Highly twisted adamantyl arotinoids: Synthesis, antiproliferative effects and RXR transactivation profiles

Article abstract of DOI:10.1016/j.ejmech.2009.01.011

Retinoid-related molecules with an adamantyl group (adamantyl arotinoids) have been described with selective activities towards the retinoid receptors as agonists for NR1B2 and NR1B3 (RARβ,γ) (CD437, MX3350-1) or RAR antagonists (MX781) that induce growth arrest and apoptosis in cancer cells. Since these molecules induce apoptosis independently of RAR transactivation, we set up to synthesize novel analogs with impaired RAR binding. Here we describe adamantyl arotinoids with 2,2′-disubstituted biaryl rings prepared using the Suzuki coupling of the corresponding fragments. Those with cinnamic and naphthoic acid end groups showed significant antiproliferative activity in several cancer cell lines, and this effect correlated with the induction of apoptosis as measured by caspase activity. Strikingly, some of these compounds, whereas devoid of RAR binding capacity, were able to activate RXR.

Full text of DOI:10.1016/j.ejmech.2009.01.011

European Journal of Medicinal Chemistry 44 (2009) 2434–2446
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Highly twisted adamantyl arotinoids: Synthesis, antiproliferative effects
and RXR transactivation profiles
a
b
a
a
´
´
´
´
Santiago Perez-Rodrıguez , Maria A. Ortiz , Raquel Pereira , Fatima Rodrıguez-Barrios ,
a,
b,
**
´
*
Angel R. de Lera , F. Javier Piedrafita
a
´
Departamento de Qu´ımica Organica, Universidade de Vigo, Lagoas-Marcosende, 36310 Vigo, Spain
b Sidney Kimmel Cancer Center, 10905 Road to the Cure, San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Retinoid-related molecules with an adamantyl group (adamantyl arotinoids) have been described with
Received 18 July 2008
Received in revised form
20 December 2008
Accepted 9 January 2009
Available online 20 January 2009
selective activities towards the retinoid receptors as agonists for NR1B2 and NR1B3 (RARb,g) (CD437,
MX3350-1) or RAR antagonists (MX781) that induce growth arrest and apoptosis in cancer cells. Since
these molecules induce apoptosis independently of RAR transactivation, we set up to synthesize novel
analogs with impaired RAR binding. Here we describe adamantyl arotinoids with 2,20-disubstituted
biaryl rings prepared using the Suzuki coupling of the corresponding fragments. Those with cinnamic
and naphthoic acid end groups showed significant antiproliferative activity in several cancer cell lines,
and this effect correlated with the induction of apoptosis as measured by caspase activity. Strikingly,
some of these compounds, whereas devoid of RAR binding capacity, were able to activate RXR.
Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords:
Adamantyl arotinoids
Retinoid-related molecules (RRMs)
RXR
Apoptosis
Synthesis
1. Introduction
conformational change in the ligand binding domain of the recep-
tors causing the dissociation of the co-repressor complex and the
Vitamin A (retinol) and its derivatives, the retinoids, are essen-
tial regulators of many biological events including cell growth and
differentiation, development, homeostasis and carcinogenesis.
Because of their antiproliferative activity, retinoids have been
proposed as cancer preventive and chemotherapeutic agents [1].
re-positioning of the activation function-2 located in helix H12. This
exposes a LXXLL motif necessary for the recruitment of chromatin-
modifying co-activators and the RNA polymerase II transcriptional
machinery to initiate transcription (see [5,6] and references therein).
Despite their promising activity in vitro, most natural and
synthetic retinoid analogs exhibit significant toxicity that has
limited their oncological therapeutic use [7]. All-trans-retinoic acid
(atRA) is used as a differentiation agent against acute promyelocytic
leukaemia [8], whereas the synthetic RXR agonist LGD1069 (Tar-
gretinÒ) has been approved for the treatment of cutaneous T cell
lymphoma [9] and is being clinically evaluated against lung and
breast cancers [10]. Other retinoid analogs are currently under
clinical evaluation with mixed results [1]. The development of
novel retinoids with selectivity towards RXRs or the different RAR
subtypes and isoforms has led to the intriguing discovery of a novel
class of derivatives with strong pro-apoptotic activity [11]. Because
they often exert their anticancer activity independently of the
retinoid receptors, they are also known as atypical retinoids or
Retinoid signals are mediated by the retinoid receptors RAR
a, b, g
(NR1B1, 2, 3) and RXR (NR2B1, 2, 3) [2,3], which belong to the
a, b, g
superfamily of nuclear hormone receptors [4]. These are ligand-
activated transcription factors that bind to specific DNA sequences
in the regulatory regions of target genes. In the absence of ligand,
RAR/RXR heterodimers interact with co-repressors and bind DNA
to inhibit transcription. Ligand binding induces
a profound
Abbreviations: 9cRA, 9-cis-retinoic acid; AdAr, adamantyl arotinoid; atRA, all-
trans-retinoic acid; IKK, kinase; JNK, cJun N-terminal kinase; LBD, ligand
binding domain; MAPK, mitogen activated protein kinase; NF
IkB
kB, nuclear factor-
kB;
RAR, retinoic acid receptor; RRM, retinoid-related molecule; RXR, retinoid
receptor; SHP, short heterodimer partner.
X
* Corresponding author. Present address: Torrey Pines Institute for Molecular
Studies, 3550 General Atomics Ct., San Diego, CA 92121, USA. Tel.: þ34 986 812316;
fax þ34 986 811940.
retinoid-related molecules (RRMs). In particular CD437, a RARb/g-
selective agonist and several analogs that contain an adamantyl
group are classified as adamantyl arotinoids (AdArs). A small
number of RRMs are known to induce apoptosis in cancer cells.
These include anhydroretinol [12,13], 4-hydroxyphenylretinamide
** Corresponding author. Tel.: þ1 858 597 3881; fax: þ1 858 455 3804.
´
E-mail addresses: qolera@uvigo.es (A.R. de Lera), jpiedrafita@tpims.org
(F.J. Piedrafita).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.01.011

S. Pe´rez-Rodr´ıguez et al. / European Journal of Medicinal Chemistry 44 (2009) 2434–2446
2435
(4-HPR) [14], some heteroarotinoids [15], and a few AdArs (MX781
9, CD2325 2 and their analogs, Fig. 1) [16].
compounds have shown the important synergistic role of the
adamantyl and phenol groups on RAR binding selectivity [49].
Moreover, the bulky adamantyl group appears to be necessary for
anticancer activity but not sufficient, since several other AdArs
exhibit low or moderate activity. The carboxylic acid might play
a role in apoptosis because its replacement by other bioisosters and
related groups led to reduction or loss of activity [50].
CD437 1 is the prototype of the RRM subfamily of RARb/g-
selective AdAr agonists [11] that includes CD2325 2, MX2870-1 3,
and MX3350-1 4 (Fig. 1). CD437 1 was first shown to induce growth
arrest and apoptosis in breast cancer cells [17] and several AdArs
were later found to inhibit growth of numerous cancer cell lines in
vitro and in animal models (see [16] and references therein). Given
that AdArs inhibit cell growth independently of RAR transactivation
[18–20], greatefforts haveaimed at characterizing the mechanism of
AdAr-induced apoptosis and, ultimately, at identifying AdAr targets
that mediate their anticancer activity. Thus, CD437 1-induced
apoptosis requires transcription/translation in a cell-type depen-
dent manner [21–24] (reviewed in [16]), and caspases are activated
via the mitochondrial pathway [22,25–27], although a role for
death-receptor signalling has also been suggested with some
compounds [28–30]. CD437 1 and AdArs alike cause a strong and
sustained activation of JNK and p38 stress kinases that precedes the
release of cytochrome c and subsequent induction of apoptosis [31];
however, contrasting results have been reported by different labo-
ratories using a variety of kinase inhibitors and cell lines [32–36]. In
contrast tothe activation of JNK/p38 MAPKs, certain apoptotic AdArs
Previous docking studies in the RAR
g LBD of 5-Cl-AHPN 5,
3-Cl-AHPC 7 and analogs have revealed that the steric clash
of the substituents located ortho to the biaryl bond (chloro, 3-
acetamidopropoxy of 8) induce a twist of that bond that displaces
the adamantyl fragment from the coplanarity with the aromatic
rings of their polar termini (naphthoic acid of CD437 or cinnamic
acid of AHPC) [47]. As a consequence, the position of helix H12 is
not appropriate for the interaction with the co-activator [51,52],
and the transactivation activities are considerably reduced [47].
Nevertheless, competition experiments with [3H]-9cRA revealed
that 3-Cl-AHPC 7 efficiently and selectively competed with the
native ligand for binding to RARg (83% displacement; cf. 31% RARa,
17% RAR ,16% RXR ). Further analysis led to the suggestion that the
b
a
conformational effect induced by 3-Cl-AHPC 7 was not sufficient
to induce dissociation of co-repressors and association of co-
activators, and therefore the ligand apparently behaved as a trans-
target the IKK/NFkB signalling pathway [20], which evokes survival
signals [37]. MX781 9 and CD2325 2 significantly inhibited kinase
activity of immunopurified IKK complex in vitro [20]; furthermore,
using purified recombinant kinases we have recently proved that
activational antagonist for RARg [47].
Based on this model, we considered to further increase the steric
interactions on that region by incorporation of two substituents (Cl
and Me) at the vicinal positions of the biaryl connection, which
would further impair binding of the ligand to the RAR receptor
subtypes. Moreover, to additionally increase the bulk of the
analogs, we considered the incorporation of a MEM substituent on
the phenol after recognizing that the combined effect of the acetal
and the adamantyl group of MX781 9 is likely associated to its RAR
antagonistic properties. In addition to the naphthoic acid deriva-
tives of CD437 1, other conformationally more flexible polar chains
of arylacetic and cinnamic acids were also considered (Scheme 1).
These AdArs were evaluated with regard to their ability to inhibit
cancer cell proliferation and induce apoptosis. Interestingly, we
found that while the anticancer effects are independent of RAR
transactivation, some of these highly twisted analogs bind and
transactivate through RXR.
MX781 9 is a selective inhibitor of IKK
been prepared with enhanced anti-IKK
b
and several analogs have
and growth inhibitory
b
activities [38]. Our findings disagree with recent reports indicating
that CD437 1 and its analog 3-Cl-AHPC 7 induced apoptosis via
activation of NFkB [39,40].
Second generation AdArs have been described with improved
anticancer activity [36,41–43]. Cinnamic acid derivative ST1926
(AHPC) 6 activates RARg and induces apoptosis in various cancer
cell lines with stronger potency as compared to CD437 1 [44,45].
Derivatives of CD437 1 lacking RAR transactivation activity, most
notably 3-Cl-AHPN (MM11453) [41], 3-Cl-AHPC 7 [43,46], and 5-Cl-
AHPN 5 [47], also elicit anticancer activity comparable to the parent
compound, whereas derivative 3-A-AHPC 8 prevented the induc-
tion of apoptosis by CD437 1 analogs but did not inhibit their effect
on cell cycle [47]. 3-Cl-AHPC 7 is an AdAr with cinnamic acid
substructure (Fig. 1) that induces cell-cycle arrest and apoptosis in
several cancer cell lines. Induction of apoptosis by 3-Cl-AHPC 7 and
some of its analogs was later shown to occur through binding to the
nuclear receptor SHP (small heterodimer partner, NR0B2) [48].
2. Chemistry
The Suzuki coupling between an arylboronic acid and an aryl
triflate, a strategy also utilized by Dawson et al. [47], was chosen as
a keystepof the syntheticsequence togeneratethe highlycongested
2,20-disubstituted biaryl bond (Scheme 1). The greater tolerance to
steric hindrance of the Suzuki reaction relative to other palladium-
catalyzed cross-coupling variants is well established [53,54].
With the exception of IKKb [20,38] and SHP [48], the cellular
targets that mediate the anticancer activity of these AdArs are
largely unknown, which represents a significant drawback for the
drug development efforts. Existing SAR studies of the RRM family of
CO2H
CO2H
CO2H
5
X
HO
HO
OH
1, X = H, CD437 (AHPN)
3, MX2870-1
2, CD2325
5, X = Cl, 5-Cl-AHPN
CO2H
CO2H
O
3
X
HO
O
CO2H
9, MX781 (CD2674)
O
O
O
6, X = H, ST1926 (AHPC)
7, X = Cl, 3-Cl-AHPC
8, X = AcNH-(CH2)3-O (3-A-AHPC)
O
4, MX3350-1
Fig. 1. Representative adamantyl arotinoids (AdArs).

2436
S. Pe´rez-Rodr´ıguez et al. / European Journal of Medicinal Chemistry 44 (2009) 2434–2446
COOR
COOR
Ad
COOR
XO
Cl
XO
Cl
COOEt
Ad
21 X =TBDMS,R = Me
22 X =H, R= Me
15X = R =H
d
e
j
Cl
TfO
16X = H, R =Me
k
XO
17X = Tf, R = Me
23 X =MEM,R = Me
24 X =MEM,R = H
25 X =H, R = H
Cl
l
14
l
g
26 X = TBDMS, R= Et
27 X = H, R = Et
28 X = MEM,R = Et
29 X = MEM,R = H
30 X = H, R =H
j
h
k
l
OH
l
Br
Ad
Br
Ad
Br
b
Ad
B
c
a
OH
HO
HO
TBDMSO
TBDMSO
10
11
12
13
i
COOR
CO2Et
CHO
e
f
Ad
TfO
XO
Cl
XO
Cl
Cl
20
31X = TBDMS, R = Et
32 X = H, R = Et
33 X = MEM,R = Et
34 X = MEM,R = H
35 X = H, R =H
j
18 R = H
19 R = Tf
k
l
l
Scheme 1. Reagents and reaction conditions: (a) adamant-1-ol, H2SO4, 25 ꢀC, 2 h, 98%. (b) Et3N, DMAP, TBDMSCl, 25 ꢀC, 1.5 h, 88%. (c) i. n-BuLi, THF, ꢁ78 ꢀC, 1 h; ii. B(OiPr)3, ꢁ78 to
25 ꢀC, 18 h; iii. HCl, 25 ꢀC, 1 h, 65%. (d) MeOH, TMSCHN2, 25 ꢀC, 1.5 h, 98%. (e) (F3CSO2)2O, pyridine, 0 ꢀC, 1.5 h, 82%. (f) NaH, (EtO)2POCH2CO2Et, 0 ꢀC, 4 h, 51%. (g), (h), (i) Suzuki
coupling (see text). (j) TBAF, THF, 0 ꢀC. (k) MEMCl, NaH, DMF, 25 ꢀC. (l) Na2CO3, MeOH, 70 ꢀC.
Incorporation of the adamantyl group using a Friedel–Crafts
alkylation of 4-bromo-3-methylphenol 10 with adamant-1-ol
proceeded uneventfully and in excellent yield (98%). Protection of
the phenol as silyl ether 12, followed by bromine–lithium exchange
(n-BuLi, THF, –78 ꢀC) and trapping the aryllithium with tri-iso-
propyl borate produced the corresponding di-isopropylboronate,
which was hydrolyzed with HCl to afford 13. This unstable boronic
acid was used in the next step without further purification. The
synthesis of triflate 14 from commercially available 6-hydrox-
ynaphthoic acid has been described previously [47]. Aryl triflate 17
[55] was in turn prepared in two steps from commercial 3-chloro-
4-hydroxyphenylacetic acid 15, via ester 16 (obtained from 15 by
treatment with TMSCHN2 at ambient temperature), which was
then treated with TFAA in pyridine to afford the desired sulphonate
(82%). In anticipation of the application of the Horner–Wadsworth–
Emmons reaction to afford the cinnamic ester, commercially
available 3-chloro-4-hydroxybenzaldehyde 18 was treated with
trifluoromethanesulphonic anhydride in pyridine to furnish triflate
19. The condensation of the latter under the conditions described
by Bates et al. [56] provided cinnamate E-20 in 55% yield as the only
geometric isomer (Scheme 1).
After exploring different conditions for the Suzuki coupling of 13
and triflate 17 as a model system, we obtained higher yields using
those of Cockerill et al. [57], which involve heating to 90 ꢀC [Et3N,
Pd(PPh3)4, DMF, 27.5 h] thoroughly degassed reaction mixtures of 13
and 17. Worthy of note, all attempts to scale-up the reaction led to
erosion in the yield of 21. The same protocol served well for the
coupling of the naphthyl triflate 14 to the same boronic acid, which
delivered 26 in 85% yield (Scheme 1). Exploiting the acceleration
effectof microwave irradiation [58] the reaction reached completion
after 15 min at 70 ꢀC, but the yield decreased (63%). The optimized
reaction conditions did not serve well for triflate 20, since it afforded
31 in disappointing yields (34%). Turning to the precedent approach
to 3-Cl-AHPC 7 by Zhang et al. [43,47], which used Pd(PPh3)4 as pre-
catalyst, Na2CO3 as base and LiCl as additive in DME, the coupled
product 31 (67%) was instead obtainedafter 22 h heatingat 80 ꢀC. No
improvement of the yield for this coupling was observed using
microwave irradiation. Deprotection of the silyl ether of all analogs
with TBAF in THF at 0 ꢀC was followed by treatment of the phenols
with MEMCl using NaH in DMF at 0 ꢀC to furnish in moderate yields
derivatives 23, 28 and 33. Finally, saponification (aq. K2CO3, MeOH,
70 ꢀC) produced the desired carboxylic acids 24, 29 and 34,
respectively. The same conditions were used for the synthesis of the
adamantyl arotinoids 25, 30 and 35.
3. Results
3.1. Inhibition of cancer cell proliferation by novel AdArs
We first evaluated the effect of the new AdAr analogs on Jurkat T
leukaemia cells, which are very sensitive to the antiproliferative
activity of RRMs. Fig. 2 shows that the parent compounds CD2325 2
and MX781 9 strongly inhibited Jurkat cell proliferation in a dose-
dependent manner, with estimated IC50 values of 0.082 and
0.287
synthesized compounds, the naphthoic acid derivatives 30 (IC50
0.435 M) and 29 (IC50 0.707 M) were the most active against
Jurkat cells, followed by the cinnamic analogs 35 (IC50 1.414 M)
and 34 (IC50 1.866 M). Compounds 24 and 25 were noticeably less
mM, respectively, after 48 h of treatment. Among the
m
m
m
m
effective and only partial inhibition was seen at higher concentra-
tions. As expected, the antiproliferative activity of the AdArs was
dependent on the time of incubation (data not shown).
We next examined the antiproliferative activity of the analogs
against a panel of cancer cell lines of diverse epithelial origin.
Higher concentrations of AdArs are usually required to inhibit
growth of those cells as compared to Jurkat cells and Table 1
summarizes the percentage of growth inhibition observed in the
presence of 4 mM of the different AdArs. As observed with the most
sensitive Jurkat T cell line, 29 and 30 were the most active of the
new derivatives, with similar or even increased activity as
compared to parent AdArs CD2325 2 and MX7819. It is evident that
34 exhibited strong antiproliferative activity with comparable or
enhanced activity than MX781 9 against ovarian (SkOV3), lung
(A549), and pancreatic (MiaPaCa-2) cancer cells (Table 1), despite
limited activity in Jurkat cells (see Fig. 2). We included 3-Cl-AHPC 7
in our cell proliferation studies for comparison and found strong
antiproliferative activity against all cell lines evaluated, which was
identical to that elicited by CD2325 2 (data not shown).
3.2. AdArs induce apoptosis in cancer cells
The activation of caspases is an early event in apoptosis [59].
To evaluate the pro-apoptotic activity of the novel AdArs we
examined the induction of DEVDase activity as a measure of

S. Pe´rez-Rodr´ıguez et al. / European Journal of Medicinal Chemistry 44 (2009) 2434–2446
2437
Fig. 2. Antiproliferative activity of AdArs against Jurkat T leukaemia cells. Jurkat cells were incubated with increasing concentrations (between 62.5 nM and 2
mM) of phenols (A) or
MEM derivatives (B) for two days, when cell proliferation was measured by MTT. The results of a typical experiment performed in triplicate are shown.
caspases 3/7 activity in Jurkat T cells after short periods of treat-
ment. Fig. 3 illustrates that AdArs CD2325 2 and MX781 9
provoked a robust induction of DEVDase activity when tested at 1
a significant effect on RAR
at the highest concentration of 4
analogs 29 and 34 and the phenol 30 inhibited RAR
32% (Fig. 4B). Lower concentrations of those derivatives had no
significant effect on atRA-mediated RAR activity. This contrasts
with the 90% inhibition elicited by the parental compound MX7819
at the same concentration and the total inhibition of RAR activity
observed even with lower concentrations of the inverse agonist
UVI2024 (BMS204493, see structure in Fig. 6) [60]. When the same
a
activity (43.7% inhibition) when tested
M, whereas the other MEM
activity by 27–
m
a
and 4
well as 1
substantial DEVDase activity but only when tested at 4
m
M. Similar activation was observed in the presence of 30 as
M 3-Cl-AHPC 7. The MEM derivative 29 also induced
M (Fig. 3).
m
a
m
Likewise, the phenols 25 and 35 activated caspases 3 and 7 only at
the highest concentration. This parallel with diminished anti-
proliferative activity observed at low concentrations of 25, 29, and
35 (see Fig. 2). In contrast to the pro-apoptotic activity of 29, other
MEM-functionalized compounds showed limited (24) or no
activity (34). In general, the induction of DEVDase activity corre-
lated well with the antiproliferative activity measured by MTT
assay described in Fig. 2. The small differences observed between
those two activities are possibly due to delays in the activation of
caspases.
experiment was performed with Gal4-RAR
new AdArs failed to inhibit atRA-mediated RAR
activation even at the 200-fold molar excess concentration, which
contrasts with the almost complete inhibition of both RAR and
RAR seen with UVI2024 at any given concentration or with MX781
9 at 4 M (data not shown).
The effect of the AdArs RXR
Fig. 5. To our surprise, phenol 35 and all new MEM-containing
AdArs (24, 29, and 34) were capable of inducing RXR -dependent
b
and Gal4-RARg, the
b
and RARg trans-
b
g
m
a
transactivation is illustrated in
a
3.3. RAR/RXR transactivation activity
luciferase activity almost as efficiently as the natural ligand 9cRA 36
(Fig. 5A), and this transactivation was inhibited by the RXR antag-
onist UVI3003 38 (Fig. 5B) [61]. Whereas 29 behaved as a partial
agonist and reached a maximum activity of w45% of that seen in
the presence of saturating concentrations of 9cRA 36 or the RXR
agonist LGD1069 37 (Fig. 6), the other analogs activated RXRa with
similar or even enhanced potency as compared to the natural ligand
(Fig. 5C). The EC50 values estimated for 29, 34, and 35 were
We used transient transfections in CV-1 cells with expression
vectors containing the Gal4 DNA binding domain fused to the C-
terminus ligand binding domain (LBD) of RARa, b, g or RXRa to
evaluate the transactivation potential of the novel AdArs. As shown
in Fig. 4A, none of the compounds was able to induce luciferase
activity in the presence of RAR
a. Likewise, all the analogs were
inactive on Gal4-RAR and Gal4-RAR
b
g
proteins (data not shown).
0.667
activated RXR
than those observed with 9cRA 36 (0.2
(0.04 M) (not shown).
m
M, 0.60
m
M, and 0.576
m
M, respectively. Compound 24
The ability to function as RAR antagonists was next evaluated in CV-
1 cells stimulated with 20 nM atRA. Testing compounds were
added in excess of 10 (0.2 mM), 40 (0.8 mM), or 200-fold (4 mM) with
a
with an EC50 of 1.137
m
M. These values were higher
mM) or LGD1069 37
m
respect to the natural ligand. Only the MEM derivative 24 had
Table 1
Inhibition of cancer cell proliferation by the novel AdArs.a
PC-3
SkOV3
A549
MiaPaCa-2
BxPC-3
T-47D
MB468
2
92.3 ꢂ 1.4
66.7 ꢂ 6.9
97.7 ꢂ 0.8
68.7 ꢂ 1.5
67.0 ꢂ 1.8
30.3 ꢂ 1.6
87.0 ꢂ 0.9
65.1 ꢂ 0.8
79.0 ꢂ 4.0
15.9 ꢂ 5.1
88.1 ꢂ 2.1
23.7 ꢂ 4.5
76.1 ꢂ 6.0
26.8 ꢂ 5.5
91.5 ꢂ 4.5
26.3 ꢂ 4.1
95.7 ꢂ 0.2
49.2 ꢂ 4.7
94.7 ꢂ 0.5
36.0 ꢂ 3.0
62.7 ꢂ 3.0
29.1 ꢂ 4.2
55.8 ꢂ 0.6
24.2 ꢂ 4.8
76.7 ꢂ 1.3
11.1 ꢂ 7.7
59.2 ꢂ 2.2
24.9 ꢂ 5.9
25
30
35
9
92.1 ꢂ 2.3
52.2 ꢂ 3.5
97.6 ꢂ 1.1
72.0 ꢂ 6.6
61.5 ꢂ 7.1
19.1 ꢂ 4.1
71.2 ꢂ 1.5
80.1 ꢂ 0.6
42.1 ꢂ 5.6
7.9 ꢂ 7.1
74.9 ꢂ 2.8
6.3 ꢂ 3.4
96.6 ꢂ 0.4
14.0 ꢂ 3.6
70.2 ꢂ 4.7
45.8 ꢂ 1.8
50.6 ꢂ 2.3
27.6 ꢂ 3.9
46.1 ꢂ 1.3
29.1 ꢂ 3.3
72.4 ꢂ 1.3
25.3 ꢂ 5.6
49.9 ꢂ 2.2
42.0 ꢂ 3.6
24
29
34
88.1 ꢂ 3.7
51.1 ꢂ 0.7
63.7 ꢂ 2.8
63.3 ꢂ 4.5
a
The effect of AdArs on cancer cell proliferation was evaluated by MTT assay. Prostate (PC-3), ovarian (SkOV3), breast (T-47D, MDA-MB468), and pancreas (MiaPaCa-2,
BxPC3) cancer cells were treated with 4 M AdArs for 48 h. NSCLC cells (A549) were treated for 72 h before measuring cell viability. All experiments were performed one to
three times with triplicate points and the average ꢂ S.D. is shown.
m

2438
S. Pe´rez-Rodr´ıguez et al. / European Journal of Medicinal Chemistry 44 (2009) 2434–2446
4. Discussion
The so called ‘‘adamantyl retinoids’’ [63,64] can structurally be
considered as arotinoids [65] (or arene-based retinoids having
some of the double bonds included in arenes) with an adamantyl
ring in the hydrophobic part of the retinoid structure. Although
some bind to and activate the nuclear receptors, the RARs seem
dispensable for their anticancer activity [1]. Some analogs show
selectivity for certain subtypes of the retinoid receptor and can bind
other non-retinoid receptors simultaneously [66,67].
Comprehensive SAR studies on AdArs other than the RAR
binding/transactivation are not available and knowledge on the
structural requirements that mediate their anticancer activity is
insufficient. Provided that AdArs and other atypical retinoids
induce apoptosis independently of RARs and RXRs, the identifica-
tion of the AdAr targets responsible for their anticancer activity will
be critical to further optimize the available scaffolds using a struc-
ture-based approach and especially focused SAR studies. One such
cellular target is IKK
analogs in vitro and in cell based assays [20,38]. Compound 30 is
also a strong inhibitor of recombinant IKK (our unpublished
b, which is inhibited by MX781 9 and several
Fig. 3. Induction of apoptosis by AdArs. Jurkat cells were incubated with 1 (grey) or 4
(black) mM of the indicated compounds for 4 h, when cells were lysed and assayed for
DEVDase activity. Control cells (no) were incubated with 0.1% (v/v) DMSO. The
experiment was performed three times in duplicate and shown here are the results of
one typical experiment. ND: not determined.
b
observations), and this might be partly responsible for the signifi-
cant antiproliferative activity found against most cancer cell lines
(Table 1), although IKK inhibition needs to be confirmed in cellular
assays. Another AdAr target is SHP, though 3-Cl-AHPC 7 can induce
significant apoptosis in cells lacking SHP [48], and the signalling
linking to apoptosis remains to be elucidated. In addition to SHP
binding, two analogs of 3-Cl-AHPC 7 were found to inhibit CD45
and SHP-2 protein tyrosine phosphatases in vitro at micromolar
concentrations [50]. Similarly, CD437 1 was shown to inhibit
recombinant MKP-1 [67]. The effect of RRMs on protein phospha-
tases in cells has yet to be demonstrated, but it is worthy to point
out that those 3-Cl-AHPC 7 analogs shown to inhibit CD45 and SHP-
2 exhibit very little pro-apoptotic and growth inhibitory activities
[50], suggesting that this effect on protein tyrosine phosphatases
might not be directly involved in the anticancer power of active
AdArs. AdAr-mediated inhibition of MKP-1 on the other hand might
be responsible for the sustained activation of JNK and p38 observed
by CD437 1 and other analogs [31], which is likely to play a causal
role in the induction of apoptosis by JNK activating stimuli, as
demonstrated by others [68–71].
This unexpected result prompted us to use Molecular Docking
studies in order to justify the rexinoid profile of the AdArs. The
complexes obtained upon replacement of 9cRA by the synthesized
AdAr into the crystal structure of human holo-RXRa (PDB code 1k74)
[62] were analyzed. Analysis of the most populated clusters found by
AutoDock for 35 [root-mean-square deviation (rmsd) < 1.0 Å] indi-
cate that it binds RXR
buried in an essentially hydrophobic pocket formed by residues
located on helices, H3, H5, H7 and H11, and the -turn. The elongated
a similarly to 9cRA 36 (Fig. 7). The ligand 35 is
b
pocket is sealed by Arg316 of helix H5 on one side and by the trans-
activationhelix H12ontheotherside. Thecarboxylategroupof35 like
that of 9cRA is sandwiched between the side chains of residues
Phe313, Ala271 and Ala272, which form a hydrophobic tunnel. The
ionic interaction between the carboxylate group and Arg316 is similar
to that seen in atRA and 9cRA RARg holo-LBD structures. At the other
side of the pocket, residues Leu433, His435, Val 342, Gly343 and
Ile345 make contacts with the adamantyl residue.
It has been pointed out that the role of the chlorine substituent
on the structure and activity of 5-Cl-AHPN 5 appears to go beyond
the classical steric effect, since the methyl analog at the same
Whereas the docking protocol provided good fits for the free
phenol (35), however, no stabilizing solutions were found for the
MEM derivatives (24, 29, 34) using AutoDock.
Fig. 4. Effect of AdArs on RAR
a
transactivation activity. (A) CV-1 cells were transfected with Gal4-RAR
a and stimulated with 1 mM atRA or 2 mM of the indicated analogs. Luciferase
activity was determined after 6 h of stimulation following standard procedures and normalized by
b-galactosidase. (B) RAR antagonist activity of AdArs. Transfected CV-1 cells were
co-stimulated with 20 nM of atRA together with increasing concentrations of the indicated analogs. Phenols and MEM derivatives are represented by closed and open symbols,
respectively. For comparison, the antagonist activity of UVI2024 and MX781 9 were also evaluated. The percentage of the maximum activity measured with respect to cells
stimulated with 20 nM of atRA alone is represented. Data are the average ꢂ standard deviation of two independent experiments performed with triplicate points.

S. Pe´rez-Rodr´ıguez et al. / European Journal of Medicinal Chemistry 44 (2009) 2434–2446
2439
Fig. 5. Activation of RXR
measured. (B) CV-1 cells were stimulated with 1
for 6 h, when luciferase and -galactosidase activities were determined. RXR activating compounds were further titrated to estimate their EC50 values (C). Luciferase activity in A
a
by AdArs. (A) Gal4-RXR
a
-transfected CV-1 cells were stimulated with 1
mM 9cRA 36 or 2
mM of the indicated analogs for 6 h, when luciferase activity was
m
M 9cRA 36 or 5 M of the indicated AdArs in the absence (black columns) or in the presence of 1
m
mM UVI3003 38 (grey columns)
b
and C represents the percentage of the maximum activity obtained in the presence of 1
mM 9cRA 36.
position is considerably less active [47]. We therefore decided to
preserve the halogen at that position of the naphthoic acid (or that
of the cinnamic acid analogs) and designed a series of analogs with
an even greater steric congestion at the biaryl ring bond. The
synthesis follows principles already described for the parent
analogs and improvements in yields and reaction times have been
developed using microwave irradiation for the Suzuki coupling
reaction. Of the novel AdArs reported here, the naphthoic deriva-
tives 29 and 30 elicited the strongest growth inhibition and
3-Cl-AHPC 7, 35 was also a strong activator of RXRa-driven lucif-
erase activity in our transient transfection assays. Our docking
studies could not find good fits of the MEM-containing rexinoids
24, 29, and 34 within the structure of holo-RXR
substantial transactivation of RXR , which was comparable to that
of 9cRA 36 with 24 and superior in the presence of 34, it is possible
that an active 24/34-RXR conformation might be stabilized by the
presence of a particular co-activator or heterodimer partner. In this
regard, the crystal structure of the constitutively active RXR -F318A
a. Given the
a
a
a
apoptotic (DEVDase) activities. Similar to 5-Cl-AHPN
3-Cl-AHPC 7, the compounds characterized here have no RAR
transactivation activity. Contrary to 5-Cl-AHPN 5 and 3-Cl-AHPC 7,
5
and
mutant, which crystallized in the presence of oleic acid, showed an
alternate position of helix H12 with the co-activator LXXLL motif
not exposed [72]; however, contrary to a pure antagonist, helix H12
was not precluded from adopting an agonist position, which could
be stabilized in the presence of co-activators. Future studies will
need to evaluate the activity of these rexinoid AdArs on full length
RXRs, the activation of different heterodimer partners, and their
effects on RXR-co-repressor/co-activator interactions. Furthermore,
in base of the recent discovery of RXR subtype preference by certain
synthetic rexinoids [73,74], it will be interesting to explore the
which allegedly function as RARg antagonists because of their
ability to bind RAR but failure to induce release of co-repressors
g
and association of co-activators [47], the synthesized analogs also
lack of significant RAR antagonistic activity, with only partial
inhibition of RARa activity observed in the presence of elevated
concentrations (200-fold excess) of the MEM analogs 24, 29, and 34
or the phenol 30. To our surprise, the MEM-containing derivatives
24, 29, and 34, activated RXR
a
. Likewise, the o-methyl analog of
activation of RXR
b
and
g
by these rexinoid AdArs. Together, all
HO
CO2H
CO2H
CO2H
O
37, LGD1069
CO2H
36,9-cis-retinoic acid
38, UVI3003
UVI2024 (BMS493)
Fig. 6. RAR and RXR (ant)agonists used as control in transactivation studies.

2440
S. Pe´rez-Rodr´ıguez et al. / European Journal of Medicinal Chemistry 44 (2009) 2434–2446
Fig. 7. Docking of adamantyl arotinoid 35 (in yellow) into the RXRa-binding pocket and close-up view highlighting the main interactions with the protein residues. For comparison,
the structure of 9cRA (in grey) is superimposed. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
these studies will certainly shed light onto the structural basis of
their rexinoid activity.
One important feature of the rexinoid AdArs characterized here
is their ability to inhibit cancer cell growth via apoptosis induction,
in particular compounds 29 and 35, although there is no clear
correlation between RXR transactivation and induction of caspase
activity. For example, 29 is a partial agonist of RXRa and the most
effective growth inhibitor of all rexinoid AdArs reported here,
whereas 35 has reduced growth inhibitory and apoptosis inducing
DNA synthesis in S phase [79] and G1 arrest [81]. All together, these
studies suggest that the differentiation activity and cell-cycle
effects contribute to the chemopreventive activity of previously
known rexinoids. In contrast, induction of apoptosis might be the
primary effect of the rexinoid AdArs described here, at least in the
case of 29, which exhibits substantial antiproliferative activity
against most cancer cell lines evaluated (see Fig. 2 and Table 1), but
reduced RXR transactivating capacity. Whether these rexinoid
AdArs cause cell death via activation of RXRs, similar to other
previously characterized rexinoids, or the induction of apoptosis is
intrinsic to the AdAr structure and independent of RXR trans-
activation remains to be elucidated.
activities but activates RXR
a almost as efficiently as 9cRA 36.
Moreover, 24 and 34 are also effective RXR transactivators but show
limited anticancer activity, as does the natural ligand 9cRA 36, in
comparison to other synthetic AdArs. Rexinoids, as well as reti-
noids, are well known for their significant chemopreventive and
therapeutic activity in cancer and rexinoids have the additional
advantage of being less toxic [1,75]. In addition to FDA approved
Bexarotene (TargretinÒ, LGD1069), which has residual RAR activity
[76], two synthetic rexinoids with no RAR activity, LGD100268 and
AGN194204 [76,77], offer improved anticancer activity in preclin-
ical models of breast, lung, and prostate cancer prevention and this
anticancer activity is significantly higher in combination with other
agents (see [75] for review and references therein). As opposed to
rexinoid AdArs 29 and 35, which produce substantial DEVDase
activity in Jurkat T cells (Fig. 3), other known rexinoids are poor
inducers of apoptosis by themselves [78–81], unless cells are
deprived of growth factors [82] or treatment is performed in
combination with the estrogen antagonist arzoxifene [80].
5. Conclusion
In this study, we intended to eliminate RAR binding and trans-
activation activity in AdArs while maintaining their growth inhib-
itory and apoptosis inducing profiles. While this goal was
accomplished, we also found that some of the AdArs elicited
significant RXR
a transactivation activity. RXR plays an important
role in the anticancer activity of other known rexinoids, which
contrasts with the RAR-independence demonstrated in the induc-
tion of apoptosis by parent AdArs. Among the four AdArs with RXR
activity reported here, only compound 29 was found to have
substantial anticancer activity against various types of cancer cells,
which paralleled the induction of apoptosis. Interestingly,
compound 29 was a partial agonist of RXRa, whereas other AdArs
Interestingly, LGD100268 seems to inhibit NF
k
B activity [80],
with enhanced RXR transactivation activity elicited limited growth
inhibitory effects. Therefore, future studies will be essential to
elucidate the role of RXRs on the anticancer activity of rexinoid
AdArs.
although a direct effect of the rexinoids on IKK activity has not been
yet reported. Contrary to RRMs in general, and particularly AdArs,
RXRs seem to mediate the growth inhibitory effects of synthetic
rexinoids, as the sensitivity of breast cancer cells to AGN194204
correlates with RXR
a
expression levels [79] and RXR antagonists
6. Experimental protocols
prevent rexinoid activity [78]. Likewise, the synergy between
rexinoids and protein kinase A activators to induce differentiation
of atRA-resistant leukemia cells requires transactivation of the RAR-
RXR heterodimer [60,83]. Gene expression analyses have demon-
strated that LGD1069 37 (Fig. 6) induces adipocyte-specific changes
in regressing rat tumors [84] and affects expression of several genes
involved in cell growth, including cyclin D1, cox-2, PTEN, and p27 in
mammary epithelial cells [85]. This correlated with inhibition of
6.1. Chemistry
Solvents were dried according to published methods and
distilled before use. HPLC grade solvents were used for the HPLC
purification. All other reagents were commercial compounds of the
highest purity available. All reactions were carried out under argon
atmosphere, and those not involving aqueous reagents were

S. Pe´rez-Rodr´ıguez et al. / European Journal of Medicinal Chemistry 44 (2009) 2434–2446
2441
carried out in oven-dried glassware. Analytical thin layer chroma-
tography (TLC) was performed on aluminium plates with Merck
Kieselgel 60F254 and visualized by UV irradiation (254 nm) or by
staining with an ethanolic solution of phosphomolibdic acid. Flash
column chromatography was carried out using Merck Kieselgel 60
(230–400 mesh) under pressure. Infrared spectra were obtained on
JASCO FTIR 4200 spectrophotometer, from a thin film deposited
onto a NaCl glass. 1H NMR spectra were recorded in CDCl3, CD2Cl2,
and CD3OD at ambient temperature on a Bruker AMX-400 spec-
trometer at 400 MHz with residual protic solvent as the internal
reference (CDCl3, dH ¼ 7.26 ppm; CD2Cl2, dH ¼ 5.30 ppm; CD3OD,
1.5 M in THF, 3.51 mmol) over 10 min. After stirring for 0.5 h tri-
isopropyl borate (2.2 mL, 9.58 mmol) was added the resulting
mixture was stirred for 2 h at ꢁ78 ꢀC and was allowed to warm at
room temperature overnight. The reaction was cooled (0 ꢀC) and
then was treated with water and a solution of 10% HCl in water. The
mixture was extracted with EtOAc (3ꢃ), washed with brine, dried
over anhydrous Na2SO4 and evaporated to afford 0.57 g (63%) of the
desired boronic acid 13, which was used in the Suzuki coupling
reactions without further purification due to its instability.
Methyl 2-(3-chloro-4-hydroxyphenyl)acetate (16) [55].
A
suspension of 15 (5.0 g, 26.77 mmol) in MeOH (38.3 mL) and
benzene (267.9 mL) was treated with a solution of (trimethylsi-
lyl)diazomethane (17.18 mL, 2 M in hexane, 34.83 mmol). After
stirring for 1.5 h at 25 ꢀC the solvent was evaporated to afford ester
dH ¼ 3.31 ppm); chemical shifts (
d) are given in parts per million
(ppm), and coupling constants (J) are given in Hertz (Hz). The
proton spectra are reported as follows:
d (multiplicity, coupling
constant J, number of protons, assignment). 13C NMR spectra were
recorded in CDCl3 and CD2Cl2 or CD3OD at ambient temperature on
the same spectrometer at 100 MHz, with the central peak of CDCl3
16 (5.3 g, 98%) as a white solid. 1H NMR (400.13 MHz, CDCl3):
d 6.97
(d, J ¼ 8.2 Hz, 1H, ArH) 6.85 (d, J ¼ 8.2 Hz, 1H, ArH), 5.43 (s, 1H, ArH),
3.64 (s, 3H), 3.48 (s, 2H) ppm.
(
dC ¼ 77.0 ppm), CD2Cl2 (dC ¼ 54.2 ppm) or CD3OD (dC ¼ 49.05 ppm)
Methyl 2-(3-chloro-4-(trifluoromethylsulfonyloxy)phenyl)acetate
(17) [86]. General procedure for the synthesis of triflates. To a solution
of phenol 16 (0.90 g, 4.44 mmol) and pyridine (1.1 mL) in CH2Cl2
(30 mL) was added dropwise trifluoromethanesulfonic anhydride
(0.92 mL, 6.84 mmol). The reaction mixture was allowed to warm
to room temperature, stirred for 12 h, washed (2 ꢃ H2O, 10% HCl,
and brine), dried (Na2SO4) and the solvent was removed to afford
1.2 g (82%) of 17 as a white solid (m.p.: 54 ꢀC, CH2Cl2/hexane). 1H
as the internal reference. DEPT135 pulse sequences were used to
aid in the assignment of signals in the 13C NMR spectra. Mass
Spectra were obtained on a Hewlett–Packard HP59970 instrument
operating at 70 eV by electron ionisation. High Resolution Mass
Spectra were taken on a VG Autospec M instrument. FAB experi-
ments were performed on a VG AutoSpec instrument, using
3-nitrobenzylalcohol or glycerol as matrices. The microwave reac-
tions were carried out in argon-flushed microwave quartz glass
vessels in a CEM model Explorer with maximum power at 300 W.
Melting points were measured in a Stuart Scientific apparatus.
2-(Adamant-1-yl)-4-bromo-5-methylphenol (11). A solution of
phenol 10 (1.90 g, 10.39 mmol) in anhydrous dichloromethane
(7 mL) was treated with 1-adamantanol (1.60 g, 10.39 mmol) and
H2SO4 (0.53 mL, 10.39 mmol). After stirring at 25 ꢀC for 2 h, the
mixture was neutralized with a 5% NaHCO3 solution and extracted
with CH2Cl2 (3ꢃ). The organic extracts were washed with NaCl,
dried over anhydrous Na2SO4 and evaporated to afford phenol 11
NMR (400.13 MHz, CDCl3):
d
7.57 (d, J ¼ 8.0 Hz, 1H, ArH), 7.23 (d,
J ¼ 8.0 Hz, 1H, ArH), 7.10 (s, 1H, H2), 3.71 (s, 3H), 3.63 (s, 2H) ppm.
2-Chloro-4-formylphenyl trifluoromethanesulfonate (19) [50]. In
accordance to general procedure described above, compound 18
(0.1 g, 0.60 mmol) was treated with pyridine (1.1 mL, 2.1 mmol) and
trifluoromethanesulfonic anhydride (0.12 mL, 0.74 mmol) to afford
19 (0.14 g, 82%) as a white solid. 1H NMR (400.13 MHz, CDCl3):
d
10.00 (s, 1H, CHO), 8.16 (d, J ¼ 1.9 Hz, 1H, H3), 7.88 (dd, J ¼ 8.2 Hz,
1.9 Hz, 1H, H6), 7.55 (d, J ¼ 8.2 Hz, 1H, H5) ppm.
(E)-Ethyl
3-(3-chloro-4-(trifluoromethylsulphonyloxy)phenyl)
(3.30 g, 98%) as a white solid. 1H NMR (400.13 MHz, CDCl3):
d
7.30
acrylate (20) [43]. To a solution of triethyl 2-phosphonoacetate
(0.11 mL, 0.54 mmol) in THF (1.1 mL) was added NaH (0.02 g, 60% in
mineral oil, 0.54 mmol) at 0 ꢀC. After stirring for 15 min, a solution
of aldehyde 19 (1.60 g, 6.58 mmol) in THF (1.2 mL) was slowly
added, via cannula, and the mixture was stirred for 4 h at the same
temperature. The mixture was poured into H2O and extracted with
AcOEt (3ꢃ). The combined organic extracts were washed with an
aqueous saturated NH4Cl solution (3ꢃ) and brine, dried with
Na2SO4, filtered and concentrated to dryness. The residue was
purified by flash chromatography (silica gel, 95:5 hexane/AcOEt) to
afford 20 (91 mg, 51% yield) as a white powder. 1H NMR (300 MHz,
(s, 1H, H6 or H3), 6.54 (s, 1H, H3 or H6), 2.28 (s, 3H, 3ꢃ AdCH), 2.07
(s, 9H, 3ꢃ AdCH2 þ ArCH3), 1.77 (s, 6H, 3ꢃ AdCH2) ppm.
2-(Adamant-1-yl)-(4-bromo-5-methylphenyl)
tert-butyldime-
thylsilyl ether (12). To a solution of phenol 11 (1.20 g, 3.73 mmol) in
DMF (5 mL) were added Et3N (0.76 mL, 5.04 mmol), DMAP (0.02 g,
0.19 mmol) and TBDMSCl (0.76 g, 5.04 mmol). After stirring for 16 h
at room temperature, the mixture was poured into ice-water and
extracted with ethyl acetate (3ꢃ). The combined organic extracts
were washed with brine, dried over anhydrous Na2SO4 and evap-
orated. The residue was purified by column chromatography on
silica gel (100% hexane) to afford bromide 12 (1.40 g, 88%) as
CDCl3):
d
7.67 (d, J ¼ 1.9 Hz, 1H, H2), 7.59 (d, J ¼ 16.0 Hz, 1H,
a white solid. 1H NMR (400.13 MHz, CDCl3):
d
7.35 (s, 1H, H6 or H3),
CH]CHCO2Et), 7.48 (dd, J ¼ 8.5, 1.8 Hz, 1H, H6), 7.38 (d, J ¼ 8.5 Hz,
1H, H5), 6.45 (d, J ¼ 16.0 Hz, 1H, CH]CHCO2Et), 4.28 (q, J ¼ 7.1 Hz,
2H, CO2CH2CH3), 1.35 (t, J ¼ 7.1 Hz, 3H, CO2CH2CH3) ppm.
6.71 (s, 1H, H3 or H6), 2.32 (s, 3H, 3ꢃ AdCH), 2.10 (s, 9H, 3ꢃ
AdCH2 þ ArCH3), 1.79 (s, 6H, 3ꢃ AdCH2), 1.08 (s, 9H, SiC(CH3)3), 0.34
(s, 6H, Si(CH3)2) ppm. 13C NMR (100.62 MHz, CDCl3):
d
153.7 (s),
Methyl
2-[50-(adamant-1-yl)-40-(tert-butyldimethylsilyloxy)-2-
139.2 (s), 135.3 (s), 130.8 (d, C6 or C3), 121.3 (d, C3 or C6), 115.6 (s),
40.3 (t, 3ꢃ AdCH2), 37.0 (t, 3ꢃ AdCH2), 36.6 (s, AdC), 29.0 (d, 3ꢃ
AdCH), 26.4 (q, 3ꢃ SiC(CH3)3), 22.4 (q, ArCH3), 18.9 (s, SiC(CH3)3),
chloro-20-methylbiphenyl-4-yl]acetate (21). General procedure for the
coupling of aryl triflates with arylboronic acids. A Schlenk tube was
charged with triflate 17 (0.05 g, 0.13 mmol), Pd(PPh3)4 (0.015 g,
0.013 mmol), arylboronic acid 13 (0.06 g, 0.19 mmol) and Et3N
(0.07 mL, 0.523 mmol) in DMF (0.4 mL). The reaction mixture was
thoroughly degassed with three freeze–thaw cycles, placed in
a 90 ꢀC oil bath and stirred for 23 h at 90 ꢀC. After cooling down to
room temperature, water was added and the mixture was extracted
with AcOEt (3ꢃ). The combined organic extracts were washed with
NH4Cl, H2O, brine, dried (Na2SO4) and the solvent was evaporated.
Purification by column chromatography on silica gel (98:2 hexane/
AcOEt) provided 21 (0.05 g, 78%) as a yellow oil. 1H NMR
-3.4 (q, 2ꢃ Si(CH3)2) ppm. IR (NaCl):
n 2972 (m, C–H), 2900 (m, C–
H), 1746 (m), 1701 (s), 1684 (m), 1650 (m), 1555 (m), 1538 (s), 1520
(s), 1512 (s), 1455 (m), 1392 (m) cmꢁ1. MS (EIþ): m/z (%) 437
([M þ 1]þ
[
81Br], 9), 436 (Mþ
[
81Br], 32), 435 ([M þ 1]þ
[
79Br], 10),
434 (Mþ 79Br], 32), 380 (14), 379 (47), 378 (14), 377 (45), 298 (26),
[
259 (12), 257 (14), 257 (25), 177 (11), 135 (100), 79 (15), 73 (41).
HRMS: calcd for C23H3579BrOSi, 434.1641 and C23H3581BrOSi,
436.1620; found, 434.1639 and 436.1641.
5-(Adamant-1-yl)-4-(tert-butyldimethylsilyloxy)-2-methylpheny
lboronic acid (13). To a cooled (ꢁ78 ꢀC) solution of bromide 12
(1.40 g, 3.19 mmol) in THF (8 mL) was slowly added n-BuLi (2.3 mL,
(400.13 MHz, CDCl3):
d
7.39 (s, 1H, H3), 7.22 (br, 2H, H6 þ H5), 6.97
(s,1H, H60 or H30), 6.70 (s, 1H, H30 or H60), 3.75 (s, 3H, COOCH3), 3.65

2442
S. Pe´rez-Rodr´ıguez et al. / European Journal of Medicinal Chemistry 44 (2009) 2434–2446
(s, 2H, CH2COOCH3), 2.11 (s, 6H, 3ꢃ AdCH2), 2.06 (s, 6H, 3ꢃ
AdCH þ ArCH3), 1.77 (br, 6H, 3ꢃ AdCH2), 1.08 (s, 9H, SiC(CH3)3), 0.39
89 ([C4H9O2], 100). HRMS: calcd for C30H3735ClO5, 512.2330 and
C30H3737ClO5, 514.2300; found, 521.2330 and 514.2303.
(s, 6H, Si(CH3)2) ppm. 13C NMR (100.62 MHz, CDCl3):
d
171.5 (s,
2-[50-(Adamant-1-yl)-2-chloro-40-[(2-methoxyethoxy)methoxy]-20-
methylbiphenyl-4-yl]acetic Acid (24). General procedure for hydrolysis
of esters. To a stirred suspension of 23 (0.02 mg, 0.03 mmol) in
MeOH (0.5 mL) was added Na2CO3 (0.14 mL, 2 M in H2O,
0.28 mmol). The mixture was stirred at 70 ꢀC for 3 h, cooled down
to room temperature, acidified (10% HCl), and extracted with EtOAc.
The combined organic extracts were washed (H2O, brine), dried
(Na2SO4), and evaporated. The residue was purified by column
chromatography on silica gel (95:5 CH2Cl2/MeOH) to provide 12 mg
CO2Me), 154.0 (s), 139.7 (s), 136.5 (s), 134.0 (s, 2ꢃ), 133.8 (s), 131.8
(d), 130.9 (s), 130.0 (d), 128.6 (d), 127.3 (d), 120.1 (d), 52.2 (q,
CH2COOCH3), 40.6 (t, 3ꢃ AdCH2), 40.4 (t, CH2COOCH3), 37.0 (t, 3ꢃ
AdCH2), 36.5 (s, AdC), 29.0 (d, 3ꢃ AdCH), 26.4 (q, 3ꢃ, SiC(CH3)3),
19.4 (q, ArCH3), 18.9 (s, SiC(CH3)3), ꢁ3.3 (q, SiCH3), ꢁ3.4 (q, SiCH3)
ppm. IR (NaCl): n 2902 (s, C–H), 2851 (m, C–H), 1740 (m, C]O), 1715
(m), 1484 (m), 1452 (m), 1391 (m), 1252 (s), 1227 (m), 1151 (m), 1018
(m), 837 (s), 779 (s) cmꢁ1. MS (EIþ): m/z (%) 541 ([M þ 1]þ
[
37Cl],16),
540 (Mþ
[
37Cl], 46), 539 ([M þ 1]þ
[
35Cl], 43), 538 (Mþ
[
35Cl], 100),
(64%) of 24 as a colourless oil. 1H NMR (400.13 MHz, CD2Cl2):
d 7.40
483 (34), 482 (33), 481 (80), 459 (21), 299 (28), 242 (23), (23), 135
(45). HRMS: calcd for C32H4335ClO3Si, 538.2670 and C32H4337ClO3Si,
540.2641; found, 538.2676 and 540.2653.
(d, J ¼ 8.0 Hz, 1.4 Hz, 1H, H3), 7.24 (d, J ¼ 8.0 Hz, 1.4 Hz, 1H, H5), 7.20
(d, J ¼ 8 Hz, 1H, H6), 7.01 (s, 1H, H60 or H30), 6.95 (s, 1H, H30 or H60),
5.32 (s, 2H, OCH2O), 3.8–3.9 (m, 2H, O(CH2)2O), 3.70 (s, 2H,
CH2COOH), 3.5–3.6 (m, 2H, OCH2O(CH2)2OCH3), 3.36 (s, 3H, OCH3),
2.07 (m, 12H, 3ꢃ AdCH þ ArCH3 þ 3ꢃ AdCH2), 1.76 (br, 6H, 3ꢃ
Methyl
2-[50-(adamant-1-yl)-2-chloro-40-hydroxy-20-methyl-
biphenyl-4-yl]acetate (22). General procedure for the cleavage of silyl
ethers. To a stirred solution of 21 (0.12 g, 0.22 mmol) in THF
(0.21 mL) was added dropwise tetrabutylammonium fluoride
(0.25 mL, 1 M in THF, 0.25 mmol) at 0 ꢀC and the mixture was
stirred for 2.5 h at 0 ꢀC. The solvent was removed in vacuo and the
residue was purified by column chromatography on silica gel
(50:50 hexane/AcOEt) to afford 0.09 g (94%) of 22 as a yellow oil. 1H
AdCH2) ppm. 13C NMR (100.62 MHz, CD2Cl2):
d 175.2 (s, COOH),
156.6 (s), 140.3 (s), 135.3 (s), 134.7 (s), 134.2 (s), 132.5 (s), 132.4 (d),
130.7 (d), 128.7 (d), 128.3 (d), 116.3 (d), 94.0 (t, OCH2O(CH2)2OCH3),
136.4 (s), 72.2 (t, OCH2O(CH2)2OCH3), 68.7 (t, OCH2O(CH2)2OCH3),
59.2 (q, OCH2O(CH2)2OCH3), 41.4 (t, ArCH2COOCH3 þ 3ꢃ AdCH2),
37.6 (t, 3ꢃ AdCH2), 37.3 (s, AdC), 29.8 (d, 3ꢃ AdCH), 19.8 (q, ArCH3)
NMR (400.13 MHz, CDCl3):
d
7.39 (s, 1H, H3), 7.20 (br, 2H, H6 þ H5),
ppm. IR (NaCl): n 3400–2500 (br, O–H), 2905 (s, C–H), 2851 (s, C–H),
6.95 (s, 1H, H60 or H30), 6.57 (s, 1H, H30 or H60), 3.75 (s, 3H, CO2CH3),
3.66 (s, 2H, CH2CO2CH3), 2.11 (s, 6H, 3ꢃ AdCH2), 2.06 (s, 3H, ArCH3
or 3ꢃ AdCH), 2.04 (s, 3H, 3ꢃ AdCH or ArCH3), 1.77 (br, 6H, 3ꢃ
1713 (s, C]O), 1604 (m), 1485 (s), 1452 (s), 1260 (s), 1215 (s), 1100
(s), 1017 (s) cmꢁ1. MS (EIþ): m/z (%) 501 ([M þ 1]þ
[
37Cl], 2), 500 (Mþ
[
37Cl], 7), 499 ([M þ 1]þ
[ [
35Cl], 6), 498 (Mþ 35Cl], 18), 424 (10), 422
AdCH2) ppm. 13C NMR (100.62 MHz, CDCl3):
d
172.1 (s, COOMe),
(23), 89 ([C4H9O2], 100). HRMS: calcd for C29H3535ClO5, 498.2173
and C29H3537ClO5, 500.2144; found, 498.2178 and 500.2145.
2-[50-(Adamant-1-yl)-2-chloro-40-hydroxy-20-methylbiphenyl-4-yl]
acetic acid (25). According to general procedure for hydrolysis of
esters, treatment of compound 22 (0.02 g, 0.05 mmol) with Na2CO3
(0.24 mL, 2 M in H2O, 0.48 mmol) for 3 h at 70 ꢀC afforded, after
purification by column chromatography on silica gel (95:5 CH2Cl2/
MeOH), 11 mg (54%) of 25 as a white solid. 1H NMR (400.13 MHz,
153.9 (s), 139.6 (s), 134.6 (s, 2ꢃ), 133.9 (s, 2ꢃ), 133.5 (s), 131.8 (d),
131.2 (s), 130.0 (d), 128.6 (d), 127.4 (d), 117.9 (d), 52.2 (q,
CH2COOCH3), 40.7 (t, 3ꢃ AdCH2 þ CH2COOCH3), 37.0 (t, 3ꢃ AdCH2),
36.4 (s, AdC), 29.0 (d, 3ꢃ AdCH), 19.1 (q, ArCH3) ppm. IR (NaCl):
n
3100–2700 (br, O–H), 2901 (s, C–H), 2848 (m, C–H), 1716 (s, C]O),
1437 (m), 1397 (s), 1258 (s), 1219 (s), 1144 (s), 1011 (m), 850 (s), 832
(s) cmꢁ1. MS (EIþ): m/z (%) 427 ([M þ 1]þ
[ [
37Cl], 2), 426 (Mþ 37Cl],
38), 425 ([M þ 1]þ
[
35Cl], 31), 424 (Mþ
[
35Cl], 100), 367 (19), 330
CDCl3):
d
7.40 (s, 1H, H3), 7.21 (br, 2H, H6 þ H5), 6.94 (s, 1H, H60 or
(11).
H30), 6.58 (s, 1H, H30 or H60), 3.68 (s, 2H, CH2COOH), 2.10 (s, 6H, 3ꢃ
AdCH2), 2.05 (s, 3H, ArCH3 or 3ꢃ AdCH), 2.03 (s, 3H, 3ꢃ AdCH or
ArCH3),1.75 (br, 6H, 3ꢃ AdCH2) ppm. 13C NMR (100.62 MHz, CDCl3):
Methyl
2-[50-(adamant-1-yl)-2-chloro-40-[(2-methoxyethox-
y)methoxy]-20-methylbiphenyl-4-yl]acetate (23). General procedure
for the protection of phenols with MEMCl. To a cooled (0 ꢀC)
suspension of 22 (0.12 g, 0.22 mmol) in DMF (1.5 mL) under Ar was
added NaH (0.004 g, 60% in mineral oil, 0.10 mmol). After 0.5 h at
0 ꢀC MEMCl (0.011 mL, 0.10 mmol) was added dropwise. The
reaction mixture was stirred for 4 h at room temperature, and was
extracted with ethyl acetate (3ꢃ). The combined organic extracts
were washed with brine, dried (Na2SO4) and evaporated. The
residue was subjected to column chromatography on silica gel
(50:50 hexane/CH2Cl2) to afford 23 (0.072 g, 64%) as a yellow oil. 1H
d
176.2 (s, CO2H), 153.9 (s), 139.8 (s, ArC), 134.7 (s), 134.0 (s), 133.6
(s), 133.6 (s), 131.8 (d), 131.3 (s), 130.2 (d), 128.6 (d), 127.5 (d), 118.0
(s), 118.0 (d), 40.7 (t, 3ꢃ AdCH2), 40.3 (t, CH2COOH), 37.0 (t, 3ꢃ
AdCH2), 36.4 (s, AdC), 29.0 (d, 3ꢃ AdCH), 19.1 (q, ArCH3) ppm. IR
(NaCl):
n 3100–2700 (br, O–H), 2898 (m, C–H), 2847 (s, C–H), 1691
(s, C]O), 1417 (m), 1398 (s), 1292 (m), 1223 (m), 1146 (s), 1067 (m),
894 (m), 877 (s), 825 (m), 668 (m) cmꢁ1. MS (EIþ): m/z (%) 413
([M þ 1]þ
[
37Cl], 12), 412 (Mþ
[
37Cl], 40), 411 ([M þ 1]þ
[
35Cl], 34),
410 (Mþ 35Cl], 100), 355 (10), 353 (25), 316 (13). HRMS: calcd for
[
NMR (400.13 MHz, CD2Cl2):
d
7.39 (s, 1H, H3), 7.19 (br, 2H, H6 þ H5),
C25H2735ClO3, 410.1649 and C25H2737ClO3, 412.1619; found, 410.1630
and 412.1625.
7.04 (s, 1H, H60 or H30), 6.96 (s, 1H, H30 or H60), 5.37 (d, J ¼ 6.6 Hz,
1H, OCH2O), 5.33 (d, J ¼ 6.6 Hz, 1H, OCH2O), 3.90 (t, J ¼ 4.6 Hz, 2H,
OCH2O(CH2)2OCH3), 3.74 (s, 3H, COOCH3), 3.6–3.5 (m, 4H,
OCH2O(CH2)2OCH3 þ CH2COOMe), 3.42 (s, 3H, OCH2O(CH2)2OCH3),
2.08 (s, 9H, ArCH3 or 3ꢃ AdCH þ 3ꢃ AdCH2), 2.04 (s, 3H, 3ꢃ AdCH
or ArCH3), 1.75 (br, 6H, 3ꢃ AdCH2) ppm. 13C NMR (100.62 MHz,
Ethyl
6-[5-(Adamant-1-yl)-4-(tert-butyldimethylsilyloxy)-2-
methylphenyl]-5-chloro-2-naphthoate (26). In accordance with the
general procedure for the coupling of aryl triflates with arylbor-
onics acids, the reaction of triflate 14 (0.05 g, 0.13 mmol) with
arylboronic acid 13 (0.06 g, 0.19 mmol) gave, after purification by
column chromatography on silica gel (98:2 hexane/AcOEt), 0.06 g
(85%) of 26 as a white solid (m.p.: 177 ꢀC, CH2Cl2/hexane). 1H NMR
CD2Cl2):
d 171.5 (s, CO2Me), 156.0 (s), 139.7 (s), 135.7 (s), 134.7 (s),
134.2 (s), 133.8 (s), 132.0 (s), 131.7 (d), 130.1 (d), 128.2 (d), 127.4 (d),
115.7 (d), 93.5 (t, OCH2O(CH2)2OCH3), 71.7 (t, OCH2O(CH2)2OCH3),
67.8 (t, OCH2O(CH2)2OCH3), 59.0 (q, OCH2O(CH2)2OCH3), 52.2 (q,
ArCH2COOCH3), 40.9 (t, 3ꢃ AdCH2), 40.4 (t, ArCH2COOCH3), 37.1 (t,
3ꢃ AdCH2), 36.8 (s, AdC), 29.1 (d, 3ꢃ AdCH), 19.5 (q, ArCH3) ppm. IR
(400.13 MHz, CDCl3):
d
8.66 (s, 1H, H1), 8.44 (d, J ¼ 8.8 Hz, 1H, ArH)
8.22 (d, J ¼ 8.8 Hz, 1H, ArH), 7.90 (d, J ¼ 8.4 Hz, 1H, ArH), 7.47 (d,
J ¼ 8.4 Hz, 1H, ArH), 7.10 (s, 1H, H60 or H30), 6.79 (s, 1H, H30 or H60),
4.50 (q, J ¼ 7.0 Hz, 2H, CO2CH2CH3), 2.16 (s, 6H, 3ꢃ AdCH2), 2.10 (s,
3H, ArCH3 or 3ꢃ AdCH), 2.08 (s, 3H, 3ꢃ AdCH or ArCH3),1.79 (br, 6H,
3ꢃ AdCH2), 1.50 (t, J ¼ 7.0 Hz, 3H, CO2CH2CH3), 1.13 (s, 9H,
SiC(CH3)3), 0.44 (s, 6H, Si(CH3)2) ppm. 13C NMR (100.62 MHz,
(NaCl): n 2904 (s, C–H), 2851 (s, C–H), 1741 (s, C]O), 1605 (d), 1485
(m), 1450 (m), 1252 (m), 1217 (m), 1155 (s), 1102 (s), 1014 (s) cmꢁ1
.
MS (EIþ): m/z (%) 515 ([M þ 1]þ
[ [
37Cl], 1), 514 (Mþ 37Cl], 2), 513
([M þ 1]þ
[
35Cl], 22), 512 (Mþ
[
35Cl], 22), 438 (11), 436 (24), 303 (10),
CDCl3): d 166.4 (s, CO2Et), 154.2 (s), 140.8 (s), 136.7 (s), 133.8 (s),

S. Pe´rez-Rodr´ıguez et al. / European Journal of Medicinal Chemistry 44 (2009) 2434–2446
2443
133.2 (s), 132.6 (s), 131.6 (s), 130.8 (d), 130.7 (s), 129.9 (d), 128.4 (d),
128.1 (s), 127.5 (d), 126.4 (d), 125.2 (d), 120.2 (d), 61.2 (t,
COOCH2CH3), 40.5 (t, 3ꢃ AdCH2), 37.0 (t, 3ꢃ AdCH2), 36.6 (s, AdC),
29.0 (d, 3ꢃ AdCH), 26.4 (q, 3ꢃ SiC(CH3)3), 19.5 (q, ArCH3), 18.9 (s,
SiC(CH3)3), 14.4 (q, COOCH2CH3), -3.3 (q, SiCH3), -3.4 (q, SiCH3)
procedure for hydrolysis of esters, treatment of compound 28
(0.01 g, 0.02 mmol) with Na2CO3 for 3 h at 70 ꢀC afforded, after
purification, 6 mg (57%) of 29 as a colourless oil. 1H NMR
(400.13 MHz, CD2Cl2):
d
8.67 (s,1H, H1), 8.38 (d, J ¼ 8.8 Hz,1H, ArH),
8.38 (d, J ¼ 8.8 Hz, 1H, ArH), 8.16 (dd, J ¼ 8.8, 1.6 Hz, 1H, ArH), 7.89
(d, J ¼ 8.2 Hz, 1H, ArH), 7.37 (d, J ¼ 8.2 Hz, 1H, ArH), 7.00 (s, 1H, H60
or H30), 6.98 (s, 1H, H30 or H60), 5.30 (d, J ¼ 6.7 Hz, 1H, OCH2O), 5.26
(d, J ¼ 6.7 Hz, 1H, OCH2O), 3.81 (m, 2H, OCH2O(CH2)2OCH3), 3.53
(m, 2H, OCH2O(CH2)2OCH3), 3.30 (s, 3H, OCH2O(CH2)2OCH3), 2.04
(s, 6H, 3ꢃ AdCH2), 2.01 (s, 3H, ArCH3 or 3ꢃ AdCH), 1.97 (s, 3H, 3ꢃ
AdCH or ArCH3),1.69 (s, 6H, 3ꢃ AdCH2) ppm. 13C NMR (100.62 MHz,
ppm. IR (NaCl):
n 2902 (m, C–H), 2850 (m, C–H), 1711 (m, C]O),
1246 (s), 1192 (m), 1097 (m), 977 (m), 885 (m), 837 (s), 778 (s), 753
(m) cmꢁ1. MS (EIþ): m/z (%) 591 ([M þ 1]þ
[
37Cl], 17), 590 (Mþ
[
37Cl],
46), 589 ([M þ 1]þ
[ [
35Cl], 46), 588 (Mþ 35Cl], 100), 534 (13), 533
(34), 532 (34), 531 (75), 411 (20), 409 (22),135 (48). HRMS: calcd for
C36H4535ClO3Si, 588.2827 and C36H4537ClO3Si, 590.2797; found,
588.2831 and 590.2819.
CD2Cl2):
d 170.7 (s, CO2H), 156.5 (s), 141.6 (s), 136.4 (s), 135.0 (s),
Ethyl 6-[5-(Adamant-1-yl)-4-hydroxy-2-methylphenyl]-5-chloro-
2-naphthoate (27). According to the general procedure for the
cleavage of the silyl ether group, treatment of compound 26 (0.14 g,
133.9 (s), 133.2 (s), 133.1 (s), 132.9 (s), 132.2 (d), 131.0 (s), 130.4 (d),
128.3 (d), 128.2 (d), 127.1 (s), 125.7 (d), 116.2 (d), 93.8 (t, OCH2O),
72.1 (t, OCH2), 68.5 (t, OCH2), 59.1 (q, OCH3), 41.2 (t, 3ꢃ AdCH2), 37.4
(t, 3ꢃ AdCH2), 37.2 (s, AdC), 29.6 (d, 3ꢃ AdCH), 19.7 (q, ArCH3) ppm.
0.23 mmol) with
a solution of TBAF (0.25 mL, 1 M in THF,
0.25 mmol) afforded, after purification by column chromatography
IR (NaCl): n 3500–2500 (br, O–H), 2962 (s, C–H), 2905 (s, C–H), 2851
on silica gel (50:50 hexane/AcOEt), 0.07 g (68%) of 27 as a white solid
(m, C–H), 1693 (m, C]O), 1472 (w), 1414 (w), 1260 (s), 1096 (s), 1018
(m.p.: 201 ꢀC, CH2Cl2/hexane). 1H NMR (400.13 MHz, CDCl3):
d
8.34
(s), 800 (s) cmꢁ1. MS (EIþ): m/z (%) 537 ([M þ 1]þ
[
37Cl], 2), 536 (Mþ
(s, 1H, H1), 8.10 (d, J ¼ 8.8 Hz, 1H, ArH), 7.89 (d, J ¼ 8.8 Hz, 1H, ArH),
7.58 (d, J ¼ 8.0 Hz, 1H, ArH), 7.12 (d, J ¼ 8.0 Hz, 1H, ArH), 6.74 (s, 1H,
H60 or H30), 6.35 (s, 1H, H30 or H60), 4.18 (q, J ¼ 7.1 Hz, 2H,
CO2CH2CH3), 1.84 (s, 6H, 3ꢃ AdCH2), 1.74 (s, 6H, ArCH3 or 3ꢃ AdCH),
[
37Cl], 6), 535 ([M þ 1]þ
[ [
35Cl], 6), 534 (Mþ 35Cl], 15), 485 (13), 89
([C4H9O2]þ, 100). HRMS: calcd for C32H3535ClO5, 534.2173 and
C32H3537ClO5, 536.2144; found, 534.2164 and 536.2170.
6-[5-(Adamant-1-yl)-4-hydroxy-2-methylbiphenyl)]-4-chloro-2-
naphthoic acid (30). According to the general procedure for hydro-
lysis of esters, the treatment of compound 27 (0.04 g, 0.09 mmol)
with Na2CO3 for 3 h at 70 ꢀC afforded, after purification, 0.034 g
(85%) of 30 as a white solid (m.p.: 236 ꢀC, CH2Cl2/hexane). 1H NMR
1.46 (br, 6H, 3ꢃ AdCH2), 1.18 (t, J ¼ 7.1 Hz, 3H, CO2CH2CH3) ppm. 13
C
NMR (100.62 MHz, CDCl3):
d 166.7 (s, CO2Et), 154.2 (s), 140.7 (s),
134.4 (s), 133.8 (s), 132.6 (s), 131.7 (s), 131.0 (d), 130.8 (s), 129.8 (d),
128.3 (d), 128.0 (s), 127.7 (d), 126.5 (d), 125.2 (d), 118.2 (d), 61.4 (t,
COOCH2CH3), 40.7 (t, 3ꢃ AdCH2), 37.0 (t, 3ꢃ AdCH2), 36.4 (s, AdC),
29.0 (d, 3ꢃ AdCH), 19.1 (q, ArCH3), 14.3 (q, COOCH2CH3) ppm. IR
(400.13 MHz, CDCl3):
d
8.76 (s, 1H, H1), 8.47 (d, J ¼ 8.8 Hz, 1H, ArH),
8.27 (d, J ¼ 8.8 Hz, 1H, ArH), 7.93 (d, J ¼ 8.4 Hz, 1H, ArH), 7.46 (d,
J ¼ 8.4 Hz, 1H, ArH), 7.06 (s, 1H, H60 or H30), 6.64 (s, 1H, H30 or H60),
2.15 (s, 6H, 3ꢃ AdCH2), 2.07 (s, 6H, 3ꢃ AdCH þ ArCH3), 1.78 (br, 6H,
(NaCl): n 3400–3100 (br, O–H), 2901 (s, C–H), 2849 (m, C–H),1682 (s,
C]O),1609 (w),1471 (w),1402 (m),1359 (m),1293 (s),1227 (s),1149
(s), 1101 (s), 819 (s), 749 (s) cmꢁ1. MS (EIþ): m/z (%) 477 ([M þ 1]þ
3ꢃ AdCH2) ppm. 13C NMR (100.62 MHz, CDCl3):
d 171.7 (s, CO2H),
[
[
37Cl], 12), 476 (Mþ
[
37Cl], 40), 475 ([M þ 1]þ
[
35Cl], 36), 474 (Mþ
154.0 (s), 141.2 (s), 134.5 (s), 133.9 (s), 132.7 (s), 132.1 (d), 131.9 (s),
130.0 (d), 128.4 (d), 127.9 (d), 127.0 (s), 126.7 (d), 125.5 (d), 118.1 (d),
40.8 (t, 3ꢃ AdCH2), 37.0 (t, 3ꢃ AdCH2), 36.5 (s, AdC), 29.0 (d, 3ꢃ
35Cl], 100), 440 (27), 417 (13). HRMS: calcd for C30H3135ClO3,
474.1962 and C30H3137ClO3, 476.1932; found, 474.1947 and 476.1931.
Ethyl
6-[5-(Adamant-1-yl)-4-[(2-methoxyethoxy)methoxy]-2-
AdCH), 19.1 (q, ArCH3) ppm. IR (NaCl): n 3200–2500 (br, O–H), 2967
methylphenyl]-5-chloro-2-naphthoate (28). According to the general
procedure for the protection of phenols with MEMCl, compound 27
(0.02 g, 0.05 mmol) was treated with NaH (0.002 g, 60% in mineral
oil, 0.05 mmol) and MEMCl (0.010 mL, 0.05 mmol) to afford, after
purification by column chromatography on silica gel (50:50
hexane/CH2Cl2), 0.012 g (46%) of 28 as a colourless oil. 1H NMR
(m, C–H), 2901 (s, C–H), 2850 (m, C–H),1698 (s, C]O),1540 (s),1509
(s),1472 (m),1456 (m),1292 (s),1253 (s),1220 (m), 975 (m), 842 (m),
818 (m), 154 (m) cmꢁ1. MS (EIþ): m/z (%) 449 ([M þ 1]þ
[
37Cl], 11),
448 (Mþ
[
37Cl], 36), 477 ([M þ 1]þ
[ [
35Cl], 30), 446 (Mþ 35Cl], 100),
389 (20), 352 (11). HRMS: calcd for C28H2735ClO3, 446.1649 and
C28H2737ClO3, 448.1619; found, 446.1645 and 448.1626.
(400.13 MHz, CDCl3):
d
8.65 (s, 1H, H1), 8.43 (d, J ¼ 8.8 Hz, 1H, ArH),
(E)-Ethyl 3-[50-(Adamant-1-yl)-40-(tert-butyldimethylsilyloxy)-2-
chloro-20-methylbiphenyl-4-yl]acrylate (31). To a mixture of triflate
20 (0.10 g, 0.27 mmol) and Pd(PPh3)4 (0.03 g, 0.03 mmol) in DMF
(2.2 mL) in a Schlenk flask was added arylboronic acid 13 (0.10 g,
0.25 mmol), Na2CO3 (0.25 mL, 0.50 mmol) and LiCl (0.02 g,
0.56 mmol). The reaction mixture was stirred for 22 h at 80 ꢀC.
After cooling down to room temperature, water was added and the
mixture was extracted with EtOAc (3ꢃ). The combined organic
extracts were washed with NH4Cl, H2O and brine, dried (Na2SO4)
and evaporated. The residue was subjected to column chromatog-
raphy (C18–SiO2, CH3CN) to afford 31 (0.10 g, 67%) as a yellow oil. 1H
8.21 (d, J ¼ 8.8 Hz, 1H, ArH), 7.90 (d, J ¼ 8.4 Hz, 1H, ArH), 7.43 (d,
J ¼ 8.4 Hz, 1H, ArH), 7.12 (s, 1H, H60 or H30), 7.07 (s, 1H, H30 or H60),
5.43 (d, J ¼ 7.0 Hz, 1H, OCH2O), 5.38 (d, J ¼ 7.0 Hz, 1H, OCH2O), 4.89
(q, J ¼ 6.9 Hz, 2H, COOCH2CH3), 3.94 (t, J ¼ 4.4 Hz, 2H, OCH2O(-
CH2)2OCH3), 3.67 (t, J ¼ 4.4 Hz, 2H, OCH2O(CH2)2OCH3), 3.45 (s, 3H,
OCH2O(CH2)2OCH3), 2.13 (s, 6H, 3ꢃ AdCH2), 2.12 (s, 3H ArCH3 or 3ꢃ
AdCH), 2.07 (s, 3H, 3ꢃ AdCH or ArCH3), 1.78 (br, 6H, 3ꢃ AdCH2), 1.49
(t, J ¼ 6.9 Hz, 3H, COOCH2CH3) ppm. 13C NMR (100.62 MHz, CDCl3):
d
166.5 (s, CO2Et), 156.2 (s), 140.7 (s), 135.9 (s), 134.6 (s), 133.3 (s),
132.7 (s), 132.8 (s), 130.9 (d), 130.7 (s), 129.8 (d), 128.3 (s), 128.0 (d),
127.7 (d), 126.6 (d), 125.3 (d), 115.9 (d), 93.5 (t, OCH2O(CH2)2OCH3),
71.7 (t, OCH2), 67.9 (t, OCH2), 61.3 (t, OCH2), 59.1 (q, OCH2O(-
CH2)2OCH3), 40.9 (t, 3ꢃ AdCH2), 37.1 (t, 3ꢃ AdCH2), 36.9 (s, AdC),
29.1 (d, 3ꢃ AdCH), 19.6 (q, ArCH3), 14.4 (q, COOCH2CH3) ppm. IR
NMR (400.13 MHz, CDCl3):
d
7.67 (d, J ¼ 16.0 Hz, 1H, H3), 7.69 (s, 1H,
H3), 7.43 (d, J ¼ 8.0 Hz, 1H, H5 or H6), 7.28 (d, J ¼ 8.0 Hz, 1H, H6 or
H5), 6.97 (s, 1H, H60), 6.71 (s, 1H, H30), 6.48 (d, J ¼ 16.0 Hz, 1H, H2),
4.29 (q, J ¼ 7.2 Hz, 2H, COOCH2CH3), 2.12 (s, 6H, 3ꢃ AdCH2), 2.07 (s,
6H, 3ꢃ AdCH þ ArCH3), 1.77 (br, 6H, 3ꢃ AdCH2), 1.36 (t, J ¼ 7.2 Hz,
3H, COOCH2CH3), 1.09 (s, 9H, SiC(CH3)3), 0.40 (s, 6H, Si(CH3)2) ppm.
(NaCl):
n 2961 (m, C–H), 2902 (m, C–H), 2849 (m, C–H), 1691 (m,
C]O), 1257 (s), 1084 (s), 1013 (s), 793 (s) cmꢁ1. MS (EIþ): m/z (%)
565 ([M þ 1]þ
[
37Cl], 3), 564 (Mþ
[
37Cl], 9), 563 ([M þ 1]þ
[
35Cl], 9),
13C NMR (100.62 MHz, CDCl3):
d 166.7 (s, CO2Et),154.3 (s), 142.9 (d),
562 (Mþ
[
35Cl], 23), 489 ([M – C2H5O2]þ
[
35Cl], 21), 89 ([C4H9O2]þ,
142.8 (s), 136.7 (s), 134.6 (s), 134.5 (s), 133.9 (s), 132.2 (d), 130.6 (s),
128.7 (d), 128.4 (d), 125.9 (d), 120.3 (d), 119.2 (d), 60.6 (q,
COOCH2CH3), 40.6 (t, 3ꢃ AdCH2), 37.0 (t, 3ꢃ AdCH2), 36.6 (s, AdC),
29.0 (d, 3ꢃ AdCH), 26.4 (d, 3ꢃ SiC(CH3)3), 19.4 (q, ArCH3), 18.9 (s,
SiC(CH3)3), 14.3 (q, COOCH2CH3), ꢁ3.3 (q, SiCH3), ꢁ3.4 (q, SiCH3)
100). HRMS: calcd for C34H3935ClO5, 562.2486 and C34H3937ClO5,
564.2457; found, 562.2475 and 564.2469.
6-[5-(Adamant-1-yl)-4-[(2-methoxyethoxy)methoxy]-2-methylph
enyl]-5-chloro-2-naphthoic acid (29). According to the general

2444
S. Pe´rez-Rodr´ıguez et al. / European Journal of Medicinal Chemistry 44 (2009) 2434–2446
ppm. IR (NaCl):
n
2902 (s, C–H), 2852 (m, C–H), 1713 (s, C]O), 1484
J ¼ 1.6 Hz, 1H, H30), 7.50 (d, J ¼ 8.0, 1.6 Hz, 1H, H50), 7.28 (d,
J ¼ 8.0 Hz, 1H, H60), 7.02 (s, 1H, H600), 6.96 (s, 1H, H300), 6.50
(d, J ¼ 16.3 Hz, 1H, H2), 5.30–5.33 (m, 2H, OCH2O), 3.86 (t,
J ¼ 4.7 Hz, 2H, O(CH2)2O), 3.59 (t, J ¼ 4.7 Hz, 2H, O(CH2)2O), 3.36
(s, 3H, OCH2O(CH2)2OCH3), 2.09 (s, 6H, 3ꢃ AdCH2), 2.08 (s, 3H,
ArCH3 or 3ꢃ AdCH), 2.03 (s, 3H, 3ꢃ AdCH or ArCH3), 1.76 (s, 6H,
(m), 1453 (m), 1305 (m), 1254 (s), 1173 (s), 1152 (s), 872 (s), 835 (s),
779 (s) cmꢁ1. MS (EIþ): m/z (%) 557 ([M þ 1]þ
[
37Cl], 16), 566 (Mþ
[
37Cl], 44), 565 ([M þ 1]þ
[ [
35Cl], 43), 564 (Mþ 35Cl], 100), 510 (14),
509 (37), 508 (36), 507 (84), 387 (26), 385 (25), 299 (19), 135 (62).
HRMS: calcd for C34H4535ClO3, 564.2827 and C34H4537ClO3,
566.2797; found, 564.2869 and 566.2820.
3ꢃ AdCH2) ppm. 13C NMR (100.62 MHz, CDCl3):
d 170.7 (s, CO2H),
(E)-Ethyl 3-[50-(Adamant-1-yl)-2-chloro-40-hydroxy-20-methyl-
biphenyl-4-yl]acrylate (32). In accordance to the general procedure
for the cleavage of silyl ethers, compound 31 (0.10 g, 0.17 mmol)
was treated with a solution of TBAF (0.19 mL, 1 M in THF,
0.19 mmol) to afford, after purification by column chromatography
(C18–SiO2, CH3CN), 0.08 g (98%) of 32 as a yellow oil. 1H NMR
156.6 (s), 145.4 (d), 143.6 (s), 136.3 (s), 135.0 (s), 134.9 (s), 134.8
(s), 132.7 (d), 131.9 (s), 129.3 (d), 128.2 (d), 126.8 (d), 118.5 (d),
116.2 (d), 93.8 (t, OCH2O), 72.0 (t, O(CH2)2O), 68.5 (t, O(CH2)2O),
59.0 (q, OCH2O(CH2)2OCH3), 41.2 (t, 3ꢃ AdCH2), 37.4 (t, 3ꢃ
AdCH2), 37.2 (s, AdC), 30.1 (t), 29.6 (d, 3ꢃ AdCH), 19.6 (q, ArCH3)
ppm. IR (NaCl):
n 3100–2500 (br, O–H), 2960 (m, C–H), 2904 (s,
(400.13 MHz, CDCl3):
d
7.65 (d, J ¼ 16.1 Hz, 1H, H3), 7.62 (s, 1H, H30),
C–H), 1641 (d, C]O), 1564 (s, C]O), 1484 (m), 1456 (m), 1363 (s),
7.43 (d, J ¼ 7.6 Hz,1H, H50 or H60), 7.26 (s, 1H, H60 or H50), 6.95 (s,1H,
H600), 6.58 (s,1H, H300), 6.46 (d, J ¼ 16.1 Hz,1H, H2), 4.82 (br,1H, OH),
4.28 (q, J ¼ 7.2 Hz, 2H, COOCH2CH3), 2.11 (s, 6H, 3ꢃ AdCH2), 2.07 (s,
3H, ArCH3 or 3ꢃ AdCH), 2.05 (s, 3H, 3ꢃ AdCH or ArCH3),1.77 (br, 6H,
3ꢃ AdCH2), 1.35 (t, J ¼ 7.2 Hz, 3H, COOCH2CH3) ppm. 13C NMR
1100 (m), 1014 (s), 747 (s) cmꢁ1. MS (EIþ): m/z (%) 513 ([M þ 1]þ
[
[
37Cl], 21), 512 (Mþ
[
37Cl], 21), 511 ([M þ 1]þ
[
35Cl], 25), 510 (Mþ
35Cl], 40), 437 (16), 436 (18), 435 (38), 434 (20), 433 (21), 429
(16), 397 (11), 395 (12), 392 (13), 391 (40), 167 (22), 155 (38), 154
(100), 151 (22).
(100.62 MHz, CDCl3):
d
167.0 (s, CO2Et), 154.5 (s), 143.1 (d), 142.9 (s),
(E)-3-[50-(Adamant-1-yl)-2-chloro-40-hydroxy-20-methylbiphenyl
-4-yl]acrylic acid (35). According to the general procedure for
hydrolysis of esters, treatment of compound 32 (0.006 g,
0.013 mmol) with Na2CO3 (0.07 mL, 2 M in H2O, 0.13 mmol) for 3 h
at 70 ꢀC provided, after purification by column chromatography on
silica gel (70:30 AcOEt/hexane), 0.006 g (99%) of 35 as a colourless
134.6 (s), 134.4 (s), 133.7 (s), 132.2 (d), 130.6 (s), 128.7 (d), 128.2 (d),
126.0 (d), 119.1 (d), 118.1 (d), 60.8 (t, COOCH2CH3), 40.6 (t, 3ꢃ
AdCH2), 37.0 (t, 3ꢃ AdCH2), 36.4 (s, AdC), 29.0 (d, 3ꢃ AdCH), 19.1 (q,
ArCH3), 14.3 (q, CO2CH2CH3) ppm. IR (NaCl): n 3400–2400 (br, O–H),
2853 (m, C–H), 1717 (s, C]O), 1639 (s), 1606 (m), 1483 (s), 1390 (s),
1259 (s), 1174 (s), 873 (s), 838 (s) cmꢁ1. MS (EIþ): m/z (%) 453
oil. 1H NMR (400.13 MHz, CDCl3):
d
7.75 (d, J ¼ 16.0 Hz, 1H, H3), 7.64
([M þ 1]þ
[
37Cl], 11), 452 (Mþ
[
37Cl], 40), 451 ([M þ 1]þ
[
35Cl], 34),
(s, 1H, H3), 7.45 (d, J ¼ 8.0 Hz, 1H, H5 or H6), 7.27 (d, J ¼ 8.0 Hz, 1H,
H6 or H5), 6.94 (s, 1H, H60), 6.58 (s, 1H, H30), 2.11 (s, 6H, 3ꢃ AdCH2),
2.04 (s, 6H, 3ꢃ AdCH þ ArCH3), 1.76 (br, 6H, 3ꢃ AdCH2) ppm. 13C
450 (Mþ
[
35Cl], 100), 393 (18), 356 (11). HRMS: calcd for
C28H3135ClO3, 450.1962 and C28H3137ClO3, 452.1932; found,
450.1944 and 452.1934.
NMR (100.62 MHz, CDCl3):
d 170.9 (s, CO2H), 154.1 (s), 145.3 (d),
(E)-Ethyl
3-[50-(adamant-1-yl)-2-chloro-40-[(2-methoxyethox-
143.2 (s), 134.7 (s), 134.5 (s), 134.3 (s), 133.8 (s), 132.3 (d), 130.9 (s),
129.0 (d), 128.4 (d), 126.3 (d), 118.1 (d, 2ꢃ), 40.7 (t, 3ꢃ AdCH2), 37.0
(t, 3ꢃ AdCH2), 36.4 (s, AdC), 29.0 (d, 3ꢃ AdCH), 19.1 (q, ArCH3) ppm.
y)methoxy]-20-methylbiphenyl-4-yl]acrylate (33). According to the
general procedure for the protection of phenols with MEMCl,
compound 32 (0.009 g, 0.02 mmol) was treated with NaH (0.001 g,
60% in mineral oil, 0.04 mmol) and MEMCl (0.01 mL, 0.02 mmol) to
afford, after purification by column chromatography (C18–SiO2,
CH3CN), 0.01 g (97%) of 33 as a yellow oil. 1H NMR (400.13 MHz,
IR (NaCl): n 3500–2400 (br, O–H), 2905 (s, C–H), 2850 (s, C–H), 1689
(s, C]O), 1631 (s), 1401 (s), 1263 (s), 1213 (s), 1144 (s) cmꢁ1. MS
(EIþ): m/z (%) 425 ([M þ 1]þ
[ [
37Cl], 10), 424 (Mþ 37Cl], 38), 423
([M þ 1]þ
[ [
35Cl], 30), 422 (Mþ 35Cl], 100), 367 (10), 365 (26), 328
CDCl3):
d
7.66 (d, J ¼ 16.0 Hz, 1H, H3), 7.61 (s, 1H, H30), 7.42 (d,
(14). HRMS: calcd for C26H2735ClO3, 422.1649 and C26H2737ClO3,
424.1619; found, 422.1658 and 424.1638.
J ¼ 8.0 Hz, 1H, H50 or H60), 7.25 (d, J ¼ 8.0 Hz, 1H, H60 or H50), 7.06 (s,
1H, H600), 6.96 (s, 1H, H300), 6.46 (d, J ¼ 16.0 Hz, 1H, H2), 5.38 (d,
J ¼ 6.4 Hz, 1H, OCH2O), 5.34 (d, J ¼ 6.4 Hz, 1H, OCH2O), 4.28 (q,
J ¼ 7.2 Hz, 2H, COOCH2CH3), 3.90 (t, J ¼ 4.4 Hz, 2H, O(CH2)2O), 3.63
(t, J ¼ 4.4 Hz, 2H, O(CH2)2O), 3.42 (s, 3H, OCH2O(CH2)2OCH3), 2.09
(s, 9H, 3ꢃ AdCH2 þ ArCH3 or 3ꢃ AdCH), 2.05 (s, 3H, 3ꢃ AdCH or
ArCH3), 1.76 (br, 6H, 3ꢃ AdCH2), 1.35 (t, J ¼ 7.2 Hz, 3H, COOCH2CH3)
6.2. Biology
6.2.1. Cell proliferation assays
All cancer cell lines were obtained from ATCC and grown
following ATCC’s recommendations. AdArs were prepared in
DMSO as 4 mM stock solutions and diluted in basic DMEM as
20ꢃ dilutions.
ppm. 13C NMR (100.62 MHz, CDCl3):
d 166.7 (s, CO2Et), 156.2 (s),
142.8 (d), 142.7 (s), 135.8 (s), 134.7 (s), 134.6 (s), 134.5 (s), 132.1 (d),
131.6 (s), 128.7 (d), 127.9 (d), 126.0 (d), 119.3 (d), 115.8 (d), 93.4 (t,
OCH2O), 71.6 (t, O(CH2)2O), 67.9 (t, O(CH2)2O), 60.6 (t, COOCH2CH3),
59.0 (q, OCH2O(CH2)2OCH3), 40.8 (t, 3ꢃ AdCH2), 37.0 (t, 3ꢃ AdCH2),
36.8 (s, AdC), 29.0 (d, 3ꢃ AdCH), 19.5 (q, ArCH3 or COOCH2CH3), 14.3
Cell proliferation assays were performed in 96 well plates. PC-
3, SkOV3, A549 cells (3000 cells per well), MiaPaCa-2, BxPC-3
(4000 cells per well), T-47D, and MDA-MB-468 (5000 cells per
well) were seeded in complete growing medium the day before
treatment and allowed to attach. Medium was changed to 0.5%
FBS containing medium just before AdAr exposure. Jurkat T cells
(10,000 cells per well) were resuspended (105 cells/mL) in RPMI
supplemented with 0.5% FBS and seeded in 96 well plates
immediately before AdAr treatment. Cells were treated with
(q, COOCH2CH3 or ArCH3) ppm. IR (NaCl):
n 2902 (s, C–H), 2850 (s,
C–H), 1712 (s, C]O), 1639 (s), 1483 (m), 1452 (m), 1252 (m), 1256
(s), 1172 (s), 1152 (s), 1017 (s), 977 (s), 861 (s), 834 (s), 755 (s) cmꢁ1
.
MS (EIþ): m/z (%) 541 ([M þ 1]þ
[ [
37Cl], 3), 540 (Mþ 37Cl], 10), 539
([M þ 1]þ
[ [
35Cl], 30), 538 (Mþ 35Cl], 25), 464 (10), 462 (22),
89 ([C4H9O2] þ, 100). HRMS: calcd for C32H3935ClO5, 538.2486 and
C32H3937ClO5, 540.2457; found, 538.2480 and 540.2458.
increasing concentrations of the AdArs and, when indicated, 10 mL
of a 5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazo-
lium bromide (MTT) solution were added. After 4 h incubation at
37 ꢀC, 100
mL 10% SDS in 10 mM HCl were added to solubilise the
formazan crystals. The absorbance at 595 nm was measured in
a Molecular Devices SpectraMax reader and the background
obtained in the absence of cells was subtracted. The percentage of
cell viability was calculated with respect to controls (100%), which
were incubated with an equivalent amount of solvent DMSO
(E)-3-[50-(Adamant-1-yl)-2-chloro-40-[(2-methoxyethoxy)metho
xy]-20-methylbiphenyl-4-yl]acrylic acid (34). In accordance to the
general procedure for hydrolysis of esters, compound 33 (0.025 g,
0.046 mmol) was treated with Na2CO3 (0.23 mL, 2 M in H2O,
0.46 mmol) to provide, after purification by column chromatog-
raphy (C18–SiO2, CH3CN), 0.023 g (97%) of 34 as a yellow oil. 1H
NMR (400.13 MHz, CD2Cl2):
d
7.75 (d, J ¼ 16.0 Hz, 1H, H3), 7.67 (d,

S. Pe´rez-Rodr´ıguez et al. / European Journal of Medicinal Chemistry 44 (2009) 2434–2446
2445
(ꢄ0.1% v/v). All experiments were performed at least twice with
References
triplicate points.
[1] L. Altucci, H. Gronemeyer, Nat. Rev. Cancer 1 (2001) 181–193.
[2] P. Germain, P. Chambon, G. Eichele, R.M. Evans, M.A. Lazar, M. Leid, A.R. de
Lera, R. Lotan, D.J. Mangelsdorf, H. Gronemeyer, Pharmacol. Rev. 58 (2006)
712–725.
[3] P. Germain, P. Chambon, G. Eichele, R.M. Evans, M.A. Lazar, M. Leid, A.R. de
Lera, R. Lotan, D.J. Mangelsdorf, H. Gronemeyer, Pharmacol. Rev. 58 (2006)
760–772.
[4] V. Laudet, H. Gronemeyer, The Nuclear Receptor Facts Book, Academic Press,
San Diego, 2002.
[5] C.K. Glass, M.G. Rosenfeld, Genes Dev. 14 (2000) 121–141.
[6] P. Germain, L. Altucci, W. Bourguet, C. Rochette-Egly, H. Gronemeyer, Pure
Appl. Chem. 75 (2003) 1619–1664.
6.2.2. DEVDase measurement
To measure caspase activity we adapted a fluorescence enzy-
matic assay [27] into a 96 well plate format. Jurkat T cells (40,000
cells per well; 106 cells/mL) resuspended in RPMI containing 0.5%
FBS were seeded and immediately treated with AdArs. After 4 h
of treatment, cells were lysed by adding 10
(25 mM PIPES pH 7, 25 mM KCl, 5 mM EGTA, 1 mM DTT, 10
cytochalasin B, 0.5% NP-40, and a mixture of protease inhibitors
consisting of 1 mM PMSF, 1 g/mL leupeptine, and g/mL
aprotinin) followed by 30 min incubation at 4 ꢀC. Cell extracts
were then diluted with 50
L 2ꢃ caspase buffer (50 mM HEPES
pH 7.4, 100 mM NaCl, 1 mM EDTA, 5 mM DTT, 0.1% CHAPS and
10% sucrose) containing 100 M of Ac-DEVD-AFC (Enzyme System
m
L 5ꢃ CE buffer
mM
[7] M.A. Ortiz, Y. Bayon, F.J. Lopez-Hernandez, F.J. Piedrafita, Drug Resist. Updat. 5
(2002) 162–175.
[8] S. Castaigne, C. Chomienne, M.T. Daniel, P. Ballerini, R. Berger, P. Fenaux,
L. Degos, Blood 76 (1990) 1704–1709.
m
1 m
m
[9] J.E. Henney, JAMA 283 (2000) 1131.
[10] L.T. Farol, K.B. Hymes, Exp. Rev. Anticancer Ther. 4 (2004) 180–188.
[11] A. Bernard, J.M. Bernardon, C. Delescluse, B. Martin, M.C. Lenoir, J. Maignan,
B. Charpentier, W.R. Pilgrim, U. Reichert, B. Shroot, Biochem. Biophys. Res.
Commun. 186 (1992) 977–983.
[12] J. Buck, F. Grun, F. Derguini, Y. Chen, S. Kimura, N. Noy, U. Hammerling, J. Exp.
Med. 178 (1993) 675–680.
[13] I. Korichneva, U. Ha¨mmerling, J. Cell Sci. 112 (1999) 2521–2528.
[14] D. Delia, A. Aiello, L. Lombardi, P.G. Pellici, F. Grignani, F. Formelli, S. Menard,
A. Costa, U. Veronesi, M.A. Pierotti, Cancer Res. 53 (1993) 6036–6041.
[15] K.H. Chun, D.M. Benbrook, K.D. Berlin, W.K. Hong, R. Lotan, Cancer Res. 63
(2003) 3826–3832.
[16] F.J. Piedrafita, M.A. Ortiz, Curr. Cancer Ther. Rev. 2 (2006) 185–198.
[17] Z.-M. Shao, M.I. Dawson, X.S. Li, A.K. Rishi, M.S. Sheikh, Q.-X. Han, V. Ordonez,
B. Shroot, J.A. Fontana, Oncogene 11 (1995) 493–504.
[18] C.A. Hsu, A.K. Rishi, X. Su-Li, T.M. Gerald, M.I. Dawson, C. Schiffer, U. Reichert,
B. Shroot, G.C. Poirer, J.A. Fontana, Blood 89 (1997) 4470–4479.
[19] S.Y. Sun, P. Yue, R.A. Chandraratna, Y. Tesfaigzi, W.K. Hong, R. Lotan, Mol.
Pharmacol. 58 (2000) 508–514.
[20] Y. Bayon, M.A. Ortiz, F.J. Lopez-Hernandez, F. Gao, M. Karin, M. Pfahl,
F.J. Piedrafita, Mol. Cell Biol. 23 (2003) 1061–1074.
[21] F.J. Piedrafita, M. Pfahl, Mol. Cell Biol. 17 (1997) 6348–6358.
[22] P. Marchetti, N. Zamzami, B. Joseph, S. Schraen-Maschke, C. Mereau-Richard,
P. Costantini, D. Metivier, S.A. Susin, G. Kroemer, P. Formstecher, Cancer Res. 59
(1999) 6257–6266.
m
Products). Reactions were incubated at 37 ꢀC for 30 min, and the
release of free AFC was measured every 2 min at 510 nm emission
upon excitation at 390 nm, using a Victor2 microplate reader
(Perkin Elmer).
6.2.3. RAR/RXR transactivation profiling
Gal4-LBD fusion proteins contain residues 1-147 encoding the
DNA binding domain of Gal4 in pSG424 [87] fused to the LBDs of
hRAR
a
(residues 156–462), hRAR
b (residues 169–448), hRARg
(residues 218–467). CV-1 cells
(residues 178–454), or mRXR
(20,000 cells per well) were transfected in 96 well plates with 10 ng
of each of the Gal4-LBD expression vectors together with 100 ng
a
UAS-luciferase reporter and 25 ng
b-galactosidase expression
vector (pCH110) using Superfect transfection reagent. 16 h after
transfection, the medium was removed and fresh medium con-
taining 5% charcoal-treated FBS was added, followed by AdAr
stimulation for 6 h (RAR
a, RARb, RXRa) or 20 h (RARg). Cells were
[23] Y. Li, B. Lin, A. Agadir, R. Liu, M.I. Dawson, J.C. Reed, J.A. Fontana, F. Bost,
P.D. Hobbs, Y. Zheng, G.-Q. Chen, B. Shroot, D. Mercola, X. Zhang, Mol. Cell Biol.
18 (1998) 4719–4731.
[24] S.Y. Sun, P. Yue, G.S. Wu, W.S. El Deiry, B. Shroot, W.K. Hong, R. Lotan, Cancer
Res. 59 (1999) 2829–2833.
washed once with PBS, lysed and assayed for luciferase and
galactosidase activities using a Dual-Light chemiluminescent assay
(Tropix).
b
-
[25] A.S. Belzacq, C. El Hamel, H.L. Vieira, I. Cohen, D. Haouzi, D. Metivier,
P. Marchetti, C. Brenner, G. Kroemer, Oncogene 20 (2001) 7579–7587.
[26] F.J. Lopez-Hernandez, M.A. Ortiz, Y. Bayon, F.J. Piedrafita, Mol. Cancer Ther. 2
(2003) 255–263.
[27] F.J. Lopez-Hernandez, M.A. Ortiz, Y. Bayon, F.J. Piedrafita, Cell Death Differ. 11
(2004) 154–164.
[28] S.Y. Sun, P. Yue, W.K. Hong, R. Lotan, Cancer Res. 60 (2000) 7149–7155.
[29] S.Y. Sun, P. Yue, W.K. Hong, R. Lotan, Cancer Res. 60 (2000) 6537–6543.
[30] K.H. Chun, M. Pfahl, R. Lotan, Oncogene 24 (2005) 3669–3677.
[31] M.A. Ortiz, F.J. Lopez-Hernandez, Y. Bayon, M. Pfahl, F.J. Piedrafita, Cancer Res.
61 (2001) 8504–8512.
[32] J. Zhang, M.C. Reedy, Y.A. Hannun, L.M. Obeid, J. Cell Biol. 145 (1999) 99–108.
[33] I. Ponzanelli, M. Gianni, R. Giavazzi, A. Garofalo, I. Nicoletti,
U. Reichert, E. Erba, A. Rambaldi, M. Terao, E. Garattini, Blood 95
(2000) 2672–2682.
6.2.4. Molecular docking
The genetic algorithm [88] implemented in AutoDock [89] and
the human RXR
9-cis-retinoic acid ligand as the target protein were used to
generate different RXR -bound conformers of 35 by randomly
a (PDB code 1k74) [62] upon removal of the
a
changing torsion angles and the overall orientation of the
molecule. A volume for exploration was defined in the shape of
a
three-dimensional cubic grid (80 ꢃ 80 ꢃ 80 Å3) [90] with
a spacing of 0.3 Å that enclosed the residues that are known to
make up the agonist binding pocket. At each grid point, the
receptor’s atomic affinity potentials for carbon, oxygen, and
hydrogen atoms present in the studied ligands were pre-
calculated for rapid intra- and intermolecular energy evaluation
of each docked conformation.
[34] M. Sakaue, H. Adachi, M. Dawson, A.M. Jetten, Cell Death Differ. 8 (2001) 411–424.
[35] W.F. Holmes, D.R. Soprano, K.J. Soprano, J. Biol. Chem. 277 (2002) 45408–
45419.
[36] E. Garattini, E. Parrella, L. Diomede, M. Gianni’, Y. Kalac, L. Merlini, D. Simoni,
R. Zanier, F.F. Ferrara, I. Chiarucci, P. Carminati, M. Terao, C. Pisano, Blood 103
(2004) 194–207.
[37] M. Karin, Y. Cao, F.R. Greten, Z.W. Li, Nat. Rev. Cancer 2 (2002) 301–310.
[38] P. Lorenzo, R. Alvarez, M.A. Ortiz, S. Alvarez, F.J. Piedrafita, A.R. de Lera, J. Med.
Chem. 51 (2008) 5431–5440.
Acknowledgements
[39] L. Farhana, M.I. Dawson, J.A. Fontana, Cancer Res. 65 (2005) 4909–4917.
[40] F. Jin, X. Liu, Z. Zhou, P. Yue, R. Lotan, F.R. Khuri, L.W. Chung, S.Y. Sun, Cancer
Res. 65 (2005) 6354–6363.
[41] M.I. Dawson, P.D. Hobbs, V.J. Peterson, M. Leid, C.W. Lange, K.C. Feng, G. Chen,
J. Gu, H. Li, S.K. Kolluri, X. Zhang, Y. Zhang, J.A. Fontana, Cancer Res. 61 (2001)
4723–4730.
[42] R.G. Keedwell, Y. Zhao, L.A. Hammond, S. Qin, K.Y. Tsang, A. Reitmair, Y. Molina,
Y. Okawa, L.I. Atangan, D.L. Shurland, K. Wen, D.M. Wallace, R. Bird,
R.A. Chandraratna, G. Brown, Cancer Res. 64 (2004) 3302–3312.
[43] Y. Zhang, M.I. Dawson, R. Mohammad, A.K. Rishi, L. Farhana, K.C. Feng, M. Leid,
V. Peterson, X.K. Zhang, M. Edelstein, D. Eilander, S. Biggar, N. Wall, U. Reichert,
J.A. Fontana, Blood 100 (2002) 2917–2925.
This work was supported by funds from the Spanish MEC
(SAF04-07131, SAF2007-63880-FEDER to A.R. de L.; Juan de la
Cierva Contract to F.R.-B.), Xunta de Galicia (PGI-
DIT05PXIC31403PN, 08CSA052383PR from DXIþDþI, Con-
´
solidacion 2006/15 from DXPCTSUG to A.R. de L.), and NIH grant
´
´
(CA55681 to F.J.P.). We thank Dr. Efren Perez and Dr. Susana
Alvarez for the UVI2024 and UVI3003 ligands, M. Ptashne for the
pSG424 expression vector, and Ming Zhao for excellent technical
assistance.

2446
S. Pe´rez-Rodr´ıguez et al. / European Journal of Medicinal Chemistry 44 (2009) 2434–2446
[44] R. Cincinelli, S. Dallavalle, L. Merlini, S. Penco, C. Pisano, P. Carminati,
G. Giannini, L. Vesci, C. Gaetano, B. Illy, V. Zuco, R. Supino, F. Zunino, J. Med.
Chem. 46 (2003) 909–912.
[45] R. Cincinelli, S. Dallavalle, R. Nannei, S. Carella, D. De Zani, L. Merlini, S. Penco,
E. Garattini, G. Giannini, C. Pisano, L. Vesci, P. Carminati, V. Zuco, C. Zanchi,
F. Zunino, J. Med. Chem. 48 (2005) 4931–4946.
[46] Y. Zhang, M.I. Dawson, Y. Ning, L. Polin, R.E. Parchment, T. Corbett,
A.N. Mohammad, K.C. Feng, L. Farhana, A.K. Rishi, D. Hogge, M. Leid,
V.J. Peterson, X.K. Zhang, R. Mohammad, J.S. Lu, C. Willman, E. VanBuren,
S. Biggar, M. Edelstein, D. Eilender, J.A. Fontana, Blood 102 (2003) 3743–3752.
[47] M.I. Dawson, D.L. Harris, G. Liu, P.D. Hobbs, C.W. Lange, L. Jong, N. Bruey-
Sedano, S.Y. James, X.K. Zhang, V.J. Peterson, M. Leid, L. Farhana, A.K. Rishi,
J.A. Fontana, J. Med. Chem. 47 (2004) 3518–3536.
[68] Y.L. Guo, K. Baysal, B. Kang, L.J. Yang, J.R. Williamson, J. Biol. Chem. 273 (1998)
4027–4034.
[69] Y.L. Hu, S. Li, J.Y. Shyy, S. Chien, Am. J. Physiol. 277 (1999) H1593–H1599.
[70] W.H. Wang, G. Gregori, R.L. Hullinger, O.M. Andrisani, Mol. Cell Biol. 24 (2004)
10352–10365.
[71] S.K. Venugopal, J. Chen, Y. Zhang, D. Clemens, A. Follenzi, M.A. Zern, J. Biol.
Chem. 282 (2007) 31900–31908.
[72] W. Bourguet, V. Vivat, J.M. Wurtz, P. Chambon, H. Gronemeyer, D. Moras, Mol.
Cell 5 (2000) 289–298.
[73] K. Takamatsu, A. Takano, N. Yakushiji, K. Morishita, N. Matsuura,
M. Makishima, H.I. Ali, E. Akaho, A. Tai, K. Sasaki, H. Kakuta, ChemMedChem 3
(2008) 454–460.
[74] K. Takamatsu, A. Takano, N. Yakushiji, K. Morohashi, K. Morishita,
N. Matsuura, M. Makishima, A. Tai, K. Sasaki, H. Kakuta, ChemMedChem 3
(2008) 780–787.
[48] L. Farhana, M.I. Dawson, M. Leid, L. Wang, D.D. Moore, G. Liu, Z. Xia,
J.A. Fontana, Cancer Res. 67 (2007) 318–325.
[49] B. Charpentier, J.M. Bernardon, J. Eustache, C. Millois, B. Martin, S. Michel,
B. Shroot, J. Med. Chem. 38 (1995) 4993–5006.
[50] M.I. Dawson, Z. Xia, G. Liu, J.A. Fontana, L. Farhana, B.B. Patel, S. Arumugarajah,
M. Bhuiyan, X.K. Zhang, Y.H. Han, W.B. Stallcup, J. Fukushi, T. Mustelin, L. Tautz,
Y. Su, D.L. Harris, N. Waleh, P.D. Hobbs, L. Jong, W.R. Chao, L.J. Schiff, B.P. Sani, J.
Med. Chem. 50 (2007) 2622–2639.
[51] H. Gronemeyer, J.A. Gustafsson, V. Laudet, Nat. Rev. Drug Discov. 3 (2004) 950–964.
[52] A.R. de Lera, W. Bourguet, L. Altucci, H. Gronemeyer, Nat. Rev. Drug Discov. 6
(2007) 811–820.
[53] S. Kotha, K. Lahiri, D. Kashinath, Tetrahedron 58 (2002) 9633–9695.
[54] H. Chaumeil, S. Signorella, C. Le Drian, Tetrahedron 56 (2000) 9655–9662.
[55] A.G. Chittiboyina, M.S. Venkatraman, C.S. Mizuno, P.V. Desai, A. Patny,
S.C. Benson, C.I. Ho, T.W. Kurtz, H.A. Pershadsingh, M.A. Avery, J. Med. Chem.
49 (2006) 4072–4084.
[75] K.T. Liby, M.M. Yore, M.B. Sporn, Nat. Rev. Cancer 7 (2007) 357–369.
[76] M.F. Boehm, L. Zhang, L. Zhi, M.R. McClurg, E. Berger, M. Wagoner, D.E. Mais,
C.M. Suto, J.A. Davies, R.A. Heyman, J. Med. Chem. 38 (1995) 3146–3155.
[77] V. Vuligonda, S.M. Thacher, R.A. Chandraratna, J. Med. Chem. 44 (2001) 2298–
2303.
[78] S. Balasubramanian, R.A. Chandraratna, R.L. Eckert, Carcinogenesis 25 (2004)
1377–1385.
[79] D.L. Crowe, R.A. Chandraratna, Breast Cancer Res. 6 (2004) R546–R555.
[80] M.H. Rendi, N. Suh, W.W. Lamph, S. Krajewski, J.C. Reed, R.A. Heyman,
A. Berchuck, K. Liby, R. Risingsong, D.B. Royce, C.R. Williams, M.B. Sporn,
Cancer Res. 64 (2004) 3566–3571.
[81] K. Wu, E. DuPre, H. Kim, U. Tin, R.P. Bissonnette, W.W. Lamph, P.H. Brown,
Breast Cancer Res. Treat. 96 (2006) 147–157.
[82] G.R. Benoit, M. Flexor, F. Besancon, L. Altucci, A. Rossin, J. Hillion, Z. Balajthy,
L. Legres, E. Segal-Bendirdjian, H. Gronemeyer, M. Lanotte, Mol. Endocrinol. 15
(2001) 1154–1169.
[83] L. Altucci, A. Rossin, O. Hirsch, A. Nebbioso, D. Vitoux, E. Wilhelm, F. Guidez,
M. De Simone, E.M. Schiavone, D. Grimwade, A. Zelent, H. de The,
H. Gronemeyer, Cancer Res. 65 (2005) 8754–8765.
[56] R.W. Bates, T. Rama-Devi, Synlett (1995) 1151–1152.
[57] S.G. Cockerill, H.J. Easterfield, J.M. Percy, S. Pintat, J. Chem. Soc., Perkin Trans. 1
(2000) 2591–2599.
[58] W. Solodenko, U. Schoen, J. Messinger, A. Glinschert, A. Kirschning, Synlett
(2004) 1699–1702.
[59] K.M. Boatright, G.S. Salvesen, Biochem. Soc. Symp. (2003) 233–242.
[60] G. Benoit, L. Altucci, M. Flexor, S. Ruchaud, J. Lillehaug, W. Raffelsberger,
H. Gronemeyer, M. Lanotte, EMBO J. 18 (1999) 7011–7018.
[61] V. Nahoum, E. Perez, P. Germain, F. Rodriguez-Barrios, F. Manzo, S. Kammerer,
G. Lemaire, O. Hirsch, C.A. Royer, H. Gronemeyer, A.R. de Lera, W. Bourguet,
Proc. Natl. Acad. Sci. U.S.A. 104 (2007) 17323–17328.
[62] H.E. Xu, M.H. Lambert, V.G. Montana, K.D. Plunket, L.B. Moore, J.L. Collins,
J.A. Oplinger, S.A. Kliewer, R.T. Gampe jr., D.D. McKee, J.T. Moore, T.M. Willson,
Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 13919–13924.
[63] M.I. Dawson, W.H. Okamura, Chemistry and Biology of Synthetic Retinoids,
CRC Press, Boca Raton, 1990.
[84] V.R. Agarwal, E.D. Bischoff, T. Hermann, W.W. Lamph, Cancer Res. 60 (2000)
6033–6038.
[85] H.T. Kim, G. Kong, D. DeNardo, Y. Li, I. Uray, S. Pal, S. Mohsin, S.G. Hilsenbeck,
R. Bissonnette, W.W. Lamph, K. Johnson, P.H. Brown, Cancer Res. 66 (2006)
12009–12018.
[86] R. Epple, R. Russo, M. Azimioara, C. Cow, Y. Xie, X. Wang, J. Wityak,
D. Karanewsky, A. Gerken, M. Iskandar, E. Saez, H.M. Seidel, S.S. Tian, Bioorg.
Med. Chem. Lett. 16 (2006) 4376–4380.
[87] I. Sadowski, J. Ma, S. Triezenberg, M. Ptashne, Nature (London) 335 (1988)
563–564.
[88] G.M. Morris, D.S. Goodsell, R.S. Halliday, R. Huey, W.E. Hart, R.K. Belew,
A.J. Olson, J. Comput. Chem. 19 (1998) 1639–1662.
[89] G.M. Morris, D.S. Goodsell, R. Huey, W.E. Hart, S. Halliday, R. Belew, A.J. Olson,
Autodock, automated docking of flexible ligands to receptors. La Jolla, CA
92037-1000, 1999.
[64] M.B. Sporn, A.B. Roberts, D.S. Goodman, The Retinoids: Biology, Chemistry,
and Medicine, Raven Press, Ltd., New York, NY, 1994.
[65] P. Loeliger, W. Bollag, H. Mayer, Eur. J. Med. Chem. 15 (1980) 9–15.
[66] J.A. Fontana, A.K. Rishi, Leukemia 16 (2002) 463–472.
[67] M. Pfahl, F.J. Piedrafita, Oncogene 22 (2003) 9058–9062.
[90] P.J. Goodford, J. Med. Chem. 28 (1985) 849–857.