Nonsteroidal anti-inflammatory drugs and their analogues as inhibitors of aldo-keto reductase AKR1C3: New lead compounds for the development of anticancer agents

Article abstract of DOI:10.1016/j.bmcl.2005.08.063

Nonsteroidal anti-inflammatory drugs (NSAIDs) like indomethacin, flufenamic acid, and related compounds have been recently identified as potent inhibitors of AKR1C3. We report that some other NSAIDs (diclofenac and naproxen) also inhibit AKR1C3, with the IC₅₀ values in the low micromolar range. In order to obtain more information about the structure-activity relationship and to identify new leads, a series of compounds designed on the basis of NSAIDs were synthesized and screened on AKR1C3. The most active compounds were 2-[(2,2-diphenylacetyl)amino]benzoic acid 4 (IC₅₀ = 11 μM) and 3-phenoxybenzoic acid 10 (IC₅₀ = 0.68 μM). These compounds represent promising starting points for the development of new anticancer agents.

Full text of DOI:10.1016/j.bmcl.2005.08.063

Bioorganic & Medicinal Chemistry Letters 15 (2005) 5170–5175
Nonsteroidal anti-inflammatory drugs and their analogues
as inhibitors of aldo-keto reductase AKR1C3: New lead
compounds for the development of anticancer agents
b
ˇa
ˇ
ˇ
Stanislav Gobec,a, Petra Brozˇic and Tea Lanisnik Rizner
*
a
ˇ
ˇ
Faculty of Pharmacy, University of Ljubljana, Askerceva 7, 1000 Ljubljana, Slovenia
bInstitute of Biochemistry, Medical Faculty, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia
Received 8 July 2005; revised 10 August 2005; accepted 22 August 2005
Available online 23 September 2005
Abstract—Nonsteroidal anti-inflammatory drugs (NSAIDs) like indomethacin, flufenamic acid, and related compounds have been
recently identified as potent inhibitors of AKR1C3. We report that some other NSAIDs (diclofenac and naproxen) also inhibit
AKR1C3, with the IC50 values in the low micromolar range. In order to obtain more information about the structure–activity rela-
tionship and to identify new leads, a series of compounds designed on the basis of NSAIDs were synthesized and screened on
AKR1C3. The most active compounds were 2-[(2,2-diphenylacetyl)amino]benzoic acid 4 (IC50 = 11 lM) and 3-phenoxybenzoic acid
10 (IC50 = 0.68 lM). These compounds represent promising starting points for the development of new anticancer agents.
ꢀ 2005 Elsevier Ltd. All rights reserved.
O
Selective control of the biological activity of steroids,
by inhibiting specific enzymes involved in their biosyn-
thesis and metabolism, has been an attractive pharma-
ceutical target over the last two decades. In many
cases, weak precursor hormones are converted into
more potent metabolites by specific enzymes, known
as molecular switches.1,2 The active forms have a high
affinity toward corresponding receptors and the inac-
tive forms have a very low affinity. The enzymes that
interconvert the active and inactive forms, and thus
act as molecular switches are pre-receptor regulatory
enzymes.2–4 Of these, we recently focused our atten-
tion on the human enzyme AKR1C3, a member of
the aldo-keto reductase superfamily.5 AKR1C3 is a
peripheral 17b-hydroxysteroid dehydrogenase (17b-
HSD type 5) that reduces a weak androgen, andro-
stenedione, to the potent androgen testosterone, and
a weak estrogen estrone to the potent estrogen 17b-
estradiol, using NADPH as a coenzyme (Fig. 1).6
AKR1C3 is thus an interesting target for the develop-
ment of agents for treating hormone dependent forms
of cancer like prostate cancer, breast cancer, and
OH
AKR1C3
O
O
Androstendione
Testosterone
O
OH
AKR1C3
HO
HO
Estrone
Estradiol
HO
HO
HO
COOH
COOH
AKR1C3
O
OH
OH
Prostaglandin D2
9α, 11β-Prostaglandin F2α
Figure 1. Reactions catalyzed by AKR1C3 in vivo.
Keywords: AKR1C3 inhibitors; Anti-inflammatory drugs; Anticancer
agents; Cancer chemoprevention.
endometrial cancer. As AKR1C3 also catalyzes the
reduction of 3-keto- and 20-ketosteroids and prosta-
glandin D2 (PGD2), it has been known variously as
*
Corresponding author. Tel.: +386 1 47 69 585; fax: +386 1 42 58
031; e-mail: gobecs@ffa.uni-lj.si
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.08.063

S. Gobec et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5170–5175
5171
3a-hydroxysteroid dehydrogenase type 27 and PGD2
non-COX-2 pathway.19 They demonstrated that inhibi-
tion of AKR1C3 by indomethacin prevented the prolif-
eration of human myeloid leukemia cells (HL-60).
AKR1C3 converts PGD2 into PGF2a, thereby prevent-
ing its conversion to 15-D12,14-PGJ2, a natural ligand
for the peroxisome proliferator-activated receptor-c
(PPARc). Inhibitors of AKR1C3 are thus potential
anti-neoplastic agents as they can indirectly activate
PPARc receptor by diverting PGD2 catabolism to the
generation of J-series prostanoids. Activation of
PPARc receptor induces differentiation, is anti-prolifer-
ative, and causes apoptosis in many cell types and can-
cers.19,20 Since many of the frequently used NSAIDs
have not been evaluated for inhibition of human
AKR1C3, we have examined the inhibitory potencies
of diclofenac, naproxen, ibuprofen, ketoprofen, acetyl-
salicylic acid, paracetamol, phenacetin, and etodolac
(Table 1).
11-ketoreductase.8
Although AKR1C3 is a promising therapeutic target,
only a few inhibitors have been reported so far.9 Devel-
opment of inhibitors that consist of nonsteroid core, and
are thus devoid of residual steroidogenic activity, would
be especially attractive. Dietary phytoestrogens (such as
coumestrol, quercetin, and biochanin) and mycoestro-
gen zearalenon were reported to inhibit the enzyme in
low micromolar concentrations,10 as well as some
other small molecule compounds like benzodiazepines,11
benzofuranes, and phenolphthalein derivatives.12 Indo-
methacin, flufenamic acid, and some related nonsteroi-
dal anti-inflammatory drugs (NSAIDs, Fig. 2) are also
very potent inhibitors, as is the cyclooxygenase
(COX)-2 selective celecoxib8,13,14 However, other NSA-
IDs (e.g., naproxen, diclofenac, salicylates, etc.) have
been evaluated only on rat liver 3a-hydroxysteroid
dehydrogenase (AKR1C9), a model for the human
AKR1C isozymes.15 Starting from mefenamic acid, a
series of very active N-phenylanthranilic acid derivatives
were synthesized (Fig. 2).13 Recently, the X-ray crystal
structures of AKR1C3 complexed with indomethacin,
flufenamic acid, PGD2, and rutin were reported, provid-
ing a structural basis for rational design of new and im-
proved inhibitors.14,16
NSAIDs, like N-phenylanthranilic acid derivatives, are
excellent starting points for the design of new inhibitors
of AKR1C3. We postulated that the structurally related
N-acylanthranilic acids could be potential inhibitors of
this enzyme. In addition, Bauman et al. reported that,
Table 1. Inhibition of AKR1C3 by nonsteroidal anti-inflammatory
drugs
NSAIDs are drugs used to control inflammatory diseas-
es by inhibiting COX and, in particular, COX-2 activi-
ty. It is generally accepted that NSAIDs also protect
against the progression of gastrointestinal tumors and
also other cancers like prostate carcinoma and leuke-
mia.17 NSAIDs are anti-proliferative against a broad
spectrum of in vivo and in vitro models of human
malignancies.18 Recently, Desmond et al. suggested that
NSAIDs could exert their anti-neoplastic activities via a
NSAID
Structure
IC50 (lM)
Cl
Cl
Diclofenac
2.6
NH
O
ONa
CH3
COOH
Naproxen
Ibuprofen
Ketoprofen
Paracetamol
Phenacetin
0.48
33
CH3O
O
CH3
CO OH
CH3
COOH
Cl
R2
R1
O
(12%)a
(8%)a
R3
CH3
O
HN
N
R4
H3C
HO
H
N
OH
O
O
O
Flufenamic acid (R1=R2=R3=H; R3=CF3)
Mefenamic acid (R1=R2=CH3; R3=R4=H)
HO
Indomethacin
H
N
O
(10%)a
O
O
O
O
Acetylsalicylic acid
NIa
COOH
R1
O
HN
HO
R2
O
NIa
O
OH
Etodolac
N
H
CH2CO OH
H3C
4-Benzoylbenzoic acid
N-Phenylanthranilic acid derivatives
CH3
a The values in parentheses are the % inhibition at 50 lM concn of
inhibitor; NI, no inhibition observed.
Figure 2. Nonsteroidal anti-inflammatory drugs and related com-
pounds that inhibit AKR1C3.

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S. Gobec et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5170–5175
besides N-phenylanthranilic acids, the benzophenone
derivative 4-benzoylbenzoic acid (Fig. 2) potently
inhibits AKR1C3.13 Benzophenone derivatives and
related compounds are also interesting because it is
known that the replacement of the nitrogen atom be-
tween the two aromatic rings by a carbonyl group al-
most completely abolishes COX inhibition, while
retaining AKR1C3 inhibition.13 Due to serious, adverse
cardiovascular effects of COX-2 inhibitors,21 design of
AKR1C3 inhibitors devoid of COX inhibitory activity
would be an advantage. In order to prepare new inhib-
itors and to obtain more information about their struc-
ture–activity relationship, we synthesized a series of
N-acylanthranilic acids, 2-benzoylbenzoic acids, benz-
ophenones, and one phenoxybenzoic acid (Table 2),
and screened their AKR1C3 inhibitory activities.
NSAIDs and related compounds synthesized in this
study were tested for their inhibitory activity against hu-
man recombinant AKR1C3. AKR1C3 catalyzed reduc-
tion of 9,10-phenanthrenequinone in the presence of
coenzyme NADPH.15 The reaction was followed spec-
trophotometrically by measuring the change in NADPH
absorbance (ek340 = 6270 Mꢀ1 cmꢀ1) in the absence and
presence of inhibitor.22 Initial velocities were calculated
and IC50 values were determined (Tables 1 and 2).23
Of the NSAIDs evaluated, diclofenac (IC50 = 2.6 lM),
naproxen
(IC50 = 0.48 lM),
and
ibuprofen
(IC50 = 33 lM) inhibited human AKR1C3 (Table 1).22
Diclofenac is a 2-(2-anilinophenyl)acetic acid derivative
which is closely related structurally to N-phenylanthra-
nilic acids, the most promising inhibitors of AKR1C3.
The good inhibitory properties of diclofenac imply that
further investigation of a series of functionalized 2-(2-
anilinophenyl)acetic acids could yield new and more po-
tent inhibitors. The naphthalene derivative, naproxen,
which belongs to the aryl-propanoic acid family of
NSAIDs, is an even better inhibitor. The inhibitory
activity of ibuprofen was in the low micromolar range
while, in our hands, ketoprofene was almost inactive.
Many members of the profene family of drugs, like
suprofen, flubiprofen, ibuprofen, and ketoprofen, have
been reported to be good inhibitors of the oxidative
reaction catalyzed by AKR1C3.8 Since AKR1C3 in vivo
catalyzes the reduction of sex hormones, our method
involving the inhibition of the reductive reaction is more
appropriate for anticancer drug design and development
purposes. The 4-aminophenol derivatives paracetamol
and phenacetin were poor inhibitors, while acetylsalicyl-
ic acid and etodolac were devoid of any inhibitory
activity.
Table 2. New inhibitors of human recombinant AKR1C3
Compound
Structure
IC50 (lM)
COOH
H
N
1
(10%)a
O
O
COOH
H
N
2
3
(28%)a
O
O
COOH
NIa
N
O
COOH
4
5
11
70
H
N
O
O
The X-ray crystal structure of AKR1C3 reveals a sub-
strate-binding site that consists mainly of hydrophobic
aromatic amino acid side chains (Tyr24, Tyr55, Leu54,
Trp227, and Phe306). The four conserved amino acids
Asp50, Tyr55, Lys84, and His117 have been proposed
to form a catalytic tetrad involved in the oxidation of
alcohol or reduction of ketone functional groups via a
Ôpush-pullÕ mechanism.25 An oxyanion hole, which is
located at the bottom of the hydrophobic pocket, is
formed by active site tyrosine (Tyr55), histidine
(His117), and the coenzymeÕs nicotinamide ring. A po-
tent AKR1C3 inhibitor, flufenamic acid, binds at the ac-
tive site of the enzyme in the vicinity of the nicotinamide
ring, with the carboxylate oxygen occupying the oxyan-
ion hole.14 A similar binding mode, where the carboxyl-
ate group is bound next to the conserved tyrosine and
histidine residues, is found in crystal structures of many
AKR enzymes that have been complexed with carboxyl-
ic acid-containing compounds.13 Another NSAID, indo-
methacin, also contains a carboxylate, but surprisingly,
this does not bind toward the oxyanion hole. Instead,
it H-bonds to the NADP+ diphosphate moiety located
at the opposite side of the active site.14
OH
O
O
OH
OH
6
7
44
39
CH3
O
O
O
Br
OMe
CH3
8
9
180
O
O
(18%)a
O
OH
10
0.68
O
In order to investigate the possible binding mode, our
best inhibitor, naproxen, was docked into the AKR1C3
active site (pdb code 1S2A), using AutoDock 3.0 with
a Values in parentheses are % inhibition at 50 lM concn of inhibitor;
NI, no inhibition observed.

S. Gobec et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5170–5175
5173
the Lamarckian genetic algorithm.26 AutoDock calcu-
lated that naproxen occupies a similar position of ac-
tive site as indomethacin (Fig. 3). It binds to the
bottom of the active siteÕs hydrophobic pocket and
on top of the coenzymeÕs nicotinamide moiety. Also,
naproxenÕs carboxylate could form H-bonds with the
oxygen atoms of the NADP+ diphosphate moiety. This
binding mode suggests that potential inhibitors, con-
sisting of two carboxylic acid groups appropriately at-
tached to the opposite sides of a lipophilic aromatic
system, should be designed and synthesized. These
hypothetical inhibitors could utilize the aromatic frag-
ment (e.g., naphthalene ring) to interact with the active
site hydrophobic pocket while the first carboxylate may
occupy the oxyanion hole and the second forms H-
bonds with oxygens of coenzymeÕs diphosphate moiety.
with a variety of substituents at different positions in or-
der to optimize binding to the enzyme with further
improvement of the inhibitory activity.
N-Phenylanthranilic acids and related compounds are
so far the most thoroughly investigated inhibitors of
AKR1C3 and isozymes. Bauman et al. recently report-
ed that movement of the carboxylic acid from the
ortho to the para position did not decrease the enzyme
inhibition, nor did replacement of the nitrogen atom
between the aromatic rings by a keto group.13 In fact,
4-benzoylbenzoic acid (Fig. 2) was one of the best
AKR1C3 inhibitors in the series. However, benzoyl-
benzoic acids with a carboxylic group at the ortho
position have not yet been evaluated. We found that
2-benzoylbenzoic acids 5–7 were fair inhibitors of
AKR1C3, with IC50 values between 39 and 70 lM.
It appears that 40-substitution with electron-donating
substituents slightly improves the inhibitory activity.
In order to investigate the influence of the carboxylic
acid group, benzophenone derivatives 8 and 9, which
lack a 2-carboxylate, were prepared. 4-Methoxybenzo-
phenone 8 is a poor inhibitor (IC50 = 180 lM) and
4-methylbenzophenone is almost devoid of any inhib-
itory activity, so we can conclude that, in this series of
compounds, a carboxylic acid moiety is essential for
good inhibition of AKR1C3.
Target anthranilamides 1, 2, and 4 were prepared
from anthranilic acid and the appropriate carboxylic
acids by the method of mixed anhydrides. For the
synthesis of compound 3, anthranilic acid was N-
phthaloylated by phthalanhydride. 2-Benzoylbenzoic
acids 5–7 were prepared by Friedel–Crafts acylation
of benzene and its derivatives with phthalanhydride.
Benzophenones 8 and 9 were synthesized by Friedel-
Crafts acylation with benzoyl chloride of anisole and
toluene, respectively. 3-Phenoxybenzoic acid 10 was
obtained by Cr2O3 oxidation of 3-phenoxybenzyl
alcohol.24
Another compound structurally related to the N-
phenylanthranilic acid inhibitors of AKR1C3 is
3-phenoxybenzoic acid 10. Here, benzoic acid is substi-
tuted at the meta-position and, in addition, the two
aromatic rings are linked together by an oxygen atom.
These structural modifications resulted in a very active
inhibitor, with IC50 in the submicromolar range.
Docking studies of this new inhibitor revealed a bind-
ing mode which is very similar to that of flufenamic
acid (pdb code 1S2C). Also, compound 10 binds to
the active site of the enzyme in the vicinity of the
coenzymeÕs nicotinamide ring, with the aromatic resi-
Of the anthranilic acid derivatives described in this pa-
per N-benzoylanthranilic acid 1 and N-(2-phenoxyace-
tyl)anthranilic acid 2 were only weak inhibitors of
human AKR1C3, while N-phthaloylanthranilic acid 3
was inactive. However, if the anthranilic acid was acyl-
ated with 2,2-diphenylacetic acid, the amide 4 was ob-
tained and found to be a very promising inhibitor
(IC50 = 11 lM). Compound 4 is thus an interesting lead
compound for developing new inhibitors of human
AKR1C3, since its aromatic rings can be substituted
Figure 3. Superimposition of the computer model of naproxen (in
yellow, carboxylate oxygens in red) on the X-ray structure of
indomethacin (in green, carboxylate oxygens in red) bound to
AKR1C3. The surfaces of the active siteÕs most important amino acid
residues and the nicotinamide diphosphate part of the coenzyme
NADP+ (NAP, in sticks) are shown.
Figure 4. Superimposition of the computer model of compound 10 (in
yellow, carboxylate oxygens in red) on the X-ray structure of
flufenamic acid (in green, carboxylate oxygens in red) bound to
AKR1C3. The surfaces of the active siteÕs most important amino acid
residues and the nicotinamide diphosphate part of the coenzyme
NADP+ (NAP, in sticks) are shown.

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S. Gobec et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5170–5175
16. Komoto, J.; Yamada, T.; Watanabe, K.; Takusagawa, F.
Biochemistry 2004, 43, 2188.
dues located in the hydrophobic pocket. The carboxyl-
ate oxygen is located in the oxyanion hole formed by
active site Tyr55, His117, and the nicotinamide ring
(Fig. 4).
17. (a) Xu, X. Anti-Cancer Drugs 2002, 13, 127; (b) Gupta, R.
A.; DuBois, R. N. Nat. Rev. Cancer 2001, 1, 11; (c)
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M.; McCracken, J. Cancer Res. 1997, 57, 4316; (f)
Wechter, W. J.; Leipold, D. D.; Murray, E. D.; Quiggle,
D.; McCracken, J. D.; Barrios, R. S.; Greenberg, N. M.
Cancer Res. 2000, 60, 2203; (g) Kasum, C. M.; Blair, C.
K.; Folsom, A. R.; Ross, J. A. Cancer Epidemiol. Biomark.
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D. K.; Myers, R. B.; Talley, L. I.; Partridge, E. E.;
Grizzle, W. E. Clin. Cancer Res. 2002, 8, 2002; (d)
Mohammed, S. I.; Bennett, P. F.; Craig, B. A.;
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Knapp, D. W. Cancer Res. 2002, 62, 356; (e) Thurnher,
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Fischer, S. M.; Lippman, S. M. Cancer Res. 2001, 61,
4879.
We have identified some new, structurally diverse, inhib-
itors of human recombinant AKR1C3. The active com-
pounds presented in this paper are promising lead
compounds for the development of new anticancer
agents. In principle, two different mechanisms of action
are possible for their antitumor activities. Inhibitors of
AKR1C3 are potential agents for treating hormone
dependent forms of cancer, since AKR1C3 catalyzes
the conversion of androstenedione and estrone to their
active metabolites. On the other hand, AKR1C3 inhibi-
tors can also have a cancer chemopreventive role, since
inhibition of AKR1C3 can lead to diversion of prosta-
glandin catabolism toward the generation of J-series
prostanoids and thereby to the activation of PPARc
receptor.
Acknowledgments
This work was supported financially by the Ministry of
Education, Science and Sport of the Republic of Slove-
nia. The authors thank Professor Dr. Roger Pain for
critical reading of the manuscript.
19. 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.
20. Wick, M.; Hurteau, G.; Dessev, C.; Chan, D.; Geraci, M.
W.; Winn, R. A.; Heasley, L. E.; Nemenoff, R. A. Mol.
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125
100
75
50
25
0
-8
-6
log conc.
-3
-9
-5
-4
-2
-7

S. Gobec et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5170–5175
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