Dendronized metallic oxide nanoparticles, a process for preparing the same and their uses
10624976 ยท 2020-04-21
Assignee
- Centre National De La Recherche Scientifique (Paris, FR)
- UNIVESITE DE STRASBOURG (Strasbourg, FR)
- Hospices Civils De Lyon (Lyons, FR)
- Universite Claude Bernard Lyon 1 (Villeurbanne, FR)
Inventors
- Sylvie Begin-Colin (Achenheim, FR)
- Delphine Felder-Flesch (Hattstatt, FR)
- Claire Billotey (Lyons, FR)
- Benoit Pichon (Strasbourg, FR)
Cpc classification
A61K49/186
HUMAN NECESSITIES
A61K41/0052
HUMAN NECESSITIES
A61K41/0038
HUMAN NECESSITIES
A61K49/1842
HUMAN NECESSITIES
A61K49/1839
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
International classification
A61K9/00
HUMAN NECESSITIES
A61K41/00
HUMAN NECESSITIES
Abstract
Dendronized metallic oxide nanoparticles, a process for preparing the same and their uses.
Claims
1. A functionalized metallic oxide nanoparticle comprising: a metallic oxide nanoparticle and at least two compounds selected from the following compounds: ##STR00086## ##STR00087## said compounds being iono-covalently bound to said metallic oxide nanoparticle via the PO.sub.3H.sub.2 groups these compounds can be functionalized by: a ligand targeting tumor cells, abnormal cells in respect to their metabolic state or their activation state, or elements constituting an extracellular matrix, a radioelement chelant; a specific molecule recognition agent, being able to form a complex with said specific molecule, optionally linked to another dendrimer, said metallic oxide nanoparticle being: a homogenous metallic oxide nanoparticle selected from the group consisting in: a metallic oxide of the following formula (II):
M.sub.xO.sub.y(II) wherein: M is Fe, x and y are positive integers such as y=(x.Math.v)/2, wherein v is the average oxidation state of M in M.sub.xO.sub.y, a metallic oxide of the following formula (III):
Fe.sub.3-yM.sub.yO.sub.4(III) wherein M is a metal selected from the group constituted of Zn, Co, Ni and Mg, y being such as 0<y1, or a core-shell metallic oxide nanoparticule, said core being selected from the group constituted of: a metallic oxide of the following formula (II):
M.sub.xO.sub.y(II) wherein: M is Fe, x and y are positive integers such as y=(x.Math.v)/2, wherein v is the average oxidation state of M in M.sub.xO.sub.y, a metallic oxide of the following formula (III):
Fe.sub.3-yM.sub.yO.sub.4(III) wherein M is a metal selected from the group constituted of Zn, Co, Ni and Mg, y being such as 0<y1, said shell being selected from the group constituted of: a metallic oxide of the following formula (II):
M.sub.xO.sub.y(II) wherein: M is Fe, x and y are positive integers such as y=(x.Math.v)/2, wherein v is the average oxidation state of M in M.sub.xO.sub.y, a metallic oxide of the following formula (III):
Fe.sub.3-yM.sub.yO.sub.4(III) wherein M is a metal selected from the group constituted of Zn, Co, Ni and Mg, y being such as 0<y1, Au, provided that: said core and said shell are not the same metallic oxide, said metallic oxide nanoparticle: being a magnetic resonance imaging contrast agent, and having a sufficient heating power for a magnetic hyperthermia treatment, the functionalized metallic oxide nanoparticle being a dendronized nanoparticle operative as both a T2 MRI contrast agent and an agent for hyperthermia therapy.
2. The functionalized metallic oxide nanoparticle according to claim 1, said nanoparticle having a r.sub.2 relaxivity value above 60 s.sup.1 mM.sup.1, and a relaxivity ratio such that the r.sub.2/r.sub.1 ratio is above 6, said r.sub.1 and r.sub.2 values being measured with a nanoparticle having a mean hydrodynamic size of about 15 nm, and under a magnetic field of 1.41 T at 37 C., and a specific absorption rate above 80 W/g, said rate being measured at a concentration of iron and/or magnetic metallic atom in said nanoparticle of 0.01 mol/L, at a field frequency of 700 kHz with a field amplitude of 27 mT and at 37 C.
3. The functionalized metallic oxide nanoparticle according to claim 2, comprising an iron oxide.
4. The functionalized metallic oxide nanoparticle according to claim 1, said nanoparticle having a r.sub.1 relaxivity value comprised from 4 to 5 s.sup.1 mM.sup.1, and a r.sub.1 relaxivity ratio such that the r.sub.2/r.sub.1 ratio is comprised from 4 to 5, r.sub.1 and r.sub.2 values being measured with a nanoparticle having a mean hydrodynamic size of about 15 nm, under a magnetic field of 1.41 T at 37 C.
5. The functionalized metallic oxide nanoparticle according to claim 1, the largest dimension of which being comprised from 5 to 30 nm.
6. The functionalized metallic oxide nanoparticle according to claim 1, said nanoparticle being: cubic, rodshaped, octopod-shaped or nanoplatelet-shaped, and/or a core-shell metallic oxide nanoparticule.
7. A chain of functionalized metallic oxide nanoparticles according to claim 1, said chain being linear.
8. A method for medical imaging comprising the use of a functionalized metallic oxide nanoparticle according to claim 1.
9. A method for the treatment of tumors or other pathological tissues comprising the use of a functionalized metallic oxide nanoparticle according to claim 1, as a hyperthermia and/or radiosensitizing agent.
10. A pharmaceutical or diagnostic composition comprising functionalized metallic oxide nanoparticles according to claim 1, as active agents and a pharmaceutically acceptable vehicle.
Description
FIGURES
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EXAMPLES
(29) In the following examples, the formula Fe.sub.3-xO.sub.4 depicts Fe.sub.3O.sub.4 nanoparticles, which are slightly oxidized on their surface due to their nanosize.
Example 1: Synthesis of Core-Shell FeO@Fe3-xO4 Cubic-Shaped Iron Oxide Nanoparticles (NC16, Mean Size: 16 nm)
(30) By FeO@Fe.sub.3-xO.sub.4 is meant a core-shell composition with a FeO core and a Fe.sub.3-xO.sub.4 shell. First of all, the iron oleate complex Fe(oleate).sub.3 was prepared from 10.8 g (40.0 mmol) FeCl.sub.3.6H.sub.2O (99%, Merck) dissolved in 60 ml H.sub.2O (Milli-Q) and 80 ml ethanol. 36.5 g (120 mmol) of sodium oleate dissolved in 140 ml hexane were then mixed with the iron (III) solution. The resulting biphasic mixture was then refluxed at 70 C. for 4 h under stirring. Then, the organic phase containing the iron complex was separated, washed three times with 30 ml of distilled water at 50 C. to extract salts and then dried over MgSO.sub.4. The evaporation of hexane led to a waxy solid.
(31) Secondly, (2.08 mmol) of Fe(oleate).sub.3, 0.705 g (2.32 mmol) of sodium oleate (>97.0%, TCI) and 0.2 ml (0.6510.sup.3 mol) of oleic acid (99%, Alfa-Aesar) were added to 15 ml of octadecene (90%, Fluka, b.p. 318 C.). The mixture was heated at 120 C. in the absence of a reflux condenser for 1 h to evaporate the traces of unwanted solvent and to dissolve the reactants. The solution was first quickly heated to 200 C. without stirring and maintained at this temperature for 10 minutes. Then the solution was heated at a rate of 1 C./min up to 320 C. and was refluxed for 60 min either under argon or air. The resultant black solution was cooled down to room temperature and the NPs were washed three times by addition of acetone and centrifugation (14000 rpm, 10 min.). The cubic-shaped NPs, were then easily suspended in chloroform and displayed a mean size of about 16 nm. They are named NC16.
Example 2: Oxidation of Core-Shell FeO@Fe3-xO4 Cubic-Shaped Iron Oxide Nanoparticles (oxNC16)
(32) 50 mg of cubic nanoparticles synthesized as described in example 1 were dispersed in toluene and heated to reflux during 48 h with bubbling air inside the suspension. After this post synthesis oxidation step, the nanoparticles were dispersed and stored in THF after evaporation of the toluene. oxNC16 nanoparticles displayed a mean size of about 16 nm.
Example 3: Synthesis of Core-Shell FeO@Fe3-xO4 Cubic-Shaped Iron Oxide Nanoparticles (Mean Size: 13 nm)
(33) The synthesis was performed in the same conditions as those of example 1, but with a heating rate adjusted to 5 C. min.sup.1 instead of 1 C. min.sup.1 and led to cubic-shaped NPs with a mean size of 13 nm.
Example 4: Synthesis of Core-Shell FeO@Fe3-xO4 Cubic-Shaped Iron Oxide Nanoparticles (Mean Size: 30 nm)
(34) The synthesis was performed in the same conditions as those of example 1, but the solvent was changed to eicosene (Aldrich, 90%) and the solution was refluxed for only 15 minutes to obtain nanocubes of 30 nm.
Example 5: Synthesis of Spherical Homogenous Fe3-xO4 Nanoparticles (Mean Size of 10.1 nm)
(35) Spherical iron oxide nanocrystals were synthesized by thermal decomposition of iron stearate in octylether. 1.38 g (2.22 mmol) of Fe(stearate).sub.2 (Strem Chemicals) and 1.24 g (4.44 mmol) of oleic acid (99%, Alfa Aesar) were added to 20 mL octyl ether (99%, Fluka, b.p. 287 C.). The mixture was heated and kept at 100 C. under stirring for 15 min in order to dissolve the reactants. The solution was heated to 287 C. with a heating rate of 5 C./min without stirring and was refluxed for 120 min at this temperature under air. A black suspension containing spherical NPs is obtained. These standard conditions lead to NPs with a mean size of 10.1 nm, labeled NP10.
Example 6: Synthesis of Spherical Homogenous Fe3-xO4 Nanoparticles (Mean Size of 5.4 nm)
(36) NP5 nanoparticles (d=5.4 nm) have been obtained by using the same procedure as the one of example 5, with hexadecene as solvent (T.sub.b=274 C.).
Example 7: Synthesis of Spherical Homogenous Fe3-xO4 Nanoparticles (Mean Size of 14.7 nm)
(37) NP15 nanoparticles (d=15.5 nm) have been obtained by using the same procedure as the one of example 5, with eicosene as solvent (T.sub.b=330 C.).
Example 8: Synthesis of Spherical Homogenous Fe3-xO4 Nanoparticles (Mean Size of 20.3 nm)
(38) NP20 nanoparticles (d=20.3 nm) have been obtained by using the same procedure as the one of example 5, except that the reactants were dissolved in docosene (Tb=355 C.), and the solution was maintained at 250 C. for 30 min, then heating quickly the solution to reflux for 2 h.
Example 9: Synthesis of Spherical Homogenous Fe3-xO4 Nanoparticles (Mean Size of 28 nm)
(39) NP30 nanoparticles (d=28 nm) have been obtained by using the same procedure as the one of example 5, except that the reactants were dissolved in docosene (Tb=355 C.), and the solution was maintained at 250 C. for 1 h, then heating quickly the solution to reflux for 2h.
Example 10: Synthesis of Spherical Homogenous Manganese Oxide Nanoparticles
(40) The same conditions of synthesis as those used for iron oxide nanoparticles were used, except that manganese acetate was used instead of iron stearate.
Example 11: Synthesis of Spherical Homogenous Doped Ferrite Nanoparticles
(41) The same conditions of synthesis as those used for iron oxide nanoparticles were used, except that a metallic complex (either metal acetate or metal(acac) or metal stearate or metal oleate or any metal precursor decomposing between 180 and 250 C.) is added with iron stearate in stoichiometric proportions according to Fe.sub.3-yM.sub.yO.sub.4.
Example 12: Synthesis of Spherical Core-Shell Fe3-xO4@MxOy Nanoparticles
(42) General Procedure
(43) In a typical synthesis, a two necked round bottom flask was charged with 1.38 g (2.22 mmol) of Fe(stearate).sub.2 (9.47% Fe, Strem Chemicals), 1.254 g (4.44 mmol) of oleic acid (99%, Alfa-Aesar) and 20 mL of octyl ether (97%, Fluka, bp 287 C.) used as solvent. The mixture was sonicated and stirred at 120 C. for 10 mn to dissolve the reactants until a clear solution was obtained. The solution was then heated to boiling temperature (287 C.) with a heating rate of 5 C./mn and kept at this temperature for 120 minutes under air. The resultant black solution was then cooled to 100 C. 10 ml of solution was taken for characterization of Fe.sub.3-xO.sub.4 nanoparticles.
(44) In a second step, 0.67 g (2.22 mmol) of the metal precursor (stearate/oleate/octanoate . . . of a metal or M(CO)x or M(acac) or any metal precursor decomposing between 180 and 250 C.)) dissolved in 20 mL of octadecene (90%, Alfa Aesar, bp 318 C.) was added to the remaining solution kept at 100 C. and the mixture containing both octyl ether and octadecene was heated again to reflux for 3 hours under argon (heating rate of 1 C./mn). After cooling down to room temperature, each types of nanoparticles (Fe.sub.3O.sub.4 and Fe.sub.3O.sub.4@MxOy nanoparticles) were precipitated by the addition of an excess of acetone and washed 3 times by a mixture of hexane/acetone (1/3) by centrifugation (14000 rpm, 10 mn). Finally the Fe.sub.3-xO.sub.4 and Fe.sub.3O.sub.4@ MxOy nanoparticles were easily suspended in chloroform.
(45) Synthesis of Core-Shell Fe.sub.3-xO.sub.4@CoO Nanoparticles
(46) Fe.sub.3-xO.sub.4@CoO nanoparticles were obtained according to the general procedure, using cobalt stearate and iron stearate as metal precursors.
(47) In particular, Fe.sub.3-xO.sub.4@CoO nanoparticles with a core having a mean diameter of 7 or 10 nm, and a shell of a mean thickness of 0.5, 1 or 2 nm were obtained.
(48) In a typical synthesis, a two necked round bottom flask was charged with 1.38 g (2.22 mmol) of Fe(stearate).sub.2 (9.47% Fe, Strem Chemicals), 1.254 g (4.44 mmol) of oleic acid (99%, Alfa-Aesar) and 20 mL of octyl ether (97%, Fluka, bp 287 C.) used as solvent. The mixture was sonicated and stirred at 120 C. for 10 mn to dissolve the reactants until a clear solution was obtained. The solution was then heated to boiling temperature (287 C.) with a heating rate of 5 C./mn and kept at this temperature for 120 minutes under air. The resultant black solution was then cooled to 100 C. 10 ml of solution was taken for characterization of Fe.sub.3O.sub.4 nanoparticles. In a second step, 0.67 g (2.22 mmol) of Co(stearate).sub.2 (9-10% Co, Strem Chemicals) dissolved in 20 mL of octadecene (90%, Alfa Aesar, bp 318 C.) was added to the remaining solution kept at 100 C. and the mixture containing both octyl ether and octadecene was heated again to reflux for 3 hours under argon (heating rate of 1 C./mn). After cooling down to room temperature, nanoparticles were precipitated by the addition of an excess of acetone and washed 3 times by a mixture of hexane/acetone (1/3) by centrifugation (14000 rpm, 10 mn). Finally the Fe.sub.3O.sub.4 and Fe.sub.3O.sub.4@CoO nanoparticles were easily suspended in chloroform.
(49) The shell thickness is tuned by varying the amount of cobalt stearate added during the second step: twice and three times the amount of cobalt stearate.
Example 13: Synthesis of Core-Shell Nanoparticles in Two Steps
(50) General Procedure (Adapted to all Types of Nanoparticles Coated with a Surfactant and Stable in Organic Solvent, to all Morphologies: Spherical, Nanocubes, Nanowires and Octopods and all Compositions: Fe.sub.3-xO.sub.4 and Also Other Types of Ferrites MFe.sub.2O.sub.4 with M=Fe, Ni, Zn, Mn or Metal Oxides MxOy with M=Mn)
(51) First Step:
(52) synthesis of the nanoparticles with a given composition and shape and formation of a stable suspension of these nanoparticles coated with surfactants in an organic solvent with a high boiling temperature.
(53) 2.sup.nd Step:
(54) In a second step, 2.22 mmol of the metal precursor (stearate/oleate/octanoate. of a metal or M(CO)x or M(acac) or any metal precursor decomposing between 180 and 250 C.)) dissolved in 20 mL of octadecene (90%, Alfa Aesar, bp 318 C.) was added to the 10 ml of the solution of pre-synthesized nanoparticles and the mixture was heated again to reflux for 3 hours under argon (heating rate of 1 C./mn). After cooling down to room temperature, the so obtained nanoparticles were precipitated by the addition of an excess of acetone and washed 3 times by a mixture of hexane/acetone (1/3) by centrifugation (14000 rpm, 10 mn). Finally the core-shell nanoparticles were easily suspended in chloroform.
(55) Synthesis of Core-Shell Fe.sub.3-xO.sub.4@MnO Nanoparticles
(56) Fe.sub.3-xO.sub.4@MnO nanoparticles were obtained according to the two steps procedure, using iron stearate and manganese acetate (Mn(acetat).sub.2) as metal precursor.
(57) Synthesis of Fe.sub.3O.sub.4 Seeds
(58) Iron oxide NPs were synthesized by thermal decomposition of an iron stearate complex in presence of oleic acid in a high boiling solvent. 1.38 (2.2 mmol) of Fe(stearate).sub.2 and 1.25 g (4.4 mmol) of oleic acid (OA) were added to 20 mL of octyl ether (b.p 288 C.). The mixture was heated at 110 C. during 30 minutes until all the reactants were completely dissolved. The solution was heated to 288 C. with a heating rate of 5 C./min and was reflux for 120 min at this temperature under air. The resulting black solution was then cooled down to room temperature and the NPs were washed 3 times by addition of ethanol and by centrifugation. The as synthesized NPs, named Fe.sub.3O.sub.4 seeds, were then easily suspended in hexane.
(59) Synthesis of Core-Shell Fe.sub.3O.sub.4@MnO Nanoparticles
(60) A MnO shell was grown on the Fe.sub.3O.sub.4 seeds previously synthesized using a seeds mediated growth method. 0.38 g (2.2 mmol) Mn(acetate).sub.2 and 1.86 g (6.6 mmol) OA were added to 15 mL octadecene. 3 ml of Fe.sub.3O.sub.4 seeds solution (4.87 mg/mL) in hexane were injected in the mixture. The solution was heated at 110 C. during 1 h until all the reactants were completely dissolved and the hexane evaporated. The solution was then slowly increased (1 C./min) to 318 C. and was reflux for 60 min. The resulting black solution was then cooled down to room temperature and the NPs were washed 3 times by addition of ethanol or acetone and by centrifugation. The as synthesized NPs, named Fe.sub.3O.sub.4@MnO, were then easily suspended in THF.
(61) In particular, Fe.sub.3-xO.sub.4@MnO nanoparticles with a core having a mean diameter of 6 nm, and a shell of a mean thickness of 6.5 nm were obtained.
(62) The shell thickness is tuned by varying the amount of Mn(acetate).sub.2 added during the second step: 0.25, 0.5 and twice times the amount of Mn(acetate).sub.2.
(63) Synthesis of Core-Shell CoFe.sub.2O.sub.4@MnO Nanoparticles
(64) CoFe.sub.2O.sub.4@MnO nanoparticles were obtained according to the general procedure, using cobalt stearate, iron stearate and manganese acetate as metal precursor.
(65) 0.38 g (2.2 mmol) Mn(acetate).sub.2 and 1.86 g (6.6 mmol) OA were dissolved in 20 mL of octadecene (90%, Alfa Aesar, bp 318 C.) was added to the 10 ml of the solution of pre-synthesized CoFe.sub.2O.sub.4 nanoparticles and the mixture was heated again to reflux for 3 hours under argon (heating rate of 1 C./mn). After cooling down to room temperature, the so obtained nanoparticles were precipitated by the addition of an excess of acetone and washed 3 times by a mixture of hexane/acetone (1/3) by centrifugation (14000 rpm, 10 mn). Finally the core-shell nanoparticles were easily suspended in chloroform.
(66) In particular, CoFe.sub.2O.sub.4@MnO nanoparticles with a core having a mean diameter of 8 nm, and a shell of a mean thickness of 2 nm were obtained.
Example 14: Synthesis of Octopods (NO24)
(67) First of all, the iron oleate complex Fe(oleate).sub.3 was prepared from 10.8 g (40.0 mmol) FeCl.sub.3.6H.sub.2O (99%, Merck) dissolved in 60 ml H.sub.2O (Milli-Q) and 80 ml ethanol. 36.5 g (120 mmol) of sodium oleate dissolved in 140 ml hexane were then mixed with the iron (III) solution. The resulting biphasic mixture was then refluxed at 70 C. for 4 h under stirring. Then, the organic phase containing the iron complex was separated, washed three times with 30 ml of distilled water at 50 C. to extract salts and then dried over MgSO4. The evaporation of hexane led to a waxy solid.
(68) Secondly, (2.08 mmol) of Fe(oleate)3, 0.705 g (2.32 mmol) of sodium oleate (>97.0%, TCI) and 0.2 ml (0.6510-3 mol) of oleic acid (99%, Alfa-Aesar) were added to 15 ml of octadecene (90%, Fluka, b.p. 318 C.). The mixture was heated at 110 C. in absence of a reflux condenser and then the solution was quickly heated to 220 C. and maintained at this temperature for 10 min. The synthesis mixture was then heated at a rate of 5 C./min and temperature was maintained at 320 C. for 1 h. The resultant black solution was then cooled to room temperature, and the NPs were washed several times by addition of ethanol and by centrifugation (14000 rpm, 10 min). The edges grown cubic NPs or octopodes with a size of 24 nm, were easily suspended in THF. They are named NO24.
Example 15: Synthesis of Core-Shell FeO@Fe3O4 Spherical Nanoparticles (NS19)
(69) Spherical core shell FeO@Fe.sub.3O.sub.4 NPs were synthesized by thermal decomposition of iron stearate complex in presence of oleic acid in a high boiling point solvent. 1.38 g (2.2 mmol) of Fe(stearate).sub.2 and 1.25 g (4.4 mmol) of oleic acid (OA) were added to 18 g of docosene (B.P 355 C.). The mixture was heated at 110 C. in absence of a reflux condenser for 1H to evaporate the traces of unwanted solvent and to dissolve the reactants. The solution was then heated to 355 C. with a heating rate of 5 C./min and refluxed for 120 min at this temperature under air. The resultant black solution was then cooled to room temperature, and the NPs were washed several times by addition of ethanol and by centrifugation (14000 rpm, 10 min). The nano spheres, named NS19, were easily suspended in THF, and displayed a mean size of about 19 nm.
Example 16: Synthesis of Dendrons
(70) ##STR00081##
Compound 1:
(71) A solution of methyl gallate (20.0 g, 108.6 mmol), benzyl bromide (14.2 mL, 119.0 mmol, 1.1 eq.), KHCO.sub.3 (32.4 g, 324.0 mmol, 3.0 eq.) and KI (0.1 g, 0.60 mmol) in DMF (100 mL) was stirred during 4 days at 30 C. The reaction mixture was poured into 1 L of water and sulfuric acid was added until obtention of a neutral pH. The aqueous layer was then extracted 3 times with 150 mL of dichloromethane. The combined organic layers were brought together, washed three times with 50 mL of brine, dried over MgSO.sub.4 and filtered. The solvent was removed by evaporation and the residue was purified by column chromatography on silica gel eluting with dichloromethane/methanol (98/2) to provide an yellow oil. The crude material was evaporated many times with dichloromethane. The obtained residue was filtered and washed petroleum ether to provide compound 1 as a white solid in 70% yield. .sup.1H NMR (300 MHz, CD.sub.3OD-d) 7.52 (d, J=7.5 Hz, 2H, Ar.sup.2-2,6-H), 7.31 (m, 3H, Ar.sup.2-3,4,5-R), 7.13 (s, 2H, Ar.sup.1-2,6-H), 5.18 (s, 2H, Ar.sup.2OCH.sub.2), 3.83 (s, 3H, COOCH.sub.3); .sup.13C NMR (75 MHz, CD.sub.3OD-d) 167.1, 150.5, 138.2, 137.2, 128.5, 128.0, 127.8, 125.0, 108.8, 73.8, 51.2.
(72) Compound 2:
(73) A solution of para-toluenesulfonyl chloride (22.3 g, 105 mmol) in THF (35 mL) was added dropwise to a solution of tetraethyleneglycol methyl ether (20.0 g, 96 mmol) and NaOH (6.7 g, 166 mmol) in a mixture of THF/H.sub.2O (135 mL/45 mL) at 0 C. After 1 hour stirring at 0 C., the reaction was allowed to warm at room temperature and was stirred 20 additional hours. The solution was then poured into 200 mL of brine and the volatiles were evaporated. The resulting mixture was extracted several times with dichloromethane and the combined organic layers were washed with brine, dried over MgSO.sub.4 and filtered. The solvent was evaporated under reduced pressure and the oil and was purified by column chromatography on silica gel eluting with dichloromethane/methanol (98/2). Compound 2 was obtained as a pale yellow oil in 94% yield. .sup.1H NMR (300 MHz, CDCl.sub.3) 7.73 (d, J=1.5 Hz, 2H, Ar-2,6-H), 7.28 (d, J=1.5 Hz, 2H, Ar-3,5-H), 4.11-4.08 (m, 2H, ArSO.sub.2OCH.sub.2), 3.64-3.47 (m, 14H, OCH.sub.2CH.sub.2O), 3.31 (s, 3H, OCH.sub.3), 2.39 (s, 3H, ArCH.sub.3); .sup.13C NMR (75 MHz, CDCl.sub.3) 144.9, 133.2, 130.0, 72.1, 70.9, 70.7, 70.6, 69.5, 68.8, 59.1, 28.1, 21.8.
(74) Compound 3:
(75) A solution of 1 (9.2 g, 33.4 mmol), 2 (26.9 g, 74.3 mmol, 2.2 eq.), K.sub.2CO.sub.3 (28.0 g, 200 mmol, 6.0 eq.) and KI (0.6 g, 3.3 mmol, 0.1 eq.) in acetone (600 mL) was stirred during 30 hours at 65 C. The reaction mixture was filtered over Celite and the solvent was evaporated. The resulting crude product was diluted in dichloromethane (200 mL) and washed twice with an aqueous saturated solution of NaHCO.sub.3 and with brine. After drying over MgSO.sub.4, filtration and evaporation of the solvent, the crude product was purified by chromatography over silica gel column (dichloromethane/methanol 98/2 to 95/5) to afford 3 as a colorless oil in 75% yield. .sup.1H NMR (300 MHz, CDCl.sub.3) 7.48 (d, J=7.7 Hz, 2H, Ar.sup.2-2,6-H), 7.28 (m, 5H, Ar.sup.2-3,4,5-H and Ar.sup.1-2,6-H), 5.12 (s, 2H, Ar.sup.2OCH.sub.2), 4.20-4.17 (t, J=4.8 Hz, 4H, Ar.sup.1OCH.sub.2), 3.90 (s, 3H, COOCH.sub.3), 3.88-3.85 (t, J=4.8 Hz, 4H, OCH.sub.2CH.sub.2O), 3.74-3.69 (m, 4H, OCH.sub.2CH.sub.2O), 3.67-3.60 (m, 16H, OCH.sub.2CH.sub.2O), 3.54-3.50 (m, 4H, OCH.sub.2CH.sub.2O), 3.35 (s, 6H, OCH.sub.2CH.sub.2OCH.sub.3); .sup.13C NMR (75 MHz, CDCl.sub.3) 166.9, 152.5, 142.2, 138.2, 128.2, 128.0, 127.8, 125.3, 109.1, 74.8, 72.3, 71.2, 71.0, 70.9, 70.8, 70.0, 69.2, 59.3, 52.5. MALDI: calculated for C.sub.33H.sub.50NaO.sub.13: 677.33. obtained: 677.03.
(76) Compound 4:
(77) To a solution of compound 3 (8.3 g, 12.7 mmol) in a mixture methanol/water 4/1 (150 mL) was added of sodium hydroxyde (5.1 g, 127.0 mmol, 10 eq.). The reaction mixture was stirred 2h at 85 C. and stopped. The mixture was concentrated in vacuo and hydrolyzed (200 mL). The pH was adjusted at 3 by HCl 12N and the aqueous solution was extracted with dichloromethane (3100 mL). The combined organic phase was washed with brine and water, dried over MgSO.sub.4, filtered and concentrated under reduced pressure purified by column chromatography on silica gel eluting with dichloromethane/methanol (95/5) to afford 4 as a colorless oil in 90% yield. .sup.1H NMR (300 MHz, CDCl.sub.3) 7.50 (d, J=7.8 Hz, 2H, Ar.sup.2-2,6-H), 7.38 (s, 2H, Ar.sup.1-2,6-H), 7.35-7.28 (m, 3H, Ar.sup.2-3,4,5-H), 5.13 (s, 2H, Ar.sup.2OCH.sub.2), 4.20-4.16 (t, J=4.8 Hz, 4H, Ar.sup.1OCH.sub.2), 3.87-3.82 (t, J=4.8 Hz, 4H, OCH.sub.2CH.sub.2O), 3.74-3.69 (m, 4H, OCH.sub.2CH.sub.2O), 3.67-3.61 (m, 16H, OCH.sub.2CH.sub.2O), 3.54-3.50 (m, 4H, OCH.sub.2CH.sub.2O), 3.37 (s, 6H, OCH.sub.2CH.sub.2OCH.sub.3); .sup.13C NMR (75 MHz, CDCl.sub.3) 169.3, 152.8, 138.2, 142.4, 128.2, 128.0, 127.8, 125.3, 109.2, 74.8, 72.3, 71.2, 71.0, 70.9, 70.8, 70.0, 69.2, 52.5. MALDI: calculated for C.sub.32H.sub.48O.sub.13: 640.31. obtained: 640.24. calculated for C.sub.29H.sub.48NaO.sub.13: 627.30. obtained: 627.13; calculated for C.sub.29H.sub.48KO.sub.13: 643.27. obtained: 643.09.
(78) ##STR00082## ##STR00083##
Compound 5:
(79) 4.20 g of dimethyl 5-hydroxyisophtalate (20.0 mmol) were dissolved in 21 mL of anhydrous THF. Then, a LiAlH.sub.4 solution, 0.5 M in THF (36.0 mmol, 1.8 eq.) was added dropwise at 0 C. After refluxing during 3 hours, the mixture was cooled to room temperature and acidified with 30 mL of a solution of H.sub.2SO.sub.4 10%. The THF was evaporated under vacuum and the resulting aqueous phase was extracted several times (at least 6 times, TLC control) with ethyl acetate. The organic phase was dried over MgSO.sub.4, filtered and concentrated under reduced pressure to afford 5 as a white solid in 94% yield. .sup.1H NMR (300 MHz, CD.sub.3OD-d) 6.82 (s, 1H, Ar-4-H), 6.71 (s, 2H, Ar-2.6-H), 4.52 (d, J=5.8 Hz, 4H, ArCH.sub.2OH); .sup.13C NMR (75 MHz, CD.sub.3OD-d) 157.2, 143.7, 115.1, 111.6, 63.0.
(80) Compound 6:
(81) 2.00 g of 5 (13.0 mmol) were dissolved in 21 ml, of acetic acid. Then, a solution of HBr 30% in acetic acid (36.0 mmol, 1.8 eq.) was added dropwise at 0 C. The mixture was stirred 24 h at room temperature, and then 80 mL of distilled water were added. A white precipitate formed and the mixture was stirred 10 minutes more. The resulting aqueous phase was extracted 3 times with 200 mL dichloromethane and the organic layer was washed twice with 120 ml, of distilled water, twice with 120 mL of a saturated solution of sodium hydrogenocarbonate, and 80 ml, of brine. The organic phase was dried over MgSO.sub.4, filtered and concentrated under reduced pressure to afford 6 as a white solid in 96% yield. .sup.1H NMR (300 MHz, CDCl.sub.3) 6.99 (t, J=1.3 Hz, 1H, Ar-4-H), 6.04 (d, J=1.3 Hz, 2H, Ar-2,6-H), 5.38 (br s, 1H, OH), 4.40 (s, 4H, ArCH.sub.2Br); .sup.13C NMR (75 MHz, CDCl.sub.3) 155.8, 140.0, 122.2, 116.2, 32.7.
(82) Compound 7:
(83) A solution of 6 (2.24 g, 8.0 mmol) in 5.0 mL of P(OEt).sub.3 (4.0 eq.), was stirred during 2 hours at 160 C. The excess of P(OEt).sub.3 was evaporated under reduced pressure at 70 C. The crude product was purified by chromatography over silica gel column (dichloromethane/methanol 95/5) to afford 7 as a white solid in 95% yield. .sup.1H NMR (300 MHz, CDCl.sub.3) 6.82 (bs, 2H, Ar-2.6-H), 6.62 (bs, 1H, Ar-4-H), 3.99 (m, 8H, PO(OCH.sub.2CH.sub.3).sub.2), 3.49 (d, J=21.9 Hz, 4H, ArCH.sub.2P), 1.23 (t, J=7.1 Hz, 12H, PO(OCH.sub.2CH.sub.3).sub.2); .sup.13C NMR (75 MHz, CDCl.sub.3) 157.9, 132.6 (J=10.6 Hz), 122.4 (J=6.7 Hz), 115.8, 62.5 (J=6.6 Hz), 33.6 (J=138.8 Hz), 16.5 (J=5.2 Hz); .sup.31P NMR (81 MHz, CDCl.sub.3) 26.72. MALDI: calculated for C.sub.16H.sub.29O.sub.7P.sub.2: 395.138. obtained: 394.963.
(84) Compound 8:
(85) To a solution of 7 (1.5 g, 3.8 mmol) in acetone (40 mL) were added (2-bromo-ethyl)carbamic acid tert-butyl ester (1.1 g, 4.95 mmol, 1.3 eq.), K.sub.2CO.sub.3 (2.1 g, 15.2 mmol, 4 eq.) and KI (0.1 g, 0.4 mmol, 0.1 eq.). The mixture was stirred during 48 h at 65 C., filtered over Celite and evaporated under reduced pressure. The resulting crude product was diluted in dichloromethane (100 mL) and washed twice with an aqueous saturated solution of NaHCO.sub.3 and with brine. After drying over MgSO.sub.4, filtration and evaporation of the solvent, the crude product was purified by chromatography over silica gel column (dichloromethane/methanol 98/2 to 95/5) to afford (Boc-amino) derivative as white solid (76%). This compound (1.2 g, 2.2 mmol) was dissolved in 30 mL of CH.sub.2Cl.sub.2 anhydre at 0 C. and trifluoroacetic acid was added dropwise 2 mL (22.0 mmol, 10.0 eq.). The reaction mixture was stirred overnight at room temperature, then the volatiles were evaporated. The crude product was dissolved in dichloromethane (20 mL) and was washed with NaOH 1N (210 mL). The organic layer was dried over MgSO.sub.4, filtered and concentrated under reduced pressure to afford 8 as a white solid in 88% yield and used without further purification. .sup.1H NMR (300 MHz, CDCl.sub.3) 6.72 (m, 3H, Ar-2,4,6-H), 5.25 (br s, 2H, OCH.sub.2CH.sub.2NH.sub.2), 4.03-3.92 (m, 10H, PO(OCH.sub.2CH.sub.3).sub.2 and OCH.sub.2CH.sub.2NH), 3.10 (d, J=21.7 Hz, 4H, ArCH.sub.2P), 3.02 (m, 2H, OCH.sub.2CH.sub.2NH), 1.25 (t, J=7.1 Hz, 12H, PO(OCH.sub.2CH.sub.3).sub.2); .sup.13C NMR (75 MHz, CDCl.sub.3) 159.0 (J=2.8 Hz), 133.1 (J=6.0 Hz), 123.8 (J=6.8 Hz), 114.5 (J=5.0 Hz), 70.0, 62.1 (J=7.0 Hz), 41.5, 33.5 (J=138.2 Hz), 16.5 (J=2.7 Hz); .sup.31P NMR (81 MHz, CDCl.sub.3) 26.24. MALDI: calculated for C.sub.18H.sub.34NO.sub.7P.sub.2: 438.17. obtained: 438.18. calculated for C.sub.18H.sub.34NaO.sub.7P.sub.2: 460.17. obtained: 460.16.
(86) Compound 9:
(87) To a solution of carboxylic acid derivative 4 (1.45 g, 2.05 mmol, 1.0 eq.) in 30 mL of distilled dichloromethane was added, under argon, coupling reagent BOP (1.2 g, 2.68 mmol, 1.3 eq.).
(88) After 5 min, were added amine derivative 8 (0.9 g, 2.05 mmol 1.0 eq.) and N,N-diisopropylethylamine (1.0 mL, 6.8 mmol, 3 eq.). The reaction mixture was stirred overnight at room temperature. 50 mL of dichloromethane was added and the organic layer was washed with a solution of sodium hydroxyde 1N (230 mL), HCl 1N (230 mL), brine (230 mL) and water (130 mL), dried over MgSO4, filtered and concentrated under reduced pressure. The crude product was purified by chromatography over silica gel column (dichloromethane/methanol 98/2 to 95/5) to afford compound 9 as colorless oil in 87% of yield. .sup.1H NMR (300 MHz, CDCl.sub.3) 7.50 (d, J=7.7 Hz, 2H, Ar.sup.3-2,6-H), 7.35-7.28 (m, 3H, Ar.sup.3-3,4,5-H), 7.07 (s, 2H, Ar.sup.2-2,6-H), 6.88 (t, J=5.7 Hz, 1H, OCH.sub.2CH.sub.2NH), 6.85-6.78 (m, 3H, Ar.sup.1-2,4,6-H), 5.07 (s, 2H, Ar.sup.3OCH.sub.2), 4.20-4.17 (t, J=4.8 Hz, 4H, Ar.sup.2OCH.sub.2), 4.15-4.11 (t, J=5.0 Hz, 2H, OCH.sub.2CH.sub.2NH), 4.08-3.96 (m, 8H, PO(OCH.sub.2CH.sub.3).sub.2), 3.88-3.78 (m, 6H, OCH.sub.2CH.sub.2NH and OCH.sub.2CH.sub.2O), 3.71-3.68 (m, 4H, OCH.sub.2CH.sub.2O), 3.65-3.58 (m, 16H, OCH.sub.2CH.sub.2O), 3.55-3.49 (m, 4H, OCH.sub.2CH.sub.2O), 3.35 (s, 6H, OCH.sub.2CH.sub.2OCH.sub.3), 3.08 (d, J=21.5 Hz, 4H, Ar.sup.1CH.sub.2P), 1.25 (t, J=7.0 Hz, 12H); .sup.13C NMR (75 MHz, CDCl.sub.3) 167.2, 158.6 (J=2.8 Hz), 152.8, 141.0, 137.8, 133.1 (J=6.0 Hz), 129.6, 128.2, 128.0, 127.8, 124.0 (J=6.8 Hz), 114.6 (J=4.8 Hz), 107.0, 74.9, 72.0, 70.8, 70.7, 70.6, 69.8, 69.1, 66.8, 62.1 (J=3.4 Hz), 58.9, 53.2, 39.5, 36.8 (J=3.9 Hz), 33.5 (J=138.3 Hz), 16.5=2.7 Hz); .sup.31P NMR (81 MHz, CDCl.sub.3) 26.08. MALDI: calculated for C.sub.50H.sub.79NaNO.sub.19P.sub.2: 1082.87. obtained: 1082.51.
(89) Compound 10:
(90) The benzylated compound 9 (2 g, 1.9 mmol) was dissolved in ethanol absolute (20 mL) and palladium activated on carbon 10% (0.5 eq.) was added. The mixture was stirred under hydrogen atmosphere at room temperature for 16 h. The product was filtered through a plug of Celite before being concentrated and purified by column chromatography on silica gel eluting with dichloromethane/methanol (98/2 to 90/10) to afford 10 as colorless oil in 87% of yield. .sup.1H NMR (300 MHz, CDCl.sub.3) 7.16 (s, 2H, Ar.sup.2-2,6-H), 6.85-6.78 (m, 3H, Ar.sup.1-2,4,6-H), 6.65 (m, 1H, OCH.sub.2CH.sub.2NH), 4.27-4.21 (t, J=4.7 Hz, 4H, Ar.sup.2OCH.sub.2), 4.15-4.10 (t, J=5.0 Hz, 2H, OCH.sub.2CH.sub.2NH), 4.08-3.98 (m, 8H, PO(OCH.sub.2CH.sub.3).sub.2), 3.88-3.78 (m, 6H, OCH.sub.2CH.sub.2NH and OCH.sub.2CH.sub.2O), 3.75-3.60 (m, 20H, OCH.sub.2CH.sub.2O), 3.56-3.51 (m, 4H, OCH.sub.2CH.sub.2O), 3.35 (s, 6H, OCH.sub.2CH.sub.2OCH.sub.3), 3.09 (d, J=22.0 Hz, 4H, Ar.sup.1CH.sub.2P), 1.26 (t, J=7.1 Hz, 12H, PO(OCH.sub.2CH.sub.3).sub.2); .sup.13C NMR (75 MHz, CDCl.sub.3) 167.3, 158.7 (J=2.8 Hz), 146.8, 141.0, 133.1 (J=6.0 Hz), 124.0, 114.6, 108.4, 72.0, 70.8, 70.7, 70.6, 69.8, 69.1, 66.8, 62.1 (J=3.4 Hz), 58.9, 39.5, 33.4 (J=138.1 Hz), 16.4 (J=2.7 Hz); .sup.31P NMR (81 MHz, CDCl.sub.3) 26.10. MALDI: calculated for C.sub.43H.sub.74NO.sub.19P.sub.2: 970.43. obtained: 970.44. calculated for C.sub.43H.sub.73NaNO.sub.19P.sub.2: 992.43. obtained: 992.44.
(91) Compound 11:
(92) To a solution of Hydroxy-dPEG.sub.8-t-butyl ester (1.00 g, 2.0 mmol) in 20 mL of CH.sub.2Cl.sub.2 at 0 C. are added sequentially 840 mL (6.0 mmol, 3.0 eq.) of NEt.sub.3 and 570 mg (3.0 mmol, 1.5 eq.) of para-toluenesulfonyl chloride. After 40 h stirring at room temperature, the reaction mixture is diluted with 70 mL of CH.sub.2Cl.sub.2. The organic phases are combined, washed with brine, dried over MgSO4, filtered and concentrated under reduced pressure. The crude product is purified by chromatography over silica gel column (ethyl acetate/methanol 95/5 to 90/10) to afford 11 as a colorless oil in 70% yield. .sup.1H NMR (300 MHz, CDCl.sub.3): (ppm) 1.44 (s, 9H), 2.45 (s, 3H), 2.50 (t, 2H, .sub.3J=6.6 Hz), 3.58-3.73 (m, 32H), 4.16 (t, 2H, .sub.3J=4.9 Hz), 7.34 (2H, AA part of an AABB system), 7.81 (2H, BB part of an AABB system). .sup.13C NMR (75 MHz, CDCl.sub.3): (ppm) 21.14, 27.65, 35.84, 66.40, 68.15, 69.00, 70.08, 79.78, 127.46, 129.47, 132.74, 144.32, 170.20. MALDI: calculated for C10H20LiO5: 227.15. obtained: 227.08. calculated for C26H44LiO12S: 587.27. obtained: 587.13.
(93) Compound 12:
(94) To an equimolar solution of phenolic derivative 10 (0.3 g, 0.31 mmol) and compound 11 (0.20 g, 0.31 mmol) in 10 mL of acetone were added K.sub.2CO.sub.3 (0.13 g, 0.93 mmol, 3 eq.) and KI (18 mg, 0.11 mmol, 0.3 eq.). The reaction mixture was stirred at 60 C. during 24 h. After filtration over Celite, the solvent was evaporated and the residue was diluted in dichloromethane (50 mL). The organic layer was washed twice with a saturated solution of NaHCO.sub.3, then with brine, filtered and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel eluting with dichloromethane/methanol (98/2 to 90/10) to afford compound 12 as a colorless oil in 90% yield after purification. .sup.1H NMR (300 MHz, CDCl.sub.3) 7.10 (s, 2H, Ar.sup.2-2,6-II), 6.87 (t, J=5.1 Hz, 1H, Ar.sup.1OCH.sub.2CH.sub.2NH), 6.80 (t, 1H, J=2.0 Hz, Ar-2-H), 6.76 (q, 2H, J=2.0 Hz, Ar.sup.1-4,6-H), 4.22-4.15 (m, 6H, Ar.sup.2OCH.sub.2CH.sub.2O), 4.12 (t, 2H, J=5.1 Hz, Ar.sup.1OCH.sub.2CH.sub.2NH), 4.05-3.95 (m, 8H, J=7.0 Hz, PO(OCH.sub.2CH.sub.3).sub.2), 3.85-3.75 (m, 8H, Ar.sup.1OCH.sub.2CH.sub.2NH and OCH.sub.2CH.sub.2O), 3.70-3.50 (m, 54H, OCH.sub.2CH.sub.2O), 3.33 (s, 6H, OCH.sub.2CH.sub.2OCH.sub.3), 3.07 (d, J=21.7 Hz, 4H, Ar.sup.1CH.sub.2P), 2.48 (t, 2H, J=6.6 Hz, Ar.sup.2OCH.sub.2CH.sub.2COOC(CH.sub.3).sub.3), 1.42 (s, 9H, Ar.sup.2OCH.sub.2CH.sub.2COOC(CH.sub.3).sub.3), 1.22 (t, J=7.0 Hz, 12H, PO(OCH.sub.2CH.sub.3).sub.2); .sup.13C NMR (75 MHz, CDCl.sub.3) 170.9, 167.1, 157.5, 152.4, 141.6, 133.2 (J=6.0 Hz), 129.4, 124.1, 114.6 (J=5.0 Hz), 107.3, 80.4, 72.2, 71.9, 70.7, 70.6, 70.5, 70.55, 70.4, 70.3, 69.7, 69.1, 66.8, 66.6, 62.1 (J=7.0 Hz), 58.9, 39.6, 36.1, 33.8 (J=137.8 Hz), 27.9, 16.4 (J=6.0 Hz); .sup.31P NMR (81 MHz, CDCl.sub.3) 26.06. MALDI: calculated for C.sub.66H.sub.117NaNO.sub.29P.sub.2: 1472.72. obtained: 1472.65.
(95) Compound 13:
(96) To a solution of ethyl phosphonate derivatives 12 (0.2 g, 0.14 mmol) in 5 mL of distillated dichloromethane at 0 C., was added dropwise 0.55 mL of TMSBr (3 mmol, 30 eq.). After stirring overnight at room temperature, the volatiles were evaporated and methanol is added to the crude product and evaporated several times. The phosphonic acid 13 was obtained as an orange oil in 94% yield without further purification. .sup.1H NMR (300 MHz, CD.sub.3OD) 7.28 (s, 2H, Ar.sup.2-2,6-H), 6.92-6.86 (m, 3H, Ar-2,4,6-H), 4.35-4.20 (m, 8H, Ar.sup.2OCH.sub.2CH.sub.2O and Ar.sup.1OCH.sub.2CH.sub.2NH), 3.92-3.82 (m, 8H, Ar.sup.1OCH.sub.2CH.sub.2NH and OCH.sub.2CH.sub.2O), 3.80-3.53 (m, 54H, OCH.sub.2CH.sub.2O), 3.38 (s, 6H, OCH.sub.2CH.sub.2OCH.sub.3), 3.18 (d, J=21.8 Hz, 4H, Ar.sup.1CH.sub.2P), 2.62 (t, 2H, J=6.0 Hz, Ar.sup.2OCH.sub.2CH.sub.2COOH); .sup.13C NMR (75 MHz, CD.sub.3OD) 172.2, 167.1, 158.8, 152.3, 141.0, 134.8 (J=6.0 Hz), 128.8, 123.8, 114.3, 106.4, 72.2, 71.7, 70.4, 70.25, 70.15, 70.1, 70.0, 69.95, 69.4, 68.8, 66.3, 66.1, 60.8, 57.8, 50.8, 39.6, 34.6, 33.6 (J=134.5 Hz); .sup.31P NMR (81 MHz, CD.sub.3OD) 25.19. MALDI: calculated for C.sub.54H.sub.94NO.sub.29P.sub.2: 1282.53. obtained: 1282.46. calculated for C.sub.54H.sub.93NaNO.sub.29P.sub.2: 1304.53. obtained: 1304.45.
(97) ##STR00084##
Compound 25:
(98) Compound 25 was obtained by the same procedure used for 8. Starting from 3,5-dihydroxybenzoic methyl ester (0.7 g, 4.3 mmol), a white foam was synthesized after purification by chromatography over silica gel column (dichloromethane/methanol 98/2 to 95/5) (65% of yield). .sup.1H NMR (300 MHz, CDCl.sub.3) 7.18 (d, J=2.5 Hz, 2H, Ar-2,6-H), 6.65 (t, J=2.4 Hz, 1H, Ar-4-H), 4.98 (m, 2H, OCH.sub.2CH.sub.2NH), 4.04 (t, J=5.0 Hz, 4H, OCH.sub.2CH.sub.2NH), 3.91 (s, 3H, COOCH.sub.3), 3.56 (m, 4H, OCH.sub.2CH.sub.2NH), 1.45 (s, 18H, COOC(CH.sub.3)); .sup.13C NMR (75 MHz, CDCl.sub.3) 166.9, 152.8, 142.4, 134.7, 125.3, 117.9, 109.2, 74.3, 72.3, 71.2, 71.0, 70.9, 70.8, 70.0, 69.2, 59.3, 52.5. MALDI: calculated for C.sub.22H.sub.34N.sub.2O.sub.8: 454.23. obtained: 454.02. calculated for C.sub.29H.sub.48NaO.sub.13: 627.30. obtained: 627.13. calculated for C.sub.29H.sub.48KO.sub.13: 643.27. obtained: 643.09.
(99) Compound 26:
(100) Compound 26 was obtained by the same procedure used for 4. Starting from 25 (0.75 g, 1.7 mmol), a white foam was synthesized, which was used without further purification (86% of yield). .sup.1H NMR (300 MHz, CDCl.sub.3) 7.22 (s, 2H, Ar-2,6-H), 6.62 (s, 1H, Ar-4-H), 5.02 (m, 2H, OCH.sub.2CH.sub.2NH), 4.03 (t, J=4.8 Hz, 4H, OCH.sub.2CH.sub.2NH), 3.52 (m, 4H, OCH.sub.2CH.sub.2NH), 1.48 (s, 18H, COOC(CH.sub.3)); .sup.13C NMR (75 MHz, CDCl.sub.3) 166.9, 152.8, 142.4, 134.7, 125.3, 117.9, 109.2, 74.3, 72.3, 71.2, 71.0, 70.9, 70.8, 70.0, 69.2, 59.3, 52.5. MALDI: calculated for C.sub.21H.sub.32N.sub.2O.sub.8: 440.22. obtained: 440.02; calculated for C.sub.29H.sub.48NaO.sub.13: 30. obtained: 627.13. calculated for C.sub.29H.sub.48KO.sub.13: 643.27. obtained: 643.09.
(101) Compound 27:
(102) To an equimolar solution of carboxylic acid derivative 26 (0.4 g, 0.9 mmol) in dichloromethane anhydre (20 mL) was added under argon coupling reagent BOP (0.5 g, 1.2 mmol, 1.3 eq.). After 5 min, were added the amine derivative 8 (0.4 g, 0.9 mmol) and diisopropylethylamine (0.45 mL, 2.7 mmol, 3 eq.). The reaction mixture was stirred overnight at room temperature. 20 mL of dichloromethane were added and the organic layer was washed with a solution of NaOH 1N (220 mL), HCl 1N (220 mL), brine (220 mL) and water (220 mL), dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The crude product was purified by chromatography over silica gel column (dichloromethane/methanol 98/2 to 95/5) to afford 27 as a colorless oil in 65% of yield. .sup.1H NMR (300 MHz, CDCl.sub.3) 6.95 (d, J=2.4 Hz, 2H, Ar.sup.1-2,6-H), 6.85-6.78 (m, 3H, Ar.sup.2-2,4,6-H), 6.69 (t, J=2.4 Hz, 1H, Ar.sup.1-4H), 6.57 (t, J=2.0 Hz, 1H, Ar.sup.1OCH.sub.2CH.sub.2NH), 5.02 (m, 2H, Ar.sup.2OCH.sub.2CH.sub.2NH), 4.13 (t, J=5.0 Hz, 2H, Ar.sup.1OCH.sub.2CH.sub.2NH), 4.07-3.97 (m, 12H, Ar.sup.2OCH.sub.2CH.sub.2NH and PO(OCH.sub.2CH.sub.3).sub.2), 3.82 (q, J=5.0 Hz, 2H, Ar.sup.1OCH.sub.2CH.sub.2NH), 3.55-3.50 (m, 4H, Ar.sup.2OCH.sub.2CH.sub.2NH), 3.08 (d, J=22.0 Hz, 4H, Ar.sup.1CH.sub.2P), 1.42 (s, 18H, COOC(CH.sub.3)); 1.25 (t, J=7.0 Hz, 12H, PO(OCH.sub.2CH.sub.3).sub.2); .sup.13C NMR (75 MHz, CDCl.sub.3) 167.2, 159.8, 158.5, 155.8, 136.8, 133.1 (J=6.0 Hz), 124.0, 114.8 (J=4.5 Hz), 106.0, 104.7, 67.5, 66.8, 62.3 (J=3.4 Hz), 39.5, 36.8 (J=4.0 Hz), 33.4 (J=138.0 Hz), 16.4 (J=2.7 Hz); .sup.31P NMR (81 MHz, CDCl.sub.3) 26.10. MALDI: calculated for C.sub.39H.sub.63N.sub.3O.sub.14P.sub.2: 859.38. obtained: 859.10. calculated for C.sub.29H.sub.53NaO.sub.14P: 679.31. obtained: 679.24.
(103) Compound 28:
(104) To a solution of 27 (0.4 g, 0.4 mmol) in 15 mL of CH.sub.2Cl.sub.2 at 0 C. was added dropwise 780 L (8.0 mmol, 20.0 eq.) of trifluoroacetic acid. The reaction mixture was stirred overnight at room temperature, then the volatiles were evaporated. The crude product was kept under the form of its difluoroacetate salt and 28 was obtained as a white solid in 98% yield and used without further purification. .sup.1H NMR (300 MHz, CDCl.sub.3) 8.74 (m, 1H, Ar.sup.1OCH.sub.2CH.sub.2NH), 8.55 (m, 4H, Ar.sup.2OCH.sub.2CH.sub.2NH.sub.2), 6.95 (m, 2H, Ar.sup.1-2,6-H), 6.85-6.78 (m, 3H, Ar.sup.2-2,4,6-H), 6.69 (m, 1H, Ar.sup.1-4-H), 4.21 (m, 2H, Ar.sup.1OCH.sub.2CH.sub.2NH), 4.05-3.90 (m, 12H, Ar.sup.2OCH.sub.2CH.sub.2NH and PO(OCH.sub.2CH.sub.3).sub.2), 3.78 (m, 2H, Ar.sup.1OCH.sub.2CH.sub.2NH), 3.58-3.50 (m, 4H, Ar.sup.2OCH.sub.2CH.sub.2NH), 3.08 (m, 4H, Ar.sup.1CH.sub.2P), 1.25 (t, J=7.0 Hz, 12H, PO(OCH.sub.2CH.sub.3).sub.2); .sup.13C NMR (75 MHz, CDCl.sub.3) 166.9, 152.8, 142.4, 134.7, 125.3, 117.9, 109.2, 74.3, 72.3, 71.2, 71.0, 70.9, 70.8, 70.0, 69.2, 59.3, 52.5; .sup.31P NMR (81 MHz, CDCl.sub.3) 26.60. MALDI: calculated for C.sub.29H.sub.47N.sub.3O.sub.14P.sub.2: 659.27. obtained: 659.02. calculated for C.sub.29H.sub.53NaO.sub.14P: 679.31. obtained: 679.24.
(105) Compound 29:
(106) Compound 29 was obtained by the same procedure used for 9. Starting from 28 (0.40 g, 0.45 mmol), a colorless oil was synthesized after purification by chromatography over silica gel column (dichloromethane/methanol 98/2 to 95/5) (70%). .sup.1H NMR (300 MHz, CDCl.sub.3) 7.50 (d, J=7.7 Hz, 2H, Ar.sup.3-2,6-H), 7.35-7.28 (m, 3H, Ar.sup.3-3,4,5-H), 7.11 (s, 4H, Ar.sup.2-2,6-H), 7.05 (t, J=5.0 Hz, 2H, Ar.sup.2OCH.sub.2CH.sub.2NH), 6.95 (d, J=2.3 Hz, 2H, Ar.sup.1-2,6-H), 6.69 (t, J=2.4 Hz, 1H, Ar.sup.1-4-H), 6.82-6.78 (m, 3H, Ar.sup.2-2,4,6-H), 6.64 (t, J=1.9 Hz, 1H, Ar.sup.1OCH.sub.2CH.sub.2NH), 5.07 (s, 4H, Ar.sup.3OCH.sub.2), 4.20-4.17 (m, 14H, Ar.sup.2OCH.sub.2CH.sub.2O, Ar.sup.1OCH.sub.2CH.sub.2NH and Ar.sup.2OCH.sub.2CH.sub.2NH), 4.08-3.96 (m, 8H, PO(OCH.sub.2CH.sub.3).sub.2), 3.83-3.78 (m, 14H, Ar.sup.1OCH.sub.2CH.sub.2NH, Ar.sup.2OCH.sub.2CH.sub.2NH and OCH.sub.2CH.sub.2O), 3.65-3.55 (m, 34H, OCH.sub.2CH.sub.2O), 3.55-3.48 (m, 8H, OCH.sub.2CH.sub.2O), 3.34 (s, 12H, OCH.sub.2CH.sub.2OCH.sub.3), 3.08 (d, J=22.0 Hz, 4H, Ar.sup.1CH.sub.2P), 1.23 (t, J=7.0 Hz, 12H, PO(OCH.sub.2CH.sub.3).sub.2); .sup.13C NMR (75 MHz, CDCl.sub.3) 167.2, 159.8, 152.5, 141.0, 137.8, 136.5, 133.1 (J=6.0 Hz), 129.5, 128.2, 128.0, 127.8, 124.0, 114.8 (J=4.8 Hz), 107.8, 107.0, 106.0, 74.9, 72.0, 70.8, 70.7, 70.6, 69.8, 69.1, 66.8, 62.1 (J=3.4 Hz), 58.9, 39.5, 35.2, 33.3 (J=138.0 Hz), 16.4 (J=2.7 Hz); .sup.31P NMR (81 MHz, CDCl.sub.3) 26.60. MALDI: calculated for C.sub.93H.sub.169N.sub.3O.sub.34P.sub.2: 1903.87. obtained: 1903.52; calculated for C.sub.29H.sub.53NaO.sub.14P: 679.31. obtained: 679.24.
(107) Compound 30:
(108) Compound 30 was obtained by the same procedure used for 10. Starting from 29 (0.65 g, 0.34 mmol), a colorless oil was obtained after purification by chromatography over silica gel column (dichloromethane/methanol 98/2 to 95/5) (76%). .sup.1H NMR (300 MHz, CDCl.sub.3) 7.62 (br s, 2H, Ar.sup.2OH), 7.20 (s, 4H, Ar.sup.2-2,6-H), 7.11 (t, J=5.5 Hz, 2H, Ar.sup.2OCH.sub.2CH.sub.2NH), 6.96 (d, J=1.7 Hz, 2H, Ar.sup.1-2,6-H), 6.80-6.74 (m, 3H, Ar.sup.2-2,4,6-H), 6.55 (t, J=1.9 Hz, 1H, Ar.sup.1-4-H), 6.44 (t, J=1.9 Hz, 1H, Ar.sup.1OCH.sub.2CH.sub.2NH), 4.17 (t, J=4.8 Hz, 8H, Ar.sup.2OCH.sub.2), 4.11 (t, J=4.6 Hz, 6H, Ar.sup.1OCH.sub.2CH.sub.2NH and Ar.sup.2OCH.sub.2CH.sub.2NH), 4.05-3.93 (m, 8H, PO(OCH.sub.2CH.sub.3).sub.2), 3.83-3.75 (m, 14H, Ar.sup.1OCH.sub.2CH.sub.2NH, Ar.sup.2OCH.sub.2CH.sub.2NH and OCH.sub.2CH.sub.2O), 3.70-3.60 (m, 34H, OCH.sub.2CH.sub.2O), 3.52-3.48 (m, 8H, OCH.sub.2CH.sub.2O), 3.34 (s, 12H, OCH.sub.2CH.sub.2OCH.sub.3), 3.06 (d, J=22.0 Hz, 4H, Ar.sup.1CH.sub.2P), 1.21 (t, J=7.0 Hz, 12H, PO(OCH.sub.2CH.sub.3).sub.2); .sup.13C NMR (75 MHz, CDCl.sub.3) 167.2, 159.8, 152.5, 140.8, 136.5, 133.1 (J=6.0 Hz), 129.5, 124.5, 114.8 (J=4.8 Hz), 108.7, 106.0, 104.8, 71.9, 70.8, 70.7, 70.6, 69.8, 69.1, 66.8, 62.1 (J=7.0 Hz), 58.9, 39.5, 35.2, 33.4 (J=137.5 Hz), 16.3 (J=2.7 Hz); .sup.31P NMR (81 MHz, CDCl.sub.3) 26.60. MALDI: calculated for C.sub.79H.sub.127N.sub.3O.sub.34P.sub.2: 1723.78. obtained: 1723.46. calculated for C.sub.29H.sub.53NaO.sub.14P: 679.31. obtained: 679.24.
Example 17: Grafting of Dendrons at the Surface of Nanoparticles Whatever their Form or Composition
(109) The as synthesized nanoparticles are coated with surfactants and stable in suspension in an organic solvent. Then the molecules may be grafted at the surface of nanoparticles either by a ligand exchange and phase transfer process in water or by direct grafting in the organic solvent and then transfer of the so functionalized NPs in water.
(110) For the dendrons which bear functional bioactive groups such as vectors or fluorescent molecules or peptides . . . the dendron bearing these functions may be directly grafted at the surface of nanoparticles or a mall dendron bearing a functional group allowing a further coupling of the bioactive molecules is grafted at the surface of NPs and then a coupling reaction is induced to couple the bioactive molecules at the surface of NPs.
Example 18: Functionalization Process by Ligand Exchange in Organic Solvents
(111) 30 ml of a NP@ligands suspension in chloroforme/THF (1 mg/ml) were put into contact with an excess of dendron molecules bearing different anchoring function (phosphonate or carboxylate). Organic suspensions were magnetically stirred for one night. A ligand exchange occurs leading to suspensions of dendronized NPs. The amount of molecules which is added is the amount necessary to coat all the surface of NPs+40% in weight.
Example 19: Functionalization Process by Ligand Exchange and Phase Transfer
(112) 10 ml of a NP@ligands suspension in hexane (1 mg/ml) were put in contact with a suspension (13 mg of molecules, 5 ml water and 2 ml methanol) of the dendron, at a pH which depend on the functional group beared by the dendron (pH=5 for dendron bearing bearing no functional group and pH 3.5 for dendrons bearing a carboxylic acid function at its periphery with a pKa value around 4). Both immiscible suspensions were magnetically stirred for one night. A ligand exchange and a phase transfer process led to an aqueous suspension of dendronized NPs.
(113) The grafted NPs were then separated from the ungrafted dendrons by ultrafiltration. This technique, well adapted to purify all functionalized aqueous NPs suspensions, involves regenerated cellulose membranes with a nominal molecular weight limit (NMWL) of 30 kDa. After at least 4 purification steps by ultrafiltration, the pH of the NPs suspension was 6.
(114) To coat the nanoparticles with different types of dendrons, a mixture of dendrons is added instead of one type of dendron: tests have to be conducted by varying the proportion of each dendron to reach the good grafting rate of each dendron.
Example 20: Functionalization Process by Ligand Exchange in an Organic Solvent
(115) 40 mg of NPs were dispersed in THF at a concentration of 1 mg/mL were mixed to 10 mg of the dendrons and magnetically stirred for 24 h. After this period, the suspensions were purified by ultrafiltration. The THF suspension was introduce in the apparatus and purification occurred by pressurizing the solution flow. The solvent and un-grafted molecules (released oleic acid and dendron molecule excess) went through the membrane while grafted nanoparticles did not. The particles were then redispersed in 20 mL. This operation was repeated 3 times. After purification, 10 mg of dendrons were added to the purified suspension, and the suspension was magnetically stirred for another 24h. The nanoparticles were then precipitated by adding hexane. After centrifugation the grafted particles were easily redispersed in distilled water.
Example 21: Characterization of Dendronized NPs
(116) The grafting of dendrons at the surface of nanoparticles after the purification step is confirmed by infra-red (IR) spectroscopy and photoemission spectroscopy (XPS). By IR spectroscopy, the characteristic IR bands of the dendron appear in the IR spectra of dendronized nanoparticles and the PO and POH bands have been modified due to the formation of a bond between phosphonate groups and the iron oxide surface. By XPS, a shift of the P 2p bands is observed after the grafting the dendron through the phosphonate group at the surface of NPs. The observed significant shift is consistent with an environment of oxygen atoms in a more shielded environment than for the single dendron, the oxygen atoms being linked not only to phosphorus but also to the NPs iron atoms. The environment of the phosphorus atoms is less electronegative, the energy required to remove an electron from 2p core levels of phosphorus atoms is thus less significant, which decreases the binding energy. The significant chemical shift shows the formation of a strong binding between phosphonate and iron oxide and the formation of at least a bi- or tri-nuclear-type complex as suggested from IR results.
(117) The grafting rate was determined by elemental analysis of iron and phosphorus in each sample, and by considering the surface covered by one dendritic molecule (the value was deduced from molecular modelling experiments), one may determined the amount of molecule/nm.sup.2 or the number of molecule/nanoparticle or the number of molecule/g of nanoparticles.
Example 22: In Vitro Evaluation
(118) I. Hyperthermia Measurements
(119) Preliminary measurements have been done on low concentrated samples and high values of specific absorption rate (SAR) or specific loss power (SLP) values were obtained with core-shell NPs. Oxidized nanocubes display low SAR values by comparison with the results of Pellegrino et al (ACS Nano 2012, 6(4), 3080-3091) but this may be attributed to our synthesis process which has led to nanocubes with a lot of defects such as APB and dislocations. At larger concentration, the SAR values strongly decreases indicating thus an effect of the concentration on heating properties. Such an effect of the concentration has already been reported and in particular with nanocubes. Indeed the heat released by magnetic NPs do not depend only on their properties, such as shape, composition and size but also on the interaction between individual NPs. The latter is an important issue intimately related to the efficiency of MH agents that has not been properly addressed in the past years. For NPs with large size (in particular near the superparamagnetic/blocked single domain size threshold), magnetic interactions are strong and NPs tend to aggregate or align in chains during magnetic field application. The role that dipolar interactions may have in SAR is not completely understood at present, and recent experimental studies have reported either an increase or decrease of SAR with interactions (Scientific reports 2013, 3, 1652 and references herein). Overall, results suggest a widely negative influence of dipole-dipole interactions on the heating power of nanoparticles. Some measurements have been performed as a function of the concentration (
(120) However quite large heating values are obtained in the range of those reported for the best spherical single-core maghemite NPs and magnetite nanocubes. Very recent evaluations of these interaction effects pointed out that chains of magnetic NPs are ideal for obtaining high heating properties. Indeed it has been demonstrated with cubic shaped NPS that the different geometrical arrangement of nanoparticles in suspensions may play some role in explaining the increase of SAR for nanocubes compared to spherical particles. Indeed chains of nanocubes formed due to the existence of strongly anisotropic dipolar forces mediating nanoparticle attachment. In the case of the present invention, the formation of chains with core-shell nanocubes was observed (
(121) II. In Vitro MRI
(122) Relaxivity measurements were performed on all dendronized NPs at 0.47 (20 MHz), 1.41 (60 MHz) and 7 T (300 MHz). The longitudinal and transverse relaxivities are given in Table 1 and compared to the commercial product Endorem and to 10 nm spherical Fe.sub.3O.sub.4 nanoparticles (NS10), the synthesis and functionalization of which have already been described (Dalton Transaction 2013, 42, 2146-2157) and which may be considered as a reference. Here the question is to investigate if the spinel iron oxide may act as a T2 contrast agent even under the form of a shell in core-shell FeO@Fe.sub.3O.sub.4 NPs and what is the impact of this structure on the relaxivity values. To be used as T2 contrast agent, the NPs have to display both a high transverse relaxivity r2 and a high ratio r2/r1.
(123) All core-shell structures NS19, NC16 and NO24 exhibit much higher r.sub.2 and r.sub.2/r.sub.1 values than NS10 at 20 MHz and 60 MHz. The same tendency is also observed at 300 MHz excepted for NO24 that seems to behave differently from the other core-shell NPs. Here, in the case of core-shell structures, only the magnetite shell contributes to the magnetization and to the r.sub.2 values. Thus the relaxivity values have also been calculated by considering only the iron amount coming from the magnetite phase. However one may notice that the ratio r.sub.2/r.sub.1 does not depend on this parameter and thus this ratio confirmed the aforementioned results. By contrast, oxNCl6 present the highest r.sub.2 values: 201, 222 and 509 s.sup.i.Math.mMol.sup.1 at 20, 60 and 300 MHz respectively. These values are much higher than those recorded for the different core-shell structures and also for the 10 nm spherical NPs.
(124) TABLE-US-00001 TABLE 1 Relaxivities r1 and r2 (s.sup.1 .Math. mMol.sup.1) calculated from the total amount of iron in each sample Relaxivities at 20 MKz Relaxivities at 60 MKz TEM DLS r1 r2 r1 r2 (mm) (mm) s.sup.1 .Math. mM.sup.1 s.sup.1 .Math. mM.sup.1 r2/r1 s.sup.1 .Math. mM.sup.1 s.sup.1 .Math. mM.sup.1 r2/r1 NS19 19 42 20.6 163.6 8.0 7.2 201.9 27.9 NC16 16 74 13.2 113.3 8.6 5.1 116.1 22.9 NO24 21 55 7.4 65.8 8.8 2.8 80.3 28.5 oxNC16 16 42 24.0 200.7 8.4 8.1 221.5 27.4 NS10 10 15 13 78 6 Endorem ND 120-130 10 141 13
(125) The mean diameter of the unfunctionalized nanoparticles is determined by measuring the diameter of at least 300 nanoparticles on Transmission Electron Microscopy (TEM) images. The size of the functionalized nanoparticles in suspension is determined by dynamic light scattering (DLS), a method for granulometric measurements.
(126) The contrast enhancement properties of all NPs were also evaluated in vitro by MRI at 7 T and ghost images (
(127) Core shell nanocubes and spherical NPs display high T2w EHC even at low iron concentration. The smaller values with NO24 may be related to their lower amount in spinel iron oxide by comparison with former NPs. The higher negative T2w EHC values obtained for oxidized nanocubes oxNC16 confirmed their high contrast power even at high magnetic field (7 T) and even at very low iron concentrations. MR images (
(128) For all NPs a hyposignal in T1w images is observed (
Example 23: In Vivo Evaluation
(129) Core-Shell Spherical NS19
(130) Core-shell spherical NS19 (example 15) diluted in human injectable water solution were injected by intra-venous route via a catheter at a concentration of 1 Mol/kg, which is a low concentration considering usual injected concentrations. No adverse effect in rats was observed following the intravenous injection, even 3 months after injection. The MRI signal in different organs was followed as a function of time and is presented in
(131) The signal in liver evolved similarly to that of blood with time suggesting that the liver signal is mainly due to blood circulation in this organ and that there is no captation by the RES. The signal is in the range [15; 5] in the kidney and bladder and the decrease of the bladder signal with time suggests an urinary elimination.
(132) The T1w signal depicted in
(133) These in vivo results confirm the in vitro one and allow us to conclude that core-shell structure with a wustite core and a magnetite shell can be used as contrast agent for MRI even at low iron concentration.
(134) Oxidized Nanocubes oxNC16
(135) In vivo studies were also conducted with dendronized oxidized nanocubes oxNC16 described in example 2, which were shown to display very interesting in vitro contrast properties. The T2w signals as function of time after IV in different organs are given in
(136) The T1w signals in organs are displayed in
(137) The enhancement of signal appears more dependent on the aggregation state of oxidized nanocubes by comparison with core-shell spherical NPs.
(138) To try to evidence an effect of the aggregation state of NPs on the observed contrast, in vivo MRI studies have been performed by varying the injected concentration.
(139) Influence of the Iron Oxide Concentration
(140) T1w and T2w EHC signals have been followed in different organs as function of the time delay after IV injection and as function of the injected concentration. Only the T1w and T2w Enhancement signal contrast in the blood (aorta) are presented on
(141) For iron concentration smaller than 2 mM/kg, a positive contrast is observed as well as a negative contrast. That suggests that for this range of concentration either T1 or T2 MRI can be performed. Such behavior for low concentration may be related to the ability of nanocubes to align in chains at low concentrations.
(142) For higher concentration, no more positive contrast is observed and only a negative contrast is noticed.
(143) Whatever the concentrations: after 1 hour, the T2w signal slightly increased and reached zero, proving the elimination of the NPs from the blood after 1 hour.
(144) Thus the different contrast evolution in the blood according to the iron oxide concentration may be related to the formation of aggregates with different sizes depending on the NPs concentration. At concentration higher than 2 mM/kg, large aggregates are formed leading to high negative and low positive contrasts. For concentration lower than 2 mM/kg, smaller aggregates should be formed leading to both high positive and negative contrast.
(145) More precisely, probably the aggregation of nanocubes in blood was a dynamic equilibrium providing aggregate sizing roughly the maximum size allowing the maximal enhancing of the longitudinal relaxivity.
(146) Noteworthy is that 1 mol of iron/kg corresponding to the 1 eqIM injected in a 298 g weighting rat is 50 time less than the usual dose used for preclinical MRI studies. A very interesting positive T1w contrast was provided in the main organs (
Example 24: Coupling with Targeting Compound ICF01102
(147) Coupling was performed with dendronised nanoparticles of the present invention, said dendrons being terminated by carboxylic acid groups.
(148) Coupling Procedure with ICF01102
(149) ICF01102 has the following formula:
(150) ##STR00085##
(151) 20 mg of EDCl were added to a suspension of dendronised nanoparticles of the present invention in water. The resulting mixture was then stirred for 30 minutes. 8 mg of ICF01102 were then added to said mixture, which was stirred for 1 hour.
(152) Colloidal stability of the dendronised nanoparticles of the present invention bearing ICF01102 (NP@dendrons+ICF) was checked by DLS.
(153) The presence of the ICF01102 does not modify the size distribution, which stays monomodal. The mean hydrodynamic diameter after coupling with ICF01102 is close to the diameter of dendronised nanoparticles of the present invention before said coupling (NP@dendrons) (
(154) Zeta potential was measured at pH 7.4 before and after coupling with ICF01102.
(155) Zeta potential after coupling increases slightly, indicating that some of the carboxylic acids groups were coupled with ICF01102 (
(156) Melanine targeting with NP@dendrons+ICF nanoparticles was followed by fluorescence.
(157) Thus, a fluorophore (Dye 647) was further coupled after coupling with ICF01102 by a method known by hose skilled in the art.
(158) This fluorophore is necessary to follow the course of the NP in optical imaging.
(159) The presence of ICF01102 and the fluorophore on the NPs surface was confirmed by UV-visible spectroscopy (
(160) Targeting of Melanine Granules
(161) A melanoma was induced by a subcutaneous injection at the side of a mouse of B16F0 cells (300,000 cells). The tumor is measurable from 12 days after injection of the tumor cells (
(162) The biodistribution of the NPs was followed at different times by optical imaging. Two hours after injection of the NPs, a fluorescence signal was observed in the urine and the gastrointestinal tract. At 4 hours, the urinary excretion was complete but a signal was still observed in the digestive tract. 24 hours after injection, all NPs have been eliminated. This once again confirms the proper elimination of the dendronized NPs, even with the targeting ligand melanin on the surface of said NPs. Rapid urinary excretion (2 hours) and then a slower hepatobiliary elimination was observed (
(163) The melanoma tumors can not be observed by optical imaging as they appear black because of their high content of melanin. Therefore, they were observed by confocal microscopy (VivaScope 1500, Caliber Inc, Rochester, N.Y., USA, distributed in France by Mavig, Munich).
(164) To show targeting of dendronized NPs in vivo, NPs coupled to ICF ligand and fluorophore Dye 647 (NP@dendrons+ICF+Dye647, emitting in the near IR) and dendronized NPs that are not bearing the ICF ligand but coupled to a fluorophore Dye 495 ((NP@dendrons+Dye495, green emitting) were injected simultaneously intravenously to mice wherein have been grafted malignant melanoma tumor cells. After injection, tumors were removed and imaged ex vivo by confocal microscopy in reflectance mode and fluorescence at 488 nm and 658 nm Melanin in the form of granules in the cytoplasm of the tumor cells has significant autofluorescence. Indeed, these granules correspond to the white dots on the reflectance image shown in
(165) While by excitation by the blue, no fluorescence corresponding to non targeting NPs (green fluorescence) is observed (
(166) The in vivo targeting is effective, important, since many NPs were internalized by tumor cells. Targeting is also specific as there is no uptake of non targeting NPs i.e. not functionalized by the ICF ligand.
(167) The presence of NPs in tumors has also been proven by imaging by TEM tumors after having calcined them (
Example 25: Synthesis of Nanoplatelets (Also Called Nanoplates)
(168) Iron Stearate Complex Synthesis.
(169) The iron stearate was prepared by ligands exchange between iron chloride and sodium stearate in water. At first, 40 mmol of FeCl.sub.3.6H.sub.2O was dissolved in distilled H.sub.2O and mixed with 80 mmol of sodium stearate under vigorous stirring. The mixture was heated, under stirring, at 70 C. for 4 h. The stearate complex was, then, washed several times with warm distilled water (50 C.) to remove the chloride traces and the formed NaCl and then conserved at 4 C.
(170) Synthesis of Nanoplates:
(171) a mixture of 2.08 g (2.32 mmol) of the synthesized stearate, 0.2 mL (0.65 mmol) of oleic acid and 0.705 g (2.32 mmol) of sodium oleate used as surfactants was added to 20 mL of octadecene (90%, Alfa Aesar, bp 318 C.). The mixture was, first, heated at 120 C. in the absence of a reflux condenser for 30 min and then to its boiling temperature (318 C.) with a heating rate of 5 C./min and refluxed for 60 min at this temperature under air. After cooling to the room temperature, the NPs were precipitated by the addition of an excess of acetone and washed 3 times by a mixture of hexane/acetone (1/3) followed by centrifugation (14000 rpm, 10 mn). Finally, the as-obtained NPs were easily suspended in organic solvents.