Synthesis and CYP26A1 inhibitory activity of 1-[benzofuran-2-yl-(4-alkyl/aryl-phenyl)-methyl]-1H-triazoles

Article abstract of DOI:10.1016/j.bmc.2006.01.018

Methodology previously described by our group was applied to the preparation of a series of 4-alkyl/aryl-substituted 1-[benzofuran-2-yl-phenylmethyl]-1H-triazoles. The [1,2,4]-triazole derivatives were prepared for a range of alkyl and aryl substituents,

Full text of DOI:10.1016/j.bmc.2006.01.018

Bioorganic & Medicinal Chemistry 14 (2006) 3643–3653
Synthesis and CYP26A1 inhibitory activity of
1-[benzofuran-2-yl-(4-alkyl/aryl-phenyl)-methyl]-1H-triazoles
Stephane Pautus,a Sook Wah Yee,a Martyn Jayne,a Michael P. Cooganb and
Claire Simonsa,*
aWelsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cardiff CF10 3XF, UK
bSchool of Chemistry, Cardiff University, Cardiff CF10 3TB, UK
Received 29 November 2005; revised 9 January 2006; accepted 10 January 2006
Available online 3 February 2006
Abstract—Methodology previously described by our group was applied to the preparation of a series of 4-alkyl/aryl-substituted 1-
[benzofuran-2-yl-phenylmethyl]-1H-triazoles. The [1,2,4]-triazole derivatives were prepared for a range of alkyl and aryl substitu-
ents, and for the 4-methyl, 4-ethyl, 4-ipropyl, 4-tbutyl, 4-phenyl and 4-chlorophenyl derivatives, the minor [1,3,4]-triazole isomer
also isolated. All the triazole derivatives were evaluated for CYP26A1 inhibitory activity using a MCF-7 cell-based assay. The 4-
ethyl and 4-phenyl-1,2,4-triazole derivatives displayed inhibitory activity (IC50 4.5 and 7 lM, respectively) comparable with that
of the CYP26 inhibitor liarozole (IC50 7 lM). Using a CYP26A1 homology model (based on CYP3A4) template, docking experi-
ments were performed with MOE with multiple hydrophobic interactions observed in addition to coordination between the triazole
nitrogen and the haem transition metal.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
efficacy. Although other CYPs are also induced by
ATRA and catalyse its metabolism, it is thought that
CYP26A1 is likely to be the most important enzyme in-
volved in its degradation7 (Fig. 1).
In humans, CYP26A1 is expressed in the liver, heart,
pituitary gland, adrenal gland, testis, brain and placen-
ta,1 and has been mapped to chromosome 10q23-q24.2
It is thought that the principal role of CYP26A1 is
homeostatic, that is, the regulation of intracellular
ATRA steady-state levels via a negative feedback loop
similar to that of CYP24 in the metabolism of cholecal-
ciferol.3 The enzyme therefore may have a protective
function, as an important regulator of differentiation
and a possible modulator of disease states indirectly by
controlling ATRA and other retinoid concentrations.4
ATRA may improve the efficacy of other treatments
such as radiation, cisplatin and interferon therapies.8,9
Retinoids have been used for some time in the treatment
of psoriasis, cystic acne, cutaneous malignancies due to
hyperkeratinisation as well as in the treatment of photo-
damaged skin.10,11 As CYP26A1 appears to be so
important in the management of intracellular levels of
ATRA and the development of resistance to its effects
in cancer, it presents a useful target in combating the
development of resistance. Inhibition of CYP26A1
would elevate normal tissue levels of ATRA or maintain
high therapeutic levels of ATRA preventing the resis-
tance that develops to treatment. This might provide a
leap forward in the prevention and treatment of a num-
ber of diseases including hormone refractory prostate
cancer and psoriasis.
Retinoic acid has been used in a number of clinical situ-
ations, especially oncology and dermatology. In oncolo-
gy, ATRA has shown spectacular success in the
treatment of acute promyelocytic leukaemia,5,6 although
remission seen is followed by relapse within 4–6 months;
this appears to be due to increased RA metabolism as a
result of RA induction so leading to decreased clinical
A number of inhibitors of ATRA metabolism have been
developed over the last fifteen years.12–15 The discovery
of the imidazoles as inhibitors led to further study into
this class of compounds, resulting in the synthesis of
Keywords: 1-[Benzofuran-2-yl-(4-alkyl/aryl-phenyl)-methyl]-1H-tria-
zoles; CYP26A1; Enzyme inhibition; MCF-7; Molecular modelling.
*
Corresponding author. Tel.: +44 02920 876307; fax: +44 02920
874149; e-mail: SimonsC@Cardiff.ac.uk
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.01.018

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S. Pautus et al. / Bioorg. Med. Chem. 14 (2006) 3643–3653
Figure 1. Metabolism of all-trans retinoic acid (RA = retinoic acid).
Figure 2. CYP26A1 metabolism inhibitors and target structures.
liarozole, which became the most studied inhibitor of
ATRA metabolism.16–18 Though more specific and effec-
tive than the other imidazoles, liarozole is still a potent
inhibitor of aromatase, preventing its use as an oral ret-
inoic acid-mimetic for sex hormone-independent can-
cers, therefore clinical development of liarozole was
discontinued.3 Further development has led to the dis-
covery of the 1H-triazole derivative, R115866, a potent
and selective inhibitor of ATRA metabolism (Fig. 2).19
2. Chemistry
The required intermediates for the preparation of the 1-
[(benzofuran-2-yl)phenylmethyl] triazoles were the cor-
responding ketones, benzo[b]furan-2-yl-phenylmetha-
nones (3). The synthesis of the ketones was achieved
by the Rap–Stoermer reaction,20 and involved reaction
of salicylaldehyde (1) with the a-bromoketones (2),21
generated by reaction of the appropriate acetophenone
with bromine and aluminium trichloride according to
literature methodology22 (Scheme 1).
As part of our ongoing research into the development of
CYP26A1 inhibitors, we evaluated a number of com-
pound classes for their inhibitory activity. The 4-alkyl-
benzofuran derivatives were promising candidates
owing to their structural similarity with liarozole as con-
firmed by molecular modelling. The synthesis,
CYP26A1 inhibitory activity and bonding interactions
at the CYP26A1 active site are described.
The ketones were then reduced to the respective racemic
carbinols (4) in quantitative yields by reaction with sodi-
um borohydride. The target triazole derivatives (5–11)
were then obtained by reaction with N,N0-thionylditriaz-
ole, which was prepared by the reaction of 1,2,4-1H-tri-
azole with thionyl chloride at 10 ꢁC for 1 h, for 4 days.

S. Pautus et al. / Bioorg. Med. Chem. 14 (2006) 3643–3653
3645
Scheme 1. Reagents and conditions: (i) Br2, AlCl3, Et2O, 1 h; (ii) NaH, DMF, 80 ꢁC, 2 h then NaOMe, 80 ꢁC, 1 h; (iii) NaBH4, dioxane, 2 h; (iv)
N,N0-thionylditriazole, K2CO3, CH3CN, 4 days.
In addition to the 1,2,4-triazole isomer, the minor 1,3,4-
isomer was obtained for some of the 1-[(benzofuran-2-
yl)phenylmethyl] triazoles. For the 1,2,4-isomers, the
triazole protons were observed as two singlets, each
integrating for one proton, at ꢀ8.2 and 8.1 ppm. The
structure of the 1,3,4-triazole isomers was confirmed by
1H NMR, which gave a singlet, integrating for two pro-
tons, at ꢀ8.30 ppm for the symmetrical triazole protons.
The 4-methyl derivative (5) has previously been evaluated
as an aromatase (CYP19) inhibitor (IC50 = 0.59 lM, cf.
anastrazole = 0.6 lM).21 The 4-ethyl (6) and 4-phenyl
(15) derivatives were also screened against CYP19 to
determine selectivity, both having an inhibitory activity
IC50 > 100 lM. The negligible CYP19 inhibitory activity
of 6 and 15 was expected from SAR studies21 which had
shown that only small substituents, for example, F, Cl,
CH3 and CN were tolerated at the 4-position of the phenyl
ring for CYP19 inhibitors.
The 4-aryl ketone derivatives (13) were prepared from
the 4-bromoketone (12) derivative using a Suzuki cou-
pling23 with the appropriate phenyl boronic acid. Subse-
quent reduction gave the alcohols (14), which were
reacted with N,N0-thionylditriazole to generate the
1,2,4- and 1,3,4-triazole isomers of the phenyl (15) and
4-chlorophenyl (16) benzofurans (Scheme 2).
4. Molecular modelling
In order to rationalise the results obtained, MOE
(Molecular Operating Environment)24 docking studies
of the inhibitors were performed using a human
CYP26A1 model,25 built using the recently crystallised
human CYP3A426 as a template. Docking interactions
at the enzyme active site were shown to be comparable
with those of the CYP26 inhibitor liarozole with the
benzofurans positioned above the haem with multiple
hydrophobic interactions (Fig. 3). The inhibitor com-
pounds are all racemic therefore both enantiomers of
all the compounds were docked to determine any prefer-
ence for (R)- or (S)-configuration; no preference was ob-
served with both enantiomers exhibiting a similar ‘fit’ in
the active site with the nitrogen of the heterocyclic ring
coordinating with the Fe3+ of the haem, therefore for
comparison Figure 3 shows the (S)-enantiomers of the
4-ethyl (6a) and the 4-phenyl (15a) 1,2,4-triazole benzo-
furan derivatives.
3. CYP26A1 inhibitory activity
The 1-[benzofuran-2-yl-(4-alkyl/aryl-phenyl)-methyl]-
1H-triazoles (5–11, 15 and 16) were evaluated for their
retinoic acid metabolism inhibitory activity using a
MCF-7 cell assay,13 using radiolabelled [11,12-3H] all-
trans retinoic acid as the substrate and liarozole and
R115866 as standards for comparison. The 4-ethyl (6a,
IC50 = 4.5 lM; 6b, IC50 = 7 lM) and 4-phenyl (15a,
IC50 = 7 lM; 15b, IC50 = 9 lM) derivatives displayed
inhibitory activity of CYP26A1 comparable with that
of liarozole (IC50 = 7 lM), and the cyclohexyl derivative
(11, IC50 = 5–15 lM) displayed moderate activity, the
remaining alkyl/aryl derivatives all poor CYP26A1
inhibitors of retinoic acid metabolism (IC50 > 20 lM)
(Table 1). All the 1-[benzofuran-2-yl-(4-alkyl/aryl-phen-
yl)-methyl]-1H-triazoles were considerably less active
than R115866 (IC50 = 5 nM).
Docking of the 4-alkyl derivatives with a chain length
of >2 indicated that steric hindrance was encountered
˚
preventing close (2.5–3.5 A) interaction with the haem.

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S. Pautus et al. / Bioorg. Med. Chem. 14 (2006) 3643–3653
Scheme 2. Reagents and conditions: (i) Pd(PPh3)4, Na2CO3(aq) toluene, 100 ꢁC, 5 h, then H2O2, rt, 1 h (60–70 %); (ii) NaBH4, dioxane, 2–6 h; (iii)
N,N0-thionylditriazole, K2CO3, CH3CN, 4 days.
Table 1. IC50 data for the novel benzofuran derivatives using CYP26A1 MCF-7 assays
R
O
N
X
Y
N
Compound
R
X
Y
IC50 (lM)
5a
5b
CH3
CH3
N
CH
N
>40
>40
4.5
CH
N
6a
6b
CH2CH3
CH2CH3
CH2CH2CH3
CH(CH3)2
CH(CH3)2
C(CH3)3
CH2CH(CH3)2
Cyclohexyl
C6H5
CH
N
CH
N
5
7
8a
CH
CH
N
50–100
20–40
50–100
10–25
40–50
5–15
7
N
8b
CH
N
9
10
CH
CH
CH
CH
N
N
11
15a
N
N
15b
16a
C6H5
CH
N
9
p-Cl–C6H5
p-Cl–C6H5
—
CH
N
20–40
20–40
12
16b
CH
—
—
—
Ketoconazole
Liarozole
R115866
—
—
—
—
7
0.005
—
IC50 values are the average ( 5%) of two experiments.
The phenyl derivative (15, IC50 = 7–9 lM) has a greater
inhibitory activity despite its size owing to favourable
conformation and enhanced hydrophobic bonding. This
may strengthen the interaction of the phenyl derivative
increasing its potency as an inhibitor of the enzyme;
however, the unsubstituted phenyl was the limit with
the introduction of the chloro substituent (16,
IC50 = 20–40 lM) resulting in a reduction of inhibitory
activity owing to steric/electronic factors.
The docking studies however fail to shed any light on
the poor results obtained for the methyl compound (5,

S. Pautus et al. / Bioorg. Med. Chem. 14 (2006) 3643–3653
3647
Figure 3. Active site region of CYP26A1 model showing (a) S-6a and (b) S-15a in ball and stick form. The distance from the haem is indicated with a
green line, transition metal interaction with a purple line. Amino acid residues identified are involved in hydrophobic interactions.
IC50 > 40 lM), which was expected to possess potency
similar to that of the ethyl derivative (6, IC50 = 4.5–
5 lM).
5.1.1.1. Benzofuran-2-yl-(4-ethyl-phenyl)-methanone
(3a, R = CH2CH3). Brown solid (34%) after recrystalli-
sation from methanol, mp 60–62 ꢁC. 1H NMR (CDCl3):
d 8.05 (d, J = 8.1 Hz, 2H, Ar), 7.78 (d, J = 7.7 Hz, 1H,
Ar), 7.70 (d, J = 7.9, 1H, Ar), 7.55 (m, 2H, Ar), 7.37
(m, 3H, Ar), 2.81 (q, J = 7.6 Hz, 2H, CH2), 1.36 (t,
J = 7.6 Hz, 3H, CH3). 13C NMR (CDCl3): d 184.56
(C@O), 156.35 (C, C-7a), 152.83 (C, C-40), 150.43 (C,
C-10), 135.22 (C, C-2), 130.19 (2· CH, CH-20 and CH-
60), 128.66 (CH, CH-6), 128.52 (2· CH, CH-30 and
CH-50), 127.48 (C, C-3a), 124.36 (CH, Ar), 123.70
(CH, Ar), 116.62 (CH, CH-3), 112.98 (CH, CH-7),
29.46 (CH2), 15.71 (CH3). Anal. Calcd for C17H14O2
(250.296): C, 81.58; H, 5.64. Found: C, 81.44; H, 5.84.
A series of 4-alkyl/aryl benzofuran phenyl triazole deriv-
atives has been prepared with the ethyl and phenyl
derivatives shown to possess comparable inhibitory
activity with that of the known CYP26 inhibitor
liarozole.
5. Experimental
5.1. Materials and methods: 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 chromatog-
raphy was performed with silica gel 60 (230–400mesh)
(Merck) and TLC was carried out on precoated silica
plates (kiesel gel 60 F254, BDH). Melting points were
determined on an electrothermal instrument and are
uncorrected.
5.1.1.2. Benzofuran-2-yl-(4-propyl-phenyl)-methanone
(3b, R = CH2CH2CH3). Cream crystalline solid (42%)
after recrystallisation from methanol, mp 64–66 ꢁC. H
1
NMR (CDCl3): d 7.92 (d, J = 8.2 Hz, 2H, Ar), 7.65 (d,
J = 7.9 Hz, 1H, Ar), 7.57 (dd, J = 0.40, 8.5 Hz, 1H,
Ar), 7.46 (d, J = 0.6 Hz, 1H, Ar), 7.42 (m, 1H, Ar),
7.26 (m, 3H, Ar), 2.62 (m, CH2, propyl), 1.64 (dt,
J = 7.4, 8.5 Hz, CH2, propyl), 0.91 (t, J = 7.3 Hz, CH3,
propyl). 13C NMR (CDCl3): d 184.09 (C@O), 155.95
(C, C-7a), 152.51 (C, C-40), 148.50 (C, C-10), 134.84
(C, C-2), 129.67 (2· CH, CH-20 and CH-60) and
128.68 (2· CH, CH-30 and CH-50), 128.19 (CH, Ar),
127.08 (C, C-3a), 123.92 (CH, Ar), 116.07 (CH, CH-
3), 112.56 (CH, CH-7), 38.12 (CH2), 24.25 (CH2),
13.75 (CH3). HRMS (EI+) m/z Calcd for C18H16O2
(M+H)+ 265.1223. Found 265.1224.
5.1.1. General method for the preparation of the ketones
3. To a solution of sodium hydride (60%, 11 mmol) in
dry N,N-dimethylformamide (10 mL) was added a solu-
tion of salicylaldehyde (1, 10 mmol) in dry N,N-dimeth-
ylformamide (6 mL) dropwise. Hydrogen gas was
liberated to give a yellow solution of the sodium salt. A
solution of the a-bromoketone 2 (10 mmol) in dry
N,N-dimethylformamide (10 mL) was then added drop-
wise and the reaction mixture was heated under nitrogen
at 80 ꢁC for 1.5 h. Sodium methoxide (2.5 mmol) was
added to the mixture and heating was continued for
another hour (TLC system: petroleum ether/ethyl acetate
4:1 v/v). After cooling, the reaction mixture was evapo-
rated to about a third of its volume, diluted with dichlo-
romethane (100 mL), washed with water (100 mL), dried
(MgSO4) and concentrated under reduced pressure.
5.1.1.3. Benzofuran-2-yl-(4-isopropyl-phenyl)-metha-
none (3c, R = CH(CH3)2). Pale orange solid (44%) after
recrystallisation from methanol, mp 93–95 ꢁC. 1H NMR
(CDCl3): d 7.85 (m, 2H, Ar), 7.53 (m, 2H, Ar), 7.35 (m,
2H, Ar), 7.34 (s, 1H, H-7), 7.32 (m, 2H, Ar) 3.05 (septet,
1H, CH, isopropyl), 1.32 (s, 6H, isopropyl). 13C NMR
(CDCl3): d 186.36 (C@O), 155.81 (C, C-7a), 154.60 (C,
C-2), 154.39 (C, C-40), 152.39 (C, C-10), 134.89 (C, C-
3a), 131.15 (2· CH, CH-20 and CH-60), 129.74 (2·
CH, CH-30 and CH-50), 126.62 (CH, Ar), 124.22 (CH,
Ar), 123.27 (CH, Ar), 115.76 (CH, CH-3), 111.20 (CH,

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S. Pautus et al. / Bioorg. Med. Chem. 14 (2006) 3643–3653
CH-7), 34.24 (CH, isopropyl), 23.82 (CH3, isopropyl).
Anal. Calcd for C18H16O2 (264.318): C, 81.79; H, 6.10.
Found: C, 81.58; H, 6.07.
acetate, 1:1 v/v, Rf: 0.63. 1H NMR (CDCl3): d 8.21
(dd, J = 1.6, 8.2 Hz, 2H, Ar), 7.82 (m, 3H, Ar), 7.73
(m, 3H, Ar), 7.65 (s, 1H, H-3), 7.46 (m, 5H, Ar). 13C
NMR (CDCl3): d 184.33 (C@O), 156.43 (C, C-7a),
152.80 (C, C-40), 146.16 (C, C-100), 140.29 (C, C-10),
136.31 (C, C-2), 130.58 (2· CH, CH-20 and CH-60),
129.46 (2· CH, CH-300 and CH-500), 128.83 (2· CH,
CH-30 and CH–50), 128.76 (CH, Ar), 127.77 (2· CH,
CH-200 and CH-600), 127.66 (CH, Ar), 127.48 (C, C-3a),
124.46 (CH, Ar), 123.78 (CH, Ar), 116.83 (CH, CH-3),
113.03 (CH, CH-7).
5.1.1.4. Benzofuran-2-yl-(4-tert-butyl-phenyl)-metha-
none (3d, R = C(CH3)3). Yellow solid (32%) after recrys-
tallisation from methanol, mp 103–105 ꢁC. 1H NMR
(CDCl3): d 7.87 (m, 2H, Ar), 7.67 (m, 2H, Ar), 7.53 (m,
2H, Ar), 7.34 (s, 1H, H-7), 7.32 (m, 2H, Ar), 1.35 (s,
9H, tbutyl). 13C NMR (CDCl3): d 184.08 (C@O),
167.76 (C, C-7a), 156.77 (C, C-2), 155.95 (C, C-40),
155.95 (C, C-10), 134.54 (C, C-3a), 130.89 (2· CH,
CH-20 and CH-60), 129.51 (2· CH, CH-30 and CH-50),
127.09 (CH, Ar), 125.55 (CH, Ar), 123.92 (CH, Ar),
116.14 (CH, CH-3), 112.57 (CH, CH-7), 68.17 (C, tbutyl),
5.1.2.2.
Benzo[b]furan-2-yl(40-chlorobiphenyl-4-
yl)methanone (13b). Light yellow solid (73%), mp 76–
78 ꢁC. TLC system: petroleum ether/ethyl acetate, 4:1
t
1
31.15 (CH3, butyl). Anal. Calcd for C19H18O2Æ0.6H2O
(289.159): C, 78.92; H, 6.69. Found: C, 79.17; H, 7.06.
v/v, Rf: 0.50. H NMR (CDCl3): d 8.25 (dd, J = 1.7,
6.6 Hz, 2H, Ar), 7.81 (m, 4H, Ar), 7.69 (m, 2H, Ar),
7.52 (m, 5H, Ar). 13C NMR (CDCl3): d 184.18 (C@O),
156.44 (C, C-7a), 152.75 (C, C-40), 144.82 (C, C-10),
138.71 (C, C-2), 136.57 (C, C-100), 134.96 (C, C-400),
130.67 (2· CH, CH-20 and CH-60), 129.64 (2· CH,
CH-300 and CH-500), 128.89 (2· CH, CH-30 and CH-50),
128.65 (CH, CH-6), 127.49 (2· CH, CH-200 and CH-
600), 127.44 (C, C-3a), 124.49 (CH, Ar), 123.79 (CH,
Ar), 116.87 (CH, CH-3), 113.02 (CH, CH-7). Anal.
Calcd for C21H13ClO2 (332.785): C, 75.79; H, 3.94.
Found: C, 76.14; H, 4.00.
5.1.1.5.
Benzofuran-2-yl-(4-isobutyl-phenyl)-metha-
none (3e, R = CH2CH(CH3)2). White solid (50%) after
recrystallisation from methanol, mp 62–63 ꢁC. 1H
NMR (CDCl3): d 7.90 (m, 2H, Ar), 7.59 (m, 2H, Ar),
7.52 (m, 2H, Ar), 7.34 (m, 2H, Ar), 7.32 (s, 1H, H-7),
2.56 (d, 2H, H-16), 1.96 (m, 1H, H-17), 1.01 (d, 6H,
H-18, H-19). 13C NMR (CDCl3): d 184.08 (C@O),
155.95 (C, C-7a), 152.50 (C, C-2), 147.55 (C, C-40),
134.86 (2· C, C-10 and C-3a), 129.63 (2· CH, CH-20
and CH-60), 129.30 (2· CH, CH-30 and CH-50), 128.19
(CH, Ar), 127.08 (CH, Ar), 123.92 (CH, Ar), 123.25
(CH, Ar), 116.09 (CH, CH-3), 112.55 (CH, CH-7),
45.48 (CH2, isobutyl), 30.16 (CH, isobutyl), 22.39
(CH3, isobutyl). Anal. Calcd for C19H18O2 (278.345):
C, 81.99; H, 6.52. Found: C, 81.55; H, 6.45.
5.1.3. General method for the preparation of the alcohols
4 and 14. To a cooled (0 ꢁC) solution of benzofuran-2-yl-
(4-alkyl/aryl-phenyl)-methanone (5 mmol) in anhydrous
dioxan (15 mL) was added sodium borohydride
(5 mmol) and then the mixture was allowed to stir at
room temperature under nitrogen for 2 h. The solvent
was concentrated under reduced pressure and aqueous
hydrochloric acid (1 M, 10 mL) was added to the resi-
due. The oil formed was extracted with diethyl ether
(2· 50 mL) and washed with water (2· 25 mL), the
organic layers were combined and dried with MgSO4
and the solvent concentrated under reduced pressure.
5.1.1.6. Benzofuran-2-yl-(4-cyclohexyl-phenyl)-metha-
none (3f, R = cC6H12).27 Light yellow solid (29%) after
1
recrystallisation from ethyl acetate. Mp 92–94 ꢁC. H
NMR (CDCl3): d 7.93 (m, 2H, Ar), 7.24 (m, 2H, Ar),
7.19 (m, 4H, Ar), 6.62 (s, 1H, H-7), 2.51 (quintet, 1H,
CH-cyclohexyl), and 1.56 (m, 10H, cyclohexyl).
5.1.2. General method for the preparation of the biphenyl
ketones 13. Two molar aqueous Na2CO3 (11.65 mL) was
added to a solution of benzo[b]furan-2-yl(4-bromophe-
nyl)methanone21 (1.0 g, 3.32 mmol) in toluene (20 mL).
The mixture was bubbled with nitrogen for one minute
and then Pd(PPh3)4 (0.20 g, 0.166 mmol) was added to
the mixture. Phenyl- or 4-phenyl-boronic acid
(6.64 mmol) in ethanol (5 mL) was added to the above
mixture and the reaction was refluxed at 100 ꢁC for 4 h.
After the reaction was complete, the residual borane
was oxidised by the addition of H2O2 (30%, 2.5 mL) at
room temperature for 1 h. The crude product was extract-
ed with CH2Cl2 (100 mL) and water (3· 100 mL). The
organic layer was dried with MgSO4, filtered and reduced
in vacuo to give a light yellow oily residue. Purification by
flash column chromatography (petroleum ether/ethyl
acetate 95:5 v/v increasing to 80:20 v/v) gave the product,
which was recrystallised from petroleum ether.
5.1.3.1.
Benzofuran-2-yl-(4-ethyl-phenyl)-methanol
(4a, R = CH2CH3). White solid (83%), mp 45–47 ꢁC.
TLC system: petroleum ether/ethyl acetate, 3:2 v/v, Rf:
0.64. 1H NMR (CDCl3): 7.60 (m, 1H, Ar), 7.50 (m,
3H, Ar), 7.32 (m, 4H, Ar), 6.61 (s, 1H, H-3), 5.96 (d,
J = 4.6 Hz, 1H, CH-OH), 2.91 (d, J = 4.6 Hz, 1H,
OH), 2.76, (q, J = 7.6 Hz, 2H, CH2), 1.34 (t,
J = 7.6 Hz, 3H, CH3). 13C NMR (CDCl3): d 160.31
(C, C-2), 159.21 (C, C-7a), 155.54 (C, C-40), 144.97 (C,
C-10), 138.12 (C, C-3a), 128.58 (2· CH, CH-30 and
CH-50), 127.34 (2· CH, CH-20 and CH-60), 124.67
(CH, Ar), 123.25 (CH, Ar), 121.58 (CH, Ar), 111.81
(CH, CH-7), 104.35 (CH, CH-3), 71.02 (CH, CH-OH),
29.08 (CH2), 16.05 (CH3). LRMS (EI+) m/z: 252.1
(M+). Anal. Calcd for C17H16O2 (252.311): C, 80.93;
H, 6.39. Found: C, 80.73; H, 6.41.
5.1.3.2. Benzofuran-2-yl-(4-propyl-phenyl)-methanol
(4b, R = CH2CH2CH3). Yellow syrup (60%). TLC sys-
1
tem: petroleum ether/ethyl acetate, 4:1 v/v, Rf: 0.64. H
5.1.2.1. Benzo[b]furan-2-yl-biphenyl-4-yl-methanone
(13a). Light yellow fine crystals (58%), mp 150–152 ꢁC
(lit.28 153–155 ꢁC). TLC system: petroleum ether/ethyl
NMR (CDCl3): d 7.50 (m, 4H, Ar), 7.26 (m, 4H, Ar),
5.70 (d, J = 9.5 Hz, 1H, CH-OH), 2.64 (m, CH2, pro-

S. Pautus et al. / Bioorg. Med. Chem. 14 (2006) 3643–3653
3649
1
pyl), 2.50 (s, 1H, OH), 1.69 (m, CH2, propyl), 0.10 (dt,
J = 3.5, 7.3 Hz, CH3, propyl). 13C NMR (CDCl3): d
157.20 (C, C-2), 155.25 (C-7a), 142.95 (C, C-40),
135.72 (C, C-10), 128.76 (2· CH, CH-30 and CH-50),
128.15 (2· CH, CH-20 and CH-60), 127.57 (C, C-3a),
126.78 (CH, Ar), 124.25 (CH, Ar), 122.82 (CH, Ar),
121.13 (CH, Ar), 111.48 (CH, CH-7), 105.09 (CH,
CH-3), 75.22 (CH, CH-OH), 37.85 (CH2), 24.51
(CH2), 13.93 (CH3). Anal. Calcd for C18H18O2
(266.339): C, 81.17; H, 6.81. Found: C, 80.87; H, 6.92.
0.63. H NMR (CDCl3): d 7.59 (m, 1H, Ar), 7.48 (m,
3H, Ar), 7.25 (m, 4H, Ar), 6.58 (s, 1H, H-7), 5.89 (s,
1H, CH-OH), 3.72 (s, 1H, OH), 2.61 (m, 1H, cyclohex-
yl), 1.58 (m, 10H, cyclohexyl). 13C NMR (CDCl3): d
159.28 (C, C-2), 155.46 (C, C-7a), 144.09 (C, C-40),
134.90 (C, C-10), 128.72 (2· CH, CH-30 and CH-50),
128.59 (2· CH, CH-20 and CH-60), 124.00 (CH, Ar),
122.77(CH, Ar), 120.91 (CH, Ar), 111.25 (CH, Ar),
103.24 (CH, Ar), 67.63 (CH, CH-OH), 45.00 (CH,
CH-cyclohexyl), 35.40 (CH2, cyclohexyl), 27.65 (CH2,
cyclohexyl), 26.95 (CH2, cyclohexyl). HRMS (EI+) m/z
Calcd for C21H22O2 (M+H)+ 358.1914. Found 358.1910.
5.1.3.3. Benzofuran-2-yl-(4-isopropyl-phenyl)-metha-
nol (4c, R = CH(CH3)2). White solid (96%), mp 69–
70 ꢁC. TLC system: petroleum ether/ethyl acetate, 4:1
5.1.3.7.
Benzo[b]furan-2-yl-biphenyl-4-yl-methanol
1
v/v, Rf: 0.74. H NMR (CDCl3): d 7.42 (m, 2H, Ar),
(14a, R = phenyl). White solid (90%), mp 121–123 ꢁC
(lit.28 123–125 ꢁC). TLC system: petroleum ether/ethyl
acetate, 1:1 v/v, Rf: 0.63. 1H NMR (CDCl3): d 7.63
(m, 6H, Ar), 7.46 (m, 4H, Ar), 7.30 (m, 3H, Ar), 6.64
(d, J = 0.6 Hz, 1H, H-3), 6.04 (d, J = 3.1 Hz, 1H, CH-
OH), 2.73, (d, J = 3.9 Hz, 1H, OH). 13C NMR (CDCl3):
d 158.84 (C, C-2), 155.57 (C, C-7a), 141.76 (C, C-10),
141.10 (C, C-100), 139.69 (C, C-40), 129.28 (2· CH,
CH-300 and CH-500), 128.47 (C, C-3a), 127.92 (2· CH,
CH-20 and CH-60), 127.83 (2· CH, CH-30 and CH-50),
127.72 (2· CH, CH-200 and CH-600), 127.60 (CH, Ar),
124.82 (CH, Ar), 123.34 (CH, Ar), 121.64 (CH, Ar),
111.83 (CH, CH-7), 104.57 (CH, CH-3), 70.91 (CH-
OH).
7.34 (m, 2H, Ar), 7.16 (m, 2H, Ar), 7.11 (s, 2H, Ar),
6.45 (s, 1H, H-7), 5.81 (s, 1H, CH-OH), 2.82 (m, 1H, iso-
propyl), 2.59 (s, 1H, OH), 1.18 (s, 6H, isopropyl). 13C
NMR (CDCl3): d 157.64 (C, C-2), 154.03 (C, C-7a),
148.10 (C, C-40), 136.71 (2· CH, CH-30 and CH-50),
127.03 (2· CH, CH-20 and CH-60), 125.81 (CH, Ar),
125.65 (CH, Ar), 123.16 (CH, Ar), 110.28 (CH, CH-7),
102.82 (CH, CH-3), 69.54 (CH, CH-OH), 32.84 (CH,
isopropyl), 22.93 (CH3, isopropyl). Anal. Calcd for
C18H19O2Æ0.5H2O (276.3545): C, 78.23; H, 7.29. Found:
C, 78.14; H, 7.05.
5.1.3.4. Benzofuran-2-yl-(4-tert-butyl-phenyl)-metha-
nol (4d, R = C(CH3)3). White solid (99%), mp 87–
88 ꢁC. TLC system: petroleum ether/ethyl acetate, 4:1
5.1.3.8.
Benzo[b]furan-2-yl-(40-chlorobiphenyl-4-
1
v/v, Rf: 0.72. H NMR (CDCl3): d 7.39 (m, 2H, Ar),
yl)methanol (14b, R = 4-chlorophenyl). White solid
(75%), mp 76–78 ꢁC. TLC system: petroleum ether/ethyl
acetate, 1:1 v/v, Rf: 0.60. H NMR (CDCl3): d 7.54 (m,
7.34 (m, 2H, Ar), 7.30 (m, 2H, Ar), 7.12 (m, 2H, Ar),
6.41 (s, 1H, H-7), 5.78 (s, 1H, CH-OH), 2.58 (s, 1H,
1
t
OH), 1.21 (s, 9H, butyl). 13C NMR (CDCl3): d 158.74
7H, Ar), 7.44 (m, 3H, Ar), 7.25 (m, 2H, Ar), 6.59 (s, 1H,
H-3), 6.00 (d, J = 4.5 Hz, 1H, CH-OH), 2.65 (d,
J = 4.6 Hz, 1H, OH). 13C NMR (CDCl3): d 158.69 (C,
C-2), 155.55 (C, C-7a), 140.48 (C, C-10), 140.31 (C, C-
40), 139.51 (C, C-100), 134.02 (C, C-400), 129.42 (2· CH,
CH-300 and CH-500), 128.81 (2· CH, CH-200 and CH-
600), 128.41 (C, C-3a), 127.80 (2· CH, CH-20 and CH-
60), 127.64 (2· CH, CH-30 and CH-50), 124.87 (CH,
Ar), 123.36 (CH, Ar), 121.65 (CH, Ar), 111.82 (CH,
CH-7), 104.59 (CH, CH-3), 70.82 (CH-OH). Anal.
Calcd for C21H15ClO2 (334.800): C, 75.20; H, 4.45.
Found: C, 75.34; H, 4.52.
(C, C-2), 155.14 (C, C-7a), 151.24 (C, C-40), 130.72 (2·
CH, CH-30 and CH-50), 129.36 (2· CH, CH-20 and
CH-60), 128.15 (CH, Ar), 126.64 (CH, Ar), 125.93
(CH, Ar), 111.39 (CH, CH-7), 103.92 (CH, CH-3),
70.57 (CH, CH-OH), 34.66 (C, tbutyl), 31.39 (CH3, tbu-
tyl). Anal. Calcd for C19H20O2 (280.361): C, 81.40; H,
7.19. Found: C, 81.22; H, 7.01.
5.1.3.5. Benzofuran-2-yl-(4-isobutyl-phenyl)-methanol
(4e, R = CH2CH(CH3)2). Yellow syrup (99%), mp 87–
88 ꢁC. TLC system: petroleum ether/ethyl acetate, 4:1
v/v, Rf: 0.73. 1H NMR (CDCl3): d 7.53 (m, 1H,
Ar), 7.48 (m, 1H, Ar), 7.41 (m, 2H, Ar), 7.20 (m,
4H, Ar), 6.57 (s, 1H, H-7), 5.95 (s, 1H, CH-OH),
3.72 (s, 1H, OH), 2.52 (s, 2H, isobutyl), 1.91 (m,
1H, isobutyl), 0.96 (s, 6H, isobutyl). 13C NMR
(CDCl3): d 158.74 (C, C-2), 155.12 (C, C-7a), 142.05
(C, C-40), 137.66 (C, C-10), 129.38 (2· CH, CH-30
and CH-50), 128.10 (CH, Ar), 126.63 (2· CH, CH-20
and CH-60), 124.23 (CH, Ar), 122.81 (CH, Ar),
121.12 (CH, Ar), 111.34 (CH, CH-7), 103.89 (CH,
CH-3), 70.66 (CH, CH-OH), 45.16 (CH2, isobutyl),
30.21 (CH, isobutyl), 22.38 (CH3, isobutyl). Anal.
Calcd for C19H20O2 (280.361): C, 81.40; H, 7.19.
Found: C, 81.07; H, 7.43.
5.1.4. General method for the preparation of the triazole
compounds 5–11, 15 and 16. Thionyl chloride (4 mmol)
in anhydrous acetonitrile (10.0 mL) was added dropwise
to a stirred solution of 1,2,4-triazole (16 mmol) in anhy-
drous acetonitrile (10.0 mL) at a temperature of 10 ꢁC.
The white suspension formed was stirred for 1 h at
10 ꢁC. A solution of benzo[b]furan-2-yl-(4-alkyl/arylphe-
nyl)methanol (4a–f, 14) (1.0 g, 4 mmol) in anhydrous
acetonitrile (10.0 mL) was added to the mixture fol-
lowed by activated potassium carbonate (1.10 g,
8 mmol). The suspension was stirred under nitrogen at
room temperature for 4 days. The resulting suspension
was filtered and the filtrate was evaporated in vacuo to
yield a light brown oil. The oil was extracted with
CH2Cl2 (150 mL) and water (3· 100 mL). The organic
layer was dried with MgSO4, filtered and reduced
in vacuo.
5.1.3.6. Benzofuran-2-yl-(4-cyclohexyl-phenyl)-metha-
nol (4f, R = cC6H12). White solid (98%), mp 67–68 ꢁC.
TLC system: petroleum ether/ethyl acetate, 4:1 v/v, Rf:

3650
S. Pautus et al. / Bioorg. Med. Chem. 14 (2006) 3643–3653
5.1.4.1. 1-[Benzo[b]furan-2-yl-(4-methylphenyl)meth-
7.57 (d, J = 6.8 Hz, 1H, Ar), 7.46 (d, J = 8.4 Hz, 1H,
Ar), 7.37–7.19 (m, 6H, Ar), 6.75 (s, 1H, H-1), 6.62 (s,
1H, H-3), 2.70 (q, J = 7.6 Hz, 2H, CH2), 1.26 (t,
J = 7.6 Hz, 3H, CH3). 13C NMR (CDCl3): d 155.76
(C, C-2), 152.90 (C, C-7a), 146.26 (C, C-40), 142.95 (2·
CH, CH-2000 and CH-5000), 132.98 (C, C-10), 129.27 (2·
CH, CH-20 and CH-60), 127.89 (2· CH, CH-30 and
CH-50), 127.67 (C, C-3a), 125.91 (CH, Ar), 123.94
(CH, Ar), 122.01 (CH, Ar), 112.01 (CH, CH-7), 108.04
(CH, CH-3), 58.28 (CH, CH-1), 28.95 (CH2), 15.83
(CH3). Anal. Calcd for C19H17N3O (303.362): C,
75.23; H, 5.65; N, 13.84. Found: C, 75.19; H, 5.74; N,
13.61.
yl]-1H-[1,2,4]triazole (5a) and 4-[benzo[b] furan-2-yl-(4-
methylphenyl)methyl]-4H-[1,3,4]triazole (5b). Purified by
column chromatography (petroleum ether/ethyl acetate,
1:1 v/v) to give the [1,2,4]triazole 5a, further elution
(dichloromethane/methanol, 95:5 v/v) gave the [1,3,4]tri-
azole 5b.
Data for 5a: orange crystals (28%), mp 106–108 ꢁC. TLC
system: petroleum ether/ethyl acetate, 4:1 v/v, Rf: 0.14.
1H NMR (CDCl3): d 8.14 (s, 1H, H-3000), 8.05 (s, 1H,
H-5000), 7.56 (d, J = 7.7 Hz, 1H, Ar), 7.48 (d,
J = 8.3 Hz, 1H, Ar), 7.34 (m, 1H, Ar), 7.25 (m, 5H,
Ar), 6.84 (s, 1H, H-1), 6.59 (s, 1H, H-3), 2.40 (s, 3H,
CH3). 13C NMR (CDCl3): d 155.34 (C, C-2), 152.93
(C, C-7a), 152.21 (CH, CH-5000), 143.21 (CH, CH-3000),
139.24 (C, C-3a), 132.61 (C, C-10), 129.80 and 127.67
(CH, Ar), 127.55 (C, C-40), 125.17, 123.30 and 121.49
(CH, Ar), 111.54 (CH, CH-7), 107.58 (CH, CH-3),
62.10 (CH, CH-1), 21.19 (CH3).
5.1.4.3. 1-[Benzo[b]furan-2-yl-(4-propylphenyl)meth-
yl]-1H-[1,2,4]triazole (7). Purified by column chromatog-
raphy (petroleum ether/ethyl acetate, 1:1 v/v) to give the
[1,2,4]triazole 7 as a yellow syrup (2%). TLC system:
petroleum ether/ethyl acetate, 4:1 v/v, Rf: 0.25. 1H
NMR (CDCl3): d 8.03 (s, 1H, H-3000), 7.94 (s, 1H, H-
5000), 7.46 (d, J = 7.7 Hz, 1H, Ar), 7.38 (d, J = 8.3 Hz,
1H, Ar), 7.23 (m, 1H, Ar), 7.16 (m, 5H, Ar), 6.74 (s,
1H, H-1), 6.49 (s, 1H, H-3), 2.53 (t, J = 7.6 Hz, CH2,
propyl), 1.57 (dt, J = 7.5, 15 Hz, CH2, propyl), 0.87 (t,
J = 7.3 Hz, CH3, propyl). 13C NMR (CDCl3): d 154.32
(C, C-2), 151.92 (C, C-7a), 151.17 (CH, CH-5000),
142.96 (C, C-3a), 131.77 (C, C-10), 128.17 (2· CH,
CH-20 and CH-60), 126.61 (2· CH, CH-30 and CH-50),
126.53 (C, C-40), 124.13, 122.27 and 120.46 (CH, Ar),
110.52 (CH, CH-7), 106.55 (CH, CH-3), 61.11 (CH,
CH-1), 36.68 (CH2), 23.34 (CH2), 12.79 (CH3). HRMS
(EI+) m/z Calcd for C20H19N3O (M+H)+ 318.1601.
Found 318.1601.
Data for 5b: white solid (12%), mp 64–66 ꢁC. TLC sys-
tem: dichloromethane/methanol, 9:1 v/v, Rf: 0.27. 1H
NMR (CDCl3): d 8.21 (s, 2H, H-2000 and H-5000), 7.57
(d, J = 7.7 Hz, 1H, Ar), 7.48 (d, J = 8.3 Hz, 1H, Ar),
7.36 (m, 1H, Ar), 7.28 (m, 3H, Ar), 7.17 (d,
J = 8.1 Hz, 2H, Ar), 6.64 (s, 1H, H-1), 6.59 (s, 1H, H-
3), 2.41 (s, 3H, CH3). 13C NMR (CDCl3): d 155.39 (C,
C-2), 152.42 (C, C-7a), 142.43 (2· CH, CH-2000 and
CH-5000), 139.72 (C, C-10), 132.27 (C, C-40),130.08 and
127.38 (CH, Ar), 127.22 (C, C-3a), 125.56, 123.56 and
121.57 (CH, Ar), 111.62 (CH, CH-7), 107.60 (CH,
CH-3), 58.00 (CH, CH-1), 21.18 (CH3). HRMS (EI+)
m/z Calcd for C18H15N3O (M+H)+ 290.1288. Found
290.1287.
5.1.4.4.
methyl]-1H-[1,2,4]triazole (8a) and 4-[benzo[b] furan-2-
yl-(4-isopropylphenyl)methyl]-4H-[1,3,4]triazole (8b).
1-[Benzo[b]furan-2-yl-(4-isopropylphenyl)-
5.1.4.2. 1-[Benzo[b]furan-2-yl-(4-ethylphenyl)methyl]-
1H-[1,2,4]triazole (6a) and 4-[benzo[b] furan-2-yl-(4-eth-
ylphenyl)methyl]-4H-[1,3,4]triazole (6b). Purified by col-
umn chromatography (petroleum ether/ethyl acetate,
7:3 v/v) to give the [1,2,4]triazole 6a, further elution
(dichloromethane/methanol, 99:1 v/v) gave the [1,3,4]tri-
azole 6b.
Purified by column chromatography (hexane/ethyl ace-
tate, 9:1 v/v) to give the [1,2,4]triazole 8a, further elution
(dichloromethane/methanol, 9:1 v/v) gave the [1,3,4]tria-
zole 8b.
Data for 8a: yellow syrup (78%). TLC system: petroleum
1
ether/ethyl acetate, 1:1 v/v, Rf: 0.72. H NMR (CDCl3):
Data for 6a: yellow syrup (67%). TLC system: petroleum
ether/ethyl acetate, 1:1 v/v, Rf: 0.42. H NMR (CDCl3):
d 8.42 (s, 1H, H-3000), 8.33 (s, 1H, H-5000), 7.31 (m, 1H,
Ar), 7.24 (m, 2H, Ar), 7.06 (m, 5H, Ar), 6.62 (s, 1H,
H-1), 6.37 (s, 1H, H-3), 2.73 (septet, 1H, isopropyl),
1.05 (d, 6H, isopropyl). 13C NMR (CDCl3): d 155.25
(C, C-2), 153.15 (CH, C-7a), 152.11 (CH, CH-5000),
149.79 (C, C-3a), 143.35 (C, C-10), 133.09 (C, C-40),
127.76 (2· CH, CH-20 and CH-60), 126.84 (2· CH,
CH-30 and CH-50), 125.04 (CH, Ar), 123.22 (CH, Ar),
112.44 (CH, Ar), 111.42 (CH, CH-7), 107.07 (CH,
CH-3), 62.03 (CH, CH-1), 34.47 (C, isopropyl), 24.16
(CH3, isopropyl). HRMS (EI+) m/z Calcd for
C20H19N3O (M)+ 318.1601. Found 318.1601.
1
d 8.19 (s, 1H, H-3000), 8.09 (s, 1H, H-5000), 7.58 (d,
J = 8.4 Hz, 1H, Ar), 7.51 (d, J = 9.3 Hz, 1H, Ar),
7.39–7.27 (m, 6H, Ar), 6.89 (s, 1H, H-1), 6.63 (s, 1H,
H-3), 2.73 (q, J = 7.6 Hz, 2H, CH2), 1.30 (t,
J = 7.6 Hz, 3H, CH3). 13C NMR (CDCl3): d 155.75
(C, C-2), 153.342 (C, C-7a), 152.65 (CH, CH-5000),
145.91 (C, C-40), 143.67 (CH, CH-3000), 133.20 (C, C-
10), 129.06 (2· CH, CH-20 and CH-60), 128.16 (2· CH,
CH-30 and CH-50), 127.96 (C, C-3a), 125.59 (CH, Ar),
123.73 (CH, Ar), 121.93 (CH, Ar), 111.97 (CH, CH-7),
108.06 (CH, CH-3), 62.51 (CH, CH-1), 28.99 (CH2),
15.85 (CH3). HRMS (EI+) m/z Calcd for C19H17N3O
(M+H)+ 304.1444. Found 304.1442.
Data for 8b: yellow syrup (7%). TLC system: petroleum
1
ether/ethyl acetate, 1:1 v/v, Rf: 0.11. H NMR (CDCl3):
d 8.12 (s, 2H, H-2000 and H-5000), 7.47 (m, 1H, Ar), 7.39
(m, 1H, Ar), 7.27 (m, 1H, Ar), 7.20 (m, 4H, Ar), 7.13
(m, 3H, Ar), 6.56 (s, 1H, H-1), 6.49 (s, 1H, H-3), 2.75
(septet, 1H, isopropyl), 1.18 (d, 6H, isopropyl). 13C
Data for 6b: White solid (13%), mp 126–128 ꢁC. TLC
system: dichloromethane/methanol, 9:1 v/v, Rf: 0.31.
1H NMR (CDCl3): d 8.25 (s, 2H, H-2000 and H-5000),

S. Pautus et al. / Bioorg. Med. Chem. 14 (2006) 3643–3653
3651
NMR (CDCl3): d 155.39 (C, C-2), 152.30 (C, C-7a),
150.63 (2· CH, CH-2000 and CH-5000), 142.47 (C, C-40),
132.46 (C, C-10), 127.72 (C, C-3a), 127.52 (2· CH,
CH-20 and CH-60), 127.21 (2· CH, CH-30 and CH-50),
125.58 (CH, Ar), 123.58 (CH, Ar), 121.59 (CH, Ar),
111.64 (CH, CH-7), 107.67 (CH, CH-3), 58.08 (CH,
CH-1), 33.89 (C, isopropyl), 23.85 (CH3, isopropyl).
HRMS (EI+) m/z Calcd for C20H19N3O (M+H)+
318.1601. Found 318.1603.
(CH, Ar), 110.89 (CH, CH-7), 101.37 (CH, CH-3),
56.50 (CH, CH-1), 45.00 (CH, cyclohexyl), 35.40 (2·
CH2, cyclohexyl), 27.65 (2· CH2, cyclohexyl), 26.95
(1· CH2, cyclohexyl). HRMS (EI+) m/z Calcd for
C23H23N3O2 (M+H)+ 358.1914. Found 358.1910.
5.1.4.8. 1-(Benzo[b]furan-2-yl-biphenyl-4-yl-methyl)-
1H-[1,2,4]triazole (15a) and 4-(benzo[b]furan-2-yl-biphe-
nyl-4-yl-methyl)-4H-[1,2,4]triazole (15b). Purified by col-
umn chromatography (petroleum/ethyl acetate, 9:1 v/v
increasing to 65:35 v/v) to give the [1,2,4]triazole 15a,
further elution (dichloromethane/methanol, 99:1 v/v)
gave the [1,3,4]triazole 15b.
5.1.4.5.
1-[Benzo[b]furan-2-yl-(4-tert-butyl-phenyl)-
methyl]-1H-[1,2,4]triazole (9). Purified by column chro-
matography (hexane/ethyl acetate, 9:1 v/v) to give the
[1,2,4]triazole 9, as a yellow syrup (62%). TLC system:
petroleum ether/ethyl acetate, 1:1 v/v, Rf: 0.61. 1H
NMR (CDCl3): d 8.15 (s, 1H, 1H, H-3000), 8.01 (s, 1H,
H-5000), 7.49 (m, 1H, Ar), 7.40 (m, 3H, Ar), 7.25 (m,
3H, Ar), 7.19 (m, 3H, Ar), 6.76 (s, 1H, H-1), 6.57 (s,
Data for 15a: White solid (53%), mp 130–132 ꢁC. TLC
system: petroleum ether/ethyl acetate, 1:1 v/v, Rf: 0.41.
1H NMR (CDCl3): d 8.24 (s, 1H, H-3000), 8.12 (s, 1H,
H-5000), 7.68 (d, J = 8.3 Hz, 2H, Ar), 7.63 (m, 3H, Ar),
7.53 (m, 3H, Ar), 7.45–7.31 (m, 5H, Ar), 6.96 (s, 1H,
H-1), 6.69 (s, 1H, H-3). 13C NMR (CDCl3): d 155.80
(C, C-2), 152.99 (C, C-7a), 152.78 (CH, CH-5000),
143.76 (CH, CH-3000), 142.6.1 (C, C-100), 140.55 (C, C-
10), 134.92 (C, C-40), 129.36 (2· CH, CH-20 and CH-
60), 128.58 (2· CH, CH-300 and CH-500), 128.27 (2·
CH, CH-200 and CH-600), 128.23 (2· CH, CH-30 and
CH-50), 127.92 (C, C-3a), 127.60 (CH, CH-400), 125.75
(CH, Ar), 123.83 (CH, Ar), 122.02 (CH, Ar), 112.02
(CH, CH-7), 108.34 (CH, CH-3), 62.41 (CH, CH-1).
Anal. Calcd for C23H17N3O (351.406): C, 78.61; H,
4.88; N, 11.95. Found: C, 78.38; H, 4.74; N, 11.89.
t
1H, H-3), 1.32 (s, 9H, butyl). 13C NMR (CDCl3): d
156.98 (C, C-2), 154.16 (CH, CH-5000), 150.30 (C,
C-7a), 148.52 (CH, CH-3000), 136.47 (C, C-40), 132.56
(2· CH, CH-20 and CH-60), 129.94 (C, C-10), 129.08
(C, C-3a), 127.92 (2· CH, CH-30 and CH-50), 123.37
(CH, Ar), 122.14 (CH, Ar), 120.55 (CH, Ar), 110.82
(CH, CH-7), 101.37 (CH, CH-3), 61.90 (CH, CH-1),
t
t
34.80 (C, butyl), 31.41 (CH3, butyl). HRMS (EI+) m/
z Calcd for C21H21N3O (M)+ 332.1757. Found 332.1761.
5.1.4.6. 1-[Benzo[b]furan-2-yl-(4-isobutylphenyl)meth-
yl]-1H-[1,2,4]triazole (10). Purified by column chroma-
tography (hexane/ethyl acetate, 9:1 v/v) to give the
[1,2,4]triazole 10 as a yellow syrup (56%). TLC system:
petroleum ether/ethyl acetate, 1:1 v/v, Rf: 0.63. 1H
NMR (CDCl3): d 8.28 (s, 1H, 1H, H-3000), 8.22 (s, 1H,
1H, H-5000), 7.60 (m, 2H, Ar), 7.40 (m, 2H, Ar), 7.31
(m, 2H, Ar), 7.23 (m, 2H, Ar), 6.82 (s, 1H, H-1), 6.54
(s, 1H, H-3), 2.54 (d, 2H, CH2, isobutyl), 1.95 (m, 1H,
CH, isobutyl), 1.02 (s, 6H, isobutyl). 13C NMR
(CDCl3): d 155.27 (C, C-2), 153.21 (CH, CH-5000),
152.11 (CH, CH-3000), 136.47 (C, C-7a), 132.51 (2· CH,
CH-20 and CH-60), 131.47 (2· CH, CH-30 and CH-50),
131.36 (C, C-40), 129.94 (C, C-10), 128.88 (C, C-3a),
123.37 (CH, Ar), 122.14 (CH, Ar), 120.55 (CH, Ar),
110.89 (CH, CH-7), 101.37 (CH, CH-3), 56.50 (CH,
CH-1), 45.54 (CH2, isobutyl), 27.57 (CH, isobutyl),
22.23 (CH3, isobutyl). Anal. Calcd for C21H21N3OÆ0.3-
H2O (336.821): C, 74.89; H, 6.46; N, 12.48. Found: C,
74.87; H, 6.45; N, 12.15.
Data for 15b: Light yellow solid (6%), mp 56–58 ꢁC.
TLC system: dichloromethane/methanol, 9:1 v/v, Rf:
1
0.31. H NMR (CDCl3): d 8.41 (s, 2H, H-2000 and H-
5000), 7.79 (d, J = 8.4 Hz, 2H, Ar), 7.72 (m, 3H, Ar),
7.61 (m, 3H, Ar), 7.58–7.40 (m, 5H, Ar), 6.87 (s, 1H,
H-1), 6.79 (s, 1H, H-3). 13C NMR (CDCl3): d 155.85
(C, C-2), 152.44 (C, C-7a), 143.02 (C, C-100), 142.93
(2· CH-2000 and CH-5000), 140.23 (C, C-10), 134.50 (C,
C-40), 129.42 (2· CH, CH-20 and CH-60), 128.51 (2·
CH, CH-300 and CH-500), 128.41 (2· CH, CH-200 and
CH-600), 128.31 (CH, CH-400), 127.59 (2· CH, CH-30
and CH-50 and C, C-3a), 126.12 (CH, Ar), 124.07
(CH, Ar), 122.09 (CH, Ar), 112.10 (CH, CH-7), 108.34
(CH, CH-3), 58.33 (CH, CH-1). Anal. Calcd for
C23H17N3O (351.406): C, 78.61; H, 4.88; N, 11.95.
Found: C, 78.77; H, 4.62; N, 11.79.
5.1.4.9. 1-[Benzo[b]furan-2-yl-(40-chloro-biphenyl-4-yl)-
methyl]-1H-[1,2,4]triazole (16a) and 4-[benzo[b]furan-2-yl-
(40chloro-biphenyl-4-yl)-methyl]-4H-[1,2,4]triazole (16b).
Purified by column chromatography (petroleum/ethyl
acetate, 9:1 v/v increasing to 65:35 v/v) to give the
[1,2,4]triazole 16a, further elution (dichloromethane/
methanol, 99:1 v/v) gave the [1,3,4]triazole 16b.
5.1.4.7.
1-[Benzo[b]furan-2-yl-(4-cyclohexylphenyl)-
methyl]-1H-[1,2,4]triazole (11). Purified by column chro-
matography (hexane/ethyl acetate, 9:1 v/v) to give the
[1,2,4]triazole 11 as a yellow syrup (14%). TLC system:
petroleum ether/ethyl acetate, 1:1 v/v, Rf: 0.70. 1H
NMR (CDCl3): d 8.28 (s, 1H, 1H, H-3000), 8.22 (s, 1H,
1H, H-5000), 7.60 (m, 2H, Ar), 7.40 (m, 2H, Ar), 7.31
(m, 2H, Ar), 7.23 (m, 2H, Ar), 6.82 (s, 1H, H-1), 6.54
(s, 1H, H-3), 2.54 (m, 1H, CH, cyclohexyl), 1.48 (m,
10H, cyclohexyl). 13C NMR (CDCl3): d 156.98 (C, C-
2), 154.16 (CH, C-7a), 148.52 (CH, CH-5000), 143.74
(CH, CH-3000), 136.47 (C, C-40), 131.56 (2· CH, CH-20
and CH-60), 129.81 (2· CH, CH-30 and CH-50), 128.64
(C, C-3a), 123.37 (CH, Ar), 122.14 (CH, Ar), 120.55
Data for 16a: white solid (67%), mp 34–36 ꢁC. TLC sys-
1
tem: petroleum ether/ethyl acetate, 1:1 v/v, Rf: 0.26. H
NMR (CDCl3): d 8.24 (s, 1H, H-3000), 8.11 (s, 1H, H-
5000), 7.64 (d, J = 8.3 Hz, 2H, Ar), 7.60–7.52 (m, 3H,
Ar), 7.49–7.29 (m, 7H, Ar), 6.95 (s, 1H, H-1), 6.69 (s,
1H, H-3). 13C NMR (CDCl3): d 155.80 (C, C-2),
152.80 (CH, CH-5000 and C, C-7a), 143.74 (CH, CH-

3652
S. Pautus et al. / Bioorg. Med. Chem. 14 (2006) 3643–3653
3000), 141.36 (C, C-10), 138.97 (C, C-100), 135.32 (C, C-40),
134.38 (C, C-400), 129.52 (2· CH, CH-20 and CH-60),
128.83 (2· CH, CH-300 and CH-500), 128.67 (2· CH,
CH-200 and CH-600), 128.10 (2· CH, CH-30 and CH-50),
127.87 (C, C-3a), 125.80 (CH, Ar), 123.86 (CH, Ar),
122.02 (CH, Ar), 112.02 (CH, CH-7), 108.40 (CH,
CH-3), 62.35 (CH, CH-1). Anal. Calcd for C23H16
ClN3O (385.852): C, 71.60; H, 4.18; N, 10.89. Found:
C, 71.45; H, 4.10; N, 10.62.
connected to a b-RAM radioactivity detector, connected
to a CompaqTM computer running Lauraꢂ data acquisi-
tion and analysis software. This enabled on-line detec-
tion 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 used to separate the metabolites which
were eluted with acetonitrile/1% ammonium acetate in
water/acetic acid (75:25:0.1 v/v/v) at a flow rate of
1.9 mL/min. EcoscintTM was used as the flow scintilla-
tion fluid.
Data for 16b: Light yellow solid (13%), mp 68–70 ꢁC.
TLC system: dichloromethane/methanol, 9:1 v/v, Rf:
1
0.50. H NMR (CDCl3): d 8.31 (s, 2H, H-2000 and H-
5.4. Molecular docking
5000), 7.65 (d, J = 8.3 Hz, 2H, Ar), 7.61–7.43 (m, 6H,
Ar), 7.41–7.31 (m, 4H, Ar), 6.79 (s, 1H, H-1), 6.70 (s,
1H, H-3). 13C NMR (CDCl3): d 155.85 (C, C-2),
152.29 (C, C-7a), 142.89 (2· CH, CH-2000 and CH-5000),
141.75 (C, C-10), 138.66 (C, C-100), 134.93 (C, C-40),
134.58 (C, C-400), 129.59 (2· CH, CH-20 and CH-60),
128.82 (2· CH, CH-300 and CH-500), 128.42 (2· CH,
CH-200 and CH-600), 128.35 (2· CH, CH-30 and CH-50),
127.56 (C, C-3a), 126.16 (CH, Ar), 124.10 (CH, Ar),
122.10 (CH, Ar), 112.10 (CH, CH-7), 108.37 (CH,
CH-3), 58.26 (CH, CH-1). Anal. Calcd for
C23H16ClN3OÆ0.1H2O (387.653): C, 71.26; H, 4.21; N,
10.83. Found: C, 71.05; H, 4.09; N, 10.75.
All molecular modelling studies were performed on a
RM Innovator Pentium IV 2.4 GHz running either Li-
nux Fedora Core 3 or Windows XP using Molecular
Operating Environment (MOE) 2004.0324 software. All
the minimisations were performed witꢁh1MOE a until a
˚
with the force-
RMSD gradient of 0.05 Kcal molꢁ1
A
field specified and the partial charges were automatically
calculated.
Ligands were docked within the active site of the
CYP26A1 homology model25 using the MOE-Dock
with simulated annealing used as the search protocol
with a total of 5 runs, 10 cycles per run, 8000 steps
per cycle and an initial temperature of 1000 K. Molecu-
lar dynamics was performed with MOE using the NVT
environment for 100 ps and constant temperature of
300 K using the MMFF94X forcefield with all other de-
fault settings in MOE-dynamics chosen. The lowest
energy conformation was selected and subjected to an
energy minimisation using the MMFF94X forcefield.
The scoring.svl script was used to identify interaction
types between ligand and protein.
5.2. MCF-7 (CYP26A1) assay for inhibition of metabo-
lism of atRA
Human MCF-7 breast cancer cells were cultured in phe-
nol red free RPMI 1640 medium supplemented with 5 %
(v/v) charcoal free foetal calf serum, antibiotics (penicillin
and streptomycin) and fungizone at the same concentra-
tion of 10 iU/mL. Cells were grown in a humidified incu-
bator (5% CO2, 95% air) at 37 ꢁC. MCF-7 cells were
seeded in 12-well cell culture plates (Cornings 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-buffered saline
(PBS) and replaced by fresh medium plus 10 lL inhibi-
tor/solvent (acetonitrile) and 10 lL of atRA (to give a fi-
nal concentration of 1 · 10ꢁ7 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 duplicate. The incubation was stopped
by addition of 1% acetic acid (100 lL/well), the medium
removed into separate glass tubes. Two hundred microli-
tres of distilled water was added to each well and the cells
were scrapped off and the contents added to the appropri-
ate glass tube. This procedure was repeated with a further
400 lL water but without scraping. Ethyl acetate con-
taining 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 con-
nected to a vacuum pump and a multitrap at ꢁ80 ꢁC.
Acknowledgments
One of us (S.W.Y.) would like to acknowledge the ORS
Awards Scheme for a United Kingdom Scholarship. We
also acknowledge the technical staff of the Tenovus Can-
cer Centre for the preparation of cells and media, and
the EPSRC Mass Spectrometry Centre, Swansea, UK,
for mass spectroscopy data.
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