Classifications

• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
  • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
  • A61K31/727—Heparin; Heparan
• • 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
  • A61K47/51—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 non-active ingredient being a modifying agent
  • A61K47/56—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 non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
  • A61K47/61—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 non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P33/00—Antiparasitic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • 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
  • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof

Definitions

• the invention relates to new compounds or neoglycoconjugates which bind to interferon-gamma.
• the invention also relates to the process for the preparation of these compounds, the complexes formed by these compounds and interferon-gamma, and the medicaments comprising these compounds or complexes.
• Interferon-gamma is a polypeptide comprising, for example, 143 amino acids in humans which is part of the family of cyto ines. Cytokines are mediators of cellular communication, which act according to a paracrine, autocrine or sometimes even endocrine process.
• IFN ⁇ first characterized, on the basis of its antiviral activity, is involved in particular in the control of the immune response and during inflammation.
• This cytokine is also cytotoxic or cytostatic for transformed cells and induces the synthesis of oxygen radicals. It regulates expression a large number of molecules in the pericellular space, in particular cell surface molecules, as well as a large number of compounds of the extracellular matrix. IFN ⁇ therefore plays an important role, among other things, in defense mechanisms, such as the immune response and inflammation, in cell growth and differentiation, in the phenomena of cell adhesion and migration (1).
• Therapeutics linked to cytokines, such as IFN ⁇ consist either in administering this type of molecule, or, on the contrary, in inhibiting their activities.
• IFN ⁇ Developing a therapy based on the use of IFN ⁇ poses technical problems important, linked in particular to its low half-life in vivo and its low bioavailability.
• the obstacle of low bioavailability of IFN ⁇ can be overcome by using local application methods, but these methods do not allow deep organs to be reached systemically, moreover, the problem of low half - life in vivo remains intact.
• such methods of local administration of IFN ⁇ mention may be made of the inhalation of IFN ⁇ for the treatment of lung cancer, its nebulization in the treatment of the allergic response, or its encapsulation in liposomes.
• the cellular response to IFN ⁇ depends on the type of cells stimulated, the local concentration of IFN ⁇ and the other regulatory factors to which the cell is exposed concomitantly.
• IFN ⁇ was also capable of binding to oligossacharides of the heparin or heparan sulphate (HS) type, with a high affinity (5 to 10 nM) (11) .
• heparan sulfate effectively fixes IFN ⁇ , and this interaction controls the plasma elimination of the cytokine, its accumulation in various organs, and its location in the tissues.
• the IFN ⁇ is eliminated by a biexponential process, during which 90% of the cytokine disappears from the blood circulation during the first 5 to 10 minutes, with a particularly short half-life time, similar 1 minute.
• hepatocytes and endothelial cells - in the renal glomeruli or in the red pulp of the spleen (13).
• heparin makes it possible to displace the cytokine accumulated in the tissues by endogenous HS, and therefore to reduce or suppress its activity.
• a heparin molecule to protect IFN ⁇ and increase its bioavailability, or on the contrary to suppress its local action, raises difficulties which arise from the activities of heparin, itself, and , in particular, its anticoagulant properties.
• HS heparan sulfates
• the interaction site for IFN ⁇ was characterized. It consists of highly sulfated octasaccharide domains separated by a larger, less sulfated 7 kD domain.
• Figure 1 shows a dimeric complex of IFN ⁇ and heparan sulfate.
• the two octasaccharide domains are represented by bold lines in FIG. 1.
• the molecules designated by A and B are fragments of depolymerization of heparin or of sparse sulfate. It may be recalled here that these molecules are characterized by very great structural heterogeneity, and that there is no method for obtaining molecules of this type of defined structure, from natural sources. The compound described in this document therefore in fact represents a mixture of molecules of various structures. Likewise for segment X which, depending on the case, may also be a fragment of depolymerization of heparan sulfate.
• Such a molecule does not necessarily have C2 symmetry (the fragments A, X and B are "in lines", or oriented in the same direction, or else arranged in a "parallel” manner) and therefore does not respect the symmetry of the protein it's supposed to bind to.
• these molecules are of animal origin and have the disadvantage of carrying possible transmissible infectious agents.
• the object of the invention is to provide a molecule capable of binding to IFN ⁇ , which meets, among other things, the needs indicated above.
• the object of the invention is also to provide a molecule capable of binding to IFN ⁇ , which does not have the drawbacks, limitations, defects and disadvantages of analogous molecules of the prior art, in particular heparin and which resolves the problems of the prior art.
• X is a divalent spacer group of sufficient length to allow the two oligosaccharide fragments A and B to each bind to one of the peptide sequences 125 to 143 of the C-terminal ends of an interferon- ⁇ homodimer (IFN ⁇ )
• n represents an integer from 0 to 10, for example and equal to 0.1, 2, 3, 4, or 5 and each R independently represents a hydrogen atom, an SO 3 "group , or a phosphate group, provided that no SO 3 "group is found in position 3 of the glucosamine units of compound (I).
• all R's represent a group
• the molecule according to the invention is new. It is not identical to any natural molecule, nor to any of the molecules synthesized in the prior art and in particular in application FR-A-2 736 832 (O-A-97/03700).
• the molecule according to the invention has a specific structure due, first of all, to the fact that the two oligosaccharide groups, placed on either side of the spacer group X, are in an arrangement which can be qualified "symmetrical” or “antiparallel” with respect to the spacer arm, while both in the natural molecules of heparin and heparan sulfate, as in synthetic heparins and in similar molecules described in the prior art, both oligosaccharide groups are in a "parallel” or "asymmetrical” arrangement. These molecules therefore do not respect the symmetry of the protein on which they are capable of binding.
• the natural molecules and the molecules of the prior art, whether they are heparin, heparan sulfate, or molecules analogous thereto ' are completely, completely asymmetrical, that is to say that they come in a form of type "1212", while the molecules of the invention are in an antiparallel form of type "1221", that is to say having a symmetry of type C2.
• the molecules of the invention are fundamentally distinguished from natural molecules and molecules of the prior art by another essential structural characteristic in the sense that the molecules according to the invention do not contain a sulfate group in position 3 of the units glucosamine.
• the molecules according to the invention therefore do not have one of the main drawbacks of the molecules of the prior art linked to their anticoagulant activity .
• the molecule according to the invention which can be defined as being a "structural mimic" of heparan sulfate or heparin, in which heparin-type oligosaccharides are linked by a hydrophilic spacer (X) of modular length, is specifically binds to IFN ⁇ like HS.
• the compound according to the invention therefore possesses, and even beyond, all the advantageous properties of heparin: namely, inter alia, the fact of protecting the IFN ⁇ molecule against attacks by proteases, the increase in bioavailability of IFN ⁇ , the ability to dissociate, by competition, an iFN ⁇ / heparan sulfate complex, without having the essential drawback: namely, the anticoagulant activity.
• the molecules according to the invention are entirely synthetic molecules, unlike molecules of the prior art, represented, for example, by the document FR-A-2 736 832, in which the "terminal" oligosaccharide fragments are of natural origin, as in most cases, as the spacer arm.
• the disadvantages presented by the molecules of document FR-A-2 736 832 have already been described above, in particular as regards the natural, animal origin of the fragments which constitute them.
• the spacer group has a length o from 15 to 150 A, preferably from 33 to 50 A.
• Affinity for the cytokine is optimal for a length of 33 to 50, for example 50 A.
• the spacer group consists of a carbon chain, preferably from 1 to 120 C, one or more of the carbon atoms of which are optionally replaced by a heteroatom chosen from N, S, P and O, an S0 group, or a aryl group, said carbon chain carrying, optionally, one or more anionic groups preferably chosen from sulphate, phosphate and carboxylic groups, etc.
• the spacer group X is derived from a polyglycol chosen, preferably, from poly (alkylene glycol) of which the alkylene group comprises from 1 to 4 C, such as poly (ethylene glycol).
• the spacer group will be able to respond to the
• n is generally an integer from 5 to 32.
• n and m have the meaning already given above.
• the invention also relates to a process for preparing the compounds corresponding to formula (II), in which the radical coupling of two compounds is carried out water soluble precursors of oligosaccharides of formula (III):
• n is an integer from 0 to 10, for example equal to 0, 1, 2, 3, 4 or 5
• R 1 and R 2 represent a protecting group for the hydroxyl group chosen, preferably, from p-methoxybenzyl groups and benzyl, with a dithiol precursor compound of the spacer group of formula:
• R 1 is preferably a p-methoxybenzyl group and R 2 is a benzyl group.
• the water-soluble compound precursor of oligosaccharides whose formula is given above (III) is prepared by the following successive steps: a) a disaccharide of formula (V):
• a disaccharide of formula (V), above is subjected to an isomerization of the allyl group to 1-propenyl, followed by a hydrolysis of the enol ether formed and an activation of the hydroxyl group in the form of trichloroacetamidate to give a “donor” disaccharide, of formula (VIb):
• the disaccharide of formula (V) is preferably prepared by a coupling reaction between a compound of formula (VIII):
• a preferred process for preparing the compound (XI) is to carry out the acetylation of the compound of formula:
• the compound of formula (XI) is obtained with a very high yield, generally greater than or equal to 95%, and high purity since the furano derivatives are only present in trace amounts in a proportion generally close to or less at 2%.
• the therapeutic uses of the compound according to the invention can use one but also several compounds according to the invention.
• the invention further relates to the compound (I) and the preferred compounds (II), and (IIa) to (Ili) for use as a medicament, generally, the compound (I) and the compounds (II) and (IIa) to (Ili) being new.
• the invention relates to the use of the compound (I) and of the compounds (II) and (IIa) to (IIi) in general for preparing a medicament.
• the compounds according to the invention can be used to modulate, for example inhibit, the activity of exogenous or endogenous interferon- ⁇ .
• the invention therefore also relates to the compound (I) and to the compounds (II) and (IIa) to (Ili) for use as a modulator, for example an inhibitor, that is to say reducing or suppressing activity endogenous or exogenous interferon- ⁇ .
• a modulator for example an inhibitor, that is to say reducing or suppressing activity endogenous or exogenous interferon- ⁇ .
• the invention relates, in addition, to compound (I) and to compounds (II) and (IIa) to (Ili) (alone (s): that is to say without other active principle of different structure) for a use in the treatment of diseases linked to, or characterized by the presence of proinflammatory cytokines such as IFN- ⁇ , these are for example autoimmune, inflammatory or degenerative diseases such as multiple sclerosis, glomerulonephritis, Crohn's disease and rheumatoid arthritis, transplant rejection, etc.
• the invention thus also relates to compound (I) and to compounds (II) and (IIa to (II)) alone for use in a treatment which complements the immunosuppressive treatments used, for example, to avoid rejection of transplants.
• the invention also relates to the use of (I) (alone) and of compounds (II) and (IIa) to (Ili) for preparing a medicament intended for the treatment of disorders, pathologies, related to the activity, in particular excessive endogenous or exogenous interferon- ⁇ and the use of (I) to prepare a medicament intended for the treatment of diseases linked to, or characterized by the presence of pro-inflammatory cytokines such as IFN- ⁇ , it s 'acts for example " autoimmune, inflammatory, or degenerative diseases such as multiple sclerosis, glomerulonephritis, Crohn's disease, and rheumatoid arthritis, transplant rejection, etc.
• pro-inflammatory cytokines such as IFN- ⁇
• the invention also relates to the use of a compound (I) and of compounds (II) and (IIa) to (Ili) for preparing a medicament intended for a treatment complementary to the immunosuppressive treatments used, for example, to avoid rejection of transplants.
• the invention further relates to a medicament containing a compound (or more compounds) of formula (I) or of formula (II) or (IIa) to (Ili), alone (s) (i.e. - say without other active compound); to a composition containing the compound (or more compounds) of formula (I), (II), (IIa) to (Ili), alone (s) and a pharmaceutically acceptable vehicle for use in the treatment of diseases linked to or characterized by the presence of pro-inflammatory cytokines such as IFN- ⁇ (these are example of autoimmune, inflammatory, or degenerative diseases such as multiple sclerosis, glomerulonephritis, Crohn's disease and rheumatoid arthritis, transplant rejection, etc .; to a composition containing the compound (or more compounds) of formula (I), (II), (IIa) to (Ili) alone and a pharmaceutically acceptable vehicle for use in a treatment complementary to the immunosuppressive treatments used, for example example, to avoid rejection of transplants
• interferon-gamma is a proinflammatory cytokine, the presence of which characterizes a certain number of pathologies linked to inflammation. In such situations, it is useful to suppress or reduce the biological activity of the endogenous interferon-gamma. In animals, experimental models have proven the advantage of such a strategy (inhibition of interferon-gamma) using inhibiting monoclonal antibodies, or a soluble form of the cytokine receptor. As an example, we can cite autoimmune or degenerative diseases (multiple sclerosis,
• interferon-gamma can be an effective complement to the immunosuppressive treatments for example by cyclosporine used for example to avoid rejection of grafts.
• Medicines containing the compound (or compounds) (I) alone (s) can be administered in doses which can be determined beforehand by routine experiments, depending in particular on the desired effect. These doses can range, for example, from 0.1 to 200 mg per individual per day, preferably from 1 to 50 mg.
• the invention further relates to a medicament containing, in addition to the compound (I) or the preferred compounds (II) from (IIa to (II)), interferon- ⁇ .
• Such a medicament contains in combination the compound (I) and the interferon- ⁇ , preferably at a rate of
• the compound (I) e.g., 0.05 to 1 mg of interferon- ⁇ and from 1 to 50 equivalents of compound (I).
• the compound (I) e.g., 0.05 to 1 mg of interferon- ⁇ and from 1 to 50 equivalents of compound (I).
• the compound (I) e.g., 0.05 to 1 mg of interferon- ⁇ and from 1 to 50 equivalents of compound (I).
• the compound (I) e.g.
• interferon- ⁇ is preferably in the form of a complex of the compound (I) and the interferon- ⁇ . Said complex makes it possible to increase the bioavailability of the cytokine and protects it from proteolytic degradations.
• the compound (I) prevents the capture of interferon- ⁇ by endogenous sparse sulfate molecules, present, for example, in the extracellular matrix and on the surface of many cells, and therefore allows its maintenance and transport in general traffic.
• the compound (I) protects the interferon- ⁇ against degradations capable of reducing or canceling its activity, and it makes it possible to maintain the interferon- ⁇ in its most active form, until the moment of its action on competent cells.
• the invention therefore relates to: the complex of compound (I) (or (IIa) to (Ili)) and of interferon- ⁇ for use as a medicament.
• this complex is new and its therapeutic use has never been mentioned; the complex, above, for use as an immunostimulant.
• Immunostimulatory effects include, for example, the antiproliferative effect in cancers and the activation of immune defenses in infectious diseases, for example viral, bacterial or parasitic, or the ability to block the synthesis of collagen in fibrosis of organs, etc.
• the invention will therefore relate to the above complex for use in the treatment of a disease chosen from cancer, infectious diseases, for example viral, bacterial or parasitic, and organ fibrosis.
• the invention relates generally to a medicament containing said complex, as well as the use of the complex for the treatment of a disease, as mentioned above.
• the invention also relates to the use of the complex, in general, to prepare a medicament and, in particular, to the use of the complex to prepare a medicament intended for the treatment of a disease, as mentioned above.
• the invention further relates to a composition containing said complex and a pharmaceutically acceptable vehicle for use in the treatment of a disease selected from cancer, infectious diseases, for example viral, bacterial or parasitic, and fibrosis of organs.
• the spacer group consists essentially of a polyglycol, in particular of a polyethylene glycol.
• FIG. 1 represents an IFN ⁇ / HS dimer complex or molecule according to the invention
• FIG. 2 is a graph which gives the% inhibition I for different molecules according to the invention defined by the length L of the spacer arm o (in A) and of the number of saccharides of the oligosaccharide groups (tetra, hexa 5 or octasaccharide ).
• the compounds according to the invention can be defined as structural mimics of heparin or heparan sulfate or also as neoglycoconjugates corresponding, in particular, to formula (II). 0
• tetra with octasaccharides, of the heparin type are linked by a hydrophilic spacer of modular length, for example a polyglycol type spacer.
• the molecules according to the invention bind specifically to IFN ⁇ , in the manner of Heparan-Sulphate, as described in FIG. 1.
• L-Iduronic acid which is a rare sugar that cannot be isolated in sufficient quantity from natural sources for synthetic needs.
• R '- CH CH 2 5a, 8a, 9a
• L-Iduronic acid is a rare sugar that it is impossible to isolate in sufficient quantity from natural sources for synthetic needs. It is therefore essential to have effective methods for preparing derivatives of this compound (25). Although the addition of organometallic to aldehyde 12 (26) is postponed to occur with a very low diastereoselectivity (27), a study has been undertaken within the framework of the invention. nucleophiles precursors of carboxylate groups on this compound (diagram 4).
• This methodology was also applied in part to the glucuronic synthon 14a, and made it possible to prepare a glucuronic acid donor having protective groups close to 26.
• the acceptor disaccharide 25 is easily prepared by oxidative cleavage of the paramethoxybenzyl group by DDQ: dichlorodycyanoquinone.
• the donor disaccharide 26 (29) is, for its part, prepared by isomerization of the allyl to 1-propenyl with an iridium-based catalyst, (30), then hydrolysis of the ethenol ether, thus formed, catalyzed by mercury salts, which allows the release of the anomeric position, which is then activated in the form of trichloroacetimidate (scheme 9).
• the protected oligosaccharides are transformed into partially deprotected and water-soluble compounds by: deacetylation, reduction of the azido function, sulfation, then saponification (diagram 11).
• This device uses an optical detection system, the surface plasmon resonance phenomenon, to measure the concentration of molecules that have reacted on the surface of a biosensor ("sensor chip").
• Biotinilated heparin is immobilized on a "sensor chip” previously activated with streptavidin. It is then possible to measure the interaction of IFN ⁇ , injected in continuous flow on the surface of this sensor, with the immobilized heparin. The interaction between heparin and IFN ⁇ causes a change in mass at the surface of the sensor, which is recorded as a function of time.
• the IFN ⁇ is previously incubated with the various synthesized products, then the complexes are injected onto the surface of the biosensor. The capacity of the products tested is then measured to inhibit the interaction of IFN ⁇ / heparin.
• the synthesis of compounds according to the invention is described: namely, neoglycoconjugates fixing the interferon, in which the group or spacer arm is derived from poly (ethylene glycol) s of variable lengths and the two oligosaccharides of ends are constituted by trisulfated disaccharides.
• the starting product of the synthesis is compound 8 ⁇ , prepared according to LUBINEAU A.; ESCHER S.; ALAIS J.; BONNAFFE D., Tetrahedron Lett. 1997, 38, 4,087 - 4,090, that is to say according to the following operating mode 1: Operating mode 1:
• glucosamine hydrochloride is peracetylated in a mixture of acetic anhydride (20 equivalents) and pyridine (30 equivalents). After evaporation of the reagents and coevaporation with toluene, the mixture is crystallized from an ethyl acetate / petroleum ether mixture to yield peracetylated glucosamine with a yield of 90%. The crystals recovered are treated with 1.1 equivalent of hydrazine acetate in anydre THF (concentration 0.3 M) for 20 h at 20 ° C.
• the THF is then evaporated at room temperature under reduced pressure, the residue coevaporated with toluene and the residual oil directly chromatographed on silica gel with an AcOEt / petroleum ether / CH 2 Cl 2 mixture (8/1/1 to 8 / 0/2).
• the free compound in the anomeric position is obtained with 75% yield. It is then dissolved in the necessary volume of CH 2 C1 2 to have a concentration of 0.5 M and added dropwise to a mixture of tetrabutylammonium bromide (2 eq.), NaH (1.5 eq) and allyl bromide (20 eq.) Cools to -20 ° C. The temperature is then allowed to rise to 20 ° C.
• Compound 8a ⁇ is treated with eight equivalents of Ba (OH) 2 in water at 100 ° C overnight, the pH is then lowered to 3 by adding sulfuric acid, the mixture centrifuged to precipitate barium sulfate , the supernatant collected, then evaporated under reduced pressure and coevaporated with water to remove the acetic acid formed.
• Salt 18 is neutralized with potassium carbonate and directly treated with TfN 3 , following the protocol described in ALPER PB; HUNG SC; ONG CH, Tetrahedron Lett. 1996, 37, 6 029-6032, that is to say according to the following procedure 2: Procedure 2: 15 equivalents of NaN 3 are dissolved in the minimum of water necessary and cooled to 0 ° C.
• the resulting solution is deposited on silica gel 70-200 ⁇ by evaporation and the compound 19 eluted with a MeOH / CH 2 Cl 2 8/2 mixture.
• the residue is dissolved in anhydrous acetonitrile and treated with benzaldehyde dimethylacetal in the presence of camphorsulfonic acid. After two hours at room temperature, the solution is neutralized by adding an aqueous solution of sodium bicarbonate and the compound extracted with diethyl ether. After purification by chromatography on silica gel (petroleum ether / AcOEt), the compound 20 is treated with benzyl bromide in the presence of NaH in DMF. After dilution with diethyl ether, washing of the organic phase with water and evaporation, the residue is treated at 50 ° C., with a 60% acetic acid solution, for two hours.
• derivative 17 is prepared according to LUBINEAU A.; GAVARD O.; ALAIS J.; BONNAFFE D., Tetrahedron Lett. 2000, 307 - 311, that is to say according to the following operating mode 3: Operating mode 3:
• a solution of tris-phenylthiomethyl-lithium is prepared in the following manner: 1.2 equivalents (relative to the aldehyde) of tris-phenylthiomethane are dissolved in the quantity of THF necessary to obtain a concentration of 0.8 M, the temperature is lowered to -78 ° C and the mixture put under mechanical stirring, then 1.1 equivalents (relative to the aldehyde) of n-butyllithium in solution in hexane are added. A yellow precipitate appears and stirring is continued at -78 ° C for 1 h 30. The aldehyde, dissolved in THF (0.6 M concentration) is then added dropwise. The mixture is stirred at -78 ° C.
• Tris-phénylethioorthoester 13c obtained above is dissolved in the volume of methanol necessary to obtain a concentration of 0.05 M was then added l / 10th volume of water and / 10th volume of dichloromethane and then 1.7 CuO equivalents and 4 CuCl 2 equivalents. After one hour at room temperature, the mixture is filtered through Celite 545 and evaporated at room temperature under reduced pressure. The residue is taken up in CH 2 C1 2 and the organic phase is washed with a saturated NaCl solution, filtered through phase separator paper and evaporated. The mixture is purified by chromatography on silica (eluent: petroleum ether / AcOEt 8/2 to 5/5), product 14 is thus obtained with a yield of 94%.
• the product 14 is then treated with a 90% trifluoroacetic acid solution for 30 minutes at room temperature (initial concentration of the reagent: 0.35 M).
• the reaction medium is evaporated under reduced pressure and then coevaporated twice with water. An oil is thus obtained which crystallizes.
• These crystals of compound 15 are suspended in dichloromethane (concentration 0.2 M) and the temperature is lowered to -40 ° C, 9 equivalents of pyridine, 0.01 equivalent of 4-dimethylaminopyridine and 5 equivalents of chloride are then added. acetyl.
• the derivative 17 is converted into donor 27 according to JACQUINET J. C.; PETITOU M.; DUCHAUSSOY P.; LEDERMAN I.; CHOAY J.; TORRI G.; SINAY P., Carbohydrate Res. 1984, 130, 221, that is to say according to the following procedure 4: Procedure 4:
• the mixture 17 is dissolved in anhydrous dichloromethane (0.1 M concentration), then 1/10 th by volume of anhydrous ethyl acetate is added and the mixture is left to react for 24 hours in the presence of 1.3 equivalents of TiBr 4 .
• the mixture is diluted with CH 2 C1 2 and washed with ice water.
• the organic phase is filtered on silicone paper and then concentrated.
• the product 27 thus obtained is used directly in the next step.
• the basic disaccharide 10 is transformed with a 95% yield into donor disaccharide 26 (synthesized by another route in TABEUR C.; MALLET JM; BONO F.; HERBERT JM; PETITOU M.; SINAY P., Bioorg., Med. Chem 1999, 7, 2 003 - 2 012, by isomerization of allyl, in the presence of C 8 H 14 MePh 2 PIr 1 PF s and cleavage of enol by mercury salts, as described by OLTVOORT JJ; VAN BOECKEL CAA; KONING JH; VAN BOOM JH, Synthesis 1981, 305 - 308, that is to say according to the following operating mode 5: Operating mode 5:
• Disaccharide 10 is dissolved in anydre THF so as to have a concentration of 0.06 M.
• the solution is degassed under vacuum, then 0.013 equivalents of C 8 H 14 MePh 2 PIr I PF 6 are added .
• the mixture is redegased then left in contact with dihydrogen for 2 minutes and finally redegased then put under an argon atmosphere.
• the mixture is evaporated and then taken up in the volume of acetone necessary to obtain a concentration of 0.05 M, 1.2 equivalents of HgO and 1.1 of HgCl 2 are then added. .
• the mixture is filtered under Celite 545, evaporated and taken up in ethyl ether.
• Disaccharide 10 treated with 1.5 equivalents of DDQ in dichloromethane saturated with water, leads after washing the organic phase with a saturated NaHCO 3 solution and chromatography on silica gel with petroleum ether / AcOEt mixture and acceptor disaccharide 25 with a yield of 95%.
• the coupling of the acceptor disaccharide 25 and the donor disaccharide 26 (1.3 equivalents) is carried out in o dichloromethane at -40 ° C. in the presence of 4 A sieve and of terbutyldimethylsilyl triflate (0.2 equivalents). After addition of the catalyst, the reaction is left for 30 minutes at -40 ° C, then the temperature is allowed to rise to 0 ° C, temperature at which the reaction is stopped by adding 0.2 equivalents of triethylamine. Chromatography on direct silica gel of the reaction mixture with the CH 2 Cl 2 / AcOEt / petroleum ether mixture makes it possible to isolate the tetrasaccharide 27 with a yield of 95%.
• tetrasaccharides acceptor 28 (90% yield) and donor 29 (95% yield) using the same reaction sequences as for disaccharide 10.
• the couplings of the donor disaccharide 26 with the acceptor tetrasaccharide 28 and donor tetrasaccharide 29 with the acceptor tetrasaccharide 28 lead to hexasaccharide 30 and to octasaccharide 31 with yields of 95%.
• the oligosaccharides 27, 30 and 31 are deacetylated in anhydrous methanol in the presence of 0.5 equivalent of anhydrous K 2 C0 3 .
• the products are purified by chromatography on silica gel and obtained with yields of 86 to 95%.
• the reduction of the azido groups is carried out with propane dithiol (2 equivalents per azido group) in methanol in the presence of triethylamine (2 equivalents per azido group).
• the residues are chromatographed on silica gel (CH 2 Cl 2 / MeOH) to yield the amino compounds with yields of 90% to 80%. These compounds are then sulfated by the pyridine complex.
• the couplings of oligosaccharides 32 to 34 (2.5 equivalents) on the dithiol spacers derived from polyethylene glycol of variable lengths: namely m 5, 10 and 32, at a concentration of 0.2 M in water, s' perform under ultraviolet irradiation at 365 nM or by heating in the presence of a radical initiator.
• the mixture is directly treated with 8 equivalents of oxone, that is to say with a 0.2 M solution, brought to pH 6 by addition of K 2 HP0 4 .
• the excess oxidant is reduced by adding a sodium thiosulfate solution and the mixture is directly purified by semi-preparative HPLC on reverse phase C18 (CH 3 CN / AcOH buffer -NEt 3 5 mM pH 7.0).
• BIORAD ® AG 50 WX 8 200 Na +, oligosaccharides are resolubilized in 100 uL 40 mM sodium phosphate buffer pH 7 and spokes under hydrogen atmospheric pressure in the presence of a mass equivalent of palladium hydroxide 20% on coal.
• OZMEN L. ROMAN D., FOUNTOULAKIS M., SCHMID G., RYFFEL B., GAROTTA GJ, Interferon Res, 1994, 14: 283 - 4.
• OZMEN L. ROMAN D., FOUNTOULAKIS M., SCHMID G ., RYFFEL B., GAROTTA G., Eu. J. Immunol. , 1995, 25: 6-12.

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Medicinal Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Veterinary Medicine (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Organic Chemistry (AREA)
• Pharmacology & Pharmacy (AREA)
• Animal Behavior & Ethology (AREA)
• Public Health (AREA)
• Engineering & Computer Science (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• General Chemical & Material Sciences (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Immunology (AREA)
• Molecular Biology (AREA)
• Epidemiology (AREA)
• Polymers & Plastics (AREA)
• Materials Engineering (AREA)
• Biochemistry (AREA)
• Oncology (AREA)
• Communicable Diseases (AREA)
• Rheumatology (AREA)
• Dermatology (AREA)
• Virology (AREA)
• Biomedical Technology (AREA)
• Pain & Pain Management (AREA)
• Neurology (AREA)
• Tropical Medicine & Parasitology (AREA)
• Neurosurgery (AREA)
• Transplantation (AREA)
• Orthopedic Medicine & Surgery (AREA)
• Physical Education & Sports Medicine (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Polysaccharides And Polysaccharide Derivatives (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Applications Claiming Priority (3)

Publications (1)

Family

ID=29719874

Family Applications (1)

Country Status (7)

Families Citing this family (6)

Family Cites Families (1)

• 2002
  • 2002-06-19 FR FR0207562A patent/FR2841250B1/fr not_active Expired - Fee Related
• 2003
  • 2003-06-18 AU AU2003260601A patent/AU2003260601A1/en not_active Abandoned
  • 2003-06-18 EP EP03760740A patent/EP1521779A1/fr not_active Withdrawn
  • 2003-06-18 JP JP2004514944A patent/JP2005533881A/ja not_active Withdrawn
  • 2003-06-18 WO PCT/FR2003/001860 patent/WO2004000887A1/fr active Application Filing
  • 2003-06-18 US US10/518,177 patent/US20060166927A1/en not_active Abandoned
  • 2003-06-18 CA CA002489561A patent/CA2489561A1/fr not_active Abandoned

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events