Synthesis of Patent Blue derivatized hydrophilic dendrons dedicated to sentinel node detection in breast cancer

Article abstract of DOI:10.1016/j.tetlet.2011.03.144

In the last decade, methods for the precise localization of sentinel lymph node (SLN) have drawn tremendous attention by oncology surgeons and researchers in the field of medical diagnosis. The accurate identification and characterization of lymph nodes by imaging has important therapeutic and prognostic significance in patients with newly diagnosed cancers. The SLN is the first lymph node that receives lymphatic drainage from the site of a primary tumor. Two biocompatible dendronized phosphonates, one bearing a Patent Blue (PB VF) dye at its periphery, where synthesized. Indeed, such a blue dye is currently injected to label the lymph node system for its per-operative detection. Therefore, developing chemistry of Patent Blue VF to optimize early diagnosis is of great current interest.

Full text of DOI:10.1016/j.tetlet.2011.03.144

Tetrahedron Letters 52 (2011) 2906–2909
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis of Patent Blue derivatized hydrophilic dendrons dedicated
to sentinel node detection in breast cancer
⇑
Marie Kueny-Stotz, Hind Mamlouk-Chaouachi, Delphine Felder-Flesch
IPCMS, UMR CNRS-Université de Strasbourg 7504, 23 rue du loess, BP 43, 67034 Strasbourg cedex 2, France
a r t i c l e i n f o
a b s t r a c t
Article history:
In the last decade, methods for the precise localization of sentinel lymph node (SLN) have drawn tremen-
dous attention by oncology surgeons and researchers in the field of medical diagnosis. The accurate iden-
tification and characterization of lymph nodes by imaging has important therapeutic and prognostic
significance in patients with newly diagnosed cancers. The SLN is the first lymph node that receives lym-
phatic drainage from the site of a primary tumor. Two biocompatible dendronized phosphonates, one
bearing a Patent Blue (PB VF) dye at its periphery, where synthesized. Indeed, such a blue dye is currently
injected to label the lymph node system for its per-operative detection. Therefore, developing chemistry
of Patent Blue VF to optimize early diagnosis is of great current interest.
Received 7 February 2011
Revised 28 March 2011
Accepted 30 March 2011
Available online 5 April 2011
Keywords:
Sulfonamide
Phosphonate
Dendronized nanoparticle
Breast cancer
Patent Blue
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
clear detection are also investigated, and in this context, we
decided to develop a new and original project based on dendritic
In the field of cancer research, the term sentinel lymph node
(SLN) refers to any node receiving lymph drainage from the tumor
site and containing most likely malignancy if the tumor has metas-
tasized. The treatment of breast cancer through SLN detection is
currently a great challenge.1 Indeed, an early detection of this node
can prevent patients with a low risk of lymph node involvement
from suffering an unnecessary axillary lymphadenectomy.2 Dyes
have been exploited in medicine for more than one century to ex-
plore the lymphatic system. Several dyes more or less toxic were
used, such as Gerota mass (Prussian blue and iron ferrocyanide
in oil), cresyl blue (amino-dimethylamino ethyldiphenazonium
chloride; C17H20N3OCl) or indigo carmine (indigo tin disulfonate
sodium) in pre-mortem, then Patent Blue V or Evans blue during
a surgery.3 These latter ones have been then replaced by fluores-
cent substances, Indian ink or activated carbon.4 The next step
has been to use radioactive gold colloids (Au 198) in diagnosis as
well as in therapy of breast cancer, then replaced by colloids la-
beled with 99mTc.5 Most medical teams using sentinel node biopsy
in the treatment of breast cancer inject either a radioactive colloid
magnetic iron oxide nanoparticles covalently connected to a blue
dye (PB VF) for magneto-optical detection. Indeed, in the field of
biology and medicine, dendrimers and their elementary unit called
dendron, are showing great promise as they combine unique
advantages such as monodispersity although they can be polyfunc-
tional, easy control of their physico-chemical characteristics as a
function of their generation, tuneable amphiphilicity (to cross bio-
logical barriers).8 In our team, which has been interested for some
years in surface engineering of super paramagnetic iron oxide
(SPIO) NPs dedicated to MRI contrast enhancement,9 new attempts
to develop original dendronized NPs have recently been investi-
gated in the field of cancer diagnosis.
2. Results and discussion
Here, we present the synthetic route to two new dendronized
phosphonates: 1 which is bearing a primary alcohol function at
its periphery, and 2, bearing a Patent Blue dye dedicated to SLN
detection (Scheme 1). Key-functionalities have to appear in the
dendritic structure: (i) a coupling agent at the focal point to ensure
a strong and stable connection to the surface of SPIO NPs; (ii) oli-
goethyleneglycol chains to reach hydrophilicity and confer bio-
compatibility to the final nano-object; (iii) in the case of 2, a blue
dye for optical detection.
(
99mTc-labeled RuS)6 and/or a blue dye7 to label the lymph node
system for its per-operative detection. Such procedure, involving
then two injections, is based on a gamma probe and/or visual color
detections, respectively. If radioisotope injection is widely used to-
day in the most advanced countries, new strategies using non-nu-
Dealing with the coupling agent, phosphonates are candidate of
choice as former studies pointed out a stronger binding and a
higher grafting rate than with carboxylate anchors.10 Moreover,
⇑
Corresponding author. Tel.: +33 3 88 107163; fax: +33 3 88 107246.
E-mail address: Delphine.Felder@ipcms.u-strasbg.fr (D. Felder-Flesch).
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.03.144

M. Kueny-Stotz et al. / Tetrahedron Letters 52 (2011) 2906–2909
2907
phosphonates are known to stabilize suspensions in water at phys-
iological pH and to preserve the magnetic properties.9,10
Compounds 1 and 2 can be obtained from key building block 3
which was synthesized from methyl gallate 4 in seven steps
(Scheme 2): para-allylated methyl gallate 7 and tosylated tetraeth-
yleneglycol monomethyl ether 6 can easily be produced in good
yields from commercially available compounds 4 and 5, respec-
tively, following described one-step-procedures.11,12 Then, a Wil-
liamson reaction in acetone at 60 °C in the presence of K2CO3 and
KI (catalytic) allows obtaining ester 8 in 70% yield. From here,
the allylated ethyl phosphonate 9c can be obtained thanks to a
three-step sequence: (i) reduction of the ester function using
LiAlH4 as reducing agent (obtention of the benzyl alcohol 9a,
97%), (ii) the treatment with PBr3 (obtention of the benzyl bromide
9b, 99%) and (iii) 2-hour refluxing of 9b at 160 °C in P(OEt)3 (obten-
tion of the allylated ethyl phosphonate 9c, 85%). Finally, deallyla-
tion by the treatment with Pd(PPh3)4/NaBH4 leads to key
phosphonate 3 in 90% yield. In order to prepare 1, a monotosyla-
tion of commercially available octaethyleneglycol 10 was first
achieved to obtain 11 in 59% yield.13 Then, 11 was subjected to
etherification with 3 to yield ethyl phosphonate 12 (75%) which
was finally converted into its corresponding phosphonic acid 1 in
a good 93% yield by a silylation/methanolyse step using an excess
of bromotrimethylsilane.14
Concerning the design of a blue dye-derivatized dendritic phos-
phonate, our first idea was to use Patent Blue V 13 (Scheme 3),
which is one of the most used Vital Blue dye in sentinel node
detection.15 Retrosynthetically, the considered strategy was the
elaboration of a Patent Blue V-derivatized dendron 14 from ether-
ification between 3 and 15. Unfortunately, our attempts to obtain
such an oligoethyleneglycol chain linked to the Patent Blue V dye
failed (Table 1): neither a Williamson reaction between 13 and
16a (entry 1), nor a Mitsonobu reaction between 13 and 16b (entry
2) succeeded and compound 17 was never isolated. In the first at-
tempt (entry 1) conversion was very low and a hard-to-purify mix-
ture of dye-derivatives was obtained, whereas, in the second
investigation (entry 2) no conversion was observed, even after sev-
eral days of reaction.
Scheme 1. Retrosyntheses to dendritic phosphonates 1 and 2.
Scheme 2. Synthesis of key-synthon 3 and final steps to dendron 1. Reagents and conditions: (a) pTsCl (1.1 equiv), NaOH (1.7 equiv), THF/H2O, 0 °C?rt, one night, 96%; (b)
allylbromide (1.0 equiv), KHCO3 (4.0 equiv), KI (0.005 equiv), DMF, 30 °C, 5 days, 85%; (c) 2 (1.0 equiv), 1 (2.2 equiv), K2CO3 (5.0 equiv), KI (0.3 equiv), acetone, 60 °C, 24 h,
70%; (d) LiAlH4 (0.5 equiv), THF, 0 °C?rt, 1 h, 97%; (e) PBr3 (1.8 equiv), CH2Cl2, 0 °C?rt, 2 h, 99%; (f) P(OEt)3, 160 °C, 2 h, 85%; (g) Pd(PPh3)4 (0.02 equiv), NaBH4 (2.5 equiv),
THF/DMF, rt, 2 h, 90%; (h) pTsCl (1.2 equiv), Ag2O (1.8 equiv), CH2Cl2, 0 °C, 15 min, 59%; (i) 3 (1.0 equiv), 11 (1.0 equiv), K2CO3 (2.5 equiv), KI (0.3 equiv), acetone, 60 °C, 24 h,
75%; (j) TMSBr (15.0 equiv), CH2Cl2, 0 °C?rt, one night, 93%.

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M. Kueny-Stotz et al. / Tetrahedron Letters 52 (2011) 2906–2909
Table 1
These results, together with the fact that we were not able to
Attempts for synthesizing a Patent Blue V-derivatized oligoethyleneglycol chain 17
find any described reaction involving 13 as a reactant or reagent,
lead us to the conclusion that the phenol function of 13 is not reac-
tive enough, certainly due to mesomeric stabilization effect of the
sulfonate functional groups in ortho and para positions. Conse-
quently, we decided to look for a new alternative and work with
Patent Blue VF 18 (Scheme 4), which has a structure similar to
the Patent Blue V, but without a phenol function on its aromatic
system. Indeed, there is the possibility to regioselectively activate
the less hindered sulfonate function of 18 into its corresponding
sulfonyl chloride, which can then react with an amine to form a
sulfonamide link.16 It should be noted that the activation of Patent
Blue V 13 into one of its sulfonyl chloride was also tested but it
seemed that in this case the activation was not regioselective at
all and that the presence of the unprotected phenol led to second-
ary reactions. In order to reach Patent Blue VF-dye derivatized
phosphonic acid 2, amine-terminated dendron 22 was first pre-
pared from key-synthon 3 and commercially available amino tetra-
ethyleneglycol 19 in 4 steps: a Boc protection of the amine function
followed by a tosylation of the alcohol function led to 20 in 80%
overall yield. Etherification between 20 and 3 allowed to obtain
21 (95% yield) which was treated with an excess of trifluoroacetic
acid to yield 22 (97%). Sulfonyl chloride 23 was prepared from Pat-
ent Blue VF 18 following Tahtaoui et al.’s procedure.16 Sulfonamide
Entry
Electrophile
Conditions
Yield (%)
a
1
2
16a R = Tos
16b R = H
K2CO3, KI, DMF, 60 °C
DEAD, PPh3, THF, rt
—
—
b
a
Hard-to-purify mixture of dye-derivatized compounds in very low yield.
No conversion observed, even after long reaction times (17 days).
b
linkage was then achieved between 23 (used without further puri-
fication) and 22 in the presence of triethylamine and 4-DMAP and
allowed obtaining dye derivatized ethyl phosphonate 24 in a mod-
erate 54% yield. Finally 24 was converted into its corresponding
phosphonic acid 2 in 95% yield by the treatment with an excess
of bromotrimethylsilane.17
The structures and purity of all compounds were confirmed by
1H and 13C NMR and mass spectrometry (see Supplementary data).
3. Conclusion
In conclusion, we have prepared two new low-generation
hydrophilic and biocompatible dendritic phosphonates 1 and 2,
either derivatized with a primary alcohol allowing further grafting
of biological effectors (for efficient targeted imaging and therapy),
or with a Vital Blue dye ensuring optical detection. The functional-
ization of 10 nm iron oxide nanoparticles with these original coat-
ings is currently under investigation in our laboratory in order to
develop highly efficient and stable (in vivo) magneto-optical
probes (Fig. 1).
Scheme 3. First considered strategy to a Patent blue V-derivatized dendron.
Figure 1. Patent Blue (PB) derivatized dendronized SPIO NPs.

M. Kueny-Stotz et al. / Tetrahedron Letters 52 (2011) 2906–2909
2909
Scheme 4. Final steps to dye-derivatized dendron 2. Reagents and conditions: (k) Boc2O (1.0 equiv), CH2Cl2, 0 °C?rt, one night, 86%; (l) pTsCl (1.1 equiv), NEt3 (2.3 equiv),
CH2Cl2, 0 °C?rt, 40 h, 93%; (m) 3 (1.0 equiv), 20 (1.0 equiv), K2CO3 (4.0 equiv), KI (0.3 equiv), acetone, 60 °C, 48 h, 95%; (n) TFA (15.0 equiv), CH2Cl2, 0 °C?rt, one night, 97%;
(o) POCl3 (20.0 equiv), rt, 72 h; (p) 22 (1.0 equiv), 23 (1.0 equiv), NEt3 (3.0 equiv), 4-DMAP (0.1 equiv), CH2Cl2/DMF 4:1, 0 °C?rt, one night, 54% (steps n + o); (q) TMSBr
(15.0 equiv), CH2Cl2, 0 °C?rt, one night, 95%.
S. Chem. Mater. 2008, 20, 5869–5875; (d) Boyer, C.; Bulmus, V.; Priyanto, P.;
Teoh, W. Y.; Amal, R.; Davis, T. P. J. Mater. Chem. 2009, 19, 111–123.
Acknowledgments
11. Freeman, A. W.; Chrisstoffels, L. A. J.; Fréchet, J. M. J. J. Org. Chem. 2000, 65,
We would like to thank Emilie Voirin and Emilie Couzigné for
their assistance. We also thank the European Community for fund-
ing through the NANOMAGDYE project CP-FP 214032-2 and Dr.
Geneviève Pourroy, coordinator of the NANOMAGDYE project.
7612–7617.
12. Snow, A. W.; Foos, E. E. Synthesis 2003, 35, 509–512.
13. Bouzide, A.; Sauvé, G. Org. Lett. 2002, 4, 2329–2332.
14. Compound 1: to a solution of 12 (250 mg, 0.25 mmol) in 15 mL of CH2Cl2 at
0 °C, is added dropwise 500 lL (3.75 mmol, 15.0 equiv) of TMSBr. After one
night stirring at room temperature, the volatiles are evaporated and methanol
is added to the crude product and evaporated several times. Compound 1 is
obtained as a dark yellow oil in 93% yield without further purification. 1H NMR
(300 MHz, CD3OD, 20 °C): d (ppm) = 3.08 (d, 2H, 2J = 21.5 Hz), 3.34 (s, 6H),
3.50–3.77 (m, 52H), 3.78 (t, 2H, 3J = 5.2 Hz), 3.84 (t, 4H, 3J = 5.2 Hz), 4.09–4.17
Supplementary data
Supplementary data (synthetic procedures, characterization
and 1H and 13C NMR spectra for compounds 1, 2, 3, 12, 21, 22,
and 24) associated with this article can be found, in the online ver-
sion, at doi:10.1016/j.tetlet.2011.03.144.
(m, 6H), 6.64 (d, 2H, 4J = 2.5 Hz). 13C NMR (75 MHz, CD3OD, 20 °C):
d
(ppm) = 35.61 (1JCP = 135.5 Hz), 59.07, 62.16, 69.93, 70.76, 71.26, 71.30,
71.46, 71.56, 71.66, 72.88, 73.43, 73.56, 110.38 (3JCP = 5.8 Hz), 129.64
(2JCP = 9.1 Hz), 138.15 (5JCP = 2.5 Hz), 153.65 (4JCP = 2.5 Hz). 31P NMR (81 MHz,
CD3OD, 20 °C): d (ppm) = 24.32. MALDI: calculated for C41H78O22P: 953.47,
obtained: 953.47; calculated for
C41H77NaO22P: 975.45, obtained: 975.46;
References and notes
calculated for
C41H77KO22P: 991.43, obtained: 991.43; calculated for
C41H76Na2O22P: 997.44, obtained: 997.44. IR (KBr, cmꢀ1): 3409, 2875, 1590,
1511, 1443, 1352, 1249, 1106, 955, 848.
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17. Compound 2: to a solution of 24 (136 mg, 0.10 mmol) in 6 mL of CH2Cl2 at 0 °C,
is added dropwise 200 lL (1.50 mmol, 15.0 equiv) of TMSBr. After one night
stirring at room temperature, the volatiles are evaporated and methanol is
added to the crude product and evaporated several times. Compound 2 is
obtained as a dark blue oil in 95% yield without further purification.1H NMR
(300 MHz, DMSO-d6, 20 °C): d (ppm) = 1.20 (t, 3J = 6.7 Hz, 12H), 2.78 (d, 2H,
2J = 21.0 Hz), 3.00 (bt, 2H, 3J = 5.8 Hz), 3.22 (s, 6H), 3.39–3.71 (m, 50H), 3.93 (bt,
3
2H, J = 5.2 Hz), 4.00–4.07 (m, 4H), 6.57 (s, 2H), 6.98 (m, 4H, AA0 part of an
3
AA0BB0 system), 7.23 (d, 1H, J = 8.1 Hz), 7.27 (m, 4H, BB0 part of an AA0BB0
system), 7.85 (dd, 1H, 3J = 8.1 Hz, 4J = 1.8 Hz), 8.03 (br s, 1H), 8.35 (d, 1H,
4J = 1.8 Hz). 13C NMR (75 MHz, DMSO-d6, 20 °C):
d (ppm) = 12.70, 35.29
(1JCP = 132.7 Hz), 42.42, 45.26, 58.02, 60.19, 68.32, 68.97, 69.12, 69.56, 69.69,
69.76, 69.81, 69.94, 71.25, 71.77, 72.30, 108.98 (3JCP = 5.8 Hz), 113.27, 125.84,
125.97, 126.08, 129.46 (2JCP = 8.5 Hz), 131.68, 136.01 (5JCP = 3.4 Hz), 139.80,
139.99, 141.38, 148.22, 151.66 (4JCP = 2.2 Hz), 154.53, 173.61. 31P NMR
(81 MHz, DMSO-d6, 20 °C):
d
(ppm) = 18.29. MALDI: calculated for
obtained: 1302.57; calculated for
60H92N3NaO22PS2: 1324.53, obtained: 1324.55. IR (KBr, cmꢀ1): 3416, 2925,
C60H93N3O22PS2:
1302.54,
C
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Vis (in H2O): kmax ) = 646 (82 800) nm (shoulder around 600 nm).
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