Second-generation testosterone-platinum(II) hybrids for site-specific treatment of androgen receptor positive prostate cancer: Design, synthesis and antiproliferative activity

Article abstract of DOI:10.1016/j.ejmech.2019.06.090

Prostate cancer is the most diagnosed type of cancer in men in Canada. One out of eight men will be stricken with this disease during the course of his life. It is noteworthy that, at initial diagnoses 80–90% of cancers are androgen dependent. Hence, the androgen receptor is a viable biological target to be considered for drug targeting. We have developed a new generation of testosterone-Pt(II) hybrids for site-specific treatment of hormone-dependent prostate cancer. The hybrid molecules are made from testosterone using an eight-step reaction sequence with about 7% overall yield. They are linked with a stronger tether chain between the testosterone moiety and the Pt(II) moiety in comparison to our first generation hybrids. The new hybrids were tested on hormone-dependent and –independent prostate cancer cell lines. The hybrid 3a presents the best antiproliferative activity and was selective on hormone-dependent prostate cancer with IC₅₀ of 2.2 μM on LNCaP (AR+) in comparison to 13.3 μM on PC3 (AR-) and 8.8 μM on DU145 (AR-) prostate cancer cells. On the same cell lines, CDDP displayed IC₅₀ of 2.1 μM, 0.5 μM and 1.0 μM, respectively. Remarkably, hybrid 3a was inactive on both colon carcinoma (HT-29) and normal human adult keratinocyte cells (HaCat) with an IC₅₀ of >25 μM. This is not the case for CDDP showing IC₅₀ of 1.3 μM and 5.1 μM on HT-29 and HaCat cells, respectively. The potential for selective activity on androgen-receptor positive prostate cancer cells is confirmed with hybrid 3a giving new hope for an efficient and less toxic platinum-based treatment of prostate cancer patients.

Full text of DOI:10.1016/j.ejmech.2019.06.090

Accepted Manuscript
Second-generation testosterone-platinum(II) hybrids for site-specific treatment of
androgen receptor positive prostate cancer: Design, synthesis and antiproliferative
activity
Vincent Ouellette, Marie-France Côté, René C. Gaudreault, Heidar-Ali Tajmir-Riahi,
Gervais Bérubé
PII:
S0223-5234(19)30617-8
DOI:
https://doi.org/10.1016/j.ejmech.2019.06.090
EJMECH 11493
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 May 2019
Revised Date: 13 June 2019
Accepted Date: 28 June 2019
Please cite this article as: V. Ouellette, M.-F. Côté, René.C. Gaudreault, H.-A. Tajmir-Riahi, G. Bérubé,
Second-generation testosterone-platinum(II) hybrids for site-specific treatment of androgen receptor
positive prostate cancer: Design, synthesis and antiproliferative activity, European Journal of Medicinal
Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.06.090.
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ACCEPTED MANUSCRIPT
Graphical abstract
Two types of testosterone-platinum(II) complexes (compounds 3a and 3b,c) were made
and tested for their antiproliferative activity. Hybrid 3a was as active as cisplatin and
showed selectivity on androgen-dependent prostate cancer cells LNCaP.
Cl-
+
OH
OH
O
N
N
O
N
N
NH
Pt
n
N
N
N
Pt
Cl Cl
N
3
a
3b, n = 1
c, n = 2
3
Cl

ACCEPTED MANUSCRIPT
Second-generation testosterone-platinum(II) hybrids for site-specific treatment of androgen
receptor positive prostate cancer: Design, synthesis and antiproliferative activity
a
b
b
a
Vincent Ouellette , Marie-France Côté , René C.-Gaudreault , Heidar-Ali Tajmir-Riahi , and
a,
Gervais Bérubé *
a
Département de Chimie, Biochimie et Physique, Université du Québec à Trois-Rivières, C.P.
b
5
00, Trois-Rivières, Québec, Canada G9A 5H7 and Axe oncologie, Centre de recherche du
CHU de Québec-Université Laval, Hôpital Saint-François d'Assise, 10, rue de l'Espinay, Québec,
Québec, Canada G1L 3L5.
e-mail: Vincent.Ouellette@uqtr.ca, Marie-France.Cote@crchudequebec.ulaval.ca, Rene.C-
Gaudreault@crchudequebec.ulaval.ca,
Gervais.Berube@uqtr.ca.
Heidar-Ali.Tajmir-Riahi@uqtr.ca,
*
Corresponding authors: Dr Gervais Bérubé; phone: 819-376-5011 ext. 3353, fax: 819-376-
084, e-mail: Gervais.Berube@uqtr.ca.
5
Abbreviation list: T, testosterone; T-Pt(II), testosterone-platinum(II) hybrid; PCa, prostate
cancer; AR, androgen receptor.
1

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Abstract
Prostate cancer is the most diagnosed type of cancer in men in Canada. One out of eight men will
be stricken with this disease during the course of his life. It is noteworthy that, at initial diagnoses
8
0-90% of cancers are androgen dependent. Hence, the androgen receptor is a viable biological
target to be considered for drug targeting. We have developed a new generation of testosterone-
Pt(II) hybrids for site-specific treatment of hormone-dependent prostate cancer. The hybrid
molecules are made from testosterone using an eight-step reaction sequence with about 7%
overall yield. They are linked with a stronger tether chain between the testosterone moiety and
the Pt(II) moiety in comparison to our first generation hybrids. The new hybrids were tested on
hormone-dependent and –independent prostate cancer cell lines. The hybrid 3a presents the best
antiproliferative activity and was selective on hormone-dependent prostate cancer with IC of
5
0
2
.2 µM on LNCaP (AR+) in comparison to 13.3 µM on PC3 (AR-) and 8.8 µM on DU145 (AR-)
prostate cancer cells. On the same cell lines, CDDP displayed IC50 of 2.1 µM, 0.5 µM and 1.0
µM, respectively. Remarkably, hybrid 3a was inactive on both colon carcinoma (HT-29) and
normal human adult keratinocyte cells (HaCat) with an IC of > 25 µM. This is not the case for
5
0
CDDP showing IC50 of 1.3 µM and 5.1 µM on HT-29 and HaCat cells, respectively. The
potential for selective activity on androgen-receptor positive prostate cancer cells is confirmed
with hybrid 3a giving new hope for an efficient and less toxic platinum-based treatment of
prostate cancer patients.
Keywords. testosterone; prostate cancer; SRB assay; hybrid anticancer agent; drug-targeting.
2

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1
. Introduction
Cancer is a difficult disease to cure if not detected and treated at its early stages. It is the second
leading cause of death globally, and is responsible for an estimated 9.6 million deaths in 2018
[1]. Worldwide, about 1 in 6 deaths is due to cancer. Prostate cancer (PCa) is a major health
problem afflicting men with an estimate of 1.28 million cases worldwide [1]. In fact, PCa is the
fourth most frequent cancer in the world with 7.1% of all cases, after lung (11.6%), breast
(11.6%), and colorectal (10.2%) cancers. In Canada, PCa is estimated to be ranked first among all
cancer cases diagnosed in the Canadian men with about 21,300 new cases [2]. The projected
mortality for PCa is about 4,100 cases and is the third most lethal cancer in men [2]. Most PCa
(74%) are diagnosed at stage I or II and are generally hormone-dependent [2,3]. Considering the
latter fact, it is theoretically possible to use the androgen receptor (AR) as a biological target to
direct an anticancer drug to PCa cells.
Today, despite the use of innovative anticancer drugs there is still a need to improve selectivity,
efficacy and, at the same time to reduce the many incapacitating side effects of cancer treatments.
Hence, there is great scientific interest in using hybrid molecules to combat several types of
diseases, including cancer. One can appreciate the power of hybridization technology used during
many drug discovery projects through recent reviews on the subject [4-9].
We have investigated the use of testosterone as a carrier molecule united to different DNA
alkylating agents [10,11]. Two types of testosterone-anticancer agent hybrids were made using
either a nitrogen-mustard or a platinum(II) complexes. For example, the testosterone-
3

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chlorambucil hybrid 1 was made and tested in our laboratory with the goal of expanding the use
®
of chlorambucil to PCa [10]. Indeed, chlroambucil (Leukeran ) is a long-standing anticancer
drug that is still in use for the treatment certain cancers such as leukemias and lymphomas. The
hybrid 1 was as active as chlorambucil and displayed selectivity on hormone-dependent PCa cells
[10]. We also developed a first generation of testosterone-platinum(II) hybrid molecules for the
treatment of PCa [11]. It was discovered that the thiazolylalanine hybrid 2 possesses the best in
vitro antiproliferative activity with IC of 1.8 µM and 1.4 µM on LNCaP (AR+) and PC3 (AR-)
5
0
cancer cells, respectively. Cisplatin, the reference drug was 3.7 and 5.3 times less potent on the
same cell lines [11]. Encouraged by these results, a second-generation testosterone-platinum
hybrids 3 was investigated and the results are presented in this manuscript (see Fig. 1). For this
second generation of hybrids, it was decided to use a 1,2,3-triazole function (3) instead of an ester
function (1 and 2) to bind the steroid moiety with the platinum(II) moiety in order to obtain
chemically stable hybrids for subsequent in vivo biological assays. In earlier studies, we
investigated using multiple spectroscopic methods the interactions of the hybrid 3a and its
precursor 6a with several bio-macromolecules (see Scheme 1). The first study showed that both
compounds interact with serum proteins (HAS, BSA and β-lactoglobulin) via ionic contacts with
BSA forming more stable conjugates than HSA and β-LG [12]. Their interaction with DNA was
also studied in our laboratory. Likewise, the drug-DNA conjugation occurs via ionic interactions
5
−1
with 3a forming more stable DNA adducts than 6a with K6a-DNA =1.80 (±0.5) x 10 M and K3a-
5
−1
DNA = 2.3 (±0.8) x 10 M [13]. Finally, it was observed that the interaction with tRNA was also
through ionic interactions with 3a forming more stable tRNA complexes in comparison to 6a:
5
-1
5
-1
K3a-tRNA = 3.2 (± 0.9) x 10 M > K
= 2.1 (± 0.7) x 10 M [14]. This manuscript describes
6a-tRNA
4

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the design, synthesis and characterization of the second-generation testosterone-platinum(II)
hybrid. It also presents their antiproliferative activity on several human cancer cell lines.
Insert Fig. 1 and its legend
2
. Design and chemistry
Testosterone was selected as the carrier molecule of the hybrids based on its inherent affinity for
prostate tissue, to optimize the pharmacokinetics and the pharmacodynamics of testosterone
towards AR and to allow the activation of AR signaling pathways. As in our earlier work, the 7α
position of testosterone was used as it is nicely located at the midway of the important binding
sites (3-C=O and 17-OH groups) involved in essential hydrogen bonds with AR. It is important
that these two key groups remain free of steric hindrances to promote strong interactions with AR
allowing its activation. As aforementioned, it was decided to construct the new hybrids using a
1
,2,3-triazole ring system allowing easy access to the final hybrid molecules via the azide-alkyne
cycloaddition coupling reaction [15,16].
The synthetic methodology for the preparation of the second-generation hybrids is described in
Scheme 1. Derivative 4 was prepared in good yield via the intermediate 7α-allyl-4-androstene-
1
7β-ol-3-one acetate using a five-step reaction sequence as reported by Bastien et al. [10]. Next,
a simple substitution of the chloride atom using sodium azide gave derivative 5 with 92% yield.
This derivative is now ready for the 1,3-dipolar Huisgen cycloaddition with an appropriate
alkyne. So, upon treatment of azide 5 with commercially available 2-ethynylpyridine (7a),
5

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sodium carbonate and copper sulfate in a mixture of tetrahydrofuran and water, the 1,2,3-triazole-
pyridine ligand 6a was obtained with 54% yield. The cycloaddition reaction was performed with
two additional alkynes, derivatives 7b and 7c. These amino-pyridine alkynes were obtained by
alkylation of picolylamine and 2-(2-aminoethyl)pyridine with propargyl bromide following a
known procedure (Scheme 2) [17]. Compounds 7b and 7c were obtained with 50% and 46%
yield, respectively. The Huisgen cycloaddition reaction with azide 5 and the relevant alkyne (7b
and 7c) gave the 1,2,3-triazole-aminopyridine ligands 6b and 6c with 29% and 60% yield,
respectively. Finally, complexation of derivatives 6a-c with potassium tetrachloroplatinate(II) in
a mixture of dimethylformamide (DMF) and water led to the testosterone-platinum(II) hybrids
(compounds 3a-c). As expected, the hybrid 3a (34%) is bound to the bidentate ligand 6a and the
hybrids 3b (77%) and 3c (60%) are obtained with tridentate ligands 6b and 6c, respectively. All
compounds were characterized by their respective infrared (IR), nuclear magnetic resonance
spectroscopy (proton and carbon NMR) and by high resolution mass analysis.
Insert scheme 1 and its legend
Insert scheme 2 and tis legend
3
3
. Results and discussion
.1 Antiproliferative activity on prostate cancer cell lines
The antiproliferative activity of compounds 3a-c and 6a-c was assessed using the sulforhodamine
B colorimetric test on three PCa cell lines: LNCaP (AR+), PC3 (AR-) and DU145 (AR-) [18,19].
6

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Also, to further verify their biological potential, our products were tested on two additional cell
lines: on colon carcinoma (HT-29) and on primary human immortalized keratinocyte cells
(HaCat). CDDP was used as a positive control drug in order to better appreciate and compare the
biological activity of the synthesized compounds. The results from the antiproliferative activity
assay are summarized in Table 1. IC is the concentration of drug inhibiting cell growth by
5
0
5
0%.
The ligands and platinum complexes were dissolved in DMF at an initial concentration of 25 µM.
This concentration was selected as the platinum derivatives 3b and 3c were not completely
soluble at higher concentrations. The solubility of the platinum complex 3a was much better. One
factor contributing to the solubility of these molecules is their chemical structure that are quite
different from one another. Indeed, platinum(II) complex 3a is obtained with a bidentate ligand
while complexes 3b and 3c are coordinated with tridentate ligands. The later complexes are
charged positively while the complex 3a is neutral. The literature describes the synthesis
estradiol-platinum(II) complexes bonded to a tridentate ligand (2,2’:6’,2’’-terpyridine) displaying
similar low solubility [20].
The results show that the ligand precursors 6a-c are active on all cell tested at the exception of
ligand 6c which is inactive on AR+ PCa cells displaying a IC50 > 25 µM. The IC50 for the
precursor ligands ranged from 6.3 to 20 µM (Table 1). There is no selective activity on any type
of cancer cells tested in the panel. This in itself is an interesting result. It is important to note that
platinum(II) complex 3a displays higher activity in comparison to its precursor ligand 6a on only
7

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two types of cells: LNCaP: 2.2 µM vs 6.3 µM, and DU145: 8.8 µM vs 12.3 µM. Derivative 3a
and 6a have similar activity on PC3 cells: 13.3 µM vs 11.0 µM. Hence, the precursor ligands are
more broadly active than the final platinum complexes. This observation can be explained by the
fact that compounds bearing a 1,2,3-triazole ring system possess an intrinsic biological activity
being used in some cases as anticancer agents [21].
Intriguing results were observed with the platinum complexes. Indeed, complexes 3b and 3c were
inactive on all cells tested showing an IC50 > 25 µM in each case. These results can be explained
by two main factors; the low solubility of the complexes and the unsuitable complexation pattern
obtained with a tridentate ligand. On the other hand, the classic platinum(II) complex 3a displays
very interesting antiproliferative activities. Indeed, it possesses an IC50 of 2.2 µM which is
essentially equal to that of CDDP displaying an IC of 2.1 µM on AR+ PCa cell LNCaP. Even
50
more interesting, complex 3a shows selectivity towards the AR+ PCa cell line. In fact, it is about
to 6 times more potent on LNCaP cell in comparison to the AR- PCa cells: DU145 (IC50 of 8.8
µM) and PC3 (13.3 µM). On the latter two types of cells, CDDP showed an IC of 1.0 µM and
4
5
0
0
.5 µM, respectively. Hence, no selectivity was observed with CDDP on any type of cells, its
inhibitory concentration varying from 0.5 to 5.1 µM on the cell tested. It is worth noting that, not
only the testosterone-platinum(II) complex 3a is selective on hormone-dependent PCa cells but,
it also displays low activity on primary human immortalized keratinocyte cells (HaCat) as well as
on the colon carcinoma (HT-29) with IC50 > 25 µM in both cases.
8

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3
. Conclusion
In this study, we prepared and characterized several testosterone-platinum(II) hybrid molecules
designed for the treatment of PCa. Two types of hybrids were made: hybrid 3a constructed with a
bidentate ligand and two hybrids 3b and 3c synthesized with tridentate ligands. They were
prepared by efficient transformation of testosterone into an azide derivative 5 (37% overall)
which was reacted with pyridine-alkyne derivatives via a cycloaddition reaction to give the
precursor 1,2,3-triazole ligands 6a-c with 48% average yield. Complexation occurred upon
treatment with potassium tetrachloroplatinate to give the final hybrids 3a-c with 57% average
yield. Antiproliferative assays showed that the ligands possess intrinsic biological activities on
the cells tested exhibiting IC ranging from 6.3 to 20 µM at the exception of ligand 6c with IC
5
0
50
>
25 µM on LNCaP cells. Unfortunately, the platinum(II) complexes 3b and 3c had low
solubility and were found to be completely inactive at the maximum dose tested. However, the
platinum(II) complex 3a exhibited promising in vitro selectivity on AR+ PCa cells LNCaP, being
from 4 and 6 times more active than on the AR- DU145 and PC3 cancer cells. Furthermore, this
hybrid was inactive on primary human immortalized keratinocyte cells (HaCaT) as well as on
colon carcinoma (HT-29). Despite the fact that the hybrids 3b and 3c were inactive, the precursor
ligands give surprising results and could potentially be used as antiandrogens. Further study are
needed to confirm this hypothesis. In conclusion, it is recommended to design classic
platinum(II) hybrid molecules such as 3a in order to obtain products with significant
antiproliferative activity and selectivity on AR+ PCa.
4
. Experimental protocols
9

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4
.1 Biological Methods
The antiproliferative activity assay of all compounds was assessed using the procedure
recommended by the National Cancer Institute for its drug-screening program with minor
modifications [22]. LNCaP androgen-sensitive human prostate adenocarcinoma, PC3 androgen-
insensitive human prostate adenocarcinoma, DU145 androgen-insensitive human prostate
adenocarcinoma, HT-29 human colon carcinoma and HaCaT primary human immortalized
keratinocyte cells were purchased from the American Type Culture Collection (Manassas, VA).
LNCap, PC3, DU145 and HT29 were cultured in RPMI medium (Hyclone, Logan, UT)
supplemented with 10% of calf serum and Penicillin-Streptomycin-Glutamine. HaCat were
cultured in DMEM medium supplemented with 10% FBS and Penicillin-Steptomycin-Glutamine.
The cells were maintained at 37 °C in a moisture saturated atmosphere containing 5% CO2.
4
.1.1 Antiproliferative activity assay
The antiproliferative activity of compounds 3a-c, 6a-c and CDDP was assessed using the SRB
assay [18,19]. Briefly, the 96-well microtiter plates were seeded with 75 µL of cells suspended
3
3
3
3
3
(
LNCaP, 12 x 10 ; PC3, 6 x 10 ; DU 145, 6 x 10 ; HT-29, 3 x 10 ; HaCat, 5 x 10 ) in medium.
Plates were incubated at 37 °C, 5% CO for 24 h. Freshly solubilized drugs in DMF were diluted
2
in fresh medium, and 75 µL aliquots containing serially diluted concentrations of the drug were
added and the plates were incubated for 72 h. Final drug concentrations ranged from 25 µM to
0
.780 µM. The final concentration of DMF in the culture media was 0.1% and was kept constant
in all experiments. Plates containing attached cells were then stained with sulforhodamine B to
determine the number of viable cells. Briefly, cells were fixed by addition of cold trichloroacetic
acid to the wells (10% (w/v) final concentration), for 30 minutes at 4 °C. Plates were washed five
1
0

ACCEPTED MANUSCRIPT
times with tap water and dried. Sulforhodamine B solution (75 µL) at 0.1% (w/v) in 1% acetic
acid was added to each well, and the plates were incubated for 15 minutes at room temperature.
Unbound dye was removed by washing five times with 1% acetic acid. Bonded dye was
solubilized in 10 mM Tris base, and the absorbance was read using a µQuant Universal
microplate spectrophotometer (Biotek, Winooski, VT) at a wavelength between 530 and 565 nm
according to color intensity. Readings obtained from treated cells were compared with
measurements from control cells plates fixed on the treatment day, and the percentage of cell
growth inhibition was then calculated for each drug. The experiments were performed three times
in triplicate at the exception of the LNCaP cells which was performed once in triplicate. The
assays were considered valid when the coefficient of variation for a given set of conditions and
within the same experiment was < 10%.
4
.2 Chemistry
Anhydrous reactions were performed under an inert atmosphere of nitrogen. The starting
material, reactant and solvents were obtained commercially and were used as such or purified and
dried by standard means [23]. Organic solutions were dried over magnesium sulfate (MgSO4),
filtered and evaporated on a rotary evaporator under reduced pressure. All reactions were
monitored by UV fluorescence. Commercial TLC plates were Sigma T 6145 (polyester silica gel
6
0Ǻ, 0.25mm). Flash column chromatography was performed according to the method of Still et
al. on Merck grade 60 silica gel, 230-400 mesh [24]. All solvents used in chromatography were
distilled prior to use.
1
1

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The infrared spectra were taken on a Nicolet Impact 420 FT-IR spectrophotometer. Mass spectral
assays were obtained using a MS model 6210, Agilent technology instrument. The high
resolution mass spectra (HRMS) were obtained by TOF (time of flight) using ESI (electrospray
ionization) using the positive mode (ESI+) (Université du Québec à Montréal and at Université
Laval). Nuclear magnetic resonance (NMR) spectra were recorded on a Varian 200 MHz NMR
apparatus. Samples were dissolved in deuterated chloroform (CDCl ) or in the cases of the
3
platinum complexes in a mixture of deuterated chloroform and methanol (CDCl + CD OD ≈ 2
3
3
drops) for data acquisition using the residual solvent signal as internal standard (chloroform, δ
1
13
7
.26 ppm for H NMR and δ 77.23 ppm for C NMR). Chemical shifts (δ) are expressed in parts
per million (ppm), the coupling constants (J) are expressed in hertz (Hz). Multiplicities are
described by the following abbreviations: s for singlet, d for doublet, t for triplet and m for
multiplet and, bs for broad singlet.
Note: The nomenclature of the testosterone derivatives reported herein was based on the
androgen skeleton (4-androsten-17β-ol-3-one) for clarity to the readers. This should facilitate the
visualization of the molecular structures, particularly for researchers working with steroids.
4
.2.1 Synthesis of testosterone-platinum(II) hybrids
4
.2.1.1 Synthesis of 7α-(4-chloro-but-2-enyl)-4-androsten-17β-ol-3-one (4)
The 7α-allyl-4-androstene-17β-ol-3-one acetate (842 mg, 2.01 mmol, 1 eq.) is dissolved in
methanol (16 mL) and the solution is stirred. 5 N HCl solution (5 mL) is added to the mixture and
stirred for 48 hours at room temperature. The solution is then diluted with ether (50 mL) and the
1
2

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organic layer is then washed with saturated NaHCO solution (50 mL). The organic layer is then
3
washed 3 times with distilled water (25 mL), dried over MgSO
4
, filtered and evaporated. The
product is purified by flash chromatography (hexane/acetone, 8:2) to give 640 mg of 4. Yield, 95
-1
%
; white solid; mp: 142 – 145 °C. IR ν (cm ): 3423 (O-H), 1669 (C=O, enone), 1614 (C=C),
1
1
2
1
250 (C-O). H NMR (200 MHz, CDCl
3
, δ ppm): 5.67 (1H, s, 4-CH), 5.56 (2H, m, 21-CH and
2-CH), 3.98 (2H, m, 23-CH ), 3.62 (1H, t, J=8.2 Hz, 17-CH), 1.17 (3H, s, 19-CH ), 0.76 (3H, s,
2
3
8-CH3).
4
.2.1.2 Synthesis of 7
α
-(4-azido-but-2-enyl)-4-androsten-17β-ol-3-one (5)
Steroid 4 (218 mg, 0.58 mmol, 1 eq.) is dissolved in methanol (10 mL). Sodium azide (226 mg,
.47 mmol, 6 eq.) is added to the solution, stirred and heated (reflux) for 2 hours. Let the mixture
3
cool down to room temperature. A liquid-liquid extraction is performed with a mix of ether and
ether (8:2). The organic layer is extracted 3 portions of water (25 mL) and one portion of
saturated NaCl solution (25 mL). The organic phase is then dried over MgSO , filtered and
4
evaporated under pressure to give 204 mg of the desired material. No purification needed for this
-1
compound. Yield, 92 %; beige solid; mp: 139 – 143 °C. IR ν (cm ): 3432 (O-H), 2945, 2915 and
1
2
873 (C-H), 2094 and 2073 (N=N=N), 1653 (C=O), 1617 (C=C), 1239 (C-O), 875 (C-Cl). H
NMR (200 MHz, CDCl
3
, δ ppm): 5.72 (1H, s, 4-CH), 5.56 (2H, m, 21-CH and 22-CH), 3.67 (3H,
1
3
m, 23-CH and 17-CH), 1.21 (3H, s, 19-CH ), 0.80 (3H, s, 18-CH ). C NMR (200 MHz, CDCl ,
2
3
3
3
δ ppm): 199.1 (C-3), 169.1 (C-5), 134.9 (C-21), 126.2 (C-22), 125.1 (C-4), 81.6 (C-17), 52.6,
4
7.2, 46.2, 42.9 (C-23), 38.9, 38.8, 36.9, 36.5, 36.4, 36.2, 34.2, 30.5, 28.8, 23.0, 21.1, 18.2, 11.2.
+
HRMS (ESI+): (M+H) calculated for C23
H
34
N
3
O
2
= 384.2646; found : 384.2662.
1
3

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4
.2.1.3 Synthesis of prop-2-ynyl-pyridin-2-ylmethylamine (7b)
2
2 3
-picolylamine (1.0 mL, 9.70 mmol, 2 eq.) and K CO (1.34 g, 9.70 mmol, 2 eq.) are dissolved in
CH CN (5 mL) and stirred for 10 minutes. Propargyl bromide (430 µL, 4.85 mmol, 1 eq.), diluted
3
in CH CN (25 mL), is added dropwise to the mixture over 2 hours and then stirred for 16 hours at
3
room temperature. The reaction is then filtered to eliminate K CO and the solvent is evaporated.
2
3
The product is purified by flash chromatography (DCM/methanol/TEA, 100:5:1) to give 7b (355
-1
mg). Yield, 50 %; orange oil. IR ν (cm ): 3293 (NH secondary), 1674, 1590, 1569, 1474, 1433,
1
1
357, 1148, 1121. H NMR (200 MHz, CDCl , δ ppm): 8.55 (1H, d, J=4.7 Hz, a-CH), 7.62 (1H,
3
td, J=7.8 Hz 1.8 Hz, c-CH), 7.30 (1H, d, J=7.8 Hz, d-CH), 7.14 (1H, m, b-CH), 3.98 (2H, s, pyr-
CH -NH-CH ), 3.48 (2H, d, J= 2.3 Hz, pyr-CH -NH-CH ), 2.23 (1H, t, J=2.5 Hz, CH alkyne),
2
2
2
2
1
3
2
7
3
.12 (1H, bs, NH). C NMR (200 MHz, CDCl , δ ppm): 159.0, 149.3, 136.4, 122.4, 122.0, 81.8,
+
1.7, 53.6, 37.7. HRMS (ESI+): (M+H) calculated for C H N = 147.0917; found : 147.0920.
9
11
2
4
.2.1.4 Synthesis of prop-2-ynyl-pyridin-2-ylethylamine (7c)
-(2-aminoethyl)pyridine (1.0 mL, 8.40 mmol, 2 eq.) and K
2
2
3
CO (1.16 g, 8.41 mmol, 2 eq.) are
dissolved in CH CN (5 mL) and stirred for 10 minutes. Propargyl bromide (370 µL, 4.2 mmol, 1
3
eq.), diluted in CH CN (25 mL), is added dropwise to the mixture over 2 hours and then stirred
3
for 16 hours at room temperature. The reaction is then filtered to eliminate K CO and the solvent
2
3
is evaporated. The product is purified by flash chromatography (DCM/methanol/ Et N, 100:5:1)
3
-1
to give 7c (309 mg). Yield, 40 %; orange oil. IR ν (cm ): 3294 (NH), 1674, 1591, 1568, 1474,
1
1
433, 1116, 749, 629. H NMR (200 MHz, CDCl , δ ppm): 8.51 (1H, d, J=4.7 Hz, a-CH), 7.59
3
(
1H, td, J=7.8 Hz and 2.0 Hz, c-CH), 7.17 (1H, d, J=7.8 Hz, d-CH), 7.10 (1H, m, b-CH), 3.44
2H, d, J=2.7 Hz, CH CH -NH-CH ), 3.10 (2H, m, CH CH -NH-CH ), 2.96 (2H, m, CH CH -
(
2
2
2
2
2
2
2
2
1
4

ACCEPTED MANUSCRIPT
1
3
NH-CH
2
), 2.18 (1H, t, J=2.5 Hz, CH alkyne), 1.81 (1H, bs, NH). C NMR (200 MHz, CDCl
3
, δ
+
ppm): 159.9, 149.2, 136.3, 123.2, 121.3, 82.0, 71.4, 48.0, 38.0 (2 CH ). HRMS (ESI+): (M+H)
2
calculated for C H N = 161.1073; found : 161.1077.
1
0
13
2
4
.2.1.5 Synthesis of 7
α-[4-(4-pyridin-2-yl-[1,2,3]triazol-1-yl)-but-2-enyl]-4-androsten-17β-ol-3-
one (6a)
Azide 5 (84 mg, 0.22 mmol, 1 eq.) and 2-ethynylpyridine (9a) (37 µL, 0.37 mmol, 1.7 eq.) are
added to a mixture of THF/water (3:1, 6 mL) and stirred. Then, sodium ascorbate (0.55 mmol,
2
4 2
.5 eq.) and CuSO • 5 H O (16.4 mg, 0.07 mmol, 0.3 eq.) are added and the mixture is stirred at
room temperature for 16 hours. The product is extracted with EtOAc (50 mL) and washed thrice
with water (3 x 30 mL). The organic phase is dried over MgSO , filtered and evaporated under
4
reduced pressure. The crude product is purified by flash chromatography (hexanes/acetone, 7:3)
-1
to give 6a (57 mg). Yield, 54 %; brownish oil. IR ν (cm ): 3391 (O-H), 3150 (C-H triazole),
1
2
944 (C-H), 1610 (C=O), 1570 (C=N), 1436, 1355, 1225, 950, 785. H NMR (200 MHz, CDCl ,
3
δ ppm): 8.56 (1H, d, J=5.2 Hz, a-CH), 8.17 (1H, d, J=7.8 Hz, d-CH), 8.13 (1H, s, 24-CH-
triazole), 7.77 (1H, td, J=7.8 Hz and 1.6 Hz, c-CH), 7.21 (1H, m, b-CH), 5.68 (3H, bs, 4-CH, 21-
CH and 22-CH), 4.98 (2H, m, 23-CH ), 3.65 (1H, t, J=8.0 Hz, 17-CH), 1.20 (3H, s, 19-CH ),
2
3
1
3
0
1
3
.79 (3H, s, 18-CH
3
). C NMR (200 MHz, CDCl
3
, δ ppm): 198.9, 168.9, 150.2, 149.3, 148.5,
36.9, 135.5, 126.1, 124.9, 122.9, 121.7, 120.3, 81.5, 52.1, 47.1, 46.2, 42.9, 38.7, 38.5, 36.3,
+
6.2, 36.1, 35.9, 33.9, 30.2, 28.6, 22.8, 20.8, 17.9, 10.9. HRMS (ESI+): (M+H) calculated for
C H N O = 487.3068; found : 487.3080.
3
0
39
4
2
4
.2.1.6 Synthesis of 7α-[4-(4-(((pyridin-2-ylmethyl)-amino)-methyl)-[1,2,3]-triazol-1-yl)-but-2-
enyl]-4-androsten-17β-ol-3-one (6b)
1
5

ACCEPTED MANUSCRIPT
Azide 5 (55 mg, 0.14 mmol, 1 eq.) and alkyne 7b (42 mg, 0.29 mmol, 2 eq.) are added to a
mixture of THF/water (1:1, 1.3 mL) and stirred. Then, sodium ascorbate (5.7 mg, 0.029 mmol,
0
4 2
.2 eq.) and CuSO • 5 H O (3.6 mg, 0.015 mmol, 0.1 eq.) are added and the mixture is stirred at
room temperature for 16 hours. The product is extracted with EtOAc (50 mL) and washed twice
with saturated NaCl solution (2 x 30 mL). Aqueous phases are combined and reextracted with
EtOAc. Organic phases are combined and dried over MgSO , filtered and evaporated under
4
reduced pressure. The crude product is purified by flash chromatography (DCM/methanol/Et N,
3
-
1
1
2
00:5:1) to give 6b (22 mg). Yield, 29 %; orange oil. IR ν (cm ): 3304 (O-H), 2939 (C-H), 2094,
1
073, 1654 (C=O), 1611 (C=C). H NMR (200 MHz, CDCl
3
, δ ppm): 8.54 (1H, m, a-CH), 7.65
(1H, m, c-CH), 7.56 (1H, s, 24-CH), 7.35 (1H, m, d-CH), 7.18 (1H, m, b-CH), 5.60 (3H, bs, 4-
CH, 21-CH and 22-CH), 4.89 (2H, m, 23-CH ), 4.03 (4H, m, 26-CH and 27-CH ), 3.64 (1H, t,
2
2
2
1
3
J=8.2 Hz, 17-CH), 1.19 (3H, s, 19-CH ), 0.79 (3H, s, 18-CH ). C NMR (200 MHz, CDCl ,
3
3
3
δ ppm): 199.0, 169.0, 158.7, 149.3, 146.4, 136.6, 135.2, 126.1, 125.1, 122.5, 122.2, 121.7, 81.5,
5
1
1.9, 47.1, 46.1, 44.1, 42.9, 38.7, 38.5, 36.4, 36.2, 36.0, 35.9, 33.9, 30.2, 29.7, 28.8, 22.8, 20.8,
+
7.9, 10.9. HRMS (ESI+): (M+H) calculated for C H N O = 530.3490; found : 530.3505.
32
44
5
2
4
.2.1.7 Synthesis of 7α-[4-(4-((2-pyridin-2-yl-ethylamino)-methyl)-[1,2,3]-triazol-1-yl)-but-2-
-ol-3-one (6c)
enyl]-4-androsten-17
β
Azide 5 (56 mg, 0.15 mmol, 1 eq.) and alkyne 7c (25 mg, 0.16 mmol, 1.1 eq.) are added to a
mixture of t-BuOH/water (1:1, 2 mL) and stirred. Then, sodium ascorbate (5.8 mg, 0.029 mmol,
0
.2 eq.) and CuSO • 5 H O (3.6 mg, 0.015 mmol, 0.1 eq.) are added and the mixture is stirred at
4 2
room temperature for 16 hours. The product is extracted with EtOAc (50 mL) and washed twice
with saturated NaCl solution (2 x 30 mL). Aqueous phases are combined and reextracted with
1
6

ACCEPTED MANUSCRIPT
EtOAc. Organic phases are combined and dried over MgSO , filtered and evaporated under
4
3
reduced pressure. The crude product is purified by flash chromatography (DCM/methanol/Et N,
-
1
1
2
00:5:1) to give 48 mg of 6c. Yield, 60 %; light brown oil. IR ν (cm ): 3306 (O-H and N-H),
1
936 (C-H), 1659 (C=O), 1613 (C=C), 1592, 1374, 1222, 1138, 1051, 750. H NMR (200 MHz,
CDCl , δ ppm): 8.47 (1H, d, J=4.7 Hz, a-CH), 7.56 (1H, td, J=7.8 Hz and 1.8 Hz, c-CH), 7.48
3
(
1H, s, 24-CH-triazole), 7.10 (2H, m, b-CH and d-CH), 5.58 (3H, bs, 4-CH, 21-CH and 22-CH),
.85 (2H, m, 23-CH ), 3.93 (2H, s, 26-CH ), 3.61 (1H, t, J=8.2 Hz, 17-CH), 3.01 (4H, m, 27-CH
and 28-CH ), 1.22 (3H, s, 19-CH ), 0.76 (3H, s, 18-CH ). C NMR (200 MHz, CDCl , δ ppm):
4
2
2
2
13
2
3
3
3
1
4
1
98.9, 168.9, 159.9, 149.2, 146.5, 136.5, 135.2, 126.1, 125.1, 123.3, 121.5, 121.4, 81.4, 51.9,
8.7, 47.1, 46.1, 44.5, 42.9, 38.7, 38.5, 36.4, 36.2, 36.0, 35.9, 33.9, 30.2, 29.7, 28.7, 22.8, 20.8,
+
7.9, 10.9. HRMS (ESI+): (M+H) calculated for C33
H
46
N
5
O
2
= 544.3646; found : 544.3662.
4
.2.1.8 Synthesis of 7α-[4-(4-pyridin-2-yl-[1,2,3]triazol-1-yl)-but-2-enyl]-4-androsten-17β-ol-3-
one dichloroplatinum(II) (3a)
Triazole compound 6a (57 mg, 0.117 mmol, 1 eq.) is added to 1.0 mL of DMF and stirred for
several minutes. Potassium tetrachloroplatinate(II) (K PtCl ) (51 mg, 0.12 mmol, 1.1 eq.) is
2
4
diluted in 0.5 mL of distilled water and added to the solution of compound 6a. The mixture is
stirred for 5 days in the dark. After 5 days, 2 mL of a saturated KCl solution and 0.2 g of solid
KCl is added to the mixture and stirred vigorously overnight (16 hours). The product is filtered
and washed with distilled water. The product is purified by flash chromatography
2 2
(CH Cl /MeOH, 95:5) to give 3a (31 mg). Yield, 34 %; yellow solid, mp: 255 – 257 °C. IR ν
-1
(
cm ): 3403 (N-H and OH), 3103, 2931 (C-H), 1653 (C=O), 1623 (C=C), 1463, 1356, 1274,
1
1
052, 985, 775. H NMR (200 MHz, CDCl , δ ppm): 9.08 (1H, m, a-CH), 8.55 (1H, s, 24-CH),
3
1
7

ACCEPTED MANUSCRIPT
8
2
1
1
3
.05 (2H, m, c-CH and d-CH), 7.26 (1H, m, b-CH), 5.92 (1H, m, 4-CH), 5.65 (2H, m, 21-CH and
2-CH), 5.12 (2H, m, 23-CH
2
), 3.62 (1H, t, J=9.0 Hz, 17-CH), 1.19 (3H, s, 19-CH
3
), 0.78 (3H, s,
1
3
8-CH3). C NMR (200 MHz, CDCl + CD OD (9 :1), δ ppm): 199.9, 170.4, 149.4, 148.4,
3
3
39.7, 139.1, 137.0, 125.8, 124.9, 122.8, 122.4, 81.1, 54.8, 50.6, 50.1, 47.1, 46.1, 42.8, 38.7,
+
8.5, 36.5, 36.1, 35.8, 33.9, 29.8, 28.9, 22.7, 20.8, 17.9, 10.9. HRMS (ESI+): (M+H) calculated
2 5 2
for C30H42Cl N O Pt = 770.2348; found : 770.2376.
4
.2.1.9 Synthesis of 7α-[4-(4-(((pyridin-2-ylmethyl)-amino)-methyl)-[1,2,3]-triazol-1-yl)-but-2-
enyl]-4-androsten-17β-ol-3-one chloroplatinum(II) chloride (3b)
Triazole compound 6b (34.2 mg, 0.06 mmol, 1 eq.) is added to 1.0 mL of DMF and stirred for
several minutes. Potassium tetrachloroplatinate(II) (K PtCl ) (28 mg, 0.07 mmol, 1.1 eq.) is
2
4
diluted in 0.5 mL of distilled water and added to the solution of compound 6b. The mixture is
stirred for 5 days in the dark. After 5 days, 2 mL of a saturated KCl solution and 0.2 g of solid
KCl is added to the mixture and stirred vigorously overnight (16 hours). The product is filtered
and washed with distilled water to give 3b (37.4 mg). Yield, 77 %; beige solid, mp: degrades at
-1
2
56 °C. IR ν (cm ): 3438 (O-H and N-H), 3080, 2931 (C-H), 2360 – 2154 (N-N=N), 1653
1
(
(
(
C=O), 1614 (C=C), 1442, 1348, 1240, 1056, 950, 771. H NMR (200 MHz, CDCl + CD OD
3
3
9 :1), δ ppm): 8.82 (1H, m, a-CH), 7.96 (2H, m, 24-CH and c-CH), 7.51 (1H, m, d-CH), 7.35
1H, m, b-CH), 5.82 – 5.45 (3 H, m, 4-CH, 21-CH and 22-CH), 4.91 (2H, m, 24-CH ), 4.59 (2H,
2
m, CH ), 4.22 – 4.00 (2H, m, CH ), 3.49 (1H, t, 17-CH), 1.12 (3H, s, 19-CH ), 0.69 (3H, s, 18-
2
2
3
+
CH ). HRMS (ESI+): (M+H) calculated for C H ClN O Pt = 760.2747; found : 760.2771.
3
32 43
5
2
1
8

ACCEPTED MANUSCRIPT
4
.2.1.10 Synthesis of 7α-[4-(4-((2-(pyridin-2-yl-ethylamino)-methyl)-[1,2,3]-triazol-1-yl)-but-2-
enyl]-4-androstene-17β-ol-3-one chloroplatinum(II) chloride (3c)
Triazole derivative 6c (53 mg, 0.1 mmol, 1 eq.) is added to 1.5 mL of DMF and stirred for
several minutes. Potassium tetrachloroplatinate(II) (K PtCl ) (42.5 mg, 0.1 mmol, 1 eq.) is
2
4
diluted in 0.75 mL of distilled water and added to the solution of compound 6c. The mixture is
stirred for 5 days in the dark. After 5 days, 2 mL of a saturated KCl solution and 0.2 g of solid
KCl is added to the mixture and stirred vigorously overnight (16 hours). The product is filtered
and washed with distilled water to give 3c (45 mg). Yield, 60 %; beige solid, mp: degrades at
-1
2
53 °C. IR ν (cm ): 3413 (N-H and O-H), 3120, 2925 and 2854 (C-H), 2361 – 2160, 1653
1
(
C=O), 1612 (C=C), 1446, 1352, 1272, 1020, 960, 747. H NMR (200 MHz, CDCl + CD OD
3
3
(
9 :1), δ ppm): 9.14 (1H, d, J=6.3 Hz, a-CH), 8.09 (1H, s, 24-CH), 7.87 (1H, t, J=7.2 Hz, c-CH),
.38 (2H, m, b-CH and d-CH), 5.75 – 5.51 (3H, m, 4-CH, 21-CH and 22-CH), 5.01 (2H, m, 23-
CH ), 4.10 – 3.83 (2H, m, 26-CH ), 3.59 (1H, t, J=8.2 Hz, 17-CH), 3.38 – 2.84 (4H, m, 27-CH
7
2
2
2
+
and 28-CH ), 1.17 (3H, s, 19-CH ), 0.76 (3H, s, 18-CH ). HRMS (ESI+): (M+H) calculated for
2
3
3
C
33
H45ClN
5
O
2
Pt = 774.2904; found : 774.2928.
Acknowledgments
The authors thank the Cancer Research Society (CRS: number 22471) and the Canadian
Institutes of Health Research for financial support (CIHR; number 392334). This work was also
supported by a grant from the Natural Sciences and Engineering Research Council of Canada
(NSERC) to H.A. Tajmir-Riahi (NSERC; number 1512). The assistance of Lily Frenette for the
biological assay is also acknowledged.
1
9

ACCEPTED MANUSCRIPT
References
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[
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[
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[
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1
1 (2016) 281–305, DOI: 10.1517/17460441.2016.1135125
2
0

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[
10] D. Bastien, R. Hanna, V. Leblanc, E. Asselin, G. Bérubé, Synthesis and preliminary in vitro
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[
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[
13]. P. Chanphai, V. Ouellette, S. K. Mandal, G. Bérubé, H.A. Tajmir-Riahi, Testo and testo-
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2
2

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Legend of Figure and Scheme
Fig. 1. Structure of testosterone-anticancer hybrids 1, 2 and 3 designed in our laboratory.
Scheme 1
Reagents and conditions: (i) NaN , CH OH, reflux, 2 h (92%), (ii) 1. Relevant pyridine-
3
3
acetylene, sodium ascorbate, copper sulfate, THF:H O (3:1), 22 °C, 16 h (48% average yield).
2
(
iii) K PtCl , DMF:H O (2:1), 22 °C, 5 days (57% average yield).
2 4 2
Scheme 2
Reagents and conditions: i) 1. 2-Picolylamine or 2-(2-aminoethyl)pyridine, K CO , CH CN,
2
3
3
addition of propargyl bromide over 2 h at 22 °C; 2. Stirring at 22°C for 16 h (48% average yield).
2
4

ACCEPTED MANUSCRIPT
Table 1
Antiproliferative activity of novel testosterone-1,2,3-triazole-pyridine ligands (compounds 6a-c)
and testosterone-platinum(II) hybrids (compounds 3a-c) and CDDP on androgen-sensitive
(
LNCaP) and androgen-insensitive (PC3 and DU145) human prostate adenocarcinoma cell lines
and on colon carcinoma (HT-29) and normal human adult keratinocyte cells (HaCat).
a
IC50 (µM)
Compound
+
-
-
LNCaP (AR )
PC3 (AR )
DU145 (AR )
HT-29
19.9
14.7
8.5
HaCat
18.0
18.3
16.4
>25
>25
>25
5.1
6
a
6.3
20.0
>25
2.2
11.0
13.3
10.5
13.3
>25
>25
0.5
12.3
13.8
14.4
8.8
6
b
6
c
3
a
>25
>25
>25
1.3
3
b
>25
>25
2.1
>25
>25
1.0
3
c
CDDP
a
Inhibitory concentration (IC ) is concentration of drug inhibiting cell growth by 50%. Cisplatin
5
0
(CDDP)
2
5

ACCEPTED MANUSCRIPT
OH
Cl
N
O
Cl
O
O
1
OAc
O
H
N
2
Cl
Pt Cl
O
O
N
2
S
OH
O
N
N
N
N
Pt
Cl Cl
3
Fig. 1.
2
6

ACCEPTED MANUSCRIPT
OH
OAc
OH
O
O
O
R1
4
, R = Cl
1
Testosterone
4-androstene-17β-ol-3-one)
7α-allyl-4-androstene-17β-ol-3-one acetate
i
(
5, R = N3
1
OH
OH
ii
iii
O
N
O
N
N
N
N
N
N
N
Pt
Cl Cl
3
a
6a
Cl-
+
OH
OH
ii
N
iii
HN
O
N
N
O
N
N
n
N
NH
Pt
Cl
n
N
3
3
b, n = 1
c, n = 2
N
6b, n = 1
6c, n = 2
Scheme 1
2
7

ACCEPTED MANUSCRIPT
H
N
n
i
NH
2
+
Br
N
N
N
n
7a
7b, n = 1
n = 1 or 2
Propargyl bromide
7
c, n = 2
Scheme 2
2
8

ACCEPTED MANUSCRIPT
Highlights
‹
‹
‹
‹
‹
Novel testosterone-platinum(II) hybrids are reported.
The best hybrid exhibits IC50 in of 2.2 µM on AR+ prostate cancer cells (LNCaP).
The best hybrid was selective on AR+ LNCaP cells and was as active as cisplatin.
The best hybrid was inactive on normal human adult keratinocyte cells (HaCaT).
The precursor ligands showed significant antiproliferative activity.