Novel bisphosphonates with antiresorptive effect in bone mineralization and osteoclastogenesis

Article abstract of DOI:10.1016/j.ejmech.2018.08.044

Bisphosphonates such as zoledronic, alendronic and risedronic acids are a class of drugs clinically used to prevent bone density loss and osteoporosis. Novel P-C-P bisphosphonates were synthesized for targeting human farnesyl pyrophosphate synthase (hFPPS) and human geranylgeranyl pyrophosphate synthase (hGGPPS), key enzymes of the mevalonate pathway, and capable of anti-proliferative action on a number of cell lines (PC3, MG63, MC3T3, RAW 264.7, J774A.1, bone marrow cells and their co-colture with PC3) involved in bone homeostasis, bone formation and death. Among sixteen compounds, [1-hydroxy-2-(pyrimidin-2-ylamino)ethane-1,1-diyl]bis(phosphonic acid) (10) was effective in reducing PC3 and RAW 264.7 cell number in crystal-violet and cell-dehydrogenase activity assays at 100 μM concentration. 10 reduced differentiated osteoclasts number similarly with zoledronic acid in osteoclastogenesis assay. At nanomolar concentrations, 10 was more effective than zoledronic acid in inducing mineralization in MC3T3 and murine bone marrow cells. Further, 10 significantly inhibited the activity of hFPPS showing an IC₅₀ of 0.31 μM and a remarkable hydroxyapatite binding of 90%. Docking calculations were performed identifying putative interactions between some representative novel bisphosphonates and both hFPPS and hGGPPS. Then, 10 was found to behave similarly or even better than zoledronic acid as a anti-resorptive agent.

Full text of DOI:10.1016/j.ejmech.2018.08.044

Accepted Manuscript
Novel bisphosphonates with antiresorptive effect in bone mineralization and
osteoclastogenesis
Salvatore Savino, Annamaria Toscano, Rosa Purgatorio, Emanuela Profilo,
Antonio Laghezza, Paolo Tortorella, Mariacristina Angelelli, Rosa Scala, Domenico
Tricarico, Carlo Marya Thomas Marobbio, Filippo Perna, Paola Vitale, Mariangela
Agamennone, Vincenzo Dimiccoli, Anna Tolomeo, Antonio Scilimati
PII:
S0223-5234(18)30715-3
10.1016/j.ejmech.2018.08.044
EJMECH 10655
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 May 2018
Revised Date: 18 July 2018
Accepted Date: 16 August 2018
Please cite this article as: S. Savino, A. Toscano, R. Purgatorio, E. Profilo, A. Laghezza, P. Tortorella,
M. Angelelli, R. Scala, D. Tricarico, C.M. Thomas Marobbio, F. Perna, P. Vitale, M. Agamennone,
V. Dimiccoli, A. Tolomeo, A. Scilimati, Novel bisphosphonates with antiresorptive effect in bone
mineralization and osteoclastogenesis, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2018.08.044.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Novel Bisphosphonates with Antiresorptive Effect in Bone
Mineralization and Osteoclastogenesis
Salvatore Savino, Annamaria Toscano, Rosa Purgatorio, Emanuela Profilo, Antonio
Laghezza, Paolo Tortorella, Mariacristina Angelelli, Rosa Scala, Domenico Tricarico,
Carlo Marya Thomas Marobbio, Filippo Perna, Paola Vitale, Mariangela
Agamennone, Vincenzo Dimiccoli, Anna Tolomeo, Antonio Scilimati*

ACCEPTED MANUSCRIPT
Novel Bisphosphonates with Antiresorptive Effect in
Bone Mineralization and Osteoclastogenesis
a
a
a
a
a
a
Salvatore Savino, Annamaria Toscano, Rosa Purgatorio, Emanuela Profilo,
a
a
a
Antonio Laghezza, Paolo Tortorella, Mariacristina Angelelli, Rosa Scala,
a
b
a
Domenico Tricarico, Carlo Marya Thomas Marobbio, Filippo Perna, Paola Vitale,
c
d
d
Mariangela Agamennone, Vincenzo Dimiccoli, Anna Tolomeo, Antonio
a,
Scilimati *
a
b
Department of Pharmacy – Pharmaceutical Sciences; Department of Biosciences, Biotechnologies
and Biopharmaceutics, University of Bari “A. Moro”, via E. Orabona 4, 70125, Bari, Italy;
c
Department of Pharmacy, University of Chieti “Gabriele d’Annunzio”, Via dei Vestini, 31, 66100,
d
Chieti (Italy); ITEL Telecomunicazioni S.r.l., Via A. Labriola, 70037 Ruvo di Puglia, Bari, Italy.
Keywords: Bisphosphonates, cancer, bone mineralization, human farnesyl diphosphate synthase,
human geranylgeranyl diphosphate synthase, cell lines, hydroxyapatite.
*Corresponding author.
Phone: +39 0805442753. Fax: +39 0805442724. E-mail address: antonio.scilimati@uniba.it (A.
Scilimati).

ACCEPTED MANUSCRIPT
Highlights
•
Bisphosphonic acids (N-BPs) were synthesized with fair to good yields
•
•
•
Pyrimidine- and quinazoline-N-BPs were tested as hFPPS and hGGPPS inhibitors
Zoledronate and pyrimidine-N-BP (py-N-BP) show a similar anti-proliferative profile
py-N-BP induces osteoblast mineralization and restorative osteoblastogenesis effect

ACCEPTED MANUSCRIPT
Abstract: Bisphosphonates such as zolendronic, alendronic and risedronic acids are a class of
drugs clinically used to prevent bone density loss and osteoporosis. Novel P-C-P bisphosphonates
were synthesized for targeting human farnesyl pyrophosphate synthase (hFPPS) and human
geranylgeranyl pyrophosphate synthase (hGGPPS), key enzymes of the mevalonate pathway, and
capable of anti-proliferative action on a number of cells (PC3, MG63, MC3T3, RAW 264.7,
J774A.1, bone marrow cells and their co-colture with PC3) involved in bone homeostasis, bone
formation and death. Among sixteen compounds, [1-Hydroxy-2-(pyrimidin-2-ylamino)ethane-1,1-
diyl]bis(phosphonic acid) (10) was effective in reducing PC3 and RAW 264.7 cell number in
crystal-violet and cell-dehydrogenase activity assays at 100 µM concentration. 10 reduced
differentiated osteoclasts number similarly with zolendronic acid in osteoclastogenesis assay. At
nanomolar concentrations, 10 was more effective than zoledronic acid in inducing mineralization in
MC3T3 and murine bone marrow cells. Further, 10 significantly inhibited the activity of hFPPS
showing an IC50 of 0.31 µM and a remarkable hydroxyapatite binding of 90%. Docking
calculations were performed identifying putative interactions between some representative novel
bisphosphonates and both hFPPS and hGGPPS. Then, 10 was found to behave similarly or even
better than as zoledronic acid as anti-resorptive agent
.

ACCEPTED MANUSCRIPT
1
. Introduction
Advanced cancers usually spread from the primary tumor site to other parts of the body. Bone
metastasis is the result of some tumors [1]. About three out of four cases of bone metastasis result
from breast, prostate, or kidney tumors [2]. However, also thyroid, lung, bladder, renal and skin
cancers, or multiple myeloma can spread to bone [3]. Almost 70% of people with advanced breast
or prostate cancer have bone metastasis [3]. Bone metastases are a major clinical concern that can
cause severe pain, bone pathological fractures, spinal cord compression, hypercalcemia, anemia,
spinal instability, decreased mobility, and rapid degradation in the quality of life [3]. Three types of
bone metastasis are known: osteolytic, characterized by destruction of normal bone, osteoblastic or
sclerotic, in which there is a deposition of new bone, and mixed if a patient has both osteolytic and
osteoblastic lesions [1]. Bisphosphonates (BPs) are primarily known for their ability to inhibit
osteoclast-mediated bone resorption [4]. They are an indispensable part of therapy for patients with
cancers that cause osteolysis. However, there is a growing body of evidence from preclinical
6
research showing that BPs also exhibit antitumor activity [5]. They can affect molecular
mechanisms of tumor cell adhesion, invasion, and proliferation; reinforce the effects of cytotoxic
agents in a synergistic manner; and exhibit antiangiogenic and immunomodulatory effects [6].
These preclinical findings reveal exciting ways of optimizing BP therapy in oncology to fully
exploit their antitumor potential. Currently, zoledronic acid (6, Figure 1) is the most used BP to
prevent and treat skeletal-related events (SREs) in metastatic castration-resistant prostate cancer
(mCRPC) patients [7]. BPs are pyrophosphate structural analogues, where a carbon atom replaces
the central oxygen atom, making the P-C-P backbone non-hydrolysable and hence more
metabolically stable [6]. Furthermore, the P-C-P backbone structure allows the BPs binding to
hydroxyapatite in bone tissue through the chelation of calcium ions [8-11]. Subsequently to bone
adsorption phase, BPs are locally internalized by bone-resorbing osteoclasts [12-14] leading to cell
apoptosis [15-19].

ACCEPTED MANUSCRIPT
Non N-BPs
N-BPs
OH
OH
OH
PO H
3
2
H C
3
PO H
H N
2
PO H
3 2
3
2
PO H
3
2
PO H
PO H
3 2
3
2
N
1
(Etidronic acid, ETI)
3 (Pamidronic acid, PAM)
5 (Risedronic acid, RIS)
OH
Cl
OH
PO H
3 2
Cl
PO H
PO H
3 2
3
2
N
H N
2
PO H
3 2
PO H
PO H
3
2
3
2
N
2
(Clodronic acid, CLO)
4 (Alendronic acid, ALE)
6 (Zoledronic acid, ZOL)
Figure 1. Representative active principle ingredients of non-N-BPs and N-BPs drugs.
Currently used BPs despite their extensive use in therapy and their recognized therapeutic benefit,
show side effects and severe reactions associated with accumulation in not target bone tissues and
unselective cytotoxicity such as for instance osteonecrosis of the jaw. Therefore, novel BPs with
improved profile are needed.
BPs are grouped into two classes, based on their biochemical targets and chemical structure [20-
22]. To the first class belongs BPs without nitrogen (non-N-BPs, Figure 1), such as 1 (etidronate)
and 2 (clodronate). They are incorporated into the corresponding non-hydrolysable β,γ-methylene
analogs of adenosine 5’-triphosphate (ATP) [16,23], causing the osteoclast apoptosis via
intracellular formation of toxic ATP-metabolites [16,24]. The second class is constituted by
nitrogen-containing bisphosphonates (N-BPs, Figure 1), such as 3 (pamidronic acid), 4 (alendronic
acid), 5 (risedronic acid) and 6 (zoledronic acid), that exert their action by suppressing bone
resorption via inhibition of human farnesyl pyrophosphate synthase (hFPPS), a mevalonate pathway
enzyme in osteoclasts [25-29].
hFPPS is active as a dimer [30], composed of 10 α-helices, and has highly conserved sequences
[
first aspartate rich motif (FARM), and second aspartate rich motif (SARM)] involved in the

ACCEPTED MANUSCRIPT
substrate binding [31]. This enzyme catalyzes the bio-synthesis of geranyl pyrophosphate (GPP)
and farnesyl pyrophosphate (FPP) by successive condensations of isopentenyl pyrophosphate (IPP)
and dimethylallyl pyrophosphate (DMAPP) [32]. The FPP is the substrate of the human
geranylgeranyl pyrophosphate synthase (hGGPPS) and it is converted into geranylgeranyl
pyrophosphate (GGPP).
The covalent attachment of the FPP or GGPP lipid tail to a highly conserved cysteine residue of a
GTPase protein imparts the membrane localization of this protein, promotes specific interactions
with other proteins and plays a crucial role in the control of intracellular trafficking. The isoprene
moieties from FPP and GGPP are post-translationally incorporated into several proteins, including
many members of the Ras family of small GTPases, which control cell growth and proliferation
[
33], and of Rho family of small GTPases, that are important mediators of cell migration [34].
Therefore, N-BPs cause the disruption of small GTPases prenylation through hFPPS and/or
hGGPPS inhibition [35], leading to inactivity and apoptosis of the osteoclasts [20-22,25].
Herein, we report the design and synthesis of a novel set of BPs, their evaluation as hFPPS and
hGGPPS inhibitors and as anti-proliferative agents of a number of cell lines (osteoblast-like murine
cell line MC3T3, osteoclast-like murine cell line RAW 264.7 and J774A.1, human osteosarcoma
cell line MG63, human prostate cancer cell line PC3, ex vivo murine bone marrow cells) involved in
bone homeostasis and mineralization. The hFPPS and hGGPPS heterologous expression and
purification and their use in the BPs inhibition assay are described in detail. Hydroxyapatite affinity
of the novel BPs is also reported together with their effect on bone marrow cells in co-colture with
PC3, as a capability test to induce bone osteoblastogenesis and mineralization. Docking calculations
were performed to rationalize the hFPPS and hGGPPS inhibitory activity of some representative
novel bisphosphonates through the identification of putative interactions between such BPs and the
two enzymes.

ACCEPTED MANUSCRIPT
2. Results and discussion
2
.1 Novel BPs Design
Structure–activity relationship studies of N-BPs confirm that the presence of two phosphonate
residues in the molecule is crucial for the binding to the hFPPS that occurs through the interaction
2
+
with the two Asp-rich motifs (FARM and SARM) via three Mg ions coordination [36]. The
complete removal or the replacement of a phosphate moiety with a bioisostere group (i.e., a
carboxylate group) leads to a dramatic decrease of hFPPS inhibition [37]. In fact, the derivative of 5
in which the (HO) OP-C-PO(OH) moiety is replaced by HOOC-C-PO(OH) was found to be
2
2
2
44,000-fold less active than 5. This is mainly due to the enzyme–inhibitor complex inability to be in
a still active isomerized state.
The hydroxy group linked to the central carbon atom of the P-C-P structure contributes to hFPPS
binding via polar interaction with Asp243. The absence of OH in the BPs molecule reduces the
ability of the N-BP to hold the complex in the active isomerized state, leading to a decrease of the
drug inhibitory activity. This result was confirmed by X-ray investigation of FPPS:6:IPP crystal,
which shows that the presence of the hydroxy improves the packing with IPP by forming a stable
ternary complex [37]. The side chain features of a number of hFPPS inhibitors led to identify two
N-BPs sub-groups [38,39]: the alkyl-amino BPs [as in pamidronic acid (3) and alendronic acid (4),
Figure 1] and the heterocyclic N-BPs [as in risedronic acid (5) and zoledronic acid (6), Figure 1].
The position of the nitrogen with respect to the P-C-P backbone represents a further crucial factor
to be taken into consideration in the attempt to optimize the interaction of N-BPs and the enzyme
binding site [40].
NMR studies and quantum mechanical calculations demonstrated that N-BPs bind to hFPPS in
their protonated form [41]. The absence of a nitrogen atom in the BP molecules leads to a reduction
of the inhibitory potency and complete loss of slow-binding kinetics [28].

ACCEPTED MANUSCRIPT
The alkyl-amino group of BPs interacts with the hydroxyl of Thr201 or Tyr204, however, only
the heterocyclic N-BPs make strong bifurcated hydrogen bonds in hFPPS binding site that explains
the heterocyclic N-BPs higher hFPPS inhibitory potency than alkyl-amino BPs [41].
With the aim of discovering novel drugs useful to treat bone related diseases including metastatic
SREs [7], we designed a new N-BPs set bearing nitrogen-containing heterocycles, such as
pyrimidine, quinazoline, benzothiazole and benzimidazole.
Preliminarly, 10-13 (Figure 2) were designed using 5 (risedronic acid) as a “lead compound”.
Then, the pyridine ring and the methylene group present in the side chain of 5 were replaced by the
pyrimidine nucleus and a NH moiety, respectively, possibly involved in additional hydrogen
bonding in the hFPPS active site. Modifications in length of the spacer between the heterocycle and
P-C-P backbone were also evaluated by compounds 11–13 (Figure 2).
Figure 2: Structure of compounds 10–28
Compound
Ar
X
n
R
Compound
Ar
X
n
R
10
11
12
13
14
15
16
NH 0
NH 1
NH 2
NH 3
NH 0
NH 1
NH 2
H
H
H
H
H
H
H
19
20
21
22
23
24
25
NH 0
NH 1
NH 2
NH 3
H
H
H
H
NH 0 CH
3
NH 0
NH 1
H
H
1
7
8
NH 3
H
26
27
S
0
H
1
NH 0 CH
3
NH 0
H
H
2
8
S
0

ACCEPTED MANUSCRIPT
The same strategy was followed for the benzimidazole 26 and benzothiazole 27 and 28
derivatives; compound 26 is mimetic of 6 (zoledronic acid), and the presence of condensed
aromatic ring would increase the molecule lipophilicity, a crucial determinant of BPs anti-resorptive
activity. To gain a deeper understanding of the isosteric modification effects, the NH group was
replaced by a sulfur atom obtaining the benzothiazole 27 and 28.
To investigate the lipophilic effect on the pyrimidine scaffold, compounds 14-18 containing a
quinazoline ring and their regioisomers 19-23 were designed.
Finally, compounds 24 and 25 (Figure 2) were projected to evaluate the steric and electronic
effects of an additional phenyl ring in position 2 on quinazolin-4-yl nucleus.
Then, a suitable synthetic strategy was identified for all new 10-28 BPs. The pure compounds were
submitted to hFPPS and hGGPPS inhibition assays and were examined for their in vitro effect on
the viability of PC3 human prostate cancer cell line, MG63 human osteosarcoma cell line,
osteoclasts like murine cell lines J774A.1 and RAW 264.7, osteoblasts like murine MC3T3-E1 cells
and ex-vivo murine bone marrow cells. The affinity for hydroxyapatite and the capability of these
new drugs to induce in vitro mineralization were also investigated. Docking of some representative
novel BPs into the active site of both hFPPS and hGGPPS was also accomplished.
2
.2 Chemistry
The synthetic strategy used to prepare the arylamino bisphosphonic acids 10-25 is depicted in
Scheme 1. In particular, N-phthaloylglycine (32) and N-phthaloylalanine (36) were commercially
available, whereas N-phthaloylamino acids 33-35 were synthesized by reacting the aminoacids 29-
31 and phthalic anhydride. 32-36 were converted into the corresponding acyl chlorides by thionyl
chloride and used as reagents in a modified Arbuzov’s reaction [42], in which the phosphorylating
agent was the tris(trimethylsilyl)phosphite (step c, Scheme 1). The silyl esters of the geminal
bisphosphonic acid intermediates were hydrolyzed to compounds 37-41. Deprotection of the amine
group, achieved by 12 N HCl at reflux, affords N-BPs 3, 4, 7-9. Finally, arylamino bisphosphonic

ACCEPTED MANUSCRIPT
acids 10-25 were prepared through the amination reaction of the free amino group of the
corresponding N-BPs and the appropriate heteroaromatic chlorides (2-chloropyrimidine, 2-
chloroquinazoline, 4-chloroquinazoline and 4-chloro-2-phenylquinazoline). Such a reaction was
performed in water and in the presence of K CO , the reaction temperature was modified from 90 to
2
3
180 °C, based on the reactivity of different starting compounds 3, 4 and 7-9 (see Experimental
3
1
section). The reaction progress was monitored using P NMR, recorded on crude reaction mixture,
3
1
by following the decreasing of the P NMR signal of each starting N-BPs. After removal of the
solvent by evaporation, pure 10-25 were obtained by washing the crude reaction residue with hot
ethanol or chloroform, followed by a crystallization (0.4 N HCl) at 5 °C.
The synthesis of benzoimidazole 26 and benzothiazole-bisphosphonic acid derivatives 27 and 28
is reported in Scheme 2. Their synthesis started from the preparation of the precursors 45 and 48,
performed through bromide nucleophilic substitution in bromoacetic acid by 2-
mercaptobenzoimidazole and 2-mercaptobenzothiazole. 2-Aminobenzothiazole acid 47 was
obtained by hydrolysis of its ethyl ester 46, in turn obtained by reacting 2-aminobenzothiazole and
ethyl bromoacetate. The benzoimidazole- 26 and benzothiazole- bisphosphonic acid 27 and 28 were
prepared from the corresponding carboxylic acids by a mixture of phosphorous acid and phosphorus
trichloride (Michaelis−Arbuzov reaction) [43], then quenched with water. The purification occurred
by treating the crude reaction mixtures with charcoal. The filtrates were kept at room temperature to
obtain white crystals of pure products.

ACCEPTED MANUSCRIPT
O
R
O
R
PO H
OH
3
2
R
O
R
PO H
OH
3
2
a
COOH b, c, d
e
N
n
N
n
PO H
3
2
H N
n
OH
H N
n
PO H
2
2
3
2
O
O
29 (n = 1; R = H)
30 (n = 2; R = H)
31 (n = 3; R = H)
32 (n = 0; R = H)
33 (n = 1; R = H)
34 (n = 2; R = H)
37 (n = 0; R = H)
38 (n = 1; R = H)
39 (n = 2; R = H)
40 (n = 3; R = H)
41 (n = 0; R = CH3)
7 (n = 0; R = H)
3 (n = 1; R = H)
4 (n = 2; R = H)
8 (n = 3; R = H)
9 (n = 0; R = CH3)
35 (n = 3; R = H)
36 (n = 0; R = CH3)
f
N
N
N
R
PO
OH
PO H
3
H
2
2
N
HN
Ar
n
3
(pyr)
(4-quin)
Ar =
1
1
0 (n = 0; R = H; Ar = pyr)
1 (n = 1; R = H; Ar = pyr)
12 (n = 2; R = H; Ar = pyr)
N
1
1
1
1
1
1
1
2
2
2
2
2
2
3 (n = 3; R = H; Ar = pyr)
N
N
Ph
4 (n = 0; R = H; Ar = 2-quin)
5 (n = 1; R = H; Ar = 2-quin)
6 (n = 2; R = H; Ar = 2-quin)
7 (n = 3; R = H; Ar = 2-quin)
N
(
2-quin)
(Ph-quin)
8 (n = 0; R = CH ; Ar = 2-quin)
3
9 (n = 0; R = H; Ar = 4-quin)
0 (n = 1; R = H; Ar = 4-quin)
1 (n = 2; R = H; Ar = 4-quin)
2 (n = 3; R = H; Ar = 4-quin)
3 (n = 0; R = CH ; Ar = 4-quin)
3
4 (n = 0; R = H; Ar = Ph-quin)
5 (n = 1; R = H; Ar = Ph-quin)
Scheme 1. Synthesis of pyrimidine- and quinazoline-BPs. Reagents and conditions: a) phthalic
anhydride, Et N(cat.), toluene, reflux, h; b) thionyl chloride, reflux, 2h; c)
5
3
tris(trimethylsilyl)phosphite, THF, 0 °C to r. t., 15 min–overnight; d) MeOH, r.t., 1h; e) 12 N HCl,
reflux, 5.5-20 h; f) ArCl, K CO , H O, 90-180 °C, 5h – 25 h.
2
3
2
PO H
OH
O
C
Z
3
2
2
N
X
N
X
N
X
c
a
Y
Y
PO H
Y
3
4
4
5 (X = NH, Y = S, Z = OH)
6 (X = S, Y = NH, Z = OEt)
2
6 (X = NH, Y = S)
27 (X = S, Y = NH)
8 (X = S, Y = S)
4
4
4
2 (X = N, Y = SH)
3 (X = S, Y = NH2)
4 (X = S, Y = SH)
b
47 (X = S, Y = NH, Z = OH)
8 (X = S, Y = S, Z = OH)
2
4
Scheme 2. Synthesis of benzoimidazole- and benzothiazole-BPs. Reagents and conditions: a) 2-
bromoacetic acid, KOH, EtOH, r.t., 3 h; or ethyl 2-bromo acetate, K CO , acetone, reflux, 16 h; b)
2
3
HCl, dioxane, reflux, 3 h; c) PCl , H PO , CH CN, reflux, 5 h.
3
3
3
3

ACCEPTED MANUSCRIPT
2
.3 Biology
2.3.1 Effects of target BPs on osteoblasts and osteoclasts cell lines and cancer cell lines
The novel synthesized compounds showed different capability in reducing cell number and cell
dehydrogenases activity in murine pre-osteoclasts like J774A.1 and RAW 264.7 cells (Figure 3A,
B), human pre-osteoblast MG63 and human tumour prostate cancer PC3 cell lines (Figure 3C, D).
The maximal anti-proliferative effect of the novel BPs was evaluated in each cell line after 72 hours
of incubation (Supplementary material, Table S4), using the crystal violet (CV) assay. The data
reported refer to the effects of the novel BPs at 100
µM. The antiproliferative effects of the
compounds were investigated at 1-500 M concentrations. From these data, we identified the 100
µ
µM concentration as the minimal saturating concentration for all these compounds capable to cause
that maximal antiproliferative effects in all cell lines. No further inhibition of proliferation was
observed at 500 µM concentration with the compounds under investigation in different cell lines.
Figure 3. Anti-proliferative effects of zoledronic acid (ZOL) and novel synthesized compounds at
100 µM on murine pre-osteoclasts like cells J774A.1 (A), murine pre-osteoclasts like cells
RAW264.7 (B), human osteosarcoma cells MG63 (C) and human prostate cancer cells PC3 (D).

ACCEPTED MANUSCRIPT
Cell viability was evaluated by crystal violet (CV) assay. Data are the mean ± E.S. of nine
replicates and expressed as percentage (%) of proliferation changes. Negative value indicates a
reduction of cell proliferation vs controls. Data were statistically different between groups as
determined by ANOVA one way (p < 0.05).
Even if a number of the new compounds affect the viability of the cell lines (Figure 3A, B, C, D),
the anti-proliferative profile of compound 10 appeared to be similar to that of zoledronic acid being
effective in all cell lines. Based on this result, the compound 10 was further investigated in cell
dehydrogenase activity (DH) assay in comparison with zoledronic acid (ZOL), alendronic acid
(ALE) and risedronic acid (RIS) used as references being the pharmaceutical active principle
ingredients of drugs already approved to treat bone metastases and staurosporine (STS) (Figure 4A,
B, C, D). STS is a well-known apoptotic agent acting as a PAN inhibitor of tyrosine kinases
including protein kinase C.
The rank order of efficacy of the drugs (100
DH activity assay was RIS > ALE > ZOL > 10 (Figure 4A) and ZOL > RIS > ALE > 10 (Figure
B), respectively. In MG63 and PC3 cells, all drugs (100 M) were less effective than
staurosporine (STS) in reducing cell number resulting in the following rank order: ZOL > ALE > 10
RIS (Figure 4C) and 10 > ZOL > ALE > RIS (Figure 4D), respectively.
µM) in J774A.1 and RAW 264.7 cell lines using the
4
µ
>

ACCEPTED MANUSCRIPT
Figure 4. Anti-proliferative effects of zoledronic acid (ZOL), alendronic acid (ALE), risedronic
acid (RIS) and 10 at 100 µM and staurosporine (STS) at 2.14 µM on murine pre-osteoclasts like
cells J774A.1 (A), murine pre-osteoclasts like cells RAW264.7 (B), human osteosarcoma cells
MG63 (C) and human prostate cancer cells PC3 (D). Cell viability was evaluated by cellular
dehydrogenase (DH) activity assay CCK8. Data are the mean ± E.S. of nine replicates and
expressed as percentage (%) of DH activity changes. Data were statistically different between
groups as determined by ANOVA one way (p < 0.05).
Therefore, 6 (ZOL) and the compound 10 showed a similar anti-proliferative profile, among the
novel compounds, being effective in all cell lines. Then, 10 was investigated for its effects on cell
proliferation and osteoclastogenesis in differentiated RAW264.7 in comparison with the 6 (ZOL), 4
(ALE) and 5 (RIS). In differentiated RAW 264.7 cells, the rank order of efficacy of the drugs (0.1
mM) using the DH assay was: ZOL > 10 > ALE > RIS (Figure 5A), suggesting that cell
differentiation enhanced the anti-proliferative response of the compound 10 reducing that of ALE
and RIS.

ACCEPTED MANUSCRIPT
Figure 5. (A) Anti-proliferative effects of zoledronic (ZOL), alendronic (ALE), risedronic acid
(RIS) and 10 at 100 µM and staurosporine (STS) at 2.14 µM on RANK ligand differentiated
RAW264.7. The cell number and viability were evaluated by crystal violet (CV) and cell
dehydrogenase (DH) activity assays. Data were statistically different between groups as determined
by ANOVA one way (p < 0.05). (B) Effects of BPs on multinucleated RANK ligand differentiated
RAW 264.7 osteoclasts evaluated by Trap assay. *Data significantly different vs controls (CTRL)
as evaluated by student t test (p < 0.05).
In the osteoclastogenesis assay performed on differentiated RAW 264.7 cell line (Figure 5B), the
compounds under investigation were capable to inhibit osteoclastogenesis in the micromolar
concentration range, after 7 days of cells incubation with the receptor activator of NF-kB ligand
(RANKL), that in turn plays a critical role in the formation of osteoclasts by stimulating precursor
cells when binds to receptor activator of NF-kB (RANK) on the cell membrane of osteoclast
precursors. The rank order of efficacy to reduce the numbers of differentiated osteoclasts was ZOL
=
10 > RIS = ALE. It appears clearly enough that 10 activates RANK, similarly to ZOL.
2
.3.2 hFPPS and hGGPPS expression and their inhibition
The novel BPs were therefore investigated for their capability to inhibit the human recombinant
enzymes. hFPPS and hGGPPS were over-expressed in BL21 E. coli cells with a hexahistidine
(6xHis) tag at the C-terminus of the proteins (Supplementary material: Figures S1-S2) to allow

ACCEPTED MANUSCRIPT
purification by immobilized metal affinity chromatography (IMAC). The proteins were efficiently
purified with Nickel-nitrilotriacetate (Ni-NTA) agarose matrix using a stepwise imidazole gradient
(Supplementary material: Figures S3-S4).
The assay of hFPPS and hGGPPS activity inhibition by the novel set of BPs was based on the
detection of pyrophosphate (PPi) or phosphate (Pi), the byproducts of hFPPS and hGGPPS
catalyzed reactions. The PPi (or Pi) reacts quantitatively with ammonium heptamolybdate, under
acidic conditions, to afford a PPi-molybdate (or Pi-molybdate) complex. These colorless complexes
can be converted to stable colored molybdate blue adducts by the addition of reducing agents
(bisulfate reagent and 2-mercaptoethanol) [44]. The colored product can be monitored using
UV/Vis spectrophotometer (absorption peaks at λ = 615 nm and 846 nm for PPi and Pi,
respectively).
A preliminary assay of the new N-BPs was performed at 100 µM to evaluate hFPPS and hGGPPS
inhibition (%) using 5 (risedronic acid) and 6 (zoledronic acid) as reference compounds (Table 1).
For the most active compounds 10 and 15, the corresponding IC values were determined by a
5
0
curve fitting of residual relative activity versus inhibitor concentration (Figure 6, Table 2).
2
.3.2.1 Structure-activity relationships of 10-28 in hFPPS and hGGPPS inhibition
Among pyrimidin-2-yl-N-BPs, compound 10 characterized by a pyrimidine ring and a
methylenamino spacer, showed a complete inhibition of hFPPS at 100 µM, whereas the elongation
of the spacer (compounds 11-13) determined a 100-fold reduction of the inhibitory activity.
Concerning hGGPPS inhibition, compounds 10-13 showed a different pattern of inhibition with
respect to that observed for hFPPS. Compound 10 showed the same inhibitory activity of 11 and 12,
but was less active than 13.
As far as the quinazolin-2-yl-N-BPs is concerned, the best hFPPS inhibitor was 15 [n = 1;
inhibition (%) = 79 ± 6], In line with this evidence, this compound showed a remarkable anti-
proliferative effect on osteoclast cell lines. The compounds 16 and 17 showed a similar efficacy of

ACCEPTED MANUSCRIPT
ZOL and 10 as anti-proliferative compounds in osteoclasts cell lines thereby suggesting that the
presence of the quinazolin nucleus and the enhanced number of methylene groups in the BPs
structure maintain the anti-proliferative activity on osteoclast cell lines. Compounds 14 and 18 of
this group were, however, less effective than reference compounds either as inhibitors of the
enzymes activity and as anti-proliferative compounds in osteoclasts, suggesting that the presence of
the spacer in the quinazolin-2-yl-N-BPs structure is critical for their activity.
Quinazolin-4-yl derivatives with one or two methylenes as a spacer afforded 19 and 20 that
showed a comparable inhibition for both enzymes. Chain elongation decreased by a half the activity
on hFPPS, and a remarkable increase against hGGPPS was observed for compound 22.
Furthermore, the isosteric substitution of a hydrogen atom in compound 19 with a methyl group
(23), resulted in the same inhibitory effect. In line with this evidence, 19 and 20 were indeed less
effective as anti-proliferative agents in osteoclasts cell lines with respect to pyrimidin-2-yl-N-BPs
and quinazolin-2-yl-N-BPs.
Finally, compounds 24 and 25, bearing a phenyl ring in position 2 on quinazolin-4-yl nucleus,
showed a low inhibitor effect on the enzyme hFPPS and found quite inactive against hGGPPS as
well as the compounds 26-28, in which the benzothiazole and benzoimidazole rings seem to be
detrimental to the both enzymes inhibition activity. The compound 24, and with minor extend the
compound 25, were however effective and selective against osteoclasts as anti-proliferative
compounds thereby suggesting that additional mechanisms may be involved.
Table 1. hFPPS and hGGPPS inhibitory activity by aryl bisphosphonic acids (Ar-N-BPs) 10-28,
risedronic (5) and zoledronic (6) acid.
hFPPS
inhibition (%)
hGGPPS
inhibition (%)
Compound
a
a
b
5
6
1
(RIS)
93.5 ± 2.5
17 ± 7
38 ± 4
20 ± 8
b
(ZOL)
91.1 ± 1.4
0
96 ±3

ACCEPTED MANUSCRIPT
1
1
1
1
1
1
1
1
1
2
2
2
2
1
2
3
4
5
6
7
8
9
0
1
2
3
54 ± 4
43 ± 7
51 ± 9
39.9 ± 1.3
79 ± 6
30 ± 6
48 ± 8
40 ± 6
52 ± 6
56 ± 9
25 ± 7
29 ± 12
56 ± 8
21 ± 6
25 ± 8
24 ± 8
23 ± 5
25 ± 8
40 ± 8
23.9 ± 2.7
c
n.a.
52.2 ± 2.8
44 ± 3
3.3 ± 2.3
25 ± 4
38 ± 7
33 ± 6
45 ± 7
24 ± 5
c
2
4
n.a.
2
2
2
2
5
6
7
8
17 ± 7
c
n.a.
c
c
n.a.
n.a.
c
26 ± 9
n.a.
a
Data are means ± SD from three independent experiments performed in triplicate; the inhibition
extent of hFPPS and hGGPPS, expressed as a percentage (%), determined in the presence of 100
µM of the inhibitors with respect to the activity of the enzymes in the absence of any inhibitor (at 1
µM and 10 µM the inhibition values (%) of 10-28 ranged from inactive to 68 or 79%, respectively;
b
hFPPS percentage of inhibition at 1 µM final concentration 5 (risedronic acid) and 6 (zoledronic
c
acid); n.a. = not active.

ACCEPTED MANUSCRIPT
hFPPS
1
25
00
1
5
6
1
1
7
5
2
5
0
5
0
0
5
0.001
0.01
0.1
1
10
100
Inhibitor / µM
Figure 6. hFPPS residual activity vs inhibitor concentration. hFPPS inhibition by 10, 15 and
reference compounds 5 and 6.
Table 2. hFPPS IC values of 5-6, 10 and 15.
5
0
a
Compound
IC (µM)
50
5
6
1
1
(RIS)
0.07 ± 0.01
0.06 ± 0.02
0.31 ± 0.07
41 ± 11
a
(ZOL)
IC50 values are
response plots as a
five independent
performed in triplicate.
from concentration-
mean ± SD from
experiments
0
5
The hFPPS IC values obtained for the reference compounds 5 and 6 were comparable (IC =
5
0
50
0
.07 µM and IC = 0.06 µM, respectively) to already reported data [37, 45-46]. Among the novel
50
Ar-N-BPs, compound 10 showed a potent inhibitor activity in the low micromolar range (IC =
5
0
0.31 µM), although it is about four times less potent than 5 and 6. Within quinazoline derivatives,
compound 15 showed IC = 41 µM, resulting approximately three orders of magnitude less active
5
0
than the reference compounds.
As far as hGGPPS inhibition is concerned, the new Ar-N-BPs showed a lower, comparable or
higher inhibitory activity than 5 and 6 which showed in our experiments IC values >100 µM
5
0

ACCEPTED MANUSCRIPT
consistent with previously published data [47-49]. Specifically, 13, 16, 17 and 22, with the longest
spacer, were found to be the most active compounds against hGGPPS.
2
.3.3 Docking calculations
In order to have an insight about the putative interactions between our newly synthesized ligands
and studied enzymes, docking calculations were carried out in the
sites (PDB IDs respectively 4H5D and 2Q80).
hFPPS and hGGPPS binding
Concerning the docking in the
followed that allowed to obtain a quantitative model for a diverse set of FPPS inhibitors. In that
case, the binding mode of N-BPs into the FPPS active site were modeled exploiting the
information from similar X-ray complexes and not taking into account sequential FPPS adaptation
and isomerization or stabilization by IPP binding. The 3D structure of FPPS and the docking
hFPPS catalytic site, a previously setup procedure has been
h
h
protocol used in the present work were setup as described in the previous article where a cross-
docking run allowed to identify the protein structure best performing in the reproduction of the
experimental binding geometry of ligands. The same procedure highlighted the role of X-ray water
2
+
molecules close to Mg ions in determining the correct binding mode and energy. The same water
molecules have been explicitly considered in the docking calculations [50].
All docked ligands occupy the DMAPP/GPP binding pocket, similarly to known N-BPs. The
2
+
bisphosphonic portion establishes very conserved interactions, coordinating the three Mg ions and
forming ionic interactions with Lys200, Lys257 and Arg112, while the OH function forms a H-
bond with the Asp243 side chain.
Main differences have been observed in the side-chain interactions. As previously stated, N-BPs
bind the
hFPPS with their protonated side chain acting as carbocation transition state analogues
[
51]. In particular, the heterocyclic N-BPs potency is due to their ability to form a bifurcated H-
bond interaction with Thr201 side chain and backbone carbonyl oxygen of Lys200 via their
protonated heterocyclic ring.

ACCEPTED MANUSCRIPT
In this respect, compound 10 locates the piperidine ring in the proximal part of the hydrophobic
DMAPP/GPP site as well as risedronate (Figure 7A, C); differently to this latter, the longer spacer
between the bisphosphonic portion and the aromatic ring allows the H-bonding to Lys200 carbonyl
oxygen, but not with Thr201. Moreover, compound 10 presents a piperidine ring with more acidic
heterocyclic nitrogen atoms (calculated pKa = 3.9) with respect to the pyridinic one of risedronate
(calculated pKa = 5.18); to account for this effect, docking calculations have been carried out in
both protonated and deprotonated forms.
Compound 15 maintains the BP interactions, its larger aromatic portion gains an interaction with
Phe99 side chain (the same interaction has been detected for minodronate), but no H-bond
involving the side chain has been detected (Figure 7B). The same result has been obtained for all
the other docked ligands that do not show any H-bond interaction with the protein: larger ligands
occupy the whole binding pocket, improving the Van der Waals interactions. However, the analysis
of the energetic contributions to the docking score reveals that the electrostatic component better
correlates to the experimental activity, confirming that H-bond/electrostatic interaction overcomes
the hydrophobic contribution in determining the binding affinity to
GGPPS has 17% sequence identity to FPPS and, probably, it shares the same catalytic
mechanism. With respect to FPPS, it presents a longer hydrophobic tunnel surrounded by
lipophilic and aromatic residues [52]. Potent GGPPS inhibitors, in fact, possess long alkyl chains,
while the presence of cationic functions is not required. GGPPS binding, therefore, differently
from FPPS, is mainly driven by hydrophobic interactions. As a matter of fact, also risedronate has
hFPPS [41].
h
h
h
h
h
h
been found to be inactive toward
hydrophobic tail [54].
hGGPPS [53], while dual binding compounds present a long
The bisphosphonic function of all docked ligands aligns well with the pyrophosphate group of co-
2
+
crystallized
interactions with Lys212, Gln185, Lys202, Arg73, Asp188 and Lys151. Most active compounds
13, 16, 17, 22) have longer linkers that bring the ligand aromatic portion in a sandwich interaction
hGGPP, providing coordination of the two Mg ions and ionic and/or H-bonding
(

ACCEPTED MANUSCRIPT
with His57 and Phe156 side chains (Figure 7D). However, they occupy the hydrophobic pocket
only partially. Ligands with a shorter linker are not able to interact with these aromatic residues and
locate the NH function in an unfavorable position.
Docking results can thus explain the opposite behavior of our compounds with respect to the
h
FPPS/
h
GGPPS inhibition, i.e. the effect of linker elongation reduces the
GGPPS.
hFPPS inhibition, while
improves the binding affinity to
h
Figure 7. Docked poses of compounds 10 (green carbon atoms, A), 15 (cyan carbon atoms, B)
and X-ray pose of risedronate (yellow carbon atoms, C) in the FPPS active site (grey cartoon), and
superimposed docked geometry of compounds 13 (light green carbon atoms), 16 (salmon carbon
atoms), 17 (blue carbon atoms) and 22 (gold carbon atoms) in the GGPPS binding site (light grey
2
+
cartoon). More relevant residues are reported as sticks, Mg ions are represented as purple spheres,
H-bonds are depicted as yellow dashed lines. Water molecules are omitted for sake of clarity.

ACCEPTED MANUSCRIPT
2
.3.4 Hydroxyapatite (HAP) Affinity Assay
BPs affinity for bone mineral represents an essential feature which allows to predict the in vivo
interaction with bone tissue and eventually the possible clinical efficacy. All BPs with P-C-P
backbone structure have a high affinity for bone [55]. The modification of their side chain could
affect the affinity of the compounds for bone tissue. Then, the bone mineral affinity of the most
active new N-BPs [10, 15 and 19] was investigated evaluating their binding to hydroxyapatite
(HAP) powder and compared with 5 (risedronic acid) and 6 (zoledronic acid). The N-BPs ranking,
according to their HAP binding affinity, was determined by ESI-MS analysis, quantifying the
residual abundances of N-BPs, in the sample, after the interaction with HAP. In particular, the rank
order of binding affinities to HAP is 6 (ZOL) > 10 > 5 (RIS) > 15
≈
19 (Table 3).
Table 3. Hydroxyapatite (HAP) binding (%) assay.
Compound
5 (RIS)
6 (ZOL)
10 15
19
HAP binding (%)
87
> 95
90 84
81
2
.3.5 Effects of novel BPs on mineralization of pre-osteblastic cell line
In the mineralization assay performed on pre-osteoblastic like cell line MC3T3-E1, the compounds
under investigation were capable to induce osteoblastogenesis and mineralization in the nanomolar
concentration range (30-500 nM) after 10-15 days of incubation of the cells with the mineralization
medium (MM). No effects were observed at micromolar concentration range. The most effective
drugs in inducing mineralization were ZOL and 10 (Figure 8A) at 30 nM concentration, whereas at
5
0 nM the compound 10 was more effective than ZOL.

ACCEPTED MANUSCRIPT
Figure 8. Percentage (%) of mineralization in the absence (CTRL MM) or in the presence of
zoledronic (ZOL), alendronic (ALE), risedronic acid (RIS) and 10 at 30 nM and 50 nM
concentrations in MC3T3-E1 murine osteoblast like cells (A) and bone marrow cells (B) and at 50
nM and 100 nM concentrations in bone marrow cells co-cultured with PC3 (C). *Data significantly
different vs controls (CTRL MM) as evaluated by student t test (p < 0.05).
2
.3.6 Ex vivo investigation on the effects of BPs on murine bone marrow cells
In the mineralization assay, performed on bone marrow cells extracted from tibia and femur of the
mice, the compounds under investigation were capable to induce osteoblastogenesis and
mineralization in the nanomolar concentration range (30-50 nM), after 10-15 days of incubation of
the cells with the MM. The rank order of efficacy in inducing mineralization at 50 nM was: 10 >
ZOL > RIS = ALE (Figure 8B). No effects were observed at micromolar concentration range.
The compounds under investigation were capable to induce osteoblastogenesis and mineralization
in the nanomolar concentration range (30-50 nM) after 10-15 days of co-incubation of the cells with
PC3 tumor prostate cancer cells. The compound 10 and ZOL were equally effective in inducing
mineralization while RIS and ALE were not effective (Figure 8C). No effects were observed at
micromolar concentration range. The enhanced efficacy of the compound 10 and ZOL vs ALE and

ACCEPTED MANUSCRIPT
RIS in the mineralization assay can be explained taking into account the differences in the
molecular structures of BPs. The geminal bisphosphates in the compounds 10 and ZOL is linked to
a
N-methylene which is lacking in the RIS and ALE compounds improving their H-bonding
capability.
To evaluate the specificity of cytotoxic action of the novel BPs, the effects of these compounds
were tested on murine bone marrow cells (30 nM-500 µM) in control condition. The incubation of
native cells with RIS and ALE for 72 hours did not produce any significant effects on proliferation
of bone marrow cells (Figure 9). The significant change in variance calculated by one-way ANOVA
was due to the proliferative effects of the structural analogue 10 at 30 nM and ZOL at 100 nM
concentrations and to the anti-proliferative effects of ZOL at 100 µM and 500 µM concentrations.
30 nM 100 nM 10 µM 100 µM500 µM
4
0
0
0
ZOL
ALE
RIS
2
-
20
40
1
0
-
Concentration
Figure 9. Effects of commercially available bisphosphonates and their novel structural analogues
0 on viability of murine bone marrow cells at different concentrations by crystal violet (CV) assay
1
after 72 hours of incubation. Data were statistically different between groups as determined by
ANOVA one way (p < 0.05).
The biological effects of 10, especially those related with the anti-proliferative effects of
osteoclasts, are mostly explained by the inhibition of the human hFPPS. Compound 10 was more
effective than reference compounds in inducing osteoblastogenesis and mineralization, observed in
the nanomolar concentration range, suggesting that the chemical structure changes may have
affected, other than binding to the main target enzyme, also other properties of the compound
related for instance to the interaction with additional targets. N-BPs can, indeed, interfere with the

ACCEPTED MANUSCRIPT
receptor activator of NF-kB ligand (RANKL) expression and with RANKL decoy receptor
osteoprotegerin (OPG) involved in osteoclastogenesis, osteoclasts differentiation and their
recruitment in the area of bone resorption. In addition, N-PBs activation of the ion channel
Connexin 43 appears to be related with proliferation of osteoblasts.
3
. Conclusion
The novel synthetized compound 10 was more effective than ZOL in inducing mineralization of
osteoblasts in native murine bone marrow cells following long term exposure of the cells (14 days)
still maintaining antiproliferative action against osteoclasts. This compound induced mineralization
of osteoblast cell line in the absence and in the presence of cancerous PC3 cells, suggesting an
interesting capability to preserve bone volume and density in bone lytic diseases associated with
SRE.
The specificity of the antiproliferative action of the compound 10 was demonstrated in cell-based
proliferation assay in which this compound was capable to reduce cell viability in all cell lines
including osteoclasts showing a large spectrum of action with respect to the other novel synthetized
compounds. A further point of interest is the fact that the compound 10 did not produce significant
anti-proliferative effects in the native murine bone marrow cells suggesting the lack of unselective
cytotoxic effects, while ZOL significantly reduced cell proliferation of about -20% in the same
cells. Therefore, the lack of unselective cytotoxicity against native cells of the compound 10
combined with the osteoblastogenesis and osteoclastogenesis actions explains the observed efficacy
in “ex vivo” experiments in murine bone marrow cells.
The compound 10 was effective in the hydroxyapatite (HAP) binding (%) assay as well as in
binding the purified
h
FPPS recombinant enzyme responsible for the anti-proliferative action of BPs.
FPPS binding site,
Docking calculations suggest a putative binding mode for compound 10 in the
h
providing indications for its optimization. These data support the use of the compound 10 as a “lead
molecule” for future investigation.

ACCEPTED MANUSCRIPT
3
3
3
. Experimental section
.1 Chemistry.
.1.1 General
Phthaloylglycine (32) and phthaloylalanine (36) were commercially available.
propionic acid (33) [56], -phthaloyl butyric acid (34) [56], -phthaloyl valeric acid (35) [57], 3
palmidronic acid) [56], 4 (alendronic acid) [42], 5 (risedronic acid) [58], 6 (zoledronic acid) [59]
were prepared according to known procedures.
N-Phthaloyl
N
N
(
All other chemicals and solvents were purchased from Sigma-Aldrich and used without any
further purification.
1
31
Proton ( H) and phosphorous ( P) NMR spectra were recorded on a 600 MHz Bruker/Advance
1
31
Spectrometer or on an Agilent Spectrometer Technologies (500 MHz for H; 202 MHz for P)
using DMSO-d , D O, or NaOD/D O as solvents. Chemical shifts are given in parts per million
6
2
2
1
13
(
ppm) (
δ relative to residual solvent peak for H and C). Absolute values of coupling constants
(J) are reported. Electrospray Mass Spectrometry (ESI-MS) experiments were performed with a
dual electrospray interface and a quadrupole time-of-flight mass spectrometer (Agilent 6530 Series
Accurate-Mass Quadrupole Time-of-Flight (Q-TOF) LC/MS, Agilent Technologies Italia S.p.A.,
Cernusco sul Naviglio, Italy). The samples were infused via a KD Scientific syringe pump at a rate
of 10 mL/min. Ionization was achieved in the negative or positive ion mode. The pressure of the
nebulizer gas was 20 psi. The drying gas was heated to 325 °C at a flow of 10 L/min. Full-scan
mass spectra were recorded in the mass/charge (m/z) range of 50–800 Da. The purity extent of the
target compounds 10-28 was found to be > 98% at a specific pH value (pH = 4-7). It was
determined by HPLC on a reverse phase C3 column (Agilent Zorbax 300SB-C3) and a mixture of
acetonitrile/sodium acetate 20:80 (10mM, pH = 4, 5, 6 and 7) as a mobile phase. The samples to be
analyzed were prepared by solubilizing 10-28 in the mobile phase (10mM, pH = 5) at 1mM final
concentration.

ACCEPTED MANUSCRIPT
3
.1.2 Procedures for 7-25 synthesis
The synthesis of the new arylamino bisphosphonic acids 10–25 (Scheme 1) has been carried out,
by converting -phthaloyl amino acid 32-36 into 37-41, by a modified Arbuzov reaction. The
hydrolysis of phthalimide moiety, with 12 N HCl, yielded the -BPs [3 (pamidronic acid), 4
aledronate), 7-9] with the necessary free amino group to be linked to heteroaromatic rings through
N
N
(
the heteroaromatic chlorides (2-chloropyrimidine, 2-chloroquinazoline, 4-chloroquinazoline and 4-
chloro-2-phenylquinazoline) to give the target 10–25. A similar procedure was followed to prepare
4
(alendronic acid) and 6 (zoledronic acid).
(2-Amino-1-hydroxyethane-1,1-diyl)bis(phosphonic acid) (7) [42]. Commercially available N-
phthaloylglycine (32, 200 mg, 0.975 mmol) was dissolved in SOCl (2.84 mL) and refluxed for 2 h.
2
After evaporation of the solvent under reduced pressure, the obtained white solid was solubilized in
THF (1.7 mL), and treated with tris(trimethylsilyl)phosphite (978 µL, 2.92 mmol) at 0 °C for 15
min, and then stirred at room temperature for 15 min. Methanol (4 mL) was added and the solution
was stirred for 1 h at room temperature. The solvent was evaporated under vacuum and the white
solid was washed three times with a toluene/hexane mixture (1:1) (4 mL), washed with Et O and
2
1
dried under vacuum, obtaining the intermediate 37. Yield: 337 mg (98.5%). H NMR (500 MHz,
3
1
DMSO-d6, δ): 4.17-4.21 (m, 2H), 7.82-7.89 (m, 4 H). P NMR (202 MHz, DMSO-d , δ): 15.6.
6
A solution of 37 (337 mg, 0.96 mmol) in 8 mL of 12 N HCl was refluxed for 5.5 h. The solvent
was removed under reduced pressure obtaining a white solid that was washed with 50 mL of
ethanol at 70 °C. The solid was then isolated by filtration of the solution, washed with warm
1
ethanol and dried under vacuum. The product 7 was isolated in 86% yield (183 mg). H NMR (500
3
1
MHz, D O, δ): 3.38 (t, 2H, J = 11.8 Hz). P NMR (202 MHz, D O, δ): 14.9.
2
2
(5-Amino-1-hydroxypentane-1,1-diyl)bis(phosphonic acid) (8).
N-Phthaloyl valeric acid [57]
(
35, 500 mg, 2.02 mmol) was solubilized in SOCl (5.9 mL) and refluxed for 3 h. The solvent was
2
evaporated under reduced pressure, affording a white solid, that in turn, was dissolved in dry THF