Arylamino methylene bisphosphonate derivatives as bone seeking matrix metalloproteinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.08.054

The complexity of matrix metalloproteinase inhibitors (MMPIs) design derives from the difficulty in carefully addressing their inhibitory activity towards the MMP isoforms involved in many pathological conditions. In particular, specific metalloproteinases, such as MMP-2 and MMP-9, are key regulators of the 'vicious cycle' occurring between tumor metastases growth and bone remodeling. In an attempt to devise new approaches to selective inhibitor derivatives, we describe novel bisphosphonate bone seeking MMP inhibitors (BP-MMPIs), capable to be selectively targeted and to overcome undesired side effects of broad spectrum MMPIs. In vitro activity (IC₅₀ values) for each inhibitor was determined against MMP-2, -8, -9 and -14, because of their relevant role in skeletal development and renewal. The results show that BP-MMPIs reached IC₅₀ values of enzymatic inhibition in the low micromolar range. Computational studies, used to rationalize some trends in the observed inhibitory profiles, suggest a possible differential binding mode in MMP-2 that explains the selective inhibition of this isoform. In addition, survival assay was conducted on J774 cell line, a well known model system used to evaluate the structure-activity relationship of BPs for inhibiting bone resorption. The resulting data, confirming the specific activity of BP-MMPIs, and their additional proved propensity to bind hydroxyapatite powder in vitro, suggest a potential use of BP-MMPIs in skeletal malignancies.

Full text of DOI:10.1016/j.bmc.2013.08.054

Accepted Manuscript
Arylamino Methylene Bisphosphonate Derivatives As Bone Seeking Matrix
Metalloproteinase Inhibitors
Marilena Tauro, Antonio Laghezza, Fulvio Loiodice, Mariangela Agamennone,
Cristina Campestre, Paolo Tortorella
PII:
S0968-0896(13)00757-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.08.054
BMC 11070
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
28 March 2013
29 July 2013
23 August 2013
Please cite this article as: Tauro, M., Laghezza, A., Loiodice, F., Agamennone, M., Campestre, C., Tortorella, P.,
Arylamino Methylene Bisphosphonate Derivatives As Bone Seeking Matrix Metalloproteinase Inhibitors,
Bioorganic & Medicinal Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bmc.2013.08.054
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ARYLAMINO METHYLENE BISPHOSPHONATE DERIVATIVES AS BONE
SEEKING MATRIX METALLOPROTEINASE INHIBITORS
Marilena Tauroa, Antonio Laghezzaa, Fulvio Loiodicea, Mariangela Agamennoneb,
Cristina Campestreb, Paolo Tortorellaa*
a Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi ”Aldo Moro”
di Bari, Via Orabona 4, 70126 Bari (Italy). Fax +39 080 5442231.
b Dipartimento di Farmacia, Università “G. d’Annunzio” Chieti, Via dei Vestini 31,
66013 Chieti (Italy)
* Corresponding author: Dipartimento di Farmacia-Scienze del Farmaco, Università
degli Studi ”Aldo Moro” di Bari, Via Orabona 4, 70126 Bari (Italy). Tel. +39
0805442735, Fax +39 0805442231. E-mail: paolo.tortorella@uniba.it
Abstract
The complexity of matrix metalloproteinases inhibitors (MMPIs) design derives from
the difficulty in carefully addressing their inhibitory activity towards the MMP
isoforms involved in many pathological conditions. In particular, specific
metalloproteinases, such as MMP-2 and MMP-9, are key regulators of the "vicious
cycle" occurring between tumor metastases growth and bone remodeling. In an
attempt to devise new approaches to selective inhibitor derivatives, we describe novel
bisphosphonate bone seeking MMP inhibitors (BP-MMPIs), capable to be selectively
targeted and to overcome undesired side effects of broad spectrum MMPIs.
In vitro activity (IC50 values) for each inhibitor was determined against MMP-2, -8, -9
and -14, because of their relevant role in skeletal development and renewal. The
results show that BP-MMPIs reached IC50 values of enzymatic inhibition in the low
micromolar range. Computational studies, used to rationalize some trends in the
observed inhibitory profiles, suggest a possible differential binding mode in MMP-2
that explains the selective inhibition of this isoform.
In addition, survival assay was conducted on J774 cell line, a well known model
system used to evaluate the structure–activity relationship of BPs for inhibiting bone
resorption. The resulting data, confirming the specific activity of BP-MMPIs, and
their additional proved propensity to bind hydroxyapatite powder in vitro, suggest a
potential use of BP-MMPIs in skeletal malignancies.
1. Introduction
Bone is a frequent site of metastases for several cancers. In 2012, the American
Cancer Society estimated that approximately 68,000 men and women will succumb to
prostate and breast cancers (www.cancer.org Cancer Facts and Figures American
Cancer Society 2012) and studies predicted that 80-90% of these patients will have
evidence of bone metastases at the time of their death. Bone metastases induce
skeletal related events (SRE), including hypercalcemia and spontaneous pathologic
bone fracture that can cause intense pain and greatly impact the patient's quality of
life.[1, 2] The bone microenvironment provides a fertile soil for the growth and
expansions of tumor cells, altering the physiological remodeling process.

Bone remodeling is a delicate balance between bone matrix synthesizing osteoblasts
and bone resorbing osteoclasts activities. Active bone metastases typically subvert
this process to generate lesions that include extensive areas of pathological
osteogenesis and osteolysis.[3] The resultant increase in bone matrix remodeling
enhances cytokine/growth factors bioavailability thus creating a vicious cycle that
stimulates tumor assessment and progression at the bone site.[4]
Recent studies have identified matrix metalloproteinases (MMPs) as key regulators of
this cycle, and pre-clinical experiments have reported the efficacy of broad spectrum
metalloproteinase inhibitors (MMPIs) in preventing the skeletal related events
secondary to prostate and breast tumors evolution.[4, 5] MMP-2 and -9 play a key
role in the progression of the vicious cycle, while the first one derives from
osteoblast, the second one is secreted by osteoblasts, osteoclasts and tumor cells. In
this way they considerably contribute to promote tumor survival at the mineral
surface of the host microenvironment, by regulating the bioavailability of TGF-β,
PTHrP and RANKL that are traditionally associated with driving that vicious
cycle.[4]
Besides, remodeling of the bone matrix is required to accommodate the expansion of
metastases and unsurprisingly, matrix metalloproteinases (MMPs) are over-expressed
at the tumor-bone interface due to their central role in the processing of type I
collagen-rich osteoid and other bone matrix components.[5, 6] MMPs also hydrolyze
a number of other proteinases and some of them are involved in invasion and
angiogenic mechanisms. Accordingly, while the selective inhibition of a limited set of
MMPs is of relevant importance at the early stages of the disease, broad-spectrum
inhibitors allow to intervene during the progression of the pathology. MMPIs are able
to disrupt the vicious cycle occurring in the spread of tumor cells to bone; their
possible therapeutic utility, however, is limited by the typical side effects of broad
spectrum activity. Nonetheless, these adverse effects could be reduced by using
molecules able to selectively target a specific tissue as proposed for bisphosphonates
whose bone accumulation is well known.[7] It is clear that these compounds could
represent an additional treatment strategy for patients with debilitating bone
metastases.
Since MMPs belong to the family of zinc-containing enzymes, they are characterized
by the presence of a zinc ion at the active site, responsible for the amide bond
hydrolysis. An effective MMPI should satisfy the following requirements: (1) the
presence of a functional group able to bind the catalytic Zn2+ ion of the enzyme; (2) at
least one functional group capable to form hydrogen bonds with the enzyme
backbone; (3) one or more moieties that can have effective van der Waals interactions
with the enzyme subsites.
Hydroxamate has been considered for a long time the most potent zinc binding group
(ZBG), able to work as a bidentate ligand with an optimum distance (1.9 – 3.3 Å)
between the oxygen atoms and the active site Zn2+ ion.[8, 9] Although pharmaceutical
companies have developed very potent hydroxamic inhibitors in the last 25 years, still
no MMPIs are present in the market as anticancer agents. The reasons for the failure
of clinical trials are multifold, mainly correlated with their relatively low selectivity,
pharmacokinetics and toxicological problems.[10, 11]
We have been studying non-hydroxamic MMPIs for a long time.[12-19] In a recent
paper we reported a series of inhibitors characterized by a bisphosphonic core (BP-
MMPIs), a well known bone seeking moiety, as ZBG.[20] The benefits deriving from
the introduction of this ZBG are twofold. Firstly, it realizes an efficient interaction
with the catalytic zinc ion, inhibiting the proteolytic activity of matrix

metalloproteinases involved in several pathological conditions. Secondly, the
molecules are more effective towards MMPs involved in the vicious cycle, since bone
targeting concentrates the pharmacological agents at the desired active site allowing a
more potent effect without increasing the administered dose.[7]
The present work stems from the demonstrated MMPs inhibitory activity of some
bisphosphonic drugs already clinically used in osteoporosis disease.[20] In particular
tiludronate and zoledronate (Fig.1) have shown a very interesting activity against
MMP-2 and MMP-8, but none of them realized a satisfactory activity against MMP-9,
a fundamental target of the vicious cycle. This prompted us to develop a new series of
analogues, capable of selective bone targeting and disrupting the vicious cycle of
bone tumor growth through MMP-2 and MMP-9 inhibition.
In this context, we report herein the synthesis of arylamino methylene
bisphosphonates (Fig.1) and their biological evaluation against various MMPs
involved in skeletal formation (Table 1).
1-21
22
Figure 1. Chemical structures of bisphosphonate derivatives. Substituents X in 1–21
are presented in Table 1
In order to evaluate the mineral bond affinity of the synthesized compounds, we
decided to assess their binding to hydroxyapatite (HAP), the main constituent of bone.
This value, in fact, is considered as a measurement of the selective targeting effect of
bisphosphonic moiety towards the bone matrix surface.[21-23] We also evaluated the
effect of these compounds directly on J774 vitality as indicative of an osteoclast
activity. Promising results were expected based on the recent paper about
tetraphenyl(phenylamino)methylene bisphosphonates showing remarkable decrease in
osteoclastic bone resorption compared to the commercial drug alendronate.[24]
2. Results and discussion
2.1 Chemistry
Reference compounds tiludronate and zoledronate were prepared as previously
reported.[20]
Arylamino methylene bisphosphonic acids 1-22 were prepared as reported in Scheme
1. The one pot condensation of the suitable arylamines (1eq.) with diethyl phosphite

(1.2 eq.) in the presence of triethyl orthoformate (3 eq.), followed by de-alkylation
with trimethylsilyl bromide in anhydrous acetonitrile of the corresponding tetraethyl
ester intermediates 1a-22a led to the final acids 1-22.
1a-22a
1-22
Scheme 1. a) 160°C, 3-4h; b) (CH3)3SiBr, CH3CN, 48-72h, MeOH.
2.2 MMP activity
In the first part of this work we decided to use tiludronate as a template and to modify
its structure by replacement of sulfur atom with an isosteric amine function and
introduction of different substituents on the aromatic ring. These substituents were
selected in order to explore the role of lipophilic ( of Hansch), electronic ( of
Hammett) and steric (Es of Taft) parameters [25] on MMP inhibiting activity.
With this aim, a set of bisphosphonates have been synthesized and tested against
MMP-2, -8, -9 and -14. These particular MMP isoforms were selected due to their
recognized critical role in skeletal development and remodeling and their function in
the progression of tumor metastases.
Table 1. MMP inhibition activity (IC50 M)
X
Entry
MMP-2
MMP-8
MMP-9
MMP-14
tiludronate
7.2±0.5
7.0±1.3
>100
32±3
17.6±4.6
>100
>100
52±6
>100
>100
>100
30±7
20±6
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
30.5±1.6
12.6±0.1
>100
zoledronate
1
2
H
4-F
28.2±1.3
>100
>100
>100
>100
>100
3
4-Cl
6±3
2.4±0.4
15±2
3.9±1.7
26±5
4
4-Br
20±10
>100
5
4-NO2
4-CH3
4-CF3
4-CN
4-OH
3-NO2
3-CF3
3-F
36±4
>100
6
15±4
>100
>100
7
36±4
>100
>100
8
6.6±0.7
15.5±0.5
20.8±1.2
42±8
>100
>100
9
>100
>100
10
11
12
13
14
15
>100
>100
>100
>100
37.4±2.1
>100
>100
>100
3-Br
25.5±0.4
>100
20±5
3,4-Cl
3-NO2, 4-F
25±3
>100

>100
>100
>100
30±5
>100
>100
>100
33.5±2.1
43±14
76±7
16
17
18
19
20
21
22
2,4-Cl
4-Ph
2.8±0.4
2.1±0.5
2.8±0.1
>100
>100
444
39.9±0.2
4-Ph-O
55±18
87±4
4-(4-Cl-PhO)
4-(4-Br-PhO)
3-Ph
43±3
49.5±2.3
40±4
52±10
61±20
>100
6.4±1.4
6.0±1.2
69±8
Although the replacement of sulfur atom of tiludronate with an amine group is not
functional to the improvement of the MMP inhibitory activity profile (compounds 3
and 1), the specific MMPs effectiveness can be restored by introduction of different
groups on the aromatic ring of 1. The obtained results show, therefore, that the
interactions at the different enzymatic active sites can be modulated by a suitable
regulation of lipophilic, electronic and steric effects.
Overall micromolar inhibition has been obtained; compound 4 shows the highest
inhibitory activity towards MMP-8 and -14 (IC50=2.4µM and 3.9µM, respectively),
whereas compounds 19 and 22 are, respectively, the most potent on MMP-2 and
MMP-9, (IC50=2.1µM and 6.0µM). These BPs exhibit an improved inhibition profile
compared to tiludronate, confirming their promising capability to be more effective
towards specific bone MMP contribution in the vicious cycle.
Notably, this series of BPs shows a quite peculiar selectivity profile, in particular
towards MMP-2 (2, 7-13, 15, 18-20). Activity on this isoform seems to be affected by
the presence of electron withdrawing groups on the phenyl ring as well as by the
increase of lipophilicity of substituents as shown by 18-20.
Actually, the same behavior is also observed for the 4-hydroxy derivative 9; in this
case, however, the effect could be ascribed to the predicted capability to form
additional H bonds with amino acid residues in S1' pocket.
In contrast, the introduction of an electron donor substituent, as in the case of
compound 6, decreases the activity on all tested MMPs, even if a residual activity on
MMP-8 still remains.
Interestingly, the substitution of the flexible phenoxybenzene substituent (18-20) with
the more rigid biphenyl moiety (17 and 21) seems to reduce the activity against
MMP-2.
A particular behavior is shown from the substitution with chlorine. The presence of
this halogen in 4 or 2,4 positions (3, 16) provides compounds devoid of any MMP
enzymatic inhibition; however, moving chlorine from 2 to 3 position (14), a shift of
selective inhibitory activity from MMP-2, as shown by all 3-substituted analogues, to
MMP-8 and -14 occurs.
In summary, the newly synthesized bisphosphonic molecules show low-micromolar
IC50 values towards MMP-2, whose pivotal role is already proven in the progression
of cancer bone metastases. However, the broad spectrum activity of compounds 4 and
5 on all the tested MMP isoforms represents an additional benefit because of the key
role of MMP-2, -8, -9 and -14 in the physiopathology of skeletal renewal.
Furthermore compound 22 shows comparable activities on both MMP-2 and-9, and it
represents the most promising BP-MMPI candidate for disrupting the vicious cycle.
Worthy of note is the effectiveness of compound 22 towards MMP-9, whose
inhibition always encounters some difficulties, as previously observed for
sulfonamide BPs,[20] for tiludronate and zoledronate.

In order to rationalize the inhibition data and in particular to explain the different
activity profiles among tested MMPs, all ligands were docked into the X-ray structure
of MMP-2, -8, -9 and -14 applying a flexible docking approach, using Glide. Best
poses were subsequently minimized exploiting the Embrace module of MacroModel
to improve the positioning of ligands and calculate the different contributions to the
binding energy (obtained energies are reported in Tables 6-9 of the Supporting
Information).
The predicted binding mode of all active ligands towards MMP-8, -9 and -14, and
compounds 4, 5, 9, 18-20 and 22 to MMP-2, is well conserved: they occupy the S1’
site, providing the - stacking interaction with the His201 side chain (MMP-2
numbering). The bisphosphonate function is not able to chelate the zinc ion, therefore
just one phosphonic group coordinates the Zn2+, while the other one forms two H-
bonds with the NH of Leu164 and Ala165 or alternatively with Pro221 CO, similarly
to what observed for sulfonamide BP.[20] In Figure 2 the conserved binding mode of
broad spectrum inhibitors 4 and 5 into the MMP-2 active site is represented.
Figure 2. Superimposition of docked poses of compounds 4 (orange C atoms) and 5
(blue C atoms) into MMP-2. MMP-2 structure is represented as grey cartoon. Zinc ion
is depicted as a purple sphere. Ligands and most relevant receptor residues are
represented as sticks. H-bonds are represented as green dashed lines.
MMP-9 inhibition by compound 22 can be due to a favorable positioning of the
ligand that forms hydrophobic interactions in the proximal region of the S1’ site with
Tyr423, Leu188, Val398, Leu418 and provides an extended - stacking interaction
with His401; moreover, the amino methylene-bisphosphonate group forms a H-bond
network with Leu188 and Pro421 (Fig. 3).

Figure 3. Docked pose of compound 22 into MMP-9.
MMP-9 structure is represented as dark grey cartoon. Zinc ion is depicted as a purple
sphere. Ligands and most relevant receptor residues are represented as sticks. H-
bonds are represented as green dashed lines.
Activity data on MMP-2 indicate a dual trend: larger and more hydrophobic
compounds and smaller and hydrophilic ligands are both active. The first behavior is
in line with the results previously discussed for the other MMPs, while docking
results reveal that smaller and more hydrophilic ligands bind MMP-2 with the
bisphosphonate function chelating the zinc ion, and the aromatic ring occupying the
S1 and S3 site, instead of the deep S1’ site (Fig. 4). The only exception is represented
by compound 9 that, as already stated, binds the S1' site. This result is quite surprising
as all MMPs share a very conserved binding mode and the hydrophobic contact in the
S1’ site is widely considered as the leading interaction for MMPs inhibition.
Figure 4. Superimposition of docked poses of compounds 10 (magenta C atoms) and
11 (yellow C atoms) into MMP-2.

MMP-2 structure is represented as grey cartoon. Zinc ion is depicted as a purple
sphere and ligands as sticks. H-bonds are represented as green dashed lines. For
clarity the molecular surface of S1 and S3 sites is shown.
Analysis of the binding poses shows that small compounds positioning in the S1 and
S3 site is allowed by favorable interactions with Tyr155, Leu163, His166 and Phe157
residues. The different behavior of inhibitors towards MMP-2 with respect to MMP-8
and -14 can be at least partially attributed to the presence of Ser151 and Thr190
instead of Tyr155 in the S1 site of MMP-8 and MMP-14 respectively, that form much
more solvent exposed S1 site. The S1 site of MMP-9 is much smaller and does not
allow the binding of these ligands.
Targeting properties of the synthesized BPs at the bone site were investigated directly
on hydroxyapatite powder (HAP), in order to predict their possible in vivo interaction.
Among the different analytic techniques already described in the literature, such as
HPLC [22, 26] or NMR [23] determinations, we decided to quantify relative binding
affinity of this new set of bisphosphonates to hydroxyapatite through UV
spectrophotometry technique.[27] This sensitive method, in fact, easily allows to
detect and compare BPs affinity with that of tiludronate as shown in Table 2. We
performed the experiment by adding hydroxyapatite powder to each BP solution and
monitoring the residual amount of free compound remaining in solution by measuring
its absorbance at the suitable wavelength.
Table 2. In vitro HAP binding affinity
Entry
% adsorbed
tiludronate
51.5±2.1
58±5
1
2
61.2±0.1
64.2±0.6
49±4
3
4
76.0±1.8
23.9±0.3
42.5±0.1
76.00.6
88.40.1
57.8±1.6
50.3±1.6
63.6±0.7
70.1±1.1
53.5±0.5
66.2±2.8
56.8±1.3
60±7
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
66.2±0.2
52.1±0.6

60.7±1.6
66.4±0.3
51.6±0.1
20
21
22
All the analyzed BPs show similar binding affinity to HAP in comparison with the
already clinically used tiludronate and, as a consequence, probable same ability in
bone targeting. However, compounds 5, 8 and 9 stand out with their higher adsorption
percentages to the mineral powder, reaching 76% and 88%, respectively. Our data
suggest that only the introduction of the methyl substituent (entry 6) significantly
reduces the BP affinity for HAP.
To provide a more definite picture of bioavailability and drug-like properties of
studied compounds, some physicochemical parameters were calculated (Table 3).
Table 3. Physicochemical properties calculated for tested compounds.
Name
Tiludronate
Zoledronate
mol MW HBAa
HBDb
AlogP
0.82
-2.61
-0.57
-0.37
0.09
0.18
-0.68
-0.09
0.37
-0.69
-0.84
-0.68
0.37
-0.37
0.18
0.76
-0.47
0.76
0.95
0.99
1.65
1.74
0.95
0.34
RBc
4
4
4
4
4
4
5
4
5
4
4
5
5
4
4
4
5
4
5
6
6
6
5
4
PSAd
165.6
170.2
152.4
152.4
152.4
152.4
195.5
152.4
152.4
176.2
172.6
195.5
152.4
152.4
152.4
152.4
195.5
152.4
152.4
161.6
161.6
161.6
152.4
152.4
QPlogSwe
-1.02
1.21
318.6
257.1
267.1
285.1
301.6
346.0
312.1
281.1
335.1
292.1
282.1
312.1
335.1
285.1
346.0
336.0
330.1
336.0
343.2
359.2
393.7
438.1
343.2
317.2
2
3
2
2
2
2
2
2
2
3
3
2
2
2
2
2
2
2
2
3
3
3
2
2
2
3
3
3
3
3
3
3
3
3
4
3
3
3
3
3
3
3
3
3
3
3
3
3
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
-0.82
-1.16
-1.51
-1.61
-0.89
-1.27
-2.10
-1.78
-0.93
-0.88
-2.18
-1.16
-1.61
-2.08
-1.11
-1.78
-2.54
-2.46
-3.18
-3.25
-2.51
-1.61
a Number of hydrogen-bond acceptor atoms; b Number of hydrogen-bond donor atoms; c Number of
rotatable bonds; d Polar Surface Area; e Log of the calculated solubility in water.
It is worth noting that all compounds comply with the Lipinski’s Rule of five,
although recent research showed that the major disadvantage of the clinically utilized
BPs is their poor oral adsorption from the gastrointestinal tract (typically <1% is
adsorbed). [28] This behavior can be rationalized considering the high PSA value
(Veber et al. define a maximum value of 140 Å2 for drug-like compounds [29]),
mainly due to the bisphosphonic moiety. This functional group, however, is
responsible for the targeting on the bone tissue and therefore is mandatory for its
activity. One strategy for improving oral absorption and permeability through cell

membrane of these types of molecules could be the development of more lipophilic
derivatives. Our molecules 18-20 and 22 show the same HAP affinity but are more
lipophilic than zoledronate or tiludronate as demonstrated by the AlogP values and,
therefore, can be considered worth of following optimization.
2.3 Cell viability
Bisphosphonates bind avidly to exposed bone mineral around resorbing osteoclasts,
resulting in very high levels of bisphosphonate in the resorption lacunae.
At a cellular level, bisphosphonates are clearly targeting the osteoclasts and may
inhibit their functions in several ways: 1) inhibition of osteoclast recruitment, 2)
reduction of the osteoclast life span by direct apoptotic effect impairing osteoclast-
mediated bone resorption, and 3) reduction of the tumor-associated osteolysis that is
initiated by the development of skeletal metastases.[30]
We started analyzing the inhibitory effect of this new class of bisphosphonic
compounds on the mouse macrophage cell line J774, that is a model system already
used to screen BPs activity on osteoclast viability and functionality.[20, 31, 32] In
order to evaluate the selective effect on J774 cell line, we treated also a non-myeloid
derived cell type, via the human liver hepatocellular carcinoma cell line HepG2.
The one way ANOVA analysis of obtained data (Fig. 5) shows a selective activity of
these new BP-MMPIs against J774 cells. The observed lack of activity on HepG2
suggests that the effects of these BPs is not due to indiscriminate cytotoxicity.
Though it is difficult to establish a correlation between the data obtained by cell-based
assays and MMPs inhibition assay, compounds 9, 18-20 showed interesting effects on
both MMPs and J774 vitality; the two mechanisms could synergically act against the
progression of bone loss associated with bone metastases.
150
J774
HepG2
100
50
0
Ctrl Til 1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22
Figure 5. Graphical representation of cell % viability derived from MTT assay of
compounds 1-22 in comparison with tiludronate at 100 µM. The side-by-side
comparison between BP-MMPIs effects on J774 mouse macrophage cell line and
HepG2 hepatocellular carcinoma underlines the selectivity of action of BPs. Data
processed by one-way analysis of variance (Bonferroni's Multiple Comparison Test)
show that each BP-MMPI presents a statistical significance of P<0.001 from control.
3. Conclusions

A new series of bisphosphonates was synthesized and tested. A good effectiveness on
MMP-2 was reached by compounds 4, 9, 18, 19, 20 and 22, with the last one having
the same IC50 value on MMP-9. The activity on these two MMPs which have a key
role in the vicious cycle occurring in bone formation metastatic process, combined
with the selective inhibitory effects on J774 cells and the proven high affinity for
HAP, allows to consider these BP-MMPIs as new promising agents for bone
malignancy treatment.
4. Experimental section
4.1 Chemistry
Melting points were determined in open capillaries on a Gallenkamp electrothermal
apparatus and are uncorrected. Mass spectra were recorded on a HP MS 6890–5973
MSD spectrometer, electron impact 70 eV, equipped with a HP ChemStation or with
an Agilent LC-MS 1100 Series LC-MSD Trap System VL spectrometer, electrospray
ionization (ESI). 1H and 31P NMR spectra were recorded using the suitable deuterated
solvent on a Varian Mercury 300 NMR Spectrometer. Chemical shifts (δ) are
expressed as parts per million (ppm) and the coupling constants (J) in Hertz (Hz).
Microanalyses of solid compounds were carried out with a Eurovector Euro EA 3000
model analyzer; the analytical results are within ± 0.4% of theoretical values. Flash
column chromatography was performed using Geduran silica gel 60 Å (45–63 m).
Chemicals were purchased from Aldrich Chemicals (Milan, Italy) and were used
without any further purification.
UV quantification was effectuated with UV/Vis Spectrometer Perkin Elmer, Lambda
Bio 20.
Tetraethyl [(arylamino)methyl]-1,1-bisphosphonates. General procedure.
Triethylorthoformate (1.2 mmol), diethyl phosphite (3 mmol) and the suitable aniline
(1 mmol) were added in a 5 mL round bottom flask fitted with a distillation apparatus.
The resulting mixture was heated at 160°C under argon atmosphere, until the
evolution of EtOH was completed and the residue was dissolved in ethyl acetate. The
solvent was distilled off affording a crude yellow oil, which was purified on a silica
gel column using ethyl acetate as eluent. The titled compounds were obtained as
white solids in 32-89% yield.
Tetraethyl [(phenylamino)methyl]-1,1-bisphosphonate (1a). 53% yield; 1HNMR
(CDCl3): δ 1.21-1.30 (m, 12H, CH3), 4.07-4.27 (m, 10H, CH2, CH, NH), 6.67-6.70,
6.74-6.79, 7.15-7.25 (m, 2H, 1H, 2H, aromatics).
Tetraethyl [(4-fluorophenylamino)methyl]-1,1-bisphosphonate (2a). 56% yield;
1HNMR (CDCl3): δ 1.23-1.31 (m, 12H, CH3), 3.99-4.26 (m, 10H, CH2, CH, NH),
6.62-6.66, 6.86-6.93(m, 2H, 2H, aromatics); MS (ESI): m/z 420 [M+Na]+; MS2 m/z
(%): 254 (100); m/z 396 [M-H]- MS2 m/z (%): 153 (100).
Tetraethyl [(4-chlorophenylamino)methyl]-1,1-bisphosphonate (3a). 56% yield;
1HNMR (CDCl3): δ 1.23-1.32 (m, 12H, CH3), 3.99-4.23 (m, 10H, CH2, CH, NH),
6.60-6.64, 7.10-7.15 (m, 2H, 2H, aromatics); GC–MS m/z (%): 413 [M]+ (16), 415
[M+2]+ (5), 276 (100).
Tetraethyl [(4-bromophenylamino) methyl]-1,1-bisphosphonate (4a). 83% yield;
mp: 132-134 °C; 1H-NMR (CDCl3): δ 1.22 -1.31 (m, 12H, CH3), 1.62-1.90(bb 1H,
NH) 4.08-4.24 (m, 9H, CH2, CH), 6.55-6.60, 7.24-7.29 (m, 2H, 2H, aromatics); GC-
MS m/z (%): 459 [M+2]+ (15), 457 [M]+ (16), 320 [C11H16BrNO3P]+(100).

Tetraethyl [(4-nitrophenylamino)methyl]-1,1-bisphosphonate (5a). 89% yield;
1HNMR ([D6]DMSO): δ 1.09-1.20 (m, 12H, CH3), 3.99-4.12 (m, 8H, CH2), 4.88-
4.99 (m, 1H, CH), 7.05-7.08, 7.94-7.97 (m, 2H, 2H, aromatics), 7.40-7.44 (d, 1H,
NH).
Tetraethyl [(4-methylphenylamino) methyl]-1,1-bisphosphonate (6a). 87% yield;
1HNMR (CDCl3): δ 1.22-1.30 (m, 12H, CH2CH3), 2.23 (s, 3H, CH3), 3.94-3.99 (m,
1H, NH), 4.00-4.24 (m, 9H, CH2, CH), 6.59-6.61, 6.97-6.99 (m, 2H, 2H, aromatics);
GC–MS m/z (%): 393 [M]+ (17.5), 256 [C12H19NO3P]+ (100).
Tetraethyl [(4-trifluoromethyl-phenylamino)methyl]-1,1-bisphosphonate (7a).
71% yield; 1HNMR (CDCl3): δ 1.22-1.33 (m, 12H, CH3), 4.06-4.33 (m, 9H, CH2,
CH), 4.50-4.60 (bb, 1H, NH), 6.70-6.73, 7.40-7.43 (m, 2H, 2H, aromatics); MS (ESI):
m/z 470 [M+Na]+; MS2 m/z (%): 304 (100); m/z 446 [M-H]- MS2 m/z (%): 280 (100).
Tetraethyl [(4-cyano-phenylamino)methyl]-1,1-bisphosphonate (8a). 78% yield;
1HNMR (CDCl3): δ 1.17-1.29 (m, 12H, CH3), 4.02-4.25 (m, 9H, CH2, CH), 4.92 (bb,
1H, NH), 6.66-6.69, 7.39-7.42 (m, 2H, 2H, aromatics). GC–MS m/z (%): 404 [M]+
(10.8), 267 [C12H16N2O3P]+ (100).
Tetraethyl [(4-hydroxy-phenylamino)methyl]-1,1-bisphosphonate (9a). 57%
yield; 1HNMR (CDCl3): δ 1.20-1.27 (m, 12H, CH3), 4.06-4.21 (m, 9H, CH2, CH),
5.89 (bb, 2H, OH, NH), 6.60-6.64, 6.70-6.74 (m, 2H, 2H, aromatics). GC–MS m/z
(%): 395 [M]+ (23.1), 258 [C11H17NO4P]+ (100).
Tetraethyl [(3-nitrophenylamino)methyl]-1,1-bisphosphonate (10a). 53% yield;
1H NMR (CDCl3): δ 1.23-1.34 (m, 12H, CH3), 4.10-4.28 (m, 9H, CH2, CH), 4.54-
4.57 (m, 1H, NH), 6.97-7.01, 7.32-7.34, 7.52, 7.60-7.62 (m, 1H, 1H, 1H, 1H,
aromatics); MS (ESI): m/z 447 [M+Na]+; MS2 m/z (%): 419 (100); m/z 423 [M-H]-
MS2 m/z (%): 257 (100).
Tetraethyl [(3-trifluoromethylphenylamino)methyl]-1,1-bisphosphonate (11a).
82% yield; 1H NMR (CDCl3): δ 1.22-1.32 (m, 12H, CH3), 4.08-4.38 (m, 9H, CH2,
CH, NH), 6.83-6.89, 7.00-7.02, 7.25-7.30 (m, 2H, 1H, 1H, aromatics); MS m/z (%):
470 [M+Na]+, 304 (100); m/z 446 [M-H]- MS2 m/z (%): 280 (100).
Tetraethyl [(3-fluorophenylamino)methyl]-1,1-bisphosphonate (12a). 55% yield;
1H NMR (CDCl3): δ 1.22-1.32 (m, 12H, CH3), 4.02-4.27 (m, 10H, CH2, CH, NH),
6.37-6.48, 7.07-7.14 (m, 3H, 1H, aromatics); MS (ESI): m/z 396[M-H]-; 420
[M+Na]+; MS2 m/z (%): 254 (100); m/z 396 [M-H]- MS2 m/z (%): 153 (100).
Tetraethyl [(3-bromophenylamino)methyl]-1,1-bisphosphonate (13a).64% yield;
1H NMR (CDCl3): δ 1.24-1.33 (m, 12H, CH3), 4.02-4.20 (m, 10H, CH2, CH, NH),
6.59-6.62, 6.84-6.90, 7.00-7.06 (m, 1H, 2H, 1H, aromatics); MS (ESI): m/z 482
[M+2+Na]+, 480 [M+Na]+, MS2 m/z (%): 314 (100); MS (ESI): m/z 458 [M+2-H]-,
456 [M-H]- , MS2 m/z (%): 153 (100).
Tetraethyl [(3,4-dichlorophenylamino)methyl]-1,1-bisphosphonate (14a). 78%
yield; 1HNMR (CDCl3): δ 1.24-1.34 (m, 12H, CH3), 3.96-4.27 (m, 10H, CH2, CH,
NH), 6.51-6.55, 6.78-6.79, 7.19-7.22 (m, 1H, 1H, 1H, aromatics); MS (GC): m/z 451
[M+4]+ (1.7), 449 [M+2]+(8.6), 447 [M]+ (15), 310 [C11H15Cl2NO3P]+ (100).
Tetraethyl [(4-fluoro-3-nitrophenylamino)methyl]-1,1-bisphosphonate (15a).
64% yield; 1H NMR (CDCl3): δ 1.25-1.35 (m, 12H, CH3), 3.98-4.27 (m, 9H, CH2,
CH), 4.40-4.55 (bb, 1H, NH), 6.91-7.6.96, 7.08-7.14, 7.26-7.35 (m, 1H, 1H, 1H,
aromatics); MS (ESI): m/z 465 [M+Na]+, MS2 m/z (%): 437 (100), 441 [M-H]- MS2
m/z (%): 275 (100);
Tetraethyl [(2,4-dichlorophenylamino)methyl]-1,1-bisphosphonate (16a). 83%
yield; 1HNMR (CDCl3): δ 1.24-1.33 (m, 12H, CH3), 4.05-4.12 (m, 10H, CH2, CH,

NH),6.64-6.67, 7.15-7.18, 7.17-7.19 (m, 1H, 1H, 1H, aromatics); GC–MS m/z
(%):451 [M+4]+ (1.3), 449 [M+2]+ (7.5), 447 [M]+ (11.5), 310 (100).
Tetraethyl [(4-biphenylamino)methyl]-1,1-bisphosphonate (17a).32% yield;
1HNMR (CDCl3): δ 1.24-1.33 (m, 12H, CH3), 4.11-4.32 (m, 10H, CH2, CH, NH),
6.75-6.78, 7.24-7.29, 7.37-7.48, 7.51-7.55 (m, 2H, 1H, 4H, 2H, aromatics); GC–MS:
m/z 455 [M]+ (39), 318 (100).
Tetraethyl [(4-phenoxyphenylamino)methyl]-1,1-bisphosphonate (18a). 66%
yield; 1HNMR (CDCl3): δ 1.25–1.33 (m, 12H, CH3), 4.00–4.29 (m, 10H, CH2, CH,
NH), 6.67-6.72, 6.88-6.92, 6.99-7.04, 7.24-7.31 (m, 2H, 4H, 1H, 2H, aromatics); MS
(ESI) 494 [M+Na]+; MS2 m/z (%): 328 (100); m/z 470 [M-H]- MS2 m/z (%): 304
(100).
Tetraethyl [(4-(4’-chlorophenoxy)phenylamino)methyl]-1,1-bisphosphonate
(19a). 53% yield; 1HNMR (CDCl3): δ 1.25-1.33 (m, 12H, CH3), 4.04-4.25 (m, 10H,
CH2, CH, NH), 6.68-6.71, 6.82-6.90, 7.20-7.26 (m, 2H, 4H, 2H, aromatics); GC–MS:
m/z 507 [M+2]+ (10), 505 [M]+ (24); 368 (100).
Tetraethyl [(4-(4’-bromophenoxy)phenylamino)methyl]-1,1-bisphosphonate
(20a). 82% yield; 1HNMR (CDCl3): δ 1.17-1.25 (m, 12H, CH3), 4.00-4.19 (m, 9H,
CH2, CH), 4.41 (bb, 1H, NH), 6.64-6.72, 6.78-6.82, 7.25-7.29 (m, 4H, 2H, 2H,
aromatics). GC–MS: m/z 550 [M+2]+ (36), 548 [M]+ (33), 414 (100), 412 (98).
Tetraethyl [3-biphenylaminomethyl]-1,1-bisphosphonate (21a). 67% yield; 1H
NMR (CDCl3): δ 1.23-1.31 (m, 12H, CH3), 4.11-4.35 (m, 10H, CH2, CH, NH), 6.67-
6.70, 6.90-6.91, 7.00-7.02, 7.23-7.31, 7.33-7.45, 7.53-7.57 (m, 1H, 1H, 1H, 1H, 1H,
2H, 2H, aromatics); MS (ESI): m/z 478 [M+Na]+; MS2 m/z (%): 312 (100); m/z 454
[M-H]- MS2 m/z (%): 288 (100).
Tetraethyl [(naphthalen-1-ylamino)methyl]-1,1-bisphosphonate (22a). 81% yield;
1H NMR (CDCl3): δ 1.19-1.29 (m, 12H, CH3), 4.07-4.26 (m, 8H, CH2), 4.34-4.52 (m,
1H, CH), 4.85-4.91 (m, 1H, NH) 6.76-6.79, 7.29-7.38, 7.44-7.50, 7.78-7.90 (m, 1H,
2H, 2H, 2H, aromatics); MS (ESI): m/z 452 [M+Na]+; MS2 m/z (%): 286 (100); m/z
400 [M-Et]- MS2 m/z (%): 153 (100).
[(Arylamino)methyl]-1,1-bisphosphonic acids. General procedure. Anhydrous
trimethylsilylbromide (8 mmol) was carefully added to a solution of the
corresponding ester in anhydrous acetonitrile (4 mL) under argon atmosphere. The
resulting mixture was stirred for 24h, then CH3OH (2 mL) was added and stirring was
continued for additional 30 min. The solvent was distilled off and the oil residue was
washed with a mixture of hexanes and Et2O (1:1) to give the desired acids as solids in
21-96% yield.
[(Phenylamino)methyl]-1,1-bisphosphonic acid (1) [33]. 54 % yield; mp 114-116
°C; 1H NMR ([D6]DMSO): δ 3.92 (t, JHP 21.46 Hz, 1H, PCHP), 6.51-6.56, 6.70-6.72,
7.01-7.07 (m, 1H, 2H, 2H, aromatics), 7.10-7.80 (bb, H, OH, NH); 31PNMR
([D6]DMSO): δ=16.98 (d, JPH 21.37 Hz, 2P, PCHP); MS (ESI): m/z 266 [M-H]- ;
MS2: m/z (%): 248 (100); Anal. calcd for C7H11NO6P2∙2 H2O: C 27.73%, H 4.99%, N
4.62%, found: C 27.30%, H 4.71%, N 4.47%.
[(4-Fluorophenylamino)methyl]-1,1-bisphosphonic acid (2). 77% yield; mp: 168-
169 °C; 1HNMR ([D6]DMSO): δ 3.86 (t, JHP 21.46 Hz, 1H, PCHP), 6.69-6.74, 6.84-
6.90 (m, 2H, 2H, aromatics), 7.60-8.60 (bb, 5H, OH, NH); 31PNMR ([D6]DMSO): δ
=17.03 (d, JPH 21.36 Hz, 2P, PCHP); MS (ESI): m/z 284 [M-H]-; MS2: m/z (%): 266
(100); Anal. calcd for C7H10FNO6P2∙1/2 H2O : C 28.56%, H 3.77%, N 4.76%, found:
C 28.56%, H 3.92%, N 4.44%.

[(4-Chlorophenylamino)methyl]-1,1-bisphosphonic acid (3) [34]. 85% yield; mp:
155-156 °C; 1HNMR ([D6]DMSO): δ 3.85 (t, JHP 21.45 Hz, 1H, PCHP), 6.72-6.74,
7.03-7.06 (m, 2H, 2H, aromatics), 6.20-7.60 (bb, OH, NH); 31PNMR ([D6]DMSO): δ
=16.50 (d, JPH 21.36 Hz, 2P, PCHP); MS (ESI): m/z 302 [M+2-H]- (32), 300 [M-H]-
(100); MS2 : m/z (%): 282 (100); Anal. calcd for C7H10ClNO6P2∙2 H2O: C 24.90%, H
4.18%, N 4.15%, found: C 24.70%, H 4.22%, N 4.10%.
[(4-Bromophenylamino)methyl] 1,1 bisphosphonic acid (4). 90% yield; mp:107-
1
111 °C; HNMR ([D6]DMSO): δ 3.87 (t, JHP 21.46 Hz 1H, PCHP), 6.67-6.70, 7.14-
7.17 (m, 2H, 2H, aromatics), 8.11 (bb, 5H, NH, OH); 31PNMR ([D6]DMSO): δ 16.84
(d, JHP 21.36 Hz, PCHP); MS (ESI): m/z (%): 344 [M-H]- ; MS2 (%): 328 (100): Anal.
calcd for C7H10 BrNO6P2: C 24.30%, H 2.91%, N 4.05%, found: C 24.42%, H 3.04%,
N 4.23%.
[(4-Nitrophenylamino)methyl]-1,1-bisphosphonic acid (5). 37% yield; mp: 218-
219 °C; 1HNMR ([D6]DMSO): δ 4.10 (t, JHP 21.46 Hz 1H, PCHP), 6.60-8.80 (bb,
5H, OH, NH), 6.85-6.88, 7.92-7.95 (m, 2H, 2H aromatics); 31P NMR ([D6]DMSO): δ
=14.89 (d, JPH 21.36 Hz, 2P, PCHP); MS (ESI): m/z 311 [M-H]- ; MS2 : m/z (%): 293
(100); Anal. calcd for C7H12N2O8P2: C 26.77%, H 3.85%, N 8.92%, found: C 25.35%,
H 3.86%, N 8.10%.
[(4-Methylphenylamino)methyl]-1,1- bisphosphonic acid (6). 96% yield; mp: 213-
215°C; 1HNMR ([D6]DMSO): δ 2.12 (s, 3H, CH3), 3.84 (t, JHP 21.45, 1H, PCHP),
6.60-6.62, 6.84-6.87 (m, 2H, 2H, aromatics), 5.80-7.60 (bb, 5H, OH, NH); 31PNMR
([D6]DMSO): δ 17.13 (d, JPH 21.36 Hz, 2P, PCHP); MS (ESI) m/z: 280 [M-H]-; MS2
m/z (%): 262 (100); Anal. calcd for C8H13NO6P2: C 34.18%, H 4.66%, N 4.98%,
found: C 34.07%, H 4.77%, N 4.75%.
[(4-Trifluoromethylphenylamino)methyl]-1,1-bisphosphonic acid (7). 21% yield;
mp:184-186 °C; 1HNMR ([D6]DMSO): δ 4.02 (t, JHP 20.08 Hz, 1H, PCHP), 5.40-
7.40 (bb, 5H, OH, NH), 6.76-6.96, 7.61-7.63 (m, 2H, 2H, aromatics); 31PNMR
([D6]DMSO): δ =16.53 (d, JPH 21.36, 2P, PCHP); MS (ESI): m/z 334 [M-H]- ; MS2 :
m/z (%): 316 (100); Anal. calcd for C8H10F3NO6P2∙2 H2O: C 25.89%, H 3.80%, N
3.77%, found: C 26.01%, H 3.99%, N 3.73%.
[(4-Cyanophenylamino)methyl]-1,1-bisphosphonic acid (8). 93% yield; mp: 215
°C (dec); 1HNMR ([D6]DMSO): δ 4.14 (t, JHP 20.10 Hz, 1H, PCHP), 6.85-6.88, 7.38-
7.41 (m, 2H, 2H, aromatics), 9.84 (bb, 5H, OH, NH); 31PNMR ([D6]DMSO): δ
=15.62 (d, JPH 21.37, 2P, PCHP); MS (ESI): m/z 291 [M-H]- ; MS2 : m/z (%): 273
(100); Anal. calcd for C8H10N2O6P2∙2 H2O: C 29.28%, H 4.30%, N 8.54%, found: C
29.19%, H 4.17%, N 8.96%.
[(4-Hydroxyphenylamino)methyl]-1,1-bisphosphonic acid (9). 92% yield; mp:
229-230 °C; 1HNMR ([D6]DMSO): δ 3.71 (t, JHP 21.30 Hz, 1H, PCHP), 6.49-6.52,
6.57-6.60 (m, 2H, 2H, aromatics), 7.51 (bb, 6H, OH, NH). 31PNMR ([D6]DMSO): δ
=17.17 (d, JPH 21.36, 2P, PCHP); MS (ESI): m/z 282 [M-H]- ; MS2 : m/z (%): 264
(100); Anal. calcd for C7H11NO7P2∙2 H2O: C 26.34%, H 4.74%, N 4.39%, found: C
26.53%, H 4.43%, N 4.55%.
[(3-Nitrophenylamino)methyl]-1,1-bisphosphonic acid (10) [35]. 64% yield; mp:
204-206 °C; 1HNMR ([D6]DMSO): δ 4.05 (t, JHP 21.46 Hz, 1H, PCHP), 7.21-7.34,
7.59 (m, 3H, 1H aromatics), 8.00-9.6 (bb, 5H, OH, NH); 31PNMR ([D6]DMSO): δ
=16.10 (d, JPH 21.36 Hz, 2P, PCHP); MS (ESI): m/z 311[M-H]- ; MS2 : m/z (%): 293
(100); Anal. calcd for C7H12N2O8P2 ∙1/2 H2O : C 26.18%, H 3.45%, N 8.72%, found:
C 26.05%, H 3.68%, N 8.30%.
[(3-Trifluoromethylphenylamino)methyl]-1,1-bisphosphonic acid (11). 70% yield;
mp: 212-214 °C; 1HNMR ([D6]DMSO): δ 3.96 (t, JHP 21.46 Hz, 1H, PCHP), 6.60-

7.80 (bb, 5H, OH, NH), 6.79-6.81, 6.97-7.06, 7.21-7.26 (m, 1H, 2H, 1H, aromatics);
31PNMR ([D6]DMSO): δ= 16.43 (d, JPH 21.37, 2P, PCHP); MS (ESI): m/z 334 [M-H]-
; MS2 : m/z (%): 316 (100); Anal. calcd for C8H10F3NO6P2: C 28.67%, H 3.01%, N
4.18%, found: C 28.90 %, H 3.21 %, N 3.98 %.
[(3-Fluorophenylamino)methyl]-1,1-bisphosphonic acid (12). 74% yield; mp:197-
198 °C; 1HNMR ([D6]DMSO): δ 3.92 (t, JHP 21.87 Hz, 1H, PCHP), 6.26-6.31, 6.53-
6.56, 6.99-7.07 (m, 1H, 2H, 1H, aromatics), 10.60-11.80 (bb, 5H, OH, NH); 31PNMR
([D6]DMSO): δ =16.55 (d, JPH 21.36, 2P, PCHP); MS (ESI): m/z 284 [M-H]- ; MS2 :
m/z (%): 266 (100); Anal. calcd for C7H10 FNO6P2: C 29.49%, H 3.54%, N 4.91%,
found: C 29.88%, H 3.31%, N 4.84%.
[(3-Bromophenylamino)methyl]-1,1-bisphosphonic acid (13) [35]. 95% yield;
mp:185-187°C; 1HNMR ([D6]DMSO): δ 3.89 (t, JHP 22.01 Hz, 1H, PCHP), 6.65-
6.74, 6.91-6.97 (m, 2H, 2H, aromatics) 10.20-11.80 (bb, 5H, OH, NH); 31PNMR
([D6]DMSO): δ =16.45 (d, JPH 21.37, 2P, PCHP); MS (ESI): m/z 346 [M+2-H]-, 344
[M-H]- ; MS2 : m/z (%): 326 (100); Anal. calcd for C7H10 BrNO6P2: C 24.30%, H
2.91%, N 4.05%, found: C 24.52%, H 3.02%, N 4.43%.
[(3,4-Dichlorophenylamino)methyl]-1,1-bisphosphonic acid (14) [34]. 74% yield;
mp:134-135 °C; 1HNMR ([D6]DMSO): δ 3.89 (t, JHP 21.45, 1 H, PCHP), 6.72–6.75,
6.96, 7.18–7.21 (m, 1H, 1H, 1H, aromatics), 8.00-9.80 (bb, 5H, OH, NH); 31PNMR
([D6]DMSO): δ =16.00 (d, JPH 21.37 Hz, 2P, PCHP); MS (ESI): m/z 334 [M-H]-;
MS2: m/z (%): 316 (100); Anal. calcd for C7H9Cl2NO6P2∙H2O: C 23.75%, H 3.13%, N
3.96%, found: C 24.16%, H 3.48%, N 4.03%.
[(4-Fluoro-3-nitrophenylamino)methyl]-1,1-bisphosphonic acid (15). 85% yield;
mp:155-156 °C (dec); 1HNMR ([D6]DMSO): δ 3.97 (t, JHP 21.45 Hz, 1H, PCHP),
7.21-7.24, 7.41-7.42 (m, 2H, 1H, aromatics), 8.00-10.00 (bb, 5H, OH, NH); 31P NMR
([D6]DMSO): δ = 16.03 (d, JPH 21.36, 2P, PCHP); MS (ESI): m/z 329[M-H]- ; MS2 :
m/z (%): 311 (100); Anal. calcd for C7H11FN2O8P2∙H2O: C 24.15%, H 3.18%, N
8.05%, found: C 24.51%, H 3.24%, N 8.24%.
[(2,4-Dichlorophenylamino)methyl]-1,1-bisphosphonic acid (16) [35]. 82 % yield;
mp: 242-243°C; 1HNMR ([D6]DMSO): δ 3.99 (t, JHP 20.11 Hz, 1H, PCHP), 4.70-
5.00 (bb, 1H, NH), 6.84-6.87, 7.15-7.18, 7.37-7.38 (m, 1H, 1H, 1H, aromatics), 8.60-
10.00 (bb, 4H, OH); 31PNMR ([D6]DMSO): δ =15.51 (d, JPH 18.31, 2P, PCHP); MS
(ESI): m/z 336 [M+2-H]- (65), 334[M-H]- (100); MS2 : m/z (%): 316 (100); Anal.
calcd for C7H9Cl2NO6P2∙1/2 H2O: C 24.37%, H 2.92%, N 4.06%, found: C 24.72%, H
2.75%, N 3.81%.
[(4-Biphenylamino)methyl]-1,1-bisphosphonic acid (17) [35]. 66% yield; mp: 232-
233°C; 1HNMR ([D6]DMSO): δ 3.97 (t, JHP 21.45 Hz, 1H, PCHP), 6.80-6.83, 7.17-
7.22, 7.33-7.40, 7.52-7.54 (m, 2H, 1H, 4H, 2H, aromatics), 6.40-8.40 (bb, 5H, OH,
NH ); 31PNMR ([D6]DMSO): δ=16.73 (d, JPH 21.36 Hz, 2P, PCHP); MS (ESI): m/z
342 [M-H]- ; MS2 : m/z (%): 324 (100); Anal. calcd for C13H15NO6P2: C 45.49%, H
4.41%, N 4.08%, found: C 45.24%, H 4.48%, N 3.97%.
[(4-Phenoxyphenylamino)methyl]-1,1-bisphosphonic acid (18). 80% yield; mp:
240°C (dec); 1HNMR ([D6]-DMSO): δ = 3.88 (t, 1H, JHP 21.40 Hz), 6.74-6.85, 6.96-
7.01, 7.25-7.31 (m, 6H, 1H, 2H, aromatics), 7.40-8.80 (bb, 5H, OH, NH); 31PNMR
([D6]-DMSO): δ = 16.93 (d, 2P, JPH = 21.36); ESI-MS, m/z: 358 [M-H]-; MS2, m/z
(%): 340 (100); Anal. calcd for C13H15NO7P2: C 43.47%, H4.21%, N 3.90%, found: C
43.09%, H 4.25%, N 3.71%.
[(4-(4’-Chlorophenoxy)phenylamino)methyl]-1,1-bisphosphonic acid (19). 83%
yield; mp: 218-219°C; 1HNMR ([D6]DMSO): δ 3.90 (t, JHP 21.45 Hz, 1H, PCHP),
6.76-6.87, 7.30-7.35 (m, 6H, 2H, aromatics), 7.60-8.40 (bb, 5H, OH, NH); 31P NMR

([D6]DMSO): δ=16.83 (d, JPH 21.37, 2P, PCHP); MS (ESI): m/z 392 [M-H]- ; MS2 :
m/z (%): 374 (100); Anal. calcd for C13H14ClNO7P2: C 39.66%, H3.58%, N 3.56%,
found: C 39.94%, H 3.74%, N 3.80%.
[(4-(4’-Bromophenoxy)phenylamino)methyl]-1,1-bisphosphonic acid (20). 98%
yield; mp:256-257 °C; 1HNMR ([D6]DMSO): δ 3.97 (t, JHP 21.60 Hz, 1H, PCHP),
6.76-6.79, 7.42-7.45 (m, 6H, 2H, aromatics), 9.14 (bb, 5H, OH, NH); 31P NMR
([D6]DMSO): δ=16.90 (d, JPH 21.56, 2P, PCHP); MS (ESI): m/z 462 [M+2+Na]+ , 460
[M+Na]+, MS2 : m/z (%): 380 (100), 378 (87); Anal. calcd for C13H14BrNO7P2∙ H2O:
C 34.23%, H 3.54%, N 3.07%, found: C 33.89%, H 3.47%, N 3.34%.
[(3-Biphenylamino)methyl]-1,1-bisphosphonic acid (21) [36]. 79% yield; mp: 212-
214 °C; 1HNMR ([D6]DMSO): δ 4.04 (t, JHP 21.46 Hz, 1H, PCHP), 5.80-7.00 (bb,
5H, OH, NH) 6.72-7.60 (m, 9H, aromatics); 31PNMR ([D6]DMSO): δ =16.93 (d, JPH
21.37 Hz, 2P, PCHP); MS (ESI): m/z 342 [M-H]- ; MS2 : m/z (%): 324 (100); Anal.
calcd for C13H15NO6P2∙2 H2O: C 41.17%, H 5.05%, N 3.69%, found: C 41.65%, H
4.97%, N 3.77%.
[(Naphthalen-1-ylamino)methyl]-1,1-bisphosphonic acid (22). 95% yield; mp 97-
99 °C; 1HNMR ([D6]DMSO): δ 4.16 (t, JHP 20.91 Hz, 1H, PCHP), 6.71-7.93 (m, 7H,
aromatics), 8.40-10.00 (bb, 5H, OH, NH); 31PNMR ([D6]DMSO): δ = 16.36(d, JPH
21.36 Hz, 2P, PCHP); MS (ESI): m/z 316 [M-H]- ; MS2 : m/z (%): 298 (100); Anal.
calcd for C11H13NO6P2∙2 H2O: C 37.41%, H 4.85%, N 3.97%, found: C 37.32%, H
4.44%, N 4.12%.
[(4-Chlorophenylthio)methyl]-1,1-bisphosphonic acid (Tiludronate). 54% yield;
mp:174–177°C; 1HNMR ([D6]DMSO): δ= 3.09 (t, JHP 19.8 Hz, 1H, PCHP), 6.20-7.80
(bb, 4H, OH), 7.30-7.33, 7.49-7.52 (m, 2H, 2H, aromatics); 31PNMR ([D6]DMSO):
δ= 14.46 (d, JPH18.30, 2P, PCHP); MS (ESI) m/z: 318 ([M+2]–, 34), 316 ([M]–, 100);
MS2m/z (%): 299 (100); Anal. calcd for C7H9ClO6P2S: C 26.39%, H 2.85%, found: C
26.73%, H 3.05%.
1-Hydroxy-2-(imidazol-1-yl)-ethylidene-1,1-bisphosphonic acid (Zoledronate).
38% yield; mp: 220–223°C; 1HNMR (D2O): δ=4.49– 4.56 (m, 2H, NCH2C), 7.21,
7.36, 8.56 (s, 1H, 1H, 1H aromatics); 31PNMR (D2O): δ=14.83 (bb, 2P, PCP). MS
(ESI) m/z: 271 [M–H]–; MS2 m/z (%): 189 (100); Anal. calcd for C5H10N2O7P2∙ H2O:
C 20.70%, H 4.17%, N 9.66, found: C 20.41%, H 3.80%, N 9.38%.
4.2 Biological Methods
MMP inhibition assays.[37] Recombinant human pro-gelatinase A (pro- MMP-2) and
enzymes consisting of the catalytic domain of MMP-9 and MMP-14 were purchased
from Calbiochem, while catalytic domain of MMP-8 was purchased from Biomol.
Pro-MMP-2 was activated immediately prior to use with p-aminophenylmercuric
acetate (APMA, 2 mM) for 1 h at 37 °C. The assays were performed in triplicate in a
total volume of 100 µL per well in 96-well microtitre plates (Corning, white, NBS).
For assay measurements, inhibitor stock solutions (DMSO, 10 mM) were diluted to
six different concentrations (0.1 nM–100 µM) in fluorometric assay buffer (50 mM
Tris-HCl, pH 7.5, 200 mM NaCl, 1 mM CaCl2, 1 µM ZnCl2, 0.05% NaN3, 0.05%
Brij-35, and 1% DMSO). Activated enzyme and inhibitor solutions were incubated in
the assay buffer for 30 min at 25 °C before the addition of the fluorogenic substrate
solution (Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2, Calbiochem, 2.5 µM final
concentration). After further incubation for 2–4 h at 37 °C, the hydrolysis was
stopped by the addition of a 3% acetic acid solution, and the fluorescence was

measured (λex=340 nm, λem= 405 nm) using a PERKIN–ELMER Victor V3 plate
reader. Control wells lacked inhibitor. The MMP inhibition activity was expressed in
relative fluorescent units (RFU). Percent of inhibition was calculated from control
reactions without the inhibitor, and IC50 values were determined using GraphPad
PRISM version 5.0 software.[38]
MTT assay for cell viability.[39, 40] The murine macrophage-like J774 A.1 and
human liver carcinoma HepG2 cell lines were obtained from the ITCC (Genova,
Italy). Cells were grown in DMEM (J774 A.1) or MEM (HepG2) medium
supplemented with 10% fetal bovine serum, 10 UmL-1 penicillin, 100 mgmL-1
streptomycin, and 2 mM L-glutamine in a 5% CO2 atmosphere at 37 °C. Cells were
seeded at a density of 1-5.104 cells/well into 96-well flat bottom culture plates
containing 50 µL of the test compounds, previously half-log serially diluted (from
0.316 µM to 100 nM), in a final volume of 100 µL. The bisphosphonates were
dissolved in DMSO (1% final concentration; DMSO carrier had no effect on cell
proliferation). Control wells lacked inhibitor. After 48 h of incubation at 37°C in a 5%
CO2 atmosphere, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide
(MTT, 5 mgmL-1 stock solution) was added to a final concentration of 0.5 mgmL-1.
As control for background absorbance, six wells of cells were lysed by adding Triton
X-100 (0.1% v/v final concentration) immediately prior to the addition of MTT
reagent. After incubation under the same conditions for further 3–4 h, the culture
medium was removed, the insoluble product dissolved by the addition of 100 µL of
solvent (50% DMSO, 50% EtOH v/v), and the absorbance of the well was measured
at 570 nm using a PERKIN–ELMER Victor V3 plate reader. Cell growth inhibition
was then calculated using Equation (1),
A  Ab
Ac  Ab
(1)
V% 
100
where V% is the percentage of cell viability, A is the absorbance of treated cultures,
Ab is the absorbance of background control, and Ac is the absorbance of control
cultures. IC50 values were determined from dose-response curves using GraphPad
PRISM version 5.0.
4.3 Hydroxyapatite affinity measurement
A solution of each synthesized bisphosphonic acid (100 µM) in Tris-HCl buffer (50
mM pH=7.5) was prepared. It was divided into two aliquots (5 ml) in order to obtain
the experiment in duplicate, added and stirred with HAP (4 mg) at 37 °C.
After 2 h, the mixture aliquots were centrifuged (10 min, 8000 rpm), the decanted
solutions were filtered (0.22 µm syringe's filter) and the absorbance of the clear
solutions was recorded and quantified by calibration lines. The resulting λ max, ε
value, angular coefficient of the calibration line (m) and the coefficient of
determination (R2) related are listed in the Supporting Information.
4.4 Computational methods
All calculations were performed on a DELL T5500 workstation, equipped with two
Intel® Xeon® E5630 2.53 GHz processors.
All compounds were manually built in Maestro version 9.3.5 [41], exploiting the Built
facility. Ligands were submitted to Epik v. 2.3 [41-43] to calculate the protonation

state at neutral pH. All structures were minimized to a derivative convergence of
0.001 kJÅ-1mol-1, using the Truncated Newton Conjugate Gradient (TNCG)
minimization algorithm, the OPLS2005 force field, and the GB/SA water solvation
model implemented in MacroModel version 9.9.[41]
Conformational searches, applying the Mixed torsional/Low-mode sampling and the
automatic set-up protocol, were carried out on all minimized ligand structures in order
to obtain the global minimum geometry of each molecule, which was then used as the
starting conformation for docking calculations with Glide version 5.8.[41, 44-46]
Three-dimensional coordinates of MMP-2 (PBD ID: 1QIB), MMP-8 (PBD ID:
1ZVX), MMP-9 (PBD ID: 1GKC) and MMP-14 (PBD ID: 3MA2), were downloaded
from the Brookhaven Protein Data Bank.[38]
MMPs 3D structures were submitted to the Protein Preparation routine in Maestro
that allows fixing of receptor structures, eliminating water molecules and possible
ligands, fixing bond orders, adding hydrogen atoms, and ionizing lysine, arginine,
glutamate, and aspartate residues. To optimize the hydrogen bond network, histidine
tautomers and ionization states were predicted, 180° rotations of the terminal angle
of Asn, Gln, and His residues were assigned, and hydroxyl and thiol hydrogen atoms
were sampled. For each structure, a brief relaxation was performed using an all-atom
constrained minimization carried out with the OPLS-2005 force field to reduce steric
clashes that may exist in the original PDB structures. The minimization was
terminated when the energy converged or the root mean square deviation (RMSD)
reached a maximum cut-off of 0.30 Å.
Glide energy grids were generated for each receptor structure using the MMP-8
crystallographic ligand as the center of the grid as a reference for all MMPs. The size
of the box was determined automatically on the basis of the ligand dimensions. The
global minimum geometry of each ligand was submitted to flexible docking
calculations in the previously prepared proteins. The van der Waals radii for non-
polar ligand atoms were scaled by a factor of 0.8, thereby decreasing penalties for
close contacts. Receptor atoms were not scaled. A first docking run was carried out
applying the Standard Precision (SP) settings of Glide. Ten poses were saved and the
best ranking pose for each ligand was submitted to minimization process applying the
Embrace module of MacroModel. Embrace performs a multiple minimization on each
pre-positioned ligand and the receptor. All structures were fully minimized to a
gradient convergence of 0.05 kJÅ-1mol-1, using the PRCG minimization algorithm,
the OPLS2005 force field and the GB/SA water solvation model. The Energy
difference mode was applied to calculate the binding energy. Physicochemical
properties were calculated using Canvas [41].
Acknowledgements
The authors are grateful to Dr. Maria Teresa Rubino for preliminary experiments.
This work was accomplished thanks to the financial support of the Ministero
dell’Istruzione, dell’Università e della Ricerca (MIUR 2007 JERJPC_003).
Keywords
Matrix metalloproteinases inhibitors, zinc binding group, bone targeting,
bisphosphonates
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Captions
Figure 1. Chemical structures of bisphosphonate derivatives. Substituents X in 1–21
are presented in Table 1
Scheme 1. a) 160°C, 3-4h; b) (CH3)3SiBr, CH3CN, 48-72h, MeOH.
Figure 2. Black and White Superimposition of docked poses of compounds 4 (light
grey C atoms) and 5 (dark grey C atoms) into MMP-2. MMP-2 structure is
represented as grey cartoon. Zinc ion is depicted as a sphere. Ligands and most
relevant receptor residues are represented as sticks. H-bonds are represented as
dashed lines.
Figure 2. Superimposition of docked poses of compounds 4 (orange C atoms) and 5
(blue C atoms) into MMP-2. MMP-2 structure is represented as grey cartoon. Zinc ion
is depicted as a purple sphere. Ligands and most relevant receptor residues are
represented as sticks. H-bonds are represented as green dashed lines.
Figure 3. Black and White Docked pose of compound 22 into MMP-9.
MMP-9 structure is represented as dark grey cartoon. Zinc ion is depicted as a sphere.
Ligands and most relevant receptor residues are represented as sticks. H-bonds are
represented as dashed lines.
Figure 3. Docked pose of compound 22 into MMP-9.
MMP-9 structure is represented as dark grey cartoon. Zinc ion is depicted as a purple
sphere. Ligands and most relevant receptor residues are represented as sticks. H-
bonds are represented as green dashed lines.
Figure 4. Black and White Superimposition of docked poses of compounds 10 (light
grey C atoms) and 11 (dark grey C atoms) into MMP-2.
MMP-2 structure is represented as grey cartoon. Zinc ion is depicted as a sphere and
ligands as sticks. H-bonds are represented as dashed lines. For clarity the molecular
surface of S1 and S3 sites is shown.
Figure 4. Superimposition of docked poses of compounds 10 (magenta C atoms) and
11 (yellow C atoms) into MMP-2.
MMP-2 structure is represented as grey cartoon. Zinc ion is depicted as a purple
sphere and ligands as sticks. H-bonds are represented as green dashed lines. For
clarity the molecular surface of S1 and S3 sites is shown.
Figure 5. Graphical representation of cell % viability derived from MTT assay of
compounds 1-22 in comparison with tiludronate at 100 µM. The side-by-side
comparison between BP-MMPIs effects on J774 mouse macrophage cell line and
HepG2 hepatocellular carcinoma underlines the selectivity of action of BPs. Data
processed by one-way analysis of variance (Bonferroni's Multiple Comparison Test)
show that each BP-MMPI presents a statistical significance of P<0.001 from control.

Table 1. MMP inhibition activity (IC50 M)
X
Entry
MMP-2
MMP-8
MMP-9
MMP-14
tiludronate
7.2±0.5
7.0±1.3
>100
32±3
17.6±4.6
>100
>100
52±6
>100
>100
>100
30±7
20±6
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
55±18
87±4
>100
6.0±1.2
30.5±1.6
12.6±0.1
>100
zoledronate
1
2
H
4-F
28.2±1.3
>100
>100
>100
>100
>100
3
4-Cl
6 ± 3
2.4±0.4
15±2
3.9±1.7
26±5
4
4-Br
20±10
>100
5
4-NO2
4-CH3
4-CF3
36±4
>100
6
15±4
>100
>100
7
36±4
>100
>100
8
4-CN
6.6±0.7
15.5±0.5
20.8±1.2
42±8
>100
>100
9
4-OH
>100
>100
10
11
12
13
14
15
16
17
18
19
20
21
22
3-NO2
3-CF3
>100
>100
>100
>100
3-F
37.4±2.1
>100
>100
>100
3-Br
25.5±0.4
>100
20±5
3,4-Cl
3-NO2, 4-F
2,4-Cl
4-Ph
25±3
>100
>100
>100
>100
>100
30±5
33.5±2.1
43±14
76±7
2.8±0.4
2.1±0.5
2.8±0.1
>100
444
39.9±0.2
43±3
4-Ph-O
4-(4-Cl-PhO)
4-(4-Br-PhO)
3-Ph
49.5±2.3
40±4
52±10
61±20
6.4±1.4
69±8

Table 2. In vitro HAP binding affinity
Entry
% adsorbed
tiludronate
51.5±2.1
58±5
1
2
61.2±0.1
64.2±0.6
49±4
3
4
76.0±1.8
23.9±0.3
42.5±0.1
76.00.6
88.40.1
57.8±1.6
50.3±1.6
63.6±0.7
70.1±1.1
53.5±0.5
66.2±2.8
56.8±1.3
60±7
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
66.2±0.2
52.1±0.6
60.7±1.6
66.4±0.3
51.6±0.1

Table 3. Physicochemical properties calculated for tested compounds.
Name
mol MW HBAa
HBDb
AlogP
0.82
-2.61
-0.57
-0.37
0.09
0.18
-0.68
-0.09
0.37
-0.69
-0.84
-0.68
0.37
-0.37
0.18
0.76
-0.47
0.76
0.95
0.99
1.65
1.74
0.95
0.34
RBc
4
4
4
4
4
4
5
4
5
4
4
5
5
4
4
4
5
4
5
6
6
6
5
4
PSAd
165.6
170.2
152.4
152.4
152.4
152.4
195.5
152.4
152.4
176.2
172.6
195.5
152.4
152.4
152.4
152.4
195.5
152.4
152.4
161.6
161.6
161.6
152.4
152.4
QPlogSwe
-1.02
1.21
Tiludronate
Zoledronate
318.6
257.1
267.1
285.1
301.6
346.0
312.1
281.1
335.1
292.1
282.1
312.1
335.1
285.1
346.0
336.0
330.1
336.0
343.2
359.2
393.7
438.1
343.2
317.2
2
3
2
2
2
2
2
2
2
3
3
2
2
2
2
2
2
2
2
3
3
3
2
2
2
3
3
3
3
3
3
3
3
3
4
3
3
3
3
3
3
3
3
3
3
3
3
3
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
-0.82
-1.16
-1.51
-1.61
-0.89
-1.27
-2.10
-1.78
-0.93
-0.88
-2.18
-1.16
-1.61
-2.08
-1.11
-1.78
-2.54
-2.46
-3.18
-3.25
-2.51
-1.61

O
P
O
P
Figure 1
OH
OH
OH
OH
S
N
OH
OH
N
OH
OH
P
P
HO
O
zoledronate
Cl
O
tiludronate
O
H
O
H
N
OH
OH
OH
OH
N
P
P
OH
OH
OH
OH
P
P
X
O
O
1-21
22

O
P
O
P
Scheme1
OEt
OEt
OEt
O
H
N
H
N
OEt
OEt
OH
OH
a
b
OEt
OEt
+
+
Ar NH2
H
H P
Ar
Ar
O P
O P
OEt
OH
OEt
OH

Figure 2 color

Figure 2 bw