Towards potential nanoparticle contrast agents: Synthesis of new functionalized PEG bisphosphonates

Article abstract of DOI:10.3762/bjoc.12.130

The use of nanotechnologies for biomedical applications took a real development during these last years. To allow an effective targeting for biomedical imaging applications, the adsorption of plasmatic proteins on the surface of nanoparticles must be prevented to reduce the hepatic capture and increase the plasmatic time life. In biologic media, metal oxide nanoparticles are not stable and must be coated by biocompatible organic ligands. The use of phosphonate ligands to modify the nanoparticle surface drew a lot of attention in the last years for the design of highly functional hybrid materials. Here, we report a methodology to synthesize bisphosphonates having functionalized PEG side chains with different lengths. The key step is a procedure developed in our laboratory to introduce the bisphosphonate from acyl chloride and tris(trimethylsilyl)phosphite in one step.

Full text of DOI:10.3762/bjoc.12.130

Towards potential nanoparticle contrast agents:
Synthesis of new functionalized PEG bisphosphonates
Souad Kachbi-Khelfallah1, Maelle Monteil1, Margery Cortes-Clerget1,
Evelyne Migianu-Griffoni1, Jean-Luc Pirat2, Olivier Gager1, Julia Deschamp1
and Marc Lecouvey*1
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2016, 12, 1366–1371.
1Université Paris 13, Sorbonne Paris Cité, Laboratoire de Chimie,
Structure, Propriétés de Biomatériaux et d’Agents Thérapeutiques
(CSPBAT), CNRS UMR 7244, F-93017 Bobigny, France and 2ICG
Montpellier-UMR 5253, Equipe AM2N, ENSCM, 8, Rue de l’Ecole
Normale, F-34296 Montpellier Cedex 5, France
doi:10.3762/bjoc.12.130
Received: 16 February 2016
Accepted: 14 June 2016
Published: 04 July 2016
Email:
Associate Editor: P. R. Hanson
Marc Lecouvey* - marc.lecouvey@univ-paris13.fr
© 2016 Kachbi-Khelfallah et al.; licensee Beilstein-Institut.
License and terms: see end of document.
* Corresponding author
Keywords:
bisphosphonate; Fe2O3 nanoparticle; polyethylene glycol derivatives;
surface ligand synthesis
Abstract
The use of nanotechnologies for biomedical applications took a real development during these last years. To allow an effective
targeting for biomedical imaging applications, the adsorption of plasmatic proteins on the surface of nanoparticles must be
prevented to reduce the hepatic capture and increase the plasmatic time life. In biologic media, metal oxide nanoparticles are not
stable and must be coated by biocompatible organic ligands. The use of phosphonate ligands to modify the nanoparticle surface
drew a lot of attention in the last years for the design of highly functional hybrid materials. Here, we report a methodology to
synthesize bisphosphonates having functionalized PEG side chains with different lengths. The key step is a procedure developed in
our laboratory to introduce the bisphosphonate from acyl chloride and tris(trimethylsilyl)phosphite in one step.
Introduction
Numerous researchers are interested in the development of possess certain chemical and biological properties as the flexi-
superparamagnetic iron oxide nanoparticles (SPIONPs) because bility, the hydrophilicity and an absence of in vivo toxicity. In
of their biocompatibility which allows there in vivo use both for addition, the nanoparticulate systems so obtained must be stable
diagnosis in magnetic resonance imaging and in therapy [1,2]. in the various biological compartments and they must be
Most often, it is necessary to modify the surface of SPIONPs to stealthy to avoid the elimination by macrophages.
increase the metabolic stability.
For this purpose, appropriate coatings have already been re-
To overcome this main drawback, the NP surface could be ported [3,4]. Some of which consist in the NP surface
derivatized by various functional groups. These ligands have to modification using hydrophilic polymers (dextran, PEG) or
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bifunctional molecules substituted by amines, thiols, carboxyl- The first method, used in the industry, allows accessing the
ates, sulfonates, phosphonates or bisphosphonates [5-7]. Partic- desired products in one step under rather harsh conditions [14].
ularly, a strong interaction between the NPs and the phos- 1-Hydroxyalkylidenebisphosphonic acids have also been ob-
phonic moiety was observed and more interestingly the best tained in good yields. However, this widely used method seems
results were obtained with bisphosphonate products [8,9]. For not to be compatible with breakable and delicate functionalized
the past years, our group has focused its interest in the synthe- substrates. In contrast, our lab has developed a new synthetic
sis of various functionalized hydroxymethylene bisphospho- strategy starting from an acid chloride and tris(trimethylsilyl)
nates (HMBPs) [10] and their applications in health science, phosphite, followed by a methanolysis step [15].
especially in antitumor therapy [11-13]. Herein, we described
the synthesis of novel bifunctional PEG-HMBP compounds in This one-pot procedure allows the synthesis of various aliphat-
order to employ them as anchoring agents for SPIONPs ic and aromatic bisphosphonic acids under mild conditions.
(Figure 1).
Moreover, reactions were very fast and pure products were ob-
tained after evaporation of the volatile fraction. The scope of
this reaction was successfully widened in aliphatic and aromat-
ic anhydride [15-21]. The introduction of the HMBP moiety in
presence of the PEG tether seems to be critical due to its high
sensitivity under harsh conditions. Wherefore, our methodolo-
gy, which exhibits a high tolerance to various functionalized
groups, appears to be an adequate way to introduce the HMBP
chain in presence of the PEG moiety.
Figure 1: Bifunctional PEG-HMBPs 1.
To obtain the PEG-HMBP 1 compound family, the synthetic
strategy consists in mono-protecting and/or mono-functional-
izing commercially available PEGs followed by the lab-made
Results and Discussion
For this family of compounds, the HMBP moiety has to be built HMBP methodology introduced previously (Scheme 2).
starting from a modified PEG chain. The HMBP introduction
could be achieved by several reported methodologies starting Starting materials, the free alcohol PEG and monomethyl ether
from an acid chloride (Scheme 1).
PEGs (compounds 3a,b) with various chain lengths (n = 4, 7
and 12) were commercially available. Firstly, the free alcohol
PEG was selectively monoprotected with a benzyl group
(Scheme 3).
Only one alcohol function was indeed deprotonated with one
equivalent of sodium hydride at −78 °C in THF after the solu-
tion was stirred for 12 hours at room temperature. The alcohol-
ate intermediate reacted smoothly with benzyl bromide at room
temperature to afford the monobenzylated PEG 2 in 77% yield
(Table 1, entry 1). Afterwards, the alcohols 2 and 3a,b have to
be oxidized to the corresponding carboxylic acids 6 and 7a,b.
First of all, the direct oxidation reported in the literature in one
step has been performed. Thus, tested oxidants were the Jones
Scheme 1: Direct methods for the 1-hydroxyalkylidenebisphosphonic
acid synthesis.
Scheme 2: Synthetic strategy of PEG-HMBPs 1.
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Beilstein J. Org. Chem. 2016, 12, 1366–1371.
Scheme 3: Synthesis of PEG-HMBPs 1 and 1’.
Table 1: Synthesis of PEG-HMBPs 1,1’and 10.
smoothly oxidized in the presence of a catalytic amount of PCC
and a the co-oxidative agent H5IO6 in acetonitrile at 0 °C. The
PEG 6 and 7a,b were obtained in good yields (Table 1, entries
5–7).
Entry Compound
R
n
Yield (%) 31P δ (ppm)
1
2
Bn
Bn
Me
Me
Bn
Me
Me
Bn
Me
Me
Bn
Me
Me
H
4
77
–
2
4
4
89
–
Finally, the optimized two-step procedure enabled us to isolate
the expected carboxylic acids 6 and 7a,b which are key inter-
mediates in the synthesis of PEG-HMBPs.
3
5a
5b
6
7
79
–
4
12
4
80
–
5
72
–
6
7a
7b
8
7
82
–
7
12
4
72
–
The carboxylic acids 6 and 7a,b reacted quantitatively with
oxalyl chloride to give acyl chlorides 8 and 9a,b at room tem-
perature after 24 hours (Table 1, entries 8–10). The completion
of the reaction was monitored by infrared spectroscopy with the
disappearance of the hydroxy absorption band and the shifting
of the carbonyl vibration band to about 1800 cm−1. The addi-
tion of two equivalents of tris(trimethylsilyl) phosphite to the
acyl chloride derivatives 8 and 9a,b yielded the corresponding
silylated PEG-HMBP.
8
quant.
quant.
quant.
78
–
9
9a
9b
1
7
–
10
11
12
13
14
12
4
–
16.8
17.2
17.2
16.2
1’a
1’b
10
7
47
12
4
43
72
aIsolated yield. bproton decoupling 31P NMR experiment.
The formation of silylated bisphosphonate was monitored by
31P NMR. After evaporation of volatile compounds under
vacuum the silylated PEG-HMBP was hydrolyzed with methan-
ol at room temperature for 24 hours. After methanol evapora-
tion, the crude PEG-HMBP containing phosphorous acid was
purified by successive washes with dry diethyl ether. The pure
targeted PEG-HMBPs 1 and 1’a,b were then obtained in moder-
ate yields (Table 1, entries 11–13). The treatment of 1 with
dihydrogen and palladium on charcoal in water allowed
cleavage of the benzyl moiety and led to the HO-PEG-HMBP
10 in 72% yield (Table 1, entry 14). The ligand 10 permitted to
obtain new gadolinium phosphate nanocrystals with lumines-
cent properties [26].
reagent [22], potassium permanganate [23], with catalytic
o-iodoxybenzoic acid (IBX) in oxone [24] and catalytic 2,2,6,6-
tetramethyl-1-piperidinyloxy (TEMPO) with bis(acetoxy)iodo-
benzene (BAIB) [25]. The first two conditions led to a PEG
chain cleavage and the recovery of benzoic acid from alcohol 2.
Besides, the mixture IBX/oxone gave the expected product
inseparable of IBX byproducts. Only oxidation using TEMPO
and BAIB furnished the pure corresponding carboxylic acid.
Nevertheless, the low obtained yields encouraged us to test a
strategy in two steps via an aldehyde. Fortunately, the following
two-step procedure was more effective. The alcohol derivatives
2 and 3a,b were treated with dimethyl sulfoxide, oxalyl chlo-
ride and triethylamine in dichloromethane at −55 °C. Under
these classical Swern conditions, the corresponding products 4
and 5a,b were isolated in excellent yields from 79% to 89%
(Table 1, entries 2–4). The aldehydes 4 and 5a,b were next
Therefore, we considered the syntheses of other compounds
which possess azido or amino functional groups. The azido
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products will allow us to perform click chemistry to introduce carboxylic acids 14a,b were isolated in quantitative yields. The
various functionalities. Moreover, the amino derivatives will be HMBP moiety was subsequently incorporated by the lab-made
easily obtained by reducing the azido group. As previously methodology previously described. The carboxylic acids 14a,b
mentioned, the first step was a selective mono-activation of were converted into the expected HMBP-PEG-N3 16a,b via the
PEG using para-toluenesulfonyl chloride in the presence of so- corresponding acyl chloride 15a,b. The reactions were moni-
dium hydroxide in a water/THF mixture (Scheme 4).
tored by 31P NMR, compounds 16a and 16b were obtained after
12 and 18 hours, respectively.
The tosylated products 11a,b were obtained after three hours at
0 °C with a 95% yields. Next, the monoactivated compounds Thus, HMBP-PEG-N3 16a,b were obtained after purification in
11a,b were substituted by sodium azide in DMF at 60 °C within 72% and 74% yield and characterized by a singlet in 31P [27]
five hours. The azido compounds 12a,b were obtained in 83% NMR at about 17 ppm. Finally, the reduction of the azido com-
and 81% yield, respectively. The alcohols 12a,b were subse- pounds 16a,b in the presence of palladium on charcoal and
quently firstly deprotonated with NaH in DMF and the gener- dihydrogen led to the targeted amino-PEG-HMBPs 17a and
ated alcoholates were stirred 16 hours with ethyl bromoacetate 17b, respectively in moderate 62% and 68% yields.
giving the expected esters 13a,b in moderate yields.
In order to access available PEG-HMBPs functionalized with a
The saponification reactions of the esters 13a,b were carried out primary amine or a carboxylic acid group usable in peptidic
with sodium hydroxide in methanol. The completion of the coupling with various molecules for example, the HMBP-PEG-
reactions was controlled by TLC. After neutralization with a COOH 23 was synthesized (Scheme 5). This compound was ob-
cationic exchange Dowex® 50WX2 resin, the corresponding tained in six steps starting from a free alcohol four-unit PEG. It
Scheme 4: Syntheses of HMBP-PEG-N3 16 and HMBP-PEG-NH3+ 17.
Scheme 5: Synthesis of HMBP-PEG-COOH 23.
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Acknowledgements
Université Paris 13, Sorbonne Paris Cité and Centre de la
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