Potent CYP19 (aromatase) 1-(benzofuran-2-yl)(phenylmethyl)pyridine, -imidazole, and -triazole inhibitors: Synthesis and biological evaluation

Article abstract of DOI:10.1021/jm0508282

The synthesis of a series of novel 1-[(benzofuran-2-yl)phenylmethyl]- pyridine, -imidazole, and -triazole derivatives is described. All the compounds were evaluated in vitro for inhibitory activity against aromatase (P450 _AROM, CYP19), using hu

Full text of DOI:10.1021/jm0508282

1016
J. Med. Chem. 2006, 49, 1016-1022
Potent CYP19 (Aromatase) 1-[(Benzofuran-2-yl)(phenylmethyl)pyridine, -imidazole, and
-triazole Inhibitors: Synthesis and Biological Evaluation
Mohammed Reza Saberi,†,§ Tai Ky Vinh,† Sook Wah Yee,† B. J. Nathan Griffiths,‡ Peter J. Evans,‡ and Claire Simons*,†
Medicinal Chemistry, Welsh School of Pharmacy, Cardiff UniVersity, King Edward VII AVenue, Cardiff CF10 3XF, U.K.;
Cardiff School of Biosciences, Cardiff UniVersity, Museum AVenue, Cardiff, CF10 3AT, U.K.
ReceiVed August 19, 2005
The synthesis of a series of novel 1-[(benzofuran-2-yl)phenylmethyl]-pyridine, -imidazole, and -triazole
derivatives is described. All the compounds were evaluated in vitro for inhibitory activity against aromatase
(P450AROM, CYP19), using human placental microsomes. The 6-methoxy- and 6-hydroxy-substituted
benzofuran derivatives were shown to be potent CYP19 inhibitors (IC50 ) 0.01-1.46 µM) with activity
greater than that observed for the unsubstituted parent compounds and inhibitory activity comparable with
or greater than the reference compound arimidex (IC50 ) 0.6 µM). Six of the benzofuran derivatives were
subjected to in vitro cytotoxicity assays, using rat liver hepatocytes with cytotoxicity determined from alteration
in cell morphology and lactate dehydrogenase enzyme retention over a period of 24 h, and selectivity (CYP17,
17â-HSD types 1 and 3, CYP24, and CYP26) determination; negligible inhibitory activity was observed,
suggesting a good selectivity for CYP19. The pyridine benzofuran 4a containing the 4-fluorophenyl group
was the most promising (IC50 ) 44 nM; LC50 >100 µM) compared with arimidex (IC50 ) 600 nM; LC50
> 200 µM).
Introduction
Breast cancer is the most commonly diagnosed cancer among
women and continues to be a major cause of cancer deaths.1
The two most important identified risk factors for breast cancer
are gender and age,2 and more than 70% of women over the
age of 50 with breast cancer do not have any other remarkable
risk factor.2 A high proportion of breast tumors in postmeno-
pausal women are dependent on estrogen for growth and after
surgery estrogen deprivation strategies are used to prevent
development of metastases.3 Aromatase inhibitors, which were
first reported in the 1970s, have been used in the clinic as second
line drugs following relapse on tamoxifen therapy for this
purpose.4,5
The third generation nonsteroidal aromatase inhibitors are
triazole derivatives, e.g. letrozole and anastrozole (arimidex)
(Figure 1), and have shown considerable advances in the
Figure 1. Potent nonsteroidal CYP19 inhibitors.
treatment of hormone-dependent breast cancer.6 In vitro studies
Fe3+ of the heme in the active site of the enzyme; therefore,
showed that letrozole significantly suppressed the endogenous
the coordination potential of the heterocyclic nitrogen is of
aromatase-induced proliferation of MCF-7 cells.7 The “ATAC”
importance.13,14 For the triazole derivatives, substitution in the
(Arimidex, Tamoxifen, Alone or in Combination) trial suggested
5-position of benzofuran ring was shown to be detrimental to
that aromatase inhibitors, specifically arimidex, may well be
inhibitory activity compared with the unsubstituted parent
superior to tamoxifen (the current first-line therapy of choice)
benzofuran.12 Molecular modeling studies indicated that intro-
for the treatment of hormone-dependent breast cancer.8,9 Studies
duction of a hydrogen donor group, e.g. OH or OCH3, in the
have also indicated the use of aromatase inhibitors for preventive
6-position of the benzofuran ring, which would also more closely
therapeutics, owing to their ability to reduce estrogen levels; a
mimic the natural substrate, androstenedione, might enhance
high level of plasma estrogen has been shown to be a high risk
binding interaction at the enzyme active site. We report here
factor for subsequent breast cancer.10
the synthesis and biological evaluation of these novel aromatase
1-[(Benzofuran-2-yl)phenylmethyl]imidazoles11 and -tria-
inhibitors.
zoles12 are potent P450AROM inhibitors, with the pyridine
derivatives displaying moderate activity (Figure 1).13 These
inhibitors bind to the active site of P450AROM through coordina-
tion of a heterocyclic nitrogen lone pair of electrons with the
Chemistry. The synthesis of the 6-methoxybenzofuran-2-
ylphenylmethanone derivatives 3 involved reaction of com-
mercially available 4-methoxysalicylaldehyde 1 with the ap-
propriate phenacyl bromide 2, which was either obtained
commercially or prepared by conventional methodology by
bromination of the precursor acetophenone with bromine/AlCl3
as previously described.15 The Rap-Stoermer reaction16,17
produced the required benzofuran ketones 3a-h in yields
ranging from 37 to 80% after purification by recrystallization.
* Corresponding author. Tel: +44-(0)2920-876307. Fax: +44-(0)-
2920-874149. E-mail SimonsC@Cardiff.ac.uk.
† Medicinal Chemistry.
‡ School of Biosciences.
§ Present address: School of Pharmacy, Mashhad University of Medical
Sciences, P.O. Box 91775-1365, Mashhad, I.R. Iran.
10.1021/jm0508282 CCC: $33.50 © 2006 American Chemical Society
Published on Web 01/11/2006

CYP19 Aromatase Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 1017
Scheme 1a
a Reagents and conditions: i. 1, NaH, DMF, 10 min, then 2, 80 °C, 2 h; ii. NaOMe, 80 °C, 1 h; iii.Mg, I2, THF, 3-bromopyridine, 70 °C, 2 h, then 3, THF,
70 °C, 1 h; iv. NaBH4, dioxane, rt, 2 h; v. SOCl2, imidazole, CH3CN, -10 °C, 1 h, then 5, CH3CN, K2CO3, rt, 4 days; vi. SOCl2, triazole, CH3CN, -10
°C, 1 h, then 5, CH3CN, K2CO3, rt, 4 days. [a, R ) F; b, R ) Cl; c, R ) OCH3; d, R ) CN; e, R ) NO2; f, R ) CH3; g, R ) CF3; h, R ) CH2CH3].
Table 1. IC50 Data for Inhibitory Activity of Benzofuran Pyridine (4), Imidazole (6), and Triazole Derivatives (7 and 11) vs Human Placental CYP19a
compound
R
yield (%)
IC50 (µM)a
compound
R
yield (%)
IC50 (µM)a
4a
4b
4c
6
7a
7b
7c
7d
F
Cl
OCH3
OCH3
F
Cl
OCH3
CN
80
80
55
85
83
80
85
73
0.044
0.049
0.16
7e
7f
7g
7h
11b
11d
11e
arimidex
NO2
30
27
28
77
76
85
86
-
0.6
0.1
CH3
CF3
CH2CH3
Cl
CN
NO2
-
0.13
1.23
1.46
0.02
0.06
0.6
0.13
0.049
0.044
0.13
0.01
a IC50 values are the average ((5%) of two experiments.
Preparation of the benzofuran-2-yl-(phenyl)-3-pyridylmetha-
nols 4a-c was achieved by reaction of the corresponding
ketones 3a-c with the Grignard reagent pyridine-3-magnesium
bromide, which was generated in situ from commercially
available pyridin-3-yl bromide and magnesium in the usual
manner (Scheme 1).13 The racemic pyridylmethanols were
obtained in moderate to good yields, after purification by column
chromatography and subsequent recrystallization.
Sodium borohydride reduction of the ketones 3 gave the
carbinols 5 in almost quantitative yields, which were converted
into the target imidazole 6 and triazoles 7 by the described
methodology12,17 (Scheme 1): the benzofuran alcohols 5 and
activated potassium carbonate in anhydrous acetonitrile were
added to a solution of azole (imidazole or triazole) and thionyl
chloride in anhydrous acetonitrile at 10 °C, and the reactions
were left stirring at 10 °C for 1 h and at room temperature for
4 days (Scheme 1).
The yields for this final reaction were variable (Table 1), and
in many cases, the reaction failed to reach completion. Low
yields of product were obtained together with recovered starting
material. After purification by flash column chromatography,
the products were confirmed by 1H NMR by the disappearance
of the OH group at approximately δ 3.7 and the presence of
two triazole protons at δ 8.2 and 8.1, respectively. In a few
reactions the 1,3,4-triazole isomer was observed by TLC running
a more polar CH2Cl2-MeOH eluent and characterized by a
singlet integrating for 2 protons at δ 8.25. The ratio of the 1,2,4-
triazole:1,3,4-triazole was found to be approximately 10:1.
Owing to the very small quantities of the isolated 1,3,4-triazole
product, only the major 1,2,4-triazole derivatives were fully
characterized and presented here.
6-hydroxy derivatives. This initially involved investigating
deprotection of the parent 6-methoxy derivative; however, this
proved unsatisfactory with either no reaction occurring or
decomposition, depending on the method applied. A different
protecting group strategy was then explored. Although 2,4-
dihydroxybenzaldehye has two hydroxy groups, only one was
selectively protected in the presence of 3,4-dihydropyran and
p-toluenesulfonic acid in diethyl ether. The reaction yielded 48%
of the 6-O-pyran-protected product 8, owing to incomplete
reaction with starting material recovered (Scheme 2).
Compound 8 was further reacted with phenacyl bromides 2b,
2d, and 2e to give required benzofurans 9 in moderate yields.
Reduction of the benzofuran ketones gave almost quantitative
yields of alcohols 10 (Scheme 2). It was anticipated that two
more steps were required to produce our target compounds, but
when the alcohols were reacted with triazole in the presence of
thionyl chloride and potassium carbonate, the pyran protecting
group was also cleaved in the process as indicated NMR.
Compounds 11b, 11d, and 11e were synthesized in excellent
yields.
CYP19 Inhibitory Activity. The pyridine (4a-c), imidazole
(6), and triazole (7a-h, 11b, 11d, and 11e) benzofuran
derivatives were evaluated for CYP19 inhibitory activity using
human placental microsomes,18 using radiolabeled [1,2,6,7-3H]-
androstenedione as the substrate and arimidex as the standard
for comparison (Table 1).
Arimidex was determined to have an IC50 value of 0.6 µM.
The pyridine benzofuran derivatives 4 were all potent inhibitors
of CYP19, with optimal activity obtained with the halogen-
substituted derivatives 4a and 4b (IC50: 4a ) 0.044 µM; 4b )
0.049 µM). The more bulky methoxy-substituted pyridine
derivative 4c was less active and was comparable with the
It was hypothesized that the 6-hydroxy derivative might have
enhanced binding interaction (H-bonding) at the enzyme active
site; therefore, attempts were made to prepare the corresponding

1018 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Saberi et al.
Scheme 2a
a Reagents and conditions: i. 8, NaH, DMF, 10 min, then 2, 80 °C, 2 h; ii. NaOMe, 80 °C, 1 h; iii. NaBH4, dioxane, rt, 2 h; v. SOCl2, triazole, CH3CN,
-10 °C, 1 h, then 10, CH3CN, K2CO3, rt, 4 days.
tions up to 20 µM observed. The enzyme preparation and assays
have been described by us previously.19-22
Toxicology. Toxicology evaluation was undertaken with six
of the 6-methoxy benzofuran inhibitor compounds, chosen to
reflect the varying heterocycle, and the reference standard
arimidex, using rat liver hepatocytes. Cytotoxicity was deter-
mined using two criteria: alteration in cell morphology and
marker enzyme retention. Drug concentrations of 100, 50, 25,
10, 5, 1, 0.5, and 0.1 µM (diluted using DMSO) were used,
with 2,4-dinitrophenol (DNP), a known hepatotoxin, used as a
positive control. In this study, samples were taken at 0, 4, 8,
and 24 h. None of the compounds caused any changes in cell
morphology or caused cell death, as determined from lactate
dehydrogenase (LDH) enzyme retention, over an 8 h period at
any of the concentrations tested (Figure 2).
However, after 24 h exposure of cells to these substances at
the highest concentration (100 µM), cell death and changes in
morphology were observed. Morphological evaluation indicated
a gradation in the cellular perturbation seen at 100 µM of the
test compounds. In order of being most disruptive: 6 > 7a >
7b and 7c > 4a > 4c > arimidex. Cytotoxicity as determined
by LDH retention was in the order: 6 and 7b > 7c > 4c and
7a > 4a > arimidex (Figure 2). The LC50 data for the
benzofuran inhibitor compounds and arimidex are shown in
Table 2. Arimidex was not found to be cytotoxic at a
concentration of 200 µM (24 h).
Figure 2. Cytotoxicity of the tested compounds (100 µM) determined
from LDH retention data.
methoxy-substituted imidazole 6 and triazole 7c benzofuran
derivatives (IC50: 4c ) 0.16 µM; 6 ) 0.13 µM; 7c ) 0.13
µM).
In the triazole benzofuran series the nitrile derivative 7d was
the most potent (IC50 ) 0.01 µM) with the halogen-substituted
derivatives 7a and 7b showing potency comparable with their
pyridine counterparts (IC50: 7a ) 0.049 µM; 7b ) 0.044 µM).
The alkyl-substituted triazole derivatives were less potent with
a substantial reduction in activity on progressing from the methyl
or trifluoromethyl (IC50: 7f ) 0.1 µM; 7g ) 0.13 µM) to the
ethyl (IC50: 7h ) 1.23 µM) group. The nitro-substituted triazole
derivative (7e) was found to have inhibitory activity of the same
order as the standard arimidex (IC50: 7e ) arimidex ) 0.6 µM).
In the 6-hydroxy triazole benzofuran series, the nitrile derivative
11d showed activity comparable with the 6-methoxy derivative
(IC50: 11d ) 0.02 µM; 7d ) 0.01 µM), the nitro derivative
11e displayed enhanced inhibitory activity compared with the
6-methoxy series (IC50: 11e ) 0.06 µM; 7e ) 0.6 µM), whereas
the chloro derivative 11b showed a loss in potency (IC50: 11b
) 1.46 µM; 7b ) 0.044 µM).
Six of the inhibitor compounds, the pyridine derivatives 4a
and 4c, the imidazole 6, and the triazoles 7a, 7b, and 7c, were
evaluated further for CYP19 selectivity and cytotoxicity (see
Toxicology, Figure 2).
The compounds were evaluated for inhibition of steroidogenic
enzymes P450 17 (CYP17, 17,20-lyase) and 17â-HSD types 1
and 3. Using 2 µM concentrations of the most potent inhibitors,
imidazole 6 and triazoles 7a-c, negligible inhibition of CYP17
was observed. No inhibition of 17â-HSD type 1 and 17â-HSD
type 3 using inhibitor concentrations of 20 µM was observed
with any of the inhibitors. The compounds were also evaluated
against the P450 enzymes CYP24 and CYP26 involved in
differentiation and proliferation, with no inhibition at concentra-
Discussion
The benzofuran inhibitors described are very potent using
the human placental aromatase assay, compared with the
reference compound arimidex (IC50 ) 0.6 µM), with nanomolar
inhibitory concentrations (Table 1). The preferred substitutions
in the 4-position of the phenyl ring were previously determined12
to be limited to small substituents/groups, and this was reflected
in this series with the larger OCH3, CH3, CF3, and CH2CH3
substituents resulting in reduced inhibitory activity compared
with the F, Cl, NO2, and CN substituted derivatives. Introduction
of the 6-methoxy or 6-hydroxy substituent into the benzofuran
ring greatly enhanced CYP19 inhibitory activity, and this was
further investigated by molecular modeling to probe for potential
bonding interactions with the enzyme active site.
Ligand docking of the inhibitor compounds with the CYP19
theoretical model (PDB 1TQA23,24) was performed using both
SYBYL FlexX25 and MOE-Dock26 software. The inhibitor

CYP19 Aromatase Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 1019
Figure 3. Active site region of CYP19 model showing the inhibitor ligand (a) S-11d and (b) S-4a enantiomers as a ball-and-stick form. Hydrogen
bonding interactions are shown as red dashed lines, and distance from the heme is indicated with a green line, and transition metal interaction with
a purple line. Amino acid residues identified are involved in hydrophobic interactions.
Table 2. Comparison of 6-Methoxy and Parent Benzofuran Inhibitors
a Reference 13. b Reference 12.
Table 3. LC50/IC50 Ratio
compounds are all racemic; therefore, both enantiomers of all
compound
IC50 (µM)
LC50 (µM) 24h
LC50/IC50
the compounds were docked to determine any preference for
(R)- or (S)-configuration; no preference was observed, with both
enantiomers exhibiting a good ‘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-methoxybenzofuran 4a and the 6-hydroxy-
benzofuran 11d.
4a
4c
6
7a
7b
7c
arimidex
0.044
0.16
0.13
0.049
0.044
0.13
0.6
>100
100
50
>2273
625
385
100
50
90
2041
1136
692
>200
>333
Ligand docking indicated that the 6-hydroxy group may form
hydrogen bonding interactions with ALA223 (Figure 3a),
allowing enhanced binding; however, this has not resulted in
substantially enhanced activity with the 6-hydroxy triazole
benzofuran series showing similar or, in the case of the 4-chloro
derivative 11b, reduced CYP19 inhibitory activity compared
with the 6-methoxy derivatives.
The 6-methoxy substituent of the pyridine series may form
hydrogen bonding interactions with HIS480, as determined from
docking studies. The pyridine substituent may allow optimal
orientation at the enzyme active site and maximize hydrophobic
interactions (Figure 3b), resulting in the enhanced inhibitory
activity observed compared with the unsubstituted parent
benzofuran (e.g. Table 2). This increase in inhibitory potency
is particularly noted in the pyridine series (4a-c).
(general trend 4 > 7 > 6). Comparison of the six compounds
with arimidex cannot be conclusive, as the in vivo concentration
of the benzofuran inhibitor compounds required to produce the
same as or greater in vivo activity compared with arimidex
would be anticipated to be less, owing to their enhanced potency.
A comparison of LC50/IC50 data is shown (Table 3) to provide
an indication of in vitro selectivity.
All the benzofuran inhibitors described are racemic; therefore,
further research would involve chiral resolution of the enan-
tiomers (chiral HPLC) for CYP19 inhibitory and cytotoxicity
assays. Likewise, further toxicology evaluation of the most
potent CYP19 inhibitors 7d and 11d (IC50 0.01 and 0.02 µM,
respectively) is warranted, in particular to consider the differ-
ence, if any, between the 6-methoxy and 6-hydroxy substituent
on cytotoxicity.
Initial toxicology data (rat hepatocytes) is promising, with
the pyridine derivatives showing the most favorable profile

1020 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Saberi et al.
(C), 161.0 (C), 158.7 (C), 148.2 (CH), 147.3 (CH), 143.7 (C), 141.1
(C), 135.7 (C), 134.4 (CH), 130.5 (CH), 129.4 (CH), 125.0 (C),
123.5 (CH), 122.8 (CH), 106.2 (CH), 106.0 (CH), 96.2 (CH, C-3),
78.0 (CH, H-1), 56.9 (CH3). Anal. (C21H16ClNO3) C, H, N.
(6-Methoxybenzofuran-2-yl)-(4-methoxyphenyl)-3-pyridyl-
methanol (4c). White crystalline solid. Yield: 55%; mp 39-43
°C; Rf 0.22 (petroleum ether/ethyl acetate 2:1). 1H NMR (CDCl3):
δ 8.58 (ψt, 1, J ) 1.7 Hz, Ar), 8.45 (dd, 1, J ) 1.6, 4.8 Hz, Ar),
7.79 (m, 1, Ar), 7.38 (d, 1, J ) 8.5 Hz, Ar), 7.27 (m, 3, Ar), 6.96
(d, 1, J ) 2.0 Hz, Ar), 6.88 (m, 3, Ar), 6.28 (d, 1, J ) 0.8 Hz,
H-3), 5.08 (s, 1, OH), 3.84 (s, 3, OCH3), 3.73 (s, 3, OCH3). 13C
NMR (CDCl3): δ 159.7 (C), 159.3 (C), 158.5 (C), 156.7 (C), 149.1
(CH), 148.8 (CH), 140.8 (C), 136.7 (C), 135.6 (CH), 129.0 (CH),
123.3 (CH), 121.8 (CH), 121.4 (C), 114.0 (CH), 112.5 (CH), 106.7
(CH), 96.5 (CH, C-3), 76.9 (CH, H-1), 56.1 (CH3), 55.7 (CH3).
Anal. (C22H19NO4) C, H, N.
General Method for the Preparation of the Carbinols 5 and
10. To a suspension of the ketone (3 or 9, 3 mmol) in dry dioxane
(7 mL) was added sodium borohydride (3 mmol) and the reaction
stirred at room temperature under nitrogen for 2 h. The reaction
was concentrated under reduced pressure and 2 M aqueous
hydrochloric acid (approximately 10 mL) added to the resulting
syrup. This solution was extracted into diethyl ether (100 mL),
washed with water (2 × 25 mL), dried (MgSO4), and concentrated
under reduced pressure to give the required product in quantitative
yield which was used in the next reaction without any further
purification. Analytical data is provided in Supporting Information.
General Procedure for the Preparation of the 1-[(6-Meth-
oxybenzofuran-2-yl)-(4-substituted phenyl)methyl]-1H-imida-
zole (6), -1H-1,2,4-triazoles (7), and 6-Hydroxybenzofuran-1H-
1,2,4-triazole Derivatives (11). To a cooled (0 °C) solution of
imidazole or 1,2,4-triazole (12.15 mmol) in anhydrous acetonitrile
(5 mL) was added thionyl chloride (0.36 g, 3.04 mmol) in anhydrous
acetonitrile (5 mL) and the reaction stirred at 10 °C for 1 h.
Potassium carbonate (3.0 mmol) was then added followed by a
solution of the carbinol (4 or 10, 3.0 mmol) in anhydrous acetonitrile
(5 mL) and the reaction allowed to stir at room temperature for 5
days. The suspension was filtered and the solution concentrated
under reduced pressure to produce a residue which was extracted
with dichloromethane (2 × 50 mL) and water (2 × 25 mL). The
organic layers were combined and dried (MgSO4), and the solvent
was evaporated to give a yellow syrup. Purification by flash column
chromatography (petroleum ether-ethyl acetate 9:1 to 6:4 v/v) gave
the required product.
Experimental Section
General Procedures. H and 13C NMR spectra were recorded
1
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 center (Swansea, UK).
Microanalyses were determined by Medac Ltd (Surrey, UK). Flash
column chromatography was performed with silica gel 60 (230-
400mesh) (Merck), and TLC was carried out on precoated silica
plates (kiesel gel 60 F254, BDH). Compounds were visualized by
illumination under UV light (254 nm) or by the use of vanillin
stain followed by charring on a 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 of Laboratory Chemicals27 and stored over 4 Å
molecular sieves, under nitrogen. [1,2,6,7-3H]Androstenedione (86.4
Ci/mmol, 37 MBq/mL) was purchased from NEN-Dupont UK
(Stevenage, Herts). Scintillation fluid was Optiphase Hisafe from
Fisons Chemicals (Loughborough, UK). The scintillation counter
used was a LKB Wallac, 1217, Rack-beta. Toxicology evaluation,
cell morphology, and marker enzyme retention assays were
performed by PrimaCell (Cardiff University). The numbering of
1
compounds for H and 13C NMR characterization and assignment
of 1H and 13C NMR for all compounds is provided in the Supporting
Information.
General Method for the Preparation of the Ketones 3 and 9.
To a solution of sodium hydride (60%, 11 mmol) in dry N,N-
dimethylformamide (10 mL) was added a solution of the methoxy-
aldehyde (1 or 8, 10 mmol) in dry N,N-dimethylformamide (6 mL)
dropwise. Hydrogen gas was liberated to give a yellow solution of
the sodium salt. A solution of the phenacyl bromide (2,10 mmol)
in dry N,N-dimethylformamide (10 mL) was then added dropwise
and the reaction heated under nitrogen at 80 °C for 1.5 h. Sodium
methoxide (2.5 mmol) was added to the mixture and heating
continued for another 1 h (TLC system: petroleum ether-ethyl
acetate 4:1 v/v). After cooling, the reaction was evaporated to about
a third of its volume, diluted with dichloromethane (100 mL),
washed with water (100 mL), dried (MgSO4), and concentrated
under reduced pressure. The resulting ketone 3 or 9 was purified
by recrystallization. Analytical data is provided in Supporting
Information.
General Method for the Preparation of Benzofuran-2-yl-
(phenyl)-3-pyridylmethanols 4a-c. To a solution of pyridine-3-
magnesium bromide (20 mmol) in THF (30 mL), generated in situ
by the reaction of pyridin-3-yl bromide (20 mmol) and dry
magnesium turnings (20 mmol) in THF (30 mL) under reflux for
1 h, was added the ketone 3 (5 mmol) and the reaction heated at
70 °C for 1 h. The solvent was removed under reduced pressure
and the resulting residue dissolved in CH2Cl2 (50 mL), washed with
H2O (30 mL), dried (MgSO4), and concentrated under reduced
pressure. The crude product was purified by flash column chro-
matography (petroleum ether-ethyl acetate 3:2 increasing to 2:3
v/v) to give the target compound.
1-[(6-Methoxybenzofuran-2-yl)-(4-methoxyphenyl)methyl]-
1H-imidazole (6). Amorphous white solid. Yield: 85%; Rf 0.20
1
(petroleum ether/ethyl acetate 1:1). H NMR (CDCl3): δ 7.66 (s,
1, H-2′′), 7.49 (d, 1, J ) 8.6 Hz, H-4′′), 7.26 (m, 3, Ar), 7.09 (d,
1, J ) 2.0 Hz, Ar), 7.01 (m, 4, Ar), 6.63 (s, 1, H-3), 6.53 (s, 1,
H-1), 3.95 (s, 3, OCH3), 3.93 (s, 3, OCH3). 13C NMR (CDCl3): δ
164.5 (C), 160.4 (C), 159.0 (C), 143.1 (CH), 136.9 (C), 132.0 (CH),
128.7 (CH), 126.9 (C), 124.3 (CH), 120.2 (CH), 116.9 (CH), 107.5
(C), 105.8 (CH), 96.9 (CH), 60.3 (CH), 55.5 (CH3), 55.0 (CH3).
Anal. (C20H18N2O3) C, H, N.
6-Methoxybenzofuran-2-yl-(4-fluorophenyl)-3-pyridylmetha-
nol (4a). White solid. Yield: 80%; mp 66-68 °C; Rf 0.14
(petroleum ether/ethyl acetate 3:1). H NMR (CDCl3): δ 8.64 (d,
1-[(6-Methoxybenzofuran-2-yl)-(4-fluorophenyl)methyl]-1H-
1,2,4-triazole (7a). Opaque syrup. Yield: 83%; Rf 0.16 (petroleum
1
1
1, J ) 2.3 Hz, Ar), 8.60 (dd, 1, J ) 1.6, 4.6 Hz, Ar), 7.78 (dt, 1,
J ) 2.0, 8.1 Hz, Ar), 7.38 (m, 4, Ar), 7.10 (m, 2, Ar), 7.02 (d, 1,
J ) 1.9 Hz, Ar), 6.93 (dd, 1, J ) 2.2, 8.6 Hz, Ar), 6.28 (d, 1, J )
0.7 Hz, Ar), 3.89 (s, 3, OCH3), 3.74 (s, 1, OH). 13C NMR
(CDCl3): δ 160.4 (C), 158.0 (C), 156.0 (C), 149.5 (CH), 149.3
(CH), 148.0 (C), 141.5 (C), 135.3 (CH), 129.6 (CH), 126.2 (CH),
123.4 (CH), 122.0 (CH), 120.5 (C), 115.8 (CH), 115.5 (CH), 112.8
(CH), 107.5 (CH), 96.4 (CH, C-3), 77.9 (CH, H-1), 56.1 (CH3).
Anal. (C21H16FNO3) C, H, N.
ether/ethyl acetate 3:1). H NMR (CDCl3): δ 8.18 (s, 1, H-3′′),
8.08 (s, 1, H-5′′), 7.46 (d, 1, J ) 8.6 Hz, H-8), 7.33 (m, 2, Ar),
7.15 (m, 2, Ar), 7.04 (d, 1, J ) 7.1 Hz, Ar), 6.94 (dd, 1, J ) 2.3,
8.6 Hz, H-7), 6.85 (s, 1, H-3), 6.55 (s, 1, H-1), 3.89 (s, 3, OCH3).
13C NMR (CDCl3): δ 165.0 (C), 159.2 (C), 156.9 (C), 152.7 (CH),
151.5 (C), 143.6 (CH), 132.1 (C), 130.0 (CH), 129.8 (CH), 122.1
(CH), 120.9 (C), 116.7 (CH), 116.4 (CH), 113.1 (CH), 108.4 (CH),
96.3 (CH), 62.0 (CH), 56.1 (CH3). HRMS (ES+) Calcd for C18H14-
FN3O2 [M]+ 323.325, found 323.100.
6-Methoxybenzofuran-2-yl-(4-chlorophenyl)-3-pyridylmetha-
nol (4b). White solid. Yield: 80%; mp 68-70 °C; Rf 0.23
(petroleum ether/ethyl acetate 3:1). 1H NMR (CDCl3): δ 8.58 (m,
2, Ar), 7.76 (dt, 1, J ) 2.0, 8.0 Hz, Ar), 7.37 (m, 7, Ar), 7.02 (s,
1, Ar), 6.93 (dd, 1, J ) 2.2, 8.5 Hz, Ar), 6.29 (d, 1, J ) 0.7 Hz,
Ar), 3.88 (s, 3, OCH3), 3.74 (s, 1, OH). 13C NMR (CDCl3): δ 161.4
1-[(6-Methoxybenzofuran-2-yl)-(4-chlorophenyl)methyl]-1H-
1,2,4-triazole (7b). Opaque syrup. Yield: 80%; Rf 0.25 (petroleum
ether/ethyl acetate 3:2). H NMR (CDCl3): δ 8.18 (s, 1, H-3′′),
8.08 (s, 1, H-5′′), 7.43 (ψt, 1, J ) 3.9, 4.6 Hz, Ar), 7.32 (m, 2,
Ar), 7.15 (m, 2, Ar), 7.13 (m, 2, Ar), 7.02 (d, 1, J ) 1.9 Hz, Ar),
6.92 (dd, 1, J ) 2.2, 8.6 Hz, H-7), 6.85 (s, 1, H-3), 6.53 (s, 1,
1

CYP19 Aromatase Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 1021
H-1), 3.85 (s, 3, OCH3). 13C NMR (CDCl3): δ 161.7 (C), 159.1
(C), 156.9 (C), 152.7 (CH), 151.5 (C), 143.6 (CH), 132.1 (C), 130.0
(CH), 129.9 (CH), 122.1 (CH), 120.9 (C), 116.6 (CH), 116.4 (CH),
113.1 (CH), 108.3 (CH), 96.3 (CH), 61.9 (CH), 56.1 (CH3). HRMS
(ES+) Calcd for C18H14ClN3O2 [M]+ 339.770, found 339.770.
2-[(4-Chlorophenyl)-[1,2,4]triazol-1-ylmethyl]benzofuran-6-
ol (11b). Yellow amorphous solid. Yield: 76%; Rf 0.55 (petroleum
ether/ethyl acetate 1:1). 1H NMR (CDCl3): δ 9.08 (s, 1, OH), 8.32
(s, 1, H-3′′), 8.20 (s, 1, H-4′′), 7.49 (m, 3, H-2′, H-6′, H-4), 7.34
(m, 3, H-3′, H-5′, H-7), 7.01 (d, 1, J ) 1.7 Hz, H-5), 6.93 (m, 1,
H-3), 6.62 (s, 1, H-1). 13C NMR (CDCl3): δ 156.91 (CH), 156.42
(CH), 151.99 (C), 150.15 (C), 143.47 (C), 135.72 (C), 134.48 (C),
131.50 (C), 129.77 (CH), 129.30 (CH), 122.41 (CH), 120.29 (CH),
113.91 (CH), 109.09 (CH), 98.62 (CH). Anal. (C17H12 ClN3O2‚
0.2H2O) C, H, N.
1-[(6-Methoxybenzofuran-2-yl)-(4-methoxyphenyl)methyl]-
1H-1,2,4-triazole (7c). Syrup. Yield: 85%; Rf 0.28 (petroleum
1
ether/ethyl acetate 2:1). H NMR (CDCl3): δ 8.09 (s, 1, H-3′′),
8.01 (s, 1, H-5′′), 7.38 (d, 1, J ) 8.6 Hz, Ar), 7.26 (d, 1, J ) 82.1
Hz, Ar), 7.23 (d, 1, J ) 2.0 Hz, Ar), 6.97 (d, 1, J ) 1.9 Hz, Ar),
6.92 (m, 2, Ar), 6.87 (dd, 1, J ) 2.2, 8.6 Hz, H-7), 6.75 (s, 1,
H-3), 6.47 (s, 1, H-1), 3.82 (s, 3, OCH3), 3.81 (s, 3, OCH3). 13C
NMR (CDCl3): δ 165.5 (C), 158.9 (C), 156.8 (C), 152.6 (CH),
152.3 (C), 143.5 (CH), 129.5 (CH), 128.1 (C), 122.0 (CH), 121.1
(C), 114.8 (CH), 112.9 (CH), 107.9 (CH), 96.3 (CH), 62.2 (CH),
56.1 (CH3), 55.8 (CH3). Anal. (C19H17N3O3) C, H, N.
4-[(6-Hydroxybenzofuran-2-yl)-[1,2,4]triazol-1-ylmethyl]ben-
zonitrile (11d). Orange amorphous solid. Yield: 85%; Rf 0.57
1
(petroleum ether/ethyl acetate 1:1). H NMR (CDCl3): δ 8.29 (s,
1, OH), 8.22 (s, 1, H-3′′), 8.17 (s, 1, H-4′′), 7.76 (d, 2, J ) 8.3 Hz,
H-2′, H-6′), 7.43 (dd, 3, J ) 3.2 Hz, H-3′, H-5′, H-4), 6.93 (m, 3,
H-7, H-5, H-3), 6.61 (s, 1, H-1). 13C NMR (CDCl3): δ 156.95
(CH), 156.40 (CH), 152.38 (C), 149.25 (C), 143.69 (C), 141.12
(C), 133.28 (CH), 128.62 (CH), 131.36 (C), 122.58 (CH), 120.27
(C), 118.43 (CH), 114.05 (CH), 113.63 (CH), 109.50 (CH). Anal.
(C18H12N4O2‚0.5H2O) C, H, N.
4-[6-Methoxybenzofuran-2-yl)-1H-1,2,4-triazol-1-ylmethyl]-
benzonitrile (7d). Yellow honeycomb solid. Yield: 73%; mp 57-
59 °C; Rf 0.63 (petroleum ether/ethyl acetate 1:1). 1H NMR
(CDCl3): δ 8.24 (s, 1, H-3′′), 8.11 (s, 1, H-4′′), 7.77 (d, 2, J ) 8.3
Hz, H-2′, H-6′), 7.42 (m, 3, H-3′, H-5′, H-4), 7.01 (d, 1, J ) 1.9
Hz, 1, H-7), 6.96 (m, 2, H-5, H-3), 6.63 (s, 1, H-1), 3.90 (s, 3,
OCH3). 13C NMR (CDCl3): δ 159.43 (CH), 158.36 (CH), 157.00
(C), 153.04 (C), 149.42 (C), 143.87 (C), 141.42 (C), 133.26 (CH),
128.64 (CH), 122.29 (CH), 120.65 (C), 118.53 (CH), 113.51 (CH),
113.46 (CH), 96.30 (CH), 61.99 (CN), 56.17 (CH3). Anal.
(C19H14N4O2‚0.2H2O) C, H, N.
2-[(4-Nitrophenyl)-[1,2,4]triazol-1-ylmethyl]benzofuran-6-
ol (11e). Pale yellow amorphous solid. Yield: 86%; Rf 0.20
1
(petroleum ether/ethyl acetate 1:1). H NMR (CDCl3): δ 8.43 (s,
1, OH), 8.32 (m, 3, H-3′′, H-4′′, H-2′), 8.20 (s, 1, H-6′), 7.43 (m,
3, H-3′, H-5′, H-4), 6.95 (m, 3, H-7, H-5, H-3), 6.65 (s, 1, H-1).
13C NMR (CDCl3): δ 156.98 (CH), 156.47 (CH), 152.42 (C),
149.08 (C), 148.67 (C), 143.73 (C), 142.85 (C), 132.68 (C), 128.89
(CH), 124.68 (CH), 122.60 (CH), 129.43 (CH), 123.33 (CH), 121.47
(CH), 114.09 (CH). Anal. (C17H12N4O4‚0.2H2O) C, H, N.
Aromatase Assay. The classical 3H2O assay18 was used to
measure the effect of the inhibitor compounds on aromatase activity
using human placental microsomes. A solution of [1,2,6,7-3H]-
androstenedione and androstenedione (0.5 µM final concentration)
was incubated in test tubes at 37 °C for 15 min with the human
placental microsomal preparation (8.24 mg/mL, 30 µL), phosphate
buffer (400 µL, 50 mM, pH 7.4), and NADPH (50 µL, 16 mM) in
the presence of inhibitor (10 µL, 1 or 5 mmol, 20 or 100 µM final
concentration, respectively) in EtOH. Control experiments were run
with EtOH (10 µL) in place of the inhibitor. The reaction was
quenched by the addition of aqueous HgCl2 (30 µL, 1 mM) followed
by an aqueous suspension of charcoal (1 mL, 1 wt %). The test
tubes were centrifuged (15 min, 3000 rpm), and then the supernatant
liquid was placed in scintillation vial to which 1 mL of scintillation
fluid was added. The 3H2O contained in each vial was then
determined using a LKB Wallac, 1217, Rack-beta scintillation
counter.
1-[(6-Methoxybenzofuran-2-yl)-(4-nitrophenyl)methyl]-1H-
1,2,4-triazole (7e). Yellow amorphous solid. Yield: 30%; Rf 0.27
1
(petroleum ether/ethyl acetate 1:1). H NMR (CDCl3): δ 8.33 (s,
1, H-3′′), 8.31 (s, 1, H-4′′), 8.25 (s, 1, H-2′), 8.12 (s, 1, H-6′), 7.49
(m, 3, H-3′, H-5′, H-4), 7.05 (d, 1, J ) 1.8 Hz, H-7), 7.00 (m, 2,
H-5, H-3), 6.65 (s, 1, H-1), 3.90 (s, 3, OCH3). 13C NMR (CDCl3):
δ 159.47 (CH), 156.87 (CH), 157.02 (C), 153.10 (C), 149.84 (C),
148.59 (C), 143.86 (C), 128.90 (CH), 124.65 (CH), 122.31 (C),
120.63 (CH), 113.50 (CH), 109.15 (CH), 96.30 (CH), 76.56 (CH),
19.04 (CH3). Anal. (C18H14N4O4) C, H, N.
1-[(6-Methoxybenzofuran-2-yl)-p-tolylmethyl]-1H-1,2,4-tria-
zole (7f). Yellow syrup. Yield: 27%; Rf 0.34 (petroleum ether/
ethyl acetate 1:1). 1H NMR (CDCl3): δ 8.16 (s, 1, H-3′′), 8.08 (s,
1, H-4′′), 7.46 (d, 1, J ) 8.6 Hz, H-4), 7.26 (dd, 4, J ) 8.5 Hz,
H-2′, H-6′, H-3′, H-5′), 7.04 (d, 1, J ) 1.9 Hz, H-7), 6.93 (dd, 1,
J ) 2.1 Hz, H-5), 6.83 (s,1, H-3), 6.55 (s, 1, H-1), 3.89 (s, 3, OCH3),
2.43 (s, 3, CH3). 13C NMR (CDCl3): δ 158.99 (CH), 156.84 (CH),
152.60 (C), 152.13 (C), 143.58 (C), 139.55 (C), 133.19 (C), 130.18
(CH), 127.97 (CH), 122.01 (CH), 121.08 (CH), 112.97 (CH), 108.07
(CH), 96.34 (CH), 62.51 (CH), 56.13 (CH3), 21.63 (CH3). Anal.
(C19H17N3O2) C, H, N.
For IC50 values the general method described for determination
of percentage inhibition was followed except that a range of
concentrations of inhibitor were used. Calculation of IC50 was
determined by plotting % inhibition versus Log [I] using Cricket
Graph III 1.5f software.28
1-[(6-Methoxybenzofuran-2-yl)-(4-trifluoromethylphenyl)-
methyl]-1H-1,2,4-triazole (7g). Yellow/brown syrup. Yield: 28%;
1
Rf 0.30 (petroleum ether/ethyl acetate 1:1). H NMR (CDCl3): δ
Toxicology. Hepatocytes were prepared and cultured from male
Sprague Dawley rats as described previously.29,30 Cells were
cultured in 96-well plates (8-well strip format, Fisher Scientific)
on an adsorbed collagen layer (type III). The coated wells were
prepared by covering the bottom of each well with sterile collagen
(2 mg/mL) in 0.1% acetic acid. The plates were left for 1 h, washed
three times with sterile water, and placed in a 37 °C incubator for
48 h before use. The plating medium was Leibovitz L-15 medium
pH 7.4 supplemented with glucose (8.3 mM), HEPES (25 mM),
insulin (0.8 µg/mL), dexamethasone (1 µM), 10% (v/v) heat-
inactivated (56 °C for 30 min) new born calf serum, and gentamycin
(50 µg/mL). Hepatocytes (4.5 × 104) were plated out in 0.2 mL.
The plates were placed in a 37 °C humidified incubator to allow
cell attachment. After 2 h the plates were removed from the
incubator and the medium aspirated off. Each well was washed
twice with 0.2 mL of serum free medium, and 0.2 mL of serum
free medium containing the appropriate concentration of the test
compounds was added to the wells. The plates were returned to
the 37 °C humidified incubator for the times indicated in the figure
legends.
8.22 (s, 1, H-3′′), 8.10 (s, 1, H-4′′), 7.73 (d, 2, J ) 8.1 Hz, H-2′,
H-6′), 7.46 (m, 3, H-3′, H-5′, H-4), 7.05 (s, 1, H-7), 6.95 (s, 2,
H-5, H-3), 6.60 (s, 1, H-1), 3.90 (s, 3, OCH3). 13C NMR (CDCl3):
δ 159.34 (CH), 156.98 (CH), 152.89 (C), 150.61 (C), 143.76 (C),
140.24 (C), 131.94 (C), 131.50 (C), 128.32 (CH), 126.51 (CH),
122.35 (CH), 122.22 (CH), 120.79 (CH), 113.34 (CH), 108.88 (CH),
96.32 (CF3), 56.14 (CH3). Anal. (C19H14F3N3O2•0.5H2O) C, H, N.
1-[(6-Methoxybenzofuran-2-yl)-(4-ethylphenyl)methyl]-1H-
1,2,4-triazole (7h). Yellow amorphous solid. Yield: 77%; Rf 0.33
1
(petroleum ether/ethyl acetate 1:1). H NMR (CDCl3): δ 8.33 (s,
1, H-3′′), 8.18 (s, 1, H-4′′), 7.43 (d, 1, J ) 8.6 Hz, H-4), 7.28 (m,
4, H-2′, H-6′, H-3′, H-5′), 7.03 (d, 1, J ) 1.9 Hz, H-7), 6.93 (dd,
1, J ) 2.2 Hz, H-5), 6.84 (s, 1, H-3), 6.65 (s, 1, H-1), 3.87 (s, 3,
OCH3), 2.72 (dd, 2, J ) 7.6 Hz, CH2), 1.30 (m, 3, CH3). 13C NMR
(CDCl3): δ 159.00 (CH), 156.85 (CH), 152.58 (C), 152.20 (C),
145.76 (C), 143.63 (C), 133.45 (C), 128.99 (CH), 128.05 (CH),
122.02 (CH), 121.12 (C), 112.97 (CH), 108.05 (CH), 96.36 (CH),
62.51 (CH), 56.10 (CH3), 28.97 (CH2), 15.82 (CH3). Anal.
(C20H19N3O2) C, H, N.

1022 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Saberi et al.
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inhibitors of human testes microsomal 17â-hydroxysteroid dehydro-
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2-substituted- 3,4-dihydro-2H-naphthalen-1-one (tetralone) deriva-
tives. Bioorg. Med. Chem. Lett. 2004, 14, 5651-5654.
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parative model: PDB 1TQA. http://www.rcsb.org/pdb/cgi/model-
Browser.cgi.
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(25) Tripos SYBYL 7.0; Tripos Inc., 1699 South Hanley Rd., St. Louis,
MO 63144, USA. http://www. tripos.com.
(26) Molecular Operating Environment (MOE). Chemical Computing
Group, Inc. Montreal, Quebec, Canada. http://www.chemcomp.com.
Code “scoring svl” obtained from SVL Exchange website http://
svl.chemcomp.com., Chemical Computing Group, Inc., Montreal,
Canada.
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Biochim. Biophys. Acta 1981, 677, 433-444.
Stock solutions (5 mM) of the test compounds were prepared in
DMSO. They were diluted to the appropriate concentrations by
serial dilution with serum free medium. All the compounds were
soluble at 100 µM, after incubating at 37 °C. Diluted stocks were
filter sterilized. Triplicate samples were taken at each time point
along with the appropriate DMSO solvent controls.
Cell morphology in the presence of test compounds was
monitored with an Olympus inverted microscope fitted with a Nikon
digital camera. Cell viability was determined using a CytoTox 96
nonradioactive cytotoxicity assay kit (Promega). Cells on compound
treated and control well strips were removed from the incubator at
0, 4, 8, and 24 h. The culture medium was aspirated away; the
cells were lysed and diluted with buffer pH 7.4, containing sucrose
(0.25 M), HEPES (5 mM), and EDTA (1 mM), until linear rates
of lactate dehydrogenase were recorded. The cytotoxicity assay was
carried out at 37 °C, in 96-well dishes, using a Tecan GENios plate
reader. The cytotoxicity of the test compounds was determined by
comparing with the appropriate DMSO solvent controls.
Molecular Docking. All molecular modeling studies were
performed on an RM Innovator Pentium IV 2.4GHz running either
Linux Fedora Core 3 or Windows XP using Molecular Operating
Environment (MOE) 2004.0326 and FlexX module in SYBYL 7.025
molecular modeling software. All the minimizations were performed
with MOE until RMSD gradient of 0.05 kcal mol-1 Å-1 with the
force field specified, and the partial charges were automatically
calculated.
Ligands were docked within the active site of the homology
model PDN 1TQA23 using both the FlexX docking program of
SYBYL and MOE-Dock. For MOE-Dock, simulated annealing was
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.
Docking with FlexX was performed with the default values.
Molecular dynamics was performed with MOE using the NVT
environment for 100 ps and constant temperature of 300 K using
the MMFF94X force field with all other default settings in MOE-
dynamics chosen. The lowest energy conformation was selected
and subjected to an energy minimization using the MMFF94X force
field. The output of FlexX docking was visualized in MOE and
the scoring.svl script was used to identify interaction types between
ligand and protein.
Acknowledgment. For M.R.S. we would like to thank
Mashhad University of Medical Sciences (MUMS) and the
Iranian Ministry of Health for the award of a Ph.D. scholarship,
for T.K.V. we acknowledge the Cardiff Partnership Fund for
the award of a research grant, and for S.W.Y. we acknowledge
the ORS Awards Scheme for a United Kingdom Scholarship.
We also acknowledge the EPSRC Mass Spectrometry Centre,
Swansea, UK for mass spectroscopy data.
Supporting Information Available: The numbering of com-
1
pounds for H and 13C NMR characterization and assignment of
1H and 13C NMR spectra for ketones 3 and 9, carbinols 4 and 10,
and 2-hydroxy-4-(tetrahydropyran-2-yloxy)benzaldehyde (8). This
material is available free of charge via the Internet at http://
pubs.acs.org.
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