2 (RCB1886), was provided by the RIKEN BRC
through the National Bio-Resource project of the Ministry of Education, Culture, Sports, Science and
Technology (MEXT), Japan. HepG2 cells were maintained in Dulbecco’s modified Eagle’s medium
(Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum, 100 µg/mL penicilin,
and 100 units/mL streptomycin (Sigma) at 37 ◦C in a humidified 5% CO2 atmosphere.
3.3. Plasmids and Recombinant Protein
pGal4-human PPAR
γ
LBD and pGal4-human PPAR
γ
LBD-Cys285Ala were constructed as
previously described [14]. Recombinant PPAR
Oyama (Yamanashi University).
γLBD protein was kindly provided by Dr. Takuji
3.4. PPARγ Reporter Assay
The PPAR
pGal4-human PPAR
Promega, Madison, WI, USA) were transfected into HepG2 cells using Hily-max (Dojindo, Kumamoto,
Japan) and PPAR agonist activity was determined from the luciferase activity using the Dual-Luciferase
γ
reporter assay was carried out as previously reported [20] with slight modifications.
γ
LBD, pUAS-tk-luc and Renilla luciferase transfection control vector (pGL4.74,
γ
Reporter Assay System (Promega) according to the manufacturer’s protocol.
3.5. Rhodamine-Maleimide Assay.
Rhodamine-Maleimide assay was performed as previously described [14]. Briefly, recombinant
PPARγ LBD (2 µM) was mixed with various ligands at the indicated concentrations in 20 mM Tris-HCl,
pH8.0, 150 mM NaCl, and 1 mM tris(2-carboxylethyl)phosphine (TCEP). The mixture was incubated
for 30 min at room temperature. SDS was added to the reaction mixture to a final concentration of
0.5%, followed by rhodamine red C2 maleimide (Life Technologies, Carlsbad, CA, USA) at a final
concentration of 1 mM. After incubation at room temperature for 30 min, samples were treated with
SDS-PAGE sample buffer containing β
-mercaptoethanol for 30 min at 55 ◦C and were separated by

Molecules 2019, 24, 2019
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SDS-PAGE. Fluorescent signals were visualized using the LAS1000 plus image analyzer (Fujifilm,
Tokyo, Japan).
3.6. LC-MS/MS Detection of the Hybrid Ligand Modification of PPARγ
LC-MS/MS detection was performed as previously described [14]. Briefly, purified recombinant
PPARγ LBD (20 µM) was incubated with ligands (40 µM) in 20 mM Tris-HCl, pH8.0, 150 mM NaCl,
and 1 mM TCEP for 30 min. Reacted proteins were denatured in SDS sample buffer and subjected
to SDS-PAGE followed by Coomassie brilliant blue (CBB) staining. After removing CBB, the excised
gel slices were incubated with 25 mM NH4HCO3 with sequencing grade-modified trypsin at 37 ◦C
for 12 h. After in-gel digestion, extracted peptides were subjected to analysis with a Q Exactive Plus
(Thermo Fisher Scientific, Waltham, MA, USA).
3.7. Docking Studies
Molecular docking of the compounds against the human PPARγLBD was performed using
Molegro Virtual Docker ver. 6.0.1 (CLC bio, Aarhus, Denmark). The ligand structures were drawn
using Marvin Sketch 5.11.5. The protein models and compounds to be docked were imported into the
docking program according to the software’s instructions. Potential ligand-binding sites of proteins
were calculated using the Molegro cavity detection algorithm. For prediction of the binding site of
the compounds, the crystal structures of the human PPAR
γ LBD and GW9662 (PDB code 3B0R) was
used for docking of 4a , and . Because the chlorine atom in the GW9662 unit of the compounds is
,
c
d
eliminated by nucleophilic substitution, we docked the ligand using a structure in which the chlorine
atom of the compounds was substituted with a hydrogen atom. Taking into account this covalent bond
formation, constraint-based docking was performed according to the software’s instructions. Briefly,
the fifth carbon of the nitrobenzene ring in the ligand was constrained to the sulfur atom of the thiol
group in Cys285 and then the constrained ligand was docked to the crystal structure of the human
PPARγ LBD.
3.8. Statistics
All data are depicted as means
and accepted at p < 0.05.
±
SD. Statistical significance was determined by Student’s t-test
4. Conclusions
In conclusion, we synthesized new derivatives of the covalent agonist
3 using an improved
synthetic protocol, allowing us to access a variety of compounds with a simplified linker moiety and
a different connection site between GW9662 and the cinnnamate unit. A cell-based assay revealed
that the synthesized ligands showed distinct profiles of transactivation of the PPARγ. Compounds
4a and 4c, in which cinnamate was linked with a diphenyl ether linker at the 20 or 30 position of
the aniline ring in GW9662, showed about 3-fold more potent agonist activities than the previously
synthesized compound 3, indicating that the close proximity of GW9662 and cinnamate may improve
the binding affinity. In contrast, compounds 4d and 16, in which the cinnamate unit was placed
at the 40 position of the aniline ring, exhibited no agonistic activity. By using docking simulation,
we reasoned that the different activation profiles are likely to depend on the interaction between the
cinnamate moiety and the Ω-loop region in the PPARγ LBD. Interestingly, docking studies predicted
that the synthesized compounds may mimic the binding modes of endogenous covalent ligands like
oxidized fatty acids with corresponding activity profiles. Because we have not yet fully investigated
what cellular responses are caused by the formation of a covalent bond with Cys285, our synthesized
ligands can serve as chemical probes to assess these biological questions. We further characterized the
synthesized compounds and found that ligands 4a and 4c partially activated PPAR
manner dependent on covalent modification with the Cys285 residue in the PPAR
of 4a and 4c to form a covalent bond could also result in two ligands being anchored in the LBP, causing
γ
transcription in a
γ
LBD. The abilities

Molecules 2019, 24, 2019
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prolonged transactivation. This feature reflects one of the advantages of covalent drugs, including
prolonged duration of action and less-frequent drug dosing [27], although the safety of compounds
that participate in covalent modification needs to be clarified using an in vivo model. Further biological
and pharmacological studies will enhance the development of new-types of PPAR
ability to form covalent bonds.
γ ligands with the
Supplementary Materials: The Supplementary Materials are available online.
Author Contributions: Conceptualization, Y.U., and Y.M.; methodology, Y.U., H.K.; validation, Y.U., H.K., A.S.A.A.,
and Y.M.; investigation, Y.U., H.K., A.S.A.A., and Y.M.; resources, Y.K., M.N., H.I., and Y.M.; writing—original
draft preparation, Y.U. and Y.M.; writing—review and editing, Y.U., Y.K., M.N., H.I., and Y.M.; supervision, Y.M.;
funding acquisition, Y.M.
Funding: This study was funded by Grants-in-Aid for Young Scientists (B) from the Japan Society for the
Promotion of Science [KAKENHI, Grant no. 17K13264 to Y.M.].
Acknowledgments: We thank Y. Watanabe (Kyoto University) for kind support with LC-FT-MS measurements.
We are grateful to T. Oyama (Yamanashi University) for providing purified recombinant PPARγ LBD protein.
This study was supported by Grants-in-Aid for Young Scientists (B) from the Japan Society for the Promotion of
Science [KAKENHI, Grant no. 17K13264 to Y.M.].
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 4a, 4b, 4c, 4d, 4e, and 16, as well as the docking data, are
available from the authors.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).