Theranostic Pt(IV) Conjugate with Target Selectivity for Androgen Receptor

Article abstract of DOI:10.1021/acs.inorgchem.8b00083

It is difficult to diagnose and treat castration-resistant prostate cancer (CRPC) which occurs due to the overexpression of androgen receptor (AR). Because there is a high level of AR in CRPC, we designed and prepared three Pt(IV)-based prodrugs targeting AR. Among them, compound 3, a three-in-one hybrid (an AR binding ligand, a cisplatin unit, and a coumarin moiety), was found to display satisfactory AR binding affinity and antagonist activity against androgen receptor, which could also be effectively internalized and visualized in LNCaP (AR+) cells. Due to its AR affinity, 3 selectively accumulated in greater quantities in LNCaP (AR+) cells than in PC-3 (AR-) cells. Moreover, compound 3 exhibited excellent anticancer activity superior to cisplatin.These results highlight the targeting theranostic application of Pt(IV) prodrugs.

Full text of DOI:10.1021/acs.inorgchem.8b00083

Article
pubs.acs.org/IC
Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX
Theranostic Pt(IV) Conjugate with Target Selectivity for Androgen
Receptor
Xiaodong Qin,†,‡,§ Lei Fang,†,‡,§ Jian Zhao,†,‡ and Shaohua Gou*,†,‡
†Pharmaceutical Research Center and School of Chemistry and Chemical Engineering and ‡Jiangsu Province Hi-Tech Key Laboratory
for Biomedical Research, Southeast University, Nanjing 211189, China
S
* Supporting Information
ABSTRACT: It is difficult to diagnose and treat castration-
resistant prostate cancer (CRPC) which occurs due to the over-
expression of androgen receptor (AR). Because there is a high
level of AR in CRPC, we designed and prepared three Pt(IV)-
based prodrugs targeting AR. Among them, compound 3, a
three-in-one hybrid (an AR binding ligand, a cisplatin unit, and
a coumarin moiety), was found to display satisfactory AR binding
affinity and antagonist activity against androgen receptor, which
could also be effectively internalized and visualized in LNCaP
(AR+) cells. Due to its AR affinity, 3 selectively accumulated in
greater quantities in LNCaP (AR+) cells than in PC-3 (AR-)
cells. Moreover, compound 3 exhibited excellent anticancer activ-
ity superior to cisplatin.These results highlight the targeting theranostic application of Pt(IV) prodrugs.
prognosis and metastatic spreading, with a median survival range
from 18 to 24 months.7 Therefore, effective diagnosis and ther-
apeutic treatments are needed to treat CRPC.
INTRODUCTION
■
Prostate cancer (PC) is now one of the leading causes of cancer
deaths in men worldwide.1,2 Depending on the diagnosis, treat-
ment may involve surgical removal of the prostate gland or andro-
gen depletion therapy (ADT). At present, nonsteroidal antiandro-
gens such as flutamide, bicalutamide, and nilutamide are the most
commonly prescribed ADT drugs whose mechanism of action
consists of competitively inhibiting androgen receptor (AR) activity
associated with PC growth, division, and survival (Figure 1).3−5
AR is a ligand-dependent transcription factor that is vital for
the normal development and maintenance of the prostate. How-
ever, AR-mediated gene expression is also believed to act as an
important driver throughout prostate cancer progression.8−10
Although the mechanisms responsible for the progression of PC to
CRPC are not well-known, it has become clear that AR protein
is essential for CRPC to adapt to the low levels of androgens.11
It has been reported that the expression level of AR is approxi-
mately 6-fold higher in castration-resistant than hormone-sensitive
prostate cancer.11,12 Hence, AR could be recognized as an attrac-
tive target for the treatment of CRPC.13 For example, Koch and
co-workers have reported an androgen receptor-targeted non-
steroidal ligand for the tumor-specific delivery of a doxorubicin−
formaldehyde conjugate.14 Oyelere et al. studied a series of hetero-
bifunctional conjugates by conjugating nonsteroidal antiandrogen
ligands with histone deacetylase inhibitors.15 Several promising
agents including enzalutamide have been applied to improve
CRPC patient survival;16 however, CRPC has evolved to be able
to reactivate AR despite continued androgen depletion therapy.17
Therefore, discovery of more potent drugs beyond only acting
on AR is needed.
Cisplatin (CDDP) is one of the most efficient drugs used for
cancer chemotherapy. It binds to nuclear DNA, and then causes
the blockage of replication and transcription, which ultimately
leads to cellular apoptosis.18,19 However, the clinical efficiency
Figure 1. Examples of nonsteroidal antiandrogens reported in the
literature.
Although these therapies are initially effective at suppressing tumor
growth and improving survival, the tumor will eventually prog-
ress and develop into castration-resistant prostate cancer (CRPC),6
which is almost always incurable and often accompanied by poor
Received: January 10, 2018
© XXXX American Chemical Society
A
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Figure 2. Chemical structures of cisplatin, cis-steroidal Pt(II), and several known Pt(IV) complexes.
of cisplatin is limited by several side effects such as nephrotox-
icity, nerotoxicity, and ototoxicity mainly due to its nonspec-
ificity.20,21 To overcome the nonspecificity of cisplatin, Rodger
et al. developed a nonconventional Pt(II) complex that conjugates
a steroidal androgen to target tumor cells (Figure 2).22 Over
the past decade, the development of inert Pt(IV) prodrugs has
been a promising strategy because the axial position of Pt(IV)
complexes could be modified by various functional groups.23−25
On this basis, Lippard et al. reported a method to deliver cisplatin
to prostate cancer cells by constructing Pt(IV)-encapsulated
prostate-specific membrane antigen (PSMA), providing an avenue
for systemic targeted therapy against this cancer.26 Even though
several Pt-based conjugates intended for prostate cancer treat-
ment have been reported thus far,22,27 there are few reports to
our knowledge describing the use of Pt-based complexes to
conjugate a nonsteroidal unit targeting AR.
In order to introduce an AR-binding ligand to the axial posi-
tion of Pt(IV) complexes and to develop selective and potent
drugs to treat CRPC, here we report a few scaffolds composed
of Pt(IV) complexes and nonsteroidal cyanonilutamide,14,15,28
which are expected to target PC via AR to endow the Pt(IV)
prodrugs with favorable accumulation in cancer cells. In addi-
tion to assessing the cellular uptake and accumulation of Pt,
a fluorescent agent (coumarin) was also introduced in a Pt(IV)
prodrug to implement real-time imaging so that the cell uptake
and targeting efficiency could be simultaneously visualized.
AR LBD).29 The binding modes of these compounds were
depicted in Figure 3 and the Surflex docking scores obtained
were summarized in Table S1. The docking scores were 5.51
for L-1, 3.64 for cyanonilutamide, and 8.15 for testosterone,
where higher scores indicated greater binding affinity. Although
weaker than testosterone, the binding affinity of L-1 was greater
than its parent compound cyanonilutamide. In Figure 3C, testos-
terone in complex with AR LBD showed that the carbonyl group
had a hydrogen bond interaction with ARG752 and the hydroxyl
group had a hydrogen bond with ASN705, contributing to the
closed conformation of AR.30 L-1, possessing a similar binding
mode to cyanonilutamide, could also fit into the binding site by
the formation of the key hydrogen bond between the cyano group
and ARG752 (Figure 3A,B). The docking results suggested that
L-1 had a competitive mechanism of action at the same site as
testosterone, which might disturb the proper positioning of
H12 and repress the activation of AR.
Synthesis. Three novel Pt(IV) prodrugs conjugated with L-1
were designed and synthesized (Scheme 1). Cyanonilutamide (6)
was prepared according to the former literature report.28 Com-
pound 7 was synthesized via an alkylation of 6 with ethyl brom-
oacetate in the presence of sodium hydride. Then L-1 was
obtained by the hydrolysis of 7. To explore the effect of ligands
on the activity, complexes 1 and 2 were axially coordinated with
chloridion and hydroxyl, respectively. Besides, a coumarin deriv-
ative (11) was introduced to the axial position of 2 to obtain a
theranostic prodrug 3. All new compounds were unambiguously
characterized by NMR spectroscopy and ESI-MS. The purity of
Pt(IV) complexes 1−3 were confirmed to be more than 95% by
HPLC.
RESULTS AND DISCUSSION
■
Design of an AR Ligand. To endow the Pt(IV) complex
with AR-targeting ability, we designed a cyanonilutamide deriv-
ative (L-1) as the ligand (Scheme 1). Initial docking studies of
L-1, cyanonilutamide and testosterone (an endogenous steroid
that binds AR) were carried out to explore the binding capa-
bility in the cavity of helix-12 (H12, at the C-terminus of the
Cyclic Voltammetry. To show how easily these compounds
can be reduced to its Pt(II) equivalents, we measured the reduc-
tion potentials of complexes 1−3. In cyclic voltammograms
(Figure S1, see Supporting Information), all complexes exhibited
B
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Scheme 1. Synthesis of Pt(IV) Conjugates
irreversible reduction processes with the reduction potentials of
1−3 being −0.435, −0.455, and −0.467 V, respectively. The
result indicated that the complex with an axial chloridion ligand
(1) possessed the lowest electrochemical reducibility and the
complex with an axial carboxyl ligand (3) possessed the highest
electrochemical reducibility.
slower than 1, indicating that an axial chlorido ligand in the
coordination sphere would facilitate the electron transfer better
than an axial hydroxyl ligand. In contrast, 3 displayed the slow-
est chemical reduction rate, demonstrating that carboxylates do
not facilitate the electron transfer from the reducing agent to
the Pt(IV) center.34 However, no peak of the supposed cis-
platin species was observed in HPLC chromatograms under the
test condition, which was due to its weak chromophore in the
UV detecting condition. It was noted that the reducing rate
trend of complexes monitored by HPLC was in accordance with
that measured by cyclic voltammetry. These observations revealed
that the Pt(IV) complexes were stable in PBS and could be
reduced to release the axial ligand(s) in the presence of ascorbic
acid.
AR Binding Affinity. In order to explore the AR binding
affinity of the new cyanonilutamide derivatives, the fluorescence
polarization (FP) based binding assay by competition with the
fluorescent tracer was carried out. As shown in Table 1, such
derivatives retained the ability to bind AR directly, while cisplatin
had no apparent binding affinity. The values of AR affinity for
cyanonilutamide and bicalutamide were 4.42 and 2.51 μM, respec-
tively, a little different from those in the previous report.15 Con-
sidering that the values were in the same order, our results were
convincing as well. L-1 showed the highest binding affinity with
an IC50 value of 3.75 μM among the new cyanonilutamide deriv-
atives. Meanwhile, 1−3 displayed a little weaker binding affinities
with IC50 values in the range from 5.49 to 7.58 μM. The tiny
decline might be owing to the bigger size of the Pt(IV)
HPLC Analyses on the Stability and Reduction of
Complexes 1−3. The solution stability and the reduction of
complexes were examined by HPLC at different times. As shown
in Figures 4A and S18−S20, all Pt(IV) complexes were stable
in medium RPMI-1640 (10% fetal bovine serum) and PBS after
24 h. To further confirm whether the complex could be reduced
to release its Pt(II) equivalent with the AR-targeting ligand and
compare the reducing rate in the presence of ascorbic acid, each
compound was investigated by HPLC in a mixture solvent of ace-
tonitrile/water containing ascorbic acid. As seen in Figure S19,
the absorption peak of 1 decreased upon reacting with VC and
the peak of the AR-targeting ligand increased simultaneously
as the time passed (T1/2: 2.3 h, Figure 4). The similar phenom-
enon was observed when 2 was incubated with VC apart from
the slower reducing rate (T1/2: 8.8 h). As for 3, the reduction
could also be observed with the longest time (T1/2: 13.9 h),
as shown by the presence of the AR-targeting ligand and cou-
marin motiety. Under the same condition, the experimental data
of complexes 1−3 were all fitted well to a monoexponential
decay function as described by previous reports.31−33 The rate
constants (kobs) of 1−3 were 0.3010 h−1, 0.0792 h−1, and
0.0501 h−1, respectively. The chemical reduction rate of 2 was
C
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Figure 3. Molecular docking of androgen receptor (AR) in complex with L-1 (A), cyanonilutamide (B), and testosterone (C).
complexes, which made them harder to insert the ligand-binding
pocket.
When excited at 320 nm, 3 (Φ = 0.072) displayed an emission
band at 397 nm. However, its fluorescence quantum yield was
weaker than the coumarin ligand 11 (Φ = 0.32). It’s well-
known that Pt(IV) complexes could usually release the axial
ligand after reduced by ascorbic acid or GSH, which were
overexpressed in cancer cells.24 Thus, we assumed that prodrug
3 could exhibit strong fluorescence after uptaken into cancer
cells.
Confocal Microscopy. With an aim to explore the poten-
tial of prodrug 3 as targeted theranostic in cells, LNCaP (AR+)
and PC-3 (AR−) were employed for cell imaging to demon-
strate the fluorescence response by laser scanning confocal micro-
scopy. As shown in Figure S21, PC-3 (AR−) cells treated with
3 or 11 both showed weak fluorescence. Significantly, LNCaP
cells exhibited strong fluorescence intensity upon incubation with
3 for 4 h, confirming the remarkable cellular uptake of 3 by
LNCaP cells (Figure 6). Even though the fluorescence in PC-3
cells was weaker than that in LNCaP cells, we could observe the
internalization of compound 3, responsible for the death of cells,
in both the cells. On the other hand, the cells showed faint
Binding of molecules to AR may lead to antagonist activity.15
To assess the antiandrogenic activity of the compounds, we eval-
uated their pharmacological properties in androgen responsive ele-
ment (ARE)-driven luciferase reporter assay in 293T cells. The AR
luciferase assays were examined in the treatment of 1 nM AR
agonist R1881 along with 10 μM tested compounds, respectively,
and the antagonist activity was measured as inhibition of R1881
induced luciferase expression. All tested compounds showed
excellent AR antagonist activity. However, there was no apparent
correlation between the effect on antagonistic activity and bind-
ing affinity of these compounds. Overall, it was determined that
L-1 and 1−3 all possessed potent binding affinity and displayed
antagonist activity against AR.
UV Absorption and Fluorescence Spectroscopy.
UV absorption and fluorescence spectra of all Pt(IV) compounds
were measured. In Figure 5, complexes 1−3 exhibited an absorp-
tion band at 224 nm. As for 3, an extra absorption band at
320 nm was observed, which is typical for the coumarin group.
D
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Figure 4. (A) Stability of 1−3 in medium RPMI-1640 (10% fetal bovine serum). Time-dependent reductions of 1 (B), 2 (C), and 3 (D) upon VC
on HPLC (1−3 were 0.2 mM and VC was 1 mM). The experimental data of complexes 1−3 were fitted well to a monoexponential decay function
(equation: y = y0 + A1 exp(−x/t1), kobs = ln(2)/t1).
Table 1. AR Binding Affinity and AR Antagonist Activity
of Bicalutamide, Cyanonilutamide, Testosterone, Cisplatin,
L-1, and complexes 1−3
the uptake of three Pt(IV) complexes did not exhibit apparent
improvement compared with that of cisplatin in PC-3 (AR−)
cells. However, in LNCaP (AR+) cells, complexes 1−3 accu-
mulated in a higher extent than cisplatin. This suggested that
the conjugation of the AR ligand with Pt(IV) units resulted in
the increased cellular uptake, which might account for their
higher cytotoxicity in AR-positive LNCaP cells. Moreover, com-
plexes 1−3 preferentially accumulated in LNCaP (AR+) cells
compared with PC-3 (AR−) cells, but in contrast, there was
almost no difference in the control group of cisplatin. The amount
of the platinum uptake of complex 3 was in accordance with the
result in cell imaging experiments. These data clearly demon-
strated that AR-targeted conjugates displayed the greater abil-
ities of accumulation and selectivity owing to the acquired AR
binding affinity.
a
compound
IC50 (μM)
inhibition % (10 μM)
bicalutamide
2.51 0.06
4.42 0.12
3.75 0.10
5.49 0.14
6.13 0.10
7.58 0.19
0.0095 0.0006
85.4
77.8
51.2
73.5
66.3
70.1
cyanonilutamide
L-1
1
2
3
b
testosterone
cisplatin
NT
c
ND
ND
a
Inhibition rate was shown as a ratio to the R1881 control and the
data represent the mean of at least three independent experiments.
NT, not tested. ND, not determined.
b
c
In Vitro Cytotoxicity. To investigate cell-type selectivity and
potency, we estimated the antiproliferative activity of these
compounds in LNCaP (AR+) and PC-3 (AR−) cells. The
in vitro cytotoxicity of the novel compounds against the two cell
lines was evaluated by the MTT assay with cisplatin, bicalutamide
and cyanonilutamide as reference controls. The correspon-
ding IC50 values (concentration required to reduce viability to
50%) obtained after 72 h exposure were summarized in Table 2.
Bicalutamide showed moderate antiproliferative activity in
LNCaP cells and no apparent activity in PC-3 cells, whereas
cyanonilutamide had no activity in both cells. Not surprisingly,
the IC50 value of cisplatin in LNCaP cells was higher than that in
PC-3 cells (SI = 0.75), which was consistent with the previous
report.26 This result indicated that cisplatin did not show any
pronounced AR dependency in its cytotoxicity to either cell lines.
However, it was gratifying to observe that all three complexes are
fluorescence under the same treatment with 11 (the precursor
of 3). As the stronger fluorescence often means higher cellular
uptake, we could deem that the uptake of 3 was more than that
of 11 in LNCaP cells. The results proved that the AR targeted
ligand could significantly increase the uptake of 3 in AR over-
expressed LNCaP cells and the conjugate could meet the require-
ment for cell imaging.
Cellular Uptake. Since the cytotoxic effect of Pt-based anti-
cancer drugs is highly dependent on cellular uptake and accumu-
lation, we explored the effect of the AR-ligand conjugation on the
accumulation of the Pt(IV) complexes. For this purpose, the
cellular uptake of the Pt(IV) complexes was determined by induc-
tively coupled plasma mass spectrometry (ICP-MS) in both AR-
positive LNCaP and AR-negative PC-3 cells. As shown in Figure 7,
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Figure 5. (A) Absorption of complexes 1−3 and 11. (B) Fluorescence spectra of 3 and 11. Compounds were dissolved in PBS containing 5% DMF
at the concentration of 0.1 mM.
Figure 6. Confocal microscopy images of LNCaP cells treated with 20 μM compound 11 (d−f), 20 μM prodrug 3 (g−i), or untreated control cells
(a−c). Fluorescence from DAPI staining nucleus appears as blue signals (Ex: 340 nm; Em: 450−470 nm) in the left column while fluorescence from
11 or 3 appears as green signals (Ex: 355 nm; Em: 490−520 nm) in the middle column. The right column was the merge of both above. Scale bar:
30 μm.
Table 2. In Vitro Cytotoxicity of Cisplatin, Bicalutamide,
Cyanonilutamide, L-1, 11, and Complexes 1−3
IC50 (μM)
a
compound
cisplatin
PC-3 (−)
LNCaP (+)
SI
7.31 0.29
>50
9.78 0.48
47.39 1.85
>50
0.75
bicalutamide
cyanonilutamide
>50
L-1
11
1
>50
>50
>50
>50
6.88 0.51
5.79 0.39
2.84 0.25
3.15 0.27
2.87 0.15
1.02 0.08
2.18
2.01
2.78
2
3
Figure 7. Cellular uptake of cisplatin and 1−3 in PC-3 and LNCaP
cells after 12 h of incubation.
a
SI (Selectivity Index) is defined as IC50 value in PC-3 cells/IC50 value
in LNCaP cells.
generally more cytotoxic against AR-dependent LNCaP cells
than them against AR-independent PC-3 cells. More significantly,
all Pt(IV) complexes displayed enhanced activity superior to
cisplatin. The results demonstrated that the ability of these
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Figure 8. Flow cytometry analysis for apoptosis (A) and cell cycle distribution (B) of LNCaP cells induced by cisplatin or 3 at 20 μM for 24 h.
AR-Pt(IV) conjugates to bind to AR has indeed conferred cell-
type selectivity and improved the antitumor effect remarkably.
Notably, the theranostic prodrug 3, conjugated with a AR-targeting
ligand and the coumarin motiety simultaneously, was 9.6× more
active than cisplatin and exhibited the best selectivity (SI = 2.78).
Cell Cycle and Apoptosis Analysis. Since prodrug 3 dis-
played the best antitumor activity in vitro, we chose 3 as the
optimal candidate for further therapeutic mechanism investigation.
To evaluate whether the inhibition of the cancer cell proliferation
was associated with apoptosis, an Annexin V-FITC/PI staining
assay was carried out in LNCaP cells. The flow cytometry ana-
lysis showed that cisplatin achieved an apoptosis rate of 71.6%
(including the early and late apoptosis). In contrast, 3 could
achieve an apoptosis rate of 83.0%, much superior to that of cis-
platin apparently (Figure 8A). Additionally, we also assessed
the effect of 3 on cell cycle by measuring DNA content. LNCaP
cells were still treated with 20 μM cisplatin and 3 for 24 h, and
then cell cycle distribution was tested. As shown in Figure 8B,
the percentage of sub-G1 phase, which is considered as a bio-
marker for DNA damage and related to the presence of apopto-
sis, increased apparently when treated with cisplatin or 3. More-
over, the cell cycle slightly changed after incubation with
cisplatin: the percentage of G0/G1 phase decreased from 51.27%
to 47.34%, the percentage of S phase increased from 30.49% to
33.71%, while the percentage of G2/M phase showed no obvious
variations, respectively. However, the LNCaP cells were signifi-
cantly arrested at S phase (40.37%) after incubation with 3, reveal-
ing that 3 possessed the stronger cell cycle arrest than cisplatin.
Western Blot Analysis. To gain more insights into the
molecular mechanisms the activity of complex 3, we measured
the level of proteins related to the regulation of apoptosis by
Western blotting. In Figure 9, the combined treatment with
Figure 9. Western blotting analysis of the expression of related
proteins.
cisplatin increased the expression of pro-apoptotic gene Bax and
suppressed the expression of antiapoptotic gene Bcl-2. Excit-
ingly, 3 showed a stronger effect on Bax and Bcl-2 genes than
cisplatin, confirming that apoptosis was conspicuously promoted
by 3. In addition, 3 also induced the proteolytic cleavage of PARP
and obviously decreased expression level of caspase-3, while
cisplatin displayed little influence on it. These results proved
that the Pt(IV) conjugate 3 might possess a unique mode of
action to kill cancer cells different from cisplatin owing to the
conjugation with both the AR-targeting ligand and coumarin
moiety.
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DMSO-d6) δ 1.45 (s, 6H), 4.14 (s, 2H), 8.04−8.07 (d, 1H, J = 8.5 Hz),
8.20 (s, 1H), 8.30−8.33 (d, 1H, J = 8.3 Hz), 12.93 (s, 1H) ppm.
Synthesis of 11. 7-Hydroxycoumarin (1.62 g, 10 mmol), potas-
sium carbonate (6.91 g, 50 mmol), bromoacetic acid (6.95 g, 50 mmol),
and acetone (250 mL) were added into a 500 mL round-bottom flask.
Then catalytic amount of potassium iodide was added. The mixture
was refluxed for 20 h. The reaction was monitored by TLC. After
reaction finished, water (200 mL) was added and the pH was adjusted
to ∼3 by 5% HCl aqueous. Then acetone was removed under reduced
pressure at room temperature. The mixture was filtered and the solid
was washed by water (30 mL × 3) and Et2O (30 mL × 3). The white
CONCLUSIONS
■
Herein, we have described a type of novel AR-targeted anti-
cancer agents produced by the conjugation of a cyanoniluta-
mide ligand to Pt(IV) complexes derived from cisplatin and
evaluated their biological activity. Electrochemical character-
istics and HPLC studies revealed that the order of the chemical
reduction rates of these complexes was 1 > 2 > 3. Competitive
experiments showed that all AR-Pt(IV) conjugates displayed
satisfactory AR binding affinity and antagonist activity against
androgen receptor. Additionally, there was greater accumu-
lation of our complexes, especially 3, in LNCaP (AR+) cells
than in PC-3 (AR−) cells. Such differences in accumulation
contributed to the selective antitumor activity in vitro. In addi-
tion, the internalization of prodrug 3 could be easily visualized
by confocal microscopy due to its fluorescence property, hinting at
its potential as a targeting theranostic agent. Further mechanistic
research revealed that 3 arrested the cell cycle at S phase and
dramaticlly increased the apoptosis.
1
solid was dried, yield 89%. H NMR (300 MHz, DMSO-d6) δ 4.82
(s, 2H), 6.82−6.32 (d, 1H, J = 9.5 Hz), 6.94−6.97 (m, 2H), 7.62−7.65
(d, 1H, J = 9.2 Hz), 7.97−8.00 (d, 1H, J = 9.5 Hz), 13.09 (s, 1H) ppm.
Synthesis of 1. A solution of TBTU (105.6 mg, 0.33 mmol) and 8
(117.2 mg, 0.33 mmol) in 10 mL of anhydrous DMF was stirred at
room temperature under N2 atmosphere. After 10 min, TEA (33.3 mg,
0.33 mmol) was added and the reaction was stirred for 15 min, c,c,
t-[Pt(NH3)2Cl3(OH)] (115.8 mg, 0.33 mmol) was then added and the
reaction mixture was stirred at room temperature for 12 h. The solvent
was then removed by evaporation under reduced pressure. Column
chromatography (eluent 15:1 DCM/methanol) gave 1 as a pale yellow
solid (111.6 mg, 49%). ESI-HRMS: calcd for m/z [M − H]−,
Our three-in-one hybid 3 with its innovative design is a
Pt(IV) prodrug that can simultaneously bind to AR and emit
fluorescence. The AR-targeted theranostic agent is able to add
the AR overpressed tumor selectivity of cisplatin and offer a
promising strategy to treat CRPC.
1
687.9946; found, 687.9851. H NMR (300 MHz, DMSO-d6) δ 1.45
(s, 6H), 4.10 (s, 2H), 6.20 (m, 6H), 8.04−8.07 (d, 1H), 8.19 (s, 1H),
8.30−8.33 (d, 1H) ppm. 13C NMR (75 MHz, DMSO-d6) δ 22.87,
42.72, 62.27, 107.38, 115.70, 121.37 (J = 274.6 Hz), 124.46 (J =
4.8 Hz), 130.44, 131.52 (J = 33.6 Hz), 136.70, 137.74, 153.23, 175.26,
175.95 ppm. 195Pt NMR (129 MHz, DMSO-d6) δ 552 ppm. Anal.
Calcd for C15H17Cl3F3N5O4Pt: C, 26.12; H, 2.48; N, 10.15%. Found:
C, 26.35; H, 2.59; N, 10.03%.
Synthesis of 2. A solution of TBTU (105.6 mg, 0.33 mmol) and 8
(117.2 mg, 0.33 mmol) in 40 mL of anhydrous DMF was stirred at
room temperature under a N2 atmosphere. After 10 min, TEA (33.3 mg,
0.33 mmol) was added and the reaction was stirred for 15 min c,c,
t-[Pt(NH3)2Cl2(OH)2] (110.3 mg, 0.33 mmol) was then added and
the reaction mixture was stirred at 55 °C for 12 h. The solvent was
then removed by evaporation under reduced pressure. Column chro-
matography (eluent 8:1 DCM/Methanol) gave 2 as a yellow solid
(50.3 mg, 22.7%). ESI-HRMS: calcd for m/z [M − H]−, 670.0285;
found, 670.0168. 1H NMR (300 MHz, DMSO-d6) δ 1.44 (s, 6H), 4.00
(s, 2H), 5.83−6.08 (m, 6H), 8.04−8.06 (d, 1H, J = 8.1 Hz), 8.19
(s, 1H), 8.31−8.33 (d, 1H, J = 8.1 Hz) ppm. 13C NMR (75 MHz,
DMSO-d6) δ 22.87, 42.78, 62.19, 107.29, 115.68, 121.35 (J = 273.3 Hz),
124.38 (J = 4.9 Hz), 130.35, 131.50 (J = 32.5 Hz), 136.66, 137.20,
153.16, 175.35, 175.95 ppm. 195Pt NMR(129 MHz, DMSO-d6) δ 1065
ppm. Anal. Calcd for C15H18Cl2F3N5O5Pt: C, 26.84; H, 2.70; N,
10.43%. Found: C, 26.65; H, 2.79; N, 10.19%.
EXPERIMENTAL SECTION
■
Materials and Instrument. All chemicals and solvents were of ana-
lytical reagent grade and used without further purification, unless noted
specifically. c,c,t-[Pt(NH3)2Cl3(OH)] and c,c,t-[Pt(NH3)2Cl2(OH)2]
were prepared according to literature reports.22 The purity of all
compounds used in the biological studies was ≥95%. All antibodies
were purchased from Santa Cruz Biotechnology. All cancer cell lines
were obtained from Jiangsu KeyGEN BioTECH company (China).
Cell cycle and apoptosis experiments were measured by flow cytom-
etry (FAC Scan, Becton Dickenson) and analyzed by Cell Quest soft-
ware. 1H NMR and 13C NMR spectra were recorded in DMSO-d6 on a
Bruker 300 MHz spectrometer. 195Pt NMR spectra were measured in
DMSO-d6 with a Bruker 600 MHz spectrometer. Platinum contents
were determined by Inductively Coupled Plasma-Mass Spectrometer
(ICP-MS, Optima 5300DV, PerkinElmer, USA). Mass spectra were
measured by an Agilent 6224 ESI/TOF MS instrument. Elemental
analyses of C, H, and N used a Vario MICRO CHNOS elemental
analyzer (Elementar).
Chemistry. Synthesis, and Characterization of 4. 4-Fluoro-2-
(trifluoromethyl)benzonitrile (4.02 g, 21.3 mmol) was added to a mix-
ture of hydantoin (13.6 g, 106.3 mmol) and potassium carbonate (4.40 g,
31.9 mmol) in DMF (60 mL) and stirred at 45 °C under argon for 48 h.
Reaction mixture was diluted in ethyl acetate and washed three times
with water. The organic layer was dried over sodium sulfate, filtered
and concentrated in vacuo. Column chromatography (eluent 30:1
Synthesis of 3. A solution of TBTU (35.3 mg, 0.11 mmol) and 11
(24.2 mg, 0.11 mmol) in 10 mL of anhydrous DMF was stirred at
room temperature under a N2 atmosphere. After 10 min, TEA (11.2 mg,
0.11 mmol) was added and the reaction was stirred for 15 min. Complex
2 (73.7 mg, 0.11 mmol) was then added and the reaction mixture was
stirred at 25 °C for 12 h. The solvent was then removed by evaporation
under reduced pressure. Column chromatography (eluent 15:1 DCM/
methanol) gave 3 as a yellow solid (52.3 mg, 54.5%). ESI-HRMS: calcd
1
DCM/Methanol) gave 6 as a white solid (4.62 g, 74%). H NMR
(300 MHz, DMSO-d6): δ 1.42 (s, 6H), 8.01−8.04 (d, 1H), 8.18 (s, 1H),
8.29−8.32 (d, 1H), 8.84 (s, 1H) ppm.
Synthesis of 8 (L-1). Compound 6 (5.00 g, 16.8 mmol) was
dissolved in DMF (40 mL) under argon, followed by addition of NaH
(60% in mineral oil, 1.00 g, 25.2 mmol) and stirring for 2 h at room
temperature. Then ethyl bromoacetate (4.22 g, 25.2 mmol) was added
and the reaction was stirred for 5 h at 55 °C. To the reaction was
added EtOAc (200 mL) and the mixture was successively washed with
brine (5 × 150 mL) and H2O (3 × 125 mL). The organic layer was
dried over sodium sulfate, filtered and concentrated in vacuo. Column
chromatography (eluent 5:1 PE/EtOAc) gave 7 as a white solid (5.21 g,
81%). Compound 7 (5.01 g, 13.1 mmol) was dissolved in MeOH
(40 mL), followed by addition of NaOH (2.0 g, 50.4 mmol) and stirring
for 2 h at 50 °C. The reaction mixture was concentrated, dissolved in
150 mL of H2O and adjusted the pH value to acidity. The precipitate
was filtered and dried in vacuo to get 8 as a white solid (4.55 g, 97%).
ESI-HRMS: m/z [M − H]− = 354.0699. 1H NMR (300 MHz,
1
for m/z [M − H]−, 872.0551; found, 872.0421. H NMR (300 MHz,
DMSO-d6) δ 1.45 (s, 6H), 4.12 (s, 2H), 4.78 (s, 2H), 6.27−6.30
(s, 1H, J = 10.0 Hz), 6.50 (m, 6H), 6.93−6.95 (d, 1H, J = 8.7 Hz),
7.01 (s, 1H), 7.59−7.61 (d, 1H, J = 8.4 Hz), 7.97−8.00 (d, 1H, J =
9.6 Hz), 8.04−8.07 (d, 1H, J = 8.5 Hz), 8.19 (s,1H), 8.31−8.33 (d, 1H, J =
8.4 Hz) ppm. 13C NMR (75 MHz, DMSO-d6) δ 22.81, 41.77, 62.24,
65.01, 102.17, 107.37, 113.02, 113.05, 113.47, 115.67, 121.34 (J =
272.6 Hz), 124.42 (J = 4.6 Hz), 129.75, 130.38, 131.51 (J = 31.5 Hz),
136.69, 137.12, 144.81, 153.22, 155.74, 160.83, 161.75, 175.20, 175.27,
176.10 ppm. 195Pt NMR(129 MHz, DMSO-d6) δ 1231 ppm. Anal.
Calcd for C26H24Cl2F3N5O9Pt: C, 35.75; H, 2.77; N, 8.02%. Found: C,
35.92; H, 2.90; N, 7.86%.
Cyclic Voltammetry. The cyclic voltammetry (CV) was measured
on PGSTAT101 (Autolab, Metrohm) in a range of potentials from
H
DOI: 10.1021/acs.inorgchem.8b00083
Inorg. Chem. XXXX, XXX, XXX−XXX

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Article
+1.0 V to −1.0 V, with a three electrode setup comprising a glassy car-
bon working electrode, platinum wire auxiliary electrode and a calomel
reference electrode. Complexes 1−3 were dissolved in PBS containing
5% DMF at the concentration of 20 mM. The scan rate was 100 mV·s−1
at 25 °C, KCl (0.1 M, pH 7.0) was used as a background electrolyte.
Stability of Complexes 1−3 in PBS Buffer and Medium
RPMI-1640. The stability of Pt(IV) complexes in a phosphate buffer
saline (PBS) and RPMI-1640 supplemented with 10% fetal bovine
serum was investigated by HPLC. The incubation was generated by
adding different complexes into PBS buffer or RPMI-1640, which was
performed at 25 °C at different times. Reversed phase HPLC was
implemented on a 250 × 4.5 mm ODS column and the HPLC profiles
were recorded on UV detection at 210 nm. Mobile phase consisted of
acetonitrile/water, and flow rate was 1.0 mL/min. The samples were
taken for HPLC analysis after filtration by 0.45 μm filter.
Reduction Ability of Pt(IV) Complexes 1−3 Treated with
Ascorbic Acid. The Reduction of Pt(IV) complexes was carried out
using ascorbic acid in acetonitrile/water and reduction products were
examined at 25 °C by HPLC. Reversed phase HPLC was implemented
on a 250 × 4.5 mm ODS column and the HPLC profiles were recorded
on UV detection at 210 nm. Mobile phase consisted of acetonitrile/
water (80:20, v/v), and flow rate was 1.0 mL/min. The samples were
taken for HPLC analysis after filtration by 0.45 μm filter.
(MatTek). The cells were treated and incubated with tested com-
pounds at 37 °C under 5% CO2 for 4 h. Then DAPI was added during
the final 15 min of the incubation. The cells were washed by phos-
phate buffered saline (PBS) and then imaged after further incubation
in colorless serum-free media for 15 min. Fluorescence images were
taken using a confocal laser scanning microscope (Zeiss LSM 700,
Zeiss, Germany). Fluorescence from 11 or 3 appears as green signals
(Ex: 355 nm; Em: 490−520 nm), while that from DAPI staining
nucleus appears as blue signals (Ex: 340 nm; Em: 450−470 nm). Scale
bar, 30 μm.
AR Ligand Binding Affinity. The fluorescence polarization
technique was used to analyze the binding of L-1, 1−3, cisplatin,
cyanonilutamide, and bicalutamide to the androgen receptor using the
PolarScreen AR Competitor Assay, Green (lifetechnologies, A15880)
according to the manufacturer’s instructions. Briefly, the assay entails
titration of the test compound against a preformed complex of Fluormone
AL Green and the AR-LBD (GST). The assay mixture was allowed to
equilibrate at room temperature in 384-well black plates for 4 h, after
which the fluorescence polarization values were measured in a SpectraMax
Paradigm Multi-Mode Detection Platform (Molecular Devices) using
an excitation wavelength of 485 nm and an emission wavelength of
535 nm. Data analysis for the ligand binding assays was performed
using Prism software (GraphPad Software, Inc.).
UV−Vis Spectral test. The UV absorption of complexes 1−3 in
PBS containing 5% DMF at the concentration of 0.1 mM was recorded
on a Shimadzu UV2600 instrument equipped with a thermostatically
controlled cell holder. The wavelength range is 210−400 nm. All the
experiments were studied at 37 0.1 °C, the solvent absorption was
deducted as the background.
Determination of Quantum Yields. Quantum yields were deter-
mined at 25 °C, Rhodamine B (Φ = 0.59) in ethanol was used as a
standard. The absorption of Rhodamine B was adjusted to the same
value (abs < 0.1) as that of fluorescent molecules. Excitation was
chosen at 320 nm; the emission spectra were corrected and integrated
from 340 to 520 nm. The quantum yields were calculated with the
following equation: Φsample = Φstandard (Fsample/Fstandard) (Asample/Astandard).
Φ is the quantum yield, F is the integration of emission intensity, and
A is the absorbance value at excitation wavelength.
Preparation of Stock Solutions for Cellular Studies. Organic
compounds and Pt(IV) complexes were dissolved in DMF to a final
concentration of 20 mM and serially diluted prior to testing. The final
DMF concentration in culture medium did not exceed 0.4%. Stock
solution of cisplatin was prepared in water and diluted directly into
culture medium.
Cell Culture. PC-3 (Human prostatic cancer cell line, AR−) and
LNCaP (Human prostatic cancer cell line, AR+) were maintained in
the logarithmic phase at 37 °C in a 5% carbon dioxide atmosphere
using the following monolayer culture media containing 10% fetal bovine
serum (FBS), 100 μg/mL of penicillin, and 100 μg/mL of streptomycin.
Molecular Docking Analysis. All the docking studies were
carried out using Sybyl-X 2.0 on a Windows workstation. The initial
coordinates for AR were taken from the crystal structure of AR in
complex with testosterone (PDB: 2AM9).24,25 The AR-targeting
ligand L-1, cyanonilutamide and the endogenous steroid testosterone
were selected for the docking studies. The 3D structures of these
selected compounds were first built using Sybyl-X 2.0 sketch followed
by energy minimization using the MMFF94 force field and Gasteiger-
Marsili charges. We employed Powell’s method for optimizing the geom-
etry with a distance dependent dielectric constant and a termination
energy gradient of 0.005 kcal/mol. All the selected compounds were
automatically docked into the binding pocket of AR by an empirical
scoring function and a patented search engine in the Surflex docking
program. Before the docking process, the natural ligand was extracted;
the water molecules were removed from the crystal structure. Sub-
sequently, the protein was prepared by using the Biopolymer module
implemented in Sybyl. The polar hydrogen atoms were added. The
automated docking manner was applied in the present work.
Confocal Microscopy. Cellular localization of tested compounds
in LNCaP and PC-3 cells was determined by using confocal micro-
scopy. 0.5 × 106 cells were seeded on the 35 mm glass bottom dishes
Luciferase Assay. Assay of androgenic activity was performed by
means of ARE luciferase reporter assay using HEK 293T cells. Cells
were sown in a 150 cm2 flask (Corning), and cultured in culture
medium (DMEM medium containing 10% Dextran Charcoal (DCC)-
Fetal Bovine Serum (FBS), 2 mM glutamine) for 24 h pcDNA3.1-AR,
pRLSV40, and pMMTV-Luc vector containing luciferase gene bound
at the downstream of an AR promoter derived from Mouse Mammary
Tumor Virus (MMTV) were cotransfected by using Lipofectamine
2000. After culturing at 37 °C in a 5% CO2 atmosphere for 4 h, these
cells were harvested and plated in a 96 well plate (10000 cells/well)
and cultured for 2 h. A total of 24 h after addition of the sample (final
concentration, 10 μM) and 1 nM R1881, cells were harvested with
20 μL of cell passive lysis buffer (Promega), and the firefly and Renilla
luciferase activities were determined with a Dual Luciferase Asaay Kit
(Promega) by measuring luminescence with a Wallac Micro-Beta scintil-
lation counter (PerkinElmer Life Sciences). The data were obtained in
triplicate and expressed as inhibition rate over the R1881 control.
Inhibition% = 1 − (RLUtest − RLUblank)/(RLUR1881 − RLUblank) ×
100%. RLU = relative light unit.
Cytotoxicity Measurement. The in vitro activity of the syn-
thesized compounds was determined by the MTT method. The cul-
tured cells with better vitality were transferred to a 96-well plate so
that the density of the cells was 5000 per well, and they were incubated
overnight. Then, the compounds were dissolved in DMF and diluted
with medium to various concentrations (the final concentration of
DMF was less than 0.4%). After being incubated at 37 °C for 72 h,
cells were stained with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-
tetrazolium bromide (MTT; 5 mg/mL) for another 4 h, and then
dissolved with 150 μL of DMSO. The UV absorption intensity was
detected with an ELISA reader at 490 nm. The IC50 values were
calculated by SPSS software after three parallel experiments.
Cellular Uptake Test. LNCaP and PC-3 cells with good activity
were seeded in 6-well plates at 37 °C in 5% CO2. After the cell density
reached 80%, cisplain or complexes 1−3 was added to each well at a
concentration of 20 μM, and the plates were incubated for 12 h. Then,
cells were collected and washed three times with ice-cold PBS, fol-
lowed by centrifugation for 10 min and resuspension in PBS (1 mL).
Then, 100 μL of suspension was taken out for measuring the cell
density. The remaining cells were digested by HNO3 (200 μL, 65%) at
65 °C for 10 min. The results were measured by ICP-MS after three
parallel experiments.
Apoptosis Analysis. LNCaP cells were used in the apoptosis
experiment. Cisplatin was the positive control at a concentration of
20 μM. Specific operations were as follows: LNCaP cells with good
activity (5 × 105 cells per plate) were transferred to six-well plates and
cultured overnight in 5% CO2 at 37 °C. Drugs were added, which were
diluted to a concentration of 20 μM. After 24 h, the cells were digested
I
DOI: 10.1021/acs.inorgchem.8b00083
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
with trypsin and washed twice with cold PBS. Then, cells were
collected by centrifugation (2000 rpm, 5 min). After that, cells were
resuspended in binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM
CaCl2, pH 7.4) and incubated with annexin V-VAPC (100 ng/mL)
and then with propidium iodide (2 μg/mL) for 15 min in the dark at
room temperature. At last, the fluorescence of cells was detected by an
annexin V-APC/7-AAD apoptosis detection kit (Roche) according to
the manufacturer’s protocol, and cells were analyzed by a computer
station running Cell Quest software.
Cell Cycle Measurement. LNCaP cells with good vitality were
transferred into six-well plates, with a density of 10000 per well, and
cultured overnight at 37 °C. Then, 20 μM of the tested compounds
were incubated with cells for 24 h. All adherent and floating cells were
collected and washed twice with PBS. Then, the cells were fixed with
70% EtOH at 4 °C for 24 h. After that, fixed cells were washed with
PBS. After being centrifuged, cells were stained with 50 μg/mL pro-
pidium iodide solution containing 100 μg/mL RNase at 37 °C for 0.5 h.
The sample (at least 1 × 104 cells) was measured by flow cytometry
(FAC Scan, Becton Dickenson) using Cell Quest software and recording
propidium iodide (PI) in the FL2 channel.
Western Blot. The active preferred LNCaP cells were seeded until
the cell density reached 80%. Then 20 μM of the compounds were
added, and the cells were cultured for 12 h at 37 °C. Proteins were
extracted by lysis buffer. The concentration of protein was measured
by the BCA (bicinchoninic acid) assay with a Varioskan multimode
microplate spectrophotometer (Thermo, Waltham, MA). Then equal
amounts of protein (20 mg/Lane) were separated by 10% sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After
that, protein samples were transferred onto polyvinylidene difluoride
(PVDF) Immobilon-P membrane (Bio-Rad) with a transblot apparatus
(Bio-Rad). The blots, blocked with 5% nonfat milk in TBST (Tris-
buffered saline plus 0.1% Tween 20) for 1 h, were incubated with
primary antibodies diluted in PBST overnight at 4 °C. After that, the
membrane was washed with PBST three times and incubated with
IRDye 800 conjugated secondary antibody for 1 h at 37 °C. Detection
was performed by an Odyssey scanning system (Li-COR, Lincoln,
Nebraska).
completing the AR binding affinity and antagonist activity stud-
ies. We would like to express our gratitude to Prof. Hengshan
Wang (Guangxi Normal University) for a license to use their
software. We would like to thank LetPub (www.letpub.com)
for providing linguistic assistance during the preparation of this
manuscript.
ABBREVIATIONS
■
DMSO dimethyl sulfoxide; DCM dichloromethane; TEA
triethylamine; DMF N,N′-dimethylformamide; PBS phosphate
buffer solution; TBTU O-(benzotriazol-1-yl)-N,N,N′,N′-tetra-
methyluronium tetrafluoroborate; ICP-MS inductively coupled
plasma mass spectrometry; VC ascorbic acid; CV cyclic
voltammetry; AR androgen receptor
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ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acs.inorgchem.8b00083.
1
Diagrams of ESI-MS, H, 13C and 195Pt NMR spectra.
Cyclic voltammograms of complexes 1−3. HPLC ana-
lyses on the stability and the released ability of Pt(IV)
complexes under reduction with ascorbic acid (PDF).
́
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Corresponding Author
*E-mail: sgou@seu.edu.cn.
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Shaohua Gou: 0000-0003-0284-5480
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§These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
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The authors are grateful to the National Natural Science Foun-
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DOI: 10.1021/acs.inorgchem.8b00083
Inorg. Chem. XXXX, XXX, XXX−XXX