Synthesis and biochemical evaluation of a range of sulfonated derivatives of 4-hydroxybenzyl imidazole as highly potent inhibitors of rat testicular 17α-hydroxylase/17,20-lyase (P-45017α)

Article abstract of DOI:10.1016/j.bmcl.2009.06.070

We report the synthesis and biochemical evaluation of a range of 4-sulfonated derivatives of 4-hydroxybenzyl imidazole which has been targetted against the two components of 17α-hydroxylase/17,20-lyase (P-450_17α), namely, 17α-hydroxylase (17α-OHase) and 17,20-lyase (lyase). The results from the biochemical testing suggest that the compounds synthesised are highly potent inhibitors possessing excellent selectivity towards the lyase component.

Full text of DOI:10.1016/j.bmcl.2009.06.070

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4698–4701
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Bioorganic & Medicinal Chemistry Letters
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Synthesis and biochemical evaluation of a range of sulfonated derivatives
of 4-hydroxybenzyl imidazole as highly potent inhibitors of rat testicular
17a-hydroxylase/17,20-lyase (P-45017 )
a
b
a
a
Sabbir Ahmed a, , Imran Shahid , Sachin Dhanani , Caroline P. Owen
*
a School of Science, University of the West of Scotland, High Street, Paisley, PA1 2BE, Scotland, UK
b School of Pharmacy and Chemistry, Kingston University, Kingston, KT1 2EE, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the synthesis and biochemical evaluation of a range of 4-sulfonated derivatives of 4-hydroxy-
benzyl imidazole which has been targetted against the two components of 17 -hydroxylase/17,20-lyase
(P-45017 ), namely, 17 -hydroxylase (17 -OHase) and 17,20-lyase (lyase). The results from the biochem-
ical testing suggest that the compounds synthesised are highly potent inhibitors possessing excellent
selectivity towards the lyase component.
Received 5 March 2009
Revised 17 June 2009
Accepted 18 June 2009
Available online 21 June 2009
a
a
a
a
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Hydroxylase
Lyase
Prostate cancer
Inhibition
Sulfonate
In the conversion of progestins and pregnanes to the corre-
sponding androgen precursors such as androstenedione (AD) and
P45017 5 and has entered Phase II of clinical trials. Indeed, we have
a
recently reported the design and synthesis of a number of benzyl
imidazole-based compounds as inhibitors of this enzyme com-
plex.6–9 In particular, we have shown previously that inhibitors
able to bind to the two hydrogen bonding groups present at the ac-
dehydroepiandrosterone (DHEA) (Fig. 1), 17a-hydroxylase/17,20-
lyase (P-45017 ) is a pivotal enzyme.1
a
This enzyme is of interest as a target in the treatment of andro-
gen-dependent prostate cancer because of its role in the biosynthe-
tive site of P-45017 possess greater inhibitory activity than com-
a
sis of these androgen precursors, however, 17
involved in the direct biosynthesis of glucocorticoids and mineral-
ocorticoids, the latter two being produced directly from the 17
a
-hydroxylase is also
pounds which only utilise one of the two hydrogen bonding
groups6 [these H-bonding groups are involved in stabilising the en-
zyme–substrate complex with each H-bonding group binding to
a-
hydroxy progestins. The overall enzyme complex is postulated to
be a bi-lobed structure with two substrate binding sites, one asso-
ciated with binding substrate for the hydroxylase reaction and the
other with binding for the lyase reaction.2,3 In particular, the over-
all conversion of progestins and pregnanes involves two sequential
oxidative steps requiring both NADPH and oxygen4—the first in-
the C(3) polar group within each C21 steroid, that is, P or 17
OHP]. Here, we report our continued efforts in the design and syn-
a-
thesis of potent and specific inhibitors of P-45017 , in particular,
a
compounds which are able to bind to both H-bonding groups with-
in the active site. As such, we report: the synthesis of a range of
sulfonate derivatives of 4-hydroxybenzyl imidazole-based com-
pounds and their biochemical evaluation (in comparison to KTZ)
against both components of rat testicular microsomal enzyme.
In the synthesis of the benzyl imidazole-based compounds, the
azole functionality was reacted with a benzyl bromide in the pres-
ence of a suitable base6–9 (Scheme 1).
However, in the synthesis of derivatives of 4-hydroxybenzyl
imidazole, the reaction outlined in Scheme 1 cannot be used since
the labile proton present within the starting material (i.e., where
R = OH) would be expected to neutralise the azolyl ion produced
if this route was followed. The method of Machin et al.10 was there-
fore utilised (Scheme 2, step a), as such, the synthesis of 4-
hydroxybenzyl imidazole11 (1) involved heating 4-hydroxybenzyl
volves an initial 17
ylase (17 -OHase) component of the enzyme complex] of the C21
steroid, followed by the cleavage of the C(17)–C(20) bond of the
17 -hydroxy intermediate [catalysed by the 17,20-lyase (lyase)
a-hydroxylation [catalysed by the 17a-hydrox-
a
a
component], to give the corresponding androgen precursor.
In the inhibition of this enzyme, a number of different com-
pounds has been synthesised and evaluated including ketocona-
zole (KTZ) and abiraterone acetate (Fig. 2)—this latter compound
has been shown to be
a selective and potent inhibitor of
* Corresponding author. Tel.: +44 141 848 3000.
E-mail address: sabbir.ahmed@uws.ac.uk (S. Ahmed).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.06.070

S. Ahmed et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4698–4701
4699
O
O
O
OH
lyase
17α-OHase
O
O
O
P
AD
17α-OHP
Figure 1. Reaction catalysed by P-45017 in the conversion of progesterone (P) to AD via 17
a
-hydroxyprogesterone (17a-OHP).
a
N
N
O
O
N
N
N
O
O
Cl
O
O
Cl
Abiraterone acetate
Ketoconazole
Figure 2. Structures of two known potent inhibitors of P45017
.
a
17a-OHase and lyase as well as the IC50 values obtained for the
two standard inhibitors used within our study—it should be noted
Br
N
a
N
that we have previously reported 4-iodobenzyl imidazole (10) as a
potent inhibitor of both 17a-OHase and lyase components, how-
R
R
ever, we have used the compound as a standard so as to allow us
to compare the novel compounds (2–9) reported here with our
previously reported inhibitors.
Scheme 1. Synthesis of azole-based derivatives (a = imidazole/K2CO3/THF/D;
R = various substituents).
Initial consideration of the inhibitory data for the compounds
shows that all of the benzyl compounds evaluated (2–9) were con-
siderably weaker inhibitors against 17a-OHase than against
OH
N
a
N
lyase—this is suggested to be a beneficial property since it is ex-
pected to result in reduced side-effects as the compounds would
not be expected to interfere with corticosteroid synthesis. As such,
compounds 2, 7 and 9 are observed to possess an excellent selec-
tivity index value with compounds 3, 4, 5 and 8 possessing good
selectivity (Table 1). Furthermore, the compounds considered
within the study were all found to possess weak inhibitory activity
HO
HO
b
N
against 17
KTZ (IC50=3.76 0.01
highly potent inhibitory activity against the lyase component in
comparison to KTZ (IC50 = 1.66 0.15 M against lyase).
a-OHase when compared to the standard compound,
O
S
N
l
M against 17 -OHase) whilst possessing
a
R
O
O
l
With regards to our own standard compound (10), the com-
pounds within the current study were found to be, in general, equi-
potent or more potent than 10 against the 17a-OHase component—
Scheme 2. Synthesis of a number of sulfonate derivatives of 4-hydroxybenzyl
imidazole (a = imidazole/ b = 4-substituted phenyl sulfonyl chloride/DCM/
R = various substituents, for example, CH3, NO2, F, Cl, and Br).
D
;
D;
only compounds 2, 4 and 9 were weaker. However, all of the com-
pounds (2–9) were found to be more potentthan 10 against the lyase
component, indeed, the most potent compound against this compo-
nent was compound 7 which was found to possess an IC50 value of
65 nM. The most potent compound (across both components) with-
in the new range of inhibitors was compound 3 which was found to
alcohol in the presence of excess imidazole (in the absence of a sol-
vent), and resulted in the 4-hydroxybenzyl imidazole in very good
yield (87%). In the synthesis of the sulfonate derivatives, the appro-
priate sulfonyl chloride was reacted with 1 in the presence of tri-
ethylamine (TEA) and anhydrous dichloromethane (DCM)
(Scheme 2, step b)—the synthesis of the p-toluenesulfonate deriv-
ative (2) is given as an example.12 In general, the reactions pro-
ceeded in moderate to good yield (ranging from 60% to 80%) and
without any major problems.
possess an IC50 value of 6.88
against lyase. Compound 5 was also found to be a potent inhibitor,
possessing an IC50 value of 6.85 M against 17 -OHase and 99 nM
lM against 17a-OHase and 85 nM
l
a
against lyase. However, in comparison to compounds 2, 7 and 9,
compounds 3 and 5 were found to possess slightly poorer selectivity.
Due to the microsomal nature of P-45017 , the crystal structure
a
The biochemical evaluation of the synthesised compounds
of this enzyme has yet to be determined, however, several model-
ling techniques exist which may be utilised to study the mode of
binding of these inhibitors.2,15–17 These studies have proposed
against both 17a-OHase and lyase was undertaken using a litera-
ture method.6–9,13,14 Table 1 shows the IC50 values obtained for
the compounds considered within the current study against both

4700
S. Ahmed et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4698–4701
Table 1
Table 2
Showing the IC50 values (n = 9) and the selectivity index (which is the ratio of [IC50
(17 -OHase)/IC50 (lyase)]) for the compounds considered within the current study
Initial screening ([I] = 10 lM) and IC50 values for the benzyl imidazole-based
compounds, including the standard AG18
a
against both the 17
a
-OHase and lyase components of the overall P-45017
a
N
N
N
O
S
N
R1
O
R
O
Compound
R
IC50 (lM)
11
12
13
14
15
AG
NO2
F
Cl
CN
CH3
—
0.13 0.19
0.88 0.03
0.07 0.5
1.20 0.08
24.5 0.12
70.6 0.1
Compound
number
R1
17
a
-OHase
Lyase [IC50 values
(nM)]
Selectivity
index
[IC50 values (lM)]
2
3
4
5
6
7
8
9
CH3
NO2
F
Cl
Br
25.16 1.01
6.88 0.33
15.93 0.72
6.85 0.20
4.84 0.02
10.01 0.04
6.00 0.01
64.91 2.12
10.06 0.66
3.76 0.01
230.2 10.2
85.0 8.1
210.4 27.4
98.7 36.2
70.4 5.7
109.4
80.9
75.9
69.9
34.6
100.1
60.0
223.8
6.4
I
65.1 4.5
OCH3
CF3
—
100.9 13.2
290.8 43.1
1580.0 100
1660.0 150
the current study would also be expected to possess inhibitory activ-
ity against other P-450 enzymes and therefore may not be suitable
drugsubstancesdueto theirpoorselectivityagainstotherP-450sys-
tems. Finally, it should be noted that the current study has utilised
rat testicular microsomal enzyme which has been shown to be dif-
10
KTZ
—
2.3
the existence of two ‘lobes’ which are utilised by the two sub-
strates of P-45017 . From the consideration of the inhibitory activ-
ferent to human P-45017 , as such, whilst these compounds are
highly potent inhibitors, they would be required to be evaluated
a
a
ity observed within this series of compounds, in comparison to
previous benzyl azole-based compounds reported by us6–9, we
hypothesise that these compounds are able to utilise additional
binding to increase potency. As such, we propose that the potency
of the inhibitors is due to two interactions: the 4-substituent on
the phenyl ring undergoes polar–polar interaction with one of
the hydrogen bonding groups at the active site, whilst the second
interaction involves the sulfonate moiety and presumably the
S@O groups which are able to interact with the active site. Both
interactions (together with the Fe–N dative covalent bond forma-
tion) therefore increase the stability of the inhibitor–enzyme com-
plex leading to an increase in the potency of the inhibitor in
comparison to compounds which are only able to interact with a
single hydrogen bonding group. That this is indeed the rationale
for the increased inhibitory activity can be observed within the
4-halogenated compounds, where the potency of the inhibitor in-
creases with a decrease in electronegativity of the halogen.
Furthermore, we observe that compounds which are not able to
undergo both interactions with the two hydrogen bonding groups
at the same time (i.e., compounds 2, 8 and 9 which lack groups on
the phenyl moiety able to undergo polar–polar interaction) possess
poor inhibitory activity in comparison to the other compounds.
This therefore adds further support to our previous study where
we have shown that the di-halogen derivatives of benzyl imidaz-
ole-based compounds possessed greater inhibitory activity than
the mono-substituted compounds.6 That is, the second polar–polar
interaction between the inhibitor and the hydrogen bonding group
at the active site results in extremely strong binding of the inhib-
itor to the active site as a result of which the catalytic activity of
the overall enzyme complex is greatly reduced.
against human P-45017 prior to further development.
a
Acknowledgements
The authors thank the EPSRC National Mass Spectrometry ser-
vice at the University of Wales College Swansea (UK) and the ele-
mental analysis service at the School of Pharmacy, University of
London (UK) for the provision of high resolution and elemental
analysis data, respectively.
References and notes
1. Ortiz de Montellano, P. R. In Cytochrome P-450: Structure Mechanism and
Biochemistry; Ortiz de Montellano, P. R., Ed.; Plenum: New York, 1986; pp 217–
272.
2. Laughton, C. A.; Neidle, S.; Zvelebil, M. J. J. M.; Sternberg, M. J. E. A. Biochem.
Biophys. Res. Commun. 1990, 171, 1160.
3. Burke, D. F.; Laughton, C. A.; Neidle, S. Anti-Cancer Drug Des. 1997, 12, 113.
4. Robichaud, P.; Wright, J. N.; Akhtar, M. J. Chem. Soc., Chem. Commun. 1994, 12,
1501.
5. Attard, G.; Reid, A. H. M.; Yap, T. A.; Raynaud, F.; Dowsett, M.; Settatree, S.;
Barrett, M.; Parker, C.; Martins, V.; Folkerd, E.; Clark, J.; Cooper, C. S.; Kaye, S. B.;
Dearnaley, D.; Lee, G.; de Bono, J. S. J. Clin. Oncol. 2008, 26, 4563.
6. Owen, C. P.; Dhanani, S.; Patel, C. H.; Shahid, I.; Ahmed, S. Bioorg. Med. Chem.
Lett. 2006, 16, 4011.
7. Patel, C. H.; Dhanani, S.; Owen, C. P.; Ahmed, S. Bioorg. Med. Chem. Lett. 2006, 16,
4752.
8. Shahid, I.; Patel, C. H.; Dhanani, S.; Owen, C. P.; Ahmed, S. J. Steroid Biochem.
Mol. Biol. 2008, 110, 18.
9. Owen, C. P.; Shahid, I.; Olusanjo, M. S.; Patel, C. H.; Dhanani, S.; Ahmed, S. J
Steroid Biochem. Mol. Biol. 2008, 111, 117.
10. Machin, P. J.; Hurst, D. N.; Bradshaw, R. M.; Blaber, L. C.; Burden, D. T.;
Melarange, R. A. J. Med. Chem. 1984, 27, 503.
11. 1-(4-Hydroxy-benzyl)-1H-imidazole (1): Imidazole (13.4 g, 200 mmol) was
mixed with 4-hydroxybenzyl alcohol (5 g, 40 mmol). The reaction mixture
was then heated at 160 °C for 30 min resulting in a dark brown oil which was
added to 500 mL of hot water resulting in a brown solid which was filtered and
vacuum dried to give a light brown solid. Column chromatography of the solid
gave 1 as a light brown solid (6.08 g, yield 87%); [mp = 209.8–210.6 °C (lit.
mp = 212.0–213.0 °C [12])]; Rf = 0.35 [50/50 (diethyl ether/petroleum ether)].
In conclusion, we have introduced novel compounds which have
been shown to be highly potent inhibitors of P-45017 , with good
a
selectivity towards the lyase component in comparison to the 17a-
OHase component, as such, they appear to be extremely good lead
compounds in the continued design and synthesis of potential drug
m
(max) (Film)cmÀ1: 3421 (OH), 1653 (Im, CH2–N), 1601 (Ar, C@C); dH (400 MHz,
DMSO): 9.45 (1H, s, Ph–OH), 7.66 (1H, s, NCHN, Im), 7.09 (1H, s, CH2–NCH, Im),
7.06 (2H, d, J = 8.79 Hz, Ph–H), 6.83 (1H, s, NCH, Im), 6.68 (2H, d, J = 8.79 Hz,
Ph–H); 4.99 (2H, s, Ph–CH2); dC (100 MHz, DMSO): 157.55 (Ar, C-OH), 137.65
(NCN), 129.65, 128.51, 115.88 (Ar, C), 129.09, 119.87 (Im, C), 49.67 (Ph–CH2) ;
GC: tR 9.43 min, LRMS (EI): m/z 174 (M+, 18%), 107 (M+ÀC3H3N2, 100%), 77
(M+ÀC4H5N2O, 14%); HRMS (EI): Found m/z 175.0866300 (M+) C10H11O1,
calculated m/z 175.0866295.
substances against P-45017 . However, whilst these compounds
a
have shown extremely favourable selectivity between the two com-
ponents of P-45017 , the compounds would be required to also pos-
a
sess selectivity against other P-450 enzymes, for example, P-45011
.
In an earlier study, we have shown that related 4-substituted benzyl
imidazole compounds possessed highly potent inhibitory activity
against aromatase (e.g., compound 13 was found to possess an IC50
of 70 nM)18 (Table 2), as such, the compounds considered within
12. 4-(1H-imidazol-1-ylmethylphenyl 4-toluenesulfonate (2): To
a mixture of 1
(1.00 g, 5.75 mmol) and triethylamine (0.70 g, 6.89 mmol) in anhydrous DCM,
4-toluene sulfonyl chloride (1.20 g, 6.32 mmol) was added and the reaction
mixture refluxed for 12 h. After cooling, the reaction mixture was poured on ice

S. Ahmed et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4698–4701
4701
(100 mL) and washed with sodium carbonate solution (Na2CO3) (2 Â 50 mL).
The organic layer was washed with water (3 Â 50 mL) and dried over
anhydrous magnesium sulfate (MgSO4), filtered and the DCM removed under
vacuum to give a yellow oil which was purified using column chromatography
to give 2 as a light yellow coloured oil (0.92 g, yield 63%); Rf = 0.37 [60/30/10
(petroleum ether/diethyl ether/methanol)]. m(max) (film)cmÀ1: 3109 (Ar, C–H),
2303 (Im, C@N), 1596 (Ar, C@C), 1371 (S@O); dH (400 MHz, CDCl3): 7.61 (1H, s,
NCHN, Im), 7.56 (2H, d, J = 8.42, Ph–H), 7.28 (2H, d, J = 8.42 Hz, Ph–H), 7.07 (2H,
d, J = 8.79 Hz, Ph–H), 6.97 (1H, s, CH2–NCH, Im), 6.86 (3H; 2H, d, J = 8.79 Hz,
Ph–H, 1H, NCH, Im), 5.08 (2H, s, Ph–CH2), 2.32 (3H, s, CH3); dC (100 MHz,
CDCl3): 149.44, 145.97 (Ar, C), 137.32 (Im, NCN), 136.31, 132.20, 129.72,
128.66, 128.30, 122.55 (Ar, C), 128.14, 119.56 (Im, C), 49.35 (Ph–CH2), 20.28
(CH3); GC: tR 28.65 min; LRMS (EI): m/z 328 (M+, 52%), 261 (M+ÀC3H3N2, 52%),
155 (M+ÀC10H9N2O, 64%), 91 (M+ÀC10H9N2O3S, 100%); HRMS (EI): found m/z
329.0955060, C17H17N2O3S, calculated m/z 329.0954395.
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 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].
14. 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
l
L), radiolabelled 17
a-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 Â 2 mL), and the organic layers
combined. The solvent was removed under a stream of nitrogen, acetone
13. 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
(30
l
L) was added to each tube and the solution spotted onto silica based TLC
-hydroxyprogesterone, testosterone and
(50 mM, pH 7.4, 905
l
L), radiolabelled progesterone as substrate (1.5
l
l
M,
M,
plates along with carrier steroids (17a
15
20
l
L), NADPH generating system (50 L) and solution of the inhibitor (10
l
androstenedione, 5 mg/mL). The TLC plates were developed using the mobile
phase DCM/ethylacetate (4:1). 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 for 3H. 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, for each compound, five or more inhibitor concentrations were used
and the inhibitory activity 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].
l
L) 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 L). The assay mixture was incubated for 15 min. The reaction
l
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 Â 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, 17 -hydroxyprogesterone, testosterone and
a
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 for 3H. Control samples with no inhibitor were incubated
simultaneously. In determining the IC50 values for the most potent compounds,
15. Ahmed, S.; Davis, P. J. Bioorg. Med. Chem. Lett. 1995, 5, 2789.
16. Ahmed, S. Bioorg. Med. Chem. Lett. 1995, 5, 2795.
17. Ahmed, S. Biochem. Biophys. Res. Commun. 2004, 316, 595.
18. Adat, S.; Owen, C. P.; Ahmed, S. Lett. Drug Des. Discovery 2007, 4, 545.