Discovery of substituted 3-(phenylamino)benzoic acids as potent and selective inhibitors of type 5 17β-hydroxysteroid dehydrogenase (AKR1C3)

Article abstract of DOI:10.1016/j.bmcl.2011.01.010

Aldo-keto reductase 1C3 (AKR1C3) also known as type 5 17β- hydroxysteroid dehydrogenase has been implicated as one of the key enzymes driving the elevated intratumoral androgen levels observed in castrate resistant prostate cancer (CRPC). AKR1C3 inhibition therefore presents a rational approach to managing CRPC. Inhibitors should be selective for AKR1C3 over other AKR1C enzymes involved in androgen metabolism. We have synthesized 2-, 3-, and 4-(phenylamino)benzoic acids and identified 3-(phenylamino)benzoic acids that have nanomolar affinity and exhibit over 200-fold selectivity for AKR1C3 versus other AKR1C isoforms. The AKR1C3 inhibitory potency of the 4′-substituted 3-(phenylamino)benzoic acids shows a linear correlation with both electronic effects of substituents and the pK_a of the carboxylic acid and secondary amine groups, which are interdependent. These compounds may be useful in treatment and/or prevention of CRPC as well as understanding the role of AKR1C3 in endocrinology.

Full text of DOI:10.1016/j.bmcl.2011.01.010

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1464–1468
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of substituted 3-(phenylamino)benzoic acids as potent and
selective inhibitors of type 5 17b-hydroxysteroid dehydrogenase (AKR1C3)
Adegoke O. Adeniji a, Barry M. Twenter b, Michael C. Byrns a, Yi Jin a, Jeffrey D. Winkler b,
,
⇑
Trevor M. Penning a,
⇑
a Department of Pharmacology and Center for Excellence in Environmental Toxicology, University of Pennsylvania, School of Medicine, 130C John Morgan Bldg., 3620 Hamilton
Walk Philadelphia, PA 19104-6084, United States
b Department of Chemistry, University of Pennsylvania, 231 S 34th Street, Philadelphia, PA 19104-6323, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Aldo-keto reductase 1C3 (AKR1C3) also known as type 5 17b-hydroxysteroid dehydrogenase has been
implicated as one of the key enzymes driving the elevated intratumoral androgen levels observed in cas-
trate resistant prostate cancer (CRPC). AKR1C3 inhibition therefore presents a rational approach to man-
aging CRPC. Inhibitors should be selective for AKR1C3 over other AKR1C enzymes involved in androgen
metabolism. We have synthesized 2-, 3-, and 4-(phenylamino)benzoic acids and identified 3-(phenylami-
no)benzoic acids that have nanomolar affinity and exhibit over 200-fold selectivity for AKR1C3 versus
other AKR1C isoforms. The AKR1C3 inhibitory potency of the 40-substituted 3-(phenylamino)benzoic
acids shows a linear correlation with both electronic effects of substituents and the pKa of the carboxylic
acid and secondary amine groups, which are interdependent. These compounds may be useful in treat-
ment and/or prevention of CRPC as well as understanding the role of AKR1C3 in endocrinology.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 11 November 2010
Revised 3 January 2011
Accepted 4 January 2011
Available online 7 January 2011
Keywords:
Aldo-keto reductase (AKR)
Castrate resistant prostate cancer (CRPC)
Non steroidal anti-inflammatory drugs
(NSAIDs)
N-Phenylanthranilic acids
Androgen biosynthesis
Prostate cancer (PC) is the second most common cancer in
American men and is responsible for about 11% of all cancer re-
lated deaths.1,2 PC is initially dependent on testicular androgens
and responsive to androgen ablation. However, the therapeutic
benefit of the androgen deprivation therapy is temporary as it is of-
ten followed by recurrence of a more aggressive metastatic disease,
known as castrate resistant prostate cancer (CRPC). CRPC is charac-
terized by elevated intratumoral androgen levels, increased andro-
gen receptor (AR) signaling and expression of pro-survival genes
despite castrate level circulating androgen concentrations.3–5 The
source of intratumoral androgens is likely dehydroepiandrosterone
(DHEA) from the adrenal, which is subsequently metabolized to
adrenal pituitary axis and prevent the undesirable build up of
the mineralocorticoid desoxycorticosterone.6,8
This intratumoral conversion of DHEA to active androgens is
driven primarily by elevated expression of key enzymes in the
androgen biosynthetic pathway.5,9,10 In particular, type 5, 17b-
hydroxysteroid dehydrogenase (HSD), also known as aldo-keto
reductase 1C3 (AKR1C3) has been shown to be among the most
highly upregulated enzymes in CRPC.10–12 This enzyme catalyzes
the reduction of the weak androgen, 4-androstene-3,17-dione to
testosterone which can subsequently be reduced by 5
a-reductases
to 5a
-DHT.13,14 (Figure 1) It represents a superior drug target to
CYP17 since it catalyzes the final step in testosterone production.
AKR1C3 is also involved in prostaglandin biosynthesis, as it cat-
alyzes the NADPH dependent formation of pro-proliferative signal-
ing molecules, prostaglandin (PG) F2 and 11b-PGF2 from PGH2
testosterone and 5
way shown in Figure 1.
Recently, there has been clinical success with the CYP17
a-dihydrotestosterone (5a-DHT) by the path-
a
-
a
a
hydroxylase/17,20-lyase (CYP17) inhibitor, abiraterone acetate in
the treatment of CRPC, endorsing the concept that intratumoral
androgen biosynthesis from DHEA plays a role.6,7 Abiraterone
acetate is generally given with a glucocorticoid to suppress the
and PGD2, respectively.15–17 The reduction of PGH2 and PGD2 by
AKR1C3 also prevents the spontaneous conversion of PGD2 to the
anti-proliferative product, 15-deoxy
D
12,14 PGJ2.18,19 The net effect
of increased AKR1C3 expression and activity as seen in CRPC, will
be an increase in cellular proliferative signaling and tumor growth.
AKR1C3 thus represents a rational therapeutic target for the man-
agement of CRPC irrespective of whether it is hormone dependent.
⇑
Corresponding authors. Tel.: +1 215 898 0052; fax: +1 215 573 6329 (J.D.W.);
AKR1C1 (20a-HSD) and AKR1C2 (type 3 3a-HSD) are closely re-
tel.: +1 215 898 9445; fax: +1 215 573 2236 (T.M.P.).
E-mail addresses: winkler@sas.upenn.edu (J.D. Winkler), penning@upenn.edu
(T.M. Penning).
lated enzymes with >86% sequence identity to AKR1C3, both of
which can act on androgens.20,21 The two enzymes catalyze the
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.010

A. O. Adeniji et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1464–1468
1465
Figure 1. Role of AKR1C1-3 in androgen biosynthesis in the prostate (SRD5A1 and SRD5A2 are type 1 and 2 5
a-reductases, respectively; AR = androgen receptor, 3b-HSD/KSI:
3b-hydroxysteroid dehydrogenase/ketosteroid isomerase, 3b-Adiol = 5 -androstane-3b,17b-diol, 3 -Adiol = 5
a
a
a
-androstane-3 ,17b-diol, ERb = estrogen receptor b)
a
conversion of 5
and -androstane-3
(Fig. 1).21,22 3b-Adiol is a pro-apoptotic ligand for estrogen receptor
b, while 3
-Adiol is an inactive androgen.22,23 Inhibition of AKR1C1
a
-DHT to 5
a-androstane-3b,17b-diol (3b-Adiol)
5a
a,17b-diol
(3a-Adiol), respectively
a
and AKR1C2 in the prostate can therefore be expected to increase
androgen dependent proliferative signaling. This makes it impera-
tive that inhibitors developed for AKR1C3 should have little or no
effect on its closely related isoforms.
Non-steroidal anti-inflammatory drugs (NSAIDs) are known to
be inhibitors of the AKR1C enzymes.24 NSAIDs based on N-pheny-
lanthranilic acids (N-PA), in particular, are known to be potent and
non-selective inhibitors of AKR1C enzymes.25 Developing N-PA-
based AKR1C3 inhibitors that are devoid of cyclooxygenase (COX)
or other AKR1C enzyme inhibitory activity is desirable since much
is known about the absorption, distribution, metabolism, excretion
and toxicity (ADMET) of this class of drugs. Based on known struc-
ture–activity relationships for COX isozymes, substitution on the
A-ring of an N-PA, or movement of the carboxylic acid from the
ortho-position would eliminate COX inhibition.25-27 Also, the
X-ray crystal structure of the AKR1C3ꢀNADP+ꢀflufenamic acid com-
plex (PDB# 1S2C) provides insights on how the prototypical N-PA,
flufenamic acid is bound. Its carboxylic acid group is anchored via
hydrogen bond interaction at the oxyanion hole of the active site,
which is formed by the cofactor and catalytic tetrad. The secondary
amine of the drug also forms hydrogen bond interactions with the
carboxamide oxygen of the nicotinamide ring of the cofactor, while
the B-ring containing the meta-trifluoromethyl group substituent
is bound in a distinct subpocket.28,29 Analysis of this subpocket
in AKR1C3 showed that it was larger, different in shape and lined
by polar amino acids (S118, S308 and Y319) relative to either
AKR1C1 or AKR1C2, where the corresponding residues are F118,
L308, and F319.25,29 This suggested that appropriate substitution
on the B-ring may provide the needed AKR1C3 selectivity.
In this study, we have systematically examined the effect of the
movement of the carboxylic acid on the A-ring of an N-PA and con-
currently measured the effect of substituents on the B-ring. This
has led to a series of 3-(phenylamino)benzoic acid derivatives that
are potent and selective inhibitors for AKR1C3 over AKR1C1 and
AKR1C2. (Fig. 2 and Table 1)
Figure 2. Synthetic scheme for 2-, 3-, and 4-(phenylamino)benzoic acid analogs,
compounds 1–12 (see Table 1): (a) Pd(OAc)2, 2,20-bis(diphenylphosphino)-1,10-
binaphthyl (BINAP), Cs2CO3, toluene, 120 °C; (b) KOH, EtOH, H2O, 100 °C.
Compounds were synthesized according to the scheme in Fig-
ure 2. The reaction involved a Buchwald–Hartwig C–N coupling
followed by methyl ester saponification.30–32 In most cases, the
product crystallizes out of the aqueous medium upon acidification
allowing the product to be collected by a simple vacuum filtration.
Compounds were characterized by NMR analysis and high resolu-
tion mass spectrometry (see Supplementary data).33
AKR1C1 and AKR1C2 share 98% sequence identity and differ in
only one amino acid at their respective active sites.20,34 It is there-
fore likely that an inhibitor of AKR1C2 will also inhibit AKR1C1.
Based on this assumption, the screening strategy adopted was to
determine the inhibitory potency of each compound against
AKR1C2 and AKR1C3 and use the ratio of IC50 value for AKR1C2:
1C50 value for AKR1C3 as an index of compound selectivity for
AKR1C3. The inhibitory potency of compounds on AKR1C2 and
AKR1C3 was determined by measuring the inhibition of the NADP+
dependent oxidation of S-tetralol.35 All assays were performed at
the KM of the respective AKR1C isoforms so that IC50 values were
directly comparable.
Our lead compound was the prototypic N-PA, flufenamic acid
(o-CO2H, 30-CF3) 1, which gave an IC50 value of 50 nM for AKR1C3
and showed a modest sevenfold selectivity for the inhibition of
AKR1C3 over AKR1C2, Table 1. We first synthesized compounds

1466
A. O. Adeniji et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1464–1468
Table 1
Compound structure and inhibitory potency on AKR1C2 and AKR1C3
Compounds
AKR1C3 IC50
0.051
(
lM)
AKR1C2 IC50
0.37
(l
M)
Ratio IC50 values AKR1C2:AKR1C3
HO2C
H
N
CF3
1
7.3
Flufenamic acid
HO2C
H
N
2
3
4
1.5
0.44
13
0.29
14
H
N
HO2C
0.94
2.8
H
N
3.0
1.1
HO2C
HO2C
H
N
5
6
7
8
9
0.036
0.054
0.062
0.13
3.4
19
15
19
18
94
NO2
COCH3
CF3
Cl
H
N
HO2C
HO2C
360
250
150
150
H
N
H
N
HO2C
HO2C
HO2C
H
N
0.12
Br
H
N
10
0.28
31
110
H
N
HO2C
HO2C
11
12
0.49
0.70
11
56
23
80
OCH3
CH3
H
N
2–4 to evaluate the effect of changing the position of the carboxylic
acid group on the A-ring on the inhibitory potency and selectivity
for AKR1C3. The B-ring unsubstituted compounds were initially
chosen to preclude substituent effects. It was found that move-
ment of the CO2H group had no remarkable effect on the inhibitory
potency for AKR1C3, thus, ortho-, meta- and para-carboxylate gave
analog of 1 was 30-fold less potent as an AKR1C3 inhibitor than 1,
suggesting that B-ring substitution was important for potency. This
loss of potency is expected given the greater distance and conse-
quently, reduced interactions between the B-ring and the subpock-
et in the absence of a substituent on the B-ring. Using compound 3
as the new lead (m-CO2H on the A-ring), eight B-ring para substi-
tuted analogs of 3, 5–12 were synthesized and evaluated to assess
the effects of different substituents on AKR1C3 potency and selec-
tivity. The p-substituents were selected since they should project
furthest into the subpocket, increasing the potential for interaction
with the relevant amino acid residues.
IC50 values of 1.5 lM, 0.94 lM and 2.8 lM for compounds 2, 3, and
4, respectively. These compounds offered two important clues to
further inhibitor development. First, compound 3 showed a 14-fold
selectivity for the inhibition of AKR1C3 over AKR1C2, indicating a
48-fold increase in AKR1C3 selectivity with movement of the car-
boxylic acid from o- to m-position (compare IC50 value ratios for
compounds 2 and 3), suggesting that the m-CO2H may be impor-
tant for selectivity. Second, compound 2, the B-ring unsubstituted
As shown in Table 1, the lowest IC50 values for AKR1C3 inhibi-
tion were obtained with an electron withdrawing group (EWG) as
the B-ring substituent, such as 5 (p-NO2) which gave a value of

A. O. Adeniji et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1464–1468
1467
36 nM. Compound 6 (p-Ac) and 7 (p-CF3) were the two most
selective inhibitors and gave IC50 values that were 360 and
250-fold selective for AKR1C3 over AKR1C2, respectively. However,
all the B-ring para substituted analogs (both EWG and EDG)
exhibited significantly better inhibitory potency for AKR1C3 and
were also more selective for AKR1C3 relative to AKR1C2 than the
parent compound 3.
Interestingly, analogs with EWG, that is, compounds 5–9, dis-
played relatively similar AKR1C3 inhibitory potency with flufen-
amic acid, 1, while those with electron donating substituents
(EDG), compounds 10–12 displayed weaker AKR1C3 inhibitory
activity than 1. In contrast, all B-ring para-substituted compounds
bearing an A-ring m-CO2H display significantly higher IC50 values
than flufenamic acid for AKR1C2, regardless of the electronic prop-
erties of the substituent. The increase in IC50 values for AKR1C2
due to the presence of the m-CO2H group results in increased
AKR1C3 selectivity.
A regression analysis of the electronic effect of the varied sub-
stituent at the para position and the inhibitory potency for AKR1C2
and AKR1C3 was then performed. The plot revealed a significant
correlation between the electronic effect and AKR1C3 potency for
the para-substituted 3-(phenylamino)benzoic acid analogs
(R2 = 0.81, p = 0.0009), Figure 3. However, no significant correlation
between the electronic effect and inhibitory potency for AKR1C2
(R2 = 0.37, p = 0.084) was noted for these compounds.
Further analysis showed there was also a significant correlation
between the AKR1C3 potency and the calculated pKa of the carbox-
ylic acid group (R2 = 0.80, p = 0.0012) and the pKa of the secondary
amine (R2 = 0.79, p = 0.0014), both of which are related to the elec-
tronic effect of the B ring substituents. Our data on the electronic
effects seen with the AKR1C3 inhibitors is consistent with these
compounds forming hydrogen bonds with AKR1C3 at the active
site. EWG or EDG on the B-ring that alter the acidity of the carbox-
ylic acid and basicity of the secondary amine group in particular
would be expected to affect the inhibitory potency of the com-
pounds. The electronic effect observed for AKR1C3 inhibition could
also be as a result of discrete interactions between residues lining
the subpocket and the B-ring substituents.
from binding in the expected subpocket in this isoform, presum-
ably as a result of the smaller size of this pocket. We speculate that
it is likely that the B-ring of these inhibitors is forced to occupy the
larger vacant steroid binding site in AKR1C2. Further X-ray crystal-
lographic studies will be required to determine the precise mech-
anism by which these compounds interact with the two enzymes
and account for the selectivity seen with m-COOH analogs.
Several AKR1C3 inhibitors of varied structural classes have been
reported in the literature.36–42 However, selectivity for AKR1C3
over AKR1C1-2 is often low or not indicated. We have developed
3-(phenylamino) benzoic acid analogs that are potent inhibitors
of AKR1C3 with orders of magnitude selectivity for AKR1C3. These
compounds highlight structural modifications on N-PA that are
essential for AKR1C3 inhibition and confer selectivity over AKR1C1
and AKR1C2. A meta arrangement of the carboxylic acid and sec-
ondary amine moieties as well as a strong electron withdrawing
group at the para position of the B-ring confers selectivity for
AKR1C3 over AKR1C2.
N-PA are known to be competitive inhibitors of AKR1C3. The
compounds in this study were also found to inhibit AKR1C3 in a
competitive manner (data not shown). Also, they are predicted to
have little or no inhibitory activity on COX enzymes since the ortho
arrangement of the –CO2H and –NH2 on the A-ring has been shown
to be critical for COX activity.26,27,43
The success of abiraterone acetate, in clinical trials for manage-
ment of CRPC makes the discovery of these AKR1C3 selective inhib-
itors promising. Because of the position of AKR1C3 in the androgen
biosynthetic pathway in the prostate, these compounds are likely
to be more specific and have less undesirable side effects as seen
with CYP17 inhibitors.
In conclusion, we have discovered inhibitors with nanomolar
potency for AKR1C3 that exhibit significant selectivity for AKR1C3
over other AKR1C enzymes. At the very least, they may be used as
probes to elucidate the role of AKR1C3 in cell culture models. More
importantly, these compounds may serve as useful therapeutic
leads in the treatment and/or prevention of CRPC as well as other
androgen/estrogen dependent malignancies.
Hydrogen bond formation at the active site is also expected in
AKR1C2 but the lack of correlation between inhibitory potency
and the electronic effect suggests other factors might be more rel-
evant for inhibitor binding. Most of the B-ring substituted com-
pounds have relatively similar inhibitory potency on AKR1C2 and
gave IC50 values which were in the low micromolar range, it is
likely that with A-ring m-CO2H analogs, the B-ring is precluded
Acknowledgment
Supported by R01-CA90744, P30-ES013508 and Prostate Cancer
Foundation Challenge grant awarded to T.M.P. We thank Jin Park
for technical assistance during the development of the enzyme
assays.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.01.010.
References and notes
1. Jemal, A.; Siegel, R.; Xu, J.; Ward, E. CA Cancer J. Clin. 2010, 60, 277.
2. Altekruse, S.F.; Krapcho, M.; Neyman, N.; Aminou, R.; Waldron, W.; Ruhl, J.;
Howlader, N.; Tatalovich, Z.; Cho, H.; Mariotto, A.; Eisner, M.P.; Lewis, D.R.;
Cronin, K.; Chen, H.S.; Feuer, E.J.; Stinchcomb, D.G.; Edwards, B.K., Eds.; SEER
Cancer Statistics Review, 1975–2007; National Cancer Institute: Bethesda, MD,
2010.
3. Knudsen, K. E.; Scher, H. I. Clin. Cancer Res. 2009, 15, 4792.
4. Knudsen, K. E.; Penning, T. M. Trends Endocrinol. Metab. 2010, 21, 315.
5. Locke, J. A.; Guns, E. S.; Lubik, A. A.; Adomat, H. H.; Hendy, S. C.; Wood, C. A.;
Ettinger, S. L.; Gleave, M. E.; Nelson, C. C. Cancer Res. 2008, 68, 6407.
6. O’Donnell, A.; Judson, I.; Dowsett, M.; Raynaud, F.; Dearnaley, D.; Mason, M.;
Harland, S.; Robbins, A.; Halbert, G.; Nutley, B.; Jarman, M. Br. J. Cancer 2004, 90,
2317.
7. Reid, A. H.; Attard, G.; Danila, D. C.; Oommen, N. B.; Olmos, D.; Fong, P. C.;
Molife, L. R.; Hunt, J.; Messiou, C.; Parker, C.; Dearnaley, D.; Swennenhuis, J. F.;
Terstappen, L. W.; Lee, G.; Kheoh, T.; Molina, A.; Ryan, C. J.; Small, E.; Scher, H.
I.; de Bono, J. S. J. Clin. Oncol. 2010, 28, 1489.
Figure 3. Correlation of AKR1C3 inhibitory potency and electronic effect (r) of
substituents.

1468
A. O. Adeniji et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1464–1468
8. Attard, G.; Reid, A. H.; A’Hern, R.; Parker, C.; Oommen, N. B.; Folkerd, E.;
Messiou, C.; Molife, L. R.; Maier, G.; Thompson, E.; Olmos, D.; Sinha, R.; Lee, G.;
Dowsett, M.; Kaye, S. B.; Dearnaley, D.; Kheoh, T.; Molina, A.; de Bono, J. S. J.
Clin. Oncol. 2009, 27, 3742.
9. Titus, M. A.; Schell, M. J.; Lih, F. B.; Tomer, K. B.; Mohler, J. L. Clin. Cancer Res.
2005, 11, 4653.
10. Montgomery, R. B.; Mostaghel, E. A.; Vessella, R.; Hess, D. L.; Kalhorn, T. F.;
Higano, C. S.; True, L. D.; Nelson, P. S. Cancer Res. 2008, 68, 4447.
11. Stanbrough, M.; Bubley, G. J.; Ross, K.; Golub, T. R.; Rubin, M. A.; Penning, T. M.;
Febbo, P. G.; Balk, S. P. Cancer Res. 2006, 66, 2815.
31. Old, D. W.; Wolfe, J. P.; Buchwald, S. L. J. Am. Soc. 1998, 120, 9722.
32. Csuk, R.; Raschke, C.; Barthel, A. Tetrahedron 2004, 60, 5737.
33. (a) To a solution of bromide (or triflate) (1 equiv) in toluene (0.1 M) was added
aniline (1.2 equiv), Cs2CO3 (1.4 equiv) BINAP (0.08 equiv), and Pd(OAc)2
(0.05 equiv) at room temperature. The reaction mixture was allowed to stir
at 120 °C for 4–48 h. Once the reaction appeared to be complete by
consumption of the bromide (or triflate) by TLC analysis, the mixture was
allowed to cool to room temperature, diluted with EtOAc, washed with 2 M aq
HCl (2ꢁ), brine, and dried over sodium sulfate. The solution was concentrated,
loaded on silica gel, and purified by silica gel chromatography.
12. Hofland, J.; van Weerden, W. M.; Dits, N. F.; Steenbergen, J.; van Leenders, G. J.;
Jenster, G.; Schroder, F. H.; de Jong, F. H. Cancer Res. 2010, 70, 1256.
13. Fung, K. M.; Samara, E. N.; Wong, C.; Metwalli, A.; Krlin, R.; Bane, B.; Liu, C. Z.;
Yang, J. T.; Pitha, J. V.; Culkin, D. J.; Kropp, B. P.; Penning, T. M.; Lin, H. K. Endocr.
Relat. Cancer 2006, 13, 169.
(b) To a solution of methyl ester (1 equiv) in EtOH (0.2 M) was added KOH
(2 equiv per ester) in water (0.2 M) at room temperature. The reaction mixture
was allowed to stir at 100 °C for 1–6 h. Once the reaction appeared complete by
TLC analysis, EtOH was evaporated from the reaction mixture; the resultant
solution was cooled to 0 °C and acidified to pH 2 w 2 M aq HCl. The resultant
precipitated product was collected by vacuum filtration and washed with
water.
14. Lin, H. K.; Jez, J. M.; Schlegel, B. P.; Peehl, D. M.; Pachter, J. A.; Penning, T. M.
Mol. Endocrinol. 1997, 11, 1971.
15. Chen, D. B.; Westfall, S. D.; Fong, H. W.; Roberson, M. S.; Davis, J. S.
Endocrinology 1998, 139, 3876.
34. Jin, Y.; Stayrook, S. E.; Albert, R. H.; Palackal, N. T.; Penning, T. M.; Lewis, M.
Biochemistry 2001, 40, 10161.
16. Matsuura, K.; Shiraishi, H.; Hara, A.; Sato, K.; Deyashiki, Y.; Ninomiya, M.; Sakai,
S. J. Biochem. 1998, 124, 940.
17. Suzuki-Yamamoto, T.; Nishizawa, M.; Fukui, M.; Okuda-Ashitaka, E.; Nakajima,
T.; Ito, S.; Watanabe, K. FEBS Lett. 1999, 462, 335.
18. Desmond, J. C.; Mountford, J. C.; Drayson, M. T.; Walker, E. A.; Hewison, M.;
Ride, J. P.; Luong, Q. T.; Hayden, R. E.; Vanin, E. F.; Bunce, C. M. Cancer Res. 2003,
63, 505.
35. The inhibitory potency of these compounds was determined using purified
homogenous recombinant enzymes. Full concentration response studies were
conducted and IC50 values calculated using Grafit 5.0 software. The compounds
were evaluated for their effects on enzyme catalyzed oxidation of S-tetralol
using a 96-well format. The reaction was fluorimetrically (exc/em; 340 nm/
460 nm) monitored by the measurement of NADPH production on a BIOTEK
Synergy 2 Multimode plate reader at 37 °C. Assay mixture consists of S-tetralol
19. Forman, B. M.; Tontonoz, P.; Chen, J.; Brun, R. P.; Spiegelman, B. M.; Evans, R. M.
Cell 1995, 83, 803.
(in DMSO), inhibitor (in DMSO), 100 mM phosphate buffer, pH 7.0, 200
NADP+, and purified recombinant enzyme (30
l) to give a total volume of
200 and 4% DMSO. The S-tetralol concentration used for AKR1C2 and
AKR1C3 inhibition assay were 22.5 M and 165 M, respectively, equal to the
lM
l
20. Burczynski, M. E.; Harvey, R. G.; Penning, T. M. Biochemistry 1998, 37, 6781.
21. Penning, T. M.; Burczynski, M. E.; Jez, J. M.; Hung, C. F.; Lin, H. K.; Ma, H.;
Moore, M.; Palackal, N.; Ratnam, K. Biochem. J. 2000, 351, 67.
22. Steckelbroeck, S.; Jin, Y.; Gopishetty, S.; Oyesanmi, B.; Penning, T. M. J. Biol.
Chem. 2004, 279, 10784.
23. Guerini, V.; Sau, D.; Scaccianoce, E.; Rusmini, P.; Ciana, P.; Maggi, A.; Martini, P.
G.; Katzenellenbogen, B. S.; Martini, L.; Motta, M.; Poletti, A. Cancer Res. 2005,
65, 5445.
ll
l
l
KM obtained for the respective isoforms under the same experimental
conditions. Initial velocity, obtained by linear regression of the progress
curve, in the presence of varying concentration of inhibitor was compared to
solvent control to give percent inhibition values. IC50 values were obtained
from a single experiment with each concentration run in quadruplicate.
36. Koda, N.; Tsutsui, Y.; Niwa, H.; Ito, S.; Woodward, D. F.; Watanabe, K. Arch.
Biochem. Biophys. 2004, 424, 128.
24. Penning, T. M.; Talalay, P. Proc. Natl. Acad. Sci. U.S.A. 1983, 80, 4504.
25. Bauman, D. R.; Rudnick, S. I.; Szewczuk, L. M.; Jin, Y.; Gopishetty, S.; Penning, T.
M. Mol. Pharmacol. 2005, 67, 60.
37. Davies, N. J.; Hayden, R. E.; Simpson, P. J.; Birtwistle, J.; Mayer, K.; Ride, J. P.;
Bunce, C. M. Cancer Res. 2009, 69, 4769.
26. Lombardino, J. G. Nonsteroidal Antiinflammatory Drugs; Wiley Interscience:
New York, NY, 1985.
38. Stefane, B.; Brozic, P.; Vehovc, M.; Rizner, T. L.; Gobec, S. Eur. J. Med. Chem.
2009, 44, 2563.
27. Scherrer, A. R. Antiinflammatory Agents: Chemistry and Pharmacology; Academic
Press: New York, NY, 1974.
28. Lovering, A. L.; Ride, J. P.; Bunce, C. M.; Desmond, J. C.; Cummings, S. M.; White,
S. A. Cancer Res. 2004, 64, 1802.
39. Bydal, P.; Luu-The, V.; Labrie, F.; Poirier, D. Eur. J. Med. Chem. 2009, 44, 632.
40. Qiu, W.; Zhou, M.; Mazumdar, M.; Azzi, A.; Ghanmi, D.; Luu-The, V.; Labrie, F.;
Lin, S. X. J. Biol. Chem. 2007, 282, 8368.
41. Poirier, D. Curr. Med. Chem. 2003, 10, 453.
29. Byrns, M. C.; Jin, Y.; Penning, T. M. JSBMB 2010. doi:10.1016/
j.jsbmb.2010.11.004.
30. Hamann, B. C.; Hartwig, J. F. J. Am. Soc. 1998, 120, 3694.
42. Poirier, D. Expert Opin. Ther. Pat. 2010, 20, 1123.
43. Selinsky, B. S.; Gupta, K.; Sharkey, C. T.; Loll, P. J. Biochemistry 2001, 40,
5172.