Synthesis and structure-activity relationships of retinoid X receptor selective diaryl sulfide analogs of retinoic acid

Article abstract of DOI:10.1021/jm960386h

Retinoids exert their biological effects by binding to and activating nuclear receptors that interact with responsive elements on DNA to promote gene transcription. There are two families of retinoid receptors, the retinoic acid receptor (RAR) family and

Full text of DOI:10.1021/jm960386h

3556
J . Med. Chem. 1996, 39, 3556-3563
Syn th esis a n d Str u ctu r e-Activity Rela tion sh ip s of Retin oid X Recep tor
Selective Dia r yl Su lfid e An a logs of Retin oic Acid
Richard L. Beard,*,† Diana F. Colon,† Tae K. Song,† Peter J . A. Davies,§ Devendra M. Kochhar,∇ and
Roshantha A. S. Chandraratna*,†,‡
Retinoid Research, Departments of Chemistry and Biology, Allergan, Incorporated, Irvine, California 92715-1599,
Department of Pathology, Anatomy and Cell Biology, Thomas J efferson University, Philadelphia, Pennsylvania 19107,
and Departments of Pharmacology and Medicine, University of Texas Medical School, Houston, Texas 77225
Received May 31, 1996X
Retinoids exert their biological effects by binding to and activating nuclear receptors that
interact with responsive elements on DNA to promote gene transcription. There are two
families of retinoid receptors, the retinoic acid receptor (RAR) family and the retinoid X receptor
(RXR) family, which are each further divided into three subclasses: RARR,â,γ and RXRR,â,γ
.
Herein we describe the synthesis and structure-activity relationships of a new series of diaryl
sulfide retinoid analogs that specifically bind and transactivate the RXRs. Furthermore, the
sulfoxide and sulfone derivatives of these analogs are partial agonists which activate the RXRs
only at high concentrations. Thus, these compounds possess a potential site of metabolic
deactivation and may have less prolonged systemic effects than other compounds with arotinoid-
like structures. We show also that these compounds have activity in nontransfected cells as
demonstrated by their ability to induce TGase activity in HL-60 cells. Finally, we corroborate
our earlier report that RXR-specific agonists may possess reduced teratogenic toxicity compared
to RAR-specific agonists since these compounds are much less potent inhibitors of chondro-
genesis than RAR-specific agonists such as TTNPB.
In tr od u ction
addition, it is known that RXRs form homodimers in
the presence of RXR ligands and regulate gene tran-
scription through promoters that are distinct from those
utilized by RAR-RXR heterodimers.13 RXRs also form
heterodimers with the thyroid, vitamin D, and other
receptors.11 Thus, the biology associated with the RXR
family is still being elucidated.
Retinoids are natural and synthetic analogs of vita-
min A that are involved in the regulation of several
biological functions including cell differentiation and
proliferation.1 Clinically, retinoids are useful for the
treatment of skin disorders2 and cancer3 and are cur-
rently being investigated in several other therapeutic
areas, including arthritis,4 dyslipidemias,5 and the
prevention of HIV-induced lymphopenia.6 However,
retinoids are of limited therapeutic value because they
cause a number of undesirable side effects such as
mucocutaneous toxicity and teratogenicity. Retinoids
are believed to function by binding to nuclear receptors
which regulate gene transcription.7 The six known
retinoid receptors are divided into two families: the
retinoic acid receptor family (RARR,â,γ)8 and the ret-
inoid X receptor family (RXRR,â,γ).9 all-trans-Retinoic
acid (RA) specifically transactivates only the RARs,
whereas 9-cis-retinoic acid (9-cis-RA) binds to and
transactivates both RXRs and RARs.10 Under physi-
ological conditions, the RARs and RXRs form het-
erodimers which can bind to the promoter regions of
genes to modulate gene transcription.11 The RAR
hormonal pathways can be activated by RAR-specific
ligands which bind to the RAR component of the RAR-
RXR heterodimer, but RXR-specific ligands appear
unable to activate these pathways by binding to the
RXR component. However, RXR ligands display syn-
ergistic activation of RA responsive genes when they are
used in combination with RAR-specific ligands.12 In
* Corresponding authors: Roshantha A. S. Chandraratna, Director,
and Richard L. Beard, Senior Scientist, Retinoid Research, Department
of Chemistry, Allergan, Inc., 2525 Dupont Dr., Irvine, CA 92715-1599.
† Department of Chemistry, Allergan, Inc.
A number of groups have reported13-17 the develop-
ment of ligands that display varying degrees of selectiv-
ity for the RXRs. One important class of RXR selective
compounds are 3-substituted stilbenes,14 such as (E)-
‡ Department of Biology, Allergan, Inc.
∇ Thomas J efferson University.
§ University of Texas Medical School.
X Abstract published in Advance ACS Abstracts, August 1, 1996.
S0022-2623(96)00386-X CCC: $12.00 © 1996 American Chemical Society

RXR Selective Diaryl Sulfide Analogs of RA
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 18 3557
Ta ble 1. Cotransfection Assay Data for Diaryl Sulfide Retinoidsa
a
NA indicates not active (i.e., EC50 > 104 nmol). EC50 values were determined from full dose-response curves ranging from 10-12 to
10-5 M. Retinoid activity was normalized to that of all-trans RA and is expressed as potency (EC50), which is the concentration of retinoid
required to produce 50% of the maximal observed response. Values represent the EC50 determination of a single experiment with triplicate
determinations. Standard errors for this assay system are, on average, approximately 15% of the mean values.
4-[2-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naph-
thalenyl)propen-1-yl]benzoic acid (3-methyl-TTNPB).
3-Methyl-TTNPB is a pan-agonist (activates both RAR
and RXR receptors), while its desmethyl analog, (E)-4-
[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphtha-
lenyl)propen-1-yl]benzoic acid (TTNPB), is a highly
potent and specific activator of the RARs. Recently, we
reported15 that substitution of the benzoate moiety of
3-methyl-TTNPB with heteroaromatic carboxylic acids
resulted in analogs with greater RXR selectivity as well
as those with pan-agonist activities. A second class of
RXR selective agonists are benzophenonecarboxylic acid
derivatives such as SR 1123716a and LGD 1069,16b,c of
which the latter is currently in phase I/IIA clinical trial
for the treatment of cancer. In this report, we discuss
the synthesis and structure-activity relationships (SAR)
of RXR-specific compounds in which the tetrahydronaph-
thalene ring and the benzoic acid moieties are bridged
by a sulfur atom. Further, we show that the sulfoxide
and sulfone derivatives of these compounds have sig-
nificantly reduced retinoid activity compared to the
parent diaryl sulfide analogs. Thus, these compounds
possess a potential site of metabolic deactivation and
thus may have fewer prolonged systemic effects than
other arotinoid-like derivatives.
Resu lts a n d Discu ssion
Ch em istr y. The analogs used in this study are
summarized in Table 1. RA was purchased from Sigma
Chemical Co. 9-cis-RA was synthesized according to
literature procedures.17 Analogs 1-5 were prepared as
depicted in Scheme 1. Thus, 5,6,7,8-tetrahydro-5,5,8,8-
tetramethylnaphthalene-2-thiol (7)18 was deprotonated
with NaH and heated in the presence of CuI and ethyl
4-iodobenzoate (9) to give the coupled product, diaryl
sulfide 11, in 27% yield. The ester of 11 was saponified
with LiOH in methanol to produce diaryl sulfide 1 in
62% yield. The same protocol was applied to 5,6,7,8-
tetrahydro-3,5,5,8,8-pentamethylnaphthalene-2-thiol (8)19
to prepare ester 12, which was saponified with KOH in
ethanol to give 2 in 50% overall yield. Sulfide 12 was
oxidized either with NaIO4 to produce sulfoxide 3 in 37%
overall yield after ester hydrolysis or with m-CPBA to

3558 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 18
Beard et al.
Sch em e 1a
RA binds to and activates each of the RAR and RXR
subtypes with high affinity. In the cotransfection assay,
the desmethyl analog 1 has no activity at RARR but
effectively activates the other five retinoid receptors,
albeit at high concentrations at RXRâ and RXRγ. In
contrast, the 3-methyl derivative 2 is an RXR-specific
transactivator which is 3-5-fold more potent than 1 at
the RXRs and with absolutely no activity at the RARs.
These results were confirmed in the competitive binding
assay in which 1 has little affinity to any of the retinoid
receptors, while 2 binds exclusively to the RXRs, with
Kd values of approximately 300 nM. The sulfoxide and
sulfone derivatives of 2, compounds 3 and 4, are lowered
in their affinity to the RXRs relative to 2. In addition,
they are lowered in both efficacy and potency in the
cotransfection assays, having only partial agonist activ-
ity at the RXRs even at high concentrations. The effects
of heterocyclic carboxylic acid substitution on receptor
binding and transactivation properties of these com-
pounds was examined using the nicotinic acid isostere
5 and the thiazolecarboxylic acid analog 6. The nicotinic
acid moiety has a favorable effect on RXR transactiva-
tion and binding since 5 is at least 5-fold more potent
than 2 in the transactivation assay and binds to the
RXRs with about 4-fold higher affinity. Thus, the RXR-
specific agonist 5 has receptor-binding and transacti-
vation properties that are comparable to those of LGD
1069.16b However, the thiazole derivative 6 is 4-8-fold
less potent than 2 in the cotransfection assays, having
EC50 values of >1300 nM at all three RXRs. Both
compounds are, however, specific for the RXRs having
no RAR activity.
a
(a) NaH, CuI, HMPA; (b) aqueous KOH, EtOH, 10% HCl; (c)
12, NaIO4; (d) 12, m-CPBA; (e) aqueous LiOH, THF, 10% HCl.
Sch em e 2a
Previous studies have established that RXR selective
retinoids induce tissue transglutaminase (TGase) in a
human promyelocytic leukemia cell line (HL-60 cdm-
1).15a,1b,22 As a measure of their activity in a nontrans-
fected cell line, we tested 2 and 5, along with selected
reference compounds, in the TGase assay (Table 3). As
reported previously,15a while the RAR-specific agonist
TTNPB is inactive in this assay, the RXR-specific diaryl
sulfides 2 and 5 induced TGase activity effectively. We
also wanted to determined the activity of these analogs
in an assay of retinoid toxicity, namely, that of inhibition
of chondrogenesis in mouse embryo limb bud mesen-
chymal cells (Table 2).23 This assay is highly predictive
of the in vivo teratogenic potential of retinoids in ICR
mice.24 We had previously reported that RXR selective
analogs appear to be reduced in their teratogenic
potential.15a In keeping with these observations and
consistent with its RXR specificity, the diaryl sulfides
2 and 5 display very little activity in this assay, being
more than 10000-fold less potent than the RAR-specific
analog TTNPB.
a
(a) n-BuLi, -78 °C, EtO2CCl; (b) I2, THF; (c) NaH, CuI, HMPA;
(d) aqueous LiOH, THF, 10% HCl.
furnish sulfone 4 (92% after hydrolysis). The nicotinic
acid derivative 5 was prepared in 39% yield by coupling
8 and ethyl 6-iodonicotinate (10) followed by saponifica-
tion of the resulting ester 14.
The synthesis of the thiazole derivative 6 is presented
in Scheme 2. Thus, 2-(trimethylsilyl)thiazole (15) was
carboxylated in the 5-position (n-BuLi, THF, -78 °C,
EtO2CCl) and the 2-trimethylsilyl group iodinated (I2,
THF) to give ethyl 2-iodo-5-thiazolecarboxylate (16).
Compound 16 was coupled to 8 and saponified as
described above to produce 6 in 32% yield.
Biologica l Stu d ies. The retinoid analogs in Table
1 were tested separately for activity at each of the six
retinoid receptors in a cotransfection assay,9a,20,21 which
measures the ability of compounds to induce transcrip-
tion in CV-1 cells transiently cotransfected with a
specific receptor gene construct and an appropriate
reporter gene. The binding affinities of the compounds
to each of the receptor subtypes were measured in
competitive binding assays13b,c and are reported in Table
2. In the cotransfection assay, RA activates each of the
RAR subtypes with high potency and each of the RXR
subtypes with low potency, consistent with its ability
to bind to each of these receptors. In contrast, 9-cis-
Discu ssion
The data presented in Tables 1 and 2 demonstrate
several interesting characteristics of these compounds.
First, all of the compounds that were tested in this
series that have a 3-methyl group on the tetrahy-
dronaphthalene ring do not bind to or transactivate any
of the RARs. Thus, it appears that a 3-methyl group
on the tetrahydronaphthalene ring is necessary for RXR
specificity and activity. This finding is consistent with
our earlier results in the stilbene series in that a
3-substituent on the tetrahydronaphthalene ring re-
sulted in significantly increased transactivational po-

RXR Selective Diaryl Sulfide Analogs of RA
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 18 3559
Ta ble 2. Competitive Binding Assay Data for Diaryl Sulfide Retinoidsa
a
NA indicates not active (i.e., Kd > 30000 nmol).
tencies at the RXRs and decreased potencies at the
RARs. Second, the oxidized derivatives of 2, sulfoxide
3 and sulfone 4, are characterized by reduced transac-
tivational potencies and lower binding affinities relative
to sulfide 2, suggesting that the sulfides have a potential
site of metabolic deactivation that is not available to
other RXR-specific compounds with arotinoid-like struc-
tures. Third, we recently reported that stilbenes sub-
stituted with nicotinic acid isosteres were of comparable
potencies in the RXR cotransfection assays to the
corresponding benzoic acid derivatives, whereas the
thiazolecarboxylic acids had dramatically increased
potencies. In the case of diaryl sulfides, however, the
effect is altered; replacement of the benzoic acid group
with a thiazolecarboxylic acid (compound 6) significantly
decreases the transactivational potencies for the RXRs,
while replacement of the benzoic acid with a nicotinic
acid increases RXR transactivational potencies. Thus,
five-membered heteroaromatic carboxylic acid groups
are detrimental to the RXR transactivational and bind-
ing properties of these compounds.
providing additional evidence that ligand-dependent
RXR activation is necessary for the induction of TGase
activity in these cells. In the chondrogenesis assay, the
RXR-specific diaryl sulfides 2 and 5 are some of the least
active retinoids tested, being about 10000-fold less
potent than the RAR-specific arotinoid TTNPB. These
data support our earlier suggestion15a,24b that the ter-
atogenic potency of RXR selective agonists is decreased
relative to RAR agonists.
Con clu sion s
In summary, we have described the synthesis and
SAR of a new series of diaryl sulfide retinoid analogs
that specifically bind to and transactivate RXRs. We
have shown that a 3-methyl substituent on the tetrahy-
dronaphthalene ring of these compounds is required for
RXR specificity in receptor cotransfection and competi-
tive binding assays. RXR transactivational potencies
and binding affinities were increased further by replace-
ment of the benzoic acid group of 2 with a nicotinic acid
group as in compound 5, a potent and specific RXR
agonist. In addition, the sulfoxide and sulfone deriva-
tives of 2, compounds 3 and 4, were shown to be partial
agonists which activate the RXRs only at high concen-
trations. Thus, these compounds possess a potential
site of metabolic deactivation and may have less pro-
The data in Table 3 corroborate our earlier inference
that RAR and RXR selective compounds possess differ-
ent biological properties in nontransfected cells. The
RXR-specific compounds 2 and 5 both appear to be able
to induce TGase activity in HL-60 cells effectively,

3560 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 18
Ta ble 3. Biological Assay Data for Representative Retinoid Analogs
Beard et al.
1.25 h. Copper(I) iodide (0.592 g, 3.1 mmol) was added, and
the mixture was stirred at 0 °C for 45 min. A solution of ethyl
4-iodobenzoate (0.839 g, 3.04 mmol) and 2.0 mL of dimethyl-
formamide was added, and the mixture was heated to 75 °C
for 48 h, cooled to room temperature, and stirred for 48 h. The
reaction mixture was then poured onto ice, and the products
were extracted with ether (4×). The organic extracts were
combined, washed with brine, dried (MgSO4), and filtered, and
the solvents were removed in vacuo to give an orange solid.
The crude product was purified by flash chromatography on
silica gel (2% ethyl acetate in hexanes) to give 11 as a clear
longed systemic effects than compounds with arotinoid-
like structures. Furthermore, the activity of these
compounds in nontransfected cells was demonstrated
by the ability of 2 and 5 to induce TGase activity in HL-
60 cells. Finally, we have shown that these compounds
are much less potent inhibitors of chondrogenesis than
RAR-specific agonists such as TTNPB, thus corroborat-
ing our earlier suggestion that RXR-specific agonists
possess reduced toxicity relative to RAR-specific ago-
nists.
oil (0.32 g, 27%): IR (salt plate) 1716 (CdO) cm-1 1H NMR
;
Exp er im en ta l Section
(300 MHz, CDCl3) δ 1.26 (s, 6 H), 1.30 (s, 6 H), 1.37 (t, 3 H, J
) 7.1 Hz), 1.70 (s, 4 H), 4.34 (q, 2 H, J ) 7.1 Hz), 7.17 (d, 2 H,
J ) 8.5 Hz), 7.22 (dd, 1 H, J ) 2.0, 8.2 Hz), 7.32 (d, 1 H, J )
8.2 Hz), 7.44 (d, 1 H, J ) 2.0 Hz), 7.89 (d, 2 H, J ) 8.5 Hz);
13C NMR (75 MHz, CDCl3) δ 14.3, 31.7, 34.3, 34.4, 34.9, 60.8,
126.7, 127.3, 128.0, 130.0, 131.0, 132.4, 137.6, 145.2, 146.0,
146.6, 166.3; MS (EI, 70 eV) m/ z 368 (M+, 58), 353 (100).
Gen er a l P r oced u r es. RA was obtained from Sigma
Chemical Co. 9-cis-RA was prepared by the procedure of J ong
et al.17 Solvents were used as purchased unless otherwise
noted. When deemed necessary, reaction flasks containing
magnetic stir bars were flame-dried, cooled under vacuum (<3
Torr), and flushed several times with dry argon before any
reagents were added. Most reactions were monitored by
analytical thin-layer chromatography (TLC) using Merck TLC
glass plates precoated with silica gel 60 F254 (0.2 mm thick).
Flash chromatography was performed using Merck silica gel
60. Melting points and boiling points are uncorrected. Unless
the use of an internal thermometer is indicated, temperatures
are reported as bath temperatures. IR spectra were obtained
on a Mattson Galaxy Series 3000 FTIR spectrophotometer.
NMR spectra were recorded on Gemini 300 (300 MHz) and
XL 300 (300 MHz) Varian spectrometers. Mass spectral
analyses were conducted on an EG 7070E organic mass
spectrometer. Elemental analyses were conducted at Robert-
son Microlit Laboratories, Inc., Madison, NJ .
Eth yl 4-[(5,6,7,8-Tetr ah ydr o-5,5,8,8-tetr am eth yl-2-n aph -
th yl)th io]ben zoa te (11). Sodium hydride (0.807 g, 60%
dispersion in oil, 20 mmol) was rinsed three times with hexane
and dried under vacuum. The vacuum was broken with dry
argon, to this was added 10.0 mL of dimethylformamide, and
the mixture was cooled to 0 °C. 5,6,7,8-Tetrahydro-5,5,8,8-
tetramethyl-2-naphthalenethiol18 (702 mg, 3.2 mmol) was
added, and the resulting mixture was stirred at 0-10 °C for
4-[(5,6,7,8-Tetr a h yd r o-5,5,8,8-tetr a m eth yl-2-n a p h th yl)-
th io]ben zoic Acid (1). To a solution of 70 mg (0.19 mmol)
of ethyl 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)-
thio]benzoate (11) and 4.0 mL of THF were added 1.0 mL of
LiOH (1.9 M aqueous solution) and 1.5 mL of MeOH. The
solution was heated at 55 °C for 3 h, cooled to room temper-
ature, and concentrated in vacuo. The residue was diluted
with water and washed with hexanes. The aqueous layer was
separated and acidified to pH ) 1 with 10% aqueous HCl, and
the products were extracted with ether (2×). The combined
organic extracts were washed with brine, dried (MgSO4), and
filtered, and the solvents were removed in vacuo. The residue
was purified by flash chromatography on silica gel (30% ethyl
acetate in hexanes) to give 1 as a white solid (40 mg, 62%):
mp 174-178 °C; IR (salt plate) 3500-2500 (COOH), 1689
1
(CdO) cm-1; H NMR (300 MHz, CDCl3) δ 1.27 (s, 6 H), 1.31
(s, 6 H), 1.71 (s, 4 H), 7.18 (d, 2 H, J ) 8.6 Hz), 7.25 (dd, 1 H,
J ) 2.0, 8.2 Hz), 7.34 (d, 1 H, J ) 8.2 Hz), 7.47 (d, 1 H, J ) 2.0
Hz), 7.95 (d, 2 H, J ) 8.6 Hz); 13C NMR (75 MHz, CDCl3) δ
31.8, 34.3, 34.5, 34.9, 126.4, 127.5, 128.1, 130.6, 131.3, 132.8,

RXR Selective Diaryl Sulfide Analogs of RA
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 18 3561
146.2, 146.8, 146.9, 166.2; MS (EI, 70 eV) 340 (M+, 55), 325
(100); HRMS calcd for C21H24O2S 340.1497, found 340.1493.
Eth yl 4-[(5,6,7,8-Tetr a h yd r o-3,5,5,8,8-p en ta m eth yl-2-
n a p h th yl)th io]ben zoa te (12). Sodium hydride (65 mg, 60%
dispersion in oil, 1.62 mmol) was rinsed three times with
hexane and dried under vacuum. The vacuum was broken
with dry argon, and 2.5 mL of HMPA and 5,6,7,8-tetrahydro-
3,5,5,8,8-pentamethyl-2-naphthalenethiol17 (0.38 g, 1.62 mmol)
were added sequentially. After 30 min at 50 °C, copper(I)
iodide (257 mg, 1.35 mmol) was added, which caused the
solution to become deep green. The solution was stirred for
15 min, and ethyl 4-iodobenzoate (373 mg, 1.35 mmol) was
added. The solution was heated to 90 °C for 5 h, the bath
was removed, and stirring was continued overnight at room
temperature. Water was added, and the products were
extracted with diethyl ether (3×). The combined ether layers
were washed with brine, dried (MgSO4), and filtered, and the
solvent was removed in vacuo. The residue was purified by
flash chromatography on silica gel (5% ethyl acetate in
hexanes) to give 12 as a light yellow solid (260 mg, 50%): mp
vacuo, the residue was treated with brine and acidified with
10% aqueous HCl, and the product was extracted with ether
(2×). The combined ether layers were dried (MgSO4) and
filtered, and the solvents were removed in vacuo to give 3 as
1
a white solid (0.39 mg, 72%): mp 235-240 °C dec; H NMR
(300 MHz, CDCl3) δ 1.22 (s, 3 H), 1.24 (s, 3 H), 1.26 (s, 3 H),
1.29 (s, 3 H), 1.66 (s, 4 H), 2.34 (s, 3 H), 7.10 (s, 1 H), 7.70 (d,
2 H, J ) 8.3 Hz), 7.76 (s, 1 H), 8.17 (d, 2 H, J ) 8.3 Hz); 13C
NMR (75 MHz, CDCl3) δ 18.2, 31.5, 31.6, 31.8, 34.3, 34.4, 34.7,
124.0, 125.6, 129.6, 130.8, 131.6, 132.9, 138.5, 144.6, 149.1,
150.2, 169.9; MS (EI, 70 eV) m/ z 370 (M+, 25), 357 (100). Anal.
(C22H26O3S) C, H.
Eth yl 4-[(5,6,7,8-Tetr a h yd r o-3,5,5,8,8-p en ta m eth yl-2-
n a p h th yl)su lfon yl]ben zoa te. To a solution of 69 mg (0.18
mmol) of 12 in 2.0 mL of methylene chloride was added a
solution of m-chloroperoxybenzoic acid (80 mg, 50-60%, 0.27
mmol) and 2.0 mL of methylene chloride. The resulting
solution was stirred at room temperature for 3 h and diluted
with water, and the products were extracted with methylene
chloride (2×). The combined organic layers were dried (Mg-
SO4) and filtered, and the solvents were removed in vacuo.
The crude product was purified by flash chromatography on
silica gel (10% ethyl acetate in hexanes) to give the title
compound as a white solid (51 mg, 94%): mp 130-131 °C; IR
106-107.5 °C; IR 1716 (CdO) cm-1 1H NMR (300 MHz,
;
CDCl3) δ 1.24 (s, 6 H), 1.30 (s, 6 H), 1.36 (t, 3 H, J ) 7.1 Hz),
1.69 (s, 4 H), 2.28 (s, 3 H), 4.33 (q, 2 H, J ) 7.1 Hz), 7.05 (d,
2 H, J ) 8.6 Hz), 7.23 (s, 1 H), 7.26 (s, 1 H), 8.87 (d, 2 H, J )
8.6 Hz); 13C NMR (75 MHz, CDCl3) δ 14.3, 20.3, 31.7, 31.8,
34.0, 34.2, 34.9, 35.0, 60.8, 125.7, 126.7, 126.9, 129.1, 129.9,
134.4, 138.8, 144.1, 145.2, 145.8, 166.3; MS (EI, 70 eV) m/ z
382 (M+, 42), 367 (67), 171 (100). Anal. (C24H30O2S) C, H, S.
4-[(5,6,7,8-Tetr a h yd r o-3,5,5,8,8-p en ta m eth yl-2-n a p h th -
yl)t h io]b en zoic Acid (2). Ethyl 4-[(5,6,7,8-tetrahydro-
3,5,5,8,8-pentamethyl-2-naphthyl)thio]benzoate (12; 170 mg,
0.44 mmol) was dissolved in ethyl alcohol (4 mL) and the
solution treated with 2 M aqueous KOH (2 mL). The solution
was heated to 50 °C for 4 h and concentrated in vacuo. The
residue was treated with diethyl ether, cooled to 0 °C, and
acidified with 10% aqueous HCl. The product was extracted
with ether, washed with water and brine, dried (MgSO4), and
filtered, and the solvent was removed under reduced pressure
to give 2 as a yellow solid (158 mg, 100%): mp 249-250 °C;
IR 3300-2400 (COOH), 1672 (CdO) cm-1; 1H NMR (300 MHz,
CDCl3) δ 1.25 (s, 6 H), 1.31 (s, 6 H), 1.69 (s, 4 H), 2.29 (s, 3 H),
7.05 (d, 2 H, J ) 8.5 Hz), 7.25 (s, 1 H), 7.26 (s, 1 H), 7.92 (d,
2 H, J ) 8.5 Hz); 13C NMR (75 MHz, CDCl3) δ 20.3, 31.7, 31.8,
34.1, 34.2, 34.9, 35.0, 125.4, 125.5, 126.3, 129.1, 130.6, 134.6,
139.0, 144.2, 146.9, 147.1, 171.0; MS (EI, 70 eV) m/ z 354 (M+,
41), 335 (67), 173 (100). Anal. (C22H26O2S) C, H, S.
Eth yl 4-[(5,6,7,8-Tetr a h yd r o-3,5,5,8,8-p en ta m eth yl-2-
n a p h th yl)su lfoxy]ben zoa te. To a solution of 12 (0.12 g, 0.31
mmol) and 4 mL of dioxane was added 1.0 mL of a 0.42 M
solution of sodium periodate (prepared by dissolving 180 mg
of sodium periodate in 0.7 mL of water and 1.3 mL of
methanol). An additional 6.0 mL of methanol was added, and
the resulting mixture was stirred at room temperature for 42
h and then heated to 50 °C for 8 days. Additional sodium
periodate (80 mg, 0.38 mmol) and 2.0 mL of dioxane were
added periodically during this time. The reaction mixture was
cooled to room temperature, brine was added, and the mixture
was extracted with ether (2×). The organic layers were dried
(MgSO4), filtered, and concentrated under reduced pressure.
The resulting oil was purified by flash chromatography on
silica gel (15% ethyl acetate in hexanes) to give the title
compound as a clear oil (65 mg, 52%): 1H NMR (300 MHz,
CDCl3) δ 1.23 (s, 3 H), 1.24 (s, 3 H), 1.26 (s, 3 H), 1.30 (s, 3 H),
1.39 (t, 3 H, J ) 7.1 Hz), 1.67 (s, 4 H), 2.31 (s, 3 H), 4.38 (q, 2
H, J ) 7.1 Hz), 7.08 (s, 1 H), 7.66 (d, 2 H, J ) 8.4 Hz), 7.76 (s,
1 H), 8.12 (d, 2 H, J ) 8.4 Hz); 13C NMR (75 MHz, CDCl3) δ
14.2, 18.2, 31.6, 31.8, 34.3, 34.4, 34.8, 61.4, 123.7, 125.4, 129.5,
130.2, 132.5, 132.7, 139.1, 144.4, 148.8, 149.8, 165.6; MS (EI,
70 eV) m/ z 398 (M+, 12), 382 (23), 367 (34), 352 (100).
1
2962, 1724 cm-1; H NMR (300 MHz, CDCl3) δ 1.25 (s, 6 H),
1.34 (s, 6 H), 1.39 (t, 3 H, J ) 7.1 Hz), 1.70 (s, 4 H), 2.33 (s, 3
H), 4.40 (q, 2 H, J ) 7.1 Hz), 7.11 (s, 1 H), 7.90 (d, 2 H, J )
8.5 Hz), 8.15 (s, 1 H), 8.16 (d, 2 H, J ) 8.5 Hz); 13C NMR (75
MHz, CDCl3) δ 14.2, 19.8, 31.5, 31.7, 34.3, 34.5, 34.7, 61.7,
127.5, 128.1, 130.1, 131.0, 134.1, 134.3, 135.0, 143.9, 145.5,
151.7, 165.1; MS (EI, 70 eV) m/ z 416 (MH+, 9), 414 (9), 399
(76), 305 (86), 77 (100). Anal. (C24H30O4S) C, H.
4-[(5,6,7,8-Tetr a h yd r o-3,5,5,8,8-p en ta m eth yl-2-n a p h th -
yl)su lfon yl]ben zoic Acid (4). To a solution of 50 mg (0.12
mmol) of ethyl 4-[(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-
naphthyl)sulfonyl]benzoate and 3.0 mL of THF was added 1.0
mL of 1 N aqueous LiOH. The solution was heated to 50 °C
for 3 h, cooled to room temperature, and concentrated in vacuo.
The residue was diluted with brine and acidified with 10%
aqueous HCl, and the products were extracted with ether (2×).
The combined ether layers were dried (MgSO4) and filtered,
and the solvents were removed in vacuo to give 4 as a white
solid (45 mg, 98%): mp 228-231 °C; IR 3300-2500, 1695 cm-1
;
1H NMR (300 MHz, CDCl3) δ 1.25 (s, 6 H), 1.34 (s, 6 H), 1.70
(s, 4 H), 2.33 (s, 3 H), 7.12 (s, 1 H), 7.95 (d, 2 H, J ) 8.4 Hz),
8.15 (s, 1 H), 8.22 (d, 2 H, J ) 8.4 Hz); 13C NMR (75 MHz,
CDCl3) δ 19.9, 31.5, 31.7, 34.3, 34.5, 34.7, 127.7, 128.2, 130.8,
131.1, 134.1, 134.8, 144.0, 146.5, 151.9, 165.0; MS (EI, 70 eV)
387 m/ z 387 (MH+, 33), 371 (100). Anal. (C22H26O4S) C, H,
S.
Eth yl 6-[(5,6,7,8-Tetr a h yd r o-3,5,5,8,8-p en ta m eth yl-2-
n a p h th yl)th io]n icotin a te (14). Sodium hydride (171 mg,
60% dispersion in oil, 4.3 mmol) was rinsed three times with
hexane and dried under vacuum. The vacuum was broken
with dry argon, and 6.6 mL of HMPA and 5,6,7,8-tetrahydro-
3,5,5,8,8-pentamethyl-2-naphthalenethiol (1.0 g, 4.27 mmol)
were added sequentially. After 30 min at 50 °C, copper(I)
iodide (678 mg, 3.56 mmol) was added, which caused the
solution to become deep green. The solution was stirred for
an additional 15 min at 50 °C, ethyl 2-iodonicotinate (986 mg,
3.56 mmol) was added, and the solution was heated to 90 °C
for 5 h. The heating bath was removed, and the reaction
mixture was stirred overnight at room temperature. Water
was added, and the products were extracted with diethyl ether
(3×). The combined ether layers were washed with brine,
dried (MgSO4), and filtered, and the solvent was removed in
vacuo. The residue was purified by flash chromatography on
silica gel (5% ethyl acetate in hexanes) to give 14 as a light
yellow solid (642 mg, 47%): mp 110-111 °C; 1H NMR (300
MHz, CDCl3) δ 1.26 (s, 6 H), 1.31 (s, 6 H), 1.37 (t, 3 H, J ) 7.1
Hz), 1.69 (s, 4 H), 2.32 (s, 3 H), 4.36 (q, 2 H, J ) 7.1 Hz), 6.68
(d, 1 H, J ) 8.0 Hz), 7.23 (s, 1 H), 7.53 (s, 1 H), 7.99 (dd, 1 H,
J ) 2.3, 8.0 Hz), 9.00 (d, 1 H, J ) 2.3 Hz); 13C NMR (75 MHz,
CDCl3) δ 14.3, 20.4, 31.7, 31.8, 34.1, 34.2, 34.9, 34.9, 61.1,
118.9, 121.8, 125.3, 129.3, 135.1, 137.2, 139.2, 144.4, 147.6,
4-[(5,6,7,8-Tetr a h yd r o-3,5,5,8,8-p en ta m eth yl-2-n a p h th -
yl)su lfoxy]ben zoic Acid (3). To a solution of 58 mg (0.15
mmol) of ethyl 4-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-
naphthyl)sulfoxy]benzoate and 4.0 mL of THF were added 1.0
mL of 2 M aqueous LiOH and 2.0 mL of MeOH. The solution
was heated at 55 °C for 2 h, cooled to room temperature, and
stirred for 8 h. The reaction mixture was concentrated in

3562 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 18
Beard et al.
150.7, 165.3, 167.9; MS (EI, 70 eV) m/ z 383 (M+, 47), 368 (83),
350 (100). Anal. (C23H29NO2S) C, H, N, S.
residue was diluted with water and acidified with 10% aqueous
HCl, and the products were extracted with ether (3×). The
combined organic layers were washed with brine, dried
(MgSO4), and filtered, and the solvents were removed in vacuo
to give 6 as a white solid (131 mg, 78%): mp 220-224 °C dec;
6-[(5,6,7,8-Tetr a h yd r o-3,5,5,8,8-p en ta m eth yl-2-n a p h th -
yl)th io]n icotin ic Acid (5). To a solution of ethyl 2-[(5,6,7,8-
tetrahydro-3,5,5,8,8-pentamethyl-2-naphthyl)thio]nicotinate (300
mg, 0.78 mmol) and ethanol (8 mL) was added 2 N KOH (2
mL), and the resulting solution was stirred at 50 °C for 34 h.
The solution was concentrated in vacuo, water was added, and
the mixture was acidified with 10% aqueous HCl. The product
was extracted with methylene chloride (3×), and the combined
organic extracts were washed with brine, dried (MgSO4),
filtered, and concentrated in vacuo. The solid residue was
recrystallized from acetonitrile/methanol (4:1) to give the title
compound (233 mg, 84%) as light yellow crystals: mp 259-
260 °C; 1H NMR (300 MHz, DMSO-d6) δ 1.21 (s, 6 H), 1.26 (s,
6 H), 1.63 (s, 4 H), 2.23 (s, 3 H), 6.81 (d, 1H, J ) 8.2 Hz), 7.39
(s, 1 H), 7.50 (s, 1 H), 7.05 (dd, 1 H, J ) 2.1, 8.2 Hz), 8.84 (d,
1H, J ) 2.1 Hz); 13C NMR (75 MHz, DMSO-d6) δ 19.9, 31.4,
31.4, 33.7, 33.9, 34.4, 119.0, 122.6, 124.8, 129.2, 134.4, 137.7,
138.9, 143.9, 147.1, 150.3, 165.7, 166.0; MS (EI, 70 eV) m/ z
355 (M+, 31), 340 (80), 322 (100). Anal. (C21H25NO2S) C, H,
N, S.
Eth yl 2-Iod o-5-th ia zoleca r boxyla te (16). To a solution
of 4.96 g (31.5 mmol) of 2-(trimethylsilyl)thiazole in 100 mL
of ether at -78 °C under argon was added n-BuLi (23.0 mL,
36.8 mmol, 1.6 M in hexanes), and the resulting mixture was
stirred at -78 °C for 30 min. Ethyl chloroformate (7.60 mL,
10.6 g, 98 mmol) was added, and the reaction mixture was
stirred at -78 °C for 30 min and then at room temperature
for 30 min. The solution was recooled to -78 °C, and a solution
of 10.8 g (42.5 mmol) of iodine and 50 mL of THF was added
via canula. The reaction mixture was warmed slowly to room
temperature and stirred for 15 h. The reaction mixture was
then recooled to -78 °C and the reaction quenched with 20%
aqueous sodium thiosulfate. The products were extracted with
ether (3×), the organic layers were combined, washed with
brine, dried (Na2SO4), and filtered, and the solvents were
removed in vacuo. The crude product was purified by flash
chromatography on silica gel (15% ethyl acetate in hexanes)
to give 16 as a yellow oil (0.89 g, 10%): 1H NMR (300 MHz,
CDCl3) δ 1.38 (t, 3 H, J ) 7.1 Hz), 4.36 (q, 2 H, J ) 7.1 Hz),
8.11 (s, 1 H); 13C NMR (75 MHz, CDCl3) δ 14.1, 61.9, 135.6,
149.4, 159.7.
Eth yl 2-[(5,6,7,8-Tetr a h yd r o-3,5,5,8,8-p en ta m eth yl-2-
n a p h t h yl)t h io]-5-t h ia zoleca r b oxyla t e. Sodium hydride
(0.057 g, 60% dispersion in oil, 2.4 mmol) was rinsed three
times with hexane and dried under vacuum. The vacuum was
broken with dry argon and the hydride was suspended in 5.0
mL of HMPA. 5,6,7,8-Tetrahydro-3,5,5,8,8-pentamethyl-2-
naphthalenethiol (445 mg, 1.9 mmol) was added, and the
resulting mixture was heated at 50 °C for 45 min. Copper(I)
iodide (0.36 g, 1.9 mmol) was added, and the mixture was
heated at 55 °C for 1.5 h. A solution of ethyl 2-iodo-5-
thiazolecarboxylate (0.65 g, 2.3 mmol) and 2.0 mL of HMPA
was added, and the mixture was heated to 95 °C for 2 h. The
reaction mixture was cooled to 0 °C, the reaction was quenched
with water, and the products were extracted with ether (2×).
The organic extracts were combined, washed with brine, dried
(MgSO4), and filtered, and the solvents were removed in vacuo.
The crude product was purified by flash chromatography on
silica gel (15% ethyl acetate in hexanes) to produce the title
IR (KBr pellet) 3400-2500 (COOH), 1702 (CdO) cm-1 1H
;
NMR (300 MHz, CD3OD) δ 1.28 (s, 6 H), 1.32 (s, 6 H), 1.73 (s,
4 H), 2.40 (s, 3 H), 7.42 (s, 1 H), 7.63 (s, 1 H), 8.12 (s, 1 H); 13
C
NMR (75 MHz, DMSO) δ 19.8, 31.4, 31.5, 31.5, 33.8, 34.1, 34.3,
125.3, 129.6, 129.7, 134.7, 138.6, 144.6, 148.5, 148.6, 161.5;
MS (EI, 70 eV) m/ z 361 (M+, 58), 345 (57), 328 (25), 249 (26),
122 (12), 81 (53), 69 (100). Anal. (C19H22NO2S2) C, H, N, S.
Recep tor Assa ys. The transactivation and binding prop-
erties of retinoid analogs were determined at Ligand Phar-
maceuticals (La J olla, CA) as previously described.14b,c
TGa se In d u ction . HL-60 cdm-1 cells were cultured under
conditions described in detail previously.22 Cells in log-phase
growth (2 × 10-5 cells/mL) in RPMI 1640 (Fisher Scientific)
supplemented with insulin, transferrin, and sodium selenide
(TIS; Sigma) were pretreated with 1.25% dimethyl sulfoxide
(DMSO) for 18 h. Cells were then sedimented and resus-
pended in RPMI-TIS containing retinoids or an equivalent
solvent control (0.1% ethanol). After culture for 24 h, cells
were again sedimented, washed once, and lysed, and the
transglutaminase activity was assayed by measuring the
covalent and Ca2+-dependent conjugation of [3H]putrescine to
N,N-dimethylcasein.22 The EC50 value is defined as the
concentration required to induce 50% of the maximal induction
of transglutaminase activity achieved with that compound.
Ch on d r ogen esis In h ibition . The in vitro bioassay em-
ployed high-density micromass cultures of day 11 embryonic
limb bud cells as described.23 Briefly, forelimb buds were
dissociated in a trypsin-EDTA solution, and the resultant
single-cell suspension was plated as 20 µL spots (200 000 cells/
spot) on plastic culture dishes. Retinoid concentrations rang-
ing from 0.3 to 3 µg/mL (1 nM-10 µΜ) were added to the
culture medium (Eagle’s MEM + 10% fetal bovine serum;
GIBCO) 24 h after initial plating. Control cultures received
only the vehicle (ethanol, concentration < 1% by volume). The
cultures were terminated 96 h after plating, at which time the
medium was removed and the cells were fixed for 1 h in 10%
formalin containing 0.5% cetylpyridinium chloride. The cul-
tures were rinsed with acetic acid and then dehydrated in
ethanol and scored for chondrogenesis under the microscope.
An absence or reduction in the number of cartilage nodules
as compared to control cultures was taken as a measure of
suppression of chondrogenesis. The number of cartilage
nodules stained in the whole spot were counted by automated
image scan using the N.I.H. Image-1.52 application. Mean
number of nodules and standard deviations were calculated
for four replicate cultures per concentration. The median
concentration of each retinoid causing 50% inhibition of
chondrogenesis compared with controls (IC50) was calculated
by logarithmic curve fitting of the dose-response data.
Ack n ow led gm en t. We are grateful to Dr. Glenn
Croston at Ligand Pharmaceuticals, La J olla, CA, for
providing the receptor transactivation and binding
assay data presented in Tables 1 and 2. We thank Mr.
Hai Nguyen for conducting the mass spectral analysis.
compound as an orange oil (0.30 g, 41%): IR 1714 (CdO) cm-1
;
1H NMR (300 MHz, CDCl3) δ 1.28 (s, 6 H), 1.32 (s, 6 H), 1.32
(t, 3 H, J ) 7.1 Hz), 1.70 (s, 4 H), 2.41 (s, 3 H), 4.29 (q, 2 H, J
) 7.1 Hz), 7.30 (s, 1 H), 7.60 (s, 1 H), 8.19 (s, 1 H); 13C NMR
(75 MHz, CDCl3) δ 14.3, 20.3, 31.7, 31.8, 34.1, 34.3, 34.8, 34.9,
61.4, 125.6, 128.5, 129.8, 135.1, 139.0, 144.9, 148.8, 148.9,
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