Hydroxytriazole derivatives as potent and selective aldo-keto reductase 1C3 (AKR1C3) inhibitors discovered by bioisosteric scaffold hopping approach

Article abstract of DOI:10.1016/j.ejmech.2017.08.046

The aldo-keto reductase 1C3 isoform (AKR1C3) plays a vital role in the biosynthesis of androgens, making this enzyme an attractive target for castration-resistant prostate cancer therapy. Although AKR1C3 is a promising drug target, no AKR1C3-targeted agen

Full text of DOI:10.1016/j.ejmech.2017.08.046

Accepted Manuscript
Hydroxytriazole derivatives as potent and selective aldo-keto reductase 1C3 inhibitors
discovered by bioisosteric scaffold hopping approach
Agnese C. Pippione, Alessandro Giraudo, Davide Bonanni, Irene M. Carnovale,
Elisabetta Marini, Clara Cena, Annalisa Costale, Daniele Zonari, Klaus Pors, Maria
Sadiq, Donatella Boschi, Simonetta Oliaro-Bosso, Marco L. Lolli
PII:
S0223-5234(17)30657-8
10.1016/j.ejmech.2017.08.046
EJMECH 9690
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 April 2017
Revised Date: 4 August 2017
Accepted Date: 21 August 2017
Please cite this article as: A.C. Pippione, A. Giraudo, D. Bonanni, I.M. Carnovale, E. Marini, C. Cena, A.
Costale, D. Zonari, K. Pors, M. Sadiq, D. Boschi, S. Oliaro-Bosso, M.L. Lolli, Hydroxytriazole derivatives
as potent and selective aldo-keto reductase 1C3 inhibitors discovered by bioisosteric scaffold hopping
approach, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.08.046.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Hydroxytriazole Derivatives as Potent and Selective
Aldo-keto Reductase 1C3 Inhibitors Discovered by
Bioisosteric Scaffold Hopping Approach
Agnese C. Pippione,1 Alessandro Giraudo,1 Davide Bonanni,1 Irene M. Carnovale,1 Elisabetta
Marini,1 Clara Cena,1 Annalisa Costale,1 Daniele Zonari,1 Klaus Pors,2 Maria Sadiq,2
Donatella Boschi,1 Simonetta Oliaro-Bosso1* and Marco L. Lolli.1*
1
Department of Science and Drug Technology, University of Torino, via Pietro Giuria 9,
10125 Torino (Italy).
2
Institute of Cancer Therapeutics, Faculty of Life Sciences, University of Bradford, West
Yorkshire BD7 1DP (UK).
Keywords
Aldo-keto reductase 1C3; AKR1C3; 17β-HSD5; Prostate cancer; CRPC; Bioisosterism;
Scaffold hopping; Inhibitors.
Abstract
The aldo-keto reductase 1C3 isoform (AKR1C3) plays a vital role in the biosynthesis of
androgens, making this enzyme an attractive target for castration-resistant prostate cancer
therapy. Although AKR1C3 is a promising drug target, no AKR1C3-targeted agent has to
date been approved for clinical use. Flufenamic acid, a non-steroidal anti-inflammatory drug,
is known to potently inhibit AKR1C3 in a non-selective manner as COX off-target effects are
also observed. To diminish off-target effects, we have applied a scaffold hopping strategy
replacing the benzoic acid moiety of flufenamic acid with an acidic hydroxyazolecarbonylic
scaffold. In particular, differently N-substituted hydroxylated triazoles were designed to
simultaneously interact with both subpockets 1 and 2 in the active site of AKR1C3, larger for
AKR1C3 than other AKR1Cs isoforms. Through computational design and iterative rounds
of synthesis and biological evaluation, novel compounds are reported, sharing high
selectivity (up to 230-fold) for AKR1C3 over 1C2 isoform and minimal COX1 and COX2
off-target inhibition. A docking study of compound 8, the most interesting compound of the
series, suggested that its methoxybenzyl substitution has the ability to fit inside subpocket 2,
being involved in π-π staking interaction with Trp227 (partial overlapping) and in a T-shape
π-π staking with Trp86. This compound was also shown to diminish testosterone production
in the AKR1C3-expressing 22RV1 prostate cancer cell line while synergistic effect was
observed when 8 was administered in combination with abiraterone or enzalutamide.
1. Introduction
Prostate cancer (PCa) is the most commonly diagnosed cancer and the second leading cause
of death in men.[1] Individuals diagnosed with high-risk PCa are typically treated with
surgery or a combination of radiation and androgen deprivation therapy (ADT). Many will
inevitably relapse and ultimately develop castration-resistant prostate cancer (CRPC), which
is responsible for the vast majority of PCa mortalities. Accordingly, there is an unmet clinical
need to develop new therapies for the treatment of CRPC patients. Although the mechanisms
of resistance are multi-factorial, the androgen axis still plays a major role in being active even
after ADT.[2] Beside evidence that androgen receptor (AR) mutations, splice variants and
increased copy number represent putative mechanisms of resistance to therapy,[3-6] the
increased expression of enzymes able to facilitate the intratumoral conversion of circulating
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adrenal androgen precursors to active AR ligands could be responsible of the CRPC surviving
mechanisms.[7] In CRPC cells the aldo-keto reductase 1C3 (AKR1C3 or 17β-HSD5) is
highly expressed.[5] This enzyme, which catalyzes the reduction of carbonyl substrates
derived from both endogenous compounds and xenobiotics,[8] is a key player in several steps
of the complex biochemical pathways leading to androgen production of potent AR ligands,
as testosterone (T) and 5α-dihydrotestosterone (DHT).[8] Moreover, AKR1C3 has also been
discovered to play roles in resistance to both hormone[5] and radiation therapy.[9] Potential
clinical use of AKR1C3 inhibitors has been demonstrated as indomethacin (Figure 1), a
potent but unselective AKR1C3 inhibitor, is able to circumvent resistance to the
steroidogenic enzyme CYP17A1 inhibitor abiraterone,[10] and to the AR antagonist
enzalutamide.[11] Few recent studies indicate controversial observations about the in vivo
effectiveness of AKR1C3-based therapies,[12-14] but other studies strongly indicate
AKR1C3 as a therapeutic target in PCa.[3, 15] Although several lead compounds have
emerged,[16-19] the pharmacopeia still lacks an AKR1C3-targeted drug with clinical
potential.
COOH
O
OH
NH
O
CF3
N
O
Cl
Indomethacin
Flufenamic acid
(FLU)
O
N
O
O
N
H
N
S
O
HO
N
O
SN33638
ASP9521
Figure 1. Chemical structures of indomethacin, flufenamic acid and AKR1C3 inhibitors
recently deep evaluated.
In order to understand several key unanswered questions related to the in vivo application of
AKR1C3 inhibitors, it is desirable to develop potent, selective and drug-like AKR1C3
inhibitors. Amongst NSAIDs, flufenamic acid (FLU, Figure 1) potently inhibits AKR1C3
although it suffers from COX off-target effects.[20, 21] Recently, we successfully applied a
scaffold hopping strategy based on the replacement of quinolinecarboxylate moiety of
brequinar with a hydroxyazolecarboxamidic scaffold for the design of new potent human
dihydroorotate dehydrogenase (hDHODH) inhibitors.[22] In the present study, we applied a
similar scaffold hopping strategy to the FLU benzoic acid moiety (Figure 2). Specifically,
three hydroxyazoles (hydroxyfurazan, hydroxythiadiazole and a series of N-substituted
hydroxy-1,2,3-triazoles) were used to design compounds 1 - 8 (Figure 2). Due to their acidic
properties, the hydroxyazole scaffolds are widely deprotonated at physiological pH and are
valid isosters of the carboxylic acid function as we recently demonstrated.[23-25] In contrast
to the hydroxyfurazan and hydroxythiadiazole scaffolds, the regiosubstitution of the nitrogen
of the hydroxytriazole ring allows the possibility to make a structural refinement, which
enables an opportunity to improve on binding pocket affinity.[26] Accordingly, the
substituents present on the triazole ring (Figure 2) were selected by their possibility of
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establishing interactions with sub-pocket 2 (SP2) of the AKR1C3 binding site[27] in an
attempt to enhance potency and target selectivity.
OH
OH
N
N
O
N
NH
NH
CF3
CF3
N
N
OH
N
O
O
S
1
8
NH
CF3
O
N
O
OH
O
N
OH
2
O
OH
NH
N
N
NH
CF3
N
X
NH
CF3
CF3
Y
7
O
Replacement of benzoic
acid moiety
FLUFENAMIC ACID
OH
N
OH
N
N
NH
CF3
N
N
NH
CF3
N
O
OH
N
OH
O
N
N
N
3
N
6
NH
CF3
NH
CF3
N
O
O
4
5
Figure 2. AKR1C3 inhibitors based on hydroxyazole scaffolds.
We here report on the design, synthesis and biological explorations of eight candidate
structures (Figure 2), which supports the findings of AKR1C3-selective compounds with no
off-target COX effect. Furthermore, the binding modes of the most representative molecules
were suggested by computational modelling.
2. Result and discussion
2.1 Chemistry.
The methodology used for the synthesis of the target compounds is described in Scheme 1.
The first part of the work consisted in acquiring the protected benzyloxyazolecarboxylic
acids (compounds 13, 16 a-f, Scheme 1a and 1b). Subsequently these acids were coupled
with 3-trifluoromethylaniline (Scheme 1c). The furazancarboxylic acid derivative was
prepared starting from 9[25] that was hydrolysed to the corresponding acid 10. This latter
was decarboxylated and dehydrated to the nitrile 11, then transformed into the methyl ester
12 by a Pinner reaction. Compound 13 was finally obtained by base-catalyzed hydrolysis of
12 (Scheme 1a). Regio-substituted hydroxytriazole carboxylic acids 16 a-f were prepared
from the N-unsubstituted triazole 14 as a common starting material.[23] This building block
presents an alkylation pattern directed toward positions N(b) and N(c) of the triazole ring,
leading to a mixture of two isomeric products. The isomeric mixtures were
chromatographically resolved and each isomer structurally assigned on the basis of the
heteronuclear 2D-NMR (HSQC and HMBC) and 13C-NMR spectra.[28] The hydrolysis of
the obtained esters 15 a-f afforded the carboxylic acids of general structure 16 (Scheme 1b).
Finally, carboxylic acids 13 and 16 a-f were converted into the corresponding acyl chlorides
and allowed to react with 3-trifluoromethylaniline to generate the amides 18 and 19 a-f
(Scheme 1c); these latter compounds were deprotected through catalytic hydrogenation to
give the desired target compounds 1, 3 – 8. Compound 2 was directly obtained from coupling
of unprotected 4-hydroxy-1,2,5-thiadiazole-3-carboxylic acid 17[29] with 3-
trifluoromethylaniline. Although this reaction occured with low yield, the protection of the
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ACCEPTED MANUSCRIPT
hydroxyl group of 17 was avoided as the final removal of benzylic group in oxythiadiazoles
is challenging[22].
1a)
HO
HO
N
N
N
OEt
OH
O
O
O
CN
a
b
c, d
O
O
N
N
N
N
N
O
O
O
9
10
11
O
N
O
N
O
O
OMe
OH
a
N
N
O
O
12
13
1b)
O
O
O
N
O
N
O
OH
OEt
O
OEt
a
e
N
R
N
N
R
N
a
N
c
N
N
H
b
16 a - f
15 a R = N(b) CH3
b R = N(c) CH3
14
c R = N(b) CH(CH2)2
d R = N(c) CH(CH2)2
e R = N(c) benzyl
f
R = N(c) 4-methoxybenzyl
1c)
O
N
CF3
CF3
O
O
O
HO
OH
HO
O
O
OH
NH
NH
f, g
h
f, g
N
N
Y
N
N
Y
Y
S
X
X
X
1 X = O, Y = N
2 X = S, Y = N
3 X = N-CH3, Y = N
18
17
13
16 a
X = O, Y = N
X = N-CH3, Y = N
X = O, Y = N
X = N-CH3, Y = N
19 a
19 b
19 c
19 d
19 e
19 f
16 b
16 c
16 d
16 e
X = N, Y = N-CH3
X = N, Y = N-CH3
4 X = N, Y = N-CH3
X = N-CH(CH2)2, Y = N
X = N, Y = N-CH(CH2)2
X = N, Y = N-CH2Ph
X = N-CH(CH2)2, Y = N
X = N, Y = N-CH(CH2)2
X = N, Y = N-CH2Ph
5 X = N-CH(CH2)2, Y = N
6 X = N, Y = N-CH(CH2)2
7 X = N, Y = N-CH2Ph
16 f X = N, Y = N-(4-methoxybenzyl)
X = N, Y = N-(4-methoxybenzyl)
8 X = N, Y = N-(4-methoxybenzyl)
Scheme 1. (a) 1) NaOH, EtOH, rt; 2) 2M HCl; (b) Ac2O, 70°C; (c) NaH, dry MeOH; (d) 2M
H2SO4; (e) RX, K2CO3, CH3CN, r.t.; (f) ClCOCOCl, dry DMF, dry THF, 0°C; (g) 3-
trifluoromethylaniline, dry pyridine, dry THF, r.t.; (h) H2, Pd/C, THF, r.t.
2.2 AKR1C3 inhibition assays
The activity of the compounds 1 - 8 and FLU as AKR1C3 inhibitors was initially tested by
incubating a bacterial suspension containing recombinant AKR1C3 with radiolabeled [14C]-
androstenedione in the presence of increasing inhibitor concentrations (Table 1). In
comparison with FLU (IC50 = 8.63 µM), the hydroxyfurazan 1, the hydroxythiadiazole 2 and
the N(b) methyl triazole 3 were found to be almost inactive while the N(c) methyl triazole 4
(IC50 = 32.79 µM) demonstrated weak inhibitory activity. Furthermore, comparing the two
cyclopropylmethyl triazoles 5 and 6, the N(c) isomer resulted in more active compound than
the N(b) analogue. As the binding site seems to better tolerate the N(c) substitution, we
designed two more N(c) analogues with substituents of increasing size (7 and 8). Indeed N(c)
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cyclopropylmethyl triazole 6 was shown to be more active than N(c) methyl triazole 4,
indicating the preference for lipophilic groups in that position. This hypothesis was
confirmed as the benzyl 7 and 4-methoxylbenzyl 8 analogues displayed activity (IC50s of
10.19 µM and 3.56 µM, respectively) comparable with FLU. Notably, compound 8 was more
potent than FLU under the conditions investigated.
Table 1. Inhibitory effect on AKR1C3 and AKR1C2 recombinant enzymes.
Bacterial suspension
Purified enzyme
AKR1C3
IC50 ± SE
(µM) a
AKR1C3
IC50 ± SE
(µM) b
AKR1C2
IC50 ± SE
(µM) b
Ratio
IC50 value
(1C2:1C3)
Compound
8.63 ± 1.70
0.44 ± 0.02
n.d.
0.53 ± 0.03
n.d.
1.2
FLU
1
>50
n.d.
(35.37% ± 3.37)c
≥ 50
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
2
3
4
5
(47.22% ± 3.16)c
>50
(18.47% ± 3.71) c
32.79±1.10
>50
(5.35% ± 1.44) c
13.22 ± 0.81
10.19±1.47
3.56±0.08
1.60 ± 0.22
0.48 ± 0.02
0.31 ± 0.01
70.63 ± 6.32
62.94 ± 5.13
73.23 ± 8.67
44
6
131
236
7
8
a) experiments performed with [14C]androstenedione as substrate; b) experiments performed with S-tetralol as
substrate; c) % of inhibition ± SE at 50 µM. n.d: not determined.
The three most active compounds assayed in the bacterial suspension assay (6 - 8) were also
assayed with AKR1C3 purified enzyme (Table 1) by following the oxidation of S-tetralol in
the presence of NADP+. The inhibition pattern observed was similar to that observed with the
bacterial suspension. Notably, compounds 7 and 8 were equipotent (IC50s 0.48 µM and 0.31
µM, respectively) with FLU (IC50 0.44 µM).
2.3. AKR1Cs selectivity and COX inhibition
In PCa therapy, selective targeting of AKR1C3 over 1C2 is considered critical [30]:
AKR1C2 share > 86% sequence identity with AKR1C3, and it is involved in
dihydrotestosterone inactivation, so its inhibition would be undesirable. Hence, the most
active compounds 6 - 8 and FLU were assayed for their inhibitory properties using purified
AKR1C2 incubated with S-tetralol (Table 1). Whereas the AKR1C2/C3 inhibition ratio of
FLU was found to be 1.2, triazole analogues 7 and 8 were found to be 131 and 236-fold more
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selective toward AKR1C3 inhibition. Next, the compounds 6 - 8 were assayed for their
inhibitor effect on COX-1 and COX-2 off-target. Their activity, compared with different
standards using ovine COX-1 (oCOX-1) and human COX-2 (hCOX-2) is reported in Table 2.
Table 2. COX-1 and COX-2 inhibitory activities of compounds 6, 7 and 8, compared with
flufenamic acid, indomethacin, celecoxib and rofecoxib.
Ratio
IC50 value
(COX1:AKR1C3) (COX2:AKR1C3)
Ratio
IC50 value
oCOX1
IC50 ± SE (µM)
hCOX2
IC50 ± SE (µM)
Compound
> 100
FLU
6
14 ± 1
32
> 227
> 63
(18% ± 2)a
> 100
> 100
> 63
> 208
(17% ± 1)a
(9.6% ± 5.2)a
> 100
> 100
> 208
7
(15% ± 4) a
(7.5% ± 7.5) a
>100
(0) a
>100
> 322
n.d.
> 322
n.d.
8
(12% ± 4) a
0.10 ± 0.01
14 ± 12
0.61 ± 0.09
0.54 ± 0.12
3.0 ± 1.0
Indomethacin
Celecoxib
Rofecoxib
n.d.
n.d.
˃100
n.d.
n.d.
(25 ± 14)a
a) % of inhibition ± SE at 100 µM. n.d: not determined.
Notably, compounds 6 - 8 did not display significant inhibitory activity on any of the two
COX isoforms at the highest concentration tested (100 µM). In contrast, FLU was shown to
be effective in inhibiting COX-1 but not COX-2. Because contradictory data are reported in
the literature for FLU and COX inhibition,[31] we validated our assay with indomethacin
(time-dependent non-selective inhibitor[31]), celecoxib and rofecoxib (non-time-dependent
for COX-1, time-dependent and selective for COX-2).[32, 33] Pleasingly, the COX 1/2 data
obtained was consistent with inhibitory potencies reported in literature.[34, 35]
2.4 Antiproliferative activity and testosterone suppression
We evaluated compounds 7, 8 and FLU in AKR1C3-expressing 22RV1 cells, which have
been shown to possess resistance to abiraterone and enzalutamide.[10-12] The presence of
AKR1C3 was confirmed using western blot (Figure 3A) before assessing the antiproliferative
activity of the three compounds using the sulforhodamine B assay. Although compounds 7, 8
and FLU appeared to be equipotent AKR1C3 inhibitors, it was observed that 7 is more
antiproliferative (Figure 3B). As AKR1C3 plays a key role in the production of testosterone
in the androgen biosynthetic pathway, therapeutic intervention of this pathway is vital to
effective treatment outcome. Given that 8 had a higher AKR1C3:AKR1C2 selectivity ratio
than 7, we decided to evaluate the former for its ability to interfere with testosterone
formation in a cell-based system. This system constitutes a more physiological environment
than a homogenate or purified enzyme. Furthermore, working with intact cells allows to
observe if the compounds are able to enter the cells. So, AKR1C3-expressing 22RV1 cells
were treated with the compound 8 and androstenedione. The formation of testosterone was
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analysed in cell supernatant by ELISA. In the presence of only androstenedione, testosterone
production was increased > 200-fold. When cells were pre-treated with 8, a significant dose-
dependent impact on testosterone levels was observed (Figure 3C). Encouragingly a
significant decrease in testosterone production was observed at 5 µM and 50 % was inhibited
at 100 µM. The lack of complete testosterone inhibition might be due to alternative
steroidogenic pathways by-passing AKR1C3 activity and/or the binding efficacy of
compound 8 to the target is insufficient to completely abolish testosterone production.
Figure 3. Antiproliferative activity and inhibition of testosterone production, evaluated for
representative compounds. Confirmation of AKR1C3 expression in 22RV1 cells by western
blot (A) and antiproliferative activity of compounds 7, 8 and FLU using the SRB assay (B).
Inhibitory effect on testosterone production of compound 8 in androstenedione (AD) treated
22RV1 cells (C) by Elisa kit.
In order to evaluate a possible synergistic effect of our compounds with abiraterone and
enzalutamide, experiments of co-treatment were performed. 22RV1 cells were treated with
60 µM compound 8 with or without 10 µM abiraterone for 72 h. The same experiment was
carried out by treating cells with or without 20 µM enzalutamide. As shown in Figure 4,
abiraterone, enzalutamide and compound 8 had limited effects on cell growth. When
compound 8 was added together with either abiraterone or enzalutamide, the cell viability
was reduced to 45 % or 50 %, respectively, suggesting a synergistic effect was achieved.
Figure 4. Effect of the co-treatment with compound 8 and abiraterone (ABI) or enzalutamide
(ENZ) on 22RV1 cells proliferation by SRB. Cells were treated with 60 µM compound 8
with or without 10 µM abiraterone (or 20 µM enzalutamide) for 72 h. Cell growth is
expressed as % T/C (mean OD of treated cells/mean OD of control cells X 100).
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2.5 Molecular modeling
To elucidate the observed activity of hydroxytriazole-based compounds, we performed a
molecular docking study starting from the crystal structure of the protein complexed with
FLU (PDB code: 1S2C).[36] A comparison of compound 8 (grey) and FLU (orange) docked
in the AKR1C3 binding site is presented in Figure 5. At first, self-docking procedure was
successfully applied on FLU well reproducing the crystallographic pose of FLU inside the
binding site (Figure S1). The binding mode of FLU shows the carboxylate moiety interacting
with the so-called oxyanion site (OS) forming hydrogen bonds with Tyr55, His117 and
NADP+ co-factor by a water molecule. The trifluoromethylphenyl moiety is directed toward
the SP1, formed by Ser118, Asn167, Phe306, Phe311, Tyr216, Met120 and Tyr319, where it
is involved in lipophilic interactions. The docking simulations performed on compound 8
revealed a high superimposition to FLU (Figure S4). In particular, the hydroxyl group present
in the triazole ring is located in the OS, thereby mimicking the carboxylic group of FLU. The
amide link present in compound 8 compared to the amine in FLU, forces the 3-
trifluoromethylphenyl moiety inside SP1. This fact could explain the selectivity of 8 forwards
AKR1C3, since the AKR1C2 isoform is characterized by a smaller SP1.[30] Interestingly,
the 4-methoxy-benzyl substituent of 8 fits inside SP2, a small pocket part of the steroid
channel (SC) left unoccupied by FLU. The docking results suggest the formation of π-π
staking and T-shape π-π staking between the N(c) 4-methoxy-benzyl substituent and Trp227
(partial overlapping) and Trp86 respectively.
Figure 5. Compound 8 (grey, A) and FLU (orange, B) docked in the AKR1C3 binding site.
NADP+ is coloured in green.
We also performed docking simulations using the X-ray structure of COX1 co-crystallized
with diclofenac (PDB code: 3N8Y) to clarify the low activity of the compounds versus
COX1. The compounds 4, 6, 7, 8 as well as FLU and diclofenac were then docked in the
active site (see material and methods for more information). FLU maintained the same
interaction of diclofenac, placing the carboxylic portion near Ser530 and Tyr385 and
establishing with both residues hydrogen bonds. Moving to compound 4, this latter was able
to mimic diclofenac and FLU binding mode (SP Fig. S5), although associated with a large
decrease in binding affinity (SP Tab S1). Instead, a completely different binding mode (data
no shown) was found for compounds 6, 7, 8, suggesting that a bulky group in position 1 of
hydroxytriazol ring makes the compounds unable to mimic the binding mode of FLU and
diclofenac. This modeling analysis shows how the replacement of the FLU benzoic acid by a
4-hydroxy-N1-substituted triazolecarbonylic moiety is a bioisosteric scaffold-hopping
replacement for AKR1C3 but not for COX1 inhibition activity.
3. Conclusions
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This study has focused on a new generation of AKR1C3 inhibitors designed by utilizing a
scaffold hopping approach to replace the benzoic acid moiety of FLU with hydroxylated
azoles. The best compound of the series, the 4-methoxybenzyl substituted analogue 8, was
found to selectively inhibit AKR1C3 activity without any significant AKR1C2 and COX1/2
off-target effects. Compound 8 was also able to inhibit the testosterone production and cell
proliferation in AKR1C3-expressing 22RV1 CRPC cells. In addition, the inhibition of
AKR1C3 activity by compound 8 partly resensitized 22RV1 cells to enzalutamide and
abiraterone treatment. Taken together, the novel chemical scaffolds provides a promising
starting point for the design of more potent AKR1C3 inhibitors with clinical potential.
4. Experimental section
4.1 Chemistry
4.1.1 General methods. All chemical reagents were obtained from commercial sources
(Sigma Aldrich, Alfa Aesar) and used without further purification. Culture media were
obtained from Sigma‑Aldrich. Restriction enzymes, DNA polymerase and T4 DNA ligase
were obtained from Promega. [4-14C]-androst-4-ene-3,17-dione (10 µCi/370 KBq, 53.6
mCi/mmol) was obtained from Perkin-Elmer. Analytical grade solvents (acetonitrile,
diisopropyl ether, diethyl ether, dichloromethane [DCM], dimethylformamide [DMF],
ethanol 99.8 % v/v, ethyl acetate, methanol [MeOH], petroleum ether b.p. 40 - 60°C
[petroleum ether]) were used without further purification. When needed, solvents were dried
on 4 Å molecular sieves. Tetrahydrofuran (THF) was distilled immediately prior to use from
Na and benzophenone under N2. Thin layer chromatography (TLC) on silica gel was carried
out on 5 x 20 cm plates with 0.25 mm layer thickness to monitor the process of reactions.
Anhydrous MgSO4 was used as a drying agent for the organic phases. Purification of
compounds was achieved with flash column chromatography on silica gel (Merck Kieselgel
60, 230-400 mesh ASTM) using the eluents indicated or by CombiFlash Rf 200 (Teledyne
Isco) with 5–200 mL/min, 200 psi (with automatic injection valve) using RediSep Rf Silica
columns (Teledyne Isco) with the eluents indicated. Purity of compounds was measured by
HPLC analyses, performed on an UHPLC chromatographic system (Perkin Elmer, Flexar).
The analytical column was an UHPLC Acquity CSH Fluoro-Phenyl (2.1x100 mm, 1.7 µm
particle size) (Waters). Compounds were dissolved in acetonitrile or methanol, and injected
through a 20 µl loop. The mobile phase consisted of acetonitrile / water with 0.1 %
trifluoroacetic acid; two gradient profiles of mobile phase were used to assay the purity of
each compound. UHPLC analysis were run at flow rates of 0.5 mL/min, and the column
effluent was monitored at 215 and 254 nm, referenced against a 360 nm wavelength. Purity
of the synthetic intermediates varied between 90 % and 99 % purity. The biological
experiments were employed on compounds with a purity of at least 95%. Melting points
(m.p.) were measured on a capillary apparatus (Büchi 540) by placing the sample at a
temperature 10° C below the m.p. and applying a heating rate of 1° C min-1. All compounds
1
1
were routinely checked by H- and 13C-NMR and mass spectrometry. H- and 13C-NMR
spectra were performed on a Bruker Avance 300 instrument. For coupling patterns, the
following abbreviations are used: br = broad, s = singlet, d = doublet, dd = doublet of
doublets, t = triplet, q = quartet, m = multiplet. Chemical shifts (δ) are given in parts per
million (ppm). MS spectra were performed on Finnigan-Mat TSQ-700 (70 eV, direct inlet for
chemical ionization [CI]) or Waters Micromass ZQ equipped with ESCi source for
electrospray ionization mass spectra. Compounds 9,[25] 15a, 15b, 15e,[23] 15c, 15d, 16a-
d,[22] 17[29]were prepared following already described procedures.
4.1.2. 2-[4-(Benzyloxy)-1,2,5-oxadiazol-3-yl](hydroxyimino)acetic acid (10). 5M NaOH (8
mL) was added to a solution of 9 (2.21 g, 7.60 mmol) in ethanol (40 ml). The reaction
9

ACCEPTED MANUSCRIPT
mixture was stirred at room temperature for 24 h, then concentrated under reduced pressure.
The crude material was dissolved in water (15 mL) and acidified with HCl until precipitation
of the desired product occurred. The white obtained solid was a mixture of the two
1
geometrical isomers in variable ratio. Yield 76 %. H-NMR (300 MHz, most abundant
13
isomer, DMSO-d6): δ 5.32 (s, 2H), 7.29 - 7.32 (m, 5H), 13.59 (s, 1H), 13.80 (br s, 1H). C-
NMR (75 MHz, most abundant isomer, DMSO-d6): δ 74.0, 128.7, 128.8, 128.9, 134.9, 136.5,
139.6, 162.6, 163.3. MS (CI) 263 [M + H]+.
4.1.3. 4-(Benzyloxy)-1,2,5-oxadiazole-3-carbonitrile (11). A solution of 10 (1.30 g, 4.94
mmol) in acetic anhydride (13 mL) was stirred at 70 °C for 2 h. The reaction mixture was
cooled to r.t. and concentrated under reduced pressure to half volume, then poured into iced
water (50 mL). The resulting mixture was stirred at room temperature for 30 min, then
extracted twice with diethyl ether. The organic layers were washed with saturated NaHCO3
and brine, dried with Na2SO4 and concentrated under reduced pressure. The oily crude was
purified by flash chromatography using DCM as eluent to afford the title compound as a
1
white solid (amorphous solid, m.p. 44.0 – 45.7 °C). Yield 92 %. H-NMR (300 MHz,
CDCl3): δ 5.35 (s, 2H), 7.34 - 7.42 (m, 5H). 13C-NMR (75 MHz, CDCl3): δ 75.7, 106.3,
125.9, 129.0, 129.1, 129.8, 133.2, 164.6. MS (CI) 202 [M + H]+.
4.1.4. Methyl 4-(benzyloxy)-1,2,5-oxadiazole-3-carboxylate (12). Sodium hydride 60 %
dispersion in mineral oil (398 mg) was added to a solution of 11 (1.00 g, 4.98 mmol) in dry
methanol (10 mL). The reaction mixture was stirred under nitrogen atmosphere at 0 oC for 15
min, then allowed to reach room temperature and further stirred for 60 min. 2M H2SO4 (10
ml) was slowly added cooling the reaction mixture at 0 °C. The resulting suspension was
extracted with DCM. The organic layers were collected, dried with brine and Na2SO4 and
concentrated under reduced pressure. The crude material was purified by flash
chromatography using petroleum ether / DCM 60:40 v/v as eluent, to afford the title
1
compound as a white solid (amorphous solid, m.p. 45 - 48 °C). Yield 98 %. H-NMR (300
MHz, CDCl3): δ 3.91 (s, 3H), 5.35 (s, 2H), 7.29-7.43 (m, 5H). 13C-NMR (75 MHz, CDCl3): δ
53.1, 74.4, 128.3, 128.6, 128.9, 134.0, 139.2, 157.4, 163.7. MS (CI) 235 [M + H]+.
4.1.5. Ethyl 4-(benzyloxy)-1-(4-methoxybenzyl)-1H-1,2,3-triazole-5-carboxylate (15f).
Cs2CO3 (2.90 g, 8.91 mmol) and 1-(chloromethyl)-4-methoxybenzene (1.23 g, 7.94 mmol)
were added to a solution of 14 (1.10 g, 4.45 mmol) in CH3CN (25 mL). The resulting mixture
was stirred at room temperature for 16 hours. When the reaction was complete, the mixture
was concentrated under reduced pressure and the crude product was partitioned between
ethyl acetate and water. The aqueous phase was extracted twice with ethyl acetate and
collected organic layers were washed with 1M HCl (30 mL), 1M NaOH (30 mL) and brine.
The organic phase was dried with Na2SO4 and concentrated under reduced pressure to afford
a colorless oil. The latter showed two spots on TLC (eluent: petroleum ether / ethyl acetate
90/10 v/v) relative to the N(b) and N(c) substituted triazole isomers. The two isomers were
separated using flash chromatography (eluent: petroleum ether / ethyl acetate 95/5 v/v). First
1
eluted isomer, white solid (powder, m.p. 76.5 - 78.7 °C). Yield 36 %. H-NMR (300 MHz,
DMSO-d6): δ 1.24 (3H, t, J = 7.1 Hz), 3.72 (3H, s), 4.27 (2H, q, J = 7.1 Hz), 5.45 (2H, s),
13
5.73 (2H, s), 6.90 (2H, d, J = 8.5 Hz), 7.18 (2H, d, J = 8.7 Hz), 7.29 - 7.50 (5H, m); C-
NMR (75 MHz, DMSO-d6): δ 13.9, 53.5, 55.1, 61.1, 71.1, 110.4, 114.1, 127.5, 127.6, 128.1,
128.4, 129.1, 136.4, 157.7, 159.1, 160.6. MS (ESI) 368 [M + H]+. The second eluted isomer
ethyl 5-(benzyloxy)-2-(4-methoxybenzyl)-2H-1,2,3-triazole-4-carboxylate was also isolated as
1
a white solid and characterized. M.p. 80.7 – 81.7 °C, from ethanol. Yield 60 %. H-NMR
(300 MHz, DMSO-d6): δ 1.26 (t, J = 7.1 Hz, 3H), 3.74 (s, 3H), 4.25 (q, J = 7.1 Hz, 2H), 5.30
(s, 2H), 5.48 (s, 2H), 6.92 (d, J = 8.6 Hz, 2H), 7.27 (d, J = 8.6 Hz, 2H), 7.31 - 7.49 (m, 5H).
13C-NMR (75 MHz, DMSO-d6): δ 14.0, 55.0, 58.3, 60.3, 71.7, 113.9, 123.0, 126.7, 127.8,
128.1, 128.3, 129.5, 135.8, 159.1, 159.4, 160.1. MS (ESI) 368 [M + H]+.
10

ACCEPTED MANUSCRIPT
4.1.6. General procedure for compounds 16e, 16f and 13.
6M NaOH (0.57 mL, 3.45 mmol) was added to a solution of the appropriate ester (1.15
mmol) in ethanol (25 mL) and the reaction mixture was stirred at room temperature until
disappearance of starting material. The resulting solution was neutralized with 2M HCl, then
concentrated under reduced pressure. 2M HCl was added until pH 1-2, observing
precipitation of a white solid. The solid was isolated by filtration to give the appropriate
carboxylic acid.
4.1.6.1 4-(Benzyloxy)-1,2,5-oxadiazole-3-carboxylic acid (13). Crystalline white solid (m.p.
106.1 -107.5 °C from hexane/diisopropyl ether). 1H-NMR (300 MHz, CDCl3): δ 5.45 (s, 2H),
7.30 - 7.50 (m, 5H), 10.91 (br s, 1H). 13C-NMR (75 MHz, CDCl3): δ 74.8, 128.7, 128.8,
129.1, 134.0, 139.1, 161.6, 163.9. MS (CI) 220 [M + H]+.
4.1.6.2 1-Benzyl-4-(benzyloxy)-1H-1,2,3-triazole-5-carboxylic acid (16e). Crystalline white
solid (m.p. 165.7 - 166.8°C). Yield 90 %. 1H- NMR (300 MHz, DMSO-d6): δ 5.44 (s, 2H),
5.82 (s, 2H), 7.14‒7.52 (m, 10H), 13.66 (br, 1H). 13C-NMR (75 MHz, DMSO-d6): δ 53.7,
71.1, 111.2, 127.4, 128.0, 128.1, 128.4, 128.7, 128.7, 135.9, 136.4, 159.1, 160,4. MS (ESI)
310 [M + H]+.
4.1.6.3 4-(Benzyloxy)-1-(4-methoxybenzyl)-1H-1,2,3-triazole-5-carboxylic acid (16f).
Crystalline white solid (m.p. 179.1 - 180.5°C). Yield 84 %. 1H-NMR (300 MHz, DMSO-d6):
δ 3.72 (s, 3H), 5.43 (s, 2H), 5.73 (s, 2H), 6.90 (d, J = 8.3 Hz, 2H), 7.18 (d, J = 8.3 Hz, 2H),
7.29 - 7.51 (m, 5 H), 13.62 (br, 1H). 13C-NMR (75 MHz, DMSO-d6): δ 53.2, 55.1, 71.0,
111.0, 114.0, 127.7, 128.0, 128.1, 128.4, 129.1, 136.4, 159.1, 159.2, 160.5. MS (ESI) 340 [M
+ H]+.
4.1.7. General procedure for synthesis of amides 18 - 19a-f:
Dry DMF (26 µL) and 2M oxalyl chloride in DCM (3.06 mmol, 1.53 mL) were added to a
cooled (0°C) solution of the appropriate carboxylic acid (13, 16 a - f, 0.901 mmol) in dry
THF (15 mL). The reaction was stirred for 3 hours at room temperature under nitrogen
atmosphere. The solvent was evaporated under reduced pressure and the residue was
dissolved in dry THF (this process was repeated for three times). The resulting acyl chloride
was dissolved in dry THF (10 mL) and used without any further purification in the next step.
Dry pyridine (219 µL, 2.70 mmol) and 3-trifluoromethylaniline (113 µL, 0.901 mmol) were
added to the described solution. The reaction mixture was stirred for 12 hours at room
temperature under nitrogen atmosphere. 0.5 M HCl was added to the resulting mixture, which
was concentrated under reduced pressure. The resulting suspension was acidified with 0.5 M
HCl to pH 2 and extracted with ethyl acetate (3 × 20 mL). The organic phases were collected,
washed with brine, dried with Na2SO4, and the solvent was evaporated. The crude product
was purified using flash chromatography (gradient of petroleum ether /ethyl acetate from
90/10 v/v to 70/30 v/v) to obtain the corresponding amide.
4.1.7.1. 4-(Benzyloxy)-N-(3-(trifluoromethyl)phenyl)-1,2,5-oxadiazole-3-carboxamide (18).
Flash chromatography eluent: petroleum ether / ethyl acetate 9:1 v/v. White solid (powder,
1
m.p. 110.1 - 111.1 °C), yield 74 %. H-NMR (300 MHz, CDCl3): δ 5.48 (s, 2H), 7.39-7.52
(7H, m), 7.80 (d, J = 7.6 Hz, 1H), 7.91 (s, 1H), 8.58 (br s, 1H). 13C-NMR (75 MHz, CDCl3):
δ 75.2, 116.9 (q, J = 3.8 Hz), 122.0 (q, J = 3.7 Hz), 123.1 (q, J = 0.8 Hz), 123.6 (q, J = 272.6
Hz), 128.7, 128.9, 129.3, 129.9, 131.70 (q, J = 32.1 Hz), 133.9, 137.0, 140.9, 153.8, 163.3.
MS (CI) 364 [M + H]+.
4.1.7.2.
5-(Benzyloxy)-2-methyl-N-(3-(trifluoromethyl)phenyl)-2H-1,2,3-triazole-4-
carboxamide (19a). White solid (powder, m.p. 110.2 - 112.3 °C). Yield 76 %. 1H NMR (300
MHz, DMSO-d6): δ 4.15 (s, 3H), 5.34 (s, 2H), 7.32‒7.62 (m, 7H), 8.01 (d, J = 8.2 Hz, 1H),
11

ACCEPTED MANUSCRIPT
13
8.26 (s, 1H), 10.48 (s, 1H). C NMR (75 MHz, DMSO-d6): δ 42.4, 72.0, 116.2 (q, J = 4.1
Hz), 119.9 (q, J = 4.1 Hz), 123.6 (q, J = 1.2 Hz), 124.1 (q, J = 272.3 Hz), 125.5, 128.0, 128.2,
128.4, 129.3 (q, J = 31.4 Hz), 129.8, 136.0, 139.4, 158.1, 159.4. MS (ESI) 377 [M + H]+.
4.1.7.3.
4-(Benzyloxy)-1-methyl-N-(3-(trifluoromethyl)phenyl)-1H-1,2,3-triazole-5-
1
carboxamide (19b). White solid (powder, m.p. 136.4 - 139.4°C). Yield 58 %. H NMR (300
MHz, DMSO-d6): δ 4.20 (s, 3H), 5.52 (s, 2H), 7.31‒7.44 (m, 3H), 7.46‒7.66 (m, 4H), 7.85
(d, J = 8.2 Hz, 1H), 8.08 (s, 1H), 9.90 (s, 1H). 13C NMR (75 MHz, DMSO-d6): δ 37.9, 71.8,
114.2, 116.1 (q, J =4.1 Hz), 120.6 (q, J = 3.8 Hz), 123.6 (q, J = 1.3 Hz), 123.9 (q, J = 272.3
Hz), 127.9, 128.2, 128.3, 129.5 (q, J = 31.8 Hz), 130.1, 136.2, 138.6, 155.9, 157.8. MS (ESI)
377 [M + H]+.
4.1.7.4.
5-(Benzyloxy)-2-(cyclopropylmethyl)-N-(3-(trifluoromethyl)phenyl)-2H-1,2,3-
1
triazole-4-carboxamide (19c). White solid (powder, m.p. 79.0 - 80.7 °C). Yield 75 %. H
NMR (300 MHz, DMSO-d6): δ 0.41‒0.49 (m, 2H), 0.54‒0.62 (m, 2H), 1.29‒1.42 (m, 1H),
4.25 (d, J = 7.2 Hz, 2H), 5.35 (s, 2H), 7.32 ‒ 7.62 (m, 7H), 8.01 (d, J = 8.2 Hz, 1H), 8.24 (s,
1H), 10.36 (s, 1H). 13C NMR (75 MHz, DMSO-d6): δ 3.6, 10.7, 59.5, 72.0, 116.2 (q, J = 4.3
Hz), 119.9 (q, J = 4.1 Hz), 123.7 (q, J = 0.7 Hz), 124.1 (q, J = 272.4 Hz), 125.6, 128.2, 128.2,
128.4, 129.3 (q, J = 31.3 Hz), 129.8, 136.0, 139.4, 158.1, 159.2. MS (ESI) 417 [M + H]+.
4.1.7.5.
4-(Benzyloxy)-1-(cyclopropylmethyl)-N-(3-(trifluoromethyl)phenyl)-1H-1,2,3-
1
triazole-5-carboxamide (19d). White solid (powder, m.p. 114.1 - 116.0 °C). Yield 63 %. H
NMR (300 MHz, DMSO-d6): δ 0.37 - 0.45 (m, 2H), 0.48 - 0.57 (m, 2H), 1.27 - 1.44 (m, 1H),
4.47 (d, J = 7.3 Hz, 2H), 5.52 (s, 2H), 7.31 - 7.67 (m, 7H), 7.85 (d, J = 8.2 Hz, 1H), 8.08 (s,
1H), 10.06 (s, 1H). 13C NMR (75 MHz, DMSO-d6): δ 3.7, 11.4, 54.9, 71.9, 113.5, 116.1 (q, J
= 4.1 Hz), 120.7 (q, J = 4.3 Hz), 123.6 (q, J = 1.2 Hz), 124.0 (q, J = 272.1 Hz), 128.1, 128.3,
128.4, 129.6 (q, J = 31.5 Hz), 130.3, 136.3, 138.7, 156.1, 158.0. MS (ESI) 417 [M + H]+.
4.1.7.6.
1-Benzyl-4-(benzyloxy)-N-(3-(trifluoromethyl)phenyl)-1H-1,2,3-triazole-5-
carboxamide (19e). White solid (powder, m.p. 152.2 - 154.2 °C). Yield 51 %. 1H NMR (300
MHz, DMSO-d6): δ 5.51 (s, 2H), 5.84 (s, 2H), 7.20 - 7.65 (m, 12H), 7.81 (d, J = 8.1 Hz, 1H),
8.00 (s, 1H), 10.00 (s, 1H). 13C NMR (75 MHz, DMSO-d6): δ 53.4, 72.0, 113. 7, 116.1 (q, J =
3.8 Hz), 120.8 (q, J = 4.3 Hz), 123.6 (q, J = 0.7 Hz), 124.0 (q, J =, 272.3 Hz), 127.7, 128.1,
128.2, 128.4, 128.5, 128.8, 129.6 (q, J = 31.7 Hz), 130.3, 135.7, 136.2, 138.6, 155.9, 158.2.
MS (ESI) 453 [M + H]+.
4.1.7.7 4-(Benzyloxy)-1-(4-methoxybenzyl)-N-(3-(trifluoromethyl)phenyl)-1H-1,2,3-triazole-
1
5-carboxamide (19f). White solid (powder, m.p. 138.9 - 140.3 °C). Yield 65 %. H NMR
(300 MHz, DMSO-d6): δ 3.70 (s, 3H), 5.49 (s, 2H), 5.75 (s, 2H), 6.89 (d, J = 7.9 Hz, 2H),
7.24 (d, J = 7.9 Hz, 2H), 7.30‒7.67 (m, 7H), 7.82 (d, J = 8.0 Hz, 1H), 8.01 (s, 1H), 10.00 (s,
13
1H). C NMR (75 MHz, DMSO-d6): 52.9, 55.1, 71.9, 113.5, 114.1, 116.1 (q, J = 3.0 Hz),
120.7 (q, J = 3.9 Hz), 123.6 (q, J = 0.5 Hz), 124.0 (q, J = 272.3 Hz) 127.5, 128.1, 128.3,
128.4, 129.5, 129.6 (q, J = 31.8 Hz), 130.3, 136.2, 138.6, 155.9, 158.2, 159.2. MS (ESI) 483
[M + H]+.
4.1.8. General hydrogenation procedure to obtain target compounds 1, 3 - 8.
The appropriate protected hydroxyazole (18, 19a-f, 0.400 mmol) dissolved in dry THF (20
mL) was hydrogenated in presence of Pd/C (45 mg) for 1 hour at atmospheric pressure. The
reaction mixture was filtered off through a short layer of celite and the solvent was
evaporated under reduced pressure yielding the desired compound.
4.1.8.1. 4-Hydroxy-N-(3-(trifluoromethyl)phenyl)-1,2,5-oxadiazole-3-carboxamide (1). White
1
solid (powder, m.p. 165.3 - 167.5 °C, from diisopropyl ether/hexane). Yield 62 %. H-NMR
12

ACCEPTED MANUSCRIPT
(300 MHz, (CD3)2CO):
Hz, 1H), 8.30 (s, 1H), 10.24 (br s, 1H). C-NMR (75 MHz, (CD3)2CO):
δ
7.56 (d, J = 7.8 Hz, 1H), 7.69 (t, J = 8.0 Hz, 1H), 8.08 (d, J = 8.1
13
δ 117.8 (q, J = 3.9
Hz), 122.3 (q, J = 3.8 Hz), 124.7, 124.9 (q, J = 272.1 Hz), 131.0, 131.5 (q, J= 32.1 Hz),
139.2, 142.2, 156.7, 163.5. ESI-HRMS (m/z) [M - H]- calcd. for C10H5F3N3O3 272.0278,
obsd. 272.0274.
4.1.8.2. 5-Hydroxy-2-methyl-N-(3-(trifluoromethyl)phenyl)-2H-1,2,3-triazole-4-carboxamide
1
(3). Crystalline white solid (m.p. 216.6 - 217.0 °C, from acetonitrile). Yield 97 %. H NMR
(300 MHz, DMSO-d6): δ 4.07 (s, 3H), 7.43 (d, J = 7.8 Hz, 1H), 7.57, (t, J = 8.0 Hz, 1H), 8.00
(d, J = 8.1 Hz, 1H), 8.29 (s, 1H), 10.31 (s, 1H), 11.26 (br, 1H). 13C NMR (75 MHz, DMSO-
d6): δ 42.1, 116.2 (q, J = 4.1 Hz), 119.8 (q, J = 3.8 Hz), 123.6 (q, J = 1.1 Hz), 124.2 (q, J =
272.3 Hz), 124.9, 129.4 (q, J = 31.6 Hz), 129.8, 139.5, 158.8, 158.9. ESI-HRMS (m/z) [M -
H]- calcd. for C11H8F3N4O2 285.0605, obsd. 285.0594.
4.1.8.3. 4-Hydroxy-1-methyl-N-(3-(trifluoromethyl)phenyl)-1H-1,2,3-triazole-5-carboxamide
(4). Crystalline white solid (m.p. 235.6 - 241.0 °C, from acetonitrile). Yield 96 %. 1H NMR
(300 MHz, DMSO-d6): δ 4.19 (s, 3H), 7.47 (d, J = 7.7 Hz, 1H), 7.59 (t, J = 8.0 Hz, 1H), 7.83
(d, J = 8.2 Hz, 1H), 8.20 (s, 1H), 9.90 (s, 1H), 12.98 (br s, 1H). 13C NMR (DMSO-d6): δ 38.6,
111.9, 116.0 (q, J = 4.1 Hz), 120.3 (q, J = 3.9 Hz), 123.6 (q, J = 1.3 Hz), 124.0 (q, J = 272.4
Hz), 129.6 (q, J = 31.7 Hz), 130.1, 138.8, 156.6, 158.1. ESI-HRMS (m/z) [M - H]- calcd. for
C11H8F3N4O2 285.0605, obsd. 285.0605.
4.1.8.4. 2-(Cyclopropylmethyl)-5-hydroxy-N-(3-(trifluoromethyl)phenyl)-2H-1,2,3-triazole-4-
carboxamide (5). Crystalline white solid (m.p. 187.3 - 188.4 °C, from acetonitrile). Yield 96
1
%. H NMR (300 MHz, DMSO-d6): δ 0.38 - 0.47 (m, 2H), 0.54 ‒ 0.64 (m, 2H), 1.25‒1.40
(m, 1H), 4.18 (d, J = 7.2 Hz, 2H), 7.43 (d, J = 7.8 Hz, 1H), 7.57 (t, J = 8.0 Hz, 1H), 8.01 (d, J
= 8.2 Hz, 1H), 8.27 (s, 1H), 10.20 (s, 1H), 11.30 (br, 1H). 13C NMR (75 MHz, DMSO-d6): δ
3.6, 10.7, 59.3, 116.2 (q, J = 3.6 Hz), 119.8 (q, J = 3.7 Hz), 123.7 (q, J = 1.4 Hz), 124.1 (q, J
= 272.3 Hz), 124.8, 129.4 (q, J = 31.5 Hz), 129.8, 139.4, 158.6, 159.0. ESI-HRMS (m/z) [M -
H]- calcd. for C14H12F3N4O2 325.0910, obsd. 325.0907.
4.1.8.5. 3-(Cyclopropylmethyl)-4-hydroxy-N-(3-(trifluoromethyl)phenyl)-1H-1,2,3-triazole-4-
carboxamide (6). Crystalline white solid (m.p. 226.9 - 229.0 °C, from acetonitrile). Yield 79
%. 1H NMR (300 MHz, DMSO-d6): δ 0.35 ‒ 0.45 (m, 2H), 0.47 ‒ 0.57 (m, 2H), 1.30 ‒ 1.45
(m, 1H), 4.47 (d, J = 7.3 Hz, 2H), 7.48 (d, J = 7.7 Hz, 1H), 7.60 (t, J = 8.0 Hz, 1H), 7.83 (d, J
= 8.2 Hz, 1H), 8.20 (s, 1H), 10.01 (s, 1H), 13.05 (br s, 1H). 13C NMR (75 MHz, DMSO-d6):
δ 3.49, 11.2, 55.3, 110.9, 115.9 (q, J = 4.0 Hz), 120.3 (q, J = 3.9 Hz), 123.5 (q, J = 1.1 Hz),
123.9 (q, J = 272.4 Hz), 129.5 (q, J = 31.6 Hz), 130.0, 138.7, 156.5, 158.2. ESI-HRMS (m/z)
[M - H]- calcd. for C14H12F3N4O2 325.0907, obsd. 325.0911.
4.1.8.6. 1-Benzyl-4-hydroxy-N-(3-(trifluoromethyl)phenyl)-1H-1,2,3-triazole-5-carboxamide
(7). Fluffy white solid (m.p. 222.2 - 224.6 °C, from acetonitrile). Yield 97 %. 1H NMR (300
MHz, DMSO-d6): δ 5.84 (s, 2H), 7.23‒7.39 (m, 5 H), 7.46 (d, J = 7.9 Hz, 1H), 7.57 (t, J = 8.0
13
Hz, 1H), 7.82 (d, J = 8.1 Hz, 1H), 8.13 (s, 1H), 9.88 (s, 1H), 12.73 (br, 1H). C NMR (75
MHz, DMSO-d6): δ 53.6, 111.3, 116.2 (q, J = 4.3 Hz), 120.5 (q, J = 4.3 Hz), 123.8 (q, J = 1.3
Hz), 124.1 (q, J = 272.4 Hz), 127.6, 128.1, 128.7, 129.6 (q, J = 31.7 Hz), 130.2, 135.8, 138.7,
156.5, 158.3. ESI-HRMS (m/z) [M - H]- calcd. for C17H12F3N4O2 361.0907, obsd. 361.0908.
4.1.8.7. 4-Hydroxy-1-(4-methoxybenzyl)-N-(3-(trifluoromethyl)phenyl)-1H-1,2,3-triazole-5-
carboxamide (8). Fluffy white solid (m.p. 215.5 - 217.7 °C, from acetonitrile). Yield 98 %.
1H NMR (300 MHz, DMSO-d6): δ 3.71 (s, 3H), 5.75 (s, 2H), 6.90 (d, J = 8.6 Hz, 2H), 7.25
(d, J = 8.6 Hz, 2H), 7.47 (d, J = 7.8 Hz, 1H), 7.58 (t, J = 7.9 Hz, 1H), 7.83 (d, J =8.2 Hz, 1H),
8.14 (s, 1H), 9.90 (s, 1H), 12.88 (br, 1H). 13C NMR (75 MHz, DMSO-d6): δ 53.2, 55.1,
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111.1, 114.1, 116.1 (q, J = 4.1 Hz), 120.5 (q, J = 3.9 Hz), 123.8 (q, J = 1.5 Hz), 124.1 (q, J =
272.1 Hz), 127.6, 129.4, 129.6 (q, J = 30.4 Hz), 130.2, 138.7, 156.5, 158.4, 159.1. ESI-
HRMS (m/z) [M - H]- calcd. for C18H14F3N4O3 391.1024, obsd. 391.1031.
4.1.9. 4-Hydroxy-N-[3-(trifluoromethyl)phenyl]-1,2,5-thiadiazole-3-carboxamide (2). The
compound was obtained following the general procedure for synthesis of amides starting
from 17.[29] Flash chromatography eluent: DCM / methanol 90/10 v/v. White solid (powder,
m.p. 177.6 – 178.9 °C, from diisopropyl ether). Yield 40 %.1H-NMR (300 MHz, CD3OD): δ
7.46 (1H, d, J = 7.9 Hz), 7.56 (1H, t, J = 8.0 Hz), 7.97 (1H, d, J = 8.2 Hz), 8.20 (1H, s). 13C-
NMR (75 MHz, CD3OD)
δ 118.3 (q, J = 4.0 Hz), 122.3 (q, J = 4.0 Hz), 125.1 (q, J = 1.3 Hz),
125.5 (q, J = 272.5 Hz), 130.8, 132.2 (q, J = 32.3 Hz), 139.7, 140.8, 160.7, 166.0. MS (CI)
290 [M + H]+. ESI-HRMS (m/z) [M - H]- calcd. for C10H5F3N3O2S 288.0049, obsd.
288.0047.
4.2. Expression and purification of recombinant human AKR1C3 and AKR1C2.
Plasmid coding for human AKR1C3 was kindly provided by Prof. J. Adamski (Institute of
Experimental Genetics, Genome Analysis Center, Neuherberg, Germany). The cDNA
showed one mutation His5Gln in comparison to the NCBI sequence. This conservative
mutation is described in the literature as a single nucleotide polymorphism (SNPs) and seems
to be very common (refSNP: rs12529). Clone RC213538 containing human AKR1C2 cDNA
was obtained by Origene. cDNA was sequenced by the C.R.I.B.I. – BMR Servizio
Sequenziamento DNA, Padova (Italy). Then, it was amplified by PCR and, subsequently,
subcloned using Xho I and Sal I into the same pGEX 2T- modified plasmid vector (kindly
provided by Prof. J. Adamski), used for AKR1C3.
Plasmids coding for AKR1C3 and AKR1C2 were transformed into Escherichia coli BL21
(DE) Codon Plus RP (Agilent Technologies). For the protein expression, bacteria cells
expressing AKR1C3 and AKR1C2 were grown in YT2X media supplemented with
ampicillin at 37 °C with continuous shaking. At OD600 nm = 0.6 the expression was induced
by IPTG (0.5 mM). Bacteria were harvested 2 h after induction by centrifugation and stored
at -20°C until use. For the AKR1C3 and AKR1C2 purification, bacteria were suspended in
PBS supplemented with lysozyme (0.1 mg/ml) and protease inhibitor (Sigma). Then, bacteria
were lysed by four freeze-thaw cycles followed by DNA-digestion with benzonase (25 U) in
presence of MgCl2 5 mM. After centrifugation of the lysate for 30 min at 4°C and 13,000 x g,
the supernatant was collected. Then, AKR1C3 and AKR1C2 were affinity purified via N-
terminal GST-tag on glutathione (GT) sepharose (GE-Healthcare) and cleaved of by
thrombin according to the manufacturer’s protocol. Expression and purification was
monitored by SDS-PAGE.
4.3
In vitro AKR1C3 and AKR1C2 inhibition assays.
The inhibition assays were carried out by using bacterial suspension or purified enzymes. In
the screening of AKR1C3 inhibitors, bacteria expressing AKR1C3 were suspended in 100
mM phosphate buffer pH 6.6 without lysis. The bacterial suspension (about 15 µg of
proteins) was incubated with [14C]androstenedione (6 X 10-4 µCi) in the presence of Tween80
(0.1 mg/mL), ATP (1 mM), MgCl2 (1 mM) and a NADPH generating system (1 mM NADP+,
3 mM glucose-6-phosphate and 3 units of G-6-P dehydrogenase) for 30 min at 30°C with
vigorous shaking. Inhibitors, when present, were added as solution in ETOH (10% v/v). The
enzymatic reaction was stopped by heating at 80 °C for 10 min. After extracting two times
with ethyl ether (1.5 mL), the solvent was evaporated and the extract was separated on TLC
silica gel plates using chloroform/ethyl acetate (4:1; v/v) as a developing system. Percent
conversion of the labeled substrate to testosterone was estimated by integration from
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radioactivity scans with a System 200 Imaging Scanner (Hewlett-Packard, Palo Alto, CA,
USA).
In order to study the selectivity versus AKR1C2 of the active inhibitors of AKR1C3, the
purified recombinant enzymes were used. The activity of the inhibitors was evaluated by
using as a substrate S-tetralol in 96-well format. The reaction was fluorimetrically (exc/em;
340 nm/ 460 nm) monitored by the measurement of NADPH production on a “Ensight” plate
reader (Perkin Elmer) at 37 °C. Assay mixture contained S-tetralol (in ETOH ), inhibitor (in
ETOH), 100 mM phosphate buffer, pH 7, 200 µM NADP+, and purified recombinant enzyme
(30 µl) in a final volume of 200 µl and 10% ETOH. The S-tetralol concentration used in the
AKR1C2 and AKR1C3 inhibition assay were 15 microM and 160 microM, respectively, the
some as the Km described for the respective isoforms under the same experimental
conditions. Percent inhibition with respect to the controls containing the same amount of
solvent, without inhibitor, was calculated from the initial velocities, obtained by linear
regression of the progress curve, at different concentrations of inhibitor. The IC50 values were
obtained using PRISM 7.0, GraphPad Software. The values are the means of two separate
experiments each carried out in triplicate.
4.4
COX1 and COX2 inhibition assays
References and selected compounds were tested for their ability to inhibit COX-1 and COX-2
using a COX (ovine/human) Inhibitor Screening Assay Kit (Cayman Chemical Co., Ann
Arbor, MI), following manufacturer’s instructions. The assay directly measured PGF2α by
SnCl2 reduction of COX-derived PGH2 produced in the COX reaction. The prostanoid
product was quantified via enzyme immunosorbent assay (ELISA); absorbance
measurements were obtained on a PerkinElmer 2030 Multilabel Reader. IC50 values were
obtained by linear regression using PRISM 7.0, GraphPad Software. Results were calculated
as mean value ± standard error (SE) of at least three experiments.
4.5
Tumor cell lines and cell culture.
22RV1 castration-resistant prostate cancer cells were used. Cells were grown in RPMI
supplemented with 10% (v/v) fetal calf serum, 2% (v/v) penicillin-streptomycin, 0.03% L-
glutamine and maintained at 37 °C in a humidified atmosphere containing 5% CO2.
4.6
Cell proliferation assay.
Cell growth inhibition was evaluated by sulforhodamine B colorimetric proliferation assay
(SRB assay) modified by Vichai and Kirtikara.[37] 22RV1 cells were seeded into 96-well
plates in RPMI containing 10% charcoal stripped serum, 2 % (v/v) penicillin-streptomycin
and 0.03% L-glutamine, at a density of 10,000 cells/well and incubated at 37 °C with 5%
CO2 for 24 hours to allow cellular adhesion. Various dilutions of inhibitors in ethanol were
added in triplicate, and incubated for 72 h. Control cells were incubated with the same final
concentration of ethanol (maximum concentration 1% v/v). The assay was done as previously
described.[38] For co-treatment experiments, 22RV1 cells were treated with abiraterone (10
µM) or enzalutamide (20 µM) with or without compound 8 (60 µM) for 72 h. The statistical
analysis were performed with PRISM 7.0, GraphPad Software. The values are the means of
two separate experiments each carried out in triplicate.
4.7
Inhibition of AKR1C3-Mediated Production of Testosterone in 22RV1 cells.
22RV1 cells were seeded into 96-well plates in RPMI media containing 10% charcoal
stripped serum, 2% (v/v) penicillin-streptomycin and 0.03% L-glutamine, at a density of
30,000 cells per well, and were incubated at 37°C with 5% CO2 for 24 hours. Compound 8
was added to the wells at 4 different concentrations and incubated for 1 hour. Equimolar
(28nM) concentration of androstenedione was then added to the wells. The plate was returned
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to the incubator for a further 24h. Cell supernatant was removed for analysis of testosterone
by ELISA following the manufacturer’s guide (Testosterone ELISA kit was purchased from
Cayman Chemical Company). The ELISA plate was read at a wavelength of 405nM on a
microplate reader. Analysis was performed using the Cayman Chemical Company’s online
available analysis tool and data was quantitated against a standard curve generated in ELISA
buffer. Cross reactivity to androstenedione was accounted for by adding these to cell-free
wells of the ELISA plate. The statistical analysis were performed with PRISM 7.0, GraphPad
Software. The values are the means of two separate experiments each carried out in triplicate.
4.8
Western blot.
25 ug of total protein lysate was loaded for analysis by western blot. For detection of
AKR1C3, 1:10,000 dilution of mouse monoclonal anti-AKR1C3 (Sigma Aldrich) was
prepared in blocking buffer and added to the membrane with an overnight incubation at 4 °C
on a shaker. Mouse B-actin (Sigma Aldrich) was used as internal control with a dilution of
1:20,000 in blocking buffer, with overnight incubation at 4 °C on a shaker. Membranes were
subsequently washed with PBST and incubated with secondary goat anti-mouse antibody
(Thermo Fisher Scientific) at a dilution of 1:500 for 1 hour at room temperature on a shaker.
Membranes were washed and exposed to UV light to detect target bands and captured using a
digital camera.
4.9 Molecular Modeling.
The structures of compounds 6, 7 and 8, as well as the structures of the lead compound
Flufenamic Acid, were built in their dissociated forms using the 2D Sketcher tool
implemented in Maestro GUI. For each compound, an advanced conformational search was
performed using OPLS_2005 as Force Field and setting 1000 maxium steps for each run.
Quantum mechanics/molecular mechanics (QM/MM) docking was performed using
Schrödinger QM-Polarized Ligand Docking protocol (QPLD).[36] For this purpose, the X-
ray crystallographic structure of AKR1C3 was retrieved from RCSB Database (PDB code:
1S2C) and the generated conformers were docked. Before docking, the crystal structure of
the protein underwent an optimization process using the Protein Preparation Wizard tool,
implemented in Maestro™ GUI. Missing hydrogen atoms were added and bond orders were
assigned. Then, DMS, non-structural water molecules and impurities (such as solvent
molecules) were removed. The water 2152 was mantained in the binding site because its
important water network,[27] also the cofactor NAP and the co-crystalized ligand Flufenamic
Acid were maintained. Reorienting automatically optimized the hydrogen bond network:
hydroxyl and thiol groups, amide groups of asparagine (Asn) and glutamine (Gln), and the
imidazole ring in histidine (His). Moreover, the protonation states prediction of His—aspartic
acid (Asp), glutamic acid (Glu), and tautomeric states of His—were accomplished using
PROPKA.™ Finally, a restrained minimization of the protein structure was accomplished by
converging heavy atoms to a 0.30 Å RMSD. A grid of 10 Å x 10 Å x 10 Å (x, y, and z) was
created and centered on the co-crystalized ligand Flufenamic acid. The ligand was extracted
from the structure and used for docking validation. The QPLD protocol was carried out using
Glide Extra Precision (XP) mode, setting QM Level to Accurate (B3LYP functional, 6-
31G*/LACVP* basis set, ultrafine SCF accuracy level). In the QPLD procedure, after the
first XP docking run, QM-derived charge is calculated for the top five poses of each
compound in the field of the receptor. Then, a new XP docking is performed with new QM
charges calculated. Finally, re-docking and re-scoring were performed, keeping the 10
highest ranked poses. Regarding docking analysis on COX the crystallographic complex of
diclofenac and COX1 was used (PDB code: 3N8Y). The same protocol discussed above were
utilized for the protein and ligands preparation. The overall of docking were obtained using
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Glide XP keeping default parameters and centered the grid on the co-crystalized ligand
Diclofenac.
AUTHOR INFORMATION
Corresponding Authors: *
Phone: +39-0116707993. Fax: +39-0116707687, e-mail: marco.lolli@unito.it.
Phone: +39-0116706864 . e-mail: simona.oliaro@unito.it.
Notes. The authors declare no competing financial interest.
ABBREVIATIONS USED
Aldo-keto reductase 1C3 isoform (AKR1C3), Prostate cancer (PCa), androgen deprivation
therapy (ADT), castration-resistant prostate cancer (CRPC), androgen receptor (AR),
flufenamic acid (FLU), sub-pocket 2 (SP2), sub-pocket 1 (SP1), cyclooxygenase (COX),
aldo-keto reductase 1C2 isoform (AKR1C2), dichloromethane (DCM), dimethylformamide
(DMF), methanol (MeOH), Quantum mechanics/molecular mechanics (QM/MM), QM-
Polarized Ligand Docking (QPLD), Palladium on carbon (Pd/C), hexadeuterodimethyl
sulfoxide (DMSO-d6), deuterochloroform (CDCl3), tetradeuteromethanol (CD3OD),
deuterated acetone (CD3)2CO).
ACKNOWLEDGEMENTS
We thank Prof Norman J. Maitland (University of York) for provision of the 22RV1 PC cell
line and Prof. J. Adamski (Institute of Experimental Genetics, Genome Analysis Center,
Neuherberg, Germany) for supplying the plasmid coding for human AKR1C3. We also thank
prof. Francesca Spyrakis (DSTF) for fruitful discussions and useful suggestions on the
modeling studies. This research was financially supported by the University of Turin
(Ricerca Locale grant 2014 and 2015) and Prostate Cancer UK grant S12-027. Authors wish
to thank dr. Livio Stevanato for performing all the NMR experiments and for maintenance of
the instrument.
Appendix A. Supplementary data
Supplementary data related to this article can be found at: XXXXXX
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Highlights
1) New AKR1C3 inhibitors were designed and synthetized using hydroxyazole scaffolds.
2) Modeling was used to speculate the interaction with the AKR1C3 binding site.
3) New compounds were assayed for AKR1C3 selectivity and cell-based activities.
4) Cpd 8 shows synergistic effect in combination with abiraterone and enzalutamide.
1