Discovery of novel and potent retinoic acid receptor α agonists: Syntheses and evaluation of benzofuranyl-pyrrole and benzothiophenyl-pyrrole derivatives

Article abstract of DOI:10.1021/jm000098s

In the course of our studies on retinoic acid receptor (RAR) agonists, we have designed and synthesized a series of benzofuran and benzothiophene derivatives. Some of these compounds (1a,b,e,f,j) markedly inhibited LPS- induced B-lymphocyte proliferation and exerted RARα selectivity. One of them, 4-[5-(4,7-dimethylbenzofuran-2-yl)pyrrol-2-yl]benzoic acid (1b), when orally administered significantly inhibited mouse antibody production and delayed type hypersensitivity (DTH) responses from a dose of 0.1 mg/kg.

Full text of DOI:10.1021/jm000098s

J . Med. Chem. 2000, 43, 2929-2937
2929
Discover y of Novel a n d P oten t Retin oic Acid Recep tor r Agon ists: Syn th eses
a n d Eva lu a tion of Ben zofu r a n yl-p yr r ole a n d Ben zoth iop h en yl-p yr r ole
Der iva tives
Hiroyuki Yoshimura, Kouichi Kikuchi, Shigeki Hibi, Katsuya Tagami, Takashi Satoh, Toshihiko Yamauchi,
Akira Ishibahi, Kenji Tai, Takayuki Hida, Naoki Tokuhara, and Mitsuo Nagai*
Tsukuba Basic Research Laboratories for Drug Discovery, Eisai Company, Ltd., 1-3, Tokodai 5-chome,
Tsukuba-shi, Ibaraki 300-2635, J apan
Received March 2, 2000
In the course of our studies on retinoic acid receptor (RAR) agonists, we have designed and
synthesized a series of benzofuran and benzothiophene derivatives. Some of these compounds
(1a ,b,e,f,j) markedly inhibited LPS-induced B-lymphocyte proliferation and exerted RARR
selectivity. One of them, 4-[5-(4,7-dimethylbenzofuran-2-yl)pyrrol-2-yl]benzoic acid (1b), when
orally administered significantly inhibited mouse antibody production and delayed type
hypersensitivity (DTH) responses from a dose of 0.1 mg/kg.
Ch a r t 1
In tr od u ction
Retinoids are potent molecules that have a variety of
biological activities, including induction of cellular
proliferation, differentiation, apoptosis, and develop-
mental changes.1 These compounds also act as modula-
tors for a variety of inflammatory and immunological
events.2 In immunologically related animal models, 13-
cis-retinoic acid inhibited disease progress in both rat
adjuvant arthritis3 and rat experimental autoimmune
encephalomyelitis,4 which are known as models of
human rheumatoid arthritis and multiple sclerosis, at
doses of 100 and 75 mg/kg, respectively. These models
are mainly dependent on T-cell activation. However, this
compound was not effective in type II collagen (CII)-
induced arthritis which is known to be an antibody-
dependent disease.5
It has been shown that the biological effects of
retinoids are mediated by activation of retinoic acid
receptors (RARs), which are ligand-dependent gene
transcription factors. There are three distinct receptor
subtypes (RARR, -â, and -γ), which possess considerable
homology in their ligand-binding domains.6
Although retinoids are thought to have great thera-
peutic potential, their clinical use is so far limited
mainly to dermatological diseases1a,7 and some cancers,
for which retinoids may have both chemotherapeutic
and chemopreventive applications.8 The main reason for
this limited use is the wide range of toxic effects of
retinoids.9 Thus, recent research has focused on the
synthesis and development of subtype-selective retinoids
in order to reduce their toxicity.10
There are few reports addressing the immunological
effects and RAR subtypes both in vitro and in vivo. Apfel
et al. reported that the RARR agonist Ro 40-6055
(Am80) (Chart 1) was a potent inhibitor of LPS-induced
murine B-lymphocyte proliferation and that there was
a correlation between the activity of RARR transcription
and its potency to inhibit B-cell activation.11 Am80 was
also shown to inhibit inflammatory cytokine IL-6 pro-
duction in vitro.12 Furthermore, Am80 inhibited the
increase of CII antibody from a dose of 0.3 mg/kg in rat
CII-induced arthritis.13 These results suggest that there
is a strong correlation between the RARR agonistic
activity and immunosuppressive effects, especially in
the inhibition of antibody production. The therapeutic
potential of RARR agonists is not only for immunological
disorders but also in the treatment of cancer14 and
dermatological diseases.15 In the course of our studies
aimed at synthesizing novel retinoid analogues,16 we
focused our attention on RARR agonists which could be
therapeutic agents of immunological disease with less
toxicity than nonselective retinoids.
We reported previously that ER-41666, which pos-
sesses a flat structural moiety and a 2,5-disubstituted
pyrrole group in the hydrophobic part and the linker,
respectively, showed highly selective transactivation
activity of the RARR receptor.16e ER-38930, which
possesses a monocyclic five-membered ring moiety in
* To whom correspondence should be addressed. Tel: 81-298-47-
5888. Fax: 81-298-47-2037. E-mail: m2-nagai@hhc.eisai.co.jp.
10.1021/jm000098s CCC: $19.00 © 2000 American Chemical Society
Published on Web 06/14/2000

2930 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15
Yoshimura et al.
Sch em e 1a
etaldehyde diethyl acetal in the presence of potassium
carbonate to afford the diethyl acetal 6. Compound 6
was then cyclized with acetic acid to give the carbalde-
hyde 7 (method B).
The benzofuran derivatives 7e,f, which possess a
strong electron-withdrawing substituent such as fluoro
and trifluoromethyl, could not be obtained by acid-
catalyzed cyclization of the diethyl acetal derivatives.
Therefore, 7e,f were synthesized by Nelson’s method18
as shown in Scheme 2. The Claisen rearrangement of
the allyl ether 9 was carried out in N,N-dimethylaniline
at 190 °C to afford the phenol 10. The construction of
the dihydrobenzofuran was achieved by epoxidation of
compound 10 with 3-chloroperoxybenzoic acid (MCPBA)
followed by intramolecular opening of the epoxide by
the phenolate anion to give the alcohol which was then
transformed to the acetate 11. Conversion of the dihy-
drobenzofuran derivative 11 to the benzofuran deriva-
tive 12 was accomplished in three steps. The dihydro-
benzofuran 11 was subjected to bromination with
N-bromosuccinimide followed by dehydrobromination to
afford the benzofuran derivative, which was deacylated
with potassium carbonate to give the alcohol 12. This
compound 12 was oxidized under Swern’s condition to
give the aldehydes 7e,f (method C).
a
Reagents: (a) bromoacetaldehyde diethyl acetal, K2CO3, DMF;
(b) polyphosphoric acid, toluene; (c) n-BuLi, THF, DMF; (d) AcOH.
the hydrophobic part, was also shown to markedly
activate transactivation of the RARR receptor.16d We
hoped that novel retinoids which possess a flat struc-
tural five,six-membered bicyclic ring, such as benzofu-
ran and benzothiophene, might be more potent and
selective than the retinoids we had previously obtained.
Here we report the syntheses, structure-activity rela-
tionships (SAR), and in vivo results of such benzofura-
nyl-pyrrole and benzothiophenyl-pyrrole derivatives.
The syntheses of the benzofuranyl-pyrrole derivatives
1a -k were performed by the method described for the
synthesis of ER-34617.16c The key intermediate diketone
14 was synthesized by condensation of the aldehyde 7
with the enone 13 in the presence of 3-benzyl-5-(2-
hydroxyethyl)-4-methylthiazolium chloride and triethyl-
amine. The pyrrole formation was conducted with
ammonium acetate in methanol to give the pyrrole
derivatives, which were then hydrolyzed to the final
target compounds 1a -k (Scheme 3).
Ch em istr y
Three alternative routes for the synthesis of the
important intermediate, the benzofuran 7, are shown
in Schemes 1 and 2. In the first method, the diethyl
acetal 3, derived from a commercially available phenol
2 and bromoacetaldehyde diethyl acetal, was treated
with polyphosphoric acid to afford the benzofuran
derivative 4. Treatment of the benzofuran derivative 4
with n-butyllithium followed by formylation with N,N-
dimethylformamide afforded the carbaldehyde 7. The
benzothiophene derivatives were prepared starting from
the thiophenol instead of the phenol (method A).
The second method17 of benzofuran synthesis involved
the treatment of the salicylaldehyde 5 with bromoac-
Resu lts a n d Discu ssion
The above novel retinoids were synthesized and
evaluated in vitro for their ability to bind to individual
RARs and to induce gene transcription in the cotrans-
fection assay. Cotransfection assays were performed as
described in the Experimental Section, and relative EC30
values are reported (see footnotes to Table 1). Binding
assays for RAR receptor subtypes were performed in a
manner similar to that described previously16c using
Sch em e 2a
a
Reagents: (a) allyl bromide, K2CO3, DMF; (b) 190 °C, N,N-dimethylaniline; (c) (i) m-CPBA, CH2Cl2, (ii) aq KOH, DMSO, (iii) Ac2O,
pyridine; (d) (i) N-bromosuccinimide, AIBN, CCl4, (ii) t-BuOK, t-BuOH, (iii) K2CO3, MeOH; (e) (COCl)2, DMSO, Et3N, CH2Cl2.

Novel and Potent RARR Agonists
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15 2931
Sch em e 3a
a
Reagents: (a) 3-benzyl-5-(2-hydroxyethyl)-4-methylthiazolium chloride, Et3N, DMF; (b) (i) AcONH4, MeOH, (ii) aq NaOH, EtOH.
Ta ble 1. Competitive Binding, Transactivation, and B-Cell Proliferation Inhibition
binding affinity,a
relative IC50
subtype-specific transactivation,e
proliferation
inhibitionh
b
f
relative EC30
i
compd R1
R2
R3
R4
RAR-R
RAR-â
ndc
RAR-γ
ndc
RAR-R
RAR-â
RAR-γ
relative IC50
1a
1b
1c
1d
1e
1f
Me
Me
Cl
Me
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Cl
OMe
F
CF3
H
1.6 ( 0.1
1.4 ( 0.3
1.5 ( 0.3
15 ( 3
2.5 ( 0.3
1.9 ( 0.4
16 ( 3
9.4 ( 5.0
12 ( 8
6.1 ( 0.4
62 ( 21
0.80 ( 0.14
0.37 ( 0.02
0.70 ( 0.15
6.0 ( 2.1
92 ( 38
25 ( 12
110 ( 30
1000 ( 300
240 ( 30
820 ( 290
0.98 ( 0.11
0.76 ( 0.28
1.3 ( 0.4
490 ( 60
690 ( 300
ndc
ndc
ndc
ndc
ndc
ndc
ndc
ndc
ndc
ndc
ndc
730 ( 340 3700 ( 210
4.7 ( 1.2
ndc
0.80 ( 0.17
0.29 ( 0.04
1.0 ( 0.4
210 ( 40
8.5 ( 0.4
380 ( 130
200 ( 70
160 ( 80
130 ( 60
1750 ( 750
110 ( 20
0.93 ( 0.11
0.37 ( 0.04
1.2 ( 0.1
F
500 ( 50
1g
1h
1i
Me
Me
Me Me
Me
Me Me
H
H
ndc
890 ( 90
480 ( 40
ndc
2.0 ( 0.5
2.2 ( 0.5
2.4 ( 0.4
0.75 ( 0.02
3.6 ( 0.2
H
Me
H
ndc
3.1 ( 1.1
2000 ( 500
840 ( 360
1j
1k
Me
Me
H
H
ndc
1.3 ( 0.6
Me
ndc
2.4 ( 0.5
1300 ( 500 2900 ( 50
ATRA
1.0
1.0
1.0
1.0
1.0
1.0
1.0
j
0.76 ( 0.11d 0.47 ( 0.05d 0.41 ( 0.04d
3.4 ( 0.4g
2.2 ( 0.2g
0.26 ( 0.03g 0.65 ( 0.10
a
Specific binding affinity was defined as total binding minus nonspecific binding, and the 50% inhibitory dose (IC50) values were
obtained from logarithmic plots. The selectivity of test compounds for each receptor is indicated as relative IC50, where the IC50 value for
b
each receptor was divided by that of the natural ligand (ATRA). Means of IC50/ATRA IC50 ( SEM. c nd: not detectable (relative IC50
>
1000). Means of ATRA IC50 (nM) ( SEM. e Subtype-specific activity is expressed in terms of relative EC30, which is the concentration
d
of retinoid required to produce 30% of the maximal observed response, normalized relative to that of ATRA. f Means of EC30/ATRA EC30
g
h
( SEM. Means of ATRA EC30 (nM) ( SEM. The inhibition of LPS-induced mouse B-lymphocyte proliferation of each compound is
i
j
indicated as relative IC50
. Means of IC50/ATRA IC50 ( SEM. Means of ATRA IC50 (nM) ( SEM.
[3H]ATRA. RXRR transactivation was also studied, but
none of these compounds activated RXRR (data not
shown). Inhibition of LPS-induced mouse B-lymphocyte
proliferation was studied by using the method reported
by Apfel.11 The results are summarized in Table 1.
We initially synthesized the 4,7-dimethylbenzofuran
derivative 1b which was thought to possess a similar
hydrophobic structure to both the benzopyranyl-pyrrole
derivative (ER-41666) and the pyrazolyl-pyrrole deriva-
tive (ER-38930). In the cotransfection assay, compound
1b was more potent than ATRA at RARR and 25-240-
fold less potent at RARâ and RARγ. It also showed very
weak affinity for RARâ and RARγ, whereas its binding
affinity at RARR was comparable to that of ATRA.
Furthermore, this compound (1b) showed stronger
inhibitory activity for LPS-induced mouse B-lymphocyte
proliferation than ATRA (Figure 1).
derivative 1c showed high selective binding affinity for
RARR, both of these compounds had less potent trans-
activation of RARR than the 4,7-dimethylbenzofuran 1b.
The 4-methoxy-7-methylbenzofuran derivative 1d, which
possesses an electron-donating substituent and a group
larger than methyl at the 4-position, had less potent
inhibitory activity on B-lymphocyte proliferation. In-
troduction of an electron-withdrawing substituent and
a smaller or equivalent size group with a methyl
substituent such as fluoride and trifluoromethyl did not
reduce the selectivity and inhibitory activity of B-
lymphocyte proliferation (1e,f). In particular, the 7-fluoro-
4-trifluoromethylbenzofuran derivative 1f showed marked
potency in inhibitory activity of B-lymphocyte prolifera-
tion. The 4,5,7-trimethylbenzofuran derivative 1h and
the 6,7-dimethylbenzofuran derivative 1i showed weak
inhibitory activity. The benzothiophene derivative 1j,
in which a sulfur atom was used to replaced an oxygen
of the benzofuran, showed comparable inhibitory activ-
ity of B-lymphocyte proliferation to the 4,7-dimethyl-
Next we examined the effects of substituents at the
4- and 7-positions. Although the 4-desmethyl-7-meth-
ylbenzofuran derivative 1a and 4,7-dichlorobenzofuran

2932 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15
Yoshimura et al.
F igu r e 2. Effect of 1b on antibody production in BALB/c mice.
BALB/c mice were immunized and treated with 1b, and their
serum antibody titers were determined as indicated in the
Experimental Section. Data represent the mean ( SEM of
serum anti-DNP IgG2a titers in 5 animals. *p < 0.05, 1b-
treated compared with vehicle control by one-way ANOVA
followed by Dunnett multiple comparison.
F igu r e 1. Inhibition of LPS-induced mouse B-cell prolifera-
tion with 1b and ATRA.
benzofuran derivative 1b. However, the 5,7-dimethyl-
benzothiophene derivative 1k had less potent RARR
activity and inhibitory activity than the 5,7-dimethyl-
benzofuran derivative 1g.
From the SAR of these benzofuranyl derivatives and
benzothiophenyl derivatives, it was found that both
types of derivatives have transactivation activity and
inhibitory activity of B-lymphocyte proliferation. The
4,7- and 5,7-disubstituents of benzofuran were found to
be important for RARR selectivity. In particular, the 4,7-
disubstituted benzofuranyl derivatives showed strong
inhibitory activity. Methyl, fluoride, and trifluoromethyl
appear to be the most suitable substituents to support
this action. Some of these benzofuranyl derivatives
(1a ,b,e,f) and the benzothiophenyl derivative (1j) in-
hibited LPS-induced B-lymphocyte proliferation more
potently than ATRA and exerted RARR selectivity.
Next, we studied the oral immunosuppressive activity
of compound 1b, which exhibited high RARR selectivity
and potent inhibitory activity of LPS-induced B-lym-
phocyte proliferation, as well as improved pharmaco-
kinetic characteristics (data not shown). The results of
inhibition of mouse antibody production19 and mouse
DTH20 for this compound are summarized in Figures 2
and 3. The former model is mainly mediated by acti-
vated B lymphocytes, and the latter one is mediated by
T lymphocytes. Compound 1b suppressed anti-DNP
IgG2a antibody production in DNP-KLH-immunized
mice dose-dependently in the antibody production model.
1b did not cause any obvious toxicity at the doses tested.
Furthermore, 1b suppressed the response in a dose-
dependent manner in the mouse DTH model. Oral
treatment of compound 1b significantly inhibited both
mouse antibody production and delayed type hypersen-
sitivity (DTH) responses from a dose of 0.1 mg/kg.
Moreover, 1b was effective in various immunological
disease models. In both adjuvant- and collagen-induced
arthritis models in rats, we observed that 1b markedly
inhibited paw swelling. It also ameliorated the disease-
related symptoms in mouse graft versus host disease.21
In addition, 1b exerted a profound improvement in
experimental and spontaneous nephritis models.22 The
details of pharmacological studies on compound 1b and
other potent compounds will be the subject of further
reports from our laboratory.
F igu r e 3. Effect of 1b on DTH responses in BALB/c mice.
BALB/c mice were immunized, treated with 1b, and challenged
with antigen as indicated in the Experimental Section. Data
represent the mean ( SEM of right foot pad swelling in 5
animals. *p < 0.05, 1b-treated compared with vehicle control
by one-way ANOVA followed by Dunnett multiple comparison.
In conclusion, we have described for the first time the
synthesis and SAR of a new series of benzofuranyl-
pyrrole derivatives and benzothiophenyl-pyrrole deriva-
tives. Some of these compounds (1a ,b,e,f,j) markedly
inhibited LPS-induced B-lymphocyte proliferation and
exerted RARR selectivity. One of them, compound 1b
(ER-38925), when orally administered significantly
inhibited DTH response at fairly low doses, i.e., less
than 0.1 mg/kg, and was effective in various im-
munological disease models. This compound (1b) and
other potent analogues (1a ,e,f,j) are currently being
evaluated with regard to immunosuppressive activity,
pharmacokinetics, and safety to select a candidate
which may be used clinically as an immunosuppressive
agent.
Exp er im en ta l Section
Ch em istr y. Reagents and solvents were purchased from
usual commercial sources. Silica gel (Kieselgel 60, Merck) was
used for column chromatography and silica gel (Kieselgel 60

Novel and Potent RARR Agonists
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15 2933
F254, Merck) for analytical thin-layer chromatography (TLC).
Compounds were detected on TLC plates by exposure to UV
light (254 nm). Melting points were measured on a Yanagimoto
micromelting point apparatus without correction. 1H NMR
spectra were recorded on a Varian Unity 400 spectrometer,
and chemical shifts are expressed in ppm downfield of tet-
ramethylsilane (TMS), which was used as an internal refer-
ence. Mass spectra (MS) were obtained on a J EOL J MS-HX100
mass spectrometer. All organic extracts were dried over
MgSO4, and the solvents were removed with a rotary evapora-
tor under reduced pressure.
Gen er a l P r oced u r e for P r ep a r a tion of Ben zofu r a n -2-
ca r ba ld eh yd e (Meth od C). Syn th esis of 7-F lu or o-4-tr i-
flu or om eth ylben zofu r a n -2-ca r ba ld eh yd e (7f). 2-Allyloxy-
1-flu or o-4-tr iflu or om eth ylben zen e (9b). To a solution of
2-fluoro-5-trifluoromethylphenol (8b) (50 g, 139 mmol) in N,N-
dimethylformamide (300 mL) were added allyl bromide (31
mL, 361 mmol) and potassium carbonate (57 g, 412 mmol) at
room temperature. The mixture was stirred for 2 h at 80 °C
under a nitrogen atmosphere. After the reaction cooled to room
temperature, brine was added and the mixture was then
extracted with ethyl acetate. The combined organic phases
were washed with brine, then dried and evaporated. The
residue was purified by flash column chromatography on silica
gel, eluting with 10% ethyl acetate in hexane to give 9b (56 g,
92%) as a pale yellow oil: 1H NMR (CDCl3) δ 4.63-4.66 (m,
2H), 5.35 (dd, J ) 1.2, 11.2 Hz, 1H), 5.45 (dd, J ) 1.6, 17.2
Hz, 1H), 6.00-6.12 (m, 1H), 7.14-7.21 (m, 3H).
2-Allyl-6-flu or o-3-tr iflu or om eth ylp h en ol (10b). 9b (56
g, 254 mmol) was dissolved in N,N-dimethylaniline (120 mL)
at room temperature, and the mixture was stirred for 28 h at
190 °C under a nitrogen atmosphere. After the reaction cooled
to 0 °C, the mixture was poured into 10% aqueous hydrochloric
acid and then extracted with ethyl acetate. The combined
organic phases were washed with brine, then dried and
evaporated. The residue was purified by flash column chro-
matography on silica gel, eluting with 10% ethyl acetate in
n-hexane to give 10b (54 g, 96%) as a pale yellow oil: 1H NMR
(CDCl3) δ 3.59 (d, J ) 6 Hz, 2H), 5.02-5.09 (m, 2H), 5.52-
5.56 (m, 1H), 5.90-6.00 (m, 1H), 7.04 (t, J ) 9.2 Hz, 1H), 7.20
(dd, J ) 4.8, 8.4 Hz, 1H).
(7-F lu or o-4-tr iflu or om eth yl-2,3-d ih yd r oben zofu r a n -2-
yl)m eth yl Aceta te (11b). To a solution of 10b (54 g, 245
mmol) in dichloromethane (300 mL) was added 3-chloroper-
oxybenzoic acid (84 g, 343 mmol) for 10 min at room temper-
ature. The resulting mixture was stirred for 6 h at the same
temperature under a nitrogen atmosphere. The mixture was
diluted with ethyl acetate, washed with saturated aqueous
sodium hydrogen carbonate, saturated aqueous sodium thio-
sulfate, and brine, and then dried and evaporated to give
6-fluoro-2-(2-oxiranylmethyl)-3-trifluoromethylphenol (53 g) as
a crude yellow oil: 1H NMR (CDCl3) δ 2.80-2.86 (m, 2H), 2.97
(d, J ) 4.0 Hz, 1H), 3.33 (dd, J ) 4.0, 7.2 Hz, 1H), 3.42-3.48
(m, 1H), 7.08 (t, J ) 8.8 Hz, 1H), 7.21 (dd, J ) 5.2, 9.2 Hz,
1H).
To a solution of 6-fluoro-2-(2-oxiranylmethyl)-3-trifluorom-
ethylphenol (53 g, 224 mmol) in dimethyl sulfoxide (280 mL)
was added potassium hydroxide (18.8 g, 269 mmol) in water
(50 mL) for 15 min. at 0 °C. The mixture was stirred for 14 h
at room temperature under a nitrogen atmosphere and then
diluted with water and extracted with ethyl acetate. The
combined organic phases were washed with brine, then dried
and evaporated to give (7-fluoro-4-trifluoromethyl-2,3-dihy-
drobenzofuran-2-yl)methanol (41 g) as a crude pale yellow
oil: 1H NMR (CDCl3) δ 3.29 (dd, J ) 7.6, 16.4 Hz, 1H), 3.44
(dd, J ) 9.2, 16.8 Hz, 1H), 3.78 (dd, J ) 5.2, 12.0 Hz, 1H),
3.96 (dd, J ) 2.0, 12.0 Hz, 1H), 5.03-5.13 (m, 1H), 6.99 (t, J
) 8.8 Hz, 1H), 7.07 (dd, J ) 4.0, 8.8 Hz, 1H).
Gen er a l P r oced u r e for P r ep a r a tion of Ben zofu r a n -2-
ca r ba ld eh yd e (Meth od A). 4,7-Dim eth ylben zofu r a n -2-
ca r ba ld eh yd e (7b). To a solution of 2,5-dimethylphenol (2b)
(10 g, 81.8 mmol) in N,N-dimethylformamide (100 mL) were
added successively potassium carbonate (22.6 g, 163.5 mmol)
and bromoacetaldehyde diethyl acetal (14.8 mL, 98.4 mmol)
at room temperature. This mixture was stirred at 140 °C for
2.5 h and then cooled to room temperature. The mixture was
diluted with water and extracted with ethyl acetate. The
organic phases were washed with brine, dried, and evaporated
to afford 2,2-diethyl-2,4-dimethylphenyl ether (18 g) as a
yellow oil: 1H NMR (CDCl3) δ 1.22-1.26 (m, 6H), 2.18 (s, 3H),
2.29 (s, 3H), 3.63-3.69 (m, 2H), 3.76-3.81 (m, 2H), 3.97-3.99
(m, 2H), 4.85 (t, J ) 5.2 Hz, 1H), 6.38 (d, J ) 1.6 Hz, 1H),
6.68 (dd, J ) 1.6, 7.2 Hz, 1H), 6.99 (d, J ) 7.2 Hz, 1H).
To a solution of this crude diethyl acetal (18 g, 75.5 mmol)
in toluene (100 mL) was added polyphosphoric acid (50 g), and
this mixture was stirred at 90 °C for 1 h under a nitrogen
atmosphere and then cooled and poured into ice-water. The
mixture was extracted with ethyl acetate, and the organic
phases were washed with brine, dried, and evaporated. The
crude residue was purified by flash column chromatography
on silica gel, eluting with n-hexane, to afford 4,7-dimethyl-
benzofuran (4b) (3.5 g) as a pale yellow oil: 1H NMR (CDCl3)
δ 2.50 (s, 6H), 6.77 (d, J ) 2.0 Hz, 1H), 6.93 (d, J ) 8.0 Hz,
1H), 6.99 (d, J ) 8.0 Hz, 1H), 7.62 (d, J ) 2 Hz, 1H).
To a solution of 4b (3.5 g, 23.9 mmol) in anhydrous
tetrahydrofuran (50 mL) was added n-butyllithium (1.6 M
solution in n-hexane; 18.4 mL, 28.7 mmol) at -30 °C under a
nitrogen atmosphere. After being stirred for 15 min at this
temperature, the mixture was treated by dropwise addition
of N,N-dimethylformamide (5.6 mL, 81.5 mmol). This mixture
was allowed to warm to room temperature for 30 min, and
then was quenched with saturated aqueous ammonium chlo-
ride and extracted with ethyl acetate. The organic extract was
washed with brine, dried, and evaporated. The resulting solid
residue was washed with n-hexane to afford 7b (2.3 g, 16%;
three steps) as a pale yellow solid: mp 84-85 °C; 1H NMR
(CDCl3) δ 2.53 (s, 6H), 7.02 (d, J ) 6.8 Hz, 1H), 7.20 (d, J )
6.8 Hz, 1H), 7.59 (s, 1H), 9.85 (s, 1H).
Gen er a l P r oced u r e for P r ep a r a tion of Ben zofu r a n -2-
ca r ba ld eh yd e (Meth od B). 4,7-Dim eth ylben zofu r a n -2-
ca r ba ld eh yd e (7b). To a solution of 3,6-dimethylsalicylalde-
hyde (5) (17.4 g, 116 mmol) in N,N-dimethylformamide (200
mL) were added bromoacetaldehyde diethyl acetal (17.8 mL,
118 mmol) and potassium carbonate (32 g, 232 mmol) at room
temperature. The mixture was stirred for 2.5 h at 150 °C. After
cooling to room temperature, the mixture was diluted with
water and then extracted with ethyl acetate. The organic
extract was washed with brine, dried, and evaporated. The
crude residue was purified by flash column chromatography
on silica gel, eluting with 10% ethyl acetate in n-hexane, to
afford 6 as a pale yellow oil (23.4 g).
To a solution of (7-fluoro-4-trifluoromethyl-2,3-dihydroben-
zofuran-2-yl)methanol (41 g, 173 mmol) in pyridine (130 mL)
was added acetic anhydride (21 mL, 225 mmol) at room
temperature, and the mixture was stirred for 4 h at the same
temperature under
a nitrogen atmosphere. The reaction
mixture was poured into 10% aqueous hydrochloric acid, and
the mixture was extracted with ethyl acetate. The combined
organic phases were washed with brine, then dried and
evaporated. The residue was purified by flash column chro-
matography on silica gel, eluting with 10% ethyl acetate in
n-hexane to give 11b (37.1 g, 66%, three steps) as a pale yellow
oil: 1H NMR (CDCl3) δ 2.08 (s, 3H), 3.20 (dd, J ) 6.8, 16.4
Hz, 1H), 3.52 (dd, J ) 9.6, 16.4 Hz, 1H), 4.30 (dd, J ) 6.4,
12.4 Hz, 1H), 4.36 (dd, J ) 3.6, 12.4 Hz, 1H), 5.14-5.24 (m,
1H), 7.02 (t, J ) 8.8 Hz, 1H), 7.10 (dd, J ) 4.8, 8.8 Hz, 1H).
A solution of 6 (23.4 g, 98.2 mmol) in AcOH (120 mL) was
refluxed for 8 h under a nitrogen atmosphere. After cooling to
room temperature, the mixture was poured into saturated
aqueous sodium hydrogen carbonate and then extracted with
ethyl acetate. The organic extract was washed with brine,
dried, and evaporated. The resulting solid residue was washed
with n-hexane to afford 7b (7.8 g, 39%) as a pale yellow solid.
The proton NMR spectra, mp and Rf value were identical with
those of 7b prepared by method A.
(7-F lu or o-4-t r iflu or om et h ylb en zofu r a n -2-yl)m et h a -
n ol (12b). To a solution of 11b (37.1 g, 133 mmol) in carbon

2934 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15
Yoshimura et al.
tetrachloride (320 mL) were added N-bromosuccinimide (23.7
g, 133 mmol) and 2,2′-azobis(isobutyronitrile) (285 mg, 1.7
mmol) at room temperature. The mixture was stirred for 105
min at 60 °C under a nitrogen atmosphere. After the reaction
mixture cooled to 0 °C, the solid was filtered through a glass
filter and was washed with hexane. The organic filtrates were
combined and concentrated under reduced pressure to afford
a residue, which was diluted with ethyl acetate, washed with
saturated aqueous sodium thiosulfate, brine, and then dried
and evaporated to give (3-bromo-7-fluoro-4-trifluoromethyl-2,3-
dihydrobenzofuran-2-yl)methyl acetate (44 g) as a brown oil.
To a stirred solution of (3-bromo-7-fluoro-4-trifluoromethyl-
2,3-dihydrobenzofuran-2-yl)methyl acetate (44 g, 123 mmol)
in 160 mL of 2-methyl-2-propanol was added potassium tert-
butoxide (1.0 M in 2-methyl-2-propanol solution; 135 mL, 135
mmol) at room temperature under a nitrogen atmosphere. This
mixture was stirred for 90 min at 60 °C. After cooling to room
temperature, the reaction mixture was poured into iced water
and extracted with ethyl acetate. The combined organic phases
were washed with brine, then dried and evaporated to give
crude (7-fluoro-4-trifluoromethylbenzofuran-2-yl)methyl ac-
etate (34 g) as a brown oil.
To a solution of (7-fluoro-4-trifluoromethylbenzofuran-2-yl)-
methyl acetate (34 g, 118 mmol) in 160 mL of methanol was
added potassium carbonate (24 g, 175 mmol) at room temper-
ature under a nitrogen atmosphere. This mixture was stirred
for 1 h at 60 °C. After cooling, the reaction mixture was diluted
with ethyl acetate, washed with brine, and then dried and
evaporated to give a crude brown oil. The residue was purified
by flash column chromatography on silica gel, eluting with 10-
20% ethyl acetate in n-hexane to give the (7-fluoro-4-trifluo-
romethylbenzofuran-2-yl)methanol (12b) 7.1 g (36%, three
steps) as a pale yellow oil: 1H NMR (CDCl3) δ 4.85 (d, J ) 7.0
Hz, 2H), 6.91 (brs, 1H), 7.10 (t, J ) 9.2 Hz, 1H), 7.47 (dd, J )
4.0, 8.8 Hz, 1H).
7-F lu or o-4-t r iflu or om e t h ylb e n zofu r a n -2-ca r b a ld e -
h yd e (7f). To a stirred solution of oxalyl chloride (7.48 mL,
85 mmol) in 300 mL of dichloromethane was added dimethyl
sulfoxide (12.1 mL, 170 mmol) for 5 min at -78 °C under a
nitrogen atmosphere. 12b (10.0 g, 43 mmol) in 20 mL of
dichloromethane was added to the reaction mixture at -78
°C, and the mixture was stirred for 40 min at the same
temperature. Triethylamine (35 mL, 251 mmol) was added to
the reaction mixture at -78 °C and stirred 15 min at the same
temperature. The reaction temperature was allowed to rise to
room temperature for 30 min. After the mixture was cooled to
-78 °C, water was added to the reaction mixture, and the
temperature was raised to room temperature. The mixture was
diluted with ethyl acetate, washed with water, and then dried
and evaporated to give a crude yellow oil (11 g). The residue
was purified by flash column chromatography on silica gel,
eluting with 10% ethyl acetate in n-hexane to give 7f (7.2 g,
73%) as a pale yellow solid: mp 58-59 °C; 1H NMR (CDCl3) δ
7.34 (t, J ) 8.8 Hz, 1H), 7.62 (ddd, J ) 0.8, 4.0, 8.4 Hz, 1H),
7.74 (dd, J ) 1.6, 2.8 Hz, 1H), 9.98 (s, 1H).
3.86 (s, 3H), 6.60 (d, J ) 8.4 Hz, 1H), 7.22 (d, J ) 8.4 Hz, 1H),
7.65 (s, 1H), 9.80 (s, 1H).
4,7-Diflu or oben zofu r an -2-car baldeh yde (7e). Compound
7e was synthesized from 2,5-difluorophenol (2e) following the
representative procedure described as method C and obtained
as a pale yellow oil in 3% yield (nine steps): 1H NMR (CDCl3)
δ 6.96 (dt, J ) 2.8, 8.8 Hz, 1H), 7.21 (ddd, J ) 4.0, 8.8, 9.6 Hz,
1H), 7.66 (d, J ) 2.4 Hz, 1H), 9.92 (s, 1H).
5,7-Dim et h ylb en zofu r a n -2-ca r b a ld eh yd e (7g). Com-
pound 7g was synthesized from 2,4-dimethylphenol (2g)
following the representative procedure described as method
A and obtained as a pale yellow solid in 32% yield (three
steps): mp 72-73 °C; 1H NMR (CDCl3) δ 2.21 (s, 3H), 2.52 (s,
3H), 7.14 (s, 1H), 7.32 (s, 1H), 7.50 (s, 1H), 9.82 (s, 1H).
4,5,7-Tr im eth ylben zofu r a n -2-ca r ba ld eh yd e (7h ). Com-
pound 7h was synthesized from 2,4,5-trimethylphenol (2h )
following the representative procedure described as method
A and obtained as a pale yellow solid in 53% yield (three
steps): mp 121-122 °C; 1H NMR (CDCl3) δ 2.33 (s, 3H), 2.43
(s, 3H), 2.50 (s, 3H), 7.13 (s, 1H), 7.57 (s, 1H), 9.83 (s, 1H).
6,7-Dim et h ylb en zofu r a n -2-ca r b a ld eh yd e (7i). Com-
pound 7i was synthesized from 2,3-dimethylphenol (2i) fol-
lowing the representative procedure described as method A
and obtained as a pale yellow solid in 9.8% yield (three
steps): mp 81-82 °C; 1H NMR (CDCl3) δ 2.42 (s, 3H), 7.24 (t,
J ) 7.6 Hz, 1H), 7.32 (d, J ) 7.6 Hz, 1H), 7.56 (s, 1H), 7.57 (d,
J ) 7.6 Hz, 1H), 9.88 (s, 1H).
4,7-Dim eth ylben zoth ioph en e-2-car baldeh yde (7j). Com-
pound 7j was synthesized from 2,5-dimethylthiophenol (2j)
following the representative procedure described as method
A and obtained as a pale yellow solid in 10% yield (three
steps): mp 97-98 °C; 1H NMR (CDCl3) δ 2.54 (s, 3H), 2.64 (s,
3H), 7.15 (d, 1H), 7.21 (d, 1H), 10.21 (s, 1H).
5,7-Dim eth ylben zoth ioph en e-2-car baldeh yde (7k). Com-
pound 7k was synthesized from 2,4-dimethylthiophenol (2k )
following the representative procedure described as method
A and obtained as a pale yellow solid in 14% yield (three
steps): mp 66-67 °C; 1H NMR (CDCl3) δ 2.44 (s, 3H), 2.56 (s,
3H), 7.14 (s, 1H), 7.58 (s, 1H), 7.99 (s, 1H), 10.16 (s, 1H).
Gen er a l P r oced u r e for P r ep a r a tion of P yr r olylben -
zoic Acid 1. Meth yl 4-[4-(4,7-Dim eth ylben zofu r a n -2-yl)-
4-oxobu ta n oyl]ben zoa te (14b). To a solution of 7b (5.5 g,
31.6 mmol) in 100 mL of N,N-dimethylformamide were added
methyl 4-acryloylbenzoate (6.0 g, 31.6 mmol) (13), triethy-
lamine (5.3 mL, 37.9 mmol) and 3-benzyl-5-(2-hydroxyethyl)-
4-methylthiazolium chloride (1.7 g, 6.3 mmol) at room tem-
perature. The mixture was stirred for 30 min at 80 °C. After
cooling to room temperature, the reaction mixture was diluted
with water, extracted with ethyl acetate, washed with brine,
and then dried and evaporated to give a crude solid. It was
recrystallized with methanol to give 14b (9.5 g, 83%) as a pale
1
yellow solid: mp 131 °C (methanol); H NMR (CDCl3) δ 2.50
(s, 3H), 2.51 (s, 3H), 3.45-3.55 (m, 4H), 3.94 (s, 3H), 7.00 (d,
J ) 6.8 Hz, 1H), 7.16 (d, J ) 6.8 Hz, 1H), 7.62 (s, 1H), 8.09 (d,
J ) 8.4 Hz, 2H), 8.14 (d, J ) 8.4 Hz, 2H).
7-Meth ylben zofu r a n -2-ca r ba ld eh yd e (7a ). Compound
7a was synthesized from 2-methylphenol (2a ) following the
representative procedure described as method A and obtained
as a pale yellow solid in 9% yield (three steps): mp 58-59 °C;
1H NMR (CDCl3) δ 2.58 (s, 3H), 7.24 (t, J ) 7.6 Hz, 1H), 7.32
(d, J ) 7.6 Hz, 1H), 7.56 (s, 1H), 7.57 (d, J ) 7.6 Hz, 1H), 9.88
(s, 1H). The proton NMR spectral data were identical with
those reported.17
4-[5-(4,7-Dim et h ylb en zofu r a n -2-yl)p yr r ol-2-yl]b en zo-
ic Acid (1b). To a suspension of 14b (9.5 g, 26 mmol) in
methanol (40 mL) was added ammonium acetate (20 g, 260
mol) at room temperature. After stirring under reflux for 2 h,
the mixture was diluted with water and cooled to 0 °C. The
precipitate was collected by filtration to give methyl 4-[5-(4,7-
dimethylbenzofuran-2-yl)pyrrol-2-yl]benzoate (8.7 g, 97%) as
1
a pale yellow solid: mp 177-178 °C; H NMR (CDCl3) δ 2.48
4,7-Dich lor ob en zofu r a n -2-ca r b a ld eh yd e (7c). Com-
pound 7c was synthesized from 2,5-dichlorophenol (2c) fol-
lowing the representative procedure described as method A
and obtained as a pale yellow solid in 31% yield (three steps):
mp 155-159 °C; H NMR (CDCl3) δ 7.29 (d, J ) 8.4 Hz, 1H),
7.46 (d, J ) 8.4 Hz, 1H), 9.84 (s, 1H).
7-Meth yl-4-m eth oxyben zofu r a n -2-ca r ba ld eh yd e (7d ).
Compound 7d was synthesized from 2-methyl-5-methoxyphe-
nol (2d ) following the representative procedure described as
method A and obtained as a pale yellow solid in 20% yield
(three steps): mp 100-101 °C; 1H NMR (CDCl3) δ 2.24 (s, 3H),
(s, 3H), 2.55 (s, 3H), 3.93 (s, 3H), 6.72-6.77 (m, 2H), 6.83 (s,
1H), 6.93 (d, J ) 6.8 Hz, 1H), 6.97 (d, J ) 6.8 Hz, 1H), 7.63 (d,
J ) 8.4 Hz, 2H), 8.07 (d, J ) 8.4 Hz, 2H).
To a solution of methyl 4-[5-(4,7-dimethylbenzofuran-2-yl)-
pyrrol-2-yl]benzoate (8.7 g, 25.2 mmol) in ethanol (100 mL)
was added 5 N aqueous sodium hydroxide (20 mL) solution at
room temperature, and this mixture was refluxed for 30 min.
After cooling to room temperature it was diluted with water
(150 mL) and acidified with 5 N aqueous hydrochloric acid.
The precipitate was collected by filtration and dried to give
crude 1b as a yellow solid (8.1 g, 98%). It was recrystallized
1

Novel and Potent RARR Agonists
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15 2935
from ethanol-water to give a yellow solid: mp 271 °C dec; 1H
NMR (DMSO-d6) δ 2.43 (s, 3H), 2.46 (s, 3H), 6.71 (t, J ) 2.4
Hz, 1H), 6.84 (t, J ) 2.4 Hz, 1H), 6.92 (d, J ) 7.2 Hz, 1H),
6.96 (d, J ) 7.2 Hz, 1H), 7.23 (s, 1H), 7.89 (d, J ) 8.4 Hz, 2H),
7.95 (d, J ) 8.4 Hz, 2H), 11.81 (brs, 1H), 12.85 (brs, 1H). Anal.
(C21H17NO3‚0.2H2O) C, H, N.
4-[5-(7-Meth ylben zofu r an -2-yl)pyr r ol-2-yl]ben zoic Acid
(1a ). Compound 1a was synthesized from 7-methylbenzofuran-
2-carbaldehyde (7a ) following the general procedure described
previously and obtained as a pale yellow solid in 53% yield
(three steps): mp 264-266 °C (ethanol-water); 1H NMR
(DMSO-d6) δ 2.52 (s, 3H), 6.71-6.74 (m, 1H), 6.83-6.86 (m,
1H), 7.06 (d, J ) 7.2 Hz, 1H), 7.12 (t, J ) 7.2 Hz, 1H), 7.18 (s,
1H), 7.43 (d, J ) 7.2 Hz, 1H), 7.89 (d, J ) 8.4 Hz, 2H), 7.95 (d,
found, 5.29. HRMS Calcd for C22H19NO3 (MH+): 345.1365.
Found: 345.1378.
4-[5-(6,7-Dim et h ylb en zofu r a n -2-yl)p yr r ol-2-yl]b en zo-
ic Acid (1i). Compound 1i was synthesized from 6,7-dimeth-
ylbenzofuran-2-carbaldehyde (7i) following the general proce-
dure described previously and obtained as a pale yellow solid
in 43% yield (three steps): mp 278-280 °C (ethanol-water);
1H NMR (DMSO-d6) δ 2.30 (s, 3H), 2.42 (s, 3H), 6.69-6.72 (m,
1H), 6.81-6.84 (m, 1H), 7.02 (d, J ) 8.4 Hz, 1H), 7.11 (s, 1H),
7.30 (d, J ) 8.4 Hz, 1H), 7.86 (d, J ) 8.4 Hz, 2H), 7.94 (d, J )
8.4 Hz, 2H), 11.78 (s, 1H), 12.80 (brs, 1H). Anal. (C21H17NO3‚
0.2H2O) C, H, N.
4-[5-(4,7-Dim eth ylben zoth ioph en e-2-yl)pyr r ol-2-yl]ben -
zoic Acid (1j). Compound 1j was synthesized from 4,7-
dimethylthiophene-2-carbaldehyde (7j) following the general
procedure described previously and obtained as a pale yellow
solid in 14% yield (three steps): mp 280-281 °C (ethanol-
water); 1H NMR (DMSO-d6) δ 2.42 (s, 3H), 2.54 (s, 3H), 6.56-
6.59 (m, 1H), 6.78-6.81 (m, 1H), 7.02 (d, J ) 6.8 Hz, 1H), 7.08
(d, J ) 6.8 Hz, 1H), 7.89 (s, 1H), 7.90 (d, J ) 8.4 Hz, 2H), 7.94
J ) 8.4 Hz, 2H), 11.83 (s, 1H), 12.82 (brs, 1H). Anal. (C20H15
NO3‚0.2H2O) C, H, N.
-
4-[5-(4,7-Dich lor ob en zofu r a n -2-yl)p yr r ol-2-yl]b en zo-
ic Acid (1c). Compound 1c was synthesized from 4,7-dichlo-
robenzofuran-2-carbaldehyde (7c) following the general pro-
cedure described previously and obtained as a yellow solid in
38% yield (three steps): mp 276-279 °C (ethanol-water); 1H
NMR (DMSO-d6) δ 6.83 (t, J ) 2.4 Hz, 1H), 6.89 (t, J ) 2.4
Hz, 1H), 7.35 (d, J ) 7.2 Hz, 1H), 7.38 (d, J ) 7.2 Hz, 1H),
7.39 (s, 1H), 7.91 (d, J ) 8.4 Hz, 2H), 7.97 (d, I ) 8.4 Hz, 2H),
12.02 (brs, 1H), 12.86 (brs, 1H). Anal. (C19H11NO3Cl2‚0.3H2O)
C, H, N.
(d, J ) 8.4 Hz, 2H), 11.76 (s, 1H), 12.83 (brs, 1H). Anal. (C21H17
NO2S‚0.2H2O) C, H, N.
-
4-[5-(5,7-Dim eth ylben zoth ioph en e-2-yl)pyr r ol-2-yl]ben -
zoic Acid (1k ). Compound 1k was synthesized from 5,7-
dimethylthiophene-2-carbaldehyde (7k ) following the general
procedure described previously and obtained as a pale yellow
solid in 26% yield (three steps): mp 261-262 °C (ethanol-
water); 1H NMR (DMSO-d6) δ 2.36 (s, 3H), 2.42 (s, 3H), 6.54-
6.56 (m, 1H), 6.77-6.79 (m, 1H), 6.96 (s, 1H), 7.43 (s, 1H), 7.71
(s, 1H), 7.88 (d, J ) 8.4 Hz, 2H), 7.93 (d, J ) 8.4 Hz, 2H),
11.76 (s, 1H), 12.76 (brs, 1H). Anal. (C21H17NO2S‚0.5H2O) C,
H, N.
4-[5-(7-Meth yl-4-m eth oxyben zofu r a n -2-yl)p yr r ol-2-yl]-
ben zoic Acid (1d ). Compound 1d was synthesized from
7-methyl-4-methoxybenzofuran-2-carbaldehyde (7d ) following
the general procedure described previously and obtained as a
yellow solid in 55% yield (three steps): mp 262-263 °C
1
(ethanol-water); H NMR (DMSO-d6) δ 2.63 (s, 3H), 3.92 (s,
Biology. 1. Bin d in g Stu d y. Binding assays were per-
formed as described in a previous report.16c Compounds were
tested in log dilutions of 5.0 × 10-7 to 5.0 × 10-11 M with
duplicate determinations at each concentration. Binding in the
presence of a 1000-fold excess of unlabeled ligand was defined
as nonspecific binding. Specific binding was defined as the total
binding minus the nonspecific binding, and the 50% inhibitory
dose (IC50) values were obtained from logarithmic plots. The
selectivity of compounds for each receptor is indicated as
relative IC50, where the IC50 value of each compound for a
receptor was divided by that of ATRA. The values given in
Table 1 are the average of at least two experiments.
2. Tr a n sa ctiva tion Assa y. Transient transactivation as-
says for each receptor were also performed by the method
previously reported.16c Compounds were tested in log dilutions
of 3.0 × 10-6 to 1.0 × 10-10 M with duplicate determinations
at each concentration. Receptor and reporter vectors were
transfected into COS-1 cells by the Lipofection method. After
4 h of incubation, the medium was replaced with DMEM
supplemented with 10% FBS and incubation was continued
for an additional 20 h. The cells were then suspended in
DMEM supplemented with 10% FBS and seeded at 3 × 104/
well in 96-well plates. After 6 h of incubation, compounds at
various concentrations were added to duplicate wells. The
plates were incubated for a further 48 h, and then the cell
supernatants were assayed for PLAP activity. The values given
in Table 1 are the average of at least two experiments.
3. In h ibition of LP S-In d u ced Mou se B-Lym p h ocyte
P r olifer a tion Assa y. Mouse splenocyte proliferation assay
induced by E. coli lipopolysaccharide (LPS), a well-known
murine B-lymphocyte proliferation inducer, was performed
according to the method previously reported with slight
modifications.11 Compounds were tested in log dilutions of 1.0
× 10-6 to 1.0 × 10-12 M with triplicate determinations at each
final concentration. BALB/c spleen cell suspensions were
placed at 1.2 × 105 cells/180 µL/well in 96-well culture plates,
in RPMI-1640 medium containing antibiotics, 50 µM 2-mer-
captoethanol, 10% FBS and 5 µg/mL LPS. Then 20 µL of each
compound serially diluted was added to the wells and the
plates were incubated for 3 days at 37 °C, 5% CO2. Splenocytes
were pulsed with 20 µL of 0.5 µCi of [3H]thymidine for further
6 h. After incubation, cells were harvested on glass fiber filters
3H), 6.77-6.82 (m, 3H), 7.24 (d, J ) 8.0 Hz, 1H), 7.86-7.95
(m, 5H), 8.13 (d, J ) 8.8 Hz, 1H), 8.28 (s, 1H), 11.62 (s, 1H).
Anal. (C21H17NO4‚0.3H2O) C, H, N.
4-[5-(4,7-Diflu or oben zofu r a n -2-yl)p yr r ol-2-yl]ben zoic
Acid (1e). Compound 1e was synthesized from 4,7-difluo-
robenzofuran-2-carbaldehyde (7e) following the general pro-
cedure described previously and obtained as a yellow solid in
47% yield (three steps): mp >300 °C (ethanol-water); 1H NMR
(DMSO-d6) δ 6.82 (dd, J ) 2.4, 3.6 Hz, 1H), 6.86 (dd, J ) 2.4,
3.6 Hz, 1H), 7.08 (dd, J ) 3.2, 8.8 Hz, 1H), 7.19 (dd, J ) 3.2,
8.8 Hz, 1H), 7.42 (d, J ) 2.4 Hz, 1H), 7.92 (d, J ) 8.4 Hz, 2H),
7.96 (d, J ) 8.4 Hz, 2H), 12.08 (s, 1H). HRMS Calcd for C19H11
-
NO3F2 (MH+): 339.0707. Found: 339.0698.
4-[5-(7-F lu or o-4-t r iflu or om et h ylb en zofu r a n -2-yl)p yr -
r ol-2-yl]ben zoic Acid (1f). Compound 1f was synthesized
from 7-fluoro-4-trifluoromethylbenzofuran-2-carbaldehyde (7f)-
following the general procedure described previously and
obtained as a pale yellow solid in 44% yield (three steps): mp
215-217 °C (acetone-water); 1H NMR (DMSO-d6) δ 6.90 (brs,
2H), 7.35 (t, J ) 8.8 Hz, 1H), 7.49 (brs, 1H), 7.63 (dd, J ) 3.6,
8.8 Hz, 1H), 7.91 (d, J ) 8.4 Hz, 2H), 7.97 (d, J ) 8.4 Hz, 2H),
12.08 (brs, 1H). Anal. (C20H11F4NO3‚0.5H2O) C, H, N.
4-[5-(5,7-Dim et h ylb en zofu r a n -2-yl)p yr r ol-2-yl]b en zo-
ic Acid (1g). Compound 1g was synthesized from 5,7-
dimethylbenzofuran-2-carbaldehyde (7g) following the general
procedure described previously and obtained as a pale yellow
solid in 41% yield (three steps): mp 262-263 °C (ethanol-
water); 1H NMR (DMSO-d6) δ 2.32 (s, 3H), 2.45 (s, 3H), 6.68-
6.71 (m, 1H), 6.80-6.83 (m, 1H), 6.88 (d, J ) 1.2 Hz, 1H), 7.10
(s, 1H), 7.20 (d, J ) 1.2 Hz, 1H), 7.86 (d, J ) 8.4 Hz, 2H), 7.93
(d, J ) 8.4 Hz, 2H), 11.78 (s, 1H), 12.80 (brs, 1H). Anal. (C21H17
NO3‚0.2H2O) C, H, N.
-
4-[5-(4,5,7-Tr im eth ylben zofu r a n -2-yl)p yr r ol-2-yl]ben -
zoic Acid (1h ). Compound 1h was synthesized from 4,5,7-
trimethylbenzofuran-2-carbaldehyde (7h ) following the general
procedure described previously and obtained as a pale yellow
solid in 71% yield (three steps): mp 267 °C dec (ethanol-
1
water); H NMR (DMSO-d6) δ 2.26 (s, 3H), 2.35 (s, 3H), 2.43
(s, 3H), 6.67-6.71 (m, 1H), 6.81-6.85 (m, 1H), 6.87 (s, 1H),
7.21 (s, 1H), 7.88 (d, J ) 8.4 Hz, 2H), 7.94 (d, J ) 8.0 Hz, 2H),
11.78 (brs, 1H). Anal. (C21H17NO3‚H2O) C, N; H: calcd, 5.85;

2936 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15
Yoshimura et al.
(7) Orfanos, C. E.; Zouboulis, C. C.; Almond-Roesler, B.; Geilen, C.
C. Current Use and Future Potential Role of Retinoids in
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and processed for â-plate counting. The 50% inhibitory con-
centration (IC50) values were calculated with a nonlinear
regression method. The inhibition of each compound is indi-
cated as relative IC50, where the IC50 value of the compound
was divided by that of ATRA. The values given in Table 1 are
means of relative IC50 ( SEM from triplicate determinations.
4. Mice. Female BALB/c mice were obtained from Charles
River J apan and used at 8-10 weeks of age. They were bred
in the company’s animal facility under SPF conditions and
allowed access to food and water ad libitum during the study.
5. An tibod y P r od u ction in Mice. BALB/c mice were
divided into groups each consisting of 5 animals. They were
immunized by intraperitoneal injection of 100 µg of dinitro-
phenyl-conjugated keyhole lympet hemocyanin (DNP-KLH)
emulsified in Freund’s complete adjuvant on day 0. Com-
pounds were orally administered to the mice at various doses
from day 0 to 9 post-immunization. Control mice were given
vehicle only during the same period. On day 10, mice were
bled from the orbital vein, and anti-DNP antibody (IgG2a
class) titer in the serum was determined by DNP-specific
ELISA assay.
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6. DNP -Sp ecific ELISA. A 96-well ELISA plate was coated
with 5 µg/mL of DNP-condjugated bovine serum albumin
(BSA) for 1 h at 37 °C. After blocking with 300 µL of blocking
buffer (1% BSA-phosphate-buffered saline (PBS)) for 2 h at
37 °C, 50 µL of either samples or standard serum, previously
diluted with ELISA buffer (1% BSA, 0.1% Tween 20-PBS), was
added and incubated for 1 h at 37 °C. After washing, plates
were added with 50 µL of peroxidase-conjugated anti-mouse
IgG2a, diluted with ELISA buffer at 1:2500, and incubated
further for 1 h at 37 °C. After washing, plates were developed
with 50 µL of 400 µg/mL o-phenylenediamine dihydrochloride.
Thirty minutes later, the reaction was stopped with 50 µL of
1 N sulfuric acid and the plates were read at 490 nm with an
automated microplate reader (BIO-TEK). The units of anti-
DNP titers in each sample were calculated with reference to
standard serum obtained from DNP-KLH-immunized mouse.
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mice were divided into groups each consisting of 5 animals.
The animals were immunized with subcutaneous injection of
1 × 108 of sheep red blood cells (SRBC) in their back.
Compounds were orally administered to the mice at various
doses from day 3 to 5 post-immunization. Control mice were
given vehicle only during the same period. On day 6, mice were
subcutaneously injected with 7 × 107 of SRBC in their right
food pad. Twenty-four hours later, thickness of both foot pads
was measured and paw swelling in each mice was calculated
by subtracting left from right foot pad thickness.
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