Preliminary investigations into triazole derived androgen receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2014.03.018

A range of 1,4-substituted-1,2,3-N-phenyltriazoles were synthesized and evaluated as non-steroidal androgen receptor (AR) antagonists. The motivation for this study was to replace the N-phenyl amide portion of small molecule antiandrogens with a 1,2,3-tri

Full text of DOI:10.1016/j.bmc.2014.03.018

Bioorganic & Medicinal Chemistry 22 (2014) 2692–2706
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Preliminary investigations into triazole derived androgen receptor
antagonists
Jarrad M. Altimari a, Birunthi Niranjan b, Gail P. Risbridger b, Stephanie S. Schweiker c, Anna E. Lohning c,
Luke C. Henderson a,d,
⇑
a Strategic Research Center for Chemistry and Biotechnology, Deakin University, Pigdons Road, Waurn Ponds Campus, Geelong 3216, Victoria, Australia
b Department of Anatomy and Developmental Biology, Faculty of Medicine, Nursing and Health Sciences, Monash University, Victoria 3800, Australia
c Faculty of Health Sciences and Medicine, Bond University, Gold Coast 4229, Queensland, Australia
d Institute for Frontier Materials, Deakin University, Pigdons Road, Waurn Ponds Campus, Geelong 3216, Victoria, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
A range of 1,4-substituted-1,2,3-N-phenyltriazoles were synthesized and evaluated as non-steroidal
androgen receptor (AR) antagonists. The motivation for this study was to replace the N-phenyl amide
portion of small molecule antiandrogens with a 1,2,3-triazole and determine effects, if any, on biological
activity. The synthetic methodology presented herein is robust, high yielding and extremely rapid. Using
this methodology a series of 17 N-aryl triazoles were synthesized from commercially available starting
Received 22 January 2014
Revised 5 March 2014
Accepted 13 March 2014
Available online 1 April 2014
materials in less than 3 h. After preliminary biological screening at 20 and 40
lM, the most promising
Keywords:
three compounds were found to display IC50 values of 40–50 M against androgen dependent (LNCaP)
l
Click chemistry
Androgen receptor
Triazole
Molecular modelling
Prostate cancer
cells and serve as a starting point for further structure–activity investigations. All compounds in this
work were the focus of an in silico study to dock the compounds into the human androgen receptor
ligand binding domain (hARLBD) and compare their predicted binding affinity with known antiandro-
gens. A comparison of receptor–ligand interactions for the wild type and T877A mutant AR revealed
two novel polar interactions. One with Q738 of the wild type site and the second with the mutated
A877 residue.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
A common structural feature of non-steroidal AR antagonists is
the extremely electron-deficient N-phenyl amide moiety. The aryl
Prostate cancer (PC) is the most commonly diagnosed cancer in
men and is a leading cause of death in the male population, in 2013
prostate cancer was estimated to account for almost 14% of all
diagnosed cancers.1 Antagonistic binding of the steroid-docking
site within the androgen receptor (AR) is the most common form
of small-molecular therapy for PC. The antagonistic binding miti-
gates cancer cell proliferation and disease progression by prevent-
ing the binding of endogenous testosterone or dihydrotestosterone
portion commonly bears a trifluoromethyl group (CF3) in addition
to a strongly deactivating group at the para-position, relative to the
amide.3–8 Typical examples of para-substitution are the nitro group
(NO2) in the case of 1 and 2, or the nitrile (CN) of 3 and MDV3100.
Also, electron deficient N-phenyl amides have found widespread
use in a variety of fields such as antimicrobials, anti-HIV therapy,
Raf inhibitors and have shown potential as antitubercular
agents.9–15
(5a-DHT) into the AR. The initial inspiration of PC chemotherapy
Recently, we reported the synthesis of several anti-androgenic
was the chemical modification of naturally occurring steroidal li-
gands. Though viable, these compounds were plagued with poor
bioavailability, hepatotoxicity and a lack of tissue specific action,
leading to their discontinued clinical use.2 As such, non-steroidal
AR antagonists, such as FlutamideÒ 1 (its active metabolite 2),
Enzalutamide (MDV3100) and Bicalutamide 3 (CasodexÒ) are the
current state of the art in androgen blockade therapy (Fig. 1).
( )-a-lipoic acid derivatives bearing meta- and para- substitution
patterns of CF3–NO2 and CF3–CN, respectively.8 Though success-
fully synthesised, we have found low-to-moderate yields (typically
<50%) of the amide formation due to the electron deficient aryl ring
despite extensive optimisation (Scheme 1).
In addition to low yields, another common problem noted by us
and others,8,16 is the tedious removal of residual deactivated ani-
line present in the crude product from the desired N-phenyl amide.
This occurs usually from the inability of the deactivated aniline to
be protonated and washed out during acidic work-up and/or their
tendency to possess poorly defined Rf values (streaking) during
⇑
Corresponding author. Tel.: +61 3 52272767; fax: +61 3 52271045.
E-mail address: luke.henderson@deakin.edu.au (L.C. Henderson).
http://dx.doi.org/10.1016/j.bmc.2014.03.018
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

J. M. Altimari et al. / Bioorg. Med. Chem. 22 (2014) 2692–2706
2693
H
N
H
N
OH
O
F
O
O
N
O2N
O2N
N
H
S
CF3
CF3
1
2
N
N
F
N
N
N
R
2 = Ph
OH
HO
O
O
R2
H
N
F
O
S
F
F
O
MDV3100
R1
NC
R1 = NO2, CF3, CN, etc
CF3
3
4
Figure 1. Enzalutamide MDV3100, flutamide 1, hydroxyflutamide 2, bicalutamide 3 and proposed triazoles 4.
formation in situ facilitated by sodium nitrite in HCl, using modified
conditions reported by Lear et al. (Table 1).28 Starting with aniline
12 (R1–3 = H), an electronically ‘neutral’ substrate, these conditions
furnished phenyl azide 14 in moderate yield (62%) and in >95%
crude purity (Table 1, entry 1). Encouraged by this result we moved
on to 3-trifluoromethyl-4-cyanoaniline (R1 = CF3, R2 = CN, R3 = H),
an aryl unit commonly used in prostate cancer therapy,8,29–33 which
proceeded much better than the previous example giving the de-
sired azide 15 in 95% yield (Table 1, entry 2).
O
H2N
R2
R1
4 CO2H
PyBrop, CHCl3
100 °C, 1 hr
4HN
+
S
S
S
R2
S
R1
5
6
8
9
R1 = NO2, R2 = CF3
R1 = NO2, R2 = F3, 46%
R1 = CN, R2 = CF3, 34%
7
R1 = CN, R2 = CF3
Scheme 1. Previously synthesised electron deficient N-arylamides.
Applying these conditions to a series of substituted anilines
gave the desired azides (16–21) in good to excellent yields (67–
95%) and were isolated analytically pure as the crude material. Pre-
sumably the azide formation occurs much slower for anilines
which were obtained in <70% yield (Table 1, entries 1, 3 and 6) than
the other examples. Nevertheless these compounds were still iso-
lated in synthetically usable yields.
With phenyl azides 14–21 in hand, our attention turned to the
application of these compounds to the copper-alkyne-azide-cyclo-
addition (CuAAc) reaction. We chose the CuAAc reaction between
21 and phenylacetylene as our model reaction of choice as this pro-
vided a simple, yet novel, triazole 22. Our initial reaction condi-
tions used a combination of copper(II) sulfate and ascorbic acid
in a water/ethanol mixture at room temperature (Table 2, entry
1) which only gave a trace (<5%) of the desired compound.
Repeating this reaction at a higher temperature (100 °C) using
microwave irradiation, we found that the reaction proceeded in
30 min (Table 2, entry 2), giving the desired compound in excellent
yield (89%). The drastic reduction of reaction duration using
microwave irradiation has been shown several times within our
group34–38 and is a phenomena commonly observed in synthetic
chemistry.39–43 A copper source which is soluble in organic solvents
column chromatography. To circumvent this problem, Sugai et al.16
used an acetylation technique (derivatising both desired com-
pound and residual starting aniline) to allow for chromatographic
resolution. Though an effective strategy, the acyl group then had
to be removed from their target compound, adding several syn-
thetic steps which is not optimal. Despite these low yields and
problematic purifications, there is no optimised methodology or
alternative functional group easily accessible for this type of com-
pound. As such we turned our attention to the use of 1,2,3-triazoles
which have been used extensively as amide bond isosteres
throughout medicinal chemistry for the past decade.17–26 Recently,
Antonella and co-workers have synthesised several triazole-
derived compounds inspired by Bicalutamide 2.27
As such, the impetus for this study was to replace the
N-phenylamide moiety of simple AR antagonists with a 1,4-substi-
tuted-1,2,3-triazole and determine if there is any retention of
biological activity against androgen dependent cells (LNCaP).
Therefore, we designed a range of potential triazole derived AR
antagonists inspired by anti-androgen Flutamide 1. The synthetic
procedures presented here are rapid, robust and require no purifi-
cation techniques. Indeed, in contrast to the electron deficient
anilines being a problematic for amide formation, these same deac-
tivated anilines, using this approach, are the best performing sub-
strates. The isosteric replacement of the amide with a triazole has
an additional benfit in that these compounds will also probe the
importance of the amide carbonyl present in many common AR
antagonists. These compounds were then evaluated in vitro for
their ability to inhibit androgen stimulated cell proliferation using
an androgen dependent (LNCaP) cell line. Following this, com-
pounds 23–39 were docked into the human ligand binding domain
(hARLBD) of wild type AR and the mutated AR present in LNCaP
cells and a comparison of interactions is presented. The latter of
which possesses a key point mutation of threonine 877 for an
alanine (T877A) allowing for correlation to in vitro studies.
Table 1
Synthesis of aryl azides
1. 1:8, H2O:MeCN then HCl
10 minutes, r.t.
2. NaNO2, 1 hr
NH2
N3
3. NaN3, 1 hr, 0 °C to r.t.
R3
R1
R3
R1
R2
R2
12
13
Entry
Compound
R1
R2
R3
Yield (%)
1
2
3
4
5
6
7
8
14
15
16
17
18
19
20
21
H
CF3
H
H
CN
Cl
CN
NO2
H
NO2
Cl
H
H
H
H
H
CF3
H
62
95
67
95
76
68
82
95
2. Results and discussion
H
CF3
CF3
H
2.1. Synthesis of 1,4-substituted-1,2,3-triazoles
We began our investigation into the conversion of anilinic
amines into phenyl azides, of general structure 13, via diazo-phenyl
CF3
H

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J. M. Altimari et al. / Bioorg. Med. Chem. 22 (2014) 2692–2706
Table 3
Reaction scope
was also used in an effort to increase the product yield within the
same timeframe. Employing copper(I) chloride (Table 2, entry 3) in
water at 100 °C gave a marginally better yield (90%) than that ob-
served with copper(II) sulfate, similarly Cu2O (Table 2, entry 4) gave
a good yield but required a longer reaction time. As such we consid-
ered copper(II) sulfate the optimal catalyst as this can be made as a
stock solution and is stable on the bench, whereas copper(I) chloride
requires frequent regeneration. We then reduced the reaction time
to 20 and 15 min resulting in slightly reduced yield (Table 2, entries
5 and 6, respectively).
Application of our optimal reaction conditions (Table 2, entry 2)
to the remaining azides gave the desired triazoles 23–39 in good to
excellent yield (75–96%), with only triazoles 24 and 26, being
isolated in moderate yield (52% and 64%, respectively).
Following the series of phenylacetylene derivatives (Table 3,
entries 1–7) we chose 2-methyl-3-butyn-2-ol as the alkyne partner
as this group bears significant resemblance to the carbonyl portion
of hydroxyflutamide 2, the active androgen receptor antagonist
which results from in vivo metabolism of Flutamide 1.
Again, under the specified conditions, all the desired triazoles
were obtained in very good yields. Of particular note are 33 and
36 which were isolated in excellent yields of 79% and 93%, respec-
tively and possess the same aryl substitution pattern present in
Flutamide 1 and Bicalutamide 3. Finally, the last alkyne partner
was Fmoc protected propargyl glycine methyl ester, giving the
corresponding triazoles 38 and 39 (Table 3, entries 16 and 17) in
excellent yield (96% and 77%, respectively) and possess the orthog-
onal reactivity of the amino acid group in the scaffold allowing for
a myriad of potential synthetic derivatisation.
It is important to note that no purification was required to obtain
analytically pure samples for all compounds presented in Table 3.
Examination of the literature showed that triazoles 23, 26 and 27
have been made by others,44–47 though in all cases longer reaction
times were required than those in this work, and ours is the only sys-
tem to use an entirely aqueous solvent system.
With a range of the desired triazoles in hand we checked the
Lipinski parameters of these compounds (refer to ESI) to which tri-
azoles 23–39 conformed to Lipinski’s rules well with only 38 and
39 having any violations, due to molecular weight. Recently, Guo
and co-workers48 synthesised several AR antagonists on the
hypothesis that small molecules possessing cLogP values <3.5
and polar surface areas (PSA) of >70 Å would demonstrate better
in vivo clearance profiles while possessing sufficient polarity with-
N3
Alkyne (1.0 eq), CuSO4 (10 mol%),
Ascorbic acid (20 mol%), H2O
R1
R3
R4
R2
N
N
MW, 100 °C, 30 min
R3
R1
N
R2
Entry
1
Product
23
R1
H
R2
R3
H
Alkyne
Yield (%)
75
H
2
24
H
NO2
NO2
Cl
H
52
3
25
CF3
H
H
87
4
26
H
64
5
27
H
CN
CN
H
H
96
6
28
CF3
CF3
CF3
H
H
95
7
29
CF3
H
92
8
30
Cl
83
OH
9
31
H
H
81
OH
OH
OH
OH
OH
OH
10
11
12
13
14
15
32
H
NO2
NO2
Cl
H
75
33
CF3
H
H
79
34
H
80
35
H
CN
CN
H
H
64
36
CF3
CF3
H
93
37
CF3
79
OH
NHFmoc
16
17
38
39
CF3
CF3
NO2
CN
H
H
96
77
CO2Me
NHFmoc
CO2Me
Table 2
Investigation into optimal reaction conditions
in the AR-LBD to discourage helix-12 closure, thus minimising AR
transactivation. With this in mind the majority of triazoles syn-
thesised in this study fulfil these criteria very well and as such
our next focus was investigating the in vitro activity of these com-
pounds in LNCaP cells.
Ph
N
N
N3
N
Ph
"Cu" (10 mol%)
F3C
F3C
2.2. Evaluation of triazoles in vitro
Cl
Cl
21
22
Initially, we undertook a preliminary biological evaluation as a
screening tool to identify compounds for further studies. This
screening process involved monitoring cell proliferation of LNCaP
cells at two static concentrations, 20 and 40 lM (Table 4). From
this data, any compounds showing promising anti-proliferative
Entry
Catalysta
Time (h)
Tempb (°C)
Solvent
Yield (%)
c
1
2
3
4
5
6
CuSO4
24
0.5
0.5
24
20 min
15 min
rt
EtOH/H2O
H2O
Trace
89
90
83
83
c
CuSO4
100
100
rt
100
100
CuCl
Cu2O
H2O
CHCl3
H2O
properties would be selected and IC50 values determined.
c
Evaluation of these compounds for their potential to stop
androgen stimulated growth in LNCaP cells gave a wide variety
of results. While the reduction in LNCaP cell growth was minimal
for the majority of compounds (typically 5–10%), relative to DHT
CuSO4
c
CuSO4
H2O
79
a
b
c
10 mol % of Cu catalyst was used in all reactions.
Heated using microwave radiation unless at rt.
Carried out with ascorbic acid (20 mol %).

J. M. Altimari et al. / Bioorg. Med. Chem. 22 (2014) 2692–2706
2695
control, compounds 32, 34, 36 and 37 displayed moderate to good
levels of growth inhibition (Table 4, entries 11, 13, 15 and 16).
Compounds 34 and 36 (Table 4, entries 13 and 15), possessing
the aryl substitution pattern for Bicalutamide 3 and Flutamide 1,
respectively, also showed very promising levels of LNCaP growth
LNCaP values), though interestingly 37 possessed an IC50 only mar-
ginally increased compared to that of the LNCaP cell line (40 M vs
42 M). The reason for the similar IC50 displayed by 37 is unknown
l
l
but is presumably due to the bis-trifluoromethyl groups on the aryl
portion. In any case this aryl moiety has been the basis of a new
series of compounds currently under investigation within our
group with the aim of developing potent PC-3 inhibitors and any
findings will be reported in due course. Despite all inhibition val-
ues for 23–39 being quite high we were pleased with these levels
of activity considering the rapid synthetic methodology to access
these compounds and that they are both the first and simplest
scaffolds to be accessed. This also suggests that the amide carbonyl
of existing AR antagonists plays an important role in binding to the
AR.
Nevertheless, given the success with which the 1,2,3-triazole
scaffold has been used to mimic an amide functionality throughout
medicinal chemistry we were curious about the low-to-moderate
activities which had been displayed by the majority of these com-
pounds. As such we turned to in silico docking of these compounds
to rationalise these results. The LNCaP cell line possesses a key
point mutation within the ligand binding domain at threonine
inhibition at both 20 and 40
Interestingly, 37 (Table 4, entry 16) showed the most promising
inhibitory activity at 40 M, despite having minimal similarity to
lM compared to the positive control.
l
existing AR antagonists. Based on these preliminary results three
of these compounds (32, 36 and 37) were selected for further
in vitro evaluation to determine IC50 values as these compounds
displayed promising activity while also were representative exam-
ples of aromatic ring substitution patterns/electronics.50 In the
interest of thoroughness, these three compounds were also
screened for IC50 values against PC-3 cells which are androgen
independent and effective inhibition of PC3 cells remains a chal-
lenge.49, 51–53
As seen from Figure 2, these compounds possessed moderate
activity for LNCaP growth inhibition, as was expected from the pre-
liminary screen, with IC50 values from 40 to 50 lM. Both 32 and 36
showed a large increase in IC50 for the PC-3 cell line (relative to
Table 4
Preliminary in vitro evaluation of compounds 23–39
Entry
1
#
3
R1
—
R2
—
R3
—
R4
—
LNCaP inhibitiona (20
28
l
M, %)
LNCaP inhibitiona (40
33
lM, %)
2
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
H
H
H
7
6
3
H
Cl
H
9
15
4
H
CN
CN
NO2
NO2
H
H
4
10
5
CF3
H
H
13
À3b
À11b
À12b
7
6
6
H
À11b
À14b
À16b
4
7
CF3
CF3
H
H
8
CF3
H
9
H
OH
OH
OH
OH
OH
OH
OH
OH
10
11
12
13
14
15
16
H
Cl
H
5
10
CF3
H
Cl
H
12
14
20
15
15
15
21
CN
CN
NO2
NO2
H
H
13
CF3
H
H
24
H
16
CF3
CF3
H
24
CF3
37
O
17
18
38
39
CF3
CF3
CN
H
H
10
8
11
16
OMe
NHFmoc
O
NO2
OMe
NHFmoc
a
Value given is relative to a positive control using 2 nM of DHT.
Negative values imply agonistic binding to the AR.
b

2696
J. M. Altimari et al. / Bioorg. Med. Chem. 22 (2014) 2692–2706
OH
OH
OH
domain.54 EM5744 40 was specifically designed to hinder the he-
lix-12 (H12) conformational change required for AF-2 transactiva-
tion function essential during its transcriptional activity, 40 has an
additional chain at C18 which extends towards the H12 site within
the hARLBD (Fig. 3). The role of H12 within transactivation is to
close over the LBD after ligand docking (DHT), this reinforces the
ligand–receptor interaction while also creating a hydrophobic cleft
which allows the recruitment of co-activators required to initiate
AR transcription.55 The crystal structure for the hARLBD with
T877A mutation was 2OZ7.pdb with bound cyproterone acetate
(CPY, 41).56
N
N
N
N
N
N
N
N
N
CF3
F3C
F3C
F3C
Cl
NO2
32
36
37
LNCaP IC50 = 49 µM
PC-3 IC50 = 69 µM
LNCaP IC50 = 45 µM
PC-3 IC50 = 63 µM
LNCaP IC50 = 40 µM
PC-3 IC50 = 42 µM
Validation for the docking method involved determining the
heavy atom root mean square deviation (RMSD) between the origi-
nal X-ray structure ligands conformation and its docked poses. An
RMSD of less than 2 Å was considered a good reproduction of the
crystal structure conformation. For 2PNU the RMSD between
EM5744 40 and its docked pose was found to be 0.482 and
0.949 Å for 2OZ7 the RMSD between CYP 41 and its docked pose.
Subsequent docking of ligands was thereafter assumed to be rea-
sonably well predicted by Surflex-Dock. Surflex-Dock was ranked
in the top three docking programs in performance in terms of
docking accuracy and in ranking known inhibitors in a virtual
screening experiment.57
Figure 2. IC50 values for compounds 32, 56 and 37 against LNCaP and PC-3 cell
lines.
877, which is replaced with an alanine residue, commonly referred
to as T877A. Thus, we carried out molecular docking of these com-
pounds into the wild type LBD and T877A mutated LBD, a compar-
ison of interactions is provided.
2.3. Molecular modelling
Our initial work in this area was to examine triazoles 23–39 and
examine them from an in silico perspective to map the energetic
landscape within the hARLBD of the T877A mutant and compare
with that of the wild type LBD. Numerous high resolution crystal
structures have been determined bound to either cognate ligands,
testosterone and dihydrotestosterone (DHT), or one of a vast array
of inhibitors, both steroidal and non-steroidal in nature. In this
study the compound library was initially docked into the hAR
structure, 2AMB.pdb,51 bound to tetrahydrogestrione (THG), a po-
tent competitive inhibitor of endogenous androgens. However,
since the ligand-based approach of Surflex-Dock uses the volume
and nature of the reference ligand to generate the protomol (i.e.,
the conformational space within which the compounds are
docked), a target protein with a larger volume ligand was prefera-
ble. For this reason 2PNU.pdb was selected as its bound ligand,
EM5744 40, covered a larger space within the ligand binding
Surflex-dock’s scoring function ranks ligands in order of highest
binding interaction (total score (pKD), note that binding affinity is
not necessarily predictive of inhibitory potency) and includes con-
tributions from (in order of import) hydrophobic complementarity,
polar complementarity and entropy with the first two dominating
the scoring function.55
We initially docked the triazoles 23–39 into the wild-type AR
receptor (2PNU) to gain insight into how these compounds may
interact with the native AR prior to several key point mutations
which commonly occur after AR-antagonistic therapy results in
the development of hormone-independent prostate cancer. It has
been suggested56 that mutations that lead to increased promiscu-
ity within the LBD binding site facilitate AF-2 transactivation via
repositioning H12. While the replacement of T877 with A877 pro-
vides space to accommodate more bulky antagonists, a key hydro-
gen bonding group is removed. Compounds 23–39 were then
Figure 3. A: 2PNU with bound steroidal antagonist, EM5744, 40 (orange), with ribbon backbone coloured on secondary structure. Position of H12 shown in cyan, key LBD
residues in blue. B: 2OZ7 with bound steroidal antagonist, CPY, 41 (orange). Ribbon backbone coloured on secondary structure. Position of H12 shown in cyan and key LBD
residues in blue.

J. M. Altimari et al. / Bioorg. Med. Chem. 22 (2014) 2692–2706
2697
Table 5
Scores for binding to the AR for both wild type (left) and T877A mutant (right)
N
N
R4
N
R3
R2
R1
Entry
1
#
3
R1
—
R2
—
R3
—
R4
2PNU score (pKD)
2OZ7 scorea (pKD)
—
6.6
5.6
3.3
2
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
H
H
H
H
H
H
H
H
3.8
4.0
3.9
3.6
3.4
2.4
3.9
4.9
4.1
3.7
5.1
4.8
4.6
4.5
4.5
3
H
Cl
3.1
3.0
3.7
2.9
3.0
1.8
4.2
3.3
3.6
3.7
3.7
3.1
3.7
4.9
4
H
CN
CN
NO2
NO2
H
5
CF3
H
6
7
CF3
CF3
H
8
CF3
H
9
H
OH
OH
OH
OH
OH
OH
OH
OH
10
11
12
13
14
15
16
H
Cl
H
CF3
H
Cl
H
CN
CN
NO2
NO2
H
H
CF3
H
H
H
CF3
CF3
H
CF3
O
17
18
19
20
38R
38S
39R
39S
CF3
CF3
CF3
CF3
CN
H
H
H
H
7.1
8.6
5.8
8.0
2.6
1.9
2.1
1.2
OMe
NHFmoc
O
CN
OMe
NHFmoc
O
NO2
NO2
OMe
NHFmoc
O
OMe
NHFmoc
21
22
23
24
40
41
1
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
12.4
—
6.3
2.7
—
9.9
4.4
0.7
MVD3100
a
LNCaP cells possess a key point mutation in the AR whereby a threonine is replaced with an alanine (T877A).
docked into the LBD of the T877A mutant, 2OZ7 to investigate the
impact within the binding site. Additionally, as 38 and 39 are the
only compounds to possess a chiral carbon, both enantiomers were
evaluated in silico, despite only the natural isomer being evaluated
in vitro. Table 5 lists the results of docking into the hARLBD of
2PNU.pdb and 2OZ7.pdb. Score (pKD) values are shown with the
higher values corresponding to higher predicted binding affinity,
with both crystal structure ligands, EM5744 40 and CYP 41, assum-
ing the top position in each run as expected.
The bulky Fmoc compounds (38R/S and 39R/S) generally scored
highest in the 2PNU site followed by the tertiary alcohols (30–37)
and then the aryl compounds (23–29) whereas for the mutated

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J. M. Altimari et al. / Bioorg. Med. Chem. 22 (2014) 2692–2706
2OZ7 site the tertiary alcohols (30–37) scored highest followed by
the aryl compounds (23–29) and then Fmoc compounds (38R/S
and 39R/S) which was surprising as it may be expected that the
bulkier groups would be less well accommodated in the smaller
site of 2PNU.
The docked poses for the aryl compounds, 23–29, were ob-
served to adopt one of two orientations within the 2PNU LBD.
The compounds in this series most highly stabilized (23, 24 and
29) were oriented to maximize stabilization by two key hydrogen
bond interactions between the triazole nitrogen and T877 (Fig. 4).
In this orientation the substituted benzene is directed into the
hydrophobic pocket flanked by H12 and W741. The substituted
ring of 29 is rotated compared to that of 23 to better accommodate
the two bulky CF3 groups while the chlorine atom of 24 is oriented
towards the polar N738.
group of 25 scored highest in this position while introducing a sec-
ond substituent caused a loss of stability due to steric interference.
Figure 6 depicts all 7 docked aryl substituted compounds,
23–29 within the 2OZ7 T877A mutant site. In this case only one
orientation was observed similar to that normally occupied by
the steroid nucleus of the cognate ligand. In all cases the substi-
tuted ring is oriented to maximize hydrogen bonding opportunities
with R752 and N711. Here 26 scores highest facilitating two hydro-
gen bonds, one each to R752 and N711, though compounds 23 and
24 are small and able to minimize steric issues within the site.
Within the tertiary alcohol series, one of two possible orienta-
tions within the 2PNU site were observed depending on position
and length of substituents on the aryl ring (Fig. 7). Those com-
pounds that had either no substituent (30), one or two atom single
substituents (31 and 33) as well as the meta di-substituted groups
(37) were positioned within the small hydrophobic flanked by the
H12 and W741.
The remaining mono and di-substituted compounds (25–28)
were docked into the site normally occupied by the steroid nucleus
of the cognate ligand achieving a degree of stabilization by hydro-
gen bonding the polar substituent to R752 (Fig. 5). The linear CN
Stabilization of this position was afforded by hydrogen bonding
between T877 and both triazole and hydroxyl groups of the
Figure 4. 2PNU–aryl compound 23 (atom colours), 24 (violet) and 29 (red).
Figure 5. 2PNU–compounds 25 (atom colours), 26 (cyan), 27 (magenta) and 28 (orange).

J. M. Altimari et al. / Bioorg. Med. Chem. 22 (2014) 2692–2706
2699
Figure 6. 2OZ7–aryl compounds 23 (atom colours), 24 (orange), 25 (cyan), 26 (green), 27 (purple), 28 (beige) and 29 (red).
Figure 7. 2PNU–tertiary alcohols 30 (atom colours), 31 (red), 33 (cyan), 34 (green), 35 (orange), 36 (brown), 37 (violet).
ligands. Otherwise those ligands with three atom, single substitu-
ents (35) and those with ortho disubstituted groups (34 and 36)
were oriented to facilitate hydrogen bonding to R752 and between
the hydroxyl group of the ligand and T877. Compound 33 had the
highest degree of stabilization in this set due to the additional sta-
bilization from the hydrogen bond to N738. The smallest of the set,
30, had the second highest total score within the 2PNU LBD. The
next three (34–36) were next in terms of score and were all stabi-
lized by the strong hydrogen bonding interactions with R752 as
well as T877. The most destabilization was seen in 32 (Fig. 8)
which was due to the steric hindrance from the bulky CF3 ortho
to the chlorine and loss of hydrogen bond to T877.
For the hARLBD T877A mutant site the tertiary alcohols were
found in a different orientation (Fig. 9), having to find alternate
arrangements due to the loss of the hydrogen bond to T877. As a
result, the hydroxyl group of compounds 30 and 31 were observed
to hydrogen bonding with L704 whereas the longer compounds 33
and 35 interacted with N705. By orienting to N705 these
compounds were able to exploit additional hydrogen bonding be-
tween their substituted group, CN or NO2 respectively, to Q711
and R752.
Di-substitution of the benzene ring results in two possible ori-
entations (Fig. 10). Compound 37 had the highest score oriented
with its bulky CF3 group towards Q711. The least bulky 32 adopted
a similar orientation within the site and was observed to make a
hydrogen bond to N705. Compounds 34 and 36 were flipped in ori-
entation with their di-substituted groups closer to L880.
Within the 2PNU binding site both 38S and 38R were observed
in an extended conformation stabilized centrally by two hydrogen
bonds between the triazole nitrogens and T877 side chain (Fig. 11,
top). The substituted benzene ring is positioned in close proximity
to the H12 within range for polar interactions with Q738, while the
Fmoc group was observed to bind in a hydrophobic site normally
occupied by the steroid nucleus of the cognate ligands. A high de-
gree of polar and non-polar stabilization contributes to the ranking
of these compounds as first and third in the group.

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J. M. Altimari et al. / Bioorg. Med. Chem. 22 (2014) 2692–2706
Figure 8. 2PNU–tertiary alcohol 32.
Figure 9. 2OZ7–tertiary alcohols 30 (atom colours), 31 (red), 33 (cyan), 35 (orange).
In contrast, a single substitution in the 2OZ7 T887A mutant
for the 2OZ7 T877A mutant where the R stereoisomers of 38 and
39 had higher scores (Fig. 12). These in silico results have explored
the conformational space around the hARLBD of both the wild type
2PNU and 2OZ7 T887A mutant by docking a set of novel com-
pounds into their respective LBDs. While both sites can accommo-
date large and flexible compounds, the removal of the threonine
side chain in the T877A mutant results in even more space avail-
able for large molecules to maneuver. This is consistent with the
findings in binding studies that the T877A mutant shows more
promiscuous ligand binding. The Fmoc group mimics the steroid
nucleus of the androgen structure binding into a similar hydropho-
bic area. For all ligands at this site, additional hydrogen bond
acceptors para- to the triazole ring of 2–3 bonds in length facilitate
a stabilizing hydrogen bonding interaction with R752 (Fig. 13).
With similar length, Bicalutamide (3) and Enzalutamide
(MVD3100) adopt similar orientations within each site. In both
cases, the di-substituted benzenes with the CN and CF3 groups
are able to hydrogen bond with Q711 and R752. Then, depending
on the conformational space available, the orientation of the other
end of the molecules is either directed towards the H12 helix (for
2PNU) or down towards the more hydrophobic pocket around L701
(for 2OZ7).
site, results in the destabilization of these two compounds into
a folded conformation where the peptide bond acts as a hinge
allowing intramolecular interactions between the two ring sys-
tems (Fig. 11, bottom). These unfavorable conformations caused
an overall destabilizing effect as reflected in the lower score
despite a high degree of hydrogen bonding. Two hydrogen bonds
stabilized 38S between its CN group and both Q711 and R752.
Two hydrogen bonds were observed between the carbonyl of
38R and both W741 and Q711 with a third between the ether
oxygen and Q711.
As for both isomers in 38, 39R also displays two hydrogen
bonds between the triazole ring and T877 of the 2PNU site whereas
no hydrogen bonding was observed for its stereoisomer (Fig. 12). In
the more accommodating 2OZ7 site however 39R made three
hydrogen bonds, one between the ester O and the Q711 and two
between the peptide carbonyl and each of W741 and Q711. For
39S a unique hydrogen bonding interaction was seen between
A877 and the CF3 group. In addition one hydrogen bond was pres-
ent between the ester carbonyl oxygen and Q711.
A preference for the S stereoisomer was observed within the
2PNU site for both 38 and 39 compared to the reverse situation

J. M. Altimari et al. / Bioorg. Med. Chem. 22 (2014) 2692–2706
2701
Figure 10. 2OZ7–tertiary alcohols 32 (atom colours), 34 (orange), 36 (violet), 37 (red).
Figure 11. Top, 2PNU 38S (atom colours), 38R (orange), bottom, 2OZ7 T887A mutant 38S (atom colours), 38R (orange).

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J. M. Altimari et al. / Bioorg. Med. Chem. 22 (2014) 2692–2706
Figure 12. Top, 2PNU 39 S (atom colours), 39 R (orange); bottom, 2OZ7 T887A mutant 39 S (atom colours), 39 R (orange).
The low docking score for MVD3100 can be explained by the
mutant where the strained ‘sandwich’ conformation resulted in
the least binding affinity of the compounds.
flexibility differences where its central thiohydantoin ring limited
its flexibility compared to bicalutamide 3 and this forced the polar
amide end into the hydrophobic pocket in the T877A mutant site.
Comparing MVD3100, 3 and 1 to the synthesized triazoles, 26,
28, 29, 32, 34, 36 and 37 reveals a prominent hydrogen bonding
interaction between the di-substituted benzene and R752 and
Q711. The presence of the two hydrogen bonding groups and the
similarity in overall molecular length governs the binding mode
for these molecules. The smaller triazoles with either no substitu-
tion or mono-substitution on the aryl portion showed a variable
binding mode due to the larger conformational space available to
them within the confines of the protomol volume.
Overall, other significant polar interactions that appear to dom-
inate the binding are with N705, T877 and pi-stacking with W741.
Two novel interactions were identified in this study. Firstly, the
interaction between Q738 side chain NH2 and hydrogen bond
acceptors such as the CN group of compound 38 and 39 in the
2PNU site. Secondly, a hydrogen bonding interaction between the
CF3 group of 39S and the peptide amide of the mutated A877 res-
idue. In the 2OZ7 T877A mutant, the removal of one of the key LBD
residues creates a larger space with less hydrogen bonding. All li-
gands were observed to bind exclusively into the steroidal binding
site with the rings of the larger compounds, 38/39, forming a ‘sand-
wich’ conformation within the site. It is worth noting here that the
S isomer of both 38 and 39 bound with the highest energy of all
compounds within the 2PNU site compared to the 2OZ7 T877A
The in silico docking results showed that the compounds that
bound with the highest binding energy within the 2PNU LBD in a
conformation that exploited both the steroidal and hydrophobic
pocket. These same compounds bound least well within the 2OZ7
T877A mutant site and limited to the steroidal binding site. If the
purpose is to specifically target the H12 region of the 2OZ7 T877A
mutant to limit transactivation by conformational change, then
these results indicate that addition of a large hydrophobic moiety
onto the alcohol scaffold could promote pi stacking with W741
within the hydrophobic site. This could be achieved by ensuring a
size greater than that which allowed the ‘sandwich’ conformation
in the steroidal site (e.g., 38/39). It is of note that the inhibition as-
say results, using the LNCaP cell lines with the T877A mutation,
showed 37, a tertiary alcohol, to have the highest percentage inhi-
bition which correlated with the highest scoring ligand in this dock-
ing experiment despite the lack of hydrogen bond interaction with
R752. The meta positioning of the CF3 groups facilitated favourable
utilization of the space, which therefore could make a reasonable
starting scaffold for a new series of potential antagonists.
3. Summary
In summary, we have developed a range of novel triazole-de-
rived AR antagonists which are easily accessible and have potential
as lead compounds for prostate cancer therapy. In this work we

J. M. Altimari et al. / Bioorg. Med. Chem. 22 (2014) 2692–2706
2703
Figure 13. Top, 2PNU MVD3100 (atom colours), 1 (red), 3 (purple); bottom, 2OZ7 T887A mutant MVD3100 (atom colours), 1 (red), 3 (purple).
have presented both the in silico and in vivo data which proved to
be variable at of predicting in vitro potency. This is most likely due
to several reasons, such as the mutated AR providing a more pro-
miscuous binding site through T877A replacement and the in silico
predicted interactions are not necessarily antagonistic in nature.
Nevertheless the docking provided insight into key interactions
and several novel interactions were found via computational
methods such as the pi-stacking interaction with W741, hydrogen
bonds to Q711 and Q738. The standout compounds of this study
were 32, 36 and 37 which will be used for further development
of novel AR antagonists and computational studies suggest that
these compounds may still be suitable for wild type AR inhibition.
Further exploration of this scaffold to develop more potent AR
antagonists is currently underway and will be reported in due
course.
standard bore (solution) as indicated. Samples were dissolved in
deuterated chloroform (CDCl3) with the residual solvent peak used
as an internal reference (CDCl3–d H 7.26 ppm). Proton spectra are
reported as follows: chemical shift d (ppm), (integral, multiplicity
(s = singlet, br s = broad singlet, d = doublet, dd = doublet of
doublets, t = triplet, q = quartet, m = multiplet), coupling constant
J (Hz), assignment).
Thin Layer Chromatography (TLC) was performed using alumin-
ium-backed Merck TLC Silica gel 60 F254 plates, and samples were
visualised using 254 nm ultraviolet (UV) light, and potassium per-
manganate/potassium carbonate oxidising dip (1:1:100 KMnO4/
K2CO3/H2O w/w). Column chromatography was performed using
silica gel 60 (70–230 mesh). All solvents used were AR grade. Spe-
cialist reagents were obtained from Sigma–Aldrich chemical com-
pany and used without further purification. Petroleum spirits
refers to the fraction boiling between 40–60 °C. HRMS was found
via a 6210 MSD TOF mass spectrometer under the conditions:
gas temperature (350 °C), vaporizer (28 °C), capillary voltage
(3.0 kV), cone voltage (40 V), nitrogen flow rate (7.0 L/min), nebul-
iser (15 psi). Samples were dissolved in MeOH. Melting points
were found on a Stuart Scientific Melting Point Apparatus SMP3,
v.5 and are uncorrected.
4. Experimental procedures
4.1. General experimental
All 1H and 13C NMR spectra were recorded on a Jeol JNM-EX
270 MHz, Jeol JNM-EX 400 MHz or Bruker AVANCE III 500 MHz

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J. M. Altimari et al. / Bioorg. Med. Chem. 22 (2014) 2692–2706
4.2. General experimental procedure for phenyl azide formation
1362, 1227, 1132, 736; HRMS, m/z calcd for (C16H10F6N3)
358.07342, found 358.07849.
To a solution of electron withdrawn aniline (20 mmol) in H2O/
MeCN (1:8, 36 mL) was added HCl (10 mL, 18 M) and stirred vigor-
ously for 10 min at room temperature. Sodium nitrite (40 mmol,
2 equiv) was added portion wise and stirred for an additional hour
at room temperature, the solution was then cooled to 0 °C and so-
dium azide (40 mmol, 2 equiv) was added portion wise. The result-
ing solution is then stirred for an additional hour and allowed to
warm to room temperature. Water (40 mL) is then added to dis-
solve and remaining solid, the solution is then reduced in vauco
to remove residual MeCN. The aqueous phase is then extracted
with CH2Cl2 (4 Â 30 mL), the layers combined and washed with
brine (1 Â 20 mL), dried (MgSO4 anhydrous) and the solvent re-
moved in vacuo to give the desired product.
4.4.4. 2-(1-(4-Chloro-3-(trifluoromethyl)phenyl)-1H-1,2,
3-triazol-4-yl)propan-2-ol 30
mp 93.7–95.8 °C; 1H NMR (CDCl3, 400 MHz): d 8.05 (1H, s, Ar-H)
8.00 (1H, d, J = 2 Hz, Ar-H) 7.83 (1H, dd, J = 8.8 Hz, 2.4 Hz, Ar-H)
7.58 (1H, d, J = 8 Hz, Ar-H) 3.69 (1H, br s, OH) 1.64 (6H, s, CH3);
13C NMR (DMSO-d6, 100 MHz): d 159.0, 136.4, 133.9, 130.6, 128.5
(q, J2C–F = 31 Hz) 125.6, 122.9 (q, J1C–F = 272 Hz) 120.0, 119.7 (q,
J3C–F = 5 Hz) 67.5, 31.1; 19F NMR (DMSO-d6, 470 MHz): d À61.5;
IR (cmÀ1): 3353, 2979, 1492, 1310, 1142, 1037, 831736; HRMS,
m/z calcd for (C12H11ClF3N3NaO) 328.04404, found 328.04952.
4.4.5. 4-(4-(2-Hydroxypropan-2-yl)-1H-1,2,3-triazol-1-
yl)benzonitrile 35
4.3. General experimental procedure for CuAAC reaction
mp 108.1–111.2 °C; 1H NMR (DMSO-d6, 400 MHz): d 8.78 (1H, s,
Ar-H) 8.17 (1H, d, J = 5.4 Hz, Ar-H) 8.08 (2H, d, J = 5.4 Hz, Ar-H) 3.35
(1H, s, OH) 1.54 (6H, s, CH3); 13C NMR (DMSO-d6, 100 MHz): d
158.0, 140.3, 134.8, 120.8, 119.7, 118.7, 111.3, 67.5, 31.1; IR
(cmÀ1): 3144, 1614, 1510, 1314, 1137, 1025, 695; HRMS m/z calcd
for (C12H13N4O) 229.10839, found 229.10718.
The azide (1 mmol) and alkyne (1 mmol) are placed in a micro-
wave reaction vessel, to this is added a solution of CuSO4 (10 mol %,
25 mg/mL aqueous solution) followed by ascorbic acid (20 mol %,
20 mg/mL aqueous solution) and the total volume brought to
3 mL with water. The reaction is then heated to 100 °C using
microwave irradiation for 30 min. The aqueous phase is then ex-
tracted with CH2Cl2 (2 Â 20 mL), the layers combined and washed
with brine (1 Â 20 mL), dried (MgSO4 anhydrous) and the solvent
removed in vacuo to give the desired product in >95% crude purity
by 1H NMR. Note that despite all compounds being >95% pure by
1H NMR spectroscopy when isolated as the crude material, the
compounds were subjected to purification prior to biological eval-
uation to ensure no residual metals or other organic impurities
were present.
4.4.6. 4-(4-(2-Hydroxypropan-2-yl)-1H-1,2,3-triazol-1-yl)-2-
(trifluoromethyl)benzonitrile 36
mp 112.4–114.2 °C; 1H NMR (DMSO-d6, 400 MHz): d 8.23 (1H, s,
Ar-H) 8.14–8.11 (2H, m, Ar-H) 8.01 (1H, d, J = 8 Hz, Ar-H) 1.69 (6H,
s, CH3) (OH not observed); 13C NMR (DMSO-d6, 100 MHz): d 158.2,
140.4, 138.0, 133.3 (q, J2C–F = 16 Hz) 124.0, 120.16, 116.4 (m),
115.6, 108.0, 67.5, 31.0; 19F NMR (DMSO-d6, 470 MHz): d À62.2;
IR (cmÀ1): 3407, 3145, 2981, 1616, 1587, 1365, 1051, 801; HRMS,
m/z calcd for (C13H12F3N4O) 297.09577, found 297.09699.
4.4. Compound data
4.4.7. 2-(1-(4-Nitro-3-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-
4-yl)propan-2-ol 33
4.4.1. 4-(4-Phenyl-1H-1,2,3-triazol-1-yl)-2-(trifluoromethyl)
benzonitrile 28
1H NMR (DMSO-d6, 400 MHz): d 8.94 (1H, s, Ar-H) 8.36 (1H, d,
J = 4 Hz, Ar-H) 8.28-8.25 (1H, m, Ar-H) 7.92 (1H, d, J = 8 Hz, Ar-H)
1.54 (1H, s, OH); 13C NMR (DMSO-d6, 100 MHz): d 157.9, 136.1,
mp 222.8–229.1 °C; 1H NMR (DMSO-d6, 400 MHz): d 9.63 (1H, s,
Ar-H) 8.58–8.46 (3H, m, Ar-H) 7.96–7.94 (2H, m, Ar-H) 7.54 (2H, t,
Ar-H) 7.44–7.41 (1H, m, Ar-H); 13C NMR (DMSO-d6, 100 MHz): d
148.5, 140.2, 138.1, 133.3 (q, J2C–F = 32 Hz) 130.2, 129.7, 129.2,
126.0, 124.2, 122.7 (q, J1C–F = 272 Hz) 120.7, 118.6, 115.6, 108.4;
19F NMR (DMSO-d6, 470 MHz): d À62.2; IR (cmÀ1): 3329, 1684,
1480, 1313, 1034, 653; HRMS, m/z calcd for (C16H10F3N4)
315.08576, found 315.08448
133.8 (m), 130.5, 128.5 (q, J2C–F = 31 Hz) 125.5, 122.8 (q, J1
=
C–F
272 Hz) 119.9, 119.5 (m) 67.5, 31.0; 19F NMR (DMSO-d6,
470 MHz): d À63.0; IR (cmÀ1): 3370, 2980, 1492, 1311, 1143,
1037, 831; HRMS m/z calcd for (C12H12F3N4O3) 317.08566, found
317.08856.
4.4.8. 2-(1-(3,5-Bis(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-
yl)propan-2-ol 37
4.4.2. 1-(4-Nitro-3-(trifluoromethyl)phenyl)-4-phenyl-1H-1,2,
3-triazole 25
mp 149.8–150.4 °C; 1H NMR (DMSO-d6, 400 MHz): d 9.02 (1H, s,
Ar-H) 8.66 (2H, s, Ar-H) 8.22 (1H, s, Ar-H) 5.32 (1H, br s, OH) 1.56
(6H, s, CH3); 13C NMR (DMSO-d6, 100 MHz): d 158.0, 144.4, 138.7,
132.4 (q, J2C–F = 34 Hz) 123.4 (q, J1C–F = 272 Hz) 122.1 (m) 121.0 (m)
120.3, 67.5, 31.0, 19F NMR (DMSO-d6, 470 MHz): d À61.5; IR
(cmÀ1): 3290, 3141, 1428, 1275, 1109, 868, 736; HRMS calcd for
(C13H12F6N3O)+ 340.08791, found 340.08950.
mp 156.6–158.2 °C; 1H NMR (DMSO-d6, 400 MHz): d 9.51 (1H, s,
Ar-H) 8.43 (1H, m, Ar-H) 8.35–8.32 (1H, m, Ar-H) 8.04 (1H, d,
J = 8 Hz, Ar-H) 7.96–7.94 (2H, m, Ar-H) 7.53 (2H, t, J = 8 Hz, Ar-H)
7.41 (1H, t, J = 8 Hz, Ar-H); 13C NMR (DMSO-d6, 100 MHz): d
148.2, 136.2, 134.0 130.9, 130.5, 129.7, 129.1, 128.5 (q,
J2C–F = 31 Hz) 125.9, 125.7, 124.3 (q, J1C–F = 272 Hz) 120.6, 119.8;
19F NMR (DMSO-d6, 470 MHz): d À63.0; IR (cmÀ1): 3124, 1494,
1309, 1135, 727; HRMS, m/z calcd for (C15H11F3N4O2) 335.0750,
found 335.0794.
4.4.9. Methyl-(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)
amino)-3-(1-(4-cyano-3-(trifluoromethyl)phenyl)-1H-1,2,
3-triazol-4-yl)propanoate 39
mp 82.5–83.9 °C; 1H NMR (DMSO-d6, 500 MHz): d 8.94 (1H, s,
ArH) 8.45–8.38 (3H, m, ArH) 7.95 (2H, d, J = 10 Hz, ArH) 7.86 (2H,
d, J = 10 Hz, ArH) 7.63 (2H, t, J = 5 Hz, ArH) 7.38 (2H, t, J = 10 Hz,
ArH) 7.27 (2H, dd, J = 5 Hz, 10 Hz, ArH) 4.53–4.48 (1H, m, CH)
4.28 (2H, d, J = 10 Hz, CH2) 4.18–4.15 (1H, m, CH) 3.67 (3H, s,
CH3); 13C NMR (DMSO-d6, 100 MHz): d 172.2, 156.5, 145.3, 144.3,
144.2, 141.3, 140.2, 138.1, 133.2 (q, J2C–F = 33 Hz) 128.2, 127.6,
125.7, 124.1, 122.7, 122.6 (q, J1C–F = 272 Hz) 120.7, 118.4, 115.6,
66.3, 54.1, 52.8, 47.1, 27.7; 19F NMR (DMSO-d6, 470 MHz): d
4.4.3. 1-(3,5-Bis(trifluoromethyl)phenyl)-4-phenyl-1H-1,2,
3-triazole 29
mp134.5–136.8 °C; 1H NMR (DMSO-d6, 400 MHz): d 9.63 (1H, s,
Ar-H) 8.68 (2H, s, Ar-H) 8.29 (1H, s, Ar-H) 7.95 (2H, t, J = 8 Hz, Ar-H)
7.42 (2H, t, J = 8 Hz, Ar-H); 13C NMR (DMSO-d6, 100 MHz): d 148.3,
138.5, 132.5 (q, J2C–F = 33 Hz) 130.3, 129.7, 129.1, 125.9, 123.4 (q,
J1C–F = 272 Hz) 122.6 (m), 122.0, 121.1 (m), 120.8; 19F NMR
(DMSO-d6, 470 MHz): d À63.0; IR (cmÀ1): 3098, 1612, 1495,

J. M. Altimari et al. / Bioorg. Med. Chem. 22 (2014) 2692–2706
2705
À61.0; IR (cmÀ1): 3367, 2954, 1719, 1509, 1467, 1147, 1032, 760;
Acknowledgements
HRMS (ES) calcd for (C29H23F3N5O4)+ 562.16967, found 562.17244.
The authors gratefully acknowledge the CASS foundation and
the Strategic Research Center for Chemistry and Biotechnology
for the funding to carry out this work. We thank Metabolomics
Australia for the use of their HRMS service. We would also like to
thank the Australian Government for an APA scholarship to J.A.
4.4.10. Methyl-(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)
amino)-3-(1-(4-nitro-3-(trifluoromethyl)phenyl)-1H-1,2,
3-triazol-4-yl)propanoate 38
mp 141.3–142.3 °C; 1H NMR (DMSO-d6, 400 MHz): d 8.79 (1H, s,
ArH) 8.29 (1H, d, J = 5 Hz, ArH) 8.22–8.20 (1H, m, ArH) 7.95 (2H, t,
J = 10 Hz, ArH) 7.87 (2H, d, J = 5 Hz, ArH) 7.64 (2H, dd, J = 5 Hz,
2.5 Hz, ArH) 7.39–7.36 (2H, m, ArH) 7.29, 7.24 (2H, m, ArH)
4.51–4.47 (1H, m, CH) 4.29 (2H, d, J = 5 Hz, CH2) 4.18 (1H, t,
J = 5 Hz CH) 3.67 (3H, s, CH3); 13C NMR (DMSO-d6, 100 MHz): d
172.3, 156.5, 145.0, 144.3, 141.3, 136.1, 133.9, 130.7, 128.5 (q,
J2C–F = 32 Hz) 128.1, 127.6, 125.7, 125.6, 122.8 (q, J1C–F = 272 Hz)
122.4, 120.7, 119.7 (m) 66.3, 54.1, 52.7, 47.1, 27.7; IR (cmÀ1):
3352, 3066, 2852, 1718, 1492, 1289, 1142, 1036, 739, 536; HRMS
(ES) calcd for (C28H23F3N5O6)+ 582.1595, found 582.1624.
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.03.018.
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