N-Benzoyl anthranilic acid derivatives as selective inhibitors of aldo-keto reductase AKR1C3

Article abstract of DOI:10.1016/j.bmcl.2012.07.062

Human aldo-keto reductases AKR1C1-AKR1C3 are involved in the biosynthesis and inactivation of steroid hormones and prostaglandins and thus represent attractive targets for the development of new drugs. We synthesized a series of N-benzoyl anthranilic acid derivatives and tested their inhibitory activity on AKR1C enzymes. Our data show that these derivatives inhibit AKR1C1-AKR1C3 isoforms with low micromolar potency. In addition, five selective inhibitors of AKR1C3 were identified. The most promising inhibitors were compounds 10 and 13, with IC₅₀ values of 0.31 μM and 0.35 μM for AKR1C3, respectively.

Full text of DOI:10.1016/j.bmcl.2012.07.062

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5948–5951
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
N-Benzoyl anthranilic acid derivatives as selective inhibitors of aldo–keto
reductase AKR1C3
a
b
c
c
ˇ b
ˇ a
Maša Sinreih , Izidor Sosic , Nataša Beranic , Samo Turk , Adegoke O. Adeniji , Trevor M. Penning ,
a
b,
⇑
ˇ
Tea Lanišnik Rizner , Stanislav Gobec
a Institute of Biochemistry, Medical Faculty, University of Ljubljana, Ljubljana, Slovenia
b Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia
c Deparment of Pharmacology and Center of Excellence in Environmental Toxicology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Human aldo–keto reductases AKR1C1–AKR1C3 are involved in the biosynthesis and inactivation of ste-
roid hormones and prostaglandins and thus represent attractive targets for the development of new
drugs. We synthesized a series of N-benzoyl anthranilic acid derivatives and tested their inhibitory activ-
ity on AKR1C enzymes. Our data show that these derivatives inhibit AKR1C1–AKR1C3 isoforms with low
micromolar potency. In addition, five selective inhibitors of AKR1C3 were identified. The most promising
Received 1 June 2012
Revised 12 July 2012
Accepted 16 July 2012
Available online 22 July 2012
inhibitors were compounds 10 and 13, with IC50 values of 0.31
l
M and 0.35
Ó 2012 Elsevier Ltd. All rights reserved.
lM for AKR1C3, respectively.
Keywords:
Anthranilic acid
AKR1C3 inhibitors
Anticancer agents
Human aldo–keto reductases AKR1C1–AKR1C4 are involved in
the biosynthesis and inactivation of steroid hormones, neuroster-
oids, prostaglandins, products of lipid peroxidation and xenobiot-
ics. These AKR1C isoenzymes reduce carbonyl containing
substrates to alcohols and also act as 3-keto, 17-keto and 20-ketos-
teroid reductases to varying extents. In this manner, they regulate
the activity of androgens, estrogens, progesterone and the occu-
pancy and activation of their corresponding nuclear receptors.1,2
They possess a broad spectrum of physiological roles, but when
differentially expressed they are also involved in different
pathophysiological conditions, such as hormone-dependent and
independent cancers for example prostate cancer,3–8 other
hormone-dependent diseases,9 depression, epilepsy and neurode-
generative diseases.2 These enzymes, and especially AKR1C1 and
AKR1C3, thus represent attractive targets for the development of
new drugs. As AKR1C enzymes share more than 86% sequence
identity at the amino acid level and interestingly, AKR1C1 and
AKR1C2 differ in only one residue in the active site (Leu/Val54),
development of specific inhibitors is a challenging task.
a model for human AKR1C isoenzymes.10 Other nonselective NSA-
IDs and selective COX-2 inhibitors also proved to be potent inhib-
itors of AKR1C isoenzymes.3,11 This inhibition of AKR1C enzymes
by NSAIDs may thus explain the known antineoplastic effects of
these drugs. To confirm the hypothesis on involvement of AKR1C
isoenzymes in COX-independent anti-proliferative effects, Penning
et al. developed selective inhibitors of AKR1Cs based on N-pheny-
lanthranilic acid and cholanic acid with IC50 values in low
lM
range.3 Recently, we synthesized a series of N-benzoyl anthranilic
acid derivatives as inhibitors of penicillin binding proteins.12 Due
to their structural similarities to several known inhibitors of AKR1C
enzymes, we tested their inhibitory activity on AKR1C en-
zymes.13,14 We demonstrated that these derivatives inhibit AKR1C
isoenzymes in the low micromolar range. In addition, 5 com-
pounds were found to be selective inhibitors of AKR1C3.
The compounds were synthesized by the formation of the
amide bond between carboxylic group of benzoic acid derivatives
and the amino moiety of C-protected anthranilic acids. The carbox-
ylic acid groups were converted into acid chlorides using SOCl2, fol-
lowed by the addition of the corresponding C-protected anthranilic
acid derivatives. The esters were subsequently deprotected with
alkaline hydrolysis and where necessary an aromatic NO2 group
was reduced into their corresponding amine by catalytic hydroge-
nation to obtain the target compounds 1–16. The general experi-
mental procedures for the synthesis of compounds presented in
Table 1 and their spectroscopic properties are described in Ref.12
The detailed synthetic procedure and the spectroscopic data of a
representative compound (11) are described in note.15
Anthranilic acid derivatives have been known as good inhibitors
of AKR1C isoenzymes, with Ki values in low micromolar or nano-
molar range. Almost 30 years ago, Penning and Talalay showed
that indomethacin and mefenamic acid, nonselective NSAIDs,
strongly inhibit rat liver hydroxysteroid dehydrogenase AKR1C9,
⇑
Corresponding authors.
E-mail address: gobecs@ffa.uni-lj.si (S. Gobec).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.07.062

M. Sinreih et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5948–5951
5949
Table 1
Inhibitory activities for compounds 1–16a
O
R1
OH
A
R2
NH
R3
R4
R5
O
B
1-16
R1
R2
R3
R4
R5
AKR1C1 inhibitionb,c(%)
AKR1C2 inhibitionb,c (%)
AKR1C3 inhibitionb,c (%)
AKR1C4 inhibitionc (%)
1
2
3
4
5
6
7
8
OH
Cl
OMe
Cl
H
H
OMe
H
H
OMe
H
H
H
H
H
Me
H
H
H
H
NO2
NO2
NO2
NO2
NH2
NH2
NH2
NO2
H
H
H
H
H
H
H
F
NI
NI
31.6d
23.4
NI
22.5
NI
20.2
28.7
33.6
1.9
3.4
3.5
NI
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
22.2
NI
8.0
18.0
9.7
53.2
H
NI
OMe
OH
Br
5.6
5.6
5.6
IC50 = 8.4
NI
lM
IC50 = 15.6
NI
lM
9
H
H
H
OH
H
42.1
n.d.
IC50 = 12.7
IC50 = 5.19
l
l
M
IC50 = >100
(>19)e
17.4
l
Mc
IC50 = 48.1
(9)
6.7
l
Mc
Mc
IC50 = 100
(>19)e
n.d.
l
Mc
e
10
11
Cl
H
H
H
OH
OH
H
H
77.8
IC50 = 2.2
IC50 = 0.31
70.2 IC50 = 5.2
l
l
M
e
IC50 = 50.8
11.8
IC50 = 86.3
l
Mc (164)
IC50 = 50.2
29.4
IC50 = 30.0
l
l
Mc (162)e
Mc
lM
IC50 = 20.1
n.d.
IC50 = 50.9 l
l
Mc (65)e
OMe
OMe
e
e
l
l
Mcc (30)
Mc (10)
IC50 = 2.9
l
Mc
Mc (18)e
12
13
NO2
Br
H
H
H
H
OH
OH
H
H
11.8
IC50 = 35.2
21.6
10.4
IC50 = 41.6
16.7
87.9 IC50 = 2.6
IC50 = 0.84
89.6
IC50 = 1.9
IC50 = 0.35
l
M
n.d.
IC50 = 32.3
n.d.
Mc (42)e
l
Mc (50)
l
Mc
l
Mc (38)
Mc (86)
e
e
lM
l
e
e
IC50 = ꢀ100
l
Mc (286)
IC50 = 63.0
l
Mc
Mc
IC50 = 30.3
n.d.
l
e
(180)
14
15
16
Br
Br
H
H
H
H
H
H
H
OEt
H
31.9
IC50 = 20.6
65.9
36.8
IC50 = 17.1
70.5
19.1
30.4
l
M
lM
OBu
OBu
H
n.d.
IC50 = 4.9
70.8
l
M
M
IC50 = 4.9
58.3
lM
NO2
47.2
IC50 = 7.5 lM
n.d.
IC50 = 3.2
l
IC50 = 6.5
l
M
NI, No inhibition observed.
n.d., Not determined.
a
The data represent the mean value of two independent experiments. Standard deviations were within 10% of these mean values.
The inhibitory activities at 10 lM of each compound and IC50 values were determined with the primary assay, first line.
The IC50 values of the selected 5 compounds were determined with the secondary assay, second line.
The compound precipitated at higher concentrations under assay conditions.
b
c
d
e
Values in parenthesis, determined on the basis of the secondary assay results, represent fold selectivity for AKR1C3.
Screening of the compounds against recombinant AKR1C1–
formed into an ethoxy (R4 = OEt, compound 14) or butoxy
(R4 = OBu, compound 15) moiety, the inhibition shifted towards
AKR1C1–C2 isoforms, whereas inhibition of AKR1C3 was almost
completely lost. The importance of the hydroxyl group was also
confirmed when we compared the inhibitory activities of com-
pounds 2 (3.4% AKR1C3 inhibition) and 3 (3.5% AKR1C3 inhibition),
bearing a NO2 group at the meta position on B ring (R4 = NO2), with
AKR1C416 gave the results presented in Table 1. We found that
compounds that possessed either NO2 (1–4) or NH2 group (5–7)
at the meta position of B ring (R4 = NO2 or NH2) and in addition
contained different substituents on the anthranilic acid (A) ring
were inactive on all AKR1C isoforms. The only exception was com-
pound 8 that had an additional fluorine atom positioned ortho with
respect to the NO2 group on the ring B (R4 = NO2, R5 = F). It exhib-
ited quite potent inhibitory activity against AKR1C1 and AKR1C2
their corresponding hydroxy analogs 10 (IC50 = 2.2
lM) and 11
(IC50 = 5.2 M). The same pattern was observed when inhibitory
l
with IC50 values of 8.4
AKR1C3 isoform inhibition was observed.
l
M and 15.6
l
M, respectively, whereas no
potency of compounds 5 and 6 were compared to the inhibitory
potency of compounds 9 and 11. The latter two compounds pos-
sessed a OH group (R4 = OH) instead of a NH2 moiety at the meta
position of B ring (R4 = NH2); again this substitution led to in-
creased AKR1C3 inhibitory potency (Table 1). We also found that
the substitution on the anthranilic acid (A) ring does not play a sig-
nificant role in AKR1C3 inhibition as the inhibitory potencies of
compounds 9–13 which bear variable substituents on this ring
AKR1C3 inhibition was, however, achieved with compounds 9–
13. On the basis of these results we were able to postulate an initial
structure–activity relationship for 1C3 isoform inhibition. We
found that the presence of the hydroxy group at the meta position
on the B ring (R4 = OH, compounds 9–13) was essential for selec-
tive AKR1C3 inhibition. However, if the hydroxyl group was trans-

5950
M. Sinreih et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5948–5951
gave comparable IC50 values ranging from 1.9 to 12.7
l
M. How-
It is interesting to note that this hypothetical binding pose is differ-
ent from binding poses of highly related N-phenylaminobenzo-
ate,22 which were shown to occupy the SP1 binding pocket
(formed by Ser118, Asn167, Phe306, Phe311, and Tyr319), simi-
larly as flufenamic acid.23
ever, due to some subtle differences in the IC50 values we hypoth-
esized that introduction of a large bromine atom to the meta
position on the A ring (R1 = Br) to yield compound 13, would pro-
duce a more potent inhibitor. Compound 13 was found to have an
IC50 value of 1.9
all three isoforms 1C1–1C3, with IC50s of 3.2, 6.5, and 7.5
respectively.
l
M for AKR1C3 (Table 1). Compound 16 inhibited
To conclude, we have evaluated a series of N-benzoyl anthra-
nilic acid derivatives for their inhibition of aldo–keto reductases
AKR1C1–C4 and discovered some new selective inhibitors of
AKR1C3, an important drug target. The results represent an impor-
tant basis for the synthesis of next generation of AKR1C3 inhibi-
tors. As the synthetic procedures to obtain structurally related
derivatives are very straightforward, we are certain that further
improvements can be achieved. Both phenyl rings (A and B) offer
several possibilities for the introduction of new functionalities at
different positions. The most promising way forward is to synthe-
size compounds which retain moieties that seem to play an impor-
tant role for selective AKR1C3 inhibition and to explore the SAR of
different compounds with varied substitution patterns on the
anthranilic acid phenyl ring. These optimized compounds will have
the potential to be further developed into drug candidates for
treatment of hormone dependent and independent forms of pros-
tate and breast cancers.
lM,
Those compounds that were selective AKR1C3 inhibitors in the
primary screen against 1C1–1C3 isoforms, were subjected to a con-
firmatory screen which included AKR1C4. These compounds
showed even better inhibitory potencies (Table 1), which we assign
to differences in assay procedures and conditions.16 As the IC50 val-
ues for compounds 9–13 were determined on all AKR1C isoforms,
we were able to calculate the range of selectivity for the most po-
tent AKR1C3 inhibitors. Here, the most promising compound was
13 with an IC50 value of 0.35 lM on the AKR1C3 isoform and it
exhibited 286-, 180- and 86-fold selectivity for AKR1C3 in compar-
ison with isoforms 1C1, 1C2 and 1C4, respectively (Table 1). Also,
compound 10 seems very promising as it was similarly selective
as compound 13. These two inhibitors of AKR1C3 are amongst
the most potent selective non-steroidal inhibitors published so
far. The only significantly more potent inhibitors were steroidal
lactones, which were active in nanomolar concentrations. How-
ever, their selectivity over other AKR1C isoforms has not been
demonstrated.17,18
To enhance our understanding of the results of enzymatic as-
says and to confirm our postulated SAR we used molecular dock-
ing19,20 to predict the hypothetical binding pose of compound 13
in the active site of AKR1C3 (PDB code 1S2A).21 The predicted bind-
ing pose of 13 showed several important interactions. With its car-
boxyl group, it was predicted to form H-bonds with the catalytic
tetrad members Tyr55 and His117 (Fig. 1). Surprisingly, the other
part of compound 13 (ring B) was predicted to bind to the SP3
binding pocket21,22 composed of Tyr24, Glu192, Ser221 and
Tyr305 which is similar to the binding mode of indomethacin.23
The 3-hydroxy group of ring B was predicted to form H-bonds with
References and notes
1. Barski, O. A.; Tipparaju, S. M.; Bhatnagar, A. Drug. Metab. Rev. 2008, 40, 553.
2. Penning, T. M.; Drury, J. E. Arch. Biochem. Biophy. 2007, 464, 241.
3. Bauman, D. R.; Rudnick, S. I.; Szewczuk, L. M.; Jin, Y.; Gopishetty, S.; Penning, T.
M. Mol. Pharmacol. 2005, 67, 60.
4. Wang, S.; Yang, Q.; Fung, K. M.; Lin, H. K. Mol. Cel. Endocrinol. 2008, 289, 60.
5. Adeniji, A. O.; Twenter, B. M.; Byrns, M. C.; Jin, Y.; Winkler, J. D.; Penning, T. M.
Bioorg. Med. Chem. Lett. 2011, 21, 1464.
6. Desmond, J. C.; Mountford, J. C.; Drayson, M. T.; Walker, E. A.; Hewison, M.;
Ride, J. P.; Loung, Q. T.; Hayden, R. E.; Vanin, E. F.; Bunce, C. M. Cancer Res. 2003,
63, 505.
7. Dozmorov, M. G.; Azzarelo, J. T.; Wren, J. D.; Fung, K. M.; Yang, Q.; Davis, J. S.;
Hurst, R. E.; Culkin, D. J.; Penning, T. M.; Lin, H. K. BMC Cancer 2010, 10, 672.
8. Lewis, M. J.; Wiebe, J. P.; Heathcote, J. G. BMC Cancer 2004, 4, 27.
ˇ
9. Hevir, N.; Vovk, K.; Pucelj, M. R.; Rizner, T. L. Chem.-Biol. Interact. 2011, 191, 217.
10. Penning, T. M.; Talalay, P. Med. Sci. 1983, 80, 4504.
ˇ ˇ
ˇ
11. Gobec, S.; Brozic, P.; Rizner, T. L. Bioorg. Med. Chem. Lett. 2005, 15, 5170.
ˇ
12. Sosic, I.; Turk, S.; Sinreih, M.; Trošt, N.; Verlaine, O.; Amoroso, A.; Zervosen, A.;
Ser221 and the backbone nitrogen of Gln222. Additional p–p inter-
Luxen, A.; Joris, B.; Gobec, S. Acta Chim. Slov. 2012, 59, 380.
actions were predicted to form between ring B and Tyr24. The
interactions with SP3 binding pocket seem to be crucial for good
AKR1C3 inhibitory activity in this series as compounds 14 and 15
with alkylated hydroxy groups exhibited lower AKR1C3 inhibition,
most probably due to the loss of H-bonds with Ser221 and Gln222.
ˇ ˇ
ˇ
13. Brozic, P.; Šmuc, T.; Gobec, S.; Rizner, T. L. Mol. Cell. Endocrinol. 2006, 259, 30.
ˇ ˇ
ˇ
14. Brozic, P.; Turk, S.; Lanišnik-Rizner, T.; Gobec, S. Curr. Med. Chem. 2011, 18,
2554.
15. Experimental
procedure
for
the
preparation
To
of
methyl
2-(3-
of 3-
hydroxybenzamido)-4,5-dimethoxybenzoate:
a
solution
hydroxybenzoic acid (138.12 mg, 1.0 mmol) in CH2Cl2 (10 mL), pyridine
(99 mg, 1.25 mmol) and SOCl2 (773 mg, 6.5 mmol) were slowly added. After
stirring at 45 °C for 2 h, the solvent was removed under reduced pressure. The
reaction mixture was dissolved in toluene, and then methyl 2-amino-4,5-
dimethoxybenzoate (264 mg, 1.25 mmol) was added. The reaction mixture
was stirred at 100 °C for 3 h. After the reaction was complete (monitored by
TLC), the solvent was evaporated, then an aqueous solution of Na2CO3 (10%,
10 mL) was added, and the aqueous layer was extracted with CH2Cl2
(3 Â 10 mL). The combined organic phases were washed with brine
(2 Â 20 mL) and dried over Na2SO4. The solvent was evaporated, and the
pure product was obtained by crystallization from EtOH. Yield, 71% (235 mg).
Experimental procedure for the preparation of 2-(3-hydroxybenzamido)-4,5-
dimethoxybenzoic acid (11): To
a stirred solution of the methyl 2-(3-
hydroxybenzamido)-4,5-dimethoxybenzoate (165 mg, 0.5 mmol) in dioxane/
THF mixture (1:1, 2 mL), 1 M NaOH (1 mL) was added, and the reaction
mixture stirred until the starting material had completely reacted (monitored
by TLC). The solvent was then evaporated under reduced pressure, the residue
diluted with H2O (10 mL), and washed with EtOAc (2 Â 10 mL). The aqueous
phase was acidified to pH
2 using 1 M HCl, and extracted with EtOAc
(3 Â 10 mL). The combined organic phases were washed with brine
(3 Â 10 mL), then dried over Na2SO4, filtered, and evaporated to dryness, to
provide compound 11. Yield, 74% (117 mg).
Spectroscopic data for 2-(3-hydroxybenzamido)-4,5-dimethoxybenzoic acid
(11): White solid; Yield: 51% (2 steps); Rf 0.49 (CH2Cl2/MeOH/AcOH = 9/1/0.1);
Mp: 245.0–248.0 °C; 1H NMR (400 MHz, DMSO-d6): d 3.79 (s, 3H, OCH3), 3.87
(s, 3H, OCH3), 6.95–7.03 (m, 1H, Ar-H), 7.32–7.39 (m, 3H, Ar-H), 7.49 (s, 1H, Ar-
H), 8.52 (s, 1H, Ar-H), 9.91 (br s, 1H, NHCO), 13.51 (br s, 1H, COOH); 13C NMR
(100 MHz, DMSO-d6): d 55.54 (2C), 102.98, 107.47, 112.83, 113.84, 117.15,
119.01, 129.95, 135.95, 136.98, 143.58, 153.25, 157.76, 164.41, 169.72; HRMS
(ESI) m/z calcd for C16H15N2O5 [M+H]+ 318.0978, found 318.0969; HPLC purity:
95.54%, retention time: 14.06 min. HPLC was performed on an Agilent Eclipse
Figure 1. The predicted binding conformation of compound 13 (blue) in the active
site of AKR1C3. Relevant enzyme residues are shown as green sticks, the catalytic
tetrad is presented as yellow sticks, the co-crystallized indomethacin as magenta
sticks and the cofactor as orange sticks. Compound 13 occupies a portion of the SP3
pocket which up until now has only been occupied by indomethacin.

M. Sinreih et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5948–5951
5951
C18 column (4.6 Â 50 mm, 5
l
m), with a flow rate of 1.0 mL/min, detection at
mixture consisted of 100 mM phosphate buffer, pH 7.0, S-tetralol (in DMSO),
inhibitor (in DMSO), 200
M NADP+, and purified recombinant enzyme to give
a total volume of 200 l, and 4% DMSO. The concentration of S-tetralol used in
the assays for each AKR1C isoform was equal to the Km value for the respective
enzyme. The Km value obtained for S-tetralol for AKR1C1, AKR1C2, AKR1C3
254 nm, and an eluent system of: A = 0.1% TFA in H2O; B = MeOH. The
following gradient was applied: 0–3 min, 40% B; 3–18 min, 40% B ? 80% B; 18–
23 min, 80% B; 23–30 min, 80% B ? 40% B. The run time was 30 min, at a
temperature of 25 °C.
l
l
16. The primary inhibition assay: Recombinant enzymes AKR1C1–AKR1C3 were
prepared as described previously (13). Inhibition of the individual isoenzyme
was monitored spectrophotometrically in the direction of oxidation, with 1-
acenaphthenol as a substrate and NAD+ as a coenzyme. Reaction was followed
and AKR1C4 under the same experimental conditions are 8
lM, 22.5 lM,
165 M, and 25 M, respectively. Enzyme concentrations used for AKR1C1,
l
l
AKR1C2, AKR1C3 and AKR1C4 were 111 nM, 86 nM, 95 nM and 184 nM,
respectively. Initial velocity from progress curves was calculated with the
GEN5 software. Inhibition data were fit using Grafit 5.0[y = (range)/(1+I/IC50)s)
+ background] to give the IC50 values.
by measuring the increase in NADH absorbance (
absence and presence of each of the compound. The assays were carried out in
a 300 L volume that included 100 mM phosphate buffer (pH 9.0), 0.005%
triton X-114 and 5% DMSO as a co-solvent. A substrate concentration of 30
(Km), 60 M (Km) and 100 M (<Km) and an enzyme concentration of 0.1
0.3 M, and 1.5 M were used for assays with AKR1C1, AKR1C2 and AKR1C3,
respectively, in the presence of 2.3 mM NAD+. Screening was performed at
10 M concentration of compounds. For compounds that showed more than
30% AKR1C1–AKR1C3 inhibition, IC50 values were determined. The
measurements were performed on Biotek PowerWave XS microplate
e
k340 = 6220 MÀ1cmÀ1) in the
l
17. Bydal, P.; Luu-The, V.; Labrie, F.; Poirier, D. Eur. J. Med. Chem. 2009, 44, 632.
18. Qiu, W.; Zhou, M.; Mazumdar, M.; Azzi, A.; Ghanmi, D.; Luu-The, V.; Labrie, F.;
Lin, S.-X. J. Biol. Chem. 2007, 282, 8368.
lM
l
l
lM,
l
l
19. For docking experiments, FlexX 3.1 (BiosolveIT GmbH) (20) was used together
with the crystal structure of AKR1C3 (PDB code 1S2A (21). The active site was
defined as the volume of the enzyme within 6 Å from co-crystallized ligand.
The cofactor NADP+ was retained in the active site. For the base placement,
Triangle Matching was used: this programme generated the maxima of 200
solutions per iteration and 200 per fragmentation. The docking procedure was
validated since FlexX was able to reproduce the conformation of co-
crystallized ligand.
l
a
readers with initial velocities calculated, and the IC50 values were
determined graphically, using GraphPad Prism Version 4.00 (GraphPad
Software, Inc.).
The secondary inhibition assay: The potency of the compounds was determined
also by measuring their ability to inhibit the NADP+ dependent oxidation of S-
(+)-1,2,3,4-tetrahydro-1-naphthol (S-tetralol) catalyzed by AKR1C1–AKR1C4
enzymes. The continuous assay was conducted using a 96-well plate format
and the reaction measured the appearance of NADPH fluorimetrically (Exc/
Emi, 340/460 nm) on a BIOTEK Synergy 2 Multimode plate reader. The assay
20. Rarey, M.; Kramer, B.; Lengauer, T.; Klebe, G. J. Mol. Biol. 1996, 261, 470.
21. Byrns, M. C.; Jin, Y.; Penning, T. M. J. Steroid. Biochem. Mol. Biol. 2011, 125, 95.
22. Chen, M.; Adeniji, A. O.; Twenter, B. M.; Winkler, J. D.; Christianson, D. W.;
Penning, T. M. Bioorg. Med. Chem. Lett. 2012, 22, 3492.
23. Lovering, A. L.; Ride, J. P.; Bunce, C. M.; Desmond, J. C.; Cummings, S. M.; White,
S. A. Cancer Res. 1802, 2004, 64.