A new class of RAR subtype saelective retinoids: Correlation of pharmacological effects with receptor activity

Article abstract of DOI:10.1016/S0968-0896(98)00212-0

The synthesis and biological activity of a series of structurally related retinoids with different RAR subtype selectivities are described. These retinoids bind to all three RAR subtypes but in functional transactivation assays, they show RARβ or RARβ,γ selectivity with weak RARα activity. The subtype selectivity of these retinoids was found to correlate with their efficacy (ODC inhibition) and toxicity (topical irritation and teratogenicity) profiles. The degree of RARγ transactivation activity correlates with their topical toxicity and teratogenicity as measured by the inhibition of chondrogenesis. Of the RARβ selective retinoids reported here, retinoid 12 is the most promising, as it is completely devoid of two common retinoid related toxicities, namely topical irritation and teratogenesis. Copyright (C) 1999 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(98)00212-0

Bioorganic & Medicinal Chemistry 7 (1999) 263±270
A New Class of RAR Subtype Selective Retinoids:
Correlation of Pharmacological E€ects with
Receptor Activity
Vidyasagar Vuligonda,a,* Yuan Lin,a Scott M. Thacher,b Andrew M. Standeven,b
Devendra M. Kochar,c and Roshantha A. S. Chandraratnaa,b,
*
aDepartment of Chemistry, Retinoid Research, Allergan Inc., Irvine, CA 92614, USA
bDepartment of Biology, Retinoid Research, Allergan Inc., Irvine, CA 92614, USA
cDepartment of Pathology, Anatomy and Cell Biology, Thomas Je€erson University, Philadelphia, PA 19107, USA
Received 15 June 1998; accepted 8 September 1998
AbstractÐThe synthesis and biological activity of a series of structurally related retinoids with di€erent RAR subtype selectivities
are described. These retinoids bind to all three RAR subtypes but in functional transactivation assays, they show RARb or RARb,g
selectivity with weak RARa activity. The subtype selectivity of these retinoids was found to correlate with their ecacy (ODC
inhibition) and toxicity (topical irritation and teratogenicity) pro®les. The degree of RARg transactivation activity correlates with
their topical toxicity and teratogenicity as measured by the inhibition of chondrogenesis. Of the RARb selective retinoids reported
here, retinoid 12 is the most promising, as it is completely devoid of two common retinoid related toxicities, namely topical irrita-
tion and teratogenesis.# 1999 Elsevier Science Ltd. All rights reserved.
Introduction
The utility of nonselective retinoids in the treatment of
human disease has been limited by the toxicity asso-
ciated with these compounds. The possibility that cer-
tain retinoid toxicities may be associated with speci®c
RAR subtypes is supported by the implication of
RARg, the major RAR subtype found in the skin,7a
with retinoid induced topical irritation.7d The fact that
the distribution of various receptor subtypes is relatively
tissue speci®c7 suggests that receptor-selective retinoids
would be more speci®c in their biological action. As a
consequence, receptor-selective retinoids could be asso-
ciated with fewer toxicities than their nonselective
counterparts and hence would be preferred therapeutic
agents. Several classes of RAR8 and RXR9 speci®c
ligands have been described, and recently RAR subtype
selectivity10 and even speci®city11 has been achieved. As
part of an ongoing program to design more selective
retinoids, we had previously investigated dihydronaph-
thalenic retinoids substituted at the C-1 sp2 carbon (1,
Figure 1) and obtained signi®cant receptor subtype and
functional selectivity.11b,12 Here we report on the struc-
ture±activity relationship (SAR) of optically pure tetra-
hydronaphthalenic retinoids substituted at the C-1 sp3
carbon (2, Figure 1), and demonstrate distinct RAR sub-
type selectivities which are dependent upon the relative
stereochemistry of the functional group at the C-1 posi-
tion. We also correlate the RAR activation pro®les of
these compounds with activity in other pharmacological
and toxicological assays.
Natural retinoids, which are derivatives of retinol (vita-
min A), regulate a variety of physiological processes
including embryogenesis, reproduction, cell prolifera-
tion and di€erentiation, vision, and immune function.1
The mechanism of action of retinoids has come under
intense study due to their therapeutic potential in sev-
eral areas including cancer,2 skin disorders,3 metabolic
disease,4 and HIV-induced lymphopenia.5 The biologi-
cal actions of retinoids result from the regulation of
gene transcription through the intermediacy of nuclear
receptors.6 There are two families of retinoid receptors,
retinoic acid receptors (RARs) and retinoid X receptors
(RXRs), each with three subtypes (a,b, and g). The
RARs and RXRs are ligand-dependent transcription
factors which belong to the superfamily of steroid
nuclear receptors. Retinoic acid (RA) (Fig. 1), the
physiological hormone for the RARs, binds with
similar anity to all the three RAR subtypes, and does
not bind to the RXRs. In contrast, the putative hor-
mone for the RXRs, 9-cis retinoic acid (9-cis RA)
(Fig. 1), binds with similar anities to the RXRs and
RARs.6c
Key words: RARs; subtype selective retinoids; ODC; retinoid toxicity;
teratogenicity.
*Corresponding authors.
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(98)00212-0

264
V. Vuligonda et al. / Bioorg. Med. Chem. 7 (1999) 263±270
Results and Discussion
were hydrolyzed to the corresponding carboxylic acids
10±15 (Scheme 3) without loss of optical purity. The opti-
cal integrity of the ®nal acids was established by obtaining
constant speci®c rotation by repeat crystallization.
Synthesis
The retinoids described in this paper were prepared
starting from the known ketone 3.12 Enantioselective
reduction of 3 gave the R (4) and S (5) alcohols (Scheme
1). The C-1 stereochemistry of the alcohols was assigned
on the basis of results using three di€erent chiral redu-
cing agents and comparison with literature reports on
reduction of related ketones.13 The enantiomeric alco-
hols 4 and 5 were prepared on multigram scale by
NaBH4 reduction of 3 and separation of the racemic
mixture by chiral HPLC. The optically pure alcohols 4
and 5 were each reacted with dihydropyran (DHP) in
the presence of a catalytic amount of p-toluene sulfonic
acid (p-TSA) and the pairs of diastereomeric tetra-
hydropyran (THP) ethers 6/7 and 8/9 were separated by
normal phase HPLC (Scheme 2). Although the absolute
stereochemistries at the anomeric carbon in the THP
ring were not de®ned, the diastereomeric pairs 6/8 and
7/9 were shown to be related as enantiomers on the
basis of their speci®c rotations. The ethyl esters 4±9
Biological evaluation
Binding anities of the retinoid analogues were deter-
mined using baculovirus expressed receptors and com-
petition with [3H]-RA for the RARs and [3H]-9-cis-RA
for the RXRs.14 The functional gene transcriptional
activities of the retiniods were measured in transactiva-
tion assays in CV-1 cells transiently cotransfected with
an individual receptor gene construct and a reporter
gene.15 Several retinoids were also tested in an in vivo
assay of ecacy, namely that of inhibition of 12-O-tet-
radecanoylphorbol-13-acetate (TPA) induced ornithine
decarboxylase (ODC) activity in hairless mouse skin.
ODC is a critical enzyme in the biosynthetic pathway to
polyamines, and is elevated in cells prior to a hyperpro-
liferative response.16 Thus, inhibition of ODC activity
could be predictive of the potential clinical utility of
Figure 1.
Scheme 1.

V. Vuligonda et al. / Bioorg. Med. Chem. 7 (1999) 263±270
265
retinoids in the treatment of hyperproliferative skin
diseases such as psoriasis. The toxicities of these reti-
noids were determined using an in vivo topical irritation
assay in hairless mice17 and an in vitro inhibition of
chondrogenesis assay in mouse embryo limb bud
mesenchymal cells.18 The latter assay is predictive of the
in vivo teratogenic potential of retinoids.19
shown). The enantiomeric alcohols 10 and 11 had rela-
tively low binding anities to the RARs. This e€ect
may be due to unfavorable interactions of the hydroxyl
group with a hydrophobic region of the receptor. Inter-
estingly, the R alcohol (10) had a 2.5- to 6-fold lower
anity to the RARs than the corresponding S alcohol
(11), indicating that the introduction of a hydroxyl
group in the former spatial orientation is specially dis-
favored. Conversion of the C-1 hydroxyl into lipophilic
moieties as in the THP ethers (12±15) resulted in increased
anity for the RARs. These compounds with increased
hydrophobicity at C-1 (12±15) are quite similar in their
The free carboxylic acids 10±15 were evaluated in the
receptor binding assay and were found to bind RARs
with di€erent anities (Table 1). However, none of the
compounds had any anity for the RXRs (data not
Scheme 2. THP = tetrahydropyranyl. Note that the absolute stereochemistry at the anomeric carbon is not de®ned. (a) RT is retention time in
minutes on a normal-phase HPLC column (Partisil-10 PAC column; hexane:ethyacetate, 19:1).
Scheme 3.

266
V. Vuligonda et al. / Bioorg. Med. Chem. 7 (1999) 263±270
relative binding anities to the three RAR subtypes. In
addition, these compounds are somewhat RARb selec-
tive, binding with 5- to 20-fold higher anity to RARb
compared to RARa and RARg. Although compounds
12±15 are quite similar in their relative binding anities
to the three RAR subtypes, they are quite distinct in
their abilities to activate gene transcription. Thus, the
THP ether compounds 14 and 15, which have S stereo-
chemistry at C-1, are quite potent at RARb and RARg
and activate RARa only at higher concentrations in the
transactivation assays (Fig. 2). In contrast, compounds
12 and 13, which have R stereochemistry at C-1, are
quite RARb selective, again activating RARa only at
higher concentrations and having little or no activity at
RARg. Interestingly, changes in the relative stereo-
chemistry at the anomeric carbon of the THP ether can
also lead to subtle changes in transactivation. Thus,
compound 12 has no detectable activity at RARg, while
compound 13 has a very low but detectable level of
activity at RARg. Also, compound 15 appears to be
more potent at RARg than the related compound 14.
The transactivation data for these closely related com-
pounds illustrate the phenomenon of functional selec-
tivity where compounds that bind to a receptor with
similar anity elicit quite di€erent functional e€ects
because they cause subtly di€erent conformational
changes in the receptor. It should however be noted that
in some cases, as in compounds 14 and 15, there is a
lack of exact correlation between binding anities and
potencies in the transactivation assays. This may be
because the binding assays are performed with baculo-
virus expressed RARs where the receptors presumably
are in a monomeric form while the transactivation
assays utilize transfected cells where the RARs can form
heterodimers. For comparison purposes, RA had trans-
activation potencies in the range 1±10 nM (data not
shown) and binding anities in the range 10±20 nM
(Table 1) at the three RARs.
Table 1. Competitive binding assay data for retinoid acids 10±15
Kd (nM) RAR
Retinoid
a
b
g
RA
10
11
12
13
14
15
9
12
1329
549
48
20
31
19
2607
571
282
188
171
208
3516
580
282
116
182
325
39
The ODC inhibition16 and topical irritation assays in
hairless mouse were carried out using the diastereomeric
ethyl ester derivatives (6±9) of compounds 12±15
(Table 2). It is expected that the ethyl esters act as pro-
drugs and are converted in vivo to the active free acid
forms during the course of the assays.20a Zorac,TM
a
currently marketed retinoid for the topical treatment of
psoriasis, is an ethyl ester derivative of the acetylenic
class of retinoids, similar to the retinoids of the present
discussion. Extensive pharmacokinetic studies on Zor-
acTM and related acetylenic retinoids have established
that the esters are readily hydrolyzed to the active free
acid forms in vivo.20 We20c and others20d have employed
synthetic retinoid esters in the ODC inhibition assay
and reached similar conclusions that benzoate ester
retinoids and their corresponding free acids are equally
active in this assay. In addition, the ethyl esters of these
acetylenic retinoids are less irritating and have better
topical therapeutic indices than the corresponding free
acids in the topical irritation assay making them ideal
pro-drugs for the study. Compounds 8 and 9, the esters
of the RARb,g active retinoids 14 and 15, are potent
Table 2. ODC inhibition and irritation assay data for retinoid esters
6±9
ODC Inhibition
Cutaneous toxicitya
Retinoid
Dose
(nmol/25 g)
% Inhibition
of ODC
Dose
(nmol/25 g)
Toxicity
score
6
7
8
9
30
3
0.3
0.3
61
70
75
79
2000
100
110
42
0
3
11
11
aCutaneous toxicity is a composite measure of ¯aking and abrasion of
the skin on hairless mice caused by the test retinoid with a maximum
possible score of 17.17
Figure 2. Dose±response curves at each RAR for retinoids 12±15 in
CV-1 cells transfected with RAR holoreceptors and a MTV-4 (R5G)-
luciferase reporter plasmid.

V. Vuligonda et al. / Bioorg. Med. Chem. 7 (1999) 263±270
267
inhibitors of ODC, giving 75 and 79% inhibition,
respectively, at a dose of 0.3 nmol/25 g (Table 2). Com-
pound 7, the free acid (13) of which is potent at RARb
and only weakly active at RARg, is about tenfold less
potent than 8 or 9. Compound 6, the acid (12) of which
is transcriptionally inactive at RARg, is about 100-fold
less potent than 8 or 9 since it gives only 61% inhibition
at a dose of 30 nmol/25 g. Thus, in this series of com-
pounds, the ability to activate RARg correlates well
with ODC inhibitory activity suggesting that RARg
plays a major role in the inhibition of ODC activity in
hairless mouse skin. Note also that 12 (active form of 6)
is weakly active at RARa, suggesting that RARa does
not play a signi®cant role in ODC inhibitory activity. It
is also interesting that compound 6, the active form of
which (compound 12) has absolutely no RARg tran-
scriptional activity, still has ODC inhibitory activity
although of reduced potency. Since 12 still binds to
RARg, it is possible that with this compound the
transactivation and ODC inhibitory activities through
RARg have been separated similar to the previously
described separation of transactivation and anti-AP1
activities.21 These data suggest that the mechanism of
retinoid inhibition of ODC involves antagonism of the
activity of a transcription factor such as AP-1 or NF-
IL622 and not direct transcriptonal activation of reti-
noid responsive genes.
in this assay having an IC50 value of >10,000 ng/mL.
These data which suggest that RARg plays a major role
in mediating the toxic e€ects of retinoids are consistent
with previous studies using receptor knockout ani-
mals.7e These results establish for the ®rst time the cor-
relation of ligand mediated RARg transactivation with
chondrogenesis inhibition. Our data also suggest that
RARb-speci®c retinoids may be devoid of teratogenic
potential, at least in those aspects of teratogenicity
which are predicted by the chondrogenesis inhibition
assay.
Conclusion
In conclusion, we have shown that the absolute stereo-
chemistry at the C-1 position of acetylenic retinoids
imparts unique transactivation pro®les to this class of
retinoids. In addition, subtle changes in the stereo-
chemistry at the C-1 asymmetric carbon can lead to
dramatic changes in the receptor transactivation pro®les
of structurally similar retinoids. Our data show that
RARg transactivation correlates with ODC inhibition
and topical irritation. Further, we show for the ®rst
time a correlation of RARg transactivation with chon-
drogenesis inhibition. Finally, in compound 12, we have
synthesized an RARb subtype selective transactivator
with complete lack of transactivation through RARg
and with weak RARa activity, which retains ODC
inhibitory activity and hence potentially anti-pro-
liferative activity. Compound 12 is greatly reduced in
topical irritation and in teratogenic potential and could
be a retinoid with superior therapeutic index in hyper-
proliferative diseases such as psoriasis. These ®ndings
demonstrate that it is possible to selectively and favor-
ably modulate the biological activities (ecacy and
toxicity) of retinoid analogues by changing their recep-
tor activation pro®les and this augurs well for the future
of retinoid therapeutics.
The ability of these retinoid esters to induce topical
irritation in hairless mice was measured using a cuta-
neous toxicity score which is an aggregate measure of
the skin ¯aking and abrasion that is induced by applied
retinoid.17 Compounds 8 and 9, the esters of the
RARb,g active retinoids are quite irritating, giving a
toxicity score of 11 at doses of 110 and 42 nmol/25 g,
respectively (Table 2). Compound 7 has an intermediate
topical irritation potential and compound 6 causes
absolutely no irritation even at a 20-fold higher dose
(Table 2). Clearly, the topical irritation potential of
these THP ether retinoids tracks with their RARg
transactivation activity. These data suggest that RARg
transactivation is associated with topical irritation and
are in agreement with a recent report.7d
Experimental
Melting points were determined using a Thomas±
Hoover melting point apparatus and are uncorrected.
1H NMR spectra were recorded using a Varian Gemini
300 spectrometer (300 MHz) and 13C NMR spectra
using a Varian XL 300 spectrometer (75 MHz) in the
solvent indicated. Enantiomeric ratio was determined
using Chiralcel OD-H column, with hexane:EtOAc (9:1)
as mobile phase. Optical rotations were recorded on
Perkin±Elmer Model 241 polarimeter. Elemental ana-
lyses were performed by Robertson Microlit Labora-
tories, Inc., Madison, NJ.
The activities of compounds 12±15 in the chondrogen-
esis inhibition assay are summarized in Table 3. Com-
pounds 14 and 15, the RARb,g active retinoids, are
highly potent in this assay having IC50 values of 0.15
and 0.045 ng/mL, respectively. In comparison, com-
pound 13, which has only weak RARg activity, is about
100-fold less potent than 14 or 15. The RARb-selective
analogue 12, which has no RARg transcriptional activ-
ity and only weak RARa activity, is essentially inactive
Table 3. Chondrogenesis inhibition assay18 data for retinoid acids
12±15
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). All reactions were carried out
under a positive pressure of argon using reagent grade
or anhydrous solvents as received. The phrase `dried
and evaporated' indicates drying over MgSO4 followed
by evaporation of the solvents under house vacuum.
Retinoid
IC50(ng/mL)
12
13
14
15
>10,000
15
0.15
0.045

268
V. Vuligonda et al. / Bioorg. Med. Chem. 7 (1999) 263±270
Enantioselective reduction of ketone 3 with ( ) DIP
chlorideTM. To a cold ( 25ꢀC) solution of ( ) DIP
chlorideTM in THF was added ketone 3 (71 mg,
0.2 mmol) in THF (1 mL). The mixture was stirred for
72 h at 25ꢀC. The solvent was removed under reduced
pressure, the residue was dissolved in ether (5 mL) and
diethanolamine (75 mg, 0.7 mmol) was added via syr-
inge. The mixture was stirred for 2 h at ambient tem-
perature. The resultant solid was ®ltered, washed with
ether, the combinded organic layer was dried and eva-
porated. Column chromatography a€orded 48 mg
(68%) of the desired alcohol: mp 120±121 ꢀC. The
enantiomeric ratio was determined by chiral HPLC. R:
alcohol 4; rt, 9.5 min (20%). [a]2d2 80.0ꢀ (c 0.0035,
CH2Cl2). Anal. calcd for C23H24O3; C, 79.31; H, 6.90;
found C, 78.92, H, 7.07. S: alcohol 5; rt, 6.6 min (80%).
[a]2d2 +81.0ꢀ (c 0.003, CH2Cl2). Anal. calcd for
C23H24O3: C, 79.31; H, 6.90. Found: C, 79.11; H, 7.09.
1H NMR (CDCl3): d 1.27 (s, 3H), 1.34 (s, 3H), 1.41 (t,
3H, J = 7.1 Hz), 1.63 (m, 1H), 1.87 (m, 2H), 2.10 (m,
1H), 4.39 (q, 2H, J = 7.1 Hz), 4.73 (brs, 1H), 7.33 (d,
1H, J = 8.1 Hz), 7.42 (dd, 1H, J = 1.8, 8.1 Hz), 7.56 (d,
2H, J = 8.4 Hz), 7.65 (d, 1H, J = 1.8 Hz), 8.03 (d, 2H,
J = 8.4 Hz); 13C NMR (CDCl3): d 14.3, 28.8, 31.2, 31.3,
34.1, 34.3, 61.1, 68.6, 88.2, 92.2, 120.2, 126.8, 128.0,
129.4, 129.6, 130.9, 131.4, 131.6, 138.2, 146.7, 166.1.
Preparation of tetrahydropyranyl ethers 6 and 7. To a
cold (0 ꢀC) solution of R alcohol 4 (272 mg, 0.78 mmol),
DHP (1 mL) in CH2Cl2 (10 mL) was added p-toluene-
sulfonic acid (p-TsOH) (10 mg) and the mixture was
stirred for 8 h at rt. The reaction was quenched with aqu-
eous NaHCO3 (10 mL) extracted with EtOAc (2Â25 mL).
The organic layer was washed with brine, dried, and
evaporated. The crude material was puri®ed by chro-
matography (hexane:EtOAc, 19:1). The two diaster-
eomers were separated by normal-phase HPLC (hexane:
EtOAc, 19:1) to a€ord 6 (138 mg) and 7 (135 mg).
Diastereomer 6: HPLC (rt), 88 min [a]2d2 +28.9ꢀ (c
0.0063, CH2Cl2). 1H NMR (CDCl3): d 1.25 (s, 3H), 1.32
(s, 3H), 1.40 (t, 3H, J = 7.1 Hz), 1.50 2.15 (m, 10H),
3.55 3.65 (m, 1H), 3.95 4.05 (m, 1H), 4.38 (q, 2H, J =
7.1 Hz), 4.64 (t, 1H, J = 4.9 Hz), 4.89 (t, 1H, J = 4.9 Hz),
7.32 (d, 1H, J = 7.3 Hz), 7.41 (dd, 1H, J = 1.8, 7.3 Hz),
7.47 (d, 1H, J = 1.8 Hz), 7.57 (d, 2H, J = 8.3 Hz), 8.01
(d, 2H, J = 8.3 Hz); 13C NMR (CDCl3): d 14.2, 19.7,
25.5, 27.4, 31.0, 31.4, 31.5, 34.0, 34.6, 61.0, 62.6, 74.4,
87.9, 92.6, 99.1, 119.6, 127.0, 128.1, 129.4, 129.6, 130.9,
131.3, 131.9, 136.1, 147.2, 166.0. Anal. calcd for
C28H32O4: C, 77.78; H, 7.41. Found: C, 78.06; H, 7.51.
Diastereomer 7: HPLC (rt), 95 min [a]2d2 89.4ꢀ (c 0.008,
CH2Cl2). 1H NMR (CDCl3): d 1.27 (s, 3H), 1.33 (s, 3H),
1.41 (t, 3H, J = 7.2 Hz), 1.50±1.70 (m, 5H), 1.72±2.10
(m, 5H), 3.55±3.67 (m, 1H), 4.02±4.10 (m, 1H), 4.39 (q,
2H, J = 7.2 Hz), 4.79 (t, 1H, J = 5.9 Hz), 4.83±4.94 (m,
1H), 7.31 (d, 1H, J = 8.1 Hz), 7.41 (dd, 1H, J = 1.8, 8.1
Hz), 7.58 (d, 2H, J = 8.3 Hz), 7.69 (d, 1H, J = 1.8 Hz),
8.02 (d, 2H, J = 8.3 Hz); 13C NMR (CDCl3): d 14.2, 19.9,
24.1, 25.4, 31.1, 31.2, 31.3, 34.0, 34.3, 61.0, 63.0, 71.1,
87.8, 92.8, 95.7, 119.9, 126.6, 128.2, 129.3, 129.5, 130.7,
131.4, 132.2, 136.5, 147.0, 166.0. Anal. calcd for
C28H32O4: C, 77.78; H, 7.41. Found: C, 77.44; H, 7.42.
Enantioselective reduction of ketone 3 with (R)-(+)-
BINAL-H. To a solution of LAH in THF (2 mL,
2 mmol) was added anhydrous EtOH (92 mg, 2 mmol) in
THF (2 mL) dropwise (10 min). To this solution (R)-
(+)-1,10-bi-2-naphthol (310 mg, 1.1 mmol) in THF
(2 mL) was added dropwise (15 min). The mixture was
stirred for additional 30 min and cooled to 78ꢀC and
ketone 3 (68 mg, 0.2 mmol) in THF (1 mL) was added.
The mixture was stirred at 50 ꢀC for 16 h. Reaction
was quenched with EtOH (1 mL), and diluted with ether
(50 mL) and EtOAc (15 mL). The mixture was washed
with water (5 mL), 3 N NaOH (3Â5 mL), water (5 mL)
and brine (5 mL). The organic layer was dried and eva-
porated. Column chromatography a€orded 62 mg of the
desired alcohol. R: alcohol 4, 85%. S: alcohol 5, 15%.
Preparation of tetrahydropyranyl ethers
8 and 9.
Employing the procedure used for the preparation of 6
and 7, compounds 8 and 9 were prepared from alcohol 5.
Diastereomer 8: HPLC (rt), 88min. [a]2d2 28.5ꢀ (c 0.0066,
CH2Cl2). Anal. calcd for C28H32O4: calcd C, 77.78; H,
7.41. Found: C, 77.53, H, 7.39. It is identical to com-
pound 6 by 1H and 13C NMR.
Enantioselective reduction of ketone 3 with (S)-( )-
BINAL-H. Ketone 3 was reduced using the same pro-
cedure as used with the (R)-(+)-BINAl-H reagent,
instead (S)-( )-BINAL-H was used as the reducing
agent. R: alcohol 4, 17%. S: alcohol 5, 83%.
Diastereomer 9: HPLC (rt), 95 min [a]2d2 +90.8ꢀ (c
0.0057, CH2Cl2). Anal. (C28H32O4): C, 77.78; H, 7.41.
Found: C, 77.53, H, 7.48. It is identical to compound 7
Enantioselective reduction of ketone 3 with (S)-( )-
OAB-BH3. To a cold ( 20ꢀC) solution of ketone 3
(300 mg, 0.9 mmol) in CH2Cl2 (15 mL) was added 2-
propanol (104 mg, 1.7 mmol), (S)-( ) B-methylox-
azoborolidine (288 mg, 1.04 mmol) and borane±THF
(1 M solution in THF) (1 mL) sequentially. The reaction
was stirred for 4 h at 20ꢀC and 16 h at ambient tem-
perature. To the reaction water (3 mL) was added and
extracted with EtOAc (2Â20 mL). The combined
organic layer was washed with water (5 mL), brine
(5 mL) dried and evaporated. The crude material was
chromatographed (hexane:EtOAc, 9:1) to a€ord the
alcohol as a white solid 267 mg (89%). R: alcohol 4,
88%. S: alcohol 5, 12%.
1
by H and 13C NMR.
Compound 10. (Standard hydrolysis procedure): To a
solution of ester 4 (46 mg, 0.11 mmol) in THF (1 mL),
MeOH (0.5 mL), was added LiOH in water (0.5 M,
1 mL). The mixture was stirred for 16 h at rt. The mix-
ture was diluted with EtOAc (30 mL), acidi®ed to pH 5
using ice-cold 10% HCl. The mixture was washed with
brine (2Â5 mL), dried, and evaporated. Recrystalliza-
tion from hexane:ether gave 10 as a white solid (35 mg).
Compound 10: [a]2d2 86.4ꢀ (c 0.0025, acetone). 1H NMR
(acetone-d6): d 1.28 (s, 3H), 1.30 (s, 3H), 1.60±1.70
(m, 1H), 1.75±1.98 (m, 2H), 2.00±2.10 (m, 1H), 4.65 (t, J

V. Vuligonda et al. / Bioorg. Med. Chem. 7 (1999) 263±270
269
= 4.8 Hz, 1H), 7.40 (brs, 2H), 7.66 (d, J = 8.2 Hz, 3H),
8.05 (d, J = 8.2 Hz, 2H); 13C NMR (acetone-d6): d
31.46, 34.82, 35.72, 68.35, 88.39, 93.26, 120.35, 127.64,
128.79, 130.64, 131.00, 132.26, 132.37, 132.63, 140.82,
147.57, 166.92. Anal. calcd for C21H20O3: C, 78.75; H,
6.25. Found: C, 78.50; H, 5.99.
treatment cells were harvested in 0.1 M K3PO4 (pH 7.8),
1.0% Triton X-100, 1.0 mM DTT, 2 mM EDTA. Luci-
ferase activity was measured using ®re¯y luciferin
(Analytical Luminescence Laboratory) and an EG&G
Berthold 96-well plate luminometer. Luciferase values
represent the mean ‹ SEM of triplicate determinations.
Compound 11. Employing the standard hydrolysis pro-
cedure, acid 11 was prepared from ester 5. Compound
11: [a]2d2 +83.9ꢀ (c 0.0023, acetone). It is identical to
Binding assay
1
compound 10, by H and 13C NMR. Anal. calcd for
Each receptor subtype (RAR aa,b,g) was expressed in
Baculovirus. Stock solutions of all compounds were
prepared as 10 mM ethanol solutions and serial dilu-
tions carried out into DMSO:glycerol (1:1), 120 mM
KCl, 8 mM Tris, 5 mM CHAPS, 4 mM DTT, and
0.24 mM PMSF at pH 7.4 at room temperature.
C21H20O3: calcd C, 78.75; H, 6.25. Found: C, 78.50 H,
6.50.
Compound 12. Employing the standard hydrolysis pro-
cedure, acid 12 was prepared from ester 6. Compound
12: [a]2d2 +21.0ꢀ (c 0.0019, CH2Cl2). 1H NMR (acetone-
d6): d 1.25 (s, 3H), 1.31 (s, 3H), 1.55±2.10 (m, 10H), 3.50
3.61 (m, 1H), 3.86±3.96 (m, 1H), 4.65 (t, 1H, J = 4.5 Hz),
4.91 (t, 1H, J = 3.1 Hz), 7.45 (d, 2H, J = 1.1 Hz), 7.50
(brs, 1H), 7.65 (dd, 2H, J = 1.9, 8.4 Hz), 8.05 (dd, 2H, J
= 8.4 Hz); 13C NMR (acetone-d6): d 20.4, 26.3, 28.0,
29.1, 30.6, 31.7, 34.6, 35.2, 62.8, 74.9, 88.5, 93.1, 99.6,
120.3, 128.1, 128.7, 130.1, 130.9, 131.7, 132.3, 133.0,
137.5, 148.2, 166.9. Anal. calcd for C26H28O4: calcd C,
77.22; H, 6.93. Found: C, 76.42;, H, 6.94.
The ®nal assay volume was 250 mL and contained 10±
40 mg of extract protein along with 5 nM of [3H] all trans
retinoic acid and varying concentrations of competing
ligand at a range from 0±10 5 M. The assays were run
using a Biomek formatted for a 96-well minitube sys-
tem. Incubations were carried out at 4 ꢀC until equili-
brium was achieved. Nonspeci®c binding was de®ned as
that binding remaining in the presence of 1000 nM of
unlabelled RA. At the end of the incubation period,
50 mL of 6.25% hydroxyapitite was added in a wash
bu€er which consisted of 100 mM KCl, 10 mM Tris,
and 0.5% Triton X-100. The mixture was vortexed and
incubated for 10 min at 4ꢀC, centrifuged and super-
natant removed. The hydroxyapitite was washed three
more times with the bu€er and the amount of receptor±
ligand complex determined by liquid scintillation
counting of the pellet. After correcting for nonspeci®c
binding, IC50 values were determined graphically from a
log±logit plot of the data. The Kd values were deter-
mined by application of the Cheng±Prussof equation to
the IC50 values, the labelled ligand concentration, and
the Kd of the labeled ligand.
Compound 13. Employing the standard hydrolysis pro-
cedure, acid 13 was prepared from ester 7. Compound
13: [a]2d2 80.0ꢀ (c 0.0015, CH2Cl2). H NMR (acetone-
1
d6): d 1.26 (s, 3H), 1.31 (s, 3H), 1.55±1.65 (m, 4H), 1.65
2.10 (m, 6H), 3.51±3.60 (m, 1H), 3.92±4.02 (m, 1H), 4.74
(t, 1H, J = 4.4 Hz), 4.88 (brs, 1H), 7.43 (brs, 2H), 7.65
(s, 1H), 7.65 (d, 2H, J = 8.4 Hz), 8.05 (d, 2H, J =
8.4 Hz); 13C NMR (acetone-d6): d 21.0, 25.2, 26.4, 31.4,
31.6, 32.1, 34.7, 35.2, 63.7, 72.3, 88.5, 93.3, 97.2, 120.5,
127.7, 128.8, 130.6, 130.9, 131.5, 132.3, 133.4, 138.1,
148.2, 166.9. Anal. calcd for C26H28O4: C, 77.22; H,
6.93. Found: C, 76.64; H, 7.00.
Compound 14. Employing the standard hydrolysis pro-
cedure, acid 14 was prepared from ester 8. Compound
14: [a]2d2 20.9ꢀ (c 0.0033, CH2Cl2). It is identical to
Acknowledgements
The authors thank M. E. Garst and A. T. Johnson for
helpful discussions and D. Mais, E. Berger, and K.
Flatten for providing receptor binding data and H.
Nguyen for the mass spectral analyses.
1
compound 12, by H and 13C NMR. Anal. calcd for
C26H28O4: C, 77.22; H, 6.93. Found: C, 76.59; H, 7.00.
Compound 15. Employing the standard hydrolysis pro-
cedure, carboxylic acid 15 was prepared from ester 9.
Compound 15: [a]d22 +79.6ꢀ (c 0.0022, CH2Cl2). It is
References and Notes
1
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