Synthesis, biochemical evaluation and rationalisation of the inhibitory activity of a series of 4-hydroxyphenyl ketones as potential inhibitors of 17β-hydroxysteroid dehydrogenase type 3 (17β-HSD3)

Article abstract of DOI:10.1016/j.bmcl.2006.06.029

We report the preliminary results of the synthesis and biochemical evaluation of a number of 4-hydroxyphenyl ketones as inhibitors of the isozyme of the enzyme 17β-hydroxysteroid dehydrogenase (17β-HSD) responsible for the conversion of androstenedione (AD) to testosterone (T), more specifically type 3 (17β-HSD3). The results of our study suggest that we have synthesised compounds which are, in general, potent inhibitors of 17β-HSD3, in particular, we discovered that 1-(4-hydroxy-phenyl)-nonan-1-one (8) was the most potent (IC₅₀ = 2.86 ± 0.03 μM). We have therefore provided good lead compounds in the synthesis of novel non-steroidal inhibitors of 17β-HSD3.

Full text of DOI:10.1016/j.bmcl.2006.06.029

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4519–4522
Synthesis, biochemical evaluation and rationalisation
of the inhibitory activity of a series of 4-hydroxyphenyl
ketones as potential inhibitors of 17b-hydroxysteroid
dehydrogenase type 3 (17b-HSD3)
Rupinder K. Lota,a Sachin Dhanani,b Caroline P. Owena,* 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 22 May 2006; revised 8 June 2006; accepted 8 June 2006
Available online 22 June 2006
Abstract—We report the preliminary results of the synthesis and biochemical evaluation of a number of 4-hydroxyphenyl ketones as
inhibitors of the isozyme of the enzyme 17b-hydroxysteroid dehydrogenase (17b-HSD) responsible for the conversion of androstene-
dione (AD) to testosterone (T), more specifically type 3 (17b-HSD3). The results of our study suggest that we have synthesised com-
pounds which are, in general, potent inhibitors of 17b-HSD3, in particular, we discovered that 1-(4-hydroxy-phenyl)-nonan-1-one
(8) was the most potent (IC50 = 2.86 0.03 lM). We have therefore provided good lead compounds in the synthesis of novel non-
steroidal inhibitors of 17b-HSD3.
ꢀ 2006 Elsevier Ltd. All rights reserved.
The biosynthesis of testosterone (T) [the precursor to
dihydrotestosterone (DHT), the most potent androgen]
involves the reduction of the C(17) carbonyl moiety
within androstenedione (AD) and is catalysed by the
type 3 isozyme of the enzyme 17b-hydroxysteroid dehy-
drogenase (17b-HSD3) (Fig. 1).
the targeting of isozymes of this enzyme in the treatment
of hormone-dependent cancers.1 In an effort to aid the
design of novel inhibitors of this enzyme, we have previ-
ously undertaken the derivation of the transition-states
(TS) of the reduction reaction catalysed by 17b-
HSD1.2 We concluded, from our study, that the carbon-
yl moiety was an important feature of any potential
inhibitor of the numerous isozymes of 17b-HSD in-
volved in the reduction (and therefore a good mimic)
of the steroid C(17)@O moiety within the natural sub-
strate. In an effort to evaluate our hypothesis, we
undertook the design of a number of compounds as
potential inhibitors, as such, we report here the initial
results of the synthesis of a range of straight alkyl chain
containing 4-hydroxyphenyl ketones and their
This enzyme has now become a potential biochemical
target1 in the fight against hormone-dependent prostate
(as well as breast) cancer, the rationale being that the
reduction of testosterone levels would result in a subse-
quent reduction in DHT, thereby leading to a loss of
stimulation of the prostate cancer cells. Several isozymes
of 17b-HSD are known to exist and are responsible for a
number of redox reactions, including the conversion of
the weak C@O containing sex steroids to the more mito-
genic 17b-hydroxy containing steroids [e.g., AD to T
catalysed by type 3 and conversion of estrone (E1) to
estradiol (E2) catalysed by type 1 (17b-HSD1)], hence
Keywords: 17b-hydroxysteroid dehydrogenase; Type 3; Inhibitors;
Androstenedione; Testosterone.
*
Corresponding authors. Tel.: +44 20 8547 2000; fax: +44 20 8547
7562 (S.A.); e-mail: S.AHMED@KINGSTON.AC.UK
Figure 1. Conversion of AD to T catalysed by 17b-HSD type 3.
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.06.029

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R. K. Lota et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4519–4522
subsequent biochemical evaluation against rat testicular
microsomal enzyme against 17b-HSD3 using radiola-
belled AD as the substrate.
the compounds synthesised, only two are found to pos-
sess poor IC50 values. Detailed consideration of
the inhibitory activity shows that compounds
8
(IC50 = 2.86 lM) and 9 (IC50 = 4.97 lM) are the most
potent compounds within the current study. As such,
8 is approximately 65 and 23 times more potent than
the two previously reported inhibitors of 17b-HSD3,
namely Baicalein (IC50 = 185.92 lM) and 7-hydroxy-
flavone (IC50 = 66.98 lM), respectively. Compounds 6
In the synthesis of the 4-hydroxyphenyl ketones consid-
ered within the current study, we utilised the reaction
outlined in Scheme 1, a reaction which has been utilised
extensively by us in the synthesis of the corresponding
sulfamate derivatives as potent inhibitors of the enzyme
estrone sulfatase.3,4 In general, the reaction involved
Friedel–Crafts acylation of phenol using the appropriate
acyl chloride in the presence of anhydrous aluminium
chloride (AlCl3) and using anhydrous dichloromethane
(DCM) as the reaction solvent. The reactions proceeded
in relatively good yield (between a range of ꢀ24% and
ꢀ56%) and without any major problems; the syntheses
of 1-(4-hydroxy-phenyl)-ethanone5 (1) and 1-(4-hy-
droxy-phenyl)-propan-1-one6 (2) are given as examples.
(IC50 = 7.84 lM), 7 (IC50 = 6.52 lM) and 10 (IC50
=
7.55 lM) are also found to possess potent inhibitory
activity in comparison to the two standard compounds
(although they are all approximately 2–3 times less
potent than 8 and may be considered to be equipotent
to 9).
From the consideration of the biological data, we initial-
ly observe that the potency of the inhibitors appears to
increase with increasing alkyl chain length and upon
consideration of the physicochemical factors for the
novel inhibitors, we observe a good correlation between
the logarithm of the calculated partition coefficient
(logP) (calculated using Quantum CaChe Project
Leader10) and IC50 (Fig. 2), resulting in an optimum
logP of approximately 4.1, corresponding to compound
8. We therefore suggest that hydrophobicity may be an
important physicochemical factor in determining the
overall inhibitory activity in the inhibition of 17b-
HSD3.
Table 1 shows the results of the initial screening, using a
modified literature-based assay,7,8 as well as the IC50
values for the compounds synthesised within the current
study. Due to the lack of compounds in the clinic for
this biochemical target, it was not possible to compare
the biological activity (and therefore relative potency)
of the synthesised compounds within the current study
against a single standard inhibitor. However, as the fla-
vonoid-based compounds have been previously evaluat-
ed and found to possess good inhibitory activity against
17b-HSD3, in particular, Baicalein and 7-hydroxyflav-
one,9 we have therefore used these for comparison.
Consideration of the steroid backbone suggests that
there are two modes of superimpositioning the inhibi-
tors such that the carbonyl moiety within the inhibitor
mimics the C(17)@O of the substrate: one, whereby
the alkyl chain extends towards the area of space nor-
mally occupied by the rings A, B and C of the steroid
substrate, placing the 4-hydroxyphenyl moiety beyond
the C(15) and C(16) position of the steroid backbone.
The alternative mode of superimpositioning involves
the 4-hydroxyphenyl being positioned towards the ste-
roid backbone whilst the alkyl chain is now positioned
such that it extends far beyond the D-ring. In a
previous study, the area of the enzyme active site cor-
responding to the D-ring of the natural substrate has
been shown to be populated with hydrogen donor and
bonding groups as well as NADPH as the reducing
agent (in the conversion of E1 to E2) and is therefore
In general, consideration of the biological activity
obtained shows that the 4-hydroxyphenyl ketone-based
compounds synthesised within the current study possess
potent inhibitory activity against 17b-HSD3. Indeed of
O
R1
a
HO
HO
Scheme 1. Synthesis of the 4-hydroxyphenyl ketones as potential
inhibitors of 17b-HSD. Reagents: (a) acid chloride/AlCl3/DCM;
R1 = C1–C11
.
Table 1. Inhibitory data obtained for the 4-hydroxyphenyl ketones
synthesised within the present study as inhibitors of 17b-HSD3
20
18
16
Compound
R
% inhibition
[I] = 100 lM
IC50 value (lM)
y = 8.8201x2 - 73.077x + 154.67
R2 = 0.95
Baicalein
7-Hydroxyflavone
—
—
NQ
NQ
185.92 12.70
66.98 0.95
1708.92 170.71
150.56 12.21
89.51 6.73
60.52 5.83
18.02 0.96
7.84 0.36
14
12
10
8
6
4
1
2
CH3
36.59 0.52
53.03 2.18
60.18 0.77
61.81 0.89
76.40 0.18
80.26 0.20
82.58 0.49
83.53 0.48
81.39 0.09
78.92 0.58
C2H5
C3H7
C4H9
C5H11
C6H13
C7H15
C8H17
C9H19
3
4
5
2
0
6
7
6.52 0.18
2.86 0.03
2.8
3.3
3.8
logP
4.3
4.8
8
9
10
4.97 0.25
7.55 0.32
C
11H23
Figure 2. Plot of IC50 versus calculated logP for a small range of the
compounds (from 5 to 10) synthesised within the current study.
NQ, not quoted.

R. K. Lota et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4519–4522
4521
constrained in the volume of space available for the
large acyl chains.2 As such, binding in such a manner
where the alkyl chain extends out beyond the D-ring
is unlikely due to the increased unfavourable steric
interactions which would therefore lead to reduced
inhibitory activity—this is contrary to what is
observed experimentally.
We therefore undertook a molecular modelling study
involving the determination of low energy conformers
and the subsequent superimpositioning of the conform-
ers of the most potent compound (namely 8) onto the
backbone of AD (such that the alkyl chain mimicked
the backbone of AD). We discovered that numerous
low energy conformers of compound 8 exist, however,
one low energy conformer was found which allows the
alkyl chain to be positioned over the steroid backbone
without a major change in energy when compared to
the global minima (DE = 0.82 kcal/mol). Occupying the
same volume of space as the steroid backbone of the
substrate AD (Fig. 3) allows the alkyl chain to utilise
any available hydrophobic interactions. We therefore
propose that the potent inhibitory activity observed
within compound 8 is not only due to the increased logP
of this compound but also due to the ability of the alkyl
chain to mimic the steroid backbone and not undergo
any unfavourable steric interactions.
Figure 4. Superimpositioning of a low energy conformer of 10 (in
green) onto the backbone of AD.
From the molecular modelling study, we also suggest
that the 4-hydroxyphenyl moiety (especially within the
larger chain containing compounds) is involved in
hydrogen bonding interactions with the active site about
the C(15) and C(16) area of the steroid backbone. Our
hypothesis may be rationalised by the large differences
in inhibitory activity observed with increasing alkyl
chain length within the synthesised compounds (e.g.,
compound 1 is found to have an IC50 value of
1708.92 170.71 lM, whereas compounds 6 and 8 are
found to possess IC50 values of 7.84 0.36 and
2.86 0.03 lM, respectively). We hypothesise therefore
that within the small alkyl chain containing compounds,
these inhibitors may bind in either of the two modes sug-
gested above. That is, as well as binding such that the 4-
hydroxyphenyl moiety is able to hydrogen bond with
groups about the C(15) and C(16), compounds 1–4
may also bind in an alternative manner such that the
acetyl moiety is positioned about the C(15) and C(16)
position of the steroid backbone resulting in decreased
interaction between the inhibitor and the enzyme lead-
ing to decreased potency (Fig. 5 shows the superimposi-
tioning of compound 2 such that the alkyl chain
occupies an area about the C(15) and C(16) area of
the androgen backbone). As a result of the two available
modes of binding, therefore, we propose that the inhib-
itor is not able to bind in an effective manner (due to de-
creased hydrogen bonding) and as a result possesses a
much greater IC50 value than would be expected in com-
parison to the other derivatives. That is, differences in
logP cannot be utilised in rationalising the large differ-
ence in potency between the small and larger com-
pounds, and we suggest that this difference is also due
to the ineffective binding of these smaller compounds
to the 17b-HSD3 active site.
That our hypothesis may have some validity can be ob-
served when the larger alkyl chain containing com-
pounds, for example 10, are superimposed onto the
steroid backbone. Detailed conformational analysis of
compound 10 resulted in the discovery of numerous
conformers and from the superimpositioning of the con-
formers onto the backbone of AD, we observed that a
conformer exists which allows the C11 containing alkyl
chain to approach the area of the active site (and there-
fore the appropriate hydrogen bonding group) which
would normally interact with the C(3)@O moiety of
the substrate (Fig. 4). We therefore suggest that an in-
crease in alkyl chain length (beyond the C8 or C9 found
in compounds 8 and 9) would result in the longer alkyl
chain containing inhibitors undergoing steric interaction
with the hydrogen bonding group at the active site cor-
responding to the C(3)@O moiety, thereby reducing the
inhibitory activity of the larger alkyl chain containing
compounds.
Figure 3. Superimpositioning of a low energy conformer of 8 (in green)
onto the backbone of AD.
Figure 5. Superimpositioning of 2 (in green) onto the backbone of AD.

4522
R. K. Lota et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4519–4522
phy of the crude solid gave 1 as a white solid (0.68 g, 47%
yield) [mp 109.8–110.1 ꢁC; Rf = 0.26 diethyl ether/petro-
leum ether 40–60 ꢁC (30:70); lit. mp 110.2–110.4 ꢁC]. m(max)
(film) cmÀ1: 3321.93 (OH), 1662.55 (C@O), 1603.51 (Ar
C@C); dH (CDCl3): 7.89 (2H, d, J = 8.79 Hz, Ph-H), 6.91
(2H, d, J = 8.97 Hz, Ph-H), 2.56 (3H, s, CH3); dC (CDCl3):
198.59 (C@O), 160.74, 131.19, 129.55, 115.51 (Ar C),
26.29 (CH3); GC: tR 6.06 min; LRMS (EI): 136 (M+,
30%), 121 (M+ÀCH3, 100%); Elemental analysis, Found:
C, 70.42%; H, 5.88%; C8H8O2 requires C, 70.58%; H,
5.92%.
As previously mentioned, due to the extensive size of the
octyl chain within compound 8 (or indeed any com-
pound possessing an alkyl chain greater than C4), it is
only able to bind such that the alkyl chain mimics the
steroid backbone, with the 4-hydroxyphenyl moiety able
to undergo hydrogen bonding with the active site, lead-
ing to increased inhibitory activity. The ability of the
compounds to undergo this favourable hydrogen bond-
ing interaction is therefore another reason for the potent
inhibitory activity observed within the larger inhibitors
of 17b-HSD3. In an effort to validate our hypothesis,
we undertook the biochemical evaluation of a range of
inhibitors (not reported here) which contained various
substituents (in particular groups which lacked any
hydrogen bonding groups) in place of the 4-hydroxy
moiety and discovered that the compounds were either
weak or non-inhibitors of this enzyme.
6. 1-(4-Hydroxy-phenyl)-propan-1-one (2): Compound 2 was
synthesised in a similar manner to 1 except that propanoyl
chloride (1.02 mL, 11.7 mmol) was used in place of acetyl
chloride. The crude solid was purified via flash chroma-
tography to give 2 as an off-white solid (0.84 g, 53% yield)
[mp 158.2–158.6 ꢁC; Rf = 0.32 diethyl ether/petroleum
ether 40–60 ꢁC (30:70); lit. mp 152–153 ꢁC]. m(max) (film)
cmÀ1: 3222.83 (OH), 1650.16 (C@O), 1605.78 (Ar C@C);
dH (CDCl3): 7.85 (2H, d, J = 8.42 Hz, Ph-H), 6.81 (2H, d,
J = 8.42 Hz, Ph-H), 2.89 (2H, q, JAB = 7.32 Hz,
JAB = 7.14 Hz, O@C–CH2), 1.15 (3H, t, JAB = 7.14 Hz,
JAB = 7.32 Hz, CH2CH3); dC (CDCl3): 198.96 (C@O),
152.58, 130.56, 115.26 (Ar C), 31.39 (CH2CH3), 8.38
(CH3); GC: tR 6.81 min; LRMS (EI): 150 (M+, 9%), 121
(M+ÀC2H5, 100%); Elemental analysis, Found: C,
71.84%; H, 6.71%; C9H10O2 requires C, 71.98%, H, 6.71%.
7. Le Lain, R.; Nicholls, P. J.; Smith, H. J.; Maharlouie, F.
H. J. Enzyme Inhib. 2001, 16, 35.
8. Preliminary screening and IC50 determinations of com-
pounds: All incubations were carried out in triplicate at
37 ꢁC in a shaking water bath. Incubation mixtures
(1 mL), containing NADPH generating system (50 lL),
inhibitor (varying concentration, 20 lL) and substrate
(1.5 lM final concentration, 15 lL), in phosphate buffer
(pH 7.4, 905 lL), were allowed to warm to 37 ꢁC. The rat
testicular microsomes were thawed and warmed to 37 ꢁC
before addition of the enzyme (0.097 mg/mL final con-
centration, 10 lL) to the assay mixture. The solutions were
incubated for 30 min at 37 ꢁC and the reaction was
quenched by the addition of ether (2 mL). The solutions
were vortexed, then left to stand over ice for 15 min. The
assay mixture was extracted with further aliquots of ether
(2· 2 mL) and organic layers combined into a clean tube
before the solvent was evaporated. Acetone (30 lL) was
added to each tube and vortexed thoroughly. Aliquots,
along with steroid carriers (A and T, 5 mg/mL, approx-
imately 10 lL), were spotted onto TLC plates and run,
using a mobile phase consisting of dichloromethane
(70 mL) and ethyl acetate (30 mL). After development,
the separated steroids were identified, using an UV lamp,
cut from the plate and placed into scintillation vials.
Acetone (1 mL) was added to each vial in order to dissolve
the steroid from the silica plate and then scintillation fluid
(Optiscint HiSafe) (3 mL) was added. The samples were
vortexed and read for tritium for 4 min per tube. In
determining the IC50 values for the compounds studied
within the current study, 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].
In conclusion, from the consideration of the inhibitory
activity of the 4-hydroxyphenyl ketones synthesised with-
in the current study, we have first produced two highly
potent inhibitors (compounds 8 and 9) of 17b-HSD3.
Furthermore, from the molecular modelling study, we
have proposed a probable mode of action of these com-
pounds and have therefore rationalised the structure–
activity relationship observed within the current range
of compounds. We have also suggested a structural mod-
ification which may allow non-steroidal inhibitors of this
enzyme to possess greater inhibitory activity.
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.
References and notes
1. Poirier, D. Curr. Med. Chem. 2003, 10, 453.
2. Owen, C. P.; Ahmed, S. Biochem. Biophys. Res. Commun.
2004, 318, 131.
3. Ahmed, S.; Owen, C.; James, K.; Patel, C. K.; Patel, M.
Bioorg. Med. Chem. Lett. 2001, 11, 2525.
4. Ahmed, S.; James, K.; Owen, C. P.; Patel, C. K.; Patel, M.
J. Steroid Biochem. Mol. Biol. 2002, 80, 419.
5. 1-(4-Hydroxy-phenyl)-ethanone (1): AlCl3 (1.50 g,
21 mmol) was added to a solution of phenol (0.50 g,
10.6 mmol) in anhydrous DCM (10 mL). The slurry was
left stirring for 30 min before acetyl chloride (0.83 mL,
11.7 mmol) was added in a dropwise manner. The solution
was then left to stir for 14 h. The reaction was quenched
using ice-cold solution of hydrochloric acid (HCl) (1 M,
30 mL) and then extracted into diethyl ether (2· 50 mL).
The combined organic layer was extracted into sodium
hydroxide (NaOH) (2 M, 2· 50 mL) and then acidified to
pH 2 using HCl (1 M). The product was extracted into
diethyl ether (2· 50 mL) and the organic layer was washed
with water (2· 50 mL) and dried over anhydrous magne-
sium sulfate (MgSO4), filtered and the solvent removed
under vacuum to give a brown solid. Flash chromatogra-
9. Le Lain, R.; Nicholls, P. J.; Smith, H. J.; Maharlouie, F.
H. J. Pharm. Pharmacol. 1999, 51(Suppl.), 23.
10. Quantum Cache Project Leader is a trademark of Oxford
Molecular Ltd (Fujitsu), Oxford Science Park, Oxford,
UK.