Novel azolyl-(phenylmethyl)]aryl/heteroarylamines: Potent CYP26 inhibitors and enhancers of all-trans retinoic acid activity in neuroblastoma cells

Article abstract of DOI:10.1016/j.bmc.2007.06.048

The synthesis and potent inhibitory activity of novel 4-[(imidazol-1-yl and triazol-1-yl)(phenyl)methyl]aryl-and heteroaryl amines versus a MCF-7 CYP26A1 cell assay is described. Biaryl imidazole ([4-(imidazol-1-yl-phenyl-methyl)-phenyl]-naphthalen-2-yl-a

Full text of DOI:10.1016/j.bmc.2007.06.048

Bioorganic & Medicinal Chemistry 16 (2008) 8301–8313
Novel azolyl-(phenylmethyl)]aryl/heteroarylamines: Potent
CYP26 inhibitors and enhancers of all-trans retinoic acid activity
in neuroblastoma cells
Mohamed Sayed Gomaa,a Jane L. Armstrong,b Beatrice Bobillon,b Gareth J. Veal,b
Andrea Brancale,a Christopher P. F. Redfernb and Claire Simonsa,*
aMedicinal Chemistry Division, Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cardiff CF10 3XF, UK
bNorthern Institute for Cancer Research, Paul O’Gorman Building, Medical School, Framlington Place,
Newcastle University, Newcastle Upon Tyne, NE2 4HH, UK
Received 30 March 2007; revised 19 June 2007; accepted 26 June 2007
Available online 29 June 2007
Abstract—The synthesis and potent inhibitory activity of novel 4-[(imidazol-1-yl and triazol-1-yl)(phenyl)methyl]aryl-and heteroaryl
amines versus a MCF-7 CYP26A1 cell assay is described. Biaryl imidazole ([4-(imidazol-1-yl-phenyl-methyl)-phenyl]-naphthalen-2-
yl-amine (8), IC50 = 0.5 lM; [4-(imidazol-1-yl-phenyl-methyl)-phenyl]-indan-5-yl-amine (9), IC50 = 1.0 lM) and heteroaryl imidazole
derivatives ((1H-benzoimidazol-2-yl)-{4-[(5H-imidazol-1-yl)-phenyl-methyl]-phenyl}-amine (15), IC50 = 2.5 lM; benzooxazol-2-yl-
{4-[(5H-imidazol-1-yl)-phenyl-methyl]-phenyl}-amine (16), IC50 = 0.9 lM; benzothiazol-2-yl-{4-[(5H-imidazol-1-yl)-phenyl-methyl]-phe-
nyl}-amine (17), IC50 = 1.5 lM) were the most potent CYP26 inhibitors. Using a CYP26A1 homology model differences in activity
were investigated. Incubation of SH-SY5Y human neuroblastoma cells with the imidazole aryl derivative 8, and the imidazole
heteroaryl derivatives 16 and 17 potentiated the atRA-induced expression of CYP26B1. These data suggest that further structure–
function studies leading to clinical development are warranted.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
The main function of RA is to modulate gene transcrip-
tion by liganding nuclear receptors that bind to their
DNA response element motifs in promoters of target
genes.4 RXRs are the heterodimeric partners of approx-
imately 1/3 of the human members of the nuclear hor-
mone receptor subfamily,5 for example, the nuclear
receptors VDR and TR, and the orphan nuclear recep-
tors PPAR, LXR, FXR and NURR1, many of these
heterodimers are activated by RXR agonists including
the endogenous ligand 9-cis-RA (Fig. 1). As such RA
(all-trans-RA and 9-cis-RA) has been used in a number
of clinical situations, especially oncology (acute promy-
elocytic leukaemia6,7) and dermatology (acne, psoria-
sis8,9). RA may improve the efficacy of other
treatments such as radiation, cisplatin and interferon
therapies.10,11 Biochemical results also support lower
retinoid supply, synthesis, impaired transport, and
hypofunction as contributing factors to late onset Alz-
heimer’s disease (LOAD)12 suggesting that increasing
endogenous levels of RA may modify memory perfor-
mance via the promotion of both adult neurogenesis
and adult synaptic plasticity.13
Retinoic acid (RA) is a naturally occurring retinoid
responsible for growth and differentiation of mamma-
lian epithelial tissues1 which exerts activity by binding
to transcription-regulatory factors in the cell nucleus
known as RAR (retinoic acid receptor) and RXR (reti-
noid X receptor), each having subtypes a, b and c.2 RA
controls transcription by binding to RAR/RXR dimers,
which locally modifies chromatin structure (Fig. 1). Syn-
thesis and degradation of RAR and RXR by protea-
somes control amount and timing of the retinoid
responses and also modulates phosphorylations.3
Abbreviations: atRA, all-trans-retinoic acid; RA, retinoic acid; RAM-
BAs, retinoic acid metabolism blocking agents.
Keywords: 4-[(Imidazol-1-yl and triazol-1-yl)(phenyl)methyl]aryl- and
heteroaryl amines; IC50 enzyme inhibition; Retinoic acid metabolism
blocking agents (RAMBAs); MCF-7; CYP26A1; Molecular model-
ling; Neuroblastoma.
*
Corresponding author. Tel.: +44 2920 876307; fax: +44 2920
874149; e-mail: SimonsC@Cardiff.ac.uk
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.06.048

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M. S. Gomaa et al. / Bioorg. Med. Chem. 16 (2008) 8301–8313
CRBP
CRABP
ALDH
ADH
SDR
Retinal
Retinoic acid
Retinol
NUCLEUS
CYP26 +
other P450s
Retinyl esters
(storage in liver)
RXR RAR
INACTIVE
metabolites
gene
TRANSCRIPTION
REGULATION
or other nuclear hormone receptors
e.g. PPAR, TR, VDR,
LXR, FXR, NURR1
Figure 1. Biosynthetic pathway of retinol and receptor activation by retinoic acid. Alcohol dehydrogenases (ADH); short-chain dehydrogenase/
reductase (SDR); aldehyde dehydrogenases (ALDH); cellular retinol-binding protein (CRBP); cellular retinoic acid-binding protein (CRABP);
retinoic acid receptor (RAR); retinoid X receptor (RXR); peroxisome-proliferator-activator receptors (PPARs), thyroid hormone receptor (TRs),
vitamin D receptor (VDR), liver X receptors (LXRs), farnesoid X receptors (FXRs) and NURR1.
atRA has a short half life and its potency is reduced
when administered systemically, owing to metabolism
by several human liver and intestine cytochrome P450s
to the inactive 4-hydroxy-RA and then by dehydrogen-
ases to the partially active 4-keto-RA and inactive polar
metabolites.14 Indeed, a relationship between induction
of atRA metabolism and drug resistance has been dem-
onstrated in acute promyelocytic leukaemia patients.15
In living tissues, all-trans-RA administration induces
another enzyme CYP2616 which recognises only RA as
its substrate, and the expression of this isozyme can be
induced by atRA both in vitro and in vivo. Three mem-
bers of the CYP26 family have now been identified:
CYP26A116 and CYP26B1,17 which metabolise atRA
in the embryo and adult and, more recently,18 CYP26C1
that may have a role in the specific metabolism of both
all-trans and 9-cis isomers of RA. The dose-dependent
induction of CYP26 mRNA has been used as a sensitive
marker of retinoid response: in SH-SY5Y neuroblas-
toma cells CYP26 mRNA is normally undetectable
but treatment with atRA results in the dose-dependent
induction of CYP26A1 and CYP26B1 mRNA and in-
creased metabolism of atRA within 24 h.19
We have recently described a series of benzofuran-2-yl-
[(4-alkyl/aryl-phenyl)methyl]triazole derivatives with
modest CYP26 inhibitory activity (Fig. 2).26 Using
SAR derived from this series with the amine moiety
common in the very potent CYP26 inhibitors described
by Janssen and OSI Pharma, a series of novel 4-[(imi-
dazol-1-yl and triazol-1-yl)(phenyl)methyl]aryl- and het-
eroaryl amines (Fig. 2) were designed, using our
CYP26A1 model,25 and synthesized. These compounds
were evaluated for their ability to inhibit CYP26 activity
and their ability to enhance the biological activity of
atRA in inducing gene expression in human SH-SY5Y
neuroblastoma cells.
2. Chemistry
The N-aryl substituted triazol- and imidazol-1-ylmethyl-
phenylamines were prepared via a three step procedure
involving a Suzuki coupling to give the 4-aminophenyl
ketones (1) via route A or B (Scheme 1), subsequent
reduction to the alcohols (2) and finally addition of
the aza ring (triazole (3–4) or imidazole (5–9)).
An inhibitor of the metabolism of endogenous RA
would be expected to have a beneficial effect on epithelial
differentiation and proliferation as a RA-mimetic, with
considerable therapeutic potential. A number of retinoic
acid metabolism blocking agents (RAMBAs) have been
described (Fig. 2) and recently reviewed by Njar et al.20
The Janssen triazoles R11586621 and R11601022 are po-
tent inhibitors of retinoic acid metabolism, and R115866,
in vivo in rats after a single oral dose, increases endoge-
nous tissue RA levels and mimics RA in several other of
its biological actions.21 Selective naphthyl derivatives
have also been described with IC50 = 3.3 nM for the most
potent compound (OSI Pharma).23 The majority of
CYP26 inhibitors contain an azolyl group, either an
imidazole or triazole heterocycle, which coordinates with
the Fe3+ of the porphyrin haem. Non-azoyl CYP26
inhibitors have also been described such as the tetralone
derivatives,24 designed using a computer generated
homology model of CYP26A1.25
The N-arylation using the Suzuki reaction followed de-
scribed methodology27,28 employing a stoichiometric
amount of copper and a tertiary amine base, pyridine
in this reaction series. The reaction was performed in
air allowing oxygen uptake and therefore more efficient
oxidation of a reduced copper intermediate.29 This was
done by using vigorous stirring in flasks with a large vol-
ume relative to that of the solvent volume. This is more
important during the first few hours of the reaction since
it was found that the reaction proceeded smoothly to
35–45% conversion in the first 4–5 h, and catalytic activ-
ity rapidly diminished over the next 20 h.30 The coupled
products were confirmed by the presence of an NH sin-
glet peak at approximately dH 5.5 (Scheme 1).
The 4-aminophenyl ketones were efficiently reduced to
their corresponding 4-aminophenylmethanol derivatives
(2) by treatment with NaBH4 in MeOH or MeOH/diox-
ane depending on solubility.

M. S. Gomaa et al. / Bioorg. Med. Chem. 16 (2008) 8301–8313
8303
N
N
N
N
N
N
N
N
O
N
S
N
S
N
N
N
HO
O
H
H
OSI Pharma
R116010
R115866
N
N
O
N
N
X
N
NH
R
H3CO
OH
O
R
Benzofuran derivatives
R = Et, IC50 4.5 µM
R = Ph, IC50 7 µM
TARGET COMPOUNDS
R = phenyl, naphthyl, indane,
benzimidazole, benzoxazole,
benzthiazole
Tetralone
Figure 2. Retinoic acid metabolism blocking agents (RAMBAs) and target compounds.
O
OH
OH
H2N R2
A
B
+
+
B
R1
O
(i)
NH R2
R1
(1)
(ii)
HO
HO
O
B
R2
NH2
R1
HO
NH R2
(iii)
R1
(2)
N
N
N
(iv)
NH R2
R1
N
3-4
N
NH R2
R1
5-9
˚
Scheme 1. Reagents and conditions: (i) CuOAc, pyridine, 4 A molecular sieves, CH2Cl2, rt, 3 days; (ii) NaBH4, MeOH/dioxane, rt, 1–18 h; (iii)
(triazol-1-yl)2SO, K2CO3, CH3CN, rt, 4 days; (iv) CDI, imidazole, CH3CN, 65 ꢁC, 1 h.
The synthesis of the triazole compounds (3–4) in-
volved the reaction of the aminophenylmethanol
derivatives (2) with the in situ prepared N,N0-di(1H-
1,2,4-triazol-1-yl)sulfoxide.31 After purification by col-
umn chromatography, the products were confirmed by
1H NMR with the disappearance of the OH group at
approximately d 1.5–2.0 and the presence of the two
singlet protons of the triazole at approximately d 8.1
and 8.2. The imidazole compounds (5–9) were pre-
pared by direct reaction of the aminophenylmethanol
derivatives (2) with 1,10-carbonyldiimidazole in the
presence of excess imidazole as previously re-
ported.23,32 Therefore, the need for the in situ
preparation of the reactive species N,N0-di(1H-imi-
dazol-1-yl)sulfoxide was eliminated. The yields for
the final triazole and imidazole derivatives (3–9) are
given in Table 1.
The N-heteroaryl substituted imidazol-1-ylmethyl-phe-
nylamines (15–17) were prepared via a four-step proce-
dure in good yields (Table 1). Aminobenzophenone
was reduced to the corresponding carbinol (10) by reac-
tion with NaBH4 in methanol, reaction of 10 with thio-
phosgene as described in the literature33 produced the
isothiocyanate (11),34 which was then cyclized using
mercury(II) oxide in the presence of a catalytic amount
of sulphur35,36 to give the cyclized products (12–14).
Treatment of 12–14 with 1,10-carbonyldiimidazole in
the presence of excess imidazole gave the imidazole
derivatives (15–17) (Scheme 2).

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M. S. Gomaa et al. / Bioorg. Med. Chem. 16 (2008) 8301–8313
Table 1. Substituents, yields and mps for triazole (X = N, 3–4) and imidazole (X = CH, 5–9, 15–17) products
N
X
N
NH R2
R1
Compound
R1
R2
Yield (%)
72
mp (ꢁC)
3
CH3
108–110
4
5
69
84
89
84
88
83
76
71
73
138–140
110–112
208–210
170–172
94–96
CH3
6
7
CH3
8
9
88–90
N
15
16
17
274–276
214–216
258–260
N
H
N
O
N
S
O
OH
OH
(ii)
(i)
NH2
NH2
NCS
(10)
(11)
(iii)
N
N
OH
(iv)
N
N
X
X
N
H
N
H
(15-17)
(12-14)
Scheme 2. Reagents and conditions: (i) NaBH4, MeOH, rt, 1 h; (ii) CSCl2, CH2Cl2, ice/H2O, 0 ꢁC, 2 h, refrigerator o/n; (iii) 2-substituted
aminobenzene, EtOH, HgO, S (cat.), reflux, 2 h; (iv) CDI, imidazole, CH3CN, 65 ꢁC, 1 h.

M. S. Gomaa et al. / Bioorg. Med. Chem. 16 (2008) 8301–8313
8305
3. CYP26A1 inhibitory activity
induced CYP26 when used alone. R116010 (IC50
8.7 nM22) at a concentration of 1 lM co-incubated
with 0.01 lM atRA induced CYP26B1 mRNA 5–6 fold
compared with 0.01 lM atRA alone; this level of
induction was comparable with treatment with a 10-
fold higher of atRA (0.1 lM) (Fig. 3). Co-incubation
of 0.01 lM atRA with 8, 16 or 17 (0.1–10 lM) for
24 h also substantially increased expression of
CYP26B1 compared with 0.01 lM atRA alone. The
imidazole 8 at 10 lM had an activity comparable to
1 lM R116010. The bicyclic heteroaryl imidazole
derivatives 16 and 17 were less effective, reaching
maximal activity of 2- to 4-fold induction of CYP26B1
for 17 and 16, respectively, over the concentration
range 0.1–10 lM (Fig. 3). The order of CYP26B1
induction activity for these inhibitors at a concentra-
tion of 10 lM (inhibitor 8 > 16 > 17) correlated with
the IC50 values determined in the MCF-7 cell assay.
Compound 4 was only tested at 1 lM but at this con-
centration, unlike 8, 16 and 17, it was ineffective at
increasing CYP26B1 expression in the presence of
0.01 lM atRA.
The imidazole and triazole derivatives were evaluated
for their retinoic acid metabolism inhibitory activity
using
a
MCF-7 cell assay,24 using radiolabelled
[11,12-3H] all-trans retinoic acid as the substrate and
liarozole (a non-selective CYP26 inhibitor20) and
R115866 as standards for comparison. With the excep-
tion of the methyl derivatives (3, 5 and 7, IC50 = >50,
35 and 15 lM, respectively), the novel 4-[(imidazol-1-yl
and triazol-1-yl)(phenyl)methyl]aryl- and heteroaryl
amines were all potent inhibitors of CYP26A1 (Table
2). The biaryl imidazole (naphthyl 8, IC50 = 0.5 lM; in-
dane 9, IC50 = 1.0 lM) and bicyclic heteroaryl imidazole
derivatives (benzimidazole 15, IC50 = 2.5 lM; benzoxaz-
ole 16, IC50 = 0.9 lM; benzothiazole 17, IC50 = 1.5 lM)
were the most potent CYP26 inhibitors. Considerable
differences in activity were observed with R1 = CH3 (3
and 5, IC50 = 50 lM) and R1 = Ph (4 and 6, IC50
5 lM). Likewise a difference in activity was observed
for R2 = Ph (4, IC50 = 5 lM) and R2 = biaryl/biheteroa-
ryl (e.g. 8, IC50 = 0.5 lM and 15, IC50 = 2.5 lM).
=
4. Enhancement of retinoic acid effects
The induction of CYP26B1 mRNA was used to evalu-
ate the ability of 8, 16 and 17 to enhance the biological
effects of atRA. CYP26B1 mRNA was chosen as it is a
more-responsive marker: we see a 15–20 fold induction
of B1 compared with 10-fold of A1 with the same con-
centration of atRA.19 None of the CYP26 inhibitors
5. Discussion
In order to rationalise the results obtained, FlexX37
docking studies of all the inhibitors were performed
using a human CYP26A1 model,25 built using the

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M. S. Gomaa et al. / Bioorg. Med. Chem. 16 (2008) 8301–8313
Table 2. IC50 data for the novel triazole (X=N, 3–4) and imidazole
(X=CH, 5–9 and 15–17) products using MCF-7 assay
N
X
N
NH R2
R1
Compound
R1
R2
MCF-7 cells
a
IC50 (lM)
3
CH3
CH3
CH3
>50
5
4
5
35
5
6
7
15
0.5
1
8
9
N
15
16
17
2.5
0.9
1.5
Figure 3. Real-time PCR analysis of CYP26B1 mRNA expression
after treatment with atRA in combination with 8, 16 and 17. SH-SY5Y
cells were treated with inhibitor (1 lM) either alone or in combination
with atRA (0.01 lM) for 24 h. Total RNA was isolated, reverse
transcribed, and subjected to real-time PCR using TaqMan probes for
CYP26B1 and b-actin. Values are normalised for b-actin levels and
expressed as fold increase relative to CYP26B1 expression in SH-SY5Y
cells treated with 0.01 lM ATRA. Data are mean values S.E.M.
(n P 3).
N
H
N
O
N
S
Liarozole
R115866
—
—
—
—
7
0.005
a IC50 values are the average ( 5%) of two experiments.
their IC50 values in the MCF7 assay. Compounds
having R1 = Ph and R2 = biaryl/biheteroaryl (8, 9,
15, 16 and 17) docked well in the active site
establishing multiple hydrophobic interactions with
different residues at the active site, in particular
TRP112, PHE374, PHE84, PHE299, VAL116 and
PRO371, thereby holding the inhibitors tightly in
the active site resulting in greater IC50 values in the
MCF7 assay and activity in the CYP26B1 induction
assay (Fig. 4).
recently crystallised human CYP3A4 as the template.
The results of the docking were only analysed visually
due to the lack of correlation between the FlexX scoring
functions and the visual compound–active site interac-
tion. The position of the triazole ring from the haem
was first evaluated. In the well-docked compounds, the
distance between the imidazole or triazole N and the
˚
iron of the haem should be 3 A or less thereby allowing
the coordination bond to be formed. Second, the side
chain R1 and R2 were assessed for their ability to form
hydrogen and/or hydrophobic bonds with different resi-
dues at the active site.
Compounds having R1 = Ph and R2 = Ph (4 and 6) were
less active as the reduced size, compared with the biaryl/
biheteroaryl derivatives, resulted in fewer hydrophobic
interaction with residues at the active site. It is worth not-
ing that only the (S)-conformation of these compounds
docked in the active site while the (R)-conformation
did not dock favourably, suggesting that the pure (S)-
enantiomer may be more active than the racemic com-
pound, this is currently under investigation.
Compounds having R1 = CH3 (3, 5 and 7) are
relatively small and have been found to move freely
in the active site with no conformation that favours
the coordination of the imidazole or the triazole N
with the iron of the haem and this correlated well with

M. S. Gomaa et al. / Bioorg. Med. Chem. 16 (2008) 8301–8313
8307
Figure 4. Active site region of CYP26A1 model showing (a) (S)—5 and (b) (S)—8 in stick form. Transition metal interaction with the haem (green
stick form with iron colored orange) is indicated with a black line. Amino acid residues identified are involved in hydrophobic interactions.
In addition to their ability to inhibit CYP26-mediated
atRA metabolism in the MCF-7 assay, compounds used
in the SH-SY5Y cell assays enhanced the biological activ-
ity of exogenous atRA. The R1 = Ph and R2 = biaryl
compound 8 had a peak activity comparable with
R116010. When used at relatively low doses (0.1 lM),
compound 8 and the N-heteroaryl-substituted benzo-
thiazole 16 were effective at enhancing the biological
activity of atRA, suggesting that in vivo studies with a
view to clinical development are warranted.
acid (30 mmol), 4-aminoacetophenone or 4-aminobenz-
ophenone or 5-aminoindane (10 mmol), anhydrous cup-
ric acetate (3.63 g, 20 mmol), pyridine (1.58 g, 30 mmol)
˚
and 250 mg activated 4 A molecular sieves under an
atmosphere of air was added dichloromethane (25 mL)
and the reaction mixture stirred under air atmosphere
at ambient temperature for 3 days. The product was iso-
lated by direct flash column chromatography of the
crude reaction mixture.
6.1.1.1. 1-(4-Phenyl)-ethanone (1a, R1 = CH3, R2 =
Ph)27. Purification by column chromatography (petro-
leum ether–ethyl acetate, 100:0 v/v increasing to 80:20 v/
v) gave 1-(4-Phenylamino-phenyl)-ethanone as a pale yel-
low crystalline solid. Yield, 1.35 g (64%); mp 118–120 ꢁC;
Rf = 0.57 (petroleum ether–ethyl acetate, 4:1). 1H NMR:
d (CDCl3): 2.57 (s, 3H, H-2), 6.58 (s, 1H, H–NH), 7.03 (d,
J = 8.7 Hz, 2H, Ar), 7.10 (t, J = 7.3 Hz, 1H, H-400), 7.21
(d, J = 7.7 Hz, 2H, Ar), 7.36 (m, 2H, Ar), 7.89 (d,
J = 8.7 Hz, 2H, Ar). 13C NMR: d (CDCl3): 26.14 (CH3,
C-2), 114.46 (CH, C-30, C-50), 120.73 (CH, C-200, C-600),
123.40 (CH, C-400), 129.15 (C, C-10), 129.55 (CH, C-300,
C-500), 130.62 (CH, C-20, C-60), 140.64 (C, C-100), 148.36
(C, C-40), 196.31 (C, C-1).
6. Experimental
6.1. Materials and methods: Chemistry
[11,12-3H] All trans-retinoic acid (37 MBq/mL) was pur-
chased from Amersham (UK). Acetic acid, ammonium
acetate and Optisafe 3 scintillation fluid were obtained
from Fisher Scientific (UK). All solvents used for chroma-
tography were HPLC grade from Fisher Scientific (UK).
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 determined by the EPSRC mass spectrometry cen-
tre (Swansea, UK). Microanalyses were determined by
Medac Ltd (Surrey, UK). Flash column chromatogra-
phy 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 visu-
alised by illumination under UV light (254 nm) or by the
use of vanillin stain followed by charring on a hotplate.
Melting points were determined on an electrothermal
instrument and are uncorrected. All solvents were dried
prior to use as described by the handbook Purification
6.1.1.2. Phenyl-(4-phenylamino-phenyl)-methanone (1b,
R1 = R2 = Ph). Purification by column chromatography
(petroleum ether–ethyl acetate, 100:0 v/v increasing to
80:20 v/v) gave phenyl-(4-phenylamino-phenyl)-metha-
none as a shiny yellow crystalline solid. Yield, 1.79 g
(65%); mp 140–142 ꢁC; Rf = 0.67 (petroleum ether–ethyl
1
acetate, 4:1). H NMR: d (CDCl3): 6.12 (s, 1H, H–NH),
6.93 (d, J = 8.5 Hz, 2H, Ar), 7.00 (t, J = 7.3 Hz, 1H,
Ar), 7.12 (d, J = 8.0, 2H, Ar), 7.26 (t, J = 7.8 Hz, 2H,
Ar), 7.38 (t, J = 7.6 Hz, 2H, Ar), 7.46 (t, J = 7.3 Hz, 1H,
Ar), 7.67 (d, J = 7.4 Hz, 2H, Ar), 7.69 (d, J = 8.6 Hz,
13
of Laboratory Chemicals38 and stored over 4 A molecu-
˚
2H, Ar). C NMR: d (CDCl3): 114.36 (CH, C-30, C-50),
lar sieves, under nitrogen.
120.72 (CH, C-200, C-600), 123.37 (CH, C-400), 128.15
(CH, C-4, C-6), 128.68 (C, C-10), 129.56 (CH, C-30, C-
50), 129.62 (CH, C-3, C-7), 131.59 (CH, C-5), 132.76
(CH, C-20, C-60), 138.72 (C, C-2), 140.66 (C, C-100),
148.24 (C, C-40), 195.21 (C, C-1). Anal. (C19H15NO) C,
H, N. Anal. Calcd for C19H15NO (273.33): C, 83.49; H,
5.53; N, 5.12. Found: C, 83.26; H, 5.61; N, 5.04.
The numbering of compounds for H and 13C NMR
characterisation is as follows:
1
6.1.1. General procedure for the Suzuki coupling prepa-
ration of the 4-aminophenyl ketones (1). To aryl boronic

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M. S. Gomaa et al. / Bioorg. Med. Chem. 16 (2008) 8301–8313
6.1.1.3. 1-[4-(Naphthalen-2-ylamino)-phenyl]-ethanone
145.82 (C, C-600), 149.23 (C, C-40), 195.13 (C, C-1). Anal.
Calcd for C22H19NO (313.39): C, 84.32; H, 6.11; N, 4.47.
Found: C, 84.12; H, 6.08; N, 4.33.
(1c, R1 = CH3, R2 = naphthyl). Purification by column
chromatography (petroleum ether–ethyl acetate,
100:0 v/v increasing to 80:20 v/v) gave 1-[4-(Naphtha-
len-2-ylamino)-phenyl]-ethanone as a yellowish brown
crystalline solid. Yield, 1.65 g (63%); mp 154–156 ꢁC;
Rf = 0.61 (petroleum ether–ethyl acetate, 4:1). 1H
NMR: d (CDCl3): 2.57 (s, 3H, H-2), 6.37 (s, 1H, H–
NH), 6.98 (d, J = 8.8 Hz, 2H, H-30, H-50), 7.22 (dd,
J1 = 2.2 Hz, J2 = 8.5 Hz, 1H, H-300), 7.3 (m, 1H, H-700),
7.37 (m, 1H, H-800), 7.49 (d, J = 2.1 Hz, 1H, H-100),
7.62 (d, J = 8.4 Hz, 1H, 400), 7.72 (t, J = 8.7 Hz, 2H, H-
600, H-900), 7.81 (d, J = 8.8 Hz, 2H, H-20, H-60). 13C
NMR: d (CDCl3): 26.20 (CH, C-2), 114.82 (CH, C-30,
C-50), 115.97 (CH, C-100), 121.37 (CH, C-300), 124.67
(CH, C-700), 126.72 (CH, C-900), 126.91 (CH, C-800),
127.75 (CH, C-600), 129.36 (C, C-500), 129.46 (CH, C-
400), 130.26 (C, C-10), 130.68 (CH, C-20, C-60), 134.30
(C, C-1000), 138.30 (C, C-200), 148.25 (C, C-40), 196.44
(C, C-1). Anal. Calcd for C18H15NOÆ0.1H2O (279.33):
C, 82.17; H, 5.75; N, 5.32. Found: C, 82.25; H, 5.78;
N, 5.17.
6.1.2. General procedure of the reduction reaction for the
preparation of the carbinol compounds (2) and (10). To a
cooled solution of 4-aminophenyl ketones (1) or 4-
aminobenzophenone (5 mmol) in anhydrous methanol
(10 mL) or a mixture of anhydrous methanol (5 mL)
and anhydrous dioxane (5 mL) was added sodium boro-
hydride (10 mmol) and the reaction mixture stirred un-
der nitrogen at room temperature for the specified
time. The solvent was evaporated in vacuo and acetone
(1 mL) was added to the residue in order to quench any
excess reducing agent. The oil that separated was ex-
tracted with dichloromethane (2· 150 mL), washed with
water (3· 100 mL), then the organic layer was dried with
MgSO4, filtered and reduced in vacuo. The residue was
then purified by flash column chromatography.
6.1.2.1. 1-(4-Phenylamino-phenyl)-ethanol (2a, R1 = CH3,
R2 = Ph). Stirring for 2 h in anhydrous methanol and purifi-
cation by column chromatography (petroleum ether–ethyl
acetate, 95:5 v/v increasing to 50:50 v/v) gave 1-(4-phenyla-
mino-phenyl)-ethanol as a white solid. Yield, 1.07 g (88%);
mp 66–68 ꢁC; Rf = 0.51 (petroleum ether–ethyl acetate,
6.1.1.4. [4-(Naphthalen-2-ylamino)-phenyl]-phenyl-meth-
anone (1d, R1 = Ph, R2 = naphthyl). Purification by col-
umn chromatography (petroleum ether–ethyl acetate,
100:0 v/v increasing to 80:20 v/v) gave [4-(naphthalen-2-
ylamino)-phenyl]-phenyl-methanone as a yellowish brown
crystalline solid. Yield, 1.98 g (61%); mp 140–142 ꢁC;
1
1:1). H NMR: d (CDCl3): 1.41 (d, J = 6.4 Hz, 3H, H-2),
1.73 (s, 1H, H–OH), 4.76 (q, J = 6.3 Hz, 1H, H–CH), 5.63
(s, 1H, H–NH), 6.84 (t, J = 7.3 Hz, 1H, H-400), 6.97 (m,
4H, Ar), 7.18 (m, 4H, Ar). 13C NMR: d (CDCl3): 24.94
(CH3, C-2), 70.12 (CH, C-1), 117.79 (CH, C-200, C-600),
117.84 (CH, C-30, C-50), 121.04 (CH, C-400), 126.62 (CH,
C-20, C-60), 129.38 (CH, C-300, C-500), 138.37 (C, C-10),
142.55 (C, C-40), 143.15 (C, C-100). Anal. Calcd for
C14H15NOÆ0.1H2O (215.08): C, 78.18; H, 7.12; N, 6.51.
Found: C, 78.19; H, 7.16; N, 6.46.
1
Rf = 0.69 (petroleum ether–ethyl acetate, 4:1). H NMR:
d (CDCl3): 6.31 (s, 1H, H–NH), 7.0 (d, J = 8.7 Hz, 2H,
H-30, H-50), 7.22 (dd, J1 = 2.2 Hz, J2 = 8.5 Hz, 1H, H-
300), 7.3 (m, 1H, H-700), 7.37 (m, 3H, H-4, H-6, H-800),
7.45 (m, 1H, H-20), 7.49 (d, J = 2.2 Hz, 1H, H-100), 7.62
(d, J = 7.9 Hz, 1H, H-400), 7.70 (m, 6H, H-3, H-5, H-7,
H-60, H-600, H-900). 13C NMR: d (CDCl3): 114.75 (CH,
C-30, C-50), 115.95 (CH, C-100), 123.39 (CH, C-300),
124.65 (CH, C-700), 126.71 (CH, C-900), 126.93 (CH, C-
800), 127.75 (CH, C-600), 128.18 (CH, C-4, C-6), 128.98
(C, C-500), 129.45 (CH, C-400), 129.65 (CH, C-3, C-7),
130.26 (C, C-10), 131.66 (CH, C-5), 132.79 (CH, C-20,
C-60), 134.32 (C, C-1000), 138.33 (C, C-2), 138.69 (C, C-
200), 148.10 (C, C-40), 195.29 (C, C-1). Anal. Calcd for
C23H17NOÆ0.2H2O (326.99): C, 84.48; H, 5.24; N, 4.28.
Found: C, 84.54; H, 5.26; N, 4.04.
6.1.2.2. Phenyl-(4-phenylamino-phenyl)-methanol (2b,
R1 = R2 = Ph). Stirring for 3 h in anhydrous methanol/
anhydrous dioxane and purification by column chroma-
tography (petroleum ether–ethyl acetate, 95:5 v/v
increasing to 50:50 v/v) gave phenyl-(4-phenylamino-
phenyl)-methanol as a brown oil. Yield, 0.95 g (86%);
Rf = 0.58 (petroleum ether–ethyl acetate, 1:1). 1H
NMR: d (CDCl3): 2.94 (s, 1H, H–OH), 5.81 (s, 1H, H-
1), 5.87 (s, 1H, H–NH), 7.04 (t, J = 7.3 Hz, 1H, Ar),
7.08 (d, J = 8.5 Hz, 2H, Ar), 7.13 (d, J = 7.6 Hz, 2H,
Ar), 7.31 (d, J = 8.4 Hz, 2H, Ar), 7.36 (m, 3H, Ar),
7.44 (t, J = 7.3 Hz, 2H, Ar), 7.49 (d, J = 7.3 Hz, 2H,
Ar). 13C NMR: d (CDCl3): 75.76 (CH, C-1), 117.44
(CH, C-200, C-600), 117.70 (CH, C-30, C-50), 121.34
(CH, C-400), 126.37 (CH, C-3, C-7), 127.48 (CH, C-5),
128.16 (CH, C-4, C-6), 128.87 (CH, C-20, C-60), 129.82
(CH, C-300, C-500), 136.51 (C, C-10), 142.69 (C, C-40),
143.15 (C, C-2), 144.24 (C, C-100). HRMS (ES+) m/z
Calcd for C19H17NO (M + H)+ 275.1305. Found:
275.1309.
6.1.1.5. [4-(Indan-5-ylamino)-phenyl]-phenyl-methanone
(1e, R1 = Ph, R2 = indanyl). Purification by column chro-
matography (petroleum ether–ethyl acetate, 100:0 v/v
increasing to 80:20 v/v) gave [4-(indan-5-ylamino)-phe-
nyl]-phenyl-methanone (13) as a shiny yellow crystalline
solid. Yield, 2.1 g (67%); mp 96–98 ꢁC; Rf = 0.73 (petro-
1
leum ether–ethyl acetate, 4:1). H NMR: d (CDCl3): 2.0
(m, 2H, H-200), 2.82 (m, 4H, H-100, H-300), 6.00 (s, 1H,
H–NH), 6.86 (d, J = 8.8, 2H, H-30, H-50), 6.90 (m, 1H,
Ar), 7.00 (s, 1H, H-500), 7.10 (d, J = 8.0 Hz, 1H, H-800),
7.37 (m, 2H, H-4, H-6), 7.44 (m, 1H, H-5), 7.67 (m, 4H,
13
Ar). C NMR: d (CDCl3): 25.69 (C, C-200), 32.36 (C, C-
100), 33.03 (C, C-300), 113.75 (CH, C-30, C-50), 117.93
(CH, C-700), 119.86 (CH, C-500), 125.07 (CH, C-800),
128.01 (CH, C-4, C-6), 128.01 (C, C-10), 129.56 (CH, C-
3, C-7), 131.44 (CH, C-5), 132.81 (CH, C-20, C-60),
138.60 (C, C-900), 138.90 (C, C-2), 139.95 (C, C-400),
6.1.2.3. 1-[4-(Naphthalen-2-ylamino)-phenyl]-ethanol
(2c, R1 = CH3, R2 = naphthyl). Stirring for 3 h in anhy-
drous methanol/anhydrous dioxane and purification
by column chromatography (petroleum ether–ethyl
acetate, 95:5 v/v increasing to 50:50 v/v) gave 1-[4-

M. S. Gomaa et al. / Bioorg. Med. Chem. 16 (2008) 8301–8313
8309
(naphthalen-2-ylamino)-phenyl]-ethanol as a brown
solid. Yield, 1.34 g (89%); mp 98–100; Rf = 0.57 (petro-
leum ether–ethyl acetate, 2:1).). H NMR: d (CDCl3):
135.62 (C, C-900), 137.64 (C, C-10), 140.99 (C, C-400),
143.78 (C, C-600), 144.12 (C, C-40), 145.59 (C, C-2).
HRMS (ES+) m/z Calcd for C23H19NO (M + H)+
316.1696. Found: 316.1695.
1
1.42 (d, J = 6.5 Hz, 1H, H-2), 1.76 (s, 1H, H–OH),
4.78 (q, J = 6.5 Hz, 1H, H-1), 5.81 (s,0 1H, H–NH),
7.04 (d, J = 8.5 Hz, 2H, H-30, H5 ), 7.11 (dd,
J1 = 2.3 Hz, J2 = 8.7 Hz, 1H, H-300), 7.2 (d,
J = 1.2 Hz, 1H, H-100), 7.22 (d, J = 8.3 Hz, 2H, H-20,
H-60), 7.31 (m, 2H, H-700, H-800), 7.54 (d, J = 8.2 Hz,
1H, H-400), 7.64 (d, J = 8.8 Hz, 2H, H-600, H-900). 13C
NMR: d (CDCl3): 24.98 (CH, C-2), 70.13 (CH, C-1),
111.57 (CH, C-100), 118.32 (CH, C-30, C-50), 120.02
(CH, C-300), 123.54 (CH, C-700), 126.50 (CH, C-600, C-
900), 126.70 (CH, C-20, C-60), 127.03 (C, C-500), 127.68
(CH, C-800), 129.23 (CH, C-400), 134.66 (C, C-10),
138.79 (C, C-1000), 140.90 (C, C-40), 142.35 (C, C-200).
Anal. Calcd for C18H17NOÆ0.3H2O (268.73): C,
80.45; H, 6.38; N, 5.21. Found: C, 80.80; H, 6.44; N,
5.13.
6.1.2.6. (4-Amino-phenyl)-phenyl-methanol (10). Stir-
ring for 1 h in anhydrous methanol gave (4-amino-phe-
nyl)-phenyl-methanol (10) as a white solid without
further purification. Yield, 1.92 g (95%); mp 116–
118 ꢁC; Rf = 0.39 (petroleum ether–ethyl acetate, 2:1).
1H NMR: d (CDCl3): 3.21 (s, 1H, H–OH), 4.83 (s, 2H,
H–NH), 5.62 (s, 1H, H-1), 6.59 (dd, J1 = 1.65 Hz,
J2 = 1.71 Hz, 2H, H-4, H-6), 7.00 (dd, J1 = 1.55 Hz,
J2 = 1.55 Hz, 2H, H-3, H-7), 7.12 (m, 2H, H-30, H-50),
7.20 (m, 1H, H-40), 7.25 (m, 2H, H-20, H-60). 13C
NMR: d (CDCl3): 76.86 (CH, C-1), 116.42 (CH, C-4,
C-6), 127.58 (CH, C-20, C-60), 127.94 (CH, C-40),
128.93 (CH, C-30, C-50), 129.13 (CH, C-3, C-7), 135.63
(C, C-2), 146.36 (C, C-10), 147.88 (C, C-5). Anal. Calcd
for C13H13NOÆ0.3H2O (204.653): C, 76.30; H, 6.40; N,
6.84. Found: C, 76.36; H, 6.43; N, 6.80.
6.1.2.4. [4-(Naphthalen-2-ylamino)-phenyl]-phenyl-metha-
nol (2d, R1 = Ph, R2 = naphthyl). Stirring for 1 h in anhy-
drous methanol/anhydrous dioxane and purification by
column chromatography (petroleum ether–ethyl acetate,
95:5 v/v increasing to 50:50 v/v) gave [4-(naphthalen-2-yla-
mino)-phenyl]-phenyl-methanol as a red-brown oil. Yield,
1.38 g (92%); Rf = 0.69 (petroleum ether–ethyl acetate,
6.1.3. (4-Isothiocyanato-phenyl)-phenyl-methanol (11).
To a solution of 10 (1.4 g, 7.03 mmol) in CH2Cl2
(20 mL) was added a mixture of ice (2 g) and water
(1 mL) and subsequently thiophosgene (0.64 mL,
8.4 mmol) dropwise with vigorous stirring. The mixture
was stirred 2 h at 0 ꢁC and kept overnight in a refriger-
ator. The organic layer was separated and extracted suc-
cessively with water (2· 50 mL), 10% NaHCO3 aq.
(50 mL) and water again (50 mL), dried (MgSO4) and
evaporated to obtain the pure product as a yellow oil.
Yield, 1.34 g (79%); Rf = 0.73 (petroleum ether–ethyl
acetate, 1:1). 1H NMR: d (CDCl3): 2.69 (s, 1H, H–
OH), 5.59 (s, 1H, H-1), 7.00 (d, J = 8.3 Hz, 2H, H-4,
H-6), 7.18 (m, 7H, H-3, H-7, H-20, H-30, H-40, H-50,
H-60). 13C NMR: d (CDCl3): 75.58 (CH, C-1), 125.50
(CH, C-4, C-6), 126.08 (CH, C-20, C-60), 127.46 (CH,
C-40), 127.89 (CH, C-30, C-50), 129.02 (CH, C-3, C-7),
130.29 (C, C-5), 135.45 (C, C-2), 143.06 (C, C-10),
143.25 (C, C-NCS). HRMS (ES+) m/z Calcd for
C14H11NOS (M + H)+ 241.0556. Found: 241.0557.
1
2:1). H NMR: d (CDCl3): 3.24 (s, 1H, H–OH), 5.81 (s,
1H, H-1), 6.01 (s, 1H, H–NH), 7.15 (d, J = 8.6 Hz, 2H,
H-30, H-50), 7.23 (dd, J1 = 2.2 Hz, J2 = 8.7 Hz, 1H, H-300),
7.35 (d, J = 8.7 Hz, 2H, H-20, H-60), 7.39 (m, 1H, H-700),
7.41 (d, J = 2.2 Hz, 1H, H-100), 7.47 (m, 6H, H-3, H-4, H-
5, H-6, H-7, H-800), 7.70 (d, J = 8.2 Hz, 1H, H-400), 7.78
(d, J = 8.8 Hz, 1H, H-900), 7.82 (d, J = 8.1 Hz, 1H, H-600).
13C NMR: d (CDCl3): 76.26 (CH, C-1), 117.84 (CH, C-
100), 118.43 (CH, C-30, C-50), 120.50 (CH, C-300), 123.37
(CH, C-700), 126.95 (CH, C-900), 127.19 (CH, C-3, C-7),
127.79 (CH, C-800), 128.00 (CH, C-600), 128.33 (CH, C-4,
C-6), 128.78 (CH, C-20, C-60), 128.88 (CH, C-5), 129.25
(CH, C-400), 129.28 (C, C-500), 134.75 (C, C-10), 136.99 (C,
C-1000), 140.92 (C, C-40), 142.46 (C, C-200), 144.37 (C, C-
2). HRMS (ES+) m/z Calcd for C23H19NO (M + H)+
326.1539. Found: 326.1538.
6.1.4. General procedure for the preparation of 2-substi-
tuted aminobenzoheterocycles. 2-Substituted aminoben-
zene (1.7 mmol) was added to a solution of 11
(1.7 mmol) in absolute ethanol (10 mL) and the mixture
stirred overnight at room temperature. To the same
flask HgO (3.3 mmol) and S (0.33 mmol) was added
and the reaction mixture refluxed for 2 h then filtered
through Celite. The solvent was evaporated in vacuo to
give an oil, which was purified by column
chromatography.
6.1.2.5. [4-(Indan-5-ylamino)-phenyl]-phenyl-methanol
(2e, R1 = Ph, R2 = indanyl). Stirring for 7 h in anhydrous
methanol/anhydrous dioxane and purification by col-
umn chromatography (petroleum ether–ethyl acetate,
95:5 v/v increasing to 50:50 v/v) gave [4-(indan-5-ylami-
no)-phenyl]-phenyl-methanol as a brown oil. Yield,
1.36 g (91%); Rf = 0.71 (petroleum ether–ethyl acetate,
1
2:1). H NMR: d (CDCl3): 1.98 (m, 2H, H-200), 2.12 (s,
1H, H–OH), 2.77 (t, J = 7.4Hz, 4H, H-100, H-300), 5.51
(s, 1H, H–NH), 5.21 (s, 1H, H-1), 6.76 (dd,
J1 = 2.0 Hz, J2 = 8.5 Hz, 1H, H-700), 6.87 (m, 3H, H-3,
H-7, H-500), 7.02 (d, J = 8.00 Hz, 1H, H-800), 7.11 (d,
J = 8.5 Hz, 2H, H-20, H-60), 7.17 (m, 1H, H-5), 7.25
(m, 2H, H-4, H-6), 7.30 (d, J = 7.5 Hz, 2H, H-30, H-
6.1.4.1. [4-(1H-Benzoimidazol-2-ylamino)-phenyl]-phe-
nyl-methanol (12). Purification by column chromatogra-
phy (dichloromethane–methanol, 100:0 v/v to 95:5 v/v)
gave [4-(1H-benzoimidazol-2-ylamino)-phenyl]-phenyl-
methanol (12) as a yellowish white solid. Yield, 0.42 g
(81%); mp 190–192 ꢁC; Rf = 0.54 (dichloromethane–
13
50). 00 C NMR: d (CDCl3): 25.71 (C, C-200), 32.38 (C,
C-1 ), 33.08 (C, C-300), 76.00 (CH, C-1), 115.28 (CH,
C-700), 116.70 (CH, C-30, C-50), 117.35 (CH, C-500),
124.89 (CH, C-5), 126.60 (CH, C-3, C-7), 127.86 (CH,
C-800), 128.11 (CH, C-4, C-6), 128.42 (CH, C-20, C-60),
1
methanol, 95:5). H NMR: d (DMSO): 5.67 (s, 1H, H-
100), 5.78 (s, 1H, H–OH), 6.98 (s, 2H, Ar), 7.18 (m, 1H,
H-500), 7.29 (m, 6H, Ar), 7.38 (d, J = 7.4 Hz, 2H, H-30,

8310
M. S. Gomaa et al. / Bioorg. Med. Chem. 16 (2008) 8301–8313
H-50), 7.66 (d, J = 8.3 Hz, 2H, H-5, C-8), 9.41 (s, 1H,
NH), 10.87 (s, 1H, NH). 13C NMR: d (DMSO): 76.73
(CH, C-100), 113.65 (CH, C-500), 119.54 (CH, C-5, C-8),
122.05 (CH, C-20, C-60), 127.71 (CH, C-6, C-7), 128.20
(CH, C-300, C-700), 129.08 (CH, C-400, C-600), 129.54 (CH,
C-30, C-50), 138.78 (C, C-40), 139.97 (C, C-4, C-9),
140.66 (C, C-2), 146.06 (C, C-10), 152.81 (C, C-200). Anal.
Calcd for C20H16N3OÆ0. 7H2O (326.973): C, 73.47; H,
5.36; N, 12.85. Found: C, 73.36; H, 5.35; N, 12.63.
MgSO4, filtered and reduced in vacuo. The residue was
then purified by flash column chromatography.
6.1.5.1. Phenyl-[4-(1-[1,2,4]triazol-1-yl-ethyl)-phenyl]-
amine (3). Purification by column chromatography
(petroleum ether–ethyl acetate, 90:10 v/v increasing to
10:90 v/v) gave [phenyl-[4-(1-[1,2,4]triazol-1-yl-ethyl)-
phenyl]-amine (3) as a light brown solid. Yield, 0.19 g
(72%); mp 108–110 ꢁC; Rf = 0.32 (petroleum ether–ethyl
1
acetate, 1:1). H NMR: d (CDCl3): 1.93 (d, J = 7.0 Hz,
6.1.4.2. [4-(Benzooxazol-2-ylamino)-phenyl]-phenyl-meth-
anol (13). Purification by column chromatography (petro-
leum ether/ethyl acetate 100:0 v/v to 75:25 v/v) gave [4-
(benzooxazol-2-ylamino)-phenyl]-phenyl-methanol (13) as
a yellow solid. Yield, 0.4 g (77%); mp 160–162 ꢁC;
3H, H-2), 5.50 (q, J = 7.0 Hz, 1H, H-1), 5.89 (s, 1H,
H–NH), 6.98 (t, J = 7.3 Hz, 1H, Ar), 7.05 (d,
J = 8.5 Hz, 2H, Ar), 7.10 (d, J = 7.9 Hz, 2H, Ar), 7.19
(d, J = 8.5 Hz, 2H, Ar), 7.30 (t, J = 7.6 Hz, 2H, Ar),
7.98 (s, 1H, H-3000), 8.03 (s, 1H, H-5000). 13C NMR: d
(CDCl3): 21.26 (CH3, C-2), 59.32 (CH, C-1), 117.26
(CH, C-30, C-50), 118.58 (CH, C-200, C-600), 121.70
(CH, C-400), 127.87 (CH, C-20, C-60), 129.42 (CH, C-
300, C-500), 131.45 (C, C-10), 141.83 (CH, C-3000), 142.39
(C, C-40), 143.75 (C, C-100), 151.81 (C, C-5000). Anal.
Calcd for C16H16N4Æ0.3H2O (269.734): C, 71.24; H,
5.97; N, 20.77. Found: C, 71.44; H, 6.03; N, 20.37.
1
Rf = 0.57 (petroleum ether–ethyl acetate, 2:1). H NMR:
d (DMSO): 5.71 (s, 1H, H-100), 5.85 (s, 1H, H–OH), 7.15
(m, 1H, H-500), 7.25 (m, 2H, H-20, H-60), 7.34 (m, 2H,
Ar), 7.41 (m, 4H, Ar), 7.49 (dd, J1 = 7.5 Hz, J2 = 7.8 Hz,
2H, H-30, C-50), 7.72 (d, J = 8.4 Hz, 2H, H-5, C-8),
10.59 (s, 1H, NH). 13C NMR: d (DMSO): 73.92 (CH,
C-100), 108.87 (CH, C-5), 116.51 (CH, C-8), 117.39 (CH,
C-20, C-60), 121.54 (CH, C-6), 123.94 (CH, C-7), 126.17
(CH, C-300, C-700), 126.58 (CH, C-500), 126.87 (CH, C-400,
C-600), 127.99 (CH, C-30, C-50), 137.31 (C, C-40), 139.59
(C, C-9), 142.44 (C, C-200), 145.84 (C, C-10), 147.00 (C,
C-4), 158.04 (C, C-2). HRMS (ES+) m/z Calcd for
C20H16N2O2 (M + H)+ 317.1285. Found: 317.1287.
6.1.5.2. Phenyl-[4-(phenyl-[1,2,4]triazol-1-yl-methyl)-phe-
nyl]-amine (4). Purification by column chromatography
(petroleum ether–ethyl acetate, 90:10 v/v increasing to
10:90 v/v) gave phenyl-[4-(phenyl-[1,2,4]triazol-1-yl-methyl)-
phenyl]-amine (4) as a light brown solid. Yield, 0.41 g (69%);
mp 138–140 ꢁC; Rf = 0.42 (petroleum ether–ethyl acetate,
1
6.1.4.3. [4-(Benzothiazol-2-ylamino)-phenyl]-phenyl-meth-
anol (14). Purification by column chromatography (petro-
leum ether/ethyl acetate 100:0 v/v to 75:25 v/v) gave [4-
(benzothiazol-2-ylamino)-phenyl]-phenyl-methanol (14) as
a greyish white solid. Yield, 0.45 g (82%); mp 138–
1:1). H NMR: d (CDCl3): 5.91 (s, 1H, H–NH), 6.72 (s,
1H, H-1), 7.04 (m, 4H, Ar), 7.13 (m, 4H, Ar), 7.31 (m,
3H, Ar), 7.39 (m, 3H, Ar), 7.97 (s, 1H, H-3000), 8.06 (s, 1H,
H-5000). 13C NMR: d (CDCl3): 67.56 (CH, C-1), 116.95
(CH, C-200, C-600), 118.83 (CH, C-30, C-50), 121.90 (CH, C-
400), 127.77 (CH, C-3, C-7), 128.39 (CH, C-5), 128.89 (CH,
C-4, C-6), 129.40 (C, C-10), 129.43 (CH, C-20, C-60),
129.57 (CH, C-300, C-500), 138.52 (C, C-40), 142.16 (C,
C-2), 143.51 (CH, C-3000), 143.93 (C, C-100), 152.27 (CH, C-
5000). Anal. Calcd for C21H18N4 (326.40): C, 77.28; H, 5.56;
N, 17.16. Found: C, 76.90; H, 5.54; N, 17.04.
1
140 ꢁC; Rf = 0.54 (petroleum ether–ethyl acetate, 2:1). H
NMR: d (DMSO): 5.68 (s, 1H, H-100), 5.81 (s, 1H, H–
OH), 7.15 (m, 1H, Ar), 7.21 (m, 1H, Ar), 7.35 (m, 7H,
Ar), 7.58 (d, J = 7.9 Hz, 1H, Ar), 7.70 (d, J = 8.3 Hz,
2H, H-30, C-50), 7.79 (d, J = 7.7 Hz, 2H, H-5, C-8),
10.51 (s, 1H, NH). 13C NMR: d (DMSO): 73.94 (CH,
C-100), 117.65 (CH, C-20, C-60), 119.10 (CH, C-5), 120.98
(CH, C-8), 122.14 (CH, C-6), 125.80 (CH, C-7), 126.16
(CH, C-300, C-700), 126.57 (CH, C-500), 126.88 (CH, C-400,
C-600), 127.99 (CH, C-30, C-50), 129.96 (C, C-9), 139.25
(C, C-40), 139.54 (C, C-10), 145.85 (C, C-200), 152.10 (C,
C-4), 161.64 (C, C-2). Anal. Calcd for C20H16N2OSÆ0.4-
H2O (339.618): Calcd C, 70.73; H, 4.99; N, 8.24. Found:
C, 70.87; H, 5.08; N, 7.84.
6.1.5.3. General procedure for the addition of the
imidazole ring. To a solution of the carbinol compound
(2) (1.8 mmol) in acetonitrile (20 mL) were added imidaz-
ole (5.5 mmol) and 1,10-carbonydiimidazole (3.5 mmol).
The mixture was then heated under reflux for 1 h. The
reaction mixture was allowed to cool and then extracted
with ethyl acetate (150 mL) and water (3· 100 mL). The
organic layer was dried with MgSO4, filtered and reduced
in vacuo. The residue was then purified by flash column
chromatography.
6.1.5. General procedure for the addition of the triazole
ring. Thionyl chloride (2 mmol) in anhydrous acetoni-
trile (10 mL) was added dropwise to a stirred solution
of 1,2,4-triazole (4 mmol) in anhydrous acetonitrile
(10 mL) at a temperature of 10 ꢁC. The white suspension
formed was stirred for 1 h at 10 ꢁC. A solution of the
carbinol compound (2) (1 mmol) in anhydrous acetoni-
trile (10 mL) was added to the mixture followed by acti-
vated potassium carbonate (2 mmol). The suspension
was stirred under nitrogen at room temperature for 4
days. The resulting suspension was filtered and the fil-
trate was evaporated in vacuo to yield a light brown
oil. The oil was extracted with ethyl acetate (150 mL)
and water (3· 100 mL). The organic layer was dried with
6.1.5.4. Phenyl-[4-(1-imidazol-1-yl-ethyl)-phenyl]-amine
(5). Purification by column chromatography (dichloro-
methane–methanol, 100:0 v/v increasing to 90:10 v/v) gave
phenyl-[4-(1-imidazol-1-yl-ethyl)-phenyl]-amine (5) as a
light brown solid. Yield, 0.52 g (84%); mp 110–112 ꢁC;
1
Rf = 0.37 (dichloromethane–methanol, 90:10). H NMR:
d (CDCl3): 1.85 (d, J = 7.0 Hz, 3H, H-2), 5.30 (q,
J = 7.0 Hz, 1H, H-1), 6.02 (s, 1H, H–NH), 6.97 (m, 2H,
Ar), 7.07 (m, 7H, Ar), 7.29 (m, 2H, Ar), 7.60 (s, 1H, H-
2000). 13C NMR: d (CDCl3): 22.07 (CH3, C-2), 56.15 (CH,
C-1), 117.35 (CH, C-30, C-50), 117.89 (CH, C-4000), 118.35

M. S. Gomaa et al. / Bioorg. Med. Chem. 16 (2008) 8301–8313
8311
(CH, C-200, C-600), 121.47 (CH, C-400, C-5000), 127.24 (CH, C-
20, C-60), 129.40 (CH, C-300, C-500), 133.28 (C, C-10), 136.05
(CH, C-2000), 142.63 (C, C-40), 143.35 (C, C-100). Anal.
Calcd for C17H17N3Æ0.1H2O (265.141): C, 77.01; H, 6.46;
N, 15.85. Found: C, 76.89; H, 6.46; N, 15.87.
127.69 (CH, C-5, C-5000), 127.80 (CH, C-30, C-7), 128.23
(CH, C-600), 128.85 (C, C-20, C-60), 129.29 (CH, C-2000),
129.45 (C, C-400), 129.51 (CH, C-4, C-6), 129.51 (C, C-
500), 131.10 (C, C-10), 134.53 (C, C-1000), 139.67 (C, C-
40), 140.00 (C, C-200), 143.45 (C, C-2). Anal. Calcd for
C26H21N3Æ0.3H2O (380.87): C, 81.99; H, 5.56; N, 11.03.
Found: C, 81.86; H, 5.59; N, 10.82.
6.1.5.5.
Phenyl-[4-(phenyl-imidazol-1-yl-methyl)-phe-
nyl]-amine (6). Purification by column chromatography
(dichloromethane–methanol, 100:0 v/v increasing to
90:10 v/v) gave [phenyl-[4-(phenyl-imidazol-1-yl-methyl)-
phenyl]-amine (6) as a white solid. Yield, 0.53 g (89%);
mp 208–210 ꢁC; Rf = 0.41 (dichloromethane–methanol,
90:10). 1H NMR: d (CDCl3): 5.80 (s, 1H, H–NH),
6.48 (s, 1H, H-1), 7.01 (m, 4H, Ar), 7.17 (m, 4H, Ar),
7.30 (m, 4H, Ar), 7.38 (m, 3H, Ar), 7.46 (s, 1H, H-
2000). 13C NMR: d (CDCl3): 64.83 (CH, C-1), 116.79
(CH, C-200, C-600), 117.06 (CH, C-400), 118.42 (CH, C-
30, C-50), 121.92 (CH, C-4000), 126.93 (CH, C-5, C-5000),
128.27 (CH, C-3, C-7), 128.87 (CH, C-4, C-6), 129.30
(CH, C-20, C-60), 129.40 (CH, C-300, C-500), 129.75 (C,
C-10), 132.54 (CH, C-2000), 139.55 (C, C-40), 143.06 (C,
C-2), 143.91 (C, C-100). Anal. Calcd for C22H19N3Æ0.3-
H2O (330.815): C, 79.87; H, 5.78; N, 12.70. Found: C,
79.72; H, 5.86; N, 12.56.
6.1.5.8. [4-(Imidazol-1-yl-phenyl-methyl)-phenyl]-indan-
5-yl-amine (9). Purification by column chromatography
(dichloromethane–methanol, 100:0 v/v increasing to
95:5 v/v) gave [4-(imidazol-1-yl-phenyl-methyl)-phenyl]-in-
dan-5-yl-amine (9) as a white solid. Yield, 0.48 g (83%); mp
88–90 ꢁC; Rf = 0.43 (dichloromethane–methanol, 90:10).
1H NMR: d (CDCl3): 1.98 (m, 2H, H-200), 2.76 (m, 4H,
H-100, H-300), 5.78 (s, 1H, H–NH), 6.35 (s, 1H, H-1), 6.79
(m, 2H, Ar), 6.87 (m, 4H, Ar), 6.92 (d, J = 1.5 Hz, 1H,
H-500), 7.01 (m, 3H, Ar), 7.04 (d, J = 8.0 Hz, 1H, H-800),
7.25 (m, 3H, Ar), 7.34 (s, 1H, H-2000). 13C NMR: d (CDCl3):
25.69 (C, C-200), 32.31 (C, C-100), 33.05 (C, C-300), 64.73 (CH,
C-1), 115.98 (CH, C-700), 116.09 (CH, C-30, C-50), 118.02
(CH, C-500), 124.57 (CH, C-4000), 127.63 (CH, C-5, C-5000),
127.97 (CH, C-800), 128.67 (CH, C-3, C-7), 129.02 (CH,
C-4, C-6), 129.19 (CH, C-20, C-60), 129.38 (C, C-900),
132.81 (CH, C-2000), 138.25 (C, C-10), 139.83 (C, C-400),
140.30 (C, C-600), 145.19 (C, C-40), 145.64 (C, C-2). Anal.
Calcd for C25H23N3Æ0.7H2O (378.08): C, 79.42; H, 6.13;
N, 11.11. Found: C, 79.37; H, 6.48; N, 11.32.
6.1.5.6. [4-(1-Imidazol-1-yl-ethyl)-phenyl]-naphthalen-
2-yl-amine (7). Purification by column chromatography
(dichloromethane–methanol, 100:0 v/v increasing to
95:5 v/v) gave 4-(1-imidazol-1-yl-ethyl)-phenyl]-naph-
thalen-2-yl-amine (7) as a white solid. Yield, 0.5 g
(84%); mp 170–172 ꢁC; Rf = 0.33 (dichloromethane–
methanol, 90:10). 1H NMR: d (CDCl3): 1.78 (d,
J = 6.4 Hz, 1H, H-2), 5.19 (q, J = 6.4 Hz, 1H, H-1),
5.87 (s, 1H, H–NH), 6.91 (d, J = 1.7 Hz, 1H, H-100),
7.05 (d, J = 8.5 Hz, 4H, H-20, H-30, H-50, H-60), 7.15
(dd, J1 = 2.1 Hz, J2 = 8.6 Hz, 1H, H-300), 7.19 (m, 1H,
H-700), 7.32 (m, 1H, H-800), 7.38 (d, J = 7.7 Hz, 2H, H-
4000, H-5000), 7.51 (s, 1H, H-2000), 7.59 (d, J = 8.4 Hz, 1H,
H-400), 7.71 (d, J = 8.8 Hz, 2H, H-600, H-900). 13C NMR:
d (CDCl3): 21.05 (CH, C-1), 52.40 (CH, C-2), 111.45
(CH, C-100), 116.79 (CH, C-30, C-50), 119.22 (CH, C-
300), 122.78 (CH, C-4000, C-700), 125.53 (CH, C-800, C-900),
126.31 (CH, C-5000, C-600), 126.65 (C, C-500), 128.30
(CH, C-20, C-60), 128.42 (C, C-400), 132.75 (C, C-10),
133.52 (C, C-1000), 135.03 (CH, C-2000), 139.18 (C, C-
40), 142.06 (C, C-200). HRMS (ES+) m/z Calcd for
C21H19N3 (M + H)+ 314.1652. Found: 314.1654.
6.1.5.9. (1H-Benzoimidazol-2-yl)-{4-[(5H-imidazol-1-
yl)-phenyl-methyl]-phenyl}-amine (15). Purification by
column chromatography (dichloromethane–methanol,
100:0 v/v increasing to 95:5 v/v) gave (1H-ben-
zoimidazol-2-yl)-{4-[(5H-imidazol-1-yl)-phenyl-methyl]-
phenyl}-amine (15) as a yellowish white solid. Yield,
0.28 g (76%); mp 274–276 ꢁC; Rf = 0.17 (dichlorometh-
1
ane–methanol, 90:10). H NMR: d (DMSO): 6.80 (s,
1H, H-100), 6.99 (m, 3H, Ar), 7.13 (m, 5H, Ar), 7.35 (m,
5H, Ar), 7.6 (s, 1H, H-2000), 7.78 (d, J = 7.4 Hz, 2H, H-
5, H-8), 9.54 (s, 1H, NH), 10.68 (s, 1H, NH). 13C
NMR: d (DMSO): 63.13 (CH, C-100), 117.14 (CH, C-5,
C-8), 119.18 (CH, C-500), 120.08 (CH, C-6, C-7), 127.48
(CH, C-300, C-700), 127.73 (CH, C-5000), 128.51 (CH, C-30,
C-50, C-400, C-600), 128.78 (CH, C-2000, C-4000), 131.58 (C,
C-40), 140.48 (C, C-4, C-9), 140.66 (C, C-2), 150.35 (C,
C-10, C-200). HRMS (ES+) m/z Calcd for C23H19N5
(M + H)+ 316.1713. Found: 316.1715.
6.1.5.7. [4-(Imidazol-1-yl-phenyl-methyl)-phenyl]-naph-
thalen-2-yl-amine (8). Purification by column chromatog-
raphy (dichloromethane–methanol, 100:0 v/v increasing
to 95:5 v/v) gave [4-(imidazol-1-yl-phenyl-methyl)-phe-
nyl]-naphthalen-2-yl-amine (8) as a pink solid. Yield,
0.51 g (88%); mp 94–96 ꢁC; Rf = 0.40 (dichloromethane–
6.1.5.10. Benzooxazol-2-yl-{4-[(5H-imidazol-1-yl)-phe-
nyl-methyl]-phenyl}-amine (16). Purification by column
chromatography (petroleum ether–ethyl acetate, 80:20 v/
v increasing to 0:100 v/v) gave benzooxazol-2-yl-{4-[(5H-
imidazol-1-yl)-phenyl-methyl]-phenyl}-amine (16) as a
yellow solid. Yield, 0.19 g (71%); mp 214–216 ꢁC;
1
1
methanol, 90:10). H NMR: d (CDCl3): 6.06 (s, 1H, H–
Rf = 0.37 (ethyl acetate, 100%). H NMR: d (DMSO):
NH), 6.38 (s, 1H, H-1), 6.81 (s, 1H, H-100), 6.94 (d,
J = 8.6 Hz, 2H, H-30, H-50), 7.03 (m, 5H, Ar), 7.15 (dd,
J1 = 2.3 Hz, J2 = 8.5 Hz, 1H, H-300), 7.23 (m, 4H, Ar),
7.32 (m, 1H, Ar), 7.38 (m, 2H, Ar), 7.57 (d, J = 8.2 Hz,
1H, H-400), 7.66 (d, J = 8.8 Hz, 2H, H-600, H-900). 13C
NMR: d (CDCl3): 64.73 (CH, C-1), 112.80 (CH, C-100),
117.36 (CH, C-30, C-50), 120.40 (CH, C-300), 123.87 (CH,
C-700), 126.56 (CH, C-900), 126.60 (CH, C-800, C-4000),
6.84 (s, 1H, H-100), 6.98 (s, 1H, Ar), 7.14 (m, 4H, Ar),
7.21 (m, 3H, Ar), 7.35 (m, 1H, H-500), 7.39 (m, 3H, Ar),
7.49 (d, J = 7.9 Hz, 1H, H-4000), 7.66 (s, 1H, H-2000), 7.77
(d, J = 8.0 Hz, 2H, H-5, H-8), 10.78 (s, 1H, NH). 13C
NMR: d (DMSO): 62.99 (CH, C-100), 108.97 (CH, C-8),
116.64 (CH, C-5), 117.75 (CH, C-20, C-60), 119.15 (CH,
C-6), 121.74 (CH, C-7), 124.01 (CH, C-500), 127.54 (CH,
C-300, C-700), 127.81 (CH, C-5000), 128.65 (CH, C-400, C-600,

8312
M. S. Gomaa et al. / Bioorg. Med. Chem. 16 (2008) 8301–8313
C-30, C-50), 128.74 (CH, C-200, C-400), 133.39 (C, C-40),
138.44 (C, C-4), 140.27 (C, C-200), 142.29 (C, C-10),
147.00 (C, C-9), 157.86 (C, C-2). HRMS (ES+) m/z Calcd
for C23H18N4O (M + H)+ 367.1553. Found: 367.1555.
(75:25:0.1 v/v/v) at a flow rate of 1.9 mL/min. The Eco-
scintTM was used as the flow scintillation fluid.
6.4. Neuroblastoma cell assays
6.1.5.11. Benzothiazol-2-yl-{4-[(5H-imidazol-1-yl)-phenyl-
methyl]-phenyl}-amine (17). After reflux for 1 h, the reaction
mixture was allowed to cool and the precipitate that formed
was filtered and washed with hot CH3CN (2· 5 mL) and
dried to give benzothiazol-2-yl-{4-[(5H-imidazol-1-yl)-
phenyl-methyl]-phenyl}-amine (17) as a yellowish white
solid. Yield, 0.29 g (73%); mp 258–260 ꢁC; Rf = 0.26 (ethyl
6.4.1. Cell lines and retinoid treatments. SH-SY5Y neu-
roblastoma cells were cultured at 37 ꢁC in RPMI 1640
medium containing foetal calf serum (10%) and L-gluta-
mine (2 mM) in a humidified atmosphere of 5% CO2 in
air. atRA was dissolved in dimethyl sulfoxide and added
to the culture medium as described by Armstrong et al.19
1
acetate, 100%). H NMR: d (DMSO): 6.83 (s, 1H, H-100),
6.4.2. Real-time PCR. RNA was reverse-transcribed
from random hexamer primers and real-time PCR per-
formed on 20 ng cDNA using TaqMan Gene Expression
products for human CYP26B1 in combination with the
TaqMan Universal PCR master mix (Applied Biosys-
tems, Warrington, UK) on a GeneAmp 5700 Sequence
Detection System as described previously for
CYP26A1.19 Appropriate controls for non-specific
amplification and contamination were included and b-
actin was measured simultaneously using the endoge-
nous control assay provided by Applied Biosystems as
an internal standard. The thermocycling program con-
sisted of one cycle at 50 ꢁC for 2 min followed by
6.97 (s, 1H, Ar), 7.15 (m, 6H, Ar), 7.34 (m, 2H, Ar), 7.40
(m, 2H, Ar), 7.59 (d, J = 7.9 Hz, 1H, H-4000), 7.65 (s, 1H,
H-2000), 7.80 (m, 3H, Ar), 10.59 (s, 1H, NH). 13C NMR: d
(DMSO): 63.01 (CH, C-100), 117.88 (CH, C-20, C-60),
119.15 (CH, C-5), 119.25 (CH, C-8), 121.05 (CH, C-6),
122.34 (CH, C-7), 125.86 (CH, C-500), 127.56 (CH, C-300,
C-700), 127.81 (CH, C-5000), 127.99 (CH, C-400, C-600), 128.62
(CH, C-30, C-50), 128.71 (CH, C-2000, C-5000), 130.02 (C, C-
9), 133.21 (C, C-40), 137.14 (C, C-200), 140.29 (C, C-10),
151.96 (C, C-4), 161.43 (C, C-2). Anal. Calcd for
C23H18N4SÆ0.3H2O (387.886): Calcd C, 71.22; H, 4.68; N,
14.44. Found: C, 71.01; H, 4.73; N, 14.18.
95 ꢁC for 10 min and 40 cycles at 95 ꢁC (15 s) and
60 ꢁC (1 min). The comparative Ct method (2ꢀDDC
was used for relative quantification of gene expression.
t
)
6.2. MCF-7 (CYP26A1) assay for inhibition of metabo-
lism of atRA
MCF-7 cells were seeded in 12-well cell culture plates (Cor-
nings Inc., New York, USA) 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 re-
moved, washed once with Phosphate Buffer Saline (PBS)
and replaced by fresh medium plus 10 lL inhibitor/solvent
(acetonitrile) and 10 lL of atRA (to give final concentra-
tion of 1 · 10ꢀ7 M atRA and 0.1 lCi [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 dupli-
cate. The incubation was stopped by addition of 1% acetic
acid (100 lL/well), the medium was removed into separate
glass tubes. Distilled water (200 lL) was added to each well
and the cells scrapped off and the contents added to the
appropriate glass tube. This procedure was repeated with
a further 400 lL 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.
6.5. Molecular docking
Ligands were docked within the active site of the
CYP26A1 homology model25 using the FlexX docking
program of SYBYL,38 performed with the default val-
ues. The active site was defined by all the amino acid res-
˚
idues within a 6.5 A distance from TRP112, VAL116,
THR304, VAL370 and GLY373, including the haem
in a heteroatom file. Subsequent manipulation and inter-
action evaluation was performed with MOE (Molecular
Operating Environment) software.39
Acknowledgments
For MG we acknowledge the Embassy of the Arab
Republic of Egypt for the award of a PhD scholarship.
We also acknowledge the technical staff of the Tenovus
Cancer Centre for the preparation of MCF-7 cells and
media, and the EPSRC Mass Spectrometry Centre,
Swansea, UK, for mass spectroscopy data. Studies on
SH-SY5Y cells were supported by SPARKS and Cancer
Research UK.
6.3. High performance liquid chromatography (HPLC)
TheHPLC systemwasequipped with a high pressure pump
(Milton-Roy pump), injector with a 50 lL loop connected
to abeta-RAM radioactivity deꢂtector, connected toa Com-
paqTM computer running Laura data acquisition and anal-
ysis software. This enabled on-line detection and
quantification of radioactive peaks. The HPLC column
(10 lM C18 lBondapakTM 3.9 · 300 mm HPLC column
from Waters, UK) operating at ambient temperature was
usedtoseparatethemetaboliteswhichwereelutedwithace-
tonitrile/1% ammonium acetate in water/acetic acid
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