Identification of highly potent retinoic acid receptor α-selective antagonists

Article abstract of DOI:10.1021/jm9703911

The syntheses and full retinoid receptor characterization of a novel series of retinoic acid receptor α (RARα) antagonists, 1-5, are described. These compounds bind with high affinity to RARα but were completely inactive in gene transactivation. They were also potent and effective antagonists of retinoic acid (RA) induced gene transcription at RARα. Compounds 1-5 exhibited varying degrees of selectivity for RARα relative to RARβ/γ, with compound 5 being the most selective in both binding and functional antagonism assays. These compounds will be invaluable tools in delineating the physiological roles of RARα in development and in the adult animal and may themselves be useful therapeutic agents in human diseases associated with RARα.

Full text of DOI:10.1021/jm9703911

J . Med. Chem. 1997, 40, 2445-2451
2445
Expedited Articles
Id en tifica tion of High ly P oten t Retin oic Acid Recep tor r-Selective An ta gon ists
Min Teng,*,†,‡ Tien T. Duong,† Alan T. J ohnson,† Elliott S. Klein,§ Liming Wang,† Berket Khalifa,§ and
Roshantha A. S. Chandraratna*,†,§
Departments of Chemistry and Biology, Retinoid Research, Allergan Incorporated, Irvine, California 92623-9534
Received J une 12, 1997X
The syntheses and full retinoid receptor characterization of a novel series of retinoic acid
receptor R (RARR) antagonists, 1-5, are described. These compounds bind with high affinity
to RARR but were completely inactive in gene transactivation. They were also potent and
effective antagonists of retinoic acid (RA) induced gene transcription at RARR. Compounds
1-5 exhibited varying degrees of selectivity for RARR relative to RARâ/γ, with compound 5
being the most selective in both binding and functional antagonism assays. These compounds
will be invaluable tools in delineating the physiological roles of RARR in development and in
the adult animal and may themselves be useful therapeutic agents in human diseases associated
with RARR.
Ch a r t 1
In tr od u ction
Retinoids are small-molecule hormones that affect a
variety of fundamental biological process such as cell
differentiation and proliferation and apoptosis.1 Ret-
inoids elicit their biological effects by regulating gene
transcription through a series of nuclear receptors which
are ligand-inducible transcription factors belonging to
the steroid receptor superfamily.2 The retinoid recep-
tors are classified into two families, the retinoic acid
receptors (RARs)3 and the retinoid X receptors (RXRs),4
each consisting of three distinct subtypes (R, â, and γ).
The RARs function in vivo as RAR-RXR heterodimers.5
all-trans-Retinoic acid (RA) (Chart 1) is the physiological
hormone for the RARs.2 RA binds with approximately
equal affinity to all three RAR subtypes and effectively
activates gene transcription through all RAR-RXR
heterodimers.
Ch a r t 2
Ch a r t 3
Retinoids are extensively used in dermatology6 and
have shown therapeutic promise in other disease areas
including oncology.7 However, the clinical use of non-
selective retinoids, compounds that activate the full
range of RAR-mediated pathways, is invariably ac-
companied by a broad spectrum of toxic side effects.8
Since the individual RAR subtypes have distinct tissue
distribution patterns9 and appear to regulate different
subsets of genes,10 compounds that are selective for
these subtypes will have more restricted pharmacologi-
cal effects and better therapeutic indices in specific
disease applications. In addition, such selective retin-
oids, both agonists and antagonists, will be invaluable
tools in understanding the physiology associated with
each RAR subtype and in identifying newer disease
applications.
41-5253 (Chart 1) is an RARR selective antagonist, but
must be present in approximately 1000-fold molar
excess to completely suppress the activity induced by
RA.12 We have previously reported the discovery of
AGN 193109 (Chart 2), a potent RAR antagonist.11a
More recently, we identified the RARR specific agonist
AGN 193836 (Chart 2).13 In this paper we present the
results of our studies which combined the structural
features responsible for RAR antagonism with those
affording RARR selectivity to produce the highly potent
and selective antagonists 1-5 (Chart 3) of RARR func-
tion. Of particular interest are compounds 4 and 5
which completely abrogate RA-induced gene transcrip-
Although several classes of RAR antagonists have
been reported in the literature,11 truly potent RAR
subtype selective antagonists remain elusive targets. Ro
† Department of Chemistry.
‡Current address: Alanex Corporation, 3550 General Atomics
Court, San Diego, CA 92121-1194.
§ Department of Biology.
X Abstract published in Advance ACS Abstracts, J uly 15, 1997.
S0022-2623(97)00391-9 CCC: $14.00 © 1997 American Chemical Society

2446 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16
Teng et al.
Sch em e 1a
Resu lts a n d Discu ssion
The binding affinities of compounds 1-5 were mea-
sured using baculovirus-expressed RARs and RXRs.14
None of the compounds showed any affinity to the RXRs
(data not shown). All of the compounds bound with high
affinity to RARR, having values comparable to or higher
than RA (Table 1). In contrast, compounds 1-5 dis-
played much lower affinity to RARâ and RARγ, and with
varying degrees of selectivity. In the dihydronaphtha-
lene series, compound 1 showed approximately 40-100-
fold higher affinity to RARR relative to RARâ and RARγ.
Introduction of a fluorine atom ortho to the carboxylic
acid as in 2 increased the affinity to RARR and displayed
greater R/â selectivity. The difluoro analog 3 was found
to be even more RARR selective, having an R/â-selectiv-
ity of approximately 100-fold, and a preference for RARR
relative to RARγ approaching 1000-fold. In the di-
methylchromene series, the monofluoro analog 4 exhib-
ited very high affinity and selectivity for RARR. The
corresponding difluoro analog 5 was the most interest-
ing in the series. In addition to an approximately 20-
fold higher affinity for RARR than the natural hormone
RA, compound 5 also exhibited true pharmacological
specificity for RARR by binding to RARR with ap-
proximate 1000-fold and 10000-fold higher affinity than
to RARâ and RARγ, respectively.
The ability of retinoids 1-5 to induce gene transcrip-
tion was determined using CV-1 cells transfected with
individual RAR holoreceptors and an MTV-4(R5G)-
luciferase reporter.15 While RA effectively transacti-
vated all three RAR subtypes, compounds 1-5 had
absolutely no activity at any of the RARs (Figure 1).
These data, when viewed with the receptor binding data
(Table 1), suggested that compounds 1-5 would act as
effective RARR-selective antagonists. As a result, func-
tional antagonism assays were performed using the
transactivation assays described above and a constant
dose (10-8 M) of RA as the test agonist (Figure 2).
Compounds 1 and 2 were effective antagonists of RA
function at RARR, inhibiting RA-induced gene tran-
scription by approximately 75% at equimolar concentra-
tions and completely abrogating RA activity at a 10-
fold molar excess. However, at higher concentrations,
compounds 1 and 2 also inhibited RA-induced activity
at RARâ and RARγ by approximately 35%. The data
for compound 3 reveal a somewhat less potent RA
antagonist at RARR, requiring ∼100-fold molar excess
to completely suppress RA activity. In the dimethyl-
chromene series, 4 and 5 were very potent antagonists
at RARR in accord with their high binding affinities to
this receptor subtype. Compound 4 was the most potent
antagonist at RARR, showing complete inhibition at a
concentration 10-fold lower than the test agonist. At
RARâ and RARγ, inhibition was observed only at higher
concentrations. Consistent with Kd values, compound
5 was also found to be a potent antagonist at RARR by
completely inhibiting RA activity at approximately
equimolar concentrations. It is also a very selective
antagonist since even at 1 µM concentration (100-fold
molar excess) it had no effect on RA activity at RARγ
and only partially blocked activity at RARâ.
a
(a) Mg0/4-bromotoluene/THF/1
h (62%); (b) p-TsOH‚H2O/
benzene/80 °C/2 h (81%); (c) t-BuLi/THF/-78 °C/CO2, then HCl/
-78 °C to room temperature (75%); (d) EDC/DMAP/DMF/4-
H2NC6H4CO2Et (10, 74%), or 2-F-4-H2NC6H3CO2Et (11, 50%), or
2,6-F2-4-H2NC6H2CO2Et (12, 19%); (e) NaOH/MeOH/H2O (1, 73%;
2, 72%; 3, 70%).
tion through RARR at 10-fold lower and equimolar
concentrations, respectively.
Ch em istr y
The synthesis of compounds 1-3 are described in
Scheme 1. Grignard reaction of p-tolylmagnesium
bromide and bromotetralone 611a gave the corresponding
alcohol 7. Dehydration of 7 using catalytic p-TsOH
afforded dihydronaphthalene 8. Compound 8 was sub-
jected to lithium-halogen exchange followed by a CO2
quench to afford the carboxylic acid 9 in good yield.
Coupling of 9 with the appropriate aminobenzoates
afforded the amides 10-12, which after hydrolysis
under basic conditions gave the corresponding acids
1-3.
Compounds 4 and 5 were prepared according to
Scheme 2. Dihydrocoumarin (13) was exposed to excess
methylmagnesium chloride and the resultant tertiary
alcohol cyclized using aqueous H2SO4 to give chroman
14. Acylation of 14 under Friedel-Crafts conditions
afforded methyl ketone 15. Oxidation of 15 using
NaOCl followed by Fisher esterification provided ethyl
ester 16 in 92% overall yield. Bromination of 16 in
acetic acid afforded 17 which was then oxidized using
CrO3 in HOAc to yield 18. Addition of p-tolylmagne-
sium bromide to ketone 18, followed by p-TsOH-
catalyzed dehydration, gave 19. Hydrolysis of the ethyl
ester followed by coupling of the resulting acid 20 with
the appropriate 4-aminobenzoate13 gave 21 and 22. A
second hydrolysis of the terminal ethyl ester group with
base afforded compounds 4 and 5.
In summary, we have identified a series of high-
affinity RARR ligands which are also highly selective
antagonists of RA-induced gene transcriptional activity
at RARR. These compounds, together with the previ-

Retinoic Acid Receptor R-Selective Antagonists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16 2447
Sch em e 2a
a
(a) MeMgCl/THF/then H2SO4/H2O/100 °C (100%); (b) CH3COCl/AlCl3/CH2Cl (54%); (c) NaOCl/NaOH/dioxane/65 °C then EtOH/
H2SO4/∆ (92%); (d) Br2/HOAc (100%); (e) CrO3/HOAc/Ac2O (76%); (f) p-tolyl-MgBr/THF then p-TsOH‚H2O/toluene/∆ (25%); (g) NaOH/
EtOH/H2O (98%); (h) EDC/DMAP/DMF/2-F-4-H2NC6H3CO2Et (21, 63%), or 2,6-difluoro-4-H2NC6H2CO2Et (22, 33%); (i) NaOH/EtOH/
H2O (4, 86%; 5, 70%).
Ta ble 1. Binding Affinity (Kd) of Retinoids to RARR/â/γa
chromatography (hexane: EtOAc, 96:4) afforded 3.4 g (62%)
of the desired product as a colorless solid: 1H NMR δ 7.36 (1H,
dd, J ) 2.1, 7.6 Hz), 7.26 (3H, m), 7.12 (3H, s), 2.34 (3H, s),
2.24-2.04 (2H, m), 1.81 (1H, m), 1.55 (1H, m), 1.35 (3H, s),
1.30 (3H, s).
7-Br om o-3,4-d ih yd r o-4,4-d im eth yl-1-(4-m eth ylp h en yl)-
n a p h th a len e (8). To a solution of compound 7 (3.4 g, 9.85
mmol) in benzene (40 mL) in a flask equipped with a Dean-
Stark trap was added catalytic amount of p-toluenesulfonic
acid. The resulting solution was heated at reflux for 2 h. The
resulting solution was cooled to room temperature, extracted
with Et2O, dried (MgSO4) and concentrated under reduced
pressure. Column chromatography (hexanes) afforded 2.63 g
(81%) of the desired compound as a colorless solid: 1H NMR
δ 7.32 (1H, dd, J ) 2.1, 8.2 Hz), 7.21 (5H, m), 7.15 (1H, d, J )
2.1 Hz), 5.98 (1H, t, J ) 4.7 Hz), 2.40 (3H, s), 2.32 (2H, d, J )
4.7 Hz), 1.30 (6H, s). Anal. (C19H19Br) C, H.
5,6-Dih yd r o-5,5-d im eth yl-8-(4-m eth ylp h en yl)-2-n a p h -
th a len eca r boxylic Acid (9). To a solution of 8 (250.0 mg,
0.764 mmol) in THF (2.0 mL) at -78 °C was added tert-
butyllithium (1.0 mL, 1.68 mmol, 1.7 M solution in pentane).
After 1 h of stirring at -78 °C, gaseous CO2 was bubbled
through the reaction for 1 h. The reaction mixture was
warmed to room temperature and quenched by the addition
of 10% aqueous HCl. Extraction with EtOAc, followed by
drying (Na2SO4) of the combined organic layers, and concen-
tration under reduced pressure afforded 167 mg (75%) of the
desired compound as a colorless solid: 1H NMR δ 7.94 (1H,
dd, J ) 1.8, 8.1 Hz), 7.76 (1H, d, J ) 1.8 Hz), 7.45 (1H, d, J )
8.1 Hz), 7.24 (4H, m), 6.01 (1H, t, J ) 4.7 Hz), 2.40 (3H, s),
2.36 (2H, d, J ) 4.7 Hz), 1.35 (6H, s). Anal. (C20H20O2) C, H.
Eth yl 4-[[[5,6-Dih ydr o-5,5-dim eth yl-8-(4-m eth ylph en yl)-
2-n a p h th a len yl]ca r bon yl]a m in o]ben zoa te (10). A solu-
tion of 9 (170.0 mg, 0.58 mmol), ethyl 4-aminobenzoate (115.0
mg, 0.70 mmol), 1-[3-(dimethylamino)propyl]-3-ethylcarbodi-
imide hydrochloride (145.0 mg, 0.76 mmol) and 4-(dimethyl-
amino)pyridine (92.4 mg, 0.76 mmol) in DMF (6.0 mL) was
stirred at room temperature for12 h. The solution was diluted
with EtOAc, washed with H2O, dried (MgSO4), and concen-
trated to give an oil. Column chromatography (hexanes:
EtOAc, 85:15) afforded 187 mg (74%) of the desired product
as a colorless solid: 1H NMR δ 8.02 (2H, d, J ) 8.7 Hz), 7.72
(2H, m), 7.65 (2H, d, J ) 8.7 Hz), 7.52 (1H, d, J ) 1.8 Hz),
7.48 (1H, d, J ) 8.0 Hz), 7.25 (4H, m), 6.15 (1H, t, J ) 4.9
Hz), 4.36 (2H, q, J ) 7.1 Hz), 2.40 (3H, s), 2.38 (2H, d, J ) 4.9
Hz), 1.39 (3H, t, J ) 7.1 Hz), 1.37 (6H, s); 13C NMR δ 166.1,
165.8, 149.8, 142.0, 138.6, 137.2, 134.7, 132.2, 130.8, 129.2,
128.4, 127.2, 126.2, 126.0, 124.4, 124.3, 119.0, 77.4, 77.2, 77.0,
76.8, 76.6, 60.9, 38.6, 34.0, 28.0, 21.2, 14.3; MS (EI) m/e 439,
275 (base peak), 232.
RAR
entry
R
â
γ
retinoic acid
15 ( 1.7
27 ( 15
13 ( 2.5
1020 ( 316
622 ( 185
2256 ( 1257
320 ( 87
18 ( 1
1
2
3
4
5
3121 ( 1457
863 ( 138
5.7 ( 0.9
32 ( 15
>30000
7258 ( 2648
10618 ( 3688
2.8 ( 1.1
1.5 ( 0.8
898 ( 362
a
Kd values (mean ( SEM of triplicate determinations) were
determined via competition of [3H]-(all-E)-retinoic acid (5 nM)
binding with unlabeled test retinoid at baculovirus expressed
RARs and application of the equation of Cheng and Prussof.14c
ously identified RARR-specific agonists,13a will be very
powerful tools in elucidating the physiological roles of
RARR in development and in the adult animal. Com-
pounds of this type could also be effective therapeutic
agents in diseases where the inappropriate activation
of RARR is pathogenic.
Exp er im en ta l Section
Gen er a l Meth od s. 1H NMR spectra were recorded using
a Varian Gemini 300 spectrometer (300 MHz) in CDCl3 unless
otherwise indicated. Mass spectra were recorded using VG-
analytical 7070E organic mass spectrometer. High-resolution
mass spectra were recorded using VG autospec 3000 mass
spectrometer. Elemental analyses were performed by Rob-
ertson Microlit Laboratories, Inc., Madison, NJ . Thin layer
chromatography (TLC) was carried out using Whattman silica
gel 60 A plates (0.25 mm). Flash chromatography was
performed using E. Merck silica gel 60 (230-400 mesh).
7-Br om o-4,4-d im eth yl-1-h yd r oxy-1-(4-m eth ylp h en yl)-
1,2,3,4-tetr a h yd r on a p h th a len e (7). To magnesium turn-
ings (648.0 mg, 27.0 mmol) in THF (25 mL) was added a
solution of 4-bromotoluene (5.40 g, 31.8 mmol) in THF (10 mL)
in two portions. The reaction was initiated by the addition of
2 mL of the solution, followed by slow addition of the remaining
solution via an addition funnel. The mixture was stirred at
room temperature for 1 h and then transferred to a second
flask through a canula. To this Grignard reagent was added
a solution of 3,4-dihydro-4,4-dimethyl-7-bromo-1(2H)-naph-
thalenone (6) (4.0 g, 15.9 mmol) in THF (15 mL). The resulting
solution was heated at reflux for 12 h, cooled to room
temperature, and quenched by slow addition of ice-cold 10%
aqueous HCl. The reaction mixture was extracted with Et2O,
and the combined organic layers were washed with H2O and
brine and dried over MgSO4. Concentration of the dry solution
under reduced pressure provided an oil which after column
Eth yl 2-Flu or o-4-[[[5,6-d ih ydr o-5,5-dim eth yl-8-(4-m eth -
ylph en yl)-2-n aph th alen yl]car bon yl]am in o]ben zoate (11).

2448 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16
Teng et al.
127.3, 126.1, 124.5, 124.4, 114.5, 114.2, 114.0, 108.0, 107.6,
61.1, 38.5, 34.0, 28.0, 21.2, 14.3; MS (EI) m/e 457, 443, 275
(base peak).
Eth yl 2,6-Diflu or o-4-[[[5,6-d ih yd r o-5,5-d im eth yl-8-(4-
m et h ylp h en yl)-2-n a p h t h a len yl]ca r b on yl]a m in o]-b en -
zoa te (12). Using the same procedure as for the synthesis of
compound 10, compound 12 was obtained in 19% yield as a
colorless solid: 1H NMR δ 7.82 (b, 1H), 7.68 (1H, dd, J ) 2.1,
7.1 Hz), 7.50 (1H, d, J ) 2.1 Hz), 7.47 (1H, d, J ) 7.1 Hz),
7.28 (2H, d, J ) 9.8 Hz), 7.23 (4H, m), 6.06 (1H, t, J ) 4.8
Hz), 4.39 (2H, q, J ) 4.1 Hz), 2.40 (3H, s), 2.37 (2H, d, J ) 4.8
Hz), 1.38 (3H, t, J ) 4.1 Hz), 1.36 (6H, s).
4-[[[5,6-Dih yd r o-5,5-d im et h yl-8-(4-m et h ylp h en yl)-2-
n a p h th a len yl]ca r bon yl]a m in o]ben zoic Acid (1). To a
solution of 10 (26.5 mg, 0.06 mmol) in EtOH (3.0 mL) and THF
(4.0 mL) was added NaOH (240.1 mg, 6.00 mmol, 3.0 mL of 2
M aqueous solution). After 72 h of stirring at room temper-
ature, the reaction was quenched by the addition of 10%
aqueous HCl. Extraction (EtOAc), drying (MgSO4), and
concentration under reduced pressure provided a solid. Re-
crystallization from CH3CN afforded 18 mg (73%) of the title
compound as a colorless solid: 1H NMR (DMSO-d6) δ 10.4 (1H,
s), 7.91-7.81 (5H, m), 7.54 (1H, d, J ) 8.1 Hz), 7.45 (1H, d, J
) 1.7 Hz), 7.23 (4H, s), 6.04 (1H, t, J ) 4.7 Hz), 2.35 (5H, s),
1.33 (6H, s); 13C NMR (DMSO-d6) δ 166.9, 166.0, 148.7, 143.2,
138.1, 136.9, 136.6, 133.5, 130.1, 129.1, 128.2, 126.7, 125.3,
125.0, 123.9, 119.2, 33.5, 27.7, 20.8; MS (EI) m/e 411, 275 (base
peak). Anal. (C27H25NO3) C, H, N.
2-F lu or o-4-[[[5,6-d ih yd r o-5,5-d im e t h yl-8-(4-m e t h yl-
p h en yl)-2-n a p h t h a len yl]ca r b on yl]a m in o]b en zoic Acid
(2). Using the same procedure as for the synthesis of 1,
compound 2 was obtained as a solid in 72% yield: 1H NMR
(acetone-d6) δ 9.84 (1H, b), 7.91 (1H, t, J ) 8.6 Hz), 7.85 (2H,
m), 7.64 (1H, d, J ) 1.9 Hz), 7.52 (2H, m), 7.23 (4H, s), 6.04
(1H, t, J ) 4.7 Hz), 2.37 (2H, d, J ) 4.7 Hz), 2.36 (3H, s), 1.35
(6H, s); 13C NMR (acetone-d6) δ 166.9, 165.0, 164.8, 164.7,
161.6, 150.3, 146.1, 145.9, 139.7, 138.2, 137.7, 135.1, 133.7,
133.2, 130.0, 129.2, 127.6, 127.3, 126.2, 124.9, 115.7, 115.6,
114.1, 113.9, 108.4, 108.0, 39.1, 34.5, 28.2, 21.1; MS (EI) m/e
429, 369 (base peak), 275, 231; HRMS found 429.1734 (M),
calcd for C27H24FNO3 429.1740 (M). Anal. (C27H24
-
FNO3‚0.6H2O) C, H, N.
2,6-Diflu or o-4-[[[5,6-d ih yd r o-5,5-d im et h yl-8-(4-m et h -
ylp h en yl)-2-n a p h th a len yl]ca r bon yl]a m in o]ben zoic Acid
(3). Using the same procedure as for the synthesis of 1,
compound 3 was obtained as a solid in 70% yield. 1H NMR δ
7.88 (1H, bs), 7.67 (1H, dd, J ) 2.1, 7.1 Hz), 7.50 (1H, d, J )
2.1 Hz), 7.47 (1H, d, J ) 7.1 Hz), 7.30 (2H, d, J ) 10.3 Hz),
7.21 (4H, m), 6.05 (1H, t, J ) 4.8 Hz), 2.39 (3H, s), 2.37 (2H,
d, J ) 4.8 Hz), 1.36 (6H, s); MS (EI) m/e 447, 403, 275; HRMS
found 447.1604 (M), calcd for C27H23F2NO3 447.1646 (M).
Anal. (C27H23F2NO3‚1.6H2O) C, H, N.
2,2-Dim eth ylch r om a n (14). To a solution of dihydrocou-
marin (13) (5.0 g, 33.78 mmol) in dry THF (50 mL) at 0 °C
was added dropwise methylmagnesium chloride (33.7 mL, 3M
solution in THF, 0.1 mol). The reaction mixture was slowly
warmed up to room temperature and stirred overnight.
Extraction (Et2O), drying (Na2SO4), and concentration under
reduced pressure afforded the diol (6.25 g) as a colorless solid.
The diol was combined with 15% aqueous H2SO4 (50 mL) in
benzene (15 mL) and the resulting mixture heated at 105 °C
for 2 h. Upon being cooled to room temperature, the mixture
was extracted with Et2O, dried (Na2SO4), and concentrated
under reduced pressure to give 6.0 g (100%) of the title
compound as a light yellow liquid: 1H NMR δ 7.08 (2H, m),
6.80 (2H, m), 2.79 (2H, t, J ) 6.8 Hz), 1.81 (2H, t, J ) 6.8 Hz),
1.35 (6H, s).
F igu r e 1. Dose-response curves for RA and retinoids 1-5
in CV-1 cells transfected with RAR holoreceptors and an MTV-
4(R5G)-luciferase reporter plasmid at each RAR. ER-RARR,
ER-RARâ, and ER-RARγ-mediated luciferase activity was
measured for RA and compounds 1-5. The vertical scale is
the luciferase activity (mean ( SEM of triplicate determina-
tions). The horizontal scale is the molar concentration of the
retinoids.
6-Acetyl-2,2-d im eth ylch r om a n (15). To a solution of 14
(3.15 g, 19.4 mmol) in dry CH2Cl2 (100 mL) was added acetyl
chloride (1.52 mL, 21.3 mmol) followed by AlCl3 (2.84 g, 21.3
mmol). The reaction mixture was stirred at room temperature
for 30 min and then poured into ice water. Extraction (CH2-
Cl2), drying (Na2SO4), and concentration under reduced pres-
sure afforded a yellow oil. Column chromatography (EtOAc:
hexanes, 1:9) yielded 2.15 g (54%) of 13 as a yellow solid: 1H
NMR δ 7.73 (2H, m), 6.80 (1H, d, J ) 8.3 Hz), 2.83 (2H, t, J )
Using the same procedure as for the synthesis of compound
10, compound 11 was obtained in 50% yield as a colorless
solid: 1H NMR δ 7.91 (1H , t, J ) 8.4 Hz), 7.80 (1H, bs), 7.70
(2H, m), 7.51 (1H, d, J ) 2.0 Hz), 7.48 (1H, d, J ) 8.1 Hz),
7.22 (m, 5H), 6.06 (1H, t, J ) 4.6 Hz), 4.37 (2H, q, J ) 7.1
Hz), 2.40 (3H, s), 2.38 (2H, d, J ) 4.6 Hz), 1.39 (3H, t, J ) 7.1
Hz), 1.37 (6H, s); 13C NMR δ 165.8, 164.4, 164.0, 160.9, 150.1,
143.5, 143.3, 138.6, 137.2, 134.8, 132.8, 131.8, 129.3, 128.4,

Retinoic Acid Receptor R-Selective Antagonists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16 2449
F igu r e 2. The antagonist effect of compounds 1-5 on transactivation activity induced by RA (10-8 M) at each RAR and a
comparison of 1-5 at RARR. In each panel, the vertical scale is the percentage transactivation activity of RA (10-8 M) in the
presence of the antagonist. The horizontal scale is the molar concentration of the antagonist.
6.9 Hz), 2.54 (3H, s), 1.84 (2H, t, J ) 6.9 Hz), 1.36 (6H, s).
Anal. (C13H16O2) C, H.
chromatography (EtOAc:hexanes, 1:3) afforded 1.98 g (92%)
of 2,2-dimethylchroman-6-carboxylic acid as a light tan solid:
1H NMR δ 7.78 (2H, m), 6.82 (1H, d, J ) 8.3 Hz), 2.83 (2H, t,
J ) 6.7, Hz), 1.85 (2H, t, J ) 6.7 Hz), 1.37 (6H, s).
To a solution of this solid (1.64 g, 7.95 mmol) in EtOH (50
mL) was added H2SO4 (0.5 mL). The mixture was heated
overnight at 95 °C, cooled to room temperature and concen-
trated under reduced pressure. Column chromatography
(EtOAc:hexanes, 1:5) afforded 1.91 g (100%) of 16 as a colorless
solid: 1H NMR δ 7.79 (2H, m), 6.79 (2H, d, J ) 8.3 Hz), 4.34
(2H, q, J ) 7.2 Hz), 2.82 (2H, t, J ) 6.7 Hz), 1.84 (2H, t, J )
Eth yl 2,2-Dim eth ylch r om a n -6-ca r boxyla te (16). A so-
lution of 15 (2.15 g, 10.5 mmol) and NaOH (2.0 g, 50 mmol) in
dioxane (50 mL) and bleach (50 mL, 5.25% NaOCl) was heated
at 65 °C for 4 days. Upon cooling to room temperature Na2-
SO3 was added until an aliquot of this solution remained
colorless color when treated with of one drop of a solution of
I2 in CCl4. The solution was acidified with H2SO4 (pH 4) and
extracted with ether. The combined organic layers were dried
(Na2SO4) and concentrated under reduced pressure. Column

2450 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16
Teng et al.
6.7 Hz), 1.38 (3H, t, J ) 7.2 Hz), 1.36 (6H, s). Anal.
(C14H18O3‚0.25H2O) C, H.
20% aqueous NaOH (2 mL). The reaction mixture was stirred
at room temperature overnight and acidified to pH 4 with 10%
aqueous HCl. The EtOH was removed under reduced pres-
sure, and ethyl acetate and water were added to the residue.
The organic layer was separated, washed with saturated
NaHCO3 and saturated aqueous NaCl, and dried over MgSO4.
Concentration under reduced pressure afforded 110 mg (86%)
of 4 as a colorless solid: 1H NMR δ (acetone-d6) δ 8.09 (1H, d,
J ) 2.1 Hz), 7.91 (1H, t, J ) 7.5 Hz), 7.68 (1H, d, J ) 2.1 Hz),
7.83 (1H, dd, J ) 12.8, 2.1 Hz), 7.50 (1H, dd, J ) 7.5, 2.1 Hz),
7.27 (4H, s), 5.87 (1H, s), 2.37 (3H, s), 1.56 (6H, s); MS (EI)
Eth yl 8-Br om o-2,2-dim eth ylch r om an -6-car boxylate (17).
To a solution of 16 (500 mg, 2.14 mmol) in HOAc (4 mL) was
added Br2 (0.11 mL, 2.14 mmol). The reaction mixture was
stirred at room temperature overnight, and then a stream of
air was passed through the reaction mixture to remove the
excess Br2. Removal of the solvent under reduced pressure
and column chromatography using the residue (EtOAc:hex-
anes, 1:9) afforded 741 mg (100%) of 17 as an oil: 1H NMR δ
8.05 (1H, d, J ) 2.2 Hz), 7.73 (1H, d, J ) 2.2 Hz), 4.34 (2H, q,
J ) 7.1 Hz), 2.84 (2H, t, J ) 6.7 Hz), 1.85 (2H, t, J ) 6.7 Hz),
m/e 511, 509, 496, 494 , 452, 450, 357, 355, 312. Anal. (C26H21
BrFNO4‚0.6H2O) C, H, N.
-
1.40 (6H, s), 1.38 (3H, t, J ) 7.2 Hz). Anal (C14H17
BrO3‚0.2H2O) C, H.
-
Eth yl 2,6-Diflu or o-4-[[[8-br om o-2,2-dim eth yl-4-(4-m eth -
ylp h en yl)-6-ch r om a n yl]ca r bon yl]a m in o]ben zoa te (22).
Using the same procedure as for the synthesis of compound
21, compound 20 (100 mg, 0.29 mmol) afforded 53 mg (33%)
of 22 as a colorless oil: 1H NMR δ 7.87 (1H, d, J ) 2.1 Hz),
7.47 (1H, d, J ) 2.1 Hz), 7.28 (2H, d, J ) 8.7 Hz), 7.19 (4H,
m), 5.70 (1H, s), 4.38 (2H, q, J ) 7.2 Hz), 2.41 (3H, s), 1.57
(6H, s), 1.38 (3H, t, J ) 7.2 Hz); MS (EI) m/e 557, 555, 542,
540, 448, 446. Anal. (C28H24BrF2NO4) C, H, N.
2,6-Diflu or o-4-[[[8-b r om o-2,2-d im e t h yl-4-(4-m e t h yl-
p h en yl)-6-ch r om a n yl)ca r bon yl]a m in o]ben zoic a cid (5).
Using the same procedure as for the synthesis of compound
4, compound 22 (53 mg, 0.1 mmol) afforded 35 mg (70%) of 5
as a colorless solid: 1H NMR δ (acetone-d6) 9.93 (1H, bs), 8.07
(1H, d, J ) 2.1 Hz), 7.66 (1H, d, J ) 2.1 Hz), 7.54 (2H, d, J )
9.9 Hz), 7.27 (4H, s), 5.88 (1H, s), 2.38 (3H, s), 1.56 (6H, s).
MS (EI) m/e 514, 512, 485, 483, 470, 468. Anal. (C26H20BrF2-
NO4) C, H, N.
Eth yl 8-Br om o-2,2-d im eth yl-4-oxoch r om a n -6-ca r boxy-
la te (18). To a solution of HOAc (15 mL) and Ac2O (7.5 mL)
at 0 °C was added CrO3 (1.18 g, 12 mmol) in small portions.
This solution was stirred for 15 min and diluted with benzene
(10 mL), and a solution of 17 (741 mg, 2.38 mmol) in benzene
(5 mL) was slowly added over several minutes. After 4 h
stirring at 0 °C, the reaction mixture was poured over ice and
extracted with EtOAc, and the combined organic layers were
dried (Na2SO4) and concentrated under reduced pressure to
give an oil. Column chromatography (EtOAc:hexanes, 1:9)
yielded 589 mg (76%) of 18 as a colorless solid: 1H NMR δ 8.5
(1H, d, J ) 2.1 Hz), 8.40 (1H, d, J ) 2.1 Hz), 4.37 (2H, q, J
)7.1 Hz), 2.80 (2H, s), 1.54 (6H, s), 1.40 (3H, t, J ) 7.1 Hz).
Anal. (C14H15BrO4) C, H, N.
Eth yl 8-Br om o-2,2-d im eth yl-4-(4-m eth ylp h en yl)-4(2H)-
ch r om a n -6-ca r boxyla te (19). To a solution of 18 (589 mg,
1.81 mmol) in THF (20 mL) under Ar at -78 °C was added
p-tolylmagnesium bromide (2.16 mL, 2.72 mmol, 1 M solution
in ether). The reaction mixture was slowly warmed to room
temperature and stirred overnight. Saturated NH4Cl solution
was added to the reaction at 0 °C and the resulting mixture
extracted with ether. The combined organic layers were
washed with saturated aqueous NaCl, dried over Na2SO4, and
concentrated under reduced pressure. Column chromatogra-
phy (EtOAc:hexanes, 1:3) afforded 297 mg of a mixture of 19
and the intermediate tertiary alcohol as an oil. This mixture
was combined with p-TsOH (20 mg) in dry toluene (15 mL)
and heated at 110 °C for 30 min. Upon being cooled to room
temperature, the solvent was removed under reduced pressure
and the product 182 mg (25%) isolated by column chromatog-
raphy (EtOAc:hexane, 1:3): 1H NMR δ 8.1 (1H, d, J ) 1.7 Hz),
7.68 (1H, d, J ) 1.7 Hz), 7.22 (4H, s), 5.67 (1H, s), 4.29 (2H, q,
J ) 7.1 Hz), 2.41(3H, s), 1.56 (6H, s), 1.33 (3H, t, J ) 7.1 Hz);
Bin d in g Assa y. Each receptor subtype was expressed in
baculovirus. Stock solutions of all compounds were prepared
as 10 mM ethanol solutions and serial dilutions carried out
into 1:1 DMSO:glycerol, 120 mM KCl, 8 mM Tris, 5 mM
CHAPS, 4 mM DTT, and 0.24 mM PMSF, at pH ) 7.4 at room
temperature.
The final assay volume was 250 µL and contained 10-40 µg
of extract protein along with 5 nM of [3H]-all-trans-retinoic
acid and varying concentrations of competing ligand that
ranged from 0 to 10-5 M. The assays were run using a Biomek
formatted for a 96-well minitube system. Incubations were
carried out at 4 °C until equilibrium was achieved. Nonspecific
binding was defined as that binding remaining in the presence
of 1000 nM of unlabeled RA. At the end of the incubation
period, 50 µL of 6.25% hydroxyapitite was added in a wash
buffer which consisted of 100 nM KCl, 10 mM Tris, and 0.5%
Triton X-100. The mixture was vortexed and incubated for
10 min at 4 °C and centrifuged and the supernatant removed.
The hydroxyapitite was washed three more times with the
buffer and the amount of receptor-ligand complex determined
by liquid scintillation counting of the pellet.
MS (EI) m/e 402, 400, 387, 385, 359, 357. Anal. (C21H21
BrO3‚0.45H2O) C, H.
-
8-Br om o-2,2-d im eth yl-4-(4-m eth ylp h en yl)-4(2H)-ch r o-
m a n -6-ca r boxylic Acid (20). To a solution of 19 (189 mg,
0.51 mmol) in EtOH (15 mL) and THF (10 mL) was added
20% aqueous NaOH (2 mL). The reaction mixture was
warmed to 45 °C for 2 h, cooled to room temperature, and
acidified to pH 4 with 10% aqueous HCl. Extraction (EtOAc),
drying (MgSO4), and concentration under reduced pressure
afforded 18 (171 mg) (98%) as a colorless solid: 1H NMR δ
8.15 (d, J ) 2.0 Hz, 1H), 7.71 (d, J ) 2.0 Hz, 1H), 7.21 (m,
4H), 5.69 (s, 1H), 2.42(s, 3H), 1.58 (s, 6H).
After correcting for nonspecific binding, IC50 values were
determined graphically from a log-logit plot of the data. The
Kd values were determined by application of the Cheng-
Prussof equation14c to the IC50 values, the labeled ligand
concentration, and the Kd of the labeled ligand.
Tr a n sfection s a n d DNA Con str u cts. Transfections were
carried out as previously described.15 Briefly, 4 × 105 CV-1
cells were transiently transfected via calcium phosphate
precipitation16 with 0.7 µg of the reporter plasmid MTV-
4(R5G)-Luc, 0.1 µg of the the â-galactosidase expression
plasmid pCH110 (Pharmacia), 0.01 µg of the plasmid pRS-
hRXRR,4a and 0.05 µg of either pRS-RARR-P-GR, pcDNA3-
RARâ-P-GR, or pcDNA3-RARγ-P-GR. Eighteen hours after
introduction of the DNA precipitants, the cells were rinsed
with phosphate-buffered saline (PBS) and fed with D-MEM
(Gibco-BRL) containing 10% activated charcoal extracted fetal
bovine serum (Gemini Bio-Products). Cells were treated for
18 h with 10 nM ATRA in conjunction with the compounds
indicated in the figure (antagonism studies). After being
rinsed with PBS, cells were lysed and luciferase activity was
measured as previously described.17 Luciferase values repre-
sent the mean ( SEM of triplicate determinations normalized
to â-galactosidase activity.
E t h yl 2-F lu or o-4-[[[8-b r om o-2,2-d im et h yl-4-(4-m et h -
ylp h en yl)-6-ch r om a n yl]ca r bon yl]a m in o]ben zoa te (21).
To a solution of 20 (100 mg, 0.29 mmol) in CH2Cl2 (8 mL) was
added DMAP (87 mg, 0.69 mmol), ethyl 4-amino-2-fluoroben-
zoate (53 mg, 0.29 mmol), and EDC (72 mg, 0.37 mmol). The
reaction mixture was stirred at room temperature overnight
and then concentrated to dryness. Column chromatography
(EtOAc:hexanes, 1:3) afforded 98 mg (63%) of 21 as a colorless
solid: 1H NMR δ 7.99 (1H, bs), 7.89 (1H, t, J ) 7.5 Hz), 7.88
(1H, d, J ) 2.1 Hz), 7.65 (1H, dd, J ) 12.8, 2.1 Hz), 7.25 (1H,
dd, J ) 7.5 Hz, 2.1 Hz), 7.19 (4H, s), 5.70 (1H, s), 4.36 (2H, q,
J ) 7.2 Hz), 2.38 (3H, s), 1.56 (6H, s), 1.39 (3H, t, J ) 7.2 Hz);
MS (EI) m/e 537, 537, 524, 526, 357, 355, 312. Anal (C28H25
-
BrFNO4) C, H, N.
2-Flu or o-4-[[[8-br om o-2,2-dim eth yl-4-(4-m eth ylph en yl)-
6-ch r om a n yl]ca r bon yl]a m in o]ben zoic Acid (4). To a
solution of 21 (135 mg, 0.25 mmol) in EtOH (5 mL) was added

Retinoic Acid Receptor R-Selective Antagonists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16 2451
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