New platinum(II) complexes conjugated at position 7α of 17β-acetyl-testosterone as new combi-molecules against prostate cancer: Design, synthesis, structure-activity relationships and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2013.08.011

Prostate cancer is a major public health problem worldwide and, more specifically, new treatments for hormone-refractory cancers are highly sought by several research groups. Although platinum(II)-based chemotherapy and other strategies grow in interest to treat castration-resistant prostate cancer (CRPC), they still exhibit modest activity on CRPC and overall patient survival. In this study, we designed and prepared new combi-molecules using 17β-acetyl-testosterone and amino acid platinum(II) complexes linked at the position 7α to target and to improve the antiproliferative activity of platinum(II)-based chemotherapy on prostate cancer cells. Twelve chemical intermediates and six new combi-molecules were prepared and characterized. Structure-activity relationships studies show that the platinum complex moiety is essential for an optimal cytocidal activity. Moreover, stereochemistry of the amino acid involved in the platinum complexes had only minor effects on the antiproliferative activity whereas pyridinyl (10a and b) and thiazolyl (10f) complexes exhibited the highest cytocidal activities that are significantly superior to that of cisplatin used as control on human prostate adenocarcinoma LNCaP (AR⁺), PC3 (AR^-) and DU145 (AR^-). Compounds 10a, b and f arrested the cell cycle progression in S-phase and induced double strand breaks as confirmed by the phosphorylation of histone H2AX into γH2AX. Compounds 10a and f showed 33 and 30% inhibition, respectively of the growth of HT-1080 tumors grafted onto chick chorioallantoic membranes. Finally, compounds 10a and 10f exhibited low toxicity on the chick embryos (18 and 21% of death, respectively), indicating that these new combi-molecules might be a promising new class of anticancer agents for prostate cancer.

Full text of DOI:10.1016/j.ejmech.2013.08.011

European Journal of Medicinal Chemistry 68 (2013) 433e443
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
New platinum(II) complexes conjugated at position 7
a of
17 -acetyl-testosterone as new combi-molecules against prostate
b
cancer: Design, synthesis, structureeactivity relationships and
biological evaluation
,
,
*
Sébastien Fortin a b, Kevin Brasseur b, Nathalie Morin a, Éric Asselin b, Gervais Bérubé a
a Département de Chimie et Physique et, Université du Québec à Trois-Rivières, C.P. 500, Trois-Rivières, Québec, Canada G9A 5H7
b Département de Biologie Médicale, Université du Québec à Trois-Rivières, C.P. 500, Trois-Rivières, Québec, Canada G9A 5H7
a r t i c l e i n f o
a b s t r a c t
Article history:
Prostate cancer is a major public health problem worldwide and, more specifically, new treatments for
hormone-refractory cancers are highly sought by several research groups. Although platinum(II)-based
chemotherapy and other strategies grow in interest to treat castration-resistant prostate cancer
(CRPC), they still exhibit modest activity on CRPC and overall patient survival. In this study, we designed
Received 19 June 2013
Received in revised form
26 July 2013
Accepted 7 August 2013
Available online 15 August 2013
and prepared new combi-molecules using 17
b-acetyl-testosterone and amino acid platinum(II) com-
plexes linked at the position 7 to target and to improve the antiproliferative activity of platinum(II)-
a
based chemotherapy on prostate cancer cells. Twelve chemical intermediates and six new combi-
molecules were prepared and characterized. Structureeactivity relationships studies show that the
platinum complex moiety is essential for an optimal cytocidal activity. Moreover, stereochemistry of the
amino acid involved in the platinum complexes had only minor effects on the antiproliferative activity
whereas pyridinyl (10a and b) and thiazolyl (10f) complexes exhibited the highest cytocidal activities
that are significantly superior to that of cisplatin used as control on human prostate adenocarcinoma
LNCaP (ARþ), PC3 (ARꢀ) and DU145 (ARꢀ). Compounds 10a, b and f arrested the cell cycle progression in
S-phase and induced double strand breaks as confirmed by the phosphorylation of histone H2AX into
Keywords:
Platinum(II) complexes
17b-Acetyl-testosterone
Combi-molecules
Prostate cancer
Anticancer agents
g
H2AX. Compounds 10a and f showed 33 and 30% inhibition, respectively of the growth of HT-1080
tumors grafted onto chick chorioallantoic membranes. Finally, compounds 10a and 10f exhibited low
toxicity on the chick embryos (18 and 21% of death, respectively), indicating that these new combi-
molecules might be a promising new class of anticancer agents for prostate cancer.
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
mediates gene expression. AR is also a member of the superfamily
of steroid and nuclear receptors [2]. In adult men, androgens and
Prostate cancer is a major public health problem worldwide. In
the United-States, prostate cancer is ranked first amongst all cancer
cases diagnosed in men and it is the second most lethal cancer. It is
estimated that in 2013, 238 590 new cases of prostate cancer will be
diagnosed in the US and that 29 720 Americans will die from that
disease [1]. Androgen receptor (AR) is a cytoplasmic receptor that
AR are responsible for secondary sexual characteristics such as
spermatogenesis, muscle mass, bone mineral density, erythropoi-
esis and the maintenance of libido [3,4]. AR activation by endoge-
nous androgens such as testosterone (1, Fig. 1) and dihydr
otestosterone (DHT, 2) leads to sequential conformations of the
receptor (required by the dissociation of chaperone proteins),
dimerization, phosphorylation and translocation into the nucleus
for transcription of the target genes [2]. AR and endogenous an-
drogens play key roles in the initiation of the differentiation, pro-
gression and the maintenance of prostate cancer [5e7].
Abbreviations: AR, androgen receptor; CRPC, castration-resistant prostate can-
cer; CDDP, cisplatin; PSA, prostate-specific antigen; CAM, chick chorioallantoic
membrane.
* Corresponding author. Tel.: þ1 819 376 5011x3353; fax: þ1 819 376 5084.
E-mail addresses: Sebastien.Fortin1@uqtr.ca (S. Fortin), Kevin.Brasseur@uqtr.ca
(K. Brasseur), nathalie_morin_1234@hotmail.com (N. Morin), Eric.Asselin@uqtr.ca
(É. Asselin), Gervais.Berube@uqtr.ca (G. Bérubé).
Anti-hormone therapies using either luteinizing hormone-
releasing analogs (e.g. leuprolide [8]), anti-androgens (bicaluta-
mide (3) [9], cyproterone acetate (4) [10]) and orchiectomy are
effective against prostate cancer at their early stages. However, it
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.08.011

434
S. Fortin et al. / European Journal of Medicinal Chemistry 68 (2013) 433e443
Fig. 1. Molecular structures of testosterone (1), dihydrotestosterone (2), bicalutamide (3), cyproterone acetate (4), CDDP (5), carboplatin (6) HE3235 (7) compounds assessed in this
study (compounds 8aef, 9aef and 10aef).
must be kept in mind that anti-hormone therapies exhibit several
significant side effects such as incontinence, erectile dysfunction,
depression, muscle loss, anemia, osteoporosis and many others
[11]. Although many patients benefit from the therapy and expe-
rience prolonged remission, 10e20% of patients are still developing
castration-resistant prostate cancer (CRPC) within 5 years of the
follow-up and 84% of those CRPC are metastatic [12]. At this point,
the first-line chemotherapy regimen approved by the FDA includes
first docetaxel and prednisone for three weeks [13]. The positive
response to the treatment does not exceed 6 month with a median
survival time of 18.9 months [14]. The second-line chemotherapy
involves several other antineoplastics such as abiraterone acetate,
cabozantinib, ipilimumab, TAK-700 and their therapeutic combi-
nations that are under clinical investigation to assess their clinical
efficacy and potential benefits [15]. Among those, platinum(II)-
based chemotherapeutics such as cisplatin (CDDP, 5) and carbo-
platin (6) are of growing interest [16] despite the belief that they
exhibit only modest activity against prostate cancers and CRPC [17].
However, the clinical benefits achieved so far remains low and
increasing the survival by only 3e6 months [15,17]. Therefore, the
development of more effective platinum(II)-based therapies to
improve both life expectancy and the quality of life of CRPC patients
is still urgently needed.
Although anti-hormone therapies are ineffective against CRPC,
several groups have shown that ARs are still expressed [18] and
they still play important roles in tumor growth of CRPC as
demonstrated by the expression of the AR regulated gene, prostate-
specific antigen (PSA) [5]. On the other hand, chemoresistance can
also be related to multifactorial causes such as increased expression
of AR, AR gene mutations and altered ligand specificity, down-
stream signaling receptor for androgens and other bypass path-
ways. The latter can result in activation of AR requiring very low
androgen concentration, activation by ligands that are not normally
agonistic or ligand-independent [19e21].
[22] and in vivo [23]. Moreover, it has been shown that high-doses
of exogenous testosterone can be safely administered to patients
with CRPC [24,25]. The authors have evidenced the antitumor ac-
tivity of the treatment and the long-term stabilization of the dis-
ease in patients exhibiting low PSA concentrations. Another
example of androgen supplementation approach is the use of
HE3235 (7), a synthetic androgen analog. HE3235 exhibited a
potent antiproliferative and proapoptotic activity both in vitro and
in vivo [26,27]. Moreover, phase I studies showed that it was safe
and well tolerated [28]. Thus, androgen therapy targeting AR-
mediated signaling seems a possible and an innovative new
approach for treatment of selected types of CRPC [29,30].
In the aim to improve the cytocidal activity, the selectivity and
the biopharmaceutical properties of anticancer drugs toward
prostate cancer tumors, we designed new combi-molecules
merging 17b-acetyl-testosterone and different new platinum(II)
complexes to target simultaneously the AR-mediated signaling
pathway and DNA. In this study, we described the design, the
preparation and the biological evaluation of three series of combi-
molecules namely: Boc amino acids, amino acids and platinum(II)
complexes that are conjugated at position 7
testosterone (17 -acetyl-testosterone-7 -Boc amino acids (com-
pounds 8aef), 17 -acetyl-testosterone-7 -amino acids (com-
pounds 9aef) and 17 -acetyl-testosterone-7 -platinum(II) com
a of the 17b-acetyl-
b
a
b
a
b
a
plexes (compounds 10aef), respectively). We assessed the anti-
proliferative activity of the Boc, amine and platinum(II) complex
moieties, respectively on androgen-sensitive human prostate
adenocarcinoma LNCaP cells and androgen-insensitive human
prostate adenocarcinoma PC3 and DU145 cells and that in regard
of the nature of the amino acid side-chain. The most potent
combi-molecules were assessed also for: (1) antiproliferative ac-
tivity on a panel of 8 cancer cell lines unrelated to prostate cancer
comprising HT-1080, HT-29, M21, MCF-7, MDA-MB-231, MDA-MB-
468, CEM and CEM-VLB cells, (2) effect on cell cycle progression,
(3) potential induction of histone H2AX phosphorylation and (4)
antitumoral activities and toxicity onto the chick chorioallantoic
membrane (CAM) tumor assays.
In such a context, treatment of CRPC with androgen supple-
mentation seems counterintuitive. However, preclinical studies
showed that testosterone inhibits tumor cell growth both in vitro

S. Fortin et al. / European Journal of Medicinal Chemistry 68 (2013) 433e443
435
2. Design and chemistry
First, 17 -acetyl-testosterone was selected as the carrier for
combi-molecules based on its natural tropism for prostate tissue, to
optimize the pharmacokinetics and the pharmacodynamics of
testosterone towards AR and to activate AR signaling pathways.
3. Results and discussion
b
3.1. Antiproliferative activity on prostate cancer cell lines and
structureeactivity relationships
The antiproliferative activity of compounds 8aef, 9aef and
10aef was assessed at first on LNCaP, an androgen-sensitive human
prostate adenocarcinoma cells, and on PC3 and DU145, that are
both androgen-insensitive human prostate adenocarcinoma cells.
The antiproliferative activity and the median lethal concentration
were evaluated in vitro using the MTT cell proliferation assay with
minor modifications [42,43]. The results are summarized in Table 1
and show antiproliferative activity (IC50) and the median lethal
concentration (LC50) of 8aef, 9aef and 10aef. IC50 is defined, as the
concentration of drug inhibiting cell growth by 50% and the LC50 is
concentration required to kill 50% of the cells. CDDP was used as
positive control.
Second, position 7a of testosterone is located away from the 3-
carbonyl and 17-hydroxyl groups that are involved in key hydrogen
interactions with AR. These two crucial groups must be free of steric
hindrances to favor optimal interactions with AR and to trigger the
signaling pathways. Finally,
methionine, -histidine and
L
- and
D-2-pyridylalanine, L- and D-
L
L-4-thiazolylalanine amino acids were
selected because they easily form platinum(II) complexes and that
they are inexpensive templates to optimize our synthetic approaches
[31,32]. We also observed in our recent studies on estrogen combi-
molecules that amino pyridine group easily form platinum(II) com-
plexes and show interesting biological activities [33e36].
The methods used for the preparation of 17
b
b
-acetyl-testos-
-acetyl-testos-
The antiproliferative activity of all three series of compounds
was slightly higher on PC3 cells followed by LNCaP and DU145
terone-7
terone-7
a
a
-Boc amino acids (compounds 8aef), 17
-amino acids (compounds 9aef) and 17
b-acetyl-
cells. The antiproliferative activity of 17
Boc amino acids (compounds 8aef) and 17
7a-amino acids (compounds 9aef) were evaluated to assess the
b
-acetyl-testosterone-7
a-
testosterone-7
a
-platinum(II) complexes (compounds 10aef) are
b-acetyl-testosterone-
described in Scheme 1. Compound 11 was prepared in excellent
yields as published elsewhere using the method described by
contribution of the platinum complex to the cytocidal activity.
Nickisch et al. [37] and Bastien et al. [38e41]. Then, 17
b-acetyl-
Compounds 8aef and 9aef were cytostatic and exhibiting IC50
testosterone-7 -Boc amino acids (compounds 8aef) were obtained
after treatment of compound 11 and the selected amino acid in the
presence of cesium carbonate in methyl ethyl ketone. Afterward,
a
between 12 and >100
100 M. Boc and amine series were clearly less active than their
platinum complex counterparts showing IC50 in the low M range
(1.4e28 M) indicating that the platinum complex moiety is
mM while their LC50 were higher than
m
m
17
b
-acetyl-testosterone-7
a
-amino acids (compounds 9aef) were
m
achieved by treatment of compounds 8aef with trifluoroacetic acid
in methylene chloride. Finally, compounds 9aef were treated with
potassium tetrachloroplatinate(II) in a mixture of dimethylforma-
crucial for the activity. Interestingly, the stereochemistry of the
amino acids used has minor effects on the activity as exemplified
by the R-pyridinyl (series a) and the S-pyridinyl (series b) as well
as the R-methionyl (series c) and the S-methionyl (series d). The
mide and water to afford 17b-acetyl-testosterone-7a-platinum(II)
complexes (compounds 10aef).
17b
-acetyl-testosterone-7a-platinum(II) complexes bearing a pyr
a
b
c
d
e
f
Series 8
(R)
Series 9
(R1)
Series
10 (R2)
Scheme 1. Reagents and conditions: (i) Boc-amino acids, Cs2CO3, MEK, 110 ꢁC, 72 h; (ii) TFA, DCM, 22 ꢁC, 4 h and (iii) K2PtCl4, DMF:H2O, 22 ꢁC, 5 days.

436
S. Fortin et al. / European Journal of Medicinal Chemistry 68 (2013) 433e443
Table 1
decreases dramatically within a short range of concentrations
suggesting that CDDP and 17 -acetyl-testosterone-7 -platinum(II)
complexes do not act completely through the same mechanism of
action.
Antiproliferative activity and median lethal concentration of novel 17
testosterone-7 -Boc amino acids (compounds 8aef), 17 -acetyl-testosterone-7
amino acids (compounds 9aef) and 17 -acetyl-testosterone-7 -platinum(II) com-
b-acetyl-
b
a
a
b
a-
b
a
plexes (compounds 10aef) and CDDP on androgen-sensitive (LNCaP) and androgen-
insensitive (PC3 and DU145) human prostate adenocarcinoma cell lines.
Compd
IC50
(
m
M)a
LC50 (m
M)b
3.2. Antiproliferative activity of compounds on a panel of cancer
cell lines unrelated to prostate cancer
LNCaP
PC3
DU145
LNCaP
PC3
DU145
(ARꢀ
(ARþ
)
(ARꢀ
)
(ARꢀ
)
(ARþ
)
(ARꢀ
)
)
8a
8b
8c
8d
8e
8f
9a
9b
9c
9d
9e
9f
10a
10b
10c
10d
10e
10f
CDDP
25
21
>100
12.5
42
32
>100
46
>100
>100
45
>100
2.5
3.1
28
21
20
1.8
6.7
14
12
19
18.1
24
17
40
33
58
43
29
58
1.9
2.3
14
11
12
1.4
7.4
34
29
>100
>100
54
>100
>100
46
12
89
49
>100
3.6
3.5
23
16
13
1.9
2.1
57
>100
>100
>100
>100
43
>100
>100
>100
>100
>100
>100
>100
3.5
4.0
31
25
34
2.6
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
96
>100
9.4
11
41
31
AR is not only expressed in male reproductive organs such as
prostate, testis and seminal vesicle but it is also expressed in a
variety of normal and cancer tissues including hepatic, thyroid,
pancreatic, gastrointestinal, renal, bone marrow, breast and many
others [44e51]. Thus, in the aim to verify that the new combi-
molecules concept can be useful to the chemotherapy of other
cancers, compounds 10a, b and f exhibiting IC50 and LC50 lower
>100
>100
>100
70
>100
>100
>100
>100
>100
67
>100
6.4
8.0
64
44
than 12
mM on LNCaP, PC3 and DU145 tumor cell lines were
assessed on eight cell lines unrelated to prostate tissues. The IC50
and the LC50 are summarized in Tables 2 and 3, respectively and
were evaluated using the MTT cell proliferation assay as described
above on human fibrosarcoma HT-1080, colon carcinoma HT-29,
skin melanoma M21, breast carcinoma MCF-7, breast adenocarci-
noma MDA-MB-231, breast adenocarcinoma MDA-MB-468, T cell
leukemia CEM and CEM-VLB cells, respectively. CEM-VLB cells are
overexpressing P-glycoprotein [52], which is responsible for the
cellular efflux of several anticancer drugs such as doxorubicin,
paclitaxel, and vinblastine. In addition P-glycoprotein is part of a
potent mechanism of resistance encountered in clinical settings
[53e55]. CDDP, colchicine and vinblastine are used as positive
controls.
80
4.7
44
41
6.4
>100
a
Inhibitory concentration (IC50) is concentration of drug inhibiting cell growth by
50%.
b
Median lethal concentration (LC50) is the concentration required to kill 50% of
the cells.
idinyl (compounds 10a and b) or a thiazolyl (compound 10f) side
Compounds 10a, b and f showed antiproliferative activities in
the same order of magnitude as previously determined on LnCAP,
chain exhibited the highest IC50 between 1.4 and 3.6
mM. The
substitution of a pyridinyl (10a and b) and thiazolyl (10f) chelating
moiety by a thiomethyl (10c and d) or by an imidazolyl moiety
(10e) decreased the IC50 by up to 10-fold. Interestingly, the LC50 for
PC3 and DU145 prostate cancer cell lines (0.39e3.1
mM). Interest-
ingly, the IC50 of 17 -acetyl-testosterone-7 -platinum(II) com-
b
a
plexes were higher than CDDP alone on MDA-MB-468, CEM and
CEM-VLB cells while they were lower on other cancer cell lines.
CEM-VLB cells exhibited low resistance to 10a, b and f (5.7-, 6.7-
and 7.2-fold) which are more than 10-times lower than for
colchicine (87-fold) and several logs lower than vinblastine (980-
fold) while CDDP was clearly unaffected by the overexpression of
P-glycoprotein. This suggests that overexpression of P-glycoprotein
compounds 10a, b and f (2.6e8
mM) were several times lower than
CDDP (44 to >100 M) indicating that 10a, b and f were much
m
more cytocidal than CDDP. On one hand, compound 10f bearing a
thiazolyl moiety exhibited the highest antiproliferative activity
with IC50 of 1.8, 1.4 and 1.9
which are up to five times more potent than CDDP alone that
exhibited IC50 of 6.7, 7.4 and 2.1 M, respectively. On the other
hand, the LC50 of 10f on the same cell lines were from 9 to over
38-times more potent than CDDP (4.7, 2.6 and 6.4 M for 10f vs.
44, >100 and >100 M for CDDP, respectively). Moreover, the
general aspects of the antiproliferative curves for 17 -acetyl-
testosterone-7 -platinum(II) complexes and CDDP are quite
different. For example, when the concentration of CDDP alone
increases, the cell growth decreases slowly while when using 17
mM on LNCaP, PC3 and DU145 cells
m
does not affect the antiproliferative activity of 17
terone-7 -platinum(II) complexes. In addition, except for CEM-VLB
cells, the LC50 of 17 -acetyl-testosterone-7 -platinum(II) com-
plexes 10a, b and f (0.64e4.0 M) were several times lower than
that of CDDP (1.3e50 M), which were similar to the results obtain
on prostate cancer cell lines. However, the IC50 and the LC50 of the
most potent 17 -acetyl-testosterone-7 -platinum(II) complexes
b-acetyl-testos-
a
m
b
a
m
m
b
m
a
b
a
b
-
10a, b and f were several times higher than colchicine and
acetyl-testosterone-7a-platinum(II) complexes the cell growth
vinblastine.
Table 2
Antiproliferative activity (IC50) of compounds 10a, b and f, CDDP, colchicine and vinblastine on selected cancer cell lines.
Compd
IC50
(
m
M)a
Ratio resistant/wild-type
CEM-VLB/CEM
HT-1080
HT-29
M21
MCF-7
MDA-MB-231
MDA-MB-468
CEM
CEM-VLBb
10a
10b
10f
CDDP
Colc
Vbld
0.77
0.72
0.52
1.8
0.014
0.00046
1.8
1.6
1.1
2.8
0.011
0.00055
1.3
1.0
0.83
3.8
0.018
0.00081
0.88
0.78
0.58
1.6
0.010
0.00047
1.3
1.0
0.83
5.0
0.018
0.0016
0.59
0.46
0.39
0.32
0.0068
0.00033
0.57
0.56
0.39
1.0
0.0071
0.00054
3.0
3.1
3.0
0.89
0.30
0.35
5.2
5.5
7.9
0.87
42
640
a
IC50 (mM) is the concentration of the tested drug inhibiting cell growth by 50%.
Multidrug-resistant leukemia CEM-VLB cells.
Col, colchicine.
b
c
d
Vbl, vinblastine.

S. Fortin et al. / European Journal of Medicinal Chemistry 68 (2013) 433e443
437
Table 3
Median lethal concentration of compounds 10a, b and f, CDDP, colchicine and vinblastine on selected cancer cell lines.
Compd
LC50
(
m
M)a
Ratio resistant/wild-type
HT-1080
HT-29
M21
MCF-7
MDA-MB-231
MDA-MB-468
CEM
CEM-VLBb
CEM-VLB/CEM
10a
10b
10f
CDDP
Colc
Vbld
1.4
1.3
1.1
8.8
0.084
0.0052
4.0
3.2
2.5
49
>0.10
>0.10
2.2
1.5
1.5
11
>0.10
>0.10
2.4
2.2
1.7
50
>0.10
>0.10
2.0
1.5
1.3
27
>0.10
>0.10
0.94
0.76
0.64
1.3
0.021
0.0089
1.2
1.0
0.90
14
0.018
0.0030
6.6
6.5
6.5
3.0
1.6
2.9
5.7
6.7
7.2
0.21
87
980
a
LC50 (mM) is the concentration of the drug required to kill 50% of the cells.
Multidrug-resistant leukemia CEM-VLB cells.
Col, colchicine.
b
c
d
Vbl, vinblastine.
Table 4
Effects of compounds 10a, b and f, and CDDP on cell cycle progression and on the phosphorylation of histone H2AX into
gH2AX.
Compd
Cell cycle progression
Conc ( M)
Phosphorylation of histone H2AXa
m
Sub-G1
G0/G1
S
G2/M
10a
9.1
9
46
18
27
10b
12
2
12
1
53
45
69
68
15
19
15
8
30
24
15
24
10f
6.9
CDDP
19
DMF
0.1%
0
a
For phosphorylation of histone H2AX experiments, PC3 cells were incubated for 24 h in the presence of the selected 17b-acetyl-testosterone-7a-platinum(II) complexes at
1.5-fold of IC50
.

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S. Fortin et al. / European Journal of Medicinal Chemistry 68 (2013) 433e443
3.3. Effect on cell cycle progression
Table 4 shows the percentage of PC3 cells that are in Sub-G1, G0/
G1, S and G2/M phases, respectively, after treatment with com-
pounds 10a, b and f, and CDDP for 24 h at optimal concentrations
regarding the arrest of cell cycle progression in S-phase. Control
cells treated with the excipient (0.1% DMF) were found in sub-G1,
G0/G1, S and G2/M phases at 0, 68, 8 and 24%, respectively. Com-
pounds 10a, b and f arrested mainly the cell cycle progression in the
S-phase as observed with CDDP where the percentage of S-phase
cells increased by 7e11%.
3.4. Phosphorylation of histone H2AX
DNA replication is the main event occurring during the S-phase
and platinum(II)-based anticancer agents are well known to form
reactive species such as cationic aqua ligand that bind to DNA,
resulting in intrastrand and interstrand cross-links [56,57], trig-
gering the formation of DNA single- and double-strand breaks that
lead to apoptosis [58,59]. Moreover, phosphorylation of Ser-139 at
Fig. 2. Effects of compounds 10a, b and f as well as CDDP on the growth of HT-1080
tumors and their toxicity on chick embryos in the CAM assay. Gray bars represent
the percentage of wet weight of tumors treated with or without excipient. Black bars
represent the percentage of chick embryo mortality.
the C-terminus of histone H2AX (
DNA double-strand breaks [60,61]. Consequently, we used the
phosphorylation of H2AX into of H2AX to assess the effect of the
platinum complex moiety in the mechanism of action and in the
activity of compounds 10a, b and f. As depicted in Table 4, com-
pounds 10a, b and f and CDDP induced H2AX phosphorylation in
gH2AX) occurs upon induction of
(100
m
L) in the CAM. The concentration of DMF was kept at 0.3% to
avoid embryo’s toxicity. CDDP (10
mg) was used as positive control.
g
As depicted in Fig. 2, compounds 10a, b and f significantly
inhibited tumor growth by 33, 21 and 30%, respectively and
exhibited toxicity in 18, 29, and 21% of the chick embryos, respec-
tively. CDDP inhibited tumor growth by 50% and exhibited toxicity
in 5% of the chick embryos. Thus, we conclude that the excipient is
not toxic for the embryos, the compounds 10a, b and f show anti-
tumoral activities on HT-1080 cells and low toxicity toward em-
bryos suggesting that this new compounds have interesting
biological activities and biopharmaceutical properties to continue
biological and in vivo investigations.
PC3 cells.
gH2AX was detected as nuclear red spots in nuclei stained
in blue by 40,6-diamidino-2-phenylindole (DAPI). As shown in
Table 4, compounds 10a, b and f induced lower level of gH2AX than
CDDP and therefore less double-strand breaks.
The induction of DNA double-strand breaks confirmed by the
phosphorylation of H2AX ( H2AX) explains at least in part the
mechanism of action of 17 -acetyl-testosterone-7 -platinum(II)
complexes. In fact, the phosphorylation of H2AX into H2AX does
not correlate to the cytotoxicity of CDDP and 17 -acetyl-testos-
terone-7 -platinum(II) complexes; 17 -acetyl-testosterone-7
g
b
a
g
b
4. Conclusion
a
b
a-
platinum(II) complexes induced a lower number of double-strand
breaks than CDDP while they were significantly more cytotoxic
than CDDP. This suggests that the platinum(II) complexes are
responsible for only a portion of the cytocidal activity. Research is in
progress to determine the contribution of each constituent of the
combi-molecules (testosterone and the various platinum com-
plexes) to the cytocidal activity.
In this study, we designed, prepared and characterized six new
combi-molecules using 17
b
D
-acetyl-testosterone and
L
- and
D
-2-
-4-
pyridylalanine, and
L-
-methionine, -histidine and
L
L
thiazolylalanine amino acid platinum(II) complexes linked at po-
sition 7 to target prostate cancer cells (17 -acetyl-testosterone-
-platinum(II) complexes 10aef). To the best of our knowledge, it
is the first time such testosterone-7 -Pt(II) combi-molecules are
a
b
7a
a
reported in the literature. They exhibited antiproliferative activity
(IC50) and median lethal concentration (LC50) at the micromolar
level on panel of prostate cancer cell lines and cancer cell lines
unrelated to prostate cancer. Structureeactivity relationships
showed that platinum complex is essential for optimal activity. The
stereochemistry of the amino acids used had only minor effects on
3.5. Chick chorioallantoic membrane (CAM) tumor assay
The CAM tumor assay is a simple and inexpensive animal model
providing a large amount of information related to the systemic
toxicity, the antineoplastic and the antiangiogenic potential of a
new drug on a human tumor grafted onto the chorioallantoic
membrane. In addition, it contributes to validate the drug formu-
lations developed for intravenous administration of the new drugs
and their future biopharmaceutical studies. However, it must be
noted that only a limited number of human cell lines can form
sizable tumors on the CAM. In that context, the human HT-1080
fibrosarcoma cell lines were selected instead of PC3 cells. HT-
1080 cells are sensitive to most anticancer agents and form solid
tumors easy to handle [62e64]. As depicted in Fig. 2 the results
were obtained from two independent experiments using 10 to 12
eggs per experiments. No systemic toxicity was observed on chick
embryos treated with the excipient alone. Sham-treated embryos
were used as negative controls and for normalization of the results.
Forty micrograms of compounds 10a, b and f freshly dissolved in
DMF were added to a mixture of cremophor ELÔ, 99% ethanol and
phosphate buffered saline (PBS) (1/1/14 v/v) were administered iv
the cytocidal activity. Moreover, L- and D-2-pyridylalanine, and L-4-
thiazolylalanine platinum(II) complexes (compounds 10a, b and f,
respectively) exhibited the most potent antiproliferative activity
and are unaffected by the overexpression of P-glycoprotein. In
addition, compounds 10a, b and f arrest the cell cycle progression in
S-phase and induce DNA double-strand breaks as confirmed by the
phosphorylation of H2AX into gH2AX. Finally, compounds 10a and
f showed antitumoral activity on HT-1080 tumors grafted onto CAM
and exhibited low toxicity on the chick embryos. The strategic
location of the platinum(II) moiety and the efficient chemical
transformations of testosterone represent an attractive strategy to
develop that type of anticancer agents. Hence, the innovative
design and the significant biological activity of 17
b-acetyl-testos-
terone-7
a
-platinum(II) complexes toward ARþ and ARꢀ prostate
cancer cells suggest that they might be members of a promising
new class of anticancer agents for prostate cancers.

S. Fortin et al. / European Journal of Medicinal Chemistry 68 (2013) 433e443
439
5. Experimental protocols
slides were treated with 1.5 mL of a fibronectin solution in PBS
(10
g/mL) for 1 h at 37 ꢁC. Slides were then rinsed twice with PBS.
m
5.1. Biological methods
PC3 cells (1.5 ꢂ 105) were seeded into the plates and incubated for
24 h. Cells were then incubated with the test compound at 1.5 and
3-times their respective IC50 for 24 h at 37 ꢁC. The control solution
consisted of DMF dissolved in culture medium at 0.1% (v/v). Cells
were fixed using 1 mL of formaldehyde at 3.7% and permeabilized
by addition of a saponin solution (0.1% in PBS) containing 3% (w/v)
bovine serum albumin (saponin-BSA). Cells were incubated with
mouse anti-H2AX pS139 antibody (Millipore, Billerica, MA). Cover
slides were next incubated for 3 h at room temperature and then
washed twice with PBS supplemented with 0.05% (v/v) Tween 20
(PBS-T). Saponin-BSA containing goat anti-mouse IgG conjugated to
AlexaFluor 594 (Invitrogen, Burlington, Ontario, Canada), and DAPI
LNCaP androgen-sensitive human prostate adenocarcinoma,
PC3 androgen-insensitive human prostate adenocarcinoma, DU145
androgen-insensitive human prostate adenocarcinoma, HT-1080
human fibrosarcoma, HT-29 human colon carcinoma, MCF7 hu-
man breast carcinoma, MDA-MB-231 human breast carcinoma and
MDA-MB-468 human breast adenocarcinoma were purchased from
the American Type Culture Collection (Manassas, VA). M21 human
skin melanoma cells were kindly provided by Dr. David Cheresh
(University of California, San Diego School of Medicine, CA). T cell
leukemia CEM and multidrug-resistant leukemia CEM-VLB were
generously provided by Dr. William T. Beck (University of Illinois at
Chicago, College of Pharmacy, IL) [53]. Cells were cultured in RPMI
medium (Hyclone, Logan, UT) supplemented with 10% of calf serum
and PenicillineStreptomycineGlutamine. The cells were main-
tained at 37 ꢁC in a moisture saturated atmosphere containing 5%
CO2.
(0.3 mg/mL) was then added. The cover slides were incubated for 2 h
at room temperature and then washed twice with PBS-T and twice
with PBS. The cover slides were mounted in darkness on a micro-
scope slide overnight with Fluoromount-Gtm (Southern Biotech no:
0100-01) before analysis using an Olympus BX51 epifluorescence
microscope. Images were captured as 8-bit tagged image format
files with a Q imaging RETIGA EXI digital camera driven by Image
Pro Express software.
5.1.1. Antiproliferative activity assay
The antiproliferative activity of compounds 8aef, 9aef, 10aef,
CDDP, colchicine and vinblastine was assessed using the MTT assay
[42,43]. Briefly, the 96-well microtiter plates were seeded with
5.1.4. CAM tumor assay
Human HT-1080 fibrosarcoma cells were used to assess the
antitumoral activity of compound 10a, b, and f in the CAM assay
[63,65,66]. Briefly, fertilized chicken eggs purchased from Couvoirs
Victoriaville (Victoriaville, Québec, Canada) were incubated for 10
days in a Pro-FI egg incubator fitted with an automatic egg turner
before being transferred to a Roll-X static incubator for the rest of
the incubation time (incubators were purchased from Lyon Electric,
Chula Vista, San Diego, CA). The eggs were kept at 37 ꢁC in a 60%
humidity atmosphere for the entire incubation period. On day 10,
by use of a hobby drill (Dremel, Racine, WI), a hole was drilled on
the side of the egg and a negative pressure was applied to create a
new air sac. A window was opened on this new air sac and was
covered with transparent adhesive tape to prevent contamination.
75 mL of a tumor cells in suspension (for LNCaP, 12 000; PC3, 6000;
DU145, 6000; HT-1080, 2500; HT-29, 3000; M21, 3000; MCF-7,
3000; MDA-MB-231, 5000; MDA-MB-468, 5000; CEM, 10 000;
CEM-VLB, 10 000) in medium. Plates were incubated at 37 ꢁC, 5%
CO2 for 24 h. Freshly solubilized drugs in DMF were diluted in fresh
medium, and 50 mL aliquots containing escalating concentrations of
the drug were added and the plates were incubated for 48 h. The
final concentration of DMF in the culture media was 0.1% and was
kept constant in all experiment. After 48 h, 10
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
(5 mg/mL in PBS) were added to the wells. Four hours later, 100
mL of 3-(4,5-
mL
of the solubilization solution (10% sodium dodecyl sulfate (SDS) in
0.01 M HCl) was added and the plate was incubated overnight
(37 ꢁC, 5% CO2). The optical density was read using a Fluostar op-
tima BMG (BMG Labtech inc., Durham, NC) at 565 nm. Readings
obtained from treated cells were compared with measurements
from control cells plates fixed on the treatment day, and the per-
centage of cell growth inhibition was then calculated for each drug.
The experiments were performed at least twice 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%.
A
freshly prepared cell suspension (40
mL) of HT-1080
(3.5 ꢂ 105 cells/egg) cells in the culture media was applied
directly onto the freshly exposed CAM tissue through the window.
On day 11, the drugs dissolved in DMF were extemporaneously
diluted in the excipient (CremophorÔ/ethanol 99%/PBS, 6.25/6.25/
87.5 v/v). The concentration of DMF in the excipient was kept at
0.3% to avoid any potential toxicity. 40
terone-7 -platinum(II) complexes and 10
100 L of excipient were injected iv into 10e12 eggs. The eggs were
m
g for 17
b-acetyl-testos-
a
mg for CDDP dissolved in
m
incubated until day-17, at which time the embryos were euthanized
at 4 ꢁC followed by decapitation. Tumors were collected, and the
tumor-wet weights were recorded. The number of dead embryos
and signs of toxicity from the different groups were recorded.
5.1.2. Cell cycle analysis
PC3 cells (2.5 ꢂ 105) were seeded into six-well plates and
incubated for 24 h. Cells were then incubated with the test com-
pound at 1.5 and 3-times their respective IC50 for 24 h at 37 ꢁC. The
control solution consisted of DMF dissolved in culture medium at
0.1% v/v. After incubation, the cells were trypsinized, washed with
5.2. Chemistry
PBS and resuspended in 250
m
L of PBS. Cells were fixed by the
Proton NMR spectra were recorded on a Varian 200 MHz NMR
or a Bruker AM-300 spectrometer. Chemical shifts (d) are reported
addition of 750
m
L of ice-cold EtOH and stored at ꢀ20 ꢁC until
analysis. Prior to FACS, the cells were washed with PBS and resus-
in parts per million. IR spectra were recorded on a Magna FT-IR
spectrometer (Nicolet Instrument Corporation, Madison, WI, U.S.).
Chemicals were supplied by Aldrich Chemicals (Milwaukee, WI,
USA) or VWR International (Mont-Royal, Qc, Canada) and used as
received. Testosterone was supplied by Steraloids Inc. (Newport, RI)
and Boc-amino acids were supplied by Peptech Corporation (Bed-
ford, MA). Uncorrected melting points were determined on an
Electrothermal melting point apparatus. Mass spectral were ob-
tained from NanoQAM (Université du Québec à Montréal) using a
Time-of-Flight LC/MS (LC/MS-TOF), Agilent Technologie, LC 1200
pended in 1 mL of PBS containing 2 m
g/mL of 40,6-diamidino-2-
phenylindole (DAPI, Sigma, Oakville, Ontario, Canada). Fixed cell
suspensions were incubated on ice for 1 h, and the cell cycle dis-
tribution was then analyzed using a BD SORP LSR II cytometer (BD
Biosciences, Franklin Lakes, NJ).
5.1.3. Immunofluorescence
Cover slides (22 mm ꢂ 22 mm) sterilized with 70% (v/v) EtOH
were placed in six-well plates. To promote cell adhesion, cover

440
S. Fortin et al. / European Journal of Medicinal Chemistry 68 (2013) 433e443
Series/6210 TOF-LCMS with electrospray ionization and positive
28.3, 27.3, 22.9, 21.1, 20.7, 17.9, 11.9. HRMS (ESIþ) m/z found
649.3835; C38H53N2O7 (Mþ þ H) requires 649.3854.
mode (ESIþ). Liquid flash chromatography was performed on silica
ꢀ
gel F60, 60 A, 40e63
mm supplied by Silicycle (Québec, Canada)
using a FPX flash purification system (Biotage, Charlottesville, VA)
and using solvent mixtures expressed as v/v ratios. Solvents and
reagents were used without purification unless specified other-
wise. The completion of all reactions was monitored by TLC on
precoated silica gel 60 F254 TLC plates (VWR). The chromatograms
were viewed under UV light at 254 and/or 265 nm.
5.3.3. (E)-4-[(7R,10R,13S,17S)-17-Acetoxy-10,13-dimethyl-3-oxo-
2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]
phenanthren-7-yl]but-2-en-1-yl (tert-butoxycarbonyl)-L-
methioninate (8c)
Compound 8c was synthesized from N-Boc-
chromatography (hexanes to hexanes/ethyl acetate (50:50)). Yield,
68%; White solid; mp: 54e57 ꢁC. IR
: 2920 (Ar), 1713 (C]O), 1672
(C]O) cmꢀ1. 1H NMR (300 MHz, CDCl3):
L-methionine. Flash
n
5.3. General procedure for the synthesis of Boc amino acid
d
5.67e5.44 (m, 3H, 3ꢂ ]
conjugates at position 7
a
of 17b
-acetyl-testosterone (compounds
CH), 5.30 (d, 1H, J ¼ 7.5 Hz, NH), 4.60e4.54 and 4.40e34.37 (m, 4H,
8aef)
CH, CHOAc and CH2), 2.52e1.07 (m, 42H, 4ꢂ CH, 10ꢂ CH2, and 6ꢂ
CH3), 0.81 (s, 3H, CH3). 13C NMR (300 MHz, CDCl3):
d 198.9, 172.1,
The appropriate Boc-amino acid (0.16 mmol, 1 eq.) and cesium
carbonate (0.16 mmol, 1 eq.) were added to a solution of the 7
(E)-4-chlorobut-2-enyl-4-androsten-17 -ol-3-one acetate (11)
(0.16 mmol, 1 eq.) in butan-2-one (2 mL). The key intermediate
-(E)-4-chlorobut-2-enyl-4-androsten-17 -ol-3-one acetate (11)
171.1, 168.7, 134.7, 126.2, 125.7, 82.3, 79.8, 65.7, 52.9, 47.0, 46.0, 42.6,
38.6, 38.3, 36.4, 36.2, 35.9, 34.0, 32.2, 30.0, 28.7, 28.3, 27.3, 22.9, 21.1,
20.7, 18.0, 15.4, 11.9. HRMS (ESIþ) m/z found 632.3592; C35H54NO7S
(Mþ þ H) requires 632.3622.
a
-
b
7
a
b
was efficiently prepared as described in an earlier study [37e41].
The reaction mixture was heated to 110 ꢁC under pressure in
sealed tube and stirred for 3 days. Afterward the mixture was
cooled to room temperature, the solvent was evaporated under
reduced pressure. The residue was solubilized by methylene
chloride (15 mL) and washed with a solution of sodium hydroxide
1 N (15 mL). The aqueous phase was extracted thrice with
methylene chloride (15 mL). The combined organic phases were
washed with brine (25 mL), dried over anhydrous sodium sulfate,
filtered, and evaporated to dryness under vacuum. The crude
compound was purified by flash chromatography on silica gel to
afford compounds 8aef.
5.3.4. (E)-4-[(7R,10R,13S,17S)-17-Acetoxy-10,13-dimethyl-3-oxo-
2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]
phenanthren-7-yl]but-2-en-1-yl (tert-butoxycarbonyl)-D-
methioninate (8d)
Compound 8d was synthesized from N-Boc-
chromatography (hexanes to hexanes/ethyl acetate (50:50)). Yield,
74%; White solid; mp: 58e61 ꢁC. IR
: 2940 (Ar), 1713 (C]O), 1671
(C]O) cmꢀ1. 1H NMR (300 MHz, CDCl3):
D-methionine. Flash
n
d
5.62e5.42 (m, 3H, 3ꢂ ]
CH), 5.22 (d, 1H, J ¼ 7.6 Hz, NH), 4.57e4.51 and 4.38e34.34 (m, 4H,
CH, CHOAc and CH2), 2.48e1.04 (m, 42H, 4ꢂ CH, 10ꢂ CH2, and 6ꢂ
CH3), 0.79 (s, 3H, CH3). 13C NMR (300 MHz, CDCl3):
d 198.8, 172.0,
171.0, 168.8,134.7, 126.2,125.6, 82.3, 79.8, 65.7, 52.8, 47.0, 45.9, 42.5,
38.6, 38.2, 36.4, 36.2, 36.0, 35.9, 33.9, 32.1, 30.0, 28.6, 28.3, 27.3,
22.9, 21.1, 20.7, 17.9, 15.4, 11.9. HRMS (ESIþ) m/z found 632.3607;
5.3.1. (E)-4-[(7R,10R,13S,17S)-17-Acetoxy-10,13-dimethyl-3-oxo-
2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]
phenanthren-7-yl]but-2-en-1-yl (2S)-2-[(tert-butoxycarbonyl)
amino]-3-(pyridin-2-yl)propanoate (8a)
C
35H54NO7S (Mþ þ H) requires 632.3622.
5.3.5. (E)-4-[(7R,10R,13S,17S)-17-Acetoxy-10,13-dimethyl-3-oxo-
2,3,6,7,8,9,10,11,12,13, 14,15,16,17-tetradecahydro-1H-cyclopenta[a]
phenanthren-7-yl]but-2-en-1-yl (tert-butoxycarbonyl)-L-histidinate
Compound 8a was synthesized from Boc-
Flash chromatography (hexanes to hexanes/ethyl acetate (50:50)).
Yield, 86%; White solid; mp: 66e68 ꢁC. IR
: 2940 (Ar), 1712 (C]O),
1672 (C]O) cmꢀ1. 1H NMR (200 MHz, CDCl3):
8.46e8.44 (m, 1H,
L-2-pyridylalanine.
n
(8e)
d
Compound 8e was synthesized from N-Boc-L-histidine. Flash
pyr), 7.61e7.52 (m, 1H, pyr), 7.14e7.08 (m, 2H, pyr), 5.92 (d, 1H,
J ¼ 8.3 Hz, NH), 5.65e5.43 (m, 3H, 3ꢂ ]CH), 4.68e4.48 and 4.17e
3.99 (m, 4H, CH, CHOAc and CH2), 3.27e3.25 (m, 2H, CH2), 2.41e
1.08 (m, 35H, 4ꢂ CH, 8ꢂ CH2, and 5ꢂ CH3), 0.81 (s, 3H, CH3). 13C
chromatography (hexanes to hexanes/ethyl acetate (50:50) to
methylene chloride to methylene chloride/methanol (90:10)).
Yield, 51%; White solid; mp: 100e104 ꢁC. IR
n
: 2937 (Ar), 1713 (C]
O), 1671 (C]O) cmꢀ1
.
1H NMR (300 MHz, CDCl3):
d
7.69 (s, 1H,
NMR (200 MHz, CDCl3):
d
198.6, 171.3, 170.7, 168.5, 156.8, 155.2,
imidazole), 6.82 (s, 1H, imidazole), 6.51 (brs, 1H, NH of imidazole),
5.95 (d, 1H, J ¼ 7.7 Hz, NH), 5.68e5.37 (m, 3H, 3ꢂ ]CH), 4.62e4.53
(m, 4H, CH, CHOAc and CH2), 3.11e3.05 (m, 2H, CH2), 2.49e1.13 (m,
35H, 4ꢂ CH, 8ꢂ CH2, and 5ꢂ CH3), 0.83 (s, 3H, CH3). 13C NMR
148.5, 136.4, 133.7, 125.8, 125.5, 123.5, 121.5, 82.0, 79.2, 65.2, 52.8,
46.6, 45.6, 42.2, 38.9, 38.2, 37.9, 36.0, 35.8, 35.6, 35.6, 33.6, 28.2,
27.9, 26.9, 22.5, 20.8, 20.4, 17.6, 11.5. HRMS (ESIþ) m/z found
649.3825; C38H53N2O7 (Mþ þ H) requires 649.3854.
(300 MHz, CDCl3):
d 200.0, 171.8, 171.2, 170.4, 155.6, 135.1, 133.9,
133.3, 126.3, 125.7, 116.4, 82.4, 79.7, 65.7, 53.7, 47.1, 45.9, 42.6, 38.7,
38.2, 36.4, 36.2, 36.0, 35.9, 33.9, 28.6, 28.3, 27.9, 27.3, 22.8, 21.1, 20.7,
17.9, 11.9. HRMS (ESIþ) m/z found 638.3783; C36H52N3O7 (Mþ þ H)
requires 638.3806.
5.3.2. (E)-4-[(7R,10R,13S,17S)-17-Acetoxy-10,13-dimethyl-3-oxo-
2,3,6,7,8,9,10,11,12,13,14,15, 16,17-tetradecahydro-1H-cyclopenta[a]
phenanthren-7-yl]but-2-en-1-yl (2R)-2-[(tert-butoxycarbonyl)
amino]-3-(pyridin-2-yl)propanoate (8b)
Compound 8b was synthesized from Boc-
Flash chromatography (hexanes to hexanes/ethyl acetate (50:50)).
Yield, 68%; White solid; mp: 60e62 ꢁC. IR
: 2928 (Ar), 1714 (C]O),
1671 (C]O) cmꢀ1. 1H NMR (300 MHz, CDCl3):
8.45e8.44 (m, 1H,
D
-2-pyridylalanine.
5.3.6. (E)-4-[(7R,10R,13S,17S)-17-Acetoxy-10,13-dimethyl-3-oxo-
2,3,6,7,8,9,10,11,12,13, 14,15,16,17-tetradecahydro-1H-cyclopenta[a]
phenanthren-7-yl]but-2-en-1-yl (2S)-2-[(tert-butoxycarbonyl)
amino]-3-(thiazol-4-yl)propanoate (8f)
n
d
Pyr), 7.59e7.54 (m, 1H, pyr), 7.14e7.08 (m, 2H, pyr), 5.92 (d, 1H,
J ¼ 8.1 Hz, NH), 5.63e5.39 (m, 3H, 3ꢂ ]CH), 4.66e4.46 and 4.09e
4.01 (m, 4H, CH, CHOAc and CH2), 3.27e3.20 (m, 2H, CH2), 2.37e
1.05 (m, 35H, 4ꢂ CH, 8ꢂ CH2, and 5ꢂ CH3), 0.79 (s, 3H, CH3). 13C
Compound 8f was synthesized from Boc-
Flash chromatography (hexanes to hexanes/ethyl acetate (50:50)).
Yield, 67%; White solid; mp: 70e75 ꢁC. IR
: 2937 (Ar), 1714 (C]O),
1670 (C]O) cmꢀ1 1H NMR (300 MHz, CDCl3):
8.75 (s, 1H, thia-
zole), 7.06 (s, 1H, thiazole), 5.73 (d, 1H, J ¼ 8.4 Hz, NH), 5.65e5.39
(m, 3H, 3ꢂ ]CH), 4.63e4.48 (m, 4H, CH, CHOAc and CH2), 3.2 (d,
2H, J ¼ 4.9 Hz, CH2), 2.38e1.07 (m, 35H, 4ꢂ CH, 8ꢂ CH2, and 5ꢂ
L-4-thiazolylalanine.
n
.
d
NMR (300 MHz, CDCl3):
d
198.8, 171.6, 171.0, 168.8, 157.2, 155.5,
148.9, 136.7, 134.0, 126.2, 125.8, 123.9, 121.8, 82.3, 79.5, 65.4, 53.1,
47.0, 46.0, 42.5, 39.2, 38.6, 38.2, 36.4, 36.1, 36.0, 35.9, 33.9, 28.6,

S. Fortin et al. / European Journal of Medicinal Chemistry 68 (2013) 433e443
441
CH3), 0.80 (s, 3H, CH3). 13C NMR (300 MHz, CDCl3):
d
198.9, 171.5,
5.4.4. (E)-4-((7R,10R,13S,17S)-17-Acetoxy-10,13-dimethyl-3-oxo-
2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]
171.1, 168.8, 155.4, 153.0, 152.6, 134.2, 126.2, 125.8, 115.6, 82.3, 79.7,
65.6, 53.3, 47.0, 46.0, 42.5, 38.6, 38.3, 36.4, 36.2, 36.0, 35.9, 34.0,
33.3, 28.6, 28.3, 27.3, 22.9, 21.1, 20.7, 18.0, 11.9. HRMS (ESIþ) m/z
found 655.3388; C36H51N2O7S (Mþ þ H) requires 655.3418.
phenanthren-7-yl)but-2-en-1-yl
Yield, 100%; Yellowish solid; mp: 85e88 ꢁC. IR
(C]O), 1670 (C]O) cmꢀ1. 1H NMR (200 MHz, CDCl3):
D
-methioninate (9d)
: 2932 (Ar), 1729
5.63e5.49
n
d
(m, 3H, 3ꢂ ]CH), 4.59e4.51 (m, 3H, CHOAc and CH2), 3.56e3.53
(m, 1H, CH), 2.59 (t, 2H, J ¼ 7.0 Hz, CH2), 2.39e1.03 (m, 33H, 4ꢂ CH,
9ꢂ CH2, 3ꢂ CH3 and NH2), 0.80 (s, 3H, CH3). 13C NMR (200 MHz,
5.4. General procedure for the synthesis of amino acid conjugates at
position 7a of 17b-acetyl-testosterone (compounds 9aef)
CDCl3):
d 199.0, 175.4, 171.1, 168.9, 134.5, 126.1, 125.9, 82.3, 65.4,
Trifluoroacetic acid (7.8 mmol, 60 eq.) was added dropwise to a
solution of the appropriate Boc amino acid (compounds 8aef,
0.13 mmol, 1 eq.) in dichloromethane (8 mL). The resulting mixture
was stirred at room temperature for 5 h. Then, mixture was alka-
linized (pH 14) with solution of NaOH (1 N, 15 mL) and the aqueous
layer was extracted thrice with methylene chloride (10 mL),
washed with brine (15 mL), dried with sodium sulfate, and the
solvent was evaporated under reduced pressure to afford the amino
53.3, 47.0, 45.9, 42.5, 38.6, 38.2, 36.4, 36.2, 35.9, 35.9, 33.9, 33.8,
30.4, 28.7, 27.3, 22.8, 21.1, 20.7, 17.9, 15.3, 11.9. HRMS (ESIþ) m/z
found 532.3075; C30H46NO5S (Mþ þ H) requires 532. 3097.
5.4.5. (E)-4-[(7R,10R,13S,17S)-17-Acetoxy-10,13-dimethyl-3-oxo-
2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]
phenanthren-7-yl]but-2-en-1-yl
Yield, 100%; Orange solid; mp: 94e96 ꢁC. IR
(C]O), 1670 (C]O) cmꢀ1. 1H NMR (200 MHz, CDCl3):
d
L
-histidinate (9e)
n
: 2935 (Ar), 1729
7.52 (s, 1H,
acid conjugate at position 7
a
of 17
b
-acetyl-testosterone (com-
pounds 9aef).
imidazole), 6.81 (s, 1H, imidazole), 5.64e5.39 (m, 3H, 3ꢂ ]CH),
4.62e4.40 (m, 3H, CHOAc and CH2), 3.79e3.72 (m, 1H, CH), 3.11e
2.82 (m, 2H, CH2), 2.45e1.04 (m, 29H, 4ꢂ CH, 8ꢂ CH2, 2ꢂ CH3, NH
5.4.1. (E)-4-[(7R,10R,13S,17S)-17-Acetoxy-10,13-dimethyl-3-oxo-
2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]
phenanthren-7-yl]but-2-en-1-yl (2S)-2-amino-3-(pyridin-2-yl)
propanoate (9a)
and NH2), 0.81 (s, 3H, CH3). 13C NMR (200 MHz, CDCl3):
d 199.6,
174.8, 171.2, 169.9, 169.9, 135.1, 134.1, 133.4, 126.3, 125.9, 82.3, 65.5,
54.6, 47.0, 45.9, 42.5, 38.7, 38.2, 36.4, 36.2, 35.9, 35.8, 33.9, 28.7, 27.3,
22.9, 21.1, 20.7, 17.9, 11.9. HRMS (ESIþ) m/z found 538.3285;
Yield, 100%; White solid; mp: 95e100 ꢁC. IR
n
: 2932 (Ar), 1729
8.49e8.47
(C]O), 1670 (C]O) cmꢀ1. 1H NMR (200 MHz, CDCl3):
d
C
31H44N3O5 (Mþ þ H) requires 538.3282.
(m, 1H, pyr), 7.61e7.52 (m, 1H, pyr), 7.18e7.06 (m, 2H, pyr), 5.64e
5.41 (m, 3H, 3ꢂ ]CH), 4.61e4.50 and 4.05e3.93 (m, 4H, CH, CHOAc
and CH2), 3.29e2.95 (m, 2H, CH2), 2.39e1.05 (m, 28H, 4ꢂ CH, 8ꢂ
CH2, 2ꢂ CH3 and NH2), 0.81 (s, 3H, CH3). 13C NMR (200 MHz, CDCl3):
5.4.6. (E)-4-[(7R,10R,13S,17S)-17-Acetoxy-10,13-dimethyl-3-oxo-
2,3,6,7,8,9,10,11,12,13, 14,15,16,17-tetradecahydro-1H-cyclopenta[a]
phenanthren-7-yl]but-2-en-1-yl (2S)-2-amino-3-(thiazol-4-yl)
propanoate (9f)
d
198.9, 174.9, 171.1, 168.9, 158.0, 149.3, 136.4, 134.2, 126.2, 126.0,
123.9, 121.6, 82.3, 65.3, 54.4, 47.0, 45.9, 42.5, 38.6, 38.2, 36.3, 36.1,
36.0, 35.9, 33.9, 29.6, 28.6, 27.3, 22.9, 21.1, 20.7, 17.9, 11.9. HRMS
(ESIþ) m/z found 549.3309; C33H45N2O5 (Mþ þ H) requires
549.3329.
Yield, 87%; Yellowish solid; mp: 85e88 ꢁC. IR
n
: 2923 (Ar), 1730
8.73 (s, 1H,
(C]O), 1670 (C]O) cmꢀ1. 1H NMR (300 MHz, CDCl3):
d
thiazole), 7.10 (s, 1H, thiazole), 5.64e5.42 (m, 3H, 3ꢂ ]CH), 4.60e
4.47 (m, 3H, CHOAc and CH2), 3.95e3.91 (m, 1H, CH), 3.32e3.08 (m,
2H, CH2), 2.39e1.02 (m, 28H, 4ꢂ CH, 8ꢂ CH2, 2ꢂ CH3 and NH2), 0.81
5.4.2. (E)-4-[(7R,10R,13S,17S)-17-Acetoxy-10,13-dimethyl-3-oxo-
2,3,6,7,8,9,10,11,12,13, 14,15,16,17-tetradecahydro-1H-cyclopenta[a]
phenanthren-7-yl]but-2-en-1-yl (2R)-2-amino-3-(pyridin-2-yl)
propanoate (9b)
(s, 3H, CH3). 13C NMR (300 MHz, CDCl3):
d 198.9, 174.3, 171.1, 168.9,
153.5, 152.8, 134.4, 126.2, 126.0, 115.4, 82.3, 65.5, 54.3, 47.0, 46.0,
42.5, 38.6, 38.3, 36.4, 36.2, 35.9, 35.8, 33.9, 29.7, 28.7, 27.3, 22.9, 21.1,
20.7, 18.0, 11.9. HRMS (ESIþ) m/z found 555.2867; C31H43N2O5S
(Mþ þ H) requires 555.2893.
Yield, 95%; Yellowish solid; mp: 76e80 ꢁC. IR
n
: 2927 (Ar), 1730
8.49e8.48
(C]O), 1670 (C]O) cmꢀ1. 1H NMR (300 MHz, CDCl3):
d
(m, 1H, Pyr), 7.61e7.56 (m, 1H, pyr), 7.18e7.09 (m, 2H, Pyr), 5.68e
5.43 (m, 3H, 3ꢂ ]CH), 4.60e4.47 and 4.06e3.99 (m, 4H, CH, CHOAc
and CH2), 3.32e3.02 (m, 2H, CH2), 2.69 (brs, 2H, NH2) 2.37e1.05 (m,
26H, 4ꢂ CH, 8ꢂ CH2, and 2ꢂ CH3), 0.81 (s, 3H, CH3). 13C NMR
5.5. General procedure for the synthesis of platinum(II) complex
conjugates at position 7a of 17b-acetyl-testosterone (17b-acetyl-
testosterone-7 -platinum(II) complexes 10aef)
a
(300 MHz, CDCl3):
d
198.9, 174.4, 171.1, 168.9, 157.9, 149.2, 136.5,
To a solution of the amino acid (compounds 9aef, 1.0 eq.,
0.10 mmol) in DMF (3 mL) at room temperature was added po-
tassium tetrachloroplatinate(II) (1.5 eq., 0.15 mmol) dissolved in
water (1.5 mL). After 5 days of stirring in darkness at room tem-
perature, a saturated KCl solution (4 mL) was added followed by
solid KCl (0.5 g). The mixture was vigorously stirred for 30 min and
the suspension was then filtered, washed with water (15 mL) and
dried under vacuum. The crude compound was purified by flash
chromatography on silica gel to afford cis-dichloroplatinum(II)
complexes (compounds 10aef).
134.3,126.2,126.0,124.0,121.7, 82.4, 65.4, 54.3, 47.0, 46.0, 42.5, 38.6,
38.3, 36.4, 36.2, 36.0, 35.9, 34.0, 29.7, 28.6, 27.3, 22.9, 21.1, 20.7, 18.0,
11.9. HRMS (ESIþ) m/z found 549.3311; C33H45N2O5 (Mþ þ H) re-
quires 549.3329.
5.4.3. (E)-4-[(7R,10R,13S,17S)-17-Acetoxy-10,13-dimethyl-3-oxo-
2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]
phenanthren-7-yl]but-2-en-1-yl
Yield, 95%; Yellowish solid; mp: 78e82 ꢁC. IR
1730 (C]O), 1672 (C]O) cmꢀ1 1H NMR (200 MHz, CDCl3):
L
-methioninate (9c)
n
: 2924 (Ar),
.
d
5.64e5.42 (m, 3H, 3ꢂ ]CH), 4.60e4.51 (m, 3H, CHOAc and
5.5.1. 17b-Acetyl-testosterone-7a-platinum(II) complex 10a
CH2), 3.59e3.53 (m, 1H, CH), 2.59 (t, 2H, J ¼ 7.1 Hz, CH2), 2.40e
1.07 (m, 33H, 4ꢂ CH, 9ꢂ CH2, 3ꢂ CH3 and NH2), 0.81 (s, 3H, CH3).
Flash chromatography (methylene chloride to methylene chlo-
ride/ethyl acetate (50:50)). Yield, 33%; Yellowish solid; mp: 215e
13C NMR (200 MHz, CDCl3):
d
198.9, 175.5, 171.1, 168.8, 134.6,
218 ꢁC. IR : 2936 (Ar), 1729 (C]O), 1657 (C]O) cmꢀ1 1H NMR
n .
126.2, 125.9, 82.3, 65.4, 53.3, 47.0, 45.9, 42.5, 38.6, 38.2, 36.4,
36.2, 35.9, 33.9, 33.9, 30.4, 28.7, 27.3, 22.9, 21.1, 20.7, 18.0, 15.4,
11.9. HRMS (ESIþ) m/z found 532.3075; C30H46NO5S (Mþ þ H)
requires 532.3097.
(200 MHz, CDCl3): d 9.24e9.21 (m, 1H, pyr), 7.88e7.80 (m, 1H, pyr),
7.31e7.27 (m, 2H, pyr), 6.14e6.05 and 5.71e5.36 (m, 3H, 3ꢂ ]CH),
4.78e4.31 (m, 4H, CH, CHOAc and CH2), 4.15e4.06 and 3.59e3.49
(m, 2H, CH2), 2.43e1.08 (m, 28H, 4ꢂ CH, 8ꢂ CH2, 2ꢂ CH3 and NH2),

442
S. Fortin et al. / European Journal of Medicinal Chemistry 68 (2013) 433e443
0.84 (s, 3H, CH3). 13C NMR (200 MHz, CDCl3):
d
199.2, 171.2, 169.4,
213 ꢁC. IR
(200 MHz, CDCl3):
n
: 2944 (Ar), 1730 (C]O), 1660 (C]O) cmꢀ1
.
1H NMR
169.0, 155.5, 154.0, 138.9, 136.2, 126.1, 125.8, 124.6, 124.4, 82.3, 67.2,
50.2, 47.1, 46.0, 42.6, 38.6, 38.3, 36.4, 36.2, 35.9, 35.8, 34.0, 28.7, 27.3,
22.9, 21.2, 20.7, 18.0, 11.9. HRMS (ESIþ) m/z found 814.2357;
d
9.63 (d, 1H, J ¼ 2.6 Hz, thiazole), 7.39 (d, 1H,
J ¼ 2.6 Hz, thiazole), 5.64e5.46 (m, 4H, 3ꢂ ]CH and NH), 4.98e4.87
and 4.64e4.57 (m, 4H, CHOAc, CH2 and NH), 4.00e3.96 (m, 1H, CH),
3.76e3.69 and 3.42e3.31 (m, 2H, CH2), 2.44e1.07 (m, 26H, 4ꢂ CH,
8ꢂ CH2 and 2ꢂ CH3), 0.84 (s, 3H, CH3). 13C NMR (200 MHz, CDCl3):
C
33H45Cl2N2O5Pt (Mþ þ H) requires 814.2354.
5.5.2. 17
b
-Acetyl-testosterone-7a-platinum(II) complex 10b
d 199.3, 171.2, 169.2, 168.9, 157.3, 149.3, 135.7, 126.2, 125.0, 117.3,
Flash chromatography (methylene chloride to methylene chlo-
82.4, 67.1, 52.0, 47.1, 46.0, 42.6, 38.6, 38.3, 36.4, 36.3, 35.9, 35.7, 34.0,
33.5, 28.9, 27.3, 22.9, 21.2, 20.7, 18.0, 11.9. HRMS (ESIþ) m/z found
820.1898; C31H43Cl2N2O5PtS (Mþ þ H) requires 820.1918.
ride/ethyl acetate (50:50)). Yield, 21%; Yellowish solid; mp: 202e
205 ꢁC. IR : 2941 (Ar), 1729 (C]O), 1659 (C]O) cmꢀ1 1H NMR
n .
(200 MHz, CDCl3): d 9.23e9.20 (m, 1H, pyr), 7.87e7.79 (m, 1H, pyr),
7.36e7.22 (m, 2H, pyr), 6.13e6.02 and 5.77e5.33 (m, 3H, 3ꢂ ]CH),
4.82e4.24 (m, 4H, CH, CHOAc and CH2), 4.11e4.05 and 3.62e3.51
(m, 2H, CH2), 2.43e1.12 (m, 28H, 4ꢂ CH, 8ꢂ CH2, 2ꢂ CH3 and NH2),
Acknowledgments
This work was supported by the Fonds de Recherche sur la
Nature et les Technologies du Québec (FQRNT, Grant #132839). S.
Fortin is the recipient of postgraduate fellowships from the Fonds
de Recherche du Québec e Santé (FRQS) and from the Canadian
Institutes of Health Research (CIHR).
0.84 (s, 3H, CH3). 13C NMR (200 MHz, CDCl3):
d 199.2, 171.2, 169.4,
169.1, 155.6, 154.0, 138.9, 136.2, 126.2, 125.9, 124.7, 124.3, 82.3, 67.3,
50.2, 47.1, 46.0, 42.6, 38.6, 38.3, 36.4, 36.3, 36.0, 35.7, 34.0, 28.8, 27.3,
22.9, 21.2, 20.7, 18.0, 11.9. HRMS (ESIþ) m/z found 814.2354;
C
33H45Cl2N2O5Pt (Mþ þ H) requires 814.2354.
Appendix A. Supplementary data
5.5.3. 17b-Acetyl-testosterone-7a-platinum(II) complex 10c
Flash chromatography (methylene chloride to methylene chlo-
ride/ethyl acetate (50:50) to methylene chloride/methanol
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.08.011.
(90:10)). Yield, 28%; White solid; mp: 192e195 ꢁC. IR
n
: 2933 (Ar),
1728 (C]O), 1660 (C]O) cmꢀ1. 1H NMR (300 MHz, CDCl3):
d
5.67e
References
5.41 (m, 4H, 3ꢂ ]C and NH), 4.77e4.52 (m, 4H, CHOAc, CH2 and
NH), 4.15e4.03 (m, 1H, CH), 3.41e3.10 and 2.68e2.62 (m, 5H, CH2
and CH3), 2.41e1.14 (m, 28H, 4ꢂ CH, 9ꢂ CH2 and 2ꢂ CH3), 0.84 (s,
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: 2942 (Ar),
1729 (C]O), 1658 (C]O) cmꢀ1. 1H NMR (300 MHz, CDCl3):
d
5.69e
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5.44 (m, 4H, 3ꢂ ]C and NH), 4.74e4.52 (m, 4H, CHOAc, CH2 and
NH), 4.12e4.03 (m, 1H, CH), 3.39e3.07 and 2.68e2.60 (m, 5H, CH2
and CH3), 2.41e1.13 (m, 28H, 4ꢂ CH, 9ꢂ CH2 and 2ꢂ CH3), 0.84 (s,
3H, CH3). 13C NMR (200 MHz, CDCl3):
d 199.2, 171.1, 169.9, 169.4,
135.6, 126.1, 125.2, 82.4, 66.9, 53.0, 47.0, 45.9, 42.6, 38.6, 38.3, 36.4,
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quires 797.2122.
5.5.5. 17b-Acetyl-testosterone-7a-platinum(II) complex 10e
Flash chromatography (methylene chloride to methylene chlo-
ride/methanol (90:10)). Yield, 36%; White solid; mp: 208e213 ꢁC. IR
n
: 2936 (Ar), 1729 (C]O), 1655 (C]O) cmꢀ1 1H NMR (300 MHz,
.
CDCl3):
d 11.6 (s, 1H, NH imidazole), 8.18 (s, 1H, imidazole), 6.95 (s,
1H, imidazole), 5.78e5.41 (m, 3H, 3ꢂ ]CH), 4.79e4.55 (m, 3H,
CHOAc and CH2), 4.11e3.99 and 3.38e3.32 (m, 1H, CH), 3.06e3.00
and 2.42e1.06 (m, 30H, 4ꢂ CH, 9ꢂ CH2, 2ꢂ CH3, NH2), 0.81 (s, 3H,
CH3). 13C NMR (300 MHz, CDCl3):
d 200.0, 171.2, 170.4, 169.5, 136.6,
135.1, 126.2, 125.9, 125.4, 125.1, 82.5, 66.7, 51.9, 47.1, 46.0, 42.6, 38.7,
38.3, 36.4, 36.3, 35.9, 35.8, 34.0, 28.8, 27.4, 22.9, 21.2, 20.7, 18.0, 11.9.
HRMS (ESIþ) m/z found 803.2301; C31H44Cl2N3O5Pt (Mþ þ H) re-
quires 803.2307.
5.5.6. 17b-Acetyl-testosterone-7a-platinum(II) complex 10f
Flash chromatography (methylene chloride to methylene chlo-
ride/ethyl acetate (50:50)). Yield, 34%; Yellowish solid; mp: 210e

S. Fortin et al. / European Journal of Medicinal Chemistry 68 (2013) 433e443
443
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