Synthesis and biochemical evaluation of a range of (4-substituted phenyl)sulfonate derivatives of 4-hydroxybenzyl imidazole-based compounds as potent inhibitors of 17α-hydroxylase/17,20-lyase (P450 17α) derived from rat testicular microsomes

Article abstract of DOI:10.1016/j.bmcl.2010.02.100

We report the synthesis, biochemical evaluation and rationalisation of the inhibitory activity of a range of (4-substituted phenyl)sulfonate derivatives of 4-hydroxybenzyl imidazole against the two components of 17α-hydroxylase/ 17,20-lyase (P450_17α), namely, 17α-hydroxylase (17α-OHase) and 17,20-lyase (lyase). The results show the compounds to be highly potent inhibitors with limited selectivity towards the lyase component [e.g., toluene-4-sulfonic acid 4-imidazol-1-ylmethyl-phenyl ester (4) possessed an IC₅₀ value of 40 nM against 17α-OHase and 30 nM against lyase].

Full text of DOI:10.1016/j.bmcl.2010.02.100

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5345–5348
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biochemical evaluation of a range of (4-substituted
phenyl)sulfonate derivatives of 4-hydroxybenzyl imidazole-based
compounds as potent inhibitors of 17
a
-hydroxylase/17,20-lyase (P45017 )
a
derived from rat testicular microsomes
Caroline P. Owen a, Imran Shahid b, Wai-Yee Lee b, Sabbir Ahmed a,
*
a School of Science, Faculty of Science and Technology, University of the West of Scotland, High Street, Paisley, PA1 2BE, Scotland, UK
b Kingston University, Penrhyn Road, Kingston, 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, biochemical evaluation and rationalisation of the inhibitory activity of a range of
(4-substituted phenyl)sulfonate derivatives of 4-hydroxybenzyl imidazole against the two components of
Received 2 December 2009
Revised 24 February 2010
Accepted 26 February 2010
Available online 3 March 2010
17a-hydroxylase/17,20-lyase (P45017 ), namely, 17a-hydroxylase (17a-OHase) and 17,20-lyase (lyase).
a
The results show the compounds to be highly potent inhibitors with limited selectivity towards the lyase
component [e.g., toluene-4-sulfonic acid 4-imidazol-1-ylmethyl-phenyl ester (4) possessed an IC50 value
of 40 nM against 17a-OHase and 30 nM against lyase].
Keywords:
Synthesis
Inhibition
Enzyme
Ó 2010 Elsevier Ltd. All rights reserved.
17
a-Hydroxylase
17,20-Lyase
The cytochrome P450 enzyme 17
a
-hydroxylase/17,20-lyase
verse effects. In more recent studies, however, KTZ has been eval-
uated against hormone-refractory prostate cancer in lower doses.4
Previously, we have outlined the synthesis and biochemical
evaluation of halogenated derivatives of benzyl azole-based com-
(P45017 ) is involved in the production of the C19 containing andro-
a
gen precursors from the C21 containing steroids such as the pregn-
anes and progestins (Fig. 1). Steroids such as androstenedione (AD)
and dehydroepiandrosterone (DHEA) are therefore synthesised as a
pounds which have been shown to be good inhibitors of P45017
a
result of the action of P45017 on progesterone and pregnenolone,
in comparison to the standard KTZ;5–9 recently we reported a range
of (4-substituted phenyl)sulfonate derivatives of 4-hydroxybenzyl
imidazole (1) which were found to possess good inhibitory activity
a
respectively,1 involving an initial 17
a
-hydroxylation [via 17
a-
hydroxylase (17a-OHase)], followed by the cleavage of the
C(17)–C(20) bond [via 17,20-lyase (lyase)]—both hydroxylation
against P45017 .
10 Indeed, compounds based on the biphenyl back-
a
and C–C cleavage steps require NADPH and oxygen.2,3
bone have also been developed and have also shown good level of
potentcy.11–13 Here, we report: the synthesis of a range of (4-alkyl-
substituted phenyl)sulfonate derivatives of 1 and their biochemical
evaluation (in comparison to KTZ) against both components of
P45017 is a potential biological target in the treatment of hor-
a
mone-dependent diseases such as prostate cancer which has been
shown to be androgen-dependent in its early stages. A number of
compounds has therefore been investigated, including the antimy-
cotic ketoconazole (KTZ) and abiraterone acetate (Fig. 2). The latter
compound is currently in clinical trials, however, KTZ, a known
P45017 in an attempt to discover potent and highly specific inhib-
a
itors of P45017 . In an effort to compare the results of our previous
a
studies, we also undertook the evaluation of the compounds previ-
azole-based P45017 inhibitor (albeit an unselective inhibitor of
ously reported by us as inhibitors of P45017 , in particular, benzyl
a
a
steroid biosynthesis and which has been shown to inhibit other
steroidogenic enzymes whilst possessing weak activity towards
imidazole (2) and 4-iodobenzyl imidazole (3) (the latter compound
was shown to possess the highest inhibitory activity within the
halogen derivatives of benzyl imidazole8) as well as three previ-
ously reported sulfonate derivatives,10 namely, compounds 4, 9
and 10 (Table 1).
P45017 ), has been used previously in the treatment of prostate
a
cancer, however, was withdrawn for this use due to serious ad-
In the synthesis of the (4-alkylsubstituted phenyl)sulfonate
derivatives of 1 (compounds 4–11), the reactions outlined in
Scheme 1 were undertaken—we have reported previously the
* Corresponding author. Tel.: +44 141 848 3000.
E-mail address: sabbir.ahmed@uws.ac.uk (S. Ahmed).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.02.100

5346
C. P. Owen et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5345–5348
O
O
O
OH
lyase
17α-OHase
O
O
O
P
AD
17α-OHP
Figure 1. Reaction catalysed by P45017 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
KTZ
Figure 2. Structures of two KTZ and abiraterone acetate.
synthesis of other derivatives of compound 1 where we have suc-
cessfully utilised the reactions outlined in Scheme 1,10 as such, no
specific methodology has been discussed within the current report.
The biochemical evaluation of the synthesised compounds
OH
N
a
N
HO
HO
(1)
against both 17a-OHase and lyase components was undertaken
using the method of Owen et al.14 and involved the use of thin layer
chromatography (TLC) in the separation and identification of the
radiolabelled substrate from the incubation mixture. Table 1 shows
the IC50 values obtained for the compounds considered within the
b
current study against both 17a-OHase and lyase. P45017 is also
indirectly involved in the biosynthesis of the glucocorticoids and
mineralocorticoids, in particular, the latter is produced directly from
a
N
O
S
N
R
O
the 17a-hydroxylated derivatives of the progestins, as such, inhibi-
O
tors of P45017 should preferentially inhibit the C–C bond cleavage
a
reaction in comparison to the hydroxylation step. This is therefore
expected to have minimal effect on the hydroxylase reaction and
therefore glucocorticoid biosynthesis.15
Scheme 1. Synthesis of a number of sulfonate derivatives of 1 and its derivatives
(a = imidazole/ ; b = 4-substituted phenyl sulfonyl chloride/DCM/ ; R = various
substituents, e.g., CH3, C2H5, C3H7, C4H9, C5H11, CF3, F and Ph).
D
D
Initial consideration of the IC50 values show that compounds 4
to 11 are more potent than KTZ (which was found to possess an
IC50 value of 2660 nM and 206 nM against the 17a-OHase and
potency against the lyase component in comparison to the 17a-
OHase component; this was also observed with our own standard
lyase components, respectively). Furthermore, with the exception
(compound 3) which was found to possess an IC50 value of 730 nM
of compound 8, all the compounds appear to possess greater
and 164 nM against the 17a-OHase and lyase components, respec-
Table 1
IC50 and log P values for compounds under consideration within the current study (log P was calculated using Project Leader from Fujitsu)
N
N
R
Compound number
R
IC50 (nM) against lyase
IC50 (nM) against 17
a
-OHase
Calculated log P
1
2
OH
H
1470
>3000
1
2496 300
>3000
1.03
—
3
I
164 50
730 80
—
4
5
6
7
8
9
10
11
KTZ
CH3–C6H4SO2O–
C2H5–C6H4SO2O–
C3H7–C6H4SO2O–
C4H9–C6H4SO2O–
C5H11–C6H4SO2O–
CF3–C6H4SO2O–
F–C6H4SO2O–
Ph–C6H4SO2O–
—
30
20
30
40 0.1
160 14
7
4
1
40
110
8
1
2.50
2.90
3.30
3.69
4.09
2.92
2.18
3.72
3.18
1210 20
70
130 30
1040 61
200 10
9
34
5
14 0.9
10 0.04
206 33
100
2660 10
1

C. P. Owen et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5345–5348
5347
tively. It should be noted that the previously reported values for
these compounds appear to be inconsistent with the current study,
however, this would be expected as different rat testicular tissues
was utilised in the previous study when compared to the current
study, as such, consistent data would not be expected.
component) was then determined using 17
lone as the substrate and the structure again minimised. The haem
and the two hydrogen bonding groups at the active site were then
a
-hydroxypregneno-
locked and 17a-hydroxypregnenolone removed to give the overall
substrate–haem complex for P45017 . That the active site consists
a
The most potent compounds within the current series of com-
pounds were therefore 4, 5, 6, 9, 10 and 11, with compound 6 pos-
sessing the best selectivity towards the lyase reaction in
comparison to the hydroxylase reaction, indeed, this compound
of two lobes where the two substrates for this enzyme bind to (at
different times) was previously proposed by Laughton et al.19 who
found that the two ‘lobes’ were orientated in such a manner so as
to approximate to the L-shape with the lyase binding site being lar-
was found to possess an IC50 value of 1210 nM against the 17
a
-
ger than the 17a-OHase binding site. The substrate–haem complex
OHase component and 30 nM against the lyase component. Com-
pound 11 showed the most potent inhibitory activity with an IC50
value of 100 nM against the 17a-OHase component and 10nM
approach also shows the lyase binding site to be the larger of the
two and that the overall orientation of the two hydrogen bonding
groups appears to be of a similar orientation as reported by Laugh-
ton et al. (with respect to the haem).
against the lyase component. In comparison, KTZ was found to pos-
sess good selectivity but weaker inhibitory activity in comparison to
compounds 3 to 11.
A detailed consideration of the IC50 values shows that com-
pounds 4 to 8 possess potent inhibitory activity against the lyase
component with an optimum activity being possessed by the ethyl
(compound 5) and phenyl (compound 11) derivatives, whereas the
butyl and the pentyl derivatives appear to possess weaker inhibi-
tory activity.
Inhibitors of P45017 can also utilise these two hydrogen bond-
a
ing groups and we have previously reported the use of both groups
by inhibitors (such as 3,5-dichlorobenzyl imidazole6) which re-
sulted in the di-substituted compounds possessing potent inhibi-
tory activity in comparison to mono-halogenated compounds.
Modelling of compound 8 using the substrate–haem complex ap-
proach shows that this compound is able to utilise either of the
two potential hydrogen bonding groups, albeit one at a time
(Fig. 3). However, from the consideration of the conformational
analysis of the alkyl moiety within 8 shows that conformers exist
which allow the alkyl chain to undergo steric interaction with
the hydrogen bonding group at the active site. We therefore pro-
pose that the increase in the steric interactions results in an overall
reduction in inhibitory activity when compared to compounds
containing smaller alkyl chains, for example, compound 7.
Since no crystal structure exists for P45017 , we developed a no-
a
vel technique to model this enzyme using the substrate–haem
complex approach. The methodology for the derivation of the sub-
strate–haem complex has been previously described,16–18 as such,
no detailed discussion is given here. In general, however, the deter-
mination of the overall representation of the P45017 active site
a
initially involved the derivation of the binding site corresponding
to the 17
a
-OHase component using pregnenolone bound to the
Our hypothesis is further supported by the inhibitory activity
observed within compounds 9 and 10, which contain CF3 and F
as the substituents, respectively, rather than the alkyl chain. These
two compounds are hypothesised to occupy reduced conforma-
tional space, as such, they do not undergo the level steric interac-
tions observed within compounds 5 to 8. Modelling of compound 9
haem moiety—the substrate was allowed to minimise to give the
approximate position of the hydrogen bonding group which would
be expected to bind to the C(3) moiety within the substrate. The
structure was ‘locked’ after the removal of the pregnenolone and
the location of the second binding site (corresponding to the lyase
Figure 3. Conformer of compound 8 bound to the haem moiety within the overall substrate–haem complex representation of P45017 showing the potential steric
a
interaction between the pentyl moiety and the hydrogen bonding group at the active site.

5348
C. P. Owen et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5345–5348
Figure 4. Compound 11 bound to the haem moiety within the overall substrate–haem complex representation of P45017
.
a
within the overall enzyme complex shows that the CF3 moiety is
far removed from the second hydrogen bonding group at the active
site to undergo any steric interaction, indeed, the nearest atom is
5.7 Å away from the hydrogen bonding group. Modelling the more
bulky derivative of 9 (more specifically compound 11, Fig. 4) with-
in the substrate–haem complex shows that as a result of the
greatly reduced conformational flexibility of this inhibitor, the
additional phenyl moiety is unable to undergo any steric interac-
tion with the active site and therefore possesses greater inhibitory
activity in comparison to 9.
References and notes
1. Ortiz de Montellano, P. R. In Cytochrome P-450: Structure Mechanism and
Biochemistry; Ortiz de Montellano, P. R., Ed.; Plenum Press: New York, 1986; pp
217–272.
2. Ahmed, S.; Owen, C. P. Bioorg. Med. Chem. Lett. 1998, 8, 1023.
3. Robichaud, P.; Wright, J. N.; Akhtar, M. J. Chem. Soc., Chem. Commun. 1994, 12,
1501.
4. Nakabayashi, M.; Xie, W.; Regan, M. M.; Jackman, D. M.; Kantoff, P. W.; Oh, W.
K. Cancer 2006, 107, 975.
5. Ahmed, S.; Smith, H. J.; Nicholls, P. J.; Whomsley, R.; Cariuk, P. Drug Des.
Discovery 1995, 13, 27.
6. Owen, C. P.; Dhanani, S.; Patel, C. H.; Shahid, I.; Ahmed, S. Bioorg. Med. Chem.
Lett. 2006, 16, 4011.
In conclusion, we have produced some highly potent inhibitors
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.
of P45017 in comparison to the standard compound KTZ. Also, due
to the limited specificity of these compounds against lyase in com-
parison to the 17a-OHase component, these compounds would be
a
expected to have a major impact on corticosteroid biosynthesis.
Furthermore, through the consideration of the modelling of these
inhibitors, we have provided an insight into the conformational
space available about the area of the active site. Although the com-
pounds have shown high levels of potency, the biochemical evalu-
ation of similar benzyl imidazole-based compounds suggests that
the use of these compounds as potential drug substances is limited
due to their ability to inhibit other cytochrome P450 enzymes such
as cholesterol side chain cleavage5 or indeed aromatase,5,20 and
which therefore suggests a lack of selectivity.
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. Ahmed, S.; Shahid, I.; Dhanani, S.; Owen, C. P. Bioorg. Med. Chem. Lett. 2009, 19,
4698.
11. Hu, Q.; Negri, M.; Jahn-Hoffmann, K.; Zhuang, Y.; Olgen, S.; Bartels, M.; Müller-
Vieira, U.; Lauterbach, T.; Hartmann, R. W. Bioorg. Med. Chem. 2008, 16, 7715.
12. Zhuang, Y.; Wachall, B. G.; Hartmann, R. W. Bioorg. Med. Chem. 2000, 8, 1245.
13. Hille, U. E.; Hu, Q.; Vock, C.; Negri, M.; Bartels, M.; Muller-Vieira, U.;
Lauterbach, T.; Hartmann, R. W. Eur. J. Med. Chem. 2009, 44, 2765.
14. Owen, C. P.; Nicholls, P. J.; Smith, H. J.; Whomsley, R. J. Pharm. Pharmacol. 1999,
51, 427.
15. Njar, V. C. O.; Brodie, A. M. H. Curr. Pharm. Des. 1999, 5, 163.
16. Ahmed, S. Drug Des. Discovery 1994, 12, 77.
17. Ahmed, S. Biochem. Biophys. Res. Commun. 2004, 316, 595.
18. Ahmed, S. Bioorg. Med. Chem. Lett. 1995, 5, 2795.
19. Laughton, C. A.; Neidle, S.; Zvelebil, M. J. J. M.; Sternberg, M. J. E. A. Biochem.
Biophys. Res. Commun. 1990, 171, 1160.
Acknowledgements
20. Adat, S.; Owen, C. P.; Ahmed, S. Lett. Drug Des. Discovery 2007, 4, 545.
The authors thank the Mass Spectrometry service at the Kings
College London (UK) and the elemental analysis service at the
School of Pharmacy, University of London (UK) for the provision
of high resolution and elemental analysis data, respectively.