1,4-Substituted Triazoles as Nonsteroidal Anti-Androgens for Prostate Cancer Treatment

Article abstract of DOI:10.1021/acs.jmedchem.7b00105

Prostate cancer (PC) is the fifth leading cause of cancer death in men, and the androgen receptor (AR) represents the primary target for PC treatment, even though the disease frequently progresses toward androgen-independent forms. Most of the commerciall

Full text of DOI:10.1021/acs.jmedchem.7b00105

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Article
1,4-Substituted Triazoles as Non-Steroidal
Antiandrogens for Prostate Cancer Treatment
Claudia Ferroni, Antonella Pepe, Yeong Sang Kim, Sunmin Lee, Andrea Guerrini, Marco
Daniele Parenti, Anna Tesei, Alice Zamagni, Michela Cortesi, Nadia Zaffaroni, Michelandrea
De Cesare, Giovanni Luca Beretta, Jane B. Trepel, Sanjay V. Malhotra, and Greta Varchi
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00105 • Publication Date (Web): 08 Mar 2017
Downloaded from http://pubs.acs.org on March 9, 2017
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1,4-Substituted Triazoles as Non-Steroidal Antiandrogens for Prostate Cancer
Treatment
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Claudia Ferroni,¶*¥Antonella Pepe,‡¥ Yeong Sang Kim,# Sunmin Lee,# Andrea Guerrini,¶ Marco
Daniele Parenti,¶ Anna Tesei,§ Alice Zamagni,§ Michela Cortesi, § Nadia Zaffaroni,†
Michelandrea De Cesare,† Giovanni Luca Beretta,† Jane B. Trepel,# Sanjay V. Malhotra∫ and
Greta Varchi¶*
¶Italian National Research Council, Institute for the Organic Synthesis and Photoreactivity Via
Gobetti 101 ꢀ 40129 Bologna, Italy
‡Purdue Center for Cancer Research, 720 Clinic Drive, West Lafayette, Indiana 47907, United
States
#Center for Cancer Research, National Cancer Institute, National Institutes of Health, Building
10 ꢀ Magnuson CC, Bethesda, Maryland 20892, United States
§Biosciences Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori
(IRST), IRCCS, Via P. Maroncelli, 40 ꢀ 47014 Meldola, Forlì, Italy
†Fondazione IRCCS Istituto Tumori Milano, Via Amadeo, 42 ꢀ 20133 Milano, Italy
∫Department of Radiation Oncology, Division of Radiation and Cancer Biology, Stanford
University School of Medicine, CA 94304, United States
¥These authors equally contributed to this work.
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KEYWORDS prostate cancer, bicalutamide, enzalutamide, antiandrogens, triazole, clickꢀ
chemistry.
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ABSTRACT
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Prostate cancer (PC) is the fifth leading cause of cancer death in men and the androgen
receptor (AR) represents the primary target for PC treatment, even though the disease frequently
progresses toward androgenꢀindependent forms. Most of the commercially available nonꢀ
steroidal antiandrogens show a common scaffold consisting of two aromatic rings connected by a
linear or a cyclic spacer. By taking advantage of a facile, oneꢀpot click chemistry reaction, we
report herein the preparation of a small library of novel 1,4ꢀsubstituted triazoles with AR
antagonistic activity. Biological and theoretical evaluation demonstrated that the introduction of
the triazole core in the scaffold of nonꢀsteroidal antiandrogens allowed the development of small
molecules with improved overall ARꢀantagonist activity. In fact, compound 14d displayed
promising in vitro antitumor activity toward three different prostate cancer cell lines and was
able to induce 60% tumor growth inhibition of the CW22Rv1 in vivo xenograft model. These
results represent a step towards the development of novel and improved AR antagonists.
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1. Introduction
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Prostate cancer (PC) is the second most frequently diagnosed cancer and the fifth leading cause
of death from malignancy in men worldwide.1 The activation of the androgen receptor (AR)
signaling has a pivotal role for PC development, progression and androgenꢀindependent
growth.2,3
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AR belongs to the nuclear receptor family of transcription factors that mediates androgen
action in cells upon ligand binding. The AR is widely distributed and is composed of a Nꢀ
terminal transcriptional activation domain, a central DNAꢀbinding domain, a shortꢀhinge region,
and a Cꢀterminal ligandꢀbinding domain (LBD), comprising a 12 helical structure.3
Endocrine therapy has been used in order to reduce androgen synthesis or block AR activation.
So far, androgenꢀdeprivation therapy (ADT) or treatment with firstꢀgeneration antiandrogens
represents the mainstay of treatment for advanced PC. However, most patients acquire resistance
to long term treatment, thus developing more aggressive androgenꢀindependent (AIPC) or
castrationꢀresistant (CRPC, formerly known as hormoneꢀrefractory prostate cancer) tumors.4 In
the past decades, research has proved that signaling through the AR continues to be fundamental
for tumor growth despite castration.5 Indeed, AR aberrations, including gene amplifications,6
point mutations7–9 as well as constitutively active splice variants, are implicated in CRPC.2,10
Antiandrogens are classified into steroidal and nonꢀsteroidal compounds. Steroidal antiꢀ
androgens were first developed in the late 1960s. Among these, cyproterone acetate (1, Fig. 1)
acts by blocking the interaction of testosterone (T) and/or dihydrotestosterone (DHT) with the
AR. Despite its effectiveness, cyproterone suffers from central and progestational side effects,
namely impotence and loss of libido.11
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First generation nonꢀsteroidal AR antagonists, such as flutamide, 2,12 hydroxyflutamide, 3,
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bicalutamide, 4, and nilutamide, 5, diminish androgenic effects by competitively inhibiting
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androgenꢀAR binding. However, after a short time of treatment (2 years), these antiandrogens
may become partial AR agonists due to the occurrence of LBD point mutations and to the
expression of active AR splice variants.14 Therefore, improved understanding of the mechanisms
underpinning resistance has led to the development of a second generation of steroidal or nonꢀ
steroidal AR targeting molecules that significantly contribute to prolonging patient survival, such
as abiraterone, 6,10 enzalutamide, 7 (formerly known as MDV3100)15,16 and 4ꢀ(7ꢀ(4ꢀcyanoꢀ3ꢀ
(trifluoromethyl)phenyl)ꢀ8ꢀoxoꢀ6ꢀthioxoꢀ5,7ꢀdiazaspiro[3.4]octanꢀ5ꢀyl)ꢀ2ꢀfluoroꢀNꢀ
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methylbenzamide, 8 (ARNꢀ509).17 Abiraterone was the firstꢀinꢀclass potent and selective CYP17
inhibitor that causes a decrease in adrenal androgens, thus inhibiting the AR signaling pathway
by blocking ligand synthesis. Despite initial responses, several patients develop resistance within
a year.18 Enzalutamide and 8 are secondꢀgeneration nonꢀsteroidal antiandrogens with high
affinity binding for the AR LBD. In particular, enzalutamide received FDA approval in 2012 for
the treatment of patients with metastatic castrationꢀresistant PC who have previously received
docetaxel.19 However, a F876L missense mutation in the AR LBD has been shown to confer
resistance to enzalutamide and 8, by switching their activity on AR from antagonist to
agonist.20,21 Recently, Moilanen and coworkers described the discovery and the preclinical
development of ODMꢀ201, 9,22 a novel full antagonist for the mutant AR that demonstrated
antitumor activity in vitro and in vivo.23 Furthermore, Purushottamachar demonstrated that a
phase 3 clinical agent, galeterone 10, and its analogues are able to disrupt AR signaling through
enhanced AR degradation.24
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Figure 1. Androgen receptor targeting therapeutics for the treatment of PC.
Based on these findings, it is evident that the development of novel antiandrogens aimed at
circumventing the mutationꢀbased resistance of PC is an urgent need.25 Commercially available
nonꢀsteroidal antiandrogens show a common scaffold composed of two aromatic rings linked by
a linear or a cyclic spacer (11, Fig. 1).
Searching for novel nonꢀsteroidal antiandrogen molecules,26–29 we have discovered a new class
of AR modulators bearing the triazole core. Triazoles are heterocyclic compounds that are often
embedded in more complex molecules with important biological activity,30 including
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antibacterial, antifungal, antiꢀinflammatory, antiꢀHIV and recently as potential androgen receptor
antagonists.31 Triazole derivatives have been extensively used as amide bond isosteres, with the
ability to increase molecule bioavailability and water solubility, and favor hydrogen bonding as
well as dipoleꢀdipole and πꢀstacking interactions. Furthermore, the 1,2,3ꢀtriazole core is
extremely stable towards hydrolysis, oxidative/reductive conditions and metabolic degradation.32
Herein we report the preparation of a diverse library of 1,4ꢀsubstituted triazoles able to reduce
the level of prostateꢀspecific antigen (PSA) and showing activity against a panel of prostate
cancer cell lines, e.g. LNCaP, LNCaPꢀAR and CW22Rv1. Based on structure−activity
relationship (SAR) study, we identified a promising hit molecule that was further tested in vivo.
2. Results and Discussion
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2.1. Chemistry
The impressive advances in the “click” chemistry field along with the promising biological
activity of 1,2,3ꢀtriazole derivatives, make them attractive targets in synthetic organic
chemistry.30 In order to obtain a large library of compounds, we performed the Huisgen 1,3ꢀ
dipolar cycloaddition33 between an azide moiety and a terminal alkyne. In particular, we used
copper (I) catalyst (copperꢀalkyneꢀazideꢀcycloaddition, CuAAC) that dramatically accelerates
the reaction rate, while making it highly regioselective and leading exclusively to the 1,4ꢀ
disubstituted isomer.34 CuAAC allowed to obtain the corresponding triazoles in high yield with a
oneꢀpot procedure starting from the aromatic amine and without azide isolation or purification.35
We synthesized substituted triazoles bearing none, one or two spacers between the aromatic
rings and the triazole core (I, II, III; Fig. 2).
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Figure 2. Classes of synthesized triazole derivatives.
Scheme 1 outlines the synthesis of derivatives of type I. Starting from commercially available
aldehydes, the terminal alkynes (2aꢀc) were obtained through the CoreyꢀFuchs reaction;36
aldehydes were easily converted to the corresponding diꢀbromo olefins (1aꢀc), which were then
treated with lithium diisopropylamide (LDA) to afford the desired alkynes in good yields.
Subsequently, 4ꢀcyanoꢀ3ꢀ(trifluoromethyl)aniline was treated with tertꢀbutyl nitrite (tꢀBuONO)
and azidotrimethylsilane (TMSN3) to generate the corresponding azide in situ. After 2 hours at
0°C, a catalytic amount of CuSO4, sodium ascorbate, as reducing agent, and the terminal alkyne
were added to the reaction mixture to afford the triazole derivatives (3aꢀc).
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Scheme 1. Synthesis of noꢀspacer triazoles 3a-c.a
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aReagents: (a) Ph3P, CBr4, DCM, from 0°C to rt, 15', 72ꢀ99%; (b) nꢀBuLi, DIPA, THF, 0°C,
1h; ꢀ78°C, 3h; from ꢀ78°C to rt, 12h, 9ꢀ51%; (c) tꢀBuONO, TMSN3, CH3CN, from 0°C to rt, 2h;
CuSO4, NaꢀLꢀascorbate, rt, 12h, 24ꢀ32%.
Compounds 5aꢀm, corresponding to scaffold II (shown in Fig. 2) were prepared as depicted in
Scheme 2. Phenols or thioꢀphenols were first alkylated by Williamson reaction with propargyl
bromide to give terminal alkynes (4aꢀj) in quantitative yield.37
Alkyne 4k was prepared in a twoꢀstep protocol, starting from 2ꢀbromoꢀ5ꢀnitrobenzotrifluoride.
The first step is the copperꢀcatalyzed hydroxylation and the second step is the alkylation of the
resulting phenol (6). The terminal alkynes (4a-k) were then used as substrates for the subsequent
cycloaddition with aromatic azides.
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Scheme 2. Synthesis of oneꢀspacer triazoles 5a-m.a
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Compounds
R1
H
H
H
H
H
H
H
H
H
F
CF3
CF3
H
R2
H
H
H
CN
H
R3
F
CN
F
H
NO2
CF3
H
F
H
R4
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
H
X
S
5a
5b
5c
5d
5e
5f
5g
5h
5i
O
O
O
O
O
O
O
O
O
O
O
O
H
CF3
CF3
H
CF3
H
H
5j
5k
5l
NO2
NO2
CN
H
H
Cl
5m
aReagents: (a) K2CO3, propargyl bromide, acetone, reflux, 24h, 79ꢀ100%; (b) tꢀBuONO,
TMSN3, CH3CN, from 0°C to rt, 2h; CuSO4, NaꢀLꢀascorbate, rt, 12h, 16ꢀ87%; (c) CuI,
phenanthroline, KOH, DMSO/H2O, 100°C, 24h, 43%.
The synthesis of compounds 8a-d, bearing linkers with variable length (n = 1, 3) is shown in
Scheme 3. 4ꢀCyanoꢀ3ꢀ(trifluoromethyl)aniline was reacted with propargyl alcohol and 4ꢀpentynꢀ
1ꢀol to yield triazoles 7a and 7b, respectively These were then reacted with different phenols
under Mitsunobu38 conditions, to provide the corresponding ethers (8aꢀ8d) in high yields.
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Scheme 3. Synthesis of oneꢀspacer triazoles 8a-d.a
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aReagents: (a) tꢀBuONO, TMSN3, CH3CN, from 0°C to rt, 2h; CuSO4, NaꢀLꢀascorbate, rt, 12h,
69ꢀ89%; (b) Ph3P, DIAD, THF, 0°C, 30'; rt, 12h, 24ꢀ77%.
In order to expand our SAR study, 1,4ꢀdisubstitued 1,2,3ꢀtriazoles were modified at position
five of the triazole ring (Scheme 4). In particular, compound 5b was reacted with nꢀBuLi and
acetaldehyde to afford derivative 10 in good yield. In addition, an alternative CuAAC protocol
was employed to prepare 5ꢀiodoꢀ1,4ꢀdisubstituted 1,2,3ꢀtriazole 11, and CuI/NBS was used to
generate in situ I+, which readily undergoes the electrophilic substitution on the 5ꢀposition of the
triazole ring.39
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Scheme 4. Introduction of 5ꢀsubstituent on 1,4ꢀsubstitued 1,2,3ꢀtriazoles. a
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aReagents: (a) nꢀBuLi, acetaldehyde, THF, ꢀ78°c to rt, 1h, 44%; (b) tꢀBuONO, TMSN3,
CH3CN, from 0°C to rt, 2h; CuI, DIPEA, NBS, THF, rt, 12h, 35%.
The twoꢀspacer triazole derivatives (12a-c) were synthesized as shown in scheme 5. Trifluoroꢀ
methanesulfonyl azide (TfN3) was prepared in situ from trifluoromethanesulfonic anhydride and
sodium azide and was reacted with glycine methyl ester hydrochloride, the alkyne (4a-c),
NaHCO3, sodium ascorbate and CuSO4. Tris(benzyltriazolylmethyl)amine (TBTA) was also
added to the mixture, to stabilize Cu(I). Upon microwave irradiation and maintaining the
temperature at 80°C, the desired products 12a-c were obtained in good yields (power 100 W, 5ꢀ
10 min).40
The methyl esters 12a-c were then hydrolyzed under basic conditions to the corresponding
carboxylic acids 13a-c and reacted with 4ꢀcyanoꢀ3ꢀ(trifluoromethyl)aniline in a PCl3ꢀmediated
microwaveꢀassisted condensation, to afford the final compounds 14a-d.41
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Scheme 5. Synthesis of twoꢀspacer triazoles 14a-d.a
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aReagents: (a) DCM, 0°C, 2h, 100%; (b) CuSO4, NaHCO3, MeOH, rt, 30'; TBTA, NaꢀLꢀ
ascorbate, 80°C, 10', mw, 76ꢀ92%; (c)LiOH, THF/MeOH, rt, 12h, 88ꢀ100%; (d) PCl3, CH3CN,
100°C, 5', mw, 56ꢀ72%.
2.2. In vitro biological activity
PSA Expression. The antiandrogen activity of the newly synthesized triazole derivatives was
initially screened in vitro using realꢀtime quantitative reverse transcriptionꢀpolymerase chain
reaction (RTꢀqPCR). The activity of the compounds was defined by testing their ability to
control and to lower PSA expression. To this purpose, we selected the ARꢀpositive LNCaP
cells,42 a cell system widely used as a model of androgenꢀresponsive growth. LNCaP cells were
treated with 10 µM compounds for 24 h at 37°C and PSA mRNA levels were measured.
Compared with untreated cells, the compounds generally reduce the PSA level (Fig. 3). In
particular, we identified 10 compounds able to reduce PSA mRNA expression over 50%. Noꢀ
spacer triazoles 3a-c, (blue bars, Fig. 3) were found to be less active with a highest inhibition of
38% (3a). Compounds 14, bearing two spacers (red bars, Fig. 3), showed modest activity, except
for 14b that produced 70% reduction in PSA mRNA expression level.
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Figure 3. Evaluation of PSA mRNA levels after exposure of LNCaP cell lines to triazoles.
Twentyꢀfour hours after seeding, cells were treated for 24 h with 10 µM compounds. Cells were
then collected, processed for mRNA extraction and analyzed using RTꢀqPCR. Blue bars: noꢀ
spacer triazoles (type I); Green bars: oneꢀspacer triazoles (type II); Red bars twoꢀspacer triazoles
(type III).
Oneꢀspacer triazoles (green bars, Figure 3) showed good activity when the B ring was benzene
(5i) or had orthoꢀ or paraꢀsubstituent (5a-c; 5e; 8a-b and 10). In particular, the PSA downꢀ
regulation was over 65% for compounds 5a, 5b, 5c, 5e, 5i, 8a, 8b and 10. It is worth noting that
a trifluoromethyl group on the B ring dramatically decreased compound activity (5f, 5g, 5h, 5j,
5k, 5l and 11). Moreover, a decreased efficacy was observed when the sulfur moiety of
derivative 5a was oxidized to sulphone (compound 9, supporting information section).
Furthermore, compound 12b, lacking the A ring, was considerably less potent, suggesting that
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this aromatic ring is crucial. Finally, further functionalization on the 5ꢀposition of the triazole
e.g. 10 and 11, did not significantly improve the ability to reduce PSA levels. Based on these
findings, we summarized (Fig. 4), the effect of triazole ring substituents on PSA mRNA
reduction.
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Figure 4. Influence of 1,4ꢀtriazole ring substituents on the PSA level reduction.
Cytotoxic Activity in vitro. On the basis of the PSA expression assay, some of the most active
compounds (5a, 5c, 5e, 5i, 7a, 8a, 8b and 10 that inhibited PSA mRNA expression over 50%)
were selected for cytotoxic experiments. Compounds 3a (representative of noꢀspacer triazoles)
and 14b and 14d (representatives of the twoꢀspacer derivatives series) were also tested.
Cytotoxic experiments were carried out using LNCaP and LNCaPꢀAR cell lines, the latter
engineered to stably express high levels of AR, a condition considered representative of clinical
AR gene amplification reported in 25−30% of patients with CRPC.26,43 Cells were exposed to
drug concentrations ranging from 0.02 ꢁM to 20 ꢁM for 144 hours using (R)ꢀbicalutamide and
enzalutamide as references. As reported in Figure 5 and Table 1, not all the compounds reached a
cytotoxic activity toward the selected human PC cells.
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A)
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Figure 5. Cytotoxic activity of novel triazole derivatives against human PC LNCaP (A) and
LNCaPꢀAR (B) cells. Twentyꢀfour hours after seeding, cells were exposed to the compounds for
144 h to the compounds and cytotoxicity was measured using a sulforhodamine B (SRB) assay.
(R)ꢀbicalutamide and enzalutamide were used as references. The mean of three independent
experiments is reported.
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Table 1. Cytotoxic activity of (R)ꢀbicalutamide, enzalutamide and triazole derivatives on LNCaP
5
6
and LNCaPꢀAR cells.a
7
8
9
GI50 value (ꢁM)b
LC50 value (ꢁM)c
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LNCaP
LNCaPꢀAR
n.r.
LNCaP
LNCaPꢀAR
(R)ꢀbicalutamide
1.8±0.53
n.r.
n.r.
n.r.
n.r.
n.r.
n.r.
n.r.
n.r.
n.r.
n.r
enzalutamide
18.88±1.89
17.01±2.02
n.r.
5a
5c
16.75±0.17
19.23±0.37
17.34±0.19
6.42±0.23
5.09±0.85
n.r.
n.r.
10
n.r
n.r.
14b
10.30±1.47
10.16±0.66
n.r.
15.70±0.22
14d
aTwentyꢀfour hours after seeding, cells were exposed for 144 h to the compounds and
b
cytotoxicity was measured using a sulforhodamine B (SRB) assay. GI50 Growth Inhibition: is
the concentration of compound producing 50% of maximal inhibition of cell proliferation; cLC50
Lethal Concentration: is the concentration of compound producing 50% cell death; n.r., not
reached. Data represent mean values ± SD of three independent experiments.
In ARꢀmutated LNCaP cells, (R)ꢀbicalutamide was the most effective compound (GI50 = 1.8 ±
0.53 ꢁM), while only 5a, 5c, 10, 14b and 14d inhibited cell proliferation with GI50 values
ranging from about 5 ꢁM to 19 ꢁM. At the tested concentrations, only 14d exhibited a cytocidal
effect in LNCaP cells (LC50 = 15.70 ± 0.22 ꢁM).
As expected, (R)ꢀbicalutamide was ineffective in inhibiting LNCaPꢀAR cell proliferation.
Conversely, 5a, 14b and 14d showed higher cell growth inhibition as compared with
enzalutamide (GI50 = 18.88 ± 1.89 ꢁM) without reaching LC50 values. In particular, compounds
14b (GI50 = 10.30 ± 1.47 ꢁM) and 14d (GI50 = 10.16 ± 0.66 ꢁM) were the most potent
derivatives of the series.
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These findings suggested that the type III triazoles displayed the strongest cytotoxic effect, and
compound 14d was the most effective of the series.
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Recently, the detection of androgen receptor splice variant 7 (ARꢀV7) mRNA in circulating
tumor cells from men with advanced prostate cancer has been reported to associate with
resistance to enzalutamide and abiraterone.44 On the basis of these findings, CW22Rv1 cells, a
prostate cancer cell model that constitutively expresses high levels of ARꢀV7,45 were used to
evaluate the antitumor activity in vitro of compound 14d. (R)ꢀbicalutamide and enzalutamide
were used as references (Fig. 6).
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Figure 6. Cytotoxic activity of 14d against human CW22Rv1 cells. Twentyꢀfour hours after
seeding, cells were exposed for 144 h to the compounds. The cytotoxic potency was measured
using a sulforhodamine B (SRB) assay. (R)ꢀbicalutamide and enzalutamide were used as
references. The mean of three independent experiments is reported.
No significant difference in antitumor activity was observed for (R)ꢀbicalutamide and
enzalutamide on CW22Rv1 cells, while our newly synthesized 14d compound resulted in 3.8ꢀ
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and 4.4ꢀfold more active than (R)ꢀbicalutamide and enzalutamide, respectively (IC50 = 8.14 ±
0.90 ꢁM). At the tested concentrations, only 14d exhibited antiproliferative and cytocidal effects
(GI50 = 8.25 ± 0.08 and LC50 = 15.70 ± 0.22 ꢁM, Table 2).
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None of the active compounds, filtered for Pan Assay Interference Compounds (PAINS)46
using FAFꢀDrug3,47 were already described.
Table 2. Cytotoxic activity of (R)ꢀbicalutamide, enzalutamide and triazole derivative 14d on
CW22Rv1.a
IC50 72 h (ꢁM)b
31.29±1.83
35.75±2.43
8.14±0.90*
GI50 value 144 h (ꢁM)c LC50 value 144 h (ꢁM)d
(R)ꢀbicalutamide
enzalutamide
14d
n.r.
n.r.
n.r.
n.r.
8.25±0.08
23.07±0.90
aTwentyꢀfour hours after seeding, CW22Rv1 cells were exposed to the compounds for 72 h
b
and 144 h. Seventyꢀtwo hours after exposure, cells were counted using a cell counter. IC50 is
c
defined as the concentration of compound causing 50% cell growth inhibition. To define GI50
(Growth Inhibition: concentration of compound producing 50% of maximal inhibition of cell
d
proliferation) and LC50 (Lethal Concentration: concentration of compound producing 50% cell
death) values, 144 h after compound exposure, cell proliferation was determined using a
sulforhodamine B (SRB) assay. n.r., not reached. Data represent mean values ± SD of three
independent experiments. *P<0.005 14d vs (R)ꢀbicalutamide and enzalutamide.
2.3. In vivo Biological Activity
Based on these results, compound 14d was selected for in vivo. CW22Rv1 cells (5x106
cells/mouse) were subcutaneously inoculated in the flank of CDꢀ1 male nude (nu/nu) mice. The
day after cell inoculation, mice were treated with 50 mg/Kg of 14d according to the schedule
qdx4ꢀ5/wx3w (every day for five days a week for three weeks). The secondꢀgeneration androgen
receptor antagonist enzalutamide was used as reference (50 mg/Kg, qdx5/wx3w). Compared
with enzalutamide, 14d was more active (Figure 7). As reported in Table 3, mice treated with
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14d showed a 60% tumor volume inhibition. Finally, the treatment with 14d was well tolerated,
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Figure 7. Antitumor activity of oral 14d and enzalutamide against human prostate CW22Rv1
carcinoma tumor xenograft. CW22Rv1 cells (5x106/mouse) were injected into the right flank of
the mice on day 0. Treatment started when tumors were not palpable (day 1). Drugs or vehicle
(for Control group), were administrated orally at the dose of 50mg/kg according the qdx5/wx3w
schedule. Tumor size was measured by caliper, and tumor volume calculated using: TV= a ×
b2/2, where a and b are the long and short diameters of the tumor, respectively. Points, mean
from five mice per group; bars, SD.
Table 3. Antitumor activity of oral 14d and enzalutamide on human prostate CW22Rv1
carcinoma cells xenografted s.c. in male nude mice.a
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TVI%maxb
43(7)
BWL%c
Toxd
0/5
enzalutamide
0
0
60*(7)
0/5
14d
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aCells (5x106/mouse) were injected into the right flank of the mice on day 0. Treatment started
when tumors were not palpable (day 1). Drugs were administrated at the dose of 50mg/kg
b
according the qdx5/wx3w schedule. The highest value of TVI%. In parentheses, the day on
c
d
which it was assessed. Body weight loss % induced by drug treatment. Dead/treated mice.
*P<0.05 by Student’s t test vs solventꢀtreated mice.
2.4. Molecular Modeling
In order to better elucidate the mechanism of compound 14d antagonism at the molecular
level, we performed molecular modeling calculations and compared them to those of (R)ꢀ
bicalutamide and enzalutamide. At first, a rigid docking simulation was performed, however this
procedure failed to provide a binding pose for both enzalutamide and compound 14d. The
analysis of the scaffolds overlaying between enzalutamide and 14d with (R)ꢀbicalutamide,
revealed that their lower molecular flexibility could prevent their suitable fitting in the active site
cavity. This finding clearly indicated the possible occurrence of conformational changes in the
active site geometry induced by the bound ligand, the soꢀcalled “induced fit” mechanism.
Therefore, to further elucidate the binding site flexibility, binding modes of active compounds
were generated through Glide/Induced Fit Docking protocol (IFD), which exhaustively considers
all possible binding modes and the associated conformational changes within the receptor active
site. Figure 7 reports the predicted binding modes of the three molecules.
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Figure 8. Putative binding mode of AR modulators. Predicted binding mode of (R)ꢀbicalutamide
(A), enzalutamide (B) and compound 14d (C). Hydrogen bonds are represented as red dotted
lines. Binding pose of (R)ꢀbicalutamide was obtained through rigid docking starting from AR
crystallographic structures (pdb code 2AXA), whereas poses of enzalutamide and 14d were
obtained from IFD simulations.
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The pose of (R)ꢀbicalutamide (Fig. 8ꢀA) is consistent with crystallographic complexes
previously reported.48 In particular, the cyano group on the A ring forms an hydrogen bond with
the side chain of Arg752, as well as with the amide nitrogen and the chiral hydroxyl group with
Leu704 and Asn705 respectively, while the B ring of the ligand is enclosed in an hydrophobic
pocket formed by Trp741, Met742, Met745 and Met895. The IFD calculation suggests that small
adjustments in the position of the side chain of Arg779, Phe876, Phe891 and Leu880 could allow
an effective binding of enzalutamide and 14d (Fig. 8ꢀB and C). In both compounds the A ring
establishes a hydrogen bond network similar to (R)ꢀbicalutamide, even if the hydrogen bond
formed by the cyano group is replaced by a halogen bond in 14d, while the B ring is located in a
different pocket, formed as a result of the conformational rearrangement of the active site.
Enzalutamide forms additional hydrogen bond interactions through its amide group on ring B
with the side chain of Arg779 and the backbone carbonyl of Phe876. Similarly, 14d keeps the
interaction between the amide nitrogen and the side chain of Leu704, and forms an additional
hydrogen bond between the side chain of Lys883 and the cyano group on ring B, and a PiꢀPi
stacking interaction with Phe876.
3. Conclusions
Herein we report the synthesis and biological in vitro evaluation of a novel class of
bicalutamideꢀlike molecules as androgen receptor antagonists. Based on the consideration that
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commercially available nonꢀsteroidal antiandrogens show a common scaffold consisting of two
aromatic rings linked by a linear or a cyclic spacer, we developed a facile and efficient CuAAC
process that allows to access to a wide library of new triazole derivatives with antagonist activity
against the androgen receptor. Collectively, the biological data demonstrate that the introduction
of the triazole ring as an amide bond isoster is a promising option toward the improvement of the
ARꢀantagonist behavior. Overall, compound 14d proved to be the most promising of the series,
being active toward three different cell prostate cancer lines, including LNCaP, LNCaPꢀAR and
CW22Rv1. Although less effective than (R)ꢀbicalutamide on the LNCaP cell line, 14d exhibited
a superior antitumor activity towards LNCaPꢀAR, a cell line overꢀexpressing the AR and
representative of 25ꢀ30% of patients with CRPC. Furthermore, 14d was more active than (R)ꢀ
bicalutamide and enzalutamide on CW22Rv1 cells, a model cell system representatives of
enzalutamide resistance associated with the expression of ARꢀV7 in men suffering from
advanced prostate cancer. Importantly, the results observed in vitro are corroborated by the
superior antitumor potency showed in vivo by 14d in comparison to enzalutamide (60 vs 43%
TVI%) on xenografted CW22Rv1 tumors. Collectively, the results of induced fit docking suggest
that the aromatic rings are able to establish interaction networks similar to (R)ꢀbicalutamide,
confirming that the triazole ring keeps a suitable conformation. In conclusion, the significant in
vitro and in vivo antitumor activity shown by 14d highlights its potential usefulness in the
treatment of advanced human prostate cancer.
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4. Experimental section
4.1 General Chemistry Methods. All reagents were used as obtained from commercial sources
unless otherwise indicated. Solvents were dried over standard drying agents and freshly distilled
prior to use. Deionized water was obtained with the MilliꢀQ Millipore Water Purification System
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and is referred to as milliQ water throughout the paper. All microwave reactions were performed
in a CEM Discover reactor. The 1H and 13C NMR spectra were recorded on Varian spectrometers
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(400 and 500 MHz for H, 100 and 125 MHz for C). Deuterated chloroform was used as the
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1
solvent for NMR experiments, unless otherwise stated. H chemical shifts values (δ) are
referenced to the residual nonꢀdeuterated components of the NMR solvents (δ = 7.26 ppm for
CHCl3 and δ = 3.31 ppm for CHD2OD, etc.). The 13C chemical shifts (δ) are referenced to CDCl3
(central peak, δ = 77.0 ppm), CD3OD (δ = 49.3 ppm), etc. as the internal standard. Optical
rotation was detected on an ADP220 Automatic polarimeter from Bellingham & Stanley
Limited. Mass spectra were measured in positive mode electrospray ionization (ESI). ESIꢀ
HRMS was acquired on an Agilent Dual ESI Q TOF 6520, in positiveꢀion mode, using methanol.
Flash chromatography was performed on Teledyne Isco CombiFlash® Rf 200 using RediSep®
Normalꢀphase Silica Flash Columns (230ꢀ400 mesh). TLC was performed on silica gel 60 F254
plastic sheets. Purity of prepared compounds was determined by HPLCꢀUV analysis (Waters 600
HPLC connected with Photodiode Array Detector 996). Pure (R)ꢀbicalutamide (the active
enantiomer of the nonꢀsteroidal antiandrogen, Casodex®) was straightforwardly synthesized by
highly diastereoselective procedure,49 and enzalutamide was purchased from VinciꢀBiochem Srl
(Florence, Italy). All compounds tested in biological assay were >95%. Purity of intermediates
was >90%, unless otherwise stated. UV/Vis spectra were registered on a Agilent Cary 100
spectrophotometer.
4.2. Synthetic procedures. 4.2.1. General procedure for Williamson reaction. Method B:
Synthesis of terminal alkynes 4b-k. A round bottom flask equipped with a magnetic stir bar was
charged with phenol (1.0 equiv), K2CO3 (4.0 equiv), propargyl bromide (80% wt toluene, 4.0
equiv) and acetone (2 mL/mmol phenol). The mixture was heated to reflux for 24 hours and
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cooled to rt. The solvent was removed under pressure and water was added. The heterogeneous
mixture was extracted twice with EtOAc and the combined organic layers were dried over
Na2SO4, filtered and concentrated. Characterization exemplified by 4ꢀ(propꢀ2ꢀynꢀ1ꢀ
yloxy)benzonitrile (4b). The title compound was prepared using 4ꢀcyanophenol. The yellow solid
didn’t need any further purification, quantitative yield. 1H NMR (400 MHz, CDCl3) δ 7.63–7.60
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(m, 2H), 7.06–7.03 (m, 2H), 4.76 (d, J = 2.4 Hz, 2H), 2.57 (t, J = 2.4, Hz, 1H). C NMR (100
MHz, CDCl3) δ 160.6, 133.9, 119.0, 115.6, 104.9, 76.6, 65.8, 55.9. HRMS (m/z): calcd for
C10H7NO [M]+, 157.0528; found: 157.0528.
4.2.2. General procedure for microwaveꢀassisted click chemistry: synthesis of triazoles (12aꢀ
c). NaN3 (6.0 equiv) was dissolved in H2O (1 mL x 0.4 g NaN3). At 0°C Tf2O (3.0 equiv) in
CH2Cl2 (1 mL x 0.4 g NaN3) was added dropwise to the vigorously stirred solution. After 2
hours at 0°C, the aqueous phase was once extracted with the same volume of CH2Cl2. The
combined organic phase was washed with saturated NaHCO3 solution and used without further
purification.
In a microwave reaction vial, glycine methyl ester hydrochloride (1.0 equiv), CuSO4·5H2O (2
mol%) and NaHCO3 (2.0 equiv) were dissolved in the same volume of water as the volume of
the TfN3 solution to be employed. The TfN3 solution was added to the mixture, followed by
methanol until the solution became homogeneous. The reaction was stirred at rt for 30 min, then
alkyne (1.0 equiv), TBTA (5 mol%) and sodium Lꢀascorbate (10 mol%) were added and the tube
was sealed and heated at 80°C for 10 min by MW irradiation. The reaction mixture was diluted
with water and then extracted with EtOAc. The crude was purified by flash chromatography.
Characterization exemplified by Methyl 2ꢀ(4ꢀ((4ꢀcyanophenoxy)methyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ1ꢀ
yl)acetate (12b). The title compound was prepared using alkyne 4b and following the general
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method. The crude material was purified on a silica gel column eluted with 0ꢀ40% ethyl acetate
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in hexane, affording a white solid, yield = 92%. H NMR (400 MHz, CDCl3) δ 7.79 (s, 1H),
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7.57–7.53 (m, 2H), 7.05–7.01 (m, 2H), 5.24 (s, 2H), 5.18 (s, 2H), 3.78 (s, 3H). C NMR (100
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MHz, CDCl3) δ 166.5, 161.3, 143.3, 133.9, 124.4, 119.0, 115.4, 104.4, 61.8, 53.04, 50.6. HRMS
(m/z): calcd for C13H12N4O3 [M]+, 272.0909; found 272.0911.
4.2.3. General procedure for hydrolysis of methyl ester (13a-c). To a solution of ester (1.0
equiv) in THF (3.3 mL/mmol ester) and MeOH (2 mL/mmol ester) was added an aqueous 1.0M
LiOH solution (2.0 equiv). The resulting mixture was stirred at room temperature overnight. The
reaction mixture was neutralized with HCl 1N. The aqueous phase was extracted with EtOAc
and the combined organic layers were washed with brine, dried and evaporated. The white solid
didn’t need any further purification. Characterization exemplified by 2ꢀ(4ꢀ((4ꢀ
cyanophenoxy)methyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ1ꢀyl)acetic acid (13b). The title compound was prepared
1
using ester 12b and following the general method, quantitative yield. H NMR (400 MHz,
13
CD3OD) δ 8.14 (s, 1H), 7.68–7.64 (m, 2H), 7.19–7.15 (m, 2H), 5.30 (s, 2H), 5.27 (s, 2H). C
NMR (100 MHz, CD3OD) δ 169.8, 163.3, 144.3, 135.4, 127.3, 120.1, 117.0, 105.4, 62.7, 51.8.
HRMS (m/z): calcd for C12H10N4O3 [M]+, 258.0753; found 258.0755.
4.2.4. General procedure for synthesis of triazoles 14a-d. To a solution of aniline (1.01 equiv)
and carboxylic acid (1.0 equiv) in dry CH3CN (10 mL/mmol aniline), PCl3 (1.0 equiv) was
added. The tube was sealed and heated at 100°C for 5 min by microwave irradiation (standard
method), then the mixture was quenched with few drops of water. The reaction mixture was
poured on a preꢀpacked basic alumina column and eluted with CH2Cl2/MeOH: 98/2.
Characterization
exemplified
by
Nꢀ(4ꢀchloroꢀ3ꢀ(trifluoromethyl)phenyl)ꢀ2ꢀ(4ꢀ((4ꢀ
cyanophenoxy)methyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ1ꢀyl)acetamide (14d). The title compound was obtained
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as a white solid, starting from acid 13b and 4ꢀchloroꢀ3ꢀ(trifluoromethyl)aniline and following the
general method (yield = 72%). 1H NMR (400 MHz, CD3OD) δ 8.20 (s, 1H), 8.08 (d, J = 2.8 Hz,
1H), 7.79 (dd, J = 8.4, 2.4 Hz, 1H), 7.69–7.65 (m, 2H), 7.55 (d, J = 8.8 Hz, 1H), 7.20–7.17 (m,
2H), 5.39 (s, 2H), 5.30 (s, 2H). 13C NMR (100 MHz, CD3OD) δ 166.3, 163.4, 144.3, 138.9,
135.4, 133.4, 129.7 (q, J = 31.7 Hz), 127.8, 127.5, 125.4, 124.2 (q, J = 271.1 Hz), 120.2, 119.9
(q, J = 5.6 Hz), 117.0, 105.5, 62.8, 53.6. HRMS (m/z): calcd C19H13ClF3N5O2 [M]+, 435.0710;
found 435.0705.
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4.3. Bioassays. 4.3.1. Cell Lines. The human prostate cancer cell line LNCaP was obtained
from the American Type Culture Collection (Manassas, VA, USA). The LNCaPꢀAR cell line,
derived from LNCaP and engineered to stably express high levels of AR, was kindly provided by
Dr. Charles L. Sawyers (Howard Hughes Medical Institute Investigator at Memorial Sloan
Kettering Cancer Center in New York, NY; U.S.A. The cells were maintained in RPMI medium
supplemented with 10% fetal bovine serum (FBS) and 1% glutamine. CW22Rv1 cells were
cultured in RPMI 1640 containing 10% FBS and 1% glutamine. Cell lines were periodically
checked for mycoplasma contamination by MycoAlert mycoplasma detection kit (Lonza).
4.3.2. Quantitative RealꢀTime ReverseꢀTranscription Polymerase Chain Reaction. LNCaP cells
were incubated with the corresponding compounds for 24 h at 37 ºC. RNA was isolated from
cells using the RNeasy mini kit (Qiagen). cDNA was generated using equal concentrations of
RNA and the Taqman High Capacity Reverse Transcription kit in the GeneAmp PCR System
9700 machine (Applied Biosystems). Diluted cDNA was combined with the forward primer,
reverse primer, SYBR Green PCR Master Mix and RNAseꢀfree water in a 96ꢀwell plate.
Analysis of mRNA expression was carried out using the ABI 7500 RealꢀTime PCR System
(Applied Biosystems). All samples were normalized to the level of 18S ribosomal RNA (rRNA).
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The threshold cycles (Ct) for the control (rRNA) and gene of interest were determined and
relative RNA levels were calculated by the comparative Ct method. Realꢀtime RTꢀqPCR
experiments were performed in triplicate with the following primers:
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PSA
forward 5’ꢀGCAGCATTGAACCAGAGGAGTTꢀ3’
reverse 5’ꢀCACGTCATTGGAAATAACATGGAꢀ3’
18S rRNA
forward 5’ꢀAGTCCCTGCCCTTTGTACACAꢀ3’
reverse 5’ꢀCGATCCGAGGGCCTCACTAꢀ3’
4.3.3. In Vitro Chemosensitivity Assay. Sulforhodamine B (SRB) assay was used according to
the method by Skehan et al.50 Briefly, cells were collected by trypsinization, counted, and plated
at a density of 5000 cells/well (LNCAP and LNCAPꢀAR cell lines) or 1700 cells/well
(CW22Rv1 cell line) in 96ꢀwell flatꢀbottomed microtiter plates (100 ꢁL of cell suspension/well).
Twentyꢀfour hours after seeding, the cells were exposed to the drugs for 144 h (concentration
range 0.02ꢀ20 ꢁM). Experiments were run in octuplicate, and each experiment was repeated
three times. The optical density of cells was determined at a wavelength of 490 nm by a
colorimetric plate reader.
Growth inhibition and cytocidal effect of drugs were calculated according to the formula
reported by Monks et al.:51 [(ODtreated − ODzero)/(ODcontrol − ODzero)] X 100%, when ODtreated is >
ODzero. If ODtreated is below ODzero, then cell killing has occurred. ODzero depicts the cell number
at the moment of drug addition, ODcontrol reflects the cell number in untreated wells, and ODtreated
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reflects the cell number in treated wells on the day of the assay. GI50 and LC50 were defined as
the concentration inhibiting cell growth by 50% and causing the 50% of cell killing, respectively.
Cytotoxic potency on CW22Rv1 was also assessed by growth inhibition assay. Twentyꢀfour
hours after seeding, cells were exposed to the drugs (concentration range 0.5−100 ꢁM) and
counted 72 h later by coulter counter. IC50 is defined as the drug concentration causing 50% cell
growth inhibition, determined by dose−response curves. Experiments were performed in
triplicate.
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4.3.4. In Vivo Experiments. Eight weekꢀold CDꢀ1 male nude (nu/nu) mice (Charles River
Laboratories, Calco, Italy) were used. Experiments were carried out at the Animal Facility of
Fondazione IRCCS Istituto Nazionale dei Tumori of Milan, Italy. The procedures were in
compliance with our institutional animal care guidelines and with international directives in
accordance with Italian law and approval by the Ministry of Health. The animals were
euthanized by cervical dislocation when tumors reached a mean volume of 800mm3. For
antitumor activity experiments, CW22Rv1 prostate cancer cells (5x106 cells/mouse in 100 ꢁL of
saline) were subcutaneously injected in the right flank of the mice. Animals were randomized,
divided in groups (5 mice per group) and, starting the day after cell injection, treated orally by
gavage with 50 mg/Kg of enzalutamide or 14d at a volume of 10mL/kg of body weight, every
day for five days a week for three weeks (qdx5/wx3w). All the compounds were dissolved in
DMSO and Cremophor and suspended in PBS (10+5+85%). Control mice were treated with
vehicle for the same treatment period. Tumor size was measured by caliper, and tumor volume
calculated using the following formula: a × b2/2, where a and b are the long and short diameters
of the tumor, respectively.
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4.4. Theoretical Calculations Method. 4.4.1. Protein Preparation. The Xꢀray coordinates of
human androgen receptor ligand binding domain in complex with Sꢀ1 were extracted from the
Protein Data Bank (PDB code 2AXA)[16129672]. The structure was then processed with the
Protein Preparation Wizard tool as included in the Schrödinger software suite (Schrödinger
Release 2014ꢀ4, Schrödinger, LLC, New York, NY, 2014); this tool automatically sets charges
and correct atom types, bond orders and the orientation of any misoriented groups (such as amide
groups of Asn and Gln). The ligand and water molecules were removed, and an exhaustive
sampling of the orientations of groups, whose hydrogen bonding network needs to be optimized,
was performed. Finally, the protein structure was refined to relieve steric clashes with a
restrained minimization with the OPLS2005 force field until a final rmsd of 0.020 Å with respect
to the input protein coordinates.
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4.4.2. Docking. Docking studies were performed using the software Glide (Schrödinger
Release 2014ꢀ4, Schrödinger, LLC, New York, NY, 2014). The protein structure, prepared as
described above, was used to build the energy grid. A docking grid was obtained by centering a
25 Å box in the centroid of the bound ligand, as present in the template crystallographic structure
of the human AR. All waters were removed from the grid. Standard precision (SP) docking was
used to perform a first round of virtual screening, and poses were further refined with the extraꢀ
precision (XP) docking.
4.4.3. Induced Fit protocol. An initial Glide SP docking of each ligand was performed by using
a softened potential, i.e., a van der Waals radius scaling factor of 0.50 for receptor/ligand atoms
with a partial atomic charge (absolute value) less than 0.15, the metal constraint, and a number of
1000 poses per ligand to be energy minimized on the OPLSꢀAA nonbondedꢀinteraction grid, as
reported above. One hundred poses were saved for each ligand and submitted to the subsequent
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