Extract
Note: Please read the complete
full text with Figures and Tables at
Introduction
Hypertriglyceridemia and hypercholesterolemia are 2
major risk factors for coronary heart disease
(CHD)[1_3], which remains the leading cause of death in the developed countries.
Fibrate drugs (Figure 1) have been widely used for the
clinical treatment of dyslipidemia by lowering serum
triglycerides and raising HDL cholesterol (HDLc), and remain the
first choice for the treatment of severe hypertriglyceridemia[4_6]. Several studies have provided evidence that the
hypolipi-demic effect of fibrate drugs is attributed to the
activation of peroxisome proliferator-activated receptors
(PPAR)α[7,8], one of the 3 isoforms (α,
γ, and δ) of the PPAR[9]. PPARα is highly expressed in the liver, heart, and muscle
with a range of fatty acids in its natural ligands. In the human
body, the activation of PPARα increases the clearance of
the TG-rich, very low-density lipoprotein (VLDL) via the
reduction of plasma levels of
ApoCIII8[10_12] and the upregula-tion of
ApoA1, which is the principal lipoprotein component
of HDL[12_14]. The elevation of HDLc levels observed with
fibrates arises partially due to the transcriptional induction
of the major HDL apolipoproteins, apoA-I and
apoA-II[15]. Regulating PPARα is also implicated as an
anti-atherosclerosis approach[16]. Agonists have been shown to
downregu-late the expression of VCAM-1, to inhibit
NF-κB and AP-1, and to mediate the reduction of plasma levels of
interleukin-6, fibrinogen, and C-reactive
protein[17].
Although fibrates are ligands for the PPARα receptor,
they only show weak agonist activity as shown in Table
1[18,19]. More potent human PPARα agonists are expected to
provide a better tool for studying the biology of
PPARα and a superior clinical profile for therapeutic intervention in
dyslipidemia and other metabolic
disorders[20,21]. These reasons prompted us to discover novel small molecules as
PPARα agonists with higher affinities.
Chalcones, with a common 1,3-diphenyl-2-propen-1-one
framework (Figure 1), have been known for over a century.
Natural chalcones exist mainly as petal pigments and have
also been found in the heartwood, bark, leaf, fruit, and root
of a variety of trees and plants. Chalcone-containing plants,
such as the Glycyrrhiza species have been used as folk
remedies for a long time. Naturally occurring and synthetic
chalcone compounds have shown interesting biological
activities as shown by antioxidant, anti-inflam-matory,
anti-cancer, or anti-infective
agents[22].
In this paper, we constructed a novel framework with the
combination of the classical fibrate "head group" (Figure
2)[23], a linker with appropriate length and a chalcone. We designed
37 compounds and they were all identified by the
structure-based virtual screening
approach[21], based on the crystal structure of
PPARα. According to the scores of virtual screening, structural similarity, and synthetic complexity, 6
compounds were selected, synthesized, and bioassayed; all
of them showed high activation activities, with 50% effective
concentration (EC50) values less than 2.5 µmol/L. Compounds
4d, 4e, and 4h are the most prominent, with
EC50 values of 0.37 µmol/L, 0.63 µmol/L, and 0.06 µmol/L, respectively.
Materials and methods
Design of analogues of compounds for virtual screening
In the first round of virtual screening, we designed
compounds DE001-DE013 (Table 2) by keeping the structures of
the fibrate "head group" (Figure 2) and chalcone, and
changed the length and the structure of the linker.
According to the result of the virtual screening (Table 2), we chose
the ethoxy group (the linker of compound DE001) as the
linker of our target compounds; the binding free energy of
compound DE001 with PPARα is -9.18 kcal/mol, which is the
most prominent among compounds DE001-DE013. In the
second round of virtual screening, we designed compounds by
keeping the fibrate "head group" and the ethoxy linker and
changed the structures and substituents of the chalcones.
By substituting position 3' of phenyl ring A of chalcone with
the ethoxy linker and introducing electronic, hydrophobic,
and steric bulky groups to phenyl ring B, we obtained
compounds DE014-DE023; by substituting the 4' position of
phenyl ring B of chalcone with the ethoxy linker and
introducing electronic, hydrophobic, and steric bulky groups to
phenyl ring A, we obtained compounds DE024-DE031; by
introducing the ethoxy linker to positions 2' and 5' of phenyl
ring A, we obtained compounds DE032-DE033; and by
introducing the ethoxy linker to positions 3, 5, 2, and 6 of phenyl
ring B, we obtained compounds DE034-DE037.
Virtual screening by molecular docking The crystal
structure of PPARα complexing with GW409544
(1K7L)[23] recovered from the Brookhaven Protein Data Bank was used
as the target for molecular docking. The docking
calculations of compounds with PPARα were performed with
the AutoDock 3.0 program (Morris et al, The Scripps Research
Institute, La Jolla, CA, USA). Up to 20 different docking
solutions were obtained for each molecule. All docking
conformations in the different receptor conformers were then
ranked with the AutoDock 3.0 scoring
function[24]. For each compound, only the conformation with the highest score
was chosen for analysis.
Synthetic procedures Figure 3 depicts the sequence of
reactions that led to the preparation of compounds 4a_h.
Compound 5 was converted to 6 by refluxing with
2-bromo-2-methyl-propionic acid ethyl ester in
CH3CN in the presence of
K2CO3[25],
and 6 was condensed with
p-hydroxy-benzaldehyde in tetrahydrofuran (THF) in the presence of
PPh3 and diisopropyl azodicarboxylate (DIAD)(Mitsunobu
reaction)[26], giving the key intermediate 7. Similarly,
compound 8 was converted to 4a and 4c through the Mitsunobu
reaction, then 4a and 4c were hydrolyzed using LiOH at room
temperature to afford the target compounds 4b and 4d,
respectively. Compound 9 was condensed with compound
7 by refluxing in CH3OH with 50% aq KOH, affording the
target compounds 4e_h[27].
Biological assay The PPARα activation activities of
the target compounds were monitored through published
methods[28,29].
The response element (UASGAL4*5) was cloned upstream of the Pgl2-sv 40-Luc reporter (Promega, Madison,
WI, USA), which contains the Simian virus early promoter
for luciferase assay. GAL4 fusions were made by fusing
human PPARc1 or PPARα ligand-binding domains (amino
acids: 174_475) to the C-terminal end of the yeast GAL4
DNA-binding domain (amino acids: 1_147) of the pM1 vector. The
pAdVAntage (Promega, Madison, WI, USA) vector was used
to enhance luciferase expression.
HEK 293T cells were grown in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 10% fetal
bovine serum (FBS) at 37 °C in 5%
CO2. At 1 d prior to trans-fection, the cells were plated to 50%_60% confluence in
DMEM containing 10% delipidated FBS (DMEM-DFBS). The
cells were transfected by Superfect (QIAGEN, Valencia, CA,
USA) as per the manufacturer's protocol. At 3 h after
trans-fection, the reagent was removed and the cells were
maintained in DMEM-DFBS. At 42 h after transfection, the cells
were placed in phenol red-free DMEM-DFBS, treated for
24 h with the test compounds, and then collected with cell
culture lysis buffer. Luciferase activity was monitored using
the luciferase assay kit (Tropix, Bedford, MA, USA)
according to the manufacturer's instructions. Light emission was
read in a Labsystems ascent fluoroskan reader (Flow
Labora-tories, Inc., Costa Mesa, CA, USA). To measure
galactosidase activity to normalize the luciferase data, 50 µL
supernatant from each transfection lysate was transferred to a new
microplate. Galactosidase assays were performed in the
microwell plates using a kit from Promega and read in a
microplate reader. The agonist rates were calculated
according to the activation data in 10 µmol/L compounds, and the
EC50 values were estimated by fitting the activation data to a
dose-dependent curve using a logistic derivative equation.
Results
Compound identification by virtual screening
In order to avoid the synthesis of meaningless compounds and
improve the efficiency of drug discovery, molecular
modeling was employed to prescreen the designed compounds.
Docking calculation was performed to rank the designed
com-pounds. The binding free energy for each compound with
PPARα is shown in Table 2. In most cases, the binding free
energy between the designed compounds and PPARα shows
a negative value less than -6 kcal/mol, indicating strong
binding activity. As mentioned earlier, we chose the ethoxy group
(the linker of compound DE001) as the linker for the target
compounds according to the virtual screening results of
compounds DE001-DE013 (Table 2). Similarly, compound
DE014 is the most potent in this class, based on the virtual
screening results of compounds DE014-DE023; compounds
DE024-DE027 are the most potent and most representative
among compounds DE024-DE031 according to the virtual
screening result; and for compounds DE032-DE037, some
also show excellent activities in terms of free energy scores.
However, considering the binding free energy value,
structural similarity, and synthetic complexity, we chose
compounds DE001, DE014, and DE024-DE027 for further
evaluation. As many drug candidates failed in clinical trials
due to their poor ADMET properties, a prior consideration
of their drug likeness would be of vital importance. The
computational results of the drug likeness in Table 3 show that
the 6 compounds meet most of the requirements in terms of
the classic Lipinski rule-of-five. In addition, they satisfy 6 of
7 drug-like criteria developed by Zheng et
al and Chen
et al[30,31], with a high satisfaction rate of 0.9. As a result, the
6 compounds were finally chosen for synthesis and
bioassay.
Analogues design and synthesis According to the
results of the virtual screening, 6 compounds (4b, and
4d_4h) were synthesized; their chemical structures are shown in
Table 4. These compounds were synthesized through the
route outlined in Figure 3, and the details of synthetic
procedures and structural characterizations are described in
Appendix I.
Activation activity towards PPARα To determine the
exact potency of the designed compounds, they were
investigated in concentration-response studies and their
EC50 values are also shown in Table 4. All showed
EC50 values less than 2.5 µmol/L; 4d, 4e, and 4h are the most prominent, with
EC50 values of 0.37 µmol/L, 0.63
µmol/L, and 0.06 µmol/L, respectively.
Discussion
To gain structural information for further structural
optimization, the 3-D binding models of the designed
compounds to PPARα were generated based on the docking
simulation. Figure 4A shows the superimposition of the 6
compounds within the binding pocket of PPARα. These
binding models for the 6 compounds match that of the agonist
GW409544 in the crystal structure[23] very well, especially
the carboxyl head group. There is a strong electric field within
the place where the head group locates, which forms strong
electrostatic interactions to fix the head group, as shown
from the polar motif of the surface in Figure 4 (4B_4D).
However, the orientations of hydrophobic tails are different;
they can be classified into 3 major conformations: tail up, tail
down, and tail in the middle. The 3 most potent agonists 4d,
4e, and 4h represent the 3 major binding orientations of the
hydrophobic tails, as shown in Figure 4 (4B_4D). This could
be due to the fact that both the upper and lower arms of the
"Y"-shaped pocket are hydrophobic, as shown from the
hydropathicity surface in Figure 4 (4B_4D). Moreover, this
result is consistent with the studies of Xu et
al[23], who proved that the EPA in the crystal structure of
PPARα also revealed the tail-up and tail-down configurations.
Subse-quently, we then mapped the detailed interaction (H-bonds
and hydrophobic interaction) between the 3 most potent
agonists 4d, 4e, and 4h and PPARα, as shown in Figure 5.
Similarly, their head groups formed quite a few H-bonds with
Ser280, Tyr314, His440, and Tyr464 in the binding pocket of
PPARα, revealing a highly polar space under the AF-2 helix.
Respectable hydrophobic interactions have been found
between the tail of agonists and residues in the pocket, and the
3 agonists share some hydrophobic interactions with
residues, such as Leu321, Val332, Phe273, and Cys276.
However, some other hydrophobic interactions are different
from each other because the orientations of the
hydrophobic tails of the 3 agonists are different. For example, agonist
4d interacts with Met220, Leu331, and Met330 because its
tail is in the upper arm of pocket while 4e does not, which
may be due to its binding within the lower arm. This series of
analogues interact with PPARα through a network of
H-bonds involved the head acid group and numerous
hydrophobic interactions by the tail.
In summary, 3 potent compounds (4d, 4e, and 4h) were
discovered by using a structure-based virtual screening
approach in conjunction with chemical synthesis and
bioassay. We constructed a novel framework with the
combination of the classical fibrate "head group" (Figure 2), a
linker with appropriate length, and a chalcone. We designed
37 compounds on the basis of the novel framework; these
compounds were identified by the structure-based virtual
screening approach based on the crystal structure of
PPARα. According to the scores of the virtual screening, structural
similarity, and synthetic complexity, 6 compounds were
chosen for synthesis and bioassay. Compounds 4d, 4e, and 4h
were proven to be potent PPARα agonists, with
EC50 values of 0.37 µmol/L, 0.63
µmol/L, and 0.06 µmol/L, respectively. To explore the binding characteristics of the designed
compounds for further structural optimization, the binding
models of compounds 4d, 4f, and 4i with PPARα were constructed
based on molecular docking simulation. Their binding
models are consistent with the binding model of agonist
GW409544 with the crystal structure of PPARα, which
demonstrates that our drug design strategy is reasonable and
successful.
Appendix I
The reagents (chemicals) were purchased from Lancaster,
Acros and Shanghai Chemical Reagent Company, and used
without further purification. Analytical thin-layer
chromatography (TLC) was HSGF 254 (150_200 µm thickness, Yantai
Huiyou Company, China). Yields were not optimized.
Melting points were measured in a capillary tube on a SGW X-4
melting point aPPARαtus without correction. Nuclear
magnetic resonance (NMR) spectra were performed on a Brucker
AMX-300 NMR (IS as TMS). Chemical shifts were reported
in parts per million (ppm, δ) downfield from tetramethylsilane.
Proton coupling patterns were described as singlet (s),
doublet (d), triplet (t), quartet (q), multiplet (m), and broad (br).
Low- and high-resolution mass spectra (HRMS) were given
with electric (EI), electrospray, and matrix-assisted laser
desorption ionization, produced by Finnigan MAT-95,
LCQ-DECA spectrometer and IonSpec 4.7 Tesla.
2-(4-[2-Hydroxy-ethyl]-phenoxy)-2-methyl-propionic
acid ethyl ester (6) 4-(2-Hydroxy-ethyl)-phenol
(6) (6.0 g, 43.2 mmol) was dissolved in
CH3CN (100 mL), then 2-bromo-2-methyl-propionic acid ethyl ester (7.2 mL, 48.0 mmol) and
K2CO3 (12 g, 87.0 mmol) were added in sequence. The
reaction mixture was refluxed overnight, and
K2CO3 was removed by filtration. Then the filtrate was condensed in vacuo and
purified by flash chromatography on silica gel, eluted with a
mixture of EtOAc/petroleum ether (1:4, v/v), to give 6 as a
yellow oil. Yield: 45.9%. 1H NMR
(CDCl3, 300 MHz): δ 1.25
(t, J=6.9 Hz, 3H), 1.57 (s, 6H), 2.79 (t,
J=6.6 Hz, 2H), 3.80 (t, J=6.6 Hz, 2H), 4.22 (q,
J=6.9 Hz, 2H), 6.79 (d, J=6.9 Hz, 2H),
7.08 (d, J=6.6 Hz, 2H); EI_MS m/z 252
(M+), 107 (100%); HRMS (EI) m/z calcd.
C14H20O4
(M+) 252.1362, found 252.1357.
2-(4-[2-{4-Formyl-phenoxy}-ethyl]-phenoxy)-2-methyl-
propionic acid ethyl ester (7) A solution of
2-(4-[2-hydroxy-ethyl]-phenoxy)-2-methyl-propionic acid ethyl ester (6; 1.2 g
4.8 mmol) and p-hydroxybenzaldehyde (600 mg, 4.8 mmol) in
THF (80 mL, redistilled from sodium) was treated at 0 °C with
a preformed solution of PPh3 and DIAD, which was formed
by the addition of DIAD (1.8 mL, 9.3 mmol) to a solution of
PPh3 (2.5 g, 9.3 mmol) in THF (50 mL) at 0 °C. The reaction
mixture was stirred at room temperature overnight, condensed
in vacuo, purified by flash chromatography on silica gel, and
eluted with a mixture of EtOAc/petroleum ether (1:8,
v/v) to give 7 as a yellow oil. Yield:
29.7%. 1H NMR (CDCl3, 300 MHz):
δ1.25 (t, J=7.5 Hz, 3H), 1.59 (s, 6H), 3.05 (t,
J=7.2 Hz, 2H), 4.22 (m, 4H), 6.79 (d,
J=6.6 Hz, 2H), 6.99 (d, J=9.0 Hz, 2H), 7.13 (d,
J=8.7 Hz, 2H), 7.81 (d, J=6.9 Hz, 2H), 9.88 (s, 1H); EI-MS
m/z 356 (M+), 121 (100%); HRMS (EI)
m/z calcd.
C21H24O5
(M+) 356.1624, found 356.1644.
2-Methyl-2-(4-[2-{4-oxo-2-phenyl-chroman-7-yloxy}-
ethyl]-phenoxy)-propionic acid ethyl ester
(4a) A solution of compound 6 (252 mg, 1.0 mmol) and
7-hydroxy-2-phenyl-chroman-4-one (240 mg, 1.0 mmol) in THF (30 mL, redistilled
from sodium) was treated at 0 °C with a preformed solution
of PPh3 and DIAD, which was formed by the addition of
DIAD (400 µL, 2.0 mmol) to a solution of
PPh3 (524 mg, 2.0 mmol) in THF (30 mL) at 0 °C. The reaction mixture was stirred
at room temperature overnight, condensed in vacuo,
purified by flash chromatography on silica gel, and eluted with a
mixture of EtOAc/petroleum ether (1:8, v/v), to give 4a as a
yellow oil. Yield: 55.3%. 1H NMR
(CDCl3, 300 MHz): δ 1.25 (t, J=7.2 Hz, 3H), 1.62 (s, 6H), 2.98 (m, 4H), 4.16
(t, J=7.2 Hz, 2H), 4.24 (q, J=7.2 Hz, 2H), 5.45 (m, 1H), 6.49 (s, 1H), 6.59
(dd, J=9.0 Hz and 2.4 Hz, 1H), 6.78 (d, J=8.4 Hz, 2H), 7.14
(d, J=8.4 Hz, 2H), 7.40 (m, 5H), 7.84 (d,
J=9.0 Hz, 1H); EI_MS m/z 474
(M+), 121 (100%); HRMS (EI) m/z calcd.
C29H30O6
(M+) 474.2042, found 474.2034.
2-(4-[2-{3-Hydroxy-4-(3-phenyl-acryloyl)-phenoxy}-
ethyl]-phenoxy)-2-methyl-propionic acid (4b)
Compound 4a (85 mg, 0.18 mmol) in a solvent of
CH3OH (10 mL) and THF (10 mL) was added to 1 mol/L aqueous LiOH solution (2.5
mL, 2.5 mmol), and the mixture was stirred at room
temperature for 6 h. CH3OH was removed in vacuo and water was
added to the mixture. Then 10% HCl was added until pH=3.
The mixture was then extracted with EtOAc (5 mL×3). The
combined organic layer was dried, filtered, and condensed
in vacuo to afford 4b as a yellow oil. Yield: 77.5%.
1H NMR (DMSO-d6, 300 MHz):
δ 1.57 (s, 6H), 3.08 (t, J=6.6 Hz, 2H),
4.21 (t, J=6.6 Hz, 2H), 6.46 (m, 2H), 6.93
(d, J=8.4 Hz, 2H), 7.21 (d, J=8.4 Hz, 2H), 7.44 (m, 3H), 7.65 (m, 3H), 7.84 (m, 2H); EI-MS
m/z 446 (M+), 121 (100%); HRMS (EI)
m/z calcd.
C27H26O6
(M+) 446.1729, found 446.1710.
2-Methyl-2-(4-[2-{4-oxo-2-phenyl-chroman-6-yloxy}-
ethyl]-phenoxy)-propionic acid ethyl ester
(4c) In the same manner as described in the preparation of 4a,
4c was prepared from
2-(4-[2-hydroxy-ethyl]-phenoxy)-2-methyl-propionic acid ethyl ester (6) and
6-hydroxy-2-phenyl-chroman-4-one as a yellow oil. Yield: 49.8%;
1H NMR (CDCl3, 300 MHz): δ 1.18 (t,
J=7.2 Hz, 3H), 1.49 (s, 6H), 2.84 (m, 4H), 4.03
(t, J=7.2 Hz, 2H), 4.12 (q, J=7.2 Hz, 2H), 5.56 (m, 1H), 6.72
(d, J=8.4 Hz, 2H), 7.04 (d, J=8.4 Hz, 1H), 7.20 (m, 4H), 7.42 (m, 3H), 7.51 (d,
J=7.5 Hz, 2H); EI_MS m/z 474
(M+), 121 (100%); HRMS (EI) m/z calcd.
C29H30O6
(M+) 474.2042, found 474.2027.
2-(4-[2-{4-Hydroxy-3-(3-phenyl-acryloyl)-phenoxy}-
ethyl]-phenoxy)-2-methyl-propionic acid (4d) In
the same manner as described in the preparation of
4b, 4d was prepared from
compound 4c as a yellow oil. Yield: 75.8%;
1H NMR (DMSO-d6, 300 MHz):
δ 1.60 (s, 6H), 3.05 (t, J=6.9 Hz, 2H), 4.14 (m, 2H), 6.92 (m, 2H), 6.99 (m, 1H), 7.10 (m, 1H), 7.21
(m, 2H), 7.26 (m, 1H), 7.42 (m, 3H) 7.53 (m, 1H) 7.65 (m, 2H)
7.89 (m, 1H); EI_MS m/z 446 (M+), 121 (100%); HRMS (EI)
m/z calcd.
C27H26O6
(M+) 446.1729, found 446.1742.
2-(4-[2-{4-(3-[2-Hydroxy-phenyl]-3-oxo-propenyl)-
phenoxy}-ethyl]-phenoxy)-2-methyl-propionic acid (4e)
1-(2-hydroxy-phenyl)-ethanone (56 mg, 0.41 mmol) and
2-(4-[2-{4-formyl-phenoxy}-ethyl]-phenoxy)-2-methyl-propionic
acid ethyl ester (7; 158 mg, 0.44 mmol) was dissolved in
CH3OH (15 mL). To this solution,
H2O (1 mL) and 50% aq. KOH (1 mL) were added successively, and the mixture was
refluxed for 1 h. After cooling, the solution was poured onto
crushed ice and acidified with 6 mol/L HCl. Then the mixture
was extracted with EtOAc (5 mL×3). The combined organic
layer was dried, filtered, and condensed in vacuo.
Then the residue was purified by preparative TLC and eluted with a
mixture of CH3OH/acetone/petroleum ether (1:30:30,
v/v) to give 4e as a yellow oil. Yield: 3.8%.
1H NMR (DMSO-d6, 300 MHz):
δ 1.60 (s, 6H), 3.07 (t, J=6.6 Hz, 2H), 4.20
(t, J=6.6 Hz, 2H), 7.00 (m, 6H), 7.21 (m, 2H), 7.51 (m, 4H), 7.91 (m, 2H);
EI_MS m/z 446 (M+), 121 (100%); HRMS (EI)
m/z calcd.
C27H26O6
(M+) 446.1729, found 446.1740.
2-[4-(2-{4-[3-(2-Hydroxy-5-methyl-phenyl)-3-oxo-
propenyl]-phenoxy}-ethyl)-phenoxy]-2-methyl-propionic acid
(4f) In the same manner as described in the preparation of
4e, 4f was prepared from
1-(2-hydroxy-5-methyl-phenyl)-ethanone and
2-(4-[2-{4-formyl-phenoxy}-ethyl]-phenoxy)-2-methyl-propionic acid ethyl ester (7) as a yellow oil. Yield:
11.0%; 1H NMR
(DMSO-d6, 300 MHz): δ 1.58 (s, 6H), 2.31 (s,
3H), 3.06 (t, J=6.9 Hz, 2H), 4.18 (t,
J=6.9 Hz, 2H), 6.90 (m, 5H), 7.16 (d,
J=8.4 Hz, 2H), 7.28 (m, 1H), 7.59 (m, 4H), 7.84 (m, 1H);
EI_MS m/z 460 (M+), 298 (100%); HRMS (EI)
m/z calcd.
C28H28O6
(M+) 460.1886, found 460.1893.
2-[4-(2-{4-[3-(2-Hydroxy-5-methoxy-phenyl)-3-oxo-
propenyl]-phenoxy}-ethyl)-phenoxy]-2-methyl-propionic acid
(4g) In the same manner as described in the preparation of
4e, 4g was prepared from
1-(2-hydroxy-5-methoxy-phenyl)-ethanone and
2-(4-[2-{4-formyl-phenoxy}-ethyl]-phenoxy)-2-methyl-propionic acid ethyl ester (7) as a yellow oil. Yield:
34.6%; 1H NMR
(DMSO-d6, 300 MHz): δ 1.59 (s, 6H), 3.06 (t,
J =6.6 Hz, 2H), 3.84 (s, 3H), 4.21 (t,
J=6.6 Hz, 2H), 6.96 (m, 5H), 7.11 (m, 3H), 7.35 (m, 1H), 7.43 (m, 1H), 7.58 (d, J=8.7 Hz, 2H),
7.82 (m, 1H); EI_MS m/z 476 (M+), 121 (100%); HRMS (EI)
m/z calcd.
C28H28O7
(M+) 476.1835, found 476.1836.
2-[4-(2-{4-[3-(5-Chloro-2-hydroxy-phenyl)-3-oxo-
propenyl]-phenoxy}-ethyl)-phenoxy]-2-methyl-propionic acid
(4h) In the same manner as described in the preparation of
4e, 4h was prepared from
1-(5-chloro-2-hydroxy-phenyl)-ethanone and
2-(4-[2-{4-formyl-phenoxy}-ethyl]-phenoxy)-2-methyl-propionic acid ethyl ester (7) as a yellow oil. Yield:
21.6%; 1H NMR
(DMSO-d6, 300 MHz): δ 1.59 (s, 6H), 3.05
(t, J=6.9 Hz, 2H), 4.20 (t, J=6.9 Hz, 2H), 6.92 (m, 5H), 7.21 (d,
J=8.4 Hz, 2H), 7.43 (m, 2H), 7.60 (d,
J=9.0 Hz, 2H), 7.85 (m, 2H); EI-MS m/z
480 (M+), 121 (100%); HRMS (EI)
m/z calcd.
C27H25ClO6
(M+) 480.1340, found 480.1335.
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