Novel tetralone-derived retinoic acid metabolism blocking agents: Synthesis and in vitro evaluation with liver microsomal and MCF-7 CYP26A1 cell assays

Article abstract of DOI:10.1021/jm0501681

The potent inhibitory activity of novel 2-benzyltetralone and 2-benzylidenetetralone derivatives vs liver microsomal retinoic acid metabolizing enzymes and a MCF-7 CYP26A1 cell assay is described. In the liver microsomal assay, the 2-biphenylmethyl-6-hydr

Full text of DOI:10.1021/jm0501681

J. Med. Chem. 2005, 48, 7123-7131
7123
Novel Tetralone-Derived Retinoic Acid Metabolism Blocking Agents: Synthesis
and in Vitro Evaluation with Liver Microsomal and MCF-7 CYP26A1 Cell Assays
Sook Wah Yee,† Laetitia Jarno,†,‡ Mohamed Sayed Gomaa,† Carole Elford,§ Li-Ling Ooi,# Michael P. Coogan,#
Richard McClelland,‡ Robert Ian Nicholson,‡ Bronwen A. J. Evans,§ Andrea Brancale,† and Claire Simons*,†
Medicinal Chemistry and Tenovus Research Centre, Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue,
Cardiff CF10 3XF, U.K., School of Chemistry, Cardiff University, Cardiff CF10 3AT, U.K., and Department of Child Health,
School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, U.K.
Received February 22, 2005
The potent inhibitory activity of novel 2-benzyltetralone and 2-benzylidenetetralone derivatives
vs liver microsomal retinoic acid metabolizing enzymes and a MCF-7 CYP26A1 cell assay is
described. In the liver microsomal assay, the 2-biphenylmethyl-6-hydroxytetralone derivatives
16a and 16b were found to be potent inhibitors (IC50 ) 0.5 and 0.8 µM) compared with the
broad spectrum P450 inhibitor ketoconazole and the retinoid mimetic R115866 (IC50 ) 18.0
and 9.0 µM, respectively). In the MCF-7 CYP26A1 cell assay, the 2-(4-hydroxybenzyl)-6-
methoxytetralone 5 and unsaturated benzylidene precursor 6 were found to be the most potent
(IC50 ) 7 and 5 µM, respectively), which was comparable with liarozole (7 µM) but considerably
less active than R115866 (IC50 ) 5 nM). With a CYP26A1 homology model, the tetralones were
shown to be positioned in a hydrophobic tunnel with additional interactions, e.g., transition
metal coordination and hydrogen-bonding interactions with GLY300, observed for the potent
4-hydroxyphenyl substituted inhibitors.
Introduction
systemically, due to metabolism by human liver and
intestine cytochrome P450s to the inactive 4-hydroxy-
RA and thence by dehydrogenases to the partially active
4-keto-RA and inactive polar metabolites (Figure 1).11
All-trans-retinoic acid (atRA) is a naturally occurring
retinoid responsible for growth and differentiation of
mammalian epithelial tissues,1 which exerts activity by
binding to transcription-regulatory factors in the cell
nucleus known as RAR (retinoic acid receptor) and RXR
(retinoid X receptor), each having subtypes R, â, and
δ.2 Upon atRA binding the activated receptor transcrip-
tionally regulates its target genes by binding to its
response element retinoic acid response element (RARE)
or retinoid X response element (RXRE) (Figure 1).3,4
Retinoic acid has been used in a number of clinical
situations, especially oncology and dermatology. In
oncology, atRA has shown spectacular success in the
treatment of acute promyelocytic leukaemia,5,6 although
the remission seen is followed by relapse within 4-6
months; this appears to be due to increased RA me-
tabolism as a result of RA induction, leading to de-
creased clinical efficacy. atRA may improve the efficacy
of other treatments such as radiation, cisplatin, and
interferon therapies.7,8 Retinoids have been used for
some time in the treatment of psoriasis, cystic acne, and
cutaneous malignancies due to hyperkeratinization as
well as in the treatment of photodamaged skin.9,10
Vitamin A (retinol) is oxidized through retinal by
dehydrogenases in the cytoplasm of target cells in low
yields to all-trans-retinoic acid (Figure 1), which is at
least 100-fold more active than retinol and accounts for
its biological action. atRA has a short half-life (ca. 1 h),
and its potency is reduced when it is administered
The specific P450s responsible for 4-hydroxylation of
atRA in the human liver are CYP2C8 as a major
contributor as well as 3A7, 3A5, 3A4, 2C9, and 1A1.12
Several CYP isozymes from different rat tissues have
been shown in vitro to be capable of metabolizing RA
via 4-hydroxylation,13 with RA metabolism by rat liver
microsomes being mainly by the 1A1/2, 2A6, and 3A4
forms. However, in living tissues, atRA administration
induces another RA-metabolizing enzyme, CYP26,14
which recognizes only atRA as its substrate, and the
expression of this isozyme can be induced by atRA both
in vitro and in vivo.15 Three members of the CYP26
family have now been identified: CYP26A114 and
CYP26B1,16 which metabolize atRA in the embryo and
adult, and more recently,17 CYP26C1, which may have
a role in the specific metabolism of both all-trans and
9-cis isomers of RA.
An inhibitor of the metabolism of endogenous atRA
would be expected to have a beneficial effect on epithe-
lial differentiation and proliferation as an RA mimetic,
with potential use as an agent for nonhormone-depend-
ent cancers and various skin conditions. Importantly,
such inhibitors should display selectivity for CYP26
because inhibition of hepatic enzymes, e.g., CYP2C8,
3A7, 3A5, 3A4, 2C9, and 1A1, may interfere with the
metabolism of other endogenous compounds or drugs.
A number of retinoic acid metabolism blocking agents
(RAMBAs) have been described (Figure 2). The imida-
zoles, ketoconazole and liarozole, were reported as
inhibitors of RA-metabolizing enzymes while being
studied as inhibitors of 17R-hydroxylase: 17,20-lyase
(P450 17R) as agents for the treatment of androgen-
* To whom correspondence should be addressed. Phone: +44-(0)-
2920-876307. Fax: +44-(0)2920-874149. E-mail: SimonsC@Cardiff.ac.uk.
† Medicinal Chemistry, Welsh School of Pharmacy.
‡ Tenovus Research Centre, Welsh School of Pharmacy.
§ School of Medicine.
# School of Chemistry.
10.1021/jm0501681 CCC: $30.25 © 2005 American Chemical Society
Published on Web 10/21/2005

7124 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23
Yee et al.
Figure 1. Alcohol dehydrogenases (ADH) and short-chain dehydrogenase/reductase catalyze the oxidation of retinol to
retinaldehyde, which is subsequently oxidized by aldehyde dehydrogenases (ALDH) to retinoic acid. Isomers of retinoic acid,
all-trans (atRA) and 9-cis (9cRA), are further metabolized by cytochrome P450 enzymes (including CYP26A1, B1, and C1) to
inactive polar metabolites resulting in retinoid excretion. Intracelluar retinol can bind with cellular retinol-binding protein (CRBP)
and retinoic acid with cellular retinoic acid-binding protein (CRABP). Retinoic acid binds to transcription-regulatory factors in
the cell nucleus known as RAR (retinoic acid receptor) and RXR (retinoid X receptor).
We have recently described several classes of RAM-
BAs in rat liver microsomal assays, other systems
(human placenta and human liver microsomes, human
skin homogenates), and RA-induced cell cultures (hu-
man male genital fibroblasts and HaCat cells).22-24 The
most potent inhibitor of retinoic acid metabolism was
the 2-(4-aminobenzyl)-6-hydroxytetralone (Figure 2).24,25
With this compound as a lead, a range of tetralone
derivatives were prepared to probe requirements for
optimal retinoic acid metabolism inhibitory activity
using two in vitro assay systems: rat liver microsomes
and human breast cancer MCF-7 cells expressing
Figure 2. Retinoic acid metabolism blocking agents (RAM-
BAs).
CYP26A1 activity.
Results
dependent prostatic cancer by lowering testosterone
levels.11 Ketoconazole is not a suitable oral agent as an
RA mimetic for sex-hormone-independent cancers be-
cause it inhibits several other P450 enzymes on the
steroidogenic pathway of androgen synthesis and, fur-
thermore, has a poor pharmacokinetics profile. Liarozole
(Liazal) inhibits testicular (but not adrenal) P450 17 and
is a potent inhibitor of aromatase (P450AROM). Both
these targets negate its potential as an oral RA mimetic
for sex-hormone-independent cancers despite its ef-
fectiveness in clinical trials (oral administration) in
psoriasis,18 as well as icthyosis and hormone-resistant
prostate cancer.19,20 The triazole R115866 has been
described as a novel inhibitor of CYP26 which, in vivo
in rats after a single oral dose, increases endogenous
tissue RA levels and mimics RA in several other of its
biological actions.15 R115866 has also shown beneficial
effects when administered topically to skin.15 A more
recent derivative, R116010, has shown potent and
selective inhibitory activity of retinoic acid metabolism
in human T47D breast cancer cells.21
Chemistry. The method employed for the prepara-
tion of the 6-methoxy-2-(phenylmethylidene)-3,4-dihy-
dro-2H-naphthalen-1-one precursors 2 involved direct
condensation of the commercially available tetralone 1
with the appropriate benzaldehyde in ethanolic KOH
solution.26 This method was successfully used in the
absence of a hydroxyl group on the tetralone or benzal-
dehyde (Scheme 1). The synthesis of hydroxyl deriva-
tives (5, 10, and 14) required initial protection of one
or both benzaldehyde and tetralone hydroxyl groups
(Schemes 1 and 2) with the tetrahydropyran (THP)
protecting group, which was stable under the basic
ethanolic KOH condensation conditions.
The 2-biphenyl substituted derivatives 15a,b were
prepared by Suzuki coupling with phenylboronic acid
in the presence of Pd(PPh3)4 catalyst27 (Scheme 2). The
reduced 2-(benzyl)-3,4-dihydro-2H-naphthalen-1-one de-
rivatives (3a, 3b, 6, 7, 11, 12, 16a, and 16b) were readily
obtained by hydrogenation with 10% Pd/C catalyst for
1 h at approximately 30 psi (Parr hydrogenator). When

RAMBAs
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23 7125
Scheme 1a
a Reagents and conditions: (i) R-C6H4CHO or THPO-C6H4CHO, 4% KOH/EtOH, room temp, 1-72 h; (ii) 10% Pd/C, H2, MeOH, room
temp, 1 h; (iii) 2 M HCl(aq), EtOAc/2-butanone (1:1 v/v), reflux, 1 h; (iv) 10% Pd/C, H2, MeOH, room temp, 2 h; (v) 48% HBr, reflux, 5 h;
(vi) 3,4-dihydropyran, AcOH, p-TsOH, Et2O, room temp, 3 h.
Scheme 2a
a Reagents and conditions: (i) Br-C6H4CHO, 4% KOH/EtOH, room temp, 1-4 h; (ii) 2 M HCl(aq), EtOAc/2-butanone (1:1 v/v), reflux,
1 h; (iii) phenylboronic acid, Pd(PPh3)4, toluene, 100 °C, 5 h; (iv) 10% Pd/C, H2, MeOH, room temp, 1 h.
the hydrogenation reaction was allowed to proceed for
2 h, deoxygenation at C1 was found to occur (7, Scheme
1).
and HMBC spectra (400 MHz) were obtained for the 2c
derivative. The HSQC spectrum confirmed that the
broad singlet did correspond to the two CH2 peaks at δ
27 and 29 in the 13C NMR spectrum. The HMBC
spectrum (used to determine the multiple-bond cou-
plings over two or three bonds) indicated that (i) the
CH2 at δ 27 correlated with H-9, (ii) the CH2 at δ 29
correlated to H-5, and (iii) the alkene proton (H-9)
correlated to the CdO signal (Figure 3a). These finding
all indicated that the 2-bromobenzaldehyde had reacted
at the R-carbon of the tetralone. A crystal structure of
The 2-bromo derivatives 2c, 13a, and 14 gave an
1
unexpected result in the H NMR (300 MHz) spectra,
with a singlet at δ 2.2-3 corresponding to the four CH2
protons of 3,4-dihydronaphthalene. In all other com-
pounds the four CH2 protons were observed as two
distinct multiplets at δ 2.5 and 3.0. To confirm that the
2-bromobenzaldehyde had reacted at the R-carbon rather
than the (unlikely) â-carbon of the tetralone, 1H HSQC

7126 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23
Yee et al.
Figure 3. (a) NMR confirmed that (i) the CH2 at δ 27 correlated with H-9, (ii) the CH2 at δ 29 correlated to H-5, and (iii) the
alkene proton (H-9) correlated to the CdO signal. (b) X-ray crystallographic structure of compound 2c.
Table 1. IC50 Data for the Novel Benzylidene Tetralone
Derivatives Using Rat Liver Microsomal and MCF-7 Assays
Figure 4. RT-PCR analysis of CYP26A1 mRNA expression
after treatment with all-trans-retinoic acid (atRA). MCF-7
human breast cancer cells were treated with 10-7 M atRA for
9 h. Total RNA was isolated and subjected to RT-PCR.
Amplified products were separated on an agarose gel contain-
ing ethidium bromide by electrophoresis.
2-[1-(2-bromophenyl)methylidene]-6-methoxy-3,4-dihy-
dronaphthalen-1-one (2c) confirmed the structure (Fig-
ure 3b). Therefore, we could conclude that the four
protons from the two CH2 atoms at carbons 3 and 4
appear as one broad singlet owing to accidental degen-
eracy.
a IC50 values are the average ((5%) of two experiments.
with the results obtained by Stoppie et al.15 With the
exception of the 2-bromo derivative, 2-[1-(2-bromophe-
nyl)methylidene]-6-hydroxy-3,4-dihydro-2H-naphthalen-
1-one (14, IC50 ) 2.4 µM), the benzylidene derivatives
were all poor inhibitors of retinoic acid metabolism (IC50
> 20 µM), using the liver microsomal assay (Table 1).
Compound 14 did not retain activity in the MCF-7
assay; however, the corresponding 6-methoxybenzylidene
derivative 2c was comparable with liarozole (IC50: 2c,
9 µM; liarozole, 7 µM) in its inhibition of atRA metabo-
lism. The 4-hydroxybenzylidene derivatives 5 and 10a
also displayed inhibitory activity (IC50: 5, 7 µM; 10a, 9
µM) comparable with liarozole; however, 2c, 5, and 10a
were considerably less active than R115866 (IC50 ) 5
nM).
The 2-benzyl derivatives, in contrast to the 2-ben-
zylidene derivatives, displayed potent inhibition of atRA
metabolism in the liver microsomal assay (Table 2). The
inhibitory activity observed using the liver microsomal
assay was not observed with the MCF-7 CYP26A1 assay
with the exception of the 4-hyroxy derivative, 2-(4-
hydroxybenzyl)-6-methoxy-3,4-dihydro-2H-naphthalen-
1-one (6) (IC50: liver microsomes, 0.4 µM; MCF-7 cells,
5 µM). The previously24 resolved enantiomers of the lead
tetralone 2-(4-aminobenzyl)-6-hydroxytetralone were
also evaluated and shown to display poor inhibitory
activity in the MCF-7 assay (39 and 43 µM).
Enzyme Inhibition. The 2-benzylidenetetralone (2a-
c, 5, 10a,b, 14, and 15a,b) and 2-benzyltetralone (3a,b,
6, 7, 11, 12, and 16a,b) derivatives were evaluated for
their retinoic acid metabolism inhibitory activity using
rat liver microsomes and a MCF-7 cell assay,28,29 using
radiolabeled [11,12-3H]-all-trans-retinoic acid as the
substrate and using ketoconazole, liarozole, and R115866
as standards for comparison.
The presence of CYP26A1 has not been confirmed in
the MCF-7 cell line; therefore, prior to commencement
of the MCF-7 cell assays, it was necessary to perform
reverse transcriptase polymerase chain reaction (RT-
PCR) analysis of CYP26A1 mRNA expression. This was
performed after treatment of MCF-7 cells with 10-7
M
atRA for 9 h (mirroring the assay induction conditions).
After isolation of RNA and RT-PCR the amplified
products were separated on a 1.5% (w/v) agarose gel
containing ethidium bromide and separated at 100 V.
UV illumination confirmed the presence of the 184 base
pair fragment of CYP26A1 cDNA (Figure 4).
The standards were evaluated using the assay sys-
tems to determine comparison IC50 values. Ketoconazole
had a similar profile in both assays (IC50: liver mi-
crosomes, 18 µM; MCF-7 cells, 12 µM). Liarozole has
previously been shown to induce atRA metabolism in
rat liver microsomes,15 a finding we also observed;
however, in the MCF-7 cell assay inhibition of atRA
metabolism does occur with IC50 activity determined as
7 µM. R115866 was not particularly active as an
inhibitor of atRA metabolism in the liver microsomal
assay (IC50 ) 9 µM); however, in the MCF-7 assay it
was a very potent RAMBA (IC50 ) 5 nM). The data
obtained for liarozole and R115866 were comparable
Discussion
The 2-benzyl derivatives displayed potent inhibitory
activity of atRA metabolism using the rat liver microso-
mal assay. Inhibition of atRA metabolism in the MCF-7
assay was observed for only one of the 2-benzyl deriva-

RAMBAs
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23 7127
Table 2. IC50 Data for the Novel Benzyltetralone Derivatives
Using Rat Liver Microsomal and MCF-7 Assays
a IC50 values are the average ((5%) of two experiments. b Data
from ref 24.
tives, 2-(4-hydroxybenzyl)-6-methoxy-3,4-dihydro-2H-
naphthalen-1-one (6), and three of the 2-benzylidene
derivatives, 2-[1-(2-bromophenyl)methylidene]-6-meth-
oxy-3,4-dihydro-2H-naphthalen-1-one (2c), 2-[1-(4-hy-
droxyphenyl)methylidene]-6-methoxy-3,4-dihydro-2H-
naphthalen-1-one (5), and 6-hydroxy-2-[1-(4-hydroxy-
phenyl)methylidene]-3,4-dihydro-2H-naphthalen-1-one
(10a); that is, inhibitory activity with the two assay
systems did not generally correlate. The lack of activity
of the 2-benzylidine derivatives in the rat microsomal
assay may be related to a lack of flexibility or electronic
effects resulting from the presence of an R,â-unsatur-
ated ketone. Inhibition of atRA metabolism in the rat
liver microsomal assay requires inhibition of the 1A1/
2, 2A6, and 3A4 CYP isoforms. However, in the MCF-7
cell assay, expression of the RA-metabolizing enzyme
CYP26A1 is induced by atRA (10-7 M); therefore,
activity in the MCF-7 cell assay is more relevant, being
a measure of human atRA metabolism by the specific
CYP26A1 enzyme. The results obtained using both
assays, particularly with reference to the potent CYP26
inhibitor R115866, would indicate that the liver mi-
crosomal assay is not a good model for human atRA
metabolism. However, a comparison of results of the two
assays can be used to consider selectivity. The inhibitory
activity of the 4-hyroxy derivative (6) was retained
(relative to standards) (IC50: liver microsomes, 0.4 µM;
MCF-7 cells, 5 µM) between the two assay systems,
suggesting a broad spectrum of inhibitory activity for
the CYP forms involved, including 1A1/2, 2A6, 3A4, and
CYP26A1 isoforms. The benzylidene derivatives 2c, 5,
and 10a (IC50: liver microsomes, >20 µM; MCF-7 cells,
7-9 µM) would appear to be more specific for CYP26A1
with minimal inhibition of the 1A1/2, 2A6, and 3A4
forms.
Figure 5. (a) Hydrogen (red) and transition metal (purple)
interactions between the 4-hydroxyphenyl substituent of
compound 5 and the active site of CYP26A1. (b) Hydrophobic
(green lines) interactions between the 2-bromotetralone de-
rivative 2c and the active site of CYP26A1.
recently crystallized human CYP3A432 as a template.
Docking interactions at the enzyme active site were
shown to be comparable with atRA with the tetralones
positioned in a hydrophobic tunnel. Additional interac-
tions were noted for the benzylidene derivatives 5 and
10a and the benzyl derivative 6 with coordinate binding
with the heme and hydrogen bonding to GLY300
through the 4-hydroxyphenyl substituent. The 2-bro-
mobenzylidene derivative 2c interacted at the active site
solely by hydrophobic interactions (Figure 5).
The enhanced activity of tetralones 5, 6, and 10a may
be due to the interaction of the 4-hydroxyphenyl sub-
stituent with GLY300 and the heme transition metal,
resulting in enhanced binding. Interaction through the
4-hydroxy substituent may mimic the interaction of the
4-hydroxylated product of the natural atRA substrate.
The activity of the 2-bromo derivative 2c probably
results from tighter binding through hydrophobic in-
teractions and the increased size of 2c due to the bulk
of the bromo substituent, inducing a tighter fit at the
active site.
To rationalize the results obtained, FlexX30 docking
studies of all the tetralone derivatives were performed
using a human CYP26A1 model,31 built using the

7128 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23
Yee et al.
reverse transcriptase, and nuclease-free water (to give a total
reaction volume of 40 µL) was incubated at 42 °C for 60 min
in a thermocycler. The newly synthesized cDNA was then used
for PCR analysis.
Experimental Section
General Procedures. Phenol red-free RPMI-1640 medium,
fetal calf serum (FCS), penicillin-streptomycin, and fungizone
were purchased from Gibco Europe Ltd. (Paisley, U.K.). Tri
Reagent was purchased from Sigma-Aldrich (MO).
To amplify a 184 base pair segment of CYP26A1 cDNA,
primers were 5′-GCTGAAGAGTAAGGGTTTAC-3′ (sense) and
5′-CTTGGGAATCTGGTATCCAT-3′ (antisense).21 â-Actin was
used as the endogenous control. Amplification was performed
using the PCR amplification kit by Promega (Promega, U.K.).
The PCR reaction mixture contained 2.5 µL of cDNA, 2.5 µL
of 10× reverse transcription 10× buffer, 0.25 µL of sense
primer (100 µM), 0.25 µL of antisense primer (100 µM), 0.5
µL of dNTP mix (10 mM), 0.2 µL of Taq DNA polymerase (5
U/µL), and nuclease-free water (to give a total reaction volume
of 25 µL). Taq DNA polymerase was activated at 94 °C for 5
min. The mixtures were then subjected to 30 cycles of
amplification. Each cycle conditions were as follows: 94 °C
for 1 min (denaturation), 55 °C for 1 min (annealing), and 72
°C for 2 min (elongation). An extra extension step was included
(72 °C for 10 min) at the end of the incubation. Negative control
of cDNA synthesis was carried out under the same experi-
mental conditions but in the absence of AMV reverse tran-
scriptase.
[11,12-3H]-All trans-retinoic acid (37 MBq/mL) was pur-
chased from Perkin-Elmer Life Science (U.K.). All-trans-
retinoic acid, NADPH, butylated hydroxyanisole, and keto-
conazole were obtained from Sigma Chemical Company (U.K.).
Acetic acid, ammonium acetate. and Optisafe 3 scintillation
fluid were obtained from Fisher Scientific (U.K.). All solvents
used for chromatography were HPLC grade from Fisher
Scientific (U.K.).
1H and 13C NMR spectra were recorded with a Brucker
Avance DPX300 spectrometer operating at 300 and 75 MHz,
with Me4Si as internal standard. Mass spectra were deter-
mined by the EPSRC mass spectrometry center (Swansea,
U.K.). Microanalyses were determined by Medac Ltd. (Surrey,
U.K.). Flash column chromatography was performed with
silica gel 60 (230-400 mesh) (Merck), and TLC was carried
out on precoated silica plates (kiesel gel 60 F254, BDH).
Compounds were visualized by illumination under UV light
(254 nm) or by the use of vanillin stain followed by charring
on a hot plate. Melting points were determined on an electro-
thermal instrument and are uncorrected. All solvents were
dried prior to use as described by the handbook Purification
of Laboratory Chemicals33 and stored over 4 Å molecular sieves
under nitrogen.
An amount of 20 µL of the PCR products was separated on
a 1.5% (w/v) agarose gel containing ethidium bromide and
separated at 100 V. The bands were visualized under UV light
illumination.
MCF-7 (CYP26A1) Assay for Inhibition of Metabolism
of atRA. MCF-7 cells were seeded in 12-well cell culture plates
(Cornings Inc., New York) at 2.5 × 105 cells per well in a total
volume of 1.5 mL. Cells were allowed to adhere to the well for
24 h. After 24 h, the medium from each well was removed,
washed once with phosphate buffered saline (PBS), and
replaced by fresh medium plus 10 µL of inhibitor/solvent
(acetonitrile) and 10 µL of atRA (to give a final concentration
of 1 × 10-7 M atRA and 0.1 µCi [11,12-3H]-all-trans retinoic
acid). The plates were foil-wrapped and incubated at 37 °C
for 9 h. Each treatment was performed in duplicate. The
incubation was stopped by addition of 1% acetic acid (100 µL/
well), the medium was removed into separate glass tubes, and
200 µL of distilled water was added to each well. The cells
were scraped off, and the contents were added to the appropri-
ate glass tube. This procedure was repeated with a further
400 µL of water but without scraping. Ethyl acetate containing
0.05% (w/v) butylated hydroxyanisole (2 × 2 mL) was added
to each tube. After vortexing for 15 s, the tubes were spun
down at 3000 rpm for 15 min. The organic layer was then
evaporated using a Christ centrifuge connected to a vacuum
pump and a multitrap at -80 °C.
High-Performance Liquid Chromatography (HPLC).
The HPLC system was equipped with a high-pressure pump
(Milton-Roy pump) and an injector with a 50 µL loop connected
to a â-RAM radioactivity detector connected to a Compaq
computer running Laura data acquisition and analysis soft-
ware. This enabled on-line detection and quantification of
radioactive peaks. The HPLC column (10 µm C18 µBondapak,
3.9 mm × 300 mm, HPLC column from Waters, U.K.) operat-
ing at ambient temperature was used to separate the metabo-
lites that were eluted with acetonitrile/1% ammonium acetate
in water/acetic acid (75:25:0.1 v/v/v) at a flow rate of 1.9 mL/
min. The Ecoscint was used as the flow scintillation fluid.
The numbering of compounds for H and 13C NMR charac-
1
terization and assignment of 1H and 13C NMR for all com-
pounds is provided in the Supporting Information.
Enzyme Assays. Microsomal Assay for Metabolism of
All-Trans Retinoic Acid. The assay performed was a modi-
fication of the general procedure previously described.28 Tubes
in duplicate, with a total volume of 500 µL, containing [11,-
12-3H]-all-trans-retinoic acid (10 µL, obtained from a stock
mixture containing 15 µL of [11,12-3H]-all trans-retinoic acid
and 1 mL of 120 µM unlabeled all-trans-retinoic acid), inhibitor
(10 µL, dissolved in acetonitrile), NADPH (4 mM, 50 µL),
phosphate buffer (50 mM, pH 7.4, 410 µL), and rat liver
microsomes (20 µL) were incubated in a shaking water bath
for 30 min at 37 °C. The reaction was terminated by the
addition of acetic acid (1% v/v, 100 µL). Then ethyl acetate (2
mL) containing 0.02% butylated hydroxylanisole was added,
and the tubes were vortexed for 10 s. The tubes were left for
5 min at room temperature before removal of the organic layer
(1.5 mL) from each tube and transfer to another set of tubes.
The organic layer was then evaporated using a Christ centri-
fuge connected to a vacuum pump and a multitrap at -80 °C.
MCF-7 Cell Culture. Human MCF-7 breast cancer cells
were cultured in phenol red-free RPMI 1640 medium supple-
mented with 5% (v/v) charcoal free fetal calf serum, antibiotics
(penicillin and streptomycin), and fungizone at the same
concentration of 10 iU/mL. Cells were grown in a humidified
incubator (5% CO2, 95% air) at 37 °C.
Reverse Transcriptase Polymerase Chain Reaction
(RT-PCR). MCF-7 cells were seeded in a 60 mm culture dish
at a concentration of 1 × 106 cells in 3 mL of medium. Cells
were allowed to adhere to plastic for 24 h. The cells were
further incubated with 10-7 M all-trans-retinoic acid for 9 h
before total cellular RNA was isolated by using Tri Reagent
following the manufacturer’s instructions. Total RNA amounts
Chemistry. General Procedure for the Condensation
of Tetralone (1 or 8) with Benzaldehyde (R-C6H4CHO
or THPO-C6H4CHO) for Preparation of the Benzylidene
Derivatives 2a, 2b, 2c, 9a, 9b, 13a, 13b. A mixture of the
tetralone (1 or 8, 4 mmol) and benzaldehyde (R-C6H4CHO or
THPO-C6H4CHO, 4 mmol) in 4% ethanolic KOH (10 mL) was
stirred at room temperature for 1-4 h. The purification is
described for each benzylidene product.
2-{1-[2,5-Di(trifluoromethyl)phenyl]methylidene}-6-
methoxy-3,4-dihydro-2H-naphthalen-1-one (2a). A mix-
ture of 6-methoxy-3,4-dihydro-2H-naphthalen-1-one (1) and
2,5-di(trifluoromethyl)benzaldehyde was stirred at room tem-
perature for 1 h. The resulting white precipitate was collected,
were quantified by measuring absorbance at 260 nm. The A260
/
A280 nm absorption ratio was greater than 1.7. Denaturing
agarose gel electrophoresis was performed to verify the
integrity of RNA. The intensity of the 28S rRNA band was
twice that of the 18S rRNA band stained by eithidium bromide.
Total RNA was used to synthesize cDNA using the manufac-
turer’s protocol provided with an AMV reverse transcriptase
(Promega, U.K.) and performed in a thermocycler (Perkin-
Elmer). Total RNA (2 µg) in the presence of 8 µL of MgCl2 (25
mM), 2 µL of oligo(dT)15 primer (500 ng/µL), 4 µL of reverse
transcription 10× buffer, 4 µL of dNTP mix (10 mM), 1 µL of
recombinant RNasin ribonuclease inhibitor, 1.6 µL of AMV

RAMBAs
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23 7129
washed with water, and finally recrystallized with acetone to
give 2-{1-[2,5-di(trifluoromethyl)phenyl]methylidene}-6-meth-
oxy-3,4-dihydro-2H-naphthalen-1-one (2a) as a white solid.
Yield, 0.40 g (24%); mp 98-100 °C; Rf ) 0.69 (petroleum ether/
ethyl acetate 3:1). Anal. (C20H14F6O2) C, H.
(51%); mp 165-167 °C [lit. mp24 166-168 °C]. Analytical data,
including assignment of 1H and 13C NMR, are provided in
Supporting Information.
6-Methoxy-2-{1-[4-(tetrahydro-2H-2-pyranyloxy)phenyl]-
methylidene}-3,4-dihydro-2H-naphthalen-1-one (4). A mix-
ture of 4-hydroxybenzaldehyde (5.0 g, 40.94 mmol), 3,4-
dihydro-2H-pyran (8.27 g, 98.26 mmol), acetic anhydride (1
mL), and p-toluenesulfonic acid (50 mg) in diethyl ether (150
mL) was stirred at room temperature for 2 h. The resulting
light-brown suspension turned into a clear-brown solution on
stirring at room temperature. The organic layer was then
washed with 2% KOH (2 × 100 mL) and water (2 × 100 mL),
dried with MgSO4, filtered, and reduced in vacuo to give a
yellow residue. Purification by column chromatography (pe-
troleum ether/ethyl acetate, 100:0 increasing to 87.5:12.5) gave
4-(tetrahydropyran-2-yloxy)benzaldehyde as a colorless syrup.
Yield, 6.37 g (76%).
A mixture of the 6-methoxy-3,4-dihydro-2H-naphthalen-1-
one (1) (3.46 g, 19.64 mmol) and 4-(tetrahydropyran-2-yloxy)-
benzaldehyde (4.05 g, 19.64 mmol) in 4% ethanolic KOH (80
mL) was stirred at room temperature for 12 h. The resulting
precipitate was collected, washed with water, and finally
recrystallized with acetone to give the product (4) as a light-
brown solid. Yield, 3.00 g (42%); mp 100-102 °C; Rf ) 0.47
(petroleum ether/ethyl acetate 3:1). Anal. (C23H24O4) C, H.
2-[1-(4-Hydroxyphenyl)methylidene]-6-methoxy-3,4-
dihydro-2H-naphthalen-1-one (5). Aqueous hydrochloric
acid (2 M, 15 mL) was added to a solution of 6-methoxy-2-{1-
[4-(tetrahydro-2H-2-pyranyloxy)phenyl]methylidene}-3,4-dihy-
dro-2H-naphthalen-1-one (4) (2 g, 5.49 mmol), in 2-butanone/
ethyl acetate (1:1 v/v, 40 mL), and the mixture was stirred at
100 °C for 1 h. The yellow solution was evaporated in vacuo,
and the resulting yellow solid was washed with water/
methanol (2:1 v/v, 40 mL) to give 2-[1-(4-hydroxyphenyl)-
methylidene]-6-methoxy-3,4-dihydro-2H-naphthalen-1-one (5)
as a yellow powder. Yield, 1.39 g (90%); mp 172-174 °C; Rf )
0.22 (petroleum ether/ethyl acetate 3:1). Anal. (C18H16O3‚
0.5H2O) C, H.
General Procedure for the Suzuki Coupling Prepara-
tion of the Benzylidene Derivatives 15a and 15b. A 2 M
aqueous Na2CO3 (13.0 mL) sample was added to a solution of
13a or 13b (3.63 mmol) in toluene (40 mL). The mixture was
bubbled with nitrogen for 1 min, and then Pd(PPh3)4 (0.18
mmol) was added to the mixture. Phenylboronic acid (7.26
mmol) in ethanol (10 mL) was added to the above mixture,
and the mixture was refluxed at 100 °C for 5 h. After the
reaction was complete, the residual borane was oxidized by
the addition of H2O2 (30%, 3.5 mL) at room temperature for 1
h. The crude product was extracted with CH2Cl2 (100 mL) and
water (3 × 100 mL). The organic layer was dried with MgSO4,
filtered, and reduced in vacuo to give an oily residue. Purifica-
tion by flash column chromatography (petroleum ether/ethyl
acetate 87.5:12.5; dichloromethane/methanol 99:1) gave 2-bi-
phenyl-2-yl- and 2-biphenyl-4-ylmethylene-6-(tetrahydropyran-
2-yloxy)-3,4-dihydro-2H-naphthalen-1-ones in 87% and 50%
yield, respectively. Full analytical data is provided in Sup-
porting Information.
Aqueous hydrochloric acid (2 M, 15 mL) was added to a
solution of the biphenyl-2-ylmethylene-6-(tetrahydropyran-2-
yloxy)-3,4-dihydro-2H-naphthalen-1-one (2.92 mmol) in 2-bu-
tanone/ethyl acetate (1:1 v/v, 40 mL), and the mixture was
stirred at 100 °C for 1 h. The yellow solution was evaporated
in vacuo, and the resulting yellow solid was washed with
water/methanol (2:1 v/v, 40 mL) to give 2-biphenyl-2-yl- and
2-biphenyl-4-yl-methylene-6-hydroxy-3,4-dihydro-2H-naphtha-
len-1-one (15a and 15b) as a yellow solid in yields of 91% and
95%, respectively.
6-Methoxy-2-[1-(2-methylphenyl)methylidene]-3,4-di-
hydro-2H-naphthlalen-1-one (2b). A mixture of 6-methoxy-
3,4-dihydro-2H-naphthalen-1-one (1) and O-tolualdehyde was
stirred at room temperature for 72 h. The resulting brown
solution was evaporated in vacuo to yield a brown syrup. The
crude product was extracted with CH2Cl2 (100 mL) and water
(2 × 100 mL). The organic layer was dried with MgSO4,
filtered, and reduced in vacuo to give a brown residue. The
brown residue was then purified by column chromatography
(petroleum ether/ethyl acetate, 100:0 increasing to 87.5:12.5)
to give 6-methoxy-2-[1-(2-methylphenyl)methylidene]-3,4-di-
hydro-2H-naphthlalen-1-one (2b) as a yellow solid. Yield, 1.57
g (71%); mp 88-90 °C; Rf ) 0.65 (system petroleum ether/
ethyl acetate 3:1). Anal. (C19H18O2) C, H.
2-[1-(2-Bromophenyl)methylidene]-6-methoxy-3,4-di-
hydro-2H-naphthalen-1-one (2c). A mixture of 6-methoxy-
3,4-dihydro-2H-naphthalen-1-one (1) and 2-bromobenzalde-
hyde was stirred at room temperature for 2 h. The resulting
precipitate was collected, washed with water, and finally
recrystallized from acetone to give 2-[1-(2-bromophenyl)meth-
ylidene]-6-methoxy-3,4-dihydro-2H-naphthalen-1-one (2c) as
white crystals. Yield, 3.22 g (68%); mp 78-80 °C; Rf ) 0.62
(petroleum ether/ethyl acetate 3:1). Anal. (C18H15BrO2) C, H.
6-(Tetrahydro-2H-2-pyranyloxy-2-{1-[4-(tetrahydro-
2H-2-pyranyloxy)phenyl]methylidene}-3,4-dihydro-2H-
naphthalen-1-one (9a). A mixture of 6-(tetrahydropyran-2-
yloxy)-3,4-dihydro-2H-naphthalen-1-one (8) and 4-(tetrahydro-
pyran-2-yloxy)benzaldehyde (see preparation of compound 4)
was stirred at room temperature for 1 h. The resulting
precipitate was collected, washed with water, and finally
purified by column chromatography (petroleum ether/ethyl
acetate, 100:0 increasing to 87.5:12.5) to give the product (9a)
as a colorless syrup. Yield, 1.00 g (41%); Rf ) 0.51 (petroleum
ether/ethyl acetate 4:1). HRMS (ES+) Calcd for C27H30O5 [M
+ H]+ 435.2166. Found 435.2165.
2-{1-[4-(Dimethylamino)phenyl]methylidene}-6-(tet-
rahydro-2H-2-pyranyloxy)-3,4-dihydro-2H-naphthalen-
1-one (9b). A mixture of 6-(tetrahydropyran-2-yloxy)-3,4-
dihydro-2H-naphthalen-1-one (8) and 4-(dimethylamino)-
benzaldehyde was stirred at room temperature for 72 h. The
resulting brown solution was evaporated in vacuo to yield a
brown syrup. The crude product was extracted with CH2Cl2
(100 mL) and water (2 × 100 mL). The organic layer was dried
with MgSO4, filtered, and reduced in vacuo to give a brown
residue. The brown residue was then purified by column
chromatography (dichloromethane/methanol, 100:0 increasing
to 99:1) to give the product (9b) as an orange solid. Yield, 1.72
g (35%); mp 103-105 °C; Rf ) 0.59 (petroleum ether/ethyl
acetate 3:1). Anal. (C24H27NO3) C, H, N.
2-[1-(2-Bromophenyl)methylidene]-6-(tetrahydro-2H-
2-pyranyloxy)-3,4-dihydro-2H-naphthalen-1-one (13a). A
mixture of 6-(tetrahydropyran-2-yloxy)-3,4-dihydro-2H-naph-
thalen-1-one (8) and 2-bromobenzaldehyde was stirred at room
temperature for 4 h. The resulting precipitate was collected,
washed with water, and finally recrystallized from acetone to
give 2-[1-(2-bromophenyl)methylidene]-6-(tetrahydro-2H-2-
pyranyloxy)-3,4-dihydro-2H-naphthalene-1-one (13a) as a white
solid. Yield, 7.60 g (87%); mp 76-78 °C; Rf ) 0.66 (petroleum
ether/ethyl acetate 3:1). Anal. (C22H21BrO3‚0.5H2O) C, H.
2-(4-Bromobenzylidene)-6-(tetrahydropyran-2-yloxy)-
3,4-dihydro-2H-naphthalen-1-one (13b). A mixture of 6-(tet-
rahydropyran-2-yloxy)-3,4-dihydro-2H-naphthalen-1-one (8)
and 4-bromobenzaldehyde was stirred at room temperature
for 1 h. The resulting precipitate was collected, washed with
water, and finally recrystallized from ethanol to give 2-(4-
bromobenzylidene)-6-(tetrahydropyran-2-yloxy)-3,4-dihydro-
2H-naphthalen-1-one (13b) as a white solid. Yield, 4.55 g
2-Biphenyl-2-ylmethylene-6-hydroxy-3,4-dihydro-2H-
naphthalen-1-one (15a). Mp 221-223 °C; Rf ) 0.05 (petro-
leum ether/ethyl acetate 3:1). Anal. (C23H18O2) C, H.
2-Biphenyl-4-ylmethylene-6-hydroxy-3,4-dihydro-2H-
naphthalen-1-one (15b). Yield, 0.38 g (95%); mp 258-260
°C; Rf ) 0.05 (petroleum ether/ethyl acetate 3:1). Anal.
(C23H18O2) C, H.

7130 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23
Yee et al.
General Procedure for the Reductive Hydrogenation
of Benzylidene Derivatives for the Preparation of Ben-
zyl Derivatives 3a, 3b, 6, 11, 12, 16a, 16b. A mixture of
benzylidene (2 mmol) and 10% palladium on charcoal (56 mg)
in methanol (200 mL) was shaken in an atmosphere of
hydrogen at room temperature for 1 h. Palladium was removed
by filtration through a bed of Celite, and the filtrate was
concentrated in vacuo. The purification is described for each
benzyl derivative.
2-[2,5-Di(trifluoromethyl)benzyl]-6-methoxy-3,4-dihy-
dro-2H-naphthalen-1-one (3a). Purification by column chro-
matography (petroleum ether/ethyl acetate, 100:0 increasing
to 87.5:12.5) gave a white residue with two close spots by TLC.
The impure compound was further purified using preparative
TLC to give 2-[2,5-di(trifluoromethyl)benzyl]-6-methoxy-3,4-
dihydro-2H-naphthalen-1-one (3a) as a white solid. Yield, 19
mg (6%); mp 48-50 °C; Rf ) 0.72 (petroleum ether/ ethyl
acetate 1:1). HRMS (ES+) Calcd for C20H16F6O2 [M + H]+
403.1127. Found 403.1121.
6-Methoxy-2-(2-methylbenzyl)-3,4-dihydro-2H-naph-
thalen-1-one (3b). Purification by column chromatography
(petroleum ether/ethyl acetate, 100:0 increasing to 80:20) gave
the product (3b) as a yellow syrup. Yield, 0.43 g (77%); Rf )
0.71 (petroleum ether/ethyl acetate 3:1). Anal. (C19H20O2‚
0.1H2O) C, H.
2-(4-Hydroxybenzyl)-6-methoxy-3,4-dihydro-2H-naph-
thalen-1-one (6). Purification by trituration with acetone gave
2-(4-hydroxybenzyl)-6-methoxy-3,4-dihydro-2H-naphthalen-1-
one (6) as a hygroscopic solid. Yield, 0.34 g (57%); Rf ) 0.30
(petroleum ether/ethyl acetate 1:1). Anal. (C18H18O3‚0.3H2O)
C, H.
6-(Hydroxy-2-(4-hydroxybenzyl)-3,4-dihydro-2H-naph-
thalen-1-one (11). Purification by column chromatography
(dichloromethane/methanol, 100:0 increasing to 97:3) gave an
off-white fluffy solid that was triturated with acetone to give
6-(hydroxy-2-(4-hydroxybenzyl)-3,4-dihydro-2H-naphthalen-1-
one (11) as a light-brown solid. Yield, 30 mg (6%); mp 189-
191 °C; Rf ) 0.05 (petroleum ether/ethyl acetate 1:1). Anal.
(C17H16O3) C, H.
2-[4-(Dimethylamino)benzyl]-6-hydroxy-3,4-dihydro-
2H-naphthalen-1-one (12). Purification by column chroma-
tography (petroleum ether/ethyl acetate, 100:0 increasing to
50:50) gave a brown residue that was triturated with acetone
to give 2-[4-(dimethylamino)benzyl]-6-hydroxy-3,4-dihydro-2H-
naphthalen-1-one (12) as a brown solid. Yield, 42 mg (6%); mp
200-202 °C; Rf ) 0.10 (petroleum ether/ethyl acetate 3:1).
Anal. (C19H21NO2‚0.1H2O) C, H, N.
2-Biphenyl-2-ylmethyl-6-hydroxy-3,4-dihydro-2H-naph-
thalen-1-one (16a). Purification by column chromatography
(petroleum ether/ethyl acetate, 90:10 increasing to 65:35) gave
the product 2-biphenyl-2-ylmethyl-6-hydroxy-3,4-dihydro-2H-
naphthalen-1-one (16a) as a light-brown solid. Yield, 0.50 g
(83%); mp 38-40 °C; Rf ) 0.05 (petroleum ether/ethyl acetate
1:1). Anal. (C23H20O2‚0.3H2O) C, H.
2-Biphenyl-4-ylmethyl-6-hydroxy-3,4-dihydro-2H-naph-
thalen-1-one (16b). Purification by trituration with methanol
gave 2-biphenyl-4-ylmethyl-6-hydroxy-3,4-dihydro-2H-naph-
thalen-1-one (16b) as a white solid. Yield, 0.20 g (63%); mp
220-222 °C; Rf ) 0.32 (petroleum ether/ethyl acetate 1:1).
Anal. (C23H20O2) C, H.
6-(Tetrahydropyran-2-yloxy)-3,4-dihydro-2H-naphtha-
len-1-one (8). 8 was prepared as previously described from
6-methoxy-3,4-dihydronaphthalen-1(2H)-one (1).24
6-Hydroxy-2-[1-(4-hydroxyphenyl)methylidene]-3,4-di-
hydro-2H-naphthalen-1-one (10a). Aqueous hydrochloric
acid (2 M, 20 mL) was added to a solution of 6-(tetrahydro-
2H-2-pyranyloxy-2-{1-[4-(tetrahydro-2H-2-pyranyloxy)phenyl]-
methylidene}-3,4-dihydro-2H-naphthalen-1-one (9a) (2 g, 4.60
mmol) in 2-butanone/ethyl acetate (1/1 v/v, 40 mL), and the
mixture was stirred at 100 °C for 1 h. The yellow solution was
evaporated in vacuo, and the resulting yellow solid was washed
with water/methanol (2:1 v/v, 40 mL) to give the product 10a
as a yellow powder. Yield, 0.67 g (55%); mp 260-262 °C; Rf )
0.05 (petroleum ether/ethyl acetate 3:1). Anal. (C17H14O3.0.1H2O)
C, H.
2-{1-[4-(Dimethylamino)phenyl]methylidene}-6-hy-
droxy-3,4-dihydro-2H-naphthalen-1-one (10b). Aqueous
hydrochloric acid (2 M, 15 mL) was added to a solution of 2-{1-
[4-(dimethylamino)phenyl]methylidene}-6-(tetrahydro-2H-2-
pyranyloxy)-3,4-dihydro-2H-naphthalen-1-one (9b) (1.5 g, 3.97
mmol) in 2-butanone/ethyl acetate (1/1 v/v, 40 mL), and the
mixture was stirred at 100 °C for 1 h. The yellow solution was
evaporated in vacuo, and the resulting yellow solid was washed
with water/methanol (2:1, 40 mL) to give 2-{1-[4-(dimethy-
lamino)phenyl]methylidene}-6-hydroxy-3,4-dihydro-2H-naph-
thalen-1-one (10b) as a yellow solid. Yield, 0.82 g (70%); mp
210-212 °C; Rf ) 0.05 (petroleum ether/ethyl acetate 3:1).
2-[1-(2-Bromophenyl)methylidene]-6-hydroxy-3,4-di-
hydro-2H-naphthalen-1-one (14). Aqueous hydrochloric acid
(2 M, 15 mL) was added to a solution of 2-[1-(2-bromophenyl)-
methylidene]-6-(tetrahydro-2H-2-pyranyloxy)-3,4-dihydro-2H-
naphthalen-1-one (13a) (0.96 g, 2.42 mmol) in 2-butanone/ethyl
acetate (1:1 v/v, 30 mL), and the mixture was stirred at 100
°C for 1 h. The yellow solution was evaporated in vacuo and
the resulting yellow solid was washed with water/methanol
(2:1 v/v, 40 mL) to give 2-[1-(2-bromophenyl)methylidene]-6-
hydroxy-3,4-dihydro-2H-naphthalen-1-one (14) as a white
solid. Yield, 0.60 g (63%); mp 178-180 °C; Rf ) 0.20 (petroleum
ether/ethyl acetate 3:1). Anal. (C17H13BrO2) C, H.
Molecular Docking. Substrates were docked within the
active site of the homology model (built using the CYP3A4
template) using the FlexX docking program of SYBYL.30
Subsequent manipulation and interaction evaluation was
performed with MOE (Molecular Operating Environment)
software.35
Acknowledgment. For S.W.Y. we acknowledge the
ORS Awards Scheme for a United Kingdom Scholarship.
For M.G. and L.J. we acknowledge the Embassy of the
Arab Republic of Egypt and Tenovus Cancer Charity,
respectively, for the award of Ph.D. scholarships. We
also acknowledge the technical staff of the Tenovus
Cancer Centre for the preparation of cells and media,
and the EPSRC Mass Spectrometry Centre, Swansea,
U.K., for mass spectrometry data.
Supporting Information Available: X-ray crystallo-
graphic data for compound 2c, the numbering of compounds
1
1
for H and 13C NMR characterization, and assignment of H
and 13C NMR for all compounds described. This material is
available free of charge via the Internet at http://pubs.acs.org.
4-[(6-Methoxy-3,4-dihydro-2H-naphthalenyl)methyl]-
phenol (7). A mixture of 2-[1-(4-hydroxyphenyl)methylidene]-
6-methoxy-3,4-dihydro-2H-naphthalen-1-one (5) (1.0 g, 3.56
mmol) and 10% palladium on charcoal (75 mg) in methanol
(200 mL) was shaken in an atmosphere of hydrogen at room
temperature for 2 h. Palladium was removed by filtration
through a bed of Celite, and the filtrate was concentrated in
vacuo to give a yellow residue that was purified by column
chromatography (petroleum ether/ethyl acetate, 100:0 increas-
ing to 70:30) to give 4-[(6-methoxy-3,4-dihydro-2H-naphtha-
lenyl)methyl]phenol (7) as a white solid. Yield, 0.60 g (63%);
mp 84-86 °C; Rf ) 0.61 (petroleum ether/ethyl acetate 3:1).
Anal. (C18H20O2) C, H.
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