Design, synthesis, and biological evaluation of novel aminobisphosphonates possessing an in vivo antitumor activity through a γδ-T lymphocytes-mediated activation mechanism

Article abstract of DOI:10.1021/jm801003y

A small series of aminobisphosphonates (N-BPs) structurally related to zoledronic acid was synthesized with the aim of improving activity toward activation of human γδ T cells and in turn their in vivo antitumor activity. The absence of the 1-OH moiety, together with the position and the different basicity of the nitrogen, appears crucial for antitumor activity. In comparison to zoledronic acid, compound 6a shows a greater ability to activate γδ T cells expression (100 times more) and a proapoptotic effect that is better than zoledronic acid. The potent activation of γδ T cells, in addition to evidence of the in vivo antitumor activity of 6a, suggests it may be a new potential drug candidate for cancer treatment.

Full text of DOI:10.1021/jm801003y

6800
J. Med. Chem. 2008, 51, 6800–6807
Design, Synthesis, and Biological Evaluation of Novel Aminobisphosphonates Possessing an in
Vivo Antitumor Activity Through a γδ-T Lymphocytes-Mediated Activation Mechanism
Daniele Simoni,*,† Nicola Gebbia,‡ Francesco Paolo Invidiata,§ Marco Eleopra,† Paolo Marchetti,† Riccardo Rondanin,†
Riccardo Baruchello,† Stefano Provera,| Carla Marchioro,| Manlio Tolomeo, Luciana Marinelli,# Vittorio Limongelli,#
Ettore Novellino,# Aaron Kwaasi, James Dunford, Simona Buccheri,O Nadia Caccamo,O and Francesco DieliO
Dipartimento di Scienze Farmaceutiche, UniVersita` di Ferrara, Italy, Via Fossato di Mortara 17/19, 44100 Ferrara, Italy, Consorzio di
Ricerca sul Rischio Biologico in Agricoltura (Co.Ri.Bi.A.), Palermo, Italy, Dipartimento Farmacochimico Tossicologico e Biologico, UniVersita`
di Palermo, Italy, GlaxoSmithKline, Medicines Research Center, Via Fleming 4, 37135 Verona, Italy, DiVisione di Ematologia e SerVizio AIDS
Policlinico, UniVersita` di Palermo, Italy, Dipartimento di Chimica Farmaceutica e Tossicologica, UniVersita` di Napoli “Federico II”, Via D.
Montesano, 49, 80131 Napoli, Italy, Nuffield Department of Orthopaedic Surgery, The Botnar Research Centre, UniVersity of Oxford, Institute
of Musculoskeletal Sciences, Nuffield Orthopaedic Centre, Headington, Oxford OX3 7LD, United Kingdom, Dipartimento di Biopatologia e
Metodologie Biomediche, UniVersita` di Palermo, Italy
ReceiVed August 8, 2008
A small series of aminobisphosphonates (N-BPs) structurally related to zoledronic acid was synthesized
with the aim of improving activity toward activation of human γδ T cells and in turn their in vivo antitumor
activity. The absence of the 1-OH moiety, together with the position and the different basicity of the nitrogen,
appears crucial for antitumor activity. In comparison to zoledronic acid, compound 6a shows a greater
ability to activate γδ T cells expression (100 times more) and a proapoptotic effect that is better than zole-
dronic acid. The potent activation of γδ T cells, in addition to evidence of the in vivo antitumor activity of
6a, suggests it may be a new potential drug candidate for cancer treatment.
Introduction
signaling cascades such as Ras2 or Rho. It also results in the
accumulation of isoprenoids, such as IPP (isopentenyl pyro-
phosphate), which induce expansion of γδ T cells. Nowadays,
there is substantial evidence that γδ T cells (which, in humans,
comprise only 5-10% of all circulating T-cells) possess potent
antitumor activities. They are capable of recognizing tumor
targets via cancer antigens or stress-induced molecules without
the requirement of classical MHC presentation. This gives broad
applicability across a large range of tumor types. Different
biological studies have demonstrated that γδ T cells are effective
against different types of cancer such as melanomas, renal
carcinoma, lymphoma, and multiple myeloma.7 Thus, γδ T cells,
or γδ T cells agonists, such as N-BPs, could play a major role
in controlling human malignancy and could be used therapeuti-
cally in combination with other anticancer agents. Indeed,
clinical studies have recently shown that adding immune therapy
to classical chemotherapy has survival benefits when compared
to chemotherapy alone.8 There is, therefore, major interest in
the development of novel N-BPs as new promising anticancer
agents.
Nitrogen-containing bisphosphonates (N-BPs), a a class of
geminal bisphosphonates (BPs), with 1 containing nitrogen in
the R2 side chain, are important drugs widely used in a variety
of bone resorption diseases, such as osteoporosis, Paget’s
disease, and hypercalcemia.1 Some also have antiparasitic and
antibacterical activity.2 They act by inhibiting the enzyme
farnesyl diphosphate synthase (FPPS),3 which plays an important
role in the mevalonate pathway and in protein prenylation.4
Recently, N-BPs have gained additional importance as they have
been shown to inhibit tumor cell adhesion, invasion, and
proliferation and to induce apoptosis of a variety of human tumor
cell lines in vitro.5 More importantly, N-BPs also have been
shown to exhibit direct antitumor activity in vivo by inhibiting
cancer growth through antiangiogenic, anti-invasive, and im-
munomodulatory actions.6 Although several mechanisms have
been proposed to explain the N-BPs’ antitumor effect, the
inhibition of FPPS surely plays an important role in their
anticancer properties. This inhibition results in the reduced
prenylation of a variety of proteins with mediator functions in
Structurally, geminal bisphosphonates (BPs) 1 are analogues
of endogenous pyrophosphate in which a carbon atom replaces
the central atom of oxygen. This substitution makes BPs resistant
to hydrolysis and allows two additional chains of variable
structure. The ability of BPs to interact with specific molecular
targets strictly depends on the BP structural properties. For
example, it is well-known that acid phosphonic groups are
essential for high affinity for bone mineral, and that this is further
increased by the hydroxyl group at the side chain on central
carbon atom position (“bone hook”).9 Consequently, BPs such
as clodronate or etidronate, which represent the “first generation”
of BPs, are rapidly adsorbed by bone and therefore have been
primarily used in bone loss therapy. To increase the activity of
the above-mentioned drugs, a “second generation” of BPs, all
containing nitrogen (N-BPs), have been developed. These
include compounds such as pamidronate or ibandronate (Figure
* To whom correspondence should be addressed: Phone: 39-(0)532-
455923. Fax: 39-(0)532-455953. E-mail: smd@unife.it.
†
Dipartimento di Scienze Farmaceutiche, Universita` di Ferrara.
Consorzio di Ricerca sul Rischio Biologico in Agricoltura.
Dipartimento Farmacochimico Tossicologico e Biologico, Universita`
‡
§
di Palermo.
|
GlaxoSmithKline, Medicines Research Center.
Divisione di Ematologia e Servizio AIDS Policlinico, Universita` di
Palermo.
#
Dipartimento di Chimica Farmaceutica e Tossicologica, Universita` di
Napoli “Federico II”.
Nuffield Department of Orthopaedic Surgery, The Botnar Research
Centre, University of Oxford, Institute of Musculoskeletal Sciences, Nuffield
Orthopaedic Centre.
O
Dipartimento di Biopatologia e Metodologie Biomediche, Universita`
di Palermo.
a Abbreviations: N-BPs, nitrogen-containing bisphosphonates; BPs,
bisphosphonates; FPPS, farnesyl diphosphate synthase; IPP, isopentenyl
pyrophosphate; TBD, 1,5,7-triazabicyclo[4.4.0]dec-5-ene.
10.1021/jm801003y CCC: $40.75
2008 American Chemical Society
Published on Web 10/21/2008

Aminobisphosphonates with in ViVo Antitumor ActiVity
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6801
approach to inducing immunological and clinical responses in
patients with metastatic carcinomas.14
Encouraged by such positive results and with the aim of
finding more potent novel γδ T cells agonists, we synthesized
a small series of modified N-BPs structurally related to
zoledronic acid (Figure 1b).
Chemistry. Bisphosphonates 6a-f were prepared from a
common starting intermediate, the methylenebisphosphonate 4,
by treatment with the proper heterocyclic nucleophile as shown
in Figure 1b. TBD (1,5,7-triazabicyclo[4.4.0]dec-5-ene) was
used as the catalyst. Additionally, bisphosphonate derivatives
were also prepared from the corresponding methylenebispho-
sphonate 4 by treatment with the proper nucleophile at 150-160
°C for 15 min in a scientific microwave oven. The bisphos-
phonate acid derivatives 6a-f were in turn obtained, starting
from the corresponding ester derivatives, by treatment with
hydrochloric acid or with iodotrimethylsilane. Compound 6e
has been fully studied by means of NMR techniques to
determine the regioisomery of the major resulting structure.
Peaks assignments were achieved for both isomers, directly on
the 90:10 mixture, using gHMQC and gHMBC experiments,
while gROESY experiments allowed the determination of the
chain position. Related results should also be valid for compound
6f.
Results and Discussion
Molecular Modeling. In the design of the novel compounds,
we benefited from the recent three-dimensional pharmacophore
model developed by Oldfield et al., who identified hydrophobic,
aromatic, and cationic features, together with two metal-binding
groups, for the creation of a BP with antitumor activity.15 We
also began with the knowledge of how our lead (zoledronate)
binds to the FPPS catalytic site. In analyzing the details of the
interactions between zoledronate and the FPPS catalytic site
(PDB codes of the X-ray structures: 1ZW5, 2F8Z, 2F9K), it
emerges that the two phosphonate groups are highly important
for the binding of zoledronate to the enzyme. This is because
they are responsible for the coordination of the three metal ions
present in the FPPS active site. Even the imidazole moiety of
zoledronate plays an important role in the binding to the enzyme.
Indeed, the imidazole in its protonated state mimics the
carbocation intermediate formed during the catalysis, and the
protonated nitrogen makes a double H-bond with the Lys200
carbonyl oxygen and the Thr201 side chain (Oγ1). As a
consequence, the only group that could be substituted without
a dramatic decrease of the FPPS inhibition would be the 1-OH
group, which, in the binary complex FPPS-zoledronate, forms
a H-bond with Asp243. Here, to test our hypothesis, we
performed molecular docking studies of zoledronate and 1-deshy-
droxyzoledronate (6a) using the FPPS (PDB code: 2F8Z) as
receptor. As expected, docking calculations provided a binding
mode of 6a largely overlapping with that of zoledronate.
Furthermore, the computed binding free energy of 6a, -10.8
kcal/mol, is comparable to that of zoledronate, -9.8 kcal/mol,
within the statistical error of the algorithm used for docking.
The only difference in the binding mode of 6a with respect to
zoledronate resides in the lack of the interaction established by
the hydroxyl group, which is absent in 6a.
Figure 1. (a) General structures of geminal bisphosphonates (BPs)
and structures of bisphosphonic acid reported in literature. (b) Synthesis
of new bisphosphonates used in current study. Reagents and conditions:
(i) Et2NH, (CHO)n, pTSA; (ii) Het(a-f), TBD; (iii) HCl conc or TMSI.
1a). The mechanism of action of the first and second generations
of BPs differs, as the former act by accumulating a toxic
analogue of adenosine triphosphate,10 while the latter lead to
the inhibition of the FPPS. Indeed, the protonated-nitrogen forms
of N-BPs closely resemble the cationic transition state/reactive
intermediate formed during the FPPS catalytic cycle. This
inhibition capacity of second generation N-BPs is also common
to the “third generation” of N-BPs, which are essentially
aromatic N-BPs and are extremely potent FPPS inhibitors. As
mentioned above, the inhibition of FPPS is of crucial importance
in the expansion and activation of γδ T cells.11 In this regard,
Wilhelm et al.12 reported the results of a pilot study of low-
dose interleukin-2 (IL-2) in combination with pamidronate in
patients with low-grade non-Hodgkin lymphoma or multiple
myeloma. It has been shown that, of nine patients, five had
significant in vivo activation/proliferation of γδ T cells. Of these,
three achieved objective responses, indicating that γδ T cells
are the main cell type responsible for the observed antilym-
phoma effect.
In line with this study, we first showed that zoledronic acid
was as able as phosphoantigen isopentenylpyrophosphate (IPP)
to induce expansion of γδ T cells in vitro in the presence of
IL-2.13 Prompted by this finding, we decided to examine the
feasibility and effects of using the γδ T cells’ agonist zoledr-
onate alone or in combination with IL-2. We therefore began a
phase I clinical trial in metastatic hormone-refractory prostate
cancer. The results clearly show that administration of zoledr-
onate together with IL-2 represents a novel, safe, and feasible
If the removal of the 1-OH does not substantially influence
the inhibition of FPPS, it should have a certain impact on
zoledronate activity in vitro and especially in vivo. In fact,
removal of this group should reduce the bone and the skeletal
retention and should increase exposure of the drug in the
peripheral blood and in the soft tissue with an increase in the

6802 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Simoni et al.
Table 1. Bioactivity of Phosphoantigens and N-BPs for Vγ9V δ2 T
Lymphocytes
Half-maximun stimulation of
Vγ9Vδ2 T lymphocytes (µM)
compound
BrHPP
IPP
Zoledronate
6a
6b
6c
6d
6e
6f
0.015-0.001
1-3
0.1-0.3
0.001-0.003
>10
1-2
>10
0.2-0.4
0.1-0.3
Table 2. Percentage ((SEa) of Apoptosis Induced by 100 µM
Compound 6a and 100 µM Zoledronic Acid
compound 6a
zoledronic acid
HUT78
K562
CEM
CEM-VBL300
CFU-GM
40 ( 5.3
15 ( 3.2
22 ( 3.1
37 ( 6.0
15 ( 2.2
24 ( 4.6
16 ( 5.1
20 ( 4.4
22 ( 3.2
18 ( 2.7
a SE ) standard error.
assays (Table 3), it becomes apparent that movement of the
nitrogen away from the position of the nitrogen in zoledronate
leads to a large decrease in potency, e.g., 6b.
Furthermore, the basicity of the nitrogens present in the ring
also plays a pivotal role in determining the different pharma-
cological profiles of the compounds under study, influencing
their strength of binding to the enzyme as well as their ability
to pass through the cellular membrane. In fact, from the enzyme
inhibition assays (Table 3), it clearly emerges that compounds
presenting a more basic nitrogen such as 6a, 6c, and 6d, show
lower IC50 values. This is probably due to a stronger interaction
of the protonable nitrogen with the nearby Lys200 and Thr201.
Moreover, although the introduction of purine system 6e,f
decreases by around 100-fold, the affinity for the enzyme, those
compounds show an activity on human γδ T cell activation
comparable to that of zoledronic acid. It seems likely that their
lipophilic character enhances the passing rate thorough the
cellular membrane.
However, as summarized in Table 1, the most surprising
finding is that the deshydroxyzoledronate 6a is approximately
100 times more capable of activating γδ T lymphocytes with
respect to zoledronate, considering that the IC50 for FPPS
inhibition for 6a is twice that of zoledronate (Table 3). In fact,
derivative 6a was extremely active in inducing activation of
human γδ T lymphocytes upon culture of peripheral blood
mononuclear cells (PBMC) in the presence of a low dose (20
U/mL final concentration) of interleukin (IL)-2. Dose response
analysis revealed that optimal stimulation and expansion of
human γδ T cells occurs at the final concentration of 1 nM. In
addition, 6a produced a phenotypic switch from na¨ıve and
central memory to effector memory cells, the latter exerting
important effector functions (such as production of TNF-R and
IFN-γ and cytotoxicity) against tumor cell lines in vitro (data
not shown).
With regard to the mechanism of action of 6a, from our initial
in vitro studies we found that: (1) as shown in Figure 2b,
treatment of PBMC with 6a and IL-2, but in the presence of
mevastatin or lovastatin, blocks the drug effects; (2) when the
above-described in vitro assay is carried out with PBMCs
depleted of monocytes, the drugs’ effects again disappear (data
not shown). Both these sets of experiments clearly indicate that
6a does not act directly on the γδ T cell receptor but may
Figure 2. (a) Ability of different N-BPs derivatives to expand in vitro
human γδ T cells. Peripheral blood mononuclear cells were incubated
in vitro with different N-BPs derivatives at the indicated final
concentrations and 20 U/mL final concentration of rIL-2, for 7 days at
37 °C. At the end of the incubation period, cells were collected and
the γδ T cell expansion factor calculated as described in Experimental
Section. (b) Mechanism of action of different N-BPs derivatives on
human γδ T cell activation. Expansion in vitro of γδ T cells was carried
out in the presence or absence of mevastatin or lovastatin, both used
at 1 µM final concentration.
in vitro/vivo antitumor activity. Moreover, with the aim of
having more lipophilic species and gaining better insight into
the SAR of zoledronate, we have additionally synthesized
compounds 6b-f.
Pharmacological Evaluation. All the synthesized compounds
have been tested in a preliminary assay for their in vitro
bioactivity toward activation-expansion of γδ T lymphocytes.
The capacity of bisphosphonates to activate, in vitro, human
γδ T lymphocytes was studied in comparison to zoledronic acid
and phosphostim (3), the bromohydrin derivative of isopentenyl
pyrophosphate, which strongly activates γδ T lymphocytes.16
Figure 2a and Table 1 report the activity of the compounds
toward expansion γδ T lymphocytes. In particular, Figure 2a
shows the results of one typical dose-response experiment out
of seven, in which peripheral blood mononuclear cells were
stimulated in vitro with different concentrations of compounds
and low doses of IL-2 for 7 days. The expansion of γδ T cells
was calculated and expressed as an expansion factor. At first
glance, it is evident that activity of the compounds seems
strongly influenced by the position of the nitrogen in the
aromatic system, with the imidazole derivative 6a being more
active than the pyrazole compound 6b by more than 5000 times.
This is in perfect line with the theory that the protonable nitrogen
has to be at a certain distance from the geminal carbon atom of
N-BPs to properly mimic the cationic transition intermediate
formed during the FPPS catalytic cycle.15,17 In the in vitro FPPS

Aminobisphosphonates with in ViVo Antitumor ActiVity
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6803
Figure 4. Effects of imatinib-zoledronic acid and imatinib-compound
6a combinations in K562 cells. Cells were incubated with 30 µM
zoledronic acid or 30 µM compound 6a in combination with concentra-
tions of imatinib ranging from 0.065 to 0.5 µM. After 48 h, the number
of living cells was determined and expressed as percentage of control.
Combination index (CI) was calculated as described in materials and
methods. Combinations were additive when CI ) 1, synergistic when
CI < 1, and antagonist for CI > 1.
Table 3. Inhibition of Recombinant Human FPPS by N-BP
compound
IC50 ( SEM (nM)
Zoledronate
4.1 ( 0.2229
6a
6b
6c
6d
6e
6f
9.03 ( 1.8
3069 ( 264
16.1 ( 1.4
28.1 ( 2.8
388.2 ( 16.2
883.1 ( 71.9
compound 6a was more active than zoledronic acid in the MDR-
expressing cells than in the parental CCRF-CEM line (Figure.
3a,b and Table 2). As shown in Figure 3a,b and Table 2,
zoledronic acid and 6a were barely active on Bcr-Abl, express-
ing K562 cells when used at concentrations lower than 50 µM.
On the basis of recent reports indicating that zoledronic acid
and imatinib mesilate combination have a synergistic activity
on Bcr-Abl expressing cells,18 we investigated the effects of
6a in combination with imatinib. K562 cells were exposed to
different concentrations of imatinib in combination with 30 µM
6a or zoledronic acid. Figure 4 shows that imatinib-6a combina-
tionsdisplayasynergicactivityhigherthantheimatinib-zoledronic
acid combination. Additionally, both zoledronic acid and
compound 6a proved to be nontoxic on normal hemopoietic
CFU-GM. Therefore, besides the potent activation of Vγ9Vδ2
T lymphocytes, compound 6a leads to a greater cytotoxic
activity than zoledronic acid.
Prompted by these startling results, we decided to test the
antitumor effects of 6a in vivo. We investigated the in vivo
tumor reactivity of human γδ T cells activated and expanded
in vitro and then adoptively transferred into SCID mice,
according to the experimental protocol described by Kabelitz
et al.,19 analyzing the optimal therapeutic efficacy against the
breast cancer BT459 cell line. SCID mice were conditioned by
irradiation and antiasialo-GM1 monoclonal antibody treatment,
and received at day 0 a single intraperitoneal injection of the
breast cancer cell line BT549 (2 × 106/mouse). The high
numbers of tumor cells were inoculated to allow for fully fledged
tumor growth within the period before graft-vs-host reactions
developed in the SCID mouse reconstituted with human
lymphocytes. Mice received concomitantly, with the tumor cells,
an intraperitoneal injection of human recombinant (r)IL-2 (300
ng), or 6a (2 µg), or zoledronate (5 µg), or 6a (2 µg) plus rIL-2
(300 ng), or zoledronate (5 µg) plus rIL-2 (300 ng). In addition,
mice received highly purified human γδ T cells (2 × 107/mouse)
where indicated. γδ T cells (2 × 107/mouse) and zoledronate
Figure 3. (a) Cell growth inhibition induced by different concentration
of compound 6a. (b) Cell growth inhibition induced by different
concentration of zoledronic acid. (c) Antitumor effect of 6a and γδ T
cells in SCID mice in vivo. SCID mice received a single intraperitoneal
injection of the breast cancer cell line BT549. Mice concomitantly
received with the tumor cells an intraperitoneal injection of human
recombinant (r)IL-2, or 6a, or zoledronate, or 6a plus rIL-2, or
zoledronate plus rIL-2. Where indicated, mice received highly purified
human γδ T cells. For a complete description of cell numbers, drug
doses, and time points, see details in Experimental Section. Data show
the mean survival time expressed in days. *p < 0.02 and **p < 0.005,
when compared to all other groups.
indirectly affect γδ T cells via IPP production in monocytes
and macrophages, which are ultimately responsible for cell
activation and differentiation. It seems likely, therefore, that 6a
behaves like other “third generation” N-BPs, although it proved
considerably more potent than zoledronic acid.
To gain better insight into the antitumor activity of 6a, we
further investigated its apoptotic effect on different tumor cell
lines and its toxicity on normal and bone marrow-derived
hematopoietic cells. The cytotoxic activity of 6a and of
zoledronate was tested on the HUT78 (human T-lymphoma),
K562 (chronic myeloid leukemia expressing the antiapoptotic
oncogene Bcr-Abl), CCRF-CEM (acute lymphoblastic), CCRF-
CEM VBL300 (P-glycoprotein, multiple drug resistant (MDR)
protein expressing acute lymphoblastic) cell lines. Interestingly,
compound 6a was endowed with antiproliferative effects and
pro-apoptotic activity higher than that of zoledronic acid in both
human lymphoma HUT78 cells and P-glycoprotein expressing
CCRF-CEM VBL300, while no significant differences were
observed in K562 and CCRF-CEM cells (Figure 3a,b). More-
over, compound 6a showed in HUT78 and CCRF-CEM
VBL300 cells a pro-apoptotic activity higher than that of
zoledronic acid (Table 2). Of note and quite surprisingly,

6804 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Simoni et al.
RESP charges were obtained by a two-stages fitting procedure,25
fitting first the polar areas by using weak hyperbolic restraints
(0.0005 au) and then fitting the remaining areas, imposing
equivalencies, and by using a stronger hyperbolic restraint (0.001
au). In each step, the charges of the standard residues ACE and
NME were constrained to their AMBER force field value.25 The
electrostatic potential used as an input by the RESP program was
sampled by adapting the Merz-Singh-Kollman scheme,26 namely
using 10 concentric layers at the default level of spacing, a surface
density of 6 points/Å2, and adopting the covalent radius of 0.86 Å
for magnesium as reported on the WebElements server (Winter M.
WebElements Periodic Table; United Kingdom: University of
Sheffield. http://www.webelements.com/). Hence, the computed
charges for ligands, cofactor, and Mg subsite were used for further
docking calculations.
Docking Setup. The docking area has been defined by a box,
centered on the mass center of the cocrystallized zoledronate. Grids
points of 40 × 40 × 40 with 0.375 Å spacing were calculated
around the docking area for all the ligand atom types using
AutoGrid4. For each ligand, 100 separate docking calculations were
performed. Each docking calculation consisted of 2.5 × 106 energy
evaluations using the Lamarckian genetic algorithm local search
(GALS) method. A low-frequency local search according to the
method of Solis and Wets was applied to docking trials to ensure
that the final solution represents a local minimum. Each docking
run was performed with a population size of 150, and 300 rounds
of Solis and Wets local search were applied with a probability of
0.06. A mutation rate of 0.02 and a crossover rate of 0.8 were used
to generate new docking trials for subsequent generations. The
GALS method evaluates a population of possible docking solutions
and propagates the most successful individuals from each generation
into the next one. The docking results from each of the 100
calculations were clustered on the basis of root-mean square
deviation (rmsd ) 1.5 Å) between the Cartesian coordinates of the
ligand atoms and were ranked on the basis of the free energy of
binding.
(5 µg), or 6a (2 µg), were injected every 15 days, while (r)IL-2
(300 ng) was given on the same day and at 3 and 6 days after
each injection of γδ T cells.
As shown in Figure 3c, neither 6a nor zoledronate or rIL-2,
given alone or in combination, improved the survival of the
mice. Survival was prolonged when γδ cells where given
together with 6a but not together with zoledronate or IL-2,
although the difference (40 ( 10 versus 28 ( 6) was not
statistically significant. The application of γδ T cells together
with zoledronate and rIL-2 significantly prolonged the mean
survival time (p < 0.02). Much longer survival was achieved,
however, when γδ T cells were given together with 6a and rIL-2
(p < 0.005). Interestingly, the difference in survival between
mice receiving γδ T cells + 6a + rIL-2 and those receiving
γδ T cells + zoledronate + rIL-2 was statistically significant
(p < 0.05), indicating that 6a is more potent than zoledronate
in activating and sustaining the antitumor activity of human γδ
T cells.
In conclusion, we have synthesized a small series of N-BPs
structurally related to zoledronate, whose antitumor activity has
been previously demonstrated, with the aim of improving its in
vivo anticancer property. From our research efforts, we identified
compound 6a, which shows a better ability to activate γδ T
cell expression and an increased proapoptotic effect compared
to the most potent γδ T cells agonist known to date (zoledr-
onate). Moreover, from our in vivo tests, where the antitumor
effect of 6a was studied in the presence of a variety of other
agents, we identified a synergic pro-apoptotic activity when it
is administered with imatinib mesylate (Gleevec). Thus, taking
into account the unique pharmacological profile of 6a, charac-
terized by a potent activation of γδ T cells, and by a remarkable
proapoptotic effect both in vitro and in vivo, it may represent
a promising anticancer drug which can be added to the
traditional chemotherapy of a wide range of tumor types.
Chemistry. General Methods and Materials. Melting points
were obtained in open capillary tubes and are uncorrected. Reactions
and product mixtures were routinely monitored by thin-layer
chromatography (TLC) on Merck silica gel precoated F254 plates.
Nuclear magnetic resonance (1H NMR, 13C NMR) spectra were
determined in CDCl3 solution unless otherwise indicated with a
Bruker AC-200 or with Varian Mercury Plus 400 spectrometer,
and peak positions are given in parts per million downfield from
tetramethylsilane as the internal standard; J values are expressed
in hertz. Compound 6e has been studied using a Varian Inova 600
MHz spectrometer in D2O solution. Light petroleum ether refers
to the 40-60 °C boiling range fractions. Column chromatography
was performed with Merck 60-200 mesh silica gel. All drying
operations were performed over anhydrous sodium sulfate. Column
chromatography (medium pressure) was carried out using the “flash”
technique. Microanalysis of all new synthesized compounds agreed
within (0.4% of calculated values. The microwave reactions were
performed in a Discover CEM, which produced controlled irradia-
tion with a power of 0-300 W.
Experimental Section
Molecular Modeling. Docking Simulations. Molecular docking
of zoledronate and 6a into the three-dimensional X-ray structure
of FPPS (PDB code: 2F8Z) was carried out using the AutoDock
software package (version 4.0) as implemented through the graphi-
cal user interface AutoDockTools (ADT 1.4.6).20
Ligands and Protein Setup. The structures of the inhibitors were
first generated from the standard fragment library of the SYBYL
software version 7.3.21 The docking parametrization of ligands, the
cofactor isopentenyl pyrophosphate (IPP), and the region of the
enzyme including the metals and its coordinating ligands (Mg
subsite) were performed by using the Gaussian03 package22 by
applying a full geometry optimization at Hartree-Fock level of
theory and then calculating the charges using the restrained
electrostatic potential (RESP) fitting procedure.23,24 For both the
optimization and the electrostatic potential (ESP) calculation, the
locally dense 6-31G* basis set was used.
Tetraethyl-ethenylidenebisphosphonate. (4). Paraformaldehyde
(520 mg, 17.3 mmol) and diethyl amine (0.36 mL, 3.46 mmol)
were combined in 10 mL of methanol, warmed until clear, then
treated with tetraethylmethylene bisphosphonate (0.86 mL, 3.46
mmol) and refluxed for 18 h. The mixture was concentrated in
vacuo, toluene was then added, and the solvent was evaporated
again (2 × 5 mL). The residue was dissolved in toluene (20 mL),
treated with p-toluensulphonic acid (cat.), and refluxed through a
Dean-Stark trap for 18 h. The sample was concentrated in vacuo,
dissolved in chloroform (20 mL), washed with water (10 mL), dried,
and concentrated in vacuo. The residue oil was used without further
purifications for the next reaction. Yellow oil; yield: 90%.
The starting geometry of the Mg subsite for ab initio calculations
was derived by a crystallographic template (PDB code: 2F8Z)
including the cocrystallized zoledronic acid necessary to get an
octahedral coordination of the ions. We treated the three magnesium
as ions in the +2 oxidation state and with a low spin state (S ) 0).
To properly describe the electrostatic protein environment, the
N and C termini of the coordinating aspartates were capped with
NME and ACE residues, respectively. Hence, the system was
geometrically optimized by constraining the atoms of the backbone
of the aspartates and using the previously reported locally dense
basis set. The model system was then optimized until reaching the
default Gaussian03 convergence criterion.
1H NMR δ: 1.17 (t, J ) 7.2, 12H), 3.90-4.03 (m, 8H), 6.63 (s,
1H), 6.70 (d, J ) 38, 1H), 7.10 (d, J ) 38, 1H).13C NMR δ: 14.5,
62.4, 121.8, 148.2.
The RESP charges were then calculated on the geometrically
minimized ligands, cofactor, and Mg subsite. First, the ESP were
calculated again by means of the Gaussian package and then the

Aminobisphosphonates with in ViVo Antitumor ActiVity
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6805
General Procedures for the Synthesis of Tetraethyl Bisphos-
phonate 5a-f. Method A. A solution of compounds a-f (0.66
mmol) and 4 (200 mg, 0.66 mmol) in THF (10 mL) was kept in a
microwave vial and heated to 150-160 °C for 15 min in a scientific
microwave oven. After cooling at room temperature, the solvent
was evaporated in vacuo, the crude material was dissolved in water
(10 mL) and extracted with dichloromethane (2 × 15 mL). The
organic layers were collected, dried, filtered, and evaporated. The
crude material was purified by flash chromatography on silica gel
with dichloromethane-methanol as eluent.
2.20-2.35 (m, 1H), 4.45-4.58 (m, 2H), 7.35-7.42 (m, 2H),
7.83-7.98 (m, 2H), 8.72 (s, 1H). 13C NMR (NaOD/D2O) δ 25.3,
34.0, 118.4, 125.9, 143.9, 151.5. Anal. (C9H12N2O6P2) C, H, N.
[2-(Imidazol-2-yl-thio)ethyl]-bisphosphonic acid (6d). Yield 40%.
1H NMR (NaOD/D2O) δ 2.89-2.98 (m, 1H), 3.99-4.06 (m, 2H),
7.01-7.03 (m, 2H). Anal. (C5H10N2O6P2S) C, H, N, S.
[2-(Purin-9-yl)ethyl]-bisphosphonic acid (6e). Yield 55%; yellow
solid. Title compound was identified by NMR study present as the
main isomer (90%). 1H NMR (D2O) δ 3.05 (m, 1H), 4.95 (m, 2H),
8.97 (s, 1H), 9.30 (s, 1H), 9.42 (s, 1H). 13C NMR (150.81 MHz,
D2O) δ 42.98, 38.38, 132.42, 140.56, 147.23, 153.35, 154.75.
[2-(Purin-7-yl)ethyl]-bisphosphonic acid was found as minor
isomer (10%). 1H NMR (D2O) δ 2.84 (m, 1H), 4.99 (m, 2H), 9.15
(s, 1H), 9.27 (s, 1H), 9.62 (s, 1H). Anal. (C7H10N4O6P2) C, H, N.
[2-(6-Chloro-purin-9-yl)ethyl]-bisphosphonic acid (6f). Yield
59%; yellow solid. Main isomer: 1H NMR (NaOD/D2O) δ
2.16-2.32 (m, 1H), 4.32-4.42 (m, 2H), 7.93 (s, 1H), 7.97 (s, 1H).
13C NMR (NaOD/D2O) δ 41.5, 44.6, 133.3, 146.7, 149.5, 150.8,
152.7. Anal. (C7H9ClN4O6P2) C, H, N.
Biology. Expansion in Vitro of γδT Cells. Heparinized blood
specimens were collected from 12 healthy volunteers (males, age
range 24-46 years), all working at the Dipartimento di Biopatologia
e Metodologie Biomediche, University of Palermo, upon informed
consent. The study was approved by the ethical committee of the
University Hospital. Peripheral blood mononuclear cells were
incubated in vitro with different N-BPs or BrHPP (at the indicated
final concentrations) and 20 U/mL final concentration of rIL-2, for
7 days at 37 °C. At the end of the incubation period, cells were
collected and the γδ T cell expansion factor calculated according
to our previously published method.27
Expansion in vitro of γδ T cells, when the mechanism of action
of different N-BPs was evaluated, was performed as indicated
previously, except that the assay was carried out in the presence or
absence of mevastatin or lovastatin, both used at 1 µM final
concentration.
Method B. A mixture of compounds a-f (0.66 mmol), 4 (200
mg, 0.66 mmol), and TBD (88 mg, 0.066 mmol) in THF (15 mL)
was stirred under reflux for 4 h, the solvent was evaporated in vacuo,
and the crude material was dissolved in water (10 mL) and extracted
with dichloromethane (2 × 10 mL). The organic layers were
collected, dried, filtered, and evaporated. The crude material was
purified by flash chromatography on silica gel with dichlo-
romethane-methanol as eluent.
Tetraethyl-[2-(imidazol-1-yl)ethyl]-bisphosphonate (5a). Yield
1
90%; yellow oil. H NMR δ 1.25-1.34 (m, 12H), 2.40-2.85 (m,
1H), 4.05-4.21 (m, 8H), 4.41-4.59 (m, 2H), 6.90 (s, 1H), 7.21
(s, 1H), 7.57 (s, 1H). 13C NMR δ 13.4, 45.3, 48.2, 61.3, 121.7,
126.9, 141.6.
Tetraethyl-[2-(pyrazol-1-yl)ethyl]-bisphosphonate (5b). Yield
1
79%; yellow oil. H NMR δ 1.25-1.32 (m, 12H), 3.16-3.43 (m,
1H), 4.02-4.20 (m, 8H), 4.56-4.73 (m, 2H), 6.17-6.19 (m, 1H),
7.48 (d, J ) 2.0, 1H), 7.52 (d, J ) 2.0, 1H).
Tetraethyl-[2-(benzoimidazol-1-yl)ethyl]-bisphosphonate (5c).
Yield 71%; yellow oil. 1H NMR δ 1.20-1.33 (m, 12H), 2.45-2.80
(m, 1H), 4.08-4.20 (m, 8H), 4.45-4.60 (m, 2H), 7.30-7.42 (m,
2H), 7.83-7.98 (m, 2H), 8.90 (s, 1H). 13C NMR δ 13.2, 25.3, 34.0,
65.3, 118.4, 125.9, 143.9, 151.5.
Tetraethyl-[2-(imidazol-2-yl-thio)ethyl]-bisphosphonate (5d). Yield
1
45%; yellow oil. H NMR δ 1.24-1.34 (m, 12H), 3.80-3.91 (m,
1H), 4.06-4.28 (m, 8H), 4.36-4.52 (m, 2H), 6.64 (d, J ) 2.0,
1H), 6.87 (d, J ) 2.0, 1H), 11.15 (br, 1H).
In Vivo Antitumor Activity. In vivo antitumor activity of γδ T
cells was evaluated in SCID mice, using the protocol described by
Kabelitz et al., with minor modifications.19
By means of the procedure utilized, 5e and 5f were obtained as
inseparable mixtures of regioisomers (ratio 9:1), whose structure
was defined by NMR experiments carried out on derivative 6e.
Tetraethyl-[2-(purin-9-yl)ethyl]-bisphosphonate (5e). Yield 48%;
yellow oil. Main isomer: 1H NMR δ 1.19-1.24 (m, 12H),
3.36-3.53 (m, 1H), 4.06-4.16 (m, 8H), 4.75-4.83 (m, 2H), 8.17
(s, 1H), 8.95 (s, 1H), 9.11 (s, 1H). 13C NMR δ 13.3, 44.5, 48.7,
62.0, 133.3, 146.6, 149.6, 150.8, 168.3.
Briefly, SCID mice (6 mice per group) were conditioned by
irradiation (300 rads from a cesium source) and antiasialo-GM1
monoclonal antibody (BD Bioscience) treatment. Mice received at
day 0 a single intraperitoneal injection of the breast cancer cell
line BT549 (2 × 106/mouse). These high numbers of tumor cells
were inoculated to allow for full-fledged tumor growth within the
period before graft-vs-host reactions developed in the SCID mouse
reconstituted with human lymphocytes. Mice received, concomi-
tantly with the tumor cells, an intraperitoneal injection of human
recombinant (r)IL-2 (300 ng), or 6a (2 µg), or zoledronate (5 µg),
or 6a (2 µg) plus rIL-2 (300 ng), or zoledronate (5 µg) plus rIL-2
(300 ng). Where indicated, mice received, in addition, highly
purified human γδ T cells (2 × 107/mouse). γδ T cells (2 × 107/
mouse) and zoledronate (5 µg), or 6a (2 µg), were injected again
every 15 days, while (r)IL-2 (300 ng) was given on the same day
and at 3 and 6 days after each injection of γδ T cells. *p < 0.02
and **p < 0.005, when compared to all other groups.
Cytotoxicity Assays. To evaluate the number of live and dead
neoplastic cells, the cells were stained with trypan blue and counted
on a hemocytometer. To determine the growth inhibitory activity
of the drugs tested, 2 × 105 cells were plated into 25 mm wells
(Costar, Cambridge, UK) in 1 mL of complete medium and treated
with different concentrations of each drug. After 48 h of incubation,
the number of viable cells was determined and expressed as the
percentage of control proliferation.
Tetraethyl-[2-(6-chloro-purin-9-yl)ethyl]-bisphosphonate (5f).
1
Yield 50%; yellow oil. Main isomer: H NMR δ 1.20-1.24 (m,
12H), 3.35-3.43 (m 1H), 4.01-4.15 (m, 8H), 4.73-4.84 (m, 2H),
8.0 (s, 1H), 8.22 (s, 1H), 8.71 (s, 1H). 13C NMR δ 13.0, 41.5,
44.6, 62.3, 133.3, 146.7, 149.5, 150.8, 152.7.
General Procedures for the Synthesis of Bisphosphonic acid
6a-f. Method A. A solution of 5a-f (0.25 mmol) in concentrated
HCl (3 mL) was stirred under reflux for 3 h and the solvent
evaporated in vacuo. The residual solid was recrystallized from
H2O-MeOH to give 6a-f.
Method B. To an ice-cooled solution of 5a-f (0.25 mmol) in
CCl4 (5 mL) was added iodotrimethylsilane (0.179 mL, 1.25 mmol)
dropwise, and the resulting mixture was stirred below 5 °C for 3 h.
The reaction mixture was quenched by adding MeOH (10 mL),
and the precipitate was collected and recrystallized by from H2O-
MeOH to give 6a-f as white or yellow solids.
[2-(Imidazol-1-yl)ethyl]-bisphosphonic acid (6a). Yield 90%;
white solid, mp >250 °C dec. 1H NMR (NaOD/D2O) δ 1.83-1.94
(m, 1H), 4.16-4.24 (m, 2H), 6.73 (s, 1H), 7.09 (s, 1H), 7.57 (s,
1H). 13C NMR (NaOD/D2O) δ 44.3, 47.0, 120.7, 126.9, 138.8.
Anal. (C5H10N2O6P2) C, H, N.
[2-(Pyrazol-1-yl)ethyl]-bisphosphonic acid (6b). Yield 79%; white
solid, mp ) 199-202 °C. 1H NMR (NaOD/D2O) δ 2.03-2.24 (m,
1H), 4.27-4.39 (m, 2H), 6.02-6.06 (m, 1H), 7.29 (s, 1H), 7.59
(s, 1H). Anal. (C5H10N2O6P2) C, H, N.
Clonal Assays. To evaluate the cytotoxic effects of biphospho-
nates on normal hemopoietic progenitor cells, a clonal assay for
CFU-GM (colony-forming units-granulocyte macrophage) was
performed. Bone marrow mononucleated cells were obtained from
bone marrow aspirates of five normal volunteers. Bone marrow
(3-5 mL) was diluted in RPMI 1640, layered over a Ficoll-Hypaque
gradient (density, 1.077), centrifuged at 400g for 30 min, and the
interface mononuclear cells were collected. The interface cells were
[2-(Benzoimidazol-1-yl)ethyl]-bisphosphonic acid (6c). Yield
67%; white solid, mp >250 °C dec. 1H NMR (NaOD/D2O) δ

6806 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Simoni et al.
washed three times in PBS, counted, and resuspended at a
concentration of 1 × 105 in MEM containing 0.9% methylcellulose,
30% FCS, 10-4 M ꢀ-mercaptoethanol, 5% medium conditioned
by leukocytes in the presence of phytohemagglutinin (PHA-LCM)
in 15 mm plastic dishes. After 7 days of culture at 37 °C in an
environment of 5% CO2 and 100% humidity, the number of CFU-
GM was evaluated.
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derivatives 6a-f. This material is available free of charge via the
Internet at http://pubs.acs.org.

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