Potent and selective aldo-keto reductase 1C3 (AKR1C3) inhibitors based on the benzoisoxazole moiety: application of a bioisosteric scaffold hopping approach to flufenamic acid

Article abstract of DOI:10.1016/j.ejmech.2018.03.040

The aldo-keto reductase 1C3 (AKR1C3) isoform plays a vital role in the biosynthesis of androgens and is considered an attractive target in prostate cancer (PCa). No AKR1C3-targeted agent has to date been approved for clinical use. Flufenamic acid and indomethacine are non-steroidal anti-inflammatory drugs known to inhibit AKR1C3 in a non-selective manner as COX off-target effects are also observed. Recently, we employed a scaffold hopping approach to design a new class of potent and selective AKR1C3 inhibitors based on a N-substituted hydroxylated triazole pharmacophore. Following a similar strategy, we designed a new series focused around an acidic hydroxybenzoisoxazole moiety, which was rationalised to mimic the benzoic acid role in the flufenamic scaffold. Through iterative rounds of drug design, synthesis and biological evaluation, several compounds were discovered to target AKR1C3 in a selective manner. The most promising compound of series (6) was found to be highly selective (up to 450-fold) for AKR1C3 over the 1C2 isoform with minimal COX1 and COX2 off-target effects. Other inhibitors were obtained modulating the best example of hydroxylated triazoles we previously presented. In cell-based assays, the most promising compounds of both series reduced the cell proliferation, prostate specific antigen (PSA) and testosterone production in AKR1C3-expressing 22RV1 prostate cancer cells and showed synergistic effect when assayed in combination with abiraterone and enzalutamide. Structure determination of AKR1C3 co-crystallized with one representative compound from each of the two series clearly identified both compounds in the androstenedione binding site, hence supporting the biochemical data.

Full text of DOI:10.1016/j.ejmech.2018.03.040

Accepted Manuscript
Potent and selective aldo-keto reductase 1C3 (AKR1C3) inhibitors based on the
benzoisoxazole moiety: Application of a bioisosteric scaffold hopping approach to
flufenamic acid
Agnese Chiara Pippione, Irene Maria Carnovale, Davide Bonanni, Marcella Sini,
Parveen Goyal, Elisabetta Marini, Klaus Pors, Salvatore Adinolfi, Daniele Zonari,
Claudio Festuccia, Weixiao Yuan Wahlgren, Rosmarie Friemann, Renzo Bagnati,
Donatella Boschi, Simonetta Oliaro-Bosso, Marco Lucio Lolli
PII:
S0223-5234(18)30283-6
10.1016/j.ejmech.2018.03.040
EJMECH 10305
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 22 December 2017
Revised Date: 13 March 2018
Accepted Date: 14 March 2018
Please cite this article as: A.C. Pippione, I.M. Carnovale, D. Bonanni, M. Sini, P. Goyal, E. Marini,
K. Pors, S. Adinolfi, D. Zonari, C. Festuccia, W.Y. Wahlgren, R. Friemann, R. Bagnati, D. Boschi, S.
Oliaro-Bosso, M.L. Lolli, Potent and selective aldo-keto reductase 1C3 (AKR1C3) inhibitors based on
the benzoisoxazole moiety: Application of a bioisosteric scaffold hopping approach to flufenamic acid,
European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.03.040.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Potent and selective Aldo-Keto Reductase 1C3 (AKR1C3) inhibitors based on the
benzoisoxazole moiety: Application of a Bioisosteric Scaffold Hopping Approach to
Flufenamic acid
Agnese Chiara Pippione,1 Irene Maria Carnovale,1 Davide Bonanni,1 Marcella Sini,2 Parveen
Goyal,4 Elisabetta Marini,1 Klaus Pors,2 Salvatore Adinolfi,1 Daniele Zonari,1 Claudio
Festuccia,3 Weixiao Yuan Wahlgren,4 Rosmarie Friemann,4 Renzo Bagnati,5 Donatella
Boschi,1 Simonetta Oliaro-Bosso1* and Marco Lucio Lolli1*
1
Department of Science and Drug Technology, University of Torino, via Pietro Giuria 9,
10125 Torino (Italy).
2 Institute of Cancer Therapeutics, School of Pharmacy and Medical Sciences, Faculty of Life
Sciences, University of Bradford, BD7 1DP West Yorkshire (UK).
3 Department of Biotechnological and Applied Clinical Sciences, Laboratory of Radiation
Biology, University of L'Aquila, via Vetoio snc, Coppito II, 67100 L'Aquila (Italy).
4 Department of Chemistry and Molecular Biology, University of Gothenburg, Box 462, S-
40530 Gothenburg (Sweden).
5 IRCCS Istituto di Ricerche Farmacologiche Mario Negri, Via La Masa 19, 20156 Milano
(Italy).
Keywords
aldo-keto reductase 1C3; AKR1C3; 17β-HSD5; Prostate cancer (PCa); CRPC; bioisosterism;
scaffold hopping; inhibitors; X-ray crystallography.
Abstract
The aldo-keto reductase 1C3 (AKR1C3) isoform plays a vital role in the biosynthesis of
androgens and is considered an attractive target in prostate cancer (PCa). No AKR1C3-
targeted agent has to date been approved for clinical use. Flufenamic acid and indomethacine
are non-steroidal anti-inflammatory drugs known to inhibit AKR1C3 in a non-selective
manner as COX off-target effects are also observed. Recently, we employed a scaffold
hopping approach to design a new class of potent and selective AKR1C3 inhibitors based on
a N-substituted hydroxylated triazole pharmacophore. Following a similar strategy, we
designed a new series focused around an acidic hydroxybenzoisoxazole moiety, which was
rationalised to mimic the benzoic acid role in the flufenamic scaffold. Through iterative
rounds of drug design, synthesis and biological evaluation, several compounds were
discovered to target AKR1C3 in a selective manner. The most promising compound of the
series (6) was found to be highly selective (up to 450-fold) for AKR1C3 over the 1C2
isoform with minimal COX1 and COX2 off-target effects. Other inhibitors were obtained
modulating the best example of hydroxylated triazoles we previously presented. In cell-based
assays, the most promising compounds of both series reduced the cell proliferation, prostate
specific antigen (PSA) and testosterone production in AKR1C3-expressing 22RV1 prostate
cancer cells and showed synergistic effect when assayed in combination with abiraterone and
enzalutamide. Structure determination of AKR1C3 co-crystallised with one representative
compound from each of the two series clearly identified both compounds in the
androstenedione binding site, hence supporting the biochemical data.
1

ACCEPTED MANUSCRIPT
1. Introduction
AKR1C3, or HSD17B5, is a soluble enzyme member of the aldo-ketoreductase family,
which is highly expressed in testes and extragonadal tissues such as basal cells of the
prostate, adrenals and liver.[1] It catalyses the NADPH dependent reduction of
androstenedione (AD) to testosterone and, compared to other HSD17B isoforms, is the most
abundant isoform with elevated levels found in agressive PCa, such as castration-resistant
prostate cancer (CRPC).[2] AKR1C3 may provide a mechanism to divert traces of androgens
that remain after androgen deprivation therapy (ADT) to the potent androgen receptor (AR)
ligand 5α-dihydrotesterone (DHT).[3] Besides evidence that AR mutations, splice variants
and increased copy number represent putative mechanisms of resistance to therapy,[4-7]
AKR1C3 has also been discovered to play a role in resistance to pharmacological[6] and
radiation[8] therapy. Potential clinical use of AKR1C3 inhibitors has been demonstrated as in
the case of indomethacin, a potent but unselective AKR1C3 inhibitor able to circumvent
resistance to both abiraterone (ABI) [9] and enzalutamide (ENZA) [10] (Figure 1). Although
few recent studies indicate controversial observations about the in vivo effectiveness of
AKR1C3-based therapies,[11-13] other studies advocate for AKR1C3 as a therapeutic target
in PCa.[2, 4] Even if several lead compounds have emerged from AKR1C3-targeting
medicinal chemistry programs,[14-17], no agent has been approved yet for clinical use.[18]
In order to understand the clinical potential of targeting AKR1C3, it is desirable to develop
more potent, selective and drug-like AKR1C3 inhibitors. Amongst NSAIDs, flufenamic acid
(FLU, Figure 1) inhibits AKR1C3 in a non-selective manner, as it suffers from
cyclooxygenase (COX) off-target effects.[19, 20] The COX active site consists of a narrow
long hydrophobic channel that ends with a charged arginine residue (Arg120), which
provides a suitable pocket for a ligand that contains a carboxylate group.[21] The carboxylic
acid moiety of all the fenamate inhibitors forms a salt bridge with Arg120, with the two
carboxylate oxygen atoms of fenamates within the proximity of 1.45 Å and 1.60 Å from one
of the guanidine nitrogens of the arginine. This binding mode affects the ionic bond
enthalpies and impacts on the overall inhibitory effect.[21] Removal of this carboxylic acid
moiety in fenamate derivatives concurs with reduction of COX1 activity as demonstrated by
us in a recent report on the design of new AKR1C3 inhibitors.[22] We successfully applied a
scaffold hopping strategy based on the replacement of the FLU benzoate moiety with three
hydroxyazoles (hydroxyfurazan, hydroxythiadiazole and a series of N-substituted hydroxyl-
1,2,3-triazoles). Through the design of these new ligands we showed that hydroxyazoles are
valid bioisosters of the carboxylic acid function. Depending on their acidic properties, they
can be deprotonated to various degrees at physiological pH.[23-26] Inside the series, we
demonstrated how the replacement of the benzoic acid of FLU with a 4-hydroxy-N1-
substituted triazolecarbonylic moiety enables a bioisosteric scaffold-hopping replacement for
AKR1C3 activity with no associated COX effect. Moreover, regiosubstitution of nitrogen
atoms of the hydroxytriazole ring allowed the possibility to perform a structural
refinement,[27] which enabled an opportunity to improve AKR1C3 binding while reduce
AKR1C2 and COX1/2 off-target binding.
2

ACCEPTED MANUSCRIPT
N
HN
F
O
F3C
NC
S
H
N
N
H
H
HO
O
Abiraterone
Enzalutamide
F3C
F3C
NH
F3C
NH
O
NH
N
OH
O
OH
N
OH
N
O
FLU
meta-
1
Flufenamic acid
(FLU)
O
Figure 1. Chemical structures of some known AKR1C3 inhibitors.
Hydroxytriazole 1[22] (Figure 1), the most active compound of our previous study, was
used as starting point to design two triazoles 2 and 3 (Figure 2) bearing two p- and m-
methoxy or one p-trifluoromethoxy substituents, respectively. In analogy to 1, the rationale
was to project its 4-methoxybenzyl moiety into the unexplored SP2 sub-pocket of AKR1C3
and thereby establishing, as the docking studies suggested, π-π staking and T-shape π-π
staking with Trp227 (partial overlapping) and Trp86[22]; the new substitutions in the phenyl
ring provided an opportunity to reinforce the binding by establishing interactions with the
polar Ser129 present in this pocket [22]. In the following, a conformational restriction
approach was employed to improve the potency and the selectivity of FLU, resulting in the
design of a small, but focused library of 3-hydroxybenzoxazole-based compounds in which
the carboxylic acid substituent was fused with the benzene ring, in order to constrain one of
FLU conformations (compounds 4 - 8, Figure 2). We report here on synthetic strategies,
biochemical and cell-based studies of the new compounds alone or in combination with ABI
and ENZA. In addition, to demonstrate selective AKR1C3 inhibition with impact on
testosterone and PSA synthesis, we also show the binding mode of compound 1 and the most
potent 3-hydroxybenzoxazole (compound 6) by high-resolution crystal structures of
AKR1C3 complexed with each of these two compounds.
3

ACCEPTED MANUSCRIPT
F3C
F3C
NH
N
NH
N
OH
OH
O
O
N
N
N
N
O
O
O
F3C
NH
2
3
OH
N
F3C
NH
OH
N
O
CF3
O
4
5
CF3
O2N
F5S
NH
NH
OH
OH
N
F3C
NH
OH
N
N
O
O
O
6
7
8
Figure 2. Chemical structures of AKR1C3 inhibitors studied in this work.
2. Result and discussion
2.1 Chemistry.
The methodology used for the synthesis of the two triazole derivatives 2 and 3 is described in
Scheme 1. The common intermediate 9 was regioselectively alkylated by the appropriate
benzylhalide using the procedure previously described.[23] The building block 9 is
susceptible to alkylation directed towards positions N(b) and N(c) of the triazole ring, leading
in each case to a mixture of two isomeric products(10 - 12 and 11 - 13, respectively). The
isomeric mixtures were chromatographically resolved and the N(c) esters 10 and 12 were
hydrolysed to the corresponding carboxylic acids 14 and 15. The latter two compounds were
converted into the corresponding acyl chlorides and allowed to react with 3-
(trifluoromethyl)aniline to afford amides 16 and 17, which were subsequently deprotected
through catalytic hydrogenation to obtain the desired target compounds 2 and 3.
4

ACCEPTED MANUSCRIPT
X = Cl or Br
X
O
O
O
OEt
O
OEt
O
N
R
2
N
N
N
N
O
OEt
R
1
N
N
+
R2
R1
a
N
c
a
N
R2
b
R1
10 R1=R2=OCH3
9
11 R1=R2=OCH3
12 R1= OCF3 R2=H
13 R1= OCF3 R2=H
CF3
CF3
O
N
O
N
O
N
HO
O
O
NH
OH
NH
b
e
c, d
10
12
N
N
N
N
N
N
R2
R2
R2
R1
R1
R1
14 R1=R2=OCH3
15 R1= OCF3 R2=H
16 R1=R2=OCH3
17 R1= OCF3 R2=H
2 R1=R2=OCH3
3 R1= OCF3 R2=H
Scheme 1: a) Cs2CO3, CH3CN, rt; b) 1) NaOH, EtOH, rt; 2) 2M HCl; c) (CO)2Cl2, dry DMF,
dry THF, rt; d) 3-(trifluoromethyl)aniline, dry pyridine, dry THF, rt; e) H2, Pd/C, dry THF, rt.
The methodology used for the synthesis of the 4-anilino-1,2-benzoxazol-3-ols derivatives 4
and 6 - 8, described in Scheme 2, follows a described procedure for substituted 1,2-
benzoxazol-3-ols.[28] Commercially available 2-bromo-6-fluorobenzoic acid was treated
with (CO)2Cl2 and dry DMF in dry THF to generate acyl chloride 18, which was coupled
with N-[(2,4,6-trimethoxyphenyl)methyl]hydroxylamine 19 to afford N-hydroxybenzamide
20. Cyclisation of compound 20 in basic condition yielded the 1,2-benzisoxazol-3-one
derivative 21, where the nitrogen atom of the ring is protected with the 2,4,6-
trimethoxybenzyl (Tmob) group. Starting from intermediate 21, four substituted anilines
were used to perform Buchwald-Hartwig coupling in which halogenated rings were coupled
with aniline derivatives in the presence of a palladium catalyst and a base. Compound 21 was
treated with the appropriate aniline, Pd(OAc)2, (±)-2,2-Bis(diphenylphosphino)-1,1-
binaphthalene (BINAP) and Cs2CO3 in dry toluene at reflux for 5 hours, affording
compounds 22 - 25. After coupling, the target compounds 4 and 6 - 8 were obtained from 22
- 25 by deprotection of Tmob through treatment with trifluoroacetic acid (TFA) and
triisopropylsilane in dry DCM.
5

ACCEPTED MANUSCRIPT
O
19
HN
OH
O
O
Br
O
O
F
Br
Br
COOH
F
COCl
F
N
a
b
c
OH
O
O
18
20
Br
R
O
NH
O
R
N
O
O
O
d
e
NH
OH
N
N
O
O
O
O
O
O
4 R = m-CF3
R = m,m'-bisCF
21
22 R = m-CF3
23 R = m,m'-bisCF3
R = m-NO
6
3
7 R = m-NO2
8 R = m-SF5
24
25 R = m-SF5
2
Scheme 2: a) (CO)2Cl2, dry DMF, dry THF, rt; b) Et3N, dry THF, rt; c) K2CO3, dry DMF,
reflux; d) substituted aniline, Cs2CO3, BINAP, Pd(OAc)2, dry toluene, reflux; e) TFA, (i-
Pr)3SiH, dry DCM, rt.
Unfortunately, the Buchwald-Hartwig coupling conditions failed when applied to affording
compound 5 from the appropriate 5-bromo isomer of 21. This behaviour is probably due to a
low reactivity of the bromide group in position 5 of the Tmob protected 1,2-benzisoxazol-3-
one. To avoid such problem, a second synthetic route was devised to obtain 5 by changing
the sequence order of the reactions (Scheme 3). The Buchwald-Hartwig coupling between the
3-trifluoromethylaniline was performed as the first reaction with the methyl 5-bromo-2-
fluorobenzoate 26. Before coupling intermediate 27 with 19, it was necessary to Boc protect
the aniline nitrogen in order to reduce its electron donor properties and thereby activate the
para-positioned fluorine to nucleophilic substitutions. The treatment of 27 with Boc2O in dry
THF afforded the Boc protected 28, which was hydrolysed to afford carboxylic acid 29. This
latter was coupled with 19 in the presence of DCC as activating agent to afford 30.
Importantly, when 30 was placed in presence of the basic conditions (K2CO3, DMF), it
cyclised to afford compound 31. This result was not observed when the analogue of 30
without Boc protection was treated in the same condition. Finally, the target compound 5 was
obtained from 31 by treatment with TFA and triisopropylsilane in order to remove both Boc
and Tmob protective groups.
6

ACCEPTED MANUSCRIPT
Boc
N
NH
COOCH 3
COOCH 3
F
Br
COOCH 3
F
a
b
F
CF3
CF3
26
27
28
Boc
N
Boc
N
O
O
COOH
d
c
F3C
N
OH
F
F
O
O
CF3
29
30
Boc
N
O
OH
N
F3C
NH
N
O
f
e
O
O
O
CF3
O
31
5
Scheme 3: a) 3-(trifluoromethyl)aniline, Cs2CO3, Pd(OAc)2, BINAP, dry toluene, reflux; b)
Boc2O, DMAP, dry THF, rt; c) KOH, EtOH, rt; d) 19, DCC, DMAP, dry DCM, rt; e) K2CO3,
dry DMF, reflux; f) TFA, (i-Pr)3SiH, dry DCM, rt.
2.2 AKR1C3 and AKR1C2 inhibitor screening.
Selective targeting of AKR1C3 over 1C2 is considered critical to effective PCa therapy.[29]
Not only does the two isoforms share 86 % sequence similarity, but AKR1C2 is also
involved in DHT inactivation and hence its inhibition is undesirable. Accordingly, the
inhibitory potencies of compounds 2 - 8 were determined for both AKR1C2 and AKR1C3
and compared with 1 and FLU as controls. To understand their selectivity the ratio of IC50
was used as indicator of AKR1C2 and AKR1C3 inhibition (a high ratio shows high
selectivity for AKR1C3, Table 1). The inhibitory activity was obtained using recombinant
purified enzymes with oxidation of S-tetralol in the presence of NADP+. Although the
triazole derivatives 1 - 3 presented IC50 values in the similar range as FLU (IC50 0.44 µM),
they presented greater than 240-290 fold selectivity for AKR1C3 over AKR1C2. The
trifluoromethoxy substituent in the para position of the benzylic moiety appeared to be
responsible for increasing activity, as 3 retained potent AKR1C3 inhibition with an IC50
value of 0.19 µM and 289-fold selectivity for AKR1C3 over AKR1C2. The inhibitory effect
of the 1,2-benzoxazoles series on AKR1C3 and C2 enzymes confirms the hypothesis that the
conformational restriction approach to FLU is able to retain potency and improve selectivity
of the over the lead compound (Table 1). Noteworthy, both 4 and FLU are equipotent in
regard to AKR1C3 inhibition (IC50 are 0.55 and 0.44, respectively), but the former is 8-fold
more selective in targeting AKR1C3 over AKR1C2. As expected, the shifting of the aniline
moiety to position 5 of the 1,2-benzoxazole to mimic the meta-FLU’s conformation led to
reduced potency but greater AKR1C3 selectivity.[20] Indeed, compound 5 displayed an IC50
value of 3.48 µM against the AKR1C3 isoform but exhibited 19-fold selectivity for AKR1C3
over AKR1C2 (AKR1C2 IC50 = 67 µM). The data support the 3-hydroxy-1,2-benzoxazole as
a promising new scaffold for the design of potent and selective AKR1C3 inhibitors.
In attempt to improve the potency and selectivity of compound 4, modulation of substitution
of the aniline moiety was performed. The modulation of FLU derivative by the introduction
of a second substituent in the aniline substructure is known[20] to improve potency and
7

ACCEPTED MANUSCRIPT
selectivity. Applying a similar strategy, the introduction of a second m-trifluoromethyl
substituent (compound 6) doubled the activity and improved drastically the selectivity for
AKR1C3 over AKR1C2 enzyme. Specifically, compound 6 displayed an IC50 value of 0.26
µM against AKR1C3 and 456-fold selectivity for AKR1C3 over AKR1C2 (IC50 119 µM).
This result indicates that the introduction of a second meta substituent in the phenyl ring of
compound 4 did not have a remarkable effect on the inhibitory potency for AKR1C3, but
decreased the affinity for AKR1C2 significantly.
Subsequently, (bio)isosteric replacements of the trifluoromethyl substituent of 4 were
performed. Between the possible isostere options, we chose the nitro and pentafluorosulfanyl
groups as they retained the activity in the FLU modulations.[20, 30] While the replacement
of the meta-trifluoromethyl substituent with the nitro group was a successful bioisosteric
modulation in the FLU scaffold,[20] when applied to our new 1,2-benzoxazole scaffold
(compound 7) it was detrimental for potency as well as for the selectivity. The IC50 values of
compound 7 were 1.41 µM and 5.70 µM for AKR1C3 and AKR1C2 respectively, showing
that 7 is 2,5-fold less active that its m-trifluoromethyl analogue 4 and 1.3-fold less selective.
On the contrary, the replacement of the m-trifluoromethyl substituent of 4 with the
pentafluorosulfanyl (SF5) moiety (compound 8) was very successful. The SF5 group, a
bioisoster of the CF3 and OCF3 groups, has been gaining greater attention and increased
reported usage in the literature.[31] While SF5-bearing building blocks are typically >5x
economically more expensive than the analogous CF3 compounds, they are particularly
attractive to medicinal chemists due to their reported effects on slowing the rates of
metabolism.[32] The SF5 group is a large, very electronegative and lipophilic group. It is also
more resistant to acid hydrolysis than either CF3 or OCF3.[31] FLU analogues bearing the
SF5 moiety in meta and para positions have been reported to retain comparable AKR1C3
inhibition activity and selectivity when compared with FLU.[30] Applying this bioisosteric
replacement on the 1,2-benzoxazole scaffold, it was evident that the SF5 derivative 8 gained
increase in AKR1C3 potency and selectivity (AKR1C3 vs AKR1C2) to afford a novel
analogue that performed similar to compound 4.
8

ACCEPTED MANUSCRIPT
Table 1. Inhibitory effect of compounds 1-8 and FLU against AKR1C3 and AKR1C2
recombinant purified enzymes.
AKR1C3
IC50 ± SE
(µM)
AKR1C2
IC50 ± SE
(µM)
Ratio
IC50 value
(1C2:1C3)
Compound
FLU
Structure
F3
C
NH
OH
0.44 ± 0.023 a 0.53 ± 0.032 a
1.2
O
O
NH
OH
F3
C
N
N
N
O
0.31 ± 0.005 a 73.23 ± 8.67 a
236
270
289
8
1
2
3
4
O
OH
F
3C
NH
N
N
N
0.40 ± 0.059
0.19 ± 0.020
0.55 ± 0.03
108.13 ± 7.42
54.87 ± 5.01
4.38 ± 0.49
O
O
O
OH
F3
C
NH
N
N
N
O
CF3
NH
F3
C
OH
N
O
OH
NH
F3
C
N
3.48 ± 0.48
0.26 ± 0.03
66.95 ± 10.67
118.55 ± 4.86
19
5
6
O
CF3
NH
NH
NH
F3
C
OH
456
N
O
O2
N
OH
N
1.41 ± 0.16
5.70 ± 0.30
4
7
8
O
F5
S
OH
N
0.31 ± 0.033
4.29 ± 0.144
14
O
a Data from ref.[22]
2.3. COX inhibition.
The compounds were also evaluated against COX1 and COX2 to ensure no off-target effects.
Compounds 2 - 8 were assayed for their inhibitor effect on COX1 and COX2 using ovine
COX1 (oCOX1) and human COX2 (hCOX2). Notably, compounds 2 - 8 did not display
significant inhibitory activity on any of the two COX isoforms at the highest concentration
evaluated (100 µM) (Table 2).
9

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Table 2. Inhibitory effect of compounds 1-8 and FLU against COX1 and COX2.
oCOX1
IC50 ± SE
(µM)
Ratio
IC50 value
(COX1:AKR1C3)
hCOX2
IC50 ± SE (µM)
Compound
> 100a
14 ± 1a
32
FLU
(18 % ± 2) a, b
>100a
(0)a, b
>100a
> 322
> 250
> 526
1
2
(12 % ± 4)a, b
> 100
(0)b
> 100
(0.87 % ± 0.87)b
> 100
(0)b
> 100
(0)b
3
4
> 100
> 100
> 182
> 29
(29 % ± 1)b
(13 % ± 4)b
> 100
> 100
(0)b
5
6
7
8
(29 % ± 3)b
> 100
> 100
(0)b
> 385
> 71
(2.1 % ± 2.1)b
> 100
(0)b
> 100
(0.37 % ± 0.37)b
> 100
(0)b
> 100
> 323
(4.3 % ± 2.3)b
a Data from ref. [22]
b % of inhibition ± SE at 100 µM.
2.4 Inhibition of cell proliferation and PSA expression
The effects of the compounds on cell proliferation were evaluated using the AKR1C3-
expressing 22RV1 PCa cell line. Prior to chemosensitivity screening, AKR1C3 protein was
confirmed using western blot (Figure 3A). The antiproliferative activity of the compounds
was initially performed using the sulforhodamine B (SRB) assay and the IC50 values are
reported in Figure 3B. All derivatives evaluated, except 7, exhibited a more pronounced
antiproliferative effect than FLU. Triazole 3 (IC50 31.28 µM) and 1,2-benzoxazole 6 (IC50
26.70 µM) were shown to be over 4-fold more potent than FLU (IC50 115 µM, Figure 3B).
Since the SRB assay does not distinguish between cytotoxic and cytostatic effects, a colony-
growing inhibition investigation was also carried out. In this assay, colonies were observed
and counted before and after treatment with selected compounds (triazole derivatives 1 - 3
and 1,2-benzoxazole derivative 6, Figure 3C-E). 22RV1 cells were incubated for 72 h in the
presence of these compounds and the number of viable and dead cells was measured with
NucleCounter. The percentage of live cells was significantly reduced in a dose-dependent
manner with all tested compounds. Interestingly, when the data for dead cells were analysed
(Figure 3D) it was evident that the decrease in cell viability correlated with cell death only
for analogues 1 and 6, while compounds 2 and 3 exhibited a more cytostatic than cytotoxic
effect (Figure 3D and 3E).
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Figure 3. Inhibition of cell proliferation. (A) Confirmation of AKR1C3 expression in
22RV1 cells by western blot. (B) IC50 values of antiproliferative activity using the SRB assay
and cLogP calculated by QikProp.[33] Direct count of live (C) and dead (D) cells by
NucleoCounter N100 after incubation for 72 h with compounds, 1-3 and 6 at different
concentrations. Each column represent the percentage of cells versus control (mean value).
Percentage of cell death (mean value) (E). Standard errors were lower than 10%. a) The
result is in agreement with our previous study[22]; b) % of inhibition ± SE at 100 µM.
In addition, the effect of the compounds on PSA expression were evaluated using western
blot and ELISA assays. Viable cells were lysed from treated 22RV cells described above and
western blots were performed. Figure 4A reveal that the PSA expression in the cell extracts
was reduced in a dose-dependent manner in cultures treated with compounds 2 and 3. High
doses of 6 also reduced PSA levels in the cell extracts, whereas only very low effects were
observed for 1. However, the differences were much more marked when the secretion of PSA
in culture supernatants were measured using ELISA. All compounds resulted in a significant
dose-dependent reduction of PSA secretion in treated cells (Figure 4B).
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Figure 4. Inhibition of PSA expression. Western blot analysis performed on cell extracts
(A) and ELISA assay performed on cell supernatants (secreted PSA) (B) of cultures treated
with 1 - 3 and 6 at three different concentrations. Standard errors for ELISA were lower of 10
%.
To explore further the effect of the novel compounds in the steroidogenic pathway,
compounds 3 and 6 were investigated for their ability to inhibit testosterone production. After
24 h incubation, androstenedione (AD) was added to each cell with or without compounds 3
and 6 at three concentrations (0.2, 2 and 20 µM). The cell culture media was collected 24 h
after treating the cells and both compounds had an effect on the synthesis of testosterone.
Compound 3 exerted a dose-dependent effect, whereas 6 had an initial effect at low dose (0.2
µM) but no improved effect at higher doses was observed (Figure 5). Even if compounds 3
and 6 possess similar AKR1C3 inhibitory activity, AKR1C3/1C2 and AKR1C3/COX
selectivity ratios, their effects on PSA and testosterone production indicate subtle differences.
This result may be related to a number of other key players in the AR dependent and
independent pathways of PCa.
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Figure 5. Inhibition of testosterone production. Evaluation of the inhibitory effect of
compound 3 and 6 in AD treated 22RV1 cells.
In order to evaluate a possible synergistic effect, compounds 3 and 6 were explored in
combination treatments with ABI and ENZA. 22RV1 cells were treated for 72 h with both
compounds at 20 µM with or without 10 µM ABI (Figure 6A). The same experiment was
carried out by treating cells with or without 20 µM ENZA (Figure 6B). At these
concentrations, ABI and ENZA had limited effects on cell growth. When compound 3 or 6
were added together with either ABI or ENZA, the cell viability was reduced by
approximately 10-25% compared with either drug alone, indicating a synergistic effect. The
22RV1 cell line was proved to have relative levels of resistance to both ABI and ENZA,[10-
12] hence the outcome of the combination experiments are encouraging and indicate the
potential of using an AKR1C3 inhibitor to intervene the steroidogenic pathway and effect
PSA and testosterone production.
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Figure 6. Effect of the co-treatment of compounds 3 and 6 with ABI (A) or ENZA (B) on
22RV1 cell proliferation using the SRB assay. Cells were treated with 20 µM of compounds
3 and 6 with or without 10 µM ABI or 20 µM ENZA for 72 h. Cell growth is expressed as %
T/C (mean OD of treated cells/mean OD of control cells X 100).
2.5 Analysis of the binding mode of compounds 1 and 6 co-crystallised in complex with
AKR1C3.
To support experimental data, co-crystallisation of AKR1C3 in complexes with compound 6
was carried out. Furthermore, we also determined the crystal structures of AKR1C3 in
complexes with compound 1, whose binding mode was speculated by us in previously
reported docking studies[22]. The structures were determined by molecular replacement and
have been refined to 1.88 Å (1, PDB ID: 6F2U) and to 1.30 Å (6, PDB ID: 6F78). X-ray data
collection and refinement statistics are summarised in Table S16. As reported
previously[34], the AKR1C3 ligand-binding pocket is composed by five compartments:
an oxyanion site (OS) formed by the cofactor NADP+ and the catalytic residues Tyr55
and His117, a steroid channel (SC) (Tyr24, Leu54, Ser129, Trp227) and three subpockets
SP1(Ser118, Asn167, Phe306, Phe311, Tyr319), SP2 (Trp86, Leu122, Ser129, Phe311)
and SP3(Tyr24, Glu192, Ser221, Tyr305). Here, the AKR1C3 inhibitors could be clearly
identified in the electron density maps in the androstenedione binding site (Figure S17).
Compound 6 was found able to establish key interactions inside the OS, involving the
hydroxyl group via a double H-bond with Tyr55 and His177, at 2.6 Å and 2.8 Å respectively
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(Fig. 7). On the other end, the 3,5-trifluoromethylphenyl moiety binds into the SP1 site,
placing one trifluoromethyl towards SP1 and the other close to the Phe311 of SP2.
In regard to the analysis of compound 1, we found an experimental binding mode that is
detached to the one previously docking hypotheses.[22] While the hydroxyl group present in
the triazole ring binds into the OS with Tyr55 and His117, producing two H-bond
interactions, as predicted by docking, the 4-methoxybenzyl moiety is unexpected found
rotated by around 180 degree respect the docking pose, showing a preferred orientation
toward SP3 deeply inside the binding pocket. More precisely, this group is located between
Phe306 and NADP, making a parallel displacing π-π and T-shape π-π interaction
respectively. However, the modulations herein used on benzyl motif in compounds 1, 2, 3
affected the activity modestly, that indicates an extended SAR analysis is required to better
understand which substitution on 4-methoxybenzyl are beneficial for the activity. Finally, the
trifluoromethylphenyl projects into the SP1 pocket, enabling the trifluoromethyl to bind
deeply within the cavity. Crystallographic data of AKR1C isoforms show that SP1 pocket in
C3 is larger and less discriminating than in C2, this differences should be behind compounds
selectivity toward the two isoforms. In order to understand the role of SP1 shape in
compound 1 selectivity, a docking study on AKR1C2 was performed. Compound 1 shows a
divergent binding mode inside C2 isoform (Figure S18), because the smaller SP1 force the
inhibitor to place mCF3-phenyl toward the SC, shifting the molecule that became unable to
maintain H-bond interaction inside OS, which is required for AKR1C activity.
The crystallographic data of compounds 1 and 6 well demonstrate the ability of both
bioisosteric approaches to effectively mimic carboxylic group into OS. The two co-
crystallized compounds demonstrate to have the same contacts as the reference FLU inside
SP1 and SP3, moreover compound 1 express a promising additional interaction through the
4-methoxybenzyl moiety that could be a key point to develop more potent and selective
AKR1C3 inhibitors.
Figure 7. AKR1C3 co-crystallized with compounds 1 in gray (PDB id: 6F2U, A) and 6 in
purple (PDB id: 6F78, B). NADP+ is represented in orange. Nitrogen, fluorine, oxygen and
sulphur atoms are depicted in blue, green, red and yellow, respectively. Molecular graphics
and analyses were performed with the UCSF Chimera package.[35]
3. Conclusions
This study has focused on a new generation of AKR1C3 inhibitors designed by application of
a scaffold hopping approach to replace the benzoic acid moiety of FLU with hydroxylated
azoles. The best compound of the first series, the 4-trifluoromethoxybenzyl substituted
analogue 3, was found to selectively inhibit AKR1C3 activity without any significant
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AKR1C2 and COX1/2 off-target effects. Although the triazole derivative 3 presented IC50
values in the similar range as FLU, it presented greater than 280-fold selectivity for AKR1C3
over AKR1C2.
The data obtained from the second series support the 3-hydroxy-1,2-benzoxazole as a
promising new scaffold for the design of potent and selective AKR1C3 inhibitors.
Specifically, compound 6 displayed a submicromolar activity against AKR1C3 and 456-fold
selectivity for AKR1C3 over AKR1C2. Compound 3 and 6 were also able to inhibit the cell
proliferation of AKR1C3-expressing 22RV1 CRPC cells as well as the PSA expression in a
dose-dependent manner. Both compounds showed an effect on the testosterone production,
but only compound 3 exerted a dose-dependent effect. These results probably show the
complexity in studying the steroidogenic pathway and its multiple parallel outcomes linked to
AR dependent and independent pathways. To better clarify this aspect, the effect of 3 and 6
on the AR dependent pathway will be the subject of further investigations. In addition, the
inhibition of AKR1C3 activity by compound 3 and 6 were shown to be synergistic in
combination with either ENZA and ABI treatment. Taken together, the novel chemical
scaffolds here reported provide a promising starting point for the design of more potent
AKR1C3 inhibitors with clinical potential use in order to intervene the steroidogenic pathway
and reduce both PSA and testosterone production. The crystal structures of the most
interesting AKR1C3 inhibitors 1 and 6 will facilitate further optimisation of these lead
compounds, aiming to discover new compounds with more drug-like properties and optimal
pharmacokinetic characteristics.
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.
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 was checked using two analytical methods. HPLC analyses were 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 and injected through a 20 µl loop. The mobile
phase consisted of acetonitrile / water with 0.1 % trifluoroacetic acid (ratio between 60 / 40
and 40 / 60, depending on the compound’s retention factor). 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
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1
of 1° C min-1. All compounds were routinely checked by H- and 13C-NMR and mass
spectrometry. 1H- 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, quint = quintuplet, m = multiplet.
Chemical shifts (δ) are given in parts per million (ppm). Accordingly with Zarantonello et
al.,[36] the 13C NMR spectra of the compounds 8 and 25 are characterized by the presence of
the C-SF5 signals as a quintet of doublets due to the coupling (2JCF 15 - 18 Hz) with the
equatorial four fluorine atoms and with the axial one. The same feature is detected for the
2
3
carbon atoms in ortho position with respect to the SF5 group (the JCF and the JCF coupling
constants values with the axial fluorine atom are not reported having a value smaller than 1
1
Hz). According to Ferraris et al.,[28] that reported H spectra of similar N-hydroxy-
1
13
benzamides, H- and C-NMR spectra of compounds 20 and 30 showed the presence of the
two amide rotamers in the analyzed solutions. 1H- and 13C-NMR spectra of final compounds
2 – 8 are shown in supplementary. 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 (ESI). HRMS spectra of final
compounds (compounds 2 – 8) were recorded on LTQ Orbitrap XL plus (Thermo Fisher
Scientific, Waltham, MA USA) equipped with an ESI ionization source, with positive or
negative ions (Spray capillary voltage: 3000 V (+), 2500 V (-)). Compound 9[23] was
prepared following already described procedure. IR spectra of final compounds (compounds
2 – 8) were recorded on FT-IR (PerkinElmer SPECTRUM BXII, KBr dispersions) using a
diffuse reflectance apparatus DRIFT ACCY (see supplementary to visualize the spectra).
4.1.2. Ethyl 4-(benzyloxy)-1-[(3,4-dimethoxyphenyl)methyl]-1H-1,2,3-triazole-5-carboxylate
(10) and ethyl 5-(benzyloxy)-2-[(4-methoxyphenyl)methyl]-2H-1,2,3-triazole-4-carboxylate
(11). Cesium carbonate (3.29 g, 10.1 mmol) and 1-(chloromethyl)-3,4-dimethoxybenzene
(1.88 g, 10.1 mmol) were added to a solution of 9 (1.00 g, 4.04 mmol) in dry DMF (20 mL).
The resulting mixture was stirred at room temperature overnight. When the reaction was
complete, the mixture was diluted with water (20 mL) and the pH adjusted to 7 with 0.5 N
HCl. The solution was concentrated to half of its volume under reduced pressure, extracted
with EtOAc (3 x 30 mL). The combined organic layer was washed with brine, dried over
MgSO4 and concentrated under reduced pressure to afford a colorless oil. The latter showed
two spots on TLC (eluent: petroleum ether /EtOAc 80/20 v/v) relative to two substituted
triazole isomers. The two isomers were separated using flash chromatography (eluent:
petroleum ether / EtOAc 80/20 v/v). First eluted isomer, 10, white solid (m.p. 80.6 – 81.7°C).
1
Yield 31 %. H-NMR (300 MHz, CDCl3): δ 1.34 (3H, t, J = 7.1 Hz), 3.83 (3H, s), 3.85 (3H,
s), 4.33 (2H, q, J = 7.1 Hz), 5.52 (2H, s), 5.75 (2H, s), 6.79 (1H, d, J = 8.2 Hz), 6.90 – 6.95
(2H, m), 7.28 – 7.39 (3H, m), 7.46 – 7.49 (2H, m); 13C-NMR (75 MHz, CDCl3):
δ 14.3, 54.4,
56.0, 61.3, 71.6, 110.7, 111.0, 111.3, 121.0, 127.6, 127.7, 128.2, 128.5, 136.5, 149.1, 149.2,
158.8, 161.3. MS (ESI) 398 [M + H]+.
Second eluted isomer, 11, white solid (m.p. 95.8 – 97.6°C). Yield 41 %. 1H-NMR (300 MHz,
CDCl3): δ 1.38 (3H, t, J = 7.1 Hz), 3.82 (3H, s), 3.87 (3H, s), 4.40 (2H, q, J = 7.1 Hz), 5.33
13
(2H, s), 5.38 (2H, s), 6.80 – 6.94 (3H, m), 7.29 – 7.38 (3H, m), 7.41 – 7.46 (2H, m); C-
NMR (75 MHz, CDCl3):
δ 14.5, 56.01, 56.03, 59.6, 61.2, 72.3, 111.1, 111.3, 121.1, 124.1,
126.9, 127.8, 128.2, 128.5, 136.1, 149.2, 149.4, 160.6, 161.0. MS (ESI) 398 [M + H]+.
4.1.3. Ethyl 4-(benzyloxy)-1-(4-methoxybenzyl)-1H-1,2,3-triazole-5-carboxylate (12) and
ethyl 5-(benzyloxy)-2-(4-methoxybenzyl)-2H-1,2,3-triazole-4-carboxylate (13). Cesium
carbonate (2.37 g, 7.28 mmol) and 1-(bromomethyl)-4-(trifluoromethoxy)benzene (1.65 g,
6.48 mmol) were added to a solution of 9 (0.90 g, 3.64 mmol) in acetonitrile (20 mL). The
resulting mixture was stirred at room temperature overnight. When the reaction was
complete, the mixture was diluted with water (20 mL) and the pH adjusted to 7 with 0.5 N
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HCl. The solution was concentrated to half of its volume under reduced pressure, extracted
with EtOAc (3 x 30 mL). The combined organic layer was washed with brine, dried over
MgSO4 and concentrated under reduced pressure to afford a colorless oil. The latter showed
two spots on TLC (eluent: petroleum ether /EtOAc 85/15 v/v) relative to two substituted
triazole isomers. The two isomers were separated using flash chromatography (eluent:
petroleum ether / EtOAc 85/15 v/v). First eluted isomer, 12, white solid (m.p. 100.0 - 102.1
°C). Yield 33 %. 1H-NMR (300 MHz, CDCl3): δ 1.33 (t, J = 7.1 Hz, 3H), 4.32 (q, J = 7.1 Hz,
2H), 5.53 (s, 2H), 5.81 (s, 2H), 7.16 (d, J = 8.0 Hz, 2H), 7.32 - 7.40 (m, 5H), 7.48 (d, J = 7.2
13
Hz, 2H). C-NMR (75 MHz, CDCl3):
δ 14.2, 53.8, 61.5, 71.7, 110.8, 120.5 (q, J = 257.5
Hz), 121.3 (q, J = 0.9 Hz), 127.7, 128.2, 128.6, 129.8, 133.8, 136.4, 149.2 (q, J = 1.8 Hz),
158.7, 161.3. MS (ESI) 422 [M + H]+.
Second eluted isomer, 13, white solid (m.p. 29.3 - 30.5 °C). Yield 45 %. 1H-NMR (300 MHz,
CDCl3): δ 1.38 (t, J = 7.1 Hz, 3H), 4.40 (q, J = 7.1 Hz, 2H), 5.34 (s, 2H), 5.44 (s, 2H), 7.17
(d, J = 8.6 Hz, 2H), 7.28 - 7.38 (m, 5H), 7.44 (d, J = 7.9, 2H). 13C-NMR (75 MHz, CDCl3):
δ
14.5, 58.8, 61.3, 72.4, 120.5 (q, J = 257.6 Hz), 121.3 (q, J = 0.9 Hz), 124.5, 127.9, 128.3,
128.5, 129.7, 133.1, 136.0, 149.4 (q, J = 1.8 Hz), 160.4, 161.1. MS (ESI) 422 [M + H]+.
4.1.4. 4-(Benzyloxy)-1-[(3,4-dimethoxyphenyl)methyl]-1H-1,2,3-triazole-5-carboxylic acid
(14). 6M NaOH (0.1 mL) was added to a solution of 10 (0.10 mmol) in ethanol (25 mL) and
the reaction mixture was stirred overnight. The resulting solution was neutralized with 2M
HCl and concentrated under reduced pressure to half of its volume. 2M HCl was added until
pH 1-2, observing precipitation of a white solid. The solid was isolated by filtration and
washed with water to give carboxylic acid 14 (m.p. 141.3 – 142.9° C). Yield 92 %. 1H-NMR
(300 MHz, DMSO-d6): δ 3.70 (s, 3H), 3.71 (s, 3H), 5.43 (s, 2H), 5.72 (s, 2H), 6.72 (dd, J =
8.2 Hz and J = 1.2 Hz, 1H), 6.89 - 6.91 (m, 2H), 7.30 - 7.47 (m, 5H), 13.69 (broad s, 1H).
13C-NMR (75 MHz, DMSO-d6):
δ 53.5, 55.4, 55.5, 71.0, 111.0, 111.6, 111.8, 120.0, 127.97,
128.04, 128.1 128.4, 136.5, 148.6, 148.7, 159.2, 160.4. MS (ESI) 370 [M + H]+.
4.1.5. 4-(Benzyloxy)-1-[[4-(trifluoromethoxy)phenyl]methyl]-1H-1,2,3-triazole-5-carboxylic
acid (15). This product was synthetized following the same procedure of 14, starting from 12.
White solid (m.p. 179.8 °C - 181.0 °C). Yield 93 %. 1H-NMR (300 MHz, DMSO-d6): δ 5.44
13
(s, 2H), 5.84 (s, 2H), 7.28 - 7.48 (m, 9H). C-NMR (75 MHz, DMSO-d6):
δ 52.9, 71.2,
111.3, 120.12 (q, J = 256.3 Hz), 121.4 (q, J = 0.9 Hz), 128.1, 128.2, 128.5, 129.5, 135.3,
136.4, 148.0, 159.1, 160.5. MS (ESI) 416 [M + Na]+.
4.1.6.
4-(Benzyloxy)-1-[(3,4-dimethoxyphenyl)methyl]-N-[3-(trifluoromethyl)phenyl]-1H-
1,2,3-triazole-5-carboxamide (16). Dry DMF (30 µL) and oxalyl chloride (2.40 mmol, 206
µL) were added to a cooled (0°C) solution of 14 (1.00 mmol, 369 mg) in dry THF (20 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 (20 mL) and used without any further purification in the next step. Dry pyridine (242
µL, 3.00 mmol) and 3-trifluoromethylaniline (177 mg, 1.10 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 (20 mL) was added to the resulting mixture, which was
concentrated under reduced pressure to half of its volume. The resulting suspension was
acidified with 0.5 M HCl to pH 2 and extracted with ethyl acetate (3 × 40 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 80/20 v/v ‰ 0/100 v/v) to obtain the amide 16 as a white solid
1
(150.5 - 151.4 °C). Yield 61%. H-NMR (300 MHz, CDCl3): δ 3.84 (s, 3H), 3.86 (s, 3H),
5.60 (s, 2H), 5.89 (s, 2H), 6.81 (d, J = 8.8 Hz, 1H), 7.04 – 7.12 (m, 2H), 7.35 – 7.62 (m, 8H),
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13
7.70 (s, 1H), 8.75 (s, 1H). C-NMR (75 MHz, CDCl3):
δ 54.4, 55.98, 56.02, 73.4, 111.1,
111.9, 112.4, 116.6 (q, J = 4.2 Hz), 121.3 (q, J = 3.4 Hz), 121.6, 122.9 (q, J = 1.3 Hz), 125.6
(q, J = 250.2 Hz), 127.6, 128.6, 129.1, 129.3, 129.8, 131.4 (q, J = 32.5 Hz), 135.3, 138.0,
149.0, 149.3, 155.6, 158.6. MS (ESI) 513 [M + H]+.
4.1.7. 4-(Benzyloxy)-1-[[4-(trifluoromethoxy)phenyl]methyl]-N-[3-(trifluoromethyl)phenyl]-
1H-1,2,3-triazole-5-carboxamide (17). This product was synthetized following the same
procedure of 16, starting from 15. White solid (m.p. 136.5 - 137.1 °C). Yield 69 %. 1H-NMR
(300 MHz, CDCl3): δ 5.62 (s, 2H), 5.96 (s, 2H), 7.18 (d, J = 8.1 Hz, 2H), 7.31 – 7.66 (m,
11H), 8.75 (s, 1H). 13C-NMR (75 MHz, CDCl3):
δ 53.7, 73.5, 112.5, 116.7 (q, J = 3.9 Hz),
120.5 (q, J = 257.4 Hz), 121.3 (q, J = 0.9 Hz), 121.4 (q, J = 3.8 Hz), 122.9 (q, J = 1.4 Hz),
126.9 (q, J = 266.5 Hz) 128.6, 129.1, 129.4, 129.9, 130.3, 131.7 (q, J = 32.7 Hz), 133.7,
135.2, 137.8, 149.4 (q, J = 1.9 Hz), 155.5, 158.6. MS (ESI) 537 [M + H]+.
4.1.8. 1-[(3,4-Dimethoxyphenyl)methyl]-4-hydroxy-N-[3-(trifluoromethyl)phenyl]-1H-1,2,3-
triazole-5-carboxamide (2). Compound 16 (0.20 mmol, 102 mg) was dissolved in dry THF
(10 mL) and hydrogenated in presence of Pd/C (5% w/w) 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. White solid (m.p. 215.1 -
216.8 °C). Yield 90 %. 1H-NMR (300 MHz, DMSO-d6): δ 3.68 (s, 3H), 3.70 (s, 3H), 5.74 (s,
2H), 6.80 (dd, J = 8.3, 1.8 Hz, 1H), 6.87 – 6.99 (m, 2H), 7.47 (d, J = 7.8 Hz, 1H), 7.59 (t, J =
7.9 Hz, 1H), 7.83 (d, J = 8.2 Hz, 1H), 8.16 (s, 1H), 9.98 (s, 1H). 13C-NMR (75 MHz,
DMSO-d6):
δ 53.5, 55.3, 55.5, 111.2, 111.7, 111.8, 116.1 (q, J = 4.1 Hz), 120.4, 120.5 (q, J
= 4.6 Hz), 123.7 (q, J = 1.1 Hz), 124.1 (q, J = 272.4 Hz), 128.0, 129.6 (q, J = 31.6 Hz),
130.20, 138.8, 148.6, 148.7, 156.7, 158.5. MS (ESI) 445 [M + Na]+. ESI-HRMS (m/z) [M +
H]+ calcd. for C19H17F3N4O4 423.1275, obsd. 423.1271. IR (KBr) ν (cm-1): 2960, 1691, 1640,
1519, 1449, 1340, 1120.
4.1.9. 4-Hydroxy-1-[[4-(trifluoromethoxy)phenyl]methyl]-N-[3-(trifluoromethyl)phenyl]-1H-
1,2,3-triazole-5-carboxamide (3). This product was synthetized following the same procedure
1
of 2, starting from 17. White solid (m.p. 197.8°C - 198.7 °C). Yield 98 %. H-NMR (300
MHz, DMSO-d6): δ 5.87 (s, 2H), 7.35 (d, J = 8.3 Hz, 2H), 7.41 (d, J = 8.8 Hz, 2H), 7.46 (d, J
= 7.8 Hz, 1H), 7.58 (t, J = 8.0 Hz, 1H), 7.82 (d, J = 8.3 Hz, 1H), 8.11 (s, 1H), 9.83 (s, 1H).
13C-NMR (75 MHz, DMSO-d6):
δ 52.8, 11.3, 116.2 (q, J = 4.1 Hz), 120.0 (q, J = 256.5 Hz),
120.5 (q, J = 3.9 Hz), 121.2 (q, J = 0.8 Hz), 123.8 (q, J = 1.3 Hz), 124.0 (q, J = 272.4 Hz),
129.5 (q, J = 31.7 Hz), 129.7, 130.0, 135.1, 138.5, 148.0, (q, J = 1.8 Hz), 156.4, 158.2. MS
(ESI) 447 [M + H]+. ESI-HRMS (m/z) [M + H]+ calcd. for C18H12F6N4O3 447.0886, obsd.
447.0888. IR (KBr) ν (cm-1): 3113, 3036, 1679, 1635, 1575, 1454, 1336, 1278, 1129.
4.1.10. 2-Bromo-6-fluoro-N-hydroxy-N-[(2,4,6-trimethoxyphenyl)methyl]benzamide (20).
Dry DMF (30 µL) and 2M oxalyl chloride in DCM (18.3 mmol, 9.13 mL) were added to a
cooled (0 °C) solution of 2-bromo-6-fluorobenzoic acid (1.60 g, 7.31 mmol) in dry THF (40
mL). The reaction was stirred for 1 hour at room temperature under nitrogen atmosphere,
then 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 18 was dissolved
in dry THF (40 mL) and used without any further purification in the next step. Et3N (3 mL)
and N-[(2,4,6-trimethoxyphenyl)methyl]hydroxylamine 19 (1.56 g, 7.31 mmol) were added
to the described solution. The reaction mixture was stirred for 2 hours at room temperature
under nitrogen atmosphere, then the solvent was evaporated under reduced pressure. The
resulting crude material was diluted with DCM, washed with water and brine, dried with
Na2SO4, and the solvent was evaporated. The crude product was purified through flash
chromatography (gradient of petroleum ether /ethyl acetate from 90/10 v/v to 70/30 v/v) to
19

ACCEPTED MANUSCRIPT
1
yield the title compound as a yellow solid (m.p. 164.7 - 165.9 °C). Yield 55 %. H-NMR
(300 MHz, DMSO-d6): δ 3.65 (s, 3H, major rotamer), 3.75 (s, 6H major and 3H minor
rotamers), 3.78 (s, 6H, minor rotamer), 4.30 (d, J = 13.7 Hz, 2H minor rotamer), 4.54 (d, J =
13.7 Hz, 2H minor rotamer), 4.75 (d, J = 13.5 Hz, 2H, major rotamer), 4.87 (d, J = 13.5 Hz,
2H, major rotamer), 6.16 (s, 1H minor rotamer), 6.23 (s, 1H, major rotamer), 7.21– 7.57 (m,
3H), 9.47 (s, 1H, major rotamer), 9.62 (s, 1H, minor rotamer). MS (ESI) 414 [M + H]+.
4.1.11. 4-Bromo-2-[(2,4,6-trimethoxyphenyl)methyl]-1,2-benzoxazol-3(2H)-one (21). K2CO3
(0.805 g, 6.28 mmol) was added to a solution of 20 (1.30 g, 3.14 mmol) in DMF (25 mL) and
°
the mixture was stirred at 120 C for 30 minutes. The reaction mixture was cooled to room
temperature and concentrated under reduced pressure. The resulting solid was partitioned
between water (50 mL) and diethyl ether (50 mL), the organic layer was separated and the
aqueous layer was extracted with diethyl ether (2 x 50 mL). The combined organic layer was
washed with brine, dried over MgSO4 and concentrated under reduced pressure to obtain 21
as a pale yellow solid (m.p. 127.3 - 130.0 °C). Yield 70 %. 1H-NMR (300 MHz, DMSO-d6):
13
δ 3.74 (s, 6H), 3.78 (s, 3H), 5.04 (s, 2H), 6.25 (s, 2H), 7.39-7.57 (m, 3H). C-NMR (75
MHz, DMSO-d6): δ 38.6, 55.3, 55.9, 90.8, 101.8, 109.8, 114.3, 116.9, 127.4, 134.8, 159.5,
159.8, 160.4, 161.4. MS (ESI) 394 [M + H]+.
4.1.12. General procedure for the preparation of substituted 2-[(2,4,6-
trimethoxyphenyl)methyl]-1,2-benzoxazol-3(2H)-ones 22-25: the appropriate aniline (1 - 1.4
eq), Cs2CO3 (0.179 g, 5.50 mmol), palladium(II) acetate (0.004 g, 0.019 mmol) and BINAP
(0.021 g, 0.031mmol) were added to a solution of 21 (0.200 g, 0.507 mmol) in dry toluene
under inert atmosphere. The reaction mixture was stirred at 110°C for 3-5 hours, then it was
allowed to reach room temperature and the solvent was evaporated. The residue was
partitioned between ethyl acetate (30 mL) and HCl 2M (30 mL). The organic layer was
separated and the aqueous layer was extracted with ethyl acetate (2 x 30 mL). The combined
organic layer was washed with brine, dried over MgSO4 and concentrated under reduced
pressure.
4.1.12.1. 4-[3-(Trifluoromethyl)anilino]-2-[(2,4,6-trimethoxyphenyl)methyl]-1,2-benzoxazol-
3(2H)-one (22). 1 Eq of 3-(trifluoromethyl)aniline was used. The crude product was purified
by flash chromatography (petroleum ether/ethyl acetate 80:20 v/v) to give the title compound
as a white solid (m.p. 121.3 – 124.5 °C). Yield 73 %. 1H-NMR (300 MHz, DMSO-d6): δ 3.76
(s, 6H), 3.79 (s, 3H), 5.00 (s, 2H), 6.26 (s, 2H), 6.72 (d, J = 8.2 Hz, 1H), 6.93 (d, J = 8.0 Hz,
1H), 7.34 (d, J = 7.3 Hz, 1H), 7.44 (t, J = 8.1 Hz, 1H), 7.55 (t, J = 7.6 Hz, 1H), 7.68 – 7.59
(m, 2H), 8.62 (s, 1H). 13C-NMR (75 MHz, DMSO-d6): δ 38.4, 55.3, 55.9, 90.8, 100.3, 102.1,
103.2, 106.3, 116.0, 122.3, 123.0, 126.1 (q, J = 272.7 Hz), 130.1 (q, J = 31.1 Hz), 130.4,
135.2, 141.4, 141.7, 159.6, 160.9, 161.4, 162.6. MS (ESI) 497 [M + Na]+.
4.1.12.2.
4-[3,5-Bis(trifluoromethyl)anilino]-2-[(2,4,6-trimethoxyphenyl)methyl]-1,2-
benzoxazol-3(2H)-one (23). 1 Eq of 3,5-bis(trifluoromethyl)aniline was used. Flash
chromatography eluent: petroleum ether/ethyl acetate 80:20 v/v. Pale yellow solid (m.p.
125.5 - 128.0 °C). Yield 41 %. 1H-NMR (300 MHz, DMSO-d6): δ 3.76 (s, 6H), 3.78 (s, 3H),
5.01 (s, 2H), 6.21 (s, 2H), 6.77 (d, J = 8.3 Hz, 1H), 6.99 (d, J = 8.0 Hz, 1H), 7.47 (s and t, J =
8.1 Hz, 2H), 7.85 (s, 2H), 8.98 (br s, 1H). 13C-NMR (75 MHz, DMSO-d6 and CDCl3): δ 39.3,
50.1, 55.6, 90.5, 101.8, 102.0, 104.5, 107.6, 118.1 (q, J = 4.2 Hz), 125.1, 131.4 (q, J = 32.6
Hz), 134.7 (q, J = 0.9 Hz), 141.8 (q, J = 270.0 Hz), 145.2, 149.9, 159.5, 160.8, 161.4, 161.9.
MS (ESI) 543 [M + H]+.
20

ACCEPTED MANUSCRIPT
4.1.12.3. 4-(3-Nitroanilino)-2-[(2,4,6-trimethoxyphenyl)methyl]-1,2-benzoxazol-3(2H)-one
(24). 1.4 Eq of 3-nitroaniline was used. The crude material was purified by two subsequent
flash chromatography separations (first eluent: petroleum ether/ethyl acetate from 80:20 to
60:40 v/v; second eluent: DCM/ethyl acetate 95:5 v/v). Yellow solid (m.p. 122.2 - 125.0 °C).
1
Yield 81 %. H-NMR (300 MHz, CDCl3): δ 3.83 (s, 3H), 3.84 (s, 6H), 5.18 (s, 2H), 6.15 (s,
2H), 6.58 (d, J = 8.3 Hz, 1H), 6.96 (d, J = 8.1 Hz, 1H), 6.58 (d, J = 8.3 Hz, 1H), 7.60 – 7.42
(m, 2H), 7.87 (d, J = 7.5 Hz, 1H), 8.20 (s, 1H), 8.50 (br s, 1H). 13C-NMR (75 MHz, CDCl3):
δ 38.6, 55.5, 56.1, 90.6, 100.6, 103.0, 103.6, 104.9, 113.8, 117.3, 125.8, 130.3, 134.7, 141.7,
142.1, 149.2, 160.1, 161.0, 161.8, 169.1. MS (ESI) 474 [M + Na]+.
4.1.12.4.
4-[3-(Pentafluorosulfanyl)anilino]-2-[(2,4,6-trimethoxyphenyl)methyl]-1,2-
benzoxazol-3(2H)-one (25). 1.2 Eq of 3-(pentafluorosulfanyl)aniline was used. Flash
chromatography eluent: petroleum ether/ethyl acetate 80:20 v/v. Yellow solid (m.p. 123.4-
1
125.2°C). Yield 79 %. H-NMR (300 MHz, CDCl3): δ 3.82 (s, 3H), 3.84 (s, 6H), 5.17 (s,
2H), 6.15 (s, 2H), 6.53 (d, J = 8.3 Hz, 1H), 6.86 (d, J = 8.1 Hz, 1H), 7.32 (t, J = 8.2 Hz, 1H),
7.49 – 7.36 (m, 3H), 7.70 (s, 1H), 8.39 (br s, 1H). 13C-NMR (75 MHz, CDCl3): δ 38.6, 55.5,
2
3
56.1, 90.6, 100.1, 103.1, 103.3, 104.3, 118.0 (quint, J CF = 5.0 Hz), 120.3 (quint, J CF = 4.8
3
Hz), 123.3, 129.6, 134.7, 140.4 (quint, J CF = 18.5 Hz) 141.1, 142.3, 160.1, 161.1, 161.8,
163.0. MS (ESI) 533 [M + H]+.
4.1.13. General procedure for the preparation of substituted 1,2-benzoxazol-3-ols 4, 6, 7, 8:
Triisopropylsilane (0.044 g, 0.276 mmol) and TFA (1.5 mL) were added to a solution of the
correspondent 2-[(2,4,6-trimethoxyphenyl)methyl]-1,2-benzoxazol-3(2H)-one (22-25, 0.256
mmol) in DCM (7.5 mL) and the mixture was stirred for 2 h. The reaction mixture was
quenched with water, the organic layer was separated and the aqueous layer was extracted
with DCM. The combined organic layer was washed with brine, dried over MgSO4 and
concentrated under reduced pressure.
4.1.13.1. 4-[3-(Trifluoromethyl)anilino]-1,2-benzoxazol-3-ol (4). White solid (m.p. 148.6 –
149.2 °C, from hexane). Yield 82 %. 1H-NMR (300 MHz, DMSO-d6): δ 7.06 – 6.84 (t and s,
2H), 7.25 (d, J = 6.4 Hz, 1H), 7.43 (t, J = 8.1 Hz, 1H), 7.61 – 7.48 (m, 3H), 8.30 (s, 1H),
13
12.55 (br s, 1H). C-NMR (75 MHz, DMSO-d6): δ 102.0, 104.8, 108.1, 114.8 (q, J = 3.2
Hz), 117.4 (q, J = 3.6 Hz), 121.8, 124.2 (q, J = 272.3 Hz), 130.0 (q, J = 31.4 Hz), 130.3,
132.5, 138.7, 143.1, 164.8, 165.4. ESI-HRMS (m/z) [M + H]+ calcd. for C14H10F3N2O2
295.0689, obsd. 295.0685. IR (KBr) ν (cm-1): 3413, 3364, 1656, 1626, 1536, 1333, 1112.
4.1.13.2. 4-[3,5-Bis(trifluoromethyl)anilino]-1,2-benzoxazol-3-ol (6). Pale pink solid (m.p.
1
215.3 - 220.1°C, from hexane). Yield 71 %. H-NMR (300 MHz, DMSO-d6): δ 7.07 (d, J =
7.8 Hz, 1H), 7.15 (d, J = 8.3 Hz, 1H), 7.47 (s, 1H), 7.51 (t, J = 8.1 Hz, 1H), 7.69 (s, 2H), 8.84
(s, 1H), 12.52 (br s, 1H). 13C-NMR (75 MHz, DMSO-d6): δ 104.1, 106.3, 111.0, 112.48 (q, J
= 3.7 Hz), 116.51 (q, J = 2.6 Hz), 123.39 (q, J = 273.0 Hz), 131.10 (q, J = 32.5 Hz), 132.4,
136.7, 145.3, 164.9, 165.0. ESI-HRMS (m/z) [M + H]+ calcd. for C15H9F6N2O2 363.0563,
obsd. 363.0560. IR (KBr) ν (cm-1): 3413, 3373, 1668, 1618, 1531, 1383, 1284, 1123.
4.1.13.3. 4-(3-Nitroanilino)-1,2-benzoxazol-3-ol (7). Yellow solid (m.p. 216.1 - 221.0 °C,
from acetonitrile). Yield 50 %. 1H-NMR (300 MHz, DMSO-d6): δ 6.97-7.11 (m, 2H), 7.47 (t,
J = 8.3 Hz, 1H), 7.54 (d, J = 8.1 Hz, 1H), 7.62 (d, J = 8.2 Hz, 1H), 7.73 (d, J = 8.0 Hz, 1H),
7.99 – 8.01 (m, 1H), 8.56 (s, 1H), 12.55 (br s, 1H). 13C-NMR (75 MHz, DMSO-d6): δ 102.9,
105.5, 109.4, 111.7, 115.1, 123.9, 130.4, 138.0, 144.1, 146.8, 148.6, 164.9, 165.2. ESI-
21

ACCEPTED MANUSCRIPT
HRMS (m/z) [M + H]+ calcd. for C13H10N3O4 272.0666, obsd. 272.0664. IR (KBr) ν (cm-1):
3390, 3352, 3094, 1660, 1623, 1534, 1349, 1060.
4.1.13.4. 4-[3-(Pentafluoro-sulfanyl)anilino]-1,2-benzoxazol-3-ol (8). White solid (m.p.
1
174.3 - 177.6°C, from diisopropyl ether). Yield 44 %. H-NMR (300 MHz, DMSO-d6): δ
6.95 (d, J = 7.8 Hz, 1H), 6.95 (d, J = 7.8 Hz, 1H), 7.56 - 7.32 (m, 4H), 7.71 (s, 1H), 8.42 (s,
1H), 12.48 (br s, 1H). 13C-NMR (75 MHz, DMSO-d6): δ 102.4, 105.1, 108.5, 115.56 (quint, J
= 5.7 Hz), 117.89 (quint, J = 5.3 Hz), 121.4, 130.0, 132.5, 138.4, 143.4, 153.73 (quint, J =
14.3 Hz), 164.9, 165.3. ESI-HRMS (m/z) [M + H]+ calcd. for C13H10F5N2O2S 353.0378,
obsd. 353.0374. IR (KBr) ν (cm-1): 3413, 3364, 2975, 1664, 1623, 1604, 1528, 1364.
4.1.14.
Methyl
2-fluoro-5-[3-(trifluoromethyl)anilino]benzoate
(27).
3-
(Trifluoromethyl)aniline (0.387 g, 2.40 mmol), Cs2CO3 (0.912 g, 2.80 mmol), palladium(II)
acetate (22.5 mg, 0.100 mmol) and BINAP (0.100 g, 0.160 mmol) were added to a solution
of methyl 5-bromo-2-fluorobenzoate 26 (0.466 g, 2.00 mmol) in dry toluene under inert
atmosphere. The reaction mixture was stirred at 110 °C overnight, then it was allowed to
reach room temperature and the solvent was evaporated. The residue was partitioned between
ethyl acetate (100 mL) and 2M HCl (100 mL). The organic layer was separated and the
aqueous layer was extracted with ethyl acetate (2 x 100 mL). The combined organic layer
was washed with brine, dried over MgSO4 and concentrated under reduced pressure. The
crude material was purified through flash chromatography (eluent: petroleum ether / ethyl
acetate 90:10 v/v) to yield the title compound 27 as a white solid. M.p. 93.7 – 95.2 °C, from
1
petroleum ether). Yield 71 %. H-NMR (300 MHz, DMSO-d6): δ 3.48 (s, 3H), 7.13 (d, J =
7.6 Hz, 1H), 7.33 – 7.18 (m, 3H), 7.50 – 7.34 (m, 2H), 7.45 (t, J = 8.0 Hz, 1H), 7.57 (dd, J =
6.1, 2.9 Hz, 1H), 8.70 (s, 1H). 13C-NMR (75 MHz, DMSO-d6): δ 52.4, 111.9 (q, J = 4.0 Hz),
115.8 (q, J = 4.0 Hz), 118.1 (q, J = 23.7 Hz), 118.5 (q, J = 11.4 Hz), 119.1, 120.2 (q, J = 0.7
Hz), 124.2 (q, J = 272.3 Hz), 124.4 (q, J = 8.4 Hz), 130.2 (q, J = 31.4 Hz), 130.5, 138.6 (q, J
= 2.7 Hz), 144.3, 155.6 (q, J = 251.7 Hz), 164.0 (q, J = 3.8 Hz).
4.1.15. Methyl 5-[(tert-butoxycarbonyl)[3-(trifluoromethyl)phenyl]amino]-2-fluorobenzoate
(28). Di-tert-butyl dicarbonate (0.818 g, 3.75 mmol) and 4-dimethylaminopyridine (0.458 g,
3.75 mmol) were added to a solution of 27 (0.783, 2.50 mmol) in dry THF (30 mL). The
reaction mixture was stirred at room temperature for 3 hour, then poured in phosphate buffer
(pH 5). The resulting solution was extracted twice with diethyl ether and the combined
organic layer was washed with brine, dried over MgSO4 and concentrated under reduced
pressure. The crude material was purified through flash chromatography (eluent: petroleum
ether / ethyl acetate 90:10 v/v) to yield the title compound as a colorless oil. Yield 70%. 1H-
NMR (300 MHz, DMSO-d6): δ 1.38 (s, 9H), 3.84 (s, 3H), 7.43 – 7.33 (m, 1H), 7.54 – 7.44
13
(m, 1H), 7.66 – 7.53 (m, 3H), 7.72 (s, 1H), 7.77 (dd, J = 6.4, 2.8 Hz, 1H). C-NMR (75
MHz, DMSO-d6): δ 27.7, 52.6, 81.5, 117.9 (q, J = 23.7 Hz), 118.6 (q, J = 11.7 Hz), 122.6 (q,
J = 3.9 Hz), 123.7 (q, J = 3.5 Hz), 123.8 (q, J = 272.5 Hz), 129.6 (q, J = 31.9 Hz), 130.2,
130.4 (q, J = 1.5 Hz), 130.8, 134.3 (q, J = 9.7 Hz), 138.3 (q, J = 3.5 Hz), 143.0, 152.4, 158.8
(q, J = 257.7 Hz), 163.44 (q, J = 3.8 Hz). MS (ESI) 436 [M + Na]+.
4.1.16. 5-[(Tert-butoxycarbonyl)[3-(trifluoromethyl)phenyl]amino]-2-fluorobenzoic acid
(29). 0.2 M KOH (20 mL) was added to a solution of 28 (0.700 g, 1.70 mmol) in EtOH (25
mL), and the reaction mixture was stirred at room temperature overnight. The mixture was
concentrated to half of its volume and acidified with 2 M HCl until pH 2 was reached. The
suspension was extracted with DCM (3x30 mL) and the combined organic layer was washed
with brine, dried over MgSO4 and concentrated under reduced pressure. White solid (m.p.
1
156.6 - 157.6° C). Yield 81%. H-NMR (300 MHz, DMSO-d6): δ 1.38 (s, 9H), 7.41 – 7.26
22

ACCEPTED MANUSCRIPT
13
(m, 1H), 7.67 – 7.41 (m, 4H), 7.80 – 7.65 (m, 2H). C-NMR (75 MHz, DMSO-d6): δ 27.8,
81.5, 117.8 (q, J = 24.6 Hz), 119.8 (q, J = 11.6 Hz), 122.6 (q, J = 4.3 Hz), 123.7 (q, J = 0.7
Hz), 123.9 (q, J = 272.5 Hz), 129.6 (q, J = 31.9 Hz), 130.2, 130.6 (q, J = 1.8 Hz), 130.8,
133.8 (q, J = 9.2 Hz), 138.2 (q, J = 3.4 Hz), 143.1, 152.5, 159.0 (q, J = 256.9 Hz), 164.5 (q, J
= 3.5 Hz). MS (ESI) 422 [M + Na]+.
4.1.17. 2-Fluoro-N-hydroxy-N-[(2,4,6-trimethoxyphenyl)methyl]-5-[(tert-butoxycarbonyl)[3-
(trifluoromethyl)phenyl]amino]benzamide (30). N,N'-Dicyclohexylcarbodiimide (0.310 g,
1.50 mmol) was added to a solution od 15 (0.600 g, 1.50 mmol) in dry DCM. After 40
minutes, N-hydroxy-1-(2,4,6-trimethoxyphenyl)methanamine (0.320, 1.50 mmol) and DMAP
(0.018 g, 0.15 mmol) were added and the reaction mixture was stirred at room temperature
overnight. The mixture was cooled to 0°C, filtered, and the solid washed with cold DCM.
The filtrate was evaporated and the resulting crude purified through flash chromatography
(eluent: DCM / ethyl acetate 90:10 v/v) to obtain a white solid (m.p. 169.6 - 171.5° C). Yield
1
54%. H-NMR (300 MHz, DMSO-d6): δ 1.37 (s, 9H), 3.58 (s, 3H, major rotamer), 3.69 (s,
6H, major and 3H minor rotamer), 3.77 (s, 6H, minor rotamer), 4.48 (s, 2H, minor rotamer),
4.78 (s, 2H, major rotamer), 6.18 - 6.21 (m, 2H), 7.18 – 7.73 (m, 7H), 9.42 (br s, 1H). MS
(ESI) 595 [M + H]+.
4.1.18.
2-[(2,4,6-Trimethoxyphenyl)methyl]-5-[(tert-butoxycarbonyl)[3-
(trifluoromethyl)phenyl] amino]-1,2-benzoxazol-3(2H)-one (31). K2CO3 (55.3 mg, 0.400
mmol) was added to a solution of 30 (119 mg, 0.200 mmol) in DMF (7 mL) and the mixture
was stirred at 120 °C for 45 minutes. The reaction mixture was cooled to r.t. and concentrated
under reduced pressure, the resulting solid was partitioned between water (30 mL) and
diethyl ether (30 mL). The organic layer was separated and the aqueous layer was extracted
with diethyl ether (2 x 30 mL). The combined organic layer was washed with brine, dried
over MgSO4 and concentrated under reduced pressure. The crude product was purified
through flash chromatography (eluent: DCM / ethyl acetate 95:5 v/v) to obtain to obtain 31 as
1
a pale yellow solid (m.p. 169.0 – 170.3 °C). Yield 83 %. H-NMR (300 MHz, DMSO-d6): δ
1.37 (s, 9H), 3.73 (s, 6H), 3.77 (s, 3H), 5.05 (s, 2H), 6.20 (s, 2H), 7.35 (d, J = 8.9 Hz, 1H),
7.41 – 7.47 (m, 1H), 7.47 – 7.58 (m, 3H), 7.58 – 7.71 (m, 2H). 13C-NMR (75 MHz, DMSO-
d6): δ 27.7, 48.0, 55.2, 55.7, 81.2, 90.6, 99.0, 101.9, 110.8, 116.6, 121.4 (q, J = 273.4 Hz),
122.9, 123.8, 129.7 (q, J = 32.3 Hz), 129.8, 133.3, 137.7, 138.2, 143.3, 152.6, 157.3, 159.4,
160.6, 161.3. MS (ESI) 575 [M + H]+.
4.1.19. 5-[3-(Trifluoromethyl)anilino]-1,2-benzoxazol-3-ol (5). Triisopropylsilane (26 mg,
0.162 mmol) and TFA (1.5 mL) were added to a solution of 31 (87 mg, 0.150 mmol) in DCM
(7.5 mL) and the mixture was stirred for 2 h. The reaction mixture was quenched with water,
the organic layer was separated and the aqueous layer was extracted twice with DCM. The
combined organic layer was washed with brine, dried over Na2SO4 and concentrated under
1
reduced pressure. Pale pink solid (m.p. 161.1 – 162.7° C, from hexane). Yield 63 %. H-
NMR (300 MHz, DMSO-d6): δ 7.08 (d, J = 7.6 Hz, 1H), 7.20 (s, 1H), 7.26 (d, J = 8.2 Hz,
13
1H), 7.32 – 7.47 (m, 3H), 7.52 (d, J = 8.9 Hz, 1H), 8.60 (s, 1H), 12.20 (br s, 1H). C-NMR
(75 MHz, DMSO-d6): δ 109.4, 111.1 (q, J = 3.9 Hz), 111.2, 115.1 (q, J = 3.8 Hz), 115.1,
118.2, 124.4 (q, J = 272.3 Hz), 124.6, 130.2 (q, J = 31.2 Hz), 130.5, 137.7, 145.5, 159.2,
165.2. MS (ESI) 293 [M - H]-. ESI-HRMS (m/z) [M + H]+ calcd. for C14H10F3N2O2
295.0689, obsd. 295.0686. IR (KBr) ν (cm-1): 3411, 1611, 1561, 1537, 1463, 1341, 1108.
4.2. Expression and purification of recombinant human AKR1C3 and AKR1C2
Escherichia coli BL21 (DE) Codon Plus RP cells expressing recombinant AKR1C3 and
AKR1C2 proteins were obtained as previously described.[22] AKR1C3 showed one mutation
23

ACCEPTED MANUSCRIPT
His5Gln in comparison to the NCBI sequence, described in the literature as a single
nucleotide polymorphism very common (refSNP: rs12529). Protein expression and
purification were performed as previously described.[22] Briefly, bacteria cells were grown
in YT2X media supplemented with ampicillin at 37°C and at OD600 nm = 0.6 the expression
was induced by IPTG (0.5 mM) at 24°C for 2 h. Then, bacteria were centrifuge and lysed
with four freeze-thaw cycles in presence of lysozyme and protease inhibitors. DNA was
digested with benzonase (25 U) in presence of MgCl2 5 mM. The lysate was centrifuged for
30 min at 13,000 x g and the supernatant was collected. 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 performed on purified recombinant enzymes as previously
described.[22] Briefly, the enzymatic 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 were added in 96-well plate. The S-tetralol
concentration used in the AKR1C2 and AKR1C3 inhibition assay were 15 µM and 160 µM,
respectively, the same 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 COX1 and COX2
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 routinely maintained
as monolayers in RPMI supplemented with 10 % (v/v) fetal calf serum, 2 % (v/v) penicillin-
streptomycin and 0.03% L-glutamine. Cells were grown 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, as previously described.[37, 38] 10.000
cells/well were seeded into 96-well plates in RPMI containing 10% charcoal stripped serum,
and incubated for 24 hours. Then, 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). For co-treatment experiments,
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22RV1 cells were treated with ABI (10 µM) or ENZA (20 µM) with or without compounds 3
and 6 (20 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. Viability/death cell determination
22RV1 cell lines cultured as above were trypsinized at 72 h and counted by using the
NucleoCounter® NC-200™ (ChemoMetech, Allerod, Denmark) and the propidium iodine
uptake to distinguish dead (uptake of propidium iodine without lysis of cells) and total cells
(after lysis). The difference between total and dead cells is the number of live cells.[39]
4.8
Western blot
Protein extraction from both untreated and treated 22RV1 cells was conducted using a RIPA
lysis buffer containing the Complete Protease Inhibitor Cocktail (Roche Molecular
Biochemicals). The protein concentration was determined by use of a Bradford assay. Protein
samples (20 µg) were denatured by Tris-Glycine SDS Sample Buffer (Biorad), aliquots of
which were loaded into polyacrylamide gel wells alongside identical volumes of β-actin.
Polyacrylamide gel electrophoresis (SDS-PAGE) was run at 120V for 2 hours. Proteins were
transferred to nitrocellulose membrane (GE Healthcare Life Sciences) by electro-blotting at
300 mA for 3 hours, then blocked using 5% dried milk powder in 0.05% Tris-buffered saline
(TBS) + Tween 20. The membrane was incubated overnight at 4°C on a shaker with specific
primary antibody diluted in 5% milk powder 0.05% TBS + Tween 20. The following
primary antibodies were used: mouse β-actin (1: 20:000, Sigma-Aldrich), mouse monoclonal
anti-AKR1C3 (1:333, Sigma Aldrich), goat polyclonal PSA (C-19) (1:500, sc-7638 Santa
Cruz Biotechnology). The membrane was washed with TBS and incubated with a HRP-
conjugated secondary antibody (Santa-Cruz Biotechnology) diluted 1:5000 in 5% dried milk
powder with 0.05% TBS + Tween 20 at room temperature. Ponceau Red Staining (Sigma-
Aldrich) was used to ensure samples were equally loaded in SDS-PAGE. Visualisation was
achieved using a chemiluminescent substrate (Amersham ECL, GE Healthcare Life Sciences)
and captured into Kodak film.
4.9
Determination of PSA expression by Elisa
22RV1 cell lines are cultured as above described. Supernatants were collected, centrifuged to
eliminate cell debris and stored at -80 °C until to use when 100 µl were analyzed by a PSA
(Total) Human ELISA Kit (Termofisher scientific (Life Technologies Italia, Monza Italy).
PSA (ng/ml) were normalized for the cell number. The statistical analysis were performed
with PRISM 7.0, GraphPad Software. Results are representative of one replicated experiment
performed in triplicate.
4.10 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, at a density of 30,000 cells per well, and were incubated at 37 °C with 5%
CO2 for 24 h. Compound 3 and Compound 6 were added to the wells at 4 different
concentrations and incubated for 1 h. Equimolar (28 nM) concentration of androstenedione
was then added to the wells. The plate was returned to the incubator for a further 24 h. 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
statistical analysis were performed with PRISM 7.0, GraphPad Software. The values are the
means of two separate experiments each carried out in triplicate.
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4.10 Protein expression, purification and crystallization
Plasmid coding for human AKR1C3[22] was modified by removing the thrombin cleavage-
site and adding the TEV (Tobacco Etch Virus) cleavage-site. The new plasmid construct was
transformed into E. coli BL21 (DE) Codon Plus RP (Agilent Technologies).
The cells were grown in LB media supplemented with ampicillin (100 µg/mL) and
chloramphenicol (34 µg/mL) at 37 °C with continuous shaking at 200 rpm. At OD600nm =
0.5 the expression was induced by IPTG (0.4 mM) and the temperature was lower to 22 °C
for overnight. The cell pellet was resuspended in the binding buffer A (10 mM Na2HPO4, 1.8
mM KH2PO4, 140 mM NaCl, 2.7 mM KCl pH 7.3) supplemented with DNase 1 (0.1 µg per
gram of wet cells), lysozyme (0.1 mg/ml). The suspension was passed through the emulsiflex
3 times at 12000 psi at 4 °C. The cell lysate was centrifuged at 40000 x g, 4 °C, 1 hour in
JA25 rotor. The clarified lysate was loaded on to 5 ml GST TRAP column (GE healthcare)
prequlibriated with the binding buffer. The loaded column was washed until no more protein
was eluted. The protein was eluted using the elution buffer 50 mM Tris-HCl pH 8.0, 10 mM
Glutathione reduced. The eluted fractions were pooled and subjected to overnight TEV
protease digestion at 4 °C to remove GST tag. TEV protease was used in 1:100 dilution for
the digestion. Next day, protein solution was loaded on to a 5 ml His TRAP column
equilibriated with buffer Tris-HCl pH 8.0, NaCl 150 mM, DTT 1mM. The flowthrough
containing cleaved AKR1C3 protein was collected and concentrated using Vivaspin 10 kDa
cutoff. For final polishing step, size exclusion chromatography was performed using
superdex 200 column (GE healthcare). The concentrated protein was applied to s 200 column
with 10 mM potassium phosphate pH 7, 1 mM EDTA and 1 mM DTT. Fractions were run
on the SDS PAGE to check the purity. Pure fractions containing AKR1C3 were pooled and
concentrated to 75 mg/ml.
Co-crystals of AKR1C3 and inhibitors were obtained using hanging vapour diffusion
method. Both NADP and inhibitor were added in final concentration of 2 mM to the protein
solution (25 mg/ml) and incubated overnight at 4 °C. For crystallization, 3 µl the overnight
incubated protein was mixed with the reservoir solution (100 mM MES pH 6.0, 12-25% PEG
8000).[40] The crystallization plates were incubated both, at 4 °C and 20 °C. Crystals for
both the inhibitors appeared in 100 mM MES pH 6.0, 25% PEG 8000. Crystals for compound
1 appeared at 20 °C while that of 6 formed at 4 °C. The crystals were cryoprotected using
20% ethylene glycol in mother liquor prior to cryo-cooling.
4.11 X-ray data collection, structure determination and refinement
X-ray diffraction data of compound 6 was collected at ID23-1 while for compound 1 was
collected at ID30A-3 beamlines of ESRF. X-ray diffraction spots were processed using XDS,
pointless and Scala.[41] PDB id1ry09[40] was used as the molecular replacement template
for the structure determination using Phaser of PHENIX suite.[42] Both structures were
refined using PHENIX. Data collection and refinement statistics are summarised in Table
S16.
4.12 Docking
The structures of compounds 1 were built in their dissociated forms using the 2D Sketcher
tool implemented in Maestro GUI. Ligand docking was performed using Schrödinger GLIDE
extra precision (XP) protocol.[43] or this purpose, the X-ray crystallographic structure of
AKR1C2 was retrieved from RCSB Database (PDB code: 4JQA). Before docking, the crystal
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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. 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.™ A grid of 10 Å x 10 Å x 10 Å (x, y, and z) was created and centred on the
co-crystalized ligand mefenamic acid.
AUTHOR INFORMATION
Corresponding Authors: *
Phone: +39-0116707180. Fax: +39-0116707162, 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), hexadeuterodimethyl sulfoxide (DMSO-d6),
deuterochloroform (CDCl3), tetradeuteromethanol (CD3OD), deuterated acetone (CD3)2CO),
PSA (prostatic serum antigen).
ACKNOWLEDGEMENTS
We thank Prof Norman J. Maitland (University of York) for provision of the 22RV1 PC cell
line. This research was financially supported by the University of Turin (Ricerca Locale
grant 2015-2017) and Prostate Cancer UK grant S12-027.
Appendix A. Supplementary data
Supplementary data related to this article can be found at: XXXXXX
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