(2-Aminobenzothiazole)-Methyl-1,1-bisphosphonic acids: Targeting matrix metalloproteinase 13 inhibition to the bone

Article abstract of DOI:10.3390/ph14020085

Matrix Metalloproteinases (MMPs) are a family of secreted and membrane-bound enzymes, of which 24 isoforms are known in humans. These enzymes degrade the proteins of the extracellular matrix and play a role of utmost importance in the physiological remodeling of all tissues. However, certain MMPs, such as MMP-2, -9, and -13, can be overexpressed in pathological states, including cancer and metastasis. Consequently, the development of MMP inhibitors (MMPIs) has been explored for a long time as a strategy to prevent and hinder metastatic growth, but the important side effects linked to promiscuous inhibition of MMPs prevented the clinical use of MMPIs. Therefore, several strategies were proposed to improve the therapeutic profile of this pharmaceutical class, including improved selectivity toward specific MMP isoforms and targeting of specific organs and tissues. Combining both approaches, we conducted the synthesis and preliminary biological evaluation of a series of (2-aminobenzothiazole)-methyl-1,1-bisphosphonic acids active as selective inhibitors of MMP-13 via in vitro and in silico studies, which could prove useful for the treatment of bone metastases thanks to the bone-targeting capabilities granted by the bisphosphonic acid group.

Full text of DOI:10.3390/ph14020085

pharmaceuticals
Communication
(2-Aminobenzothiazole)-Methyl-1,1-Bisphosphonic Acids:
Targeting Matrix Metalloproteinase 13 Inhibition to the Bone
Antonio Laghezza 1 , Luca Piemontese 1 , Leonardo Brunetti 1 , Alessia Caradonna 1,
Mariangela Agamennone 2 , Fulvio Loiodice 1,* and Paolo Tortorella 1,
*
1
Department of Pharmacy and Pharmaceutical Sciences, University of Bari “A. Moro”, via E. Orabona 4,
70125 Bari, Italy; antonio.laghezza@uniba.it (A.L.); luca.piemontese@uniba.it (L.P.);
leonardo.brunetti@uniba.it (L.B.); a.caradonna@hotmail.it (A.C.)
Department of Pharmacy, University “G. d’Annunzio” of Chieti-Pescara, Via Dei Vestini, 31, 66100 Chieti,
Italy; magamennone@unich.it
2
*
Correspondence: fulvio.loiodice@uniba.it (F.L.); paolo.tortorella@uniba.it (P.T.)
Abstract: Matrix Metalloproteinases (MMPs) are a family of secreted and membrane-bound enzymes,
of which 24 isoforms are known in humans. These enzymes degrade the proteins of the extracellular
matrix and play a role of utmost importance in the physiological remodeling of all tissues. However,
certain MMPs, such as MMP-2, -9, and -13, can be overexpressed in pathological states, including
cancer and metastasis. Consequently, the development of MMP inhibitors (MMPIs) has been explored
for a long time as a strategy to prevent and hinder metastatic growth, but the important side effects
linked to promiscuous inhibition of MMPs prevented the clinical use of MMPIs. Therefore, several
strategies were proposed to improve the therapeutic profile of this pharmaceutical class, including
improved selectivity toward specific MMP isoforms and targeting of specific organs and tissues.
Combining both approaches, we conducted the synthesis and preliminary biological evaluation
of a series of (2-aminobenzothiazole)-methyl-1,1-bisphosphonic acids active as selective inhibitors
of MMP-13 via in vitro and in silico studies, which could prove useful for the treatment of bone
metastases thanks to the bone-targeting capabilities granted by the bisphosphonic acid group.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Laghezza, A.; Piemontese,
L.; Brunetti, L.; Caradonna, A.;
Agamennone, M.; Loiodice, F.;
Tortorella, P. (2-Aminobenzothiazole)-
Methyl-1,1-Bisphosphonic Acids:
Targeting Matrix Metalloproteinase
13 Inhibition to the Bone.
Keywords: Matrix Metalloproteinase inhibitors; bone targeting; bisphosphonates; antitumor agents;
Pharmaceuticals 2021, 14, 85.
skeletal malignancies
https://doi.org/10.3390/ph14020085
Academic Editor:
Salvatore Santamaria
1. Introduction
Received: 18 December 2020
Accepted: 20 January 2021
Published: 24 January 2021
The remodeling of the extracellular matrix (ECM) is an essential process for the devel-
opment and maintenance of numerous organs and tissues, such as bone. This process is
regulated by a variety of mediators and enzymes, among which Matrix Metalloproteinases
(MMPs) are widely considered to be the most important players [1–3]. Their expression
is finely regulated, and their activity is also modulated by tissue inhibitors of MMPs
(TIMPs) [3].
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
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iations.
In bone, MMPs are physiologically involved in several processes, including cellular
differentiation of osteoblasts, bone formation, bone resorption, and osteoclast recruitment
and migration. During the metastatic process, MMPs are involved in the so-called “vicious
cycle” established by malignant cells. In order to carve out the metastatic niche, these cells
trigger RANK-Ligand (RANKL) production by osteoblasts, which promotes osteoclast
differentiation and bone matrix resorption; the degradation of the extracellular matrix
results in the release of growth factors, driving forward tumor growth and fueling the
vicious cycle [4–6].
Bone metastases are linked to so-called skeletal-related events, which include spinal
cord compression and bone fractures. Moreover, they cause chronic pain and bone marrow
aplasia and impair patient mobility, resulting in a severe reduction in quality of life. A
wide range of treatments is available for bone metastasis (e.g., radiotherapy, chemotherapy,
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
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Pharmaceuticals 2021, 14, 85. https://doi.org/10.3390/ph14020085
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Pharmaceuticals 2021, 14, 85
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endocrine treatments, and orthopedic intervention). In this context, bisphosphonates, such
as zoledronic acid, were standard-of-care for more than a decade and currently share
the spotlight with RANKL-targeted monoclonal antibodies, such as Denosumab, for the
treatment of this condition. Regrettably, although these therapeutic options significantly
reduce the morbidity related to bone metastases, they are generally palliative and do not
lead to tumor eradication [7–10].
Likewise, the ongoing research efforts toward the development of Matrix Metallopro-
teinase Inhibitors (MMPIs) have not yet resulted in satisfying clinical applications, mostly
due to a lack of selectivity caused by the hydroxamic zinc-binding group (ZBG), typical
of the first generation of MMPIs. Indeed, indiscriminate inhibition of MMPs and other
zinc proteases leads to severe adverse reactions, ranging from pain to musculoskeletal syn-
dromes, since zinc ions are essential to an enormous variety of biological processes [1,11].
Therefore, in the last decade, most research efforts focused on curbing the adverse effects
of these compounds by acting on their selectivity or by targeting them to specific tissues,
such as bone. This was achieved either by designing nonzinc-binding MMPIs, which target
allosteric sites, or by developing compounds containing novel, more selective ZBGs [1,3,12].
Bisphosphonic acids belong to the latter class of compounds due to the bone-seeking
nature of the bisphosphonic moiety itself. In the last few years, they proved to be a reliable
and versatile scaffold for the development of novel bone-seeking MMPIs [13–16]. Lead
compound ML115 showed high potency and was also endowed with antiosteoclastic
activity, which could reduce skeletal disease burden in patients with conditions involving
abnormal bone resorption [
that modifications on the arylbisphosphonic scaffold could afford compounds which
selectively inhibit MMP-2 [ 15 17] and MMP-9 [18], and that also exhibit significant
potential for bone malignancy therapy, being superior at promoting cancer apoptosis
than standard-of-care bisphosphonates, such as zoledronic acid [ 17]. Moreover, these
8,17]. Further developments along this line of research showed
8, ,
8,
bisphosphonic MMPIs (BMMPIs) showed no particular side effects in vivo at therapeutic
dosages [8].
The significant inhibitory potency of ML115 toward MMP-2 and MMP-8 can be
explained by the increased hydrophobic interactions that the biphenyl group forms with
0
the deep S1 sites of such MMP isoforms [19]. However, inhibition of MMP-13, an enzyme
with an equally deep S10 pocket [20], has not yet been investigated as part of the activity
spectrum of bisphosphonic acids. This MMP isoform is strongly overexpressed in the
metastatic microenvironment, where it plays a central role by activating MMP-9 and
osteolysis, resulting in the release of growth factors that further stimulate cancer cell
proliferation. Indeed, in recent years, various studies validated MMP-13 as a therapeutic
target for the treatment of bone metastases [21–24]. Following this particular line of
research, we report the development of a series of (2-aminobenzothiazole)methyl-1,1-
bisphosphonic acids with particular attention to their activity as MMP-13 inhibitors, and
provide a rationalization of their activity profile via computational studies.
2. Results and Discussion
Compounds 1–12 (reported in Table 1) were specifically developed to evaluate how
the substitution of the biphenylsulfonamide of ML115 with a 2-aminobenzothiazole moiety
could modify the MMP inhibition profile of these compounds, and whether substitutions
on the benzothiazole scaffold could be used to further modulate their activity.

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Table 1. Compounds 1–12.
1: R = H
2: R = F
3: R = Cl
4: R = NO2
ML115
5: R = H
6: R = Br
8
7: R = NO2
9
10
12
11
Treatment of the appropriate commercially available 2-aminobenzothiazole with
triethyl orthoformate and diethyl phosphite afforded tetraethyl-1,1-bisphosphonates 1a–
4a. These bisphosphonic esters were dealkylated with trimethylsilyl bromide (TMBS) in
anhydrous acetonitrile, affording bisphosphonic acids 1–4. Intermediate 4a, bearing a nitro
group, was also separately reduced to compound 5b, which was the key to the synthesis of
compounds 5–11. For compounds 5–8, the precursor was acylated with the appropriate
acyl chloride and subsequently dealkylated with TMBS. Compounds 9–11 were instead
obtained via condensation with phenyl isocyanate, phthalic anhydride or Boc-L-Ala and
subsequent dealkylation of the bisphosphonic ester intermediates with TMBS (Figure 1).
Compound 12 required a different synthetic route, involving the preparation of
a tetraethyl ethenyliden-bisphosphonate (12b), which was condensed with 2-amino-6-
chlorobenzothiazole, affording intermediate 12a, whose dealkylation in acidic conditions
resulted in the desired product 12 (Figure 2).

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Figure 1. Synthesis of compounds 1–11. Reagents and conditions: ( ◦
a
) HC(OEt)3, HP(OEt)2, 160◦C, 6 h (15–53%); (
rt, 3–12 h (82%); ( ) phenyl isocyanate, toluene
dry, reflux, 2 h (57%); (e) phthalic anhydride, glacial AcOH, reflux, 4 h (54%); (f) EDC, DMAP, Boc-L-Ala, CHCl3, rt, 12 h
b) H2,
Pd/C 10%, EtOH, 3 bar, rt, 12 h (77%); (
c
) RCOOCl, NEt3, THF dry, 0 C
→
d
(47%); (g) TMBS, CH2Cl2 dry, rt, 24–48 h (40–96%).
Figure 2. Synthesis of compound 12. Reagents and conditions: (
a
) diethylamine, MeOH, reflux 24 h, (
b) p-TsA, toluene,
reflux, 6 h (yield a, b 81%); (c d) 3N HCl, reflux, 17 h
(96%).
) 2-amino-6-chlorobenzothiazole, CHCl3 dry, N2, 40 ◦C, 27 h (65%); (
Compounds
1–12 were evaluated in an enzyme inhibition assay against MMP-2, -8, -9,
and -13, whose results are reported in Table 2, with ML 115 as a reference compound.

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Table 2. In vitro inhibitory activity was evaluated by fluorometric assay using commercially available catalytic domains
of MMP-2, -8, -9, and -13. IC50 M values are reported as the mean SEM (standard error of the mean) of at least 3
independent experiments, which were performed in triplicate.
µ
±
Compound
R
n
MMP-2
MMP-8
MMP-9
MMP-13
ML 115
0.14 ± 0.04 a
12.7 ± 0.8
9.0 ± 0.9
0.40 ± 0.03 a
84.3 ± 2.1
32.1 ± 1.9
14.6 ± 0.2
9.0 ± 0.4
9.0 ± 0.3
60 ± 2.6
>100 a
>100
26.5 ± 2.7
18.7 ± 2.9
7.7 ± 1.1
6.3 ± 0.9
>100
0.6 ± 0.3
6.5 ± 1.2
1
2
3
4
5
6
7
8
9
H
F
Cl
0
0
0
0
0
0
0
0
0
0
0
1
4.25 ± 0.35
1.23 ± 0.12
2.25 ± 0.15
0.670 ± 0.025
6.7 ± 0.5
2.64 ± 0.04
4.5 ± 0.5
NO2
NHCOPh
NHCOPh-4-Br
NHCOPh-4-NO2
NHCOCH2Ph
NHCONHPh
N(CO)2Ph
(S)-NHCOCH(CH3)NH2
Cl
1.16 ± 0.18
13.8 ± 0.9
0.98 ± 0.16
2.4 ± 0.5
6.7 ± 0.8
10.8 ± 3.1
15 ± 5
5.4 ± 1.1
0.50 ± 0.01
9.3 ± 0.7
22 ± 8
3.0 ± 0.4
13.6 ± 1.4
5.4 ± 1.6
>100
11.5 ± 1.9
10.9 ± 2.4
61.9 ± 1.3
0.82 ± 0.06
10
11
12
1.69 ± 0.08
9.4 ± 2.0
12.0 ± 0.4
54 ± 6
4.7 ± 1.1
21.3 ± 1.7
36.6 ± 1.8
a
Inhibition values from [19].
At a first glance, the most apparent characteristic of these compounds, when compared
to ML115, was reduced activity toward MMP-2 and MMP-8. Interestingly, such a decrease
occurred more markedly for MMP-8, which is a known antitarget in the treatment of metas-
tasis [
ring offered the opportunity to improve inhibition of MMP-2. The most potent compounds
were and , with IC50 values of 1.16 M and 0.98 M, respectively; these two showed to
1,3]. Our results show that adding a substituent in position 6 of the benzothiazole
5
7
µ
µ
be among the most selective toward MMP-8 (7.76 and 6.84, respectively).
Compared to ML-115, our novel benzothiazole derivatives gained activity against
MMP-9 influenced by the steric and electronic effects of the various substituents. Starting
from analogues 1–7, an increase in activity was observed as the substituents became bulkier
and more electron-withdrawing, with the notable exception of compound
dropped sharply.
6, whose potency
The most significant result of these compounds was, however, their activity as in-
hibitors of MMP-13. This was not surprising, seeing as, along with MMP-2 and MMP-8,
this enzyme isoform presents a deep S10 specificity pocket [20]. Indeed, the length of the
substituent seems to be a crucial parameter for the activity of these compounds; shorter
compounds, such as 1–4, were two to ten times less potent than longer derivatives
5 and 7,
which instead showed the optimal length for MMP-13 inhibition. Compound 11, with an
(L)-Ala residue, showed a stark decrease in potency, probably due to the polarity of the
free amino group and the more flexible nature of this substituent. Even longer substituents,
as seen with compounds
potency.
6,
8
, and
9, or more rigid ones (10), also led to a marked loss of
Compound 12 showed an interesting behavior. With respect to its homologue
3
, the
longer spacer between the BP group and the aryl portion improved activity toward MMP-
13 while reducing potency toward MMP-2, -8, and -9 with optimal Ligand Efficiency (LE)
and selectivity (especially toward MMP-8, as seen in Table 3).

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Table 3. Selectivity and ligand efficiency calculated on the basis of the IC50 values of Table 2.
Selectivity
Ligand Efficiency
MMP-8 MMP-9
Compound
MMP-2/13
MMP-8/13
MMP-9/13
MMP-2
MMP-13
ML115
0.23
1.95
2.12
2.15
2.00
1.73
2.06
1.96
0.26
0.26
0.16
0.15
5.73
0.67
12.97
7.55
11.87
4.00
13.43
8.96
13.40
1.16
1.30
1.10
0.87
25.98
>166.67
>15.4
6.24
15.20
3.42
0.38
0.36
0.35
0.38
0.33
0.29
0.22
0.27
0.27
0.26
0.26
0.28
0.35
0.35
0.30
0.31
0.33
0.31
0.25
0.19
0.23
0.23
0.23
0.23
0.23
0.31
-
-
0.34
0.38
0.37
0.41
0.35
0.30
0.24
0.28
0.24
0.23
0.23
0.23
0.40
1
2
3
4
5
6
7
8
9
0.31
0.32
0.32
0.25
-
0.23
0.22
0.23
0.24
-
9.40
>14.92
10.80
2.37
1.18
0.50
>1.62
44.63
10
11
12
0.29
The higher selectivity of these compounds toward MMP-13 is not unwelcome for the
development of novel BMMPIs. Indeed, MMP-13 favors the formation of metastasis by
modeling the premetastatic niche and stimulating the processes of angiogenesis, thereby re-
sulting in enhanced survival and growth of cancer cells in their metastatic environment [22].
Furthermore, MMP-13 is highly expressed in lung and prostate malignancies [1,3], is a
validated therapeutic target for a variety of pathologies that imply abnormal tissue degra-
dation [21], and is not involved in the adverse effects of broad-spectrum MMPIs. Selective
inhibitors of MMP-13 were developed over the years that, instead of binding the zinc
ion, exploit the interaction with an accessory pocket named S10* [
3,25]. Although the
latest research efforts in the field of MMP-13 inhibitors focused on the development of
nonzinc-binding ligands, a recent approach, involving the introduction of a zinc-binding
group to an otherwise nonzinc-binding class of compounds, yielded compounds capable
of selectively inhibiting this enzyme in the submicromolar range [26].
Docking studies confirmed that these compounds inhibit MMPs thanks to both 0their
ZBG (the bisphosphonic function) and their aromatic moiety that interacts with the S1 site,
a hydrophobic pocket that influences ligand selectivity of MMP inhibitors.
The main difference between ML115 and the benzothiazole series is the lack of the
sulfonamide function. This group is known to be a key H-bond acceptor that, by binding
to NH of Leu164 and Ala165 (MMP-13 numbering), addresses the aromatic portion in the
S10 site of MMPs [27].
The absence of the sulfonamide moiety causes the linker between the bisphosphonate
and the aromatic portion to be one atom shorter, making the contemporary binding of both
functions more difficult and accounting for the lower potency measured for this class of
ligand with respect to sulfonamide bisphosphonates.
For this reason, moreover, the binding mode obtained for these ligands in all MMPs is
not well conserved; one phosphonic group coordinates the zinc ion following the crystallo-
graphic tetrahedral geometry, while the other can line up to the binding mode observed in
the X-ray complex [18] for some ligands. At the same time, for other ligands, the second
phosphonic group reaches Leu164 and Ala165 NHs, providing key H-bonds and allowing
the aromatic group to reach deeper in the S10 site, as observed for arylamino-derivatives
that possess the same structural frame [15]. The alpha NH often forms a H-bond with the
Ala165 CO.
The better activity observed for these molecules toward MMP-13 can be attributed to
the larger flexibility of the S10 loop of this isoform with respect to other MMPs [28], giving
relief to the conformational strain paid by ligands to adapt to the binding site, even if the
observed interactions in the docked poses were almost the same.

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The binding of compound
7, the most active inhibitor of MMP-13 in the series, to the
binding site of this enzyme is reported as an example (Figure 3).
Figure 3. Docked pose of compound
7 (stick, green C atoms) in the MMP-13 binding site (grey
cartoon). Most relevant residues are represented as lines and H-bonds are represented by yellow
dashed lined.
In order to further validate the obtained binding geometries, additional docking
calculations were carried out for the studied ligands using Autodock 4.2 [29]. Applied
methods and results are reported in the Supplementary Material, and the similarity of the
poses obtained with both programs is shown, once again using compound
in Figure S1.
7 as an example,
3. Materials and Methods
3.1. MMP Inhibition Assays
Catalytic domains of MMP-2, -8, -9, and -13 were obtained from Enzo Life Sciences.
96-well white microtiter plates (Corning, NBS) were used to carry out the assays (in
triplicate). The assay measurements were performed by preparing dilutions to six different
concentrations (1 nM–100
µM) of each inhibitor in a fluorometric assay buffer (50 mM
Tris HCl pH 7.5, 200 mM NaCl, 1 mM CaCl2, 1
·
µ
M ZnCl2, 0.05% Brij-35, and 1% DMSO).
Incubation of the enzyme and inhibitor solutions occurred for 15 min at room temperature;
fluorogenic substrate solution (OmniMMP® = Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2,
Enzo Life Sciences, 2.5
Cha-Abu-Smc-His-Ala-Dab (6 -TAMRA)-Ala-Lys-NH2, Enzo Life Sciences, and 1
µ
M final concentration or OmniMMP®RED = TQ3-GABA-Pro-
0
µ
M final
◦
concentration) was subsequently added. The assay was incubated for 2–4 h at 37 C,
after which a Perkin–Elmer Victor V3 plate reader was used to measure fluorescence
(λex = 340 nm, λem = 405 nm or λex = 545 nm, λem = 572 nm). Included in the assay were
control wells lacking any inhibitor. MMP activity was thus determined and expressed in
relative fluorescence units (RFU). Percent inhibition was calculated from control wells and
IC50 values were determined using GraphPad Prism 5.0 and are shown as mean
at least three independent measurements, which were performed in triplicate.
±
SEM of
3.2. Chemical Methods
All reagents were purchased from Sigma Aldrich Chemicals (Milan, Italy) and were
used without purification. The reactions were monitored by TLC (silica gel, UV254) with
UV light (short wave ultraviolet 254 nm and long wave ultraviolet 365 nm). All an-
hydrous reactions were performed under argon or nitrogen atmosphere. The column

Pharmaceuticals 2021, 14, 85
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˙
chromatography was performed using Fluka silica gel 60
60 (40–63
A
(63–200
µ
m) or silica gel Si
µ
m). Mass spectra were recorded on an HP MS 6890-5973 MDS spectrometer,
electron impact 70 eV, equipped with an HP ChemStation or with an Agilent 6530 Se-
ries Accurate-Mass Quadrupole Time-of-FLIGHT (Q-TOF) LC/MS. High-resolution mass
spectrometry (HRMS) analyses were performed using a Bruker microTOF QII mass spec-
trometer equipped with an electrospray ion source (ESI). 1H NMR was recorded using the
suitable deuterated solvent on a Varian Mercury 300 or 500 NMR Spectrometer. Chemical
shift (δ) was expressed as parts per million (ppm) and the coupling constant (J) in Hertz
(Hz). Melting points were determined in open capillaries on a Gallenkamp electrothermal
apparatus and were uncorrected.
3.2.1. General Procedure for the Preparation of Tetraethyl Bisphosphonates (1a–4a)
Triethyl orthoformate, diethyl phosphite and the opportune 2-aminobenzothiazole
were added to a round-bottom flask fitted with a distillation apparatus in a 1.2:3:1 stoichio-
metric ratio. The resulting mixture was then heated to 160 ◦C until all EtOH was distilled
away; the residue was dissolved in ethyl acetate, and evaporated away under vacuum,
affording a crude yellow oil, which was further purified via column chromatography over
silica gel (eluent EtOAc/MeOH 9:1). The desired compounds were obtained as white solids
in 15–53% yields.
Tetraethyl [(benzo[d]thiazol-2-amino)methyl]-1,1-bisphosphonate (1a): White solid, 42%
yield. 1H NMR (500 MHz, CDCl3):
δ = 1.26–1.32 (m, 12H, CH3), 4.17–4.30 (m, 8H, CH2),
5.26 (t, JHP = 21.52, 1H, PCHP), 6.17 (bs, 1H, NH), 7.12 (t, J = 7.83,1H, aromatic), 7.30 (t,
J = 7.83,1H, aromatic), 7.55–7.59 (m, 2H, aromatics). MS (ESI): m/z: 437[M+H]+; MS2 m/z
(%): 299(46), 271 (88), 243(100), 225(66), 161 (39).
Tetraethyl [(6-fluorobenzo[d]thiazol-2-amino)methyl]-1,1-bisphosphonate (2a): White
solid, 53% yield. 1H NMR (500 MHz, CDCl3):
δ = 1.26–1.31 (m, 12H, CH3), 4.18–4.27
(m, 8H, CH2), 5.19 (t, 1H, PCHP), 5.95 (bs, 1H, NH), 7.00–7.03 (m, 1H, aromatic), 7.26–7.29
(m, 1H, aromatic), 7.45–7.48 (m, 1H, aromatic). MS(ESI): m/z: 455 [M+H]+; MS2: m/z (%):
317(38), 289(89), 261(100), 243 (75), 179(39).
Tetraethyl [(6-chlorobenzo[d]thiazol-2-amino)methyl]-1,1-bisphosphonate (3a): White
solid, 15% yield. 1H NMR(500 MHz, CDCl3):
δ = 1.26–1.31 (m, 12 H, CH3), 4.16–4.29
(m, 8H, CH2), 5.19 (t, JHP = 21.53, 1H, PCHP), 5.80 (bs, 1H, NH), 7.24–7.27 (dd, J1 = 8.81,
J2 = 2.45, 1H, aromatic), 7.44 (d, J = 8.81, 1H, aromatic), 7.54 (d, J = 2.45, 1H, aromatic).
MS(ESI): m/z: 495 [M+2+Na]+, 493[M+Na]+, 473[M+2+H]+, 471[M+H]+; MS2(471): m/z
(%): 335 (27), 333(43), 307(49), 305(86), 279 (45), 277(100), 197 (18), 195 (41).
Tetraethyl [(6-nitrobenzo[d]thiazol-2-amino)methyl]-1,1-bisphosphonate (4a): Yellow solid,
40% yield. 1H NMR(500 MHz, CDCl3):
δ = 1.26 (t, J = 7.095, 6H, CH3), 1.33 (t, J = 7.095,
6H, CH3), 1.7(bs, 1H, NH), 4.18–4.32 (m, 8H, CH2), 5.29 (t, JHP = 21.53, 1H, PCHP), 7.55 (d,
J = 8.81, 1H, aromatic), 8.19–8.21(dd, J1 = 8.81, J2 = 2.45, 1H, aromatic), 8.48 (d, J = 2.45, 1H,
aromatic). MS(ESI): m/z: 482[M+H]+; MS2: m/z (%): 344(18), 316.01(69), 288 (100), 272 (12),
206 (25), 139 (16).
Tetraethyl [(6-aminobenzo[d]thiazol-2-amino)methyl]-1,1-bisphosphonate (5b): Ten per-
cent Pd/C (0.20 mmol) was added to the solution of the nitro compound (0.532 mmol) in
8.5 mL EtOH, and the mixture was hydrogenated at room temperature at a pressure of 3
bar for 12 h. The reaction mixture was filtered through a pad of celite and the filtrate was
evaporated in vacuo to give an oil. The residue was purified by chromatography on silica
gel (eluent: CHCl3/ MeOH 9.5: 0.5 v/v) to give the desired amino derivate. White solid,
77% yield. 1H NMR (CDCl3, 500 MHz):
δ = 1.25–1.30 (m, 12H, CH3), 3.64 (b, 2H, NH2),
4.09–4.27 (m, 8H, CH2), 5.15 (t, JHP = 21.53, 1H, PCHP), 5.52 (bs, 1H, NH), 6.67 (dd, J1 = 8.32,
J2 = 2.45, 1H, aromatic), 6.91 (d, J = 2.45, 1H, aromatic), 7.35 (d, J = 8.32, 1H, aromatic).
MS(ESI): m/z: 474[M+Na]+; MS2: m/z (%): 336 (100), 308 (73), 262 (32), 174 (24), 146 (22).

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3.2.2. General Procedure for N-Acylation of Compound 5b
The appropriate acyl chloride (1.1–2 mmol) and triethylamine (2 mmol) were added to
the solution of amino derivate (5b) (1 mmol) in anhydrous THF and the mixture was stirred
at room temperature on nitrogen or argon for 3–12 h. After the given time, the eluent was
evaporated in vacuo and the residue was partitioned between EtOAc and NaHCO3 and the
layers were separated. The organic phase was washed with HCl 1N, NH4Cl ss, brine, dried
over anhydrous Na2SO4, filtered and the filtrate was evaporated in vacuo. The residue
was purified by chromatography on silica gel (eluent: CHCl3/MeOH 98:2 v/v or AcOEt
/MeOH 9:1 v/v) or crystalized with AcOEt to give the desired product.
Tetraethyl [(6-(benzamido)benzo[d]thiazol-2-amino)methyl]-1,1-bisphosphonate: White
solid, 61% yield (chromatography, eluent: CHCl3/MeOH 98: 2 v/v). 1H NMR (500 MHz,
[D6] DMSO):
δ = 1.13–1.97 (m, 12H, CH3), 4.06–4.12 (m, 8H, CH2), 5.09–5.21 (td, JHP = 22.5,
JHH = 9.79, 1H, PCHP), 7.40 (d, J = 8.81, 1H, aromatic), 7.50–7.53 (m, 4H, aromatics), 7.92–
7.94 (m, 2H, aromatics), 8.16 (d, J = 1.96, 1H, aromatic), 8.73 (d, J = 9.78, 1H, NH), 10.25 (s,
1H, NH). MS (ESI): m/z: 578 [M+Na]+, 556 [M+H]+; MS2(556): m/z (%): 418 (100), 390 (92),
372 (33), 381 (33), 362 (57), 344 (73), 280 (29). 554 [M-H]−.
Tetraethyl [(6-(4-bromobenzamido)benzo[d]thiazol-2-amino)methyl]-1,1-bisphosphonate:
White solid, 82% yield, (AcOEt). 1H NMR(500 MHz, [D6] DMSO): δ = 1.13–1.21 (m, 12H,
CH3), 4.07–4.10 (m, 8H, CH2), 5.15 (td, JHP = 22.5, JHH = 9.30, 1H, PCHP), 7.40 (d, J = 8.81,
1H, aromatic), 7.51 (d, J = 8.81, 1H, aromatic), 7.73 (d, J = 8.32, 2H, aromatics), 7.91(d,
J = 8.32, 2H, aromatics), 8.16 (s, 1H, aromatic), 8.75 (d, J = 9.30, NH), 10.37 (s, 1H, NH).
MS(ESI): m/z: 658[M+2+Na]+, 656[M+Na]+, 636[M+2+H]+, 634 [M+H]+; MS2(634): m/z
(%): 498(100), 496 (70), 470 (55), 468 (62), 452 (40), 450 (32), 424 (55), 422 (47).
Tetraethyl [(6-(4-nitrobenzamido)benzo[d]thiazol-2-amino)methyl]-1,1-bisphosphonate:
Yellow solid, 64% yield, (AcOEt). 1H NMR (500 MHz, [D6] DMSO):
δ = 1.15–1.20 (m, 12H,
CH3), 4.07–4.12 (m, 8H, CH2), 5.15 (t, JHP = 22.5, 1H, PCHP), 7.43 (d, J = 8.81, 1H, aromatic),
7.51–7.53 (dd, J1 = 8.32, J2 = 1.96, 1H, aromatic), 8.16–8.18 (m, 3H, aromatics), 8.68 (m, 2H,
aromatics), 8.8 (bs, 1H, NH), 10.59 (s, 1H, NH). MS(ESI): m/z: 623[M+Na]+, 601[M+H]+;
MS2: m/z (%): 555(24), 463 (100), 436(18.25), 389 (69), 325 (25).
Tetraethyl [(6-(2-phenylacetamido)benzo[d]thiazol-2-amino)methyl]-1,1-bisphosphonate:
White solid, 56% yield, (AcOEt). 1H NMR (500 MHz, [D6] DMSO): δ = 1.12–1.18 (m, 12H,
CH3), 3.60 (s, 2H, CH2), 3.97–4.17 (m, 8H, CH2), 5.13 (td, JHP = 22.25, JHH = 9.78, 1H, PCHP),
7.22–7.36 (m, 7H, aromatics), 8.03 (s, 1H, aromatic), 8.67 (d, J = 9.78, 1H, NH), 10.15 (s, 1H,
NH). MS (ESI): m/z: 592[M+Na]+, 570[M+H]+; MS2 (570): m/z (%): 432(100), 404 (81), 386
(30), 358(61), 294(24). 568[M-H]−; MS2: m/z (%): 430(89), 339 (90), 310 (100), 287 (49), 259
(27), 190 (42), 185 (16), 137 (97), 107 (33).
Tetraethyl [(6-(3-phenylureido)benzo[d]thiazol-2-amino)methyl]-1,1-bisphosphonate: A so-
lution of phenyl isothiocyanate (1.2 mmol) in anhydrous toluene (2 mL) was added to the
suspension of 5b (1 mmol) in anhydrous toluene (2 mL) and the mixture was heated to
reflux for 2 h. After the given time, the eluent was evaporated in vacuo and the residue was
triturated with AcOEt and filtered to afford the desired product: white solid, 57% yield. 1H
NMR(300 MHz, [D6]DMSO): δ = 1.12–1.20 (m, 12H, CH3), 4.06–4.13 (m, 8H, CH2), 5.05–5.13
(td, JHP = 22.26, JHH = 9.96, 1H, PCHP), 6.91–9.95 (t, J = 7.02, 1H, aromatic), 7.12–7.28 (m,
3H, aromatics), 7.34 (d, J = 8.787, 1H, aromatic), 7.43 (d, J = 8.20, 2H, aromatics), 7.88 (d,
J = 2.34, 1H, aromatic), 8.62 (d, J = 9.96, 1H, NH), 8.72 (s, 1H, NH), 8.75 (s, 1H, NH). MS(ESI):
m/z: 593[M+Na]+, 571[M+H]+; MS2: m/z (%): 525 (17), 433 (100), 405 (77), 378 (32), 359
(58).
Tetraethyl [(6-(1,3-dioxoisoindolin-2-yl)benzothiazol-2-amino)methyl]-1,1-bisphosphonate:
A
mixture of 5b (1 mmol) and phthalic anhydride (1.07 mmol) in 6 mL of glacial acetic acid
(AcOH) was refluxed for 4 h. Then, the eluent was evaporated in vacuo; the residue
was diluted with EtOAc and a solution of 6 M NaOH was added until pH = 6, and the

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layers were separated. The organic phase was washed with brine, dried over anhydrous
Na2SO4, filtered, and the filtrate was evaporated in vacuo. The residue was purified by
chromatography on silica gel (eluent: CHCl3/MeOH 9: 1 v/v) to give the desired product:
green solid, 54% yield. 1H NMR(500 MHz, [D6]DMSO):
δ = 1.15–1.21 (m, 12H, CH3),
4.07- 4.13 (m, 8H, CH2), 5.14–5.25 (td, 1H, JHP = 22.25, JHH = 8.07, 1H, PCHP), 7.28–7.30
(dd, J1 = 8.33, J2 = 1.96, 1H, aromatic), 7.54(d, J = 8.33, 1H, aromatic), 7.76 (d, J = 1.96,
1H, aromatic), 7.88–7.97 (m, 4H, aromatics), 8.95 (d, J = 8.07, 1H, NH). MS (ESI): m/z:
604[M+Na]+, 582[M+H]+; MS2(582): m/z (%): 536 (23), 444 (100), 417 (23), 416 (80), 398
(30), 388 (57), 370 (72), 306 (24), 297 (11). 580[M-H]−; MS2: m/z (%): 137 (100), 108 (32).
Tetraethyl [(6-((L) N-Boc 2 aminopropanamido)benzothiazol-2-amino)methyl]-1,1-bisph
osphonate: Boc-L-Ala (1 mmol), EDC (1 mmol) and DMAP (2 mmol) were added to the
solution of 5b (1 mmol) in CHCl3 (4 mL) and the mixture was stirred at room temperature
on nitrogen for 12 h. After the given time, the eluent was evaporated in vacuo, the residue
was diluted with EtOAc, the organic phase was washed with brine, dried over anhydrous
Na2SO4, and filtered, and the filtrate was evaporated in vacuo. The residue was triturated
with AcOEt and filtered to afford the final compound: white solid, 47% yield. 1H NMR(500
MHz, [D6]DMSO):
δ = 1.12–1.20 (m, 12H, CH3), 1.23(d, J = 6.85, 3H, CH3), 1.36 (s, 9H,
-(CH3)3), 4.07–4.13 (m, 9H, CH2, CH), 5.07–5.19 (td, 1H, JHP = 22.25, JHH = 9.78, 1H, PCHP),
7.02 (d, J = 6.85, 1H, CH), 7.32 (dd, J1 = 8.32, J2 = 1.47, 1H, aromatic), 7.35 (d, J = 8.32,
1H, aromatic), 8.03 (d, J = 1.47, 1H, aromatic), 8.67 (d, J = 9.78, 1H, NH), 9.88 (s, 1H, NH).
MS(ESI): m/z: 645[M+Na]+, 623[M+H]+; MS2 (623): m/z (%): 521(62), 430(18), 401 (19), 385
(21), 383 (25), 355 (18), 286 (12).
3.2.3. General Procedure for the Preparation of 1,1-Bisphosphonic Acids (1–12)
Method A: A solution of the appropriate tetraethyl bisphosphonate (1 mmol) in 4 mL
2N HCl solution was kept at reflux for 12–24 h. After removal of the aqueous phase
under reduced pressure, the crude bisphosphonic acids were triturated with the opportune
solvent and filtered to afford the final compounds as white solids in 20–96% yield.
Method B: Anhydrous trimethylsilylbromide (17–32 mmol) was carefully added to a
solution of the corresponding tetraethyl bisphosphonate (1 mmol) in anhydrous acetonitrile
(6 mL) at 0 ◦C under argon and the resulting mixture was stirred at room temperature for
24–48 h. After the given time, 2 mL of MeOH was added and the mixture was stirred for
5 min. The solvent was distilled off and the crude bisphosphonic acids were triturated
with the opportune solvent and filtered to afford the desired compounds as white solids in
40–100% yield.
(Benzo[d]thiazol-2-amino)methyl-1,1-bisphosphonic acid (1): Method A; white solid, 72%
◦
yield; mp: >250 C (Et2O). 1H NMR (500 MHz, NaOD):
δ = 2.8–4.00 (br, 6H, NH, OH,
PCHP), 6.95 (t, 1H, J = 7.58, aromatic), 7.16 (t, J = 7.58, 1H, aromatic), 7.23 (d, J = 7.83, 1H,
−
aromatic), 7.51 (d, J = 7.83, 1H, aromatic). MS(ESI): m/z: 323[M-H] ; MS2: m/z (%): 287(26),
241 (100), 177 (33), 149 (10).
(6-fluorobenzo[d]thiazol-2-amino)methyl-1,1-bisphosphonic acid (2): Method A; white
solid, 26% yield; mp: >250 ◦C (EtOAc and Acetone). 1H NMR (500 MHz, [D6] DMSO):
δ
= 3.67–5.45 (br, 4H, OH), 4.77 (t, JHP = 21.28, 1H, PCHP), 7.06 (m, 1H, aromatic), 7.33–7.36
(m, 1H, aromatic), 7.59 (m, 1H, aromatic), 8.26–9.17 (b, 1H, NH). 31P NMR(500 MHz, [D6]
DMSO):
= 14.04 (d, JPH= 16.79, 2P, PCHP). MS (ESI): m/z: 341 [M-H]−; MS2: m/z (%):
δ
305(32), 259 (100), 195 (40), 167 (13).
(6-chlorobenzo[d]thiazol-2-amino)methyl-1,1-bisphosphonic acid (3): Method A; white
◦
solid, 68% yield; mp: > 250 C (Acetone/AcOEt). 1H NMR (500 MHz, [D6] DMSO):
δ
= 4.00–
6.00 (br, 4H, OH), 4.80 (t, JHP = 20.79, 1H, PCHP), 7.23 (dd, J1 = 8.81, J2 = 1.46, 1H, aromatic),
7.34 (d, J = 8.81, 1H, aromatic), 7.77 (d, J = 1.46, 1H, aromatic), 7.86–8.26 (bs, 1H, NH). 31
NMR (500 MHz, [D6] DMSO):
= 14.074 (d, 2P, PCHP). MS(ESI): m/z: 359[M+2-H]−, 357
[M-H]−; MS2: m/z (%): 323(24), 321 (41), 277 (43), 275 (100), 213 (21), 211 (44).
P
δ

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(6-chlorobenzo[d]thiazol-2-amino)ethyl-1,1-bisphosphonic acid (12): Method A; white
solid, 96% yield; mp: > 250 ◦C (AcOEt). 1H NMR (500 MHz, [D6] DMSO):
δ
= 3.6–4.00 (br,
8H, OH, PCHP, NH, CH2), 7.22 (d, J = 8.56, 1H, aromatic), 7.34 (d, J = 8.56, 1H, aromatic),
7.79 (s, 1H, aromatic). 31P NMR (500 MHz, [D6] DMSO):
δ = 17.40 (d, JPH= 22.86, 2P,
PCHP). MS (ESI): m/z: 373[M+2-H]−, 371[M-H]−; MS2 m/z (%): 337(13), 335(26), 247(40),
245(100).
(6-nitrobenzo[d]thiazol-2-amino)methyl-1,1-bisphosphonic acid (4): Method B; white solid,
◦
76% yield; mp: >250 C (MeOH). 1H NMR (500 MHz, [D6] DMSO):
δ = 4.1–5.5 (br, 5H, OH,
PCHP), 7.44 (d, J = 8.81, 1H, aromatic), 8.08 (d, J = 8.81, 1H, aromatic), 8.65 (s, 1H, aromatic),
8.65 (bs, 1H, NH). 31P NMR (500 MHz, [D6] DMSO):
δ = 13.64 (d, J = 21.45, PCHP). MS
(ESI): m/z: 368[M-H]−; MS2: m/z (%): 332 (45), 305 (25), 286 (58), 222 (100).
(6-benzamidobenzo[d]thiazol-2-amino)methyl-1,1-bisphosphonic acid (5): Method B; white
◦
solid, 40% yield; mp: >250 C(Acetone/ MeOH 3:1 v/v). 1H NMR (500 MHz, [D6] DMSO):
δ
= 4.80 (t, JHP = 20.5, 1H, PCHP), 5.00–6.4 (br, 4H, OH), 7.38 (d, J = 8.32, 1H, aromatic),
7.49–7.58 (m, 4H, aromatics), 7.93 (dd, J1 = 7.34, J2 = 1.46, 1H, aromatics), 8.16 (d, J = 1.46,
1H, aromatic), 8.95 (bs, 1H, NH), 10.24 (s, 1H, NH). 31P NMR (500 MHz, [D6] DMSO):
−
δ
= 13.69 (d, J = 16.78, PCHP). MS (ESI): m/z: 442 [M-H] ; MS2: m/z (%): 407 (15), 406 (62),
361 (22), 360 (100), 343 (18), 342 (69), 296 (25).
(6-(4-bromobenzamido)benzo[d]thiazol-2-amino)methyl-1,1-bisphosphonic acid (6): Method
B; white solid, 78% yield; mp: >250 ◦C ISO). 1H NMR(500 MHz, [D6] DMSO): 2.00–3.5 (br,
6H, OH, NH, PCHP), 7.22–7.88 (m, 6H, aromatics), 8.11 (s, 1H, aromatic), 10.24 (s, 1H, NH).
31P NMR (500 MHz, [D6] DMSO): = 14.40 (d, 2P, PCHP). MS (ESI): m/z: 522[M+2-H]−,
δ
520 [M-H]−; MS2: m/z (%): 486 (91), 484 (16), 440 (10), 438 (66), 422 (95.5), 420 (72).
(6-(4-nitrobenzamido)benzo[d]thiazol-2-amino)methyl-1,1-bisphosphonic acid (7): Method
◦
B; white solid, 96% yield; mp: > 250 C (MeOH). 1H NMR (300 MHz, [D6] DMSO):
δ = 4.73
(t, JHP = 20.33, 1H, PCHP), 4.86- 6.09 (br, 5H, OH, NH), 7.45 (d, J = 7.91, 1H, aromatic), 7.63
(d, J = 7.91, 1H, aromatic), 8.17 (d, J = 8.79, 2H, aromatic), 8.29 (s, 1H, aromatic), 8.36 (d,
J = 8.79, 2H, aromatics), 10.89 (s, 1H, NH). 31P NMR (500 MHz, [D6] DMSO): δ = 12.76 (d,
JPH = 21.24, 2P, PCHP). MS (ESI): m/z: 487[M-H]−; MS2: m/z (%): 452 (13), 451 (53), 406
(17), 405 (78), 388 (24), 387 (100), 341 (25), 339 (20), 233 (14).
(6-(2-phenylacetamido)benzo[d]thiazol-2-amino)methyl-1,1-bisphosphonic acid (8): Method
B; white solid, 69% yield; mp: >250 ◦C (ISO). 1H NMR (500 MHz, [D6] DMSO):
δ = 4.43 (s,
2H, CH2), 3.90–5.10 (br, 5H, OH, PCHP), 8.01–8.14 (m, 8H, aromatics), 8.81 (s, 1H, NH),
10.17 (s, 1H, NH). MS (ESI): m/z: 456[M-H]−; MS2: m/z (%): 420 (55), 375 (24), 374 (100),
357 (12), 356 (49), 310 (19.5).
(6-(3-phenylureido)benzo[d]thiazol-2-amino)methyl-1,1-bisphosphonic acid (9): Method B;
white solid, 69% yield; mp:> 250 ◦C (ISO). 1H NMR (300 MHz, [D6] DMSO):
δ = 4.76 (t,
JHP = 19.62, 1H, PCHP), 5.00–6.00 (br, 5H, OH, NH), 6.94 (t, J = 7.03, 1H, aromatic), 7.22–7.44
(m, 6H, aromatics), 7.92 (s, 1H, aromatic), 8.66 (s, 1H, NH), 8.74 (s, 1H, NH). 31P NMR (500
MHz, [D6] DMSO): 13.02 (d, 2P, PCHP). MS (ESI): m/z: 457[M-H]−; MS2: m/z (%): 421
(58), 376 (24), 375 (100), 357 (20), 311 (24), 238 (94).
(6-(1,3-dioxoisoindolin-2-yl)benzo[d]thiazol-2-amino)methyl-1,1-bisphosphonic acid (10):
Method B; white solid, 58% yield; mp: >250 ◦C (ISO). 1H NMR(500 MHz, [D6] DMSO):
δ
= 4.20–6.00 (br, 5H, OH, PCHP), 6.97–7.25 (m, 1H, aromatic), 7.33–7.47 (m, 3H, aromatics),
7.71 (s, 1H, aromatic), 7.83–7.95 (m, 2H, aromatics), 8.7 (br, 1H, NH). 31P NMR (500 MHz,
[D6] DMSO):
= 13.02 (d, 2P, PCHP). MS (ESI): m/z: 468[M-H]−; MS2: m/z (%): 432 (69),
δ
386 (100), 322 (20), 197 (18).
6-((L) 2-aminopropanamido)benzo[d]thiazo◦l-2-amino)methyl-1,1-bisphosphonic acid (11):
Method B; white solid, 94% yield; mp: >250 C (ISO). 1H NMR (500 MHz, NaOD):
δ = 1.19
(d, J = 6.85, 3H, CH3), 3.43–3.47 (q, J = 6.85, 1H, CH), 4.49–4.88 (br, 9H, OH, PCHP, NH,
NH, NH2), 7.08 (dd, J1 = 8.80, J2 = 1.96, 1H, aromatic), 7.20 (d, J = 8.8, 1H, aromatic), 7.54 (d,

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J = 1.96, 1H, aromatic). 31P (500 MHz, NaOD):
δ
= 14.84 (d, J = 18.31, 2P, PCHP). MS (ESI):
m/z: 409 [M-H]−; MS2: m/z (%): 373 (41), 327 (100), 309 (51), 263 (27). Procedure for the
preparation of Tetraethyl (2-methoxyethane-1,1-diyl)bisphosphonate (12c): Diethylamine (1
mmol) was added to the suspension of paraformaldehyde (1 mmol) in 13 mL of methanol.
The system was left under reflux until complete solubilization of the suspension. Subse-
quently, the solution obtained was stirred at room temperature and a solution of tetraethyl
methylenebisphosphonate (1.6 mmol) in 2 mL of methanol was added. The system was
kept at reflux for 24 h. After the given time, the solvent was evaporated in vacuo and 3 mL
of toluene was added and subsequently evaporated away three times, obtaining a yellow
oil which was used in the next reaction without further purification. 1H NMR(500 MHz
CDCl3):
δ = 1.2 (t, J = 7.1, 12H, OCH2CH3), 2.52 (t, JHP = 24.00, JHH = 6.00, 1H, PCHP), 3.2
(s, 3H, OCH3), 3.63 (m, 2H, CH3OCH2), 4.02 (m, J = 7.30, 8H, OCH2CH3). GC-MS: m/z (%):
332 (0.3), 195 (100).
Procedure for the preparation of tetraethyl ethenylidenebisphosphonate (12b): p-TsA
monohydrate (0.26 mmol) was added to the solution of tetraethyl (2-methoxyethyl)bisphosphonate
12c (5 mmol) in 7 mL of anhydrous toluene and the mixture was refluxed for 6 h. After the
given time, the solvent was evaporated in vacuo. The residue was partitioned between
CHCl3 and NaHCO3 and the layers were separated. The organic layer was washed with
brine, dried over anhydrous Na2SO4, filtered, and the filtrate was evaporated in vacuo,
obtaining a brown oil which was used in the next reaction without further purification:
81% yield. 1H NMR (500 MHz, CDCl3):
δ = 1.34 (t, J = 7.1, 12H, CH3), 4.02 (m, 8H, CH2),
6.98 (d, 2H, C=CH2). GC-MS: m/z (%): 301 (3.5), 171 (100), 163 (96).
Tetraethyl (((2-(6-chlorobenzo[d]thiazol-2-yl)amino)ethane-1,1-diyl)bisphosphonate)
(12a): Tetraethyl ethenylidenebisphosphonate 12b (1.2 mmol) was added to the solution of
2-amino-6-chlorobenzothiazole (1.62 mmol) in 8 mL of anhydrous CHCl3 and the system
◦
was stirred, under nitrogen atmosphere, at 40 C for 27 h. After the given time, the solvent
was evaporated in vacuo and the residue was purified by chromatography on silica gel
(eluent: EtOAc) to give the desired product: white solid, 65% yield. 1H NMR (500 MHz,
CDCl3):
δ = 1.24–1.76 (m, 12H, CH3), 2.84 (tt, JHP = 23, JHH = 6.36, 1H, CH), 3.99–4.07 (td,
J1 = 16.00, J2 = 6.36, 2H, CH2), 4.08–4.26 (m, 8H, CH2), 6.38 (bs, 1H, NH), 7.22–7.24 (dd,
J1 = 8.81, J2 = 1.96, 1H, aromatic), 7.41 (d, J = 8.81, 1H, aromatic), 7.55 (d, J = 1.96, 1H,
aromatic).
3.3. Docking Studies
All calculations were carried out using the Schrodinger Suite 2019-3 (Schrodinger, New
York, NY, USA) [30]. Ligand structures were built using Maestro in a fully deprotonated
form, minimized with MacroModel using the OPLS2005 force field, the PRCG algorithm,
at a convergence gradient of 0.05. A Monte Carlo Multiple Minimum/Low Mode Con-
formational Search (MCMM/LMCS) protocol was applied for the conformational search
using the automatic setup, performing 200 steps per rotatable bond. The global minimum
geometry was used to follow the docking studies carried out on the four studied MMPs
following the previously described procedure [13].
4. Conclusions
Over the years, inhibition of MMPs has been a much sought-after target for the
treatment of bone metastases, showing promise in stopping the vicious cycle of metastatic
cell growth and bone matrix degradation. Although this therapeutic strategy is plagued
by numerous and severe side effects, such as musculoskeletal syndromes, recent research
showed that targeting to a specific tissue, such as bone, and targeting specific MMPs
which are overexpressed in the metastatic microenvironment, such as MMP-13, are viable
strategies to minimize them.
Following both of these approaches, we demonstrated that a benzothiazole scaffold
coupled with a bisphosphonic moiety is a versatile starting point for the development of
bone-targeted MMP-13 inhibitors, with the possibility of tailoring their activity profile in

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order to also target other validated MMPs, mainly MMP-2, allowing for a mostly favorable
therapeutic profile for the treatment of bone malignancies.
Supplementary Materials: The following are available online at https://www.mdpi.com/1424-8
247/14/2/85/s1: Figure S1. Docked pose of compound 7 (stick) in the MMP-13 binding site (grey
cartoon) obtained with Glide (green C atoms) and Autodock (cyan C atoms).
Author Contributions: Conceptualization, P.T.; investigation, A.L., A.C., L.B., L.P., and M.A.;
writing—original draft preparation, L.B. and L.P.; writing—review and editing, M.A., P.T., and
F.L.; supervision, P.T. and F.L.; project administration, P.T. All authors have read and agreed to the
published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained in the article.
Conflicts of Interest: The authors declare no conflict of interest.
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