Synthesis and CYP24A1 inhibitory activity of (E)-2-(2-substituted benzylidene)- and 2-(2-substituted benzyl)-6-methoxy-tetralones

Article abstract of DOI:10.1016/j.ejmech.2010.07.001

A series of (E)-2-(2-substituted benzylidene)- and 2-(2-substituted benzyl)-6-methoxy-tetralones were prepared, using an efficient synthetic scheme, and evaluated for their inhibitory activity against cytochrome P450C24A1 (CYP24A1) hydroxylase. In general the reduced benzyl tetralones were more active than the parent benzylidene tetralones. The 2-ethyl and 2-trifluoromethyl benzyl tetralone derivatives (4c and 4b) showed optimal activity in this series with IC₅₀ values of 1.92 μM and 2.08 μM, respectively compared with the standard ketoconazole IC₅₀ 0.52 μM. The 2-bromobenzyl tetralone (4d) showed a preference for CYP27A1 (IC₅₀ 59 nM) over CYP24A1 (IC50 16.3 μM) and may be a useful lead in CYP27A1 inhibition studies. The 2-ethylphenyl benzyl derivative (9c), which showed weak activity against the wild type CYP24A1 (IC₅₀ 25.57 μM), exhibited enhanced inhibitory activity towards L148F and M416T mutants, this difference in activity for the L148F mutant has been explained using molecular modelling.

Full text of DOI:10.1016/j.ejmech.2010.07.001

European Journal of Medicinal Chemistry 45 (2010) 4427e4434
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and CYP24A1 inhibitory activity of (E)-2-(2-substituted benzylidene)-
and 2-(2-substituted benzyl)-6-methoxy-tetralones
,
Ahmed S. Aboraia a 1, Bart Makowski b, Alba Bahja a, David Prosser b, Andrea Brancale a, Glenville Jones b,
,
Claire Simons a
*
a Medicinal Chemistry, Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cardiff CF10 3NB, UK
b Department of Biochemistry, Queens University, Kingston, Ontario, Canada K7L 3N6
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of (E)-2-(2-substituted benzylidene)- and 2-(2-substituted benzyl)-6-methoxy-tetralones were
prepared, using an efficient synthetic scheme, and evaluated for their inhibitory activity against cyto-
chrome P450C24A1 (CYP24A1) hydroxylase. In general the reduced benzyl tetralones were more active
than the parent benzylidene tetralones. The 2-ethyl and 2-trifluoromethyl benzyl tetralone derivatives
Received 12 February 2010
Received in revised form
20 May 2010
Accepted 1 July 2010
Available online 8 July 2010
(4c and 4b) showed optimal activity in this series with IC50 values of 1.92
compared with the standard ketoconazole IC50 0.52 M. The 2-bromobenzyl tetralone (4d) showed
a preference for CYP27A1 (IC50 59 nM) over CYP24A1 (IC50 16.3 M) and may be a useful lead in
CYP27A1 inhibition studies. The 2-ethylphenyl benzyl derivative (9c), which showed weak activity
against the wild type CYP24A1 (IC50 25.57 M), exhibited enhanced inhibitory activity towards L148F and
mM and 2.08 mM, respectively
m
m
Keywords:
Benzylidene tetralones
Benzyl tetralones
CYP24A1
m
M416T mutants, this difference in activity for the L148F mutant has been explained using molecular
modelling.
CYP27A1
Enzyme inhibition
Molecular modelling
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
and derivatives [1,3]. Inhibitors of CYP24A1 are expected to extend
the t1/2 of calcitriol thereby enhancing endogenous levels of cal-
Vitamin D3 is converted to 25-hydroxycholecalciferol or calci-
diol in the liver, and then metabolised by the kidney to the active
form 1a,25-dihydroxycholecalciferol (1a,25(OH)2D3) or calcitriol
citriol, or vitamin D analogues, which may result in stabilised
CYP24A1 and enhanced stability of vitamin D compounds.
The challenge in the design of CYP24A1 inhibitors is designing
selective CYP24A1 inhibitors with respect to other CYPs including
CYP27A1 and CYP27B1.
CYP inhibitors fall in to two main categories, azole and non-
azole inhibitors. Azole inhibitors have a nitrogen heterocyclic ring,
commonly an imidazole or triazole, within the structure and it is
through a nitrogen of the heterocyclic ring that binding occurs with
the Fe3þ of the haem. Non-azole inhibitors lack this nitrogen
heterocycle with binding within the active site involving hydrogen
bonding and hydrophobic interactions, however the strength of
these interactions are usually less than that observed with azole
inhibitors resulting in less potent inhibitors. However, the potential
benefit of non-azole inhibitors is greater selectivity against other
CYP enzymes compared with azole inhibitors, which makes non-
azole inhibitors a valid area to explore.
(Fig. 1) [1]. Calcitriol then binds to the vitamin D receptor (VDR),
which mediates tissue-specific effects including enhancement of
intestinal calcium transport, maintenance of skeletal health and
epidermal integrity and immune cell regulation. Replacement cal-
citriol therapy has been used in chronic kidney disease, hyper-
proliferative skin diseases and osteoporosis, with clinical trials for
various forms of cancer and autoimmune conditions ongoing [2].
Although the potential of vitamin D as an antiproliferative drug
has been realised in psoriasis and parathyroid cell hyperplasia, an
anticancer treatment incorporating vitamin D remains a challenge
owing to increased drug resistance. Evidence is growing that this
resistance is caused by upregulation of the cytochrome P450
enzyme, CYP24A1, resulting in accelerated metabolism of calcitriol
Ketoconazole, a broad spectrum CYP inhibitor is an azole
compound with CYP24A1 inhibitory activity and approximately 4-
fold selectivity for CYP24A1 vs CYP27B1 [4]. Schuster et al. [4]
described the azole compound (R)-VID400 (Fig. 2), which dis-
played a 40-fold selectivity for CYP24A1 over CYP27B1. Calcitriol
* Corresponding author. Tel.: þ44 2920 876307; fax: þ44 2920 874149.
E-mail address: simonsc@cardiff.ac.uk (C. Simons).
URL: http://www.cardiff.ac.uk/phrmy/staffinfo/cs/homepage/cs.html
Present address: Medicinal Chemistry Department, Faculty of Pharmacy, Assiut
1
University, Assiut, Egypt.
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.07.001

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A.S. Aboraia et al. / European Journal of Medicinal Chemistry 45 (2010) 4427e4434
GI Absorption
Skin Synthesis
Vitamin D
Liver
CYP27A1
25(OH)D3
Skin, Prostate,
Colon, Breast
kidney
CYP27B1
Local
Cellular
Systemic
Calcium
1,25(OH)2D3
1,25(OH)2D3
Regulation
Homeostasis
CYP24
Paracrine,
Autocrine
Endocrine
Inactivated
Fig. 1. The active form of Vitamin D (calcitriol/1a,25-(OH)2D3) interacts with the VDR in an endocrine or autocrine fashion.
derivatives with sulfoximine modification of the 24-hydroxy group
(Fig. 2) resulted in compounds with CYP24A1 selectivity (75-fold
selectivity CYP24A1/CYP27B1) and low calcemic activity, deter-
mined using an in vivo rat model [5].
most potent tetralones were the 2-methyl and 2-phenyl-
substituted benzyl tetralones with IC50 values of 0.9 and 2.1
m
M
respectively (Fig. 3). This paper describes the further development
of these lead compounds to explore the effect of varying the 2-
substituents on IC50. A slight preference for a 6-methoxy rather
than a 6-hydroxy substituent in the naphthalene ring was noted;
therefore the 6-methoxy has been included.
The effects of CYP24 inhibitors at a molecular level with non-
azole compounds have been described by two groups. At Cardiff [6]
novel tetralone CYP24A1 inhibitors, combined with calcitriol, dis-
played enhanced antiproliferative activity in DU145 cells and
upregulation of VDR genes CYP24A1, p21waf1/cip1 and GADD45a
(Fig. 2). Swaimi et al. [7] studied the phytoestrogen genestein,
which, in combination with calcitriol, increased the t1/2 of calcitriol
and VDR levels resulting in significant enhancement of calcitriol-
mediated functional responses including upregulation of mRNA
levels of p21/WAF1, p21 and IGFBP-3 gene.
The benzylidene tetralones were found to be very poor inhibi-
tors in the rat kidney mitochondrial assay compared with the
saturated benzyl tetralones [8], this was in contrast to previous
studies with benzylidene 2-(pyridylmethylene)-1-tetralones which
showed potent inhibitory activity against aromatase (CYP19) [9]. As
a cell based CYP24A1 assay using Chinese hamster lung fibroblasts
(V79-4) stably expressing CYP24A1 was employed in this research
[10], both the benzylidene precursors and final benzyl derivatives
(Fig. 1) were evaluated, to determine any differences (species and
mitochondrial versus cell). For some of the compounds selectivity
was assessed by determining the inhibitory activity against
CYP27A1, and a preliminary study to investigate the inhibitory
activity against mutant CYP24A1 strains was also performed.
We have previously described a series of 2-substituted-3,4-
dihydro-2H-naphthalen-1-one (tetralone) derivatives with prom-
ising CYP24 inhibitory activity in a rat kidney mitochondrial assay,
compared with the standard ketoconazole (IC50 ¼ 20
mM) [8]. The
OH
OH
O
O
Lead compounds
HO
O
H3CO
OH
O
CH3
O
2
tetralone
genistein
6
O
H3CO
HO
R2
S
NH
R1
IC50 = 0.9
M
IC50 = 2.1 M
H
R1
Cl
N
Target compounds
N
H
N
H
O
R
O
R
2
2
R3
O
6
6
R3
R4
(R)-VID 400
H3CO
H3CO
HO
benzyl tetralones
benzylidene tetralones
24-sulfoximine vit D3 analogues
Fig. 2. Described CYP24A1 inhibitors.
Fig. 3. Lead and target 2-substituted tetralone derivatives.

A.S. Aboraia et al. / European Journal of Medicinal Chemistry 45 (2010) 4427e4434
4429
Docking experiments were performed to rationalise some of the
data obtained.
were more potent than ketoconazole (average IC50 ¼ 0. 52
m
M). The
IC50 values for the tetralones are summarised in Table 1, with the 2-
ethyl-substituted benzyl tetralone 4c having the highest potency
with an IC50 of 1.92 mM. A preliminary docking of the inhibitors
2. Results and discussion
reported here was performed using PLANTS [14] and a homology
model of CYP24A1 [15], shown in Fig. 4.
2.1. Chemistry
An assay assessing selectivity using the CYP27A1 cell line was
performed and the IC50 values from these assays are summarised in
Table 2. A preliminary assay assessing the inhibitory effect of some
of the inhibitors on mutants of CYP24A1 is shown in Fig. 5.
All the syntheses of the tetralone derivatives were performed by
the general methods outlined in Schemes 1 and 2. The synthesis
was based on the procedure described by our group [8,11].
The method employed for the preparation of the (E)-2-(2-
substituted benzylidene)-6-methoxytetralone derivatives (3aec)
involved direct condensation of commercially available tetralone
(1) with the appropriate benzaldehyde (2) in ethanolic KOH solu-
tion (Scheme 1) [12]. The reduced 2-(benzyl)-tetralones (4aec)
were readily obtained by hydrogenation with 10% Pd/C catalyst for
1 h, at approximately 30 psi (Parr hydrogenator) (Scheme 1).
Preparation of the bromo derivative 4d followed a three-step
procedure involving first the preparation of the ethyl carboxylate
(5) by reaction of 6-methoxy-1-tetralone (1) with NaH to abstract
2.2.1. CYP24A1 inhibitory data
Pairs of inhibitor structures e.g. 3 and 4, show a systematic
variation between a double and a single bond at the position of
attachment between the tetralone and benzyl ring. The inclusion of
a double bond at this position appeared to reduce the inhibitory
effect in the inhibitors, which is consistent with our previous study
[8]. For example, 4c (IC50 ¼ 1.92
m
M) was more potent than its
M) (Table 1). The double
double bond counterpart, 3c (IC50 ¼ 10.76
m
bond conjugates with the adjacent phenyl group resulting in
decreased conformational flexibility of the structure.
the acidic
reaction of the carbanion with diethyl carbonate. Introduction of
the bromophenyl used NaH to again abstract the acidic -proton of
a-proton of the tetralone, and subsequent nucleophilic
This loss of flexibility could inhibit the entrance of the inhibitor
through the access channel of CYP24A1, thus decreasing its access
to the enzyme cavity where it would exert its inhibitory effect.
Alternatively, the loss of conformational flexibility could prevent
a favourable conformation that stabilises the inhibitor inside the
enzyme cavity. Using a homology model of CYP24A1 [16] a molec-
ular docking was performed using PLANTS [14], and single bond
variants were found to have the capability of occupying the
hydrophobic region on the enzyme cavity defined by Leu148, Ile242
and Val391 (Fig. 4), while the oxygen atom of the methoxy group is
coordinating the haem iron. Furthermore, the phenyl moiety in the
S-enantiomers establishes another series of interactions with
Trp134, Pro392 and Ile149. Such hydrophobic interactions could
stabilise the inhibitor conformation within the enzyme, thus
increasing the inhibition capacity. It should be noted that both the
R-enantiomer of compounds 4 and the double bonds derivatives (3)
did not dock with the methoxy group coordinated with the haem-
Fe and these results were not considered for further examination.
The two most potent inhibitors had an ethyl (4c) and a tri-
fluoromethyl (4d) at the 2-position. When the 2-position is
substituted with a methyl (4a) or a phenyl (9a) group, the inhibitory
a
the tetralone (5) followed by reaction with 2-bromobenzyl
bromide. Decarboxylation of (6) was achieved on reaction with HBr
and acetic acid to give the required 2-bromobenzyl tetralone (4d),
however only in 29% yield (Scheme 1). The low yield resulted from
demethylation of the 6-methoxy group, however this by-product
was quantitatively converted to the required methoxy product (4d)
on reaction with potassium carbonate and methyl iodide.
The 6-aryl substituted benzylidene derivatives (8a and 8b) were
prepared by Suzuki coupling of the 2-bromo-benzylidene tetralone
(7) [8] with arylboronic acid in the presence of Pd(PPh3)4 catalyst
(Scheme 2) [13]. Reduction of 8a and 8b by hydrogenation with 10%
Pd/C catalyst for 1 h, at approximately 30 psi (Parr hydrogenator),
gave the reduced 2-(benzyl)-tetralones (9a) and (9c). The 2-styr-
ylbenzyl tetralone (9b) was prepared from 4d by Suzuki coupling as
described for the 2-aryl benzylidene derivatives 8a and 8b.
2.2. Biological results
Tetralones 3, 4, 7, 8 and 9 were tested at 6 h incubation times on
activity decreases (IC50 126.9 mM and 127.6 mM respectively). Such
the wild type CYP24A1 expressing cell line. None of the inhibitors
O
R
H
2
O
R
O
R
O
ii
i
H3CO
H3CO
H3CO
1
3a R = CH3
3b R = CF3
3c R = CH2CH3
4a R = CH3
4b R = CF3
4c R = CH2CH3
iii
O
O
O
Br
CO2Et
CO2Et
v
iv
H3CO
H3CO
RO
5
Br
6
4d R = CH3
4e R = OH
Scheme 1. Reagents and conditions: (i) 4% ethanolic KOH, 2e72 h (ii) 10% Pd/C, H2, methanol, 30 psi,1 h (iii) NaH, diethyl carbonate, toluee, reflux, 18 h (iv) NaH, 2-bromobenzyl
bromide, DMF, 60 ꢀC, 8 h (v) HBr, AcOH, 120 ꢀC, 1.5 h.

4430
A.S. Aboraia et al. / European Journal of Medicinal Chemistry 45 (2010) 4427e4434
O
R
O
R
O
Br
iii
i
H3CO
H3CO
H3CO
7
8a R = Ph
9a R = Ph
9b R = CH=CHPh
9c R = CH2CH2Ph
8b R = CH=CHPh
i
O
Br
H3CO
4d
Scheme 2. Reagents and conditions: (i) (a) Arylboronic acid, Pd(PPh3)4, toluene, 100 ꢀC, 5 h (b) H2O2, room temperature, 1 h (ii) 10% Pd/C, H2, methanol, 1 atm,1 h (iii) 10% Pd/C, H2,
methanol, 30 psi,1 h.
a result could indicate that the ethyl group has a stabilising effect on
the binding of the inhibitor, but unfortunately the docking studies
are inconclusive on this matter and it is not possible to identify
a specific structural justification to the observed activity profile.
The strong inhibitory capacity of 4b could reflect a trend
observed in inhibitor design; the addition of fluorine groups
increases the inhibitory capacity of a compound. It has been sug-
gested that this increase is not caused by a stronger enzyme-
einhibitor complex, but by increasing the cell-membrane
penetrating capacity and thus increasing the bioavailability.
However, many of the compounds appear to be hydrophobic and
should have little problem penetrating cell membranes. As such,
the fluorination may have a stabilising effect on the inhibitor inside
the enzyme resulting in more effective inhibition.
substituted benzyl tetralone (4d) displayed a much greater
potency towards CYP27A1 than CYP24A1 (276-fold selectivity),
likewise the 2-bromo-substituted benzylidene tetralone (7),
although considerably less potent, displayed a 28-fold selectivity
towards CYP27A1 (Table 2); such an outlier could be quite useful
in future inhibition studies of CYP27A1.
2.2.3. Mutant studies
A preliminary assay was performed to screen for the possibility
of a mutated residue causing a weak inhibitor to be more potent. It
was found that the 2-phenylethyl-substituted benzyl tetralone (9c)
was significantly more potent towards two mutants relative to the
wild type form of the enzyme (L148F and M416T) (Fig. 5). L148F is
a mutant strain characterised by a shift towards C24 metabolism,
coupled with an increase in calcitroic acid metabolism. The Leu148
residue is located in the hydrophobic pocket described earlier that
2.2.2. CYP27A1 assay
A potent and specific inhibitor of CYP24A1 would display a low
IC50 value towards CYP24A1 and a high IC50 value towards related
cytochrome P450s. CYP27A1 is a good enzyme to test for specificity
because of its structural similarity to CYP24A1, as well as the
structural similarity of their respective substrates (1
,25-(OH)2D3).
Upon comparison of IC50 values between CYP24A1 and
a-OH-D3 and
1
a
CYP27A1 (Table 2), it is clear that none of the most potent tetra-
lone inhibitors of CYP24A1 are particularly specific, i.e. they have
similar IC50 values for CYP27A1. Surprisingly, the 2-bromo-
Table 1
CYP24A1 inhibitory (IC50) data for the tetralone derivatives.
No.
R
IC50
(m
M)a
No.
R
IC50 (m
M)a
3a
3b
3c
7
8a
8b
CH3
CF3
CH2CH3
Br
Ph
480.1
11.22
10.76
122.0
111.1
5.08
4a
4b
4c
4d
9a
9b
9c
CH3
CF3
CH2CH3
Br
Ph
126.9
2.08
1.92
16.3
127.6
342.8
25.57
CH]CHPh
CH]CHPh
CH2CH2Ph
Fig. 4. Preliminary Docking of 4c into the CYP24A1 Homology Model Enzyme Cavity.
The phenyl group is able to occupy the hydrophobic region within the cavity. This
cavity is proximal to the hydrophobic residues Leu148, Ile 131 and Leu 129.
a
IC50 values are the average (ꢁ5%) of two experiments. Ketoconazole (IC50
M) was used as the standard for comparison.
0.52
m

A.S. Aboraia et al. / European Journal of Medicinal Chemistry 45 (2010) 4427e4434
4431
Table 2
CYP24A1 and CYP27A1 IC50 data for the tetralone derivatives.
Tetralone
IC50
(m
M)a CYP27A1
IC50 (m
M)a CYP24A1
7
4.33
122.0
16.3
2.08
1.92
5.08
4d
4b
4c
8b
0.059
5.34
8.23
5.69
a
IC50 values are the average (ꢁ5%) of two experiments.
could be responsible for inhibitor stabilization. In a wild-type
setting the Leu148 residue is thought to interact with the A-ring of
1
a,25-(OH)2D3 [15].
To investigate the increase in activity of 9c in the L148F mutant,
we have introduced the appropriate mutations in our model using
the rotamer explorer function in MOE [17]. From the docking
results of 9c it is possible to observe (Fig. 6) that the phenylalanine
side chain could establish a pep interaction with the aromatic ring
connected to the tetralone scaffold (in the S-configuration), leading
to an increased binding affinity of this compound in this specific
mutant.
Fig. 6. Docking of 9c into the ‘mutated’ L148F CYP24A1 Homology Model Enzyme
Cavity.
The M416T mutant also shows increased calcitroic acid
production capabilities, however the residue is thought to be
located near the access channel and in our model, it is just in
contact with the terminal aromatic ring of 9c. In this case, the
model with the appropriate mutation does not show a clear
structural advantage for the binding of this analogue, as with the
previous mutant. It is possible that the bulkier tetralone 9c (relative
to the other tetralones) might have a higher inhibitory capacity
with respect to this mutant because of a wider access channel. As
a result, 9c may be able to access the CYP24A1 enzyme cavity with
greater ease and allow the interaction of its bulky R group with the
internal residues of the cavity.
3. Conclusions
The most potent benzyl tetralones displayed moderate CYP24A1
inhibitory activity in the described assay. Very different results
were obtained for the 2-methyl substituted benzyl tetralone (Fig. 3,
4a), with the promising CYP24A1 inhibitory activity (IC50 0.9
compared with ketoconazole (IC50 20 M) previously seen in the rat
microsomal assay [8], not translating to the cell based assay
employed here (4a IC50 126.9 M; ketoconazole IC50 0.52 M),
suggesting than the enzyme active site cavity in rat is probably
different enough to affect IC50, this is perhaps supported by the
mutant study performed here with one amino acid mutation
resulting in notable differences in inhibitory activity (Fig. 5).
The inhibition of CYP24A1 by tetralones is still not as well
characterised as that of azole-based inhibitors, however with the
information gained from the docking and mutant experiments, it
may be possible to refine the compounds to increase the potency
and/or specificity of this class of inhibitors. The use of tetralones to
inhibit CYP24A1 may have unforeseen advantages over some of the
current published inhibitors, and it is worthwhile to pursue future
generations of such inhibitors. Not only could such inhibitors be
mM)
m
m
m
There appears to be an anomaly with the 2-phenyl-substituted
benzylidene (8a) and benzyl (9a) tetralones in that they do not
inhibit CYP24A1 activity in this assay. This could be a result of these
two inhibitors being so poor that the initial IC50 values were
actually a reflection of the decrease in cell viability and not of their
inhibitory capacity. In the CYP24A1 assays, a large amount of
ethanol was used at the 10ꢂ4 M data point (w50
mL/mL of medium)
because the inhibitor needed 1 mL of ethanol to dissolve 1 mg. This
may have led to a lower apparent IC50 value that was not carried
through in the mutant experiment.
Fig. 5. Inhibitor Capacity of Weak Novel Inhibitors on CYP24A1 Mutant Strains. An anomaly is apparent with MCC 179 (8a) and MCC180 (9a) where the activity is higher than the
non-inhibited wild type. The interesting result comes from an increase in potency of ketoconazole and 9c (MCC 183) towards L148F and M416T mutants.

4432
A.S. Aboraia et al. / European Journal of Medicinal Chemistry 45 (2010) 4427e4434
useful in a clinical setting, but they could also advance the under-
standing of the structure and binding mechanism of CYP24A1.
4.2. Chemistry
1H and 13C NMR spectra were recorded with a Brucker Avance
DPX500 spectrometer operating at 500 and 125 MHz, with Me4Si as
internal standard. Mass spectra were determined by the EPSRC mass
spectrometry centre (Swansea, UK). Microanalyses were determined
by Medac Ltd (Surrey, UK). Flash column chromatography was per-
formed with silica gel 60 (230e400 mesh) (Merck) and TLC was
carried out onprecoated silica plates (kiesel gel 60F254, BDH). Melting
points were determined on an electrothermal instrument and are
uncorrected. Compounds were visualised by illumination under UV
light (254 nm) or by the use of vanillin stain followed by charring on
a hotplate. All solvents were dried prior to use as-described by the
handbook Purification of Laboratory Chemicals [18] and stored over
4. Experimental
4.1. Pharmacology
4.1.1. CYP24A1and CYP27A1 preparation and assay
Cell culture: Chinese hamster lung fibroblasts (V79-4) stably
expressing either CYP24A1 (WT, L148F, M252L, V391L, or M416T)
[10] or CYP27A1 (WT) [16] were maintained and grown using
Dulbecco’s Modified Eagle Medium (DMEM) purchased from Invi-
trogen Life Technologies. The growth medium was supplemented
with fetal calf serum (FCS) as well as antibiotics (1% v/v) and
hygromycin (0.2% v/v) and glucose (1 g/500 mL). Cells were plated
on standard 100 mm cell culture dishes and were maintained in
a humidified atmosphere of 5% CO2 in air.
ꢀ
4 A molecular sieves, under nitrogen. We have previously described
compounds 3a, 4a, 5, 7a and 8a [8,19]. Compound 5 was prepared
according to the literature method [20].
Inhibitor preparations: Milligram quantities of the inhibitors
were dissolved in 100% ethanol, while ketoconazole was dissolved
in 0.05 M HCl. A 1 mg/mL stock solution was made up for each
inhibitor to be tested. Serial 1/10 dilutions were then made to give
stock solutions suitable for the low concentration incubations.
Incubations: Prior to incubation, cells were grown on P-150
plates until reaching w100% confluence. The cells were then sub-
cultured onto 6-well plates in the FCS-supplemented DMEM for
24 h. The incubation medium for the experiments was BSA sup-
plemented (1% w/v) DMEM containing the appropriate substrate;
The numbering of compounds for NMR characterisation is as
follows:
4.2.1. General procedure for the synthesis of (E)-2-(2-substituted
benzylidene)-6-methoxy-tetralone derivatives (3)
100,000 CPM per well of [1
containing cells and 10 M 1
cells. Each well also contained 1
b
a
-3H]1
-OH-D3 for the CYP27A1-containing
L of DPPD (100 M) to act as an
a,25-(OH)2D3 for the CYP24A1-
m
A mixture of the 6-methoxytetralone (1) (2.0 g, 11 mmol) and 2-
substituted benzaldehyde (2) (11 mmol) in 4% ethanolic KOH
(100 mL) was stirred at room temperature for 1 h. The resulting
precipitate was collected, washed with water and finally recrys-
tallised from ethanol.
m
m
anti-oxidant. Each assay was conducted with a ‘no inhibitor’, ‘dead
cell’ and ‘no cell’ control. The ‘dead cell’ control was prepared by
microwaving the plate of cells for 30 s prior to incubation and each
well contained 1 mL of incubation medium. Each inhibitor was
tested at 4 different concentrations; 1 ꢃ 10ꢂ4 M, 1 ꢃ 10ꢂ5 M,
1 ꢃ10ꢂ6 M, and 1 ꢃ10ꢂ7 M. The CYP24A1 assays were carried out at
an optimized 6 h time interval, while the CYP27A1 assay was
carried out at a 24 h interval which had been previously optimized.
The incubations were arrested by the addition of 2.5 mL of meth-
anol to each well.
4.2.1.1. (E)-2-(2-Trifluoromethylbenzylidene)-6-methoxy-tetralone (3b).
A cream crystalline solid was obtained. Yield: 2.48 g (68%), Rf 0.54
(petroleum ethereEtOAc 3:1 v/v); m.p. 81e83 ꢀC; 1H NMR:
d 8.17 (d,
J¼ 8.8 Hz,1H, H-8), 7.96 (s,1H, eC]CH-Ph), 7.75 (d, J¼ 7.9 Hz, 1H, H-30),
7.58 (t, J¼ 7.6 Hz, 1H, H-60), 7.47 (t, J¼ 7.7 Hz, 1H, H-50), 7.34 (d, J¼ 7.6 Hz,
1H, H-40), 6.91 (dd, J ¼ 2.5, 8.8 Hz, 1H, H-7), 6.72 (d, J ¼ 2.4 Hz, 1H, H-5),
Extraction: The incubation medium was extracted using
a modified Bligh and Dyer extraction [16]. For samples destined for
HPLC analysis (mainly samples taken from CYP27A1 assays), 2 mg of
3.89 (s, 3H, OCH3), 2.91 (m, 2H, CH2), 2.85 (m, 2H, CH2).13C NMR:
d189.2
(C, C]O), 163.8 (C, C-6), 146.0 (C), 138.2 (C), 135.2 (C), 132.3 (CH), 131.5
(CH), 130.9 (CH), 130.4 (CH), 129.34 (C, CeF), 129.1 (C, CeF), 127.9 (CH),
126.8 (C),126.1 (CH),125.0 (C),122.9 (C, CeF),113.5(CH),112.4(CH), 55.5
(CH3), 29.5 (CH2), 27.4 (CH2). Anal. Calcd. for C19H15O2F3$0.1H2O
(334.123): C, 68.30%, H, 4.59%. Found: C, 68.24%, H, 4.45%.
25-OH-D3 was added to each sample as an internal recovery stan-
dard for subsequent normalization of metabolite quantification.
The samples from the CYP24A1 assays were analysed using scin-
tillation counting of 0.5 mL of the aqueous phase (from the Bligh
and Dyer extraction). Beckman Coulter Ready SafeÔ Liquid Scin-
tillation Cocktail for Aqueous Samples (5 mL) was added to the
0.5 mL samples, and scintillation counting was performed using
a Beckman Coulter L6500 Multipurpose Scintillation Counter.
High performance liquid chromatography: To determine the
amount of metabolite formed in the CYP27A1 incubations, HPLC
paired with a photodiode array detector (Waters 996 PDA) or
4.2.1.2. (E)-2-(2-Ethylbenzylidene)-6-methoxy-tetralone (3c). A light
yellow crystalline solid was obtained. Yield: 2.33 g (72%), Rf 0.74
(petroleum ethereEtOAc 3:1 v/v); m.p. 78e80 ꢀC; 1H NMR:
d 8.18 (d,
J ¼ 8.7 Hz,1H, H-8), 7.97 (s,1H, eC]CH-Ph), 7.27 (m, 4H, Ar), 6.91 (dd,
J ¼ 2.5, 8.8 Hz,1H, H-7), 6.73 (d, J ¼ 2.4 Hz,1H, H-5), 3.90 (s, 3H, OCH3),
2.97 (m, 2H, CH2), 2.91 (m, 2H, CH2), 2.72 (q, J ¼ 7.6 Hz, 2H, CH2CH3),
1.21 (t, J ¼ 7.6 Hz, 3H, CH2CH3).13C NMR:
d 186.8 (C, C]O),163.6 (C, C-
a
RadioFlow Detector (LB509; EG&G Berthold, Bad Wilbad,
6),146.0 (C),143.8 (C),136.4 (C),135.0 (CH),134.6 (C),130.8 (CH),129.1
(CH), 128.5 (CH), 127.1 (C), 125.5 (CH), 113.4 (CH), 112.4 (CH), 55.5
(CH3), 29.7 (CH2), 27.3 (CH2), 26.7 (CH2CH3), 15.1 (CH2CH3). Anal.
Calcd. for C20H20O2 (292.377): C, 82.16%, H, 6.89%. Found: C, 82.25%,
H, 7.15%.
Germany) was used. The HPLC uses a Zorbax-Sil column (3 m;
m
6.2 ꢃ 80 mm) coupled to a Waters 1695 Separation Module, and
a solvent system of 91% hexane (v/v), 7% isopropanol (v/v), 2%
methanol (v/v) at a flow rate of 1.0 mL/min for a 30 min run time.
The organic phase of the Bligh and Dyer extraction was dried down
under N2 gas, dried with 400
m
L of ethanol, and then prepared in
4.2.2. General procedure for the synthesis of 2-(2-substituted
benzyl)-6-methoxy-tetralone derivatives (4 and 9c)
110 L of HPLC solvent. Empower software (Waters Corp.) was used
m
to integrate the peaks and determine the amount of metabolite
formed (ng) by analysing the peaks characteristic for vitamin D
A mixture of the (E)-2-(2-substituted benzylidene)-6-methoxy-
tetralone (3 or 8b) (2.0 g, 6 mmol) and 10% Pd on charcoal (160 mg)
in methanol (200 mL), was shaken in an atmosphere of hydrogen at
(lmax ¼ 265 nm, lmin ¼ 228 nm, and
3
¼ 18,300).

A.S. Aboraia et al. / European Journal of Medicinal Chemistry 45 (2010) 4427e4434
4433
room temperature for 1 h. Pd was removed by filtration over a bed
of celite and the filtrate was concentrated in vacuo to give a yellow
residue. Purification by flash column chromatography (petroleum
ethereEtOAc 90:10 v/v) gave the product.
Purification by flash column chromatography (petroleum ether-
eEtOAc 100:0 v/v increasing to 80:20 v/v) gave the product as
a thick yellow oil. Yield: 2.9 g (69%), Rf 0.69 (petroleum ether-
eEtOAc 3:1 v/v); 1H NMR:
d
8.09 (d, J ¼ 8.8 Hz, 1H, H-8), 7.55 (dd,
J ¼ 1.3, 7.9 Hz, 1H, Ar), 7.30 (dd, J ¼ 1.7, 7.6 Hz, 1H, Ar), 7.19 (m, 1H,
Ar), 7.06 (m, 1H, Ar), 6.85 (dd, J ¼ 2.5, 8.8 Hz, 1H, H-7), 6.62 (d,
J ¼ 2.5 Hz, 1H, H-5), 4.16 (m, 2H, OCH2CH3), 3.85 (s, 3H, OCH3), 3.69
(dd, J ¼ 14.2, 24.4 Hz, CeCH2eAr), 3.06 (m, 1H, H-4), 2.78 (m, 1H, H-
4), 2.61 (m, 1H, H-3), 2.05 (m, 1H, H-3), 1.17 (t, J ¼ 7.1 Hz, 2H,
4.2.2.1. 2-(2-Trifluoromethylbenzyl)-6-methoxy-tetralone
cream crystalline solid was obtained. Yield: 1.03 g (53%), Rf 0.60
(petroleum ethereEtOAc 3:1 v/v); m.p. 82e84 ꢀC; 1H NMR:
8.08
(4b). A
d
(d, J ¼ 8.8 Hz, 1H, H-8), 7.68 (d, J ¼ 7.9 Hz, 1H, H-30), 7.51 (t,
J ¼ 7.6 Hz, 1H, H-60), 7.42 (d, J ¼ 7.7 Hz, 1H, H-50), 7.34 (t, J ¼ 7.6 Hz,
1H, H-40), 6.87 (dd, J ¼ 2.5, 8.8 Hz, 1H, H-7), 6.69 (d, J ¼ 2.4 Hz, 1H,
H-5), 3.88 (s, 3H, OCH3), 3.80 (m, 1H, Hx), 2.93 (m, 2H, CHaHb-Ph),
CH2CH3). 13C NMR:
d
193.2 (C]O, C-1), 171.3 (C]O, C-10), 163.7 (C,
C-6), 145.8 (C), 136.9 (C), 132.9 (CH), 132.2 (CH), 130.7 (CH), 128.3
(CH), 127.3 (CH), 125.4 (C), 125.9 (C), 113.5 (CH), 112.3 (CH), 61.6
(CH2, ethyl), 58.7 (C, C-2), 55.4 (CH3, OCH3), 38.4 (CH2, CH2Ph), 30.1
(CH2, C-4), 26.5 (CH2, H-3), 14.0 (CH3, ethyl). LRMS (CIþ) m/z: 419.3
and 417.2 (81/79Br) [M þ H]þ, 339.2 and 337.3 (81/79Br) [M ꢂ Br]þ,
2.82 (m, 2H, CH2, H-3), 2.06 (m, 1H, CHAHB), 1.86 (m, 1H, CHAHB). 13
C
NMR: d 197.6 (C, C]O),163.5 (C, C-6),146.4 (C),139.3 (C),131.7 (CH),
131.6 (CH), 130.0 (CH), 129.3 (CeF), 129.1 (C, CeF), 126.2 (CH), 126.15
(CH), 126.1 (C), 125.7 (CeF), 123.5 (CeF), 113.2 (CH), 112.4 (CH), 55.4
(CH3), 49.0 (CH, CHx), 32.3 (CH2, CHaHbPh) 29.4 (CH2, C-3), 28.2
(CH2, CHAHB). Anal. Calcd. for C19H17O2F3 (334.337): C, 68.26%, H,
5.12%. Found: C, 68.03%, H, 5.13%.
249.2 and 247.2 (81/79Br) [M ꢂ 2-bromo C6H5CH2]þ. HRMS (CIþ):
79
Calc C21
H
BrO4 [M þ H]þ 417.0696. Found 417.0694.
22
4.2.4. 2-(2-Bromobenzyl)-6-methoxy-tetralone (4d)
A suspension of tetralone (6) (2.6 g, 6.23 mmol), 48% HBr
(13.76 mL) and 96% AcOH (11.5 mL) was stirred at 120 ꢀC for 1.5 h.
The mixture was cooled to room temperature and diluted with H2O
(5 mL). The reaction mixture was extracted with Et2O (3 ꢃ 50 mL);
the combined organic extracts were dried (MgSO4) and concen-
trated in vacuo. Purification by flash column chromatography
(petroleum ethereEtOAc 100:0 v/v increasing to 80:20 v/v) gave
the 2-(2-bromobenzyl)-6-methoxy-tetralone (4d) as the minor
product and the demethylated 2-(2-bromobenzyl)-6-hydroxy-tet-
ralone (4e) as the major product.
4.2.2.2. 2-(2-Ethylbenzyl)-6-methoxy-tetralone (4c). A dark orange
syrup was obtained. Yield: 1.49 g (64%), Rf 0.71 (petroleum ether-
eEtOAc 3:1 v/v); 1H NMR:
d
8.09 (d, J ¼ 8.8 Hz, 1H, H-8), 7.29e7.12
(m, 4H, Ar), 6.87 (dd, J ¼ 2.5, 8.8 Hz, 1H, H-7), 6.70 (d, J ¼ 2.4 Hz, 1H,
H-5), 3.88 (s, 3H, OCH3), 3.69 (dd, J ¼ 3.7, 14.2 Hz, 1H, CHaHb-Ph),
2.92 (m, 2H, CH2, H-3), 2.72 (q, J ¼ 7.4 Hz, 2H, CH2CH3) overlapping
(m, 1H, Hx), 2.55 (dd, J ¼ 10.5, 14.2 Hz, 1H, CHaHb-Ph), 2.12 (m, 1H,
CHAHB), 1.84 (m, 1H, CHAHB), 1.27 (t, J ¼ 7.6 Hz, 3H, CH2CH3). 13C
NMR: d 198.2 (C, C]O), 163.5 (C, C-6), 146.5 (C), 142.5 (C), 137.8 (C),
130.2 (CH), 130.0 (CH), 128.6 (CH), 126.5 (CH), 126.4 (C), 126.2 (CH),
113.2 (CH), 112.5 (CH), 55.4 (CH3), 48.7 (CH, CHx), 33.7 (CH2,
CHaHbPh) 29.3 (CH2, C-3), 28.0 (CH2, CHAHB), 25.5 (CH2CH3), 15.4
(CH2CH3). LRMS (EIþ) m/z: 295.4 [M þ H]þ, 294.3 [M]þ, 176.3
([M þ H] ꢂ [CH2ePheEt])þ, 175.31 ([M] ꢂ [CH2ePheEt])þ, 91.1
[C6H5CH2]þ, 77.2 [C6H5]þ. HRMS (ESþ): Calc C20H22O2 [M þ H]þ
295.1693. Found 295.1695.
4.2.5. 2-(2-Bromobenzyl)-6-methoxy-tetralone (4d)
A yellow oil was obtained. Yield: 0.55 g (26%), Rf 0.82 (petroleum
ethereEtOAc 3:1 v/v); 1H NMR:
d
8.07 (d, J ¼ 8.8 Hz, 1H, H-8), 7.58
(dd, J ¼ 1.0, 7.9 Hz,1H, Ar), 7.29 (m, 2H, Ar), 7.11 (m,1H, Ar), 6.85 (dd,
J ¼ 2.5, 8.8 Hz, 1H, H-7), 6.69 (d, J ¼ 2.5 Hz, 1H, H-5), 3.87 (s, 3H,
OCH3), 3.72 (dd, J ¼ 4.4, 13.9 Hz,1H, CHaHb-Ph), 2.90 (m, 2H, CH2, H-
3), 2.87 (m, 1H, Hx), 2.76 (dd, J ¼ 9.5, 13.8 Hz, 1H, CHaHb-Ph), 2.08
4.2.2.3. 2-(2-Phenethylbenzyl)-6-methoxy-tetralone (9c). A pale
orange waxy solid was obtained. Yield: 1.27 g (63%), Rf 0.78
(m, 1H, CHAHB), 1.88 (m, 1H, CHAHB). 13C NMR:
d 197.8 (C, C]O),
163.5 (C, C-6), 146.5 (C), 139.9 (C), 133.0 (CH), 131.7 (CH), 130.0 (CH),
127.9 (CH), 127.3 (CH), 126.2 (C), 124.9 (C), 113.2 (CH), 112.5 (CH),
55.4 (CH3), 47.7 (CH, CHx), 36.1 (CH2, CHaHb-Ph) 29.3 (CH2, C-3), 28.1
(CH2, CHAHB). LRMS (CIþ) m/z: 347.1 and 345.2 (81/79Br) [M þ H]þ,
(petroleum ethereEtOAc 4:1 v/v); 1H NMR:
d
8.09 (d, J ¼ 8.8 Hz, 1H,
H-8), 7.31e7.20 (m, 9H, Ar), 6.88 (dd, J ¼ 2.3, 8.8 Hz, 1H, H-5), 6.70
(d, J ¼ 2.3 Hz, 1H, H-7), 3.88 (s, 3H, OCH3), 3.69 (dd, J ¼ 3.0, 13.6 Hz,
1H, CHaHb-Ph), 3.05e2.83 (m, 6H, 3 ꢃ CH2), 2.59 (m, 2H, CH2), 2.11
267.2 and 265.3 (81/79Br) [M ꢂ Br]þ,177.4 and 175.3 (81/79Br) [M ꢂ 2-
79
(m, 1H, CHAHB), 1.83 (m, 1H, CHAHB). 13C NMR:
d
198.0 (C, C]O),
bromo C6H5CH2]þ. HRMS (CIþ): Calc C18
345.0485. Found 345.0484.
H
BrO2 [M
þ
H]þ
18
163.5 (C, C-6),146.5 (C),141.8 (C),136.9 (C),140.2 (C),138.1 (C),130.3
(CH), 130.0 (CH), 129.9 (CH), 129.5 (CH), 129.4 (CH), 129.3 (CH), 127.1
(CH), 126.7 (C), 126.4 (CH), 126.2 (CH), 113.2 (CH), 112.5 (CH), 55.4
(CH3), 48.8 (CH, CHx), 37.4 (CH2), 34.8 (CH2), 32.4 (CH2, CHaHbPh),
29.3 (CH2, C-3), 28.1 (CH2, CHAHB). LRMS (EIþ) m/z: 371.4.4
[M þ H]þ, 370.3 [M]þ, 176.3 ([M þ H] ꢂ [CH2ePheEt])þ, 175.31
([M] ꢂ [CH2ePheEt])þ, 105.3 [C6H5CH2CH2]þ, 91.1 [C6H5CH2]þ, 77.2
[C6H5]þ. HRMS (ESþ): Calc C26H26O2 [M þ H]þ 371.2006. Found
371.2004.
4.2.6. 2-(2-Bromobenzyl)-6-hydroxy-tetralone (4e)
A white solid was obtained. Yield: 0.8 g (39%), Rf 0.61 (petroleum
ethereEtOAc 3:1 v/v); m.p. 171e174 ꢀC;
10.32 (s, 1H, OH), 7.78 (d,
d
J ¼ 8.6 Hz, 1H, H-8), 7.58 (d, J ¼ 8.0 Hz, 1H, Ar), 7.32 (m, 2H, Ar), 7.16
(dd, J ¼ 7.0, 7.9 Hz, 1H, Ar), 6.73 (d, J ¼ 8.6 Hz, 1H, H-7), 6.62 (s, 1H,
H-5), 3.48 (dd, J ¼ 4.2, 13.8 Hz, 1H, CHaHb-Ph), 2.79 (m, 3H, CH2, H-3
and CHx), 2.61 (dd, J ¼ 9.8, 13.8 Hz, 1H, CHaHb-Ph), 1.87 (m, 1H,
CHAHB), 1.71 (m, 1H, CHAHB).
4.2.3. Ethyl 2-(2-bromobenzyl)-6-methoxy-1-oxo-1,2,3,4-
13C NMR:
d 196.8 (C, C]O), 161.8 (C, C-6), 146.8 (C), 139.2 (C),
tetrahydro-2-naphthalene carboxylate (6)
132.6 (CH), 131.8 (CH), 129.4 (CH), 128.3 (CH), 127.6 (CH), 124.2 (C),
124.0 (C),114.3 (CH),114.0 (CH), 46.6 (CH, CHx), 35.4 (CH2, CHaHbPh)
28.1 (CH2, C-3), 27.4 (CH2, CHAHB). Anal. Calcd. for C17H15BrO2
(331.20): C, 61.65%, H, 4.56%. Found: C, 61.59%, H, 4.58%.
A mixture of NaH (60% dispersion in mineral oil, 0.45 g,
10.37 mmol), ethyl-6-methoxy-1-tetralone carboxylate (5) [20]
(2.5 g, 10.0 mmol) in DMF (30 mL) was heated at 60 ꢀC for 1 h. A
solution of 2-bromobenzyl bromide (2.67 g, 10.7 mmol) in DMF
(10 mL) was added and the reaction heated at 60 ꢀC for a further 8 h.
On completion of the reaction, a few drops of H2O were added and
the resulting suspension extracted with Et2O (3 ꢃ 50 mL). The
combined organic extracts were washed with brine (50 mL), dried
(MgSO4) and concentrated in vacuo to give an oily residue.
4.2.7. General procedure for the synthesis of (E)-2-(2-aryl/alkylary-
benzylidene)-andbenzyl-6-methoxy-tetralonederivatives(8band9b)
2 M aqueous Na2CO3 (7 mL) was added to a solution of (E)-2-(2-
bromobenzylidene)-6-methoxy-tetralone (5) (2 mmol) in toluene
(15 mL), then Pd(PPh3)4 (0.1 mmol) was added to the mixture.

4434
A.S. Aboraia et al. / European Journal of Medicinal Chemistry 45 (2010) 4427e4434
ꢀꢂ1
Arylboronic acid (4 mmol) in ethanol (4 mL) was added to the
above mixture and the reaction was refluxed at 100 ꢀC for 5 h. After
the reaction was complete, the residual borane was oxidised by the
addition of H2O2 (30%, 2 mL) at room temperature for 1 h. The crude
product was extracted with CH2Cl2 (100 mL) and water
(3 ꢃ 100 mL), then the organic layer was dried (MgSO4) and
concentrated in vacuo. Purification by flash column chromatog-
raphy (petroleum ethereEtOAc 100:0 v/v increasing to 90:10 v/v)
gave the product, which was recrystallised with EtOH.
MMFF94x forcefield until a RMSD gradient of 0.05 kcal molꢂ1
A
was reached. The resulting structures were used in the docking
simulations. PLANTS software was used with the default settings
(aco_ants 20; aco_evap 0.15; aco_sigma 5.0), defining the binding
site as a sphere of 12 A around the alpha carbon of Ile242. Docking
results were imported in a MOE database and examined using the
ligand interaction function.
ꢀ
Acknowledgments
4.2.7.1. (E)-2-(2-(E)-Styrylbenzylidene)-6-methoxy-tetralone (8b). A
light brown solid was obtained. Yield: 51%, Rf 0.68 (petroleum
For ASA we acknowledge the Embassy of the Arab Republic of
Egypt for the award of a PhD scholarship. We also acknowledge the
EPSRC Mass Spectrometry Centre, Swansea, UK for mass spectros-
copy data.
ethereEtOAc 4:1 v/v); m.p. 120e122 ꢀC; 1H NMR:
d 8.20 (d,
J ¼ 8.7 Hz, 1H, H-8), 8.06 (s, 1H, -C]CHePh), 7.76 (d, J ¼ 7.8 Hz, 1H,
Ar), 7.51 (d, J ¼ 7.3 Hz, 1H, Ar), 7.41e7.26 (m, 8H, Ar), 7.10 (d,
J ¼ 16.1 Hz, 1H, trans eCH]CHe), 6.92 (dd, J ¼ 2.5, 8.7 Hz, 1H, H-5),
6.72 (d, J ¼ 2.5 Hz, 1H, H-7), 3.89 (s, 3H, OCH3), 2.91 (s, 4H, 2ꢃ CH2).
References
13C NMR:
d 186.6 (C, C]O), 163.7 (C, C-6), 146.1 (C), 147.3 (C), 136.9
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(CH), 125.7 (CH), 113.5 (CH), 112.4 (CH), 55.5 (CH3), 29.6 (CH2), 27.5
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4.2.7.2. 2-(2-(E)-Styrylbenzyl)-6-methoxy-tetralone (9b). A colour-
less oil was obtained. Yield: 95%, Rf 0.82 (petroleum ethereEtOAc
4:1 v/v); 1H NMR:
d
8.11 (d, J ¼ 8.8 Hz, 1H, H-8), 7.73 (d, J ¼ 7.4 Hz,
1H, Ar), 7.55 (t, J ¼ 7.3 Hz, 1H, Ar), 7.39 (t, J ¼ 7.5 Hz, 1H, Ar),
7.32e7.24 (m, 5H, Ar), 7.07 (d, J ¼ 16.1 Hz, 1H, trans eCH]CHe),
6.87 (dd, J ¼ 2.6, 8.8 Hz, 1H, H-5), 6.69 (d, J ¼ 2.5 Hz, 1H, H-7), 3.93
(dd, J ¼ 3.2, 13.6 Hz, 1H, CHaHb-Ph), 3.87 (s, 3H, OCH3), 2.90 (m, 2H,
CH2, H-3), 2.70 (m, 2H, Hx and CHaHb-Ph), 2.09 (m, 1H, CHAHB), 1.84
(m, 1H, CHAHB). 13C NMR:
d 197.9 (C, C]O), 163.5 (C, C-6), 146.6 (C),
138.3 (C), 137.5 (C), 136.4 (C), 126.0 (C), 134.7 (CH), 130.9 (CH), 130.5
(CH), 130.1 (CH), 129.3 (CH), 128.4 (CH), 127.7 (CH), 127.0 (CH), 126.8
(CH),126.3 (CH),125.9 (CH), 113.3 (CH),112.46 (CH), 55.4 (CH3), 48.8
(CH, C-2), 33.45 (CH2), 29.2 (CH2), 28.0 (CH2). LRMS (CIþ) m/z: 369.3
[M þ H]þ, 177.2 [M ꢂ {6-methoxy-1-tetralone}]þ. HRMS (EI): Calcd.
C26H25O2 [M þ H]þ 369.1849. Found 369.1843.
4.3. Docking studies
[18] D.D. Perrin, W.L.F. Armarengo, Purification of Laboratory Chemicals, third ed.
Pergamon Press, New York, 1988.
[19] S.W. Yee, L. Jarno, M.S. Gomaa, C. Elford, L.-L. Ooi, M.P. Coogan, R. McClelland,
R.I. Nicholson, B.A.J. Evans, A. Brancale, C. Simons, J. Med. Chem. 48 (2005)
7123e7131.
All molecular modelling studies were performed on a MacPro
dual 2.66 GHz Xeon running Ubuntu 8. Docking simulations were
performed using PLANTS [13] and the results were visualised in
MOE [16]. Ligand structures were built in MOE minimised using the
[20] B.T. Cho, S.K. Kang, S.H. Shin, Tetrahedron: Asymmetry 13 (2002) 1209e1217.