Synthesis and biochemical evaluation of a range of potent benzyl imidazole-based compounds as potential inhibitors of the enzyme complex 17α-hydroxylase/17,20-lyase (P45017α)

Article abstract of DOI:10.1016/j.bmcl.2006.05.070

The cytochrome P-450 enzyme, 17α-hydroxylase/17,20-lyase (P450_17α), is a potential target in hormone-dependent cancers. Here, we report the synthesis and biochemical evaluation of a range of benzyl imidazole-based compounds which have been targeted against the two components of this enzyme, that is, 17α-hydroxylase (17α-OHase) and 17,20-lyase (lyase). The results from the biochemical testing suggest that the compounds synthesised are good inhibitors, with N-4-iodobenzyl imidazole (5) (IC₅₀ = 10.06 μM against 17α-OHase and IC₅₀ = 1.58 μM against lyase) showing equipotent activity against lyase compared to the standard compound, ketoconazole (KTZ) (IC₅₀ = 3.76 ± 0.01 μM against 17α-OHase and IC₅₀ = 1.66 ± 0.15 μM against lyase). Furthermore, the compounds tested are less potent towards the 17α-OHase component, a desirable property in the development of novel inhibitors of P450_17α.

Full text of DOI:10.1016/j.bmcl.2006.05.070

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4011–4015
Synthesis and biochemical evaluation of a range of potent
benzyl imidazole-based compounds as potential inhibitors
of the enzyme complex 17a-hydroxylase/17,20-lyase (P45017a)
Caroline P. Owen,a,* Sachin Dhanani,b Chirag H. Patel,a
Imran Shahida and Sabbir Ahmeda,*
aDepartment of Pharmacy, School of Pharmacy and Chemistry, Kingston University, Penrhyn Road,
Kingston upon Thames, Surrey KT1 2EE, UK
bSchool of Life Sciences, Kingston University, Penrhyn Road, Kingston upon Thames, Surrey KT1 2EE, UK
Received 14 March 2006; revised 3 May 2006; accepted 3 May 2006
Available online 5 June 2006
Abstract—The cytochrome P-450 enzyme, 17a-hydroxylase/17,20-lyase (P45017a), is a potential target in hormone-dependent can-
cers. Here, we report the synthesis and biochemical evaluation of a range of benzyl imidazole-based compounds which have been
targeted against the two components of this enzyme, that is, 17a-hydroxylase (17a-OHase) and 17,20-lyase (lyase). The results from
the biochemical testing suggest that the compounds synthesised are good inhibitors, with N-4-iodobenzyl imidazole (5)
(IC50 = 10.06 lM against 17a-OHase and IC50 = 1.58 lM against lyase) showing equipotent activity against lyase compared to
the standard compound, ketoconazole (KTZ) (IC50 = 3.76 0.01 lM against 17a-OHase and IC50 = 1.66 0.15 lM against lyase).
Furthermore, the compounds tested are less potent towards the 17a-OHase component, a desirable property in the development of
novel inhibitors of P45017a
.
ꢀ 2006 Elsevier Ltd. All rights reserved.
The enzyme complex 17a-hydroxylase/17,20-lyase
(P45017a) is a pivotal enzyme in the conversion of prog-
estins and pregnanes to androgen precursors such as
androstenedione and dehydroepiandrosterone.1 It is a
cytochrome P450 enzyme, requiring both NADPH and
oxygen for the sequential oxidative steps,2 which initial-
ly involves 17a-hydroxylation [via 17a-hydroxylase
(17a-OHase)] followed by the subsequent cleavage of
the C(17)–C(20) bond [via 17,20-lyase (lyase)] (Fig. 1).
P45017a is responsible for the second step in the steroidal
cascade, leading to the biosynthesis of the sex hormones,
glucocorticoids and mineralocorticoids, the latter
two being produced directly from the 17a-hydroxy
progestins.
sesses an active site consisting of a bilobed structure
with two substrate binding sites, one associated with
binding substrate for the hydroxylase reaction and the
other with binding for the lyase reaction.3,4 However,
a more recent molecular modelling study suggests only
a single substrate-binding pocket is present.5
This enzyme is of interest as a target in the treatment of
androgen-dependent prostate cancer because of its role
in the biosynthesis of androgen precursors. However,
ideally, potential inhibitors of this enzyme should pref-
erentially inhibit the lyase reaction and have minimal ef-
fect on the hydroxylase reaction and therefore
corticosteroid biosynthesis.6 Ketoconazole (KTZ), a
known azole-based P45017a inhibitor, has been used in
the treatment of prostate cancer, however, was with-
drawn for this use due to serious adverse effects. Here,
we report: the synthesis of a range of imidazole-based
compounds and their biochemical evaluation (in com-
parison to KTZ) against both components of this
enzyme.
P45017a is a membrane-bound enzyme and therefore no
crystal structure for it has been reported. Homology
modelling studies have suggested that the enzyme pos-
Keywords: Lyase; Hydroxylase; P-45017a; Inhibitors; Substrate–haem
complex.
In the synthesis of the proposed inhibitors, the azole
functionality was reacted with a phenyl alkyl halide in
*
Corresponding authors. Tel.: +44 208 547 2000; fax: +44 208 547
7562; e-mail: s.ahmed@kingston.ac.uk
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.05.070

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C. P. Owen et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4011–4015
O
O
O
OH
lyase
17α-OHase
O
O
O
P
AD
17α-OHP
Figure 1. Reaction catalysed by P45017a in the conversion of progesterone (P) to androstenedione (AD) via 17a-hydroxyprogesterone (17a-OHP).
the presence of a suitable base (Scheme 1)—the synthesis
of N-1-benzyl-imidazole (1) is given as an example.7 In
general, the reactions proceeded in moderate to good
yield (ranging from 25% to 70%) and without any major
problems. However, in the synthesis of 4-hydroxybenzyl
imidazole, the method of Machin et al.8 was used due to
the labile proton present within the starting material
(which would be expected to neutralise any azolyl ion
produced). This involved heating 4-hydroxybenzyl alco-
hol in the presence of excess imidazole (in the absence of
solvent), and resulted in the 4-hydroxybenzyl imidazole
in very good yield (80%).
(IC50 = 86.58 5.21 lM against 17a-OHase and
IC50 = 18.44 0.17 lM against lyase). Considering the
4-substituted series of compounds alone, we do not see
a clear structure–activity relationship.
Consideration of the di-substituted compounds shows
that these inhibitors are, in general, more potent than
the 4-substituted mono-derivatives against both compo-
nents of P45017a. For example, the mono-fluoro deriva-
tive (2) is weaker than both disubstituted derivatives,
11 (IC50 = 83.10 7.05 lM against 17a-OHase and
IC50 = 11.80 0.41 lM against lyase) and 12 (IC50
70.66 6.72 lM against 17a-OHase and IC50
=
=
The biochemical evaluation of the synthesised com-
pounds against both 17a-OHase and lyase was under-
taken using a modified literature method of Li et al.9
involving the use of an alternative mobile phase in the
separation and identification of the radiolabelled sub-
strate from the incubation mixture.10,11 Tables 1a and
1b show the IC50 values obtained for the compounds un-
der consideration against both 17a-OHase and lyase.
9.60 0.14 lM against lyase), with greater potency being
seen when the substitution is in the meta- and para-
positions. This is also observed for the chloro derivatives,
where the 3,4-dichloro derivative (14) is the most potent
against both 17a-OHase and lyase. The modelling of the
di-substituted compounds using the substrate–haem
complex approach12 shows that the di-substituted com-
pounds are able to increase the interaction they undergo
with the active site in comparison to the 4-substituted
mono-derivatives, which are only able to undergo a single
interaction with the active site. For example, when 14 is
modelled within the combined substrate–haem complex
as a representation of the overall P45017a active site, it is
found that this compound is able to undergo interaction
with the hydrogen bonding groups within both compo-
nents of the active site, whereas the mono-substituted
compound is only able to undergo interaction with one
of the components (Figs. 2a and b). That is, from the mod-
elling study, we propose that the disubstituted (in partic-
ular, the 3,4-disubstituted) compounds are able to utilise
two differently situated hydrogen bonding groups within
the active site which would be utilised by the C(3) hydro-
gen bonding groups of the two natural substrates in the
overall reaction (e.g., progesterone and 17a-hydroxypro-
gesterone) to bind to the active site. As such, this is the first
study to suggest that inhibitors may be designed which are
able to utilise both hydrogen bonding groups, and the
results of the current study would appear to support the
bilobed nature of the P45017a active site.
Initial consideration of the inhibitory data for the com-
pounds shows that all of the benzyl compounds tested
were weaker inhibitors against 17a-OHase than against
lyase, and, when compared to the standard compound,
KTZ, they were also found to possess weaker inhibitory
activity. The most potent compound was found to be
4-iodobenzyl imidazole (5), which had an IC50
=
10.06 0.96 lM against 17a-OHase and an IC50 value
of 1.58 0.17 lM against lyase, compared to KTZ
(IC50 = 3.76 0.01 lM
against
17a-OHase
and
IC50=1.66 0.15 lM against lyase).
Detailed consideration of the mono- and di-substituted
compounds does not show a clear structure–activity
relationship, other than that all the substituted inhibi-
tors (except for 10) show greater potency against both
17a-OHase and lyase in comparison to the non-substi-
tuted derivative. For example, the unsubstituted deriva-
tive, 1 (IC50 = 214.58 19.67 lM against 17a-OHase
and IC50 = 39.06 1.22 lM against lyase), is found to
be ꢀ2.5 and ꢀ2 times weaker, respectively, than the
least
active
mono-substituted
derivative,
2
In summary, whilst the compounds within the current
study have been shown, in general, to possess weaker
inhibitory activity against the lyase components of the
overall enzyme complex of P45017a in comparison to the
standard compound KTZ, all are significantly weaker
inhibitors against the 17a-OHase component and
therefore would be expected to have less of an effect on
corticosteroid biosynthesis. The greatest selectivity is seen
Br
N
a
N
R1
R1
Scheme 1. Synthesis of azole-based derivatives (a) imidazole/K2CO3/
THF/D; R1 = various substituents).

C. P. Owen et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4011–4015
4013
Table 1a. Showing the IC50 values (n = 9) of the mono-substituted derivatives of benzyl imidazole against both the 17a-OHase and lyase component
of the overall P-45017a
Compound
Structure
17a-OHase [IC50 values (lM)]
Lyase [IC50 values (lM)]
N
1
214.58 19.67
39.06 1.22
N
N
2
3
4
5
6
7
8
9
86.58 5.21
31.63 3.86
53.40 4.40
10.06 0.96
40.26 3.49
25.38 1.65
50.56 8.01
72.05 3.95
18.44 0.17
2.81 0.27
6.76 0.36
1.58 0.17
7.67 0.05
7.17 0.13
8.81 0.43
8.22 0.97
N
F
N
N
Cl
Br
I
N
N
N
N
N
N
N
N
N
O2N
N
N
H3C
HO
N
N
Table 1b. Showing the IC50 values (n = 9) of the di-substituted derivatives of benzyl imidazole against both the 17a-OHase and lyase component of
the overall P-45017a
Compound
Structure
17a-OHase [IC50 values (lM)]
17,20-lyase [IC50 values (lM)]
F3C
N
10
244.85 24.45
19.46 0.75
N
CF3
F
F
N
N
11
12
13
14
83.10 7.05
70.66 6.72
22.56 0.34
12.22 0.88
11.80 0.41
9.60 0.14
3.34 0.11
2.07 0.07
N
F
N
F
Cl
N
N
Cl
Cl
N
N
N
N
Cl
Br
15
25.95 0.91
3.76 0.01
3.16 0.11
1.66 0.15
Br
KTZ

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C. P. Owen et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4011–4015
Figure 2. (a) Binding of 3 (in green) into the substrate–haem complex for the overall enzyme complex. (b) Binding of 14 (in green) into the substrate–
haem complex for the overall enzyme complex.
(NaHCO3) and then extracted into DCM (2· 40 mL).
The combined DCM layer was washed with water (3·
50 mL), dried over anhydrous magnesium sulfate
(MgSO4) and filtered. Removal of DCM under vacuum
gave 1 as a yellow solid (0.93 g, yield 40%) (mp 71.5–
72.5 ꢁC). m(max) (film) cm À1: 3387.3 (NCN imidazole),
2938.9 (CH aliphatic); dH (CDCl3): 7.46 (1H, s, NCHN
imidazole), 7.18 (5H, m, ArH), 7.00 (1H, s, NCH
imidazole), 6.81 (1H, s, NCH imidazole), 5.02 (2H, s,
PhCH2); dC (CDCl3): 137.43 (NCN), 129.78, 128.97,
128.24, 127.26 (ArC), 136.18, 119.30 (ImC), 50.76
(PhCH2); GCMS tR 8.24 min. m/z 158 (M+), 91 (base
peak).
for compounds 3 and 10 which show over 10-fold differ-
ence in activity between the two components. They are
thus good lead compounds in the design and synthesis
of more potent inhibitors of this biochemical target.
Acknowledgments
The authors thank the EPSRC National Mass Spec-
trometry service at the University of Wales College
Swansea (UK) and the elemental analysis service at
the School of Pharmacy, University of London (UK),
for the provision of high resolution and elemental anal-
ysis data, respectively.
8. Machin, P. J.; Hurst, D. N.; Bradshaw, R. M.; Blaber, L.
C.; Burden, D. T.; Melarange, R. A. J. Med. Chem. 1984,
27, 503.
9. Li, J. S.; Li, Y.; Son, C.; Brodie, A. M. H. J. Med. Chem.
1996, 39, 4335.
References and notes
10. 17a-OHase assay using rat microsomal enzyme: Rat
testicular microsomal suspension was thawed under cold
running water and vortexed. The final incubation assay
mixture (1 mL) consisted of sodium phosphate buffer
(50 mM, pH 7.4, 905 lL), radiolabelled progesterone as
substrate (1.5 lM, 15 lL), NADPH generating system
(50 lL) and solution of the inhibitor (10 lM, 20 lL) in
absolute ethanol. Tubes were warmed to 37 ꢁC for 5 min
and the assay initiated by the addition of microsomal
enzyme (final concentration 0.16 mg/mL, 10 lL). The
assay mixture was incubated for 15 min. The reaction
was quenched by the addition of ether (2 mL), vortexed
and placed on ice. The organic layer was then placed into a
separate tube. The assay mixture was further extracted
with ether (2· 2 mL), and the organic layers combined.
The solvent was removed under a stream of nitrogen,
acetone (30 lL) was added to each tube and the solution
spotted onto silica-based TLC plates along with carrier
steroids (progesterone, 17a-hydroxyprogesterone, testos-
terone and androstenedione, 5 mg/mL). The TLC plates
were developed using the mobile phase DCM/ethylacetate
(7:3). The separated spots were identified under UV light
and each spot cut out and placed into scintillation vials.
Acetone (1 mL) and scintillation fluid (Cocktail T) (3 mL)
were added to each vial, vortexed and counted for 3 min
1. Ortiz de Montellano, P. R. In Cytochrome P-450: Struc-
ture Mechanism and Biochemistry; Ortiz de Montellano, P.
R., Ed.; Plenum: New York, 1986; p 217.
2. Robichaud, P.; Wright, J. N.; Akhtar, M. J. Chem. Soc.,
Chem. Commun. 1994, 12, 1501.
3. Laughton, C. A.; Neidle, S.; Zvelebil, M. J. J. M.;
Sternberg, M. J. E. A. Biochem. Biophys. Res. Commun.
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Drug Des. 1997, 12, 113.
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6. Njar, V. C. O.; Brodie, A. M. H. Curr. Pharm. Des. 1999,
5, 163.
7. N-1-Benzyl-imidazole (1): Imidazole (2 g, 29.4 mmol) was
added to anhydrous potassium carbonate (K2CO3)
(1.02 g, 7.34 mmol) and anhydrous tetrahydrofuran
(THF) (50 mL). The mixture was stirred at room temper-
ature for 10 min prior to the addition of benzyl bromide
(2.51 g, 14.7 mmol). The mixture was then stirred under
reflux for 24 h. After filtration, the THF was removed
under vacuum to leave a yellow solid which was dissolved
in dichloromethane (DCM) (40 mL) and washed with
water (3· 50 mL). The organic layer was then extracted
using hydrochloric acid (HCl) (2 M, 3· 30 mL) followed
by water (2· 50 mL). The combined acid layer was
neutralised with solid saturated sodium bicarbonate
3
for H. Control samples with no inhibitor were incubated
simultaneously. In determining the IC50 values for the
most potent compounds, the inhibitory activity was
determined using the method outlined above, however,

C. P. Owen et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4011–4015
4015
for each compound, five or more inhibitor concentrations
were used and the inhibitory activity was determined at
each concentration (in triplicate); the IC50 was then
determined from a graph (using linear regression analysis)
of the inhibitory activity versus log[I].
stream of nitrogen, acetone (30 lL) was added to each
tube and the solution spotted onto silica-based TLC
plates along with carrier steroids (17a-hydroxyproges-
terone, testosterone and androstenedione, 5 mg/mL).
The TLC plates were developed using the mobile phase
DCM/ethylacetate (4:1). The separated spots were iden-
tified under UV light and each spot cut out and placed
into scintillation vials. Acetone (1 mL) and scintillation
fluid (Cocktail T) (3 mL) were added to each vial,
11. Lyase assay using rat microsomal enzyme: Rat testicular
microsomal suspension was thawed under cold running
water and vortexed. The final incubation assay mixture
(1 mL) consisted of sodium phosphate buffer (50 mM,
pH 7.4, 905 lL), radiolabelled 17a-hydroxyprogesterone
as substrate (1 lM, 10 lL), NADPH generating system
(50 lL) and solution of the inhibitor (10 lM, 20 lL) in
absolute ethanol. Tubes were warmed to 37 ꢁC for 5 min
and the assay initiated by the addition of microsomal
enzyme (final concentration 0.23 mg/mL, 15 lL). The
assay mixture was incubated for 30 min. The reaction
was quenched by the addition of ether (2 mL), vortexed
and placed on ice. The organic layer was then removed
and placed into a separate tube. The assay mixture was
further extracted with ether (2· 2 mL), and the organic
layers combined. The solvent was removed under a
3
vortexed and counted for 3 min for H. Control samples
with no inhibitor were incubated simultaneously. In
determining the IC50 values for the most potent com-
pounds, the inhibitory activity was determined using the
method outlined above, however, for each compound,
five or more inhibitor concentrations were used and the
inhibitory activity was determined at each concentration
(in triplicate); the IC50 was then determined from a
graph (using linear regression analysis) of the inhibitory
activity versus log[I].
12. Ahmed, S.; Davis, P. J. Bioorg. Med. Chem. Lett. 1995, 5,
2789.