Identification of a retinoic acid receptor α subtype specific agonist

Full text of DOI:10.1021/jm9603532

© Copyright 1996 by the American Chemical Society
Volume 39, Number 16
August 2, 1996
Communications to the Editor
only in those tissues in which the receptor is substan-
Id en tifica tion of a Retin oic Acid
tially expressed. Such receptor specific retinoids will
possess much better therapeutic indices than their
nonselective counterparts, although each of the specific
retinoids is likely to be effective in a narrower range of
diseases.
Recep tor r Su btyp e Sp ecific Agon ist
Min Teng,*,† Tien T. Duong,† Elliott S. Klein,‡
Mary E. Pino,‡ and
Roshantha A. S. Chandraratna*,†,‡
Several classes of molecules that are specific for the
RAR11 or RXR12 families of receptors have been de-
scribed. However, only moderate selectivity has been
achieved in terms of discriminating between the recep-
tor subtypes.13 An important advance was made with
the internal amide analog Am 580 (Chart 1), which
Retinoid Research, Departments of Chemistry and Biology,
Allergan Incorporated, Irvine, California 92713-9534
Received May 15, 1996
Retinoids are potent molecules that can effect a
variety of fundamental biological processes including
cell differentiation and proliferation and apoptosis.1
However, in spite of their great therapeutic promise, the
clinical use of retinoids is still restricted largely to
dermatology2 and some cancers.3 This is largely due
to the wide range of toxic effects that are associated with
the currently available retinoids.4 The more extensive
clinical use of retinoids will occur only if new synthetic
analogs with vastly improved therapeutic indices can
be developed. Recent advances in the understanding
of the molecular mechanisms of action of retinoids
suggest that it would be possible to design such retinoids
with improved therapeutic indices. Retinoids elicit their
biological effects by activating a series of nuclear
receptors which are ligand-inducible transcription fac-
tors belonging to the steroid receptor superfamily. The
retinoid receptors are classified under two families, the
retinoic acid receptors (RARs)5 and the retinoid X
receptors (RXRs),6 each consisting of three distinct
subtypes (R, â, and γ). The RARs function in vivo as
RAR-RXR heterodimers7 which bind to the promoter
regions of retinoid responsive genes and mediate gene
transcription upon ligand binding to the RARs.8 It
appears that each of the RARs regulates distinct genes,
although there is some overlap.9 Also, each of the RAR
subtypes has a unique tissue distribution pattern.10
Thus, a retinoid agonist which binds specifically to one
RAR subtype would have limited biological effects
because it would regulate only a subset of retinoid
responsive genes, and these effects would be observed
exhibited moderately selective (∼30-fold) binding affin-
14
ity for RARR relative to RARâ/γ
.
In this paper, we
report on the identification of two retinoid analogs, 1
(AGN 193835) and 2 (AGN 193836), which have very
greatly increased selectivity for the RARR subtype
(Chart 2). Compound 2, which has >2000-fold higher
binding affinity for RARR relative to RARâ with no
measurable binding affinity for RARγ, is the first
example of a true pharmacologically retinoid receptor
subtype specific analog. These compounds will greatly
facilitate the elucidation of the biology associated with
RARR and the identification of RARR agonist responsive
diseases. Also, these are the first examples of a new
generation of receptor subtype specific retinoids that are
likely to significantly advance the role of retinoid drugs
in clinical medicine.
Compounds 1 and 2 were synthesized as outlined in
Scheme 1. Bromination of 315 under acidic conditions
gave the bromophenol 4. Compound 4 was protected
as its MOM ether and then subjected to metal halogen
exchange followed by CO2 quench to afford the car-
boxylic acid 6. Bromination of 6 under acidic conditions
gave the bromo-substituted acid 7. Compound 7 was
protected as its MOM ether and subsequently coupled
with either the mono- or difluoro-substituted amino-
benzoate 13 or 16 to give the amide 9 or 10. Compounds
9 and 10 were subjected to the same sequences of base
hydrolysis of ester followed by acid-catalyzed deprotec-
tion of the MOM group to afford the retinoids 1 and 2,
respectively. The aminobenzoate 13 was synthesized
as shown in Scheme 2 where the nitrotoluene 11
(Aldrich) was oxidized with Na2Cr2O7 under acidic
† Department of Chemistry.
‡ Department of Biology.
S0022-2623(96)00353-6 CCC: $12.00 © 1996 American Chemical Society

3036 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16
Communications to the Editor
Ch a r t 1
Sch em e 2a
a
(a) Na2Cr2O7, HOAc, H2SO4, 90 °C; (b) SOCl2, 80 °C/EtOH,
Py, CH2Cl2; (c) H2, Pd/C, ethyl acetate.
Sch em e 3a
a
(a) SOCl2, 80 °C, 30 min/EtOH, triethylamine; (b) NaN3, H2O,
CH3CN, 80 °C, 72 h; (c) H2, Pd/C, ethyl acetate, 6 h.
Ta ble 1. Binding Affinity Kd (nM) of Retinoids to RAR R/â/γ
RAR
entry
R
â
γ
Ch a r t 2
retinoic acid
TTNPB
Am 580
1 (AGN 193835)
2 (AGN 193836)
15 ( 1.7
72 ( 37
36 ( 1.5
4.4 ( 2.3
13 ( 2.5
5 ( 2
18 ( 1
26 ( 20
1361 ( 321
3037 ( 859
3824 ( 2644
>30 000
8.4 ( 1.3 17 374 ( 6347 >30 000
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.16c
Sch em e 1a
baculovirus-expressed RARs and RXRs16 are shown in
Table 1. Retinoic acid, the natural hormone for the
RARs, binds with approximately equal affinity to all
three RAR subtypes. In contrast, Am 580 binds with
approximately 30-fold selectivity to RARR relative to
RARâ/γ. However, Am 580 still has measurable affinity
to RARâ and RARγ with Kd values around 1 µM. Thus,
pharmacological doses of Am 580 will activate RARâ and
RARγ as well as RARR, and the biology of Am 580 will
be accordingly complicated. Compound 1 is much more
selective for RARR having around 700-fold higher affin-
ity for RARR than for RARâ with no measurable binding
affinity for RARγ. Interestingly, compound 1 binds to
RARR with slightly higher affinity than the natural
hormone retinoic acid. Compound 2 shows true phar-
macological specificity in that it binds with greater than
2000-fold higher affinity to RARR than to RARâ and has
no measurable binding affinity to RARγ. Am 580, 1, and
2 showed no measurable binding affinity to any of the
RXR subtypes (data not shown).
The gene transcriptional properties of these com-
pounds were determined in transactivation assays using
an estrogen receptor-RAR chimera (ER-RAR). These
chimeric receptor assays provide a very clean readout
of only the transfected receptor subtype by circumvent-
ing contribution by the endogenous RARs.17 Am 580
transactivates effectively through all three RARs with
some selectivity for RARR (Figure 1A). However, Am
580 has essentially full agonist activity at all three
RARs at 1 µM concentration. In contrast, compound 1
strongly transactivates only through RARR, having
activity at RARâ only at 1 µM and being inactive at
RARγ (Figure 1B). Compound 2 is completely inactive
at RARâ/γ and transactivates exclusively through RARR,
albeit with somewhat reduced efficacy (Figure 1C).
It is likely that the H-bond donor properties of the
internal amide linkage play an important role in the
a
(a) Br2, HOAc; (b) chloromethyl methyl ether, diisopropyl-
ethylamine, Bu4NBr, CH2Cl2, rt; (c) (i) t-BuLi, THF, -78 °C, (ii)
CO2, THF, -78 °C, (iii) 10% HCl, -78 °C; (d) Br2, HOAc; (e)
chloromethyl methyl ether, diisopropylethylamine, Bu4NBr, CH2Cl2,
rt; (f) NaOH, EtOH, rt, 2 h; (g) 13 or 16, EDC, DMAP, CH2Cl2; (h)
NaOH, EtOH; (i) HCl (conctd), MeOH (20% for 1 from 9, 54% for
2 from 10).
conditions and the resulting carboxylic acid then esteri-
fied to afford 12. Palladium-catalyzed hydrogenation
of 12 gave 13 in quantitative yield. The synthesis of
the corresponding difluoroaminobenzoate 16 is outlined
in Scheme 3. The benzoic acid 14 (Aldrich) was first
esterified, and the 4-fluoro group of 15 was replaced
with azide by refluxing with aqueous NaN3 in aceto-
nitrile. Palladium-catalyzed hydrogenation of the azide
gave the desired aminobenzoate 16.
The binding affinities of the retinoids measured using

Communications to the Editor
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16 3037
RARR selectivity of these analogs. The introduction of
the polar, hydrophilic amide linkage into the tether
region of the arotinoid skeleton of TTNPB as in Am 580
would be expected to lead to reduced affinity for retinoid
receptors. This is indeed observed for RARâ and RARγ
which bind Am 580 with much lower affinity than they
bind TTNPB. However, in spite of its reduced lipophi-
licity, Am 580 binds equally well to RARR as does
TTNPB, suggesting that the internal amide of Am 580
selectively stabilizes its interaction with RARR. One
possible explanation for these data is that a H-bond is
formed between the amide proton of Am 580 and a
residue in the binding pocket of RARR, while such
interactions are not possible for RARâ and RARγ. We
systematically explored substitutions on the aromatic
rings that could potentially affect the H-bond donor
properties of the internal amide linkage and found that
the substitutions present in 1 and 2 were optimal in
achieving RARR selectivity.
In summary, we have identified retinoid analogs that
bind with high affinity and great selectivity to RARR.
Compound 2 is the first example of a truly retinoid
receptor subtype specific (>1000-fold selectivity in bind-
ing) analog. These analogs will be invaluable tools in
determining the physiological role of RARR and in
identifying therapeutic applications that are unique to
RARR agonists. In addition, such subtype selective
agonists are very likely to be associated with fewer toxic
effects and hence would be useful drugs of improved
therapeutic index in the treatment of a subset of retinoid
responsive diseases.
Ack n ow led gm en t. We thank D. Mais, E. Berger,
and K. Flatten of Ligand Pharmaceuticals for the ligand
binding assays.
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures and analytical data for compounds 1, 2, 4-10, 12, 13,
15, and 16 along with descriptions of binding and trans-
activation assays (4 pages). Ordering information is given on
any current masthead page.
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F igu r e 1. Dose-response curves for Am 580, 1, and 2. CV-1
cells were transiently transfected with chimeric ER-RAR
receptor expression plasmids and an ER responsive luciferase
reporter plasmid as indicated in methods. ER-RARR-, ER-
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