Stilbene analogs as inducers of apolipoprotein-I transcription

Article abstract of DOI:10.1016/j.ejmech.2009.12.076

Based on the naturally occurring stilbene, Resveratrol, a series of novel stilbene derivatives were synthesized, which have the ability to induce the expression of the ApoA-I gene. Several compounds equally or more potent than Resveratrol were identified. trans-4,4′-Dihydroxy-2-methoxystilbene was the most potent (4.6× more potent than Resveratrol). These compounds provide an early lead into new drugs to treat atherosclerosis.

Full text of DOI:10.1016/j.ejmech.2009.12.076

European Journal of Medicinal Chemistry 45 (2010) 2018–2023
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Stilbene analogs as inducers of apolipoprotein-I transcription
,
a
a
a
b
Henrik C. Hansen a , Fabrizio S. Chiacchia , Reena Patel , Norman C.W. Wong , Vladimir Khlebnikov ,
*
Renata Jankowska b, Kalpesh Patel b, M. Madhava Reddy b
a Resverlogix Corp., Suite 202, 279 Midpark Way SE, Calgary, Alberta, T2X 1M2, Canada
b NAEJA Pharmaceuticals Inc., 4290-91A Street, Edmonton, Alberta, T6E 5V2, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on the naturally occurring stilbene, Resveratrol, a series of novel stilbene derivatives were
synthesized, which have the ability to induce the expression of the ApoA-I gene. Several compounds
equally or more potent than Resveratrol were identified. trans-4,40-Dihydroxy-2-methoxystilbene was
the most potent (4.6ꢀ more potent than Resveratrol). These compounds provide an early lead into new
drugs to treat atherosclerosis.
Received 13 October 2009
Received in revised form
26 December 2009
Accepted 30 December 2009
Available online 14 January 2010
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
Stilbene derivatives
Transcription
High-density lipoprotein
Apolipoprotein-I
Cholesterol transport
Atherosclerosis
1. Introduction
(Fig. 1) [8], a stilbene, and Genistein [9], an isoflavonoid, have been
linked to increased HDL and ApoA-I levels.
Recent advances in understanding the preventive role of high-
density lipoprotein (HDL) and apolipoproteinA-I (ApoA-I) has
resulted in considerable interest to develop therapies capable of
elevating ApoA-I/HDL [1,2]. HDL is responsible for the transport of
cholesterol from peripheral tissues to the liver for excretion as fecal
sterols and bile acids, preventing the build-up of atherosclerotic
plaque. Epidemiological data has demonstrated an inverse
relationship between blood levels of HDL and the incidence of
clinically significant atherosclerosis [3,4]. ApoA-I is a critical
component of HDL particles [5]. Increasing the levels of ApoA-I, e.g.
by infusion, results in a significant reduction in coronary plaque
over a short period of time [6], suggesting that increasing the
production of endogenous ApoA-I would be an effective way to
treat atherosclerosis.
The protective role of polyphenols, and Resveratrol in particular,
prompted us to investigate stilbene analogs capable of upregulating
the transcription of ApoA-I.
2. Results and discussion
Early on in the program, the stilbene trans-4,40-dihydroxy-
stilbene (2, Fig. 1) was identified from our screening library as
a compound with activity better than Resveratrol (Table 1). In this
paper, we report the synthesis of various trans-4,40-dihydroxy-
stilbene analogs capable of upregulating the transcription of
endogenous ApoA-I, focusing on key positions that can be favorably
substituted, as well as alternatives to the trans-double bond.
Naturally occurring polyphenols belong to a class of compounds
known to have cardio-protective effects by increasing HDL-c levels
[7]. The phenomenon known as the French Paradox, where
moderate consumption of red wine has been associated with
a reduced risk of cardiovascular diseases, is believed to be due to
polyphenols found in wine. Specific polyphenols, like Resveratrol
2.1. Chemistry
2.1.1. Synthesis of derivatives
Based on early screening data, we were interested in exploring
analogs of stilbene 2 with substituents in the 2- and 3-positions.
A series of these analogs were synthesized via a Heck coupling [10]
between substituted aryl bromides (5a-e) and a protected styrene
analog (3 or 4; Scheme 1). As a general procedure, to a solution of
the aryl bromide in acetonitrile were added a styrene analog,
* Corresponding author. Tel.: þ1 1403 254 9252; fax: þ1 1403 256 8495.
E-mail address: henrik@resverlogix.com (H.C. Hansen).
0223-5234/$ – see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.12.076

H.C. Hansen et al. / European Journal of Medicinal Chemistry 45 (2010) 2018–2023
2019
Table 1
OH
HO
OH
Induction of ApoA-I promotor in Caco-2 cells.
2
HO
3
Entry
Compound
Fold
Fold induction
induction
relative to 1
6
4
1
2
3
4
1 (Resveratrol)
2
7
2.6–3.6
5.1
12.5
2.0
1.1
9.4
3.2
0.9
2.0
1.2
1.1
2.7
1.1
3.9
3.4
1.9
1.7
1.7
1.3
3.7
1.9
2.2
1.4
1
5
1
2
OH
1.6
4.6
0.5
0.2
2.8
0.9
0.3
0.7
0.4
0.4
0.7
0.4
1.0
1.3
0.7
0.5
0.6
0.4
0.9
0.5
0.6
0.5
Fig. 1. Resveratrol (1) and trans-4,40-dihydroxystilbene (2).
8
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
33
34
35
36
diisopropylethylamine (DIEA), biphenyl-2-yl-di-tert-butyl phos-
phine, and palladium acetate. The reaction mixture was stirred at
80 ꢁC for 48 h, giving the stilbene products (6a–e) in 30-70% yield.
A series of functional group transformations (FGT) were used to
provide the desired products (7–16; Scheme 1). In each case, the
reactions proceeded as expected and the products were obtained in
reasonable yields. Additional details can be found in the experi-
mental section.
An analog of compound 2, containing a nitrogen in the aromatic
ring (17; Fig. 2) was synthesized in two steps from (4-methoxy-
benzyl)triphenylphosphonium chloride and 6-methoxypyridine-3-
carbaldehyde via Wittig olefination, using sodium methoxide as
base. The di-methoxy analog was deprotected using thiophenol and
K2CO3, giving compound 17 in in 31% overall yield.
In addition to the structure-activity relationship (SAR) around
the 2- and 3-positions of trans-4,40-dihydroxystilbene (2), we were
also interested in replacing the trans double bond with an alter-
native unit that would be more likely to possess improved drug-like
properties. Initially, a series of potential isosteres, including satu-
rated alkyl (ethyl), azo, and amide analogs (18–23; Fig. 2), were
synthesized using standard methodology. This allowed us to assess
the importance of the double bond on the ability of the analogs to
induce ApoA-I transcription.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
All compounds screened were >93% pure by HPLC. All compounds, including the
positive control, Resveratrol, were tested at 15
M.
m
The screening data are given in Table 1 above. For each
compound the fold induction is reported as relative to untreated
cells and relative to Resveratrol, which was included as a positive
control in each experiment. Resveratrol typically gave fold induc-
tions between 2.6- and 3.6-fold.
Second, a series of 2,5-diaryl-1,3,4-oxadiazole (33–36; Scheme 2)
analogs was prepared by cyclization of the corresponding
di-aroylhydrazines (29-32) in the presence of SOCl2 in pyridine or
Ac2O in pyridine [11]. The desired compounds were obtained in good
yields (40–54%).
The 2-position was explored by synthesizing a number of
analogs containing both electron withdrawing and electron
donating groups, as well as compounds with some variation of the
size of the substituent. Smaller groups (OMe, NO2, NH2; entries 3, 6,
and 7), providing less sterical hindrance, were preferred, and
compound 7, trans-4,40-dihydroxy-2-methoxystilbene, was the
most potent in the series made, being 4.6x more potent than
Resveratrol (1) and 2.9x more potent than the screening hit 2.
Larger groups were less potent, including amides (entries 5 & 8)
and the sulfone (entry 9). The hydroxymethyl analog (8) was
significantly less potent than the methoxy analog (7), suggesting
that the nature of the hydrogen bonding is important. The elec-
tronic properties of the substituent in the 2-position were not very
important for the activity, as the nitro analog (10; entry 6) also was
active.
2.2. Biological activity
The compounds (7–23 and 33–36) were evaluated in a cell-
based assay for their ability to induce ApoA-I expression [12].
Briefly, Caco-2 cells transfected with an ApoA-I promoter operably
linked to
a luciferase reporter gene were incubated with
compounds for 48 h prior to harvesting. The cells were lysed and
the luciferase activity was measured with a luminometer. The
luciferase activity of cells treated with the test compounds were
normalized to that of untreated cells to calculate the observed fold
induction.
Varying the substituent in the 3-position quickly appeared to be
less fruitful than the 2-position (Table 1, entries 14–16). Only the
OH
Br
or
OMe
4
OAc
R2
R1
b
R1
3
a
R
6
5
HO
6a to 7: R = 2-OMe
6b to 8: R = 2-CH2OH
a: R1 = 2-OMe, 4-OAc
a: R1 = 2-OMe, 4-OAc; R2 = 4'-OAc
b: R1 = 2-CO2Me, 4-OMe; R2 = 4'-OMe
c: R1 = 2-NO2, 4-OMe; R2 = 4'-OMe
d: R1 = 2-SO2Me, 4-OMe; R2 = 4'-OAc
e: R1 = 3-CO2Me, 4-OAc; R2 = 4'-OAc
6b to 9: R = 2-CONMe2
6c to 10: R = 2-NO2
10 to 11: R = 2-NH2
11 to 12: R = 2-NHAc
6d to 13: R = 2-SO2Me
6e to 14: R = 3-CO2H
6e to 15: R = 3-CO2Et
6e to 16: R = 3-CONH2
b: R1 = 2-CO2Me, 4-OMe
c: R1 = 2-NO2, 4-OMe
d: R1 = 2-SO2Me, 4-OMe
e: R1 = 3-CO2Me, 4-OAc
Scheme 1. Synthesis of stilbene analogs. a: Cat. Pd(OAc)2, cat. biphenyl-2-yl-di-tertbutyl phosphine, N,N-diisopropylethylamine, N2, 80 ꢁC, 15-48 h; b: Various Functional Group
Transformation (FGT; see experimental section for details).

2020
H.C. Hansen et al. / European Journal of Medicinal Chemistry 45 (2010) 2018–2023
4. Experimental procedures
OH
OH
4.1. General procedures
N
17
18
HO
HO
All starting materials, solvents and reagents were used as
purchased from commercial suppliers, unless otherwise noted.
Anhydrous THF was purchased from Sigma-Aldrich. Yields refer to
chromatographically and spectroscopically (1H-NMR) homoge-
neous materials, unless otherwise stated. Reactions were
monitored by thin-layer chromatography (TLC) carried out on
Macherey-Nagel (MN) Alugram SIL G/UV254 silica gel 60 with
fluorescent indicator UV254 plates (catalogue # 818 133) or on
Sigma-Aldrich silica gel plates (catalogue # Z193291-1PAK) using
phosphomolybdic acid in absolute ethanol and heat as a developing
agent. NMR spectra were recorded on Varian Mercury 400 NMR
instrument using residual undeuterated solvent as an internal
reference. Low resolution mass spectra (MS) were recorded on
a Water Micromass ZQ using electrospray ionization-ion trap (ESI).
Sodium sulfate (Na2SO4) was used as drying reagent, unless
otherwise noted. All column chromatography was performed on
silica gel (SILICYCLE, 230-400 mesh, cat# R12030B). The solvent
volumes included in the experimental details are the volumes need
to elute the compound of interest. Compounds 18 [13], 19 [14], 20
[15], 21 [16], 22 [17], 34 [11], and 35 [18] were synthesized as
described or by a slightly modified procedure. In all cases, the
analytical data was in agreement with reported values.
OH
O
R2
N
N
N
H
R1
19
HO
HO
20 R1 = 4-OH; R2 = 4-OH
21 R1 = 4-OH; R2 = H
22 R1 = H; R2 = 4-OH
OH
O
S
O
N
H
23
Fig. 2. Synthesis of double bond analogs.
carboxamide (16) approached potency comparable to Resveratrol
(1) and it was significantly less potent than compound 2. Also, the
pyridyl analog (17) was less potent than both 1 and 2. For these
reasons this series was abandoned, and we focused on the 2-
position and the double bond replacement analogs.
To better understand the pharmacophore and the importance of
the double bond connecting the two aryl rings, a series of common
isosteres was prepared and tested (14–19; entries 18–23). The
saturated analog (18) as well as the azo compound (19) had potency
similar to Resveratrol. The more polar amides (20–22) and
sulfonamide (23) were less potent. These data indicated that the
double bond itself is not essential for activity, and that other groups
are acceptable, including more flexible alkyl chains. In addition,
a series of 2,5-diaryl oxadiazoles were tested. Compound 33 with
both hydroxyl substituents intact proved to have potency similar to
Resveratrol, suggesting that 2,5-diaryl oxadiazoles could be a viable
substitute for the double bond during optimization of potency and
the drug-like properties.
4.1.1. (E)-4-[2-(4-Hydroxyphenyl)-vinyl]-3-methoxyphenol (7)
In a round-bottomed flask fitted with condenser were placed
4-bromo-3-methoxyphenol (2.0 g, 9.9 mmol), Ac2O (1.21 g,
11.8 mmol), Et3N (1.20 g, 11.8 mmol) and a catalytic amount of
4-dimethylaminopyridine (DMAP). The reaction mixture was stir-
red for 24 h at room temperature before poured into water and
extracted with ethyl acetate. The organic layer was washed with
water, dried, and concentrated to give 4-bromo-3-methoxyphenyl
acetate (5a; 2.4 g, 99%). To a stirred solution of 5a (3.55 g,
14.5 mmol) in acetonitrile (60 mL) were added 4-acetoxystyrene
(3; 2.47 g, 15.2 mmol), N,N-diisopropylethylamine (5.61 g,
43.5 mmol), biphenyl-2-yl-di-tert-butyl phosphine (259 mg,
0.870 mmol) and palladium acetate (195 mg, 0.870 mmol) under
N2. The reaction mixture was heated at 80 ꢁC for 48 h and then
cooled to room temperature. Water was added and the mixture was
extracted with ethyl acetate. The organic layer was washed with
water, brine, dried, and concentrated to give the crude product,
which was purified by column chromatography (hexane/EtOAc
9:1), to give (E)-4-[2-(4-acetoxy-2-methoxyphenyl)-vinyl]-phenyl
acetate (6a). To a solution of 6a (220 mg, 0.675 mmol) in methanol-
THF (10:4) was added K2CO3 (466 mg, 3.37 mmol), and the reaction
mixture was stirred for 25 min at room temperature. The reaction
mixture was neutralized with dilute HCl and extracted with ethyl
acetate. The combined organic layers were washed with water,
brine, dried, and concentrated. Purification by column chroma-
tography (hexane/EtOAc 1:1), gave the title compound 7 (140 mg,
85%); Rf ¼ 0.60 (hexane/ethyl acetate, 1:1); mp. 192 – 194 ꢁC; 1H
3. Conclusion
In summary, exploration of the SAR around Resveratrol for the
ability to induce the expression of the ApoA-I protein, led to the
identification of more potent analogs, as well as isosteres of
the double bond, providing access to more drug-like compounds.
Compound 7 was 4.6 fold more potent than Resveratrol and 2.9 fold
more potent than the screening hit trans-4,40-dihydroxystilbene (2).
In addition, the double bond connecting the two aryl rings were not
essential for activity and could be replaced by other, preferably non-
polar isosteres. Also, 2,5-di-substituted oxadiazoles were tolerated.
O
O
NH2
a
N
H
OH
R1
R2
24: R1= 4-OH
25: R1= H
26: R2 = 3-OH
27: R2 = H
28: R2 = CH2OH
NMR (400 MHz, CD3OD)
(d, J ¼ 16.4 Hz, 1 H), 6.87 (d, J ¼ 16.7 Hz, 1 H), 6.73 (m, 2 H), 6.46 –
6.32 (m, 2 H), 3.83 (s, 3 H); 13C NMR (100 MHz, CD3OD)
158.1,
d
7.37 (d, J ¼ 8.5 Hz, 1 H), 7.30 (m, 2 H), 7.16
O
H
b
R2
O
N
N
R1
d
R2
N
H
N
158.0, 156.5, 130.4, 127.1, 126.6, 125.6, 120.4, 118.5, 115.2, 107.3, 98.6,
54.6; MS (ESþ) m/z 243.11 (M þ 1); Anal. Calcd for
C15H14O3$0.2H2O: C 73.27; H 5.90. Found: C73.29; H 5.87.
O
R1
29: R1 = 4-OH; R2 = 3-OH
30: R1 = 4-OH; R2 = H
31: R1 = H; R2 = 3-OH
33: R1 = 4-OH; R2 = 3-OH
34: R1 = 4-OH; R2 = H
35: R1 = H; R2 = 3-OH
4.1.2. (E)-3-Hydroxymethyl-4-[2-(4-hydroxyphenyl)-vinyl]-
phenol (8)
32: R1 = OH; R2 = 3-CH2OH 36: R1 = OH; R2 = 3-CH2OH
The intermediate (E)-5-methoxy-2-[2-(4-methoxyphenyl)-
vinyl]-benzoic acid methyl ester (6b) was synthesized from methyl
Scheme 2. Synthesis of 2,5-diaryl-1,3,4-oxadiazoles. a: EDCI, DMF. b: SOCl2, pyridine
or Ac2O, pyridine.

H.C. Hansen et al. / European Journal of Medicinal Chemistry 45 (2010) 2018–2023
2021
2-bromo-5-methoxybenzoate (5b) and 4-methoxystyrene (4)
essentially as described for compound 6a in 30% yield after puri-
fication by column chromatography (hexane/EtOAc 6:1). To
a suspension of LiAH4 (106 mg, 2.82 mmol) in THF (20 mL) was
added 6b (700 mg, 2.34 mmol) in THF (10 mL) at 0 ꢁC. The resulting
reaction mixture was stirred at 0 ꢁC for 2 h, then quenched with 1 N
aqueous NaOH solution at 0 ꢁC. The resulting mixture was extracted
with ethyl acetate, and the combined organic layers were washed
with water, brine, dried, and concentrated. The crude product was
purified by column chromatography (hexane/EtOAc 2:1), to give
the primary alcohol (572 mg, 90%). To a solution of the primary
alcohol (572 mg, 2.12 mmol) and thiophenol (513 mg, 4.66 mmol)
in NMP (10 mL), was added K2CO3 (29 mg, 0.21 mmol). The reaction
mixture was heated at 190 ꢁC for 7 h. The mixture was diluted with
water, made alkaline using NaOH (1 N), and was extracted
with ethyl acetate. The combined organic layers were washed with
water, brine, dried, and concentrated. The crude product was
purified by column chromatography (EtOAc/MeOH 20:1), to give
the title compound 8 (120 mg, 23%); Rf ¼ 0.10 (hexane/ethyl
4.1.4. (E)-4-[2-(4-Hydroxyphenyl)-vinyl]-3-nitro-phenol (10)
The intermediate (E)-4-methoxy-1-[2-(4-methoxyphenyl)-
vinyl]-2-nitrobenzene (6c) was synthesized from 2-bromo-5-
methoxynitrobenzene (5c) and 4-methoxystyrene (4) essentially as
described for compound 6a in 72% yield after purification by
column chromatography (hexane/EtOAc 20:1 to 9:1). Compound 6c
(1.45 g, 5.09 mmol) and pyridinium hydrochloride (8.82 g,
76.3 mmol) were mixed, and the reaction mixture was heated to
190 ꢁC for 2 h. After cooling, water (50 mL) was added and the
mixture was extracted with ethyl acetate (3 ꢀ 80 mL). The
combined organic layers were washed with water and brine, dried
(MgSO4), and concentrated. The crude product was purified by
column chromatography (hexane/EtOAc 9:1, 400 mL to 4:1,
400 mL), to afford the title compound 10; (480 mg, 37%); Rf ¼ 0.40
(hexane/ethyl acetate, 4:1); mp. 237 – 240 ꢁC; 1H NMR (400 MHz,
DMSO-d6)
d
10.41 (s, 1 H), 9.64 (s, 1 H), 7.75 (d, J ¼ 8.8 Hz, 1 H), 7.38
(d, J ¼ 8.5 Hz, 2 H), 7.29 (d, J ¼ 2.6 Hz, 1 H), 7.20 - 6.98 (m, 3 H), 6.78
(d, J ¼ 8.5 Hz, 2 H); MS (ESþ) m/z: 255.85 (M þ 1); Anal. Calcd for
C14H11NO4: C 65.37; H 4.31; N 5.44. Found: C 65.50; H 4.48; N 5.62.
acetate, 1:1); mp. 223–225 ꢁC; 1H NMR (400 MHz, CD3OD)
d 7.48 (d,
J ¼ 8.6 Hz, 1 H), 7.36 (d, J ¼ 8.6 Hz, 2 H), 7.19 (d, J ¼ 16.0 Hz, 1 H), 6.89
4.1.5. (E)-3-Amino-4-[2-(4-hydroxyphenyl)-vinyl]-phenol (11)
To a solution of compound 10 (216 mg, 0.84 mmol) in acetone
(6 mL) was added a solution of ammonium chloride (89.9 mg,
1.68 mmol) in water (2 mL). The mixture was heated to reflux, and
zinc dust (166 mg, 2.50 mmol) was added in small portions,
maintaining a moderate reaction. After the reaction subsided, an
additional portion of zinc dust (82.4 mg, 1.25 mmol) was added and
the reaction mixture was heated at reflux for an additional 2 h. The
solids were removed by filtration, washed with acetone (2 ꢀ 20 mL)
and ethyl acetate (2 ꢀ 30 mL). The combined washings were
concentrated in vacuo and the crude product was purified by
column chromatography (CH2Cl2/MeOH 20:1) to afford the title
compound 11 (155 mg, 72% yield); Rf ¼ 0.37 (CH2Cl2/MeOH, 20:1);
– 6.79 (m, 2 H), 6.78–6.67 (m, 3 H), 4.67 (s, 2 H); 13C NMR (100 MHz,
CD3OD):
d 156.9, 156.7, 139.5, 129.9, 128.0, 127.8, 127.5, 126.5, 122.2,
115.2, 114.8, 114.5, 62.1; MS (ESþ) m/z: 241.12 (M ꢂ 1); Anal. Calcd
for C15H14O3: C 74.36; H 5.82. Found: C 74.13; H 5.99.
4.1.3. (E)-5-Hydroxy-2-[2-(4-hydroxyphenyl)-vinyl]-N,N-
dimethylbenzamide (9)
To a solution of compound 6b (485 mg, 1.60 mmol) in mixture
of THF and acetonitrile (1:1, 20 mL) was added LiOH$H2O (102 mg,
2.40 mmol) in water (1 mL). The reaction mixture was stirred at
room temperature for 16 h. Solvent was removed in vacuo, water
(10 mL) was added and pH was adjusted to 3 by the addition of
2 N aq HCl. The mixture was extracted with ethyl acetate (3 ꢀ
30 mL), the combined extracts were washed with water, brine,
dried (MgSO4), and concentrated. Purification by column
chromatography (CH2Cl2/MeOH 20:1) gave (E)-5-methoxy-2-[2-
(4-methoxyphenyl)-vinyl]-benzoic acid (462 mg, quant.). (E)-5-
methoxy-2-[2-(4-methoxyphenyl)-vinyl]-benzoic acid (462 mg,
1.6 mmol) was dissolved in anhydrous dichloromethane (10 mL),
and oxalyl chloride (227 mg, 1.79 mmol) was added at room
temperature under N2. The reaction mixture was stirred for 2 h,
then triethylamine (247 mg, 2.44 mmol) and dimethylamine (2 M
solution in THF, 1.6 mL) were added. The solution was stirred at
room temperature overnight. The reaction mixture was concen-
trated, water was added and the mixture was extracted with ethyl
acetate. The organic layer was separated, washed with water,
brine, dried (MgSO4), and concentrated to give the crude (E)-5-
methoxy-2-[2-(4-methoxyphenyl)-vinyl]-N,N-dimethylbenzamide
(510 mg, used in the next step without further purification). The
crude amide (510 mg, 1.6 mmol) and pyridinium hydrochloride
(2.82 g, 24.4 mmol) were heated at 190 ꢁC for 3 h. After cooling,
water (20 mL) was added and the product was extracted with
ethyl acetate (3 ꢀ 50 mL). The combined organic layers were
washed with water, brine, dried (MgSO4), and concentrated.
Purification by column chromatography (CH2Cl2/MeOH 20:1)
afforded the title compound 9 (210 mg, 46% yield); Rf ¼ 0.35
(CH2Cl2/MeOH, 20:1); mp. 185–187 ꢁC; 1H NMR (400 MHz, CD3OD)
mp. 229–232 ꢁC; 1H NMR (400 MHz, DMSO-d6)
d 9.38 (s, 1 H), 9.01
(s, 1 H), 7.37 (d, J ¼ 8.5 Hz, 2 H), 7.18 (d, J ¼ 8.5 Hz, 1 H), 7.06 (d,
J ¼ 16.1 Hz, 1 H), 6.79 - 6.58 (m, 3 H), 6.07 (d, J ¼ 2.3 Hz, 1 H), 6.00
(dd, J ¼ 8.5, 2.3 Hz, 1 H), 5.17 (s, 2 H); 13C NMR (100 MHz, DMSO-d6)
d
158.3, 156.9, 147.9, 130.2, 127.9, 126.9, 124.5, 122.0, 116.0, 114.2,
105.4, 102.2; MS (ESþ) m/z: 228.11 (M þ 1); Anal. Calcd for
C14H13NO2: C 73.99; H 5.77; N 6.16. Found: C 74.24; H 5.75; N 6.27.
4.1.6. (E)-N-{5-Hydroxy-2-[2-(4-hydroxyphenyl)-vinyl]-phenyl}-
acetamide (12)
To the solution of compound 11 (100 mg, 0.44 mmol) in anhy-
drous THF were added Et3N (223 mg, 2.20 mmol), followed by
acetic anhydride (225 mg, 2.20 mmol), and the reaction mixture
was stirred at room temperature for 16 h. The mixture was
concentrated and dissolved in methanol (30 mL), then water
(20 mL) and K2CO3 (197 mg, 1.42 mmol) were added. The reaction
mixture was vigorously stirred at room temperature for 1 h. The
mixture was concentrated in vacuo and the residue was purified by
column chromatography on silica gel (CH2Cl2/MeOH 20:1) to give
the title compound 12 (55 mg, 46% for two steps); Rf ¼ 0.46
(CH2Cl2/MeOH, 20:1); mp. 227–229 ꢁC; 1H NMR (400 MHz, CD3OD)
d
7.51 (d, J ¼ 8.5 Hz, 1 H), 7.35 (d, J ¼ 8.8 Hz, 2 H), 7.04–6.97 (m, 1 H),
6.88 (d, J ¼ 6.2 Hz,1 H), 6.79–6.72 (m, 2 H), 6.69 (dd, J ¼ 8.5, 2.6 Hz,1
H), 2.17 (s, 3 H); 13C NMR (100 MHz, CD3OD)
171.2, 157.0,156.9,
d
135.3, 129.8, 127.9, 127.5, 126.5, 124.5, 120.6, 115.3, 113.9, 112.9, 22.0;
MS (ESþ) m/z: 270.14 (M þ 1); Anal. Calcd for C16H15NO3: C 71.36; H
5.61; N 5.20. Found: C 70.55; H 6.10; N 4.83.
d
7.58 (d, J ¼ 8.6 Hz, 1 H), 7.29 (d, J ¼ 9.0 Hz, 2 H), 6.88–6.97 (m, 1
H), 6.85 (dd, J ¼ 8.6, 2.34 Hz, 1 H), 6.69 - 6.79 (m, 3 H), 6.62 (d,
J ¼ 2.7 Hz, 1 H), 3.14 (s, 3 H), 2.84 (s, 3 H); 13C NMR (100 MHz,
CD3OD)
d
172.4, 157.3, 157.0, 136.1, 129.3, 128.7, 127.5, 126.9, 125.9,
4.1.7. (E)-4-[2-(4-Hydroxyphenyl)-vinyl]-3-methanesulfonylphenol
(13)
To a solution of 3-methoxythioanisole (3.1 g, 20 mmol) in acetone
(300 mL) was added a solution of oxone (30.7 g, 50.0 mmol) in water
121.4, 116.7, 115.3, 112.5, 37.8, 33.9; MS (ESþ) m/z: 281.92 (M ꢂ 1);
Anal. Calcd for C17H17NO3: C 72.07; H 6.05; N 4.94. Found: C 71.95;
H 6.17; N 4.92.

2022
H.C. Hansen et al. / European Journal of Medicinal Chemistry 45 (2010) 2018–2023
(125 mL). After 16 h, the solid was filtered off and washed with
acetone. The filtrate was concentrated to remove acetone, and the
aqueous layer was extracted with ethyl acetate (2 ꢀ 200 mL), dried,
and concentrated to give 1-methanesulfonyl-3-methoxybenzene as
an oil (3.68 g, 98%). The oil was dissolved in glacial acetic acid
(90 mL) and water (100 mL). A solution of bromine (4.79 g, 30 mmol)
in acetic acid (10 mL) was added dropwise at 10 ꢁC and the reaction
mixture was stirred at room temperature for 15 h. The mixture was
then neutralized with aqueous NaOH solution, extracted with ethyl
acetate (3 ꢀ 200 mL), dried, and concentrated. The crude compound
was purified by column chromatography (hexane/EtOAc 4:1) to give
1-bromo-2-methanesulfonyl-4-methoxybenzene (5d; 2.4 g, 45%).
The intermediate (E)-4-[2-(2-methanesulfonyl-4-methoxyphenyl)-
vinyl]-phenyl acetate (6d) was synthesized from 5d and 4-acetox-
ystyrene (3) essentially as described for compound 6a in 43% yield
after purification by column chromatography (hexane/EtOAc 4:1).
A mixture of compound 6d (1.21 g, 3.50 mmol) and pyridinium
hydrochloride (6.07 g, 52.5 mmol) were stirred at 190 ꢁC for 15 h,
then cooled and washed with water. The residue was purified
by column chromatography (CH2Cl2/MeOH 3:1) to give the
title compound 13 (350 mg, 33%) as an off-white solid; Rf ¼ 0.47
(CH2Cl2/MeOH, 20:1); mp. 244 – 245 ꢁC; 1H NMR (400 MHz, CD3OD)
Compound 15: Rf ¼ 0.82 (hexane/ethyl acetate, 1:1); mp. 269–
271 ꢁC; 1H NMR (400 MHz, DMSO-d6)
10.58 (s, 1 H), 9.55 (s, 1 H),
d
7.88 (d, J ¼ 2.3 Hz, 1 H), 7.78 (dd, J ¼ 8.8, 2.15 Hz, 1 H), 7.41 (m, 2 H),
7.13–6.89 (m, 3 H), 6.76 (d, J ¼ 8.6 Hz, 2 H), 4.39 (q, J ¼ 7.0 Hz, 2 H),
1.37 (t, J ¼ 7.0 Hz, 3 H); 13C NMR (100 MHz, DMSO-d6)
d 169.5, 159.9,
157.8, 133.2, 129.9, 128.8, 128.4, 128.3, 127.83, 124.5, 118.6, 116.2,
113.9, 62.1, 14.7; MS (ESþ) m/z: 283.14 (M ꢂ 1); Anal. Calcd for
C17H16O4: C 71.82; H 5.67. Found: C 71.66; H 5.60. Compound 16:
Rf ¼ 0.30 (hexane/ethyl acetate, 1:1); mp. 272–274 ꢁC; 1H NMR
(400 MHz, DMSO-d6)
d 13.01 (s, 1 H), 9.54 (s, 1 H), 8.47 (br. s, 1 H),
8.07 (d, J ¼ 2.3 Hz, 1 H), 7.96 (br. s, 1 H), 7.57 (dd, J ¼ 8.8, 2.15 Hz, 1
H), 7.36 (m, 2 H), 7.15 - 6.99 (m, 1 H), 6.97 - 6.84 (m, 2 H), 6.77 (d,
J ¼ 8.6 Hz, 2 H); 13C NMR (100 MHz, DMSO-d6)
d 172.7, 160.9, 157.7,
132.8, 129.0, 128.9, 128.1, 127.4, 125.9, 125.0, 118.3, 116.3, 115.1; MS
(ESþ) m/z: 254.07 (M ꢂ 1); Anal. Calcd for C15H13NO3: C 70.58; H
5.13; N 5.49. Found: C 69.85; H 5.29; N 5.31.
4.1.10. (E)-5-[2-(4-Hydroxyphenyl)-vinyl]-pyridin-2-ol (17)
To a suspension of (4-methoxybenzyl)triphenylphosphonium
chloride (4.79 g, 11.4 mmol) in dry methanol (50 mL), sodium
methoxide (25% solution in methanol, 4.94 g, 22.8 mmol) was
added. The mixture was stirred at room temperature under
nitrogen for 30 minutes. 6-Methoxypyridine-3-carbaldehyde
(1.57 g, 11.4 mmol) was added and the stirring was continued for
16 h. Excess of sodium methoxide was quenched by addition of
water, the pH of the mixture was adjusted to 7.5-8.0 by addition of
1 N HCl and saturated aqueous NaHCO3. The mixture was extracted
with ethyl acetate (500 mL), the organic layer was washed with
water, brine, dried, and concentrated. The crude product was
purified by column chromatography (hexane/EtOAc 20:1, 1000 mL
to 9:1, 800 mL) to give (E)-2-Methoxy-5-[2-(4-methoxyphenyl)-
vinyl]-pyridine (1.27 g, 46%). The vinyl pyridine (1.17 g, 4.85 mmol)
was dissolved in dry NMP (12 mL), then thiophenol (1.1 g,
9.7 mmol) and K2CO3 (67 mg, 0.48 mmol) were added. The reaction
mixture was heated at 195 ꢁC for 8 h under N2, then cooled to room
temperature. Aqueous 1 N NaOH solution was added until pH ¼ 10.
The mixture was extracted with ether (3 ꢀ 50 mL). The aqueous
layer was acidified to pH 4 with aqueous 1 N HCl, stirred at room
temperature for 30 min, then extracted with ether (2 ꢀ 50 mL). The
pH was adjusted to pH 8 with saturated aqueous NaHCO3 solution.
The precipitated solid was isolated by filtration, washed with water
(100 mL), ethyl acetate (50 mL), ether (30 mL), and air-dried to give
the title compound 17 (715 mg, 69%); Rf ¼ 0.42 (hexane/ethyl
d
7.76 – 7.68 (m, 2 H), 7.43 (d, J ¼ 2.3 Hz, 1 H), 7.38 (d, J ¼ 8.6 Hz, 2 H),
7.07 (dd, J ¼ 8.4, 2.5 Hz, 1 H), 6.98 (d, J ¼ 16.4 Hz, 1 H), 6.78 (d,
J ¼ 8.6 Hz, 2 H), 3.07 (s, 3 H); 13C NMR (100 MHz, CD3OD)
d 157.8,
157.1, 137.9, 131.8, 129.2, 129.0, 128.9, 127.8, 121.0, 120.5, 115.4, 114.9,
41.5; MS (ESþ) m/z: 289.07 (M ꢂ 1); Anal. Calcd for C15H14O4S: C
62.05; H 4.86. Found: C 62.15; H 4.91.
4.1.8. (E)-2-Hydroxy-5-[2-(4-hydroxyphenyl)-vinyl]-benzoic acid
(14)
A mixture of methyl 5-bromosalicylate (5.00 g, 21.6 mmol),
Ac2O (2.65 g, 26.0 mmol), Et3N (2.63 g, 26.0 mmol) and catalytic
amount of DMAP was stirred for 24 h at room temperature. The
reaction mixture was poured into water and extracted with ethyl
acetate. The organic layer was washed with water, dried, and
concentrated to give 2-acetoxy-5-bromobenzoic acid methyl ester
(5e; 5.9 g, 98%). The intermediate (E)-2-acetoxy-5-[2-(4-acetoxy-
phenyl)-vinyl]-benzoic acid methyl ester 6e was synthesized from
5e and 4-acetoxystyrene (3) essentially as described for compound
6a in 42% yield after purification by column chromatography
(hexane/EtOAc 4:1). To a solution of 6e (500 mg, 1.41 mmol) in
acetonitrile:THF (1:1, 20 mL) was added NaOH (388 mg,
8.47 mmol). The reaction mixture was stirred for 24 h at room
temperature. It was neutralized with 1 N aqueous HCl and extrac-
ted with ethyl acetate. The organic layer was washed with water,
brine, dried, and concentrated. The crude product was purified by
crystallization from hexane – ethyl acetate to give the title
compound 14 (250 mg, 69%); Rf ¼ 0.15 (EtOAc/MeOH, 20:1); mp.
acetate, 9:1); mp. > 300 ꢁC; 1H NMR (400 MHz, DMSO-d6)
d 11.64
(br. s, 1 H), 9.50 (s, 1 H), 7.85 (dd, J ¼ 9.8, 2.7 Hz, 1 H), 7.44 (d,
J ¼ 2.3 Hz, 1 H), 7.31 (m, 2 H), 6.82 (d, J ¼ 1.6 Hz, 2 H), 6.76 (m, 2 H),
6.38 (d, J ¼ 9.4 Hz, 1 H); 13C NMR (100 MHz, DMSO-d6)
d 162.4,
157.5, 138.2, 129.0, 127.9, 116.9, 116.2; MS (ESþ) m/z: 214.08 (M þ 1);
Anal. Calcd for C13H11NO2$0.5H2O: C 70.26; H 5.44; N 6.30. Found: C
70.69; H 5.15; N 6.34.
241–243 ꢁC; 1H NMR (400 MHz, CD3OD)
d
7.94 (d, J ¼ 2.3 Hz, 1 H),
7.68 (dd, J ¼ 8.6, 2.34 Hz, 1 H), 7.37 (m, 2 H), 6.92 (m, 3 H), 6.76 (d,
J ¼ 8.6 Hz, 2 H); 13C NMR (100 MHz, CD3OD)
d
172.3, 161.1, 157.1,
4.1.11. 4-Hydroxy-N-(4-hydroxyphenyl)benzenesulfonamide (23)
A solution of thionyl chloride (9.2 mL, 126.1 mmol) in dry DMF
(0.16 mL) was added to sodium 4-hydroxybenzenesulfonate (4.9 g,
25 mmol). The resulting mixture was stirred at 60 ꢁC for 3.5 h. The
solution was poured into ice. The aqueous solution was extracted
(dichloromethane), dried, and concentrated to give crude
4-hydroxy-benzenesulfonyl chloride (4.7 g, 97%) as an oil. The
crude oil (w1.9 mmol) and 4-aminophenol (207 mg, 1.90 mmol)
were stirred in pyridine (6 mL) at room temperature for 2 h before
concentrated. The residue was purified by column chromatography
(hexane/acetone 3:1, 200 mL to 1:1, 200 mL and CH2Cl2/MeOH
50:1, 250 mL to 15:1, 300 mL) to afford the title compound 23
(140 mg, 28%) as a brownish solid; Rf ¼ 0.45 (CH2Cl2/MeOH 20:1);
132.7, 129.6, 129.3, 128.0, 127.5, 127.1, 124.3, 117.4, 115.3, 112.7; MS
(ESþ) m/z: 255.05 (M ꢂ 1); Anal Calcd for C15H12O4: C 70.31; H 4.72.
Found: C 69.90; H 4.42.
4.1.9. (E)-2-Hydroxy-5-[2-(4-hydroxyphenyl)-vinyl]-benzoic acid
ethyl ester (15) and (E)-2-hydroxy-5-[2-(4-hydroxyphenyl)-vinyl]-
benzamide (16)
A mixture of compound 6e (500 mg, 1.41 mmol) and ammonia
(2 M solution in ethanol, 15 mL) was heated at 115 ꢁC for 12 h in
a sealed tube. The reaction mixture was cooled to room tempera-
ture and concentrated and the crude product was purified by
column chromatography on silica gel (hexane/EtOAc 1:1, 850 mL)
to give the title compounds 15 (200 mg, 50%) and 16 (75 mg, 20%);
1H NMR (200 MHz, DMSO-d6)
d 10.4 (br s, 1 H), 9.5 (br s, 1 H), 9.25

H.C. Hansen et al. / European Journal of Medicinal Chemistry 45 (2010) 2018–2023
2023
(br. s,1H), 7.5 (m, 2 H), 6.8 (m, 4 H), 6.6 (m, 2 H); 13C NMR (200 MHz,
DMSO-d6)
266.09; Anal. Calcd for C12H11NSO4: C 54.33; H 4.18; N 5.28. Found:
C 54.32; H 4.83; N 5.17.
the crude product was purified by column chromatography on
silica gel (CH2Cl2/MeOH 20:1) to give the title compound 36
(154 mg, 85%); Rf ¼ 0.25 (CH2Cl2/MeOH 20:1); mp. 246 – 249 ꢁC; 1H
d
161.0, 155.0, 130.0, 129.0, 124.0, 116.0; MS (ESþ) m/z:
NMR (400 MHz, DMSO-d6)
d
10.35 (br s, 1 H), 8.06 (d, J ¼ 8.5 Hz, 2
H), 7.97 (m, 2 H), 7.56 (m, 2 H), 7.03–6.93 (m, 2 H), 5.41 (br s, 1 H),
4.1.12. 3,40-(1,3,4-Oxadiazole-2,5-diyl)diphenol (33)
4.61 (d, J ¼ 3.5 Hz, 2 H); 13C NMR (100 MHz, DMSO-d6)
d 164.8,
To
a
solution of 4-hydroxybenzhydrazide (24; 1.49 g,
164.1, 161.6, 147.4, 129.4, 127.7, 127.0, 122.5, 116.9, 114.8, 63.1; MS
(ESþ) m/z: 268.95 (M þ 1); Anal. Calcd for C15H12N2O3$0.3 H2O: C
65.69; H 4.65; N 10.21. Found: C 65.69; H 4.65; N 10.05.
9.80 mmol) and 3-hydroxybenzoic acid (26; 1.13 g, 8.2 mmol) in
dry DMF (20 mL) was added EDCI hydrochloride (1.57 g,
8.20 mmol). The reaction mixture was stirred at room temperature
under nitrogen for 16 h. The mixture was concentrated and water
(80 mL) was added. The white precipitate was filtered, washed with
water, 25% aqueous sodium bicarbonate, 1 N aq HCl, water, and air-
dried to give 4-hydroxybenzoic acid N0-(3-hydroxybenzoyl)-
hydrazide (29; 1.32 g, 59% yield), which was dried under vacuum
and used in the next step without further purification. Compound
29 (1.1 g, 4.0 mmol) was added in small portions to a stirred
mixture of thionyl chloride (7.2 g, 61 mmol) and pyridine (128 mg,
1.60 mmol). The reaction mixture was heated at 80 ꢁC for 5 h, then
cooled and diluted with water (20 mL). The yellowish precipitate
was filtered off, washed with water and air-dried. Recrystallization
from ethanol afforded the title compound 33 (550 mg, 54%) as
a white solid; Rf ¼ 0.64 (CH2Cl2/MeOH 10:1); mp. 291–293 ꢁC; 1H
4.2. Biology screening
4.2.1. ApoA-I promotor induction in Caco-2 cells
The promotor region of the human ApoA-I gene was isolated
and ligated upstream of the firefly luciferase gene to construct the
reporter plasmid pAI.474-Luc. This reporter plasmid and pRSV-b-
galactosidase (as a control for transfection efficiency) were co-
transfected into Caco-2 cells. The Caco-2 cells were then incubated
in MEM selection media (20% calf serum with 0.5 mg/mL G418;
Gibco) to establish stable strains that express the reporter gene. The
strains were seeded in 6-well culture plates and incubated for 24 h
at 37 ꢁC under 5% carbon dioxide. Cells were starved for 24 h in
MEM selection media containing 0.5% FBS. The test compounds
were added typically dissolved in 0.01% DMSO in MEM selection
media containing 0.5% FBS. After incubation for 48 h, the cells were
harvested and lysed using Reporter Lysis Buffer (PROMEGA E3971),
NMR (400 MHz, DMSO-d6)
J ¼ 8.8 Hz, 2 H), 7.46 - 7.56 (m, 2 H), 7.42 (t, J ¼ 7.9 Hz, 1 H), 6.94 -
7.07 (m, 3 H); 13C NMR (100 MHz, DMSO-d6)
164.8, 164.0, 161.5,
d 10.33 (br s,1 H), 9.97 (br s,1 H), 7.95 (d,
d
158.6, 131.3, 129.3, 125.2, 119.6, 117.9, 116.9, 114.8, 113.5; MS (ESþ)
m/z: 254.95 (M þ 1); Anal. Calcd for C14H10N2O3: C 66.14; H 3.96; N
11.02. Found: C 66.22; H 3.93; N 11.02.
and 50 mL of luciferase assay reagent (PROMEGA E4550 Luciferase
Reporter 1000 assay system) was added to measure luciferase
activity with a luminometer (Fluoroskan Ascent FL, Thermo Elec-
tron Corp.). Luciferase activity was normalized to lysate protein
concentrations, measured using Bradford Reagent (BioRad Protein
Assay reagent Cat# 500-0006). The luciferase activity of cells
treated with various concentrations of compounds was compared
to that of untreated control samples.
4.1.13. 4-[5-(3-Hydroxymethylphenyl)-[1,3,4]oxadiazol-2-yl]-
phenol (36)
To
a
solution of 4-hydroxybenzhydrazide (24; 1.49 g,
9.80 mmol) and 3-hydroxymethylbenzoic acid (28; 1.25 g,
8.2 mmol) in dry DMF (20 mL) was added EDCI hydrochloride
(1.57 g, 8.2 mmol). The reaction mixture was stirred at room
temperature under nitrogen for 16 h. Solvent was removed under
reduced pressure and water (80 mL) was added. The white
precipitate was filtered, washed with water, 25% aqueous NaHCO3,
aqueous 1 N HCl, water, and air-dried. The isolated 4-hydroxy-
benzoic acid N0-(3-hydroxymethylphenyl)-hydrazide (32; 1.2 g,
42%) was dried under vacuum and used without further purifica-
tion. To a solution of 32 (1.2 g, 4.2 mmol) in anhydrous pyridine
(8 mL) was added acetic anhydride (1.07 g, 10.5 mmol) and the
reaction mixture was stirred at room temperature for 16 h. Water
was added (50 mL) and the mixture was extracted with ethyl
acetate (3 ꢀ 100 mL). The combined organic layers were washed
with water, aqueous 1 N HCl, brine, dried (MgSO4), and concen-
trated to give 3-[N0-(4-acetoxybenzoyl)-hydrazino]-benzyl acetate
(800 mg, 52%) as a white solid. To 3-[N0-(4-acetoxybenzoyl)-
hydrazino]-benzyl acetate (400 mg, 1.08 mmol) were added thionyl
chloride (1.67 g, 14.5 mmol) and pyridine (0.04 mL, 0.4 mmol) and
the reaction mixture was heated at 80 ꢁC for 5 h. After cooling
to room temperature, the reaction was quenched slowly with ice-
water. The precipitated product was filtered, washed with water,
diethyl ether, and air-dried. Recrystallization from ethanol afforded
Appendix. Supplementary material
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.ejmech.2009.12.076.
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MeOH/THF/water (1:1:1, 10 mL), potassium carbonate (207 mg,
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