Dendrimers ended by non-symmetrical azadiphosphonate groups: Synthesis and immunological properties

Article abstract of DOI:10.1016/j.bmcl.2009.03.003

The synthesis and characterization of new series of phosphorus-containing dendrimers ended by non-symmetrical azamonophosphonates, or azadiphosphonates, or azadiphosphonic acid salts are reported. The sodium salts of the non-symmetrical azadiphosphonic de

Full text of DOI:10.1016/j.bmcl.2009.03.003

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3963–3966
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Dendrimers ended by non-symmetrical azadiphosphonate groups: Synthesis
and immunological properties
Patrice Marchand a,b, Laurent Griffe a,b, Mary Poupot c, Cédric-Olivier Turrin a,b, Gérard Bacquet d,
c,
a,b,
*
Jean-Jacques Fournié c, Jean-Pierre Majoral a,b, , Rémy Poupot , Anne-Marie Caminade
*
*
a CNRS, LCC (Laboratoire de Chimie de Coordination), 205 route de Narbonne, F-31077 Toulouse, France
b Université de Toulouse, UPS, INPT, LCC, F-31077 Toulouse, France
c INSERM, U.563, Centre de Physiopathologie de Toulouse-Purpan, Toulouse, F-31300 France and Université Paul-Sabatier, Toulouse, F-31400, France
d Rhodia, Centre de Recherches d’Aubervilliers, 52 rue de la Haie Coq, 93308 Aubervilliers Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and characterization of new series of phosphorus-containing dendrimers ended by non-
symmetrical azamonophosphonates, or azadiphosphonates, or azadiphosphonic acid salts are reported.
The sodium salts of the non-symmetrical azadiphosphonic dendrimers are soluble in water. Their influ-
ence towards human immune blood cells is assayed ex vivo.
Received 11 February 2009
Revised 27 February 2009
Accepted 3 March 2009
Available online 6 March 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Phosphonates
Synthesis
Immunology
PBMC
Activation of monocytes
Peripheral blood immune cells are easily accessible and wide-
spread in the whole body, thus they are a target of choice for trying
to increase their fighting efficiency against bacterial, viral or para-
sitic infections, and even against cancers. It is known that the surface
of cells is composed of complex and dynamic micro-domains, and
that the activation of cells often occurs with an increased efficiency
if multivalent interactions can be induced.1 For this purpose, multi-
valent and perfectly defined macromolecules such as dendrimers
have received a tremendous attention for testing their biological
properties since one decade.2 Very few of these tests concern the
influence of dendrimers towards immune blood cells, but we have
already demonstrated that some phosphorus-containing dendri-
mers capped with azadiphosphonic acids are able to promote hu-
man monocytes activation,3 to increase the efficiency of the IL2-
dependent proliferation of human Natural Killer (NK) cells,4 and
possess anti-inflammatory properties for the human myeloid cell
lineage.5 In this Letter, we report the synthesis of new series of phos-
phorus-containing dendrimers capped with non-symmetrical
azadiphosphonic acids, and their ability to activate human mono-
cytes isolated from the peripheral blood of healthy individuals.
The azadiphosphonate derivatives can be linked to the dendri-
mer through the central nitrogen affording symmetrical terminal
Figure 1. Types of grafting of azadiphosphonate groups to the surface of dendri-
mers. Left: symmetrical grafting through nitrogen; right: non-symmetrical grafting
through carbon.
groups, or through one carbon affording non-symmetrical terminal
groups. This latter anchorage produces racemic compounds, but of-
fers the possibility to vary easily the nature of the R substituent,
thus allowing a structure/activity relationship study (Fig. 1).
We have already reported the synthesis of the azadiphospho-
nate dendrimer where R = methyl;3a we report here the synthesis
of new dendrimers where R has an increasing number of bonds
(R = allyl (a), butyl (b), decyl (c)), to determine the influence of
an increased hydrophobicity on the biological properties.
One of the simplest way to graft functional groups to the surface
of the phosphorus-containing dendrimers consists in reacting phe-
nols with P(S)Cl2 terminal groups. Starting from 4-hydroxybenzal-
dehyde, azadiphosphonate groups are obtained in 3 steps. The first
step is a condensation reaction of the aldehyde with primary amines
bearing the R substituent, to afford compounds 1a–c. They are gen-
erally not isolated but used directly to react with dimethylphosphite
* Corresponding authors. Fax: +33 5 61 55 30 03.
E-mail addresses: jean-pierre.majoral@lcc-toulouse.fr (J.-P. Majoral), remy.pou
pot@inserm.fr (R. Poupot), anne-marie.caminade@lcc-toulouse.fr (A.-M. Caminade).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.003

3964
P. Marchand et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3963–3966
sponding to P(S)Cl2. When the reaction has gone to completion,
both signals have totally disappeared, on behalf of a singlet at
65 ppm. Obviously, the singlets corresponding to the core
(11.4 ppm) and to both types of phosphonates (28 and 31 ppm)
are also observed. Such reaction can be also applied to larger gen-
erations, as shown in particular by the synthesis of the second gen-
eration 5b-[G2].
A potentially alternative to this method for synthesizing com-
pounds 5-[G1] consists in carrying on a dendrimer the same reac-
tions than those carried out on 4-hydroxybenzaldehyde. In this
case, dendrimers ended by benzaldehyde are needed, such as com-
pound 6-[G1].7 The condensation with allylamine affords dendri-
mer 7a-[G1], characterized in particular by the disappearance of
the signal corresponding to the aldehydes in 1H and 13C NMR
and IR spectra. Addition of dimethylphosphite on the imines af-
fords dendrimer 8a-[G1] ended by monophosphonate derivatives.
The completion of the reaction is shown in particular by the disap-
pearance of the signal corresponding to the N@C(sp2)H functions
on behalf of N-C(sp3)H functions by 1H NMR, from 8.17 to
4.06 ppm. Furthermore, this signal is a doublet (coupling with
phosphonate (2JHP = 19.8 Hz)). The last step to obtain dendrimer
5a-[G1] from 8a-[G1] should consist in Kabachnik–Field reactions
with formaldehyde in water and dimethylphosphite. However, this
reaction affords numerous compounds (multiple signals in 31P
NMR), thus no attempt was made to isolate the expected com-
pound, since we have a straightforward way to access to the same
compound (Scheme 2).
The last step for obtaining the water-soluble dendrimers con-
sists in the deprotection of the phosphonates, to generate the phos-
phonic acids, then their sodium salts, in a multi-step one-pot
procedure. Addition of bromotrimethylsilane changes the O–Me
groups to O–SiMe3, which are highly sensitive to hydrolysis to af-
ford O–H groups. Addition of 1 equiv of NaOH per P(O)(OH)2
groups affords the P(O)(OH)(ONa) derivatives 9a-[G1]9 and 9b-
[G1] which are water-soluble (Scheme 2). This transformation is
in particular characterized by 31P NMR, with a shielding of the
Scheme 1. Synthesis of phenol functionalized by azamonophosphonates (2a–c)
and azadiphosphonates (3a–c).
in the second step, to afford the monophosphonate derivatives 2a–c,
isolated in 65–92% yield. The third step is a Kabachnik–Fields proce-
dure: formaldehyde in water is first added to compounds 2a–c, then
dimethylphosphite. Non-symmetrical azadiphosphonates 3a–c are
isolated in moderate yields (28–60%) (Scheme 1). A typical proce-
dure of synthesis is given for compound 3a.6 These compounds are
characterized in particular by 31P NMR which displays two different
signals at about 29 and 31 ppm, emphasizing the presence of two
non-equivalent phosphonate groups.
The next step consists in grafting these multifunctional phenols
to the surface of dendrimers ended by P(S)Cl2 groups, in particular
to the first generation dendrimer7 4-[G1], with Cs2CO3 as base. The
reaction affords dendrimers 5a-[G1], 5b-[G1], or 5c-[G1] in 76%,
65%, or 85% yield after work-up, respectively8 (Scheme 2).
The reaction is monitored by 31P NMR, which displays first the
appearance of an intermediate singlet at about 70 ppm, corre-
sponding to the substitution of one Cl on the P(S)Cl2 terminal
groups, together with the decrease of the signal at 65.5 ppm corre-
Scheme 2. Synthesis of dendrimers ended by azamonophosphonates (8a-[G1]), or non-symmetrical azadiphosphonates (5a-[G1], 5a-[G2], 5b-[G1], 5c-[G1]), or the sodium salt
of non-symmetrical azadiphosphonic acids (9a-[G1], 9b-[G1]). Schematization of dendrimers 9-[G1]. Compound 9d-[G1] was previously reported.3a

P. Marchand et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3963–3966
3965
Figure 2. (A) Morphological changes induced by dendrimer on monocytes enable their selection in culture of PBMC (gating). (B) Down-regulation of the expression of CD14
on monocytes activated by dendrimer (dotted line: isotype-matched control, continuous line: staining with anti-CD14 monoclonal antibody, mAb). (C) Comparison of the bio-
activity of three dendrimers (at 2
Control corresponds to untreated monocytes. Results shown were obtained from one healthy donor and are representative of the three donors tested. (D) Dose-dependent
bio-activity of dendrimer 9a-[G1] on human monocytes (mfi-R of HLA-DR and CD14 with 0, 0.02, 0.2, 2 and 20 M from dark to white bars of the histograms).
lM, gray bars, and 20 lM, white bars) on monocytes measured by the mfi-ratio (mfi-R) of two membrane markers: HLA-DR and CD14.
l
signals of the P(O) groups from about 28 ppm (P(O)(OMe)2) to
about 11–13 ppm. Both compounds were engaged in biological
tests regarding the activation of human monocytes present in
peripheral blood mononuclear cells (PBMC). Upon activation by
phosphorylated dendrimers, human monocytes undergo both
morphological and phenotypic changes.3 They become bigger and
more granular (Fig. 2A). Expression of membrane markers such
as CD14 (Fig. 2B) and HLA-DR are down regulated. We compare
the bio-activity of dendrimers 9a-[G1] and 9b-[G1] to the bio-activ-
ity of a non-symmetrical azadiphosphonate dendrimer in which
R = Methyl (9d-[G1]).3a We have already reported that the bio-
activity of this N-methylated dendrimer is very close to the
bioactivity of symmetrical azadiphosphonate capped dendri-
mers.3a The three dendrimers were added in cultures of PBMC at
it can be said that the ‘dose-dependent’ bio-activity is the same
for both proteins. As dendrimer 9b-[G1] led to cell death in
long-lasting cultures, we only test dendrimer 9a-[G1] to assess its
dose-dependent effect between 0.02 and 20 lM (Fig. 2D).
In conclusion, we have shown that non-symmetrical azadi-
phosponate-capped dendrimers with various substituents on the
nitrogen atom (methyl, allyl or butyl) are able to activate human
monocytes in short term in vitro cultures, even at 0.02 lM. Never-
theless, the dendrimer 9b-[G1] with the longest hydrophobic por-
tion of the series displays a cytotoxic effect in long term cultures.
Ongoing studies will assess the particular interaction between this
molecule and cell membrane
Acknowledgments
2 and 20 lM, and after 4 days the down-regulation of CD14 and
HLA-DR was analyzed. This down-regulation is quantified by the
mfi-R10 a low mfi-R indicates a strong activation of monocytes,
and vice versa (Fig. 2C). HLA-DR and CD14 are two independent
proteins which differ in their respective levels of expression. Thus,
Rhodia, the CNRS, INSERM, the University Paul-Sabatier, the
Institut National du Cancer (contract # PL 06-87) and Region
Midi-Pyrenees (Biotherapy program) are acknowledged for finan-
cial support.

3966
P. Marchand et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3963–3966
7. (a) Launay, N.; Caminade, A. M.; Lahana, R.; Majoral, J. P. Angew. Chem., Int. Ed.
Supplementary data
Engl. 1994, 33, 1589; (b) Launay, N.; Caminade, A. M.; Majoral, J. P. J. Organomet.
Chem. 1997, 529, 51.
Supplementary data (Syntheses and characterization of all com-
pounds) associated with this article can be found, in the online ver-
sion, at doi:10.1016/j.bmcl.2009.03.003.
8. Typical synthesis of 5a-[G1]: Compound 3a (600 mg) and Cs2CO3 (596 mg) were
added to a solution of 4-[G1] (223 mg) in THF (3 mL). This suspension was
stirred up to the full substitution of Cl. After decantation, the solution was
recovered, then the solid was washed with THF and this solution was added to
the previous one, then concentrated under reduced pressure then precipitated
with pentane. The resulting solid was washed with THF/pentane then with
THF/ether, to afford 5a-[G1] in 76% yield as a white solid.
References and notes
9. Typical synthesis of 9a-[G1]: Bromo-trimethylsilane (260 lL) was slowly added
1. Kiessling, L. L.; Gestwicki, J. E.; Strong, L. E. Curr. Opin. Chem. Biol. 2000, 4, 696.
2. For reviews about biological uses of dendrimers, see: (a) Cloninger, M. J. Curr.
Opin. Chem. Biol. 2002, 6, 742; (b) Boas, U.; Heegaard, P. M. H. Chem. Soc. Rev.
2004, 33, 43; (c) Kofoed, J.; Reymond, J. L. Curr. Opin. Chem. Biol. 2005, 9, 656;
(d) Lee, C. C.; MacKay, J. A.; Fréchet, J. M. J.; Szoka, F. C. Nat. Biotechnol. 2005, 23,
1517; (e) Svenson, S.; Tomalia, D. A. Adv. Drug Delivery Rev. 2005, 57, 2106; (f)
Wolinsky, J. B.; Grinstaff, M. W. Adv. Drug. Delivery Rev. 2008, 60, 1037; (g)
Caminade, A. M.; Turrin, C. O.; Majoral, J. P. Chem. Eur. J. 2008, 14, 7422.
3. (a) Poupot, M.; Griffe, L.; Marchand, P.; Maraval, A.; Rolland, O.; Martinet, L.;
L’Faqihi-Olive, F. E.; Turrin, C. O.; Caminade, A. M.; Fournié, J. J.; Majoral, J. P.;
Poupot, R. FASEB J. 2006, 2339; (b) Rolland, O.; Griffe, L.; Poupot, M.; Maraval,
A.; Ouali, A.; Coppel, Y.; Fournié, J. J.; Bacquet, G.; Turrin, C. O.; Caminade, A. M.;
Majoral, J. P.; Poupot, R. Chem. Eur. J. 2008, 14, 4836.
4. Griffe, L.; Poupot, M.; Marchand, P.; Maraval, A.; Turrin, C. O.; Rolland, O.;
Métivier, P.; Bacquet, G.; Fournié, J. J.; Caminade, A. M.; Poupot, R.; Majoral, J. P.
Angew. Chem., Int. Ed. 2007, 46, 2523.
5. Fruchon, S.; Poupot, M.; Martinet, L.; Turrin, C. O.; Majoral, J. P.; Fournié, J. J.;
Caminade, A. M.; Poupot, R. J. Leukocyte Biol. 2009, 85, 553.
6. Typical sequence of reactions for the synthesis of 3a: MgSO4 (15 g) and allylamine
(7.5 mL) were added to a solution of 4-hydroxy-benzaldehyde (11 g) in CH2Cl2
(25 mL) at 0 °C, to afford 1a after one night at rt in situ addition of (MeO)2P(O)H
(9 mL) gave a solution stirred for 3 days, then poured into water (100 mL), and
extracted by CH2Cl2 (3 Â 100 mL). The organic phases were dried (MgSO4) and
evaporated; the residue was washed with ether to afford 2a as very viscous
pale yellow oil in 90% yield. CH2O at 37% in water (1.06 mL) was added to a
to a solution of 5a-[G1] (200 mg) in CH3CN at 0 °C. The solution was stirred for
18 h at rt then evaporated to dryness. MeOH (5 mL) was added, and the
mixture was vigorously stirred for 2 h. After evaporation to dryness, the
residue was washed with ether, then with water and MeOH. The residue was
dried under reduced pressure. A solution of NaOH in water (0.1966 M, 3.99 mL)
was slowly added to the solid, to afford a solution which was filtered then
lyophilized. Compound 9a-[G1] was isolated as a white powder in 97% yield.
10. Blood samples, cells and cell cultures. PBMC purification and culture: Fresh blood
samples were collected from healthy adult donors, PBMC were prepared on a
Ficoll-Paque density gradient (Amersham Biosciences AB, Upsalla, Sweden) by
centrifugation (800 g, 30 min at room temperature). Collected PBMC were
washed twice and finally diluted at 1.5 million cells per mL in complete RPMI
1640 medium, that is, supplemented with penicillin and streptomycin, both at
100 U per mL (Cambrex Bio Science, Verviers, Belgium), 1 mM sodium
pyruvate and 10% heat-inactivated fetal calf serum (both from Invitrogen
Corporation, Paisley, UK). PBMC (4.5 millions) were cultured in 3 mL of
complete RPMI 1640 medium in 6-well plates. Sterile filtered solutions of the
specified dendrimers were added to cultures at the desired final concentration.
Flow cytometry and microscopy: Flow cytometry was performed on a LSR-II
cytometer (BD Biosciences, San Jose, CA, USA). All cell stainings were done
using fluorochrome-conjugated mAb from BD Biosciences, San Jose, CA, USA:
clone Tü36 for anti-HLA-DR (coupled to FITC fluorochrome) and clone M5E2 for
anti-CD14 (coupled to PE-Pc5 fluorochrome). Monocytes were selected upon
morphological criteria. To compare the surface densities of various molecules
at the surface of monocytes, we calculated the mean fluorescence intensity
ratio (mfi-R), that is, the ratio between the mfi of cells stained with the selected
mAb and that of cells stained with the isotype-matched control (negative
control).3
solution of 2a (970 mg) in THF (5 mL). (MeO)2P(O)H (492 lL) was added after
30 min. The solution was stirred for 72 h then poured into water (30 mL) and
extracted with CH2Cl2. After drying (MgSO4) and evaporating the organic
phases, the residue was washed with ether to afford viscous oil purified by
column chromatography (silica gel; eluent: AcOEt/MeOH, 95/5). Compound 3a
was isolated as a white powder in 28% yield.