Conformationally defined 6-s-trans-retinoic acid analogs. 3. Structure- activity relationships for nuclear receptor binding, transcriptional activity, and cancer chemopreventive activity

Article abstract of DOI:10.1021/jm9603126

We recently demonstrated that conformationally defined 6-s-trans- retinoic acid (RA) analogs were effective in the prevention of skin papillomas (Vaezi et al. J. Med. Chem. 1994, 37, 44994507) and selective agonists for nuclear receptor binding and activation (Alam et al. J. Med. Chem. 1995, 38, 2302-2310). In order to probe important structure-activity relationships, we evaluated a homologous series of four 6-s-trans-retinoids that are 8-(2'-cyclohexen-1'-ylidene)3,7-dimethyl-2,4,6-octatrienoic acids with different substituents at 2' (R₂) and 3' (R₁) positions on the cyclohexene ring. UAB1 (R₁ = R₂ = H), UAB4 (R₁ = R₂ = Me), UAB7 (R₁ = Me, R₂ = iPr), and UAB8 (R₁ = Et, R₂ = iPr) contain alkyl R groups that mimic, to different extents, portions of the trimethylcyclohexenyl ring of RA. Both 9Z- and all-E-isomers of these retinoids were evaluated in binding assays for cellular retinoic acid-binding proteins (CRABP-I and CRABP-II), a nuclear retinoic acid receptor (RARα), and a nuclear retinoid X receptor (RXRα). The all-E-isomers of UAB retinoids bound tightly to CRABPs and RARα, the binding affinity of the all-E-isomer increased systematically from UAB1 to UAB8, and binding for the latter was comparable to that of all-E-RA. In contrast to RA, the (9Z)-UAB retinoids were at least 200-fold less active than the all-E-isomers in binding to RARα. The (9Z)-UAB isomers exhibited increasingly stronger binding to RXRα, and (9Z)-UAB8 was nearly as effective as (9Z)-RA in binding affinity. The retinoids were also evaluated in gene expression assays mediated by RARα and RXRα homodimers or RARα/RXRα heterodimers. Consistent with the binding affinities, the (all-E)-UAB retinoids activated gene transcription mediated by RARer homodimers or RARα/RXRα heterodimers, while the (9Z)-UAB isomers activated only the RXRα homodimer-mediated transcription. The all-E- and 9Z-isomers of the UAB retinoids were further evaluated for their capacity to prevent the induction of mouse skin papillomas. When compared to RA, only the (all-E)-UAB retinoids containing bulky R₁ and R₂ groups were effective in this chemoprevention assay. (9Z)-RA displayed equal capacity as RA to prevent papillomas, while the 9Z-isomers of the UAB retinoids were much less effective. Taken together, these studies demonstrate that the cyclohexenyl ring substituents of 6-s- trans-UAB retinoids are important for their biological activities and that the chemopreventive effect of the all-E-isomers of these retinoids correlates well with their capacity to bind to RARs and activate RAR/RXR-mediated transcription.

Full text of DOI:10.1021/jm9603126

J . Med. Chem. 1996, 39, 3625-3635
3625
Con for m a tion a lly Defin ed 6-s-tr a n s-Retin oic Acid An a logs. 3.
Str u ctu r e-Activity Rela tion sh ip s for Nu clea r Recep tor Bin d in g,
Tr a n scr ip tion a l Activity, a n d Ca n cer Ch em op r even tive Activity
Donald D. Muccio,*,† Wayne J . Brouillette,*,† Muzaffar Alam,† Michael F. Vaezi,† Brahma P. Sani,‡
Pratap Venepally,‡ Lakshmi Reddy,‡ Ellen Li,§ Andrew W. Norris,§ Linda Simpson-Herren,‡ and Donald L. Hill‡
Department of Chemistry, University of Alabama at Birmingham, Birmingham, Alabama 35294, Southern Research Institute,
2000 Ninth Avenue South, Birmingham, Alabama 35205, and Departments of Medicine and of Biochemistry and Molecular
Biophysics, Washington University, St. Louis, Missouri 63110
Received April 26, 1996X
We recently demonstrated that conformationally defined 6-s-trans-retinoic acid (RA) analogs
were effective in the prevention of skin papillomas (Vaezi et al. J . Med. Chem. 1994, 37, 4499-
4507) and selective agonists for nuclear receptor binding and activation (Alam et al. J . Med.
Chem. 1995, 38, 2302-2310). In order to probe important structure-activity relationships,
we evaluated a homologous series of four 6-s-trans-retinoids that are 8-(2′-cyclohexen-1′-ylidene)-
3,7-dimethyl-2,4,6-octatrienoic acids with different substituents at 2′ (R2) and 3′ (R1) positions
on the cyclohexene ring. UAB1 (R1 ) R2 ) H), UAB4 (R1 ) R2 ) Me), UAB7 (R1 ) Me, R2 )
iPr), and UAB8 (R1 ) Et, R2 ) iPr) contain alkyl R groups that mimic, to different extents,
portions of the trimethylcyclohexenyl ring of RA. Both 9Z- and all-E-isomers of these retinoids
were evaluated in binding assays for cellular retinoic acid-binding proteins (CRABP-I and
CRABP-II), a nuclear retinoic acid receptor (RARR), and a nuclear retinoid X receptor (RXRR).
The all-E-isomers of UAB retinoids bound tightly to CRABPs and RARR, the binding affinity
of the all-E-isomer increased systematically from UAB1 to UAB8, and binding for the latter
was comparable to that of all-E-RA. In contrast to RA, the (9Z)-UAB retinoids were at least
200-fold less active than the all-E-isomers in binding to RARR. The (9Z)-UAB isomers exhibited
increasingly stronger binding to RXRR, and (9Z)-UAB8 was nearly as effective as (9Z)-RA in
binding affinity. The retinoids were also evaluated in gene expression assays mediated by
RARR and RXRR homodimers or RARR/RXRR heterodimers. Consistent with the binding
affinities, the (all-E)-UAB retinoids activated gene transciption mediated by RARR homodimers
or RARR/RXRR heterodimers, while the (9Z)-UAB isomers activated only the RXRR homodimer-
mediated transcription. The all-E- and 9Z-isomers of the UAB retinoids were further evaluated
for their capacity to prevent the induction of mouse skin papillomas. When compared to RA,
only the (all-E)-UAB retinoids containing bulky R1 and R2 groups were effective in this
chemoprevention assay. (9Z)-RA displayed equal capacity as RA to prevent papillomas, while
the 9Z-isomers of the UAB retinoids were much less effective. Taken together, these studies
demonstrate that the cyclohexenyl ring substituents of 6-s-trans-UAB retinoids are important
for their biological activities and that the chemopreventive effect of the all-E-isomers of these
retinoids correlates well with their capacity to bind to RARs and activate RAR/RXR-mediated
transcription.
In tr od u ction
Since RA has a role in controlling gene expression,
its use as a cancer chemopreventive agent has been
investigated using animal models.10 Even though stud-
ies have shown that RA may be effective in cancer
prevention, its clinical use has been limited due to
toxicity11 and teratogenicity.12 It has been suggested
that both the therapeutic and toxicologic effects of RA
may be mediated by RARs, RXRs, and binding pro-
teins.13 In order to improve the therapeutic ratio of RA,
several groups have designed new retinoid analogs that
selectively activate individual nuclear receptors, either
RAR-selective14 or RXR-selective agonists.15 Hopefully
these receptor-selective retinoids will provide the ben-
eficial effects of RA with reduced toxicity.10b
We designed16 a series of 6-s-trans-retinoids based on
the observation that certain proteins17 selectively bind
this conformation of vitamin A derivatives.18 Previously
we reported19 the synthesis of one 6-s-trans-retinoid
(UAB7) and showed that it was effective in inhibiting
the chemical induction of mouse skin papillomas and
that it also had reduced toxicity. Subsequently, we
generated20 a more elaborate example (UAB8) and
Retinoic acid (RA) is essential for diverse biological
processes, including the control of cellular differentia-
tion and development.1,2 The pleiotropic effects of RA
appear to be related to its interactions with a large
family of nuclear receptors. Two classes of nuclear
retinoic acid receptors have been identified (RARs and
RXRs), and each class contains several different sub-
types. Collectively these nuclear receptors, which are
related to the steroid/thyroid hormone superfamily of
receptors,3 act as ligand-dependent transcription factors
for different genes.4 Two RA configurational isomers
((all-E)-RA and (9Z)-RA) are important in its natural
function. Both isomers of RA bind to RARs and activate
transcription mediated by RAR homodimers or RAR/
RXR heterodimers,5,6 but (9Z)-RA is the only known
natural ligand for RXRs.7-9
* Authors to whom correspondence should be addressed.
† University of Alabama at Birmingham.
‡ Southern Research Institute.
§ Washington University.
X Abstract published in Advance ACS Abstracts, August 15, 1996.
S0022-2623(96)00312-3 CCC: $12.00 © 1996 American Chemical Society

3626 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 19
Muccio et al.
Sch em e 1
Sch em e 2
demonstrated selectivity in RAR and RXR nuclear
receptor binding and activation. Here we report a
thorough comparison of the all-E- and 9Z-isomers for
four synthetic homologs of these retinoids (UAB1,
UAB4, UAB7, and UAB8). These studies demonstrate
the importance of large R1 and R2 substituents in the
UAB retinoids for enhanced biological activity and
suggest a correlation between nuclear receptor binding/
activation and the cancer chemopreventive effects in
skin.
and UAB8,19,20 except that a different starting enone
(1 in Scheme 1) was used. Some key points of the
synthesis are reported here. Unlike previous re-
ports,19,21 a δ-lactone intermediate was not detected but
was assumed to be an intermediate in the production
of (9Z)-2. (9Z)-5 and (9Z,13Z)-5 were preparatively
separated by HPLC on silica gel (0.5% Et2O, 0.1% THF
in hexane) using methods similar to those we previously
described.19,20,22 Individual isomers of ester 5 were
hydrolyzed to the corresponding acids in KOH, without
E/Z-isomerization.23 In order to produce (all-E)-UAB4,
intermediate aldehyde (9Z)-4 was isomerized using I2
to yield a 1:2 ratio of all-E- and 9Z-isomers. As for (9Z)-
4, a Horner-Emmons condensation using (all-E)-4
produced a 2:1 mixture of all-E- and 13Z-ester 5. These
two isomers were also separated by HPLC on silica gel
(1% Et2O, 0.5% THF in hexane). The isomeric purities
for the final acids (>97%) were determined by NMR and
reverse-phase HPLC.20 Experimental yields and se-
lected data for the intermediates and products in
Scheme 1 are summarized in Table 1. The chemical
shift assignments (Table 2) for the UAB4 isomers were
made using methods previously described.19,24,25
As shown in Scheme 2, the synthesis of UAB1 was
accomplished by a modification of the approach in
Scheme 1. Since Horner-Emmons condensations on
2-cyclohexenone gave only 1,4-addition products, we
first generated ester 7, a dihydro precursor to UAB1.
Ch em istr y
Scheme 1 summarizes the methods employed to
prepare UAB4. This approach was identical with that
which we previously reported for the syntheses of UAB7

Conformationally Defined 6-s-trans-RA Analogs
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 19 3627
Ta ble 1. Selected Data for New Compounds Produced in
Schemes 1 and 2
modimers, or RXRR/RARR heterodimers using a CAT
reporter gene containing the TREpal.30 The UAB
retinoids were evaluated in a skin antipapilloma assay
which measured the capacity of retinoids to prevent
chemically induced papillomas in mice.31
UV/visb
λmax
IRc
CdO CdC (m/z)
yield
(%) Rf
GC/MS
a
compd
(9Z)-2
(9Z)-3
(9Z)-4
e
79
93
87
51
0.28 320 11 500 1672 1580
0.25 266 13 500
0.46 298 7500
206
192
190
190
300
300
300
300
272
272
272
272
206
164
162
272
272
272
244
244
1630
1672 1607
Resu lts
(all-E)-4
0.38 324 16 000 1660 1577
UAB retinoids employ a dimethylene bridge between
C18 and C7 of the polyene chain to maintain a 6-s-trans-
conformation. When the polyene chain of RA is overlaid
with that of the UAB retinoids, the two methylene
groups in the bridge do not occupy space which is in
common with that of RA.19 R1 and R2 alkyl substituents
are used to mimic the steric volume of the carbon atoms
within the trimethylcyclohexenyl ring of RA. UAB1 (R1
) R2 ) H) thus represents a minimal structure without
any R groups to mimic the alkyl portion of the RA ring.
The homologous series of UAB4 (R1 ) R2 ) Me), UAB7
(R1 ) Me, R2 ) iPr), and UAB8 (R1 ) Et, R2 ) iPr) adds
hydrophobic groups to the terminal end of the polyene
chain which may mimic the RA ring.
(9Z)-5f
81e 0.69 329 23 000 1706 1602
81e 0.71 331 22 000 1708 1605
75e 0.67 362 27 500 1706 1598
75e 0.73 369 24 000 1709 1604
(9Z,13Z)-5f
(all-E)-5f
(13Z)-5f
(9Z)-UAB4
98
0.34 341 19 000 1672 1594
0.29 338 17 500 1684 1601
0.20 378 26 000 1672 1598
0.24 378 21 000 1673 1596
0.80 298 17 000 1712 1633
(9Z,13Z)-UAB4 95
(all-E)-UAB4
(13Z)-UAB4
(all-E)-8
98
96
63
91
78
(all-E)-9
0.21 272 NDd
0.39 326 ND
1600
1650 1580
(all-E)-10
(all-E)-11g
(7Z)-11g
80e 0.68 368 39 000 1702 1605
80e 0.68 366 36 000 1712 1605
80e 0.68 370 29 000 1709 1607
(13Z)-11g
(all-E)-UAB1
(7Z)-UAB1
99
94
0.25 370 32 000 1682 1595
0.25 366 25 000 1675 1593
a
Com p u ta tion a l Stu d ies. Molecular mechanics cal-
culations were used to evaluate the structural similarity
of the UAB retinoids to RA. Using the MM3(94) force
field, the low-energy structures of (all-E)- or (9Z)-UAB
retinoids containing an 8-s-trans-conformation were
computed (Table 3). The ψ5,6,7,8 torsional angles were
nearly planar for either (all-E)- or (9Z)-UAB1. As the
size of R1 and R2 increased, this torsional angle became
increasingly less planar (about 35° out of plane for
UAB8). The ψ7,8,9,10 torsional angles were nonplanar
by about 40°, due to steric interactions between the C2′
methylene and the C19 methyl group. For the 8-s-cis-
conformer, the ψ5,6,7,8 torsional angles were nearly
identical with those of the 8-s-trans-conformer and the
ψ7,8,9,10 angles were nonplanar by about 50°. Further,
the 8-s-cis-conformers were slightly more stable (∆G ≈
1 kcal/mol) than the corresponding 8-s-trans-conformers.
RA adopts primarily a 6-s-cis-conformation in solu-
tion,18 a conformation17f,g found for the vitamin bound
to CRABP-I/CRABP-II/RAR-γ. The C7-C15 portion of
the polyene chain adopts an s-trans-conformation for the
low-energy structure.18 Structures of (all-E)- and (9Z)-
RA were calculated using torsional angles from the
X-ray crystal structures32 of (all-E)-RA and (9Z)-retinal,
both of which crystallized in a 6-s-cis-conformation. The
Values obtained on silica gel using either diethyl ether or ethyl
acetate in hexane as an eluent: 10% Et2O (4 and 5), 20% Et2O (2,
b
UAB4, 11, and UAB1), 30% Et2O (3), 10% EtOAc (8-10). The
wavelength maximum (nm) and extinction coefficients (M-1 cm-1
)
were obtained in cyclohexane (2-5, 8, 11, and UAB1), chloroform
(UAB4), and MeOH (9 and 10) at room temperature. c The IR
stretching frequencies (cm-1) were obtained as thin films on NaCl
plates. The hydroxyl group frequency was observed at 3326 cm-1
for (9Z)-3 and at 3340 cm-1 for (all-E)-9. ND ) not determined.
d
e Yield for the E,Z-product mixture. f The separation of retinoid 5
isomers was performed on a Whatman Partisil 10 M20/50 column
(500 × 22 mm i.d.). Retention times (min): 91 (all-E), 83 ((9Z)-
and (9Z,13Z)-isomer mixture), 80.5 (13Z) using 1% Et2O, 0.5%
THF in hexane with a flow rate of 5 mL/min. Retention times
(min): 181 (9Z); 175 (9Z,13Z) using 0.5% Et2O, 0.1% THF in
hexane with a flow rate of 5 mL/min. g The ratio of (all-E)- to (13Z)-
11 was 6:1 with a trace amount of (7Z)-11. The separation of
retinoid 11 isomers was performed on a Whatman Partisil 10 M20/
50 column (500 × 22 mm i.d.). Retention times (min): 33 (13Z),
53 (7Z), 56 (all-E) using 0.5% Et2O, 0.2% THF in hexane with a
flow rate of 10 mL/min.
This was accomplished using a Horner-Emmons con-
densation between cyclohexanone and diethyl 3-(ethoxy-
carbonyl)-2-methylprop-2-enylphosphonate, which we
previously reported in modest yield during a reactivity
study.21 The modified procedure described here utilized
HMPA as cosolvent, produced much higher yields, and
gave nearly exclusively (9E)-7. The cyclohexenyl double
bond was then introduced via regioselective bromination
with NBS followed by dehydrobromination to give (all-
E)-8; this was carried on as in Scheme 1 to give UAB1.
ψ5,6,7,8 torsional angles of either (all-E)- or (9Z)-RA were
substantially different than those found in the UAB
retinoids; this is due to the methylene bridge used in
the latter compounds to maintain 6-s-trans-geometry.
The ψ7,8,9,10 torsional angles of the RAs were nearly 180°,
which was significantly different from those of the UAB
retinoids (Table 3). Unlike UAB retinoids, the energies
for 8-s-cis-RA conformers were about 1-2 kcal/mol
higher than those for the corresponding 8-s-trans-
conformers. While (all-E)-RA adopted a completely
planar 8-s-cis-orientation, the corresponding UAB com-
pounds had ψ7,8,9,10 torsional angles that were skewed
by about 50°. This is presumably due to steric interac-
tions between the H10 methine and H2′ methylene
protons. Taken together, these studies suggest that
UAB compounds adopt very similar structures to RA
along the polyene chain between C9 and C15, but they
differ in the terminal end of the molecule (C5-C9).
In order to evaluate electrostatic properties, the dipole
moments of RA and UAB retinoids were calculated. The
Biology
The IC50 and Kd′ values were determined for the E/ Z-
isomers of UAB retinoids with CRABPs, RARs, and
RXRs. The IC50 values were evaluated in a chick skin
CRABP radioligand binding assay.26 The Kd′ values
were determined using a fluorometric titration of ligand
and apo-CRABP-I or apo-CRABP-II.27 This approach
was also used to determine Kd′ values to the ligand-
binding (DEF) domain of human RXRR.28 The IC50
values for each E/ Z-isomer of the UAB retinoids were
next determined in an assay for the inhibition of the
binding of (all-E)-RA to RARR.8a Similar studies were
also performed for the inhibition of the binding of (9Z)-
RA to RXRR.29 Each isomer was evaluated for the
efficiency of activating RARR homodimers, RXRR ho-

3628 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 19
Muccio et al.
Ta ble 2. 1H Chemical Shifts (ppm, TMS) of UAB1 and UAB4 Retinoids and Intermediates in CDCl3
compd
(9Z)-2
(9Z)-3
(9Z)-4
(all-E)-4
(9Z)-5
(9Z,13Z)-5
(13Z)-5
(all-E)-5
(9Z)-UAB4
(9Z,13Z)-UAB4
(13Z)-UAB4
(all-E)-UAB4
H-1′
H-2′
H-1
H-4
H-8
H-10
H-11
H-12
H-14
H-18
H-19
H-20
1.65
1.64
1.65
1.65
1.62
1.62
1.64
1.63
1.62
1.62
1.65
1.65
2.35
2.15
2.26
2.53
2.14
2.18
2.53
2.54
2.16
2.18
2.56
2.54
1.81
1.79
1.84
1.82
1.81
1.81
1.81
1.81
1.82
1.81
1.82
1.82
1.84
1.79
1.82
1.84
1.85
1.84
1.81
1.81
1.86
1.85
1.82
1.82
6.50
5.74
5.93
5.88
5.84
5.84
5.89
5.91
5.84
5.84
5.91
5.91
5.72
5.47
5.92
5.93
6.03
6.13
6.20
6.09
6.04
6.15
6.22
6.11
2.13
2.13
2.14
2.17
2.15
2.15
2.13
2.14
2.15
2.13
2.13
2.14
2.06
1.79
2.01
2.28
1.91
1.91
1.99
1.99
1.91
1.92
2.01
2.01
4.00
9.54
10.04
6.57
6.57
6.93
6.93
6.62
6.62
6.97
6.98
6.18
7.67
7.74
6.23
6.21
7.64
7.69
6.26
5.74
5.59
5.62
5.76
5.76
5.62
5.64
5.79
2.27
1.98
2.07
2.35
2.27
2.01
2.10
2.37
H-1′
H-2′
H-5
H-6
H-8
H-10
H-11
H-12
H-14
H-18
H-19
H-20
(all-E)-8
(all-E)-9
1.74
1.70
1.75
1.71
1.77
1.70
1.77
2.59
2.55
2.60
2.64
2.35
2.64
2.36
5.96
5.83
5.95
5.88
5.91
5.89
5.92
6.08
6.05
6.10
6.09
6.67
6.09
6.67
5.71
5.65
5.75
5.79
5.69
5.79
5.70
5.71
5.56
5.97
6.16
6.14
6.17
6.16
2.15
2.14
2.15
2.16
2.19
2.15
2.18
2.28
1.82
2.30
2.04
2.02
2.05
2.03
4.25
10.05
6.92
6.92
6.98
6.98
(all-E)-10
(all-E)-11
(7Z)-11
(all-E)-UAB1
(7Z)-UAB1
6.26
6.26
6.28
6.29
5.78
5.78
5.79
5.78
2.35
2.35
2.36
2.36
Ta ble 3. Torsional Angles and Relative Energies of 8-s-trans/8-s-cis-Conformers of the UAB Retinoids Using Molecular Mechanics
Calculations (MM3(94) Force Field)
8-s-trans
8-s-cis
retinoid
RA
(all-E)-UAB8
(all-E)-UAB7
(all-E)-UAB4
(all-E)-UAB1
(9Z)-RA
(9Z)-UAB8
(9Z)-UAB7
(9Z)-UAB4
(9Z)-UAB1
ψ5,6,7,8
ψ7,8,9,10
energya
ψ5,6,7,8
ψ7,8,9,10
energya
∆G° b
µ (D)c
1.6
-59
-146
-150
-159
-175
-60
-146
-149
-159
-176
-180
-137
-139
-140
-143
176
-130
-129
-135
-138
25.1
34.5
26.8
23.7
18.3
24.9
34.5
26.8
23.8
18.6
-59
-146
-150
-159
-175
-56
-146
-149
-158
-174
-4
52
50
49
48
-46
62
62
62
26.3
33.9
26.3
26.3
17.8
26.5
33.6
26.0
23.3
18.3
1.9
-0.9
-0.9
-1.0
-0.9
1.2
-1.2
-1.1
-1.1
-1.0
1.5, 1.6
1.5, 1.6
1.5, 1.6
1.2, 1.3
1.6, 1.7
1.7, 1.8
1.6, 1.8
1.6, 1.8
1.9, 2.1
64
a
b
The strain energy is calculated in kcal/mol. ∆G° is the difference in free energy (kcal/mol) between 8-s-cis- and 8-s-trans-conformers
(G°trans - G°cis) calculated at 300 K. c Dipole moments are measured in Debye (D); two values appear for 8-s-cis- and 8-s-trans-conformers.
Ta ble 4. Structure-Activity Relationships for UAB Retinoids
dipole moments of UAB4, UAB7, and UAB8 were very
and Binding to Mouse and Chick Skin CRABPs
similar (Table 3) and close to that of RA. The dipole
moment of UAB1 was slightly smaller for the all-E-
chick skin CRABP
Kd (nM)
% inhibtn at 100- IC50
isomer. The Connolly surface area of these structures
was also determined using a 1.4 Å probe. For the (all-
E)-UAB retinoids, the surface area increased system-
atically: 265 (UAB1), 296 (UAB4), 324 (UAB7), and 338
(UAB8) Å2 as compared to 325 Å2 for RA. The 9Z-
isomers had similar values and identical trends.
CRABP Bin d in g Affin ity. UAB retinoids were
evaluated in the CRABP binding assay26 which utilizes
[3H]-(all-E)-RA as the radioligand. The IC50 values of
the UAB retinoids are compared to those of (all-E)- and
(9Z)-RA (Table 4). At 100-fold excess, (all-E)-UAB1
inhibited the binding of [3H]RA by only 50%. (all-E)-
UAB7 and UAB8, with larger R1 and R2 substituents,
had improved binding profiles relative to UAB1 and had
IC50 values similar to (all-E)-RA in this assay. (9Z)-
RA competes weakly for the CRABP binding site,
inhibiting only 25% of the binding of labeled RA at 100-
fold excess.33 Likewise, the (9Z)-UAB retinoids were not
effective binders to these proteins. Interestingly (7Z)-
UAB1 was as effective as (all-E)-UAB1.
retinoid
fold excess liganda (µM) CRABP-I CRABP-II
(all-E)-RA
100
100
100
90
50
25
0.6 0.4 ( 0.3 2 ( 1
0.7 0.3 ( 0.02 1.4 ( 0.2
(all-E)-UAB8
(all-E)-UAB7
(all-E)-UAB4
(all-E)-UAB1
(9Z)-RA
(9Z)-UAB8
(9Z)-UAB7
(9Z)-UAB4
(7Z)-UAB1
0.6 1 ( 0.6
1.3 1.2 ( 1
14 ( 2
13 ( 5
13 ( 4
>200
>10
3 ( 2
>200
>200
>200
>200
3 ( 1
>10
>10
>10
>10
>10
15
43
10
45
>200
>200
>200
10 ( 2
a
The percent inhibition of binding of 100-fold excess of test
retinoids with [3H]-(all-E)-RA (n ) 1). IC50 values were deter-
mined from five doses run in duplicate and have error of less than
20% of their mean.
200 nM). Due to the poor solubility of retinoids in
aqueous solutions,34 accurate Kd′ values can not be
measured for weakly bound ligands. Fluorometric
titrations of the tight binding retinoid isomers were
performed at 10-25 nM protein concentrations to more
accurately determine Kd′ values (Table 4).35 The Kd′
values for the protein-ligand complex with (all-E)-
UAB8 were 0.3 nM (mCRABP-I) and 1.4 nM (mCRABP-
II), which were in excellent agreement with previous
reports on RA complexes27 (Table 4). For CRABP-I, the
Fluorescence titrations27 of the all-E- and 9Z-isomers
were measured with CRABP-I and CRABP-II (mouse)
at micromolar protein concentrations to determine the
stoichiometry of the ligand-protein complex (1:1). The
9Z-isomers were poor binders to these proteins (Kd′ >

Conformationally Defined 6-s-trans-RA Analogs
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 19 3629
Ta ble 5. Structure-Activity Relationships for the Binding of
UAB Retinoid Isomers to Mouse Retinoic Acid Receptors
(mRAR), Mouse Retinoid X Receptors (mRXR), and Human
Retinoid X Receptor DEF Domain (hRXR-(DEF))
RARR
IC50
inhibtna (nM)a inhibtnb (nM)b
RXRR
retinoid
isomer
%
%
IC50 hRXRR-(DEF)
Kd (nM)c
(all-E)-RA
100
98
100
90
55
95
42
37
10
45
6
11
8
55
600
10
0
28
0
>2000
>2000
>2000
>2000
>2000
82
1000
620
>2000
>2000
>200
>200
>200
>200
>200
3 ( 0.5
16 ( 0.6
33 ( 5
120
(all-E)-UAB8
(all-E)-UAB7
(all-E)-UAB4
(all-E)-UAB1
(9Z)-RA
(9Z)-UAB8
(9Z)-UAB7
(9Z)-UAB4
(7Z)-UAB1
5
31
100
50
98
40
25
>1000
>1000
>2000
>2000
>200
a
The percent inhibition was determined by competition of 100-
fold excess of unlabeled retinoid with 5 nM [3H]-(all-E)-RA (n )
1). The IC50 values were determined from nine doses run in
duplicate as reported by Alam et al.20 The standard deviation was
b
less than 10% of the IC50 value. The percent inhibition was
determined by competition of 100-fold excess of unlabeled retinoid
with 20 nM [3H]-(9Z)-RA (n ) 1). The IC50 values were determined
from nine doses run in duplicate as reported in Alam et al.20 c Kd
values were determined by the method of Cheng et al.28
binding affinity decreased 3-fold in going from UAB7
to UAB1; a 10-fold decrease in binding affinity occurred
between UAB8 and UAB7 for CRABP-II. The binding
affinity of (7Z)-UAB1 to either protein was much better
than that of the (9Z)-UAB compounds and comparable
to that of (all-E)-UAB1.
Nu clea r Recep tor Bin d in g Affin ity. Each isomer
was evaluated for its capacity to inhibit the binding of
[3H]-(all-E)- and [3H]-(9Z)-RA to the RARR and RXRR
subtypes. To survey the inhibition process, RARR was
first exposed to 1000 nM UAB retinoids and 5 nM [3H]-
(all-E)-RA. This was compared to a positive control
using 1000 nM unlabeled (all-E)-RA and a control using
no retinoid. (all-E)-UAB1 displaced the radioactive
ligand only 55% as well as (all-E)-RA, and the IC50 was
100-fold greater than that of (all-E)-RA. The percent
inhibition of binding increased and the IC50 values
decreased systematically as alkyl substituents were
added to this minimal structure (Table 5). The binding
affinities of (all-E)-UAB7 and (all-E)-UAB8 were in the
same range as that of (all-E)-RA. Therefore, as ob-
served for binding to CRABPs (Table 4), large alkyl
groups at R1 and R2 are important for the efficient
binding of UAB retinoids to RARR. Unlike (9Z)-RA, the
(9Z)-UAB retinoids bound weakly to RARR (IC50 > 1000
nM). (9Z)-UAB4 was the weakest binder to RARR; its
IC50 value was over 100-fold higher than that for (9Z)-
RA. (7Z)-UAB1 also displayed poor binding affinity to
RARR.
F igu r e 1. (A) Evaluation of RARR receptor-mediated tran-
scriptional activation by the all-E-isomers of RA, UAB1, UAB4,
UAB7, and UAB8 at 10-6-10-9 M concentrations. (B) Evalu-
ation of RARR receptor-mediated transcriptional activation by
(9Z)-RA, (7Z)-UAB1, (9Z)-UAB4, (9Z)-UAB7, and (9Z)-UAB8
at 10-6-10-9 M concentrations. The measurements were made
in triplicate at each dose. Error was estimated at 10%.
values was observed for the (9Z)-UAB retinoids; an
increased binding affinity occurred for retinoids with
large alkyl groups at R1 and R2 (Table 5). The dissocia-
tion constant for (9Z)-UAB8 was only 5-fold greater than
that observed for (9Z)-RA.
Nu clea r Recep tor Tr a n scr ip tion a l Activity. To
determine if the UAB retinoids exhibited functional
activity within the cell, their ability to induce RAR- and
RXR-mediated transcriptional activity was examined
and compared to those of (all-E)- and (9Z)-RA.36 (all-
E)-UAB8 was equivalent to or better than (all-E)-RA
in inducing the receptor-activated transcription of the
reporter gene at 10-6 M (Figure 1A). For the all-E-
isomers, activation of transcriptional activity decreased
in going from UAB7 to UAB1, consistent with the IC50
values found for the binding to the RARs (Table 5).
Activation of RARs by (9Z)-RA was slightly better than
by (all-E)-RA (Figure 1B). This is consistent with
previous studies6 reporting that (9Z)-RA had equal or
better activity than (all-E)-RA in inducing transcrip-
tional activation in GAL4-RAR chimeric constructs. In
contrast, (9Z)-UAB4-UAB8 at 10-7 M and lower were
dramatically less effective than (9Z)- or (all-E)-RA.
Even at micromolar doses, these retinoids were much
The UAB retinoids were evaluated for their capacity
to inhibit the binding of [3H]-(9Z)-RA to RXRR.
A
preliminary screen was performed using 2000 nM
retinoid and 20 nM [3H]-(9Z)-RA, which was efficiently
inhibited by unlabeled (9Z)-RA at 100-fold excess. None
of the all-E-isomers had appreciable affinity for RXRR,
consistent with the results observed for (all-E)-RA.
Among the 9Z-isomers, (9Z)-UAB4 was the poorest
binder. (9Z)-UAB7 and (9Z)-UAB8 had significant
activity in this assay but less than that of (9Z)-RA.
These retinoids were also evaluated in a binding assay
for the apo-hRXRR-(DEF) domain using fluorometric
titrations of the ligand. A systematic trend in the Kd′

3630 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 19
Muccio et al.
F igu r e 2. (A) Evaluation of RARR/RXRR receptor-mediated
transcriptional activation by the all-E-isomers of RA, UAB1,
UAB4, UAB7, and UAB8 at 10-6-10-9 M concentrations. (B)
Evaluation of RARR/RXRR receptor-mediated transcriptional
activation by (9Z)-RA, (7Z)-UAB1, (9Z)-UAB4, (9Z)-UAB7, and
(9Z)-UAB8 at 10-6-10-9 M concentrations. The measurements
were made in triplicate at each dose.
F igu r e 3. (A) Evaluation of RXRR receptor-mediated tran-
scriptional activation by the all-E-isomers of RA, UAB1, UAB4,
UAB7, and UAB8 at 10-6-10-9 M concentrations. (B) Evalu-
ation of RXRR receptor-mediated transcriptional activation by
(9Z)-RA, (7Z)-UAB1, (9Z)-UAB4, (9Z)-UAB7, and (9Z)-UAB8
at 10-6-10-9 M concentrations. The measurements were made
in triplicate at each dose.
at concentrations as low as 10-8 M. The trends in the
transactivational data are consistent with the larger Kd′
values and IC50 values, relative to (9Z)-RA, reported for
(9Z)-UAB retinoids (Table 5). Taken together, these
results demonstrate that, unlike (9Z)-RA, (9Z)-UAB
retinoids, especially UAB7 and UAB8, are RXR-selective
ligands.
Mou se Sk in An tip a p illom a Assa y. The UAB re-
tinoids were next evaluated for their activity in prevent-
ing the chemical induction of papillomas on mouse
skin.31 (all-E)-RA was very effective in this chemopre-
ventive assay (Table 6). At a 45.9 nmol dose, it
prevented 95% of tumor formation. The ED50 value
obtained in the present study was 3 nmol, which is
consistent with previously reported values.37 (all-E)-
UAB7 and UAB8 were as effective as RA in this assay
(Table 6). Other (all-E)-UAB retinoids were much less
effective in this assay. (all-E)-UAB4 prevented only
32% of tumor formation (45.9 nmol dose); the ED50 value
was estimated at greater than 100 nmol. In comparison
to RA, (all-E)-UAB1 was only marginally effective at the
45.9 nmol dose.
less effective than RA in inducing the RARR-mediated
response.
The activity of UAB retinoids on the transcription
mediated by RARR/RXRR heterodimers was evaluated.
Both (all-E)-UAB8 and (all-E)-UAB7 were nearly as
efficient as (all-E)-RA in activating transcription medi-
ated by RARR/RXRR heterodimers (Figure 2A). As
observed for RARR activation, the other (all-E)-UAB
retinoids displayed decreased activity. Further, induc-
tion of the reporter gene by (9Z)-UAB8 was apparent
only at 10-6 M, unlike (9Z)-RA (Figure 2B). Thus, the
(9Z)-UAB retinoids are extremely poor binders and
activators of both RARR homodimer and RARR/RXRR
heterodimers. When the effect on RXRR homodimer-
mediated transcriptional activity was studied, (all-E)-
RA did not efficiently activate the reporter gene (Figure
3A), nor did any of the (all-E)-UAB retinoids. In
contrast, (9Z)-RA efficiently activated this reporter gene
(Figure 3B), which is consistent with previous observa-
tions.9 In this assay, the (9Z)-UAB retinoids were also
highly active in inducing transcription at 10-6 M.
However, relative to (9Z)-RA, transcriptional activation
by (9Z)-UAB retinoids was less efficient at doses less
than 10-6 M. Only (9Z)-RA caused significant activation
This assay was also used to evaluate (9Z)-RA. As
suggested from RAR and RXR binding and transcrip-

Conformationally Defined 6-s-trans-RA Analogs
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 19 3631
protein-ligand complex (nanomolar Kd′ values).
Ta ble 6. Structure-Activity Comparisons for the Activities of
UAB Retinoids in the Prevention of Mouse Skin Papillomas
A
positive electrostatic potential near the carboxylate
binding site is thought, at least for RARs, to be involved
in the proper docking of RA. (all-E)-RA adopts a
surprisingly similar conformation in each site. The
polyene chain of RA (C6-C15) is flat, and the conforma-
tion is in an extended s-trans-conformation. The trim-
ethylcyclohexenyl ring adopts a distorted 6-s-cis-geom-
etry relative to the chain. For CRABP-I/II the C5-C6-
C7-C8 torsional angle of RA is -33°. (This torsional
angle was not reported by Moras and co-workers,17g but
RA was depicted in a 6-s-cis-geometry.) This is slightly
more planar than the torsional angle reported in the
structures of (all-E)-RA crystallized in a triclinic crystal32a
and for other related 6-s-cis-retinoids.32c
% papilloma reductiona
at 45.9 nmol
ED50 (papilloma)b
(nmol)
retinoid
(all-E)-RA
(all-E)-UAB8
(all-E)-UAB7
(all-E)-UAB4
(all-E)-UAB1
(9Z)-RA
(9Z)-UAB8
(9Z)-UAB7
(9Z)-UAB4
(13Z)-RA
95
99
95
32
22
95
43
30
50
3.0
2.5
5.0
490
>1000
2.1
>100
>100
>50
64c
(13Z)-UAB8
81
4.7
a
Percentage of papilloma reduction from the application of 45.9
nmol dose of test retinoid 1 h prior to TPA application (n ) 1).
The percent papilloma reduction was calculated as (1 - tumor
suppression) × 100, where tumor suppression is defined as (mean
number of tumors in retinoid-treated animals)/(mean number of
Through the incorporation of a dimethylene bridge,
the UAB retinoids were designed to maintain a 6-s-
trans-conformation between terminal double bonds of
the polyene chain. Even with this structural constraint,
UAB7 and UAB8 are very active in several biological
assays (comparable to RA). Since RA adopts a 6-s-cis-
geometry in these binding sites, the high affinity of the
UAB retinoids is unexpected in the protein binding
assays. In order to explore possible explanations for the
high activity of UAB7 and UAB8 in these assays, their
low-energy structures were calculated from MM3(94)
(Table 3) and overlaid with that of 6-s-cis-RA (Figure
4). When 8-s-trans-UAB8 was overlaid with RA, the
ethyl group at R1 in UAB8 occupies similar space as the
C3 and C4 of the RA ring and the isopropyl group at R2
is near C1 and the gem-dimethyl groups of the RA ring.
The correspondence of these groups is due to the
distorted nature of the polyene chain at the C7-C8-
C9-C10 bond (-140° torsional angle) in the UAB
retinoids and the nonplanar orientation about the C5-
C6-C7-C8 bond (-59° torsional angle) in 6-s-cis-RA.
When the structure of 8-s-cis-UAB8 is overlaid with that
of RA, there is little agreement between the positions
of R1 and R2 and the RA ring.
b
tumors in control). ED50 values were determined by a probit
analysis of a dose-response curve. Estimated standard error is
20% of the mean. c Value determined by Dawson et al.37
tional activational studies, (9Z)-RA also prevented pap-
illoma formation as well as RA (Table 6). In constrast,
(9Z)-UAB retinoids were uniformily poor in papilloma
reduction; they prevented only 30-50% of tumor forma-
tion at the 45.9 nmol dose. Unlike (9Z)-RA, (all-E)-RA,
or (all-E)-UAB retinoids, the (9Z)-UAB retinoids did not
respond in a dose-dependent manner; their ED50 values
are estimated to be 10-fold greater than that of (9Z)-
RA (Table 6). Considering the trends noted above for
the (all-E)-UAB retinoids and RA, the low activities of
the (9Z)-UAB compounds correlate with their poor
capacity to activate RARs and not with their RXR-
selective actions.
Interestingly, (13Z)-UAB8 exhibited very high activity
in the antipapilloma assay, comparable to that for RAs
and (all-E)-UAB8 (Table 6). Previously it was re-
ported37 that (13Z)-RA is about 12-fold less active than
RA in this assay. The surprisingly high activity of
(13Z)-UAB8 in the antipapilloma assay correlates well
with its capacity to bind to and activate RARs, as we
previously demonstrated.20
The structure of (9Z)-RA bound to RXRs has not been
determined. Using ligand-based design on conforma-
tionally rigid retinoids and resulting structure-activity
studies, Dawson et al.15d suggested that RXR-selective
ligands need to maintain a 10 Å distance between the
C15 carboxylate and C1 and C4 of the RA ring. Longer
and more planar retinoids tended to also bind to RARs,
and shorter ones were not very active as RXR agonists.
They also identified, among other effects, that retinoids
like SR11217 with nonplanar structures (corresponding
to C8-C11 of the polyene chain of RA) were potent and
selective RXR agonists. We determined these distances
in (9Z)-UAB7 and (9Z)-UAB8, two retinoids which also
display RXR selectivity (Table 4). In the 8-s-trans-UAB
retinoids, the C1-C15 and C4-C15 distances were 9.7
and 10.8 Å, respectively. For the 8-s-cis-UAB retinoids,
these distances were slightly shorter (9.6 and 10.3 Å).
Due to steric interactions, both the 8-s-cis- and 8-s-trans-
conformations are expected to adopt twisted low-energy
conformations about the C8-C9 bond (Table 3). The
structural properties of the UAB retinoids with bulky
R1 and R2 groups are consistent with known properties
of other RXR-selective ligands.
Discu ssion
We report here a strong correlation between the
biological activities of the UAB retinoids and the
increased size of the R1 and R2 substituents in their
structures. These assays included CRABP-I, CRABP-
II, RARR, and RXRR. Recently, J ones and co-workers17f
determined the X-ray crystal structures of (all-E)-RA
bound to both CRABP-I and CRABP-II, and Moras and
co-workers17g reported the structure of this hormone
bound to the RARγ ligand binding domain. Despite
differences in sequence homology and folds, the RA
binding sites in CRABPs and RARs are primarily
composed of hydrophobic amino acids. Positively charged
amino acids (K and R) occupy one end of the RA binding
pocket, and these residues form salt bridges and hy-
drogen bonds with the RA carboxylate group. The RA
ring is in a well-defined hydrophobic binding site at the
other end of the pocket. The RA binding pocket for
RXRR and the anticipated RA binding sites in other
RARs are expected to have similar features due to the
high sequence homology of these proteins.17g Together
the hydrophobic and ionic forces stabilize RA in a tight
Since (9Z)-RA also binds tightly to RARs, interest has
centered on how this configuration of RA interacts with
the receptors. Moras and co-workers17g were not able

3632 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 19
Muccio et al.
F igu r e 4. Stereoview (relaxed) for (all-E)-RA (top) in a 6-s-cis-conformation with (all-E)-UAB8 (bottom) in an 8-s-trans-
conformation. The structures were energy minimized using MM3(94), and the C9-C15 atoms were fit onto one another. See text
and Table 3 for details.
to determine the X-ray structure of (9Z)-RA bound to
RARγ. However, by allowing similar ionic interactions
of the carboxylate group of (9Z)-RA and minimizing
steric interactions, they suggested that (9Z)- and (all-
E)-RA occupy similar binding sites in RARs. On the
basis of these criteria, the rings of (9Z)- and (all-E)-RA
were suggested to occupy similar positions, and the
single bonds for (9Z)-RA were shown in an extended
s-trans-conformation like in (all-E)-RA. If (9Z)-RA, like
(all-E)-RA, adopts a planar s-trans-conformation in this
region, then retinoids like (9Z)-UAB8, which are sig-
nificantly skewed about the C8-C9 bond (Table 3), may
introduce steric interactions with the receptor not
present in (9Z)-RA. Indeed, Moras and co-workers17g
suggested a close contact between the C19 methyl group
in (9Z)-RA and the H5 helix in RARγ. These steric
interactions may be more significant in the (9Z)-UAB
retinoids resulting in their poor binding affinity relative
to (9Z)-RA and their RXR-selective characteristics.
The activation of RARs and RXRs by retinoids is a
powerful tool for evaluating their capacity to act as novel
pharmacological agents. However, to evaluate their
potential in cancer chemoprevention, the mouse skin
antipapilloma model is often used.37 Beard et al.15c

Conformationally Defined 6-s-trans-RA Analogs
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 19 3633
(E)-E t h yl 3-Met h yl-4-cycloh exylid en eb u t -2-en oa t e
recently reported that RAR-selective retinoids, but not
RXR-selective retinoids, are active as antiproliferative
agents in skin. Chandranatna et al.14e showed that
RARâ- and RARγ-selective retinoids are excellent in-
hibitors of ODC activity in skin, an assay which cor-
relates well with the skin antipapilloma assay.31 We
demonstrate here that (9Z)-RA is as effective as RA for
the prevention of skin papillomas, and we report that
(all-E)-UAB7 and UAB8 are as active as (all-E)- and
(9Z)-RA in this assay. We also show that RXR-selective
retinoids, like (9Z)-UAB8, are much less effective in skin
cancer prevention. Zhang et al.38 demonstrated that
this effect is observed in other cancer models. (all-E)-
UAB8 is as effective as RA and (9Z)-RA in inhibiting
the growth of hormone-dependent breast cancer cells,
presumably through RARâ activation; however, (9Z)-
UAB8 was ineffective in these actions.
((9E)-7). NaH (60% dispersion in mineral oil, 1.24 g, 31.0
mmol) was washed under N2 with dry THF (3 × 10 mL) to
remove the mineral oil. Anhydrous THF (10 mL) was added,
the suspension was cooled to 0 °C, and freshly distilled diethyl
3-(ethoxycarbonyl)-2-methylprop-2-enylphosphonate (5.40 g,
20.4 mmol) was added. After stirring for 30 min, HMPA (7.30
g, 40.7 mmol) followed by cyclohexanone (2.00 g, 20.4 mmol)
was introduced and stirring was continued at 0 °C for 2 h. At
that time the reaction was carefully quenched by adding,
dropwise, ice-cold water (10 mL). The resulting mixture was
extracted with ether (3 × 20 mL); the combined ether layers
were washed once with brine (15 mL), dried (Na2SO4), and
concentrated under vacuum to give crude ester 7 (4.32 g). A
hexane solution of crude 7 was purified by flash chromatog-
raphy on silica gel (400 g, 2% Et2O in hexane eluent, Rf 0.64)
to provide pure 7 (4.10 g, 96.0%) as a pale yellow oil: UV (λmax
)
1
275 nm; H NMR, IR, and mass spectra were identical with
those previously reported21 for 7.
(2E,4E)-Eth yl 4-(2′-Cycloh exen -1′-ylid en e)-3-m eth yl-2-
bu ten oa te ((a ll-E)-8). To a solution of (9E)-7 (3.00 g, 14.4
mmol) in anhydrous CH2Cl2 (30 mL), under N2 at 0 °C, were
added NBS (2.80 g, 15.7 mmol), CaO (0.660 g, 11.8 mmol), and
NaHCO3 (2.18 g, 26.0 mmol). The cooling bath was removed
and the reaction mixture stirred at room temperature for 8 h.
The mixture was filtered through a sintered glass funnel to
remove succinimide, and the filtrate was concentrated under
vacuum. Freshly distilled quinoline (1.97 g, 15.2 mmol) was
added to the residue and the mixture was stirred at 100 °C
for 2 h. The brownish mixture was cooled to room temperature
and dissolved in ether (250 mL), and the resulting solution
was washed with 5% H2SO4 (2 × 150 mL), water (100 mL),
and saturated NaHCO3 (150 mL). The ether solution was then
dried (Na2SO4) and concentrated under vacuum to provide (all-
E)-8 (1.88 g, 63.0%) as an oil. While (all-E)-8 was sufficiently
pure to carry on in this form, a sample (0.48 g) was further
purified by flash chromatography (1% Et2O/hexane) to give
pure (all-E)-8 (0.40 g, Rf 0.80, 10% EtOAc/hexane).
(2E,4E)-4-(2′-Cycloh exen -1′-ylid en e)-3-m eth yl-2-bu ten -
1-ol ((a ll-E)-9). To a solution of (all-E)-8 (1.33 g, 6.45 mmol)
in anhydrous ether (10 mL) under N2 at -78 °C was added,
dropwise, a solution of 1 M LiAlH4 in ether (6.50 mL, 6.50
mmol). The mixture was allowed to warm to 0 °C, stirred for
1 h, and cooled to -78 °C. To this mixture was added,
dropwise, methanol (5 mL) followed by 5% H2SO4 (20 mL). The
reaction mixture was allowed to warm to room temperature
and extracted with ether (2 × 20 mL). The combined ether
layers were washed with brine (30 mL), dried (Na2SO4), and
concentrated under vacuum to provide (all-E)-9 (0.98 g, 91%)
as an oil. This alcohol was not further purified but im-
mediately oxidized to the aldehyde.
Biology. The chick skin CRABP binding assay measured
IC50 values for retinoid binding to CRABP-II using a radiola-
beled competition assay.26 The Kd′ values used a fluorescence
titration method27,28 to measure the affinities of the retinoids
to recombinant mCRABP-I, mCRABP-II, and hRXRR-(DEF).
The IC50 values for retinoids with RARs and RXRs were
measured with a radioligand competition assay.20 The nuclear
receptor transcriptional activity assays were performed using
CV-1 cells. Transient transfection of these cells with a DNA
plasmid was performed essentially as described in Alam et al.20
The mouse skin antipapilloma assay measured the ED50 values
for retinoid tumor inhibition on the dorsal skin of mice. The
inhibition of mouse skin papilloma by retinoids was performed
according to a modification of the procedure developed by
Verma and Boutwell31 as reported previously.39
Molecu la r Mod elin g. Retinoid structures were generated
with Sybyl version 6.2 (Tripos Inc., St. Louis, MO) on a Silicon
Graphics Indigo 2 workstation. RA structures were built using
the x-ray crystal structure torsional angles.32 The RA ring is
in a half-chair conformation with C1, C6, C5, and C4 es-
sentially planar and C2 and C3 above and below this plane.32a
Another ring conformation occurs in solution due to conversion
to the other half-chair conformer. Since ring inversion does
not occur in the minimization, the calculated RA structures
contained the half-chair conformation reported in the crystal
Con clu sion s
We synthesized two new examples (UAB1 and UAB4)
from a new class of conformationally constrained 6-s-
trans-retinoids to form a homologous series: UAB1,
UAB4, UAB7, and UAB8. We find that both protein
affinity and nuclear receptor activation improve with
increasing size at R1 and R2. Assuming that these R
groups mimic the steric space of the RA ring, these
results demonstrate the importance of this ring in
achieving high biological activities in several assays. We
also show that the structural changes contained in the
UAB retinoids generate either RAR- or RXR-selective
retinoids and that only RAR-selective ligands are effec-
tive agents for skin cancer prevention. If RA mediates
its cancer chemoprevention and toxicity effects through
nuclear receptors, the similar activity and lower toxic-
ity20,39 of UAB7 and UAB8 (relative to RA) may result
from their nuclear receptor-selective actions.
Exp er im en ta l Section
Ch em ist r y. Gen er a l Met h od s. 1H NMR spectra were
obtained at 300.1 MHz (Bruker ARX spectrometer) and NOE
experiments were performed on degassed samples.19 UV/vis
spectra were recorded on an AVIV 14DS spectrophotometer
in cyclohexane solution (Fisher, Spectrograde). IR spectra
were recorded using a Nicolet FT IR spectrometer on films.
Electron-impact mass spectra were obtained on a Hewlett
Packard 5985 GC/MS instrument with an ultraperformance
fused silica gel column. HPLC separations were performed
on a Gilson HPLC gradient system using 25 mL pump heads
and an ISCO V′4 variable wavelength detector. The column
employed was a Whatman Partisil 10 M20/50 (500 × 22 mm
i.d.). Reverse-phase HPLC separations on carboxylic acids
were performed as described previously20 using a Chromanet-
ics Spherisorb ODS 5 µm column (250 × 4.6 mm i.d.). TLC
chromatography was performed on precoated 250 µm silica gel
GF glass plates (Analtech, Inc.; 5 × 10 cm). Solvents and
liquid starting materials were distilled prior to use. Reactions
and purifications were conducted with deoxygenated solvents,
under inert gas (N2) and subdued lighting.
Diethyl 3-(ethoxycarbonyl)-2-methylprop-2-enylphosphonate
was synthesized according to Iqbal et al.40 2,3-Dimethyl-2-
cyclohexenone (1) (Scheme 1) was prepared from 1,3-dimethox-
ybenzene using the method of J ung et al.41 All synthetic
procedures for the conversion of 1 to UAB4 (Scheme 1) were
the same as those we previously reported in detail for UAB7
and UAB8.19,20 Referring to Scheme 2, the preparation of ester
(9E)-7 was reported by us in modest yields.21 A modified
procedure giving much better yield is described here. All
procedures involved in converting alcohol (all-E)-9 to UAB1
were the same as those utilized in Scheme 1 for comparable
transformations.

3634 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 19
Muccio et al.
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structure. The structures were minimized using Maximin
(Sybyl), MM2, MM2(91), and MM3(94). Allinger’s MM3(94)
force field produced the structure that most closely cor-
responded to the X-ray structures. The 8-s-cis-RA structures
were generated by rotating just the C7-C8-C9-C10 bond to
the s-cis-conformation followed by energy minimization. The
(9Z)-RA structure was obtained by changing the configuration
of this bond to Z followed by energy minimization. The
resulting torsional angles agreed well with those found in the
X-ray structure of (9Z)-retinal. The 8-s-cis-conformers were
generated directly from the 8-s-trans-conformers.
The structures of the UAB retinoids were generated in a
similar manner. Since X-ray crystal structures of final
compounds are not available, a closely related synthetic
intermediate which had a solved structure (manuscript in
preparation) was chosen for validation of the structure calcula-
tion. This energy-minimized structure was used to generate
the corresponding UAB retinoids as described for RA.
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structure were calculated at 300 K by MM3(94) using a full-
matrix diagonalization. To generate overlapping structures,
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UAB retinoids were fit onto each other. The Connolly surface
area of each structure was obtained using a 1.4 Å probe and
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Ack n ow led gm en t. These studies were supported in
part by grants PO1 CA34968 (D.D.M., W.J .B., D.L.H.,
B.P.S.), DK40172 (E.L.), RCDADK02072 (E.L.), AICR
93B39 (B.P.S.), and RO1 CA59446 (B.P.S.).
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