New cyclopentane derivatives as inhibitors of steroid metabolizing enzymes AKR1C1 and AKR1C3

Article abstract of DOI:10.1016/j.ejmech.2009.01.028

A series of cyclopentane derivatives was synthesized and evaluated for inhibition of the steroid metabolizing enzymes AKR1C1 and AKR1C3. Selective inhibitors that are active in the low micromolar range were identified. These compounds represent promising starting points in the development of new anticancer agents for the treatment of hormone-dependent forms of cancer and other diseases where AKR1C1 and AKR1C3 are involved.

Full text of DOI:10.1016/j.ejmech.2009.01.028

European Journal of Medicinal Chemistry 44 (2009) 2563–2571
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
New cyclopentane derivatives as inhibitors of steroid metabolizing enzymes
AKR1C1 and AKR1C3
a
,
b
c
b
c,
**
ˇ
*
ꢀ
ꢀ
ˇ
ˇ
Bogdan Stefane , Petra Brozic , Matej Vehovc , Tea Lanisnik Rizner , Stanislav Gobec
a
ˇ
ˇ
Faculty of Chemistry and Chemical Technology, University of Ljubljana, Askerceva 5, 1000 Ljubljana, Slovenia
b Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia
c
ˇ
ˇ
Faculty of Pharmacy, University of Ljubljana, Askerceva 7, 1000 Ljubljana, Slovenia
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of cyclopentane derivatives was synthesized and evaluated for inhibition of the steroid
metabolizing enzymes AKR1C1 and AKR1C3. Selective inhibitors that are active in the low micromolar
range were identified. These compounds represent promising starting points in the development of new
anticancer agents for the treatment of hormone-dependent forms of cancer and other diseases where
AKR1C1 and AKR1C3 are involved.
Received 18 September 2007
Received in revised form
4 January 2009
Accepted 24 January 2009
Available online 5 February 2009
Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords:
AKR1C1 inhibitors
AKR1C3 inhibitors
Anticancer agents
Cancer chemoprevention
Cyclopentane derivatives
1. Introduction
for the treatment of endometrial cancers, premenstrual syndrome,
catamenial epilepsy, and depressive disorders, and for the main-
Hydroxysteroid dehydrogenases (HSDs) are crucial enzymes
that are involved in both the synthesis and inactivation of all classes
of steroid hormones. HSDs belong to two distinct protein super-
families: the short-chain dehydrogenase/reductase (SDR) [1,2]
superfamily and the aldo–keto reductase (AKR) superfamily [3].
Human AKR HSDs belong to the AKR1C subfamily and regulate the
concentrations of active and inactive androgens, estrogens and
progestins in target tissues, and thus regulate ligand occupancy and
trans-activation of the nuclear steroid hormone receptors [4–6].
tenance of pregnancy [9,10].
AKR1C3 is a peripheral 17
androgen androstenedione into the potent androgen testosterone,
and the weak estrogen estrone into the potent estrogen
b
-HSD (type 5) that reduces the weak
17b-estradiol (Fig. 1) [11]. AKR1C3 is thus an interesting target for
the development of agents for treating hormone-dependent forms
of cancer, such as prostate, breast and endometrial cancers. AKR1C3
also catalyzes the reduction of 3-ketosteroids, 20-ketosteroids and
prostaglandin D2 (PGD2), so it has been known variously as 3
type 2 [12] and PGD2 11-ketoreductase [13]. By converting PGD2
into PGF2a, AKR1C3 prevents the conversion of PGD2 into 15-D12,14
PGJ2, which is a natural ligand for the peroxisome-proliferator-
activated receptor- (PPAR ). Inhibitors of AKR1C3 are thus
potential antineoplastic agents also, because they can indirectly
activate the PPAR receptor by diverting PGD2 catabolism to the
generation of J-series prostanoids. Activation of the PPAR receptor
a-HSD
AKR1C1 acts preferentially as
a
20a-HSD and inactivates
progesterone by converting it to 20
a
-hydroxyprogesterone (20
a
-
-
OHP; Fig. 1), which has a low affinity for progesterone receptors [6].
AKR1C1 converts 3 ,5 -tetrahydroprogesterone (5 -THP), an allo-
steric modulator of the -aminobutyric acid (GABA)A receptor into
the inactive 5 -pregnane-3 ,20 -diol. It thereby exhibits anaes-
a
a
a
g
g
g
a
a
a
g
thetic, analgesic, anxiolytic and anticonvulsant effects [6–8].
Inhibitors of AKR1C1 are thus very interesting as potential agents
g
induces differentiation, is anti-proliferative, and causes apoptosis in
many cell types and cancers [14,15].
Although both AKR1C1 and AKR1C3 are promising therapeutic
targets, only a few inhibitors have been reported to date. Dietary
phytoestrogens were shown to inhibit both of these enzymes in low
micromolar concentrations [16,17], as were some other small mole-
cule compounds, including benzodiazepines [18], benzofuranes and
* Corresponding author. Tel.: þ386 1 24 19 264; fax: þ386 1 24 19 220.
** Corresponding author. Tel.: þ386 1 47 69 585; fax: þ386 1 42 58 031.
ˇ
E-mail addresses: bogdan.stefane@fkkt.uni-lj.si (B. Stefane), gobecs@ffa.uni-lj.si
(S. Gobec).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.01.028

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B. Stefane et al. / European Journal of Medicinal Chemistry 44 (2009) 2563–2571
2564
were synthesized according to Scheme 1. 2-(4-Chlorobenzy-
lidene)cyclopentanone (2) was obtained by base-promoted aldol
condensation of cyclopentanone and 4-chlorobenzaldehyde
O
OH
1
(Scheme 1). Further, reductions under Luche conditions [29] were
carried out at room temperature in methanol or ethanol, giving
the allylic ethers 3 and 4, respectively. Treatment of 1 with LDA at
ꢀ5 ꢁC with the subsequent addition of benzyl bromide resulted in
the formation of a mixture of benzyl-substituted cyclopentanones
(5–8) that were partially separated by column chromatography
(SiO2). An inseparable mixture of isomers 6 and 7 (formed in the
ratio 5:1) was taken directly into the next step. Benzylation of 1
was followed by reduction under Luche conditions, producing the
desired cyclopentanol derivatives 9–11 in reasonable yields
(Scheme 1).
AKR1C1
AKR1C1
O
O
20α-Hydroxyprogesterone
Progesterone
O
OH
The common precursors of several substrates were aziridine 12,
which was easily accessible by Sharpless aziridination [30], and the
epoxy derivative 13 (Scheme 2). Tosylamino functionalised cyclo-
pentane derivatives 14 and 15 were prepared from 12 via nucleo-
philic aziridine ring opening, providing these products at good
yields [31]. The selective hydrolysis of 14 resulted in the formation
of the corresponding amide 16 and carboxylic acid 17 [32] (Scheme
2). Treatment of the mixture of the epoxide 13 and aniline with
equimolar amounts of ZrCl4 proved to be an efficient method for
producing the desired amino alcohol 18 at good yield [33].
However, use of BiCl3 [34] as a Lewis acid was much less efficient
for this epoxide ring opening, giving 18 only at a 53% yield.
The preparation of 3-phenylcyclopentanecarboxylic acid (20) is
presented in Scheme 3 and it is based on peroxyselenium-
promoted 4-phenylcyclohexanone (19) ring contraction [35],
deriving the desired 20 at moderate yields.
HO
HO
H
H
3α,5α-Tetrahydroprogesterone
5α-Pregnane-3α,20α-diol
O
OH
AKR1C3
O
O
Androstenedione
Testosterone
OH
O
AKR1C3
AKR1C3
3. Results and discussion
HO
HO
Estrone
OH
Estradiol
OH
In our search for new inhibitors of the AKR1C1 and AKR1C3
isoforms as potential anticancer agents, we decided to investigate
only compounds that consist of a non-steroid core, and are thus
devoid of residual steroidogenic activity. Since one of the substrates
of AKR1C3 is a cyclopentane-containing PGD2 (Fig. 1), we focused
our attention on derivatives of cyclopentane. Preliminary docking
experiments revealed that correctly substituted cyclopentanes
should bind to the active sites of AKR1C1 and AKR1C3 and inhibit
their enzymatic activities. To confirm this hypothesis, a series of
cyclopentane derivatives was synthesized and evaluated for inhi-
bition of AKR1C1 and AKR1C3.
HO
HO
HO
O
COOH
COOH
Prostaglandin D2
9α,11β-Prostaglandin F2α
Fig. 1. Reactions catalyzed by AKR1C1 and AKR1C3 in vivo.
phenolphthalein derivatives [12]. Indomethacin, flufenamic acid,
N-phenylanthranilic acid derivatives and some related non-steroidal
anti-inflammatory agents are very potent inhibitors [13,19–21].
Steroid carboxylates [19], benzoyl benzoic acids [21], 3-phenox-
ybenzoic acid [21] and cinnamic acids [22] have also been reported to
inhibit AKR1C3. Recently, structurally diverse inhibitors of AKR1C1
were also discovered by two virtual high-throughput screenings
[23,24] and synthetic efforts [25]. The X-ray crystal structures of
Most of the compounds synthesized in the present study were
evaluated for their inhibitory activities against recombinant human
AKR1C1 and AKR1C3 (Table 1). Of the 4-chlorobenzylidene cyclo-
pentane derivatives evaluated, ketone 2 was the most active
inhibitor of AKR1C1, with a Ki value of 17.2
mM, Ki value for AKR1C3
was 33.4 M. In a kinetic experiment, competitive inhibition
m
pattern was obtained for AKR1C1 with compound 2 (Section 5.2.2).
For the remainder of the compounds Ki values were calculated as
described under Experimental protocols. Methyl ether 3 was the
best inhibitor of AKR1C3 arising from this study with Ki value of
16.2 mM. As this compound has a lower affinity towards AKR1C1 it
represents an excellent starting point for development of selective
inhibitors of AKR1C3. Ethyl ether 4 inhibited both AKR1C1 and
AKR1C3, with Ki values of 30.5 mM and 20.5 mM, respectively.
Cyclopentanone derivatives 5 and 8 were insoluble. Cyclo-
pentanol derivatives 9–11, tosylate 12 and aminocyclopentanes
14–18 were poor inhibitors of both AKR1C1 and AKR1C3. The only
exceptions here are the compounds 10 and 11, which inhibited
AKR1C1 in complex with 20a-OHP [26] and AKR1C3 complexed with
indomethacin [20a], flufenamic acid [20], PGD2 [27], rutin [27] and
androstenedione [28] were reported, providing an excellent basis for
the structure-based design of new and improved inhibitors.
Furthermore, an indomethacin analogue as a selective inhibitor of
aldo–keto reductase 1C3 (type 2 3a-HSD, type 5 17b-HSD, and
prostaglandin F synthase) have been discussed by Penning et al.
[20b].
2. Chemistry
AKR1C3 for 67% and 46% at 50 mM, respectively. However, due to
The synthesis of all of these compounds is presented in
Schemes 1–3. The benzyl-substituted cyclopentane derivatives
poor solubility of the compounds at higher concentrations we were
unable to determine the IC50 and Ki values. Interesting inhibitory

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B. Stefane et al. / European Journal of Medicinal Chemistry 44 (2009) 2563–2571
2565
OH
CH2Ph
cis-9 (42%)
trans-9 (24%)
d
O
O
CH2Ph
PhH2C
PhH2C
CH2Ph
CH2Ph
O
O
a
8 (1%)
5 (40%)
b
O
O
CH2Ph
CH2Ph
1
2 (40%)
CH2Ph
Cl
c
7
6
6 : 7 = 5:1 (15%)
OR
d
OH
OH
CH2Ph
CH2Ph
10 (56%)
Cl
+
3: R; Me (93%)
4: R; Et (60%)
PhH2C
CH2Ph
11 (13%)
Scheme 1. a) NaOH, 4-chlorobenzaldehyde, H2O, 25 ꢁC, 14 h; b) LDA, BnBr, THF, ꢀ5 ꢁC, 1 h, 25 ꢁC, 24 h; c) NaBH4, CeCl3 ꢂ 6H2O, ROH, 25 ꢁC, 2 h then HCl (1 M), 15 h 25 ꢁC; d) NaBH4,
CeCl3 ꢂ 6H2O, MeOH, 25 ꢁC, 1.5 h.
activities were also seen for 3-phenylcyclopentanecarboxylic acid
(20) and its isomer, the commercially available 1-phenyl-
cyclopentanecarboxylic acid (21). The former inhibited both
Lamarckian genetic algorithm [37]. According to the docking
calculations, inhibitor 21 should occupy the bottom of the hydro-
phobic pocket of the active site, making hydrophobic interactions
with Leu54, Trp227 and Leu308 (Fig. 2). The carboxylate moiety of
inhibitor is pointing towards the catalytic tetrade and the nicotin-
amide moiety of the coenzyme (which form the oxyanion hole).
This highest ranked docking pose had a mean docked energy of
ꢀ6.53 kcal/mol and was found six times out of 100 docking runs.
Compound 3 was docked into the active site of AKR1C3 (pdb
code 1RY0, co-crystal structure with prostaglandin D2). The dock-
ing position is presented in Fig. 3. Also inhibitor 3 is located at the
bottom of the enzyme active site where it makes hydrophobic
interactions with Tyr24, Tyr55 and Trp227. This docking pose was
found as the lowest energy pose with the predicted docked energy
of ꢀ7.91 kcal/mol and was found eight times out of 100 runs.
AKR1C1 and AKR1C3 (Ki values of 48.5
mM and 69.6
mM, respec-
tively), while the latter was inhibitor of AKR1C1 (Ki ¼ 35.8
mM) with
no affinity towards AKR1C3.
The X-ray crystal structures of AKR1C1 and AKR1C3 reveal
substrate-binding sites that consist mainly of hydrophobic
aromatic amino acid side chains (Leu54, His222, Trp227, Thr307
and Leu308 for AKR1C1; Tyr24, Tyr55, Leu54, Trp227 and Phe306
for AKR1C3). The four conserved amino acids Asp50, Tyr55, Lys84
and His117 have been proposed to form a catalytic tetrad that is
involved in the oxidation of alcohol or reduction of ketone func-
tional groups via a ‘‘push–pull’’ mechanism [36]. To investigate the
possible binding mode of the best inhibitor of AKR1C1, compound
21 was docked into the AKR1C1 active site (pdb code 1MRQ, co-
crystal structure with 20
a
-OHP) using AutoDock 3.0 with the
4. Conclusion
To conclude, we have presented the synthesis and biochemical
evaluation of new selective inhibitors of the steroid metabolizing
enzymes AKR1C1 and AKR1C3 that were based on the cyclopentane
scaffold. These compounds represent promising starting points for
development of agents for treating hormone-dependent forms of
cancers and other diseases where AKR1C1 and AKR1C3 are
involved.
CONH2
b
NHTs
16 (90%)
OH
a
e
CN
X
NHPh
12: X = NTs
13: X = O
NHTs
18 (86%)
c
14 (85%)
O
d
CO2H
Ph
CO2H
20 (43%)
a
Br
NHTs
17 (72%)
NHTs
Ph
19
15 (53%)
Scheme 2. a) TMSCN, TBAF, THF, 25 ꢁC, 4 days; b) HCl (conc.), 25 ꢁC, 21 h; c) HCl (conc.),
80 ꢁC, 9 h; d) PPh3, CBr4, MeOH, MW, 130 ꢁC, 10 min.; e) PhNH2, ZrCl4, 25 ꢁC, 0.5 h.
Scheme 3. a) (PhSe)2, H2O2 (30%), t-BuOH, reflux, 4 days.

ˇ
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B. Stefane et al. / European Journal of Medicinal Chemistry 44 (2009) 2563–2571
Table 1
Inhibitors of AKR1C1 and AKR1C3.
Compound
Structure
AKR1C1 inhibition (%) ꢃ SDa
77.8 ꢃ 0.4
AKR1C3 inhibition (%) ꢃ SDb
88.8 ꢃ 2.6
O
IC50 ¼ 35.0
Ki ¼ 17.2
m
Mc
IC50 ¼ 36.7
Ki ¼ 33.4
m
Mc
m
M
mM
2
Cl
Cl
Cl
87.4 ꢃ 1.1
IC50 ¼ 17.8
OMe
OEt
m
mM
Mc
Ki ¼ 16.2
3
40.1 ꢃ 0.8
52.9 ꢃ 3.9
IC50 ¼ 62.1
83.9 ꢃ 5.1
m
M
Mc
IC50 ¼ 22.5
m
Mc
Ki ¼ 30.5
m
Ki ¼ 20.5
mM
4
OH
CH2Ph
CH2Ph
cis-9
trans-9
10
9.7 ꢃ 5.3
NI
NI
OH
OH
13.8 ꢃ 4.0
15.9 ꢃ 1.2d
CH2Ph
CH2Ph
67.3 ꢃ 1.8e
OH
PhH2C
CH2Ph
11
12
14
11.5 ꢃ 1.3d
2.1 ꢃ 1.5
46.6 ꢃ 0.8e
2.7 ꢃ 1.1
NI
N
Ts
CN
15.9 ꢃ 5.4
NHTs
CONH2
15
12.9 ꢃ 3.4
NI
NHTs

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B. Stefane et al. / European Journal of Medicinal Chemistry 44 (2009) 2563–2571
2567
Table 1 (continued )
Compound
Structure
AKR1C1 inhibition (%) ꢃ SDa
AKR1C3 inhibition (%) ꢃ SDb
CO2H
16
17
18
26.1 ꢃ 4.5
NI
NHTs
Br
20.3 ꢃ 0.8
9.2 ꢃ 1.7
NHTs
OH
14.7 ꢃ 1.3
NI
NHPh
55.4 ꢃ 0.9
52.7 ꢃ 2.3
Ph
CO2H
IC50 ¼ 98.6
m
M
Mc
IC50 ¼ 76,6
m
Mc
20
21
Ki ¼ 48.5
m
Ki ¼ 69.6
mM
53.5 ꢃ 2.3
IC50 ¼ 72.9
Ph
CO2H
m
mM
Mc
5.8 ꢃ 0.8
Ki ¼ 35.8
NI – no inhibition detected.
a
Percentage of enzyme inhibition at 30
Percentage of enzyme inhibition at 100
m
m
M acenaphthenol and 100
M acenaphthenol and 100
IC50 values were determined as described.
m
m
M of each inhibitor. The results of at least three independent experiments are shown as means ꢃ SD.
b
c
M of each inhibitor. The results of at least three independent experiments are shown as means ꢃ SD.
d
e
Percentage of enzyme inhibition at 30
Percentage of enzyme inhibition at 100
m
m
M acenaphthenol and 50
m
M of each inhibitor. The results of at least three independent experiments are shown as means ꢃ SD.
M acenaphthenol and 50
m
M of each inhibitor. The results of at least three independent experiments are shown as means ꢃ SD.
5. Experimental protocols
dry solvents. Light petroleum refers to the fraction with the distil-
lation range 40–60 ꢁC. TLC was carried out on Fluka silica-gel TLC-
cards. All mps were determined on a hot stage and are uncorrected.
IR spectra were recorded on a BioRad FTS 3000MX instrument.
5.1. Chemistry
Solvents and starting compounds (including compounds 13 and
21) were obtained from commercial sources (Fluka, Sigma and
Aldrich). All reactions requiring dry conditions were carried out in
NMR spectra were recorded on a Bruker Avance 300 DPX spec-
trometer at 302 K. Chemical shifts are reported in
d ppm, refer-
enced to an internal TMS standard for 1H NMR, chloroform-d
Fig. 2. Superimposition of the computer model of compound 21 (white) on the X-ray
Fig. 3. Superimposition of the computer model of compound 3 (white) on the X-ray
structure of prostaglandine D2 (yellow) and NADPþ (magenta) bound to AKR1C3. The
highest ranked position of the inhibitor is presented (as calculated by AutoDock 3.0).
(For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
structure of 20a
-hydroxyprogesterone (yellow) and NADþ (magenta) bound to
AKR1C1. The highest ranked position of the inhibitor is presented (as calculated by
AutoDock 3.0). (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)

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B. Stefane et al. / European Journal of Medicinal Chemistry 44 (2009) 2563–2571
2568
(
d
77.0), DMSO-d6
(
d
39.5) for 13C NMR. The 1H–13C HMBC spectra
temperature overnight. The precipitated material was filtered off
and mother-liquid treated with water (300 mL) and extracted with
Et2O (2 ꢂ 200 mL). The combined organic extracts were washed
with HCl (1 M, 250 mL), saturated NaHCO3 (250 mL), and brine
(250 mL), dried over MgSO4 and evaporated under reduced pres-
sure. The residue, colourless oil, was purified by SiO2 column
chromatography (light petroleum:EtOAc ¼ 20:1). 2-Benzyl cyclo-
pentanon (5); colourless oil, 2.39 g (40%); Rf 0.18 (light petro-
leum:EtOAc ¼ 20:1); IR (film, cmꢀ1): 3454, 3061, 3028, 2963, 2876,
1950, 1881, 1805, 1738, 1639, 1603, 1495, 1450, 1405, 1341, 1310,
1267,1219,1154,1111,1067,1028,1002, 924, 845, 802, 760, 725, 700;
were obtained with 512 time increments and 32 scans per t1
increment. Microanalyses were performed on a Perkin–Elmer 2400
series II CHNS/O analyser. Mass spectra and high-resolution mass
measurements were performed on a VG-Analytical Autospec EQ
instrumet.
5.1.1. Synthesis
5.1.1.1. 2-(4-Chlorobenzylidene)cyclopentanone (2) [38]. A mixture
of cyclopentanone (4.00 g, 47.6 mmol) and 4-chlorobenzaldehyde
(3.37 g, 24 mmol) was treated with NaOH (0.2 M, 350 mL) at 25 ꢁC.
The reaction mixture was stirred at 25 ꢁC for 14 h, diluted with
water (350 mL), acidified with HCl (5%, 50 mL) pH ¼ 5, and
extracted with CH2Cl2 (3 ꢂ 150 mL). The combined extracts were
dried over MgSO4 and evaporated under reduced pressure. The
residue was purified by SiO2 column chromatography (light
petroleum:EtOAc ¼ 20:1) yielding title compound 2 as white crys-
tals, 1.98 g (40%). Mp ¼ 74–76 ꢁC; IR (KBr, cmꢀ1): 2983, 2947, 1713,
1623, 1584, 1487, 1460, 1405, 1301, 1275, 1230, 1172,1089, 1005, 905,
1H NMR (300 MHz, CDCl3):
d 1.48–1.62 (m, 1H), 1.67–1.79 (m, 1H),
1.89–2.00 (m, 1H), 2.04–2.16 (m, 2H), 2.28–2.40 (m, 2H), 2.54 (dd,
J ¼ 9.5, 14.0 Hz, 1H), 3.15 (dd, J ¼ 4.0, 14.0 Hz, 1H), 7.15–7.30 (m, 5H).
EI-MS, m/z (relative intensity) 174 (13%); HRMS calcd for C12H14O:
174.1045; found 174.1049. Mixture of isomers 2,2-dibenzylcyclo-
pentanone (6) and 2,5-dibenzylcyclopentanone (7); yellowish oil,
1.43 g (15% overall); ratio of isomers 6:7 ¼ 5:1; Rf 0.42 (light
petroleum:EtOAc ¼ 7:1); EI-MS: m/z ¼ 264 (16%); HRMS calcd for
C19H20O: 264.1514; found 264.1521. 2,2,5-Tribenzylcyclopentanone
(8); colourless solid, 173 mg (1%); Rf 0.47 (light petroleum:-
EtOAc ¼ 7:1); mp ¼ 94 ꢀ 96 ꢁC; IR (KBr, cmꢀ1): 3443, 3028, 2935,
2908, 1961, 1896, 1827, 1731, 1638, 1599, 1494, 1454, 1342, 1320,
1306, 1236, 1167, 1125, 1075, 1028, 976, 913, 861, 822, 755, 728, 706.
871, 817. 1H NMR (300 MHz, CDCl3):
2H); 2.41 (t, J ¼ 7.5 Hz, 2H); 2.95 (dt, J ¼ 2.5 Hz, 2H); 7.33
(t, J ¼ 2.5 Hz, 1H); 7.36–7.40 (m, 2H); 7.45–7.48 (m, 2H).
d
2.05 (tt, J ¼ 7.5 Hz, 15.0 Hz,
5.1.1.2. General procedure for 2-(4-chlorobenzylidene)cyclopentyl
methyl ether (3) and 2-(4-chlorobenzylidene)cyclopentyl ethyl ether
(4). A mixture of 2-(4-chlorobenzylidene)cyclopentanone (2)
(200 mg, 0.97 mmol) and CeCl3 ꢂ 6H2O (687 mg, 1.94 mmol) in
MeOH (10 mL) or EtOH (10 mL) was treated with NaBH4 (74.0 mg,
1.94 mmol). After stirring of the reaction mixture at 25 ꢁC for
45 min, HCl (1 M, 25 mL) was added and the reaction mixture
stirred for the additional 15 h. The reaction mixture was than
extracted with EtOAc (3 ꢂ 25 mL). Combined organic layers were
washed with NaHCO3 (5%, 2 ꢂ 50 mL), H2O (50 mL), brine (50 mL),
dried over MgSO4 and evaporated under reduced pressure. The
residue, colourless liquid, was purified by column chromatography
on SiO2 (light petroleum/EtOAc ¼ 10:1) yielding 200 mg (93%) of
product 3. 2-(4-Chlorobenzylidene)cyclopentyl methyl ether (3);
colourless liquid; Rf 0.57 (light petroleum:EtOAc ¼ 5:1); IR (NaCl,
cmꢀ1): 3029, 2963, 2884, 2817, 1491, 1462, 1406, 1339, 1276, 1196,
1150, 1088, 1055, 1012, 926, 885, 821; 1H NMR (300 MHz, CDCl3):
1H NMR (300 MHz, CDCl3):
d 0.72–0.88 (m, 1H), 1.44–1.52 (m, 1H),
1.68–1.76 (m, 1H), 1.78–1.90 (m, 2H), 2.05 (dd, J ¼ 10.5, 14.0 Hz, 1H),
2.58 (dd, J ¼ 10.5, 13.0 Hz, 2H), 2.92 (d, J ¼ 13.0 Hz, 1H), 3.02
(dd, J ¼ 4.0, 14.0 Hz, 1H), 3.10 (d, J ¼ 13.0 Hz, 1H), 6.94–6.96 (m, 2H),
7.05–7.31 (m, 13H). EI-MS, m/z (relative intensity) 354 (3.3%); HRMS
calcd for C26H26O: 354.1984; found 354.1990.
5.1.1.4. cis- and trans-2-Benzylcyclopentanol (cis- and trans-9)
[39]. To
a mixture of 2-benzylcyclopentanone (5) (150 mg,
0.86 mmol) and CeCl3 ꢂ 6H2O (581 mg, 1.56 mmol) in MeOH
(10 mL), NaBH4 (62.8 mg, 1.65 mmol) was added. The reaction
mixture was stirred at 25 ꢁC for 1.5 h and then treated with HCl
(1 M, 50 mL), extracted with EtOAc (3 ꢂ 50 mL), dried over MgSO4
and evaporated under reduced pressure. The residue was purified
by SiO2 column chromatography (light petroleum:EtOAc ¼ 5:1)
yielding cis and trans isomer in ratio 1:1. cis-2-Benzylcyclopentanol
(cis-9); colourless oil; 64 mg (42%); Rf 0.26 (light petroleum:-
EtOAC ¼ 5:1); IR (film, cmꢀ1): 3564, 3387, 3062, 3026, 2957, 2872,
1944, 1873, 1805, 1603, 1494, 1454, 1404, 1305, 1199, 1145, 1109,
d
1.69–1.79 (m, 2H), 1.81–1.91 (m, 1H), 1.94–2.04 (m, 1H), 2.43–2.55
(m,1H), 2.58–2.69 (m, 1H), 3.38 (s, 3H), 4.07–4.10 (m, 1H), 6.48–6.50
(m, 1H), 7.28 (bs, 4H); 13C NMR (100 MHz, CDCl3):
145.1, 136.3,
d
132.1, 129.7, 128.4, 123.8, 86.0, 56.1, 31.3, 28.9, 22.9. EI-MS, m/z
(relative intensity) 222 (46%); HRMS calcd for C13H15ClO: 222.0811;
found 222.0817. 2-(4-Chlorobenzylidene)cyclopentyl ethyl ether
(4); colourless liquid; Rf 0.54 (light petroleum:EtOAc ¼ 7:1); IR
(film, cmꢀ1): 3407, 2967, 2873, 1491, 1442, 1403, 1327, 1300, 1078,
1030, 986, 908, 751, 700. 1H NMR (300 MHz, CDCl3):
d 1.46–1.61
(m, 3H), 1.63–1.74 (m, 2H), 1.67–1.91 (m, 2H), 1.95–2.08 (m, 1H),
2.68 (dd, J ¼ 8.0, 13.5 Hz, 1H), 2.85 (dd, J ¼ 8.0, 13.5 Hz, 1H), 4.09 (br,
1H), 7.16–7.31 (m, 5H). EI-MS, m/z (relative intensity) 176 (7.9%);
HRMS calcd for C12H16O: 176.1201; found 176.1206. trans-2-Ben-
zylcyclopentanol (trans-9); colourless oil; 44 mg (29%); Rf 0.17
(light petroleum:EtOAc ¼ 5:1); IR (film, cmꢀ1): 3345, 3062, 3026,
2955, 2872, 2402, 1944, 1873, 1804, 1603, 1495, 1453, 1343, 1257,
1170, 1071, 1031, 967, 913, 746, 700. 1H NMR (300 MHz, CDCl3):
882, 820; 1H NMR (300 MHz, CDCl3):
d
1.24 (t, J ¼ 7.0 Hz, 3H), 1.64–
1.75 (m, 2H), 1.80–2.03 (m, 2H), 2.43–2.54 (m, 1H), 2.57–2.65
(m,1H), 3.52–3.59 (m, 2H), 4.19 (bs, 1H), 6.48 (bs, 1H), 7.24–7.28
(m, 4H); 13C NMR (100 MHz, CDCl3): 145.5,136.3, 131.9,129.6,128.2,
123.2, 84.1, 63.9, 31.7, 28.9, 22.7, 15.5; EI-MS, m/z (relative intensity)
236 (36%); HRMS calcd for C14H17ClO: 236.0968; found 236.0961.
d
1.21–1.33 (m, 2H), 1.50–2.09 (m, 6H), 2.55 (dd, J ¼ 8.0, 13.5 Hz, 1H),
2.76 (dd, J ¼ 8.0, 13.5 Hz, 1H), 3.88–3.94 (m, 1H), 7.16–7.31 (m, 5H).
13C NMR (75 MHz, CDCl3):
141.1, 128.8, 128.4, 126.0, 78.6, 49.9,
d
5.1.1.3. Synthesis of compounds 5–8. To
a
solution of cyclo-
39.8, 34.2, 29.9, 21.5. EI-MS, m/z (relative intensity) 176 (8.0%).
pentanone (3.00 g, 35.7 mmol) in THF (50 mL) cooled to 0 ꢁC LDA
(18 mL, 35.7 mmol) was added drop-wise. After being stirred at
ꢀ5 ꢁC for 20 min, the solution of benzyl bromide (30.50 g,
178.5 mmol) in THF (10 mL) was added and the reaction mixture
was stirred at room temperature for 24 h. After the reaction was
complete, the mixture was treated with HCl (0.5 M, 300 mL) and
extracted with EtOAc (3 ꢂ 200 mL). The combined organic extracts
were dried (MgSO4) and evaporated under reduced pressure. The
residue was dissolved in pyridine (50 mL) and stirred at room
5.1.1.5. 2,2-Dibenzylcyclopentanol (10) and 2,5-dibenzylcyclopenta-
nol (11). To a mixture of isomers 7 and 8 (223 mg, 0.85 mmol) and
CeCl3 ꢂ 6H2O (599 mg, 1.69 mmol) in MeOH (10 mL), NaBH4
(63.9 mg, 1.69 mmol) was added. The reaction mixture was stirred at
25 ꢁC for 1.5 h and then treated with HCl (1 M, 50 mL), extracted with
EtOAc (3 ꢂ 50 mL), dried over MgSO4 and evaporated under reduced
pressure. The residue was purified by SiO2 column chromatography
(light petroleum:EtOAc ¼ 10:1). 2,2-Dibenzylcyclopentanol (10);

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B. Stefane et al. / European Journal of Medicinal Chemistry 44 (2009) 2563–2571
2569
colourless oil; 105 mg (56%); Rf 0.32 (light petroleum:EtOAc ¼ 7:1); IR
(film, cmꢀ1): 3582, 3399, 3060, 3026, 2953, 2875, 1949, 1881, 1810,
1601, 1494, 1454, 1397, 1338, 1072, 1031, 975, 913, 865, 753, 703. 1H
1.38–1.54 (m, 4H), 1.74–1.88 (m, 1H), 2.38 (s, 3H), 2.42–2.47 (m, 1H),
3.63–3.72 (m, 1H), 6.70 (s, 1H), 7.11 (s, 1H), 7.35–7.37 (m, 2H), 7.59
(bd, J ¼ 8.0 Hz, 1H), 7.65–7.67 (m, 2H).
NMR (300 MHz, CDCl3): d 1.19–1.29 (m, 1H), 1.39–1.52 (m, 2H), 1.54–
1.67 (m, 2H),1.69–1.83 (m,1H),1.90–2.02 (m,1H), 2.52 (d, J ¼ 13.0 Hz,
5.1.1.10. 2-(4-Methylphenylsulfonamido)cyclopentanecarboxylic acid
(17) [32]. N-(2-Cyanocyclopentyl)-4-methylbenzenesulfonamide
(50 mg, 0.19 mmol) was dissolved in concentrated HCl (2 mL). After
the reaction mixture was heated at 80 ꢁC for 9 h it was diluted with
H2O (5 mL). The precipitated material was filtered off, washed with
H2O (3 mL) and dried under the vacuum providing the pure product
17, 36 mg (72%). White solid; Rf 0.10 (light petroleum:EtOAc ¼ 5:3);
mp ¼ 120–122 ꢁC; IR (KBr, cmꢀ1): IR (KBr, cmꢀ1): 3302, 3035, 2959,
2873, 2654, 1690, 1599, 1454, 1331, 1290, 1220, 1160, 1093, 1050,
1H), 2.73 (dd, J ¼ 5.0,14.0 Hz, 3H), 3.89 (t, J ¼ 7.5 Hz,1H), 7.09–7.12 (m,
2H), 7.16–7.33 (m, 8H).13C NMR (75 MHz, CDCl3):
d 139.3, 138.4, 130.8,
130.8, 128.0, 127.9, 126.0, 126.0, 76.5, 49.1, 40.6, 37.4, 31.2, 30.0, 18.5.
EI-MS, m/z (relative intensity) 266 (2%); HRMS calcd for C19H22O:
266.1671; found 266.1677. 2,5-Dibenzylcyclopentanol (11); white
solid; 24 mg (13%); Rf 0.38 (light petroleum:EtOAc ¼ 7:1); mp ¼ 49–
51 ꢁC; IR (KBr, cmꢀ1): 3592, 3325, 3024, 2949, 2919, 2850,1604,1495,
1450, 1345, 1121, 1060, 1030, 936, 880, 739, 698. 1H NMR (300 MHz,
CDCl3):
d
1.15–1.27 (m, 2H),1.43–1.56 (m,1H),1.65–1.75 (m,1H),1.86–
1017, 930, 814; 1H NMR (300 MHz, CDCl3):
d 1.41–1.53 (m, 1H),
1.97 (m,1H), 2.14–2.27 (m, 2H), 2.48–2.64 (m, 2H), 2.68–2.76 (m,1H),
1.55–1.74 (m, 2H). 1.77–1.89 (m, 1H), 1.95–2.10 (m, 2H), 2.42 (s, 3H),
2.69–2.77 (m, 1H), 3.73–3.83 (m, 1H), 4.83 (bd, J ¼ 6.0 Hz, 1H), 7.29–
7.32 (m, 2H), 7.75–7.77 (m, 2H); EI-MS, m/z (relative intensity) 284
(0.3%); HRMS calcd for C13H18NO4S: 284.0957; found 284.0968.
2.88 (dd, J ¼ 7.0, 13.5 Hz, 1H), 3.84–3.85 (m, 1H), 7.15–7.30 (m, 10H).
13C NMR (75 MHz, CDCl3):
d 141.7,141.0,128.8,128.7 (2C),128.3,125.9,
125.8, 78.8, 49.5, 45.4, 40.7, 35.3, 29.1, 28.9. EI-MS, m/z (relative
intensity) 266 (0.5%); HRMS calcd for C19H22O: 266.1671; found
266.1680.
5.1.1.11. 2-(Phenylamino)cyclopentanol (18) [33,34]. White solid;
175 mg (86%); Rf 0.10 (light petroleum:EtOAc ¼ 5:1); mp ¼ 57–58 ꢁC;
IR (KBr, cmꢀ1): IR (NaCl, cmꢀ1) 3394, 3087, 3051, 3021, 2961, 2874,
1921, 1830, 1603, 1505, 1470, 1432, 1317, 1284, 1180, 1154, 1107, 1076,
5.1.1.6. 6-Tosyl-6-azacicyclo[3.1.0]hexane (12) [30]. White solid;
2.78 g (80%); Rf 0.22 (light petroleum:EtOAc ¼ 5:1); mp ¼ 69–71 ꢁC;
IR (KBr, cmꢀ1): 3353, 3260, 3048, 2958, 2856, 2412, 1950, 1682,
1595, 1491, 1437, 1400, 1367, 1341, 1317, 1200, 1154, 1092, 1012, 972,
1041, 978, 871, 837, 750, 694; 1H NMR (300 MHz, CDCl3):
d 1.35–1.47
(m,1H),1.59–1.91 (m, 4H),1.94–2.05 (m,1H), 2.23–2.34 (m,1H), 3.59–
3.65 (m, 1H), 4.05–4.10 (m, 1H), 6.64–6.73 (m, 3H), 7.14–7.21 (m, 2H).
929, 870, 828, 722, 676; 1H NMR (300 MHz, CDCl3):
d 1.52–1.68
(m, 4H); 1.91–1.98 (m, 2H); 2.44 (s, 3H); 3.33 (s, 2H); 7.31–7.33 (m,
2H); 7.80–7.82 (m, 2H).
5.1.1.12. 3-Phenylcyclopentanecarboxylic acid (20) [35]. To the stir-
red mixture of the corresponding cyclohexanone 19 (1.394 g,
8 mmol), diphenyldiselenide (25 mg, 0.08 mmol), and t-BuOH
(4 mL), H2O2 (30%, 6 mL, 48 mmol) was added. The reaction was
heated in sealed tube for 4 days. After the reaction was complete,
Pd/C (10%, 50 mg) was added and the solvent was distilled off. The
residue was treated with aqueous Na2CO3 (10%, 100 mL) and
washed with CH2Cl2 (3 ꢂ10 mL). The aqueous phase was adjusted
to pH 1 with HCl (1%, ca. 170 mL) and extracted with CH2Cl2
(3 ꢂ 25 mL), dried with Na2SO4, and evaporated under reduced
pressure. The residue, colourless oil, was distilled under the
vacuum (7 ꢂ 10ꢀ3 mbar, 110 ꢁC) yielding colourless oil, mixture of
isomers cis and trans, 1.10 g (70%); Rf 0.15 (light petroleum:-
EtOAc ¼ 5:3); IR (film, cmꢀ1): 3028, 2963, 2932, 2670, 1708, 1602,
1493, 1452, 1413, 1287, 1246, 1169, 1072, 1030, 935, 760, 701; 1H
5.1.1.7. N-(2-Cyanocyclopentyl)-4-methylbenzenesulfonamide (14)
[31]. White solid; 380 mg (85%); Rf 0.32 (light petroleum:-
EtOAc ¼ 5:3); mp ¼ 106–108 ꢁC; IR (KBr, cmꢀ1): 3906, 3251, 3036,
2954, 2879, 2244, 1927, 1813, 1600, 1452, 1401, 1333, 1269, 1159,
1093, 1019, 937, 896, 820, 674; 1H NMR (300 MHz, CDCl3):
d 1.42–
1.54 (m, 1H),1.70–1.80 (m, 2H), 1.84–1.95 (m, 1H), 1.98–2.17 (m, 2H),
2.45 (s, 3H), 2.81–2.87 (m, 1H), 3.72–3.81 (m, 1H), 5.18 (bd,
J ¼ 6.5 Hz, 1H), 7.31–7.36 (m, 2H), 7.75–7.82 (m, 2H).
5.1.1.8. N-(2-Bromocyclopentyl)-4-methylbenzenesulfonamide (15)
[40]. Aziridine 12 (237 mg, 1 mmol) was dissolved in methanol
(5 mL), Ph3P (525 mg, 2 mmol), and CBr4 (667 mg, 2 mmol) were
added. The mixture was then heated under microwave conditions
at 130 ꢁC for 10 min. After this time the solvent was evaporated and
the crude material was purified by SiO2 column chromatography
(light petroleum:EtOAc ¼ 10:1) to provide the product 15 (167 mg,
53%) as a white solid; Rf 0.10 (light petroleum:EtOAc ¼ 7:1);
mp ¼ 82–85 ꢁC; IR (KBr, cmꢀ1): IR (KBr, cmꢀ1): 3438, 3263, 2973,
2877, 1597, 1439, 1412, 1324, 1162, 1095, 1077, 1036, 901, 847, 806,
NMR (300 MHz, CDCl3):
d 1.62–1.88 (m, 1H), 1.90–2.20 (m, 4H),
2.36–2.46 (m, 1H), 2.90–2.31 (m, 2H), 7.10–7.32 (m, 5H), 11.30 (bs,
1H); EI-MS, m/z (relative intensity) 190 (69%); HRMS calcd for
C12H14NO2: 190.0994; found 190.0986.
5.2. Pharmacology
665; 1H NMR (300 MHz, CDCl3):
d 1.36–1.47 (m,1H), 1.66–1.89 (m,
2H), 1.91–2.02 (m, 1H), 2.15–2.32 (m, 2H), 2.44 (s, 3H), 3.62–3.70
(m, 1H), 4.06–4.11 (m, 1H), 4.62 (bd, J ¼ 6.0 Hz, 1H), 7.32–7.34 (m,
2H), 7.77–7.79 (m, 2H). EI-MS, m/z (relative intensity) 319 (30%);
HRMS calcd for C12H26BrNSO2: 317.0085; found 317.0095.
5.2.1. Expression and purification of recombinant AKR1C1
and AKR1C3
pGex-AKR1C1 (constructed from a pcDNA3-AKR1C1 vector
provided by Dr. Trevor M. Penning) [17] and pGex-AKR1C3
(provided by Dr. Jerzy Adamski) were transferred into the BL21
Escherichia coli strain. The cells were then grown in Luria-Bertani
5.1.1.9. 2-(4-Methylphenylsulfonamido)cyclopentanecarboxamide
(16) [32]. N-(2-Cyanocyclopentyl)-4-methylbenzenesulfonamide
(50 mg, 0.19 mmol) was dissolved in concentrated HCl (2 mL). After
the reaction mixture was stirred at 25 ꢁC for 21 h it was diluted
with H2O (10 mL) and the product was extracted with EtOAc
(5 ꢂ15 mL), which was dried (Na2SO4), filtered, and evaporated
under reduced pressure yielding pure product 16, 48 mg (90%).
White solid; Rf 0.18 (light petroleum:EtOAc ¼ 5:3); mp ¼ 174–
175 ꢁC; IR (KBr, cmꢀ1): IR (KBr, cmꢀ1): 3398, 3291, 3190, 3117, 2977,
2883, 2790, 2739, 1929, 1668, 1623, 1445, 1351, 1308, 1149, 1096,
medium containing 100 m
g/ml ampicilin, at 37 ꢁC in a rotary shaker,
until an OD600 of 1.0 had been reached. Expression of AKR1C1 and
AKR1C3 was induced by IPTG at a final concentration of 1 mM and
0.5 mM, respectively, and the incubations were continued for 16 h
at 24 ꢁC and 3 h at 37 ꢁC, respectively [16,41]. The preparation of
cell extracts, the purification of glutathione-S-transferase (GST)-
fusion proteins by affinity binding to glutathione-Sepharose, and
the cleavage of these with thrombin were all performed as
described in the GST Gene Fusion System Handbook (Amersham
Biosciences). Protein concentrations were determined using the
955, 918, 822, 708; 1H NMR (300 MHz, DMSO):
d 1.16–1.27 (m, 1H),

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B. Stefane et al. / European Journal of Medicinal Chemistry 44 (2009) 2563–2571
2570
Bradford method, with bovine serum albumin as standard, and the
homogeneity of the proteins was checked by SDS PAGE followed by
Coomassie Blue staining.
algorithm method, implemented in the program AutoDock 3.0, was
employed [37]. The structures of inhibitors were prepared using
HyperChem 7.5 (HyperChem, version 7.5 for Windows. Hypercube,
Inc.: Gainesville, FL, 2002). The crystal structures of AKR1C1 and
AKR1C3 were retrieved from the RCSB protein database (PDB codes
1MRQ and 1RY0, respectively) and all water molecules and
substrates removed. Polar hydrogen atoms were added and Koll-
man charges [42], atomic solvation parameters and fragmental
volumes were assigned to the protein using AutoDock Tools (ADT).
For docking calculations, Gasteiger–Marsili partial charges [43]
were assigned to the ligands and cofactors and nonpolar hydrogen
atoms were merged. All torsions were allowed to rotate during
docking. The grid map, which was large enough to cover the
inhibitors and the enzyme’s active site, was generated with Auto-
Grid. Lennard–Jones parameters 12-10 and 12-6, supplied with the
program, were used for modeling H-bonds and van der Waals
interactions, respectively. The distance-dependent dielectric
permittivity of Mehler and Solmajer [44] was used to calculate the
electrostatic grid maps. Random starting points, random orienta-
tion, and torsions were used for all ligands. The translation,
quaternion, and torsion steps were taken from default values in
AutoDock 3.0. The Lamarckian genetic algorithm and the pseudo-
Soils and Wets methods were applied for minimization, using
default parameters. The number of docking runs was 100, the
population in the genetic algorithm was 250, the number of energy
evaluations was 500,000, and the maximum number of iterations
27,000.
5.2.2. Inhibition assays
Human recombinant AKR1C1 and AKR1C3 catalyze the oxida-
tion of the 1-acenaphthenol in the presence of the coenzyme NADþ.
The reaction was followed spectrophotometrically by measuring
the increase in NADH absorbance (3l ¼ 6220 Mꢀ1 cmꢀ1) in the
340
absence and presence of each of the compound. The assays for
AKR1C1 were carried out in a 0.6-mL volume that included 100 mM
phosphate buffer (pH 6.5), 0.005% triton X-114 and 1.5% N,N-di-
methylformamide (DMF) as a co-solvent. A substrate concentration
of 30
AKR1C1 converted 1-acenaphthenol with a specific activity of
1.3 mol 1-acenaphthenol oxidised/min/mg. The concentrations of
compounds ranged from 1.2 M to 300 M. The assays for AKR1C3
mM was used, with 2.3 mM coenzyme and 0.8 mM enzyme.
m
m
m
were carried out in a 0.6-mL volume that included 100 mM sodium
phosphate buffer (pH 9.0), 0.005% triton X-114 and 1.7% DMF as
a co-solvent. A substrate concentration of 100
2.3 mM coenzyme and 1.0 M enzyme. AKR1C3 converted 1-ace-
naphthenol with a specific activity of 208 nmol 1-acenaphthenol
oxidised/min/mg at 100 M 1-acenaphtehenol. The concentrations
of compounds ranged from 4.5 M to 300 M. The measurements
mM was used, with
m
m
m
m
were performed on a Beckman DU7500 spectrophotometer, initial
reaction velocities were calculated, and the IC50 values were
determined graphically from plots of log10 [inhibitor concentration]
versus % inhibition, using GraphPad Prism Version 4.00 (GraphPad
Software, Inc.). Representative inhibition pattern was determined
for AKR1C1 with compound 2. Here, the concentrations of the
inhibitor were in the range 0.5–2.0 ꢂ IC50 and that of the substrate
in the range 0.5–5.0 ꢂ Km. Initial velocity data were fitted to
competitive, non-competitive and uncompetitive inhibition
models using SigmaPlot 8.0 software (Fig. 4). Using the Cheng–
Prusoff relationship, Ki values were calculated for the remainder of
the compounds.
Acknowledgements
This work was supported by the Ministry of Higher Education,
Science and Technology of the Republic of Slovenia and the
Slovenian Research Agency (P1-0230-0103). The authors thank
Dr. T.M. Penning (University of Pennsylvania, School of Medicine,
Philadelphia, PA, USA) for the pcDNA3-AKR1C1 construct, Dr. Jerzy
Adamski (GSF, National Research Centre for Health and Environ-
ment, Institute of Experimental Genetics, Neuherberg, Germany)
for the pGex-AKR1C3 construct and Dr. Chris Berrie for critical
reading of the manuscript.
5.3. Molecular docking
Automated docking was used to determine the orientation of
inhibitors bound in the active site of AKR1C1 and AKR1C3. A genetic
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