WO1995021870A1 - Method for the preparation of branched cyclomaltooligosaccharides, in particular, branched cyclodextrines - Google Patents

Method for the preparation of branched cyclomaltooligosaccharides, in particular, branched cyclodextrines Download PDF

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WO1995021870A1
WO1995021870A1 PCT/FR1995/000162 FR9500162W WO9521870A1 WO 1995021870 A1 WO1995021870 A1 WO 1995021870A1 FR 9500162 W FR9500162 W FR 9500162W WO 9521870 A1 WO9521870 A1 WO 9521870A1
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reducing
cyclomaltooligosaccharide
hydrogen fluoride
branched
anhydrous hydrogen
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PCT/FR1995/000162
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French (fr)
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Jacques Defaye
José Manuel GARCIA FERNANDEZ
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Commissariat A L'energie Atomique
Centre National De La Recherche Scientifique (Cnrs)
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Publication of WO1995021870A1 publication Critical patent/WO1995021870A1/en

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6949—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
    • A61K47/6951—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes using cyclodextrin
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
    • C08B37/0012—Cyclodextrin [CD], e.g. cycle with 6 units (alpha), with 7 units (beta) and with 8 units (gamma), large-ring cyclodextrin or cycloamylose with 9 units or more; Derivatives thereof

Definitions

  • the present invention relates to a process for the preparation of branched cyclo altooligossaccharides, in particular branched cyclodextrins.
  • Cyclomaltooligosaccharides such as cyclodextrins
  • cyclodextrins are cyclic oligosaccharides comprising, in the case of cyclodextrins ⁇ , ⁇ and ⁇ , 6, 7 or 8 D-glucopyranose units linked to ⁇ - (l- »4).
  • These oligosaccharide macrocycles have the property of including in their cavities various molecules, of size adapted to that of the host structure.
  • the generally apolar nature of these associations leads to preferentially including structures of the hydrophobic type, allowing in particular the solubilization in water of compounds poorly or not soluble in these media, thanks to the solubility in water of the cyclodextrins.
  • branched cyclodextrins comprising one or more substituents ⁇ -D-glucopyranosyl, ⁇ -maltosyl and ⁇ -maltotriosyl on the primary hydroxyl positions of the cyclodextrin.
  • branched cyclodextrins have a high solubility in water ranging from 970 g / 1 (750 mmol / 1) for 6-O- ⁇ -D-glucopyranosyl-cyclomaltoheptaose, to 1700 g / 1 (1165 mmol / 1) for 6-O-maltosyl-cyclomaltoheptaose.
  • These branched cyclodextrins therefore have interesting properties, but unfortunately their preparation by biotechnological routes has many drawbacks.
  • EP-A-0-036366 also discloses a process for the chemical synthesis of branched derivatives of cyclodextrins, 6-S-glycosyl-6-thiocyclodextrins starting from a
  • 1-thioglucose and ⁇ -, ⁇ - and ⁇ -cyclodextrins suitably activated, which makes it possible to obtain these branched derivatives in one step with good yields.
  • the present invention specifically relates to a process for the preparation of other branched derivatives of cyclomaltooligosaccharides, which allows these derivatives to be easily obtained by chemical synthesis.
  • the process for the preparation of branched cyclomaltooligosaccharides is characterized in that a cyclomaltooligosaccharide is reacted with at least one reducing compound chosen from monosaccharides, reducing oligosaccharides, reducing polysaccharides and their derivatives, in the presence of anhydrous hydrogen fluoride, operating at least at the start of the reaction under conditions such as fluoride anhydrous hydrogen is liquid, and using 0.1 to 1 ml of anhydrous hydrogen fluoride per gram of the mixture of cyclomaltooligosaccharide and reducing compound (s), and in that the products are then separated of the reaction of hydrogen fluoride.
  • anhydrous hydrogen fluoride operating at least at the start of the reaction under conditions such as fluoride anhydrous hydrogen is liquid
  • cyclodextrin as alcohol to be added to the cation formed by the action of a protonating agent on the reducing compound chosen from monosaccharides, oligosaccharides reducing agents and reducing polysaccharides.
  • This reaction can take place on different hydroxyls, preferably in the primary hydroxyl position of the cyclomaltooligosaccharide which, as we know, is the most reactive. Also, at the end of the reaction, it is possible to obtain several branched cyclomaltooligosaccharides having different substitution rates for C-6.
  • the process of the invention it is possible to intimately mix, preferably by kneading, the cyclomaltooligosaccharide considered and the monosaccharide, the reducing oligosaccharide or the reducing polysaccharide which it is desired to graft onto the cyclodextrin, in a suitable container. , for example Teflon, polyethylene or steel. It is then possible to add to the mixture, at a temperature such that the anhydrous hydrogen fluoride is liquid, for example by cooling, in an acetone-dry ice bath, an amount of hydrogen fluoride sufficient to obtain a homogeneous paste, then then allow the mixture to return to room temperature or slightly below while maintaining good agitation.
  • a suitable container for example Teflon, polyethylene or steel.
  • reaction mixture is again cooled, for example in a dry ice acetone bath, and the reaction is stopped by addition of ether.
  • the anhydrous hydrogen fluoride and the ether are then separated by decantation.
  • the insoluble part which corresponds to the reaction products can be washed several times with ether, then dried and used as it is or separated into its various constituents by conventional methods.
  • the products obtained can also be purified before separating them, for example to eliminate the presence of traces of residual fluorine, hydrogen fluoride or glycosyl fluoride. This purification can be carried out by dissolving the reaction products in water followed by the addition of calcium carbonate to the solution, to neutralize the residual acidity and cause hydrolysis of any traces of glycosyl fluoride.
  • anhydrous hydrogen fluoride alone or dilute it in a nonaqueous solvent.
  • the amounts of cyclomaltooligosaccharides and of reducing compounds used are chosen according to the result which it is desired to obtain.
  • the stoichiometry corresponding to the number of hydroxyls due to cyclomaltooligossacharide capable of being substituted by the reducing compound is not exceeded.
  • the molar ratio of the reducing compound chosen from monosaccharides, oligosaccharides and polysaccharides to cyclomaltooligosaccharide is
  • the cyclomaltooligosaccharides used in the process of the invention can in particular be the cyclodextrins ⁇ , ⁇ and ⁇ .
  • the reducing compound used can be a monosaccharide such as glucose, galactose, mannose, a disaccharide such as maltose, lactose, as well as oligosaccharides and small reducing polysaccharides.
  • the quantity of hydrogen fluoride added must be sufficient for a homogeneous paste to be obtained in order to obtain a substitution rate for the regular cyclomaltooligosaccharide, but it must not be too large because in the presence of a high proportion of hydrogen fluoride relative to the cyclomaltooligosaccharide and reducing compound (s), there is a significant hydrolysis of the cyclomaltooligosaccharide.
  • an amount of anhydrous hydrogen fluoride ranging from 0.1 to 1 ml per gram of mixture of cyclomaltooligosaccharide and reducing compound (s) is used.
  • the best results are obtained in particular when a ratio (cyclomaltooligosaccharides + reducing compound (s) / anhydrous hydrogen fluoride in P / V of about 2) is used.
  • Good results are also obtained when using 0 , 4 ml of anhydrous hydrogen fluoride per g of mixture
  • the yield of branched cyclomaltooligosaccharide drops considerably, due to the lack of solubilization of the reactants and of protonation of the reducing compound to be grafted.
  • the chain length of the substituents corresponding to the reducing compounds used also plays an important role in the solubility of the branched products obtained. Indeed, when replaces D-glucose with maltose and equimolecular proportions of cyclomaltoheptaose and maltose are used, the solubility in water of the product mixture obtained reaches 1,200 g / 1 (803 mmol / 1), whereas it is 91 g / 1 in the case of D-glucose.
  • the products obtained by the process of the invention can therefore have many applications because of their improved solubility in water.
  • they can be used to dissolve active substances in water, in particular pharmaceutical substances such as anti-tumor agents, in particular those of the taxol family, such as the taxotere.
  • the properties of the substituents grafted onto the cyclomaltooligosaccharide can also be used for other applications, for example its possibility of recognition by its environment.
  • oligosaccharide recognition signals can be grafted onto a cyclodextrin allowing the transport and vectorization of products for therapeutic use.
  • Example 1 To a mixture, carefully ground, of cyclomaltoheptaose (3.15 g, 2.8 mmol) and D-glucose (2.8 mmol) in a polyethylene container placed in an acetone-dry ice bath, anhydrous hydrogen fluoride (1.8 ml) is added. The pasty mixture is homogenized using a steel spatula until a syrup is obtained. The container is then closed and stored at room temperature while maintaining magnetic stirring for 1 hour. After this time, the container is again cooled in liquid nitrogen and added with ether. The resulting white precipitate is separated from the supernatant by decantation, washed again with ether (3x50 ml), filtered and dried, which leads to a white pulverulent substance (3.6 g).
  • the mixture obtained is analyzed by mass spectrometry by the ionization technique using the bombardment of accelerated atoms in a glycerol / thioglycerol matrix with sodium iodide as cationization agent.
  • the cationized or protonated molecular ions of the glycosylcyclodextrin constituents of the mixture obtained represent the majority signals if not exclusive of this spectrum, which allows an excellent characterization of the mass distribution of the oligosaccharide constituents of the crude mixture (as described in (article by C. Bosso, J. Defaye, A. Heyraud and J. Ulrich.
  • Example 2 The same procedure is followed as in Example 1, starting from a mixture of cyclomaltoheptaose (3.15 g, 2.8 mmol) and D-glucose (1.5 g, 8.4 mmol) which is supplemented with anhydrous hydrogen fluoride (2.3 ml).
  • the mass distribution of the mixture of branched cyclodextrins is directly proportional to the amount of glucose reacted.
  • Example 3 The procedure of Example 1 is applied to a mixture of cyclomaltoheptaose (3.15 g, 2.8 mmol) and maltose (IH2O, 1.0 g, 2.8 mmol), which is added with anhydrous hydrogen fluoride (1.8 ml).
  • Example 2 After the series of operations in Example 1, 4.0 g of a white pulverulent substance are obtained which shows in mass spectrometry (FAB + ; glycerol-thioglycerol; INa), the following series of signals at m / z 1157 (100, [CD + Na] + ) 1481 (40, [CDMalt + Na] + ), 1805 (20, [CDMalt 2 + Na] + ), 2129 (10, [CDMalt 3 + Na] + ), 2453 (5, [CDMalt 4 + Na + ), 2777 (2, [CDMalt 5 + Na] + ), 3101 (1, [CDMalt 6 + Na]) + ).
  • FAB + mass spectrometry
  • Example 4 The procedure of Example 1 is applied to a mixture of cyclomaltohexaose (2.7 g, 2.8 mmol) and maltose (1H 2 0, 1.0 g, 2.8 mmol) to which we add anhydrous hydrogen fluoride (1.8 ml).
  • Example 1 The procedure of Example 1 is applied to a mixture of cyclomaltooctaose (3.6 g, 2.8 mmol) and maltose (1 H 2 0, 1.0 g, 2.8 mmol), to which the anhydrous hydrogen fluoride (2.3 ml).
  • Example 2 After the series of operations in Example 1, 4.4 g of a white pulverulent substance are obtained which shows in mass spectrometry (FAB + , glycerol-thioglycerol, ICs), the following series of signals at m / z 1429 (100, [CD + CSJ +), 1753 (40, [CDMalt + Cs] + ), 2077 (30, [CDMalt + Cs] + ), 2401 (15, ⁇ CDMalt 3 + Cs ⁇ + ), 2725 ( 10, ⁇ CDMalt + Cs ⁇ + ), 3049 (5, CDMalt 5 + Cs ⁇ + ), 3373 (3, [CDMaltg + Cs] + ), 3697 (2, [CDMalt 7 + Cs] + ), 4021 (1 , [CDMalt 8 + Cs] + ).
  • FAB + mass spectrometry
  • Example 2 The procedure of Example 1 is applied to a mixture of cyclomaltoheptaose (3.15 g, 2.8 mmol) and D-mannose (1.5 g, 8.4 mmol) to which hydrogen fluoride is added. anhydrous (2.3 ml).
  • Example 2 After the series of operations of Example 1, 4.4 g of a white pulverulent substance are obtained which shows, in mass spectrometry (FAB + , glycerol-thioglycerol, INa), the following series of signals at __ / z 1267 (100, [CD + Cs] +), 1429 (80, [CDMan + Cs] + ), 1591 (60, [CDMan 2 + Cs] + ), 1753 (50, [CDMan 3 + Cs] + ), 1915 (40, [CDMan + Cs] + ), 2077 (30, [CDMan 5 + Cs] + ), 2239 (25, [CDMan 6 + Cs] + ), 2401 (20, [CDMan 7 + Cs] + ), 2563 (17, [CDMan 8 + Cs] + ), 2725 (15, [CDMan 9 + Cs] + ), 2887 (12, [CDMan 10 + Cs] + ), 3049 (10, [CDMan 1: L + Cs] + ), 3211
  • the solubility of this substance in water is -1100 g / 1.
  • Example 8 The operating protocol of Example 1 is repeated, but with cyclomaltoheptaose alone (3.15 g. 2.8 mmol) which is added with anhydrous hydrogen fluoride (6.3 ml).
  • Example 2 After the series of operations of Example 1, a pulverulent substance (2.7 g) is obtained which, examined by C NMR in deuterium oxide, leads to a complex spectrum which is noted in particular in the anomeric carbon region of the 92.9 and 96.7 ppm signals attributable to Cl of ⁇ -and- ⁇ -Dglucopyranose reducing units respectively (K. Bock and C. Pedersen, Adv. Carbohydr. Chem. Biochem., 41, 27-66, 1983) as well as a broad doublet at -108 ppm (J-222 Hz) indicating the presence of ⁇ -D-glucopyranosyl fluoride (K. Bock and C. Pedersen, Acta Chem. Scand. , Ser.
  • Example 1 The operating protocol of Example 1 is repeated with the pulverulent mixture, and homogenized by grinding, of cyclomaltoheptaose (3.15 g, 2.8 mmol) and of D-glucose (2.8 mmol) which is added with anhydrous hydrogen fluoride (6.3 ml).
  • cyclomaltoheptaose 3.15 g, 2.8 mmol
  • D-glucose 2.8 mmol
  • anhydrous hydrogen fluoride 6.3 ml
  • Example 10 The operating protocol of Example 1 is repeated with cyclomaltoheptaose alone (3.15 g, 2.8 mmol) which is supplemented with anhydrous hydrogen fluoride (2.3 ml). According to the series of operations of Example 1, a pulverulent substance is obtained which has the same characteristics (Pf [ ⁇ ] D , NMR i3 C) as the starting cyclomaltoheptaose.
  • Example 11 The operating protocol of Example 1 is repeated with cyclomaltoheptaose alone (3.15 g, 2.8 mmol) which is supplemented with anhydrous hydrogen fluoride (2.3 ml). According to the series of operations of Example 1, a pulverulent substance is obtained which has the same characteristics (Pf [ ⁇ ] D , NMR i3 C) as the starting cyclomaltoheptaose.
  • Example 11 The operating protocol of Example 1 is repeated with cyclomaltoheptaose alone (3.15 g, 2.8 mmol
  • Example 7 The operating protocol of Example 7 is followed, with the mixture of cyclomaltoheptaose (2.56 g, 2.26 mmol) and 2-acetamido-2-deoxy-D-glucose (1.0 g, 4.52 mmol ) which is supplemented with anhydrous hydrogen fluoride (7 ml). After the series of operations in Example 7, a pulverulent substance (3.45 g) is obtained which shows in mass pectrometry a complex set of signals in which we do not find those described in Example 7 for the 2 -acetamido-2-deoxy-D-glucosylcyclodextrin.
  • the 3 C NMR spectrum is also complex and there are signals at 92.9 and 96.7 ppm for reducing units of ⁇ - and ⁇ -D-glucopyranose, a broad doublet at - 108 ppm for fluoride of ⁇ -D-glucopyranosyl, as well as a signal at 102.4 ppm indicating the presence of 2-acetamido-2-deoxy- ⁇ -D-glucopyranosyl units (J. Defaye, A. Gadelle and C. Pedersen, Carbohydr. Res. 186, 177-188, 1989).
  • Example 12 which follows illustrates the use of branched cyclomaltooligosaccharides obtained in accordance with the invention for the solubilization of active principles.
  • Example 12 Solubilization of Taxotere in the mixture of glucosyl-cyclomaltoheptaoses prepared in Example 1.
  • Taxotere is added, with vigorous stirring, to an aqueous solution (5 ml) containing 65 g / l of glucosylcyclomaltoheptaoses prepared in Example 1, and this until the solution remains clear. It is found that the solubility of Taxotere in water under these conditions, in the presence of the product of Example 1 (50 mmol / 1) is - 1.5 g / 1 while the solubility of Taxotere alone in water n is only 0.004 g / 1.

Abstract

Method for the preparation of branched cyclomaltooligosaccharides, in particular, branched cyclodextrines. The method comprises the steps of reacting a cyclomaltooligosaccharide, for example, a cyclodextrine, with at least one reducing agent selected from monosaccharides, reducing oligosaccharides, reducing polysaccharides and derivatives thereof, in the presence of anhydrous hydrogen fluoride, in order to substitute the hydroxyl groups in the cyclomaltooligosaccharide with the reducing agent, for example, glucose, maltose, mannose and 2-acetoamido-2-deoxy-D-glucose. According to the invention, the solubility in water of the starting cyclooligossacharide is enhanced by this method, while conferring to said substance useful environment-recognizable properties, for conveying therapeutic agents to the required site.

Description

PROCEDE DE PREPARATION DE CYCLOMALTOOLIGOSACCHARIDES RAMIFIES, EN PARTICULIER DE CYCLODEXTRINES RAMIFIEES. PROCESS FOR THE PREPARATION OF BRANCHED CYCLOMALTOOLIGOSACCHARIDES, IN PARTICULAR BRANCHED CYCLODEXTRINS.
La présente invention a pour objet un procédé de préparation de cyclo altooligossaccharides ramifiés, en particulier de cyclodextrines ramifiées.The present invention relates to a process for the preparation of branched cyclo altooligossaccharides, in particular branched cyclodextrins.
Les cyclomaltooligosaccharides tels que les cyclodextrines, sont des oligosaccharides cycliques comportant, dans le cas des cyclodextrines α, β et γ, 6, 7 ou 8 unités D-glucopyranose liées α-(l-»4). Ces macrocycles oligosaccharidiques ont la propriété d'inclure dans leurs cavités des molécules diverses, de taille adaptée à celle de la structure hôte. Le caractère généralement apolaire de ces associations conduit à inclure préférentiellement des structures de type hydrophobe, permettant notamment la solubilisation dans 1'eau de composés peu ou pas solubles dans ces milieux, grâce à la solubilité dans l'eau des cyclodextrines. De ce fait, on a développé récemment l'utilisation des cyclodextrines dans les domaines de l'agro-alimentaire, de l'agrochimie, des cosmétiques et de la pharmacie pour améliorer la solubilité, la stabilité et la biodisponibilité de substances d'intérêt telles que des principes actifs. Toutefois, la solubilité relativement faible dans l'eau des cyclodextrines, et notamment de la plus accessible d'entre elles sur le plan économique, la β-cyclodextrine qui présente une hydrosolubilité de 18,5 g/1 à 25°C, limite l'utilisation des cyclodextrines dans ces différents domaines, d'autant plus que les constantes de stabilité des complexes d'inclusion formés avec les substances d'intérêt sont généralement peu élevées.Cyclomaltooligosaccharides, such as cyclodextrins, are cyclic oligosaccharides comprising, in the case of cyclodextrins α, β and γ, 6, 7 or 8 D-glucopyranose units linked to α- (l- »4). These oligosaccharide macrocycles have the property of including in their cavities various molecules, of size adapted to that of the host structure. The generally apolar nature of these associations leads to preferentially including structures of the hydrophobic type, allowing in particular the solubilization in water of compounds poorly or not soluble in these media, thanks to the solubility in water of the cyclodextrins. As a result, the use of cyclodextrins has recently been developed in the food, agrochemical, cosmetic and pharmaceutical fields to improve the solubility, stability and bioavailability of substances of interest such as only active ingredients. However, the relatively low solubility in water of cyclodextrins, and in particular of the most economically accessible of them, β-cyclodextrin which has a water solubility of 18.5 g / l at 25 ° C, limit l use of cyclodextrins in these different fields, especially since the stability constants of the inclusion complexes formed with the substances of interest are generally low.
En conséquence, de nombreuses recherches ont été entreprises pour modifier les cyclodextrines en vue d'améliorer leur solubilité dans l'eau. Dans ce but, on les a dotées de substituants éther et on a ainsi pu obtenir par synthèse chimique le 2,6-di-O-méthylcyclomaltoheptaose, leAs a result, much research has been undertaken to modify cyclodextrins to improve their solubility in water. In this But, they were endowed with ether substituents and it was thus possible to obtain by chemical synthesis 2,6-di-O-methylcyclomaltoheptaose,
2,3,6-tri-O-méthylcyclomaltoheptaose et les 2- et 3-hydroxypropylcyclomaltooligosaccharides. Parmi ces dérivés hydrophiles des cyclodextrines, les 2-hydroxypropyl cyclodextrines présentent un certain intérêt comme il est décrit par Szente et Strattan dans New Trends in Cyclodextrines and Derivatives, D. Duchêne Ed, Editions de Santé, Paris 1991, pp. 57-96.2,3,6-tri-O-methylcyclomaltoheptaose and 2- and 3-hydroxypropylcyclomaltooligosaccharides. Among these hydrophilic cyclodextrin derivatives, the 2-hydroxypropyl cyclodextrins are of some interest as described by Szente and Strattan in New Trends in Cyclodextrins and Derivatives, D. Duchêne Ed, Editions de Santé, Paris 1991, pp. 57-96.
Pour améliorer la solubilité dans l'eau des cyclodextrines, on a aussi envisagé de réaliser des cyclodextrines ramifiées comportant un ou plusieurs substituants α-D-glucopyranosyle, α-maltosyle et α -maltotriosyle sur les positions hydroxyle primaires de la cyclodextrine. Cependant, la préparation de ces cyclodextrines ramifiées fait intervenir des procédés biotechnologiques, à savoir l'action de cyclomaltodextrines glucanotransférases sur l'amidon ou l'amylopectine, ou encore l'action de la pullulanase ou d'une isoamylase sur un mélange de maltose ou de maltooligossacharides et de la cyclodextrine correspondante, comme il est décrit par H.Hashimoto dans New Trends in Cyclodextrines and Derivatives, D. Duchêne Ed, Editions de Santé, Paris 1991, pp. 99-156. Ces cyclodextrines ramifiées ont une solubilité élevée dans l'eau allant de 970 g/1 (750 mmol/1) pour le 6-O-α -D-glucopyranosyl-cyclomaltoheptaose, à 1700 g/1 (1165 mmol/1) pour le 6-O-maltosyl-cyclomaltoheptaose. Ces cyclodextrines ramifiées ont donc des propriétés intéressantes, mais malheureusement leur préparation par des voies biotechnologiques présente de nombreux inconvénients. En effet, les rendements sont faibles ; le procédé conduit à des mélanges complexes de dérivés acycliques et cycliques incluant du maltose, du maltotriose, du maltotétraose, des α-, des β- et γ -cyclodextrines libres qui accompagnent les dérivés correspondants mono et di-substitués par de l'a -D-glucose ainsi que de l'α-maltose.To improve the water solubility of cyclodextrins, it has also been envisaged to produce branched cyclodextrins comprising one or more substituents α-D-glucopyranosyl, α-maltosyl and α -maltotriosyl on the primary hydroxyl positions of the cyclodextrin. However, the preparation of these branched cyclodextrins involves biotechnological processes, namely the action of cyclomaltodextrins glucanotransferases on starch or amylopectin, or even the action of pullulanase or an isoamylase on a mixture of maltose or maltooligossacharides and the corresponding cyclodextrin, as described by H. Hashimoto in New Trends in Cyclodextrines and Derivatives, D. Duchêne Ed, Editions de Santé, Paris 1991, pp. 99-156. These branched cyclodextrins have a high solubility in water ranging from 970 g / 1 (750 mmol / 1) for 6-O-α -D-glucopyranosyl-cyclomaltoheptaose, to 1700 g / 1 (1165 mmol / 1) for 6-O-maltosyl-cyclomaltoheptaose. These branched cyclodextrins therefore have interesting properties, but unfortunately their preparation by biotechnological routes has many drawbacks. Indeed, yields are low; the process leads to complex mixtures of derivatives acyclic and cyclic including maltose, maltotriose, maltotetraose, α-, β- and γ-free cyclodextrins which accompany the corresponding derivatives mono and di-substituted by a -D-glucose as well as α -maltose.
Des procédés de synthèse chimique ont toutefois été récemment publiés pour la préparation d' α-D-glucopyranosyl-cyclomaltohexaose et d'α -D-glucopyranosyl-cyclomaltoheptaose comme il est décrit par Takeo et al dans J. Carbohydr. Chem. 7 (1988) 293-308 et par Fugedi et al. dans Carboydr. Res., 175 (1988) 173-181. Cependant, ces procédés de synthèse chimiques font intervenir au moins cinq étapes de chimie fine, aboutissent à des rendements globaux de l'ordre de 13 %, et ne sont pas commodément transposables sur un plan préparatif.Chemical synthesis methods have however recently been published for the preparation of α-D-glucopyranosyl-cyclomaltohexaose and of α-D-glucopyranosyl-cyclomaltoheptaose as described by Takeo et al in J. Carbohydr. Chem. 7 (1988) 293-308 and by Fugedi et al. in Carboydr. Res., 175 (1988) 173-181. However, these chemical synthesis methods involve at least five stages of fine chemistry, result in overall yields of the order of 13%, and are not conveniently transposable to a preparatory level.
On connaît également, par le document EP-A-0- 03 366, un procédé de synthèse chimique de dérivés ramifiés de cyclodextrines, les 6-S-glycosyl-6-thiocyclodextrines en partant d'unEP-A-0-036366 also discloses a process for the chemical synthesis of branched derivatives of cyclodextrins, 6-S-glycosyl-6-thiocyclodextrins starting from a
1-thioglucose et des α-, β- et γ-cyclodextrines convenablement activées, qui permet d'obtenir ces dérivés ramifiés en une étape avec de bons rendements.1-thioglucose and α-, β- and γ-cyclodextrins suitably activated, which makes it possible to obtain these branched derivatives in one step with good yields.
La présente invention a précisément pour objet un procédé de préparation d'autres dérivés ramifiés des cyclomaltooligosaccharides, qui permet d'obtenir aisément ces dérivés par synthèse chimique.The present invention specifically relates to a process for the preparation of other branched derivatives of cyclomaltooligosaccharides, which allows these derivatives to be easily obtained by chemical synthesis.
Selon l'invention, le procédé de préparation de cyclomaltooligosaccharides ramifiés, se caractérise en ce que l'on fait réagir un cyclomaltooligosaccharide avec au moins un composé réducteur choisi parmi les monosaccharides, les oligosaccharides réducteurs, les polysaccharides réducteurs et leurs dérivés, en présence de fluorure d'hydrogène anhydre, en opérant au moins au début de la réaction dans des conditions telles que le fluorure d'hydrogène anhydre soit liquide, et en utilisant 0,1 à 1 ml de fluorure d'hydrogène anhydre par gramme du mélange de cyclomaltooligosaccharide et de composé (s) réducteur (s), et en ce que l'on sépare ensuite les produits de la réaction du fluorure d'hydrogène.According to the invention, the process for the preparation of branched cyclomaltooligosaccharides, is characterized in that a cyclomaltooligosaccharide is reacted with at least one reducing compound chosen from monosaccharides, reducing oligosaccharides, reducing polysaccharides and their derivatives, in the presence of anhydrous hydrogen fluoride, operating at least at the start of the reaction under conditions such as fluoride anhydrous hydrogen is liquid, and using 0.1 to 1 ml of anhydrous hydrogen fluoride per gram of the mixture of cyclomaltooligosaccharide and reducing compound (s), and in that the products are then separated of the reaction of hydrogen fluoride.
On savait depuis les travaux de Fischer en 1895, comme il est rapporté à la page 281 de l'ouvrage : "The Carbohydrate, Chemistry and Biochemistry" W. PIGMAN et D. HORTON (Ed. ) Académie Press, 1972, vol. 1A, p. 279-353, que les oses ont la possibilité d'additionner un alcool sur le cation formé en position anomérique par action d'un agent protonant, mais que cette réaction était réversible et conduisait dans le cas de disaccharides à générer les constituants monosaccharidiques par hydrolyse. La possibilité d'addition d'un alcool est mise à profit dans le procédé de 1'invention en utilisant la cyclodextrine comme alcool à additionner sur le cation formé par action d'un agent protonant sur le composé réducteur choisi parmi les monosaccharides, les oligosaccharides réducteurs et les polysaccharides réducteurs.We have known since Fischer's work in 1895, as it is reported on page 281 of the book: "The Carbohydrate, Chemistry and Biochemistry" W. PIGMAN and D. HORTON (Ed.) Académie Press, 1972, vol. 1A, p. 279-353, that the dares have the possibility of adding an alcohol to the cation formed in the anomeric position by the action of a protonating agent, but that this reaction was reversible and led in the case of disaccharides to generate the monosaccharide constituents by hydrolysis . The possibility of adding an alcohol is taken advantage of in the process of the invention by using cyclodextrin as alcohol to be added to the cation formed by the action of a protonating agent on the reducing compound chosen from monosaccharides, oligosaccharides reducing agents and reducing polysaccharides.
Cette réaction correspond au schéma réactionnel suivant lorsque le composé réducteur est le D-glucose :This reaction corresponds to the following reaction scheme when the reducing compound is D-glucose:
Figure imgf000006_0001
HF anhydre
Figure imgf000006_0001
HF anhydrous
Figure imgf000007_0001
Cette réaction peut intervenir sur différents hydroxyles préférentiellement en position hydroxyle primaire du cyclomaltooligosaccharide qui, on le sait, est la plus réactive. Aussi, en fin de réaction, on- peut obtenir plusieurs cyclomaltooligosaccharides ramifiés ayant des taux de substitution différents en C-6.
Figure imgf000007_0001
This reaction can take place on different hydroxyls, preferably in the primary hydroxyl position of the cyclomaltooligosaccharide which, as we know, is the most reactive. Also, at the end of the reaction, it is possible to obtain several branched cyclomaltooligosaccharides having different substitution rates for C-6.
La substitution en position hydroxyle primaire du cyclomaltooligosaccharide est confirmée par le spectre de RMN du *3C des cyclodextrines ramifiées obtenues par le procédé de l'invention, qui montre le déplacement à bas champ attendu pour les C-6 substitués.The substitution in the primary hydroxyl position of the cyclomaltooligosaccharide is confirmed by the NMR spectrum of * 3 C of the branched cyclodextrins obtained by the process of the invention, which shows the low-field displacement expected for the substituted C-6s.
L'utilisation dans le procédé de l'invention d'un agent protonant particulier, constitué par le fluorure d'hydrogène anhydre permet d'éviter les inconvénients habituels du procédé décrit par Fischer, c'est-à-dire l'hydrolyse des disaccharides et polysaccharides en leurs constituants monosaccharidiques et l'obtention de très mauvais rendements, ce qui était le cas lorsqu'on utilisait comme agent protonant les acides chlorhydrique et sulfurique ou des acides organiques. En revanche, en utilisant selon l'invention du fluorure d'hydrogène anhydre comme solvant et comme catalyseur de glycosidation, on peut obtenir des cyclomaltooligosaccharides ramifiés, c'est-à-dire substitués par des monosaccharides, des oligosaccharides ou des polysaccharides, en une seule étape avec de bons rendements.The use in the process of the invention of a particular protonating agent, constituted by anhydrous hydrogen fluoride makes it possible to avoid the usual drawbacks of the process described by Fischer, that is to say the hydrolysis of disaccharides and polysaccharides in their monosaccharide constituents and obtaining very poor yields, which was the case when using as protonating agent hydrochloric and sulfuric acids or organic acids. On the other hand, by using anhydrous hydrogen fluoride according to the invention as solvent and as glycosidation catalyst, it is possible to obtain branched cyclomaltooligosaccharides, that is to say substituted with monosaccharides, oligosaccharides or polysaccharides, in a single step with good yields.
Pour mettre en oeuvre le procédé de l'invention, on peut mélanger intimement, de préférence par malaxage, le cyclomaltooligosaccharide considéré et le monosaccharide, 1'oligosaccharide réducteur ou le polysaccharide réducteur que 1'on désire greffer sur la cyclodextrine, dans un récipient approprié, par exemple en Téflon, en polyéthylène ou en acier. On peut ensuite ajouter au mélange, à une température telle que le fluorure d'hydrogène anhydre soit liquide, par exemple en refroidissant, dans un bain d'acétone-carboglace, une quantité de fluorure d'hydrogène suffisante pour obtenir une pâte homogène, puis laisser ensuite le mélange revenir à la température ambiante ou légèrement en-dessous tout en maintenant une bonne agitation. Après un temps de réaction approprié, par exemple de 1 à 15 h, on refroidit à nouveau le mélange réactionnel, par exemple dans un bain d'acétone carboglace, et on arrête la réaction par addition d'éther. On sépare ensuite le fluorure d'hydrogène anhydre et l'éther par décantation. La partie insoluble qui correspond aux produits de la réaction peut être lavée plusieurs fois par l'éther, puis séchée et utilisée telle quelle ou séparée en ses divers constituants par des procédés classiques. On peut également purifier les produits obtenus avant de les séparer, par exemple pour éliminer la présence de traces de fluor résiduel, de fluorure d'hydrogène ou de fluorure de glycosyle. Cette purification peut être effectuée par dissolution dans l'eau des produits de la réaction suivie d'une addition de carbonate de calcium à la solution, pour neutraliser l'acidité résiduelle et provoquer l'hydrolyse des traces éventuelles de fluorure de glycosyle.To carry out the process of the invention, it is possible to intimately mix, preferably by kneading, the cyclomaltooligosaccharide considered and the monosaccharide, the reducing oligosaccharide or the reducing polysaccharide which it is desired to graft onto the cyclodextrin, in a suitable container. , for example Teflon, polyethylene or steel. It is then possible to add to the mixture, at a temperature such that the anhydrous hydrogen fluoride is liquid, for example by cooling, in an acetone-dry ice bath, an amount of hydrogen fluoride sufficient to obtain a homogeneous paste, then then allow the mixture to return to room temperature or slightly below while maintaining good agitation. After an appropriate reaction time, for example from 1 to 15 h, the reaction mixture is again cooled, for example in a dry ice acetone bath, and the reaction is stopped by addition of ether. The anhydrous hydrogen fluoride and the ether are then separated by decantation. The insoluble part which corresponds to the reaction products can be washed several times with ether, then dried and used as it is or separated into its various constituents by conventional methods. The products obtained can also be purified before separating them, for example to eliminate the presence of traces of residual fluorine, hydrogen fluoride or glycosyl fluoride. This purification can be carried out by dissolving the reaction products in water followed by the addition of calcium carbonate to the solution, to neutralize the residual acidity and cause hydrolysis of any traces of glycosyl fluoride.
Pour séparer les produits de la réaction, on peut utiliser les procédés décrits en particulier par Hashimoto dans New Trends in Cyclodextrines andTo separate the reaction products, the methods described in particular by Hashimoto in New Trends in Cyclodextrins and can be used.
Derivatives, D. Duchêne, Editions de Santé, Paris,Derivatives, D. Duchêne, Editions de Santé, Paris,
1991, p. 99-156 ainsi que les techniques de purification par exclusion de gel.1991, p. 99-156 as well as purification techniques by gel exclusion.
Pour mettre en oeuvre le procédé de l'invention, on peut utiliser le fluorure d'hydrogène anhydre seul ou le diluer dans un solvant non aqueux.To carry out the process of the invention, one can use anhydrous hydrogen fluoride alone or dilute it in a nonaqueous solvent.
A titre d'exemples de tels solvants, on peut citer le dioxyde de soufre, le dioxane, la pyridine, etc.As examples of such solvents, mention may be made of sulfur dioxide, dioxane, pyridine, etc.
Selon l'invention, les quantités de cyclomaltooligosaccharides et de composés réducteurs utilisées sont choisies en fonction du résultat que l'on veut obtenir. De préférence, on ne dépasse pas la stoechiométrie correspondant au nombre d'hydroxyles dû cyclomaltooligossacharide susceptibles d'être substitués par le composé réducteur. Généralement, le rapport molaire du composé réducteur choisi parmi les monosaccharides, les oligosaccharides et les polysaccharides au cyclomaltooligosaccharide est deAccording to the invention, the amounts of cyclomaltooligosaccharides and of reducing compounds used are chosen according to the result which it is desired to obtain. Preferably, the stoichiometry corresponding to the number of hydroxyls due to cyclomaltooligossacharide capable of being substituted by the reducing compound is not exceeded. Generally, the molar ratio of the reducing compound chosen from monosaccharides, oligosaccharides and polysaccharides to cyclomaltooligosaccharide is
0,5 à 50 et de préférence de 1 à 10. Les cyclomaltooligosaccharides utilisés dans le procédé de 1'invention peuvent être en particulier les cyclodextrines α, β et γ.0.5 to 50 and preferably from 1 to 10. The cyclomaltooligosaccharides used in the process of the invention can in particular be the cyclodextrins α, β and γ.
Le composé réducteur utilisé peut être un monosaccharide tel que le glucose, le galactose, le mannose, un disaccharide tel que le maltose, le lactose, ainsi que des oligosaccharides et petits polysaccharides réducteurs.The reducing compound used can be a monosaccharide such as glucose, galactose, mannose, a disaccharide such as maltose, lactose, as well as oligosaccharides and small reducing polysaccharides.
On peut aussi utiliser des dérivés de ces monosaccharides, oligosaccharides et polysaccharides, c'est-à-dire les dérivés substitués de ces composés qui conservent la propriété de former un cation par action d'un agent protonant et de pouvoir ainsi réagir avec le cyclomaltooligosaccharide.It is also possible to use derivatives of these monosaccharides, oligosaccharides and polysaccharides, that is to say the substituted derivatives of these compounds which retain the property of forming a cation by action of a protonating agent and thus being able to react with the cyclomaltooligosaccharide.
A titre d'exemple de tels dérivés, on peut citer le 2-acétamido-2-désoxy-D-glucose. Selon l'invention, la quantité de fluorure d'hydrogène ajouté doit être suffisante pour que l'on obtienne une pâte homogène afin d'obtenir un taux de substitution du cyclomaltooligosaccharide régulier, mais elle ne doit pas être trop importante car en présence d'une forte proportion de fluorure d'hydrogène par rapport à l'ensemble cyclomaltooligosaccharide et composé(s) réducteur(s), il se produit une hydrolyse importante du cyclomaltooligosaccharide. Ausi, on utilise une quantité de fluorure d'hydrogène anhydre allant de 0,1 à 1 ml par gramme de mélange de cyclomaltooligosaccharide et de composé(s) réducteur(s) . Les meilleurs résultats sont en particulier obtenus lorsqu'on utilise un rapport (cyclomaltooligosaccharides + composé(s) réducteur(s)/fluorure d'hydrogène anhydre en P/V d'environ 2. On obtient aussi de bons résultats lorsqu'on utilise 0,4 ml de fluorure d'hydrogène anhydre par g de mélange. En présence d'une proportion de HF trop faible, le rendement en cyclomaltooligosaccharide ramifiée chute considérablement, par défaut de solubilisation des réactants et de protonation du composé réducteur à greffer. Le procédé de l'invention conduit à un mélange de cyclomaltooligosaccharides ramifiés, mais de façon tout à fait intéressante et inattendue, on n'a pas mis en évidence dans les produits de la réaction la présence d'oligosaccharides linéaires, ce qui confirme l'absence d'hydrolyse du cyclomaltooligosaccharide, à condition bien entendu de ne pas dépasser la stoechiométrie correspondant au nombre d'hydroxyles pouvant être substitués. Par ailleurs, on a observé que cette réaction conduit à introduire presque exclusivement des substituants d'anomérie α lorsque le D-glucose est le monosaccharide utilisé. En effet, la technique de RMN du 13C, appliquée au produit brut de la réaction entre la β-cyclodextrine et le D-glucose, indique que cette réaction conduit à introduire presque exclusivement des substituants d'anomérie a , puisque le signal prédominant est à 96,5 ppm, ce qui correspond au C-l du substituant glucopyranoside et que l'on obtient un signal diffus vers 103 ppm attribuable à la présence de glucopyranoside d'anomérie β, qui ne dépasse pas 5 % de la valeur de l'intégrale du signal précédent. De plus, l'absence de substituant glucofuranosidique est confirmée par l'absence de signaux anomériques à plus bas champs.By way of example of such derivatives, mention may be made of 2-acetamido-2-deoxy-D-glucose. According to the invention, the quantity of hydrogen fluoride added must be sufficient for a homogeneous paste to be obtained in order to obtain a substitution rate for the regular cyclomaltooligosaccharide, but it must not be too large because in the presence of a high proportion of hydrogen fluoride relative to the cyclomaltooligosaccharide and reducing compound (s), there is a significant hydrolysis of the cyclomaltooligosaccharide. Also, an amount of anhydrous hydrogen fluoride ranging from 0.1 to 1 ml per gram of mixture of cyclomaltooligosaccharide and reducing compound (s) is used. The best results are obtained in particular when a ratio (cyclomaltooligosaccharides + reducing compound (s) / anhydrous hydrogen fluoride in P / V of about 2) is used. Good results are also obtained when using 0 , 4 ml of anhydrous hydrogen fluoride per g of mixture In the presence of too low a proportion of HF, the yield of branched cyclomaltooligosaccharide drops considerably, due to the lack of solubilization of the reactants and of protonation of the reducing compound to be grafted. of the invention leads to a mixture of branched cyclomaltooligosaccharides, but quite interestingly and unexpectedly, the presence of linear oligosaccharides has not been demonstrated in the reaction products, which confirms the absence of hydrolysis of cyclomaltooligosaccharide, provided of course not to exceed the stoichiometry corresponding to the number of hydroxyls which can be substituted. Furthermore, it has been observed that this reaction leads to the introduction almost exclusively of α-anomerism substituents when D-glucose is the monosaccharide used. Indeed, the 13 C NMR technique, applied to the crude product of the reaction between β-cyclodextrin and D-glucose, indicates that this reaction leads to almost exclusively introducing substituents of anomerism a, since the predominant signal is at 96.5 ppm, which corresponds to the Cl of the glucopyranoside substituent and that a diffuse signal is obtained around 103 ppm attributable to the presence of glucopyranoside with β anomerism, which does not exceed 5% of the value of the integral of the previous signal. In addition, the absence of glucofuranoside substituent is confirmed by the absence of lower field anomeric signals.
Les mélanges de cyclomaltooligosaccharides ramifiés obtenus 'par le procédé de 1'invention sont très intéressants, car ils ont une solubilité dans l'eau élevée. En effet, dans le cas de la réaction impliquant une proportion équimoléculaire de cyclomaltoheptaose et de D-glucose, la solubilité dans l'eau des produits obtenus atteint 91 g/1 à 25°C (69 mmol/litre) alors que la solubilité du cyclomaltoheptaose de départ n'est que de 19 g/1. Cette solubilité croît très rapidement avec le taux de substitution du cyclomaltooligosaccharide, puisque, pour 3 équivalents moléculaires de D-glucose par rapport au cyclomaltoheptaose, on obtient une solubilité de 1 320 g/1, ce qui est très élevé par rapport aux 19 g/1 du cyclomaltoheptaose de départ.Mixtures of branched cyclomaltooligosaccharides obtained 'by the 1'invention method are very interesting, because they have a high solubility in water. Indeed, in the case of the reaction involving an equimolecular proportion of cyclomaltoheptaose and D-glucose, the solubility in water of the products obtained reaches 91 g / 1 at 25 ° C (69 mmol / liter) while the solubility of starting cyclomaltoheptaose is only 19 g / 1. This solubility increases very rapidly with the substitution rate of cyclomaltooligosaccharide, since, for 3 molecular equivalents of D-glucose compared to cyclomaltoheptaose, a solubility of 1320 g / 1 is obtained, which is very high compared to 19 g / 1 of the starting cyclomaltoheptaose.
La longueur de chaîne des substituants correspondant aux composés réducteurs utilisés joue également un rôle important dans la solubilité des produits ramifiés obtenus. En effet, lorsqu'on remplace le D-glucose par le maltose et qu'on utilise des proportions équimoléculaires de cyclomaltoheptaose et de maltose, la solubilité dans l'eau du mélange de produits obtenu atteint 1 200 g/1 (803 mmol/1), alors qu'elle est de 91 g/1 dans le cas du D-glucose.The chain length of the substituents corresponding to the reducing compounds used also plays an important role in the solubility of the branched products obtained. Indeed, when replaces D-glucose with maltose and equimolecular proportions of cyclomaltoheptaose and maltose are used, the solubility in water of the product mixture obtained reaches 1,200 g / 1 (803 mmol / 1), whereas it is 91 g / 1 in the case of D-glucose.
Les produits obtenus par le procédé de 1'invention peuvent donc avoir de nombreuses applications en raison de leur solubilité dans 1'eau améliorée. Ainsi, on peut les utiliser pour solubiliser dans l'eau des substances actives, notamment des substances pharmaceutiques telles que des agents antitumoraux, en particulier ceux de la famille du taxol, comme le taxotère. On peut aussi utiliser les propriétés des substituants greffés sur le cyclomaltooligosaccharide pour d'autres applications, par exemple sa possibilité de reconnaissance par son environnement. Ainsi, on peut greffer sur une cyclodextrine des signaux de reconnaissance oligosaccharidiques permettant le transport et la vectorisation de produits à usage thérapeutique. A titre d'exemple de telles possibilités, on peut citer le greffage de 2-acétamido-2-désoxy-D-glucose et de D-mannose qui sont des composants fréquemment associés aux glycoprotéines membranaires des cellules et permettent ainsi de transporter préférentiellement des produits à usage thérapeutique vers ces glycoprotéines membranaires. D'autres caractéristiques et avantages deThe products obtained by the process of the invention can therefore have many applications because of their improved solubility in water. Thus, they can be used to dissolve active substances in water, in particular pharmaceutical substances such as anti-tumor agents, in particular those of the taxol family, such as the taxotere. The properties of the substituents grafted onto the cyclomaltooligosaccharide can also be used for other applications, for example its possibility of recognition by its environment. Thus, oligosaccharide recognition signals can be grafted onto a cyclodextrin allowing the transport and vectorization of products for therapeutic use. By way of example of such possibilities, mention may be made of the grafting of 2-acetamido-2-deoxy-D-glucose and of D-mannose which are components frequently associated with the membrane glycoproteins of cells and thus make it possible to preferentially transport products for therapeutic use towards these membrane glycoproteins. Other features and benefits of
1'invention apparaîtront mieux à la lecture des exemples suivants, donnés bien entendu à titre illustratif et non limitatif. Exemple 1 : A un mélange, broyé avec soin, de cyclomaltoheptaose (3,15 g, 2,8 mmol) et de D-glucose (2,8 mmol) dans un récipient en polyéthylène placé dans un bain d'acétone-carboglace, on ajoute le fluorure d'hydrogène anhydre (1,8 ml). Le mélange pâteux est homogénéisé à l'aide d'une spatule en acier jusqu'à l'obtention d'un sirop. Le récipient est ensuite fermé et conservé à la température ambiante tout en maintenant une agitation magnétique pendant 1 heure. Après ce laps de temps, le récipient est à nouveau refroidi dans 1'azote liquide et additionné d'éther. Le précipité blanc résultant est séparé du liquide surnageant par décantation, lavé à nouveau avec de l'éther (3x50 ml), filtré et séché, ce qui conduit à une substance pulvérulente blanche (3,6 g).1'invention will appear better on reading the following examples, given of course by way of illustration and not limitation. Example 1: To a mixture, carefully ground, of cyclomaltoheptaose (3.15 g, 2.8 mmol) and D-glucose (2.8 mmol) in a polyethylene container placed in an acetone-dry ice bath, anhydrous hydrogen fluoride (1.8 ml) is added. The pasty mixture is homogenized using a steel spatula until a syrup is obtained. The container is then closed and stored at room temperature while maintaining magnetic stirring for 1 hour. After this time, the container is again cooled in liquid nitrogen and added with ether. The resulting white precipitate is separated from the supernatant by decantation, washed again with ether (3x50 ml), filtered and dried, which leads to a white pulverulent substance (3.6 g).
On analyse le mélange obtenu par spectrométrie de masse par la technique d'ionisation utilisant le bombardement d'atomes accélérés dans une matrice de glycérol/thioglycérol avec l'iodure de sodium comme agent de cationisation. Avec cette technique, les ions moléculaires cationisés ou protonés des constituants glycosylcyclodextrines du mélange obtenu représentent les signaux majoritaires sinon exclusifs de ce spectre, ce qui permet une excellente caractérisation de la distribution en masse des constituants oligosaccharidiques du mélange brut (comme il est décrit dans l'article de C. Bosso, J. Defaye, A. Heyraud et J. Ulrich. "Fast atom bombardment-mass spectrometry for the characterization of cello- and malto-oligosaccharides" paru dans Carbohydr Res. 125 (1984) 309-317). Cette analyse par spectrométrie de masse du produit obtenu (FAB+ ; matrice glycérol-thioglycérol additionnée d'INa) donne la série de signaux suivants à m/i H57 (100, [CD+Na+]), 1319 (10, [CDGlc+Na]+), 1481 (10, [CDGlc2+Na]+), 1643(4, [CDGlc3+Na]+), 1805 (1, [CDGlc +Na]+). Ainsi, pour une proportion 1:1 de D-glucose et de cyclomaltoheptaose, on note la présence sur le spectre, indépendamment d'un ion à m/z 1157 ([M+Na]+ du cyclomaltoheptaose n'ayant pas réagi), d'ions à m/z 1319, 1481, 1643 et 1805 correspondant respectivement aux ions moléculaires cationisés d'un monoglucosylcyclomaltoheptaose ainsi que de di-, tri- et tétraglucosylcyclomaltoheptaoses.The mixture obtained is analyzed by mass spectrometry by the ionization technique using the bombardment of accelerated atoms in a glycerol / thioglycerol matrix with sodium iodide as cationization agent. With this technique, the cationized or protonated molecular ions of the glycosylcyclodextrin constituents of the mixture obtained represent the majority signals if not exclusive of this spectrum, which allows an excellent characterization of the mass distribution of the oligosaccharide constituents of the crude mixture (as described in (article by C. Bosso, J. Defaye, A. Heyraud and J. Ulrich. "Fast atom bombardment-mass spectrometry for the characterization of cello- and malto-oligosaccharides" published in Carbohydr Res. 125 (1984) 309-317) . This mass spectrometric analysis of the product obtained (FAB + ; glycerol-thioglycerol matrix supplemented with INa) gives the following series of signals at m / i H57 (100, [CD + Na + ]), 1319 (10, [CDGlc + Na] + ), 1481 (10, [CDGlc 2 + Na] + ), 1643 (4, [CDGlc 3 + Na] + ), 1805 (1, [CDGlc + Na] + ). Thus, for a 1: 1 proportion of D-glucose and of cyclomaltoheptaose, the presence on the spectrum is noted, independently of an ion at m / z 1157 ([M + Na] + of unreacted cyclomaltoheptaose), ions at m / z 1319, 1481, 1643 and 1805 respectively corresponding to the cationized molecular ions of a monoglucosylcyclomaltoheptaose as well as of di-, tri- and tetraglucosylcyclomaltoheptaoses.
La solubilité de cette substance dans l'eau est de ~91 g/1 (69 mmol/1). Exemple 2 :The solubility of this substance in water is ~ 91 g / 1 (69 mmol / 1). Example 2:
On suit le même mode opératoire que dans l'Exemple 1, en partant d'un mélange de cyclomaltoheptaose (3,15 g, 2,8 mmol) et de D-glucose (1,5 g, 8,4 mmol) qui est additionné de fluorure d'hydrogène anhydre (2,3 ml). On obtient ,après la série d'opérations de l'Exemple 1, une substance pulvérulente blanche (4,5 g) qui montre en spectrométrie de masse (FAB+ ; glycérol-thioglycérol ; INa) la série de signaux suivants à m/z 1157(100), 1319(90), 1481(60), 1643(50), 1805(45), 1967 (35, [CdGlc5+Na]+), 2129(30, [CDGlc6+Na]+) , 2291 (25, [CDGlc7+Na]+), 2453 (25, [CDGlc8+Na]+) , 2615 (20, [CDGlCg+Na]"1"), 2777 (10, [CDGlc10+Na]+), 2939 (5, [CDGlcu+Na]"1"), 3101 (1, [CDGlc12+Na]+), 3263 (1, [CDGlc13+Na]+), 3425 (1, [CDGlc1 +Na]+) , 3587 (>1, [CDGlc15+Na]+) .The same procedure is followed as in Example 1, starting from a mixture of cyclomaltoheptaose (3.15 g, 2.8 mmol) and D-glucose (1.5 g, 8.4 mmol) which is supplemented with anhydrous hydrogen fluoride (2.3 ml). After the series of operations in Example 1, a white pulverulent substance (4.5 g) is obtained which shows in mass spectrometry (FAB + ; glycerol-thioglycerol; INa) the following series of signals at m / z 1157 (100), 1319 (90), 1481 (60), 1643 (50), 1805 (45), 1967 (35, [CdGlc 5 + Na] + ), 2129 (30, [CDGlc 6 + Na] + ) , 2291 (25, [CDGlc 7 + Na] + ), 2453 (25, [CDGlc 8 + Na] + ), 2615 (20, [CDGlCg + Na] "1" ), 2777 (10, [CDGlc 10 + Na ] + ), 2939 (5, [CDGlcu + Na] "1" ), 3101 (1, [CDGlc 12 + Na] + ), 3263 (1, [CDGlc 13 + Na] + ), 3425 (1, [CDGlc 1 + Na] + ), 3587 (> 1, [CDGlc 15 + Na] + ).
Si l'on compare ces résultats avec ceux de l'exemple 1, on observe que lorsque la proportion D-glucose/cyclomaltoheptaose est de 3, la distribution en masse telle qu'elle apparaît dans le spectre est modifiée puisqu'on note la présence, indépendamment de celle des ions précédents de l'exemple 1 encore plus intenses, d'ions à m/z 1967, 2129, 2291, 2453, 2615, 2777, 2939, 3101, 3263, 3425 et 3587 indicatifs de la présence associée des tri-jusqu'au pentadécaglucosylcyclomaltoheptaoses.If we compare these results with those of Example 1, we observe that when the proportion D-glucose / cyclomaltoheptaose is 3, the mass distribution as it appears in the spectrum is modified since we note the presence , independently of that of the preceding ions of Example 1, which are even more intense, of ions at m / z 1967, 2129, 2291, 2453, 2615, 2777, 2939, 3101, 3263, 3425 and 3587 indicative of the associated presence of tri-to pentadecaglucosylcyclomaltoheptaoses.
Ainsi, la distribution en masse du mélange de cyclodextrines ramifiées est directement proportionnelle à la quantité de glucose mis en réaction.Thus, the mass distribution of the mixture of branched cyclodextrins is directly proportional to the amount of glucose reacted.
La solubilité dans 1'eau de cette substance est de ~1320 g/1 (~894 mmol/1). Exemple 3 : On applique le mode opératoire de l'Exemple 1 à un mélange de cyclomaltoheptaose (3,15 g, 2,8 mmol) et de maltose (IH2O, 1,0 g, 2,8 mmol), qui est additionné de fluorure d'hydrogène anhydre (1,8 ml). On obtient, après la série d'opérations de l'Exemple 1, 4,0 g d'une substance pulvérulente blanche qui montre en spectrométrie de masse (FAB+ ; glycérol-thioglycérol ; INa), la série de signaux suivants à m/z 1157 (100, [CD+Na]+) 1481 (40, [CDMalt+Na]+), 1805 (20, [CDMalt2+Na]+) , 2129 (10, [CDMalt3+Na]+), 2453 (5, [CDMalt4+Na+), 2777 (2, [CDMalt5+Na]+), 3101 (1, [CDMalt6+Na] )+) .The solubility in water of this substance is ~ 1320 g / 1 (~ 894 mmol / 1). EXAMPLE 3 The procedure of Example 1 is applied to a mixture of cyclomaltoheptaose (3.15 g, 2.8 mmol) and maltose (IH2O, 1.0 g, 2.8 mmol), which is added with anhydrous hydrogen fluoride (1.8 ml). After the series of operations in Example 1, 4.0 g of a white pulverulent substance are obtained which shows in mass spectrometry (FAB + ; glycerol-thioglycerol; INa), the following series of signals at m / z 1157 (100, [CD + Na] + ) 1481 (40, [CDMalt + Na] + ), 1805 (20, [CDMalt 2 + Na] + ), 2129 (10, [CDMalt 3 + Na] + ), 2453 (5, [CDMalt 4 + Na + ), 2777 (2, [CDMalt 5 + Na] + ), 3101 (1, [CDMalt 6 + Na]) + ).
La solubilité dans 1'eau de cette substance est de ~1200 g/1 (~803 mmol/1). Exemple 4 : On applique le mode opératoire de l'Exemple 1 à un mélange de cyclomaltohexaose (2,7 g, 2,8 mmol) et de maltose (1H20, 1,0 g, 2,8 mmol) auquel on ajoute le fluorure d'hydrogène anhydre (1,8 ml). On obtient, en répétant la série d'opérations de l'Exemple 1, 3,6 g d'une substance pulvérulente blanche qui montre en spectrométrie de masse (FAB+ ; glycérol-thioglycérol ; ICs), la série de signaux suivants à m/z 1105 (100, [CD+Cs]+), 1429 (30, [CDMalt+Cs]+), 1753 (15, [CDMalt2+Cs]+) , 2077 (10, [CDMalt3+Cs]+) , 2401 (7, [CDMalt4+Cs]+), 2725 (5, [CDMalt5+Cs]+) , 3049 (3, [CDMalt6+Cs]+), 3373 (2 , [CDMalt7+Cs]+) , 3697 (1, [CDMalt8+Cs]+) .The solubility in water of this substance is ~ 1200 g / 1 (~ 803 mmol / 1). Example 4: The procedure of Example 1 is applied to a mixture of cyclomaltohexaose (2.7 g, 2.8 mmol) and maltose (1H 2 0, 1.0 g, 2.8 mmol) to which we add anhydrous hydrogen fluoride (1.8 ml). By repeating the series of operations in Example 1, 3.6 g of a white powdery substance are obtained which shows in mass spectrometry (FAB + ; glycerol-thioglycerol; ICs), the following series of signals at m / z 1105 (100, [CD + Cs] + ), 1429 (30, [CDMalt + Cs] + ), 1753 (15, [CDMalt 2 + Cs] + ), 2077 (10, [CDMalt 3 + Cs] + ), 2401 (7, [CDMalt 4 + Cs] + ), 2725 (5, [CDMalt 5 + Cs] + ), 3049 (3, [CDMalt 6 + Cs] + ), 3373 (2, [CDMalt 7 + Cs] + ), 3697 (1, [CDMalt 8 + Cs] + ).
La solubilité dans l'eau de cette substance est de -1450 g/1 (-1088 mmol/1). Exemple 5 :The solubility in water of this substance is -1450 g / 1 (-1088 mmol / 1). Example 5:
On applique le mode opératoire de 1'Exemple 1 à un mélange de cyclomaltooctaose (3,6 g, 2,8 mmol) et de maltose (1 H20, 1,0 g, 2,8 mmol), auquel on ajoute le fluorure d'hydrogène anhydre (2,3 ml). On obtient, après la série d'opérations de l'Exemple 1, 4,4 g d'une substance pulvérulente blanche qui montre en spectrométrie de masse (FAB+, glycérol-thioglycérol, ICs), la série de signaux suivants à m/z 1429 (100, [CD+CSJ+), 1753 (40, [CDMalt+Cs]+), 2077 (30, [CDMalt +Cs]+), 2401 (15, {CDMalt3+Cs}+) , 2725 (10, {CDMalt +Cs}+), 3049 (5, CDMalt5+Cs}+), 3373 (3, [CDMaltg+Cs]+), 3697 (2, [CDMalt7+Cs]+) , 4021 (1, [CDMalt8+Cs]+).The procedure of Example 1 is applied to a mixture of cyclomaltooctaose (3.6 g, 2.8 mmol) and maltose (1 H 2 0, 1.0 g, 2.8 mmol), to which the anhydrous hydrogen fluoride (2.3 ml). After the series of operations in Example 1, 4.4 g of a white pulverulent substance are obtained which shows in mass spectrometry (FAB + , glycerol-thioglycerol, ICs), the following series of signals at m / z 1429 (100, [CD + CSJ +), 1753 (40, [CDMalt + Cs] + ), 2077 (30, [CDMalt + Cs] + ), 2401 (15, {CDMalt 3 + Cs} + ), 2725 ( 10, {CDMalt + Cs} + ), 3049 (5, CDMalt 5 + Cs} + ), 3373 (3, [CDMaltg + Cs] + ), 3697 (2, [CDMalt 7 + Cs] + ), 4021 (1 , [CDMalt 8 + Cs] + ).
La solubilité de cette substance dans 1'eau est de -1580 g/1 (954 mmol/1). Exemple 6 :The solubility of this substance in water is -1580 g / 1 (954 mmol / 1). Example 6:
On applique le mode opératoire de 1'exemple 1 à un mélange de cyclomaltoheptaose (3,15 g, 2,8 mmol) et de D-mannose (1,5 g, 8,4 mmol) auquel on ajoute le fluorure d'hydrogène anhydre (2,3 ml). On obtient, après la série d'opérations de l'exemple 1, 4,4 g d'une substance pulvérulente blanche qui montre, en spectrométrie de masse (FAB+, glycérol-thioglycérol, INa), la série de signaux suivants à __/z 1267 (100, [CD+Cs]+), 1429 (80, [CDMan+Cs]+), 1591 (60, [CDMan2+Cs]+), 1753 (50, [CDMan3+Cs]+), 1915(40, [CDMan +Cs]+), 2077 (30, [CDMan5+Cs]+), 2239 (25, [CDMan6+Cs]+), 2401 (20, [CDMan7+Cs]+), 2563 (17, [CDMan8+Cs]+), 2725 (15, [CDMan9+Cs]+), 2887 (12, [CDMan10+Cs]+), 3049 (10, [CDMan1:L+Cs]+) , 3211 (7, [CDMan12+Cs]+), 3373 (5,[CDMan13+Cs]+), 3535 (3, [CDMan14+Cs]+), 3697 (2, [CDMan15+Cs]+) , 3859 (1, [CDMan16+Cs]+), 9021 (1, [CDMan17+Cs]+) .The procedure of Example 1 is applied to a mixture of cyclomaltoheptaose (3.15 g, 2.8 mmol) and D-mannose (1.5 g, 8.4 mmol) to which hydrogen fluoride is added. anhydrous (2.3 ml). After the series of operations of Example 1, 4.4 g of a white pulverulent substance are obtained which shows, in mass spectrometry (FAB + , glycerol-thioglycerol, INa), the following series of signals at __ / z 1267 (100, [CD + Cs] +), 1429 (80, [CDMan + Cs] + ), 1591 (60, [CDMan 2 + Cs] + ), 1753 (50, [CDMan 3 + Cs] + ), 1915 (40, [CDMan + Cs] + ), 2077 (30, [CDMan 5 + Cs] + ), 2239 (25, [CDMan 6 + Cs] + ), 2401 (20, [CDMan 7 + Cs] + ), 2563 (17, [CDMan 8 + Cs] + ), 2725 (15, [CDMan 9 + Cs] + ), 2887 (12, [CDMan 10 + Cs] + ), 3049 (10, [CDMan 1: L + Cs] + ), 3211 (7, [CDMan 12 + Cs] + ), 3373 (5, [CDMan 13 + Cs] + ), 3535 (3, [CDMan 14 + Cs] + ), 3697 (2, [CDMan 15 + Cs] + ), 3859 (1, [CDMan 16 + Cs] + ), 9021 (1, [CDMan 17 + Cs] + ).
La solubilité dans l'eau de cette substance est de -1400 g/1 (-853 rnmol/1). Exemple 7 :The solubility in water of this substance is -1400 g / 1 (-853 rnmol / 1). Example 7:
A un mélange, broyé avec soin, de cyclomaltoheptaose (2,56 g, 2,26 mmol) et de 2-acétamido-2-désoxy-D-glucose (1,0 g, 4,52 mmol), dans un récipient en polyéthylène placé dans un bain d'acétone-carboglace, on ajoute le fluorure d'hydrogène anhydre (2 ml). Le mélange pâteux est homogénéisé à l'aide d'une spatule en acier jusqu'à l'obtention d'un sirop. Le récipient non clos est ensuite amené à la température ambiante tout en maintenant l'agitation et ce, jusqu'à ce qu'elle s'arrête d'elle-même par suite de 1'évaporation de HF. Après 15 heures, le résidu est dissous dans l'eau (30 ml) et additionné jusqu'à neutralité de CaCo3 pulvérulent. La suspension est alors débarrassée des sels insolubles par filtration et lyophilisée, ce qui conduit à une substance pulvérulente blanche (3,4 g) qui montre en spectrométrie de masse (FAB+ ; matrice glycérol-thioglycérol ; ICs) une série de signaux à m/z 1267 (100, [CD+Cs]+), 1470 (60, [CDGleNAc+Cs]+), 1673 (15, [CD(GleNac)2+Cs]+), 1876 (1, [CD(GleNAc)3+Cs]+) .To a carefully ground mixture of cyclomaltoheptaose (2.56 g, 2.26 mmol) and 2-acetamido-2-deoxy-D-glucose (1.0 g, 4.52 mmol), in a container polyethylene placed in an acetone-dry ice bath, anhydrous hydrogen fluoride (2 ml) is added. The pasty mixture is homogenized using a steel spatula until a syrup is obtained. The unclosed container is then brought to ambient temperature while maintaining stirring until it stops by itself as a result of the evaporation of HF. After 15 hours, the residue is dissolved in water (30 ml) and added until the powdered CaCo 3 is neutral. The suspension is then freed of the insoluble salts by filtration and lyophilized, which leads to a white pulverulent substance (3.4 g) which shows in mass spectrometry (FAB + ; glycerol-thioglycerol matrix; ICs) a series of signals at m / z 1267 (100, [CD + Cs] + ), 1470 (60, [CDGleNAc + Cs] + ), 1673 (15, [CD (GleNac) 2 + Cs] + ), 1876 (1, [CD (GleNAc ) 3 + Cs] + ).
La solubilité de cette substance dans 1'eau est de -1100 g/1.The solubility of this substance in water is -1100 g / 1.
Les exemples 8 à 11 qui suivent sont donnés à titre comparatif pour montrer que l'on ne peut obtenir un cyclomaltooligosaccharide ramifié lorsque la quantité de fluorure d'hydrogène utilisé dans le procédé de 1'invention est trop importante. Exemple 8 Le protocole opératoire de l'Exemple 1 est répété, mais avec le cyclomaltoheptaose seul (3,15 g. 2,8 mmol) qui est additionné de fluorure d'hydrogène anhydre (6,3 ml). Après la série d'opérations de l'exemple 1, on obtient une substance pulvérulente (2,7 g) qui, examinée en RMN du C dans l'oxyde de deutérium, conduit à un spectre complexe où l'on note en particulier dans la région des carbones ano ères des signaux à 92,9 et 96,7 ppm attribuables au C-l d'unités réductrices α-et-β-Dglucopyranose respectivement (K. Bock et C. Pedersen, Adv. Carbohydr. Chem. Biochem., 41, 27-66, 1983) ainsi qu'un doublet large à - 108 ppm (J-222 Hz) indiquant la présence de fluorure d'α-D-glucopyranosyle (K. Bock et C. Pedersen, Acta Chem. Scand., Ser. B, 29, 682- 686, 1975). Un spectre pratiquement identique est obtenu par fluorolyse de l'amidon dans les mêmes conditions (J. Defaye, A. Gadelle et C. Pedersen, 100, 217-227, 1982) et ceci confirme une dégradation importante de la cyclodextrine dans ces conditions de dilution dans HF, par coupure des liaisons glycosidiques.Examples 8 to 11 which follow are given by way of comparison to show that a branched cyclomaltooligosaccharide cannot be obtained when the quantity of hydrogen fluoride used in the process of the invention is too large. Example 8 The operating protocol of Example 1 is repeated, but with cyclomaltoheptaose alone (3.15 g. 2.8 mmol) which is added with anhydrous hydrogen fluoride (6.3 ml). After the series of operations of Example 1, a pulverulent substance (2.7 g) is obtained which, examined by C NMR in deuterium oxide, leads to a complex spectrum which is noted in particular in the anomeric carbon region of the 92.9 and 96.7 ppm signals attributable to Cl of α-and-β-Dglucopyranose reducing units respectively (K. Bock and C. Pedersen, Adv. Carbohydr. Chem. Biochem., 41, 27-66, 1983) as well as a broad doublet at -108 ppm (J-222 Hz) indicating the presence of α-D-glucopyranosyl fluoride (K. Bock and C. Pedersen, Acta Chem. Scand. , Ser. B, 29, 682-686, 1975). A practically identical spectrum is obtained by fluorolysis of starch under the same conditions (J. Defaye, A. Gadelle and C. Pedersen, 100, 217-227, 1982) and this confirms a significant degradation of cyclodextrin under these conditions. dilution in HF, by cutting glycosidic bonds.
Lorsque le cyclomaltohexaose et le cyclomaltoheptaose sont traités par HF dans les mêmes conditions de concentration, un spectre de RMN du 13C du produit obtenu montre des caractéristiques pratiquement identiques, indiquant également une dégradation importante. Exemple 9When cyclomaltohexaose and cyclomaltoheptaose are treated with HF under the same concentration conditions, a 1 3 C NMR spectrum of the product obtained shows practically identical characteristics, also indicating significant degradation. Example 9
Le protocole opératoire de 1'Exemple 1 est répété avec le mélange pulvérulent, et homogénéisé par broyage, de cyclomaltoheptaose (3,15 g, 2,8 mmol) et de D-glucose (2,8 mmol) que l'on additionne de fluorure d'hydrogène anhydre (6,3 ml). Après la série d'opérations de l'exemple 1, on obtient une substance pulvérulente (3,5g) qui, examinée en RMN du 13C, conduit à un spectre globalement identique à celui de 1'exemple 8, indiquant une dégradation importante de la cyclodextrine. Le spectre de masse n'indique pas la présence des signaux caractéristiques des mono-, di-, tri-, et tétra glucosylcyclomaltoheptaoses. Exemple 10 : Le protocole opératoire de 1'exemple 1 est répété avec le cyclomaltoheptaose seul (3,15 g, 2,8 mmol) qui est additionné de fluorure d'hydrogène anhydre (2,3 ml). On obtient, d'après la série d'opérations de l'exemple 1, une substance pulvérulente qui a les mêmes caractéristiques (P.f. [α]D, RMN i3C) que le cyclomaltoheptaose de départ. Exemple 11The operating protocol of Example 1 is repeated with the pulverulent mixture, and homogenized by grinding, of cyclomaltoheptaose (3.15 g, 2.8 mmol) and of D-glucose (2.8 mmol) which is added with anhydrous hydrogen fluoride (6.3 ml). After the series of operations of Example 1, a pulverulent substance (3.5 g) is obtained which, examined by 13 C NMR, leads to a spectrum which is broadly identical to that of Example 8, indicating a significant degradation of cyclodextrin. The mass spectrum does not indicate the presence of the characteristic signals of mono-, di-, tri-, and tetra glucosylcyclomaltoheptaoses. Example 10: The operating protocol of Example 1 is repeated with cyclomaltoheptaose alone (3.15 g, 2.8 mmol) which is supplemented with anhydrous hydrogen fluoride (2.3 ml). According to the series of operations of Example 1, a pulverulent substance is obtained which has the same characteristics (Pf [α] D , NMR i3 C) as the starting cyclomaltoheptaose. Example 11
Le protocole opératoire de l'exemple 7 est suivi, avec le mélange de cyclomaltoheptaose (2,56 g, 2,26 mmol) et de 2-acétamido-2-désoxy-D-glucose (1,0 g, 4,52 mmol) qui est additionné de fluorure d'hydrogène anhydre (7 ml). Après la série d'opérations de l'exemple 7, on obtient une substance pulvérulente (3,45 g) qui montre en pectrométrie de masse un ensemble complexe de signaux dans lesquels on ne retrouve pas ceux décrits dans 1'exemple 7 pour la 2-acétamido-2-désoxy-D-glucosylcyclodextrine. Le spectre de RMN du 3C est également complexe et on y retrouve des signaux à 92,9 et 96,7 ppm pour des unités réductrices d'α- et β-D-glucopyranose, un doublet large à - 108 ppm pour le fluorure d'α-D- glucopyranosyle, ainsi qu'un signal à 102,4 ppm indiquant la présence d'unités 2-acétamido-2-désoxy-β- D-glucopyranosyle (J. Defaye, A. Gadelle et C. Pedersen, Carbohydr. Res. 186, 177-188, 1989).The operating protocol of Example 7 is followed, with the mixture of cyclomaltoheptaose (2.56 g, 2.26 mmol) and 2-acetamido-2-deoxy-D-glucose (1.0 g, 4.52 mmol ) which is supplemented with anhydrous hydrogen fluoride (7 ml). After the series of operations in Example 7, a pulverulent substance (3.45 g) is obtained which shows in mass pectrometry a complex set of signals in which we do not find those described in Example 7 for the 2 -acetamido-2-deoxy-D-glucosylcyclodextrin. The 3 C NMR spectrum is also complex and there are signals at 92.9 and 96.7 ppm for reducing units of α- and β-D-glucopyranose, a broad doublet at - 108 ppm for fluoride of α-D-glucopyranosyl, as well as a signal at 102.4 ppm indicating the presence of 2-acetamido-2-deoxy-β-D-glucopyranosyl units (J. Defaye, A. Gadelle and C. Pedersen, Carbohydr. Res. 186, 177-188, 1989).
L'exemple 12 qui suit illustre l'utilisation de cyclomaltooligosaccharides ramifiés obtenus conformément à 1'invention pour la solubilisation de principes actifs. Exemple 12 : Solubilisation du Taxotère dans le mélange de glucosyl-cyclomaltoheptaoses préparé dans 1'exemple 1.Example 12 which follows illustrates the use of branched cyclomaltooligosaccharides obtained in accordance with the invention for the solubilization of active principles. Example 12: Solubilization of Taxotere in the mixture of glucosyl-cyclomaltoheptaoses prepared in Example 1.
On ajoute, sous forte agitation, du Taxotère à une solution aqueuse (5 ml) contenant 65 g/1 de glucosylcyclomaltoheptaoses préparés dans l'exemple 1, et cela jusqu'à ce que la solution reste claire. On constate que la solubilité du Taxotère dans 1'eau dans ces conditions, en présence du produit de l'exemple 1 (50 mmol/1) est - 1,5 g/1 alors que la solubilité du Taxotère seul dans l'eau n'est que de 0,004 g/1. Taxotere is added, with vigorous stirring, to an aqueous solution (5 ml) containing 65 g / l of glucosylcyclomaltoheptaoses prepared in Example 1, and this until the solution remains clear. It is found that the solubility of Taxotere in water under these conditions, in the presence of the product of Example 1 (50 mmol / 1) is - 1.5 g / 1 while the solubility of Taxotere alone in water n is only 0.004 g / 1.

Claims

REVENDICATIONS
1. Procédé de préparation de cyclomaltooligosaccharides ramifiés, caractérisé en ce que 1'on fait réagir un cyclomaltooligosaccharide avec au moins un composé réducteur choisi parmi les monosaccharides, les oligosaccharides réducteurs, les polysaccharides réducteurs et leurs dérivés, en présence de fluorure d'hydrogène anhydre, en opérant au moins au début de la réaction dans des conditions telles que le fluorure d'hydrogène anhydre soit liquide, et en utilisant 0,1 à 1 ml de fluorure d'hydrogène anhydre par gramme du mélange de cyclomaltooligosaccharide et de composé(s) réducteur(s) , et en ce que l'on sépare ensuite les produits de la réaction du fluorure d'hydrogène.1. Process for the preparation of branched cyclomaltooligosaccharides, characterized in that a cyclomaltooligosaccharide is reacted with at least one reducing compound chosen from monosaccharides, reducing oligosaccharides, reducing polysaccharides and their derivatives, in the presence of anhydrous hydrogen fluoride , operating at least at the start of the reaction under conditions such that the anhydrous hydrogen fluoride is liquid, and using 0.1 to 1 ml of anhydrous hydrogen fluoride per gram of the mixture of cyclomaltooligosaccharide and of compound (s ) reducing agent (s), and in that the reaction products of hydrogen fluoride are then separated.
2. Procédé selon la revendication 1, caractérisé en ce que l'on purifie ensuite les produits de la réaction par dissolution dans 1'eau suivie d'une addition de carbonate de calcium à la solution pour éliminer les traces de fluor résiduelles, d'acide fluorhydrique et/ou de fluorure du composé réducteur.2. Method according to claim 1, characterized in that the reaction products are then purified by dissolution in water followed by addition of calcium carbonate to the solution to remove traces of residual fluorine, hydrofluoric acid and / or fluoride of the reducing compound.
3. Procédé selon 1'une quelconque des revendications 1 et 2, caractérisé en ce que le fluorure d'hydrogène anhydre est mélangé à un solvant non aqueux.3. Method according to any one of claims 1 and 2, characterized in that the anhydrous hydrogen fluoride is mixed with a non-aqueous solvent.
4. Procédé selon la revendication 3, caractérisé en ce que le solvant non aqueux est choisi parmi le dioxyde de soufre, le dioxane et la pyridine. 5. Procédé selon l'une quelconque des revendications 1 à 4, caractérisé en ce que le rapport molaire du composé réducteur au cyclomaltooligosaccharide est de 0,4. Method according to claim 3, characterized in that the non-aqueous solvent is chosen from sulfur dioxide, dioxane and pyridine. 5. Method according to any one of claims 1 to 4, characterized in that the molar ratio of the reducing compound to the cyclomaltooligosaccharide is 0,
5 à 50.5 to 50.
6. Procédé selon l'une quelconque des revendications 1 à 5, caractérisé en ce que le cyclomaltooligosaccharide est choisi parmi les cyclodextrines α, β et γ.6. Method according to any one of claims 1 to 5, characterized in that the cyclomaltooligosaccharide is chosen from the cyclodextrins α, β and γ.
7. Procédé selon l'une quelconque des revendications 1 à 6, caractérisé en ce que le composé réducteur est choisi parmi le D-glucose, le maltose, le D-mannose et le 2-acétamido-2-désoxy-D-glucose.7. Method according to any one of claims 1 to 6, characterized in that the reducing compound is chosen from D-glucose, maltose, D-mannose and 2-acetamido-2-deoxy-D-glucose.
8. Procédé selon l'une quelconque des revendications 1 à 7, caractérisé en ce que la rapport du mélange de cyclomaltooligosaccharide et de composé(s) réducteur(s) au fluorure d'hydrogène anhydre (en P/V) est d'environ 2.8. Method according to any one of claims 1 to 7, characterized in that the ratio of the mixture of cyclomaltooligosaccharide and compound (s) reducing (s) to anhydrous hydrogen fluoride (in W / V) is about 2.
9. Procédé pour améliorer la solubilité dans l'eau d'un cyclomaltooligosaccharide, caractérisé en ce qu'il consiste à substituer le cyclomaltooligosaccharide par au moins un composé réducteur choisi parmi les monosaccharides, les oligosaccharides réducteurs, les polysaccharides réducteurs et leurs dérivés en mettant en oeuvre le procédé selon 1'une quelconque des revendications 1 à 8.9. Process for improving the solubility in water of a cyclomaltooligosaccharide, characterized in that it consists in replacing the cyclomaltooligosaccharide by at least one reducing compound chosen from monosaccharides, reducing oligosaccharides, reducing polysaccharides and their derivatives by putting implementing the method according to any one of claims 1 to 8.
10. Utilisation des cyclomaltooligosaccharides ramifiés obtenus par le procédé selon l'une quelconque des revendications 1 à 8 pour la solubilisation dans 1'eau de substances actives. 10. Use of branched cyclomaltooligosaccharides obtained by the process according to any one of claims 1 to 8 for the solubilization in water of active substances.
11. Utilisation selon la revendication 10, caractérisée en ce que la substance active est le Taxotère. 11. Use according to claim 10, characterized in that the active substance is Taxotere.
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US6080733A (en) * 1996-03-14 2000-06-27 Centre National De La Recherche Scientifique Thioureido-cyclodextrins, utilized in particular to solubilize anti-tumor, and antiparasitic agents and their preparation processes
US6570009B1 (en) 1998-05-26 2003-05-27 Centre National De La Recherche Scientifique Region-selective method for preparing cyclodextrin C-6 monosulphonyl derivatives
KR100407043B1 (en) * 1996-07-11 2004-05-31 엔스이코 세이토 가부시키가이샤 Branched cyclodextrin and preparation method thereof
FR2862649A1 (en) * 2003-11-26 2005-05-27 Centre Nat Rech Scient New dimers of cyclodextrins used to solubilize pharmacologically active molecules, especially anti-tumorals such as Taxol and its derivatives
JP2006521440A (en) * 2003-03-28 2006-09-21 サントル・ナショナル・ドゥ・ラ・ルシェルシュ・シャンティフィク Novel cyclodextrin derivatives, processes for their preparation and their use for solubilizing pharmacologically active substances
WO2014095427A1 (en) 2012-12-20 2014-06-26 L'oreal Water-insoluble cyclodextrin polycondensate; uses as a capturing agent
WO2014095426A1 (en) 2012-12-20 2014-06-26 L'oreal Composition which makes possible the release of a beneficial agent comprising a water-insoluble cyclodextrin polycondensate and at least one beneficial agent

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6080733A (en) * 1996-03-14 2000-06-27 Centre National De La Recherche Scientifique Thioureido-cyclodextrins, utilized in particular to solubilize anti-tumor, and antiparasitic agents and their preparation processes
KR100407043B1 (en) * 1996-07-11 2004-05-31 엔스이코 세이토 가부시키가이샤 Branched cyclodextrin and preparation method thereof
US6570009B1 (en) 1998-05-26 2003-05-27 Centre National De La Recherche Scientifique Region-selective method for preparing cyclodextrin C-6 monosulphonyl derivatives
JP2006521440A (en) * 2003-03-28 2006-09-21 サントル・ナショナル・ドゥ・ラ・ルシェルシュ・シャンティフィク Novel cyclodextrin derivatives, processes for their preparation and their use for solubilizing pharmacologically active substances
FR2862649A1 (en) * 2003-11-26 2005-05-27 Centre Nat Rech Scient New dimers of cyclodextrins used to solubilize pharmacologically active molecules, especially anti-tumorals such as Taxol and its derivatives
WO2005054303A2 (en) * 2003-11-26 2005-06-16 Centre National De La Recherche Scientifique Cyclodextrin dimers and derivatives thereof, methods for preparing them and their use, in particular, for solubilizing pharmacologically active substances
WO2005054303A3 (en) * 2003-11-26 2005-08-04 Centre Nat Rech Scient Cyclodextrin dimers and derivatives thereof, methods for preparing them and their use, in particular, for solubilizing pharmacologically active substances
US7781417B2 (en) 2003-11-26 2010-08-24 Centre National De La Recherche Scientifique Cyclodextrin dimers and derivatives thereof, methods for preparing them and their use, in particular, for the solubilizing pharmacologically active substances
WO2014095427A1 (en) 2012-12-20 2014-06-26 L'oreal Water-insoluble cyclodextrin polycondensate; uses as a capturing agent
WO2014095426A1 (en) 2012-12-20 2014-06-26 L'oreal Composition which makes possible the release of a beneficial agent comprising a water-insoluble cyclodextrin polycondensate and at least one beneficial agent

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FR2716200B1 (en) 1996-04-26

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