WO2012052596A1 - Ganglioside derivatives and use thereof as inhibitors of tumour cell division - Google Patents
Ganglioside derivatives and use thereof as inhibitors of tumour cell division Download PDFInfo
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- WO2012052596A1 WO2012052596A1 PCT/ES2011/070732 ES2011070732W WO2012052596A1 WO 2012052596 A1 WO2012052596 A1 WO 2012052596A1 ES 2011070732 W ES2011070732 W ES 2011070732W WO 2012052596 A1 WO2012052596 A1 WO 2012052596A1
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Classifications
-
- 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
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/02—Acyclic radicals, not substituted by cyclic structures
- C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
- C07H15/10—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical containing unsaturated carbon-to-carbon bonds
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y203/00—Acyltransferases (2.3)
- C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
- C12Y203/01045—N-Acetylneuraminate 7-O (or 9-O)-acetyltransferase (2.3.1.45)
Definitions
- the present invention relates to new inhibitors of tumor cell division.
- Said inhibitors are semi-synthetic derivatives of neurostatin.
- the present invention relates to a new synthesis procedure.
- inhibitors and neurostatin refers to a procedure for obtaining O-acylated gangliosides specifically in terminal sialics. It also refers to the use of these inhibitors for the manufacture of a medicament for the treatment of brain tumors.
- the invention is part of the pharmaceutical sector, and is applicable in the medical sector for the treatment of brain tumors.
- Glioblastoma multiforme is the most common primary brain tumor in adults and also the most deadly. In the United States, only half of the patients receiving standard treatment survive one year after diagnosis. Less than one in ten survives more than five years. Usually
- Brain tumors are the first cause of death
- Treatment depends on the location and grade of the tumor; Surgery is applied when the tumor is accessible and there is no danger of damaging vital structures. Radiation therapy is used to stop tumor growth or to
- Gangliosides are glycolipids formed by a ceramide chain (which is composed of a residue of the aminoalcoholesphingosine bound to a fatty acid) linked to an oligosaccharide chain. Gangliosides have as their structural characteristic, the presence of one or more sialic acid residues in the oligosaccharide chain.
- Neurostatin is characterized by being a ganglioside of the series b, GD1 b, with a modification in the form of O-acetylation in one of the hydroxyls of its terminal sialic (Romero-Ramirez L, Nieto-Sampedro M. Inhibiting human astrocytomagrowth: structure - activityrelationships in neurostatinrelatedglycolipids. Journal of Medicinal Chemistry 2004; 47 (21): 4983-4.).
- the position of this O-acetylation is preferably in the hydroxyl of carbon 9, since although O-acetylations in carbons 7 and 8 have also been described, under physiological pH conditions the O-acetyls in those carbons migrate to position 9 O-acetylation is the most frequent modification in nature and is preferably found in the sialics of gangliosides linked in links to (2,8) and (2,3) to the glycidic chain.
- Neurostatin is a natural ganglioside that is expressed in very low concentration in the central nervous system of mammals (Abad-Rodr ⁇ guez J, Bernabé M, Romero-Ramirez L, Vallejo-Cremades M, Fernandez- Mayoralas A, Nieto-Sampedro M. Purification and structure of neurostatin, an inhibitor of astrocyted vision of mammalianbrain, Journal of Neurochemistry 2000; 74 (6): 2547-56.).
- the present invention describes a new series of gangliosides with inhibitory activity (of the neurostatin family) of tumor cell division and glioma growth.
- the present invention describes a novel method of obtaining O-acetylated gangliosides and neurostatin which is characterized by its simplicity, specificity and high performance. This procedure has a commercial interest in the field of glycobiology for the production of these O-acetylated gangliosides in large quantities.
- O-propionylated and O-malonylated gangliosides are very interesting products for clinical use as antitumor agents.
- a first aspect of the present invention relates to a method of synthesizing a compound, specifically a ganglioside, of the general formula.
- Z is selected from a group of formula Zi:
- Z 2 is a group of formula (Z 2 ):
- Xi is selected from -CH 2 -O-CO-X 2 and -CH 2 OH;
- X 2 is selected from a Ci-C 2 and -CH 2 COOH alkyl group
- X3 is selected from H, OH, a group of formula (II) or a group of formula
- X5 is selected from an OH group or a -0-COXe group
- Xese selects from a C 1 -C 2 or -CH 2 COOH alkyl group; Except when:
- - Z is a group of formula (Z 1 ) or (Z 2 ), X 1 is -CH 2 -OH and X 3 is H;
- - Z is a group of formula (Zi) or (Z 2 ), Xi is -CH 2 -OH, X 3 is the group of formula (III) and X4 is H;
- - Z is a group of formula (Z1) or (Z 2 ), X1 is -CH 2 -OH, X 3 is the group of formula (III) and X4 is the group of formula (IV) and X 5 is OH;
- - Z is a group of formula (Z1) or (Z 2 ), X1 is -CH 2 -OH, X 3 is the group of formula (III) and X4 is the group of formula (V) and X 5 is OH; comprising the steps of: a) dissolving a ganglioside in a buffer solution; b) add an emulsifier to the mixture of step a) c) add to the mixture of step b) the enzyme o-acetyltransferase; and d) stop the reaction by adding methanol to the mixture obtained in step c).
- the ganglioside of the general formula (I) is selected from the group consisting of O-acetyl-GM3, O-acetyl-GM2, O-acetyl-GD3, O-acetyl-GD2, Neurostatin, AO-acetyl-GT1 b, AO-acetyl-GQ1 b, O-propionyl-GD1 b, O-propionyl-GD3, O-propionyl-GD2, AO-propionyl-GT1 b, BO-propionyl-GT1 b, BO-acetyl-GT1 b, di -O-acetyl-GT1 b, O-Propionyl-GM3, O-Malonil-GM3, O- Propionyl-GM2, O-Malonil-GM2, O-Malonyl-GD3, O-Malonil-GD2, O-Propionyl- GD1 a , O-Malonil-GD1
- Y is any ceramide, which is composed of a sphingosine and a fatty acid.
- sphingosine comprises 18 to 20 carbon atoms and has a number of unsaturations of 0 to 2.
- the fatty acid may contain 8 to 24 carbon atoms and with a number of unsaturations of 0 to 4;
- GD1 b, GD3, GD2, GT1 b, GQ1 b, GM3, GM2, GM1 a or GD1 a refer to gangliosides of the same name known in the state of the art by any person skilled in the art.
- these gangliosides comprise among other elements, a ceramide (Y) which in turn comprises a sphingosine and a fatty acid.
- Sphingosine is preferably 18 carbon atoms with an unsaturation or 20 carbon atoms with an unsaturation.
- the fatty acid is 18 carbon atoms without any unsaturation or unsaturation.
- the compound of general formula I has the formula (la):
- Xi is -CH 2 -O-CO-X 2 ;
- X2 is a C1-C2 alkyl group
- X3 is selected from H, OH, a group of formula (II) or a group of formula (III):
- X 5 is selected from an OH group or a - ⁇ -COXe group; and ⁇ is a C1-C2 alkyl group;
- - Xi is -CH2-O-CO-X2, X2 is a group CH 3 , X3 is the group of formula (III) and X4 is H;
- - Xi is -CH2-O-CO-X2, X2 is a CH 3 group, X3 is the group of formula (II);
- - Xi is -CH2-O-CO-X2
- X2 is a group CH 3
- X3 is the group of formula (III)
- X4 is the group of formula (IV)
- X 5 is OH
- - Xi is -CH2-O-CO-X2, X2 is a group CH 3 , X3 is the group of formula (la) and X4 is OH; comprising the steps of: a) dissolving a ganglioside in a buffer solution; b) add an emulsifier to the mixture of step a) c) add to the mixture of step b) the enzyme o-acetyltransferase; and d) stop the reaction by adding methanol to the mixture obtained in step c).
- the ganglioside is selected from the group consisting of GD1 b, GD3, GD2 and GT1 b, as they have been defined above.
- Said gangliosides GD1 b, GD3, GD2 and GT1 b, as mentioned above, comprise among other elements, a ceramide (Y) as defined above, which in turn comprises a sphingosine and a fatty acid.
- Sphingosine is preferably 18 carbon atoms with an unsaturation or 20 carbon atoms with an unsaturation.
- the fatty acid is 18 carbon atoms without any unsaturation or unsaturation.
- a ganglioside concentration between 50 to 600 pmoles / L reaction, preferably between 300 and 600 pmoles / L and more preferably 500 moles / L is added
- the buffer solution is added in a concentration from 1 to 250 mmol / L of reaction preferably from 0 to 100 mM, more preferably at a concentration of 50 mM, at a pH from 6 to 9 , preferably at a pH between 7 and 8, more preferably at a pH of 7.0.
- the buffer solution is from TRIS (tris (hydroxymethyl) aminomethane), MES (2- (N-morpholino) ethanesulfonic acid), phosphate, etc., preferably from MES.
- MgC buffer (10 mmol / L) and DTT (Dithiothreitol) (1 mmol / L) are added to the solution.
- a buffer and a coenzyme A derivative selected from Acetyl-Coenzyme A, Propionyl-Coenzyme A and Malonyl-Coenzyme A, more preferably Acetyl-Coenzyme A or Propionyl-Coenzyme A are added to the solution at a concentration from 0.1 to 10 mmol / L reaction, preferably between 0.5 and 2 mmol / L more preferably at 1 mmol / L.
- the emulsifier is a bile acid or bile salt at a preferable concentration between 0.05% and 0.2% total weight / volume of the reaction, more preferably 0.1% in Total weight / volume of the reaction. That is, the emulsifier is a bile acid or bile salt at a preferable concentration between 0.05% and 0.2% by weight with respect to the total volume of the reaction, more preferably 0.1% by weight with respect to the total volume of the reaction.
- the bile acid is selected from the group consisting of colic acid, deoxycholic acid, taurocolic, glycocolic, ursodeoxycholic, lithocolic acid, etc. as its sodium and potassium salts, (example: cholate, taurocholate, deoxycholate, ursodeoxycholate, glycocholate, etc. ).
- the emulsifier is the sodium bile colata salt.
- the emulsifier reduces the micellation of gangliosides, increasing the accessibility of terminal sialics to the enzyme O-acetyltransferase.
- the enzyme O-acetyltransferase is added at a concentration of between 2 and 30 pg / ⁇ reaction, preferably between 15 and 25 pg / ⁇ , more preferably at 20 Mg / ⁇ -
- the process of the present invention uses as an catalyst an O-acetyltransferase obtained from the bacteria Campylobacterjejunni, whose cloning, sequencing and activity have been patented (WO 2007016792 20070215) and published by Houliston R. et al (Houliston RS, Endtz HP, Yuki N, et al. The Journal of Biological Chemistry 2006; 281 (17): 11480-6).
- the reaction of step c) was performed between 30-40 ° C, preferably between 35-38 ° C, more preferably at a temperature of 37 ° C.
- the reaction of step c) was carried out during a reaction time between 1 hour and 24 hours, preferably between 5 and 12 hours, more preferably for 7 hours, and with stirring. After stopping the reaction with methanol in step d), the following steps were carried out:
- the procedure described in the present invention is a specific protocol for ganglioside O-acetylation. Unlike the procedure described in (Houliston RS, Endtz HP, Yuki N, et al. Identification of a sialate O-acetyltransferase from Campylobacter jejuni: demonstration of direct transfer to the C-9 position of terminalalpha-2, 8-linked sialic acid The Journal of Biological Chemistry 2006; 281 (17): 11480-6) and in the patent (WO 2007016792), in the process of the present invention an emulsifier, which can be a bile acid or a salt, is added to the reaction mixture bile, which in combination of the pH reduces the micellation of the gangliosides, increasing the accessibility of the terminal sialics to the enzyme O-acetyltransferase.
- the pH of the reaction must be equal to or greater than 6.5 for the sodium colata to be soluble in the buffer and can act on gangliosides
- the procedure has a much higher performance than those described above.
- the yield is 90%, compared to the scarce 10-15% of other procedures described in the prior art. Thanks to this performance, neurostatin can now be considered as an interesting product as an antitumor in patients and, therefore, with possible commercial use.
- the process described in the present invention also allows to obtain natural O-acetylated products of the neurostatin family, such as O-acetyl-GD2 and O-acetyl-GT1 b, which have not been described in the scientific literature as antitumor; and others, such as di-O-acetyl-GT1 b, which have not been described in nature, nor as antitumor agents.
- O-acetylated gangliosides the process described in the present invention can be used to prepare O-propionylated and O-malonylated gangliosides.
- Both the O-acetylated, O-propionylated or O-malonylated products may refer to derivatives with one or two acyl groups (acetyl, propionyl or malonyl).
- These O-propionylated products such as O-propionyl-GM3, O-propionyl-GM2, O-propionyl-GD3, O-propionyl-GD2, O-propionyl-GD1 b, O-propionyl-GT1 by O-propionyl-GQ1 b , and their respective O-malonylates have never been described in the literature, nor are they found in nature.
- these O-propionylated products have greater inhibitory activity of the proliferation of the C6 glioma line (Table 1) and could be more resistant to hydrolysis by glycosidases, so that their effect would be more lasting.
- a second aspect of the present invention relates to a compound (in hereinafter also referred to as ganglioside or compound of the invention), which has the general formula (I) according to as defined above, except when:
- - Z is a group of formula (Zi), Xi is -CH2-O-CO-X2, X2 is a group CH 3 , X3 is the group of formula (III) and X4 is H;
- - Z is a group of formula (Z1), X1 is -CH2-O-CO-X2, X2 is a group CH 3 , X3 is the group of formula (II);
- - Z is a group of formula (Z1), X1 is -CH2-O-CO-X2, X2 is a group CH 3 , X3 is the group of formula (III), X4 is the group of formula (IV) and X 5 is OH;
- - Z is a group of formula (Z1), X1 is -CH2-O-CO-X2, X2 is a group CH 3 , X3 is OH;
- - Z is a group of formula (Z1), X1 is -CH2-O-CO-X2, X2 is a group CH 3 , X3 is the group of formula (III), X4 is the group of formula (V), X 5 is -OH;
- - Z is a group of formula (Z 2 ), X1 is -CH2-O-CO-X2, X2 is a group CH 3 , X3 is OH;
- - Z is a group of formula (Z 2 ), X1 is -CH2-O-CO-X2, X2 is a group CH 3 , X3 is the group of formula (II);
- - Z is a group of formula (Z 2 )
- X1 is -CH 2 -OH
- X 3 is the group of formula (III)
- X4 is the group of formula (IV)
- X 5 is -O-CO-X6 and ⁇ is a CH 3 group.
- the compound of the invention has the formula
- X2 is a C1-C2 alkyl group
- X3 is selected from H, OH, a compound of general formula (II) or a compound of general formula (III)
- X ⁇ is selected from H and a compound of general formula (IV):
- X 5 is selected from an OH group or a -0-COXe group; ⁇ is a C1-C2 alkyl group;
- - Xi is -CH 2 -O-CO-X2, X2 is methyl, X3 is the compound of general formula (III) and X4 is hydrogen;
- Xies -CH2-O-CO-X2, X2 is methyl
- X3 is the compound of general formula (III) where X4 is the compound of general formula (IV) where X 5 is OH;
- - X1 is - CH2-O- CO-X2,
- X 2 is a methyl,
- X 3 is OH;
- - Xi is -CH2-O-CO-X2, X 2 is methyl, X 3 is OH;
- - Xi is -CH2-O-CO-X2, X2 is methyl, X3 is the group of formula (II);
- - Xi is -CH2-O-CO-X2, X2 is methyl, X3 is the group of formula (III), X4 is the group of formula (V) and X 5 is OH;
- Y is a ceramide comprising a sphingosine and a natural or synthetic fatty acid.
- sphingosine comprises 18 to 20 carbon atoms and has an unsaturation number of 0 to 2.
- the fatty acid comprises 8 to 24 carbon atoms and has an unsaturation number of 0 to 4;
- X 2 is a methyl group.
- X 2 is an ethyl group.
- X 2 is a -CH 2 COOH group.
- X 3 is H.
- X 3 is OH.
- X 3 is a group of formula (II).
- X 3 is a compound of general formula (III)
- X4 is H.
- X4 is a compound of general formula (IV)
- X4 is a compound of general formula (V).
- X 5 is an OH group.
- X 5 is a group - ⁇ -COXe.
- ⁇ is a methyl. According to another preferred embodiment, ⁇ is an ethyl.
- ⁇ is a -CH 2 COOH group.
- the compounds of the present invention are selected from the following list: O-propionyl-GD1b, O-propionyl-GD3, O-propionyl-GD2, AO-propionyl-GT1b, BO-propionyl-GT1b, di-O -propionyl-GT1b, BO-acetyl-GT1b, di-O-acetyl-GTIb, O-Propionyl-GM3, O-Malonyl-GM3, O- Propionyl-GM2, O-Malonyl-GM2, O-Malonil-GD3, O -Malonil-GD2, O-Propionyl- GD1a, O-Malonil-GD1a, O-Malonil-GD1b, AO-Malonil-GT1b, BO-Malonil- GT1b, di-O-Malonil-GT1b, BO
- B-0-propionyl-GT1 b where Y is any ceramide, which is composed of a sphingosine and a fatty acid.
- sphingosine comprises 18 to 20 carbon atoms and has a number of unsaturations of 0 to 2.
- the fatty acid may contain 8 to 24 carbon atoms and with a number of unsaturations of 0 to 4;
- GD1 b, GD3, GD2, GT1 b, GQ1 b, GM3, GM2, GM1 a or GD1 a refer to gangliosides of the same name known in the state of the art by any person skilled in the art.
- these gangliosides comprise among other elements, a ceramide (Y) which in turn comprises a sphingosine and a fatty acid.
- Sphingosine is preferably 18 carbon atoms with a unsaturation or 20 carbon atoms with unsaturation.
- the fatty acid is 18 carbon atoms without any unsaturation or unsaturation.
- Another aspect of the invention relates to a compound synthesized according to the process of the invention.
- Another aspect of the present invention relates to the compound of general formula (I) for use as a medicament.
- a third aspect of the present invention relates to a pharmaceutical composition
- a pharmaceutical composition comprising at least the compound of general formula (I), or a compound of formula (la), and at least one pharmaceutically acceptable carrier, adjuvant and / or vehicle.
- the pharmaceutical composition further comprises another active ingredient.
- a fourth aspect of the present invention relates to the use of the ganglioside of general formula (I), or a ganglioside of formula (la), or any of its salts, isomers or solvates, wherein; Y, Ac, Xi, X2, X3 , X4, X5 and ⁇ are defined as defined above for the preparation of a medicine.
- a preferred embodiment refers to the use for the preparation of a medicament for the treatment or prevention of tumors, preferably for the treatment or prevention of astrocytomas, glioblastomas, oligodendrogliomas, neuroblastomas or meningiomas.
- a fifth aspect of the present invention relates to the use of the ganglioside of general formula (I), or of the ganglioside of formula (la), or any of its salts, isomers or solvates, where: Y, Ac, X1, X2, X3 , X4, X5 and ⁇ are defined as defined above, as a reagent in biological assays.
- the compound described in the present invention, its salts, prodrugs and / or solvates as well as the pharmaceutical, cosmetic and nutritional compositions containing them can be used together with other drugs, or active ingredients, additional to provide a combination therapy.
- Said additional drugs may be part of the same pharmaceutical composition or, alternatively, they may be provided in the form of a separate composition for simultaneous or non-simultaneous administration to the pharmaceutical composition comprising the compound described above, or a salt, prodrug or solvate. of the same.
- the pharmaceutical compositions are suitable for oral administration, in solid or liquid form.
- Possible forms for oral administration are tablets, capsules, syrups or solutions and may contain conventional excipients known in the pharmaceutical field, as aggregating agents (eg syrup, acacia, gelatin, sorbitol, tragacanth or polyvinylpyrrolidone), fillers (eg lactose, sugar , corn starch, calcium phosphate, sorbitol or glycine), disintegrants (eg starch, polyvinylpyrrolidone or microcrystalline cellulose) or a pharmaceutically acceptable surfactant such as sodium lauryl sulfate.
- aggregating agents eg syrup, acacia, gelatin, sorbitol, tragacanth or polyvinylpyrrolidone
- fillers eg lactose, sugar , corn starch, calcium phosphate, sorbitol or glycine
- disintegrants eg starch, polyvinyl
- compositions for oral administration can be prepared by conventional methods of Galenic Pharmacy, as mixing and dispersion.
- the tablets can be coated following methods known in the pharmaceutical industry.
- compositions can be adapted for parenteral administration, as sterile solutions, suspensions, or lyophilized products of the invention, using the appropriate dose.
- Suitable excipients such as pH buffering agents or surfactants, can be used.
- the aforementioned formulations can be prepared using conventional methods, such as those described in the Pharmacopoeias of different countries and in other reference texts.
- the administration of the compounds or compositions of the present invention can be performed by any suitable method, such as intravenous infusion and oral, intraperitoneal or intravenous routes. Oral administration is preferred for the convenience of patients and for the chronic nature of the diseases to be treated.
- the amount administered of a compound of the present invention will depend on the relative efficacy of the compound chosen, the severity of the disease to be treated and the weight of the patient. However, the compounds of this invention will be administered one or more times a day, for example 1, 2, 3 or 4 times daily, with a total dose between 0.1 and 1000 mg / kg / day. It is important to keep in mind that it may be necessary to introduce variations in the dose, depending on the age and condition of the patient, as well as modifications in the route of administration.
- the compounds and compositions of the present invention can be used together with other medicaments in combination therapies.
- the other drugs may be part of the same composition or of a different composition, for administration at the same time or at different times.
- alkyl refers in the present invention to aliphatic, linear or branched chains, having 1 to 3 carbon atoms or 1 to 2 carbon atoms, for example, methyl, ethyl, n-propyl, i-propyl , etc.
- the alkyl group has between 1 and 2 carbon atoms or preferably the alkyl group is a methyl.
- the alkyl radicals may be optionally substituted by one or more substituents such as halogen, hydroxyl, azide, carboxylic acid or a substituted or unsubstituted group selected from amino, amido, carboxylic ester, ether, thiol, acylamino or carboxamido.alkoxide, thiol , amino, acylamino, cyano, carboxylate, carboxamide, carboxy ester, aryl or heteroaryl or combinations of these groups.
- substituents such as halogen, hydroxyl, azide, carboxylic acid or a substituted or unsubstituted group selected from amino, amido, carboxylic ester, ether, thiol, acylamino or carboxamido.alkoxide, thiol , amino, acylamino, cyano, carboxylate, carboxamide, carboxy ester, aryl or heteroaryl or combinations of these groups.
- the O-acetylation reaction of GD1 b resulted in a reaction product whose mobility in thin layer chromatography (TLC) was different from GD1 b.
- TLC thin layer chromatography
- MALDI TOF-MS Through flight time spectrometry
- Electrospray it was characterized as Neurostatin, for presenting a single O-acetylation in the terminal sialic.
- the reaction yield was 90%.
- the O-acetylation reaction of GT1 b gave rise to two reaction products whose mobility in thin layer chromatography was different from that of GT1 b.
- the compound with the greatest mobility in thin layer chromatography presented 2 O-acetylations, each in one of the two terminal sialics and was called di-O-acetyl-GT1 b.
- the second product presented a single O-acetylation.
- A-O-acetyl-GT1 b which presents the O-acetylation in the terminal sialic which is bound to another sialic
- B-O-acetyl-GT1 b which is mono-O-acetylated in the sialic not bound to another sialic.
- the reaction yield was 60% for mono-O-acetylated species and 20% for di-O-acetylated species.
- the O-propionylation reaction of GD1 b resulted in a reaction product whose mobility in thin layer chromatography was different from that of GD1 b.
- the reaction yield was 10%.
- Table 1 Inhibition of division in C6 cells (48 h) in average values of ID 50 (nM). The symbol "-" indicates that the compound weakly inhibited proliferation at the maximum concentration tested (4 ⁇ ), but not enough to calculate the value of your ID 5 or-
- Table 1 we present the activity of gangliosides GD1 b, GT1 b, and their modified derivatives.
- Ganglioside GD1 b has no proliferative inhibitory activity of the C6 glioma line. While the compounds derived from GD1 b, Neurostatin (O-acetyl-GD1 b), and O-propionyl-GD1 b have activity in the nanomolar range. The ganglioside O-propionyl-GD1 b is slightly more active than Neurostatin.
- Ganglioside GT1 b has a proliferation inhibitory activity.
- the compound O-acetyl-GT1 b almost twice improves its inhibitory activity with respect to GT1 b.
- the ganglioside GT1 b is modified with two O-acetylations (di-O-acetyl-GT1 b) it also improves its activity twice as much as the O-acetyl-GT1 b and 4 times with respect to the GT1 b.
- the addition of O-acetyls to the ganglioside GT1 b progressively increases its proliferation inhibitory activity in C6 cells.
- O-propionylated gangliosides have higher inhibitory activity (O-propionyl-GD1 b and O-propionyl-GT1 b) than their respective mono-O-acetylated gangliosides (O-acetyl-GD1 b and O-acetyl-GT1 b). These O-propionylated compounds could be more resistant to hydrolysis by glycosidases, so their effect would be more lasting.
- the O-acetylation reaction of GM3 resulted in a reaction product whose mobility in thin layer chromatography was different from that of GM3.
- MALDITOF-MS flight time spectrometry
- electrospray it was characterized as an O-acetylated mono derivative in the sialic we call O-acetyl-GM3.
- the reaction yield was 90%.
- the O-acetylation reaction of GD3 resulted in a reaction product whose mobility in thin layer chromatography was different from that of GD3.
- the reaction yield was 80%.
- the enzyme O-acetyltransferase was added at a concentration of 20 ⁇ g ⁇ l in a total reaction volume of 432 ⁇ . The reaction was carried out at 37 ° C for 7 hours with stirring (300 rpm). After time, the reaction was stopped by the addition of methanol.
- the O-acetylation reaction of GD1 a resulted in a reaction product whose mobility in thin layer chromatography was different from that of GD1 a.
- MALDITOF-MS Through flight time spectrometry
- electrospray it was characterized as an O-acetylated mono derivative in the sialic linked in alpha (2-3) to the galactose-N-acetyl-galactosamine radical that we call O-acetyl-GD1 to.
- the reaction yield was 70%.
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Abstract
The invention relates to novel inhibitors of tumour cell division. These inhibitors are semi-synthetic derivatives of neurostatin. The invention also relates to a novel method for the synthesis of the inhibitors and neurostatin. In addition, the invention relates to the use of said inhibitors for the production of a drug for the treatment of brain tumours.
Description
DERIVADOS DE GANGLIOSIDOS Y SU USO COMO INHIBIDORES DE LA DIVISION DE CÉLULAS TUMORALES GANGLIOSID DERIVATIVES AND THEIR USE AS INHIBITORS OF THE TUMOR CELL DIVISION
La presente invención se refiere a nuevos inhibidores de la división de células tumorales. Dichos inhibidores, son derivados semisintéticos de la neurostatina. The present invention relates to new inhibitors of tumor cell division. Said inhibitors are semi-synthetic derivatives of neurostatin.
5 Además la presente invención se refiere a un nuevo procedimiento de síntesis In addition, the present invention relates to a new synthesis procedure.
de los inhibidores y de la neurostatina. Y también en general se refiere a un procedimiento para obtener gangliósidos O-acilados específicamente en los siálicos terminales. Además se refiere al uso de estos inhibidores para la fabricación de un medicamento para el tratamiento de tumores cerebrales. of inhibitors and neurostatin. And also in general it refers to a procedure for obtaining O-acylated gangliosides specifically in terminal sialics. It also refers to the use of these inhibitors for the manufacture of a medicament for the treatment of brain tumors.
10 10
La invención se encuadra en el sector farmacéutico, y es aplicable en el sector médico para el tratamiento de tumores cerebrales. The invention is part of the pharmaceutical sector, and is applicable in the medical sector for the treatment of brain tumors.
ESTADO DE LA TÉCNICA STATE OF THE TECHNIQUE
15 fifteen
El glioblastoma multiforme es el tumor cerebral primario más frecuente en adultos y también el más mortal. En Estados Unidos, solo la mitad de los pacientes que reciben el tratamiento estándar sobreviven un año después del diagnóstico. Menos de uno de cada diez sobrevive más de cinco años. Suele Glioblastoma multiforme is the most common primary brain tumor in adults and also the most deadly. In the United States, only half of the patients receiving standard treatment survive one year after diagnosis. Less than one in ten survives more than five years. Usually
20 presentarse en personas mayores de cuarenta años, con un máximo de 20 show up in people over forty, with a maximum of
incidencia entre los 50 y 55 años y es más frecuente entre los varones. En adultos, hay unos 17.000 nuevos casos de tumores cerebrales cada año incidence between 50 and 55 years and is more frequent among men. In adults, there are about 17,000 new cases of brain tumors every year
(además, otros tipos de cáncer pueden metastatizar al cerebro), lo que supone unas 14000 muertes. Los tumores cerebrales son la primera causa de muerte (In addition, other types of cancer can metastasize to the brain), which is about 14,000 deaths. Brain tumors are the first cause of death
25 por cáncer en niños y menores de 20 años. 25 due to cancer in children and children under 20 years.
El tratamiento depende de la localización y del grado del tumor; se aplica cirugía cuando el tumor es accesible y no hay peligro de dañar estructuras vitales. La radioterapia se utiliza para detener el crecimiento del tumor o para Treatment depends on the location and grade of the tumor; Surgery is applied when the tumor is accessible and there is no danger of damaging vital structures. Radiation therapy is used to stop tumor growth or to
30 hacer que disminuya de tamaño y la quimioterapia destruye las células 30 make it shrink and chemotherapy destroys cells
tumorales que quedan después de la cirugía y la radioterapia. La quimioterapia más habitual (BCNU, CCNU) no parece tener un efecto significativo, aunque en algunas ocasiones se ha conseguido aumentar algunos meses la
supervivencia. Por tanto, el desarrollo de nuevas moléculas capaces de detener la proliferación de gliomas es de gran interés para el tratamiento de esta enfermedad. Los gangliósidos, son glicolípidos formados por una cadena de ceramida (que se compone de unresiduo del aminoalcoholesfingosina unido a un ácido graso) unida a una cadena de oligosacárido. Los gangliósidos tienen como característica estructural, la presencia de uno o varios residuos de ácido siálico en la cadena de oligosacárido. La Neurostatina se caracteriza por ser un gangliósido de la serie b, GD1 b, con una modificación en forma de O- acetilación en uno de los hidroxilos de su siálico terminal (Romero-Ramirez L, Nieto-Sampedro M. Inhibiting human astrocytomagrowth: structure- activityrelationships in neurostatinrelatedglycolipids. Journal of Medicinal Chemistry 2004;47(21):4983-4.). La posición de esta O-acetilación es preferentemente en el hidroxilo del carbono 9, ya que aunque también han sido descritas O-acetilaciónes en los carbonos 7 y 8, en condiciones de pH fisiológico los O-acetilos en esos carbonos migran a la posición 9. La O- acetilación es la modificación más frecuente en la naturaleza y se encuentran preferentemente en los siálicos de los gangliósidos unidos en enlaces a(2,8) y a(2,3) a la cadena glicídica. tumor that remain after surgery and radiotherapy. The most common chemotherapy (BCNU, CCNU) does not seem to have a significant effect, although in some cases it has been possible to increase the number of months survival. Therefore, the development of new molecules capable of stopping the proliferation of gliomas is of great interest for the treatment of this disease. Gangliosides are glycolipids formed by a ceramide chain (which is composed of a residue of the aminoalcoholesphingosine bound to a fatty acid) linked to an oligosaccharide chain. Gangliosides have as their structural characteristic, the presence of one or more sialic acid residues in the oligosaccharide chain. Neurostatin is characterized by being a ganglioside of the series b, GD1 b, with a modification in the form of O-acetylation in one of the hydroxyls of its terminal sialic (Romero-Ramirez L, Nieto-Sampedro M. Inhibiting human astrocytomagrowth: structure - activityrelationships in neurostatinrelatedglycolipids. Journal of Medicinal Chemistry 2004; 47 (21): 4983-4.). The position of this O-acetylation is preferably in the hydroxyl of carbon 9, since although O-acetylations in carbons 7 and 8 have also been described, under physiological pH conditions the O-acetyls in those carbons migrate to position 9 O-acetylation is the most frequent modification in nature and is preferably found in the sialics of gangliosides linked in links to (2,8) and (2,3) to the glycidic chain.
La Neurostatina es un gangliósido natural que se expresa en concentración muy baja en el sistema nervioso central de los mamíferos (Abad-Rodríguez J, Bernabé M, Romero-Ramirez L, Vallejo-Cremades M, Fernandez-Mayoralas A, Nieto-Sampedro M. Purification and structure of neurostatin, aninhibitor of astrocyted ¡visión of mammalianbrain. Journal of Neurochemistry 2000;74(6):2547-56.). El procedimiento de purificación de la neurostatinay de otros gangliósidos O-acetiladosa partir de cerebros de mamífero es muy laborioso y el rendimiento obtenido hace inviable su usopara el estudio del papel biológico de esta modificación, en la experimentación animal o para su posible uso en clínica. Neurostatin is a natural ganglioside that is expressed in very low concentration in the central nervous system of mammals (Abad-Rodríguez J, Bernabé M, Romero-Ramirez L, Vallejo-Cremades M, Fernandez-Mayoralas A, Nieto-Sampedro M. Purification and structure of neurostatin, an inhibitor of astrocyted vision of mammalianbrain, Journal of Neurochemistry 2000; 74 (6): 2547-56.). The procedure of purification of the neurostatin and other O-acetylated gangliosides from mammalian brains is very laborious and the yield obtained makes its use unfeasible for the study of the biological role of this modification, in animal experimentation or for its possible use in clinic.
Recientemente, se han desarrollado una serie de procedimientos para la
obtención de gangliósidos O-acetilados mediante diferentes métodos de O- acetilación del gangliósido comercial GD1 b y de otros gangliósidos( Va//e-/4rgos B, Gomez-Nicola D, Nieto-Sampedro M. Synthesis and characterization of neurostatin-relatedcompoundswithhighinhibitoryactivity of glioma growth. EuropeanJournal of Medicinal Chemistry; 2010, 45(5):2034-43.). Aunque los procedimientos químicos reducen los pasos para la obtención de gangliósidos O-acetilados, son reacciones inespecíficas donde no se controlan ni el número, ni la posición de las O-acetilaciones. En consecuencia, se obtiene una gran variedad de productos que dificultan su purificación a homogeneidad y reducen considerablemente el rendimiento de la reacción. A pesar de que este procedimiento reduce los pasos necesarios para producir neurostatina, el producto obtenido sigue siendo insuficiente para su posible uso clínico. Además, dada la poca abundancia de estas sustancias en la naturaleza, el estudio de su actividad biológica hace necesaria la búsqueda de nuevos procedimientos para su obtención. Recently, a series of procedures have been developed for the Obtaining O-acetylated gangliosides by different methods of O-acetylation of the commercial ganglioside GD1 by and other gangliosides (Va // e- / 4rgos B, Gomez-Nicola D, Nieto-Sampedro M. Synthesis and characterization of neurostatin-relatedcompoundswithhighinhibitoryactivity of glioma growth European Journal of Medicinal Chemistry; 2010, 45 (5): 2034-43.). Although chemical procedures reduce the steps for obtaining O-acetylated gangliosides, they are nonspecific reactions where neither the number nor the position of the O-acetylations are controlled. Consequently, a wide variety of products are obtained that hinder their purification to homogeneity and considerably reduce the yield of the reaction. Although this procedure reduces the steps necessary to produce neurostatin, the product obtained remains insufficient for possible clinical use. In addition, given the low abundance of these substances in nature, the study of their biological activity makes it necessary to find new procedures to obtain them.
Por otro lado el procedimiento descrito en la publicación (Houliston RS, Endtz HP, Yuki N, et al. Identification of a sialate O-acetyltransferase from Campylobacter jejuni: demonstration of direct transfer to the C-9 position of terminalalpha-2, 8-linked sialic acid. TheJournal of BiologicalChemistry 2006;281(17):11480-6) consigue O-acetilar polisacáridos de glicolípidos derivados del éster del ácido hexanoico6-(5-Fluorescein-carboxamido)- (FCHASE) y glicoproteínas, pero no gangliósidos. On the other hand, the procedure described in the publication (Houliston RS, Endtz HP, Yuki N, et al. Identification of a sialate O-acetyltransferase from Campylobacter jejuni: demonstration of direct transfer to the C-9 position of terminalalpha-2, 8- linked sialic acid The Journal of Biological Chemistry 2006; 281 (17): 11480-6) achieves O-acetylate polysaccharides of glycolipids derived from the hexanoic acid ester 6- (5-Fluorescein-carboxamido) - (FCHASE) and glycoproteins, but not gangliosides.
El procedimiento descrito en la patente (WO 2007016792) es bastante general. Los rangos de pH que describe son bastante amplios (de 5 a 8), habla de un donador de grupos acetilo y un aceptor de los grupos acetilo, donde se prevé el uso de sustancias tampón y la reacción se lleva a cabo entre 0 y 40 °C, preferentemente entre 20 y 37 °C, en medio acuoso. También describe que la mezcla de reacción puede contener cationes metálicos divalentes (tales como magnesio o manganeso) y también puede contener detergentes solubilizantes o disolventes orgánicos, si fuese necesario. Este procedimiento no detalla
ningún protocolo específico para O-acetilargangliósidos y siguiéndolo no se consigue O-acetilargangliósidos. The procedure described in the patent (WO 2007016792) is quite general. The pH ranges he describes are quite wide (from 5 to 8), he talks about an acetyl group donor and an acetyl group acceptor, where the use of buffer substances is anticipated and the reaction is carried out between 0 and 40 ° C, preferably between 20 and 37 ° C, in aqueous medium. It also describes that the reaction mixture may contain divalent metal cations (such as magnesium or manganese) and may also contain solubilizing detergents or organic solvents, if necessary. This procedure does not detail no specific protocol for O-acetylgangliosides and following it is not possible to obtain O-acetylaryngiosides.
En el procedimiento descrito por Houliston, R. et al. (Houliston RS, Endtz HP, Yuki N, et al. Identification of a sialate O-acetyltransferase from Campyiobacter jejuni: demonstration of direct transfer to the C-9 position of terminalalpha-2, 8- linked sialic acid. The Journal of BiologicalChemistry 2006;281(17):11480-6) que utiliza una O-acetiltransferasa de la bacteria Campylobacterjejuni. Este procedimiento fue utilizado para O-acetilar específicamente los ácidos siálicos terminales de las cadenas de polisacaridos de glicolípidos derivados del éster succidimil del ácido hexanoico 6-(5-Fluorescein-carboxamido) (FCHASE) y también glicoproteínas. El uso de este enzima para O-acetilar oligosacáridos se encuentra patentado (WO 2007016792 20070215). El procedimiento descrito por estos investigadores no pudo O-acetilar con éxito gangliósidos. Los gangliosidos en solución acuosa se agrupan en micelas que impiden el acceso de la O-acetiltransferase al siálico terminal. In the procedure described by Houliston, R. et al. (Houliston RS, Endtz HP, Yuki N, et al. Identification of a sialate O-acetyltransferase from Campyiobacter jejuni: demonstration of direct transfer to the C-9 position of terminalalpha-2, 8-linked sialic acid. The Journal of Biological Chemistry 2006 ; 281 (17): 11480-6) which uses an O-acetyltransferase of the Campylobacterjejuni bacteria. This procedure was used to specifically o-acetylate the terminal sialic acids of the polysaccharide chains of glycolipids derived from the succinate ester of hexanoic acid 6- (5-Fluorescein-carboxamide) (FCHASE) and also glycoproteins. The use of this enzyme for O-acetylated oligosaccharides is patented (WO 2007016792 20070215). The procedure described by these researchers failed to successfully O-acetylate gangliosides. Gangliosides in aqueous solution are grouped in micelles that prevent the access of the O-acetyltransferase to the terminal sialic.
DESCRIPCIÓN DE LA INVENCIÓN DESCRIPTION OF THE INVENTION
La presente invención describe una nueva serie de gangliósidos con actividad inhibidora (de la familia de la neurostatina) de la división de células tumorales y del crecimiento de gliomas. Además la presente invención describe un novedoso procedimiento de obtención de gangliósidos O-acetilados y de la neurostatina que se caracteriza por su sencillez, especificidad y alto rendimiento. Este procedimiento tiene un interés comercial en el campo de la glicobiología para la producción de estos gangliósidos O-acetilados en grandes cantidades. Utilizando el mismo procedimiento que se describe hemos obtenido nuevos gangliósidos de la familia de la neurostatina O-propionilados y O-malonilados, no descritos en la naturaleza y que tienen mayor actividad inhibidora de la proliferación de la línea de glioma C6 (Tabla 1 ). Estos compuestos podrían ser más resistentes a la hidrólisis por glicosidasas, por lo que su efecto sería más
duradero. The present invention describes a new series of gangliosides with inhibitory activity (of the neurostatin family) of tumor cell division and glioma growth. In addition, the present invention describes a novel method of obtaining O-acetylated gangliosides and neurostatin which is characterized by its simplicity, specificity and high performance. This procedure has a commercial interest in the field of glycobiology for the production of these O-acetylated gangliosides in large quantities. Using the same procedure as described, we have obtained new gangliosides from the O-propionylated and O-malonylated neurostatin family, not described in nature and that have greater inhibitory activity on the proliferation of the C6 glioma line (Table 1). These compounds could be more resistant to hydrolysis by glycosidases, so their effect would be more long lasting.
Por lo tanto, estos gangliósidos O-propionilados y O-maloniladosson unos productos muy interesantes para su uso clínico como antitumorales. Therefore, these O-propionylated and O-malonylated gangliosides are very interesting products for clinical use as antitumor agents.
Teniendo en cuenta la gravedad de los glioblastomas y la escasa efectividad de los tratamientos disponibles, el desarrollo de nuevas moléculas capaces de detener la proliferación de gliomas es de enorme interés. Taking into account the severity of glioblastomas and the limited effectiveness of available treatments, the development of new molecules capable of stopping the proliferation of gliomas is of great interest.
Por lo tanto un primer aspecto de la presente invención se refiere a un procedimiento de síntesis de un compuesto, concretamente un gangliósido, de fórmula gener Therefore, a first aspect of the present invention relates to a method of synthesizing a compound, specifically a ganglioside, of the general formula.
(l) (l)
o cualquiera de sus sales, isómeros o solvatos, or any of its salts, isomers or solvates,
donde: where:
Y es una ceramida; And it is a ceramide;
Ac es un grupo acetilo; Ac is an acetyl group;
Z se selecciona entre un grupo de fórmula Zi : Z is selected from a group of formula Zi:
(Zi)
de manera ue la fórmula general (I) corresponde a la fórmula (la): (Zi) so that the general formula (I) corresponds to the formula (la):
(la) (the)
o Z es un grupo de fórmula (Z2):
or Z is a group of formula (Z 2 ):
(¾) (¾)
de manera que la fórmula general (I) corresponde a la fórmula (Ib): so that the general formula (I) corresponds to the formula (Ib):
(Ib) (Ib)
donde tanto para la fórmula la como para la formula Ib: where for both formula and formula Ib:
Xi se selecciona entre -CH2-O-CO-X2 y -CH2OH; Xi is selected from -CH 2 -O-CO-X 2 and -CH 2 OH;
X2 se selecciona entre un grupo alquilo Ci-C2 y-CH2COOH; X 2 is selected from a Ci-C 2 and -CH 2 COOH alkyl group;
X3 se selecciona entre H, OH, un grupo de fórmula (II) o un grupo de fórmula
X3 is selected from H, OH, a group of formula (II) or a group of formula
(II) (i») donde en la fórmula III, X4 se selecciona entre H, un grupo de fórmula (IV) o un grupo de fórmula (V): (II) (i ») where in formula III, X4 is selected from H, a group of formula (IV) or a group of formula (V):
(V) (V)
donde tanto para la fórmula IV como para la fórmula V: where for both formula IV and formula V:
X5 se selecciona entre un grupo OH o un grupo -0-COXe; X5 is selected from an OH group or a -0-COXe group;
Xese selecciona entre un grupo alquilo C1-C2 o -CH2COOH; excepto cuando: Xese selects from a C 1 -C 2 or -CH 2 COOH alkyl group; Except when:
- Z es un grupo de fórmula (Z1) o (Z2), X1 es -CH2-OH y X3 es H; - Z is a group of formula (Z 1 ) or (Z 2 ), X 1 is -CH 2 -OH and X 3 is H;
- Z es un grupo de fórmula (Z1) o (Z2), X1 es -CH2-OH y X3 es OH;
- Z es un grupo de fórmula (Zi) o (Z2), Xi es -CH2-OH y X3 es el grupo de fórmula (II); - Z is a group of formula (Z 1 ) or (Z 2 ), X 1 is -CH 2 -OH and X 3 is OH; - Z is a group of formula (Zi) or (Z 2 ), Xi is -CH 2 -OH and X 3 is the group of formula (II);
- Z es un grupo de fórmula (Zi) o (Z2), Xi es -CH2-OH, X3 es el grupo de fórmula (III) y X4 es H; - Z is a group of formula (Zi) or (Z 2 ), Xi is -CH 2 -OH, X 3 is the group of formula (III) and X4 is H;
- Z es un grupo de fórmula (Z1) o (Z2), X1 es -CH2-OH, X3 es el grupo de fórmula (III) y X4 es el grupo de fórmula (IV) y X5 es OH; - Z is a group of formula (Z1) or (Z 2 ), X1 is -CH 2 -OH, X 3 is the group of formula (III) and X4 is the group of formula (IV) and X 5 is OH;
- Z es un grupo de fórmula (Z1) o (Z2), X1 es -CH2-OH, X3 es el grupo de fórmula (III) y X4 es el grupo de fórmula (V) y X5 es OH; que comprende las etapas de: a) disolver un gangliósido en una solución tampón; b) adicionar un emulgente a la mezcla de la etapa a) c) adicionar a la mezcla de la etapa b) la enzima o-acetiltransferasa; y d) parar la reacción por adición de metanol a la mezcla obtenida en la etapa c). - Z is a group of formula (Z1) or (Z 2 ), X1 is -CH 2 -OH, X 3 is the group of formula (III) and X4 is the group of formula (V) and X 5 is OH; comprising the steps of: a) dissolving a ganglioside in a buffer solution; b) add an emulsifier to the mixture of step a) c) add to the mixture of step b) the enzyme o-acetyltransferase; and d) stop the reaction by adding methanol to the mixture obtained in step c).
En adelante, dicho procedimiento puede ser referido como procedimiento de la invención. Hereinafter, said process may be referred to as the method of the invention.
Según una realización preferida, el gangliósido de fórmula general (I) se selecciona del grupo formado por O-acetil-GM3, O-acetil-GM2, O-acetil-GD3, O-acetil-GD2, Neurostatina, A-O-acetil-GT1 b, A-O-acetil-GQ1 b, O-propionil- GD1 b, O-propionil-GD3, O-propionil-GD2, A-O-propionil-GT1 b, B-O-propionil- GT1 b, B-O-acetil-GT1 b, di-O-acetil-GT1 b, O-Propionil-GM3, O-Malonil-GM3, O- Propionil-GM2, O-Malonil-GM2, O-Malonil-GD3, O-Malonil-GD2, O-Propionil- GD1 a, O-Malonil-GD1 a, O-Malonil-GD1 b, A-O-Malonil-GT1 b, B-O-Malonil- GT1 b, di-O-Malonil-GT1 b, B-O-Acetil-GQ1 b, di-O-Acetil-GQ1 b, A-O-Propionil- GQ1 b, B-O-Propionil-GQ1 b, di-O-Propionil-GQ1 b, A-O-Malonil-GQ1 b, B-O- Malonil-GQ1 b y di-O-Malonil-GQ1 b, según se definen a continuación:
According to a preferred embodiment, the ganglioside of the general formula (I) is selected from the group consisting of O-acetyl-GM3, O-acetyl-GM2, O-acetyl-GD3, O-acetyl-GD2, Neurostatin, AO-acetyl-GT1 b, AO-acetyl-GQ1 b, O-propionyl-GD1 b, O-propionyl-GD3, O-propionyl-GD2, AO-propionyl-GT1 b, BO-propionyl-GT1 b, BO-acetyl-GT1 b, di -O-acetyl-GT1 b, O-Propionyl-GM3, O-Malonil-GM3, O- Propionyl-GM2, O-Malonil-GM2, O-Malonyl-GD3, O-Malonil-GD2, O-Propionyl- GD1 a , O-Malonil-GD1 a, O-Malonil-GD1 b, AO-Malonil-GT1 b, BO-Malonil-GT1 b, di-O-Malonil-GT1 b, BO-Acetyl-GQ1 b, di-O-Acetyl -GQ1 b, AO-Propionyl- GQ1 b, BO-Propionyl-GQ1 b, di-O-Propionyl-GQ1 b, AO-Malonil-GQ1 b, BO- Malonil-GQ1 by di-O-Malonil-GQ1 b, according They are defined below:
0-propionil-GD1b
0-propionyl-GD1b
di-0-acetil-GT1b di-0-acetyl-GT1b
A-0-propionil-GT1 b A-0-propionyl-GT1 b
B-0-propionil-GT1b
B-0-propionyl-GT1b
di-0-propionil-GT1 b di-0-propionyl-GT1 b
0-acetil-GD3 0-acetyl-GD3
A-0-acetil-GT1b
A-0-acetyl-GT1b
Q-Acetil-GM2
Q-Acetyl-GM2
0-Acetil-GD1a
0-Acetyl-GD1a
A-0-Acetil-GQ1 b A-0-Acetyl-GQ1 b
Q-Propionil-GM3 Q-Propionil-GM3
Q-Malonil-GM3 Q-Malonil-GM3
Q-Propionil-GM2
Q-Propionil-GM2
0-Malonil-GM2
0-Malonil-GM2
0-Malonil-GD2
0-Malonil-GD2
0-Propionil-GD1a
0-Propionyl-GD1a
0-Malonil-GD1a 0-Malonil-GD1a
0-Malonil-GD1b 0-Malonil-GD1b
A-0-Malonil-GT1b
A-0-Malonil-GT1b
B-0-Malonil-GT1b B-0-Malonil-GT1b
di-0-Malonil-GT1b di-0-Malonil-GT1b
B-0-Acetil-GQ1b
B-0-Acetyl-GQ1b
B-0-Propionil-GQ1b
B-0-Propionyl-GQ1b
di-0-Propionil-GQ1b di-0-Propionyl-GQ1b
A-0-Malonil-GQ1b A-0-Malonil-GQ1b
B-0-Malonil-GQ1b
B-0-Malonil-GQ1b
di-0-Malonil-GQ1b di-0-Malonil-GQ1b
donde Y es cualquier ceramida, que está compuesta por una esfingosina y un ácido graso. De manera preferida la esfingosina comprende de 18 a 20 átomos de carbono y tiene un número de insaturaciones de 0 a 2. De manera preferida el ácido graso puede contener de 8 a 24 átomos de carbono y con un número de insaturaciones de 0 a 4; where Y is any ceramide, which is composed of a sphingosine and a fatty acid. Preferably, sphingosine comprises 18 to 20 carbon atoms and has a number of unsaturations of 0 to 2. Preferably, the fatty acid may contain 8 to 24 carbon atoms and with a number of unsaturations of 0 to 4;
En la presente invención los términos GD1 b, GD3, GD2, GT1 b, GQ1 b, GM3, GM2, GM1 a o GD1 a, hacen referencia a gangliosidos del mismo nombre conocidos en el estado de la técnica por cualquier experto en la materia. En este mismo sentido estos gangliosidos comprenden entre otros elementos, una ceramida (Y) que a su vez comprende una esfingosina y un ácido graso. La esfingosina es preferentemente de 18 de átomos de carbono con una insaturación o de 20 átomos de carbono con una insaturación. De manera preferente el ácido graso es de 18 átomos de carbono sin ninguna insaturación o con una insaturación.
En otra realización preferida del procedimiento de la presente invención, el com uesto de fórmula general I presenta la fórmula (la): In the present invention the terms GD1 b, GD3, GD2, GT1 b, GQ1 b, GM3, GM2, GM1 a or GD1 a, refer to gangliosides of the same name known in the state of the art by any person skilled in the art. In this same sense, these gangliosides comprise among other elements, a ceramide (Y) which in turn comprises a sphingosine and a fatty acid. Sphingosine is preferably 18 carbon atoms with an unsaturation or 20 carbon atoms with an unsaturation. Preferably, the fatty acid is 18 carbon atoms without any unsaturation or unsaturation. In another preferred embodiment of the process of the present invention, the compound of general formula I has the formula (la):
(la) (the)
o cualquiera de sus sales, isómeros o solvatos, or any of its salts, isomers or solvates,
donde: where:
Y es una ceramida; And it is a ceramide;
Ac es un grupo acetilo; Ac is an acetyl group;
Xi es -CH2-O-CO-X2; Xi is -CH 2 -O-CO-X 2 ;
X2 es un grupo alquilo C1-C2; X2 is a C1-C2 alkyl group;
X3 se selecciona entre H, OH, un grupo de fórmula (II) o un grupo de fórmula (III): X3 is selected from H, OH, a group of formula (II) or a group of formula (III):
(II) (III) donde en la fórmula III, X4 se selecciona entre H o un grupo de fórmula (IV)
(II) (III) where in formula III, X4 is selected from H or a group of formula (IV)
(IV) donde: X5 se selecciona entre un grupo OH o un grupo -Ο-COXe; y ΧΘ es un grupo alquilo C1-C2; (IV) where: X 5 is selected from an OH group or a -Ο-COXe group; and ΧΘ is a C1-C2 alkyl group;
excepto cuando: Except when:
- Xi es -CH2-O-CO-X2, X2 es un grupo CH3, X3 es el grupo de fórmula (III) y X4 es H; - Xi is -CH2-O-CO-X2, X2 is a group CH 3 , X3 is the group of formula (III) and X4 is H;
- Xi es -CH2-O-CO-X2, X2 es un grupo CH3, X3 es el grupo de fórmula (II); - Xi is -CH2-O-CO-X2, X2 is a CH 3 group, X3 is the group of formula (II);
- Xi es -CH2-O-CO-X2, X2 es un grupo CH3, X3 es el grupo de fórmula (III), X4 es el grupo de fórmula (IV) y X5 es OH; - Xi is -CH2-O-CO-X2, X2 is a group CH 3 , X3 is the group of formula (III), X4 is the group of formula (IV) and X 5 is OH;
- Xi es -CH2-O-CO-X2, X2 es un grupo CH3, X3 es el grupo de fórmula (la) y X4 es OH; que comprende las etapas de: a) disolver un gangliósido en una solución tampón; b) adicionar un emulgente a la mezcla de la etapa a) c) adicionar a la mezcla de la etapa b) la enzima o-acetiltransferasa; y d) parar la reacción por adición de metanol a la mezcla obtenida en la etapa c). - Xi is -CH2-O-CO-X2, X2 is a group CH 3 , X3 is the group of formula (la) and X4 is OH; comprising the steps of: a) dissolving a ganglioside in a buffer solution; b) add an emulsifier to the mixture of step a) c) add to the mixture of step b) the enzyme o-acetyltransferase; and d) stop the reaction by adding methanol to the mixture obtained in step c).
En una realización preferida de cualquiera de las anteriores, el gangliósido se selecciona del grupo formado por GD1 b, GD3, GD2 y GT1 b, según se han
definido anteriormente. Dichos gangliósidos GD1 b, GD3, GD2 y GT1 b, como se menciona anteriormente, comprenden entre otros elementos, una ceramida (Y) según se definió anteriormente, que a su vez comprende una esfingosina y un ácido graso. La esfingosina es preferentemente de 18 de átomos de carbono con una insaturación o de 20 átomos de carbono con una insaturación. De manera preferente el ácido graso es de 18 átomos de carbono sin ninguna insaturación o con una insaturación. In a preferred embodiment of any of the above, the ganglioside is selected from the group consisting of GD1 b, GD3, GD2 and GT1 b, as they have been defined above. Said gangliosides GD1 b, GD3, GD2 and GT1 b, as mentioned above, comprise among other elements, a ceramide (Y) as defined above, which in turn comprises a sphingosine and a fatty acid. Sphingosine is preferably 18 carbon atoms with an unsaturation or 20 carbon atoms with an unsaturation. Preferably, the fatty acid is 18 carbon atoms without any unsaturation or unsaturation.
En una realización preferida de cualquiera de las anteriores, se añade una concentración de gangliósido entre 50 a 600 pmoles/L de reacción, preferentemente entre 300 y 600 pmoles/L y más preferiblemente de 500 moles/L In a preferred embodiment of any of the above, a ganglioside concentration between 50 to 600 pmoles / L reaction, preferably between 300 and 600 pmoles / L and more preferably 500 moles / L is added
En otra realización preferida de cualquiera de las anteriores, se añade la solución tampón en una concentración desde 1 hasta 250 mmoles/L de reacción preferiblemente desdel O hasta 100 mM, más preferiblemente a una concentración de 50 mM, a un pH desde 6 a 9, preferiblemente a un pH entre 7 y 8, más preferiblemente a un pH de 7.0. De manera preferida la solución tampón es de TRIS(tris(hidroximetil)aminometano), MES (ácido 2-(N- morfolino)etanosulfonico), fosfato, etc, preferiblemente de MES. In another preferred embodiment of any of the above, the buffer solution is added in a concentration from 1 to 250 mmol / L of reaction preferably from 0 to 100 mM, more preferably at a concentration of 50 mM, at a pH from 6 to 9 , preferably at a pH between 7 and 8, more preferably at a pH of 7.0. Preferably, the buffer solution is from TRIS (tris (hydroxymethyl) aminomethane), MES (2- (N-morpholino) ethanesulfonic acid), phosphate, etc., preferably from MES.
Según una realización preferida se añade a la solución tampón MgC (10 mmoles/L) y DTT (Ditiotreitol) (1 mmoles/L). Según una realización preferida se añade a la solución un tampón y un derivado de la coenzima A seleccionado entre Acetil-Coenzima A, Propionil- Coenzima A y Malonil-Coenzima A, más preferiblemente Acetil-Coenzima A ó Propionil-Coenzima A, a una concentración desde 0.1 a 10 mmoles/L reacción, preferiblemente entre 0,5 y 2 mmoles/L más preferiblemente a 1 mmoles/L. According to a preferred embodiment, MgC buffer (10 mmol / L) and DTT (Dithiothreitol) (1 mmol / L) are added to the solution. According to a preferred embodiment, a buffer and a coenzyme A derivative selected from Acetyl-Coenzyme A, Propionyl-Coenzyme A and Malonyl-Coenzyme A, more preferably Acetyl-Coenzyme A or Propionyl-Coenzyme A, are added to the solution at a concentration from 0.1 to 10 mmol / L reaction, preferably between 0.5 and 2 mmol / L more preferably at 1 mmol / L.
En otra realización preferida de cualquiera de las anteriores, el emulgente es un ácido biliar o sal biliar a una concentración preferible entre 0,05% y 0,2% peso/volumen total de la reacción, más preferiblemente a 0,1 % en
peso/volumen total de la reacción. Es decir, el emulgente es un ácido biliar o sal biliar a una concentración preferible entre 0,05% y 0,2% en peso respecto del volumen total de la reacción, más preferiblemente a 0,1 % en peso respecto del volumen total de la reacción. In another preferred embodiment of any of the above, the emulsifier is a bile acid or bile salt at a preferable concentration between 0.05% and 0.2% total weight / volume of the reaction, more preferably 0.1% in Total weight / volume of the reaction. That is, the emulsifier is a bile acid or bile salt at a preferable concentration between 0.05% and 0.2% by weight with respect to the total volume of the reaction, more preferably 0.1% by weight with respect to the total volume of the reaction.
De manera preferida, el ácido biliar se selecciona del grupo formado por el ácido cólico, ácido deoxicólico, taurocólico, glicocólico, ursodeosicólico, litocólico, etc como sus sales sódicas y potásicas, (ejemplo: colato, taurocolato, deoxicolato,ursodeoxicolato, glicocolato, etcétera). De manera preferida el emulgente es la sal biliar colato sódico. Preferably, the bile acid is selected from the group consisting of colic acid, deoxycholic acid, taurocolic, glycocolic, ursodeoxycholic, lithocolic acid, etc. as its sodium and potassium salts, (example: cholate, taurocholate, deoxycholate, ursodeoxycholate, glycocholate, etc. ). Preferably, the emulsifier is the sodium bile colata salt.
El emulgente reduce el micelado de los gangliósidos, aumentando la accesibilidad de los siálicos terminales al enzima O-acetiltransferasa. En otra realización preferida de cualquiera de las anteriores, en la etapa c), se añade la enzima O-acetiltransferasa a una concentración de entre 2 y 30 pg/μΙ de reacción, preferiblemente entre 15 y 25 pg/μΙ, más preferiblemente a 20 Mg/μΙ- El procedimiento de la presente invención utiliza como catalizador una O- acetiltransferasa obtenida de la bacteria Campylobacterjejunni, cuya clonación, secuenciación y actividad han sido patentadas (WO 2007016792 20070215) y publicadas por Houliston R. et al (Houliston RS, Endtz HP, Yuki N, et al. TheJournal of BiologicalChemistry 2006;281(17):11480-6).
The emulsifier reduces the micellation of gangliosides, increasing the accessibility of terminal sialics to the enzyme O-acetyltransferase. In another preferred embodiment of any of the above, in step c), the enzyme O-acetyltransferase is added at a concentration of between 2 and 30 pg / μΙ reaction, preferably between 15 and 25 pg / μΙ, more preferably at 20 Mg / μΙ- The process of the present invention uses as an catalyst an O-acetyltransferase obtained from the bacteria Campylobacterjejunni, whose cloning, sequencing and activity have been patented (WO 2007016792 20070215) and published by Houliston R. et al (Houliston RS, Endtz HP, Yuki N, et al. The Journal of Biological Chemistry 2006; 281 (17): 11480-6).
En otra realización preferida de cualquiera de las anteriores, la reacción de la etapa c) se realizó entre 30-40°C, preferiblemente entre 35-38°C, más preferiblemente a una temperatura de 37°C. En otra realización preferida de cualquiera de las anteriores, la reacción de la etapa c) se realizó durante un tiempo de reacción entre 1 hora y 24 horas, preferiblemente entre 5 y 12 horas, más preferiblemente durante 7 horas, y en agitación. Tras parar la reacción con metanol en la etapa d), se llevaron a cabo las siguientes etapas: In another preferred embodiment of any of the above, the reaction of step c) was performed between 30-40 ° C, preferably between 35-38 ° C, more preferably at a temperature of 37 ° C. In another preferred embodiment of any of the above, the reaction of step c) was carried out during a reaction time between 1 hour and 24 hours, preferably between 5 and 12 hours, more preferably for 7 hours, and with stirring. After stopping the reaction with methanol in step d), the following steps were carried out:
- desalar los productos obtenidos tras la etapa d) utilizando un cartucho de fase reversa Sep-Pak C18 (Waters), - desalt the products obtained after step d) using a Sep-Pak C18 (Waters) reverse phase cartridge,
- desecar la muestra en vacío (Speed-Vac). - Dry the sample in a vacuum (Speed-Vac).
- purificar los diferentes productos por TLC preparativa o HPLC preparativa en una columna amino. - purify the different products by preparative TLC or preparative HPLC on an amino column.
- calcular el peso molecular de los productos mediante espectrometría de masas de tiempo de vuelo (MALDITOF-MS). - calculate the molecular weight of the products by means of flight time mass spectrometry (MALDITOF-MS).
- caracterización de la posición de las O-acetilaciones u O- propionilaciones mediante secuenciación por espectrometría de masas - characterization of the position of the O-acetylations or O-propionylations by sequencing by mass spectrometry
"electrospray". "electrospray".
El procedimiento descrito en la presente invención, es un protocolo específico para la O-acetilación de gangliósidos. A diferencia del procedimiento descrito en (Houliston RS, Endtz HP, Yuki N, et al. Identification of a sialate O- acetyltransferase from Campylobacter jejuni: demonstration of direct transfer to the C-9 position of terminalalpha-2, 8-linked sialic acid. TheJournal of BiologicalChemistry 2006;281(17):11480-6) y en la patente (WO 2007016792), en el procedimiento de la presente invención se añade a la mezcla de reacción un emulgente, que puede ser un ácido biliar o una sal biliar, que en combinación del pHreduce el micelado de los gangliósidos, aumentando la accesibilidad de los siálicos terminales al enzima O-acetiltransferasa.EI pH de la reacción tiene que ser igual o superior a 6,5 para que el colato sódico sea
soluble en el tampón y pueda actuar sobre los gangliósidos. The procedure described in the present invention is a specific protocol for ganglioside O-acetylation. Unlike the procedure described in (Houliston RS, Endtz HP, Yuki N, et al. Identification of a sialate O-acetyltransferase from Campylobacter jejuni: demonstration of direct transfer to the C-9 position of terminalalpha-2, 8-linked sialic acid The Journal of Biological Chemistry 2006; 281 (17): 11480-6) and in the patent (WO 2007016792), in the process of the present invention an emulsifier, which can be a bile acid or a salt, is added to the reaction mixture bile, which in combination of the pH reduces the micellation of the gangliosides, increasing the accessibility of the terminal sialics to the enzyme O-acetyltransferase. The pH of the reaction must be equal to or greater than 6.5 for the sodium colata to be soluble in the buffer and can act on gangliosides.
El procedimiento descrito en la presente invención es específico para los siálicos terminales y no para cualquier grupo hidroxilo O-acetilable, como ocurre en los otros procedimientos del estado de la técnica anterior. The process described in the present invention is specific for terminal sialics and not for any O-acetylable hydroxyl group, as in the other prior art processes.
Por lo tanto, el número de productos de reacción se reduce, favoreciendo la purificación de los productos. Además, el procedimiento tiene un rendimiento muy superior a los descritos anteriormente. En el caso de la reacción para obtener neurostatina (O-acetilGD1 b) a partir de GD1 b, el rendimiento es del 90%, frente al escaso 10-15% de otros procedimientos descritos en el estado de la técnica anterior. Gracias a este rendimiento, la neurostatina puede considerarse ahora como un producto interesante como antitumoral en pacientes y, por lo tanto, con posible uso comercial. Therefore, the number of reaction products is reduced, favoring the purification of the products. In addition, the procedure has a much higher performance than those described above. In the case of the reaction to obtain neurostatin (O-acetyl GD1 b) from GD1 b, the yield is 90%, compared to the scarce 10-15% of other procedures described in the prior art. Thanks to this performance, neurostatin can now be considered as an interesting product as an antitumor in patients and, therefore, with possible commercial use.
El procedimiento descrito en la presente invención también permite obtener productos naturales O-acetilados de la familia de la neurostatina, como O- acetil-GD2 y O-acetil-GT1 b, que no han sido descritos en la literatura científica como antitumorales; y otros, como el di-O-acetil-GT1 b, que no han sido descritos ni en la naturaleza, ni como antitumorales. Además de gangliósidos O-acetilados, el procedimiento descrito en la presente invención, puede ser utilizado para preparar gangliósidos O-propionilados y O-malonilados. Tanto los productos O-acetilados, O-propionilados u O-malonilados pueden referirse a los derivados con uno o dos grupos acilo (acetilo, propionilo o malonilo). Estos productos O-propionilados, como O-propionil-GM3, O-propionil-GM2, O- propionil-GD3, O-propionil-GD2, O-propionil-GD1 b, O-propionil-GT1 b y O- propionil-GQ1 b, y sus respectivos O-malonilados nunca han sido descritos en la literatura, ni se encuentran en la naturaleza. Además, estos productos O- propionilados tienen mayor actividad inhibidora de la proliferación de la línea de glioma C6 (Tabla 1 ) y podrían ser más resistentes a la hidrólisis por glicosidasas, por lo que su efecto sería más duradero. The process described in the present invention also allows to obtain natural O-acetylated products of the neurostatin family, such as O-acetyl-GD2 and O-acetyl-GT1 b, which have not been described in the scientific literature as antitumor; and others, such as di-O-acetyl-GT1 b, which have not been described in nature, nor as antitumor agents. In addition to O-acetylated gangliosides, the process described in the present invention can be used to prepare O-propionylated and O-malonylated gangliosides. Both the O-acetylated, O-propionylated or O-malonylated products may refer to derivatives with one or two acyl groups (acetyl, propionyl or malonyl). These O-propionylated products, such as O-propionyl-GM3, O-propionyl-GM2, O-propionyl-GD3, O-propionyl-GD2, O-propionyl-GD1 b, O-propionyl-GT1 by O-propionyl-GQ1 b , and their respective O-malonylates have never been described in the literature, nor are they found in nature. In addition, these O-propionylated products have greater inhibitory activity of the proliferation of the C6 glioma line (Table 1) and could be more resistant to hydrolysis by glycosidases, so that their effect would be more lasting.
Un segundo aspecto de la presente invención se refiere a un compuesto (en
adelante también referido como gangliósido o compuesto de la invención), que presenta la fórmula general (I) de acuerdo a como se definió anteriormente, excepto cuando: A second aspect of the present invention relates to a compound (in hereinafter also referred to as ganglioside or compound of the invention), which has the general formula (I) according to as defined above, except when:
- Z es un grupo de fórmula (Zi), Xi es -CH2-O-CO-X2, X2 es un grupo CH3, X3 es el grupo de fórmula (I I I) y X4 es H ; - Z is a group of formula (Zi), Xi is -CH2-O-CO-X2, X2 is a group CH 3 , X3 is the group of formula (III) and X4 is H;
- Z es un grupo de fórmula (Z1 ), X1 es -CH2-O-CO-X2, X2 es un grupo CH3, X3 es el grupo de fórmula (I I); - Z is a group of formula (Z1), X1 is -CH2-O-CO-X2, X2 is a group CH 3 , X3 is the group of formula (II);
- Z es un grupo de fórmula (Z1 ), X1 es -CH2-O-CO-X2, X2 es un grupo CH3, X3 es el grupo de fórmula (I I I), X4 es el grupo de fórmula (IV) y X5 es OH ; - Z is a group of formula (Z1), X1 is -CH2-O-CO-X2, X2 is a group CH 3 , X3 is the group of formula (III), X4 is the group of formula (IV) and X 5 is OH;
- Z es un grupo de fórmula (Z1 ), X1 es -CH2-O-CO-X2, X2 es un grupo CH3, X3 es OH ; - Z is a group of formula (Z1), X1 is -CH2-O-CO-X2, X2 is a group CH 3 , X3 is OH;
- Z es un grupo de fórmula (Z1 ), X1 es -CH2-O-CO-X2, X2 es un grupo CH3, X3 es el grupo de fórmula (I I I), X4 es el grupo de fórmula (V), X5 es -OH ; - Z is a group of formula (Z1), X1 is -CH2-O-CO-X2, X2 is a group CH 3 , X3 is the group of formula (III), X4 is the group of formula (V), X 5 is -OH;
- Z es un grupo de fórmula (Z2), X1 es -CH2-O-CO-X2, X2 es un grupo CH3, X3 es OH ; - Z is a group of formula (Z 2 ), X1 is -CH2-O-CO-X2, X2 is a group CH 3 , X3 is OH;
- Z es un grupo de fórmula (Z2), X1 es -CH2-O-CO-X2, X2 es un grupo CH3, X3 es el grupo de fórmula (I I); - Z is a group of formula (Z 2 ), X1 is -CH2-O-CO-X2, X2 is a group CH 3 , X3 is the group of formula (II);
- Z es un grupo de fórmula (Z2), X1 es -CH2-OH , X3 es el grupo de fórmula (I I I), X4 es el grupo de fórmula (IV), X5 es -O-CO-X6 y ΧΘ es un grupo CH3. - Z is a group of formula (Z 2 ), X1 is -CH 2 -OH, X 3 is the group of formula (III), X4 is the group of formula (IV), X 5 is -O-CO-X6 and ΧΘ is a CH 3 group.
En una realización preferida, el compuesto de la invención presenta la fórmula In a preferred embodiment, the compound of the invention has the formula
(la)
o cualquiera de sus sales, isómeros o solvatos, (the) or any of its salts, isomers or solvates,
donde; where;
Y es una ceramida,; And it is a ceramide;
Ac es un grupo acetilo; Ac is an acetyl group;
Xies un grupo -CH2-O-CO-X2 Xies a group -CH2-O-CO-X2
X2 es un grupo alquilo C1-C2; X2 is a C1-C2 alkyl group;
X3 se selecciona entre H, OH, un compuesto de fórmula general (II) o un compuesto de fórmula general (III) X3 is selected from H, OH, a compound of general formula (II) or a compound of general formula (III)
(II) (III) (II) (III)
X^se selecciona entre H y un compuesto de fórmula general (IV): X ^ is selected from H and a compound of general formula (IV):
o cualquiera de sus sales, isómeros o solvatos, or any of its salts, isomers or solvates,
X5 se selecciona entre un grupo OH o un grupo -0-COXe;
Χβ es un grupo alquilo C1-C2; X 5 is selected from an OH group or a -0-COXe group; Χβ is a C1-C2 alkyl group;
excepto cuando: Except when:
- Xi es-CH2-O-CO-X2, X2 es metilo, X3 es el compuesto de fórmula general (III) y X4 es hidrógeno; - Xi is -CH 2 -O-CO-X2, X2 is methyl, X3 is the compound of general formula (III) and X4 is hydrogen;
-Xies -CH2-O-CO-X2, X2 es metilo, y X3 es el compuesto (II); y -Xies -CH2-O-CO-X2, X2 is methyl, and X3 is the compound (II); Y
- Xies -CH2-O-CO-X2, X2 es metilo,X3 es el compuesto de fórmula general (III) donde X4 es el compuesto de fórmula general (IV) donde X5 es OH;- X1 es - CH2-O-CO-X2, X2 es un metilo, X3 es OH; - Xies -CH2-O-CO-X2, X2 is methyl, X3 is the compound of general formula (III) where X4 is the compound of general formula (IV) where X 5 is OH; - X1 is - CH2-O- CO-X2, X 2 is a methyl, X 3 is OH;
- Xi es -CH2-O-CO-X2, X2 es metilo, X3 es OH; - Xi is -CH2-O-CO-X2, X 2 is methyl, X 3 is OH;
- Xi es -CH2-O-CO-X2, X2 es metilo, X3 es el grupo de fórmula (II); - Xi is -CH2-O-CO-X2, X2 is methyl, X3 is the group of formula (II);
- Xi es -CH2-O-CO-X2, X2 es metilo, X3 es el grupo de fórmula (III), X4 es el grupo de fórmula (V) y X5 es OH; - Xi is -CH2-O-CO-X2, X2 is methyl, X3 is the group of formula (III), X4 is the group of formula (V) and X 5 is OH;
Según una realización preferida, Y es una ceramida que comprende una esfingosina y un ácido graso natural o sintético. De manera preferida la esfingosina comprende de 18 a 20 átomos de carbono y tiene un número de insaturaciones de 0 a 2. De manera preferida el ácido graso comprende de 8 a 24 átomos de carbono y tiene un número de insaturaciones de 0 a 4; According to a preferred embodiment, Y is a ceramide comprising a sphingosine and a natural or synthetic fatty acid. Preferably, sphingosine comprises 18 to 20 carbon atoms and has an unsaturation number of 0 to 2. Preferably, the fatty acid comprises 8 to 24 carbon atoms and has an unsaturation number of 0 to 4;
Según otra rea ización preferida, X2 es un grupo metilo. According to another preferred embodiment, X 2 is a methyl group.
Según otra rea ización preferida, X2 es un grupo etilo. According to another preferred embodiment, X 2 is an ethyl group.
Según otra rea ización preferida, X2 es un grupo -CH2COOH. According to another preferred embodiment, X 2 is a -CH 2 COOH group.
Según otra rea ización preferida, X3 es H. According to another preferred embodiment, X 3 is H.
Según otra rea ización preferida, X3 es OH. According to another preferred embodiment, X 3 is OH.
Según otra rea ización preferida, X3 es un grupo de fórmula (II). According to another preferred embodiment, X 3 is a group of formula (II).
Según otra rea ización preferida, X3 es un compuesto de fórmula general (III)According to another preferred embodiment, X 3 is a compound of general formula (III)
Según otra rea ización preferida, X4 es H. According to another preferred embodiment, X4 is H.
Según otra rea ización preferida, X4 es un compuesto de fórmula general (IV) According to another preferred embodiment, X4 is a compound of general formula (IV)
Según otra rea ización preferida, X4 es un compuesto de fórmula general (V).According to another preferred embodiment, X4 is a compound of general formula (V).
Según otra rea ización preferida, X5 es un grupo OH. According to another preferred embodiment, X 5 is an OH group.
Según otra rea ización preferida, X5 es un grupo -Ο-COXe. According to another preferred embodiment, X 5 is a group -Ο-COXe.
Según otra realización preferida, Χβ es un metilo. Según otra realización preferida, Χβ es un etilo. According to another preferred embodiment, Χβ is a methyl. According to another preferred embodiment, Χβ is an ethyl.
Según otra realización preferida, Χβ es un grupo -CH2COOH.
Según otra realización preferida los compuestos de la presente invención se seleccionan de la siguiente lista: O-propionil-GD1b, O-propionil-GD3, O- propionil-GD2, A-O-propionil-GT1b, B-O-propionil-GT1b, di-O-propionil-GT1b, B-O-acetil-GT1b, di-O-acetil-GTIb, O-Propionil-GM3, O-Malonil-GM3, O- Propionil-GM2, O-Malonil-GM2, O-Malonil-GD3, O-Malonil-GD2, O-Propionil- GD1a, O-Malonil-GD1a, O-Malonil-GD1b, A-O-Malonil-GT1b, B-O-Malonil- GT1b, di-O-Malonil-GT1b, B-O-Acetil-GQ1b, di-O-Acetil-GQ1b, A-O-Propionil- GQ1b, B-O-Propionil-GQ1b, di-O-Propionil-GQ1b, A-O-Malonil-GQ1b, B-O- Malonil-GQ1b y di-O-Malonil-GQ1b, según se definieron anteriormente en la presente memoria., According to another preferred embodiment, Χβ is a -CH 2 COOH group. According to another preferred embodiment the compounds of the present invention are selected from the following list: O-propionyl-GD1b, O-propionyl-GD3, O-propionyl-GD2, AO-propionyl-GT1b, BO-propionyl-GT1b, di-O -propionyl-GT1b, BO-acetyl-GT1b, di-O-acetyl-GTIb, O-Propionyl-GM3, O-Malonyl-GM3, O- Propionyl-GM2, O-Malonyl-GM2, O-Malonil-GD3, O -Malonil-GD2, O-Propionyl- GD1a, O-Malonil-GD1a, O-Malonil-GD1b, AO-Malonil-GT1b, BO-Malonil- GT1b, di-O-Malonil-GT1b, BO-Acetyl-GQ1b, di -O-Acetyl-GQ1b, AO-Propionyl- GQ1b, BO-Propionyl-GQ1b, di-O-Propionyl-GQ1b, AO-Malonyl-GQ1b, BO- Malonil-GQ1b and di-O-Malonyl-GQ1b, as defined previously herein.,
Según otra realización preferida los compuestos de la presente invención se seleccionan de la siguiente lista: According to another preferred embodiment the compounds of the present invention are selected from the following list:
0-propionil-GD3 0-propionyl-GD3
0-propionil-GD2
0-propionyl-GD2
0-propionil-GD1b 0-propionyl-GD1b
B-0-acetil-GT1b B-0-acetyl-GT1b
di-0-acetil-GT1b
di-0-acetyl-GT1b
B-0-propionil-GT1 b donde Y es cualquier ceramida, que está compuesta por una esfingosina y un ácido graso. De manera preferida la esfingosina comprende de 18 a 20 átomos de carbono y tiene un número de insaturaciones de 0 a 2. De manera preferida el ácido graso puede contener de 8 a 24 átomos de carbono y con un número de insaturaciones de 0 a 4; B-0-propionyl-GT1 b where Y is any ceramide, which is composed of a sphingosine and a fatty acid. Preferably, sphingosine comprises 18 to 20 carbon atoms and has a number of unsaturations of 0 to 2. Preferably, the fatty acid may contain 8 to 24 carbon atoms and with a number of unsaturations of 0 to 4;
En la presente invención los términos GD1 b, GD3, GD2, GT1 b, GQ1 b, GM3, GM2, GM1 a o GD1 a, hacen referencia a gangliosidos del mismo nombre conocidos en el estado de la técnica por cualquier experto en la materia. In the present invention the terms GD1 b, GD3, GD2, GT1 b, GQ1 b, GM3, GM2, GM1 a or GD1 a, refer to gangliosides of the same name known in the state of the art by any person skilled in the art.
En este mismo sentido estos gangliosidos comprenden entre otros elementos, una ceramida (Y) que a su vez comprende una esfingosina y un ácido graso. La esfingosina es preferentemente de 18 de átomos de carbono con una
insaturación o de 20 átomos de carbono con una insaturación. De manera preferente el ácido graso es de 18 átomos de carbono sin ninguna insaturación o con una insaturación. Otro aspecto de la invención se refiere a un compuesto sintetizado según el procedimiento de la invención. In this same sense, these gangliosides comprise among other elements, a ceramide (Y) which in turn comprises a sphingosine and a fatty acid. Sphingosine is preferably 18 carbon atoms with a unsaturation or 20 carbon atoms with unsaturation. Preferably, the fatty acid is 18 carbon atoms without any unsaturation or unsaturation. Another aspect of the invention relates to a compound synthesized according to the process of the invention.
Otro aspecto de la presente invención se refiere al compuesto de fórmula general (I) para su uso como medicamento. Another aspect of the present invention relates to the compound of general formula (I) for use as a medicament.
Un tercer aspecto de la presente invención se refiere a una composición farmacéutica que comprende al menos al compuesto de fórmula general (I), o un compuesto de fórmula (la), y al menos un transportador farmacéuticamente aceptable, adyuvante y/o vehículo. A third aspect of the present invention relates to a pharmaceutical composition comprising at least the compound of general formula (I), or a compound of formula (la), and at least one pharmaceutically acceptable carrier, adjuvant and / or vehicle.
En una realización preferida, la composición farmacéutica además comprende otro principio activo. In a preferred embodiment, the pharmaceutical composition further comprises another active ingredient.
Un cuarto aspecto de la presente invención se refiere al uso del gangliósido de fórmula general (I), o un gangliósido de fórmula (la), o cualquiera de sus sales, isómeros o solvatos, donde;Y, Ac, Xi, X2, X3, X4, X5 y ΧΘ se definen como se definieron anteriormente para la elaboración de un medicamento. A fourth aspect of the present invention relates to the use of the ganglioside of general formula (I), or a ganglioside of formula (la), or any of its salts, isomers or solvates, wherein; Y, Ac, Xi, X2, X3 , X4, X5 and ΧΘ are defined as defined above for the preparation of a medicine.
Una realización preferida se refiere al uso para la elaboración de un medicamento para el tratamiento o prevención de tumores, preferiblemente para el tratamiento o prevención de astrocitomas, glioblastomas, oligodendrogliomas, neuroblastomas o meningiomas. A preferred embodiment refers to the use for the preparation of a medicament for the treatment or prevention of tumors, preferably for the treatment or prevention of astrocytomas, glioblastomas, oligodendrogliomas, neuroblastomas or meningiomas.
Un quinto aspecto de la presente invención se refiere al uso del gangliósido de fórmula general (I), o del gangliósido de fórmula (la), o cualquiera de sus sales, isómeros o solvatos, donde: Y, Ac, X1 , X2, X3, X4, X5 y ΧΘ se definen como se definieron anteriormente, como reactivo en ensayos biológicos.
El compuesto descrito en la presente invención, sus sales, profármacos y/o solvatos así como las composiciones farmacéuticas, cosméticas y nutricionales que los contienen pueden ser utilizados junto con otros fármacos, o principios activos, adicionales para proporcionar una terapia de combinación. Dichos fármacos adicionales pueden formar parte de la misma composición farmacéutica o, alternativamente, pueden ser proporcionados en forma de una composición separada para su administración simultánea o no a la de la composición farmacéutica que comprende el compuesto anteriormente descrito, o una sal, profármaco o solvato del mismo. A fifth aspect of the present invention relates to the use of the ganglioside of general formula (I), or of the ganglioside of formula (la), or any of its salts, isomers or solvates, where: Y, Ac, X1, X2, X3 , X4, X5 and ΧΘ are defined as defined above, as a reagent in biological assays. The compound described in the present invention, its salts, prodrugs and / or solvates as well as the pharmaceutical, cosmetic and nutritional compositions containing them can be used together with other drugs, or active ingredients, additional to provide a combination therapy. Said additional drugs may be part of the same pharmaceutical composition or, alternatively, they may be provided in the form of a separate composition for simultaneous or non-simultaneous administration to the pharmaceutical composition comprising the compound described above, or a salt, prodrug or solvate. of the same.
En una realización preferida de la presente invención, las composiciones farmacéuticas son adecuadas para la administración oral, en forma sólida o líquida. Las posibles formas para la administración oral son tabletas, cápsulas, siropes o soluciones y pueden contener excipientes convencionales conocidos en el ámbito farmacéutico, como agentes agregantes (p.e. sirope, acacia, gelatina, sorbitol, tragacanto o polivinilpirrolidona), rellenos (p.e. lactosa, azúcar, almidón de maíz, fosfato de calcio, sorbitol o glicina), disgregantes (p.e. almidón, polivinilpirrolidona o celulosa microcristalina) o un surfactante farmacéuticamente aceptable como el lauril sulfato de sodio. In a preferred embodiment of the present invention, the pharmaceutical compositions are suitable for oral administration, in solid or liquid form. Possible forms for oral administration are tablets, capsules, syrups or solutions and may contain conventional excipients known in the pharmaceutical field, as aggregating agents (eg syrup, acacia, gelatin, sorbitol, tragacanth or polyvinylpyrrolidone), fillers (eg lactose, sugar , corn starch, calcium phosphate, sorbitol or glycine), disintegrants (eg starch, polyvinylpyrrolidone or microcrystalline cellulose) or a pharmaceutically acceptable surfactant such as sodium lauryl sulfate.
Las composiciones para administración oral pueden ser preparadas por los métodos convencionales de Farmacia Galénica, como mezcla y dispersión. Las tabletas se pueden recubrir siguiendo métodos conocidos en la industria farmacéutica. Compositions for oral administration can be prepared by conventional methods of Galenic Pharmacy, as mixing and dispersion. The tablets can be coated following methods known in the pharmaceutical industry.
Las composiciones farmacéuticas se pueden adaptar para la administración parenteral, como soluciones estériles, suspensiones, o liofilizados de los productos de la invención, empleando la dosis adecuada. Se pueden emplear excipientes adecuados, como agentes tamponadores del pH o surfactantes. The pharmaceutical compositions can be adapted for parenteral administration, as sterile solutions, suspensions, or lyophilized products of the invention, using the appropriate dose. Suitable excipients, such as pH buffering agents or surfactants, can be used.
Las formulaciones anteriormente mencionadas pueden ser preparadas usando métodos convencionales, como los descritos en las Farmacopeas de diferentes países y en otros textos de referencia.
La administración de los compuestos o composiciones de la presente invención puede ser realizada mediante cualquier método adecuado, como la infusión intravenosa y las vías oral, intraperitoneal o intravenosa. La administración oral es la preferida por la conveniencia de los pacientes y por el carácter crónico de las enfermedades a tratar. The aforementioned formulations can be prepared using conventional methods, such as those described in the Pharmacopoeias of different countries and in other reference texts. The administration of the compounds or compositions of the present invention can be performed by any suitable method, such as intravenous infusion and oral, intraperitoneal or intravenous routes. Oral administration is preferred for the convenience of patients and for the chronic nature of the diseases to be treated.
La cantidad administrada de un compuesto de la presente invención dependerá de la relativa eficacia del compuesto elegido, la severidad de la enfermedad a tratar y el peso del paciente. Sin embargo, los compuestos de esta invención serán administrados una o más veces al día, por ejemplo 1 , 2, 3 ó 4 veces diarias, con una dosis total entre 0.1 y 1000 mg/Kg/día. Es importante tener en cuenta que puede ser necesario introducir variaciones en la dosis, dependiendo de la edad y de la condición del paciente, así como modificaciones en la vía de administración. The amount administered of a compound of the present invention will depend on the relative efficacy of the compound chosen, the severity of the disease to be treated and the weight of the patient. However, the compounds of this invention will be administered one or more times a day, for example 1, 2, 3 or 4 times daily, with a total dose between 0.1 and 1000 mg / kg / day. It is important to keep in mind that it may be necessary to introduce variations in the dose, depending on the age and condition of the patient, as well as modifications in the route of administration.
Los compuestos y composiciones de la presente invención pueden ser empleados junto con otros medicamentos en terapias combinadas. Los otros fármacos pueden formar parte de la misma composición o de otra composición diferente, para su administración al mismo tiempo o en tiempos diferentes. The compounds and compositions of the present invention can be used together with other medicaments in combination therapies. The other drugs may be part of the same composition or of a different composition, for administration at the same time or at different times.
El término "alquilo" se refiere en la presente invención a cadenas alifáticas, lineales o ramificadas, que tienen de 1 a3 átomos de carbono o de 1 a 2 átomos de carbono, por ejemplo, metilo, etilo, n-propilo, i-propilo, etc. Preferiblemente el grupo alquilo tiene entre 1 y 2 átomos de carbono o preferiblemente el grupo alquilo es un metilo. Los radicales alquilo pueden estar opcionalmente sustituidos por uno o más sustituyentes tales como halógeno, hidroxilo,azida, ácido carboxílico o un grupo sustituido o no sustituido seleccionado de entre amino, amido, éster carboxílico, éter, tiol, acilamino o carboxamido.alcóxido, tiol, amino, acilamino, ciano, carboxilato, carboxamida, carboxiéster, arilo o heteroarilo o combinaciones de estos grupos. Cuando el grupo alquilo está sustituido, lo está preferentemente por uno o varios grupos amina, amida o éter, que a su vez pueden estar o no sustituidos por grupos
alquilo, amida, cicloalquilo o éteres y estos a su vez, pueden estar igualmente sustituidos o no. The term "alkyl" refers in the present invention to aliphatic, linear or branched chains, having 1 to 3 carbon atoms or 1 to 2 carbon atoms, for example, methyl, ethyl, n-propyl, i-propyl , etc. Preferably the alkyl group has between 1 and 2 carbon atoms or preferably the alkyl group is a methyl. The alkyl radicals may be optionally substituted by one or more substituents such as halogen, hydroxyl, azide, carboxylic acid or a substituted or unsubstituted group selected from amino, amido, carboxylic ester, ether, thiol, acylamino or carboxamido.alkoxide, thiol , amino, acylamino, cyano, carboxylate, carboxamide, carboxy ester, aryl or heteroaryl or combinations of these groups. When the alkyl group is substituted, it is preferably substituted by one or more amine, amide or ether groups, which in turn may or may not be substituted by groups. alkyl, amide, cycloalkyl or ethers and these in turn may be equally substituted or not.
A lo largo de la descripción y las reivindicaciones la palabra "comprende" y sus variantes no pretenden excluir otras características técnicas, aditivos, componentes o pasos. Para los expertos en la materia, otros objetos, ventajas y características de la invención se desprenderán en parte de la descripción y en parte de la práctica de la invención. Los siguientes ejemplos se proporcionan a modo de ilustración, y no se pretende que sean limitativos de la presente invención. Throughout the description and claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples are provided by way of illustration, and are not intended to be limiting of the present invention.
EJEMPLOS EXAMPLES
A) Preparación de Neurostatina (0-acetil-GD1 b), A) Preparation of Neurostatin (0-acetyl-GD1 b),
A una solución del gangliósido GD1 b (400 g) en tampón MES (ácido 2- morfolinoetanosulfónico), 50 mM, pH=7.0, se añadió MgC (10 mM), DTT (1 mM), Acetil-Coenzima A (1 mM) y 0.1 % en peso/volumen de colato sódico. Finalmente, se añadió el enzima O-acetiltransferasa a una concentración de 20 μg μΙ, en un volumen de reacción total de 432 μΙ. La reacción se realizó a 37°C durante 7 horas con agitación (600 rpm). Transcurrido el tiempo, la reacción se detuvo por adición de metanol . To a solution of the ganglioside GD1 b (400 g) in MES buffer (2- morpholinoethanesulfonic acid), 50 mM, pH = 7.0, MgC (10 mM), DTT (1 mM), Acetyl-Coenzyme A (1 mM) was added and 0.1% by weight / volume of sodium cholate. Finally, the enzyme O-acetyltransferase was added at a concentration of 20 μg μΙ, in a total reaction volume of 432 μΙ. The reaction was carried out at 37 ° C for 7 hours with stirring (600 rpm). After time, the reaction was stopped by the addition of methanol.
La reacción de O-acetilación de GD1 b dio lugar a un producto de reacción cuya movilidad en cromatografía en capa fina (TLC) fue diferente a GD1 b. Mediante espectrometría de tiempo de vuelo (MALDI TOF-MS) y Electrospray se caracterizó como Neurostatina, por presentar una única O-acetilación en el siálico terminal. El rendimiento de la reacción fue del 90%. The O-acetylation reaction of GD1 b resulted in a reaction product whose mobility in thin layer chromatography (TLC) was different from GD1 b. Through flight time spectrometry (MALDI TOF-MS) and Electrospray, it was characterized as Neurostatin, for presenting a single O-acetylation in the terminal sialic. The reaction yield was 90%.
B) Preparación de 0-acetil-GT1 b y di-0-acetil-GT1 b B) Preparation of 0-acetyl-GT1 b and di-0-acetyl-GT1 b
El gangliósido GT1 b (1000 g) en un tampón 50 mM de MES (ácido 2- morfolino etanosulfónico) pH=7.0, al que se añadió MgC^ O O mM), DTT (1 mM), Acetil-Coenzima A (1 mM) y 0.1 % en peso/volumen de colato sódico.
Finalmente, se añadió la enzima O-acetiltransferasa a una concentración de 20 μg/μ\ en un volumen de reacción total de 432 μΙ. La reacción se realizó a 37°C durante 7 horas con agitación (300 rpm). Transcurrido el tiempo, la reacción se detuvo por adición de metanol . Ganglioside GT1 b (1000 g) in a 50 mM buffer of MES (2- morpholino ethanesulfonic acid) pH = 7.0, to which was added MgC ^ OO mM), DTT (1 mM), Acetyl-Coenzyme A (1 mM) and 0.1% by weight / volume of sodium cholate. Finally, the enzyme O-acetyltransferase was added at a concentration of 20 µg / µ \ in a total reaction volume of 432 µΙ. The reaction was carried out at 37 ° C for 7 hours with stirring (300 rpm). After time, the reaction was stopped by the addition of methanol.
La reacción de O-acetilación de GT1 b dio lugar a dos productos de reacción cuya movilidad en cromatografía en capa fina fue diferente a la de GT1 b. Mediante espectrometría de tiempo de vuelo (MALDITOF-MS) y electrospray el compuesto de mayor movilidad en cromatografía en capa fina presentó 2 O- acetilaciones, cada una en uno de los dos siálicos terminales y fue denominado di-O-acetil-GT1 b. El segundo producto presentó una sola O-acetilación. La caracterización de este compuesto dio lugar a dos especies: el compuesto denominado A-O-acetil-GT1 b, que presenta la O-acetilación en el siálico terminal que se encuentra unido a otro siálico; y el compuesto denominado B- O-acetil-GT1 b que se encuentra mono O-acetilado en el siálico no unido a otro siálico. El rendimiento de la reacción fue del 60% para las especies mono-O- acetiladas y del 20% para la especie di-O-acetilada. The O-acetylation reaction of GT1 b gave rise to two reaction products whose mobility in thin layer chromatography was different from that of GT1 b. By means of flight time spectrometry (MALDITOF-MS) and electrospray, the compound with the greatest mobility in thin layer chromatography presented 2 O-acetylations, each in one of the two terminal sialics and was called di-O-acetyl-GT1 b. The second product presented a single O-acetylation. The characterization of this compound gave rise to two species: the compound called A-O-acetyl-GT1 b, which presents the O-acetylation in the terminal sialic which is bound to another sialic; and the compound called B-O-acetyl-GT1 b which is mono-O-acetylated in the sialic not bound to another sialic. The reaction yield was 60% for mono-O-acetylated species and 20% for di-O-acetylated species.
C) Preparación de 0-propionil-GD1 b C) Preparation of 0-propionyl-GD1 b
El gangliósido GD1 b (400 g) en un tampón 50 mM de MES (ácido 2- morfolinoetanosulfónico) pH=7.0, al que se añadió MgC^ CI O mM), DTT (1 mM), Propionil-Coenzima A (1 mM) y 0.1 % en peso/volumen de colato sódico. Finalmente, se añadió la enzima O-acetiltransferasa a una concentración de 20 μg μΙ en un volumen de reacción total de 432 μΙ. La reacción se realizó a 37°C durante 7 horas con agitación (600 rpm). Transcurrido el tiempo, la reacción se detuvo por adición de metanol .
La reacción de O-propionilación de GD1 b dio lugar a un producto de reacción cuya movilidad en cromatografía en capa fina fue diferente a la de GD1 b. Mediante espectrometría de tiempo de vuelo (MALDITOF-MS) y electrosprayse caracterizó como un derivado mono O-propionilado en el siálico terminal que denominamos O-propionil-GD1 b. El rendimiento de la reacción fue del 10%. Ganglioside GD1 b (400 g) in a 50 mM MES buffer (2- morpholinoethanesulfonic acid) pH = 7.0, to which MgC ^ CI O mM), DTT (1 mM), Propionyl-Coenzyme A (1 mM) was added and 0.1% by weight / volume of sodium cholate. Finally, the enzyme O-acetyltransferase was added at a concentration of 20 μg μΙ in a total reaction volume of 432 μΙ. The reaction was carried out at 37 ° C for 7 hours with stirring (600 rpm). After time, the reaction was stopped by the addition of methanol. The O-propionylation reaction of GD1 b resulted in a reaction product whose mobility in thin layer chromatography was different from that of GD1 b. Through flight time spectrometry (MALDITOF-MS) and electrospray it was characterized as a mono-derivative O-propionylated derivative in the terminal sialic we call O-propionyl-GD1 b. The reaction yield was 10%.
D) Preparación de 0-propionil-GT1 b D) Preparation of 0-propionyl-GT1 b
Siguiendo el mismo procedimiento que para el O-propionil-GD1 b, pero partiendo del gangliósido GT1 b se obtuvo un producto de reacción cuya movilidad en cromatografía de capa fina (TLC) fue diferente a la de GT1 b. Dicho producto fue caracterizado mediante espectrometría de tiempo de vuelo (MALDITOF-MS) y Electrospray, como el gangliósido GT1 b con una O- propionilación en el siálico terminal que se encuentra unido a otro siálico y fue denominado O-propionil-GT1 b. El rendimiento de la reacción fue del 20%. Following the same procedure as for O-propionyl-GD1 b, but starting from the GT1 b ganglioside, a reaction product was obtained whose mobility in thin layer chromatography (TLC) was different from that of GT1 b. This product was characterized by flight time spectrometry (MALDITOF-MS) and Electrospray, such as the ganglioside GT1 b with an O-propionylation in the terminal sialic which is attached to another sialic and was called O-propionyl-GT1 b. The reaction yield was 20%.
E) Actividad inhibitoria de los compuestos obtenidos anteriormente La actividad inhibitoria de la división de células tumorales de los gangliósidos y sus derivados O-acetilados y O-propionilados obtenidos fue determinada mediante en ensayo MTT que mide la actividad mitocondrial, en cultivos de la línea tumoral C6 (glioma de rata) durante 48 horas de tratamiento, utilizando 10 ng/ml de FGF básico como mitógeno E) Inhibitory activity of the compounds obtained above The inhibitory activity of the tumor cell division of gangliosides and their O-acetylated and O-propionylated derivatives obtained was determined by MTT assay measuring mitochondrial activity, in tumor line cultures C6 (rat glioma) for 48 hours of treatment, using 10 ng / ml of basic FGF as a mitogen
Tabla 1 . Inhibición de la división en células C6 (48 h) en valores promedio de ID50 (nM).EI símbolo "-"indica que el compuesto inhibió débilmente la proliferación a la concentración máxima ensayada (4 μΜ), pero no lo suficiente como para calcular el valor de su ID5o-
En la tabla 1 presentamos la actividad de los gangliósidos GD1 b, GT1 b, y sus derivados modificados. El gangliósido GD1 b no tiene actividad inhibitoria de la proliferación de la línea de glioma C6. Mientras que los compuestos derivados de GD1 b, Neurostatina (O-acetil-GD1 b), y el O-propionil-GD1 b presentan actividad en el rango nanomolar. El gangliósido O-propionil-GD1 b es ligeramente más activo que la Neurostatina. Table 1 . Inhibition of division in C6 cells (48 h) in average values of ID 50 (nM). The symbol "-" indicates that the compound weakly inhibited proliferation at the maximum concentration tested (4 μΜ), but not enough to calculate the value of your ID 5 or- In table 1 we present the activity of gangliosides GD1 b, GT1 b, and their modified derivatives. Ganglioside GD1 b has no proliferative inhibitory activity of the C6 glioma line. While the compounds derived from GD1 b, Neurostatin (O-acetyl-GD1 b), and O-propionyl-GD1 b have activity in the nanomolar range. The ganglioside O-propionyl-GD1 b is slightly more active than Neurostatin.
El gangliósido GT1 b tiene una actividad inhibidora de la proliferación. El compuesto O-acetil-GT1 b mejora casi al doble su actividad inhibidora respecto al GT1 b. Cuando el gangliósido GT1 b se modifica con dos O-acetilaciones (di- O-acetil-GT1 b) también mejora su actividad al doble respecto al O-acetil-GT1 b y 4 veces respecto al GT1 b. La adición de O-acetilos al gangliósido GT1 b aumenta progresivamente su actividad inhibidora de la proliferación en las células C6. Ganglioside GT1 b has a proliferation inhibitory activity. The compound O-acetyl-GT1 b almost twice improves its inhibitory activity with respect to GT1 b. When the ganglioside GT1 b is modified with two O-acetylations (di-O-acetyl-GT1 b) it also improves its activity twice as much as the O-acetyl-GT1 b and 4 times with respect to the GT1 b. The addition of O-acetyls to the ganglioside GT1 b progressively increases its proliferation inhibitory activity in C6 cells.
Los gangliósidos O-propionilados tienen mayor actividad inhibitoria (O- propionil-GD1 b y O-propionil-GT1 b) que sus respectivos gangliósidos mono-O- acetilados (O-acetil-GD1 b y O-acetil-GT1 b). Estos compuestos O-propionilados podrían ser más resistentes a la hidrólisis por glicosidasas, por lo que su efecto sería más duradero. O-propionylated gangliosides have higher inhibitory activity (O-propionyl-GD1 b and O-propionyl-GT1 b) than their respective mono-O-acetylated gangliosides (O-acetyl-GD1 b and O-acetyl-GT1 b). These O-propionylated compounds could be more resistant to hydrolysis by glycosidases, so their effect would be more lasting.
F) Preparación de 0-malonil-GD1 b El gangliósido GD1 b (400 g) en un tampón 50 mM de MES (ácido 2- moríolino etanosulfónico) pH=7.0, al que se añadió MgCI2 (10 mM), DTT (1 mM), Malonil- Coenzima A (1 mM) y 0.1 % en peso/volumen de colato sódico. Finalmente, se añadió la enzima O-acetiltransferasa a una concentración de 20 μg μl en un volumen de reacción total de 432 μΙ. La reacción se realizó a 37°C durante 7 horas con agitación (300 rpm). Transcurrido el tiempo, la reacción se detuvo por adición de metanol. F) Preparation of 0-malonyl-GD1 b Ganglioside GD1 b (400 g) in a 50 mM buffer of MES (2-ethano sulfine ethanesulfonic acid) pH = 7.0, to which was added MgCl 2 (10 mM), DTT (1 mM), Malonyl-Coenzyme A (1 mM) and 0.1% by weight / volume of sodium cholate. Finally, the enzyme O-acetyltransferase was added at a concentration of 20 μg μl in a total reaction volume of 432 μΙ. The reaction was carried out at 37 ° C for 7 hours with stirring (300 rpm). After time, the reaction was stopped by the addition of methanol.
La reacción de O-malonilación de GD1 b dio lugar a un producto de reacción cuya movilidad en cromatografía en capa fina fue diferente a la de GD1 b.
Mediante espectrometría de tiempo de vuelo (MALDITOF-MS) y electrospray se caracterizó como un derivado mono O-malonilado en el siálico terminal que denominamos O-malonil-GD1 b. El rendimiento de la reacción fue del 5%. G) Preparación de 0-acetil-GM3 The O-malonylation reaction of GD1 b gave rise to a reaction product whose mobility in thin layer chromatography was different from that of GD1 b. Through flight time spectrometry (MALDITOF-MS) and electrospray, it was characterized as an O-malonylated mono derivative in the terminal sialic we call O-malonyl-GD1 b. The reaction yield was 5%. G) Preparation of 0-acetyl-GM3
El gangliósido GM3 (1000 g) en un tampón 50 mM de MES (ácido 2- moríolino etanosulfónico) pH=7.0, al que se añadió MgCI2 (10 mM), DTT (1 mM), Acetil- Coenzima A (1 mM) y 0.1 % en peso/volumen de colato sódico. Finalmente, se añadió la enzima O-acetiltransferasa a una concentración de 20 μg μl en un volumen de reacción total de 432 μΙ. La reacción se realizó a 37°C durante 7 horas con agitación (300 rpm). Transcurrido el tiempo, la reacción se detuvo por adición de metanol. La reacción de O-acetilación de GM3 dio lugar a un producto de reacción cuya movilidad en cromatografía en capa fina fue diferente a la de GM3. Mediante espectrometría de tiempo de vuelo (MALDITOF-MS) y electrospray se caracterizó como un derivado mono O-acetilado en el siálico que denominamos O-acetil-GM3. El rendimiento de la reacción fue del 90%. The GM3 ganglioside (1000 g) in a 50 mM MES buffer (2-ethano sulfonic acid ethanesulfonic acid) pH = 7.0, to which was added MgCl 2 (10 mM), DTT (1 mM), Acetyl-Coenzyme A (1 mM) and 0.1% by weight / volume of sodium cholate. Finally, the enzyme O-acetyltransferase was added at a concentration of 20 μg μl in a total reaction volume of 432 μΙ. The reaction was carried out at 37 ° C for 7 hours with stirring (300 rpm). After time, the reaction was stopped by the addition of methanol. The O-acetylation reaction of GM3 resulted in a reaction product whose mobility in thin layer chromatography was different from that of GM3. Through flight time spectrometry (MALDITOF-MS) and electrospray, it was characterized as an O-acetylated mono derivative in the sialic we call O-acetyl-GM3. The reaction yield was 90%.
H) Preparación de 0-acetil-GD3 El gangliósido GD3 (400 g) en un tampón 50 mM de MES (ácido 2- morfolino etanosulfónico) pH=7.0, al que se añadió MgCI2 (10 mM), DTT (1 mM), Acetil- Coenzima A (1 mM) y 0.1 % en peso/volumen de colato sódico. Finalmente, se añadió la enzima O-acetiltransferasa a una concentración de 20 μg μl en un volumen de reacción total de 432 μΙ. La reacción se realizó a 37°C durante 7 horas con agitación (300 rpm). Transcurrido el tiempo, la reacción se detuvo por adición de metanol. H) Preparation of 0-acetyl-GD3 Ganglioside GD3 (400 g) in a 50 mM buffer of MES (2- morpholino ethanesulfonic acid) pH = 7.0, to which MgCl 2 (10 mM), DTT (1 mM) was added , Acetyl-Coenzyme A (1 mM) and 0.1% by weight / volume of sodium cholate. Finally, the enzyme O-acetyltransferase was added at a concentration of 20 μg μl in a total reaction volume of 432 μΙ. The reaction was carried out at 37 ° C for 7 hours with stirring (300 rpm). After time, the reaction was stopped by the addition of methanol.
La reacción de O-acetilación de GD3 dio lugar a un producto de reacción cuya movilidad en cromatografía en capa fina fue diferente a la de GD3. Mediante espectrometría de tiempo de vuelo (MALDITOF-MS) y electrospray se
caracterizó como un derivado mono O-acetilado en el siálico terminal que denominamos O-acetil-GD3. El rendimiento de la reacción fue del 80%. The O-acetylation reaction of GD3 resulted in a reaction product whose mobility in thin layer chromatography was different from that of GD3. Through flight time spectrometry (MALDITOF-MS) and electrospray, characterized as an O-acetylated mono derivative in the terminal sialic we call O-acetyl-GD3. The reaction yield was 80%.
I) Preparación de 0-acetil-GD1a I) Preparation of 0-acetyl-GD1a
El gangliósido GD1 a (1000 g) en un tampón 50 mM de MES (ácido 2- morfolino etanosulfónico) pH=7.0, al que se añadió MgC^ CI O mM), DTT (1 mM), Acetil-Coenzima A (1 mM) y 0.1 % en peso/volumen de colato sódico. Finalmente, se añadió la enzima O-acetiltransferasa a una concentración de 20 μg μl en un volumen de reacción total de 432 μΙ. La reacción se realizó a 37°C durante 7 horas con agitación (300 rpm). Transcurrido el tiempo, la reacción se detuvo por adición de metanol. Ganglioside GD1 a (1000 g) in a 50 mM buffer of MES (2- morpholino ethanesulfonic acid) pH = 7.0, to which MgC ^ CI O mM was added), DTT (1 mM), Acetyl-Coenzyme A (1 mM ) and 0.1% by weight / volume of sodium cholate. Finally, the enzyme O-acetyltransferase was added at a concentration of 20 μg μl in a total reaction volume of 432 μΙ. The reaction was carried out at 37 ° C for 7 hours with stirring (300 rpm). After time, the reaction was stopped by the addition of methanol.
La reacción de O-acetilación de GD1 a dio lugar a un producto de reacción cuya movilidad en cromatografía en capa fina fue diferente a la de GD1 a. Mediante espectrometría de tiempo de vuelo (MALDITOF-MS) y electrospray se caracterizó como un derivado mono O-acetilado en el siálico unido en enlace alpha(2-3) al radical galactosa-N-acetil-galactosamina que denominamos O- acetil-GD1 a. El rendimiento de la reacción fue del 70%.
The O-acetylation reaction of GD1 a resulted in a reaction product whose mobility in thin layer chromatography was different from that of GD1 a. Through flight time spectrometry (MALDITOF-MS) and electrospray, it was characterized as an O-acetylated mono derivative in the sialic linked in alpha (2-3) to the galactose-N-acetyl-galactosamine radical that we call O-acetyl-GD1 to. The reaction yield was 70%.
Claims
1. Un procedimiento de síntesis de un compuesto de fórmula general (I): 1. A synthesis procedure of a compound of general formula (I):
(I) (YO)
o cualquiera de sus sales, isómeros o solvatos, or any of its salts, isomers or solvates,
donde: where:
Y es una ceramida; And it is a ceramide;
Ac es un grupo acetilo; Ac is an acetyl group;
Z se selecciona entre un grupo de fórmula (Zi): Z is selected from a group of formula (Zi):
(Zl) (Zl)
de manera que la fórmula general (I) corresponde a la fórmula (la): so that the general formula (I) corresponds to the formula (la):
HOJA DE REEMPLAZO (Regla 23)
o Z es un grupo de fórmula (Z2):
REPLACEMENT SHEET (Rule 23) o Z is a group of formula (Z 2 ):
(Z2) ( Z2 )
de manera que la fórmula general (I) corresponde a la fórmula (Ib): so that the general formula (I) corresponds to the formula (Ib):
(Ib) (ib)
donde tanto para la fórmula (la) como para la formula (Ib): where for both formula (la) and formula (Ib):
Xi se selecciona entre -CH2-O-CO-X2 y -CH2OH; Xi is selected from -CH 2 -O-CO-X 2 and -CH 2 OH;
X2 se selecciona entre un grupo alquilo Ci-C2 y-CH2COOH; X 2 is selected from a Ci-C 2 alkyl group and -CH 2 COOH;
X3 se selecciona entre H, OH, un grupo de fórmula (II) o un grupo de fórmula X3 is selected from H, OH, a group of formula (II) or a group of formula
(III): (III):
HOJA DE REEMPLAZO (Regla 23}
REPLACEMENT SHEET (Rule 23}
donde en la fórmula III, X4 se selecciona entre H, un grupo de fórmula (IV) o un grupo de fórmula (V): where in formula III, X4 is selected from H, a group of formula (IV) or a group of formula (V):
(V) (V)
donde tanto para la fórmula IV como para la fórmula V: where for both formula IV and formula V:
X5 se selecciona entre un grupo OH o un grupo -0-COXe; X 5 is selected from an OH group or a -0-COXe group;
Χβββ selecciona entre un grupo alquilo C1-C2 o -CH2COOH; excepto cuando: Χβββ selects from a C1-C2 alkyl group or -CH 2 COOH; Except when:
- Z es un grupo de fórmula (Z^ o (Z2), X1 es -CH2-OH y X3 es H; - Z is a group of formula (Z^ or (Z2), X1 is -CH 2 -OH and X3 is H;
- Z es un grupo de fórmula (ΖΊ) o (Z2), X1 es -CH2-OH y X3 es OH; - Z is a group of formula (Ζ Ί ) or (Z2), X1 is -CH2-OH and X3 is OH;
HOJA DE REEMPLAZO (Regla 23)
- Z es un grupo de fórmula (Zi) o (Z2), Xi es -CH2-OH y X3 es el grupo de fórmula (II); REPLACEMENT SHEET (Rule 23) - Z is a group of formula (Zi) or (Z 2 ), Xi is -CH2-OH and X3 is the group of formula (II);
- Z es un grupo de fórmula (Z-i) o (Z2), X1 es -CH2-OH, X3 es el grupo de fórmula (III) y X4 es H; - Z is a group of formula (Zi) or (Z 2 ), X1 is -CH 2 -OH, X 3 is the group of formula (III) and X4 is H;
- Z es un grupo de fórmula (Z1) o (Z2), X1 es -CH2-OH, X3 es el grupo de fórmula (III) y X4 es el grupo de fórmula (IV) y X5 es OH; - Z is a group of formula (Z1) or (Z 2 ), X1 is -CH 2 -OH, X 3 is the group of formula (III) and X4 is the group of formula (IV) and X 5 is OH;
- Z es un grupo de fórmula (Z1) o (Z2), X1 es -CH2-OH, X3 es el grupo de fórmula (III) y X4 es el grupo de fórmula (V) y X5 es OH; que comprende las etapas de: a) disolver un gangliósido en una solución tampón; b) adicionar un emulgente a la mezcla de la etapa a) c) adicionar a la mezcla de la etapa b) la enzima o-acetiltransferasa; y d) parar la reacción por adición de metanol a la mezcla obtenida en la etapa c). - Z is a group of formula (Z1) or (Z 2 ), X1 is -CH 2 -OH, X3 is the group of formula (III) and X 4 is the group of formula (V) and X 5 is OH; which comprises the steps of: a) dissolving a ganglioside in a buffer solution; b) add an emulsifier to the mixture of step a) c) add to the mixture of step b) the o-acetyltransferase enzyme; and d) stopping the reaction by adding methanol to the mixture obtained in step c).
HOJA DE REEMPLAZO Re ia 23
o cualquiera de sus sales, isómeros o solvatos, REPLACEMENT SHEET Re ia 23 or any of its salts, isomers or solvates,
donde: where:
Y es una ceramida; And it is a ceramide;
Ac es un grupo acetilo; Ac is an acetyl group;
X2 es un grupo alquilo C1-C2; X 2 is a C1-C2 alkyl group;
X3 se selecciona entre H, OH, un grupo de fórmula (II) o un grupo de fórmula (III): X3 is selected from H, OH, a group of formula (II) or a group of formula (III):
(II) (III) donde en la fórmula III, X4 se selecciona entre H o un grupo de fórmula (IV): (II) (III) where in formula III, X4 is selected from H or a group of formula (IV):
excepto cuando: Except when:
- Xi es -CH2-O-CO-X2, X2 es un grupo CH3, X3 es el grupo de fórmula (III) y X4 es H; - Xi is -CH2-O-CO-X2, X2 is a CH 3 group, X3 is the group of formula (III) and X4 is H;
- X! es -CH2-O-CO-X2, X2 es un grupo CH3, X3 es el grupo de fórmula (II); - X! is -CH2-O-CO-X2, X2 is a CH 3 group, X 3 is the group of formula (II);
HOJA DE REEMPLAZO (Regla 23)
- Xi es -CH2-O-CO-X2, X2 es un grupo CHU, X3 es el grupo de fórmula (III), X4 es el grupo de fórmula (IV) y X5 es OH; que comprende las etapas de: a) disolver un gangliósido en una solución tampón; b) adicionar un emulgente a la mezcla de la etapa a) c) adicionar a la mezcla de la etapa b) la enzima o-acetiltransferasa; y d) parar la reacción por adición de metanol a la mezcla obtenida en la etapa c). REPLACEMENT SHEET (Rule 23) - Xi is -CH 2 -O-CO-X 2 , X 2 is a CHU group, X 3 is the group of formula (III), X 4 is the group of formula (IV) and X 5 is OH; which comprises the steps of: a) dissolving a ganglioside in a buffer solution; b) add an emulsifier to the mixture of step a) c) add to the mixture of step b) the o-acetyltransferase enzyme; and d) stopping the reaction by adding methanol to the mixture obtained in step c).
3. El procedimiento según la reivindicación 1 , donde en la etapa a) el gangliósido se selecciona del grupo formado por GD1 b, GD3, GD2, GT1 b, GQ1b, G 3, GM2, GM1a y GD1a. 3. The procedure according to claim 1, where in step a) the ganglioside is selected from the group consisting of GD1 b, GD3, GD2, GT1 b, GQ1b, G 3, GM2, GM1a and GD1a.
4. El procedimiento según la reivindicación 2, donde en la etapa a) el gangliósido se selecciona del grupo formado por GD1b, GD3, GD2 y GT1 b. 4. The procedure according to claim 2, where in step a) the ganglioside is selected from the group consisting of GD1b, GD3, GD2 and GT1 b.
5. El procedimiento según una cualquiera de las reivindicaciones 1 a 4, donde en la etapa a) se añade una concentración de gangliósido entre 50 a 600 Mmoles/L reacción. 5. The procedure according to any one of claims 1 to 4, where in step a) a concentration of ganglioside between 50 to 600 Mmol/L is added to the reaction.
6. El procedimiento según la reivindicación 5, donde en la etapa a) se añade una concentración de gangliósido entre 300 y 600 moles/L de reacción.. 6. The procedure according to claim 5, wherein in step a) a concentration of ganglioside between 300 and 600 moles/L of reaction is added.
7. El procedimiento según la reivindicación 6, donde en la etapa a) se añade una concentración de gangliósido de 500 Mmoles(L de reacción. 7. The procedure according to claim 6, wherein in step a) a ganglioside concentration of 500 mmol (L of reaction) is added.
HOJA DE REEMPLAZO (Regla 23}
REPLACEMENT SHEET (Rule 23}
8. El procedimiento según una cualquiera de las reivindicaciones 1 a 7, donde la solución tampón está en una concentración desde 1 hasta 250 mmoles/L de reacción. 8. The procedure according to any one of claims 1 to 7, wherein the buffer solution is in a concentration of from 1 to 250 mmol/L of reaction.
9. El procedimiento según la reivindicación 8, donde la solución tampón está en una concentración desdelO hasta 100 mmoles/L de reacción. 9. The process according to claim 8, wherein the buffer solution is in a concentration of from 10 to 100 mmol/L of reaction.
10. El procedimiento según la reivindicación 9, donde la solución tampón está en una concentración de 50 mmoles/L de reacción.. 10. The procedure according to claim 9, wherein the buffer solution is at a concentration of 50 mmol/L of reaction.
1 1. El procedimiento según una cualquiera de las reivindicaciones 8 a 10, donde la solución tampón se selecciona del grupo formado por TRIS, MES, o fosfato. 1 1. The method according to any one of claims 8 to 10, wherein the buffer solution is selected from the group consisting of TRIS, MES, or phosphate.
12. El procedimiento según la reivindicación 11 , donde la solución tampón es MES. 12. The method according to claim 11, wherein the buffer solution is MES.
13. El procedimiento según una cualquiera de las reivindicaciones 8 a 12, donde la solución tampón tiene un pH desde 6 a 9. 13. The method according to any one of claims 8 to 12, wherein the buffer solution has a pH from 6 to 9.
14. El procedimiento según la reivindicación 13, donde el pH de la solución tampón está entre 7 y 8. 14. The method according to claim 13, wherein the pH of the buffer solution is between 7 and 8.
15. El procedimiento según la reivindicación 14, donde el pH de la solución tampón es de 7.0. 15. The method according to claim 14, wherein the pH of the buffer solution is 7.0.
16. El procedimiento según una cualquiera de las reivindicaciones 8 a 15, donde se añade a la solución tampón MgC 10 mM y DTT 1mM, y además un derivado de la coenzimaA seleccionado entre Acetil-Coenzima A, Propionil- Coenzima A y Malonil-CoenzimaA, a una concentración entre 0.1 a 10 mmoles/L de reacción.. 16. The procedure according to any one of claims 8 to 15, wherein 10 mM MgC and 1 mM DTT are added to the buffer solution, and also a derivative of coenzyme A selected from Acetyl-Coenzyme A, Propionyl-Coenzyme A and Malonyl-Coenzyme A. , at a concentration between 0.1 to 10 mmol/L of reaction.
HOJA DE REEMPLAZO (Regla 23)
REPLACEMENT SHEET (Rule 23)
17. El procedimiento según la reivindicación 16, donde el derivado de Coenzima A se selecciona entre Acetil-Coenzima A ó Propionil-Coenzima A. 17. The procedure according to claim 16, wherein the Coenzyme A derivative is selected from Acetyl-Coenzyme A or Propionyl-Coenzyme A.
18. El procedimiento según una de las reivindicaciones 16 ó 17, donde el derivado de Coenzima A está a una concentración entre 0.5 y 2 mmoles/L de reacción. 18. The procedure according to one of claims 16 or 17, wherein the Coenzyme A derivative is at a concentration between 0.5 and 2 mmol/L of reaction.
19. El procedimiento según la reivindicación 18, donde el derivado de Coenzima A está a una concentración de 1 mmoles/L de reacción. 19. The procedure according to claim 18, wherein the Coenzyme A derivative is at a concentration of 1 mmol/L of reaction.
20. El procedimiento según una cualquiera de las reivindicaciones 3 a 19, donde el emulgente es un ácido biliar o sal biliar a una concentración entre 0.05% y 0.2% peso/volumen total de reacción. 20. The procedure according to any one of claims 3 to 19, wherein the emulsifier is a bile acid or bile salt at a concentration between 0.05% and 0.2% weight/total reaction volume.
21. El procedimiento según la reivindicación 20, donde el emulgente tiene una concentración del 0.1 % en peso/volumen total de reacción. 21. The process according to claim 20, wherein the emulsifier has a concentration of 0.1% by weight/total reaction volume.
22. El procedimiento según una cualquiera de las reivindicaciones 20 ó 21 , donde el ácido biliar se selecciona del grupo formado por el ácido cólico, ácido deoxicólico, taurocólico, glicocólico, ursodeosicólico, litocólico, o sus sales sódicas y potásicas, dentro del grupo formado porcolatos, taurocolatos, deoxicolatos, ursodeoxicolatos o glicocolatos. 22. The procedure according to any one of claims 20 or 21, wherein the bile acid is selected from the group formed by cholic acid, deoxycholic acid, taurocholic acid, glycocholic acid, ursodeosicolic acid, lithocholic acid, or its sodium and potassium salts, within the group formed porcholates, taurocholates, deoxycholates, ursodeoxycholates or glycocholates.
23. El procedimiento según la reivindicación 22, donde el emulgente es la sal biliar colato sódico. 23. The process according to claim 22, wherein the emulsifier is sodium cholate bile salt.
24. El procedimiento según una cualquiera de las reivindicaciones 3 a 23, donde en la etapa c), se añade la enzima O-acetiltransferasa una concentración de entre 2 y 30 pg/μΙ de reacción. 24. The procedure according to any one of claims 3 to 23, wherein in step c), the O-acetyltransferase enzyme is added at a concentration of between 2 and 30 pg/μΙ of reaction.
25. El procedimiento según la reivindicación 24, donde la enzima se añade a una concentración entre 15 y 25 pg/μΙ de reacción. 25. The procedure according to claim 24, wherein the enzyme is added at a concentration between 15 and 25 pg/μΙ of reaction.
HOJA DE REEMPLAZO (Regía 23)
REPLACEMENT SHEET (Rule 23)
26. El procedimiento según la reivindicación 25, donde la enzima se añade a una concentración de 20 [ig/ i\ de reacción. 26. The process according to claim 25, wherein the enzyme is added at a concentration of 20 [ig/i\ reaction.
27. El procedimiento según una cualquiera de las reivindicaciones 3 a 26, donde la reacción de la etapa c) se lleva a cabo a una temperatura entre 30- 40°C. 27. The procedure according to any one of claims 3 to 26, wherein the reaction of step c) is carried out at a temperature between 30-40°C.
28. El procedimiento según la reivindicación 27, donde la reacción de la etapa c) se lleva a cabo a una temperatura entre 35-38 °C. 28. The procedure according to claim 27, wherein the reaction of step c) is carried out at a temperature between 35-38 °C.
29. El procedimiento según la reivindicación 28, donde la reacción de la etapa c) se lleva a cabo a una temperatura de 37 °C. 29. The process according to claim 28, wherein the reaction of step c) is carried out at a temperature of 37 °C.
30. El procedimiento según una cualquiera de las reivindicaciones 3 a 29, donde adicionalmente tras la etapa d) se llevan a cabo las siguientes etapas: 30. The procedure according to any one of claims 3 to 29, wherein additionally after step d) the following steps are carried out:
- desalar los productos obtenidos tras la etapa d) utilizando un cartucho de fase reversa Sep-Pak C18, - desalt the products obtained after step d) using a Sep-Pak C18 reverse phase cartridge,
- desecar la muestra con los productos desalados anteriores en vacío; - dry the sample with the previous desalted products in a vacuum;
- purificar los diferentes productos desecados por TLC preparativa o HPLC preparativa en una columna amino; - purify the different dried products by preparative TLC or preparative HPLC on an amino column;
- calcular el peso molecular de los productos de la etapa anterior mediante espectrometría de masas de tiempo de vuelo; - calculate the molecular weight of the products of the previous stage by time-of-flight mass spectrometry;
- caracterización de la posición de las O-acetilaciones u O- propionilaciones mediante secuenciación por espectrometría de masas "electrospray". - characterization of the position of O-acetylations or O-propionylations by sequencing by electrospray mass spectrometry.
31. Compuesto definido por la fórmula general (I), excepto cuando: 31. Compound defined by general formula (I), except when:
- Z es un grupo de fórmula (Zi), Xi es -CH2-O-CO-X2, X2 es un grupo CH3, X3 es el grupo de fórmula (III) y X4 es H; - Z is a group of formula (Zi), Xi is -CH2-O-CO-X2, X2 is a CH 3 group, X 3 is the group of formula (III) and X4 is H;
- Z es un grupo de fórmula (Z1), X1 es -CH2-0-CO-X2, X2 es un grupo CH3, X3 es el grupo de fórmula (II); - Z is a group of formula (Z1), X1 is -CH 2 -0-CO-X 2 , X2 is a group CH 3 , X 3 is the group of formula (II);
- Z es un grupo de fórmula (Z^, X1 es -CH2-O-CO-X2, X2 es un grupo CH3) X3 es el grupo de fórmula (III), X4 es el grupo de fórmula (IV) y X5 es OH; - Z is a group of formula (Z^, X1 is -CH2-O-CO-X2, X2 is a group CH 3 ) X 5 is OH;
OJA ü>&. ^ ^lAZ ( lcg!& 2c)
- Z es un grupo de fórmula (Zi), Xi es -CH2-0-CO-X2, X2 es un grupo CH3, X3 es OH; OJA ü>&. ^ ^lAZ ( lcg!& 2c) - Z is a group of formula (Zi), Xi is -CH 2 -0-CO-X 2 , X2 is a CH 3 group, X 3 is OH;
- Z es un grupo de fórmula (Zi), X1 es -CH2-O-CO-X2, X2 es un grupo CH3, X3 es el grupo de fórmula (III), X4 es el grupo de fórmula (V), X5 es -OH; - Z is a group of formula (Zi), X1 is -CH 2 -O -CO - X2, ), X 5 is -OH;
- Z es un grupo de fórmula (Z2), X1 es -CH2-O-CO-X2, X2 es un grupo CH3, X3 es OH; - Z is a group of formula (Z 2 ), X1 is -CH 2 -O-CO-X 2 , X 2 is a CH 3 group, X 3 is OH;
- Z es un grupo de fórmula (Z2), X1 es -CH2-O-CO-X2, X2 es un grupo CH3, X3 es el grupo de fórmula (II); - Z is a group of formula (Z 2 ), X1 is -CH 2 -O-CO-X 2 , X 2 is a group CH 3 , X 3 is the group of formula (II);
- Z es un grupo de fórmula (Z2), X1 es -CH2-OH, X3 es el grupo de fórmula (III), X4 es el grupo de fórmula (IV), X5 es -O-CO-X6 y Χβ es un grupo CH3. - Z is a group of formula (Z 2 ), X1 is -CH 2 -OH, X 3 is the group of formula (III), X4 is the group of formula (IV), X 5 is -O-CO-X 6 and Χβ is a CH 3 group.
32. Compuesto definido por la fórmula (la), excepto cuando: 32. Compound defined by formula (la), except when:
- Xi es -CH2-O-CO-X2, X2 es un grupo CH3, X3 es el grupo de fórmula (III) y X4 es H; - Xi is -CH 2 -O-CO-X 2 , X 2 is a CH 3 group, X 3 is the group of formula (III) and X4 is H;
- Xi es -CH2-O-CO-X2, X2 es un grupo CH3, X3 es el grupo de fórmula (II); - Xi is -CH 2 -O-CO-X 2 , X 2 is a CH 3 group, X 3 is the group of formula (II);
- Xi es -CH2-O-CO-X2, X2 es un grupo CH3, X3 es el grupo de fórmula (III), X4 es el grupo de fórmula (IV) y X5 es OH. - Xi is -CH 2 -O-CO-X 2 , X 2 is a CH 3 group, X 3 is the group of formula (III), X4 is the group of formula (IV) and X 5 is OH.
33. El compuesto según una de las reivindicaciones 31 ó 32, donde Y es una ceramida que comprende una esfingosina y un ácido graso natural o sintético. 33. The compound according to one of claims 31 or 32, wherein Y is a ceramide comprising a sphingosine and a natural or synthetic fatty acid.
34. El compuesto según la reivindicación 33, donde la esfingosina comprende de 18 a 20 átomos de carbono y de 0 a 2 insaturaciones. 34. The compound according to claim 33, wherein sphingosine comprises 18 to 20 carbon atoms and 0 to 2 unsaturations.
35. El compuesto según una cualquiera de las reivindicaciones 33 ó 34, donde el ácido graso comprende de 8 a 24 átomos de carbono y con un número de insaturaciones de 0 a 4. 35. The compound according to any one of claims 33 or 34, wherein the fatty acid comprises from 8 to 24 carbon atoms and with a number of unsaturations from 0 to 4.
36. El compuesto según una cualquiera de las reivindicaciones 31 a 35, donde X2 es un grupo metilo. 36. The compound according to any one of claims 31 to 35, wherein X 2 is a methyl group.
37. El compuesto según una cualquiera de las reivindicaciones 31 a 35, donde X2 es un grupo etilo. 37. The compound according to any one of claims 31 to 35, wherein X 2 is an ethyl group.
HOJA DE REEMPLAZO (Regla 23)
REPLACEMENT SHEET (Rule 23)
38. El compuesto según una cualquiera de las reivindicaciones 31 a 35, donde X2 es un grupo -CH2COOH. 38. The compound according to any one of claims 31 to 35, wherein X 2 is a -CH 2 COOH group.
39. El compuesto según una cualquiera de las reivindicaciones 31 a 38, donde X3 es H. 39. The compound according to any one of claims 31 to 38, where X 3 is H.
40. El compuesto según una cualquiera de las reivindicaciones 31 a 38, .donde X3 es OH. 40. The compound according to any one of claims 31 to 38, where X 3 is OH.
41. El compuesto según una cualquiera de las reivindicaciones 31 a 38, donde X3 es un grupo de fórmula (II). 41. The compound according to any one of claims 31 to 38, where X 3 is a group of formula (II).
42. El compuesto según una cualquiera de las reivindicaciones 31 a 38, donde X3es un grupo de fórmula (III). 42. The compound according to any one of claims 31 to 38, where X 3 is a group of formula (III).
43. El compuesto según la reivindicación 42, donde X es H. 43. The compound according to claim 42, where X is H.
44. El compuesto según la reivindicación 42, donde X4 es un compuesto de fórmula general (IV). 44. The compound according to claim 42, wherein X4 is a compound of general formula (IV).
45. El compuesto según la reivindicación 42, donde X4 es un compuesto de fórmula general (V). 45. The compound according to claim 42, wherein X4 is a compound of general formula (V).
46. El compuesto según una de las reivindicación 44 ó 45, donde X5 es un grupo OH. 46. The compound according to one of claims 44 or 45, wherein X5 is an OH group.
47. El compuesto según una de las reivindicación 44 ó 45, dondeXs es un grupo -O-COX6. 47. The compound according to one of claims 44 or 45, where Xs is a -O-COX 6 group.
48. El compuesto según la reivindicación 47, donde XQ es un metilo. 48. The compound according to claim 47, wherein XQ is a methyl.
49. El compuesto según la reivindicación 47, donde XQ es un etilo.
49. The compound according to claim 47, wherein XQ is an ethyl.
50. El compuesto según la reivindicación 47, donde Χβ es un grupo - CH2COOH. 50. The compound according to claim 47, where Χβ is a -CH 2 COOH group.
51. El compuesto según una de las reivindicaciones 31 ó 32, que se selecciona de la siguiente lista: 0-propionil-GD1b, 0-propionil-GD3, 0-propionil-GD2, A-O- propionil-GT1b, B-0-prop¡on¡l-GT1b, di-0-propionil-GT1b, B-0-acetil-GT1b, di- 0-acetil-GT1 b, 0-Propionil-GM3, 0-Malonil-GM3, 0-Propionil-GM2, O-Malonil- GM2, 0-Malonil-GD3, 0-Malonil-GD2, 0-Propionil-GD1a, 0-Malonil-GD1a, O- Malonil-GD1b, A-0-Malonil-GT1b, B-0-Malonil-GT1b, di-0-Malonil-GT1b, B-O- Acetil-GQ1b, di-0-Acetil-GQ1b, A-0-Propionil-GQ1b, B-0-Propionil-GQ1b, di- 0-Propionil-GQ1b, A-0-Malonil-GQ b, B-0-Malonil-GQ1b y di-0-Malonil-GQ1b. 51. The compound according to one of claims 31 or 32, which is selected from the following list: 0-propionyl-GD1b, 0-propionyl-GD3, 0-propionyl-GD2, A-O-propionyl-GT1b, B-0-prop ¡on¡l-GT1b, di-0-propionyl-GT1b, B-0-acetyl-GT1b, di- 0-acetyl-GT1 b, 0-Propionyl-GM3, 0-Malonyl-GM3, 0-Propionyl-GM2, O-Malonyl- GM2, 0-Malonyl-GD3, 0-Malonyl-GD2, 0-Propionyl-GD1a, 0-Malonyl-GD1a, O- Malonyl-GD1b, A-0-Malonyl-GT1b, B-0-Malonyl- GT1b, di-0-Malonyl-GT1b, B-O- Acetyl-GQ1b, di-0-Acetyl-GQ1b, A-0-Propionyl-GQ1b, B-0-Propionyl-GQ1b, di- 0-Propionyl-GQ1b, A- 0-Malonyl-GQ b, B-0-Malonyl-GQ1b and di-0-Malonyl-GQ1b.
52. El compuesto según la reivindicación 32, que se selecciona de la siguiente lista: 52. The compound according to claim 32, which is selected from the following list:
0-propionil-GD3 0-propionyl-GD3
HOJA DE REEMPLAZO (Regla 23)
REPLACEMENT SHEET (Rule 23)
0-propionil-GD1b
0-propionyl-GD1b
0-propionil-GT1b
0-propionyl-GT1b
B-0-acetil-GT1b donde Y es cualquier ceramida. B-0-acetyl-GT1b where Y is any ceramide.
53. Compuesto sintetizado según el procedimiento de una de las reivindicaciones 1 a 30. 53. Compound synthesized according to the procedure of one of claims 1 to 30.
54. Composición farmacéutica que comprende al menos el compuesto según la reivindicación 31 y al menos un transportador farmacéuticamente aceptable, adyuvante y/o vehículo. 54. Pharmaceutical composition comprising at least the compound according to claim 31 and at least one pharmaceutically acceptable carrier, adjuvant and/or vehicle.
55. Composición farmacéutica que comprende al menos el compuesto según la reivindicación 32 y al menos un transportador farmacéuticamente aceptable, adyuvante y/o vehículo. 55. Pharmaceutical composition comprising at least the compound according to claim 32 and at least one pharmaceutically acceptable carrier, adjuvant and/or vehicle.
56. La composición farmacéutica definida en una de las reivindicaciones 54 ó 55, que además comprende otro principio activo. 56. The pharmaceutical composition defined in one of claims 54 or 55, which further comprises another active ingredient.
57. Uso del compuesto de una de las reivindicaciones 31 a 53, o de la composición farmacéutica de una de las reivindicaciones 54 a 56, para la elaboración de un medicamento. 57. Use of the compound of one of claims 31 to 53, or of the pharmaceutical composition of one of claims 54 to 56, for the preparation of a medicine.
58. Uso del compuesto de la reivindicación 32, o de la composición farmacéutica de una de las reivindicaciones 55 ó 56, para la elaboración de un medicamento. 58. Use of the compound of claim 32, or of the pharmaceutical composition of one of claims 55 or 56, for the preparation of a medicine.
HOJA DE REEMPLAZO (Regía 23)
REPLACEMENT SHEET (Rule 23)
59. Uso del compuesto según una de las reivindicaciones 56 ó 57, para la elaboración de un medicamento para el tratamiento o prevención de tumores. 59. Use of the compound according to one of claims 56 or 57, for the preparation of a medicine for the treatment or prevention of tumors.
60. Uso del compuesto según la reivindicación 58, donde el tumor se selecciona de la lista que comprende: astrocitoma, glioblastoma, oligodendroglioma, neuroblastoma o meningioma. 60. Use of the compound according to claim 58, wherein the tumor is selected from the list comprising: astrocytoma, glioblastoma, oligodendroglioma, neuroblastoma or meningioma.
61. Uso del compuesto de una de las reivindicaciones 31 a 53, como reactivo en ensayos biológicos.
61. Use of the compound of one of claims 31 to 53, as a reagent in biological assays.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ES201031552A ES2380864B1 (en) | 2010-10-22 | 2010-10-22 | INHIBITORS OF THE DIVISION OF TUMOR CELLS. |
| ESP201031552 | 2010-10-22 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2012052596A1 true WO2012052596A1 (en) | 2012-04-26 |
Family
ID=45974738
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/ES2011/070732 WO2012052596A1 (en) | 2010-10-22 | 2011-10-21 | Ganglioside derivatives and use thereof as inhibitors of tumour cell division |
Country Status (2)
| Country | Link |
|---|---|
| ES (1) | ES2380864B1 (en) |
| WO (1) | WO2012052596A1 (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2662697A1 (en) * | 1990-06-01 | 1991-12-06 | Snow Brand Milk Products Co Ltd | New ganglioside from milk and process for producing it |
| US5102663A (en) * | 1988-10-18 | 1992-04-07 | Sloan-Kettering Instutute For Cancer Research | Vaccine for stimulating or enhancing production of antibodies against 9-O-acetyl GD3 |
| WO1994014825A1 (en) * | 1992-12-18 | 1994-07-07 | Merck Patent Gmbh | Process for preparing gd2 ganglioside and o-acetyl-gd2 ganglioside |
| US5766887A (en) * | 1996-08-26 | 1998-06-16 | The Scripps Research Institute | Synthesis of 9-0-acetyl N-acetylneuraminic acid oligosaccharides |
-
2010
- 2010-10-22 ES ES201031552A patent/ES2380864B1/en not_active Expired - Fee Related
-
2011
- 2011-10-21 WO PCT/ES2011/070732 patent/WO2012052596A1/en active Application Filing
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5102663A (en) * | 1988-10-18 | 1992-04-07 | Sloan-Kettering Instutute For Cancer Research | Vaccine for stimulating or enhancing production of antibodies against 9-O-acetyl GD3 |
| FR2662697A1 (en) * | 1990-06-01 | 1991-12-06 | Snow Brand Milk Products Co Ltd | New ganglioside from milk and process for producing it |
| WO1994014825A1 (en) * | 1992-12-18 | 1994-07-07 | Merck Patent Gmbh | Process for preparing gd2 ganglioside and o-acetyl-gd2 ganglioside |
| US5766887A (en) * | 1996-08-26 | 1998-06-16 | The Scripps Research Institute | Synthesis of 9-0-acetyl N-acetylneuraminic acid oligosaccharides |
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| ABAD-RODRIGUEZ, J. ET AL.: "Purification and structure of neurostatin, an inhibitor of astrocyte division of mammalian brain.", JOURNAL OF NEUROCHEMISTRY., vol. 74, no. 6, 2000, pages 2547 - 2556 * |
| HOULISTON, R.S. ET AL.: "Identification of a Sialate O-Acetyltransferase from Campylobacter jejuni. Demonstration of direct transfer to the C-9 position of terminal -2, 8-linked sialic acid.", J. BIOL. CHEM., vol. 281, no. 17, 2006, pages 11480 - 11486, XP003008560, DOI: doi:10.1074/jbc.M512183200 * |
| IGARASHI, M. ET AL.: "Characteristics of gangliosides including O-acetylated species in growth cone membranes at several developmental stages in rat forebrain.", DEVELOPMENTAL BRAIN RESEARCH., vol. 78, no. 1, 1994, pages 17 - 24, XP024334455, DOI: doi:10.1016/0165-3806(94)90004-3 * |
| NIETO-SAMPEDRO, M. ET AL.: "Inhibitors of Glioma Growth that Reveal the Tumour to the Immune System.", CLINICAL MEDICINE INSIGHTS ONCOLOGY., vol. 5, 21 September 2011 (2011-09-21), pages 265 - 314 * |
| ROMERO-RAMIREZ, L. ET AL.: "Inhibiting Human Astrocytoma Growth: Structure -Activity Relationships in Neurostatin Related Glycolipids.", J. MED. CHEM., vol. 47, no. 21, 2004, pages 4983 - 4984 * |
| VALLE-ARGOS. B ET AL.: "Synthesis and characterization of neurostatin-related compounds with high inhibitory activity of glioma growth.", EUROPEAN JOURNAL OF MEDICINAL CHEMISTRY, vol. 45, no. 5, May 2010 (2010-05-01), pages 2034 - 43, XP026976527 * |
Also Published As
| Publication number | Publication date |
|---|---|
| ES2380864B1 (en) | 2013-04-15 |
| ES2380864A1 (en) | 2012-05-21 |
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