EP0652887A1 - Neuraminsäure-derivate - Google Patents

Neuraminsäure-derivate

Info

Publication number
EP0652887A1
EP0652887A1 EP93918621A EP93918621A EP0652887A1 EP 0652887 A1 EP0652887 A1 EP 0652887A1 EP 93918621 A EP93918621 A EP 93918621A EP 93918621 A EP93918621 A EP 93918621A EP 0652887 A1 EP0652887 A1 EP 0652887A1
Authority
EP
European Patent Office
Prior art keywords
acid
group
carboxylic
derived
amide according
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP93918621A
Other languages
English (en)
French (fr)
Inventor
Aurelio Romeo
Gunter Kirschner
Hari Manev
Martino Trimarco
Gino Toffano
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Fidia SpA
Original Assignee
Fidia SpA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Fidia SpA filed Critical Fidia SpA
Publication of EP0652887A1 publication Critical patent/EP0652887A1/de
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
    • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
    • C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
    • 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
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
    • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
    • C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
    • C07H13/06—Fatty acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
    • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
    • C07H13/08—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals directly attached to carbocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
    • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
    • C07H13/10—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals directly attached to heterocyclic rings
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06026—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K9/00—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof
    • C07K9/001—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof the peptide sequence having less than 12 amino acids and not being part of a ring structure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to new derivatives of neuraminic acid, especially carboxylic amides of the following formula,
  • Ac represents an acyl residue of a carboxylic acid of the aliphatic, araliphatic, aromatic, alicyclic or heterocyclic series, comprising the carboxylic amides, their 2-hydrocarbyl-glycosides, and their peracylated derivatives at the hydroxy groups of both these series of amides.
  • These compounds have interesting pharmacological properties, especially a protective effect against the neurotoxicity induced by excitatory amino acids of the of glutamic acid type, and can therefore be used in therapies of the central nervous system, such as those following cerebral degenerations or lesions, e.g., ischemia, hypoxia, epilepsy, trauma or compressions, metabolic dysfunctions, aging, toxic-infective and chronic neurodegenerative diseases, like Alzheimer's, Parkinson's, and Huntington's diseases.
  • cerebral degenerations or lesions e.g., ischemia, hypoxia, epilepsy, trauma or compressions, metabolic dysfunctions, aging, toxic-infective and chronic neurodegenerative diseases, like Alzheimer's, Parkinson's, and Huntington's diseases.
  • the present invention also provides pharmacological preparations containing the aforesaid derivatives for therapeutic use.
  • a third object of the present invention concerns the therapeutic use of these preparations.
  • a final object of the present invention concerns procedures for the production of these new derivatives.
  • the contents of each of the references cited herein are incorporated by reference in their entirety.
  • the amides and their derivatives according to the present invention can derive from both possible anomeric forms in position 2 of neuraminic acid, and therefore all the new compounds can be of a type at that position.
  • the steric configuration of the other carbon atoms of the neuraminic residue is the same as that of the natural acid.
  • the acyl group Ac on the nitrogen of the neuraminic acid residue in the aforesaid formula has at least 4 and not more than 24 carbon atoms, and derives from non- substituted or substituted acids, preferably from 1 to 3 functions selected from the group consisting of halogen atoms; free, esterified, or etherified hydroxylic or mercapto groups; free or esterified carboxylic or sulfonic groups, or such groups transformed into amides; and free hydrocarbylic groups or hydrocarbylic groups substituted aminic groups.
  • These acids can be interrupted by -SO-, -SO.,-, or phenylene groups in the carbon atom chain o ⁇ the hydrocarbylic residue.
  • the halogen atoms are preferentially fluorine, bromine, or chlorine.
  • Esterified hydroxylic or mercapto groups can derive from one of the acids mentioned regarding the Ac group, but they preferentially derive from aliphatic or aromatic acids with not more than 8 carbon atoms. Moreover, they can derive from inorganic acids such as, for example, sulfuric or phosphoric acid, or especially from their partial esters with mono- or polyvalent aliphatic alcohols, eventually with hydroxylic groups or aminic functions substituting in the hyrdocarbylic residues. Finally, they can derive from hydrocarbylsulfonic acids.
  • Ac groups containing functionally modified hydroxy, mercapto, or amino groups can also be present in the form of hydrocarbylic residues of the Ac acyl group, interrupted in the carbon atom chain by the heteroatoms -0-, -S-, or -NH-, and, in the particular case of esters of hydroxy or mercapto groups with partially esterified sulfuric or phosphoric acid, by groups of the type
  • hydrocarbylic residue Ac can be blocked by sulfoxide or sulfonyl residues.
  • converted carboxylic or sulfonic groups preferentially derive from lower aliphatic amines with not more than 4 carbon atoms, or from araliphatic amines with only one benzene ring and one or two carbon atoms in alkenyl residue.
  • the acids from which the Ac groups of the aliphatic series derive can be saturated or unsaturated, and in this case, they preferentially have only one double bond, and can have linear or branched chains.
  • acids from which the Ac groups of the aliphatic series derive can be saturated or unsaturated, and in this case, they preferentially have only one double bond, and can have linear or branched chains.
  • acids from which the Ac groups of the aliphatic series derive can be saturated or unsaturated, and in this case, they preferentially have only one double bond, and can have linear or branched chains.
  • acids from which the Ac groups of the aliphatic series derive can be saturated or unsaturated, and in this case, they preferentially have only one double bond, and can have linear or branched chains.
  • butyric, valeric, particularly normal valeric and isovaleric trimethylacetic (pivalic acid) , caproic, isocaproic, enantic, caprylic, pelargonic
  • levulinic acid must be mentioned; among dicarboxylic acids, succinic acid; and among natural amino acids, e.g., valine, leucine, phenylalanine, tryptophan, aminobutyric acid, methionine, lysine, aspartic acid, glutamic acid, proline, hydroxyproline; among the acids substituted with halogens, mono- and dichloroacetic acid, trichlorobutyric acid, and dibromobutyric acid.
  • dicarboxylic acids succinic acid
  • natural amino acids e.g., valine, leucine, phenylalanine, tryptophan, aminobutyric acid, methionine, lysine, aspartic acid, glutamic acid, proline, hydroxyproline
  • acids substituted with halogens mono- and dichloroacetic acid, trichlorobutyric acid, and dibromobutyric acid.
  • the Ac group of formula I can also derive from natural or synthetic peptides preferentially having not more than 12 amino acids, selected from naturally occurring amino acids, e.g., those aforesaid.
  • Acids from which derive an Ac group of araliphatic nature, are e.g., phenylacetic, cinnamic, phenylpropionic or atropic acid.
  • aromatic acids benzoic acid and its methylated homologues, salicylic acid, anthranilic acid, trimethoxybenzoic acid, phthalic or terephthalic acid, o,o' -dicarbonic acid, chlorobenzoic acid, vanillic acid, and veriatric or piperonilic acid must be mentioned.
  • cyclohexane- and cyclopentane-carbonic acids hexahydrophthalic, hexahydroisophhtalic and hexahydroterephthalic acids, camphoric and apocamphoric acid, and, among acids with a higher carbon atom content, prostaglandins and steroidic acids such as, for example, cholanic or cholic acid.
  • Ac represents an acyl group belonging to an acid of the heterocyclic series
  • this can be one of the following acids: nicotinic or isonicotinic, cinconninic, lysergic, isolysergic, dihydrolysergi c , 2-bromo-lysergic, 2-bromo-dihydrolysergic, 1-methyl-lysergic , 1-methyl -dihydro-lysergic, 1-methyl-2-bromo-lysergic or teophyllinacetic.
  • the carboxylamido functions according to the present invention can derive from ammonia (and in this case it is the non-substituted amide, -CONH 2 ) , or from primary or secondary aliphatic, aromatic, araliphatic, alicyclic or heterocyclic amines, which can also be substituted in the hydrocarbylic residue by one to three functions selected from the group consisting of free, esterified, or etherified hydroxylic or mercapto groups, halogens, free, esterified, or amide-modified carboxylic or sulfonic groups, and free or hydrocarbyl-substituted amino groups, wherein the hydrocarbyl group is blocked with an -SO- or-S0 2 - group.
  • These amines have no more than 24 carbon atoms.
  • Aliphatic amines can have an open, saturated, unsaturated, linear, branched or cyclic chain.
  • alkyl- and dialkylamines having from 1 to 12 carbon atoms, such as, for example, methylamine, ethylamine, propylamine, hexylamine, diethylamine, dimethylamine, diisopropylamine, dihexylamine and alkylenylamines having from 3 to 6 cyclic carbon atoms, wherein the rings are substituted or non-substituted, preferentially between one and three Cl-14 alkyl groups, e.g., methyl groups, like pyrrolidine, piperidine, and azepine.
  • hydrocarbylic chains can also be blocked with heteroatoms such as, for example, -0-, -S-, or -NH- groups, or they can be substituted, as already mentioned, with different functions, particularly alcoholic, amino, mercapto, carboxylic, and sulfonic functions, or by their functionally modified forms, such as esters, ethers, or alkylated derivatives.
  • heteroatoms such as, for example, -0-, -S-, or -NH- groups
  • aliphatic diamines like ethylenediamine, trimethylenediamine, tetramethylenediamine, penta- and hexamethylenediamine, piperazine and its N-alkyl or C-alkyl derivatives having a Cl-4 alkyl; aminoalcohols like aminoethanol or aminopropanol; aminomercaptanes like mercaptoethylamine; aliphatic aminoacids like all those mentioned for the Ac group of formula I; and aminosulfonic acids like taurine.
  • morpholine and thiomorpholine and their alkylated derivatives such as, for example, those which are N- or C-methylated, are also useful.
  • the amide groups eventually can derive from peptides, such as those mentioned for The Ac group.
  • the carboxylic group of the N-acyl-neuraminic acids can contain saturated or unsaturated aminic groups having between 14 and 24 carbon atoms, or bases present in the following lipids: phosphatidylethanolamine, phosphatidylserine, sphingosine, dihydrosphingosine , psychosine, dihydropsychosine, phosphorylcholine-sphingosine, phosphorylcholine-dihydrosphingosine, and phytosphingosine.
  • the carboxylamides of the present invention can also derive from aromatic or araliphatic amines, but preferentially from those having only one aromatic ring, which can be substituted with 1 to 3 functional groups selected from the group consisting of halogens, hydroxylic or methoxylic groups, carboxylic or sulfonic groups, or Cl-4 lower aliphatic hydrocarbylic groups such as, for example, aniline, anthranilic acid, 1-amino-4-sulfonic acid, and benzylamide.
  • a phenyl group can be present to block the carbon atom chain.
  • Amines which can be used for the conversion into amides according to the present invention include, for example, amines of pyrimidines such as cyanmethine, i.e. , 2 , 4-dimethyl-6-amino-pyrimidine, purine derivatives such as adenine, 4-aminouracil, and guanine, and alkaloids such as ephedrine, tyramine, and adrenalin.
  • pyrimidines such as cyanmethine, i.e. , 2 , 4-dimethyl-6-amino-pyrimidine
  • purine derivatives such as adenine, 4-aminouracil, and guanine
  • alkaloids such as ephedrine, tyramine, and adrenalin.
  • the 2-hydrocarbyl-glycosides of the aforesaid amides of neuraminic acids of formula I derive from alcohols of the aliphatic, cycloaliphatic, aromatic, araliphatic or heterocyclic series, particularly from alcohols of the aliphatic series having not more than 12 carbon atoms, or from the araliphatic series having preferentially only one benzene ring, eventually substituted with 1 - 3 lower Cl-4 alkyl groups, for example methyl groups, and not more than 4 carbon atoms in the aliphatic chain, or from alcohols of the alicyclic or aliphatic-alicyclic series having only one cycloaliphatic ring and not more than 14 carbon atoms, or from the heterocyclic series having not more than 12, and especially 6 carbon atoms, and only one heterocyclic ring containing 1 or 2 heteroatoms selected from the group consisting of -NH- , -O- and -S- .
  • These alcohols can also be substituted, particularly with functions selected from the group consisting of hydroxy, amino, and alkoxy groups having not more than 4 carbon atoms, and carboxylic and carbalkoxy groups having not more than 4 carbon atoms in the alkyl residues.
  • the aforesaid alcohols can be mono- and polyvalent, particularly bivalent.
  • alcohols of the aliphatic series of particular interest are lower alcohols having not more than 6 carbon atoms such as, for example, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, and tert-butyl alcohol, and ethyleneglycol and propyleneglycol, among divalent alcohols.
  • alcohols of the araliphatic series of particular interest are those having only one benzene residue, like benzyl and phenetyl alcohol; among alcohols of the alicyclic series, preferred are those having only one cycloaliphatic ring, like cyclohexylic alcohol.
  • steroid alcohols such as, for example, those of the pregnane group, like corticosteroids, and among these methylprednisolone, must be mentioned.
  • alcohols of the heterocyclic series there must be mentioned tetrahydrofuranol, tetrahydropyranol, furfuryl alcohol and pyridylcarbinol.
  • peracylated derivatives of the amides and their 2-hydrocarbyl-glycosides the hydroxy groups in position 2,4,7,8 and 9 are acylated with acids belonging to the aliphatic, aromatic, araliphatic, alicyclic and heterocyclic series.
  • Peracylated derivatives derive preferentially from acids of the aliphatic series having not more than 10 carbon atoms, like formic, acetic, and butyric acid and their isomers; valeric acids, like normal valeric, or pivalic acid; and capronic or capric acid.
  • these acids can also be substituted, and the peracylated derivatives can therefore derive from hydroxyacids like lactic acid, from amino acids like glycine, or from dibasic acids like succinic, malonic or maleic acid.
  • aromatic acids there must be mentioned those with only one benzene ring, particularly benzoic acid and its derivatives with methyl, hydroxy, amino or carboxylic groups such as, for example, p-aminobenzoic, salicylic and phthalic acid.
  • the new compounds according to present invention can eventually be transformed into their acidic addition salts or into metallic salts with organic bases, if the corresponding basic or acidic functions are present.
  • These salts can also be used for the therapeutic purposes described infra. With resepect to this equivalence between salts and amides in free form, it is obvious that what will be described for the compounds in free form, especially their pharmaceutical and medical applications, is also true for the corresponding salts, provided that these salts are therapeutically acceptable, and therefore they also form an object of the present invention.
  • These salts can also be used for the purification of the amides, and in this case also, therapeutically non-acceptable bases and acids can be used, such as salts of picric and picrolonic acid.
  • compounds according to the present invention include the amide, methylamide, ethylamide, dimethylamide, diethylamide, propylamide, glycine amide, L-serine amide, aminobutyric amide, L-cysteine amide, taurine amide, the amide of cysteic acid, homocysteic acid, N-palmitoyl-neuraminic acid, N-stearoyl-neuraminic acid, N-acetyl-neuraminic acid, N-propionyl-neuraminic acid, N-pivaloyl-neuraminic acid, N-valeroyl-neuraminic acid, N-caproyl-neuraminic acid, N-lauroyl-neuraminic acid, N-succinyl-neuraminic acid, phenylacetyl- neuraminic acid, benzoyl-neuraminic acid, trimethoxy- benzoyl-
  • Other compounds of the present invention include the amide, methylamide, ethylamide, dimethylamide, diethylamide, propylamide, glycine amide, L-serine amide, aminobutyric amide, L-cysteine amide, taurine amide of N-acylneuraminic acids having an acyl group deriving from one of the following acids: aminobutyric, methionine, lysine, aspartic acid, glutamic acid, proline, tryptophan, or from an acyl residue deriving from a peptide present in the thymus, and their 2-glycosides deriving from one of the following alcohols: methyl alcohol, ethyl alcohol, propyl alcohol, isopropyl alcohol, butyl alcohol, isobutyl alcohol, tertbutyl alcohol, ethyleneglycol, propyleneglycol, benzyl alcohol, methyl-prednisolone, tetrahydrofuran
  • Another group of interesting compounds according to the present invention is formed by the amides deriving from pyrrolidine, piperidine, azepine, ethylenediamine, trimethylenediamine, hexamethylenediamine, piperazine or N-methyl or N-ethyl-piperazine, aminoethanol, aminopropanol , mercaptoethylamine, morpholine, tiomorpholine, or peptides like those present in the thymus, and from phosphatidylethanolamine, phosphatidylserine , sphingosine, psychosine, dihydropsychosine, sphingosylphosphorylcholine, dihydrosphingosylphosphorylcholine, or from the phytosphingosine of one of the following N-acyl-neuraminic acids: N-palmitoyl-neuraminic acid, N-stearoyl-neuraminic acid, N-acetyl-neuraminic
  • derivatives are those peracylated at the hydroxy groups of N-acyl-neuraminic acid, and particularly peracetates, perpropionates, perbutyrates, pervalerianates, perpivalates, persuccinates and perbenzoates.
  • the present invention also comprises processes for the preparation of the new amides of N-acyl-neuraminic acids, their 2-hydrocarbylglycosides, and their salts. These processes are already known, and consist of the stepwise introduction of the amine function and eventually of the 2-glycosidic group into an N-acyl-neuraminic acid, and eventually of acyl groups "into the hydroxy groups and the final formation of their salts.
  • the carboxylic group of an N-acyl-neuraminic acid in which the acyl group is desired in the final compound, or its 2-hydrocarbyl-glycosidic derivatives, is transformed to an amide group and, if desired, the 2-hydrocarbyl group can be eliminated, again forming the hydroxy group; if desired, the obtained compound is converted into a peracylated derivative at the hydroxy functions.
  • the carboxy group of the neuraminic acid can be converted into the amide, and eventually the 2-hydroxy group in its hydrocarbyl derivatives in both sequences and acylate the free amino group with the desired acid and, if desired, peracylate the free hydroxy groups, or perform this peracylation in every step of the processes, e.g., at the beginning.
  • the conversion of the carboxylic group of N-acyl derivatives of neuraminic acid or of their 2-hydrocarbyl-glycosides to the corresponding amide can be performed directly starting from the acid, or from its metal or organic base salt, or indirectly preparing first the ester of the acid, an anhydride, or a halogenide of the acid, and then converting these compounds into the amide.
  • a preferred method consists of activating the carboxylic group and then reacting the intermediate with the desired amine, utilizing methods known in peptide chemistry, avoiding methods utilizing acidic or basic conditions. If metal salts of the acid, like sodium, are used, it is convenient to treat the salt with an ion exchange resin of the Dowex type or a similar resin. As an example, it is possible to use the condensation method in presence of carbodiimides, e.g., dicyclohexylcarbodiimide, benzylisopropylcarbodiimideor benzylethylcarbodiimide, in the presence of 1-hydroxybenzo-triazol, or the condensation in the presence of N,N' carbonyl-diimidazol.
  • carbodiimides e.g., dicyclohexylcarbodiimide, benzylisopropylcarbodiimideor benzylethylcarbodiimide
  • the transformation into the amide is carried out by direct treatment with the desired amine at relatively low temperature, e.g., room temperature or -5 °C to 10 °C, or higher temperatures, e.g., between 30 and 120 °C.
  • relatively low temperature e.g., room temperature or -5 °C to 10 °C, or higher temperatures, e.g., between 30 and 120 °C.
  • Ketones, aromatic hydrocarbides, dimethylformamide, dimethylsulfoxide, dioxane or tetrahydrofuran can be used as solvents.
  • the starting esters can be aliphatic esters, e.g., ethyl or methyl esters, or aromatic esters, e.g., phenols.
  • the 2-0-hydrocarbyl derivatives of the starting compounds or of the compounds already possessing the amine function are prepared according to the conditions known for the acetylation of aldehydes or ketones, or for the preparation of glycosides.
  • the 2-hydrocarbyl groups of the glycosides can be transformed at the hydroxy group at every step by hydrolysis with acids under mild conditions.
  • acylation of the amine group of neuraminic acid is performed at the end of the procedure, e.g., after amide or glycoside formation
  • known acylation methods are used, e.g., treatment of the compound with acid halogenides or anhydrides, eventually in the presence of inorganic or organic bases, like pyridine or collidine. This acylation can be performed contemporaneously with the acylation of hydroxy groups.
  • the aforesaid procedure according to the present invention also comprises all variations in which the procedure is stopped at every step, or in which the starting compound is an intermediate, or in which the starting compounds are prepared m situ.
  • the synthesis of the compounds of the present invention is illustrated by the following examples.
  • Rf 0.25, chloroform/methyl alcohol/water, 110:40:6.
  • Rf 0.50, chloroform/methyl alcohol/water, 110:40:6;
  • Example 6 ⁇ -2-0-ethylcrlycoside of the dimethylamide of N-acetylneuraminic acid 3.65 g (10 mM) of the ⁇ -2-O-ethylglycoside of N-acetylneuraminic acid ethyl ester were solubilized in 50 ml of anhydrous methyl alcohol; 4.5 g (100 mM) of dimethylamine were added. The mixture was stirred overnight at 25 °C. The solution was evaporated under vacuum and the residue was purified by silica gel chromatography, using as solvent a mixture of methylene chloride/methyl alcohol/water, 80:20:2.
  • Rf 0.38, chloroform/methyl alcohol/water, 110:40:6.
  • Example 8 ⁇ -2-O-ethylglvcoside of the dimethylaminopropylamide of N-palmitoylneuraminic acid (maleic acid salt) 5.62 g (10 mM) of the ⁇ -2-O-ethylglycoside of N-palmitoylneuraminic acid ethyl ester were solubilized in 50 ml of anhydrous methyl alcohol; 10.2 g (100 mM) of dimethylaminopropylamine were added. The mixture was stirred overnight at 25 °C. The solution was evaporated under vacuum and the residue was purified by silica gel chromatography, using as solventt a mixture of methylene chloride/methyl alcohol/water, 110:40:6.
  • Rf 0.12, chloroform/methyl alcohol/2.5N NH 4 0H, 40:60:15.
  • Rf 0.40, chloroform/methyl alcohol/0.3% CaCl 2 , 60:40:9.
  • N-palmitoylneuraminic acid maleic acid salt
  • N-palmitoylneuraminic acid ethyl ester were solubilized in 50 ml of anhydrous methyl alcohol; 10.2 g (100 mM) of dimethylaminopropylamine were added. The mixture was stirred overnight at 25 °C. The solution was evaporated under vacuum and the residue was purified by silica gel chromatography, using as solvent a mixture of methylene chloride/methyl alcohol/water, 110:40:6. The fractions containing the ⁇ .-2-O-ethylglycoside of the dimethylammopropylamide of N-palmitoyl-neuraminic acid were gathered and evaporated under vacuum. The residue was dissolved in 50 ml of water, a stoichiometric amount of maleic acid was added, and the material was lyophilized. Yield: 70%.
  • Rf 0.40, chloroform/methyl alcohol/0.3% CaCl 2 , 60:40:9.
  • Rf 0.69, chloroform/methyl alcohol/water, 110:40:6.
  • Rf 0.53, chloroform/methyl alcohol/2.5N NH 4 OH, 40:60:15.
  • Rf 0.21, chloroform/methyl alcohol/2.5N NH 4 0H, 40:60:15.
  • Example 16 o ⁇ -2-O-ethylqlvcoside of N-acetylneuraminic acid amide with L-alanine-D-isoglutamine 3.65 g (10 mM) of the o;-2-0-ethylglycoside of N-acetylneuraminic acid ethyl ester were solubilized in 40 ml of water, and 10 ml (10 mM) of NaOH were added. The solution was maintained at 25 °C for 30 minutes, neutralized with IN HCI, and eluted with water from a column containing 30 ml of Dowex 50x8 resin, pyridinium form.
  • the eluate was lyophilized and the residue was solubilized in 100 ml of anhydrous pyridine.
  • 1.15 g (10 mM) of N-hydroxysuccinimide and 4.13 g (20 mM) of N,N' -dicyclohexylcarbodiimide were added at -10 °C. After 15 minutes, the temperature was raised to 25 °C, and the mixture was stirred for 5 hours.
  • 5.14 g (15 mM) of L-alanine-D-isoglutamine benzyl ester hydrochloride (prepared according to Le Francier and Kusumoto) were added at 25 °C. The mixture was stirred overnight and then evaporated under vacuum.
  • the obtained residue was dissolved in 200 ml of a mixture of n-butanol/water/acetic acid, 4:1:1, and hydrogenated in an H 2 current in the presence of BaS0 4 -supported palladium. After filtration, the solution was evaporated, and the residue was purified by silica gel chromatography, using as solvent a mixture of methylene chloride/methyl alcohol/water, 60:35:8. The fractions containing the ⁇ _-2-0-ethylglycoside of N-acetyl ⁇ neuraminic acid amide with L-alanine-D-isoglutamine were gathered and evaporated under vacuum. The residue was dissolved in 200 ml of water and lyophilized. Yield: 60%.
  • Example 17 Peracetylated ⁇ -2-O-ethylglvcoside of N-palmitoyl ⁇ neuraminic acid amide with L-alanine-D-isoglutamine 5.56 g (10 mM) of the ⁇ .-2-O-ethylglycoside of N-palmitoylneuraminic acid, sodium salt, were solubilized in 50 ml of anhydrous N,N' -dimethylformamide at 25 °C; 3.06 g (11 mM) of p-bromophenacyl bromide were added and the solution was stirred overnight.
  • the residue was extracted three times with 200 ml of ethyl acetate, washed with 100 ml of cold IN HCI and twice with 50 ml of water.
  • the organic phases were anhydrified with anhydrous sodium sulfate, gathered, and evaporated under vacuum.
  • the residue was dissolved in 30 ml of water and eluted with water from a column containing 30 ml of Dowex 50x8 resin, pyridinium form.
  • the eluate was lyophilized and the residue was solubilized in 100 ml of anhydrous pyridine.
  • the solution was evaporated under vacuum and the residue was dissolved with 100 ml of water and extracted three times with 200 ml of methylene chloride.
  • the organic phases were washed twice with 50 ml of water and then gathered, anhydrified with anhydrous sodium sulfate, and evaporated under vacuum.
  • the obtained residue was solubilized in 50 ml of anhydrous N,N' -dimethylformamide at 25 °C; 2.64 g (20 mM) of sodium thiophenate were added and the mixture was stirred for 4 hours.
  • the solution was evaporated under high vacuum.
  • the residue was extracted three times with 200 ml of ethyl acetate, washed with 100 ml of cold IN HCI and twice with 50 ml of water.
  • the organic phases were anhydrified with anhydrous sodium sulfate, gathered, and evaporated under vacuum.
  • the residue was dissolved in 30 ml of water and eluted with water from a column containing 30 ml of Dowex 50x8 resin, pyridinium form.
  • the eluate was lyophilized and the residue was solubilized in 100 ml of anhydrous pyridine.
  • 1.15 g (10 mM) of N-hydroxysuccinimide and 4.13 g (20 mM) of N,N' -dicyclohexylcarbodiimide were added at -10 °C. After 15 minutes, the temperature was raised to 25 °C and the mixture was stirred for 5 hours.
  • the solution was stirred for 15 minutes at 25 °C and eluted with water from a column containing 30 ml of Dowex 50x8 resin, H + form.
  • the eluate was lyophilized, and purified by silica gel chromatography, using as solvent a mixture of methylene chloride/methyl alcohol/water, 60:40:9.
  • the fractions containing the j ⁇ -2-O-ethylglycoside of N-acetyl-neuraminic acid amide with L-alanine-D-isoglutamine were gathered and evaporated under vacuum. The residue was dissolved in 50 ml of water and lyophilized. Yield: 60%.
  • Rf ⁇ 0.12, chloroform/methyl alcohol/2.5N NH 4 OH, 60:35:8; 0.10, chloroform/methyl alcohol/0.3% CaCl 2/
  • Example 19 ⁇ -2-O-ethylglycoside of N-palmitoylneuraminic acid amide with L-alanine-D-isoglutamine 5.56 g (10 mM) of the j ⁇ -2-0-ethylglycoside of N-palmitoylneuraminic acid, sodium salt, were solubilized in 50 ml of anhydrous N,N' -dimethylformamide at 25 °C; 3.06 g (11 mM) of p-bromophenacyl bromide were added and the solution was stirred overnight. 18 ml of anhydrous pyridine and 10.2 g of acetic anhydride were added and stirring was conducted for 24 hours at 35 °C.
  • the solution was evaporated under vacuum and the residue was dissolved with 100 ml of water and extracted three times with 200 ml of methylene chloride.
  • the organic phases were washed twice with 50 ml of water and then gathered, anhydrified with anhydrous sodium sulfate and evaporated under vacuum.
  • the obtained residue was solubilized in 50 ml of anhydrous N,N' -dimethylformamide at 25 °C; 2.64 g (20 mM) of sodium thiophenate were added and the mixture was stirred for 4 hours.
  • the solution was evaporated under high vacuum.
  • the residue was extracted three times with 200 ml of ethyl acetate, washed with 100 ml of cold IN HCI and twice with 50 ml of water.
  • N-hydroxysuccinimide and 4.13 g (20 mM) of N,N' -dicyclohexylcarbodiimide were added at -10 °C. After 15 minutes, the temperature was raised to 25 °C and the mixture was stirred for 5 hours. 5.14 g (15 mM) of L-alanine-D-isoglutamine benzyl ester hydrochloride (prepared according to Le Francier and Kusumoto) were added at 25 °C. The mixture was stirred overnight and then evaporated under vacuum. The obtained residue was dissolved in 60 ml of anhydrous methanol at 25 °C, 100 mg of potassium terbutylate were added, and the mixture was stirred for 30 minutes.
  • N-acetylneuraminic acid ethyl ester prepared according to van der Vlengel et al . , Carbohydr. Res. 102, 121 (1982) , were solubilized in 40 ml of water and 10 ml (10 mM) of NaOH were added. The solution was maintained at 25 °C for 30 minutes, neutralized with IN HCI, and eluted with water from a column containing 30 ml of Dowex 50x8 resin, pyridinium form. The eluate was lyophilized and the residue was solubilized in 100 ml of anhydrous pyridine.
  • Example 21 i ⁇ -2-O-ethylglycoside of N-acetylneuraminic acid amide with arginine 3.59 g (10 mM) of the 3-2-O-ethylglycoside of
  • N-acetylneuraminic acid, sodium salt were solubilized in 50 ml of anhydrous N,N' -dimethylformamide at 25 °C;
  • the obtained residue was solubilized in 50 ml of anhydrous N,N' -dimethylformamide at 25 °C; 2.64 g (20 mM) of sodium thiophenate were added and the mixture was stirred for 4 hours. The solution was evaporated under high vacuum. The residue was extracted three times with 200 ml of ethyl acetate, washed with 100 ml of cold IN HCI and twice with 50 ml of water. The organic phases were anhydrified with anhydrous sodium sulfate, gathered, and evaporated under vacuum.
  • the obtained residue was dissolved in 60 ml of anhydrous methanol at 25 °C. 100 mg of potassium terbutylate were added, and the mixture was stirred for 30 minutes. 5 ml of anhydrous Dowex 50x8 resin, H + form, were added. The solution was filtered and evaporated under vacuum, and the residue was dissolved in 100 ml of a mixture of N-butanol/water/acetic acid, 4:1:1, and hydrogenated in an H 2 current in the presence of BaS0 4 -supported palladium. After filtration, the solution was evaporated, and the residue was purified by silica gel chromatography, using as solvent a mixture of methylene chloride/methyl alcohol/water, 110:40:6.
  • Example 22 jS-2-O-ethylglvcoside of N-palmitoylneuraminic acid amide with arginine 5.56 g (10 mM) of the j ⁇ -2-O-ethylglycoside of N-palmitoyl-neuraminic acid, sodium salt, were solubilized in 50 ml of anhydrous N,N' -dimethylformamide at 25 °C; 3.06 g (11 mM) of p-bromophenacyl bromide were added and the solution was stirred overnight. 18 ml of anhydrous pyridine and 10.2 g of acetic anhydride were added and stirring was conducted for 24 hours at 35 °C.
  • the solution was evaporated under vacuum and the residue was dissolved with 100 ml of water and extracted three times with 200 ml of methylene chloride.
  • the organic phases were washed twice with 50 ml of water and then gathered, anhydrified with anhydrous sodium sulfate, and evaporated under vacuum.
  • the obtained residue was solubilized in 50 ml of anhydrous N,N' -dimethylformamide at 25 ⁇ C; 2.64 g (20 mM) of sodium thiophenate were added and the mixture was stirred for 4 hours.
  • the solution was evaporated under high vacuum.
  • the residue was extracted three times with 200 ml of ethyl acetate, washed with 100 ml of cold IN HCI and twice with 50 ml of water.
  • the organic phases were anhydrified with anhydrous sodium sulfate, gathered, and evaporated under vacuum. The residue was dissolved in 30 ml of water and eluted with water from a column containing 30 ml of a Dowex 50x8 Dowex resin, pyridinium form. The eluate was lyophilized and then solubilized in 100 ml of anhydrous pyridine; 1.15 g (10 mM) of N-hydroxysuccinimide, 4.13 g (20 mM) of N,N' -dicyclohexylcarbodiimide and 11.6 g (10 mM) of pyridinium chloride were added at -10 °C.
  • Rf 0.14, chloroform/methyl alcohol/H 2 0, 110:40:6; 0.32, chloroform/methyl alcohol/2.5N NH 4 OH,
  • Rf 0.63, chloroform/methyl alcohol/H 2 0, 110:40:6.
  • Rf 0.30, chloroform/methyl alcohol/H 2 0, 80:20:2; 0.62, chloroform/methyl alcohol/2.5N NH 4 OH, 110:40:6.
  • Example 27 Dimethylaminopropylamide of N-palmitoyl-neuraminic acid 5.34 g (10 mM) of the ⁇ .-2-O-ethylglycoside of N-palmitoyl-neuraminic acid were suspended in a mixture of 100 ml of 0.1M H 2 S0 4 /ethanol, 4:1, at 60 °C, and stirred for 16 hours. The product was extracted once with 200 ml of ethyl acetate and then twice with 100 ml of ethyl acetate; the organic phases were washed three times with 50 ml of water, gathered, and evaporated under vacuum. The obtained residue was dissolved in 200 ml of anhydrous methanol at 25 °C. 20 ml of anhydrous Dowex 50x8 resin, H + form, were added. The mixture was stirred for 2 hours. To the filtered solution 10.2 g
  • Example 29 ⁇ -2-0-ethylglvcoside butylamide of N-palmitoylneuraminic acid 5.62 g (10 mM) of 3-2-O-ethylglycoside N-palmitoylneuraminic acid ethyl ester were solubilized in 50 ml of anhydrous methanol; 3.66 g (50 mM) of 2-butylamine were added. The mixture was stirred for 5 hours at 40 °C. The solution was evaporated under vacuum and the residue was purified by silica gel chromatography, using as solvent a mixture of methylene chloride/methyl alcohol/water, 80:10:1.
  • Rf 0.71, chloroform/methyl alcohol, 80:20;
  • N-palmitoylneuraminic acid were solubilized in 50 ml of pyridine; 2.3 g (20 mM) of pyridinium chloride and 4.12 g (20 mM) of N,N' -dicyclo-hexylcarbodiimide were added. The mixture was stirred for 2 hours at 25 °C. 8.8 g (100 mM) of dimethylaminoethylamine were added and the reaction was conducted overnight at 25 °C. The solution was evaporated under vacuum and the residue was purified by silica gel chromatography, using as solvent a mixture of methylene chloride/methyl alcohol/2.5N NH 4 OH, 80:20 :2.
  • Rf 0.11, chloroform/methyl alcohol, 70:30;
  • Rf 0.37, chloroform/methyl alcohol/2.5N NH 4 OH, 40:60:15.
  • Rf 0.1, chloroform/methyl alcohol/0.3% CaCl 2 , 55:45:10.
  • Rf 0.44, chloroform/methyl alcohol/2.5N NH 4 OH, 110:40:6.
  • Example 36 o.-2-O-ethylglycoside ethanolamide of N-palmitoyl- neuraminic acid
  • the residue was solubilized in 100 ml of ethanol/water, 1:1, and 11 ml (10 mM) of IM NaOH were added. The solution was maintained at 25 °C for 30 minutes. 2 ml of anhydrous Dowex 50x8 resin, H + form, were added. The filtered solution was evaporated under vacuum. The residue was solubilized in 50 ml of anhydrous pyridine. 2.3 g (20 mM) of pyridinium chloride and 4.12 g (20 mM) of N,N' -dicyclohexylcarbodiimide were added. The mixture was stirred for 2 hours at 25 °C.
  • Rf 0.42, chloroform/methyl alcohol/H 2 0, 80:20:2.
  • Rf 0.60, chloroform/methyl alcohol/H 2 0, 80:20:2.
  • Rf 0.56, chloroform/methyl alcohol/H 2 0, 80:20:2.
  • Example 44 jg-2-O-ethylglvcoside dimethylamide of N-lauroyl- neuraminic acid 3.23 g (10 mM) of the 3-2-O-ethylglycoside of neuraminic acid ethyl ester were solubilized at 5 °C in 100 ml of anhydrous methanol and 50 ml of anhydrous methylene chloride. 8.25 g (40 mM) of N,N' -dicyclohexylcarbodiimide, 2.0 g (20 mM) of triethylamine, and 4.57 g (20 mM) of lauric acid were added, and the mixture was stirred overnight at 5 °C.
  • Rf 0.54, chloroform/methyl alcohol/H 2 0, 80:20:2.
  • Example 45 ⁇ -2-O-ethylglvcoside dimethylamide of N-nicotinoyl- neuraminic acid 3.23 g (10 mM) of the /3-2-O-ethylglycoside of neuraminic acid ethyl ester were solubilized at 5 °C in 100 ml of anhydrous methanol and 50 ml of anhydrous methylene chloride. 8.25 g (40 mM) of N,N' -dicyclohexylcarbodiimide, 2.0 g (20 mM) of triethylamine, and 2.46 g (20 mM) of nicotinic acid were added, and the mixture was stirred overnight at 5 °C.
  • Rf 0.27, chloroform/methyl alcohol/H 2 0, 80:20:2.
  • Example 46 ⁇ -2-O-ethylglvcoside dimethylamide of N-trimethoxy- benzoylneuraminic acid 3.23 g (10 mM) of the ⁇ -2-O-ethylglycoside of neuraminic acid ethyl ester were solubilized at 5 °C in 100 ml of anhydrous methanol and 50 ml of anhydrous methylene chloride.
  • Rf 0.52, chloroform/methyl alcohol/H 2 0, 80:20:2.
  • Example 47 ⁇ -2-O-ethylglvcoside pyrrolidylamide of N-palmitoyl ⁇ neuraminic acid 5.56 g (10 mM) of the /3-2-O-ethylglycoside of N-palmitoylneuraminic acid, sodium salt, were solubilized in 50 ml of anhydrous pyridine. 2.3 g (20 mM) of pyridinium chloride and 4.12 g (20 mM) of N,N' -dicyclohexylcarbodiimide were added. The mixture was stirred for 2 hours at 25 °C. 7.17 g (100 mM) of pyrrolidine were added and the reaction was conducted overnight at 25 °C.
  • Rf 0.14, methylene chloride/methyl alcohol/H 2 0, 110:10:6.
  • the residue was solubilized in 150 ml of water and the product was extracted once with 300 ml chloroform and then twice with 150 ml of chloroform; the organic phases were washed three times with 150 ml of water, gathered, and evaporated.
  • the residue was purified by silica gel chromatography, using as solvent a mixture of methylene chloride/methyl alcohol, 9:1. The fractions containing the c ⁇ -2-O-ethylglycoside of N-palmitoylneuraminic methyl ester were gathered and evaporated under vacuum. The residue was crystallized from 100 ml of tert-butyl-ether. Yield: 70%.
  • Rf 0.25, chloroform/methyl alcohol/H 2 0, 110:10:6.
  • BIOLOGICAL STUDIES The antineuronotoxic activities of the new amides of the neuraminic acids of the present invention are demonstrated by the following experimental studies conducted with the j ⁇ -2-0-ethylglycoside of the dimethylamide of N-palmitoylneuraminic acid of the formula: identified as ND37.
  • Example 53 Antmeuronotoxic effect of ND37 in vitro on cerebellar granule cells: protective effect on exogenous glutamate induced neurotoxicity
  • Neurons were grown in 35 mm plates for 11-13 days and maintained in a humid environment (95% air and 5% C0 2 ) .
  • Cultures (3xl0 6 cells/plate) are mainly formed by granule cells (95%) with a small amount (5%) of glial cells (Gallo V. et al. : Selective release of glutamate from cerebellar granule cells differentiating in culture. Proc. Natl . Acad. Sci. USA 79, 7919-23, 1982) .
  • Derivative ND37 was solubilized at a concentration of 1x10 ⁇ ⁇ M in dimethylsulfoxide (1% DMSO) and then diluted at various concentrations in Locke's solution
  • Treated cells were washed (3 x 2 ml) with Locke's solution + 10% fetal calf serum, then washed (3 x 2 ml) with Locke's solution without Mg ++ . 100 ⁇ M glutamate (1.5 ml) in Locke's solution (-Mg ++ ) or culture medium
  • ND37 has a marked antineuronotoxic activity: the presence of the free compound in the incubation medium during the exposure to the glutamate toxin is not necessary.
  • the neuroprotective effect of ND37 is very high: at a concentration of lxlO -5 M there is a protection of about 63%, and the highest protection
  • Antineuronotoxic effect of ND37 in vitro in cerebellar granule cells protective effect on exogenous glutamate- induced neurotoxicity during cotreatment of cells with the active compound MATERIALS AND METHODS
  • Derivative ND37 was solubilized at a concentration of lxlO "2 M in dimethylsulfoxide (1% DMSO) , and then diluted at various concentrations in Locke's solution
  • the cell culture medium was aspirated from the plates (and correctly maintained) . Plates were washed (3 x 2 ml) with Locke's solution without Mg ++ , and solutions
  • Treated cells were washed (3 x 2 ml) with Locke's solution + 10% fetal calf serum, then washed (3 x 2 ml) with Locke's solution without Mg ++ .
  • 1.5 ml of Locke's solution (-Mg ++ ) or 50 ⁇ M of glutamate ⁇ the test compound (concentrations between 1 and 4xl0 "5 M) in 1.5 ml of Locke's solution (-Mg ++ ) were added.
  • the incubation was conducted for 15 minutes (37 °C) . Glutamate and the compound were removed.
  • the plates were washed with Locke's solution (2 x 2 ml) , and then incubated in the presence of the starting medium (correctly maintained) for 24 hours at 37 °C in an incubator (5% C0 2 ) .
  • cellular viability was assayed via quantification by the fluorescein diacetate (FDA) and propidium iodide (PI) colorimetric test (Manev H. et al . : Glutamate induced neuronal death in primary cultures of cerebellar granule cells : protection by synthetic derivatives of endogenous sphingolipids; J. Pharm. Exp. Ther. 252,1 419-427, 1990) .
  • FDA fluorescein diacetate
  • PI propidium iodide
  • Monolayers were washed with Locke's solution and stained for 3 minutes at 22 °C with a solution containing 36 ⁇ M FDA and 7 ⁇ M of PI .
  • the stained cells where immediately analyzed using a standard fluorescence microscope for epiillumination (Vanox Olympus, 450 nm excitation, 520 nm emission) .
  • FDA a non polar ester, crosses cell membranes and is hydrolyzed by intracellular esterases with the consequent production of a yellow greenish color. Neuronal damage influences the FDA-induced color and allows the permeation of PI, which is a polar compound capable of interacting with nuclear DNA, producing a brilliant red fluorescence.
  • ND37 protects neurons at a dose of 10-40 ⁇ M, even if administered contemporaneously with the application of the toxin. This shows the rapid mode of action of ND37.
  • Table 2 Antineuronotoxic effect of ND37 during cotreatment of exogenous glutamate on cerebellar granule cells (pro ⁇ tective effect)
  • Example 55 In vivo effect of ND37 on cerebral damage induced by intracerebroventricular (icv) injection of . N-methyl-D-aspartate in neonatal rats
  • the compound was administered subcutaneously (sc) after suspension in 1% DMSO (experiment No.l) or in 0.5% tragacanth (experiment No.2) .
  • the compound was tested at the following doses: 1-3-5 mg/kg sc.
  • the treatment was conducted performing two administrations:
  • mice saline (1 ⁇ l) + saline (1 ⁇ l) NMDA (25 nmoles/ ⁇ l) + saline (1 ⁇ l) NMDA (25 nmoles/ ⁇ l) + ND37 (1 mg/kg/ml) NMDA (25 nmoles/ ⁇ l) + ND37 (3 mg/kg/ml) NMDA (25 nmoles/ ⁇ l) + ND37 (5 mg/kg/ml)
  • the number of animals utilized in each experimental group (corresponding to the total number of animals, i.e., experiment 1 + experiment 2, according to Table 3) is indicated in brackets.
  • Animals were sacrificed on the 12th day (i.e., 5 days after NMDA injection) for the evaluation of the in toto brain weight, defined in mg.
  • Results show that: treatment with ND37 is effective in reducing the brain damage induced by the excitotoxin (evaluated as the lowering of total brain weight;
  • ND37 is significatively effective (p ⁇ 0.01) at a dose of 1 mg/kg sc.
  • brackets is the indication of the number of animals.
  • Compounds under examination were administered at doses of 1-3-5 mg/kg sc 1 hours before NMDA injection (25 nmoles/1 ul) and immediately after NMDA. Brain weight is expressed in mg.
  • Neurons were grown in 35 mm plates for 11-13 days and maintained in a humid environment (95% air and 5% C0 2 ) .
  • Cultures (3xl0 6 cells/plate) are mainly formed by granule cells (95%) . with a small amount (5%) of glial cells (Gallo V. et al. : Selective release of glutamate from cerebellar granule cells differentiating in culture. Proc. Natl. Acad. Sci. USA 79, 7919-23, 1982) .
  • Glial proliferation was prevented by arabinofuranoside cytosine.
  • Derivative ND37 was solubilized at a concentration of 10 M in dimethylsulfoxide (1% DMSO) , and then diluted at various concentrations (0.1 - 1 - 10 - 20 ⁇ M) in Locke's solution (154 mM NaCl/5.6 mM KCl/ 3.6 mM NaHC0 3 /2.3 mM CaCl 2 /l mM MgCl 2 /5.6 mM glucose/5 mM HEPES, pH 7.4) .
  • the cell culture medium was aspirated from the plates. Plates were washed (3 x 2 ml) with Locke's solution, and solutions (750 ⁇ l) containing the compound to be tested (concentrations from 0.1 to 20 ⁇ M) were added and incubated for 2 hours in an incubator at 37 °C (5% C0 2 ) both in the presence and absence of different depolarizing concentrations of KCl (between 5 and 50 mM) . The incubation medium was gathered, filtered through a 0.2 ⁇ filter, and processed (220 ⁇ l) for the analysis of the amino acid content.
  • the glutamate and aspartate content (expressed as ⁇ M) was evaluated by HPLC (high pressure liquid chromatography) , according to Bidlingmeryer B.A. et al . , J. Chromatogr. 336, 93-104, 1984.
  • ND37 is able to diminish in cerebellar granule cells, in a dose-dependent manner (Table 4) , the potassium-induced increase of glutamate and aspartate (Table 5) .
  • ND 37 completely abolished the glutamate increase induced by KCl in the culture medium at a dose of 20 ⁇ M (Table 5) .
  • ND37 may reside in the release and/or uptake of endogenous compounds.
  • Table 5 Effect of ND37 (20 uM) on extracellular glutamate and aspartate content in cerebellar granule cells in depo ⁇ larizing conditions induced by different concentrations of KCl (15 - 50 mM) .
  • Neurons were grown on 35 mm plates for 7-8 days and maintained in a humid environment (95% air and 5% C0 2 ) .
  • Cultures (2.5xl0 6 cells/plate) were mainly formed by granule cells (95%) , v.'ith a small amount (5%) of glial cells (Gallo V. et al. : Selective release of glutamate from cerebellar granule cells differentiating in culture. Proc. Natl. Acad. Sci. USA 79, 7919-23, 1982) .
  • Glial proliferation was prevented by arabinofuranoside cy osine.
  • Cultures were grown on 35 mm plates, to which was added 10 ⁇ g/ml of poly-L-lysine, at a density of 5xl0 5 cortical neurons/plate. Cultures were prepared on basal Eagle medium (Gibco) containing 10% fetal calf serum
  • ND37 Cortical neurons were cultivated for 3 weeks.
  • Derivative ND37 was solubilized at a concentration of lxlO -2 M in dimethylsulfoxide (1% DMSO) , and then diluted at various concentrations in Locke's solution (-Mg ++ ) (154 mM NaCl / 5.6 mM KCl / 3.6 mM NaHC0 3 / 2.3 mM CaCl 2 / 5.6 mM glucose / 5 mM HEPES, pH 7.4) .
  • ND37 was analyzed at concentrations of 20 and 30 ⁇ M.
  • Glutamate (50 ⁇ M) able to activate currents directed inside, was released by pressure on voltage-clamped cell bodies of cerebellar and cortical neurons. Because these experiments were performed in the absence of Mg ++ , the response to glutamate was probably mediated by the NMDA and non-NMDA glutamate receptor. The combined administration of ND37 (30 ⁇ M) and glutamate (50 ⁇ M) , does not influence the control response, which is not different from the controls (mean 200 pA at voltage - 50 mV) in the three different conditions in 3 cerebellar and 5 cortical preparations . Results
  • Table 6 Effect of ND37 on glutamate controlled cationic channels activity in cerebellar granule neurons and cortical neurons in culture.
  • Group n 10 Channel conductivity Opening frequence
  • ND37 a compound protecting neurons from glutamate receptor-mediated toxicity, does not seem to produce this effect blocking the ionotropic glutamate receptors, as can be seen from the absence of effects of cationic glutamate stimulated channels.
  • Example 57 The neuritogenic activity of the new compounds of the present invention can be shown by experiments performed with the aforesaid compound ND37 , and with the 2-ethylglycoside of ⁇ -palmitoyl-neuraminic acid dimethylaminopropylamide, which will be subsequently referred to as ⁇ D35.
  • C1300 mouse neuroblastoma cells, Neuro-2a clone, (American Cell Type Culture Collection, Bethesda, MD) were grown at a density of 10,000 cells/well in a culture medium containing Dulbecco's modified Eagle medium (DMEM, Flow) , 10% fetal calf serum (FCS, batch IP 02, Seromed) , penicillin (100 units per ml, Irvine) , and L-glutamine (2 mM, Sigma) . Cells were incubated at 37 °C for 24 hours, and medium was removed and substituted with 350 ⁇ l of fresh medium plus compounds to be tested.
  • DMEM Dulbecco's modified Eagle medium
  • FCS 10% fetal calf serum
  • PC penicillin
  • L-glutamine (2 mM, Sigma
  • Neuritogenic activity (cell number with neurites, optical microscopy) Culture plates were incubated with compounds to be tested and analyzed using a phase contrast microscope (250x) . Nine fields with prefixed coordinates were chosen and photographed. Then the total number of cells and those with neurites (length at least double the cell diameter) were counted in blind in each photograph. The percentage of cells with neurites was determined after counting of at least 100 cells (Facci L. et al . : Promotion of neuritogenesis in mouse neuroblastoma cells by exogenous ganglioside GM1. J. Neurochem. Raven Press, New York, 299-305,1984) .
  • results show that derivatives ND35 and ND37 both induce neuritogenesis in vitro.
  • the neuritogenic effect was already significant at a concentration of 5xl0 ⁇ 5 M (p ⁇ 0.01), with the highest efficacy (about 48% of cells with neurites) at a concentration of lxlO -4 M.
  • Table 7 Neuritogenic effect of ND35 and ND37 in N2a neuroblastoma cells.
  • the new derivatives of neuraminic acid can be used in pathologies related to the excitotoxic effect of excititatory amino acids. It has been shown that these amino acids, e.g., glutamic acid and aspartic acid, besides their important functions in several physiological processes such as, for example, synaptogenesis and plasticity, are involved in the ethiogenesis and/or evolution of different pathologies related to neuronal evolution and/or death.
  • neuronal damage can have several causes, neuronal disfunctions excite a cascade of cellular events, such as the activation of Ca ++ ion-dependent enzymes, the influx of Ca ++ ions, and the activation of second messengers, which cause neuronal death.
  • Oxidative Damage to the nervous system due to excititatory amino acids is present in ischaemia, hypoxia, epilepsy, trauma, compressions, metabolic disfunctions, aging, and toxic-infective and chronic neurodegenerative diseases, like Alzeimer's and Huntington's diseases.
  • these new compounds Because of the modulatory effect of the new derivatives on the processes of release and/or uptake, and of the increase in the intracellular space, of neurotransmitter amino acids, these new compounds have therapeutic relevance in neuro-psychiatric disorders, where the pathological event derives from the imbalance of the aforesaid processes. Owing to the fact that the protective action of the derivatives of invention against the toxicity of excititatory amino acids occurs through the activation of glutamate receptors, the use of these compounds does not have the disadvantages of other known derivatives, which block these receptors (see Olney J.W. et al. : "Pathological changes induced in cerebrocortical neurons by phencyclidine and related drugs", Science 224, 1360-1362, 1989; Olney J.W. et al. : "NMDA antagonist neurotoxicity: mechanism and prevention, Science 254, 1515-1518, 1991) .
  • the new compounds of the present invention because of their neuritogenic activity, are valuable in therapies related to the recovery of nervous functions in pathologies associated with neuronal damage, like peripheral neuropathies.
  • Objects of the present invention also include pharmaceutical preparations having, as active ingredients, one or more of the new aforesaid derivatives and, particularly, those especially mentioned or those described in the foregoing examples.
  • These pharmaceutical preparations can used for oral, rectal, parenteral, local or intradermic use. They can therefore be in solid or semisolid form, e.g., pills, tablets, gelatineous soft capsules, capsules, soft gelatin suppositories, etc.
  • parenteral use it is possible to use formulations for intramuscular, subcutaneous, or transdermic use, or suitable for infusions or intravenous injections, and they can therefore be prepared as solutions of active components or as a lyophilized powder of the active components, eventually to be added to one or more excipients or pharmaceutically acceptable solvents, which are usable for the aforesaid purpose, and which are osmolar with physiological fluids.
  • spray preparations e.g, . nasal sprays
  • ointments for topical use, or plasters for transdermal administration can be used.
  • the preparations of the present invention can be used both in man and animals. Preferably, they contain between about 0.01 % and 10 % of the active components for solutions, sprays, ointments and creams, and between 1 % and 100 %, preferably between 5 % and 50 % of the active component, for solid form preparations.
  • the dosage will vary according to the indication, the desired effect, and the route of administration.
  • the dosage varies preferentially between 0.05 and 10 mg per kg body weight per day, and especially between 0.05 and 2 mg per kg body weight per day.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Neurosurgery (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Biomedical Technology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Neurology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Psychiatry (AREA)
  • Cardiology (AREA)
  • Hospice & Palliative Care (AREA)
  • Urology & Nephrology (AREA)
  • Vascular Medicine (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Saccharide Compounds (AREA)
EP93918621A 1992-08-03 1993-08-03 Neuraminsäure-derivate Withdrawn EP0652887A1 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
ITPD920146 1992-08-03
ITPD920146A IT1260156B (it) 1992-08-03 1992-08-03 Derivati dell'acido neuraminico
PCT/US1993/007307 WO1994003469A1 (en) 1992-08-03 1993-08-03 New derivatives of neuraminic acid

Publications (1)

Publication Number Publication Date
EP0652887A1 true EP0652887A1 (de) 1995-05-17

Family

ID=11390038

Family Applications (1)

Application Number Title Priority Date Filing Date
EP93918621A Withdrawn EP0652887A1 (de) 1992-08-03 1993-08-03 Neuraminsäure-derivate

Country Status (6)

Country Link
EP (1) EP0652887A1 (de)
JP (1) JPH08502953A (de)
AU (1) AU4801493A (de)
CA (1) CA2141679C (de)
IT (1) IT1260156B (de)
WO (1) WO1994003469A1 (de)

Families Citing this family (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5783564A (en) * 1993-12-24 1998-07-21 Mitsubishi Chemical Corporation Sialic acid derivatives
TW479061B (en) * 1993-12-24 2002-03-11 Mitsubishi Chem Corp Sialic acid derivatives
IT1271624B (it) * 1994-03-21 1997-06-04 Lifegroup Spa Derivati glucosidici di n-acil alchilamine capaci di esercitare effetto o neuroprotettivo utilizzabili nelle patologie acute e croniche del sistema nervoso centrale correlate ad eccitotossicita'
US5849709A (en) * 1995-05-10 1998-12-15 Glycomed Incorporated Saccharopeptides and derivatives thereof
JP4094662B2 (ja) * 1995-06-23 2008-06-04 三菱化学株式会社 シアル酸誘導体
US5824662A (en) * 1996-09-27 1998-10-20 Guilford Pharmaceuticals Inc. Treatment of global and focal ischemia using naaladase inhibitors
PL332413A1 (en) 1996-09-27 1999-09-13 Guilford Pharm Inc Compositions containing inhibitors of naaladase as well as methods of treating glutamatic anomaly and influencing neuronic functions among animals
WO1999052931A1 (fr) 1998-04-10 1999-10-21 Mitsubishi Chemical Corporation Dispersion solide contenant un derive d'acide sialique
WO2002094302A1 (en) * 2001-05-23 2002-11-28 Gil-Ja Jhon Compositions comprising extract of ganoderma lucidum, oleamide and its structural analogue as an effective component for preventing or treating dementia
JP4005115B1 (ja) * 2007-02-08 2007-11-07 日本臓器製薬株式会社 疼痛疾患治療剤
JP5327839B2 (ja) * 2008-06-09 2013-10-30 国立大学法人埼玉大学 シアル酸誘導体の製造方法とインフルエンザウィルス阻害剤としての利用
JP5298390B2 (ja) * 2009-11-19 2013-09-25 学校法人福岡大学 フルオラス化シアル酸誘導体およびその分析方法
TWI599354B (zh) 2016-08-10 2017-09-21 國立臺灣師範大學 四氫吡喃醇衍生化合物用於製備Tau蛋白病變類疾病之醫藥組成物上之用途

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3014027A (en) * 1956-01-20 1961-12-19 Ciba Pharm Prod Inc N-acylamino compounds
IL41409A (en) * 1972-02-04 1977-04-29 Richter Gedeon Vegyeszet Lysergic acid amides and process for their preparation
JPS60231690A (ja) * 1984-05-01 1985-11-18 Kanto Ishi Pharma Co Ltd Ｎ―置換ノイラミン酸誘導体
HU906341D0 (en) * 1986-10-13 1991-04-29 Sandoz Ag Process for producing peptonic derivatives modified with sugar and pharmaceutical preparatives containing these compounds as active substance
JPH03151398A (ja) * 1989-11-06 1991-06-27 Mect Corp シアル酸結合生理活性ペプチド及びその製造方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9403469A1 *

Also Published As

Publication number Publication date
ITPD920146A0 (it) 1992-08-03
JPH08502953A (ja) 1996-04-02
CA2141679A1 (en) 1994-02-17
ITPD920146A1 (it) 1994-02-03
CA2141679C (en) 2000-09-19
WO1994003469A1 (en) 1994-02-17
IT1260156B (it) 1996-03-28
AU4801493A (en) 1994-03-03

Similar Documents

Publication Publication Date Title
EP1071702B1 (de) Fettsäurederivate von gallensäure und gallensäurederivaten
CA2141679C (en) New derivatives of neuraminic acid
EP0373038B1 (de) Lysosphingolipid-Derivate
NZ236057A (en) N-acyl-, n'-acyl- and n,n'-diacyl-n,n'-di-lysogangliosides and derivatives thereof, preparation and pharmaceutical compositions thereof.
CS205027B2 (en) Method of producing glucosamine derivatives
EP0599908B1 (de) Neue ganglioside-derivaten
US5677285A (en) Derivatives of neuraminic acid
FI92324C (fi) Menetelmä lääkeaineina käyttökelpoisten 2'-deoksiuridiinijohdannaisten valmistamiseksi
DE69031448T2 (de) Halbsynthetische Gangliosidanaloge
Bulusu et al. Acyclic analogs of lipid A: synthesis and biological activities.
HU205129B (en) Process for producing lysoganglioside derivatives, as well as pharmaceutical compositions comprising such derivatives as active ingredient
PT88568B (pt) Processo para a preparacao de derivados de sacaridos
EP0410883B1 (de) Di-lysogangliosidderivate
US6407072B1 (en) Lysoganglioside derivatives
US5795869A (en) Sulfated lyso-ganglioside derivatives
US5849717A (en) O-sulfated gangliosides and lyso-ganglioside derivatives
EP0688330B1 (de) Neue o-sulfatierte ganglioside und lyso-ganglioside-derivaten
AU685787B2 (en) New sulfated lyso-ganglioside derivatives

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19950227

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LI LU MC NL PT SE

17Q First examination report despatched

Effective date: 19970806

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20020301