Retinoid receptor subtype-selective modulators through synthetic modifications of RARγ agonists

Article abstract of DOI:10.1016/j.bmc.2009.05.035

A series of retinoids designed to interfere with the repositioning of H12 have been synthesized to identify novel RARγ antagonists based on the structure of known RARγ agonists. The transcriptional activities of the novel ligands were revealed by cell-based reporting assays, using engineered cells containg RAR subtype-selective fusions of the RAR ligand-binding domains with the yeast GAL4 activator DNA-binding domain and the cognate luciferase reporter gene. Whereas none of the ligands exhibited features of a selective RARγ antagonist, some of them are endowed with interesting activities. In particular 24a acts as a pan-RAR agonist that induces at high concentration a higher transactivation potential on RARα than TTNPB and synergizes at low concentration with TTNPB-bound RARα but not RARβ or RARγ. Similarly, 24c synergizes with TTNPB-bound RARγ and exhibits RARα,β antagonist activity. Compounds 24b and 25b are strong RARα,β-selective antagonists without agonist or antagonist activities for RARγ. Compounds 24b and 24c display weak RXR antagonist activity. In addition several pan-antagonists and partial agonist/antagonists have been defined.

Full text of DOI:10.1016/j.bmc.2009.05.035

Bioorganic & Medicinal Chemistry 17 (2009) 4345–4359
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Retinoid receptor subtype-selective modulators through synthetic
modifications of RARc agonists
Susana Álvarez a, Rosana Álvarez a, Harshal Khanwalkar b, Pierre Germain b, Géraldine Lemaire b,
a,
Fátima Rodríguez-Barrios a, Hinrich Gronemeyer b, , Ángel R. de Lera
*
*
a Departamento de Química Orgánica, Universidade de Vigo, 36310 Vigo, Spain
b Department of Cancer Biology—Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC)/CNRS/INSERM/ULP, BP 163, 67404 Illkirch Cedex, C.U. de Strasbourg, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 December 2008
Revised 6 May 2009
Accepted 11 May 2009
Available online 18 May 2009
A series of retinoids designed to interfere with the repositioning of H12 have been synthesized to identify
novel RARc antagonists based on the structure of known RARc agonists. The transcriptional activities of
the novel ligands were revealed by cell-based reporting assays, using engineered cells containg RAR sub-
type-selective fusions of the RAR ligand-binding domains with the yeast GAL4 activator DNA-binding
domain and the cognate luciferase reporter gene. Whereas none of the ligands exhibited features of a
selective RAR
as a pan-RAR agonist that induces at high concentration a higher transactivation potential on RAR
TTNPB and synergizes at low concentration with TTNPB-bound RAR but not RARb or RAR . Similarly,
24c synergizes with TTNPB-bound RAR and exhibits RAR ,b antagonist activity. Compounds 24b and
25b are strong RAR ,b-selective antagonists without agonist or antagonist activities for RAR . Com-
c
antagonist, some of them are endowed with interesting activities. In particular 24a acts
Keywords:
Retinoid receptor subtypes
Antagonists
Arotinoids
Synthesis
a
than
a
c
c
a
a
c
pounds 24b and 24c display weak RXR antagonist activity. In addition several pan-antagonists and partial
Molecular modeling
agonist/antagonists have been defined.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
tion of the biological effects that are under the control of certain
RXR–NR heterodimers is increasingly recognized.10
Retinoids, that is, the natural and synthetic derivatives of vita-
min A, are powerful signaling molecules that exert their actions
on cell growth and differentiation, carcinogenesis, homeostasis
and development primarily through binding to the retinoid recep-
tors RARs and RXRs,1–3 members of the nuclear receptor superfam-
ily.4,5 Accumulating structural data has provided detailed
information on the binding characteristics of agonists, inverse ago-
nists, antagonists and partial agonists, to the point that retinoid
receptors are perhaps the best known members of the nuclear
receptor (NR) superfamily.6
RAR subtypes, expressed from three isotypic genes (
differ in only three residues of the ligand-binding pocket (LBP).11,12
RAR and RARb differ by just one residue in helix 3 (S232 in RAR
A225 in RARb), whereas RARb and RAR diverge in two residues in
helices H5 (I263 in RARb; M272 in RAR ) and H11 (V398 in RARb; A397
in RAR ). H3 and H5 interact with the polyene side-chain, whereas
a, b and c),
a
a;
c
c
c
H11 contacts the hydrophobic ring of the retinoid ligand.
Based on numerous structural and functional studies, general
guidelines for the design of RAR subtype-selective agonists and
antagonists have been established.12,6,13 RAR
c-selective retinoids
The retinoic acid receptors are important drug targets for cancer
therapy and prevention.7 All-trans-retinoic acid (ATRA), the natural
ligand for RAR is used as a differentiation agent against acute pro-
myelocytic leukemia. A synthetic RXR agonist LGD1069 (bexaro-
tene, TargretinÒ) has been approved for the treatment of
cutaneous T cell lymphoma8 and is undergoing clinical trials for
other indications.9 Moreover, the potential of RXR ligands (rexi-
noids) for the therapy of metabolic diseases through the modula-
exhibit the longest connectors between the hydrophobic and the
polar ends rings of the arotinoid (or aromatic retinoid) skeleton.
The size of the linker directs the hydrophobic ring into the corre-
sponding pocket close to H11 and this causes a decrease in the
affinity for RARa/b. Moreover, the RARc-specific Met272 which
shows a highly conserved conformation in all solved crystal struc-
tures, can engage in hydrogen bonding interactions with suitable
functional groups such as those of the RARc
-selective ligands 2,14
3,14 4,15 5,16 6,17 and 718 (see Fig. 1).6,19–21
The RARc subtype is highly expressed in the epidermis and has
* Corresponding authors. Tel.: +33 3 88 65 3473; fax: +33 3 88 65 3437 (H.G.),
tel.: +34 986 812316; fax: +34 986 811940 (A.D.L.).
E-mail addresses: hg@igbmc.u-strasbg.fr (H. Gronemeyer), qolera@uvigo.es (Á.R.
de Lera).
been shown to contribute to primitive endodermal differentiation
of F9 murine embryonal carcinoma cells.22 A partial23a and a selec-
tive antagonist of RARc
have been reported23b but the structure of
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.05.035

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S. Álvarez et al. / Bioorg. Med. Chem. 17 (2009) 4345–4359
CO2H
We report herein the synthesis and characterization of novel
retinoids derived from agonists 6–10 as well as their characteriza-
tion using transient transactivation assays. Unexpectedly, no selec-
CO2H
OH
tive RAR
c antagonist could be identified within the series of
1,
2
all-trans-retinoic acid
retinoids prepared. However, some of the analogues displayed sub-
type-selectivities ranging from pan-antagonists to partial agonists/
antagonists and ‘super-agonists’.
CO2H
2. Chemistry
3
HO
N
OH
The structure of the target arotinoids called for key disconnec-
tions involving the generation of the functional groups (chalcone/
allyl alcohol/dihydroisoxazole on the one hand, keto/hydrox-
yalkylamide on the other). The previously described lengthy syn-
thesis of 6-bromodihydronaphthalenone 17 that starts with the
Friedel–Crafts acylation of bromobenzene and succinic anhydride
1130 was followed (see Supplementary data). Clemensen reduction
(Zn, HgCl2, H2O, HCl, 100 °C) of ketone 12 afforded carboxylic acid
13 (60%), which was converted into the methyl ester 14 (p-TsOH,
MeOH, benzene, 90 °C, 80%) and the latter transformed into ter-
tiary alcohol 15 using MeMgBr in ether (Scheme 1).
CO2H
CO2H
HO
4, SR11254
5, CD1530
O
H
OH
H
N
N
5''
O
F
5''
5''
O
CO2H
F
CO2H
CO2H
6, BMS189961
7,
BMS190914
O
Friedel–Crafts alkylation and tetrahydronaphthalene construc-
tion providing 16 (79% yield) took place upon treating 15 with
polyphosphoric acid at 50 °C. Finally, a highly efficient (96%) ben-
zylic oxidation of 16 (CrO3, AcOH, Ac2O, benzene, 0 °C) afforded
OH
5''
CO2H
6-bromo-3,4-dihydro-4,4-dimethylnaphthalene-1(2H)-one
17.
8, CD666
9, Ch80
Addition of Grignard reagents PhMgBr and p-tolylMgBr to 17 pro-
vided benzyl alcohols 18a and b in 80% and 75% yield, respectively.
Likewise, addition of the lithium acetylide derived from ethynyl-
benzene produced propargylic alcohol 18c in 77% yield. These
alcohols were then efficiently (92–96%) dehydrated to dihydro-
naphthalenes 19a–c upon refluxing with p-TsOH in benzene. Bro-
mine–lithium exchange in 19a–c was followed by trapping the
organolithium with DMF to produce aldehydes 20a–c (72% yield),
which then were converted into ketones 22a–c in a two-step se-
quence involving addition of MeLi to 20a–c (77–88%) and oxida-
tion of the benzyl alcohols 21a–c with MnO2 under basic
conditions (87–99%).
O
N
5''
CO2H
10, MMI1391
Figure 1. RAR
c-selective retinoids (2–5, 6, 8), and RAR-pan agonists (1, 7, 9, 10).
The last five retinoids have been selected as leads for the design of RAR
c-selective
antagonists.
Chalcones 24a–c were acquired by the Claisen–Schmidt con-
densation of ketones 22a–c and ethyl 4-formylbenzoate 23 follow-
ing the protocol described for the parent system 9 (NaOH, MeOH,
25 °C).16 The basic reaction conditions produced the saponification
of the esters, and therefore the desired carboxylic acids 24a–c were
obtained as final products. Allyl alcohols 25a–c were obtained in
good yields by treatment of enones 24a–c with Luche’s reagent
(NaBH4, CeCl3ꢀ7H2O, MeOH).31 Finally, dihydroisoxazole deriva-
tives 26a–c were straightforwardly prepared by reaction of chal-
cones 24a–c with hydroxylamine (NH2OHꢀHCl, NaOAc, MeOH,
70 °C).
the latter was not disclosed. In order to have access to such a phar-
macologically relevant tool, we integrated the principles of nuclear
receptor antagonism24 within the structure of some of the best
characterized RAR
c
agonists.6,12,25 The simplest approach is to
place at appropriate positions of the agonist structures substitu-
ents that hinder H12 re-positioning. This in turn will impede the
release of co-repressors and/or the recruitment of co-activators,
two of the key processes linked to transcriptional activation.24,25
In the present study we have focused on RARc-selective ago-
nists with hydroxyalkylamide (BMS189961,17 6) and allyl alcohol
(CD666,16 8) functional groups and envisioned synthetic modifica-
tions of the hydrophobic region for attaching the ‘antagonist’ sub-
stituents. Through molecular modeling studies (vide infra) position
C500 of the tetramethyltetrahydronaphthalene core suggested itself
as optimal for attaching substituents oriented towards H12, an ap-
proach pioneered by chemists at Allergan and Bristol–Myers–
Squibb.26,17 Relative to more flexible groups, the rigidity of aryl
and alkynyl substituents enforce the directionality. By systemati-
cally increasing the length of these groups (phenyl, p-tolyl, phenyl-
ethynyl), we expected to monitor the transition from agonists to
antagonists, in the same manner described for RXR in our previous
studies.27 Along the synthetic sequence analogues of ketoamide 7
and chalcone 928 (the oxidized precursors of 6 and 8) having the
same substitution pattern at C500 were prepared. Finally, the reac-
tivity of chalcone 9 allows for further diversification, and the
C500-substituted dihydroisoxazole analogues of parent MMI1391
1029 were also prepared.
The synthesis of the analogues of selective RAR
c agonist
BMS189961 6 is depicted on Scheme 2. The capture of the organo-
lithium resulting from the bromine–lithium exchange reaction of
19a–c with dimethyl oxalate installed the
a-ketoester group of
27a–c (52–63%). Due to their instability, ketoesters 27a–c were
treated with LiOH to produce 28a–c in moderate to good yields
(75–83%).
In situ preparation of the corresponding acid chlorides using
doubly-distilled oxalyl chloride was followed by treatment with
methyl 4-amino-3-fluorobenzoate 32 (itself obtained from 3-flu-
oro-4-nitrotoluene 29 via 30 and 31 as described32) to afford the
ketoamides 33a–c in 51–55% yields. These were reduced with so-
dium borohydride to the corresponding hydroxyamides 35a–c
(80–84%). Lastly, esters 33a–c and 35a–c were converted into the
carboxylic acids 34a–c and 36a–c in excellent yields (89–98%)
using NaOH in MeOH.

S. Álvarez et al. / Bioorg. Med. Chem. 17 (2009) 4345–4359
4347
O
HO2C
Br
RO2C
Br
HO
Br
b
Br
a
d
e
f
O
O
12
13
14
, R= H
O
11
c
15
16
, R= Me
OH
Br
O
Br
X
g
h
j
k
HO
R
O
17
R
18
R
22
21
OHC
R
19
, X= Br
a, R = Ph
i
20
, X= CHO
b
, R = p-Tol
c, R = phenylethynyl
COOEt
l
n
23
O
O
N
OH
m
CO2H
CO2H
CO2H
R
R
R
24
26
25
Scheme 1. Reagents and conditions: (a) bromobenzene, AlCl3, 96 h, 93%; (b) Zn–Hg, HCl, H2O, reflux, 15 h, 60%; (c) p-TsOH, C6H6, MeOH, reflux, 18 h, 95%; (d) MeMgBr, Et2O,
24 h, 97%; (e) PPA, 50 °C, 9 h, 71%; (f) CrO3, Ac2O, AcOH, 5 h, 70%; (g) PhMgBr, THF, 20 h, 87%; p-TolMgBr, THF, 20 h, 80%; n-BuLi, ethynylbenzene, THF, 15 h, 77%; (h) p-TsOH,
C6H6, reflux, 2 h (19a, 96%; 19b, 94%; 19c, 92%); (i) t-BuLi, DMF, THF, ꢁ78 °C, 5 h (20a, 72%; 20b, 72%; 20c, 71%); (j) MeLi, THF, 0 °C (21a, 77%; 21b, 88%; 21c, 83%); (k) MnO2,
Na2CO3, CH2Cl2 (22a, 99%; 22b, 87%; 22c, 66%); (l) ethyl 4-formylbenzoate 23, NaOH, THF (24a, 63%; 24b, 72%; 24c, 68%); (m) NaBH4, CeCl3ꢀ7H2O, MeOH (25a, 81%; 25b, 92%;
25c, 90%); (n) NH2OHꢀHCl, NaOAc, MeOH, 70 °C (26a, 65%; 26b, 70%; 26c, 74%).
various compounds was compared with the activity of the pan-
RAR agonist TTNPB 37 and the RXR-agonist 9-cis-retinoic acid 38
(see Fig. 4) as positive controls for RAR (see Supplementary data)
and RXR (Fig. 4), respectively. Note that this reporter system is
insensitive to endogenous receptors, which cannot recognize the
GAL4-binding site.
3. Transcriptional activation studies
To evaluate the effects of the described retinoids on RARa,
RARb, RARc, and RXRb- mediated transactivation, a reporter assay
with genetically engineered HeLa cell lines33 that had been stably
cotransfected with a chimeric receptor construct and the cognate
reporter gene was used. Since the aminoacid residues that consti-
tute the ligand-binding pockets of the RXR subtypes do not differ,
the single RXRb reporter is assumed to reveal the ligand respon-
siveness as readout for all three RXRs. This reporter assay is based
on the generation of a fusion protein, which consists of the ligand-
binding domain of the corresponding receptor and the DNA-bind-
ing domain of the yeast GAL4 transcription factor. The cells also
contain a stably integrated luciferase reporter, which is controlled
by five Gal4 response elements in front of a b-globin promoter; this
is termed ‘(17m)5-bG-Luc’.34,35 The transcriptional activity of the
The analysis reveals a number of interesting features of some of
the new retinoids. It is remarkable that 24a and 24c act at high
concentration as highly potent agonists for RAR
a and RARc,
respectively, if compared with TTNPB (Fig. 2).36 This appears not
to be due to residual RXR agonist activity (which might have pos-
sibly originated from traces of endogenous RXR that is well-estab-
lished to synergize with retinoids in RAR–RXR heterodimers4,37), as
24c does not display any RXR agonist activity but rather has weak
RXR antagonist activity, which is even more pronounced for 24a
(see Supplementary data). While 25b has no significant RAR
c
O
O
Br
a
OMe
OH
b
O
O
R
R
R
19
27
28
a, R = Ph
F
R
b, R= p-Tol
F
R
e
c, R = phenylethynyl
f
O2N
H2N
32
33
29, R= CH3
c
d
30, R= CO2H
31, R= CO2Me
O
F
H
N
OH
F
H
h
N
O
CO2R
O
CO2R
R
35
, R= Me
, R= Me
R
g
g
36, R= H
3 , R= H
Scheme 2. Reagents and conditions: (a) t-BuLi, (MeOCO)2, THF, 10 h (27a, 52%; 27b, 59%; 27c, 63%); (b) LiOH, THF/H2O, 80 °C, 2 h (28a, 83%; 28b, 81%; 28c, 85%); (c) KMnO4,
H2O, 100 °C, 50%; (d) p-TsOH, MeOH, reflux, 16 h, 70%; (e) H2, Pd/C, 45 psi, 45 min, 75%; (f) (ClCO)2, CH2Cl2, DMF, Et3N, AcOEt, 80 °C (33a, 52%; 33b, 51%; 33c, 55%); (g) 10 N
NaOH, MeOH, 23 °C (34a, 90%; 34b, 91%; 34c, 89%; 36a, 98%; 36b, 90%; 36c, 91%); (h) NaBH4, MeOH, AcOEt (35a, 84%; 35b, 80%; 35c, 80%).

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S. Álvarez et al. / Bioorg. Med. Chem. 17 (2009) 4345–4359
Figure 2. Dose–response luciferase reporter cell assays (fold-induction) revealing the RAR subtype agonist activities of the retinoids 24–26. For details on the assay system,
see Section 5. The data are derived from at least three independent experiments; the standard deviations are indicated.
activity, it is a strong RAR
50% inhibition of 10nM TTNPB-induced activity at 400nM) and dis-
plays also significant RARb antagonist activity (RARb, IC50 = 2 M)
(Figs. and 3). Virtually the same is true for 24b (RAR
IC50 < 300nM; RARb, IC50 = 400nM). It is interesting to note from
the perspective of RAR , that 26c is a moderate (IC50 = 3 M) and
26a a weak (IC50 > 10 M) antagonist, while 26b is inactive.
The 34a,b,c series corresponds to pan-RAR antagonists (see
Supplementary data) which however display high RXR agonist
activity (see Supplementary data); in contrast 36a is a partial ago-
nist with lower activity than TTNPB (see Supplementary data) and
(similarly to 36c) a weak pan-RAR antagonist with no RXR agonist
activity detected. 36b is neither agonist nor antagonist and may
not significantly bind to RARs or RXR. In contrast to 36a analog
36c does not possess any significant RAR agonist activity (see Sup-
plementary data).
a
antagonist (RAR
a
, IC50 = 400nM, that is,
agonists and antagonists.12,6,13 For example, RAR
a agonists are
characterized by the presence of amide bonds in the linker be-
tween a hydrophobic ring and a benzoic acid terminus, which
are considered to participate in hydrogen bonding interactions
with the subtype-determining Ser232. In addition, small halogen
l
2
a,
c
l
l
atoms (F) at the ortho position of the benzoic acid improve RARa
selectivity. RARb selectivity38 is expected for retinoids with bulkier
hydrophobic rings capable of filling the enlarged ligand-binding
pocket between H5 and H10 that results from the orientation of
Ile263 away from the ligand, as observed in the crystal structure,
and is further increased by halogen atoms (Cl) at the meta position
of the benzoic acid.13 On the other hand, RAR
c selectivity is exhib-
ited by retinoids with longer (one additional atom) connectors be-
tween the hydrophobic and the polar ends of the skeleton in
particular if these functional groups have hydrogen bond donors.
The gain in RARc selectivity is considered to result from the hydro-
gen bonding interactions with the subtype-specific Met272 resi-
due, a structural feature that is highly conserved in all crystal
structures determined.6,19–21
Although an RAR
the structure of this compound has not been disclosed. Moreover,
4. Discussion
c
-selective antagonist has been described,23b
A great number of structural and functional studies have pro-
vided a series of guidelines for the design of RAR subtype-selective

S. Álvarez et al. / Bioorg. Med. Chem. 17 (2009) 4345–4359
4349
RARα
RARα
RARα
35
45
40
35
30
25
20
15
10
5
40
25a 25b 25c
26a 26b 26c
24a 24b 24c
30
25
20
15
10
5
35
30
25
20
15
10
5
0
0
0
Concentration [nM]
Conccentration [nM]
Concentration [nM]
RARβ
RARβ
β
RARβ
14
12
10
8
RAR
14
25a
25b
25c
26a 26b 26c
12
24a 24b 24c
12
10
8
10
8
6
6
6
4
4
4
2
2
2
0
0
0
Concentration [nM]
Concentration [nM]
Concentration [nM]
RARγ
RARγ
RARγ
RAR
25a 25b 25c
26a 26b 26c
8
7
6
5
4
3
2
1
0
10
24a 24b 24c
7
6
5
4
3
2
1
0
γ
RAR
9
8
7
6
5
4
3
2
1
0
12
10
8
β
24a 24b 24c
6
4
2
0
Concentration [nM]
Concentration [nM]
Concentration [nM]
Figure 3. Dose–response luciferase reporter cell assays revealing the RAR subtype antagonist activities of the retinoids 24–26. The agonist TTNPB (10 nM) (compound 37,
Fig. 4) was challenged with increasing amounts of the various retinoids and luciferase activity was determined. The decreased activity relative to TTNPB reveals antagonism.
For further details on the assay system, see Section 5. The data are derived from at least three independent experiments; the standard deviations are indicated.
no structure of an antagonist-bound complex with resolution at
the atomic level is currently available. Only the selective antago-
nist BMS195614 (39, Fig. 4) bound to RAR
a
when forming a hetero-
F318A bound
9
CO2H
dimer with the constitutively active mutant mRXR
a
to oleic acid (PDB code: 1dkf) has yielded a crystal structure.39
To aid the synthetic design this complex was used as a starting
structure for modeling the ligand-binding domain of the human
CO2H
RAR
c
-antagonist-bound conformation. The protocol is described
38 9-cis-retinoic acid
37 TTNPB (Ro13-7410)
in detail in Section 5. Docking known RAR
c-selective agonists
(BMS189961 6, CD666 8; see Fig. 1), in this model suggested that
the ‘antagonist’ substituent should be placed at the C500 position
of the tetrahydronaphthalene ring. Based on this assumption, a col-
lection of fifteen retinoids having in common an ‘antagonist’ sub-
stituent at C500 of increasing size and defined orientation (phenyl,
p-tolyl and 4-phenylethynyl) but differing in the nature of the con-
nector and polar groups were synthesized.
N
CO2H
O
N
H
Transient transactivation studies of these ligands afforded
activity profiles which are difficult to reconcile with the modeling
and design principles. First of all, none of the retinoids could be
39 BMS195,614
Figure 4. Structures of the RAR agonist (37) and RXR agonist (38) used as standards
in the transactivation assays, and the RAR -selective antagonist (39).
classified as RARc-selective antagonist. The greatest discrimination
a

4350
S. Álvarez et al. / Bioorg. Med. Chem. 17 (2009) 4345–4359
of RAR
26. Whereas 26a is a RAR
c
vs its paralogues was found for the dihydroisoxazole series
antagonist of moderate potency, 26c is a
c
stronger antagonist, whereas 26b is inactive. This selectivity is con-
sistent with molecular modeling studies in which analogue 26a
was docked into the antagonist-bound RARc structure constructed
by homology and then minimized. Since the ligand was synthe-
sized as a racemate, both enantiomers of 26a were docked into
the RARc binding pocket. Only the S enantiomer afforded reason-
able energy values upon minimization, and molecular dynamics
studies were carried on with this enantiomer. The agonist core of
(S)-26a is buried in a predominantly hydrophobic pocket corre-
sponding essentially to the all-trans-retinoic acid binding niche
in the holo-RAR
c
LBD structure.40,41 Residues located in H1, H3,
H5, the b turn, loop L6–7, and H11 form the agonist cavity. The
C500-phenyl moiety of (S)-26a, which is almost perpendicular to
the dihydronaphthalene part of the ligand, protrudes from the ago-
nist pocket between H12 (Leu411, Ile412), H11 (Ala394, Ala397),
L11–12 (Met408), and H3 (Trp227, Ser231, Ala234, Thr235,
Ile238). All these residues make van der Waals contacts with the
ligand, particularly with the phenyl group at C500. The resulting
model of RARc in complex with (S)-26a was refined using energy
minimization and its dynamic behavior was simulated using unre-
strained Molecular Dynamics to assess the feasibility of the pro-
posed complexes. As seen in Figure 5, the bulky phenyl group at
the C500 position is directed towards the H11–H12 region, thus sta-
bilizing the antagonist conformation of the receptor. Moreover, a
comparison between the reported crystal structure of RAR
BMS195614 (39) and the model of RAR -(S)-26a justifies the
experimentally determined moderate subtype selectivity of the
latter as a racemate. The small Ala397 group in RAR is replaced
and Val388 in RARb, which interact
a-
c
c
by the bulkier Val395 in RAR
a
with the added substituent at C500 (see Fig. 5). Nevertheless the
antagonist does not maintain the hydrogen bonding to the sub-
type-discriminating Met272 due to the different occupancy of the
binding pocket, and thus the RARc-subtype selectivity is eroded.
On the other hand, the ketoamides derived from 3-fluorobenzo-
ic acid (34a–c) proved to be pan-RAR-antagonists, a profile shared
by the corresponding hydroxyalkylamides although at higher con-
centrations. It is puzzling that the parent retinoid BMS189961 (6,
as a racemate) is a potent and selective RARc agonist, an activity
due to the (R)-enantiomer BMS270394,41 whereas its derivatives
36 are unselective antagonists.
The series of compounds 25 formally derive from the structure
Figure 5. Top: Minimized structure obtained after docking (S)-26a in the RARc-
binding pocket; the side chains of the aminoacids that make contact with the ligand
are highlighted. Bottom: Close-up view of the superimposed minimized structures
of allyl alcohol CD666 (8), another known RAR
pound 25b, with a p-tolyl group at C500 behaves as a dual RAR
RARb antagonist, with greater potency for the former, while devoid
of RAR agonistic activity. The same properties are exhibited by
c
agonist.42,20 Com-
a
and
of the complexes (S)-26a-RAR
c
(gray) and BMS614 (9)-RAR
a (green). The subtype-
specific Ala397 (RAR ) and Val395 (RAR
c
a
) residues at H11 are labeled.
c
the corresponding chalcone 24b with the same C500-substituent,
formally derived from pan-agonist Ch80 (9). Interestingly, the ana-
logues of 24b with phenyl (24a) and phenylethynyl (24c) groups
displayed the remarkable property of inducing higher maximum
transactivation at high concentration than the powerful pan-ago-
gand. In this case long-lived TTNPB-bound RAR
cient at coactivator recruitment, may take over the pre-established
complexes from the 24a-bound RAR , resulting in apparent ligand
a, which is less effi-
a
synergy on a single receptor species. Towards higher concentra-
tions of the strong agonist increasingly less TTNPB-bound recep-
tors are available such that only 24a-bound receptors drive the
recruitment process. Retinoid 24c shows the same property with
TTNPB-bound RARc, and moreover has RARa,b-antagonist activity.
This selectivity profile is unprecedented in retinoid receptor
nist TTNPB (37) in RARa and RARc, respectively. This could be ex-
plained by a scenario in which 24a has a lower affinity to RAR
a
than TTNPB but is more efficient at inducing a conformation that
allows coactivator recruitment and/or facilitates corepressor re-
lease. A similar rationale for 24c would explain its strong agonist
ligands.
activity for RARc at high concentrations. It is remarkable that these
We do not believe that the effects of chalcones 24 are due to the
synergistic activation of RXR.36 RXR ligands or rexinoids on their
own activate ‘permissive’ heterodimers (e.g., PPAR/RXR) but, due
to a phenomenon referred to as ‘RXR subordination’ generally
not ‘non-permissive’ heterodimers (e.g. RXR/RAR).35 Selective
RXR modulators, that is, rexinoid ligands that activate a particular
subset of heterodimers, have been described for PPAR and RAR het-
erodimers.36,6 Certain rexinoids act as synergists36 for transactiva-
two ligands at low concentration enhance the transactivation in-
duced by TTNPB for those receptors where they exhibit the en-
hanced agonist activity at high concentration. While we have no
immediate explanation for this phenomenon, we speculate that
at low concentration (300 nM in Fig. 3) they may be a coexistence
of TTNPB and 24a-bound RARa. The 24a-bound receptors may effi-
ciently recruit coactivator complexes but display a rapid release
from the target promoter due to high dissociation rate of the li-

S. Álvarez et al. / Bioorg. Med. Chem. 17 (2009) 4345–4359
4351
tion through RAR and enhance the cell differentiation induced by
synthetic retinoids.43–46 However, the synergistic activities of 24a
and 24c seem to be unrelated to RXR transactivation, since they be-
have rather as antagonists of RXR. More subtle and possibly kinetic
phenomena are likely operating in this case as suggested above.
The effect of ligands on the mechanism of transactivation must
in fact be analyzed in the context of the relative stability of the li-
gand–nuclear receptor complexes with their co-regulators (co-
repressors or co-activators). This complex is an ensemble of pro-
teins and enzymes that includes histone deacetylases and histone
acetyl transferases that in turn induces chromatin remodeling
and transcriptional activation.47
DEPT135 pulse sequences were used to aid in the assignment of sig-
nals in the 13C NMR spectra. Mass Spectra were obtained on a instru-
ment operating at 70 eV by electron ionization. FAB experiments
were performed on a instrument using 3-nitrobenzylalcohol or glyc-
erol as matrices.
5.2. 6-Bromo-4,4-dimethyl-1,2,3,4-tetrahydro-1-phenyl-(1H)-
naphthalen-1-ol (18a)
To a solution of 6-bromo-4,4-dimethyl-1,2,3,4-tetrahydro-(1H)-
naphthalen-1-one 1730 (1.5 g, 5.9 mmol) in THF (19 mL) was added
phenylmagnesium bromide (9.0 mL, 1 M in THF, 9.0 mmol) and the
mixture was stirred for 22 h at 25 °C. A 5% aqueous HCl solution
was added and the mixture was extracted with t-BuOMe (3ꢂ).
The combined organic layers were washed with H2O (3ꢂ) and
brine (3ꢂ) and dried (Na2S2O3) and the solvent was removed.
The residue was purified by chromatography (silica gel, 95:5 hex-
ane/AcOEt) to afford 1.71 g (87%) of a white solid identified as 18a.
Mp: 105 °C (Et2O/hexane). Elemental Anal. Calcd for C18H19BrO: C,
65.27; H, 5.78. Found: C, 65.09; H, 5.79. 1H NMR (400.16 MHz,
To conclude, strong agonists selective for the RAR
a and RARc
subtypes have been discovered from transactivation studies of a
collection of retinoids designed to acquire antagonistic properties
starting from the structure of RARc-selective agonists. Whereas
in general the synthetic retinoids displayed antagonistic profiles
with moderate or no selectivity, a series (24a–c) are endowed with
the ability to activate transcription through a specific RAR
RAR subtype at higher values than pan-agonist TTNPB (37). It is
interesting to note that the crystal structure of parent pan-agonist
chalcone Ch80 (9, Fig. 1)20 bound to RAR
reveals a s-trans confor-
a or
c
CDCl3):
d 7.54 (d, J = 2.0 Hz, 1H), 7.4–7.2 (m, 6H), 6.97 (d,
c
J = 8.4 Hz, 1H), 2.23 (ddd, J = 13.5, 10.1, 3.1 Hz, 1H), 2.12 (ddd,
J = 12.3, 8.2, 3.0 Hz, 1H), 1.88 (ddd, J = 13.3, 10.0, 2.9 Hz, 1H), 1.58
(ddd, J = 11.3, 8.2, 3.1 Hz, 1H), 1.40 (s, 3H), 1.28 (s, 3H) ppm. 13C
NMR (100.62 MHz, CDCl3): d 148.6 (s), 148.2 (s), 139.8 (s), 130.6
(d), 129.5 (d), 129.2 (d), 127.8 (d, 2ꢂ), 126.8 (d), 126.3 (d, 2ꢂ),
121.9 (s), 75.4 (s), 37.3 (t), 34.5 (t), 34.2 (s), 31.3 (q), 31.2 (q)
ppm. MS (EI+): m/z (%) 332 (M+, 16), 330 (M+, 16), 255 (13), 253
(25), 252 (100), 251 (13), 219 (10), 218 (45), 115 (12), 105 (10),
91 (11), 77 (23). HRMS (EI+): calcd for C18H1979BrO, 330.0619;
mation about the bond joining the carbonyl group to the double
bond.20 The increase in the number of rotatable bonds in the cen-
tral connector region of the retinoids 24, 25, 34 and 36 increases
the conformational freedom of the ligand relative to more rigid
analogues (such as, for example, compounds 2–5). The increase
in flexibility makes the computational-based prediction of the li-
gand activity less accurate, as shown in this work with the RARc-
inspired antagonists 24–26, 34 and 36. Computational chemists
are increasingly paying attention to the importance of the target
flexibility in the description of receptor-ligand interactions.48 At
present we are still facing the difficulty that the current structural
tools (X-ray crystallography, NMR spectroscopy and molecular
dynamics) still provide too limited information to accurately de-
scribe ligand–receptor complexes and their biological readouts
which adds uncertainty to the prediction of the properties (ago-
nists, antagonist) of ligands as exemplified by the present report.
found, 330.0633. IR (NaCl):
m .
3416 (w, OH), 2959 (w, C–H) cmꢁ1
UV (MeOH): kmax 230 nm.
5.3. 6-Bromo-4,4-dimethyl-1,2,3,4-tetrahydro-1-p-tolyl-(1H)-
naphthalen-1-ol (18b)
Following the above procedure, the reaction of 17 (1.5 g,
5.9 mmol) and p-tolylmagnesium bromide (9 mL, 1 M in THF,
8.8 mmol) in THF (19 mL) afforded, after purification by chroma-
tography (silica gel, 95:5 hexane/AcOEt), 1.63 g (80%) of a white so-
lid identified as 18b. Mp: 101 °C (Et2O/hexane). Elemental Anal.
Calcd for C19H21BrO: C, 66.09; H, 6.13. Found: C, 66.20; H, 6.15.
1H NMR (400.16 MHz, CDCl3): d 7.29 (dd, J = 8.5, 2.0 Hz, 1H), 7.19
(s, 1H), 7.17 (d, J = 8.3 Hz, 1H), 7.2–7.0 (m, 4H), 2.54 (s, 3H), 2.11
(ddd, J = 13.5, 9.1, 3.2 Hz, 1H), 2.09 (ddd, J = 12.1, 9.0, 3.1 Hz, 1H),
1.72 (ddd, J = 13.8, 9.2, 3.1 Hz, 1H), 1.47 (ddd, J = 12.3, 9.0, 3.2 Hz,
1H), 1.25 (s, 3H), 1.22 (s, 3H) ppm. 13C NMR (100.62 MHz, CDCl3):
d 145.6 (s), 145.5 (s), 143.8 (s), 137.0 (s), 131.7 (d), 131.4 (d), 128.8
(d, 2ꢂ), 128.7 (d), 126.8 (d, 2ꢂ), 120.2 (s), 76.0 (s), 37.9 (t), 35.1 (t),
34.3 (s), 31.8 (q), 31.7 (q), 21.4 (q). MS (EI+): m/z (%) 346 (M+, 2),
344 (M+, 2), 328 (52), 326 (58), 313 (26), 311 (31), 283 (48), 255
(42), 253 (48), 232 (100), 217 (53), 215 (51), 202 (50), 115 (18),
81 (46), 69 (53). HRMS (EI+): calcd for C19H2179BrO, 344.0776;
5. Experimental
5.1. General
Solvents were dried accordingto publishedmethodsand distilled
before use. HPLC grade solvents were used for the HPLC purification.
All other reagents were commercial compounds of the highest pur-
ity available. All reactions were carried out under argon atmosphere,
and those not involving aqueous reagents were carried out in oven-
dried glassware. Analytical thin layer chromatography (TLC) was
performed on aluminium plates with Merck Kieselgel 60F254 and
visualized by UV irradiation (254 nm) or by staining with an etha-
nolic solution of phosphomolibdic acid. Flash column chromatogra-
phy was carried out using Merck Kieselgel 60 (230–400 mesh) under
pressure. Infrared spectra were obtained on a 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 spectrometer at 400 MHz with residual protic solvent as the inter-
nal reference [CDCl3, dH = 7.26 ppm; CD2Cl2, dH = 5.30 ppm; CD3OD,
dH = 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 at 100 MHz,
with the central peak of CDCl3 (dC = 77.0 ppm), CD2Cl2
(dC = 54.2 ppm) or CD3OD (dC = 49.05 ppm) as the internal reference.
found, 344.0785. IR (NaCl):
m .
3432 (w, OH), 3058–2863 (s) cmꢁ1
UV (MeOH): kmax 232 nm.
5.4. 6-Bromo-4,4-dimethyl-1,2,3,4-tetrahydro-1-(2-
phenylethynyl)-(1H)-naphthalen-1-ol (18c)
To
9.5 mmol) in THF (60 mL) was added n-BuLi (6.5 mL, 1.45 M in
hexane, 9.46 mmol). After 30 min, solution of 17 (2.0 g,
a cooled (0 °C) solution of 1-ethynylbenzene (0.96 g,
a
7.88 mmol) in THF (40 mL) was added and the mixture was stirred
for 20 h at 25 °C. After cooling down to 0 °C, H2O was added and
the mixture was extracted with t-BuOMe (3ꢂ). The combined
organic layers were dried (Na2SO4) and the solvent was removed.

4352
S. Álvarez et al. / Bioorg. Med. Chem. 17 (2009) 4345–4359
The residue was purified by chromatography (silica gel, 95:5 hex-
ane/AcOEt) to afford 2.16 g (77%) of a white solid identified as 18c.
Mp: 106 °C (Et2O/hexane). Elemental Anal. Calcd for C20H19BrO: C,
67.61; H, 5.39. Found: C, 67.91; H, 5.69. 1H NMR (400.16 MHz,
CDCl3): d 7.73 (dd, J = 8.5, 1.0 Hz, 1H), 7.5–7.3 (m, 7H), 2.36 (ddd,
J = 10.5, 9.9, 2.9 Hz, 1H), 2.3–2.2 (m, 1H), 1.99 (ddd, J = 11.0, 9.3,
3.0 Hz, 1H), 1.87 (ddd, J = 7.0, 5.4, 3.1 Hz, 1H), 1.36 (s, 3H), 1.33
(s, 3H) ppm. 13C NMR (100.62 MHz, CDCl3): d 146.9 (s), 137.1 (s),
131.5 (d, 2ꢂ), 129.6 (d), 129.5 (d), 129.5 (d), 128.4 (d, 2ꢂ), 128.1
(d), 122.5 (s), 122.3 (s), 92.8 (s), 84.6 (s), 35.2 (t), 34.2 (t), 34.1
(s), 31.2 (q), 31.1 (q) ppm. MS (EI+): m/z (%) 356 (M+, 5), 354 (M+,
5), 338 (32), 336 (30), 323 (34), 321 (32), 243 (25), 242 (100),
241 (43), 240 (16), 239 (37), 228 (15), 226 (24), 215 (26), 115
(20). HRMS (EI+): calcd for C20H1979BrO, 354.0619; found,
gel, hexane), 1.54 g (92%) of a white solid identified as 19c. M.p.:
68 °C (Et2O/hexane). Elemental Anal. Calcd. for C20H17Br: C,
71.23; H, 5.08. Found: C, 71.80; H, 5.00. 1H NMR (400.16 MHz,
CDCl3): d 7.52 (d, J = 8.2 Hz, 1H), 7.78 (d, J = 7.7 Hz, 1H), 7.4–7.2
(m, 5H), 7.21 (s, 1H), 6.44 (t, J = 4.8 Hz, 1H), 2.29 (d, J = 4.8 Hz,
2H), 1.20 (s, 6H) ppm. 13C NMR (100.62 MHz, CDCl3): d 142.6 (s),
135.5 (d), 133.5 (s), 131.6 (d, 2ꢂ), 130.9 (d), 128.5 (d, 2ꢂ), 128.4
(d), 128.3 (d), 125.6 (d), 123.2 (s), 120.5 (s), 120.2 (s), 90.8 (s),
86.6 (s), 38.9 (t), 33.3 (s), 28.4 (q, 2ꢂ) ppm. MS (EI+): m/z (%) 338
(M+, 59), 336 (M+, 61), 323 (44), 322 (12), 321 (44), 295 (16), 292
(18), 257 (15), 243 (24), 242 (100), 241 (35), 229 (27), 68 (14).
HRMS (EI+): calcd for C20H1779Br, 336.0514; found, 336.0515. IR
(NaCl):
m
2961 (m, C–H) cmꢁ1. UV (MeOH): kmax 258, 267 nm.
354.0622. IR (NaCl):
m
3396 (w, OH), 3083 (s, C–H) cmꢁ1. UV
5.8. 7,8-Dihydro-8,8-dimethyl-5-phenylnaphthalene-2-
carbaldehyde (20a)
(MeOH): kmax 235 nm.
5.5. 6-Bromo-3,4-dihydro-4,4-dimethyl-1-phenylnaphthalene
(19a)
5.8.1. General procedure for formylation of organolithium
intermediates derived from arylbromides
To a cooled (ꢁ78 °C) solution of 19a (0.4 g, 1.53 mmol) in THF
(22 mL) was added t-BuLi (1.8 mL, 1.7 M in pentane, 3.06 mmol).
After 30 min, DMF (0.35 mL, 4.59 mmol) was added and the mix-
ture was stirred for a further 5 h at ꢁ78 °C. After cooling down to
ambient temperature, a saturated aqueous solution of NH4Cl was
added and the mixture was extracted with Et2O (3ꢂ). The combined
organic layers were dried (Na2SO4) and the solvent was removed.
The residue was purified by chromatography (silicagel, 95:5 hex-
ane/AcOEt) to afford 0.29 g (72%) of a yellow solid identified as
20a. Mp: 77 °C (Et2O/hexane). 1H NMR (400.16 MHz, CDCl3): d
9.99 (s, 1H), 7.90 (s, 1H), 7.61 (d, J = 7.9 Hz, 1H), 7.4–7.3 (m, 5H),
7.19 (d, J = 7.9 Hz, 1H), 6.18 (t, J = 4.6 Hz, 1H), 2.44 (d, J = 4.6 Hz,
2H), 1.42 (s, 6H) ppm. 13C NMR (100.62 MHz, CDCl3): d 191.9 (d),
145.8 (s), 140.0 (s), 139.7 (s), 139.0 (s), 135.3 (s), 130.0 (d), 128.5
(d, 2ꢂ), 128.3 (d), 128.3 (d, 2ꢂ), 127.3 (d), 126.4 (d), 124.5 (d),
38.7 (t), 33.6 (s), 27.9 (q, 2ꢂ) ppm. MS (EI+): m/z (%) 262 (M+, 36),
247 (19), 234 (49), 233 (34), 220 (20), 219 (100), 218 (32), 217
(11), 215 (11), 205 (13), 204 (41), 203 (28), 202 (28), 192 (10).
HRMS (EI+): calcd for C19H18O, 262.1358; found, 262.1352. IR
5.5.1. General procedure for the acid-catalyzed dehydration
To a solution of 18a (1.71 g, 5.16 mmol) in C6H6 (41 mL) placed
in a flask equiped with a Dean–Stark apparatus was added p-tolu-
enesulfonic acid (0.01 g, 0.052 mmol). After 2 h refluxing, H2O was
added and the mixture was extracted with Et2O (3ꢂ). The com-
bined organic layers were dried (Na2SO4) and the solvent was re-
moved. The residue was purified by chromatography (silica gel,
hexane) to afford 1.24 g (96%) of a white solid identified as 19a.
Mp: 71 °C (Et2O/hexane). 1H NMR (400.16 MHz, CDCl3): d 7.73 (d,
J = 1.7 Hz, 1H), 7.7–7.6 (m, 5H), 7.47 (dd, J = 8.3, 1.9 Hz, 1H), 7.14
(d, J = 8.3 Hz, 1H), 6.25 (t, J = 4.6 Hz, 1H), 2.60 (d, J = 4.6 Hz, 2H),
1.81 (s, 6H) ppm. 13C NMR (100.62 MHz, CDCl3): d 147.3 (s),
140.4 (s), 138.6 (s), 132.7 (s), 128.6 (d, 2ꢂ), 128.5 (d), 128.2 (d,
2ꢂ), 127.6 (d), 127.1 (d), 126.9 (d), 126.6 (d), 121.2 (s), 38.6 (t),
33.7 (s), 27.9 (q, 2ꢂ) ppm. MS (EI+): m/z (%) 314 (M+, 57), 312
(M+, 57), 299 (20), 297 (20), 270 (20), 268 (19), 233 (13), 219
(19), 218 (100), 217 (16), 215 (15), 203 (20), 202 (25). HRMS
(EI+): calcd for C18H1779Br, 312.0514; found, 312.0514. IR (NaCl):
m
2925 (m, C–H) cmꢁ1. UV (MeOH): kmax 263 nm.
(NaCl): m
3055 (w, C–H) cmꢁ1. UV (MeOH): kmax 234, 278 nm.
5.6. 6-Bromo-3,4-dihydro-4,4-dimethyl-1-p-tolylnaphthalene
5.9. 7,8-Dihydro-8,8-dimethyl-5-p-tolyl-naphthalene-2-
(19b)
carbaldehyde (20b)
Following the general procedure, the reaction of 18b (1.25 g,
3.62 mmol) and p-toluenesulfonic acid (0.008 g, 0.040 mmol) in
C6H6 (29 mL) afforded, after purification by chromatography (silica
gel, hexane), 1.12 g (94%) of a white solid identified as 19b. Mp:
80 °C (Et2O/hexane). Elemental Anal. Calcd. for C19H19Br: C,
69.73; H, 5.95. Found: C, 69.57; H, 5.92. 1H NMR (400.16 MHz,
CDCl3): d 7.37 (s, 1H), 7.2–7.1 (m, 5H), 6.81 (d, J = 8.6 Hz, 1H),
5.87 (t, J = 4.6 Hz, 1H), 2.30 (s, 3H), 2.23 (d, J = 4.6 Hz, 2H), 1.23
(s, 6H) ppm. 13C NMR (100.62 MHz, CDCl3): d 147.3 (s), 138.5 (s),
137.4 (s), 136.8 (s), 132.8 (s), 128.9 (d, 2ꢂ), 128.6 (d), 128.4 (d,
2ꢂ), 127.7 (d), 126.9 (d), 126.1 (d), 121.2 (s), 38.6 (t), 33.7 (s),
27.9 (q, 2ꢂ), 21.0 (q) ppm. MS (EI+): m/z (%) 328 (M+, 54), 326
(M+, 57), 313 (20), 311 (20), 284 (32), 283 (32), 247 (12), 233
(23), 232 (100), 217 (22), 215 (24), 202 (21). HRMS (EI+): calcd
Following the general procedure, the reaction of 19b (0.3 g,
0.92 mmol), t-BuLi (1.1 mL, 1.7 M in pentane, 1.84 mmol) and
DMF (0.21 mL, 2.76 mmol) in THF (13 mL) afforded, after purifica-
tion by chromatography (silica gel, 95:5 hexane/AcOEt), 0.18 g
(72%) of a white solid identified as 20b. Mp: 83 °C (Et2O/hexane).
Elemental Anal. Calcd for C20H19O: C, 86.92; H, 7.29. Found: C,
86.90; H, 7.29. 1H NMR (400.16 MHz, CDCl3): d 10.21 (s, 1H),
8.11 (d, J = 1.3 Hz, 1H), 7.83 (dd, J = 8.2, 1.6 Hz, 1H), 7.5–7.4 (m,
4H), 7.43 (d, J = 8.0 Hz, 1H), 6.38 (t, J = 4.6 Hz, 1H), 2.65 (s, 3H),
2.44 (d, 2H), 1.63 (s, 6H) ppm. 13C NMR (100.62 MHz, CDCl3): d
191.9 (d), 145.9 (s), 139.8 (s), 138.9 (s), 137.1 (s), 137.0 (s), 135.2
(s), 129.6 (d), 128.9 (d, 2ꢂ), 128.4 (d, 2ꢂ), 128.3 (d), 126.4 (d),
124.5 (d), 38.7 (t), 33.6 (s), 27.9 (q, 2ꢂ), 21.0 (q) ppm. MS (EI+):
m/z (%) 276 (M+, 100), 261 (34), 234 (10), 234 (21), 233 (49), 233
(55), 218 (27), 217 (15), 215 (18), 203 (13), 204 (19). HRMS (EI+):
for C19H1979Br, 326.0670; found, 326.0670. IR (NaCl):
m 2927 (m,
C–H) cmꢁ1. UV (MeOH): kmax 273 nm.
Calcd for C20H20O, 276.1514; found, 276.1507. IR (NaCl): m 3024
(w, C–H), 1696 (s, C@O) cmꢁ1. UV (MeOH): kmax 247 nm.
5.7. 6-Bromo-3,4-dihydro-4,4-dimethyl-1-(2-phenylethynyl)-
naphthalene (19c)
5.10. 7,8-Dihydro-8,8-dimethyl-5-(2-phenylethynyl)-
naphthalene-2-carbaldehyde (20c)
Following the general procedure, the reaction of 18c (1.78 g,
5.15 mmol) and p-toluenesulfonic acid (0.01 g, 0.051 mmol) in
C6H6 (41 mL) afforded, after purification by chromatography (silica
Following the general procedure, the reaction of 19c (0.3 g,
0.89 mmol), t-BuLi (1.0 mL, 1.7 M in pentane, 1.78 mmol) and

S. Álvarez et al. / Bioorg. Med. Chem. 17 (2009) 4345–4359
4353
DMF (0.20 mL, 2.67 mmol) in THF (13 mL) afforded, after purifica-
tion by chromatography (silica gel, 95:5 hexane/AcOEt), 0.18 g
(71%) of a white solid identified as 20c. Mp: 87 °C (Et2O/hexane).
Elemental Anal. Calcd for C21H18O: C, 88.08; H, 6.34. Found: C,
87.64; H, 6.33. 1H NMR (400.16 MHz, CDCl3): d 10.02 (s, 1H),
7.89 (d, J = 7.7 Hz, 1H), 7.87 (s, 1H), 7.78 (dd, J = 7.8, 1.5 Hz, 1H),
7.6–7.4 (m, 5H), 6.67 (t, J= 4.8 Hz, 1H), 2.45 (d, J = 4.8 Hz, 2H),
1.38 (s, 6H) ppm. 13C NMR (100.62 MHz, CDCl3): d 192.0 (d),
144.6 (s), 137.8 (d), 137.2 (s), 136.0 (s), 131.6 (d, 2ꢂ), 128.9 (d),
128.4 (d, 3ꢂ), 126.3 (d), 124.5 (d), 123.0 (s), 121.1 (s), 91.0 (s),
86.5 (s), 39.0 (t), 33.5 (s), 28.4 (q, 2ꢂ) ppm. MS (EI+): m/z (%) 286
(M+, 100), 272 (13), 271 (60), 244 (11), 243 (30), 243 (18), 242
(16), 241 (18), 239 (19), 229 (11), 228 (35), 115 (13) 91 (11). HRMS
(7 mL) afforded, after purification by chromatography (silica gel,
80:17:3 hexane/AcOEt/Et3N), 0.10 g (83%) of a yellow oil identified
as 21c. 1H NMR (400.16 MHz, CDCl3): d 7.71 (d, J = 7.9 Hz, 1H), 7.55
(dd, J = 7.9, 2.6 Hz, 1H), 7.5–7.2 (m, 6H), 6.49 (t, J = 4.9 Hz, 1H), 4.93
(q, J = 6.3 Hz), 2.66 (d, J = 6.7 Hz, 3H), 2.39 (d, J = 4.9 Hz, 2H), 1.33
(s, 6H) ppm. 13C NMR (100.62 MHz, (CD3)2CO): d 147.2 (s), 144.0
(s), 136.2 (d), 133.2 (d, 2ꢂ), 132.7 (s), 130.5 (d, 2ꢂ), 130.2 (s),
127.4 (d), 125.4 (d), 125.3 (d), 124.6 (d), 123.4 (s), 92.1 (s), 89.3
(s), 70.3 (d), 40.8 (t), 34.8 (s), 27.2 (q, 2ꢂ), 27.0 (q) ppm. MS
(EI+): m/z (%) 302 (M+, 19), 287 (18), 286 (18), 285 (22), 284 (72),
271 (23), 270 (26), 269 (100), 254 (49), 241 (70), 239 (55), 228
(28), 86 (34). HRMS (EI+): Calcd for C22H22O, 302.1671; found,
302.1670. IR (NaCl):
(MeOH): kmax 252, 323 nm.
m
3462 (br, OH), 2966 (s, C–H) cmꢁ1. UV
(EI+): Calcd for C21H18O, 286.1358; found, 286.1357. IR (NaCl):
m
3053 (w, C–H), 1698 (s, C@O) cmꢁ1. UV (MeOH): kmax 234, 277 nm.
5.14. 1-(7,8-Dihydro-8,8-dimethyl-5-phenyl-naphthalen-2-yl)-
ethan-1-one (22a)
5.11. 1-(7,8-Dihydro-8,8-dimethyl-5-phenyl-naphthalen-2-yl)-
ethan-2-ol (21a)
5.14.1. General procedure for the oxidation of benzylic alcohols
To a solution of 21a (0.071 g, 0.26 mmol) in CH2Cl2 (2 mL) were
sequentially added MnO2 (0.49 g, 4.68 mmol) and Na2CO3 (0.40 g,
4.68 mmol) and the reaction was stirred for 2 h. The mixture was
filtered through CeliteÒ and the solvent was removed. The residue
was purified by chromatography (silicagel, 90:7:3 hexane/AcOEt/
Et3N) to afford 0.071 g (99%) of a white solid identified as 22a.
Mp: 71 °C (Et2O/hexane). Elemental Anal. Calcd for C20H20O: C,
86.92; H, 7.28. Found: C, 86.64; H, 7.26. 1H NMR (400.16 MHz,
CDCl3): d 7.85 (dd, J = 8.1, 1.7 Hz, 1H), 7.65 (d, J = 1.5 Hz, 1H),
7.47 (d, J = 8.1 Hz, 1H), 7.4–7.3 (m, 5H), 6.06 (t, J = 4.6 Hz, 1H),
2.46 (s, 3H), 2.40 (d, J = 4.7 Hz, 2H), 1.38 (s, 6H) ppm. 13C NMR
(100.62 MHz, CDCl3): d 197.8 (s), 150.6 (s), 140.2 (s), 138.9 (s),
134.9 (s), 134.0 (s), 128.4 (d, 2ꢂ), 128.3 (d, 2ꢂ), 127.5 (d), 127.3
(d), 127.1 (d), 125.7 (d), 123.9 (d), 38.5 (t), 33.9 (s), 27.8 (q), 26.4
(q, 2ꢂ) ppm. MS (EI+): m/z (%) 276 (M+, 100), 262 (12), 261 (53),
234 (25), 233 (50), 219 (37), 218 (20), 217 (15), 215 (18), 204
(19), 203 (33), 202 (58), 191 (23), 189 (15). HRMS (EI+): Calcd for
5.11.1. General procedure for the synthesis of alcohols by the
addition of MeLi to aldehydes
To a solution of 20a (0.1 g, 0.38 mmol) in THF (6 mL) was added
MeLi (0.26 mL, 1.6 M in Et2O, 0.42 mmol) and the mixture was stir-
red for 2 h. An aqueous saturated NH4Cl solution was added, the
reaction was extracted with Et2O (3ꢂ), the combined organic lay-
ers were dried (Na2SO4) and the solvent was removed. The residue
was purified by chromatography (silicagel, 80:17:3 hexane/AcOEt/
Et3N), to afford 0.081 g (77%) of a white solid identified as 21a. Mp:
80 °C (Et2O/hexane). 1H NMR (400.16 MHz, CDCl3): d 7.44 (dd,
J = 8.3, 1.3 Hz, 1H), 7.4–7.3 (m, 6H), 7.06 (s, 1H), 6.03 (t,
J = 4.6 Hz, 1H), 4.77 (q, J = 6.4 Hz, 1H), 2.38 (d, J = 4.6 Hz, 2H),
1.43 (d, J = 6.5 Hz, 3H), 1.38 (s, 3H), 1.37 (s, 3H) ppm. 13C NMR
(100.62 MHz, CDCl3): d 144.5 (s), 143.2 (s), 140.9 (s), 139.3 (s),
133.9 (s), 128.6 (d, 2ꢂ), 128.3 (d, 2ꢂ), 128.1 (d), 126.8 (d), 124.4
(d), 124.0 (d), 123.4 (d), 70.3 (d), 38.9 (t), 33.5 (s), 28.2 (q), 28.2
(q), 24.9 (q) ppm. MS (EI+): m/z (%) 278 (M+, 22), 260 (21), 245
(66), 230 (17), 219 (100), 217 (14), 215 (21), 204 (23), 203 (17),
202 (21), 85 (11), 84 (18). HRMS (EI+): Calcd for C20H22O,
C20H20O, 276.1514; found, 276.1510. IR (NaCl):
m 2959 (w, C–H),
1682 (s, C@O) cmꢁ1. UV (MeOH): kmax 248 nm.
278.1671; found, 278.1676. IR (NaCl):
m
3060 (w, OH) cmꢁ1. UV
(MeOH): kmax 276 nm.
5.15. 1-(7,8-Dihydro-8,8-dimethyl-5-p-tolyl-naphthalen-2-yl)-
ethan-1-one (22b)
5.12. 1-(7,8-Dihydro-8,8-dimethyl-5-p-tolyl-naphthalen-2-yl)-
ethan-2-ol (21b)
Following the general procedure, the reaction of 21b (0.043 g,
0.15 mmol), MnO2 (0.29 g, 2.7 mmol) and Na2CO3 (0.23 g,
2.7 mmol) in CH2Cl2 (2 mL) afforded, after purification by chroma-
tography (silica gel, 90:7:3 hexane/AcOEt/Et3N), 0.038 g (87%) of a
white solid identified as 22b. Mp: 77 °C (Et2O/hexane). Elemental
Anal. Calcd for C21H22O: C, 86.85; H, 7.64. Found: C, 86.50; H,
7.65. 1H NMR (400.16 MHz, CDCl3): d 7.84 (dd, J = 8.1, 1.8 Hz,
1H), 7.67 (d, J = 1.8 Hz, 1H), 7.46 (d, J = 8.1 Hz, 1H), 7.3–7.2 (m,
4H), 6.04 (t, J = 4.7 Hz, 1H), 2.47 (s, 3H), 2.42 (s, 3H), 2.38 (d,
J = 4.7 Hz, 2H), 1.37 (s, 6H) ppm. 13C NMR (100.62 MHz, CDCl3): d
197.9 (s), 145.5 (s), 138.9 (s), 138.5 (s), 137.4 (s), 137.0 (s), 129.0
(d, 2ꢂ), 128.9 (d, 2ꢂ), 128.8 (s), 128.5 (d), 126.5 (d), 126.0 (d),
123.6 (d), 38.9 (t), 33.7 (s), 30.4 (q), 28.1 (q), 26.6 (q, 2ꢂ) ppm.
MS (EI+): m/z (%) 290 (M+, 100), 276 (17), 275 (41), 248 (20), 247
(92), 233 (52), 232 (19), 218 (21), 217 (20), 216 (19), 115 (40),
202 (37). HRMS (EI+): Calcd for C21H22O, 290.1671; found,
Following the general procedure, the reaction of 20b (0.14 g,
0.51 mmol) and MeLi (0.35 mL, 1.6 M in Et2O, 0.56 mmol) in THF
(8 mL) afforded, after purification by chromatography (silica gel,
80:17:3 hexane/AcOEt/Et3N), 0.13 g (88%) of a yellow oil identified
as 21b. 1H NMR (400.16 MHz, CDCl3): d 7.29 (d, J = 1.4 Hz, 1H), 7.2–
7.1 (m, 4H), 7.02 (dd, J = 7.9, 1.5 Hz, 1H), 6.94 (d, J = 7.9 Hz, 1H),
5.86 (t, J = 4.7 Hz, 1H), 4.81 (q, J = 6.4 Hz, 1H), 2.32 (s, 3H), 2.26
(d, J = 4.7 Hz, 2H), 1.43 (d, J = 6.4 Hz, 3H), 1.30 (s, 6H) ppm. 13C
NMR (100.62 MHz, CDCl3): d 154.4 (s), 144.8 (s), 138.9 (s), 137.9
(s), 136.6 (s), 133.2 (s), 128.8 (d, 2ꢂ), 128.4 (d, 2ꢂ), 126.1 (d),
125.7 (d), 122.5 (d), 120.8 (d), 70.5 (d), 38.8 (t), 33.7 (s), 28.1 (q,
2ꢂ), 24.9 (q), 21.1 (q) ppm. MS (EI+): m/z (%) 292 (M+, 23), 274
(85), 259 (100), 244 (33), 233 (43), 232 (20), 231 (79), 229 (35),
215 (31), 202 (26), 81 (24), 69 (49), 67 (12). HRMS (EI+): Calcd
for C21H24O, 292.1827; found, 292.1833. IR (NaCl):
m
3030 (w,
290.1670. IR (NaCl):
(MeOH): kmax 248 nm.
m
3023 (w, C–H), 1728 (s, C@O) cmꢁ1. UV
OH) cmꢁ1. UV (MeOH): kmax 272 nm.
5.13. 1-[7,8-Dihydro-8,8-dimethyl-5-(2-phenylethynyl)-
naphthalen-2-yl]-ethan-1-ol (21c)
5.16. 1-[7,8-Dihydro-8,8-dimethyl-5-(2-phenylethynyl)-
naphthalen-2-yl]-ethan-1-one (22c)
Following the general procedure, the reaction of 20c (0.12 g,
Following the general procedure, the reaction of 21c (0.1 g,
0.42 mmol) and MeLi (0.3 mL, 1.6 M in Et2O, 0.46 mmol) in THF
0.37 mmol), MnO2 (0.70 g, 6.7 mmol) and Na2CO3 (0.58 g,

4354
S. Álvarez et al. / Bioorg. Med. Chem. 17 (2009) 4345–4359
6.7 mmol) in CH2Cl2 (5 mL) afforded, after purification by chroma-
tography (silica gel, 90:7:3 hexane/AcOEt/Et3N), 0.096 g (86%) of a
white solid identified as 22c. Mp: 75 °C (Et2O/hexane). Elemental
Anal. Calcd for C22H20O: C, 87.96; H, 6.71. Found: C, 87.74; H,
6.67. 1H NMR (400.16 MHz, CDCl3): d 8.24 (d, J = 1.8 Hz, 1H), 7.80
(dd, J = 8.1, 1.9 Hz, 1H), 7.5–7.4 (m, 2H), 7.34 (d, J = 8.1 Hz, 1H),
7.3–7.2 (m, 3H), 6.47 (t, J= 4.9 Hz, 1H), 2.56 (s, 3H), 2.33 (d,
J = 4.9 Hz, 2H), 1.25 (s, 6H) ppm. 13C NMR (100.62 MHz, CDCl3): d
197.6 (s), 149.0 (s), 135.3 (s), 134.8 (d), 131.5 (s), 131.4 (d, 2ꢂ),
128.3 (d, 3ꢂ), 128.2 (d), 128.0 (d), 125.7 (s), 123.9 (d), 120.8 (s),
91.0 (s), 86.6 (s), 38.7 (t), 33.7 (s), 28.2 (q, 2ꢂ), 26.4 (q) ppm. MS
(EI+): m/z (%) 300 (M+, 100), 286 (22), 285 (94), 257 (38), 243
(29), 242 (46), 241 (29), 240 (18), 239 (38), 226 (29). HRMS (EI+):
5.19. 4-[(1E)-3-(7,8-Dihydro-8,8-dimethyl-5-(2-phenylethynyl)-
naphthalen-2-yl)-3-oxoprop-1-en-1-yl]-benzoic Acid (24c)
Following the general procedure, the reaction of 22c (0.050 g,
0.17 mmol), ethyl 4-formylbenzoate 23 (0.030 g, 0.17 mmol) and a
1 N aqueous NaOH solution (1 mL) in MeOH (1.5 mL) afforded, after
purification by chromatography (silicagel, 95:5 CH2Cl2/MeOH),
0.057 g (78%) of a yellow solid identified as 24c. Mp: 117 °C (AcOEt/
hexane). Elemental Anal. Calcd for C30H24O3ꢀH2O: C, 79.90; H, 5.82.
Found: C, 80.00; H, 5.50. 1H NMR (400.16 MHz, (CD3)2CO): d 8.1–
8.0 (m, 3H), 7.96 (d, J = 8.2 Hz, 2H), 7.89 (d, J = 15.7 Hz, 1H), 7.83 (d,
J = 8.2 Hz, 2H), 7.76 (d, J = 8.5 Hz, 2H), 7.71 (d, J = 15.6 Hz, 1H), 7.5–
7.2 (m, 3H), 6.55 (t, J = 4.8 Hz, 1H), 2.36 (d, J = 4.8 Hz, 2H), 1.25 (s,
6H) ppm. 13C NMR (100.62 MHz, (CD3)2CO): d 190.3 (s), 168.1 (s),
146.2 (s), 144.2 (d), 141.3 (s), 139.6 (s), 139.4 (d), 137.4 (s), 133.3
(d, 2ꢂ), 131.9 (d), 131.3 (d), 131.2 (d), 130.5 (d, 2ꢂ), 130.5 (d),
130.4 (d), 129.0 (d), 127.6 (d), 126.2 (d), 125.9 (d), 124.9 (s), 122.7
(s), 92.6 (s), 88.3 (s), 40.6 (t), 35.2 (s), 29.6 (q, 2ꢂ) ppm. MS (EI+): m/
z (%) 432 (M+, 100), 422 (10), 418 (19), 417 (60), 408 (24), 390 (14),
389 (13), 257 (11), 175 (53), 103 (21). HRMS (EI+): Calcd for
Calcd for C22H20O, 300.1514; found, 300.1517. IR (NaCl):
m 3057
(w, C–H), 1682 (s, C@O) cmꢁ1. UV (MeOH): kmax 252 nm.
5.17. 4-[(1E)-3-(7,8-Dihydro-8,8-dimethyl-5-phenyl-
naphthalen-2-yl)-3-oxoprop-1-en-1-yl]-benzoic Acid (24a)
5.17.1. General procedure for the Claisen–Schmidt
condensation
C30H24O3, 432.1725; found, 432.1727. IR (NaCl):
m 3431 (br, OH),
To a solution of 22a (0.035 g, 0.13 mmol) in MeOH (1.5 mL) was
added ethyl 4-formylbenzoate 23 (0.020 g, 0.13 mmol) and a 1 N
aqueous NaOH solution (0.8 mL). After stirring for 15 h, the solvent
was removed and a 10% aqueous HCl solution was added until
acidic pH. The mixture was extracted with CH2Cl2 (3ꢂ), the com-
bined organic layers were dried (Na2SO4) and the solvent was re-
moved. The residue was purified by chromatography (silicagel,
95:5 CH2Cl2/MeOH) to afford 0.034 g (63%) of a white solid identi-
fied as 24a. Mp: 106 °C (acetone/hexane). 1H NMR (400.16 MHz,
1690 (s, C@O), 1606 (s, C@O) cmꢁ1. UV (MeOH): kmax 299 nm.
5.20. rac-4-[(1E)-3-(7,8-Dihydro-8,8-dimethyl-5-phenylnaphtha-
len2-yl)-3-hydroxy-prop-1-en-1-yl]-benzoic Acid (25a)
5.20.1. General procedure for the Luche reduction
A solution of 24a (0.062 g, 0.15 mmol) and CeCl3ꢀ7H2O (0.051 g,
0.19 mmol) in MeOH (3 mL) was treated at 0 °C with NaBH4
(0.006 g, 0.15 mmol) for 1 h. A 10% aqueous HCl solution was
added, the mixture was extracted with CH2Cl2, the combined or-
ganic layers were dried with Na2SO4 and the solvent was removed.
Purification by column chromatography (silica gel, 90:10 CH2Cl2/
MeOH) afforded 0.050 g (81%) of a white solid identified as 25a.
Mp: 107 °C (AcOEt/hexane). Elemental Anal. Calcd for C28H26O3:
C, 81.92; H, 6.38. Found: C, 82.34; H, 6.73. 1H NMR (400.16 MHz,
(CD3)2CO): d 8.21 (s, 1H), 8.17 (d, J = 8.1 Hz), 8.1–7.8 (m, 6H),
7.82 (d, J = 15.6 Hz, 1H), 7.3–7.2 (m, 4H), 7.17 (d, J = 8.1 Hz, 1H),
6.21 (t, J = 4.5 Hz, 1H), 5.20 (s, 1H), 2.43 (d, J = 4.5 Hz, 2H), 2.41
(s, 3H), 1.43 (s, 6H) ppm. 13C NMR (100.62 MHz, (CD3)2CO): d
167.3 (s), 147.5 (s), 144.1 (d), 142.1 (s), 140.1 (s), 140.0 (s), 138.9
(s), 131.9 (d, 2ꢂ), 131.2 (d), 130.9 (d), 130.5 (s), 130.9 (s), 130.4
(d), 130.3 (d, 2ꢂ), 129.2 (d), 129.2 (d), 128.6 (d), 127.8 (d), 126.9
(d), 126.2 (d), 125.9 (d), 73.1 (d), 40.4 (t), 34.5 (s), 30.1 (q, 2ꢂ),
23.2 (q) ppm. MS (EI+): m/z (%) 410 (M+, 100), 409 (10), 408 (16),
406 (31), 380 (25), 379 (40), 360 (19), 318 (15), 317 (40), 247
(11), 215 (14), 202 (11), 175 (49), 103 (21). HRMS (EI+): Calcd for
(CD3)2CO):
d 8.17 (s, 1H), 8.11 (d, J = 8.2 Hz, 2H), 8.01 (d,
J = 15.7 Hz, 1H), 7.96 (d, J = 8.0 Hz, 2H), 7.84 (d, J = 15.7 Hz, 1H),
7.5–7.4 (m, 6H), 7.13 (d, J = 8.0 Hz, 1H), 6.19 (t, J = 4.7 Hz, 1H),
2.46 (d, J = 4.7 Hz, 2H), 1.43 (s, 6H) ppm. 13C NMR (100.62 MHz,
(CD3)2CO): d 190.2 (s), 167.3 (s), 147.5 (s), 144.0 (d), 142.2 (s),
141.0 (s), 140.0 (s), 138.9 (s), 133.3 (d, 2ꢂ), 131.9 (d), 131.2 (d),
130.9 (d), 130.5 (s), 130.4 (d, 2ꢂ), 130.3 (d, 2ꢂ), 129.3 (d), 129.2
(s), 128.5 (d), 127.8 (d), 127.7 (d), 126.9 (d), 40.4 (t), 35.4 (s),
29.3 (q, 2ꢂ) ppm. MS (EI+): m/z (%) 408 (M+, 89), 407 (10), 394
(12), 393 (35), 379 (12), 366 (21), 365 (19), 202 (16), 180 (29),
175 (68), 131 (12), 131 (48), 118 (31), 103 (24). HRMS (EI+): Calcd
for C28H24O3, 408.1725; found, 408.1718. IR (NaCl):
m 3028 (br,
OH), 1687 (s, C@O), 1606 (s, C@O) cmꢁ1. UV (MeOH): kmax 319 nm.
5.18. 4-[(1E)-3-(7,8-Dihydro-8,8-dimethyl-5-p-tolyl-
naphthalen-2-yl)-3-oxoprop-1-en-1-yl]-benzoic Acid (24b)
Following the general procedure, the reaction of 22b (0.038 g,
0.13 mmol), ethyl 4-formylbenzoate 23 (0.021 g, 0.13 mmol) and a
1 N aqueous NaOH solution (1 mL) in MeOH (2 mL) afforded, after
purification by chromatography (silica gel, 95:5 CH2Cl2/MeOH),
0.040 g (72%) of a yellow solid identified as 24b. Mp: 115 °C
(AcOEt/hexane). Elemental Anal. Calcd for C29H26O3ꢀH2O: C, 79.07;
H, 6.41. Found: C, 78.60; H, 5.99. 1H NMR (400.16 MHz, (CD3)2CO):
d 8.19 (s, 1H), 8.15 (d, J = 8.1 Hz), 8.1–7.9 (m, 5H), 7.88 (d,
J = 15.7 Hz, 1H’), 7.3–7.2 (m, 4H), 7.19 (d, J = 8.1 Hz, 1H), 6.19 (t,
J = 4.6 Hz, 1H), 2.48 (d, J = 4.5 Hz, 2H), 2.43 (s, 3H), 1.46 (s, 6H) ppm.
13C NMR (100.62 MHz, (CD3)2CO): d 190.2 (s), 171.8 (s), 147.5 (s),
144.1 (d), 140.9 (s), 140.1 (s), 139.2 (s), 138.8 (s), 131.9 (d), 131.3
(d), 131.2 (d), 131.2 (s), 130.9 (d, 2ꢂ), 130.7 (d), 130.4 (d, 2ꢂ), 130.3
(d), 130.3 (d), 128.5 (s), 127.8 (s), 126.0 (d), 125.9 (d), 40.4 (t), 35.4
(s), 29.3 (q, 2ꢂ), 22.2 (q) ppm. MS (EI+): m/z (%) 422 (M+, 100), 408
(16), 407 (35), 380 (25), 379 (40), 360 (19), 318 (15), 317 (45), 247
(11), 215 (14), 202 (11), 175 (49), 103 (19). HRMS (EI+): Calcd for
C28H26O3, 410.1920; found, 410.2002. IR (NaCl):
m 3017 (br, OH),
1683 (s, C@O), 1606 (s, C@O) cmꢁ1. UV (MeOH): kmax 277 nm.
5.21. rac-4-[(1E)-3-(7,8-Dihydro-8,8-dimethyl-5-p-tolyl-
naphthalen-2-yl)-3-hydroxy-prop-1-en-1-yl]-benzoic Acid
(25b)
Following the general procedure, the reaction of 24b (0.040 g,
0.094 mmol) with CeCl3ꢀ7H2O (0.033 g, 0.12 mmol) and NaBH4
(0.003 g, 0.094 mmol) in MeOH (2 mL) afforded, after purification
by column chromatography (silica gel, 90:10 CH2Cl2/MeOH),
0.037 g (92%) of a yellow solid identified as 25b. Mp: 110 °C
(AcOEt/hexane). Elemental Anal. Calcd for C29H28O3: C, 82.05; H,
6.65. Found: C, 82.34; H, 6.73. 1H NMR (400.16 MHz, (CD3)2CO):
d 8.21 (s, 1H), 8.17 (d, J = 8.1 Hz’), 8.1–7.9 (m, 5H), 7.9 (d,
J = 15.6 Hz, 1H), 7.31 (s, 4H), 7.23 (d, J = 8.2 Hz, 1H), 6.21 (t,
J = 4.6 Hz, 1H), 5.32 (s, 1H), 2.48 (d, J = 4.5 Hz, 2H), 2.42 (s, 3H),
1.43 (s, 6H) ppm. 13C NMR (100.62 MHz, (CD3)2CO): d 168.1 (s),
C29H26O3, 422.1882; found, 422.1897. IR (NaCl):
m 3017 (br, OH),
1683 (s, C@O), 1606 (s, C@O) cmꢁ1. UV (MeOH): kmax 318 nm.

S. Álvarez et al. / Bioorg. Med. Chem. 17 (2009) 4345–4359
4355
152.6 (s), 143.9 (d), 141.3 (s), 140.8 (s), 139.4 (s), 138.8 (s), 137.7
(s), 136.3 (s), 133.7 (s), 131.9 (d, 2ꢂ), 131.3 (d, 2ꢂ), 130.9 (d),
130.3 (d, 2ꢂ), 130.1 (d), 128.7 (d), 128.6 (d), 127.6 (d), 126.5 (d),
126.1 (d), 72.1 (d), 40.4 (t), 35.7 (s), 29.2 (q, 2ꢂ), 22.2 (q) ppm.
MS (EI+): m/z (%) 424 (M+, 100), 423 (32), 422 (20), 408 (16), 406
(31), 380 (25), 379 (40), 360 (19), 318 (15), 317 (40), 247 (11),
215 (14), 202 (11), 175 (49), 103 (21). HRMS (EI+): Calcd for
(0.02 g, 0.21 mmol) in MeOH (2 mL) afforded, after purification
by column chromatography (silica gel, 90:10 CH2Cl2/MeOH),
0.048 g (93%) of a yellow solid identified as 26b. Mp: 113 °C
(AcOEt/hexane). 1H NMR (400.16 MHz, (CD3)2CO): d 8.20 (s, 1H),
8.16 (d, J = 8.1 Hz), 8.1–7.8 (m, 4H), 7.31 (s, 4H), 7.23 (d,
J = 8.1 Hz, 1H), 6.22 (t, J = 4.5 Hz, 1H), 5.03 (t, J = 4.5 Hz, 1H), 3.85
(m, 1H), 3.60 (m, 1H), 2.48 (d, J = 4.5 Hz, 2H), 2.42 (s, 3H), 1.45 (s,
6H) ppm. 13C NMR (100.62 MHz, (CD3)2CO): d 169.3 (s), 156.2 (s),
151.9 (s), 139.4 (s), 139.2 (s), 137.0 (s), 137.6 (s), 135.2 (s), 133.8
(s), 132.5 (s), 130.4 (d, 2ꢂ), 129.7 (d), 129.1 (d, 2ꢂ), 128.9 (d,
2ꢂ), 126.3 (d, 2ꢂ), 126.2 (d), 124.9 (d), 123.9 (d), 80.9 (d), 48.4
(s), 45.9 (t), 42.0 (t), 29.2 (q, 2ꢂ), 24.3 (q) ppm. MS (EI+): m/z (%)
437 (M+, 100), 423 (32), 422 (20), 408 (16), 406 (31), 380 (25),
379 (40), 360 (19), 318 (15), 317 (40), 247 (11), 215 (14), 202
(11), 175 (49), 103 (21). HRMS (EI+): Calcd for C29H27NO3,
C29H28O3, 424.2002; found, 424.2003. IR (NaCl):
m 3017 (br, OH),
1683 (s, C@O), 1606 (s, C@O) cmꢁ1. UV (MeOH): kmax 267 nm.
5.22. rac-4-[(1E)-3-(7,8-Dihydro-8,8-dimethyl-5-(2-
phenylethynyl)-naphthalen-2-yl)-3-hydroxyprop-1-
enyl]benzoic Acid (25c)
Following the general procedure, the reaction of 24c (0.040 g,
0.093 mmol) with CeCl3ꢀ7H2O (0.033 g, 0.12 mmol) and NaBH4
(0.003 g, 0.094 mmol) in MeOH (2 mL) afforded, after purification
by column chromatography (silica gel, 90:10 CH2Cl2/MeOH),
0.036 g (90%) of a yellow solid identified as 25c. M. p.: 109 °C
(AcOEt/hexane). Elemental Anal. Calcd for C29H28O3: C, 82.05; H,
6.65. Found: C, 82.34; H, 6.73. 1H NMR (400.16 MHz, (CD3)2CO):
d 8.1–7.9 (m, 3H), 7.98 (d, J = 8.1 Hz, 2H), 7.90 (d, J = 15.5 Hz, 1H),
7.82 (d, J = 8.2 Hz, 2H), 7.73 (d, J = 8.5 Hz, 2H), 7.69 (d, J = 15.6 Hz,
1H), 7.5–7.2 (m, 3H), 6.49 (t, J = 4.8 Hz, 1H), 5.20 (s, 1H), 2.36 (d,
J = 4.9 Hz, 2H), 1.23 (s, 6H) ppm. 13C NMR (100.62 MHz, (CD3)2CO):
d 168.1 (s), 146.2 (s), 144.2 (d), 141.3 (s), 139.6 (s), 139.4 (d), 133.3
(d, 2ꢂ), 132.0 (d, 2ꢂ), 131.3 (s), 131.2 (d), 130.5 (d, 2ꢂ), 130.5 (d),
130.4 (s), 129.0 (d), 127.6 (d), 126.2 (d), 125.9 (d), 125.0 (s), 122.7
(s), 91.4 (s), 87.2 (s), 73.1 (d), 40.6 (t), 34.3(s), 28.9 (q, 2ꢂ) ppm. MS
(EI+): m/z (%) 434 (M+, 100), 422 (10), 418 (19), 417 (60), 408 (24),
390 (14), 389 (13), 257 (11), 175 (53), 103 (21). HRMS (EI+): Calcd
437.2102; found, 437.2103. IR (NaCl):
m 3007 (br, OH), 1693 (s,
C@O), 1616 (s, C@O) cmꢁ1. UV (MeOH): kmax 260 nm.
5.25. rac-4-[3-(8,8-Dimethyl-5-(phenylethynyl)-7,8-dihydrona-
phthalen-2-yl)-4,5-dihydroisoxazol-5-yl]benzoic Acid (26c)
Following the general procedure, the reaction of 24c (0.040 g,
0.08 mmol) with NH2OHꢀHCl (0.007 g, 0.096 mmol) and NaOAc
(0.02 g, 0.14 mmol) in MeOH (1 mL) afforded, after purification
by column chromatography (silica gel, 90:10 CH2Cl2/MeOH),
0.020 g (93%) of a yellow solid identified as 26c. Mp: 113 °C
(AcOEt/hexane). 1H NMR (400.16 MHz, (CD3)2CO): d 8.1–7.9 (m,
3H), 7.98 (d, J = 8.1 Hz, 2H), 7.75 (d, J = 8.2 Hz, 2H), 7.73 (d,
J = 8.5 Hz, 2H), 7.5–7.2 (m, 3H), 6.49 (t, J = 4.8 Hz, 1H), 5.12 (t,
J = 4.6 Hz, 1H), 3.91 (m, 1H), 3.71 (m, 1H), 2.50 (d, J = 4.7 Hz, 2H’),
1.23 (s, 6H) ppm. 13C NMR (100.62 MHz, (CD3)2CO): d 169.3 (s),
156.2 (s), 151.9 (s), 141.3 (s), 139.6 (s), 133.5 (s), 133.3 (d, 2ꢂ),
131.4 (d, 2ꢂ), 128.5 (d, 3ꢂ), 128.3 (d), 127.0 (s), 126.2 (s), 124.9
(d), 123.9 (d), 122.7 (s), 93.1 (s), 89.2 (s), 80.9 (d), 47.5 (s), 45.1
(t), 42.0 (t), 29.2 (q, 2ꢂ) ppm. MS (EI+): m/z (%) 447 (M+, 100),
422 (10), 418 (19), 417 (60), 408 (24), 390 (14), 389 (13), 257
(11), 175 (53), 103 (21). HRMS (EI+): Calcd for C30H25NO3,
for C30H26O3, 434.1925; found, 434.1927. IR (NaCl):
m 3431 (br,
OH), 1690 (s, C@O), 1606 (s, C@O) cmꢁ1. UV (MeOH): kmax 299 nm.
5.23. rac-4-[(3-(8,8-Dimethyl-5-phenyl-7,8-dihydronaphthalen-
2-yl)-4,5-dihydroisoxazol-5-yl)]benzoic Acid (26a)
5.23.1. General procedure for the addition of hydroxylamine to
chalcones
447.1915; found, 434.1927. IR (NaCl): m 3431 (br, OH), 1690 (s,
C@O), 1606 (s, C@O) cmꢁ1. UV (MeOH): kmax 244 nm.
To a solution of NH2OHꢀHCl (0.07 g, 0.085 mmol) and NaOAc
(0.01 g, 0.13 mmol) in MeOH (0.2 mL) was added a solution of
24a (0.035 g, 0.09 mmol) in MeOH (0.5 mL) and the mixture was
heated to 70 °C for 16 h. The solvent was removed and brine was
added. The mixture was then extracted with CH2Cl2 (3ꢂ), the com-
bined organic layers were dried (Na2SO4) and the solvent was re-
moved. The residue was purified by chromatography (silica gel,
90:10 CH2Cl2/MeOH), to afford 0.027 g (75%) of a white solid iden-
tified as 26a. M. p.: 115 °C (acetone/hexane). 1H NMR (400.16 MHz,
(CD3)2CO): d 8.19 (s, 1H), 8.15 (d, J = 8.3 Hz, 2H), 7.97 (d, J = 8.0 Hz,
2H), 7.4–7.3 (m, 6H), 7.13 (d, J = 8.0 Hz, 1H), 6.19 (t, J = 4.5 Hz, 1H),
5.93 (t, J = 3.6 Hz, 1H), 3.9–3.6 (m, 2H), 2.45 (d, J = 4.6 Hz, 2H), 1.41
(s, 6H) ppm. 13C NMR (100.62 MHz, (CD3)2CO): d 189.1 (s), 167.3 (s),
156.2 (s), 147.5 (s), 144.0 (d), 142.2 (s), 141.0 (s), 139.2 (s), 138.9 (s),
133.3 (d, 2ꢂ), 131.9 (d), 131.2 (d), 130.9 (d), 130.5 (s), 130.4 (d, 2ꢂ),
130.3 (d, 2ꢂ), 129.3 (d), 129.2 (s), 128.5 (d), 127.8 (d), 127.7 (d),
126.9 (d), 80.9 (s), 42.0 (d), 40.3 (t), 35.4 (s), 29.3 (q, 2ꢂ) ppm. MS
(EI+): m/z (%) 423 (M+, 100), 407 (10), 394 (12), 393 (35), 180 (29),
175 (68), 131 (48), 118 (31), 103 (24). HRMS (EI+): Calcd for
5.26. Methyl 2-(7,8-Dihydro-8,8-dimethyl-5-phenylnaphtha-
len-2-yl)-2-oxoacetate (27a)
5.26.1. General procedure for the synthesis of a-ketoesters
To a solution of 19a (0.25 g, 0.79 mmol) in THF (5 mL) at ꢁ 78 °C
was added t-BuLi (1 mL, 1.7 M in pentane, 1.7 mmol). After 30 min,
a solution of dimethyloxalate (0.93 g, 7.9 mmol) in THF (6 mL) was
added and the mixture was stirred for 30 min at ꢁ78 °C and for 4 h
at 25 °C. The mixture was treated with a saturated aqueous solu-
tion of NH4Cl, and extracted with Et2O (3ꢂ). The combined organic
layers were washed with H2O, dried (Na2SO4) and the solvent was
removed. The residue was purified by chromatography (silicagel,
95:5 hexane/AcOEt), to afford 0.19 g (75%) of a yellow oil identified
as 27a. 1H NMR (400.16 MHz, CDCl3): d 8.03 (d, J = 1.7 Hz, 1H), 7.71
(dd, J = 8.1, 1.7 Hz, 1H), 7.4–7.3 (m, 5H), 7.14 (d, J = 8.1 Hz, 1H),
6.19 (t, J = 4.6 Hz, 1H), 3.97 (s, 3H), 2.42 (d, J = 4.6 Hz, 2H), 1.41
(s, 6H) ppm. 13C NMR (100.62 MHz, CDCl3): d 185.5 (s), 164.2 (s),
145.8 (s), 140.2 (s), 139.8 (s), 138.9 (s), 131.1 (s), 130.7 (d), 128.5
(d, 2ꢂ), 128.5 (d), 128.3 (d, 2ꢂ), 127.4 (d), 126.2 (d), 125.1 (d),
52.5 (q), 38.7 (t), 33.7 (s), 27.9 (q, 2ꢂ) ppm. MS (EI+): m/z (%) 320
(M+, 16), 309 (11), 293 (34), 281 (19), 280 (100), 278 (15), 277
(69), 275 (18), 262 (16), 261 (80), 247 (12), 233 (15), 221 (30),
215 (14), 203 (16), 202 (16), 191 (11), 189 (14), 165 (14), 115
(11), 113 (30), 105 (38), 91 (13), 86 (13), 84 (20). HRMS (EI+): calcd
C28H25NO3, 423.1725; found, 423.1719. IR (NaCl):
m 3030 (br, OH),
1685 (s, C@O), 1616 (s, C@O) cmꢁ1. UV (MeOH): kmax 257 nm.
5.24. rac-4-[(3-(8,8-Dimethyl-5-p-tolyl-7,8-dihydronaphthalen-
2-yl)-4,5-dihydroisoxazol-5-yl)]benzoic Acid (26b)
Following the general procedure, the reaction of 24b (0.051 g,
0.12 mmol) with NH2OHꢀHCl (0.009 g, 0.14 mmol) and NaOAc
for C21H20O3, 320.1412; found, 320.1398. IR (NaCl):
m 2829 (m, C–
H), 1737 (s, C@O), 1679 (s, C@O) cmꢁ1. UV (MeOH): kmax 330 nm.

4356
S. Álvarez et al. / Bioorg. Med. Chem. 17 (2009) 4345–4359
5.27. Methyl 2-(7,8-Dihydro-8,8-dimethyl-5-p-tolylnaphthalen-
2-yl)-2-oxoacetate (27b)
gel, 80:20 hexane/AcOEt), to afford 0.021 g (58%) of a yellow solid
identified as 33a.
To a solution of 33a (0.026 g, 0.057 mmol) in THF (0.5 mL) and
Following the general procedure, the reaction of 19b (0.25 g,
0.76 mmol), t-BuLi (1.3 mL, 1.7 M in pentane, 2.25 mmol) and
dimethyloxalate (0.89 g, 7.6 mmol) in THF (12 mL) afforded, after
purification by chromatography (silica gel, 95:5 hexane/AcOEt),
MeOH (0.3 mL) was added
a
solution of LiOHꢀH2O (0.007 g,
0.17 mmol) in THF (0.5 mL) and H2O (1 mL), and the reaction
was stirred for 4 h at 25 °C. The mixture was diluted with H2O, a
10% aqueous HCl solution was added and then extracted with
AcOEt (3ꢂ). The combined organic layers were dried (Na2SO4)
and the solvent was removed. The residue was purified by chroma-
tography (silica gel, 95:5 CH2Cl2/MeOH), to afford 0.10 g (78%) of a
yellow solid identified as 34a. 1H NMR (400.16 MHz, (CD3)2CO): d
9.89 (s, 1H), 8.52 (d, J = 7.9 Hz, 1H), 8.32 (s, 1H), 8.07 (d,
0.21 g (83%) of
a
yellow oil identified as 27b. 1H NMR
(400.16 MHz, CDCl3): d 8.20 (s, 1H), 7.87 (dd, J = 8.1, 1.5 Hz, 1H),
7.4–7.3 (m, 4H), 7.34 (d, J = 7.9 Hz, 1H), 6.35 (t, J = 4.4 Hz, 1H),
4.15 (s, 3H), 2.58 (d, J = 4.3 Hz, 2H), 1.61 (s, 6H2) ppm. 13C NMR
(100.62 MHz, CDCl3): d 185.6 (s), 164.3 (s), 145.8 (s), 140.3 (s),
138.8 (s), 137.1 (s), 136.9 (s), 131.0 (s), 130.3 (d), 129.0 (d), 128.5
(d, 2ꢂ), 128.4 (d, 2ꢂ), 126.2 (d), 125.1 (d), 52.5 (q), 38.7 (t), 33.7
(s), 27.9 (q, 2ꢂ), 21.1 (q) ppm. MS (EI+): m/z (%) 334 (M+, 30),
323 (10), 309 (12), 308 (15), 307 (64), 294 (45), 291 (31), 289
(28), 279 (19), 276 (23), 275 (100), 261 (19), 215 (14), 203 (11),
202 (14), 119 (39), 91 (17). HRMS (EI+): calcd for C22H22O3,
3
J = 9.0 Hz, 1H), 7.94 (d, J = 7.9 Hz, 1H), 7.83 (d, JH–F = 11.3 Hz,
1H), 7.5–7.4 (m, 5H), 7.15 (d, J = 8.5 Hz, 1H), 6.26 (t, J = 4.6 Hz,
1H’), 2.49 (d, J = 4.7 Hz, 2H), 1.48 (s, 6H) ppm. 13C NMR
(100.62 MHz, (CD3)2CO): d 170.1 (s), 169.3 (s), 167.5 (s), 152.3 (s,
1JC–F = 239.8 Hz), 147.4 (s), 145.2 (s), 138.0 (d), 132.5 (d, 2ꢂ),
131.0 (s), 130.4 (d, 2ꢂ), 129.4 (d), 128.6 (d), 128.3 (d), 127.9 (d),
3
334.1569; found, 334.1581. IR (NaCl):
m
2956 (m, C–H), 1737 (s,
127.8 (d, JC–F = 20.1 Hz), 124.9 (s), 124.5 (d), 123.8 (s), 123.1 (s),
C@O), 1685 (s, C@O) cmꢁ1. UV (MeOH): kmax 267 nm.
123.2 (s), 117.9 (d, JC–F = 20.8 Hz), 40.3 (t), 35.4 (s), 29.3 (q, 2ꢂ)
2
ppm. MS (FAB+): m/z (%) 439 (M+, 18), 438 (53), 437 (25), 422
(12), 391 (10), 307 (27), 289 (15), 166 (10), 155 (32), 154 (100).
HRMS (FAB+): calcd for C27H22FNO4, 443.4701; found, 443.4711.
5.28. Methyl 2-(7,8-dihydro-8,8-dimethyl-5-(2-phenylethynyl)-
naphthalen-2-yl)-2-oxoacetate (27c)
IR (NaCl):
m 2999 (w, OH), 2923 (s, C–H), 1659 (s, C@O), 1631 (s,
C@O) cmꢁ1. UV (MeOH): kmax 283 nm.
Following the general procedure, the reaction of 19c (0.25 g,
0.74 mmol) with t-BuLi (0.96 mL, 1.7 M in pentane, 1.63 mmol)
and dimethyloxalate (0.87 g, 7.4 mmol) in THF (11 mL) afforded,
after purification by chromatography (silica gel, 95:5 hexane/
AcOEt), 0.21 g (79%) of a yellow oil identified as 27c. 1H NMR
(400.16 MHz, CDCl3): d 8.10 (d, J = 1.7 Hz, 1H), 7.79 (dd, J = 8.3,
1.7 Hz, 1H), 7.5–7.4 (m, 5H,), 7.16 (d, J = 8.5 Hz, 1H’), 6.23 (t,
J = 4.6 Hz, 1H), 3.97 (s, 3H), 2.45 (d, J = 4.8 Hz, 2H), 1.40 (s, 6H)
ppm. 13C NMR (100.62 MHz, CDCl3): d 185.9 (s), 165.2 (s), 145.7
(s), 141.2 (s), 139.0 (s), 138.9 (s), 131.3 (s), 130.6 (d), 129.5 (d,
2ꢂ), 128.5 (d), 128.3 (d, 2ꢂ), 127.4 (d), 126.2 (d), 124.1 (d), 91.1
(s), 89.3 (s), 52.5 (q), 38.7 (t), 33.7 (s), 27.5 (q, 2ꢂ) ppm. MS (EI+):
m/z (%) 344 (M+, 20), 310 (15), 290 (35), 282 (20), 280 (100), 277
(69), 261 (80), 221 (30), 113 (30), 105 (38), 90 (13), 86 (13), 84
(20). HRMS (EI+): calcd for C23H20O3, 344.1001; found, 344.1111.
5.30. 4-[2-(8,8-Dimethyl-5-p-tolyl-7,8-dihydronaphthalen-2-
yl)-2-oxoacetamido]-3-fluorobenzoic Acid (34b)
Following the general procedure, the reaction of 27b (0.34 g,
1.02 mmol) and LiOHꢀH2O (0.064 g, 1.53 mmol) in THF (9 mL) and
H2O (8 mL) afforded, after purification by chromatography (silica gel,
95:5 CH2Cl2/MeOH), 0.24 g (75%) of a yellow solid identified as 28b.
The reaction of 28b (0.049 g, 0.15 mmol), oxalyl chloride
(0.040 mL, 0.45 mmol), methyl 4-amino-3-fluorobenzoate 32
(0.033 g, 0.18 mmol) and Et3N (0.063 mL, 0.45 mmol) in AcOEt
(2 ml), CH2Cl2 (2 mL) and DMF (0.23 mL) afforded, after purifica-
tion by chromatography (silica gel, 80:20 hexane/AcOEt), 0.045 g
(64%) of a yellow solid identified as 33b.
IR (NaCl):
m
2958 (s, C–H), 1740 (s, C@O), 1681 (s, C@O) cmꢁ1
.
Thereaction of 33b (0.045 g, 0.095 mmol) and LiOHꢀH2O (0.011 g,
0.28 mmol) in THF (1 mL), H2O (1 mL) and MeOH (0.5 mL) afforded,
after purification by chromatography (silica gel, 95:5 CH2Cl2/
MeOH), 0.038 g (88%) of a white solid identified as 34b. 1H NMR
(400.16 MHz, (CD3)2CO): d 9.33 (s, 1H), 8.51 (t, J = 8.2 Hz, 1H), 8.31
(d, J = 1.7 Hz, 1H), 8.11 (dd, J = 8.2, 1.7 Hz, 1H), 7.82 (d, J = 8.3 Hz,
1H), 7.74 (dd, J = 1.6 Hz, JH–F = 11.2 Hz, 1H), 7.4–7.2 (m, 4H), 7.07
(d, J = 8.2 Hz, 1H), 6.12 (t, J = 4.7 Hz, 1H), 3.63 (s, 3H), 2.35 (d,
J = 4.7 Hz, 2H), 1.45 (s, 6H2) ppm. 13C NMR (100.62 MHz, (CD3)2CO):
d 170.7 (s), 167.3 (s), 165.8 (s), 154.8 (s, 1JC–F = 240.5 Hz), 144.6 (s),
139.9 (s), 137.0 (d), 132.2 (d, 2ꢂ), 133.1 (s), 130.4 (d, 2ꢂ), 130.1
UV (MeOH): kmax 330 nm.
5.29. 4-[2-(7,8-Dihydro-8,8-dimethyl-5-phenylnaphthalen-2-
yl)-2-oxoacetamido]-3-fluorobenzoic Acid (34a)
5.29.1. General procedure for the synthesis of 2-oxo-
acetamidobenzoic acids
To a solution of 27a (0.13 g, 0.42 mmol) in THF (1 mL) was
added a solution of LiOHꢀH2O (0.026 g, 0.63 mmol) in THF (3 mL)
and H2O (3 mL), and the reaction was stirred to 80 °C for 2 h. The
mixture was diluted with H2O, a 10% aqueous HCl solution was
added and the mixture was extracted with AcOEt (3ꢂ). The com-
bined organic layers were dried (Na2SO4) and the solvent was re-
moved. The residue was purified by chromatography (silica gel,
95:5 CH2Cl2/MeOH), to afford 0.10 g (78%) of a yellow solid identi-
fied as 28a. To a solution of 28a (0.024 g, 0.078 mmol) in CH2Cl2
(1 mL) and DMF (0.12 mL) was added oxalyl chloride (0.020 mL,
0.23 mmol). After stirring for 1 h, the solvent was removed and
the residue was diluted with AcOEt (1 mL) and treated with a solu-
tion of methyl 4-amino-3-fluorobenzoate 32 (0.017 g, 0.094 mmol)
in AcOEt (1 mL) and Et3N (0.033 mL, 0.23 mmol) and the mixture
was stirred for 16 h at 25 °C. An aqueous saturated Na2CO3 solution
was added and the mixture was extracted with AcOEt (3ꢂ). The
combined organic layers were dried (Na2SO4) and the solvent
was removed. The residue was purified by chromatography (silica
3
(d), 129.2 (d), 128.3 (d), 125.7 (d, JC–F = 19.7 Hz), 125.1 (s), 125.0
2
(s), 123.9 (s), 123.8 (s), 123.1 (d), 118.0 (d, JC–F = 22.1 Hz), 115.3
(s), 53.0 (q), 40.6 (t), 44.1 (s), 28.9 (q, 2ꢂ) ppm. MS (FAB+): m/z (%)
457 (M+, 11), 327 (11), 314 (11), 307 (15), 289 (20), 285 (24), 240
(20), 228 (20), 226 (21), 167 (26), 166 (25), 165 (37), 155 (30), 154
(100). HRMS (FAB+): calcd for C28H24FNO4, 457.4901; found,
457.4910. IR (NaCl):
m 3000 (w, OH), 1702 (s, C–H), 1698 (s), 1629
(s) cmꢁ1. UV (MeOH): kmax 276 nm.
5.31. 4-[2-(8,8-Dimethyl-5-(2-phenylethynyl)-7,8-dihydronaph-
thalen-2-yl)-2-oxoacetamido]-3-fluorobenzoic acid (34c)
Following the general procedure, the reaction of 27c (0.14 g,
0.39 mmol) and LiOHꢀH2O (0.025 g, 0.58 mmol) in THF (4 mL) and
H2O (3 mL) afforded, after purification by chromatography (silica

S. Álvarez et al. / Bioorg. Med. Chem. 17 (2009) 4345–4359
4357
gel, 95:5 CH2Cl2/MeOH), 0.11 g (82%) of a yellow solid identified as
28c.
(silica gel, 80:20 hexane/AcOEt), 0.012 g (80%) of a white solid
identified as 35b. Following the general procedure, the reaction
of 35b (0.012 g, 0.022 mmol) and LiOHꢀH2O (0.003 g, 0.066 mmol)
in THF (0.5 mL), H2O (0.5 mL) and MeOH (0.2 mL) afforded, after
purification by chromatography (silica gel, 90:10 CH2Cl2/MeOH),
The reaction of 28c (0.12 g, 0.36 mmol), oxalyl chloride
(0.096 mL, 1.08 mmol), methyl 4-amino-3-fluorobenzoate 32
(0.080 g, 0.43 mmol) and Et3N (0.15 mL, 1.08 mmol) in AcOEt
(4 mL), CH2Cl2 (4 mL) and DMF (0.43 mL) afforded, after purifica-
tion by chromatography (silica gel, 80:20 hexane/AcOEt), 0.11 g
(65%) of a yellow solid identified as 33c.
0.010 g (98%) of
a
white solid identified as 36b. 1H NMR
(400.16 MHz, CDCl3): d 9.65 (s, 1H), 8.63 (d, J = 8.2 Hz, 1H), 7.98
(s, 1H), 7.87 (d, J = 8.3 Hz, 1H), 7.79 (d, JH–F= 11.9 Hz, 1H), 7.4–7.3
(m, 4H), 6.49 (t, J = 4.4 Hz, 1H), 5.38 (s, 1H), 2.43 (d, J = 4.6 Hz,
2H), 2.40 (s, 3H), 1.35 (s, 6H) ppm. 13C NMR (100.62 MHz,
The reaction of 33c (0.032 g, 0.066 mmol) and LiOHꢀH2O
(0.008 g, 0.20 mmol) in THF (1 mL), H2O (1 mL) and MeOH
(0.3 mL) afforded, after purification by chromatography (silica
gel, 95:5 CH2Cl2/MeOH), 0.025 g (81%) of a white solid identified
as 34c. 1H NMR (400.16 MHz, (CD3)2CO): d 9.89 (s, 1H), 8.52 (d,
J = 7.9 Hz, 1H), 8.32 (s, 1H), 8.07 (d, J = 9.0 Hz, 1H), 7.94 (d,
J = 7.9 Hz, 1H), 7.83 (d, JH–F= 11.3 Hz, 1H), 7.5–7.4 (m, 5H), 7.15
(d, J = 8.5 Hz, 1H), 6.26 (t, J = 4.6 Hz, 1H), 2.49 (d, J = 4.7 Hz, 2H),
1.48 (s, 6H) ppm. 13C NMR (100.62 MHz, (CD3)2CO): d 172.7 (s),
1
(CD3)2CO): d 172.5 (s), 167.5 (s), 155.3 (s, JC–F = 243.1 Hz), 146.6
(s), 139.7 (s), 137.7 (d, 2ꢂ), 133.2 (d), 133.1 (s), 131.3 (d, 2ꢂ),
3
130.5 (d), 128.2 (d), 128.1 (d), 128.0 (s), 126.7 (d, JC–F = 19.6 Hz),
2
125.3 (s), 122.9 (s), 122.8 (s), 122.1 (d), 116.9 (d, JC–F = 22.0 Hz),
116.3 (s), 77.4 (d), 53.4 (q), 41.5 (t), 43.9 (s), 31.1 (q, 2ꢂ) ppm.
MS (FAB+): m/z (%) 460 (M++1, 11), 459 (M+, 20), (20), 285 (24),
240 (20), 228 (20), 226 (21), 215 (20), 202 (22), 189 (22), 180
(24), 167 (26), 166 (25), 165 (37), 154 (100). HRMS (FAB+): calcd
1
166.3 (s), 165.8 (s), 153.8 (s, JC–F = 242.5 Hz), 145.6 (s), 139.9 (s),
136.9 (d), 133.2 (d, 2ꢂ), 133.1 (s), 130.4 (d, 2ꢂ), 130.2 (d), 128.2
for C28H26FNO4, 459.4901; found, 459.4910. IR (NaCl): m 3450 (w,
3
(d), 128.3 (d), 125.7 (d, JC–F = 19.1 Hz), 125.1 (s), 122.9 (s), 123.1
OH), 3026–2852 (m), 1719 (s, C@O), 1603 (s, C@O) cmꢁ1. UV
(MeOH): kmax 249, 326 nm.
2
(d), 123.0 (s), 120.4 (d), 117.8 (d, JC–F = 21.9 Hz), 115.3 (s), 92.3
(s), 88.9 (s), 40.6 (t), 43.9 (s), 29.6 (q, 2ꢂ) ppm. MS (FAB+): m/z
(%) 468 (M++1, 25), 467 (M+, 53), 454 (20), 453 (40), 452 (50),
287 (68), 285 (40), 282 (44), 258 (100), 257 (38), 255 (47), 253
(32), 243 (87), 242 (46), 241 (47), 239 (56), 229 (55), 228 (54),
215 (52), 185 (39), 165 (39). HRMS (FAB+): calcd for C29H22FNO4,
5.34. rac-4-[2-(8,8-Dimethyl-5-(2-phenylethynyl)-7,8-
dihydronaphthalen-2-yl)-2-hydroxyacetamido]-3-
fluorobenzoic Acid (36c)
467.4901; found, 467.4910. IR (NaCl):
m
2956–2850 (m), 1730 (s,
Following the general procedure, the reaction of 33c (0.015 g,
0.031 mmol) and NaBH4 (0.001 g, 0.016 mmol) in MeOH (0.6 mL)
and AcOEt (0.3 ml) afforded, after purification by chromatography
(silica gel, 80:20 hexane/AcOEt), 0.011 g (80%) of a white solid
identified as 35c. Following the general procedure, the reaction
of 35c (0.011 g, 0.022 mmol) and LiOHꢀH2O (0.003 g, 0.066 mmol)
in THF (0.5 mL), H2O (0.5 mL) and MeOH (0.2 mL) afforded, after
purification by chromatography (silica gel, 90:10 CH2Cl2/MeOH),
C@O) cmꢁ1. UV (MeOH): kmax 295, 324 nm.
5.32. rac-4-[2-(8,8-Dimethyl-5-phenyl-7,8-dihydronaphthalen-
2-yl)-2-hydroxy-acetamido]-3-fluorobenzoic acid (36a)
5.32.1. General procedure for the synthesis
of
a-hydroxyacetamidobenzoic acids
To a solution of 33a (0.012 g, 0.026 mmol) in MeOH (0.5 mL) and
0.010 g (91%) of
a
white solid identified as 36c. 1H NMR
AcOEt(0.2 mL) was addedNaBH4 (0.001 g, 0.015 mmol)and the reac-
tion was stirred for 15 min at 0 °C. A 10% aqueous HCl solution was
added and the organic layer was extracted with AcOEt. The combined
organic layers were washed with brine and dried (Na2SO4) and the
solvent was removed. The residue was purified by chromatography
(silicagel, 80:20 hexane/AcOEt), to afford 0.010 g (84%) of a white so-
lid identified as 35a. Following the general procedure for the hydro-
lysis of methyl esters with LiOH, the reaction of 35a (0.010 g,
0.022 mmol) and LiOHꢀH2O (0.003 g, 0.066 mmol) in THF (0.5 mL),
H2O (0.5 mL) and MeOH (0.2 mL) afforded, after purification by chro-
matography (silica gel, 90:10 CH2Cl2/MeOH), 0.010 g (98%) of a white
solid identified as 36a. 1H NMR (400.16 MHz, CDCl3): d 9.55 (s, 1H),
8.51 (d, J = 8.0 Hz, 1H), 7.94 (s, 1H), 8.87 (d, J = 8.2 Hz, 1H), 7.80 (d,
3JH–F= 11.6 Hz, 1H), 7.5–7.4 (m, 6H, HPh), 6.53 (t, J = 4.5 Hz, 1H),
5.40 (s, 1H), 2.40 (d, J = 4.6 Hz, 2H), 1.30 (s, 6H) ppm. 13C NMR
(400.16 MHz, (CD3)2CO): d 9.54 (s, 1H), 8.50 (t, J = 8.1 Hz, 1H),
7.93 (d, J = 1.6 Hz, 1H), 7.85 (d, J = 8.7 Hz, 1H), 7.78 (d, JH–
3
F = 11.6 Hz, 1H), 7.6–7.5 (m, 2H,’), 7.4–7.3 (m, 5H), 6.51 (t,
J = 4.9 Hz, 1H), 5.39 (s, 1H), 2.39 (d, J = 4.9 Hz, 2H), 1.29 (s, 6H)
ppm. 13C NMR (100.62 MHz, (CD3)2CO): d 172.7 (s), 167.3 (s),
1
153.8 (s, JC–F = 243.5 Hz), 145.6 (s), 139.9 (s), 136.7 (d), 133.2 (d,
2ꢂ), 133.1 (s), 130.4 (d, 2ꢂ), 130.2 (d), 128.2 (d), 128.3 (d), 125.7
3
(d, JC–F = 18.1 Hz), 125.1 (s), 122.9 (s), 122.8 (s), 122.1 (d), 117.8
2
(d, JC–F = 20.9 Hz), 115.3 (s), 92.3 (s), 88.9 (s), 75.9 (d), 40.6 (t),
43.9 (s), 29.6 (q, 2ꢂ) ppm. MS (FAB+): m/z (%) 470 (M++1, 53),
469 (M+, 29), 454 (20), 453 (40), 452 (50), 287 (68), 285 (40),
282 (44), 258 (100), 257 (38), 255 (47), 243 (87), 242 (46), 241
(47), 239 (56), 229 (55), 228 (54), 215 (52). HRMS (FAB+): calcd
for C29H24FNO4, 469.5001; found, 469.5010. IR (NaCl):
m 3000–
2860 (w), 1660 (s), 1558 (s) cmꢁ1. UV (MeOH): kmax 249, 327 nm.
1
(100.62 MHz, (CD3)2CO): d 168.0 (s), 158.5 (s), 147.4 (s, JC–
F = 241.3 Hz), 142.7 (s), 141.3 (s), 139.3 (s), 135.9 (s), 133.9 (s),
5.35. Computational methods
133.6 (s), 133.4 (s), 132.3 (d, 2ꢂ), 130.5 (d, 2ꢂ), 130.3 (d), 129.1 (d),
3
128.5 (d), 127.9 (d), 127.7 (d), 126.3 (d), 125.9 (d, JC–F = 20.0 Hz),
5.35.1. Quantum mechanics calculations
2
115.9 (d, JC–F = 21.3 Hz), 40.5 (t), 35.3 (s), 29.4 (q, 2ꢂ) ppm. MS
The (S)-26a ligand was model-built in Insight II49 using stan-
dard bond lengths and angles. The geometry of the ligand was then
(FAB+): m/z (%) 446 (M++1, 100), 445 (M+, 45), 437 (11), 436 (28),
318 (14), 276 (42), 154 (19). HRMS (FAB+): calcd for C27H24FNO4,
fully optimized at the DFT level with the 6-31G basis set using the
*
445.4811; found, 445.4810. IR (NaCl):
m
3376 (w, OH), 2960–2850
ab initio quantum chemistry program GAUSSIAN 98.50 Charge distri-
(s), 1722 (s, C@O), 1618 (m, C@O) cmꢁ1. UV (MeOH): kmax 314 nm.
bution for the ligand was calculated by fitting the DFT/6-31G elec-
*
trostatic potential to atom centers using the RESP procedure51 and
van der Waals parameters for ligand atoms were transferred from
those defined for related atoms in the AMBER force field
(parm99).52 Covalent and nonbonded parameters for the ligand
atoms were derived, by analogy or through interpolation, from
those already present in the same force field (parm99) or consis-
tently derived, as explained in more detail elsewhere.53
5.33. rac-4-[2-(8,8-Dimethyl-5-p-tolyl-7,8-dihydronaphthalen-
2-yl)-2-hydroxy-acetamido]-3-fluorobenzoic Acid (36b)
Following the general procedure, the reaction of 33b (0.015 g,
0.032 mmol) and NaBH4 (0.001 g, 0.016 mmol) in MeOH (0.6 mL)
and AcOEt (0.3 ml) afforded, after purification by chromatography

4358
S. Álvarez et al. / Bioorg. Med. Chem. 17 (2009) 4345–4359
5.35.2. Construction and refinement of human RAR
antagonist-bound conformation
c
in the
lution of the rms deviation (rmsd) of (S)-26a, with respect to the
initial structure, it can be clearly seen that the rmsd value of the
antagonist was maintained around 0.5 Å. The simulation resulted
in a stable trajectory and the relationships between secondary
structures, together with the respective interactions, were also
maintained, lending further support to the proposed models. All
calculations were performed on the SGI R14000 Origin 3800 at CIE-
MAT (Madrid), on the SGI 1.5 GHz Itanium2 at CESGA (Santiago de
Compostela) and locally on SGI R12000 Octane workstations.
The crystal structure of RAR
1.38 Å resolution (PDB code: 1fd0)19 and the crystal structure of
human RAR in complex with the antagonist BMS614 (39) at
2.50 Å resolution (PDB code: 1dkf)39 were used to model the hu-
man RAR in the antagonist-bound conformation in complex with
(S)-26a. The ligand-binding domain of RAR shares a 70% amino
acid sequence identity with LBD of RAR , and the core structure
of these two receptors has a similar overall fold. To drive the RAR
into the antagonist-bound conformation, helix H12 was manually
reoriented to make it adopt the known antagonist form of RAR
c in complex with SR11254 (4) at
a
c
a
c
c
5.36. Transient transactivation assays
a
.
A short optimization run restraining all non-H atoms in the resi-
dues to their initial coordinates then allowed readjustment of
covalent bonds and van der Waals contacts without changing the
overall conformation of the receptor. Then (S)-26a was positioned
in the binding pocket on the basis of the crystallographic structures
of the complexes of RAR with several ligands.13,19,39–41,20,21
Determination of RAR/RXR agonistic activity. HeLa reporter
cells were stably transfected with an (17m)5-bG-Luc-Neo reporter
and with Gal4-mRARa (resp. b, c) or Gal4-hRXRb plasmids. They
were maintained in DMEM that contained 5% fetal calf serum
(FCS), supplemented with geneticin G418 (0.8 mg mLꢁ1), puromy-
cin (0.3
only for the Gal4-hRXRb-engineered HeLa cell line), and gentamy-
cin (40
g mLꢁ1). The ligand-binding assays were performed add-
ing the ligands to the cell culture in DMEM medium without
phenol red with 5% charcoal-treated FCS. To determine the RAR
RARb, and RAR induction potential of the ligands, equal aliquots
l
g mLꢁ1), hygromycin (0.2 mg mLꢁ1; added additionally
5.35.3. Molecular dynamics simulations
l
The RARc-(S)-26a complex was then refined using the second
generation AMBER force field52 and 3000 steps of steepest descent
energy minimization and 6000 steps of conjugate gradient of only
sidechain of the protein and those atoms belonging to the bound
ligand. This procedure allowed readjustment of covalent bonds
and van der Waals contacts without changing the overall confor-
mation of the complex.
a,
c
(160,000 cells/well) of the corresponding cell line were seeded in
a 24-well plate, and 12 h later the medium was replaced by a solu-
tion of the corresponding ligand in medium. The cells were incu-
bated at 37 °C in 5% CO2 for 12 h. After that, the cells were
The molecular system was neutralized by addition of the appro-
priate number of sodium ions,54 placed in positions of negative
electrostatic potential and immersed in a rectangular box of
ꢃ8020 transferable intermolecular potential three point model
water molecules.55 Each water box extended 8 Å away from any
solute atom, and the cutoff distance for the nonbonded interac-
tions was 9 Å. Periodic boundary conditions were used, and elec-
trostatic interactions were represented using the smooth particle
mesh Ewald method with a grid spacing of ꢃ1 Å. Unrestrained
molecular dynamics (MD) simulations at 300 K and 1 atm were
then run for 6 ns using the SANDER module in AMBER 8.56 The con-
stants for the temperature and pressure baths were 1.0 and 0.2 ps,
respectively. SHAKE57 was applied to all bonds involving hydro-
gens, and an integration step of 2 fs was used throughout. The non-
bonded pair list was updated every 10 steps. The simulation
protocol involving a series of progressive energy minimizations
followed by a 20 ps heating phase and a 70 ps equilibration period
before data collection. System coordinates were saved every 2 ps
for further analysis.
washed (PBS) and lysed (50
phate (pH 7.8), 2 mM EDTA, 1 mM DTT, 10% glycerol, and 1% Triton
X-100) for 15 min. Equal aliquots (50 L) of the cell lysates were
transferred in an Optiplate-96, and the luminescence in RLU (rela-
tive luminescence units) was determined on a MicroLumat LB96P
lL of lysis buffer: 25 mM Tris phos-
l
luminometer (‘Berthold’) after automatic injection of 50 lL of lucif-
erin buffer (20 mM Tris phosphate (pH 7.8), 1.07 mM MgCl2,
2.67 mM MgSO4, 0.1 mM EDTA, 33.3 mM DTT, 0.53 mM ATP,
0.47 mM luciferin, and 0.27 mM coenzyme A). The receptor activa-
tion potential of each compound was presented as fold induction
measured as ratio of RLU of the compound over the RLU of the
vehicle control.
Acknowledgments
The authors are grateful to the European Union (Anticancer Ret-
inoids, QLK3-2002-02029), the MEC-Spain (SAF2004-07131, Con-
tract to S.A.; SAF2007-63880-FEDER; Juan de la Cierva Contract
to F.R.B.), Xunta de Galicia (Grant 08CSA052383PR from DXI+D+i;
Consolidación 2006/15 from DXPCTSUG), the INCa, the ANR and
the Ligue National Contre le Cancer (H.G., équipe labelisé) for
financial support.
5.35.4. Analysis of the molecular dynamics trajectories and
electrostatic energy calculations
Three-dimensional structures and trajectories were visually in-
spected using the computer graphics program InsightII. Root-
mean-square (rms) deviations from both, the initial structures
and the average structures, interatomic distances, and snapshot
geometries were obtained using the PTRAJ module in AMBER.
Intermolecular van der Waals and electrostatic energies for indi-
vidual residues were calculated with the ANAL module. After the
equilibration period, the progression of the root-mean-square
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.05.035.
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