Bifunctional bisphosphonate derivatives and platinum complexes with high affinity for bone hydroxyapatite

Article abstract of DOI:10.1016/j.bmcl.2016.12.050

A series of ethylenediamine/1,3-propanediamine derivatives containing bifunctional bisphosphonate substituents and their corresponding dichloroplatinum(II) complexes have been synthesized and characterized by elemental analysis, ¹H NMR, ¹³C NMR, ³¹P NMR, and HRMS spectra. Based on WST-8 assay with CCK-8, in general, the newly synthesized dichloroplatinum complexes 1–6 showed higher in vitro antitumor activity than platinum-free compounds L1–L6 against three tumor cell lines (especially osteosarcoma MG-63). According to hydroxyapatite binding experiment, complexes 2, 3, and 6 showed much higher affinity (K′ = 3.7, 4.0, and 3.0, respectively) for bone hydroxyapatite than cisplatin (K′ < 0.1), comparable to zoledronate (K′ = 2.8). It can be found that representative complex 2 with high cytotoxicity and in vitro antiproliferative activity against osteosarcoma cell line, as well as promising hydroxyapatite binding ability has been screened as a potential bone-targeting antitumor agent for subsequent in vivo study. In addition, flow cytometry experiment was applied to investigate the mode of action of representative complex 2.

Full text of DOI:10.1016/j.bmcl.2016.12.050

Accepted Manuscript
Bifunctional bisphosphonate derivatives and platinum complexes with high af-
finity for bone hydroxyapatite
Yanyan Sun, Lei Chen, Xiwen Wu, Qian Ding
PII:
S0960-894X(16)31330-0
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.12.050
BMCL 24539
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Bioorganic & Medicinal Chemistry Letters
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15 December 2016
20 December 2016
Please cite this article as: Sun, Y., Chen, L., Wu, X., Ding, Q., Bifunctional bisphosphonate derivatives and platinum
complexes with high affinity for bone hydroxyapatite, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://
dx.doi.org/10.1016/j.bmcl.2016.12.050
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Yanyan Sun, Lei Chen, Xiwen Wu and Qian Ding

Bioorganic & Medicinal Chemistry Letters
Bifunctional bisphosphonate derivatives and platinum complexes with high
affinity for bone hydroxyapatite
Yanyan Suna, , Lei Chena, Xiwen Wua and Qian Dinga
a School of Chemistry Biology and Material Engineering, Suzhou University of Science and Technology, Suzhou, 215009, China
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
A
series of ethylenediamine/1,3-propanediamine derivatives containing bifunctional
bisphosphonate substituents and their corresponding dichloroplatinum(II) complexes have been
synthesized and characterized by elemental analysis, 1H NMR, 13C NMR, 31P NMR, and HRMS
spectra. Based on WST-8 assay with CCK-8, in general, the newly synthesized dichloroplatinum
complexes 1-6 showed higher in vitro antitumor activity than platinum-free compounds L1-L6
against three tumor cell lines (especially osteosarcoma MG-63). According to hydroxyapatite
binding experiment, complexes 2, 3, and 6 showed much higher affinity (K’ = 3.7, 4.0, and 3.0,
respectively) for bone hydroxyapatite than cisplatin (K’ < 0.1), comparable to zoledronate (K’ =
2.8). It can be found that representative complex 2 with high cytotoxicity and in vitro
antiproliferative activity against osteosarcoma cell line, as well as promising hydroxyapatite
binding ability has been screened as a potential bone-targeting antitumor agent for subsequent in
vivo study. In addition, flow cytometry experiment was applied to investigate the mode of action
of representative complex 2.
Keywords:
Bisphosphonate
Platinum(II) complexes
Cytotoxicity
Hydroxyapatite binding
Apoptosis
2009 Elsevier Ltd. All rights reserved.
In clinic, cisplatin is known as one of the most active agents in
solid tumor chemotherapy, which was approved for the treatment
of testicular and ovarian cancer in 1978.1,2 However, the clinical
applications of cisplatin are confined by its drawbacks including
side effects and drug resistance due to the poor tumor-selectivity
of cisplatin.3-5 Therefore, many novel platinum-based complexes
with improved target distribution and tumor-selectivity have been
designed and prepared in an attempt to overcome these
limitations.6-9
Cisplatin is also widely used as one of the primary choices for
the treatment of osteosarcoma despite its significant side
effects.10 Therefore, it is necessary to develop novel platinum-
based drugs with bone-targeting ability in order to reduce the side
effects and improve the tumor-selectivity towards osteosarcoma
which is considered as one of the most untreatable and painful
malignant tumors. And the introduction of bone-targeting group
in the non-leaving or leaving moieties of platinum drugs has been
exploited as a useful strategy to design novel platinum agents.
Geminal bisphosphonates (Figure 1, BPs), which show high
affinity for bone mineral hydroxyapatite (HAP) and other
calcified tissues, are known for their bone-targeting properties
and are in clinical use for several bone-related diseases including
osteoporosis, myeloma, hypercalcemia and Paget’s disease.11-14
BPs has also been reported to exhibit significant inhibition to
osteoclastic resorption or antitumor effects.15 Besides, BPs has
been used as a moiety (non-leaving or leaving group) in
combination with platinum agents to increase the accumulation
of drugs in the bone tissue and consequently reduce the toxicity
and improve the biological activity. In the early 1990s, Keppler
and co-workers have reported platinum agents linked with
functional phosphonates which showed high activity towards
transplantable osteosarcoma in vitro and in vivo.16 Natile et al.
have synthesized a series of platinum complexes by introducing
bisphosphonate in the leaving group and found a different
Figure 1. Structures of pyrophosphate, bisphosphonate, zoledronate,
and cisplatin.
———
 Corresponding author. Tel.: +86 512 68418430; e-mail: sunyy0628@163.com

cytotoxic mechanism from that of cisplatin.17-19 Bose and co-
workers have demonstrated that platinum–pyrophosphato
complexes showed non-DNA-binding ability towards human
ovarian cancer cells.20 However, the platinum-phosphato
complexes mentioned above may lose the leaving group
phosphate moieties in the process of physiological metabolism
before reaching the bone tissue or targeting receptor. Therefore,
Guo et al. and Liang et al. have designed a series of
dichloroplatinum complexes containing pyridine derivative as
carrier ligand (non-leaving group) with bisphosphonate and
monophosphonate substituent, respectively, and some of these
complexes showed cytotoxicity against human osteosarcoma cell
line (MG-63).21-23
Nevertheless, based on the previous studies by other
researchers, it has not been reported that diamine derivatives with
two bisphosphonate groups attached to two nitrogen atoms,
symmetrically, coordinates with platinum ion to form a novel
bifunctional Pt-bisphosphonate complex. In this paper, we report
the preparation and cytotoxicity of
a
series of
ethylenediamine/1,3-propanediamine derivatives containing
N,N’-dibisphosphonate substituents and their corresponding
dichloroplatinum complexes bearing diamine derivatives as non-
leaving groups.
The preparation of N,N’-dibisphosphonate substituted
ethylenediamine/1,3-propanediamine derivatives (L1-L6) was
carried out starting from ethylenediamine/1,3-propanediamine
and monochloroacetic acid via several synthetic steps (Scheme
1). Several methods have been reported to synthesize
ethylenediamine-N,N’-diacetic acid (EDDA, M1) in the past
decades. In 1930s, Reinhold Fick first reported the preparation of
EDDA by the reaction of ethylenediamine with formaldehyde
and hydrogen cyanide.24 Bersworth have applied sodium cyanide
in place of hydrogen cyanide in the reaction mentioned
above.25,26 In 1960s, Hoechst reported that ethylenediamine and
monochloroacetic acid were used as starting materials and
aluminum hydroxide as catalyst.27 Gorelov have obtained EDDA
by decarboxylic reaction of ethylenediamine-N,N’-dimalonic
acid.28 In 1980s, Anton et al. have developed the reaction of
ethylene oxide with glycine to synthesize EDDA.29
Figure 2. Proposed structure-activity relationship of newly
synthesized platinum complexes.
Scheme 1. General synthetic route to ligands L1-L6.
1
In our study, ethylenediamine and monochloroacetic acid have
been chosen as starting materials and aluminum hydroxide as
catalyst to prepare EDDA (M1),27 followed by the reactions of
the carboxyl group with phosphorus trichloride and
hydrolyzation to give target compound L1.13,30-32 The other
compounds L2-L6 were synthesized by the similar method. All
the compounds (L1-L6) were characterized by elemental
analysis, 1H NMR, 13C NMR, 31P NMR, and HRMS spectra.
spectra. In the H NMR spectra of complexes 1-6, the broad
signals of hydrogen atoms belonging to amino groups appear in
the range of 4.60-4.79 ppm due to amine coordination with
platinum(II), shifting high-field relative to the metal-free ligands
(L1-L6) in the range of 5.45-5.68 ppm. Besides, the signals of C-
H protons connected to the amino groups occur in the range of
2.38-2.70 ppm as a multiplet, shifting high-field relative to the
corresponding signals (2.58-2.87 ppm) in the platinum-free
ligands (L1-L6). These chemical shifts demonstrate the
coordination sphere around the metal center as shown in Scheme
2. The coordination of ligands with platinum ion through
nitrogen atoms of aminobisphosphonate can also be judged by
31P NMR spectroscopy. In the phosphorus spectra, the free ligand
resonates at 17.40-17.45 ppm. After coordination of the
bisphosphonate substituted amine ligands, chemical shifts
between 21.36-21.62 ppm can be found.
Corresponding platinum(II) complexes 1-6 were obtained by
the reaction of potassium tetrachloroplatinate with compounds
L1-L6 in chloroform (Scheme 2) and the resulting complexes
1
were characterized by elemental analysis, H NMR, 13C NMR,
31P NMR, and HRMS spectra. NMR spectroscopy of both ligands
L1-L6 and the corresponding platinum(II) complexes 1-6 was
performed with d6-DMSO as the solvent, and the expected
1
signals were observed in H NMR, 13C NMR, and 31P NMR

Scheme 2. General synthetic route to platinum(II) complexes 1-6.
All the platinum(II) complexes show 100% of [M-Cl]- peaks
According to the IC50 values in Table 1, the cytotoxicity of
platinum-based complexes 1-6 seemed to be generally higher
than platinum-free ligands L1-L6 against tested cell lines,
indicating that the bifunctional N,N’-dibisphosphonate groups
and platinum moiety may have synergic effect on tumor cells. It
can be noted from Table 1 that complexes 1-3 (m=2) were more
effective in vitro than complexes 4-6 (m=3) towards three tumor
cell lines, while complexes 1-3 showed lower cytotoxicity than
that of cisplatin, with the IC50 values of 10.21-54.72 μM, 1.2-7.1-
fold less potent than cisplatin (IC50 = 6.52-11.83 μM). Especially
for MG-63 cell line, complexes 1-3 (m=2) containing
in the HRMS spectra, of which have more than three protonated
ion peaks owing to the presence of platinum and chlorine
isotopes. The structures of the target complexes were further
confirmed by elemental analysis.
The in vitro cytotoxic potency of free ligands L1-L6 and
corresponding platinum(II) complexes 1-6 has been tested by
WST-8 assay with CCK-8 against human tumor cell lines (A549
non-small cell lung cancer, HCT116 colorectal cancer, and MG-
63 osteosarcoma) after 48 h incubation. Cisplatin and zoledronate
were chosen as positive controls. The cytotoxicity results were in
terms of IC50 values presented in Table 1. It was notable that
ethylenediamine derivatives L1-L3 (m=2) with N,N’-
dibisphosphonate groups showed selective cytotoxic effect
against A549 and MG-63 cell lines, which were obviously lower
than those of cisplatin and zoledronate. Besides, 1,3-
propanediamine derivatives L4-L6 (m=3) hardly exhibited
cytotoxicity towards three tested cell lines. HCT-116 was the
least sensitive cell line to all ligands L1-L6.
ethylenediamine moiety exhibited in vitro cytotoxicity (IC50
=
10.21-26.53 μM) comparable to that of zoledronate (IC50 = 12.91
μM), 1.2-3.1-fold less potent than that of cisplatin (IC50 = 8.6
μM). On account of these IC50 data, it can be concluded that the
ethylenediamine skeleton of resulting platinum-based complexes
may have more influence on suppressing tumor cell proliferation
in vitro than 1,3-propanediamine skeleton.
Table 1. IC50 values of compounds L1-L6 and 1-6 tested by WST-8 assay with Cell Counting Kit-8 against human tumor cell lines.
Compd
m
n
IC50 (μM)a
A549b
HCT116c
> 100
MG-63d
L1
2
2
2
3
3
3
2
2
2
3
3
3
1
2
3
1
2
3
1
2
3
1
2
3
45.92 ± 2.10
31.94 ± 1.52
67.66 ± 4.19
> 100
63.84 ± 5.38
45.45 ± 3.42
> 100
L2
> 100
L3
> 100
L4
> 100
70.36 ± 4.67
> 100
L5
> 100
> 100
L6
> 100
> 100
> 100
1
35.63 ± 1.82
18.17 ± 1.04
46.48 ± 3.27
> 100
40.58 ± 2.36
23.54 ± 2.02
54.72 ± 3.49
> 100
26.53 ± 1.81
10.21 ± 0.59
19.67 ± 1.22
61.44 ± 4.53
33.46 ± 2.18
40.39 ± 3.03
8.56 ± 0.51
12.91 ± 0.77
2
3
4
5
67.20 ± 4.71
> 100
44.64 ± 2.88
58.05 ± 5.47
11.83 ± 0.82
15.57 ± 1.44
6
Cisplatin
Zoledronate
6.52 ± 0.40
14.09 ± 1.13
a Values represent the mean ± SD from three independent experiments; IC50 is defined as the drug concentration required to inhibit 50% of cell
growth determined by WST-8 assay (WST-8 = water-soluble tetrazolium salt) with Cell Counting Kit-8 (CCK-8).
b Human non-small cell lung cancer cell line.
c Human colorectal cancer cell line.
d Human osteosarcoma cell line.

When the length of linear alkyl linking amino and
bisphosphonate group was modified from one- to three-carbon
(n=1-3), the in vitro antitumor activity of complexes 1-6 had a
convex parabolic change. Complexes 2 (m=2, n=2) and 5 (m=3,
n=2) with two-carbon linker between amino and bisphosphonate
group were more active in vitro (complexes 2: IC50 = 10.21-23.54
μM, complexes 5: 33.46-67.20 μM) against three cell lines than
other compounds, suggesting that the linear alkyl with moderate-
length (n=2) linking ethylenediamine/1,3-propanediamine
skeleton and bisphosphonate groups may play a more important
role in biological activity than those with one- or three-carbon
alkyl linkers.
potent than cisplatin in terms of IC50 values against three cell
lines.
Based on the cytotoxicity results above, MG-63 was selected
as the target to test the inhibition of cell growth by all platinum
compounds. For comparison, cisplatin and zoledronate were also
investigated as positive controls, and the result of cell counting at
different concentrations of compounds was shown in Figure 3. It
was noted that the inhibition effect of all compounds on the cell
proliferation was concentration-dependent. Cisplatin and
zoledronate at low concentration of 5 μM completely inhibited
cell proliferation in MG-63. Complexes 4-5 had an obvious effect
of inhibiting cell growth on MG-63 at the concentration of 10-25
μM within the culture duration of 6 days, while complexes 1 and
3 showed in vitro antiproliferative activity at the concentration of
5-10 μM in the same cell line. Among the tested compounds,
On basis of the results in Table 1, complex 2 with
ethylenediamine skeleton and two-carbon linker between amino
and bisphosphonate groups has been screened to be the most
effective antitumor agent against three cell lines among the
newly synthesized compounds, with the IC50 values of 10.21-
23.54 μM, which showed promising cytotoxicity comparable to
those of zoledronate (IC50 = 12.91-15.57 μM), 1.2-2.8-fold less
complex
2 was significantly efficient in inhibiting cell
proliferation at the concentration of 1-5 μM comparable to
cisplatin and zoledronate, which is consistent with the result of in
vitro cytotoxicity.
Figure 3. Inhibition of cell proliferation of MG-63 by platinum compounds and positive controls at different concentrations for 6 days of
incubation (0-50 μM).

It has been demonstrated that the bisphosphonate moiety
shows binding affinity for bone hydroxyapatite, which was used
as a significant functional group in a lot of agents used clinically
to target bone tissue and treat some bone-related diseases
including osteoporosis.33,34 In this study, the in vitro bone affinity
of complexes 1-6 was evaluated by developing a bone-binding
model as compared to zoledronate and cisplatin. According to the
PI), late apoptotic or necrotic cells (upper right, positive for
annexin and PI) and apoptotic cells (lower right, negative for PI
and positive for annexin).
previous report by Bohacek et al.,
a
microcrystalline
hydroxyapatite adsorption column with phosphate gradient
elution has been applied to test the hydroxyapatite affinity of
bisphosphonate group belonging to complexes 1-6 by HPLC
assay.33 The retention time in the chromatography was expressed
in terms of capacity factor K’.
K’ = (tR-t0)/t0
Where tR is the measured retention time of tested compound
and t0 is the retention time of void column.
Table 2. K’ values of complexes 1-6 by hydroxyapatite
chromatography.
Figure 4. Cisplatin, zoledronate and complex 2 induce apoptosis on
MG-63 cells; cells were treated with these compounds at 50 μM for
24 h and then stained with annexin V–FITC and PI, and analyzed by
FACScan.
IC50
K’
Compd
(MG-63, μM)
26.53 ± 1.81
10.21 ± 0.59
19.67 ± 1.22
61.44 ± 4.53
33.46 ± 2.18
40.39 ± 3.03
8.56 ± 0.51
12.91 ± 0.77
((tR-t0)/t0)a
1.82 ± 0.13
3.68 ± 0.21
4.03 ± 0.26
1.56 ± 0.10
2.24 ± 0.15
3.01 ± 0.20
< 0.1
1
2
3
4
5
6
Cisplatin
Zoledronate
2.77 ± 0.13
a
Values represent the mean ± SD from three independent
experiments; tR is the measured retention time of tested compound
and t0 is the retention time of void column.
Figure 5. Cisplatin, zoledronate and complex 2 induce apoptosis on
A549 cells; cells were treated with these compounds at 50 μM for 24
h and then stained with annexin V–FITC and PI, and analyzed by
FACScan.
In Table 2, the hydroxyapatite chromatography results
revealed that cisplatin barely had affinity for bone hydroxyapatite
(K’ < 0.1). We found that compounds 1 (m=2, n=1), 4 (m=3,
n=1), and 5 (m=3, n=2) exhibited slightly lower hydroxyapatite
In Figure 4, it was obvious that complex 2 showed higher
apoptosis effect on MG-63 cells than zoledronate after 24 h
incubation, with apoptotic population of 26.7%, 2.1-fold more
than zoledronate (12.7%). Nevertheless, the apoptosis rate of
MG-63 cells produced by complex 2 was lower than positive
cisplatin (41.4%) at 50 μM after 24 h incubation, 1.5-fold less
than that of cisplatin.
binding efficacy (K’
= 1.56-2.24) than positive control,
zoledronate (K’ = 2.77). On the other hand, it was worth noting
that compounds 2 (m=2, n=2), 3 (m=2, n=3), and 6 (m=3, n=3)
were more efficient (K’
= 3.01-4.03) in binding bone
hydroxyapatite than zoledronate (K’ = 2.77). And the different
hydroxyapatite binding behaviors of cisplatin and compounds 1-6
indicated that the bifunctional bisphosphonate moieties of
compounds 1-6 were responsible for their affinity for bone
hydroxyapatite.
With respect to A549 cell line in Figure 5, complex 2 was
more effective in producing cell death by apoptosis than
zoledronate at 50 μM after 24 h incubation, with apoptotic
population of 28.3%, 1.5-fold more than zoledronate (19.2%),
while the apoptosis rate generated by complex 2 was obviously
lower than positive cisplatin (41.4%), 1.5-fold less than that of
cisplatin.
Considering that platinum-based complex 2 as a potential
antitumor agent displayed promising in vitro cytotoxicity and
antiproliferative activity on MG-63 cell line comparable to
cisplatin and higher hydroxyapatite affinity than zoledronate, a
flow cytometry study was performed for complex 2, cisplatin and
zoledronate to investigate the mechanism action of producing cell
death (necrosis or apoptosis). The resulting phase diagrams were
shown in Figure 4 and Figure 5 after MG-63 and A549 cells were
respectively treated with these compounds at 50 μM for 24 h.
Four areas in the diagrams represent four different cell states:
necrotic cells (upper left, positive for PI and negative for
annexin/FITC), living cells (lower left, negative for annexin and
Based on the results above, both complex 2 and cisplatin
produced higher apoptotic cell populations than platinum-free
zoledronate at the same concentration, suggesting that platinum-
based complex 2 induced cell death by apoptosis effect similar to
cisplatin.
In conclusion, six ethylenediamine/1,3-propanediamine
derivatives containing bifunctional N,N’-dibisphosphonate
substituents and six corresponding dichloroplatinum(II)

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three cell lines, suggesting that the bifunctional bisphosphonate
moieties and dichloroplatinum skeleton may have synergistic
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with
1,3-propanediamine
skeleton,
indicating
that
ethylenediamine moiety may play a more important role in
biological activity in vitro than 1,3-propanediamine moiety.
According to the result of hydroxyapatite binding assay for
positive agents and platinum-based complexes, it was testified
that complexes 2, 3, and 6 showed significant affinity for bone
hydroxyapatite superior to zoledronate and cisplatin.
Furthermore, complex 2 has been proved to be a promising
antitumor candidate with high cytotoxic efficacy against three
tumor cell lines, especially against MG-63, and hydroxyapatite
binding efficacy. Flow cytometry assay for complex 2 has been
carried out, demonstrating that representative complex
2
produced cell death of A549 and MG-63 by apoptotic effect,
similar to cisplatin.
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The authors are grateful to the National Natural Science
Foundation of China (21401137) for financial support to this
study. Chen, Wu and Ding would like to thank the College
Students’ Innovation and Entrepreneurship Project of Jiangsu
Province (201510332057X). We also appreciate very much the
support from the College and Institute’s High-level Elite Project
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Supplementary Material
Supplementary data include procedures for preparation of
ethylenediamine/1,3-propanediamine
dibisphosphonates, preparation of platinum complexes,
cytotoxicity assay (IC50 values), cell growth assay, flow
cytometry, and hydroxyapatite binding assay.
derivatives
with
10. Pasello, M.; Michelacci, F.; Scionti, I.; Hattinger, C. M.; Zuntini,
M.; Caccuri, A. M.; Scotlandi, K.; Picci, P.; Serra, M. Cancer Res.
2008, 68, 6661.
11. Sanders, J. M.; Song, Y.; Chan, J. M. W.; Zhang, Y.; Jennings, S.;
Kostzowski, T.; Odeh, S.; Flessner, R.; Schwerdtfeger, C.;
Kotsikorou, E.; Meints, G. A.; Gomez, A. O.; González-
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Highlights
1. Pt(II) complexes with a series of bifunctional
bisphosphonate derivatives as ligands.
2. Pt(II) complexes showed cytotoxicity and
antiproliferative activity against MG-63.
3. Synergistic effect on tumor cells by
bisphosphonate moieties and dichloroplatinum
skeleton.
4. Typical complexes showed hydroxyapatite
affinity superior to zoledronate and cisplatin
5. Representative compound induced apoptosis
analogous to cisplatin.